US20230078265A1 - Methods and compositions for editing nucleotide sequences - Google Patents

Abstract

The present disclosure provides compositions and methods for conducting prime editing of a target DNA molecule (e.g., a genome) that enables the incorporation of a nucleotide change and/or targeted mutagenesis. The nucleotide change can include a single-nucleotide change (e.g., any transition or any transversion), an insertion of one or more nucleotides, or a deletion of one or more nucleotides. More in particular, the disclosure provides fusion proteins comprising nucleic acid programmable DNA binding proteins (napDNAbp) and a polymerase (e.g., reverse transcriptase), which is guided to a specific DNA sequence by a modified guide RNA, named an PEgRNA. The PEgRNA has been altered (relative to a standard guide RNA) to comprise an extended portion that provides a DNA synthesis template sequence which encodes a single strand DNA flap, which is homologous to a strand of the targeted endogenous DNA sequence to be edited, but which contains the desired one or more nucleotide changes and which, following synthesis by the polymerase (e.g., reverse transcriptase), becomes incorporated into the target DNA molecule. Also disclosed herein are various methods that leverage prime editing, including treating trinucleotide repeat contraction diseases, installing targeted peptide tags, treating prion disease through the installation of protection mutations, manipulating RNA-encoding genes for the installation of RNA tags for controlling the function and expression of RNA, using prime editing to construct sophisticated gene libraries, using prime editing to insert immunoepitopes into proteins, use of prime editing to insert inducible dimerization domains into protein targets, and delivery methods, among others.

Description

RELATED APPLICATIONS AND INCORPORATION BY REFERENCE
• This U.S. Provisional application refers to and incorporates by reference the following applications, namely, U.S. Provisional Application No. 62/820,813, filed Mar. 19, 2019 (Attorney Docket No. B1195.70074US00), U.S. Provisional Application No. 62/858,958 (Attorney Docket No. B1195.70074US01), filed Jun. 7, 2019, U.S. Provisional Application No. 62/889,996 (Attorney Docket No. B1195.70074US02), filed Aug. 21, 2019, U.S. Provisional Application No. 62/922,654, filed Aug. 21, 2019 (Attorney Docket No. B1195.70083US00), U.S. Provisional Application No. 62/913,553 (Attorney Docket No. B1195.70074US03), filed Oct. 10, 2019, U.S. Provisional Application No. 62/973,558 (Attorney Docket No. B1195.70083US01), filed Oct. 10, 2019, U.S. Provisional Application No. 62/931,195 (Attorney Docket No. B1195.70074US04), filed Nov. 5, 2019, U.S. Provisional Application No. 62/944,231 (Attorney Docket No. B1195.70074US05), filed Dec. 5, 2019, U.S. Provisional Application No. 62/974,537 (Attorney Docket No. B1195.70083US02), filed Dec. 5, 2019, U.S. Provisional Application No. 62/991,069 (Attorney Docket No. B1195.70074US06), filed Mar. 17, 2020, and U.S. Provisional application No. (serial number not available as of this filing) (Attorney Docket No. B1195.70083US03), filed Mar. 17, 2020.
GOVERNMENT SUPPORT
• This invention was made with government support under grant numbers U01AI142756, RM1HG009490, R01EB022376, and R35GM118062 awarded by the National Institutes of Health. The government has certain rights in the invention.
BACKGROUND OF THE INVENTION
• Pathogenic single nucleotide mutations contribute to approximately 50% of human diseases for which there is a genetic component,⁷ according to some estimates. Unfortunately, treatment options for patients with these genetic disorders remain extremely limited, despite decades of gene therapy exploration⁸. Perhaps the most parsimonious solution to this therapeutic challenge is direct correction of single nucleotide mutations in patient genomes, which would address the root cause of disease and would likely provide lasting benefit. Although such a strategy was previously unthinkable, recent improvements in genome editing capabilities brought about by the advent of the CRISPR/Cas system⁹ have now brought this therapeutic approach within reach. By straightforward design of a guide RNA (gRNA) sequence that contains ˜20 nucleotides complementary to the target DNA sequence, nearly any conceivable genomic site can be specifically accessed by CRISPR associated (Cas) nucleases^1,2. To date, several monomeric bacterial Cas nuclease systems have been identified and adapted for genome editing applications¹⁰. This natural diversity of Cas nucleases, along with a growing collection of engineered variants^11-14, offers fertile ground for developing new genome editing technologies.
• While gene disruption with CRISPR is now a mature technique, precision editing of single base pairs in the human genome remains a major challenge³. Homology directed repair (HDR) has long been used in human cells and other organisms to insert, correct, or exchange DNA sequences at sites of double strand breaks (DSBs) using donor DNA repair templates that encode the desired edits¹⁵. However, traditional HDR has very low efficiency in most human cell types, particularly in non-dividing cells, and competing non-homologous end joining (NHEJ) leads predominantly to insertion-deletion (indel) byproducts¹⁶. Other issues relate to the generation of DSBs, which can give rise to large chromosomal rearrangements and deletions at target loci¹⁷, or activate the p53 axis leading to growth arrest and apoptosis^18,19.
• Several approaches have been explored to address these drawbacks of HDR. For example, repair of single-stranded DNA breaks (nicks) with oligonucleotide donors has been shown to reduce indel formation, but yields of desired repair products remain low²⁰. Other strategies attempt to bias repair toward HDR over NHEJ using small molecule and biologic reagents^21-23. However, the effectiveness of these methods is likely cell-type dependent, and perturbation of the normal cell state could lead to undesirable and unforeseeable effects.
• Recently, the inventors, led by Prof. David Liu et al., developed base editing as a technology that edits target nucleotides without creating DSBs or relying on HDR^4-6,24-27. Direct modification of DNA bases by Cas-fused deaminase enzymes allows for C●G to T●A, or A●T to G●C, base pair conversions in a short target window (˜5-7 bases) with very high efficiency. As a result, base editors have been rapidly adopted by the scientific community. However, the following factors limit their generality for precision genome editing: (1) “bystander editing” of non-target C or A bases within the target window are observed; (2) target nucleotide product mixtures are observed; (3) target bases must be located 15±2 nucleotides upstream of a PAM sequence; and (5) repair of small insertion and deletion mutations is not possible.
• Therefore, the development of programmable editors that are flexibly capable of introducing any desired single nucleotide change and/or which could install base pair insertions or deletions (e.g., at least 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more base pair insertions or deletions) and/or which could alter or modify the nucleotide sequence at a target site with high specificity and efficiency would substantially expand the scope and therapeutic potential of genome editing technologies based on CRISPR.
SUMMARY OF THE INVENTION
• The present invention describes an entirely new platform for genome editing called “prime editing.” Prime editing is a versatile and precise genome editing method that directly writes new genetic information into a specified DNA site using a nucleic acid programmable DNA binding protein (“napDNAbp”) working in association with a polymerase (i.e., in the form of a fusion protein or otherwise provided in trans with the napDNAbp), wherein the prime editing system is programmed with a prime editing (PE) guide RNA (“PEgRNA”) that both specifies the target site and templates the synthesis of the desired edit in the form of a replacement DNA strand by way of an extension (either DNA or RNA) engineered onto a guide RNA (e.g., at the 5′ or 3′ end, or at an internal portion of a guide RNA). The replacement strand containing the desired edit (e.g., a single nucleobase substitution) shares the same sequence as the endogenous strand of the target site to be edited (with the exception that it includes the desired edit). Through DNA repair and/or replication machinery, the endogenous strand of the target site is replaced by the newly synthesized replacement strand containing the desired edit. In some cases, prime editing may be thought of as a “search-and-replace” genome editing technology since the prime editors, as described herein, not only search and locate the desired target site to be edited, but at the same time, encode a replacement strand containing a desired edit which is installed in place of the corresponding target site endogenous DNA strand.
• The prime editors of the present disclosure relate, in part, to the discovery that the mechanism of target-primed reverse transcription (TPRT) or “prime editing” can be leveraged or adapted for conducting precision CRISPR/Cas-based genome editing with high efficiency and genetic flexibility (e.g., as depicted in various embodiments of FIGS. 1A-1F). TPRT is naturally used by mobile DNA elements, such as mammalian non-LTR retrotransposons and bacterial Group II introns^28,29. The inventors have herein used Cas protein-reverse transcriptase fusions or related systems to target a specific DNA sequence with a guide RNA, generate a single strand nick at the target site, and use the nicked DNA as a primer for reverse transcription of an engineered reverse transcriptase template that is integrated with the guide RNA. However, while the concept begins with prime editors that use reverse transcriptases as the DNA polymerase component, the prime editors described herein are not limited to reverse transcriptases but may include the use of virtually and DNA polymerase. Indeed, while the application throughout may refer to prime editors with “reverse transcriptases,” it is set forth here that reverse transcriptases are only one type of DNA polymerase that may work with prime editing. Thus, where ever the specification mentions “reverse transcriptases,” the person having ordinary skill in the art should appreciate that any suitable DNA polymerase may be used in place of the reverse transcriptase. Thus, in one aspect, the prime editors may comprise Cas9 (or an equivalent napDNAbp) which is programmed to target a DNA sequence by associating it with a specialized guide RNA (i.e., PEgRNA) containing a spacer sequence that anneals to a complementary protospacer in the target DNA. The specialized guide RNA also contains new genetic information in the form of an extension that encodes a replacement strand of DNA containing a desired genetic alteration which is used to replace a corresponding endogenous DNA strand at the target site. To transfer information from the PEgRNA to the target DNA, the mechanism of prime editing involves nicking the target site in one strand of the DNA to expose a 3′-hydroxyl group. The exposed 3′-hydroxyl group can then be used to prime the DNA polymerization of the edit-encoding extension on PEgRNA directly into the target site. In various embodiments, the extension—which provides the template for polymerization of the replacement strand containing the edit—can be formed from RNA or DNA. In the case of an RNA extension, the polymerase of the prime editor can be an RNA-dependent DNA polymerase (such as, a reverse transcriptase). In the case of a DNA extension, the polymerase of the prime editor may be a DNA-dependent DNA polymerase.
• The newly synthesized strand (i.e., the replacement DNA strand containing the desired edit) that is formed by the herein disclosed prime editors would be homologous to the genomic target sequence (i.e., have the same sequence as) except for the inclusion of a desired nucleotide change (e.g., a single nucleotide change, a deletion, or an insertion, or a combination thereof). The newly synthesized (or replacement) strand of DNA may also be referred to as a single strand DNA flap, which would compete for hybridization with the complementary homologous endogenous DNA strand, thereby displacing the corresponding endogenous strand. In certain embodiments, the system can be combined with the use of an error-prone reverse transcriptase enzyme (e.g., provided as a fusion protein with the Cas9 domain, or provided in trans to the Cas9 domain). The error-prone reverse transcriptase enzyme can introduce alterations during synthesis of the single strand DNA flap. Thus, in certain embodiments, error-prone reverse transcriptase can be utilized to introduce nucleotide changes to the target DNA. Depending on the error-prone reverse transcriptase that is used with the system, the changes can be random or non-random.
• Resolution of the hybridized intermediate (comprising the single strand DNA flap synthesized by the reverse transcriptase hybridized to the endogenous DNA strand) can include removal of the resulting displaced flap of endogenous DNA (e.g., with a 5′ end DNA flap endonuclease, FEN1), ligation of the synthesized single strand DNA flap to the target DNA, and assimilation of the desired nucleotide change as a result of cellular DNA repair and/or replication processes. Because templated DNA synthesis offers single nucleotide precision for the modification of any nucleotide, including insertions and deletions, the scope of this approach is very broad and could foreseeably be used for myriad applications in basic science and therapeutics.
• In one aspect, the specification provides a fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a reverse transcriptase. In various embodiments, the fusion protein is capable of carrying out genome editing by target-primed reverse transcription in the presence of an extended guide RNA.
• In certain embodiments, the napDNAbp has a nickase activity. The napDNAbp may also be a Cas9 protein or functional equivalent thereof, such as a nuclease active Cas9, a nuclease inactive Cas9 (dCas9), or a Cas9 nickase (nCas9).
• In certain embodiments, the napDNAbp is selected from the group consisting of: Cas9, Cas12e, Cas12d, Cas12a, Cas12b1, Cas13a, Cas12c, and Argonaute and optionally has a nickase activity.
• In other embodiments, the fusion protein when complexed with an extended guide RNA is capable of binding to a target DNA sequence.
• In still other embodiments, the target DNA sequence comprises a target strand and a complementary non-target strand.
• In other embodiments, the binding of the fusion protein complexed to the extended guide RNA forms an R-loop. The R-loop can comprise (i) an RNA-DNA hybrid comprising the extended guide RNA and the target strand, and (ii) the complementary non-target strand.
• In still other embodiments, the complementary non-target strand is nicked to form a reverse transcriptase priming sequence having a free 3′ end.
• In various embodiments, the extended guide RNA comprises (a) a guide RNA and (b) an RNA extension at the 5′ or the 3′ end of the guide RNA, or at an intramolecular location in the guide RNA. The RNA extension can comprise (i) a reverse transcription template sequence comprising a desired nucleotide change, (ii) a reverse transcription primer binding site, and (iii) optionally, a linker sequence. In various embodiments, the reverse transcription template sequence may encode a single-strand DNA flap that is complementary to an endogenous DNA sequence adjacent to the nick site, wherein the single-strand DNA flap comprises the desired nucleotide change.
• In various embodiments, the RNA extension is at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides, at least 22 nucleotides, at least 23 nucleotides, at least 24 nucleotides, or at least 25 nucleotides in length.
• In still other embodiments, the single-strand DNA flap may hybridize to the endogenous DNA sequence adjacent to the nick site, thereby installing the desired nucleotide change. In still other embodiments, the single-stranded DNA flap displaces the endogenous DNA sequence adjacent to the nick site and which has a free 5′ end. In certain embodiments, the displaced endogenous DNA having the 5′ end is excised by the cell.
• In various embodiments, the cellular repair of the single-strand DNA flap results in installation of the desired nucleotide change, thereby forming a desired product.
• In various other embodiments, the desired nucleotide change is installed in an editing window that is between about −4 to +10 of the PAM sequence.
• In still other embodiments, the desired nucleotide change is installed in an editing window that is between about −5 to +5 of the nick site, or between about −10 to +10 of the nick site, or between about −20 to +20 of the nick site, or between about −30 to +30 of the nick site, or between about −40 to +40 of the nick site, or between about −50 to +50 of the nick site, or between about −60 to +60 of the nick site, or between about −70 to +70 of the nick site, or between about −80 to +80 of the nick site, or between about −90 to +90 of the nick site, or between about −100 to +100 of the nick site, or between about −200 to +200 of the nick site.
• In various embodiments, the napDNAbp comprises an amino acid sequence of SEQ ID NO: 18. In various other embodiments, the napDNAbp comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 26-39, 42-61, 75-76, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487 (Cas9); (SpCas9); SEQ ID NO: 77-86 (CP-Cas9); SEQ ID NO: 18-25 and 87-88 (SpCas9); and SEQ ID NOs: 62-72 (Cas12)
• In other embodiments, the reverse transcriptase of the disclosed fusion proteins and/or compositions may comprise any one of the amino acid sequences of SEQ ID NOs: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, 662, 700-716, 739-742, and 766. In still other embodiments, the reverse transcriptase may comprise an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, 662, 700-716, 739-742, and 766. These sequences may be naturally occurring reverse transcriptase sequences, e.g., from a retrovirus or a retrotransposon, of the sequences may be recombinant.
• In various other embodiments, the fusion proteins herein disclosed may comprise various structural configurations. For example, the fusion proteins may comprise the structure NH₂-[napDNAbp]-[reverse transcriptase]-COOH; or NH₂-[reverse transcriptase]-[napDNAbp]-COOH, wherein each instance of “]-[” indicates the presence of an optional linker sequence.
• In various embodiments, the linker sequence comprises an amino acid sequence of SEQ ID NOs: 127, 165-176, 446, 453, and 767-769, or an amino acid sequence that this at least 80%, 85%, or 90%, or 95%, or 99% identical to any one of the linker amino acid sequence of SEQ ID NOs: 127, 165-176, 446, 453, and 767-769.
• In various embodiments, the desired nucleotide change that is incorporated into the target DNA can be a single nucleotide change (e.g., a transition or transversion), an insertion of one or more nucleotides, or a deletion of one or more nucleotides.
• In certain cases, the insertion is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, or at least 500 nucleotides in length.
• In certain other cases, the deletion is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, or at least 500 nucleotides in length.
• In another aspect, the present disclosure provides an extended guide RNA comprising a guide RNA and at least one RNA extension. The RNA extension can be positioned at the 3′ end of the guide RNA. In other embodiments, the RNA extension can be positioned at the 5′ of the guide RNA. In still other embodiments, the RNA extension can be positioned at an intramolecular position within the guide RNA, however, preferable, the intramolecular positioning of the extended portion does not disrupt the functioning of the protospacer.
• In various embodiments, the extended guide RNA is capable of binding to a napDNAbp and directing the napDNAbp to a target DNA sequence. The target DNA sequence can comprise a target strand and a complementary non-target strand, wherein the guide RNA hybridizes to the target strand to form an RNA-DNA hybrid and an R-loop.
• In various embodiments of the extended guide RNA, the at least one RNA extension can comprise a reverse transcription template sequence. In various other embodiment, the RNA extension may further comprises a reverse transcription primer binding site. In still further embodiments, the RNA extension may comprise a linker or spacer sequence that joins the RNA extension to the guide RNA.
• In various embodiments, the RNA extension can be at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides, at least 22 nucleotides, at least 23 nucleotides, at least 24 nucleotides, at least 25 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides, at least 80 nucleotides, at least 90 nucleotides, at least 100 nucleotides, at least 150 nucleotides, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, or at least 500 nucleotides in length.
• In other embodiments, the reverse transcription template sequence is at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides, at least 80 nucleotides, at least 90 nucleotides, at least 100 nucleotides, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, or at least 500 nucleotides in length.
• In still other embodiments, wherein the reverse transcription primer binding site sequence is at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides, at least 80 nucleotides, at least 90 nucleotides, at least 100 nucleotides, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, or at least 500 nucleotides in length.
• In other embodiments, the optional linker or spacer sequence is at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides, at least 80 nucleotides, at least 90 nucleotides, at least 100 nucleotides, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, or at least 500 nucleotides in length.
• In various embodiments of the extended guide RNAs, the reverse transcription template sequence may encode a single-strand DNA flap that is complementary to an endogenous DNA sequence adjacent to a nick site, wherein the single-strand DNA flap comprises a desired nucleotide change. The single-stranded DNA flap may displace an endogenous single-strand DNA at the nick site. The displaced endogenous single-strand DNA at the nick site can have a 5′ end and form an endogenous flap, which can be excised by the cell. In various embodiments, excision of the 5′ end endogenous flap can help drive product formation since removing the 5′ end endogenous flap encourages hybridization of the single-strand 3′ DNA flap to the corresponding complementary DNA strand, and the incorporation or assimilation of the desired nucleotide change carried by the single-strand 3′ DNA flap into the target DNA.
• In various embodiments of the extended guide RNAs, the cellular repair of the single-strand DNA flap results in installation of the desired nucleotide change, thereby forming a desired product.
• In certain embodiments, the PEgRNA comprises the nucleotide sequence of SEQ ID NOs: 131, 222, 394, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 641, 642, 643, 644, 645, 646, 647, 648, 649, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 738, 2997, 2998, 2999, 3000, 3001, 3002, 3003, 3004, 3005, 3006, 3007, 3008, 3009, 3010, 3011, 3012, 3013, 3014, 3015, 3016, 3017, 3018, 3019, 3020, 3021, 3022, 3023, 3024, 3025, 3026, 3027, 3028, 3029, 3030, 3031, 3032, 3033, 3034, 3035, 3036, 3037, 3038, 3039, 3040, 3041, 3042, 3043, 3044, 3045, 3046, 3047, 3048, 3049, 3050, 3051, 3052, 3053, 3054, 3055, 3056, 3057, 3058, 3059, 3060, 3061, 3062, 3063, 3064, 3065, 3066, 3067, 3068, 3069, 3070, 3071, 3072, 3073, 3074, 3075, 3076, 3077, 3078, 3079, 3080, 3081, 3082, 3083, 3084, 3085, 3086, 3087, 3088, 3089, 3090, 3091, 3092, 3093, 3094, 3095, 3096, 3097, 3098, 3099, 3100, 3101, 3102, 3103, 3113, 3114, 3115, 3116, 3117, 3118, 3119, 3120, 3121, 3305, 3306, 3307, 3308, 3309, 3310, 3311, 3312, 3313, 3314, 3315, 3316, 3317, 3318, 3319, 3320, 3321, 3322, 3323, 3324, 3325, 3326, 3327, 3328, 3329, 3330, 3331, 3332, 3333, 3334, 3335, 3336, 3337, 3338, 3339, 3340, 3341, 3342, 3343, 3344, 3345, 3346, 3347, 3348, 3349, 3350, 3351, 3352, 3353, 3354, 3355, 3356, 3357, 3358, 3359, 3360, 3361, 3362, 3363, 3364, 3365, 3366, 3367, 3368, 3369, 3370, 3371, 3372, 3373, 3374, 3375, 3376, 3377, 3378, 3379, 3380, 3381, 3382, 3383, 3384, 3385, 3386, 3387, 3388, 3389, 3390, 3391, 3392, 3393, 3394, 3395, 3396, 3397, 3398, 3399, 3400, 3401, 3402, 3403, 3404, 3405, 3406, 3407, 3408, 3409, 3410, 3411, 3412, 3413, 3414, 3415, 3416, 3417, 3418, 3419, 3420, 3421, 3422, 3423, 3424, 3425, 3426, 3427, 3428, 3429, 3430, 3431, 3432, 3433, 3434, 3435, 3436, 3437, 3438, 3439, 3440, 3441, 3442, 3443, 3444, 3445, 3446, 3447, 3448, 3449, 3450, 3451, 3452, 3453, 3454, 3455, 3479, 3480, 3481, 3482, 3483, 3484, 3485, 3486, 3487, 3488, 3489, 3490, 3491, 3492, 3493, 3522, 3523, 3524, 3525, 3526, 3527, 3528, 3529, 3530, 3531, 3532, 3533, 3534, 3535, 3536, 3537, 3538, 3539, 3540, 3549, 3550, 3551, 3552, 3553, 3554, 3555, 3556, 3628, 3629, 3630, 3631, 3632, 3633, 3634, 3635, 3636, 3637, 3638, 3639, 3640, 3641, 3642, 3643, 3644, 3645, 3646, 3647, 3648, 3649, 3650, 3651, 3652, 3653, 3654, 3655, 3656, 3657, 3658, 3659, 3660, 3661, 3662, 3663, 3664, 3665, 3666, 3667, 3668, 3669, 3670, 3671, 3672, 3673, 3674, 3675, 3676, 3677, 3678, 3679, 3680, 3681, 3682, 3683, 3684, 3685, 3686, 3687, 3688, 3689, 3690, 3691, 3692, 3693, 3694, 3695, 3696, 3697, 3698, 3755, 3756, 3757, 3758, 3759, 3760, 3761, 3762, 3763, 3764, 3765, 3766, 3767, 3768, 3769, 3770, 3771, 3772, 3773, 3774, 3775, 3776, 3777, 3778, 3779, 3780, 3781, 3782, 3783, 3784, 3785, 3786, 3787, 3788, 3789, 3790, 3791, 3792, 3793, 3794, 3795, 3796, 3797, 3798, 3799, 3800, 3801, 3802, 3803, 3804, 3805, 3806, 3807, 3808, 3809, and 3810, or a nucleotide sequence having at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99% sequence identity with any one of SEQ ID NOs: 131, 222, 394, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 641, 642, 643, 644, 645, 646, 647, 648, 649, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 738, 2997, 2998, 2999, 3000, 3001, 3002, 3003, 3004, 3005, 3006, 3007, 3008, 3009, 3010, 3011, 3012, 3013, 3014, 3015, 3016, 3017, 3018, 3019, 3020, 3021, 3022, 3023, 3024, 3025, 3026, 3027, 3028, 3029, 3030, 3031, 3032, 3033, 3034, 3035, 3036, 3037, 3038, 3039, 3040, 3041, 3042, 3043, 3044, 3045, 3046, 3047, 3048, 3049, 3050, 3051, 3052, 3053, 3054, 3055, 3056, 3057, 3058, 3059, 3060, 3061, 3062, 3063, 3064, 3065, 3066, 3067, 3068, 3069, 3070, 3071, 3072, 3073, 3074, 3075, 3076, 3077, 3078, 3079, 3080, 3081, 3082, 3083, 3084, 3085, 3086, 3087, 3088, 3089, 3090, 3091, 3092, 3093, 3094, 3095, 3096, 3097, 3098, 3099, 3100, 3101, 3102, 3103, 3113, 3114, 3115, 3116, 3117, 3118, 3119, 3120, 3121, 3305, 3306, 3307, 3308, 3309, 3310, 3311, 3312, 3313, 3314, 3315, 3316, 3317, 3318, 3319, 3320, 3321, 3322, 3323, 3324, 3325, 3326, 3327, 3328, 3329, 3330, 3331, 3332, 3333, 3334, 3335, 3336, 3337, 3338, 3339, 3340, 3341, 3342, 3343, 3344, 3345, 3346, 3347, 3348, 3349, 3350, 3351, 3352, 3353, 3354, 3355, 3356, 3357, 3358, 3359, 3360, 3361, 3362, 3363, 3364, 3365, 3366, 3367, 3368, 3369, 3370, 3371, 3372, 3373, 3374, 3375, 3376, 3377, 3378, 3379, 3380, 3381, 3382, 3383, 3384, 3385, 3386, 3387, 3388, 3389, 3390, 3391, 3392, 3393, 3394, 3395, 3396, 3397, 3398, 3399, 3400, 3401, 3402, 3403, 3404, 3405, 3406, 3407, 3408, 3409, 3410, 3411, 3412, 3413, 3414, 3415, 3416, 3417, 3418, 3419, 3420, 3421, 3422, 3423, 3424, 3425, 3426, 3427, 3428, 3429, 3430, 3431, 3432, 3433, 3434, 3435, 3436, 3437, 3438, 3439, 3440, 3441, 3442, 3443, 3444, 3445, 3446, 3447, 3448, 3449, 3450, 3451, 3452, 3453, 3454, 3455, 3479, 3480, 3481, 3482, 3483, 3484, 3485, 3486, 3487, 3488, 3489, 3490, 3491, 3492, 3493, 3522, 3523, 3524, 3525, 3526, 3527, 3528, 3529, 3530, 3531, 3532, 3533, 3534, 3535, 3536, 3537, 3538, 3539, 3540, 3549, 3550, 3551, 3552, 3553, 3554, 3555, 3556, 3628, 3629, 3630, 3631, 3632, 3633, 3634, 3635, 3636, 3637, 3638, 3639, 3640, 3641, 3642, 3643, 3644, 3645, 3646, 3647, 3648, 3649, 3650, 3651, 3652, 3653, 3654, 3655, 3656, 3657, 3658, 3659, 3660, 3661, 3662, 3663, 3664, 3665, 3666, 3667, 3668, 3669, 3670, 3671, 3672, 3673, 3674, 3675, 3676, 3677, 3678, 3679, 3680, 3681, 3682, 3683, 3684, 3685, 3686, 3687, 3688, 3689, 3690, 3691, 3692, 3693, 3694, 3695, 3696, 3697, 3698, 3755, 3756, 3757, 3758, 3759, 3760, 3761, 3762, 3763, 3764, 3765, 3766, 3767, 3768, 3769, 3770, 3771, 3772, 3773, 3774, 3775, 3776, 3777, 3778, 3779, 3780, 3781, 3782, 3783, 3784, 3785, 3786, 3787, 3788, 3789, 3790, 3791, 3792, 3793, 3794, 3795, 3796, 3797, 3798, 3799, 3800, 3801, 3802, 3803, 3804, 3805, 3806, 3807, 3808, 3809, and 3810.
• In yet another aspect of the invention, the specification provides for complexes comprising a fusion protein described herein and any extended guide RNA described above.
• In still other aspects of the invention, the specification provides a complex comprising a napDNAbp and an extended guide RNA. The napDNAbp can be a Cas9 nickase, or can be an amino acid sequence of SEQ ID NOs: 42-57 (Cas9 nickase) and 65 (AsCas12a nickase), or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 42-57 (Cas9 nickase) and 65 (AsCas12a nickase).
• In various embodiments involving a complex, the extended guide RNA is capable of directing the napDNAbp to a target DNA sequence. In various embodiments, a reverse transcriptase may be provided in trans, i.e., provided from a different source than the complex itself. For example, a reverse transcriptase could be provided to the same cell having the complex by introducing a separate vector separately encoding the reverse transcriptase.
• In yet another aspect, the specification provides polynucleotides. In certain embodiments, the polynucleotides may encode any of the fusion proteins disclosed herein. In certain other embodiments, the polynucleotides may encode any of the napDNAbps disclosed herein. In still further embodiments, the polynucleotides may encode any of the reverse transcriptases disclosed herein. In yet other embodiments, the polynucleotides may encode any of the extended guide RNAs disclosed herein, any of the reverse transcription template sequences, or any of the reverse transcription primer sites, or any of the optional linker sequences.
• In still other aspects, the specification provides vectors comprising the polynucleotides described herein. Thus, in certain embodiments, the vectors comprise polynucleotides for encoding the fusion proteins comprising a napDNAbp and a reverse transcriptase. In other embodiments, the vectors comprise polynucleotides that separately encode a napDNAbp and reverse transcriptase. In still other embodiments, the vectors may comprise polynucleotides that encode the extended guide RNAs. In various embodiments, the vectors may comprise one or more polynucleotides that encode napDNAbps, reverse transcriptase, and extended guide RNAs on the same or separate vectors.
• In still other aspects, the specification provides cells comprising a fusion protein as described herein and an extended guide RNA. The cells may be transformed with the vectors comprising the fusion proteins, napDNAbps, reverse transcriptase, and extended guide RNAs. These genetic elements may be comprised on the same vectors or on different vectors.
• In yet another aspect, the specification provides pharmaceutical compositions. In certain embodiments, the pharmaceutical compositions comprise one or more of a napDNAbp, a fusion protein, a reverse transcriptase, and an extended guide RNA. In certain embodiments, the fusion protein described herein and a pharmaceutically acceptable excipient. In other embodiments, the pharmaceutical compositions comprise any extend guide RNA described herein and a pharmaceutically acceptable excipient. In still other embodiments, the pharmaceutical compositions comprise any extend guide RNA described herein in combination with any fusion protein described herein and a pharmaceutically acceptable excipient. In yet other embodiments, the pharmaceutical compositions comprise any polynucleotide sequence encoding one or more of a napDNAbp, a fusion protein, a reverse transcriptase, and an extended guide RNA. In still other embodiments, the various components disclosed herein may be separated into one or more pharmaceutical compositions. For example, a first pharmaceutical composition may comprise a fusion protein or a napDNAbp, a second pharmaceutical compositions may comprise a reverse transcriptase, and a third pharmaceutical composition may comprise an extended guide RNA.
• In still a further aspect, the present disclosure provides kits. In one embodiment, the kit comprises one or more polynucleotides encoding one or more components, including a fusion protein, a napDNAbp, a reverse transcriptase, and an extended guide RNA. The kits may also comprise vectors, cells, and isolated preparations of polypeptides, including any fusion protein, napDNAbp, or reverse transcriptase disclosed herein.
• In yet another aspect, the present disclosure provides for methods of using the disclosed compositions of matter.
• In one embodiment, the methods relate to a method for installing a desired nucleotide change in a double-stranded DNA sequence. The method first comprises contacting the double-stranded DNA sequence with a complex comprising a fusion protein and an extended guide RNA, wherein the fusion protein comprises a napDNAbp and a reverse transcriptase and wherein the extended guide RNA comprises a reverse transcription template sequence comprising the desired nucleotide change. Next, the method involves nicking the double-stranded DNA sequence on the non-target strand, thereby generating a free single-strand DNA having a 3′ end. The method then involves hybridizing the 3′ end of the free single-strand DNA to the reverse transcription template sequence, thereby priming the reverse transcriptase domain. The method then involves polymerizing a strand of DNA from the 3′ end, thereby generating a single-strand DNA flap comprising the desired nucleotide change. Then, the method involves replacing an endogenous DNA strand adjacent the cut site with the single-strand DNA flap, thereby installing the desired nucleotide change in the double-stranded DNA sequence.
• In other embodiments, the disclosure provides for a method for introducing one or more changes in the nucleotide sequence of a DNA molecule at a target locus, comprising contacting the DNA molecule with a nucleic acid programmable DNA binding protein (napDNAbp) and a guide RNA which targets the napDNAbp to the target locus, wherein the guide RNA comprises a reverse transcriptase (RT) template sequence comprising at least one desired nucleotide change. Next, the method involves forming an exposed 3′ end in a DNA strand at the target locus and then hybridizing the exposed 3′ end to the RT template sequence to prime reverse transcription. Next, a single strand DNA flap comprising the at least one desired nucleotide change based on the RT template sequence is synthesized or polymerized by reverse transcriptase. Lastly, the at least one desired nucleotide change is incorporated into the corresponding endogenous DNA, thereby introducing one or more changes in the nucleotide sequence of the DNA molecule at the target locus.
• In still other embodiments, the disclosure provides a method for introducing one or more changes in the nucleotide sequence of a DNA molecule at a target locus by target-primed reverse transcription, the method comprising: (a) contacting the DNA molecule at the target locus with a (i) fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a reverse transcriptase and (ii) a guide RNA comprising an RT template comprising a desired nucleotide change; (b) conducting target-primed reverse transcription of the RT template to generate a single strand DNA comprising the desired nucleotide change; and (c) incorporating the desired nucleotide change into the DNA molecule at the target locus through a DNA repair and/or replication process.
• In certain embodiments, the step of replacing the endogenous DNA strand comprises: (i) hybridizing the single-strand DNA flap to the endogenous DNA strand adjacent the cut site to create a sequence mismatch; (ii) excising the endogenous DNA strand; and (iii) repairing the mismatch to form the desired product comprising the desired nucleotide change in both strands of DNA.
• In various embodiments, the desired nucleotide change can be a single nucleotide substitution (e.g., and transition or a transversion change), a deletion, or an insertion. For example, the desired nucleotide change can be (1) a G to T substitution, (2) a G to A substitution, (3) a G to C substitution, (4) a T to G substitution, (5) a T to A substitution, (6) a T to C substitution, (7) a C to G substitution, (8) a C to T substitution, (9) a C to A substitution, (10) an A to T substitution, (11) an A to G substitution, or (12) an A to C substitution.
• In other embodiments, the desired nucleoid change can convert (1) a G:C basepair to a T:A basepair, (2) a G:C basepair to an A:T basepair, (3) a G:C basepair to C:G basepair, (4) a T:A basepair to a G:C basepair, (5) a T:A basepair to an A:T basepair, (6) a T:A basepair to a C:G basepair, (7) a C:G basepair to a G:C basepair, (8) a C:G basepair to a T:A basepair, (9) a C:G basepair to an A:T basepair, (10) an A:T basepair to a T:A basepair, (11) an A:T basepair to a G:C basepair, or (12) an A:T basepair to a C:G basepair.
• In still other embodiments, the method introduces a desired nucleotide change that is an insertion. In certain cases, the insertion is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, or at least 500 nucleotides in length.
• In other embodiments, the method introduces a desired nucleotide change that is a deletion. In certain other cases, the deletion is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, or at least 500 nucleotides in length.
• In various embodiments, the desired nucleotide change corrects a disease-associated gene. The disease-associated gene can be associated with a monogenetic disorder selected from the group consisting of: Adenosine Deaminase (ADA) Deficiency; Alpha-1 Antitrypsin Deficiency; Cystic Fibrosis; Duchenne Muscular Dystrophy; Galactosemia; Hemochromatosis; Huntington's Disease; Maple Syrup Urine Disease; Marfan Syndrome; Neurofibromatosis Type 1; Pachyonychia Congenita; Phenylkeotnuria; Severe Combined Immunodeficiency; Sickle Cell Disease; Smith-Lemli-Opitz Syndrome; and Tay-Sachs Disease. In other embodiments, the disease-associated gene can be associated with a polygenic disorder selected from the group consisting of: heart disease; high blood pressure; Alzheimer's disease; arthritis; diabetes; cancer; and obesity.
• The methods disclosed herein may involve fusion proteins having a napDNAbp that is a nuclease dead Cas9 (dCas9), a Cas9 nickase (nCas9), or a nuclease active Cas9. In other embodiments, a napDNAbp and reverse transcriptase are not encoded as a single fusion protein, but rather can be provided in separate constructs. Thus, in some embodiments, the reverse transcriptase can be provided in trans relative to the napDNAbp (rather than by way of a fusion protein).
• In various embodiments involving methods, the napDNAbp may comprise an amino acid sequence of SEQ ID NOs: 26-61, 75-76, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487 (Cas9); (SpCas9); SEQ ID NO: 77-86 (CP-Cas9); SEQ ID NO: 18-25 and 87-88 (SpCas9); and SEQ ID NOs: 62-72 (Cas12). The napDNAbp may also comprise an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 26-61, 75-76, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487 (Cas9); (SpCas9); SEQ ID NO: 77-86 (CP-Cas9); SEQ ID NO: 18-25 and 87-88 (SpCas9); and SEQ ID NOs: 62-72 (Cas12).
• In various embodiments involving methods, the reverse transcriptase may comprise any one of the amino acid sequences of SEQ ID NOs: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, 662, 700-716, 739-742, and 766. The reverse transcriptase may also comprise an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, 662, 700-716, 739-742, and 766.
• The methods may involve the use of a PEgRNA comprising a nucleotide sequence of SEQ ID NOs: 131, 222, 394, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 641, 642, 643, 644, 645, 646, 647, 648, 649, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 738, 2997, 2998, 2999, 3000, 3001, 3002, 3003, 3004, 3005, 3006, 3007, 3008, 3009, 3010, 3011, 3012, 3013, 3014, 3015, 3016, 3017, 3018, 3019, 3020, 3021, 3022, 3023, 3024, 3025, 3026, 3027, 3028, 3029, 3030, 3031, 3032, 3033, 3034, 3035, 3036, 3037, 3038, 3039, 3040, 3041, 3042, 3043, 3044, 3045, 3046, 3047, 3048, 3049, 3050, 3051, 3052, 3053, 3054, 3055, 3056, 3057, 3058, 3059, 3060, 3061, 3062, 3063, 3064, 3065, 3066, 3067, 3068, 3069, 3070, 3071, 3072, 3073, 3074, 3075, 3076, 3077, 3078, 3079, 3080, 3081, 3082, 3083, 3084, 3085, 3086, 3087, 3088, 3089, 3090, 3091, 3092, 3093, 3094, 3095, 3096, 3097, 3098, 3099, 3100, 3101, 3102, 3103, 3113, 3114, 3115, 3116, 3117, 3118, 3119, 3120, 3121, 3305, 3306, 3307, 3308, 3309, 3310, 3311, 3312, 3313, 3314, 3315, 3316, 3317, 3318, 3319, 3320, 3321, 3322, 3323, 3324, 3325, 3326, 3327, 3328, 3329, 3330, 3331, 3332, 3333, 3334, 3335, 3336, 3337, 3338, 3339, 3340, 3341, 3342, 3343, 3344, 3345, 3346, 3347, 3348, 3349, 3350, 3351, 3352, 3353, 3354, 3355, 3356, 3357, 3358, 3359, 3360, 3361, 3362, 3363, 3364, 3365, 3366, 3367, 3368, 3369, 3370, 3371, 3372, 3373, 3374, 3375, 3376, 3377, 3378, 3379, 3380, 3381, 3382, 3383, 3384, 3385, 3386, 3387, 3388, 3389, 3390, 3391, 3392, 3393, 3394, 3395, 3396, 3397, 3398, 3399, 3400, 3401, 3402, 3403, 3404, 3405, 3406, 3407, 3408, 3409, 3410, 3411, 3412, 3413, 3414, 3415, 3416, 3417, 3418, 3419, 3420, 3421, 3422, 3423, 3424, 3425, 3426, 3427, 3428, 3429, 3430, 3431, 3432, 3433, 3434, 3435, 3436, 3437, 3438, 3439, 3440, 3441, 3442, 3443, 3444, 3445, 3446, 3447, 3448, 3449, 3450, 3451, 3452, 3453, 3454, 3455, 3479, 3480, 3481, 3482, 3483, 3484, 3485, 3486, 3487, 3488, 3489, 3490, 3491, 3492, 3493, 3522, 3523, 3524, 3525, 3526, 3527, 3528, 3529, 3530, 3531, 3532, 3533, 3534, 3535, 3536, 3537, 3538, 3539, 3540, 3549, 3550, 3551, 3552, 3553, 3554, 3555, 3556, 3628, 3629, 3630, 3631, 3632, 3633, 3634, 3635, 3636, 3637, 3638, 3639, 3640, 3641, 3642, 3643, 3644, 3645, 3646, 3647, 3648, 3649, 3650, 3651, 3652, 3653, 3654, 3655, 3656, 3657, 3658, 3659, 3660, 3661, 3662, 3663, 3664, 3665, 3666, 3667, 3668, 3669, 3670, 3671, 3672, 3673, 3674, 3675, 3676, 3677, 3678, 3679, 3680, 3681, 3682, 3683, 3684, 3685, 3686, 3687, 3688, 3689, 3690, 3691, 3692, 3693, 3694, 3695, 3696, 3697, 3698, 3755, 3756, 3757, 3758, 3759, 3760, 3761, 3762, 3763, 3764, 3765, 3766, 3767, 3768, 3769, 3770, 3771, 3772, 3773, 3774, 3775, 3776, 3777, 3778, 3779, 3780, 3781, 3782, 3783, 3784, 3785, 3786, 3787, 3788, 3789, 3790, 3791, 3792, 3793, 3794, 3795, 3796, 3797, 3798, 3799, 3800, 3801, 3802, 3803, 3804, 3805, 3806, 3807, 3808, 3809, and 3810, or a nucleotide sequence having at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 99% sequence identity thereto The methods may comprise the use of extended guide RNAs that comprise an RNA extension at the 3′ end, wherein the RNA extension comprises the reverse transcription template sequence.
• The methods may comprise the use of extended guide RNAs that comprise an RNA extension at the 5′ end, wherein the RNA extension comprises the reverse transcription template sequence.
• The methods may comprise the use of extended guide RNAs that comprise an RNA extension at an intramolecular location in the guide RNA, wherein the RNA extension comprises the reverse transcription template sequence.
• The methods may comprise the use of extended guide RNAs having one or more RNA extensions that are at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, or at least 500 nucleotides in length.
• It should be appreciated that the foregoing concepts, and additional concepts discussed below, may be arranged in any suitable combination, as the present disclosure is not limited in this respect. Further, other advantages and novel features of the present disclosure will become apparent from the following detailed description of various non-limiting embodiments when considered in conjunction with the accompanying figures.
BRIEF DESCRIPTION OF THE DRAWINGS
• The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
• FIG. 1A provides a schematic of an exemplary process for introducing a single nucleotide change, and/or insertion, and/or deletion into a DNA molecule (e.g., a genome) using a fusion protein comprising a reverse transcriptase fused to a Cas9 protein in complex with an extended guide RNA molecule. In this embodiment, the guide RNA is extended at the 3′ end to include a reverse transcriptase template sequence. The schematic shows how a reverse transcriptase (RT) fused to a Cas9 nickase, in a complex with a guide RNA (gRNA), binds the DNA target site and nicks the PAM-containing DNA strand adjacent to the target nucleotide. The RT enzyme uses the nicked DNA as a primer for DNA synthesis from the gRNA, which is used as a template for the synthesis of a new DNA strand that encodes the desired edit. The editing process shown may be referred to as target-primed reverse transcription editing (TRT editing) or equivalently, “prime editing.”
• FIG. 1B provides the same representation as in FIG. 1A, except that the prime editor complex is represented more generally as [napDNAbp]-[P]:PEgRNA or [P]-[napDNAbp]:PEgRNA, wherein “P” refers to any polymerase (e.g., a reverse transcriptase), “napDNAbp” refers to a nucleic acid programmable DNA binding protein (e.g., SpCas9), and “PEgRNA” refers to a prime editing guide RNA, and “]-[” refers to an optional linker. As described elsewhere, e.g., FIGS. 3A-3G, the PEgRNA comprises an 5′ extension arm comprising a primer binding site and a DNA synthesis template. Although not shown, it is contemplated that the extension arm of the PEgRNA (i.e., which comprises a primer binding site and a DNA synthesis template) can be DNA or RNA. The particular polymerase contemplated in this configuration will depend upon the nature of the DNA synthesis template. For instance, if the DNA synthesis template is RNA, then the polymerase case be an RNA-dependent DNA polymerase (e.g., reverse transcriptase). If the DNA synthesis template is DNA, then the polymerase can be a DNA-dependent DNA polymerase.
• FIG. 1C provides a schematic of an exemplary process for introducing a single nucleotide change, and/or insertion, and/or deletion into a DNA molecule (e.g., a genome) using a fusion protein comprising a reverse transcriptase fused to a Cas9 protein in complex with an extended guide RNA molecule. In this embodiment, the guide RNA is extended at the 5′ end to include a reverse transcriptase template sequence. The schematic shows how a reverse transcriptase (RT) fused to a Cas9 nickase, in a complex with a guide RNA (gRNA), binds the DNA target site and nicks the PAM-containing DNA strand adjacent to the target nucleotide. The RT enzyme uses the nicked DNA as a primer for DNA synthesis from the gRNA, which is used as a template for the synthesis of a new DNA strand that encodes the desired edit. The editing process shown may be referred to as target-primed reverse transcription editing (TRT editing) or equivalently, “prime editing.”
• FIG. 1D provides the same representation as in FIG. 1C, except that the prime editor complex is represented more generally as [napDNAbp]-[P]:PEgRNA or [P]-[napDNAbp]:PEgRNA, wherein “P” refers to any polymerase (e.g., a reverse transcriptase), “napDNAbp” refers to a nucleic acid programmable DNA binding protein (e.g., SpCas9), and “PEgRNA” refers to a prime editing guide RNA, and “]-[” refers to an optional linker. As described elsewhere, e.g., FIGS. 3A-3G, the PEgRNA comprises an 3′ extension arm comprising a primer binding site and a DNA synthesis template. Although not shown, it is contemplated that the extension arm of the PEgRNA (i.e., which comprises a primer binding site and a DNA synthesis template) can be DNA or RNA. The particular polymerase contemplated in this configuration will depend upon the nature of the DNA synthesis template. For instance, if the DNA synthesis template is RNA, then the polymerase case be an RNA-dependent DNA polymerase (e.g., reverse transcriptase). If the DNA synthesis template is DNA, then the polymerase can be a DNA-dependent DNA polymerase. In various embodiments, the PEgRNA can be engineered or synthesized to incorporate a DNA-based DNA synthesis template.
• FIG. 1E is a schematic depicting an exemplary process of how the synthesized single strand of DNA (which comprises the desired nucleotide change) becomes resolved such that the desired nucleotide change is incorporated into the DNA. As shown, following synthesis of the edited strand (or “mutagenic strand”), equilibration with the endogenous strand, flap cleavage of the endogenous strand, and ligation leads to incorporation of the DNA edit after resolution of the mismatched DNA duplex through the action of endogenous DNA repair and/or replication processes.
• FIG. 1F is a schematic showing that “opposite strand nicking” can be incorporated into the resolution method of FIG. 1E to help drive the formation of the desired product versus the reversion product. In opposite strand nicking, a second Cas9/gRNA complex is used to introduce a second nick on the opposite strand from the initial nicked strand. This induces the endogenous cellular DNA repair and/or replication processes to preferentially replace the unedited strand (i.e., the strand containing the second nick site).
• FIG. 1G provides another schematic of an exemplary process for introducing a single nucleotide change, and/or insertion, and/or deletion into a DNA molecule (e.g., a genome) of a target locus using a nucleic acid programmable DNA binding protein (napDNAbp) complexed with an extended guide RNA. This process may be referred to as an embodiment of prime editing. The extended guide RNA comprises an extension at the 3′ or 5′ end of the guide RNA, or at an intramolecular location in the guide RNA. In step (a), the napDNAbp/gRNA complex contacts the DNA molecule and the gRNA guides the napDNAbp to bind to the target locus. In step (b), a nick in one of the strands of DNA (the R-loop strand, or the PAM-containing strand, or the non-target DNA strand, or the protospacer strand) of the target locus is introduced (e.g., by a nuclease or chemical agent), thereby creating an available 3′ end in one of the strands of the target locus. In certain embodiments, the nick is created in the strand of DNA that corresponds to the R-loop strand, i.e., the strand that is not hybridized to the guide RNA sequence. In step (c), the 3′ end DNA strand interacts with the extended portion of the guide RNA in order to prime reverse transcription. In certain embodiments, the 3′ ended DNA strand hybridizes to a specific RT priming sequence on the extended portion of the guide RNA. In step (d), a reverse transcriptase is introduced which synthesizes a single strand of DNA from the 3′ end of the primed site towards the 3′ end of the guide RNA. This forms a single-strand DNA flap comprising the desired nucleotide change (e.g., the single base change, insertion, or deletion, or a combination thereof). In step (e), the napDNAbp and guide RNA are released. Steps (f) and (g) relate to the resolution of the single strand DNA flap such that the desired nucleotide change becomes incorporated into the target locus. This process can be driven towards the desired product formation by removing the corresponding 5′ endogenous DNA flap that forms once the 3′ single strand DNA flap invades and hybridizes to the complementary sequence on the other strand. The process can also be driven towards product formation with second strand nicking, as exemplified in FIG. 1F. This process may introduce at least one or more of the following genetic changes: transversions, transitions, deletions, and insertions.
• FIG. 1H is a schematic depicting the types of genetic changes that are possible with the prime editing processes described herein. The types of nucleotide changes achievable by prime editing include deletions (including short and long deletions), single-nucleotide changes (including transitions and transversions), inversions, and insertions (including short and long deletions).
• FIG. 1I is a schematic depicting temporal second strand nicking exemplified by PE3b (PE3b=PE2 prime editor fusion protein+PEgRNA+second strand nicking guide RNA). Temporal second strand nicking is a variant of second strand nicking in order to facilitate the formation of the desired edited product. The “temporal” term refers to the fact that the second-strand nick to the unedited strand occurs only after the desired edit is installed in the edited strand. This avoids concurrent nicks on both strands to lead to double-stranded DNA breaks.
• FIG. 1J depicts a variation of prime editing contemplated herein that replaces the napDNAbp (e.g., SpCas9 nickase) with any programmable nuclease domain, such as zinc finger nucleases (ZFN) or transcription activator-like effector nucleases (TALEN). As such, it is contemplated that suitable nucleases do not necessarily need to be “programmed” by a nucleic acid targeting molecule (such as a guide RNA), but rather, may be programmed by defining the specificity of a DNA-binding domain, such as and in particular, a nuclease. Just as in prime editing with napDNAbp moieties, it is preferable that such alternative programmable nucleases be modified such that only one strand of a target DNA is cut. In other words, the programmable nucleases should function as nickases, preferably. Once a programmable nuclease is selected (e.g., a ZFN or a TALEN), then additional functionalities may be engineered into the system to allow it to operate in accordance with a prime editing-like mechanism. For example, the programmable nucleases may be modified by coupling (e.g., via a chemical linker) an RNA or DNA extension arm thereto, wherein the extension arm comprises a primer binding site (PBS) and a DNA synthesis template. The programmable nuclease may also be coupled (e.g., via a chemical or amino acid linker) to a polymerase, the nature of which will depend upon whether the extension arm is DNA or RNA. In the case of an RNA extension arm, the polymerase can be an RNA-dependent DNA polymerase (e.g., reverse transcriptase). In the case of a DNA extension arm, the polymerase can be a DNA-dependent DNA polymerase (e.g., a prokaryotic polymerase, including Pol I, Pol II, or Pol III, or a eukaryotic polymerase, including Pol a, Pol b, Pol g, Pol d, Pol e, or Pol z). The system may also include other functionalities added as fusions to the programmable nucleases, or added in trans to facilitate the reaction as a whole (e.g., (a) a helicase to unwind the DNA at the cut site to make the cut strand with the 3′ end available as a primer, (b) a flap endonuclease (e.g., FEN1) to help remove the endogenous strand on the cut strand to drive the reaction towards replacement of the endogenous strand with the synthesized strand, or (c) a nCas9:gRNA complex to create a second site nick on the opposite strand, which may help drive the integration of the synthesize repair through favored cellular repair of the non-edited strand). In an analogous manner to prime editing with a napDNAbp, such a complex with an otherwise programmable nuclease could be used to synthesize and then install a newly synthesized replacement strand of DNA carrying an edit of interest permanently into a target site of DNA.
• FIG. 1K depicts, in one embodiment, the anatomical features of a target DNA that may be edited by prime editing. The target DNA comprises a “non-target strand” and a “target strand.” The target-strand is the strand that becomes annealed to the spacer of a PEgRNA of a prime editor complex that recognizes the PAM site (in this case, NGG, which is recognized by the canonical SpCas9-based prime editors) The target strand may also be referred to as the “non-PAM strand” or the “non-edit strand.” By contrast, the non-target strand (i.e., the strand containing the protospacer and the PAM sequence of NGG) may be referred to as the “PAM-strand” or the “edit strand.” In various embodiments, the nick site of the PE complex will be in the protospacer on the PAM-strand (e.g., with the SpCas9-based PE). The location of the nick will be characteristic of the particular Cas9 that forms the PE. For example, with an SpCas9-based PE, the nick site in the phosphodiester bond between bases three (“−3” position relative to the position 1 of the PAM sequence) and four (“−4” position relative to position 1 of the PAM sequence). The nick site in the protospacer forms a free 3′ hydroxyl group, which as seen in the following figures, complexes with the primer binding site of the extension arm of the PEgRNA and provides the substrate to begin polymerization of a single strand of DNA code for by the DNA synthesis template of the extension arm of the PEgRNA. This polymerization reaction is catalyzed by the polymerase (e.g., reverse transcriptase) of the PE fusion protein in the 5′ to 3′ direction. Polymerization terminates before reaching the gRNA core (e.g., by inclusion of a polymerization termination signal, or secondary structure, which functions to terminate the polymerization activity of PE), producing a single strand DNA flap that is extended from the original 3′ hydroxyl group of the nicked PAM strand. The DNA synthesis template codes for a single strand DNA that is homologous to the endogenous 5′-ended single strand of DNA that immediately follows the nick site on the PAM strand and incorporates the desired nucleotide change (e.g., single base substitution, insertion, deletion, inversion). The position of the desired edit can be in any position following downstream of the nick site on the PAM strand, which can include position +1, +2, +3, +4 (the start of the PAM site), +5 (position 2 of the PAM site), +6 (position 3 of the PAM site), +7, +8, +9, +10, +11, +12, +13, +14, +15, +16, +17, +18, +19, +20, +21, +22, +23, +24, +25, +26, +27, +28, +29, +30, +31, +32, +33, +34, +35, +36, +37, +38, +39, +40, +41, +42, +43, +44, +45, +46, +47, +48, +49, +50, +51, +52, +53, +54, +55, +56, +57, +58, +59, +60, +61, +62, +63, +64, +65, +66, +67, +68, +69, +70, +71, +72, +73, +74, +75, +76, +77, +78, +79, +80, +81, +82, +83, +84, +85, +86, +87, +88, +89, +90, +91, +92, +93, +94, +95, +96, +97, +98, +99, +100, +101, +102, +103, +104, +105, +106, +107, +108, +109, +110, +111, +112, +113, +114, +115, +116, +117, +118, +119, +120, +121, +122, +123, +124, +125, +126, +127, +128, +129, +130, +131, +132, +133, +134, +135, +136, +137, +138, +139, +140, +141, +142, +143, +144, +145, +146, +147, +148, +149, or +150, or more (relative to the downstream position of the nick site). Once the 3′end single stranded DNA (containing the edit of interest) replaces the endogenous 5′ end single stranded DNA, the DNA repair and replication processes will result in permanent installation of the edit site on the PAM strand, and then correction of the mismatch on the non-PAM strand that will exist at the edit site. In this way, the edit will extend to both strands of DNA on the target DNA site. It will be appreciated that reference to “edited strand” and “non-edited” strand only intends to delineate the strands of DNA involved in the PE mechanism. The “edited strand” is the strand that first becomes edited by replacement of the 5′ ended single strand DNA immediately downstream of the nick site with the synthesized 3′ ended single stranded DNA containing the desired edit. The “non-edited” strand is the strand pair with the edited strand, but which itself also becomes edited through repair and/or replication to be complementary to the edited strand, and in particular, the edit of interest.
• FIG. 1L depicts the mechanism of prime editing showing the anatomical features of the target DNA, prime editor complex, and the interaction between the PEgRNA and the target DNA. First, a prime editor comprising a fusion protein having a polymerase (e.g., reverse transcriptase) and a napDNAbp (e.g., SpCas9 nickase, e.g., a SpCas9 having a deactivating mutation in an HNH nuclease domain (e.g., H840A) or a deactivating mutation in a RuvC nuclease domain (D10A)) is complexed with a PEgRNA and DNA having a target DNA to be edited. The PEgRNA comprises a spacer, gRNA core (aka gRNA scaffold or gRNA backbone) (which binds to the napDNAbp), and an extension arm. The extension arm can be at the 3′ end, the 5′ end, or somewhere within the PEgRNA molecule. As shown, the extension arm is at the 3′ end of the PEgRNA. The extension arm comprises in the 3′ to 5′ direction a primer binding site and a DNA synthesis template (comprising both an edit of interest and regions of homology (i.e., homology arms) that are homologous with the 5′ ended single stranded DNA immediately following the nick site on the PAM strand. As shown, once the nick is introduced thereby producing a free 3′ hydroxyl group immediately upstream of the nick site, the region immediately upstream of the nick site on the PAM strand anneals to a complementary sequence at the 3′ end of the extension arm referred to as the “primer binding site,” creating a short double-stranded region with an available 3′ hydroxyl end, which forms a substrate for the polymerase of the prime editor complex. The polymerase (e.g., reverse transcriptase) then polymerase as strand of DNA from the 3′ hydroxyl end to the end of the extension arm. The sequence of the single stranded DNA is coded for by the DNA synthesis template, which is the portion of the extension arm (i.e., excluding the primer binding site) that is “read” by the polymerase to synthesize new DNA. This polymerization effectively extends the sequence of the original 3′ hydroxyl end of the initial nick site. The DNA synthesis template encodes a single strand of DNA that comprises not only the desired edit, but also regions that are homologous to the endogenous single strand of DNA immediately downstream of the nick site on the PAM strand. Next, the encoded 3′ ended single strand of DNA (i.e., the 3′ single strand DNA flap) displaces the corresponding homologous endogenous 5′-ended single strand of DNA immediately downstream of the nick site on the PAM strand, forming a DNA intermediate having a 5′-ended single strand DNA flap, which is removed by the cell (e.g., by a flap endonuclease). The 3′-ended single strand DNA flap, which anneals to the complement of the endogenous 5′-ended single strand DNA flap, is ligated to the endogenous strand after the 5′ DNA flap is removed. The desired edit in the 3′ ended single strand DNA flap, now annealed and ligate, forms a mismatch with the complement strand, which undergoes DNA repair and/or a round of replication, thereby permanently installing the desired edit on both strands.
• FIG. 2 shows three Cas complexes (SpCas9, SaCas9, and LbCas12a) that can be used in the herein described prime editors and their PAM, gRNA, and DNA cleavage features. The figure shows designs for complexes involving SpCas9, SaCas9, and LbCas12a.
• FIGS. 3A-3F show designs for engineered 5′ prime editor gRNA (FIG. 3A), 3′ prime editor gRNA (FIG. 3B), and an intramolecular extension (FIG. 3C). The extended guide RNA (or extended gRNA) may also be referred to herein as PEgRNA or “prime editing guide RNA.” FIG. 3D and FIG. 3E provide additional embodiments of 3′ and 5′ prime editor gRNAs (PEgRNAs), respectively. FIG. 3F illustrates the interaction between a 3′ end prime editor guide RNA with a target DNA sequence. The embodiments of FIGS. 3A-3C depict exemplary arrangements of the reverse transcription template sequence (i.e., or more broadly referred to as a DNA synthesis template, as indicated, since the RT is only one type of polymerase that may be used in the context of prime editors), the primer binding site, and an optional linker sequence in the extended portions of the 3′, 5′, and intramolecular versions, as well as the general arrangements of the spacer and core regions. The disclosed prime editing process is not limited to these configurations of extended guide RNAs. The embodiment of FIG. 3D provides the structure of an exemplary PEgRNA contemplated herein. The PEgRNA comprises three main component elements ordered in the 5′ to 3′ direction, namely: a spacer, a gRNA core, and an extension arm at the 3′ end. The extension arm may further be divided into the following structural elements in the 5′ to 3′ direction, namely: a primer binding site (A), an edit template (B), and a homology arm (C). In addition, the PEgRNA may comprise an optional 3′ end modifier region (e1) and an optional 5′ end modifier region (e2). Still further, the PEgRNA may comprise a transcriptional termination signal at the 3′ end of the PEgRNA (not depicted). These structural elements are further defined herein. The depiction of the structure of the PEgRNA is not meant to be limiting and embraces variations in the arrangement of the elements. For example, the optional sequence modifiers (e1) and (e2) could be positioned within or between any of the other regions shown, and not limited to being located at the 3′ and 5′ ends. The PEgRNA could comprise, in certain embodiments, secondary RNA structure, such as, but not limited to, hairpins, stem/loops, toe loops, RNA-binding protein recruitment domains (e.g., the MS2 aptamer which recruits and binds to the MS2cp protein). For instance, such secondary structures could be position within the spacer, the gRNA core, or the extension arm, and in particular, within the e1 and/or e2 modifier regions. In addition to secondary RNA structures, the PEgRNAs could comprise (e.g., within the e1 and/or e2 modifier regions) a chemical linker or a poly(N) linker or tail, where “N” can be any nucleobase. In some embodiments (e.g., as shown in FIG. 72(c)), the chemical linker may function to prevent reverse transcription of the sgRNA scaffold or core. In addition, in certain embodiments (e.g., see FIG. 72(c)), the extension arm (3) could be comprised of RNA or DNA, and/or could include one or more nucleobase analogs (e.g., which might add functionality, such as temperature resilience). Still further, the orientation of the extension arm (3) can be in the natural 5′-to-3′ direction, or synthesized in the opposite orientation in the 3′-to-5′ direction (relative to the orientation of the PEgRNA molecule overall). It is also noted that one of ordinary skill in the art will be able to select an appropriate DNA polymerase, depending on the nature of the nucleic acid materials of the extension arm (i.e., DNA or RNA), for use in prime editing that may be implemented either as a fusion with the napDNAbp or as provided in trans as a separate moiety to synthesize the desired template-encoded 3′ single-strand DNA flap that includes the desired edit. For example, if the extension arm is RNA, then the DNA polymerase could be a reverse transcriptase or any other suitable RNA-dependent DNA polymerase. However, if the extension arm is DNA, then the DNA polymerase could be a DNA-dependent DNA polymerase. In various embodiments, provision of the DNA polymerase could be in trans, e.g., through the use of an RNA-protein recruitment domain (e.g., an MS2 hairpin installed on the PEgRNA (e.g., in the e1 or e2 region, or elsewhere and an MS2cp protein fused to the DNA polymerase, thereby co-localizing the DNA polymerase to the PEgRNA). It is also noted that the primer binding site does not generally form a part of the template that is used by the DNA polymerase (e.g., reverse transcriptase) to encode the resulting 3′ single-strand DNA flap that includes the desired edit. Thus, the designation of the “DNA synthesis template” refers to the region or portion of the extension arm (3) that is used as a template by the DNA polymerase to encode the desired 3′ single-strand DNA flap containing the edit and regions of homology to the 5′ endogenous single strand DNA flap that is replaced by the 3′ single strand DNA strand product of prime editing DNA synthesis. In some embodiments, the DNA synthesis template includes the “edit template” and the “homology arm”, or one or more homology arms, e.g., before and after the edit template. The edit template can be as small as a single nucleotide substitution, or it may be an insertion, or an inversion of DNA. In addition, the edit template may also include a deletion, which can be engineered by encoding homology arm that contains a desired deletion. In other embodiments, the DNA synthesis template may also include the e2 region or a portion thereof. For instance, if the e2 region comprises a secondary structure that causes termination of DNA polymerase activity, then it is possible that DNA polymerase function will be terminated before any portion of the e2 region is actual encoded into DNA. It is also possible that some or even all of the e2 region will be encoded into DNA. How much of e2 is actually used as a template will depend on its constitution and whether that constitution interrupts DNA polymerase function.
• The embodiment of FIG. 3E provides the structure of another PEgRNA contemplated herein. The PEgRNA comprises three main component elements ordered in the 5′ to 3′ direction, namely: a spacer, a gRNA core, and an extension arm at the 3′ end. The extension arm may further be divided into the following structural elements in the 5′ to 3′ direction, namely: a primer binding site (A), an edit template (B), and a homology arm (C). In addition, the PEgRNA may comprise an optional 3′ end modifier region (e1) and an optional 5′ end modifier region (e2). Still further, the PEgRNA may comprise a transcriptional termination signal on the 3′ end of the PEgRNA (not depicted). These structural elements are further defined herein. The depiction of the structure of the PEgRNA is not meant to be limiting and embraces variations in the arrangement of the elements. For example, the optional sequence modifiers (e1) and (e2) could be positioned within or between any of the other regions shown, and not limited to being located at the 3′ and 5′ ends. The PEgRNA could comprise, in certain embodiments, secondary RNA structures, such as, but not limited to, hairpins, stem/loops, toe loops, RNA-binding protein recruitment domains (e.g., the MS2 aptamer which recruits and binds to the MS2cp protein). These secondary structures could be positioned anywhere in the PEgRNA molecule. For instance, such secondary structures could be position within the spacer, the gRNA core, or the extension arm, and in particular, within the e1 and/or e2 modifier regions. In addition to secondary RNA structures, the PEgRNAs could comprise (e.g., within the e1 and/or e2 modifier regions) a chemical linker or a poly(N) linker or tail, where “N” can be any nucleobase. In some embodiments (e.g., as shown in FIG. 72(c)), the chemical linker may function to prevent reverse transcription of the sgRNA scaffold or core. In addition, in certain embodiments (e.g., see FIG. 72(c)), the extension arm (3) could be comprised of RNA or DNA, and/or could include one or more nucleobase analogs (e.g., which might add functionality, such as temperature resilience). Still further, the orientation of the extension arm (3) can be in the natural 5′-to-3′ direction, or synthesized in the opposite orientation in the 3′-to-5′ direction (relative to the orientation of the PEgRNA molecule overall). It is also noted that one of ordinary skill in the art will be able to select an appropriate DNA polymerase, depending on the nature of the nucleic acid materials of the extension arm (i.e., DNA or RNA), for use in prime editing that may be implemented either as a fusion with the napDNAbp or as provided in trans as a separate moiety to synthesize the desired template-encoded 3′ single-strand DNA flap that includes the desired edit. For example, if the extension arm is RNA, then the DNA polymerase could be a reverse transcriptase or any other suitable RNA-dependent DNA polymerase. However, if the extension arm is DNA, then the DNA polymerase could be a DNA-dependent DNA polymerase. In various embodiments, provision of the DNA polymerase could be in trans, e.g., through the use of an RNA-protein recruitment domain (e.g., an MS2 hairpin installed on the PEgRNA (e.g., in the e1 or e2 region, or elsewhere and an MS2cp protein fused to the DNA polymerase, thereby co-localizing the DNA polymerase to the PEgRNA). It is also noted that the primer binding site does not generally form a part of the template that is used by the DNA polymerase (e.g., reverse transcriptase) to encode the resulting 3′ single-strand DNA flap that includes the desired edit. Thus, the designation of the “DNA synthesis template” refers to the region or portion of the extension arm (3) that is used as a template by the DNA polymerase to encode the desired 3′ single-strand DNA flap containing the edit and regions of homology to the 5′ endogenous single strand DNA flap that is replaced by the 3′ single strand DNA strand product of prime editing DNA synthesis. In some embodiments, the DNA synthesis template includes the “edit template” and the “homology arm”, or one or more homology arms, e.g., before and after the edit template. The edit template can be as small as a single nucleotide substitution, or it may be an insertion, or an inversion of DNA. In addition, the edit template may also include a deletion, which can be engineered by encoding homology arm that contains a desired deletion. In other embodiments, the DNA synthesis template may also include the e2 region or a portion thereof. For instance, if the e2 region comprises a secondary structure that causes termination of DNA polymerase activity, then it is possible that DNA polymerase function will be terminated before any portion of the e2 region is actual encoded into DNA. It is also possible that some or even all of the e2 region will be encoded into DNA. How much of e2 is actually used as a template will depend on its constitution and whether that constitution interrupts DNA polymerase function.
• The schematic of FIG. 3F depicts the interaction of a typical PEgRNA with a target site of a double stranded DNA and the concomitant production of a 3′ single stranded DNA flap containing the genetic change of interest. The double strand DNA is shown with the top strand (i.e., the target strand) in the 3′ to 5′ orientation and the lower strand (i.e., the PAM strand or non-target strand) in the 5′ to 3′ direction. The top strand comprises the complement of the “protospacer” and the complement of the PAM sequence and is referred to as the “target strand” because it is the strand that is target by and anneals to the spacer of the PEgRNA. The complementary lower strand is referred to as the “non-target strand” or the “PAM strand” or the “protospacer strand” since it contains the PAM sequence (e.g., NGG) and the protospacer. Although not shown, the PEgRNA depicted would be complexed with a Cas9 or equivalent domain of a prime editor fusion protein. As shown in the schematic, the spacer of the PEgRNA anneals to the complementary region of the protospacer on the target strand. This interaction forms as DNA/RNA hybrid between the spacer RNA and the complement of the proto spacer DNA, and induces the formation of an R loop in the protospacer. As taught elsewhere herein, the Cas9 protein (not shown) then induces a nick in the non-target strand, as shown. This then leads to the formation of the 3′ ssDNA flap region immediately upstream of the nick site which, in accordance with *z*, interacts with the 3′ end of the PEgRNA at the primer binding site. The 3′ end of the ssDNA flap (i.e., the reverse transcriptase primer sequence) anneals to the primer binding site (A) on the PEgRNA, thereby priming reverse transcriptase. Next, reverse transcriptase (e.g., provided in trans or provided cis as a fusion protein, attached to the Cas9 construct) then polymerizes a single strand of DNA which is coded for by the DNA synthesis template (including the edit template (B) and homology arm (C)). The polymerization continues towards the 5′ end of the extension arm. The polymerized strand of ssDNA forms a ssDNA 3′ end flap which, as describe elsewhere (e.g., as shown in FIG. 1G), invades the endogenous DNA, displacing the corresponding endogenous strand (which is removed as a 5′ ended DNA flap of endogenous DNA), and installing the desired nucleotide edit (single nucleotide base pair change, deletions, insertions (including whole genes) through naturally occurring DNA repair/replication rounds.
• FIG. 3G depicts yet another embodiment of prime editing contemplated herein. In particular, the top schematic depicts one embodiment of a prime editor (PE), which comprises a fusion protein of a napDNAbp (e.g., SpCas9) and a polymerase (e.g., a reverse transcriptase), which are joined by a linker. The PE forms a complex with a PEgRNA by binding to the gRNA core of the PEgRNA. In the embodiment shown, the PEgRNA is equipped with a 3′ extension arm that comprises, beginning at the 3′ end, a primer binding site (PBS) followed by a DNA synthesis template. The bottom schematic depicts a variant of a prime editor, referred to as a “trans prime editor (tPE).” In this embodiment, the DNA synthesis template and PBS are decoupled from the PEgRNA and presented on a separate molecule, referred to as a trans prime editor RNA template (“tPERT”), which comprises an RNA-protein recruitment domain (e.g., a MS2 hairpin). The PE itself is further modified to comprise a fusion to a rPERT recruiting protein (“RP”), which is a protein which specifically recognizes and binds to the RNA-protein recruitment domain. In the example where the RNA-protein recruitment domain is an MS2 hairpin, the corresponding rPERT recruiting protein can be MS2cp of the MS2 tagging system. The MS2 tagging system is based on the natural interaction of the MS2 bacteriophage coat protein (“MCP” or “MS2cp”) with a stem-loop or hairpin structure present in the genome of the phage, i.e., the “MS2 hairpin” or “MS2 aptamer.” In the case of trans prime editing, the RP-PE:gRNA complex “recruits” a tPERT having the appropriate RNA-protein recruitment domain to co-localize with the PE:gRNA complex, thereby providing the PBS and DNA synthesis template in trans for use in prime editing, as shown in the example depicted in FIG. 3H.
• FIG. 3H depicts the process of trans prime editing. In this embodiment, the trans prime editor comprises a “PE2” prime editor (i.e., a fusion of a Cas9(H840A) and a variant MMLV RT) fused to an MS2cp protein (i.e., a type of recruiting protein that recognizes and binds to an MS2 aptamer) and which is complexed with an sgRNA (i.e., a standard guide RNA as opposed to a PEgRNA). The trans prime editor binds to the target DNA and nicks the nontarget strand. The MS2cp protein recruits a tPERT in trans through the specific interaction with the RNA-protein recruitment domain on the tPERT molecule. The tPERT becomes co-localized with the trans prime editor, thereby providing the PBS and DNA synthesis template functions in trans for use by the reverse transcriptase polymerase to synthesize a single strand DNA flap having a 3′ end and containing the desired genetic information encoded by the DNA synthesis template.
• FIGS. 4A-4E demonstrate in vitro TPRT assays (i.e., prime editing assays). FIG. 4A is a schematic of fluorescently labeled DNA substrates gRNA templated extension by an RT enzyme, PAGE. FIG. 4B shows TPRT (i.e., prime editing) with pre-nicked substrates, dCas9, and 5′-extended gRNAs of differing synthesis template length. FIG. 4C shows the RT reaction with pre-nicked DNA substrates in the absence of Cas9. FIG. 4D shows TPRT (i.e., prime editing) on full dsDNA substrates with Cas9(H840A) and 5′-extended gRNAs. FIG. 4E shows a 3′-extended gRNA template with pre-nicked and full dsDNA substrates. All reactions are with M-MLV RT.
• FIG. 5 shows in vitro validations using 5′-extended gRNAs with varying length synthesis templates. Fluorescently labeled (Cy5) DNA targets were used as substrates, and were pre-nicked in this set of experiments. The Cas9 used in these experiments is catalytically dead Cas9 (dCas9), and the RT used is Superscript III, a commercial RT derived from the Moloney-Murine Leukemia Virus (M-MLV). dCas9:gRNA complexes were formed from purified components. Then, the fluorescently labeled DNA substrate was added along with dNTPs and the RT enzyme. After 1 hour of incubation at 37° C., the reaction products were analyzed by denaturing urea-polyacrylamide gel electrophoresis (PAGE). The gel image shows extension of the original DNA strand to lengths that are consistent with the length of the reverse transcription template.
• FIG. 6 shows in vitro validations using 5′-extended gRNAs with varying length synthesis templates, which closely parallels those shown in FIG. 5 . However, the DNA substrates are not pre-nicked in this set of experiments. The Cas9 used in these experiments is a Cas9 nickase (SpyCas9 H840A mutant) and the RT used is Superscript III, a commercial RT derived from the Moloney-Murine Leukemia Virus (M-MLV). The reaction products were analyzed by denaturing urea-polyacrylamide gel electrophoresis (PAGE). As shown in the gel, the nickase efficiently cleaves the DNA strand when the standard gRNA is used (gRNA_0, lane 3).
• FIG. 7 demonstrates that 3′ extensions support DNA synthesis and do not significantly effect Cas9 nickase activity. Pre-nicked substrates (black arrow) are near-quantitatively converted to RT products when either dCas9 or Cas9 nickase is used (lanes 4 and 5). Greater than 50% conversion to the RT product (red arrow) is observed with full substrates (lane 3). Cas9 nickase (SpyCas9 H840A mutant), catalytically dead Cas9 (dCas9) and Superscript III, a commercial RT derived from the Moloney-Murine Leukemia Virus (M-MLV) are used.
• FIG. 8 demonstrates dual color experiments that were used to determine if the RT reaction preferentially occurs with the gRNA in cis (bound in the same complex). Two separate experiments were conducted for 5′-extended and 3′-extended gRNAs. Products were analyzed by PAGE. Product ratio calculated as (Cy3cis/Cy3trans)/(Cy5trans/Cy5cis).
• FIGS. 9A-9D demonstrates a flap model substrate. FIG. 9A shows a dual-FP reporter for flap-directed mutagenesis. FIG. 9B shows stop codon repair in HEK cells. FIG. 9C shows sequenced yeast clones after flap repair. FIG. 9D shows testing of different flap features in human cells.
• FIG. 10 demonstrates prime editing on plasmid substrates. A dual-fluorescent reporter plasmid was constructed for yeast (S. cerevisiae) expression. Expression of this construct in yeast produces only GFP. The in vitro prime editing reaction introduces a point mutation, and transforms the parent plasmid or an in vitro Cas9(H840A) nicked plasmid into yeast. The colonies are visualized by fluorescence imaging. Yeast dual-FP plasmid transformants are shown. Transforming the parent plasmid or an in vitro Cas9(H840A) nicked plasmid results in only green GFP expressing colonies. The prime editing reaction with 5′-extended or 3′-extended gRNAs produces a mix of green and yellow colonies. The latter express both GFP and mCherry. More yellow colonies are observed with the 3′-extended gRNA. A positive control that contains no stop codon is shown as well.
• FIG. 11 shows prime editing on plasmid substrates similar to the experiment in FIG. 10 , but instead of installing a point mutation in the stop codon, prime editing installs a single nucleotide insertion (left) or deletion (right) that repairs a frameshift mutation and allows for synthesis of downstream mCherry. Both experiments used 3′ extended gRNAs.
• FIG. 12 shows editing products of prime editing on plasmid substrates, characterized by Sanger sequencing. Individually colonies from the TRT transformations were selected and analyzed by Sanger sequencing. Precise edits were observed by sequencing select colonies. Green colonies contained plasmids with the original DNA sequence, while yellow colonies contained the precise mutation designed by the prime editing gRNA. No other point mutations or indels were observed.
• FIG. 13 shows the potential scope for the new prime editing technology is shown and compared to deaminase-mediated base editor technologies.
• FIG. 14 shows a schematic of editing in human cells.
• FIG. 15 demonstrates the extension of the primer binding site in gRNA.
• FIG. 16 shows truncated gRNAs for adjacent targeting.
• FIGS. 17A-17C are graphs displaying the % T to A conversion at the target nucleotide after transfection of components in human embryonic kidney (HEK) cells. FIG. 17A shows data, which presents results using an N-terminal fusion of wild type MLV reverse transcriptase to Cas9(H840A) nickase (32-amino acid linker). FIG. 17B is similar to FIG. 17A, but for C-terminal fusion of the RT enzyme. FIG. 17C is similar to FIG. 17A but the linker between the MLV RT and Cas9 is 60 amino acids long instead of 32 amino acids.
• FIG. 18 shows high purity T to A editing at HEK3 site by high-throughput amplicon sequencing. The output of sequencing analysis displays the most abundant genotypes of edited cells.
• FIG. 19 shows editing efficiency at the target nucleotide (blue bars) alongside indel rates (orange bars). WT refers to the wild type MLV RT enzyme. The mutant enzymes (M1 through M4) contain the mutations listed to the right. Editing rates were quantified by high throughput sequencing of genomic DNA amplicons.
• FIG. 20 shows editing efficiency of the target nucleotide when a single strand nick is introduced in the complementary DNA strand in proximity to the target nucleotide. Nicking at various distances from the target nucleotide was tested (triangles). Editing efficiency at the target base pair (blue bars) is shown alongside the indel formation rate (orange bars). The “none” example does not contain a complementary strand nicking guide RNA. Editing rates were quantified by high throughput sequencing of genomic DNA amplicons.
• FIG. 21 demonstrates processed high throughput sequencing data showing the desired T to A transversion mutation and general absence of other major genome editing byproducts.
• FIG. 22 provides a schematic of an exemplary process for conducting targeted mutagenesis with an error-prone reverse transcriptase on a target locus using a nucleic acid programmable DNA binding protein (napDNAbp) complexed with an extended guide RNA, i.e., prime editing with an error-prone RT. This process may be referred to as an embodiment of prime editing for targeted mutagenesis. The extended guide RNA comprises an extension at the 3′ or 5′ end of the guide RNA, or at an intramolecular location in the guide RNA. In step (a), the napDNAbp/gRNA complex contacts the DNA molecule and the gRNA guides the napDNAbp to bind to the target locus to be mutagenized. In step (b), a nick in one of the strands of DNA of the target locus is introduced (e.g., by a nuclease or chemical agent), thereby creating an available 3′ end in one of the strands of the target locus. In certain embodiments, the nick is created in the strand of DNA that corresponds to the R-loop strand, i.e., the strand that is not hybridized to the guide RNA sequence. In step (c), the 3′ end DNA strand interacts with the extended portion of the guide RNA in order to prime reverse transcription. In certain embodiments, the 3′ ended DNA strand hybridizes to a specific RT priming sequence on the extended portion of the guide RNA. In step (d), an error-prone reverse transcriptase is introduced which synthesizes a mutagenized single strand of DNA from the 3′ end of the primed site towards the 3′ end of the guide RNA. Exemplary mutations are indicated with an asterisk “*”. This forms a single-strand DNA flap comprising the desired mutagenized region. In step (e), the napDNAbp and guide RNA are released. Steps (f) and (g) relate to the resolution of the single strand DNA flap (comprising the mutagenized region) such that the desired mutagenized region becomes incorporated into the target locus. This process can be driven towards the desired product formation by removing the corresponding 5′ endogenous DNA flap that forms once the 3′ single strand DNA flap invades and hybridizes to the complementary sequence on the other strand. The process can also be driven towards product formation with second strand nicking, as exemplified in FIG. 1F. Following endogenous DNA repair and/or replication processes, the mutagenized region becomes incorporated into both strands of DNA of the DNA locus.
• FIG. 23 is a schematic of gRNA design for contracting trinucleotide repeat sequences and trinucleotide repeat contraction with TPRT genome editing (i.e., prime editing). Trinucleotide repeat expansion is associated with a number of human diseases, including Huntington's disease, Fragile X syndrome, and Friedreich's ataxia. The most common trinucleotide repeat contains CAG triplets, though GAA triplets (Friedreich's ataxia) and CGG triplets (Fragile X syndrome) also occur. Inheriting a predisposition to expansion, or acquiring an already expanded parental allele, increases the likelihood of acquiring the disease. Pathogenic expansions of trinucleotide repeats could hypothetically be corrected using prime editing. A region upstream of the repeat region can be nicked by an RNA-guided nuclease, then used to prime synthesis of a new DNA strand that contains a healthy number of repeats (which depends on the particular gene and disease). After the repeat sequence, a short stretch of homology is added that matches the identity of the sequence adjacent to the other end of the repeat (red strand). Invasion of the newly synthesized strand, and subsequent replacement of the endogenous DNA with the newly synthesized flap, leads to a contracted repeat allele.
• FIG. 24 is a schematic showing precise 10-nucleotide deletion with prime editing. A guide RNA targeting the HEK3 locus was designed with a reverse transcription template that encodes a 10-nucleotide deletion after the nick site. Editing efficiency in transfected HEK cells was assessed using amplicon sequencing.
• FIG. 25 is a schematic showing gRNA design for peptide tagging genes at endogenous genomic loci and peptide tagging with TPRT genome editing (i.e., prime editing). The FlAsH and ReAsH tagging systems comprise two parts: (1) a fluorophore-biarsenical probe, and (2) a genetically encoded peptide containing a tetracysteine motif, exemplified by the sequence FLNCCPGCCMEP (SEQ ID NO: 1). When expressed within cells, proteins containing the tetracysteine motif can be fluorescently labeled with fluorophore-arsenic probes (see ref: J. Am. Chem. Soc., 2002, 124 (21), pp 6063-6076. DOI: 10.1021/ja017687n). The “sortagging” system employs bacterial sortase enzymes that covalently conjugate labeled peptide probes to proteins containing suitable peptide substrates (see ref: Nat. Chem. Biol. 2007 November; 3(11):707-8. DOI: 10.1038/nchembio.2007.31). The FLAG-tag (DYKDDDDK (SEQ ID NO: 2)), V5-tag (GKPIPNPLLGLDST (SEQ ID NO: 3)), GCN4-tag (EELLSKNYHLENEVARLKK (SEQ ID NO: 4)), HA-tag (YPYDVPDYA (SEQ ID NO: 5)), and Myc-tag (EQKLISEEDL (SEQ ID NO: 6)) are commonly employed as epitope tags for immunoassays. The pi-clamp encodes a peptide sequence (FCPF) that can by labeled with a pentafluoro-aromatic substrates (ref: Nat. Chem. 2016 February; 8(2):120-8. doi: 10.1038/nchem.2413).
• FIG. 26A shows precise installation of a His₆-tag and a FLAG-tag into genomic DNA. A guide RNA targeting the HEK3 locus was designed with a reverse transcription template that encodes either an 18-nt His-tag insertion or a 24-nt FLAG-tag insertion. Editing efficiency in transfected HEK cells was assessed using amplicon sequencing. Note that the full 24-nt sequence of the FLAG-tag is outside of the viewing frame (sequencing confirmed full and precise insertion). FIG. 26B shows a schematic outlining various applications involving protein/peptide tagging, including (a) rendering proteins soluble or insoluble, (b) changing or tracking the cellular localization of a protein, (c) extending the half-life of a protein, (d) facilitating protein purification, and (e) facilitating the detection of proteins.
• FIG. 27 shows an overview of prime editing by installing a protective mutation in PRNP that prevents or halts the progression of prion disease. The PEgRNA sequences correspond to SEQ ID NO: 351 on the left (i.e., 5′ of the sgRNA scaffold) and SEQ ID NO 3864 on the right (i.e., 3′ of the sgRNA scaffold).
• FIG. 28A is a schematic of PE-based insertion of sequences encoding RNA motifs.
• FIG. 28B is a list (not exhaustive) of some example motifs that could potentially be inserted, and their functions.
• FIG. 29A is a depiction of a prime editor. FIG. 29B shows possible modifications to genomic, plasmid, or viral DNA directed by a PE. FIG. 29C shows an example scheme for insertion of a library of peptide loops into a specified protein (in this case GFP) via a library of PEgRNAs. FIG. 29D shows an example of possible programmable deletions of codons or N-, or C-terminal truncations of a protein using different PEgRNAs. Deletions would be predicted to occur with minimal generation of frameshift mutations.
• FIG. 30 shows a possible scheme for iterative insertion of codons in a continual evolution system, such as PACE.
• FIG. 31 is an illustration of an engineered gRNA showing the gRNA core, ˜20 nt spacer matching the sequence of the targeted gene, the reverse transcription template with immunogenic epitope nucleotide sequence and the primer binding site matching the sequence of the targeted gene.
• FIG. 32 is a schematic showing using prime editing as a means to insert known immunogenicity epitopes into endogenous or foreign genomic DNA, resulting in modification of the corresponding proteins.
• FIG. 33 is a schematic showing PEgRNA design for primer binding sequence insertions and primer binding insertion into genomic DNA using prime editing for determining off-target editing. In this embodiment, prime editing is conducted inside a living cell, a tissue, or an animal model. As a first step, an appropriate PEgRNA is designed. The top schematic shows an exemplary PEgRNA that may be used in this aspect. The spacer in the PEgRNA (labeled “protospacer”) is complementary to one of the strands of the genomic target. The PE:PEgRNA complex (i.e., the PE complex) installs a single stranded 3′ end flap at the nick site which contains the encoded primer binding sequence and the region of homology (coded by the homology arm of the PEgRNA) that is complementary to the region just downstream of the cut site (in red). Through flap invasion and DNA repair/replication processes, the synthesized strand becomes incorporated into the DNA, thereby installing the primer binding site. This process can occur at the desired genomic target, but also at other genomic sites that might interact with the PEgRNA in an off-target manner (i.e., the PEgRNA guides the PE complex to other off-target sites due to the complementarity of the spacer region to other genomic sites that are not the intended genomic site). Thus, the primer binding sequence may be installed not only at the desired genomic target, but at off-target genomic sites elsewhere in the genome. In order to detect the insertion of these primer binding sites at both the intended genomic target sites and the off-target genomic sites, the genomic DNA (post-PE) can be isolated, fragmented, and ligated to adapter nucleotides (shown in red). Next, PCR may be carried out with PCR oligonucleotides that anneal to the adapters and to the inserted primer binding sequence to amplify on-target and off-target genomic DNA regions into which the primer binding site was inserted by PE. High throughput sequencing then me be conducted to and sequence alignments to identify the insertion points of PE-inserted primer binding sequences at either the on-target site or at off-target sites.
• FIG. 34 is a schematic showing the precise insertion of a gene with PE.
• FIG. 35A is a schematic showing the natural insulin signaling pathway. FIG. 35B is a schematic showing FKBP12-tagged insulin receptor activation controlled by FK1012.
• FIG. 36 shows small-molecule monomers. References: bumped FK506 mimic (2)¹⁰⁷
• FIG. 37 shows small-molecule dimers. References: FK1012 4^95,96; FK1012 5¹⁰⁸; FK1012 6¹⁰⁷; AP1903 7¹⁰⁷; cyclosporin A dimer 8⁹⁸; FK506-cyclosporin A dimer (FkCsA) 9¹⁰⁰.
• FIGS. 38A-38F provide an overview of prime editing and feasibility studies in vitro and in yeast cells. FIG. 38A shows the 75,122 known pathogenic human genetic variants in ClinVar (accessed July, 2019), classified by type. FIG. 38B shows that a prime editing complex consists of a prime editor (PE) protein containing an RNA-guided DNA-nicking domain, such as Cas9 nickase, fused to an engineered reverse transcriptase domain and complexed with a prime editing guide RNA (PEgRNA). The PE:PEgRNA complex binds the target DNA site and enables a large variety of precise DNA edits at a wide range of DNA positions before or after the target site's protospacer adjacent motif (PAM). FIG. 38C shows that upon DNA target binding, the PE:PEgRNA complex nicks the PAM-containing DNA strand. The resulting free 3′ end hybridizes to the primer-binding site of the PEgRNA. The reverse transcriptase domain catalyzes primer extension using the RT template of the PEgRNA, resulting in a newly synthesized DNA strand containing the desired edit (the 3′ flap). Equilibration between the edited 3′ flap and the unedited 5′ flap containing the original DNA, followed by cellular 5′ flap cleavage and ligation, and DNA repair or replication to resolve the heteroduplex DNA, results in stably edited DNA. FIG. 38D shows in vitro 5′-extended PEgRNA primer extension assays with pre-nicked dsDNA substrates containing 5′-Cy5 labeled PAM strands, dCas9, and a commercial M-MLV RT variant (RT, Superscript III). dCas9 was complexed with PEgRNAs containing RT template of varying lengths, then added to DNA substrates along with the indicated components. Reactions were incubated at 37° C. for 1 hour, then analyzed by denaturing urea PAGE and visualized for Cy5 fluorescence. FIG. 38E shows primer extension assays performed as in FIG. 38D using 3′-extended PEgRNAs pre-complexed with dCas9 or Cas9 H840A nickase, and pre-nicked or non-nicked 5′-Cy5-labeled dsDNA substrates. FIG. 38F shows yeast colonies transformed with GFP-mCherry fusion reporter plasmids edited in vitro with PEgRNAs, Cas9 nickase, and RT. Plasmids containing nonsense or frameshift mutations between GFP and mCherry were edited with 5′-extended or 3′-extended PEgRNAs that restore mCherry translation via transversion mutation, 1-bp insertion, or 1-bp deletion. GFP and mCherry double-positive cells (yellow) reflect successful editing.
• FIGS. 39A-39D show prime editing of genomic DNA in human cells by PE1 and PE2. FIG. 39A shows PEgRNAs contain a spacer sequence, a sgRNA scaffold, and a 3′ extension containing a primer-binding site (green) and a reverse transcription (RT) template (purple), which contains the edited base(s) (red). The primer-binding site hybridizes to the PAM-containing DNA strand immediately upstream of the site of nicking. The RT template is homologous to the DNA sequence downstream of the nick, with the exception of the encoded edit. FIG. 39B shows an installation of a T●A-to-A●T transversion edit at the HEK3 site in HEK293T cells using Cas9 H840A nickase fused to wild-type M-MLV reverse transcriptase (PE1) and PEgRNAs of varying primer-binding site lengths. FIG. 39C shows the use of an engineered pentamutant M-MLV reverse transcriptase (D200N, L603W, T306K, W313F, T330P) in PE2 substantially improves prime editing transversion efficiencies at five genomic sites in HEK293T cells, and small insertion and small deletion edits at HEK3. FIG. 39D is a comparison of PE2 editing efficiencies with varying RT template lengths at five genomic sites in HEK293T cells. Values and error bars reflect the mean and s.d. of three independent biological replicates.
• FIGS. 40A-40C show PE3 and PE3b systems nick the non-edited strand to increase prime editing efficiency. FIG. 40A is an overview of the prime editing by PE3. After initial synthesis of the edited strand, DNA repair will remove either the newly synthesized strand containing the edit (3′ flap excision) or the original genomic DNA strand (5′ flap excision). 5′ flap excision leaves behind a DNA heteroduplex containing one edited strand and one non-edited strand. Mismatch repair machinery or DNA replication could resolve the heteroduplex to give either edited or non-edited products. Nicking the non-edited strand favors repair of that strand, resulting in preferential generation of stable duplex DNA containing the desired edit. FIG. 40B shows the effect of complementary strand nicking on PE3-mediated prime editing efficiency and indel formation. “None” refers to PE2 controls, which do not nick the complementary strand. FIG. 40C is a comparison of editing efficiencies with PE2 (no complementary strand nick), PE3 (general complementary strand nick), and PE3b (edit-specific complementary strand nick). All editing yields reflect the percentage of total sequencing reads that contain the intended edit and do not contain indels among all treated cells, with no sorting. Values and error bars reflect the mean and s.d. of three independent biological replicates.
• FIGS. 41A-41K show targeted insertions, deletions, and all 12 types of point mutations with PE3 at seven endogenous human genomic loci in HEK293T cells. FIG. 41A is a graph showing all 12 types of single-nucleotide transition and transversion edits from position +1 to +8 (counting the location of the PEgRNA-induced nick as between position +1 and −1) of the HEK3 site using a 10-nt RT template. FIG. 41B is a graph showing long-range PE3 transversion edits at the HEK3 site using a 34-nt RT template. FIGS. 41C-41H are graphs showing all 12 types of transition and transversion edits at various positions in the prime editing window for (FIG. 41C) RNF2, (FIG. 41D) FANCF, (FIG. 41E) EMX1, (FIG. 41F) RUNX1, (FIG. 41G) VEGFA, and (FIG. 41H) DNMT1. FIG. 41I is a graph showing targeted 1- and 3-bp insertions, and 1- and 3-bp deletions with PE3 at seven endogenous genomic loci. FIG. 41J is a graph showing the targeted precise deletions of 5 to 80 bp at the HEK3 target site. FIG. 41K is a graph showing a combination edits of insertions and deletions, insertions and point mutations, deletions and point mutations, and double point mutations at three endogenous genomic loci. All editing yields reflect the percentage of total sequencing reads that contain the intended edit and do not contain indels among all treated cells, with no sorting. Values and error bars reflect the mean and s.d. of three independent biological replicates.
• FIGS. 42A-42H show the comparison of prime editing and base editing, and off-target editing by Cas9 and PE3 at known Cas9 off-target sites. FIG. 42A shows total C●G-to-T●A editing efficiency at the same target nucleotides for PE2, PE3, BE2max, and BE4max at endogenous HEK3, FANCF, and EMX1 sites in HEK293T cells. FIG. 42B shows indel frequency from treatments in FIG. 42A. FIG. 42C shows the editing efficiency of precise C●G-to-T●A edits (without bystander edits or indels) for PE2, PE3, BE2max, and BE4max at HEK3, FANCF, and EMX1. For EMX1, precise PE combination edits of all possible combinations of C●G-to-T●A conversion at the three targeted nucleotides are also shown.
• FIG. 42D shows the total A●T-to-G●C editing efficiency for PE2, PE3, ABEdmax, and ABEmax at HEK3 and FANCF. FIG. 42E shows the precise A●T-to-G●C editing efficiency without bystander edits or indels for at HEK3 and FANCF. FIG. 42F shows indel frequency from treatments in FIG. 42D. FIG. 42G shows the average triplicate editing efficiencies (percentage sequencing reads with indels) in HEK293T cells for Cas9 nuclease at four on-target and 16 known off-target sites. The 16 off-target sites examined were the top four previously reported off-target sites^118,159 for each of the four on-target sites. For each on-target site, Cas9 was paired with a sgRNA or with each of four PEgRNAs that recognize the same protospacer. FIG. 42H shows the average triplicate on-target and off-target editing efficiencies and indel efficiencies (below in parentheses) in HEK293T cells for PE2 or PE3 paired with each PEgRNA in (FIG. 42G). On-target editing yields reflect the percentage of total sequencing reads that contain the intended edit and do not contain indels among all treated cells, with no sorting. Off-target editing yields reflect off-target locus modification consistent with prime editing. Values and error bars reflect the mean and s.d. of three independent biological replicates.
• FIGS. 43A-43I show prime editing in various human cell lines and primary mouse cortical neurons, installation and correction of pathogenic transversion, insertion, or deletion mutations, and comparison of prime editing and HDR. FIG. 43A is a graph showing the installation (via T●A-to-A●T transversion) and correction (via A●T-to-T●A transversion) of the pathogenic E6V mutation in HBB in HEK293T cells. Correction either to wild-type HBB, or to HBB containing a silent mutation that disrupts the PEgRNA PAM, is shown. FIG. 43B is a graph showing the installation (via 4-bp insertion) and correction (via 4-bp deletion) of the pathogenic HEXA 1278+TATC allele in HEK293T cells. Correction either to wild-type HEXA, or to HEXA containing a silent mutation that disrupts the PEgRNA PAM, is shown.
• FIG. 43C is a graph showing the installation of the protective G127V variant in PRNP in HEK293T cells via G●C-to-T●A transversion. FIG. 43D is a graph showing prime editing in other human cell lines including K562 (leukemic bone marrow cells), U2OS (osteosarcoma cells), and HeLa (cervical cancer cells). FIG. 43E is a graph showing the installation of a G●C-to-T●A transversion mutation in DNMT1 of mouse primary cortical neurons using a dual split-intein PE3 lentivirus system, in which the N-terminal half is Cas9 (1-573) fused to N-intein and through a P2A self-cleaving peptide to GFP-KASH, and the C-terminal half is the C-intein fused to the remainder of PE2. PE2 halves are expressed from a human synapsin promoter that is highly specific for mature neurons. Sorted values reflect editing or indels from GFP-positive nuclei, while unsorted values are from all nuclei. FIG. 43F is a comparison of PE3 and Cas9-mediated HDR editing efficiencies at endogenous genomic loci in HEK293T cells. FIG. 43G is a comparison of PE3 and Cas9-mediated HDR editing efficiencies at endogenous genomic loci in K562, U2OS, and HeLa cells. FIG. 43H is a comparison of PE3 and Cas9-mediated HDR indel byproduct generation in HEK293T, K562, U2OS, and HeLa cells. FIG. 43I shows targeted insertion of a His6 tag (18 bp), FLAG epitope tag (24 bp), or extended LoxP site (44 bp) in HEK293T cells by PE3. All editing yields reflect the percentage of total sequencing reads that contain the intended edit and do not contain indels among all treated cells. Values and error bars reflect the mean and s.d. of three independent biological replicates.
• FIGS. 44A-44G show in vitro prime editing validation studies with fluorescently labeled DNA substrates. FIG. 44A shows electrophoretic mobility shift assays with dCas9, 5′-extended PEgRNAs and 5′-Cy5-labeled DNA substrates. PEgRNAs 1 through 5 contain a 15-nt linker sequence (linker A for PEgRNA 1, linker B for PEgRNAs 2 through 5) between the spacer and the PBS, a 5-nt PBS sequence, and RT templates of 7 nt (PEgRNAs 1 and 2), 8 nt (PEgRNA 3), 15 nt (PEgRNA 4), and 22 nt (PEgRNA 5). PEgRNAs are those used in FIGS. 44E and 44F; full sequences are listed in Tables 2A-2C. FIG. 44B shows in vitro nicking assays of Cas9 H840A using 5′-extended and 3′-extended PEgRNAs. FIG. 44C shows Cas9-mediated indel formation in HEK293T cells at HEK3 using 5′-extended and 3′-extended PEgRNAs. FIG. 44D shows an overview of prime editing in vitro biochemical assays. 5′-Cy5-labeled pre-nicked and non-nicked dsDNA substrates were tested. sgRNAs, 5′-extended PEgRNAs, or 3′-extended PEgRNAs were pre-complexed with dCas9 or Cas9 H840A nickase, then combined with dsDNA substrate, M-MLV RT, and dNTPs. Reactions were allowed to proceed at 37° C. for 1 hour prior to separation by denaturing urea PAGE and visualization by Cy5 fluorescence. FIG. 44E shows primer extension reactions using 5′-extended PEgRNAs, pre-nicked DNA substrates, and dCas9 lead to significant conversion to RT products. FIG. 44F shows primer extension reactions using 5′-extended PEgRNAs as in FIG. 44B, with non-nicked DNA substrate and Cas9 H840A nickase. Product yields are greatly reduced by comparison to pre-nicked substrate. FIG. 44G shows an in vitro primer extension reaction using a 3′-PEgRNA generates a single apparent product by denaturing urea PAGE. The RT product band was excised, eluted from the gel, then subjected to homopolymer tailing with terminal transferase (TdT) using either dGTP or dATP. Tailed products were extended by poly-T or poly-C primers, and the resulting DNA was sequenced. Sanger traces indicate that three nucleotides derived from the gRNA scaffold were reverse transcribed (added as the final 3′ nucleotides to the DNA product). Note that in mammalian cell prime editing experiments, PEgRNA scaffold insertion is much rarer than in vitro (FIGS. 56A-56D), potentially due to the inability of the tethered reverse transcriptase to access the Cas9-bound guide RNA scaffold, and/or cellular excision of mismatched 3′ ends of 3′ flaps containing PEgRNA scaffold sequences.
• FIGS. 45A-45G show cellular repair in yeast of 3′ DNA flaps from in vitro prime editing reactions. FIG. 45A shows that dual fluorescent protein reporter plasmids contain GFP and mCherry open reading frames separated by a target site encoding an in-frame stop codon, a +1 frameshift, or a −1 frameshift. Prime editing reactions were carried out in vitro with Cas9 H840A nickase, PEgRNA, dNTPs, and M-MLV reverse transcriptase, and then transformed into yeast. Colonies that contain unedited plasmids produce GFP but not mCherry. Yeast colonies containing edited plasmids produce both GFP and mCherry as a fusion protein. FIG. 45B shows an overlay of GFP and mCherry fluorescence for yeast colonies transformed with reporter plasmids containing a stop codon between GFP and mCherry (unedited negative control, top), or containing no stop codon or frameshift between GFP and mCherry (pre-edited positive control, bottom). FIGS. 45C-45F show a visualization of mCherry and GFP fluorescence from yeast colonies transformed with in vitro prime editing reaction products. FIG. 45C shows a stop codon correction via T●A-to-A●T transversion using a 3′-extended PEgRNA, or a 5′-extended PEgRNA, as shown in FIG. 45D. FIG. 45E shows a +1 frameshift correction via a 1-bp deletion using a 3′-extended PEgRNA. FIG. 45F shows a −1 frameshift correction via a 1-bp insertion using a 3′-extended PEgRNA. FIG. 45G shows Sanger DNA sequencing traces from plasmids isolated from GFP-only colonies in FIG. 45B and GFP and mCherry double-positive colonies in FIG. 45C.
• FIGS. 46A-46F show correct editing versus indel generation with PE1. FIG. 46A shows T●A-to-A●T transversion editing efficiency and indel generation by PE1 at the +1 position of HEK3 using PEgRNAs containing 10-nt RT templates and a PBS sequences ranging from 8-17 nt. FIG. 46B shows G●C-to-T●A transversion editing efficiency and indel generation by PE1 at the +5 position of EMX1 using PEgRNAs containing 13-nt RT templates and a PBS sequences ranging from 9-17 nt. FIG. 46C shows G●C-to-T●A transversion editing efficiency and indel generation by PE1 at the +5 position of FANCF using PEgRNAs containing 17-nt RT templates and a PBS sequences ranging from 8-17 nt.
• FIG. 46D shows C●G-to-A●T transversion editing efficiency and indel generation by PE1 at the +1 position of RNF2 using PEgRNAs containing 11-nt RT templates and a PBS sequences ranging from 9-17 nt. FIG. 46E shows G●C-to-T●A transversion editing efficiency and indel generation by PE1 at the +2 position of HEK4 using PEgRNAs containing 13-nt RT templates and a PBS sequences ranging from 7-15 nt. FIG. 46F shows PE1-mediated+1 T deletion, +1 A insertion, and +1 CTT insertion at the HEK3 site using a 13-nt PBS and 10-nt RT template. Sequences of PEgRNAs are those used in FIG. 39C (see Tables 3A-3R). Values and error bars reflect the mean and s.d. of three independent biological replicates.
• FIGS. 47A-47S show the evaluation of M-MLV RT variants for prime editing. FIG. 47A shows the abbreviations for prime editor variants used in this figure. FIG. 47B shows targeted insertion and deletion edits with PE1 at the HEK3 locus. FIGS. 47C-47H show a comparison of 18 prime editor constructs containing M-MLV RT variants for their ability to install a +2 G●C-to-C●G transversion edit at HEK3 as shown in FIG. 47C, a 24-bp FLAG insertion at HEK3 as shown in FIG. 47D, a +1 C●G-to-A●T transversion edit at RNF2 as shown in FIG. 47E, a +1 G●C-to-C●G transversion edit at EMX1 as shown in FIG. 47F, a +2 T●A-to-A●T transversion edit at HBB as shown in FIG. 47G, and a +1 G●C-to-C●G transversion edit at FANCF as shown in FIG. 47H. FIGS. 47I-47N show a comparison of four prime editor constructs containing M-MLV variants for their ability to install the edits shown in FIGS. 47C-47H in a second round of independent experiments. FIGS. 47O-47S show PE2 editing efficiency at five genomic loci with varying PBS lengths. FIG. 47O shows a +1 T●A-to-A●T variation at HEK3. FIG. 47P shows a +5 G●C-to-T●A variation at EMX1. FIG. 47Q shows a +5 G●C-to-T●A variation at FANCF. FIG. 47R shows a +1 C●G-to-A●T variation at RNF2. FIG. 47S shows a +2 G●C-to-T●A variation at HEK4. Values and error bars reflect the mean and s.d. of three independent biological replicates.
• FIGS. 48A-48C show design features of PEgRNA PBS and RT template sequences. FIG. 48A shows PE2-mediated+5 G●C-to-T●A transversion editing efficiency (blue line) at VEGFA in HEK293T cells as a function of RT template length. Indels (gray line) are plotted for comparison. The sequence below the graph shows the last nucleotide templated for synthesis by the PEgRNA. G nucleotides (templated by a C in the PEgRNA) are highlighted; RT templates that end in C should be avoided during PEgRNA design to maximize prime editing efficiencies. FIG. 48B shows +5 G●C-to-T●A transversion editing and indels for DNMT1 as in FIG. 48A. FIG. 48C shows +5 G●C-to-T●A transversion editing and indels for RUNX1 as in FIG. 48A. Values and error bars reflect the mean and s.d. of three independent biological replicates.
• FIGS. 49A-49B show the effects of PE2, PE2 R110S K103L, Cas9 H840A nickase, and dCas9 on cell viability. HEK293T cells were transfected with plasmids encoding PE2, PE2 R110S K103L, Cas9 H840A nickase, or dCas9, together with a HEK3-targeting PEgRNA plasmid. Cell viability was measured every 24 hours post-transfection for 3 days using the CellTiter-Glo 2.0 assay (Promega). FIG. 49A shows viability, as measured by luminescence, at 1, 2, or 3 days post-transfection. Values and error bars reflect the mean and s.e.m. of three independent biological replicates each performed in technical triplicate. FIG. 49B shows percent editing and indels for PE2, PE2 R110S K103L, Cas9 H840A nickase, or dCas9, together with a HEK3-targeting PEgRNA plasmid that encodes a +5 G to A edit. Editing efficiencies were measured on day 3 post-transfection from cells treated alongside of those used for assaying viability in FIG. 49A. Values and error bars reflect the mean and s.d. of three independent biological replicates.
• FIGS. 50A-50B show PE3-mediated HBB E6V correction and HEXA 1278+TATC correction by various PEgRNAs. FIG. 50A shows a screen of 14 PEgRNAs for correction of the HBB E6V allele in HEK293T cells with PE3. All PEgRNAs evaluated convert the HBB E6V allele back to wild-type HBB without the introduction of any silent PAM mutation. FIG. 50B shows a screen of 41 PEgRNAs for correction of the HEXA 1278+TATC allele in HEK293T cells with PE3 or PE3b. Those PEgRNAs labeled HEXAs correct the pathogenic allele by a shifted 4-bp deletion that disrupts the PAM and leaves a silent mutation. Those PEgRNAs labeled HEXA correct the pathogenic allele back to wild-type. Entries ending in “b” use an edit-specific nicking sgRNA in combination with the PEgRNA (the PE3b system). Values and error bars reflect the mean and s.d. of three independent biological replicates.
• FIGS. 51A-51F show a PE3 activity in human cell lines and a comparison of PE3 and Cas9-initiated HDR. Efficiency of generating the correct edit (without indels) and indel frequency for PE3 and Cas9-initiated HDR in HEK293T cells as shown in FIG. 51A, K562 cells as shown in FIG. 51B, U2OS cells as shown in FIG. 51C, and HeLa cells as shown in FIG. 51D. Each bracketed editing comparison installs identical edits with PE3 and Cas9-initiated HDR. Non-targeting controls are PE3 and a PEgRNA that targets a non-target locus. FIG. 51E shows control experiments with non-targeting PEgRNA+PE3, and with dCas9+sgRNA, compared with wild-type Cas9 HDR experiments confirming that ssDNA donor HDR template, a common contaminant that artificially elevates apparent HDR efficiencies, does not contribute to the HDR measurements in FIGS. 51A-51D. FIG. 51F shows example HEK3 site allele tables from genomic DNA samples isolated from K562 cells after editing with PE3 or with Cas9-initiated HDR. Alleles were sequenced on an Illumina MiSeq and analyzed with CRISPResso2¹⁷⁸. The reference HEK3 sequence from this region is at the top. Allele tables are shown for a non-targeting PEgRNA negative control, a +1 CTT insertion at HEK3 using PE3, and a +1 CTT insertion at HEK3 using Cas9-initiated HDR. Allele frequencies and corresponding Illumina sequencing read counts are shown for each allele. All alleles observed with frequency ≥0.20% are shown. Values and error bars reflect the mean and s.d. of three independent biological replicates.
• FIGS. 52A-52D show distribution by length of pathogenic insertions, duplications, deletions, and indels in the ClinVar database. The ClinVar variant summary was downloaded from NCBI Jul. 15, 2019. The lengths of reported insertions, deletions, and duplications were calculated using reference and alternate alleles, variant start and stop positions, or appropriate identifying information in the variant name. Variants that did not report any of the above information were excluded from the analysis. The lengths of reported indels (single variants that include both insertions and deletions relative to the reference genome) were calculated by determining the number of mismatches or gaps in the best pairwise alignment between the reference and alternate alleles.
• FIGS. 53A-53B show FACS gating examples for GFP-positive cell sorting. Below are examples of original batch analysis files outlining the sorting strategy used for generating HEXA 1278+TATC and HBB E6V HEK293T cell lines. The image data was generated on a Sony LE-MA900 cytometer using Cell Sorter Software v. 3.0.5. Graphic 1 shows gating plots for cells that do not express GFP. Graphic 2 shows an example sort of P2A-GFP-expressing cells used for isolating the HBB E6V HEK293T cell lines. HEK293T cells were initially gated on population using FSC-A/BSC-A (Gate A), then sorted for singlets using FSC-A/FSC-H (Gate B). Live cells were sorted for by gating DAPI-negative cells (Gate C). Cells with GFP fluorescence levels that were above those of the negative-control cells were sorted for using EGFP as the fluorochrome (Gate D). FIG. 53A shows HEK293T cells (GFP-negative). FIG. 53B shows a representative plot of FACS gating for cells expressing PE2-P2A-GFP. FIG. 53C shows the genotypes for HEXA 1278+TATC homozygote HEK293T cells. FIG. 53D shows allele tables for HBB E6V homozygote HEK293T cell lines.
• FIG. 54 is a schematic which summarizes the PEgRNA cloning procedure.
• FIGS. 55A-55G are schematics of PEgRNA designs. FIG. 55A shows a simple diagram of PEgRNA with domains labeled (left) and bound to nCas9 at a genomic site (right). FIG. 55B shows various types of modifications to PEgRNA which are anticipated to increase activity. FIG. 55C shows modifications to PEgRNA to increase transcription of longer RNAs via promoter choice and 5′, 3′ processing and termination. FIG. 55D shows the lengthening of the P1 system, which is an example of a scaffold modification. FIG. 55E shows that the incorporation of synthetic modifications within the template region, or elsewhere within the PEgRNA, could increase activity. FIG. 55F shows that a designed incorporation of minimal secondary structure within the template could prevent formation of longer, more inhibitory, secondary structure. FIG. 55G shows a split PEgRNA with a second template sequence anchored by an RNA element at the 3′ end of the PEgRNA (left). Incorporation of elements at the 5′ or 3′ ends of the PEgRNA could enhance RT binding.
• FIGS. 56A-56D show the incorporation of PEgRNA scaffold sequence into target loci. HTS data were analyzed for PEgRNA scaffold sequence insertion as described in FIGS. 60A-60B. FIG. 56A shows an analysis for the EMX1 locus. Shown is the % of total sequencing reads containing one or more PEgRNA scaffold sequence nucleotides within an insertion adjacent to the RT template (left); the percentage of total sequencing reads containing a PEgRNA scaffold sequence insertion of the specified length (middle); and the cumulative total percentage of PEgRNA insertion up to and including the length specified on the X axis. FIG. 56B shows the same as FIG. 56A, but for FANCF. FIG. 56C shows the same as in FIG. 56A but for HEK3. FIG. 56D shows the same as FIG. 56A but for RNF2. Values and error bars reflect the mean and s.d. of three independent biological replicates.
• FIGS. 57A-57I show the effects of PE2, PE2-dRT, and Cas9 H840A nickase on transcriptome-wide RNA abundance. Analysis of cellular RNA, depleted for ribosomal RNA, isolated from HEK293T cells expressing PE2, PE2-dRT, or Cas9 H840A nickase and a PRNP-targeting or HEXA-targeting PEgRNA. RNAs corresponding to 14,410 genes and 14,368 genes were detected in PRNP and HEXA samples, respectively. FIGS. 57A-57F show Volcano plot displaying the −log 10 FDR-adjusted p-value vs. log 2-fold change in transcript abundance for Aeach RNA, comparing (FIG. 57A) PE2 vs. PE2-dRT with PRNP-targeting PEgRNA, (FIG. 57B) PE2 vs. Cas9 H840A with PRNP-targeting PEgRNA, (FIG. 57C) PE2-dRT vs. Cas9 H840A with PRNP-targeting PEgRNA, (FIG. 57D) PE2 vs. PE2-dRT with HEXA-targeting PEgRNA, (FIG. 57E) PE2 vs. Cas9 H840A with HEXA-targeting PEgRNA, (FIG. 57F) PE2-dRT vs. Cas9 H840A with HEXA-targeting PEgRNA. Red dots indicate genes that show ≥2-fold change in relative abundance that are statistically significant (FDR-adjusted p<0.05). FIGS. 57G-57I are Venn diagrams of upregulated and downregulated transcripts (≥2-fold change) comparing PRNP and HEXA samples for (FIG. 57G) PE2 vs PE2-dRT, (FIG. 57H) PE2 vs. Cas9 H840A, and (FIG. 57I) PE2-dRT vs. Cas9 H840A.
• FIG. 58 shows representative FACS gating for neuronal nuclei sorting. Nuclei were sequentially gated on the basis of DyeCycle Ruby signal, FSC/SSC ratio, SSC-Width/SSC-height ratio, and GFP/DyeCycle ratio.
• FIGS. 59A-59F show the protocol for cloning 3′-extended PEgRNAs into mammalian U6 expression vectors by Golden Gate assembly. FIG. 59A shows the cloning overview.
• FIG. 59B shows ‘Step 1: Digest pU6-PEgRNA-GG-Vector plasmid (component 1)’. FIG. 59C shows ‘Steps 2 and 3: Order and anneal oligonucleotide parts ( components 2, 3, and 4)’.
• FIG. 59D shows ‘Step 2.b.ii.: sgRNA scaffold phosphorylation (unnecessary if oligonucleotides were purchased phosphorylated)’. FIG. 59E shows ‘Step 4: PEgRNA assembly’. FIG. 59F shows ‘Steps 5 and 6: Transformation of assembled plasmids’. FIG. 59G shows a diagram summarizing the PEgRNA cloning protocol.
• FIGS. 60A-60B show the Python script for quantifying PEgRNA scaffold integration. A custom python script was generated to characterize and quantify PEgRNA insertions at target genomic loci. The script iteratively matches text strings of increasing length taken from a reference sequence (guide RNA scaffold sequence) to the sequencing reads within fastq files, and counts the number of sequencing reads that match the search query. Each successive text string corresponds to an additional nucleotide of the guide RNA scaffold sequence. Exact length integrations and cumulative integrations up to a specified length were calculated in this manner. At the start of the reference sequence, 5 to 6 bases of the 3′ end of the new DNA strand synthesized by the reverse transcriptase are included to ensure alignment and accurate counting of short slices of the sgRNA.
• FIG. 61 is a graph showing the percent of total sequencing reads with the specified edit for SaCas9(N580A)-MMLV RT HEK3+6 C>A. The values for the correct edits as well as indels are shown.
• FIGS. 62A-62B show the importance of the protospacer for efficient installation of a desired edit at a precise location with prime editing. FIG. 62A is a graph showing the percent of total sequencing reads with target T●A base pairs converted to A●T for various HEK3 loci. FIG. 62B is a sequence analysis showing the same.
• FIG. 63 is a graph showing SpCas9 PAM variants in PAM editing (N=3). The percent of total sequencing reads with the targeted PAM edit is shown for SpCas9(H840A)-VRQR-MMLV RT, where NGA >NTA, and for SpCas9(H840A)-VRER-MMLV RT, where NGCG >NTCG. The PEgRNA primer binding site (PBS) length, RT template (RT) length, and PE system used are listed.
• FIG. 64 is a schematic showing the introduction of various site-specific recombinase (SSR) targets into the genome using PE. (a) provides a general schematic of the insertion of a recombinase target sequence by a prime editor. (b) shows how a single SSR target inserted by PE can be used as a site for genomic integration of a DNA donor template. (c) shows how a tandem insertion of SSR target sites can be used to delete a portion of the genome. (d) shows how a tandem insertion of SSR target sites can be used to invert a portion of the genome. (e) shows how the insertion of two SSR target sites at two distal chromosomal regions can result in chromosomal translocation. (f) shows how the insertion of two different SSR target sites in the genome can be used to exchange a cassette from a DNA donor template. See Example 17 for further details.
• FIG. 65 shows in 1) the PE-mediated synthesis of a SSR target site in a human cell genome and 2) the use of that SSR target site to integrate a DNA donor template comprising a GFP expression marker. Once successfully integrated, the GFP causes the cell to fluoresce. See Example 17 for further details.
• FIG. 66 depicts one embodiment of a prime editor being provided as two PE half proteins which regenerate as whole prime editor through the self-splicing action of the split-intein halves located at the end or beginning of each of the prime editor half proteins.
• FIG. 67 depicts the mechanism of intein removal from a polypeptide sequence and the reformation of a peptide bond between the N-terminal and the C-terminal extein sequences. (a) depicts the general mechanism of two half proteins each containing half of an intein sequence, which when in contact within a cell result in a fully-functional intein which then undergoes self-spicing and excision. The process of excision results in the formation of a peptide bond between the N-terminal protein half (or the “N extein”) and the C-terminal protein half (or the “C extein”) to form a whole, single polypeptide comprising the N extein and the C extein portions. In various embodiments, the N extein may correspond to the N-terminal half of a split prime editor fusion protein and the C extein may correspond to the C-terminal half of a split prime editor. (b) shows a chemical mechanism of intein excision and the reformation of a peptide bond that joins the N extein half (the red-colored half) and the C extein half (the blue-colored half). Excision of the split inteins (i.e., the N intein and the C intein in the split intein configuration) may also be referred to as “trans splicing” as it involves the splicing action of two separate components provided in trans.
• FIG. 68A demonstrates that delivery of both split intein halves of SpPE (SEQ ID NO: 762) at the linker maintains activity at three test loci when co-transfected into HEK293T cells.
• FIG. 68B demonstrates that delivery of both split intein halves of SaPE2 (e.g., SEQ ID NO: 443 and SEQ ID NO: 450) recapitulate activity of full length SaPE2 (SEQ ID NO: 134) when co-transfected into HEK293T cells. Residues indicated in quotes are the sequence of amino acids 741-743 in SaCas
• 9 (first residues of the C-terminal extein) which are important for the intein trans splicing reaction. ‘SMP’ are the native residues, which we also mutated to the ‘CFN’ consensus splicing sequence. The consensus sequence is shown to yield the highest reconstitution as measured by prime editing percentage.
• FIG. 68C provides data showing that various disclosed PE ribonucleoprotein complexes (PE2 at high concentration, PE3 at high concentration and PE3 at low concentration) can be delivered in this manner.
• FIG. 69 shows a bacteriophage plaque assay to determine PE effectiveness in PANCE. Plaques (dark circles) indicate phage able to successfully infect E. coli. Increasing concentration of L-rhamnose results in increased expression of PE and an increase in plaque formation. Sequencing of plaques revealed the presence of the PE-installed genomic edit.
• FIGS. 70A-70I provide an example of an edited target sequence as an illustration of a step-by-step instruction for designing PEgRNAs and nicking-sgRNAs for prime editing. FIG. 70A: Step 1. Define the target sequence and the edit. Retrieve the sequence of the target DNA region (˜200 bp) centered around the location of the desired edit (point mutation, insertion, deletion, or combination thereof). FIG. 70B: Step 2. Locate target PAMs. Identify PAMs in proximity to the edit location. Be sure to look for PAMs on both strands. While PAMs close to the edit position are preferred, it is possible to install edits using protospacers and PAMs that place the nick ≥30 nt from the edit position. FIG. 70C: Step 3. Locate the nick sites. For each PAM being considered, identify the corresponding nick site. For Sp Cas9 H840A nickase, cleavage occurs in the PAM-containing strand between the 3^rd and 4^th bases 5′ to the NGG PAM. All edited nucleotides must exist 3′ of the nick site, so appropriate PAMs must place the nick 5′ to the target edit on the PAM-containing strand. In the example shown below, there are two possible PAMs. For simplicity, the remaining steps will demonstrate the design of a PEgRNA using PAM 1 only. FIG. 70D: Step 4. Design the spacer sequence. The protospacer of Sp Cas9 corresponds to the 20 nucleotides 5′ to the NGG PAM on the PAM-containing strand. Efficient Pol III transcription initiation requires a G to be the first transcribed nucleotide. If the first nucleotide of the protospacer is a G, the spacer sequence for the PEgRNA is simply the protospacer sequence. If the first nucleotide of the protospacer is not a G, the spacer sequence of the PEgRNA is G followed by the protospacer sequence. FIG. 70E: Step 5. Design a primer binding site (PBS). Using the starting allele sequence, identify the DNA primer on the PAM-containing strand. The 3′ end of the DNA primer is the nucleotide just upstream of the nick site (i.e. the 4^th base 5′ to the NGG PAM for Sp Cas9). As a general design principle for use with PE2 and PE3, a PEgRNA primer binding site (PBS) containing 12 to 13 nucleotides of complementarity to the DNA primer can be used for sequences that contain ˜40-60% GC content. For sequences with low GC content, longer (14- to 15-nt) PBSs should be tested. For sequences with higher GC content, shorter (8- to 11-nt) PBSs should be tested. Optimal PBS sequences should be determined empirically, regardless of GC content. To design a length-p PBS sequence, take the reverse complement of the first p nucleotides 5′ of the nick site in the PAM-containing strand using the starting allele sequence. FIG. 70F: Step 6. Design an RT template. The RT template encodes the designed edit and homology to the sequence adjacent to the edit. Optimal RT template lengths vary based on the target site. For short-range edits (positions +1 to +6), it is recommended to test a short (9 to 12 nt), a medium (13 to 16 nt), and a long (17 to 20 nt) RT template. For long-range edits (positions +7 and beyond), it is recommended to use RT templates that extend at least 5 nt (preferably 10 or more nt) past the position of the edit to allow for sufficient 3′ DNA flap homology. For long-range edits, several RT templates should be screened to identify functional designs. For larger insertions and deletions (≥5 nt), incorporation of greater 3′ homology (˜20 nt or more) into the RT template is recommended. Editing efficiency is typically impaired when the RT template encodes the synthesis of a G as the last nucleotide in the reverse transcribed DNA product (corresponding to a C in the RT template of the PEgRNA). As many RT templates support efficient prime editing, avoidance of G as the final synthesized nucleotide is recommended when designing RT templates. To design a length-r RT template sequence, use the desired allele sequence and take the reverse complement of the first r nucleotides 3′ of the nick site in the strand that originally contained the PAM. Note that compared to SNP edits, insertion or deletion edits using RT templates of the same length will not contain identical homology. FIG. 70G: Step 7. Assemble the full PEgRNA sequence. Concatenate the PEgRNA components in the following order (5′ to 3′): spacer, scaffold, RT template and PBS. FIG. 70H: Step 8. Designing nicking-sgRNAs for PE3. Identify PAMs on the non-edited strand upstream and downstream of the edit. Optimal nicking positions are highly locus-dependent and should be determined empirically. In general, nicks placed 40 to 90 nucleotides 5′ to the position across from the PEgRNA-induced nick lead to higher editing yields and fewer indels. A nicking sgRNA has a spacer sequence that matches the 20-nt protospacer in the starting allele, with the addition of a 5′-G if the protospacer does not begin with a G. FIG. 70I: Step 9. Designing PE3b nicking-sgRNAs. If a PAM exists in the complementary strand and its corresponding protospacer overlaps with the sequence targeted for editing, this edit could be a candidate for the PE3b system. In the PE3b system, the spacer sequence of the nicking-sgRNA matches the sequence of the desired edited allele, but not the starting allele. The PE3b system operates efficiently when the edited nucleotide(s) falls within the seed region (˜10 nt adjacent to the PAM) of the nicking-sgRNA protospacer. This prevents nicking of the complementary strand until after installation of the edited strand, preventing competition between the PEgRNA and the sgRNA for binding the target DNA. PE3b also avoids the generation of simultaneous nicks on both strands, thus reducing indel formation significantly while maintaining high editing efficiency. PE3b sgRNAs should have a spacer sequence that matches the 20-nt protospacer in the desired allele, with the addition of a 5′ G if needed.
• FIG. 71A shows the nucleotide sequence of a SpCas9 PEgRNA molecule (top) which terminates at the 3′ end in a “UUU” and does not contain a toeloop element. The lower portion of the figure depicts the same SpCas9 PEgRNA molecule but is further modified to contain a toeloop element having the sequence 5′-“GAAANNNNN”-3′ inserted immediately before the “UUU” 3′ end. The “N” can be any nucleobase.
• FIG. 71B shows the results of Example 18, which demonstrates that the efficiency of prime editing in HEK cells or EMX cells is increased using PEgRNA containing toeloop elements, whereas the percent of indel formation is largely unchanged.
• FIG. 72 depicts alternative PEgRNA configurations that can be used in prime editing. (a) Depicts the PE2:PEgRNA embodiment of prime editing. This embodiment involves a PE2 (a fusion protein comprising a Cas9 and a reverse transcriptase) complexed with a PEgRNA (as also described in FIGS. 1A-1I and/or FIG. 3A-3E). In this embodiment, the template for reverse transcription is incorporated into a 3′ extension arm on the sgRNA to make the PEgRNA, and the DNA polymerase enzyme is a reverse transcriptase (RT) fused directly to Cas9. (b) Depicts the MS2cp-PE2:sgRNA+tPERT embodiment. This embodiment comprises a PE2 fusion (Cas9+a reverse transcriptase) that is further fused to the MS2 bacteriophage coat protein (MS2cp) to form the MS2cp-PE2 fusion protein. To achieve prime editing, the MS2cp-PE2 fusion protein is complexed with an sgRNA that targets the complex to a specific target site in the DNA. The embodiment then involves the introduction of a trans prime editing RNA template (“tPERT”), which operates in place of a PEgRNA by providing a primer binding site (PBS) and an DNA synthesis template on separate molecule, i.e., the tPERT, which is also equipped with a MS2 aptamer (stem loop). The MS2cp protein recruits the tPERT by binding to the MS2 aptamer of the molecule. (c) Depicts alternative designs for PEgRNAs that can be achieved through known methods for chemical synthesis of nucleic acid molecules. For example, chemical synthesis can be used to synthesize a hybrid RNA/DNA PEgRNA molecule for use in prime editing, wherein the extension arm of the hybrid PEgRNA is DNA instead of RNA. In such an embodiment, a DNA-dependent DNA polymerase can be used in place of a reverse transcriptase to synthesize the 3′ DNA flap comprising the desired genetic change that is formed by prime editing. In another embodiment, the extension arm can be synthesized to include a chemical linker that prevents the DNA polymerase (e.g., a reverse transcriptase) from using the sgRNA scaffold or backbone as a template. In still another embodiment, the extension arm may comprise a DNA synthesis template that has the reverse orientation relative to the overall orientation of the PEgRNA molecule. For example, and as shown for a PEgRNA in the 5′-to-3′ orientation and with an extension attached to the 3′ end of the sgRNA scaffold, the DNA synthesis template is orientated in the opposite direction, i.e., the 3′-to-5′ direction. This embodiment may be advantageous for PEgRNA embodiments with extension arms positioned at the 3′ end of a gRNA. By reverse the orientation of the extension arm, the DNA synthesis by the polymerase (e.g., reverse transcriptase) will terminate once it reaches the newly orientated 5′ of the extension arm and will thus, not risk using the gRNA core as a template.
• FIG. 73 demonstrates prime editing with tPERTs and the MS2 recruitment system (aka MS2 tagging technique). An sgRNA targeting the prime editor protein (PE2) to the target locus is expressed in combination with a tPERT containing a primer binding site (a13-nt or 17-nt PBS), an RT template encoding a His6 tag insertion and a homology arm, and an MS2 aptamer (located at the 5′ or 3′ end of the tPERT molecule). Either prime editor protein (PE2) or a fusion of the MS2cp to the N-terminus of PE2 was used. Editing was carried out with or without a complementary-strand nicking sgRNA, as in the previously developed PE3 system (designated in the x-axis as labels “PE2+nick” or “PE2”, respectively). This is also referred to and defined herein as “second-strand nicking.”
• FIG. 74 demonstrates that the MS2 aptamer expression of the reverse transcriptase in trans and its recruitment with the MS2 aptamer system. The PEgRNAPEgRNA contains the MS2 RNA aptamer inserted into either one of two sgRNA scaffold hairpins. The wild-type M-MLV reverse transcriptase is expressed as an N-terminal or C-terminal fusion to the MS2 coat protein (MCP). Editing is at the HEK3 site in HEK293T cells.
• FIG. 75 provides a bar graph comparing the efficiency (i.e., “% of total sequencing reads with the specified edit or indels”) of PE2, PE2-trunc, PE3, and PE3-trunc over different target sites in various cell lines. The data shows that the prime editors comprising the truncated RT variants were about as efficient as the prime editors comprising the non-truncated RT proteins.
• FIG. 76 demonstrates the editing efficiency of intein-split prime editors of Example 20. HEK239T cells were transfected with plasmids encoding full-length PE2 or intein-split PE2, PEgRNA and nicking guide RNA. Consensus sequence (most amino-terminal residues of C terminal extein) are indicated. Percent editing at two sites in shown: HEK3+1 CTT insertion and PRNP +6 G to T. Replicate n=3 independent transfections. See Example 20.
• FIG. 77 demonstrates the editing efficiency of intein-split prime editors of Example 20. Editing assessed by targeted deep sequencing in bulk cortex and GFP+ subpopulation upon delivery of 5E10vg per SpPE3 half and a small amount 1E10 of nuclear-localized GFP:KASH to P0 mice by ICV injection. Editors and GFP were packaged in AAV9 with EFS promoter. Mice were harvested three weeks post injection and GFP+ nuclei were isolated by flow cytometry. Individual data points are shown, with 1-2 mice per condition analyzed. See Example 20.
• FIG. 78 demonstrates the editing efficiency of intein-split prime editors of Example 20. Specifically, the figures depicts the AAV split-SpPE3 constructs using in Example 20. Co-transduction by AAV particles separately expressing SpPE3-N and SpPE3-C recapitulates PE3 activity. Note N-terminal genome contains a U6-sgRNA cassette expressing the nicking sgRNA, and the C-terminal genome contains a U6-PEgRNA cassette expressing the PEgRNA. See Example 20.
• FIG. 79 shows the editing efficiency of certain optimized linkers as discussed in Example 21. In particular, the data shows the editing efficiency of the PE2 construct with the current linker (noted as PE2—white box) compared to various versions with the linker replaced with a sequence as indicated at the HEK3, EMX1, FANCF, RNF2 loci for representative PEgRNAs for transition, transversion, insertion, and deletion edits. The replacement linkers are referred to as “1×SGGS”, “2×SGGS”, “3×SGGS”, “1×XTEN”, “no linker”, “1×Gly”, “1×Pro”, “1×EAAAK”, “2×EAAAK”, and “3×EAAAK”. The editing efficiency is measured in bar graph format relative to the “control” editing efficiency of PE2. The linker of PE2 is SGGSSGGSSGSETPGTSESATPESSGGSSGGSS (SEQ ID NO: 127). All editing was done in the context of the PE3 system, i.e., which refers the PE2 editing construct plus the addition of the optimal secondary sgRNA nicking guide. See Example 21.
• FIG. 80 . Taking the average fold efficacy relative to PE2 yields the graph shown, indicating that use of a 1×XTEN linker sequence improves editing efficiency by 1.14 fold on average (n=15). See Example 21.
• FIG. 81 depicts the transcription level of PEgRNAs from different promoters, as described in Example 22.
• FIG. 82 As depicted in Example 22, impact of different types of modifications on PEgRNA structure on editing efficiency relative to unmodified PEgRNA.
• FIG. 83 Depicts a PE experiment that targeted editing of the HEK3 gene, specifically targeting the insertion of a 10 nt insertion at position +1 relative to the nick site and using PE3. See Example 22.
• FIG. 84A depicts an exemplary PEgRNA having a spacer, gRNA core, and an extension arm (RT template+primer binding site), which is modified at the 3′ end of the PEgRNA with a tRNA molecule, coupled through a UCU linker. The tRNA includes various post-transcriptional modifications. Said modification are not required, however.
• FIG. 84B depicts structure of tRNA that can be used to modify PEgRNA structures. See Example 22. The P1 can be variable in length. The P1 can be extended to help prevent RNAseP processing of the PEgRNA-tRNA fusion.
• FIG. 85 depicts a PE experiment that targeted editing of the FANCF gene, specifically targeting a G-to-T conversion at position +5 relative to the nick site and using PE3 construct. See Example 22.
• FIG. 86 depicts a PE experiment that targeted editing of the HEK3 gene, specifically targeting the insertion of a 71 nt FLAG tag insertion at position +1 relative to the nick site and using PE3 construct. See Example 22.
• FIG. 87 results from a screen in N2A cells where the pegRNA installs 1412Adel, with details about the primer binding site (PBS) length and reverse transcriptase (RT) template length. (Shown with and without indels). See Example 23.
• FIG. 88 results from a screen in N2A cells where the pegRNA installs 1412Adel, with details about the primer binding site (PBS) length and reverse transcriptase (RT) template length. (Shown with and without indels). See Example 23.
• FIG. 89 depicts results of editing at a proxy locus in the β-globin gene and at HEK3 in healthy HSCs, varying the concentration of editor to pegRNA and nicking gRNA. See Example 23.
DEFINITIONS
• Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them unless specified otherwise.
Antisense Strand
• In genetics, the “antisense” strand of a segment within double-stranded DNA is the template strand, and which is considered to run in the 3′ to 5′ orientation. By contrast, the “sense” strand is the segment within double-stranded DNA that runs from 5′ to 3′, and which is complementary to the antisense strand of DNA, or template strand, which runs from 3′ to 5′. In the case of a DNA segment that encodes a protein, the sense strand is the strand of DNA that has the same sequence as the mRNA, which takes the antisense strand as its template during transcription, and eventually undergoes (typically, not always) translation into a protein. The antisense strand is thus responsible for the RNA that is later translated to protein, while the sense strand possesses a nearly identical makeup to that of the mRNA. Note that for each segment of dsDNA, there will possibly be two sets of sense and antisense, depending on which direction one reads (since sense and antisense is relative to perspective). It is ultimately the gene product, or mRNA, that dictates which strand of one segment of dsDNA is referred to as sense or antisense.
Bi-Specific Ligand
• The term “bi-specific ligand” or “bi-specific moiety,” as used herein, refers to a ligand that binds to two different ligand-binding domains. In certain embodiments, the ligand is a small molecule compound, or a peptide, or a polypeptide. In other embodiments, ligand-binding domain is a “dimerization domain,” which can be install as a peptide tag onto a protein. In various embodiments, two proteins each comprising the same or different dimerization domains can be induced to dimerize through the binding of each dimerization domain to the bi-specific ligand. As used herein, “bi-specific ligands” may be equivalently refer to “chemical inducers of dimerization” or “CIDs”.
• Cas9
• The term “Cas9” or “Cas9 nuclease” refers to an RNA-guided nuclease comprising a Cas9 domain, or a fragment thereof (e.g., a protein comprising an active or inactive DNA cleavage domain of Cas9, and/or the gRNA binding domain of Cas9). A “Cas9 domain” as used herein, is a protein fragment comprising an active or inactive cleavage domain of Cas9 and/or the gRNA binding domain of Cas9. A “Cas9 protein” is a full length Cas9 protein. A Cas9 nuclease is also referred to sometimes as a casn1 nuclease or a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)-associated nuclease. CRISPR is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements, and conjugative plasmids). CRISPR clusters contain spacers, sequences complementary to antecedent mobile elements, and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems correct processing of pre-crRNA requires a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc) and a Cas9 domain. The tracrRNA serves as a guide for ribonuclease 3-aided processing of pre-crRNA. Subsequently, Cas9/crRNA/tracrRNA endonucleolytically cleaves linear or circular dsDNA target complementary to the spacer. The target strand not complementary to crRNA is first cut endonucleolytically, then trimmed 3′-5′ exonucleolytically. In nature, DNA-binding and cleavage typically requires protein and both RNAs. However, single guide RNAs (“sgRNA”, or simply “gNRA”) can be engineered so as to incorporate aspects of both the crRNA and tracrRNA into a single RNA species. See, e.g., Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J. A., Charpentier E. Science 337:816-821(2012), the entire contents of which are hereby incorporated by reference. Cas9 recognizes a short motif in the CRISPR repeat sequences (the PAM or protospacer adjacent motif) to help distinguish self versus non-self. Cas9 nuclease sequences and structures are well known to those of skill in the art (see, e.g., “Complete genome sequence of an M1 strain of Streptococcus pyogenes.” Ferretti et al., J. J., McShan W. M., Ajdic D. J., Savic D. J., Savic G., Lyon K., Primeaux C., Sezate S., Suvorov A. N., Kenton S., Lai H. S., Lin S. P., Qian Y., Jia H. G., Najar F. Z., Ren Q., Zhu H., Song L., White J., Yuan X., Clifton S. W., Roe B. A., McLaughlin R. E., Proc. Natl. Acad. Sci. U.S.A. 98:4658-4663(2001); “CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.” Deltcheva E., Chylinski K., Sharma C. M., Gonzales K., Chao Y., Pirzada Z. A., Eckert M. R., Vogel J., Charpentier E., Nature 471:602-607(2011); and “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.” Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J. A., Charpentier E. Science 337:816-821(2012), the entire contents of each of which are incorporated herein by reference). Cas9 orthologs have been described in various species, including, but not limited to, S. pyogenes and S. thermophilus. Additional suitable Cas9 nucleases and sequences will be apparent to those of skill in the art based on this disclosure, and such Cas9 nucleases and sequences include Cas9 sequences from the organisms and loci disclosed in Chylinski, Rhun, and Charpentier, “The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems” (2013) RNA Biology 10:5, 726-737; the entire contents of which are incorporated herein by reference. In some embodiments, a Cas9 nuclease comprises one or more mutations that partially impair or inactivate the DNA cleavage domain.
• A nuclease-inactivated Cas9 domain may interchangeably be referred to as a “dCas9” protein (for nuclease-“dead” Cas9). Methods for generating a Cas9 domain (or a fragment thereof) having an inactive DNA cleavage domain are known (see, e.g., Jinek et al., Science. 337:816-821(2012); Qi et al., “Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression” (2013) Cell. 28; 152(5):1173-83, the entire contents of each of which are incorporated herein by reference). For example, the DNA cleavage domain of Cas9 is known to include two subdomains, the HNH nuclease subdomain and the RuvC1 subdomain. The HNH subdomain cleaves the strand complementary to the gRNA, whereas the RuvC1 subdomain cleaves the non-complementary strand. Mutations within these subdomains can silence the nuclease activity of Cas9. For example, the mutations D10A and H840A completely inactivate the nuclease activity of S. pyogenes Cas9 (Jinek et al., Science. 337:816-821(2012); Qi et al., Cell. 28; 152(5):1173-83 (2013)). In some embodiments, proteins comprising fragments of Cas9 are provided. For example, in some embodiments, a protein comprises one of two Cas9 domains: (1) the gRNA binding domain of Cas9; or (2) the DNA cleavage domain of Cas9. In some embodiments, proteins comprising Cas9 or fragments thereof are referred to as “Cas9 variants.” A Cas9 variant shares homology to Cas9, or a fragment thereof. For example, a Cas9 variant is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, at least about 99.8% identical, or at least about 99.9% identical to wild type Cas9 (e.g., SpCas9 of SEQ ID NO: 18). In some embodiments, the Cas9 variant may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more amino acid changes compared to wild type Cas9 (e.g., SpCas9 of SEQ ID NO: 18). In some embodiments, the Cas9 variant comprises a fragment of SEQ ID NO: 18 Cas9 (e.g., a gRNA binding domain or a DNA-cleavage domain), such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of wild type Cas9 (e.g., SpCas9 of SEQ ID NO: 18). In some embodiments, the fragment is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid length of a corresponding wild type Cas9 (e.g., SpCas9 of SEQ ID NO: 18).
• cDNA
• The term “cDNA” refers to a strand of DNA copied from an RNA template. cDNA is complementary to the RNA template.
Circular Permutant
• As used herein, the term “circular permutant” refers to a protein or polypeptide (e.g., a Cas9) comprising a circular permutation, which is change in the protein's structural configuration involving a change in order of amino acids appearing in the protein's amino acid sequence. In other words, circular permutants are proteins that have altered N- and C-termini as compared to a wild-type counterpart, e.g., the wild-type C-terminal half of a protein becomes the new N-terminal half. Circular permutation (or CP) is essentially the topological rearrangement of a protein's primary sequence, connecting its N- and C-terminus, often with a peptide linker, while concurrently splitting its sequence at a different position to create new, adjacent N- and C-termini. The result is a protein structure with different connectivity, but which often can have the same overall similar three-dimensional (3D) shape, and possibly include improved or altered characteristics, including, reduced proteolytic susceptibility, improved catalytic activity, altered substrate or ligand binding, and/or improved thermostability. Circular permutant proteins can occur in nature (e.g., concanavalin A and lectin). In addition, circular permutation can occur as a result of posttranslational modifications or may be engineered using recombinant techniques.
Circularly Permuted Cas9
• The term “circularly permuted Cas9” refers to any Cas9 protein, or variant thereof, that has been occurs as a circular permutant, whereby its N- and C-termini have been topically rearranged. Such circularly permuted Cas9 proteins (“CP-Cas9”), or variants thereof, retain the ability to bind DNA when complexed with a guide RNA (gRNA). See, Oakes et al., “Protein Engineering of Cas9 for enhanced function,” Methods Enzymol, 2014, 546: 491-511 and Oakes et al., “CRISPR-Cas9 Circular Permutants as Programmable Scaffolds for Genome Modification,” Cell, Jan. 10, 2019, 176: 254-267, each of are incorporated herein by reference. The instant disclosure contemplates any previously known CP-Cas9 or use a new CP-Cas9 so long as the resulting circularly permuted protein retains the ability to bind DNA when complexed with a guide RNA (gRNA). Exemplary CP-Cas9 proteins are SEQ ID NOs: 77-86.
CRISPR
• CRISPR is a family of DNA sequences (i.e., CRISPR clusters) in bacteria and archaea that represent snippets of prior infections by a virus that have invaded the prokaryote. The snippets of DNA are used by the prokaryotic cell to detect and destroy DNA from subsequent attacks by similar viruses and effectively compose, along with an array of CRISPR-associated proteins (including Cas9 and homologs thereof) and CRISPR-associated RNA, a prokaryotic immune defense system. In nature, CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In certain types of CRISPR systems (e.g., type II CRISPR systems), correct processing of pre-crRNA requires a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc) and a Cas9 protein. The tracrRNA serves as a guide for ribonuclease 3-aided processing of pre-crRNA. Subsequently, Cas9/crRNA/tracrRNA endonucleolytically cleaves linear or circular dsDNA target complementary to the RNA. Specifically, the target strand not complementary to crRNA is first cut endonucleolytically, then trimmed 3′-5′ exonucleolytically. In nature, DNA-binding and cleavage typically requires protein and both RNAs. However, single guide RNAs (“sgRNA”, or simply “gNRA”) can be engineered so as to incorporate aspects of both the crRNA and tracrRNA into a single RNA species—the guide RNA. See, e.g., Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J. A., Charpentier E. Science 337:816-821(2012), the entire contents of which is hereby incorporated by reference. Cas9 recognizes a short motif in the CRISPR repeat sequences (the PAM or protospacer adjacent motif) to help distinguish self versus non-self. CRISPR biology, as well as Cas9 nuclease sequences and structures are well known to those of skill in the art (see, e.g., “Complete genome sequence of an M1 strain of Streptococcus pyogenes.” Ferretti et al., J. J., McShan W. M., Ajdic D. J., Savic D. J., Savic G., Lyon K., Primeaux C., Sezate S., Suvorov A. N., Kenton S., Lai H. S., Lin S. P., Qian Y., Jia H. G., Najar F. Z., Ren Q., Zhu H., Song L., White J., Yuan X., Clifton S. W., Roe B. A., McLaughlin R. E., Proc. Natl. Acad. Sci. U.S.A. 98:4658-4663(2001); “CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.” Deltcheva E., Chylinski K., Sharma C. M., Gonzales K., Chao Y., Pirzada Z. A., Eckert M. R., Vogel J., Charpentier E., Nature 471:602-607(2011); and “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.” Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J. A., Charpentier E. Science 337:816-821(2012), the entire contents of each of which are incorporated herein by reference). Cas9 orthologs have been described in various species, including, but not limited to, S. pyogenes and S. thermophilus. Additional suitable Cas9 nucleases and sequences will be apparent to those of skill in the art based on this disclosure, and such Cas9 nucleases and sequences include Cas9 sequences from the organisms and loci disclosed in Chylinski, Rhun, and Charpentier, “The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems” (2013) RNA Biology 10:5, 726-737; the entire contents of which are incorporated herein by reference.
• In certain types of CRISPR systems (e.g., type II CRISPR systems), correct processing of pre-crRNA requires a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and a Cas9 protein. The tracrRNA serves as a guide for ribonuclease 3-aided processing of pre-crRNA. Subsequently, Cas9/crRNA/tracrRNA endonucleolytically cleaves linear or circular nucleic acid target complementary to the RNA. Specifically, the target strand not complementary to crRNA is first cut endonucleolytically, then trimmed 3′-5′ exonucleolytically. In nature, DNA-binding and cleavage typically requires protein and both RNAs. However, single guide RNAs (“sgRNA”, or simply “gRNA”) can be engineered so as to incorporate embodiments of both the crRNA and tracrRNA into a single RNA species—the guide RNA.
• In general, a “CRISPR system” refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated (“Cas”) genes, including sequences encoding a Cas gene, a tracr (trans-activating CRISPR) sequence (e.g. tracrRNA or an active partial tracrRNA), a tracr mate sequence (encompassing a “direct repeat” and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system), a guide sequence (also referred to as a “spacer” in the context of an endogenous CRISPR system), or other sequences and transcripts from a CRISPR locus. The tracrRNA of the system is complementary (fully or partially) to the tracr mate sequence present on the guide RNA.
DNA Synthesis Template
• As used herein, the term “DNA synthesis template” refers to the region or portion of the extension arm of a PEgRNA that is utilized as a template strand by a polymerase of a prime editor to encode a 3′ single-strand DNA flap that contains the desired edit and which then, through the mechanism of prime editing, replaces the corresponding endogenous strand of DNA at the target site. In various embodiments, the DNA synthesis template is shown in FIG. 3A (in the context of a PEgRNA comprising a 5′ extension arm), FIG. 3B (in the context of a PEgRNA comprising a 3′ extension arm), FIG. 3C (in the context of an internal extension arm), FIG. 3D (in the context of a 3′ extension arm), and FIG. 3E (in the context of a 5′ extension arm). The extension arm, including the DNA synthesis template, may be comprised of DNA or RNA. In the case of RNA, the polymerase of the prime editor can be an RNA-dependent DNA polymerase (e.g., a reverse transcriptase). In the case of DNA, the polymerase of the prime editor can be a DNA-dependent DNA polymerase. In various embodiments (e.g., as depicted in FIGS. 3D-3E), the DNA synthesis template (4) may comprise the “edit template” and the “homology arm”, and all or a portion of the optional 5′ end modifier region, e2. That is, depending on the nature of the e2 region (e.g., whether it includes a hairpin, toeloop, or stem/loop secondary structure), the polymerase may encode none, some, or all of the e2 region, as well. Said another way, in the case of a 3′ extension arm, the DNA synthesis template (3) can include the portion of the extension arm (3) that spans from the 5′ end of the primer binding site (PBS) to 3′ end of the gRNA core that may operate as a template for the synthesis of a single-strand of DNA by a polymerase (e.g., a reverse transcriptase). In the case of a 5′ extension arm, the DNA synthesis template (3) can include the portion of the extension arm (3) that spans from the 5′ end of the PEgRNA molecule to the 3′ end of the edit template. Preferably, the DNA synthesis template excludes the primer binding site (PBS) of PEgRNAs either having a 3′ extension arm or a 5′ extension arm. Certain embodiments described here (e.g, FIG. 71A) refer to an “an RT template,” which is inclusive of the edit template and the homology arm, i.e., the sequence of the PEgRNA extension arm which is actually used as a template during DNA synthesis. The term “RT template” is equivalent to the term “DNA synthesis template.”
• In the case of trans prime editing (e.g., FIG. 3G and FIG. 3H), the primer binding site (PBS) and the DNA synthesis template can be engineered into a separate molecule referred to as a trans prime editor RNA template (tPERT).
Dimerization Domain
• The term “dimerization domain” refers to a ligand-binding domain that binds to a binding moiety of a bi-specific ligand. A “first” dimerization domain binds to a first binding moiety of a bi-specific ligand and a “second” dimerization domain binds to a second binding moiety of the same bi-specific ligand. When the first dimerization domain is fused to a first protein (e.g., via PE, as discussed herein) and the second dimerization domain (e.g., via PE, as discussed herein) is fused to a second protein, the first and second protein dimerize in the presence of a bi-specific ligand, wherein the bi-specific ligand has at least one moiety that binds to the first dimerization domain and at least another moiety that binds to the second dimerization domain.
Downstream
• As used herein, the terms “upstream” and “downstream” are terms of relativity that define the linear position of at least two elements located in a nucleic acid molecule (whether single or double-stranded) that is orientated in a 5′-to-3′ direction. In particular, a first element is upstream of a second element in a nucleic acid molecule where the first element is positioned somewhere that is 5′ to the second element. For example, a SNP is upstream of a Cas9-induced nick site if the SNP is on the 5′ side of the nick site. Conversely, a first element is downstream of a second element in a nucleic acid molecule where the first element is positioned somewhere that is 3′ to the second element. For example, a SNP is downstream of a Cas9-induced nick site if the SNP is on the 3′ side of the nick site. The nucleic acid molecule can be a DNA (double or single stranded). RNA (double or single stranded), or a hybrid of DNA and RNA. The analysis is the same for single strand nucleic acid molecule and a double strand molecule since the terms upstream and downstream are in reference to only a single strand of a nucleic acid molecule, except that one needs to select which strand of the double stranded molecule is being considered. Often, the strand of a double stranded DNA which can be used to determine the positional relativity of at least two elements is the “sense” or “coding” strand. In genetics, a “sense” strand is the segment within double-stranded DNA that runs from 5′ to 3′, and which is complementary to the antisense strand of DNA, or template strand, which runs from 3′ to 5′. Thus, as an example, a SNP nucleobase is “downstream” of a promoter sequence in a genomic DNA (which is double-stranded) if the SNP nucleobase is on the 3′ side of the promoter on the sense or coding strand.
Edit Template
• The term “edit template” refers to a portion of the extension arm that encodes the desired edit in the single strand 3′ DNA flap that is synthesized by the polymerase, e.g., a DNA-dependent DNA polymerase, RNA-dependent DNA polymerase (e.g., a reverse transcriptase). Certain embodiments described here (e.g., FIG. 71A) refer to “an RT template,” which refers to both the edit template and the homology arm together, i.e., the sequence of the PEgRNA extension arm which is actually used as a template during DNA synthesis. The term “RT edit template” is also equivalent to the term “DNA synthesis template,” but wherein the RT edit template reflects the use of a prime editor having a polymerase that is a reverse transcriptase, and wherein the DNA synthesis template reflects more broadly the use of a prime editor having any polymerase.
Effective Amount
• The term “effective amount,” as used herein, refers to an amount of a biologically active agent that is sufficient to elicit a desired biological response. For example, in some embodiments, an effective amount of a prime editor (PE) may refer to the amount of the editor that is sufficient to edit a target site nucleotide sequence, e.g., a genome. In some embodiments, an effective amount of a prime editor (PE) provided herein, e.g., of a fusion protein comprising a nickase Cas9 domain and a reverse transcriptase may refer to the amount of the fusion protein that is sufficient to induce editing of a target site specifically bound and edited by the fusion protein. As will be appreciated by the skilled artisan, the effective amount of an agent, e.g., a fusion protein, a nuclease, a hybrid protein, a protein dimer, a complex of a protein (or protein dimer) and a polynucleotide, or a polynucleotide, may vary depending on various factors as, for example, on the desired biological response, e.g., on the specific allele, genome, or target site to be edited, on the cell or tissue being targeted, and on the agent being used.
Error-Prone Reverse Transcriptase
• As used herein, the term “error-prone” reverse transcriptase (or more broadly, any polymerase) refers to a reverse transcriptase (or more broadly, any polymerase) that occurs naturally or which has been derived from another reverse transcriptase (e.g., a wild type M-MLV reverse transcriptase) which has an error rate that is less than the error rate of wild type M-MLV reverse transcriptase. The error rate of wild type M-MLV reverse transcriptase is reported to be in the range of one error in 15,000 (higher) to 27,000 (lower). An error rate of 1 in 15,000 corresponds with an error rate of 6.7×10⁻⁵. An error rate of 1 in 27,000 corresponds with an error rate of 3.7×10⁻⁵. See Boutabout et al. (2001) “DNA synthesis fidelity by the reverse transcriptase of the yeast retrotransposon Ty1,” Nucleic Acids Res 29(11):2217-2222, which is incorporated herein by reference. Thus, for purposes of this application, the term “error prone” refers to those RT that have an error rate that is greater than one error in 15,000 nucleobase incorporation (6.7×10⁻⁵ or higher), e.g., 1 error in 14,000 nucleobases (7.14×10⁻⁵ or higher), 1 error in 13,000 nucleobases or fewer (7.7×10⁻⁵ or higher), 1 error in 12,000 nucleobases or fewer (7.7×10⁻⁵ or higher), 1 error in 11,000 nucleobases or fewer (9.1×10⁻⁵ or higher), 1 error in 10,000 nucleobases or fewer (1×10⁴ or 0.0001 or higher), 1 error in 9,000 nucleobases or fewer (0.00011 or higher), 1 error in 8,000 nucleobases or fewer (0.00013 or higher) 1 error in 7,000 nucleobases or fewer (0.00014 or higher), 1 error in 6,000 nucleobases or fewer (0.00016 or higher), 1 error in 5,000 nucleobases or fewer (0.0002 or higher), 1 error in 4,000 nucleobases or fewer (0.00025 or higher), 1 error in 3,000 nucleobases or fewer (0.00033 or higher), 1 error in 2,000 nucleobase or fewer (0.00050 or higher), or 1 error in 1,000 nucleobases or fewer (0.001 or higher), or 1 error in 500 nucleobases or fewer (0.002 or higher), or 1 error in 250 nucleobases or fewer (0.004 or higher).
Extein
• The term “extein,” as used herein, refers to an polypeptide sequence that is flanked by an intein and is ligated to another extein during the process of protein splicing to form a mature, spliced protein. Typically, an intein is flanked by two extein sequences that are ligated together when the intein catalyzes its own excision. Exteins, accordingly, are the protein analog to exons found in mRNA. For example, a polypeptide comprising an intein may be of the structure extein(N)-intein-extein(C). After excision of the intein and splicing of the two exteins, the resulting structures are extein(N)-extein(C) and a free intein. In various configurations, the exteins may be separate proteins (e.g., half of a Cas9 or PE fusion protein), each fused to a split-intein, wherein the excision of the split inteins causes the splicing together of the extein sequences.
Extension Arm
• The term “extension arm” refers to a nucleotide sequence component of a PEgRNA which provides several functions, including a primer binding site and an edit template for reverse transcriptase. In some embodiments, e.g., FIG. 3D, the extension arm is located at the 3′ end of the guide RNA. In other embodiments, e.g., FIG. 3E, the extension arm is located at the 5′ end of the guide RNA. In some embodiments, the extension arm also includes a homology arm. In various embodiments, the extension arm comprises the following components in a 5′ to 3′ direction: the homology arm, the edit template, and the primer binding site. Since polymerization activity of the reverse transcriptase is in the 5′ to 3′ direction, the preferred arrangement of the homology arm, edit template, and primer binding site is in the 5′ to 3′ direction such that the reverse transcriptase, once primed by an annealed primer sequence, polymerases a single strand of DNA using the edit template as a complementary template strand. Further details, such as the length of the extension arm, are described elsewhere herein.
• The extension arm may also be described as comprising generally two regions: a primer binding site (PBS) and a DNA synthesis template, as shown in FIG. 3G (top), for instance. The primer binding site binds to the primer sequence that is formed from the endogenous DNA strand of the target site when it becomes nicked by the prime editor complex, thereby exposing a 3′ end on the endogenous nicked strand. As explained herein, the binding of the primer sequence to the primer binding site on the extension arm of the PEgRNA creates a duplex region with an exposed 3′ end (i.e., the 3′ of the primer sequence), which then provides a substrate for a polymerase to begin polymerizing a single strand of DNA from the exposed 3′ end along the length of the DNA synthesis template. The sequence of the single strand DNA product is the complement of the DNA synthesis template. Polymerization continues towards the 5′ of the DNA synthesis template (or extension arm) until polymerization terminates. Thus, the DNA synthesis template represents the portion of the extension arm that is encoded into a single strand DNA product (i.e., the 3′ single strand DNA flap containing the desired genetic edit information) by the polymerase of the prime editor complex and which ultimately replaces the corresponding endogenous DNA strand of the target site that sits immediate downstream of the PE-induced nick site. Without being bound by theory, polymerization of the DNA synthesis template continues towards the 5′ end of the extension arm until a termination event. Polymerization may terminate in a variety of ways, including, but not limited to (a) reaching a 5′ terminus of the PEgRNA (e.g., in the case of the 5′ extension arm wherein the DNA polymerase simply runs out of template), (b) reaching an impassable RNA secondary structure (e.g., hairpin or stem/loop), or (c) reaching a replication termination signal, e.g., a specific nucleotide sequence that blocks or inhibits the polymerase, or a nucleic acid topological signal, such as, supercoiled DNA or RNA.
Flap Endonuclease (e.g., FEN1)
• As used herein, the term “flap endonuclease” refers to an enzyme that catalyzes the removal of 5′ single strand DNA flaps. These are naturally occurring enzymes that process the removal of 5′ flaps formed during cellular processes, including DNA replication. The prime editing methods herein described may utilize endogenously supplied flap endonucleases or those provided in trans to remove the 5′ flap of endogenous DNA formed at the target site during prime editing. Flap endonucleases are known in the art and can be found described in Patel et al., “Flap endonucleases pass 5′-flaps through a flexible arch using a disorder-thread-order mechanism to confer specificity for free 5′-ends,” Nucleic Acids Research, 2012, 40(10): 4507-4519, Tsutakawa et al., “Human flap endonuclease structures, DNA double-base flipping, and a unified understanding of the FEN1 superfamily,” Cell, 2011, 145(2): 198-211, and Balakrishnan et al., “Flap Endonuclease 1,” Annu Rev Biochem, 2013, Vol 82: 119-138 (each of which are incorporated herein by reference). An exemplary flap endonuclease is FEN1, which can be represented by the following amino acid sequence:

• DESCRIPTION	SEQUENCE	SEQ ID NO:
  FEN1	MGIQGLAKLIADVAPSAIRENDIKSYFGRKVAIDASMSI	SEQ ID NO: 7
  WILD	YQFLIAVRQGGDVLQNEEGETTSHLMGMFYRTIRMME
  TYPE	NGIKPVYVFDGKPPQLKSGELAKRSERRAEAEKQLQQ
  	AQAAGAEQEVEKFTKRLVKVTKQHNDECKHLLSLMGI
  	PYLDAPSEAEASCAALVKAGKVYAAATEDMDCLTFGS
  	PVLMRHLTASEAKKLPIQEFHLSRILQELGLNQEQFVDL
  	CILLGSDYCESIRGIGPKRAVDLIQKHKSIEEIVRRLDPN
  	KYPVPENWLHKEAHQLFLEPEVLDPESVELKWSEPNE
  	EELIKFMCGEKQFSEERIRSGVKRLSKSRQGSTQGRLD
  	DFFKVTGSLSSAKRKEPEPKGSTKKKAKTGAAGKFKR
  	GK

Functional Equivalent
• The term “functional equivalent” refers to a second biomolecule that is equivalent in function, but not necessarily equivalent in structure to a first biomolecule. For example, a “Cas9 equivalent” refers to a protein that has the same or substantially the same functions as Cas9, but not necessarily the same amino acid sequence. In the context of the disclosure, the specification refers throughout to “a protein X, or a functional equivalent thereof.” In this context, a “functional equivalent” of protein X embraces any homolog, paralog, fragment, naturally occurring, engineered, mutated, or synthetic version of protein X which bears an equivalent function.
Fusion Protein
• The term “fusion protein” as used herein refers to a hybrid polypeptide which comprises protein domains from at least two different proteins. One protein may be located at the amino-terminal (N-terminal) portion of the fusion protein or at the carboxy-terminal (C-terminal) protein thus forming an “amino-terminal fusion protein” or a “carboxy-terminal fusion protein,” respectively. A protein may comprise different domains, for example, a nucleic acid binding domain (e.g., the gRNA binding domain of Cas9 that directs the binding of the protein to a target site) and a nucleic acid cleavage domain or a catalytic domain of a nucleic-acid editing protein. Another example includes a Cas9 or equivalent thereof to a reverse transcriptase. Any of the proteins provided herein may be produced by any method known in the art. For example, the proteins provided herein may be produced via recombinant protein expression and purification, which is especially suited for fusion proteins comprising a peptide linker. Methods for recombinant protein expression and purification are well known, and include those described by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4^th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)), the entire contents of which are incorporated herein by reference.
Gene of Interest (GOI)
• The term “gene of interest” or “GOT” refers to a gene that encodes a biomolecule of interest (e.g., a protein or an RNA molecule). A protein of interest can include any intracellular protein, membrane protein, or extracellular protein, e.g., a nuclear protein, transcription factor, nuclear membrane transporter, intracellular organelle associated protein, a membrane receptor, a catalytic protein, and enzyme, a therapeutic protein, a membrane protein, a membrane transport protein, a signal transduction protein, or an immunological protein (e.g., an IgG or other antibody protein), etc. The gene of interest may also encode an RNA molecule, including, but not limited to, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA), antisense RNA, guide RNA, microRNA (miRNA), small interfering RNA (siRNA), and cell-free RNA (cfRNA).
• Guide RNA (“gRNA”)
• As used herein, the term “guide RNA” is a particular type of guide nucleic acid which is mostly commonly associated with a Cas protein of a CRISPR-Cas9 and which associates with Cas9, directing the Cas9 protein to a specific sequence in a DNA molecule that includes complementarity to protospacer sequence of the guide RNA. However, this term also embraces the equivalent guide nucleic acid molecules that associate with Cas9 equivalents, homologs, orthologs, or paralogs, whether naturally occurring or non-naturally occurring (e.g., engineered or recombinant), and which otherwise program the Cas9 equivalent to localize to a specific target nucleotide sequence. The Cas9 equivalents may include other napDNAbp from any type of CRISPR system (e.g., type II, V, VI), including Cpf1 (a type-V CRISPR-Cas systems), C2c1 (a type V CRISPR-Cas system), C2c2 (a type VI CRISPR-Cas system) and C2c3 (a type V CRISPR-Cas system). Further Cas-equivalents are described in Makarova et al., “C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector,” Science 2016; 353(6299), the contents of which are incorporated herein by reference. Exemplary sequences are and structures of guide RNAs are provided herein. In addition, methods for designing appropriate guide RNA sequences are provided herein. As used herein, the “guide RNA” may also be referred to as a “traditional guide RNA” to contrast it with the modified forms of guide RNA termed “prime editing guide RNAs” (or “PEgRNAs”) which have been invented for the prime editing methods and composition disclosed herein.
• Guide RNAs or PEgRNAs may comprise various structural elements that include, but are not limited to:
• Spacer sequence—the sequence in the guide RNA or PEgRNA (having about 20 nts in length) which binds to the protospacer in the target DNA.
• gRNA core (or gRNA scaffold or backbone sequence)—refers to the sequence within the gRNA that is responsible for Cas9 binding, it does not include the 20 bp spacer/targeting sequence that is used to guide Cas9 to target DNA.
• Extension arm—a single strand extension at the 3′ end or the 5′ end of the PEgRNA which comprises a primer binding site and a DNA synthesis template sequence that encodes via a polymerase (e.g., a reverse transcriptase) a single stranded DNA flap containing the genetic change of interest, which then integrates into the endogenous DNA by replacing the corresponding endogenous strand, thereby installing the desired genetic change.
• Transcription terminator—the guide RNA or PEgRNA may comprise a transcriptional termination sequence at the 3′ of the molecule.
Homology Arm
• The term “homology arm” refers to a portion of the extension arm that encodes a portion of the resulting reverse transcriptase-encoded single strand DNA flap that is to be integrated into the target DNA site by replacing the endogenous strand. The portion of the single strand DNA flap encoded by the homology arm is complementary to the non-edited strand of the target DNA sequence, which facilitates the displacement of the endogenous strand and annealing of the single strand DNA flap in its place, thereby installing the edit. This component is further defined elsewhere. The homology arm is part of the DNA synthesis template since it is by definition encoded by the polymerase of the prime editors described herein.
Host Cell
• The term “host cell,” as used herein, refers to a cell that can host, replicate, and express a vector described herein, e.g., a vector comprising a nucleic acid molecule encoding a fusion protein comprising a Cas9 or Cas9 equivalent and a reverse transcriptase.
Inteins
• As used herein, the term “intein” refers to auto-processing polypeptide domains found in organisms from all domains of life. An intein (intervening protein) carries out a unique auto-processing event known as protein splicing in which it excises itself out from a larger precursor polypeptide through the cleavage of two peptide bonds and, in the process, ligates the flanking extein (external protein) sequences through the formation of a new peptide bond. This rearrangement occurs post-translationally (or possibly co-translationally), as intein genes are found embedded in frame within other protein-coding genes. Furthermore, intein-mediated protein splicing is spontaneous; it requires no external factor or energy source, only the folding of the intein domain. This process is also known as cis-protein splicing, as opposed to the natural process of trans-protein splicing with “split inteins.” Inteins are the protein equivalent of the self-splicing RNA introns (see Perler et al., Nucleic Acids Res. 22:1125-1127 (1994)), which catalyze their own excision from a precursor protein with the concomitant fusion of the flanking protein sequences, known as exteins (reviewed in Perler et al., Curr. Opin. Chem. Biol. 1:292-299 (1997); Perler, F. B. Cell 92(1):1-4 (1998); Xu et al., EMBO J. 15(19):5146-5153 (1996)).
• As used herein, the term “protein splicing” refers to a process in which an interior region of a precursor protein (an intein) is excised and the flanking regions of the protein (exteins) are ligated to form the mature protein. This natural process has been observed in numerous proteins from both prokaryotes and eukaryotes (Perler, F. B., Xu, M. Q., Paulus, H. Current Opinion in Chemical Biology 1997, 1, 292-299; Perler, F. B. Nucleic Acids Research 1999, 27, 346-347). The intein unit contains the necessary components needed to catalyze protein splicing and often contains an endonuclease domain that participates in intein mobility (Perler, F. B., Davis, E. O., Dean, G. E., Gimble, F. S., Jack, W. E., Neff, N., Noren, C. J., Thomer, J., Belfort, M. Nucleic Acids Research 1994, 22, 1127-1127). The resulting proteins are linked, however, not expressed as separate proteins. Protein splicing may also be conducted in trans with split inteins expressed on separate polypeptides spontaneously combine to form a single intein which then undergoes the protein splicing process to join to separate proteins.
• The elucidation of the mechanism of protein splicing has led to a number of intein-based applications (Comb, et al., U.S. Pat. No. 5,496,714; Comb, et al., U.S. Pat. No. 5,834,247; Camarero and Muir, J. Amer. Chem. Soc., 121:5597-5598 (1999); Chong, et al., Gene, 192:271-281 (1997), Chong, et al., Nucleic Acids Res., 26:5109-5115 (1998); Chong, et al., J. Biol. Chem., 273:10567-10577 (1998); Cotton, et al. J. Am. Chem. Soc., 121:1100-1101 (1999); Evans, et al., J. Biol. Chem., 274:18359-18363 (1999); Evans, et al., J. Biol. Chem., 274:3923-3926 (1999); Evans, et al., Protein Sci., 7:2256-2264 (1998); Evans, et al., J. Biol. Chem., 275:9091-9094 (2000); Iwai and Pluckthun, FEBS Lett. 459:166-172 (1999); Mathys, et al., Gene, 231:1-13 (1999); Mills, et al., Proc. Natl. Acad. Sci. USA 95:3543-3548 (1998); Muir, et al., Proc. Natl. Acad. Sci. USA 95:6705-6710 (1998); Otomo, et al., Biochemistry 38:16040-16044 (1999); Otomo, et al., J. Biolmol. NMR 14:105-114 (1999); Scott, et al., Proc. Natl. Acad. Sci. USA 96:13638-13643 (1999); Severinov and Muir, J. Biol. Chem., 273:16205-16209 (1998); Shingledecker, et al., Gene, 207:187-195 (1998); Southworth, et al., EMBO J. 17:918-926 (1998); Southworth, et al., Biotechniques, 27:110-120 (1999); Wood, et al., Nat. Biotechnol., 17:889-892 (1999); Wu, et al., Proc. Natl. Acad. Sci. USA 95:9226-9231 (1998a); Wu, et al., Biochim Biophys Acta 1387:422-432 (1998b); Xu, et al., Proc. Natl. Acad. Sci. USA 96:388-393 (1999); Yamazaki, et al., J. Am. Chem. Soc., 120:5591-5592 (1998)). Each reference is incorporated herein by reference.
Ligand-Dependent Intein
• The term “ligand-dependent intein,” as used herein refers to an intein that comprises a ligand-binding domain. Typically, the ligand-binding domain is inserted into the amino acid sequence of the intein, resulting in a structure intein (N)-ligand-binding domain-intein (C). Typically, ligand-dependent inteins exhibit no or only minimal protein splicing activity in the absence of an appropriate ligand, and a marked increase of protein splicing activity in the presence of the ligand. In some embodiments, the ligand-dependent intein does not exhibit observable splicing activity in the absence of ligand but does exhibit splicing activity in the presence of the ligand. In some embodiments, the ligand-dependent intein exhibits an observable protein splicing activity in the absence of the ligand, and a protein splicing activity in the presence of an appropriate ligand that is at least 5 times, at least 10 times, at least 50 times, at least 100 times, at least 150 times, at least 200 times, at least 250 times, at least 500 times, at least 1000 times, at least 1500 times, at least 2000 times, at least 2500 times, at least 5000 times, at least 10000 times, at least 20000 times, at least 25000 times, at least 50000 times, at least 100000 times, at least 500000 times, or at least 1000000 times greater than the activity observed in the absence of the ligand. In some embodiments, the increase in activity is dose dependent over at least 1 order of magnitude, at least 2 orders of magnitude, at least 3 orders of magnitude, at least 4 orders of magnitude, or at least 5 orders of magnitude, allowing for fine-tuning of intein activity by adjusting the concentration of the ligand. Suitable ligand-dependent inteins are known in the art, and in include those provided below and those described in published U.S. Patent Application U.S. 2014/0065711 A1; Mootz et al., “Protein splicing triggered by a small molecule.” J. Am. Chem. Soc. 2002; 124, 9044-9045; Mootz et al., “Conditional protein splicing: a new tool to control protein structure and function in vitro and in vivo.” J. Am. Chem. Soc. 2003; 125, 10561-10569; Buskirk et al., Proc. Natl. Acad. Sci. USA. 2004; 101, 10505-10510); Skretas & Wood, “Regulation of protein activity with small-molecule-controlled inteins.” Protein Sci. 2005; 14, 523-532; Schwartz, et al., “Post-translational enzyme activation in an animal via optimized conditional protein splicing.” Nat. Chem. Biol. 2007; 3, 50-54; Peck et al., Chem. Biol. 2011; 18 (5), 619-630; the entire contents of each are hereby incorporated by reference. Exemplary sequences are as follows:

• NAME	SEQUENCE OF LIGAND-DEPENDENT INTEIN
  2-4 INTEIN:	CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAAAKDGTLLARPVV
  	SWFDQGTRDVIGLRIAGGAIVWATPDHKVLTEYGWRAAGELRKGD
  	RVAGPGGSGNSLALSLTADQMVSALLDAEPPILYSEYDPTSPFSEAS
  	MMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQAHLLECAWLEI
  	LMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLAT
  	SSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRA
  	LDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEH
  	LYSMKYKNVVPLYDLLLEMLDAHRLHAGGSGASRVQAFADALDD
  	KFLHDMLAEELRYSVIREVLPTRRARTFDLEVEELHTLVAEGVVVH
  	NC (SEQ ID NO: 8)
  3-2 INTEIN	CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAVAKDGTLLARPVVS
  	WFDQGTRDVIGLRIAGGAIVWATPDHKVLTEYGWRAAGELRKGDR
  	VAGPGGSGNSLALSLTADQMVSALLDAEPPILYSEYDPTSPFSEASM
  	MGLLTNLADRELVHMINWAKRVPGFVDLTLHDQAHLLECAWLEIL
  	MIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATS
  	SRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRAL
  	DKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHL
  	YSMKYTNVVPLYDLLLEMLDAHRLHAGGSGASRVQAFADALDDK
  	FLHDMLAEELRYSVIREVLPTRRARTFDLEVEELHTLVAEGVVVHN
  	C (SEQ ID NO: 9)
  30R3-1 INTEIN	CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAAAKDGTLLARPVV
  	SWFDQGTRDVIGLRIAGGATVWATPDHKVLTEYGWRAAGELRKG
  	DRVAGPGGSGNSLALSLTADQMVSALLDAEPPIPYSEYDPTSPFSEA
  	SMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQAHLLECAWL
  	EILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLL
  	ATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIH
  	RALDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGM
  	EHLYSMKYKNVVPLYDLLLEMLDAHRLHAGGSGASRVQAFADAL
  	DDKFLHDMLAEGLRYSVIREVLPTRRARTFDLEVEELHTLVAEGVV
  	VHNC (SEQ ID NO: 10)
  30R3-2 INTEIN	CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAAAKDGTLLARPVV
  	SWFDQGTRDVIGLRIAGGATVWATPDHKVLTEYGWRAAGELRKG
  	DRVAGPGGSGNSLALSLTADQMVSALLDAEPPILYSEYDPTSPFSEA
  	SMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQAHLLECAWL
  	EILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLL
  	ATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIH
  	RALDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGM
  	EHLYSMKYKNVVPLYDLLLEMLDAHRLHAGGSGASRVQAFADAL
  	DDKFLHDMLAEELRYSVIREVLPTRRARTFDLEVEELHTLVAEGVV
  	VHNC (SEQ ID NO: 11)
  30R3-3 INTEIN	CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAAAKDGTLLARPVV
  	SWFDQGTRDVIGLRIAGGATVWATPDHKVLTEYGWRAAGELRKG
  	DRVAGPGGGNSLALSLTADQMVSALLDAEPPIPYSEYDPTSPFSEA
  	SMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQAHLLECAWL
  	EILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLL
  	ATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIH
  	RALDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGM
  	EHLYSMKYKNVVPLYDLLLEMLDAHRLHAGGSGASRVQAFADAL
  	DDKFLHDMLAEELRYSVIREVLPTRRARTFDLEVEELHTLVAEGVV
  	VHNC (SEQ ID NO: 12)
  37R3-1 INTEIN	CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAAAKDGTLLARPVV
  	SWFDQGTRDVIGLRIAGGATVWATPDHKVLTEYGWRAAGELRKG
  	DRVAGPGGSGNSLALSLTADQMVSALLDAEPPILYSEYNPTSPFSEA
  	SMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQAHLLERAWL
  	EILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLL
  	ATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIH
  	RALDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGM
  	EHLYSMKYKNVVPLYDLLLEMLDAHRLHAGGSGASRVQAFADAL
  	DDKFLHDMLAEGLRYSVIREVLPTRRARTFDLEVEELHTLVAEGVV
  	VHNC ((SEQ ID NO: 13)
  37R3-2 INTEIN	CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAAAKDGTLLARPVV
  	SWFDQGTRDVIGLRIAGGAIVWATPDHKVLTEYGWRAAGELRKGD
  	RVAGPGGSGNSLALSLTADQMVSALLDAEPPILYSEYDPTSPFSEAS
  	MMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQAHLLERAWLEI
  	LMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLAT
  	SSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRA
  	LDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEH
  	LYSMKYKNVVPLYDLLLEMLDAHRLHAGGSGASRVQAFADALDD
  	KFLHDMLAEGLRYSVIREVLPTRRARTFDLEVEELHTLVAEGVVVH
  	NC (SEQ ID NO: 14)
  37R3-3 INTEIN	CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAVAKDGTLLARPVVS
  	WFDQGTRDVIGLRIAGGATVWATPDHKVLTEYGWRAAGELRKGD
  	RVAGPGGSGNSLALSLTADQMVSALLDAEPPILYSEYDPTSPFSEAS
  	MMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQAHLLERAWLEI
  	LMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLAT
  	SSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRA
  	LDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEH
  	LYSMKYKNVVPLYDLLLEMLDAHRLHAGGSGASRVQAFADALDD
  	KFLHDMLAEELRYSVIREVLPTRRARTFDLEVEELHTLVAEGVVVH
  	NC (SEQ ID NO: 15)

Linker
• The term “linker,” as used herein, refers to a molecule linking two other molecules or moieties. The linker can be an amino acid sequence in the case of a linker joining two fusion proteins. For example, a Cas9 can be fused to a reverse transcriptase by an amino acid linker sequence. The linker can also be a nucleotide sequence in the case of joining two nucleotide sequences together. For example, in the instant case, the traditional guide RNA is linked via a spacer or linker nucleotide sequence to the RNA extension of a prime editing guide RNA which may comprise a RT template sequence and an RT primer binding site. In other embodiments, the linker is an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker is 5-100 amino acids in length, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30-35, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-150, or 150-200 amino acids in length. Longer or shorter linkers are also contemplated.
Isolated
• “Isolated” means altered or removed from the natural state. For example, a nucleic 20 acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.
• In some embodiments, a gene of interest is encoded by an isolated nucleic acid. As used herein, the term “isolated,” refers to the characteristic of a material as provided herein being removed from its original or native environment (e.g., the natural environment if it is naturally occurring). Therefore, a naturally-occurring polynucleotide or protein or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated by human intervention from some or all of the coexisting materials in the natural system, is isolated. An artificial or engineered material, for example, a non-naturally occurring nucleic acid construct, such as the expression constructs and vectors described herein, are, accordingly, also referred to as isolated. A material does not have to be purified in order to be isolated. Accordingly, a material may be part of a vector and/or part of a composition, and still be isolated in that such vector or composition is not part of the environment in which the material is found in nature.
MS2 Tagging Technique
• In various embodiments (e.g., as depicted in the embodiments of FIGS. 72-73 and in Example 19), the term “MS2 tagging technique” refers to the combination of an “RNA-protein interaction domain” (aka “RNA-protein recruitment domain or protein”) paired up with an RNA-binding protein that specifically recognizes and binds to the RNA-protein interaction domain, e.g., a specific hairpin structure. These types of systems can be leveraged to recruit a variety of functionalities to a prime editor complex that is bound to a target site. The MS2 tagging technique is based on the natural interaction of the MS2 bacteriophage coat protein (“MCP” or “MS2cp”) with a stem-loop or hairpin structure present in the genome of the phage, i.e., the “MS2 hairpin.” In the case of prime editing, the MS2 tagging technique comprises introducing the MS2 hairpin into a desired RNA molecule involved in prime editing (e.g., a PEgRNA or a tPERT), which then constitutes a specific interactable binding target for an RNA-binding protein that recognizes and binds to that structure. In the case of the MS2 hairpin, it is recognized and bound by the MS2 bacteriophage coat protein (MCP). And, if MCP is fused to another protein (e.g., a reverse transcriptase or other DNA polymerase), then the MS2 hairpin may be used to “recruit” that other protein in trans to the target site occupied by the prime editing complex.
• The prime editors described herein may incorporate as an aspect any known RNA-protein interaction domain to recruit or “co-localize” specific functions of interest to a prime editor complex. A review of other modular RNA-protein interaction domains are described in the art, for example, in Johansson et al., “RNA recognition by the MS2 phage coat protein,” Sem Virol., 1997, Vol. 8(3): 176-185; Delebecque et al., “Organization of intracellular reactions with rationally designed RNA assemblies,” Science, 2011, Vol. 333: 470-474; Mali et al., “Cas9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering,” Nat. Biotechnol., 2013, Vol. 31: 833-838; and Zalatan et al., “Engineering complex synthetic transcriptional programs with CRISPR RNA scaffolds,” Cell, 2015, Vol. 160: 339-350, each of which are incorporated herein by reference in their entireties. Other systems include the PP7 hairpin, which specifically recruits the PCP protein, and the “corn” hairpin, which specifically recruits the Com protein. See Zalatan et al.
• The nucleotide sequence of the MS2 hairpin (or equivalently referred to as the “MS2 aptamer”) is:

• 	(SEQ ID NO: 763)
  	GCCAACATGAGGATCACCCATGTCTGCAGGGCC.

• The amino acid sequence of the MCP or MS2cp is:

• (SEQ ID NO: 764)

  GSASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSV

  RQSSAQNRKYTIKVEVPKVATQTVGGEELPVAGWRSYLNMELTIPIFATN

  SDCELIVKAMQGLLKDGNPIPSAIAANSGIY.

• The MS2 hairpin (or “MS2 aptamer”) may also be referred to as a type of “RNA effector recruitment domain” (or equivalently as “RNA-binding protein recruitment domain” or simply as “recruitment domain”) since it is a physical structure (e.g., a hairpin) that is installed into a PEgRNA or tPERT that effectively recruits other effector functions (e.g., RNA-binding proteins having various functions, such as DNA polymerases or other DNA-modifying enzymes) to the PEgRNA or rPERT that is so modified, and thus, co-localizing effector functions in trans to the prime editing machinery. This application is not intended to be limited in any way to any particular RNA effector recruitment domains and may include any available such domain, including the MS2 hairpin. Example 19 and FIG. 72(b) depicts the use of the MS2 aptamer joined to a DNA synthesis domain (i.e., the tPERT molecule) and a prime editor that comprises an MS2cp protein fused to a PE2 to cause the co-localization of the prime editor complex (MS2cp-PE2:sgRNA complex) bound to the target DNA site and the DNA synthesis domain of the tPERT molecule to effectuate the
• napDNAbp
• As used herein, the term “nucleic acid programmable DNA binding protein” or “napDNAbp,” of which Cas9 is an example, refer to a proteins which use RNA:DNA hybridization to target and bind to specific sequences in a DNA molecule. Each napDNAbp is associated with at least one guide nucleic acid (e.g., guide RNA), which localizes the napDNAbp to a DNA sequence that comprises a DNA strand (i.e., a target strand) that is complementary to the guide nucleic acid, or a portion thereof (e.g., the protospacer of a guide RNA). In other words, the guide nucleic-acid “programs” the napDNAbp (e.g., Cas9 or equivalent) to localize and bind to a complementary sequence.
• Without being bound by theory, the binding mechanism of a napDNAbp-guide RNA complex, in general, includes the step of forming an R-loop whereby the napDNAbp induces the unwinding of a double-strand DNA target, thereby separating the strands in the region bound by the napDNAbp. The guide RNA protospacer then hybridizes to the “target strand.” This displaces a “non-target strand” that is complementary to the target strand, which forms the single strand region of the R-loop. In some embodiments, the napDNAbp includes one or more nuclease activities, which then cut the DNA leaving various types of lesions. For example, the napDNAbp may comprises a nuclease activity that cuts the non-target strand at a first location, and/or cuts the target strand at a second location. Depending on the nuclease activity, the target DNA can be cut to form a “double-stranded break” whereby both strands are cut. In other embodiments, the target DNA can be cut at only a single site, i.e., the DNA is “nicked” on one strand. Exemplary napDNAbp with different nuclease activities include “Cas9 nickase” (“nCas9”) and a deactivated Cas9 having no nuclease activities (“dead Cas9” or “dCas9”). Exemplary sequences for these and other napDNAbp are provided herein.
Nickase
• The term “nickase” refers to a Cas9 with one of the two nuclease domains inactivated. This enzyme is capable of cleaving only one strand of a target DNA.
Nuclear Localization Sequence (NLS)
• The term “nuclear localization sequence” or “NLS” refers to an amino acid sequence that promotes import of a protein into the cell nucleus, for example, by nuclear transport. Nuclear localization sequences are known in the art and would be apparent to the skilled artisan. For example, NLS sequences are described in Plank et al., international PCT application, PCT/EP2000/011690, filed Nov. 23, 2000, published as WO/2001/038547 on May 31, 2001, the contents of which are incorporated herein by reference for its disclosure of exemplary nuclear localization sequences. In some embodiments, a NLS comprises the amino acid sequence

• 	(SEQ ID NO: 16)
  	PKKKRKV
  	or
  	(SEQ ID NO: 17)
  	MDSLLMNRRKFLYQFKNVRWAKGRRETYLC.

Nucleic Acid Molecule
• The term “nucleic acid,” as used herein, refers to a polymer of nucleotides. The polymer may include natural nucleosides (i.e., adenosine, thymidine, guano sine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine), nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C5 bromouridine, C5 fluorouridine, C5 iodouridine, C5 propynyl uridine, C5 propynyl cytidine, C5 methylcytidine, 7 deazaadenosine, 7 deazaguanosine, 8 oxoadenosine, 8 oxoguanosine, O(6) methylguanine, 4-acetylcytidine, 5-(carboxyhydroxymethyl)uridine, dihydrouridine, methylpseudouridine, 1-methyl adenosine, 1-methyl guanosine, N6-methyl adenosine, and 2-thiocytidine), chemically modified bases, biologically modified bases (e.g., methylated bases), intercalated bases, modified sugars (e.g., 2′-fluororibose, ribose, 2′-deoxyribose, 2′-O-methylcytidine, arabinose, and hexose), or modified phosphate groups (e.g., phosphorothioates and 5′ N phosphoramidite linkages).
PEgRNA
• As used herein, the terms “prime editing guide RNA” or “PEgRNA” or “extended guide RNA” refers to a specialized form of a guide RNA that has been modified to include one or more additional sequences for implementing the prime editing methods and compositions described herein. As described herein, the prime editing guide RNA comprise one or more “extended regions” of nucleic acid sequence. The extended regions may comprise, but are not limited to, single-stranded RNA or DNA. Further, the extended regions may occur at the 3′ end of a traditional guide RNA. In other arrangements, the extended regions may occur at the 5′ end of a traditional guide RNA. In still other arrangements, the extended region may occur at an intramolecular region of the traditional guide RNA, for example, in the gRNA core region which associates and/or binds to the napDNAbp. The extended region comprises a “DNA synthesis template” which encodes (by the polymerase of the prime editor) a single-stranded DNA which, in turn, has been designed to be (a) homologous with the endogenous target DNA to be edited, and (b) which comprises at least one desired nucleotide change (e.g., a transition, a transversion, a deletion, or an insertion) to be introduced or integrated into the endogenous target DNA. The extended region may also comprise other functional sequence elements, such as, but not limited to, a “primer binding site” and a “spacer or linker” sequence, or other structural elements, such as, but not limited to aptamers, stem loops, hairpins, toe loops (e.g., a 3′ toeloop), or an RNA-protein recruitment domain (e.g., MS2 hairpin). As used herein the “primer binding site” comprises a sequence that hybridizes to a single-strand DNA sequence having a 3′ end generated from the nicked DNA of the R-loop.
• In certain embodiments, the PEgRNAs are represented by FIG. 3A, which shows a PEgRNA having a 5′ extension arm, a spacer, and a gRNA core. The 5′ extension further comprises in the 5′ to 3′ direction a reverse transcriptase template, a primer binding site, and a linker. As shown, the reverse transcriptase template may also be referred to more broadly as the “DNA synthesis template” where the polymerase of a prime editor described herein is not an RT, but another type of polymerase.
• In certain other embodiments, the PEgRNAs are represented by FIG. 3B, which shows a PEgRNA having a 5′ extension arm, a spacer, and a gRNA core. The 5′ extension further comprises in the 5′ to 3′ direction a reverse transcriptase template, a primer binding site, and a linker. As shown, the reverse transcriptase template may also be referred to more broadly as the “DNA synthesis template” where the polymerase of a prime editor described herein is not an RT, but another type of polymerase.
• In still other embodiments, the PEgRNAs are represented by FIG. 3D, which shows a PEgRNA having in the 5′ to 3′ direction a spacer (1), a gRNA core (2), and an extension arm (3). The extension arm (3) is at the 3′ end of the PEgRNA. The extension arm (3) further comprises in the 5′ to 3′ direction a “primer binding site” (A), an “edit template” (B), and a “homology arm” (C). The extension arm (3) may also comprise an optional modifier region at the 3′ and 5′ ends, which may be the same sequences or different sequences. In addition, the 3′ end of the PEgRNA may comprise a transcriptional terminator sequence. These sequence elements of the PEgRNAs are further described and defined herein.
• In still other embodiments, the PEgRNAs are represented by FIG. 3E, which shows a PEgRNA having in the 5′ to 3′ direction an extension arm (3), a spacer (1), and a gRNA core (2). The extension arm (3) is at the 5′ end of the PEgRNA. The extension arm (3) further comprises in the 3′ to 5′ direction a “primer binding site” (A), an “edit template” (B), and a “homology arm” (C). The extension arm (3) may also comprise an optional modifier region at the 3′ and 5′ ends, which may be the same sequences or different sequences. The PEgRNAs may also comprise a transcriptional terminator sequence at the 3′ end. These sequence elements of the PEgRNAs are further described and defined herein.
PE1
• As used herein, “PE1” refers to a PE complex comprising a fusion protein comprising Cas9(H840A) and a wild type MMLV RT having the following structure: [NLS]-[Cas9(H840A)]-[linker]-[MMLV_RT(wt)]+a desired PEgRNA, wherein the PE fusion has the amino acid sequence of SEQ ID NO: 123, which is shown as follows;

• (SEQ ID NO: 123)

  MKRTADGSEFESPKKKRKV DKKYSIGLDIGTNSVGWAVITDEYKVPSKKF

  KVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYL

  QEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKY

  PTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVD

  KLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGE

  KKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI

  GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDL

  TLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEK

  MDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPF

  LKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVV

  DKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTE

  GMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISG

  VEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMI

  EERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDF

  LKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPA

  IKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMK

  RIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINR

  LSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNY

  WRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVA

  QILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYH

  HAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKA

  TAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT

  VRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYG

  GFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFL

  EAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKY

  VNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVIL

  ADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTID

  RKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD SGGSSGGSSGSETP

  GTSESATPESSGGSSGGSS TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQ

  AWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRL

  LDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPN

  PYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISG

  QLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAAT

  SELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLT

  EARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTG

  TLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVL

  TQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQP

  LVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPA

  TLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQE

  GQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVY

  TDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLS

  IIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP SGGS

  KRTADGSEFEPKKKRKV

  KEY:

  NUCLEAR LOCALIZATION SEQUENCE (NLS)

  TOP:(SEQ ID NO: 124), BOTTOM: (SEQ ID NO: 133)

  CAS9(H840A) (SEQ ID NO: 126)

  33-AMINO ACID LINKER (SEQ ID NO: 127)

  M-MLV reverse transcriptase (SEQ ID NO: 128).

PE2
• As used herein, “PE2” refers to a PE complex comprising a fusion protein comprising Cas9(H840A) and a variant MMLV RT having the following structure: [NLS]-[Cas9(H840A)]-[linker]-[MMLV_RT(D200N)(T330P)(L603W)(T306K)(W313F)]+a desired PEgRNA, wherein the PE fusion has the amino acid sequence of SEQ ID NO: 134, which is shown as follows:

• (SEQ ID NO: 134)

  MKRTADGSEFESPKKKRKV DKKYSIGLDIGTNSVGWAVITDEYKVPSKKF

  KVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYL

  QEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKY

  PTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVD

  KLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGE

  KKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI

  GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDL

  TLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEK

  MDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPF

  LKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVV

  DKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTE

  GMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISG

  VEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMI

  EERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDF

  LKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPA

  IKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMK

  RIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINR

  LSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNY

  WRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVA

  QILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYH

  HAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKA

  TAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT

  VRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYG

  GFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFL

  EAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKY

  VNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVIL

  ADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTID

  RKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD SGGSSGGSSGSETP

  GTSESATPESSGGSSGGSS TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQ

  AWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRL

  LDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPN

  PYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISG

  QLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAAT

  SELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLT

  EARKETVMGQPTPKTPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPG

  TLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVL

  TQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQP

  LVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPA

  TLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQE

  GQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVY

  TDSRYAFATAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPKRLS

  IIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP SGGS

  KRTADGSEFEPKKKRKV

  KEY:

  NUCLEAR LOCALIZATION SEQUENCE (NLS)

  TOP:(SEQ ID NO: 124), BOTTOM: (SEQ ID NO: 133)

  CAS9(H840A) (SEQ ID NO: 137)

  33-AMINO ACID LINKER (SEQ ID NO: 127)

  M-MLV reverse transcriptase (SEQ ID NO: 139).

PE3
• As used herein, “PE3” refers to PE2 plus a second-strand nicking guide RNA that complexes with the PE2 and introduces a nick in the non-edited DNA strand in order to induce preferential replacement of the edited strand.
PE3b
• As used herein, “PE3b” refers to PE3 but wherein the second-strand nicking guide RNA is designed for temporal control such that the second strand nick is not introduced until after the installation of the desired edit. This is achieved by designing a gRNA with a spacer sequence that matches only the edited strand, but not the original allele. Using this strategy, referred to hereafter as PE3b, mismatches between the protospacer and the unedited allele should disfavor nicking by the sgRNA until after the editing event on the PAM strand takes place.
PE-Short
• As used herein, “PE-short” refers to a PE construct that is fused to a C-terminally truncated reverse transcriptase, and has the following amino acid sequence:

• (SEQ ID NO: 765)

  MKRTADGSEFESPKKKRKV DKKYSIGLDIGTNSVGWAVITDEYKVPSKKF

  KVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYL

  QEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKY

  PTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVD

  KLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGE

  KKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI

  GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDL

  TLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEK

  MDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPF

  LKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVV

  DKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTE

  GMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISG

  VEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMI

  EERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDF

  LKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPA

  IKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMK

  RIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINR

  LSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNY

  WRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVA

  QILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYH

  HAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKA

  TAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT

  VRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYG

  GFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFL

  EAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKY

  VNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVIL

  ADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTID

  RKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD SGGSSGGSSGSETP

  GTSESATPESSGGSSGGSS TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQ

  AWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRL

  LDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPN

  PYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISG

  QLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAAT

  SELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLT

  EARKETVMGQPTPKTPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPG

  TLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVL

  TQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQP

  LVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPA

  TLLPLPEEGLQHNCLDNSRLIN SGGSKRTADGSEFEPKKKRKV

  KEY:

  NUCLEAR LOCALIZATION SEQUENCE (NLS)

  TOP:(SEQ ID NO: 124), BOTTOM: (SEQ ID NO: 133)

  CAS9(H840A) (SEQ ID NO: 157)

  33-AMINO ACID LINKER 1 (SEQ ID NO: 127)

  M-MLV TRUNCATED REVERSE TRANSCRIPTASE

  (SEQ ID NO: 766)

Peptide Tag
• The term “peptide tag” refers to a peptide amino acid sequence that is genetically fused to a protein sequence to impart one or more functions onto the proteins that facilitate the manipulation of the protein for various purposes, such as, visualization, purification, solubilization, and separation, etc. Peptide tags can include various types of tags categorized by purpose or function, which may include “affinity tags” (to facilitate protein purification), “solubilization tags” (to assist in proper folding of proteins), “chromatography tags” (to alter chromatographic properties of proteins), “epitope tags” (to bind to high affinity antibodies), “fluorescence tags” (to facilitate visualization of proteins in a cell or in vitro).
Polymerase
• As used herein, the term “polymerase” refers to an enzyme that synthesizes a nucleotide strand and which may be used in connection with the prime editor systems described herein. The polymerase can be a “template-dependent” polymerase (i.e., a polymerase which synthesizes a nucleotide strand based on the order of nucleotide bases of a template strand). The polymerase can also be a “template-independent” polymerase (i.e., a polymerase which synthesizes a nucleotide strand without the requirement of a template strand). A polymerase may also be further categorized as a “DNA polymerase” or an “RNA polymerase.” In various embodiments, the prime editor system comprises a DNA polymerase. In various embodiments, the DNA polymerase can be a “DNA-dependent DNA polymerase” (i.e., whereby the template molecule is a strand of DNA). In such cases, the DNA template molecule can be a PEgRNA, wherein the extension arm comprises a strand of DNA. In such cases, the PEgRNA may be referred to as a chimeric or hybrid PEgRNA which comprises an RNA portion (i.e., the guide RNA components, including the spacer and the gRNA core) and a DNA portion (i.e., the extension arm). In various other embodiments, the DNA polymerase can be an “RNA-dependent DNA polymerase” (i.e., whereby the template molecule is a strand of RNA). In such cases, the PEgRNA is RNA, i.e., including an RNA extension. The term “polymerase” may also refer to an enzyme that catalyzes the polymerization of nucleotide (i.e., the polymerase activity). Generally, the enzyme will initiate synthesis at the 3′-end of a primer annealed to a polynucleotide template sequence (e.g., such as a primer sequence annealed to the primer binding site of a PEgRNA), and will proceed toward the 5′ end of the template strand. A “DNA polymerase” catalyzes the polymerization of deoxynucleotides. As used herein in reference to a DNA polymerase, the term DNA polymerase includes a “functional fragment thereof”. A “functional fragment thereof” refers to any portion of a wild-type or mutant DNA polymerase that encompasses less than the entire amino acid sequence of the polymerase and which retains the ability, under at least one set of conditions, to catalyze the polymerization of a polynucleotide. Such a functional fragment may exist as a separate entity, or it may be a constituent of a larger polypeptide, such as a fusion protein.
Prime Editing
• As used herein, the term “prime editing” refers to a novel approach for gene editing using napDNAbps, a polymerase (e.g., a reverse transcriptase), and specialized guide RNAs that include a DNA synthesis template for encoding desired new genetic information (or deleting genetic information) that is then incorporated into a target DNA sequence. Certain embodiments of prime editing are described in the embodiments of FIGS. 1A-1H and FIG. 72(a)-72(c), among other figures.
• Prime editing represents an entirely new platform for genome editing that is a versatile and precise genome editing method that directly writes new genetic information into a specified DNA site using a nucleic acid programmable DNA binding protein (“napDNAbp”) working in association with a polymerase (i.e., in the form of a fusion protein or otherwise provided in trans with the napDNAbp), wherein the prime editing system is programmed with a prime editing (PE) guide RNA (“PEgRNA”) that both specifies the target site and templates the synthesis of the desired edit in the form of a replacement DNA strand by way of an extension (either DNA or RNA) engineered onto a guide RNA (e.g., at the 5′ or 3′ end, or at an internal portion of a guide RNA). The replacement strand containing the desired edit (e.g., a single nucleobase substitution) shares the same (or is homologous to) sequence as the endogenous strand (immediately downstream of the nick site) of the target site to be edited (with the exception that it includes the desired edit). Through DNA repair and/or replication machinery, the endogenous strand downstream of the nick site is replaced by the newly synthesized replacement strand containing the desired edit. In some cases, prime editing may be thought of as a “search-and-replace” genome editing technology since the prime editors, as described herein, not only search and locate the desired target site to be edited, but at the same time, encode a replacement strand containing a desired edit which is installed in place of the corresponding target site endogenous DNA strand. The prime editors of the present disclosure relate, in part, to the discovery that the mechanism of target-primed reverse transcription (TPRT) or “prime editing” can be leveraged or adapted for conducting precision CRISPR/Cas-based genome editing with high efficiency and genetic flexibility (e.g., as depicted in various embodiments of FIGS. 1A-1F). TPRT is naturally used by mobile DNA elements, such as mammalian non-LTR retrotransposons and bacterial Group II introns^28,29. The inventors have herein used Cas protein-reverse transcriptase fusions or related systems to target a specific DNA sequence with a guide RNA, generate a single strand nick at the target site, and use the nicked DNA as a primer for reverse transcription of an engineered reverse transcriptase template that is integrated with the guide RNA. However, while the concept begins with prime editors that use reverse transcriptase as the DNA polymerase component, the prime editors described herein are not limited to reverse transcriptases but may include the use of virtually any DNA polymerase. Indeed, while the application throughout may refer to prime editors with “reverse transcriptases,” it is set forth here that reverse transcriptases are only one type of DNA polymerase that may work with prime editing. Thus, where ever the specification mentions a “reverse transcriptase,” the person having ordinary skill in the art should appreciate that any suitable DNA polymerase may be used in place of the reverse transcriptase. Thus, in one aspect, the prime editors may comprise Cas9 (or an equivalent napDNAbp) which is programmed to target a DNA sequence by associating it with a specialized guide RNA (i.e., PEgRNA) containing a spacer sequence that anneals to a complementary protospacer in the target DNA. The specialized guide RNA also contains new genetic information in the form of an extension that encodes a replacement strand of DNA containing a desired genetic alteration which is used to replace a corresponding endogenous DNA strand at the target site. To transfer information from the PEgRNA to the target DNA, the mechanism of prime editing involves nicking the target site in one strand of the DNA to expose a 3′-hydroxyl group. The exposed 3′-hydroxyl group can then be used to prime the DNA polymerization of the edit-encoding extension on PEgRNA directly into the target site. In various embodiments, the extension—which provides the template for polymerization of the replacement strand containing the edit—can be formed from RNA or DNA. In the case of an RNA extension, the polymerase of the prime editor can be an RNA-dependent DNA polymerase (such as, a reverse transcriptase). In the case of a DNA extension, the polymerase of the prime editor may be a DNA-dependent DNA polymerase. The newly synthesized strand (i.e., the replacement DNA strand containing the desired edit) that is formed by the herein disclosed prime editors would be homologous to the genomic target sequence (i.e., have the same sequence as) except for the inclusion of a desired nucleotide change (e.g., a single nucleotide change, a deletion, or an insertion, or a combination thereof). The newly synthesized (or replacement) strand of DNA may also be referred to as a single strand DNA flap, which would compete for hybridization with the complementary homologous endogenous DNA strand, thereby displacing the corresponding endogenous strand. In certain embodiments, the system can be combined with the use of an error-prone reverse transcriptase enzyme (e.g., provided as a fusion protein with the Cas9 domain, or provided in trans to the Cas9 domain). The error-prone reverse transcriptase enzyme can introduce alterations during synthesis of the single strand DNA flap. Thus, in certain embodiments, error-prone reverse transcriptase can be utilized to introduce nucleotide changes to the target DNA. Depending on the error-prone reverse transcriptase that is used with the system, the changes can be random or non-random. Resolution of the hybridized intermediate (comprising the single strand DNA flap synthesized by the reverse transcriptase hybridized to the endogenous DNA strand) can include removal of the resulting displaced flap of endogenous DNA (e.g., with a 5′ end DNA flap endonuclease, FEN1), ligation of the synthesized single strand DNA flap to the target DNA, and assimilation of the desired nucleotide change as a result of cellular DNA repair and/or replication processes. Because templated DNA synthesis offers single nucleotide precision for the modification of any nucleotide, including insertions and deletions, the scope of this approach is very broad and could foreseeably be used for myriad applications in basic science and therapeutics.
• In various embodiments, prime editing operates by contacting a target DNA molecule (for which a change in the nucleotide sequence is desired to be introduced) with a nucleic acid programmable DNA binding protein (napDNAbp) complexed with a prime editing guide RNA (PEgRNA). In reference to FIG. 1G, the prime editing guide RNA (PEgRNA) comprises an extension at the 3′ or 5′ end of the guide RNA, or at an intramolecular location in the guide RNA and encodes the desired nucleotide change (e.g., single nucleotide change, insertion, or deletion). In step (a), the napDNAbp/extended gRNA complex contacts the DNA molecule and the extended gRNA guides the napDNAbp to bind to a target locus. In step (b), a nick in one of the strands of DNA of the target locus is introduced (e.g., by a nuclease or chemical agent), thereby creating an available 3′ end in one of the strands of the target locus. In certain embodiments, the nick is created in the strand of DNA that corresponds to the R-loop strand, i.e., the strand that is not hybridized to the guide RNA sequence, i.e., the “non-target strand.” The nick, however, could be introduced in either of the strands. That is, the nick could be introduced into the R-loop “target strand” (i.e., the strand hybridized to the protospacer of the extended gRNA) or the “non-target strand” (i.e., the strand forming the single-stranded portion of the R-loop and which is complementary to the target strand). In step (c), the 3′ end of the DNA strand (formed by the nick) interacts with the extended portion of the guide RNA in order to prime reverse transcription (i.e., “target-primed RT”). In certain embodiments, the 3′ end DNA strand hybridizes to a specific RT priming sequence on the extended portion of the guide RNA, i.e., the “reverse transcriptase priming sequence” or “primer binding site” on the PEgRNA. In step (d), a reverse transcriptase (or other suitable DNA polymerase) is introduced which synthesizes a single strand of DNA from the 3′ end of the primed site towards the 5′ end of the prime editing guide RNA. The DNA polymerase (e.g., reverse transcriptase) can be fused to the napDNAbp or alternatively can be provided in trans to the napDNAbp. This forms a single-strand DNA flap comprising the desired nucleotide change (e.g., the single base change, insertion, or deletion, or a combination thereof) and which is otherwise homologous to the endogenous DNA at or adjacent to the nick site. In step (e), the napDNAbp and guide RNA are released. Steps (f) and (g) relate to the resolution of the single strand DNA flap such that the desired nucleotide change becomes incorporated into the target locus. This process can be driven towards the desired product formation by removing the corresponding 5′ endogenous DNA flap that forms once the 3′ single strand DNA flap invades and hybridizes to the endogenous DNA sequence. Without being bound by theory, the cells endogenous DNA repair and replication processes resolves the mismatched DNA to incorporate the nucleotide change(s) to form the desired altered product. The process can also be driven towards product formation with “second strand nicking,” as exemplified in FIG. 1F. This process may introduce at least one or more of the following genetic changes: transversions, transitions, deletions, and insertions.
• The term “prime editor (PE) system” or “prime editor (PE)” or “PE system” or “PE editing system” refers the compositions involved in the method of genome editing using target-primed reverse transcription (TPRT) describe herein, including, but not limited to the napDNAbps, reverse transcriptases, fusion proteins (e.g., comprising napDNAbps and reverse transcriptases), prime editing guide RNAs, and complexes comprising fusion proteins and prime editing guide RNAs, as well as accessory elements, such as second strand nicking components (e.g., second strand sgRNAs) and 5′ endogenous DNA flap removal endonucleases (e.g., FEN1) for helping to drive the prime editing process towards the edited product formation.
• Although in the embodiments described thus far the PEgRNA constitutes a single molecule comprising a guide RNA (which itself comprises a spacer sequence and a gRNA core or scaffold) and a 5′ or 3′ extension arm comprising the primer binding site and a DNA synthesis template (e.g., see FIG. 3D, the PEgRNA may also take the form of two individual molecules comprised of a guide RNA and a trans prime editor RNA template (tPERT), which essentially houses the extension arm (including, in particular, the primer binding site and the DNA synthesis domain) and an RNA-protein recruitment domain (e.g., MS2 aptamer or hairpin) in the same molecule which becomes co-localized or recruited to a modified prime editor complex that comprises a tPERT recruiting protein (e.g., MS2cp protein, which binds to the MS2 aptamer). See FIG. 3G and FIG. 3H as an example of a tPERT that may be used with prime editing.
Prime Editor
• The term “prime editor” refers to the herein described fusion constructs comprising a napDNAbp (e.g., Cas9 nickase) and a reverse transcriptase and is capable of carrying out prime editing on a target nucleotide sequence in the presence of a PEgRNA (or “extended guide RNA”). The term “prime editor” may refer to the fusion protein or to the fusion protein complexed with a PEgRNA, and/or further complexed with a second-strand nicking sgRNA. In some embodiments, the prime editor may also refer to the complex comprising a fusion protein (reverse transcriptase fused to a napDNAbp), a PEgRNA, and a regular guide RNA capable of directing the second-site nicking step of the non-edited strand as described herein. In other embodiments, the reverse transcriptase component of the “primer editor” may be provided in trans.
Primer Binding Site
• The term “primer binding site” or “the PBS” refers to the nucleotide sequence located on a PEgRNA as component of the extension arm (typically at the 3′ end of the extension arm) and serves to bind to the primer sequence that is formed after Cas9 nicking of the target sequence by the prime editor. As detailed elsewhere, when the Cas9 nickase component of a prime editor nicks one strand of the target DNA sequence, a 3′-ended ssDNA flap is formed, which serves a primer sequence that anneals to the primer binding site on the PEgRNA to prime reverse transcription. FIGS. 27 and 28 show embodiments of the primer binding site located on a 3′ and 5′ extension arm, respectively.
Promoter
• The term “promoter” is art-recognized and refers to a nucleic acid molecule with a sequence recognized by the cellular transcription machinery and able to initiate transcription of a downstream gene. A promoter can be constitutively active, meaning that the promoter is always active in a given cellular context, or conditionally active, meaning that the promoter is only active in the presence of a specific condition. For example, a conditional promoter may only be active in the presence of a specific protein that connects a protein associated with a regulatory element in the promoter to the basic transcriptional machinery, or only in the absence of an inhibitory molecule. A subclass of conditionally active promoters are inducible promoters that require the presence of a small molecule “inducer” for activity. Examples of inducible promoters include, but are not limited to, arabinose-inducible promoters, Tet-on promoters, and tamoxifen-inducible promoters. A variety of constitutive, conditional, and inducible promoters are well known to the skilled artisan, and the skilled artisan will be able to ascertain a variety of such promoters useful in carrying out the instant invention, which is not limited in this respect.
Protospacer
• As used herein, the term “protospacer” refers to the sequence (˜20 bp) in DNA adjacent to the PAM (protospacer adjacent motif) sequence. The protospacer shares the same sequence as the spacer sequence of the guide RNA. The guide RNA anneals to the complement of the protospacer sequence on the target DNA (specifically, one strand thereof, i.e., the “target strand” versus the “non-target strand” of the target DNA sequence). In order for Cas9 to function it also requires a specific protospacer adjacent motif (PAM) that varies depending on the bacterial species of the Cas9 gene. The most commonly used Cas9 nuclease, derived from S. pyogenes, recognizes a PAM sequence of NGG that is found directly downstream of the target sequence in the genomic DNA, on the non-target strand. The skilled person will appreciate that the literature in the state of the art sometimes refers to the “protospacer” as the ˜20-nt target-specific guide sequence on the guide RNA itself, rather than referring to it as a “spacer.” Thus, in some cases, the term “protospacer” as used herein may be used interchangeably with the term “spacer.” The context of the description surrounding the appearance of either “protospacer” or “spacer” will help inform the reader as to whether the term is in reference to the gRNA or the DNA target.
Protospacer Adjacent Motif (PAM)
• As used herein, the term “protospacer adjacent sequence” or “PAM” refers to an approximately 2-6 base pair DNA sequence that is an important targeting component of a Cas9 nuclease. Typically, the PAM sequence is on either strand, and is downstream in the 5′ to 3′ direction of Cas9 cut site. The canonical PAM sequence (i.e., the PAM sequence that is associated with the Cas9 nuclease of Streptococcus pyogenes or SpCas9) is 5′-NGG-3′ wherein “N” is any nucleobase followed by two guanine (“G”) nucleobases. Different PAM sequences can be associated with different Cas9 nucleases or equivalent proteins from different organisms. In addition, any given Cas9 nuclease, e.g., SpCas9, may be modified to alter the PAM specificity of the nuclease such that the nuclease recognizes alternative PAM sequence.
• For example, with reference to the canonical SpCas9 amino acid sequence is SEQ ID NO: 18, the PAM sequence can be modified by introducing one or more mutations, including (a) D1135V, R1335Q, and T1337R “the VQR variant”, which alters the PAM specificity to NGAN or NGNG, (b) D1135E, R1335Q, and T1337R “the EQR variant”, which alters the PAM specificity to NGAG, and (c) D1135V, G1218R, R1335E, and T1337R “the VRER variant”, which alters the PAM specificity to NGCG. In addition, the D1135E variant of canonical SpCas9 still recognizes NGG, but it is more selective compared to the wild type SpCas9 protein.
• It will also be appreciated that Cas9 enzymes from different bacterial species (i.e., Cas9 orthologs) can have varying PAM specificities. For example, Cas9 from Staphylococcus aureus (SaCas9) recognizes NGRRT or NGRRN. In addition, Cas9 from Neisseria meningitis (NmCas) recognizes NNNNGATT. In another example, Cas9 from Streptococcus thermophilus (StCas9) recognizes NNAGAAW. In still another example, Cas9 from Treponema denticola (TdCas) recognizes NAAAAC. These are example are not meant to be limiting. It will be further appreciated that non-SpCas9s bind a variety of PAM sequences, which makes them useful when no suitable SpCas9 PAM sequence is present at the desired target cut site. Furthermore, non-SpCas9s may have other characteristics that make them more useful than SpCas9. For example, Cas9 from Staphylococcus aureus (SaCas9) is about 1 kilobase smaller than SpCas9, so it can be packaged into adeno-associated virus (AAV). Further reference may be made to Shah et al., “Protospacer recognition motifs: mixed identities and functional diversity,” RNA Biology, 10(5): 891-899 (which is incorporated herein by reference).
Recombinase
• The term “recombinase,” as used herein, refers to a site-specific enzyme that mediates the recombination of DNA between recombinase recognition sequences, which results in the excision, integration, inversion, or exchange (e.g., translocation) of DNA fragments between the recombinase recognition sequences. Recombinases can be classified into two distinct families: serine recombinases (e.g., resolvases and invertases) and tyrosine recombinases (e.g., integrases). Examples of serine recombinases include, without limitation, Hin, Gin, Tn3, β-six, CinH, ParA, γδ, Bxb1, φC31, TP901, TG1, φBT1, R4, φRV1, φFC1, MR11, A118, U153, and gp29. Examples of tyrosine recombinases include, without limitation, Cre, FLP, R, Lambda, HK101, HK022, and pSAM2. The serine and tyrosine recombinase names stem from the conserved nucleophilic amino acid residue that the recombinase uses to attack the DNA and which becomes covalently linked to the DNA during strand exchange. Recombinases have numerous applications, including the creation of gene knockouts/knock-ins and gene therapy applications. See, e.g., Brown et al., “Serine recombinases as tools for genome engineering.” Methods. 2011; 53(4):372-9; Hirano et al., “Site-specific recombinases as tools for heterologous gene integration.” Appl. Microbiol. Biotechnol. 2011; 92(2):227-39; Chavez and Calos, “Therapeutic applications of the ΦC31 integrase system.” Curr. Gene Ther. 2011; 11(5):375-81; Turan and Bode, “Site-specific recombinases: from tag-and-target-to tag-and-exchange-based genomic modifications.” FASEB J. 2011; 25(12):4088-107; Venken and Bellen, “Genome-wide manipulations of Drosophila melanogaster with transposons, Flp recombinase, and (I)C31 integrase.” Methods Mol. Biol. 2012; 859:203-28; Murphy, “Phage recombinases and their applications.” Adv. Virus Res. 2012; 83:367-414; Zhang et al., “Conditional gene manipulation: Cre-ating a new biological era.” J. Zhejiang Univ. Sci. B. 2012; 13(7):511-24; Karpenshif and Bernstein, “From yeast to mammals: recent advances in genetic control of homologous recombination.” DNA Repair (Amst). 2012; 1; 11(10):781-8; the entire contents of each are hereby incorporated by reference in their entirety. The recombinases provided herein are not meant to be exclusive examples of recombinases that can be used in embodiments of the invention. The methods and compositions of the invention can be expanded by mining databases for new orthogonal recombinases or designing synthetic recombinases with defined DNA specificities (See, e.g., Groth et al., “Phage integrases: biology and applications.” J. Mol. Biol. 2004; 335, 667-678; Gordley et al., “Synthesis of programmable integrases.” Proc. Natl. Acad. Sci. USA. 2009; 106, 5053-5058; the entire contents of each are hereby incorporated by reference in their entirety). Other examples of recombinases that are useful in the methods and compositions described herein are known to those of skill in the art, and any new recombinase that is discovered or generated is expected to be able to be used in the different embodiments of the invention. In some embodiments, the catalytic domains of a recombinase are fused to a nuclease-inactivated RNA-programmable nuclease (e.g., dCas9, or a fragment thereof), such that the recombinase domain does not comprise a nucleic acid binding domain or is unable to bind to a target nucleic acid (e.g., the recombinase domain is engineered such that it does not have specific DNA binding activity). Recombinases lacking DNA binding activity and methods for engineering such are known, and include those described by Klippel et al., “Isolation and characterisation of unusual gin mutants.” EMBO J. 1988; 7: 3983-3989: Burke et al., “Activating mutations of Tn3 resolvase marking interfaces important in recombination catalysis and its regulation. Mol Microbiol. 2004; 51: 937-948; Olorunniji et al., “Synapsis and catalysis by activated Tn3 resolvase mutants.” Nucleic Acids Res. 2008; 36: 7181-7191; Rowland et al., “Regulatory mutations in Sin recombinase support a structure-based model of the synaptosome.” Mol Microbiol. 2009; 74: 282-298; Akopian et al., “Chimeric recombinases with designed DNA sequence recognition.” Proc Natl Acad Sci USA. 2003; 100: 8688-8691; Gordley et al., “Evolution of programmable zinc finger-recombinases with activity in human cells. J Mol Biol. 2007; 367: 802-813; Gordley et al., “Synthesis of programmable integrases.” Proc Natl Acad Sci USA. 2009; 106: 5053-5058; Arnold et al., “Mutants of Tn3 resolvase which do not require accessory binding sites for recombination activity.” EMBO J. 1999; 18: 1407-1414; Gaj et al., “Structure-guided reprogramming of serine recombinase DNA sequence specificity.” Proc Natl Acad Sci USA. 2011; 108(2):498-503; and Proudfoot et al., “Zinc finger recombinases with adaptable DNA sequence specificity.” PLoS One. 2011; 6(4):e19537; the entire contents of each are hereby incorporated by reference. For example, serine recombinases of the resolvase-invertase group, e.g., Tn3 and γ6 resolvases and the Hin and Gin invertases, have modular structures with autonomous catalytic and DNA-binding domains (See, e.g., Grindley et al., “Mechanism of site-specific recombination.” Ann Rev Biochem. 2006; 75: 567-605, the entire contents of which are incorporated by reference). The catalytic domains of these recombinases are thus amenable to being recombined with nuclease-inactivated RNA-programmable nucleases (e.g., dCas9, or a fragment thereof) as described herein, e.g., following the isolation of ‘activated’ recombinase mutants which do not require any accessory factors (e.g., DNA binding activities) (See, e.g., Klippel et al., “Isolation and characterisation of unusual gin mutants.” EMBO J. 1988; 7: 3983-3989: Burke et al., “Activating mutations of Tn3 resolvase marking interfaces important in recombination catalysis and its regulation. Mol Microbiol. 2004; 51: 937-948; Olorunniji et al., “Synapsis and catalysis by activated Tn3 resolvase mutants.” Nucleic Acids Res. 2008; 36: 7181-7191; Rowland et al., “Regulatory mutations in Sin recombinase support a structure-based model of the synaptosome.” Mol Microbiol. 2009; 74: 282-298; Akopian et al., “Chimeric recombinases with designed DNA sequence recognition.” Proc Natl Acad Sci USA. 2003; 100: 8688-8691). Additionally, many other natural serine recombinases having an N-terminal catalytic domain and a C-terminal DNA binding domain are known (e.g., phiC31 integrase, TnpX transposase, IS607 transposase), and their catalytic domains can be co-opted to engineer programmable site-specific recombinases as described herein (See, e.g., Smith et al., “Diversity in the serine recombinases.” Mol Microbiol. 2002; 44: 299-307, the entire contents of which are incorporated by reference). Similarly, the core catalytic domains of tyrosine recombinases (e.g., Cre, λ integrase) are known, and can be similarly co-opted to engineer programmable site-specific recombinases as described herein (See, e.g., Guo et al., “Structure of Cre recombinase complexed with DNA in a site-specific recombination synapse.” Nature. 1997; 389:40-46; Hartung et al., “Cre mutants with altered DNA binding properties.” J Biol Chem 1998; 273:22884-22891; Shaikh et al., “Chimeras of the Flp and Cre recombinases: Tests of the mode of cleavage by Flp and Cre. J Mol Biol. 2000; 302:27-48; Rongrong et al., “Effect of deletion mutation on the recombination activity of Cre recombinase.” Acta Biochim Pol. 2005; 52:541-544; Kilbride et al., “Determinants of product topology in a hybrid Cre-Tn3 resolvase site-specific recombination system.” J Mol Biol. 2006; 355:185-195; Warren et al., “A chimeric cre recombinase with regulated directionality.” Proc Natl Acad Sci USA. 2008 105:18278-18283; Van Duyne, “Teaching Cre to follow directions.” Proc Natl Acad Sci USA. 2009 Jan. 6; 106(1):4-5; Numrych et al., “A comparison of the effects of single-base and triple-base changes in the integrase arm-type binding sites on the site-specific recombination of bacteriophage λ.” Nucleic Acids Res. 1990; 18:3953-3959; Tirumalai et al., “The recognition of core-type DNA sites by λ integrase.” J Mol Biol. 1998; 279:513-527; Aihara et al., “A conformational switch controls the DNA cleavage activity of λ integrase.” Mol Cell. 2003; 12:187-198; Biswas et al., “A structural basis for allosteric control of DNA recombination by λ integrase.” Nature. 2005; 435:1059-1066; and Warren et al., “Mutations in the amino-terminal domain of λ-integrase have differential effects on integrative and excisive recombination.” Mol Microbiol. 2005; 55:1104-1112; the entire contents of each are incorporated by reference).
Recombinase Recognition Sequence
• The term “recombinase recognition sequence”, or equivalently as “RRS” or “recombinase target sequence”, as used herein, refers to a nucleotide sequence target recognized by a recombinase and which undergoes strand exchange with another DNA molecule having a the RRS that results in excision, integration, inversion, or exchange of DNA fragments between the recombinase recognition sequences.
Recombine or Recombination
• The term “recombine,” or “recombination,” in the context of a nucleic acid modification (e.g., a genomic modification), is used to refer to the process by which two or more nucleic acid molecules, or two or more regions of a single nucleic acid molecule, are modified by the action of a recombinase protein (e.g., an inventive recombinase fusion protein provided herein). Recombination can result in, inter alia, the insertion, inversion, excision, or translocation of nucleic acids, e.g., in or between one or more nucleic acid molecules. recombinase recognition sequences
Reverse Transcriptase
• The term “reverse transcriptase” describes a class of polymerases characterized as RNA-dependent DNA polymerases. All known reverse transcriptases require a primer to synthesize a DNA transcript from an RNA template. Historically, reverse transcriptase has been used primarily to transcribe mRNA into cDNA which can then be cloned into a vector for further manipulation. Avian myoblastosis virus (AMV) reverse transcriptase was the first widely used RNA-dependent DNA polymerase (Verma, Biochim. Biophys. Acta 473:1 (1977)). The enzyme has 5′-3′ RNA-directed DNA polymerase activity, 5′-3′ DNA-directed DNA polymerase activity, and RNase H activity. RNase H is a processive 5′ and 3′ ribonuclease specific for the RNA strand for RNA-DNA hybrids (Perbal, A Practical Guide to Molecular Cloning, New York: Wiley & Sons (1984)). Errors in transcription cannot be corrected by reverse transcriptase because known viral reverse transcriptases lack the 3′-5′ exonuclease activity necessary for proofreading (Saunders and Saunders, Microbial Genetics Applied to Biotechnology, London: Croom Helm (1987)). A detailed study of the activity of AMV reverse transcriptase and its associated RNase H activity has been presented by Berger et al., Biochemistry 22:2365-2372 (1983). Another reverse transcriptase which is used extensively in molecular biology is reverse transcriptase originating from Moloney murine leukemia virus (M-MLV). See, e.g., Gerard, G. R., DNA 5:271-279 (1986) and Kotewicz, M. L., et al., Gene 35:249-258 (1985). M-MLV reverse transcriptase substantially lacking in RNase H activity has also been described. See, e.g., U.S. Pat. No. 5,244,797. The invention contemplates the use of any such reverse transcriptases, or variants or mutants thereof.
• In addition, the invention contemplates the use of reverse transcriptases which are error-prone, i.e., which may be referred to as error-prone reverse transcriptases or reverse transcriptases which do not support high fidelity incorporation of nucleotides during polymerization. During synthesis of the single-strand DNA flap based on the RT template integrated with the guide RNA, the error-prone reverse transcriptase can introduce one or more nucleotides which are mismatched with the RT template sequence, thereby introducing changes to the nucleotide sequence through erroneous polymerization of the single-strand DNA flap. These errors introduced during synthesis of the single strand DNA flap then become integrated into the double strand molecule through hybridization to the corresponding endogenous target strand, removal of the endogenous displaced strand, ligation, and then through one more round of endogenous DNA repair and/or sequencing processes.
Reverse Transcription
• As used herein, the term “reverse transcription” indicates the capability of enzyme to synthesize DNA strand (that is, complementary DNA or cDNA) using RNA as a template. In some embodiments, the reverse transcription can be “error-prone reverse transcription,” which refers to the properties of certain reverse transcriptase enzymes which are error-prone in their DNA polymerization activity.
PACE
• The term “phage-assisted continuous evolution (PACE),” as used herein, refers to continuous evolution that employs phage as viral vectors. The general concept of PACE technology has been described, for example, in International PCT Application, PCT/US2009/056194, filed Sep. 8, 2009, published as WO 2010/028347 on Mar. 11, 2010; International PCT Application, PCT/US2011/066747, filed Dec. 22, 2011, published as WO 2012/088381 on Jun. 28, 2012; U.S. application, U.S. Pat. No. 9,023,594, issued May 5, 2015, International PCT Application, PCT/US2015/012022, filed Jan. 20, 2015, published as WO 2015/134121 on Sep. 11, 2015, and International PCT Application, PCT/US2016/027795, filed Apr. 15, 2016, published as WO 2016/168631 on Oct. 20, 2016, the entire contents of each of which are incorporated herein by reference.
Phage
• The term “phage,” as used herein interchangeably with the term “bacteriophage,” refers to a virus that infects bacterial cells. Typically, phages consist of an outer protein capsid enclosing genetic material. The genetic material can be ssRNA, dsRNA, ssDNA, or dsDNA, in either linear or circular form. Phages and phage vectors are well known to those of skill in the art and non-limiting examples of phages that are useful for carrying out the PACE methods provided herein are λ (Lysogen), T2, T4, T7, T12, R17, M13, MS2, G4, P1, P2, P4, Phi X174, N4, Φ6, and Φ29. In certain embodiments, the phage utilized in the present invention is M13. Additional suitable phages and host cells will be apparent to those of skill in the art and the invention is not limited in this aspect. For an exemplary description of additional suitable phages and host cells, see Elizabeth Kutter and Alexander Sulakvelidze: Bacteriophages: Biology and Applications. CRC Press; 1st edition (December 2004), ISBN: 0849313368; Martha R. J. Clokie and Andrew M. Kropinski: Bacteriophages: Methods and Protocols, Volume 1: Isolation, Characterization, and Interactions (Methods in Molecular Biology) Humana Press; 1st edition (December, 2008), ISBN: 1588296822; Martha R. J. Clokie and Andrew M. Kropinski: Bacteriophages: Methods and Protocols, Volume 2: Molecular and Applied Aspects (Methods in Molecular Biology) Humana Press; 1st edition (December 2008), ISBN: 1603275649; all of which are incorporated herein in their entirety by reference for disclosure of suitable phages and host cells as well as methods and protocols for isolation, culture, and manipulation of such phages).
Protein, Peptide, and Polypeptide
• The terms “protein,” “peptide,” and “polypeptide” are used interchangeably herein, and refer to a polymer of amino acid residues linked together by peptide (amide) bonds. The terms refer to a protein, peptide, or polypeptide of any size, structure, or function. Typically, a protein, peptide, or polypeptide will be at least three amino acids long. A protein, peptide, or polypeptide may refer to an individual protein or a collection of proteins. One or more of the amino acids in a protein, peptide, or polypeptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc. A protein, peptide, or polypeptide may also be a single molecule or may be a multi-molecular complex. A protein, peptide, or polypeptide may be just a fragment of a naturally occurring protein or peptide. A protein, peptide, or polypeptide may be naturally occurring, recombinant, or synthetic, or any combination thereof. Any of the proteins provided herein may be produced by any method known in the art. For example, the proteins provided herein may be produced via recombinant protein expression and purification, which is especially suited for fusion proteins comprising a peptide linker. Methods for recombinant protein expression and purification are well known, and include those described by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)), the entire contents of which are incorporated herein by reference.
Protein Splicing
• The term “protein splicing,” as used herein, refers to a process in which a sequence, an intein (or split inteins, as the case may be), is excised from within an amino acid sequence, and the remaining fragments of the amino acid sequence, the exteins, are ligated via an amide bond to form a continuous amino acid sequence. The term “trans” protein splicing refers to the specific case where the inteins are split inteins and they are located on different proteins.
Second-Strand Nicking
• The resolution of heteroduplex DNA (i.e., containing one edited and one non-edited strand) formed as a result of prime editing determines long-term editing outcomes. In words, a goal of prime editing is to resolve the heteroduplex DNA (the edited strand paired with the endogenous non-edited strand) formed as an intermediate of PE by permanently integrating the edited strand into the complement, endogenous strand. The approach of “second-strand nicking” can be used herein to help drive the resolution of heteroduplex DNA in favor of permanent integration of the edited strand into the DNA molecule. As used herein, the concept of “second-strand nicking” refers to the introduction of a second nick at a location downstream of the first nick (i.e., the initial nick site that provides the free 3′ end for use in priming of the reverse transcriptase on the extended portion of the guide RNA), preferably on the unedited strand. In certain embodiments, the first nick and the second nick are on opposite strands. In other embodiments, the first nick and the second nick are on opposite strands. In yet another embodiment, the first nick is on the non-target strand (i.e., the strand that forms the single strand portion of the R-loop), and the second nick is on the target strand. In still other embodiments, the first nick is on the edited strand, and the second nick is on the unedited strand. The second nick can be positioned at least 5 nucleotides downstream of the first nick, or at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 or more nucleotides downstream of the first nick. The second nick, in certain embodiments, can be introduced between about 5-150 nucleotides on the unedited strand away from the site of the PEgRNA-induced nick, or between about 5-140, or between about 5-130, or between about 5-120, or between about 5-110, or between about 5-100, or between about 5-90, or between about 5-80, or between about 5-70, or between about 5-60, or between about 5-50, or between about 5-40, or between about 5-30, or between about 5-20, or between about 5-10. In one embodiment, the second nick is introduced between 14-116 nucleotides away from the PEgRNA-induced nick. Without being bound by theory, the second nick induces the cell's endogenous DNA repair and replication processes towards replacement or editing of the unedited strand, thereby permanently installing the edited sequence on both strands and resolving the heteroduplex that is formed as a result of PE. In some embodiments, the edited strand is the non-target strand and the unedited strand is the target strand. In other embodiments, the edited strand is the target strand, and the unedited strand is the non-target strand.
Sense Strand
• In genetics, a “sense” strand is the segment within double-stranded DNA that runs from 5′ to 3′, and which is complementary to the antisense strand of DNA, or template strand, which runs from 3′ to 5′. In the case of a DNA segment that encodes a protein, the sense strand is the strand of DNA that has the same sequence as the mRNA, which takes the antisense strand as its template during transcription, and eventually undergoes (typically, not always) translation into a protein. The antisense strand is thus responsible for the RNA that is later translated to protein, while the sense strand possesses a nearly identical makeup to that of the mRNA. Note that for each segment of dsDNA, there will possibly be two sets of sense and antisense, depending on which direction one reads (since sense and antisense is relative to perspective). It is ultimately the gene product, or mRNA, that dictates which strand of one segment of dsDNA is referred to as sense or antisense.
• In the context of a PEgRNA, the first step is the synthesis of a single-strand complementary DNA (i.e., the 3′ ssDNA flap, which becomes incorporated) oriented in the 5′ to 3′ direction which is templated off of the PEgRNA extension arm. Whether the 3′ ssDNA flap should be regarded as a sense or antisense strand depends on the direction of transcription since it well accepted that both strands of DNA may serve as a template for transcription (but not at the same time). Thus, in some embodiments, the 3′ ssDNA flap (which overall runs in the 5′ to 3′ direction) will serve as the sense strand because it is the coding strand. In other embodiments, the 3′ ssDNA flap (which overall runs in the 5′ to 3′ direction) will serve as the antisense strand and thus, the template for transcription.
Spacer Sequence
• As used herein, the term “spacer sequence” in connection with a guide RNA or a PEgRNA refers to the portion of the guide RNA or PEgRNA of about 20 nucleotides which contains a nucleotide sequence that is complementary to the protospacer sequence in the target DNA sequence. The spacer sequence anneals to the protospacer sequence to form a ssRNA/ssDNA hybrid structure at the target site and a corresponding R loop ssDNA structure of the endogenous DNA strand that is complementary to the protospacer sequence.
Subject
• The term “subject,” as used herein, refers to an individual organism, for example, an individual mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human mammal. In some embodiments, the subject is a non-human primate. In some embodiments, the subject is a rodent. In some embodiments, the subject is a sheep, a goat, a cattle, a cat, or a dog. In some embodiments, the subject is a vertebrate, an amphibian, a reptile, a fish, an insect, a fly, or a nematode. In some embodiments, the subject is a research animal. In some embodiments, the subject is genetically engineered, e.g., a genetically engineered non-human subject. The subject may be of either sex and at any stage of development.
Split Intein
• Although inteins are most frequently found as a contiguous domain, some exist in a naturally split form. In this case, the two fragments are expressed as separate polypeptides and must associate before splicing takes place, so-called protein trans-splicing.
• An exemplary split intein is the Ssp DnaE intein, which comprises two subunits, namely, DnaE-N and DnaE-C. The two different subunits are encoded by separate genes, namely dnaE-n and dnaE-c, which encode the DnaE-N and DnaE-C subunits, respectively. DnaE is a naturally occurring split intein in Synechocytis sp. PCC6803 and is capable of directing trans-splicing of two separate proteins, each comprising a fusion with either DnaE-N or DnaE-C.
• Additional naturally occurring or engineered split-intein sequences are known in the or can be made from whole-intein sequences described herein or those available in the art. Examples of split-intein sequences can be found in Stevens et al., “A promiscuous split intein with expanded protein engineering applications,” PNAS, 2017, Vol. 114: 8538-8543; Iwai et al., “Highly efficient protein trans-splicing by a naturally split DnaE intein from Nostoc punctiforme, FEBS Lett, 580: 1853-1858, each of which are incorporated herein by reference. Additional split intein sequences can be found, for example, in WO 2013/045632, WO 2014/055782, WO 2016/069774, and EP2877490, the contents each of which are incorporated herein by reference.
• In addition, protein splicing in trans has been described in vivo and in vitro (Shingledecker, et al., Gene 207:187 (1998), Southworth, et al., EMBO J. 17:918 (1998); Mills, et al., Proc. Natl. Acad. Sci. USA, 95:3543-3548 (1998); Lew, et al., J. Biol. Chem., 273:15887-15890 (1998); Wu, et al., Biochim. Biophys. Acta 35732:1 (1998b), Yamazaki, et al., J. Am. Chem. Soc. 120:5591 (1998), Evans, et al., J. Biol. Chem. 275:9091 (2000); Otomo, et al., Biochemistry 38:16040-16044 (1999); Otomo, et al., J. Biolmol. NMR 14:105-114 (1999); Scott, et al., Proc. Natl. Acad. Sci. USA 96:13638-13643 (1999)) and provides the opportunity to express a protein as to two inactive fragments that subsequently undergo ligation to form a functional product, e.g., as shown in FIGS. 66 and 67 with regard to the formation of a complete PE fusion protein from two separately-expressed halves.
Target Site
• The term “target site” refers to a sequence within a nucleic acid molecule that is edited by a prime editor (PE) disclosed herein. The target site further refers to the sequence within a nucleic acid molecule to which a complex of the prime editor (PE) and gRNA binds.
• tPERT
• See definition for “trans prime editor RNA template (tPERT).”
Temporal Second-Strand Nicking
• As used herein, the term “temporal second-strand nicking” refers to a variant of second strand nicking whereby the installation of the second nick in the unedited strand occurs only after the desired edit is installed in the edited strand. This avoids concurrent nicks on both strands that could lead to double-stranded DNA breaks. The second-strand nicking guide RNA is designed for temporal control such that the second strand nick is not introduced until after the installation of the desired edit. This is achieved by designing a gRNA with a spacer sequence that matches only the edited strand, but not the original allele. Using this strategy, mismatches between the protospacer and the unedited allele should disfavor nicking by the sgRNA until after the editing event on the PAM strand takes place.
Trans Prime Editing
• As used herein, the term “trans prime editing” refers to a modified form of prime editing that utilizes a split PEgRNA, i.e., wherein the PEgRNA is separated into two separate molecules: an sgRNA and a trans prime editing RNA template (tPERT). The sgRNA serves to target the prime editor (or more generally, to target the napDNAbp component of the prime editor) to the desired genomic target site, while the tPERT is used by the polymerase (e.g., a reverse transcriptase) to write new DNA sequence into the target locus once the tPERT is recruited in trans to the prime editor by the interaction of binding domains located on the prime editor and on the tPERT. In one embodiment, the binding domains can include RNA-protein recruitment moieties, such as a MS2 aptamer located on the tPERT and an MS2cp protein fused to the prime editor. An advantage of trans prime editing is that by separating the DNA synthesis template from the guide RNA, one can potentially use longer length templates.
• An embodiment of trans prime editing is shown in FIGS. 3G and 3H. FIG. 3G shows the composition of the trans prime editor complex on the left (“RP-PE:gRNA complex), which comprises an napDNAbp fused to each of a polymerase (e.g., a reverse transcriptase) and a rPERT recruiting protein (e.g., MS2sc), and which is complexed with a guide RNA.
• FIG. 3G further shows a separate tPERT molecule, which comprises the extension arm features of a PEgRNA, including the DNA synthesis template and the primer binding sequence. The tPERT molecule also includes an RNA-protein recruitment domain (which, in this case, is a stem loop structure and can be, for example, MS2 aptamer). As depicted in the process described in FIG. 3H, the RP-PE:gRNA complex binds to and nicks the target DNA sequence. Then, the recruiting protein (RP) recruits a tPERT to co-localize to the prime editor complex bound to the DNA target site, thereby allowing the primer binding site to bind to the primer sequence on the nicked strand, and subsequently, allowing the polymerase (e.g., RT) to synthesize a single strand of DNA against the DNA synthesis template up through the 5′ of the tPERT.
• While the tPERT is shown in FIG. 3G and FIG. 3H as comprising the PBS and DNA synthesis template on the 5′ end of the RNA-protein recruitment domain, the tPERT in other configurations may be designed with the PBS and DNA synthesis template located on the 3′ end of the RNA-protein recruitment domain. However, the tPERT with the 5′ extension has the advantage that synthesis of the single strand of DNA will naturally terminate at the 5′ end of the tPERT and thus, does not risk using any portion of the RNA-protein recruitment domain as a template during the DNA synthesis stage of prime editing.
• Trans Prime Editor RNA Template (tPERT)
• As used herein, a “trans prime editor RNA template (tPERT)” refers to a component used in trans prime editing, a modified version of prime editing which operates by separating the PEgRNA into two distinct molecules: a guide RNA and a tPERT molecule. The tPERT molecule is programmed to co-localize with the prime editor complex at a target DNA site, bringing the primer binding site and the DNA synthesis template to the prime editor in trans. For example, see FIG. 3G for an embodiment of a trans prime editor (tPE) which shows a two-component system comprising (1) an RP-PE:gRNA complex and (2) a tPERT that includes the primer binding site and the DNA synthesis template joined to an RNA-protein recruitment domain, wherein the RP (recruiting protein) component of the RP-PE:gRNA complex recruits the tPERT to a target site to be edited, thereby associating the PBS and DNA synthesis template with the prime editor in trans. Said another way, the tPERT is engineered to contain (all or part of) the extension arm of a PEgRNA, which includes the primer binding site and the DNA synthesis template.
Transitions
• As used herein, “transitions” refer to the interchange of purine nucleobases (A↔G) or the interchange of pyrimidine nucleobases (C↔T). This class of interchanges involves nucleobases of similar shape. The compositions and methods disclosed herein are capable of inducing one or more transitions in a target DNA molecule. The compositions and methods disclosed herein are also capable of inducing both transitions and transversion in the same target DNA molecule. These changes involve A↔G, G↔A, C↔T, or T↔C. In the context of a double-strand DNA with Watson-Crick paired nucleobases, transversions refer to the following base pair exchanges: A:T↔G:C, G:G↔A:T, C:G↔T:A, or T:A↔C:G. The compositions and methods disclosed herein are capable of inducing one or more transitions in a target DNA molecule. The compositions and methods disclosed herein are also capable of inducing both transitions and transversion in the same target DNA molecule, as well as other nucleotide changes, including deletions and insertions.
Transversions
• As used herein, “transversions” refer to the interchange of purine nucleobases for pyrimidine nucleobases, or in the reverse and thus, involve the interchange of nucleobases with dissimilar shape. These changes involve T↔A, T↔G, C↔G, C↔A, A↔T, A↔C, G↔C, and G↔T. In the context of a double-strand DNA with Watson-Crick paired nucleobases, transversions refer to the following base pair exchanges: T:A↔A:T, T:A↔G:C, C:G↔G:C, C:G↔A:T, A:T↔T:A, A:T↔C:G, G:C↔C:G, and G:C↔T:A. The compositions and methods disclosed herein are capable of inducing one or more transversions in a target DNA molecule. The compositions and methods disclosed herein are also capable of inducing both transitions and transversion in the same target DNA molecule, as well as other nucleotide changes, including deletions and insertions.
Treatment
• The terms “treatment,” “treat,” and “treating,” refer to a clinical intervention aimed to reverse, alleviate, delay the onset of, or inhibit the progress of a disease or disorder, or one or more symptoms thereof, as described herein. As used herein, the terms “treatment,” “treat,” and “treating” refer to a clinical intervention aimed to reverse, alleviate, delay the onset of, or inhibit the progress of a disease or disorder, or one or more symptoms thereof, as described herein. In some embodiments, treatment may be administered after one or more symptoms have developed and/or after a disease has been diagnosed. In other embodiments, treatment may be administered in the absence of symptoms, e.g., to prevent or delay onset of a symptom or inhibit onset or progression of a disease. For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and/or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example, to prevent or delay their recurrence.
Trinucleotide Repeat Disorder
• As used herein, a “trinucleotide repeat disorder” (or alternatively, “expansion repeat disorder” or “repeat expansion disorder”) refers to a set of genetic disorders which are cause by “trinucleotide repeat expansion,” which is a kind of mutation where a certain trinucleotide repeats in certain genes or introns. Trinucleotide repeats were once thought to be commonplace iterations in the genome, but the 1990s clarified these disorders. These apparently ‘benign’ stretches of DNA can sometimes expand and cause disease. Several defining features are shared amongst disorders caused by trinucleotide repeat expansions. First, the mutant repeats show both somatic and germline instability and, more frequently, they expand rather than contract in successive transmissions. Secondly, an earlier age of onset and increasing severity of phenotype in subsequent generations (anticipation) generally are correlated with larger repeat length. Finally, the parental origin of the disease allele can often influence anticipation, with paternal transmissions carrying a greater risk of expansion for many of these disorders.
• Triplet expansion is thought to be caused by slippage during DNA replication. Due to the repetitive nature of the DNA sequence in these regions ‘loop out’ structures may form during DNA replication while maintaining complementary base pairing between the parent strand and daughter strand being synthesized. If the loop out structure is formed from sequence on the daughter strand this will result in an increase in the number of repeats. However, if the loop out structure is formed on the parent strand a decrease in the number of repeats occurs. It appears that expansion of these repeats is more common than reduction. Generally the larger the expansion the more likely they are to cause disease or increase the severity of disease. This property results in the characteristic of anticipation seen in trinucleotide repeat disorders. Anticipation describes the tendency of age of onset to decrease and severity of symptoms to increase through successive generations of an affected family due to the expansion of these repeats.
• Nucleotide repeat disorders may include those in which the triplet repeat occurs in a non-coding region (i.e., a non-coding trinucleotide repeat disorder) or in a coding region
• The prime editor (PE) system described herein may use to treat nucleotide repeat disorders, which may include fragile X syndrome (FRAXA), fragile XE MR (FRAXE), Freidreich ataxia (FRDA), myotonic dystrophy (DM), spinocerebellar ataxia type 8 (SCA8), and spinocerebellar ataxia type 12 (SCA12), among others.
Upstream
• As used herein, the terms “upstream” and “downstream” are terms of relativity that define the linear position of at least two elements located in a nucleic acid molecule (whether single or double-stranded) that is orientated in a 5′-to-3′ direction. In particular, a first element is upstream of a second element in a nucleic acid molecule where the first element is positioned somewhere that is 5′ to the second element. For example, a SNP is upstream of a Cas9-induced nick site if the SNP is on the 5′ side of the nick site. Conversely, a first element is downstream of a second element in a nucleic acid molecule where the first element is positioned somewhere that is 3′ to the second element. For example, a SNP is downstream of a Cas9-induced nick site if the SNP is on the 3′ side of the nick site. The nucleic acid molecule can be a DNA (double or single stranded). RNA (double or single stranded), or a hybrid of DNA and RNA. The analysis is the same for single strand nucleic acid molecule and a double strand molecule since the terms upstream and downstream are in reference to only a single strand of a nucleic acid molecule, except that one needs to select which strand of the double stranded molecule is being considered. Often, the strand of a double stranded DNA which can be used to determine the positional relativity of at least two elements is the “sense” or “coding” strand. In genetics, a “sense” strand is the segment within double-stranded DNA that runs from 5′ to 3′, and which is complementary to the antisense strand of DNA, or template strand, which runs from 3′ to 5′. Thus, as an example, a SNP nucleobase is “downstream” of a promoter sequence in a genomic DNA (which is double-stranded) if the SNP nucleobase is on the 3′ side of the promoter on the sense or coding strand.
Variant
• As used herein the term “variant” should be taken to mean the exhibition of qualities that have a pattern that deviates from what occurs in nature, e.g., a variant Cas9 is a Cas9 comprising one or more changes in amino acid residues as compared to a wild type Cas9 amino acid sequence. The term “variant” encompasses homologous proteins having at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 99% percent identity with a reference sequence and having the same or substantially the same functional activity or activities as the reference sequence. The term also encompasses mutants, truncations, or domains of a reference sequence, and which display the same or substantially the same functional activity or activities as the reference sequence.
Vector
• The term “vector,” as used herein, refers to a nucleic acid that can be modified to encode a gene of interest and that is able to enter into a host cell, mutate and replicate within the host cell, and then transfer a replicated form of the vector into another host cell. Exemplary suitable vectors include viral vectors, such as retroviral vectors or bacteriophages and filamentous phage, and conjugative plasmids. Additional suitable vectors will be apparent to those of skill in the art based on the instant disclosure.
Wild Type
• As used herein the term “wild type” is a term of the art understood by skilled persons and means the typical form of an organism, strain, gene or characteristic as it occurs in nature as distinguished from mutant or variant forms.
5′ Endogenous DNA Flap
• As used herein, the term “5′ endogenous DNA flap” refers to the strand of DNA situated immediately downstream of the PE-induced nick site in the target DNA. The nicking of the target DNA strand by PE exposes a 3′ hydroxyl group on the upstream side of the nick site and a 5′ hydroxyl group on the downstream side of the nick site. The endogenous strand ending in the 3′ hydroxyl group is used to prime the DNA polymerase of the prime editor (e.g., wherein the DNA polymerase is a reverse transcriptase). The endogenous strand on the downstream side of the nick site and which begins with the exposed 5′ hydroxyl group is referred to as the “5′ endogenous DNA flap” and is ultimately removed and replaced by the newly synthesized replacement strand (i.e., “3′ replacement DNA flap”) the encoded by the extension of the PEgRNA.
5′ Endogenous DNA Flap Removal
• As used herein, the term “5′ endogenous DNA flap removal” or “5′ flap removal” refers to the removal of the 5′ endogenous DNA flap that forms when the RT-synthesized single-strand DNA flap competitively invades and hybridizes to the endogenous DNA, displacing the endogenous strand in the process. Removing this endogenous displaced strand can drive the reaction towards the formation of the desired product comprising the desired nucleotide change. The cell's own DNA repair enzymes may catalyze the removal or excision of the 5′ endogenous flap (e.g., a flap endonuclease, such as EXO1 or FEN1). Also, host cells may be transformed to express one or more enzymes that catalyze the removal of said 5′ endogenous flaps, thereby driving the process toward product formation (e.g., a flap endonuclease). Flap endonucleases are known in the art and can be found described in Patel et al., “Flap endonucleases pass 5′-flaps through a flexible arch using a disorder-thread-order mechanism to confer specificity for free 5′-ends,” Nucleic Acids Research, 2012, 40(10): 4507-4519 and Tsutakawa et al., “Human flap endonuclease structures, DNA double-base flipping, and a unified understanding of the FEN1 superfamily,” Cell, 2011, 145(2): 198-211 (each of which are incorporated herein by reference).
3′ Replacement DNA Flap
• As used herein, the term “3′ replacement DNA flap” or simply, “replacement DNA flap,” refers to the strand of DNA that is synthesized by the prime editor and which is encoded by the extension arm of the prime editor PEgRNA. More in particular, the 3′ replacement DNA flap is encoded by the polymerase template of the PEgRNA. The 3′ replacement DNA flap comprises the same sequence as the 5′ endogenous DNA flap except that it also contains the edited sequence (e.g., single nucleotide change). The 3′ replacement DNA flap anneals to the target DNA, displacing or replacing the 5′ endogenous DNA flap (which can be excised, for example, by a 5′ flap endonuclease, such as FEN1 or EXO1) and then is ligated to join the 3′ end of the 3′ replacement DNA flap to the exposed 5′ hydroxyl end of endogenous DNA (exposed after excision of the 5′ endogenous DNA flap, thereby reforming a phosphodiester bond and installing the 3′ replacement DNA flap to form a heteroduplex DNA containing one edited strand and one unedited strand. DNA repair processes resolve the heteroduplex by copying the information in the edited strand to the complementary strand permanently installs the edit in to the DNA. This resolution process can be driven further to completion by nicking the unedited strand, i.e., by way of “second-strand nicking,” as described herein.
DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS
• Adoption of the clustered regularly interspaced short palindromic repeat (CRISPR) system for genome editing has revolutionized the life sciences^1-3. Although gene disruption using CRISPR is now routine, the precise installation of single nucleotide edits remains a major challenge, despite being necessary for studying or correcting a large number of disease-causative mutations. Homology directed repair (HDR) is capable of achieving such edits, but suffers from low efficiency (often <5%), a requirement for donor DNA repair templates, and deleterious effects of double-stranded DNA break (DSB) formation. Recently, Prof. David Liu et al.'s laboratory developed base editing, which achieves efficient single nucleotide editing without DSBs. Base editors (BEs) combine the CRISPR system with base-modifying deaminase enzymes to convert target C●G or A●T base pairs to A●T or G●C, respectively^4-6. Although already widely used by researchers worldwide, current BEs enable only four of the twelve possible base pair conversions and are unable to correct small insertions or deletions. Moreover, the targeting scope of base editing is limited by the editing of non-target C or A bases adjacent to the target base (“bystander editing”) and by the requirement that a PAM sequence exist 15±2 bp from the target base. Overcoming these limitations would therefore greatly broaden the basic research and therapeutic applications of genome editing.
• The present disclosure proposes a new precision editing approach that offers many of the benefits of base editing—namely, avoidance of double strand breaks and donor DNA repair templates—while overcoming its major limitations. The proposed approach described herein achieves the direct installation of edited DNA strands at target genomic sites using target-primed reverse transcription (TPRT). In the design discussed herein, CRISPR guide RNA (gRNA) will be engineered to carry a reverse transcriptase (RT) template sequence encoding a single-stranded DNA comprising a desired nucleotide change. The CRISPR nuclease (Cas9)-nicked target site DNA will serve as the primer for reverse transcription of the template sequence on the modified gRNA, allowing for direct incorporation of any desired nucleotide edit.
• Accordingly, the present invention relates in part to the discovery that the mechanism of target-primed reverse transcription (TPRT) can be leveraged or adapted for conducting precision CRISPR/Cas-based genome editing with high efficiency and genetic flexibility (e.g., as depicted in various embodiments of FIGS. 1A-1F). The inventors have proposed herein to use Cas protein-reverse transcriptase fusions to target a specific DNA sequence with a modified guide RNA (“an extended guide RNA”), generate a single strand nick at the target site, and use the nicked DNA as a primer for reverse transcription of an engineered reverse transcriptase template that is integrated into the extended guide RNA. The newly synthesized strand would be homologous to the genomic target sequence except for the inclusion of a desired nucleotide change (e.g., a single nucleotide change, a deletion, or an insertion, or a combination thereof). The newly synthesize strand of DNA may be referred to as a single strand DNA flap, which would compete for hybridization with the complementary homologous endogenous DNA strand, thereby displacing the corresponding endogenous strand. Resolution of this hybridized intermediate can include removal of the resulting displaced flap of endogenous DNA (e.g., with a 5′ end DNA flap endonuclease, FEN1), ligation of the synthesized single strand DNA flap to the target DNA, and assimilation of the desired nucleotide change as a result of cellular DNA repair and/or replication processes. Because templated DNA synthesis offers single nucleotide precision, the scope of this approach is very broad and could foreseeably be used for myriad applications in basic science and therapeutics.
• [1] napDNAbp
• The prime editors and trans prime editors described herein may comprise a nucleic acid programmable DNA binding protein (napDNAbp).
• In one aspect, a napDNAbp can be associated with or complexed with at least one guide nucleic acid (e.g., guide RNA or a PEgRNA), which localizes the napDNAbp to a DNA sequence that comprises a DNA strand (i.e., a target strand) that is complementary to the guide nucleic acid, or a portion thereof (e.g., the spacer of a guide RNA which anneals to the protospacer of the DNA target). In other words, the guide nucleic-acid “programs” the napDNAbp (e.g., Cas9 or equivalent) to localize and bind to complementary sequence of the protospacer in the DNA.
• Any suitable napDNAbp may be used in the prime editors described herein. In various embodiments, the napDNAbp may be any Class 2 CRISPR-Cas system, including any type II, type V, or type VI CRISPR-Cas enzyme. Given the rapid development of CRISPR-Cas as a tool for genome editing, there have been constant developments in the nomenclature used to describe and/or identify CRISPR-Cas enzymes, such as Cas9 and Cas9 orthologs. This application references CRISPR-Cas enzymes with nomenclature that may be old and/or new. The skilled person will be able to identify the specific CRISPR-Cas enzyme being referenced in this Application based on the nomenclature that is used, whether it is old (i.e., “legacy”) or new nomenclature. CRISPR-Cas nomenclature is extensively discussed in Makarova et al., “Classification and Nomenclature of CRISPR-Cas Systems: Where from Here?,” The CRISPR Journal, Vol. 1. No. 5, 2018, the entire contents of which are incorporated herein by reference. The particular CRISPR-Cas nomenclature used in any given instance in this Application is not limiting in any way and the skilled person will be able to identify which CRISPR-Cas enzyme is being referenced.
• For example, the following type II, type V, and type VI Class 2 CRISPR-Cas enzymes have the following art-recognized old (i.e., legacy) and new names. Each of these enzymes, and/or variants thereof, may be used with the prime editors described herein:

• 	Legacy nomenclature	Current nomenclature*
  	type H CRISPR-Cas enzymes
  	Cas9	same
  	type V CRISPR-Cas enzymes
  	Cpfl	Cas12a
  	CasX	Cas12e
  	C2c1	Cas12b1
  	Cas12b2	same
  	C2c3	Cas12c
  	CasY	Cas12d
  	C2c4	same
  	C2c8	same
  	C2c5	same
  	C2c10	same
  	C2c9	same
  	type VI CRISPR-Cas enzymes
  	C2c2	Cas13a
  	Cas13d	same
  	C2c7	Cas13c
  	C2c6	Cas13b
  	*See Makarova et al., The CRISPR Journal, Vol. 1, No. 5, 2018

• Without being bound by theory, the mechanism of action of certain napDNAbp contemplated herein includes the step of forming an R-loop whereby the napDNAbp induces the unwinding of a double-strand DNA target, thereby separating the strands in the region bound by the napDNAbp. The guide RNA spacer then hybridizes to the “target strand” at the protospacer sequence. This displaces a “non-target strand” that is complementary to the target strand, which forms the single strand region of the R-loop. In some embodiments, the napDNAbp includes one or more nuclease activities, which then cut the DNA leaving various types of lesions. For example, the napDNAbp may comprises a nuclease activity that cuts the non-target strand at a first location, and/or cuts the target strand at a second location. Depending on the nuclease activity, the target DNA can be cut to form a “double-stranded break” whereby both strands are cut. In other embodiments, the target DNA can be cut at only a single site, i.e., the DNA is “nicked” on one strand. Exemplary napDNAbp with different nuclease activities include “Cas9 nickase” (“nCas9”) and a deactivated Cas9 having no nuclease activities (“dead Cas9” or “dCas9”).
• The below description of various napDNAbps which can be used in connection with the presently disclose prime editors is not meant to be limiting in any way. The prime editors may comprise the canonical SpCas9, or any ortholog Cas9 protein, or any variant Cas9 protein—including any naturally occurring variant, mutant, or otherwise engineered version of Cas9—that is known or which can be made or evolved through a directed evolutionary or otherwise mutagenic process. In various embodiments, the Cas9 or Cas9 variants have a nickase activity, i.e., only cleave of strand of the target DNA sequence. In other embodiments, the Cas9 or Cas9 variants have inactive nucleases, i.e., are “dead” Cas9 proteins. Other variant Cas9 proteins that may be used are those having a smaller molecular weight than the canonical SpCas9 (e.g., for easier delivery) or having modified or rearranged primary amino acid structure (e.g., the circular permutant formats).
• The prime editors described herein may also comprise Cas9 equivalents, including Cas12a (Cpf1) and Cas12b1 proteins which are the result of convergent evolution. The napDNAbps used herein (e.g., SpCas9, Cas9 variant, or Cas9 equivalents) may also may also contain various modifications that alter/enhance their PAM specifities. Lastly, the application contemplates any Cas9, Cas9 variant, or Cas9 equivalent which has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.9% sequence identity to a reference Cas9 sequence, such as a references SpCas9 canonical sequence or a reference Cas9 equivalent (e.g., Cas12a (Cpf1)).
• The napDNAbp can be a CRISPR (clustered regularly interspaced short palindromic repeat)-associated nuclease. As outlined above, CRISPR is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements and conjugative plasmids). CRISPR clusters contain spacers, sequences complementary to antecedent mobile elements, and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems correct processing of pre-crRNA requires a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc) and a Cas9 protein. The tracrRNA serves as a guide for ribonuclease 3-aided processing of pre-crRNA. Subsequently, Cas9/crRNA/tracrRNA endonucleolytically cleaves linear or circular dsDNA target complementary to the spacer. The target strand not complementary to crRNA is first cut endonucleolytically, then trimmed 3′-5′ exonucleolytically. In nature, DNA-binding and cleavage typically requires protein and both RNAs. However, single guide RNAs (“sgRNA”, or simply “gRNA”) can be engineered so as to incorporate aspects of both the crRNA and tracrRNA into a single RNA species. See, e.g., Jinek M. et al., Science 337:816-821(2012), the entire contents of which is hereby incorporated by reference.
• In some embodiments, the napDNAbp directs cleavage of one or both strands at the location of a target sequence, such as within the target sequence and/or within the complement of the target sequence. In some embodiments, the napDNAbp directs cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of a target sequence. In some embodiments, a vector encodes a napDNAbp that is mutated to with respect to a corresponding wild-type enzyme such that the mutated napDNAbp lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence. For example, an aspartate-to-alanine substitution (D10A) in the RuvC I catalytic domain of Cas9 from S. pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (cleaves a single strand). Other examples of mutations that render Cas9 a nickase include, without limitation, H840A, N854A, and N863A in reference to the canonical SpCas9 sequence, or to equivalent amino acid positions in other Cas9 variants or Cas9 equivalents.
• As used herein, the term “Cas protein” refers to a full-length Cas protein obtained from nature, a recombinant Cas protein having a sequences that differs from a naturally occurring Cas protein, or any fragment of a Cas protein that nevertheless retains all or a significant amount of the requisite basic functions needed for the disclosed methods, i.e., (i) possession of nucleic-acid programmable binding of the Cas protein to a target DNA, and (ii) ability to nick the target DNA sequence on one strand. The Cas proteins contemplated herein embrace CRISPR Cas 9 proteins, as well as Cas9 equivalents, variants (e.g., Cas9 nickase (nCas9) or nuclease inactive Cas9 (dCas9)) homologs, orthologs, or paralogs, whether naturally occurring or non-naturally occurring (e.g., engineered or recombinant), and may include a Cas9 equivalent from any Class 2 CRISPR system (e.g., type II, V, VI), including Cas12a (Cpf1), Cas12e (CasX), Cas12b1 (C2c1), Cas12b2, Cas12c (C2c3), C2c4, C2c8, C2c5, C2c10, C2c9 Cas13a (C2c2), Cas13d, Cas13c (C2c7), Cas13b (C2c6), and Cas13b. Further Cas-equivalents are described in Makarova et al., “C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector,” Science 2016; 353(6299) and Makarova et al., “Classification and Nomenclature of CRISPR-Cas Systems: Where from Here?,” The CRISPR Journal, Vol. 1. No. 5, 2018, the contents of which are incorporated herein by reference.
• The terms “Cas9” or “Cas9 nuclease” or “Cas9 moiety” or “Cas9 domain” embrace any naturally occurring Cas9 from any organism, any naturally-occurring Cas9 equivalent or functional fragment thereof, any Cas9 homolog, ortholog, or paralog from any organism, and any mutant or variant of a Cas9, naturally-occurring or engineered. The term Cas9 is not meant to be particularly limiting and may be referred to as a “Cas9 or equivalent.” Exemplary Cas9 proteins are further described herein and/or are described in the art and are incorporated herein by reference. The present disclosure is unlimited with regard to the particular Cas9 that is employed in the prime editor (PE) of the invention.
• As noted herein, Cas9 nuclease sequences and structures are well known to those of skill in the art (see, e.g., “Complete genome sequence of an M1 strain of Streptococcus pyogenes.” Ferretti et al., J. J., McShan W. M., Ajdic D. J., Savic D. J., Savic G., Lyon K., Primeaux C., Sezate S., Suvorov A. N., Kenton S., Lai H. S., Lin S. P., Qian Y., Jia H. G., Najar F. Z., Ren Q., Zhu H., Song L., White J., Yuan X., Clifton S. W., Roe B. A., McLaughlin R. E., Proc. Natl. Acad. Sci. U.S.A. 98:4658-4663(2001); “CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.” Deltcheva E., Chylinski K., Sharma C. M., Gonzales K., Chao Y., Pirzada Z. A., Eckert M. R., Vogel J., Charpentier E., Nature 471:602-607(2011); and “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.” Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J. A., Charpentier E. Science 337:816-821(2012), the entire contents of each of which are incorporated herein by reference).
• Examples of Cas9 and Cas9 equivalents are provided as follows; however, these specific examples are not meant to be limiting. The primer editor of the present disclosure may use any suitable napDNAbp, including any suitable Cas9 or Cas9 equivalent.
• A. Wild Type Canonical SpCas9
• In one embodiment, the primer editor constructs described herein may comprise the “canonical SpCas9” nuclease from S. pyogenes, which has been widely used as a tool for genome engineering and is categorized as the type II subgroup of enzymes of the Class 2 CRISPR-Cas systems. This Cas9 protein is a large, multi-domain protein containing two distinct nuclease domains. Point mutations can be introduced into Cas9 to abolish one or both nuclease activities, resulting in a nickase Cas9 (nCas9) or dead Cas9 (dCas9), respectively, that still retains its ability to bind DNA in a sgRNA-programmed manner. In principle, when fused to another protein or domain, Cas9 or variant thereof (e.g., nCas9) can target that protein to virtually any DNA sequence simply by co-expression with an appropriate sgRNA. As used herein, the canonical SpCas9 protein refers to the wild type protein from Streptococcus pyogenes having the following amino acid sequence:

• Description	Sequence	SEQ ID NO:
  SpCas9	MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGN	SEQ ID NO:
  Streptococcus	TDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRR		18
  pyogenes	KNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKH
  M1	ERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADL
  SwissProt	RLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQ
  Accession	TYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQL
  No.	PGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLS
  Q99ZW2	KDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDI
  Wild type	LRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQL
  	PEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEK
  	MDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELH
  	AILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
  	SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTN
  	FDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGM
  	RKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKI
  	ECFDSVETSGVEDRFNASLGTYHDLLKIIKDKDFLDNEE
  	NEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMK
  	QLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDG
  	FANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIA
  	NLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEM
  	ARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPV
  	ENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYD
  	VDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEV
  	VKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSEL
  	DKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDK
  	LIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHD
  	AYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIA
  	KSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLI
  	ETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV
  	QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPT
  	VAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEK
  	NPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRML
  	ASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPED
  	NEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKV
  	LSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDT
  	TIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD
  SpCas9	ATGGATAAAAAATATAGCATTGGCCTGGATATTGGC	SEQ ID NO:
  Reverse	ACCAACAGCGTGGGCTGGGCGGTGATTACCGATGAA		19
  translation	TATAAAGTGCCGAGCAAAAAATTTAAAGTGCTGGGC
  of	AACACCGATCGCCATAGCATTAAAAAAAACCTGATT
  SwissProt	GGCGCGCTGCTGTTTGATAGCGGCGAAACCGCGGAA
  Accession	GCGACCCGCCTGAAACGCACCGCGCGCCGCCGCTAT
  No.	ACCCGCCGCAAAAACCGCATTTGCTATCTGCAGGAA
  Q99ZW2	ATTTTTAGCAACGAAATGGCGAAAGTGGATGATAGC
  Streptococcus	TTTTTTCATCGCCTGGAAGAAAGCTTTCTGGTGGAAG
  pyogenes	AAGATAAAAAACATGAACGCCATCCGATTTTTGGCA
  	ACATTGTGGATGAAGTGGCGTATCATGAAAAATATC
  	CGACCATTTATCATCTGCGCAAAAAACTGGTGGATA
  	GCACCGATAAAGCGGATCTGCGCCTGATTTATCTGG
  	CGCTGGCGCATATGATTAAATTTCGCGGCCATTTTCT
  	GATTGAAGGCGATCTGAACCCGGATAACAGCGATGT
  	GGATAAACTGTTTATTCAGCTGGTGCAGACCTATAA
  	CCAGCTGTTTGAAGAAAACCCGATTAACGCGAGCGG
  	CGTGGATGCGAAAGCGATTCTGAGCGCGCGCCTGAG
  	CAAAAGCCGCCGCCTGGAAAACCTGATTGCGCAGCT
  	GCCGGGCGAAAAAAAAAACGGCCTGTTTGGCAACCT
  	GATTGCGCTGAGCCTGGGCCTGACCCCGAACTTTAA
  	AAGCAACTTTGATCTGGCGGAAGATGCGAAACTGCA
  	GCTGAGCAAAGATACCTATGATGATGATCTGGATAA
  	CCTGCTGGCGCAGATTGGCGATCAGTATGCGGATCT
  	GTTTCTGGCGGCGAAAAACCTGAGCGATGCGATTCT
  	GCTGAGCGATATTCTGCGCGTGAACACCGAAATTAC
  	CAAAGCGCCGCTGAGCGCGAGCATGATTAAACGCTA
  	TGATGAACATCATCAGGATCTGACCCTGCTGAAAGC
  	GCTGGTGCGCCAGCAGCTGCCGGAAAAATATAAAG
  	AAATTTTTTTTGATCAGAGCAAAAACGGCTATGCGG
  	GCTATATTGATGGCGGCGCGAGCCAGGAAGAATTTT
  	ATAAATTTATTAAACCGATTCTGGAAAAAATGGATG
  	GCACCGAAGAACTGCTGGTGAAACTGAACCGCGAA
  	GATCTGCTGCGCAAACAGCGCACCTTTGATAACGGC
  	AGCATTCCGCATCAGATTCATCTGGGCGAACTGCAT
  	GCGATTCTGCGCCGCCAGGAAGATTTTTATCCGTTTC
  	TGAAAGATAACCGCGAAAAAATTGAAAAAATTCTG
  	ACCTTTCGCATTCCGTATTATGTGGGCCCGCTGGCGC
  	GCGGCAACAGCCGCTTTGCGTGGATGACCCGCAAAA
  	GCGAAGAAACCATTACCCCGTGGAACTTTGAAGAAG
  	TGGTGGATAAAGGCGCGAGCGCGCAGAGCTTTATTG
  	AACGCATGACCAACTTTGATAAAAACCTGCCGAACG
  	AAAAAGTGCTGCCGAAACATAGCCTGCTGTATGAAT
  	ATTTTACCGTGTATAACGAACTGACCAAAGTGAAAT
  	ATGTGACCGAAGGCATGCGCAAACCGGCGTTTCTGA
  	GCGGCGAACAGAAAAAAGCGATTGTGGATCTGCTGT
  	TTAAAACCAACCGCAAAGTGACCGTGAAACAGCTGA
  	AAGAAGATTATTTTAAAAAAATTGAATGCTTTGATA
  	GCGTGGAAATTAGCGGCGTGGAAGATCGCTTTAACG
  	CGAGCCTGGGCACCTATCATGATCTGCTGAAAATTA
  	TTAAAGATAAAGATTTTCTGGATAACGAAGAAAACG
  	AAGATATTCTGGAAGATATTGTGCTGACCCTGACCC
  	TGTTTGAAGATCGCGAAATGATTGAAGAACGCCTGA
  	AAACCTATGCGCATCTGTTTGATGATAAAGTGATGA
  	AACAGCTGAAACGCCGCCGCTATACCGGCTGGGGCC
  	GCCTGAGCCGCAAACTGATTAACGGCATTCGCGATA
  	AACAGAGCGGCAAAACCATTCTGGATTTTCTGAAAA
  	GCGATGGCTTTGCGAACCGCAACTTTATGCAGCTGA
  	TTCATGATGATAGCCTGACCTTTAAAGAAGATATTC
  	AGAAAGCGCAGGTGAGCGGCCAGGGCGATAGCCTG
  	CATGAACATATTGCGAACCTGGCGGGCAGCCCGGCG
  	ATTAAAAAAGGCATTCTGCAGACCGTGAAAGTGGTG
  	GATGAACTGGTGAAAGTGATGGGCCGCCATAAACCG
  	GAAAACATTGTGATTGAAATGGCGCGCGAAAACCA
  	GACCACCCAGAAAGGCCAGAAAAACAGCCGCGAAC
  	GCATGAAACGCATTGAAGAAGGCATTAAAGAACTG
  	GGCAGCCAGATTCTGAAAGAACATCCGGTGGAAAA
  	CACCCAGCTGCAGAACGAAAAACTGTATCTGTATTA
  	TCTGCAGAACGGCCGCGATATGTATGTGGATCAGGA
  	ACTGGATATTAACCGCCTGAGCGATTATGATGTGGA
  	TCATATTGTGCCGCAGAGCTTTCTGAAAGATGATAG
  	CATTGATAACAAAGTGCTGACCCGCAGCGATAAAAA
  	CCGCGGCAAAAGCGATAACGTGCCGAGCGAAGAAG
  	TGGTGAAAAAAATGAAAAACTATTGGCGCCAGCTGC
  	TGAACGCGAAACTGATTACCCAGCGCAAATTTGATA
  	ACCTGACCAAAGCGGAACGCGGCGGCCTGAGCGAA
  	CTGGATAAAGCGGGCTTTATTAAACGCCAGCTGGTG
  	GAAACCCGCCAGATTACCAAACATGTGGCGCAGATT
  	CTGGATAGCCGCATGAACACCAAATATGATGAAAAC
  	GATAAACTGATTCGCGAAGTGAAAGTGATTACCCTG
  	AAAAGCAAACTGGTGAGCGATTTTCGCAAAGATTTT
  	CAGTTTTATAAAGTGCGCGAAATTAACAACTATCAT
  	CATGCGCATGATGCGTATCTGAACGCGGTGGTGGGC
  	ACCGCGCTGATTAAAAAATATCCGAAACTGGAAAGC
  	GAATTTGTGTATGGCGATTATAAAGTGTATGATGTG
  	CGCAAAATGATTGCGAAAAGCGAACAGGAAATTGG
  	CAAAGCGACCGCGAAATATTTTTTTTATAGCAACAT
  	TATGAACTTTTTTAAAACCGAAATTACCCTGGCGAA
  	CGGCGAAATTCGCAAACGCCCGCTGATTGAAACCAA
  	CGGCGAAACCGGCGAAATTGTGTGGGATAAAGGCC
  	GCGATTTTGCGACCGTGCGCAAAGTGCTGAGCATGC
  	CGCAGGTGAACATTGTGAAAAAAACCGAAGTGCAG
  	ACCGGCGGCTTTAGCAAAGAAAGCATTCTGCCGAAA
  	CGCAACAGCGATAAACTGATTGCGCGCAAAAAAGA
  	TTGGGATCCGAAAAAATATGGCGGCTTTGATAGCCC
  	GACCGTGGCGTATAGCGTGCTGGTGGTGGCGAAAGT
  	GGAAAAAGGCAAAAGCAAAAAACTGAAAAGCGTGA
  	AAGAACTGCTGGGCATTACCATTATGGAACGCAGCA
  	GCTTTGAAAAAAACCCGATTGATTTTCTGGAAGCGA
  	AAGGCTATAAAGAAGTGAAAAAAGATCTGATTATTA
  	AACTGCCGAAATATAGCCTGTTTGAACTGGAAAACG
  	GCCGCAAACGCATGCTGGCGAGCGCGGGCGAACTG
  	CAGAAAGGCAACGAACTGGCGCTGCCGAGCAAATA
  	TGTGAACTTTCTGTATCTGGCGAGCCATTATGAAAA
  	ACTGAAAGGCAGCCCGGAAGATAACGAACAGAAAC
  	AGCTGTTTGTGGAACAGCATAAACATTATCTGGATG
  	AAATTATTGAACAGATTAGCGAATTTAGCAAACGCG
  	TGATTCTGGCGGATGCGAACCTGGATAAAGTGCTGA
  	GCGCGTATAACAAACATCGCGATAAACCGATTCGCG
  	AACAGGCGGAAAACATTATTCATCTGTTTACCCTGA
  	CCAACCTGGGCGCGCCGGCGGCGTTTAAATATTTTG
  	ATACCACCATTGATCGCAAACGCTATACCAGCACCA
  	AAGAAGTGCTGGATGCGACCCTGATTCATCAGAGCA
  	TTACCGGCCTGTATGAAACCCGCATTGATCTGAGCC
  	AGCTGGGCGGCGAT

• The prime editors described herein may include canonical SpCas9, or any variant thereof having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with a wild type Cas9 sequence provided above. These variants may include SpCas9 variants containing one or more mutations, including any known mutation reported with the SwissProt Accession No. Q99ZW2 (SEQ ID NO: 18) entry, which include:

• SpCas9 mutation (relative to the amino	Function/Characteristic (as reported) (see
  acid sequence of the canonical SpCas9	UniProtKB-Q99ZW2 (CAS9_STRPT1) entry-
  sequence, SEQ ID NO: 18)	incorporated herein by reference)
  D10A	Nickase mutant which cleaves the protospacer
  	strand (but no cleavage of non-protospacer strand)
  S15A	Decreased DNA cleavage activity
  R66A	Decreased DNA cleavage activity
  R70A	No DNA cleavage
  R74A	Decreased DNA cleavage
  R78A	Decreased DNA cleavage
  97-150 deletion	No nuclease activity
  R165A	Decreased DNA cleavage
  175-307 deletion	About 50% decreased DNA cleavage
  312-409 deletion	No nuclease activity
  E762A	Nickase
  H840A	Nickase mutant which cleaves the non-protospacer
  	strand but does not cleave the protospacer strand
  N854A	Nickase
  N863A	Nickase
  H982A	Decreased DNA cleavage
  D986A	Nickase
  1099-1368 deletion	No nuclease activity
  R1333A	Reduced DNA binding

• Other wild type SpCas9 sequences that may be used in the present disclosure, include:

• Description	Sequence	SEQ ID NO:
  SpCas9	ATGGATAAGAAATACTCAATAGGCTTAGATATCGGCA	SEQ ID NO:
  Streptococcus	CAAATAGCGTCGGATGGGCGGTGATCACTGATGATTA	20
  pyogenes	TAAGGTTCCGTCTAAAAAGTTCAAGGTTCTGGGAAAT
  MGAS1882	ACAGACCGCCACAGTATCAAAAAAAATCTTATAGGGG
  wild type	CTCTTTTATTTGGCAGTGGAGAGACAGCGGAAGCGAC
  NC_017053.1	TCGTCTCAAACGGACAGCTCGTAGAAGGTATACACGT
  	CGGAAGAATCGTATTTGTTATCTACAGGAGATTTTTTC
  	AAATGAGATGGCGAAAGTAGATGATAGTTTCTTTCATC
  	GACTTGAAGAGTCTTTTTTGGTGGAAGAAGACAAGAA
  	GCATGAACGTCATCCTATTTTTGGAAATATAGTAGATG
  	AAGTTGCTTATCATGAGAAATATCCAACTATCTATCAT
  	CTGCGAAAAAAATTGGCAGATTCTACTGATAAAGCGG
  	ATTTGCGCTTAATCTATTTGGCCTTAGCGCATATGATT
  	AAGTTTCGTGGTCATTTTTTGATTGAGGGAGATTTAAA
  	TCCTGATAATAGTGATGTGGACAAACTATTTATCCAGT
  	TGGTACAAATCTACAATCAATTATTTGAAGAAAACCCT
  	ATTAACGCAAGTAGAGTAGATGCTAAAGCGATTCTTTC
  	TGCACGATTGAGTAAATCAAGACGATTAGAAAATCTC
  	ATTGCTCAGCTCCCCGGTGAGAAGAGAAATGGCTTGTT
  	TGGGAATCTCATTGCTTTGTCATTGGGATTGACCCCTA
  	ATTTTAAATCAAATTTTGATTTGGCAGAAGATGCTAAA
  	TTACAGCTTTCAAAAGATACTTACGATGATGATTTAGA
  	TAATTTATTGGCGCAAATTGGAGATCAATATGCTGATT
  	TGTTTTTGGCAGCTAAGAATTTATCAGATGCTATTTTA
  	CTTTCAGATATCCTAAGAGTAAATAGTGAAATAACTA
  	AGGCTCCCCTATCAGCTTCAATGATTAAGCGCTACGAT
  	GAACATCATCAAGACTTGACTCTTTTAAAAGCTTTAGT
  	TCGACAACAACTTCCAGAAAAGTATAAAGAAATCTTT
  	TTTGATCAATCAAAAAACGGATATGCAGGTTATATTGA
  	TGGGGGAGCTAGCCAAGAAGAATTTTATAAATTTATC
  	AAACCAATTTTAGAAAAAATGGATGGTACTGAGGAAT
  	TATTGGTGAAACTAAATCGTGAAGATTTGCTGCGCAA
  	GCAACGGACCTTTGACAACGGCTCTATTCCCCATCAAA
  	TTCACTTGGGTGAGCTGCATGCTATTTTGAGAAGACAA
  	GAAGACTTTTATCCATTTTTAAAAGACAATCGTGAGAA
  	GATTGAAAAAATCTTGACTTTTCGAATTCCTTATTATG
  	TTGGTCCATTGGCGCGTGGCAATAGTCGTTTTGCATGG
  	ATGACTCGGAAGTCTGAAGAAACAATTACCCCATGGA
  	ATTTTGAAGAAGTTGTCGATAAAGGTGCTTCAGCTCAA
  	TCATTTATTGAACGCATGACAAACTTTGATAAAAATCT
  	TCCAAATGAAAAAGTACTACCAAAACATAGTTTGCTTT
  	ATGAGTATTTTACGGTTTATAACGAATTGACAAAGGTC
  	AAATATGTTACTGAGGGAATGCGAAAACCAGCATTTC
  	TTTCAGGTGAACAGAAGAAAGCCATTGTTGATTTACTC
  	TTCAAAACAAATCGAAAAGTAACCGTTAAGCAATTAA
  	AAGAAGATTATTTCAAAAAAATAGAATGTTTTGATAG
  	TGTTGAAATTTCAGGAGTTGAAGATAGATTTAATGCTT
  	CATTAGGCGCCTACCATGATTTGCTAAAAATTATTAAA
  	GATAAAGATTTTTTGGATAATGAAGAAAATGAAGATA
  	TCTTAGAGGATATTGTTTTAACATTGACCTTATTTGAA
  	GATAGGGGGATGATTGAGGAAAGACTTAAAACATATG
  	CTCACCTCTTTGATGATAAGGTGATGAAACAGCTTAAA
  	CGTCGCCGTTATACTGGTTGGGGACGTTTGTCTCGAAA
  	ATTGATTAATGGTATTAGGGATAAGCAATCTGGCAAA
  	ACAATATTAGATTTTTTGAAATCAGATGGTTTTGCCAA
  	TCGCAATTTTATGCAGCTGATCCATGATGATAGTTTGA
  	CATTTAAAGAAGATATTCAAAAAGCACAGGTGTCTGG
  	ACAAGGCCATAGTTTACATGAACAGATTGCTAACTTA
  	GCTGGCAGTCCTGCTATTAAAAAAGGTATTTTACAGAC
  	TGTAAAAATTGTTGATGAACTGGTCAAAGTAATGGGG
  	CATAAGCCAGAAAATATCGTTATTGAAATGGCACGTG
  	AAAATCAGACAACTCAAAAGGGCCAGAAAAATTCGCG
  	AGAGCGTATGAAACGAATCGAAGAAGGTATCAAAGA
  	ATTAGGAAGTCAGATTCTTAAAGAGCATCCTGTTGAA
  	AATACTCAATTGCAAAATGAAAAGCTCTATCTCTATTA
  	TCTACAAAATGGAAGAGACATGTATGTGGACCAAGAA
  	TTAGATATTAATCGTTTAAGTGATTATGATGTCGATCA
  	CATTGTTCCACAAAGTTTCATTAAAGACGATTCAATAG
  	ACAATAAGGTACTAACGCGTTCTGATAAAAATCGTGG
  	TAAATCGGATAACGTTCCAAGTGAAGAAGTAGTCAAA
  	AAGATGAAAAACTATTGGAGACAACTTCTAAACGCCA
  	AGTTAATCACTCAACGTAAGTTTGATAATTTAACGAAA
  	GCTGAACGTGGAGGTTTGAGTGAACTTGATAAAGCTG
  	GTTTTATCAAACGCCAATTGGTTGAAACTCGCCAAATC
  	ACTAAGCATGTGGCACAAATTTTGGATAGTCGCATGA
  	ATACTAAATACGATGAAAATGATAAACTTATTCGAGA
  	GGTTAAAGTGATTACCTTAAAATCTAAATTAGTTTCTG
  	ACTTCCGAAAAGATTTCCAATTCTATAAAGTACGTGAG
  	ATTAACAATTACCATCATGCCCATGATGCGTATCTAAA
  	TGCCGTCGTTGGAACTGCTTTGATTAAGAAATATCCAA
  	AACTTGAATCGGAGTTTGTCTATGGTGATTATAAAGTT
  	TATGATGTTCGTAAAATGATTGCTAAGTCTGAGCAAGA
  	AATAGGCAAAGCAACCGCAAAATATTTCTTTTACTCTA
  	ATATCATGAACTTCTTCAAAACAGAAATTACACTTGCA
  	AATGGAGAGATTCGCAAACGCCCTCTAATCGAAACTA
  	ATGGGGAAACTGGAGAAATTGTCTGGGATAAAGGGCG
  	AGATTTTGCCACAGTGCGCAAAGTATTGTCCATGCCCC
  	AAGTCAATATTGTCAAGAAAACAGAAGTACAGACAGG
  	CGGATTCTCCAAGGAGTCAATTTTACCAAAAAGAAAT
  	TCGGACAAGCTTATTGCTCGTAAAAAAGACTGGGATC
  	CAAAAAAATATGGTGGTTTTGATAGTCCAACGGTAGC
  	TTATTCAGTCCTAGTGGTTGCTAAGGTGGAAAAAGGG
  	AAATCGAAGAAGTTAAAATCCGTTAAAGAGTTACTAG
  	GGATCACAATTATGGAAAGAAGTTCCTTTGAAAAAAA
  	TCCGATTGACTTTTTAGAAGCTAAAGGATATAAGGAA
  	GTTAAAAAAGACTTAATCATTAAACTACCTAAATATA
  	GTCTTTTTGAGTTAGAAAACGGTCGTAAACGGATGCTG
  	GCTAGTGCCGGAGAATTACAAAAAGGAAATGAGCTGG
  	CTCTGCCAAGCAAATATGTGAATTTTTTATATTTAGCT
  	AGTCATTATGAAAAGTTGAAGGGTAGTCCAGAAGATA
  	ACGAACAAAAACAATTGTTTGTGGAGCAGCATAAGCA
  	TTATTTAGATGAGATTATTGAGCAAATCAGTGAATTTT
  	CTAAGCGTGTTATTTTAGCAGATGCCAATTTAGATAAA
  	GTTCTTAGTGCATATAACAAACATAGAGACAAACCAA
  	TACGTGAACAAGCAGAAAATATTATTCATTTATTTACG
  	TTGACGAATCTTGGAGCTCCCGCTGCTTTTAAATATTT
  	TGATACAACAATTGATCGTAAACGATATACGTCTACA
  	AAAGAAGTTTTAGATGCCACTCTTATCCATCAATCCAT
  	CACTGGTCTTTATGAAACACGCATTGATTTGAGTCAGC
  	TAGGAGGTGACTGA
  SpCas9	MDKKYSIGLDIGTNSVGWAVITDDYKVPSKKFKVLGNT	SEQ ID NO:
  Streptococcus	DRHSIKKNLIGALLFGSGETAEATRLKRTARRRYTRRKN	21
  pyogenes	RICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHP
  MGAS1882	IFGNIVDEVAYHEKYPTIYHLRKKLADSTDKADLRLIYLA
  wild type	LAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQIYNQLFE
  NC_017053.1	ENPINASRVDAKAILSARLSKSRRLENLIAQLPGEKRNGL
  	FGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLD
  	NLLAQIGDQYADLFLAAKNLSDAILLSDILRVNSEITKAP
  	LSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQS
  	KNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLN
  	REDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLK
  	DNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITP
  	WNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLL
  	YEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLF
  	KTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLG
  	AYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRGMIE
  	ERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIR
  	DKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKA
  	QVSGQGHSLHEQIANLAGSPAIKKGILQTVKIVDELVKV
  	MGHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKE
  	LGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQEL
  	DINRLSDYDVDHIVPQSFIKDDSIDNKVLTRSDKNRGKSD
  	NVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERG
  	GLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDE
  	NDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHA
  	HDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMI
  	AKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLI
  	ETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQ
  	TGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVA
  	YSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPID
  	FLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGE
  	LQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLF
  	VEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHR
  	DKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTST
  	KEVLDATLIHQSITGLYETRIDLSQLGGD
  SpCas9	ATGGATAAAAAGTATTCTATTGGTTTAGACATCGGCAC	SEQ ID NO:
  Streptococcus	TAATTCCGTTGGATGGGCTGTCATAACCGATGAATACA	22
  pyogenes	AAGTACCTTCAAAGAAATTTAAGGTGTTGGGGAACAC
  wild type	AGACCGTCATTCGATTAAAAAGAATCTTATCGGTGCCC
  SWBC2D7W014	TCCTATTCGATAGTGGCGAAACGGCAGAGGCGACTCG
  	CCTGAAACGAACCGCTCGGAGAAGGTATACACGTCGC
  	AAGAACCGAATATGTTACTTACAAGAAATTTTTAGCA
  	ATGAGATGGCCAAAGTTGACGATTCTTTCTTTCACCGT
  	TTGGAAGAGTCCTTCCTTGTCGAAGAGGACAAGAAAC
  	ATGAACGGCACCCCATCTTTGGAAACATAGTAGATGA
  	GGTGGCATATCATGAAAAGTACCCAACGATTTATCAC
  	CTCAGAAAAAAGCTAGTTGACTCAACTGATAAAGCGG
  	ACCTGAGGTTAATCTACTTGGCTCTTGCCCATATGATA
  	AAGTTCCGTGGGCACTTTCTCATTGAGGGTGATCTAAA
  	TCCGGACAACTCGGATGTCGACAAACTGTTCATCCAGT
  	TAGTACAAACCTATAATCAGTTGTTTGAAGAGAACCCT
  	ATAAATGCAAGTGGCGTGGATGCGAAGGCTATTCTTA
  	GCGCCCGCCTCTCTAAATCCCGACGGCTAGAAAACCT
  	GATCGCACAATTACCCGGAGAGAAGAAAAATGGGTTG
  	TTCGGTAACCTTATAGCGCTCTCACTAGGCCTGACACC
  	AAATTTTAAGTCGAACTTCGACTTAGCTGAAGATGCCA
  	AATTGCAGCTTAGTAAGGACACGTACGATGACGATCT
  	CGACAATCTACTGGCACAAATTGGAGATCAGTATGCG
  	GACTTATTTTTGGCTGCCAAAAACCTTAGCGATGCAAT
  	CCTCCTATCTGACATACTGAGAGTTAATACTGAGATTA
  	CCAAGGCGCCGTTATCCGCTTCAATGATCAAAAGGTA
  	CGATGAACATCACCAAGACTTGACACTTCTCAAGGCC
  	CTAGTCCGTCAGCAACTGCCTGAGAAATATAAGGAAA
  	TATTCTTTGATCAGTCGAAAAACGGGTACGCAGGTTAT
  	ATTGACGGCGGAGCGAGTCAAGAGGAATTCTACAAGT
  	TTATCAAACCCATATTAGAGAAGATGGATGGGACGGA
  	AGAGTTGCTTGTAAAACTCAATCGCGAAGATCTACTGC
  	GAAAGCAGCGGACTTTCGACAACGGTAGCATTCCACA
  	TCAAATCCACTTAGGCGAATTGCATGCTATACTTAGAA
  	GGCAGGAGGATTTTTATCCGTTCCTCAAAGACAATCGT
  	GAAAAGATTGAGAAAATCCTAACCTTTCGCATACCTTA
  	CTATGTGGGACCCCTGGCCCGAGGGAACTCTCGGTTCG
  	CATGGATGACAAGAAAGTCCGAAGAAACGATTACTCC
  	ATGGAATTTTGAGGAAGTTGTCGATAAAGGTGCGTCA
  	GCTCAATCGTTCATCGAGAGGATGACCAACTTTGACA
  	AGAATTTACCGAACGAAAAAGTATTGCCTAAGCACAG
  	TTTACTTTACGAGTATTTCACAGTGTACAATGAACTCA
  	CGAAAGTTAAGTATGTCACTGAGGGCATGCGTAAACC
  	CGCCTTTCTAAGCGGAGAACAGAAGAAAGCAATAGTA
  	GATCTGTTATTCAAGACCAACCGCAAAGTGACAGTTA
  	AGCAATTGAAAGAGGACTACTTTAAGAAAATTGAATG
  	CTTCGATTCTGTCGAGATCTCCGGGGTAGAAGATCGAT
  	TTAATGCGTCACTTGGTACGTATCATGACCTCCTAAAG
  	ATAATTAAAGATAAGGACTTCCTGGATAACGAAGAGA
  	ATGAAGATATCTTAGAAGATATAGTGTTGACTCTTACC
  	CTCTTTGAAGATCGGGAAATGATTGAGGAAAGACTAA
  	AAACATACGCTCACCTGTTCGACGATAAGGTTATGAA
  	ACAGTTAAAGAGGCGTCGCTATACGGGCTGGGGACGA
  	TTGTCGCGGAAACTTATCAACGGGATAAGAGACAAGC
  	AAAGTGGTAAAACTATTCTCGATTTTCTAAAGAGCGAC
  	GGCTTCGCCAATAGGAACTTTATGCAGCTGATCCATGA
  	TGACTCTTTAACCTTCAAAGAGGATATACAAAAGGCA
  	CAGGTTTCCGGACAAGGGGACTCATTGCACGAACATA
  	TTGCGAATCTTGCTGGTTCGCCAGCCATCAAAAAGGGC
  	ATACTCCAGACAGTCAAAGTAGTGGATGAGCTAGTTA
  	AGGTCATGGGACGTCACAAACCGGAAAACATTGTAAT
  	CGAGATGGCACGCGAAAATCAAACGACTCAGAAGGG
  	GCAAAAAAACAGTCGAGAGCGGATGAAGAGAATAGA
  	AGAGGGTATTAAAGAACTGGGCAGCCAGATCTTAAAG
  	GAGCATCCTGTGGAAAATACCCAATTGCAGAACGAGA
  	AACTTTACCTCTATTACCTACAAAATGGAAGGGACATG
  	TATGTTGATCAGGAACTGGACATAAACCGTTTATCTGA
  	TTACGACGTCGATCACATTGTACCCCAATCCTTTTTGA
  	AGGACGATTCAATCGACAATAAAGTGCTTACACGCTC
  	GGATAAGAACCGAGGGAAAAGTGACAATGTTCCAAGC
  	GAGGAAGTCGTAAAGAAAATGAAGAACTATTGGCGGC
  	AGCTCCTAAATGCGAAACTGATAACGCAAAGAAAGTT
  	CGATAACTTAACTAAAGCTGAGAGGGGTGGCTTGTCT
  	GAACTTGACAAGGCCGGATTTATTAAACGTCAGCTCGT
  	GGAAACCCGCCAAATCACAAAGCATGTTGCACAGATA
  	CTAGATTCCCGAATGAATACGAAATACGACGAGAACG
  	ATAAGCTGATTCGGGAAGTCAAAGTAATCACTTTAAA
  	GTCAAAATTGGTGTCGGACTTCAGAAAGGATTTTCAAT
  	TCTATAAAGTTAGGGAGATAAATAACTACCACCATGC
  	GCACGACGCTTATCTTAATGCCGTCGTAGGGACCGCAC
  	TCATTAAGAAATACCCGAAGCTAGAAAGTGAGTTTGT
  	GTATGGTGATTACAAAGTTTATGACGTCCGTAAGATGA
  	TCGCGAAAAGCGAACAGGAGATAGGCAAGGCTACAG
  	CCAAATACTTCTTTTATTCTAACATTATGAATTTCTTTA
  	AGACGGAAATCACTCTGGCAAACGGAGAGATACGCAA
  	ACGACCTTTAATTGAAACCAATGGGGAGACAGGTGAA
  	ATCGTATGGGATAAGGGCCGGGACTTCGCGACGGTGA
  	GAAAAGTTTTGTCCATGCCCCAAGTCAACATAGTAAA
  	GAAAACTGAGGTGCAGACCGGAGGGTTTTCAAAGGAA
  	TCGATTCTTCCAAAAAGGAATAGTGATAAGCTCATCGC
  	TCGTAAAAAGGACTGGGACCCGAAAAAGTACGGTGGC
  	TTCGATAGCCCTACAGTTGCCTATTCTGTCCTAGTAGT
  	GGCAAAAGTTGAGAAGGGAAAATCCAAGAAACTGAA
  	GTCAGTCAAAGAATTATTGGGGATAACGATTATGGAG
  	CGCTCGTCTTTTGAAAAGAACCCCATCGACTTCCTTGA
  	GGCGAAAGGTTACAAGGAAGTAAAAAAGGATCTCATA
  	ATTAAACTACCAAAGTATAGTCTGTTTGAGTTAGAAAA
  	TGGCCGAAAACGGATGTTGGCTAGCGCCGGAGAGCTT
  	CAAAAGGGGAACGAACTCGCACTACCGTCTAAATACG
  	TGAATTTCCTGTATTTAGCGTCCCATTACGAGAAGTTG
  	AAAGGTTCACCTGAAGATAACGAACAGAAGCAACTTT
  	TTGTTGAGCAGCACAAACATTATCTCGACGAAATCATA
  	GAGCAAATTTCGGAATTCAGTAAGAGAGTCATCCTAG
  	CTGATGCCAATCTGGACAAAGTATTAAGCGCATACAA
  	CAAGCACAGGGATAAACCCATACGTGAGCAGGCGGAA
  	AATATTATCCATTTGTTTACTCTTACCAACCTCGGCGCT
  	CCAGCCGCATTCAAGTATTTTGACACAACGATAGATCG
  	CAAACGATACACTTCTACCAAGGAGGTGCTAGACGCG
  	ACACTGATTCACCAATCCATCACGGGATTATATGAAAC
  	TCGGATAGATTTGTCACAGCTTGGGGGTGACGGATCCC
  	CCAAGAAGAAGAGGAAAGTCTCGAGCGACTACAAAG
  	ACCATGACGGTGATTATAAAGATCATGACATCGATTA
  	CAAGGATGACGATGACAAGGCTGCAGGA
  SpCas9	MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTD	SEQ ID NO:
  Streptococcus	RHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRI	23
  pyogenes	CYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIF
  wild type	GNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLAL
  Encoded	AHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEE
  product of	NPINTASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLF
  SWBC2D7W014	GNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDN
  	LLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLS
  	ASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKN
  	GYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRE
  	DLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDN
  	REKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPW
  	NFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLY
  	EYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFK
  	TNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGT
  	YHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE
  	RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRD
  	KQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQ
  	VSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVM
  	GRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKE
  	LGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQEL
  	DINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKS
  	DNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAER
  	GGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYD
  	ENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINTNYHH
  	AHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRK
  	MIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRP
  	LIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV
  	QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTV
  	AYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPI
  	DFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAG
  	ELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQL
  	FVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKH
  	RDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTS
  	TKEVLDATLIHQSITGLYETRIDLSQLGGDGSPKKKRKVS
  	SDYKDHDGDYKDHDIDYKDDDDKAAG
  SpCas9	ATGGATAAGAAATACTCAATAGGCTTAGATATCGGCA	SEQ ID NO:
  Streptococcus	CAAATAGCGTCGGATGGGCGGTGATCACTGATGAATA	24
  pyogenes	TAAGGTTCCGTCTAAAAAGTTCAAGGTTCTGGGAAAT
  M1GAS wild	ACAGACCGCCACAGTATCAAAAAAAATCTTATAGGGG
  type	CTCTTTTATTTGACAGTGGAGAGACAGCGGAAGCGAC
  NC_002737.2	TCGTCTCAAACGGACAGCTCGTAGAAGGTATACACGT
  	CGGAAGAATCGTATTTGTTATCTACAGGAGATTTTTTC
  	AAATGAGATGGCGAAAGTAGATGATAGTTTCTTTCATC
  	GACTTGAAGAGTCTTTTTTGGTGGAAGAAGACAAGAA
  	GCATGAACGTCATCCTATTTTTGGAAATATAGTAGATG
  	AAGTTGCTTATCATGAGAAATATCCAACTATCTATCAT
  	CTGCGAAAAAAATTGGTAGATTCTACTGATAAAGCGG
  	ATTTGCGCTTAATCTATTTGGCCTTAGCGCATATGATT
  	AAGTTTCGTGGTCATTTTTTGATTGAGGGAGATTTAAA
  	TCCTGATAATAGTGATGTGGACAAACTATTTATCCAGT
  	TGGTACAAACCTACAATCAATTATTTGAAGAAAACCCT
  	ATTAACGCAAGTGGAGTAGATGCTAAAGCGATTCTTTC
  	TGCACGATTGAGTAAATCAAGACGATTAGAAAATCTC
  	ATTGCTCAGCTCCCCGGTGAGAAGAAAAATGGCTTATT
  	TGGGAATCTCATTGCTTTGTCATTGGGTTTGACCCCTA
  	ATTTTAAATCAAATTTTGATTTGGCAGAAGATGCTAAA
  	TTACAGCTTTCAAAAGATACTTACGATGATGATTTAGA
  	TAATTTATTGGCGCAAATTGGAGATCAATATGCTGATT
  	TGTTTTTGGCAGCTAAGAATTTATCAGATGCTATTTTA
  	CTTTCAGATATCCTAAGAGTAAATACTGAAATAACTAA
  	GGCTCCCCTATCAGCTTCAATGATTAAACGCTACGATG
  	AACATCATCAAGACTTGACTCTTTTAAAAGCTTTAGTT
  	CGACAACAACTTCCAGAAAAGTATAAAGAAATCTTTT
  	TTGATCAATCAAAAAACGGATATGCAGGTTATATTGAT
  	GGGGGAGCTAGCCAAGAAGAATTTTATAAATTTATCA
  	AACCAATTTTAGAAAAAATGGATGGTACTGAGGAATT
  	ATTGGTGAAACTAAATCGTGAAGATTTGCTGCGCAAG
  	CAACGGACCTTTGACAACGGCTCTATTCCCCATCAAAT
  	TCACTTGGGTGAGCTGCATGCTATTTTGAGAAGACAAG
  	AAGACTTTTATCCATTTTTAAAAGACAATCGTGAGAAG
  	ATTGAAAAAATCTTGACTTTTCGAATTCCTTATTATGTT
  	GGTCCATTGGCGCGTGGCAATAGTCGTTTTGCATGGAT
  	GACTCGGAAGTCTGAAGAAACAATTACCCCATGGAAT
  	TTTGAAGAAGTTGTCGATAAAGGTGCTTCAGCTCAATC
  	ATTTATTGAACGCATGACAAACTTTGATAAAAATCTTC
  	CAAATGAAAAAGTACTACCAAAACATAGTTTGCTTTAT
  	GAGTATTTTACGGTTTATAACGAATTGACAAAGGTCAA
  	ATATGTTACTGAAGGAATGCGAAAACCAGCATTTCTTT
  	CAGGTGAACAGAAGAAAGCCATTGTTGATTTACTCTTC
  	AAAACAAATCGAAAAGTAACCGTTAAGCAATTAAAAG
  	AAGATTATTTCAAAAAAATAGAATGTTTTGATAGTGTT
  	GAAATTTCAGGAGTTGAAGATAGATTTAATGCTTCATT
  	AGGTACCTACCATGATTTGCTAAAAATTATTAAAGATA
  	AAGATTTTTTGGATAATGAAGAAAATGAAGATATCTT
  	AGAGGATATTGTTTTAACATTGACCTTATTTGAAGATA
  	GGGAGATGATTGAGGAAAGACTTAAAACATATGCTCA
  	CCTCTTTGATGATAAGGTGATGAAACAGCTTAAACGTC
  	GCCGTTATACTGGTTGGGGACGTTTGTCTCGAAAATTG
  	ATTAATGGTATTAGGGATAAGCAATCTGGCAAAACAA
  	TATTAGATTTTTTGAAATCAGATGGTTTTGCCAATCGC
  	AATTTTATGCAGCTGATCCATGATGATAGTTTGACATT
  	TAAAGAAGACATTCAAAAAGCACAAGTGTCTGGACAA
  	GGCGATAGTTTACATGAACATATTGCAAATTTAGCTGG
  	TAGCCCTGCTATTAAAAAAGGTATTTTACAGACTGTAA
  	AAGTTGTTGATGAATTGGTCAAAGTAATGGGGCGGCA
  	TAAGCCAGAAAATATCGTTATTGAAATGGCACGTGAA
  	AATCAGACAACTCAAAAGGGCCAGAAAAATTCGCGAG
  	AGCGTATGAAACGAATCGAAGAAGGTATCAAAGAATT
  	AGGAAGTCAGATTCTTAAAGAGCATCCTGTTGAAAAT
  	ACTCAATTGCAAAATGAAAAGCTCTATCTCTATTATCT
  	CCAAAATGGAAGAGACATGTATGTGGACCAAGAATTA
  	GATATTAATCGTTTAAGTGATTATGATGTCGATCACAT
  	TGTTCCACAAAGTTTCCTTAAAGACGATTCAATAGACA
  	ATAAGGTCTTAACGCGTTCTGATAAAAATCGTGGTAA
  	ATCGGATAACGTTCCAAGTGAAGAAGTAGTCAAAAAG
  	ATGAAAAACTATTGGAGACAACTTCTAAACGCCAAGT
  	TAATCACTCAACGTAAGTTTGATAATTTAACGAAAGCT
  	GAACGTGGAGGTTTGAGTGAACTTGATAAAGCTGGTT
  	TTATCAAACGCCAATTGGTTGAAACTCGCCAAATCACT
  	AAGCATGTGGCACAAATTTTGGATAGTCGCATGAATA
  	CTAAATACGATGAAAATGATAAACTTATTCGAGAGGT
  	TAAAGTGATTACCTTAAAATCTAAATTAGTTTCTGACT
  	TCCGAAAAGATTTCCAATTCTATAAAGTACGTGAGATT
  	AACAATTACCATCATGCCCATGATGCGTATCTAAATGC
  	CGTCGTTGGAACTGCTTTGATTAAGAAATATCCAAAAC
  	TTGAATCGGAGTTTGTCTATGGTGATTATAAAGTTTAT
  	GATGTTCGTAAAATGATTGCTAAGTCTGAGCAAGAAA
  	TAGGCAAAGCAACCGCAAAATATTTCTTTTACTCTAAT
  	ATCATGAACTTCTTCAAAACAGAAATTACACTTGCAAA
  	TGGAGAGATTCGCAAACGCCCTCTAATCGAAACTAAT
  	GGGGAAACTGGAGAAATTGTCTGGGATAAAGGGCGAG
  	ATTTTGCCACAGTGCGCAAAGTATTGTCCATGCCCCAA
  	GTCAATATTGTCAAGAAAACAGAAGTACAGACAGGCG
  	GATTCTCCAAGGAGTCAATTTTACCAAAAAGAAATTC
  	GGACAAGCTTATTGCTCGTAAAAAAGACTGGGATCCA
  	AAAAAATATGGTGGTTTTGATAGTCCAACGGTAGCTTA
  	TTCAGTCCTAGTGGTTGCTAAGGTGGAAAAAGGGAAA
  	TCGAAGAAGTTAAAATCCGTTAAAGAGTTACTAGGGA
  	TCACAATTATGGAAAGAAGTTCCTTTGAAAAAAATCC
  	GATTGACTTTTTAGAAGCTAAAGGATATAAGGAAGTT
  	AAAAAAGACTTAATCATTAAACTACCTAAATATAGTCT
  	TTTTGAGTTAGAAAACGGTCGTAAACGGATGCTGGCT
  	AGTGCCGGAGAATTACAAAAAGGAAATGAGCTGGCTC
  	TGCCAAGCAAATATGTGAATTTTTTATATTTAGCTAGT
  	CATTATGAAAAGTTGAAGGGTAGTCCAGAAGATAACG
  	AACAAAAACAATTGTTTGTGGAGCAGCATAAGCATTA
  	TTTAGATGAGATTATTGAGCAAATCAGTGAATTTTCTA
  	AGCGTGTTATTTTAGCAGATGCCAATTTAGATAAAGTT
  	CTTAGTGCATATAACAAACATAGAGACAAACCAATAC
  	GTGAACAAGCAGAAAATATTATTCATTTATTTACGTTG
  	ACGAATCTTGGAGCTCCCGCTGCTTTTAAATATTTTGA
  	TACAACAATTGATCGTAAACGATATACGTCTACAAAA
  	GAAGTTTTAGATGCCACTCTTATCCATCAATCCATCAC
  	TGGTCTTTATGAAACACGCATTGATTTGAGTCAGCTAG
  	GAGGTGACTGA
  SpCas9	MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTD	SEQ ID NO:
  Streptococcus	RHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRI	25
  pyogenes	CYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIF
  M1GAS wild	GNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLAL
  type	AHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEE
  Encoded	NPINTASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLF
  product of	GNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDN
  NC_002737.2	LLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLS
  (100%	ASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKN
  identical to	GYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRE
  the canonical	DLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDN
  Q99ZW2	REKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPW
  wild type)	NFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLY
  	EYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFK
  	TNRKVTVKQLKEDYFKKIECFDSVETSGVEDRFNASLGT
  	YHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE
  	RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRD
  	KQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQ
  	VSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVM
  	GRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKE
  	LGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQEL
  	DINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKS
  	DNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAER
  	GGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYD
  	ENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINTNYHH
  	AHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRK
  	MIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRP
  	LIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV
  	QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTV
  	AYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPI
  	DFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAG
  	ELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQL
  	FVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKH
  	RDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTS
  	TKEVLDATLIHQSITGLYETRIDLSQLGGD

• The prime editors described herein may include any of the above SpCas9 sequences, or any variant thereof having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto.
• B. Wild Type Cas9 Orthologs
• In other embodiments, the Cas9 protein can be a wild type Cas9 ortholog from another bacterial species different from the canonical Cas9 from S. pyogenes. For example, the following Cas9 orthologs can be used in connection with the prime editor constructs described in this specification. In addition, any variant Cas9 orthologs having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to any of the below orthologs may also be used with the present prime editors.

• Description	Sequence
  LfCas9	MKEYHIGLDIGTSSIGWAVTDSQFKLMRIKGKTAIGVRLFEEGKTAAERR
  Lactobacillus	TFRTTRRRLKRRKWRLHYLDEIFAPHLQEVDENFLRRLKQSNIHPEDPTK
  fermentum	NQAFIGKLLFPDLLKKNERGYPTLIKMRDELPVEQRAHYPVMNIYKLRE
  wild type	AMINEDRQFDLREVYLAVHHIVKYRGHFLNNASVDKFKVGRIDFDKSFN
  GenBank:	VLNEAYEELQNGEGSFTIEPSKVEKIGQLLLDTKMRKLDRQKAVAKLLE
  SNX31424.11	VKVADKEETKRNKQIATAMSKLVLGYKADFATVAMANGNEWKIDLSS
  	ETSEDEIEKFREELSDAQNDILTEITSLFSQIMLNEIVPNGMSISESMMDRY
  	WTHERQLAEVKEYLATQPASARKEFDQVYNKYIGQAPKERGFDLEKGL
  	KKILSKKENWKEIDELLKAGDFLPKQRTSANGVIPHQMHQQELDRIIEKQ
  	AKYYPWLATENPATGERDRHQAKYELDQLVSFRIPYYVGPLVTPEVQK
  	ATSGAKFAWAKRKEDGEITPWNLWDKIDRAESAEAFIKRMTVKDTYLL
  	NEDVLPANSLLYQKYNVLNELNNVRVNGRRLSVGIKQDIYTELFKKKKT
  	VKASDVASLVMAKTRGVNKPSVEGLSDPKKFNSNLATYLDLKSIVGDK
  	VDDNRYQTDLENIIEWRSVFEDGEIFADKLTEVEWLTDEQRSALVKKRY
  	KGWGRLSKKLLTGIVDENGQRIIDLMWNTDQNFKEIVDQPVFKEQIDQL
  	NQKAITNDGMTLRERVESVLDDAYTSPQNKKAIWQVVRVVEDIVKAVG
  	NAPKSISIEFARNEGNKGEITRSRRTQLQKLFEDQAHELVKDTSLTEELEK
  	APDLSDRYYFYFTQGGKDMYTGDPINFDEISTKYDIDHILPQSFVKDNSL
  	DNRVLTSRKENNKKSDQVPAKLYAAKMKPYWNQLLKQGLITQRKFEN
  	LTKDVDQNIKYRSLGFVKRQLVETRQVIKLTANILGSMYQEAGTEIIETR
  	AGLTKQLREEFDLPKVREVNDYHHAVDAYLTTFAGQYLNRRYPKLRSF
  	FVYGEYMKFKHGSDLKLRNFNFFHELMEGDKSQGKVVDQQTGELITTR
  	DEVAKSFDRLLNMKYMLVSKEVHDRSDQLYGATIVTAKESGKLTSPIEI
  	KKNRLVDLYGAYTNGTSAFMTIIKFTGNKPKYKVIGIPTTSAASLKRAGK
  	PGSESYNQELHRIIKSNPKVKKGFEIVVPHVSYGQLIVDGDCKFTLASPTV
  	QHPATQLVLSKKSLETISSGYKILKDKPAIANERLIRVFDEVVGQMNRYF
  	TIFDQRSNRQKVADARDKFLSLPTESKYEGAKKVQVGKTEVITNLLMGL
  	HANATQGDLKVLGLATFGFFQSTTGLSLSEDTMIVYQSPTGLFERRICLK
  	DI (SEQ ID NO: 26)
  SaCas9	MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIG
  Staphylococcus	ALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFH
  aureus	RLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKA
  wild type	DLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEEN
  GenBank:	PlNASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP
  AYD60528.1	NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSD
  	AILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYK
  	EIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDL
  	LRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY
  	YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFD
  	KNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAI
  	VDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDL
  	LKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVM
  	KQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLI
  	HDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDEL
  	VKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQIL
  	KEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQ
  	SFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLI
  	TQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNT
  	KYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
  	AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFY
  	SNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSM
  	PQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPT
  	VAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYK
  	EVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLY
  	LASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANL
  	DKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYT
  	STKEVLDATLIHQSITGLYETRIDLSQLGGD (SEQ ID NO: 27)
  SaCas9	MGKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRS
  Staphylococcus	KRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQ
  aureus	KLSEEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEE
  	KYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQ
  	SFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELR
  	SVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKP
  	TLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENA
  	ELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSL
  	KAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSP
  	VVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNR
  	QTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAlPLEDLLNNP
  	FNYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKISY
  	ETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRY
  	ATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYK
  	HHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQ
  	EYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRKLINDTLYSTRKDDKGN
  	TLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQ
  	YGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDIT
  	DDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEV
  	NSKCYEEAKKLKKISNQAEFIASFYKNDLIKINGELYRVIGVNNDLLNRIE
  	VNMIDITYREYLENMNDKRPPHIIKTIASKTQSIKKYSTDILGNLYEVKSK
  	KHPQIIKK
  	(SEQ ID NO: 28)
  StCas9	MLFNKCIIISINLDFSNKEKCMTKPYSIGLDIGTNSVGWAVITDNYKVPSK
  Streptococcus	KMKVLGNTSKKYIKKNLLGVLLFDSGITAEGRRLKRTARRRYTRRRNRI
  thermophilus	LYLQEIFSTEMATLDDAFFQRLDDSFLVPDDKRDSKYPIFGNLVEEKVYH
  UniProtKB/	DEFPTIYHLRKYLADSTKKADLRLVYLALAHMIKYRGHFLIEGEFNSKN
  Swiss-Prot:	NDIQKNFQDFLDTYNAIFESDLSLENSKQLEEIVKDKISKLEKKDRILKLF
  G3ECR1.2	PGEKNSGIFSEFLKLIVGNQADFRKCFNLDEKASLHFSKESYDEDLETLL
  Wild type	GYIGDDYSDVFLKAKKLYDAILLSGFLTVTDNETEAPLSSAMIKRYNEH
  	KEDLALLKEYIRNISLKTYNEVFKDDTKNGYAGYIDGKTNQEDFYVYLK
  	NLLAEFEGADYFLEKIDREDFLRKQRTFDNGSIPYQIHLQEMRAILDKQA
  	KFYPFLAKNKERIEKILTFRIPYYVGPLARGNSDFAWSIRKRNEKITPWNF
  	EDVIDKESSAEAFINRMTSFDLYLPEEKVLPKHSLLYETFNVYNELTKVR
  	FIAESMRDYQFLDSKQKKDIVRLYFKDKRKVTDKDIIEYLHAIYGYDGIE
  	LKGIEKQFNSSLSTYHDLLNIINDKEFLDDSSNEAIIEEIIHTLTIFEDREMIK
  	QRLSKFENIFDKSVLKKLSRRHYTGWGKLSAKLINGIRDEKSGNTILDYLI
  	DDGISNRNFMQLIHDDALSFKKKIQKAQIIGDEDKGNIKEVVKSLPGSPAI
  	KKGILQSIKIVDELVKVMGGRKPESIVVEMARENQYTNQGKSNSQQRLK
  	RLEKSLKELGSKILKEMPAKLSKIDNNALQNDRLYLYYLQNGKDMYTG
  	DDLDIDRLSNYDIDHIIPQAFLKDNSIDNKVLVSSASNRGKSDDFPSLEVV
  	KKRKTFWYQLLKSKLISQRKFDNLTKAERGGLLPEDKAGFIQRQLVETR
  	QITKHVARLLDEKFNNKKDENNRAVRTVKIITLKSTLVSQFRKDFELYK
  	VREINDFHHAHDAYLNAVIASALLKKYPKLEPEFVYGDYPKYNSFRERK
  	SATEKVYFYSNIMNIFKKSISLADGRVIERPLIEVNEETGESVWNKESDLA
  	TVRRVLSYPQVNVVKKVEEQNHGLDRGKPKGLFNANLSSKPKPNSNEN
  	LVGAKEYLDPKKYGGYAGISNSFAVLVKGTIEKGAKKKITNVLEFQGISI
  	LDRINYRKDKLNFLLEKGYKDIELIIELPKYSLFELSDGSRRMLASILSTN
  	NKRGEIHKGNQIFLSQKFVKLLYHAKRISNTINENHRKYVENHKKEFEEL
  	FYYILEFNENYVGAKKNGKLLNSAFQSWQNHSIDELCSSFIGPTGSERKG
  	LFELTSRGSAADFEFLGVKIPRYRDYTPSSLLKDATLIHQSVTGLYETRID
  	LAKLGEG (SEQ ID NO: 29)
  LcCas9	MKIKNYNLALTPSTSAVGHVEVDDDLNILEPVHHQKAIGVAKFGEGETA
  Lactobacillus	EARRLARSARRTTKRRANRINHYFNEIMKPEIDKVDPLMFDRIKQAGLSP
  crispatus	LDERKEFRTVIFDRPNIASYYHNQFPTIWHLQKYLMITDEKADIRLIYWA
  NCBI	LHSLLKHRGHFFNTTPMSQFKPGKLNLKDDMLALDDYNDLEGLSFAVA
  Reference	NSPEIEKVIKDRSMHKKEKIAELKKLIVNDVPDKDLAKRNNKIITQIVNAI
  Sequence:	MGNSFHLNFIFDMDLDKLTSKAWSFKLDDPELDTKFDAISGSMTDNQIGI
  WP_133478044.1	FETLQKIYSAISLLDILNGSSNVVDAKNALYDKHKRDLNLYFKFLNTLPD
  Wild type	EIAKTLKAGYTLYIGNRKKDLLAARKLLKVNVAKNFSQDDFYKLINKEL
  	KSIDKQGLQTRFSEKVGELVAQNNFLPVQRSSDNVFIPYQLNAITFNKILE
  	NQGKYYDFLVKPNPAKKDRKNAPYELSQLMQFTIPYYVGPLVTPEEQV
  	KSGIPKTSRFAWMVRKDNGAITPWNFYDKVDIEATADKFIKRSIAKDSY
  	LLSELVLPKHSLLYEKYEVFNELSNVSLDGKKLSGGVKQILFNEVFKKTN
  	KVNTSRILKALAKHNIPGSKITGLSNPEEFTSSLQTYNAWKKYFPNQIDNF
  	AYQQDLEKMIEWSTVFEDHKILAKKLDEIEWLDDDQKKFVANTRLRGW
  	GRLSKRLLTGLKDNYGKSIMQRLETTKANFQQIVYKPEFREQIDKISQAA
  	AKNQSLEDILANSYTSPSNRKAIRKTMSVVDEYIKLNHGKEPDKIFLMFQ
  	RSEQEKGKQTEARSKQLNRILSQLKADKSANKLFSKQLADEFSNAIKKS
  	KYKLNDKQYFYFQQLGRDALTGEVIDYDELYKYTVLHIIPRSKLTDDSQ
  	NNKVLTKYKIVDGSVALKFGNSYSDALGMPIKAFWTELNRLKLIPKGKL
  	LNLTTDFSTLNKYQRDGYIARQLVETQQIVKLLATIMQSRFKHTKIIEVR
  	NSQVANIRYQFDYFRIKNLNEYYRGFDAYLAAVVGTYLYKVYPKARRL
  	FVYGQYLKPKKTNQENQDMHLDSEKKSQGFNFLWNLLYGKQDQIFVN
  	GTDVIAFNRKDLITKMNTVYNYKSQKISLAIDYHNGAMFKATLFPRNDR
  	DTAKTRKLIPKKKDYDTDIYGGYTSNVDGYMLLAEIIKRDGNKQYGFYG
  	VPSRLVSELDTLKKTRYTEYEEKLKEIIKPELGVDLKKIKKIKILKNKVPF
  	NQVIIDKGSKFFITSTSYRWNYRQLILSAESQQTLMDLVVDPDFSNHKAR
  	KDARKNADERLIKVYEEILYQVKNYMPMFVELHRCYEKLVDAQKTFKS
  	LKISDKAMVLNQILILLHSNATSPVLEKLGYHTRFTLGKKHNLISENAVL
  	VTQSITGLKENHVSIKQML (SEQ ID NO: 30)
  PdCas9	MTNEKYSIGLDIGTSSIGFAVVNDNNRVIRVKGKNAIGVRLFDEGKAAA
  Pedicoccus	DRRSFRTTRRSFRTTRRRLSRRRWRLKLLREIFDAYITPVDEAFFIRLKES
  damnosus	NLSPKDSKKQYSGDILFNDRSDKDFYEKYPTIYHLRNALMTEHRKFDVR
  NCBI	EIYLAIHHIMKFRGHFLNATPANNFKVGRLNLEEKFEELNDIYQRVFPDE
  Reference	SIEFRTDNLEQIKEVLLDNKRSRADRQRTLVSDIYQSSEDKDIEKRNKAV
  Sequence:	ATEILKASLGNKAKLNVITNVEVDKEAAKEWSITFDSESIDDDLAKIEGQ
  WP_062913273.1	MTDDGHEIIEVLRSLYSGITLSAIVPENHTLSQSMVAKYDLHKDHLKLFK
  Wild type	KLINGMTDTKKAKNLRAAYDGYIDGVKGKVLPQEDFYKQVQVNLDDS
  	AEANEIQTYIDQDIFMPKQRTKANGSIPHQLQQQELDQIIENQKAYYPWL
  	AELNPNPDKKRQQLAKYKLDELVTFRVPYYVGPMITAKDQKNQSGAEF
  	AWMIRKEPGNITPWNFDQKVDRMATANQFIKRMTTTDTYLLGEDVLPA
  	QSLLYQKFEVLNELNKIRIDHKPISIEQKQQIFNDLFKQFKNVTIKHLQDY
  	LVSQGQYSKRPLIEGLADEKRFNSSLSTYSDLCGIFGAKLVEENDRQEDL
  	EKIIEWSTIFEDKKIYRAKLNDLTWLTDDQKEKLATKRYQGWGRLSRKL
  	LVGLKNSEHRNIMDILWITNENFMQIQAEPDFAKLVTDANKGMLEKTDS
  	QDVINDLYTSPQNKKAIRQILLVVHDIQNAMHGQAPAKIHVEFARGEER
  	NPRRSVQRQRQVEAAYEKVSNELVSAKVRQEFKEAINNKRDFKDRLFL
  	YFMQGGIDIYTGKQLNIDQLSSYQIDHILPQAFVKDDSLTNRVLTNENQV
  	KADSVPIDIFGKKMLSVWGRMKDQGLISKGKYRNLTMNPENISAHTENG
  	FINRQLVETRQVIKLAVNILADEYGDSTQIISVKADLSHQMREDFELLKN
  	RDVNDYHHAFDAYLAAFIGNYLLKRYPKLESYFVYGDFKKFTQKETKM
  	RRFNFIYDLKHCDQVVNKETGEILWTKDEDIKYIRHLFAYKKILVSHEVR
  	EKRGALYNQTIYKAKDDKGSGQESKKLIRIKDDKETKIYGGYSGKSLAY
  	MTIVQITKKNKVSYRVIGIPTLALARLNKLENDSTENNGELYKIIKPQFTH
  	YKVDKKNGEIIETTDDFKIVVSKVRFQQLIDDAGQFFMLASDTYKNNAQ
  	QLVISNNALKAINNTNITDCPRDDLERLDNLRLDSAFDEIVKKMDKYFSA
  	YDANNFREKIRNSNLIFYQLPVEDQWENNKITELGKRTVLTRILQGLHAN
  	ATTTDMSIFKIKTPFGQLRQRSGISLSENAQLIYQSPTGLFERRVQLNKIK
  	(SEQ ID NO: 31)
  FnCas9	MKKQKFSDYYLGFDIGTNSVGWCVTDLDYNVLRFNKKDMWGSRLFEE
  Fusobaterium	AKTAAERRVQRNSRRRLKRRKWRLNLLEEIFSNEILKIDSNFFRRLKESSL
  nucleatum	WLEDKSSKEKFTLFNDDNYKDYDFYKQYPTIFHLRNELIKNPEKKIARLV
  NCBI	YLAIHSIFKSRGHFLFEGQNLKEIKNFETLYNNLIAFLEDNGINKIIDKNNI
  Reference	EKLEKIVCDSKKGLKDKEKEFKEIFNSDKQLVAIFKLSVGSSVSLNDLFD
  Sequence:	TDEYKKGEVEKEKISFREQIYEDDKPIYYSILGEKIELLDIAKTFYDFMVL
  WP_060798984.1	NNILADSQYISEAKVKLYEEHKKDLKNLKYIIRKYNKGNYDKLFKDKNE
  	NNYSAYIGLNKEKSKKEVIEKSRLKIDDLIKNIKGYLPKVEEIEEKDKAIF
  	NKILNKIELKTILPKQRISDNGTLPYQIHEAELEKILENQSKYYDFLNYEE
  	NGIITKDKLLMTFKFRIPYYVGPLNSYHKDKGGNSWIVRKEEGKILPWNF
  	EQKVDIEKSAEEFIKRMTNKCTYLNGEDVIPKDTFLYSEYVILNELNKVQ
  	VNDEFLNEENKRKIIDELFKENKKVSEKKFKEYLLVKQIVDGTIELKGVK
  	DSFNSNYISYIRFKDIFGEKLNLDIYKEISEKSILWKCLYGDDKKIFEKKIK
  	NEYGDILTKDEIKKINTFKFNNWGRLSEKLLTGIEFINLETGECYSSVMDA
  	LRRTNYNLMELLSSKFTLQESINNENKEMNEASYRDLIEESYVSPSLKRAI
  	FQTLKIYEEIRKITGRVPKKVFIEMARGGDESMKNKKIPARQEQLKKLYD
  	SCGNDIANFSIDIKEMKNSLISYDNNSLRQKKLYLYYLQFGKCMYTGREI
  	DLDRLLQNNDTYDIDHIYPRSKVIKDDSFDNLVLVLKNENAEKSNEYPV
  	KKEIQEKMKSFWRFLKEKNFISDEKYKRLTGKDDFELRGFMARQLVNV
  	RQTTKEVGKILQQIEPEIKIVYSKAEIASSFREMFDFIKVRELNDTHHAKD
  	AYLNIVAGNVYNTKFTEKPYRYLQEIKENYDVKKIYNYDIKNAWDKEN
  	SLEIVKKNMEKNTVNITRFIKEKKGQLFDLNPIKKGETSNEIISIKPKVYN
  	GKDDKLNEKYGYYKSLNPAYFLYVEHKEKNKRIKSFERVNLVDVNNIK
  	DEKSLVKYLIENKKLVEPRVIKKVYKRQVILINDYPYSIVTLDSNKLMDF
  	ENLKPLFLENKYEKILKNVIKFLEDNQGKSEENYKFIYLKKKDRYEKNET
  	LESVKDRYNLEFNEMYDKFLEKLDSKDYKNYMNNKKYQELLDVKEKFI
  	KLNLFDKAFTLKSFLDLFNRKTMADFSKVGLTKYLGKIQKISSNVLSKNE
  	LYLLEESVTGLFVKKIKL (SEQ ID NO: 32)
  EcCas9	RRKQRIQILQELLGEEVLKTDPGFFHRMKESRYVVEDKRTLDGKQVELP
  Enterococcus	YALFVDKDYTDKEYYKQFPTINHLIVYLMTTSDTPDIRLVYLALHYYMK
  cecorum	NRGNFLHSGDINNVKDINDILEQLDNVLETFLDGWNLKLKSYVEDIKNIY
  NCBI	NRDLGRGERKKAFVNTLGAKTKAEKAFCSLISGGSTNLAELFDDSSLKEI
  Reference	ETPKIEFASSSLEDKIDGIQEALEDRFAVIEAAKRLYDWKTLTDILGDSSS
  Sequence:	LAEARVNSYQMHHEQLLELKSLVKEYLDRKVFQEVFVSLNVANNYPAY
  WP_047338501.1	IGHTKINGKKKELEVKRTKRNDFYSYVKKQVIEPIKKKVSDEAVLTKLSE
  Wild type	IESLIEVDKYLPLQVNSDNGVIPYQVKLNELTRIFDNLENRIPVLRENRDK
  	IIKTFKFRIPYYVGSLNGVVKNGKCTNWMVRKEEGKIYPWNFEDKVDLE
  	ASAEQFIRRMTNKCTYLVNEDVLPKYSLLYSKYLVLSELNNLRIDGRPLD
  	VKIKQDIYENVFKKNRKVTLKKIKKYLLKEGIITDDDELSGLADDVKSSL
  	TAYRDFKEKLGHLDLSEAQMENIILNITLFGDDKKLLKKRLAALYPFIDD
  	KSLNRIATLNYRDWGRLSERFLSGITSVDQETGELRTIIQCMYETQANLM
  	QLLAEPYHFVEAIEKENPKVDLESISYRIVNDLYVSPAVKRQIWQTLLVIK
  	DIKQVMKHDPERIFIEMAREKQESKKTKSRKQVLSEVYKKAKEYEHLFE
  	KLNSLTEEQLRSKKIYLYFTQLGKCMYSGEPIDFENLVSANSNYDIDHIYP
  	QSKTIDDSFNNIVLVKKSLNAYKSNHYPIDKNIRDNEKVKTLWNTLVSK
  	GLITKEKYERURSTPFSDEELAGFIARQLVETRQSTKAVAEILSNWFPESE
  	IVYSKAKNVSNFRQDFEILKVRELNDCHHAHDAYLNIVVGNAYHTKFTN
  	SPYRFIKNKANQEYNLRKLLQKVNKIESNGVVAWVGQSENNPGTIATVK
  	KVIRRNTVLISRMVKEVDGQLFDLTLMKKGKGQVPIKSSDERLTDISKY
  	GGYNKATGAYFTFVKSKKRGKVVRSFEYVPLHLSKQFENNNELLKEYIE
  	KDRGLTDVEILIPKVLINSLFRYNGSLVRITGRGDTRLLLVHEQPLYVSNS
  	FVQQLKSVSSYKLKKSENDNAKLTKTATEKLSNIDELYDGLLRKLDLPIY
  	SYWFSSIKEYLVESRTKYIKLSIEEKALVIFEILHLFQSDAQVPNLKILGLS
  	TKPSRIRIQKNLKDTDKMSIIHQSPSGIFEHEIELTSL (SEQ ID NO: 33)
  AhCas9	MQNGFLGITVSSEQVGWAVTNPKYELERASRKDLWGVRLFDKAETAED
  Anaerostipes	RRMFRTNRRLNQRKKNRIHYLRDIFHEEVNQKDPNFFQQLDESNFCEDD
  hadrus	RTVEFNFDTNLYKNQFPTVYHLRKYLMETKDKPIARLVYLAFSKFMKN
  NCBI	RGHFLYKGNLGEVMDFENSMKGFCESLEKFNIDFPTLSDEQVKEVRDIL
  Reference	CDHKIAKTVKKKNIITITKVKSKTAKAWIGLFCGCSVPVKVLFQDIDEEIV
  Sequence:	TDPEKISFEDASYDDYIANIEKGVGIYYEAIVSAKMLFDWSILNEILGDHQ
  WP_044924278.1	LLSDAMIAEYNKHHDDLKRLQKIIKGTGSRELYQDIFINDVSGNYVCYV
  Wild type	GHAKTMSSADQKQFYTFLKNRLKNVNGISSEDAEWIDTEIKNGTLLPKQ
  	TKRDNSVIPHQLQLREFELILDNMQEMYPFLKENREKLLKIFNFVIPYYV
  	GPLKGVVRKGESTNWMVPKKDGVIHPWNFDEMVDKEASAECFISRMT
  	GNCSYLFNEKVLPKNSLLYETFEVLNELNPLKINGEPISVELKQRIYEQLF
  	LTGKKVTKKSLTKYLIKNGYDKDIELSGIDNEFHSNLKSHIDFEDYDNLS
  	DEEVEQIILRITVFEDKQLLKDYLNREFVKLSEDERKQICSLSYKGWGNL
  	SEMLLNGITVTDSNGVEVSVMDMLWNTNLNLMQILSKKYGYKAEIEHY
  	NKEHEKTIYNREDLMDYLNIPPAQRRKVNQLITIVKSLKKTYGVPNKIFF
  	KISREHQDDPKRTSSRKEQLKYLYKSLKSEDEKHLMKELDELNDHELSN
  	DKVYLYFLQKGRCIYSGKKLNLSRLRKSNYQNDIDYIYPLSAVNDRSMN
  	NKVLTGIQENRADKYTYFPVDSEIQKKMKGFWMELVLQGFMTKEKYFR
  	LSRENDFSKSELVSFIEREISDNQQSGRMIASVLQYYFPESKIVFVKEKLIS
  	SFKRDFHLISSYGHNHLQAAKDAYITIVVGNVYHTKFTMDPAIYFKNHK
  	RKDYDLNRLFLENISRDGQIAWESGPYGSIQTVRKEYAQNHIAVTKRVV
  	EVKGGLFKQMPLKKGHGEYPLKTNDPRFGNIAQYGGYTNVTGSYFVLV
  	ESMEKGKKRISLEYVPVYLHERLEDDPGHKLLKEYLVDHRKLNHPKILL
  	AKVRKNSLLKIDGFYYRLNGRSGNALILTNAVELIMDDWQTKTANKISG
  	YMKRRAIDKKARVYQNEFHIQELEQLYDFYLDKLKNGVYKNRKNNQA
  	ELIHNEKEQFMELKTEDQCVLLTEIKKLFVCSPMQADLTLIGGSKHTGMI
  	AMSSNVTKADFAVIAEDPLGLRNKVIYSHKGEK (SEQ ID NO: 34)
  KvCas9	MSQNNNKIYNIGLDIGDASVGWAVVDEHYNLLKRHGKHMWGSRLFTQ
  Kandleria	ANTAVERRSSRSTRRRYNKRRERIRLLREIMEDMVLDVDPTFFIRLANVS
  vitulina	FLDQEDKKDYLKENYHSNYNLFIDKDFNDKTYYDKYPTIYHLRKHLCES
  NCBI	KEKEDPRLIYLALHHIVKYRGNFLYEGQKFSMDVSNIEDKMIDVLRQFN
  Reference	EINLFEYVEDRKKIDEVLNVLKEPLSKKHKAEKAFALFDTTKDNKAAYK
  Sequence:	ELCAALAGNKFNVTKMLKEAELHDEDEKDISFKFSDATFDDAFVEKQPL
  WP_031589969.1	LGDCVEFIDLLHDIYSWVELQNILGSAHTSEPSISAAMIQRYEDHKNDLK
  Wild type	LLKDVIRKYLPKKYFEVFRDEKSKKNNYCNYINHPSKTPVDEFYKYIKK
  	LIEKIDDPDVKTILNKIELESFMLKQNSRTNGAVPYQMQLDELNKILENQ
  	SVYYSDLKDNEDKIRSILTFRIPYYFGPLNITKDRQFDWIIKKEGKENERIL
  	PWNANEIVDVDKTADEFIKRMRNFCTYFPDEPVMAKNSLTVSKYEVLN
  	EINKLRINDHLIKRDMKDKMLHTLFMDHKSISANAMKKWLVKNQYFSN
  	TDDIKIEGFQKENACSTSLTPWIDFTKIFGKINESNYDFIEKIIYDVTVFED
  	KKILRRRLKKEYDLDEEKIKKILKLKYSGWSRLSKKLLSGIKTKYKDSTR
  	TPETVLEVMERTNMNLMQVINDEKLGFKKTIDDANSTSVSGKFSYAEVQ
  	ELAGSPAIKRGIWQALLIVDEIKKIMKHEPAHVYIEFARNEDEKERKDSF
  	VNQMLKLYKDYDFEDETEKEANKHLKGEDAKSKIRSERLKLYYTQMG
  	KCMYTGKSLDIDRLDTYQVDHIVPQSLLKDDSIDNKVLVLSSENQRKLD
  	DLVIPSSIRNKMYGFWEKLFNNKIISPKKFYSLIKTEFNEKDQERFINRQIV
  	ETRQITKHVAQIIDNHYENTKVVTVRADLSHQFRERYHIYKNRDINDFHH
  	AHDAYIATILGTYIGHRFESLDAKYIYGEYKRIFRNQKNKGKEMKKNND
  	GFILNSMRNIYADKDTGEIVWDPNYIDRIKKCFYYKDCFVTKKLEENNG
  	TFFNVTVLPNDTNSDKDNTLATVPVNKYRSNVNKYGGFSGVNSFIVAIK
  	GKKKKGKKVIEVNKLTGIPLMYKNADEEIKINYLKQAEDLEEVQIGKEIL
  	KNQLIEKDGGLYYIVAPTEIINAKQLILNESQTKLVCEIYKAMKYKNYDN
  	LDSEKIIDLYRLLINKMELYYPEYRKQLVKKFEDRYEQLKVISIEEKCNII
  	KQILATLHCNSSIGKIMYSDFKISTTIGRLNGRTISLDDISFIAESPTGMYSK
  	KYKL (SEQ ID NO: 35)
  EfCas9	MRLFEEGHTAEDRRLKRTARRRISRRRNRLRYLQAFFEEAMTDLDENFF
  Enterococcus	ARLQESFLVPEDKKWHRHPIFAKLEDEVAYHETYPTIYHLRKKLADSSE
  faecalis	QADLRLIYLALAHIVKYRGHFLIEGKLSTENTSVKDQFQQFMVIYNQTFV
  NCBI	NGESRLVSAPLPESVLIEEELTEKASRTKKSEKVLQQFPQEKANGLFGQF
  Reference	LKLMVGNKADFKKVFGLEEEAKITYASESYEEDLEGILAKVGDEYSDVF
  Sequence:	LAAKNVYDAVELSTILADSDKKSHAKLSSSMIVRFTEHQEDLKKFKRFIR
  WP_016631044.1	ENCPDEYDNLFKNEQKDGYAGYIAHAGKVSQLKFYQYVKKIIQDIAGAE
  Wild type	YFLEKIAQENFLRKQRTFDNGVIPHQIHLAELQAIIHRQAAYYPFLKENQE
  	KIEQLVTFRIPYYVGPLSKGDASTFAWLKRQSEEPIRPWNLQETVDLDQS
  	ATAFIERMTNFDTYLPSEKVLPKHSLLYEKFMVFNELTKISYTDDRGIKA
  	NFSGKEKEKIFDYLFKTRRKVKKKDIIQFYRNEYNTEIVTLSGLEEDQFN
  	ASFSTYQDLLKCGLTRAELDHPDNAEKLEDIIKILTIFEDRQRIRTQLSTFK
  	GQFSAEVLKKLERKHYTGWGRLSKKLINGIYDKESGKTILDYLVKDDGV
  	SKHYNRNFMQLINDSQLSFKNAIQKAQSSEHEETLSETVNELAGSPAIKK
  	GIYQSLKIVDELVAIMGYAPKRIVVEMARENQTTSTGKRRSIQRLKIVEK
  	AMAEIGSNLLKEQPTTNEQLRDTRLFLYYMQNGKDMYTGDELSLHRLS
  	HYDIDHIIPQSFMKDDSLDNLVLVGSTENRGKSDDVPSKEVVKDMKAY
  	WEKLYAAGLISQRKFQRLTKGEQGGLTLEDKAHFIQRQLVETRQITKNV
  	AGILDQRYNAKSKEKKVQIITLKASLTSQFRSIFGLYKVREVNDYHHGQD
  	AYLNCVVATTLLKVYPNLAPEFVYGEYPKFQTFKENKATAKAIIYTNLL
  	RFFTEDEPRFTKDGEILWSNSYLKTIKKELNYHQMNIVKKVEVQKGGFS
  	KESIKPKGPSNKLIPVKNGLDPQKYGGFDSPVVAYTVLFTHEKGKKPLIK
  	QEILGITIMEKTRFEQNPILFLEEKGFLRPRVLMKLPKYTLYEFPEGRRRL
  	LASAKEAQKGNQMVLPEHLLTLLYHAKQCLLPNQSESLAYVEQHQPEF
  	QEILERVVDFAEVHTLAKSKVQQIVKLFEANQTADVKEIAASFIQLMQFN
  	AMGAPSTFKFFQKDIERARYTSIKEIFDATIIYQSPTGLYETRRKVVD
  	(SEQ ID NO: 36)
  Staphylococcus	KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKR
  aureus	GARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKL
  Cas9	SEEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKY
  	VAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFI
  	DTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSV
  	KYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTL
  	KQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAEL
  	LDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAI
  	NLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVK
  	RSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTN
  	ERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNY
  	EVDHIIPRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKISYETF
  	KKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATR
  	GLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHA
  	EDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYK
  	EIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIV
  	NNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDE
  	KNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYP
  	NSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKC
  	YEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMI
  	DITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQ
  	IIKKG
  	(SEQ ID NO: 37)
  Geobacillus	MKYKIGLDIGITSIGWAVINLDIPRIEDLGVRIFDRAENPKTGESLALPRRL
  thermodenitrificans	ARSARRRLRRRKHRLERIRRLFVREGILTKEELNKLFEKKHEIDVWQLRV
  Cas9	EALDRKLNNDELARILLHLAKRRGFRSNRKSERTNKENSTMLKHIEENQ
  	SILSSYRTVAEMVVKDPKFSLHKRNKEDNYTNTVARDDLEREIKLIFAKQ
  	REYGNIVCTEAFEHEYISIWASQRPFASKDDIEKKVGFCTFEPKEKRAPK
  	ATYTFQSFTVWEHINKLRLVSPGGIRALTDDERRLIYKQAFHKNKITFHD
  	VRTLLNLPDDTRFKGLLYDRNTTLKENEKVRFLELGAYHKIRKAIDSVY
  	GKGAAKSFRPIDFDTFGYALTMFKDDTDIRSYLRNEYEQNGKRMENLA
  	DKVYDEELIEELLNLSFSKFGHLSLKALRNILPYMEQGEVYSTACERAGY
  	TFTGPKKKQKTVLLPNIPPIANPVVMRALTQARKVVNAIIKKYGSPVSIHI
  	ELARELSQSFDERRKMQKEQEGNRKKNETAIRQLVEYGLTLNPTGLDIV
  	KFKLWSEQNGKCAYSLQPIEIERLLEPGYTEVDHVIPYSRSLDDSYTNKV
  	LVLTKENREKGNRTPAEYLGLGSERWQQFETFVLTNKQFSKKKRDRLLR
  	LHYDENEENEFKNRNLNDTRYISRFLANFIREHLKFADSDDKQKVYTVN
  	GRITAHLRSRWNFNKNREESNLHHAVDAAIVACTTPSDIARVTAFYQRR
  	EQNKELSKKTDPQFPQPWPHFADELQARLSKNPKESIKALNLGNYDNEK
  	LESLQPVFVSRMPKRSITGAAHQETLRRYIGIDERSGKIQTVVKKKLSEIQ
  	LDKTGHFPMYGKESDPRTYEAIRQRLLEHNNDPKKAFQEPLYKPKKNGE
  	LGPIIRTIKIIDTTNQVIPLNDGKTVAYNSNIVRVDVFEKDGKYYCVPIYTI
  	DMMKGILPNKAIEPNKPYSEWKEMTEDYTFRFSLYPNDLIRIEFPREKTIK
  	TAVGEEIKIKDLFAYYQTIDSSNGGLSLVSHDNNFSLRSIGSRTLKRFEKY
  	QVDVLGNIYKVRGEKRVGVASSSHSKAGETIRPL
  	(SEQ ID NO: 38)
  ScCas9	MEKKYSIGLDIGTNSVGWAVITDDYKVPSKKFKVLGNTNRKSIKKNLM
  S. canis	GALLFDSGETAEATRLKRTARRRYTRRKNRIRYLQEIFANEMAKLDDSF
  1375 AA	FQRLEESFLVEEDKKNERHPIFGNLADEVAYHRNYPTIYHLRKKLADSPE
  159.2 kDa	KADLRLIYLALAHIIKFRGHFLIEGKLNAENSDVAKLFYQLIQTYNQLFEE
  	SPLDEIEVDAKGILSARLSKSKRLEKLIAVFPNEKKNGLFGNIIALALGLTP
  	NFKSNFDLTEDAKLQLSKDTYDDDLDELLGQIGDQYADLFSAAKNLSDA
  	ILLSDILRSNSEVTKAPLSASMVKRYDEHHQDLALLKTLVRQQFPEKYAE
  	IFKDDTKNGYAGYVGIGIKHRKRTTKLATQEEFYKFIKPILEKMDGAEEL
  	LAKLNRDDLLRKQRTFDNGSIPHQIHLKELHAILRRQEEFYPFLKENREKI
  	EKILTFRIPYYVGPLARGNSRFAWLTRKSEEAITPWNFEEVVDKGASAQS
  	FIERMTNFDEQLPNKKVLPKHSLLYEYFTVYNELTKVKYVTERMRKPEF
  	LSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEIIGVEDRFNA
  	SLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA
  	HLFDDKVMKQLKRRHYTGWGRLSRKMINGIRDKQSGKTILDFLKSDGF
  	SNRNFMQLIHDDSLTFKEEIEKAQVSGQGDSLHEQIADLAGSPAIKKGIL
  	QTVKIVDELVKVMGHKPENIVIEMARENQTTTKGLQQSRERKKRIEEGIK
  	ELESQILKENPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDV
  	DHIVPQSFIKDDSIDNKVLTRSVENRGKSDNVPSEEVVKKMKNYWRQLL
  	NAKLITQRKFDNLTKAERGGLSEADKAGFIKRQLVETRQITKHVARILDS
  	RMNTKRDKNDKPIREVKVITLKSKLVSDFRKDFQLYKVRDINNYHHAH
  	DAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKAT
  	AKRFFYSNIMNFFKTEVKLANGEIRKRPLIETNGETGEVVWNKEKDFAT
  	VRKVLAMPQVNIVKKTEVQTGGFSKESILSKRESAKLIPRKKGWDTRKY
  	GGFGSPTVAYSILVVAKVEKGKAKKLKSVKVLVGITIMEKGSYEKDPIGF
  	LEAKGYKDIKKELIFKLPKYSLFELENGRRRMLASATELQKANELVLPQ
  	HLVRLLYYTQNISATTGSNNLGYIEQHREEFKEIFEKIIDFSEKYILKNKV
  	NSNLKSSFDEQFAVSDSILLSNSFVSLLKYTSFGASGGFTFLDLDVKQGRL
  	RYQTVTEVLDATLIYQSITGLYETRTDLSQLGGD (SEQ ID NO: 39)

• The prime editors described herein may include any of the above Cas9 ortholog sequences, or any variants thereof having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto.
• The napDNAbp may include any suitable homologs and/or orthologs or naturally occurring enzymes, such as, Cas9. Cas9 homologs and/or orthologs have been described in various species, including, but not limited to, S. pyogenes and S. thermophilus. Preferably, the Cas moiety is configured (e.g, mutagenized, recombinantly engineered, or otherwise obtained from nature) as a nickase, i.e., capable of cleaving only a single strand of the target doubpdditional suitable Cas9 nucleases and sequences will be apparent to those of skill in the art based on this disclosure, and such Cas9 nucleases and sequences include Cas9 sequences from the organisms and loci disclosed in Chylinski, Rhun, and Charpentier, “The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems” (2013) RNA Biology 10:5, 726-737; the entire contents of which are incorporated herein by reference. In some embodiments, a Cas9 nuclease has an inactive (e.g., an inactivated) DNA cleavage domain, that is, the Cas9 is a nickase. In some embodiments, the Cas9 protein comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of a Cas9 protein as provided by any one of the variants of Table 3. In some embodiments, the Cas9 protein comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the amino acid sequence of a Cas9 protein as provided by any one of the Cas9 orthologs in the above tables.
• C. Dead Cas9 Variant
• In certain embodiments, the prime editors described herein may include a dead Cas9, e.g., dead SpCas9, which has no nuclease activity due to one or more mutations that inactive both nuclease domains of Cas9, namely the RuvC domain (which cleaves the non-protospacer DNA strand) and HNH domain (which cleaves the protospacer DNA strand). The nuclease inactivation may be due to one or mutations that result in one or more substitutions and/or deletions in the amino acid sequence of the encoded protein, or any variants thereof having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto.
• As used herein, the term “dCas9” refers to a nuclease-inactive Cas9 or nuclease-dead Cas9, or a functional fragment thereof, and embraces any naturally occurring dCas9 from any organism, any naturally-occurring dCas9 equivalent or functional fragment thereof, any dCas9 homolog, ortholog, or paralog from any organism, and any mutant or variant of a dCas9, naturally-occurring or engineered. The term dCas9 is not meant to be particularly limiting and may be referred to as a “dCas9 or equivalent.” Exemplary dCas9 proteins and method for making dCas9 proteins are further described herein and/or are described in the art and are incorporated herein by reference.
• In other embodiments, dCas9 corresponds to, or comprises in part or in whole, a Cas9 amino acid sequence having one or more mutations that inactivate the Cas9 nuclease activity. In other embodiments, Cas9 variants having mutations other than D10A and H840A are provided which may result in the full or partial inactivate of the endogenous Cas9 nuclease activity (e.g., nCas9 or dCas9, respectively). Such mutations, by way of example, include other amino acid substitutions at D10 and H820, or other substitutions within the nuclease domains of Cas9 (e.g., substitutions in the HNH nuclease subdomain and/or the RuvC1 subdomain) with reference to a wild type sequence such as Cas9 from Streptococcus pyogenes (NCBI Reference Sequence: NC_017053.1). In some embodiments, variants or homologues of Cas9 (e.g., variants of Cas9 from Streptococcus pyogenes (NCBI Reference Sequence: NC_017053.1 (SEQ ID NO: 20))) are provided which are at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to NCBI Reference Sequence: NC_017053.1. In some embodiments, variants of dCas9 (e.g., variants of NCBI Reference Sequence: NC_017053.1 (SEQ ID NO: 20)) are provided having amino acid sequences which are shorter, or longer than NC_017053.1 (SEQ ID NO: 20) by about 5 amino acids, by about 10 amino acids, by about 15 amino acids, by about 20 amino acids, by about 25 amino acids, by about 30 amino acids, by about 40 amino acids, by about 50 amino acids, by about 75 amino acids, by about 100 amino acids or more.
• In one embodiment, the dead Cas9 may be based on the canonical SpCas9 sequence of Q99ZW2 and may have the following sequence, which comprises a D10X and an H810X, wherein X may be any amino acid, substitutions (underlined and bolded), or a variant be variant of SEQ ID NO: 40 having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto.
• In one embodiment, the dead Cas9 may be based on the canonical SpCas9 sequence of Q99ZW2 and may have the following sequence, which comprises a D10A and an H810A substitutions (underlined and bolded), or be a variant of SEQ ID NO: 41 having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto.

• Description	Sequence	SEQ ID NO:
  dead Cas9 or	MDKKYSIGL X IGTNSVGWAVITDEYKVPSKKFKVLGNTD	SEQ ID NO:
  dCas9	RHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRI	40
  Streptococcus	CYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIF
  pyogenes	GNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLAL
  Q99ZW2	AHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEE
  Cas9 with	NPINTASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLF
  D10 X  and	GNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDN
  H810 X	LLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLS
  Where “X” is	ASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKN
  any amino	GYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRE
  acid	DLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDN
  	REKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPW
  	NFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLY
  	EYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFK
  	TNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGT
  	YHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE
  	RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRD
  	KQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQ
  	VSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVM
  	GRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKE
  	LGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQEL
  	DINRLSDYDVD X IVPQSFLKDDSIDNKVLTRSDKNRGKS
  	DNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAER
  	GGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYD
  	ENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINTNYHH
  	AHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRK
  	MIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRP
  	LIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV
  	QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTV
  	AYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPI
  	DFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAG
  	ELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQL
  	FVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKH
  	RDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTS
  	TKEVLDATLIHQSITGLYETRIDLSQLGGD
  dead Cas9 or	MDKKYSIGL A IGTNSVGWAVITDEYKVPSKKFKVLGNTD	SEQ ID NO:
  dCas9	RHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRI	41
  Streptococcus	CYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIF
  pyogenes	GNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLAL
  Q99ZW2	AHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEE
  Cas9 with	NPINTASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLF
  D10 A  and	GNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDN
  H810 A	LLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLS
  	ASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKN
  	GYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRE
  	DLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDN
  	REKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPW
  	NFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLY
  	EYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFK
  	TNRKVTVKQLKEDYFKKIECFDSVETSGVEDRFNASLGT
  	YHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE
  	RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRD
  	KQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQ
  	VSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVM
  	GRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKE
  	LGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQEL
  	DINRLSDYDVD A IVPQSFLKDDSIDNKVLTRSDKNRGKS
  	DNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAER
  	GGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYD
  	ENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINTNYHH
  	AHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRK
  	MIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRP
  	LIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV
  	QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTV
  	AYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPI
  	DFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAG
  	ELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQL
  	FVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKH
  	RDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTS
  	TKEVLDATLIHQSITGLYETRIDLSQLGGD

• D. Cas9 Nickase Variant
• In one embodiment, the prime editors described herein comprise a Cas9 nickase. The term “Cas9 nickase” of “nCas9” refers to a variant of Cas9 which is capable of introducing a single-strand break in a double strand DNA molecule target. In some embodiments, the Cas9 nickase comprises only a single functioning nuclease domain. The wild type Cas9 (e.g., the canonical SpCas9) comprises two separate nuclease domains, namely, the RuvC domain (which cleaves the non-protospacer DNA strand) and HNH domain (which cleaves the protospacer DNA strand). In one embodiment, the Cas9 nickase comprises a mutation in the RuvC domain which inactivates the RuvC nuclease activity. For example, mutations in aspartate (D) 10, histidine (H) 983, aspartate (D) 986, or glutamate (E) 762, have been reported as loss-of-function mutations of the RuvC nuclease domain and the creation of a functional Cas9 nickase (e.g., Nishimasu et al., “Crystal structure of Cas9 in complex with guide RNA and target DNA,” Cell 156(5), 935-949, which is incorporated herein by reference). Thus, nickase mutations in the RuvC domain could include D10X, H983X, D986X, or E762X, wherein X is any amino acid other than the wild type amino acid. In certain embodiments, the nickase could be D10A, of H983A, or D986A, or E762A, or a combination thereof.
• In various embodiments, the Cas9 nickase can having a mutation in the RuvC nuclease domain and have one of the following amino acid sequences, or a variant thereof having an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto.

• Description	Sequence	SEQ ID NO:
  Cas9 nickase	MDKKYSIGL X IGTNSVGWAVITDEYKVPSKKFKVLGNTD	SEQ ID NO:
  Streptococcus	RHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRI	42
  pyogenes	CYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIF
  Q99ZW2	GNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLAL
  Cas9 with	AHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEE
  D10 X ,	NPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLF
  wherein X is	GNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDN
  any alternate	LLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLS
  amino acid	ASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKN
  	GYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRE
  	DLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDN
  	REKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPW
  	NFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLY
  	EYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFK
  	TNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGT
  	YHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE
  	RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRD
  	KQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQ
  	VSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVM
  	GRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKE
  	LGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQEL
  	DINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKS
  	DNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAER
  	GGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYD
  	ENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHH
  	AHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRK
  	MIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRP
  	LIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV
  	QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTV
  	AYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPI
  	DFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAG
  	ELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQL
  	FVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKH
  	RDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTS
  	TKEVLDATLIHQSITGLYETRIDLSQLGGD
  Cas9 nickase	MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTD	SEQ ID NO:
  Streptococcus	RHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRI	43
  pyogenes	CYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIF
  Q99ZW2	GNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLAL
  Cas9 with	AHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEE
  E762X,	NPINTASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLF
  wherein X is	GNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDN
  any alternate	LLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLS
  amino acid	ASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKN
  	GYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRE
  	DLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDN
  	REKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPW
  	NFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLY
  	EYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFK
  	TNRKVTVKQLKEDYFKKIECFDSVETSGVEDRFNASLGT
  	YHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE
  	RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRD
  	KQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQ
  	VSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVM
  	GRHKPENIVI X MARENQTTQKGQKNSRERMKRIEEGIKE
  	LGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQEL
  	DINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKS
  	DNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAER
  	GGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYD
  	ENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINTNYHH
  	AHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRK
  	MIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRP
  	LIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV
  	QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTV
  	AYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPI
  	DFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAG
  	ELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQL
  	FVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKH
  	RDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTS
  	TKEVLDATLIHQSITGLYETRIDLSQLGGD
  Cas9 nickase	MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTD	SEQ ID NO:
  Streptococcus	RHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRI	44
  pyogenes	CYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIF
  Q99ZW2	GNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLAL
  Cas9 with	AHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEE
  H983X,	NPINTASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLF
  wherein X is	GNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDN
  any alternate	LLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLS
  amino acid	ASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKN
  	GYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRE
  	DLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDN
  	REKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPW
  	NFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLY
  	EYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFK
  	TNRKVTVKQLKEDYFKKIECFDSVETSGVEDRFNASLGT
  	YHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE
  	RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRD
  	KQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQ
  	VSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVM
  	GRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKE
  	LGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQEL
  	DINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKS
  	DNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAER
  	GGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYD
  	ENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINTNYH X
  	AHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRK
  	MIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRP
  	LIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV
  	QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTV
  	AYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPI
  	DFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAG
  	ELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQL
  	FVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKH
  	RDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTS
  	TKEVLDATLIHQSITGLYETRIDLSQLGGD
  Cas9 nickase	MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTD	SEQ ID NO:
  Streptococcus	RHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRI	45
  pyogenes	CYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIF
  Q99ZW2	GNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLAL
  Cas9 with	AHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEE
  D986X,	NPINTASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLF
  wherein X is	GNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDN
  any alternate	LLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLS
  amino acid	ASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKN
  	GYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRE
  	DLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDN
  	REKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPW
  	NFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLY
  	EYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFK
  	TNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGT
  	YHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE
  	RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRD
  	KQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQ
  	VSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVM
  	GRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKE
  	LGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQEL
  	DINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKS
  	DNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAER
  	GGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYD
  	ENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINTNYHH
  	AH X AYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRK
  	MIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRP
  	LIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV
  	QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTV
  	AYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPI
  	DFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAG
  	ELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQL
  	FVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKH
  	RDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTS
  	TKEVLDATLIHQSITGLYETRIDLSQLGGD
  Cas9 nickase	MDKKYSIGL A IGTNSVGWAVITDEYKVPSKKFKVLGNTD	SEQ ID NO:
  Streptococcus	RHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRI	46
  pyogenes	CYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIF
  Q99ZW2	GNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLAL
  Cas9 with	AHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEE
  D10 A	NPINTASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLF
  	GNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDN
  	LLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLS
  	ASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKN
  	GYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRE
  	DLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDN
  	REKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPW
  	NFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLY
  	EYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFK
  	TNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGT
  	YHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE
  	RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRD
  	KQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQ
  	VSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVM
  	GRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKE
  	LGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQEL
  	DINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKS
  	DNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAER
  	GGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYD
  	ENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINTNYHH
  	AHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRK
  	MIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRP
  	LIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV
  	QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTV
  	AYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPI
  	DFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAG
  	ELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQL
  	FVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKH
  	RDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTS
  	TKEVLDATLIHQSITGLYETRIDLSQLGGD
  Cas9 nickase	MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTD	SEQ ID NO:
  Streptococcus	RHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRI	47
  pyogenes	CYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIF
  Q99ZW2	GNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLAL
  Cas9 with	AHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEE
  E762A	NPINTASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLF
  	GNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDN
  	LLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLS
  	ASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKN
  	GYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRE
  	DLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDN
  	REKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPW
  	NFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLY
  	EYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFK
  	TNRKVTVKQLKEDYFKKIECFDSVETSGVEDRFNASLGT
  	YHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE
  	RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRD
  	KQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQ
  	VSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVM
  	GRHKPENIVI A MARENQTTQKGQKNSRERMKRIEEGIKE
  	LGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQEL
  	DINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKS
  	DNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAER
  	GGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYD
  	ENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINTNYHH
  	AHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRK
  	MIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRP
  	LIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV
  	QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTV
  	AYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPI
  	DFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAG
  	ELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQL
  	FVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKH
  	RDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTS
  	TKEVLDATLIHQSITGLYETRIDLSQLGGD
  Cas9 nickase	MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTD	SEQ ID NO:
  Streptococcus	RHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRI	48
  pyogenes	CYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIF
  Q99ZW2	GNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLAL
  Cas9 with	AHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEE
  H983A	NPINTASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLF
  	GNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDN
  	LLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLS
  	ASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKN
  	GYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRE
  	DLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDN
  	REKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPW
  	NFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLY
  	EYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFK
  	TNRKVTVKQLKEDYFKKIECFDSVETSGVEDRFNASLGT
  	YHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE
  	RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRD
  	KQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQ
  	VSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVM
  	GRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKE
  	LGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQEL
  	DINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKS
  	DNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAER
  	GGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYD
  	ENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINTNYH A
  	AHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRK
  	MIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRP
  	LIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV
  	QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTV
  	AYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPI
  	DFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAG
  	ELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQL
  	FVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKH
  	RDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTS
  	TKEVLDATLIHQSITGLYETRIDLSQLGGD
  Cas9 nickase	MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTD	SEQ ID NO:
  Streptococcus	RHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRI	49
  pyogenes	CYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIF
  Q99ZW2	GNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLAL
  Cas9 with	AHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEE
  D986A	NPINTASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLF
  	GNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDN
  	LLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLS
  	ASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKN
  	GYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRE
  	DLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDN
  	REKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPW
  	NFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLY
  	EYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFK
  	TNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGT
  	YHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE
  	RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRD
  	KQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQ
  	VSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVM
  	GRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKE
  	LGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQEL
  	DINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKS
  	DNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAER
  	GGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYD
  	ENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINTNYHH
  	AH A AYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRK
  	MIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRP
  	LIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV
  	QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTV
  	AYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPI
  	DFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAG
  	ELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQL
  	FVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKH
  	RDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTS
  	TKEVLDATLIHQSITGLYETRIDLSQLGGD

• In another embodiment, the Cas9 nickase comprises a mutation in the HNH domain which inactivates the HNH nuclease activity. For example, mutations in histidine (H) 840 or asparagine (R) 863 have been reported as loss-of-function mutations of the HNH nuclease domain and the creation of a functional Cas9 nickase (e.g., Nishimasu et al., “Crystal structure of Cas9 in complex with guide RNA and target DNA,” Cell 156(5), 935-949, which is incorporated herein by reference). Thus, nickase mutations in the HNH domain could include H840X and R863X, wherein X is any amino acid other than the wild type amino acid. In certain embodiments, the nickase could be H840A or R863A or a combination thereof.
• In various embodiments, the Cas9 nickase can have a mutation in the HNH nuclease domain and have one of the following amino acid sequences, or a variant thereof having an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto.

• Description	Sequence	SEQ ID NO:
  Cas9 nickase	MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTD	SEQ ID NO:
  Streptococcus	RHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRI	50
  pyogenes	CYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIF
  Q99ZW2	GNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLAL
  Cas9 with	AHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEE
  H840 X ,	NPINTASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLF
  wherein X is	GNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDN
  any alternate	LLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLS
  amino acid	ASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKN
  	GYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRE
  	DLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDN
  	REKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPW
  	NFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLY
  	EYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFK
  	TNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGT
  	YHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE
  	RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRD
  	KQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQ
  	VSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVM
  	GRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKE
  	LGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQEL
  	DINRLSDYDVD X IVPQSFLKDDSIDNKVLTRSDKNRGKS
  	DNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAER
  	GGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYD
  	ENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINTNYHH
  	AHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRK
  	MIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRP
  	LIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV
  	QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTV
  	AYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPI
  	DFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAG
  	ELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQL
  	FVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKH
  	RDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTS
  	TKEVLDATLIHQSITGLYETRIDLSQLGGD
  Cas9 nickase	MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTD	SEQ ID NO:
  Streptococcus	RHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRI	51
  pyogenes	CYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIF
  Q99ZW2	GNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLAL
  Cas9 with	AHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEE
  H840 A	NPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLF
  	GNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDN
  	LLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLS
  	ASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKN
  	GYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRE
  	DLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDN
  	REKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPW
  	NFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLY
  	EYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFK
  	TNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGT
  	YHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE
  	RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRD
  	KQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQ
  	VSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVM
  	GRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKE
  	LGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQEL
  	DINRLSDYDVD A IVPQSFLKDDSIDNKVLTRSDKNRGKS
  	DNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAER
  	GGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYD
  	ENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHH
  	AHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRK
  	MIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRP
  	LIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV
  	QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTV
  	AYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPI
  	DFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAG
  	ELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQL
  	FVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKH
  	RDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTS
  	TKEVLDATLIHQSITGLYETRIDLSQLGGD
  Cas9 nickase	MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTD	SEQ ID NO:
  Streptococcus	RHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRI	52
  pyogenes	CYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIF
  Q99ZW2	GNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLAL
  Cas9 with	AHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEE
  R863X,	NPINTASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLF
  wherein X is	GNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDN
  any alternate	LLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLS
  amino acid	ASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKN
  	GYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRE
  	DLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDN
  	REKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPW
  	NFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLY
  	EYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFK
  	TNRKVTVKQLKEDYFKKIECFDSVETSGVEDRFNASLGT
  	YHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE
  	RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRD
  	KQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQ
  	VSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVM
  	GRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKE
  	LGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQEL
  	DINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKN X GKS
  	DNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAER
  	GGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYD
  	ENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINTNYHH
  	AHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRK
  	MIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRP
  	LIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV
  	QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTV
  	AYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPI
  	DFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAG
  	ELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQL
  	FVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKH
  	RDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTS
  	TKEVLDATLIHQSITGLYETRIDLSQLGGD
  Cas9 nickase	MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTD	SEQ ID NO:
  Streptococcus	RHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRI	53
  pyogenes	CYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIF
  Q99ZW2	GNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLAL
  Cas9 with	AHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEE
  R863 A	NPINTASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLF
  	GNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDN
  	LLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLS
  	ASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKN
  	GYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRE
  	DLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDN
  	REKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPW
  	NFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLY
  	EYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFK
  	TNRKVTVKQLKEDYFKKIECFDSVETSGVEDRFNASLGT
  	YHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE
  	RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRD
  	KQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQ
  	VSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVM
  	GRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKE
  	LGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQEL
  	DINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKN A GKS
  	DNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAER
  	GGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYD
  	ENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINTNYHH
  	AHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRK
  	MIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRP
  	LIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV
  	QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTV
  	AYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPI
  	DFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAG
  	ELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQL
  	FVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKH
  	RDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTS
  	TKEVLDATLIHQSITGLYETRIDLSQLGGD

• In some embodiments, the N-terminal methionine is removed from a Cas9 nickase, or from any Cas9 variant, ortholog, or equivalent disclosed or contemplated herein. For example, methionine-minus Cas9 nickases include the following sequences, or a variant thereof having an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto.

• Description	Sequence
  Cas9 nickase	DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIG
  (Met minus)	ALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSF
  Streptococcus	FHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDST
  pyogenes	DKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQ
  Q99ZW2	LFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIAL
  Cas9 with	SLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLA
  H840 X ,	AKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
  wherein X is	QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELL
  any alternate	VKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREK
  amino acid	IEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASA
  	QSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMR
  	KPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVETSGVE
  	DRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE
  	RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILD
  	FLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAG
  	SPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNS
  	RERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYV
  	DQELDINRLSDYDVD X IVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
  	EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKR
  	QLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFR
  	KDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYK
  	VYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIE
  	TNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILP
  	KRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLK
  	SVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELEN
  	GRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQK
  	QLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIRE
  	QAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITG
  	LYETRIDLSQLGGD (SEQ ID NO: 54)
  Cas9 nickase	DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIG
  (Met minus)	ALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSF
  Streptococcus	FHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDST
  pyogenes	DKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQ
  Q99ZW2	LFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIAL
  Cas9 with	SLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLA
  H840 A	AKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
  	QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELL
  	VKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREK
  	IEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASA
  	QSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMR
  	KPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVETSGVE
  	DRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE
  	RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILD
  	FLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAG
  	SPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNS
  	RERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYV
  	DQELDINRLSDYDVD A IVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
  	EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKR
  	QLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFR
  	KDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYK
  	VYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIE
  	TNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILP
  	KRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLK
  	SVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELEN
  	GRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQK
  	QLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIRE
  	QAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITG
  	LYETRIDLSQLGGD (SEQ ID NO: 55)
  Cas9 nickase	DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIG
  (Met minus)	ALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSF
  Streptococcus	FHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDST
  pyogenes	DKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQ
  Q99ZW2	LFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIAL
  Cas9 with	SLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLA
  R863X,	AKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
  wherein X is	QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELL
  any alternate	VKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREK
  amino acid	IEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASA
  	QSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMR
  	KPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVETSGVE
  	DRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE
  	RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILD
  	FLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAG
  	SPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNS
  	RERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYV
  	DQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKN X GKSDNVPS
  	EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKR
  	QLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFR
  	KDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYK
  	VYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIE
  	TNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILP
  	KRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLK
  	SVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELEN
  	GRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQK
  	QLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIRE
  	QAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITG
  	LYETRIDLSQLGGD(SEQ ID NO: 56)
  Cas9 nickase	DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIG
  (Met minus)	ALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSF
  Streptococcus	FHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDST
  pyogenes	DKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQ
  Q99ZW2	LFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIAL
  Cas9 with	SLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLA
  R863 A	AKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
  	QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELL
  	VKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREK
  	IEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASA
  	QSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMR
  	KPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVETSGVE
  	DRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE
  	RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILD
  	FLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAG
  	SPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNS
  	RERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYV
  	DQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKN A GKSDNVPS
  	EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKR
  	QLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFR
  	KDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYK
  	VYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIE
  	TNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILP
  	KRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLK
  	SVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELEN
  	GRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQK
  	QLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIRE
  	QAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITG
  	LYETRIDLSQLGGD (SEQ ID NO: 57)

• E. Other Cas9 Variants
• Besides dead Cas9 and Cas9 nickase variants, the Cas9 proteins used herein may also include other “Cas9 variants” having at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to any reference Cas9 protein, including any wild type Cas9, or mutant Cas9 (e.g., a dead Cas9 or Cas9 nickase), or fragment Cas9, or circular permutant Cas9, or other variant of Cas9 disclosed herein or known in the art. In some embodiments, a Cas9 variant may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acid changes compared to a reference Cas9. In some embodiments, the Cas9 variant comprises a fragment of a reference Cas9 (e.g., a gRNA binding domain or a DNA-cleavage domain), such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of wild type Cas9. In some embodiments, the fragment is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid length of a corresponding wild type Cas9 (e.g., SEQ ID NO: 18).
• In some embodiments, the disclosure also may utilize Cas9 fragments which retain their functionality and which are fragments of any herein disclosed Cas9 protein. In some embodiments, the Cas9 fragment is at least 100 amino acids in length. In some embodiments, the fragment is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, or at least 1300 amino acids in length.
• In various embodiments, the prime editors disclosed herein may comprise one of the Cas9 variants described as follows, or a Cas9 variant thereof having at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to any reference Cas9 variants.
• F. Small-Sized Cas9 Variants
• In some embodiments, the prime editors contemplated herein can include a Cas9 protein that is of smaller molecular weight than the canonical SpCas9 sequence. In some embodiments, the smaller-sized Cas9 variants may facilitate delivery to cells, e.g., by an expression vector, nanoparticle, or other means of delivery. In certain embodiments, the smaller-sized Cas9 variants can include enzymes categorized as type II enzymes of the Class 2 CRISPR-Cas systems. In some embodiments, the smaller-sized Cas9 variants can include enzymes categorized as type V enzymes of the Class 2 CRISPR-Cas systems. In other embodiments, the smaller-sized Cas9 variants can include enzymes categorized as type VI enzymes of the Class 2 CRISPR-Cas systems.
• The canonical SpCas9 protein is 1368 amino acids in length and has a predicted molecular weight of 158 kilodaltons. The term “small-sized Cas9 variant”, as used herein, refers to any Cas9 variant—naturally occurring, engineered, or otherwise—that is less than at least 1300 amino acids, or at least less than 1290 amino acids, or than less than 1280 amino acids, or less than 1270 amino acid, or less than 1260 amino acid, or less than 1250 amino acids, or less than 1240 amino acids, or less than 1230 amino acids, or less than 1220 amino acids, or less than 1210 amino acids, or less than 1200 amino acids, or less than 1190 amino acids, or less than 1180 amino acids, or less than 1170 amino acids, or less than 1160 amino acids, or less than 1150 amino acids, or less than 1140 amino acids, or less than 1130 amino acids, or less than 1120 amino acids, or less than 1110 amino acids, or less than 1100 amino acids, or less than 1050 amino acids, or less than 1000 amino acids, or less than 950 amino acids, or less than 900 amino acids, or less than 850 amino acids, or less than 800 amino acids, or less than 750 amino acids, or less than 700 amino acids, or less than 650 amino acids, or less than 600 amino acids, or less than 550 amino acids, or less than 500 amino acids, but at least larger than about 400 amino acids and retaining the required functions of the Cas9 protein. The Cas9 variants can include those categorized as type II, type V, or type VI enzymes of the Class 2 CRISPR-Cas system.
• In various embodiments, the prime editors disclosed herein may comprise one of the small-sized Cas9 variants described as follows, or a Cas9 variant thereof having at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to any reference small-sized Cas9 protein.

• Description	Sequence	SEQ ID NO:
  SaCas9	MGKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEA	SEQ ID NO:
  Staphylococcus	NVENNEGRRSKRGARRLKRRRRHRIQRVKKLLFDYNLL	58
  aureus	TDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRG
  1053 AA	VHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLE
  123 kDa	RLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLD
  	QSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEML
  	MGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDE
  	NEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIK
  	GYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIA
  	KILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHN
  	LSLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQK
  	EIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIEL
  	AREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENA
  	KYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDH
  	IIPRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDS
  	KISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSV
  	QKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSI
  	NGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIF
  	KEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEI
  	FITPHQIKHIKDFKDYKYSHRVDKKPNRKLINDTLYSTRK
  	DDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYH
  	HDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYS
  	KKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLS
  	LKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKC
  	YEEAKKLKKISNQAEFIASFYKNDLIKINGELYRVIGVNN
  	DLLNRIEVNMIDITYREYLENMNDKRPPHIIKTIASKTQSI
  	KKYSTDILGNLYEVKSKKHPQIIKK
  NmeCas9	MAAFKPNSINYILGLDIGIASVGWAMVEIDEEENPIRLIDL	SEQ ID NO:
  N. meningitidis	GVRVFERAEVPKTGDSLAMARRLARSVRRLTRRRAHRL	59
  1083 AA	LRTRRLLKREGVLQAANFDENGLIKSLPNTPWQLRAAAL
  124.5 kDa	DRKLTPLEWSAVLLHLIKHRGYLSQRKNEGETADKELGA
  	LLKGVAGNAHALQTGDFRTPAELALNKFEKESGHIRNQR
  	SDYSHTFSRKDLQAELILLFEKQKEFGNPHVSGGLKEGIE
  	TLLMTQRPALSGDAVQKMLGHCTFEPAEPKAAKNTYTA
  	ERFIWLTKLNNLRILEQGSERPLTDTERATLMDEPYRKSK
  	LTYAQARKLLGLEDTAFFKGLRYGKDNAEASTLMEMKA
  	YHAISRALEKEGLKDKKSPLNLSPELQDEIGTAFSLFKTD
  	EDITGRLKDRIQPEILEALLKHISFDKFVQISLKALRRIVPL
  	MEQGKRYDEACAEIYGDHYGKKNTEEKIYLPPIPADEIR
  	NPVVLRALSQARKVINGVVRRYGSPARIHIETAREVGKSF
  	KDRKEIEKRQEENRKDREKAAAKFREYFPNFVGEPKSKD
  	ILKLRLYEQQHGKCLYSGKEINLGRLNEKGYVEIDAALPF
  	SRTWDDSFNNKVLVLGSENQNKGNQTPYEYFNGKDNSR
  	EWQEFKARVETSRFPRSKKQRILLQKFDEDGFKERNLND
  	TRYVNRFLCQFVADRMRLTGKGKKRVFASNGQITNLLR
  	GFWGLRKVRAENDRHHALDAVVVACSTVAMQQKITRF
  	VRYKEMNAFDGKTIDKETGEVLHQKTHFPQPWEFFAQE
  	VMIRVFGKPDGKPEFEEADTLEKLRTLLAEKLSSRPEAVH
  	EYVTPLFVSRAPNRKMSGQGHMETVKSAKRLDEGVSVL
  	RVPLTQLKLKDLEKMVNREREPKLYEALKARLEAHKDD
  	PAKAFAEPFYKYDKAGNRTQQVKAVRVEQVQKTGVWV
  	RNHNGIADNATMVRVDVFEKGDKYYLVPIYSWQVAKGI
  	LPDRAVVQGKDEEDWQLIDDSFNFKFSLHPNDLVEVITK
  	KARMFGYFASCHRGTGNINIRIHDLDHKIGKNGILEGIGV
  	KTALSFQKYQIDELGKEIRPCRLKKRPPVR
  CjCas9	MARILAFDIGISSIGWAFSENDELKDCGVRIFTKVENPKT	SEQ ID NO:
  C. jejuni	GESLALPRRLARSARKRLARRKARLNHLKHLIANEFKLN	60
  984 AA	YEDYQSFDESLAKAYKGSLISPYELRFRALNELLSKQDFA
  114.9 kDa	RVILHIAKRRGYDDIKNSDDKEKGAILKAIKQNEEKLAN
  	YQSVGEYLYKEYFQKFKENSKEFTNVRNKKESYERCIAQ
  	SFLKDELKLIFKKQREFGFSFSKKFEEEVLSVAFYKRALK
  	DFSHLVGNCSFFTDEKRAPKNSPLAFMFVALTRIINLLNN
  	LKNTEGILYTKDDLNALLNEVLKNGTLTYKQTKKLLGLS
  	DDYEFKGEKGTYFIEFKKYKEFIKALGEHNLSQDDLNEIA
  	KDITLIKDEIKLKKALAKYDLNQNQIDSLSKLEFKDHLNIS
  	FKALKLVTPLMLEGKKYDEACNELNLKVAINEDKKDFLP
  	AFNETYYKDEVTNPVVLRAIKEYRKVLNALLKKYGKVH
  	KINIELAREVGKNHSQRAKIEKEQNENYKAKKDAELECE
  	KLGLKINSKNILKLRLFKEQKEFCAYSGEKIKISDLQDEK
  	MLEIDHIYPYSRSFDDSYMNKVLVFTKQNQEKLNQTPFE
  	AFGNDSAKWQKIEVLAKNLPTKKQKRILDKNYKDKEQK
  	NFKDRNLNDTRYIARLVLNYTKDYLDFLPLSDDENTKLN
  	DTQKGSKVHVEAKSGMLTSALRHTWGFSAKDRNNHLH
  	HAIDAVIIAYANNSIVKAFSDFKKEQESNSAELYAKKISEL
  	DYKNKRKFFEPFSGFRQKVLDKIDEIFVSKPERKKPSGAL
  	HEETFRKEEEFYQSYGGKEGVLKALELGKIRKVNGKIVK
  	NGDMFRVDIFKHKKTNKFYAVPIYTMDFALKVLPNKAV
  	ARSKKGEIKDWILMDENYEFCFSLYKDSLILIQTKDMQEP
  	EFVYYNAFTSSTVSLIVSKHDNKFETLSKNQKILFKNANE
  	KEVIAKSIGIQNLKVFEKYIVSALGEVTKAEFRQREDFKK
  GeoCas9	MRYKIGLDIGITSVGWAVMNLDIPRIEDLGVRIFDRAENP	SEQ ID NO:
  G.	QTGESLALPRRLARSARRRLRRRKHRLERIRRLVIREGILT	61
  stearothermophilus	KEELDKLFEEKHEIDVWQLRVEALDRKLNNDELARVLL
  1087 AA	HLAKRRGFKSNRKSERSNKENSTMLKHIEENRAILSSYRT
  127 kDa	VGEMIVKDPKFALHKRNKGENYTNTIARDDLEREIRLIFS
  	KQREFGNMSCTEEFENEYITIWASQRPVASKDDIEKKVGF
  	CTFEPKEKRAPKATYTFQSFIAWEHINKLRLISPSGARGLT
  	DEERRLLYEQAFQKNKITYHDIRTLLHLPDDTYFKGIVYD
  	RGESRKQNENIRFLELDAYHQIRKAVDKVYGKGKSSSFL
  	PIDFDTFGYALTLFKDDADIHSYLRNEYEQNGKRMPNLA
  	NKVYDNELIEELLNLSFTKFGHLSLKALRSILPYMEQGEV
  	YSSACERAGYTFTGPKKKQKTMLLPNIPPIANPVVMRAL
  	TQARKVVNAIIKKYGSPVSIHIELARDLSQTFDERRKTKK
  	EQDENRKKNETAIRQLMEYGLTLNPTGHDIVKFKLWSEQ
  	NGRCAYSLQPIEIERLLEPGYVEVDHVIPYSRSLDDSYTN
  	KVLVLTRENREKGNRIPAEYLGVGTERWQQFETFVLTNK
  	QFSKKKRDRLLRLHYDENEETEFKNRNLNDTRYISRFFA
  	NFIREHLKFAESDDKQKVYTVNGRVTAHLRSRWEFNKN
  	REESDLHHAVDAVIVACTTPSDIAKVTAFYQRREQNKEL
  	AKKTEPHFPQPWPHFADELRARLSKHPKESIKALNLGNY
  	DDQKLESLQPVFVSRMPKRSVTGAAHQETLRRYVGIDER
  	SGKIQTVVKTKLSEIKLDASGHFPMYGKESDPRTYEAIRQ
  	RLLEHNNDPKKAFQEPLYKPKKNGEPGPVIRTVKIIDTKN
  	QVIPLNDGKTVAYNSNIVRVDVFEKDGKYYCVPVYTMD
  	IMKGILPNKAIEPNKPYSEWKEMTEDYTFRFSLYPNDLIRI
  	ELPREKTVKTAAGEEINVKDVFVYYKTIDSANGGLELISH
  	DHRFSLRGVGSRTLKRFEKYQVDVLGNIYKVRGEKRVG
  	LASSAHSKPGKTIRPLQSTRD
  LbaCas12a	MSKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVED	SEQ ID NO:
  L. bacterium	EKRAEDYKGVKKLLDRYYLSFINDVLHSIKLKNLNNYIS	62
  1228 AA	LFRKKTRTEKENKELENLEINLRKEIAKAFKGNEGYKSLF
  143.9 kDa	KKDIIETILPEFLDDKDEIALVNSFNGFTTAFTGFFDNREN
  	MFSEEAKSTSIAFRCINENLTRYISNMDIFEKVDAIFDKHE
  	VQEIKEKILNSDYDVEDFFEGEFFNFVLTQEGIDVYNAIIG
  	GFVTESGEKIKGLNEYINLYNQKTKQKLPKFKPLYKQVL
  	SDRESLSFYGEGYTSDEEVLEVFRNTLNKNSEIFSSIKKLE
  	KLFKNFDEYSSAGIFVKNGPAISTISKDIFGEWNVIRDKW
  	NAEYDDIHLKKKAVVTEKYEDDRRKSFKKIGSFSLEQLQ
  	EYADADLSVVEKLKEIIIQKVDEIYKVYGSSEKLFDADFV
  	LEKSLKKNDAVVAIMKDLLDSVKSFENYIKAFFGEGKET
  	NRDESFYGDFVLAYDILLKVDHIYDAIRNYVTQKPYSKD
  	KFKLYFQNPQFMGGWDKDKETDYRATILRYGSKYYLAI
  	MDKKYAKCLQKIDKDDVNGNYEKINYKLLPGPNKMLPK
  	VFFSKKWMAYYNPSEDIQKIYKNGTFKKGDMFNLNDCH
  	KLIDFFKDSISRYPKWSNAYDFNFSETEKYKDIAGFYREV
  	EEQGYKVSFESASKKEVDKLVEEGKLYMFQIYNKDFSDK
  	SHGTPNLHTMYFKLLFDENNHGQIRLSGGAELFMRRASL
  	KKEELVVHPANSPIANKNPDNPKKTTTLSYDVYKDKRFS
  	EDQYELHIPIAINKCPKNIFKINTEVRVLLKHDDNPYVIGI
  	DRGERNLLYIVVVDGKGNIVEQYSLNEIINNFNGIRIKTD
  	YHSLLDKKEKERFEARQNWTSIENIKELKAGYISQVVHKI
  	CELVEKYDAVIALEDLNSGFKNSRVKVEKQVYQKFEKM
  	LIDKLNYMVDKKSNPCATGGALKGYQITNKFESFKSMST
  	QNGFIFYIPAWLTSKIDPSTGFVNLLKTKYTSTADSKKFISS
  	FDRIMYVPEEDLFEFALDYKNFSRTDADYIKKWKLYSYG
  	NRIRIFRNPKKNNVFDWEEVCLTSAYKELFNKYGINYQQ
  	GDIRALLCEQSDKAFYSSFMALMSLMLQMRNSITGRTDV
  	DFLISPVKNSDGIFYDSRNYEAQENAILPKNADANGAYNI
  	ARKVLWAIGQFKKAEDEKLDKVKIAISNKEWLEYAQTS
  	VKH
  BhCas12b	MATRSFILKIEPNEEVKKGLWKTHEVLNHGIAYYMNILK	SEQ ID NO:
  B. hisashii	LIRQEAIYEHHEQDPKNPKKVSKAEIQAELWDFVLKMQK	63
  1108 AA	CNSFTHEVDKDEVFNILRELYEELVPSSVEKKGEANQLSN
  130.4 kDa	KFLYPLVDPNSQSGKGTASSGRKPRWYNLKIAGDPSWEE
  	EKKKWEEDKKKDPLAKILGKLAEYGLIPLFIPYTDSNEPI
  	VKEIKWMEKSRNQSVRRLDKDMFIQALERFLSWESWNL
  	KVKEEYEKVEKEYKTLEERIKEDIQALKALEQYEKERQE
  	QLLRDTLNTNEYRLSKRGLRGWREIIQKWLKMDENEPSE
  	KYLEVFKDYQRKHPREAGDYSVYEFLSKKENHFIWRNH
  	PEYPYLYATFCEIDKKKKDAKQQATFTLADPINHPLWVR
  	FEERSGSNLNKYRILTEQLHTEKLKKKLTVQLDRLIYPTE
  	SGGWEEKGKVDIVLLPSRQFYNQIFLDIEEKGKHAFTYK
  	DESIKFPLKGTLGGARVQFDRDHLRRYPHKVESGNVGRI
  	YFNMTVNIEPTESPVSKSLKIHRDDFPKVVNFKPKELTEW
  	IKDSKGKKLKSGIESLEIGLRVMSIDLGQRQAAAASIFEV
  	VDQKPDIEGKLFFPIKGTELYAVHRASFNIKLPGETLVKS
  	REVLRKAREDNLKLMNQKLNFLRNVLHFQQFEDITEREK
  	RVTKWISRQENSDVPLVYQDELIQIRELMYKPYKDWVAF
  	LKQLHKRLEVEIGKEVKHWRKSLSDGRKGLYGISLKNID
  	EIDRTRKFLLRWSLRPTEPGEVRRLEPGQRFAIDQLNHLN
  	ALKEDRLKKMANTIIMHALGYCYDVRKKKWQAKNPAC
  	QIILFEDLSNYNPYEERSRFENSKLMKWSRREIPRQVALQ
  	GEIYGLQVGEVGAQFSSRFHAKTGSPGIRCSVVTKEKLQ
  	DNRFFKNLQREGRLTLDKIAVLKEGDLYPDKGGEKFISLS
  	KDRKCVTTHADINAAQNLQKRFWTRTHGFYKVYCKAY
  	QVDGQTVYIPESKDQKQKIIEEFGEGYFILKDGVYEWVN
  	AGKLKIKKGSSKQSSSELVDSDILKDSFDLASELKGEKLM
  	LYRDPSGNVFPSDKWMAAGVFFGKLERILISKLTNQYSIS
  	TIEDDSSKQSM

• G. Cas9 Equivalents
• In some embodiments, the prime editors described herein can include any Cas9 equivalent. As used herein, the term “Cas9 equivalent” is a broad term that encompasses any napDNAbp protein that serves the same function as Cas9 in the present prime editors despite that its amino acid primary sequence and/or its three-dimensional structure may be different and/or unrelated from an evolutionary standpoint. Thus, while Cas9 equivalents include any Cas9 ortholog, homolog, mutant, or variant described or embraced herein that are evolutionarily related, the Cas9 equivalents also embrace proteins that may have evolved through convergent evolution processes to have the same or similar function as Cas9, but which do not necessarily have any similarity with regard to amino acid sequence and/or three dimensional structure. The prime editors described here embrace any Cas9 equivalent that would provide the same or similar function as Cas9 despite that the Cas9 equivalent may be based on a protein that arose through convergent evolution. For instance, if Cas9 refers to a type II enzyme of the CRISPR-Cas system, a Cas9 equivalent can refer to a type V or type VI enzyme of the CRISPR-Cas system.
• For example, Cas12e (CasX) is a Cas9 equivalent that reportedly has the same function as Cas9 but which evolved through convergent evolution. Thus, the Cas12e (CasX) protein described in Liu et al., “CasX enzymes comprises a distinct family of RNA-guided genome editors,” Nature, 2019, Vol. 566: 218-223, is contemplated to be used with the prime editors described herein. In addition, any variant or modification of Cas12e (CasX) is conceivable and within the scope of the present disclosure.
• Cas9 is a bacterial enzyme that evolved in a wide variety of species. However, the Cas9 equivalents contemplated herein may also be obtained from archaea, which constitute a domain and kingdom of single-celled prokaryotic microbes different from bacteria.
• In some embodiments, Cas9 equivalents may refer to Cas12e (CasX) or Cas12d (CasY), which have been described in, for example, Burstein et al., “New CRISPR-Cas systems from uncultivated microbes.” Cell Res. 2017 Feb. 21. doi: 10.1038/cr.2017.21, the entire contents of which is hereby incorporated by reference. Using genome-resolved metagenomics, a number of CRISPR-Cas systems were identified, including the first reported Cas9 in the archaeal domain of life. This divergent Cas9 protein was found in little-studied nanoarchaea as part of an active CRISPR-Cas system. In bacteria, two previously unknown systems were discovered, CRISPR-Cas12e and CRISPR-Cas12d, which are among the most compact systems yet discovered. In some embodiments, Cas9 refers to Cas12e, or a variant of Cas12e. In some embodiments, Cas9 refers to a Cas12d, or a variant of Cas12d. It should be appreciated that other RNA-guided DNA binding proteins may be used as a nucleic acid programmable DNA binding protein (napDNAbp), and are within the scope of this disclosure. Also see Liu et al., “CasX enzymes comprises a distinct family of RNA-guided genome editors,” Nature, 2019, Vol. 566: 218-223. Any of these Cas9 equivalents are contemplated.
• In some embodiments, the Cas9 equivalent comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally-occurring Cas12e (CasX) or Cas12d (CasY) protein. In some embodiments, the napDNAbp is a naturally-occurring Cas12e (CasX) or Cas12d (CasY) protein. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a wild-type Cas moiety or any Cas moiety provided herein.
• In various embodiments, the nucleic acid programmable DNA binding proteins include, without limitation, Cas9 (e.g., dCas9 and nCas9), Cas12e (CasX), Cas12d (CasY), Cas12a (Cpf1), Cas12b1 (C2c1), Cas13a (C2c2), Cas12c (C2c3), Argonaute, and Cas12b1. One example of a nucleic acid programmable DNA-binding protein that has different PAM specificity than Cas9 is Clustered Regularly Interspaced Short Palindromic Repeats from Prevotella and Francisella 1 (i.e, Cas12a (Cpf1)). Similar to Cas9, Cas12a (Cpf1) is also a Class 2 CRISPR effector, but it is a member of type V subgroup of enzymes, rather than the type II subgroup. It has been shown that Cas12a (Cpf1) mediates robust DNA interference with features distinct from Cas9. Cas12a (Cpf1) is a single RNA-guided endonuclease lacking tracrRNA, and it utilizes a T-rich protospacer-adjacent motif (TTN, TTTN, or YTN). Moreover, Cpf1 cleaves DNA via a staggered DNA double-stranded break. Out of 16 Cpf1-family proteins, two enzymes from Acidaminococcus and Lachnospiraceae are shown to have efficient genome-editing activity in human cells. Cpf1 proteins are known in the art and have been described previously, for example Yamano et al., “Crystal structure of Cpf1 in complex with guide RNA and target DNA.” Cell (165) 2016, p. 949-962; the entire contents of which is hereby incorporated by reference.
• In still other embodiments, the Cas protein may include any CRISPR associated protein, including but not limited to, Cas12a, Cas12b1, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, homologs thereof, or modified versions thereof, and preferably comprising a nickase mutation (e.g., a mutation corresponding to the D10A mutation of the wild type Cas9 polypeptide of SEQ ID NO: 18).
• In various other embodiments, the napDNAbp can be any of the following proteins: a Cas9, a Cas12a (Cpf1), a Cas12e (CasX), a Cas12d (CasY), a Cas12b1 (C2c1), a Cas13a (C2c2), a Cas12c (C2c3), a GeoCas9, a CjCas9, a Cas12g, a Cas12h, a Cas12i, a Cas13b, a Cas13c, a Cas13d, a Cas14, a Csn2, an xCas9, an SpCas9-NG, a circularly permuted Cas9, or an Argonaute (Ago) domain, or a variant thereof.
• Exemplary Cas9 equivalent protein sequences can include the following:

• Description	Sequence
  AsCas12a	MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKEL
  (previously	KPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQA
  known as	TYRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTV
  Cpf1)	TTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPK
  	FKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQLLTQ
  	TQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIP
  Acidaminoc	LFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNE
  occus sp.	LNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKE
  (strain	KVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTL
  BV3L6)	KKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEM
  	EPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAI
  UniProtKB	LFVKNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAK
  U2UMQ6	MIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKF
  	QTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDLSSLRPSSQY
  	KDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKG
  	HHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHR
  	LGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVI
  	TKEVSHEIIKDRRFTSDKFFFHVPITLNYQAANSPSKFNQRVNAYLKEHP
  	ETPIIGIDRGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERV
  	AARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVLENLNFGFK
  	SKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQ
  	FTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFL
  	EGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQF
  	DAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSN
  	ILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGV
  	CFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISN
  	QDWLAYIQELRN (SEQ ID NO: 64)
  AsCas12a	MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKEL
  nickase	KPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQA
  (e.g.,	TYRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTV
  R1226A)	TTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPK
  	FKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQLLTQ
  	TQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIP
  	LFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNE
  	LNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKE
  	KVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTL
  	KKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEM
  	EPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAI
  	LFVKNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAK
  	MIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKF
  	QTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDLSSLRPSSQY
  	KDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKG
  	HHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHR
  	LGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVI
  	TKEVSHEIIKDRRFTSDKFFFHVPITLNYQAANSPSKFNQRVNAYLKEHP
  	ETPIIGIDRGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERV
  	AARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVLENLNFGFK
  	SKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQ
  	FTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFL
  	EGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQF
  	DAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSN
  	ILPKLLENDDSHAIDTMVALIRSVLQMANSNAATGEDYINSPVRDLNGV
  	CFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISN
  	QDWLAYIQELRN (SEQ ID NO: 65)
  LbCas12a	MNYKTGLEDFIGKESLSKTLRNALIPTESTKIHMEEMGVIRDDELRAEKQ
  (previously	QELKEIMDDYYRTFIEEKLGQIQGIQWNSLFQKMEETMEDISVRKDLDKI
  known as	QNEKRKEICCYFTSDKRFKDLFNAKLITDILPNFIKDNKEYTEEEKAEKE
  Cpf1)	QTRVLFQRFATAFTNYFNQRRNNFSEDNISTAISFRIVNENSEIHLQNMR
  	AFQRIEQQYPEEVCGMEEEYKDMLQEWQMKHIYSVDFYDRELTQPGIE
  Lachnospir	YYNGICGKINEHMNQFCQKNRINKNDFRMKKLHKQILCKKSSYYEIPFR
  aceae	FESDQEVYDALNEFIKTMKKKEIIRRCVHLGQECDDYDLGKIYISSNKYE
  bacterium	QISNALYGSWDTIRKCIKEEYMDALPGKGEKKEEKAEAAAKKEEYRSIA
  GAM79	DIDKIISLYGSEMDRTISAKKCITEICDMAGQISIDPLVCNSDIKLLQNKEK
  	TTEIKTILDSFLHVYQWGQTFIVSDIIEKDSYFYSELEDVLEDFEGITTLYN
  Ref Seq.	HVRSYVTQKPYSTVKFKLHFGSPTLANGWSQSKEYDNNAILLMRDQKF
  WP_11962	YLGIFNVRNKPDKQIIKGHEKEEKGDYKKMIYNLLPGPSKMLPKVFITSR
  3382.1	SGQETYKPSKHILDGYNEKRHIKSSPKFDLGYCWDLIDYYKECIHKHPD
  	WKNYDFHFSDTKDYEDISGFYREVEMQGYQIKWTYISADEIQKLDEKG
  	QIFLFQIYNKDFSVHSTGKDNLHTMYLKNLFSEENLKDIVLKLNGEAELF
  	FRKASIKTPIVHKKGSVLVNRSYTQTVGNKEIRVSIPEEYYTEIYNYLNHI
  	GKGKLSSEAQRYLDEGKIKSFTATKDIVKNYRYCCDHYFLHLPITINFKA
  	KSDVAVNERTLAYIAKKEDIHIIGIDRGERNLLYISVVDVHGNIREQRSFN
  	IVNGYDYQQKLKDREKSRDAARKNWEEIEKIKELKEGYLSMVIHYIAQL
  	VVKYNAVVAMEDLNYGFKTGRFKVERQVYQKFETMLIEKLHYLVFKD
  	REVCEEGGVLRGYQLTYIPESLKKVGKQCGFIFYVPAGYTSKIDPTTGFV
  	NLFSFKNLTNRESRQDFVGKFDEIRYDRDKKMFEFSFDYNNYIKKGTILA
  	STKWKVYTNGTRLKRIVVNGKYTSQSMEVELTDAMEKMLQRAGIEYH
  	DGKDLKGQIVEKGIEAEIIDIFRLTVQMRNSRSESEDREYDRLISPVLNDK
  	GEFFDTATADKTLPQDADANGAYCIALKGLYEVKQIKENWKENEQFPR
  	NKLVQDNKTWFDFMQKKRYL (SEQ ID NO: 66)
  PcCas 12a -	MAKNFEDFKRLYSLSKTLRFEAKPIGATLDNIVKSGLLDEDEHRAASYV
  previously	KVKKLIDEYHKVFIDRVLDDGCLPLENKGNNNSLAEYYESYVSRAQDE
  known at	DAKKKFKEIQQNLRSVIAKKLTEDKAYANLFGNKLIESYKDKEDKKKII
  Cpf1	DSDLIQFINTAESTQLDSMSQDEAKELVKEFWGFVTYFYGFFDNRKNMY
  	TAEEKSTGIAYRLVNENLPKFIDNIEAFNRAITRPEIQENMGVLYSDFSEY
  Prevotella	LNVESIQEMFQLDYYNMLLTQKQIDVYNAIIGGKTDDEHDVKIKGINEYI
  copri	NLYNQQHKDDKLPKLKALFKQILSDRNAISWLPEEFNSDQEVLNAIKDC
  	YERLAENVLGDKVLKSLLGSLADYSLDGIFIRNDLQLTDISQKMFGNWG
  Ref Seq.	VIQNAIMQNIKRVAPARKHKESEEDYEKRIAGIFKKADSFSISYINDCLNE
  WP_11922	ADPNNAYFVENYFATFGAVNTPTMQRENLFALVQNAYTEVAALLHSDY
  7726.1	PTVKHLAQDKANVSKIKALLDAIKSLQHFVKPLLGKGDESDKDERFYGE
  	LASLWAELDTVTPLYNMIRNYMTRKPYSQKKIKLNFENPQLLGGWDAN
  	KEKDYATIILRRNGLYYLAIMDKDSRKLLGKAMPSDGECYEKMVYKFF
  	KDVTTMIPKCSTQLKDVQAYFKVNTDDYVLNSKAFNKPLTITKEVFDLN
  	NVLYGKYKKFQKGYLTATGDNVGYTHAVNVWIKFCMDFLNSYDSTCI
  	YDFSSLKPESYLSLDAFYQDANLLLYKLSFARASVSYINQLVEEGKMYL
  	FQIYNKDFSEYSKGTPNMHTLYWKALFDERNLADVVYKLNGQAEMFY
  	RKKSIENTHPTHPANHPILNKNKDNKKKESLFDYDLIKDRRYTVDKFMF
  	HVPITMNFKSVGSENINQDVKAYLRHADDMHIIGIDRGERHLLYLVVIDL
  	QGNIKEQYSLNEIVNEYNGNTYHTNYHDLLDVREEERLKARQSWQTIEN
  	IKELKEGYLSQVIHKITQLMVRYHAIVVLEDLSKGFMRSRQKVEKQVYQ
  	KFEKMLIDKLNYLVDKKTDVSTPGGLLNAYQLTCKSDSSQKLGKQSGF
  	LFYIPAWNTSKIDPVTGFVNLLDTHSLNSKEKIKAFFSKFDAIRYNKDKK
  	WFEFNLDYDKFGKKAEDTRTKWTLCTRGMRIDTFRNKEKNSQWDNQE
  	VDLTTEMKSLLEHYYIDIHGNLKDAISAQTDKAFFTGLLHILKLTLQMRN
  	SITGTETDYLVSPVADENGIFYDSRSCGNQLPENADANGAYNIARKGLM
  	LIEQIKNAEDLNNVKFDISNKAWLNFAQQKPYKNG (SEQ ID NO: 67)
  ErCas 12a -	MFSAKLISDILPEFVIHNNNYSASEKEEKTQVIKLFSRFATSFKDYFKNRA
  previously	NCFSANDISSSSCHRIVNDNAEIFFSNALVYRRIVKNLSNDDINKISGDMK
  known at	DSLKEMSLEEIYSYEKYGEFITQEGISFYNDICGKVNLFMNLYCQKNKEN
  Cpf1	KNLYKLRKLHKQILCIADTSYEVPYKFESDEEVYQSVNGFLDNISSKHIV
  	ERLRKIGENYNGYNLDKIYIVSKFYESVSQKTYRDWETINTALEIHYNNI
  Eubacterium	LPGNGKSKADKVKKAVKNDLQKSITEINELVSNYKLCPDDNIKAETYIH
  rectale	EISHILNNFEAQELKYNPEIHLVESELKASELKNVLDVIMNAFHWCSVFM
  	TEELVDKDNNFYAELEEIYDEIYPVISLYNLVRNYVTQKPYSTKKIKLNF
  Ref Seq.	GIPTLADGWSKSKEYSNNAIILMRDNLYYLGIFNAKNKPDKKIIEGNTSE
  WP_11922	NKGDYKKMIYNLLPGPNKMPKVFLSSKTGVETYKPSAYILEGYKQNKH
  3642.1	LKSSKDFDITFCHDLIDYFKNCIAIHPEWKNFGFDFSDTSTYEDISGFYRE
  	VELQGYKIDWTYISEKDIDLLQEKGQLYLFQIYNKDFSKKSSGNDNLHT
  	MYLKNLFSEENLKDIVLKLNGEAEIFFRKSSIKNPIIHKKGSILVNRTYEA
  	EEKDQFGNIQIVRKTIPENIYQELYKYFNDKSDKELSDEAAKLKNVVGH
  	HEAATNIVKDYRYTYDKYFLHMPITINFKANKTSFINDRILQYIAKEKDL
  	HVIGIDRGERNLIYVSVIDTCGNIVEQKSFNIVNGYDYQIKLKQQEGARQI
  	ARKEWKEIGKIKEIKEGYLSLVIHEISKMVIKYNAIIAMEDLSYGFKKGRF
  	KVERQVYQKFETMLINKLNYLVFKDISITENGGLLKGYQLTYIPDKLKN
  	VGHQCGCIFYVPAAYTSKIDPTTGFVNIFKFKDLTVDAKREFIKKFDSIRY
  	DSDKNLFCFTFDYNNFITQNTVMSKSSWSVYTYGVRIKRRFVNGRFSNE
  	SDTIDITKDMEKTLEMTDINWRDGHDLRQDIIDYEIVQHIFEIFKLTVQM
  	RNSLSELEDRDYDRLISPVLNENNIFYDSAKAGDALPKDADANGAYCIA
  	LKGLYEIKQITENWKEDGKFSRDKLKISNKDWFDFIQNKRYL (SEQ ID
  	NO: 68)
  CsCas 12a -	MNYKTGLEDFIGKESLSKTLRNALIPTESTKIHMEEMGVIRDDELRAEKQ
  previously	QELKEIMDDYYRAFIEEKLGQIQGIQWNSLFQKMEETMEDISVRKDLDKI
  known at	QNEKRKEICCYFTSDKRFKDLFNAKLITDILPNFIKDNKEYTEEEKAEKE
  Cpf1	QTRVLFQRFATAFTNYFNQRRNNFSEDNISTAISFRIVNENSEIHLQNMR
  	AFQRIEQQYPEEVCGMEEEYKDMLQEWQMKHIYLVDFYDRVLTQPGIE
  Clostridium	YYNGICGKINEHMNQFCQKNRINKNDFRMKKLHKQILCKKSSYYEIPFR
  sp. AF34-	FESDQEVYDALNEFIKTMKEKEIICRCVHLGQKCDDYDLGKIYISSNKYE
  10BH	QISNALYGSWDTIRKCIKEEYMDALPGKGEKKEEKAEAAAKKEEYRSIA
  	DIDKIISLYGSEMDRTISAKKCITEICDMAGQISTDPLVCNSDIKLLQNKE
  Ref Seq.	KTTEIKTILDSFLHVYQWGQTFIVSDIIEKDSYFYSELEDVLEDFEGITTLY
  WP_11853	NHVRSYVTQKPYSTVKFKLHFGSPTLANGWSQSKEYDNNAILLMRDQK
  8418.1	FYLGIFNVRNKPDKQIIKGHEKEEKGDYKKMIYNLLPGPSKMLPKVFITS
  	RSGQETYKPSKHILDGYNEKRHIKSSPKFDLGYCWDLIDYYKECIHKHP
  	DWKNYDFHFSDTKDYEDISGFYREVEMQGYQIKWTYISADEIQKLDEK
  	GQIFLFQIYNKDFSVHSTGKDNLHTMYLKNLFSEENLKDIVLKLNGEAEL
  	FFRKASIKTPVVHKKGSVLVNRSYTQTVGDKEIRVSIPEEYYTEIYNYLN
  	HIGRGKLSTEAQRYLEERKIKSFTATKDIVKNYRYCCDHYFLHLPITINFK
  	AKSDIAVNERTLAYIAKKEDIHIIGIDRGERNLLYISVVDVHGNIREQRSF
  	NIVNGYDYQQKLKDREKSRDAARKNWEEIEKIKELKEGYLSMVIHYIAQ
  	LVVKYNAVVAMEDLNYGFKTGRFKVERQVYQKFETMLIEKLHYLVFK
  	DREVCEEGGVLRGYQLTYIPESLKKVGKQCGFIFYVPAGYTSKIDPTTGF
  	VNLFSFKNLTNRESRQDFVGKFDEIRYDRDKKMFEFSFDYNNYIKKGTM
  	LASTKWKVYTNGTRLKRIVVNGKYTSQSMEVELTDAMEKMLQRAGIE
  	YHDGKDLKGQIVEKGIEAEIIDIFRLTVQMRNSRSESEDREYDRLISPVLN
  	DKGEFFDTATADKTLPQDADANGAYCIALKGLYEVKQIKENWKENEQF
  	PRNKLVQDNKTWFDFMQKKRYL (SEQ ID NO: 69)
  BhCas 12b	MATRSFILKIEPNEEVKKGLWKTHEVLNHGIAYYMNILKLIRQEAIYEHH
  	EQDPKNPKKVSKAEIQAELWDFVLKMQKCNSFTHEVDKDEVFNILREL
  Bacillus	YEELVPSSVEKKGEANQLSNKFLYPLVDPNSQSGKGTASSGRKPRWYNL
  hisashii	KIAGDPSWEEEKKKWEEDKKKDPLAKILGKLAEYGLIPLFIPYTDSNEPI
  	VKEIKWMEKSRNQSVRRLDKDMFIQALERFLSWESWNLKVKEEYEKVE
  Ref Seq.	KEYKTLEERIKEDIQALKALEQYEKERQEQLLRDTLNTNEYRLSKRGLR
  WP_09514	GWREIIQKWLKMDENEPSEKYLEVFKDYQRKHPREAGDYSVYEFLSKK
  2515.1	ENHFIWRNHPEYPYLYATFCEIDKKKKDAKQQATFTLADPINHPLWVRF
  	EERSGSNLNKYRILTEQLHTEKLKKKLTVQLDRLIYPTESGGWEEKGKV
  	DIVLLPSRQFYNQIFLDIEEKGKHAFTYKDESIKFPLKGTLGGARVQFDR
  	DHLRRYPHKVESGNVGRIYFNMTVNIEPTESPVSKSLKIHRDDFPKVVNF
  	KPKELTEWIKDSKGKKLKSGIESLEIGLRVMSIDLGQRQAAAASIFEVVD
  	QKPDIEGKLFFPIKGTELYAVHRASFNIKLPGETLVKSREVLRKAREDNL
  	KLMNQKLNFLRNVLHFQQFEDITEREKRVTKWISRQENSDVPLVYQDEL
  	IQIRELMYKPYKDWVAFLKQLHKRLEVEIGKEVKHWRKSLSDGRKGLY
  	GISLKNIDEIDRTRKFLLRWSLRPTEPGEVRRLEPGQRFAIDQLNHLNALK
  	EDRLKKMANTIIMHALGYCYDVRKKKWQAKNPACQIILFEDLSNYNPY
  	EERSRFENSKLMKWSRREIPRQVALQGEIYGLQVGEVGAQFSSRFHAKT
  	GSPGIRCSVVTKEKLQDNRFFKNLQREGRLTLDKIAVLKEGDLYPDKGG
  	EKFISLSKDRKCVTTHADINAAQNLQKRFWTRTHGFYKVYCKAYQVDG
  	QTVYIPESKDQKQKIIEEFGEGYFILKDGVYEWVNAGKLKIKKGSSKQSS
  	SELVDSDILKDSFDLASELKGEKLMLYRDPSGNVFPSDKWMAAGVFFG
  	KLERILISKLTNQYSISTIEDDSSKQSM (SEQ ID NO: 70)
  ThCas12b	MSEKTTQRAYTLRLNRASGECAVCQNNSCDCWHDALWATHKAVNRG
  	AKAFGDWLLTLRGGLCHTLVEMEVPAKGNNPPQRPTDQERRDRRVLLA
  Thermomon	LSWLSVEDEHGAPKEFIVATGRDSADDRAKKVEEKLREILEKRDFQEHEI
  as	DAWLQDCGPSLKAHIREDAVWVNRRALFDAAVERIKTLTWEEAWDFL
  hydrotherm	EPFFGTQYFAGIGDGKDKDDAEGPARQGEKAKDLVQKAGQWLSARFGI
  alis	GTGADFMSMAEAYEKIAKWASQAQNGDNGKATIEKLACALRPSEPPTL
  	DTVLKCISGPGHKSATREYLKTLDKKSTVTQEDLNQLRKLADEDARNC
  Ref Seq.	RKKVGKKGKKPWADEVLKDVENSCELTYLQDNSPARHREFSVMLDHA
  WP_07275	ARRVSMAHSWIKKAEQRRRQFESDAQKLKNLQERAPSAVEWLDRFCES
  4838	RSMTTGANTGSGYRIRKRAIEGWSYVVQAWAEASCDTEDKRIAAARKV
  	QADPEIEKFGDIQLFEALAADEAICVWRDQEGTQNPSILIDYVTGKTAEH
  	NQKRFKVPAYRHPDELRHPVFCDFGNSRWSIQFAIHKEIRDRDKGAKQD
  	TRQLQNRHGLKMRLWNGRSMTDVNLHWSSKRLTADLALDQNPNPNPT
  	EVTRADRLGRAASSAFDHVKIKNVFNEKEWNGRLQAPRAELDRIAKLE
  	EQGKTEQAEKLRKRLRWYVSFSPCLSPSGPFIVYAGQHNIQPKRSGQYA
  	PHAQANKGRARLAQLILSRLPDLRILSVDLGHRFAAACAVWETLSSDAF
  	RREIQGLNVLAGGSGEGDLFLHVEMTGDDGKRRTVVYRRIGPDQLLDN
  	TPHPAPWARLDRQFLIKLQGEDEGVREASNEELWTVHKLEVEVGRTVP
  	LIDRMVRSGFGKTEKQKERLKKLRELGWISAMPNEPSAETDEKEGEIRSI
  	SRSVDELMSSALGTLRLALKRHGNRARIAFAMTADYKPMPGGQKYYFH
  	EAKEASKNDDETKRRDNQIEFLQDALSLWHDLFSSPDWEDNEAKKLWQ
  	NHIATLPNYQTPEEISAELKRVERNKKRKENRDKLRTAAKALAENDQLR
  	QHLHDTWKERWESDDQQWKERLRSLKDWIFPRGKAEDNPSIRHVGGLS
  	ITRINTISGLYQILKAFKMRPEPDDLRKNIPQKGDDELENFNRRLLEARDR
  	LREQRVKQLASRIIEAALGVGRIKIPKNGKLPKRPRTTVDTPCHAVVIESL
  	KTYRPDDLRTRRENRQLMQWSSAKVRKYLKEGCELYGLHFLEVPANYT
  	SRQCSRTGLPGIRCDDVPTGDFLKAPWWRRAINTAREKNGGDAKDRFL
  	VDLYDHLNNLQSKGEALPATVRVPRQGGNLFIAGAQLDDTNKERRAIQ
  	ADLNAAANIGLRALLDPDWRGRWWYVPCKDGTSEPALDRIEGSTAFND
  	VRSLPTGDNSSRRAPREIENLWRDPSGDSLESGTWSPTRAYWDTVQSRV
  	IELLRRHAGLPTS (SEQ ID NO: 71)
  LsCas12b	MSIRSFKLKLKTKSGVNAEQLRRGLWRTHQLINDGIAYYMNWLVLLRQ
  	EDLFIRNKETNEIEKRSKEEIQAVLLERVHKQQQRNQWSGEVDEQTLLQ
  Laceyella	ALRQLYEEIVPSVIGKSGNASLKARFFLGPLVDPNNKTTKDVSKSGPTPK
  sacchari	WKKMKDAGDPNWVQEYEKYMAERQTLVRLEEMGLIPLFPMYTDEVG
  	DIHWLPQASGYTRTWDRDMFQQAIERLLSWESWNRRVRERRAQFEKKT
  WP_13222	HDFASRFSESDVQWMNKLREYEAQQEKSLEENAFAPNEPYALTKKALR
  1894.1	GWERVYHSWMRLDSAASEEAYWQEVATCQTAMRGEFGDPAIYQFLAQ
  	KENHDIWRGYPERVIDFAELNHLQRELRRAKEDATFTLPDSVDHPLWVR
  	YEAPGGTNIHGYDLVQDTKRNLTLILDKFILPDENGSWHEVKKVPFSLA
  	KSKQFHRQVWLQEEQKQKKREVVFYDYSTNLPHLGTLAGAKLQWDRN
  	FLNKRTQQQIEETGEIGKVFFNISVDVRPAVEVKNGRLQNGLGKALTVL
  	THPDGTKIVTGWKAEQLEKWVGESGRVSSLGLDSLSEGLRVMSIDLGQ
  	RTSATVSVFEITKEAPDNPYKFFYQLEGTEMFAVHQRSFLLALPGENPPQ
  	KIKQMREIRWKERNRIKQQVDQLSAILRLHKKVNEDERIQAIDKLLQKV
  	ASWQLNEEIATAWNQALSQLYSKAKENDLQWNQAIKNAHHQLEPVVG
  	KQISLWRKDLSTGRQGIAGLSLWSIEELEATKKLLTRWSKRSREPGVVK
  	RIERFETFAKQIQHHINQVKENRLKQLANLIVMTALGYKYDQEQKKWIE
  	VYPACQVVLFENLRSYRFSFERSRRENKKLMEWSHRSIPKLVQMQGELF
  	GLQVADVYAAYSSRYHGRTGAPGIRCHALTEADLRNETNIIHELIEAGFI
  	KEEHRPYLQQGDLVPWSGGELFATLQKPYDNPRILTLHADINAAQNIQK
  	RFWHPSMWFRVNCESVMEGEIVTYVPKNKTVHKKQGKTFRFVKVEGS
  	DVYEWAKWSKNRNKNTFSSITERKPPSSMILFRDPSGTFFKEQEWVEQK
  	TFWGKVQSMIQAYMKKTIVQRMEE (SEQ ID NO: 72)
  DtCas 12b	MVLGRKDDTAELRRALWTTHEHVNLAVAEVERVLLRCRGRSYWTLDR
  	RGDPVHVPESQVAEDALAMAREAQRRNGWPVVGEDEEILLALRYLYEQ
  Dsulfonatro	IVPSCLLDDLGKPLKGDAQKIGTNYAGPLFDSDTCRRDEGKDVACCGPF
  num	HEVAGKYLGALPEWATPISKQEFDGKDASHLRFKATGGDDAFFRVSIEK
  thiodismuta	ANAWYEDPANQDALKNKAYNKDDWKKEKDKGISSWAVKYIQKQLQL
  ns	GQDPRTEVRRKLWLELGLLPLFIPVFDKTMVGNLWNRLAVRLALAHLL
  	SWESWNHRAVQDQALARAKRDELAALFLGMEDGFAGLREYELRRNESI
  WP_03138	KQHAFEPVDRPYVVSGRALRSWTRVREEWLRHGDTQESRKNICNRLQD
  6437	RLRGKFGDPDVFHWLAEDGQEALWKERDCVTSFSLLNDADGLLEKRK
  	GYALMTFADARLHPRWAMYEAPGGSNLRTYQIRKTENGLWADVVLLS
  	PRNESAAVEEKTFNVRLAPSGQLSNVSFDQIQKGSKMVGRCRYQSANQ
  	QFEGLLGGAEILFDRKRIANEQHGATDLASKPGHVWFKLTLDVRPQAPQ
  	GWLDGKGRPALPPEAKHFKTALSNKSKFADQVRPGLRVLSVDLGVRSF
  	AACSVFELVRGGPDQGTYFPAADGRTVDDPEKLWAKHERSFKITLPGEN
  	PSRKEEIARRAAMEELRSLNGDIRRLKAILRLSVLQEDDPRTEHLRLFME
  	AIVDDPAKSALNAELFKGFGDDRFRSTPDLWKQHCHFFHDKAEKVVAE
  	RFSRWRTETRPKSSSWQDWRERRGYAGGKSYWAVTYLEAVRGLILRW
  	NMRGRTYGEVNRQDKKQFGTVASALLHHINQLKEDRIKTGADMIIQAA
  	RGFVPRKNGAGWVQVHEPCRLILFEDLARYRFRTDRSRRENSRLMRWS
  	HREIVNEVGMQGELYGLHVDTTEAGFSSRYLASSGAPGVRCRHLVEEDF
  	HDGLPGMHLVGELDWLLPKDKDRTANEARRLLGGMVRPGMLVPWDG
  	GELFATLNAASQLHVIHADINAAQNLQRRFWGRCGEAIRIVCNQLSVDG
  	STRYEMAKAPKARLLGALQQLKNGDAPFHLTSIPNSQKPENSYVMTPTN
  	AGKKYRAGPGEKSSGEEDELALDIVEQAEELAQGRKTFFRDPSGVFFAP
  	DRWLPSEIYWSRIRRRIWQVTLERNSSGRQERAEMDEMPY
  	(SEQ ID NO:73)

• The prime editors described herein may also comprise Cas12a (Cpf1) (dCpf1) variants that may be used as a guide nucleotide sequence-programmable DNA-binding protein domain. The Cas12a (Cpf1) protein has a RuvC-like endonuclease domain that is similar to the RuvC domain of Cas9 but does not have a HNH endonuclease domain, and the N-terminal of Cas12a (Cpf1) does not have the alfa-helical recognition lobe of Cas9. It was shown in Zetsche et al., Cell, 163,759-771,2015 (which is incorporated herein by reference) that, the RuvC-like domain of Cas12a (Cpf1) is responsible for cleaving both DNA strands and inactivation of the RuvC-like domain inactivates Cas12a (Cpf1) nuclease activity.
• In some embodiments, the napDNAbp is a single effector of a microbial CRISPR-Cas system. Single effectors of microbial CRISPR-Cas systems include, without limitation, Cas9, Cas12a (Cpf1), Cas12b1 (C2c1), Cas13a (C2c2), and Cas12c (C2c3). Typically, microbial CRISPR-Cas systems are divided into Class 1 and Class 2 systems. Class 1 systems have multisubunit effector complexes, while Class 2 systems have a single protein effector. For example, Cas9 and Cas12a (Cpf1) are Class 2 effectors. In addition to Cas9 and Cas12a (Cpf1), three distinct Class 2 CRISPR-Cas systems (Cas12b1, Cas13a, and Cas12c) have been described by Shmakov et al., “Discovery and Functional Characterization of Diverse Class 2 CRISPR Cas Systems”, Mol. Cell, 2015 Nov. 5; 60(3): 385-397, the entire contents of which are hereby incorporated by reference.
• Effectors of two of the systems, Cas12b1 and Cas12c, contain RuvC-like endonuclease domains related to Cas12a. A third system, Cas13a contains an effector with two predicated HEPN RNase domains. Production of mature CRISPR RNA is tracrRNA-independent, unlike production of CRISPR RNA by Cas12b1. Cas12b1 depends on both CRISPR RNA and tracrRNA for DNA cleavage. Bacterial Cas13a has been shown to possess a unique RNase activity for CRISPR RNA maturation distinct from its RNA-activated single-stranded RNA degradation activity. These RNase functions are different from each other and from the CRISPR RNA-processing behavior of Cas12a. See, e.g., East-Seletsky, et al., “Two distinct RNase activities of CRISPR-Cas13a enable guide-RNA processing and RNA detection”, Nature, 2016 Oct. 13; 538(7624):270-273, the entire contents of which are hereby incorporated by reference. In vitro biochemical analysis of Cas13a in Leptotrichia shahii has shown that Cas13a is guided by a single CRISPR RNA and can be programed to cleave ssRNA targets carrying complementary protospacers. Catalytic residues in the two conserved HEPN domains mediate cleavage. Mutations in the catalytic residues generate catalytically inactive RNA-binding proteins. See e.g., Abudayyeh et al., “C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector”, Science, 2016 Aug. 5; 353(6299), the entire contents of which are hereby incorporated by reference.
• The crystal structure of Alicyclobacillus acidoterrastris Cas12b1 (AacC2c1) has been reported in complex with a chimeric single-molecule guide RNA (sgRNA). See e.g., Liu et al., “C2c1-sgRNA Complex Structure Reveals RNA-Guided DNA Cleavage Mechanism”, Mol. Cell, 2017 Jan. 19; 65(2):310-322, the entire contents of which are hereby incorporated by reference. The crystal structure has also been reported in Alicyclobacillus acidoterrestris C2c1 bound to target DNAs as ternary complexes. See e.g., Yang et al., “PAM-dependent Target DNA Recognition and Cleavage by C2C1 CRISPR-Cas endonuclease”, Cell, 2016 Dec. 15; 167(7):1814-1828, the entire contents of which are hereby incorporated by reference. Catalytically competent conformations of AacC2c1, both with target and non-target DNA strands, have been captured independently positioned within a single RuvC catalytic pocket, with C2c1-mediated cleavage resulting in a staggered seven-nucleotide break of target DNA. Structural comparisons between C2c1 ternary complexes and previously identified Cas9 and Cpf1 counterparts demonstrate the diversity of mechanisms used by CRISPR-Cas9 systems.
• In some embodiments, the napDNAbp may be a C2c1, a C2c2, or a C2c3 protein. In some embodiments, the napDNAbp is a C2c1 protein. In some embodiments, the napDNAbp is a Cas13a protein. In some embodiments, the napDNAbp is a Cas12c protein. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally-occurring Cas12b1 (C2c1), Cas13a (C2c2), or Cas12c (C2c3) protein. In some embodiments, the napDNAbp is a naturally-occurring Cas12b1 (C2c1), Cas13a (C2c2), or Cas12c (C2c3) protein.
• H. Cas9 Circular Permutants
• In various embodiments, the prime editors disclosed herein may comprise a circular permutant of Cas9.
• The term “circularly permuted Cas9” or “circular permutant” of Cas9 or “CP-Cas9”) refers to any Cas9 protein, or variant thereof, that occurs or has been modify to engineered as a circular permutant variant, which means the N-terminus and the C-terminus of a Cas9 protein (e.g., a wild type Cas9 protein) have been topically rearranged. Such circularly permuted Cas9 proteins, or variants thereof, retain the ability to bind DNA when complexed with a guide RNA (gRNA). See, Oakes et al., “Protein Engineering of Cas9 for enhanced function,” Methods Enzymol, 2014, 546: 491-511 and Oakes et al., “CRISPR-Cas9 Circular Permutants as Programmable Scaffolds for Genome Modification,” Cell, Jan. 10, 2019, 176: 254-267, each of are incorporated herein by reference. The instant disclosure contemplates any previously known CP-Cas9 or use a new CP-Cas9 so long as the resulting circularly permuted protein retains the ability to bind DNA when complexed with a guide RNA (gRNA).
• Any of the Cas9 proteins described herein, including any variant, ortholog, or naturally occurring Cas9 or equivalent thereof, may be reconfigured as a circular permutant variant.
• In various embodiments, the circular permutants of Cas9 may have the following structure:
• N-terminus-[original C-terminus]-[optional linker]-[original N-terminus]-C-terminus.
• As an example, the present disclosure contemplates the following circular permutants of canonical S. pyogenes Cas9 (1368 amino acids of UniProtKB—Q99ZW2 (CAS9_STRP1) (numbering is based on the amino acid position in SEQ ID NO: 18)):
• N-terminus-[1268-1368]-[optional linker]-[1-1267]-C-terminus;
  N-terminus-[1168-1368]-[optional linker]-[1-1167]-C-terminus;
  N-terminus-[1068-1368]-[optional linker]-[1-1067]-C-terminus;
  N-terminus-[968-1368]-[optional linker]-[1-967]-C-terminus;
  N-terminus-[868-1368]-[optional linker]-[1-867]-C-terminus;
  N-terminus-[768-1368]-[optional linker]-[1-767]-C-terminus;
  N-terminus-[668-1368]-[optional linker]-[1-667]-C-terminus;
  N-terminus-[568-1368]-[optional linker]-[1-567]-C-terminus;
  N-terminus-[468-1368]-[optional linker]-[1-467]-C-terminus;
  N-terminus-[368-1368]-[optional linker]-[1-367]-C-terminus;
  N-terminus-[268-1368]-[optional linker]-[1-267]-C-terminus;
  N-terminus-[168-1368]-[optional linker]-[1-167]-C-terminus;
  N-terminus-[68-1368]-[optional linker]-[1-67]-C-terminus; or
  N-terminus-[10-1368]-[optional linker]-[1-9]-C-terminus, or the corresponding circular permutants of other Cas9 proteins (including other Cas9 orthologs, variants, etc).
• In particular embodiments, the circular permutant Cas9 has the following structure (based on S. pyogenes Cas9 (1368 amino acids of UniProtKB—Q99ZW2 (CAS9_STRP1) (numbering is based on the amino acid position in SEQ ID NO: 18):
• N-terminus-[102-1368]-[optional linker]-[1-101]-C-terminus;
  N-terminus-[1028-1368]-[optional linker]-[1-1027]-C-terminus;
  N-terminus-[1041-1368]-[optional linker]-[1-1043]-C-terminus;
  N-terminus-[1249-1368]-[optional linker]-[1-1248]-C-terminus; or
  N-terminus-[1300-1368]-[optional linker]-[1-1299]-C-terminus, or the corresponding circular permutants of other Cas9 proteins (including other Cas9 orthologs, variants, etc).
• In still other embodiments, the circular permutant Cas9 has the following structure (based on S. pyogenes Cas9 (1368 amino acids of UniProtKB—Q99ZW2 (CAS9_STRP1) (numbering is based on the amino acid position in SEQ ID NO: 18):
• N-terminus-[103-1368]-[optional linker]-[1-102]-C-terminus;
  N-terminus-[1029-1368]-[optional linker]-[1-1028]-C-terminus;
  N-terminus-[1042-1368]-[optional linker]-[1-1041]-C-terminus;
  N-terminus-[1250-1368]-[optional linker]-[1-1249]-C-terminus; or
  N-terminus-[1301-1368]-[optional linker]-[1-1300]-C-terminus, or the corresponding circular permutants of other Cas9 proteins (including other Cas9 orthologs, variants, etc).
• In some embodiments, the circular permutant can be formed by linking a C-terminal fragment of a Cas9 to an N-terminal fragment of a Cas9, either directly or by using a linker, such as an amino acid linker. In some embodiments, The C-terminal fragment may correspond to the C-terminal 95% or more of the amino acids of a Cas9 (e.g., amino acids about 1300-1368), or the C-terminal 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% or more of a Cas9 (e.g., any one of SEQ ID NOs: 77-86). The N-terminal portion may correspond to the N-terminal 95% or more of the amino acids of a Cas9 (e.g., amino acids about 1-1300), or the N-terminal 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% or more of a Cas9 (e.g., of SEQ ID NO: 18).
• In some embodiments, the circular permutant can be formed by linking a C-terminal fragment of a Cas9 to an N-terminal fragment of a Cas9, either directly or by using a linker, such as an amino acid linker. In some embodiments, the C-terminal fragment that is rearranged to the N-terminus, includes or corresponds to the C-terminal 30% or less of the amino acids of a Cas9 (e.g., amino acids 1012-1368 of SEQ ID NO: 18). In some embodiments, the C-terminal fragment that is rearranged to the N-terminus, includes or corresponds to the C-terminal 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the amino acids of a Cas9 (e.g., the Cas9 of SEQ ID NO: 18). In some embodiments, the C-terminal fragment that is rearranged to the N-terminus, includes or corresponds to the C-terminal 410 residues or less of a Cas9 (e.g., the Cas9 of SEQ ID NO: 18). In some embodiments, the C-terminal portion that is rearranged to the N-terminus, includes or corresponds to the C- terminal 410, 400, 390, 380, 370, 360, 350, 340, 330, 320, 310, 300, 290, 280, 270, 260, 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 residues of a Cas9 (e.g., the Cas9 of SEQ ID NO: 18). In some embodiments, the C-terminal portion that is rearranged to the N-terminus, includes or corresponds to the C- terminal 357, 341, 328, 120, or 69 residues of a Cas9 (e.g., the Cas9 of SEQ ID NO: 18).
• In other embodiments, circular permutant Cas9 variants may be defined as a topological rearrangement of a Cas9 primary structure based on the following method, which is based on S. pyogenes Cas9 of SEQ ID NO: 18: (a) selecting a circular permutant (CP) site corresponding to an internal amino acid residue of the Cas9 primary structure, which dissects the original protein into two halves: an N-terminal region and a C-terminal region; (b) modifying the Cas9 protein sequence (e.g., by genetic engineering techniques) by moving the original C-terminal region (comprising the CP site amino acid) to precede the original N-terminal region, thereby forming a new N-terminus of the Cas9 protein that now begins with the CP site amino acid residue. The CP site can be located in any domain of the Cas9 protein, including, for example, the helical-II domain, the RuvCIII domain, or the CTD domain. For example, the CP site may be located (relative the S. pyogenes Cas9 of SEQ ID NO: 18) at original amino acid residue 181, 199, 230, 270, 310, 1010, 1016, 1023, 1029, 1041, 1247, 1249, or 1282. Thus, once relocated to the N-terminus, original amino acid 181, 199, 230, 270, 310, 1010, 1016, 1023, 1029, 1041, 1247, 1249, or 1282 would become the new N-terminal amino acid. Nomenclature of these CP-Cas9 proteins may be referred to as Cas9-CP¹⁸¹, Cas9-CP¹⁹⁹, Cas9-CP²³⁰, Cas9-CP²⁷⁰, Cas9-CP³¹⁰, Cas9-CP¹⁰¹⁰, Cas9-CP¹⁰¹⁶, Cas9-CP¹⁰²³, Cas9-CP¹⁰²⁹, Cas9_CP¹⁰⁴¹, Cas9-CP¹²⁴⁷, Cas9_CP¹²⁴⁹, and Cas9-CP¹²⁸², respectively. This description is not meant to be limited to making CP variants from SEQ ID NO: 18, but may be implemented to make CP variants in any Cas9 sequence, either at CP sites that correspond to these positions, or at other CP sites entirely. This description is not meant to limit the specific CP sites in any way. Virtually any CP site may be used to form a CP-Cas9 variant.
• Exemplary CP-Cas9 amino acid sequences, based on the Cas9 of SEQ ID NO: 18, are provided below in which linker sequences are indicated by underlining and optional methionine (M) residues are indicated in bold. It should be appreciated that the disclosure provides CP-Cas9 sequences that do not include a linker sequence or that include different linker sequences. It should be appreciated that CP-Cas9 sequences may be based on Cas9 sequences other than that of SEQ ID NO: 18 and any examples provided herein are not meant to be limiting. Exemplary CP-Cas9 sequences are as follows:

• 		SEQ
  		ID
  CP name	Sequence	NO:
  CP1012	DYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE	SEQ
  	ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS	ID
  	MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDW	NO:
  	DPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKEL	77
  	LGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSL
  	FELENGRKRMLASAGELQKGNELALPSKYVNFLYLAS
  	HYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKR
  	VILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNL
  	GAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYE
  	TRIDLSQLGGDGGSGGSGGSGGSGGSGGSGGDKKYSIG
  	LAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
  	LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEI
  	FSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVD
  	EVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMI
  	KFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPI
  	NASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFG
  	NLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDN
  	LLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAP
  	LSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQ
  	SKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVK
  	LNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYP
  	FLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSE
  	ETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVL
  	PKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQK
  	KAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGV
  	EDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLT
  	LTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGW
  	GRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIH
  	DDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGI
  	LQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQ
  	KNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
  	YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD
  	SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLL
  	NAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETR
  	QITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVS
  	DFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKY
  	PKLESEFVYG
  CP 1028	EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNG	SEQ
  	ETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGG	ID
  	FSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYS	NO:
  	VLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDF	78
  	LEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGE
  	LQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQ
  	LFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYN
  	KHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRK
  	RYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDGGSGG
  	SGGSGGSGGSGGSGGMDKKYSIGLAIGTNSVGWAVIT
  	DEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAE
  	ATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFF
  	HRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYH
  	LRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLN
  	PDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILS
  	ARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNF
  	KSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADL
  	FLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEH
  	HQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDG
  	GASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQR
  	TFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKI
  	LTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEV
  	VDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFT
  	VYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTN
  	RKVTVKQLKEDYFKKIECFDSVETSGVEDRFNASLGTY
  	HDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE
  	RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIR
  	DKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQ
  	KAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDEL
  	VKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRI
  	EEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRD
  	MYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTR
  	SDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQR
  	KFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQ
  	ILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQ
  	FYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEF
  	VYGDYKVYDVRKMIAKSEQ
  CP1041	NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRD	SEQ
  	FATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSD	ID
  	KLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGK	NO:
  	SKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKK	79
  	DLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPS
  	KYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLD
  	EIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQA
  	ENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDA
  	TLIHQSITGLYETRIDLSQLGGDGGSGGSGGSGGSGGSG
  	GSGGDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVL
  	GNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYT
  	RRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDK
  	KHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKA
  	DLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQL
  	VQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIA
  	QLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQ
  	LSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLS
  	DILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
  	QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPIL
  	EKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGE
  	LHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLAR
  	GNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIER
  	MTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVT
  	EGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDY
  	FKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFL
  	DNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDK
  	VMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFL
  	KSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSL
  	HEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPE
  	NIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQIL
  	KEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINR
  	LSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNV
  	PSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERG
  	GLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYD
  	ENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYH
  	HAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDV
  	RKMIAKSEQEIGKATAKYFFYS
  CP1249	PEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANL	SEQ
  	DKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFK	ID
  	YFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQL	NO:
  	GGDGGSGGSGGSGGSGGSGGSGGMDKKYSIGLAIGTN	80
  	SVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALL
  	FDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEM
  	AKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAY
  	HEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRG
  	HFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASG
  	VDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIAL
  	SLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
  	GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS
  	MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKN
  	GYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNR
  	EDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLK
  	DNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETI
  	TPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPK
  	HSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKA
  	IVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVED
  	RFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLT
  	LFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWG
  	RLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHD
  	DSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGIL
  	QTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQ
  	KNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
  	LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKD
  	DSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQ
  	LLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVE
  	TRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSK
  	LVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALI
  	KKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAK
  	YFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVW
  	DKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILP
  	KRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAK
  	VEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGY
  	KEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
  	LALPSKYVNFLYLASHYEKLKGS
  CP1300	KPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTST	SEQ
  	KEVLDATLIHQSITGLYETRIDLSQLGGDGGSGGSGGS	ID
  	GGSGGSGGSGGDKKYSIGLAIGTNSVGWAVITDEYKV	NO:
  	PSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLK	78
  	RTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEE
  	SFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKK
  	LVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNS
  	DVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLS
  	KSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNF
  	DLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAA
  	KNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDL
  	TLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQE
  	EFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNG
  	SIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIP
  	YYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGA
  	SAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNEL
  	TKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTV
  	KQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI
  	IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTY
  	AHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSG
  	KTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVS
  	GQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVM
  	GRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIK
  	ELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQ
  	ELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNR
  	GKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL
  	TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSR
  	MNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKV
  	REINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGD
  	YKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI
  	TLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS
  	MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDW
  	DPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKEL
  	LGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSL
  	FELENGRKRMLASAGELQKGNELALPSKYVNFLYLAS
  	HYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKR
  	VILADANLDKVLSAYNKHRD

• The Cas9 circular permutants that may be useful in the prime editing constructs described herein. Exemplary C-terminal fragments of Cas9, based on the Cas9 of SEQ ID NO: 18, which may be rearranged to an N-terminus of Cas9, are provided below. It should be appreciated that such C-terminal fragments of Cas9 are exemplary and are not meant to be limiting. These exemplary CP-Cas9 fragments have the following sequences:

• 			SEQ
  			ID
  	CP name	Sequence	NO:
  	CP1012	DYKVYDVRKMIAKSEQEIGKATAKYFFYSNI	SEQ
  	C-	MNFFKTEITLANGEIRKRPLIETNGETGEIV	ID
  	terminal	WDKGRDFATVRKVLSMPQVNIVKKTEVQTGG	NO:
  	fragment	FSKESILPKRNSDKLIARKKDWDPKKYGGFD	82
  		SPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
  		TIMERSSFEKNPIDFLEAKGYKEVKKDLIIK
  		LPKYSLFELENGRKRMLASAGELQKGNELAL
  		PSKYVNFLYLASHYEKLKGSPEDNEQKQLFV
  		EQHKHYLDEIIEQISEFSKRVILADANLDKV
  		LSAYNKHRDKPIREQAENIIHLFTLTNLGAP
  		AAFKYFDTTIDRKRYTSTKEVLDATLIHQSI
  		TGLYETRIDLSQLGGD
  	CP1028	EIGKATAKYFFYSNIMNFFKTEITLANGEIR	SEQ
  	C-	KRPLIETNGETGEIVWDKGRDFATVRKVLSM	ID
  	terminal	PQVNIVKKTEVQTGGFSKESILPKRNSDKLI	NO:
  	fragment	ARKKDWDPKKYGGFDSPTVAYSVLVVAKVEK	83
  		GKSKKLKSVKELLGITIMERSSFEKNPIDFL
  		EAKGYKEVKKDLIIKLPKYSLFELENGRKRM
  		LASAGELQKGNELALPSKYVNFLYLASHYEK
  		LKGSPEDNEQKQLFVEQHKHYLDEIIEQISE
  		FSKRVILADANLDKVLSAYNKHRDKPIREQA
  		ENIIHLFTLTNLGAPAAFKYFDTTIDRKRYT
  		STKEVLDATLIHQSITGLYETRIDLSQLGGD
  	CP1041	NIMNFFKTEITLANGEIRKRPLIETNGETGE	SEQ
  	C-	IVWDKGRDFATVRKVLSMPQVNIVKKTEVQT	ID
  	terminal	GGFSKESILPKRNSDKLIARKKDWDPKKYGG	NO:
  	fragment	FDSPTVAYSVLVVAKVEKGKSKKLKSVKELL	84
  		GITIMERSSFEKNPIDFLEAKGYKEVKKDLI
  		IKLPKYSLFELENGRKRMLASAGELQKGNEL
  		ALPSKYVNFLYLASHYEKLKGSPEDNEQKQL
  		FVEQHKHYLDEIIEQISEFSKRVILADANLD
  		KVLSAYNKHRDKPIREQAENIIHLFTLTNLG
  		APAAFKYFDTTIDRKRYTSTKEVLDATLIHQ
  		SITGLYETRIDLSQLGGD
  	CP1249	PEDNEQKQLFVEQHKHYLDEIIEQISEFSKR	SEQ
  	C-	VILADANLDKVLSAYNKHRDKPIREQAENII	ID
  	terminal	HLFTLTNLGAPAAFKYFDTTIDRKRYTSTKE	NO:
  	fragment	VLDATLIHQSITGLYETRIDLSQLGGD	85
  	CP1300	KPIREQAENIIHLFTLTNLGAPAAFKYFDTT	SEQ
  	C-	IDRKRYTSTKEVLDATLIHQSITGLYETRID	ID
  	terminal	LSQLGGD	NO:
  	fragment		86

• I. Cas9 Variants with Modified PAM Specificities
• The prime editors of the present disclosure may also comprise Cas9 variants with modified PAM specificities. Some aspects of this disclosure provide Cas9 proteins that exhibit activity on a target sequence that does not comprise the canonical PAM (5′-NGG-3′, where N is A, C, G, or T) at its 3′-end. In some embodiments, the Cas9 protein exhibits activity on a target sequence comprising a 5′-NGG-3′ PAM sequence at its 3′-end. In some embodiments, the Cas9 protein exhibits activity on a target sequence comprising a 5′-NNG-3′ PAM sequence at its 3′-end. In some embodiments, the Cas9 protein exhibits activity on a target sequence comprising a 5′-NNA-3′ PAM sequence at its 3′-end. In some embodiments, the Cas9 protein exhibits activity on a target sequence comprising a 5′-NNC-3′ PAM sequence at its 3′-end. In some embodiments, the Cas9 protein exhibits activity on a target sequence comprising a 5′-NNT-3′ PAM sequence at its 3′-end. In some embodiments, the Cas9 protein exhibits activity on a target sequence comprising a 5′-NGT-3′ PAM sequence at its 3′-end. In some embodiments, the Cas9 protein exhibits activity on a target sequence comprising a 5′-NGA-3′ PAM sequence at its 3′-end. In some embodiments, the Cas9 protein exhibits activity on a target sequence comprising a 5′-NGC-3′ PAM sequence at its 3′-end. In some embodiments, the Cas9 protein exhibits activity on a target sequence comprising a 5′-NAA-3′ PAM sequence at its 3′-end. In some embodiments, the Cas9 protein exhibits activity on a target sequence comprising a 5′-NAC-3′ PAM sequence at its 3′-end. In some embodiments, the Cas9 protein exhibits activity on a target sequence comprising a 5′-NAT-3′ PAM sequence at its 3′-end. In still other embodiments, the Cas9 protein exhibits activity on a target sequence comprising a 5′-NAG-3′ PAM sequence at its 3′-end.
• It should be appreciated that any of the amino acid mutations described herein, (e.g., A262T) from a first amino acid residue (e.g., A) to a second amino acid residue (e.g., T) may also include mutations from the first amino acid residue to an amino acid residue that is similar to (e.g., conserved) the second amino acid residue. For example, mutation of an amino acid with a hydrophobic side chain (e.g., alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan) may be a mutation to a second amino acid with a different hydrophobic side chain (e.g., alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan). For example, a mutation of an alanine to a threonine (e.g., a A262T mutation) may also be a mutation from an alanine to an amino acid that is similar in size and chemical properties to a threonine, for example, serine. As another example, mutation of an amino acid with a positively charged side chain (e.g., arginine, histidine, or lysine) may be a mutation to a second amino acid with a different positively charged side chain (e.g., arginine, histidine, or lysine). As another example, mutation of an amino acid with a polar side chain (e.g., serine, threonine, asparagine, or glutamine) may be a mutation to a second amino acid with a different polar side chain (e.g., serine, threonine, asparagine, or glutamine). Additional similar amino acid pairs include, but are not limited to, the following: phenylalanine and tyrosine; asparagine and glutamine; methionine and cysteine; aspartic acid and glutamic acid; and arginine and lysine. The skilled artisan would recognize that such conservative amino acid substitutions will likely have minor effects on protein structure and are likely to be well tolerated without compromising function. In some embodiments, any amino of the amino acid mutations provided herein from one amino acid to a threonine may be an amino acid mutation to a serine. In some embodiments, any amino of the amino acid mutations provided herein from one amino acid to an arginine may be an amino acid mutation to a lysine. In some embodiments, any amino of the amino acid mutations provided herein from one amino acid to an isoleucine, may be an amino acid mutation to an alanine, valine, methionine, or leucine. In some embodiments, any amino of the amino acid mutations provided herein from one amino acid to a lysine may be an amino acid mutation to an arginine. In some embodiments, any amino of the amino acid mutations provided herein from one amino acid to an aspartic acid may be an amino acid mutation to a glutamic acid or asparagine. In some embodiments, any amino of the amino acid mutations provided herein from one amino acid to a valine may be an amino acid mutation to an alanine, isoleucine, methionine, or leucine. In some embodiments, any amino of the amino acid mutations provided herein from one amino acid to a glycine may be an amino acid mutation to an alanine. It should be appreciated, however, that additional conserved amino acid residues would be recognized by the skilled artisan and any of the amino acid mutations to other conserved amino acid residues are also within the scope of this disclosure.
• In some embodiments, the Cas9 protein comprises a combination of mutations that exhibit activity on a target sequence comprising a 5′-NAA-3′ PAM sequence at its 3′-end. In some embodiments, the combination of mutations are present in any one of the clones listed in Table 1. In some embodiments, the combination of mutations are conservative mutations of the clones listed in Table 1. In some embodiments, the Cas9 protein comprises the combination of mutations of any one of the Cas9 clones listed in Table 1.

• TABLE 1
  NAA PAM Clones
  Mutations from wild-type SpCas9 (e.g., SEQ ID NO: 18)

  D177N, K218R, D614N, D1135N, P1137S, E1219V, A1320V, A1323D, R1333K

  D177N, K218R, D614N, D1135N, E1219V, Q1221H, H1264Y, A1320V, R1333K

  A10T, I322V, S409I, E427G, G715C, D1135N, E1219V, Q1221H, H1264Y, A1320V,

  R1333K

  A367T, K710E, R1114G, D1135N, P11375, E1219V, Q1221H, H1264Y, A1320V, R1333K

  A10T, I322V, S409I, E427G, R753G, D861N, D1135N, K1188R, E1219V, Q1221H,

  H1264H, A1320V, R1333K

  A10T, I322V, S409I, E427G, R654L, V743I, R753G, M1021T, D1135N, D1180G, K1211R,

  E1219V, Q1221H, H1264Y, A1320V, R1333K

  A10T, I322V, S409I, E427G, V743I, R753G, E762G, D1135N, D1180G, K1211R, E1219V,

  Q1221H, H1264Y, A1320V, R1333K

  A10T, I322V, S409I, E427G, R753G, D1135N, D1180G, K1211R, E1219V, Q1221H,

  H1264Y, S1274R, A1320V, R1333K

  A10T, I322V, S409I, E427G, A5895, R753G, D1135N, E1219V, Q1221H, H1264H,

  A1320V, R1333K

  A10T, I322V, S409I, E427G, R753G, E757K, G865G, D1135N, E1219V, Q1221H, H1264Y,

  A1320V, R1333K

  A10T, I322V, S409I, E427G, R654L, R753G, E757K, D1135N, E1219V, Q1221H, H1264Y,

  A1320V, R1333K

  A10T, I322V, S409I, E427G, K599R, M631A, R654L, K673E, V743I, R753G, N758H,

  E762G, D1135N, D1180G, E1219V, Q1221H, Q1256R, H1264Y, A1320V, A1323D,

  R1333K

  A10T, I322V, S409I, E427G, R654L, K673E, V743I, R753G, E762G, N8695, N1054D,

  R1114G, D1135N, D1180G, E1219V, Q1221H, H1264Y, A1320V, A1323D, R1333K

  A10T, I322V, S409I, E427G, R654L, L727I, V743I, R753G, E762G, R859S, N946D,

  F1134L, D1135N, D1180G, E1219V, Q1221H, H1264Y, N1317T, A1320V, A1323D,

  R1333K

  A10T, I322V, S409I, E427G, R654L, K673E, V743I, R753G, E762G, N803S, N869S,

  Y1016D, G1077D, R1114G, F1134L, D1135N, D1180G, E1219V, Q1221H, H1264Y,

  V1290G, L13185, A1320V, A1323D, R1333K

  A10T, I322V, S4091, E427G, R654L, K673E, V7431, R753G, E762G, N803S, N869S,

  Y1016D, G1077D, R1114G, F1134L, D1135N, K1151E, D1180G, E1219V, Q1221H,

  H1264Y, V1290G, L1318S, A1320V, R1333K

  A10T, I322V, S4091, E427G, R654L, K673E, V743I, R753G, E762G, N803S, N869S,

  Y1016D, G1077D, R1114G, F1134L, D1135N, D1180G, E1219V, Q1221H, H1264Y,

  V1290G, L1318S, A1320V, A1323D, R1333K

  A10T, I322V, S409I, E427G, R654L, K673E, F693L, V7431, R753G, E762G, N803S, N869S,

  L921P, Y1016D, G1077D, F1080S, R1114G, D1135N, D1180G, E1219V, Q1221H, H1264Y,

  L1318S, A1320V, A1323D, R1333K

  A10T, I322V, S409I, E427G, E630K, R654L, K673E, V743I, R753G, E762G, Q768H,

  N803S, N869S, Y1016D, G1077D, R1114G, F1134L, D1135N, D1180G, E1219V, Q1221H,

  H1264Y, L1318S, A1320V, R1333K

  A10T, I322V, S4091, E427G, R654L, K673E, F693L, V743I, R753G, E762G, Q768H,

  N803S, N869S, Y1016D, G1077D, R1114G, F1134L, D1135N, D1180G, E1219V, Q1221H,

  G1223S, H1264Y, L1318S, A1320V, R1333K

  A10T, I322V, S409I, E427G, R654L, K673E, F693L, V743I, R753G, E762G, N803S, N869S,

  L921P, Y1016D, G1077D, F1801S, R1114G, D1135N, D1180G, E1219V, Q1221H, H1264Y,

  L1318S, A1320V, A1323D, R1333K

  A10T, I322V, S409I, E427G, R654L, V743I, R753G, M1021T, D1135N, D1180G, K1211R,

  E1219V, Q1221H, H1264Y, A1320V, R1333K

  A10T, I322V, S409I, E427G, R654L, K673E, V743I, R753G, E762G, M673I, N803S, N869S,

  G1077D, R1114G, D1135N, V1139A, D1180G, E1219V, Q1221H, A1320V, R1333K

  A10T, I322V, S409I, E427G, R654L, K673E, V743I, R753G, E762G, N803S, N869S,

  R1114G, D1135N, E1219V, Q1221H, A1320V, R1333K

• In some embodiments, the Cas9 protein comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of a Cas9 protein as provided by any one of the variants of Table 1. In some embodiments, the Cas9 protein comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the amino acid sequence of a Cas9 protein as provided by any one of the variants of Table 1.
• In some embodiments, the Cas9 protein exhibits an increased activity on a target sequence that does not comprise the canonical PAM (5′-NGG-3′) at its 3′ end as compared to Streptococcus pyogenes Cas9 as provided by SEQ ID NO: 18. In some embodiments, the Cas9 protein exhibits an activity on a target sequence having a 3′ end that is not directly adjacent to the canonical PAM sequence (5′-NGG-3′) that is at least 5-fold increased as compared to the activity of Streptococcus pyogenes Cas9 as provided by SEQ ID NO: 18 on the same target sequence. In some embodiments, the Cas9 protein exhibits an activity on a target sequence that is not directly adjacent to the canonical PAM sequence (5′-NGG-3′) that is at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1,000-fold, at least 5,000-fold, at least 10,000-fold, at least 50,000-fold, at least 100,000-fold, at least 500,000-fold, or at least 1,000,000-fold increased as compared to the activity of Streptococcus pyogenes as provided by SEQ ID NO: 2 on the same target sequence. In some embodiments, the 3′ end of the target sequence is directly adjacent to an AAA, GAA, CAA, or TAA sequence. In some embodiments, the Cas9 protein comprises a combination of mutations that exhibit activity on a target sequence comprising a 5′-NAC-3′ PAM sequence at its 3′-end. In some embodiments, the combination of mutations are present in any one of the clones listed in Table 2. In some embodiments, the combination of mutations are conservative mutations of the clones listed in Table 2. In some embodiments, the Cas9 protein comprises the combination of mutations of any one of the Cas9 clones listed in Table 2.

• TABLE 2
  NAC PAM Clones
  MUTATIONS FROM WILD-TYPE SPCAS9 (E.G., SEQ ID NO: 18)

  T472I, R753G, K890E, D1332N, R1335Q, T1337N

  I1057S, D1135N, P1301S, R1335Q, T1337N

  T4721, R753G, D1332N, R1335Q, T1337N

  D1135N, E1219V, D1332N, R1335Q, T1337N

  T472I, R753G, K890E, D1332N, R1335Q, T1337N

  I1057S, D1135N, P1301S, R1335Q, T1337N

  T4721, R753G, D1332N, R1335Q, T1337N

  T472I, R753G, Q771H, D1332N, R1335Q, T1337N

  E627K, T638P, K652T, R753G, N8035, K959N, R1114G, D1135N, E1219V, D1332N,

  R1335Q, T1337N

  E627K, T638P, K652T, R753G, N803S, K959N, R1114G, D1135N, K1156E, E1219V,

  D1332N, R1335Q, T1337N

  E627K, T638P, V647I, R753G, N803S, K959N, G1030R, 11055E, R1114G, D1135N, E1219V,

  D1332N, R1335Q, T1337N

  E627K, E630G, T638P, V647A, G687R, N767D, N803S, K959N, R1114G, D1135N, E1219V,

  D1332G, R1335Q, T1337N

  E627K, T638P, R753G, N803S, K959N, R1114G, D1135N, E1219V, N1266H, D1332N,

  R1335Q, T1337N

  E627K, T638P, R753G, N8035, K959N, I1057T, R1114G, D1135N, E1219V, D1332N,

  R1335Q, T1337N

  E627K, T638P, R753G, N803S, K959N, R1114G, D1135N, E1219V, D1332N, R1335Q,

  T1337N

  E627K, M631I, T638P, R753G, N803S, K959N, Y1036H, R1114G, D1135N, E1219V,

  D1251G, D1332G, R1335Q, T1337N

  E627K, T638P, R753G, N803S, V875I, K959N, Y1016C, R1114G, D1135N, E1219V,

  D1251G, D1332G, R1335Q, T1337N, I1348V

  K608R, E627K, T638P, V647I, R654L, R753G, N803S, T804A, K848N, V922A, K959N,

  R1114G D1135N, E1219V, D1332N, R1335Q, T1337N

  K608R, E627K, T638P, V647I, R753G, N803S, V922A, K959N, K1014N, V1015A, R1114G,

  D1135N, K1156N, E1219V, N1252D, D1332N, R1335Q, T1337N

  K608R, E627K, R629G, T638P, V647I, A711T, R753G, K775R, K789E, N803S, K959N,

  V1015A, Y1036H, R1114G, D1135N, E1219V, N1286H, D1332N, R1335Q, T1337N

  K608R, E627K, T638P, V647I, T740A, R753G, N803S, K948E, K959N, Y1016S, R1114G,

  D1135N, E1219V, N1286H, D1332N, R1335Q, T1337N

  K608R, E627K, T638P, V647I, T740A, N803S, K948E, K959N, Y1016S, R1114G, D1135N,

  E1219V, N1286H, D1332N, R1335Q, T1337N

  I670S, K608R, E627K, E630G, T638P, V647I, R653K, R753G, I795L, K797N, N803S,

  K866R, K890N, K959N, Y1016C, R1114G, D1135N, E1219V, D1332N, R1335Q, T1337N

  K608R, E627K, T638P, V647I, T740A, G752R, R753G, K797N, N803S, K948E, K959N,

  V1015A, Y1016S, R1114G, D1135N, E1219V, N1266H, D1332N, R1335Q, T1337N

  I570T, A589V, K608R, E627K, T638P, V6471, R654L, Q716R, R753G, N803S, K948E,

  K959N, Y1016S, R1114G, D1135N, E1207G, E1219V, N1234D, D1332N, R1335Q, T1337N

  K608R, E627K, R629G, T638P, V647I, R654L, Q740R, R753G, N803S, K959N, N990S,

  T995S, V1015A, Y1036D, R1114G, D1135N, E1207G, E1219V, N1234D, N1266H, D1332N,

  R1335Q, T1337N

  I562F, V565D, I570T, K608R, L625S, E627K, T638P, V647I, R654I, G752R, R753G, N803S,

  N808D, K959N, M1021L, R1114G, D1135N, N1177S, N1234D, D1332N, R1335Q, T1337N

  I562F, I570T, K608R, E627K, T638P, V647I, R753G, E790A, N803S, K959N, V1015A,

  Y1036H, R1114G, D1135N, D1180E, A1184T, E1219V, D1332N, R1335Q, T1337N

  I570T, K608R, E627K, T638P, V647I, R654H, R753G, E790A, N803S, K959N, V1015A,

  R1114G, D1127A, D1135N, E1219V, D1332N, R1335Q, T1337N

  I570T, K608R, L625S, E627K, T638P, V6471, R6541, T703P, R753G, N803S, N808D, K959N,

  M1021L, R1114G, D1135N, E1219V, D1332N, R1335Q, T1337N

  I570S, K608R, E627K, E630G, T638P, V6471, R653K, R753G, I795L, N803S, K866R,

  K890N, K959N, Y1016C, R1114G, D1135N, E1219V, D1332N, R1335Q, T1337N

  I570T, K608R, E627K, T638P, V6471, R654H, R753G, E790A, N803S, K959N, V1016A,

  R1114G, D1135N, E1219V, K1246E, D1332N, R1335Q, T1337N

  K608R, E627K, T638P, V647I, R654L, K673E, R753G, E790A, N803S, K948E, K959N,

  R1114G, D1127G, D1135N, D1180E, E1219V, N1286H, D1332N, R1335Q, T1337N

  K608R, L625S, E627K, T638P, V647I, R6541, 1670T, R753G, N803S, N808D, K959N,

  M1021L, R1114G, D1135N, E1219V, N1286H, D1332N, R1335Q, T1337N

  E627K, M631V, T638P, V6471, K710E, R753G, N803S, N808D, K948E, M1021L, R1114G,

  D1135N, E1219V, D1332N, R1335Q, T1337N, S1338T, H1349R

• In some embodiments, the Cas9 protein comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of a Cas9 protein as provided by any one of the variants of Table 2. In some embodiments, the Cas9 protein comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the amino acid sequence of a Cas9 protein as provided by any one of the variants of Table 2.
• In some embodiments, the Cas9 protein exhibits an increased activity on a target sequence that does not comprise the canonical PAM (5′-NGG-3′) at its 3′ end as compared to Streptococcus pyogenes Cas9 as provided by SEQ ID NO: 18. In some embodiments, the Cas9 protein exhibits an activity on a target sequence having a 3′ end that is not directly adjacent to the canonical PAM sequence (5′-NGG-3′) that is at least 5-fold increased as compared to the activity of Streptococcus pyogenes Cas9 as provided by SEQ ID NO: 18 on the same target sequence. In some embodiments, the Cas9 protein exhibits an activity on a target sequence that is not directly adjacent to the canonical PAM sequence (5′-NGG-3′) that is at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1,000-fold, at least 5,000-fold, at least 10,000-fold, at least 50,000-fold, at least 100,000-fold, at least 500,000-fold, or at least 1,000,000-fold increased as compared to the activity of Streptococcus pyogenes as provided by SEQ ID NO: 18 on the same target sequence. In some embodiments, the 3′ end of the target sequence is directly adjacent to an AAC, GAC, CAC, or TAC sequence.
• In some embodiments, the Cas9 protein comprises a combination of mutations that exhibit activity on a target sequence comprising a 5′-NAT-3′ PAM sequence at its 3′-end. In some embodiments, the combination of mutations are present in any one of the clones listed in Table 3. In some embodiments, the combination of mutations are conservative mutations of the clones listed in Table 3. In some embodiments, the Cas9 protein comprises the combination of mutations of any one of the Cas9 clones listed in Table 3.

• TABLE 3
  NAT PAM Clones
  MUTATIONS FROM WILD-TYPE SPCAS9 (E.G., SEQ ID NO: 18)

  K961E, H985Y, D1135N, K1191N, E1219V, Q1221H, A1320A, P1321S, R1335L

  D1135N, G1218S, E1219V, Q1221H, P1249S, P1321S, D1322G, R1335L

  V743I, R753G, E790A, D1135N, G1218S, E1219V, Q1221H, A1227V, P1249S, N1286K,

  A1293T, P1321S, D1322G, R1335L, T1339I

  F575S, M631L, R654L, V748I, V743I, R753G, D853E, V922A, R1114G D1135N, G1218S,

  E1219V, Q1221H, A1227V, P1249S, N1286K, A1293T, P1321S, D1322G, R1335L, T1339I

  F575S, M631L, R654L, R664K, R753G, D853E, V922A, R1114G D1135N, D1180G, G1218S,

  E1219V, Q1221H, P1249S, N1286K, P1321S, D1322G, R1335L

  M631L, R654L, R753G, K797E, D853E, V922A, D1012A, R1114G D1135N, G1218S,

  E1219V, Q1221H, P1249S, N1317K, P1321S, D1322G, R1335L

  F575S, M631L, R654L, R664K, R753G, D853E, V922A, R1114G, Y1131C, D1135N,

  D1180G, G1218S, E1219V, Q1221H, P1249S, P1321S, D1322G, R1335L

  F575S, M631L, R654L, R664K, R753G, D853E, V922A, R1114G, Y1131C, D1135N,

  D1180G, G1218S, E1219V, Q1221H, P1249S, P1321S, D1322G, R1335L

  F575S, D596Y, M631L, R654L, R664K, R753G, D853E V922A, R1114G, Y1131C, D1135N,

  D1180G, G1218S, E1219V, Q1221H, P1249S, Q1256R, P1321S, D1322G, R1335L

  F575S, M631L, R654L, R664K, K710E, V750A, R753G, D853E, V922A, R1114G, Y1131C,

  D1135N, D1180G, G12185, E1219V, Q1221H, P1249S, P1321S, D1322G, R1335L

  F575S, M631L, K649R, R654L, R664K, R753G, D853E, V922A, R1114G, Y1131C, D1135N,

  K1156E, D1180G, G12185, E1219V, Q1221H, P1249S, P1321S, D1322G, R1335L

  F575S, M631L, R654L, R664K, R753G, D853E, V922A, R1114G, Y1131C, D1135N,

  D1180G, G1218S, E1219V, Q1221H, P1249S, P1321S, D1322G, R1335L

  F575S, M631L, R654L, R664K, R753G, D853E, V922A, I1057G, R1114G, Y1131C, D1135N,

  D1180G, G1218S, E1219V, Q1221H, P1249S, N1308D, P1321S, D1322G, R1335L

  M631L, R654L, R753G, D853E, V922A, R1114G, Y1131C, D1135N, E1150V, D1180G,

  G1218S, E1219V, Q1221H, P1249S, P1321S, D1332G, R1335L

  M631L, R654L, R664K, R753G, D853E, I1057V, Y1131C, D1135N, D1180G, G1218S,

  E1219V, Q1221H, P1249S, P1321S, D1332G, R1335L

  M631L, R654L, R664K, R753G, I1057V, R1114G, Y1131C, D1135N, D1180G, G1218S,

  E1219V, Q1221H, P1249S, P1321S, D1332G, R1335L

• The above description of various napDNAbps which can be used in connection with the presently disclose prime editors is not meant to be limiting in any way. The prime editors may comprise the canonical SpCas9, or any ortholog Cas9 protein, or any variant Cas9 protein—including any naturally occurring variant, mutant, or otherwise engineered version of Cas9—that is known or which can be made or evolved through a directed evolutionary or otherwise mutagenic process. In various embodiments, the Cas9 or Cas9 variants have a nickase activity, i.e., only cleave of strand of the target DNA sequence. In other embodiments, the Cas9 or Cas9 variants have inactive nucleases, i.e., are “dead” Cas9 proteins. Other variant Cas9 proteins that may be used are those having a smaller molecular weight than the canonical SpCas9 (e.g., for easier delivery) or having modified or rearranged primary amino acid structure (e.g., the circular permutant formats). The prime editors described herein may also comprise Cas9 equivalents, including Cas12a/Cpf1 and Cas12b proteins which are the result of convergent evolution. The napDNAbps used herein (e.g., SpCas9, Cas9 variant, or Cas9 equivalents) may also may also contain various modifications that alter/enhance their PAM specifities. Lastly, the application contemplates any Cas9, Cas9 variant, or Cas9 equivalent which has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.9% sequence identity to a reference Cas9 sequence, such as a references SpCas9 canonical sequences or a reference Cas9 equivalent (e.g., Cas12a/Cpf1).
• In a particular embodiment, the Cas9 variant having expanded PAM capabilities is SpCas9 (H840A) VRQR (SEQ ID NO: 87), which has the following amino acid sequence (with the V, R, Q, R substitutions relative to the SpCas9 (H840A) of SEQ ID NO: 51 being show in bold underline. In addition, the methionine residue in SpCas9 (H840) was removed for SpCas9 (H840A) VRQR):

• 	(SEQ ID NO: 87)
  	DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRH
  	SIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICY
  	LQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGN
  	IVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHM
  	IKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPI
  	NASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNL
  	IALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
  	IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASM
  	IKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAG
  	YIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRK
  	QRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIE
  	KILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEV
  	VDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVY
  	NELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTV
  	KQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKII
  	KDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAH
  	LFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILD
  	FLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLH
  	EHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVI
  	EMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPV
  	ENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAI
  	VPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKN
  	YWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQL
  	VETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSK
  	LVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKY
  	PKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSN
  	IMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFA
  	TVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIA
  	RKKDWDPKKYGGF V SPTVAYSVLVVAKVEKGKSKKLKSVK
  	ELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKY
  	SLFELENGRKRMLASA R ELQKGNELALPSKYVNFLYLASH
  	YEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVI
  	LADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAP
  	AAFKYFDTTIDRK Q Y R STKEVLDATLIHQSITGLYETRID
  	LSQLGGD

• In another particular embodiment, the Cas9 variant having expanded PAM capabilities is SpCas9 (H840A) VRER, which has the following amino acid sequence (with the V, R, E, R substitutions relative to the SpCas9 (H840A) of SEQ ID NO: 51 being shown in bold underline. In addition, the methionine residue in SpCas9 (H840) was removed for SpCas9 (H840A) VRER):

• 	(SEQ ID NO: 88)
  	DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRH
  	SIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICY
  	YLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFG
  	NIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAH
  	MIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP
  	INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGN
  	LIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLA
  	QIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS
  	MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYA
  	GYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
  	KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKI
  	EKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEE
  	VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTV
  	YNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVT
  	VKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI
  	IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA
  	HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTIL
  	DFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSL
  	HEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP
  	VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDA
  	IVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQ
  	LVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS
  	KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK
  	YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYS
  	NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDF
  	ATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLI
  	ARKKDWDPKKYGGF V SPTVAYSVLVVAKVEKGKSKKLKSV
  	KELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK
  	YSLFELENGRKRMLASA R ELQKGNELALPSKYVNFLYLAS
  	HYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRV
  	ILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGA
  	PAAFKYFDTTIDRK E Y R STKEVLDATLIHQSITGLYETRI
  	DLSQLGGD

• In some embodiments, the napDNAbp that functions with a non-canonical PAM sequence is an Argonaute protein. One example of such a nucleic acid programmable DNA binding protein is an Argonaute protein from Natronobacterium gregoryi (NgAgo). NgAgo is a ssDNA-guided endonuclease. NgAgo binds 5′ phosphorylated ssDNA of ˜24 nucleotides (gDNA) to guide it to its target site and will make DNA double-strand breaks at the gDNA site. In contrast to Cas9, the NgAgo-gDNA system does not require a protospacer-adjacent motif (PAM). Using a nuclease inactive NgAgo (dNgAgo) can greatly expand the bases that may be targeted. The characterization and use of NgAgo have been described in Gao et al., Nat Biotechnol., 2016 July; 34(7):768-73. PubMed PMID: 27136078; Swarts et al., Nature. 507(7491) (2014):258-61; and Swarts et al., Nucleic Acids Res. 43(10) (2015):5120-9, each of which is incorporated herein by reference.
• In some embodiments, the napDNAbp is a prokaryotic homolog of an Argonaute protein. Prokaryotic homologs of Argonaute proteins are known and have been described, for example, in Makarova K., et al., “Prokaryotic homologs of Argonaute proteins are predicted to function as key components of a novel system of defense against mobile genetic elements”, Biol Direct. 2009 Aug. 25; 4:29. doi: 10.1186/1745-6150-4-29, the entire contents of which is hereby incorporated by reference. In some embodiments, the napDNAbp is a Marinitoga piezophila Argunaute (MpAgo) protein. The CRISPR-associated Marinitoga piezophila Argunaute (MpAgo) protein cleaves single-stranded target sequences using 5′-phosphorylated guides. The 5′ guides are used by all known Argonautes. The crystal structure of an MpAgo-RNA complex shows a guide strand binding site comprising residues that block 5′ phosphate interactions. This data suggests the evolution of an Argonaute subclass with noncanonical specificity for a 5′-hydroxylated guide. See, e.g., Kaya et al., “A bacterial Argonaute with noncanonical guide RNA specificity”, Proc Natl Acad Sci USA. 2016 Apr. 12; 113(15):4057-62, the entire contents of which are hereby incorporated by reference). It should be appreciated that other argonaute proteins may be used, and are within the scope of this disclosure.
• Some aspects of the disclosure provide Cas9 domains that have different PAM specificities. Typically, Cas9 proteins, such as Cas9 from S. pyogenes (spCas9), require a canonical NGG PAM sequence to bind a particular nucleic acid region. This may limit the ability to edit desired bases within a genome. In some embodiments, the base editing fusion proteins provided herein may need to be placed at a precise location, for example where a target base is placed within a 4 base region (e.g., a “editing window”), which is approximately 15 bases upstream of the PAM. See Komor, A. C., et al., “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage” Nature 533, 420-424 (2016), the entire contents of which are hereby incorporated by reference. Accordingly, in some embodiments, any of the fusion proteins provided herein may contain a Cas9 domain that is capable of binding a nucleotide sequence that does not contain a canonical (e.g., NGG) PAM sequence. Cas9 domains that bind to non-canonical PAM sequences have been described in the art and would be apparent to the skilled artisan. For example, Cas9 domains that bind non-canonical PAM sequences have been described in Kleinstiver, B. P., et al., “Engineered CRISPR-Cas9 nucleases with altered PAM specificities” Nature 523, 481-485 (2015); and Kleinstiver, B. P., et al., “Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition” Nature Biotechnology 33, 1293-1298 (2015); the entire contents of each are hereby incorporated by reference.
• For example, a napDNAbp domain with altered PAM specificity, such as a domain with at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with wild type Francisella novicida Cpf1 (D917, E1006, and D1255) (SEQ ID NO: 74), which has the following amino acid sequence:

• 	(SEQ ID NO: 74)
  	MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDD
  	EKRAKDYKKAKQIIDKYHQFFIEEILSSVCISEDLLQNYS
  	DVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFK
  	NLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDIT
  	DIDEALEIIKSFKGWTTYFKGFHENRKNVYSSNDIPTSII
  	YRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAE
  	ELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITK
  	FNTIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYK
  	MSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIA
  	AFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLT
  	DLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQEL
  	IAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILA
  	NFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAE
  	DDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEH
  	FYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNF
  	ENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKI
  	FDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIK
  	FYNPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKF
  	IDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQ
  	GYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGR
  	PNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKK
  	ITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFF
  	HCPITINFKSSGANKFNDEINLLLKEKANDVHILSIDRGE
  	RHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAI
  	EKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYN
  	AIVVFEDLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVF
  	KDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAG
  	FTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLD
  	KGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKN
  	HNWDTREVYPTKELEKLLKDYSIEYGHGGECIKAAICGES
  	DKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGN
  	FFDSRQAPKNMPQDADANGAYHIGLKGLMLLGRIKNNQEG
  	KKLNLVIKNEEYFEFVQNRNN

• An additional napDNAbp domain with altered PAM specificity, such as a domain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with wild type Geobacillus thermodenitrificans Cas9 (SEQ ID NO: 75), which has the following amino acid sequence:

• 	(SEQ ID NO: 75)
  	MKYKIGLDIGITSIGWAVINLDIPRIEDLGVRIFDRAENP
  	KTGESLALPRRLARSARRRLRRRKHRLERIRRLFVREGIL
  	TKEELNKLFEKKHEIDVWQLRVEALDRKLNNDELARILLH
  	LAKRRGFRSNRKSERTNKENSTMLKHIEENQSILSSYRTV
  	AEMVVKDPKFSLHKRNKEDNYTNTVARDDLEREIKLIFAK
  	QREYGNIVCTEAFEHEYISIWASQRPFASKDDIEKKVGFC
  	TFEPKEKRAPKATYTFQSFTVWEHINKLRLVSPGGIRALT
  	DDERRLIYKQAFHKNKITFHDVRTLLNLPDDTRFKGLLYD
  	RNTTLKENEKVRFLELGAYHKIRKAIDSVYGKGAAKSFRP
  	IDFDTFGYALTMFKDDTDIRSYLRNEYEQNGKRMENLADK
  	VYDEELIEELLNLSFSKFGHLSLKALRNILPYMEQGEVYS
  	TACERAGYTFTGPKKKQKTVLLPNIPPIANPVVMRALTQA
  	RKVVNAIIKKYGSPVSIHIELARELSQSFDERRKMQKEQE
  	GNRKKNETAIRQLVEYGLTLNPTGLDIVKFKLWSEQNGKC
  	AYSLQPIEIERLLEPGYTEVDHVIPYSRSLDDSYTNKVLV
  	LTKENREKGNRTPAEYLGLGSERWQQFETFVLTNKQFSKK
  	KRDRLLRLHYDENEENEFKNRNLNDTRYISRFLANFIREH
  	LKFADSDDKQKVYTVNGRITAHLRSRWNFNKNREESNLHH
  	AVDAAIVACTTPSDIARVTAFYQRREQNKELSKKTDPQFP
  	QPWPHFADELQARLSKNPKESIKALNLGNYDNEKLESLQP
  	VFVSRMPKRSITGAAHQETLRRYIGIDERSGKIQTVVKKK
  	LSEIQLDKTGHFPMYGKESDPRTYEAIRQRLLEHNNDPKK
  	AFQEPLYKPKKNGELGPIIRTIKIIDTTNQVIPLNDGKTV
  	AYNSNIVRVDVFEKDGKYYCVPIYTIDMMKGILPNKAIEP
  	NKPYSEWKEMTEDYTFRFSLYPNDLIRIEFPREKTIKTAV
  	GEEIKIKDLFAYYQTIDSSNGGLSLVSHDNNFSLRSIGSR
  	TLKRFEKYQVDVLGNIYKVRGEKRVGVASSSHSKAGETIR
  	PL

• In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) is a nucleic acid programmable DNA binding protein that does not require a canonical (NGG) PAM sequence. In some embodiments, the napDNAbp is an argonaute protein. One example of such a nucleic acid programmable DNA binding protein is an Argonaute protein from Natronobacterium gregoryi (NgAgo). NgAgo is a ssDNA-guided endonuclease. NgAgo binds 5′ phosphorylated ssDNA of ˜24 nucleotides (gDNA) to guide it to its target site and will make DNA double-strand breaks at the gDNA site. In contrast to Cas9, the NgAgo-gDNA system does not require a protospacer-adjacent motif (PAM). Using a nuclease inactive NgAgo (dNgAgo) can greatly expand the bases that may be targeted. The characterization and use of NgAgo have been described in Gao et al., Nat Biotechnol., 34(7): 768-73 (2016), PubMed PMID: 27136078; Swarts et al., Nature, 507(7491): 258-61 (2014); and Swarts et al., Nucleic Acids Res. 43(10) (2015): 5120-9, each of which is incorporated herein by reference. The sequence of Natronobacterium gregoryi Argonaute is provided in SEQ ID NO: 76.
• The disclosed fusion proteins may comprise a napDNAbp domain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with wild type Natronobacterium gregoryi Argonaute (SEQ ID NO: 76), which has the following amino acid sequence:

• 	(SEQ ID NO: 76)
  	MTVIDLDSTTTADELTSGHTYDISVTLTGVYDNTDEQHPR
  	MSLAFEQDNGERRYITLWKNTTPKDVFTYDYATGSTYIFT
  	NIDYEVKDGYENLTATYQTTVENATAQEVGTTDEDETFAG
  	GEPLDHHLDDALNETPDDAETESDSGHVMTSFASRDQLPE
  	WTLHTYTLTATDGAKTDTEYARRTLAYTVRQELYTDHDAA
  	PVATDGLMLLTPEPLGETPLDLDCGVRVEADETRTLDYTT
  	AKDRLLARELVEEGLKRSLWDDYLVRGIDEVLSKEPVLTC
  	DEFDLHERYDLSVEVGHSGRAYLHINFRHRFVPKLTLADI
  	DDDNIYPGLRVKTTYRPRRGHIVWGLRDECATDSLNTLGN
  	QSVVAYHRNNQTPINTDLLDAIEAADRRVVETRRQGHGDD
  	AVSFPQELLAVEPNTHQIKQFASDGFHQQARSKTRLSASR
  	CSEKAQAFAERLDPVRLNGSTVEFSSEFFTGNNEQQLRLL
  	YENGESVLTFRDGARGAHPDETFSKGIVNPPESFEVAVVL
  	PEQQADTCKAQWDTMADLLNQAGAPPTRSETVQYDAFSSP
  	ESISLNVAGAIDPSEVDAAFVVLPPDQEGFADLASPTETY
  	DELKKALANMGIYSQMAYFDRFRDAKIFYTRNVALGLLAA
  	AGGVAFTTEHAMPGDADMFIGIDVSRSYPEDGASGQINIA
  	ATATAVYKDGTILGHSSTRPQLGEKLQSTDVRDIMKNAIL
  	GYQQVTGESPTHIVIHRDGFMNEDLDPATEFLNEQGVEYD
  	IVEIRKQPQTRLLAVSDVQYDTPVKSIAAINQNEPRATVA
  	TFGAPEYLATRDGGGLPRPIQIERVAGETDIETLTRQVYL
  	LSQSHIQVHNSTARLPITTAYADQASTHATKGYLVQTGAF
  	ESNVGFL

• In addition, any available methods may be utilized to obtain or construct a variant or mutant Cas9 protein. The term “mutation,” as used herein, refers to a substitution of a residue within a sequence, e.g., a nucleic acid or amino acid sequence, with another residue, or a deletion or insertion of one or more residues within a sequence. Mutations are typically described herein by identifying the original residue followed by the position of the residue within the sequence and by the identity of the newly substituted residue. Various methods for making the amino acid substitutions (mutations) provided herein are well known in the art, and are provided by, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)). Mutations can include a variety of categories, such as single base polymorphisms, microduplication regions, indel, and inversions, and is not meant to be limiting in any way. Mutations can include “loss-of-function” mutations which is the normal result of a mutation that reduces or abolishes a protein activity. Most loss-of-function mutations are recessive, because in a heterozygote the second chromosome copy carries an unmutated version of the gene coding for a fully functional protein whose presence compensates for the effect of the mutation. Mutations also embrace “gain-of-function” mutations, which is one which confers an abnormal activity on a protein or cell that is otherwise not present in a normal condition. Many gain-of-function mutations are in regulatory sequences rather than in coding regions, and can therefore have a number of consequences. For example, a mutation might lead to one or more genes being expressed in the wrong tissues, these tissues gaining functions that they normally lack. Because of their nature, gain-of-function mutations are usually dominant.
• Mutations can be introduced into a reference Cas9 protein using site-directed mutagenesis. Older methods of site-directed mutagenesis known in the art rely on sub-cloning of the sequence to be mutated into a vector, such as an M13 bacteriophage vector, that allows the isolation of single-stranded DNA template. In these methods, one anneals a mutagenic primer (i.e., a primer capable of annealing to the site to be mutated but bearing one or more mismatched nucleotides at the site to be mutated) to the single-stranded template and then polymerizes the complement of the template starting from the 3′ end of the mutagenic primer. The resulting duplexes are then transformed into host bacteria and plaques are screened for the desired mutation. More recently, site-directed mutagenesis has employed PCR methodologies, which have the advantage of not requiring a single-stranded template. In addition, methods have been developed that do not require sub-cloning. Several issues must be considered when PCR-based site-directed mutagenesis is performed. First, in these methods it is desirable to reduce the number of PCR cycles to prevent expansion of undesired mutations introduced by the polymerase. Second, a selection must be employed in order to reduce the number of non-mutated parental molecules persisting in the reaction. Third, an extended-length PCR method is preferred in order to allow the use of a single PCR primer set. And fourth, because of the non-template-dependent terminal extension activity of some thermostable polymerases it is often necessary to incorporate an end-polishing step into the procedure prior to blunt-end ligation of the PCR-generated mutant product.
• Mutations may also be introduced by directed evolution processes, such as phage-assisted continuous evolution (PACE) or phage-assisted noncontinuous evolution (PANCE). The term “phage-assisted continuous evolution (PACE),” as used herein, refers to continuous evolution that employs phage as viral vectors. The general concept of PACE technology has been described, for example, in International PCT Application, PCT/US2009/056194, filed Sep. 8, 2009, published as WO 2010/028347 on Mar. 11, 2010; International PCT Application, PCT/US2011/066747, filed Dec. 22, 2011, published as WO 2012/088381 on Jun. 28, 2012; U.S. application, U.S. Pat. No. 9,023,594, issued May 5, 2015, International PCT Application, PCT/US2015/012022, filed Jan. 20, 2015, published as WO 2015/134121 on Sep. 11, 2015, and International PCT Application, PCT/US2016/027795, filed Apr. 15, 2016, published as WO 2016/168631 on Oct. 20, 2016, the entire contents of each of which are incorporated herein by reference. Variant Cas9s may also be obtain by phage-assisted non-continuous evolution (PANCE),” which as used herein, refers to non-continuous evolution that employs phage as viral vectors. PANCE is a simplified technique for rapid in vivo directed evolution using serial flask transfers of evolving ‘selection phage’ (SP), which contain a gene of interest to be evolved, across fresh E. coli host cells, thereby allowing genes inside the host E. coli to be held constant while genes contained in the SP continuously evolve. Serial flask transfers have long served as a widely-accessible approach for laboratory evolution of microbes, and, more recently, analogous approaches have been developed for bacteriophage evolution. The PANCE system features lower stringency than the PACE system.
• Any of the references noted above which relate to Cas9 or Cas9 equivalents are hereby incorporated by reference in their entireties, if not already stated so.
• J. Divided napDNAbp Domains for Split PE Delivery
• In various embodiments, the prime editors described herein may be delivered to cells as two or more fragments which become assembled inside the cell (either by passive assembly, or by active assembly, such as using split intein sequences) into a reconstituted prime editor. In some cases, the self assembly may be passive whereby the two or more prime editor fragments associate inside the cell covalently or non-covalently to reconstitute the prime editor. In other cases, the self-assembly may be catalyzed by dimerization domains installed on each of the fragments. Examples of dimerization domains are described herein. In still other cases, the self-assembly may be catalyzed by split intein sequences installed on each of the prime editor fragments.
• Split PE delivery may be advantageous to address various size constraints of different delivery approaches. For example, delivery approaches may include virus-based delivery methods, messenger RNA-based delivery methods, or RNP-based delivery (ribonucleoprotein-based delivery). And, each of these methods of delivery may be more efficient and/or effective by dividing up the prime editor into smaller pieces. Once inside the cell, the smaller pieces can assemble into a functional prime editor. Depending on the means of splitting, the divided prime editor fragments can be reassembled in a non-covalent manner or a covalent manner to reform the prime editor. In one embodiment, the prime editor can be split at one or more split sites into two or more fragments. The fragments can be unmodified (other than being split). Once the fragments are delivered to the cell (e.g., by direct delivery of a ribonucleoprotein complex or by nucleic delivery—e.g., mRNA delivery or virus vector based delivery), the fragments can reassociate covalently or non-covalently to reconstitute the prime editor. In another embodiment, the prime editor can be split at one or more split sites into two or more fragments. Each of the fragments can be modified to comprise a dimerization domain, whereby each fragment that is formed is coupled to a dimerization domain. Once delivered or expressed within a cell, the dimerization domains of the different fragments associate and bind to one another, bringing the different prime editor fragments together to reform a functional prime editor. In yet another embodiment, the prime editor fragment may be modified to comprise a split intein. Once delivered or expressed within a cell, the split intein domains of the different fragments associate and bind to one another, and then undergo trans-splicing, which results in the excision of the split-intein domains from each of the fragments, and a concomitant formation of a peptide bond between the fragments, thereby restoring the prime editor.
• In one embodiment, the prime editor can be delivered using a split-intein approach.
• The location of the split site can be positioned between any one or more pair of residues in the prime editor and in any domains therein, including within the napDNAbp domain, the polymerase domain (e.g., RT domain), linker domain that joins the napDNAbp domain and the polymerase domain.
• In one embodiment, depicted in FIG. 66 , the prime editor (PE) is divided at a split site within the napDNAbp.
• In certain embodiments, the napDNAbp is a canonical SpCas9 polypeptide of SEQ ID NO: 18, as follows:

• SpCas9	MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGN	SEQ
  Strepto	TDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRR	ID
  coccus	KNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKH	NO:
  pyogenes	ERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADL	18
  M1	RLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQ
  SwissProt	TYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQL
  Accession	PGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLS
  No.	KDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDI
  Q99ZW2	LRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQL
  Wild type	PEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEK
  	MDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELH
  1368 AA	AILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
  	SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTN
  	FDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGM
  	RKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKI
  	ECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEE
  	NEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMK
  	QLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDG
  	FANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIA
  	NLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEM
  	ARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPV
  	ENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYD
  	VDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEV
  	VKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSEL
  	DKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDK
  	LIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHD
  	AYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIA
  	KSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLI
  	ETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV
  	QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPT
  	VAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEK
  	NPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRML
  	ASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPED
  	NEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKV
  	LSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDT
  	TIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD

• In certain embodiments, the SpCas9 is split into two fragments at a split site located between residues 1 and 2, or 2 and 3, or 3 and 4, or 4 and 5, or 5 and 6, or 6 and 7, or 7 and 8, or 8 and 9, or 9 and 10, or between any two pair of residues located anywhere between residues 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, 1000-1100, 1100-1200, 1200-1300, or 1300-1368 of canonical SpCas9 of SEQ ID NO: 18.
• In certain embodiments, a napDNAbp is split into two fragments at a split site that is located at a pair of residue that corresponds to any two pair of residues located anywhere between positions 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, 1000-1100, 1100-1200, 1200-1300, or 1300-1368 of canonical SpCas9 of SEQ ID NO: 18.
• In certain embodiments, the SpCas9 is split into two fragments at a split site located between residues 1 and 2, or 2 and 3, or 3 and 4, or 4 and 5, or 5 and 6, or 6 and 7, or 7 and 8, or 8 and 9, or 9 and 10, or between any two pair of residues located anywhere between residues 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, 1000-1100, 1100-1200, 1200-1300, or 1300-1368 of canonical SpCas9 of SEQ ID NO: 18. In certain embodiments, the split site is located one or more polypeptide bond sites (i.e., a “split site or split-intein split site”), fused to a split intein, and then delivered to cells as separately-encoded fusion proteins. Once the split-intein fusion proteins (i.e., protein halves) are expressed within a cell, the proteins undergo trans-splicing to form a complete or whole PE with the concomitant removal of the joined split-intein sequences.
• For example, as shown in FIG. 66 , the N-terminal extein can be fused to a first split-intein (e.g., N intein) and the C-terminal extein can be fused to a second split-intein (e.g., C intein). The N-terminal extein becomes fused to the C-terminal extein to reform a whole prime editor fusion protein comprising an napDNAbp domain and a polymerase domain (e.g., RT domain) upon the self-association of the N intein and the C intein inside the cell, followed by their self-excision, and the concomitant formation of a peptide bond between the N-terminal extein and C-terminal extein portions of a whole prime editor (PE).
• To take advantage of a split-PE delivery strategy using split-inteins, the prime editor needs to be divided at one or more split sites to create at least two separate halves of a prime editor, each of which may be rejoined inside a cell if each half is fused to a split-intein sequence.
• In certain embodiments, the prime editor is split at a single split site. In certain other embodiments, the prime editor is split at two split sites, or three split sites, or four split sites, or more.
• In a preferred embodiment, the prime editor is split at a single split site to create two separate halves of a prime editor, each of which can be fused to a split intein sequence
• An exemplary split intein is the Ssp DnaE intein, which comprises two subunits, namely, DnaE-N and DnaE-C. The two different subunits are encoded by separate genes, namely dnaE-n and dnaE-c, which encode the DnaE-N and DnaE-C subunits, respectively. DnaE is a naturally occurring split intein in Synechocytis sp. PCC6803 and is capable of directing trans-splicing of two separate proteins, each comprising a fusion with either DnaE-N or DnaE-C.
• Additional naturally occurring or engineered split-intein sequences are known in the or can be made from whole-intein sequences described herein or those available in the art. Examples of split-intein sequences can be found in Stevens et al., “A promiscuous split intein with expanded protein engineering applications,” PNAS, 2017, Vol. 114: 8538-8543; Iwai et al., “Highly efficient protein trans-splicing by a naturally split DnaE intein from Nostoc punctiforme, FEBS Lett, 580: 1853-1858, each of which are incorporated herein by reference. Additional split intein sequences can be found, for example, in WO 2013/045632, WO 2014/055782, WO 2016/069774, and EP2877490, the contents each of which are incorporated herein by reference.
• In addition, protein splicing in trans has been described in vivo and in vitro (Shingledecker, et al., Gene 207:187 (1998), Southworth, et al., EMBO J. 17:918 (1998); Mills, et al., Proc. Natl. Acad. Sci. USA, 95:3543-3548 (1998); Lew, et al., J. Biol. Chem., 273:15887-15890 (1998); Wu, et al., Biochim. Biophys. Acta 35732:1 (1998b), Yamazaki, et al., J. Am. Chem. Soc. 120:5591 (1998), Evans, et al., J. Biol. Chem. 275:9091 (2000); Otomo, et al., Biochemistry 38:16040-16044 (1999); Otomo, et al., J. Biolmol. NMR 14:105-114 (1999); Scott, et al., Proc. Natl. Acad. Sci. USA 96:13638-13643 (1999)) and provides the opportunity to express a protein as to two inactive fragments that subsequently undergo ligation to form a functional product, e.g., as shown in FIGS. 66 and 67 with regard to the formation of a complete PE fusion protein from two separately-expressed halves.
• In various embodiments described herein, the continuous evolution methods (e.g., PACE) may be used to evolve a first portion of a base editor. A first portion could include a single component or domain, e.g., a Cas9 domain, a deaminase domain, or a UGI domain. The separately evolved component or domain can be then fused to the remaining portions of the base editor within a cell by separately express both the evolved portion and the remaining non-evolved portions with split-intein polypeptide domains. The first portion could more broadly include any first amino acid portion of a base editor that is desired to be evolved using a continuous evolution method described herein. The second portion would in this embodiment refer to the remaining amino acid portion of the base editor that is not evolved using the herein methods. The evolved first portion and the second portion of the base editor could each be expressed with split-intein polypeptide domains in a cell. The natural protein splicing mechanisms of the cell would reassemble the evolved first portion and the non-evolved second portion to form a single fusion protein evolved base editor. The evolved first portion may comprise either the N- or C-terminal part of the single fusion protein. In an analogous manner, use of a second orthogonal trans-splicing intein pair could allow the evolved first portion to comprise an internal part of the single fusion protein.
• Thus, any of the evolved and non-evolved components of the base editors herein described may be expressed with split-intein tags in order to facilitate the formation of a complete base editor comprising the evolved and non-evolved component within a cell.
• The mechanism of the protein splicing process has been studied in great detail (Chong, et al., J. Biol. Chem. 1996, 271, 22159-22168; Xu, M-Q & Perler, F. B. EMBO Journal, 1996, 15, 5146-5153) and conserved amino acids have been found at the intein and extein splicing points (Xu, et al., EMBO Journal, 1994, 13 5517-522). The constructs described herein contain an intein sequence fused to the 5′-terminus of the first gene (e.g., the evolved portion of the base editor). Suitable intein sequences can be selected from any of the proteins known to contain protein splicing elements. A database containing all known inteins can be found on the World Wide Web (Perler, F. B. Nucleic Acids Research, 1999, 27, 346-347). The intein sequence is fused at the 3′ end to the 5′ end of a second gene. For targeting of this gene to a certain organelle, a peptide signal can be fused to the coding sequence of the gene. After the second gene, the intein-gene sequence can be repeated as often as desired for expression of multiple proteins in the same cell. For multi-intein containing constructs, it may be useful to use intein elements from different sources. After the sequence of the last gene to be expressed, a transcription termination sequence must be inserted. In one embodiment, a modified intein splicing unit is designed so that it can both catalyze excision of the exteins from the inteins as well as prevent ligation of the exteins. Mutagenesis of the C-terminal extein junction in the Pyrococcus species GB-D DNA polymerase was found to produce an altered splicing element that induces cleavage of exteins and inteins but prevents subsequent ligation of the exteins (Xu, M-Q & Perler, F. B. EMBO Journal, 1996, 15, 5146-5153). Mutation of serine 538 to either an alanine or glycine induced cleavage but prevented ligation. Mutation of equivalent residues in other intein splicing units should also prevent extein ligation due to the conservation of amino acids at the C-terminal extein junction to the intein. A preferred intein not containing an endonuclease domain is the Mycobacterium xenopi GyrA protein (Telenti, et al. J. Bacteriol. 1997, 179, 6378-6382). Others have been found in nature or have been created artificially by removing the endonuclease domains from endonuclease containing inteins (Chong, et al. J. Biol. Chem. 1997, 272, 15587-15590). In a preferred embodiment, the intein is selected so that it consists of the minimal number of amino acids needed to perform the splicing function, such as the intein from the Mycobacterium xenopi GyrA protein (Telenti, A., et al., J. Bacteriol. 1997, 179, 6378-6382). In an alternative embodiment, an intein without endonuclease activity is selected, such as the intein from the Mycobacterium xenopi GyrA protein or the Saccharomyces cerevisiae VMA intein that has been modified to remove endonuclease domains (Chong, 1997). Further modification of the intein splicing unit may allow the reaction rate of the cleavage reaction to be altered allowing protein dosage to be controlled by simply modifying the gene sequence of the splicing unit.
• Inteins can also exist as two fragments encoded by two separately transcribed and translated genes. These so-called split inteins self-associate and catalyze protein-splicing activity in trans. Split inteins have been identified in diverse cyanobacteria and archaea (Caspi et al, Mol Microbiol. 50: 1569-1577 (2003); Choi J. et al, J Mol Biol. 556: 1093-1106 (2006.); Dassa B. et al, Biochemistry. 46:322-330 (2007.); Liu X. and Yang J., J Biol Chem. 275:26315-26318 (2003); Wu H. et al.
• Proc Natl Acad Sci USA. £5:9226-9231 (1998.); and Zettler J. et al, FEBS Letters. 553:909-914 (2009)), but have not been found in eukaryotes thus far. Recently, a bioinformatic analysis of environmental metagenomic data revealed 26 different loci with a novel genomic arrangement. At each locus, a conserved enzyme coding region is interrupted by a split intein, with a freestanding endonuclease gene inserted between the sections coding for intein subdomains. Among them, five loci were completely assembled: DNA helicases (gp41-1, gp41-8); Inosine-5′-monophosphate dehydrogenase (IMPDH-1); and Ribonucleotide reductase catalytic subunits (NrdA-2 and NrdJ-1). This fractured gene organization appears to be present mainly in phages (Dassa et al, Nucleic Acids Research. 57:2560-2573 (2009)).
• The split intein Npu DnaE was characterized as having the highest rate reported for the protein trans-splicing reaction. In addition, the Npu DnaE protein splicing reaction is considered robust and high-yielding with respect to different extein sequences, temperatures from 6 to 37° C., and the presence of up to 6M Urea (Zettler J. et al, FEBS Letters. 553:909-914 (2009); Iwai I. et al, FEBS Letters 550: 1853-1858 (2006)). As expected, when the Cys1 Ala mutation at the N-domain of these inteins was introduced, the initial N to S-acyl shift and therefore protein splicing was blocked. Unfortunately, the C-terminal cleavage reaction was also almost completely inhibited. The dependence of the asparagine cyclization at the C-terminal splice junction on the acyl shift at the N-terminal scissile peptide bond seems to be a unique property common to the naturally split DnaE intein alleles (Zettler J. et al. FEBS Letters. 555:909-914 (2009)).
• The mechanism of protein splicing typically has four steps [29-30]: 1) an N—S or N—O acyl shift at the intein N-terminus, which breaks the upstream peptide bond and forms an ester bond between the N-extein and the side chain of the intein's first amino acid (Cys or Ser); 2) a transesterification relocating the N-extein to the intein C-terminus, forming a new ester bond linking the N-extein to the side chain of the C-extein's first amino acid (Cys, Ser, or Thr); 3) Asn cyclization breaking the peptide bond between the intein and the C-extein; and 4) a S—N or O—N acyl shift that replaces the ester bond with a peptide bond between the N-extein and C-extein.
• Protein trans-splicing, catalyzed by split inteins, provides an entirely enzymatic method for protein ligation [31]. A split-intein is essentially a contiguous intein (e.g. a mini-intein) split into two pieces named N-intein and C-intein, respectively. The N-intein and C-intein of a split intein can associate non-covalently to form an active intein and catalyze the splicing reaction essentially in same way as a contiguous intein does. Split inteins have been found in nature and also engineered in laboratories [31-35]. As used herein, the term “split intein” refers to any intein in which one or more peptide bond breaks exists between the N-terminal and C-terminal amino acid sequences such that the N-terminal and C-terminal sequences become separate molecules that can non-covalently reassociate, or reconstitute, into an intein that is functional for trans-splicing reactions. Any catalytically active intein, or fragment thereof, may be used to derive a split intein for use in the methods of the invention. For example, in one aspect the split intein may be derived from a eukaryotic intein. In another aspect, the split intein may be derived from a bacterial intein. In another aspect, the split intein may be derived from an archaeal intein. Preferably, the split intein so-derived will possess only the amino acid sequences essential for catalyzing trans-splicing reactions.
• As used herein, the “N-terminal split intein (In)” refers to any intein sequence that comprises an N-terminal amino acid sequence that is functional for trans-splicing reactions. An In thus also comprises a sequence that is spliced out when trans-splicing occurs. An In can comprise a sequence that is a modification of the N-terminal portion of a naturally occurring intein sequence. For example, an In can comprise additional amino acid residues and/or mutated residues so long as the inclusion of such additional and/or mutated residues does not render the In non-functional in trans-splicing. Preferably, the inclusion of the additional and/or mutated residues improves or enhances the trans-splicing activity of the In.
• As used herein, the “C-terminal split intein (Ic)” refers to any intein sequence that comprises a C-terminal amino acid sequence that is functional for trans-splicing reactions. In one aspect, the Ic comprises 4 to 7 contiguous amino acid residues, at least 4 amino acids of which are from the last β-strand of the intein from which it was derived. An Ic thus also comprises a sequence that is spliced out when trans-splicing occurs. An Ic can comprise a sequence that is a modification of the C-terminal portion of a naturally occurring intein sequence. For example, an Ic can comprise additional amino acid residues and/or mutated residues so long as the inclusion of such additional and/or mutated residues does not render the In non-functional in trans-splicing. Preferably, the inclusion of the additional and/or mutated residues improves or enhances the trans-splicing activity of the Ic.
• In some embodiments of the invention, a peptide linked to an Ic or an In can comprise an additional chemical moiety including, among others, fluorescence groups, biotin, polyethylene glycol (PEG), amino acid analogs, unnatural amino acids, phosphate groups, glycosyl groups, radioisotope labels, and pharmaceutical molecules. In other embodiments, a peptide linked to an Ic can comprise one or more chemically reactive groups including, among others, ketone, aldehyde, Cys residues and Lys residues. The N-intein and C-intein of a split intein can associate non-covalently to form an active intein and catalyze the splicing reaction when an “intein-splicing polypeptide (ISP)” is present. As used herein, “intein-splicing polypeptide (ISP)” refers to the portion of the amino acid sequence of a split intein that remains when the Ic, In, or both, are removed from the split intein. In certain embodiments, the In comprises the ISP. In another embodiment, the Ic comprises the ISP. In yet another embodiment, the ISP is a separate peptide that is not covalently linked to In nor to Ic.
• Split inteins may be created from contiguous inteins by engineering one or more split sites in the unstructured loop or intervening amino acid sequence between the −12 conserved beta-strands found in the structure of mini-inteins [25-28]. Some flexibility in the position of the split site within regions between the beta-strands may exist, provided that creation of the split will not disrupt the structure of the intein, the structured beta-strands in particular, to a sufficient degree that protein splicing activity is lost.
• In protein trans-splicing, one precursor protein consists of an N-extein part followed by the N-intein, another precursor protein consists of the C-intein followed by a C-extein part, and a trans-splicing reaction (catalyzed by the N- and C-inteins together) excises the two intein sequences and links the two extein sequences with a peptide bond. Protein trans-splicing, being an enzymatic reaction, can work with very low (e.g. micromolar) concentrations of proteins and can be carried out under physiological conditions.
[2] Other Programmable Nucleases
• In various embodiments described herein, the prime editors comprise a napDNAbp, such as a Cas9 protein. These proteins are “programmable” by way of their becoming complexed with a guide RNA (or a PEgRNA, as the case may be), which guides the Cas9 protein to a target site on the DNA which possess a sequence that is complementary to the spacer portion of the gRNA (or PEgRNA) and also which possesses the required PAM sequence. However, in certain embodiment envisioned here, the napDNAbp may be substituted with a different type of programmable protein, such as a zinc finger nuclease or a transcription activator-like effector nuclease (TALEN).
• FIG. 1J depicts such a variation of prime editing contemplated herein that replaces the napDNAbp (e.g., SpCas9 nickase) with any programmable nuclease domain, such as zinc finger nucleases (ZFN) or transcription activator-like effector nucleases (TALEN). As such, it is contemplated that suitable nucleases do not necessarily need to be “programmed” by a nucleic acid targeting molecule (such as a guide RNA), but rather, may be programmed by defining the specificity of a DNA-binding domain, such as and in particular, a nuclease. Just as in prime editing with napDNAbp moities, it is preferable that such alternative programmable nucleases be modified such that only one strand of a target DNA is cut. In other words, the programmable nucleases should function as nickases, preferably. Once a programmable nuclease is selected (e.g., a ZFN or a TALEN), then additional functionalities may be engineered into the system to allow it to operate in accordance with a prime editing-like mechanism. For example, the programmable nucleases may be modified by coupling (e.g., via a chemical linker) an RNA or DNA extension arm thereto, wherein the extension arm comprises a primer binding site (PBS) and a DNA synthesis template. The programmable nuclease may also be coupled (e.g., via a chemical or amino acid linker) to a polymerase, the nature of which will depend upon whether the extension arm is DNA or RNA. In the case of an RNA extension arm, the polymerase can be an RNA-dependent DNA polymerase (e.g., reverse transcriptase). In the case of a DNA extension arm, the polymerase can be a DNA-dependent DNA polymerase (e.g., a prokaryotic polymerase, including Pol I, Pol II, or Pol III, or a eukaryotic polymerase, including Pol a, Pol b, Pol g, Pol d, Pol e, or Pol z). The system may also include other functionalities added as fusions to the programmable nucleases, or added in trans to facilitate the reaction as a whole (e.g., (a) a helicase to unwind the DNA at the cut site to make the cut strand with the 3′ end available as a primer, (b) a FEN1 to help remove the endogenous strand on the cut strand to drive the reaction towards replacement of the endogenous strand with the synthesized strand, or (c) a nCas9:gRNA complex to create a second site nick on the opposite strand, which may help drive the integration of the synthesize repair through favored cellular repair of the non-edited strand). In an analogous manner to prime editing with a napDNAbp, such a complex with an otherwise programmable nuclease could be used to synthesize and then install a newly synthesized replacement strand of DNA carrying an edit of interest permanently into a target site of DNA.
• Suitable alternative programmable nucleases are well known in the art which may be used in place of a napDNAbp:gRNA complex to construct an alternative prime editor system that can be programmed to selectively bind a target site of DNA, and which can be further modified in the manner described above to co-localize a polymerase and an RNA or DNA extension arm comprising a primer binding site and a DNA synthesis template to specific nick site. For example, and as represented in FIG. 1J, Transcription Activator-Like Effector Nucleases (TALENs) may be used as the programmable nuclease in the prime editing methods and compositions of matter described herein. TALENS are artificial restriction enzymes generated by fusing the TAL effector DNA binding domain to a DNA cleavage domain. These reagents enable efficient, programmable, and specific DNA cleavage and represent powerful tools for genome editing in situ. Transcription activator-like effectors (TALEs) can be quickly engineered to bind practically any DNA sequence. The term TALEN, as used herein, is broad and includes a monomeric TALEN that can cleave double stranded DNA without assistance from another TALEN. The term TALEN is also used to refer to one or both members of a pair of TALENs that are engineered to work together to cleave DNA at the same site. TALENs that work together may be referred to as a left-TALEN and a right-TALEN, which references the handedness of DNA. See U.S. Ser. No. 12/965,590; U.S. Ser. No. 13/426,991 (U.S. Pat. No. 8,450,471); U.S. Ser. No. 13/427,040 (U.S. Pat. No. 8,440,431); U.S. Ser. No. 13/427,137 (U.S. Pat. No. 8,440,432); and U.S. Ser. No. 13/738,381, all of which are incorporated by reference herein in their entirety. In addition, TALENS are described in WO 2015/027134, U.S. Pat. No. 9,181,535, Boch et al., “Breaking the Code of DNA Binding Specificity of TAL-Type III Effectors”, Science, vol. 326, pp. 1509-1512 (2009), Bogdanove et al., TAL Effectors: Customizable Proteins for DNA Targeting, Science, vol. 333, pp. 1843-1846 (2011), Cade et al., “Highly efficient generation of heritable zebrafish gene mutations using homo- and heterodimeric TALENs”, Nucleic Acids Research, vol. 40, pp. 8001-8010 (2012), and Cermak et al., “Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting”, Nucleic Acids Research, vol. 39, No. 17, e82 (2011), each of which are incorporated herein by reference.
• As represented in FIG. 1J, zinc finger nucleases may also be used as alternative programmable nucleases for use in prime editing in place of napDNAbps, such as Cas9 nickases. Like with TALENS, the ZFN proteins may be modified such that they function as nickases, i.e., engineering the ZFN such that it cleaves only one strand of the target DNA in a manner similar to the napDNAbp used with the prime editors described herein. ZFN proteins have been extensively described in the art, for example, in Carroll et al., “Genome Engineering with Zinc-Finger Nucleases,” Genetics, August 2011, Vol. 188: 773-782; Dural et al., “Zinc finger nucleases: custom-designed molecular scissors for genome engineering of plant and mammalian cells,” Nucleic Acids Res, 2005, Vol. 33: 5978-90; and Gaj et al., “ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering,” Trends Biotechnol. 2013, Vol. 31: 397-405, each of which are incorporated herein by reference in their entireties.
[3] Polymerases (e.g., Reverse Transcriptases)
• In various embodiments, the prime editor (PE) system disclosed herein includes a polymerase (e.g., DNA-dependent DNA polymerase or RNA-dependent DNA polymerase, such as, reverse transcriptase), or a variant thereof, which can be provided as a fusion protein with a napDNAbp or other programmable nuclease, or provide in trans.
• Any polymerase may be used in the prime editors disclosed herein. The polymerases may be wild type polymerases, functional fragments, mutants, variants, or truncated variants, and the like. The polymerases may include wild type polymerases from eukaryotic, prokaryotic, archaeal, or viral organisms, and/or the polymerases may be modified by genetic engineering, mutagenesis, directed evolution-based processes. The polymerases may include T7 DNA polymerase, T5 DNA polymerase, T4 DNA polymerase, Klenow fragment DNA polymerase, DNA polymerase III and the like. The polymerases may also be thermostable, and may include Taq, Tne, Tma, Pfu, Tfl, Tth, Stoffel fragment, VENT® and DEEPVENT® DNA polymerases, KOD, Tgo, JDF3, and mutants, variants and derivatives thereof (see U.S. Pat. Nos. 5,436,149; 4,889,818; 4,965,185; 5,079,352; 5,614,365; 5,374,553; 5,270,179; 5,047,342; 5,512,462; WO 92/06188; WO 92/06200; WO 96/10640; Barnes, W. M., Gene 112:29-35 (1992); Lawyer, F. C., et al., PCR Meth. Appl. 2:275-287 (1993); Flaman, J.-M, et al., Nuc. Acids Res. 22(15):3259-3260 (1994), each of which are incorporated by reference). For synthesis of longer nucleic acid molecules (e.g, nucleic acid molecules longer than about 3-5 Kb in length), at least two DNA polymerases can be employed. In certain embodiments, one of the polymerases can be substantially lacking a 3′ exonuclease activity and the other may have a 3′ exonuclease activity. Such pairings may include polymerases that are the same or different. Examples of DNA polymerases substantially lacking in 3′ exonuclease activity include, but are not limited to, Taq, Tne(exo-), Tma(exo-), Pfu(exo-), Pwo(exo-), exo-KOD and Tth DNA polymerases, and mutants, variants and derivatives thereof.
• Preferably, the polymerase usable in the prime editors disclosed herein are “template-dependent” polymerase (since the polymerases are intended to rely on the DNA synthesis template to specify the sequence of the DNA strand under synthesis during prime editing. As used herein, the term “template DNA molecule” refers to that strand of a nucleic acid from which a complementary nucleic acid strand is synthesized by a DNA polymerase, for example, in a primer extension reaction of the DNA synthesis template of a PEgRNA.
• As used herein, the term “template dependent manner” is intended to refer to a process that involves the template dependent extension of a primer molecule (e.g., DNA synthesis by DNA polymerase). The term “template dependent manner” refers to polynucleotide synthesis of RNA or DNA wherein the sequence of the newly synthesized strand of polynucleotide is dictated by the well-known rules of complementary base pairing (see, for example, Watson, J. D. et al., In: Molecular Biology of the Gene, 4th Ed., W. A. Benjamin, Inc., Menlo Park, Calif. (1987)). The term “complementary” refers to the broad concept of sequence complementarity between regions of two polynucleotide strands or between two nucleotides through base-pairing. It is known that an adenine nucleotide is capable of forming specific hydrogen bonds (“base pairing”) with a nucleotide which is thymine or uracil. Similarly, it is known that a cytosine nucleotide is capable of base pairing with a guanine nucleotide. As such, in the case of prime editing, it can be said that the single strand of DNA synthesized by the polymerase of the prime editor against the DNA synthesis template is said to be “complementary” to the sequence of the DNA synthesis template.
• A. Exemplary Polymerases
• In various embodiments, the prime editors described herein comprise a polymerase. The disclosure contemplates any wild type polymerase obtained from any naturally-occurring organism or virus, or obtained from a commercial or non-commercial source. In addition, the polymerases usable in the prime editors of the disclosure can include any naturally-occurring mutant polymerase, engineered mutant polymerase, or other variant polymerase, including truncated variants that retain function. The polymerases usable herein may also be engineered to contain specific amino acid substitutions, such as those specifically disclosed herein. In certain preferred embodiments, the polymerases usable in the prime editors of the disclosure are template-based polymerases, i.e., they synthesize nucleotide sequences in a template-dependent manner.
• A polymerase is an enzyme that synthesizes a nucleotide strand and which may be used in connection with the prime editor systems described herein. The polymerases are preferrably “template-dependent” polymerases (i.e., a polymerase which synthesizes a nucleotide strand based on the order of nucleotide bases of a template strand). In certain configurations, the polymerases can also be a “template-independent” (i.e., a polymerase which synthesizes a nucleotide strand without the requirement of a template strand). A polymerase may also be further categorized as a “DNA polymerase” or an “RNA polymerase.” In various embodiments, the prime editor system comprises a DNA polymerase. In various embodiments, the DNA polymerase can be a “DNA-dependent DNA polymerase” (i.e., whereby the template molecule is a strand of DNA). In such cases, the DNA template molecule can be a PEgRNA, wherein the extension arm comprises a strand of DNA. In such cases, the PEgRNA may be referred to as a chimeric or hybrid PEgRNA which comprises an RNA portion (i.e., the guide RNA components, including the spacer and the gRNA core) and a DNA portion (i.e., the extension arm). In various other embodiments, the DNA polymerase can be an “RNA-dependent DNA polymerase” (i.e., whereby the template molecule is a strand of RNA). In such cases, the PEgRNA is RNA, i.e., including an RNA extension. The term “polymerase” may also refer to an enzyme that catalyzes the polymerization of nucleotide (i.e., the polymerase activity). Generally, the enzyme will initiate synthesis at the 3′-end of a primer annealed to a polynucleotide template sequence (e.g., such as a primer sequence annealed to the primer binding site of a PEgRNA), and will proceed toward the 5′ end of the template strand. A “DNA polymerase” catalyzes the polymerization of deoxynucleotides. As used herein in reference to a DNA polymerase, the term DNA polymerase includes a “functional fragment thereof”. A “functional fragment thereof” refers to any portion of a wild-type or mutant DNA polymerase that encompasses less than the entire amino acid sequence of the polymerase and which retains the ability, under at least one set of conditions, to catalyze the polymerization of a polynucleotide. Such a functional fragment may exist as a separate entity, or it may be a constituent of a larger polypeptide, such as a fusion protein.
• In some embodiments, the polymerases can be from bacteriophage. Bacteriophage DNA polymerases are generally devoid of 5′ to 3′ exonuclease activity, as this activity is encoded by a separate polypeptide. Examples of suitable DNA polymerases are T4, T7, and phi29 DNA polymerase. The enzymes available commercially are: T4 (available from many sources e.g., Epicentre) and T7 (available from many sources, e.g. Epicentre for unmodified and USB for 3′ to 5′ exo T7 “Sequenase” DNA polymerase).
• The other embodiments, the polymerases are archaeal polymerases. There are 2 different classes of DNA polymerases which have been identified in archaea: 1. Family B/pol I type (homologs of Pfu from Pyrococcus furiosus) and 2. pol II type (homologs of P. furiosus DP1/DP2 2-subunit polymerase). DNA polymerases from both classes have been shown to naturally lack an associated 5′ to 3′ exonuclease activity and to possess 3′ to 5′ exonuclease (proofreading) activity. Suitable DNA polymerases (pol I or pol II) can be derived from archaea with optimal growth temperatures that are similar to the desired assay temperatures.
• Thermostable archaeal DNA polymerases are isolated from Pyrococcus species (furiosus, species GB-D, woesii, abysii, horikoshii), Thermococcus species (kodakaraensis KOD1, litoralis, species 9 degrees North-7, species JDF-3, gorgonarius), Pyrodictium occultum, and Archaeoglobus fulgidus.
• Polymerases may also be from eubacterial species. There are 3 classes of eubacterial DNA polymerases, pol I, II, and III. Enzymes in the Pol I DNA polymerase family possess 5′ to 3′ exonuclease activity, and certain members also exhibit 3′ to 5′ exonuclease activity. Pol II DNA polymerases naturally lack 5′ to 3′ exonuclease activity, but do exhibit 3′ to 5′ exonuclease activity. Pol III DNA polymerases represent the major replicative DNA polymerase of the cell and are composed of multiple subunits. The pol III catalytic subunit lacks 5′ to 3′ exonuclease activity, but in some cases 3′ to 5′ exonuclease activity is located in the same polypeptide.
• There are a variety of commercially available Pol I DNA polymerases, some of which have been modified to reduce or abolish 5′ to 3′ exonuclease activity.
• Suitable thermostable pol I DNA polymerases can be isolated from a variety of thermophilic eubacteria, including Thermus species and Thermotoga maritima such as Thermus aquaticus (Taq), Thermus thermophilus (Tth) and Thermotoga maritima (Tma UlTma).
• Additional eubacteria related to those listed above are described in Thermophilic Bacteria (Kristjansson, J. K., ed.) CRC Press, Inc., Boca Raton, Fla., 1992.
• The invention further provides for chimeric or non-chimeric DNA polymerases that are chemically modified according to methods disclosed in U.S. Pat. Nos. 5,677,152, 6,479,264 and 6,183,998, the contents of which are hereby incorporated by reference in their entirety.
• Additional archaea DNA polymerases related to those listed above are described in the following references: Archaea: A Laboratory Manual (Robb, F. T. and Place, A. R., eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1995 and Thermophilic Bacteria (Kristjansson, J. K., ed.) CRC Press, Inc., Boca Raton, Fla., 1992.
• B. Exemplarily Reverse Transcriptases
• In various embodiments, the prime editors described herein comprise a reverse transcriptase as the polymerase. The disclosure contemplates any wild type reverse transcriptase obtained from any naturally-occurring organism or virus, or obtained from a commercial or non-commercial source. In addition, the reverse transcriptases usable in the prime editors of the disclosure can include any naturally-occurring mutant RT, engineered mutant RT, or other variant RT, including truncated variants that retain function. The RTs may also be engineered to contain specific amino acid substitutions, such as those specifically disclosed herein.
• Reverse transcriptases are multi-functional enzymes typically with three enzymatic activities including RNA- and DNA-dependent DNA polymerization activity, and an RNaseH activity that catalyzes the cleavage of RNA in RNA-DNA hybrids. Some mutants of reverse transcriptases have disabled the RNaseH moiety to prevent unintended damage to the mRNA. These enzymes that synthesize complementary DNA (cDNA) using mRNA as a template were first identified in RNA viruses. Subsequently, reverse transcriptases were isolated and purified directly from virus particles, cells or tissues. (e.g., see Kacian et al., 1971, Biochim. Biophys. Acta 46: 365-83; Yang et al., 1972, Biochem. Biophys. Res. Comm. 47: 505-11; Gerard et al., 1975, J. Virol. 15: 785-97; Liu et al., 1977, Arch. Virol. 55 187-200; Kato et al., 1984, J. Virol. Methods 9: 325-39; Luke et al., 1990, Biochem. 29: 1764-69 and Le Grice et al., 1991, J. Virol. 65: 7004-07, each of which are incorporated by reference). More recently, mutants and fusion proteins have been created in the quest for improved properties such as thermostability, fidelity and activity. Any of the wild type, variant, and/or mutant forms of reverse transcriptase which are known in the art or which can be made using methods known in the art are contemplated herein.
• The reverse transcriptase (RT) gene (or the genetic information contained therein) can be obtained from a number of different sources. For instance, the gene may be obtained from eukaryotic cells which are infected with retrovirus, or from a number of plasmids which contain either a portion of or the entire retrovirus genome. In addition, messenger RNA-like RNA which contains the RT gene can be obtained from retroviruses. Examples of sources for RT include, but are not limited to, Moloney murine leukemia virus (M-MLV or MLVRT); human T-cell leukemia virus type 1 (HTLV-1); bovine leukemia virus (BLV); Rous Sarcoma Virus (RSV); human immunodeficiency virus (HIV); yeast, including Saccharomyces, Neurospora, Drosophila; primates; and rodents. See, for example, Weiss, et al., U.S. Pat. No. 4,663,290 (1987); Gerard, G. R., DNA: 271-79 (1986); Kotewicz, M. L., et al., Gene 35:249-58 (1985); Tanese, N., et al., Proc. Natl. Acad. Sci. (USA): 4944-48 (1985); Roth, M. J., at al., J. Biol. Chem. 260:9326-35 (1985); Michel, F., et al., Nature 316:641-43 (1985); Akins, R. A., et al., Cell 47:505-16 (1986), EMBO J. 4:1267-75 (1985); and Fawcett, D. F., Cell 47:1007-15 (1986) (each of which are incorporated herein by reference in their entireties).
• Wild type RTs
• Exemplary enzymes for use with the herein disclosed prime editors can include, but are not limited to, M-MLV reverse transcriptase and RSV reverse transcriptase. Enzymes having reverse transcriptase activity are commercially available. In certain embodiments, the reverse transcriptase provided in trans to the other components of the prime editor (PE) system. That is, the reverse transcriptase is expressed or otherwise provided as an individual component, i.e., not as a fusion protein with a napDNAbp.
• A person of ordinary skill in the art will recognize that wild type reverse transcriptases, including but not limited to, Moloney Murine Leukemia Virus (M-MLV); Human Immunodeficiency Virus (HIV) reverse transcriptase and avian Sarcoma-Leukosis Virus (ASLV) reverse transcriptase, which includes but is not limited to Rous Sarcoma Virus (RSV) reverse transcriptase, Avian Myeloblastosis Virus (AMV) reverse transcriptase, Avian Erythroblastosis Virus (AEV) Helper Virus MCAV reverse transcriptase, Avian Myelocytomatosis Virus MC29 Helper Virus MCAV reverse transcriptase, Avian Reticuloendotheliosis Virus (REV-T) Helper Virus REV-A reverse transcriptase, Avian Sarcoma Virus UR2 Helper Virus UR2AV reverse transcriptase, Avian Sarcoma Virus Y73 Helper Virus YAV reverse transcriptase, Rous Associated Virus (RAV) reverse transcriptase, and Myeloblastosis Associated Virus (MAV) reverse transcriptase may be suitably used in the subject methods and composition described herein.
• Exemplary wild type RT enzymes are as follows:

• DESCRIPTION	SEQUENCE
  REVERSE	TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLA
  TRANSCRIPTASE	VRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGIL
  (M-MLV RT) WILD	VPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTV
  TYPE	PNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEW
  	RDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHP
  MOLONEY MURINE	DLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAK
  LEUKEMIA VIRUS	KAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPR
  	QLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQ
  USED IN PE1 (PRIME	QKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQ
  EDITOR
  1 FUSION	KLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGK
  PROTEIN	LTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT
  DISCLOSED	DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLT
  HEREIN)	DQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKA
  	LPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIH
  	GEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQ
  	KGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP(SEQ
  	ID NO: 89)
  REVERSE	AFPLERPDWDYTTQAGRNHLVHYRQLLLAGLQNAGRSPTNL
  TRANSCRIPTASE	AKVKGITQGPNESPSAFLERLKEAYRRYTPYDPEDPGQETNVS
  	MSFIWQSAPDIGRKLGRLEDLKSKTLGDLVREAEKIFNKRETP
  MOLONEY MURINE	EEREERIRRETEEKEERRRTVDEQKEKERDRRRHREMSKLLAT
  LEUKEMIA VIRUS	VVIGQEQDRQEGERKRPQLDKDQCAYCKEKGHWAKDCPKKP
  	RGPRGPRPQTSLLTLGDXGGQGQDPPPEPRITLKVGGQPVTFL
  REF SEQ.	VDTGAQHSVLTQNPGPLSDKSAWVQGATGGKRYRWTTDRKV
  AAA66622.1	HLATGKVTHSFLHVPDCPYPLLGRDLLTKLKAQIHFEGSGAQV
  	VGPMGQPLQVLTLNIEDEYRLHETSKEPDVSLGFTWLSDFPQA
  	WAESGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKP
  	HIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREV
  	NKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRL
  	HPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEAL
  	HRDLADFR(SEQ ID NO: 90)
  REVERSE	TLQLEEEYRLFEPESTQKQEMDIWLKNFPQAWAETGGMGTAH
  TRANSCRIPTASE	CQAPVLIQLKATATPISIRQYPMPHEAYQGIKPHIRRMLDQGIL
  	KPCQSPWNTPLLPVKKPGTEDYRPVQDLREVNKRVEDIHPTV
  FELINE LEUKEMIA	PNPYNLLSTLPPSHPWYTVLDLKDAFFCLRLHSESQLLFAFEW
  VIRUS	RDPEIGLSGQLTWTRLPQGFKNSPTLFDEALHSDLADFRVRYP
  	ALVLLQYVDDLLLAAATRTECLEGTKALLETLGNKGYRASAK
  REF SEQ. NP955579.1	KAQICLQEVTYLGYSLKDGQRWLTKARKEAILSIPVPKNSRQV
  	REFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLA
  	FEDIKKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGP
  	WKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLG
  	QPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVH
  	FGPTVSLNPATLLPLPSGGNHHDCLQILAETHGTRPDLTDQPLP
  	DADLTWYTDGSSFIRNGEREAGAAVTTESEVIWAAPLPPGTSA
  	QRAELIALTQALKMAEGKKLTVYTDSRYAFATTHVHGEIYRRR
  	GLLTSEGKEIKNKNEILALLEALFLPKRLSIIHCPGHQKGDSPQ
  	AKGNRLADDTAKKAATETHSSLTVL
  	SEQ ID NO: 91)
  REVERSE	PISPIETVPVKLKPGMDGPKVKQWPLTEEKIKALVEICTEMEKE
  TRANSCRIPTASE	GKISKIGPENPYNTPVFAIKKKDSTKWRKLVDFRELNKRTQDF
  	WEVQLGIPHPAGLKKKKSVTVLDVGDAYFSVPLDEDFRKYTA
  HIV-1 RT, CHAIN A	FTIPSINNETPGIRYQYNVLPQGWKGSPAIFQSSMTKILEPFRKQ
  	NPDIVIYQYMDDLYVGSDLEIGQHRTKIEELRQHLLRWGLTTP
  REF SEQ. ITL3-A	DKKHQKEPPFLWMGYELHPDKWTVQPIVLPEKDSWTVNDIQ
  	KLVGKLNWASQIYPGIKVRQLXKLLRGTKALTEVIPLTEEAEL
  	ELAENREILKEPVHGVYYDPSKDLIAEIQKQGQGQWTYQIYQ
  	EPFKNLKTGKYARMRGAHTNDVKQLTEAVQKITTESIVIWGK
  	TPKFKLPIQKETWETWWTEYWQATWIPEWEFVNTPPLVKLW
  	YQLEKEPIVGAETFYVDGAANRETKLGKAGYVTNRGRQKVV
  	TLTDTTNQKTELQAIYLALQDSGLEVNIVTDSQYALGIIQAQPD
  	QSESELVNQIIEQLIKKEKVYLAWVPAHKGIGGNEQVDKLVSA
  	GIRKV(SEQ ID NO: 92)
  	SEE MARTINELLI ET AL., VIROLOGY, 1990, 174(1): 135-144,
  	WHICH IS INCORPORATED BY REFERENCE
  REVERSE	PISPIETVPVKLKPGMDGPKVKQWPLTEEKIKALVEICTEMEKE
  TRANSCRIPTASE	GKISKIGPENPYNTPVFAIKKKDSTKWRKLVDFRELNKRTQDF
  	WEVQLGIPHPAGLKKKKSVTVLDVGDAYFSVPLDEDFRKYTA
  HIV-1 RT, CHAIN B	FTIPSINNETPGIRYQYNVLPQGWKGSPAIFQSSMTKILEPFRKQ
  	NPDIVIYQYMDDLYVGSDLEIGQHRTKIEELRQHLLRWGLTTP
  REF SEQ. ITL3-B	DKKHQKEPPFLWMGYELHPDKWTVQPIVLPEKDSWTVNDIQ
  	KLVGKLNWASQIYPGIKVRQLCKLLRGTKALTEVIPLTEEAELE
  	LAENREILKEPVHGVYYDPSKDLIAEIQKQGQGQWTYQIYQEP
  	FKNLKTGKYARMRGAHTNDVKQLTEAVQKITTESIVIWGKTP
  	KFKLPIQKETWETWWTEYWQATWIPEWEFVNTPPLVKLWYQ
  	LEKEPIVGAETF(SEQ ID NO: 93)
  	SEE STAMMERS ET AL., J. MOL. BIOL., 1994, 242(4): 586-588,
  	WHICH IS INCORPORATED BY REFERENCE
  REVERSE	TVALHLAIPLKWKPNHTPVWIDQWPLPEGKLVALTQLVEKEL
  TRANSCRIPTASE	QLGHIEPSLSCWNTPVFVIRKASGSYRLLHDLRAVNAKLVPFG
  	AVQQGAPVLSALPRGWPLMVLDLKDCFFSIPLAEQDREAFAF
  ROUS SARCOMA	TLPSVNNQAPARRFQWKVLPQGMTCSPTICQLIVGQILEPLRL
  VIRUS RT	KHPSLRMLHYMDDLLLAASSHDGLEAAGEEVISTLERAGFTIS
  	PDKVQKEPGVQYLGYKLGSTYAAPVGLVAEPRIATLWDVQKL
  REF SEQ. ACL14945	VGSLQWLRPALGIPPRLRGPFYEQLRGSDPNEAREWNLDMK
  	MAWREIVQLSTTAALERWDPALPLEGAVARCEQGAIGVLGQG
  	LSTHPRPCLWLFSTQPTKAFTAWLEVLTLLITKLRASAVRTFGK
  	EVDILLLPACFRDELPLPEGILLALRGFAGKIRSSDTPSIFDIARP
  	LHVSLKVRVTDHPVPGPTVFTDASSSTHKGVVVWREGPRWEI
  	KEIADLGASVQQLEARAVAMALLLWPTTPTNVVTDSAFVAKM
  	LLKMGQEGVPSTAAAFILEDALSQRSAMAAVLHVRSHSEVPG
  	FFTEGNDVADSQATFQAYPLREAKDLHTALHIGPRALSKACNI
  	SMQQAREVVQTCPHCNSAPALEAGVNPRGLGPLQIWQTDFTL
  	EPRMAPRSWLAVTVDTASSAIVVTQHGRVTSVAAQHHWATVI
  	AVLGRPKAIKTDNGSCFTSKSTREWLARWGIAHTTGIPGNSQG
  	QAMVERANRLLKDKIRVLAEGDGFMKRIPTSKQGELLAKAM
  	YALNHFERGENTKTPIQKHWRPTVLTEGPPVKIRIETGEWEKG
  	WNVLVWGRGYAAVKNRDTDKVIWVPSRKVKPDIAQKDEVT
  	KKDEASPLFA(SEQ ID NO: 94)
  	SEE YASUKAWA ET AL., J. BIOCHEM. 2009, 145(3): 315-324,
  	WHICH IS INCORPORATED BY REFERENCE
  REVERSE	MMDHLLQKTQIQNQTEQVMNITNPNSIYIKGRLYFKGYKKIEL
  TRANSCRIPTASE	HCFVDTGASLCIASKFVIPEEHWINAERPIMVKIADGSSITINKV
  	CRDIDLIIAGEIFHIPTVYQQESGIDFIIGNNFCQLYEPFIQFTDRV
  CAULIFLOWER	TTKDRTYPVHIAKLTRAVRVGTEGFLESMKKRSKTQQPEPVNI
  MOSAIC VIRUS RT	STNKIAILSEGRRLSEEKLFITQQRMQKIEELLEKVCSENPLDP
  	NKTKQWMKASIKLSDPSKAIKVKPMKYSPMDREEFDKQIKEL
  REF SEQ. AGT42196	LDLKVIKPSKSPHMAPAFLVNNEAEKRRGKKRMVVNYKAMN
  	KATVGDAYNLPNKDELLTLIRGKKIFSSFDCKSGFWQVLLDQD
  	SRPLTAFTCPQGHYEWNVVPFGLKQAPSIFQRHMDEAFRVFR
  	KFCCVYVDDILVFSNNEEDHLLHVAMILQKCNQHGIILSKKKA
  	QLFKKKINFLGLEIDEGTHKPQGHILEHINKFPDTLEDKKQLQR
  	FLGILTYASDYIPKLAQIRKPLQAKLKENVPWKWTKEDTLYM
  	QKVKKNLQGFPPLHHPLPEEKLIIETDASDDYWGGMLKAIKIN
  	EGTNTELICRYASGSFKAAEKNYHSNDKETLAVINTIKKFSIYL
  	TPVHFLIRTDNTHFKSFVNLNYKGDSKLGRNIRWQAWLSHYS
  	FDVEHIKGTDNHFADFLSREFNRVNS(SEQ ID NO: 95)
  	SEE FARZADFAR ET AL., VIRUS GENES, 2013, 47(2): 347-356,
  	WHICH IS INCORPORATED BY REFERENCE
  REVERSE	MKEKISKIDKNFYTDIFIKTSFQNEFEAGGVIPPIAKNQVSTISN
  TRANSCRIPTASE	KNKTFYSLAHSSPHYSIQTRIEKFLLKNIPLSASSFAFRKERSYL
  	HYLEPHTQNVKYCHLDIVSFFHSIDVNIVRDTFSVYFSDEFLVK
  KLEBSIELLA	EKQSLLDAFMASVTLTAELDGVEKTFIPMGFKSSPSISNIIFRKI
  PNEUMONIA	DILIQKFCDKNKITYTRYADDLLFSTKKENNILSSTFFINEISSIL
  	SINTKFKLNKSKYLYKEGTISLGGYVIENILKDNSSGNIRLSSSKL
  REF SEQ. RFF81513.1	NPLYKALYEIKKGSSSKHICIKVFNLKLKRFIYKKNKEKFEAKF
  	YSSQLKNKLLGYRSYLLSFVIFHKKYKCINPIFLEKCVFLISEIE
  	SIMNRKF(SEQ ID NO: 96)
  REVERSE	MKITSNNVTAVINGKGWHSINWKKCHQHVKTIQTRIAKAACN
  TRANSCRIPTASE	QQWRTVGRLQRLLVRSFSARALAVKRVTENSGRKTPGVDGQI
  	WSTPESKWEATKLRRKGYKPLPLKRVFIPKSNGKKRPLGIPV
  ESCERICHIA COLI	MLDRAMQALHLLGLEPVSETNADHNSYGFRPARCTADAIQQ
  RT	VCNMYSSRNASKWVLEGDIKGCFEHISHEWLLENIPMDKQIL
  	RNWLKAGIIEKSIFSKTLSGTPQGGIISPVLANMALDGLERLLQ
  REF SEQ. TGH57013	NRFGRNRLI(SEQ ID NO: 97)
  REVERSE	MSKIKINYEKYHIKPFPHFDQRIKVNKKVKENLQNPFYIAAHS
  TRANSCRIPTASE	FYPFIHYKKISYKFKNGTLSSPKERDIFYSGHMDGYIYKHYGEI
  	LNHKYNNTCIGKGIDHVSLAYRNNKMGKSNIHFAAEVINFISE
  BACILLUS SUBTILIS	QQQAFIFVSDFSSYFDSLDHAILKEKLIEVLEEQDKLSKDWWN
  RT	VFKHITRYNWVEKEEVISDLECTKEKIARDKKSRERYYTPAEF
  	REFRKRVNIKSNDTGVGIPQGTAISAVLANVYAIDLDQKLNQY
  REF SEQ. QBJ66766	ALKYGGIYRRYSDDIIMVLPMTSDGQDPSNDHVSFIKSVVKRN
  	KVTMGDSKTSVLYYANNNIYEDYQRKRESKMDYLGFSFDGM
  	TVKIREKSLFKYYHRTYKKINSINWASVKKEKKVGRKKLYLL
  	YSHLGRNYKGHGNFISYCKKAHAVFEGNKKIESLINQQIKRH
  	WKKIQKRLVDV(SEQ ID NO: 98)
  EUBACTERIUM	DTSNLMEQILSSDNLNRAYLQVVRNKGAEGVDGMKYTELKE
  RECTALE GROUP II	HLAKNGETIKGQLRTRKYKPQPARRVEIPKPDGGVRNLGVPT
  INTRON RT	VTDRFIQQAIAQVLTPIYEEQFHDHSYGFRPNRCAQQAILTALN
  	IMNDGNDWIVDIDLEKFFDTVNHDKLMTLIGRTIKDGDVISIV
  	RKYLVSGIMIDDEYEDSIVGTPQGGNLSPLLANIMLNELDKEM
  	EKRGLNFVRYADDCIIMVGSEMSANRVMRNISRFIEEKLGLKV
  	NMTKSKVDRPSGLKYLGFGFYFDPRAHQFKAKPHAKSVAKF
  	KKRMKELTCRSWGVSNSYKVEKLNQLIRGWINYFKIGSMKTL
  	CKELDSRIRYRLRMCIWKQWKTPQNQEKNLVKLGIDRNTARR
  	VAYTGKRIAYVCNKGAVNVAISNKRLASFGLISMLDYYIEKCV
  	TC(SEQ ID NO: 99)
  GEOBACILLUS	ALLERILARDNLITALKRVEANQGAPGIDGVSTDQLRDYIRAH
  STEAROTHERMOPH	WSTIHAQLLAGTYRPAPVRRVEIPKPGGGTRQLGIPTVVDRLIQ
  ILUS GROUP II	QAILQELTPIFDPDFSSSSFGFRPGRNAHDAVRQAQGYIQEGYR
  INTRON RT	YVVDMDLEKFFDRVNHDILMSRVARKVKDKRVLKLIRAYLQA
  	GVMIEGVKVQTEEGTPQGGPLSPLLANILLDDLDKELEKRGL
  	KFCRYADDCNIYVKSLRAGQRVKQSIQRFLEKTLKLKVNEEKS
  	AVDRPWKRAFLGFSFTPERKARIRLAPRSIQRLKQRIRQLTNPN
  	WSISMPERIHRVNQYVMGWIGYFRLVETPSVLQTIEGWIRRRL
  	RLCQWLQWKRVRTRIRELRALGLKETAVMEIANTRKGAWRTT
  	KTPQLHQALGKTYWTAQGLKSLTQR(SEQ ID NO: 100)

Variant and Error-Prone RTs
• Reverse transcriptases are essential for synthesizing complementary DNA (cDNA) strands from RNA templates. Reverse transcriptases are enzymes composed of distinct domains that exhibit different biochemical activities. The enzymes catalyze the synthesis of DNA from an RNA template, as follows: In the presence of an annealed primer, reverse transcriptase binds to an RNA template and initiates the polymerization reaction. RNA-dependent DNA polymerase activity synthesizes the complementary DNA (cDNA) strand, incorporating dNTPs. RNase H activity degrades the RNA template of the DNA:RNA complex. Thus, reverse transcriptases comprise (a) a binding activity that recognizes and binds to a RNA/DNA hybrid, (b) an RNA-dependent DNA polymerase activity, and (c) an RNase H activity. In addition, reverse transcriptases generally are regarded as having various attributes, including their thermostability, processivity (rate of dNTP incorporation), and fidelity (or error-rate). The reverse transcriptase variants contemplated herein may include any mutations to reverse transcriptase that impacts or changes any one or more of these enzymatic activities (e.g., RNA-dependent DNA polymerase activity, RNase H activity, or DNA/RNA hybrid-binding activity) or enzyme properties (e.g., thermostability, processivity, or fidelity). Such variants may be available in the art in the public domain, available commercially, or may be made using known methods of mutagenesis, including directed evolutionary processes (e.g., PACE or PANCE).
• In various embodiments, the reverse transcriptase may be a variant reverse transcriptase. As used herein, a “variant reverse transcriptase” includes any naturally occurring or genetically engineered variant comprising one or more mutations (including singular mutations, inversions, deletions, insertions, and rearrangements) relative to a reference sequences (e.g., a reference wild type sequence). RT naturally have several activities, including an RNA-dependent DNA polymerase activity, ribonuclease H activity, and DNA-dependent DNA polymerase activity. Collectively, these activities enable the enzyme to convert single-stranded RNA into double-stranded cDNA. In retroviruses and retrotransposons, this cDNA can then integrate into the host genome, from which new RNA copies can be made via host-cell transcription. Variant RT's may comprise a mutation which impacts one or more of these activities (either which reduces or increases these activities, or which eliminates these activities all together). In addition, variant RTs may comprise one or more mutations which render the RT more or less stable, less prone to aggregation, and facilitates purification and/or detection, and/or other the modification of properties or characteristics.
• A person of ordinary skill in the art will recognize that variant reverse transcriptases derived from other reverse transcriptases, including but not limited to Moloney Murine Leukemia Virus (M-MLV); Human Immunodeficiency Virus (HIV) reverse transcriptase and avian Sarcoma-Leukosis Virus (ASLV) reverse transcriptase, which includes but is not limited to Rous Sarcoma Virus (RSV) reverse transcriptase, Avian Myeloblastosis Virus (AMV) reverse transcriptase, Avian Erythroblastosis Virus (AEV) Helper Virus MCAV reverse transcriptase, Avian Myelocytomatosis Virus MC29 Helper Virus MCAV reverse transcriptase, Avian Reticuloendotheliosis Virus (REV-T) Helper Virus REV-A reverse transcriptase, Avian Sarcoma Virus UR2 Helper Virus UR2AV reverse transcriptase, Avian Sarcoma Virus Y73 Helper Virus YAV reverse transcriptase, Rous Associated Virus (RAV) reverse transcriptase, and Myeloblastosis Associated Virus (MAV) reverse transcriptase may be suitably used in the subject methods and composition described herein.
• One method of preparing variant RTs is by genetic modification (e.g., by modifying the DNA sequence of a wild-type reverse transcriptase). A number of methods are known in the art that permit the random as well as targeted mutation of DNA sequences (see for example, Ausubel et. al. Short Protocols in Molecular Biology (1995) 3.sup.rd Ed. John Wiley & Sons, Inc.). In addition, there are a number of commercially available kits for site-directed mutagenesis, including both conventional and PCR-based methods. Examples include the QuikChange Site-Directed Mutagenesis Kits (AGILENT®), the Q5® Site-Directed Mutagenesis Kit (NEW ENGLAND BIOLABS®), and GeneArt™ Site-Directed Mutagenesis System (THERMOFISHER SCIENTIFIC®).
• In addition, mutant reverse transcriptases may be generated by insertional mutation or truncation (N-terminal, internal, or C-terminal insertions or truncations) according to methodologies known to one skilled in the art. The term “mutation,” as used herein, refers to a substitution of a residue within a sequence, e.g., a nucleic acid or amino acid sequence, with another residue, or a deletion or insertion of one or more residues within a sequence. Mutations are typically described herein by identifying the original residue followed by the position of the residue within the sequence and by the identity of the newly substituted residue. Various methods for making the amino acid substitutions (mutations) provided herein are well known in the art, and are provided by, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)). Mutations can include a variety of categories, such as single base polymorphisms, microduplication regions, indel, and inversions, and is not meant to be limiting in any way. Mutations can include “loss-of-function” mutations which is the normal result of a mutation that reduces or abolishes a protein activity. Most loss-of-function mutations are recessive, because in a heterozygote the second chromosome copy carries an unmutated version of the gene coding for a fully functional protein whose presence compensates for the effect of the mutation. Mutations also embrace “gain-of-function” mutations, which is one which confers an abnormal activity on a protein or cell that is otherwise not present in a normal condition. Many gain-of-function mutations are in regulatory sequences rather than in coding regions, and can therefore have a number of consequences. For example, a mutation might lead to one or more genes being expressed in the wrong tissues, these tissues gaining functions that they normally lack. Because of their nature, gain-of-function mutations are usually dominant.
• Older methods of site-directed mutagenesis known in the art rely on sub-cloning of the sequence to be mutated into a vector, such as an M13 bacteriophage vector, that allows the isolation of single-stranded DNA template. In these methods, one anneals a mutagenic primer (i.e., a primer capable of annealing to the site to be mutated but bearing one or more mismatched nucleotides at the site to be mutated) to the single-stranded template and then polymerizes the complement of the template starting from the 3′ end of the mutagenic primer. The resulting duplexes are then transformed into host bacteria and plaques are screened for the desired mutation.
• More recently, site-directed mutagenesis has employed PCR methodologies, which have the advantage of not requiring a single-stranded template. In addition, methods have been developed that do not require sub-cloning. Several issues must be considered when PCR-based site-directed mutagenesis is performed. First, in these methods it is desirable to reduce the number of PCR cycles to prevent expansion of undesired mutations introduced by the polymerase. Second, a selection must be employed in order to reduce the number of non-mutated parental molecules persisting in the reaction. Third, an extended-length PCR method is preferred in order to allow the use of a single PCR primer set. And fourth, because of the non-template-dependent terminal extension activity of some thermostable polymerases it is often necessary to incorporate an end-polishing step into the procedure prior to blunt-end ligation of the PCR-generated mutant product.
• Methods of random mutagenesis, which will result in a panel of mutants bearing one or more randomly situated mutations, exist in the art. Such a panel of mutants may then be screened for those exhibiting the desired properties, for example, increased stability, relative to a wild-type reverse transcriptase.
• An example of a method for random mutagenesis is the so-called “error-prone PCR method.” As the name implies, the method amplifies a given sequence under conditions in which the DNA polymerase does not support high fidelity incorporation. Although the conditions encouraging error-prone incorporation for different DNA polymerases vary, one skilled in the art may determine such conditions for a given enzyme. A key variable for many DNA polymerases in the fidelity of amplification is, for example, the type and concentration of divalent metal ion in the buffer. The use of manganese ion and/or variation of the magnesium or manganese ion concentration may therefore be applied to influence the error rate of the polymerase.
• In various aspects, the RT of the prime editors may be an “error-prone” reverse transcriptase variant. Error-prone reverse transcriptases that are known and/or available in the art may be used. It will be appreciated that reverse transcriptases naturally do not have any proofreading function; thus the error rate of reverse transcriptase is generally higher than DNA polymerases comprising a proofreading activity. The error-rate of any particular reverse transcriptase is a property of the enzyme's “fidelity,” which represents the accuracy of template-directed polymerization of DNA against its RNA template. An RT with high fidelity has a low-error rate. Conversely, an RT with low fidelity has a high-error rate. The fidelity of M-MLV-based reverse transcriptases are reported to have an error rate in the range of one error in 15,000 to 27,000 nucleotides synthesized. See Boutabout et al., “DNA synthesis fidelity by the reverse transcriptase of the yeast retrotransposon Ty1,” Nucleic Acids Res, 2001, 29: 2217-2222, which is incorporated by reference. Thus, for purposes of this application, those reverse transcriptases considered to be “error-prone” or which are considered to have an “error-prone fidelity” are those having an error rate that is less than one error in 15,000 nucleotides synthesized.
• Error-prone reverse transcriptase also may be created through mutagenesis of a starting RT enzyme (e.g., a wild type M-MLV RT). The method of mutagenesis is not limited and may include directed evolution processes, such as phage-assisted continuous evolution (PACE) or phage-assisted noncontinuous evolution (PANCE). The term “phage-assisted continuous evolution (PACE),” as used herein, refers to continuous evolution that employs phage as viral vectors. The general concept of PACE technology has been described, for example, in International PCT Application, PCT/US2009/056194, filed Sep. 8, 2009, published as WO 2010/028347 on Mar. 11, 2010; International PCT Application, PCT/US2011/066747, filed Dec. 22, 2011, published as WO 2012/088381 on Jun. 28, 2012; U.S. application, U.S. Pat. No. 9,023,594, issued May 5, 2015, International PCT Application, PCT/US2015/012022, filed Jan. 20, 2015, published as WO 2015/134121 on Sep. 11, 2015, and International PCT Application, PCT/US2016/027795, filed Apr. 15, 2016, published as WO 2016/168631 on Oct. 20, 2016, the entire contents of each of which are incorporated herein by reference.
• Error-prone reverse transcriptases may also be obtain by phage-assisted non-continuous evolution (PANCE),” which as used herein, refers to non-continuous evolution that employs phage as viral vectors. PANCE is a simplified technique for rapid in vivo directed evolution using serial flask transfers of evolving ‘selection phage’ (SP), which contain a gene of interest to be evolved, across fresh E. coli host cells, thereby allowing genes inside the host E. coli to be held constant while genes contained in the SP continuously evolve. Serial flask transfers have long served as a widely-accessible approach for laboratory evolution of microbes, and, more recently, analogous approaches have been developed for bacteriophage evolution. The PANCE system features lower stringency than the PACE system.
• Other error-prone reverse transcriptases have been described in the literature, each of which are contemplated for use in the herein methods and compositions. For example, error-prone reverse transcriptases have been described in Bebenek et al., “Error-prone Polymerization by HIV-1 Reverse Transcriptase,” J Biol Chem, 1993, Vol. 268: 10324-10334 and Sebastian-Martin et al., “Transcriptional inaccuracy threshold attenuates differences in RNA-dependent DNA synthesis fidelity between retroviral reverse transcriptases,” Scientific Reports, 2018, Vol. 8: 627, each of which are incorporated by reference. Still further, reverse transcriptases, including error-prone reverse transcriptases can be obtained from a commercial supplier, including ProtoScript® (II) Reverse Transcriptase, AMV Reverse Transcriptase, WarmStart® Reverse Transcriptase, and M-MuLV Reverse Transcriptase, all from NEW ENGLAND BIOLABS®, or AMV Reverse Transcriptase XL, SMARTScribe Reverse Transcriptase, GPR ultra-pure MMLV Reverse Transcriptase, all from TAKARA BIO USA, INC. (formerly CLONTECH).
• The herein disclosure also contemplates reverse transcriptases having mutations in RNaseH domain. As mentioned above, one of the intrinsic properties of reverse transcriptases is the RNase H activity, which cleaves the RNA template of the RNA:cDNA hybrid concurrently with polymerization. The RNase H activity can be undesirable for synthesis of long cDNAs because the RNA template may be degraded before completion of full-length reverse transcription. The RNase H activity may also lower reverse transcription efficiency, presumably due to its competition with the polymerase activity of the enzyme. Thus, the present disclosure contemplates any reverse transcriptase variants that comprise a modified RNaseH activity.
• The herein disclosure also contemplates reverse transcriptases having mutations in the RNA-dependent DNA polymerase domain. As mentioned above, one of the intrinsic properties of reverse transcriptases is the RNA-dependent DNA polymerase activity, which incorporates the nucleobases into the nascent cDNA strand as coded by the template RNA strand of the RNA:cDNA hybrid. The RNA-dependent DNA polymerase activity can be increased or decreased (i.e., in terms of its rate of incorporation) to either increase or decrease the processivity of the enzyme. Thus, the present disclosure contemplates any reverse transcriptase variants that comprise a modified RNA-dependent DNA polymerase activity such that the processivity of the enzyme of either increased or decreased relative to an unmodified version.
• Also contemplated herein are reverse transcriptase variants that have altered thermostability characteristics. The ability of a reverse transcriptase to withstand high temperatures is an important aspect of cDNA synthesis. Elevated reaction temperatures help denature RNA with strong secondary structures and/or high GC content, allowing reverse transcriptases to read through the sequence. As a result, reverse transcription at higher temperatures enables full-length cDNA synthesis and higher yields, which can lead to an improved generation of the 3′ flap ssDNA as a result of the prime editing process. Wild type M-MLV reverse transcriptase typically has an optimal temperature in the range of 37-48° C.; however, mutations may be introduced that allow for the reverse transcription activity at higher temperatures of over 48° C., including 49° C., 50° C., 51° C., 52° C., 53° C., 54° C., 55° C., 56° C., 57° C., 58° C., 59° C., 60° C., 61° C., 62° C., 63° C., 64° C., 65° C., 66° C., and higher.
• The variant reverse transcriptases contemplated herein, including error-prone RTs, thermostable RTs, increase-processivity RTs, can be engineered by various routine strategies, including mutagenesis or evolutionary processes. In some cases, the variants can be produced by introducing a single mutation. In other cases, the variants may require more than one mutation. For those mutants comprising more than one mutation, the effect of a given mutation may be evaluated by introduction of the identified mutation to the wild-type gene by site-directed mutagenesis in isolation from the other mutations borne by the particular mutant. Screening assays of the single mutant thus produced will then allow the determination of the effect of that mutation alone.
• Variant RT enzymes used herein may also include other “RT variants” having at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to any reference RT protein, including any wild type RT, or mutant RT, or fragment RT, or other variant of RT disclosed or contemplated herein or known in the art.
• In some embodiments, an RT variant may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or up to 100, or up to 200, or up to 300, or up to 400, or up to 500 or more amino acid changes compared to a reference RT. In some embodiments, the RT variant comprises a fragment of a reference RT, such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of the reference RT. In some embodiments, the fragment is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid length of a corresponding wild type RT (M-MLV reverse transcriptase) (e.g., SEQ ID NO: 89) or to any of the reverse transcriptases of SEQ ID NOs: 90-100.
• In some embodiments, the disclosure also may utilize RT fragments which retain their functionality and which are fragments of any herein disclosed RT proteins. In some embodiments, the RT fragment is at least 100 amino acids in length. In some embodiments, the fragment is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, or up to 600 or more amino acids in length.
• In still other embodiments, the disclosure also may utilize RT variants which are truncated at the N-terminus or the C-terminus, or both, by a certain number of amino acids which results in a truncated variant which still retains sufficient polymerase function. In some embodiments, the RT truncated variant has a truncation of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 amino acids at the N-terminal end of the protein. In other embodiments, the RT truncated variant has a truncation of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 amino acids at the C-terminal end of the protein. In still other embodiments, the RT truncated variant has a truncation at the N-terminal and the C-terminal end which are the same or different lengths.
• For example, the prime editors disclosed herein may include a truncated version of M-MLV reverse transcriptase. In this embodiment, the reverse transcriptase contains 4 mutations (D200N, T306K, W313F, T330P; noting that the L603W mutation present in PE2 is no longer present due to the truncation). The DNA sequence encoding this truncated editor is 522 bp smaller than PE2, and therefore makes its potentially useful for applications where delivery of the DNA sequence is challenging due to its size (i.e., adeno-associated virus and lentivirus delivery). This embodiment is referred to as MMLV-RT(trunc) and has the following amino acid sequence:

• MMLV-	TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAE
  RT	ETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQ
  (TRUNC)	EARLGIKPHIQRLLDQGILVPCQSPTLNIEDEYRL
  	HETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVR
  	QAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHI
  	QRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPV
  	QDLREVNKRVEDIHPTVPNPYNLLSGLPTVLDLK
  	DAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWT
  	RLPQGFKNSPTLFNEALHRDLADFRIQHPDLILL
  	QYVDDLLLAATSELDCQQGTRALLQTLGNLGYRA
  	SAKKAQICQKQVKYLGYLLKEGQRWLTEARKETV
  	MGQPTPKTPRQLREFLGKAGFCRLFIPGFAEMA
  	APLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPA
  	LGLPDLTKPFELFVDEKQGYAKKGVLTQKLGPWR
  	RPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDA
  	GKLTMGQPLVILAPHAVEALVKQPPDRWLMTHYQ
  	ALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNC
  	LDNSRLIN (SEQ ID NO: 766)

• In various embodiments, the prime editors disclosed herein may comprise one of the RT variants described herein, or a RT variant thereof having at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to any reference Cas9 variants.
• In still other embodiments, the present methods and compositions may utilize a DNA polymerase that has been evolved into a reverse transcriptase, as described in Effefson et al., “Synthetic evolutionary origin of a proofreading reverse transcriptase,” Science, Jun. 24, 2016, Vol. 352: 1590-1593, the contents of which are incorporated herein by reference.
• In certain other embodiments, the reverse transcriptase is provided as a component of a fusion protein also comprising a napDNAbp. In other words, in some embodiments, the reverse transcriptase is fused to a napDNAbp as a fusion protein.
• In various embodiments, variant reverse transcriptases can be engineered from wild type M-MLV reverse transcriptase as represented by SEQ ID NO: 89.
• In various embodiments, the prime editors described herein (with RT provided as either a fusion partner or in trans) can include a variant RT comprising one or more of the following mutations: P51L, S67K, E69K, L139P, T197A, D200N, H204R, F209N, E302K, E302R, T306K, F309N, W313F, T330P, L345G, L435G, N454K, D524G, E562Q, D583N, H594Q, L603W, E607K, or D653N in the wild type M-MLV RT of SEQ ID NO: 89 or at a corresponding amino acid position in another wild type RT polypeptide sequence.
• Some exemplary reverse transcriptases that can be fused to napDNAbp proteins or provided as individual proteins according to various embodiments of this disclosure are provided below. Exemplary reverse transcriptases include variants with at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the following wild-type enzymes or partial enzymes:

• Description	Sequence (variant substitutions relative to wild type)
  Reverse	TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAP
  transcriptase	LIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPL
  (M-MLV RT)	LPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWY
  wild type	TVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKN
  	SPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRAL
  	LQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVM
  	GQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKPGTLFNWG
  	PDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKL
  moloney	GPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQP
  murine	LVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVAL
  leukemia	NPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGS
  	SLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMA
  virus	EGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLK
  	ALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLL
  	IENSSP (SEQ ID NO: 89)
  Used in PE1
  (prime editor
  1 fusion
  protein
  disclosed
  herein)
  M-MLV RT	TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAP
  D200N	LIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPL
  	LPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWY
  	TVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKN
  	SPTLF N EALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRAL
  	LQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVM
  	GQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWG
  	PDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKL
  	GPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQP
  	LVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVAL
  	NPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGS
  	SLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMA
  	EGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLK
  	ALFLPKRLS IIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLL
  	IENSSP (SEQ ID NO: 701)
  M-MLV RT	TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAP
  D200N	LIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPL
  T330P	LPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWY
  L603W	TVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKN
  	SPTLF N EALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRAL
  	LQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVM
  	GQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTK P GTLFNWG
  	PDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKL
  	GPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQP
  	LVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVAL
  	NPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGS
  	SLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMA
  	EGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLK
  	ALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLL
  	IENSSP (SEQ ID NO: 702)
  M-MLV RT	TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAP
  	LIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPL
  D200N	LPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWY
  T330P	TVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKN
  	SPTLF N EALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRAL
  L603W	LQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVM
  	GQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTK P GTLFNWG
  	PDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKL
  	GPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQP
  	LVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVAL
  	NPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGS
  	SLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMA
  	EGKKLNVYTDSRYAFATAHIHGEIYRRRG W LTSEGKEIKNKDEILALL
  	KALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTST
  	LLIENSSP (SEQ ID NO: 740)
  M-MLV RT	TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAP
  D200N	LIIPLKATSTPVSIKQYPMSQ K ARLGIKPHIQRLLDQGILVPCQSPWNTP
  T330P	LLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQW
  L603W	YTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFK
  E69K	NSPTLF N EALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRA
  	LLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETV
  	MGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTK P GTLFNW
  	GPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQ
  	KLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMG
  	QPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVV
  	ALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTD
  	GSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALK
  	MAEGKKLNVYTDSRYAFATAHIHGEIYRRRG W LTSEGKEIKNKDEILA
  	LLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDT
  	STLLIENSSP (SEQ ID NO: 703)
  M-MLV RT	TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAP
  D200N	LIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPL
  T330P	LPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWY
  L603W	TVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKN
  E302R	SPTLF N EALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRAL
  	LQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVM
  	GQPTPKTPRQLR R FLGTAGFCRLWIPGFAEMAAPLYPLTK P GTLFNWG
  	PDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKL
  	GPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQP
  	LVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVAL
  	NPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGS
  	SLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMA
  	EGKKLNVYTDSRYAFATAHIHGEIYRRRG W LTSEGKEIKNKDEILALL
  	KALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTST
  	LLIENSSP(SEQ ID NO: 704)
  M-MLV RT	TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAP
  D200N	LIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPL
  T330P	LPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWY
  L603W	TVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKN
  E607K	SPTLF N EALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRAL
  	LQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVM
  	GQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTK P GTLFNWG
  	PDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKL
  	GPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQP
  	LVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVAL
  	NPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGS
  	SLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMA
  	EGKKLNVYTDSRYAFATAHIHGEIYRRRG W LTS K GKEIKNKDEILALL
  	KALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTST
  	LLIENSSP (SEQ ID NO: 705)
  M-MLV RT	TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAP
  D200N	LIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPL
  T330P	LPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSG P PPSHQWY
  L603W	TVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKN
  L139P	SPTLF N EALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRAL
  	LQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVM
  	GQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTK P GTLFNWG
  	PDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKL
  	GPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQP
  	LVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVAL
  	NPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGS
  	SLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMA
  	EGKKLNVYTDSRYAFATAHIHGEIYRRRG W LTSEGKEIKNKDEILALL
  	KALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTST
  	LLIENSSP (SEQ ID NO: 706)
  M-MLV RT	TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAP
  D200N	LIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPL
  T330P	LPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWY
  L603W	TVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKN
  L435G	SPTLF N EALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRAL
  	LQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVM
  	GQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTK P GTLFNWG
  	PDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKL
  	GPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQP
  	LVI G APHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVAL
  	NPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGS
  	SLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMA
  	EGKKLNVYTDSRYAFATAHIHGEIYRRRG W LTSEGKEIKNKDEILALL
  	KALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTST
  	LLIENSSP (SEQ ID NO: 707)
  M-MLV RT	TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAP
  D200N	LIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPL
  T330P	LPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWY
  L603W	TVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKN
  N454K	SPTLF N EALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRAL
  	LQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVM
  	GQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTK P GTLFNWG
  	PDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKL
  	GPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQP
  	LVILAPHAVEALVKQPPDRWLS K ARMTHYQALLLDTDRVQFGPVVAL
  	NPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGS
  	SLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMA
  	EGKKLNVYTDSRYAFATAHIHGEIYRRRG W LTSEGKEIKNKDEILALL
  	KALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTST
  	LLIENSSP (SEQ ID NO: 708)
  M-MLV RT	TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAP
  D200N	LIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPL
  T330P	LPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWY
  L603W	TVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKN
  T306K	SPTLF N EALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRAL
  	LQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVM
  	GQPTPKTPRQLREFLG K AGFCRLWIPGFAEMAAPLYPLTK P GTLFNWG
  	PDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKL
  	GPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQP
  	LVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVAL
  	NPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGS
  	SLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMA
  	EGKKLNVYTDSRYAFATAHIHGEIYRRRG W LTSEGKEIKNKDEILALL
  	KALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTST
  	LLIENSSP (SEQ ID NO: 709)
  M-MLV RT	TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAP
  D200N	LIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPL
  T330P	LPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWY
  L603W	TVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKN
  W313F	SPTLF N EALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRAL
  	LQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVM
  	GQPTPKTPRQLREFLGTAGFCRL F IPGFAEMAAPLYPLTK P GTLFNWGP
  	DQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKL
  	GPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQP
  	LVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVAL
  	NPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGS
  	SLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMA
  	EGKKLNVYTDSRYAFATAHIHGEIYRRRG W LTSEGKEIKNKDEILALL
  	KALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTST
  	LLIENSSP (SEQ ID NO: 710)
  M-MLV RT	TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAP
  D200N	LIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPL
  T330P	LPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWY
  L603W	TVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKN
  D524G	SPTLF N EALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRAL
  E562Q	LQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVM
  D583N	GQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTK P GTLFNWG
  	PDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKL
  	GPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQP
  	LVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVAL
  	NPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYT G GS
  	SLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRA Q LIALQALKMA
  	EGKKLNVYT N SRYAFATAHIHGEIYRRRG W LTSEGKEIKNKDEILALL
  	KALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTST
  	LLIENSSP (SEQ ID NO: 711)
  M-MLV RT	TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAP
  D200N	LIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPL
  T330P	LPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWY
  L603W	TVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKN
  E302R	SPTLF N EALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRAL
  W313F	LQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVM
  	GQPTPKTPRQLR R FLGTAGFCRL F IPGFAEMAAPLYPLTK P GTLFNWG
  	PDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKL
  	GPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQP
  	LVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVAL
  	NPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGS
  	SLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMA
  	EGKKLNVYTDSRYAFATAHIHGEIYRRRG W LTSEGKEIKNKDEILALL
  	KALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTST
  	LLIENSSP (SEQ ID NO: 712)
  M-MLV RT	TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAP
  D200N	LIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPL
  T330P	LPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSG P PPSHQWY
  L603W	TVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKN
  E607K	SPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRAL
  L139P	LQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVM
  	GQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTK P GTLFNWG
  	PDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKL
  	GPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQP
  	LVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVAL
  	NPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGS
  	SLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMA
  	EGKKLNVYTDSRYAFATAHIHGEIYRRRG W LTS K GKEIKNKDEILALL
  	KALFLPKRLS IIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTST
  	LLIENSSP (SEQ ID NO: 713)
  M-MLV RT	TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAP
  P51L S67K	LII L LKATSTPVSI K QYPMKQEARLGIKPHIQRLLDQGILVPCQSPWNTP
  T197A	LLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQW
  H204R	YTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFK
  E302K	NSP A LFDEAL R RDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTR
  F309N	ALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKET
  W313F	VMGQPTPKTPRQLR K FLGTAG N CRL F IPGFAEMAAPLYPLTK P GTLFN
  T330P	WGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLT
  L435G	QKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTM
  N454K	GQPLVIGAPHAVEALVKQPPDRWLS K ARMTHYQALLLDTDRVQFGPV
  D524G	VALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYT
  D583N	G GSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALK
  H594Q	MAEGKKLNVYT N SRYAFATAHI Q GEIYRRRGLLTSEGKEIKNKDEILA
  D653N	LLKALFLPKRLSIIHCPGHQKGHSAEARGNRMA N QAARKAAITETPDT
  	STLLIENSSP (SEQ ID NO: 714)
  M-MLV RT	TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAP
  D200N P51L	LII L LKATSTPVSI K QYPMKQEARLGIKPHIQRLLDQGILVPCQSPWNTP
  S67K T197A	LLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQW
  H204R	YTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFK
  E302K	NSP A LF N EAL R RDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTR
  F309N	ALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKET
  W313F	VMGQPTPKTPRQLR K FLGTAG N CRL F IPGFAEMAAPLYPLTK P GTLFN
  T330P L345G	WGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLT
  N454K	QKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTM
  D524G	GQPLVI G APHAVEALVKQPPDRWLS K ARMTHYQALLLDTDRVQFGPV
  D583N	VALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYT
  H594Q	G GSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALK
  D653N	MAEGKKLNVYT N SRYAFATAHI Q GEIYRRRGLLTSEGKEIKNKDEILA
  	LLKALFLPKRLSIIHCPGHQKGHSAEARGNRMA N QAARKAAITETPDT
  	STLLIENSSP (SEQ ID NO: 715)
  M-MLV RT	TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAP
  D200N	LIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPL
  T330P	LPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWY
  L603W	TVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKN
  T306K	SPTLF N EALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRAL
  W313F	LQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVM
  	GQPTPKTPRQLREFLG K AGFCRL F IPGFAEMAAPLYPLTK P GTLFNWG
  in PE2	PDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKL
  	GPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQP
  	LVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVAL
  	NPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGS
  	SLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMA
  	EGKKLNVYTDSRYAFATAHIHGEIYRRRG W LTSEGKEIKNKDEILALL
  	KALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTST
  	LLIENSSP (SEQ ID NO: 716)

• In various other embodiments, the prime editors described herein (with RT provided as either a fusion partner or in trans) can include a variant RT comprising one or more of the following mutations: P51X, S67X, E69X, L139X, T197X, D200X, H204X, F209X, E302X, T306X, F309X, W313X, T330X, L345X, L435X, N454X, D524X, E562X, D583X, H594X, L603X, E607X, or D653X in the wild type M-MLV RT of SEQ ID NO: 89 or at a corresponding amino acid position in another wild type RT polypeptide sequence, wherein “X” can be any amino acid.
• In various other embodiments, the prime editors described herein (with RT provided as either a fusion partner or in trans) can include a variant RT comprising a P51X mutation in the wild type M-MLV RT of SEQ ID NO: 89 or at a corresponding amino acid position in another wild type RT polypeptide sequence, wherein “X” can be any amino acid. In certain embodiments, X is L.
• In various other embodiments, the prime editors described herein (with RT provided as either a fusion partner or in trans) can include a variant RT comprising a S67X mutation in the wild type M-MLV RT of SEQ ID NO: 89 or at a corresponding amino acid position in another wild type RT polypeptide sequence, wherein “X” can be any amino acid. In certain embodiments, X is K.
• In various other embodiments, the prime editors described herein (with RT provided as either a fusion partner or in trans) can include a variant RT comprising a E69X mutation in the wild type M-MLV RT of SEQ ID NO: 89 or at a corresponding amino acid position in another wild type RT polypeptide sequence, wherein “X” can be any amino acid. In certain embodiments, X is K.
• In various other embodiments, the prime editors described herein (with RT provided as either a fusion partner or in trans) can include a variant RT comprising a L139X mutation in the wild type M-MLV RT of SEQ ID NO: 89 or at a corresponding amino acid position in another wild type RT polypeptide sequence, wherein “X” can be any amino acid. In certain embodiments, X is P.
• In various other embodiments, the prime editors described herein (with RT provided as either a fusion partner or in trans) can include a variant RT comprising a T197X mutation in the wild type M-MLV RT of SEQ ID NO: 89 or at a corresponding amino acid position in another wild type RT polypeptide sequence, wherein “X” can be any amino acid. In certain embodiments, X is A.
• In various other embodiments, the prime editors described herein (with RT provided as either a fusion partner or in trans) can include a variant RT comprising a D200X mutation in the wild type M-MLV RT of SEQ ID NO: 89 or at a corresponding amino acid position in another wild type RT polypeptide sequence, wherein “X” can be any amino acid. In certain embodiments, X is N.
• In various other embodiments, the prime editors described herein (with RT provided as either a fusion partner or in trans) can include a variant RT comprising a H204X mutation in the wild type M-MLV RT of SEQ ID NO: 89 or at a corresponding amino acid position in another wild type RT polypeptide sequence, wherein “X” can be any amino acid. In certain embodiments, X is R.
• In various other embodiments, the prime editors described herein (with RT provided as either a fusion partner or in trans) can include a variant RT comprising a F209X mutation in the wild type M-MLV RT of SEQ ID NO: 89 or at a corresponding amino acid position in another wild type RT polypeptide sequence, wherein “X” can be any amino acid. In certain embodiments, X is N.
• In various other embodiments, the prime editors described herein (with RT provided as either a fusion partner or in trans) can include a variant RT comprising a E302X mutation in the wild type M-MLV RT of SEQ ID NO: 89 or at a corresponding amino acid position in another wild type RT polypeptide sequence, wherein “X” can be any amino acid. In certain embodiments, X is K.
• In various other embodiments, the prime editors described herein (with RT provided as either a fusion partner or in trans) can include a variant RT comprising a E302X mutation in the wild type M-MLV RT of SEQ ID NO: 89 or at a corresponding amino acid position in another wild type RT polypeptide sequence, wherein “X” can be any amino acid. In certain embodiments, X is R.
• In various other embodiments, the prime editors described herein (with RT provided as either a fusion partner or in trans) can include a variant RT comprising a T306X mutation in the wild type M-MLV RT of SEQ ID NO: 89 or at a corresponding amino acid position in another wild type RT polypeptide sequence, wherein “X” can be any amino acid. In certain embodiments, X is K.
• In various other embodiments, the prime editors described herein (with RT provided as either a fusion partner or in trans) can include a variant RT comprising a F309X mutation in the wild type M-MLV RT of SEQ ID NO: 89 or at a corresponding amino acid position in another wild type RT polypeptide sequence, wherein “X” can be any amino acid. In certain embodiments, X is N.
• In various other embodiments, the prime editors described herein (with RT provided as either a fusion partner or in trans) can include a variant RT comprising a W313X mutation in the wild type M-MLV RT of SEQ ID NO: 89 or at a corresponding amino acid position in another wild type RT polypeptide sequence, wherein “X” can be any amino acid. In certain embodiments, X is F.
• In various other embodiments, the prime editors described herein (with RT provided as either a fusion partner or in trans) can include a variant RT comprising a T330X mutation in the wild type M-MLV RT of SEQ ID NO: 89 or at a corresponding amino acid position in another wild type RT polypeptide sequence, wherein “X” can be any amino acid. In certain embodiments, X is P.
• In various other embodiments, the prime editors described herein (with RT provided as either a fusion partner or in trans) can include a variant RT comprising a L345X mutation in the wild type M-MLV RT of SEQ ID NO: 89 or at a corresponding amino acid position in another wild type RT polypeptide sequence, wherein “X” can be any amino acid. In certain embodiments, X is G.
• In various other embodiments, the prime editors described herein (with RT provided as either a fusion partner or in trans) can include a variant RT comprising a L435X mutation in the wild type M-MLV RT of SEQ ID NO: 89 or at a corresponding amino acid position in another wild type RT polypeptide sequence, wherein “X” can be any amino acid. In certain embodiments, X is G.
• In various other embodiments, the prime editors described herein (with RT provided as either a fusion partner or in trans) can include a variant RT comprising a N454X mutation in the wild type M-MLV RT of SEQ ID NO: 89 or at a corresponding amino acid position in another wild type RT polypeptide sequence, wherein “X” can be any amino acid. In certain embodiments, X is K.
• In various other embodiments, the prime editors described herein (with RT provided as either a fusion partner or in trans) can include a variant RT comprising a D524X mutation in the wild type M-MLV RT of SEQ ID NO: 89 or at a corresponding amino acid position in another wild type RT polypeptide sequence, wherein “X” can be any amino acid. In certain embodiments, X is G.
• In various other embodiments, the prime editors described herein (with RT provided as either a fusion partner or in trans) can include a variant RT comprising a E562X mutation in the wild type M-MLV RT of SEQ ID NO: 89 or at a corresponding amino acid position in another wild type RT polypeptide sequence, wherein “X” can be any amino acid. In certain embodiments, X is Q.
• In various other embodiments, the prime editors described herein (with RT provided as either a fusion partner or in trans) can include a variant RT comprising a D583X mutation in the wild type M-MLV RT of SEQ ID NO: 89 or at a corresponding amino acid position in another wild type RT polypeptide sequence, wherein “X” can be any amino acid. In certain embodiments, X is N.
• In various other embodiments, the prime editors described herein (with RT provided as either a fusion partner or in trans) can include a variant RT comprising a H594X mutation in the wild type M-MLV RT of SEQ ID NO: 89 or at a corresponding amino acid position in another wild type RT polypeptide sequence, wherein “X” can be any amino acid. In certain embodiments, X is Q.
• In various other embodiments, the prime editors described herein (with RT provided as either a fusion partner or in trans) can include a variant RT comprising a L603X mutation in the wild type M-MLV RT of SEQ ID NO: 89 or at a corresponding amino acid position in another wild type RT polypeptide sequence, wherein “X” can be any amino acid. In certain embodiments, X is W.
• In various other embodiments, the prime editors described herein (with RT provided as either a fusion partner or in trans) can include a variant RT comprising a E607X mutation in the wild type M-MLV RT of SEQ ID NO: 89 or at a corresponding amino acid position in another wild type RT polypeptide sequence, wherein “X” can be any amino acid. In certain embodiments, X is K.
• In various other embodiments, the prime editors described herein (with RT provided as either a fusion partner or in trans) can include a variant RT comprising a D653X mutation in the wild type M-MLV RT of SEQ ID NO: 89 or at a corresponding amino acid position in another wild type RT polypeptide sequence, wherein “X” can be any amino acid. In certain embodiments, X is N.
• Some exemplary reverse transcriptases that can be fused to napDNAbp proteins or provided as individual proteins according to various embodiments of this disclosure are provided below. Exemplary reverse transcriptases include variants with at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity to the following wild-type enzymes or partial enzymes:

• DESCRIPTION	SEQUENCE (VARIANT SUBSTITUTIONS RELATIVE TO WILD TYPE)
  REVERSE	TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPL
  TRANSCRIP	IIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLL
  TASE (M-	PVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYT
  MLV RT)	VLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSP
  WILD TYPE	TLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ
  	TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQ
  MOLONEY	PTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQ
  MURINE	QKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPW
  LEUKEMIA	RRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILA
  VIRUS	PHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLL
  	PLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEG
  USED IN	QRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLN
  PE1 (PRIME	VYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKR
  EDITOR 1	LSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP
  FUSION	(SEQ ID NO: 89)
  PROTEIN
  DISCLOSED
  HEREIN)
  M-MLV RT	TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPL
  D200N	IIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLL
  	PVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYT
  	VLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSP
  	TLF N EALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ
  	TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQ
  	PTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQ
  	QKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPW
  	RRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILA
  	PHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLL
  	PLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEG
  	QRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLN
  	VYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKR
  	LSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP
  	(SEQ ID NO: 106)
  M-MLV RT	TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPL
  D200N	IIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLL
  T330P	PVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYT
  	VLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSP
  	TLF N EALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ
  	TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQ
  	PTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTK P GTLFNWGPDQ
  	QKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPW
  	RRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILA
  	PHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLL
  	PLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEG
  	QRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLN
  	VYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKR
  	LSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP
  	(SEQ ID NO: 107)
  M-MLV RT	TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPL
  D200N	IIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLL
  T330P	PVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYT
  L603W	VLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSP
  	TLF N EALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ
  	TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQ
  	PTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTK P GTLFNWGPDQ
  	QKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPW
  	RRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILA
  	PHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLL
  	PLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEG
  	QRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLN
  	VYTDSRYAFATAHIHGEIYRRRG W LTSEGKEIKNKDEILALLKALFLPKR
  	LSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP
  	(SEQ ID NO: 108)
  M-MLV RT	TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPL
  E69K	IIPLKATSTPVSIKQYPMSQ K ARLGIKPHIQRLLDQGILVPCQSPWNTPLL
  D200N	PVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYT
  T330P	VLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSP
  L603W	TLF N EALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ
  	TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQ
  	PTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTK P GTLFNWGPDQ
  	QKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPW
  	RRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILA
  	PHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLL
  	PLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEG
  	QRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLN
  	VYTDSRYAFATAHIHGEIYRRRG W LTSEGKEIKNKDEILALLKALFLPKR
  	LSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP
  	(SEQ ID NO: 109)
  M-MLV RT	TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPL
  D200N	IIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLL
  T330P	PVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYT
  L603W	VLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSP
  E302R	TLF N EALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ
  	TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQ
  	PTPKTPRQLR R FLGTAGFCRLWIPGFAEMAAPLYPLTK P GTLFNWGPDQ
  	QKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPW
  	RRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILA
  	PHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLL
  	PLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEG
  	QRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLN
  	VYTDSRYAFATAHIHGEIYRRRG W LTSEGKEIKNKDEILALLKALFLPKR
  	LSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP
  	(SEQ ID NO: 110)
  M-MLV RT	TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPL
  D200N	IIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLL
  T330P	PVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYT
  L603W	VLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSP
  E607K	TLF N EALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ
  	TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQ
  	PTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTK P GTLFNWGPDQ
  	QKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPW
  	RRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILA
  	PHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLL
  	PLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEG
  	QRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLN
  	VYTDSRYAFATAHIHGEIYRRRG W LTS K GKEIKNKDEILALLKALFLPK
  	RLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP
  	(SEQ ID NO: 111)
  M-MLV RT	TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPL
  D200N	IIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLL
  T330P	PVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGPPPSHQWYT
  L603W	VLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSP
  L139P	TLF N EALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ
  	TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQ
  	PTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTK P GTLFNWGPDQ
  	QKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPW
  	RRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILA
  	PHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLL
  	PLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEG
  	QRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLN
  	VYTDSRYAFATAHIHGEIYRRRG W LTSEGKEIKNKDEILALLKALFLPKR
  	LSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP
  	(SEQ ID NO: 112)
  M-MLV RT	TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPL
  D200N	IIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLL
  T330P	PVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYT
  L603W	VLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSP
  L435G	TLF N EALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ
  	TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQ
  	PTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTK P GTLFNWGPDQ
  	QKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPW
  	RRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVI G A
  	PHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLL
  	PLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEG
  	QRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLN
  	VYTDSRYAFATAHIHGEIYRRRG W LTSEGKEIKNKDEILALLKALFLPKR
  	LSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP
  	(SEQ ID NO: 113)
  M-MLV RT	TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPL
  D200N	IIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLL
  T330P	PVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYT
  L603W	VLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSP
  N454K	TLF N EALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ
  	TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQ
  	PTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTK P GTLFNWGPDQ
  	QKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPW
  	RRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILA
  	PHAVEALVKQPPDRWLS K ARMTHYQALLLDTDRVQFGPVVALNPATLL
  	PLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEG
  	QRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLN
  	VYTDSRYAFATAHIHGEIYRRRG W LTSEGKEIKNKDEILALLKALFLPKR
  	LSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP
  	(SEQ ID NO: 114)
  M-MLV RT	TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPL
  D200N	IIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLL
  T330P	PVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYT
  L603W	VLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSP
  T306K	TLF N EALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ
  	TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQ
  	PTPKTPRQLREFLG K AGFCRLWIPGFAEMAAPLYPLTK P GTLFNWGPD
  	QQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGP
  	WRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVIL
  	APHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPAT
  	LLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQ
  	EGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKL
  	NVYTDSRYAFATAHIHGEIYRRRG W LTSEGKEIKNKDEILALLKALFLP
  	KRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSS
  	P (SEQ ID NO: 115)
  M-MLV RT	TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPL
  D200N	IIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLL
  T330P	PVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYT
  L603W	VLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSP
  W313F	TLF N EALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ
  	TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQ
  	PTPKTPRQLREFLGTAGFCRL F IPGFAEMAAPLYPLTK P GTLFNWGPDQ
  	QKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPW
  	RRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILA
  	PHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLL
  	PLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEG
  	QRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLN
  	VYTDSRYAFATAHIHGEIYRRRG W LTSEGKEIKNKDEILALLKALFLPKR
  	LSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP
  	(SEQ ID NO: 116)
  M-MLV RT	TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPL
  D200N	IIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLL
  T330P	PVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYT
  L603W	VLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSP
  D524G	TLF N EALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ
  E562Q	TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQ
  D583N	PTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTK P GTLFNWGPDQ
  	QKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPW
  	RRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILA
  	PHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLL
  	PLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYT G GSSLLQEG
  	QRKAGAAVTTETEVIWAKALPAGTSAQRA Q LIALTQALKMAEGKKLN
  	VYT N SRYAFATAHIHGEIYRRRG W LTSEGKEIKNKDEILALLKALFLPKR
  	LSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP
  	(SEQ ID NO: 117)
  M-MLV RT	TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPL
  D200N	IIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLL
  T330P	PVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYT
  L603W	VLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSP
  E302R	TLF N EALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ
  W313F	TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQ
  	PTPKTPRQLR R FLGTAGFCRL F IPGFAEMAAPLYPLTK P GTLFNWGPDQ
  	QKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPW
  	RRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILA
  	PHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLL
  	PLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEG
  	QRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLN
  	VYTDSRYAFATAHIHGEIYRRRG W LTSEGKEIKNKDEILALLKALFLPKR
  	LSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP
  	(SEQ ID NO: 118)
  M-MLV RT	TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPL
  D200N	IIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLL
  T330P	PVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSG P PPSHQWYT
  L603W	VLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSP
  E607K	TLF N EALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ
  L139P	TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQ
  	PTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTK P GTLFNWGPDQ
  	QKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPW
  	RRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILA
  	PHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLL
  	PLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEG
  	QRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLN
  	VYTDSRYAFATAHIHGEIYRRRG W LTS K GKEIKNKDEILALLKALFLPK
  	RLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP
  	(SEQ ID NO: 119)
  M-MLV RT	TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPL
  P51L S67K	II L LKATSTPVSIKQYPM K QEARLGIKPHIQRLLDQGILVPCQSPWNTPL
  T197A	LPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWY
  H204R	TVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKN
  E302K	SP A LFDEAL R RDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRAL
  F309N	LQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVM
  W313F	GQPTPKTPRQLR K FLGTAG N CRL F IPGFAEMAAPLYPLTK P GTLFNWGP
  T330P	DQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLG
  L435G	PWRRPVAYLS KKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVI
  N454K	GAPHAVEALVKQPPDRWLS K ARMTHYQALLLDTDRVQFGPVVALNPA
  D524G	TLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYT G GSSLL
  D583N	QEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGK
  H594Q	KLNVYT N SRYAFATAHI Q GEIYRRRGLLTSEGKEIKNKDEILALLKALFL
  D653N	PKRLSIIHCPGHQKGHSAEARGNRMA N QAARKAAITETPDTSTLLIENS
  	SP (SEQ ID NO: 120)
  M-MLV RT	TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPL
  P51L S67K	II L LKATSTPVSI K QYPMKQEARLGIKPHIQRLLDQGILVPCQSPWNTPL
  T197A	LPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWY
  D200N	TVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKN
  H204R	SP A LF N EAL R RDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRAL
  E302K	LQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVM
  F309N	GQPTPKTPRQLR K FLGTAG N CRL F IPGFAEMAAPLYPLTK P GTLFNWGP
  W313F	DQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLG
  T330P L345G	PWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVI
  N454K	G APHAVEALVKQPPDRWLS K ARMTHYQALLLDTDRVQFGPVVALNPA
  D524G	TLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYT G GSSLL
  D583N	QEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGK
  H594Q	KLNVYT N SRYAFATAHI Q GEIYRRRGLLTSEGKEIKNKDEILALLKALFL
  D653N	PKRLSIIHCPGHQKGHSAEARGNRMA N QAARKAAITETPDTSTLLIENS
  	SP (SEQ ID NO: 121)
  M-MLV RT	TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPL
  D200N T330P	IIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLL
  L603W	PVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYT
  T306K	VLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSP
  W313F	TLF N EALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ
  	TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQ
  IN PE2	PTPKTPRQLREFLG K AGFCRL F IPGFAEMAAPLYPLTK P GTLFNWGPDQ
  	QKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPW
  	RRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILA
  	PHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLL
  	PLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEG
  	QRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLN
  	VYTDSRYAFATAHIHGEIYRRRG W LTSEGKEIKNKDEILALLKALFLPKR
  	LSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP
  	(SEQ ID NO: 122)

• The prime editor (PE) system described here contemplates any publicly-available reverse transcriptase described or disclosed in any of the following U.S. patents (each of which are incorporated by reference in their entireties): U.S. Pat. Nos. 10,202,658; 10,189,831; 10,150,955; 9,932,567; 9,783,791; 9,580,698; 9,534,201; and 9,458,484, and any variant thereof that can be made using known methods for installing mutations, or known methods for evolving proteins. The following references describe reverse transcriptases in art. Each of their disclosures are incorporated herein by reference in their entireties.
• Herzig, E., Voronin, N., Kucherenko, N. & Hizi, A. A Novel Leu92 Mutant of HIV-1 Reverse Transcriptase with a Selective Deficiency in Strand Transfer Causes a Loss of Viral Replication. J. Virol. 89, 8119-8129 (2015).
• Mohr, G. et al. A Reverse Transcriptase-Cas1 Fusion Protein Contains a Cas6 Domain Required for Both CRISPR RNA Biogenesis and RNA Spacer Acquisition. Mol. Cell 72, 700-714.e8 (2018).
• Zhao, C., Liu, F. & Pyle, A. M. An ultraprocessive, accurate reverse transcriptase encoded by a metazoan group II intron. RNA 24, 183-195 (2018).
• Zimmerly, S. & Wu, L. An Unexplored Diversity of Reverse Transcriptases in Bacteria. Microbiol Spectr 3, MDNA3-0058-2014 (2015).
• Ostertag, E. M. & Kazazian Jr, H. H. Biology of Mammalian L1 Retrotransposons. Annual Review of Genetics 35, 501-538 (2001).
• Perach, M. & Hizi, A. Catalytic Features of the Recombinant Reverse Transcriptase of Bovine Leukemia Virus Expressed in Bacteria. Virology 259, 176-189 (1999).
• Lim, D. et al. Crystal structure of the moloney murine leukemia virus RNase H domain. J. Virol. 80, 8379-8389 (2006).
• Zhao, C. & Pyle, A. M. Crystal structures of a group II intron maturase reveal a missing link in spliceosome evolution. Nature Structural & Molecular Biology 23, 558-565 (2016).
• Griffiths, D. J. Endogenous retroviruses in the human genome sequence. Genome Biol. 2, REVIEWS1017 (2001).
• Baranauskas, A. et al. Generation and characterization of new highly thermostable and processive M-MuLV reverse transcriptase variants. Protein Eng Des Sel 25, 657-668 (2012).
• Zimmerly, S., Guo, H., Perlman, P. S. & Lambowltz, A. M. Group II intron mobility occurs by target DNA-primed reverse transcription. Cell 82, 545-554 (1995).
• Feng, Q., Moran, J. V., Kazazian, H. H. & Boeke, J. D. Human L1 retrotransposon encodes a conserved endonuclease required for retrotransposition. Cell 87, 905-916 (1996).
• Berkhout, B., Jebbink, M. & Zsíros, J. Identification of an Active Reverse Transcriptase Enzyme Encoded by a Human Endogenous HERV-K Retrovirus. Journal of Virology 73, 2365-2375 (1999).
• Kotewicz, M. L., Sampson, C. M., D'Alessio, J. M. & Gerard, G. F. Isolation of cloned Moloney murine leukemia virus reverse transcriptase lacking ribonuclease H activity. Nucleic Acids Res 16, 265-277 (1988).
• Arezi, B. & Hogrefe, H. Novel mutations in Moloney Murine Leukemia Virus reverse transcriptase increase thermostability through tighter binding to template-primer. Nucleic Acids Res 37, 473-481 (2009).
• Blain, S. W. & Goff, S. P. Nuclease activities of Moloney murine leukemia virus reverse transcriptase. Mutants with altered substrate specificities. J. Biol. Chem. 268, 23585-23592 (1993).
• Xiong, Y. & Eickbush, T. H. Origin and evolution of retroelements based upon their reverse transcriptase sequences. EMBO J 9, 3353-3362 (1990).
• Herschhorn, A. & Hizi, A. Retroviral reverse transcriptases. Cell. Mol. Life Sci. 67, 2717-2747 (2010).
• Taube, R., Loya, S., Avidan, O., Perach, M. & Hizi, A. Reverse transcriptase of mouse mammary tumour virus: expression in bacteria, purification and biochemical characterization. Biochem. J. 329 (Pt 3), 579-587 (1998).
• Liu, M. et al. Reverse Transcriptase-Mediated Tropism Switching in Bordetella Bacteriophage. Science 295, 2091-2094 (2002).
• Luan, D. D., Korman, M. H., Jakubczak, J. L. & Eickbush, T. H. Reverse transcription of R2Bm RNA is primed by a nick at the chromosomal target site: a mechanism for non-LTR retrotransposition. Cell 72, 595-605 (1993).
• Nottingham, R. M. et al. RNA-seq of human reference RNA samples using a thermostable group II intron reverse transcriptase. RNA 22, 597-613 (2016).
• Telesnitsky, A. & Goff, S. P. RNase H domain mutations affect the interaction between Moloney murine leukemia virus reverse transcriptase and its primer-template. Proc. Natl. Acad. Sci. U.S.A. 90, 1276-1280 (1993).
• Halvas, E. K., Svarovskaia, E. S. & Pathak, V. K. Role of Murine Leukemia Virus Reverse Transcriptase Deoxyribonucleoside Triphosphate-Binding Site in Retroviral Replication and In Vivo Fidelity. Journal of Virology 74, 10349-10358 (2000).
• Nowak, E. et al. Structural analysis of monomeric retroviral reverse transcriptase in complex with an RNA/DNA hybrid. Nucleic Acids Res 41, 3874-3887 (2013).
• Stamos, J. L., Lentzsch, A. M. & Lambowitz, A. M. Structure of a Thermostable Group II Intron Reverse Transcriptase with Template-Primer and Its Functional and Evolutionary Implications. Molecular Cell 68, 926-939.e4 (2017).
• Das, D. & Georgiadis, M. M. The Crystal Structure of the Monomeric Reverse Transcriptase from Moloney Murine Leukemia Virus. Structure 12, 819-829 (2004).
• Avidan, O., Meer, M. E., Oz, I. & Hizi, A. The processivity and fidelity of DNA synthesis exhibited by the reverse transcriptase of bovine leukemia virus. European Journal of Biochemistry 269, 859-867 (2002).
• Gerard, G. F. et al. The role of template-primer in protection of reverse transcriptase from thermal inactivation. Nucleic Acids Res 30, 3118-3129 (2002).
• Monot, C. et al. The Specificity and Flexibility of L1 Reverse Transcription Priming at Imperfect T-Tracts. PLOS Genetics 9, e1003499 (2013).
• Mohr, S. et al. Thermostable group II intron reverse transcriptase fusion proteins and their use in cDNA synthesis and next-generation RNA sequencing. RNA 19, 958-970 (2013).
• Any of the references noted above which relate to reverse transcriptases are hereby incorporated by reference in their entireties, if not already stated so.
[4] PE Fusion Proteins
• The prime editor (PE) system described herein contemplate fusion proteins comprising a napDNAbp and a polymerase (e.g., DNA-dependent DNA polymerase or RNA-dependent DNA polymerase, such as, reverse transcriptase), and optionally joined by a linker. The application contemplates any suitable napDNAbp and polymerase (e.g., DNA-dependent DNA polymerase or RNA-dependent DNA polymerase, such as, reverse transcriptase) to be combined in a single fusion protein. Examples of napDNAbps and polymerases (e.g., DNA-dependent DNA polymerase or RNA-dependent DNA polymerase, such as, reverse transcriptase) are each defined herein. Since polymerases are well-known in the art, and the amino acid sequences are readily available, this disclosure is not meant in any way to be limited to those specific polymerases identified herein.
• In various embodiments, the fusion proteins may comprise any suitable structural configuration. For example, the fusion protein may comprise from the N-terminus to the C-terminus direction, a napDNAbp fused to a polymerase (e.g., DNA-dependent DNA polymerase or RNA-dependent DNA polymerase, such as, reverse transcriptase). In other embodiments, the fusion protein may comprise from the N-terminus to the C-terminus direction, a polymerase (e.g., a reverse transcriptase) fused to a napDNAbp. The fused domain may optionally be joined by a linker, e.g., an amino acid sequence. In other embodiments, the fusion proteins may comprise the structure NH₂-[napDNAbp]-[polymerase]-COOH; or NH₂-[polymerase]-[napDNAbp]-COOH, wherein each instance of “]-[” indicates the presence of an optional linker sequence. In embodiments wherein the polymerase is a reverse transcriptase, the fusion proteins may comprise the structure NH₂-[napDNAbp]-[RT]-COOH; or NH₂-[RT]-[napDNAbp]-COOH, wherein each instance of “]-[” indicates the presence of an optional linker sequence.
• An exemplary fusion protein is depicted in FIG. 14 , which shows a fusion protein comprising an MLV reverse transcriptase (“MLV-RT”) fused to a nickase Cas9 (“Cas9(H840A)”) via a linker sequence. This example is not intended to limit scope of fusion proteins that may be utilized for the prime editor (PE) system described herein.
• In various embodiments, the prime editor fusion protein may have the following amino acid sequence (referred to herein as “PE1”), which includes a Cas9 variant comprising an H840A mutation (i.e., a Cas9 nickase) and an M-MLV RT wild type, as well as an N-terminal NLS sequence (19 amino acids) and an amino acid linker (32 amino acids) that joins the C-terminus of the Cas9 nickase domain to the N-terminus of the RT domain. The PE1 fusion protein has the following structure: [NLS]-[Cas9(H840A)]-[linker]-[MMLV_RT(wt)]. The amino acid sequence of PE1 and its individual components are as follows:

• DESCRIPTION	SEQUENCE
  PE1 FUSION	MKRTADGSEFESPKKKRKV DKKYSIGLDIGTNSVGWAVITDEYKVPS
  PROTEIN	KKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRR
  	KNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI
  	VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHF
  CAS9(H840A	LIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSAR
  )-	LSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDA
  MMLV_RT(	KLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRV
  WT)	NTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQ
  	SKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
  	KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFR
  	IPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIE
  	RMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPA
  	FLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVE
  	DRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREM
  	IEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSG
  	KTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHE
  	HIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQ
  	TTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
  	YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTR
  	SDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKA
  	ERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDK
  	LIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVG
  	TALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYS
  	NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKV
  	LSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKY
  	GGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNP
  	IDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKG
  	NELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDE
  	IIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLT
  	NLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLS
  	QLGGD SGGSSGGSSGSETPGTSESATPESSGGSSGGSS TLNIEDEYRLH
  	ETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQ
  	YPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQD
  	LREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQ
  	PLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHP
  	DLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQV
  	KYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPG
  	FAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFE
  	LFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAV
  	LTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTD
  	RVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADH
  	TWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALK
  	MAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKAL
  	FLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSS
  	P SGGSKRTADGSEFEPKKKRKV (SEQ ID NO: 123)
  	KEY:
  	NUCLEAR LOCALIZATION SEQUENCE (NLS) TOP:(SEQ ID NO: 124),
  	BOTTOM: (SEQ ID NO: 133)
  	CAS9(H840A) (SEQ ID NO: 126)
  	33-AMINO ACID LINKER  (SEQ ID NO: 127)
  	M-MLV REVERSE TRANSCRIPTASE (SEQ ID NO: 128)
  PE1 - N-	MKRTADGSEFESPKKKRKV (SEQ ID NO: 124)
  TERMINAL
  NLS
  PE1 - CAS 9	DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA
  (H840A)	LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFH
  (MET	RLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDK
  MINUS)	ADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFE
  	ENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLG
  	LTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKN
  	LSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPE
  	KYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLN
  	REDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKIL
  	TFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIE
  	RMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLS
  	GEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVETSGVEDRFNA
  	SLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTY
  	AHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSD
  	GFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKK
  	GILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMK
  	RIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDIN
  	RLSDYDVD A IVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKM
  	KNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQI
  	TKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKV
  	REINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMI
  	AKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIV
  	WDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIA
  	RKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIM
  	ERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAG
  	ELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHY
  	LDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLT
  	NLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLG
  	GD (SEQ ID NO: 130)
  PE1 -	SGGSSGGSSGSETPGTSESATPESSGGSSGGSS (SEQ ID NO: 127)
  LINKER
  BETWEEN
  CAS 9
  DOMAIN
  AND RT
  DOMAIN (33
  AMINO
  ACIDS)
  PE1 - M-	TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPL
  MLV RT	IIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLL
  	PVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYT
  	VLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSP
  	TLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ
  	TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQ
  	PTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQ
  	QKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPW
  	RRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILA
  	PHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLL
  	PLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEG
  	QRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLN
  	VYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKR
  	LSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP
  	(SEQ ID NO: 132)
  PE1 - C-	SGGSKRTADGSEFEPKKKRKV (SEQ ID NO: 133)
  TERMINAL
  NLS

• In another embodiment, the prime editor fusion protein may have the following amino acid sequence (referred to herein as “PE2”), which includes a Cas9 variant comprising an H840A mutation (i.e., a Cas9 nickase) and an M-MLV RT comprising mutations D200N, T330P, L603W, T306K, and W313F, as well as an N-terminal NLS sequence (19 amino acids) and an amino acid linker (33 amino acids) that joins the C-terminus of the Cas9 nickase domain to the N-terminus of the RT domain. The PE2 fusion protein has the following structure: [NLS]-[Cas9(H840A)]-[linker]-[MMLV_RT(D200N)(T330P)(L603W)(T306K)(W313F)]. The amino acid sequence of PE2 is as follows:

• PE2 FUSION	MKRTADGSEFESPKKKRKV DKKYSIGLDIGTNSVGWAVITDEYKVPS
  PROTEIN	KKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRR
  	KNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI
  CAS9(H840A	VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHF
  )-MMLV_RT	LIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSAR
  D200N	LSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDA
  T330P	KLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRV
  L603W	NTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQ
  T306K	SKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
  W313F	KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFR
  	IPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIE
  	RMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPA
  	FLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVE
  	DRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREM
  	IEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSG
  	KTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHE
  	HIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQ
  	TTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
  	YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTR
  	SDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKA
  	ERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDK
  	LIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVG
  	TALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYS
  	NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKV
  	LSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKY
  	GGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNP
  	IDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKG
  	NELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDE
  	IIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLT
  	NLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLS
  	QLGGD SGGSSGGSSGSETPGTSESATPESSGGSSGGSS TLNIEDEYRLH
  	ETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQ
  	YPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQD
  	LREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQ
  	PLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHP
  	DLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQV
  	KYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGKAGFCRLFIPG
  	FAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFE
  	LFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAV
  	LTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTD
  	RVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADH
  	TWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALK
  	MAEGKKLNVYTDSRYAFATAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKA
  	LFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENS
  	SP SGGSKRTADGSEFEPKKKRKV (SEQ ID NO: 134)
  	KEY:
  	NUCLEAR LOCALIZATION SEQUENCE (NLS) TOP:(SEQ ID NO: 124),
  	BOTTOM: (SEQ ID NO: 133)
  	CAS9(H840A) (SEQ ID NO: 137)
  	33-AMINO ACID LINKER  (SEQ ID NO: 127)
  	M-MLV REVERSE TRANSCRIPTASE (SEQ ID NO: 139)
  PE2 - N-	MKRTADGSEFESPKKKRKV (SEQ ID NO: 124)
  TERMINAL
  NLS
  PE2 - CAS 9	DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA
  (H840A)	LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFH
  (MET	RLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDK
  MINUS)	ADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFE
  	ENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLG
  	LTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKN
  	LSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPE
  	KYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLN
  	REDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKIL
  	TFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIE
  	RMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLS
  	GEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNA
  	SLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTY
  	AHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSD
  	GFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKK
  	GILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMK
  	RIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDIN
  	RLSDYDVD A IVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKM
  	KNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQI
  	TKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKV
  	REINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMI
  	AKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIV
  	WDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIA
  	RKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIM
  	ERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAG
  	ELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHY
  	LDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLT
  	NLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLG
  	GD (SEQ ID NO: 141)
  PE2 -	SGGSSGGSSGSETPGTSESATPESSGGSSGGSS (SEQ ID NO: 127)
  LINKER
  BETWEEN
  CAS 9
  DOMAIN
  AND RT
  DOMAIN (33
  AMINO
  ACIDS)
  PE2 -	TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPL
  MMLV_RT	IIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLL
  D200N	PVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYT
  T330P	VLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSP
  L603W	TLF N EALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ
  T306K	TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQ
  W313F	PTPKTPRQLREFLG K AGFCRL F IPGFAEMAAPLYPLTK P GTLFNWGPDQ
  	QKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPW
  	RRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILA
  	PHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLL
  	PLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEG
  	QRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLN
  	VYTDSRYAFATAHIHGEIYRRRG W LTSEGKEIKNKDEILALLKALFLPKR
  	LSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP
  	(SEQ ID NO: 143)
  PE2 - C-	SGGSKRTADGSEFEPKKKRKV (SEQ ID NO: 133)
  TERMINAL
  NLS

• In still other embodiments, the prime editor fusion protein may have the following amino acid sequences:

• PE FUSION	MKRTADGSEFESPKKKRKV TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQ
  PROTEIN	AWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLD
  	QGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYN
  MMLV_RT(	LLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLT
  WT)-32AA-	WTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELD
  CAS9(H840A	CQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARK
  )	ETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNW
  	GPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGP
  	WRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPH
  	AVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEE
  	GLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAA
  	VTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAH
  	IHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAE
  	ARGNRMADQAARKAAITETPDTSTLLIENSSP SGGSSGGSSGSETPGTSES
  	ATPESSGGSSGGSSDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKV
  	LGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRIC
  	YLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVA
  	YHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGD
  	LNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSR
  	RLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQL
  	SKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEIT
  	KAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNG
  	YAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRT
  	FDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYV
  	GPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTN
  	FDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSG
  	EQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFN
  	ASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
  	LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTIL
  	DFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIAN
  	LAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQ
  	KGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYL
  	QNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDK
  	NRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERG
  	GLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIRE
  	VKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALI
  	KKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIM
  	NFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSM
  	PQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGF
  	DSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDF
  	LEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
  	ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQ
  	ISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLG
  	APAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLG
  	GD SGGSKRTADGSEFEPKKKRKV (SEQ ID NO: 145)
  	KEY:
  	NUCLEAR LOCALIZATION SEQUENCE (NLS) TOP:(SEQ ID NO: 124),
  	BOTTOM: (SEQ ID NO: 133)
  	CAS9(H840A) (SEQ ID NO: 147)
  	33-AMINO ACID LINKER  (SEQ ID NO: 127)
  	M-MLV REVERSE TRANSCRIPTASE (SEQ ID NO: 149)
  PE FUSION	MKRTADGSEFESPKKKRKV TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQ
  PROTEIN	AWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLD
  	QGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYN
  MMLV_RT(	LLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLT
  WT)-60AA-	WTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELD
  CAS 9(H840A	CQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARK
  )	ETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNW
  	GPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGP
  	WRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPH
  	AVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEE
  	GLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAA
  	VITETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAH
  	IHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAE
  	ARGNRMADQAARKAAITETPDTSTLLIENSSPS GGSSGGSSGSETPGTSES
  	ATPESAGSYPYDVPDYAGSAAPAAKKKKLDGSGSGGSSGGSDKKYS
  	IGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLF
  	DSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFH
  	RLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDST
  	DKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTY
  	NQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLF
  	GNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGD
  	QYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQD
  	LTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIK
  	PILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAIL
  	RRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKS
  	EETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLY
  	EYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVT
  	VKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFL
  	DNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKR
  	RRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHD
  	DSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVD
  	ELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKE
  	LGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSD
  	YDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKN
  	YWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQ
  	ITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQF
  	YKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYD
  	VRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIE
  	TNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKES
  	ILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSK
  	KLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYS
  	LFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKG
  	SPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAY
  	NKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKE
  	VLDATLIHQSITGLYETRIDLSQLGGD SGGSKRTADGSEFEPKKKRKV
  	(SEQ ID NO: 150)
  	KEY:
  	NUCLEAR LOCALIZATION SEQUENCE (NLS) TOP:(SEQ ID NO: 124),
  	BOTTOM: (SEQ ID NO: 133)
  	CAS9(H840A)(SEQ ID NO: 153)
  	AMINO ACID LINKER
  	M-MLV REVERSE TRANSCRIPTASE
  PE FUSION	MKRTADGSEFESPKKKRKV DKKYSIGLDIGTNSVGWAVITDEYKVPS
  PROTEIN	KKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRR
  	KNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI
  CAS 9 (H840A	VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHF
  )-FEN1-	LIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSAR
  MMLV_RT	LS KSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDA
  D200N	KLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRV
  T330P	NTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQ
  L603W	SKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
  T306K	KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFR
  W313F	IPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIE
  	RMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPA
  	FLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVE
  	DRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREM
  	IEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSG
  	KTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHE
  	HIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQ
  	TTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
  	YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTR
  	SDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKA
  	ERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDK
  	LIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVG
  	TALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYS
  	NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKV
  	LSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKY
  	GGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNP
  	IDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKG
  	NELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDE
  	IIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLT
  	NLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLS
  	QLGGD SGGSSGGSSGSETPGTSESATPESSGGSSGGSS GIQGLAKLIA
  	DVAPSAIRENDIKSYFGRKVAIDASMSIYQFLIAVRQGGDVLQNEEGETT
  	SHLMGMFYRTIRMMENGIKPVYVFDGKPPQLKSGELAKRSERRAEAE
  	KQLQQAQAAGAEQEVEKFTKRLVKVTKQHNDECKHLLSLMGIPYLDA
  	PSEAEASCAALVKAGKVYAAATEDMDCLTFGSPVLMRHLTASEAKKLP
  	IQEFHLSRILQELGLNQEQFVDLCILLGSDYCESIRGIGPKRAVDLIQKHK
  	SIEEIVRRLDPNKYPVPENWLHKEAHQLFLEPEVLDPESVELKWSEPNE
  	EELIKFMCGEKQFSEERIRSGVKRLSKSRQGSTQGRLDDFFKVTGSLSS
  	AKRKEPEPKGSTKKKAKTGAAGKFKRGK SGGSSGGSSGSETPGTSES
  	ATPESSGGSSGGSS TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETG
  	GMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPC
  	QSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPP
  	SHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQG
  	FKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRA
  	LLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQP
  	TPKTPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQK
  	AYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVA
  	YLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALV
  	KQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHN
  	CLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTET
  	EVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEI
  	YRRRGWLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGN
  	RMADQAARKAAITETPDTSTLLIENSSP SGGSKRTADGSEFEPKKKRKV
  	(SEQ ID NO: 154)
  	KEY:
  	NUCLEAR LOCALIZATION SEQUENCE (NLS) TOP:(SEQ ID NO: 124),
  	BOTTOM: (SEQ ID NO: 133)
  	CAS9(H840A) (SEQ ID NO: 157)
  	33-AMINO ACID LINKER 1  (SEQ ID NO: 127)
  	M-MLV REVERSE TRANSCRIPTASE (SEQ ID NO: 159)
  	33-AMINO ACID LINKER 2  (SEQ ID NO: 127)
  	FEN1 (SEQ ID NO: 161)

• In other embodiments, the prime editor fusion proteins can be based on SaCas9 or on SpCas9 nickases with altered PAM specificities, such as the following exemplary sequences:

• SACAS9-M-MLV	MKRTADGSEFESPKKKRKVGKRNYILGLDIGITSVGYGIIDYETR
  RT PRIME EDITOR	DVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQRVKK
  	LLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLA
  	KRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLE
  	RLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFID
  	TYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPE
  	ELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQIIEN
  	VFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYH
  	DIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQE
  	EIEQISNLKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKL
  	VPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYG
  	LPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGK
  	ENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHII
  	PRSVSFDNSFNNKVLVKQEEASKKGNRTPFQYLSSSDSKISYETF
  	KKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVD
  	TRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFK
  	KERNKGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMF
  	EEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKK
  	PNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLIN
  	KSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGN
  	YLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVK
  	LSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYE
  	EAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIE
  	VNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNL
  	YEVKSKKHPQIIKKGSGGSSGGSSGSETPGTSESATPESSGGSSG
  	GSSTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGL
  	AVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGIL
  	VPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVP
  	NPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWR
  	DPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDL
  	ILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQ
  	ICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREF
  	LGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEI
  	KQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRR
  	PVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVI
  	LAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVV
  	ALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHT
  	WYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAEL
  	IALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSE
  	GKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRM
  	ADQAARKAAITETPDTSTLLIENSSPSGGSKRTADGSEFEPKKKR
  	KV (SEQ ID NO: 162)
  SPCAS9(H840A)-	MKRTADGSEFESPKKKRKVDKKYSIGLDIGTNSVGWAVITDEY
  VRQR-MALONEY	KVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTAR
  MURINE	RRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKH
  LEUKEMIA VIRUS	ERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLAL
  REVERSE	AHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPIN
  TRANSCRIPTASE	ASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSL
  PRIME EDITOR	GLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADL
  	FLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTL
  	LKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPI
  	LEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAIL
  	RRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTR
  	KSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKH
  	SLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKT
  	NRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLL
  	KIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFD
  	DKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDG
  	FANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSP
  	AIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQ
  	KNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQN
  	GRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSD
  	KNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKA
  	ERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDEND
  	KLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNA
  	VVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATA
  	KYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRD
  	FATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARK
  	KDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
  	TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRK
  	RMLASARELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQ
  	KQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHR
  	DKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRSTKEVL
  	DATLIHQSITGLYETRIDLSQLGGDSGGSSGGSSGSETPGTSESAT
  	PESSGGSSGGSSTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQA
  	WAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKP
  	HIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVN
  	KRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPT
  	SQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDL
  	ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLG
  	YRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPT
  	PKTPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWG
  	PDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVL
  	TQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAG
  	KLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT
  	DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTD
  	QPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPA
  	GTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIY
  	RRRGWLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHS
  	AEARGNRMADQAARKAAITETPDTSTLLIENSSPSGGSKRTADG
  	SEFEPKKKRKV (SEQ ID NO: 163)
  SPCAS9(H840A)-	MKRTADGSEFESPKKKRKVDKKYSIGLDIGTNSVGWAVITDEY
  VRER-MALONEY	KVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTAR
  MURINE	RRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKH
  LEUKEMIA VIRUS	ERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLAL
  REVERSE	AHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPIN
  TRANSCRIPTASE	ASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSL
  PRIME EDITOR	GLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADL
  	FLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTL
  	LKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPI
  	LEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAIL
  	RRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTR
  	KSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKH
  	SLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKT
  	NRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLL
  	KIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFD
  	DKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDG
  	FANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSP
  	AIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQ
  	KNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQN
  	GRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSD
  	KNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKA
  	ERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDEND
  	KLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNA
  	VVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATA
  	KYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRD
  	FATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARK
  	KDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
  	TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRK
  	RMLASARELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQ
  	KQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHR
  	DKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKEYRSTKEVL
  	DATLIHQSITGLYETRIDLSQLGGDSGGSSGGSSGSETPGTSESAT
  	PESSGGSSGGSSTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQA
  	WAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKP
  	HIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVN
  	KRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPT
  	SQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDL
  	ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLG
  	YRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPT
  	PKTPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWG
  	PDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVL
  	TQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAG
  	KLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT
  	DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTD
  	QPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPA
  	GTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIY
  	RRRGWLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHS
  	AEARGNRMADQAARKAAITETPDTSTLLIENSSPSGGSKRTADG
  	SEFEPKKKRKV (SEQ ID NO: 164)

• In yet other embodiments, the prime editor fusion proteins contemplated herein may include a Cas9 nickase (e.g., Cas9 (H840A)) fused to a truncated version of M-MLV reverse transcriptase. In this embodiment, the reverse transcriptase also contains 4 mutations (D200N, T306K, W313F, T330P; noting that the L603W mutation present in PE2 is no longer present due to the truncation). The DNA sequence encoding this truncated editor is 522 bp smaller than PE2, and therefore makes its potentially useful for applications where delivery of the DNA sequence is challenging due to its size (i.e. adeno-associated virus and lentivirus delivery). This embodiment is referred to as Cas9(H840A)-MMLV-RT(trunc) or “PE2-short” or “PE2-trunc” and has the following amino acid sequence:

• CAS9(H840A)-	MKRTADGSEFESPKKKRKV DKKYSIGLDIGTNSVGWAVITDE
  MMLV-	YKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLK
  RT(TRUNC) OR	RTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLV
  PE2-SHORT	EEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDK
  	ADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQ
  	TYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGE
  	KKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDD
  	DLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKA
  	PLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSK
  	NGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRED
  	LLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREK
  	IEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEV
  	VDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYN
  	ELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVK
  	QLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDK
  	DFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDK
  	VMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSD
  	GFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANL
  	AGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQ
  	TTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNE
  	KLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDD
  	SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAK
  	LITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
  	AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQF
  	YKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDY
  	KVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLAN
  	GEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIV
  	KKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDS
  	PTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKN
  	PIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAG
  	ELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFV
  	EQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKP
  	IREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
  	DATLIHQSITGLYETRIDLSQLGGD SGGSSGGSSGSETPGTSE
  	SATPESSGGSSGGSS TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQ
  	AWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHI
  	QRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVE
  	DIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFA
  	FEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQH
  	PDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQI
  	CQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGK
  	AGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQAL
  	LTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSK
  	KLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEA
  	LVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLP
  	EEGLQHNCLDNSRLIN SGGSKRTADGSEFEPKKKRKV (SEQ ID NO: 765)
  	KEY:
  	NUCLEAR LOCALIZATION SEQUENCE (NLS) TOP:(SEQ ID NO:
  	124), BOTTOM: (SEQ ID NO: 133)
  	CAS9(H840A) (SEQ ID NO: 157)
  	33-AMINO ACID LINKER 1  (SEQ ID NO: 127)
  	M-MLV TRUNCATED REVERSE TRANSCRIPTASE (SEQ ID NO: 766)

• See FIG. 75 , which provides a bar graph comparing the efficiency (i.e., “% of total sequencing reads with the specified edit or indels”) of PE2, PE2-trunc, PE3, and PE3-trunc over different target sites in various cell lines. The data shows that the prime editors comprising the truncated RT variants were about as efficient as the prime editors comprising the non-trunctated RT proteins.
• In various embodiments, the prime editor fusion proteins contemplated herein may also include any variants of the above-disclosed sequences having an amino acid sequence that is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to PE1, PE2, or any of the above indicated prime editor fusion sequences.
• In certain embodiments, linkers may be used to link any of the peptides or peptide domains or moieties of the invention (e.g., a napDNAbp linked or fused to a reverse transcriptase).
[5] Linkers and Other Domains
• The PE fusion proteins may comprise various other domains besides the napDNAbp (e.g., Cas9 domain) and the polymerase domain (e.g., RT domain). For example, in the case where the napDNAbp is a Cas9 and the polymerase is a RT, the PE fusion proteins may comprise one or more linkers that join the Cas9 domain with the RT domain. The linkers may also join other functional domains, such as nuclear localization sequences (NLS) or a FEN1 (or other flap endonuclease) to the PE fusion proteins or a domain thereof.
• In addition, in embodiments involving trans prime editing, linkers may be used to link tPERT recruitment protein to a prime editor, e.g., between the tPERt recruitment protein and the napDNAbp. See e.g., FIG. 3G for an exemplary schematic of a trans prime editor (tPE) that includes linkers to separately fuse a polymerase domain and a recruiting protein domain to a napDNAbp.
• A. Linkers
• As defined above, the term “linker,” as used herein, refers to a chemical group or a molecule linking two molecules or moieties, e.g., a binding domain and a cleavage domain of a nuclease. In some embodiments, a linker joins a gRNA binding domain of an RNA-programmable nuclease and the catalytic domain of a polymerase (e.g., a reverse transcriptase). In some embodiments, a linker joins a dCas9 and reverse transcriptase. Typically, the linker is positioned between, or flanked by, two groups, molecules, or other moieties and connected to each one via a covalent bond, thus connecting the two. In some embodiments, the linker is an amino acid or a plurality of amino acids (e.g., a peptide or protein). In some embodiments, the linker is an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker is 5-100 amino acids in length, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30-35, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-150, or 150-200 amino acids in length. Longer or shorter linkers are also contemplated.
• The linker may be as simple as a covalent bond, or it may be a polymeric linker many atoms in length. In certain embodiments, the linker is a polypeptide or based on amino acids. In other embodiments, the linker is not peptide-like. In certain embodiments, the linker is a covalent bond (e.g., a carbon-carbon bond, disulfide bond, carbon-heteroatom bond, etc.). In certain embodiments, the linker is a carbon-nitrogen bond of an amide linkage. In certain embodiments, the linker is a cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic or heteroaliphatic linker. In certain embodiments, the linker is polymeric (e.g., polyethylene, polyethylene glycol, polyamide, polyester, etc.). In certain embodiments, the linker comprises a monomer, dimer, or polymer of aminoalkanoic acid. In certain embodiments, the linker comprises an aminoalkanoic acid (e.g., glycine, ethanoic acid, alanine, beta-alanine, 3-aminopropanoic acid, 4-aminobutanoic acid, 5-pentanoic acid, etc.). In certain embodiments, the linker comprises a monomer, dimer, or polymer of aminohexanoic acid (Ahx). In certain embodiments, the linker is based on a carbocyclic moiety (e.g., cyclopentane, cyclohexane). In other embodiments, the linker comprises a polyethylene glycol moiety (PEG). In other embodiments, the linker comprises amino acids. In certain embodiments, the linker comprises a peptide. In certain embodiments, the linker comprises an aryl or heteroaryl moiety. In certain embodiments, the linker is based on a phenyl ring. The linker may included funtionalized moieties to facilitate attachment of a nucleophile (e.g., thiol, amino) from the peptide to the linker. Any electrophile may be used as part of the linker. Exemplary electrophiles include, but are not limited to, activated esters, activated amides, Michael acceptors, alkyl halides, aryl halides, acyl halides, and isothiocyanates.
• In some other embodiments, the linker comprises the amino acid sequence (GGGGS)_n (SEQ ID NO: 165), (G)_n (SEQ ID NO: 166), (EAAAK)_n (SEQ ID NO: 167), (GGS)_n (SEQ ID NO: 168), (SGGS)_n (SEQ ID NO: 169), (XP)_n (SEQ ID NO: 170), or any combination thereof, wherein n is independently an integer between 1 and 30, and wherein X is any amino acid. In some embodiments, the linker comprises the amino acid sequence (GGS)N(SEQ ID NO: 176), wherein n is 1, 3, or 7. In some embodiments, the linker comprises the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 171). In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 172). In some embodiments, the linker comprises the amino acid sequence SGGSGGSGGS (SEQ ID NO: 173). In some embodiments, the linker comprises the amino acid sequence SGGS (SEQ ID NO: 174). In other embodiments, the linker comprises the amino acid sequence

• (SEQ ID NO: 175, 60AA)

  SGGSSGGSSGSETPGTSESATPESAGSYPYDVPDYAGSAAPAAKKKKLDG

  SGSGGSSGGS.

• In certain embodiments, linkers may be used to link any of the peptides or peptide domains or moieties of the invention (e.g., a napDNAbp linked or fused to a reverse transcriptase).
• As defined above, the term “linker,” as used herein, refers to a chemical group or a molecule linking two molecules or moieties, e.g., a binding domain and a cleavage domain of a nuclease. In some embodiments, a linker joins a gRNA binding domain of an RNA-programmable nuclease and the catalytic domain of a recombinase. In some embodiments, a linker joins a dCas9 and reverse transcriptase. Typically, the linker is positioned between, or flanked by, two groups, molecules, or other moieties and connected to each one via a covalent bond, thus connecting the two. In some embodiments, the linker is an amino acid or a plurality of amino acids (e.g., a peptide or protein). In some embodiments, the linker is an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker is 5-100 amino acids in length, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30-35, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-150, or 150-200 amino acids in length. Longer or shorter linkers are also contemplated.
• The linker may be as simple as a covalent bond, or it may be a polymeric linker many atoms in length. In certain embodiments, the linker is a polypeptide or based on amino acids. In other embodiments, the linker is not peptide-like. In certain embodiments, the linker is a covalent bond (e.g., a carbon-carbon bond, disulfide bond, carbon-heteroatom bond, etc.). In certain embodiments, the linker is a carbon-nitrogen bond of an amide linkage. In certain embodiments, the linker is a cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic or heteroaliphatic linker. In certain embodiments, the linker is polymeric (e.g., polyethylene, polyethylene glycol, polyamide, polyester, etc.). In certain embodiments, the linker comprises a monomer, dimer, or polymer of aminoalkanoic acid. In certain embodiments, the linker comprises an aminoalkanoic acid (e.g., glycine, ethanoic acid, alanine, beta-alanine, 3-aminopropanoic acid, 4-aminobutanoic acid, 5-pentanoic acid, etc.). In certain embodiments, the linker comprises a monomer, dimer, or polymer of aminoHEXAnoic acid (Ahx). In certain embodiments, the linker is based on a carbocyclic moiety (e.g., cyclopentane, cycloHEXAne). In other embodiments, the linker comprises a polyethylene glycol moiety (PEG). In other embodiments, the linker comprises amino acids. In certain embodiments, the linker comprises a peptide. In certain embodiments, the linker comprises an aryl or heteroaryl moiety. In certain embodiments, the linker is based on a phenyl ring. The linker may included funtionalized moieties to facilitate attachment of a nucleophile (e.g., thiol, amino) from the peptide to the linker. Any electrophile may be used as part of the linker. Exemplary electrophiles include, but are not limited to, activated esters, activated amides, Michael acceptors, alkyl halides, aryl halides, acyl halides, and isothiocyanates.
• In some other embodiments, the linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 165), (G)n (SEQ ID NO: 166), (EAAAK)_n (SEQ ID NO: 167), (GGS)_n (SEQ ID NO: 168), (SGGS)n (SEQ ID NO: 169), (XP)n (SEQ ID NO: 170), or any combination thereof, wherein n is independently an integer between 1 and 30, and wherein X is any amino acid. In some embodiments, the linker comprises the amino acid sequence (GGS)N(SEQ ID NO: 176), wherein n is 1, 3, or 7. In some embodiments, the linker comprises the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 171). In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 172). In some embodiments, the linker comprises the amino acid sequence SGGSGGSGGS (SEQ ID NO: 173). In some embodiments, the linker comprises the amino acid sequence SGGS (SEQ ID NO: 174).
• In particular, the following linkers can be used in various embodiments to join prime editor domains with one another:

• (SEQ ID NO: 767)

  GGS;

  (SEQ ID NO: 768)

  GGSGGS;

  (SEQ ID NO: 769)

  GGSGGSGGS;

  (SEQ ID NO: 127)

  SGGSSGGSSGSETPGTSESATPESSGGSSGGSS;

  (SEQ ID NO: 171)

  SGSETPGTSESATPES;

  (SEQ ID NO: 175)

  SGGSSGGSSGSETPGTSESATPESAGSYPYDVPDYAGSAAPAAKKKKLDG

  SGSGGSSGG S.

• B. Nuclear Localization Sequence (NLS)
• In various embodiments, the PE fusion proteins may comprise one or more nuclear localization sequences (NLS), which help promote translocation of a protein into the cell nucleus. Such sequences are well-known in the art and can include the following examples:

• DESCRIPTION	SEQUENCE	SEQ ID NO:
  NLS OF	PKKKRKV	SEQ ID NO: 16
  SV40
  LARGE T-
  AG
  NLS	MKRTADGSEFESPKKKRKV	SEQ ID NO: 124
  NLS	MDSLLMNRRKFLYQFKNVRWAKGRRETYLC	SEQ ID NO: 17
  NLS OF	AVKRPAATKKAGQAKKKKLD	SEQ ID NO: 190
  NUCLEOPLASMIN
  NLS OF	MSRRRKANPTKLSENAKKLAKEVEN	SEQ ID NO: 191
  EGL-13
  NLS OF C-	PAAKRVKLD	SEQ ID NO: 192
  MYC
  NLS OF	KLKIKRPVK	SEQ ID NO: 193
  TUS-
  PROTEIN
  NLS OF	VSRKRPRP	SEQ ID NO: 194
  POLYOMA
  LARGE T-
  AG
  NLS OF	EGAPPAKRAR	SEQ ID NO: 195
  HEPATITIS
  D VIRUS
  ANTIGEN
  NLS OF	PPQPKKKPLDGE	SEQ ID NO: 196
  MURINE
  P53
  NLS OF	SGGSKRTADGSEFEPKKKRKV	SEQ ID NO: 133
  PE1 AND
  PE2

• The NLS examples above are non-limiting. The PE fusion proteins may comprise any known NLS sequence, including any of those described in Cokol et al., “Finding nuclear localization signals,” EMBO Rep., 2000, 1(5): 411-415 and Freitas et al., “Mechanisms and Signals for the Nuclear Import of Proteins,” Current Genomics, 2009, 10(8): 550-7, each of which are incorporated herein by reference.
• In various embodiments, the prime editors and constructs encoding the prime editors disclosed herein further comprise one or more, preferably, at least two nuclear localization signals. In certain embodiments, the prime editors comprise at least two NLSs. In embodiments with at least two NLSs, the NLSs can be the same NLSs or they can be different NLSs. In addition, the NLSs may be expressed as part of a fusion protein with the remaining portions of the prime editors. In some embodiments, one or more of the NLSs are bipartite NLSs (“bpNLS”). In certain embodiments, the disclosed fusion proteins comprise two bipartite NLSs. In some embodiments, the disclosed fusion proteins comprise more than two bipartite NLSs.
• The location of the NLS fusion can be at the N-terminus, the C-terminus, or within a sequence of a prime editor (e.g., inserted between the encoded napDNAbp component (e.g., Cas9) and a polymerase domain (e.g., a reverse transcriptase domain).
• The NLSs may be any known NLS sequence in the art. The NLSs may also be any future-discovered NLSs for nuclear localization. The NLSs also may be any naturally-occurring NLS, or any non-naturally occurring NLS (e.g., an NLS with one or more desired mutations).
• The term “nuclear localization sequence” or “NLS” refers to an amino acid sequence that promotes import of a protein into the cell nucleus, for example, by nuclear transport. Nuclear localization sequences are known in the art and would be apparent to the skilled artisan. For example, NLS sequences are described in Plank et al., International PCT application PCT/EP2000/011690, filed Nov. 23, 2000, published as WO/2001/038547 on May 31, 2001, the contents of which are incorporated herein by reference. In some embodiments, an NLS comprises the amino acid sequence PKKKRKV (SEQ ID NO: 16), MDSLLMNRRKFLYQFKNVRWAKGRRETYLC (SEQ ID NO: 17), KRTADGSEFESPKKKRKV (SEQ ID NO: 3864), or KRTADGSEFEPKKKRKV (SEQ ID NO: 13). In other embodiments, NLS comprises the amino acid sequences

• 	(SEQ ID NO: 3865)
  	NLSKRPAAIKKAGQAKKKK,
  	(SEQ ID NO: 192)
  	PAAKRVKLD,
  	(SEQ ID NO: 3866)
  	RQRRNELKRSF,
  	(SEQ ID NO: 3867)
  	NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY.

• In one aspect of the disclosure, a prime editor may be modified with one or more nuclear localization signals (NLS), preferably at least two NLSs. In certain embodiments, the prime editors are modified with two or more NLSs. The disclosure contemplates the use of any nuclear localization signal known in the art at the time of the disclosure, or any nuclear localization signal that is identified or otherwise made available in the state of the art after the time of the instant filing. A representative nuclear localization signal is a peptide sequence that directs the protein to the nucleus of the cell in which the sequence is expressed. A nuclear localization signal is predominantly basic, can be positioned almost anywhere in a protein's amino acid sequence, generally comprises a short sequence of four amino acids (Autieri & Agrawal, (1998) J. Biol. Chem. 273: 14731-37, incorporated herein by reference) to eight amino acids, and is typically rich in lysine and arginine residues (Magin et al., (2000) Virology 274: 11-16, incorporated herein by reference). Nuclear localization signals often comprise proline residues. A variety of nuclear localization signals have been identified and have been used to effect transport of biological molecules from the cytoplasm to the nucleus of a cell. See, e.g., Tinland et al., (1992) Proc. Natl. Acad. Sci. U.S.A. 89:7442-46; Moede et al., (1999) FEBS Lett. 461:229-34, which is incorporated by reference. Translocation is currently thought to involve nuclear pore proteins.
• Most NLSs can be classified in three general groups: (i) a monopartite NLS exemplified by the SV40 large T antigen NLS (PKKKRKV (SEQ ID NO: 16)); (ii) a bipartite motif consisting of two basic domains separated by a variable number of spacer amino acids and exemplified by the Xenopus nucleoplasmin NLS (KRXXXXXXXXXXKKKL (SEQ ID NO: 3868)); and (iii) noncanonical sequences such as M9 of the hnRNP A1 protein, the influenza virus nucleoprotein NLS, and the yeast Gal4 protein NLS (Dingwall and Laskey 1991).
• Nuclear localization signals appear at various points in the amino acid sequences of proteins. NLS's have been identified at the N-terminus, the C-terminus and in the central region of proteins. Thus, the disclosure provides prime editors that may be modified with one or more NLSs at the C-terminus, the N-terminus, as well as at in internal region of the prime editor. The residues of a longer sequence that do not function as component NLS residues should be selected so as not to interfere, for example tonically or sterically, with the nuclear localization signal itself. Therefore, although there are no strict limits on the composition of an NLS-comprising sequence, in practice, such a sequence can be functionally limited in length and composition.
• The present disclosure contemplates any suitable means by which to modify a prime editor to include one or more NLSs. In one aspect, the prime editors may be engineered to express a prime editor protein that is translationally fused at its N-terminus or its C-terminus (or both) to one or more NLSs, i.e., to form a prime editor-NLS fusion construct. In other embodiments, the prime editor-encoding nucleotide sequence may be genetically modified to incorporate a reading frame that encodes one or more NLSs in an internal region of the encoded prime editor. In addition, the NLSs may include various amino acid linkers or spacer regions encoded between the prime editor and the N-terminally, C-terminally, or internally-attached NLS amino acid sequence, e.g, and in the central region of proteins. Thus, the present disclosure also provides for nucleotide constructs, vectors, and host cells for expressing fusion proteins that comprise a prime editor and one or more NLSs.
• The prime editors described herein may also comprise nuclear localization signals which are linked to a prime editor through one or more linkers, e.g., and polymeric, amino acid, nucleic acid, polysaccharide, chemical, or nucleic acid linker element. The linkers within the contemplated scope of the disclosure are not indented to have any limitations and can be any suitable type of molecule (e.g., polymer, amino acid, polysaccharide, nucleic acid, lipid, or any synthetic chemical linker domain) and be joined to the prime editor by any suitable strategy that effectuates forming a bond (e.g., covalent linkage, hydrogen bonding) between the prime editor and the one or more NLS s.
• C. Flap Endonucleases (e.g., FEN1)
• In various embodiments, the PE fusion proteins may comprise one or more flap endonucleases (e.g., FEN1), which refers to an enzyme that catalyzes the removal of 5′ single strand DNA flaps. These are naturally occurring enzymes that process the removal of 5′ flaps formed during cellular processes, including DNA replication. The prime editing methods herein described may utilize endogenously supplied flap endonucleases or those provided in trans to remove the 5′ flap of endogenous DNA formed at the target site during prime editing. Flap endonucleases are known in the art and can be found described in Patel et al., “Flap endonucleases pass 5′-flaps through a flexible arch using a disorder-thread-order mechanism to confer specificity for free 5′-ends,” Nucleic Acids Research, 2012, 40(10): 4507-4519 and Tsutakawa et al., “Human flap endonuclease structures, DNA double-base flipping, and a unified understanding of the FEN1 superfamily,” Cell, 2011, 145(2): 198-211 (each of which are incorporated herein by reference). An exemplary flap endonuclease is FEN1, which can be represented by the following amino acid sequence:

• Description	Sequence	SEQ ID NO:
  FEN1	MGIQGLAKLIADVAPSAIRENDIKSYFGRKVAIDASMSI	SEQ ID NO:
  Wild type	YQFLIAVRQGGDVLQNEEGETTSHLMGMFYRTIRMME	198
  (wt)	NGIKPVYVFDGKPPQLKSGELAKRSERRAEAEKQLQQ
  	AQAAGAEQEVEKFTKRLVKVTKQHNDECKHLLSLMGI
  	PYLDAPSEAEASCAALVKAGKVYAAATEDMDCLTFGS
  	PVLMRHLTASEAKKLPIQEFHLSRILQELGLNQEQFVD
  	LCILLGSDYCESIRGIGPKRAVDLIQKHKSIEEIVRRLDP
  	NKYPVPENWLHKEAHQLFLEPEVLDPESVELKWSEPN
  	EEELIKFMCGEKQFSEERIRSGVKRLSKSRQGSTQGRLD
  	DFFKVTGSLSSAKRKEPEPKGSTKKKAKTGAAGKFKR
  	GK

• The flap endonucleases may also include any FEN1 variant, mutant, or other flap endonuclease ortholog, homolog, or variant. Non-limiting FEN1 variant examples are as follows:

• Description	Sequence	SEQ ID NO:
  FEN1	MGIQGLAKLIADVAPSAIRENDIKSYFGRKVAIDASMSI	SEQ ID NO:
  K168R	YQFLIAVRQGGDVLQNEEGETTSHLMGMFYRTIRMME	199
  (relative to	NGIKPVYVFDGKPPQLKSGELAKRSERRAEAEKQLQQ
  FEN1 wt)	AQAAGAEQEVEKFTKRLVKVTKQHNDECKHLLSLMGI
  	PYLDAPSEAEASCAALV R AGKVYAAATEDMDCLTFGS
  	PVLMRHLTASEAKKLPIQEFHLSRILQELGLNQEQFVD
  	LCILLGSDYCESIRGIGPKRAVDLIQKHKSIEEIVRRLDP
  	NKYPVPENWLHKEAHQLFLEPEVLDPESVELKWSEPN
  	EEELIKFMCGEKQFSEERIRSGVKRLSKSRQGSTQGRLD
  	DFFKVTGSLSSAKRKEPEPKGSTKKKAKTGAAGKFKR
  	GK
  FEN1	MGIQGLAKLIADVAPSAIRENDIKSYFGRKVAIDASMSI	SEQ ID NO:
  S187A	YQFLIAVRQGGDVLQNEEGETTSHLMGMFYRTIRMME	200
  (relative to	NGIKPVYVFDGKPPQLKSGELAKRSERRAEAEKQLQQ
  FEN1 wt)	AQAAGAEQEVEKFTKRLVKVTKQHNDECKHLLSLMGI
  	PYLDAPSEAEASCAALVKAGKVYAAATEDMDCLTFG
  	A PVLMRHLTASEAKKLPIQEFHLSRILQELGLNQEQFV
  	DLCILLGSDYCESIRGIGPKRAVDLIQKHKSIEEIVRRLD
  	PNKYPVPENWLHKEAHQLFLEPEVLDPESVELKWSEP
  	NEEELIKFMCGEKQFSEERIRSGVKRLSKSRQGSTQGRL
  	DDFFKVTGSLSSAKRKEPEPKGSTKKKAKTGAAGKFK
  	RGK
  FEN1	MGIQGLAKLIADVAPSAIRENDIKSYFGRKVAIDASMSI	SEQ ID NO:
  K354R	YQFLIAVRQGGDVLQNEEGETTSHLMGMFYRTIRMME	201
  (relative to	NGIKPVYVFDGKPPQLKSGELAKRSERRAEAEKQLQQ
  FEN1 wt)	AQAAGAEQEVEKFTKRLVKVTKQHNDECKHLLSLMGI
  	PYLDAPSEAEASCAALVKAGKVYAAATEDMDCLTFGS
  	PVLMRHLTASEAKKLPIQEFHLSRILQELGLNQEQFVD
  	LCILLGSDYCESIRGIGPKRAVDLIQKHKSIEEIVRRLDP
  	NKYPVPENWLHKEAHQLFLEPEVLDPESVELKWSEPN
  	EEELIKFMCGEKQFSEERIRSGVKRLSKSRQGSTQGRLD
  	DFFKVTGSLSSA R RKEPEPKGSTKKKAKTGAAGKFKR
  	GK
  GEN1	MGVNDLWQILEPVKQHIPLRNLGGKTIAVDLSLWVCE	SEQ ID NO:
  	AQTVKKMMGSVMKPHLRNLFFRISYLTQMDVKLVFV	202
  	MEGEPPKLKADVISKRNQSRYGSSGKSWSQKTGRSHF
  	KSVLRECLHMLECLGIPWVQAAGEAEAMCAYLNAGG
  	HVDGCLTNDGDTFLYGAQTVYRNFTMNTKDPHVDCY
  	TMSSIKSKLGLDRDALVGLAILLGCDYLPKGVPGVGKE
  	QALKLIQILKGQSLLQRFNRWNETSCNSSPQLLVTKKL
  	AHCSVCSHPGSPKDHERNGCRLCKSDKYCEPHDYEYC
  	CPCEWHRTEHDRQLSEVENNIKKKACCCEGFPFHEVIQ
  	EFLLNKDKLVKVIRYQRPDLLLFQRFTLEKMEWPNHY
  	ACEKLLVLLTHYDMIERKLGSRNSNQLQPIRIVKTRIRN
  	GVHCFEIEWEKPEHYAMEDKQHGEFALLTIEEESLFEA
  	AYPEIVAVYQKQKLEIKGKKQKRIKPKENNLPEPDEVM
  	SFQSHMTLKPTCEIFHKQNSKLNSGISPDPTLPQESISAS
  	LNSLLLPKNTPCLNAQEQFMSSLRPLAIQQIKAVSKSLI
  	SESSQPNTSSHNISVIADLHLSTIDWEGTSFSNSPAIQRN
  	TFSHDLKSEVESELSAIPDGFENIPEQLSCESERYTANIK
  	KVLDEDSDGISPEEHLLSGITDLCLQDLPLKERIFTKLSY
  	PQDNLQPDVNLKTLSILSVKESCIANSGSDCTSHLSKDL
  	PGIPLQNESRDSKILKGDQLLQEDYKVNTSVPYSVSNT
  	VVKTCNVRPPNTALDHSRKVDMQTTRKILMKKSVCLD
  	RHSSDEQSAPVFGKAKYTTQRMKHSSQKHNSSHFKES
  	GHNKLSSPKIHIKETEQCVRSYETAENEESCFPDSTKSS
  	LSSLQCHKKENNSGTCLDSPLPLRQRLKLRFQST
  ERCC5	MGVQGLWKLLECSGRQVSPEALEGKILAVDISIWLNQ	SEQ ID NO:
  	ALKGVRDRHGNSIENPHLLTLFHRLCKLLFFRIRPIFVF	203
  	DGDAPLLKKQTLVKRRQRKDLASSDSRKTTEKLLKTF
  	LKRQAIKTAFRSAKRDEALPSLTQVRRENDLYVLPPLQE
  	EEKHSSEEEDEKEWQERMNQKQALQEEFFHNPQAIDIE
  	SEDFSSLPPEVKHEILTDMKEFTKRRRTLFEAMPEESDD
  	FSQYQLKGLLKKNYLNQHIEHVQKEMNQQHSGHIRRQ
  	YEDEGGFLKEVESRRVVSEDTSHYILIKGIQAKTVAEV
  	DSESLPSSSKMHGMSFDVKSSPCEKLKTEKEPDATPPSP
  	RTLLAMQAALLGSSSEEELESENRRQARGRNAPAAVD
  	EGSISPRTLSAIKRALDDDEDVKVCAGDDVQTGGPGAE
  	EMRINSSTENSDEGLKVRDGKGIPFTATLASSSVNSAEE
  	HVASTNEGREPTDSVPKEQMSLVHVGTEAFPISDESMI
  	KDRKDRLPLESAVVRHSDAPGLPNGRELTPASPTCTNS
  	VSKNETHAEVLEQQNELCPYESKFDSSLLSSDDETKCK
  	PNSASEVIGPVSLQETSSIVSVPSEAVDNVENVVSFNAK
  	EHENFLETIQEQQTTESAGQDLISIPKAVEPMEIDSEESE
  	SDGSFIEVQSVISDEELQAEFPETSKPPSEQGEEELVGTR
  	EGEAPAESESLLRDNSERDDVDGEPQEAEKDAEDSLHE
  	WQDINLEELETLESNLLAQQNSLKAQKQQQERIAATVT
  	GQMFLESQELLRLFGIPYIQAPMEAEAQCAILDLTDQTS
  	GTITDDSDIWLFGARHVYRNFFNKNKFVEYYQYVDFH
  	NQLGLDRNKLINLAYLLGSDYTEGIPTVGCVTAMEILN
  	EFPGHGLEPLLKFSEWWHEAQKNPKIRPNPHDTKVKK
  	KLRTLQLTPGFPNPAVAEAYLKPVVDDSKGSFLWGKP
  	DLDKIREFCQRYFGWNRTKTDESLFPVLKQLDAQQTQ
  	LRIDSFFRLAQQEKEDAKRIKSQRLNRAVTCMLRKEKE
  	AAASEIEAVSVAMEKEFELLDKAKRKTQKRGITNTLEE
  	SSSLKRKRLSDSKRKNTCGGFLGETCLSESSDGSSSEDA
  	ESSSLMNVQRRTAAKEPKTSASDSQNSVKEAPVKNGG
  	ATTSSSSDSDDDGGKEKMVLVTARSVFGKKRRKLRRA
  	RGRKRKT

• In various embodiments, the prime editor fusion proteins contemplated herein may include any flap endonuclease variant of the above-disclosed sequences having an amino acid sequence that is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to any of the above sequences.
• Other endonucleases that may be utilized by the instant methods to facilitate removal of the 5′ end single strand DNA flap include, but are not limited to (1) trex 2, (2) exol endonuclease (e.g., Keijzers et al., Biosci Rep. 2015, 35(3): e00206)

• Trex 2

  3′ three prime repair exonuclease 2 (TREX2)-

  human

  Accession No. NM_080701

  (SEQ ID NO: 3865)

  MSEAPRAETFVFLDLEATGLPSVEPEIAELSLFAVHRSSLENPEHDESGA

  LVLPRVLDKLTLCMCPERPFTAKASEITGLSSEGLARCRKAGFDGAVVRT

  LQAFLSRQAGPICLVAHNGFDYDFPLLCAELRRLGARLPRDTVCLDTLPA

  LRGLDRAHSHGTRARGRQGYSLGSLFHRYFRAEPSAAHSAEGDVHTLLLI

  FLHRAAELLAWADEQARGWAHIEPMYLPPDDPSLEA.

  3′ three prime repair exonuclease 2 (TREX2)-

  mouse

  Accession No. NM_011907

  (SEQ ID NO: 3866)

  MSEPPRAETFVFLDLEATGLPNMDPEIAEISLFAVHRSSLENPERDDSGS

  LVLPRVLDKLTLCMCPERPFTAKASEITGLSSESLMHCGKAGFNGAVVRT

  LQGFLSRQEGPICLVAHNGFDYDFPLLCTELQRLGAHLPQDTVCLDTLPA

  LRGLDRAHSHGTRAQGRKSYSLASLFHRYFQAEPSAAHSAEGDVHTLLLI

  FLHRAPELLAWADEQARSWAHIEPMYVPPDGPSLEA.

  3′ three prime repair exonuclease 2 (TREX2)-rat

  Accession No. NM_001107580

  (SEQ ID NO: 3867)

  MSEPLRAETFVFLDLEATGLPNMDPEIAEISLFAVHRSSLENPERDDSGS

  LVLPRVLDKLTLCMCPERPFTAKASEITGLSSEGLMNCRKAAFNDAVVRT

  LQGFLSRQEGPICLVAHNGFDYDFPLLCTELQRLGAHLPRDTVCLDTLPA

  LRGLDRVHSHGTRAQGRKSYSLASLFHRYFQAEPSAAHSAEGDVNTLLLI

  FLHRAPELLAWADEQARSWAHIEPMYVPPDGPSLEA.

ExoI
• Human exonuclease 1 (EXO1) has been implicated in many different DNA metabolic processes, including DNA mismatch repair (MMR), micro-mediated end-joining, homologous recombination (HR), and replication. Human EXO1 belongs to a family of eukaryotic nucleases, Rad2/XPG, which also include FEN1 and GEN1. The Rad2/XPG family is conserved in the nuclease domain through species from phage to human. The EXO1 gene product exhibits both 5′ exonuclease and 5′ flap activity. Additionally, EXO1 contains an intrinsic 5′ RNase H activity. Human EXO1 has a high affinity for processing double stranded DNA (dsDNA), nicks, gaps, pseudo Y structures and can resolve Holliday junctions using its inherit flap activity. Human EXO1 is implicated in MMR and contain conserved binding domains interacting directly with MLH1 and MSH2. EXO1 nucleolytic activity is positively stimulated by PCNA, MutSa (MSH2/MSH6 complex), 14-3-3, MRN and 9-1-1 complex.

• exonuclease 1 (EXO1) Accession No. NM_003686

  (Homo sapiens exonuclease 1 (EXO1), transcript

  variant 3)-isoform A

  (SEQ ID NO: 3868)

  MGIQGLLQFIKEASEPIHVRKYKGQVVAVDTYCWLHKGAIACAEKLAKGE

  PTDRYVGFCMKFVNMLLSHGIKPILVFDGCTLPSKKEVERSRRERRQANL

  LKGKQLLREGKVSEARECFTRSINITHAMAHKVIKAARSQGVDCLVAPYE

  ADAQLAYLNKAGIVQAIITEDSDLLAFGCKKVILKMDQFGNGLEIDQARL

  GMCRQLGDVFTEEKFRYMCILSGCDYLSSLRGIGLAKACKVLRLANNPDI

  VKVIKKIGHYLKMNITVPEDYINGFIRANNTFLYQLVFDPIKRKLIPLNA

  YEDDVDPETLSYAGQYVDDSIALQIALGNKDINTFEQIDDYNPDTAMPAH

  SRSHSWDDKTCQKSANVSSIWHRNYSPRPESGTVSDAPQLKENPSTVGVE

  RVISTKGLNLPRKSSIVKRPRSAELSEDDLLSQYSLSFTKKTKKNSSEGN

  KSLSFSEVFVPDLVNGPTNKKSVSTPPRTRNKFATFLQRKNEESGAVVVP

  GTRSRFFCSSDSTDCVSNKVSIQPLDETAVTDKENNLHESEYGDQEGKRL

  VDTDVARNSSDDIPNNHIPGDHIPDKATVFTDEESYSFESSKFTRTISPP

  TLGTLRSCFSWSGGLGDFSRTPSPSPSTALQQFRRKSDSPTSLPENNMSD

  VSQLKSEESSDDESHPLREEACSSQSQESGEFSLQSSNASKLSQCSSKDS

  DSEESDCNIKLLDSQSDQTSKLRLSHFSKKDTPLRNKVPGLYKSSSADSL

  STTKIKPLGPARASGLSKKPASIQKRKHHNAENKPGLQIKLNELWKNFGF

  KKF.

  exonuclease 1 (EXO1) Accession No. NM_006027

  (Homo sapiens exonuclease 1 (EXO1), transcript

  variant 3)-isoform B

  (SEQ ID NO: 3869)

  MGIQGLLQFIKEASEPIHVRKYKGQVVAVDTYCWLHKGAIACAEKLAKGE

  PTDRYVGFCMKFVNMLLSHGIKPILVFDGCTLPSKKEVERSRRERRQANL

  LKGKQLLREGKVSEARECFTRSINITHAMAHKVIKAARSQGVDCLVAPYE

  ADAQLAYLNKAGIVQAIITEDSDLLAFGCKKVILKMDQFGNGLEIDQARL

  GMCRQLGDVFTEEKFRYMCILSGCDYLSSLRGIGLAKACKVLRLANNPDI

  VKVIKKIGHYLKMNITVPEDYINGFIRANNTFLYQLVFDPIKRKLIPLNA

  YEDDVDPETLSYAGQYVDDSIALQIALGNKDINTFEQIDDYNPDTAMPAH

  SRSHSWDDKTCQKSANVSSIWHRNYSPRPESGTVSDAPQLKENPSTVGVE

  RVISTKGLNLPRKSSIVKRPRSAELSEDDLLSQYSLSFTKKTKKNSSEGN

  KSLSFSEVFVPDLVNGPTNKKSVSTPPRTRNKFATFLQRKNEESGAVVVP

  GTRSRFFCSSDSTDCVSNKVSIQPLDETAVTDKENNLHESEYGDQEGKRL

  VDTDVARNSSDDIPNNHIPGDHIPDKATVFTDEESYSFESSKFTRTISPP

  TLGTLRSCFSWSGGLGDFSRTPSPSPSTALQQFRRKSDSPTSLPENNMSD

  VSQLKSEESSDDESHPLREEACSSQSQESGEFSLQSSNASKLSQCSSKDS

  DSEESDCNIKLLDSQSDQTSKLRLSHFSKKDTPLRNKVPGLYKSSSADSL

  STTKIKPLGPARASGLSKKPASIQKRKHHNAENKPGLQIKLNELWKNFGF

  KKDSEKLPPCKKPLSPVRDNIQLTPEAEEDIFNKPECGRVQRAIFQ.

  exonuclease 1 (EXO1) Accession No. NM_001319224

  (Homo sapiens exonuclease 1 (EXO1), transcript

  variant 4)-isoform C

  (SEQ ID NO: 3870)

  MGIQGLLQFIKEASEPIHVRKYKGQVVAVDTYCWLHKGAIACAEKLAKGE

  PTDRYVGFCMKFVNMLLSHGIKPILVFDGCTLPSKKEVERSRRERRQANL

  LKGKQLLREGKVSEARECFTRSINITHAMAHKVIKAARSQGVDCLVAPYE

  ADAQLAYLNKAGIVQAIITEDSDLLAFGCKKVILKMDQFGNGLEIDQARL

  GMCRQLGDVFTEEKFRYMCILSGCDYLSSLRGIGLAKACKVLRLANNPDI

  VKVIKKIGHYLKMNITVPEDYINGFIRANNTFLYQLVFDPIKRKLIPLNA

  YEDDVDPETLSYAGQYVDDSIALQIALGNKDINTFEQIDDYNPDTAMPAH

  SRSHSWDDKTCQKSANVSSIWHRNYSPRPESGTVSDAPQLKENPSTVGVE

  RVISTKGLNLPRKSSIVKRPRSELSEDDLLSQYSLSFTKKTKKNSSEGNK

  SLSFSEVFVPDLVNGPTNKKSVSTPPRTRNKFATFLQRKNEESGAVVVPG

  TRSRFFCSSDSTDCVSNKVSIQPLDETAVTDKENNLHESEYGDQEGKRLV

  DTDVARNSSDDIPNNHIPGDHIPDKATVFTDEESYSFESSKFTRTISPPT

  LGTLRSCFSWSGGLGDFSRTPSPSPSTALQQFRRKSDSPTSLPENNMSDV

  SQLKSEESSDDESHPLREEACSSQSQESGEFSLQSSNASKLSQCSSKDSD

  SEESDCNIKLLDSQSDQTSKLRLSHFSKKDTPLRNKVPGLYKSSSADSLS

  TTKIKPLGPARASGLSKKPASIQKRKHHNAENKPGLQIKLNELWKNFGFK

  KDSEKLPPCKKPLSPVRDNIQLTPEAEEDIFNKPECGRVQRAIFQ.

• D. Inteins and Split-Inteins
• It will be understood that in some embodiments (e.g., delivery of a prime editor in vivo using AAV particles), it may be advantageous to split a polypeptide (e.g., a deaminase or a napDNAbp) or a fusion protein (e.g., a prime editor) into an N-terminal half and a C-terminal half, delivery them separately, and then allow their colocalization to reform the complete protein (or fusion protein as the case may be) within the cell. Separate halves of a protein or a fusion protein may each comprise a split-intein tag to facilitate the reformation of the complete protein or fusion protein by the mechanism of protein trans splicing.
• Protein trans-splicing, catalyzed by split inteins, provides an entirely enzymatic method for protein ligation. A split-intein is essentially a contiguous intein (e.g. a mini-intein) split into two pieces named N-intein and C-intein, respectively. The N-intein and C-intein of a split intein can associate non-covalently to form an active intein and catalyze the splicing reaction essentially in same way as a contiguous intein does. Split inteins have been found in nature and also engineered in laboratories. As used herein, the term “split intein” refers to any intein in which one or more peptide bond breaks exists between the N-terminal and C-terminal amino acid sequences such that the N-terminal and C-terminal sequences become separate molecules that can non-covalently reassociate, or reconstitute, into an intein that is functional for trans-splicing reactions. Any catalytically active intein, or fragment thereof, may be used to derive a split intein for use in the methods of the invention. For example, in one aspect the split intein may be derived from a eukaryotic intein. In another aspect, the split intein may be derived from a bacterial intein. In another aspect, the split intein may be derived from an archaeal intein. Preferably, the split intein so-derived will possess only the amino acid sequences essential for catalyzing trans-splicing reactions.
• As used herein, the “N-terminal split intein (In)” refers to any intein sequence that comprises an N-terminal amino acid sequence that is functional for trans-splicing reactions. An In thus also comprises a sequence that is spliced out when trans-splicing occurs. An In can comprise a sequence that is a modification of the N-terminal portion of a naturally occurring intein sequence. For example, an In can comprise additional amino acid residues and/or mutated residues so long as the inclusion of such additional and/or mutated residues does not render the In non-functional in trans-splicing. Preferably, the inclusion of the additional and/or mutated residues improves or enhances the trans-splicing activity of the In.
• As used herein, the “C-terminal split intein (Ic)” refers to any intein sequence that comprises a C-terminal amino acid sequence that is functional for trans-splicing reactions. In one aspect, the Ic comprises 4 to 7 contiguous amino acid residues, at least 4 amino acids of which are from the last β-strand of the intein from which it was derived. An Ic thus also comprises a sequence that is spliced out when trans-splicing occurs. An Ic can comprise a sequence that is a modification of the C-terminal portion of a naturally occurring intein sequence. For example, an Ic can comprise additional amino acid residues and/or mutated residues so long as the inclusion of such additional and/or mutated residues does not render the In non-functional in trans-splicing. Preferably, the inclusion of the additional and/or mutated residues improves or enhances the trans-splicing activity of the Ic.
• In some embodiments of the invention, a peptide linked to an Ic or an In can comprise an additional chemical moiety including, among others, fluorescence groups, biotin, polyethylene glycol (PEG), amino acid analogs, unnatural amino acids, phosphate groups, glycosyl groups, radioisotope labels, and pharmaceutical molecules. In other embodiments, a peptide linked to an Ic can comprise one or more chemically reactive groups including, among others, ketone, aldehyde, Cys residues and Lys residues. The N-intein and C-intein of a split intein can associate non-covalently to form an active intein and catalyze the splicing reaction when an “intein-splicing polypeptide (ISP)” is present. As used herein, “intein-splicing polypeptide (ISP)” refers to the portion of the amino acid sequence of a split intein that remains when the Ic, In, or both, are removed from the split intein. In certain embodiments, the In comprises the ISP. In another embodiment, the Ic comprises the ISP. In yet another embodiment, the ISP is a separate peptide that is not covalently linked to In nor to Ic.
• Split inteins may be created from contiguous inteins by engineering one or more split sites in the unstructured loop or intervening amino acid sequence between the −12 conserved beta-strands found in the structure of mini-inteins. Some flexibility in the position of the split site within regions between the beta-strands may exist, provided that creation of the split will not disrupt the structure of the intein, the structured beta-strands in particular, to a sufficient degree that protein splicing activity is lost.
• In protein trans-splicing, one precursor protein consists of an N-extein part followed by the N-intein, another precursor protein consists of the C-intein followed by a C-extein part, and a trans-splicing reaction (catalyzed by the N- and C-inteins together) excises the two intein sequences and links the two extein sequences with a peptide bond. Protein trans-splicing, being an enzymatic reaction, can work with very low (e.g. micromolar) concentrations of proteins and can be carried out under physiological conditions.
• Exemplary sequences are as follows:

• NAME	SEQUENCE OF LIGAND-DEPENDENT INTEIN
  2-4 INTEIN:	CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAAAKDGTLLARPVV
  	SWFDQGTRDVIGLRIAGGAIVWATPDHKVLTEYGWRAAGELRKGD
  	RVAGPGGSGNSLALSLTADQMVSALLDAEPPILYSEYDPTSPFSEAS
  	MMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQAHLLECAWLEI
  	LMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLAT
  	SSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRA
  	LDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEH
  	LYSMKYKNVVPLYDLLLEMLDAHRLHAGGSGASRVQAFADALDD
  	KFLHDMLAEELRYSVIREVLPTRRARTFDLEVEELHTLVAEGVVVH
  	NC (SEQ ID NO: 8)
  3-2 INTEIN	CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAVAKDGTLLARPVVS
  	WFDQGTRDVIGLRIAGGAIVWATPDHKVLTEYGWRAAGELRKGDR
  	VAGPGGSGNSLALSLTADQMVSALLDAEPPILYSEYDPTSPFSEASM
  	MGLLTNLADRELVHMINWAKRVPGFVDLTLHDQAHLLECAWLEIL
  	MIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATS
  	SRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRAL
  	DKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHL
  	YSMKYTNVVPLYDLLLEMLDAHRLHAGGSGASRVQAFADALDDK
  	FLHDMLAEELRYSVIREVLPTRRARTFDLEVEELHTLVAEGVVVHN
  	C (SEQ ID NO: 9)
  30R3-1 INTEIN	CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAAAKDGTLLARPVV
  	SWFDQGTRDVIGLRIAGGATVWATPDHKVLTEYGWRAAGELRKG
  	DRVAGPGGSGNSLALSLTADQMVSALLDAEPPIPYSEYDPTSPFSEA
  	SMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQAHLLECAWL
  	EILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLL
  	ATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIH
  	RALDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGM
  	EHLYSMKYKNVVPLYDLLLEMLDAHRLHAGGSGASRVQAFADAL
  	DDKFLHDMLAEGLRYSVIREVLPTRRARTFDLEVEELHTLVAEGVV
  	VHNC (SEQ ID NO: 10)
  30R3-2 INTEIN	CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAAAKDGTLLARPVV
  	SWFDQGTRDVIGLRIAGGATVWATPDHKVLTEYGWRAAGELRKG
  	DRVAGPGGSGNSLALSLTADQMVSALLDAEPPILYSEYDPTSPFSEA
  	SMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQAHLLECAWL
  	EILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLL
  	ATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIH
  	RALDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGM
  	EHLYSMKYKNVVPLYDLLLEMLDAHRLHAGGSGASRVQAFADAL
  	DDKFLHDMLAEELRYSVIREVLPTRRARTFDLEVEELHTLVAEGVV
  	VHNC (SEQ ID NO: 11)
  30R3-3 INTEIN	CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAAAKDGTLLARPVV
  	SWFDQGTRDVIGLRIAGGATVWATPDHKVLTEYGWRAAGELRKG
  	DRVAGPGGSGNSLALSLTADQMVSALLDAEPPIPYSEYDPTSPFSEA
  	SMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQAHLLECAWL
  	EILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLL
  	ATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIH
  	RALDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGM
  	EHLYSMKYKNVVPLYDLLLEMLDAHRLHAGGSGASRVQAFADAL
  	DDKFLHDMLAEELRYSVIREVLPTRRARTFDLEVEELHTLVAEGVV
  	VHNC (SEQ ID NO: 12)
  37R3-1 INTEIN	CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAAAKDGTLLARPVV
  	SWFDQGTRDVIGLRIAGGATVWATPDHKVLTEYGWRAAGELRKG
  	DRVAGPGGSGNSLALSLTADQMVSALLDAEPPILYSEYNPTSPFSEA
  	SMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQAHLLERAWL
  	EILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLL
  	ATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIH
  	RALDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGM
  	EHLYSMKYKNVVPLYDLLLEMLDAHRLHAGGSGASRVQAFADAL
  	DDKFLHDMLAEGLRYSVIREVLPTRRARTFDLEVEELHTLVAEGVV
  	VHNC ((SEQ ID NO: 13)
  37R3-2 INTEIN	CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAAAKDGTLLARPVV
  	SWFDQGTRDVIGLRIAGGAIVWATPDHKVLTEYGWRAAGELRKGD
  	RVAGPGGSGNSLALSLTADQMVSALLDAEPPILYSEYDPTSPFSEAS
  	MMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQAHLLERAWLEI
  	LMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLAT
  	SSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRA
  	LDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEH
  	LYSMKYKNVVPLYDLLLEMLDAHRLHAGGSGASRVQAFADALDD
  	KFLHDMLAEGLRYSVIREVLPTRRARTFDLEVEELHTLVAEGVVVH
  	NC (SEQ ID NO: 14)
  37R3-3 INTEIN	CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAVAKDGTLLARPVVS
  	WFDQGTRDVIGLRIAGGATVWATPDHKVLTEYGWRAAGELRKGD
  	RVAGPGGSGNSLALSLTADQMVSALLDAEPPILYSEYDPTSPFSEAS
  	MMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQAHLLERAWLEI
  	LMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLAT
  	SSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRA
  	LDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEH
  	LYSMKYKNVVPLYDLLLEMLDAHRLHAGGSGASRVQAFADALDD
  	KFLHDMLAEELRYSVIREVLPTRRARTFDLEVEELHTLVAEGVVVH
  	NC (SEQ ID NO: 15)

• Although inteins are most frequently found as a contiguous domain, some exist in a naturally split form. In this case, the two fragments are expressed as separate polypeptides and must associate before splicing takes place, so-called protein trans-splicing.
• An exemplary split intein is the Ssp DnaE intein, which comprises two subunits, namely, DnaE-N and DnaE-C. The two different subunits are encoded by separate genes, namely dnaE-n and dnaE-c, which encode the DnaE-N and DnaE-C subunits, respectively. DnaE is a naturally occurring split intein in Synechocytis sp. PCC6803 and is capable of directing trans-splicing of two separate proteins, each comprising a fusion with either DnaE-N or DnaE-C.
• Additional naturally occurring or engineered split-intein sequences are known in the or can be made from whole-intein sequences described herein or those available in the art. Examples of split-intein sequences can be found in Stevens et al., “A promiscuous split intein with expanded protein engineering applications,” PNAS, 2017, Vol. 114: 8538-8543; Iwai et al., “Highly efficient protein trans-splicing by a naturally split DnaE intein from Nostoc punctiforme, FEBS Lett, 580: 1853-1858, each of which are incorporated herein by reference. Additional split intein sequences can be found, for example, in WO 2013/045632, WO 2014/055782, WO 2016/069774, and EP2877490, the contents each of which are incorporated herein by reference.
• In addition, protein splicing in trans has been described in vivo and in vitro (Shingledecker, et al., Gene 207:187 (1998), Southworth, et al., EMBO J. 17:918 (1998); Mills, et al., Proc. Natl. Acad. Sci. USA, 95:3543-3548 (1998); Lew, et al., J. Biol. Chem., 273:15887-15890 (1998); Wu, et al., Biochim. Biophys. Acta 35732:1 (1998b), Yamazaki, et al., J. Am. Chem. Soc. 120:5591 (1998), Evans, et al., J. Biol. Chem. 275:9091 (2000); Otomo, et al., Biochemistry 38:16040-16044 (1999); Otomo, et al., J. Biolmol. NMR 14:105-114 (1999); Scott, et al., Proc. Natl. Acad. Sci. USA 96:13638-13643 (1999)) and provides the opportunity to express a protein as to two inactive fragments that subsequently undergo ligation to form a functional product, e.g., as shown in FIGS. 66 and 67 with regard to the formation of a complete PE fusion protein from two separately-expressed halves.
• E. RNA-Protein Interaction Domain
• In various embodiments, two separate protein domains (e.g., a Cas9 domain and a polymerase domain) may be colocalized to one another to form a functional complex (akin to the function of a fusion protein comprising the two separate protein domains) by using an “RNA-protein recruitment system,” such as the “MS2 tagging technique.” Such systems generally tag one protein domain with an “RNA-protein interaction domain” (aka “RNA-protein recruitment domain”) and the other with an “RNA-binding protein” that specifically recognizes and binds to the RNA-protein interaction domain, e.g., a specific hairpin structure. These types of systems can be leveraged to colocalize the domains of a prime editor, as well as to recruitment additional functionalities to a prime editor, such as a UGI domain. In one example, the MS2 tagging technique is based on the natural interaction of the MS2 bacteriophage coat protein (“MCP” or “MS2cp”) with a stem-loop or hairpin structure present in the genome of the phage, i.e., the “MS2 hairpin.” In the case of the MS2 hairpin, it is recognized and bound by the MS2 bacteriophage coat protein (MCP). Thus, in one exemplarily scenario a deaminase-MS2 fusion can recruit a Cas9-MCP fusion.
• A review of other modular RNA-protein interaction domains are described in the art, for example, in Johansson et al., “RNA recognition by the MS2 phage coat protein,” Sem Virol., 1997, Vol. 8(3): 176-185; Delebecque et al., “Organization of intracellular reactions with rationally designed RNA assemblies,” Science, 2011, Vol. 333: 470-474; Mali et al., “Cas9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering,” Nat. Biotechnol., 2013, Vol. 31: 833-838; and Zalatan et al., “Engineering complex synthetic transcriptional programs with CRISPR RNA scaffolds,” Cell, 2015, Vol. 160: 339-350, each of which are incorporated herein by reference in their entireties. Other systems include the PP7 hairpin, which specifically recruits the PCP protein, and the “com” hairpin, which specifically recruits the Com protein. See Zalatan et al.
• The nucleotide sequence of the MS2 hairpin (or equivalently referred to as the “MS2 aptamer”) is

• (SEQ ID NO: 3872)

  GSASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSV

  RQSSAQNRKYTIKVEVPKVATQTVGGEELPVAGWRSYLNMELTIPIFATN

  SDCELIVKAMQGLLKDGNPIPSAIAANSGIY.

• The amino acid sequence of the MCP or MS2cp is:

• 	(SEQ ID NO: 3871)
  	GCCAACATGAGGATCACCCATGTCTGCAGGGCC.

• F. UGI Domain
• In other embodiments, the prime editors described herein may comprise one or more uracil glycosylase inhibitor domains. The term “uracil glycosylase inhibitor (UGI)” or “UGI domain,” as used herein, refers to a protein that is capable of inhibiting a uracil-DNA glycosylase base-excision repair enzyme. In some embodiments, a UGI domain comprises a wild-type UGI or a UGI as set forth in SEQ ID NO: 3873. In some embodiments, the UGI proteins provided herein include fragments of UGI and proteins homologous to a UGI or a UGI fragment. For example, in some embodiments, a UGI domain comprises a fragment of the amino acid sequence set forth in SEQ ID NO: 3873. In some embodiments, a UGI fragment comprises an amino acid sequence that comprises at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid sequence as set forth in SEQ ID NO: 3873. In some embodiments, a UGI comprises an amino acid sequence homologous to the amino acid sequence set forth in SEQ ID NO: 3873, or an amino acid sequence homologous to a fragment of the amino acid sequence set forth in SEQ ID NO: 3873. In some embodiments, proteins comprising UGI or fragments of UGI or homologs of UGI or UGI fragments are referred to as “UGI variants.” A UGI variant shares homology to UGI, or a fragment thereof. For example a UGI variant is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, or at least 99.9% identical to a wild type UGI or a UGI as set forth in SEQ ID NO: 3873. In some embodiments, the UGI variant comprises a fragment of UGI, such that the fragment is at least 70% identical, at least 80% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, or at least 99.9% to the corresponding fragment of wild-type UGI or a UGI as set forth in SEQ ID NO: 3873. In some embodiments, the UGI comprises the following amino acid sequence:
• Uracil-DNA glycosylase inhibitor:

• >sp|P14739|UNGI_BPPB2

  (SEQ ID NO: 3873)

  MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDES

  TDENVMLLTSDAPEYKPWALVIQDSNGENKIKML.

• The prime editors described herein may comprise more than one UGI domain, which may be separated by one or more linkers as described herein.
• G. Additional PE Elements
• In certain embodiments, the prime editors described herein may comprise an inhibitor of base repair. The term “inhibitor of base repair” or “IBR” refers to a protein that is capable in inhibiting the activity of a nucleic acid repair enzyme, for example a base excision repair enzyme. In some embodiments, the IBR is an inhibitor of OGG base excision repair. In some embodiments, the IBR is an inhibitor of base excision repair (“iBER”). Exemplary inhibitors of base excision repair include inhibitors of APE1, Endo III, Endo IV, Endo V, Endo VIII, Fpg, hOGG1, hNEIL1, T7 EndoI, T4PDG, UDG, hSMUG1, and hAAG. In some embodiments, the IBR is an inhibitor of Endo V or hAAG. In some embodiments, the IBR is an iBER that may be a catalytically inactive glycosylase or catalytically inactive dioxygenase or a small molecule or peptide inhibitor of an oxidase, or variants thereof. In some embodiments, the IBR is an iBER that may be a TDG inhibitor, MBD4 inhibitor or an inhibitor of an AlkBH enzyme. In some embodiments, the IBR is an iBER that comprises a catalytically inactive TDG or catalytically inactive MBD4. An exemplary catalytically inactive TDG is an N140A mutant of SEQ ID NO: 3872 (human TDG).
• Some exemplary glycosylases are provided below. The catalytically inactivated variants of any of these glycosylase domains are iBERs that may be fused to the napDNAbp or polymerase domain of the prime editors provided in this disclosure.

• OGG (human)

  (SEQ ID NO: 3869)

  MPARALLPRRMGHRTLASTPALWASIPCPRSELRLDLVLPSGQSFRWREQ

  SPAHWSGVLADQVWTLTQTEEQLHCTVYRGDKSQASRPTPDELEAVRKYF

  QLDVTLAQLYHHWGSVDSHFQEVAQKFQGVRLLRQDPIECLFSFICSSNN

  NIARITGMVERLCQAFGPRLIQLDDVTYHGFPSLQALAGPEVEAHLRKLG

  LGYRARYVSASARAILEEQGGLAWLQQLRESSYEEAHKALCILPGVGTKV

  ADCICLMALDKPQAVPVDVHMWHIAQRDYSWHPTTSQAKGPSPQTNKELG

  NFFRSLWGPYAGWAQAVLFSADLRQSRHAQEPPAKRRKGSKGPEG

  MPG (human)

  (SEQ ID NO: 3870)

  MVTPALQMKKPKQFCRRMGQKKQRPARAGQPHSSSDAAQAPAEQPHSSSD

  AAQAPCPRERCLGPPTTPGPYRSIYFSSPKGHLTRLGLEFFDQPAVPLAR

  AFLGQVLVRRLPNGTELRGRIVETEAYLGPEDEAAHSRGGRQTPRNRGMF

  MKPGTLYVYIIYGMYFCMNISSQGDGACVLLRALEPLEGLETMRQLRSTL

  RKGTASRVLKDRELCSGPSKLCQALAINKSFDQRDLAQDEAVWLERGPLE

  PSEPAVVAAARVGVGHAGEWARKPLRFYVRGSPWVSVVDRVAEQDTQA

  MBD4 (human)

  (SEQ ID NO: 3871)

  MGTTGLESLSLGDRGAAPTVTSSERLVPDPPNDLRKEDVAMELERVGEDE

  EQMMIKRSSECNPLLQEPIASAQFGATAGTECRKSVPCGWERVVKQRLFG

  KTAGRFDVYFISPQGLKFRSKSSLANYLHKNGETSLKPEDFDFTVLSKRG

  IKSRYKDCSMAALTSHLQNQSNNSNWNLRTRSKCKKDVFMPPSSSSELQE

  SRGLSNFTSTHLLLKEDEGVDDVNFRKVRKPKGKVTILKGIPIKKTKKGC

  RKSCSGFVQSDSKRESVCNKADAESEPVAQKSQLDRTVCISDAGACGETL

  SVTSEENSLVKKKERSLSSGSNFCSEQKTSGIINKFCSAKDSEHNEKYED

  TFLESEEIGTKVEVVERKEHLHTDILKRGSEMDNNCSPTRKDFTGEKIFQ

  EDTIPRTQIERRKTSLYFSSKYNKEALSPPRRKAFKKWTPPRSPFNLVQE

  TLFHDPWKLLIATIFLNRTSGKMAIPVLWKFLEKYPSAEVARTADWRDVS

  ELLKPLGLYDLRAKTIVKFSDEYLTKQWKYPIELHGIGKYGNDSYRIFCV

  NEWKQVHPEDHKLNKYHDWLWENHEKLSLS

  TDG (human)

  (SEQ ID NO: 3872)

  MEAENAGSYSLQQAQAFYTFPFQQLMAEAPNMAVVNEQQMPEEVPAPAPA

  QEPVQEAPKGRKRKPRTTEPKQPVEPKKPVESKKSGKSAKSKEKQEKITD

  TFKVKRKVDRFNGVSEAELLTKTLPDILTFNLDIVIIGINPGLMAAYKGH

  HYPGPGNHFWKCLFMSGLSEVQLNHMDDHTLPGKYGIGFTNMVERTTPGS

  KDLSSKEFREGGRILVQKLQKYQPRIAVFNGKCIYEIFSKEVFGVKVKNL

  EFGLQPHKIPDTETLCYVMPSSSARCAQFPRAQDKVHYYIKLKDLRDQLK

  GIERNMDVQEVQYTFDLQLAQEDAKKMAVKEEKYDPGYEAAYGGAYGENP

  CSSEPCGFSSNGLIESVELRGESAFSGIPNGQWMTQSFTDQIPSFSNHCG

  TQEQEEESHA

• In some embodiments, the fusion proteins described herein may comprise one or more heterologous protein domains (e.g., about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more domains in addition to the prime editor components). A fusion protein may comprise any additional protein sequence, and optionally a linker sequence between any two domains. Other exemplary features that may be present are localization sequences, such as cytoplasmic localization sequences, export sequences, such as nuclear export sequences, or other localization sequences, as well as sequence tags that are useful for solubilization, purification, or detection of the fusion proteins.
• Examples of protein domains that may be fused to a prime editor or component thereof (e.g., the napDNAbp domain, the polymerase domain, or the NLS domain) include, without limitation, epitope tags, and reporter gene sequences. Non-limiting examples of epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Examples of reporter genes include, but are not limited to, glutathione-5-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), beta-galactosidase, beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluorescent proteins including blue fluorescent protein (BFP). A prime editor may be fused to a gene sequence encoding a protein or a fragment of a protein that bind DNA molecules or bind other cellular molecules, including, but not limited to, maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD) fusions, GAL4 DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions. Additional domains that may form part of a prime editor are described in US Patent Publication No. 2011/0059502, published Mar. 10, 2011 and incorporated herein by reference in its entirety.
• In an aspect of the disclosure, a reporter gene which includes, but is not limited to, glutathione-5-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT) beta-galactosidase, beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluorescent proteins including blue fluorescent protein (BFP), may be introduced into a cell to encode a gene product which serves as a marker by which to measure the alteration or modification of expression of the gene product. In certain embodiments of the disclosure the gene product is luciferase. In a further embodiment of the disclosure the expression of the gene product is decreased.
• Suitable protein tags provided herein include, but are not limited to, biotin carboxylase carrier protein (BCCP) tags, myc-tags, calmodulin-tags, FLAG-tags, hemagglutinin (HA)-tags, polyhistidine tags, also referred to as histidine tags or His-tags, maltose binding protein (MBP)-tags, nus-tags, glutathione-S-transferase (GST)-tags, green fluorescent protein (GFP)-tags, thioredoxin-tags, S-tags, Softags (e.g., Softag 1, Softag 3), strep-tags, biotin ligase tags, FlAsH tags, V5 tags, and SBP-tags. Additional suitable sequences will be apparent to those of skill in the art. In some embodiments, the fusion protein comprises one or more His tags.
• In some embodiments of the present disclosure, the activity of the prime editing system may be temporally regulated by adjusting the residence time, the amount, and/or the activity of the expressed components of the PE system. For example, as described herein, the PE may be fused with a protein domain that is capable of modifying the intracellular half-life of the PE. In certain embodiments involving two or more vectors (e.g., a vector system in which the components described herein are encoded on two or more separate vectors), the activity of the PE system may be temporally regulated by controlling the timing in which the vectors are delivered. For example, in some embodiments a vector encoding the nuclease system may deliver the PE prior to the vector encoding the template. In other embodiments, the vector encoding the PEgRNA may deliver the guide prior to the vector encoding the PE system. In some embodiments, the vectors encoding the PE system and PEgRNA are delivered simultaneously. In certain embodiments, the simultaneously delivered vectors temporally deliver, e.g., the PE, PEgRNA, and/or second strand guide RNA components. In further embodiments, the RNA (such as, e.g., the nuclease transcript) transcribed from the coding sequence on the vectors may further comprise at least one element that is capable of modifying the intracellular half-life of the RNA and/or modulating translational control. In some embodiments, the half-life of the RNA may be increased. In some embodiments, the half-life of the RNA may be decreased. In some embodiments, the element may be capable of increasing the stability of the RNA. In some embodiments, the element may be capable of decreasing the stability of the RNA. In some embodiments, the element may be within the 3′ UTR of the RNA. In some embodiments, the element may include a polyadenylation signal (PA). In some embodiments, the element may include a cap, e.g., an upstream mRNA or PEgRNA end. In some embodiments, the RNA may comprise no PA such that it is subject to quicker degradation in the cell after transcription. In some embodiments, the element may include at least one AU-rich element (ARE). The AREs may be bound by ARE binding proteins (ARE-BPs) in a manner that is dependent upon tissue type, cell type, timing, cellular localization, and environment. In some embodiments the destabilizing element may promote RNA decay, affect RNA stability, or activate translation. In some embodiments, the ARE may comprise 50 to 150 nucleotides in length. In some embodiments, the ARE may comprise at least one copy of the sequence AUUUA. In some embodiments, at least one ARE may be added to the 3′ UTR of the RNA. In some embodiments, the element may be a Woodchuck Hepatitis Virus (WHP).
• Posttranscriptional Regulatory Element (WPRE), which creates a tertiary structure to enhance expression from the transcript. In further embodiments, the element is a modified and/or truncated WPRE sequence that is capable of enhancing expression from the transcript, as described, for example in Zufferey et al., J Virol, 73(4): 2886-92 (1999) and Flajolet et al., J Virol, 72(7): 6175-80 (1998). In some embodiments, the WPRE or equivalent may be added to the 3′ UTR of the RNA. In some embodiments, the element may be selected from other RNA sequence motifs that are enriched in either fast- or slow-decaying transcripts.
• In some embodiments, the vector encoding the PE or the PEgRNA may be self-destroyed via cleavage of a target sequence present on the vector by the PE system. The cleavage may prevent continued transcription of a PE or a PEgRNA from the vector. Although transcription may occur on the linearized vector for some amount of time, the expressed transcripts or proteins subject to intracellular degradation will have less time to produce off-target effects without continued supply from expression of the encoding vectors.
[6] PEgRNAs
• The prime editing system described herein contemplates the use of any suitable PEgRNAs. The inventors have discovered that the mechanism of target-primed reverse transcription (TPRT) can be leveraged or adapted for conducting precision and versatile CRISPR/Cas-based genome editing through the use of a specially configured guide RNA comprising a reverse transcription (RT) template sequence that codes for the desired nucleotide change. The application refers to this specially configured guide RNA as an “extended guide RNA” or a “PEgRNA” since the RT template sequence can be provided as an extension of a standard or traditional guide RNA molecule. The application contemplates any suitable configuration or arrangement for the extended guide RNA.
• PEgRNA Architecture
• FIG. 3A shows one embodiment of an extended guide RNA usable in the prime editing system disclosed herein whereby a traditional guide RNA (the green portion) includes a ˜20 nt protospacer sequence and a gRNA core region, which binds with the napDNAbp. In this embodiment, the guide RNA includes an extended RNA segment at the 5′ end, i.e., a 5′ extension. In this embodiment, the 5′extension includes a reverse transcription template sequence, a reverse transcription primer binding site, and an optional 5-20 nucleotide linker sequence. As shown in FIGS. 1A-1B, the RT primer binding site hybrizes to the free 3′ end that is formed after a nick is formed in the non-target strand of the R-loop, thereby priming reverse transcriptase for DNA polymerization in the 5′-3′ direction.
• FIG. 3B shows another embodiment of an extended guide RNA usable in the prime editing system disclosed herein whereby a traditional guide RNA (the green portion) includes a ˜20 nt protospacer sequence and a gRNA core, which binds with the napDNAbp. In this embodiment, the guide RNA includes an extended RNA segment at the 3′ end, i.e., a 3′ extension. In this embodiment, the 3′extension includes a reverse transcription template sequence, and a reverse transcription primer binding site. As shown in FIGS. 1C-1D, the RT primer binding site hybrizes to the free 3′ end that is formed after a nick is formed in the non-target strand of the R-loop, thereby priming reverse transcriptase for DNA polymerization in the 5′-3′ direction.
• FIG. 3C shows another embodiment of an extend guide RNA usable in the prime editing system disclosed herein whereby a traditional guide RNA (the green portion) includes a ˜20 nt protospacer sequence and a gRNA core, which binds with the napDNAbp. In this embodiment, the guide RNA includes an extended RNA segment at an intermolecular position within the gRNA core, i.e., an intramolecular extension. In this embodiment, the intramolecular extension includes a reverse transcription template sequence, and a reverse transcription primer binding site. The RT primer binding site hybrizes to the free 3′ end that is formed after a nick is formed in the non-target strand of the R-loop, thereby priming reverse transcriptase for DNA polymerization in the 5′-3′ direction.
• In one embodiment, the position of the intermolecular RNA extension is not in the protospacer sequence of the guide RNA. In another embodiment, the position of the intermolecular RNA extension in the gRNA core. In still another embodiment, the position of the intermolecular RNA extension is any with the guide RNA molecule except within the protospacer sequence, or at a position which disrupts the protospacer sequence.
• In one embodiment, the intermolecular RNA extension is inserted downstream from the 3′ end of the protospacer sequence. In another embodiment, the intermolecular RNA extension is inserted at least 1 nucleotide, at least 2 nucleotides, at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides, at least 22 nucleotides, at least 23 nucleotides, at least 24 nucleotides, at least 25 nucleotides downstream of the 3′ end of the protospacer sequence.
• In other embodiments, the intermolecular RNA extension is inserted into the gRNA, which refers to the portion of the guide RNA corresponding or comprising the tracrRNA, which binds and/or interacts with the Cas9 protein or equivalent thereof (i.e, a different napDNAbp). Preferably the insertion of the intermolecular RNA extension does not disrupt or minimally disrupts the interaction between the tracrRNA portion and the napDNAbp.
• The length of the RNA extension can be any useful length. In various embodiments, the RNA extension is at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides, at least 22 nucleotides, at least 23 nucleotides, at least 24 nucleotides, at least 25 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides, at least 80 nucleotides, at least 90 nucleotides, at least 100 nucleotides, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, or at least 500 nucleotides in length.
• The RT template sequence can also be any suitable length. For example, the RT template sequence can be at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides, at least 80 nucleotides, at least 90 nucleotides, at least 100 nucleotides, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, or at least 500 nucleotides in length.
• In still other embodiments, wherein the reverse transcription primer binding site sequence is at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides, at least 80 nucleotides, at least 90 nucleotides, at least 100 nucleotides, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, or at least 500 nucleotides in length.
• In other embodiments, the optional linker or spacer sequence is at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides, at least 80 nucleotides, at least 90 nucleotides, at least 100 nucleotides, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, or at least 500 nucleotides in length.
• The RT template sequence, in certain embodiments, encodes a single-stranded DNA molecule which is homologous to the non-target strand (and thus, complementary to the corresponding site of the target strand) but includes one or more nucleotide changes. The least one nucleotide change may include one or more single-base nucleotide changes, one or more deletions, and one or more insertions.
• As depicted in FIG. 1G, the synthesized single-stranded DNA product of the RT template sequence is homologous to the non-target strand and contains one or more nucleotide changes. The single-stranded DNA product of the RT template sequence hybridizes in equilibrium with the complementary target strand sequence, thereby displacing the homologous endogenous target strand sequence. The displaced endogenous strand may be referred to in some embodiments as a 5′ endogenous DNA flap species (e.g., see FIG. 1E). This 5′ endogenous DNA flap species can be removed by a 5′ flap endonuclease (e.g., FEN1) and the single-stranded DNA product, now hybridized to the endogenous target strand, may be ligated, thereby creating a mismatch between the endogenous sequence and the newly synthesized strand. The mismatch may be resolved by the cell's innate DNA repair and/or replication processes.
• In various embodiments, the nucleotide sequence of the RT template sequence corresponds to the nucleotide sequence of the non-target strand which becomes displaced as the 5′ flap species and which overlaps with the site to be edited.
• In various embodiments of the extended guide RNAs, the reverse transcription template sequence may encode a single-strand DNA flap that is complementary to an endogenous DNA sequence adjacent to a nick site, wherein the single-strand DNA flap comprises a desired nucleotide change. The single-stranded DNA flap may displace an endogenous single-strand DNA at the nick site. The displaced endogenous single-strand DNA at the nick site can have a 5′ end and form an endogenous flap, which can be excised by the cell. In various embodiments, excision of the 5′ end endogenous flap can help drive product formation since removing the 5′ end endogenous flap encourages hybridization of the single-strand 3′ DNA flap to the corresponding complementary DNA strand, and the incorporation or assimilation of the desired nucleotide change carried by the single-strand 3′ DNA flap into the target DNA.
• In various embodiments of the extended guide RNAs, the cellular repair of the single-strand DNA flap results in installation of the desired nucleotide change, thereby forming a desired product.
• In still other embodiments, the desired nucleotide change is installed in an editing window that is between about −5 to +5 of the nick site, or between about −10 to +10 of the nick site, or between about −20 to +20 of the nick site, or between about −30 to +30 of the nick site, or between about −40 to +40 of the nick site, or between about −50 to +50 of the nick site, or between about −60 to +60 of the nick site, or between about −70 to +70 of the nick site, or between about −80 to +80 of the nick site, or between about −90 to +90 of the nick site, or between about −100 to +100 of the nick site, or between about −200 to +200 of the nick site.
• In other embodiments, the desired nucleotide change is installed in an editing window that is between about +1 to +2 from the nick site, or about +1 to +3, +1 to +4, +1 to +5, +1 to +6, +1 to +7, +1 to +8, +1 to +9, +1 to +10, +1 to +11, +1 to +12, +1 to +13, +1 to +14, +1 to +15, +1 to +16, +1 to +17, +1 to +18, +1 to +19, +1 to +20, +1 to +21, +1 to +22, +1 to +23, +1 to +24, +1 to +25, +1 to +26, +1 to +27, +1 to +28, +1 to +29, +1 to +30, +1 to +31, +1 to +32, +1 to +33, +1 to +34, +1 to +35, +1 to +36, +1 to +37, +1 to +38, +1 to +39, +1 to +40, +1 to +41, +1 to +42, +1 to +43, +1 to +44, +1 to +45, +1 to +46, +1 to +47, +1 to +48, +1 to +49, +1 to +50, +1 to +51, +1 to +52, +1 to +53, +1 to +54, +1 to +55, +1 to +56, +1 to +57, +1 to +58, +1 to +59, +1 to +60, +1 to +61, +1 to +62, +1 to +63, +1 to +64, +1 to +65, +1 to +66, +1 to +67, +1 to +68, +1 to +69, +1 to +70, +1 to +71, +1 to +72, +1 to +73, +1 to +74, +1 to +75, +1 to +76, +1 to +77, +1 to +78, +1 to +79, +1 to +80, +1 to +81, +1 to +82, +1 to +83, +1 to +84, +1 to +85, +1 to +86, +1 to +87, +1 to +88, +1 to +89, +1 to +90, +1 to +90, +1 to +91, +1 to +92, +1 to +93, +1 to +94, +1 to +95, +1 to +96, +1 to +97, +1 to +98, +1 to +99, +1 to +100, +1 to +101, +1 to +102, +1 to +103, +1 to +104, +1 to +105, +1 to +106, +1 to +107, +1 to +108, +1 to +109, +1 to +110, +1 to +111, +1 to +112, +1 to +113, +1 to +114, +1 to +115, +1 to +116, +1 to +117, +1 to +118, +1 to +119, +1 to +120, +1 to +121, +1 to +122, +1 to +123, +1 to +124, or +1 to +125 from the nick site.
• In still other embodiments, the desired nucleotide change is installed in an editing window that is between about +1 to +2 from the nick site, or about +1 to +5, +1 to +10, +1 to +15, +1 to +20, +1 to +25, +1 to +30, +1 to +35, +1 to +40, +1 to +45, +1 to +50, +1 to +55, +1 to +100, +1 to +105, +1 to +110, +1 to +115, +1 to +120, +1 to +125, +1 to +130, +1 to +135, +1 to +140, +1 to +145, +1 to +150, +1 to +155, +1 to +160, +1 to +165, +1 to +170, +1 to +175, +1 to +180, +1 to +185, +1 to +190, +1 to +195, or +1 to +200, from the nick site.
• In various aspects, the extended guide RNAs are modified versions of a guide RNA. Guide RNAs maybe naturally occurring, expressed from an encoding nucleic acid, or synthesized chemically. Methods are well known in the art for obtaining or otherwise synthesizing guide RNAs and for determining the appropriate sequence of the guide RNA, including the protospacer sequence which interacts and hybridizes with the target strand of a genomic target site of interest.
• In various embodiments, the particular design aspects of a guide RNA sequence will depend upon the nucleotide sequence of a genomic target site of interest (i.e., the desired site to be edited) and the type of napDNAbp (e.g., Cas9 protein) present in prime editing systems described herein, among other factors, such as PAM sequence locations, percent G/C content in the target sequence, the degree of microhomology regions, secondary structures, etc.
• In general, a guide sequence is any polynucleotide sequence having sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of a napDNAbp (e.g., a Cas9, Cas9 homolog, or Cas9 variant) to the target sequence. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence, when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non-limiting example of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g., the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net). In some embodiments, a guide sequence is about or more than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length.
• In some embodiments, a guide sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides in length. The ability of a guide sequence to direct sequence-specific binding of a prime editor (PE) to a target sequence may be assessed by any suitable assay. For example, the components of a prime editor (PE), including the guide sequence to be tested, may be provided to a host cell having the corresponding target sequence, such as by transfection with vectors encoding the components of a prime editor (PE) disclosed herein, followed by an assessment of preferential cleavage within the target sequence, such as by Surveyor assay as described herein. Similarly, cleavage of a target polynucleotide sequence may be evaluated in a test tube by providing the target sequence, components of a prime editor (PE), including the guide sequence to be tested and a control guide sequence different from the test guide sequence, and comparing binding or rate of cleavage at the target sequence between the test and control guide sequence reactions. Other assays are possible, and will occur to those skilled in the art.
• A guide sequence may be selected to target any target sequence. In some embodiments, the target sequence is a sequence within a genome of a cell. Exemplary target sequences include those that are unique in the target genome. For example, for the S. pyogenes Cas9, a unique target sequence in a genome may include a Cas9 target site of the form MMMMMMMMNNNNNNNNNNNNXGG (SEQ ID NO: 204) where NNNNNNNNNNNNXGG (SEQ ID NO: 205) (N is A, G, T, or C; and X can be anything). A unique target sequence in a genome may include an S. pyogenes Cas9 target site of the form MMMMMMMMMNNNNNNNNNNNXGG (SEQ ID NO: 206) where NNNNNNNNNNNXGG (SEQ ID NO: 207) (N is A, G, T, or C; and X can be anything). For the S. thermophilus CRISPR1Cas9, a unique target sequence in a genome may include a Cas9 target site of the form MMMMMMMMNNNNNNNNNNNNXXAGAAW (SEQ ID NO: 208) where NNNNNNNNNNNNXXAGAAW (SEQ ID NO: 209) (N is A, G, T, or C; X can be anything; and W is A or T). A unique target sequence in a genome may include an S. thermophilus CRISPR 1 Cas9 target site of the form MMMMMMMMMNNNNNNNNNNNXXAGAAW (SEQ ID NO: 210) where NNNNNNNNNNNXXAGAAW (SEQ ID NO: 211) (N is A, G, T, or C; X can be anything; and W is A or T). For the S. pyogenes Cas9, a unique target sequence in a genome may include a Cas9 target site of the form MMMMMMMMNNNNNNNNNNNNXGGXG (SEQ ID NO: 212) where NNNNNNNNNNNNXGGXG (SEQ ID NO: 213) (N is A, G, T, or C; and X can be anything). A unique target sequence in a genome may include an S. pyogenes Cas9 target site of the form MMMMMMMMMNNNNNNNNNNNXGGXG (SEQ ID NO: 214) where NNNNNNNNNNNXGGXG (SEQ ID NO: 215) (N is A, G, T, or C; and X can be anything). In each of these sequences “M” may be A, G, T, or C, and need not be considered in identifying a sequence as unique.
• In some embodiments, a guide sequence is selected to reduce the degree of secondary structure within the guide sequence. Secondary structure may be determined by any suitable polynucleotide folding algorithm. Some programs are based on calculating the minimal Gibbs free energy. An example of one such algorithm is mFold, as described by Zuker and Stiegler (Nucleic Acids Res. 9 (1981), 133-148). Another example folding algorithm is the online webserver RNAfold, developed at Institute for Theoretical Chemistry at the University of Vienna, using the centroid structure prediction algorithm (see e.g. A. R. Gruber et al., 2008, Cell 106(1): 23-24; and PA Carr and GM Church, 2009, Nature Biotechnology 27(12): 1151-62). Further algorithms may be found in U.S. application Ser. No. 61/836,080; Broad Reference BI-2013/004A); incorporated herein by reference.
• In general, a tracr mate sequence includes any sequence that has sufficient complementarity with a tracr sequence to promote one or more of: (1) excision of a guide sequence flanked by tracr mate sequences in a cell containing the corresponding tracr sequence; and (2) formation of a complex at a target sequence, wherein the complex comprises the tracr mate sequence hybridized to the tracr sequence. In general, degree of complementarity is with reference to the optimal alignment of the tracr mate sequence and tracr sequence, along the length of the shorter of the two sequences. Optimal alignment may be determined by any suitable alignment algorithm, and may further account for secondary structures, such as self-complementarity within either the tracr sequence or tracr mate sequence. In some embodiments, the degree of complementarity between the tracr sequence and tracr mate sequence along the length of the shorter of the two when optimally aligned is about or more than about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99%, or higher. In some embodiments, the tracr sequence is about or more than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or more nucleotides in length. In some embodiments, the tracr sequence and tracr mate sequence are contained within a single transcript, such that hybridization between the two produces a transcript having a secondary structure, such as a hairpin. Preferred loop forming sequences for use in hairpin structures are four nucleotides in length, and most preferably have the sequence GAAA. However, longer or shorter loop sequences may be used, as may alternative sequences. The sequences preferably include a nucleotide triplet (for example, AAA), and an additional nucleotide (for example C or G). Examples of loop forming sequences include CAAA and AAAG. In an embodiment of the invention, the transcript or transcribed polynucleotide sequence has at least two or more hairpins. In preferred embodiments, the transcript has two, three, four or five hairpins. In a further embodiment of the invention, the transcript has at most five hairpins. In some embodiments, the single transcript further includes a transcription termination sequence; preferably this is a polyT sequence, for example six T nucleotides. Further non-limiting examples of single polynucleotides comprising a guide sequence, a tracr mate sequence, and a tracr sequence are as follows (listed 5′ to 3′), where “N” represents a base of a guide sequence, the first block of lower case letters represent the tracr mate sequence, and the second block of lower case letters represent the tracr sequence, and the final poly-T sequence represents the transcription terminator:

• (1)

  (SEQ ID NO: 216)

  NNNNNNNNGTTTTTGTACTCTCAAGATTTAGAAATAAATCTTGCAGAAGC

  TACAAAGATAAGGCTTCATGCCGAAATCAACACCCTGTCATTTTATGGCA

  GGGTGTTTTCGTTATTTAATTTTTT;

  (2)

  (SEQ ID NO: 217)

  NNNNNNNNNNNNNNNNNNGTTTTTGTACTCTCAGAAATGCAGAAGCTACA

  AAGATAAGGCTTCATGCCGAAATCAACACCCTGTCATTTTATGGCAGGGT

  GTTTTCGTTATTTAATTTTTT;

  (3)

  (SEQ ID NO: 218)

  NNNNNNNNNNNNNNNNNNNNGTTTTTGTACTCTCAGAAATGCAGAAGCTA

  CAAAGATAAGGCTTCATGCCGAAATCAACACCCTGTCATTTTATGGCAGG

  GTGTTTTTT;

  (4)

  (SEQ ID NO: 219)

  NNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAATAGCAAGTTAAAAT

  AAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTT

  TT;

  (5)

  (SEQ ID NO: 220)

  NNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAATAGCAAGTTAAAAT

  AAGGCTAGTCCGTTATCAACTTGAAAAAGTGTTTTTTT;

  AND

  (6)

  (SEQ ID NO: 221)

  NNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAATAGCAAGTTAAAAT

  AAGGCTAGTCCGTTATCATTTTTTTT.

• In some embodiments, sequences (1) to (3) are used in combination with Cas9 from S. thermophilus CRISPR1. In some embodiments, sequences (4) to (6) are used in combination with Cas9 from S. pyogenes. In some embodiments, the tracr sequence is a separate transcript from a transcript comprising the tracr mate sequence.
• It will be apparent to those of skill in the art that in order to target any of the fusion proteins comprising a Cas9 domain and a single-stranded DNA binding protein, as disclosed herein, to a target site, e.g., a site comprising a point mutation to be edited, it is typically necessary to co-express the fusion protein together with a guide RNA, e.g., an sgRNA. As explained in more detail elsewhere herein, a guide RNA typically comprises a tracrRNA framework allowing for Cas9 binding, and a guide sequence, which confers sequence specificity to the Cas9:nucleic acid editing enzyme/domain fusion protein.
• In some embodiments, the guide RNA comprises a structure 5′-[guide sequence]-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAAGGCUAGUCCGUUAUCAACU UGAAAAAGUGGCACCGAGUCGGUGCUUUUU-3′ (SEQ ID NO: 222), wherein the guide sequence comprises a sequence that is complementary to the target sequence. The guide sequence is typically 20 nucleotides long. The sequences of suitable guide RNAs for targeting Cas9:nucleic acid editing enzyme/domain fusion proteins to specific genomic target sites will be apparent to those of skill in the art based on the instant disclosure. Such suitable guide RNA sequences typically comprise guide sequences that are complementary to a nucleic sequence within 50 nucleotides upstream or downstream of the target nucleotide to be edited. Some exemplary guide RNA sequences suitable for targeting any of the provided fusion proteins to specific target sequences are provided herein. Additional guide sequences are well known in the art and can be used with the prime editor (PE) described herein.
• In other embodiments, the PEgRNAs include those depicted in FIG. 3D.
• In still other embodiments, the PEgRNAs may include those depicted in FIG. 3E.
• FIG. 3D provides the structure of an embodiment of a PEgRNA contemplated herein and which may be designed in accordance with the methodology defined in Example 2. The PEgRNA comprises three main component elements ordered in the 5′ to 3′ direction, namely: a spacer, a gRNA core, and an extension arm at the 3′ end. The extension arm may further be divided into the following structural elements in the 5′ to 3′ direction, namely: a primer binding site (A), an edit template (B), and a homology arm (C). In addition, the PEgRNA may comprise an optional 3′ end modifier region (e1) and an optional 5′ end modifier region (e2). Still further, the PEgRNA may comprise a transcriptional termination signal at the 3′ end of the PEgRNA (not depicted). These structural elements are further defined herein. The depiction of the structure of the PEgRNA is not meant to be limiting and embraces variations in the arrangement of the elements. For example, the optional sequence modifiers (e1) and (e2) could be positioned within or between any of the other regions shown, and not limited to being located at the 3′ and 5′ ends.
• FIG. 3E provides the structure of another embodiment of a PEgRNA contemplated herein and which may be designed in accordance with the methodology defined in Example 2. The PEgRNA comprises three main component elements ordered in the 5′ to 3′ direction, namely: a spacer, a gRNA core, and an extension arm at the 3′ end. The extension arm may further be divided into the following structural elements in the 5′ to 3′ direction, namely: a primer binding site (A), an edit template (B), and a homology arm (C). In addition, the PEgRNA may comprise an optional 3′ end modifier region (e1) and an optional 5′ end modifier region (e2). Still further, the PEgRNA may comprise a transcriptional termination signal on the 3′ end of the PEgRNA (not depicted). These structural elements are further defined herein. The depiction of the structure of the PEgRNA is not meant to be limiting and embraces variations in the arrangement of the elements. For example, the optional sequence modifiers (e1) and (e2) could be positioned within or between any of the other regions shown, and not limited to being located at the 3′ and 5′ ends.
• PEgRNA Improvements
• The PEgRNAs may also include additional design improvements that may modify the properties and/or characteristics of PEgRNAs thereby improving the efficacy of prime editing. In various embodiments, these improvements may belong to one or more of a number of different categories, including but not limited to: (1) designs to enable efficient expression of functional PEgRNAs from non-polymerase III (pol III) promoters, which would enable the expression of longer PEgRNAs without burdensome sequence requirements; (2) improvements to the core, Cas9-binding PEgRNA scaffold, which could improve efficacy; (3) modifications to the PEgRNA to improve RT processivity, enabling the insertion of longer sequences at targeted genomic loci; and (4) addition of RNA motifs to the 5′ or 3′ termini of the PEgRNA that improve PEgRNA stability, enhance RT processivity, prevent misfolding of the PEgRNA, or recruit additional factors important for genome editing.
• In one embodiment, PEgRNA could be designed with polIII promoters to improve the expression of longer-length PEgRNA with larger extension arms. sgRNAs are typically expressed from the U6 snRNA promoter. This promoter recruits pol III to express the associated RNA and is useful for expression of short RNAs that are retained within the nucleus. However, pol III is not highly processive and is unable to express RNAs longer than a few hundred nucleotides in length at the levels required for efficient genome editing. Additionally, pol III can stall or terminate at stretches of U's, potentially limiting the sequence diversity that could be inserted using a PEgRNA. Other promoters that recruit polymerase II (such as pCMV) or polymerase I (such as the U1 snRNA promoter) have been examined for their ability to express longer sgRNAs. However, these promoters are typically partially transcribed, which would result in extra sequence 5′ of the spacer in the expressed PEgRNA, which has been shown to result in markedly reduced Cas9:sgRNA activity in a site-dependent manner. Additionally, while pol III-transcribed PEgRNAs can simply terminate in a run of 6-7 U's, PEgRNAs transcribed from pol II or pol I would require a different termination signal. Often such signals also result in polyadenylation, which would result in undesired transport of the PEgRNA from the nucleus. Similarly, RNAs expressed from pol II promoters such as pCMV are typically 5′-capped, also resulting in their nuclear export.
• Previously, Rinn and coworkers screened a variety of expression platforms for the production of long-noncoding RNA-(lncRNA) tagged sgRNAs¹⁸³. These platforms include RNAs expressed from pCMV and that terminate in the ENE element from the MALAT1 ncRNA from humans¹⁸⁴, the PAN ENE element from KSHV¹⁸⁵, or the 3′ box from U1 snRNA¹⁸⁶. Notably, the MALAT1 ncRNA and PAN ENEs form triple helices protecting the polyA-tail^{184, 187}. These constructs could also enhance RNA stability. It is contemplated that these expression systems will also enable the expression of longer PEgRNAs.
• In addition, a series of methods have been designed for the cleavage of the portion of the pol II promoter that would be transcribed as part of the PEgRNA, adding either a self-cleaving ribozyme such as the hammerhead¹⁸⁸, pistol¹⁸⁹, hatchet¹⁸⁹, hairpin¹⁹⁰, VS¹⁹¹, twister¹⁹², or twister sister¹⁹² ribozymes, or other self-cleaving elements to process the transcribed guide, or a hairpin that is recognized by Csy4¹⁹³ and also leads to processing of the guide. Also, it is hypothesized that incorporation of multiple ENE motifs could lead to improved PEgRNA expression and stability, as previously demonstrated for the KSHV PAN RNA and element¹⁸⁵. It is also anticipated that circularizing the PEgRNA in the form of a circular intronic RNA (ciRNA) could also lead to enhanced RNA expression and stability, as well as nuclear localization¹⁹⁴.
• In various embodiments, the PEgRNA may include various above elements, as exemplified by the following sequence.

• Non-limiting example 1-PEgRNA expression platform

  consisting of pCMV, Csy4 hairpin, the PEgRNA, and

  MALAT1 ENE

  (SEQ ID NO: 223)

  TAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATA

  TGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCG

  CCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGT

  AACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGT

  AAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCC

  CCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTA

  CATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCA

  TCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGA

  TAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAA

  TGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTA

  ACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAG

  GTCTATATAAGCAGAGCTGGTTTAGTGAACCGTCAGATCGTTCACTGCCG

  TATAGGCAGGGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGAAATAGCA

  AGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTC

  GGTCCTCTGCCATCAAAGCGTGCTCAGTCTGTTTTAGGGTCATGAAGGTT

  TTTCTTTTCCTGAGAAAACAACACGTATTGTTTTCTCAGGTTTTGCTTTT

  TGGCCTTTTTCTAGCTTAAAAAAAAAAAAAGCAAAAGATGCTGGTGGTTG

  GCACTCCTGGTTTCCAGGACGGGGTTCAAATCCCTGCGGCGTCTTTGCTT

  TGACT

  Non-limiting example 2-PEgRNA expression platform

  consisting of pCMV, Csy4 hairing, the PEgRNA, and

  PAN ENE

  (SEQ ID NO: 224)

  TAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATA

  TGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCG

  CCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGT

  AACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGT

  AAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCC

  CCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTA

  CATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCA

  TCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGA

  TAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAA

  TGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTA

  ACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAG

  GTCTATATAAGCAGAGCTGGTTTAGTGAACCGTCAGATCGTTCACTGCCG

  TATAGGCAGGGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGAAATAGCA

  AGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTC

  GGTCCTCTGCCATCAAAGCGTGCTCAGTCTGTTTTGTTTTGGCTGGGTTT

  TTCCTTGTTCGCACCGGACACCTCCAGTGACCAGACGGCAAGGTTTTTAT

  CCCAGTGTATATTGGAAAAACATGTTATACTTTTGACAATTTAACGTGCC

  TAGAGCTCAAATTAAACTAATACCATAACGTAATGCAACTTACAACATAA

  ATAAAGGTCAATGTTTAATCCATAAAAAAAAAAAAAAAAAAA

  Non-limiting example 3-PEgRNA expression platform

  consisting of pCMV, Csy4 hairing, the PEgRNA, and

  3xPAN ENE

  (SEQ ID NO: 225)

  TAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATA

  TGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCG

  CCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGT

  AACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGT

  AAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCC

  CCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTA

  CATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCA

  TCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGA

  TAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAA

  TGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTA

  ACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAG

  GTCTATATAAGCAGAGCTGGTTTAGTGAACCGTCAGATCGTTCACTGCCG

  TATAGGCAGGGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGAAATAGCA

  AGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTC

  GGTCCTCTGCCATCAAAGCGTGCTCAGTCTGTTTTGTTTTGGCTGGGTTT

  TTCCTTGTTCGCACCGGACACCTCCAGTGACCAGACGGCAAGGTTTTTAT

  CCCAGTGTATATTGGAAAAACATGTTATACTTTTGACAATTTAACGTGCC

  TAGAGCTCAAATTAAACTAATACCATAACGTAATGCAACTTACAACATAA

  ATAAAGGTCAATGTTTAATCCATAAAAAAAAAAAAAAAAAAAACACACTG

  TTTTGGCTGGGTTTTTCCTTGTTCGCACCGGACACCTCCAGTGACCAGAC

  GGCAAGGTTTTTATCCCAGTGTATATTGGAAAAACATGTTATACTTTTGA

  CAATTTAACGTGCCTAGAGCTCAAATTAAACTAATACCATAACGTAATGC

  AACTTACAACATAAATAAAGGTCAATGTTTAATCCATAAAAAAAAAAAAA

  AAAAAATCTCTCTGTTTTGGCTGGGTTTTTCCTTGTTCGCACCGGACACC

  TCCAGTGACCAGACGGCAAGGTTTTTATCCCAGTGTATATTGGAAAAACA

  TGTTATACTTTTGACAATTTAACGTGCCTAGAGCTCAAATTAAACTAATA

  CCATAACGTAATGCAACTTACAACATAAATAAAGGTCAATGTTTAATCCA

  TAAAAAAAAAAAAAAAAAAA

  Non-limiting example 4-PEgRNA expression platform

  consisting of pCMV, Csy4 hairing, the PEgRNA, and

  3′ box

  (SEQ ID NO: 226)

  TAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATA

  TGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCG

  CCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGT

  AACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGT

  AAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCC

  CCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTA

  CATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCA

  TCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGA

  TAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAA

  TGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTA

  ACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAG

  GTCTATATAAGCAGAGCTGGTTTAGTGAACCGTCAGATCGTTCACTGCCG

  TATAGGCAGGGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGAAATAGCA

  AGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTC

  GGTCCTCTGCCATCAAAGCGTGCTCAGTCTGTTTGTTTCAAAAGTAGACT

  GTACGCTAAGGGTCATATCTTTTTTTGTTTGGTTTGTGTCTTGGTTGGCG

  TCTTAAA

  Non-limiting example 5-PEgRNA expression platform

  consisting of pU1, Csy4 hairping, the PEgRNA, and

  3′ box

  (SEQ ID NO: 227)

  CTAAGGACCAGCTTCTTTGGGAGAGAACAGACGCAGGGGCGGGAGGGAAA

  AAGGGAGAGGCAGACGTCACTTCCCCTTGGCGGCTCTGGCAGCAGATTGG

  TCGGTTGAGTGGCAGAAAGGCAGACGGGGACTGGGCAAGGCACTGTCGGT

  GACATCACGGACAGGGCGACTTCTATGTAGATGAGGCAGCGCAGAGGCTG

  CTGCTTCGCCACTTGCTGCTTCACCACGAAGGAGTTCCCGTGCCCTGGGA

  GCGGGTTCAGGACCGCTGATCGGAAGTGAGAATCCCAGCTGTGTGTCAGG

  GCTGGAAAGGGCTCGGGAGTGCGCGGGGCAAGTGACCGTGTGTGTAAAGA

  GTGAGGCGTATGAGGCTGTGTCGGGGCAGAGGCCCAAGATCTCAGTTCAC

  TGCCGTATAGGCAGGGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGAAA

  TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACC

  GAGTCGGTCCTCTGCCATCAAAGCGTGCTCAGTCTGTTTCAGCAAGTTCA

  GAGAAATCTGAACTTGCTGGATTTTTGGAGCAGGGAGATGGAATAGGAGC

  TTGCTCCGTCCACTCCACGCATCGACCTGGTATTGCAGTACCTCCAGGAA

  CGGTGCACCCACTTTCTGGAGTTTCAAAAGTAGACTGTACGCTAAGGGTC

  ATATCTTTTTTTGTTTGGTTTGTGTCTTGGTTGGCGTCTTAAA.

• In various other embodiments, the PEgRNA may be improved by introducing improvements to the scaffold or core sequences. This can be done by introducing known
• The core, Cas9-binding PEgRNA scaffold can likely be improved to enhance PE activity. Several such approaches have already been demonstrated. For instance, the first pairing element of the scaffold (P1) contains a GTTTT-AAAAC pairing element. Such runs of Ts have been shown to result in pol III pausing and premature termination of the RNA transcript. Rational mutation of one of the T-A pairs to a G-C pair in this portion of P1 has been shown to enhance sgRNA activity, suggesting this approach would also be feasible for PEgRNAs¹⁹⁵. Additionally, increasing the length of P1 has also been shown to enhance sgRNA folding and lead to improved activity¹⁹⁵, suggesting it as another avenue for the improvement of PEgRNA activity. Example improvements to the core can include:

• PEgRNA containing a 6 nt extension to P1

  (SEQ ID NO: 228)

  GGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGCTCATGAAAATGAGCTA

  GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGA

  GTCGGTCCTCTGCCATCAAAGCGTGCTCAGTCTGTTTTTTT

  PEgRNA containing a T-A to G-C mutation within P1

  (SEQ ID NO: 229)

  GGCCCAGACTGAGCACGTGAGTTTGAGAGCTAGAAATAGCAAGTTTAAAT

  AAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCCTCTG

  CCATCAAAGCGTGCTCAGTCTGTTTTTTT

• In various other embodiments, the PEgRNA may be improved by introducing modifications to the edit template region. As the size of the insertion templated by the PEgRNA increases, it is more likely to be degraded by endonucleases, undergo spontaneous hydrolysis, or fold into secondary structures unable to be reverse-transcribed by the RT or that disrupt folding of the PEgRNA scaffold and subsequent Cas9-RT binding. Accordingly, it is likely that modification to the template of the PEgRNA might be necessary to affect large insertions, such as the insertion of whole genes. Some strategies to do so include the incorporation of modified nucleotides within a synthetic or semi-synthetic PEgRNA that render the RNA more resistant to degradation or hydrolysis or less likely to adopt inhibitory secondary structures¹⁹⁶. Such modifications could include 8-aza-7-deazaguanosine, which would reduce RNA secondary structure in G-rich sequences; locked-nucleic acids (LNA) that reduce degradation and enhance certain kinds of RNA secondary structure; 2′-O-methyl, 2′-fluoro, or 2′-O-methoxyethoxy modifications that enhance RNA stability. Such modifications could also be included elsewhere in the PEgRNA to enhance stability and activity. Alternatively or additionally, the template of the PEgRNA could be designed such that it both encodes for a desired protein product and is also more likely to adopt simple secondary structures that are able to be unfolded by the RT. Such simple structures would act as a thermodynamic sink, making it less likely that more complicated structures that would prevent reverse transcription would occur. Finally, one could also split the template into two, separate PEgRNAs. In such a design, a PE would be used to initiate transcription and also recruit a separate template RNA to the targeted site via an RNA-binding protein fused to Cas9 or an RNA recognition element on the PEgRNA itself such as the MS2 aptamer. The RT could either directly bind to this separate template RNA, or initiate reverse transcription on the original PEgRNA before swapping to the second template. Such an approach could enable long insertions by both preventing misfolding of the PEgRNA upon addition of the long template and also by not requiring dissociation of Cas9 from the genome for long insertions to occur, which could possibly be inhibiting PE-based long insertions.
• In still other embodiments, the PEgRNA may be improved by introducing additional RNA motifs at the 5′ and 3′ termini of the PEgRNAs, or even at positions therein between (e.g., in the gRNA core region, or the spacer). Several such motifs—such as the PAN ENE from KSHV and the ENE from MALAT1 were discussed above as possible means to terminate expression of longer PEgRNAs from non-pol III promoters. These elements form RNA triple helices that engulf the polyA tail, resulting in their being retained within the nucleus^184,187 However, by forming complex structures at the 3′ terminus of the PEgRNA that occlude the terminal nucleotide, these structures would also likely help prevent exonuclease-mediated degradation of PEgRNAs.
• Other structural elements inserted at the 3′ terminus could also enhance RNA stability, albeit without enabling termination from non-pol III promoters. Such motifs could include hairpins or RNA quadruplexes that would occlude the 3′ terminus¹⁹⁷, or self-cleaving ribozymes such as HDV that would result in the formation of a 2′-3′-cyclic phosphate at the 3′ terminus and also potentially render the PEgRNA less likely to be degraded by exonucleases¹⁹⁸. Inducing the PEgRNA to cyclize via incomplete splicing—to form a ciRNA—could also increase PEgRNA stability and result in the PEgRNA being retained within the nucleus¹⁹⁴.
• Additional RNA motifs could also improve RT processivity or enhance PEgRNA activity by enhancing RT binding to the DNA-RNA duplex. Addition of the native sequence bound by the RT in its cognate retroviral genome could enhance RT activity¹⁹⁹. This could include the native primer binding site (PBS), polypurine tract (PPT), or kissing loops involved in retroviral genome dimerization and initiation of transcription¹⁹⁹.
• Addition of dimerization motifs—such as kissing loops or a GNRA tetraloop/tetraloop receptor pair²⁰⁰—at the 5′ and 3′ termini of the PEgRNA could also result in effective circularization of the PEgRNA, improving stability. Additionally, it is envisioned that addition of these motifs could enable the physical separation of the PEgRNA spacer and primer, prevention occlusion of the spacer which would hinder PE activity. Short 5′ extensions or 3′ extensions to the PEgRNA that form a small toehold hairpin in the spacer region or along the primer binding site could also compete favorably against the annealing of intracomplementary regions along the length of the PEgRNA, e.g., the interaction between the spacer and the primer binding site that can occur. Finally, kissing loops could also be used to recruit other template RNAs to the genomic site and enable swapping of RT activity from one RNA to the other. As exemplary embodiments of various secondary structures, the PEgRNA depicted in FIG. 3D and FIG. 3E list a number secondary RNA structures that may be engineered into any region of the PEgRNA, including in the terminal portions of the extension arm (i.e., eland e2), as shown.
• Example improvements include, but are not limited to:

• PEgRNA-HDV fusion

  GGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGAAATAGCAAGTTAAA

  ATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCC

  TCTGCCATCAAAGCGTGCTCAGTCTGGGCCGGCATGGTCCCAGCCTCC

  TCGCTGGCGCCGGCTGGGCAACATGCTTCGGCATGGCGAATGGGACTT

  TTTTT (SEQ ID NO: 230)

  PEgRNA-MMLV kissing loop

  GGTGGGAGACGTCCCACCGGCCCAGACTGAGCACGTGAGTTTTAGAGC

  TAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAA

  GTGGGACCGAGTCGGTCCTCTGCCATCAAAGCTTCGACCGTGCTCAGT

  CTGGTGGGAGACGTCCCACCTTTTTTT (SEQ ID NO: 231)

  PEgRNA-VS ribozyme kissing loop

  GAGCAGCATGGCGTCGCTGCTCACGGCCCAGACTGAGCACGTGAGTTT

  TAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTT

  GAAAAAGTGGGACCGAGTCGGTCCTCTGCCATCAAAGCTTCGACCGTG

  CTCAGTCTCCATCAGTTGACACCCTGAGGTTTTTTT

  (SEQ ID NO: 232)

  PEgRNA-GNRA tetraloop/tetraloop receptor

  GCAGACCTAAGTGGUGACATATGGTCTGGGCCCAGACTGAGCACGTGA

  GTTTTAGAGCTAUACGTAGCAAGTTAAAATAAGGCTAGTCCGTTATCA

  ACTTUACGAAGTGGGACCGAGTCGGTCCTCTGCCATCAAAGCTTCGAC

  CGTGCTCAGTCTGCATGCGATTAGAAATAATCGCATGTTTTTTT

  (SEQ ID NO: 233)

  PEgRNA template switching

  secondary RNA-HDV fusion

  TCTGCCATCAAAGCTGCGACCGTGCTCAGTCTGGTGGGAGACGTCCCA

  CCGGCCGGCATGGTCCCAGCCTCCTCGCTGGCGCCGGCTGGGCAACAT

  GCTTCGGCATGGCGAATGGGACTTTTTTT (SEQ ID NO: 234)

• PEgRNA scaffold could be further improved via directed evolution, in an analogous fashion to how SpCas9 and prime editor (PE) have been improved. Directed evolution could enhance PEgRNA recognition by Cas9 or evolved Cas9 variants. Additionally, it is likely that different PEgRNA scaffold sequences would be optimal at different genomic loci, either enhancing PE activity at the site in question, reducing off-target activities, or both. Finally, evolution of PEgRNA scaffolds to which other RNA motifs have been added would almost certainly improve the activity of the fused PEgRNA relative to the unevolved, fusion RNA. For instance, evolution of allosteric ribozymes composed of c-di-GMP-I aptamers and hammerhead ribozymes led to dramatically improved activity²⁰², suggesting that evolution would improve the activity of hammerhead-PEgRNA fusions as well. In addition, while Cas9 currently does not generally tolerate 5′ extension of the sgRNA, directed evolution will likely generate enabling mutations that mitigate this intolerance, allowing additional RNA motifs to be utilized.
• The present disclosure contemplates any such ways to further improve the efficacy of the prime editing systems disclosed here.
• In various embodiments, it may be advantageous to limit the appearance of consecutive sequence of Ts from the extension arm as consecutive series of T's may limit the capacity of the PEgRNA to be transcribed. For example, strings of at least consecutive three T's, at least consecutive four T's, at least consecutive five T's, at least consecutive six T's, at least consecutive seven T's, at least consecutive eight T's, at least consecutive nine T's, at least consecutive ten T's, at least consecutive eleven T's, at least consecutive twelve T's, at least consecutive thirteen T's, at least consecutive fourteen T's, or at least consecutive fifteen T's should be avoided when designing the PEgRNA, or should be at least removed from the final designed sequence. In one embodiment, one can avoid the includes of unwanted strings of consecutive T's in PEgRNA extension arms but avoiding target sites that are rich in consecutive A:T nucleobase pairs.
• Split PEgRNA Designs for Trans Prime Editing
• The instant disclosure also contemplates trans prime editing, which refers to a modified version of prime editing which operates by separating the PEgRNA into two distinct molecules: a guide RNA and a tPERT molecule. The tPERT molecule is programmed to co-localize with the prime editor complex at a target DNA site, bringing the primer binding site and the DNA synthesis template to the prime editor in trans. For example, see FIG. 3G for an embodiment of a trans prime editor (tPE) which shows a two-component system comprising (1) an recruiting protein (RP)-PE:gRNA complex and (2) a tPERT that includes a primer binding site and a DNA synthesis template joined to an RNA-protein recruitment domain (e.g., stem loop or hairpin), wherein the recruiting protein component of the RP-PE:gRNA complex recruits the tPERT to a target site to be edited, thereby associating the PBS and DNA synthesis template with the prime editor in trans. Said another way, the tPERT is engineered to contain (all or part of) the extension arm of a PEgRNA, which includes the primer binding site and the DNA synthesis template. One advantage of this approach is to separate the extension arm of a PEgRNA from the guide RNA, thereby minimizing annealing interactions that tend to occur between the PBS of the extension arm and the spacer sequence of the guide RNA.
• A key feature of trans prime editing is the ability of the trans prime editor to recruit the tPERT to the site of DNA editing, thereby effectively co-localizing all of the functions of a PEgRNA at the site of prime editing. Recruitment can be achieve by installing an RNA-protein recruitment domain, such as a MS2 aptamer, into the tPERT and fusing a corresponding recruiting protein to the prime editor (e.g., via a linker to the napDNAbp or via a linker to the polymerase) that is capable of specifically binding to the RNA-protein recruitment domain, thereby recruiting the tPERT molecule to the prime editor complex. As depicted in the process described in FIG. 3H, the RP-PE:gRNA complex binds to and nicks the target DNA sequence. Then, the recruiting protein (RP) recruits a tPERT to co-localize to the prime editor complex bound to the DNA target site, thereby allowing the primer binding site, located on the tPERT, to bind to the primer sequence on the nicked strand, and subsequently, allowing the polymerase (e.g., RT) to synthesize a single strand of DNA against the DNA synthesis template, located on the tPERT, up through the 5′ end of the tPERT.
• While the tPERT is shown in FIG. 3G and FIG. 3H as comprising the PBS and DNA synthesis template on the 5′ end of the RNA-protein recruitment domain, the tPERT in other configurations may be designed with the PBS and DNA synthesis template located on the 3′ end of the RNA-protein recruitment domain. However, the tPERT with the 5′ extension has the advantage that synthesis of the single strand of DNA will naturally terminate at the 5′ end of the tPERT and thus, does not risk using any portion of the RNA-protein recruitment domain as a template during the DNA synthesis stage of prime editing.
• PEgRNA Design Method
• The present disclosure also relates to methods for designing PEgRNAs.
• In one aspect of design, the design approach can take into account the particular application for which prime editing is being used. For instance, and as exemplified and discussed herein, prime editing can be used, without limitation, to (a) install mutation-correcting changes to a nucleotide sequence, (b) install protein and RNA tags, (c) install immunoepitopes on proteins of interest, (d) install inducible dimerization domains in proteins, (e) install or remove sequences to alter that activity of a biomolecule, (f) install recombinase target sites to direct specific genetic changes, and (g) mutagenesis of a target sequence by using an error-prone RT. In addition to these methods which, in general, insert, change, or delete nucleotide sequences at target sites of interest, prime editors can also be used to construct highly programmable libraries, as well as to conduct cell data recording and lineage tracing studies. In these various uses, there may be as described herein particular design aspects pertaining to the preparation of a PEgRNA that is particularly useful for any given of these applications.
• When designing a PEgRNA for any particular application or use of prime editing, a number of considerations may be taken into account, which include, but are not limited to:
• (a) the target sequence, i.e., the nucleotide sequence in which one or more nucleobase modifications are desired to be installed by the prime editor;
  (b) the location of the cut site within the target sequence, i.e., the specific nucleobase position at which the prime editor will induce a single-stand nick to create a 3′ end RT primer sequence on one side of the nick and the 5′ end endogenous flap on the other side of the nick (which ultimately is removed by FEN1 or equivalent thereto and replaced by the 3′ ssDNA flap. The cut site is analogous to the “edit location” since this what creates the 3′ end RT primer sequence which becomes extended by the RT during RNA-depending DNA polymerization to create the 3′ ssDNA flap containing the desired edit, which then replaces the 5′ endogenous DNA flap in the target sequence.
  (c) the available PAM sequences (including the canonical SpCas9 PAM sites, as well as non-canonical PAM sites recognized by Cas9 variants and equivalents with expanded or differing PAM specificities);
  (d) the spacing between the available PAM sequences and the location of the cut site in the target sequence;
  (e) the particular Cas9, Cas9 variant, or Cas9 equivalent of the prime editor being used;
  (f) the sequence and length of the primer binding site;
  (g) the sequence and length of the edit template;
  (h) the sequence and length of the homology arm;
  (i) the spacer sequence and length; and
  (j) the core sequence.
• The instant disclosure discusses these aspects above.
• In one embodiment, an approach to designing a suitable PEgRNA, and optionally a nicking-sgRNA design guide for second-site nicking, is hereby provided. This embodiment provides a step-by-step set of instructions for designing PEgRNAs and nicking-sgRNAs for prime editing which takes into account one or more of the above considerations. The steps reference the examples shown in FIGS. 70A-70I.
• • 1. Define the target sequence and the edit. Retrieve the sequence of the target DNA region (˜200 bp) centered around the location of the desired edit (point mutation, insertion, deletion, or combination thereof). See FIG. 70A.
  • 2. Locate target PAMs. Identify PAMs in the proximity to the desired edit location. PAMs can be identified on either strand of DNA proximal to the desired edit location. While PAMs close to the edit position are preferred (i.e., wherein the nick site is less than 30 nt from the edit position, or less than 29 nt, 28 nt, 27 nt, 26 nt, 25 nt, 24 nt, 23 nt, 22 nt, 21 nt, 20 nt, 19 nt, 18 nt, 17 nt, 16 nt, 15 nt, 14 nt, 13 nt, 12 nt, 11 nt, 10 nt, 9 nt, 8 nt, 7 nt, 6 nt, 5 nt, 4 nt, 3 nt, or 2 nt from the edit position to the nick site), it is possible to install edits using protospacers and PAMs that place the nick ≥30 nt from the edit position. See FIG. 70B.
  • 3. Locate the nick sites. For each PAM being considered, identify the corresponding nick site and on which strand. For Sp Cas9 H840A nickase, cleavage occurs in the PAM-containing strand between the 3^rd and 4^th bases 5′ to the NGG PAM. All edited nucleotides must exist 3′ of the nick site, so appropriate PAMs must place the nick 5′ to the target edit on the PAM-containing strand. In the example shown below, there are two possible PAMs. For simplicity, the remaining steps will demonstrate the design of a PEgRNA using PAM 1 only. See FIG. 70C.
  • 4. Design the spacer sequence. The protospacer of Sp Cas9 corresponds to the 20 nucleotides 5′ to the NGG PAM on the PAM-containing strand. Efficient Pol III transcription initiation requires a G to be the first transcribed nucleotide. If the first nucleotide of the protospacer is a G, the spacer sequence for the PEgRNA is simply the protospacer sequence. If the first nucleotide of the protospacer is not a G, the spacer sequence of the PEgRNA is G followed by the protospacer sequence. See FIG. 70D.
  • 5. Design a primer binding site (PBS). Using the starting allele sequence, identify the DNA primer on the PAM-containing strand. The 3′ end of the DNA primer is the nucleotide just upstream of the nick site (i.e. the 4^th base 5′ to the NGG PAM for Sp Cas9). As a general design principle for use with PE2 and PE3, a PEgRNA primer binding site (PBS) containing 12 to 13 nucleotides of complementarity to the DNA primer can be used for sequences that contain ˜40-60% GC content. For sequences with low GC content, longer (14- to 15-nt) PBSs should be tested. For sequences with higher GC content, shorter (8- to 11-nt) PBSs should be tested. Optimal PBS sequences should be determined empirically, regardless of GC content. To design a length-p PBS sequence, take the reverse complement of the first p nucleotides 5′ of the nick site in the PAM-containing strand using the starting allele sequence. See FIG. 70E.
  • 6. Design an RT template (or DNA synthesis template). The RT template (or DNA synthesis template where the polymerase is not reverse transcriptase) encodes the designed edit and homology to the sequence adjacent to the edit. In one embodiment, these regions correspond to the DNA synthesis template of FIG. 3D and FIG. 3E, wherein the DNA synthesis template comprises the “edit template” and the “homology arm.” Optimal RT template lengths vary based on the target site. For short-range edits (positions +1 to +6), it is recommended to test a short (9 to 12 nt), a medium (13 to 16 nt), and a long (17 to 20 nt) RT template. For long-range edits (positions +7 and beyond), it is recommended to use RT templates that extend at least 5 nt (preferably 10 or more nt) past the position of the edit to allow for sufficient 3′ DNA flap homology. For long-range edits, several RT templates should be screened to identify functional designs. For larger insertions and deletions (≥5 nt), incorporation of greater 3′ homology (˜20 nt or more) into the RT template is recommended. Editing efficiency is typically impaired when the RT template encodes the synthesis of a G as the last nucleotide in the reverse transcribed DNA product (corresponding to a C in the RT template of the PEgRNA). As many RT templates support efficient prime editing, avoidance of G as the final synthesized nucleotide is recommended when designing RT templates. To design a length-r RT template sequence, use the desired allele sequence and take the reverse complement of the first r nucleotides 3′ of the nick site in the strand that originally contained the PAM. Note that compared to SNP edits, insertion or deletion edits using RT templates of the same length will not contain identical homology. See FIG. 70F.
  • 7. Assemble the full PEgRNA sequence. Concatenate the PEgRNA components in the following order (5′ to 3′): spacer, scaffold, RT template and PBS. See FIG. 70G.
  • 8. Designing nicking-sgRNAs for PE3. Identify PAMs on the non-edited strand upstream and downstream of the edit. Optimal nicking positions are highly locus-dependent and should be determined empirically. In general, nicks placed 40 to 90 nucleotides 5′ to the position across from the PEgRNA-induced nick lead to higher editing yields and fewer indels. A nicking sgRNA has a spacer sequence that matches the 20-nt protospacer in the starting allele, with the addition of a 5′-G if the protospacer does not begin with a G. See FIG. 70H.
  • 9. Designing PE3b nicking-sgRNAs. If a PAM exists in the complementary strand and its corresponding protospacer overlaps with the sequence targeted for editing, this edit could be a candidate for the PE3b system. In the PE3b system, the spacer sequence of the nicking-sgRNA matches the sequence of the desired edited allele, but not the starting allele. The PE3b system operates efficiently when the edited nucleotide(s) falls within the seed region (˜10 nt adjacent to the PAM) of the nicking-sgRNA protospacer. This prevents nicking of the complementary strand until after installation of the edited strand, preventing competition between the PEgRNA and the sgRNA for binding the target DNA. PE3b also avoids the generation of simultaneous nicks on both strands, thus reducing indel formation significantly while maintaining high editing efficiency. PE3b sgRNAs should have a spacer sequence that matches the 20-nt protospacer in the desired allele, with the addition of a 5′ G if needed. See FIG. 70I.
• The above step-by-step process for designing a suitable PEgRNA and a second-site nicking sgRNA is not meant to be limiting in any way. The disclosure contemplates variations of the above-described step-by-step process which would be derivable therefrom by a person of ordinary skill in the art.
[7] Applications Utilizing Prime Editing
• In addition to the development of the prime editing system described herein as a new “search-and-replace” genome editing technology that mediates targeted insertions, deletions, and all 12 possible base-to-base conversions at targeted loci in human cells without requiring double-stranded DNA breaks, or donor DNA templates, the inventors have also contemplated the use of the prime editors in a wide-array of specific applications. For example, and as exemplified and discussed herein, prime editing can be used to (a) install mutation-correcting changes to a nucleotide sequence, (b) install protein and RNA tags, (c) installation of immunoepitopes on proteins of interest, (d) install inducible dimerization domains in proteins, (e) install or remove sequences to alter that activity of a biomolecule, (f) install recombinase target sites to direct specific genetic changes, and (g) mutagenesis of a target sequence by using an error-prone RT. In addition to these methods which, in general, insert, change, or delete nucleotide sequences at target sites of interest, prime editors can also be used to construct highly programmable libraries, as well as to conduct cell data recording and lineage tracing studies. The inventors have also contemplated additional design features of PEgRNAs that are aimed to improve the efficacy of prime editing. Still further, the inventors have conceived of methods for successfully delivering prime editors using vector delivery systems and which involve splitting the napDNAbp using intein domains.
• These specific exemplary uses of prime editing are in no way intended to be limiting. The present Application contemplates any use for prime editing which involves, in general, some form of the installation, removal, and/or modification of one or more nucleobases at a target site in a nucleotide sequence, e.g., a genomic DNA.
• For any of the exemplified uses for prime editing, one may use any prime editor disclosed herein, including PE1, PE2, PE3, and PE3b, or PE-short.
• A. Prime Editing Mechanism
• In various embodiments, prime editing (or “prime editing”) operates by contacting a target DNA molecule (for which a change in the nucleotide sequence is desired to be introduced) with a nucleic acid programmable DNA binding protein (napDNAbp) complexed with an extended guide RNA. In reference to FIG. 1G, the extended guide RNA comprises an extension at the 3′ or 5′ end of the guide RNA, or at an intramolecular location in the guide RNA and encodes the desired nucleotide change (e.g., single nucleotide change, insertion, or deletion). In step (a), the napDNAbp/extended gRNA complex contacts the DNA molecule and the extended gRNA guides the napDNAbp to bind to a target locus. In step (b), a nick in one of the strands of DNA of the target locus is introduced (e.g., by a nuclease or chemical agent), thereby creating an available 3′ end in one of the strands of the target locus. In certain embodiments, the nick is created in the strand of DNA that corresponds to the R-loop strand, i.e., the strand that is not hybridized to the guide RNA sequence, i.e., the “non-target strand.” The nick, however, could be introduced in either of the strands. That is, the nick could be introduced into the R-loop “target strand” (i.e., the strand hybridized to the protospacer sequence of the extended gRNA) or the “non-target strand” (i.e, the strand forming the single-stranded portion of the R-loop and which is complementary to the target strand). In step (c), the 3′ end of the DNA strand (formed by the nick) interacts with the extended portion of the guide RNA in order to prime reverse transcription (i.e, “target-primed RT”). In certain embodiments, the 3′ end DNA strand hybridizes to a specific RT priming sequence on the extended portion of the guide RNA, i.e, the “reverse transcriptase priming sequence.” In step (d), a reverse transcriptase is introduced (as a fusion protein with the napDNAbp or in trans) which synthesizes a single strand of DNA from the 3′ end of the primed site towards the 5′ end of the extended guide RNA. This forms a single-strand DNA flap comprising the desired nucleotide change (e.g., the single base change, insertion, or deletion, or a combination thereof) and which is otherwise homologous to the endogenous DNA at or adjacent to the nick site. In step (e), the napDNAbp and guide RNA are released. Steps (f) and (g) relate to the resolution of the single strand DNA flap such that the desired nucleotide change becomes incorporated into the target locus. This process can be driven towards the desired product formation by removing the corresponding 5′ endogenous DNA flap (e.g., by FEN1 or similar enzyme that is provide in trans, as a fusion with the prime editor, or endogenously provided) that forms once the 3′ single strand DNA flap invades and hybridizes to the endogenous DNA sequence. Without being bound by theory, the cells endogenous DNA repair and replication processes resolves the mismatched DNA to incorporate the nucleotide change(s) to form the desired altered product. The process can also be driven towards product formation with “second strand nicking,” as exemplified in FIG. 1G, or “temporal second strand nicking,” as exemplified in FIG. 11 and discussed herein.
• The process of prime editing may introduce at least one or more of the following genetic changes: transversions, transitions, deletions, and insertions. In addition, prime editing may be implemented for specific applications. For example, and as exemplified and discussed herein, prime editing can be used to (a) install mutation-correcting changes to a nucleotide sequence, (b) install protein and RNA tags, (c) installation of immunoepitopes on proteins of interest, (d) install inducible dimerization domains in proteins, (e) install or remove sequences to alter that activity of a biomolecule, (f) install recombinase target sites to direct specific genetic changes, and (g) mutagenesis of a target sequence by using an error-prone RT. In addition to these methods which, in general, insert, change, or delete nucleotide sequences at target sites of interest, prime editors can also be used to construct highly programmable libraries, as well as to conduct cell data recording and lineage tracing studies. The inventors have also contemplated additional design features of PEgRNAs that are aimed to improve the efficacy of prime editing. Still further, the inventors have conceived of methods for successfully delivering prime editors using vector delivery systems and which involve splitting the napDNAbp using intein domains.
• The term “prime editing system” or “prime editor (PE)” refers the compositions involved in the method of genome editing using target-primed reverse transcription (TPRT) describe herein, including, but not limited to the napDNAbps, reverse transcriptases, fusion proteins (e.g., comprising napDNAbps and reverse transcriptases), extended guide RNAs, and complexes comprising fusion proteins and extended guide RNAs, as well as accessory elements, such as second strand nicking components and 5′ endogenous DNA flap removal endonucleases (e.g., FEN1) for helping to drive the prime editing process towards the edited product formation.
• In another embodiment, the schematic of FIG. 3F depicts the interaction of a typical PEgRNA with a target site of a double stranded DNA and the concomitant production of a 3′ single stranded DNA flap containing the genetic change of interest. The double strand DNA is shown with the top strand in the 3′ to 5′ orientation and the lower strand in the 5′ to 3′ direction. The top strand comprises the “protospacer” and the PAM sequence and is referred to as the “target strand.” The complementary lower strand is referred to as the “non-target strand.” Although not shown, the PEgRNA depicted would be complexed with a Cas9 or equivalent. As shown in the schematic, the spacer of the PEgRNA anneals to a complementary region on the target strand, which is referred to as the protospacer, which is located just downstream of the PAM sequence is approximately 20 nucleotides in length. This interaction forms as DNA/RNA hybrid between the spacer RNA and the protospacer DNA, and induces the formation of an R loop in the region opposite the protospacer. As taught elsewhere herein, the Cas9 protein (not shown) then induces a nick in the non-target strand, as shown. This then leads to the formation of the 3′ ssDNA flap region which, in accordance with *z*, interacts with the 3′ end of the PEgRNA at the primer binding site. The 3′ end of the ssDNA flap (i.e., the reverse transcriptase primer sequence) anneals to the primer binding site (A) on the PEgRNA, thereby priming reverse transcriptase. Next, reverse transcriptase (e.g., provided in trans or provided cis as a fusion protein, attached to the Cas9 construct) then polymerizes a single strand of DNA which is coded for by the edit template (B) and homology arm (C). The polymerization continues towards the 5′ end of the extension arm. The polymerized strand of ssDNA forms a ssDNA 3′ end flap which, as describe elsewhere (e.g., as shown in FIG. 1G), invades the endogenous DNA, displacing the corresponding endogenous strand (which is removed as a 5′ DNA flap of endogenous DNA), and installing the desired nucleotide edit (single nucleotide base pair change, deletions, insertions (including whole genes) through naturally occurring DNA repair/replication rounds.
• This application of prime editing can be further described in Example 1.
• B. Mutagenesis Using Prime Editing with Error-Prone RT
• In various embodiments, the prime editing system (i.e., prime editing system) may include the use of an error-prone reverse transcriptase for performing targeted mutagenesis, i.e., to mutate only a well-defined stretch of DNA in a genome or other DNA element in a cell. FIG. 22 provides a schematic of an exemplary process for introducing conducting targeted mutagenesis with an error-prone reverse transcriptase on a target locus using a nucleic acid programmable DNA binding protein (napDNAbp) complexed with an extended guide RNA. This process may be referred to as an embodiment of prime editing for targeted mutagenesis. The extended guide RNA comprises an extension at the 3′ or 5′ end of the guide RNA, or at an intramolecular location in the guide RNA. In step (a), the napDNAbp/gRNA complex contacts the DNA molecule and the gRNA guides the napDNAbp to bind to the target locus to be mutagenized. In step (b), a nick in one of the strands of DNA of the target locus is introduced (e.g., by a nuclease or chemical agent), thereby creating an available 3′ end in one of the strands of the target locus. In certain embodiments, the nick is created in the strand of DNA that corresponds to the R-loop strand, i.e., the strand that is not hybridized to the guide RNA sequence. In step (c), the 3′ end DNA strand interacts with the extended portion of the guide RNA in order to prime reverse transcription. In certain embodiments, the 3′ ended DNA strand hybridizes to a specific RT priming sequence on the extended portion of the guide RNA. In step (d), an error-prone reverse transcriptase is introduced which synthesizes a mutagenized single strand of DNA from the 3′ end of the primed site towards the 3′ end of the guide RNA. Exemplary mutations are indicated with an asterisk “*”. This forms a single-strand DNA flap comprising the desired mutagenized region. In step (e), the napDNAbp and guide RNA are released. Steps (f) and (g) relate to the resolution of the single strand DNA flap (comprising the mutagenized region) such that the desired mutagenized region becomes incorporated into the target locus. This process can be driven towards the desired product formation by removing the corresponding 5′ endogenous DNA flap that forms once the 3′ single strand DNA flap invades and hybridizes to the complementary sequence on the other strand. The process can also be driven towards product formation with second strand nicking, as exemplified in FIG. 1F. Following endogenous DNA repair and/or replication processes, the mutagenized region becomes incorporated into both strands of DNA of the DNA locus.
• This application of prime editing can be further described in Example 2.
• Error-prone or mutagenic RT enzymes are known in the art. As used herein, the term “error-prone” reverse transcriptase refers to a reverse transcriptase enzyme that occurs naturally or which has been derived from another reverse transcriptase (e.g., a wild type M-MLV reverse transcriptase) which has an error rate that is less than the error rate of wild type M-MLV reverse transcriptase. The error rate of wild type M-MLV reverse transcriptase is reported to be in the range of one error in 15,000 to 27,000 nucleobase incorporations. See Boutabout et al. (2001) “DNA synthesis fidelity by the reverse transcriptase of the yeast retrotransposon Ty1,” Nucleic Acids Res 29(11):2217-2222, which is incorporated herein by reference. Thus, for purposes of this application, the term “error prone” refers to those RT that have an error rate that is greater than one error in 15,000 nucleobase incorporation (6.7×10⁻⁵ or higher), e.g., 1 error in 14,000 nucleobases (7.14×10⁻⁵ or higher), 1 error in 13,000 nucleobases or fewer (7.7×10⁻⁵ or higher), 1 error in 12,000 nucleobases or fewer (7.7×10⁻⁵ or higher), 1 error in 11,000 nucleobases or fewer (9.1×10⁻⁵ or higher), 1 error in 10,000 nucleobases or fewer (1×10⁻⁴ or 0.0001 or higher), 1 error in 9,000 nucleobases or fewer (0.00011 or higher), 1 error in 8,000 nucleobases or fewer (0.00013 or higher) 1 error in 7,000 nucleobases or fewer (0.00014 or higher), 1 error in 6,000 nucleobases or fewer (0.00016 or higher), 1 error in 5,000 nucleobases or fewer (0.0002 or higher), 1 error in 4,000 nucleobases or fewer (0.00025 or higher), 1 error in 3,000 nucleobases or fewer (0.00033 or higher), 1 error in 2,000 nucleobase or fewer (0.00050 or higher), or 1 error in 1,000 nucleobases or fewer (0.001 or higher), or 1 error in 500 nucleobases or fewer (0.002 or higher), or 1 error in 250 nucleobases or fewer (0.004 or higher).
• A variety of mutagenic RTs could be envisioned for generation of mutagenized sequences using prime editing. Two such examples are the mutagenic reverse transcriptases from Bordetella phage (see Handa, S., et al. Nucl Acids Res 9711-25 (2018), which is incorporated herein by reference) and Legionella pneumophila (see Arambula, D., et al. Proc Natl Acad Sci USA 8212-7 (2013), which is incorporated by reference). In the case of the RT from Bordetella phage (brt), an accessory protein might need to also be added (bavd) to Cas9—or delivered in trans—as well as additional RNA sequences to the PEgRNA to improve binding of the mutagenic RT to the target site (see Handa, S., et al. Nucl Acids Res 9711-25 (2018)). When using mutagenic RTs, the template region of the PEgRNA might be enriched in adenosines or AAY codons to enhance diversity.
• The amino acid sequence of the mutagenic RT from Bordetella phage is provided as follows Like other RTs disclosed herein, the Brt protein may be fused to a napDNAbp as a fusion protein to form a functional PE.

• Name	Sequence
  brt mutagenic	MGKRHRNLIDQITTWENLLDAYRKTSHGKRRTWGY
  rt	LEFKEYDLANLLALQAELKAGNYERGPYREFLVYE
  	PKPRLISALEFKDRLVQHALCNIVAPIFEAGLLPY
  	TYACRPDKGTHAGVCHVQAELRRTRATHFLKSDFS
  	KFFPSIDRAALYAMIDKKIHCAATRRLLRVVLPDE
  	GVGIPIGSLTSQLFANVYGGAVDRLLHDELKQRHW
  	ARYMDDIVVLGDDPEELRAVFYRLRDFASERLGLK
  	ISHWQVAPVSRGINFLGYRIWPTHKLLRKSSVKRA
  	KRKVANFIKHGEDESLQRFLASWSGHAQWADTHNL
  	FTWMEEQYGIACH (SEQ ID NO: 235)

• In the case of Brt from Bordetella, the PE fusion may also include an additional accessory protein (Bavd). The accessory protein may be fused to the PE fusion protein or provided in trans. The amino acid sequence of Bavd accessory protein is provided as follows:

• Name	Sequence
  bavd accessory	MEPIEEATKCYDQMLIVERYERVISYLYPIAQSI
  protein to brt	PRKHGVAREMFLKCLLGQVELFIVAGKSNQVSKL
  	YAADAGLAMLRFWLRFLAGIQKPHAMTPHQVETA
  	QVLIAEVGRILGSWIARVNRKGQAGK
  	(SEQ ID NO: 236)

• In the case of Brt from Bordetella, the PEgRNA may comprise an additional nucleotide sequence added a PEgRNA, e.g., to the 5′ or 3′ end. Exemplary sequence is as follows, which is originally from the Bordetella phage genome:

• NAME	SEQUENCE
  PEGRNA-	ACCUUCUUGCAUGGCUCUGCCAACGCUACGGCUUGGCGG
  ADDITION	GCUGGCCUUUCCUCAAUAGGUGGUCAGCCGGUUCUGUCC
  1	UGCUUCGGCGAACACGUUACACGGUUCGGCAAAACGUCG
  	AUUACUGAAAAUGGAAAGGCGGGGCCGACUUCAAGGGCA
  	GGCUGGGAAAUAA (SEQ ID NO: 237)

• This PEgRNA addition sequence can be reduced in various ways to shorten the length. For example, the PEgRNA-addition 1 sequence could be reduced to the following exemplary alternative addition sequences:

• NAME	SEQUENCE
  PEGRNA-	ACCUUCUUGCAUGGCUCUGCCAACGCUACGGCUUGGCGG
  ADDITION	GCUGGCCUUUCCUCAAUAGGUGGUCAGCCGGUUCUGUCC
  2	UGCUUCGGCGAACACGUUACACGGUUCGGCAAAACGUCG
  	AUUACUGAAAAUGGAAAGGCGGGGCCGACUUC
  	(SEQ ID NO: 238)
  PEGRNA-	ACCUUCUUGCAUGGCUCUGCCAACGCUACGGCUUGGCGG
  ADDITION	GCUGGCCUUUCCUCAAUAGGUGGUCAAAGGGCAGGCUGG
  3	GAAAUAA (SEQ ID NO: 239)
  PEGRNA-	ACCUUCUUGCAUGGCUCUGCCAACGCUACGGCUUGGCGG
  ADDITION	GCUGGCCUUUCCUCAAUAGGUGGUCA
  4	(SEQ ID NO: 277)
  PEGRNA-	CAUGGCUCUGCCAACGCUACGGCUUGGCGGGCUGGCCUU
  ADDITION	UCCUCAAUAGGUGGUCAGCCGGUUCUGUCCUGCUUCGGC
  5	GAACACGUUACACGGUUCGGCAAAACGUCGAUUACUGAA
  	AAUGGAAAGGCGGGGCCGACUUCAAGGGCAGGCUGGGAA
  	AUAA (SEQ ID NO: 240)
  PEGRNA-	CAUGGCUCUGCCAACGCUACGGCUUGGCGGGCUGGCCUU
  ADDITION	UCCUCAAUAGGUGGUCAGCCGGUUCUGUCCUGCUUCGGC
  6	GAACACGUUACACGGUUCGGCAAAACGUCGAUUACUGAA
  	AAUGGAAAGGCGGGGCCGACUUC
  	(SEQ ID NO: 241)
  PEGRNA-	CAUGGCUCUGCCAACGCUACGGCUUGGCGGGCUGGCCUU
  ADDITION	UCCUCAAUAGGUGGUCAAAGGGCAGGCUGGGAAAUAA
  7	(SEQ ID NO: 242)
  PEGRNA-	CAUGGCUCUGCCAACGCUACGGCUUGGCGGGCUGGCCUU
  ADDITION	UCCUCAAUAGGUGGUCA
  8	(SEQ ID NO: 243)

• In other embodiments, the PEgRNA addition sequence can be also be mutated. For example, the PEgRNA-addition 1 sequence could be mutated to the following exemplary alternative addition sequence:

• NAME	SEQUENCE
  PEGRNA-	ACCUUCUUGCAUGGCUCUGCCAACGCUACGGCUUGGCGG
  ADDITION	GCUGGCCUUUCCUCAAUAGAUGAGCCGCCGGUUCUGUCC
  1	UGCUUCGGCGAACACGUUACACGGUUCGGCAAAACGUCG
  MUTATED	AUUACUGAAAAUGGAAAGGCGGGGCCGACUUCAAGGGCA
  	GGCUGGGAAAUAA (SEQ ID NO: 244)

• In various embodiments relating to the use of PE for introducing mutations, special PEgRNA considerations may apply. For example, without wishing to be bound by theory, the additional PEgRNA sequences described above might be needed to enable efficient mutagenesis via mutagenic RTs.
• Any mutagenic RT may be used with the prime editors disclosed herein. For example, the error-prone RT described in the following references may be used and are incorporated herein by reference:
• Bebenek et al., “Error-prone polymerization by HIV-1 reverse transcriptase. Contribution of template-primer misalignment, miscoding, and termination probability to mutational hot spots,” J. Biol Chem, 1993, 268: 10324-34; and
• Menendez-Arias, “Mutation rates and intrinsic fidelity of retroviral reverse transcriptases,” 2009, Viruses, 1(3): 1137-1165.
• Various error-prone RTs can include, but are not limited to, the following enzymes disclosed in Table 1 of Menendez-Arias et al. (the entire contents of the reference of which are incorporated by reference), as follows:

• 	REPORTED ERROR-RATE
  ERROR-PRONE RT	RANGE
  HIV-1 RT (GROUP M, SUBTYPE B)	0.6 × 10 − 4 TO 2.0 × 10 − 4
  HIV-1 RT (GROUP O)	5.5 × 10 − 5
  SIV AGM RT	2.9 × 10 − 5
  SIV MNE RT	1.6 × 10 − 5 TO 1.2 × 10 − 4
  PFV RT	1.7 × 10 − 4
  FIV RT	6.2 × 10 − 5
  AMV RT	5.9 × 10 − 5
  MO-MLV RT	2.7 × 10 − 5 TO 3.3 × 10 − 5

• C. Use of Prime Editing for Treating Triplet Expansion Disorders
• The prime editing system or prime editing (PE) system described herein may be used to contract trinucleotide repeat mutations (or “triplet expansion diseases”) to treating conditions such as Huntington's disease and other trinucleotide repeat disorders. Trinucleotide repeat expansion disorders are complex, progressive disorders that involve developmental neurobiology and often affect cognition as well as sensori-motor functions. The disorders show genetic anticipation (i.e. increased severity with each generation). The DNA expansions or contractions usually happen meiotically (i.e. during the time of gametogenesis, or early in embryonic development), and often have sex-bias meaning that some genes expand only when inherited through the female, others only through the male. In humans, trinucleotide repeat expansion disorders can cause gene silencing at either the transcriptional or translational level, which essentially knocks out gene function. Alternatively, trinucleotide repeat expansion disorders can cause altered proteins generated with large repetitive amino acid sequences that either abrogate or change protein function, often in a dominant-negative manner (e.g. poly-glutamine diseases).
• Without wishing to be bound by theory, triplet expansion is caused by slippage during DNA replication or during DNA repair synthesis. Because the tandem repeats have identical sequence to one another, base pairing between two DNA strands can take place at multiple points along the sequence. This may lead to the formation of “loop out” structures during DNA replication or DNA repair synthesis. This may lead to repeated copying of the repeated sequence, expanding the number of repeats. Additional mechanisms involving hybrid RNA:DNA intermediates have been proposed. Prime editing may be used to reduce or eliminate these triplet expansion regions by deletion one or more or the offending repeat codon triplets. In an embodiment of this use, FIG. 23 , provides a schematic of a PEgRNA design for contracting or reducing trinucleotide repeat sequences with prime editing.
• Prime editing may be implemented to contract triplet expansion regions by nicking a region upstream of the triplet repeat region with the prime editor comprising a PEgRNA appropriated targeted to the cut site. The prime editor then synthesizes a new DNA strand (ssDNA flap) based on the PEgRNA as a template (i.e., the edit template thereof) that codes for a healthy number of triplet repeats (which depends on the particular gene and disease). The newly synthesized ssDNA strand comprising the healthy triplet repeat sequence also is synthesized to include a short stretch of homology (i.e., the homology arm) that matches the sequence adjacent to the other end of the repeat (red strand). Invasion of the newly synthesized strand, and subsequent replacement of the endogenous DNA with the newly synthesized ssDNA flap, leads to a contracted repeat allele.
• Depending on the particular trinucleotide expansion disorder, the defect-inducing triplet expansions may occur in “trinucleotide repeat expansion proteins.” Trinucleotide repeat expansion proteins are a diverse set of proteins associated with susceptibility for developing a trinucleotide repeat expansion disorder, the presence of a trinucleotide repeat expansion disorder, the severity of a trinucleotide repeat expansion disorder or any combination thereof. Trinucleotide repeat expansion disorders are divided into two categories determined by the type of repeat. The most common repeat is the triplet CAG, which, when present in the coding region of a gene, codes for the amino acid glutamine (Q). Therefore, these disorders are referred to as the polyglutamine (polyQ) disorders and comprise the following diseases: Huntington Disease (HD); Spinobulbar Muscular Atrophy (SBMA); Spinocerebellar Ataxias ( SCA types 1, 2, 3, 6, 7, and 17); and Dentatorubro-Pallidoluysian Atrophy (DRPLA). The remaining trinucleotide repeat expansion disorders either do not involve the CAG triplet or the CAG triplet is not in the coding region of the gene and are, therefore, referred to as the non-polyglutamine disorders. The non-polyglutamine disorders comprise Fragile X Syndrome (FRAXA); Fragile XE Mental Retardation (FRAXE); Friedreich Ataxia (FRDA); Myotonic Dystrophy (DM); and Spinocerebellar Ataxias (SCA types 8, and 12).
• The proteins associated with trinucleotide repeat expansion disorders can be selected based on an experimental association of the protein associated with a trinucleotide repeat expansion disorder to a trinucleotide repeat expansion disorder. For example, the production rate or circulating concentration of a protein associated with a trinucleotide repeat expansion disorder may be elevated or depressed in a population having a trinucleotide repeat expansion disorder relative to a population lacking the trinucleotide repeat expansion disorder. Differences in protein levels may be assessed using proteomic techniques including but not limited to Western blot, immunohistochemical staining, enzyme linked immunosorbent assay (ELISA), and mass spectrometry. Alternatively, the proteins associated with trinucleotide repeat expansion disorders may be identified by obtaining gene expression profiles of the genes encoding the proteins using genomic techniques including but not limited to DNA microarray analysis, serial analysis of gene expression (SAGE), and quantitative real-time polymerase chain reaction (Q-PCR).
• Non-limiting examples of proteins associated with trinucleotide repeat expansion disorders which can be corrected by prime editing include AR (androgen receptor), FMR1 (fragile X mental retardation 1), HTT (huntingtin), DMPK (dystrophia myotonica-protein kinase), FXN (frataxin), ATXN2 (ataxin 2), ATN1 (atrophin 1), FEN1 (flap structure-specific endonuclease 1), TNRC6A (trinucleotide repeat containing 6A), PABPN1 (poly(A) binding protein, nuclear 1), JPH3 (junctophilin 3), MED15 (mediator complex subunit 15), ATXN1 (ataxin 1), ATXN3 (ataxin 3), TBP (TATA box binding protein), CACNA1A (calcium channel, voltage-dependent, P/Q type, alpha 1A subunit), ATXN80S (ATXN8 opposite strand (non-protein coding)), PPP2R2B (protein phosphatase 2, regulatory subunit B, beta), ATXN7 (ataxin 7), TNRC6B (trinucleotide repeat containing 6B), TNRC6C (trinucleotide repeat containing 6C), CELF3 (CUGBP, Elav-like family member 3), MAB21L1 (mab-21-like 1 (C. elegans)), MSH2 (mutS homolog 2, colon cancer, nonpolyposis type 1 (E. coli)), TMEM185A (transmembrane protein 185A), SIX5 (SIX homeobox 5), CNPY3 (canopy 3 homolog (zebrafish)), FRAXE (fragile site, folic acid type, rare, fra(X)(q28) E), GNB2 (guanine nucleotide binding protein (G protein), beta polypeptide 2), RPL14 (ribosomal protein L14), ATXN8 (ataxin 8), INSR (insulin receptor), TTR (transthyretin), EP400 (E1A binding protein p400), GIGYF2 (GRB10 interacting GYF protein 2), OGG1 (8-oxoguanine DNA glycosylase), STC1 (stanniocalcin 1), CNDP1 (carnosine dipeptidase 1 (metallopeptidase M20 family)), C10orf2 (chromosome 10 open reading frame 2), MAML3 mastermind-like 3 (Drosophila), DKC1 (dyskeratosis congenita 1, dyskerin), PAXIP1 (PAX interacting (with transcription-activation domain) protein 1), CASK (calcium/calmodulin-dependent serine protein kinase (MAGUK family)), MAPT (microtubule-associated protein tau), SP1 (Sp1 transcription factor), POLG (polymerase (DNA directed), gamma), AFF2 (AF4/FMR2 family, member 2), THBS1 (thrombospondin 1), TP53 (tumor protein p53), ESR1 (estrogen receptor 1), CGGBP1 (CGG triplet repeat binding protein 1), ABT1 (activator of basal transcription 1), KLK3 (kallikrein-related peptidase 3), PRNP (prion protein), JUN (jun oncogene), KCNN3 (potassium intermediate/small conductance calcium-activated channel, subfamily N, member 3), BAX (BCL2-associated X protein), FRAXA (fragile site, folic acid type, rare, fra(X)(q27.3) A (macroorchidism, mental retardation)), KBTBD10 (kelch repeat and BTB (POZ) domain containing 10), MBNL1 (muscleblind-like (Drosophila)), RAD51 (RAD51 homolog (RecA homolog, E. coli) (S. cerevisiae)), NCOA3 (nuclear receptor coactivator 3), ERDA1 (expanded repeat domain, CAG/CTG 1), TSC1 (tuberous sclerosis 1), COMP (cartilage oligomeric matrix protein), GCLC (glutamate-cysteine ligase, catalytic subunit), RRAD (Ras-related associated with diabetes), MSH3 (mutS homolog 3 (E. coli)), DRD2 (dopamine receptor D2), CD44 (CD44 molecule (Indian blood group)), CTCF (CCCTC-binding factor (zinc finger protein)), CCND1 (cyclin D1), CLSPN (claspin homolog (Xenopus laevis)), MEF2A (myocyte enhancer factor 2A), PTPRU (protein tyrosine phosphatase, receptor type, U), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), TRIM22 (tripartite motif-containing 22), WT1 (Wilms tumor 1), AHR (aryl hydrocarbon receptor), GPX1 (glutathione peroxidase 1), TPMT (thiopurine S-methyltransferase), NDP (Norrie disease (pseudoglioma)), ARX (aristaless related homeobox), MUS81 (MUS81 endonuclease homolog (S. cerevisiae)), TYR (tyrosinase (oculocutaneous albinism IA)), EGR1 (early growth response 1), UNG (uracil-DNA glycosylase), NUMBL (numb homolog (Drosophila)-like), FABP2 (fatty acid binding protein 2, intestinal), EN2 (engrailed homeobox 2), CRYGC (crystallin, gamma C), SRP14 (signal recognition particle 14 kDa (homologous Alu RNA binding protein)), CRYGB (crystallin, gamma B), PDCD1 (programmed cell death 1), HOXA1 (homeobox A1), ATXN2L (ataxin 2-like), PMS2 (PMS2 postmeiotic segregation increased 2 (S. cerevisiae)), GLA (galactosidase, alpha), CBL (Cas-Br-M (murine) ecotropic retroviral transforming sequence), FTH1 (ferritin, heavy polypeptide 1), IL12RB2 (interleukin 12 receptor, beta 2), OTX2 (orthodenticle homeobox 2), HOXA5 (homeobox A5), POLG2 (polymerase (DNA directed), gamma 2, accessory subunit), DLX2 (distal-less homeobox 2), SIRPA (signal-regulatory protein alpha), OTX1 (orthodenticle homeobox 1), AHRR (aryl-hydrocarbon receptor repressor), MANF (mesencephalic astrocyte-derived neurotrophic factor), TMEM158 (transmembrane protein 158 (gene/pseudogene)), and ENSG00000078687.
• The prime editors herein disclosed may be used to contract triplet repeat expansion regions in any of the above-indicated disease proteins, including following polyglutamine triplet expansion disease genes (which show the particular location of the pathogenic repeats that may be removed wholly or in part by prime editing):

• 		POSITION
  		OF
  		PATHO-
  	AFFECTED	GENIC
  TRIPLET EXPANSION DISEASE	GENE	REPEATS
  DENTATORUBROPALLIDOLUYSIAN	ATN1	49-88
  ATROPHY	ATROPHIN-1
  HUNTINGTON'S DISEASE	HTT	36-250
  	THE
  	HUNTINGTIN
  	GENE
  SPINAL AND BULBAR MUSCULAR	AR	38-62
  ATROPHY	ANDROGEN
  	RECEPTOR
  SPINOCEREBELLAR ATAXIA TYPE	ATXN1	49-88
  1	ATAXIN 1
  SPINOCEREBELLAR ATAXIA TYPE	ATXN2	33-77
  2	ATAXIN 2
  SPINOCEREBELLAR ATAXIA TYPE	ATXN3	55-86
  3	ATAXIN 3
  SPINOCEREBELLAR ATAXIA TYPE	CACNA1A	21-30
  6
  SPINOCEREBELLAR ATAXIA TYPE	ATXN7	38-120
  7	ATAXIN 7
  SPINOCEREBELLAR ATAXIA TYPE	TBP	47-63
  17	TATA-
  	BINDING
  	PROTEIN

• The prime editors herein disclosed may also be used to contract triplet repeat expansion regions typically found in the following non-polyglutamine triplet expansion disease genes:

• 		POSITION
  		OF
  		PATHO-
  TRIPLET EXPANSION		GENIC
  DISEASE	AFFECTED GENE	REPEATS

  FRAXA (FRAGILE X	FMR1	230+
  SYNDROME)	FRAGILE X
  	MENTAL
  	RETARDATION
  	PROTEIN
  FXTAS (FRAGILE X-	FMR1	55-200
  ASSOCIATED
  TREMOR/ATAXIA
  SYNDROME)
  FRAXE (FRAGILE XE MENTAL	AFF2		200+
  RETARDATION)
  FRDA (FRIEDREICH'S ATAXIA)	FXN	100+
  	FRATAXIN
  DM1 (MYOTONIC	DMPK		50+
  DYSTROPHY TYPE 1)	MYOTONIN-
  	PROTEIN KINASE
  SCA8 (SPINOCEREBELLAR	SCA8	110-250
  ATAXIA TYPE 8)	ATAXIN 8
  SCA12 (SPINOCEREBELLAR	PPP2R2B	66-78
  ATAXIA TYPE 12)	SERINE/THREONIN
  	PROTEIN
  	PHOSPHATASE
  2A

• Prime editing may be implemented to contract triplet expansion regions using a PEgRNA with an edit template that is designed to delete at least one codon of a triplet expansion region. In other embodiments, the PEgRNAs for using in prime editing for this used to delete at least 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20, or 21, or 22, or 23, or 24, or 25, or 26, or 27, or 28, or 29, or 30, or 31, or 32, or 33, or 34, or 35, or 36, or 37, or 38, or 39, or 40, or 41, or 42, or 43, or 44, or 45, or 46, or 47, or 48, or 49, or 50, or 51, or 52, or 53, or 54, or 55, or 56, or 57, or 58, or 59, or 60, or 61, or 62, or 63, or 64, or 65, or 66, or 67, or 68, or 69, or 70, or 71, or 72, or 73, or 74, or 75, or 76, or 77, or 78, or 79, or 80, or 81, or 82, or 83, or 84, or 85, or 86, or 87, or 88, or 89, or 90, or 91, or 92, or 93, or 94, or 95, or 96, or 97, or 98, or 99, or 100, or more codons from a triplet expansion region in order to arrive at a healthy (i.e., not associated with producing the disease) number of triplet repeats.
• In other embodiments, the PEgRNAs for using in prime editing for this used to delete at least 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20 or more codons from a triplet expansion region in order to arrive at a healthy (i.e., not associated with producing the disease) number of triplet repeats.
• In other embodiments, the PEgRNAs for using in prime editing for this used to delete at least 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or more codons from a triplet expansion region in order to arrive at a healthy (i.e., not associated with producing the disease) number of triplet repeats.
• In other embodiments, the PEgRNAs for using in prime editing for this used to delete at least 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or more codons from a triplet expansion region in order to arrive at a healthy (i.e., not associated with producing the disease) number of triplet repeats.
• Prime editing may be configured to correct any triplet expansion region, such as those described in Budworth et al., “A Brief History of Triplet Repeat Diseases,” Methods Mol Biol, 2013, 1010: 3-17, US 20011/00165540 A1 (Genome editing of genes associated with trinucleotide repeat expansion disorders in animals), US 2016/0355796 A1 (Compositions and methods of use of crispr-cas systems in nucleotide repeat disorders
• In various embodiments, the disclosure provides a prime editing construct suitable for use in a cell having a trinucleotide repeat expansion region in a defective gene comprising (a) a prime editor fusion comprising a napDNAbp and a reverse transcriptase, (b) a PEgRNA comprising a spacer sequence that targets the trinucleotide repeat expansion region and an extension arm comprising an edit template that codes for the removal of the trinucleotide repeat expansion region.
• In various other embodiments, the disclosure provides a method for deleting all or a portion of a trinucleotide repeat expansion region in a defective gene in a cell using prime editing comprising contacting the cell with a prime editor fusion comprising a napDNAbp and a reverse transcriptase and a PEgRNA comprising a spacer sequence that targets the trinucleotide repeat expansion region and an extension arm comprising an edit template that codes for the removal of the trinucleotide repeat expansion region.
• In various embodiments, the trinucleotide repeat comprises repeating CTG, CAG, CGG, CCG, GAA, or TTC trinucleotides.
• In various other embodiments, the tetranucleotide repeats, pentanucleotide repeats, or hexanucleotide repeats.
• D. Use of Prime Editing for Peptide Tagging
• In another aspect, the disclosure provides a method of using the herein described prime editors for genetically grafting one or more peptide tags onto a protein using prime editing, More in particular, the disclosure provides a method for genetically installing one or more peptide tags onto a protein comprising: contacting a target nucleotide sequence encoding the protein with a prime editor configured to insert therein a second nucleotide sequence encoding the one or more peptide tags to result in a recombinant nucleotide sequence that encodes a fusion protein comprising the protein fused to the protein tag.
• In other embodiments, the disclosure provides a method for making a fusion protein comprising a peptide of interest and one or more peptide tags, the method comprising: contacting a target nucleotide sequence encoding the protein with a prime editor configured to insert therein a second nucleotide sequence encoding the one or more peptide tags to result in a recombinant nucleotide sequence that encodes the fusion protein comprising the protein fused to the protein tag.
• In various embodiments, the target nucleotide sequence is a specific gene of interest in a genomic DNA. The gene of interest may encode a protein of interest (e.g., a receptor, an enzyme, a therapeutic protein, a membrane protein, a transport protein, a signal transduction protein, or an immunological protein, etc.). The gene of interest may also encode an RNA molecule, including, but not limited to, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA), antisense RNA, guide RNA, microRNA (miRNA), small interfering RNA (siRNA), and cell-free RNA (cfRNA).
• The peptide tag may be any peptide tag or variant thereof which imparts one or more functions onto a protein for purposes such as separation, purification, visualization, solubilization, or detection. The peptides tags can include “affinity tags” (to facilitate protein purification), “solubilization tags” (to assist in proper folding of proteins), “chromatography tags” (to alter chromatographic properties of proteins), “epitope tags” (to bind to high affinity antibodies), and “fluorescence tags” (to facilitate visualization of proteins in a cell or in vitro). Examples of peptide tags include, but are not limited to the following tags:

• NAME	AMINO ACID SEQUENCE	SEQ ID NO:
  AVITAG TM	GLNDIFEAQKIEWHE	SEQ ID NO: 245
  C-TAG	EPEA	SEQ ID NO: 246
  CALMODULIN-TAG	KRRWKKNFIAVSAAN	SEQ ID NO: 247
  	RFKKISSSGAL
  POLYGLUTAMATE	EEEEEE	SEQ ID NO: 248
  TAG
  E-TAG	GAPVPYPDPLEPR	SEQ ID NO: 249
  FLAG-TAG	DYKDDDDK	SEQ ID NO: 2
  HA-TAG	YPYDVPDYA	SEQ ID NO: 5
  HIS-TAG	H (HIS1)	SEQ ID NO: 252
  	HH (HIS2)	SEQ ID NO: 253
  	HHH (HIS4)	SEQ ID NO: 254
  	HHHH (HIS5)	SEQ ID NO: 255
  	HHHHH (HIS5)	SEQ ID NO: 256
  	HHHHHH (HIS6)	SEQ ID NO: 257
  	HHHHHHH (HIS7)	SEQ ID NO: 258
  	HHHHHHHH (HIS8)	SEQ ID NO: 259
  	HHHHHHHHH (HIS9)	SEQ ID NO: 260
  	HHHHHHHHHH (HIS10)	SEQ ID NO: 261
  	HHHHHHHHHH...H...	SEQ ID NO: 262
  	(HISN,
  	WHEREIN N =1-25)
  MYC-TAG	EQKLISEEDL	SEQ ID NO: 6
  NE-TAG	TKENPRSNQEESYDDNES	SEQ ID NO: 264
  RHO1D4-TAG	TETSQVAPA	SEQ ID NO: 265
  S-TAG	KETAAAKFERQHMDS	SEQ ID NO: 266
  SBP-TAG	MDEKTTGWRGGHVVE	SEQ ID NO: 267
  	GLAGELEQ
  	LRARLEHHPQGQREP
  SOFTAG- 1	SLAELLNAGLGGS	SEQ ID NO: 268
  SOFTAG-2	TQDPSRVG	SEQ ID NO: 269
  SPOT-TAG	PDRVRAVSHWSS	SEQ ID NO: 270
  STREP-TAG	WSHPQFEK	SEQ ID NO: 271
  TC TAG	CCPGCC	SEQ ID NO: 272
  TY TAG	EVHTNQDPLD	SEQ ID NO: 273
  V5 TAG	GKPIPNPLLGLDST	SEQ ID NO: 3
  VSV-TAG	YTDIEMNRLGK	SEQ ID NO: 275
  XPRESS TAG	DLYDDDDK	SEQ ID NO: 276

• Peptide tags may also be the following affinity tags (for separation and/or purification of proteins) (as described in Table 9.9.1 of Kimple et al., “Overview of Affinity Tags for Protein Purification,” Curr Protoc Protein Sci, 2013, 73: Unit-9.9, which is incorporated herein by reference).

• NAME	AMINO ACID SEQUENCE
  AU1 EPITOPE	DTYRYI	SEQ ID NO: 278
  AU5 EPITOPE	TDFYLK	SEQ ID NO: 279
  BACTERIOPHAGE	MASMTGGQQMG	SEQ ID NO: 280
  T7 EPITOPE
  (T7-TAG)
  BLUETONGUE	QYPALT	SEQ ID NO: 281
  VIRUS TAG
  (B-TAG)
  E2 EPITOPE	SSTSSDFRDR	SEQ ID NO: 282
  HISTIDINE	KDHLIHNVHKE	SEQ ID NO: 283
  AFFINITY	FHAHAHNK
  TAG(HAT)
  HSV EPITOPE	QPELAPED	SEQ ID NO: 284
  POLYARGININE	RRRRR	SEQ ID NO: 285
  (ARG-TAG)
  POLYASPARTATE	CCCC	SEQ ID NO: 286
  (ASP-TAG)
  POLYPHENYL-	FFFFFFFFFFF	SEQ ID NO: 287
  ALANINE
  (PHE-TAG)
  S1-TAG	NANNPDWDF	SEQ ID NO: 288
  S-TAG	KETAAAKFERQHMDS	SEQ ID NO: 266
  VSV-G	YTDIEMNRLGK	SEQ ID NO: 275

• In particular embodiments, the peptide tags may include a His⁶ tag, FLAG-tag, V5-tag, GCN4-tag, HA-tag, Myc-Tag, FIAsH/ReAsH-tag, Sortase substrate, pi-clamp.
• In various embodiments, the peptide tags may be used for applications that include protein fluorescent labeling, immunoprecipitation, immunoblotting, immunohistochemistry, protein recruitment, inducible protein degrons, and genome-wide screening.
• In various other embodiments, the peptide tag may include an intein sequence to install protein self-splicing function. As used herein, the term “intein” refers to auto-processing polypeptide domains found in organisms from all domains of life. An intein (intervening protein) carries out a unique auto-processing event known as protein splicing in which it excises itself out from a larger precursor polypeptide through the cleavage of two peptide bonds and, in the process, ligates the flanking extein (external protein) sequences through the formation of a new peptide bond. This rearrangement occurs post-translationally (or possibly co-translationally), as intein genes are found embedded in frame within other protein-coding genes. Furthermore, intein-mediated protein splicing is spontaneous; it requires no external factor or energy source, only the folding of the intein domain. This process is also known as cis-protein splicing, as opposed to the natural process of trans-protein splicing with “split inteins.” Inteins are the protein equivalent of the self-splicing RNA introns (see Perler et al., Nucleic Acids Res. 22:1125-1127 (1994)), which catalyze their own excision from a precursor protein with the concomitant fusion of the flanking protein sequences, known as exteins (reviewed in Perler et al., Curr. Opin. Chem. Biol. 1:292-299 (1997); Perler, F. B. Cell 92(1):1-4 (1998); Xu et al., EMBO J. 15(19):5146-5153 (1996)).
• The mechanism of the protein splicing process has been studied in great detail (Chong, et al., J. Biol. Chem. 1996, 271, 22159-22168; Xu, M-Q & Perler, F. B. EMBO Journal, 1996, 15, 5146-5153) and conserved amino acids have been found at the intein and extein splicing points (Xu, et al., EMBO Journal, 1994, 13 5517-522).
• Inteins can also exist as two fragments encoded by two separately transcribed and translated genes. These so-called split inteins self-associate and catalyze protein-splicing activity in trans. Split inteins have been identified in diverse cyanobacteria and archaea (Caspi et al, Mol Microbiol. 50: 1569-1577 (2003); Choi J. et al, J Mol Biol. 556: 1093-1106 (2006.); Dassa B. et al, Biochemistry. 46:322-330 (2007.); Liu X. and Yang J., J Biol Chem. 275:26315-26318 (2003); Wu H. et al. Proc Natl Acad Sci USA. £5:9226-9231 (1998.); and Zettler J. et al, FEBS Letters. 553:909-914 (2009)), but have not been found in eukaryotes thus far. Recently, a bioinformatic analysis of environmental metagenomic data revealed 26 different loci with a novel genomic arrangement. At each locus, a conserved enzyme coding region is interrupted by a split intein, with a freestanding endonuclease gene inserted between the sections coding for intein subdomains. Among them, five loci were completely assembled: DNA helicases (gp41-1, gp41-8); Inosine-5′-monophosphate dehydrogenase (IMPDH-1); and Ribonucleotide reductase catalytic subunits (NrdA-2 and NrdJ-1). This fractured gene organization appears to be present mainly in phages (Dassa et al, Nucleic Acids Research. 57:2560-2573 (2009)).
• In certain embodiments, the prime editors described herein can be used to insert split-intein tags in two different proteins, causing their intracellular ligation when co-expressed to form a fusion protein. In protein trans-splicing, one precursor protein consists of an N-extein part followed by the N-intein, another precursor protein consists of the C-intein followed by a C-extein part, and a trans-splicing reaction (catalyzed by the N- and C-inteins together) excises the two intein sequences and links the two extein sequences with a peptide bond. Protein trans-splicing, being an enzymatic reaction, can work with very low (e.g., micromolar) concentrations of proteins and can be carried out under physiological conditions.
• The split intein Npu DnaE was characterized as having the highest rate reported for the protein trans-splicing reaction. In addition, the Npu DnaE protein splicing reaction is considered robust and high-yielding with respect to different extein sequences, temperatures from 6 to 37° C., and the presence of up to 6M Urea (Zettler J. et al, FEBS Letters. 553:909-914 (2009); Iwai I. et al, FEBS Letters 550: 1853-1858 (2006)). As expected, when the Cys1 Ala mutation at the N-domain of these inteins was introduced, the initial N to S-acyl shift and therefore protein splicing was blocked. Unfortunately, the C-terminal cleavage reaction was also almost completely inhibited. The dependence of the asparagine cyclization at the C-terminal splice junction on the acyl shift at the N-terminal scissile peptide bond seems to be a unique property common to the naturally split DnaE intein alleles (Zettler J. et al. FEBS Letters. 555:909-914 (2009)).
• Protein trans-splicing, catalyzed by split inteins, provides an entirely enzymatic method for protein ligation. A split-intein is essentially a contiguous intein (e.g. a mini-intein) split into two pieces named N-intein and C-intein, respectively. The N-intein and C-intein of a split intein can associate non-covalently to form an active intein and catalyze the splicing reaction essentially in same way as a contiguous intein does. Split inteins have been found in nature and also engineered in laboratories. As used herein, the term “split intein” refers to any intein in which one or more peptide bond breaks exists between the N-terminal and C-terminal amino acid sequences such that the N-terminal and C-terminal sequences become separate molecules that can non-covalently reassociate, or reconstitute, into an intein that is functional for trans-splicing reactions. Any catalytically active intein, or fragment thereof, may be used to derive a split intein for use in the methods of the invention. For example, in one aspect the split intein may be derived from a eukaryotic intein. In another aspect, the split intein may be derived from a bacterial intein. In another aspect, the split intein may be derived from an archaeal intein. Preferably, the split intein so-derived will possess only the amino acid sequences essential for catalyzing trans-splicing reactions.
• Split inteins may be created from contiguous inteins by engineering one or more split sites in the unstructured loop or intervening amino acid sequence between the −12 conserved beta-strands found in the structure of mini-inteins. Some flexibility in the position of the split site within regions between the beta-strands may exist, provided that creation of the split will not disrupt the structure of the intein, the structured beta-strands in particular, to a sufficient degree that protein splicing activity is lost.
• The prime editors described herein may incorporate peptide tags (including inteins) into the C-terminal end of a protein of interest. In other embodiments, the peptide tags (including inteins) may be incorporated into the N-terminal end of a protein of interest. The peptide tags may also be incorporated into the interior of a protein of interest. The resulting fusion proteins created by the herein described prime editors may have the following structures:
• [protein of interest]-[peptide tag];
• [peptide tag]-[protein of interest]; or
• [protein of interest -N-terminal region]-[peptide tag]-[protein of interest -C-terminal region].
• The principles of guide RNA design for use in peptide tagging throughout may be applied to peptide tagging. For example, in one embodiment, the PEgRNA structure for peptide tagging may have the following structure: 5′-[spacer sequence]-[gRNA core or scaffold]-[extension arm]-3′, wherein the extension arm comprises in the 5′ to 3′ direction, a homology arm, edit template (comprising the sequence that encodes the peptide tag), and a primer binding site. This configuration is depicted in FIG. 3D and in FIG. 24 .
• In another embodiment, the PEgRNA structure for peptide tagging may have the following structure: 5′-[extension arm]-[spacer sequence]-[gRNA core or scaffold]-3′, wherein the extension arm comprises in the 5′ to 3′ direction, a homology arm, edit template (comprising the sequence that encodes the peptide tag), and a primer binding site. This configuration is depicted in FIG. 3E.
• Embodiments of peptide tagging using prime editing is depicted in FIGS. 25 and 26 and described in Example 4.
• E. Use of Prime Editing for Preventing or Treating Prion Disease
• Prime editing can also be used to prevent or halt the progression of prion disease through the installation of one or more protective mutations into prion proteins (PRNP) which become misfolded during the course of disease. Prion diseases or transmissible spongiform encephalopathies (TSEs) are a family of rare progressive neurodegenerative disorders that affect both humans and animals. They are distinguished by long incubation periods, characteristic spongiform changes associated with neuronal loss, and a failure to induce inflammatory response.
• In humans, prion disease includes Creutzfeldt-Jakob Disease (CJD), Variant Creutzfeldt-Jakob Disease (vCJD), Gerstmann-Straussler-Scheinker Syndrome, Fatal Familial Insomnia, and Kuru. In animals, prion disease includes Bovine Spongiform Encephalopathy (BSE or “mad cow disease”), Chronic Wasting Disease (CWD), Scrapie, Transmissible Mink Encephalopathy, Feline Spongiform Encephalopathy, and Ungulate Spongiform Encephalopathy. Prime editing may be used to install protective point mutations into a prion protein in order to prevent or halt the progression of any one of these prion diseases.
• Classic CJD is a human prion disease. It is a neurodegenerative disorder with characteristic clinical and diagnostic features. This disease is rapidly progressive and always fatal. Infection with this disease leads to death usually within 1 year of onset of illness. CJD is a rapidly progressive, invariably fatal neurodegenerative disorder believed to be caused by an abnormal isoform of a cellular glycoprotein known as the prion protein. CJD occurs worldwide and the estimated annual incidence in many countries, including the United States, has been reported to be about one case per million population. The vast majority of CJD patients usually die within 1 year of illness onset. CJD is classified as a transmissible spongiform encephalopathy (TSE) along with other prion diseases that occur in humans and animals. In about 85% of patients, CJD occurs as a sporadic disease with no recognizable pattern of transmission. A smaller proportion of patients (5 to 15%) develop CJD because of inherited mutations of the prion protein gene. These inherited forms include Gerstmann-Straussler-Scheinker syndrome and fatal familial insomnia. No treatment is currently known for CJD.
• Variant Creutzfeldt-Jakob disease (vCJD) is a prion disease that was first described in 1996 in the United Kingdom. There is now strong scientific evidence that the agent responsible for the outbreak of prion disease in cows, bovine spongiform encephalopathy (BSE or ‘mad cow’ disease), is the same agent responsible for the outbreak of vCJD in humans. Variant CJD (vCJD) is not the same disease as classic CJD (often simply called CJD). It has different clinical and pathologic characteristics from classic CJD. Each disease also has a particular genetic profile of the prion protein gene. Both disorders are invariably fatal brain diseases with unusually long incubation periods measured in years, and are caused by an unconventional transmissible agent called a prion. No treatment is currently known for vCJD.
• BSE (bovine spongiform encephalopathy or “mad cow disease”) is a progressive neurological disorder of cattle that results from infection by an unusual transmissible agent called a prion. The nature of the transmissible agent is not well understood. Currently, the most accepted theory is that the agent is a modified form of a normal protein known as prion protein. For reasons that are not yet understood, the normal prion protein changes into a pathogenic (harmful) form that then damages the central nervous system of cattle. There is increasing evidence that there are different strains of BSE: the typical or classic BSE strain responsible for the outbreak in the United Kingdom and two atypical strains (H and L strains). No treatment is currently known for BSE.
• Chronic wasting disease (CWD) is a prion disease that affects deer, elk, reindeer, sika deer and moose. It has been found in some areas of North America, including Canada and the United States, Norway and South Korea. It may take over a year before an infected animal develops symptoms, which can include drastic weight loss (wasting), stumbling, listlessness and other neurologic symptoms. CWD can affect animals of all ages and some infected animals may die without ever developing the disease. CWD is fatal to animals and there are no treatments or vaccines.
• The causative agents of TSEs are believed to be prions. The term “prions” refers to abnormal, pathogenic agents that are transmissible and are able to induce abnormal folding of specific normal cellular proteins called prion proteins that are found most abundantly in the brain. The functions of these normal prion proteins are still not completely understood. The abnormal folding of the prion proteins leads to brain damage and the characteristic signs and symptoms of the disease. Prion diseases are usually rapidly progressive and always fatal.
• As used herein, the term “prion” shall mean an infectious particle known to cause diseases (spongiform encephalopathies) in humans and animals. The term “prion” is a contraction of the words “protein” and “infection” and the particles are comprised largely if not exclusively of PRNP^Sc molecules encoded by a PRNP gene which expresses PRNP^C which changes conformation to become PRNP^Sc. Prions are distinct from bacteria, viruses and viroids. Known prions include those which infect animals to cause scrapie, a transmissible, degenerative disease of the nervous system of sheep and goats as well as bovine spongiform encephalopathies (BSE) or mad cow disease and feline spongiform encephalopathies of cats. Four prion diseases, as discussed above, known to affect humans are (1) kuru, (2) Creutzfeldt-Jakob Disease (CJD), (3) Gerstmann-Strassler-Scheinker Disease (GSS), and (4) fatal familial insomnia (FFI). As used herein prion includes all forms of prions causing all or any of these diseases or others in any animals used—and in particular in humans and in domesticated farm animals.
• In general, and without wishing to be bound by theory, prior diseases are caused by misfolding of prion proteins. Such diseases—often called deposition diseases—the misfolding of the prion proteins can be accounted for as follows. If A is the normally synthesized gene product that carries out an intended physiologic role in a monomeric or oligomeric state, A* is a conformationally activated form of A that is competent to undergo a dramatic conformational change, B is the conformationally altered state that prefers multimeric assemblies (i.e., the misfolded form which forms depositions) and B_n is the multimeric material that is pathogenic and relatively difficult to recycle. For the prion diseases, PRNP^C and PRNP^Sc correspond to states A and B_n where A is largely helical and monomeric and B_n is β-rich and multimeric.
• It is known that certain mutations in prion proteins can be associated with increased risk of prior disease. Conversely, certain mutations in prion proteins can be protective in nature. See Bagynszky et al., “Characterization of mutations in PRNP (prion) gene and their possible roles in neurodegenerative diseases,” Neuropsychiatr Dis Treat., 2018; 14: 2067-2085, the contents of which are incorporated herein by reference.
• PRNP (NCBI RefSeq No. NP_000302.1 (SEQ ID NO: 291))—the human prion protein—is encoded by a 16 kb long gene, located on chromosome 20 (4686151-4701588). It contains two exons, and the exon 2 carries the open reading frame which encodes the 253 amino acid (AA) long PrP protein. Exon 1 is a noncoding exon, which may serve as transcriptional initiation site. The post-translational modifications result in the removal of the first 22 AA N-terminal fragment (NTF) and the last 23 AA C-terminal fragment (CTF). The NTF is cleaved after PrP transport to the endoplasmic reticulum (ER), while the CTF (glycosylphosphatidylinositol [GPI] signal peptide [GPI-SP]) is cleaved by the GPI anchor. GPI anchor could be involved in PrP protein transport. It may also play a role of attachment of prion protein into the outer surface of cell membrane. Normal PrP is composed of a long N-terminal loop (which contains the octapeptide repeat region), two short (3 sheets, three a helices, and a C-terminal region (which contains the GPI anchor). Cleavage of PrP results in a 208 AA long glyocoprotein, anchored in the cell membrane.
• The amino acid sequence of PRNP (NP_000302.1) is as follows:

• 	MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPG
  	GNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWG
  	QGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMS
  	RPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCV
  	NITIKQHTVTTTTKGENFTETDVKMMERVVEQMCITQYERESQAY
  	YQRGSSMVLFSSPPVILLISFLIFLIVG
  	(SEQ ID NO: 291).

• The amino acid sequence of PRNP (NP_000302.1) is encoded by the following nucleotide sequence (NCBI Ref. Seq No. NM_000311.5, “Homo sapiens prion protein (PRNP), transcript variant 1, mRNA), is as follows:

• 	GCGAACCTTGGCTGCTGGATGCTGGTTCTCTTTGTGGCCACATGG
  	AGTGACCTGGGCCTCTGCAAGAAGCGCCCGAAGCCTGGAGGATGG
  	AACACTGGGGGCAGCCGATACCCGGGGCAGGGCAGCCCTGGAGGC
  	AACCGCTACCCACCTCAGGGCGGTGGTGGCTGGGGGCAGCCTCAT
  	GGTGGTGGCTGGGGGCAGCCTCATGGTGGTGGCTGGGGGCAGCCC
  	CATGGTGGTGGCTGGGGACAGCCTCATGGTGGTGGCTGGGGTCAA
  	GGAGGTGGCACCCACAGTCAGTGGAACAAGCCGAGTAAGCCAAAA
  	ACCAACATGAAGCACATGGCTGGTGCTGCAGCAGCTGGGGCAGTG
  	GTGGGGGGCCTTGGCGGCTACATGCTGGGAAGTGCCATGAGCAGG
  	CCCATCATACATTTCGGCAGTGACTATGAGGACCGTTACTATCGT
  	GAAAACATGCACCGTTACCCCAACCAAGTGTACTACAGGCCCATG
  	GATGAGTACAGCAACCAGAACAACTTTGTGCACGACTGCGTCAAT
  	ATCACAATCAAGCAGCACACGGTCACCACAACCACCAAGGGGGAG
  	AACTTCACCGAGACCGACGTTAAGATGATGGAGCGCGTGGTTGAG
  	CAGATGTGTATCACCCAGTACGAGAGGGAATCTCAGGCCTATTAC
  	CAGAGAGGATCGAGCATGGTCCTCTTCTCCTCTCCACCTGTGATC
  	CTCCTGATCTCTTTCCTCATCTTCCTGATAGTGGGATGAGGAAGG
  	TCTTCCTGTTTTCACCATCTTTCTAATCTTTTTCCAGCTTGAGGG
  	AGGCGGTATCCACCTGCAGCCCTTTTAGTGGTGGTGTCTCACTCT
  	TTCTTCTCTCTTTGTCCCGGATAGGCTAATCAATACCCTTGGCAC
  	TGATGGGCACTGGAAAACATAGAGTAGACCTGAGATGCTGGTCAA
  	GCCCCCTTTGATTGAGTTCATCATGAGCCGTTGCTAATGCCAGGC
  	CAGTAAAAGTATAACAGCAAATAACCATTGGTTAATCTGGACTTA
  	TTTTTGGACTTAGTGCAACAGGTTGAGGCTAAAACAAATCTCAGA
  	ACAGTCTGAAATACCTTTGCCTGGATACCTCTGGCTCCTTCAGCA
  	GCTAGAGCTCAGTATACTAATGCCCTATCTTAGTAGAGATTTCAT
  	AGCTATTTAGAGATATTTTCCATTTTAAGAAAACCCGACAACATT
  	TCTGCCAGGTTTGTTAGGAGGCCACATGATACTTATTCAAAAAAA
  	TCCTAGAGATTCTTAGCTCTTGGGATGCAGGCTCAGCCCGCTGGA
  	GCATGAGCTCTGTGTGTACCGAGAACTGGGGTGATGTTTTACTTT
  	TCACAGTATGGGCTACACAGCAGCTGTTCAACAAGAGTAAATATT
  	GTCACAACACTGAACCTCTGGCTAGAGGACATATTCACAGTGAAC
  	ATAACTGTAACATATATGAAAGGCTTCTGGGACTTGAAATCAAAT
  	GTTTGGGAATGGTGCCCTTGGAGGCAACCTCCCATTTTAGATGTT
  	TAAAGGACCCTATATGTGGCATTCCTTTCTTTAAACTATAGGTAA
  	TTAAGGCAGCTGAAAAGTAAATTGCCTTCTAGACACTGAAGGCAA
  	ATCTCCTTTGTCCATTTACCTGGAAACCAGAATGATTTTGACATA
  	CAGGAGAGCTGCAGTTGTGAAAGCACCATCATCATAGAGGATGAT
  	GTAATTAAAAAATGGTCAGTGTGCAAAGAAAAGAACTGCTTGCAT
  	TTCTTTATTTCTGTCTCATAATTGTCAAAAACCAGAATTAGGTCA
  	AGTTCATAGTTTCTGTAATTGGCTTTTGAATCAAAGAATAGGGAG
  	ACAATCTAAAAAATATCTTAGGTTGGAGATGACAGAAATATGATT
  	GATTTGAAGTGGAAAAAGAAATTCTGTTAATGTTAATTAAAGTAA
  	AATTATTCCCTGAATTGTTTGATATTGTCACCTAGCAGATATGTA
  	TTACTTTTCTGCAATGTTATTATTGGCTTGCACTTTGTGAGTATT
  	CTATGTAAAAATATATATGTATATAAAATATATATTGCATAGGAC
  	AGACTTAGGAGTTTTGTTTAGAGCAGTTAACATCTGAAGTGTCTA
  	ATGCATTAACTTTTGTAAGGTACTGAATACTTAATATGTGGGAAA
  	CCCTTTTGCGTGGTCCTTAGGCTTACAATGTGCACTGAATCGTTT
  	CATGTAAGAATCCAAAGTGGACACCATTAACAGGTCTTTGAAATA
  	TGCATGTACTTTATATTTTCTATATTTGTAACTTTGCATGTTCTT
  	GTTTTGTTATATAAAAAAATTGTAAATGTTTAATATCTGACTGAA
  	ATTAAACGAGCGAAGATGAGCACCA (SEQ ID NO: 292)

• Mutation sites relative to PRNP (NP_000302.1) which are linked to CJD and FFI are reported are as follows. These mutations can be removed or installed using the prime editors disclosed herein.

• 	AMINO ACID SEQUENCE OF MUTANT PRNP LINKED TO CJD PRION
  	DISEASE (SEE TABLE 1 OF BAGYNSZKY ET AL., 2018) (RELATIVE TO
  MUTATION	SEQ ID NO: 291 OF PRNP NP_000302.1)
  D178N	MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNR
  	YPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGG
  	GTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHF
  	GSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHNCVNITIKQHTVT
  	TTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPV
  	ILLISFLIFLIVG (SEQ ID NO: 293)
  T188K	MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNR
  	YPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGG
  	GTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHF
  	GSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHKVT
  	TTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPV
  	ILLISFLIFLIVG (SEQ ID NO: 294)
  E196K	MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNR
  	YPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGG
  	GTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHF
  	GSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVT
  	TTTKGKNFTETDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPV
  	ILLISFLIFLIVG (SEQ ID NO: 295)
  E196A	MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNR
  	YPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGG
  	GTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHF
  	GSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVT
  	TTTKGANFTETDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPV
  	(SEQ ID NO: 296)
  E200K	MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNR
  	YPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGG
  	GTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHF
  	GSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVT
  	TTTKGENFTKTDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPV
  	ILLISFLIFLIVG (SEQ ID NO: 297)
  E200G	MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNR
  	YPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGG
  	GTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHF
  	GSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVT
  	TTTKGENFTGTDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPV
  	(SEQ ID NO: 298)
  V203I	MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNR
  	YPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGG
  	GTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHF
  	GSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVT
  	TTTKGENFTETDIKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPVI
  	LLISFLIFLIVG (SEQ ID NO: 299)
  R208H	MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNR
  	YPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGG
  	GTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHF
  	GSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVT
  	TTTKGENFTETDVKMMEHVVEQMCITQYERESQAYYQRGSSMVLFSSPPV
  	ILLISFLIFLIVG (SEQ ID NO: 300)
  V210I	MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNR
  	YPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGG
  	GTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHF
  	GSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVT
  	TTTKGENFTETDVKMMERVIEQMCITQYERESQAYYQRGSSMVLFSSPPVI
  	LLISFLIFLIVG (SEQ ID NO: 301)
  E211Q	MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNR
  	YPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGG
  	GTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHF
  	GSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVT
  	TTTKGENFTETDVKMMERVVQQMCITQYERESQAYYQRGSSMVLFSSPPV
  	ILLISFLIFLIVG (SEQ ID NO: 302)
  M232R	MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNR
  	YPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGG
  	GTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHF
  	GSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVT
  	TTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQRGSSRVLFSSPPVI
  	LLISFLIFLIVG (SEQ ID NO: 303)

• Mutation sites relative to PRNP (NP_000302.1) (SEQ ID NO: 291) which are linked to GSS are reported, as follows:

• 	AMINO ACID SEQUENCE OF MUTANT PRNP LINKED TO GSS PRION
  	DISEASE (SEE TABLE 2 OF BAGYNSZKY ET AL., 2018) (RELATIVE TO
  MUTATION	SEQ ID NO: 291 OF PRNP NP 000302.1)
  P102L	MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNR
  	YPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGG
  	GTHSQWNKLSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHF
  	GSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVT
  	TTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPV
  	ILLISFLIFLIVG (SEQ ID NO: 304)
  P105L	MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNR
  	YPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGG
  	GTHSQWNKPSKLKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHF
  	GSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVT
  	TTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPV
  	ILLISFLIFLIVG (SEQ ID NO: 305)
  A117V	MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNR
  	YPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGG
  	GTHSQWNKPSKPKTNMKHMAGAAVAGAVVGGLGGYMLGSAMSRPIIHF
  	GSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVT
  	TTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPV
  	ILLISFLIFLIVG (SEQ ID NO: 306)
  G131V	MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNR
  	YPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGG
  	GTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLVSAMSRPIIHF
  	GSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVT
  	TTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPV
  	ILLISFLIFLIVG (SEQ ID NO: 307)
  V176G	MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNR
  	YPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGG
  	GTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHF
  	GSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFGHDCVNITIKQHTVT
  	TTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPV
  	ILLISFLIFLIVG (SEQ ID NO: 308)
  H187R	MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNR
  	YPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGG
  	GTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHF
  	GSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQRTVT
  	TTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPV
  	ILLISFLIFLIVG (SEQ ID NO: 309)
  	MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNR
  	YPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGG
  	GTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHF
  	GSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVT
  	TTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPV
  	ILLISFLIFLIVG (SEQ ID NO: 291)
  F198S	MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNR
  	YPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGG
  	GTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHF
  	GSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVT
  	TTTKGENSTETDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPV
  	ILLISFLIFLIVG (SEQ ID NO: 311)
  D202N	MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNR
  	YPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGG
  	GTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHF
  	GSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVT
  	TTTKGENFTETNVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPV
  	ILLISFLIFLIVG (SEQ ID NO: 312)
  Q212P	MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNR
  	YPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGG
  	GTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHF
  	GSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVT
  	TTTKGENFTETDVKMMERVVEPMCITQYERESQAYYQRGSSMVLFSSPPV
  	ILLISFLIFLIVG (SEQ ID NO: 313)
  Q217R	MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNR
  	YPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGG
  	GTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHF
  	GSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVT
  	TTTKGENFTETDVKMMERVVEQMCITRYERESQAYYQRGSSMVLFSSPPV
  	ILLISFLIFLIVG (SEQ ID NO: 314)
  M232T	MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNR
  	YPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGG
  	GTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHF
  	GSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVT
  	TTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQRGSSTVLFSSPPVI
  	LLISFLIFLIVG (SEQ ID NO: 315)

• Mutation sites relative to PRNP (NP_000302.1) (SEQ ID NO: 291) which are linked to a possible protective nature against prion disease, as follows:

• 	AMINO ACID SEQUENCE OF MUTANT PRNP LINKED TO A PROTECTIVE
  	NATURE AGAINST PRION DISEASE (SEE TABLE 4 OF BAGYNSZKY ET
  MUTATION	AL., 2018) (RELATIVE TO SEQ ID NO: 291 OF PRNP NP_000302.1)
  G1275	MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNR
  	YPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGG
  	GTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGSYMLGSAMSRPIIHF
  	GSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVT
  	TTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPV
  	ILLISFLIFLIVG (SEQ ID NO: 316)
  G127V	MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNR
  	YPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGG
  	GTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGVYMLGSAMSRPIIHF
  	GSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVT
  	TTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPV
  	ILLISFLIFLIVG (SEQ ID NO: 317)
  M129V	MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNR
  	YPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGG
  	GTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYVLGSAMSRPIIHF
  	GSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVT
  	TTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPV
  	ILLISFLIFLIVG (SEQ ID NO: 318)
  D167G	MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNR
  	YPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGG
  	GTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHF
  	GSDYEDRYYRENMHRYPNQVYYRPMGEYSNQNNFVHDCVNITIKQHTVT
  	TTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPV
  	ILLISFLIFLIVG (SEQ ID NO: 319)
  D167N	MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNR
  	YPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGG
  	GTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHF
  	GSDYEDRYYRENMHRYPNQVYYRPMNEYSNQNNFVHDCVNITIKQHTVT
  	TTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPV
  	ILLISFLIFLIVG (SEQ ID NO: 320)
  N171S	MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNR
  	YPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGG
  	GTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHF
  	GSDYEDRYYRENMHRYPNQVYYRPMDEYSSQNNFVHDCVNITIKQHTVT
  	TTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPV
  	ILLISFLIFLIVG (SEQ ID NO: 321)
  E219K	MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNR
  	YPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGG
  	GTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHF
  	GSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVT
  	TTTKGENFTETDVKMMERVVEQMCITQYKRESQAYYQRGSSMVLFSSPPV
  	ILLISFLIFLIVG (SEQ ID NO: 322)
  P238S	MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNR
  	YPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGG
  	GTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHF
  	GSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVT
  	TTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSSPV
  	ILLISFLIFLIVG (SEQ ID NO: 323)

• Thus, in various embodiments, prime editing may be used to remove a mutation in PRNP that is linked to prion disease or install a mutation in PRNP that is considered to be protective against prion disease. For example, prime editing may be use to remove or restore a D178N, V180I, T188K, E196K, E196A, E200K, E200G, V203I, R208H, V210I, E211Q, I215V, or M232R mutation in the PRNP protein (relative to PRNP of NP_000302.1) (SEQ ID NO: 291). In other embodiments, prime editing may be use to remove or restore a P102L, P105L, A117V, G131V, V176G, H187R, F198S, D202N, Q212P, Q217R, or M232T mutation in the PRNP protein (relative to PRNP of NP_000302.1) (SEQ ID NO: 291). By removing or correcting for the presence of such mutations in PRNP using prime editing, the risk of prion disease may be reduced or eliminated.
• In other embodiments, prime editing may be used to install a protective mutation in PRNP that is linked to a protective effect against one or more prion diseases. For example, prime editing may be used to install a G127S, G127V, M129V, D167G, D167N, N171S, E219K, or P238S protective mutation in PRNP (relative to PRNP of NP_000302.1) (SEQ ID NO: 291). In still other embodiments, the protective mutation may be any alternate amino acid installed at G127, G127, M129, D167, D167, N171, E219, or P238 in PRNP (relative to PRNP of NP_000302.1) (SEQ ID NO: 291).
• In particular embodiments, prime editing may be used to install a G127V protective mutation in PRNP, as illustrated in FIG. 27 and discussed in Example 5.
• In another embodiment, prime editing may be used to install an E219K protective mutation in PRNP.
• The PRNP protein and the protective mutation site are conserved in mammals, so in addition to treating human disease it could also be used to generate cows and sheep that are immune to prion disease, or even help cure wild populations of animals that are suffering from prion disease. Prime editing has already been used to achieve ˜25% installation of a naturally occurring protective allele in human cells, and previous mouse experiments indicate that this level of installation is sufficient to cause immunity from most prion diseases. This method is the first and potentially only current way to install this allele with such high efficiency in most cell types. Another possible strategy for treatment is to use prime editing to reduce or eliminate the expression of PRNP by installing an early stop codon in the gene.
• Using the principles described herein for PEgRNA design, appropriate PEgRNAs may be designed for installing desired protective mutations, or for removing prion disease-associated mutations from PRNP. For example, the below list of PEgRNAs can be used to install the G127V protective allele and the E219K protective allele in human PRNP, as well as the G127V protective allele in PRNP of various animals.

• HUMAN	GCAGTGGTGGGGGGCCTTGGGTTTTAGAGCTAGAA
  PEGRNA FOR	ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG
  INSTALLATION	AAAAAGTGGCACCGAGTCGGTGCATGTAGACGCCAA
  OF G127V	GGCCCCCCAC(SEQ ID NO: 324)
  IN HUMAN PRNP:
  HUMAN	TGTGTATCACCCAGTACGAGGTTTTAGAGCTAGAAA
  PEGRNA FOR	TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA
  INSTALLATION	AAAAGTGGCACCGAGTCGGTGCAGATTCTCTCTT
  OFE219K	GTACTGGGTGA(SEQ ID NO: 325)
  IN HUMAN PRNP
  COW (BOS	GCAGTGGTAGGGGGCCTTGGGTTTTAGAGCTAGAA
  TAURUS)	ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG
  PEGRNA FOR	AAAAAGTGGCACCGAGTCGGTGCATGTAGACACCAA
  INSTALLATION	GGCCCCCTAC(SEQ ID NO: 326)
  OF G127V
  IN COW PRNP
  HAMSTER	GCCGTGGTGGGGGGCCTTGGGTTTTAGAGCTAGAA
  (MESOCRICETUS	ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG
  AURATUS)	AAAAAGTGGCACCGAGTCGGTGCATGTAGACACCAA
  PEGRNA FOR	GGCCCCCCAC(SEQ ID NO: 327)
  INSTALLATION
  OF G127V IN
  HAMSTER PRNP
  MOUSE (MUS	GCAGTAGTGGGGGGCCTTGGGTTTTAGAGCTAGAA
  MUSCULUS)	ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG
  PEGRNA FOR	AAAAAGTGGCACCGAGTCGGTGCATGTAGACACCAA
  INSTALLATION	GGCCCCCCAC(SEQ ID NO: 328)
  OF G127V IN
  MOUST PRNP
  DEER	GCAGTGGTAGGGGGCCTTGGGTTTTAGAGCTAGAA
  (ODOCOILEUS	ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG
  VIRGINIANUS)
  PEGRNA FOR	AAAAAGTGGCACCGAGTCGGTGCATGTAGACACCAA
  INSTALLATION	GGCCCCCTAC(SEQ ID NO: 329)
  OF G127V IN
  DEER PNRP
  FERRET (MUSTELA	GCGGTTGTGGGGGGCCTGGGGTTTTAGAGCTAGAA
  PUTORIUS FURO)	ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG
  PEGRNA FOR	AAAAAGTGGCACCGAGTCGGTGCATGTAGACGCCCA
  INSTALLATION	GGCCCCCCAC(SEQ ID NO: 330)
  OF G127V IN
  IN FERRET PRNP
  KEY:
  SPACER IS BOLDED.
  SGRNA SCAFFOLD IS NORMAL TEXT.
  RT TEMPLATE IS ONCE UNDERLINED.
  PBS IS TWICE UNDERLINED.

• F. Use of Prime Editing for RNA Tagging
• Prime editing may also be used to manipulate, alter, and otherwise modify the sequences of DNA encoding RNA functions through RNA tagging, and in this way provides a means to indirectly modify the structure and function of RNA. For example, PE can be used to insert motifs that are functional at the RNA level (hereafter RNA motifs) to tag or otherwise manipulate non-coding RNAs or mRNAs. These motifs could serve to increase gene expression, decrease gene expression, alter splicing, change post-transcriptional modification, affect the sub-cellular location of the RNA, enable isolation or determination of the intra- or extra-cellular location of the RNA (using, for instance, fluorescent RNA aptamers such as Spinach, Spinach2, Baby Spinach, or Broccoli), recruit endogenous or exogenous protein or RNA binders, introduce sgRNAs, or induce processing of the RNA, by either self-cleavage or RNAses (see FIG. 28B and Example 6 for further details).
• The following RNA tags or motifs may be inserted into a gene of interest using prime editing with an appropriate PEgRNA (designed using the guidance provided herein) to affect various properties of RNA, including RNA transport, expression level, splicing, and detection.

• 			EXEMPLARY
  			PEGRNA FOR PRIME
  			EDITING INSERTION
  			OF RNA MOTIF INTO
  		FUNCTION/	THE EXEMPLARY
  RNA MOTIF	SEQUENCE OF RNA MOTIF	EFFECT	HEXA GENE*
  POLYOMAVIR	AACTTGTTTATTGCAGCTT	TERMINATION	ATCCTTCCAGTCAG
  US SIMIAN	ATAATGGTTACAAATAAAG	OF	GGCCATGTTTGAGA
  VIRUS 40	CAATAGCATCACAAATTTC	TRANSCRIPTION	GCTAGAAATAGCAA
  (5V40) TYPE1	ACAAATAAAGCATTTTTTT	ON OF THE	GTTTAAATAAGGCT
  	CACTGCATTCTAGTTGTGG	TAGGED	AGTCCGTTATCAAC
  	TTTGTCCAAACTCATCAAT	GENE;	TTGAAAAAGTGGG
  	GTATCTTA (SEQ ID NO:	TRANSPORT	ACCGAGTCGGTCC A
  	331)	OF MRNA	CCTGAACCGTATATC
  		INTO	TAAGATACATTGAT
  		CYTOSOL;	GAGTTTGGACAAA
  		INCREASED	CCACAACTAGAAT
  		RNA	GCAGTGAAAAAAA
  		STABILITY	TGCTTTATTTGTG
  		AND	AAATTTGTGATGC
  		EXPRESSION	TATTGCTTTATTTG
  		OF ENCODED	TAACCATTATAAGC
  		PROTEIN	TGCAATAAACAAG
  			TT CTATGGCCCTGA
  			CTGGAA
  			(SEQ ID NO: 332)
  POLYOMAVIRUS	CCATGGCCCAACTTGTTTA	TERMINATION	ATCCTTCCAGTCAG
  SIMIAN	TTGCAGCTTATAATGGTTA	OF	GGCCATGTTTGAGA
  VIRUS 40	CAAATAAAGCAATAGCAT	TRANSCRIPTION	GCTAGAAATAGCAA
  (SV40) TYPE2	CACAAATTTCACAAATAA	ON OF THE	GTTTAAATAAGGCT
  	AGCATTTTTTTCACTGCAT	TAGGED	AGTCCGTTATCAAC
  	TCTAGTTGTGGTTTGTCCA	GENE;	TTGAAAAAGTGGG
  	AACTCATCAATGTATCTTA	TRANSPORT	ACCGAGTCGGTCC A
  	TCATGTCTGGATCTC (SEQ OF	MRNA	CCTGAACCGTATATC
  	ID NO: 333)	INTO	GAGATCCAGACAT
  		CYTOSOL;	GATAAGATACATT
  		INCREASED	GATGAGTTTGGAC
  		RNA	AAACCACAACTAG
  		STABILITY	AATGCAGTGAAAA
  		AND	AAATGCTTTATTT
  		EXPRESSION	GTGAAATTTGTGA
  		OF ENCODED	TGCTATTGCTTTAT
  		PROTEIN	TTGTAACCATTATA
  			AGCTGCAATAAAC
  			AAGTTGGGCCATG
  			G CTATGGCCCTGAC
  			TGGAA
  			(SEQ ID NO: 334)
  POLYOMAVIRUS	TGATCATAATCAAGCCATA	TERMINATION	ATCCTTCCAGTCAG
  SIMIAN	TCACATCTGTAGAGGTTTA	OF	GGCCATGTTTGAGA
  VIRUS 40	CTTGCTTTAAAAAACCTC	TRANSCRIPTION	GCTAGAAATAGCAA
  (SV40) TYPE3	CACACCTCCCCCTGAACC	ON OF THE	GTTTAAATAAGGCT
  	TGAAACATAAAATGAATG	TAGGED	AGTCCGTTATCAAC
  	CAATTGTTGTTGTTAACTT	GENE;	TTGAAAAAGTGGG
  	GTTTATTGCAGCTTATAAT	TRANSPORT	ACCGAGTCGGTCC A
  	GGTTACAAATAAAGCAAT	OF MRNA	CCTGAACCGTATATC
  	AGCATCACAAATTTCACA	INTO	GCAGATCCAGACA
  	AATAAAGCATTTTTTTCAC	CYTOSOL;	TGATAAGATACATT
  	TGCATTCTAGTTGTGGTTT	INCREASED	GATGAGTTTGGAC
  	GTCCAAACTCATCAATGTA	RNA	AAACCACAACTAG
  	TCTTATCATGTCTGGATCT	STABILITY	AATGCAGTGAAAA
  	GC (SEQ ID NO: 335)	AND	AAATGCTTTATTT
  		EXPRESSION	GTGAAATTTGTGA
  		OF ENCODED	TGCTATTGCTTTAT
  		PROTEIN	TTGTAACCATTATA
  			AGCTGCAATAAAC
  			AAGTTAACAACAA
  			CAATTGCATTCAT
  			TTTATGTTTCAGG
  			TTCAGGGGGAGG
  			TGTGGAGGTTTTT
  			TAAAGCAAGTAAA
  			CCTCTACAGATGT
  			GATATGGCTTGAT
  			TATGATCA CTATGG
  			CCCTGACTGGAA
  			(SEQ ID NO: 336)
  HUMAN	ACGGGTGGCATCCCTGTG	TRANSPORT	ATCCTTCCAGTCAG
  GROWTH	ACCCCTCCCCAGTGCCTC	OF RNA INTO	GGCCATGTTTGAGA
  HORMONE	TCCTGGCCCTGGAAGTTG	CYTOPLASM;	GCTAGAAATAGCAA
  (HGH)	CCACTCCAGTGCCCACCA	ENHANCED	GTTTAAATAAGGCT
  	GCCTTGTCCTAATAAAATT	RNA	AGTCCGTTATCAAC
  	AAGTTGCATCATTTTGTCT	STABILITY	TTGAAAAAGTGGG
  	GACTAGGTGTCCTTCTATA	AND	ACCGAGTCGGTCC A
  	ATATTATGGGGTGGAGGG	EXPRESSION	CCTGAACCGTATATC
  	GGGTGGTATGGAGCAAGG	OF ENCODED	AAGGACAGGGAA
  	GGCAAGTTGGGAAGACA	PROTEIN	GGGAGCAGTGGT
  	ACCTGTAGGGCCTGCGGG		TCACGCCTGTAAT
  	GTCTATTGGGAACCAAGC		CCCAGCAATTTGG
  	TGGAGTGCAGTGGCACAA		GAGGCCAAGGTG
  	TCTTGGCTCACTGCAATCT		GGTAGATCACCTG
  	CCGCCTCCTGGGTTCAAG		AGATTAGGAGTTG
  	CGATTCTCCTGCCTCAGCC		GAGACCAGCCTG
  	TCCCGAGTTGTTGGGATT		GCCAATATGGTGA
  	CCAGGCATGCATGACCAG		AACCCCGTCTCTA
  	GCTCAGCTAATTTTTGTTT		CCAAAAAAACAAA
  	TTTTGGTAGAGACGGGGT		AATTAGCTGAGCC
  	TTCACCATATTGGCCAGGC		TGGTCATGCATGC
  	TGGTCTCCAACTCCTAATC		CTGGAATCCCAAC
  	TCAGGTGATCTACCCACCT		AACTCGGGAGGCT
  	TGGCCTCCCAAATTGCTG		GAGGCAGGAGAAT
  	GGATTACAGGCGTGAACC		CGCTTGAACCCAG
  	ACTGCTCCCTTCCCTGTCC		GAGGCGGAGATTG
  	TT (SEQ ID NO: 337)		CAGTGAGCCAAGA
  			TTGTGCCACTGCA
  			CTCCAGCTTGGTT
  			CCCAATAGACCCC
  			GCAGGCCCTACAG
  			GTTGTCTTCCCAA
  			CTTGCCCCTTGCT
  			CCATACCACCCCC
  			CTCCACCCCATAA
  			TATTATAGAAGGA
  			CACCTAGTCAGAC
  			AAAATGATGCAAC
  			TTAATTTTATTAGG
  			ACAAGGCTGGTG
  			GGCACTGGAGTG
  			GCAACTTCCAGGG
  			CCAGGAGAGGCA
  			CTGGGGAGGGGT
  			CACAGGGATGCCA
  			CCCGT CTATGGCCC
  			TGACTGGAA
  			(SEQ ID NO: 338)
  BOVINE	CGACTGTGCCTTCTAGTTG	TRANSPORT	ATCCTTCCAGTCAG
  GROWTH	CCAGCCATCTGTTGTTTGC	OF RNA INTO	GGCCATGTTTGAGA
  HORMONE	CCCTCCCCCGTGCCTTCCT	CYTOPLASM;	GCTAGAAATAGCAA
  (BGH)	TGACCCTGGAAGGTGCCA	ENHANCED	GTTTAAATAAGGCT
  	CTCCCACTGTCCTTTCCTA	RNA	AGTCCGTTATCAAC
  	ATAAAATGAGGAAATTGC	STABILITY	TTGAAAAAGTGGG
  	ATCGCATTGTCTGAGTAGG	AND	ACCGAGTCGGTCC A
  	TGTCATTCTATTCTGGGGG	EXPRESSION	CCTGAACCGTATATC
  	GTGGGGTGGGGCAGGAC	OF ENCODED	CCATAGAGCCCAC
  	AGCAAGGGGGAGGATTG	PROTEIN	CGCATCCCCAGCA
  	GGAAGACAATAGCAGGCA		TGCCTGCTATTGT
  	TGCTGGGGATGCGGTGGG		CTTCCCAATCCTC
  	CTCTATGG (SEQ ID NO: 339		CCCCTTGCTGTCC
  			TGCCCCACCCCAC
  			CCCCCAGAATAGA
  			ATGACACCTACTC
  			AGACAATGCGATG
  			CAATTTCCTCATTT
  			TATTAGGAAAGGA
  			CAGTGGGAGTGG
  			CACCTTCCAGGGT
  			CAAGGAAGGCAC
  			GGGGGAGGGGCA
  			AACAACAGATGGC
  			TGGCAACTAGAAG
  			GCACAGTCG CTAT
  			GGCCCTGACTGGAA
  			(SEQ ID NO: 340)

  RABBIT	TTCACTCCTCAGGTGCAG	TRANSPORT	ATCCTTCCAGTCAG
  BETA-	GCTGCCTATCAGAAGGTG	OF RNA INTO	GGCCATGTTTGAGA
  GLOBIN	GTGGCTGGTGTGGCCAAT	CYTOPLASM;	GCTAGAAATAGCAA
  (RBGLOB)	GCCCTGGCTCACAAATAC	ENHANCED	GTTTAAATAAGGCT
  	CACTGAGATCTTTTTCCCT	RNA	AGTCCGTTATCAAC
  	CTGCCAAAAATTATGGGG	STABILITY	TTGAAAAAGTGGG
  	ACATCATGAAGCCCCTTG	AND	ACCGAGTCGGTCC A
  	AGCATCTGACTTCTGGCTA	EXPRESSION	CCTGAACCGTATATC
  	ATAAAGGAAATTTATTTTC	OF ENCODED	GATCTCCATAAGA
  	ATTGCAATAGTGTGTTGGA	PROTEIN	GAAGAGGGACAG
  	ATTTTTTGTGTCTCTCACT		CTATGACTGGGAG
  	CGGAAGGACATATGGGAG		TAGTCAGGAGAGG
  	GGCAAATCATTTAAAACAT		AGGAAAAATCTGG
  	CAGAATGAGTATTTGGTTT		CTAGTAAAACATG
  	AGAGTTTGGCAACATATG		TAAGGAAAATTTT
  	CCCATATGCTGGCTGCCAT		AGGGATGTTAAAG
  	GAACAAAGGTTGGCTATA		AAAAAAATAACAC
  	AAGAGGTCATCAGTATATG		AAAACAAAATATA
  	AAACAGCCCCCTGCTGTC		AAAAAAATCTAAC
  	CATTCCTTATTCCATAGAA		CTCAAGTCAAGGC
  	AAGCCTTGACTTGAGGTT		TTTTCTATGGAATA
  	AGATTTTTTTTATATTTTGT		AGGAATGGACAGC
  	TTTGTGTTATTTTTTTCTTT		AGGGGGCTGTTTC
  	AACATCCCTAAAATTTTCC		ATATACTGATGAC
  	TTACATGTTTTACTAGCCA		CTCTTTATAGCCA
  	GATTTTTCCTCCTCTCCTG		ACCTTTGTTCATG
  	ACTACTCCCAGTCATAGCT		GCAGCCAGCATAT
  	GTCCCTCTTCTCTTATGGA		GGGCATATGTTGC
  	GATC (SEQ ID NO: 341)		CAAACTCTAAACC
  			AAATACTCATTCT
  			GATGTTTTAAATG
  			ATTTGCCCTCCCA
  			TATGTCCTTCCGA
  			GTGAGAGACACAA
  			AAAATTCCAACAC
  			ACTATTGCAATGA
  			AAATAAATTTCCTT
  			TATTAGCCAGAAG
  			TCAGATGCTCAAG
  			GGGCTTCATGATG
  			TCCCCATAATTTTT
  			GGCAGAGGGAAA
  			AAGATCTCAGTGG
  			TATTTGTGAGCCA
  			GGGCATTGGCCAC
  			ACCAGCCACCACC
  			TTCTGATAGGCAG
  			CCTGCACCTGAGG
  			AGTGAA CTATGGCC
  			CTGACTGGAA
  			(SEQ ID NO: 342)
  THYMIDINE	GGGGGAGGCTAACTGAAA	TRANSPORT	ATCCTTCCAGTCAG
  KINASE (TK)	CACGGAAGGAGACAATAC	OF RNA INTO	GGCCATGTTTGAGA
  	CGGAAGGAACCCGCGCTA	CYTOPLASM;	GCTAGAAATAGCAA
  	TGACGGCAATAAAAAGAC	ENHANCED	GTTTAAATAAGGCT
  	AGAATAAAACGCACGGGT	RNA	AGTCCGTTATCAAC
  	GTTGGGTCGTTTGTTCATA	STABILITY	TTGAAAAAGTGGG
  	AACGCGGGGTTCGGTCCC	AND	ACCGAGTCGGTCC A
  	AGGGCTGGCACTCTGTCG	EXPRESSION	CCTGAACCGTATATC
  	ATACCCCACCGAGACCCC	OF ENCODED	CTATGGCAGGGCC
  	ATTGGGGCCAATACGCCC	PROTEIN	TGCCGCCCCGACG
  	GCGTTTCTTCCTTTTCCCC		TTGGCTGCGAGCC
  	ACCCCACCCCCCAAGTTC		CTGGGCCTTCACC
  	GGGTGAAGGCCCAGGGCT		CGAACTTGGGGG
  	CGCAGCCAACGTCGGGGC		GTGGGGTGGGGA
  	GGCAGGCCCTGCCATAG		AAAGGAAGAAAC
  	(SEQ ID NO: 343)		GCGGGCGTATTGG
  			CCCCAATGGGGTC
  			TCGGTGGGGTATC
  			GACAGAGTGCCA
  			GCCCTGGGACCG
  			AACCCCGCGTTTA
  			TGAACAAACGACC
  			CAACACCCGTGCG
  			TTTTATTCTGTCTT
  			TTTATTGCCGTCA
  			TAGCGCGGGTTCC
  			TTCCGGTATTGTC
  			TCCTTCCGTGTTT
  			CAGTTAGCCTCCC
  			CCCTATGGCCCTGA
  			CTGGAA
  			(SEQ ID NO: 344)
  MALAT1 ENE-	TAGGGTCATGAAGGTTTTT	RESULTS IN	ATCCTTCCAGTCAG
  MASCRNA	CTTTTCCTGAGAAAACAA	RETENTION	GGCCATGTTTGAGA
  	CACGTATTGTTTTCTCAGG	OF RNA IN	GCTAGAAATAGCAA
  	TTTTGCTTTTTGGCCTTTT	NUCLEUS,	GTTTAAATAAGGCT
  	TCTAGCTTAAAAAAAAAA	TRANSCRIPT	AGTCCGTTATCAAC
  	AAAGCAAAAGATGCTGGT	TERMINATION	TTGAAAAAGTGGG
  	GGTTGGCACTCCTGGTTT	AND	ACCGAGTCGGTCC A
  	CCAGGACGGGGTTCAAAT	STABILIZATION	CCTGAACCGTATATC
  	CCCTGCGGCGTCTTTGCTT		AGTCAAAGCAAAG
  	TGACT (SEQ ID NO: 345)		ACGCCGCAGGGAT
  			TTGAACCCCGTCC
  			TGGAAACCAGGA
  			GTGCCAACCACCA
  			GCATCTTTTGCTT
  			TTTTTTTTTTTAAG
  			CTAGAAAAAGGCC
  			AAAAAGCAAAACC
  			TGAGAAAACAATA
  			CGTGTTGTTTTCT
  			CAGGAAAAGAAAA
  			ACCTTCATGACCC
  			TA CTATGGCCCTGA
  			CTGGAA
  			(SEQ ID NO: 346)
  KSHV PAN	TGTTTTGGCTGGGTTTTTC	RESULTS IN	ATCCTTCCAGTCAG
  ENE	CTTGTTCGCACCGGACAC	RETENTION	GGCCATGTTTGAGA
  	CTCCAGTGACCAGACGGC	OF RNA IN	GCTAGAAATAGCAA
  	AAGGTTTTTATCCCAGTGT	NUCLEUS,	GTTTAAATAAGGCT
  	ATATTGGAAAAACATGTTA	TRANSCRIPT	AGTCCGTTATCAAC
  	TACTTTTGACAATTTAACG	TERMINATION	TTGAAAAAGTGGG
  	TGCCTAGAGCTCAAATTA	AND	ACCGAGTCGGTCC A
  	AACTAATACCATAACGTAA	STABILIZATION	CCTGAACCGTATATC
  	TGCAACTTACAACATAAAT		TTTTTTTTTTTTTT
  	AAAGGTCAATGTTTAATCC		TTTTTATGGATTAA
  	ATAAAAAAAAAAAAAAA		ACATTGACCTTTA
  	AAAA (SEQ ID NO: 347)		TTTATGTTGTAAG
  			TTGCATTACGTTAT
  			GGTATTAGTTTAAT
  			TTGAGCTCTAGGC
  			ACGTTAAATTGTC
  			AAAAGTATAACAT
  			GTTTTTCCAATATA
  			CACTGGGATAAAA
  			ACCTTGCCGTCTG
  			GTCACTGGAGGTG
  			TCCGGTGCGAACA
  			AGGAAAAACCCAG
  			CCAAAACA CTATG
  			GCCCTGACTGGAA
  			(SEQ ID NO: 348)
  THREE,	TGTTTTGGCTGGGTTTTTC	RESULTS IN	ATCCTTCCAGTCAG
  SEQUENTIAL	CTTGTTCGCACCGGACAC	RETENTION	GGCCATGTTTGAGA
  KSHV PAN	CTCCAGTGACCAGACGGC	OF RNA IN	GCTAGAAATAGCAA
  ENES WITH	AAGGTTTTTATCCCAGTGT	NUCLEUS,	GTTTAAATAAGGCT
  SHORT,	ATATTGGAAAAACATGTTA	TRANSCRIPT	AGTCCGTTATCAAC
  UNCONSERVED	TACTTTTGACAATTTAACG	TERMINATION	TTGAAAAAGTGGG
  RNA	TGCCTAGAGCTCAAATTA	AND	ACCGAGTCGGTCC A
  LINKERS	AACTAATACCATAACGTAA	STABILIZATION	CCTGAACCGTATATC
  	TGCAACTTACAACATAAAT	ON,	TTTTTTTTTTTTTT
  	AAAGGTCAATGTTTAATCC	PREDICTED	TTTTTATGGATTAA
  	ATAAAAAAAAAAAAAAA	TO BE	ACATTGACCTTTA
  	AAAAACACACTGTTTTGG	GREATER	TTTATGTTGTAAG
  	CTGGGTTTTTCCTTGTTCG	THAN A	TTGCATTACGTTAT
  	CACCGGACACCTCCAGTG	SINGLE PAN	GGTATTAGTTTAAT
  	ACCAGACGGCAAGGTTTT	ENE	TTGAGCTCTAGGC
  	TATCCCAGTGTATATTGGA		ACGTTAAATTGTC
  	AAAACATGTTATACTTTTG		AAAAGTATAACAT
  	ACAATTTAACGTGCCTAG		GTTTTTCCAATATA
  	AGCTCAAATTAAACTAATA		CACTGGGATAAAA
  	CCATAACGTAATGCAACTT		ACCTTGCCGTCTG
  	ACAACATAAATAAAGGTC		GTCACTGGAGGTG
  	AATGTTTAATCCATAAAAA		TCCGGTGCGAACA
  	AAAAAAAAAAAAAATCTC		AGGAAAAACCCAG
  	TCTGTTTTGGCTGGGTTTT		CCAAAACAGAGAG
  	TCCTTGTTCGCACCGGAC		ATTTTTTTTTTTTT
  	ACCTCCAGTGACCAGACG		TTTTTTATGGATTA
  	GCAAGGTTTTTATCCCAGT		AACATTGACCTTT
  	GTATATTGGAAAAACATGT		ATTTATGTTGTAAG
  	TATACTTTTGACAATTTAA		TTGCATTACGTTAT
  	CGTGCCTAGAGCTCAAAT		GGTATTAGTTTAAT
  	TAAACTAATACCATAACGT		TTGAGCTCTAGGC
  	AATGCAACTTACAACATA		ACGTTAAATTGTC
  	AATAAAGGTCAATGTTTA		AAAAGTATAACAT
  	ATCCATAAAAAAAAAAAA		GTTTTTCCAATATA
  	AAAAAAA(SEQ ID NO: 349)		CACTGGGATAAAA
  			ACCTTGCCGTCTG
  			GTCACTGGAGGTG
  			TCCGGTGCGAACA
  			AGGAAAAACCCAG
  			CCAAAACAGTGTG
  			TTTTTTTTTTTTTT
  			TTTTTTATGGATTA
  			AACATTGACCTTT
  			ATTTATGTTGTAAG
  			TTGCATTACGTTAT
  			GGTATTAGTTTAAT
  			TTGAGCTCTAGGC
  			ACGTTAAATTGTC
  			AAAAGTATAACAT
  			GTTTTTCCAATATA
  			CACTGGGATAAAA
  			ACCTTGCCGTCTG
  			GTCACTGGAGGTG
  			TCCGGTGCGAACA
  			AGGAAAAACCCAG
  			CCAAAACA CTATG
  			GCCCTGACTGGAA
  			(SEQ ID NO: 350)
  SMBOX/U1	CAGCAAGTTCAGAGAAAT	RESULTS IN	ATCCTTCCAGTCAG
  SNRNA BOX	CTGAACTTGCTGGATTTTT	RETENTION	GGCCATGTTTGAGA
  	GGAGCAGGGAGATGGAAT	OF RNA IN	GCTAGAAATAGCAA
  	AGGAGCTTGCTCCGTCCA	NUCLEUS	GTTTAAATAAGGCT
  	CTCCACGCATCGACCTGG	AND	AGTCCGTTATCAAC
  	TATTGCAGTACCTCCAGG	TRANSCRIPT	TTGAAAAAGTGGG
  	AACGGTGCACCCACTTTC	TERMINATION	ACCGAGTCGGTCC A
  	TGGAGTTTCAAAAGTAGA	N	CCTGAACCGTATATC
  	CTGTACGCTAAGGGTCATA		TTTAAGACGCCAA
  	TCTTTTTTTGTTTGGTTTG		CCAAGACACAAAC
  	TGTCTTGGTTGGCGTCTTA		CAAACAAAAAAAG
  	AA (SEQ ID NO: 351)		ATATGACCCTTAG
  			CGTACAGTCTACT
  			TTTGAAACTCCAG
  			AAAGTGGGTGCAC
  			CGTTCCTGGAGGT
  			ACTGCAATACCAG
  			GTCGATGCGTGGA
  			GTGGACGGAGCA
  			AGCTCCTATTCCA
  			TCTCCCTGCTCCA
  			AAAATCCAGCAAG
  			TTCAGATTTCTCT
  			GAACTTGCTGCTA
  			TGGCCCTGACTGGA
  			A
  			(SEQ ID NO: 352)
  Ul SNRNA 3 +40	GTTTCAAAAGTAGACTGT	RESULTS IN	ATCCTTCCAGTCAG
  BOX	ACGCTAAGGGTCATATCTT	RETENTION	GGCCATGTTTGAGA
  	TTTTTGTTTGGTTTGTGTC	OF RNA IN	GCTAGAAATAGCAA
  	TTGGTTGGCGTCTTAAA	NUCLEUS	GTTTAAATAAGGCT
  	(SEQ ID NO: 353)	AND	AGTCCGTTATCAAC
  		TRANSCRIPT	TTGAAAAAGTGGG
  		TERMINATION	ACCGAGTCGGTCC A
  			CCTGAACCGTATATC
  			TTTAAGACGCCAA
  			CCAAGACACAAAC
  			CAAACAAAAAAAG
  			ATATGACCCTTAG
  			CGTACAGTCTACT
  			TTTGAAACCTATGG
  			CCCTGACTGGAA
  			(SEQ ID NO: 354)
  TRNA-LYSINE	GCCCGGCTAGCTCAGTCG	REPORTED	ATCCTTCCAGTCAG
  	GTAGAGCATGAGACTCTT	TO ENABLE	GGCCATGTTTGAGA
  	AATCTCAGGGTCGTGGGT	TRANSPORT	GCTAGAAATAGCAA
  	TCGAGCCCCACWIGGGC	OF RNA TO	GTTTAAATAAGGCT
  	G (SEQ ID NO: 355)	MITOCHONDRIA	AGTCCGTTATCAAC
  			TTGAAAAAGTGGG
  			ACCGAGTCGGTCC A
  			CCTGAACCGTATATC
  			CGCCCAACGTGG
  			GGCTCGAACCCAC
  			GACCCTGAGATTA
  			AGAGTCTCATGCT
  			CTACCGACTGAGC
  			TAGCCGGGCCTAT
  			GGCCCTGACTGGAA
  			(SEQ ID NO: 356)
  BROCCOLI	GAGACGGTCGGGTCCAGA	VISUALIZATION	ATCCTTCCAGTCAG
  APTAMER	TATTCGTATCTGTCGAGTA	(FLUORESCE	GGCCATGTTTGAGA
  	GAGTGTGGGCTC (SEQ ID	NCE)	GCTAGAAATAGCAA
  	NO: 357)		GTTTAAATAAGGCT
  			AGTCCGTTATCAAC
  			TTGAAAAAGTGGG
  			ACCGAGTCGGTCC A
  			CCTGAACCGTATATC
  			GAGCCCACACTCT
  			ACTCGACAGATAC
  			GAATATCTGGACC
  			CGACCGTCTCCTA
  			TGGCCCTGACTGGA
  			A
  			(SEQ ID NO: 358)
  SPINACH	GACGCAACTGAATGAAAT	VISUALIZATION	ATCCTTCCAGTCAG
  APTAMER	GGTGAAGGACGGGTCCA	(FLUORESCE	GGCCATGTTTGAGA
  	GGTGTGGCTGCTTCGGCA	NCE)	GCTAGAAATAGCAA
  	GTGCAGCTTGTTGAGTAG		GTTTAAATAAGGCT
  	AGTGTGAGCTCCGTAACT		AGTCCGTTATCAAC
  	AGTCGCGTC (SEQ ID NO:		TTGAAAAAGTGGG
  	359)		ACCGAGTCGGTCC A
  			CCTGAACCGTATATC
  			GACGCGACTAGTT
  			ACGGAGCTCACAC
  			TCTACTCAACAAG
  			CTGCACTGCCGAA
  			GCAGCCACACCTG
  			GACCCGTCCTTCA
  			CCATTTCATTCAG
  			TTGCGTCCTATGGC
  			CCTGACTGGAA
  			(SEQ ID NO: 360)
  SPINACH2	GATGTAACTGAATGAAAT	VISUALIZATION	ATCCTTCCAGTCAG
  APTAMER	GGTGAAGGACGGGTCCA	(FLUORESCE	GGCCATGTTTGAGA
  	GTAGGCTGCTTCGGCAGC	NCE)	GCTAGAAATAGCAA
  	CTACTTGTTGAGTAGAGT		GTTTAAATAAGGCT
  	GTGAGCTCCGTAACTAGT		AGTCCGTTATCAAC
  	TACATC (SEQ ID NO: 361)		TTGAAAAAGTGGG
  			ACCGAGTCGGTCC A
  			CCTGAACCGTATATC
  			GATGTAACTAGTT
  			ACGGAGCTCACAC
  			TCTACTCAACAAG
  			TAGGCTGCCGAAG
  			CAGCCTACTGGAC
  			CCGTCCTTCACCA
  			TTTCATTCAGTTA
  			CATCCTATGGCCCT
  			GACTGGAA
  			(SEQ ID NO: 362)
  MANGO	GGCACGTACGAAGGGACG	VISUALIZATION	ATCCTTCCAGTCAG
  APTAMER	GTGCGGAGAGGAGAGTAC	(FLUORESCE	GGCCATGTTTGAGA
  	GTGC (SEQ ID NO: 363)	NCE)	GCTAGAAATAGCAA
  			GTTTAAATAAGGCT
  			AGTCCGTTATCAAC
  			TTGAAAAAGTGGG
  			ACCGAGTCGGTCC A
  			CCTGAACCGTATATC
  			GCACGTACTCTCC
  			TCTCCGCACCGTC
  			CCTTCGTACGTGC
  			CCTATGGCCCTGAC
  			TGGAA
  			(SEQ ID NO: 364)
  HDV	GGCCGGCATGGTCCCAGC	3 ′END RNA	ATCCTTCCAGTCAG
  RIBOZYME	CTCCTCGCTGGCGCCGGC	PROCESSING	GGCCATGTTTGAGA
  	TGGGCAACATGCTTCGGC		GCTAGAAATAGCAA
  	ATGGCGAATGGGAC		GTTTAAATAAGGCT
  	(SEQ ID NO: 365)		AGTCCGTTATCAAC
  			TTGAAAAAGTGGG
  			ACCGAGTCGGTCC A
  			CCTGAACCGTATATC
  			GTCCCATTCGCCA
  			TGCCGAAGCATGT
  			TGCCCAGCCGGC
  			GCCAGCGAGGAG
  			GCTGGGACCATGC
  			CGGCCCTATGGCCC
  			TGACTGGAA
  			(SEQ ID NO: 366)
  N6-	GGACTCTAGGACTGACT	TARGET FOR	ATCCTTCCAGTCAG
  METHYLADE	TTGGACT (SEQ ID NO: 367)	METHYLATION	GGCCATGTTTGAGA
  NOSINE		(UNDERLINE	GCTAGAAATAGCAA
  MARKER		D A'S ARE	GTTTAAATAAGGCT
  (M6A)		METHYLATE	AGTCCGTTATCAAC
  		D). M6A	TTGAAAAAGTGGG
  		METHYLATION	ACCGAGTCGGTCC A
  		CAN	CCTGAACCGTATATC
  		RESULT IN	AGTCCAAAGTCCA
  		ENHANCED	GTCCTAGAGTCCC
  		RNA	TATGGCCCTGACTG
  		STABILITY	GAA
  		AND	(SEQ ID NO: 368)
  		EXPRESSION,
  		BUT IS NOT
  		YET FULLY
  		UNDERSTOOD
  FIG. 3F as designated by font type, as follows: 5 +40-spacer sequence (normal font)-gRNA core (underlined sequence)-homology arm (italicized)-RT template (bolded font)- primer binding site (italicized)-3 +40.

• The PEgRNAs of the above table are designed to site-specifically insert examples of the above motifs into the HEXA gene (defective in Tay-Sachs disease) (e.g., GenBank No. KR710351.1 (SEQ ID NO: 369), however, this is only for purposes of illustration. The use of prime editing in RNA tagging is not limited to the HEXA gene and indeed may be any. The HEXA mRNA has the following nucleotide sequence:

• (SEQ ID NO: 369)

  GTTCGTTGCAACAAATTGATGAGCAATGCTTTTTTATAATGCCAACTTTG

  TACAAAAAAGTTGGCATGACAAGTTCCAGGCTTTGGTTTTCGCTGCTGCT

  GGCGGCAGCGTTCGCAGGACGGGCGACGGCCCTCTGGCCCTGGCCTCAGA

  ACTTCCAAACCTCCGACCAGCGCTACGTCCTTTACCCGAACAACTTTCAA

  TTCCAGTACGATGTCAGCTCGGCCGCGCAGCCCGGCTGCTCAGTCCTCGA

  CGAGGCCTTCCAGCGCTATCGTGACCTGCTTTTCGGTTCCGGGTCTTGGC

  CCCGTCCTTACCTCACAGGGAAACGGCATACACTGGAGAAGAATGTGTTG

  GTTGTCTCTGTAGTCACACCTGGATGTAACCAGCTTCCTACTTTGGAGTC

  AGTGGAGAATTATACCCTGACCATAAATGATGACCAGTGTTTACTCCTCT

  CTGAGACTGTCTGGGGAGCTCTCCGAGGTCTGGAGACTTTTAGCCAGCTT

  GTTTGGAAATCTGCTGAGGGCACATTCTTTATCAACAAGACTGAGATTGA

  GGACTTTCCCCGCTTTCCTCACCGGGGCTTGCTGTTGGATACATCTCGCC

  ATTACCTGCCACTCTCTAGCATCCTGGACACTCTGGATGTCATGGCGTAC

  AATAAATTGAACGTGTTCCACTGGCATCTGGTAGATGATCCTTCCTTCCC

  ATATGAGAGCTTCACTTTTCCAGAGCTCATGAGAAAGGGGTCCTACAACC

  CTGTCACCCACATCTACACAGCACAGGATGTGAAGGAGGTCATTGAATAC

  GCACGGCTCCGGGGTATCCGTGTGCTTGCAGAGTTTGACACTCCTGGCCA

  CACTTTGTCCTGGGGACCAGGTATCCCTGGATTACTGACTCCTTGCTACC

  CTGGGTCTGAGCCCTCTGGCACCTTTGGACCAGTGAATCCCAGTCTCAAT

  AATACCTATGAGTTCATGAGCACATTCTTCTTAGAAGTCAGCTCTGTCTT

  CCCAGATTTTTATCTTCATCTTGGAGGAGATGAGGTTGATTTCACCTGCT

  GGAAGTCCAACCCAGAGATCCAGGACTTTATGAGGAAGAAAGGCTTCGGT

  GAGGACTTCAAGCAGCTGGAGTCCTTCTACATCCAGACGCTGCTGGACAT

  CGTCTCTTCTTATGGCAAGGGCTATGTGGTGTGGCAGGAGGTGTTTGATA

  ATAAAGTAAAGATTCAGCCAGACACAATCATACAGGTGTGGCGAGAGGAT

  ATTCCAGTGAACTATATGAAGGAGCTGGAACTGGTCACCAAGGCCGGCTT

  CCGGGCCCTTCTCTCTGCCCCCTGGTACCTGAACCGTATATCCTATGGCC

  CTGACTGGAAGGATTTCTACGTAGTGGAACCCCTGGCATTTGAAGGTACC

  CCTGAGCAGAAGGCTCTGGTGATTGGTGGAGAGGCTTGTATGTGGGGAGA

  ATATGTGGACAACACAAACCTGGTCCCCAGGCTCTGGCCCAGAGCAGGGG

  CTGTTGCCGAAAGGCTGTGGAGCAACAAGTTGACATCTGACCTGACATTT

  GCCTATGAACGTTTGTCACACTTCCGCTGTGAGTTGCTGAGGCGAGGTGT

  CCAGGCCCAACCCCTCAATGTAGGCTTCTGTGAGCAGGAGTTTGAACAGA

  CCTGCCCAACTTTCTTGTACAAAGTTGGCATTATAAGAAAGCATTGCTTA

  TCAATTTGTTGCAACGAAC.

• The corresponding HEXA protein has the following amino acid sequence:

• (SEQ ID NO: 370)

  MTSSRLWFSLLLAAAFAGRATALWPWPQNFQTSDQRYVLYPNNFQFQYDV

  SSAAQPGCSVLDEAFQRYRDLLFGSGSWPRPYLTGKRHTLEKNVLVVSVV

  TPGCNQLPTLESVENYTLTINDDQCLLLSETVWGALRGLETFSQLVWKSA

  EGTFFINKTEIEDFPRFPHRGLLLDTSRHYLPLSSILDTLDVMAYNKLNV

  FHWHLVDDPSFPYESFTFPELMRKGSYNPVTHIYTAQDVKEVIEYARLRG

  IRVLAEFDTPGHTLSWGPGIPGLLTPCYPGSEPSGTFGPVNPSLNNTYEF

  MSTFFLEVSSVFPDFYLHLGGDEVDFTCWKSNPEIQDFMRKKGFGEDFKQ

  LESFYIQTLLDIVSSYGKGYVVWQEVFDNKVKIQPDTIIQVWREDIPVNY

  MKELELVTKAGFRALLSAPWYLNRISYGPDWKDFYVVEPLAFEGTPEQKA

  LVIGGEACMWGEYVDNTNLVPRLWPRAGAVAERLWSNKLTSDLTFAYERL

  SHFRCELLRRGVQAQPLNVGFCEQEFEQT.

• Notably, the resulting RNA motifs would be included within the translated region of the HEXA gene, disrupting the function of the protein coding gene. Inserted polyadenylation motifs would result in premature transcript termination. This site is merely illustrative of the potential PEgRNAs that could result in insertion of the listed RNA motifs of the above table within a genomic site that is transcribed and thus which would produce an RNA product.
• PEgRNAs for use with PE for RNA tagging could be expressed from a U6 promoter (in which case a single guanosine would be added to the 5′ end of the PEgRNA for guides that include proto spacers that do not begin with a G and 6-7 thymine would be added to the 3′ end) or a pol II promoter such as pCMV (in which case it might be necessary to remove the intrinsically transcribed sequence of this promoter from the 5′ end of the RNA via a self-cleaving element or Csy4 motif, and a termination motif would need to be added to the 3′ end of the RNA that does not result in export of the RNA from the nucleus, such as the 3′ box motif listed above. Note that this motif would not be inserted into the genome as a result of PE, as it would be 3′ of the annealing region). The core PEgRNA scaffold is underlined, the homology and annealing regions are italicized, and the inserted sequence is bolded. Note that the sequence inserted is the reverse complement of the above examples—as described below and therefore these PEgRNAs would need to be targeted to the coding strand.
• Also, note that self-cleaving ribozymes other than HDV need in some embodiments to be tailored to the given target site; that is, while HDV cleaves the encoded transcript immediately 5′ to itself, the cut sites for all other self-cleaving ribozymes are within the ribozyme itself. Therefore, the first and last roughly 5-10 nucleotides (and in some instances potentially more than 10) would actually be a part of the encoded sequence. As an example, to cleave the sequence 5′NNNNNTCATCCTGATAAACTGCAAA3′ (SEQ ID NO: 371) after the 5 Ns, where N is any nucleotide, using a hammerhead self-cleaving ribozyme, the following sequence would be inserted, where the underlined sequences form an imperfect RNA pairing element.

• (SEQ ID NO: 372)

  5′NNNNNCAGTTTGTACGGATGACTGATGAGTCCCAAATAGGACGAAACG

  CGCTTCGGTGCGTCTCATCCTGATAAACTGCAAA-3′.

• There is significant flexibility in terms of the length and nature of this pairing element, and this would be true for any of the non-HDV self-cleaving ribozymes listed in the original submission. To install a hammerhead ribozyme to cleave the hexA mRNA using a PEgRNA with the same protospacer as the above listed constructs, the following PEgRNA sequence could be used (labels same as above):

• 5′ACCTGAACCGTATATCGACGCACCGAAGCGCGTTTCGTCCTATTTGGG

  ACTCATCAGGATATACGGTTCAGGTGATATACGGTTCAGGTGACGCACCG

  AAGCGCGTTTCGTCCTATTTGGGACTCATCAGACCTGAACCGTATATCAT

  CCTTCCAGTCAGGGCCATGTTTGAGAGCTAGAAATAGCAAGTTTAAATAA

  GGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCCACCTGA

  ACCGTATATCGACGCACCGAAGCGCGTTTCGTCCTATTTGGGACTCATCA

  GGATATACGGTTCAGGTCTATGGCCCTGACTGGAA-3′

  
  (wherein the core PEgRNA scaffold is underlined, the homology and annealing regions are italicized, and the inserted sequence is bolded) (SEQ ID NO: 373).
• Designing other PEgRNA for insertion of RNA motifs may follow the general principle described herein. However, it is noted that many of RNA motifs are potentially highly structured, which could make it difficult for them to be reverse-transcribed and inserted into the genome. Although for some RNA sequences, such as simple hairpins, both the RNA sequence itself and its complement are structured. However, that is unlikely to be true for the sequences noted above. Therefore, when inserting these motifs, it would most likely be best for the PEgRNA to encode the reverse complement of these sequences, resulting in the insertion of the DNA sequence actually encoding the motif into the genome. Similarly, inclusion of a self-cleaving ribozyme in the PEgRNA template region would result in processing and inefficient activity, while inclusion of its reverse complement would not. Thus, these PEgRNAs will likely have to target the coding strand, whereas PEgRNAs encoding other types of insertions (such as therapeutic correction) would be able to theoretically target either strand.
• Also, note that for many of the inserted motifs, the resulting PEgRNA might not be able to be transcribed from the U6 promoter, necessitating use of other promoters, such as pCMV. Similarly, longer PEgRNAs could also be less stable. Shorter motifs, such as m⁶A markers, would not have this challenge.
• G. Use of Prime Editing for Generation of Sophisticated Gene Libraries
• Prime editing may also be used to generate sophisticated libraries of protein- or RNA-coding genes with defined or variable insertions, deletions, or defined amino acid/nucleotide conversions, and their use in high-throughput screening and directed evolution is described herein. This application of prime editing can be further described in Example 7.
• The generation of variable genetic libraries has most commonly been accomplished through mutagenic PCR (see Cadwell R C and Joyce G F. PCR Methods Appl. 1992). This method relies on either using reaction conditions that reduce the fidelity of DNA polymerase, or using modified DNA polymerases with higher mutation rates. As such, biases in these polymerases are reflected in the library product (e.g. a preference for transition mutations versus transversions). An inherent limitation of this approach to library construction is a relative inability to affect the size of the gene being varied. Most DNA polymerases have extremely low rates of indel mutations (insertions or deletions), and most of these will result in frameshift mutations in protein-coding regions, rendering members of the library unlikely to pass any downstream selection (See McInerney P, Adams P, and Hadi M Z. Mol Biol Int. 2014).
• Additionally, biases in PCR and cloning can make it difficult to generate single libraries consisting of genes of different sizes. These limitations can severely limit the efficacy of directed evolution to enhance existing or engineer novel protein functions. In natural evolution, large changes in protein function or efficacy are typically associated with insertion and deletion mutations that are unlikely to occur during canonical library generation for mutagenesis. Furthermore, these mutations most commonly occur in regions of the protein in question that are predicted to form loops, as opposed to the hydrophobic core. Thus, most indels generated using a traditional unbiased approach are likely to either be deleterious or ineffective.
• Libraries that could bias such mutations to the sites within the protein where they would be most likely to be beneficial, e.g., loop regions, would have a significant advantage over traditional libraries given that all libraries access only a fraction of the possible mutation space. Finally, although it is possible to generate genetic libraries with site-specific indel mutations through multistep PCR and clonal assembly using NNK primers or via DNA shuffling, these libraries cannot undergo additional rounds of ‘indelgenesis’ in continual evolution. Continuous evolution is a type of directed evolution with minimal user intervention. One such example is PACE (see Esvelt K M, Carlson J C, and Liu D R. Nature. 2011). Because continuous evolution occurs with minimal user intervention, any increase in library diversity during the evolution must occur using the native replication machinery. As such, although libraries of genes with inserted or removed codons as specific loci can be generated and screened in PACE, additional rounds of ‘indelgenesis’ are not possible.
• It is envisioned that the programmability of prime editing (PE) can be leveraged to generate highly sophisticated, programmed genetic libraries for use in high-throughput screening and directed evolution (see FIG. 29A). PE can insert, change or remove defined numbers of nucleotides from specified genetic loci using information encoded in a prime editing guide RNA (PEgRNA) (see FIG. 29B). This enables the generation of targeted libraries with one or more amino acids inserted or removed from the loop regions wherein mutations are most likely to give rise to changes in function, without background introduction of nonfunctional frameshift mutations (see FIG. 29C). PE can be used to install specific sets of mutations without regard for biases inherent in either DNA polymerase or the sequence being mutated.
• For instance, while converting a CCC codon to a stop codon would be an unlikely occurrence via canonical library generation because it would require three consecutive mutations, including two transversions, PE could be used to convert any given, targeted codon to a TGA stop codon in one step. They could also be used to install programmed diversity at given positions, for instance by incorporating codons encoding any hydrophobic amino acid at a given site, while not encoding any others. Furthermore, because of the programmability of PE, multiple PEgRNAs could be utilized to generate multiple different edits at multiple sites simultaneously, enabling the generation of highly programmed libraries (see FIG. 29D). Additionally, it is possible to use reverse transcriptases with lower fidelity to generate regions of mutagenesis within an otherwise invariable library (such as the HIV-I reverse transcriptase or Bordetella phage reverse transcriptase) (see Naorem S S, Hin J, Wang S, Lee W R, Heng X, Miller J F, Guo H. Proc Natl Acad Sci 2017 and Martinez M A, Vartanian J P, Wain-Hobson S. Proc Natl Acad Sci USA 1994).
• The possibility of iterative rounds of PE on the same site is also envisioned, allowing—for instance—the repeated insertion of codons at a single site, e.g., in a loop region. Also, it is envisioned that all of the above described approaches can be incorporated into continual evolution, enabling the generation of novel in situ evolving libraries (see FIG. 30 ). They could also be used to construct these libraries within other cell types where it would otherwise be difficult to assemble large libraries, for instance within mammalian cells. Generation of PE-encoding bacterial strains that have been optimized for directed evolution would be a useful additional tool for the identification of proteins and RNAs with improved or novel functionality. All of these uses of PE are non-obvious due to the novel nature of PEs. In conclusion, library generation via PE would be a highly useful tool in synthetic biology and directed evolution, as well as for high-throughput screening of protein and RNA combinatorial mutants.
Competing Approaches
• The chief method by which diverse libraries are currently generated is by mutagenic PCR (see Cadwell R C and Joyce G F. PCR Methods Appl. 1992), described above. Insertions or deletions can be introduced via degenerated NNK primers at defined sites during PCR, although introducing such mutations at multiple sites requires multiple rounds of iterative PCR and cloning before constructing a more diverse library via mutagenic PCR, rendering the method slow. An alternative, complementary method is DNA shuffling, where fragments of a library of genes generated via DNase treatment are introduced into a PCR reaction without primers, resulting in the annealing of different fragments to each other and the rapid generation of more diverse libraries than via mutagenic PCR alone (see Meyer A J, Ellefson J W, Ellington A D. Curr Protoc Mol Biol. 2014). Although this approach can theoretically generate indel mutations, it more often results in frameshift mutations that destroy gene function. Furthermore, DNA shuffling requires a high degree of homology between gene fragments.
• Both of these methods must be done in vitro, with the resulting library transformed into cells, while libraries generated by PE can be constructed in situ, enabling their use in continual evolution. While libraries can be constructed in situ through in vivo mutagenesis, these libraries rely on the host cellular machinery and exhibit biases against indels. Similarly, although traditional cloning methods can be used to generate site-specific mutational profiles, they cannot be used in situ and are generally assembled one at a time in vitro before being transformed into cells. The efficiency and broad functionality of PE in both prokaryotic and eukaryotic cell types further suggests that these libraries could be constructed directly in the cell type of interest, as opposed to being cloned into a model organism such as E. coli and then transferred into the cell or organism of interest. Another competing approach for targeted diversification is automated multiplex genome engineering, or MAGE, wherein multiple single-stranded DNA oligonucleotides can be incorporated within replication forks and result in programmable mutations. However, MAGE requires significant modification of the host strain and can lead to a 100-fold increase in off-target or background mutations (see Nyerges Á et al. Proc Natl Acad Sci USA. 2016), whereas PE is more highly programmed and anticipated to result in fewer off-target effects. Additionally, MAGE has not been demonstrated in a wide variety of cell types, including mammalian cells.
• By contrast, prime editing is a novel and non-obvious complementary technique for library generation.
Use of PE for Constructing Gene Libraries
• PE may be used to construct gene libraries in a programmable manner.
• In one example, PE can be used in a directed evolution experiment to introduce protein variants into gene libraries during a continual evolution experiment using PACE, permitting iterative accumulation of both point mutations and indels in a manner not possible via traditional approaches.
• It has already been shown that PE can site-specifically and programmably insert nucleotides into a genetic sequence in E. coli. Directed evolution can be used to identify monobodies with improved binding to a specific epitope via a modified two-hybrid protein:protein binding PACE selection. Specific and highly variable loops within these monobodies contribute significantly to affinity and specificity. Improved monobody binding might be obtained rapidly in PACE by varying the length and composition of these loops in a targeted fashion. However, varying sequence length is not an established functionality of PACE. While library of varied loop sizes might be used as a starting point for PACE, no subsequent improvements to length would arise throughout the PACE selection, barring access to beneficial synergistic combinations of point mutations and indel mutations.
• In various embodiments, PE can be used to improve the PACE selection by enabling the in situ generation and evolution of monobodies with varying loop lengths. To do so, the PACE E. coli strain may be introduced to an additional PE plasmid, which encodes the PE enzyme and one or more PEgRNAs. Expression of PE enzyme and PEgRNAs in the E. coli would be under the control of a small molecule delivered to the PACE lagoon at a rate selected by the experimenter.
• In various embodiments, the PEgRNA components would contain a spacer directing the PE to the site of interest on the selection phage and would be designed such that a multiple of three nucleotides could be inserted at the target site such that a new PEgRNA binding site would be introduced, enabling the iterative insertion of one or more codons at the targeted site.
• In parallel, another host E. coli strain might include PEgRNAs that would template the removal of one or more codons, enabling loop size to shrink during the evolution. A PACE experiment might utilize a mixture of both strains or alternate the two to permit the slow and controlled addition or removal of loop sequences.
• In addition to the use of PE and PACE to create monobody libraries, this technique can also be applied to the evolution of antibodies using PE and PACE. The binding principles governing antibodies are very similar to those governing monobodies: the length of antibody complement-determining region loops is critical to their binding function. Further, longer loop lengths have been found to be critical in the development of rare antibodies with broadly protective activity against HIV-1 and other viral infections (see Mascola J R, Haynes B F. Immunol Rev. 2013). Application of PE as described above to an antibody or antibody-derived molecule would permit the generation of antibodies with diverse loop length and varied loop sequence. In combination with PACE, such an approach would permit enhanced binding through loop geometries not accessible to standard PACE, and thus permit evolution of highly functional antibodies.
• As a non-limiting example, the following PEgRNAs could be used to programmably modify the genome of a bacteriophage used in a continuous evolution experiment:

• MODIFICATION	PEGRNA SEQUENCE
  CCA	UACACCAUCACGGUCUAUGCGUUUUAGAGCUAGAAA
  INSERTION	UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU
  	GAAAAAGUGGCACCGAGUCGGUGCUAUCUUCGCCCC
  	AUGCAUAGACCGUGAUGG (SEQ ID NO: 101)
  1 NT DELETION	CGCGUCGCGCUCGUCAGAUCGUUUUAGAGCUAGAAA
  	UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU
  	GAAAAAGUGGCACCGAGUCGGUGCUUUCCGCCUACC
  	UGCAUCUGACGAGCGCGA (SEQ ID NO: 102)
  POINT	AUCGGAGAAUACAUGAACAUGUUUUAGAGCUAGAAA
  MUTATION	UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU
  	GAAAAAGUGGCACCGAGUCGGUGCCGGAGAAUACAU
  	GAACAUCGGACCCGCGCUAUCUUC (SEQ ID NO:
  	103)
  NNN	UACACCAUCACGGUCUAUGCGUUUUAGAGCUAGAAA
  INSERTION	UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU
  (N = A/T/G/C)	GAAAAAGUGGCACCGAGUCGGUGCUAUCUUCGCCNN
  	NUGCAUAGACCGUGAUGG (SEQ ID NO: 104)
  ITERATIVE	UACACCAUCACGGUCUAUGCGUUUUAGAGCUAGAAA
  GGG	UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU
  INSERTION	GAAAAAGUGGCACCGAGUCGGUGCGGGGGGGGGGGG
  	GUGCAUAGACCGUGAUGG (SEQ ID NO: 105)

• In various embodiments, the use of PE for constructing gene libraries may make the use of the mutagenic activity of error-prone reverse transcriptases. The use of such mutagenic reverse transcriptase may facilitate the generation of mutagenized programmable libraries due to the lower fidelity of the error-prone RTs. As used herein, the term “error-prone” reverse transcriptase refers to a reverse transcriptase enzyme that occurs naturally or which has been derived from another reverse transcriptase (e.g., a wild type M-MLV reverse transcriptase) which has an error rate that is less than the error rate of wild type M-MLV reverse transcriptase. The error rate of wild type M-MLV reverse transcriptase is reported to be in the range of one error in 15,000 to 27,000 nucleobase incorporations. See Boutabout et al. (2001) “DNA synthesis fidelity by the reverse transcriptase of the yeast retrotransposon Ty1,” Nucleic Acids Res 29(11):2217-2222, which is incorporated herein by reference.
• Thus, for purposes of this application, the term “error prone” refers to those RT that have an error rate that is greater than one error in 15,000 nucleobase incorporation (6.7×10⁻⁵ or higher), e.g., 1 error in 14,000 nucleobases (7.14×10⁻⁵ or higher), 1 error in 13,000 nucleobases or fewer (7.7×10⁻⁵ or higher), 1 error in 12,000 nucleobases or fewer (7.7×10⁻⁵ or higher), 1 error in 11,000 nucleobases or fewer (9.1×10⁻⁵ or higher), 1 error in 10,000 nucleobases or fewer (1×10⁻⁴ or 0.0001 or higher), 1 error in 9,000 nucleobases or fewer (0.00011 or higher), 1 error in 8,000 nucleobases or fewer (0.00013 or higher) 1 error in 7,000 nucleobases or fewer (0.00014 or higher), 1 error in 6,000 nucleobases or fewer (0.00016 or higher), 1 error in 5,000 nucleobases or fewer (0.0002 or higher), 1 error in 4,000 nucleobases or fewer (0.00025 or higher), 1 error in 3,000 nucleobases or fewer (0.00033 or higher), 1 error in 2,000 nucleobase or fewer (0.00050 or higher), or 1 error in 1,000 nucleobases or fewer (0.001 or higher), or 1 error in 500 nucleobases or fewer (0.002 or higher), or 1 error in 250 nucleobases or fewer (0.004 or higher).
• A variety of mutagenic RTs could be envisioned for generation of highly mutagenized programmable libraries. Two such examples are the mutagenic reverse transcriptases from Bordetella phage (see Handa, S., et al. Nucl Acids Res 9711-25 (2018), which is incorporated herein by reference) and Legionella pneumophila (see Arambula, D., et al. Proc Natl Acad Sci USA 8212-7 (2013), which is incorporated by reference). In the case of the RT from Bordetella phage (brt), an accessory protein might need to also be added (bavd) to Cas9—or delivered in trans—as well as additional RNA sequences to the PEgRNA to improve binding of the mutagenic RT to the target site (see Handa, S., et al. Nucl Acids Res 9711-25 (2018)). When using mutagenic RTs, the template region of the PEgRNA might be enriched in adenosines or AAY codons to enhance diversity.
• The amino acid sequence of the mutagenic RT from Bordetella phage is provided as follows Like other RTs disclosed herein, the Brt protein may be fused to a napDNAbp as a fusion protein to form a functional PE.

• Name	Sequence
  brt	MGKRHRNLIDQITTWENLLDAYRKTSHGKRRTWGYLEFKE
  mutagenic	YDLANLLALQAELKAGNYERGPYREFLVYEPKPRLISALEF
  rt	KDRLVQHALCNIVAPIFEAGLLPYTYACRPDKGTHAGVCHV
  	QAELRRTRATHFLKSDFSKFFPSIDRAALYAMIDKKIHCAA
  	TRRLLRVVLPDEGVGIPIGSLTSQLFANVYGGAVDRLLHDE
  	LKQRHWARYMDDIVVLGDDPEELRAVFYRLRDFASERLGLK
  	ISHWQVAPVSRGINFLGYRIWPTHKLLRKSSVKRAKRKVAN
  	FIKHGEDESLQRFLASWSGHAQWADTHNLFTWMEEQYGIAC
  	H (SEQ ID NO: 129)

• In the case of Brt from Bordetella, the PE fusion may also include an additional accessory protein (Bavd). The accessory protein may be fused to the PE fusion protein or provided in trans. The amino acid sequence of Bavd accessory protein is provided as follows:

• Name	Sequence
  bavd accessory	MEPIEEATKCYDQMLIVERYERVISYLYPIAQSI
  protein to brt	PRKHGVAREMFLKCLLGQVELFIVAGKSNQVSKL
  	YAADAGLAMLRFWLRFLAGIQKPHAMTPHQVETA
  	QVLIAEVGRILGSWIARVNRKGQAGK (SEQ ID
  	NO: 236)

• In the case of Brt from Bordetella, the PEgRNA may comprise an additional nucleotide sequence added a PEgRNA, e.g., to the 5′ or 3′ end. Exemplary sequence is as follows, which is originally from the Bordetella phage genome:

• NAME	SEQUENCE
  PEGRNA-	ACCUUCUUGCAUGGCUCUGCCAACGCUACGGCUUGGCGGG
  ADDITION	CUGGCCUUUCCUCAAUAGGUGGUCAGCCGGUUCUGUCCUG
  1	CUUCGGCGAACACGUUACACGGUUCGGCAAAACGUCGAUU
  	ACUGAAAAUGGAAAGGCGGGGCCGACUUCAAGGGCAGGCU
  	GGGAAAUAA (SEQ ID NO: 237)

• This PEgRNA addition sequence can be reduced in various ways to shorten the length. For example, the PEgRNA-addition 1 sequence could be reduced to the following exemplary alternative addition sequences:

• NAME	SEQUENCE
  PEGRNA-	ACCUUCUUGCAUGGCUCUGCCAACGCUACGGCUUGGCGGGC
  ADDITION	UGGCCUUUCCUCAAUAGGUGGUCAGCCGGUUCUGUCCUGCU
  2	UCGGCGAACACGUUACACGGUUCGGCAAAACGUCGAUUACU
  	GAAAAUGGAAAGGCGGGGCCGACUUC (SEQ ID NO:
  	238)
  PEGRNA-	ACCUUCUUGCAUGGCUCUGCCAACGCUACGGCUUGGCGGGC
  ADDITION	UGGCCUUUCCUCAAUAGGUGGUCAAAGGGCAGGCUGGGAAA
  3	UAA (SEQ ID NO: 239)
  PEGRNA-	ACCUUCUUGCAUGGCUCUGCCAACGCUACGGCUUGGCGGGC
  ADDITION	UGGCCUUUCCUCAAUAGGUGGUCA (SEQ ID NO: 277)
  4
  PEGRNA-	CAUGGCUCUGCCAACGCUACGGCUUGGCGGGCUGGCCUUUC
  ADDITION	CUCAAUAGGUGGUCAGCCGGUUCUGUCCUGCUUCGGCGAAC
  5	ACGUUACACGGUUCGGCAAAACGUCGAUUACUGAAAAUGGA
  	AAGGCGGGGCCGACUUCAAGGGCAGGCUGGGAAAUAA
  	(SEQ ID NO: 240)
  PEGRNA-	CAUGGCUCUGCCAACGCUACGGCUUGGCGGGCUGGCCUUUC
  ADDITION	CUCAAUAGGUGGUCAGCCGGUUCUGUCCUGCUUCGGCGAAC
  6	ACGUUACACGGUUCGGCAAAACGUCGAUUACUGAAAAUGGA
  	AAGGCGGGGCCGACUUC (SEQ ID NO: 241)
  PEGRNA-	CAUGGCUCUGCCAACGCUACGGCUUGGCGGGCUGGCCUUUC
  ADDITION	CUCAAUAGGUGGUCAAAGGGCAGGCUGGGAAAUAA
  7	(SEQ ID NO: 242)
  PEGRNA-	CAUGGCUCUGCCAACGCUACGGCUUGGCGGGCUGGCCUUUC
  ADDITION	CUCAAUAGGUGGUCA
  8	(SEQ ID NO: 243)

• In other embodiments, the PEgRNA addition sequence can be also be mutated. For example, the PEgRNA-addition 1 sequence could be mutated to the following exemplary alternative addition sequence:

• NAME	SEQUENCE
  PEGRNA-	ACCUUCUUGCAUGGCUCUGCCAACGCUACGGCUUGGCGGGC
  ADDITION	UGGCCUUUCCUCAAUAGAUGAGCCGCCGGUUCUGUCCUGCU
  1	UCGGCGAACACGUUACACGGUUCGGCAAAACGUCGAUUACU
  MUTATED	GAAAAUGGAAAGGCGGGGCCGACUUCAAGGGCAGGCUGGGA
  	AAUAA (SEQ ID NO: 244)

• In various embodiments relating to the use of PE for designing gene libraries, special PEgRNAs considerations may apply. For example, without wishing to be bound by theory, the additional PEgRNA sequences described above might be needed to enable efficient mutagenesis via mutagenic RTs. In another embodiment, iterative codon insertion using PE may required specific PEgRNA designs. For example, to insert a GGG (glycine) codon iteratively, the entire homology region of the PEgRNA might need to be composed of Gs, as shown above. This would mean that only certain sites could go iterative insertion. Additionally, the PAM sequence would not be able to be disrupted by the PE.
• H. Use of Prime Editing for Insertion of Immunoepitopes
• Prime editors may also be used as a means to insert known immunogenicity epitopes into endogenous or foreign genomic DNA, resulting in modification of the corresponding proteins for therapeutic or biotechnological applications (see FIGS. 31 and 32 ). Prior to the invention of prime editing, such insertions could be achieved only inefficiently and with high rates of indel formation from DSBs. prime editing solves the problem of high indel formation from insertion edits while generally offering higher efficiency than HDR. This lower rate of indel formation presents a major advantage of prime editing over HDR as a method for targeted DNA insertions, especially in the described application of inserting immunogenicity epitopes. The length of epitopes is in a range from few bases to hundreds of bases. Prime editor is an efficient approach to achieve such targeted insertions in mammalian cells.
• The key concept of the invention is the use of prime editors to insert a nucleotide sequence containing previously described immunogenicity epitopes into endogenous or foreign genomic DNA for the downregulation and/or destruction of their protein products and/or expressing cell types. Nucleotide sequences for immunogenic epitope insertion would be targeted to genes in a manner to produce fusion proteins of the targeted gene's coded protein and the inserted immunogenic epitope's corresponding protein translation. Patient's immune systems will have been previously trained to recognize these epitopes as a result of standard prior immunization from routine vaccination against, for example, tetanus or diphtheria or measles. As a result of the immunogenic nature of the fused epitopes, patient's immune systems would be expected to recognize and disable the prime edited protein (not just the inserted epitope) and potentially the cells from which it was expressed.
• Precise genome targeting technologies using the CRISPR/Cas system have recently been explored in a wide range of applications, including the insertion of engineered DNA sequences into targeted genomic loci. Previously, homology-directed repair (HDR) has been used for this application, requiring an ssDNA donor template and repair initiation by means of a double-stranded DNA break (DSB). This strategy offers the broadest range of possible changes to be made in cells and is the only method available to insert large DNA sequences into mammalian cells. However, HDR is hampered by undesired cellular side effects stemming from its initiating DSB, such as high levels of indel formation, DNA translocations, large deletions, and P53 activation. In addition to these drawbacks, HDR is limited by low efficiency in many cell types (T cells are a notable exception to this observation). Recent efforts to overcome these drawbacks include fusing human Rad51 mutants to a Cas9 D10A nickase (RDN), resulting in a DSB-free HDR system that features improved HDR product:indel ratios and lower off target editing, but is still hampered by cell-type dependencies and only modest HDR editing efficiency.
• Recently developed fusions of Cas9 to reverse transcriptases (“Prime editors”) coupled with PEgRNAs represent a novel genome editing technology that offers a number of advantages over existing genome editing methods, including the ability to install any single nucleotide substitution, and to insert or delete any short stretch of nucleotides (up to at least several dozen bases) in a site-specific manner. Notably, PE edits are achieved with generally low rates of unintended indels. As such, PE enables targeted insertion-based editing applications that have been previously impossible or impractical.
• This particular aspect describes a method for using prime editing as a means to insert known immunogenicity epitopes into endogenous or foreign genomic DNA, resulting in modification of the corresponding proteins for therapeutic or biotechnological applications (see FIGS. 31 and 32 ). Prior to the invention of prime editing, such insertions could be achieved only inefficiently and with high rates of indel formation from DSBs. prime editing solves the problem of high indel formation from insertion edits while generally offering higher efficiency than HDR. This lower rate of indel formation presents a major advantage of prime editing over HDR as a method for targeted DNA insertions, especially in the described application of inserting immunogenicity epitopes. The length of epitopes is in a range from few bases to hundreds of bases. Prime editor is the most efficient and cleanest technology to achieve such targeted insertions in mammalian cells.
• The key concept of this aspect is the use of prime editors to insert a nucleotide sequence containing previously described immunogenicity epitopes into endogenous or foreign genomic DNA for the downregulation and/or destruction of their protein products and/or expressing cell types. Nucleotide sequences for immunogenic epitope insertion would be targeted to genes in a manner to produce fusion proteins of the targeted gene's coded protein and the inserted immunogenic epitope's corresponding protein translation. Patient's immune systems will have been previously trained to recognize these epitopes as a result of standard prior immunization from routine vaccination against, for example, tetanus or diphtheria or measles. As a result of the immunogenic nature of the fused epitopes, patient's immune systems would be expected to recognize and disable the prime edited protein (not just the inserted epitope) and potentially the cells from which it was expressed.
• Fusions to targeted genes would be engineered as needed to ensure the inserted epitope protein translation is exposed for immune system recognition. This could include targeted nucleotide insertions resulting in protein translations yielding C-terminal fusions of immunogenicity epitopes to targeted genes, N-terminal fusions of immunogenicity epitopes to targeted genes, or the insertion of nucleotides into genes so that immunogenicity epitopes are coded within surfaced-exposed regions of protein structure.
• Protein linkers encoded as nucleotides inserted between the target gene sequence and the inserted immunogenicity epitope nucleotide sequence may need to be engineered as part of this invention to facilitate immune system recognition, cellular trafficking, protein function, or protein folding of the targeted gene. These inserted nucleotide-encoded protein linkers may include (but are not limited to) variable lengths and sequences of the XTEN linker or variable lengths and sequences of Glycine-Serine linkers. These engineered linkers have been previous used to successfully facilitate protein fusions. Exemplary linkers may include any of those described herein, including the amino acid sequence (GGGGS)n (SEQ ID NO: 165), (G)n (SEQ ID NO: 166), (EAAAK)n (SEQ ID NO: 167), (GGS)n (SEQ ID NO: 168), (SGGS)n (SEQ ID NO: 169), (XP)n (SEQ ID NO: 170), or any combination thereof, wherein n is independently an integer between 1 and 30, and wherein X is any amino acid. In some embodiments, the linker comprises the amino acid sequence (GGS)n (SEQ ID NO: 176), wherein n is 1, 3, or 7. In some embodiments, the linker comprises the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 171). In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 172). In some embodiments, the linker comprises the amino acid sequence SGGSGGSGGS (SEQ ID NO: 173). In some embodiments, the linker comprises the amino acid sequence SGGS (SEQ ID NO: 174).
• Distinguishing features of this aspect include the ability to use previously acquired immune responses to specific amino acid sequences as a means to induce an immune response against otherwise non-immunogenic proteins. Another distinguishing feature is the ability to insert the nucleotide sequences of these immunogenic epitopes in a targets manner that does not induce high levels of unwanted indels as a by-product editing and is efficient in its insertion. This specific application of PE has the ability to combine cell type-specific delivery methods (such as AAV serotypes) to insert epitopes in cell types that are of interest to trigger an immune response to.
• Prime editing as a means of inserting immunogenic epitopes into pathogenic genes could be used to program the patient's immune system to fight a wide variety of diseases (not limited to cancer as with immuno-oncology strategies). An immediately relevant use of this technology would be as a cancer therapeutic as it could undermine a tumor's immune escape mechanism by causing an immune response to a relevant oncogene like HER2 or growth factors like EGFR. Such an approach could seem similar to T-cell engineering, but one novel advance of this approach is that it can be utilized in many cell types and for diseases beyond cancer, without needed to generate and introduce engineered T-cells into patients.
• Using PE to insert an immunogenicity epitope which most people are already vaccinated against (tetanus, pertussis, diphtheria, measles, mumps, rubella, etc.) into a foreign or endogenous gene that drives a disease, so the patient's immune system learns to disable that protein.
• Diseases that stand to have a potential therapeutic benefit from the aforementioned strategy include those caused by aggregation of toxic proteins, such as in fatal familial insomnia. Other diseases that could benefit include those caused by pathogenic overexpression of an otherwise nontoxic endogenous protein, and those caused by foreign pathogens.
• Primary therapeutic indications include those mentioned above such as therapeutics for cancer, prion and other neurodegenerative diseases, infectious diseases, and preventative medicine. Secondary therapeutic indications may include preventative care for patients with late-onset genetic diseases. It is expected that current standard of care medicines may be used in conjunction with prime editing for some diseases, like particularly aggressive cancers, or in cases where medications help alleviate disease symptoms until the disease completely cured. Below are examples of immunoepitopes that may be inserted into genes by the herein disclosed prime editors:

• 		EPITOPE AMINO	EXAMPLE NUCLEIC ACID
  VACCINE	DISEASE	ACID SEQUENCE	SEQUENCE (8)

  1	TETANUS	QYIKANSKFIGITE	CATGATATAAAAGCAAATTCTAAATTTA
  	TOXOID	L (SEQ ID NO: 396)	TAGGTATAACTGAACTA (SEQ ID NO:
  			397)
  2	DIPHTHERIA	GADDVVDSSKSF	GGCGCCGACGACGTGGTGGACAGCAG
  	TOXIN	VMENFSSYHGTK	CAAGAGCTTCGTGATGGAGAACTTCA
  	MUTANT	PGYVDSIQKGIQK	GCAGCTACCACGGCACCAAGCCCGGC
  	CRM197	PKSGTQGNYDDD	TACGTGGACAGCATCCAGAAGGGCAT
  		WKEFYSTDNKYD	CCAGAAGCCCAAGAGCGGCACCCAGG
  		AAGYSVDNENPL	GCAACTACGACGACGACTGGAAGGAG
  		SGKAGGVVKVTY	TTCTACAGCACCGACAACAAGTACGA
  		PGLTKVLALKVD	CGCCGCCGGCTACAGCGTGGACAACG
  		NAETIKKELGLSL	AGAACCCCCTGAGCGGCAAGGCCGGC
  		TEPLMEQVGTEEF	GGCGTGGTGAAGGTGACCTACCCCGG
  		IKRFGDGASRVVL	CCTGACCAAGGTGCTGGCCCTGAAGG
  		SLPFAEGSSSVEYI	TGGACAACGCCGAGACCATCAAGAAG
  		NNWEQAKALSVE	GAGCTGGGCCTGAGCCTGACCGAGCC
  		LEINFETRGKRGQ	CCTGATGGAGCAGGTGGGCACCGAGG
  		DAMYEYMAQAC	AGTTCATCAAGAGGTTCGGCGACGGC
  		AGNRVRRSVGSSL	GCCAGCAGGGTGGTGCTGAGCCTGCC
  		SCINLDWDVIRDK	CTTCGCCGAGGGCAGCAGCAGCGTGG
  		TKTKIESLKEHGPI	AGTACATCAACAACTGGGAGCAGGCC
  		KNKMSESPNKTV	AAGGCCCTGAGCGTGGAGCTGGAGAT
  		SEEKAKQYLEEFH	CAACTTCGAGACCAGGGGCAAGAGGG
  		QTALEHPELSELK	GCCAGGACGCCATGTACGAGTACATGG
  		TVTGTNPVFAGA	CCCAGGCCTGCGCCGGCAACAGGGTG
  		NYAAWAVNVAQV	AGGAGGAGCGTGGGCAGCAGCCTGAG
  		IDSETADNLEKTT	CTGCATCAACCTGGACTGGGACGTGAT
  		AALSILPGIGSVM	CAGGGACAAGACCAAGACCAAGATCG
  		GIADGAVHHNTEE	AGAGCCTGAAGGAGCACGGCCCCATC
  		IVAQSIALSSLMVA	AAGAACAAGATGAGCGAGAGCCCCAA
  		QAIPLVGELVDIGF	CAAGACCGTGAGCGAGGAGAAGGCC
  		AAYNFVESIINLFQ	AAGCAGTACCTGGAGGAGTTCCACCA
  		VVHNSYNRPAYSP	GACCGCCCTGGAGCACCCCGAGCTGA
  		GHKTQPFLHDGY	GCGAGCTGAAGACCGTGACCGGCACC
  		AVSWNTVEDSIIR	AACCCCGTGTTCGCCGGCGCCAACTA
  		TGFQGESGHDIKI	CGCCGCCTGGGCCGTGAACGTGGCCC
  		TAENTPLPIAGVL	AGGTGATCGACAGCGAGACCGCCGAC
  		LPTIPGKLDVNKS	AACCTGGAGAAGACCACCGCCGCCCT
  		KTHISVNGRKIRM	GAGCATCCTGCCCGGCATCGGCAGCGT
  		RCRAIDGDVTFCR	GATGGGCATCGCCGACGGCGCCGTGC
  		PKSPVYVGNGVH	ACCACAACACCGAGGAGATCGTGGCC
  		ANLHVAFHRSSSE	CAGAGCATCGCCCTGAGCAGCCTGAT
  		KIHSNEISSDSIGV	GGTGGCCCAGGCCATCCCCCTGGTGG
  		LGYQKTVDHTKV	GCGAGCTGGTGGACATCGGCTTCGCC
  		NSKLSLFFEIKS	GCCTACAACTTCGTGGAGAGCATCATC
  		(SEQ ID NO: 398)	AACCTGTTCCAGGTGGTGCACAACAG
  			CTACAACAGGCCCGCCTACAGCCCCG
  			GCCACAAGACCCAGCCCTTCCTGCAC
  			GACGGCTACGCCGTGAGCTGGAACAC
  			CGTGGAGGACAGCATCATCAGGACCG
  			GCTTCCAGGGCGAGAGCGGCCACGAC
  			ATCAAGATCACCGCCGAGAACACCCC
  			CCTGCCCATCGCCGGCGTGCTGCTGCC
  			CACCATCCCCGGCAAGCTGGACGTGA
  			ACAAGAGCAAGACCCACATCAGCGTG
  			AACGGCAGGAAGATCAGGATGAGGTG
  			CAGGGCCATCGACGGCGACGTGACCT
  			TCTGCAGGCCCAAGAGCCCCGTGTAC
  			GTGGGCAACGGCGTGCACGCCAACCT
  			GCACGTGGCCTTCCACAGGAGCAGCA
  			GCGAGAAGATCCACAGCAACGAGATC
  			AGCAGCGACAGCATCGGCGTGCTGGG
  			CTACCAGAAGACCGTGGACCACACCA
  			AGGTGAACAGCAAGCTGAGCCTGTTC
  			TTCGAGATCAAGAGC (SEQ ID NO:
  			399)
  3	MUMPS	GTYRLIPNARANL	GGCACCTACAGGCTGATCCCCAACGC
  	IMMUNOEPITOPE	TA (SEQ ID NO:	CAGGGCCAACCTGACCGCC (SEQ ID
  	1	400)	NO: 401)
  4	MUMPS	PSKFFTISDSATFA	CCGAGCAAATTTTTTACCATTAGCGAT
  	IMMUNOEPITOPE	PGPVSNA (SEQ ID	AGCGCGACCTTTGCGCCGGGCCCGGT
  	2	NO: 402)	GAGCAACGCG (SEQ ID NO: 403)
  	MUMPS	PSKLFIMLDNATF	CCGAGCAAACTGTTTATTATGCTGGAT
  	IMMUNOEPITOPE	APGPVVNA (SEQ	AACGCGACCTTTGCGCCGGGCCCGGT
  	1	ID NO: 404)	GGTGAACGCG (SEQ ID NO: 405)
  			SELECTED EXAMPLES FROM
  			HEMAGGLUTININ-NEURAMINIDASE
  			(HN) DIVERSITY AMONG OUTBREAK
  			STRAINS (TABLE 1) DIVERGENCE
  			BETWEEN VACCINE STRAIN JL5 AND
  			OUTBREAK STRAINS (TABLE 2)
  5	RUBELLA	TPPPYQVSCGGES	ACCCCCCCCCCCTACCAGGTGAGCTGC
  	VIRUS	DRASARVIDPAAQ	GGCGGCGAGAGCGACAGGGCCAGCG
  	(RV)	S (SEQ ID NO: 406)	CCAGGGTGATCGACCCCGCCGCCCAG
  			AGC (SEQ ID NO: 407)
  6	HEMAGGLUTININ	PEYAYKIVKNKK	CCCGAGTACGCCTACAAGATCGTGAA
  		MEDGFLQGMVD	GAACAAGAAGATGGAGGACGGCTTCC
  		GWYGHHSNEQGS	TGCAGGGCATGGTGGACGGCTGGTAC
  		GLMENERTLDKA	GGCCACCACAGCAACGAGCAGGGCAG
  		NPNNDLCSWSDH	CGGCCTGATGGAGAACGAGAGGACCC
  		EASSNNTNQEDLL	TGGACAAGGCCAACCCCAACAACGAC
  		QRESRRKKRIGTS	CTGTGCAGCTGGAGCGACCACGAGGC
  		TLNQRGNCNTKC	CAGCAGCAACAACACCAACCAGGAGG
  		QTEEARLKREEVS	ACCTGCTGCAGAGGGAGAGCAGGAGG
  		LVKSDQCSNGSLQ	AAGAAGAGGATCGGCACCAGCACCCT
  		CRANNSTEQVD	GAACCAGAGGGGCAACTGCAACACCA
  		(SEQ ID NO: 408)	AGTGCCAGACCGAGGAGGCCAGGCTG
  			AAGAGGGAGGAGGTGAGCCTGGTGA
  			AGAGCGACCAGTGCAGCAACGGCAGC
  			CTGCAGTGCAGGGCCAACAACAGCAC
  			CGAGCAGGTGGAC (SEQ ID NO: 409)
  7	NEURAMINIDASE	TKSTNSRSGGISG	ACCAAGAGCACCAACAGCAGGAGCG
  		PDNEAPVGEAPSP	GCGGCATCAGCGGCCCCGACAACGAG
  		YGDNPRPNDGNN	GCCCCCGTGGGCGAGGCCCCCAGCCC
  		IRIGSKGYNGIITD	CTACGGCGACAACCCCAGGCCCAACG
  		TIEESCSCYPDAK	ACGGCAACAACATCAGGATCGGCAGC
  		VVKSVELDSTIWT	AAGGGCTACAACGGCATCATCACCGA
  		SGSSPNQKIITIGW	CACCATCGAGGAGAGCTGCAGCTGCT
  		DPNGWTGTPMSP	ACCCCGACGCCAAGGTGGTGAAGAGC
  		NGAYGTDGPSNG	GTGGAGCTGGACAGCACCATCTGGAC
  		QANQHQAESISA	CAGCGGCAGCAGCCCCAACCAGAAGA
  		GNSSLCPIRDNWH	TCATCACCATCGGCTGGGACCCCAACG
  		GSNRSWSWPDGA	GCTGGACCGGCACCCCCATGAGCCCC
  		E (SEQ ID NO: 410)	AACGGCGCCTACGGCACCGACGGCCC
  			CAGCAACGGCCAGGCCAACCAGCACC
  			AGGCCGAGAGCATCAGCGCCGGCAAC
  			AGCAGCCTGTGCCCCATCAGGGACAA
  			CTGGCACGGCAGCAACAGGAGCTGGA
  			GCTGGCCCGACGGCGCCGAG (SEQ ID
  			NO: 411)
  8	TAP1	EKIVLLLAMMEKI	GAGAAGATCGTGCTGCTGCTGGCCATG
  	(TRANSPORT	VLLLAKCQTPMG	ATGGAGAAGATCGTGCTGCTGCTGGCC
  	ANTIGEN	AIKAVDGVTNKCP	AAGTGCCAGACCCCCATGGGCGCCAT
  	PRESENTATION)	YLGSPSF (SEQ ID	CAAGGCCGTGGACGGCGTGACCAACA
  	ON	NO: 412)	AGTGCCCCTACCTGGGCAGCCCCAGC
  	H5N1		TTC (SEQ ID NO: 413)
  	VIRUS
  	HEMAGGLUTININ
  9	TAP2	IRPCFWVELNPNQ	ATCAGGCCCTGCTTCTGGGTGGAGCTG
  	(TRANSPORT	KIITIRPCFWVELI	AACCCCAACCAGAAGATCATCACCATC
  	ANTIGEN	CYPDAGEIT (SEQ	AGGCCCTGCTTCTGGGTGGAGCTGATC
  	PRESENTATION)	ID NO: 414)	TGCTACCCCGACGCCGGCGAGATCAC
  	ON		C (SEQ ID NO: 415)
  	H5N1
  	VIRUS
  	NEURAMINIDASE
  10	HEMAGGLUTININ	MEKIVLLLAEKIV	ATGGAGAAGATCGTGCTGCTGCTGGCC
  	EPITOPES	LLLAMCPYLGSPS	GAGAAGATCGTGCTGCTGCTGGCCATG
  	TOWARD	FKCQTPMGAIKAV	TGCCCCTACCTGGGCAGCCCCAGCTTC
  	CLASS I	DGVTNK (SEQ ID	AAGTGCCAGACCCCCATGGGCGCCAT
  	HLA	NO: 416)	CAAGGCCGTGGACGGCGTGACCAACA
  			AG (SEQ ID NO: 417)
  11	NEURAMINIDASE	NPNQKIITICYPDAG	AACCCCAACCAGAAGATCATCACCATCT
  	EPITOPES	EITIRPCFWVELRPC	GCTACCCCGACGCCGGCGAGATCACCAT
  	TOWARD	FWVELI (SEQ ID	CAGGCCCTGCTTCTGGGTGGAGCTGAG
  	CLASS I	NO: 418)	GCCCTGCTTCTGGGTGGAGCTGATC
  	HLA		(SEQ ID NO: 419)
  12	HEMAGGLUTININ	MVSLVKSDQIGTST	ATGGTGAGCCTGGTGAAGAGCGACCAG
  	EPITOPES	LNQR (SEQ ID NO:	ATCGGCACCAGCACCCTGAACCAGAGG
  	TOWARD	420)	(SEQ ID NO: 421)
  	CLASS II
  	HLA
  13	NEURAMINIDASE	YNGIITDTI (SEQ ID	TACAACGGCATCATCACCGACACCATC
  	EPITOPES	NO: 422)	(SEQ ID NO: 423)
  	TOWARD
  	CLASS II
  	HLA
  14	HEMAGGLUTININ	MEKIVLLLAEKIVL	ATGGAGAAGATCGTGCTGCTGCTGGCC
  	EPITOPE	LLAMMVSLVKSDQ	GAGAAGATCGTGCTGCTGCTGGCCATG
  	H5N1-	CPYLGSPSFIGTSTL	ATGGTGAGCCTGGTGAAGAGCGACCAG
  	BOUND	NQRKCQTPMGAIK	TGCCCCTACCTGGGCAGCCCCAGCTTCA
  	CLASS I	AVDGVTNK (SEQ	TCGGCACCAGCACCCTGAACCAGAGG
  	AND	ID NO: 424)	(SEQ ID NO: 425)
  	CLASS II
  	HLA
  15	NEURAMINIDASE	NPNQKIITIYNGIIT	AACCCCAACCAGAAGATCATCACCATCT
  	EPITOPE	DTICYPDAGEITIRP	ACAACGGCATCATCACCGACACCATCTG
  	H5N1-	CFWVELRPCFWVE	CTACCCCGACGCCGGCGAGATCACCATC
  	BOUND	LI (SEQ ID NO: 426)	AGGCCCTGCTTCTGGGTGGAGCTGAGG
  	CLASS I		CCCTGCTTCTGGGTGGAGCTGATC (SEQ
  	AND		ID NO: 427)
  	CLASS II
  	HLA

• Additional immunoepitopes may also be installed which are known in the art. Any of the immunoepitopes available from the Immune Epitope Database and Analysis Resource (iedb.org/epitopedetails_v13.php) (the contents of which are incorporated herein by reference) may be installed by the prime editors disclosed herein.
• In some embodiments, the immunoepitopes which may be installed by the prime editors disclosed herein may include any of the following epitopes:

• 			SEQ
  			ID	ACCESS.
  NO.	DISEASE	EPITOPE AMINO ACID SEQUENCE	NO	NO.

  1	TETANUS	QYIKANSKFIGITEL	396	NA
  	TOXOID
  2	DIPHTHERIA	GADDVVDSSKSFVMENFSSYHGTKP	428	NA
  	TOXIN MUTANT	GYVDSIQKGIQKPKSGTQGNYDDDW
  	CRM197	KEFYSTDNKYDAAGYSVDNENPLSG
  		KAGGVVKVTYPGLTKVLALKVDNA
  		ETIKKELGLSLTEPLMEQVGTEEFIKR
  		FGDGASRVVLSLPFAEGSSSVEYINN
  		WEQAKALSVELEINFETRGKRGQDA
  		MYEYMAQACAGNRVRRSVGSSLSCI
  		NLDWDVIRDKTKTKIESLKEHGPIKN
  		KMSESPNKTVSEEKAKQYLEEFHQT
  		ALEHPELSELKTVTGTNPVFAGANYA
  		AWAVNVAQVIDSETADNLEKTTAALS
  		ILPGIGSVMGIADGAVHHNTEEIVAQS
  		IALSSLMVAQAIPLVGELVDIGFAAYN
  		FVESIINLFQVVHNSYNRPAYSPGHKT
  		QPFLHDGYAVSWNTVEDSIIRTGFQG
  		ESGHDIKITAENTPLPIAGVLLPTIPGK
  		LDVNKSKTHISVNGRKIRMRCRAIDG
  		DVTFCRPKSPVYVGNGVHANLHVAF
  		HRSSSEKIHSNEISSDSIGVLGYQKTV
  		DHTKVNSKLSLFFEIKS
  3	MUMPS	GTYRLIPNARANLTA	SEQ	NA
  			ID
  			NO:
  			400
  4	MUMPS	PSKFFTISDSATFAPGPVSNA;	402;	NA
  		PSKLFIMLDNATFAPGPVVNA	404
  5	RUBELLA VIRUS	TPPPYQVSCGGESDRASARVIDPAAQ	406	NA
  	(RV)	S
  6	HEMAGGLUTININ	PEYAYKIVKNKKMEDGFLQGMVDG	408	NA
  		WYGHHSNEQGSGLMENERTLDKAN
  		PNNDLCSWSDHEASSNNTNQEDLLQ
  		RESRRKKRIGTSTLNQRGNCNTKCQT
  		EEARLKREEVSLVKSDQCSNGSLQCR
  		ANNSTEQVD
  7	NEURAMINIDASE	TKSTNSRSGGISGPDNEAPVGEAPSP	410	NA
  		YGDNPRPNDGNNIRIGSKGYNGIITD
  		TIEESCSCYPDAKVVKSVELDSTIWTS
  		GSSPNQKIITIGWDPNGWTGTPMSPN
  		GAYGTDGPSNGQANQHQAESISAGN
  		SSLCPIRDNWHGSNRSWSWPDGAE
  8	TAP (TRANSPORT	EKIVLLLAMMEKIVLLLAKCQTPMG	412	NA
  	ANTIGEN	AIKAVDGVTNKCPYLGSPSF
  	PRESENTATION)
  	ON H5N1 VIRUS
  	HEMAGGLUTININ
  9	TAP (TRANSPORT	IRPCFWVELNPNQKIITIRPCFWVELIC	414	NA
  	ANTIGEN	YPDAGEIT
  	PRESENTATION)
  	ON H5N1 VIRUS
  	NEURAMINIDASE
  10	HEMAGGLUTININ	MEKIVLLLAEKIVLLLAMCPYLGSPS	416	NA
  	EPITOPES	FKCQTPMGAIKAVDGVTNK
  	TOWARD CLASS I
  	HLA
  11	NEURAMINIDASE	NPNQKIITICYPDAGEITIRPCFWVELR	418	NA
  	EPITOPES	PCFWVELI
  	TOWARD CLASS I
  	HLA
  12	HEMAGGLUTININ	MVSLVKSDQIGTSTLNQR	420	NA
  	EPITOPES
  	TOWARD CLASS
  	II HLA
  13	NEURAMINIDASE	YNGIITDTI	422	NA
  	EPITOPES
  	TOWARD CLASS
  	II HLA
  14	HEMAGGLUTININ	MEKIVLLLAEKIVLLLAMMVSLVKSD	424	NA
  	EPITOPE H5N1-	QCPYLGSPSFIGTSTLNQRKCQTPMG
  	BOUND CLASS I	AIKAVDGVTNK
  	AND CLASS II
  	HLA
  15	NEURAMINIDASE	NPNQKIITIYNGIITDTICYPDAGEITIR	426	NA
  	EPITOPE H5N1-	PCFWVELRPCFWVELI
  	BOUND CLASS I
  	AND CLASS II
  	HLA
  16	CORYNEBACTERI	AACAGNRVRRSVGSSLKC	899	SRC280292
  	UM DIPHTHERIAE
  17	MEASLES VIRUS	AADHCPVVEVNGVTI	900	P69353.1
  	STRAIN
  	EDMONSTON
  18	MEASLES VIRUS	AAHLPTGTPLDID	901	P04851.1
  	STRAIN
  	EDMONSTON
  19	BORDETELLA	AALAVWAGLAVQ	902	Q00879.1
  	PERTUSSIS
  20	MEASLES VIRUS	AALGVATAAQITAGI	903	P69353.1
  	STRAIN
  	EDMONSTON
  21	RUBELLA VIRUS	AALLNTPPPYQVSCGGESDRATAR	904	P07566.1
  	STRAIN THERIEN
  22	RUBELLA VIRUS	AAQSFTGVVYGTHTT	905	BAA28178.1
  23	RUBELLA VIRUS	ACEVEPAFGHSDAAC	906	BAA28178.1
  24	RUBELLA VIRUS	ACTFWAVNAYSSGGY	907	BAA28178.1
  25	RUBELLA VIRUS	ADDPLLR	908	BAA19893.1
  26	RUBELLA VIRUS	ADDPLLRT	909	CAJ88851.1
  27	MEASLES VIRUS	AEMICDIDTYIVEAG	910	P04851.1
  	STRAIN
  	EDMONSTON
  28	MEASLES VIRUS	AEMICDIDTYIVEAGLASFI	911	P04851.1
  	STRAIN
  	EDMONSTON
  29	MEASLES VIRUS	AEPLLSC	912	P04851.1
  	STRAIN
  	EDMONSTON
  30	BORDETELLA	AFVSTSSSRRYTEVY	913	CAD44970.1
  	PERTUSSIS
  31	BORDETELLA	AGFIYRETFCITTIYKTGQPAADHYYS	914	P04979.1
  	PERTUSSIS	KVTA
  32	RUBELLA VIRUS	AGLLACCAKCLYYLR	915	BAA28178.1
  33	RUBELLA VIRUS	AHTTSDPWHPPG	916	BAA19893.1
  34	MEASLES VIRUS	AIAKLEDAKELLESS	917	P69353.1
  	STRAIN
  	EDMONSTON
  35	MEASLES VIRUS	AIDNLRASLETTNQA	918	P69353.1
  	STRAIN
  	EDMONSTON
  36	BORDETELLA	AKGVEFR	919	ACI16088.1
  	PERTUSSIS
  37	MEASLES VIRUS	AKWAVPTTRTDDKLR	920	P08362.1
  	STRAIN
  	EDMONSTON
  38	MEASLES VIRUS	ALAEVLKKPV	921	ABO69699.1
  	STRAIN
  	EDMONSTON
  39	MEASLES VIRUS	ALGVINTLEWIPRFK	922	P08362.1
  	STRAIN
  	EDMONSTON
  40	MEASLES VIRUS	ALHQSMLNSQAIDNL	923	P69353.1
  	STRAIN
  	EDMONSTON
  41	MEASLES VIRUS	ALIGILSLFV	924	ABI54110.1
  	STRAIN
  	EDMONSTON
  42	RUBELLA VIRUS	ALLNTPPPYQVSCGGESDRA	925	CAJ88851.1
  43	RUBELLA VIRUS	ALLNTPPPYQVSCGGESDRASARV	926	CAJ88851.1
  	STRAIN M33
  44	RUBELLA VIRUS	ALVEGLAPGGGNCHL	927	BAA28178.1
  45	BORDETELLA	AMAAWSERAGEA	928	P04977.1
  	PERTUSSIS
  46	MEASLES VIRUS	ANCASILCKCYTTGT	929	P69353.1
  	STRAIN
  	EDMONSTON
  47	BORDETELLA	ANPNPYTSRRSV	930	P04977.1
  	PERTUSSIS
  48	RUBELLA VIRUS	APGPGEVW	931	CAJ88851.1
  49	RUBELLA VIRUS	APLPPHTTERIETRSARHPWRIR	932	ABD64214.1
  50	RUBELLA VIRUS	APPMPPQPPRAHGQHYGHHHHQLPF	933	CAA33016.1
  	VACCINE STRAIN	LG
  	RA27/3
  51	RUBELLA VIRUS	APPTLPQPPCAHGQHYGHHHHQLPF	934	P07566.1
  	STRAIN THERIEN	LG
  52	RUBELLA VIRUS	APPTLPQPPRAHGQHYGHHHHQLPF	935	ABD64214.1
  		LG
  53	BORDETELLA	APQPGPQPPQPPQPQPEAPAPQ	936	P14283.3
  	PERTUSSIS
  54	RUBELLA VIRUS	AQLASYFNPGGSYYK	937	BAA28178.1
  55	MEASLES VIRUS	ARAAHLPTGTPLD	938	P04851.1
  	STRAIN
  	EDMONSTON
  56	MEASLES VIRUS	ARLVSEIAMHTTEDK	939	P04851.1
  	STRAIN
  	EDMONSTON
  57	MEASLES VIRUS	ARLVSEIAMHTTEDKISRAV	940	P04851.1
  	STRAIN
  	EDMONSTON
  58	MEASLES VIRUS	ASDVETAEGGEIHELLR	941	P03422.1
  	STRAIN
  	EDMONSTON-B
  59	MEASLES	ASDVETAEGGEIHELLRLQ	942	ABO69699.1
  	MORBILLIVIRUS
  60	MEASLES VIRUS	ASDVETAEGGEIHELLRLQSR	943	P03422.1
  	STRAIN
  	EDMONSTON-B
  61	MEASLES	ASDVETAEGGEIHKLLRLQ	944	AAA63285.1
  	MORBILLIVIRUS
  62	RUBELLA VIRUS	ASDVLPGHWLQG	945	NP_740663.1
  	STRAIN M33
  63	MEASLES VIRUS	ASELGITAEDARLVS	946	P04851.1
  	STRAIN
  	EDMONSTON
  64	MEASLES VIRUS	ASELGITAEDARLVSEIAMH	947	P04851.1
  	STRAIN
  	EDMONSTON
  65	MEASLES VIRUS	ASESSQDPQDSRR	948	P04851.1
  	STRAIN
  	EDMONSTON
  66	MEASLES VIRUS	ASILCKCYTTGTIIN	949	P69353.1
  	STRAIN
  	EDMONSTON
  67	RUBELLA VIRUS	ASPVCQRHSPDCSRL	950	BAA28178.1
  68	BORDETELLA	ASQARWTGATRA	951	BAF35031.1
  	PERTUSSIS
  69	RUBELLA VIRUS	ASYFNPGGSYYKQYHPTACEVEPAFG	952	P07566.1
  	STRAIN THERIEN	HS
  70	MEASLES VIRUS	ASYKVMTRSSHQSLV	953	P69353.1
  	STRAIN
  	EDMONSTON
  71	BORDETELLA	ASYVKKPKEDVD	954	ACI16088.1
  	PERTUSSIS
  72	MEASLES VIRUS	ATAAQITAGIALHQS	955	P69353.1
  	STRAIN
  	EDMONSTON
  73	RUBELLA VIRUS	ATPERPRL	956	CAJ88851.1
  74	MEASLES VIRUS	AVCLGGLIGIPALIC	957	P69353.1
  	STRAIN
  	EDMONSTON
  75	MEASLES VIRUS	AVGPRQAQVSF	958	P04851.1
  	STRAIN
  	EDMONSTON
  76	RUBELLA VIRUS	AVNAYSSGGYAQLAS	959	BAA28178.1
  77	RUBELLA VIRUS	AVSETRQTWAEWAAA	960	BAA28178.1
  78	MEASLES VIRUS	AVTAPDTAADSELRR	961	P04851.1
  	STRAIN
  	EDMONSTON
  79	MEASLES VIRUS	AVTAPDTAADSELRRWIKYT	962	P04851.1
  	STRAIN
  	EDMONSTON
  80	BORDETELLA	AYGGIIKDAPPGAGFIYRETFC	963	P04979.1
  	PERTUSSIS
  81	RUBELLA VIRUS	CALPLAGLLACCAKC	964	BAA28178.1
  82	RUBELLA VIRUS	CARIWNGTQRACTFW	965	BAA28178.1
  83	MEASLES VIRUS	CARTLVSGSFGNRFI	966	P69353.1
  	STRAIN
  	EDMONSTON
  84	BORDETELLA	CASPYEGRYRDMYDALRBRLLY	967	SRC280066
  	PERTUSSIS
  85	BORDETELLA	CAVFVRSGQPVIGA	968	AAA83981.1
  	PERTUSSIS
  86	RUBELLA VIRUS	CCAKCLYYLRGAIAPR	969	BAA28178.1
  87	MEASLES VIRUS	CCRGRCNKKGEQVGM	970	P69353.1
  	STRAIN
  	EDMONSTON
  88	RUBELLA VIRUS	CEIPTDVSCEGLGAW	971	BAA28178.1
  89	BORDETELLA	CFGKDLKRPGSSPMEV	972	P0A3R5.1
  	PERTUSSIS
  90	MEASLES	CFQQACKGKIQALCE	973	P06830.1
  	MORBILLIVIRUS
  91	MEASLES	CFQQACKGKIQALCENPEWAPLKDN	974	AAR89413.1
  	MORBILLIVIRUS	RIPS
  92	RUBELLA VIRUS	CGGESDRASARVIDP	975	BAA28178.1
  93	BORDETELLA	CITTIYKTGQPAADHYYSKVTA	976	P04979.1
  	PERTUSSIS
  94	MEASLES	CKGKIQALCENPEWA	977	AAR89413.1
  	MORBILLIVIRUS
  95	MEASLES VIRUS	CKPWQESRKNKAQ	978	P04851.1
  	STRAIN
  	EDMONSTON
  96	MEASLES VIRUS	CNKKGEQVGMSRPGL	979	P69353.1
  	STRAIN
  	EDMONSTON
  97	RUBELLA VIRUS	CNVTTEHPFCNTPHG	980	BAA28178.1
  98	BORDETELLA	CQVGSSNSAF	981	P04977.1
  	PERTUSSIS
  99	MEASLES	CSGPTTIRGQFS	982	P08362.1
  	MORBILLIVIRUS
  100	BORDETELLA	CTSPYDGKYWSMYSRL	983	AAA83981.1
  	PERTUSSIS
  101	MEASLES VIRUS	CVLADSESGGHITHS	984	P08362.1
  	STRAIN
  	EDMONSTON
  102	MEASLES	CYTTGTIINQDPDKILTYIAADHC	985	AAF02706.1
  	MORBILLIVIRUS
  103	RUBELLA VIRUS	DADDPLLR	986	CAJ88851.1
  104	MEASLES VIRUS	DARAAHLPTGTPLDI	987	P04851.1
  	STRAIN
  	EDMONSTON
  105	MEASLES VIRUS	DARAAHLPTGTPLDIDTASE	988	P04851.1
  	STRAIN
  	EDMONSTON
  106	MEASLES VIRUS	DCHAPTYLPAEVDGD	989	P08362.1
  	STRAIN
  	EDMONSTON
  107	RUBELLA VIRUS	DCSRLVGATPERPRL	990	BAA28178.1
  108	MEASLES VIRUS	DDKLRMETCFQQACK	991	P08362.1
  	STRAIN
  	EDMONSTON
  109	RUBELLA VIRUS	DDPLLRTA	992	CAJ88851.1
  110	RUBELLA VIRUS	DDPLLRTAPGPGEVW	993	BAA28178.1
  111	BORDETELLA	DDPPATVYRYD	994	P04977.1
  	PERTUSSIS
  112	BORDETELLA	DDPPATVYRYDSRPPED	995	CAD44970.1
  	PERTUSSIS
  113	BORDETELLA	DDPPATVYRYDSRPPEDV	996	ACI04548.1
  	PERTUSSIS
  114	MEASLES	DEVGLRTPQRFTDLV	997	P06830.1
  	MORBILLIVIRUS
  115	MEASLES VIRUS	DHCPVVEVNGVTIQV	998	P69353.1
  	STRAIN
  	EDMONSTON
  116	MEASLES VIRUS	DIDTASESSQDPQ	999	P04851.1
  	STRAIN
  	EDMONSTON
  117	MEASLES VIRUS	DINKVLEKLGYSGGD	1000	P69353.1
  	STRAIN
  	EDMONSTON
  118	BORDETELLA	DLIAYKQ	1001	ACI16088.1
  	PERTUSSIS
  119	MEASLES VIRUS	DLIGQKLGLKLLRYY	1002	P69353.1
  	STRAIN
  	EDMONSTON
  120	RUBELLA VIRUS	DLQKALEAQSRALRAELAA	1003	P07566.1
  121	MEASLES	DLQYVLATYDTSRVE	1004	P06830.1
  	MORBILLIVIRUS
  122	MEASLES VIRUS	DLSLRRFMV	1005	P04851.1
  	STRAIN
  	EDMONSTON
  123	MEASLES VIRUS	DLSNCMVALGELKLA	1006	P08362.1
  	STRAIN
  	EDMONSTON
  124	RUBELLA VIRUS	DLVEYIMNYTGNQQSRWGLGSPNC	1007	CAJ88851.1
  125	MEASLES	DLVKFISDKIKFLNP	1008	AAR89413.1
  	MORBILLIVIRUS
  126	MEASLES	DLVKFISTKIKFLNP	1009	SRC280117
  	MORBILLIVIRUS
  127	MEASLES VIRUS	DLYKSNHNNV	1010	P08362.1
  	STRAIN
  	EDMONSTON
  128	BORDETELLA	DNVLDHLTGR	1011	ACI04548.1
  	PERTUSSIS
  129	BORDETELLA	DNVLDHLTGRSC	1012	P04977.1
  	PERTUSSIS
  130	BORDETELLA	DNVLDHLTGRSCQ	1013	P04977.1
  	PERTUSSIS
  131	MEASLES	DPDKILTYIAA	1014	AAF02706.1
  	MORBILLIVIRUS
  132	RUBELLA VIRUS	DPGDLVEYIMNYTGNQQSR	1015	P07566.1
  	STRAIN THERIEN
  133	RUBELLA VIRUS	DPLLRTAP	1016	CAJ88851.1
  134	RUBELLA VIRUS	DPLLRTAPGPGEVWVTPVIGSQ	1017	CAJ88851.1
  135	MEASLES VIRUS	DPQDSRRSAEPLL	1018	P04851.1
  	STRAIN
  	EDMONSTON
  136	MEASLES VIRUS	DPVIDRLYLSSHRGV	1019	P08362.1
  	STRAIN
  	EDMONSTON
  137	MEASLES VIRUS	DQILRSMKGLSSTSI	1020	P69353.1
  	STRAIN
  	EDMONSTON
  138	MEASLES	DQYCADVAAEELMNA	1021	P06830.1
  	MORBILLIVIRUS
  139	MEASLES VIRUS	DSESGGHITH	1022	P08362.1
  	STRAIN
  	EDMONSTON
  140	MEASLES VIRUS	DTASESSQDPQDS	1023	P04851.1
  	STRAIN
  	EDMONSTON
  141	RUBELLA VIRUS	DTVMSVFALASYVQH	1024	BAA28178.1
  142	BORDETELLA	DVFQNGFTAWGNND	1025	P04977.1
  	PERTUSSIS
  143	RUBELLA VIRUS	DVGAVPPGKFVTAAL	1026	BAA28178.1
  144	CORYNEBACTERIUM	DVNKSKTHISVNGRKI	1027	CAE11230.1
  	DIPHTHERIAE
  145	RUBELLA VIRUS	DVSCEGLGAWVPAAP	1028	BAA28178.1
  146	RUBELLA VIRUS	DWASPVCQRHSPDCSRLVGATPERPR	1029	P07566.1
  	STRAW THERIEN	L
  147	MEASLES VIRUS	EARESYRETGPSR	1030	P04851.1
  	STRAIN
  	EDMONSTON
  148	BORDETELLA	EAVEAERAGRGTG	1031	ACI04548.1
  	PERTUSSIS
  149	MEASLES VIRUS	EDAKELLESSDQILR	1032	P69353.1
  	STRAIN
  	EDMONSTON
  150	MEASLES VIRUS	EDRRVKQSRGEAR	1033	P04851.1
  	STRAIN
  	EDMONSTON
  151	MEASLES VIRUS	EDSITIPYQGSGKGV	1034	P08362.1
  	STRAIN
  	EDMONSTON
  152	RUBELLA VIRUS	EEAFTYLCTAPGCAT	1035	BAA28178.1
  153	MEASLES VIRUS	EGFNMILGTILAQIW	1036	P04851.1
  	STRAIN
  	EDMONSTON
  154	MEASLES VIRUS	EGFNMILGTILAQIWVLLAK	1037	P04851.1
  	STRAIN
  	EDMONSTON
  155	RUBELLA VIRUS	EHPFCNTPHGQLEVQ	1038	BAA28178.1
  156	MEASLES VIRUS	EISDIEVQDPEGFNM	1039	P04851.1
  	STRAIN
  	EDMONSTON
  157	MEASLES VIRUS	EISDIEVQDPEGFNMILGTI	1040	P04851.1
  	STRAIN
  	EDMONSTON
  158	MEASLES VIRUS	EKPNLSSKRSE	1041	P08362.1
  	STRAIN
  	EDMONSTON
  159	MEASLES VIRUS	ELKLAALCHGEDSIT	1042	P08362.1
  	STRAIN
  	EDMONSTON
  160	MEASLES	ELMNALVNSTLLETR	1043	P06830.1
  	MORBILLIVIRUS
  161	MEASLES VIRUS	ELPRL	1044	P04851.1
  	STRAIN
  	EDMONSTON
  162	MEASLES	ENPEWAPLKDNRIPSYGVLSVDL	1045	AAR89413.1
  	MORBILLIVIRUS
  163	MEASLES VIRUS	EPIRDALNAMTQNIR	1046	P69353.1
  	STRAIN
  	EDMONSTON
  164	MEASLES VIRUS	EQVGMSRPGLKPDLT	1047	P69353.1
  	STRAIN
  	EDMONSTON
  165	RUBELLA VIRUS	ERPRLRLV	1048	CAJ88851.1
  166	RUBELLA VIRUS	ERPRLRLVDADDPLL	1049	BAA28178.1
  167	MEASLES VIRUS	ESPGQLIQRITDDPDVS	1050	P04851.1
  	CAM/RB
  168	MEASLES VIRUS	ESRGIKARITHVDTE	1051	P69353.1
  	STRAIN
  	EDMONSTON
  169	MEASLES VIRUS	ESSCTFMPEGTVCSQ	1052	P69353.1
  	STRAIN
  	EDMONSTON
  170	MEASLES VIRUS	ESSQDPQDSRRSA	1053	P04851.1
  	STRAIN
  	EDMONSTON
  171	MEASLES VIRUS	ETRTTNQFLAVSKGN	1054	P08362.1
  	STRAIN
  	EDMONSTON
  172	MEASLES VIRUS	EVDGDVKLSSNLVIL	1055	P08362.1
  	STRAIN
  	EDMONSTON
  173	MEASLES VIRUS	EVNGVTIQV	1056	P26031.1
  	STRAIN
  	EDMONSTON-B
  174	RUBELLA VIRUS	EVWVTPVI	1057	CAJ88851.1
  175	RUBELLA VIRUS	EVWVTPVIGSQA	1058	BAA19893.1
  176	RUBELLA VIRUS	EWAAAHWWQLTLGAT	1059	BAA28178.1
  177	MEASLES	EWIPRFKVSPYLFTV	1060	P06830.1
  	MORBILLIVIRUS
  178	BORDETELLA	FEYVDTYGDNAG	1061	P04977.1
  	PERTUSSIS
  179	MEASLES	FGPLITHGSGMDLYK	1062	P06830.1
  	MORBILLIVIRUS
  180	MEASLES VIRUS	FIFDALAEV	1063	ABK40531.1
  	STRAIN
  	EDMONSTON
  181	MEASLES VIRUS	FISDKIKFL	1064	P08362.1
  	STRAIN
  	EDMONSTON
  182	MEASLES VIRUS	FKRNKDKPPITSGSG	1065	P04851.1
  	STRAIN
  	EDMONSTON
  183	MEASLES VIRUS	FKRNKDKPPITSGSGGAIRG	1066	P04851.1
  	STRAIN
  	EDMONSTON
  184	RUBELLA VIRUS	FKTVRPVALPRTLAP	1067	BAA28178.1
  185	MEASLES VIRUS	FLMDRHIIV	1068	ABK40531.1
  	STRAIN
  	EDMONSTON
  186	MEASLES VIRUS	FMAVLLTLQTPTGQI	1069	P69353.1
  	STRAIN
  	EDMONSTON
  187	MEASLES VIRUS	FMPEGTVCSQNALYP	1070	P69353.1
  	STRAIN
  	EDMONSTON
  188	MEASLES	FMYMSLLGV	1071	AAN09804.1
  	MORBILLIVIRUS
  189	MEASLES VIRUS	FNVPIKEAGEDCHAP	1072	P08362.1
  	STRAIN
  	EDMONSTON
  190	MEASLES	FRDLTWCINPPERIK	1073	AAC35876.2
  	MORBILLIVIRUS
  191	MEASLES VIRUS	FSHDDPISSDQSRFG	1074	P04851.1
  	STRAIN
  	EDMONSTON
  192	MEASLES VIRUS	FSHDDPISSDQSRFGWFENK	1075	P04851.1
  	STRAIN
  	EDMONSTON
  193	MEASLES	FTDLVKFISDKIKFL	1076	P06830.1
  	MORBILLIVIRUS
  194	MEASLES	FTWDQKLWCRHFCVL	1077	P06830.1
  	MORBILLIVIRUS
  195	BORDETELLA	FVRDGQSVIGACASPYEGRYRDLYD	1078	SRC280066
  	PERTUSSIS	ALRRLLY
  196	BORDETELLA	FVRSGQPVIGACTSPYDGKYWSILYS	1079	SRC280066
  	PERTUSSIS	RLRKMLY
  197	MEASLES	FYKDNPHPKGSRIVI	1080	P06830.1
  	MORBILLIVIRUS
  198	BORDETELLA	GAASSYFEYVDTYG	1081	ACI04548.1
  	PERTUSSIS
  199	BORDETELLA	GAFDLKTTFCIMTTRNTGQPA	1082	AAA83981.1
  	PERTUSSIS
  200	MEASLES VIRUS	GALIGILSLFVESPG	1083	P04851.1
  	STRAIN
  	EDMONSTON
  201	MEASLES VIRUS	GALIGILSLFVESPGQLIQR	1084	P04851.1
  	STRAIN
  	EDMONSTON
  202	RUBELLA VIRUS	GATPERPR	1085	CAJ88851.1
  203	BORDETELLA	GAYGRCPNGTRALTVAELRGNAEL	1086	P04979.1
  	PERTUSSIS
  204	RUBELLA VIRUS	GCFAPWDLEATGACI	1087	BAA28178.1
  205	MEASLES	GDINKVLEKLGYS	1088	BAB60865.1
  	MORBILLIVIRUS
  206	MEASLES	GDINKVLEKLGYSGGDLLG	1089	AAL29688.1
  	MORBILLIVIRUS
  207	RUBELLA VIRUS	GDLRAVHHRPVPA	1090	CAA28880.1
  208	RUBELLA VIRUS	GDLVEYIMNYTGNQQ	1091	BAA28178.1
  209	MEASLES VIRUS	GDSSITTRSRLLDRL	1092	P04851.1
  	STRAIN
  	EDMONSTON
  210	MEASLES VIRUS	GDSSITTRSRLLDRLVRLIG	1093	P04851.1
  	STRAIN
  	EDMONSTON
  211	MEASLES	GEDCHAPTYLPAEVD	1094	P06830.1
  	MORBILLIVIRUS
  212	MEASLES VIRUS	GELSTLESLMNLYQQ	1095	P04851.1
  	STRAIN
  	EDMONSTON
  213	MEASLES VIRUS	GELSTLESLMNLYQQMGKPA	1096	P04851.1
  	STRAIN
  	EDMONSTON
  214	MUMPS	GEQARYLALLEA	1097	P21186.1
  	RUBULAVIRUS
  215	RUBELLA VIRUS	GEVWVT	1098	BAA19893.1
  216	RUBELLA VIRUS	GEVWVTPV	1099	CAJ88851.1
  217	RUBELLA VIRUS	GEVWVTPVIGSQAR	1100	BAA19893.1
  218	BORDETELLA	GEYGGVIKDGTPGGA	1101	AAA83981.1
  	PERTUSSIS
  219	RUBELLA VIRUS	GFLSGVGPMRLRHGADT	1102	SRC265968
  220	MEASLES VIRUS	GFRASDVETAEGGEIHELLRLQ	1103	P03422.1
  	STRAIN
  	EDMONSTON-B
  221	BORDETELLA	GGAVPGGAVPGGAVPGGFGPGGFGP	1104	P14283.3
  	PERTUSSIS
  222	BORDETELLA	GGAVPGGAVPGGFGPGGFGPGGFGP	1105	CAA09475.1
  	PERTUSSIS
  223	BORDETELLA	GGAVPGGAVPGGFGPGGFGPGGFGP	1106	CAA09474.1
  	PERTUSSIS	GGFGP
  224	MEASLES	GGHITHSGMVGMGVS	1107	P06830.1
  	MORBILLIVIRUS
  225	MEASLES	GILESRGIKARITHVDTESY	1108	P26032.1
  	MORBILLIVIRUS
  226	BORDETELLA	GITGETTTTEYSNARYV	1109	CAD44970.1
  	PERTUSSIS
  227	MEASLES VIRUS	GKEDRRVKQSRGE	1110	P04851.1
  	STRAIN
  	EDMONSTON
  228	BORDETELLA	GKVTNGS	1111	ACI16088.1
  	PERTUSSIS
  229	RUBELLA VIRUS	GLGAWVPAAPCARIW	1112	BAA28178.1
  230	MEASLES VIRUS	GLIGIPALICCCRGR	1113	P69353.1
  	STRAIN
  	EDMONSTON
  231	RUBELLA VIRUS	GLLACCAKCLYYLRGAIAPR	1114	P07566.1
  	STRAIN THERIEN
  232	MEASLES	GLLAIAGIRLHRAAI	1115	P06830.1
  	MORBILLIVIRUS
  233	RUBELLA VIRUS	GLQPRADMAAPPTLPQ	1116	NP_740663.1
  234	MEASLES	GMGVSCTVTREDGTNRR	1117	AAR89413.1
  	MORBILLIVIRUS
  235	MEASLES	GMYGGTYLVEKP	1118	AAR89413.1
  	MORBILLIVIRUS
  236	BORDETELLA	GNAELQTYLRQITPGWSIYGLYDGTY	1119	P04979.1
  	PERTUSSIS	LG
  237	RUBELLA VIRUS	GNCHLTVNGEDVGAV	1120	BAA28178.1
  238	BORDETELLA	GNNDNVLDHLTGR	1121	P04977.1
  	PERTUSSIS
  239	BORDETELLA	GNNDNVLDHLTGRSC	1122	P04977.1
  	PERTUSSIS
  240	MEASLES VIRUS	GNRFILSQGNLIANC	1123	P69353.1
  	STRAIN
  	EDMONSTON
  241	RUBELLA VIRUS	GNRGRGQRRDWSRAPPPPEERQETR	1124	P07566.1
  		SQTPAPKPS
  242	RUBELLA VIRUS	GPGEVWVT	1125	CAJ88851.1
  243	RUBELLA VIRUS	GPMRLRHGADTRCGRLI	1126	P07566.1
  	STRAIN THERIEN
  244	BORDETELLA	GPNHTKV	1127	ACI16083.1
  	PERTUSSIS
  245	MEASLES VIRUS	GPRQAQVSF	1128	P10050.1
  	STRAIN HALLE
  246	MEASLES VIRUS	GPRQAQVSFLQGDQS	1129	P04851.1
  	STRAIN
  	EDMONSTON
  247	MEASLES VIRUS	GPRQAQVSFLQGDQSENELP	1130	P04851.1
  	STRAIN
  	EDMONSTON
  248	MEASLES	GRGYNVSSIVTMTSQ	1131	P06830.1
  	MORBILLIVIRUS
  249	BORDETELLA	GRTPFII	1132	ACI16083.1
  	PERTUSSIS
  250	RUBELLA VIRUS	GSPNCHGPDWASPVC	1133	BAA28178.1
  251	RUBELLA VIRUS	GSQARKCGLHIRAGP	1134	BAA28178.1
  252	BORDETELLA	GSSNSAFVSTSSSRR	1135	P04977.1
  	PERTUSSIS
  253	MEASLES VIRUS	GSTKSCARTLVSGSF	1136	P69353.1
  	STRAIN
  	EDMONSTON
  254	RUBELLA VIRUS	GSYYKQYHPTACEVE	1137	BAA28178.1
  255	RUBELLA VIRUS	GTHTTAVSETRQTWA	1138	BAA28178.1
  256	MEASLES VIRUS	GTIINQDPDKILTYI	1139	P69353.1
  	STRAIN
  	EDMONSTON
  257	BORDETELLA	GTLVRMAPVIG	1140	ADA85124.1
  	PERTUSSIS
  258	MEASLES VIRUS	GTPLDIDTASESS	1141	P04851.1
  	STRAIN
  	EDMONSTON
  259	BORDETELLA	GTYLGQAYGGIIKDAPPGAGFIYRETF	1142	P04979.1
  	PERTUSSIS	C
  260	BORDETELLA	GVATKGLGVHAKSSDWG	1143	P15318.2
  	PERTUSSIS
  261	CORYNEBACTERIUM	GVLLPTIPGKLDVNKSKTHI	1144	AAV70486.1
  	DIPHTHERIAE
  262	MEASLES	GVLSVDLSLTVELKI	1145	P06830.1
  	MORBILLIVIRUS
  263	MEASLES	GVPIELQVECFTWDQ	1146	P06830.1
  	MORBILLIVIRUS
  264	MEASLES VIRUS	GVSCTVTREDGTNRR	1147	P08362.1
  	STRAIN
  	EDMONSTON
  265	MEASLES VIRUS	GVSYNIGSQEWYTTV	1148	P69353.1
  	STRAIN
  	EDMONSTON
  266	MEASLES VIRUS	GYNVSSIVTMTSQGM	1149	P08362.1
  	STRAIN
  	EDMONSTON
  267	MEASLES	HFCVLADSESGGHIT	1150	P06830.1
  	MORBILLIVIRUS
  268	MEASLES	HGEDSITIPYQGSGK	1151	P06830.1
  	MORBILLIVIRUS
  269	RUBELLA VIRUS	HGPDWASP	1152	BAA19893.1
  270	RUBELLA VIRUS	HGPDWASPVCQRHSP	1153	BAA28178.1
  271	RUBELLA VIRUS	HGPDWASPVCQRHSPDCSRLVG	1154	CAJ88851.1
  272	RUBELLA VIRUS	HGPDWASPVCQRHSPDCSRLVGATPE	1155	CAJ88851.1
  	STRAIN M33	RPRLRLV
  273	MEASLES VIRUS	HITHSGMEGMGVSCT	1156	P08362.1
  	STRAIN
  	EDMONSTON
  274	MEASLES	HKSLSTNLDVTNSIE	1157	P06830.1
  	MORBILLIVIRUS
  275	MEASLES VIRUS	HLMIDRPYV	1158	P08362.1
  	STRAIN
  	EDMONSTON
  276	MEASLES VIRUS	HLPTGTPLDIDTA	1159	P04851.1
  	STRAIN
  	EDMONSTON
  277	MEASLES VIRUS	HLPTGTPLDIDTATESSQDPQDSR	1160	Q77M43.1
  	STRAIN
  	EDMONSTON-B
  278	MEASLES	HMTNYLEQPVSNDLS	1161	P06830.1
  	MORBILLIVIRUS
  279	MEASLES VIRUS	HQSLVIKLMPNITLL	1162	P69353.1
  	STRAIN
  	EDMONSTON
  280	MEASLES	HRAAIYTAEIHKSLS	1163	P06830.1
  	MORBILLIVIRUS
  281	BORDETELLA	HRMQEAVEAERAGRGTGH	1164	P04977.1
  	PERTUSSIS
  282	MEASLES VIRUS	HVDTESYFIVLSIAY	1165	P69353.1
  	STRAIN
  	EDMONSTON
  283	MEASLES VIRUS	HWGNLSKIGVVGIGS	1166	P69353.1
  	STRAIN
  	EDMONSTON
  284	RUBELLA VIRUS	HWWQLTLGATCALPL	1167	BAA28178.1
  285	RUBELLA VIRUS	HYRNASDVLPGHWLQGGWGCYNL	1168	NP_740663.1
  286	MEASLES VIRUS	IDLGPPISLERLDVG	1169	P69353.1
  	STRAIN
  	EDMONSTON
  287	MEASLES VIRUS	IEAIRQAGQEMILAV	1170	P69353.1
  	STRAIN
  	EDMONSTON
  288	RUBELLA VIRUS	IETRSARHP	1171	CAA28880.1
  	STRAIN M33
  289	MEASLES VIRUS	IGSQEWYTTVPKYVA	1172	P69353.1
  	STRAIN
  	EDMONSTON
  290	MEASLES VIRUS	IKGVIVHRLEGVSYN	1173	P69353.1
  	STRAIN
  	EDMONSTON
  291	MEASLES VIRUS	IKHIIIVPIPGDSSI	1174	P04851.1
  	STRAIN
  	EDMONSTON
  292	MEASLES VIRUS	IKHIIIVPIPGDSSITTRSR	1175	P04851.1
  	STRAIN
  	EDMONSTON
  293	BORDETELLA	IKLKDCP	1176	ACI16083.1
  	PERTUSSIS
  294	MEASLES VIRUS	IKLMPNITLLNNCTR	1177	P69353.1
  	STRAIN
  	EDMONSTON
  295	MEASLES	ILLERLDVGT	1178	AAF85664.1
  	MORBILLIVIRUS
  296	MEASLES	ILPGQDLQYV	1179	P08362.1
  	MORBILLIVIRUS
  297	MEASLES VIRUS	ILTYIAADHCPVVEV	1180	P69353.1
  	STRAIN
  	EDMONSTON
  298	MEASLES	INQDPDKILTY	1181	AAL29688.1
  	MORBILLIVIRUS
  299	MEASLES VIRUS	IPRFKVSPYLFNVPI	1182	P08362.1
  	STRAIN
  	EDMONSTON
  300	MEASLES VIRUS	IQALSYALGGDINKV	1183	P69353.1
  	STRAIN
  	EDMONSTON
  301	RUBELLA VIRUS	IRAGPYGHATVEMPE	1184	BAA28178.1
  302	BORDETELLA	IRMGTDK	1185	ACI16088.1
  	PERTUSSIS
  303	MEASLES VIRUS	ISNFDESSCTFMPEG	1186	P69353.1
  	STRAIN
  	EDMONSTON
  304	MEASLES VIRUS	ITAGIALHQSMLNSQ	1187	P69353.1
  	STRAIN
  	EDMONSTON
  305	MEASLES VIRUS	ITDDPDVSIRLLEVV	1188	P04851.1
  	STRAIN
  	EDMONSTON
  306	MEASLES VIRUS	ITDDPDVSIRLLEVVQSDQS	1189	P04851.1
  	STRAIN
  	EDMONSTON
  307	BORDETELLA	ITTYV	1190	ACI16083.1
  	PERTUSSIS
  308	MEASLES VIRUS	IVEAGLASFILTIKF	1191	P04851.1
  	STRAIN
  	EDMONSTON
  309	MEASLES VIRUS	IVEAGLASFILTIKFGIETM	1192	P04851.1
  	STRAIN
  	EDMONSTON
  310	MEASLES VIRUS	IVEAGLASFILTIKFGIETMYPALG	1193	P04851.1
  	STRAIN
  	EDMONSTON
  311	RUBELLA VIRUS	KALEAQSRALRAELAA	1194	P07566.1
  312	MEASLES VIRUS	KARITHVDTESYFIV	1195	P69353.1
  	STRAIN
  	EDMONSTON
  313	RUBELLA VIRUS	KCGLHIRAGPYGHAT	1196	BAA28178.1
  314	MEASLES VIRUS	KCYTTGTIINQDPDK	1197	P69353.1
  	STRAIN
  	EDMONSTON
  315	MEASLES VIRUS	KDNPHPKGSR	1198	P08362.1
  	STRAIN
  	EDMONSTON
  316	MEASLES VIRUS	KDNRIPSYGVLSVDL	1199	P08362.1
  	STRAIN
  	EDMONSTON
  317	MEASLES	KFLNPDREYDFRDLT	1200	AAC35876.2
  	MORBILLIVIRUS
  318	RUBELLA VIRUS	KFVTAALLN	1201	BAA28178.1
  319	MEASLES VIRUS	KGNCSGPTTIR	1202	P08362.1
  	STRAIN
  	EDMONSTON
  320	MEASLES	KIKFLNPDREYDFRD	1203	P06830.1
  	MORBILLIVIRUS
  321	RUBELLA VIRUS	KIVDGGCFAPWDLEA	1204	BAA28178.1
  322	MEASLES VIRUS	KLGLKLLRYYTEILS	1205	P69353.1
  	STRAIN
  	EDMONSTON
  323	MEASLES VIRUS	KLGVWKSPTDMQSWV	1206	P08362.1
  	STRAIN
  	EDMONSTON
  324	BORDETELLA	KLKECPQ	1207	ACI16088.1
  	PERTUSSIS
  325	MEASLES	KLLRYYTEI	1208	P26031.1
  	MORBILLIVIRUS
  326	MEASLES VIRUS	KLMPFSGDFV	1209	ABK40531.1
  	STRAIN
  	EDMONSTON
  327	MEASLES	KLMPNITLL	1210	P26031.1
  	MORBILLIVIRUS
  328	MEASLES	KLRMETCFQQACKGKIQALCENPEW	1211	AAR89413.1
  	MORBILLIVIRUS	A
  329	MEASLES	KLWCRHFCV	1212	P08362.1
  	MORBILLIVIRUS
  330	MEASLES VIRUS	KLWCRHFCVL	1213	P08362.1
  	STRAIN
  	EDMONSTON
  331	MEASLES	KLWESPQEI	1214	BAB60863.1
  	MORBILLIVIRUS
  332	MEASLES VIRUS	KMSSAVGFV	1215	ABO69699.1
  	STRAIN
  	EDMONSTON
  333	MEASLES VIRUS	KMSSAVGFVPDTGPASR	1216	P03422.1
  	STRAIN
  	EDMONSTON-B
  334	BORDETELLA	KMVYATN	1217	ACI16083.1
  	PERTUSSIS
  335	MEASLES VIRUS	KPDLTGTSKSYVRSL	1218	P69353.1
  	STRAIN
  	EDMONSTON
  336	MEASLES	KPNLSSKRSELSQLS	1219	P08362.1
  	MORBILLIVIRUS
  337	MEASLES VIRUS	KPNLSSKRSELSQLSMYRVF	1220	P08362.1
  	STRAIN
  	EDMONSTON
  338	MEASLES VIRUS	KQSRGEARESYRETG	1221	P04851.1
  	STRAIN
  	EDMONSTON
  339	MEASLES VIRUS	KQSRGEARESYRETGPSRAS	1222	P04851.1
  	STRAIN
  	EDMONSTON
  340	MEASLES VIRUS	KRFAGVVLAGAALGV	1223	P69353.1
  	STRAIN
  	EDMONSTON
  341	MEASLES VIRUS	KRTPGNKPRIAEMIC	1224	P04851.1
  	STRAIN
  	EDMONSTON
  342	MEASLES VIRUS	KRTPGNKPRIAEMICDIDTY	1225	P04851.1
  	STRAIN
  	EDMONSTON
  343	MEASLES	KSNHNNVYWLTIPPMKNLALGVINT	1226	AAR89413.1
  	MORBILLIVIRUS	L
  344	MEASLES	KVSPYLFNV	1227	P08362.1
  	MORBILLIVIRUS
  345	BORDETELLA	KVVQLPKISKNALKANG	1228	ACI16083.1
  	PERTUSSIS
  346	BORDETELLA	KVVQLPKISKNALRNDG	1229	ACI16087.1
  	PERTUSSIS
  347	BORDETELLA	LAHRRIPPENIR	1230	P04977.1
  	PERTUSSIS
  348	BORDETELLA	LALALWAGFALS	1231	P11092.1
  	PERTUSSIS
  349	RUBELLA VIRUS	LAPGGGNCHLTVNGE	1232	BAA28178.1
  350	MEASLES VIRUS	LAQIWVLLAKAVTAP	1233	P04851.1
  	STRAIN
  	EDMONSTON
  351	MEASLES VIRUS	LAQIWVLLAKAVTAPDTAAD	1234	P04851.1
  	STRAIN
  	EDMONSTON
  352	RUBELLA VIRUS	LASYFNPGGSYYKQYHPTACEVEPAF	1235	BAA19893.1
  		GHS
  353	MEASLES	LAVSKGNCSGPTTIR	1236	P06830.1
  	MORBILLIVIRUS
  354	MEASLES VIRUS	LCENPEWAPLKDNRI	1237	P08362.1
  	STRAIN
  	EDMONSTON
  355	MEASLES	LDRLVRLIG	1238	ABI54110.1
  	MORBILLIVIRUS
  356	CORYNEBACTERIUM	LEEEGVTPL	1239	P33120.2
  	DIPHTHERIAE
  357	BORDETELLA	LEHRMQEAVEAERAGRGTGHFI	1240	CAD44970.1
  	PERTUSSIS
  358	MEASLES VIRUS	LEKLGYSGGDLLGIL	1241	P69353.1
  	STRAIN
  	EDMONSTON
  359	MEASLES VIRUS	LEQPVSNDLS	1242	P08362.1
  	STRAIN
  	EDMONSTON
  360	MEASLES VIRUS	LERKWLDVVRNIIAE	1243	P04851.1
  	STRAIN
  	EDMONSTON
  361	MEASLES VIRUS	LERKWLDVVRNIIAEDLSLR	1244	P04851.1
  	STRAIN
  	EDMONSTON
  362	MEASLES VIRUS	LFGPSLRDPISAEIS	1245	P69353.1
  	STRAIN
  	EDMONSTON
  363	MEASLES	LGELKLAALCHGEDS	1246	P06830.1
  	MORBILLIVIRUS
  364	MEASLES VIRUS	LGGKEDRRVKQSR	1247	P04851.1
  	STRAIN
  	EDMONSTON
  365	RUBELLA VIRUS	LGHDGHHGGTLRVGQHHRNASDVL	1248	ABD64214.1
  366	RUBELLA VIRUS	LGSPNCHGPDWASPVCQRHS	1249	P07566.1
  	STRAIN THERIEN
  367	RUBELLA VIRUS	LGSPNCHGPDWASPVCQRHSPDCSRL	1250	P07566.1
  	STRAIN THERIEN	V
  368	RUBELLA VIRUS	LHDPDTEAPTEACVTSWL	1251	ABD64214.1
  369	MEASLES VIRUS	LIANCASILCKCYTT	1252	P69353.1
  	STRAIN
  	EDMONSTON
  370	MEASLES	LIGLLAIAGIRLHRAAIYTAEIHK	1253	AAR89413.1
  	MORBILLIVIRUS
  371	MEASLES VIRUS	LIPSMNQLSCDLIGQ	1254	P69353.1
  	STRAIN
  	EDMONSTON
  372	MEASLES VIRUS	LKIKIASGFGPLITH	1255	P08362.1
  	STRAIN
  	EDMONSTON
  373	MEASLES	LKIKIASGFGPLITHGSGMDLYK	1256	AAR89413.1
  	MORBILLIVIRUS
  374	BORDETELLA	LKLYFEP	1257	ACI16088.1
  	PERTUSSIS
  375	MEASLES VIRUS	LLAVLFVMFL	1258	P08362.1
  	STRAIN
  	EDMONSTON
  376	MEASLES VIRUS	LLDRLVRLIGNPDVS	1259	P04851.1
  	STRAIN
  	EDMONSTON
  377	MEASLES VIRUS	LLDRLVRLIGNPDVSGPKLT	1260	P04851.1
  	STRAIN
  	EDMONSTON
  378	MEASLES VIRUS	LLESSDQILRSMKGL	1261	P69353.1
  	STRAIN
  	EDMONSTON
  379	MEASLES	LLETRTTNQFLAVSK	1262	P06830.1
  	MORBILLIVIRUS
  380	MEASLES VIRUS	LLEVVQSDQSQSGLT	1263	P04851.1
  	STRAIN
  	EDMONSTON
  381	MEASLES VIRUS	LLEVVQSDQSQSGLTFASR	1264	P04851.1
  	STRAIN
  	EDMONSTON
  382	MEASLES VIRUS	LLEVVQSDQSQSGLTFASRG	1265	P04851.1
  	STRAIN
  	EDMONSTON
  383	MEASLES	LLGILESRGIKARIT	1266	AAL29688.1
  	MORBILLIVIRUS
  384	RUBELLA VIRUS	LLRTAPGP	1267	CAJ88851.1
  385	MEASLES VIRUS	LLRYYTEILSLFGPS	1268	P69353.1
  	STRAIN
  	EDMONSTON
  386	RUBELLA VIRUS	LLVPWVLIFMVCRRACRRRG	1269	P07566.1
  	STRAIN THERIEN
  387	MEASLES VIRUS	LLWRSRCKIV	1270	ABK40528.1
  	STRAIN
  	EDMONSTON
  388	MEASLES	LLWSYAMGV	1271	P04851.1
  	MORBILLIVIRUS
  389	MEASLES VIRUS	LLWSYAMGVGVELEN	1272	P04851.1
  	STRAIN
  	EDMONSTON
  390	MEASLES VIRUS	LLWSYAMGVGVELENSMGGL	1273	P04851.1
  	STRAIN
  	EDMONSTON
  391	MEASLES	LMIDRPYVL	1274	P08362.1
  	MORBILLIVIRUS
  392	MEASLES VIRUS	LNAMTQNIRPVQSVA	1275	P69353.1
  	STRAIN
  	EDMONSTON
  393	RUBELLA VIRUS	LNTPPPYQVSCGGES	1276	BAA28178.1
  394	MEASLES VIRUS	LRDPISAEISIQALS	1277	P69353.1
  	STRAIN
  	EDMONSTON
  395	BORDETELLA	LRGSGDLQEYLRHVTR	1278	AAA83981.1
  	PERTUSSIS
  396	RUBELLA VIRUS	LRLVDADD	1279	CAJ88851.1
  397	RUBELLA VIRUS	LRLVDADDPLLR	1280	BAA19893.1
  398	RUBELLA VIRUS	LRLVDADDPLLRTAPGPGEVWVTPVI	1281	BAA19893.1
  		GSQAR
  399	BORDETELLA	LRRLLYMIYMSGLAVRVHVSKEEQY	1282	P04979.1
  	PERTUSSIS	YDY
  400	RUBELLA VIRUS	LRTAPGPG	1283	CAJ88851.1
  401	RUBELLA VIRUS	LRVGQHYRNASDVLPGHWLQ	1284	NP_740663.1
  402	BORDETELLA	LRYLA	1285	ACI16088.1
  	PERTUSSIS
  403	MEASLES VIRUS	LSCKPWQESRKNK	1286	P04851.1
  	STRAIN
  	EDMONSTON
  404	MEASLES	LSEIKGVIVHRLEGV	1287	AAL29688.1
  	MORBILLIVIRUS
  405	MEASLES VIRUS	LSIAYPTLSEIKGVI	1288	P69353.1
  	STRAIN
  	EDMONSTON
  406	MEASLES VIRUS	LSLLDLYLGRGYNVS	1289	P08362.1
  	STRAIN
  	EDMONSTON
  407	MEASLES VIRUS	LSQGNLIANCASILC	1290	P69353.1
  	STRAIN
  	EDMONSTON
  408	MEASLES	LSSHRGVIADNQAKW	1291	P06830.1
  	MORBILLIVIRUS
  409	MEASLES VIRUS	LSVDLSLTVELKIKI	1292	P08362.1
  	STRAIN
  	EDMONSTON
  410	BORDETELLA	LTGISICNPGSSLC	1293	AAA83981.1
  	PERTUSSIS
  411	MEASLES VIRUS	LTIKFGIETMYPALG	1294	P04851.1
  	STRAIN
  	EDMONSTON
  412	MEASLES VIRUS	LTIKFGIETMYPALGLHEFA	1295	P04851.1
  	STRAIN
  	EDMONSTON
  413	MEASLES VIRUS	LTLQTPTGQIHWGNL	1296	P69353.1
  	STRAIN
  	EDMONSTON
  414	RUBELLA VIRUS	LVDADDPL	1297	CAJ88851.1
  415	RUBELLA VIRUS	LVDADDPLLR	1298	BAA19893.1
  416	MEASLES VIRUS	LVEKPNLSSKRSELS	1299	P08362.1
  	STRAIN
  	EDMONSTON
  417	RUBELLA VIRUS	LVGATPE	1300	BAA19893.1
  418	RUBELLA VIRUS	LVGATPER	1301	CAJ88851.1
  419	MEASLES	LVKLGVWKSPTGMQS	1302	P06830.1
  	MORBILLIVIRUS
  420	MEASLES VIRUS	LVSGSFGNRFILSQGNLI	1303	P26031.1
  	STRAIN
  	EDMONSTON-B
  421	MEASLES VIRUS	LYKSNHNNVYWLTIP	1304	P08362.1
  	STRAIN
  	EDMONSTON
  422	MEASLES VIRUS	LYPMSPLLQECLRGSTKSCARTLVS	1305	P69353.1
  	STRAIN
  	EDMONSTON
  423	RUBELLA VIRUS	MASTTPITMEDLQKALEA	1306	P07566.1
  424	RUBELLA VIRUS	MASTTPITMEDLQKALEAQSR	1307	ABD64200.1
  425	RUBELLA VIRUS	MASTTPITMEDLQKALEAQSRALRA	1308	P07566.1
  	STRAIN THERIEN	ELAA
  426	RUBELLA VIRUS	MASTTPITMEDLQKALEAQSRALRA	1309	ABD64200.1
  		GLAA
  427	RUBELLA VIRUS	MASTTPITMEDLQKALETQSRVLRAG	1310	CAA33016.1
  	VACCINE STRAIN	LTA
  	RA27/3
  428	MEASLES VIRUS	MATLLRSLALFKRNK	1311	P04851.1
  	STRAIN
  	EDMONSTON
  429	MEASLES VIRUS	MATLLRSLALFKRNKDKPPI	1312	P04851.1
  	STRAIN
  	EDMONSTON
  430	MEASLES	MDLYKSNHNNVYWLT	1313	P06830.1
  	MORBILLIVIRUS
  431	RUBELLA VIRUS	MEDLQKALEAQSRA	1314	P07566.1
  432	RUBELLA VIRUS	MEDLQKALEAQSRALRAELAA	1315	P07566.1
  433	MEASLES VIRUS	MGLKVNVSAIFMAVL	1316	P69353.1
  	STRAIN
  	EDMONSTON
  434	MEASLES	M1DRPYVLLAVLFVM	1317	P06830.1
  	MORBILLIVIRUS
  435	MEASLES VIRUS	MILAVQGVQDYINNE	1318	P69353.1
  	STRAIN
  	EDMONSTON
  436	MEASLES VIRUS	MLNSQAIDNLRASLE	1319	P69353.1
  	STRAIN
  	EDMONSTON
  437	MEASLES VIRUS	MNALVNSTLLETRTT	1320	P08362.1
  	STRAIN
  	EDMONSTON
  438	RUBELLA VIRUS	MNYTGNQQSRWGLGSPNCHGPDWA	1321	BAA19893.1
  		SPVCQRHS
  439	MEASLES VIRUS	MQSWVPLSTDDPVID	1322	P08362.1
  	STRAIN
  	EDMONSTON
  440	MEASLES	MSLSLLDLYLGRGYN	1323	P06830.1
  	MORBILLIVIRUS
  441	MEASLES VIRUS	MSPLLQECLRGSTKS	1324	P69353.1
  	STRAIN
  	EDMONSTON
  442	MEASLES	MSPQRDRINAFYKDN	1325	P06830.1
  	MORBILLIVIRUS
  443	MEASLES	MYRVFEVSVIRNPGL	1326	P06830.1
  	MORBILLIVIRUS
  444	MEASLES VIRUS	NALYPMSPLLQECLR	1327	P69353.1
  	STRAIN
  	EDMONSTON
  445	RUBELLA VIRUS	NCHGPDWASPVCQRHSPDCSRLVGA	1328	P07566.1
  	STRAIN THERIEN	T
  446	MEASLES VIRUS	NFGRSYFDPAYFRLG	1329	P04851.1
  	STRAIN
  	EDMONSTON
  447	MEASLES VIRUS	NFGRSYFDPAYFRLGQEMVR	1330	P04851.1
  	STRAIN
  	EDMONSTON
  448	RUBELLA VIRUS	NGTQRACTFWAVNAY	1331	BAA28178.1
  449	MEASLES VIRUS	NGVTIQVGSRRYPDA	1332	P69353.1
  	STRAIN
  	EDMONSTON
  450	MEASLES VIRUS	NIIAEDLSLRRFMVA	1333	P04851.1
  	STRAIN
  	EDMONSTON
  451	MEASLES VIRUS	NIIAEDLSLRRFMVALILDI	1334	P04851.1
  	STRAIN
  	EDMONSTON
  452	MEASLES VIRUS	NITLLNNCTRVEIAE	1335	P69353.1
  	STRAIN
  	EDMONSTON
  453	MEASLES	NLALGVINTLEWIPR	1336	P06830.1
  	MORBILLIVIRUS
  454	CORYNEBACTERIUM	NLFQVVHWSYNRPAYSPG	1337	SRC280292
  	DIPHTHERIAE
  455	MEASLES VIRUS	NLVILPGQDLQYVLA	1338	P08362.1
  	STRAIN
  	EDMONSTON
  456	MEASLES VIRUS	NLYQQMGKPAPYMVN	1339	P04851.1
  	STRAIN
  	EDMONSTON
  457	MEASLES VIRUS	NLYQQMGKPAPYMVNLENSI	1340	P04851.1
  	STRAIN
  	EDMONSTON
  458	MEASLES VIRUS	NNCTRVEIAEYRRLL	1341	P69353.1
  	STRAIN
  	EDMONSTON
  459	MEASLES VIRUS	NPDREYDFRD	1342	P08362.1
  	STRAIN
  	EDMONSTON
  460	MEASLES VIRUS	NPDVSGPKL	1343	P10050.1
  	STRAIN HALLE
  461	MEASLES VIRUS	NPDVSGPKLTGALIG	1344	P04851.1
  	STRAIN
  	EDMONSTON
  462	MEASLES VIRUS	NPDVSGPKLTGALIGILSLF	1345	P04851.1
  	STRAIN
  	EDMONSTON
  463	MEASLES	NPPERIKLDYDQYCA	1346	P06830.1
  	MORBILLIVIRUS
  464	MEASLES	NQAKWAVPTTRTDDK	1347	P06830.1
  	MORBILLIVIRUS
  465	MEASLES	NQDPDKILTYIAADH	1348	AAF02706.1
  	MORBILLIVIRUS
  466	MEASLES VIRUS	NQLSCDLIGQKLGLK	1349	P69353.1
  	STRAIN
  	EDMONSTON
  467	RUBELLA VIRUS	NQQSRWGLGSPNCHGPDWASPVCQR	1350	ABD64214.1
  		HS
  468	MUMPS	NSTLGVKSAREF	1351	ABP48111.1
  	RUBULAVIRUS
  469	RUBELLA VIRUS	NTPHGQLEVQVPPDP	1352	BAA28178.1
  470	MEASLES VIRUS	NVSAIFMAVLLTLQT	1353	P69353.1
  	STRAIN
  	EDMONSTON
  471	MEASLES	PAEVDGDVKLSSNLV	1354	P06830.1
  	MORBILLIVIRUS
  472	RUBELLA VIRUS	PAFGHSDAACWGFPT	1355	BAA28178.1
  473	MEASLES VIRUS	PALICCCRGRCNKKG	1356	P69353.1
  	STRAIN
  	EDMONSTON
  474	MEASLES	PDKILTYIAADHC	1357	AAF02706.1
  	MORBILLIVIRUS
  475	MEASLES VIRUS	PERIKLDYDQYCADV	1358	P08362.1
  	STRAIN
  	EDMONSTON
  476	RUBELLA VIRUS	PERPRLRL	1359	CAJ88851.1
  477	RUBELLA VIRUS	PGCATQAPVPVRLAG	1360	BAA28178.1
  478	RUBELLA VIRUS	PGCATQAPVPVRLAGVRFESKIVDGG	1361	CAJ88851.1
  	VACCINE STRAIN	CFA
  	RA27/3
  479	RUBELLA VIRUS	PGEVWVTP	1362	CAJ88851.1
  480	RUBELLA VIRUS	PGEVWVTPVIGSQAR	1363	BAA28178.1
  481	CORYNEBACTERIUM	PGKLDVNKSKTHISVN	1364	CAE11230.1
  	DIPHTHERIAE
  482	MEASLES VIRUS	PGLGAPVFHMTNYLE	1365	P08362.1
  	STRAIN
  	EDMONSTON
  483	RUBELLA VIRUS	PGPGEVWV	1366	CAJ88851.1
  484	RUBELLA VIRUS	PHKTVRVKFHTETRT	1367	BAA28178.1
  485	MEASLES	PIELQVECFTWDQKL	1368	AAR89413.1
  	MORBILLIVIRUS
  486	MEASLES VIRUS	PISLERLDVG	1369	P26031.1
  	STRAIN
  	EDMONSTON-B
  487	MEASLES VIRUS	PISLERLDVGTNLGN	1370	P69353.1
  	STRAIN
  	EDMONSTON
  488	BORDETELLA	PKALFTQQGGAYGRC	1371	P04979.1
  	PERTUSSIS
  489	MEASLES VIRUS	PKYVATQGYLISNFD	1372	P69353.1
  	STRAIN
  	EDMONSTON
  490	MEASLES VIRUS	PLDIDTASESSQD	1373	P04851.1
  	STRAIN
  	EDMONSTON
  491	RUBELLA VIRUS	PLGLKFKTVRPVALP	1374	BAA28178.1
  492	MEASLES VIRUS	PLITHGSGMDLYKSN	1375	P08362.1
  	STRAIN
  	EDMONSTON
  493	MEASLES	PLKDNRIPSYGVLSV	1376	P06830.1
  	MORBILLIVIRUS
  494	RUBELLA VIRUS	PLLRTAPG	1377	CAJ88851.1
  495	MEASLES VIRUS	PLLSCKPWQESRK	1378	P04851.1
  	STRAIN
  	EDMONSTON
  496	BORDETELLA	PPATVYRYDSRPPE	1379	P04977.1
  	PERTUSSIS
  497	MEASLES	PPISLERLDVGT	1380	AAL29688.1
  	MORBILLIVIRUS
  498	RUBELLA VIRUS	PPPPEERQETRSQTPAPKPS	1381	P07566.1
  499	BORDETELLA	PQEQITQHGSPYGRC	1382	AAA83981.1
  	PERTUSSIS
  500	BORDETELLA	PQEQITQHGSPYGRCANK	1383	AAA83981.1
  	PERTUSSIS
  501	BORDETELLA	PQPGPQPPQPPQPQPEAPAPQPPAG	1384	P14283.3
  	PERTUSSIS
  502	MEASLES VIRUS	PRLGGKEDRRVKQ	1385	P04851.1
  	STRAIN
  	EDMONSTON
  503	RUBELLA VIRUS	PRLRLVDA	1386	CAJ88851.1
  504	RUBELLA VIRUS	PRNVRVTGCYQCGTP	1387	BAA28178.1
  505	MEASLES VIRUS	PSRASDARAAHLP	1388	P04851.1
  	STRAIN
  	EDMONSTON
  506	MEASLES VIRUS	PTGQIHWGNLSKIGV	1389	P69353.1
  	STRAIN
  	EDMONSTON
  507	MEASLES VIRUS	PTGTPLDIDTASE	1390	P04851.1
  	STRAIN
  	EDMONSTON
  508	MEASLES VIRUS	PTLSEIKGVIVHRLE	1391	P69353.1
  	STRAIN
  	EDMONSTON
  509	MEASLES	PTTIRGQFSNMSLSL	1392	P06830.1
  	MORBILLIVIRUS
  510	MEASLES	PTTRTDDKLR	1393	AAR89413.1
  	MORBILLIVIRUS
  511	MEASLES	PTTRTDDKLRMETCFQQACKG	1394	AAR89413.1
  	MORBILLIVIRUS
  512	RUBELLA VIRUS	PVALPRTLAPPRNVR	1395	BAA28178.1
  513	CORYNEBACTERIUM	PVFAGANYAAWAVNVAQVI	1396	AAV70486.1
  	DIPHTHERIAE
  514	RUBELLA VIRUS	PVIGSQAR	1397	CAJ88851.1
  515	MEASLES	PVVEVNGVTIQVGSR	1398	AAL29688.1
  	MORBILLIVIRUS
  516	RUBELLA VIRUS	PWELVVLTARPEDGWTCRGV	1399	P07566.1
  	STRAIN THERIEN
  517	MEASLES VIRUS	PWQESRKNKAQTR	1400	P04851.1
  	STRAIN
  	EDMONSTON
  518	MEASLES VIRUS	PYMVNLENSIQNKFS	1401	P04851.1
  	STRAIN
  	EDMONSTON
  519	MEASLES VIRUS	PYMVNLENSIQNKFSAGSYP	1402	P04851.1
  	STRAIN
  	EDMONSTON
  520	MEASLES VIRUS	PYQGSGKGVS	1403	P08362.1
  	STRAIN
  	EDMONSTON
  521	RUBELLA VIRUS	PYQVSCGGESDRASA	1404	BAA28178.1
  522	MEASLES	QACKGKIQALCEN	1405	P08362.1
  	MORBILLIVIRUS
  523	MEASLES VIRUS	QAGQEMILAVQGVQD	1406	P69353.1
  	STRAIN
  	EDMONSTON
  524	MEASLES	QALCENPECVPLKDN	1407	P06830.1
  	MORBILLIVIRUS
  525	BORDETELLA	QALGALK	1408	ACI16088.1
  	PERTUSSIS
  526	RUBELLA VIRUS	QAPVPVRLAGVRFES	1409	BAA28178.1
  527	RUBELLA VIRUS	QCGTPALVEGLAPGG	1410	BAA28178.1
  528	MEASLES VIRUS	QDPDKILTYIAADHC	1411	P69353.1
  	STRAIN
  	EDMONSTON
  529	MEASLES VIRUS	QECLRGSTKSCARTL	1412	P69353.1
  	STRAIN
  	EDMONSTON
  530	BORDETELLA	QEQITQHGSPYGRC	1413	AAA83981.1
  	PERTUSSIS
  531	MEASLES VIRUS	QESRKNKAQTRTP	1414	P04851.1
  	STRAIN
  	EDMONSTON
  532	MEASLES VIRUS	QGDQSENELPRLGGK	1415	P04851.1
  	STRAIN
  	EDMONSTON
  533	MEASLES VIRUS	QGDQSENELPRLGGKEDRRV	1416	P04851.1
  	STRAIN
  	EDMONSTON
  534	CORYNEBACTERIUM	QGESGHDIKITAENTPLPIA	1417	AAV70486.1
  	DIPHTHERIAE
  535	MEASLES	QGSGKGVSFQLVKLG	1418	P06830.1
  	MORBILLIVIRUS
  536	MEASLES VIRUS	QGVQDYINNELIPSM	1419	P69353.1
  	STRAIN
  	EDMONSTON
  537	RUBELLA VIRUS	QLEVQVPPDPGDLVE	1420	BAA28178.1
  538	MEASLES	QLPEATFMV	1421	ABK40528.1
  	MORBILLIVIRUS
  539	RUBELLA VIRUS	QLPFLGHDGHHGGTLRVGQHYRNAS	1422	NP_740663.1
  540	MEASLES VIRUS	QLSMYRVFEV	1423	P08362.1
  	STRAIN
  	EDMONSTON
  541	BORDETELLA	QLSNIT	1424	ACI16083.1
  	PERTUSSIS
  542	MEASLES VIRUS	QNIRPVQSVASSRRH	1425	P69353.1
  	STRAIN
  	EDMONSTON
  543	MEASLES VIRUS	QNKFSAGSYPLLWSY	1426	P04851.1
  	STRAIN
  	EDMONSTON
  544	MEASLES VIRUS	QNKFSAGSYPLLWSYAMGVG	1427	P04851.1
  	STRAIN
  	EDMONSTON
  545	MEASLES VIRUS	QNKFSAGSYPLLWSYAMGVGVELEN	1428	P04851.1
  	STRAIN
  	EDMONSTON
  546	MEASLES VIRUS	QQACKGKIQALCENP	1429	P08362.1
  	STRAIN
  	EDMONSTON
  547	MEASLES VIRUS	QQRRVVGEFRLERKW	1430	P04851.1
  	STRAIN
  	EDMONSTON
  548	MEASLES VIRUS	QQRRVVGEFRLERKWLDVVR	1431	P04851.1
  	STRAIN
  	EDMONSTON
  549	BORDETELLA	QQTRANPNPYTSRRSVAS	1432	P04977.1
  	PERTUSSIS
  550	RUBELLA VIRUS	QRHSPDCSRLVGATP	1433	BAA28178.1
  551	MEASLES VIRUS	QSGLTFASRGTNMED	1434	P04851.1
  	STRAIN
  	EDMONSTON
  552	MEASLES VIRUS	QSGLTFASRGTNMEDEADQY	1435	P04851.1
  	STRAIN
  	EDMONSTON
  553	CORYNEBACTERIUM	QSIALSSLMVAQAIPLVGEL	1436	AAV70486.1
  	DIPHTHERIAE
  554	MEASLES VIRUS	QSRFGWFENKEISDI	1437	P04851.1
  	STRAIN
  	EDMONSTON
  555	MEASLES VIRUS	QSRFGWFENKEISDIEVQDP	1438	P04851.1
  	STRAIN
  	EDMONSTON
  556	MEASLES VIRUS	QSRGEAR	1439	P04851.1
  	STRAIN
  	EDMONSTON
  557	MEASLES VIRUS	QSRGEARESYRETGPSRA	1440	P04851.1
  	STRAIN
  	EDMONSTON
  558	RUBELLA VIRUS	QTGRGGSAPRPELGPPTN	1441	P07566.1
  	STRAIN THERIEN
  559	RUBELLA VIRUS	QTPAPKPSRAPPQQPQPPRMQTGRG	1442	P07566.1
  	STRAIN THERIEN
  560	MEASLES VIRUS	QVGSRRYPDAVYLHR	1443	P69353.1
  	STRAIN
  	EDMONSTON
  561	MEASLES VIRUS	QVSFLQGDQSENE	1444	P04851.1
  	STRAIN
  	EDMONSTON
  562	RUBELLA VIRUS	QYHPTACEVEPAFGH	1445	BAA28178.1
  563	MEASLES VIRUS	QYVLATYDTSRVEHA	1446	P08362.1
  	STRAIN
  	EDMONSTON
  564	BORDETELLA	RANPNPYTSRRSV	1447	ACI04548.1
  	PERTUSSIS
  565	MEASLES VIRUS	RASDARAAHLPTG	1448	P04851.1
  	STRAIN
  	EDMONSTON
  566	MEASLES VIRUS	RASLETTNQAIEAIR	1449	P69353.1
  	STRAIN
  	EDMONSTON
  567	RUBELLA VIRUS	RCGRLICGLSTTAQYPPTRF	1450	P07566.1
  	STRAIN THERIEN
  568	BORDETELLA	RDGQSVIGACASPYEGRYR	1451	P04979.1
  	PERTUSSIS
  569	MEASLES VIRUS	RESYRETGPSRAS	1452	P04851.1
  	STRAIN
  	EDMONSTON
  570	MEASLES VIRUS	RETGPSRASDARA	1453	P04851.1
  	STRAIN
  	EDMONSTON
  571	MUMPS	RFAKYQQQGRLEAR	1454	P21186.1
  	RUBULAVIRUS
  572	RUBELLA VIRUS	RFGAPQAFLAGLLLATVAVGTARA	1455	P07566.1
  	STRAIN THERIEN
  573	MEASLES VIRUS	RFMVALILDIKRTPG	1456	P04851.1
  	STRAIN
  	EDMONSTON
  574	MEASLES VIRUS	RFMVALILDIKRTPGNKPRI	1457	P04851.1
  	STRAIN
  	EDMONSTON
  575	MEASLES VIRUS	RGEARESYRETGP	1458	P04851.1
  	STRAIN
  	EDMONSTON
  576	RUBELLA VIRUS	RGTTPPAYG	1459	CAA28880.1
  577	RUBELLA VIRUS	RIETRSARH	1460	ABD64214.1
  	STRAIN M33
  578	BORDETELLA	RILAGALATYQ	1461	P04977.1
  	PERTUSSIS
  579	BORDETELLA	RIPPENIRRVT	1462	ACI04548.1
  	PERTUSSIS
  580	BORDETELLA	RISNLND	1463	ACI16083.1
  	PERTUSSIS
  581	BORDETELLA	RLANLNG	1464	ACI16088.1
  	PERTUSSIS
  582	MEASLES VIRUS	RLDVGTNLGNAIAKL	1465	P69353.1
  	STRAIN
  	EDMONSTON
  583	MEASLES VIRUS	RLERKWLDV	1466	P04851.1
  	STRAIN
  	EDMONSTON
  584	MEASLES VIRUS	RLGGKEDRRVKQSRG	1467	P04851.1
  	STRAIN
  	EDMONSTON
  585	MEASLES VIRUS	RLGGKEDRRVKQSRGEARES	1468	P04851.1
  	STRAIN
  	EDMONSTON
  586	MEASLES VIRUS	RLLDRLVRL	1469	ABI54110.1
  	STRAIN
  	EDMONSTON
  587	RUBELLA VIRUS	RLRLVDAD	1470	CAJ88851.1
  588	RUBELLA VIRUS	RLRLVDADDPLLR	1471	BAA19893.1
  589	RUBELLA VIRUS	RLRLVDADDPLLRTAPGPGEVWVTP	1472	BAA19893.1
  		VIGSQA
  590	RUBELLA VIRUS	RLRLVQDADDPLLRIAPGPGEVWVTP	1473	SRC265968
  		VIGSQA
  591	MEASLES VIRUS	RLSDNGYYTV	1474	ABK40528.1
  	STRAIN
  	EDMONSTON
  592	RUBELLA VIRUS	RLVDADDP	1475	CAJ88851.1
  593	RUBELLA VIRUS	RLVDADDPLLRTAPG	1476	BAA28178.1
  594	RUBELLA VIRUS	RLVGATPE	1477	CAJ88851.1
  595	RUBELLA VIRUS	RMQTGRGGSAPRPELGPPTNPFQAAV	1478	ABD64216.1
  		A
  596	MEASLES	RMSKGVFKV	1479	ABY21184.1
  	MORBILLIVIRUS
  597	MEASLES	RNPGLGAPVFHMTNY	1480	P06830.1
  	MORBILLIVIRUS
  598	RUBELLA VIRUS	RPRLRLVD	1481	CAJ88851.1
  599	BORDETELLA	RQAESSEAMAAWSERAGEA	1482	P04977.1
  	PERTUSSIS
  600	RUBELLA VIRUS	RQTWAEWAAAHWWQL	1483	BAA28178.1
  601	MEASLES VIRUS	RRVKQSRGEARES	1484	P04851.1
  	STRAIN
  	EDMONSTON
  602	MEASLES	RRYPDAVYL	1485	ACA09725.1
  	MORBILLIVIRUS
  603	MEASLES VIRUS	RRYPDAVYLHRIDLG	1486	P69353.1
  	STRAIN
  	EDMONSTON
  604	MEASLES VIRUS	RSAGKVSSTLASELG	1487	P04851.1
  	STRAIN
  	EDMONSTON
  605	MEASLES VIRUS	RSAGKVSSTLASELGITAED	1488	P04851.1
  	STRAIN
  	EDMONSTON
  606	MEASLES VIRUS	RSELSQLS	1489	P08362.1
  	STRAIN
  	EDMONSTON
  607	MEASLES VIRUS	RSELSQLSMYRVFEV	1490	P08362.1
  	STRAIN
  	EDMONSTON
  608	RUBELLA VIRUS	RSQTPAPKPSRAPPQQPQPPRMQT	1491	ABD64214.1
  609	RUBELLA VIRUS	RTAPGPGE	1492	CAJ88851.1
  610	RUBELLA VIRUS	RTAPGPGEVWVTPVI	1493	BAA28178.1
  611	MEASLES	RTDDKLRMETCFQQA	1494	P06830.1
  	MORBILLIVIRUS
  612	RUBELLA VIRUS	RTLAPPRNVRVTGCY	1495	BAA28178.1
  613	MEASLES VIRUS	RTVLEPIRDALNAMT	1496	P69353.1
  	STRAIN
  	EDMONSTON
  614	MEASLES VIRUS	RVEHAVVYYVYSPSR	1497	P08362.1
  	STRAIN
  	EDMONSTON
  615	MEASLES VIRUS	RVFEVGVIRNPGLGA	1498	P08362.1
  	STRAIN
  	EDMONSTON
  616	BORDETELLA	RVHVSKEEQYYDYEDATFE	1499	P04978.2
  	PERTUSSIS
  617	RUBELLA VIRUS	RVIDPAAQ	1500	BAA28178.1
  618	RUBELLA VIRUS	RVKFHTETRTVWQLS	1501	BAA28178.1
  619	BORDETELLA	RVYHNGITGET	1502	ACI04548.1
  	PERTUSSIS
  620	BORDETELLA	RYDSRPPEDVF	1503	ACI04548.1
  	PERTUSSIS
  621	MEASLES VIRUS	RYPDAVYLHRIDLGP	1504	P69353.1
  	STRAIN
  	EDMONSTON
  622	MEASLES VIRUS	SAEISIQALSYALGG	1505	P69353.1
  	STRAIN
  	EDMONSTON
  623	MEASLES VIRUS	SAEPLLSCKPWQESR	1506	P04851.1
  	STRAIN
  	EDMONSTON
  624	MEASLES VIRUS	SAEPLLSCKPWQESRKNKAQ	1507	P04851.1
  	STRAIN
  	EDMONSTON
  625	MEASLES	SAGKVSSTLASELG	1508	P04851.1
  	MORBILLIVIRUS
  626	MEASLES VIRUS	SAGKVSSTLASELGITAEDARLVS	1509	P04851.1
  	STRAIN
  	EDMONSTON
  627	MEASLES VIRUS	SCTVTREDGT	1510	P08362.1
  	STRAIN
  	EDMONSTON
  628	RUBELLA VIRUS	SDAACWGFPTDTVMS	1511	BAA28178.1
  629	MEASLES VIRUS	SDARAAHLPTGTP	1512	P04851.1
  	STRAIN
  	EDMONSTON
  630	RUBELLA VIRUS	SDWHQGTHVCHTKHMDFWCVEHD	1513	P07566.1
  	STRAIN THERIEN
  631	MEASLES VIRUS	SELRRWIKYTQQRRV	1514	P04851.1
  	STRAIN
  	EDMONSTON
  632	MEASLES VIRUS	SELRRWIKYTQQRRVVGEFR	1515	P04851.1
  	STRAIN
  	EDMONSTON
  633	MEASLES VIRUS	SELSQL	1516	P08362.1
  	STRAIN
  	EDMONSTON
  634	MEASLES VIRUS	SELSQLS	1517	P08362.1
  	STRAIN
  	EDMONSTON
  635	BORDETELLA	SEYLAHRRIPPENIRRVTRV	1518	CAD44970.1
  	PERTUSSIS
  636	MEASLES VIRUS	SFLQGDQSENELP	1519	P04851.1
  	STRAIN
  	EDMONSTON
  637	MEASLES VIRUS	SGKGVSFQLVKLGVW	1520	P08362.1
  	STRAIN
  	EDMONSTON
  638	MEASLES VIRUS	SHRGVIADNQAKWAV	1521	P08362.1
  	STRAIN
  	EDMONSTON
  639	MEASLES VIRUS	SIEHQVKDVLTPLFK	1522	P08362.1
  	STRAIN
  	EDMONSTON
  640	MEASLES VIRUS	SKIGVVGIGSASYKV	1523	P69353.1
  	STRAIN
  	EDMONSTON
  641	MEASLES	SKRSELSQLSMYRVF	1524	P06830.1
  	MORBILLIVIRUS
  642	MEASLES	SLFVESPGQLIQRITDDPDVS	1525	ABI54110.1
  	MORBILLIVIRUS
  643	MEASLES VIRUS	SLSTNLDVTNSIEHQ	1526	P08362.1
  	STRAIN
  	EDMONSTON
  644	MEASLES VIRUS	SLSTNLDVTNSIEHQVKDVLTPLFK	1527	P08362.1
  	STRAIN
  	EDMONSTON
  645	MEASLES VIRUS	SLWGSGLLML	1528	BAE98296.1
  	STRAIN
  	EDMONSTON
  646	MEASLES VIRUS	SMKGLSSTSIVYILI	1529	P69353.1
  	STRAIN
  	EDMONSTON
  647	MEASLES	SMYRVFEVGV	1530	P08362.1
  	MORBILLIVIRUS
  648	MEASLES	SNDLSNCMVALGELK	1531	P06830.1
  	MORBILLIVIRUS
  649	MEASLES VIRUS	SPGQLIQR	1532	P10050.1
  	STRAIN HALLE
  650	MEASLES VIRUS	SQDPQDSRRSAEP	1533	P04851.1
  	STRAIN
  	EDMONSTON
  651	MEASLES	SRIVINREHLMIDRP	1534	P06830.1
  	MORBILLIVIRUS
  652	MEASLES VIRUS	SRKNKAQTRTPLQ	1535	P04851.1
  	STRAIN
  	EDMONSTON
  653	RUBELLA VIRUS	SRLVGATP	1536	CAJ88851.1
  654	RUBELLA VIRUS	SRLVGATPERPRLRLVDADDPLLR	1537	CAJ88851.1
  655	MEASLES VIRUS	SRPGLKPDLTGTSKS	1538	P69353.1
  	STRAIN
  	EDMONSTON
  656	BORDETELLA	SRRSVASIVGTLVRM	1539	CAD44970.1
  	PERTUSSIS
  657	RUBELLA VIRUS	SRWGLGSPNCHGPDW	1540	BAA28178.1
  658	BORDETELLA	SSATK	1541	ACI16088.1
  	PERTUSSIS
  659	RUBELLA VIRUS	SSGGYAQLASYFNPG	1542	BAA28178.1
  660	BORDETELLA	SSLGNGV	1543	ACI16083.1
  	PERTUSSIS
  661	MEASLES	SSNLVILPGQDLQYV	1544	P06830.1
  	MORBILLIVIRUS
  662	MEASLES VIRUS	SSQDPQDSRRSAEPL	1545	P04851.1
  	STRAIN
  	EDMONSTON
  663	MEASLES VIRUS	SSQDPQDSRRSAEPLLSCKP	1546	P04851.1
  	STRAIN
  	EDMONSTON
  664	MEASLES VIRUS	SSRRHKRFAGVVLAG	1547	P69353.1
  	STRAIN
  	EDMONSTON
  665	MEASLES VIRUS	SSTSIVYILIAVCLG	1548	P69353.1
  	STRAIN
  	EDMONSTON
  666	BORDETELLA	STPGIVI	1549	AAA83981.1
  	PERTUSSIS
  667	BORDETELLA	STPGIVIPPQEQITQHGSPYGRC	1550	AAA83981.1
  	PERTUSSIS
  668	BORDETELLA	STSSSRRYTEVY	1551	P04977.1
  	PERTUSSIS
  669	MEASLES VIRUS	SYFIVLSIAYPTLSE	1552	P69353.1
  	STRAIN
  	EDMONSTON
  670	MEASLES VIRUS	SYRETGPSRASDA	1553	P04851.1
  	STRAIN
  	EDMONSTON
  671	BORDETELLA	SYVK	1554	ACI16083.1
  	PERTUSSIS
  672	RUBELLA VIRUS	SYVQHPHKTVRVKFH	1555	BAA28178.1
  673	RUBELLA VIRUS	TAPGPGEV	1556	CAJ88851.1
  674	BORDETELLA	TATRLLSSTNSRLC	1557	AAA83981.1
  	PERTUSSIS
  675	MEASLES	TDDPVIDRLYLSSHR	1558	P06830.1
  	MORBILLIVIRUS
  676	MEASLES VIRUS	TEILSLFGPSLRDPI	1559	P69353.1
  	STRAIN
  	EDMONSTON
  677	RUBELLA VIRUS	TETRTVWQLSVAGVS	1560	BAA28178.1
  678	BORDETELLA	TEVYLEHRMQEAVE	1561	P04977.1
  	PERTUSSIS
  679	MEASLES VIRUS	TFMPEGTVCSQNALY	1562	P69353.1
  	STRAIN
  	EDMONSTON
  680	RUBELLA VIRUS	TGACICEIPTDVSCE	1563	BAA28178.1
  681	BORDETELLA	TGDLRAY	1564	ACI16083.1
  	PERTUSSIS
  682	MEASLES	TGMQSWVPLSTDDPV	1565	P06830.1
  	MORBILLIVIRUS
  683	RUBELLA VIRUS	TGNQQSRWGLGSPNC	1566	BAA28178.1
  684	MEASLES VIRUS	TGPSRASDARAAH	1567	P04851.1
  	STRAIN
  	EDMONSTON
  685	MEASLES	TGTIINQDPDKILTY	1568	AAF02706.1
  	MORBILLIVIRUS
  686	RUBELLA VIRUS	TGVVYGTHTTAVSET	1569	BAA28178.1
  687	MEASLES VIRUS	TIRGQFSNMSLSLLD	1570	P08362.1
  	STRAIN
  	EDMONSTON
  688	RUBELLA VIRUS	TLGATCALPLAGLLA	1571	BAA28178.1
  689	MEASLES	TLLNNCTRV	1572	P26031.1
  	MORBILLIVIRUS
  690	MEASLES VIRUS	TLNVPPPPDPGR	1573	P03422.1
  	STRAIN
  	EDMONSTON-B
  691	MEASLES VIRUS	TLNVPPPPDPGRASTSGTPIKK	1574	P03422.1
  	STRAIN
  	EDMONSTON-B
  692	MEASLES	TMTSQGMYGGTYPVE	1575	P06830.1
  	MORBILLIVIRUS
  693	MEASLES VIRUS	TNLGNAIAKLEDAKE	1576	P69353.1
  	STRAIN
  	EDMONSTON
  694	MEASLES VIRUS	TNMEDEADQYFSHDD	1577	P04851.1
  	STRAIN
  	EDMONSTON
  695	MEASLES VIRUS	TNMEDEADQYFSHDDPISSD	1578	P04851.1
  	STRAIN
  	EDMONSTON
  696	RUBELLA VIRUS	TNPFQAAVARGLRPP	1579	CAA28880.1
  	STRAIN M33
  697	MEASLES	TNSIEHQVKDVLTPL	1580	P06830.1
  	MORBILLIVIRUS
  698	MEASLES VIRUS	TNYLEQPVSNDLSNC	1581	P08362.1
  	STRAIN
  	EDMONSTON
  699	MEASLES	TNYLEQPVSNDLSNCMVALGELKLA	1582	AAR89413.1
  	MORBILLIVIRUS	AL
  700	RUBELLA VIRUS	TPERPRLR	1583	CAJ88851.1
  701	RUBELLA VIRUS	TPERPRLRLVDADDPLLRTA	1584	P07566.1
  	STRAIN THERIEN
  702	MEASLES VIRUS	TPGNKPRIA	1585	P10050.1
  	STRAIN HALLE
  703	MEASLES VIRUS	TPLDIDTASESSQDP	1586	P04851.1
  	STRAIN
  	EDMONSTON
  704	MEASLES VIRUS	TPLDIDTASESSQDPQDSRR	1587	P04851.1
  	STRAIN
  	EDMONSTON
  705	MEASLES VIRUS	TPLFKIIGDEVGLRT	1588	P08362.1
  	STRAIN
  	EDMONSTON
  706	MEASLES VIRUS	TPLQCTM	1589	P04851.1
  	STRAIN
  	EDMONSTON
  707	RUBELLA VIRUS	TPVIGSQA	1590	CAJ88851.1
  708	RUBELLA VIRUS	TPVIGSQARK	1591	BAA19893.1
  709	MEASLES VIRUS	TQGYLISNFDESSCT	1592	P69353.1
  	STRAIN
  	EDMONSTON
  710	BORDETELLA	TRANPNPYTSRRSVASIVGTLVRM	1593	P04977.1
  	PERTUSSIS
  711	RUBELLA VIRUS	TRFGCAMRWGLPP	1594	NP_740663.1
  712	BORDETELLA	TRNTGQPATDHYYSNV	1595	AAA83981.1
  	PERTUSSIS
  713	MEASLES VIRUS	TRTPLQCTMTEIF	1596	P04851.1
  	STRAIN
  	EDMONSTON
  714	RUBELLA VIRUS	TRWHRLLRMPVR	1597	ABD64216.1
  715	MEASLES VIRUS	TSGSGGAIRGIKHII	1598	P04851.1
  	STRAIN
  	EDMONSTON
  716	MEASLES VIRUS	TSGSGGAIRGIKHIIIVPIP	1599	P04851.1
  	STRAIN
  	EDMONSTON
  717	MEASLES VIRUS	TSQGMYGGTYLVEKP	1600	P08362.1
  	STRAIN
  	EDMONSTON
  718	MEASLES	TSRVEHAVVYYVYSP	1601	P06830.1
  	MORBILLIVIRUS
  719	BORDETELLA	TSSSRRYTEVYL	1602	ACI04548.1
  	PERTUSSIS
  720	BORDETELLA	TSYVG	1603	ACI16088.1
  	PERTUSSIS
  721	MEASLES VIRUS	TTEDKISRAVGPRQA	1604	P04851.1
  	STRAIN
  	EDMONSTON
  722	MEASLES VIRUS	TTEDKISRAVGPRQAQVSFL	1605	P04851.1
  	STRAIN
  	EDMONSTON
  723	RUBELLA VIRUS	TTERIETRSARHP	1606	ABD64214.1
  	STRAIN M33
  724	MEASLES VIRUS	TTNQAIEAIRQAGQE	1607	P69353.1
  	STRAIN
  	EDMONSTON
  725	RUBELLA VIRUS	TTSDPWHPPGPLGLK	1608	BAA28178.1
  726	MEASLES VIRUS	TVCSQNALYPMSPLL	1609	P69353.1
  	STRAIN
  	EDMONSTON
  727	RUBELLA VIRUS	TVNGEDVGAVPPGKF	1610	BAA28178.1
  728	MEASLES	TYPVEKPNLSSKRSE	1611	P06830.1
  	MORBILLIVIRUS
  729	RUBELLA VIRUS	VAGVSCNVTTEHPFC	1612	BAA28178.1
  730	BORDETELLA	VAPGIVIPPKALFTQQGGAYGRC	1613	P04979.1
  	PERTUSSIS
  731	RUBELLA VIRUS	VCHTKHMDFWCVEHDRPPPATPTPL	1614	NP_740663.1
  732	RUBELLA VIRUS	VCQRHSPDCSRLVGATPER	1615	BAA19893.1
  733	RUBELLA VIRUS	VDADDPLL	1616	CAJ88851.1
  734	RUBELLA VIRUS	VDADDPLLRTAPGPGEVWVT	1617	BAA19893.1
  735	CORYNEBACTERIUM	VDIGFAAYNFVESIINLFQV	1618	AAV70486.1
  	DIPHTHERIAE
  736	MEASLES VIRUS	VEIAEYRRLLRTVLE	1619	P69353.1
  	STRAIN
  	EDMONSTON
  737	MEASLES VIRUS	VELENSMGGLNFGRS	1620	P04851.1
  	STRAIN
  	EDMONSTON
  738	MEASLES VIRUS	VELENSMGGLNFGRSYFDPA	1621	P04851.1
  	STRAIN
  	EDMONSTON
  739	MEASLES	VELKIKIASGFGPLI	1622	P06830.1
  	MORBILLIVIRUS
  740	RUBELLA VIRUS	VEMDEWIHAHTTSD	1623	SRC265968
  741	RUBELLA VIRUS	VEMPEWIHAHTTSDP	1624	BAA28178.1
  742	CORYNEBACTERIUM	VERRLVKVL	1625	P33120.2
  	DIPHTHERIAE
  743	MEASLES	VESPGQLI	1626	ABI54110.1
  	MORBILLIVIRUS
  744	MEASLES VIRUS	VESPGQLIQRITDDP	1627	P04851.1
  	STRAIN
  	EDMONSTON
  745	MEASLES VIRUS	VESPGQLIQRITDDPDVSIR	1628	P04851.1
  	STRAIN
  	EDMONSTON
  746	RUBELLA VIRUS	VFALASYVQHPHKTV	1629	BAA28178.1
  747	RUBELLA VIRUS	VGATPERP	1630	CAJ88851.1
  748	RUBELLA VIRUS	VGATPERPRL	1631	BAA19893.1
  749	RUBELLA VIRUS	VGATPERPRLRLVDA	1632	BAA28178.1
  750	MEASLES VIRUS	VGIGSASYKVMTRSS	1633	P69353.1
  	STRAIN
  	EDMONSTON
  751	MEASLES VIRUS	VGLRTPQRFTDLVKF	1634	P08362.1
  	STRAIN
  	EDMONSTON
  752	CORYNEBACTERIUM	VHHNTEEIVAQSIALSSLMV	1635	AAV70486.1
  	DIPHTHERIAE
  753	MEASLES VIRUS	VHRLEGVSYNIGSQE	1636	P69353.1
  	STRAIN
  	EDMONSTON
  754	RUBELLA VIRUS	VIGSQARK	1637	CAJ88851.1
  755	BORDETELLA	VITGSI	1638	ACI16088.1
  	PERTUSSIS
  756	BORDETELLA	VITGTI	1639	ACI16083.1
  	PERTUSSIS
  757	MEASLES VIRUS	VKQSRGEA	1640	P04851.1
  	STRAIN
  	EDMONSTON
  758	MEASLES VIRUS	VLFVMFLSLI	1641	P08362.1
  	STRAIN
  	EDMONSTON
  759	MEASLES	VLFVMFLSLIGLLAI	1642	P06830.1
  	MORBILLIVIRUS
  760	MEASLES	VLTPLFKIIGDEVGL	1643	P06830.1
  	MORBILLIVIRUS
  761	MEASLES VIRUS	VMTRSSHQSLVIKLM	1644	P69353.1
  	STRAIN
  	EDMONSTON
  762	RUBELLA VIRUS	VPAAPCARIWNGTQR	1645	BAA28178.1
  763	RUBELLA VIRUS	VPPDPGDLVEYIMNY	1646	BAA28178.1
  764	MEASLES VIRUS	VQSVASSRRHKRFAG	1647	P69353.1
  	STRAIN
  	EDMONSTON
  765	BORDETELLA	VQTGGTSRTVTMRYLAS	1648	ACI16083.1
  	PERTUSSIS
  766	BORDETELLA	VQVRI	1649	ACI16083.1
  	PERTUSSIS
  767	RUBELLA VIRUS	VRAYNQPAGDV	1650	NP_740662.1
  768	RUBELLA VIRUS	VRFESKIVDGGCFAP	1651	BAA28178.1
  769	RUBELLA VIRUS	VRLAGVRFESKIVDG	1652	BAA28178.1
  770	MEASLES VIRUS	VSGSFGNRFILSQGN	1653	P69353.1
  	STRAIN
  	EDMONSTON
  771	BORDETELLA	VSKEEQYYDYEDAT	1654	AAA83981.1
  	PERTUSSIS
  772	MEASLES VIRUS	VSKGNCSGPTTIRGQ	1655	P08362.1
  	STRAIN
  	EDMONSTON
  773	RUBELLA VIRUS	VTAALLNTPPPYQVS	1656	BAA28178.1
  774	RUBELLA VIRUS	VTGCYQCGTPALVEG	1657	BAA28178.1
  775	RUBELLA VIRUS	VTPVIGSQ	1658	CAJ88851.1
  776	RUBELLA VIRUS	VTTEHPFCNTPHGQLEVQVPPD	1659	P07566.1
  	STRAIN THERIEN
  777	MEASLES VIRUS	VVLAGAALGVATAAQ	1660	P69353.1
  	STRAIN
  	EDMONSTON
  778	RUBELLA VIRUS	VWQLSVAGVSCNVTT	1661	BAA28178.1
  779	RUBELLA VIRUS	VWVTPVIG	1662	CAJ88851.1
  780	RUBELLA VIRUS	VWVTPVIGSQAR	1663	BAA19893.1
  781	MEASLES VIRUS	VYILIAVCLGGLIGI	1664	P69353.1
  	STRAIN
  	EDMONSTON
  782	MEASLES VIRUS	VYLHRIDLGPPISLE	1665	P69353.1
  	STRAIN
  	EDMONSTON
  783	BORDETELLA	VYRYDSRP	1666	P04977.1
  	PERTUSSIS
  784	BORDETELLA	VYRYDSRPPEDV	1667	P04977.1
  	PERTUSSIS
  785	MEASLES	VYWLTIPPMKNLALG	1668	P06830.1
  	MORBILLIVIRUS
  786	RUBELLA VIRUS	WDLEATGACICEIPT	1669	BAA28178.1
  787	MEASLES	WDQKLWCRHFCVLAD	1670	AAR89413.1
  	MORBILLIVIRUS
  788	RUBELLA VIRUS	WGFPTDTVMSVFALA	1671	BAA28178.1
  789	RUBELLA VIRUS	WHPPGPLGLKFKTVR	1672	BAA28178.1
  790	RUBELLA VIRUS	WIHAHTTSDPWHPPG	1673	BAA28178.1
  791	MEASLES VIRUS	WLTIPPMKNLALGVI	1674	P08362.1
  	STRAIN
  	EDMONSTON
  792	MEASLES VIRUS	WQESRKNKAQTRTPLQCTMT	1675	P04851.1
  	STRAIN
  	EDMONSTON
  793	RUBELLA VIRUS	WVCIFMVCRRACR	1676	SRC265968
  794	RUBELLA VIRUS	WVTPVIGS	1677	CAJ88851.1
  795	MEASLES VIRUS	WYTTVPKYVATQGYL	1678	P69353.1
  	STRAIN
  	EDMONSTON
  796	MEASLES VIRUS	YALGGDINKVLEKLG	1679	P69353.1
  	STRAIN
  	EDMONSTON
  797	MEASLES VIRUS	YAMGVGVELE	1680	P04851.1
  	STRAIN
  	EDMONSTON
  798	MEASLES	YAMGVGVELEN	1681	ABI54110.1
  	MORBILLIVIRUS
  799	MEASLES	YCADVAAEELMNALV	6821	AAR89413.1
  	MORBILLIVIRUS
  800	MEASLES	YDFRDLTWCINPPER	1683	P06830.1
  	MORBILLIVIRUS
  801	BORDETELLA	YFEPGPT	1684	ACI16083.1
  	PERTUSSIS
  802	RUBELLA VIRUS	YFNPGGSYYKQYHPT	1685	BAA28178.1
  803	MEASLES VIRUS	YFRLGQEMVRRSAGK	1686	P04851.1
  	STRAIN
  	EDMONSTON
  804	MEASLES VIRUS	YFRLGQEMVRRSAGKVSSTL	1687	P04851.1
  	STRAIN
  	EDMONSTON
  805	MEASLES	YFYPFRLPIKGVPIE	1688	P06830.1
  	MORBILLIVIRUS
  806	BORDETELLA	YGDNAGRILAGALAT	1689	P04977.1
  	PERTUSSIS
  807	RUBELLA VIRUS	YGHATVEMPEWIHAH	1690	BAA28178.1
  808	RUBELLA VIRUS	YIMNYTGNQQSRWGL	1691	BAA28178.1
  809	MEASLES VIRUS	YINNELIPSMNQLSC	1692	P69353.1
  	STRAIN
  	EDMONSTON
  810	RUBELLA VIRUS	YLCTAPGCATQAPVP	1693	BAA28178.1
  811	MEASLES	YLFTVPIKEAGEDCH	1694	P06830.1
  	MORBILLIVIRUS
  812	MEASLES VIRUS	YLHDPEFNL	1695	ABK40531.1
  	STRAIN
  	EDMONSTON
  813	MEASLES VIRUS	YLNMSRLFV	1696	ABK40531.1
  	STRAIN
  	EDMONSTON
  814	MEASLES VIRUS	YPALGLHEF	1697	P10050.1
  	STRAIN HALLE
  815	MEASLES	YPALGLHEFA	1698	ABI54110.1
  	MORBILLIVIRUS
  816	MEASLES VIRUS	YPALGLHEFAGELST	1699	P04851.1
  	STRAIN
  	EDMONSTON
  817	MEASLES VIRUS	YPALGLHEFAGELSTLESLM	1700	P04851.1
  	STRAIN
  	EDMONSTON
  818	MEASLES VIRUS	YPFRLPIKGVPIELQ	1701	P08362.1
  	STRAIN
  	EDMONSTON
  819	MEASLES VIRUS	YPLLWSYAM	1702	P10050.1
  	STRAIN HALLE
  820	MUMPS	YQQQGRL	1703	P21186.1
  	RUBULAVIRUS
  821	BORDETELLA	YQSEYLAHRR	1704	P04977.1
  	PERTUSSIS
  822	MEASLES VIRUS	YRETGPSRASDARAA	1705	P04851.1
  	STRAIN
  	EDMONSTON
  823	MEASLES VIRUS	YRETGPSRASDARAAHLPTG	1706	P04851.1
  	STRAIN
  	EDMONSTON
  824	RUBELLA VIRUS	YRNASDVLPGHWLQGGWGCYNLSD	1707	NP_740663.1
  		W
  825	MEASLES VIRUS	YRRLLRTVLEPIRDA	1708	P69353.1
  	STRAIN
  	EDMONSTON
  826	BORDETELLA	YRYDSRPP	1709	P04977.1
  	PERTUSSIS
  827	MEASLES VIRUS	YSGGDLLGILESRGI	1710	P69353.1
  	STRAIN
  	EDMONSTON
  828	BORDETELLA	YSKVTATBLLASTNSRLCAVFVRDG	1711	SRC280066
  	PERTUSSIS
  829	MEASLES VIRUS	YSPSRSFSYFYPFRL	1712	P08362.1
  	STRAIN
  	EDMONSTON
  830	RUBELLA VIRUS	YTGNQQSRWGLGSPNCHGPDWASPV	1713	P07566.1
  	STRAIN THERIEN
  831	MEASLES	YVLLAVLFV	1714	P08362.1
  	MORBILLIVIRUS
  832	MEASLES	YVYSPGRSFSYFYPF	1715	P06830.1
  	MORBILLIVIRUS
  833	BORDETELLA	YYDYEDATFQTYALTGISLCNPAASIC	1716	P04979.1
  	PERTUSSIS
  834	MEASLES	GDLLGILESRGIKAR	1717	AAF02706.1
  	MORBILLIVIRUS
  835	MEASLES	TVPKYVATQGYLISN	1718	AAL29688.1
  	MORBILLIVIRUS
  836	MEASLES	KPWDSPQEI	1719	P26035.1
  	MORBILLIVIRUS
  837	MEASLES	KPWESPQEI	1720	CAA34579.1
  	MORBILLIVIRUS
  838	BORDETELLA	ATYQSEYLAHRRIPP	1721	ACI04548.1
  	PERTUSSIS
  839	BORDETELLA	CMARQAESSEAMAAWSERAGEAMV	1722	ACI04548.1
  	PERTUSSIS	LVYYESIAYSF
  840	BORDETELLA	CQVGSSNSAFVSTSSSRRYTEVYL	1723	ACI04548.1
  	PERTUSSIS
  841	BORDETELLA	DDPPATVYRYDSRPP	1724	ACI04548.1
  	PERTUSSIS
  842	BORDETELLA	GALATYQSEYLAHRRIPP	1725	ACI04548.1
  	PERTUSSIS
  843	BORDETELLA	MAAWSERAGEAMVLVYYESIAYSF	1726	ACI04548.1
  	PERTUSSIS
  844	BORDETELLA	MVLVYYESIAYSF	1727	ACI04548.1
  	PERTUSSIS
  845	BORDETELLA	PATVYRYDSRPPEDV	1728	ACI04548.1
  	PERTUSSIS
  846	BORDETELLA	YDSRPPEDV	1729	ACI04548.1
  	PERTUSSIS
  847	BORDETELLA	EPGITTNYDT	1730	ACI16087.1
  	PERTUSSIS
  848	BORDETELLA	GDLRAYKMVYATNPQTQLSN	1731	ACI16083.1
  	PERTUSSIS
  849	BORDETELLA	KNGDVEASAITTYVGFSVVYP	1732	ACI16083.1
  	PERTUSSIS
  850	BORDETELLA	KVTNGSKSYTLRYLASYVK	1733	ACI16088.1
  	PERTUSSIS
  851	BORDETELLA	QALGALKLYFEPGITTNYDTGDLIAY	1734	ACI16088.1
  	PERTUSSIS	KQTYNASGN
  852	BORDETELLA	YATNPQTQLS	1735	ACI16083.1
  	PERTUSSIS
  853	CORYNEBACTERIUM	DNENPLSGKAGGVVKVTYPGLTKV	1736	AAV70486.1
  	DIPHTHERIAE
  854	CORYNEBACTERIUM	ENFSSYHGTKPGYVDSI	1737	AAV70486.1
  	DIPHTHERIAE
  855	CORYNEBACTERIUM	KVDNAETIKKELGLSLTEP	1738	AAV70486.1
  	DIPHTHERIAE
  856	RUBELLA VIRUS	MEDLQKALEAQSRALRAGLAA	1739	CAA28880.1
  	STRAIN M33
  857	CORYNEBACTERIUM	QKGIQKPKSGTQGNYDDDWKGFY	1740	AAV70486.1
  	DIPHTHERIAE
  858	RUBELLA VIRUS	RTGAWQRKDWSRAPPPPEERQESRS	1741	CAA28880.1
  	STRAIN M33	QTPAPKPSR
  859	RUBELLA VIRUS	AAGASQSRRPRPPRHARAQHLPEMT	1742	SRC265968
  		PAVT
  860	RUBELLA VIRUS	CVTSWLWSEGEGAVFYRVDLHFINL	1743	CAA28880.1
  		GTP
  861	RUBELLA VIRUS	FRVGGTRWHRLLRMPVRGLDGDTAP	1744	CAA28880.1
  		LP
  862	CORYNEBACTERIUM	GRKIRMRCRAIDGDVTFCRPKSPVYV	1745	1007216A
  	DIPHTHERIAE	GN
  863	RUBELLA VIRUS	GTPPLDEDGRWDPALMYNPCGPEPPA	1746	CAA28880.1
  		HV
  864	CORYNEBACTERIUM	GVHANLHVAFHRSSSEKIHSNEISSDS	1747	AAV70486.1
  	DIPHTHERIAE	IGVLGYQKTVDHTKVNSKLSLFFEIK
  		S
  865	RUBELLA VIRUS	MASTTPITMEDLQKALEAQSRALRA	1748	ABD64200.1
  		GLAAG
  866	RUBELLA VIRUS	PELGPPTNPFQAAVARGLRPPLHDPD	1749	CAA28880.1
  		TEAPTEAC
  867	RUBELLA VIRUS	PLPPHTTERIETRSARHPWR1RFGAP	1750	CAA28880.1
  868	RUBELLA VIRUS	SRAPPPPEERQESRSQTPAPKPSRAPP	1751	CAA28880.1
  869	RUBELLA VIRUS	SRAPPQQPQPPRMQTGRGGSAPRPEL	1752	CAA28880.1
  		GP
  870	RUBELLA VIRUS	TPAVTPEGPAPPRTGAWQRKDWSRAP	1753	CAA28880.1
  		P
  871	RUBELLA VIRUS	VRAYNQPAGDVRGVWGKGERTYAE	1754	CAA28880.1
  		QDFRV
  872	RUBELLA VIRUS	AFGHSDAACWGFPTDTVMSV	1755	CAA28880.1
  873	RUBELLA VIRUS	CARIWNGTQRACTFWAVNAYS	1756	CAA28880.1
  874	RUBELLA VIRUS	EEAFTYLCTAPGCATQTPVPVR	1757	CAA28880.1
  875	RUBELLA VIRUS	FAPWDLEATGACICEIPTDV	1758	CAA28880.1
  876	RUBELLA VIRUS	GEDVGAFPPGKFVTAAL	1759	CAA28880.1
  877	RUBELLA VIRUS	GEVWVTPVIGSQARKCGLHI	1760	CAA28880.1
  878	RUBELLA VIRUS	GQLEVQVPPDPGDLVEYIMN	1761	CAA28880.1
  879	RUBELLA VIRUS	GSYYKQYHPTACEVEPAFGH	1762	CAA28880.1
  880	RUBELLA VIRUS	IHAHTTSDPWHPPGPLGLKF	1763	CAA28880.1
  881	RUBELLA VIRUS	IMNYTGNQQSRWGLGSPNCH	1764	CAA28880.1
  882	RUBELLA VIRUS	LHIRAGPYGHATVEMPEWIH	1765	CAA28880.1
  883	RUBELLA VIRUS	LKFKTVRPVALPRALAPPRN	1766	CAA28880.1
  884	RUBELLA VIRUS	LNTPPPYQVSCGGESDRASAGH	1767	CAA28880.1
  885	RUBELLA VIRUS	NCHGPDWASPVCQRHSPDCS	1768	CAA28880.1
  886	RUBELLA VIRUS	PDCSRLVGATPERPRLRLVD	1769	CAA28880.1
  887	RUBELLA VIRUS	PRNVRVTGCYQCGTPALVEG	1770	CAA28880.1
  888	RUBELLA VIRUS	PTDVSCEGLGAWVPTAPCARI	1771	CAA28880.1
  889	RUBELLA VIRUS	RLVDADDPLLRTAPGPGEVW	1772	CAA28880.1
  890	RUBELLA VIRUS	SVFALASYVQHPHKTVRVKF	1773	CAA28880.1
  891	RUBELLA VIRUS	VEGLAPGGGNCHLTVNGEDV	1774	CAA28880.1
  892	RUBELLA VIRUS	VKFHTETRTVWQLSVAGVSC	1775	CAA28880.1
  893	RUBELLA VIRUS	VPVRLAGVGFESKIVDGGCF	1776	CAA28880.1
  894	RUBELLA VIRUS	VSCNVTTEHPFCNTPHGQLE	1777	CAA28880.1
  895	BORDETELLA	AAASSPDAHVPF	1778	AAA22983.1
  	PERTUSSIS
  896	BORDETELLA	AASSPDA	1779	AAA22983.1
  	PERTUSSIS
  897	BORDETELLA	AKLGAAASSPDA	1780	AAA22983.1
  	PERTUSSIS
  898	BORDETELLA	AMKPYEVTPTRM	1781	AAA22983.1
  	PERTUSSIS
  899	BORDETELLA	AMTHLSPALADVPYVLVKTNMVVTS	1782	AAA22983.1
  	PERTUSSIS
  900	BORDETELLA	ASSPDAHVPFCF	1783	AAA22983.1
  	PERTUSSIS
  901	BORDETELLA	ASSPDAHVPFCFGKDLKRPGSSPME	1784	AAA22983.1
  	PERTUSSIS
  902	BORDETELLA	CFGKDLKRPGSS	1785	AAA22983.1
  	PERTUSSIS
  903	BORDETELLA	CFGKDLKRPGSSPMEVMLRAVFMQQ	1786	AAA22983.1
  	PERTUSSIS
  904	BORDETELLA	CGIAAKLGAAAS	1787	AAA22983.1
  	PERTUSSIS
  905	BORDETELLA	CGIAAKLGAAASSPDAHVPFCFGKD	1788	AAA22983.1
  	PERTUSSIS
  906	BORDETELLA	DAHVPFCFGKDL	1789	AAA22983.1
  	PERTUSSIS
  907	BORDETELLA	DLKRPGSSPMEV	1790	AAA22983.1
  	PERTUSSIS
  908	BORDETELLA	DVPYVLVKTNMV	1791	AAA22983.1
  	PERTUSSIS
  909	BORDETELLA	DVPYVLVKTNMVVTSVAMKPYEVTP	1792	AAA22983.1
  	PERTUSSIS	T
  910	BORDETELLA	EVMLRAVFMQQR	1793	AAA22983.1
  	PERTUSSIS
  911	BORDETELLA	FEGKPALELIRM	1794	AAA22983.1
  	PERTUSSIS
  912	BORDETELLA	FLGPKQLTFEGK	1795	AAA22983.1
  	PERTUSSIS
  913	BORDETELLA	FLGPKQLTFEGKPALELIRMVECSG	1796	AAA22983.1
  	PERTUSSIS
  914	BORDETELLA	FMQQRPLRMFLGPKQLT	1797	AAA22983.1
  	PERTUSSIS
  915	BORDETELLA	GKDLKRPGSSPM	1798	AAA22983.1
  	PERTUSSIS
  916	BORDETELLA	GKDLKRPGSSPME	1799	AAA22983.1
  	PERTUSSIS
  917	BORDETELLA	GKPALELIRMVE	1800	AAA22983.1
  	PERTUSSIS
  918	BORDETELLA	GPKQLTFEGKPA	1801	AAA22983.1
  	PERTUSSIS
  919	BORDETELLA	HVPFCFGKDLKR	1802	AAA22983.1
  	PERTUSSIS
  920	BORDETELLA	IAAKLGAAASSP	1803	AAA22983.1
  	PERTUSSIS
  921	BORDETELLA	KPYEVTPTRMLV	1804	AAA22983.1
  	PERTUSSIS
  922	BORDETELLA	KQLTFEGKPALE	1805	AAA22983.1
  	PERTUSSIS
  923	BORDETELLA	KRPGSSPMEVML	1806	AAA22983.1
  	PERTUSSIS
  924	BORDETELLA	LELIRMVECSGK	1807	AAA22983.1
  	PERTUSSIS
  925	BORDETELLA	LGAAASSPDAHV	1808	AAA22983.1
  	PERTUSSIS
  926	BORDETELLA	LIRMVECSGKQD	1809	AAA22983.1
  	PERTUSSIS
  927	BORDETELLA	LVCGIAAKLGAA	1810	AAA22983.1
  	PERTUSSIS
  928	BORDETELLA	MKPYEVTPTRM	1811	AAA22983.1
  	PERTUSSIS
  929	BORDETELLA	MLRAVFMQQRPL	1812	AAA22983.1
  	PERTUSSIS
  930	BORDETELLA	MQQRPLRM	1813	AAA22983.1
  	PERTUSSIS
  931	BORDETELLA	MQQRPLRMFLGP	1814	AAA22983.1
  	PERTUSSIS
  932	BORDETELLA	MVVTSVAMKPYE	1815	AAA22983.1
  	PERTUSSIS
  933	BORDETELLA	MVVTSVAMKPYEVTPTRMLVCGIAA	1816	AAA22983.1
  	PERTUSSIS
  934	BORDETELLA	PALELIRMVECS	1817	AAA22983.1
  	PERTUSSIS
  935	BORDETELLA	PALELIRMVECSGK	1818	AAA22983.1
  	PERTUSSIS
  936	BORDETELLA	PFCFGKDLKRPG	1819	AAA22983.1
  	PERTUSSIS
  937	BORDETELLA	PGSSPMEVMLRA	1820	AAA22983.1
  	PERTUSSIS
  938	BORDETELLA	PGSSPMEVMLRAVF	1821	AAA22983.1
  	PERTUSSIS
  939	BORDETELLA	PKQLTFEGK	1822	AAA22983.1
  	PERTUSSIS
  940	BORDETELLA	PLRMFLGPKQLT	1823	AAA22983.1
  	PERTUSSIS
  941	BORDETELLA	PTRMLVCGIAAK	1824	AAA22983.1
  	PERTUSSIS
  942	BORDETELLA	PYVLVKTNMVVT	1825	AAA22983.1
  	PERTUSSIS
  943	BORDETELLA	QLTFEGKPALELIRMVECSGKQDCP	1826	AAA22983.1
  	PERTUSSIS
  944	BORDETELLA	QRPLRMFLGPKQ	1827	AAA22983.1
  	PERTUSSIS
  945	BORDETELLA	RAVFMQQRPLRM	1828	AAA22983.1
  	PERTUSSIS
  946	BORDETELLA	RMFLGPKQLTFE	1829	AAA22983.1
  	PERTUSSIS
  947	BORDETELLA	RMLVCGIAAKLG	1830	AAA22983.1
  	PERTUSSIS
  948	BORDETELLA	RMVECSGKQDCP	1831	AAA22983.1
  	PERTUSSIS
  949	BORDETELLA	SPDAHVPFCFGK	1832	AAA22983.1
  	PERTUSSIS
  950	BORDETELLA	SSPMEVMLRAVF	1833	AAA22983.1
  	PERTUSSIS
  951	BORDETELLA	SSPMEVMLRAVFMQQRPLRMFLGPK	1834	AAA22983.1
  	PERTUSSIS
  952	BORDETELLA	SVAMKPYEVTPT	1835	AAA22983.1
  	PERTUSSIS
  953	BORDETELLA	VECSGKQDCP	1836	AAA22983.1
  	PERTUSSIS
  954	BORDETELLA	VFMQQRPLRMFL	1837	AAA22983.1
  	PERTUSSIS
  955	BORDETELLA	VFMQQRPLRMFLGPKQLTFEGKPAL	1838	AAA22983.1
  	PERTUSSIS
  956	BORDETELLA	VKTNMVVTSVAM	1839	AAA22983.1
  	PERTUSSIS
  957	BORDETELLA	VLVKTNMVVTSV	1840	AAA22983.1
  	PERTUSSIS
  958	BORDETELLA	VTPTRMLVCGIA	1841	AAA22983.1
  	PERTUSSIS
  959	BORDETELLA	VTSVAMKPYEVT	1842	AAA22983.1
  	PERTUSSIS
  960	BORDETELLA	YEVTPTRMLVCG	1843	AAA22983.1
  	PERTUSSIS
  961	BORDETELLA	YEVTPTRMLVCGIAAKLGAAASSPD	1844	AAA22983.1
  	PERTUSSIS
  962	BORDETELLA	CASPYEGRYRDMYDALR	1845	P04979.1
  	PERTUSSIS
  963	BORDETELLA	CAVFVRDGQSV	1846	P04979.1
  	PERTUSSIS
  964	BORDETELLA	CITTIYKTG	1847	P04979.1
  	PERTUSSIS
  965	BORDETELLA	CPNGTRALTV	1848	P04979.1
  	PERTUSSIS
  966	BORDETELLA	DALRRLLYMIYMSG	1849	P04979.1
  	PERTUSSIS
  967	RUBELLA VIRUS	GNRGRGQRRDWSRAPPPPEERQETR	1850	P07566.1
  	STRAIN THERIEN	S
  968	BORDETELLA	GQPAADHYYSKVT	1851	P04979.1
  	PERTUSSIS
  969	RUBELLA VIRUS	GSPNCHGPDWASPVCQRHS	1852	ABD64214.1
  970	MEASLES VIRUS	HKSLSTNLDVTNSIEHQ	1853	P08362.1
  	STRAIN
  	EDMONSTON
  971	BORDETELLA	LFTQQGGAYGRC	1854	P04979.1
  	PERTUSSIS
  972	MEASLES VIRUS	LIGLLAIAGIRLHRAAIYTAEI	1855	P08362.1
  	STRAIN
  	EDMONSTON
  973	MEASLES	PDTAADSELRRWIKY	1856	ABI54110.1
  	MORBILLIVIRUS
  974	RUBELLA VIRUS	PNCHGPDWASPVCQRHS	1857	P07566.1
  	STRAIN THERIEN
  975	RUBELLA VIRUS	QTPAPKPSRAPPQQPQPPRMQTGR	1858	ABD64216.1
  976	RUBELLA VIRUS	RAGLTAGASQSRRPRPPR	1859	CAA33016.1
  	VACCINE STRAIN
  	RA27/3
  977	RUBELLA VIRUS	RFGAPQAFLAGLLLAAVAVGTARA	1860	ABD64214.1
  	VACCINE STRAIN
  	RA27/3
  978	BORDETELLA	RGNAELQTYLRQITPG	1861	P04979.1
  	PERTUSSIS
  979	BORDETELLA	RVHVSKEEQYYDYED	1862	P04979.1
  	PERTUSSIS
  980	BORDETELLA	SIYGLYDGTYL	1863	P04979.1
  	PERTUSSIS
  981	BORDETELLA	SKVTATRLLASTNS	1864	P04979.1
  	PERTUSSIS
  982	BORDETELLA	TQQGGAYGRCPNGTRA	1865	P04979.1
  	PERTUSSIS
  983	BORDETELLA	VAPGIVIPPKAL	1866	P04979.1
  	PERTUSSIS
  984	BORDETELLA	DSRPPEDVFQNGFTAWG	1867	ACI04548.1
  	PERTUSSIS
  985	BORDETELLA	EHRMQEAVEAERAGR	1868	ACI04548.1
  	PERTUSSIS
  986	MEASLES VIRUS	ETCFQQACKGKIQALCENPEWA	1869	P08362.1
  	STRAIN
  	EDMONSTON
  987	BORDETELLA	EYVDTYGDNAGRILAGALATYQ	1870	ACI04548.1
  	PERTUSSIS
  988	BORDETELLA	HRRIPPENIRRVTR	1871	ACI04548.1
  	PERTUSSIS
  989	BORDETELLA	MARQAESSE	1872	ACI04548.1
  	PERTUSSIS
  990	BORDETELLA	MQEAVEAERAGR	1873	ACI04548.1
  	PERTUSSIS
  991	BORDETELLA	SQQTRANPNPYTSRR	1874	ACI04548.1
  	PERTUSSIS
  992	BORDETELLA	TRANPNPYTSRRSVASIVGTLVHG	1875	SRC280066
  	PERTUSSIS
  993	BORDETELLA	TVYRYDSRPPED	1876	ACI04548.1
  	PERTUSSIS
  994	MEASLES	NDRNLLD	1877	P10050.1
  	MORBILLIVIRUS
  995	MEASLES	NMEDEADQYFSHDDPISSDQSRFGW	1878	P04851.1
  	MORBILLIVIRUS	FENK
  996	MEASLES	SRASDARAAHLPTGTPLDID	1879	P04851.1
  	MORBILLIVIRUS
  997	BORDETELLA	EDVFQNGFTAW	1880	ACI04548.1
  	PERTUSSIS
  998	CORYNEBACTERIUM	AEGSSSVEYINNWEQAK	1881	AAV70486.1
  	DIPHTHERIAE
  999	CORYNEBACTERIUM	GPIKNKMSESPNKT	1882	AAV70486.1
  	DIPHTHERIAE
  1000	MEASLES VIRUS	GPKLTGALIGILSLFVESPGQLIQRITD	1883	P10050.1
  	STRAIN HALLE	DPDV
  1001	CORYNEBACTERIUM	GYQKTVDHTKVNSK	1884	AAV70486.1
  	DIPHTHERIAE
  1002	CORYNEBACTERIUM	KTVDH	1885	AAV70486.1
  	DIPHTHERIAE
  1003	CORYNEBACTERIUM	SESPNK	1886	AAV70486.1
  	DIPHTHERIAE
  1004	CORYNEBACTERIUM	AEGSSSVEYINNWEQAKALS	1887	AAV70486.1
  	DIPHTHERIAE
  1005	CORYNEBACTERIUM	AQAIPLVGELVDIGFAAYNF	1888	AAV70486.1
  	DIPHTHERIAE
  1006	CORYNEBACTERIUM	ASRVVLSLPFAEGSSSVEYI	1889	AAV70486.1
  	DIPHTHERIAE
  1007	CORYNEBACTERIUM	CINLDWDVIRDKTKTKIESL	1890	AAV70486.1
  	DIPHTHERIAE
  1008	CORYNEBACTERI	CRAIDGDVTFCRPKSPVYVG	1891	AAV70486.1
  	DIPHTHERIAE
  1009	CORYNEBACTERIUM	CRPKSPVYVGNGVHANLHVA	1892	AAV70486.1
  	DIPHTHERIAE
  1010	CORYNEBACTERIUM	DAAGYSVDNENPLSGKAGGV	1893	AAV70486.1
  	DIPHTHERIAE
  1011	CORYNEBACTERIUM	DKTKTKIESLKEHGPIKNKM	1894	AAV70486.1
  	DIPHTHERIAE
  1012	CORYNEBACTERIUM	EEFIKRFGDGASRVVLSLPF	1895	AAV70486.1
  	DIPHTHERIAE
  1013	CORYNEBACTERIUM	EKAKQYLEEFHQTALEHPEL	1896	AAV70486.1
  	DIPHTHERIAE
  1014	CORYNEBACTERIUM	FHRSSSEKIHSNEISSDSIG	1897	AAV70486.1
  	DIPHTHERIAE
  1015	CORYNEBACTERIUM	GADDVVDSSKSFVMENFSSY	1898	CAE11230.1
  	DIPHTHERIAE
  1016	CORYNEBACTERIUM	GKRGQDAMYEYMAQACAGNR	1899	AAV70486.1
  	DIPHTHERIAE
  1017	CORYNEBACTERIUM	GSVMGIADGAVHHNTEEIVA	1900	AAV70486.1
  	DIPHTHERIAE
  1018	CORYNEBACTERIUM	GTQGNYDDDWKGFYSTDNKY	1901	AAV70486.1
  	DIPHTHERIAE
  1019	CORYNEBACTERIUM	HDGYAVSWNTVEDSIIRTGF	1902	AAV70486.1
  	DIPHTHERIAE
  1020	CORYNEBACTERIUM	HGTKPGYVDSIQKGIQKPKS	1903	AAV70486.1
  	DIPHTHERIAE
  1021	CORYNEBACTERIUM	HQTALEHPELSELKTVTGTN	1904	AAV70486.1
  	DIPHTHERIAE
  1022	CORYNEBACTERIUM	IQKGIQKPKSGTQGNYDDDW	1905	AAV70486.1
  	DIPHTHERIAE
  1023	CORYNEBACTERIUM	KEHGPIKNKMSESPNKTVSE	1906	AAV70486.1
  	DIPHTHERIAE
  1024	CORYNEBACTERIUM	KGFYSTDNKYDAAGYSVDNE	1907	AAV70486.1
  	DIPHTHERIAE
  1025	CORYNEBACTERIUM	LDVNKSKTHISVNGRKIRMR	1908	AAV70486.1
  	DIPHTHERIAE
  1026	RUBELLA VIRUS	MASTIPITMEDLQKALEA	1909	SRC265968
  1027	CORYNEBACTERIUM	NGVHANLHVAFHRSSSEKIH	1910	AAV70486.1
  	DIPHTHERIAE
  1028	CORYNEBACTERIUM	NNWEQAKALSVELEINFETR	1911	AAV70486.1
  	DIPHTHERIAE
  1029	CORYNEBACTERI	NPLSGKAGGVVKVTYPGLTK	1912	AAV70486.1
  	DIPHTHERIAE
  1030	CORYNEBACTERIUM	PVFAGANYAAWAVNVAQVID	1913	AAV70486.1
  	DIPHTHERIAE
  1031	CORYNEBACTERIUM	SELKTVTGTNPVFAGANYAA	1914	AAV70486.1
  	DIPHTHERIAE
  1032	CORYNEBACTERIUM	SESPNKTVSEEKAKQYLEEF	1915	AAV70486.1
  	DIPHTHERIAE
  1033	CORYNEBACTERIUM	SETADNLEKTTAALSILPGI	1916	AAV70486.1
  	DIPHTHERIAE
  1034	CORYNEBACTERIUM	SFVMENFSSYHGTKPGYVDS	1917	AAV70486.1
  	DIPHTHERIAE
  1035	CORYNEBACTERIUM	SNEISSDSIGVLGYQKTVDH	1918	AAV70486.1
  	DIPHTHERIAE
  1036	CORYNEBACTERIUM	SPGHKTQPFLHDGYAVSWNT	1919	AAV70486.1
  	DIPHTHERIAE
  1037	CORYNEBACTERIUM	SVNGRKIRMRCRAIDGDVTF	1920	AAV70486.1
  	DIPHTHERIAE
  1038	CORYNEBACTERIUM	TAALSILPGIGSVMGIADGA	1921	AAV70486.1
  	DIPHTHERIAE
  1039	CORYNEBACTERIUM	TAENTPLPIAGVLLPTIPGK	1922	AAV70486.1
  	DIPHTHERIAE
  1040	CORYNEBACTERIUM	TEPLMEQVGTEEFIKRFGDG	1923	AAV70486.1
  	DIPHTHERIAE
  1041	CORYNEBACTERIUM	TIKKELGLSLTEPLMEQVGT	1924	AAV70486.1
  	DIPHTHERIAE
  1042	CORYNEBACTERIUM	TKVNSKLSLFFEIKS	1925	AAV70486.1
  	DIPHTHERIAE
  1043	CORYNEBACTERIUM	VEDSIIRTGFQGESGHDIKI	1926	AAV70486.1
  	DIPHTHERIAE
  1044	CORYNEBACTERIUM	VELEINFETRGKRGQDAMYE	1927	AAV70486.1
  	DIPHTHERIAE
  1045	CORYNEBACTERI	VESIINLFQVVHNSYNRPAY	1928	AAV70486.1
  	DIPHTHERIAE
  1046	CORYNEBACTERIUM	VHNSYNRPAYSPGHKTQPFL	1929	AAV70486.1
  	DIPHTHERIAE
  1047	CORYNEBACTERIUM	VKVTYPGLTKVLALKVDNAE	1930	AAV70486.1
  	DIPHTHERIAE
  1048	CORYNEBACTERIUM	VLALKVDNAETIKKELGLSL	1931	AAV70486.1
  	DIPHTHERIAE
  1049	CORYNEBACTERIUM	VLGYQKTVDHTKVNSKLSLF	1932	AAV70486.1
  	DIPHTHERIAE
  1050	CORYNEBACTERIUM	VRRSVGSSLSCINLDWDVIR	1933	AAV70486.1
  	DIPHTHERIAE
  1051	CORYNEBACTERIUM	WAVNVAQVIDSETADNLEKT	1934	AAV70486.1
  	DIPHTHERIAE
  1052	CORYNEBACTERIUM	YMAQACAGNRVRRSVGSSLS	1935	AAV70486.1
  	DIPHTHERIAE
  1053	BORDETELLA	AKAPPAPKPAPQPGP	1936	ABO77783.13.1
  	PERTUSSIS
  1054	BORDETELLA	APKPAPQPGP	1937	ABO77783.13.1
  	PERTUSSIS
  1055	BORDETELLA	APKPAPQPGPQPPQP	1938	ABO77783.13.1
  	PERTUSSIS
  1056	MEASLES VIRUS	AQTRTPLQCTMTEIF	1939	P04851.1
  	STRAIN
  	EDMONSTON
  1057	MEASLES VIRUS	ASRGTNMEDEADQYFSHDD	1940	P04851.1
  	STRAIN
  	EDMONSTON
  1058	BORDETELLA	ATIRR	1941	ABO77783.13.1
  	PERTUSSIS
  1059	BORDETELLA	DNRAG	1942	ABO77783.13.1
  	PERTUSSIS
  1060	BORDETELLA	EAPAPQPPAGRELSA	1943	ABO77783.13.1
  	PERTUSSIS
  1061	MEASLES VIRUS	EMVRRSAGKVSSTLASELGI	1944	P04851.1
  	STRAIN
  	EDMONSTON
  1062	BORDETELLA	GASEL	1945	ABO77783.13.1
  	PERTUSSIS
  1063	BORDETELLA	GDALAGGAVP	1946	AAA22980.1
  	PERTUSSIS
  1064	BORDETELLA	GDAPAGGAVP	1947	ABO77783.13.1
  	PERTUSSIS
  1065	BORDETELLA	GDTWDDD	1948	ABO77783.13.1
  	PERTUSSIS
  1066	BORDETELLA	GERQH	1949	ABO77783.13.1
  	PERTUSSIS
  1067	BORDETELLA	GGAVP	1950	ABO77783.13.1
  	PERTUSSIS
  1068	BORDETELLA	GGFGP	1951	P14283.3
  	PERTUSSIS
  1069	BORDETELLA	GGFGPGGFGP	1952	BAF35031.1
  	PERTUSSIS
  1070	BORDETELLA	GGFGPVLDGW	1953	ABO77783.13.1
  	PERTUSSIS
  1071	MEASLES VIRUS	GGKEDRRVKQSRGEARESYR	1954	P04851.1
  	STRAIN
  	EDMONSTON
  1072	BORDETELLA	GILLEN	1955	ABO77783.13.1
  	PERTUSSIS
  1073	BORDETELLA	GIRRFL	1956	ABO77783.13.1
  	PERTUSSIS
  1074	MEASLES VIRUS	HDDPISSDQSRFGWFENKEI	1957	P04851.1
  	STRAIN
  	EDMONSTON
  1075	MEASLES VIRUS	HEFAGELSTLESLMNLY	1958	P04851.1
  	STRAIN
  	EDMONSTON
  1076	BORDETELLA	HLGGLAGY	1959	ABO77783.13.1
  	PERTUSSIS
  1077	MEASLES VIRUS	HTTEDKISRAVGPRQAQVSFL	1960	P04851.1
  	STRAIN
  	EDMONSTON
  1078	MEASLES VIRUS	ICDIDTYIVEAGLASFILTI	1961	P04851.1
  	STRAIN
  	EDMONSTON
  1079	MEASLES VIRUS	IIIVPIPGDSSITTRSRLLD	1962	P04851.1
  	STRAIN
  	EDMONSTON
  1080	MEASLES VIRUS	ILDIKRTPGNKPRIAEMICD	1963	P04851.1
  	STRAIN
  	EDMONSTON
  1081	BORDETELLA	KALLYR	1964	ABO77783.13.1
  	PERTUSSIS
  1082	MEASLES VIRUS	KPPITSGSGGAIRGIKHIII	1965	P04851.1
  	STRAIN
  	EDMONSTON
  1083	BORDETELLA	LAGSGL	1966	ABO77783.13.1
  	PERTUSSIS
  1084	MEASLES VIRUS	LGITAEDARLVSEIAMHTTE	1967	P04851.1
  	STRAIN
  	EDMONSTON
  1085	MEASLES VIRUS	LGTILAQIWVLLAKAVTA	1968	P04851.1
  	STRAIN
  	EDMONSTON
  1086	MEASLES VIRUS	LPTGTPLDIDTASESSQD	1969	P04851.1
  	STRAIN
  	EDMONSTON
  1087	MEASLES VIRUS	MATLLRSLALFKRNKDK	1970	P04851.1
  	STRAIN
  	EDMONSTON
  1088	BORDETELLA	PAPQPP	1971	ABO77783.13.1
  	PERTUSSIS
  1089	MEASLES VIRUS	PDTAADSELRRWIKYTQQRR	1972	P04851.1
  	STRAIN
  	EDMONSTON
  1090	MEASLES VIRUS	PKLTGALIGILSLFVESPGQ	1973	P04851.1
  	STRAIN
  	EDMONSTON
  1091	BORDETELLA	PQP	1974	ABO77783.13.1
  	PERTUSSIS
  1092	BORDETELLA	PQPGP	1975	ABO77783.13.1
  	PERTUSSIS
  1093	BORDETELLA	PQPGPQPPQPPQPQP	1976	ABO77783.13.1
  	PERTUSSIS
  1094	MEASLES VIRUS	QDPQDSRRSAEPLLSCKPWQ	1977	P04851.1
  	STRAIN
  	EDMONSTON
  1095	MEASLES VIRUS	QRRVVGEFRLERKWLDVVR	1978	P04851.1
  	STRAIN
  	EDMONSTON
  1096	BORDETELLA	RELSA	1979	ABO77783.13.1
  	PERTUSSIS
  1097	BORDETELLA	RFAPQ	1980	ABO77783.13.1
  	PERTUSSIS
  1098	MEASLES VIRUS	SIQNKFSAGSYPLLWSYAMG	1981	P04851.1
  	STRAIN
  	EDMONSTON
  1099	BORDETELLA	SITLQAGAH	1982	ABO77783.13.1
  	PERTUSSIS
  1100	BORDETELLA	SLQPED	1983	ABO77783.13.1
  	PERTUSSIS
  1101	BORDETELLA	SNALSKRL	1984	ABO77783.13.1
  	PERTUSSIS
  1102	MEASLES VIRUS	SPGQLIQRITDDPDVSIRLL	1985	P04851.1
  	STRAIN
  	EDMONSTON
  1103	BORDETELLA	TELPSIPG	1986	ABO77783.13.1
  	PERTUSSIS
  1104	BORDETELLA	TFTLANK	1987	ABO77783.13.1
  	PERTUSSIS
  1105	BORDETELLA	TWDDD	1988	ABO77783.13.1
  	PERTUSSIS
  1106	MEASLES VIRUS	VSFLQGDQSENELPRLGGKE	1989	P04851.1
  	STRAIN
  	EDMONSTON
  1107	MEASLES VIRUS	WQESRKNKAQTRTPLQC	1990	P04851.1
  	STRAIN
  	EDMONSTON
  1108	MEASLES VIRUS	YAMGVGVELENSMGGLNFGR	1991	P04851.1
  	STRAIN
  	EDMONSTON
  1109	MEASLES VIRUS	YQQMGKPAPYMVNLENSI	1992	P04851.1
  	STRAIN
  	EDMONSTON
  1110	MEASLES VIRUS	MTRSSHQSLVIKLMP	1993	P69355.1
  	STRAIN HALLE
  1111	MEASLES VIRUS	PIRDALNAMTQNIRP	1994	P69355.1
  	STRAIN HALLE
  1112	RUBELLA VIRUS	ALLNTPPPYQVSCGGESDRASAGH	1995	CAA28880.1
  	STRAIN M33
  1113	RUBELLA VIRUS	GLGSPNCHGPDWASPVCQRHS	1996	P07566.1
  	STRAIN THERIEN
  1114	RUBELLA VIRUS	GLGSPNCHGPDWASPVCQRHSPDCS	1997	P07566.1
  	STRAIN THERIEN	RLV
  1115	RUBELLA VIRUS	NYTGNQQSRWGLGSPNCHGPDWASP	1998	P07566.1
  	STRAIN THERIEN	V
  1116	RUBELLA VIRUS	TLPQPPCAHGQHYGHHHHQL	1999	P07566.1
  	STRAIN THERIEN
  1117	RUBELLA VIRUS	TVRVKFHTETRTVWQLSVAGVSCNV	2000	P07566.1
  	STRAIN THERIEN	T
  1118	RUBELLA VIRUS	ASYFNPGGSYYKQYH	2001	BAA28178.1
  1119	RUBELLA VIRUS	FALASYVQHPHKTVR	2002	BAA28178.1
  1120	RUBELLA VIRUS	GGESDRASARVIDPAAQSFTG	2003	BAA28178.1
  1121	RUBELLA VIRUS	GPGEVWVTPVIGSQARKC	2004	BAA28178.1
  1122	RUBELLA VIRUS	GSQARKCGLHIRAG	2005	BAA28178.1
  1123	RUBELLA VIRUS	LVGATPERPRLRLVDADDPLLRTAP	2006	BAA28178.1
  1124	RUBELLA VIRUS	TAPGPGEVWVTPVI	2007	BAA28178.1
  1125	MEASLES	FIVLSIAYPTLSEIK	2008	AAL29688.1
  	MORBILLIVIRUS
  1126	MEASLES VIRUS	ETCFQQACKGKIQALCENPEWAPLK	2009	P08362.1
  	STRAIN	DNRIPSY
  	EDMONSTON-
  	ZAGREB
  1127	MEASLES	LPRLGGKEDR	2010	P04851.1
  	MORBILLIVIRUS
  1128	MEASLES	MSKTEWNASQ	2011	SRC280080
  	MORBILLIVIRUS
  1129	MEASLES	SRFGWFENKE	2012	P04851.1
  	MORBILLIVIRUS
  1130	RUBELLA VIRUS	EACVTSWLWSEGEGAVFYRVDLHFI	2013	CAA28880.1
  		NLGT
  1131	RUBELLA VIRUS	MDFWCVEHDRPPPATPTSLTT	2014	CAA33016.1
  1132	RUBELLA VIRUS	PFLGHDGHHGGTLRVGQHHRNASDV	2015	CAA33016.1
  1133	RUBELLA VIRUS	RVKFHTETRTVWQLSVAGVSC	2016	BAA19893.1
  1134	MEASLES	AEEQARHVKNGLE	2017	ABO69699.1
  	MORBILLIVIRUS
  1135	MEASLES	ESPQEISKHQALG	2018	SRC280080
  	MORBILLIVIRUS
  1136	MEASLES	GVGVELENSMGGLNF	2019	ABI54110.1
  	MORBILLIVIRUS
  1137	MEASLES	IKGANDLAKFHQMLMKIIMK	2020	ABO69699.1
  	MORBILLIVIRUS
  1138	MEASLES	MSKTLHAQLGFKKT	2021	ABK40528.1
  	MORBILLIVIRUS
  1139	MEASLES	NASGLSRPSPSAH	2022	BAE98296.1
  	MORBILLIVIRUS
  1140	MEASLES	VRVIDPSLGDRKDE	2023	SRC280148
  	MORBILLIVIRUS
  1141	BORDETELLA	DLSDGDLLV	2024	AAC31207.1
  	PERTUSSIS
  1142	BORDETELLA	EAERAGRGTG	2025	ACI04548.1
  	PERTUSSIS
  1143	BORDETELLA	YRYDSRPPEDV	2026	ACI04548.1
  	PERTUSSIS
  1144	BORDETELLA	YVDTYGDNAG	2027	ACI04548.1
  	PERTUSSIS
  1145	MEASLES	SFSYFYPFR	2028	CAB43772.1
  	MORBILLIVIRUS
  1146	MUMPS	DIFIVSPR	2029	ADF49557.1
  	RUBULAVIRUS
  1147	MEASLES	QDSRRSADALLRLQAMAGISEEQGS	2030	CAA59302.1
  	MORBILLIVIRUS	DTDTPIVYNDRN
  1148	MEASLES	SAEALLRLQA	2031	BAH22350.1
  	MORBILLIVIRUS
  1149	MEASLES	RIVINREHL	2032	BAB39835.1
  	MORBILLIVIRUS
  1150	MEASLES VIRUS	IPRFK	2033	BAB39848.1
  	GENOTYPE A
  1151	BORDETELLA	AAALSPMEI	2034	P15318.2
  	PERTUSSIS
  1152	BORDETELLA	AAASVVGAPV	2035	P15318.2
  	PERTUSSIS
  1153	BORDETELLA	AALGRQDSI	2036	P15318.2
  	PERTUSSIS
  1154	BORDETELLA	AAQRLVHAIA	2037	P15318.2
  	PERTUSSIS
  1155	BORDETELLA	AAVEAAEL	2038	P15318.2
  	PERTUSSIS
  1156	BORDETELLA	AGANVLNGL	2039	P15318.2
  	PERTUSSIS
  1157	BORDETELLA	AGYANAAD	2040	P15318.2
  	PERTUSSIS
  1158	BORDETELLA	AGYEQFEFRV	2041	P15318.2
  	PERTUSSIS
  1159	BORDETELLA	AITGNADNL	2042	P15318.2
  	PERTUSSIS
  1160	BORDETELLA	AKEKNATLM	2043	P15318.2
  	PERTUSSIS
  1161	BORDETELLA	AKGVFLSL	2044	P15318.2
  	PERTUSSIS
  1162	BORDETELLA	APHEYGFGI	2045	P15318.2
  	PERTUSSIS
  1163	BORDETELLA	ARQGNDLEI	2046	P15318.2
  	PERTUSSIS
  1164	BORDETELLA	ASVVGAPV	2047	P15318.2
  	PERTUSSIS
  1165	BORDETELLA	ATLMFRLV	2048	P15318.2
  	PERTUSSIS
  1166	BORDETELLA	AVAAAQRL	2049	P15318.2
  	PERTUSSIS
  1167	BORDETELLA	DAGANVLNGL	2050	P15318.2
  	PERTUSSIS
  1168	BORDETELLA	DALLAQLYR	2051	P15318.2
  	PERTUSSIS
  1169	BORDETELLA	DANGVLKHSI	2052	P15318.2
  	PERTUSSIS
  1170	BORDETELLA	DGDMNIGVI	2053	P15318.2
  	PERTUSSIS
  1171	BORDETELLA	DHVKNIENL	2054	P15318.2
  	PERTUSSIS
  1172	BORDETELLA	DIDMFAIM	2055	P15318.2
  	PERTUSSIS
  1173	BORDETELLA	DMFAIMPHL	2056	P15318.2
  	PERTUSSIS
  1174	BORDETELLA	DNVRNVENV	2057	P15318.2
  	PERTUSSIS
  1175	BORDETELLA	DNVRNVENVI	2058	P15318.2
  	PERTUSSIS
  1176	BORDETELLA	DTVDYSAM	2059	P15318.2
  	PERTUSSIS
  1177	BORDETELLA	DTVDYSAMI	2060	P15318.2
  	PERTUSSIS
  1178	BORDETELLA	DYLRQAGL	2061	P15318.2
  	PERTUSSIS
  1179	BORDETELLA	DYYDNVRNV	2062	P15318.2
  	PERTUSSIS
  1180	BORDETELLA	EFTTFVEI	2063	P15318.2
  	PERTUSSIS
  1181	BORDETELLA	EFTTFVEIV	2064	P15318.2
  	PERTUSSIS
  1182	BORDETELLA	EGYVFYEN	2065	P15318.2
  	PERTUSSIS
  1183	BORDETELLA	ENVQYRHV	2066	P15318.2
  	PERTUSSIS
  1184	BORDETELLA	EQLANSDGL	2067	P15318.2
  	PERTUSSIS
  1185	BORDETELLA	FGVGYGHDTI	2068	P15318.2
  	PERTUSSIS
  1186	BORDETELLA	FSPDVLETVP	2069	P15318.2
  	PERTUSSIS
  1187	BORDETELLA	FSVDHVKNI	2070	P15318.2
  	PERTUSSIS
  1188	BORDETELLA	GDDTYLFGV	2071	P15318.2
  	PERTUSSIS
  1189	BORDETELLA	GDDVFLQDL	2072	P15318.2
  	PERTUSSIS
  1190	BORDETELLA	GEDGNDIFL	2073	P15318.2
  	PERTUSSIS
  1191	BORDETELLA	GERFNVRKQL	2074	P15318.2
  	PERTUSSIS
  1192	BORDETELLA	GGAGNDTLV	2075	P15318.2
  	PERTUSSIS
  1193	BORDETELLA	GGDDFEAV	2076	P15318.2
  	PERTUSSIS
  1194	BORDETELLA	GKSEFTTFV	2077	P15318.2
  	PERTUSSIS
  1195	BORDETELLA	GKSLFDDGL	2078	P15318.2
  	PERTUSSIS
  1196	BORDETELLA	GNADNLKSV	2079	P15318.2
  	PERTUSSIS
  1197	BORDETELLA	GQLVEVDTL	2080	P15318.2
  	PERTUSSIS
  1198	BORDETELLA	GRSKFSPDV	2081	P15318.2
  	PERTUSSIS
  1199	BORDETELLA	GSSAYDTV	2082	P15318.2
  	PERTUSSIS
  1200	BORDETELLA	GTVEKWPAL	2083	P15318.2
  	PERTUSSIS
  1201	BORDETELLA	GVDYYDNV	2084	P15318.2
  	PERTUSSIS
  1202	BORDETELLA	GYEQFEFRV	2085	P15318.2
  	PERTUSSIS
  1203	BORDETELLA	HAVGAQDVV	2086	P15318.2
  	PERTUSSIS
  1204	BORDETELLA	IAAGRIGLGI	2087	P15318.2
  	PERTUSSIS
  1205	BORDETELLA	IGDAQANTL	2088	P15318.2
  	PERTUSSIS
  1206	BORDETELLA	IGLGILADL	2089	P15318.2
  	PERTUSSIS
  1207	BORDETELLA	IGNAAGIPL	2090	P15318.2
  	PERTUSSIS
  1208	BORDETELLA	IGTSMKDVL	2091	P15318.2
  	PERTUSSIS
  1209	BORDETELLA	IGVITDFEL	2092	P15318.2
  	PERTUSSIS
  1210	BORDETELLA	IPLTADIDM	2093	P15318.2
  	PERTUSSIS
  1211	BORDETELLA	ISKSALEL	2094	P15318.2
  	PERTUSSIS
  1212	BORDETELLA	ITGNADNL	2095	P15318.2
  	PERTUSSIS
  1213	BORDETELLA	KIFVVSAT	2096	P15318.2
  	PERTUSSIS
  1214	BORDETELLA	KQLNNANVYR	2097	P15318.2
  	PERTUSSIS
  1215	BORDETELLA	KVIGNAAGI	2098	P15318.2
  	PERTUSSIS
  1216	BORDETELLA	LAKVVSQL	2099	P15318.2
  	PERTUSSIS
  1217	BORDETELLA	LANDYARKI	2100	P15318.2
  	PERTUSSIS
  1218	BORDETELLA	LDYLRQAGL	2101	P15318.2
  	PERTUSSIS
  1219	BORDETELLA	LGKGFASL	2102	P15318.2
  	PERTUSSIS
  1220	BORDETELLA	LGKGFASLM	2103	P15318.2
  	PERTUSSIS
  1221	BORDETELLA	LGVDYYDN	2104	P15318.2
  	PERTUSSIS
  1222	BORDETELLA	LGVDYYDNV	2105	P15318.2
  	PERTUSSIS
  1223	BORDETELLA	LKHSIKLDVI	2106	P15318.2
  	PERTUSSIS
  1224	BORDETELLA	LQAGYIPV	2107	P15318.2
  	PERTUSSIS
  1225	BORDETELLA	LQLTGGTVE	2108	P15318.2
  	PERTUSSIS
  1226	BORDETELLA	LSAAVFGL	2109	P15318.2
  	PERTUSSIS
  1227	BORDETELLA	LSLGKGFASL	2110	P15318.2
  	PERTUSSIS
  1228	BORDETELLA	LSPMEIYGL	2111	P15318.2
  	PERTUSSIS
  1229	BORDETELLA	NAHDNFLAGG	2112	P15318.2
  	PERTUSSIS
  1230	BORDETELLA	NANVYREGV	2113	P15318.2
  	PERTUSSIS
  1231	BORDETELLA	NDTLYGGL	2114	P15318.2
  	PERTUSSIS
  1232	BORDETELLA	NGLAGNDVL	2115	P15318.2
  	PERTUSSIS
  1233	BORDETELLA	NNANVYREGV	2116	P15318.2
  	PERTUSSIS
  1234	BORDETELLA	NTVSYAAL	2117	P15318.2
  	PERTUSSIS
  1235	BORDETELLA	NVLRNIENAV	2118	P15318.2
  	PERTUSSIS
  1236	BORDETELLA	PALTFITPL	2119	P15318.2
  	PERTUSSIS
  1237	BORDETELLA	PETSNVLRNI	2120	P15318.2
  	PERTUSSIS
  1238	BORDETELLA	PMEIYGLV	2121	P15318.2
  	PERTUSSIS
  1239	BORDETELLA	PQAYFEKNL	2122	P15318.2
  	PERTUSSIS
  1240	BORDETELLA	PVNPNLSKL	2123	P15318.2
  	PERTUSSIS
  1241	BORDETELLA	QAGWNASSV	2124	P15318.2
  	PERTUSSIS
  1242	BORDETELLA	QAGWNASSVI	2125	P15318.2
  	PERTUSSIS
  1243	BORDETELLA	QDAANAGNL	2126	P15318.2
  	PERTUSSIS
  1244	BORDETELLA	QDAANAGNLL	2127	P15318.2
  	PERTUSSIS
  1245	BORDETELLA	QDSGYDSL	2128	P15318.2
  	PERTUSSIS
  1246	BORDETELLA	QQSHYADQL	2129	P15318.2
  	PERTUSSIS
  1247	BORDETELLA	RALQGAQAV	2130	P15318.2
  	PERTUSSIS
  1248	BORDETELLA	RGGLGLDTL	2131	P15318.2
  	PERTUSSIS
  1249	BORDETELLA	RKQLNNANV	2132	P15318.2
  	PERTUSSIS
  1250	BORDETELLA	RQDSGYDSL	2133	P15318.2
  	PERTUSSIS
  1251	BORDETELLA	RQFRYDGDM	2134	P15318.2
  	PERTUSSIS
  1252	BORDETELLA	RSKFSPDVL	2135	P15318.2
  	PERTUSSIS
  1253	BORDETELLA	SAGAAAGAL	2136	P15318.2
  	PERTUSSIS
  1254	BORDETELLA	SAHWGQRAL	2137	P15318.2
  	PERTUSSIS
  1255	BORDETELLA	SAMIHPGRI	2138	P15318.2
  	PERTUSSIS
  1256	BORDETELLA	SAMIHPGRIV	2139	P15318.2
  	PERTUSSIS
  1257	BORDETELLA	SAYDTVSGI	2140	P15318.2
  	PERTUSSIS
  1258	BORDETELLA	SAYGYEGD	2141	P15318.2
  	PERTUSSIS
  1259	BORDETELLA	SGGAGDDVL	2142	P15318.2
  	PERTUSSIS
  1260	BORDETELLA	SGLQVAGA	2143	P15318.2
  	PERTUSSIS
  1261	BORDETELLA	SGYDSLDGV	2144	P15318.2
  	PERTUSSIS
  1262	BORDETELLA	SLLTGALNGI	2145	P15318.2
  	PERTUSSIS
  1263	BORDETELLA	SPMEIYGL	2146	P15318.2
  	PERTUSSIS
  1264	BORDETELLA	SQMLTRGQL	2147	P15318.2
  	PERTUSSIS
  1265	BORDETELLA	SSAYDTVSGI	2148	P15318.2
  	PERTUSSIS
  1266	BORDETELLA	SSLAHGHTA	2149	P15318.2
  	PERTUSSIS
  1267	BORDETELLA	SSVTSGDSV	2150	P15318.2
  	PERTUSSIS
  1268	BORDETELLA	SVIGVQTTEI	2151	P15318.2
  	PERTUSSIS
  1269	BORDETELLA	TNTVSYAAL	2152	P15318.2
  	PERTUSSIS
  1270	BORDETELLA	TSLIAEGV	2153	P15318.2
  	PERTUSSIS
  1271	BORDETELLA	TSLLTGAL	2154	P15318.2
  	PERTUSSIS
  1272	BORDETELLA	TVPASPGL	2155	P15318.2
  	PERTUSSIS
  1273	BORDETELLA	VAKEKNATL	2156	P15318.2
  	PERTUSSIS
  1274	BORDETELLA	VAPHEYGFGI	2157	P15318.2
  	PERTUSSIS
  1275	BORDETELLA	VAVVTSLL	2158	P15318.2
  	PERTUSSIS
  1276	BORDETELLA	VFYENRAYG	2159	P15318.2
  	PERTUSSIS
  1277	BORDETELLA	VFYENRAYGV	2160	P15318.2
  	PERTUSSIS
  1278	BORDETELLA	VITDFELEV	2161	P15318.2
  	PERTUSSIS
  1279	BORDETELLA	VNPHSTSL	2162	P15318.2
  	PERTUSSIS
  1280	BORDETELLA	VNPHSTSLI	2163	P15318.2
  	PERTUSSIS
  1281	BORDETELLA	VNPNLSKL	2164	P15318.2
  	PERTUSSIS
  1282	BORDETELLA	VNPNLSKLF	2165	P15318.2
  	PERTUSSIS
  1283	BORDETELLA	VQQPIIEKL	2166	P15318.2
  	PERTUSSIS
  1284	BORDETELLA	VQYRHVEL	2167	P15318.2
  	PERTUSSIS
  1285	BORDETELLA	VSIAAAASV	2168	P15318.2
  	PERTUSSIS
  1286	BORDETELLA	VSIAAAASVV	2169	P15318.2
  	PERTUSSIS
  1287	BORDETELLA	VTSLLTGAL	2170	P15318.2
  	PERTUSSIS
  1288	BORDETELLA	VVLANASRI	2171	P15318.2
  	PERTUSSIS
  1289	BORDETELLA	WPALNLFSV	2172	P15318.2
  	PERTUSSIS
  1290	BORDETELLA	WVRKASAL	2173	P15318.2
  	PERTUSSIS
  1291	BORDETELLA	YAVQYRRKGG	2174	P15318.2
  	PERTUSSIS
  1292	BORDETELLA	YGGLGDDTL	2175	P15318.2
  	PERTUSSIS
  1293	BORDETELLA	YGLVQQSHYA	2176	P15318.2
  	PERTUSSIS
  1294	BORDETELLA	YGYEGDALL	2177	P15318.2
  	PERTUSSIS
  1295	BORDETELLA	YIPVNPNL	2178	P15318.2
  	PERTUSSIS
  1296	BORDETELLA	YSAMIHPGRI	2179	P15318.2
  	PERTUSSIS
  1297	BORDETELLA	YSQTGAHAGI	2180	P15318.2
  	PERTUSSIS
  1298	CORYNEBACTERIUM	AYNFVESIINLFQVVHNSYN	2181	CAE11230.1
  	DIPHTHERIAE
  1299	BORDETELLA	SGTTIK	2182	BAF35031.1
  	PERTUSSIS
  1300	BORDETELLA	RGHTLESAEGRKIFG	2183	AAA22974.1
  	PERTUSSIS
  1301	BORDETELLA	AGAMTVRDVAAAADLALQAGDA	2184	AAA22974.1
  	PERTUSSIS
  1302	BORDETELLA	AGAMTVRDVAAAADLALQAGDAL	2185	AAA22974.1
  	PERTUSSIS
  1303	BORDETELLA	ALAAVLVNPHIFTRIGAAQTSLADGA	2186	AAA22974.1
  	PERTUSSIS	AGPA
  1304	BORDETELLA	ALSIDSMTALGA	2187	AAA22974.1
  	PERTUSSIS
  1305	BORDETELLA	DLSAARGADISGEGR	2188	AAA22974.1
  	PERTUSSIS
  1306	BORDETELLA	DQNRYEYIWGLY	2189	AAA22974.1
  	PERTUSSIS
  1307	BORDETELLA	DYTVSADAIALA	2190	AAA22974.1
  	PERTUSSIS
  1308	BORDETELLA	GPIVVEAGELVSHAGG	2191	AAA22974.1
  	PERTUSSIS
  1309	BORDETELLA	GRPEGLKIGAHSATSVSGSFDAL	2192	AAA22974.1
  	PERTUSSIS
  1310	BORDETELLA	ITVTSRGGFDNEGKMESNK	2193	AAA22974.1
  	PERTUSSIS
  1311	BORDETELLA	LDQNRYEYIWGLYP	2194	AAA22974.1
  	PERTUSSIS
  1312	BORDETELLA	LSAARGADISG	2195	AAA22974.1
  	PERTUSSIS
  1313	BORDETELLA	NKIRLMGPLQ	2196	AAA22974.1
  	PERTUSSIS
  1314	BORDETELLA	NKLGRIRAGEDM	2197	AAA22974.1
  	PERTUSSIS
  1315	BORDETELLA	NKLGRIRAGEDMHLDAPRIE	2198	AAA22974.1
  	PERTUSSIS
  1316	BORDETELLA	PHLRNTGQVVAG	2199	AAA22974.1
  	PERTUSSIS
  1317	BORDETELLA	QVDLHDLSAARGADISG	2200	AAA22974.1
  	PERTUSSIS
  1318	BORDETELLA	RDVAAAADLALQ	2201	AAA22974.1
  	PERTUSSIS
  1319	BORDETELLA	SAARGADISGEG	2202	AAA22974.1
  	PERTUSSIS
  1320	BORDETELLA	TKGEMQIAGKGGGSP	2203	AAA22974.1
  	PERTUSSIS
  1321	BORDETELLA	TVSADAIALAAQ	2204	AAA22974.1
  	PERTUSSIS
  1322	BORDETELLA	VVAGHDIHI	2205	AAA22974.1
  	PERTUSSIS
  1323	BORDETELLA	PSGPNHTKVVQLPKISKNALKANG	2206	CAD12823.1
  	PERTUSSIS
  1324	RUBELLA VIRUS	LVGATPERPRLRLVDADDPLLRTAPG	2207	BAA19902.1
  		PGEVWVTPVIGSQAR
  1325	RUBELLA VIRUS	QQSRWGLGSPNCHGPDWASPVCQRH	2208	BAA19902.1
  		SP
  1326	BORDETELLA	AGEAMVLVYYESIAYSF	2209	ACI04548.1
  	PERTUSSIS
  1327	BORDETELLA	GGVGLASTLWYAESNALSKRLGEL	2210	AAZ74322.1
  	PERTUSSIS
  1328	BORDETELLA	GTLVRIAPVIGACMARQA	2211	ACI04548.1
  	PERTUSSIS
  1329	BORDETELLA	IRRVTRVYHNGITGETTT	2212	ACI04548.1
  	PERTUSSIS
  1330	BORDETELLA	IVKTGERQHGIHIQGSDP	2213	AAZ74322.1
  	PERTUSSIS
  1331	BORDETELLA	IVKTGERQHGIHIQGSDPGGVRTA	2214	AAZ74338.1
  	PERTUSSIS
  1332	BORDETELLA	LRDTNVTAVPASGAPAAVSVLGAS	2215	AAZ74338.1
  	PERTUSSIS
  1333	BORDETELLA	PEAPAPQPPAGRELSAAANAAVNT	2216	AAZ74322.1
  	PERTUSSIS
  1334	BORDETELLA	AAADFAHAE	2217	WP_019247158.1
  	PERTUSSIS
  1335	BORDETELLA	AAAEVAGAL	2218	WP_019249248.1
  	PERTUSSIS
  1336	BORDETELLA	AAESTFESY	2219	WP_019247158.1
  	PERTUSSIS
  1337	BORDETELLA	AAGFDPEVQ	2220	WP_019248145.1
  	PERTUSSIS
  1338	BORDETELLA	AALGRGHSL	2221	AGS56996.1
  	PERTUSSIS
  1339	BORDETELLA	AAMQGAVVH	2222	AGT50936.1
  	PERTUSSIS
  1340	BORDETELLA	AAPAAHADW	2223	AG556996.1
  	PERTUSSIS
  1341	BORDETELLA	AAQATVVQR	2224	WP_019247158.1
  	PERTUSSIS
  1342	BORDETELLA	AARVAGDNY	2225	WP_019249248.1
  	PERTUSSIS
  1343	BORDETELLA	AAVALLNKL	2226	WP_019249248.1
  	PERTUSSIS
  1344	BORDETELLA	ADDPPATVY	2227	AAW72734.1
  	PERTUSSIS
  1345	BORDETELLA	AEAGRFKVL	2228	AGS56996.1
  	PERTUSSIS
  1346	BORDETELLA	AEATQLVTA	2229	WP_019247158.1
  	PERTUSSIS
  1347	BORDETELLA	AEGGATLGA	2230	WP_019249248.1
  	PERTUSSIS
  1348	BORDETELLA	AEHGEVSIQ	2231	WP_019249248.1
  	PERTUSSIS
  1349	BORDETELLA	AEIAFYPKE	2232	WP_019249248.1
  	PERTUSSIS
  1350	BORDETELLA	AEKVTTPAV	2233	WP_019247158.1
  	PERTUSSIS
  1351	BORDETELLA	AELQTYLRQ	2234	1BCP_C
  	PERTUSSIS
  1352	BORDETELLA	AEQSLIEVG	2235	WP_019249248.1
  	PERTUSSIS
  1353	BORDETELLA	AESNALSKR	2236	AGS56996.1
  	PERTUSSIS
  1354	BORDETELLA	AESSEAMAA	2237	AFK26302.1
  	PERTUSSIS
  1355	BORDETELLA	AEVKVGYRA	2238	WP_019247158.1
  	PERTUSSIS
  1356	BORDETELLA	AEVTDTSPS	2239	WP_019249248.1
  	PERTUSSIS
  1357	BORDETELLA	AGKSLKKKN	2240	WP_019247158.1
  	PERTUSSIS
  1358	BORDETELLA	AGLAGPSAV	2241	WP_019249248.1
  	PERTUSSIS
  1359	BORDETELLA	AIRVGRGAR	2242	AGT50936.1
  	PERTUSSIS
  1360	BORDETELLA	ALAAIASAA	2243	WP_019248145.1
  	PERTUSSIS
  1361	BORDETELLA	ALADVPYVL	2244	AAA22983.1
  	PERTUSSIS
  1362	BORDETELLA	ALANDGTIV	2245	WP_019248658.1
  	PERTUSSIS
  1363	BORDETELLA	ALGRGHSLY	2246	AGS56996.1
  	PERTUSSIS
  1364	BORDETELLA	ALILAASPV	2247	WP_019248658.1
  	PERTUSSIS
  1365	BORDETELLA	ALMLACTGL	2248	AAA22974.1
  	PERTUSSIS
  1366	BORDETELLA	AMQGAVVHL	2249	AGT50936.1
  	PERTUSSIS
  1367	BORDETELLA	AMTHLSPAL	2250	AAA22983.1
  	PERTUSSIS
  1368	BORDETELLA	AMYGKHITL	2251	WP_019249248.1
  	PERTUSSIS
  1369	BORDETELLA	ANEANALLW	2252	WP_019249248.1
  	PERTUSSIS
  1370	BORDETELLA	APLSITLQA	2253	AGT50936.1
  	PERTUSSIS
  1371	BORDETELLA	APNALAWAL	2254	AAA22974.1
  	PERTUSSIS
  1372	BORDETELLA	APPAPKPAP	2255	AGS56996.1
  	PERTUSSIS
  1373	BORDETELLA	APPGAGFIY	2256	1BCP_C
  	PERTUSSIS
  1374	BORDETELLA	APQAAPLSI	2257	AGT50936.1
  	PERTUSSIS
  1375	BORDETELLA	APRIENTAK	2258	WP_019249248.1
  	PERTUSSIS
  1376	BORDETELLA	AQGKALLYR	2259	AGT50936.1
  	PERTUSSIS
  1377	BORDETELLA	AQITSYVGF	2260	WP_019248658.1
  	PERTUSSIS
  1378	BORDETELLA	AQLEVRGQR	2261	WP_019249248.1
  	PERTUSSIS
  1379	BORDETELLA	AQQLKQADR	2262	WP_019247699.1
  	PERTUSSIS
  1380	BORDETELLA	AQVTVAGRY	2263	WP_019249248.1
  	PERTUSSIS
  1381	BORDETELLA	ARRSRVRAL	2264	NP_882284.1
  	PERTUSSIS
  1382	BORDETELLA	ASPRRARRA	2265	WP_019249248.1
  	PERTUSSIS
  1383	BORDETELLA	ASSPDAHVP	2266	AAA22983.1
  	PERTUSSIS
  1384	BORDETELLA	ASVSNPGTF	2267	WP_019249248.1
  	PERTUSSIS
  1385	BORDETELLA	ATWNFQSTY	2268	WP_019249248.1
  	PERTUSSIS
  1386	BORDETELLA	ATYIADSGF	2269	AGS56996.1
  	PERTUSSIS
  1387	BORDETELLA	AVAAPAVGA	2270	WP_019249248.1
  	PERTUSSIS
  1388	BORDETELLA	AVFMQQRPL	2271	AAA22983.1
  	PERTUSSIS
  1389	BORDETELLA	AVLVNPHIF	2272	WP_019249248.1
  	PERTUSSIS
  1390	BORDETELLA	CFGKDLKRP	2273	AAA22983.1
  	PERTUSSIS
  1391	BORDETELLA	CPSSLGNGV	2274	WP_019248145.1
  	PERTUSSIS
  1392	BORDETELLA	DAGHEHDTW	2275	AAA22984.1
  	PERTUSSIS
  1393	BORDETELLA	DAKHDLTVT	2276	WP_019249248.1
  	PERTUSSIS
  1394	BORDETELLA	DASGQHRLW	2277	AGS56996.1
  	PERTUSSIS
  1395	BORDETELLA	DATFETYAL	2278	YP_006628018.1
  	PERTUSSIS
  1396	BORDETELLA	DATFQTYAL	2279	1BCP_C
  	PERTUSSIS
  1397	BORDETELLA	DATLVGAKF	2280	WP_019247158.1
  	PERTUSSIS
  1398	BORDETELLA	DDEVDVSGR	2281	AAA22974.1
  	PERTUSSIS
  1394	BORDETELLA	DENGKPQTY	2282	WP_019247158.1
  	PERTUSSIS
  1400	BORDETELLA	DGPPSRPTT	2283	WP_019247158.1
  	PERTUSSIS
  1401	BORDETELLA	DHLTGRSCQ	2284	AAW72734.1
  	PERTUSSIS
  1402	BORDETELLA	DNEGKMESN	2285	WP_019249248.1
  	PERTUSSIS
  1403	BORDETELLA	DPPATVYRY	2286	AAW72734.1
  	PERTUSSIS
  1404	BORDETELLA	EATEGDATL	2287	WP_019247158.1
  	PERTUSSIS
  1405	BORDETELLA	EATQQAAGF	2288	WP_019248145.1
  	PERTUSSIS
  1406	BORDETELLA	ECSGKQDCP	2289	AAA22983.1
  	PERTUSSIS
  1407	BORDETELLA	EGGKLRGKD	2290	AAA22974.1
  	PERTUSSIS
  1408	BORDETELLA	EGKMESNKD	2291	WP_019249248.1
  	PERTUSSIS
  1409	BORDETELLA	EHRMQEAVE	2292	AAW72734.1
  	PERTUSSIS
  1410	BORDETELLA	EKRLDIDDA	2293	WP_019249248.1
  	PERTUSSIS
  1411	BORDETELLA	EPASANTLL	2294	AGS56996.1
  	PERTUSSIS
  1412	BORDETELLA	EPQAELAVF	2295	AGS56996.1
  	PERTUSSIS
  1413	BORDETELLA	EPVKLTLTG	2296	AGT50936.1
  	PERTUSSIS
  1414	BORDETELLA	ESAEGRKIF	2297	WP_019249248.1
  	PERTUSSIS
  1415	BORDETELLA	ESYSESHNF	2298	WP_019247158.1
  	PERTUSSIS
  1416	BORDETELLA	ETFCITTIY	2299	1BCP_C
  	PERTUSSIS
  1417	BORDETELLA	EVAGALELS	2300	WP_019249248.1
  	PERTUSSIS
  1418	BORDETELLA	EVAKVEVVP	2301	WP_019247158.1
  	PERTUSSIS
  1419	BORDETELLA	EVDGIIQEF	2302	WP_019249248.1
  	PERTUSSIS
  1420	BORDETELLA	EVRADNNFY	2303	WP_019248344.1
  	PERTUSSIS
  1421	BORDETELLA	FAILSSTTE	2304	WP_019247158.1
  	PERTUSSIS
  1422	BORDETELLA	FAISAYALK	2305	AAA22984.1
  	PERTUSSIS
  1423	BORDETELLA	FALYDGTYL	2306	AFK26303.1
  	PERTUSSIS
  1424	BORDETELLA	FDTMLGFAI	2307	AAA22984.1
  	PERTUSSIS
  1425	BORDETELLA	FEGKPALEL	2308	AAA22983.1
  	PERTUSSIS
  1426	BORDETELLA	FELGADHAV	2309	AGS56996.1
  	PERTUSSIS
  1427	BORDETELLA	FEPGITTNY	2310	WP_019248658.1
  	PERTUSSIS
  1428	BORDETELLA	FETYALTGI	2311	YP_006628018.1
  	PERTUSSIS
  1429	BORDETELLA	FIYRETFCI	2312	1BCP_C
  	PERTUSSIS
  1430	BORDETELLA	FPTRTTAPG	2313	NP_882284.1
  	PERTUSSIS
  1431	BORDETELLA	FQTYALTGI	2314	1BCP_C
  	PERTUSSIS
  1432	BORDETELLA	FTHADGWFL	2315	AGS56996.1
  	PERTUSSIS
  1433	BORDETELLA	FVRDGQSVI	2316	1BCP_C
  	PERTUSSIS
  1434	BORDETELLA	FVRSGQPVI	2317	YP_006628018.1
  	PERTUSSIS
  1435	BORDETELLA	FVWYVDTVI	2318	WP_019248866.1
  	PERTUSSIS
  1436	BORDETELLA	GAASSRQAL	2319	WP_019249248.1
  	PERTUSSIS
  1437	BORDETELLA	GAASSYFEY	2320	AAW72734.1
  	PERTUSSIS
  1438	BORDETELLA	GAFDLKTTF	2321	AFK26303.1
  	PERTUSSIS
  1439	BORDETELLA	GAPAAVSVL	2322	AGS56996.1
  	PERTUSSIS
  1440	BORDETELLA	GATRAVDSL	2323	AGS56996.1
  	PERTUSSIS
  1441	BORDETELLA	GAVPGGAVP	2324	AGT50936.1
  	PERTUSSIS
  1442	BORDETELLA	GEAMVLVYY	2325	AFK26302.1
  	PERTUSSIS
  1443	BORDETELLA	GEIALGDAT	2326	WP_019249248.1
  	PERTUSSIS
  1444	BORDETELLA	GELMAAQVA	2327	WP_019247158.1
  	PERTUSSIS
  1445	BORDETELLA	GGVPGGAVP	2328	AAZ74338.1
  	PERTUSSIS
  1446	BORDETELLA	GHEHDTWFD	2329	AAA22984.1
  	PERTUSSIS
  1447	BORDETELLA	GIGALKAGA	2330	WP_019249248.1
  	PERTUSSIS
  1448	BORDETELLA	GIVIPPKAL	2331	1BCP_C
  	PERTUSSIS
  1449	BORDETELLA	GKDLKRPGS	2332	AAA22983.1
  	PERTUSSIS
  1450	BORDETELLA	GKLPKPVTV	2333	WP_019247158.1
  	PERTUSSIS
  1451	BORDETELLA	GKSLKKKNQ	2334	WP_019247158.1
  	PERTUSSIS
  1452	BORDETELLA	GLDVQQGTV	2335	WP_019249248.1
  	PERTUSSIS
  1453	BORDETELLA	GLTDGVSRI	2336	WP_019249248.1
  	PERTUSSIS
  1454	BORDETELLA	GLYPTYTEW	2337	WP_019249248.1
  	PERTUSSIS
  1455	BORDETELLA	GLYQTYTEW	2338	YP_006626470.1
  	PERTUSSIS
  1456	BORDETELLA	GPPSRPTTP	2339	WP_019247158.1
  	PERTUSSIS
  1457	BORDETELLA	GPSAVAAPA	2340	WP_019249248.1
  	PERTUSSIS
  1458	BORDETELLA	GVAPTAQQL	2341	WP_019248866.1
  	PERTUSSIS
  1459	BORDETELLA	GVGLASTLW	2342	AGS56996.1
  	PERTUSSIS
  1460	BORDETELLA	GYEAGFSLG	2343	WP_019247158.1
  	PERTUSSIS
  1461	BORDETELLA	HADDGTIVI	2344	WP_019248145.1
  	PERTUSSIS
  1462	BORDETELLA	HADWNNQSI	2345	AGS56996.1
  	PERTUSSIS
  1463	BORDETELLA	HAEHEKDVR	2346	WP_019247158.1
  	PERTUSSIS
  1464	BORDETELLA	HANHYGTRI	2347	WP_019247158.1
  	PERTUSSIS
  1465	BORDETELLA	HAQGKALLY	2348	AGT50936.1
  	PERTUSSIS
  1466	BORDETELLA	HFIGYIYEV	2349	AAW72734.1
  	PERTUSSIS
  1467	BORDETELLA	HLSPALADV	2350	AAA22983.1
  	PERTUSSIS
  1468	BORDETELLA	HSLYASYEY	2351	AGS56996.1
  	PERTUSSIS
  1469	BORDETELLA	HVRGMLVPV	2352	AAA22974.1
  	PERTUSSIS
  1470	BORDETELLA	HVSKEEQYY	2353	YP_006628018.1
  	PERTUSSIS
  1471	BORDETELLA	HVTRGWSIF	2354	AFK26303.1
  	PERTUSSIS
  1472	BORDETELLA	IADSGFYLD	2355	AGS56996.1
  	PERTUSSIS
  1473	BORDETELLA	IAHRTELRG	2356	AGS56996.1
  	PERTUSSIS
  1474	BORDETELLA	IENTAKLSG	2357	WP_019249248.1
  	PERTUSSIS
  1475	BORDETELLA	IESKISQSV	2358	WP_019249248.1
  	PERTUSSIS
  1476	BORDETELLA	IETGGARRF	2359	AGT50936.1
  	PERTUSSIS
  1477	BORDETELLA	IIKDAPPGA	2360	1BCP_C
  	PERTUSSIS
  1478	BORDETELLA	IIQEFAADL	2361	WP_019249248.1
  	PERTUSSIS
  1479	BORDETELLA	ILAGALATY	2362	AAW72734.1
  	PERTUSSIS
  1480	BORDETELLA	ILLENPAAE	2363	AGS56996.1
  	PERTUSSIS
  1481	BORDETELLA	ILPILVLAL	2364	NP_882286.1
  	PERTUSSIS
  1482	BORDETELLA	IPFQRALRL	2365	WP_019248145.1
  	PERTUSSIS
  1483	BORDETELLA	ISVRVHVSK	2366	YP_006628018.1
  	PERTUSSIS
  1484	BORDETELLA	ITNETGKTY	2367	WP_019247158.1
  	PERTUSSIS
  1485	BORDETELLA	ITNKRAALI	2368	WP_019249248.1
  	PERTUSSIS
  1486	BORDETELLA	ITSYVGFSV	2369	WP_019248658.1
  	PERTUSSIS
  1487	BORDETELLA	ITVTSRGGF	2370	WP_019249248.1
  	PERTUSSIS
  1488	BORDETELLA	IVIPPKALF	2371	1BCP_C
  	PERTUSSIS
  1489	BORDETELLA	IVVEAGELV	2372	WP_019249248.1
  	PERTUSSIS
  1490	BORDETELLA	KAAKSVNLM	2373	WP_019247158.1
  	PERTUSSIS
  1491	BORDETELLA	KAAPLRRTT	2374	AGS56996.1
  	PERTUSSIS
  1492	BORDETELLA	KAGKLSATG	2375	WP_019249248.1
  	PERTUSSIS
  1493	BORDETELLA	KAGTIAAPW	2376	WP_019249248.1
  	PERTUSSIS
  1494	BORDETELLA	KAKSLTTEI	2377	WP_019249248.1
  	PERTUSSIS
  1495	BORDETELLA	KATVTTVQV	2378	WP_019247158.1
  	PERTUSSIS
  1496	BORDETELLA	KDYRDKDGG	2379	WP_019247158.1
  	PERTUSSIS
  1497	BORDETELLA	KEAATIVAA	2380	WP_019249248.1
  	PERTUSSIS
  1498	BORDETELLA	KEDVDAAQI	2381	WP_019248658.1
  	PERTUSSIS
  1499	BORDETELLA	KEVDGIIQE	2382	WP_019249248.1
  	PERTUSSIS
  1500	BORDETELLA	KGPKLAMPW	2383	AGS56996.1
  	PERTUSSIS
  1501	BORDETELLA	KLASGGGAV	2384	WP_019249248.1
  	PERTUSSIS
  1502	BORDETELLA	KLKGKNQEF	2385	AAA22984.1
  	PERTUSSIS
  1503	BORDETELLA	KLLHHILPI	2386	NP_882286.1
  	PERTUSSIS
  1504	BORDETELLA	KPAPQPGPQ	2387	AGS56996.1
  	PERTUSSIS
  1505	BORDETELLA	KPAPTAPPM	2388	WP_019249248.1
  	PERTUSSIS
  1506	BORDETELLA	KPAVSVKVA	2389	WP_019249248.1
  	PERTUSSIS
  1507	BORDETELLA	KPDRAARVA	2390	WP_019249248.1
  	PERTUSSIS
  1508	BORDETELLA	KPLADIAVI	2391	YP_006626470.1
  	PERTUSSIS
  1509	BORDETELLA	KPLADIAVV	2392	WP_019249248.1
  	PERTUSSIS
  1510	BORDETELLA	KPLPKPLPV	2393	WP_019247158.1
  	PERTUSSIS
  1511	BORDETELLA	KQADRDFVW	2394	WP_019247699.1
  	PERTUSSIS
  1512	BORDETELLA	KSLPGGKLP	2395	WP_019247158.1
  	PERTUSSIS
  1513	BORDETELLA	KSYTLRYLA	2396	WP_019248658.1
  	PERTUSSIS
  1514	BORDETELLA	KTNMVVTSV	2397	AAA22983.1
  	PERTUSSIS
  1515	BORDETELLA	KVLAPRLYL	2398	AAA22974.1
  	PERTUSSIS
  1516	BORDETELLA	KVLSTKTTL	2399	WP_019247158.1
  	PERTUSSIS
  1517	BORDETELLA	LAAGAGLTL	2400	WP_019249248.1
  	PERTUSSIS
  1518	BORDETELLA	LAAIASAAH	2401	WP_019248145.1
  	PERTUSSIS
  1519	BORDETELLA	LAANGNGQW	2402	AGS56996.1
  	PERTUSSIS
  1520	BORDETELLA	LAAQVTQRG	2403	WP_019249248.1
  	PERTUSSIS
  1521	BORDETELLA	LAARGDGAL	2404	AAA22974.1
  	PERTUSSIS
  1522	BORDETELLA	LAAVLVNPH	2405	WP_019249248.1
  	PERTUSSIS
  1523	BORDETELLA	LAGSGLFRM	2406	AGS56996.1
  	PERTUSSIS
  1524	BORDETELLA	LAKALSAAL	2407	WP_019249248.1
  	PERTUSSIS
  1525	BORDETELLA	LALQAGDAL	2408	WP_019249248.1
  	PERTUSSIS
  1526	BORDETELLA	LAMPWTFHA	2409	AGS56996.1
  	PERTUSSIS
  1527	BORDETELLA	LANDGTIVI	2410	WP_019248658.1
  	PERTUSSIS
  1528	BORDETELLA	LAPTVGVAF	2411	WP_019247158.1
  	PERTUSSIS
  1529	BORDETELLA	LASDGSVDF	2412	AGS56996.1
  	PERTUSSIS
  1530	BORDETELLA	LATYQSEYL	2413	AAW72734.1
  	PERTUSSIS
  1531	BORDETELLA	LAWALMLAC	2414	AAA22974.1
  	PERTUSSIS
  1532	BORDETELLA	LEAGRRFTH	2415	AGS56996.1
  	PERTUSSIS
  1533	BORDETELLA	LFTQQGGAY	2416	1BCP_C
  	PERTUSSIS
  1534	BORDETELLA	LKRPGSSPM	2417	AAA22983.1
  	PERTUSSIS
  1535	BORDETELLA	LLGSHVARA	2418	AFK26303.1
  	PERTUSSIS
  1536	BORDETELLA	LLHHILPIL	2419	NP_882286.1
  	PERTUSSIS
  1537	BORDETELLA	LLNAGGTLL	2420	WP_019249248.1
  	PERTUSSIS
  1538	BORDETELLA	LMGPLQVNA	2421	WP_019249248.1
  	PERTUSSIS
  1539	BORDETELLA	LNDSKITMG	2422	WP_019248145.1
  	PERTUSSIS
  1540	BORDETELLA	LPEPVKLTL	2423	AGT50936.1
  	PERTUSSIS
  1541	BORDETELLA	LPILVLALL	2424	NP_882286.1
  	PERTUSSIS
  1542	BORDETELLA	LPKISKNAL	2425	WP_019248145.1
  	PERTUSSIS
  1543	BORDETELLA	LPKPVTVKL	2426	WP_019247158.1
  	PERTUSSIS
  1544	BORDETELLA	LPLKANPMH	2427	NP_882285.1
  	PERTUSSIS
  1545	BORDETELLA	LPPRPVVAE	2428	WP_019247158.1
  	PERTUSSIS
  1546	BORDETELLA	LPS1PGTSI	2429	AGS56996.1
  	PERTUSSIS
  1547	BORDETELLA	LPTHLYKNF	2430	AAA22984.1
  	PERTUSSIS
  1548	BORDETELLA	LPVRGVALV	2431	WP_019249248.1
  	PERTUSSIS
  1549	BORDETELLA	LPVSLTALD	2432	WP_019249248.1
  	PERTUSSIS
  1550	BORDETELLA	LQGGNKVPV	2433	WP_019249248.1
  	PERTUSSIS
  1551	BORDETELLA	LSAALGADW	2434	WP_019249248.1
  	PERTUSSIS
  1552	BORDETELLA	LSDAGHEHD	2435	AAA22984.1
  	PERTUSSIS
  1553	BORDETELLA	LSGEVQRKG	2436	WP_019249248.1
  	PERTUSSIS
  1554	BORDETELLA	LSSPSAITV	2437	WP_019249248.1
  	PERTUSSIS
  1555	BORDETELLA	LTWLAILAV	2438	AAW72734.1
  	PERTUSSIS
  1556	BORDETELLA	LVFSHVRGM	2439	AAA22974.1
  	PERTUSSIS
  1557	BORDETELLA	LVSDAGADL	2440	WP_019249248.1
  	PERTUSSIS
  1558	BORDETELLA	LVYYESIAY	2441	AFK26302.1
  	PERTUSSIS
  1559	BORDETELLA	MAAESTFES	2442	WP_019247158.1
  	PERTUSSIS
  1560	BORDETELLA	MAAGHDATL	2443	WP_019249248.1
  	PERTUSSIS
  1561	BORDETELLA	MAAWSERAG	2444	AFK26302.1
  	PERTUSSIS
  1562	BORDETELLA	MALGALGAA	2445	AGS56996.1
  	PERTUSSIS
  1563	BORDETELLA	MAPVMGACM	2446	ADA85123.1
  	PERTUSSIS
  1564	BORDETELLA	MATKGEMQI	2447	WP_019249248.1
  	PERTUSSIS
  1565	BORDETELLA	MDAKGGTLL	2448	WP_019249248.1
  	PERTUSSIS
  1566	BORDETELLA	MESNKDIVI	2449	WP_019249248.1
  	PERTUSSIS
  1567	BORDETELLA	MEVMLRAVF	2450	AAA22983.1
  	PERTUSSIS
  1568	BORDETELLA	MEYFKTPLP	2451	WP_019249248.1
  	PERTUSSIS
  1569	BORDETELLA	MHTIASILL	2452	AAA22984.1
  	PERTUSSIS
  1570	BORDETELLA	MIYMSGLAV	2453	1BCP_C
  	PERTUSSIS
  1571	BORDETELLA	MLACTGLPL	2454	AAA22974.1
  	PERTUSSIS
  1572	BORDETELLA	MPIDRKTLC	2455	AFK26303.1
  	PERTUSSIS
  1573	BORDETELLA	MPKAPELDL	2456	WP_019249248.1
  	PERTUSSIS
  1574	BORDETELLA	MQRQAGLPL	2457	NP_882285.1
  	PERTUSSIS
  1575	BORDETELLA	NALAWALML	2458	AAA22974.1
  	PERTUSSIS
  1576	BORDETELLA	NITNKRAAL	2459	WP_019249248.1
  	PERTUSSIS
  1577	BORDETELLA	NLMAAESTF	2460	WP_019247158.1
  	PERTUSSIS
  1578	BORDETELLA	NNETMSGRQ	2461	WP_019249248.1
  	PERTUSSIS
  1579	BORDETELLA	NPGSLIAEV	2462	WP_019249248.1
  	PERTUSSIS
  1580	BORDETELLA	NPMHTIASI	2463	NP_882285.1
  	PERTUSSIS
  1581	BORDETELLA	NPQTQLSNI	2464	WP_019248145.1
  	PERTUSSIS
  1582	BORDETELLA	NPYTSRRSV	2465	AFK26302.1
  	PERTUSSIS
  1583	BORDETELLA	PAPTAPPMP	2466	WP_019249248.1
  	PERTUSSIS
  1584	BORDETELLA	PASANTLLL	2467	AGS56996.1
  	PERTUSSIS
  1585	BORDETELLA	PAVALPRPL	2468	WP_019249248.1
  	PERTUSSIS
  1586	BORDETELLA	PDAHVPFCF	2469	AAA22983.1
  	PERTUSSIS
  1587	BORDETELLA	PELGAAIRV	2470	AGT50936.1
  	PERTUSSIS
  1588	BORDETELLA	PFIIKLKDC	2471	WP_019248145.1
  	PERTUSSIS
  1589	BORDETELLA	PGPQPPQPP	2472	AGS56996.1
  	PERTUSSIS
  1590	BORDETELLA	PGPQPPQPQ	2473	AAZ74338.1
  	PERTUSSIS
  1591	BORDETELLA	PGPTTDYST	2474	WP_019248145.1
  	PERTUSSIS
  1592	BORDETELLA	PGTFTAGKD	2475	WP_019249248.1
  	PERTUSSIS
  1593	BORDETELLA	PGTPGDLLE	2476	AAA22984.1
  	PERTUSSIS
  1594	BORDETELLA	PKPKPKAER	2477	WP_019247158.1
  	PERTUSSIS
  1595	BORDETELLA	PKPKPKPKA	2478	WP_019247158.1
  	PERTUSSIS
  1596	BORDETELLA	PKPKPKPKP	2479	WP_019247158.1
  	PERTUSSIS
  1597	BORDETELLA	PLPPRPVVA	2480	WP_019247158.1
  	PERTUSSIS
  1598	BORDETELLA	PPAPKPAPQ	2481	AGS56996.1
  	PERTUSSIS
  1599	BORDETELLA	PPKPAPVAK	2482	WP_019247158.1
  	PERTUSSIS
  1600	BORDETELLA	PPRPVAAQV	2483	WP_019247158.1
  	PERTUSSIS
  1601	BORDETELLA	PPRPVVAEK	2484	WP_019247158.1
  	PERTUSSIS
  1602	BORDETELLA	PPSRPTTPP	2485	WP_019247158.1
  	PERTUSSIS
  1603	BORDETELLA	PRRARRALR	2486	WP_019249248.1
  	PERTUSSIS
  1604	BORDETELLA	QAAPLSITL	2487	AGT50936.1
  	PERTUSSIS
  1605	BORDETELLA	QADRDFVWY	2488	WP_019247699.1
  	PERTUSSIS
  1606	BORDETELLA	QAIVVGKDL	2489	WP_019249248.1
  	PERTUSSIS
  1607	BORDETELLA	QALGALKLY	2490	ACI16088.1
  	PERTUSSIS
  1608	BORDETELLA	QELALKLKG	2491	AAA22984.1
  	PERTUSSIS
  1609	BORDETELLA	QITQHGGPY	2492	NP_882283.1
  	PERTUSSIS
  1610	BORDETELLA	QITQHGSPY	2493	AFK26303.1
  	PERTUSSIS
  1611	BORDETELLA	QPLPPRPVA	2494	WP_019247158.1
  	PERTUSSIS
  1612	BORDETELLA	QPPAGRELS	2495	AGS56996.1
  	PERTUSSIS
  1613	BORDETELLA	QQLKQADRD	2496	WP_019247699.1
  	PERTUSSIS
  1614	BORDETELLA	QQVQVLQRQ	2497	WP_019247158.1
  	PERTUSSIS
  1615	BORDETELLA	QSIVEAPEL	2498	AGT50936.1
  	PERTUSSIS
  1616	BORDETELLA	QVGSSNSAF	2499	AAW72734.1
  	PERTUSSIS
  1617	BORDETELLA	RAGLSPATW	2500	WP_019249248.1
  	PERTUSSIS
  1618	BORDETELLA	RARRALRQD	2501	WP_019249248.1
  	PERTUSSIS
  1619	BORDETELLA	RASASRARI	2502	WP_019249248.1
  	PERTUSSIS
  1620	BORDETELLA	RELSAAANA	2503	AGS56996.1
  	PERTUSSIS
  1621	BORDETELLA	RETFCITTI	2504	1BCP_C
  	PERTUSSIS
  1622	BORDETELLA	RGFAQRQQL	2505	AGS56996.1
  	PERTUSSIS
  1623	BORDETELLA	RGSAATFTL	2506	WP_019248295.1
  	PERTUSSIS
  1624	BORDETELLA	RGWSIFALY	2507	AFK26303.1
  	PERTUSSIS
  1625	BORDETELLA	RKMLYLIYV	2508	YP_006628018.1
  	PERTUSSIS
  1626	BORDETELLA	RLRKMLYLI	2509	YP_006628018.1
  	PERTUSSIS
  1627	BORDETELLA	RPQITDAVT	2510	WP_019249248.1
  	PERTUSSIS
  1628	BORDETELLA	RPSVNGGRI	2511	WP_019249248.1
  	PERTUSSIS
  1629	BORDETELLA	RRFTHADGW	2512	AGS56996.1
  	PERTUSSIS
  1630	BORDETELLA	RSGARATSL	2513	AAA22974.1
  	PERTUSSIS
  1631	BORDETELLA	RSRVRALAW	2514	NP_882284.1
  	PERTUSSIS
  1632	BORDETELLA	RSRVRALSW	2515	YP_006628019.1
  	PERTUSSIS
  1633	BORDETELLA	RTHGVGASL	2516	AGS56996.1
  	PERTUSSIS
  1634	BORDETELLA	RTRGQARSG	2517	AAA22974.1
  	PERTUSSIS
  1635	BORDETELLA	RVAPPAVAL	2518	WP_019249248.1
  	PERTUSSIS
  1636	BORDETELLA	RVLPEPVKL	2519	AGT50936.1
  	PERTUSSIS
  1637	BORDETELLA	RVRALAWLL	2520	NP_882284.1
  	PERTUSSIS
  1638	BORDETELLA	RVRALSWLL	2521	YP_006628019.1
  	PERTUSSIS
  1639	BORDETELLA	RVTVSGGSL	2522	AGT50936.1
  	PERTUSSIS
  1640	BORDETELLA	SEAMAAWSE	2523	AFK26302.1
  	PERTUSSIS
  1641	BORDETELLA	SESHNFHAS	2524	WP_019247158.1
  	PERTUSSIS
  1642	BORDETELLA	SGEGRVNIG	2525	WP_019249248.1
  	PERTUSSIS
  1643	BORDETELLA	SGLAVRVHV	2526	1BCP_C
  	PERTUSSIS
  1644	BORDETELLA	SLADISLGA	2527	WP_019249248.1
  	PERTUSSIS
  1645	BORDETELLA	SLFAILSST	2528	WP_019247158.1
  	PERTUSSIS
  1646	BORDETELLA	SLFAPHGNV	2529	AAZ74338.1
  	PERTUSSIS
  1647	BORDETELLA	SLSIDNATW	2530	AGS56996.1
  	PERTUSSIS
  1648	BORDETELLA	SPMEVMLRA	2531	AAA22983.1
  	PERTUSSIS
  1649	BORDETELLA	SPQPIRATV	2532	WP_019247158.1
  	PERTUSSIS
  1650	BORDETELLA	SPRRARRAL	2533	WP_019249248.1
  	PERTUSSIS
  1651	BORDETELLA	SPSRLAGTL	2534	WP_019249248.1
  	PERTUSSIS
  1652	BORDETELLA	SSTPLGSLF	2535	WP_019247158.1
  	PERTUSSIS
  1653	BORDETELLA	STYELLDYL	2536	WP_019249248.1
  	PERTUSSIS
  1654	BORDETELLA	SVAMKPYEV	2537	AAA22983.1
  	PERTUSSIS
  1655	BORDETELLA	SVAPNALAW	2538	AAA22974.1
  	PERTUSSIS
  1656	BORDETELLA	SVKVAKKLF	2539	WP_019249248.1
  	PERTUSSIS
  1657	BORDETELLA	TAFMSGRSL	2540	AAA22984.1
  	PERTUSSIS
  1658	BORDETELLA	TAGATPFDI	2541	WP_019248658.1
  	PERTUSSIS
  1659	BORDETELLA	TAPVTSPAW	2542	AAW72734.1
  	PERTUSSIS
  1660	BORDETELLA	TARTGWLTW	2543	AAW72734.1
  	PERTUSSIS
  1661	BORDETELLA	TEAQGVQVR	2544	WP_019248145.1
  	PERTUSSIS
  1662	BORDETELLA	TEVYLEHRM	2545	AAW72734.1
  	PERTUSSIS
  1663	BORDETELLA	TFEGKPALE	2546	AAA22983.1
  	PERTUSSIS
  1664	BORDETELLA	TFTGKVTNG	2547	WP_019248658.1
  	PERTUSSIS
  1665	BORDETELLA	TGDGGGHTD	2548	AGS56996.1
  	PERTUSSIS
  1666	BORDETELLA	TLAKALSAA	2549	WP_019249248.1
  	PERTUSSIS
  1667	BORDETELLA	TLANVGDTW	2550	AGS56996.1
  	PERTUSSIS
  1668	BORDETELLA	TLNASNLTL	2551	WP_019249248.1
  	PERTUSSIS
  1669	BORDETELLA	TLSSAHGNV	2552	WP_019249248.1
  	PERTUSSIS
  1670	BORDETELLA	TPFDIKLKE	2553	WP_019248658.1
  	PERTUSSIS
  1671	BORDETELLA	TPFIIKLKD	2554	WP_019248145.1
  	PERTUSSIS
  1672	BORDETELLA	TPGWSIYGL	2555	1BCP_C
  	PERTUSSIS
  1673	BORDETELLA	TPLGSAATF	2556	AGS56996.1
  	PERTUSSIS
  1674	BORDETELLA	TPLGSLFAI	2557	WP_019247158.1
  	PERTUSSIS
  1675	BORDETELLA	TPLPVSLTA	2558	WP_019249248.1
  	PERTUSSIS
  1676	BORDETELLA	TRQGIMDQY	2559	YP_006626873.1
  	PERTUSSIS
  1677	BORDETELLA	TSKQDERNY	2560	WP_019247158.1
  	PERTUSSIS
  1678	BORDETELLA	TSPYDGKYW	2561	YP_006628018.1
  	PERTUSSIS
  1679	BORDETELLA	TSRRSVASI	2562	AFK26302.1
  	PERTUSSIS
  1680	BORDETELLA	TSRTVTMRY	2563	NP_879898.1
  	PERTUSSIS
  1681	BORDETELLA	TTEYPNARY	2564	ADA85123.1
  	PERTUSSIS
  1682	BORDETELLA	TTEYSNARY	2565	AFK26302.1
  	PERTUSSIS
  1683	BORDETELLA	TTLGLEQTF	2566	WP_019247158.1
  	PERTUSSIS
  1684	BORDETELLA	TVLAAGAGL	2567	WP_019249248.1
  	PERTUSSIS
  1685	BORDETELLA	TVQELALKL	2568	AAA22984.1
  	PERTUSSIS
  1686	BORDETELLA	TVVQRNKHW	2569	WP_019247158.1
  	PERTUSSIS
  1687	BORDETELLA	VAAAADLAL	2570	WP_019249248.1
  	PERTUSSIS
  1688	BORDETELLA	VALASQARW	2571	AG556996.1
  	PERTUSSIS
  1689	BORDETELLA	VAMKPYEVT	2572	AAA22983.1
  	PERTUSSIS
  1690	BORDETELLA	VARLVKLQG	2573	WP_019249248.1
  	PERTUSSIS
  1691	BORDETELLA	VAVAGGRWH	2574	AG556996.1
  	PERTUSSIS
  1692	BORDETELLA	VEASAITTY	2575	WP_019248145.1
  	PERTUSSIS
  1693	BORDETELLA	VEDIGGKNY	2576	WP_019247158.1
  	PERTUSSIS
  1694	BORDETELLA	VEVSSPPPV	2577	WP_019247158.1
  	PERTUSSIS
  1695	BORDETELLA	VGAGGYEAG	2578	WP_019247158.1
  	PERTUSSIS
  1696	BORDETELLA	VGGGEHGRW	2579	WP_019249248.1
  	PERTUSSIS
  1697	BORDETELLA	VHVSKEEQY	2580	YP_006628018.1
  	PERTUSSIS
  1698	BORDETELLA	VIDGQKVLA	2581	AAA22974.1
  	PERTUSSIS
  1699	BORDETELLA	VIGACTSPY	2582	YP_006628018.1
  	PERTUSSIS
  1700	BORDETELLA	VKLGGVYEA	2583	AAA22974.1
  	PERTUSSIS
  1701	BORDETELLA	VLAPRLYLT	2584	AAA22974.1
  	PERTUSSIS
  1702	BORDETELLA	VLVKTNMVV	2585	AAA22983.1
  	PERTUSSIS
  1703	BORDETELLA	VPASGAPAA	2586	AGS56996.1
  	PERTUSSIS
  1704	BORDETELLA	VPFCFGKDL	2587	AAA22983.1
  	PERTUSSIS
  1705	BORDETELLA	VPVSEHCTV	2588	AAA22974.1
  	PERTUSSIS
  1706	BORDETELLA	VPVTPPKVE	2589	WP_019247158.1
  	PERTUSSIS
  1707	BORDETELLA	VRTVSAMEY	2590	WP_019249248.1
  	PERTUSSIS
  1708	BORDETELLA	VSGGSLSAP	2591	AGT50936.1
  	PERTUSSIS
  1709	BORDETELLA	VSSATKAKG	2592	WP_019248658.1
  	PERTUSSIS
  1710	BORDETELLA	VSSPPPVSV	2593	WP_019247158.1
  	PERTUSSIS
  1711	BORDETELLA	VSVKVAKKL	2594	WP_019249248.1
  	PERTUSSIS
  1712	BORDETELLA	VTMRYLASY	2595	WP_019248145.1
  	PERTUSSIS
  1713	BORDETELLA	VTSVAMKPY	2596	AAA22983.1
  	PERTUSSIS
  1714	BORDETELLA	VVAEKVTTP	2597	WP_019247158.1
  	PERTUSSIS
  1715	BORDETELLA	VVDGPPSRP	2598	WP_019247158.1
  	PERTUSSIS
  1716	BORDETELLA	VVETAQPLP	2599	WP_019247158.1
  	PERTUSSIS
  1717	BORDETELLA	WLTWLAILA	2600	AAW72734.1
  	PERTUSSIS
  1718	BORDETELLA	WTFHAGYRY	2601	AGS56996.1
  	PERTUSSIS
  1719	BORDETELLA	WVMTDNSNV	2602	AGS56996.1
  	PERTUSSIS
  1720	BORDETELLA	YAEHGEVSI	2603	WP_019249248.1
  	PERTUSSIS
  1721	BORDETELLA	YAIDGTAAG	2604	WP_019249248.1
  	PERTUSSIS
  1722	BORDETELLA	YALKSRIAL	2605	AAA22984.1
  	PERTUSSIS
  1723	BORDETELLA	YATNPQTQL	2606	WP_019248145.1
  	PERTUSSIS
  1724	BORDETELLA	YDTGDLIAY	2607	WP_019248658.1
  	PERTUSSIS
  1725	BORDETELLA	YEAGFSLGS	2608	WP_019247158.1
  	PERTUSSIS
  1726	BORDETELLA	YEDATFETY	2609	YP_006628018.1
  	PERTUSSIS
  1727	BORDETELLA	YENKSSTPL	2610	WP_019247158.1
  	PERTUSSIS
  1728	BORDETELLA	YEVTPTRML	2611	AAA22983.1
  	PERTUSSIS
  1729	BORDETELLA	YEYIWGLYP	2612	WP_019249248.1
  	PERTUSSIS
  1730	BORDETELLA	YEYIWGLYQ	2613	YP_006626470.1
  	PERTUSSIS
  1731	BORDETELLA	YEYSKGPKL	2614	AGS56996.1
  	PERTUSSIS
  1732	BORDETELLA	YFEPGPTTD	2615	WP_019248145.1
  	PERTUSSIS
  1733	BORDETELLA	YIWGLYPTY	2616	WP_019249248.1
  	PERTUSSIS
  1734	BORDETELLA	YIWGLYQTY	2617	YP_006626470.1
  	PERTUSSIS
  1735	BORDETELLA	YLRQITPGW	2618	1BCP_C
  	PERTUSSIS
  1736	BORDETELLA	YMIYMSGLA	2619	1BCP_C
  	PERTUSSIS
  1737	BORDETELLA	YPALRAALI	2620	WP_019248658.1
  	PERTUSSIS
  1738	BORDETELLA	YPGTPGDLL	2621	AAA22984.1
  	PERTUSSIS
  1739	BORDETELLA	YPTYTEWSV	2622	WP_019249248.1
  	PERTUSSIS
  1740	BORDETELLA	YQTYTEWSV	2623	YP_006626470.1
  	PERTUSSIS
  1741	BORDETELLA	YSTGDLRAY	2624	WP_019248145.1
  	PERTUSSIS
  1742	BORDETELLA	YTLRYLASY	2625	WP_019248658.1
  	PERTUSSIS
  1743	BORDETELLA	YVLVKTNMV	2626	AAA22983.1
  	PERTUSSIS
  1744	BORDETELLA	YYDYEDATF	2627	YP_006628018.1
  	PERTUSSIS
  1745	BORDETELLA	AAFIALYPNSQLAPT	2628	Q7VU05
  	PERTUSSIS 509
  1746	BORDETELLA	GGAEYNLALGQRRA	2629	Q7VU04
  	PERTUSSIS 509
  1747	BORDETELLA	GGAEYNLALGQRRADA	2630	Q7VU04
  	PERTUSSIS 509
  1748	BORDETELLA	IALYPNSQLAPT	2631	Q7VU05
  	PERTUSSIS 509
  1749	BORDETELLA	KPDQGEVVAVGPGKKTED	2632	P0A339.1
  	PERTUSSIS 509
  1750	BORDETELLA	KPDQGEVVAVGPGKKTEDG	2633	P0A339.1
  	PERTUSSIS 509
  1751	BORDETELLA	LAEVLDYHNFVLTQ	2634	Q7VWM1.1
  	PERTUSSIS 509
  1752	CORYNEBACTERIUM	QSIALSSLMVAQAIP	2635	AAV70486.1
  	DIPHTHERIAE
  1753	CORYNEBACTERIUM	SIGVLGYQKTVDHTKVNSKLSLF	2636	AAV70486.1
  	DIPHTHERIAE
  1754	BORDETELLA	AAHADWNNQSIVKT	2637	ABO77783.13.1
  	PERTUSSIS
  1755	BORDETELLA	AALGRG	2638	ABO77783.13.1
  	PERTUSSIS
  1756	BORDETELLA	AALGRGHSLYASYE	2639	ABO77783.13.1
  	PERTUSSIS
  1757	BORDETELLA	AAPLRRTTLAMALG	2640	ABO77783.13.1
  	PERTUSSIS
  1758	BORDETELLA	AAPLSITLQAGAHA	2641	ABO77783.13.1
  	PERTUSSIS
  1759	BORDETELLA	ADAQGDIVATELPS	2642	ABO77783.13.1
  	PERTUSSIS
  1760	BORDETELLA	ADSGFYLDATLRAS	2643	ABO77783.13.1
  	PERTUSSIS
  1761	BORDETELLA	AELA	2644	ABO77783.13.1
  	PERTUSSIS
  1762	BORDETELLA	AELAVFRAGGGAYR	2645	ABO77783.13.1
  	PERTUSSIS
  1763	BORDETELLA	AELQFRNGSVTSSG	2646	ABO77783.13.1
  	PERTUSSIS
  1764	BORDETELLA	AGGRWHLGGLAGYT	2647	ABO77783.13.1
  	PERTUSSIS
  1765	BORDETELLA	AGVAAMQGAVVHLQ	2648	ABO77783.13.1
  	PERTUSSIS
  1766	BORDETELLA	AGYTRGDRGFTGDG	2649	ABO77783.13.1
  	PERTUSSIS
  1767	BORDETELLA	ALASQARWTGATRA	2650	ABO77783.13.1
  	PERTUSSIS
  1768	BORDETELLA	AMPWTFHAGYRYSW	2651	ABO77783.13.1
  	PERTUSSIS
  1769	BORDETELLA	AMQGAVVHLQRATIRRGDAP	2652	ABO77783.13.1
  	PERTUSSIS
  1770	BORDETELLA	ANGLRVRDE	2653	ABO77783.13.1
  	PERTUSSIS
  1771	BORDETELLA	ANGLRVRDEGGSSV	2654	ABO77783.13.1
  	PERTUSSIS
  1772	BORDETELLA	ANKDGKVDIGTYRY	2655	ABO77783.13.1
  	PERTUSSIS
  1773	BORDETELLA	APAAVSVLGASELT	2656	ABO77783.13.1
  	PERTUSSIS
  1774	BORDETELLA	APPAPKPAPQPGPQ	2657	ABO77783.13.1
  	PERTUSSIS
  1775	BORDETELLA	AQGILLENPAAELQ	2658	ABO77783.13.1
  	PERTUSSIS
  1776	BORDETELLA	ARWTGATRAVDSLS	2659	ABO77783.13.1
  	PERTUSSIS
  1777	BORDETELLA	ASLEAGRRFTHADG	2660	ABO77783.13.1
  	PERTUSSIS
  1778	BORDETELLA	ASYEYSKGPKLAMP	2661	ABO77783.13.1
  	PERTUSSIS
  1779	BORDETELLA	ATFTLANKD	2662	ABO77783.13.1
  	PERTUSSIS
  1780	BORDETELLA	ATFTLANKDGKVDI	2663	ABO77783.13.1
  	PERTUSSIS
  1781	BORDETELLA	ATRAVDSLSIDNAT	2664	ABO77783.13.1
  	PERTUSSIS
  1782	BORDETELLA	DDDGIALYVAGEQAQ	2665	ABO77783.13.1
  	PERTUSSIS
  1783	BORDETELLA	DDGIALYVAGEQAQ	2666	ABO77783.13.1
  	PERTUSSIS
  1784	BORDETELLA	DGGHITGGRAAGVA	2667	ABO77783.1
  	PERTUSSIS
  1785	BORDETELLA	DGIRRFLGTVTVKAGK	2668	ABO77783.1
  	PERTUSSIS
  1786	BORDETELLA	DGSVDFQQPAEAGR	2669	ABO77783.1
  	PERTUSSIS
  1787	BORDETELLA	DGYAVKGKYRTHGV	2670	ABO77783.1
  	PERTUSSIS
  1788	BORDETELLA	DIVATELPSIPGTS	2671	ABO77783.1
  	PERTUSSIS
  1789	BORDETELLA	DKLVVMQDASGQHR	2672	ABO77783.1
  	PERTUSSIS
  1790	BORDETELLA	DLGLSDKLVVMQDA	2673	ABO77783.1
  	PERTUSSIS
  1791	BORDETELLA	DNATWVMTDNSNVGA	2674	ABO77783.1
  	PERTUSSIS
  1792	BORDETELLA	DNATWVMTDNSNVGALRLA	2675	ABO77783.1
  	PERTUSSIS
  1793	BORDETELLA	DNRAGRRFDQKVAG	2676	ABO77783.1
  	PERTUSSIS
  1794	BORDETELLA	EAGRFKVLTVNTLA	2677	ABO77783.1
  	PERTUSSIS
  1795	BORDETELLA	ELAQSIVEAPELGA	2678	ABO77783.1
  	PERTUSSIS
  1796	BORDETELLA	ELGAAIRVGRGARV	2679	ABO77783.1
  	PERTUSSIS
  1797	BORDETELLA	ELGADHAVAVAGGR	2680	ABO77783.1
  	PERTUSSIS
  1798	BORDETELLA	ELPSIPGTSIGPLD	2681	ABO77783.1
  	PERTUSSIS
  1799	BORDETELLA	EPVKLTLTGGADAQ	2682	ABO77783.1
  	PERTUSSIS
  1800	BORDETELLA	EQAQASIADSTLQG	2683	ABO77783.1
  	PERTUSSIS
  1801	BORDETELLA	ERGANVTVQRSAIV	2684	ABO77783.1
  	PERTUSSIS
  1802	BORDETELLA	ERQHGIHIQGSDPG	2685	ABO77783.1
  	PERTUSSIS
  1803	BORDETELLA	EVGKRIELAGGRQV	2686	ABO77783.1
  	PERTUSSIS
  1804	BORDETELLA	FDGAGTVHTNGIAH	2687	ABO77783.1
  	PERTUSSIS
  1805	BORDETELLA	FQQPAEAGRFKVLT	2688	ABO77783.1
  	PERTUSSIS
  1806	BORDETELLA	FRAGGGAYRAANGL	2689	ABO77783.1
  	PERTUSSIS
  1807	BORDETELLA	GAHAQGKALLYRVL	2690	ABO77783.1
  	PERTUSSIS
  1808	BORDETELLA	GARVTVSGGSLSAP	2691	ABO77783.1
  	PERTUSSIS
  1809	BORDETELLA	GAYRAANGLRVRDE	2692	ABO77783.1
  	PERTUSSIS
  1810	BORDETELLA	GDAPAGGAVPGGAV	2693	ABO77783.1
  	PERTUSSIS
  1811	BORDETELLA	GGAVPGGAVPGGFG	2694	ABO77783.1
  	PERTUSSIS
  1812	BORDETELLA	GGAVPGGFGPVLDG	2695	ABO77783.1
  	PERTUSSIS
  1813	BORDETELLA	GGFGPVLDGWYGVD	2696	ABO77783.1
  	PERTUSSIS
  1814	BORDETELLA	GGLHIGALQSLOPE	2697	ABO77783.1
  	PERTUSSIS
  1815	BORDETELLA	GGVQIERGANVTVQ	2698	ABO77783.1
  	PERTUSSIS
  1816	BORDETELLA	GHSLYASYEYSKGP	2699	ABO77783.1
  	PERTUSSIS
  1817	BORDETELLA	GHTDSVHVGGYATY	2700	ABO77783.1
  	PERTUSSIS
  1818	BORDETELLA	GIAHRTELRGTRAE	2701	ABO77783.1
  	PERTUSSIS
  1819	BORDETELLA	GKALLYRVLPEPVK	2702	ABO77783.1
  	PERTUSSIS
  1820	BORDETELLA	GLGMAAALGRGHSL	2703	ABO77783.1
  	PERTUSSIS
  1821	BORDETELLA	GNVIETGGARRFAP	2704	ABO77783.1
  	PERTUSSIS
  1822	BORDETELLA	GPLDVALASQARWT	2705	ABO77783.1
  	PERTUSSIS
  1823	BORDETELLA	GQHRLWVRN	2706	ABO77783.1
  	PERTUSSIS
  1824	BORDETELLA	GRGFAQRQQLDNRA	2707	ABO77783.1
  	PERTUSSIS
  1825	BORDETELLA	GRLGLEVGKRIELA	2708	ABO77783.1
  	PERTUSSIS
  1826	BORDETELLA	GRQVQPYIKASVLQ	2709	ABO77783.1
  	PERTUSSIS
  1827	BORDETELLA	GRRFTHADGWFLEPQAELA	2710	ABO77783.1
  	PERTUSSIS
  1828	BORDETELLA	GSEPASANTLLLVQ	2711	ABO77783.1
  	PERTUSSIS
  1829	BORDETELLA	GSSVLGRLGLEVGK	2712	ABO77783.1
  	PERTUSSIS
  1830	BORDETELLA	GTTIKVSGRQAQGI	2713	ABO77783.1
  	PERTUSSIS
  1831	BORDETELLA	GTVTVKAGKLVADH	2714	ABO77783.1
  	PERTUSSIS
  1832	BORDETELLA	HAVAVAGGRWHLGG	2715	ABO77783.1
  	PERTUSSIS
  1833	BORDETELLA	IELAGGRQVQPYIK	2716	ABO77783.1
  	PERTUSSIS
  1834	BORDETELLA	IHIQGSDPGGVRTA	2717	ABO77783.1
  	PERTUSSIS
  1835	BORDETELLA	IRRFLGTVTVKAGK	2718	ABO77783.1
  	PERTUSSIS
  1836	BORDETELLA	IRVGRGARVTVSGG	2719	ABO77783.1
  	PERTUSSIS
  1837	BORDETELLA	ITLQAGAHA	2720	ABO77783.1
  	PERTUSSIS
  1838	BORDETELLA	ITLQAGAHAQGKAL	2721	ABO77783.1
  	PERTUSSIS
  1839	BORDETELLA	IVEAPELGAAIRVG	2722	ABO77783.1
  	PERTUSSIS
  1840	BORDETELLA	IVKTGERQHGIHIQ	2723	ABO77783.1
  	PERTUSSIS
  1841	BORDETELLA	KAGKLVADHATLAN	2724	ABO77783.1
  	PERTUSSIS
  1842	BORDETELLA	KGKYRTHGVGASLE	2725	ABO77783.1
  	PERTUSSIS
  1843	BORDETELLA	KPAPQPGPQPPQPP	2726	ABO77783.1
  	PERTUSSIS
  1844	BORDETELLA	KVAGFELGADHAVA	2727	ABO77783.1
  	PERTUSSIS
  1845	BORDETELLA	KVAGSDGYAVKGKY	2728	ABO77783.1
  	PERTUSSIS
  1846	BORDETELLA	KVDIGTYRYRLAAN	2729	ABO77783.1
  	PERTUSSIS
  1847	BORDETELLA	KVLTVNTLAGSGLF	2730	ABO77783.1
  	PERTUSSIS
  1848	BORDETELLA	LAANGNGQWSLVGA	2731	ABO77783.1
  	PERTUSSIS
  1849	BORDETELLA	LAMPWTFHAGYRYS	2732	ABO77783.1
  	PERTUSSIS
  1850	BORDETELLA	LASTLWYAESNALS	2733	ABO77783.1
  	PERTUSSIS
  1851	BORDETELLA	LENDFKVAGSDGYA	2734	ABO77783.1
  	PERTUSSIS
  1852	BORDETELLA	LENPAAELQFRNGS	2735	ABO77783.1
  	PERTUSSIS
  1853	BORDETELLA	LGAAPAAHADWNNQ	2736	ABO77783.1
  	PERTUSSIS
  1854	BORDETELLA	LGGLAGYTRGDRGFTGDG	2737	ABO77783.1
  	PERTUSSIS
  1855	BORDETELLA	LLENP	2738	ABO77783.1
  	PERTUSSIS
  1856	BORDETELLA	LLVQTPLGSAATFT	2739	ABO77783.1
  	PERTUSSIS
  1857	BORDETELLA	LPPSRVVLRDTNVT	2740	ABO77783.1
  	PERTUSSIS
  1858	BORDETELLA	LQPEDLPPS	2741	ABO77783.1
  	PERTUSSIS
  1859	BORDETELLA	LQPEDLPPSRVVLR	2742	ABO77783.1
  	PERTUSSIS
  1860	BORDETELLA	LRASRLENDFKVAG	2743	ABO77783.1
  	PERTUSSIS
  1861	BORDETELLA	LRLASDGSVDFQQP	2744	ABO77783.1
  	PERTUSSIS
  1862	BORDETELLA	LSAAANAAVNTGGV	2745	ABO77783.1
  	PERTUSSIS
  1863	BORDETELLA	LSAPHGNVIETGGA	2746	ABO77783.1
  	PERTUSSIS
  1864	BORDETELLA	LSDDGIRRFLGTVT	2747	ABO77783.1
  	PERTUSSIS
  1865	BORDETELLA	LVGAKAPPAPKPAP	2748	ABO77783.1
  	PERTUSSIS
  1866	BORDETELLA	LYVAGEQAQASIAD	2749	ABO77783.1
  	PERTUSSIS
  1867	BORDETELLA	MALGALGAAPAAHA	2750	ABO77783.1
  	PERTUSSIS
  1868	BORDETELLA	MNMSLSRIVKAAPL	2751	ABO77783.1
  	PERTUSSIS
  1869	BORDETELLA	MQDASGQHR	2752	ABO77783.1
  	PERTUSSIS
  1870	BORDETELLA	MQGAVVHLQRATIR	2753	ABO77783.1
  	PERTUSSIS
  1871	BORDETELLA	NAAVNTGGVGLAST	2754	ABO77783.1
  	PERTUSSIS
  1872	BORDETELLA	NALSKRLGELRLNP	2755	ABO77783.1
  	PERTUSSIS
  1873	BORDETELLA	NGQWSLVGAKAPPA	2756	ABO77783.1
  	PERTUSSIS
  1874	BORDETELLA	NTLAGSGLFRMNVF	2757	ABO77783.1
  	PERTUSSIS
  1875	BORDETELLA	PAGRELSAAANAAV	2758	ABO77783.1
  	PERTUSSIS
  1876	BORDETELLA	PAPQPPAGRELSAA	2759	ABO77783.1
  	PERTUSSIS
  1877	BORDETELLA	PGPQPPQPPQPQPE	2760	ABO77783.1
  	PERTUSSIS
  1878	BORDETELLA	PGTSIGPLDVALAS	2761	ABO77783.1
  	PERTUSSIS
  1879	BORDETELLA	PLGSAATFTLANKD	2762	ABO77783.1
  	PERTUSSIS
  1880	BORDETELLA	PQPEAPAPQPPAGR	2763	ABO77783.1
  	PERTUSSIS
  1881	BORDETELLA	PQPPQPQPEAPAPQ	2764	ABO77783.1
  	PERTUSSIS
  1882	BORDETELLA	PYIKASVLQEFDGA	2765	ABO77783.1
  	PERTUSSIS
  1883	BORDETELLA	RFAPQAAPLSITLQ	2766	ABO77783.1
  	PERTUSSIS
  1884	BORDETELLA	RLGELRLNPDAGGA	2767	ABO77783.1
  	PERTUSSIS
  1885	BORDETELLA	RLNPDAGGAWGRGF	2768	ABO77783.1
  	PERTUSSIS
  1886	BORDETELLA	RNGSVTSSGQLSDD	2769	ABO77783.1
  	PERTUSSIS
  1887	BORDETELLA	RRFDQKVAGFELGA	2770	ABO77783.1
  	PERTUSSIS
  1888	BORDETELLA	RTTLAMALGALGAA	2771	ABO77783.1
  	PERTUSSIS
  1889	BORDETELLA	SAIVDGGLHIGALQ	2772	ABO77783.1
  	PERTUSSIS
  1890	BORDETELLA	SANTLLLVQTPLGS	2773	ABO77783.1
  	PERTUSSIS
  1891	BORDETELLA	SDPGGVRTASGTTI	2774	ABO77783.1
  	PERTUSSIS
  1892	BORDETELLA	SELTLDGGHITGGR	2775	ABO77783.1
  	PERTUSSIS
  1893	BORDETELLA	SGLFRMNVF	2776	ABO77783.1
  	PERTUSSIS
  1894	BORDETELLA	SGLFRMNVFADLGL	2777	ABO77783.1
  	PERTUSSIS
  1895	BORDETELLA	SGSSVELAQSIVEA	2778	ABO77783.1
  	PERTUSSIS
  1896	BORDETELLA	SIADSTLQGAGGVQ	2779	ABO77783.1
  	PERTUSSIS
  1897	BORDETELLA	SKGPKLAMPWTFHA	2780	ABO77783.1
  	PERTUSSIS
  1898	BORDETELLA	SNVGALRLASDGSV	2781	ABO77783.1
  	PERTUSSIS
  1899	BORDETELLA	SRIVKAAPLRRTTL	2782	ABO77783.1
  	PERTUSSIS
  1900	BORDETELLA	SVLGASELTLDGGH	2783	ABO77783.1
  	PERTUSSIS
  1901	BORDETELLA	SVLQEFDGA	2784	ABO77783.1
  	PERTUSSIS
  1902	BORDETELLA	SVLQEFDGAGTVHT	2785	ABO77783.1
  	PERTUSSIS
  1903	BORDETELLA	TELR	2786	ABO77783.1
  	PERTUSSIS
  1904	BORDETELLA	TELRGTRAELGLGM	2787	ABO77783.1
  	PERTUSSIS
  1905	BORDETELLA	TGDGGGHTDSVHVG	2788	ABO77783.1
  	PERTUSSIS
  1906	BORDETELLA	TGGARRFAPQAAPL	2789	ABO77783.1
  	PERTUSSIS
  1907	BORDETELLA	TGGRAAGVAAMQGA	2790	ABO77783.1
  	PERTUSSIS
  1908	BORDETELLA	TGGVGLASTLWYAE	2791	ABO77783.1
  	PERTUSSIS
  1909	BORDETELLA	THGVGASLEAGRRF	2792	ABO77783.1
  	PERTUSSIS
  1910	BORDETELLA	TIRRGDAPA	2793	ABO77783.1
  	PERTUSSIS
  1911	BORDETELLA	TLANVGDTWDDDGI	2794	ABO77783.1
  	PERTUSSIS
  1912	BORDETELLA	TLQGAGGVQIERGA	2795	ABO77783.1
  	PERTUSSIS
  1913	BORDETELLA	TLTGGADAQGDIVA	2796	ABO77783.1
  	PERTUSSIS
  1914	BORDETELLA	TNVTAVPASGAPAA	2797	ABO77783.1
  	PERTUSSIS
  1915	BORDETELLA	TRAELGLGMAAALG	2798	ABO77783.1
  	PERTUSSIS
  1916	BORDETELLA	TSSGQLSDDGIRRF	2799	ABO77783.1
  	PERTUSSIS
  1917	BORDETELLA	TVHTNGIAHRTELR	2800	ABO77783.1
  	PERTUSSIS
  1918	BORDETELLA	TYRYRLAANGNGQW	2801	ABO77783.1
  	PERTUSSIS
  1919	BORDETELLA	VADHATLANVGDTW	2802	ABO77783.1
  	PERTUSSIS
  1920	BORDETELLA	VHVGGYATYIADSG	2803	ABO77783.1
  	PERTUSSIS
  1921	BORDETELLA	VLDGWYGVD	2804	ABO77783.1
  	PERTUSSIS
  1922	BORDETELLA	VPASGAPAAVSVLG	2805	ABO77783.1
  	PERTUSSIS
  1923	BORDETELLA	VRDEGGSSVLGRLG	2806	ABO77783.1
  	PERTUSSIS
  1924	BORDETELLA	VRTASGTTIKVSGR	2807	ABO77783.1
  	PERTUSSIS
  1925	BORDETELLA	VSGGSLSAPHGNVI	2808	ABO77783.1
  	PERTUSSIS
  1926	BORDETELLA	VSGRQAQGILLENP	2809	ABO77783.1
  	PERTUSSIS
  1927	BORDETELLA	VTVQRSAIVDGGLH	2810	ABO77783.1
  	PERTUSSIS
  1928	BORDETELLA	VVLRDTNVTAVPAS	2811	ABO77783.1
  	PERTUSSIS
  1929	BORDETELLA	WNNQSIVKTGERQH	2812	ABO77783.1
  	PERTUSSIS
  1930	BORDETELLA	WVRNSGSEPASANT	2813	ABO77783.1
  	PERTUSSIS
  1931	BORDETELLA	WYAESNALSKRLGE	2814	ABO77783.1
  	PERTUSSIS
  1932	BORDETELLA	YATYIADSGFYLDA	2815	ABO77783.1
  	PERTUSSIS
  1933	BORDETELLA	YGVDVSGSS	2816	ABO77783.1
  	PERTUSSIS
  1934	BORDETELLA	YGVDVSGSSVELAQ	2817	ABO77783.1
  	PERTUSSIS
  1935	BORDETELLA	YLDATLRASRLEND	2818	ABO77783.1
  	PERTUSSIS
  1936	BORDETELLA	YRVLPEPVKLTLTG	2819	ABO77783.1
  	PERTUSSIS
  1937	BORDETELLA	VKAQNITNKRAALIEA	2820	AAA22974.1
  	PERTUSSIS
  1938	BORDETELLA	YYSNVTATRLLSSTNS	2821	AAA83981.1
  	PERTUSSIS
  1939	BORDETELLA	SPNLTDERAAQAGVT	2822	CPP72976.1
  	PERTUSSIS
  1940	MEASLES	SSRASDERAAHLPTS	2823	BAA33867.1
  	MORBILLIVIRUS
  1941	CORYNEBACTERIUM	QVVHNSYNRPAYSPG	2824	1007216A
  	DIPHTHERIAE
  1942	MEASLES VIRUS	AEGGEIHEL	2825	AAF85692.1
  	STRAIN
  	EDMONSTON-B
  1943	MEASLES VIRUS	AENLISNGIGKY	2826	AAF85698.1
  	STRAIN
  	EDMONSTON-B
  1944	MEASLES VIRUS	AEVDGDVKL	2827	CAB43772.1
  	STRAIN
  	EDMONSTON-B
  1945	MEASLES VIRUS	AIYTAEIHK	2828	AAF85697.1
  	STRAIN
  	EDMONSTON-B
  1946	MEASLES VIRUS	APVFHMTNY	2829	CAB43772.1
  	STRAIN
  	EDMONSTON-B
  1947	MEASLES VIRUS	APVFHMTNYLEQPVSN	2830	AAR89413.1
  	STRAIN
  	EDMONSTON-B
  1948	MEASLES VIRUS	AQRLNEIY	2831	AAF85698.1
  	STRAIN
  	EDMONSTON-B
  1949	MEASLES VIRUS	ARVPHAYSL	2832	AAF85698.1
  	STRAIN
  	EDMONSTON-B
  1950	MEASLES VIRUS	AVRDLERAM	2833	P03424.1
  	STRAIN
  	EDMONSTON-B
  1951	MEASLES VIRUS	AVRDLERAMTTLK	2834	P03424.1
  	STRAIN
  	EDMONSTON-B
  1952	MEASLES VIRUS	DALLRLQAM	2835	Q89933.1
  	STRAIN
  	EDMONSTON-B
  1953	MEASLES VIRUS	DIKEKVINL	2836	AAF85698.1
  	STRAIN
  	EDMONSTON-B
  1954	MEASLES VIRUS	DQGLFKVL	2837	AAF85695.1
  	STRAIN
  	EDMONSTON-B
  1955	MEASLES VIRUS	DTGVDTRIW	2838	Q9EMA9.1
  	STRAIN
  	EDMONSTON-B
  1956	MEASLES VIRUS	EPIGSLAIEEAM	2839	AAF85692.1
  	STRAIN
  	EDMONSTON-B
  1957	MEASLES VIRUS	EPIRDALNAM	2840	P69354.1
  	STRAIN
  	EDMONSTON-B
  1958	MEASLES VIRUS	FPKLGKTL	2841	AAF85692.1
  	STRAIN
  	EDMONSTON-B
  1959	MEASLES VIRUS	FRSVNAVAF	2842	AAF85695.1
  	STRAIN
  	EDMONSTON-B
  1960	MEASLES VIRUS	GKIIDNTEQL	2843	AAF85695.1
  	STRAIN
  	EDMONSTON-B
  1961	MEASLES VIRUS	GLNEKLVFY	2844	AAF85695.1
  	STRAIN
  	EDMONSTON-B
  1962	MEASLES VIRUS	GMYGGTYLVEK	2845	AAC35876.2
  	STRAIN
  	EDMONSTON-B
  1963	MEASLES VIRUS	GPPISLERLDVGTN	2846	P69354.1
  	STRAIN
  	EDMONSTON-B
  1964	MEASLES VIRUS	GPRQAQVSFL	2847	Q89933.1
  	STRAIN
  	EDMONSTON-B
  1965	MEASLES VIRUS	GRLVPQVRVID	2848	AAF85695.1
  	STRAIN
  	EDMONSTON-B
  1966	MEASLES VIRUS	GSAPISMGFR	2849	AAF85692.1
  	STRAIN
  	EDMONSTON-B
  1967	MEASLES VIRUS	HILAKSTAL	2850	AAF85698.1
  	STRAIN
  	EDMONSTON-B
  1968	MEASLES VIRUS	HYREVNLVY	2851	AAF85698.1
  	STRAIN
  	EDMONSTON-B
  1969	MEASLES VIRUS	IPPMKNLAL	2852	AAC35876.2
  	STRAIN
  	EDMONSTON-B
  1970	MEASLES VIRUS	IPYQGSGKGVSF	2853	CAB43772.1
  	STRAIN
  	EDMONSTON-B
  1971	MEASLES VIRUS	ISKESQHVY	2854	AAF85698.1
  	STRAIN
  	EDMONSTON-B
  1972	MEASLES VIRUS	IVSSHFFVY	2855	AAF85698.1
  	STRAIN
  	EDMONSTON-B
  1973	MEASLES VIRUS	KEIKETGRLF	2856	AAF85698.1
  	STRAIN
  	EDMONSTON-B
  1974	MEASLES VIRUS	KESQHVYYL	2857	AAF85698.1
  	STRAIN
  	EDMONSTON-B
  1975	MEASLES VIRUS	KIIDNTEQL	2858	AAF85695.1
  	STRAIN
  	EDMONSTON-B
  1976	MEASLES VIRUS	KKQINRQN	2859	AAA63285.1
  	STRAIN
  	EDMONSTON-B
  1977	MEASLES VIRUS	KKVDTNFIYQ	2860	AAF85698.1
  	STRAIN
  	EDMONSTON-B
  1978	MEASLES VIRUS	KLIDGFFPA	2861	AAF85698.1
  	STRAIN
  	EDMONSTON-B
  1979	MEASLES VIRUS	KPNLSSKRSEL	2862	BAB39848.1
  	STRAIN
  	EDMONSTON-B
  1980	MEASLES VIRUS	KVSPYLFTV	2863	AAR89413.1
  	STRAIN
  	EDMONSTON-B
  1981	MEASLES VIRUS	LETRTTNQFL	2864	CAB43772.1
  	STRAIN
  	EDMONSTON-B
  1982	MEASLES VIRUS	LLKEATEL	2865	AAF85695.1
  	STRAIN
  	EDMONSTON-B
  1983	MEASLES VIRUS	LLKKGNSLY	2866	AAF85698.1
  	STRAIN
  	EDMONSTON-B
  1984	MEASLES VIRUS	LPAPIGGMNY	2867	AAF85698.1
  	STRAIN
  	EDMONSTON-B
  1985	MEASLES VIRUS	MPEETLHQVM	2868	AAF85698.1
  	STRAIN
  	EDMONSTON-B
  1986	MEASLES VIRUS	PTTIRGQFS	2869	CAB43772.1
  	STRAIN
  	EDMONSTON-B
  1987	MEASLES VIRUS	QEISRHQALGY	2870	P03424.1
  	STRAIN
  	EDMONSTON-B
  1988	MEASLES VIRUS	RITHVDTESY	2871	P69354.1
  	STRAIN
  	EDMONSTON-B
  1989	MEASLES VIRUS	RPGLKPDL	2872	P69354.1
  	STRAIN
  	EDMONSTON-B
  1990	MEASLES VIRUS	RPIYGLEV	2873	AAF85698.1
  	STRAIN
  	EDMONSTON-B
  1991	MEASLES VIRUS	RQAGQEMILAV	2874	P69354.1
  	STRAIN
  	EDMONSTON-B
  1992	MEASLES VIRUS	SAVRIATVY	2875	AAF85698.1
  	STRAIN
  	EDMONSTON-B
  1993	MEASLES VIRUS	SLMPEETLHQV	2876	AAF85698.1
  	STRAIN
  	EDMONSTON-B
  1994	MEASLES VIRUS	SMIDLVTKF	2877	AAF85698.1
  	STRAIN
  	EDMONSTON-B
  1995	MEASLES VIRUS	SMLNSQAIDNLRA	2878	P69354.1
  	STRAIN
  	EDMONSTON-B
  1996	MEASLES VIRUS	SMYRVFEV	2879	CAB43772.1
  	STRAIN
  	EDMONSTON-B
  1997	MEASLES VIRUS	SQQGMFHAY	2880	AAF85698.1
  	STRAIN
  	EDMONSTON-B
  1998	MEASLES VIRUS	TDTPIVYNDRNLLD	2881	Q89933.1
  	STRAIN
  	EDMONSTON-B
  1999	MEASLES VIRUS	VIINDDQGLFKV	2882	AAF85695.1
  	STRAIN
  	EDMONSTON-B
  2000	MEASLES VIRUS	YESGVRIASL	2883	AAF85698.1
  	STRAIN
  	EDMONSTON-B
  2001	MEASLES VIRUS	YLKDKALA	2884	AAF85698.1
  	STRAIN
  	EDMONSTON-B
  2002	MEASLES VIRUS	YVYDHSGEAVK	2885	AAF85692.1
  	STRAIN
  	EDMONSTON-B
  2003	RUBELLA VIRUS	ARVIDPAAQSFTGVV	2886	BAA28178.1
  2004	RUBELLA VIRUS	SDRASARVIDPAAQS	2887	BAA28178.1
  2005	RUBELLA VIRUS	VPPGKFVTAALLNTP	2888	BAA28178.1
  2006	RUBELLA VIRUS	WVTPVIGSQARKCGL	2889	BAA28178.1
  2007	MUMPS	GTYRLIPNARANLTA	400	AGC97176.1
  	RUBULAVIRUS

• I. Delivery of Prime Editors
• In another aspect, the present disclosure provides for the delivery of prime editors in vitro and in vivo using various strategies, including on separate vectors using split inteins and as well as direct delivery strategies of the ribonucleoprotein complex (i.e., the prime editor complexed to the PEgRNA and/or the second-site gRNA) using techniques such as electroporation, use of cationic lipid-mediated formulations, and induced endocytosis methods using receptor ligands fused to the ribonucleoprotein complexes. Any such methods are contemplated herein.
Overview of Delivery Options
• In some aspects, the invention provides methods comprising delivering one or more prime editor-encoding polynucleotides, such as or one or more vectors as described herein encoding one or more components of the prime editing system described herein, one or more transcripts thereof, and/or one or proteins transcribed therefrom, to a host cell. In some aspects, the invention further provides cells produced by such methods, and organisms (such as animals, plants, or fungi) comprising or produced from such cells. In some embodiments, a prime editor as described herein in combination with (and optionally complexed with) a guide sequence is delivered to a cell. Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids in mammalian cells or target tissues. Such methods can be used to administer nucleic acids encoding components of a prime editor to cells in culture, or in a host organism. Non-viral vector delivery systems include DNA plasmids, RNA (e.g. a transcript of a vector described herein), naked nucleic acid, and nucleic acid complexed with a delivery vehicle, such as a liposome. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. For a review of gene therapy procedures, see Anderson, Science 256:808-813 (1992); Nabel & Felgner, TIBTECH 11:211-217 (1993); Mitani & Caskey, TIBTECH 11:162-166 (1993); Dillon, TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992); Van Brunt, Biotechnology 6(10):1149-1154 (1988); Vigne, Restorative Neurology and Neuroscience 8:35-36 (1995); Kremer & Perricaudet, British Medical Bulletin 51(1):31-44 (1995); Haddada et al., in Current Topics in Microbiology and Immunology Doerfler and Bihm (eds) (1995); and Yu et al., Gene Therapy 1:13-26 (1994).
• Methods of non-viral delivery of nucleic acids include lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., Transfectam™ and Lipofectin™) Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Feigner, WO 91/17424; WO 91/16024. Delivery can be to cells (e.g. in vitro or ex vivo administration) or target tissues (e.g. in vivo administration).
• The preparation of lipid:nucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to one of skill in the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese et al., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res. 52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).
• The use of RNA or DNA viral based systems for the delivery of nucleic acids take advantage of highly evolved processes for targeting a virus to specific cells in the body and trafficking the viral payload to the nucleus. Viral vectors can be administered directly to patients (in vivo) or they can be used to treat cells in vitro, and the modified cells may optionally be administered to patients (ex vivo). Conventional viral based systems could include retroviral, lentivirus, adenoviral, adeno-associated and herpes simplex virus vectors for gene transfer. Integration in the host genome is possible with the retrovirus, lentivirus, and adeno-associated virus gene transfer methods, often resulting in long term expression of the inserted transgene. Additionally, high transduction efficiencies have been observed in many different cell types and target tissues.
• The tropism of a viruses can be altered by incorporating foreign envelope proteins, expanding the potential target population of target cells. Lentiviral vectors are retroviral vectors that are able to transduce or infect non-dividing cells and typically produce high viral titers. Selection of a retroviral gene transfer system would therefore depend on the target tissue. Retroviral vectors are comprised of cis-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimum cis-acting LTRs are sufficient for replication and packaging of the vectors, which are then used to integrate the therapeutic gene into the target cell to provide permanent transgene expression. Widely used retroviral vectors include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immuno deficiency virus (SIV), human immuno deficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol. 66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992); Sommnerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol. 63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991); PCT/US94/05700). In applications where transient expression is preferred, adenoviral based systems may be used. Adenoviral based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. With such vectors, high titer and levels of expression have been obtained. This vector can be produced in large quantities in a relatively simple system. Adeno-associated virus (“AAV”) vectors may also be used to transduce cells with target nucleic acids, e.g., in the in vitro production of nucleic acids and peptides, and for in vivo and ex vivo gene therapy procedures (see, e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368; WO 93/24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351 (1994). Construction of recombinant AAV vectors are described in a number of publications, including U.S. Pat. No. 5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985); Tratschin, et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81:6466-6470 (1984); and Samulski et al., J. Virol. 63:03822-3828 (1989).
• Packaging cells are typically used to form virus particles that are capable of infecting a host cell. Such cells include 293 cells, which package adenovirus, and ψ2 cells or PA317 cells, which package retrovirus. Viral vectors used in gene therapy are usually generated by producing a cell line that packages a nucleic acid vector into a viral particle. The vectors typically contain the minimal viral sequences required for packaging and subsequent integration into a host, other viral sequences being replaced by an expression cassette for the polynucleotide(s) to be expressed. The missing viral functions are typically supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy typically only possess ITR sequences from the AAV genome which are required for packaging and integration into the host genome. Viral DNA is packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences. The cell line may also be infected with adenovirus as a helper. The helper virus promotes replication of the AAV vector and expression of AAV genes from the helper plasmid. The helper plasmid is not packaged in significant amounts due to a lack of ITR sequences. Contamination with adenovirus can be reduced by, e.g., heat treatment to which adenovirus is more sensitive than AAV. Additional methods for the delivery of nucleic acids to cells are known to those skilled in the art. See, for example, US20030087817, incorporated herein by reference.
• In various embodiments, the PE constructs (including, the split-constructs) may be engineered for delivery in one or more rAAV vectors. An rAAV as related to any of the methods and compositions provided herein may be of any serotype including any derivative or pseudotype (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 2/1, 2/5, 2/8, 2/9, 3/1, 3/5, 3/8, or 3/9). An rAAV may comprise a genetic load (i.e., a recombinant nucleic acid vector that expresses a gene of interest, such as a whole or split PE fusion protein that is carried by the rAAV into a cell) that is to be delivered to a cell. An rAAV may be chimeric.
• As used herein, the serotype of an rAAV refers to the serotype of the capsid proteins of the recombinant virus. Non-limiting examples of derivatives and pseudotypes include rAAV2/1, rAAV2/5, rAAV2/8, rAAV2/9, AAV2-AAV3 hybrid, AAVrh.10, AAVhu.14, AAV3a/3b, AAVrh32.33, AAV-HSC15, AAV-HSC17, AAVhu.37, AAVrh.8, CHt-P6, AAV2.5, AAV6.2, AAV2i8, AAV-HSC15/17, AAVM41, AAV9.45, AAV6(Y445F/Y731F), AAV2.5T, AAV-HAE1/2, AAV clone 32/83, AAVShH10, AAV2 (Y->F), AAV8 (Y733F), AAV2.15, AAV2.4, AAVM41, and AAVr3.45. A non-limiting example of derivatives and pseudotypes that have chimeric VP1 proteins is rAAV2/5-1VP1u, which has the genome of AAV2, capsid backbone of AAV5 and VP1u of AAV1. Other non-limiting example of derivatives and pseudotypes that have chimeric VP1 proteins are rAAV2/5-8VP1u, rAAV2/9-1VP1u, and rAAV2/9-8VP1u.
• AAV derivatives/pseudotypes, and methods of producing such derivatives/pseudotypes are known in the art (see, e.g., Mol Ther. 2012 April; 20(4):699-708. doi: 10.1038/mt.2011.287. Epub 2012 Jan. 24. The AAV vector toolkit: poised at the clinical crossroads. Asokan A I, Schaffer D V, Samulski R J.). Methods for producing and using pseudotyped rAAV vectors are known in the art (see, e.g., Duan et al., J. Virol., 75:7662-7671, 2001; Halbert et al., J. Virol., 74:1524-1532, 2000; Zolotukhin et al., Methods, 28:158-167, 2002; and Auricchio et al., Hum. Molec. Genet., 10:3075-3081, 2001).
• Methods of making or packaging rAAV particles are known in the art and reagents are commercially available (see, e.g., Zolotukhin et al. Production and purification of serotype 1, 2, and 5 recombinant adeno-associated viral vectors. Methods 28 (2002) 158-167; and U.S. Patent Publication Numbers US20070015238 and US20120322861, which are incorporated herein by reference; and plasmids and kits available from ATCC and Cell Biolabs, Inc.). For example, a plasmid comprising a gene of interest may be combined with one or more helper plasmids, e.g., that contain a rep gene (e.g., encoding Rep78, Rep68, Rep52 and Rep40) and a cap gene (encoding VP1, VP2, and VP3, including a modified VP2 region as described herein), and transfected into a recombinant cells such that the rAAV particle can be packaged and subsequently purified.
• Recombinant AAV may comprise a nucleic acid vector, which may comprise at a minimum: (a) one or more heterologous nucleic acid regions comprising a sequence encoding a protein or polypeptide of interest or an RNA of interest (e.g., a siRNA or microRNA), and (b) one or more regions comprising inverted terminal repeat (ITR) sequences (e.g., wild-type ITR sequences or engineered ITR sequences) flanking the one or more nucleic acid regions (e.g., heterologous nucleic acid regions). Herein, heterologous nucleic acid regions comprising a sequence encoding a protein of interest or RNA of interest are referred to as genes of interest.
• Any one of the rAAV particles provided herein may have capsid proteins that have amino acids of different serotypes outside of the VP1u region. In some embodiments, the serotype of the backbone of the VP1 protein is different from the serotype of the ITRs and/or the Rep gene. In some embodiments, the serotype of the backbone of the VP1 capsid protein of a particle is the same as the serotype of the ITRs. In some embodiments, the serotype of the backbone of the VP1 capsid protein of a particle is the same as the serotype of the Rep gene. In some embodiments, capsid proteins of rAAV particles comprise amino acid mutations that result in improved transduction efficiency.
• In some embodiments, the nucleic acid vector comprises one or more regions comprising a sequence that facilitates expression of the nucleic acid (e.g., the heterologous nucleic acid), e.g., expression control sequences operatively linked to the nucleic acid. Numerous such sequences are known in the art. Non-limiting examples of expression control sequences include promoters, insulators, silencers, response elements, introns, enhancers, initiation sites, termination signals, and poly(A) tails. Any combination of such control sequences is contemplated herein (e.g., a promoter and an enhancer).
• Final AAV constructs may incorporate a sequence encoding the PEgRNA. In other embodiments, the AAV constructs may incorporate a sequence encoding the second-site nicking guide RNA. In still other embodiments, the AAV constructs may incorporate a sequence encoding the second-site nicking guide RNA and a sequence encoding the PEgRNA.
• In various embodiments, the PEgRNAs and the second-site nicking guide RNAs can be expressed from an appropriate promoter, such as a human U6 (hU6) promoter, a mouse U6 (mU6) promoter, or other appropriate promoter. The PEgRNAs and the second-site nicking guide RNAs can be driven by the same promoters or different promoters.
• In some embodiments, a rAAV constructs or the herein compositions are administered to a subject enterally. In some embodiments, a rAAV constructs or the herein compositions are administered to the subject parenterally. In some embodiments, a rAAV particle or the herein compositions are administered to a subject subcutaneously, intraocularly, intravitreally, subretinally, intravenously (IV), intracerebro-ventricularly, intramuscularly, intrathecally (IT), intracisternally, intraperitoneally, via inhalation, topically, or by direct injection to one or more cells, tissues, or organs. In some embodiments, a rAAV particle or the herein compositions are administered to the subject by injection into the hepatic artery or portal vein.
Split PE Vector-Based Strategies
• In this aspect, the prime editors can be divided at a split site and provided as two halves of a whole/complete prime editor. The two halves can be delivered to cells (e.g., as expressed proteins or on separate expression vectors) and once in contact inside the cell, the two halves form the complete prime editor through the self-splicing action of the inteins on each prime editor half. Split intein sequences can be engineered into each of the halves of the encoded prime editor to facilitate their transplicing inside the cell and the concomitant restoration of the complete, functioning PE.
• These split intein-based methods overcome several barriers to in vivo delivery. For example, the DNA encoding prime editors is larger than the rAAV packaging limit, and so requires special solutions. One such solution is formulating the editor fused to split intein pairs that are packaged into two separate rAAV particles that, when co-delivered to a cell, reconstitute the functional editor protein. Several other special considerations to account for the unique features of prime editing are described, including the optimization of second-site nicking targets and properly packaging prime editors into virus vectors, including lentiviruses and rAAV.
• In this aspect, the prime editors can be divided at a split site and provided as two halves of a whole/complete prime editor. The two halves can be delivered to cells (e.g., as expressed proteins or on separate expression vectors) and once in contact inside the cell, the two halves form the complete prime editor through the self-splicing action of the inteins on each prime editor half. Split intein sequences can be engineered into each of the halves of the encoded prime editor to facilitate their transplicing inside the cell and the concomitant restoration of the complete, functioning PE.
• FIG. 66 depicts one embodiment of a prime editor being provided as two PE half proteins which regenerate as whole prime editor through the self-splicing action of the split-intein halves located at the end or beginning of each of the prime editor half proteins. As used herein, the term “PE N-terminal half” refers to the N-terminal half of a complete prime editor and which comprises the “N intein” at the C-terminal end of the PE N-terminal half (i.e., the N-terminal extein) of the complete prime editor. The “N intein” refers to the N-terminal half of a complete, fully-formed split-intein moiety. As used herein, the term “PE C-terminal half” refers to the C-terminal half of a complete prime editor and which comprises the “C intein” at the N-terminal end of the C-terminal half (i.e., the C-terminal extein) of a complete prime editor. When the two half proteins, i.e., the PE N-terminal half and the PE C-terminal half, come into contact with one another, e.g., within the cell, the N intein and the C intein undergo the simultaneous process of self-excision and the formation of a peptide bond between the C-terminal end of the PE N-terminal half and the N-terminal end of the PE C-terminal half to reform the complete prime editor protein comprising the complete napDNAbp domain (e.g., Cas9 nickase) and the RT domain. Although not shown in the drawing, the prime editor may also comprise additional sequences including NLS at the N-terminus and/or C-terminus, as well as amino acid linkers sequences joining each domain.
• In various embodiments, the prime editors may be engineered as two half proteins (i.e., a PE N-terminal half and a PE C-terminal half) by “splitting” the whole prime editor as a “split site.” The “split site” refers to the location of insertion of split intein sequences (i.e., the N intein and the C intein) between two adjacent amino acid residues in the prime editor. More specifically, the “split site” refers to the location of dividing the whole prime editor into two separate halves, wherein in each halve is fused at the split site to either the N intein or the C intein motifs. The split site can be at any suitable location in the prime editor fusion protein, but preferably the split site is located at a position that allows for the formation of two half proteins which are appropriately sized for delivery (e.g., by expression vector) and wherein the inteins, which are fused to each half protein at the split site termini, are available to sufficiently interact with one another when one half protein contacts the other half protein inside the cell.
• In some embodiments, the split site is located in the napDNAbp domain. In other embodiments, the split site is located in the RT domain. In other embodiments, the split site is located in a linker that joins the napDNAbp domain and the RT domain.
• In various embodiments, split site design requires finding sites to split and insert an N- and C-terminal intein that are both structurally permissive for purposes of packaging the two half prime editor domains into two different AAV genomes. Additionally, intein residues necessary for trans splicing can be incorporated by mutating residues at the N terminus of the C terminal extein or inserting residues that will leave an intein “scar.”
• Exemplary split configurations of split prime editors comprising either the SpCas9 nickase or the SaCas9 nickase are as follows.

• S. PYOGENES PE, SPLIT AT LINKER, N TERMINAL

  PORTION STRUCTURE: [N EXTEIN]-[N INTEIN]

  (SEQ ID NO: 443)

  MKRTADGSEFESPKKKRKV DKKYSIGLDIGTNSVGWAVITDEYKVPSKKF

  KVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYL

  QEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKY

  PTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVD

  KLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGE

  KKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI

  GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDL

  TLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEK

  MDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPF

  LKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVV

  DKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTE

  GMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISG

  VEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMI

  EERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDF

  LKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPA

  IKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMK

  RIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINR

  LSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNY

  WRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVA

  QILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYH

  HAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKA

  TAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT

  VRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYG

  GFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFL

  EAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKY

  VNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVIL

  ADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTID

  RKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDSGGSSGGSCLSYET

  EILTVEYGLLPIGKIVEKRIECTVYSVDNNGNIYTQPVAQWHDRGEQEVF

  EYCLEDGSLIRATKDHKFMTVDGQMLPIDEIFERELDLMRVDNLPNSGGS

  KRTADGSEFEPKKKRKV

  KEY:

  (SEQ ID NO: 124, 125)

  NLS

  (SEQ ID NO: 445)

  CAS9

  (SEQ ID NO: 446)

  LINKER

  (SEQ ID NO: 447)

  NPUN INTEIN

  S. PYOGENES PE, SPLIT AT LINKER, C TERMINAL

  PORTION STRUCTURE: C [INTEIN]-[C EXTEIN]

  (SEQ ID NO: 450)

  MKRTADGSEFESPKKKRKV IKIATRKYLGKQNVYDIGVERDHNFALKNGF

  IASN CFNSGSETPGTSESATPESSGGSSGGSS TLNIEDEYRLHETSKEPD

  VSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQ

  EARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREV

  NKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLF

  AFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLI

  LLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKY

  LGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFA

  EMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFEL

  FVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAV

  LTKDAGKLTMGQPLVILAPHAVEALVICQPPDRWLSNARMTHYQALLLDT

  DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDA

  DHTWYTDGSSLWEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQ

  ALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEIL

  ALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTS

  TLLIENSSP SGGSKRTADGSEFEPKKKRKV

  KEY:

  (SEQ ID NO: 124, 125)

  NLS

  (SEQ ID NO: 453)

  LINKER 1

  (SEQ ID NO: 174)

  LINKER 2

  (SEQ ID NO: 452)

  NPUC INTEIN

  (SEQ ID NO: 454)

  RT

  S. AUREUS PE, SPLIT BETWEEN RESIDUES 740/741, N

  TERMINAL PORTION STRUCTURE: [N EXTEIN]-[N INTEIN]

  (SEQ ID NO: 458)

  MKRTADGSEFESPKKKRKV GKRNYILGLAIGITSVGYGIIDYETRDVIDA

  GVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHS

  ELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTGNE

  LSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAK

  QLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYE

  MLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKF

  QIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHD

  IKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIE

  QISNLKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDLS

  QQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELARE

  KNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQE

  GKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQEENS

  KKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERD

  INRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFT

  SFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQ

  MFEEKQAECLSYETEILTVEYGLLPIGKIVEKRIECTVYSVDNNGNIYTQ

  PVAQWHDRGEQEVFEYCLEDGSLIRATKDHKFMTVDGQMLPIDEIFEREL

  DLMRVDNLPN SGGSKRTADGSEFEPKKKRKV

  KEY:

  (SEQ ID NO: 124, 125)

  NLS

  (SEQ ID NO: 460)

  CAS9

  (SEQ ID NO: 174)

  LINKER

  (SEQ ID NO: 462)

  NPUN INTEIN

  S. AUREUS PE, SPLIT BETWEEN RESIDUES 740/741, C

  TERMINAL PORTION STRUCTURE: [C INTEIN]-[C EXTEIN]

  (SEQ ID NO: 465)

  MKRTADGSEFESPKKKRKV IKIATRKYLGKQNVYDIGVERDHNFALKNGF

  IASN CFN EIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRKLIN

  DTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQ

  TYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGN

  KLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIK

  KENYYEVNSKCYEEAKKLKKISNQAEFIASFYKNDLIKINGELYRVIGVN

  NDLLNRIEVNMIDITYREYLENMNDKRPPHIIKTIASKTQSIKKYSTDIL

  GNLYEVKSKKHPQIIKKG SGGSSGGSSGSETPGTSESATPESSGGSSGGS

  S TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLI

  IPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLL

  PVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVL

  DLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLF

  DEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGN

  LGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQ

  LREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQ

  ALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKL

  DPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVICQPP

  DRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDI

  LAEAHGTRPDLTDQPLPDADHTWYTDGSSLWEGQRKAGAAVTTETEVIWA

  KALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYR

  RRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGN

  RMADQAARKAAITETPDTSTLLIENSSP SGGSKRTADGSEFEPKKKRKV

  KEY:

  (SEQ ID NO: 124, 125)

  NLS

  (SEQ ID NO: 467)

  CAS9

  (SEQ ID NO: 127)

  LINKER 1

  (SEQ ID NO: 174)

  LINKER 2

  (SEQ ID NO: 452)

  NPUC INTEIN

  (SEQ ID NO: 471)

  RT

• In various embodiments, using SpCas9 nickase (SEQ ID NO: 18, 1368 amino acids) as an example, the split can between any two amino acids between 1 and 1368. Preferred splits, however, will be located between the central region of the protein, e.g., from amino acids 50-1250, or from 100-1200, or from 150-1150, or from 200-1100, or from 250-1050, or from 300-1000, or from 350-950, or from 400-900, or from 450-850, or from 500-800, or from 550-750, or from 600-700 of SEQ ID NO: 18. In specific exemplary embodiments, the split site may be between 740/741, or 801/802, or 1010/1011, or 1041/1042. In other embodiments the split site may be between 1/2, 2/3, 3/4, 4/5, 5/6, 6/7, 7/8, 8/9, 9/10, 10/11, 12/13, 14/15, 15/16, 17/18, 19/20, 20/21, 21/22, 22/23, 23/24, 24/25, 25/26, 26/27, 27/28, 28/29, 29/30, 30/31, 31/32, 32/33, 33/34, 34/35, 35/36, 36/37, 38/39, 39/40, 41/42, 42/43, 43/44, 44/45, 45/46, 46/47, 47/48, 48/49, 49/50, 51/52, 52/53, 53/54, 54/55, 55/56, 56/57, 57/58, 58/59, 59/60, 61/62, 62/63, 63/64, 64/65, 65/66, 66/67, 67/68, 68/69, 69/70, 71/72, 72/73, 73/74, 74/75, 75/76, 76/77, 77/78, 78/79, 79/80, 81/82, 82/83, 83/84, 84/85, 85/86, 86/87, 87/88, 88/89, 89/90, or between any two pairs of adjacent residues between 90-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 450-500, 500-550, 550-600, 600-650, 650-700, 700-750, 750-800, 800-850, 850-900, 900-950, 950-1000, 1000-1050, 1050-1100, 1100-1150, 1150-1200, 1200-1250, 1250-1300, 1300-1350, and 1350-1368, relative to SpCas9 of SEQ ID NO: 18, at between any two corresponding residues in an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 99.9% sequence identity with SEQ ID NO: 18, or between any two corresponding residues in a variant or equivalent of SpCas9 of any of amino acid sequences SEQ ID NOs. 19-88, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 99.9% sequence identity with any of SEQ ID NOs: 19-88.
• In various embodiments, the split intein sequences can be engineered by from the following intein sequences.

• NAME	SEQUENCE OF LIGAND-DEPENDENT INTEIN
  2-4 INTEIN:	CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAAA
  	KDGTLLARPVVSWFDQGTRDVIGLRIAGGAIVWAT
  	PDHKVLTEYGWRAAGELRKGDRVAGPGGSGNSLAL
  	SLTADQMVSALLDAEPPILYSEYDPTSPFSEASMM
  	GLLTNLADRELVHMINWAKRVPGFVDLTLHDQAHL
  	LECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRN
  	QGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCL
  	KSIILLNSGVYTFLSSTLKSLEEKDHIHRALDKIT
  	DTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMS
  	NKGMEHLYSMKYKNVVPLYDLLLEMLDAHRLHAGG
  	SGASRVQAFADALDDKFLHDMLAEELRYSVIREVL
  	PTRRARTFDLEVEELHTLVAEGVVVHNC
  	(SEQ ID NO: 472)
  3-2 INTEIN	CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAVA
  	KDGTLLARPVVSWFDQGTRDVIGLRIAGGAIVWAT
  	PDHKVLTEYGWRAAGELRKGDRVAGPGGSGNSLAL
  	SLTADQMVSALLDAEPPILYSEYDPTSPFSEASMM
  	GLLTNLADRELVHMINWAKRVPGFVDLTLHDQAHL
  	LECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRN
  	QGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCL
  	KSIILLNSGVYTFLSSTLKSLEEKDHIHRALDKIT
  	DTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMS
  	NKGMEHLYSMKYTNVVPLYDLLLEMLDAHRLHAGG
  	SGASRVQAFADALDDKFLHDMLAEELRYSVIREVL
  	PTRRARTFDLEVEELHTLVAEGVVVHNC
  	(SEQ ID NO: 473)
  30R3-1 INTEIN	CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAAA
  	KDGTLLARPVVSWFDQGTRDVIGLRIAGGATVWAT
  	PDHKVLTEYGWRAAGELRKGDRVAGPGGSGNSLAL
  	SLTADQMVSALLDAEPPIPYSEYDPTSPFSEASMM
  	GLLTNLADRELVHMINWAKRVPGFVDLTLHDQAHL
  	LECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRN
  	QGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCL
  	KSIILLNSGVYTFLSSTLKSLEEKDHIHRALDKIT
  	DTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMS
  	NKGMEHLYSMKYKNVVPLYDLLLEMLDAHRLHAGG
  	SGASRVQAFADALDDKFLHDMLAEGLRYSVIREVL
  	PTRRARTFDLEVEELHTLVAEGVVVHNC
  	(SEQ ID NO: 474)
  30R3-2 INTEIN	CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAAA
  	KDGTLLARPVVSWFDQGTRDVIGLRIAGGATVWAT
  	PDHKVLTEYGWRAAGELRKGDRVAGPGGSGNSLAL
  	SLTADQMVSALLDAEPPILYSEYDPTSPFSEASMM
  	GLLTNLADRELVHMINWAKRVPGFVDLTLHDQAHL
  	LECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRN
  	QGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCL
  	KSIILLNSGVYTFLSSTLKSLEEKDHIHRALDKIT
  	DTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMS
  	NKGMEHLYSMKYKNVVPLYDLLLEMLDAHRLHAGG
  	SGASRVQAFADALDDKFLHDMLAEELRYSVIREVL
  	PTRRARTFDLEVEELHTLVAEGVVVHNC
  	(SEQ ID NO: 475)
  30R3-3 INTEIN	CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAAA
  	KDGTLLARPVVSWFDQGTRDVIGLRIAGGATVWAT
  	PDHKVLTEYGWRAAGELRKGDRVAGPGGSGNSLAL
  	SLTADQMVSALLDAEPPIPYSEYDPTSPFSEASMM
  	GLLTNLADRELVHMINWAKRVPGFVDLTLHDQAHL
  	LECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRN
  	QGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCL
  	KSIILLNSGVYTFLSSTLKSLEEKDHIHRALDKIT
  	DTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMS
  	NKGMEHLYSMKYKNVVPLYDLLLEMLDAHRLHAGG
  	SGASRVQAFADALDDKFLHDMLAEELRYSVIREVL
  	PTRRARTFDLEVEELHTLVAEGVVVHNC
  	(SEQ ID NO: 476)
  37R3-1 INTEIN	CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAAA
  	KDGTLLARPVVSWFDQGTRDVIGLRIAGGATVWAT
  	PDHKVLTEYGWRAAGELRKGDRVAGPGGSGNSLAL
  	SLTADQMVSALLDAEPPILYSEYNPTSPFSEASMM
  	GLLTNLADRELVHMINWAKRVPGFVDLTLHDQAHL
  	LERAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRN
  	QGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCL
  	KSIILLNSGVYTFLSSTLKSLEEKDHIHRALDKIT
  	DTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMS
  	NKGMEHLYSMKYKNVVPLYDLLLEMLDAHRLHAGG
  	SGASRVQAFADALDDKFLHDMLAEGLRYSVIREVL
  	PTRRARTFDLEVEELHTLVAEGVVVHNC
  	((SEQ ID NO: 477)
  37R3-2 INTEIN	CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAAA
  	KDGTLLARPVVSWFDQGTRDVIGLRIAGGAIVWAT
  	PDHKVLTEYGWRAAGELRKGDRVAGPGGSGNSLAL
  	SLTADQMVSALLDAEPPILYSEYDPTSPFSEASMM
  	GLLTNLADRELVHMINWAKRVPGFVDLTLHDQAHL
  	LERAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRN
  	QGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCL
  	KSIILLNSGVYTFLSSTLKSLEEKDHIHRALDKIT
  	DTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMS
  	NKGMEHLYSMKYKNVVPLYDLLLEMLDAHRLHAGG
  	SGASRVQAFADALDDKFLHDMLAEGLRYSVIREVL
  	PTRRARTFDLEVEELHTLVAEGVVVHNC
  	(SEQ ID NO: 478)
  37R3-3 INTEIN	CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAVA
  	KDGTLLARPVVSWFDQGTRDVIGLRIAGGATVWAT
  	PDHKVLTEYGWRAAGELRKGDRVAGPGGSGNSLAL
  	SLTADQMVSALLDAEPPILYSEYDPTSPFSEASMM
  	GLLTNLADRELVHMINWAKRVPGFVDLTLHDQAHL
  	LERAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRN
  	QGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCL
  	KSIILLNSGVYTFLSSTLKSLEEKDHIHRALDKIT
  	DTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMS
  	NKGMEHLYSMKYKNVVPLYDLLLEMLDAHRLHAGG
  	SGASRVQAFADALDDKFLHDMLAEELRYSVIREVL
  	PTRRARTFDLEVEELHTLVAEGVVVHNC
  	(SEQ ID NO: 479)

• In various other embodiments, the split intein sequences can be used as follows:

• INTEIN-N	INTEIN-C
  NPU-N	NPU-C
  CLSYETEILTVEYGLLPIGKIVEKRI	IKIATRKYLGKQNVY
  ECTVYSVDNNGNIYTQPVAQWHDRGE	DIGVERDHNFALKNG
  QEVFEYCLEDGSLIRATKDHKFMTVD	FIASN
  GQMLPIDEIFERELDLMRVDNLPNSG	(SEQ ID NO: 452)
  GS
  (SEQ ID NO: 447)

• In various embodiments, the split inteins can be used to separately deliver separate portions of a complete PE fusion protein to a cell, which upon expression in a cell, become reconstituted as a complete PE fusion protein through the trans splicing.
• In some embodiments, the disclosure provides a method of delivering a PE fusion protein to a cell, comprising:
• (a) constructing a first expression vector encoding an N-terminal fragment of the PE fusion protein fused to a first split intein sequence;
• (b) constructing a second expression vector encoding a C-terminal fragment of the PE fusion protein fused to a second split intein sequence;
• (c) delivering the first and second expression vectors to a cell,
  wherein the N-terminal and C-terminal fragment are reconstituted as the PE fusion protein in the cell as a result of trans splicing activity causing self-excision of the first and second split intein sequences.
• The split site in some embodiments can be anywhere in the prime editor fusion, including the napDNAbp domain, the linker, or the reverse transcriptase domain.
• In other embodiments, the split site is in the napDNAbp domain.
• In still other embodiments, the split site is in the reverse transcriptase or polymerase domain.
• In yet other embodiments, the split site is in the linker.
• In various embodiments, the present disclosure provides prime editors comprising a napDNAbp (e.g., a Cas9 domain) and a reverse transcriptase wherein one or both of the napDNAbp and/or the reverse transcriptase comprise an intein, for example, a ligand-dependent intein. Typically the intein is a ligand-dependent intein which exhibits no or minimal protein splicing activity in the absence of ligand (e.g., small molecules such as 4-hydroxytamoxifen, peptides, proteins, polynucleotides, amino acids, and nucleotides). Ligand-dependent inteins are known, and include those described in U.S. patent application U.S. Ser. No. 14/004,280, published as U.S. 2014/0065711 A1, the entire contents of which are incorporated herein by reference. In addition, use of split-Cas9 architecture In some embodiments, the intein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-15, 447, 452, 462, and 472-479.
• In various embodiments, the napDNAbp domains are smaller-sized napDNAbp domains as compared to the canonical SpCas9 domain of SEQ ID NO: 18.
• The canonical SpCas9 protein is 1368 amino acids in length and has a predicted molecular weight of 158 kilodaltons. The term “small-sized Cas9 variant”, as used herein, refers to any Cas9 variant—naturally occurring, engineered, or otherwise—that is less than at least 1300 amino acids, or at least less than 1290 amino acids, or than less than 1280 amino acids, or less than 1270 amino acid, or less than 1260 amino acid, or less than 1250 amino acids, or less than 1240 amino acids, or less than 1230 amino acids, or less than 1220 amino acids, or less than 1210 amino acids, or less than 1200 amino acids, or less than 1190 amino acids, or less than 1180 amino acids, or less than 1170 amino acids, or less than 1160 amino acids, or less than 1150 amino acids, or less than 1140 amino acids, or less than 1130 amino acids, or less than 1120 amino acids, or less than 1110 amino acids, or less than 1100 amino acids, or less than 1050 amino acids, or less than 1000 amino acids, or less than 950 amino acids, or less than 900 amino acids, or less than 850 amino acids, or less than 800 amino acids, or less than 750 amino acids, or less than 700 amino acids, or less than 650 amino acids, or less than 600 amino acids, or less than 550 amino acids, or less than 500 amino acids, but at least larger than about 400 amino acids and retaining the required functions of the Cas9 protein.
• In one embodiment, as depicted in Example 20, the specification embraces the following split-intein PE constructs, which are split between residues 1024 and 1025 of the canonical SpCas9 (SEQ ID NO: 18) (or which may be referred to as residues 1023 and 1024, respectively, relative to a Met-minus SEQ ID NO: 18).
• First, the amino acid sequence of SEQ ID NO: 18 is shown as follows, indicating the location of the split site between 1024 (“K”) and 1025 (“S”) residues:

• Description	Sequence	SEQ ID NO:
  SpCas9	M DKKYSIGLDIGTNSVGWAVI	SEQ ID NO:
  Streptococcus	TDEYKVPSKKFKVLGNTDRH	18,
  pyogenes	SIKKNLIGALLFDSGETAEA	indicated
  M1	TRLKRTARRRYTRRKNRICY	with
  SwissProt	LQEIFSNEMAKVDDSFFHRL	split site
  Accession	EESFLVEEDKKHERHPIFGN	1024/1025
  No.	IVDEVAYHEKYPTIYHLRKK	in
  Q99ZW2	LVDSTDKADLRLIYLALAHM	bold
  Wild type	IKFRGHFLIEGDLNPDNSDV	The M at
  	DKLFIQLVQTYNQLFEENPI	position
  	NASGVDAKAILSARLSKSRR
  	1 is not
  	LENLIAQLPGEKKNGLFGNL	necessarily
  	IALSLGLTPNFKSNFDLAED	present
  	AKLQLSKDTYDDDLDNLLAQ	in the
  	IGDQYADLFLAAKNLSDAIL	PE fusion
  	LSDILRVNTEITKAPLSASM	protein in
  	IKRYDEHHQDLTLLKALVRQ	certain
  	QLPEKYKEIFFDQSKNGYAG	embodiments.
  	YIDGGASQEEFYKFIKPILE	Thus, the
  	KMDGTEELLVKLNREDLLRK	numbering
  	QRTFDNGSIPHQIHLGELHA	of
  	ILRRQEDFYPFLKDNREKIE	the split
  	KILTFRIPYYVGPLARGNSR	site is
  	FAWMTRKSEETITPWNFEEV	1023/1024
  	VDKGASAQSFIERMTNFDKN	in
  	LPNEKVLPKHSLLYEYFTVY	the case
  	NELTKVKYVTEGMRKPAFLS	that the
  	GEQKKAIVDLLFKTNRKVTV	amino acid
  	KQLKEDYFKKIECFDSVEIS	sequence
  	GVEDRFNASLGTYHDLLKII	excludes
  	KDKDFLDNEENEDILEDIVL	Met at
  	TLTLFEDREMIEERLKTYAH	position	1.
  	LFDDKVMKQLKRRRYTGWGR
  	LSRKLINGIRDKQSGKTILD
  	FLKSDGFANRNFMQLIHDDS
  	LTFKEDIQKAQVSGQGDSLH
  	EHIANLAGSPAIKKGILQTV
  	KVVDELVKVMGRHKPENIVI
  	EMARENQTTQKGQKNSRERM
  	KRIEEGIKELGSQILKEHPV
  	ENTQLQNEKLYLYYLQNGRD
  	MYVDQELDINRLSDYDVDHI
  	VPQSFLKDDSIDNKVLTRSD
  	KNRGKSDNVPSEEVVKKMKN
  	YWRQLLNAKLITQRKFDNLT
  	KAERGGLSELDKAGFIKRQL
  	VETRQITKHVAQILDSRMNT
  	KYDENDKLIREVKVITLKSK
  	LVSDFRKDFQFYKVREINNY
  	HHAHDAYLNAVVGTALIKKY
  	PKLESEFVYGDYKVYDVRKM
  	IA KS EQEIGKATAKYFFYSN
  	IMNFFKTEITLANGEIRKRP
  	LIETNGETGEIVWDKGRDFA
  	TVRKVLSMPQVNIVKKTEVQ
  	TGGFSKESILPKRNSDKLIA
  	RKKDWDPKKYGGFDSPTVAY
  	SVLVVAKVEKGKSKKLKSVK
  	ELLGITIMERSSFEKNPIDF
  	LEAKGYKEVKKDLIIKLPKY
  	SLFELENGRKRMLASAGELQ
  	KGNELALPSKYVNFLYLASH
  	YEKLKGSPEDNEQKQLFVEQ
  	HKHYLDEIIEQISEFSKRVI
  	LADANLDKVLSAYNKHRDKP
  	IREQAENIIHLFTLTNLGAP
  	AAFKYFDTTIDRKRYTSTKE
  	VLDATLIHQSITGLYETRID
  	LSQLGGD

• In this configuration, the amino acid sequence of N-terminal half (amino acids 1-1024) is as follows:

• 	(SEQ ID NO: 3877)
  	MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVL
  	GNTDRHSIKKNLIGALLFDSGETAEATRLKRTARR
  	RYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
  	LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRK
  	KLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLN
  	PDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKA
  	ILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALS
  	LGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLA
  	QIGDQYADLFLAAKNLSDAILLSDILRVNTEITKA
  	PLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI
  	FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDG
  	TEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELH
  	AILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPL
  	ARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQS
  	FIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELT
  	KVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVT
  	VKQLKEDYFKKIECFDSVETSGVEDRFNASLGTYH
  	DLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRE
  	MIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRK
  	LINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD
  	SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKK
  	GILQTVKVVDELVKVMGRHKPENIVIEMARENQTT
  	QKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQ
  	LQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDA
  	IVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEV
  	VKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSE
  	LD K AGFIKRQLVETRQITKHVAQILDSRMNTKYDE
  	NDKLIREVKVITLKSKLVSDFRKDFQFYKVREINN
  	YHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKV
  	YDVRKMIAK.

• In this configuration, the amino acid sequence of N-terminal half (amino acids 1-1023) (where the protein is Met-minus at position 1) is as follows:

• 	(SEQ ID NO: 3878)
  	DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLG
  	NTDRHSIKKNLIGALLFDSGETAEATRLKRTARRR
  	YTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFL
  	VEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKK
  	LVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNP
  	DNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAI
  	LSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSL
  	GLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
  	IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAP
  	LSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIF
  	FDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGT
  	EELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHA
  	ILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLA
  	RGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSF
  	IERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTK
  	VKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTV
  	KQLKEDYFKKIECFDSVETSGVEDRFNASLGTYHD
  	LLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREM
  	IEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKL
  	INGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDS
  	LTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKG
  	ILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQ
  	KGQKNSRERMKRIEEGIKELGSQILKEHPVENTQL
  	QNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAI
  	VPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVV
  	KKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSEL
  	DKAGFIKRQLVETRQITKHVAQILDSRMNTKYDEN
  	DKLIREVKVITLKSKLVSDFRKDFQFYKVREINNY
  	HHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVY
  	DVRKMIA K .

• In this configuration, the amino acid sequence of C-terminal half (amino acids 1024-1368 (or counted as amino acids 1023-1367 in a Met-minus Cas9) is as follows:

• 	(SEQ ID NO: 3879)
  	S EQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKR
  	PLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIV
  	KKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKK
  	YGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLG
  	ITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK
  	YSLFELENGRKRMLASAGELQKGNELALPSKYVNF
  	LYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEII
  	EQISEFSKRVILADANLDKVLSAYNKHRDKPIREQ
  	AENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTK
  	EVLDATLIHQSITGLYETRIDLSQLGGD.

• As shown in Example 20, the PE2 (which is based on SpCas9 of SEQ ID NO: 18) construct was split at position 1023/1024 (relative to a Met-minus SEQ ID NO: 18) into two separate constructs, as follows:
• SpPE2 Split at 1023/1024 N Terminal Half

• (SEQ ID NO: 3875)

  MKRTADGSEFESPKKKRKVDKKYSIGLDIGTNSVGWAVITDEYKVPSKKF

  KVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYL

  QEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKY

  PTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVD

  KLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGE

  KKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI

  GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDL

  TLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEK

  MDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPF

  LKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVV

  DKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTE

  GMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISG

  VEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMI

  EERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDF

  LKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPA

  IKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMK

  RIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINR

  LSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNY

  WRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVA

  QILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYH

• SpPE2 split at 1023/1024 C terminal half

• (SEQ ID NO: 3876)

  EIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLI

  ARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIME

  RSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGEL

  QKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEII

  EQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGA

  SLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQE

  ARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVN

  KRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFA

  FEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLIL

  LQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYL

  GYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGKAGFCRLFIPGFAE

  MAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELF

  VDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVL

  TKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDR

  VQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADH

  TWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQA

  LKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGWLTSEGKEIKNKDEILA

  LLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTST

• The present disclosure also contemplates methods of delivering split-intein prime editors to cells and/or treating cells with split-intein prime editors.
• In some embodiments, the disclosure provides a method of delivering a PE fusion protein to a cell, comprising:
• • (a) constructing a first expression vector encoding an N-terminal fragment of the PE fusion protein fused to a first split intein sequence;
  • (b) constructing a second expression vector encoding a C-terminal fragment of the PE fusion protein fused to a second split intein sequence;
  • (c) delivering the first and second expression vectors to a cell,
    wherein the N-terminal and C-terminal fragment are reconstituted as the PE fusion protein in the cell as a result of trans splicing activity causing self-excision of the first and second split intein sequences.
• In certain embodiments, the N-terminal fragment of the PE fusion protein fused to a first split intein sequence is SEQ ID NO: 3875, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99.9% sequence identity with SEQ ID NO: 3875.
• In other embodiments, the C-terminal fragment of the PE fusion protein fused to a first split intein sequence is SEQ ID NO: 3876, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99.9% sequence identity with SEQ ID NO: 3876.
• In other embodiments, the disclosure provides a method of editing a target DNA sequence within a cell, comprising:
• • (a) constructing a first expression vector encoding an N-terminal fragment of the PE fusion protein fused to a first split intein sequence;
  • (b) constructing a second expression vector encoding a C-terminal fragment of the PE fusion protein fused to a second split intein sequence;
  • (c) delivering the first and second expression vectors to a cell,
    wherein the N-terminal and C-terminal fragment are reconstituted as the PE fusion protein in the cell as a result of trans splicing activity causing self-excision of the first and second split intein sequences.
• In certain embodiments, the N-terminal fragment of the PE fusion protein fused to a first split intein sequence is SEQ ID NO: 3875, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99.9% sequence identity with SEQ ID NO: 3875.
• In other embodiments, the C-terminal fragment of the PE fusion protein fused to a first split intein sequence is SEQ ID NO: 3876, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99.9% sequence identity with SEQ ID NO: 3876.
Delivery of PE Ribonucleoprotein Complexes
• In this aspect, the prime editors may be delivered by non-viral delivery strategies involving delivery of a prime editor complexed with a PEgRNA (i.e., a PE ribonucleoprotein complex) by various methods, including electroporation and lipid nanoparticles. Methods of non-viral delivery of nucleic acids include lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Feigner, WO 91/17424; WO 91/16024. Delivery can be to cells (e.g. in vitro or ex vivo administration) or target tissues (e.g. in vivo administration).
• The preparation of lipid:nucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to one of skill in the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese et al., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res. 52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).
• Additional reference may be made to the following references that discuss approaches for non-viral delivery of ribonucleoprotein complexes, each of which are incorporated herein by reference.
• Chen, Sean, et al. “Highly efficient mouse genome editing by CRISPR ribonucleoprotein electroporation of zygotes.” Journal of Biological Chemistry (2016): jbc-M116. PubMed
• Zuris, John A., et al. “Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo.” Nature biotechnology 33.1 (2015): 73. PubMed
• Rouet, Romain, et al. “Receptor-Mediated Delivery of CRISPR-Cas9 Endonuclease for Cell-Type-Specific Gene Editing.” Journal of the American Chemical Society 140.21 (2018): 6596-6603. PubMed.
• FIG. 68C provides data showing that various disclosed PE ribonucleoprotein complexes (PE2 at high concentration, PE3 at high concentration and PE3 at low concentration) can be delivered in this manner.
• Delivery of PE by mRNA
• Another method that may be employed to deliver prime editors and/or PEgRNAs to cells in which prime editing-based genome editing is desired is by employing the use of messenger RNA (mRNA) delivery methods and technologies. Examples of mRNA delivery methods and compositions that may be utilized in the present disclosure including, for example, PCT/US2014/028330, U.S. Pat. No. 8,822,663B2, NZ700688A, ES2740248T3, EP2755693A4, EP2755986A4, WO2014152940A1, EP3450553B1, BR112016030852A2, and EP3362461A1, each of which are incorporated herein by reference in their entireties. Additional disclosure hereby incorporated by reference can be found in Kowalski et al., “Delivering the Messenger: Advances in Technologies for Therapeutic mRNA Delivery,” Mol Therap., 2019; 27(4): 710-728.
• In contrast to DNA vector encoding prime editors, the use of RNA as delivery agent for prime editors has the advantage that the genetic material does not have to enter the nucleus to perform its function. The delivered mRNA may be directly translated in the cytoplasm into the desired protein (e.g., prime editor fusion protein) and nucleic acid products (e.g., PEgRNA). However, in order to be more stable (e.g., resist RNA-degrading enzymes in the cytoplasm), it is in some embodiments necessary to stabilize the mRNA to improve delivery efficiency. Certain delivery carriers such as cationic lipids or polymeric delivery carriers can also help protect the transfected mRNA from endogenous RNase enzymes that might otherwise degrade the therapeutic mRNA encoding the desired prime editor fusion proteins. In addition, despite the increased stability of modified mRNA, delivery of mRNA, particularly mRNA encoding full-length protein, to cells in vivo in a manner that allows therapeutic levels of protein production remains a challenge.
• With some exceptions, the intracellular delivery of mRNA is generally more challenging than that of small oligonucleotides, and it requires encapsulation into a delivery nanoparticle, in part due to the significantly larger size of mRNA molecules (300-5,000 kDa, ˜1-15 kb) as compared to other types of RNAs (small interfering RNAs [siRNAs], ˜14 kDa; antisense oligonucleotides [ASOs], 4-10 kDa).
• mRNA must cross the cell membrane in order to reach the cytoplasm. The cell membrane is a dynamic and formidable barrier to intracellular delivery. It is made up primarily of a lipid bilayer of zwitterionic and negatively charged phospholipids, where the polar heads of the phospholipids point toward the aqueous environment and the hydrophobic tails form a hydrophobic core.
• In some embodiments, the mRNA compositions of the disclosure comprise mRNA (encoding a prime editor and/or PEgRNA), a transport vehicle, and optionally an agent that facilitates contact with the target cell and subsequent transfection.
• In some embodiments, the mRNA can include one or more modifications that confer stability to the mRNA (eg, compared to the wild-type or native version of the mRNA) and is involved in the associated abnormal expression of the protein. One or more modifications to the wild type that correct the defect may also be included. For example, the nucleic acids of the invention can include modifications of one or both of a 5′ untranslated region or a 3′ untranslated region. Such modifications may include the inclusion of sequences encoding a partial sequence of the cytomegalovirus (CMV) immediate early 1 (IE1) gene, poly A tail, Cap1 structure, or human growth hormone (hGH). In some embodiments, the mRNA is modified to reduce mRNA immunogenicity.
• In one embodiment, the “prime editor” mRNA in the composition of the invention can be formulated in a liposome transfer vehicle to facilitate delivery to target cells. Contemplated transfer vehicles can include one or more cationic lipids, non-cationic lipids, and/or PEG-modified lipids. For example, the transfer vehicle can include at least one of the following cationic lipids: C12-200, DLin-KC2-DMA, DODAP, HGT4003, ICE, HGT5000, or HGT5001. In embodiments, the transfer vehicle comprises cholesterol (chol) and/or PEG modified lipids. In some embodiments, the transfer vehicle comprises DMG-PEG2K. In certain embodiments, the transfer vehicle has the following lipid formulation: C12-200, DOPE, chol, DMG-PEG2K; DODAP, DOPE, cholesterol, DMG-PEG2K; HGT5000, DOPE, chol, DMG-PEG2K, HGT5001, DOPE, chol, one of DMG-PEG2K.
• The present disclosure also provides compositions and methods useful for facilitating transfection of target cells with one or more PE-encoding mRNA molecules. For example, the compositions and methods of the present invention contemplate the use of targeting ligands that can increase the affinity of the composition for one or more target cells. In one embodiment, the targeting ligand is apolipoprotein B or apolipoprotein E, and the corresponding target cells express low density lipoprotein receptors and thus promote recognition of the targeting ligand. A vast number of target cells can be preferentially targeted using the methods and compositions of the present disclosure. For example, contemplated target cells include hepatocytes, epithelial cells, hematopoietic cells, epithelial cells, endothelial cells, lung cells, bone cells, stem cells, mesenchymal cells, nerve cells, heart cells, adipocytes, vascular smooth muscle Includes cells, cardiomyocytes, skeletal muscle cells, beta cells, pituitary cells, synovial lining cells, ovarian cells, testis cells, fibroblasts, B cells, T cells, reticulocytes, leukocytes, granulocytes, and tumor cells However, it is not limited to these.
• In some embodiments, the PE-encoding mRNA may optionally have chemical or biological modifications which, for example, improve the stability and/or half-life of such mRNA or which improve or otherwise facilitate protein production. Upon transfection, a natural mRNA in the compositions of the invention may decay with a half-life of between 30 minutes and several days. The mRNAs in the compositions of the disclosure may retain at least some ability to be translated, thereby producing a functional protein or enzyme. Accordingly, the invention provides compositions comprising and methods of administering a stabilized mRNA. In some embodiments, the activity of the mRNA is prolonged over an extended period of time. For example, the activity of the mRNA may be prolonged such that the compositions of the present disclosure are administered to a subject on a semi-weekly or bi-weekly basis, or more preferably on a monthly, bi-monthly, quarterly or an annual basis. The extended or prolonged activity of the mRNA of the present invention is directly related to the quantity of protein or enzyme produced from such mRNA. Similarly, the activity of the compositions of the present disclosure may be further extended or prolonged by modifications made to improve or enhance translation of the mRNA. Furthermore, the quantity of functional protein or enzyme produced by the target cell is a function of the quantity of mRNA delivered to the target cells and the stability of such mRNA. To the extent that the stability of the mRNA of the present invention may be improved or enhanced, the half-life, the activity of the produced protein or enzyme and the dosing frequency of the composition may be further extended.
• Accordingly, in some embodiments, the mRNA in the compositions of the disclosure comprise at least one modification which confers increased or enhanced stability to the nucleic acid, including, for example, improved resistance to nuclease digestion in vivo. As used herein, the terms “modification” and “modified” as such terms relate to the nucleic acids provided herein, include at least one alteration which preferably enhances stability and renders the mRNA more stable (e.g., resistant to nuclease digestion) than the wild-type or naturally occurring version of the mRNA. As used herein, the terms “stable” and “stability” as such terms relate to the nucleic acids of the present invention, and particularly with respect to the mRNA, refer to increased or enhanced resistance to degradation by, for example nucleases (i.e., endonucleases or exonucleases) which are normally capable of degrading such mRNA. Increased stability can include, for example, less sensitivity to hydrolysis or other destruction by endogenous enzymes (e.g., endonucleases or exonucleases) or conditions within the target cell or tissue, thereby increasing or enhancing the residence of such mRNA in the target cell, tissue, subject and/or cytoplasm. The stabilized mRNA molecules provided herein demonstrate longer half-lives relative to their naturally occurring, unmodified counterparts (e.g. the wild-type version of the mRNA). Also contemplated by the terms “modification” and “modified” as such terms related to the mRNA of the present invention are alterations which improve or enhance translation of mRNA nucleic acids, including for example, the inclusion of sequences which function in the initiation of protein translation (e.g., the Kozak consensus sequence). (Kozak, M., Nucleic Acids Res 15 (20): 8125-48 (1987)).
• In some embodiments, the mRNAs used in the compositions of the disclosure have undergone a chemical or biological modification to render them more stable. Exemplary modifications to an mRNA include the depletion of a base (e.g., by deletion or by the substitution of one nucleotide for another) or modification of a base, for example, the chemical modification of a base. The phrase “chemical modifications” as used herein, includes modifications which introduce chemistries which differ from those seen in naturally occurring mRNA, for example, covalent modifications such as the introduction of modified nucleotides, (e.g., nucleotide analogs, or the inclusion of pendant groups which are not naturally found in such mRNA molecules).
• Other suitable polynucleotide modifications that may be incorporated into the PE-encoding mRNA used in the compositions of the disclosure include, but are not limited to, 4′-thio-modified bases: 4′-thio-adenosine, 4′-thio-guanosine, 4′-thio-cytidine, 4′-thio-uridine, 4′-thio-5-methyl-cytidine, 4′-thio-pseudouridine, and 4′-thio-2-thiouridine, pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladeno sine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine, inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine, and combinations thereof. The term modification also includes, for example, the incorporation of non-nucleotide linkages or modified nucleotides into the mRNA sequences of the present invention (e.g., modifications to one or both of the 3′ and 5′ ends of an mRNA molecule encoding a functional protein or enzyme). Such modifications include the addition of bases to an mRNA sequence (e.g., the inclusion of a poly A tail or a longer poly A tail), the alteration of the 3′ UTR or the 5′ UTR, complexing the mRNA with an agent (e.g., a protein or a complementary nucleic acid molecule), and inclusion of elements which change the structure of an mRNA molecule (e.g., which form secondary structures).
• In some embodiments, PE-encoding mRNAs include a 5′ cap structure. A 5′ cap is typically added as follows: first, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5′ nucleotide, leaving two terminal phosphates; guanosine triphosphate (GTP) is then added to the terminal phosphates via a guanylyl transferase, producing a 5′5′5 triphosphate linkage; and the 7-nitrogen of guanine is then methylated by a methyltransferase. Examples of cap structures include, but are not limited to, m7G(5′)ppp (5′(A,G(5′)ppp(5′)A and G(5′)ppp(5′)G. Naturally occurring cap structures comprise a 7-methyl guanosine that is linked via a triphosphate bridge to the 5′-end of the first transcribed nucleotide, resulting in a dinucleotide cap of m7G(5′)ppp(5′)N, where N is any nucleoside. In vivo, the cap is added enzymatically. The cap is added in the nucleus and is catalyzed by the enzyme guanylyl transferase. The addition of the cap to the 5′ terminal end of RNA occurs immediately after initiation of transcription. The terminal nucleoside is typically a guanosine, and is in the reverse orientation to all the other nucleotides, i.e., G(5′)ppp(5′)GpNpNp.
• Additional cap analogs include, but are not limited to, a chemical structures selected from the group consisting of m7GpppG, m7GpppA, m7GpppC; unmethylated cap analogs (e.g., GpppG); dimethylated cap analog (e.g., m2,7GpppG), trimethylated cap analog (e.g., m2,2,7GpppG), dimethylated symmetrical cap analogs (e.g., m7Gpppm7G), or anti reverse cap analogs (e.g., ARCA; m7,2′OmeGpppG, m72′dGpppG, m7,3′OmeGpppG, m7,3′dGpppG and their tetraphosphate derivatives) (see, e.g., Jemielity, J. et al., “Novel ‘anti-reverse’ cap analogs with superior translational properties”, RNA, 9: 1108-1122 (2003)).
• Typically, the presence of a “tail” serves to protect the mRNA from exonuclease degradation. A poly A or poly U tail is thought to stabilize natural messengers and synthetic sense RNA. Therefore, in certain embodiments a long poly A or poly U tail can be added to an mRNA molecule thus rendering the RNA more stable. Poly A or poly U tails can be added using a variety of art-recognized techniques. For example, long poly A tails can be added to synthetic or in vitro transcribed RNA using poly A polymerase (Yokoe, et al. Nature Biotechnology. 1996; 14: 1252-1256). A transcription vector can also encode long poly A tails. In addition, poly A tails can be added by transcription directly from PCR products. Poly A may also be ligated to the 3′ end of a sense RNA with RNA ligase (see, e.g., Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1991 edition)).
• Typically, the length of a poly A or poly U tail can be at least about 10, 50, 100, 200, 300, 400 at least 500 nucleotides. In some embodiments, a poly-A tail on the 3′ terminus of mRNA typically includes about 10 to 300 adenosine nucleotides (e.g., about 10 to 200 adenosine nucleotides, about 10 to 150 adenosine nucleotides, about 10 to 100 adenosine nucleotides, about 20 to 70 adenosine nucleotides, or about 20 to 60 adenosine nucleotides). In some embodiments, mRNAs include a 3′ poly(C) tail structure. A suitable poly-C tail on the 3′ terminus of mRNA typically include about 10 to 200 cytosine nucleotides (e.g., about 10 to 150 cytosine nucleotides, about 10 to 100 cytosine nucleotides, about 20 to 70 cytosine nucleotides, about 20 to 60 cytosine nucleotides, or about 10 to 40 cytosine nucleotides). The poly-C tail may be added to the poly-A or poly U tail or may substitute the poly-A or poly U tail.
• PE-encoding mRNAs according to the present disclosure may be synthesized according to any of a variety of known methods. For example, mRNAs according to the present invention may be synthesized via in vitro transcription (IVT). Briefly, IVT is typically performed with a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7 or SP6 RNA polymerase), DNAse I, pyrophosphatase, and/or RNAse inhibitor. The exact conditions will vary according to the specific application.
• In embodiments involving mRNA delivery, the ratio of the mRNA encoding the PE fusion protein to the PEgRNA may be important for efficient editing. In certain embodiments, the weight ratio of mRNA (encoding the PE fusion protein) to PEgRNA is 1:1. In certain other embodiments, the weight ratio of mRNA (encoding the PE fusion protein) to PEgRNA is 2:1. In still other embodiments, the weight ratio of mRNA (encoding the PE fusion protein) to PEgRNA is 1:2. In still further embodiments, the weight ratio of mRNA (encoding the PE fusion protein) to PEgRNA is selected from the group consisting of about 1:1000, 1:900; 1:800; 1:700; 1:600; 1:500; 1:400; 1:300; 1:200; 1:100; 1:90; 1:80; 1:70; 1:60; 1:50; 1:40; 1:30; 1:20; 1:10; and 1:1. In other embodiments, the weight ratio of mRNA (encoding the PE fusion protein) to PEgRNA is selected from the group consisting of about 1:1000, 1:900; 800:1; 700:1; 600:1; 500:1; 400:1; 300:1; 200:1; 100:1; 90:1; 80:1; 70:1; 60:1; 50:1; 40:1; 30:1; 20:1; 10:1; and 1:1.
• J. Use of Prime Editing for Identifying Off-Target Editing in an Unbiased Manner
• Like other genome editors, there exists some risk that PE may introduce its programmed genetic alterations at unintended sites around the genome, i.e., “off-target” sites. However, there are currently no described methods to detect off-target editing with prime editors. Such methods would allow the identification of potential sites of off-target editing using prime editors.
• The key concept of this aspect is the idea of using prime editing to insert the same adapter sequence and/or primer binding site at on-target and off-target sites, templated from the same PEgRNA, to enable the rapid identification of genomic off-target modification sites of napDNAbp nucleases or prime editors. This method is distinguished from other techniques that identify nuclease off-target sites because the adapter and/or primer binding sequence is inserted in the same event as DNA binding and nicking by the napDNAbp, simplifying the downstream processing.
• FIG. 33 illustrates the basic principle of off-target identification. The figure is a schematic showing PEgRNA design for primer binding sequence insertions and primer binding insertion into genomic DNA using prime editing for determining off-target editing. In this embodiment, prime editing is conducted inside a living cell, a tissue, or an animal model. As a first step, an appropriate PEgRNA is designed. The top schematic shows a exemplary PEgRNA that may be used in this aspect. The spacer in the PEgRNA (labeled as “protospacer”) is complementary to one of the strands of the genomic target. The PE:PEgRNA complex (i.e., the PE complex) installs a single stranded 3′ end flap at the nick site which contains the encoded primer binding sequence and the region of homology (coded by the homology arm of the PEgRNA) that is complementary to the region just downstream of the cut site (in red). Through flap invasion and DNA repair/replication processes, the synthesized strand becomes incorporated into the DNA, thereby installing the primer binding site. This process can occur at the desired genomic target, but also at other genomic sites that might interact with the PEgRNA in an off-target manner (i.e., the PEgRNA guides the PE complex to other off-target sites due to the complementarity of the spacer region to other genomic sites that are not the intended genomic site). Thus, the primer binding sequence may be installed not only at the desired genomic target, but at off-target genomic sites elsewhere in the genome. In order to detect the insertion of these primer binding sites at both the intended genomic target sites and the off-target genomic sites, the genomic DNA (post-PE) can be isolated, fragmented, and ligated to adapter nucleotides (shown in red). Next, PCR may be carried out with PCR oligonucleotides that anneal to the adapters and to the inserted primer binding sequence to amplify on-target and off-target genomic DNA regions into which the primer binding site was inserted by PE. High throughput sequencing then may be conducted, as well as sequence alignments, to identify the insertion points of PE-inserted primer binding sequences at either the on-target site or at off-target sites.
• Thus, FIG. 33 illustrates one aspect regarding the identification of off-target editing sites when editing inside a living cell, in tissue culture or animal models. To conduct this method, a PEgRNA is generated that has an identical spacer to the final desired prime editor (and, if looking at prime editing off-targets, an identical primer-binding site sequence to the final desired editor), but includes the necessary sequences to install an adapter or primer binding site after reverse transcription by prime editing. In vivo editing is conducted using a prime editor or RT-fused nuclease, and isolate genomic DNA. The genomic DNA is fragmented by enzymatic or mechanical means and append a different adapter to sites of DNA fragmentation. PCR is used to amplify from one adapter to the adapter installed via PEgRNA. The resulting product is deep-sequenced to identify all modified sites.
• In another aspect, evaluation of off-target editing by PE may be conducted in vitro. In this aspect, PE may be used during in vitro modification of genomic DNA identification of off-target editing sites using in vitro modification of genomic DNA. To conduct this method, ribonucleoprotein (RNP) of purified prime editor fusion protein and a PEgRNA (i.e., the PE complex) is assembled that is configured to install an adapter or primer binding sequence at a target site, but is otherwise the same as the PEgRNA of interest. This RNP (i.e., PE complex) is incubated with extracted genomic DNA before or after fragmentation of the DNA. After fragmentation, different adapters sequences are ligated to the ends of the fragment DNA. PCR is used to amplify those genomic sites that span the inserted adapter sequence (i.e., inserted by EP) and the adapters ligated to fragment ends. High throughput sequencing between the adapters sequences can identify genomic sites of modification that are on-target and off-target. This in vitro editing method should enhance the sensitivity of detection because cellular DNA repair will not eliminate the reverse-transcribed DNA adapter added by the prime editor.
• These methods could be used to identify off-target editing for any prime editor, or any genome editor that uses a guide RNA to recognize a target cut site (most Cas nucleases).
• These methods could be applied to all genetic diseases for which genome editors are considered for use in treatment.
• Exemplary adapter and/or primer binding sequences that may be installed by PE include, but are not limited to:

• ADAPTER 1	5′-CGGTGGACCGATGATCT-3′ (SEQ ID NO: 177)
  ADAPTER 2	5′-GCCACCTGGCTACTAGA-3′ (SEQ ID NO: 178)
  ADAPTER 3	5′-AGATCATCGGTCCACCG-3′ (SEQ ID NO: 179)
  ADAPTER 4	5′-TCTAGTAGCCAGGTGGC-3′ (SEQ ID NO: 180)

• These adapter and/or primer binding sequences may also be used in the ligation step after genomic DNA fragmentation as outlined above.
• Exemplary PEgRNA designs that illustrate the use of the herein described method to evaluate off-target editing, and their edit target locus, are as follows:

• HEK3 TEST	GGCCCAGACTGAGCACGTGA GTTTTAGAGCTAGAAATA
  LOCUS	GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA
  OFF-	AAAGTGGCACCGAGTCGGTGCTCTGCCATCA CGGTG
  TARGET	GACCGATGATCTCGTGCTCAGTCTG
  DISCOVERY:	(SEQ ID NO: 181)
  HEK4 TEST	GGCACTGCGGCTGGAGGTGGGTTTTAGAGCTAGAAAT
  LOCUS	AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA
  OFF-	AAAAGTGGCACCGAGTCGGTGCACACAGCACCAGAG
  TARGET	TCTCCGCTTTAACCCCCA GCCACCTGGCTACTAGACC
  DISCOVERY:	TCCAGCC (SEQ lD NO: 182)
  SICKLE CELL	GCATGGTGCACCTGACTCCTGGTTTTAGAGCTAGAAA
  CORRECTION	TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG
  OFF-	AAAAAGTGGCACCGAGTCGGTGCAGACTTCTCTTCA
  TARGET	G GCCACCTGGCTACTAGAGAGTCAGGTGCAC
  DISCOVERY:	(SEQ ID NO: 183)
  KEY
  PBS Spacer
  sgRNA scaffold
  DNA synthesis template
  Adapter
  PBS

• K. Use of Prime Editing for Insertion of Inducible Dimerization Domains
• The prime editors described herein may also be used to install dimerization domains into one or more protein targets. The dimerization domains may facilitate inducible regulation of the activity associated with the dimerization of the one or more protein targets via a linking moiety (e.g., a small molecule, peptide, or protein) that binds in a bi-specific manner. In various aspects, the dimerization domains, when installed on different proteins (e.g., the same type or different proteins), each bind to the same bi-specific moiety (e.g., a bi-specific small molecule, peptide, or polypeptide having a least two regions that separately bind to the dimerization domains), thereby causing the dimerization of the proteins through their common interaction to the bi-specific ligand. In this manner, the bi-specific ligand functions as an “inducer” of dimerization of two proteins. In some cases, the bi-specific ligand or compound will have two targeting moieties that are the same. In other embodiments, the bi-specific ligand or compound will have targeting moieties that are each different from the other. The bi-specific ligand or compound having the same two targeting moieties will be able to target the same dimerization domain installed on different protein targets. The bi-specific ligand or compound having different targeting moieties will be able to target different dimerization domains installed on different protein targets.
• As used herein, the term “dimerization domain” refers to a ligand-binding domain that binds to a binding moiety of a bi-specific ligand. A “first” dimerization domain binds to a first binding moiety of a bi-specific ligand and a “second” dimerization domain binds to a second binding moiety of the same bi-specific ligand. When the first dimerization domain is fused to a first protein (e.g., via PE, as discussed herein) and the second dimerization domain (e.g., via PE, as discussed herein) is fused to a second protein, the first and second protein dimerize in the presence of a bi-specific ligand, wherein the bi-specific ligand has at least one moeity that binds to the first dimerization domain and at least another moiety that binds to the second dimerization domain.
• The term “bi-specific ligand” or “bi-specific moiety,” as used herein, refers to a ligand that binds to two different ligand-binding domains. In various embodiments, the bi-specific moiety itself is a dimer of two of same or two different chemical moieties, wherein each moiety specifically and tightly binds to a dimerization domain. In certain embodiments, the ligand is a small molecule compound, or a peptide, or a polypeptide. In other embodiments, ligand-binding domain is a “dimerization domain,” which can be install as a peptide tag onto a protein. In various embodiments, two proteins each comprising the same or different dimerization domains can be induced to dimerize through the binding of each dimerization domain to the bi-specific ligand. These molecules may also be referred to as “chemical inducers of dimerization” or CIDs. In addition, the bi-specific ligands may be prepared by coupling (e.g., through standardize chemical linkages) two of the same moieties together, or two different moieties together, wherein each moiety tightly and specifically binds to a dimerization domain.
• In various aspects, the dimerization domains installed by PE can be the same or different.
• For example, the dimerization domains can be FKBP12, which has the following amino acid sequence:

• 	FKBP12	MGVQVETISPGDGRTFPKRG
  		QTCVVHYTGMLEDGKKFDS
  		SRDRNKPFKFMLGKQEVIR
  		GWEEGVAQMSVGQRAKLTIS
  		PDYAYGATGHPGIIPPHAT
  		LVFDVELLKLE
  		(SEQ ID NO: 488)

• In another example, the dimerization domain can be a mutant of FKBP12 referred to as FKBP12-F36V, a mutant of FKBP12 with an engineered hole that binds a synthetic bumped FK506 mimic (2, FIG. 3 )¹⁰⁷:

• 	FKBP12-F36V	MGVQVETISPGDGRTFPKRG
  		QTCVVHYTGMLEDGKKVD
  		SSRDRNKPFKFMLGKQEVI
  		RGWEEGVAQMSVGQRAKLT
  		ISPDYAYGATGHPGIIPP
  		HATLVFDVELLKLE
  		(SEQ ID NO: 489)

• In another example, the dimerization domain can be cyclophilin, as follows:

• 	CYCLOPHILIN	MVNPTVFFDIAVDGEPLGRV
  		SFELFADKVPKTAENFRA
  		LSTGEKGFGYKGSCFHRII
  		PGFMCQGGDFTRHNGTGGK
  		SIYGEKFEDENFILKHTG
  		PGILSMANAGPNTNGSQFF
  		ICTAKTEWLDGKHVVFGKV
  		KEGMNIVEAMERFGSRNG
  		KTSKKITIADCGQLE
  		(SEQ ID NO: 490)

• In various embodiments, the amino acid sequences of these dimerization domains may be altered in order to optimize binding or to improve binding orthogonality to native targets. The nucleic acid sequences of the genes encoding small-molecule binding proteins may be altered in order to optimize the efficiency of the PE process, such as by reducing PEgRNA secondary structure.
• Other examples of suitable dimerization domains and a cognate small molecule compound which binds thereto are provided as follows. Note that the cognate small molecule compound could be coupled (e.g., via a chemical linker) to a second small molecule compound (either the same compound or a different compound) in order to form a bi-specific ligand that may bind two dimerization domains. In some cases, such as FK506 and cyclosporin A, dimerization of each (e.g., FK506-FK506 or cyclosporin A-cyclosporin A) reduces or eliminates immunosuppressive activity of the monomeric compounds.

• SMALL MOLECULE—BINDS TO
  THE DIMERIZATION DOMAIN—A DIMER OF
  THESE MOLECULES WOULD CONSTITUTE A BI-SPECIFIC LIGAND
  THAT WOULD BIND TWO DIMERIZATION DOMAINS	DIMERIZATION DOMAIN(S)
  FK506	AMINO ACID SEQUENCE OF FKBP12: MGVQVETISPGDGRTFPKRGQTCVVH YTGMLEDGKKFDSSRDRNKPFKFMLG KQEVIRGWEEGVAQMSVGQRAKLTISP DYAYGATGHPGIIPPHATLVFDVELLKL E (SEQ ID NO: 491) CALCINEURIN:
  FK-506
  FKBP12 Kd 0.4 nM
  PNAS
  1990, 87, 9231.
  TARGETS: FKBP12 + CALCINEURIN
  CYCLOSPORIN A	AMINO ACID SEQUENCE OF
  TARGETS: CYCLOPHILIN + CALCINEURIN	HUMAN CYCLOPHILIN A:
  	MVNPTVFFDIAVDGEPLGRVSFELF
  	ADKVPKTAENFRALSTGEKGFGYK
  	GSCFHRIIPGFMCQGGDFTRHNGTG
  	GKSIYGEKFEDENFILKHTGPGILSM
  	ANAGPNTNGSQFFICTAKTEWLDG
  	KHVVFGKVKEGMNIVEAMERFGS
  	RNGKTSKKITIADCGQLE (SEQ ID
  	NO: 490)
  	AMINO ACID SEQUENCE OF
  	HUMAN CYCLOPHILIN B:
  	MLRLSERNMKVLLAAALIAGSVFF
  	LLLPGPSAADEKKKGPKVTVKVYF
  	DLRIGDEDVGRVIFGLFGKTVPKTV
  	DNFVALATGEKGFGYKNSKFHRVIK
  	DFMIQGGDFTRGDGTGGKSIYGER
  	FPDENFKLKHYGPGWVSMANAGK
  	DTNGSQFFITTVKTAWLDGKHVVF
  	GKVLEGMEVVRKVESTKTDSRDKP
  	LKDVIIADCGKIEVEKPFAIAKE
  	(SEQ ID NO: 493)
  	AMINO ACID SEQUENCE OF
  	MURINE CYCLOPHILIN C:
  	MSPGPRLLLPAVLCLGLGALVSSSG
  	SSGVRKRGPSVTDKVFFDVRIGDK
  	DVGRIVIGLFGNVVPKTVENFVALA
  	TGEKGYGYKGSIFHRVIKDFMIQGG
  	DFTARDGTGGMSIYGETFPDENFKL
  	KHYGIGWVSMANAGPDTNGSQFFI
  	TLTKPTWLDGKHVVFGKVLDGMT
  	VVHSIELQATDGHDRPLTDCTIVNS
  	GKIDVKTPFVVEVPDW
  	(SEQ ID NO: 494)
  AP1867	AMINO ACID SEQUENCE OF FKBP12-F36V MGVQVETISPGDGRTFPKRGQTCVVH YTGMLEDGKKVDSSRDRNKPFKFMLG KQEVIRGWEEGVAQMSVGQRAKLT ISPDYAYGATGHPGIIPPHATLVFDVELL KLE (SEQ ID NO: 495)
  AP1867
  FK-506 mimic
  FKBP12 F36V Kd 94 pM
  FKBP12 Kd
  67 nM
  PNAS 1998, 95, 10437.
  TARGET(S): FKBP12
  METHOTREXATE	AMINO ACID SEQUENCE OF HUMAN DIHYDROFOLATE REDUCTASE MVGSLNCIVAVSQNMGIGKNGDLPWP PLRNEFRYFQRMTTTSSVEGKQNLVIM GKKTWFSIPEKNRPLKGRINLVLSRELK EPPQGAHFLSRSLDDALKLTEQPELAN KVDMVWIVGGSSVYKEAMNHPGHLK LFVTRIMQDFESDTFFPEIDLEKYKLLP EYPGVLSDVQEEKGIKYKFEVYEKND (SEQ ID NO: 496)
  methotrexate
  Human DHFR Kd < 10 nM
  J. Biol. Chem. 1988, 263, 10304.
  E. coli DHFR Kd 9.5 nM
  PNAS 2002, 99, 13481.
  TARGET(S): DIHYDROFOLATE REDUCTASE
  TRIMETHOPRIM	AMINO ACID SEQUENCE FOR E. COLI DIHYDROFOLATE REDUCTASE MIS LIAALAVDRVIGMENAMPWNLPAD LAWFKRNTLNKPVIMGRHTWESIGRPL PGRKNIILSSQPGTDDRVTWVKSVDEAI AACGDVPEIMVIGGGRVYEQFLPKAQ KLYLTHIDAEVEGDTHFPDYEPDDWES VFSEFHDADAQNSHSYCFEILERR (SEQ ID NO: 497)
  trimethoprim
  E. coli DHFR Kl 1.3 nM
  Biochemistry
  1982, 21, 5068.
  TARGET(S): DIHYDROFOLATE REDUCTASE
  DEXAMETHOSONE     dexamethasone Human GR Kd 4.6 nM Mol. Endocrin. 1999, 13, 1855.	AMINO ACID SEQUENCE FOR HUMAN GLUCOCORTICOID RECEPTOR MDSKESLTPGREENPSSVLAQERGDV MDFYKTLRGGATVKVSASSPSLAVASQ SDSKQRRLLVDFPKGSVSNAQQPDLSK AVSLSMGLYMGETETKVMGNDLGFPQ QGQISLSSGETDLKLLEESIANLNRSTS VPENPKSSASTAVSAAPTEKEF PKTHSDVSSEQQHLKGQTGTNGGNVK LYTTDQSTFDILQDLEFSSGSPGKETNE SPWRSDLLIDENCLLSPLAGEDDSFLLE GNSNEDCKPLILPDTKPKIKDNGDLVLS SPSNVTLPQVKTEKEDFIELCTPGVIKQ EKLGTVYCQASFPGANIIGNKMSAISV HGVSTSGGQMYHYDMNTASLSQQQD QKPIFNVIPPIPVGSENWNRCQGSGDD NLTSLGTLNFPGRTVFSNGYSSPSMRPD VSSPPSSSSTATTGPPPKLCLVCSDEASG CHYGVLTCGSCKVFFKRAVEGQHNYL
  	CAGRNDCIIDKIRRKNCPACRYRKCLQ
  	AGMNLEARKTKKKIKGIQQATTGVSQ
  	ETSENPGNKTIVPATLPQLTPTLVSLLEV
  	IEPEVLYAGYDSSVPDSTWRIMTTLNM
  	LGGRQVIAAVKWAKAIPGFRNLHLDD
  	QMTLLQYSWMFLMAFALGWRSYRQS
  	SANLLCFAPDLIINEQRMTLPCMYDQC
  	KHMLYVSSELHRLQVSYEEYLCMKTL
  	LLLSSVPKDGLKSQELFDEIRMTYIKEL
  	GKAIVKREGNSSQNWQRFYQLTKLLD
  	SMHEVVENLLNYCFQTFLDKTMSIEFP
  	EMLAEIITNQIPKYSNGNIKKLLFHQK
  	(SEQ ID NO: 498)
  RAPAMYCIN	AMINO ACID SEQUENCE OF FKBP12: MGVQVETISPGDGRTFPKRGQTCVVH YTGMLEDGKKFDSSRDRNKPFKFMLG KQEVIRGWEEGVAQMSVGQRAKLTISP DYAYGATGHPGIIPPHATLVFDVELLKL E (SEQ ID NO: 491)

• Other examples of naturally occurring bifunctional molecules and their dual target receptors are as follows. Prime editing may be used to install the dual target receptors into different proteins. Once the different proteins are modified by PE to contain a bifunctional molecule receptor, the bifunctional molecules may be introduced, thereby causing the dimerization of the proteins modified to comprise the different dimerization domains. Examples of pairings of (1) a bifunctional molecule and (2) their dual target receptors are as follows:

• 	TARGET
  	RECEPTORS
  	OF THE
  	BIOFUNCTIONAL
  NATURALLY OCCURRING BIOFUNCTIONAL MOLECULES	MOLECULE
  auxin	Target receptor 1: auxin receptor Target receptor 2: TIR1 E3 ligase
  methyl jasmonate	Target receptor 1: JAZ receptor Target receptor 2: Col1 E3 ligase
  brefeldin A	target receptor 1: GBF1 target receptor 2: GTPase Arf1p
  abscisic acid	target receptor 1: PYR receptor target receptor 2: phosphoprotein phosphatase 2C
  Forskolin	target receptor 1: adenylyl cyclase monomers target receptor 2: adenylyl cyclase monomers
  fusicoccin A	target receptor 1: 14-3-3 proteins target receptor 2: H+-ATPase
  Rapamycin	target receptor 1: FKBP12 target receptor 2: mTOR
  sanglifehrin A     Sanglifehrin A (SFA)	target receptor 1: cyclophilin target receptor 2: IMP dehydrogenase 2
  cyclosporin A	target receptor 1:
  	cyclophilin
  	target receptor 2:
  	calcineurin

• Examples of other bifunctional molecules that can be used with this aspect of prime editing are as follows:
• 

  
• Synstab A, paclitaxel, and discodermolide are microtubule stabilizers. Thus, these compounds could be used to dimerize proteins modified by PE to comprise microtubule proteins. GNE-0011, ARV-825, and dBET1 comprise a BRD4 binding motif and a CRBN binding motif. Thus, these compounds could be used to dimerize proteins modified by PE to comprise these targeting domains.
• The PEgRNAs for installing dimerization domains may comprising the following structures (in reference to FIG. 3D):
• 5′-[spacer]-[gRNA core]-[extension arm]-3′, wherein the extension arm comprises 5′-[homology arm]-[edit template]-[primer binding site]-3′; or
  5′-[extension arm]-[spacer]-[gRNA core]-3′, wherein the extension arm comprises 5′-[homology arm]-[edit template]-[primer binding site]-3′, and wherein with either configuration the “edit template” comprises a nucleotide sequence of a dimerization domain.
• In one example, the PEgRNA for insertion of the FKBP12 dimerization domain at the C-terminal end of human insulin receptor (spacer underlined, gRNA core plain, flap homology bold, FKBP12 insertion in italics, annealing region bold italics):

• 	PEGRNA	CACGGUAGGCACUGUUAGGAGUUUUAG
  	FOR	AGCUAGAAAUAGCAAGUUAAAAUAAG
  	INSTALLING	GCUAGUCCGUUAUCAACUUGAAAAAGU
  	FKBP12	GGCACCGAGUCGGUGCUUGCCUCGGU
  	IN	CCAAUCCUUCC GGAGUGCAGGUGGAAA
  	HUMAN	CCAUCUCCCCAGGAGACGGGCGCACCUU
  	INSULIN	CCCCAAGCGCGGCCAGACCUGCGUGGUG
  	RECEPTOR	CACUACACCGGGAUGCUUGAAGAUGGAA
  		AGAAAUUUGAUUCCUCCCGGGACAGAAA
  		CAAGCCCUUUAAGUUUAUGCUAGGCAAG
  		CAGGAGGUGAUCCGAGGCUGGGAAGAAG
  		GGGUUGCCCAGAUGAGUGUGGGUCAGAG
  		AGCCAAACUGACUAUAUCUCCAGAUUA
  		UGCCUAUGGUGCCACUGGGCACCCAGGC
  		AUCAUCCCACCACAUGCCACUCUCGUCU
  		UCGAUGUGGAGCUUCUAAAACUGGAA
  		(SEQ ID NO: 499)

• In another example, the PEgRNA for insertion of the FKBP12 dimerization domain at the HEK3 locus (for optimization):

• 	PEGRNA	GGCCCAGACTGAGCACGTGAGTTTTAGA
  	FOR	GCTAGAAATAGCAAGTTAAAATAAGGC
  	INSTALLING	TAGTCCGTTATCAACTTGAAAAAGTGGC
  	FKBP12	ACCGAGTCGGTGCTGGAGGAAGCAGGG
  	IN	CTTCCTTTCCTCTGCCATCATTCCAGTT
  	HEK3	TTAGAAGCTCCACATCGAAGACGAGAGT
  		GGCATGTGGTGGGATGATGCCTGGGTGC
  		CCAGTGGCACCATAGGCATAATCTGGAG
  		ATATAGTCAGTTTGGCTCTCTGACCCAC
  		ACTCATCTGGGCAACCCCTTCTTCCCAG
  		CCTCGGATCACCTCCTGCTTGCCTAGCA
  		TAAACTTAAAGGGCTTGTTTCTGTCCCG
  		GGAGGAATCAAATTTCTTTCCATCTTCA
  		AGCATCCCGGTGTAGTGCACCACGCAGG
  		TCTGGCCGCGCTTGGGGAAGGTGCGCCC
  		GTCTCCTGGGGAGATGGTTTCCACCTGC
  		ACTCCCGTGCTCAGTCTG
  		(SEQ ID NO: 500)

• The target proteins for installing dimerization domains are not particularly limited; however, it is advantageous their dimerization (once modified by PE) in the presence of a bi-specific ligand produces some advantageous biological effect, e.g., a signaling pathway, decreased immunoresponsiveness, etc. In various aspects, the target proteins that are to be dimerized through the PE-dependent installation of dimerization domains can be the same protein or different proteins. Preferably, the proteins, when dimerized, trigger one or more downstream biological cascades, e.g., a signal transduction cascade, phosphorylation, etc. Exemplary target proteins into which PE may be used to install dimerization domains, include, but are not limited to:

• MEMBRANDE-	KINASE
  BOUND	DOMAIN	CID	SIGNALING
  RECEPTOR	FUSED TO	EMPLOYED	CASCADE	REFERENCE
  T-CELL	FKBP12	FK1012	T-CELL	SCIENCE 262, 1019-24
  RECEPTOR		(FK506	RECEPTOR	(1993)
  		DIMER)	SIGNALING
  				CHEM. BIOL. 1, 163-172
  				(1994).
  FAS RECEPTOR	MURINE	CYCLOSP	FAS	CHEM. BIOL. 3,731-738
  	CYCLOPHILIN	ORIN A	PATHWAY	(1996).
  	C	DIMER	FOR
  			APOPTOSIS
  INSULIN	FKBP12	FK1012	INSULIN	CURR. BIOL. 8, 11-18
  RECEPTOR		(FK506	SIGNALING	(1998).
  		DIMER)
  PLATELET-	FKBP12	FK1012	PDGF	CURR. BIOL. 8, 11-18
  DERIVED		(FK506	MESODERM	(1998).
  GROWTH		DIMER)	FORMATION
  FACTOR (PDGF)			SIGNALING
  BETA
  ERYTHROPOIETIN	FKBP12	FK1012	EPOR-	PROC. NATL. ACAD. SCL
  RECEPTOR		(FK506	MEDIATED	94, 3076-3081(2002).
  (EPOR)		DIMER)	PROLIFER-
  			ATIVE
  			SIGNALING

• In one aspect, prime editors described herein may be used to install sequences encoding dimerization domains into one or more genes encoding target proteins of interest in a living cell or patient. This may be referred to as the “prime editing-CID system,” wherein the CID is the bi-specific ligand that induced dimerization of target proteins, each fused to a dimerization domain installed by PE. This edit alone should have no physiological effect. Upon administration of a bi-specific ligand, which typically is a dimeric small molecule that can simultaneously bind to two dimerization domain each of which is fused to a copy of the target protein, the bi-specific ligand causes dimerization of the targeted protein. This target protein dimerization event then induces a biological signaling event, such as erythropoiesis or insulin signaling. A new method to place dimerization-induced biological processes, such as receptor signaling, under control of a convenient small-molecule drug (i.e., the bi-specific ligand) by the genomic integration of genes encoding small-molecule binding proteins (i.e., the dimerization domains) with prime editing is described herein.
• Protein dimerization is a ubiquitous biological process. Notably, homodimerization of many membrane-bound receptors is known to initiate signaling cascades, often with profound biological consequences. A number of small-molecule natural products approved for use as drugs act as chemical inducers of protein dimerization as part of their mechanism of action.⁹² For example, FK506 binds tightly to FKBP12, and the resulting small molecule-protein complex then binds the phosphatase calcineurin, thereby inhibiting a step in T cell receptor signaling.⁹³ Likewise, cyclosporin A induces dimerization of cyclophilin and calcineurin, and rapamycin induces dimerization of FKBP and mTOR.^93,94
• In one embodiment, leveraging the selective, high-affinity binding of the FK506:FKBP12 and cyclosporin A:cyclophilin small molecule:protein binding interaction, synthetic chemical inducers of dimerization have also been developed. In an example, a small molecule comprised of two units of FK506, termed FK1012, was shown to effect signal transduction when the cytoplasmic domains of signaling receptors were tagged with FKBP12.⁹⁵ Chemical inducers of dimerization (CIDs) have since been used to control a number of signaling pathways.^96-103
• While useful tools for studying biological processes, one challenge facing synthetic CIDs for therapeutic applications is that introduction of the FKBP12- or cyclophilin-target protein chimeras into patients is challenging.
• The present disclosure brings together two concepts to create a previously inaccessible therapeutic process. The first concept is prime editing, described herein, which allows for precise genome editing, including targeted insertions, in living cells. The second concept is chemical-induced dimerization, a powerful tool that has enabled small-molecule control over signaling and oligomerization processes in cell culture.
• Specific cases in which chemical control over protein dimerization may have had a beneficial therapeutic effect have been identified.
• The insulin receptor is a heterotetrameric transmembrane protein that responds to insulin binding to the extracellular domain by phosphorylation of the cytoplasmic kinase domain.¹⁰⁴ An engineered chimeric protein composed of a membrane-localization component, the C-terminal kinase domain of the insulin receptor, and three copies of FKBP12 responds to FK1012 and initiates the insulin response in cell culture.⁹⁹ Similarly, it is expected that the fusion of FKBP12 to the C-terminal end of the kinase domain of the native insulin receptor in patient cells should allow for FK1012-dependent phosphorylation and initiation of the insulin signaling cascade. This system could replace or complement insulin use in patients who cannot make insulin (e.g., type-1 diabetics), or who respond weakly to insulin (e.g., type-2 diabetics).
• Additionally, erythropoietin stimulates erythrocyte proliferation by binding to the erythropoietin receptor (EpoR), either inducing dimerization or a conformational change in a preformed receptor dimer which results in activation of the Jak/STAT signaling cascade.¹⁰⁵ It has been demonstrated that FK1012-induced oligomerization of the membrane-anchored cytoplasmic domain of EpoR tagged with FKBP12 is sufficient to initiate the signaling Jak/STAT signaling cascade and promote cell proliferation.¹⁰⁶ It is anticipated that fusing FKBP12 to native EpoR by prime editing in patient cells will allow for FK1012-induced control over erythrocyte proliferation (erythropoiesis). This system could be used to trigger red blood cell growth in anemic patients. FK1012-inducible EpoR could also be employed as an in vivo selectable marker for blood cells that have undergone ex vivo engineering.
• In principle, any receptor tyrosine kinase could be viable target for a prime editing-CID therapeutic. The table below includes a list of all receptor tyrosine kinases in the human genome.¹¹⁰

• Family	Receptor	Synonyms	NT Accession	PROT Accession	Chromosome
  ALK family	ALK		NM_004304	NP_004295	2p23
  	LTK	TYK1	NM_002344	NP_002335	15q15.1-q21.1
  AKL family	AXL	UFO,	NM_001699	NP_	19q3.1
  	MER	MERTK, NYK,	NM_006343	NP_	2q14.1
  	TYRC3	RSE, SKY, BRT, ,	NM_	NP_	15q15.1-q21.1
  DDR family	DDR1	CAK, TRKE, NEP, NTRK4,	NM_013893	NP_001945	8p21.3
  		EDDR1, PTK3
  	DDR2	TKT, TYRO10,	NM_006182	NP_006173	1q21-q22
  EGFR family	EGFR	ERBB, ERBB1	NM_005228	NP_005219	7p12
  	ERBB2	HER2, ,	NM_004448	NP_	17q11.2-q12
  	ERBB3	HER3	NM_001962	NP_	12q13
  	ERBB4	HER4	NM_005235	NP_005226	2q33.3-q34
  EPH family	EPHA1	EPH, EPHT	NM_006232	NP_	7q32-q34
  	EPHA2	ECK, ,	NM_004431	NP_004422	1p34
  	EPHA3	HEK, ETK1, , ,	NM_005233	NP_	3q11.2
  	EPHA4	HEK8, , ,	NM_004435	NP_004429
  	EPHA5	HEK7, , ,	L3864
  	EPHA6
  	EPHA7	HEK11, , , ,	NM_004445	NP_	8q21
  	EPHA8	HEK3, KIAA1459, ,			1q23-q24
  	EPHB1	NET, EPHT2, HEK6, ,	NM_004441	NP_	3q21-q23
  	EPHB2	HEK5, ERK, DRT, EPHT3,			1p38.1-p38
  		, , ,
  	EPHB3	HEK2, , ,	NM_004443	NP_004434	3q21-qter
  	EPHB4	, , ,	NM_004444	NP_004435
  	EPHB5	HEP, ,	NM_004445	NP_004436	7q33-q35
  FGFR family	FGFR1	FLT2, , FLG,			5p11.2
  	FGFR2	KGFR, K-SAM, , , ,	NM_000141	NP_000132
  	FGFR3	HBGFR, ACH,	NM_000142	NP_000133	4p18.3
  	FGFR4		NM_003011	NP_002002	5q35.1-qter
  INSR family	IGF1R	JTK13	NM_000875	NP_000886	15q25-q28
  	INSR	IR	NM_000208	NP_000199	19p13.3-p13.2
  	INSRR	IRR			19p13.3-p13.2
  MET family	MET	HGFR	NM_000245	NP_000236	7q31
  	RDN	, , , ,	NM_002447	NP_	3p21.3
  MUSK family	MUSK	Nak2 , Mk1 , Mk2	NM_	NP_
  PDGFR family	CSF1R	FMS, CFMS, CD115	NM_005211	NP_005202	5q31--q32
  	FLT3	FLK2, STK1, CD135	NM_004119	NP_	13q12
  	KIT	,	NM_000222	NP_000213	4q11-q12
  	PDGFRA		NM_006206	NP_	4q11-q13
  	PDGFRB	PDGFR, JTK12	NM_002609	NP_002800	5q31-q32
  PTK7 family	PTK7	,	NM_002521	NP_002912	6p21.1-p12.2
  RET family	RET	, HSCR1, MTC1			10q11.2
  ROR family	ROR1	NTRKR1	NM_005012	NP_	1p32-p31
  	ROR2	NTRKR2	NM_004560	NP_
  ROS family	ROS1	MCF3	NM_002944	NP_	8q22
  RYK family	RYK	,			3q22
  TIE family	TEK	TIE2	NM_	NP_000450	9p21
  	TIE	TIE1, JTK14	NM_	NP_	1p34-p33
  TRK family	NTRK1	TRK, TRKA	NM_002529	NP_002520	1q21-q22
  	NTRK2	TRKB	NM_006180	NP_	9q22.1
  	NTRK3	TRKC	NM_	NP_002521	15q25
  VEGFR family	VEGFR1	FLT1	NM_002019	NP_	13q12
  	VEGFR2	KDR, FLK1			4q11-q12
  	VEGFR3	FLT4, PCL	NM_002020	NP_	5q34-q36
  AATYK family	AATYK1	AATK, KIAA0641	NM_	NP_004911	17q25.3
  	AATYK2	KIAA1079	NM_	NP_	7q21-q22
  	AATYK3				15q13.2-q13.3
  indicates data missing or illegible when filed

• There are numerous advantages to the prime editing-CID system. One such advantage is that it can replace endogenous ligands, which are typically proteins that pose complications in manufacturing, cost, delivery, production, or storage, with drug-like small-molecules that can be orally administered instead of administered by IV or injection, are readily prepared from FDA-approved drugs (or are themselves already drugs), and do not incur special production or storage costs typically associated with protein drugs. Another advantage is that the edit alone should have no physiological effect. The amount of target protein dimerization can be controlled by dosing the small-molecule CID. Further, target protein dimerization is readily and rapidly reversible by adding the monomeric form of the CID. Yet another advantage is that in instances where a single ligand targets multiple receptors, selectivity can be achieved by prime-editing only one receptor. Finally, depending on the delivery method used for prime editing, it may also be possible to restrict editing to a localized tissue or organ, allowing for inducible receptor activation only in specific areas.
• If editing efficiencies are high enough with prime editing that two separate editing events could occur at high levels, it would also be possible to tag two proteins of interest with different small-molecule binding domains (such as FKBP and cyclophilin) and induce heterodimerizations with small molecule heterodimers (such as an FK506-cyclosporin A dimer).
• The fusion of FKBP12 or other small-molecule binding proteins to native proteins has been accomplished, generally by overexpression from plasmid in tissue culture. Subsequent chemical-induced dimerization has been demonstrated to induce phenotypic changes to cells producing the fusion proteins.
• The following references are cited above in the Section G and are incorporated herein by reference.
• 1. Crabtree, G. R. & Schreiber, S. L. Three-part inventions: intracellular signaling and induced proximity. Trends Biochem. Sci. 21, 418-22 (1996).
• 2. Liu, J. et al. Calcineurin Is a Common Target of A and FKBP-FK506 Complexes. Cell 66, 807-815 (1991).
• 3. Keith, C. T. et al. A mammalian protein targeted by G1-arresting rapamycin-receptor complex. Nature 369, 756-758 (2003).
• 4. Spencer, D. M., Wandless, T. J., Schreiber, S. L. S. & Crabtree, G. R. Controlling signal transduction with synthetic ligands. Science 262, 1019-24 (1993).
• 5. Pruschy, M. N. et al. Mechanistic studies of a signaling pathway activated by the organic dimerizer FK1012. Chem. Biol. 1, 163-172 (1994).
• 6. Spencer, D. M. et al. Functional analysis of Fas signaling in vivo using synthetic inducers of dimerization. Curr. Biol. 6, 839-847 (1996).
• 7. Belshaw, P. J., Spencer, D. M., Crabtree, G. R. & Schreiber, S. L. Controlling programmed cell death with a cyclophilin-cyclosporin-based chemical inducer of dimerization. Chem. Biol. 3, 731-738 (1996).
• 8. Yang, J. X., Symes, K., Mercola, M. & Schreiber, S. L. Small-molecule control of insulin and PDGF receptor signaling and the role of membrane attachment. Curr. Biol. 8, 11-18 (1998).
• 9. Belshaw, P. J., Ho, S. N., Crabtree, G. R. & Schreiber, S. L. Controlling protein association and subcellular localization with a synthetic ligand that induces heterodimerization of proteins. Proc. Natl. Acad. Sci. 93, 4604-4607 (2002).
• 10. Stockwell, B. R. & Schreiber, S. L. Probing the role of homomeric and heteromeric receptor interactions in TGF-β signaling using small molecule dimerizers. Curr. Biol. 8, 761-773 (2004).
• 11. Spencer, D. M., Graef, I., Austin, D. J., Schreiber, S. L. & Crabtree, G. R. A general strategy for producing conditional alleles of Src-like tyrosine kinases. Proc. Natl. Acad. Sci. 92, 9805-9809 (2006).
• 12. Holsinger, L. J., Spencer, D. M., Austin, D. J., Schreiber, S. L. & Crabtree, G. R. Signal transduction in T lymphocytes using a conditional allele of Sos. Proc. Natl. Acad. Sci. 92, 9810-9814 (2006).
• 13. Myers, M. G. Insulin Signal Transduction and the IRS Proteins. Annu. Rev. Pharmacol. Toxicol. 36, 615-658 (1996).
• 14. Watowich, S. S. The erythropoietin receptor: Molecular structure and hematopoietic signaling pathways. J. Investig. Med. 59, 1067-1072 (2011).
• 15. Blau, C. A., Peterson, K. R., Drachman, J. G. & Spencer, D. M. A proliferation switch for genetically modified cells. Proc. Natl. Acad. Sci. 94, 3076-3081 (2002).
• 16. Clackson, T. et al. Redesigning an FKBP-ligand interface to generate chemical dimerizers with novel specificity. Proc. Natl. Acad. Sci. 95, 10437-10442 (1998).
• 17. Diver, S. T. & Schreiber, S. L. Single-step synthesis of cell-permeable protein dimerizers that activate signal transduction and gene expression. J. Am. Chem. Soc. 119, 5106-5109 (1997).
• 18. Guo, Z. F., Zhang, R. & Liang, F. Sen. Facile functionalization of FK506 for biological studies by the thiol-ene ‘click’ reaction. RSC Adv. 4, 11400-11403 (2014).
• 19. Robinson, D. R., Wu, Y.-M. & Lin, S.-F. The protein tyrosine kinase family of the human genome. Oncogene 19, 5548-5557 (2000).
• L. Use of Prime Editing for Cell Data Recording
• The prime editors and the resulting genomic modifications can also be used to study and record cellular processes and development. For example, the prime editors described herein may be used to record the presence and duration of a stimulus to a cell by providing to the cell a first nucleic acid sequence that encodes a fusion protein with a nucleic acid programmable DNA binding protein (napDNAbp) and a reverse transcriptase, and providing the cell at least a second nucleic acid sequence that encodes a PEgRNA. Either the first, the second, or both nucleic acid sequences are operably linked to inducible promoters that are responsive to the cell stimulus such that it induces expression of the fusion protein and/or the PEgRNA thereby causing the modification of a target sequence within the cell.
• The prime editors described herein can also be used for cellular barcoding and lineage tracing. For example, by barcoding each cell with a unique genomic barcode, the prime editor can help reveal the cell lineage map by allowing the construction of phylogenetic trees based on the modifications made in one or more target sequence. Starting from progenitor cells, the prime editor system can enable building a cell-fate map for single cells in a whole organism, which can be deciphered by analyzing the modifications made in one or more target sequence. The method for tracing the linage of cells can include providing a nucleic acid encoding a fusion protein with a napDNAbp and a reverse transcriptase, and providing at least one second nucleic acid encoding a PEgRNA. A unique cellular barcode can be generated using the fusion protein and the PEgRNA to create one or more modifications in one or more target sequence, thereby allowing the linage of any cell that arises from the first cell to be traced using the unique cellular barcode. The use of prime editors for both cell data recording and linage tracing is further described in Example 13.
• The prime editors can do perform both lineage tracing and cellular signaling recording by modifying genomic target sequences or integrated pre-designed sequences. Prime editors use a synthetic fusion protein comprising a Cas9 nickase fragment (including but not limited to the SpCas9 H840A variant) and a reverse transcriptase domain, along with an engineered prime editing guide RNA (PEgRNA). Together, these components target a specific genomic sequence or integrated pre-designed sequence and install a pre-determined edit. Because the PEgRNA specifies both the target genomic sequence and the edited outcome, highly specific and controlled genome modification can be achieved simultaneously using multiple PEgRNA within the same cell. Accessible genome modifications include all single nucleotide substitutions, small- to medium-sized sequence insertions, and small- to medium-sized deletions. The versatility of this genome editing technology can enable temporally coupled, signal-specific recording within cells.
• The use of prime editors for cell data recording can include compositions (e.g., nucleic acids), cells, systems, kits, and methods for recording the strength and/or duration of endogenous or exogenous stimuli over the course of a cell's lifetime. The cell data recording system can include a fusion protein consisting of a napDNAbp (e.g., a Cas9 domain) and a reverse transcriptase operably linked to a promoter that induces the expression of the fusion protein to induce changes by creating targeted and sequence-specified genomic insertions, deletions, or mutations in response to a stimulus or change in the cell. In contrast to digital memory devices that store information (e.g., the presence or absence of a stimulus) in one of two distinct states (i.e., “on” or “off”), these cell data recorders can induce permanent marks in cellular DNA in a manner that reflects both the strength (i.e., amplitude) and duration of one or more stimuli. Thus, in some aspects, cell data recording systems have the ability to simultaneously record multiple cell states, including, for example, exposure to a small molecule, a protein, a peptide, an amino acid, a metabolite, an inorganic molecule, an organometallic molecule, an organic molecule, a drug or drug candidate, a sugar, a lipid, a metal, a nucleic acid, a molecule produced during the activation of an endogenous or an exogenous signaling cascade, light, heat, sound, pressure, mechanical stress, shear stress, or a virus or other microorganism. These cell data recorders can employ sequencing technologies (e.g., high-throughput sequencing) to measure readout (e.g., changes in cellular DNA) and are not dependent on large cell populations for both the recording of a stimulus or the readout of the change(s) in cellular DNA induced by the stimulus.
• In general, the cell data recorder systems provided herein for use in a cell comprise a fusion protein consisting of a napDNAbp and a reverse transcriptase, wherein the nucleic acid sequence encoding the fusion plasmid is operably linked to a promoter (e.g., an inducible promoter or a constitutive promoter). When a stimulus is present, or a change in cell state occurs, the stimulus induces the expression of the fusion protein. Also present within the cell are one or more nucleic acids encoding at least one PEgRNA that associate with the napDNAbp and directs the napDNAbp or the fusion protein to a target sequence (i.e., the PEgRNA is complementary to a target sequence). The nucleic acid encoding PEgRNA may also be, or may alternatively be, operably linked to a promoter (e.g., an inducible promoter or a constitutive promoter). Under the correct stimulus, or correct set of stimuli, both the fusion protein and the PEgRNA are expressed in the cell, and the PEgRNA associates with the fusion protein to direct it to a target sequence. This target sequence records the activity of the prime editor, thereby recording the presence of a stimulus, or a set of stimuli, or a change in cell state. More than one PEgRNA sequence can also be present in the cell, and these additional PEgRNA sequences, which can direct the fusion protein to distinct target sequences, can each be operably linked to a promoter that senses the presence of a different stimulus, allowing complex cell data recorder systems to be constructed for the ordered recording of the presence and duration of a stimulus, or set of stimuli. In some cases, one or more of the components of the cell data recorder system (e.g., fusion protein and PEgRNA) may be constitutively expressed in the cell. Exemplary components of the cell data recorder system for use with the compositions are described herein. Additional suitable combinations of components provided herein will be apparent to a person of ordinary skill in the art based on this disclosure and knowledge in the field, and thus are embraced by the scope of this disclosure.
• Repeated modification of a DNA target that can be sequenced by targeted amplicon sequencing and/or RNA sequencing (which is particular value for single cell recording experiments) can be used to record a host of important biological processes, including activation of signaling cascades, metabolic states, and cellular differentiation programs. Connecting internal and external cellular signals to sequence modification in the genome is possible for any signal for which a signal responsive promotor exists. In some embodiments, the promoter is a promoter suitable for use in a prokaryotic system (i.e., a bacterial promoter). In some embodiments, the promoter is a promoter suitable for use in a eukaryotic system (i.e., a eukaryotic promoter). In some embodiments, the promoter is a promoter suitable for use in a mammalian (e.g., human) system (i.e., a mammalian promoter). In some embodiments, the promoter is induced by a stimulus (i.e., an inducible promoter). In some embodiments, the stimulus is a small molecule, a protein, a peptide, an amino acid, a metabolite, an inorganic molecule, an organometallic molecule, an organic molecule, a drug or drug candidate, a sugar, a lipid, a metal, a nucleic acid, a molecule produced during the activation of an endogenous or an exogenous signaling cascade, light, heat, sound, pressure, mechanical stress, shear stress, or a virus or other microorganism, change in pH, or change in oxidation/reduction state. In some embodiments, the stimulus is a light. In some embodiments, the stimulus is a virus. In some embodiments, the stimulus is a small molecule. In some embodiments, the stimulus is an antibiotic. In some embodiments, the stimulus is anhydrotetracycline or doxycycline. In some embodiments, the stimulus is a sugar. In some embodiments, the stimulus is arabinose, rhamnose, or IPTG. In some embodiments, the stimulus is a signaling molecule produced during an activated signaling cascade (e.g., beta-catenin produced during an activated Wnt signaling cascade). Additional promoters that detect signaling molecules can be generated to induce the expression of the nucleic acid sequence operably linked to the promoter, for example, promoters that record an endogenous pathway, including immune response (IL-2 promoter), a cAMP responsive element (CREB), NFκB signaling, interferon response, P53 (DNA damage), Sox2, TGF-β signaling (SMAD), Erk (e.g., from an activated Ras/Raf/Mek/Erk cascade), PI3K/AKT (e.g., from an activated Ras/PI3K/Akt cascade), heat shock, Notch signaling, Oct4, an aryl hydrocarbon receptor, or an AP-1 transcription factor. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is a promoter listed in Table 3. Additional suitable promoters for use in both prokaryotic and eukaryotic systems will be apparent to those of ordinary skill in the art based on this disclosure and knowledge in the field, and are within the scope of the present disclosure.
• Prime editors can also be used to trace cellular lineages. Repeated sequence modifications can be used to generate unique cellular barcodes to track individual cells. The arrays of barcodes, their order, and size can all be used to infer cellular lineages. For example, the insertion of homology sequences (i.e., sequences 3′ of the Cas9 nick location), and in particular homology sequences with associated barcodes, appear to be particularly useful lineage prime editor strategies. These systems can be designed such that successive rounds of editing result in the insertion of a barcode from a PEgRNA cassette that cannot be modified by other PEgRNA editing events in the same cell. The barcoding system can utilize multiple barcodes that can be associated with a given stimulus. This system can preserve the majority of the target protospacers but alter the seed sequence, PAM, and downstream adjacent nucleotides. This enables multiple signals to be connected to one editing locus without significant re-designing of the PEgRNAs being used. The strategy can enable multiplexed barcode insertions in response to a large number of cellular stimuli (either internal or external) at a single locus. It could enable the recording of intensity, duration, and order of as many signals as there exist unique barcodes (which can be designed with multiple N nucleotides to generate 4{circumflex over ( )}N possible barcodes, for example a 5-nt barcode would enable recording of 4{circumflex over ( )}5 or 1024 unique signals at once). This system can be used both in vitro and in vivo.
• M. Use of Prime Editing to Modulate Biomolecule Activity
• The use of prime editors described herein may also be used to regulate the subcellular localization and modification states of biomolecules, such as DNA, RNA, and proteins. Specific biological functions, like transcriptional control, cellular metabolism, and signal transduction cascades, are carefully orchestrated in particular locations within the cell. The ability to traffic proteins to these and other unique cellular compartments could provide an opportunity to alter a number of biological processes.
• Accordingly, prime editing can be used to install genetically encoded handles that will allow for altered modification states and the subcellular trafficking of biomolecules with a genetically encoded signal (e.g. proteins, lipids, sugars, and nucleic acids). In various embodiments, the target biomolecules for prime editor-mediated medication are DNA. For example, DNA could be modified by installing a number of DNA sequences that change the accessibility of the target locus, which could lead to either increased or decreased transcription of desired sequences. In other embodiments, the target biomolecules for prime editor-mediated medication are RNA. For example, the activity of RNA can be modified by changing its cellular localization, interacting partners, structural dynamics, or thermodynamics of folding. In yet other embodiments, the target biomolecules for prime editor-mediated medication are protein. Proteins can be modified to impact post-translational modifications, protein motifs can be installed to change the subcellular localization of the protein, or proteins can be modified to either create or destroy their ability to exist within protein-protein complexation events.
• This application of prime editing can be further described in Example 14.
DNA Modifications
• One target biomolecule for PE-mediated modification is DNA. Modifications to DNA could be made to install a number of DNA sequences that change the accessibility of the target locus. Chromatin accessibility controls gene transcriptional output. Installation of marks to recruit chromatin compacting enzymes should decrease the transcriptional output of neighboring genes, while installation of sequences associated with chromatin opening should make regions more accessible and in turn increase transcription. Installation of more complex sequence motifs that mirror native regulatory sequences should provide more nuanced and biologically sensitive control than the currently available dCas9 fusions to different epigenetic reader, writer, or eraser enzymes—tools that typically install large numbers of a single type of mark that may not have a particular biological antecedent. Installation of sequences that will bring two loci into close proximity, or bring loci into contact with the nuclear membrane, should also alter the transcriptional output of those loci as has been demonstrated in the burgeoning field of 3-D genomic architecture.
RNA Modifications
• Modifications to RNAs can also be made to alter their activity by changing their cellular localization, interacting partners, structural dynamics, or thermodynamics of folding. Installation of motifs that will cause translational pausing or frameshifting could change the abundance of mRNA species through various mRNA processing mechanisms. Modifying consensus splice sequences would also alter the abundance and prevalence of different RNA species. Changing the relative ratio of different splice isoforms would predictably lead to a change in the ratio of protein translation products, and this could be used to alter many biological pathways. For instance, shifting the balance of mitochondrial versus nuclear DNA repair proteins would alter the resilience of different cancers to chemotherapeutic reagents. Furthermore, RNAs could be modified with sequences that enable binding to novel protein targets. A number of RNA aptamers have been developed that bind with high affinity to cellular proteins. Installation of one of these aptamers could be used to either sequester different RNA species through binding to a protein target that will prevent their translation, biological activity, or to bring RNA species to specific subcellular compartments. Biomolecule degradation is another class of localization modification.
• For example, RNA methylation is used to regulate RNAs within the cell. Consensus motifs for methylation could be introduced into target RNA coding sequences with PE. RNAs could also be modified to include sequences that direct nonsense mediated decay machinery or other nucleic acid metabolism pathways to degrade the target RNA species would change the pool of RNAs in a cell. Additionally, RNA species could be modified to alter their aggregation state. Sequences could be installed on single RNAs of interest or multiple RNAs to generate RNA tangles that would render them ineffective substrates for translation or signaling.
Protein Modifications
• Modifications to proteins via post-translational modification (PTM) also represent an important class of biomolecule manipulation that can be carried out with PE. As with RNA species, changing the abundance of proteins in a cell is an important capability of PE. Editing can be done to install stop codons in an open reading frame—this will eliminate full-length product from being produced by the edited DNA sequence. Alternatively, peptide motifs can be installed that cause the rate of protein degradation to be altered for a target protein. Installation of degradation tags into a gene body could be used to alter the abundance of a protein in a cell. Moreover, introduction of degrons that are induced by small molecules could enable temporal control over protein degradation. This could have important implications for both research and therapeutics as researchers could readily assess whether small molecule-mediated therapeutic protein degradation of a given target was a viable therapeutic strategy. Protein motifs could also be installed to change the subcellular localization of a protein. Amino acid motifs can be installed to preferentially traffic proteins to a number of subcellular compartments including the nucleus, mitochondria, cell membrane, peroxisome, lysosome, proteasome, exosome, and others.
• Installing or destroying motifs modified by PTM machinery can alter protein post-translational modifications. Phosphorylation, ubiquitylation, glycosylation, lipidation (e.g. farnesylation, myristoylation, palmitoylation, prenylation, GPI anchors), hydroxylation, methylation, acetylation, crotonylation, SUMOylation, disulfide bond formations, side chain bond cleavage events, polypeptide backbone cleavage events (proteolysis), and a number of other protein PTMs have been identified. These PTMs change protein function, often by changing subcellular localization. Indeed, kinases often activate downstream signaling cascades via phosphorylation events. Removal of the target phosphosite would prevent signal transduction. The ability to site-specifically ablate or install any PTM motif while retaining full-length protein expression would be an important advance for both basic research and therapeutics. The sequence installation scope and target window of PE make it well suited for broad PTM modification space.
• Removal of lipidation sites should prevent the trafficking of proteins to cell membranes. A major limitation to current therapeutics that target post-translational modification processes is their specificity. Farnesyl transferase inhibitors have been tested extensively for their ability to eliminate KRAS localization at cell membranes. Unfortunately, global inhibition of farnesylation comes with numerous off target effects that have prevented broad use of these small molecules. Similarly, specific inhibition of protein kinases with small molecules can be very challenging due to the large size of the human genome and similarities between various kinases. PE offers a potential solution to this specificity problem, as it enables inhibition of modification of the target protein by ablation of the modification site instead of global enzyme inhibition. For example, removal of the lapidated peptide motif in KRAS would be a targeted approach that could be used in place of farnesyl transferase inhibition. This approach is the functional inverse of inhibiting a target protein activity by installing a lipid-targeting motif on a protein not designed to be membrane bound.
• PE can also be used to instigate protein-protein complexation events. Proteins often function within complexes to execute their biological activity. PE can be used to either create or destroy the ability of proteins to exist within these complexes. To eliminate complex formation events, amino acid substitutions or insertions along the protein: protein interface could be installed to disfavor complexation. SSX18 is a protein component of the BAF complex, an important histone-remodeling complex. Mutations in SSX18 drive synovial sarcomas. PE could be used to install side chains that prevent SSX18 from binding to its protein partners in the complex to prevent its oncogenic activity. PE could also be used to remove the pathogenic mutations to restore WT activity of this protein. Alternatively, PE could be used to keep proteins within either their native complex or to drag them to participate in interactions that are unrelated to their native activity to inhibit their activity. Forming complexes that maintain one interaction state over another could represent an important therapeutic modality. Altering protein: protein interfaces to decrease the Kd of the interaction would keep those proteins stuck to one another longer. As protein complexes can have multiple signaling complexes, like n-myc driving neuroblastoma signaling cascades in disease but otherwise participating in healthy transcriptional control in other cells. PE could be used to install mutations that drive n-myc association with healthy interactions partners and decrease its affinity for oncogenic interaction partners.
REFERENCES CITED IN SECTION I
• Each of the following references are incorporated herein by reference.
• 1. Selective Target Protein Degradation via Phthalimide Conjugation. Winter et al. Science. Author manuscript; available in PMC 2016 Jul. 8.
• 2. Reversible disruption of mSWI/SNF (BAF) complexes by the SS18-SSX oncogenic fusion in synovial sarcoma. Kadoch and Crabtree. Cell. 2013 Mar. 28; 153(1):71-85. doi: 10.1016/j.cell.2013.02.036.
• 3. Ribosomal frameshifting and transcriptional slippage: From genetic steganography and cryptography to adventitious use. Atkins et al. Nucleic Acids Research, Volume 44, Issue 15, 6 Sep. 2016, Pages 7007-7078.
• 4. Transcriptional Regulation and its Misregulation in Disease. Lee and Young. Cell. Author manuscript; available in PMC 2014 Mar. 14.
• 5. Protein localization in disease and therapy. Mien-Chie Hung, Wolfgang Link Journal of Cell Science 2011 124: 3381-3392.
• 6. Loss of post-translational modification sites in disease. Li et al. Pac Symp Biocomput. 2010:337-47. PTMD: A Database of Human Disease-associated Post-translational Modifications. Xu et al. Genomics Proteomics Bioinformatics. 2018 August; 16(4):244-251. Epub 2018 Sep. 21.
• 7. Post-transcriptional gene regulation by mRNA modifications. Zhao et al. Nature Reviews Molecular Cell Biology volume 18, pages 31-42 (2017).
• N. Improved Design Aspects of PEgRNAs for Prime Editing
• In other embodiments, the prime editing system may include the use of PEgRNA designs and strategies that can improve prime editing efficiency. These strategies seek to overcome some issues that exist because of the multi-step process required for prime editing. For example, unfavorable RNA structures that can form within the PEgRNA can result in the inhibition of DNA edits being copied from the PEgRNA into the genomic locus. These limitations could be overcome through the redesign and engineering of the PEgRNA component. These redesigns could improve prime editor efficiency, and could allow the installation of longer inserted sequences into the genome.
• Accordingly, in various embodiments, the PEgRNA designs can result in longer PEgRNAs by enabling efficient expression of functional PEgRNAs from non-polymerase III (pol III) promoters, which would avoid the need for burdensome sequence requirements. In other embodiments, the core, Cas9-binding PEgRNA scaffold can be improved to improve efficacy of the system. In yet other embodiments, modifications can be made to the PEgRNA to improve reverse transcriptase (RT) processivity, which would enable the insertion of longer sequences at the targeted genomic loci. In other embodiments, RNA motifs can be added to the 5′ and/or 3′ termini of the PEgRNA to improve stability, enhance RT processivity, prevent misfolding of the PEgRNA, and/or recruit additional factors important for genome editing. In yet another embodiment, a platform is provided for the evolution of PEgRNAs for a given sequence target that could improve the PEgRNA scaffold and enhance prime editor efficiency. These designs could be used to improve any PEgRNA recognized by any Cas9 or evolved variant thereof.
• This application of prime editing can be further described in Example 15.
• The PEgRNAs may include additional design improvements that may modify the properties and/or characteristics of PEgRNAs thereby improving the efficacy of prime editing. In various embodiments, these improvements may belong to one or more of a number of different categories, including but not limited to: (1) designs to enable efficient expression of functional PEgRNAs from non-polymerase III (pol III) promoters, which would enable the expression of longer PEgRNAs without burdensome sequence requirements; (2) improvements to the core, Cas9-binding PEgRNA scaffold, which could improve efficacy; (3) modifications to the PEgRNA to improve RT processivity, enabling the insertion of longer sequences at targeted genomic loci; and (4) addition of RNA motifs to the 5′ or 3′ termini of the PEgRNA that improve PEgRNA stability, enhance RT processivity, prevent misfolding of the PEgRNA, or recruit additional factors important for genome editing.
• In one embodiment, PEgRNA could be designed with polIII promoters to improve the expression of longer-length PEgRNA with larger extension arms. sgRNAs are typically expressed from the U6 snRNA promoter. This promoter recruits pol III to express the associated RNA and is useful for expression of short RNAs that are retained within the nucleus. However, pol III is not highly processive and is unable to express RNAs longer than a few hundred nucleotides in length at the levels required for efficient genome editing. Additionally, pol III can stall or terminate at stretches of U's, potentially limiting the sequence diversity that could be inserted using a PEgRNA. Other promoters that recruit polymerase II (such as pCMV) or polymerase I (such as the U1 snRNA promoter) have been examined for their ability to express longer sgRNAs. However, these promoters are typically partially transcribed, which would result in extra sequence 5′ of the spacer in the expressed PEgRNA, which has been shown to result in markedly reduced Cas9:sgRNA activity in a site-dependent manner. Additionally, while pol III-transcribed PEgRNAs can simply terminate in a run of 6-7 U's, PEgRNAs transcribed from pol II or pol I would require a different termination signal. Often such signals also result in polyadenylation, which would result in undesired transport of the PEgRNA from the nucleus. Similarly, RNAs expressed from pol II promoters such as pCMV are typically 5′-capped, also resulting in their nuclear export.
• Previously, Rinn and coworkers screened a variety of expression platforms for the production of long-noncoding RNA-(lncRNA) tagged sgRNAs¹⁸³. These platforms include RNAs expressed from pCMV and that terminate in the ENE element from the MALAT1 ncRNA from humans¹⁸⁴, the PAN ENE element from KSHV¹⁸⁵, or the 3′ box from U1 snRNA¹⁸⁶. Notably, the MALAT1 ncRNA and PAN ENEs form triple helices protecting the polyA-tail 184, 187. These constructs could also enhance RNA stability. It is contemplated that these expression systems will also enable the expression of longer PEgRNAs.
• In addition, a series of methods have been designed for the cleavage of the portion of the pol II promoter that would be transcribed as part of the PEgRNA, adding either a self-cleaving ribozyme such as the hammerhead¹⁸⁸, pistol¹⁸⁹, hatchet¹⁸⁹, hairpin¹⁹⁰, VS¹⁹¹, twister¹⁹², or twister sister¹⁹² ribozymes, or other self-cleaving elements to process the transcribed guide, or a hairpin that is recognized by Csy4¹⁹³ and also leads to processing of the guide. Also, it is hypothesized that incorporation of multiple ENE motifs could lead to improved PEgRNA expression and stability, as previously demonstrated for the KSHV PAN RNA and element¹⁸⁵. It is also anticipated that circularizing the PEgRNA in the form of a circular intronic RNA (ciRNA) could also lead to enhanced RNA expression and stability, as well as nuclear localization¹⁹⁴.
• In various embodiments, the PEgRNA may include various above elements, as exemplified by the following sequence.

• 	Non-limiting example 1
  	PEgRNA expression platform consisting
  	of pCMV, Csy4 hairpin, the PEgRNA,
  	and MALAT1 ENE
  	(SEQ ID NO: 501)
  	TAGTTATTAATAGTAATCAATTACGGGGTCATTAG
  	TTCATAGCCCATATATGGAGTTCCGCGTTACATAA
  	CTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAA
  	CGACCCCCGCCCATTGACGTCAATAATGACGTATG
  	TTCCCATAGTAACGCCAATAGGGACTTTCCATTGA
  	CGTCAATGGGTGGAGTATTTACGGTAAACTGCCCA
  	CTTGGCAGTACATCAAGTGTATCATATGCCAAGTA
  	CGCCCCCTATTGACGTCAATGACGGTAAATGGCCC
  	GCCTGGCATTATGCCCAGTACATGACCTTATGGGA
  	CTTTCCTACTTGGCAGTACATCTACGTATTAGTCA
  	TCGCTATTACCATGGTGATGCGGTTTTGGCAGTAC
  	ATCAATGGGCGTGGATAGCGGTTTGACTCACGGGG
  	ATTTCCAAGTCTCCACCCCATTGACGTCAATGGGA
  	GTTTGTTTTGGCACCAAAATCAACGGGACTTTCCA
  	AAATGTCGTAACAACTCCGCCCCATTGACGCAAAT
  	GGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAA
  	GCAGAGCTGGTTTAGTGAACCGTCAGATCGTTCAC
  	TGCCGTATAGGCAGGGCCCAGACTGAGCACGTGAG
  	TTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCT
  	AGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTC
  	GGTCCTCTGCCATCAAAGCGTGCTCAGTCTGTTTT
  	AGGGTCATGAAGGTTTTTCTTTTCCTGAGAAAACA
  	ACACGTATTGTTTTCTCAGGTTTTGCTTTTTGGCC
  	TTTTTCTAGCTTAAAAAAAAAAAAAGCAAAAGATG
  	CTGGTGGTTGGCACTCCTGGTTTCCAGGACGGGGT
  	TCAAATCCCTGCGGCGTCTTTGCTTTGACT
  	Non-limiting example 2
  	PEgRNA expression platform consisting
  	of pCMV, Csy4 hairing, the
  	PEgRNA, and PAN ENE
  	(SEQ ID NO: 502)
  	TAGTTATTAATAGTAATCAATTACGGGGTCATTAG
  	TTCATAGCCCATATATGGAGTTCCGCGTTACATAA
  	CTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAA
  	CGACCCCCGCCCATTGACGTCAATAATGACGTATG
  	TTCCCATAGTAACGCCAATAGGGACTTTCCATTGA
  	CGTCAATGGGTGGAGTATTTACGGTAAACTGCCCA
  	CTTGGCAGTACATCAAGTGTATCATATGCCAAGTA
  	CGCCCCCTATTGACGTCAATGACGGTAAATGGCCC
  	GCCTGGCATTATGCCCAGTACATGACCTTATGGGA
  	CTTTCCTACTTGGCAGTACATCTACGTATTAGTCA
  	TCGCTATTACCATGGTGATGCGGTTTTGGCAGTAC
  	ATCAATGGGCGTGGATAGCGGTTTGACTCACGGGG
  	ATTTCCAAGTCTCCACCCCATTGACGTCAATGGGA
  	GTTTGTTTTGGCACCAAAATCAACGGGACTTTCCA
  	AAATGTCGTAACAACTCCGCCCCATTGACGCAAAT
  	GGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAA
  	GCAGAGCTGGTTTAGTGAACCGTCAGATCGTTCAC
  	TGCCGTATAGGCAGGGCCCAGACTGAGCACGTGAG
  	TTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCT
  	AGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTC
  	GGTCCTCTGCCATCAAAGCGTGCTCAGTCTGTTTT
  	GTTTTGGCTGGGTTTTTCCTTGTTCGCACCGGACA
  	CCTCCAGTGACCAGACGGCAAGGTTTTTATCCCAG
  	TGTATATTGGAAAAACATGTTATACTTTTGACAAT
  	TTAACGTGCCTAGAGCTCAAATTAAACTAATACCA
  	TAACGTAATGCAACTTACAACATAAATAAAGGTCA
  	ATGTTTAATCCATAAAAAAAAAAAAAAAAAAA
  	Non-limiting example 3
  	PEgRNA expression platform consisting
  	of pCMV, Csy4 hairing, the PEgRNA,
  	and 3xPAN ENE
  	(SEQ ID NO: 503)
  	TAGTTATTAATAGTAATCAATTACGGGGTCATTAG
  	TTCATAGCCCATATATGGAGTTCCGCGTTACATAA
  	CTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAA
  	CGACCCCCGCCCATTGACGTCAATAATGACGTATG
  	TTCCCATAGTAACGCCAATAGGGACTTTCCATTGA
  	CGTCAATGGGTGGAGTATTTACGGTAAACTGCCCA
  	CTTGGCAGTACATCAAGTGTATCATATGCCAAGTA
  	CGCCCCCTATTGACGTCAATGACGGTAAATGGCCC
  	GCCTGGCATTATGCCCAGTACATGACCTTATGGGA
  	CTTTCCTACTTGGCAGTACATCTACGTATTAGTCA
  	TCGCTATTACCATGGTGATGCGGTTTTGGCAGTAC
  	ATCAATGGGCGTGGATAGCGGTTTGACTCACGGGG
  	ATTTCCAAGTCTCCACCCCATTGACGTCAATGGGA
  	GTTTGTTTTGGCACCAAAATCAACGGGACTTTCCA
  	AAATGTCGTAACAACTCCGCCCCATTGACGCAAAT
  	GGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAA
  	GCAGAGCTGGTTTAGTGAACCGTCAGATCGTTCAC
  	TGCCGTATAGGCAGGGCCCAGACTGAGCACGTGAG
  	TTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCT
  	AGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTC
  	GGTCCTCTGCCATCAAAGCGTGCTCAGTCTGTTTT
  	GTTTTGGCTGGGTTTTTCCTTGTTCGCACCGGACA
  	CCTCCAGTGACCAGACGGCAAGGTTTTTATCCCAG
  	TGTATATTGGAAAAACATGTTATACTTTTGACAAT
  	TTAACGTGCCTAGAGCTCAAATTAAACTAATACCA
  	TAACGTAATGCAACTTACAACATAAATAAAGGTCA
  	ATGTTTAATCCATAAAAAAAAAAAAAAAAAAAACA
  	CACTGTTTTGGCTGGGTTTTTCCTTGTTCGCACCG
  	GACACCTCCAGTGACCAGACGGCAAGGTTTTTATC
  	CCAGTGTATATTGGAAAAACATGTTATACTTTTGA
  	CAATTTAACGTGCCTAGAGCTCAAATTAAACTAAT
  	ACCATAACGTAATGCAACTTACAACATAAATAAAG
  	GTCAATGTTTAATCCATAAAAAAAAAAAAAAAAAA
  	ATCTCTCTGTTTTGGCTGGGTTTTTCCTTGTTCGC
  	ACCGGACACCTCCAGTGACCAGACGGCAAGGTTTT
  	TATCCCAGTGTATATTGGAAAAACATGTTATACTT
  	TTGACAATTTAACGTGCCTAGAGCTCAAATTAAAC
  	TAATACCATAACGTAATGCAACTTACAACATAAAT
  	AAAGGTCAATGTTTAATCCATAAAAAAAAAAAAAA
  	AAAAA
  	Non-limiting example 4
  	PEgRNA expression platform consisting
  	of pCMV, Csy4 hairing, the PEgRNA,
  	and 3′ box
  	(SEQ ID NO: 504)
  	TAGTTATTAATAGTAATCAATTACGGGGTCATTAG
  	TTCATAGCCCATATATGGAGTTCCGCGTTACATAA
  	CTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAA
  	CGACCCCCGCCCATTGACGTCAATAATGACGTATG
  	TTCCCATAGTAACGCCAATAGGGACTTTCCATTGA
  	CGTCAATGGGTGGAGTATTTACGGTAAACTGCCCA
  	CTTGGCAGTACATCAAGTGTATCATATGCCAAGTA
  	CGCCCCCTATTGACGTCAATGACGGTAAATGGCCC
  	GCCTGGCATTATGCCCAGTACATGACCTTATGGGA
  	CTTTCCTACTTGGCAGTACATCTACGTATTAGTCA
  	TCGCTATTACCATGGTGATGCGGTTTTGGCAGTAC
  	ATCAATGGGCGTGGATAGCGGTTTGACTCACGGGG
  	ATTTCCAAGTCTCCACCCCATTGACGTCAATGGGA
  	GTTTGTTTTGGCACCAAAATCAACGGGACTTTCCA
  	AAATGTCGTAACAACTCCGCCCCATTGACGCAAAT
  	GGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAA
  	GCAGAGCTGGTTTAGTGAACCGTCAGATCGTTCAC
  	TGCCGTATAGGCAGGGCCCAGACTGAGCACGTGAG
  	TTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCT
  	AGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTC
  	GGTCCTCTGCCATCAAAGCGTGCTCAGTCTGTTTG
  	TTTCAAAAGTAGACTGTACGCTAAGGGTCATATCT
  	TTTTTTGTTTGGTTTGTGTCTTGGTTGGCGTCTTA
  	AA
  	Non-limiting example 5
  	PEgRNA expression platform consisting
  	of pU1, Csy4 hairping, the PEgRNA,
  	and 3′ box
  	(SEQ ID NO: 505)
  	CTAAGGACCAGCTTCTTTGGGAGAGAACAGACGCA
  	GGGGCGGGAGGGAAAAAGGGAGAGGCAGACGTCAC
  	TTCCCCTTGGCGGCTCTGGCAGCAGATTGGTCGGT
  	TGAGTGGCAGAAAGGCAGACGGGGACTGGGCAAGG
  	CACTGTCGGTGACATCACGGACAGGGCGACTTCTA
  	TGTAGATGAGGCAGCGCAGAGGCTGCTGCTTCGCC
  	ACTTGCTGCTTCACCACGAAGGAGTTCCCGTGCCC
  	TGGGAGCGGGTTCAGGACCGCTGATCGGAAGTGAG
  	AATCCCAGCTGTGTGTCAGGGCTGGAAAGGGCTCG
  	GGAGTGCGCGGGGCAAGTGACCGTGTGTGTAAAGA
  	GTGAGGCGTATGAGGCTGTGTCGGGGCAGAGGCCC
  	AAGATCTCAGTTCACTGCCGTATAGGCAGGGCCCA
  	GACTGAGCACGTGAGTTTTAGAGCTAGAAATAGCA
  	AGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA
  	AAGTGGGACCGAGTCGGTCCTCTGCCATCAAAGCG
  	TGCTCAGTCTGTTTCAGCAAGTTCAGAGAAATCTG
  	AACTTGCTGGATTTTTGGAGCAGGGAGATGGAATA
  	GGAGCTTGCTCCGTCCACTCCACGCATCGACCTGG
  	TATTGCAGTACCTCCAGGAACGGTGCACCCACTTT
  	CTGGAGTTTCAAAAGTAGACTGTACGCTAAGGGTC
  	ATATCTTTTTTTGTTTGGTTTGTGTCTTGGTTGGC
  	GTCTTAAA

• In various other embodiments, the PEgRNA may be improved by introducing improvements to the scaffold or core sequences. This can be done by introducing known
• The core, Cas9-binding PEgRNA scaffold can likely be improved to enhance PE activity. Several such approaches have already been demonstrated. For instance, the first pairing element of the scaffold (P1) contains a GTTTT-AAAAC pairing element. Such runs of Ts have been shown to result in pol III pausing and premature termination of the RNA transcript. Rational mutation of one of the T-A pairs to a G-C pair in this portion of P1 has been shown to enhance sgRNA activity, suggesting this approach would also be feasible for PEgRNAs¹⁹⁵. Additionally, increasing the length of P1 has also been shown to enhance sgRNA folding and lead to improved activity¹⁹⁵, suggesting it as another avenue for the improvement of PEgRNA activity. Example improvements to the core can include:

• 	PEgRNA containing a 6 nt extension to P1
  	(SEQ ID NO: 228)
  	GGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGCT
  	CATGAAAATGAGCTAGCAAGTTAAAATAAGGCTAG
  	TCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGG
  	TCCTCTGCCATCAAAGCGTGCTCAGTCTGTTTTTT
  	T
  	PEgRNA containing a T-A to G-C
  	mutation within P1
  	(SEQ ID NO: 229)
  	GGCCCAGACTGAGCACGTGAGTTTGAGAGCTAGAA
  	ATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAAC
  	TTGAAAAAGTGGGACCGAGTCGGTCCTCTGCCATC
  	AAAGCGTGCTCAGTCTGTTTTTTT

• In various other embodiments, the PEgRNA may be improved by introducing modifications to the edit template region. As the size of the insertion templated by the PEgRNA increases, it is more likely to be degraded by endonucleases, undergo spontaneous hydrolysis, or fold into secondary structures unable to be reverse-transcribed by the RT or that disrupt folding of the PEgRNA scaffold and subsequent Cas9-RT binding. Accordingly, it is likely that modification to the template of the PEgRNA might be necessary to affect large insertions, such as the insertion of whole genes. Some strategies to do so include the incorporation of modified nucleotides within a synthetic or semi-synthetic PEgRNA that render the RNA more resistant to degradation or hydrolysis or less likely to adopt inhibitory secondary structures¹⁹⁶. Such modifications could include 8-aza-7-deazaguanosine, which would reduce RNA secondary structure in G-rich sequences; locked-nucleic acids (LNA) that reduce degradation and enhance certain kinds of RNA secondary structure; 2′-O-methyl, 2′-fluoro, or 2′-O-methoxyethoxy modifications that enhance RNA stability. Such modifications could also be included elsewhere in the PEgRNA to enhance stability and activity. Alternatively or additionally, the template of the PEgRNA could be designed such that it both encodes for a desired protein product and is also more likely to adopt simple secondary structures that are able to be unfolded by the RT. Such simple structures would act as a thermodynamic sink, making it less likely that more complicated structures that would prevent reverse transcription would occur. Finally, one could also split the template into two, separate PEgRNAs. In such a design, a PE would be used to initiate transcription and also recruit a separate template RNA to the targeted site via an RNA-binding protein fused to Cas9 or an RNA recognition element on the PEgRNA itself such as the MS2 aptamer. The RT could either directly bind to this separate template RNA, or initiate reverse transcription on the original PEgRNA before swapping to the second template. Such an approach could enable long insertions by both preventing misfolding of the PEgRNA upon addition of the long template and also by not requiring dissociation of Cas9 from the genome for long insertions to occur, which could possibly be inhibiting PE-based long insertions.
• In still other embodiments, the PEgRNA may be improved by introducing additional RNA motifs at the 5′ and 3′ termini of the PEgRNAs. Several such motifs—such as the PAN ENE from KSHV and the ENE from MALAT1 were discussed above as possible means to terminate expression of longer PEgRNAs from non-pol III promoters. These elements form RNA triple helices that engulf the polyA tail, resulting in their being retained within the nucleus^184,187. However, by forming complex structures at the 3′ terminus of the PEgRNA that occlude the terminal nucleotide, these structures would also likely help prevent exonuclease-mediated degradation of PEgRNAs.
• Other structural elements inserted at the 3′ terminus could also enhance RNA stability, albeit without enabling termination from non-pol III promoters. Such motifs could include hairpins or RNA quadruplexes that would occlude the 3′ terminus¹⁹⁷, or self-cleaving ribozymes such as HDV that would result in the formation of a 2′-3′-cyclic phosphate at the 3′ terminus and also potentially render the PEgRNA less likely to be degraded by exonucleases¹⁹⁸. Inducing the PEgRNA to cyclize via incomplete splicing—to form a ciRNA—could also increase PEgRNA stability and result in the PEgRNA being retained within the nucleus¹⁹⁴.
• Additional RNA motifs could also improve RT processivity or enhance PEgRNA activity by enhancing RT binding to the DNA-RNA duplex. Addition of the native sequence bound by the RT in its cognate retroviral genome could enhance RT activity¹⁹⁹. This could include the native primer binding site (PBS), polypurine tract (PPT), or kissing loops involved in retroviral genome dimerization and initiation of transcription¹⁹⁹.
• Addition of dimerization motifs—such as kissing loops or a GNRA tetraloop/tetraloop receptor pair²⁰⁰— at the 5′ and 3′ termini of the PEgRNA could also result in effective circularization of the PEgRNA, improving stability. Additionally, it is envisioned that addition of these motifs could enable the physical separation of the PEgRNA spacer and primer, prevention occlusion of the spacer which would hinder PE activity. Short 5′ or 3′ extensions to the PEgRNA that form a small toehold hairpin in the spacer region could also compete favorably against the annealing region of the PEgRNA binding the spacer. Finally, kissing loops could also be used to recruit other template RNAs to the genomic site and enable swapping of RT activity from one RNA to the other. Example improvements include, but are not limited to:

• 	PEgRNA-HDV fusion
  	(SEQ ID NO: 230)
  	GGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGAA
  	ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAAC
  	TTGAAAAAGTGGGACCGAGTCGGTCCTCTGCCATC
  	AAAGCGTGCTCAGTCTGGGCCGGCATGGTCCCAGC
  	CTCCTCGCTGGCGCCGGCTGGGCAACATGCTTCGG
  	CATGGCGAATGGGACTTTTTTT
  	PEgRNA-MMLV kissing loop
  	(SEQ ID NO: 231)
  	GGTGGGAGACGTCCCACCGGCCCAGACTGAGCACG
  	TGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAA
  	GGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCG
  	AGTCGGTCCTCTGCCATCAAAGCTTCGACCGTGCT
  	CAGTCTGGTGGGAGACGTCCCACCTTTTTTT
  	PEgRNA-VS ribozyme kissing loop
  	(SEQ ID NO: 232)
  	GAGCAGCATGGCGTCGCTGCTCACGGCCCAGACTG
  	AGCACGTGAGTTTTAGAGCTAGAAATAGCAAGTTA
  	AAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTG
  	GGACCGAGTCGGTCCTCTGCCATCAAAGCTTCGAC
  	CGTGCTCAGTCTCCATCAGTTGACACCCTGAGGTT
  	TTTTT
  	PEgRNA-GNRA tetraloop/tetraloop
  	receptor
  	(SEQ ID NO: 233)
  	GCAGACCTAAGTGGUGACATATGGTCTGGGCCCAG
  	ACTGAGCACGTGAGTTTTAGAGCTAUACGTAGCAA
  	GTTAAAATAAGGCTAGTCCGTTATCAACTTUACGA
  	AGTGGGACCGAGTCGGTCCTCTGCCATCAAAGCTT
  	CGACCGTGCTCAGTCTGCATGCGATTAGAAATAAT
  	CGCATGTTTTTTT
  	PEgRNA template switching secondary
  	RNA-HDV fusion
  	(SEQ ID NO: 234)
  	TCTGCCATCAAAGCTGCGACCGTGCTCAGTCTGGT
  	GGGAGACGTCCCACCGGCCGGCATGGTCCCAGCCT
  	CCTCGCTGGCGCCGGCTGGGCAACATGCTTCGGCA
  	TGGCGAATGGGACTTTTTTT

• PEgRNA scaffold could be further improved via directed evolution, in an analogous fashion to how SpCas9 and prime editor (PE) have been improved. Directed evolution could enhance PEgRNA recognition by Cas9 or evolved Cas9 variants. Additionally, it is likely that different PEgRNA scaffold sequences would be optimal at different genomic loci, either enhancing PE activity at the site in question, reducing off-target activities, or both. Finally, evolution of PEgRNA scaffolds to which other RNA motifs have been added would almost certainly improve the activity of the fused PEgRNA relative to the unevolved, fusion RNA. For instance, evolution of allosteric ribozymes composed of c-di-GMP-I aptamers and hammerhead ribozymes led to dramatically improved activity²⁰², suggesting that evolution would improve the activity of hammerhead-PEgRNA fusions as well. In addition, while Cas9 currently does not generally tolerate 5′ extension of the sgRNA, directed evolution will likely generate enabling mutations that mitigate this intolerance, allowing additional RNA motifs to be utilized.
• The present disclosure contemplates any such ways to further improve the efficacy of the prime editing systems disclosed here.
• O. Use of Prime Editing with Expanded Targeting Scope
• Prime editing (PE) using Streptococcus pyogenes Cas9 (SpCas9) can efficiently install all single base substitutions, insertions, deletions, and combinations thereof at genomic loci where there is a suitably-placed NGG protospacer adjacent motif (PAM) that SpCas9 can efficiently bind. However, in another aspect The methods described herein broaden the targeting capability of PE by expanding the accessible PAMs and, therefore, the targetable genomic loci accessible for efficient PE. Prime editors using RNA-guided DNA binding proteins other than SpCas9 enable an expanded targetable scope of genomic loci by allowing access to different PAMs. In addition, use of RNA-guided DNA binding proteins smaller than SpCas9 also allows for more efficient viral delivery. PE with Cas proteins or other RNA-guided DNA binding proteins beyond SpCas9 will allow for high efficiency therapeutic edits that were either inaccessible or inefficient using SpCas9-based PE.
• This is expected to be used in situations where SpCas9-based PE is either inefficient due to non-ideal spacing of an edit to relative to an NGG PAM or the overall size of the SpCas9-based construct is prohibitive for cellular expression and/or delivery. Specific disease-relevant loci such as the Huntingtin gene, which has few and poorly located NGG PAMs for SpCas9 near the target region, can easily be targeted using different Cas proteins in the PE system such as SpCas9-VRQR which recognizes an NGA PAM. Smaller Cas proteins will be used to generate smaller PE constructs that can be packaged into AAV vectors more efficiently, enabling better delivery to target tissues. FIG. 61 shows the reduction to practice of prime editing using Staphylococcus aureus CRISPR-Cas as the RNA-guided DNA binding protein. NT is untreated control.
• FIGS. 62A-62B provide a demonstration of the importance of the protospacer for efficient installation of a desired edit at a precise location with prime editing. This highlights the importance of alternate PAMs and protospacers as novel features of this technology. “n.d.” in FIG. 62A is “not detected.”
• FIG. 63 shows the reduction to practice of PE using SpCas9(H840A)-VRQR and SpCas9(H840A)-VRER as the RNA-guided DNA binding protein in a prime editor system. The SpCas9(H840A)-VRQR napDNAbp is disclosed herein as SEQ ID NO: 87. The SpCas9(H840A)-VRER napDNAbp is disclosed herein as SEQ ID NO: 88. The SpCas9(H840A)-VRER-MMLV RT fusion protein is disclosed herein as SEQ ID NO: 516, wherein the MMLV RT comprises the D200N, L603W, T330P, T306K, and W313F substitutions relative to the wild type MMLV RT. The SpCas9(H840A)-VRQR-MMLV RT fusion protein is disclosed herein as SEQ ID NO: 515, wherein the MMLV RT comprises the D200N, L603W, T330P, T306K, and W313F substitutions relative to the wild type MMLV RT. Seven different loci in the human genome are targeted: 4 with the SpCas9(H840A)-VRQR-MMLV RT prime editor system and 3 with the SpCas9(H840A)-VRER-MMLV RT system. The amino acid sequences of the tested constructs are as follows:

• SACAS9-M-MLV	MKRTADGSEFESPKKKRKVGKRNYILGLDIGITSVGYGIIDYETR
  RT PRIMER EDITOR	DVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQRVKKLL
  	FDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRR
  	GVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKD
  	GEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLE
  	TRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAY
  	NADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTL
  	KQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEI
  	IENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNL
  	KGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDLS
  	QQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIII
  	ELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYLI
  	EKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFD
  	NSFNNKVLVKQEEASKKGNRTPFQYLSSSDSKISYETFKKHILNL
  	AKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGL
  	MNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHH
  	AEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIET
  	EQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYST
  	RKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQT
  	YQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIK
  	YYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFV
  	TVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDL
  	IKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPRI
  	IKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKGSGGSSGGS
  	SGSETPGTSESATPESSGGSSGGSSTLNIEDEYRLHETSKEPDVS
  	LGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYP
  	MSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYR
  	PVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAF
  	FCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFD
  	EALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ
  	TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMG
  	QPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFN
  	WGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGV
  	LTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGK
  	LTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRV
  	QFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLP
  	DADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQR
  	AELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLT
  	SEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNR
  	MADQAARKAAITETPDTSTLLIENSSPSGGSKRTADGSEFEPKKK
  	RKV (SEQ ID NO: 514)
  SPCAS9(H840A)-	MKRTADGSEFESPKKKRKVDKKYSIGLDIGTNSVGWAVITDEYKV
  VRQR-MALONEY	PSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRY
  MURINE	TRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHP
  LEUKEMIA VIRUS	IFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMI
  REVERSE	KFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVD
  TRANSCRIPTASE	AKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNF
  PRIMER EDITOR	KSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLS
  	DAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQ
  	LPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTE
  	ELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPF
  	LKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWN
  	FEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYN
  	ELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKED
  	YFKKIECFDSVETSGVEDRFNASLGTYHDLLKIIKDKDFLDNEEN
  	EDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYT
  	GWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSL
  	TFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDEL
  	VKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELG
  	SQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYD
  	VDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNY
  	WRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQI
  	TKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQF
  	YKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYD
  	VRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPL
  	IETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
  	ESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVAKVEKG
  	KSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIK
  	LPKYSLFELENGRKRMLASARELQKGNELALPSKYVNFLYLASHY
  	EKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANL
  	DKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTID
  	RKQYRSTKEVLDATLIHQSITGLYETRIDLSQLGGDSGGSSGGSS
  	GSETPGTSESATPESSGGSSGGSSTLNIEDEYRLHETSKEPDVSL
  	GSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPM
  	SQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRP
  	VQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFF
  	CLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNE
  	ALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQT
  	LGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQ
  	PTPKTPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNW
  	GPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVL
  	TQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKL
  	TMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQ
  	FGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPD
  	ADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRA
  	ELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGWLTS
  	EGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRM
  	ADQAARKAAITETPDTSTLLIENSSPSGGSKRTADGSEFEPKKKR
  	KV (SEQ ID NO: 515)
  SPCAS9(H840A)-	MKRTADGSEFESPKKKRKVDKKYSIGLDIGTNSVGWAVITDEYKV
  VRER-MALONEY	PSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRY
  MURINE	TRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHP
  LEUKEMIA VIRUS	IFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMI
  REVERSE	KFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVD
  TRANSCRIPTASE	AKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNF
  PRIME EDITOR	KSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLS
  	DAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQ
  	LPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTE
  	ELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPF
  	LKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWN
  	FEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYN
  	ELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKED
  	YFKKIECFDSVETSGVEDRFNASLGTYHDLLKIIKDKDFLDNEEN
  	EDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYT
  	GWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSL
  	TFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDEL
  	VKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELG
  	SQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYD
  	VDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNY
  	WRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQI
  	TKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQF
  	YKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYD
  	VRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPL
  	IETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
  	ESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVAKVEKG
  	KSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIK
  	LPKYSLFELENGRKRMLASARELQKGNELALPSKYVNFLYLASHY
  	EKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANL
  	DKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTID
  	RKEYRSTKEVLDATLIHQSITGLYETRIDLSQLGGDSGGSSGGSS
  	GSETPGTSESATPESSGGSSGGSSTLNIEDEYRLHETSKEPDVSL
  	GSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPM
  	SQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRP
  	VQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFF
  	CLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNE
  	ALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQT
  	LGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQ
  	PTPKTPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNW
  	GPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVL
  	TQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKL
  	TMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQ
  	FGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPD
  	ADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRA
  	ELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGWLTS
  	EGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRM
  	ADQAARKAAITETPDTSTLLIENSSPSGGSKRTADGSEFEPKKKR
  	KV (SEQ ID NO: 516)

• As shown in FIG. 63 , the SpCas9(H840A)-VRQR-MMLV RT was operational at PAM sites that included “AGAG” and “GGAG”, with some editing activity at “GGAT” and “AGAT” PAM sequences. The SpCas9(H840A)-VRER-MMLV RT was operational at PAM sites that included “AGCG” and “GGCG”, with some editing activity at “TGCG.”
• The data demonstrates that prime editing may be conducted using napDNAbps which bear different PAM specificities, such as those Cas9 variant described herein.
• In various embodiments, the napDNAbp (e.g., Cas9) with altered PAM specificities comprise a combination of mutations that exhibit activity on a target sequence comprising a 5′-NAA-3′ PAM sequence at its 3′-end. In some embodiments, the combination of mutations are present in any one of the clones listed in Table 1. In some embodiments, the combination of mutations are conservative mutations of the clones listed in Table 1. In some embodiments, the Cas9 protein comprises the combination of mutations of any one of the Cas9 clones listed in Table 1.

• TABLE 1
  NAA PAM Clones
  Mutations from wild-type SpCas9 (e.g., SEQ ID NO: 18)

  D177N, K218R, D614N, D1135N, P1137S, E1219V, A1320V, A1323D, R1333K

  D177N, K218R, D614N, D1135N, E1219V, Q1221H, H1264Y, A1320V, R1333K

  A10T, I322V, S409I, E427G, G715C, D1135N, E1219V, Q1221H, H1264Y, A1320V,

  R1333K

  A367T, K710E, R1114G, D1135N, P1137S, E1219V, Q1221H, H1264Y, A1320V, R1333K

  A10T, I322V, S409I, E427G, R753G, D861N, D1135N, K1188R, E1219V, Q1221H,

  H1264H, A1320V, R1333K

  A10T, I322V, S409I, E427G, R654L, V743I, R753G, M1021T, D1135N, D1180G, K1211R,

  E1219V, Q1221H, H1264Y, A1320V, R1333K

  A10T, I322V, S409I, E427G, V743I, R753G, E762G, D1135N, D1180G, K1211R, E1219V,

  Q1221H, H1264Y, A1320V, R1333K

  A10T, I322V, S409I, E427G, R753G, D1135N, D1180G, K1211R, E1219V, Q1221H,

  H1264Y, S1274R, A1320V, R1333K

  A10T, I322V, S409I, E427G, A5895, R753G, D1135N, E1219V, Q1221H, H1264H,

  A1320V, R1333K

  A10T, I322V, S409I, E427G, R753G, E757K, G865G, D1135N, E1219V, Q1221H, H1264Y,

  A1320V, R1333K

  A10T, I322V, S409I, E427G, R654L, R753G, E757K, D1135N, E1219V, Q1221H, H1264Y,

  A1320V, R1333K

  A10T, I322V, S409I, E427G, K599R, M631A, R654L, K673E, V743I, R753G, N758H,

  E762G, D1135N, D1180G, E1219V, Q1221H, Q1256R, H1264Y, A1320V, A1323D,

  R1333K

  A10T, I322V, S409I, E427G, R654L, K673E, V743I, R753G, E762G, N8695, N1054D,

  R1114G, D1135N, D1180G, E1219V, Q1221H, H1264Y, A1320V, A1323D, R1333K

  A10T, I322V, S409I, E427G, R654L, L727I, V743I, R753G, E762G, R8595, N946D,

  F1134L, D1135N, D1180G, E1219V, Q1221H, H1264Y, N1317T, A1320V, A1323D,

  R1333K

  A10T, I322V, S409I, E427G, R654L, K673E, V743I, R753G, E762G, N803S, N869S,

  Y1016D, G1077D, R1114G, F1134L, D1135N, D1180G, E1219V, Q1221H, H1264Y,

  V1290G, L1318S, A1320V, A1323D, R1333K

  A10T, I322V, S409I, E427G, R654L, K673E, V743I, R753G, E762G, N803S, N869S,

  Y1016D, G1077D, R1114G, F1134L, D1135N, K1151E, D1180G, E1219V, Q1221H,

  H1264Y, V1290G, L1318S, A1320V, R1333K

  A10T, I322V, S409I, E427G, R654L, K673E, V743I, R753G, E762G, N803S, N869S,

  Y1016D, G1077D, R1114G, F1134L, D1135N, D1180G, E1219V, Q1221H, H1264Y,

  V1290G, L1318S, A1320V, A1323D, R1333K

  A10T, I322V, S409I, E427G, R654L, K673E, F693L, V743I, R753G, E762G, N803S, N869S,

  L921P, Y1016D, G1077D, F1080S, R1114G, D1135N, D1180G, E1219V, Q1221H, H1264Y,

  L1318S, A1320V, A1323D, R1333K

  A10T, I322V, S409I, E427G, E630K, R654L, K673E, V743I, R753G, E762G, Q768H,

  N803S, N869S, Y1016D, G1077D, R1114G, F1134L, D1135N, D1180G, E1219V, Q1221H,

  H1264Y, L1318S, A1320V, R1333K

  A10T, I322V, S409I, E427G, R654L, K673E, F693L, V743I, R753G, E762G, Q768H,

  N803S, N869S, Y1016D, G1077D, R1114G, F1134L, D1135N, D1180G, E1219V, Q1221H,

  G1223S, H1264Y, L1318S, A1320V, R1333K

  A10T, I322V, S409I, E427G, R654L, K673E, F693L, V743I, R753G, E762G, N803S, N869S,

  L921P, Y1016D, G1077D, F1801S, R1114G, D1135N, D1180G, E1219V, Q1221H, H1264Y,

  L1318S, A1320V, A1323D, R1333K

  A10T, I322V, S409I, E427G, R654L, V743I, R753G, M1021T, D1135N, D1180G, K1211R,

  E1219V, Q1221H, H1264Y, A1320V, R1333K

  A10T, I322V, S4091, E427G, R654L, K673E, V743I, R753G, E762G, M673I, N803S, N869S,

  G1077D, R1114G, D1135N, V1139A, D1180G, E1219V, Q1221H, A1320V, R1333K

  A10T, I322V, S409I, E427G, R654L, K673E, V743I, R753G, E762G, N803S, N869S,

  R1114G, D1135N, E1219V, Q1221H, A1320V, R1333K

• In some embodiments, the Cas9 protein comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of a Cas9 protein as provided by any one of the variants of Table 1. In some embodiments, the Cas9 protein comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the amino acid sequence of a Cas9 protein as provided by any one of the variants of Table 1.
• In some embodiments, the Cas9 protein exhibits an increased activity on a target sequence that does not comprise the canonical PAM (5′-NGG-3′) at its 3′ end as compared to Streptococcus pyogenes Cas9 as provided by SEQ ID NO: 18. In some embodiments, the Cas9 protein exhibits an activity on a target sequence having a 3′ end that is not directly adjacent to the canonical PAM sequence (5′-NGG-3′) that is at least 5-fold increased as compared to the activity of Streptococcus pyogenes Cas9 as provided by SEQ ID NO: 18 on the same target sequence. In some embodiments, the Cas9 protein exhibits an activity on a target sequence that is not directly adjacent to the canonical PAM sequence (5′-NGG-3′) that is at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1,000-fold, at least 5,000-fold, at least 10,000-fold, at least 50,000-fold, at least 100,000-fold, at least 500,000-fold, or at least 1,000,000-fold increased as compared to the activity of Streptococcus pyogenes as provided by SEQ ID NO: 2 on the same target sequence. In some embodiments, the 3′ end of the target sequence is directly adjacent to an AAA, GAA, CAA, or TAA sequence. In some embodiments, the Cas9 protein comprises a combination of mutations that exhibit activity on a target sequence comprising a 5′-NAC-3′ PAM sequence at its 3′-end. In some embodiments, the combination of mutations are present in any one of the clones listed in Table 2. In some embodiments, the combination of mutations are conservative mutations of the clones listed in Table 2. In some embodiments, the Cas9 protein comprises the combination of mutations of any one of the Cas9 clones listed in Table 2.

• TABLE 2
  NAC PAM Clones
  MUTATIONS FROM WILD-TYPE SPCAS9 (E.G., SEQ ID NO: 18)

  T472I, R753G, K890E, D1332N, R1335Q, T1337N

  I1057S, D1135N, P1301S, R1335Q, T1337N

  T472I, R753G, D1332N, R1335Q, T1337N

  D1135N, E1219V, D1332N, R1335Q, T1337N

  T472I, R753G, K890E, D1332N, R1335Q, T1337N

  I1057S, D1135N, P1301S, R1335Q, T1337N

  T472I, R753G, D1332N, R1335Q, T1337N

  T472I, R753G, Q771H, D1332N, R1335Q, T1337N

  E627K, T638P, K652T, R753G, N803S, K959N, R1114G, D1135N, E1219V, D1332N,

  R1335Q, T1337N

  E627K, T638P, K652T, R753G, N8035, K959N, R1114G, D1135N, K1156E, E1219V,

  D1332N, R1335Q, T1337N

  E627K, T638P, V647I, R753G, N803S, K959N, G1030R, I1055E, R1114G, D1135N, E1219V,

  D1332N, R1335Q, T1337N

  E627K, E630G, T638P, V647A, G687R, N767D, N803S, K959N, R1114G, D1135N, E1219V,

  D1332G, R1335Q, T1337N

  E627K, T638P, R753G, N803S, K959N, R1114G, D1135N, E1219V, N1266H, D1332N,

  R1335Q, T1337N

  E627K, T638P, R753G, N8035, K959N, I1057T, R1114G, D1135N, E1219V, D1332N,

  R1335Q, T1337N

  E627K, T638P, R753G, N803S, K959N, R1114G, D1135N, E1219V, D1332N, R1335Q,

  T1337N

  E627K, M631I, T638P, R753G, N803S, K959N, Y1036H, R1114G, D1135N, E1219V,

  D1251G, D1332G, R1335Q, T1337N

  E627K, T638P, R753G, N803S, V875I, K959N, Y1016C, R1114G, D1135N, E1219V,

  D1251G, D1332G, R1335Q, T1337N, I1348V

  K608R, E627K, T638P, V647I, R654L, R753G, N803S, T804A, K848N, V922A, K959N,

  R1114G D1135N, E1219V, D1332N, R1335Q, T1337N

  K608R, E627K, T638P, V647I, R753G, N803S, V922A, K959N, K1014N, V1015A, R1114G,

  D1135N, K1156N, E1219V, N1252D, D1332N, R1335Q, T1337N

  K608R, E627K, R629G, T638P, V647I, A711T, R753G, K775R, K789E, N8035, K959N,

  V1015A, Y1036H, R1114G, D1135N, E1219V, N1286H, D1332N, R1335Q, T1337N

  K608R, E627K, T638P, V647I, T740A, R753G, N803S, K948E, K959N, Y1016S, R1114G,

  D1135N, E1219V, N1286H, D1332N, R1335Q, T1337N

  K608R, E627K, T638P, V647I, T740A, N803S, K948E, K959N, Y1016S, R1114G, D1135N,

  E1219V, N1286H, D1332N, R1335Q, T1337N

  I670S, K608R, E627K, E630G, T638P, V647I, R653K, R753G, I795L, K797N, N803S,

  K866R, K890N, K959N, Y1016C, R1114G, D1135N, E1219V, D1332N, R1335Q, T1337N

  K608R, E627K, T638P, V647I, T740A, G752R, R753G, K797N, N803S, K948E, K959N,

  V1015A, Y1016S, R1114G, D1135N, E1219V, N1266H, D1332N, R1335Q, T1337N

  I570T, A589V, K608R, E627K, T638P, V647I, R654L, Q716R, R753G, N803S, K948E,

  K959N, Y1016S, R1114G, D1135N, E1207G, E1219V, N1234D, D1332N, R1335Q, T1337N

  K608R, E627K, R629G, T638P, V647I, R654L, Q740R, R753G, N803S, K959N, N990S,

  T995S, V1015A, Y1036D, R1114G, D1135N, E1207G, E1219V, N1234D, N1266H, D1332N,

  R1335Q, T1337N

  I562F, V565D, I570T, K608R, L625S, E627K, T638P, V647I, R654I, G752R, R753G, N803S,

  N808D, K959N, M1021L, R1114G, D1135N, N1177S, N1234D, D1332N, R1335Q, T1337N

  I562F, I570T, K608R, E627K, T638P, V647I, R753G, E790A, N803S, K959N, V1015A,

  Y1036H, R1114G, D1135N, D1180E, A1184T, E1219V, D1332N, R1335Q, T1337N

  I570T, K608R, E627K, T638P, V647I, R654H, R753G, E790A, N803S, K959N, V1015A,

  R1114G, D1127A, D1135N, E1219V, D1332N, R1335Q, T1337N

  I570T, K608R, L625S, E627K, T638P, V647I, R654I, T703P, R753G, N803S, N808D, K959N,

  M1021L, R1114G, D1135N, E1219V, D1332N, R1335Q, T1337N

  I570S, K608R, E627K, E630G, T638P, V647I, R653K, R753G, I795L, N803S, K866R,

  K890N, K959N, Y1016C, R1114G, D1135N, E1219V, D1332N, R1335Q, T1337N

  I570T, K608R, E627K, T638P, V647I, R654H, R753G, E790A, N803S, K959N, V1016A,

  R1114G, D1135N, E1219V, K1246E, D1332N, R1335Q, T1337N

  K608R, E627K, T638P, V647I, R654L, K673E, R753G, E790A, N803S, K948E, K959N,

  R1114G, D1127G, D1135N, D1180E, E1219V, N1286H, D1332N, R1335Q, T1337N

  K608R, L625S, E627K, T638P, V647I, R654I, I670T, R753G, N803S, N808D, K959N,

  M1021L, R1114G, D1135N, E1219V, N1286H, D1332N, R1335Q, T1337N

  E627K, M631V, T638P, V647I, K710E, R753G, N803S, N808D, K948E, M1021L, R1114G,

  D1135N, E1219V, D1332N, R1335Q, T1337N, S1338T, H1349R

• In some embodiments, the Cas9 protein comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of a Cas9 protein as provided by any one of the variants of Table 2. In some embodiments, the Cas9 protein comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the amino acid sequence of a Cas9 protein as provided by any one of the variants of Table 2.
• In some embodiments, the Cas9 protein exhibits an increased activity on a target sequence that does not comprise the canonical PAM (5′-NGG-3′) at its 3′ end as compared to Streptococcus pyogenes Cas9 as provided by SEQ ID NO: 18. In some embodiments, the Cas9 protein exhibits an activity on a target sequence having a 3′ end that is not directly adjacent to the canonical PAM sequence (5′-NGG-3′) that is at least 5-fold increased as compared to the activity of Streptococcus pyogenes Cas9 as provided by SEQ ID NO: 18 on the same target sequence. In some embodiments, the Cas9 protein exhibits an activity on a target sequence that is not directly adjacent to the canonical PAM sequence (5′-NGG-3′) that is at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1,000-fold, at least 5,000-fold, at least 10,000-fold, at least 50,000-fold, at least 100,000-fold, at least 500,000-fold, or at least 1,000,000-fold increased as compared to the activity of Streptococcus pyogenes as provided by SEQ ID NO: 18 on the same target sequence. In some embodiments, the 3′ end of the target sequence is directly adjacent to an AAC, GAC, CAC, or TAC sequence.
• In some embodiments, the Cas9 protein comprises a combination of mutations that exhibit activity on a target sequence comprising a 5′-NAT-3′ PAM sequence at its 3′-end. In some embodiments, the combination of mutations are present in any one of the clones listed in Table 3. In some embodiments, the combination of mutations are conservative mutations of the clones listed in Table 3. In some embodiments, the Cas9 protein comprises the combination of mutations of any one of the Cas9 clones listed in Table 3.

• TABLE 3
  NAT PAM Clones
  MUTATIONS FROM WILD-TYPE SPCAS9 (E.G., SEQ ID NO: 18)

  K961E, H985Y, D1135N, K1191N, E1219V, Q1221H, A1320A, P1321S, R1335L

  D1135N, G12185, E1219V, Q1221H, P1249S, P1321S, D1322G, R1335L

  V743I, R753G, E790A, D1135N, G1218S, E1219V, Q1221H, A1227V, P1249S, N1286K,

  A1293T, P1321S, D1322G, R1335L, T1339I

  F575S, M631L, R654L, V748I, V743I, R753G, D853E, V922A, R1114G, D1135N, G1218S,

  E1219V, Q1221H, A1227V, P1249S, N1286K, A1293T, P1321S, D1322G, R1335L, T1339I

  F575S, M631L, R654L, R664K, R753G, D853E, V922A, R1114G D1135N, D1180G, G1218S,

  E1219V, Q1221H, P1249S, N1286K, P1321S, D1322G, R1335L

  M631L, R654L, R753G, K797E, D853E, V922A, D101A, R1114G, D1135N, G1218S,

  E1219V, Q1221H, P1249S, N1317K, P1321S, D1322G, R1335L

  F575S, M631L, R654L, R664K, R753G, D853E, V922A, R1114G, Y1131C, D1135N,

  D1180G, G1218S, E1219V, Q1221H, P1249S, P1321S, D1322G, R1335L

  F575S, M631L, R654L, R664K, R753G, D853E, V922A, R1114G, Y1131C, D1135N,

  D1180G, G12185, E1219V, Q1221H, P1249S, P1321S, D1322G, R1335L

  F575S, D596Y, M631L, R654L, R664K, R753G, D853E, V922A, R1114G, Y1131C, D1135N,

  D1180G, G1218S, E1219V, Q1221H, P1249S, Q1256R, P1321S, D1322G, R1335L

  F575S, M631L, R654L, R664K, K710E, V750A, R753G, D853E, V922A, R1114G, Y1131C,

  D1135N, D1180G, G1218S, E1219V, Q1221H, P1249S, P1321S, D1322G, R1335L

  F575S, M631L, K649R, R654L, R664K, R753G, D853E, V922A, R1114G, Y1131C, D1135N,

  K1156E, D1180G, G1218S, E1219V, Q1221H, P1249S, P1321S, D1322G, R1335L

  F575S, M631L, R654L, R664K, R753G, D853E, V922A, R1114G, Y1131C, D1135N,

  D1180G, G1218S, E1219V, Q1221H, P1249S, P1321S, D1322G, R1335L

  F575S, M631L, R654L, R664K, R753G, D853E, V922A, I1057G, R1114G, Y1131C, D1135N,

  D1180G, G1218S, E1219V, Q1221H, P1249S, N1308D, P1321S, D1322G, R1335L

  M631L, R654L, R753G, D853E, V922A, R1114G, Y1131C, D1135N, E1150V, D1180G,

  G1218S, E1219V, Q1221H, P1249S, P1321S, D1332G, R1335L

  M631L, R654L, R664K, R753G, D853E, I1057V, Y1131C, D1135N, D1180G, G1218S,

  E1219V, Q1221H, P1249S, P1321S, D1332G, R1335L

  M631L, R654L, R664K, R753G, I1057V, R1114G, Y1131C, D1135N, D1180G, G1218S,

  E1219V, Q1221H, P1249S, P1321S, D1332G, R1335L

• Any of the above Cas9 variants displaying differential PAM specificities as compared to the canonical SpCas9 may be used in the herein disclosed prime editors.
• P. Use of Prime Editing for Inserting Recombinase Target Sites
• In another aspect, prime editing may be used to insert recombinase sites (or “recombinase recognition sequences”) into a desired genomic site. Insertion of recombinase sites provides a programmed location for effecting site-specific genetic changes in a genome. Such genetic changes can include, for example, genomic integration of a plasmid, genomic deletion or insertion, chromosomal translocations, and cassette exchanges, among other genetic changes. These exemplary types of genetic changes are illustrated in FIG. 64(b)-(f). The installed recombinase recognition sequences may then be used to conduct site-specific recombination at that site to effectuate a variety of recombination outcomes, such as, excision, integration, inversion, or exchange of DNA fragments. For example, FIG. 65 illustrates the installation of a recombinase site that can then be used to integrate a DNA donor template comprising a GFP expression marker. Cells containing the integrated GFP expression system into the recombinase site will fluoresce.
• The mechanism of installing a recombinase site into the genome is analogous to installing other sequences, such as peptide/protein and RNA tags, into the genome. A schematic exemplifying the installation of a recombinase target sequence is shows in FIG. 64(a). The process begins with selecting a desired target locus into which the recombinase target sequence will be introduced. Next, a prime editor fusion is provided (“RT-Cas9:gRNA”). Here, the “gRNA” refers to a PEgRNA, which can be designed using the principles described herein. The PEgRNA in various embodiments will comprise an architecture corresponding to FIG. 3D (5′-[˜20-nt spacer]-[gRNA core]-[extension arm]-3′, wherein the extension arm comprises in the 3′ to 5′ direction, a primer binding site (“A”), an edit template (“B”), and a homology arm (“C”). The edit template (“B”) will comprise a sequence corresponding to a recombinase site, i.e., a single strand RNA of the PEgRNA that codes for a complementary single strand DNA that is either the sense or the antisense strand of the recombinase site and which is incorporated into the genomic DNA target locus through the prime editing process.
• In various aspects, the present disclosure provides for the use of a PE to introduce recombinase recognition sequences at high-value loci in human or other genomes, which, after exposure to site-specific recombinase(s), will direct precise and efficient genomic modifications (FIG. 64 ). In various embodiments show in FIG. 64 , PE may be used to (b) insert a single SSR target for use as a site for genomic integration of a DNA donor template. (c) shows how a tandem insertion of SSR target sites can be used to delete a portion of the genome. (d) shows how a tandem insertion of SSR target sites can be used to invert a portion of the genome. (e) shows how the insertion of two SSR target sites at two distal chromosomal regions can result in chromosomal translocation. (f) shows how the insertion of two different SSR target sites in the genome can be used to exchange a cassette from a DNA donor template. Each of the types of genome modifications are envisioned by using PE to insert SSR targets, but this list also is not meant to be limiting.
• PE-mediated introduction of recombinase recognition sequences could be particularly useful for the treatment of genetic diseases which are caused by large-scale genomic defects, such as gene loss, inversion, or duplication, or chromosomal translocation^1-7 (Table 6). For example, Williams-Beuren syndrome is a developmental disorder caused by a deletion of 24 in chromosome 721. No technology exists currently for the efficient and targeted insertion of multiple entire genes in living cells (the potential of PE to do such a full-length gene insertion is currently being explored but has not yet been established); however, recombinase-mediated integration at a target inserted by PE offers one approach towards a permanent cure for this and other diseases. In addition, targeted introduction of recombinase recognition sequences could be highly enabling for applications including generation of transgenic plants, animal research models, bioproduction cell lines, or other custom eukaryotic cell lines. For example, recombinase-mediated genomic rearrangement in transgenic plants at PE-specific targets could overcome one of the bottlenecks to generating agricultural crops with improved properties^8,9.

• TABLE 6
  Examples of genetic diseases linked to large-scale
  genomic modifications that could be repaired through
  PE-based installation of recombinase recognition sequences.

  	DISEASE	CAUSE
  	TRISOMY 17P	GENE DUPLICATION
  	CHARCOT-MARIE-	GENE DUPLICATION
  	TOOTH DISEASE TYPE I
  	SMITH-MAGENIS	GENE DELETION
  	SYNDROME
  	WILLIAMS-BEUREN	GENE DELETION
  	SYNDROME
  	DE LA CHAPELLE	CHROMOSOMAL
  	SYNDROME	TRANSLOCATION
  	DOWN SYNDROME	CHROMOSOMAL
  	(SOME FORMS)	TRANSLOCATION
  	HEMOPHILIA A	GENE INVERSION
  	HUNTER SYNDROME	GENE INVERSION

• A number of SSR family members have been characterized and their recombinase recognition sequences described, including natural and engineered tyrosine recombinases (Table 7), large serine integrases (Table 8), serine resolvases (Table 9), and tyrosine integrases (Table 10). Modified target sequences that demonstrate enhanced rates of genomic integration have also been described for several SSRs^22-30. In addition to natural recombinases, programmable recombinases with distinct specificities have been developed^31-40. Using PE, one or more of these recognition sequences could be introduced into the genomic at a specified location, such as a safe harbor locus^41-43, depending on the desired application.
• For example, introduction of a single recombinase recognition sequence in the genome by prime editing would result in integrative recombination with a DNA donor template (FIG. 64 b ). Serine integrases, which operate robustly in human cells, may be especially well-suited for gene integration^44,45.
• Additionally, introduction of two recombinase recognition sequences could result in deletion of the intervening sequence, inversion of the intervening sequence, chromosomal translocation, or cassette exchange, depending on the identity and orientation of the targets (FIG. 64 c-f ). By choosing endogenous sequences that already closely resemble recombinase targets, the scope of editing required to introduce the complete recombinase target would be reduced.
• Finally, several recombinases have been demonstrated to integrate into human or eukaryotic genomes at natively occurring pseudosites^46-64. PE editing could be used to modify these loci to enhance rates of integration at these natural pseudosites, or alternatively, to eliminate pseudosites that may serve as unwanted off-target sequences.
• This disclosure describes a general methodology for introducing recombinase target sequences in eukaryotic genomes using PE, the applications of which are nearly limitless. The genome editing reactions are intended for use with “prime editor,” a chimeric fusion of a CRISPR/Cas9 protein and a reverse-transcriptase domain, which utilizes a custom prime editing guide RNA (PEgRNA). By extension, Cas9 tools and homology-directed repair (HDR) pathways may also be exploited to introduce recombinase recognition sequences through DNA templates by lowering the rates of indels using several techniques^65-67. A proof-of-concept experiment in human cell culture is shown in FIG. 65 .
• The following several tables are cited in the above description relating to PE-directed installation of recombinase recognition sequences and provide a listing of exemplary recombinases that may be used, and their cognate recombinase recognition sequences that may be installed by PE.

• TABLE 7
  Tyrosine recombinases and SSR target sequences.

  Recombinase	Recombinase recognition sequence	Name
  Cre	ATAACTTCGTATAGCATACATTATACGAAGTTAT	loxP
  	(SEQ ID NO: 517)
  Dre	TAACTTTAAATAATGCCAATTATTTAAAGTTA	rox
  	(SEQ ID NO: 518)
  VCre	TCAATTTCTGAGAACTGTCATTCTCGGAAATTGA	loxV
  	(SEQ ID NO: 519)
  SCre	CTCGTGTCCGATAACTGTAATTATCGGACATGAT	loxS
  	(SEQ ID NO: 520)
  Flp	GAAGTTCCTATTCTCTAGAAAGTATAGGAACTTC	FRT
  	(SEQ ID NO: 521)
  B2	GAGTTTCATTAAGGAATAACTAATTCCCTAATGAAACTC	loxB
  	(SEQ ID NO: 522)
  B3	GGTTGCTTAAGAATAAGTAATTCTTAAGCAACC	loxB3
  	(SEQ ID NO: 523)
  Kw	ACGAAAAATGGTAAGGAATAGACCATTCCTTACCATTTTTGGT
  	(SEQ ID NO: 524)
  R	TTGATGAAAGAATAACGTATTCTTTCATCAA	RS
  	(SEQ ID NO: 525)
  TD1-40	GTGCGTCAAATAATAACGTATTATTTGACACTT	TDRS
  	(SEQ ID NO: 526)
  Vika	AATAGGTCTGAGAACGCCCATTCTCAGACGTATT	vox
  	(SEQ ID NO: 527)
  Nigri	TGAATGTCCTATAATTACACTTATAGGACATTCA	nox
  	(SEQ ID NO: 528)
  Panto	GAAACTTTAAATAATAAGTCTTATTTAAAGTTTC	pox
  	(SEQ ID NO: 529)
  Kd	AAACGATATCAGACATTTGTCTGATAATGCTTCATTATCAGACAAATGT	loxK
  	CTGATATCGTTT (SEQ ID NO: 530)
  Fre	ATATATACGTATATAGACATATATACGTATATAT	loxH
  	(SEQ ID NO: 531)
  CreALSHG	ATAACTCTATATAATGTATGCTATATAGAGTTAT	loxM7
  	(SEQ ID NO: 532)
  Tre	ACAACATCCTATTACACCCTATATGCCAACATGG	loxLTR
  	(SEQ ID NO: 533)
  Brec1	AACCCACTGCTTAAGCCTCAATAAAGCTTGCCTT	loxBTR
  	(SEQ ID NO: 534)
  Cre-R3M3	GATACAACGTATATACCTTTCTATACGTTGTTTA	loxK2
  	(SEQ ID NO: 535)

• TABLE 8
  Large serine integrases and SSR target sequences.

  	Recombinase recognition sequence	Recombinase recognition sequence
  Recombinase	Left	Right
  Bxb1	GGTTTGTCTGGTCAACCACCGCGGTCTCAGTGG	GGCTTGTCGACGACGGCGGTCTCCGTCGTCAG
  	TGTACGGTACAAACC (SEQ ID NO: 536)	GATCAT (SEQ ID NO: 537)
  phiC31	GTGCCCCAACTGGGGTAACTTTGAGTTCTCTCA	TGCGGGTGCCAGGGCGTGCCCTTGGGCTCCCC
  	GTTGGGGG (SEQ ID NO: 538)	GGGCGCGTACTCC (SEQ ID NO: 539)
  R4	TGTTCCCCAAAGCGATACCACTTGAAGCAGTGG	GCATGTTCCCCAAAGCGATACCACTTGAAGCA
  	TACTGCTTGTGGGTACA	GTGGTACTGCTTGTGGGTACACTCTGCGGGTG
  	(SEQ ID NO: 540)	(SEQ ID NO: 541)
  phiBT1	GGTGCTGGGTTGTTGTCTCTGGACAGTGATCCA	CAGGTTTTTGACGAAAGTGATCCAGATGATCC
  	TGGGAAACTACTCAGCACC	AG (SEQ ID NO: 543)
  	(SEQ ID NO: 542)
  MJ1 (phiFC1)	ATTTTAGGTATATGATTTTGTTTATTAGTGTAA	CAAAGGATCACTGAATCAAAAGTATTGCTCAT
  	ATAACACTATGTACCTAAAAT	CCACGCGAAA (SEQ ID NO: 545)
  	(SEQ ID NO: 544)
  MR11	TTTGTGCGGAACTACGAACAGTTCATTAATACG	CGAAAATGTATGGAGGCACTTGTATCAATATA
  	AAGTGTACAAACTTCCATACAA	GGATGTATACCTTCGAAGACACTT
  	(SEQ ID NO: 546)	(SEQ ID NO: 547)
  TP901-1	GAGTTTTTATTTCGTTTATTTCAATTAAGGTAA	ATGCCAACACAATTAACATCTCAATCAAGGTA
  	CTAAAAAACTCCTTTTAAGG	AATGCTTTTTGCTTTTTTTGC
  	(SEQ ID NO: 548)	(SEQ ID NO: 549)
  A118	TTCCTCGTTTTCTCTCGTTGGAAGAAGAAGAAA	TTTCGGATCAAGCTATGAAGGACGCAAAGAGG
  	CGAGAAA (SEQ ID NO: 550)	GAACTAAA (SEQ ID NO: 551)
  U153	TTCCTCGTTTTCTCTCGTTGGACGGAAACGAAT	TTTCGGATCAAGCTATGAAGGACGCAAAGAGG
  	CGAGAAA (SEQ ID NO: 552)	GAACTAAA (SEQ ID NO: 553)
  phiRV1	GTAGTGTATCTCACAGGTCCACGGTTGGCCGTG	GAAGGTGTTGGTGCGGGGTTGGCCGTGGTCGA
  	GACTGCTGAAGAACATTCC	GGTGGGGT (SEQ ID NO: 555)
  	(SEQ ID NO: 554)
  phi370.1	AAAAAAATACAGCGTTTTTCATGTACAACTATA	TTGTAAAGGAGACTGATAATGGCATGTACAAC
  	CTAGTTGTAGTGCCTAAAA	TATACTCGTCGGTAAAAAGGCA
  	(SEQ ID NO: 556)	(SEQ ID NO: 557)
  TG1	TCCAGCCCAACAGTGTTAGTCTTTGCTCTTACC	GATCAGCTCCGCGGGCAAGACCTTTCTCCTTC
  	CAGTTGGGCGGGA (SEQ ID NO: 558)	ACGGGGTGGAAGGTC (SEQ ID NO: 559)
  WB	CTAGTTTTAAAGTTGGTTATTAGTTACTGTGAT	CGGAAGGTAGCGTCAACGATAGGTGTAACTGT
  	ATTTATCACGGTACCCAATAACCAATGAAT	CGTGTTTGTAACGGTACTTCCAACAGCTGGCG
  	(SEQ ID NO: 560)	CCGCCAC (SEQ ID NO: 561)
  BL3	CAATGAAAAACTAGGCATGTAGAAGTTGTTTGT	TTTCCACAGACAACTCACGTGGAGGTAGTCAC
  	(SEQ ID NO: 562)	(SEQ ID NO: 563)
  SprA	TGTAGTAAGTATCTTAATATACAGCTTTATCTG	CACCCATTGTGTTCACAGGAGATACAGCTTTA
  	TTTTTTAAGATACTTACTACTTT	TCTGTACTGATATTAATGACATGCTG
  	(SEQ ID NO: 564)	(SEQ ID NO: 565)
  phiJoe	AGTTGTGGCCATGTGTCCATCTGGGGGCAGATG	ATCTGGATGTGGGTGTCCATCTGCGGGCAGAC
  	GAGACGGGGTCACA (SEQ ID NO: 566)	GCCGCAGTCGAAGCACGG
  		(SEQ ID NO: 567)
  —	—	ACCTTGATCTCGGTGTCCATCGCCGGGCAGAC
  		GCCGCAGTCGAAGCACGG
  		(SEQ ID NO: 568)
  phiK38	CCCTAATACGCAAGTCGATAACTCTCCTGGGAG	GAGCGCCGGATCAGGGAGTGGACGGCCTGGGA
  	CGTTGACAACTTGCGCACCCTGATCTG	GCGCTACACGCTGTGGCTGCGGTCGGTGC
  	(SEQ ID NO: 569)	(SEQ ID NO: 570)
  Int2	GCTCATGTATGTGTCTACGCGAGATTCTCGCCC	GGACGGCGCAGAAGGGGAGTAGCTCTTCGCCG
  	GAGAACTTCTGCAAGGACTGCTCTTGGCT	GACCGTCGACATACTGCTCAGCTCGTC
  	(SEQ ID NO: 571)	(SEQ ID NO: 572)
  Int3	ATGGATAAAAAAATACAGCGTTTTTCATGTACA	GTTTGTAAAGGAGACTGATAATGGCATGTACA
  	ACTATACTAGTTGTAGTGCCTAAATAATGCTT	ACTATACTCGTCGGTAAAAAGGCATCTTAT
  	(SEQ ID NO: 573)	(SEQ ID NO: 574)
  Int4	AAAAATTACAAAGTTTTCAACCCTTGATTTGAA	TTCCAAAGAGCGCCCAACGCGACCTGAAATTT
  	TTAGCGGTCAAATAATTTGTAATTCGTTT	GAATAAGACTGCTGCTTGTGTAAAGGCGATGA
  	(SEQ ID NO: 575)	TT (SEQ ID NO: 576)
  Int7	GTGTTATAAACCTGTGTGAGAGTTAAGTTTACA	AGACGAGAAACGTTCCGTCCGTCTGGGTCAGT
  	TGCCTAACCTTAACTTTTACGCAGGTTCAGCTT	TGGGCAAAGTTGATGACCGGGTCGTCCGTT
  	(SEQ ID NO: 577)	(SEQ ID NO: 578)
  Int8	TTAATAAACTATGGAAGTATGTACAGTCTTGCA	CAATCATCAGATAACTATGGCGGCACGTGCAT
  	ATGTTGAGTGAACAAACTTCCATAATAAAAT	TAACCACGGTTGTATCCCGTCTAAAGTACTCG
  	(SEQ ID NO: 579)	T (SEQ ID NO: 580)
  Int9	GTGGTTGTTTTTGTTGGAAGTGTGTATCAGGTA	TTTATATTGCGAAAAATAATTGGCGAACGAGG
  	TCTGCATAGTTATTCCGAACTTCCAATTA	TAACTGGATACCTCATCCGCCAATTAAAATTT
  	(SEQ ID NO: 581)	G (SEQ ID NO: 582)
  Int10	GGAAAATATAAATAATTTTAGTAACCTACATCT	AGCACGCTGATAATCAGCAAGACCACCAACAT
  	CAATCAAGGATAGTAAAACTCTCACTCTT	TTCCACCAATGTAAAAGCTTTAACCTTAGC
  	(SEQ ID NO: 583)	(SEQ ID NO: 584)
  Int11	GTTTATATGTTTACTAATAAGACGCTCTCAACC	ATGGATTTTGCAGATTCCCAGATGCCCCTACA
  	CATAAAGTCTTATTAGTAAACATATTTCAACT	GAAAGAGGTACAAAACATTTATTGGAATTAAT
  	(SEQ ID NO: 585)	T (SEQ ID NO: 586)
  Int12	TTTTTGTATGTTAGTTGTGTCACTGGGTAGACC	GTTCGTGGTAACTATGGGTGGTACAGGTGCCA
  	TAAATAGTGACACAACTGCTATTAAAATTTAA	CATTAGTTGTACCATTTATGTTTATGTGGTTA
  	(SEQ ID NO: 587)	AC (SEQ ID NO: 588)
  Int13	CAATAACGGTTGTATTTGTAGAACTTGACCAGT	GCATACATTGTTGTTGTTTTTCCAGATCCAGT
  	TGTTTTAGTAACATAAATACAACTCCGAATA	TGGTCCTGTAAATATAAGCAATCCATGTGAGT
  	(SEQ ID NO: 589)	(SEQ ID NO: 590)
  LI	GTTTAGTATCTCGTTATCTCTCGTTGGAGGGAG	TAACTTTTTCGGATCGAGTTATGATGGACGTA
  	AAGAAACGGGATACCAAAA	AAGAGGGAACAAAGCATCTA
  	(SEQ ID NO: 591)	(SEQ ID NO: 592)
  Peaches	TAGTTTCCAATGTTACAGGAACTGCTGGCAGAA	CGGTCTCCATCGGGATCTGCTGATCGAGCAGC
  	TCCAACACATTGGAAGTCG	ATGCCGACCA (SEQ ID NO: 594)
  	(SEQ ID NO: 593)
  Bxz2	TAACCGCAAGTGTACATCCCTCGGCTGGCCGAG	CGGTCTCCATCGGGATCTGCTGATCGAGCAGC
  	ACAAGTACAGTTGCGACAG	ATGCCGACCA (SEQ ID NO: 596)
  	(SEQ ID NO: 595)
  SV1	ATGTGGTCCTTTAGATCCACTGACGTGGGTCAG	CATCAGGGCGGTCAGGCCGTAGATGTGGAAGA
  	TGTCTCTAAAGGACTCGCG	AACGGCAGCACGGCGAGGACG
  	(SEQ ID NO: 597)	(SEQ ID NO: 598)

• TABLE 9
  Serine resolvases and SSR target sequences.

  	Recombinase recognition sequence	Recombinase recognition sequence
  Resolvase	Left	Right
  Gin	CGTTTCCTGTAAACCGAGGTTTTGGATAAACA	CGTTTCCTGTAAACCGAGGTTTTGGATAATGG
  	C (SEQ ID NO: 599)	(SEQ ID NO: 600)
  Cin	GAGTTCTCTTAAACCAAGGTTTAGGATTGAAA	GAGTTCTCTTAAACCAAGGTATTGGATAACAG
  	(SEQ ID NO: 601)	(SEQ ID NO: 602)
  Hin	TGGTTCTTGAAAACCAAGGTTTTTGATAAAGC	AAATTTTCCTTTTTGGAAGGTTTTTGATAACCA
  	(SEQ ID NO: 603)	(SEQ ID NO: 604)
  Min	GCCTTCCCCTAAACCAACGTTTTTATGCCGCC	GCCTTCCCCCAAACCAAGGTAATCAAGAACGC
  	(SEQ ID NO: 605)	(SEQ ID NO: 606)
  Sin	TTGTGAAATTTGGGTACACCCTAATCATACAA	CGTATGATTAGGGTGTATATTAATTT
  	(SEQ ID NO: 607)	(SEQ ID NO: 608)

• TABLE 10
  Tyrosine integrases and target sequences.

  Integrase	attP	attB
  HK022	CAAATGATTTTATTTTGACTAATAATGACC	GCACTTTAGGTGAAAAAGGTT
  	TACTTACATTAATTTACTGATAATTAAAGA	(SEQ ID NO: 610)
  	GATTTTAAATATACAACTTATTCACCTAAA
  	GGATGACAAAA (SEQ ID NO: 609)
  	TAACATTAATCACTTAAAAATCATCGCATT
  	ACACTAATCTGTGGTTAAATGATAGACTAC
  	ATAATGCGACAAAACGCAACATATCCAGTC
  	ACTATGAATCAACTACTTAGATAGTATTAG
  	TGACCT (SEQ ID NO: 611)
  P22	CTAAGTGGTTTGGGACAAAAATGGGACATA	GCAGCGCATTCGTAATGCGAAGGT
  	CAAATCTTTGCATCGGTTTGCAAGGCTTTG	CGT (SEQ ID NO: 613)
  	CATGTCTTTCGAAGATGGGACGTGTGAGCG
  	CAGGTATGACGTGGTATGTGTTGACTTAAA
  	AGGTAGTTCTTATAATTCGTAATGCGAAGG
  	TCGTAGGTTCGACTCCTATTATCGGCACCA
  	GTTAAATCAAATACTTACGTATTATTCGTG
  	CCTTCCTTATTTTTACTGTGGGACATATTT
  	GGGACAGAAGTACCAAAAA
  	(SEQ ID NO: 612)
  L5	GCGATCCCCATCCGCGACGTGCCAACTAGG	GAGCGGGCGACGGGAATCGAACCC
  	TCTCCTCTCGTCGTGAACAAGGCTACCGGG	GCGTAGCTAGTTTGGAAGA
  	TTGCAACTCCTGTGCAACTCTCAGGCTTCA	(SEQ ID NO: 615)
  	ACGCGCTTCTACGACCTGCAATTTCTTTCC
  	ACTTAGAGGATGCAGCCGAGAGGGGTAAAA
  	ACCTATCTTGACCGGCCCATATGTGGTCGG
  	CAGACACCCATTCTTCCAAACTAGCTACGC
  	GGGTTCGATTCCCGTCGCCCGCTCCGCTGG
  	TCAGAGGGTGTTTTCGCCCTCTGGCCATTT
  	TTCTTTCCAGGGGTCTGCAACTCTTGTGCG
  	ACTCTTCTGACCTGGGCATACGCGGTTGCA
  	ACGCATCCCTGATCTGGCTACTTTCGATGC
  	TGACAAACGAATAGAGCCCCCCGCCTGCGC
  	GAACAGACGAGGGGCATTCACA
  	(SEQ ID NO: 614)

• In various other aspects, the present disclosure relates to methods of using PE to install one or more recombinase recognition sequence and their use in site-specific recombination.
• In some embodiments, the site-specific recombination may effectuate a variety of recombination outcomes, such as, excision, integration, inversion, or exchange of DNA fragments.
• In some embodiments, the methods are useful for inducing recombination of or between two or more regions of two or more nucleic acid (e.g., DNA) molecules. In other embodiments, the methods are useful for inducing recombination of or between two or more regions in a single nucleic acid molecule (e.g., DNA).
• In some embodiments, the disclosure provides a method for integrating a donor DNA template by site-specific recombination, comprising: (a) installing a recombinase recognition sequence at a genomic locus by prime editing; (b) contacting the genomic locus with a DNA donor template that also comprises the recombinase recognition sequence in the presence of a recombinase.
• In other embodiments, the disclosure provides a method for deleting a genomic region by site-specific recombination, comprising: (a) installing a pair of recombinase recognition sequences at a genomic locus by prime editing; (b) contacting the genomic locus with a recombinase, thereby catalyzing the deletion of the genomic region between the pair of recombinase recognition sequences.
• In yet other embodiments, the disclosure provides a method for inverting a genomic region by site-specific recombination, comprising: (a) installing a pair of recombinase recognition sequences at a genomic locus by prime editing; (b) contacting the genomic locus with a recombinase, thereby catalyzing the inversion of the genomic region between the pair of recombinase recognition sequences.
• In still other embodiments, the disclosure provides a method for inducing chromosomal translocation between a first genomic site and a second genomic site, comprising: (a) installing a first recombinase recognition sequence at a first genomic locus by prime editing; (b) installing a second recombinase recognition sequence at a second genomic locus by prime editing; (c) contacting the first and the second genomic loci with a recombinase, thereby catalyzing the chromosomal translocation of the first and second genomic loci.
• In other embodiments, the disclosure provides a method for inducing cassette exchange between a genomic locus and a donor DNA comprising a cassette, comprising: (a) installing a first recombinase recognition sequence at a first genomic locus by prime editing; (b) installing a second recombinase recognition sequence at a second genomic locus by prime editing; (c) contacting the first and the second genomic loci with a donor DNA comprising a cassette that is flanked by the first and second recombinase recognition sequences and a recombinase, thereby catalyzing the exchange of the flanked genomic locus and the cassette in the DNA donor.
• In various embodiments involving the insertion of more than one recombinase recognition sequences in the genome, the recombinase recognition sequences can be the same or different. In some embodiments, the recombinase recognition sequences are the same. In other embodiments, that recombinase recognition sequences are different.
• In various embodiments, the recombinase can be a tyrosine recombinase, such as Cre, Dre, Vcre, Scre, Flp, B2, B3, Kw, R, TD1-40, Vika, Nigri, Panto, Kd, Fre, Cre(ALSHG), Tre, Brec1, or Cre-R3M3, as shown in Table 7. In such embodiments, the recombinase recognition sequence may be an RRS of Table 7 that corresponds to the recombinase under use.
• In various other embodiments, the recombinase can be a large serine recombinase, such as Bxb1, PhiC31, R4, phiBT1, MJ1, MR11, TP901-1, A118, V153, phiRV1, phi370.1, TG1, WB, BL3, SprA, phiJoe, phiK38, Int2, Int3, Int4, Int1, Int8, Int9, Int10, Int11, Int12, Int13, L1, peaches, Bxz2, or SV1, as shown in Table 8. In such embodiments, the recombinase recognition sequence may be an RRS of Table 8 that corresponds to the recombinase under use.
• In still other embodiments, the recombinase can be a serine recombinase, such as Bxb1, PhiC31, R4, phiBT1, MJ1, MR11, TP901-1, A118, V153, phiRV1, phi370.1, TG1, WB, BL3, SprA, phiJoe, phiK38, Int2, Int3, Int4, Int1, Int8, Int9, Int10, Int11, Int12, Int13, L1, peaches, Bxz2, or SV1, as shown in Table 8. In such embodiments, the recombinase recognition sequence may be an RRS of Table 8 that corresponds to the recombinase under use.
• In other embodiments, the recombinase can be a serine resolvase, such as Gin, Cin, Hin, Min, or Sin, as shown in Table 9. In such embodiments, the recombinase recognition sequence may be an RRS of Table 9 that corresponds to the recombinase under use.
• In various other embodiments, the recombinase can be a tyrosine integrase, such as HK022, P22, or L5, as shown in Table 10. In such embodiments, the recombinase recognition sequence may be an RRS of Table 10 that corresponds to the recombinase under use.
• In some embodiments, any of the methods for site-specific recombination with PE can be performed in vivo or in vitro. In some embodiments, any of the methods for site-specific recombination are performed in a cell (e.g., recombine genomic DNA in a cell). The cell can be prokaryotic or eukaryotic. The cell, such as a eukaryotic cell, can be in an individual, such as a subject, as described herein (e.g., a human subject). The methods described herein are useful for the genetic modification of cells in vitro and in vivo, for example, in the context of the generation of transgenic cells, cell lines, or animals, or in the alteration of genomic sequence, e.g., the correction of a genetic defect, in a cell in a subject.
REFERENCES CITED FOR SECTION L
• Each of the following references are cited in Example 17, each of which are incorporated herein by reference.
• 1. Feuk, L. Inversion variants in the human genome: role in disease and genome architecture. Genome Med 2, 11 (2010).
• 2. Zhang, F., Gu, W., Hurles, M. E. & Lupski, J. R. Copy number variation in human health, disease, and evolution. Annu Rev Genomics Hum Genet 10, 451-481 (2009).
• 3. Shaw, C. J. & Lupski, J. R. Implications of human genome architecture for rearrangement-based disorders: the genomic basis of disease. Hum Mol Genet 13 Spec No 1, R57-64 (2004).
• 4. Carvalho, C. M., Zhang, F. & Lupski, J. R. Evolution in health and medicine Sackler colloquium: Genomic disorders: a window into human gene and genome evolution. Proc Natl Acad Sci USA 107 Suppl 1, 1765-1771 (2010).
• 5. Rowley, J. D. Chromosome translocations: dangerous liaisons revisited. Nat Rev Cancer 1, 245-250 (2001).
• 6. Aplan, P. D. Causes of oncogenic chromosomal translocation. Trends Genet 22, 46-55 (2006).
• 7. McCarroll, S. A. & Altshuler, D. M. Copy-number variation and association studies of human disease. Nat Genet 39, S37-42 (2007).
[8] Methods of Treatment
• The instant disclosure provides methods for the treatment of a subject diagnosed with a disease associated with or caused by a point mutation, or other mutations (e.g., deletion, insertion, inversion, duplication, etc.) that can be corrected by the prime editing system provided herein, as exemplified, but not limited to prion disease (e.g., Example 5 herein), trinucleotide repeat expansion disease (e.g., Example 3 herein), or CDKL5 Deficiency Disorder (CDD) (e.g., Example 23 herein).
• Virtually any disease-causing genetic defect may be repaired by using prime editing, which includes the selection of an appropriate prime editor fusion protein (including a napDNAbp and a polymerase (e.g., a reverse transcriptase), and designing of an appropriate PEgRNA designed to (a) target the appropriate target DNA containing an edit site, and (b) provide a template for the synthesis of a single strand of DNA from the 3′ end of the nick site that includes the desired edit which displaces and replaces the endogenous strand immediately downstream of the nick site. Prime editing can be used, without limitation, to (a) install mutation-correcting changes to a nucleotide sequence, (b) install protein and RNA tags, (c) install immunoepitopes on proteins of interest, (d) install inducible dimerization domains in proteins, (e) install or remove sequences to alter that activity of a biomolecule, (f) install recombinase target sites to direct specific genetic changes, and (g) mutagenesis of a target sequence by using an error-prone RT.
• The method of treating a disorder can involve as an early step the design of an appropriate PEgRNA and prime editor fusion protein in accordance with the methods described herein, which include a number of considerations that may be taken into account, such as:
• (a) the target sequence, i.e., the nucleotide sequence in which one or more nucleobase modifications are desired to be installed by the prime editor;
  (b) the location of the cut site within the target sequence, i.e., the specific nucleobase position at which the prime editor will induce a single-stand nick to create a 3′ end RT primer sequence on one side of the nick and the 5′ end endogenous flap on the other side of the nick (which ultimately is removed by FEN1 or equivalent thereto and replaced by the 3′ ssDNA flap. The cut site creates the 3′ end primer sequence which becomes extended by the polymerase of the PE fusion protein (e.g., a RT enzyme) during RNA-dependent DNA polymerization to create the 3′ ssDNA flap containing the desired edit, which then replaces the 5′ endogenous DNA flap in the target sequence.
  (c) the available PAM sequences (including the canonical SpCas9 PAM sites, as well as non-canonical PAM sites recognized by Cas9 variants and equivalents with expanded or differing PAM specificities);
  (d) the spacing between the available PAM sequences and the location of the cut site in the PAM strand;
  (e) the particular Cas9, Cas9 variant, or Cas9 equivalent of the prime editor available to be used (which in part is dictated by the available PAM);
  (f) the sequence and length of the primer binding site;
  (g) the sequence and length of the edit template;
  (h) the sequence and length of the homology arm;
  (i) the spacer sequence and length; and
  (j) the gRNA core sequence.
• A suitable PEgRNA, and optionally a nicking-sgRNA design guide for second-site nicking, can be designed by way of the following exemplarily step-by-step set of instructions which takes into account one or more of the above considerations. The steps reference the examples shown in FIGS. 70A-70I.
• 1. Define the target sequence and the edit. Retrieve the sequence of the target DNA region (˜200 bp) centered around the location of the desired edit (point mutation, insertion, deletion, or combination thereof). See FIG. 70A.
  2. Locate target PAMs. Identify PAMs in the proximity to the desired edit location. PAMs can be identified on either strand of DNA proximal to the desired edit location. While PAMs close to the edit position are preferred (i.e., wherein the nick site is less than 30 nt from the edit position, or less than 29 nt, 28 nt, 27 nt, 26 nt, 25 nt, 24 nt, 23 nt, 22 nt, 21 nt, 20 nt, 19 nt, 18 nt, 17 nt, 16 nt, 15 nt, 14 nt, 13 nt, 12 nt, 11 nt, 10 nt, 9 nt, 8 nt, 7 nt, 6 nt, 5 nt, 4 nt, 3 nt, or 2 nt from the edit position to the nick site), it is possible to install edits using protospacers and PAMs that place the nick ≥30 nt from the edit position. See FIG. 70B.
  3. Locate the nick sites. For each PAM being considered, identify the corresponding nick site and on which strand. For Sp Cas9 H840A nickase, cleavage occurs in the PAM-containing strand between the 3^rd and 4^th bases 5′ to the NGG PAM. All edited nucleotides must exist 3′ of the nick site, so appropriate PAMs must place the nick 5′ to the target edit on the PAM-containing strand. In the example shown below, there are two possible PAMs. For simplicity, the remaining steps will demonstrate the design of a PEgRNA using PAM 1 only. See FIG. 70C.
  4. Design the spacer sequence. The protospacer of SpCas9 corresponds to the 20 nucleotides 5′ to the NGG PAM on the PAM-containing strand. Efficient Pol III transcription initiation requires a G to be the first transcribed nucleotide. If the first nucleotide of the protospacer is a G, the spacer sequence for the PEgRNA is simply the protospacer sequence. If the first nucleotide of the protospacer is not a G, the spacer sequence of the PEgRNA is G followed by the protospacer sequence. See FIG. 70D.
  5. Design a primer binding site (PBS). Using the starting allele sequence, identify the DNA primer on the PAM-containing strand. The 3′ end of the DNA primer is the nucleotide just upstream of the nick site (i.e. the 4^th base 5′ to the NGG PAM for Sp Cas9). As a general design principle for use with PE2 and PE3, a PEgRNA primer binding site (PBS) containing 12 to 13 nucleotides of complementarity to the DNA primer can be used for sequences that contain ˜40-60% GC content. For sequences with low GC content, longer (14- to 15-nt) PBSs should be tested. For sequences with higher GC content, shorter (8- to 11-nt) PBSs should be tested. Optimal PBS sequences should be determined empirically, regardless of GC content. To design a length-p PBS sequence, take the reverse complement of the first p nucleotides 5′ of the nick site in the PAM-containing strand using the starting allele sequence. See FIG. 70E.
  6. Design an RT template (or DNA synthesis template). The RT template (or DNA synthesis template where the polymerase is not reverse transcriptase) encodes the designed edit and homology to the sequence adjacent to the edit. In one embodiment, these regions correspond to the DNA synthesis template of FIG. 3D and FIG. 3E, wherein the DNA synthesis template comprises the “edit template” and the “homology arm.” Optimal RT template lengths vary based on the target site. For short-range edits (positions +1 to +6), it is recommended to test a short (9 to 12 nt), a medium (13 to 16 nt), and a long (17 to 20 nt) RT template. For long-range edits (positions +7 and beyond), it is recommended to use RT templates that extend at least 5 nt (preferably 10 or more nt) past the position of the edit to allow for sufficient 3′ DNA flap homology. For long-range edits, several RT templates should be screened to identify functional designs. For larger insertions and deletions (≥5 nt), incorporation of greater 3′ homology (˜20 nt or more) into the RT template is recommended. Editing efficiency is typically impaired when the RT template encodes the synthesis of a G as the last nucleotide in the reverse transcribed DNA product (corresponding to a C in the RT template of the PEgRNA). As many RT templates support efficient prime editing, avoidance of G as the final synthesized nucleotide is recommended when designing RT templates. To design a length-r RT template sequence, use the desired allele sequence and take the reverse complement of the first r nucleotides 3′ of the nick site in the strand that originally contained the PAM. Note that compared to SNP edits, insertion or deletion edits using RT templates of the same length will not contain identical homology. See FIG. 70F.
  7. Assemble the full PEgRNA sequence. Concatenate the PEgRNA components in the following order (5′ to 3′): spacer, scaffold, RT template and PBS. See FIG. 70G.
  8. Designing nicking-sgRNAs for PE3. Identify PAMs on the non-edited strand upstream and downstream of the edit. Optimal nicking positions are highly locus-dependent and should be determined empirically. In general, nicks placed 40 to 90 nucleotides 5′ to the position across from the PEgRNA-induced nick lead to higher editing yields and fewer indels. A nicking sgRNA has a spacer sequence that matches the 20-nt protospacer in the starting allele, with the addition of a 5′-G if the protospacer does not begin with a G. See FIG. 70H.
  9. Designing PE3b nicking-sgRNAs. If a PAM exists in the complementary strand and its corresponding protospacer overlaps with the sequence targeted for editing, this edit could be a candidate for the PE3b system. In the PE3b system, the spacer sequence of the nicking-sgRNA matches the sequence of the desired edited allele, but not the starting allele. The PE3b system operates efficiently when the edited nucleotide(s) falls within the seed region (˜10 nt adjacent to the PAM) of the nicking-sgRNA protospacer. This prevents nicking of the complementary strand until after installation of the edited strand, preventing competition between the PEgRNA and the sgRNA for binding the target DNA. PE3b also avoids the generation of simultaneous nicks on both strands, thus reducing indel formation significantly while maintaining high editing efficiency. PE3b sgRNAs should have a spacer sequence that matches the 20-nt protospacer in the desired allele, with the addition of a 5′ G if needed. See FIG. 70I.
• The above step-by-step process for designing a suitable PEgRNA and a second-site nicking sgRNA is not meant to be limiting in any way. The disclosure contemplates variations of the above-described step-by-step process which would be derivable therefrom by a person of ordinary skill in the art.
• Once a suitable PEgRNA and PE fusion protein are selected/designed, they may be administered by a suitable methodology, such as by vector-based transfection (in which one or more vectors comprising DNA encoding the PEgRNA and the PE fusion protein and which are expressed within a cell upon transfection with the vectors), direct delivery of the PE fusion protein complexed with the PEgRNA (e.g., RNP delivery) in a delivery format (e.g., lipid particles, nanoparticles), or by a mRNA-based delivery system. Such methods are described herein in the present disclosure and any know method may be utilized.
• The PEgRNA and PE fusion protein (or together, referred to as the PE complex) can be delivered to a cell in a therapeutically effective amount such that upon contacting the target DNA of interest, the desired edit becomes installed therein.
• Any disease is conceivably treatable by such methods so long as delivery to the appropriate cells is feasible. The person having ordinary skill in the art will be able to choose and/or select a PE delivery methodology to suit the intended purpose and the intended target cells.
• For example, in some embodiments, a method is provided that comprises administering to a subject having such a disease, e.g., a cancer associated with a point mutation as described above, an effective amount of the prime editing system described herein that corrects the point mutation or introduces a deactivating mutation into a disease-associated gene as mediated by homology-directed repair in the presence of a donor DNA molecule comprising desired genetic change. In some embodiments, a method is provided that comprises administering to a subject having such a disease, e.g., a cancer associated with a point mutation as described above, an effective amount of the prime editing system described herein that corrects the point mutation or introduces a deactivating mutation into a disease-associated gene. In some embodiments, the disease is a proliferative disease. In some embodiments, the disease is a genetic disease. In some embodiments, the disease is a neoplastic disease. In some embodiments, the disease is a metabolic disease. In some embodiments, the disease is a lysosomal storage disease. Other diseases that can be treated by correcting a point mutation or introducing a deactivating mutation into a disease-associated gene will be known to those of skill in the art, and the disclosure is not limited in this respect.
• The instant disclosure provides methods for the treatment of additional diseases or disorders, e.g., diseases or disorders that are associated or caused by a point mutation that can be corrected by TPRT-mediated gene editing. Some such diseases are described herein, and additional suitable diseases that can be treated with the strategies and fusion proteins provided herein will be apparent to those of skill in the art based on the instant disclosure. Exemplary suitable diseases and disorders are listed below. It will be understood that the numbering of the specific positions or residues in the respective sequences depends on the particular protein and numbering scheme used. Numbering might be different, e.g., in precursors of a mature protein and the mature protein itself, and differences in sequences from species to species may affect numbering. One of skill in the art will be able to identify the respective residue in any homologous protein and in the respective encoding nucleic acid by methods well known in the art, e.g., by sequence alignment and determination of homologous residues. Exemplary suitable diseases and disorders include, without limitation: 2-methyl-3-hydroxybutyric aciduria; 3 beta-Hydroxysteroid dehydrogenase deficiency; 3-Methylglutaconic aciduria; 3-Oxo-5 alpha-steroid delta 4-dehydrogenase deficiency; 46,XY sex reversal, type 1, 3, and 5; 5-Oxoprolinase deficiency; 6-pyruvoyl-tetrahydropterin synthase deficiency; Aarskog syndrome; Aase syndrome; Achondrogenesis type 2; Achromatopsia 2 and 7; Acquired long QT syndrome; Acrocallosal syndrome, Schinzel type; Acrocapitofemoral dysplasia; Acrodysostosis 2, with or without hormone resistance; Acroerythrokeratoderma; Acromicric dysplasia; Acth-independent macronodular adrenal hyperplasia 2; Activated PI3K-delta syndrome; Acute intermittent porphyria; deficiency of Acyl-CoA dehydrogenase family, member 9; Adams-Oliver syndrome 5 and 6; Adenine phosphoribosyltransferase deficiency; Adenylate kinase deficiency; hemolytic anemia due to Adenylosuccinate lyase deficiency; Adolescent nephronophthisis; Renal-hepatic-pancreatic dysplasia; Meckel syndrome type 7; Adrenoleukodystrophy; Adult junctional epidermolysis bullosa; Epidermolysis bullosa, junctional, localisata variant; Adult neuronal ceroid lipofuscinosis; Adult neuronal ceroid lipofuscinosis; Adult onset ataxia with oculomotor apraxia; ADULT syndrome; Afibrinogenemia and congenital Afibrinogenemia; autosomal recessive Agammaglobulinemia 2; Age-related macular degeneration 3, 6, 11, and 12; Aicardi Goutieres syndromes 1, 4, and 5; Chilbain lupus 1; Alagille syndromes 1 and 2; Alexander disease; Alkaptonuria; Allan-Herndon-Dudley syndrome; Alopecia universalis congenital; Alpers encephalopathy; Alpha-1-antitrypsin deficiency; autosomal dominant, autosomal recessive, and X-linked recessive Alport syndromes; Alzheimer disease, familial, 3, with spastic paraparesis and apraxia; Alzheimer disease, types, 1, 3, and 4; hypocalcification type and hypomaturation type, IIA1 Amelogenesis imperfecta; Aminoacylase 1 deficiency; Amish infantile epilepsy syndrome; Amyloidogenic transthyretin amyloidosis; Amyloid Cardiomyopathy, Transthyretin-related; Cardiomyopathy; Amyotrophic lateral sclerosis types 1, 6, 15 (with or without frontotemporal dementia), 22 (with or without frontotemporal dementia), and 10; Frontotemporal dementia with TDP43 inclusions, TARDBP-related; Andermann syndrome; Andersen Tawil syndrome; Congenital long QT syndrome; Anemia, nonspherocytic hemolytic, due to G6PD deficiency; Angelman syndrome; Severe neonatal-onset encephalopathy with microcephaly; susceptibility to Autism, X-linked 3; Angiopathy, hereditary, with nephropathy, aneurysms, and muscle cramps; Angiotensin i-converting enzyme, benign serum increase; Aniridia, cerebellar ataxia, and mental retardation; Anonychia; Antithrombin III deficiency; Antley-Bixler syndrome with genital anomalies and disordered steroidogenesis; Aortic aneurysm, familial thoracic 4, 6, and 9; Thoracic aortic aneurysms and aortic dissections; Multisystemic smooth muscle dysfunction syndrome; Moyamoya disease 5; Aplastic anemia; Apparent mineralocorticoid excess; Arginase deficiency; Argininosuccinate lyase deficiency; Aromatase deficiency; Arrhythmogenic right ventricular cardiomyopathy types 5, 8, and 10; Primary familial hypertrophic cardiomyopathy; Arthrogryposis multiplex congenita, distal, X-linked; Arthrogryposis renal dysfunction cholestasis syndrome; Arthrogryposis, renal dysfunction, and cholestasis 2; Asparagine synthetase deficiency; Abnormality of neuronal migration; Ataxia with vitamin E deficiency; Ataxia, sensory, autosomal dominant; Ataxia-telangiectasia syndrome; Hereditary cancer-predisposing syndrome; Atransferrinemia; Atrial fibrillation, familial, 11, 12, 13, and 16; Atrial septal defects 2, 4, and 7 (with or without atrioventricular conduction defects); Atrial standstill 2; Atrioventricular septal defect 4; Atrophia bulborum hereditaria; ATR-X syndrome; Auriculocondylar syndrome 2; Autoimmune disease, multisystem, infantile-onset; Autoimmune lymphoproliferative syndrome, type 1a; Autosomal dominant hypohidrotic ectodermal dysplasia; Autosomal dominant progressive external ophthalmoplegia with mitochondrial DNA deletions 1 and 3; Autosomal dominant torsion dystonia 4; Autosomal recessive centronuclear myopathy; Autosomal recessive congenital ichthyosis 1, 2, 3, 4A, and 4B; Autosomal recessive cutis laxa type IA and 1B; Autosomal recessive hypohidrotic ectodermal dysplasia syndrome; Ectodermal dysplasia 11b; hypohidrotic/hair/tooth type, autosomal recessive; Autosomal recessive hypophosphatemic bone disease; Axenfeld-Rieger syndrome type 3; Bainbridge-Ropers syndrome; Bannayan-Riley-Ruvalcaba syndrome; PTEN hamartoma tumor syndrome; Baraitser-Winter syndromes 1 and 2; Barakat syndrome; Bardet-Biedl syndromes 1, 11, 16, and 19; Bare lymphocyte syndrome type 2, complementation group E; Bartter syndrome antenatal type 2; Bartter syndrome types 3, 3 with hypocalciuria, and 4; Basal ganglia calcification, idiopathic, 4; Beaded hair; Benign familial hematuria; Benign familial neonatal seizures 1 and 2; Seizures, benign familial neonatal, 1, and/or myokymia; Seizures, Early infantile epileptic encephalopathy 7; Benign familial neonatal-infantile seizures; Benign hereditary chorea; Benign scapuloperoneal muscular dystrophy with cardiomyopathy; Bernard-Soulier syndrome, types A1 and A2 (autosomal dominant); Bestrophinopathy, autosomal recessive; beta Thalassemia; Bethlem myopathy and Bethlem myopathy 2; Bietti crystalline corneoretinal dystrophy; Bile acid synthesis defect, congenital, 2; Biotinidase deficiency; Birk Barel mental retardation dysmorphism syndrome; Blepharophimosis, ptosis, and epicanthus inversus; Bloom syndrome; Borjeson-Forssman-Lehmann syndrome; Boucher Neuhauser syndrome; Brachydactyly types A1 and A2; Brachydactyly with hypertension; Brain small vessel disease with hemorrhage; Branched-chain ketoacid dehydrogenase kinase deficiency; Branchiootic syndromes 2 and 3; Breast cancer, early-onset; Breast-ovarian cancer, familial 1, 2, and 4; Brittle cornea syndrome 2; Brody myopathy; Bronchiectasis with or without elevated sweat chloride 3; Brown-Vialetto-Van laere syndrome and Brown-Vialetto-Van Laere syndrome 2; Brugada syndrome; Brugada syndrome 1; Ventricular fibrillation; Paroxysmal familial ventricular fibrillation; Brugada syndrome and Brugada syndrome 4; Long QT syndrome; Sudden cardiac death; Bull eye macular dystrophy; Stargardt disease 4; Cone-rod dystrophy 12; Bullous ichthyosiform erythroderma; Burn-Mckeown syndrome; Candidiasis, familial, 2, 5, 6, and 8; Carbohydrate-deficient glycoprotein syndrome type I and II; Carbonic anhydrase VA deficiency, hyperammonemia due to; Carcinoma of colon; Cardiac arrhythmia; Long QT syndrome, LQT1 subtype; Cardioencephalomyopathy, fatal infantile, due to cytochrome c oxidase deficiency; Cardiofaciocutaneous syndrome; Cardiomyopathy; Danon disease; Hypertrophic cardiomyopathy; Left ventricular noncompaction cardiomyopathy; Carnevale syndrome; Carney complex, type 1; Carnitine acylcarnitine translocase deficiency; Carnitine palmitoyltransferase I, II, II (late onset), and II (infantile) deficiency; Cataract 1, 4, autosomal dominant, autosomal dominant, multiple types, with microcornea, coppock-like, juvenile, with microcornea and glucosuria, and nuclear diffuse nonprogressive; Catecholaminergic polymorphic ventricular tachycardia; Caudal regression syndrome; Cd8 deficiency, familial; Central core disease; Centromeric instability of chromosomes 1,9 and 16 and immunodeficiency; Cerebellar ataxia infantile with progressive external ophthalmoplegi and Cerebellar ataxia, mental retardation, and dysequilibrium syndrome 2; Cerebral amyloid angiopathy, APP-related; Cerebral autosomal dominant and recessive arteriopathy with subcortical infarcts and leukoencephalopathy; Cerebral cavernous malformations 2; Cerebrooculofacioskeletal syndrome 2; Cerebro-oculo-facio-skeletal syndrome; Cerebroretinal microangiopathy with calcifications and cysts; Ceroid lipofuscinosis neuronal 2, 6, 7, and 10; Ch\xc3\xa9diak-Higashi syndrome, Chediak-Higashi syndrome, adult type; Charcot-Marie-Tooth disease types 1B, 2B2, 2C, 2F, 2I, 2U (axonal), 1C (demyelinating), dominant intermediate C, recessive intermediate A, 2A2, 4C, 4D, 4H, IF, IVF, and X; Scapuloperoneal spinal muscular atrophy; Distal spinal muscular atrophy, congenital nonprogressive; Spinal muscular atrophy, distal, autosomal recessive, 5; CHARGE association; Childhood hypophosphatasia; Adult hypophosphatasia; Cholecystitis; Progressive familial intrahepatic cholestasis 3; Cholestasis, intrahepatic, of pregnancy 3; Cholestanol storage disease; Cholesterol monooxygenase (side-chain cleaving) deficiency; Chondrodysplasia Blomstrand type; Chondrodysplasia punctata 1, X-linked recessive and 2 X-linked dominant; CHOPS syndrome; Chronic granulomatous disease, autosomal recessive cytochrome b-positive, types 1 and 2; Chudley-McCullough syndrome; Ciliary dyskinesia, primary, 7, 11, 15, 20 and 22; Citrullinemia type I; Citrullinemia type I and II; Cleidocranial dysostosis; C-like syndrome; Cockayne syndrome type A; Coenzyme Q10 deficiency, primary 1, 4, and 7; Coffin Siris/Intellectual Disability; Coffin-Lowry syndrome; Cohen syndrome; Cold-induced sweating syndrome 1; COLE-CARPENTER SYNDROME 2; Combined cellular and humoral immune defects with granulomas; Combined d-2- and 1-2-hydroxyglutaric aciduria; Combined malonic and methylmalonic aciduria; Combined oxidative phosphorylation deficiencies 1, 3, 4, 12, 15, and 25; Combined partial and complete 17-alpha-hydroxylase/17,20-lyase deficiency; Common variable immunodeficiency 9; Complement component 4, partial deficiency of, due to dysfunctional cl inhibitor; Complement factor B deficiency; Cone monochromatism; Cone-rod dystrophy 2 and 6; Cone-rod dystrophy amelogenesis imperfecta; Congenital adrenal hyperplasia and Congenital adrenal hypoplasia, X-linked; Congenital amegakaryocytic thrombocytopenia; Congenital aniridia; Congenital central hypoventilation; Hirschsprung disease 3; Congenital contractural arachnodactyly; Congenital contractures of the limbs and face, hypotonia, and developmental delay; Congenital disorder of glycosylation types 1B, 1D, 1G, 1H, 1J, 1K, 1N, 1P, 2C, 2J, 2K, IIm; Congenital dyserythropoietic anemia, type I and II; Congenital ectodermal dysplasia of face; Congenital erythropoietic porphyria; Congenital generalized lipodystrophy type 2; Congenital heart disease, multiple types, 2; Congenital heart disease; Interrupted aortic arch; Congenital lipomatous overgrowth, vascular malformations, and epidermal nevi; Non-small cell lung cancer; Neoplasm of ovary; Cardiac conduction defect, nonspecific; Congenital microvillous atrophy; Congenital muscular dystrophy; Congenital muscular dystrophy due to partial LAMA2 deficiency; Congenital muscular dystrophy-dystroglycanopathy with brain and eye anomalies, types A2, A7, A8, A11, and A14; Congenital muscular dystrophy-dystroglycanopathy with mental retardation, types B2, B3, B5, and B15; Congenital muscular dystrophy-dystroglycanopathy without mental retardation, type B5; Congenital muscular hypertrophy-cerebral syndrome; Congenital myasthenic syndrome, acetazolamide-responsive; Congenital myopathy with fiber type disproportion; Congenital ocular coloboma; Congenital stationary night blindness, type 1A, 1B, 1C, 1E, 1F, and 2A; Coproporphyria; Cornea plana 2; Corneal dystrophy, Fuchs endothelial, 4; Corneal endothelial dystrophy type 2; Corneal fragility keratoglobus, blue sclerae and joint hypermobility; Cornelia de Lange syndromes 1 and 5; Coronary artery disease, autosomal dominant 2; Coronary heart disease; Hyperalphalipoproteinemia 2; Cortical dysplasia, complex, with other brain malformations 5 and 6; Cortical malformations, occipital; Corticosteroid-binding globulin deficiency; Corticosterone methyloxidase type 2 deficiency; Costello syndrome; Cowden syndrome 1; Coxa plana; Craniodiaphyseal dysplasia, autosomal dominant; Craniosynostosis 1 and 4; Craniosynostosis and dental anomalies; Creatine deficiency, X-linked; Crouzon syndrome; Cryptophthalmos syndrome; Cryptorchidism, unilateral or bilateral; Cushing symphalangism; Cutaneous malignant melanoma 1; Cutis laxa with osteodystrophy and with severe pulmonary, gastrointestinal, and urinary abnormalities; Cyanosis, transient neonatal and atypical nephropathic; Cystic fibrosis; Cystinuria; Cytochrome c oxidase i deficiency; Cytochrome-c oxidase deficiency; D-2-hydroxyglutaric aciduria 2; Darier disease, segmental; Deafness with labyrinthine aplasia microtia and microdontia (LAMM); Deafness, autosomal dominant 3a, 4, 12, 13, 15, autosomal dominant nonsyndromic sensorineural 17, 20, and 65; Deafness, autosomal recessive 1A, 2, 3, 6, 8, 9, 12, 15, 16, 18b, 22, 28, 31, 44, 49, 63, 77, 86, and 89; Deafness, cochlear, with myopia and intellectual impairment, without vestibular involvement, autosomal dominant, X-linked 2; Deficiency of 2-methylbutyryl-CoA dehydrogenase; Deficiency of 3-hydroxyacyl-CoA dehydrogenase; Deficiency of alpha-mannosidase; Deficiency of aromatic-L-amino-acid decarboxylase; Deficiency of bisphosphoglycerate mutase; Deficiency of butyryl-CoA dehydrogenase; Deficiency of ferroxidase; Deficiency of galactokinase; Deficiency of guanidinoacetate methyltransferase; Deficiency of hyaluronoglucosaminidase; Deficiency of ribose-5-phosphate isomerase; Deficiency of steroid 11-beta-monooxygenase; Deficiency of UDPglucose-hexose-1-phosphate uridylyltransferase; Deficiency of xanthine oxidase; Dejerine-Sottas disease; Charcot-Marie-Tooth disease, types ID and IVF; Dejerine-Sottas syndrome, autosomal dominant; Dendritic cell, monocyte, B lymphocyte, and natural killer lymphocyte deficiency; Desbuquois dysplasia 2; Desbuquois syndrome; DFNA 2 Nonsyndromic Hearing Loss; Diabetes mellitus and insipidus with optic atrophy and deafness; Diabetes mellitus, type 2, and insulin-dependent, 20; Diamond-Blackfan anemia 1, 5, 8, and 10; Diarrhea 3 (secretory sodium, congenital, syndromic) and 5 (with tufting enteropathy, congenital); Dicarboxylic aminoaciduria; Diffuse palmoplantar keratoderma, Bothnian type; Digitorenocerebral syndrome; Dihydropteridine reductase deficiency; Dilated cardiomyopathy 1A, 1AA, 1C, 1G, 1BB, 1DD, 1FF, 1HH, 1I, 1KK, 1N, 1S, 1Y, and 3B; Left ventricular noncompaction 3; Disordered steroidogenesis due to cytochrome p450 oxidoreductase deficiency; Distal arthrogryposis type 2B; Distal hereditary motor neuropathy type 2B; Distal myopathy Markesbery-Griggs type; Distal spinal muscular atrophy, X-linked 3; Distichiasis-lymphedema syndrome; Dominant dystrophic epidermolysis bullosa with absence of skin; Dominant hereditary optic atrophy; Donnai Barrow syndrome; Dopamine beta hydroxylase deficiency; Dopamine receptor d2, reduced brain density of; Dowling-degos disease 4; Doyne honeycomb retinal dystrophy; Malattia leventinese; Duane syndrome type 2; Dubin-Johnson syndrome; Duchenne muscular dystrophy; Becker muscular dystrophy; Dysfibrinogenemia; Dyskeratosis congenita autosomal dominant and autosomal dominant, 3; Dyskeratosis congenita, autosomal recessive, 1, 3, 4, and 5; Dyskeratosis congenita X-linked; Dyskinesia, familial, with facial myokymia; Dysplasminogenemia; Dystonia 2 (torsion, autosomal recessive), 3 (torsion, X-linked), 5 (Dopa-responsive type), 10, 12, 16, 25, 26 (Myoclonic); Seizures, benign familial infantile, 2; Early infantile epileptic encephalopathy 2, 4, 7, 9, 10, 11, 13, and 14; Atypical Rett syndrome; Early T cell progenitor acute lymphoblastic leukemia; Ectodermal dysplasia skin fragility syndrome; Ectodermal dysplasia-syndactyly syndrome 1; Ectopia lentis, isolated autosomal recessive and dominant; Ectrodactyly, ectodermal dysplasia, and cleft lip/palate syndrome 3; Ehlers-Danlos syndrome type 7 (autosomal recessive), classic type, type 2 (progeroid), hydroxylysine-deficient, type 4, type 4 variant, and due to tenascin-X deficiency; Eichsfeld type congenital muscular dystrophy; Endocrine-cerebroosteodysplasia; Enhanced s-cone syndrome; Enlarged vestibular aqueduct syndrome; Enterokinase deficiency; Epidermodysplasia verruciformis; Epidermolysa bullosa simplex and limb girdle muscular dystrophy, simplex with mottled pigmentation, simplex with pyloric atresia, simplex, autosomal recessive, and with pyloric atresia; Epidermolytic palmoplantar keratoderma; Familial febrile seizures 8; Epilepsy, childhood absence 2, 12 (idiopathic generalized, susceptibility to) 5 (nocturnal frontal lobe), nocturnal frontal lobe type 1, partial, with variable foci, progressive myoclonic 3, and X-linked, with variable learning disabilities and behavior disorders; Epileptic encephalopathy, childhood-onset, early infantile, 1, 19, 23, 25, 30, and 32; Epiphyseal dysplasia, multiple, with myopia and conductive deafness; Episodic ataxia type 2; Episodic pain syndrome, familial, 3; Epstein syndrome; Fechtner syndrome; Erythropoietic protoporphyria; Estrogen resistance; Exudative vitreoretinopathy 6; Fabry disease and Fabry disease, cardiac variant; Factor H, VII, X, v and factor viii, combined deficiency of 2, xiii, a subunit, deficiency; Familial adenomatous polyposis 1 and 3; Familial amyloid nephropathy with urticaria and deafness; Familial cold urticarial; Familial aplasia of the vermis; Familial benign pemphigus; Familial cancer of breast; Breast cancer, susceptibility to; Osteosarcoma; Pancreatic cancer 3; Familial cardiomyopathy; Familial cold autoinflammatory syndrome 2; Familial colorectal cancer; Familial exudative vitreoretinopathy, X-linked; Familial hemiplegic migraine types 1 and 2; Familial hypercholesterolemia; Familial hypertrophic cardiomyopathy 1, 2, 3, 4, 7, 10, 23 and 24; Familial hypokalemia-hypomagnesemia; Familial hypoplastic, glomerulocystic kidney; Familial infantile myasthenia; Familial juvenile gout; Familial Mediterranean fever and Familial mediterranean fever, autosomal dominant; Familial porencephaly; Familial porphyria cutanea tarda; Familial pulmonary capillary hemangiomatosis; Familial renal glucosuria; Familial renal hypouricemia; Familial restrictive cardiomyopathy 1; Familial type 1 and 3 hyperlipoproteinemia; Fanconi anemia, complementation group E, I, N, and O; Fanconi-Bickel syndrome; Favism, susceptibility to; Febrile seizures, familial, 11; Feingold syndrome 1; Fetal hemoglobin quantitative trait locus 1; FG syndrome and FG syndrome 4; Fibrosis of extraocular muscles, congenital, 1, 2, 3a (with or without extraocular involvement), 3b; Fish-eye disease; Fleck corneal dystrophy; Floating-Harbor syndrome; Focal epilepsy with speech disorder with or without mental retardation; Focal segmental glomerulosclerosis 5; Forebrain defects; Frank Ter Haar syndrome; Borrone Di Rocco Crovato syndrome; Frasier syndrome; Wilms tumor 1; Freeman-Sheldon syndrome; Frontometaphyseal dysplasia 1 and 3; Frontotemporal dementia; Frontotemporal dementia and/or amyotrophic lateral sclerosis 3 and 4; Frontotemporal Dementia Chromosome 3-Linked and Frontotemporal dementia ubiquitin-positive; Fructose-biphosphatase deficiency; Fuhrmann syndrome; Gamma-aminobutyric acid transaminase deficiency; Gamstorp-Wohlfart syndrome; Gaucher disease type 1 and Subacute neuronopathic; Gaze palsy, familial horizontal, with progressive scoliosis; Generalized dominant dystrophic epidermolysis bullosa; Generalized epilepsy with febrile seizures plus 3, type 1, type 2; Epileptic encephalopathy Lennox-Gastaut type; Giant axonal neuropathy; Glanzmann thrombasthenia; Glaucoma 1, open angle, e, F, and G; Glaucoma 3, primary congenital, d; Glaucoma, congenital and Glaucoma, congenital, Coloboma; Glaucoma, primary open angle, juvenile-onset; Glioma susceptibility 1; Glucose transporter type 1 deficiency syndrome; Glucose-6-phosphate transport defect; GLUT1 deficiency syndrome 2; Epilepsy, idiopathic generalized, susceptibility to, 12; Glutamate formiminotransferase deficiency; Glutaric acidemia IIA and IIB; Glutaric aciduria, type 1; Gluthathione synthetase deficiency; Glycogen storage disease 0 (muscle), II (adult form), IXa2, IXc, type 1A; type II, type IV, IV (combined hepatic and myopathic), type V, and type VI; Goldmann-Favre syndrome; Gordon syndrome; Gorlin syndrome; Holoprosencephaly sequence; Holoprosencephaly 7; Granulomatous disease, chronic, X-linked, variant; Granulosa cell tumor of the ovary; Gray platelet syndrome; Griscelli syndrome type 3; Groenouw corneal dystrophy type I; Growth and mental retardation, mandibulofacial dysostosis, microcephaly, and cleft palate; Growth hormone deficiency with pituitary anomalies; Growth hormone insensitivity with immunodeficiency; GTP cyclohydrolase I deficiency; Hajdu-Cheney syndrome; Hand foot uterus syndrome; Hearing impairment; Hemangioma, capillary infantile; Hematologic neoplasm; Hemochromatosis type 1, 2B, and 3; Microvascular complications of diabetes 7; Transferrin serum level quantitative trait locus 2; Hemoglobin H disease, nondeletional; Hemolytic anemia, nonspherocytic, due to glucose phosphate isomerase deficiency; Hemophagocytic lymphohistiocytosis, familial, 2; Hemophagocytic lymphohistiocytosis, familial, 3; Heparin cofactor II deficiency; Hereditary acrodermatitis enteropathica; Hereditary breast and ovarian cancer syndrome; Ataxia-telangiectasia-like disorder; Hereditary diffuse gastric cancer; Hereditary diffuse leukoencephalopathy with spheroids; Hereditary factors II, IX, VIII deficiency disease; Hereditary hemorrhagic telangiectasia type 2; Hereditary insensitivity to pain with anhidrosis; Hereditary lymphedema type I; Hereditary motor and sensory neuropathy with optic atrophy; Hereditary myopathy with early respiratory failure; Hereditary neuralgic amyotrophy; Hereditary Nonpolyposis Colorectal Neoplasms; Lynch syndrome I and II; Hereditary pancreatitis; Pancreatitis, chronic, susceptibility to; Hereditary sensory and autonomic neuropathy type IIB amd IIA; Hereditary sideroblastic anemia; Hermansky-Pudlak syndrome 1, 3, 4, and 6; Heterotaxy, visceral, 2, 4, and 6, autosomal; Heterotaxy, visceral, X-linked; Heterotopia; Histiocytic medullary reticulosis; Histiocytosis-lymphadenopathy plus syndrome; Holocarboxylase synthetase deficiency; Holoprosencephaly 2, 3, 7, and 9; Holt-Oram syndrome; Homocysteinemia due to MTHFR deficiency, CBS deficiency, and Homocystinuria, pyridoxine-responsive; Homocystinuria-Megaloblastic anemia due to defect in cobalamin metabolism, cblE complementation type; Howel-Evans syndrome; Hurler syndrome; Hutchinson-Gilford syndrome; Hydrocephalus; Hyperammonemia, type III; Hypercholesterolaemia and Hypercholesterolemia, autosomal recessive; Hyperekplexia 2 and Hyperekplexia hereditary; Hyperferritinemia cataract syndrome; Hyperglycinuria; Hyperimmunoglobulin D with periodic fever; Mevalonic aciduria; Hyperimmunoglobulin E syndrome; Hyperinsulinemic hypoglycemia familial 3, 4, and 5; Hyperinsulinism-hyperammonemia syndrome; Hyperlysinemia; Hypermanganesemia with dystonia, polycythemia and cirrhosis; Hyperornithinemia-hyperammonemia-homocitrullinuria syndrome; Hyperparathyroidism 1 and 2; Hyperparathyroidism, neonatal severe; Hyperphenylalaninemia, bh4-deficient, a, due to partial pts deficiency, BH4-deficient, D, and non-pku; Hyperphosphatasia with mental retardation syndrome 2, 3, and 4; Hypertrichotic osteochondrodysplasia; Hypobetalipoproteinemia, familial, associated with apob32; Hypocalcemia, autosomal dominant 1; Hypocalciuric hypercalcemia, familial, types 1 and 3; Hypochondrogenesis; Hypochromic microcytic anemia with iron overload; Hypoglycemia with deficiency of glycogen synthetase in the liver; Hypogonadotropic hypogonadism 11 with or without anosmia; Hypohidrotic ectodermal dysplasia with immune deficiency; Hypohidrotic X-linked ectodermal dysplasia; Hypokalemic periodic paralysis 1 and 2; Hypomagnesemia 1, intestinal; Hypomagnesemia, seizures, and mental retardation; Hypomyelinating leukodystrophy 7; Hypoplastic left heart syndrome; Atrioventricular septal defect and common atrioventricular junction; Hypospadias 1 and 2, X-linked; Hypothyroidism, congenital, nongoitrous, 1; Hypotrichosis 8 and 12; Hypotrichosis-lymphedema-telangiectasia syndrome; I blood group system; Ichthyosis bullosa of Siemens; Ichthyosis exfoliativa; Ichthyosis prematurity syndrome; Idiopathic basal ganglia calcification 5; Idiopathic fibrosing alveolitis, chronic form; Dyskeratosis congenita, autosomal dominant, 2 and 5; Idiopathic hypercalcemia of infancy; Immune dysfunction with T-cell inactivation due to calcium entry defect 2; Immunodeficiency 15, 16, 19, 30, 31C, 38, 40, 8, due to defect in cd3-zeta, with hyper IgM type 1 and 2, and X-Linked, with magnesium defect, Epstein-Barr virus infection, and neoplasia; Immunodeficiency-centromeric instability-facial anomalies syndrome 2; Inclusion body myopathy 2 and 3; Nonaka myopathy; Infantile convulsions and paroxysmal choreoathetosis, familial; Infantile cortical hyperostosis; Infantile GM1 gangliosidosis; Infantile hypophosphatasia; Infantile nephronophthisis; Infantile nystagmus, X-linked; Infantile Parkinsonism-dystonia; Infertility associated with multi-tailed spermatozoa and excessive DNA; Insulin resistance; Insulin-resistant diabetes mellitus and acanthosis nigricans; Insulin-dependent diabetes mellitus secretory diarrhea syndrome; Interstitial nephritis, karyomegalic; Intrauterine growth retardation, metaphyseal dysplasia, adrenal hypoplasia congenita, and genital anomalies; Iodotyrosyl coupling defect; IRAK4 deficiency; Iridogoniodysgenesis dominant type and type 1; Iron accumulation in brain; Ischiopatellar dysplasia; Islet cell hyperplasia; Isolated 17,20-lyase deficiency; Isolated lutropin deficiency; Isovaleryl-CoA dehydrogenase deficiency; Jankovic Rivera syndrome; Jervell and Lange-Nielsen syndrome 2; Joubert syndrome 1, 6, 7, 9/15 (digenic), 14, 16, and 17, and Orofaciodigital syndrome xiv; Junctional epidermolysis bullosa gravis of Herlitz; Juvenile GM>1<gangliosidosis; Juvenile polyposis syndrome; Juvenile polyposis/hereditary hemorrhagic telangiectasia syndrome; Juvenile retinoschisis; Kabuki make-up syndrome; Kallmann syndrome 1, 2, and 6; Delayed puberty; Kanzaki disease; Karak syndrome; Kartagener syndrome; Kenny-Caffey syndrome type 2; Keppen-Lubinsky syndrome; Keratoconus 1; Keratosis follicularis; Keratosis palmoplantaris striata 1; Kindler syndrome; L-2-hydroxyglutaric aciduria; Larsen syndrome, dominant type; Lattice corneal dystrophy Type III; Leber amaurosis; Zellweger syndrome; Peroxisome biogenesis disorders; Zellweger syndrome spectrum; Leber congenital amaurosis 11, 12, 13, 16, 4, 7, and 9; Leber optic atrophy; Aminoglycoside-induced deafness; Deafness, nonsyndromic sensorineural, mitochondrial; Left ventricular noncompaction 5; Left-right axis malformations; Leigh disease; Mitochondrial short-chain Enoyl-CoA Hydratase 1 deficiency; Leigh syndrome due to mitochondrial complex I deficiency; Leiner disease; Leri Weill dyschondrosteosis; Lethal congenital contracture syndrome 6; Leukocyte adhesion deficiency type I and III; Leukodystrophy, Hypomyelinating, 11 and 6; Leukoencephalopathy with ataxia, with Brainstem and Spinal Cord Involvement and Lactate Elevation, with vanishing white matter, and progressive, with ovarian failure; Leukonychia totalis; Lewy body dementia; Lichtenstein-Knorr Syndrome; Li-Fraumeni syndrome 1; Lig4 syndrome; Limb-girdle muscular dystrophy, type 1B, 2A, 2B, 2D, C1, C5, C9, C14; Congenital muscular dystrophy-dystroglycanopathy with brain and eye anomalies, type A14 and B14; Lipase deficiency combined; Lipid proteinosis; Lipodystrophy, familial partial, type 2 and 3; Lissencephaly 1, 2 (X-linked), 3, 6 (with microcephaly), X-linked; Subcortical laminar heterotopia, X-linked; Liver failure acute infantile; Loeys-Dietz syndrome 1, 2, 3; Long QT syndrome 1, 2, 2/9, 2/5, (digenic), 3, 5 and 5, acquired, susceptibility to; Lung cancer; Lymphedema, hereditary, id; Lymphedema, primary, with myelodysplasia; Lymphoproliferative syndrome 1, 1 (X-linked), and 2; Lysosomal acid lipase deficiency; Macrocephaly, macrosomia, facial dysmorphism syndrome; Macular dystrophy, vitelliform, adult-onset; Malignant hyperthermia susceptibility type 1; Malignant lymphoma, non-Hodgkin; Malignant melanoma; Malignant tumor of prostate; Mandibuloacral dysostosis; Mandibuloacral dysplasia with type A or B lipodystrophy, atypical; Mandibulofacial dysostosis, Treacher Collins type, autosomal recessive; Mannose-binding protein deficiency; Maple syrup urine disease type 1A and type 3; Marden Walker like syndrome; Marfan syndrome; Marinesco-Sj\xc3\xb6gren syndrome; Martsolf syndrome; Maturity-onset diabetes of the young, type 1, type 2, type 11, type 3, and type 9; May-Hegglin anomaly; MYH9 related disorders; Sebastian syndrome; McCune-Albright syndrome; Somatotroph adenoma; Sex cord-stromal tumor; Cushing syndrome; McKusick Kaufman syndrome; McLeod neuroacanthocytosis syndrome; Meckel-Gruber syndrome; Medium-chain acyl-coenzyme A dehydrogenase deficiency; Medulloblastoma; Megalencephalic leukoencephalopathy with subcortical cysts 1 and 2a; Megalencephaly cutis marmorata telangiectatica congenital; PIK3CA Related Overgrowth Spectrum; Megalencephaly-polymicrogyria-polydactyly-hydrocephalus syndrome 2; Megaloblastic anemia, thiamine-responsive, with diabetes mellitus and sensorineural deafness; Meier-Gorlin syndromes 1 and 4; Melnick-Needles syndrome; Meningioma; Mental retardation, X-linked, 3, 21, 30, and 72; Mental retardation and microcephaly with pontine and cerebellar hypoplasia; Mental retardation X-linked syndromic 5; Mental retardation, anterior maxillary protrusion, and strabismus; Mental retardation, autosomal dominant 12, 13, 15, 24, 3, 30, 4, 5, 6, and 9; Mental retardation, autosomal recessive 15, 44, 46, and 5; Mental retardation, stereotypic movements, epilepsy, and/or cerebral malformations; Mental retardation, syndromic, Claes-Jensen type, X-linked; Mental retardation, X-linked, nonspecific, syndromic, Hedera type, and syndromic, wu type; Merosin deficient congenital muscular dystrophy; Metachromatic leukodystrophy juvenile, late infantile, and adult types; Metachromatic leukodystrophy; Metatrophic dysplasia; Methemoglobinemia types I and 2; Methionine adenosyltransferase deficiency, autosomal dominant; Methylmalonic acidemia with homocystinuria; Methylmalonic aciduria cb1B type; Methylmalonic aciduria due to methylmalonyl-CoA mutase deficiency; METHYLMALONIC ACIDURIA, mut(0) TYPE; Microcephalic osteodysplastic primordial dwarfism type 2; Microcephaly with or without chorioretinopathy, lymphedema, or mental retardation; Microcephaly, hiatal hernia and nephrotic syndrome; Microcephaly; Hypoplasia of the corpus callosum; Spastic paraplegia 50, autosomal recessive; Global developmental delay; CNS hypomyelination; Brain atrophy; Microcephaly, normal intelligence and immunodeficiency; Microcephaly-capillary malformation syndrome; Microcytic anemia; Microphthalmia syndromic 5, 7, and 9; Microphthalmia, isolated 3, 5, 6, 8, and with coloboma 6; Microspherophakia; Migraine, familial basilar; Miller syndrome; Minicore myopathy with external ophthalmoplegia; Myopathy, congenital with cores; Mitchell-Riley syndrome; mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase deficiency; Mitochondrial complex I, II, III, III (nuclear type 2, 4, or 8) deficiency; Mitochondrial DNA depletion syndrome 11, 12 (cardiomyopathic type), 2, 4B (MNGIE type), 8B (MNGIE type); Mitochondrial DNA-depletion syndrome 3 and 7, hepatocerebral types, and 13 (encephalomyopathic type); Mitochondrial phosphate carrier and pyruvate carrier deficiency; Mitochondrial trifunctional protein deficiency; Long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency; Miyoshi muscular dystrophy 1; Myopathy, distal, with anterior tibial onset; Mohr-Tranebjaerg syndrome; Molybdenum cofactor deficiency, complementation group A; Mowat-Wilson syndrome; Mucolipidosis III Gamma; Mucopolysaccharidosis type VI, type VI (severe), and type VII; Mucopolysaccharidosis, MPSI-H/S, MPS-II, MPS-III-A, MPS-III-B, MPS-III-C, MPS-IV-A, MPS-IV-B; Retinitis Pigmentosa 73; Gangliosidosis GM1 type1 (with cardiac involvement) 3; Multicentric osteolysis nephropathy; Multicentric osteolysis, nodulosis and arthropathy; Multiple congenital anomalies; Atrial septal defect 2; Multiple congenital anomalies-hypotonia-seizures syndrome 3; Multiple Cutaneous and Mucosal Venous Malformations; Multiple endocrine neoplasia, types 1 and 4; Multiple epiphyseal dysplasia 5 or Dominant; Multiple gastrointestinal atresias; Multiple pterygium syndrome Escobar type; Multiple sulfatase deficiency; Multiple synostoses syndrome 3; Muscle AMP guanine oxidase deficiency; Muscle eye brain disease; Muscular dystrophy, congenital, megaconial type; Myasthenia, familial infantile, 1; Myasthenic Syndrome, Congenital, 11, associated with acetylcholine receptor deficiency; Myasthenic Syndrome, Congenital, 17, 2A (slow-channel), 4B (fast-channel), and without tubular aggregates; Myeloperoxidase deficiency; MYH-associated polyposis; Endometrial carcinoma; Myocardial infarction 1; Myoclonic dystonia; Myoclonic-Atonic Epilepsy; Myoclonus with epilepsy with ragged red fibers; Myofibrillar myopathy 1 and ZASP-related; Myoglobinuria, acute recurrent, autosomal recessive; Myoneural gastrointestinal encephalopathy syndrome; Cerebellar ataxia infantile with progressive external ophthalmoplegia; Mitochondrial DNA depletion syndrome 4B, MNGIE type; Myopathy, centronuclear, 1, congenital, with excess of muscle spindles, distal, 1, lactic acidosis, and sideroblastic anemia 1, mitochondrial progressive with congenital cataract, hearing loss, and developmental delay, and tubular aggregate, 2; Myopia 6; Myosclerosis, autosomal recessive; Myotonia congenital; Congenital myotonia, autosomal dominant and recessive forms; Nail-patella syndrome; Nance-Horan syndrome; Nanophthalmos 2; Navajo neurohepatopathy; Nemaline myopathy 3 and 9; Neonatal hypotonia; Intellectual disability; Seizures; Delayed speech and language development; Mental retardation, autosomal dominant 31; Neonatal intrahepatic cholestasis caused by citrin deficiency; Nephrogenic diabetes insipidus, Nephrogenic diabetes insipidus, X-linked; Nephrolithiasis/osteoporosis, hypophosphatemic, 2; Nephronophthisis 13, 15 and 4; Infertility; Cerebello-oculo-renal syndrome (nephronophthisis, oculomotor apraxia and cerebellar abnormalities); Nephrotic syndrome, type 3, type 5, with or without ocular abnormalities, type 7, and type 9; Nestor-Guillermo progeria syndrome; Neu-Laxova syndrome 1; Neurodegeneration with brain iron accumulation 4 and 6; Neuroferritinopathy; Neurofibromatosis, type 1 and type 2; Neurofibrosarcoma; Neurohypophyseal diabetes insipidus; Neuropathy, Hereditary Sensory, Type IC; Neutral 1 amino acid transport defect; Neutral lipid storage disease with myopathy; Neutrophil immunodeficiency syndrome; Nicolaides-Baraitser syndrome; Niemann-Pick disease type C1, C2, type A, and type C1, adult form; Non-ketotic hyperglycinemia; Noonan syndrome 1 and 4, LEOPARD syndrome 1; Noonan syndrome-like disorder with or without juvenile myelomonocytic leukemia; Normokalemic periodic paralysis, potassium-sensitive; Norum disease; Epilepsy, Hearing Loss, And Mental Retardation Syndrome; Mental Retardation, X-Linked 102 and syndromic 13; Obesity; Ocular albinism, type I; Oculocutaneous albinism type 1B, type 3, and type 4; Oculodentodigital dysplasia; Odontohypophosphatasia; Odontotrichomelic syndrome; Oguchi disease; Oligodontia-colorectal cancer syndrome; Opitz G/BBB syndrome; Optic atrophy 9; Oral-facial-digital syndrome; Ornithine aminotransferase deficiency; Orofacial cleft 11 and 7, Cleft lip/palate-ectodermal dysplasia syndrome; Orstavik Lindemann Solberg syndrome; Osteoarthritis with mild chondrodysplasia; Osteochondritis dissecans; Osteogenesis imperfecta type 12, type 5, type 7, type 8, type I, type III, with normal sclerae, dominant form, recessive perinatal lethal; Osteopathia striata with cranial sclerosis; Osteopetrosis autosomal dominant type 1 and 2, recessive 4, recessive 1, recessive 6; Osteoporosis with pseudoglioma; Oto-palato-digital syndrome, types I and II; Ovarian dysgenesis 1; Ovarioleukodystrophy; Pachyonychia congenita 4 and type 2; Paget disease of bone, familial; Pallister-Hall syndrome; Palmoplantar keratoderma, nonepidermolytic, focal or diffuse; Pancreatic agenesis and congenital heart disease; Papillon-Lef\xc3\xa8vre syndrome; Paragangliomas 3; Paramyotonia congenita of von Eulenburg; Parathyroid carcinoma; Parkinson disease 14, 15, 19 (juvenile-onset), 2, 20 (early-onset), 6, (autosomal recessive early-onset, and 9; Partial albinism; Partial hypoxanthine-guanine phosphoribosyltransferase deficiency; Patterned dystrophy of retinal pigment epithelium; PC-K6a; Pelizaeus-Merzbacher disease; Pendred syndrome; Peripheral demyelinating neuropathy, central dysmyelination; Hirschsprung disease; Permanent neonatal diabetes mellitus; Diabetes mellitus, permanent neonatal, with neurologic features; Neonatal insulin-dependent diabetes mellitus; Maturity-onset diabetes of the young, type 2; Peroxisome biogenesis disorder 14B, 2A, 4A, 5B, 6A, 7A, and 7B; Perrault syndrome 4; Perry syndrome; Persistent hyperinsulinemic hypoglycemia of infancy; familial hyperinsulinism; Phenotypes; Phenylketonuria; Pheochromocytoma; Hereditary Paraganglioma-Pheochromocytoma Syndromes; Paragangliomas 1; Carcinoid tumor of intestine; Cowden syndrome 3; Phosphoglycerate dehydrogenase deficiency; Phosphoglycerate kinase 1 deficiency; Photosensitive trichothiodystrophy; Phytanic acid storage disease; Pick disease; Pierson syndrome; Pigmentary retinal dystrophy; Pigmented nodular adrenocortical disease, primary, 1; Pilomatrixoma; Pitt-Hopkins syndrome; Pituitary dependent hypercortisolism; Pituitary hormone deficiency, combined 1, 2, 3, and 4; Plasminogen activator inhibitor type 1 deficiency; Plasminogen deficiency, type I; Platelet-type bleeding disorder 15 and 8; Poikiloderma, hereditary fibrosing, with tendon contractures, myopathy, and pulmonary fibrosis; Polycystic kidney disease 2, adult type, and infantile type; Polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy; Polyglucosan body myopathy 1 with or without immunodeficiency; Polymicrogyria, asymmetric, bilateral frontoparietal; Polyneuropathy, hearing loss, ataxia, retinitis pigmentosa, and cataract; Pontocerebellar hypoplasia type 4; Popliteal pterygium syndrome; Porencephaly 2; Porokeratosis 8, disseminated superficial actinic type; Porphobilinogen synthase deficiency; Porphyria cutanea tarda; Posterior column ataxia with retinitis pigmentosa; Posterior polar cataract type 2; Prader-Willi-like syndrome; Premature ovarian failure 4, 5, 7, and 9; Primary autosomal recessive microcephaly 10, 2, 3, and 5; Primary ciliary dyskinesia 24; Primary dilated cardiomyopathy; Left ventricular noncompaction 6; 4, Left ventricular noncompaction 10; Paroxysmal atrial fibrillation; Primary hyperoxaluria, type I, type, and type III; Primary hypertrophic osteoarthropathy, autosomal recessive 2; Primary hypomagnesemia; Primary open angle glaucoma juvenile onset 1; Primary pulmonary hypertension; Primrose syndrome; Progressive familial heart block type 1B; Progressive familial intrahepatic cholestasis 2 and 3; Progressive intrahepatic cholestasis; Progressive myoclonus epilepsy with ataxia; Progressive pseudorheumatoid dysplasia; Progressive sclerosing poliodystrophy; Prolidase deficiency; Proline dehydrogenase deficiency; Schizophrenia 4; Properdin deficiency, X-linked; Propionic academia; Proprotein convertase 1/3 deficiency; Prostate cancer, hereditary, 2; Protan defect; Proteinuria; Finnish congenital nephrotic syndrome; Proteus syndrome; Breast adenocarcinoma; Pseudoachondroplastic spondyloepiphyseal dysplasia syndrome; Pseudohypoaldosteronism type 1 autosomal dominant and recessive and type 2; Pseudohypoparathyroidism type 1A, Pseudopseudohypoparathyroidism; Pseudoneonatal adrenoleukodystrophy; Pseudoprimary hyperaldosteronism; Pseudoxanthoma elasticum; Generalized arterial calcification of infancy 2; Pseudoxanthoma elasticum-like disorder with multiple coagulation factor deficiency; Psoriasis susceptibility 2; PTEN hamartoma tumor syndrome; Pulmonary arterial hypertension related to hereditary hemorrhagic telangiectasia; Pulmonary Fibrosis And/Or Bone Marrow Failure, Telomere-Related, 1 and 3; Pulmonary hypertension, primary, 1, with hereditary hemorrhagic telangiectasia; Purine-nucleoside phosphorylase deficiency; Pyruvate carboxylase deficiency; Pyruvate dehydrogenase E1-alpha deficiency; Pyruvate kinase deficiency of red cells; Raine syndrome; Rasopathy; Recessive dystrophic epidermolysis bullosa; Nail disorder, nonsyndromic congenital, 8; Reifenstein syndrome; Renal adysplasia; Renal carnitine transport defect; Renal coloboma syndrome; Renal dysplasia; Renal dysplasia, retinal pigmentary dystrophy, cerebellar ataxia and skeletal dysplasia; Renal tubular acidosis, distal, autosomal recessive, with late-onset sensorineural hearing loss, or with hemolytic anemia; Renal tubular acidosis, proximal, with ocular abnormalities and mental retardation; Retinal cone dystrophy 3B; Retinitis pigmentosa; Retinitis pigmentosa 10, 11, 12, 14, 15, 17, and 19; Retinitis pigmentosa 2, 20, 25, 35, 36, 38, 39, 4, 40, 43, 45, 48, 66, 7, 70, 72; Retinoblastoma; Rett disorder; Rhabdoid tumor predisposition syndrome 2; Rhegmatogenous retinal detachment, autosomal dominant; Rhizomelic chondrodysplasia punctata type 2 and type 3; Roberts-SC phocomelia syndrome; Robinow Sorauf syndrome; Robinow syndrome, autosomal recessive, autosomal recessive, with brachy-syn-polydactyly; Rothmund-Thomson syndrome; Rapadilino syndrome; RRM2B-related mitochondrial disease; Rubinstein-Taybi syndrome; Salla disease; Sandhoff disease, adult and infantil types; Sarcoidosis, early-onset; Blau syndrome; Schindler disease, type 1; Schizencephaly; Schizophrenia 15; Schneckenbecken dysplasia; Schwannomatosis 2; Schwartz Jampel syndrome type 1; Sclerocornea, autosomal recessive; Sclerosteosis; Secondary hypothyroidism; Segawa syndrome, autosomal recessive; Senior-Loken syndrome 4 and 5; Sensory ataxic neuropathy, dysarthria, and ophthalmoparesis; Sepiapterin reductase deficiency; SeSAME syndrome; Severe combined immunodeficiency due to ADA deficiency, with microcephaly, growth retardation, and sensitivity to ionizing radiation, atypical, autosomal recessive, T cell-negative, B cell-positive, NK cell-negative of NK-positive; Severe congenital neutropenia; Severe congenital neutropenia 3, autosomal recessive or dominant; Severe congenital neutropenia and 6, autosomal recessive; Severe myoclonic epilepsy in infancy; Generalized epilepsy with febrile seizures plus, types 1 and 2; Severe X-linked myotubular myopathy; Short QT syndrome 3; Short stature with nonspecific skeletal abnormalities; Short stature, auditory canal atresia, mandibular hypoplasia, skeletal abnormalities; Short stature, onychodysplasia, facial dysmorphism, and hypotrichosis; Primordial dwarfism; Short-rib thoracic dysplasia 11 or 3 with or without polydactyly; Sialidosis type I and II; Silver spastic paraplegia syndrome; Slowed nerve conduction velocity, autosomal dominant; Smith-Lemli-Opitz syndrome; Snyder Robinson syndrome; Somatotroph adenoma; Prolactinoma; familial, Pituitary adenoma predisposition; Sotos syndrome 1 or 2; Spastic ataxia 5, autosomal recessive, Charlevoix-Saguenay type, 1,10, or 11, autosomal recessive; Amyotrophic lateral sclerosis type 5; Spastic paraplegia 15, 2, 3, 35, 39, 4, autosomal dominant, 55, autosomal recessive, and 5A; Bile acid synthesis defect, congenital, 3; Spermatogenic failure 11, 3, and 8; Spherocytosis types 4 and 5; Spheroid body myopathy; Spinal muscular atrophy, lower extremity predominant 2, autosomal dominant; Spinal muscular atrophy, type II; Spinocerebellar ataxia 14, 21, 35, 40, and 6; Spinocerebellar ataxia autosomal recessive 1 and 16; Splenic hypoplasia; Spondylocarpotarsal synostosis syndrome; Spondylocheirodysplasia, Ehlers-Danlos syndrome-like, with immune dysregulation, Aggrecan type, with congenital joint dislocations, short limb-hand type, Sedaghatian type, with cone-rod dystrophy, and Kozlowski type; Parastremmatic dwarfism; Stargardt disease 1; Cone-rod dystrophy 3; Stickler syndrome type 1; Kniest dysplasia; Stickler syndrome, types 1 (nonsyndromic ocular) and 4; Sting-associated vasculopathy, infantile-onset; Stormorken syndrome; Sturge-Weber syndrome, Capillary malformations, congenital, 1; Succinyl-CoA acetoacetate transferase deficiency; Sucrase-isomaltase deficiency; Sudden infant death syndrome; Sulfite oxidase deficiency, isolated; Supravalvar aortic stenosis; Surfactant metabolism dysfunction, pulmonary, 2 and 3; Symphalangism, proximal, 1b; Syndactyly Cenani Lenz type; Syndactyly type 3; Syndromic X-linked mental retardation 16; Talipes equinovarus; Tangier disease; TARP syndrome; Tay-Sachs disease, B1 variant, Gm2-gangliosidosis (adult), Gm2-gangliosidosis (adult-onset); Temtamy syndrome; Tenorio Syndrome; Terminal osseous dysplasia; Testosterone 17-beta-dehydrogenase deficiency; Tetraamelia, autosomal recessive; Tetralogy of Fallot; Hypoplastic left heart syndrome 2; Truncus arteriosus; Malformation of the heart and great vessels; Ventricular septal defect 1; Thiel-Behnke corneal dystrophy; Thoracic aortic aneurysms and aortic dissections; Marfanoid habitus; Three M syndrome 2; Thrombocytopenia, platelet dysfunction, hemolysis, and imbalanced globin synthesis; Thrombocytopenia, X-linked; Thrombophilia, hereditary, due to protein C deficiency, autosomal dominant and recessive; Thyroid agenesis; Thyroid cancer, follicular; Thyroid hormone metabolism, abnormal; Thyroid hormone resistance, generalized, autosomal dominant; Thyrotoxic periodic paralysis and Thyrotoxic periodic paralysis 2; Thyrotropin-releasing hormone resistance, generalized; Timothy syndrome; TNF receptor-associated periodic fever syndrome (TRAPS); Tooth agenesis, selective, 3 and 4; Torsades de pointes; Townes-Brocks-branchiootorenal-like syndrome; Transient bullous dermolysis of the newborn; Treacher collins syndrome 1; Trichomegaly with mental retardation, dwarfism and pigmentary degeneration of retina; Trichorhinophalangeal dysplasia type I; Trichorhinophalangeal syndrome type 3; Trimethylaminuria; Tuberous sclerosis syndrome; Lymphangiomyomatosis; Tuberous sclerosis 1 and 2; Tyrosinase-negative oculocutaneous albinism; Tyrosinase-positive oculocutaneous albinism; Tyrosinemia type I; UDPglucose-4-epimerase deficiency; Ullrich congenital muscular dystrophy; Ulna and fibula absence of with severe limb deficiency; Upshaw-Schulman syndrome; Urocanate hydratase deficiency; Usher syndrome, types 1, 1B, 1D, 1G, 2A, 2C, and 2D; Retinitis pigmentosa 39; UV-sensitive syndrome; Van der Woude syndrome; Van Maldergem syndrome 2; Hennekam lymphangiectasia-lymphedema syndrome 2; Variegate porphyria; Ventriculomegaly with cystic kidney disease; Verheij syndrome; Very long chain acyl-CoA dehydrogenase deficiency; Vesicoureteral reflux 8; Visceral heterotaxy 5, autosomal; Visceral myopathy; Vitamin D-dependent rickets, types 1 and 2; Vitelliform dystrophy; von Willebrand disease type 2M and type 3; Waardenburg syndrome type 1, 4C, and 2E (with neurologic involvement); Klein-Waardenberg syndrome; Walker-Warburg congenital muscular dystrophy; Warburg micro syndrome 2 and 4; Warts, hypogammaglobulinemia, infections, and myelokathexis; Weaver syndrome; Weill-Marchesani syndrome 1 and 3; Weill-Marchesani-like syndrome; Weis senbacher-Zweymuller syndrome; Werdnig-Hoffmann disease; Charcot-Marie-Tooth disease; Werner syndrome; WFS1-Related Disorders; Wiedemann-Steiner syndrome; Wilson disease; Wolfram-like syndrome, autosomal dominant; Worth disease; Van Buchem disease type 2; Xeroderma pigmentosum, complementation group b, group D, group E, and group G; X-linked agammaglobulinemia; X-linked hereditary motor and sensory neuropathy; X-linked ichthyosis with steryl-sulfatase deficiency; X-linked periventricular heterotopia; Oto-palato-digital syndrome, type I; X-linked severe combined immunodeficiency; Zimmermann-Laband syndrome and Zimmermann-Laband syndrome 2; and Zonular pulverulent cataract 3.
• The target nucleotide sequence may comprise a target sequence (e.g., a point mutation) associated with a disease, disorder, or condition. The target sequence may comprise a T to C (or A to G) point mutation associated with a disease, disorder, or condition, and wherein the deamination of the mutant C base results in mismatch repair-mediated correction to a sequence that is not associated with a disease, disorder, or condition. The target sequence may comprise a G to A (or C to T) point mutation associated with a disease, disorder, or condition, and wherein the deamination of the mutant A base results in mismatch repair-mediated correction to a sequence that is not associated with a disease, disorder, or condition. The target sequence may encode a protein, and where the point mutation is in a codon and results in a change in the amino acid encoded by the mutant codon as compared to a wild-type codon. The target sequence may also be at a splice site, and the point mutation results in a change in the splicing of an mRNA transcript as compared to a wild-type transcript. In addition, the target may be at a non-coding sequence of a gene, such as a promoter, and the point mutation results in increased or decreased expression of the gene.
• Thus, in some aspects, the deamination of a mutant C results in a change of the amino acid encoded by the mutant codon, which in some cases can result in the expression of a wild-type amino acid. In other aspects, the deamination of a mutant A results in a change of the amino acid encoded by the mutant codon, which in some cases can result in the expression of a wild-type amino acid.
• The methods described herein involving contacting a cell with a composition or rAAV particle can occur in vitro, ex vivo, or in vivo. In certain embodiments, the step of contacting occurs in a subject. In certain embodiments, the subject has been diagnosed with a disease, disorder, or condition.
• In some embodiments, the methods disclosed herein involve contacting a mammalian cell with a composition or rAAV particle. In particular embodiments, the methods involve contacting a retinal cell, cortical cell or cerebellar cell.
• The split Cas9 protein or split prime editor delivered using the methods described herein preferably have comparable activity compared to the original Cas9 protein or prime editor (i.e., unsplit protein delivered to a cell or expressed in a cell as a whole). For example, the split Cas9 protein or split prime editor retains at least 50% (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%) of the activity of the original Cas9 protein or prime editor. In some embodiments, the split Cas9 protein or split prime editor is more active (e.g., 2-fold, 5-fold, 10-fold, 100-fold, 1000-fold, or more) than that of an original Cas9 protein or prime editor.
• The compositions described herein may be administered to a subject in need thereof in a therapeutically effective amount to treat and/or prevent a disease or disorder the subject is suffering from. Any disease or disorder that maybe treated and/or prevented using CRISPR/Cas9-based genome-editing technology may be treated by the split Cas9 protein or the split prime editor described herein. It is to be understood that, if the nucleotide sequences encoding the split Cas9 protein or the prime editor does not further encode a gRNA, a separate nucleic acid vector encoding the gRNA may be administered together with the compositions described herein.
• Exemplary suitable diseases, disorders or conditions include, without limitation the disease or disorder is selected from the group consisting of: cystic fibrosis, phenylketonuria, epidermolytic hyperkeratosis (EHK), chronic obstructive pulmonary disease (COPD), Charcot-Marie-Toot disease type 4J, neuroblastoma (NB), von Willebrand disease (vWD), myotonia congenital, hereditary renal amyloidosis, dilated cardiomyopathy, hereditary lymphedema, familial Alzheimer's disease, prion disease, chronic infantile neurologic cutaneous articular syndrome (CINCA), congenital deafness, Niemann-Pick disease type C (NPC) disease, and desmin-related myopathy (DRM). In particular embodiments, the disease or condition is Niemann-Pick disease type C (NPC) disease.
• In some embodiments, the disease, disorder or condition is associated with a point mutation in an NPC gene, a DNMT1 gene, a PCSK9 gene, or a TMC1 gene. In certain embodiments, the point mutation is a T3182C mutation in NPC, which results in an I1061T amino acid substitution.
• In certain embodiments, the point mutation is an A545G mutation in TMC1, which results in a Y182C amino acid substitution. TMC1 encodes a protein that forms mechanosensitive ion channels in sensory hair cells of the inner ear and is required for normal auditory function. The Y182C amino acid substitution is associated with congenital deafness.
• In some embodiments, the disease, disorder or condition is associated with a point mutation that generates a stop codon, for example, a premature stop codon within the coding region of a gene.
• Additional exemplary diseases, disorders and conditions include cystic fibrosis (see, e.g., Schwank et al., Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosis patients. Cell stem cell. 2013; 13: 653-658; and Wu et. al., Correction of a genetic disease in mouse via use of CRISPR-Cas9. Cell stem cell. 2013; 13: 659-662, neither of which uses a deaminase fusion protein to correct the genetic defect); phenylketonuria—e.g., phenylalanine to serine mutation at position 835 (mouse) or 240 (human) or a homologous residue in phenylalanine hydroxylase gene (T>C mutation)—see, e.g., McDonald et al., Genomics. 1997; 39:402-405; Bernard-Soulier syndrome (BSS)—e.g., phenylalanine to serine mutation at position 55 or a homologous residue, or cysteine to arginine at residue 24 or a homologous residue in the platelet membrane glycoprotein IX (T>C mutation)—see, e.g., Noris et al., British Journal of Haematology. 1997; 97: 312-320, and Ali et al., Hematol. 2014; 93: 381-384; epidermolytic hyperkeratosis (EHK)—e.g., leucine to proline mutation at position 160 or 161 (if counting the initiator methionine) or a homologous residue in keratin 1 (T>C mutation)—see, e.g., Chipev et al., Cell. 1992; 70: 821-828, see also accession number P04264 in the UNIPROT database at www[dot]uniprot[dot]org; chronic obstructive pulmonary disease (COPD)—e.g., leucine to proline mutation at position 54 or 55 (if counting the initiator methionine) or a homologous residue in the processed form of α₁-antitrypsin or residue 78 in the unprocessed form or a homologous residue (T>C mutation)—see, e.g., Poller et al., Genomics. 1993; 17: 740-743, see also accession number P01011 in the UNIPROT database; Charcot-Marie-Toot disease type 4J—e.g., isoleucine to threonine mutation at position 41 or a homologous residue in FIG. 4 (T>C mutation)—see, e.g., Lenk et al., PLoS Genetics. 2011; 7: e1002104; neuroblastoma (NB)—e.g., leucine to proline mutation at position 197 or a homologous residue in Caspase-9 (T>C mutation)—see, e.g., Kundu et al., 3 Biotech. 2013, 3:225-234; von Willebrand disease (vWD)—e.g., cysteine to arginine mutation at position 509 or a homologous residue in the processed form of von Willebrand factor, or at position 1272 or a homologous residue in the unprocessed form of von Willebrand factor (T>C mutation)—see, e.g., Lavergne et al., Br. J. Haematol. 1992, see also accession number P04275 in the UNIPROT database; 82: 66-72; myotonia congenital—e.g., cysteine to arginine mutation at position 277 or a homologous residue in the muscle chloride channel gene CLCN1 (T>C mutation)—see, e.g., Weinberger et al., The J. of Physiology. 2012; 590: 3449-3464; hereditary renal amyloidosis—e.g., stop codon to arginine mutation at position 78 or a homologous residue in the processed form of apolipoprotein AII or at position 101 or a homologous residue in the unprocessed form (T>C mutation)—see, e.g., Yazaki et al., Kidney Int. 2003; 64: 11-16; dilated cardiomyopathy (DCM)—e.g., tryptophan to Arginine mutation at position 148 or a homologous residue in the FOXD4 gene (T>C mutation), see, e.g., Minoretti et. al., Int. J. of Mol. Med. 2007; 19: 369-372; hereditary lymphedema—e.g., histidine to arginine mutation at position 1035 or a homologous residue in VEGFR3 tyrosine kinase (A>G mutation), see, e.g., Irrthum et al., Am. J. Hum. Genet. 2000; 67: 295-301; familial Alzheimer's disease—e.g., isoleucine to valine mutation at position 143 or a homologous residue in presenilin 1 (A>G mutation), see, e.g., Gallo et. al., J. Alzheimer's disease. 2011; 25: 425-431; Prion disease—e.g., methionine to valine mutation at position 129 or a homologous residue in prion protein (A>G mutation)—see, e.g., Lewis et. al., J. of General Virology. 2006; 87: 2443-2449; chronic infantile neurologic cutaneous articular syndrome (CINCA)—e.g., Tyrosine to Cysteine mutation at position 570 or a homologous residue in cryopyrin (A>G mutation)—see, e.g., Fujisawa et. al. Blood. 2007; 109: 2903-2911; and desmin-related myopathy (DRM)—e.g., arginine to glycine mutation at position 120 or a homologous residue in αβ crystallin (A>G mutation)—see, e.g., Kumar et al., J. Biol. Chem. 1999; 274: 24137-24141. The entire contents of all references and database entries is incorporated herein by reference.
Trinucleotide Repeat Expansion Disease
• Trinucleotide repeat expansion is associated with a number of human diseases, including Huntington's Disease, Fragile X syndrome, and Friedreich's ataxia. The most common trinucleotide repeat contains CAG triplets, though GAA triplets (Friedreich's ataxia) and CGG triplets (Fragile X syndrome) also occur. Inheriting a predisposition to expansion, or acquiring an already expanded parental allele, increases the likelihood of acquiring the disease. Pathogenic expansions of trinucleotide repeats could hypothetically be corrected using prime editing.
• A region upstream of the repeat region can be nicked by an RNA-guided nuclease, then used to prime synthesis of a new DNA strand that contains a healthy number of repeats (which depends on the particular gene and disease), in accordance with the general mechanism outlined in FIG. 1G or FIG. 22 . After the repeat sequence, a short stretch of homology is added that matches the identity of the sequence adjacent to the other end of the repeat (red strand). Invasion of the newly synthesized strand by the TPRT system, and subsequent replacement of the endogenous DNA with the newly synthesized flap, leads to a contracted repeat allele. The term “contracted” refers to a shortening of the length of the nucleotide repeat region, thereby resulting in repairing the trinucleotide repeat region.
• The prime editing system or prime editing (PE) system described herein may be used to contract trinucleotide repeat mutations (or “triplet expansion diseases”) to treating conditions such as Huntington's disease and other trinucleotide repeat disorders. Trinucleotide repeat expansion disorders are complex, progressive disorders that involve developmental neurobiology and often affect cognition as well as sensori-motor functions. The disorders show genetic anticipation (i.e. increased severity with each generation). The DNA expansions or contractions usually happen meiotically (i.e. during the time of gametogenesis, or early in embryonic development), and often have sex-bias meaning that some genes expand only when inherited through the female, others only through the male. In humans, trinucleotide repeat expansion disorders can cause gene silencing at either the transcriptional or translational level, which essentially knocks out gene function. Alternatively, trinucleotide repeat expansion disorders can cause altered proteins generated with large repetitive amino acid sequences that either abrogate or change protein function, often in a dominant-negative manner (e.g. poly-glutamine diseases).
• Without wishing to be bound by theory, triplet expansion is caused by slippage during DNA replication or during DNA repair synthesis. Because the tandem repeats have identical sequence to one another, base pairing between two DNA strands can take place at multiple points along the sequence. This may lead to the formation of “loop out” structures during DNA replication or DNA repair synthesis. This may lead to repeated copying of the repeated sequence, expanding the number of repeats. Additional mechanisms involving hybrid RNA:DNA intermediates have been proposed. Prime editing may be used to reduce or eliminate these triplet expansion regions by deletion one or more or the offending repeat codon triplets. In an embodiment of this use, FIG. 23 , provides a schematic of a PEgRNA design for contracting or reducing trinucleotide repeat sequences with prime editing.
• Prime editing may be implemented to contract triplet expansion regions by nicking a region upstream of the triplet repeat region with the prime editor comprising a PEgRNA appropriated targeted to the cut site. The prime editor then synthesizes a new DNA strand (ssDNA flap) based on the PEgRNA as a template (i.e., the edit template thereof) that codes for a healthy number of triplet repeats (which depends on the particular gene and disease). The newly synthesized ssDNA strand comprising the healthy triplet repeat sequence also is synthesized to include a short stretch of homology (i.e., the homology arm) that matches the sequence adjacent to the other end of the repeat (red strand). Invasion of the newly synthesized strand, and subsequent replacement of the endogenous DNA with the newly synthesized ssDNA flap, leads to a contracted repeat allele.
• Depending on the particular trinucleotide expansion disorder, the defect-inducing triplet expansions may occur in “trinucleotide repeat expansion proteins.” Trinucleotide repeat expansion proteins are a diverse set of proteins associated with susceptibility for developing a trinucleotide repeat expansion disorder, the presence of a trinucleotide repeat expansion disorder, the severity of a trinucleotide repeat expansion disorder or any combination thereof. Trinucleotide repeat expansion disorders are divided into two categories determined by the type of repeat. The most common repeat is the triplet CAG, which, when present in the coding region of a gene, codes for the amino acid glutamine (Q). Therefore, these disorders are referred to as the polyglutamine (polyQ) disorders and comprise the following diseases: Huntington Disease (HD); Spinobulbar Muscular Atrophy (SBMA); Spinocerebellar Ataxias ( SCA types 1, 2, 3, 6, 7, and 17); and Dentatorubro-Pallidoluysian Atrophy (DRPLA). The remaining trinucleotide repeat expansion disorders either do not involve the CAG triplet or the CAG triplet is not in the coding region of the gene and are, therefore, referred to as the non-polyglutamine disorders. The non-polyglutamine disorders comprise Fragile X Syndrome (FRAXA); Fragile XE Mental Retardation (FRAXE); Friedreich Ataxia (FRDA); Myotonic Dystrophy (DM); and Spinocerebellar Ataxias (SCA types 8, and 12).
• The proteins associated with trinucleotide repeat expansion disorders can be selected based on an experimental association of the protein associated with a trinucleotide repeat expansion disorder to a trinucleotide repeat expansion disorder. For example, the production rate or circulating concentration of a protein associated with a trinucleotide repeat expansion disorder may be elevated or depressed in a population having a trinucleotide repeat expansion disorder relative to a population lacking the trinucleotide repeat expansion disorder. Differences in protein levels may be assessed using proteomic techniques including but not limited to Western blot, immunohistochemical staining, enzyme linked immunosorbent assay (ELISA), and mass spectrometry. Alternatively, the proteins associated with trinucleotide repeat expansion disorders may be identified by obtaining gene expression profiles of the genes encoding the proteins using genomic techniques including but not limited to DNA microarray analysis, serial analysis of gene expression (SAGE), and quantitative real-time polymerase chain reaction (Q-PCR).
• Non-limiting examples of proteins associated with trinucleotide repeat expansion disorders which can be corrected by prime editing include AR (androgen receptor), FMR1 (fragile X mental retardation 1), HTT (huntingtin), DMPK (dystrophia myotonica-protein kinase), FXN (frataxin), ATXN2 (ataxin 2), ATN1 (atrophin 1), FEN1 (flap structure-specific endonuclease 1), TNRC6A (trinucleotide repeat containing 6A), PABPN1 (poly(A) binding protein, nuclear 1), JPH3 (junctophilin 3), MED15 (mediator complex subunit 15), ATXN1 (ataxin 1), ATXN3 (ataxin 3), TBP (TATA box binding protein), CACNA1A (calcium channel, voltage-dependent, P/Q type, alpha 1A subunit), ATXN80S (ATXN8 opposite strand (non-protein coding)), PPP2R2B (protein phosphatase 2, regulatory subunit B, beta), ATXN7 (ataxin 7), TNRC6B (trinucleotide repeat containing 6B), TNRC6C (trinucleotide repeat containing 6C), CELF3 (CUGBP, Elav-like family member 3), MAB21L1 (mab-21-like 1 (C. elegans)), MSH2 (mutS homolog 2, colon cancer, nonpolyposis type 1 (E. coli)), TMEM185A (transmembrane protein 185A), SIX5 (SIX homeobox 5), CNPY3 (canopy 3 homolog (zebrafish)), FRAXE (fragile site, folic acid type, rare, fra(X)(q28) E), GNB2 (guanine nucleotide binding protein (G protein), beta polypeptide 2), RPL14 (ribosomal protein L14), ATXN8 (ataxin 8), INSR (insulin receptor), TTR (transthyretin), EP400 (E1A binding protein p400), GIGYF2 (GRB10 interacting GYF protein 2), OGG1 (8-oxoguanine DNA glycosylase), STC1 (stanniocalcin 1), CNDP1 (carnosine dipeptidase 1 (metallopeptidase M20 family)), C10orf2 (chromosome 10 open reading frame 2), MAML3 mastermind-like 3 (Drosophila), DKC1 (dyskeratosis congenita 1, dyskerin), PAXIP1 (PAX interacting (with transcription-activation domain) protein 1), CASK (calcium/calmodulin-dependent serine protein kinase (MAGUK family)), MAPT (microtubule-associated protein tau), SP1 (Sp1 transcription factor), POLG (polymerase (DNA directed), gamma), AFF2 (AF4/FMR2 family, member 2), THBS1 (thrombospondin 1), TP53 (tumor protein p53), ESR1 (estrogen receptor 1), CGGBP1 (CGG triplet repeat binding protein 1), ABT1 (activator of basal transcription 1), KLK3 (kallikrein-related peptidase 3), PRNP (prion protein), JUN (jun oncogene), KCNN3 (potassium intermediate/small conductance calcium-activated channel, subfamily N, member 3), BAX (BCL2-associated X protein), FRAXA (fragile site, folic acid type, rare, fra(X)(q27.3) A (macroorchidism, mental retardation)), KBTBD10 (kelch repeat and BTB (POZ) domain containing 10), MBNL1 (muscleblind-like (Drosophila)), RAD51 (RAD51 homolog (RecA homolog, E. coli) (S. cerevisiae)), NCOA3 (nuclear receptor coactivator 3), ERDA1 (expanded repeat domain, CAG/CTG 1), TSC1 (tuberous sclerosis 1), COMP (cartilage oligomeric matrix protein), GCLC (glutamate-cysteine ligase, catalytic subunit), RRAD (Ras-related associated with diabetes), MSH3 (mutS homolog 3 (E. coli)), DRD2 (dopamine receptor D2), CD44 (CD44 molecule (Indian blood group)), CTCF (CCCTC-binding factor (zinc finger protein)), CCND1 (cyclin D1), CLSPN (claspin homolog (Xenopus laevis)), MEF2A (myocyte enhancer factor 2A), PTPRU (protein tyrosine phosphatase, receptor type, U), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), TRIM22 (tripartite motif-containing 22), WT1 (Wilms tumor 1), AHR (aryl hydrocarbon receptor), GPX1 (glutathione peroxidase 1), TPMT (thiopurine S-methyltransferase), NDP (Norrie disease (pseudoglioma)), ARX (aristaless related homeobox), MUS81 (MUS81 endonuclease homolog (S. cerevisiae)), TYR (tyrosinase (oculocutaneous albinism IA)), EGR1 (early growth response 1), UNG (uracil-DNA glycosylase), NUMBL (numb homolog (Drosophila)-like), FABP2 (fatty acid binding protein 2, intestinal), EN2 (engrailed homeobox 2), CRYGC (crystallin, gamma C), SRP14 (signal recognition particle 14 kDa (homologous Alu RNA binding protein)), CRYGB (crystallin, gamma B), PDCD1 (programmed cell death 1), HOXA1 (homeobox A1), ATXN2L (ataxin 2-like), PMS2 (PMS2 postmeiotic segregation increased 2 (S. cerevisiae)), GLA (galactosidase, alpha), CBL (Cas-Br-M (murine) ecotropic retroviral transforming sequence), FTH1 (ferritin, heavy polypeptide 1), IL12RB2 (interleukin 12 receptor, beta 2), OTX2 (orthodenticle homeobox 2), HOXA5 (homeobox A5), POLG2 (polymerase (DNA directed), gamma 2, accessory subunit), DLX2 (distal-less homeobox 2), SIRPA (signal-regulatory protein alpha), OTX1 (orthodenticle homeobox 1), AHRR (aryl-hydrocarbon receptor repressor), MANF (mesencephalic astrocyte-derived neurotrophic factor), TMEM158 (transmembrane protein 158 (gene/pseudogene)), and ENSG00000078687.
• In a particular aspect, the instant disclosure provides TPRT-based methods for the treatment of a subject diagnosed with an expansion repeat disorder (also known as a repeat expansion disorder or a trinucleotide repeat disorder). Expansion repeat disorders occur when microsatellite repeats expand beyond a threshold length. Currently, at least 30 genetic diseases are believed to be caused by repeat expansions. Scientific understanding of this diverse group of disorders came to lights in the early 1990's with the discovery that trinucleotide repeats underlie several major inherited conditions, including Fragile X, Spinal and Bulbar Muscular Atrophy, Myotonic Dystrophy, and Huntington's disease (Nelson et al, “The unstable repeats—three evolving faces of neurological disease,” Neuron, Mar. 6, 2013, Vol. 77; 825-843, which is incorporated herein by reference), as well as Haw River Syndrome, Jacobsen Syndrome, Dentatorubral-pallidoluysian atrophy (DRPLA), Machado-Joseph disease, Synpolydactyly (SPD II), Hand-foot genital syndrome (HFGS), Cleidocranial dysplasia (CCD), Holoprosencephaly disorder (HPE), Congenital central hypventilation syndrome (CCHS), ARX-nonsyndromic X-linked mental retardation (XLMR), and Oculopharyngeal muscular dystrophy (OPMD) (see. Microsatellite repeat instability was found to be a hallmark of these conditions, as was anticipation—the phenomenon in which repeat expansion can occur with each successive generation, which leads to a more severe phenotype and earlier age of onset in the offspring. Repeat expansions are believed to cause diseases via several different mechanisms. Namely, expansions may interfere with cellular functioning at the level of the gene, the mRNA transcript, and/or the encoded protein. In some conditions, mutations act via a loss-of-function mechanism by silencing repeat-containing genes. In others, disease results from gain-of-function mechanisms, whereby either the mRNA transcript or protein takes on new, aberrant functions.
• In one embodiment, a method of treating a trinucleotide repeat disorder is depicted in FIG. 23 . In general, the approach involves using TPRT genome editing (i.e., prime editing) in combination with an extended gRNA that comprises a region that encodes a desired and healthy replacement trinucleotide repeat sequence that is intended to replace the endogenous diseased trinucleotide repeat sequence through the mechanism of the prime editing process. A schematic of an exemplary gRNA design for contracting trinucleotide repeat sequences and trinucleotide repeat contraction with TPRT genome editing (i.e., prime editing) is shown in FIG. 23 .
Prion Disease
• Prime editing can also be used to prevent or halt the progression of prion disease through the installation of one or more protective mutations into prion proteins (PRNP) which become misfolded during the course of disease. Prion diseases or transmissible spongiform encephalopathies (TSEs) are a family of rare progressive neurodegenerative disorders that affect both humans and animals. They are distinguished by long incubation periods, characteristic spongiform changes associated with neuronal loss, and a failure to induce inflammatory response.
• In humans, prion disease includes Creutzfeldt-Jakob Disease (CJD), Variant Creutzfeldt-Jakob Disease (vCJD), Gerstmann-Straussler-Scheinker Syndrome, Fatal Familial Insomnia, and Kuru. In animals, prion disease includes Bovine Spongiform Encephalopathy (BSE or “mad cow disease”), Chronic Wasting Disease (CWD), Scrapie, Transmissible Mink Encephalopathy, Feline Spongiform Encephalopathy, and Ungulate Spongiform Encephalopathy. Prime editing may be used to install protective point mutations into a prion protein in order to prevent or halt the progression of any one of these prion diseases.
• Classic CJD is a human prion disease. It is a neurodegenerative disorder with characteristic clinical and diagnostic features. This disease is rapidly progressive and always fatal. Infection with this disease leads to death usually within 1 year of onset of illness. CJD is a rapidly progressive, invariably fatal neurodegenerative disorder believed to be caused by an abnormal isoform of a cellular glycoprotein known as the prion protein. CJD occurs worldwide and the estimated annual incidence in many countries, including the United States, has been reported to be about one case per million population. The vast majority of CJD patients usually die within 1 year of illness onset. CJD is classified as a transmissible spongiform encephalopathy (TSE) along with other prion diseases that occur in humans and animals. In about 85% of patients, CJD occurs as a sporadic disease with no recognizable pattern of transmission. A smaller proportion of patients (5 to 15%) develop CJD because of inherited mutations of the prion protein gene. These inherited forms include Gerstmann-Straussler-Scheinker syndrome and fatal familial insomnia. No treatment is currently known for CJD.
• Variant Creutzfeldt-Jakob disease (vCJD) is a prion disease that was first described in 1996 in the United Kingdom. There is now strong scientific evidence that the agent responsible for the outbreak of prion disease in cows, bovine spongiform encephalopathy (BSE or ‘mad cow’ disease), is the same agent responsible for the outbreak of vCJD in humans. Variant CJD (vCJD) is not the same disease as classic CJD (often simply called CJD). It has different clinical and pathologic characteristics from classic CJD. Each disease also has a particular genetic profile of the prion protein gene. Both disorders are invariably fatal brain diseases with unusually long incubation periods measured in years, and are caused by an unconventional transmissible agent called a prion. No treatment is currently known for vCJD.
• BSE (bovine spongiform encephalopathy or “mad cow disease”) is a progressive neurological disorder of cattle that results from infection by an unusual transmissible agent called a prion. The nature of the transmissible agent is not well understood. Currently, the most accepted theory is that the agent is a modified form of a normal protein known as prion protein. For reasons that are not yet understood, the normal prion protein changes into a pathogenic (harmful) form that then damages the central nervous system of cattle. There is increasing evidence that there are different strains of BSE: the typical or classic BSE strain responsible for the outbreak in the United Kingdom and two atypical strains (H and L strains). No treatment is currently known for BSE.
• Chronic wasting disease (CWD) is a prion disease that affects deer, elk, reindeer, sika deer and moose. It has been found in some areas of North America, including Canada and the United States, Norway and South Korea. It may take over a year before an infected animal develops symptoms, which can include drastic weight loss (wasting), stumbling, listlessness and other neurologic symptoms. CWD can affect animals of all ages and some infected animals may die without ever developing the disease. CWD is fatal to animals and there are no treatments or vaccines.
• The causative agents of TSEs are believed to be prions. The term “prions” refers to abnormal, pathogenic agents that are transmissible and are able to induce abnormal folding of specific normal cellular proteins called prion proteins that are found most abundantly in the brain. The functions of these normal prion proteins are still not completely understood. The abnormal folding of the prion proteins leads to brain damage and the characteristic signs and symptoms of the disease. Prion diseases are usually rapidly progressive and always fatal.
• As used herein, the term “prion” shall mean an infectious particle known to cause diseases (spongiform encephalopathies) in humans and animals. The term “prion” is a contraction of the words “protein” and “infection” and the particles are comprised largely if not exclusively of PRNP^Sc molecules encoded by a PRNP gene which expresses PRNP^C which changes conformation to become PRNP^Sc. Prions are distinct from bacteria, viruses and viroids. Known prions include those which infect animals to cause scrapie, a transmissible, degenerative disease of the nervous system of sheep and goats as well as bovine spongiform encephalopathies (BSE) or mad cow disease and feline spongiform encephalopathies of cats. Four prion diseases, as discussed above, known to affect humans are (1) kuru, (2) Creutzfeldt-Jakob Disease (CJD), (3) Gerstmann-Strassler-Scheinker Disease (GSS), and (4) fatal familial insomnia (FFI). As used herein prion includes all forms of prions causing all or any of these diseases or others in any animals used—and in particular in humans and in domesticated farm animals.
• In general, and without wishing to be bound by theory, prior diseases are caused by misfolding of prion proteins. Such diseases—often called deposition diseases—the misfolding of the prion proteins can be accounted for as follows. If A is the normally synthesized gene product that carries out an intended physiologic role in a monomeric or oligomeric state, A* is a conformationally activated form of A that is competent to undergo a dramatic conformational change, B is the conformationally altered state that prefers multimeric assemblies (i.e., the misfolded form which forms depositions) and B_n is the multimeric material that is pathogenic and relatively difficult to recycle. For the prion diseases, PRNP^C and PRNP^Sc correspond to states A and B_n where A is largely helical and monomeric and B_n is β-rich and multimeric.
• It is known that certain mutations in prion proteins can be associated with increased risk of prior disease. Conversely, certain mutations in prion proteins can be protective in nature. See Bagynszky et al., “Characterization of mutations in PRNP (prion) gene and their possible roles in neurodegenerative diseases,” Neuropsychiatr Dis Treat., 2018; 14: 2067-2085, the contents of which are incorporated herein by reference.
• PRNP (NCBI RefSeq No. NP_000302.1 (SEQ ID NO: 291))—the human prion protein—is encoded by a 16 kb long gene, located on chromosome 20 (4686151-4701588). It contains two exons, and the exon 2 carries the open reading frame which encodes the 253 amino acid (AA) long PrP protein. Exon 1 is a noncoding exon, which may serve as transcriptional initiation site. The post-translational modifications result in the removal of the first 22 AA N-terminal fragment (NTF) and the last 23 AA C-terminal fragment (CTF). The NTF is cleaved after PrP transport to the endoplasmic reticulum (ER), while the CTF (glycosylphosphatidylinositol [GPI] signal peptide [GPI-SP]) is cleaved by the GPI anchor. GPI anchor could be involved in PrP protein transport. It may also play a role of attachment of prion protein into the outer surface of cell membrane. Normal PrP is composed of a long N-terminal loop (which contains the octapeptide repeat region), two short β sheets, three a helices, and a C-terminal region (which contains the GPI anchor). Cleavage of PrP results in a 208 AA long glyocoprotein, anchored in the cell membrane.
• The 253 amino acid sequence of PRNP (NP_000302.1) is as follows:

• (SEQ ID NO: 291)

  MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYP

  PQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWN

  KPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHFGSDYEDRYY

  RENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTE

  TDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPV.

• The 253 amino acid sequence of PRNP (NP_000302.1) is encoded by the following nucleotide sequence (NCBI Ref. Seq No. NM_000311.5, “Homo sapiens prion protein (PRNP), transcript variant 1, mRNA), is as follows:

• (SEQ ID NO: 292)

  GCGAACCTTGGCTGCTGGATGCTGGTTCTCTTTGTGGCCACATGGAGTGA

  CCTGGGCCTCTGCAAGAAGCGCCCGAAGCCTGGAGGATGGAACACTGGGG

  GCAGCCGATACCCGGGGCAGGGCAGCCCTGGAGGCAACCGCTACCCACCT

  CAGGGCGGTGGTGGCTGGGGGCAGCCTCATGGTGGTGGCTGGGGGCAGCC

  TCATGGTGGTGGCTGGGGGCAGCCCCATGGTGGTGGCTGGGGACAGCCTC

  ATGGTGGTGGCTGGGGTCAAGGAGGTGGCACCCACAGTCAGTGGAACAAG

  CCGAGTAAGCCAAAAACCAACATGAAGCACATGGCTGGTGCTGCAGCAGC

  TGGGGCAGTGGTGGGGGGCCTTGGCGGCTACATGCTGGGAAGTGCCATGA

  GCAGGCCCATCATACATTTCGGCAGTGACTATGAGGACCGTTACTATCGT

  GAAAACATGCACCGTTACCCCAACCAAGTGTACTACAGGCCCATGGATGA

  GTACAGCAACCAGAACAACTTTGTGCACGACTGCGTCAATATCACAATCA

  AGCAGCACACGGTCACCACAACCACCAAGGGGGAGAACTTCACCGAGACC

  GACGTTAAGATGATGGAGCGCGTGGTTGAGCAGATGTGTATCACCCAGTA

  CGAGAGGGAATCTCAGGCCTATTACCAGAGAGGATCGAGCATGGTCCTCT

  TCTCCTCTCCACCTGTGATCCTCCTGATCTCTTTCCTCATCTTCCTGATA

  GTGGGATGAGGAAGGTCTTCCTGTTTTCACCATCTTTCTAATCTTTTTCC

  AGCTTGAGGGAGGCGGTATCCACCTGCAGCCCTTTTAGTGGTGGTGTCTC

  ACTCTTTCTTCTCTCTTTGTCCCGGATAGGCTAATCAATACCCTTGGCAC

  TGATGGGCACTGGAAAACATAGAGTAGACCTGAGATGCTGGTCAAGCCCC

  CTTTGATTGAGTTCATCATGAGCCGTTGCTAATGCCAGGCCAGTAAAAGT

  ATAACAGCAAATAACCATTGGTTAATCTGGACTTATTTTTGGACTTAGTG

  CAACAGGTTGAGGCTAAAACAAATCTCAGAACAGTCTGAAATACCTTTGC

  CTGGATACCTCTGGCTCCTTCAGCAGCTAGAGCTCAGTATACTAATGCCC

  TATCTTAGTAGAGATTTCATAGCTATTTAGAGATATTTTCCATTTTAAGA

  AAACCCGACAACATTTCTGCCAGGTTTGTTAGGAGGCCACATGATACTTA

  TTCAAAAAAATCCTAGAGATTCTTAGCTCTTGGGATGCAGGCTCAGCCCG

  CTGGAGCATGAGCTCTGTGTGTACCGAGAACTGGGGTGATGTTTTACTTT

  TCACAGTATGGGCTACACAGCAGCTGTTCAACAAGAGTAAATATTGTCAC

  AACACTGAACCTCTGGCTAGAGGACATATTCACAGTGAACATAACTGTAA

  CATATATGAAAGGCTTCTGGGACTTGAAATCAAATGTTTGGGAATGGTGC

  CCTTGGAGGCAACCTCCCATTTTAGATGTTTAAAGGACCCTATATGTGGC

  ATTCCTTTCTTTAAACTATAGGTAATTAAGGCAGCTGAAAAGTAAATTGC

  CTTCTAGACACTGAAGGCAAATCTCCTTTGTCCATTTACCTGGAAACCAG

  AATGATTTTGACATACAGGAGAGCTGCAGTTGTGAAAGCACCATCATCAT

  AGAGGATGATGTAATTAAAAAATGGTCAGTGTGCAAAGAAAAGAACTGCT

  TGCATTTCTTTATTTCTGTCTCATAATTGTCAAAAACCAGAATTAGGTCA

  AGTTCATAGTTTCTGTAATTGGCTTTTGAATCAAAGAATAGGGAGACAAT

  CTAAAAAATATCTTAGGTTGGAGATGACAGAAATATGATTGATTTGAAGT

  GGAAAAAGAAATTCTGTTAATGTTAATTAAAGTAAAATTATTCCCTGAAT

  TGTTTGATATTGTCACCTAGCAGATATGTATTACTTTTCTGCAATGTTAT

  TATTGGCTTGCACTTTGTGAGTATTCTATGTAAAAATATATATGTATATA

  AAATATATATTGCATAGGACAGACTTAGGAGTTTTGTTTAGAGCAGTTAA

  CATCTGAAGTGTCTAATGCATTAACTTTTGTAAGGTACTGAATACTTAAT

  ATGTGGGAAACCCTTTTGCGTGGTCCTTAGGCTTACAATGTGCACTGAAT

  CGTTTCATGTAAGAATCCAAAGTGGACACCATTAACAGGTCTTTGAAATA

  TGCATGTACTTTATATTTTCTATATTTGTAACTTTGCATGTTCTTGTTTT

  GTTATATAAAAAAATTGTAAATGTTTAATATCTGACTGAAATTAAACGAG

  CGAAGATGAGCACCA

• Mutation sites relative to PRNP (NP_000302.1) which are linked to CJD and FFI are reported are as follows. These mutations can be removed or installed using the prime editors disclosed herein.

• 	AMINO ACID SEQUENCE OF MUTANT PRNP LINKED
  	TO CJD PRION DISEASE (SEE TABLE 1 OF
  	BAGYNSZKY ET AL., 2018) (RELATIVE TO
  MUTATION	SEQ ID NO: 291 OF PRNP NP_000302.1)
  D178N	MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPG
  	QGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGG
  	WGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGA
  	VVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQ
  	VYYRPMDEYSNQNNFVHNCVNITIKQHTVTTTTKGENFTE
  	TDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPV
  	(SEO ID NO: 293)
  T188K	MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPG
  	QGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGG
  	WGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGA
  	VVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQ
  	VYYRPMDEYSNQNNFVHDCVNITIKQHKVTTTTKGENFTE
  	TDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPV
  	(SEQ ID NO: 294)
  E196K	MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPG
  	QGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGG
  	WGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGA
  	VVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQ
  	VYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGKNFTE
  	TDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPV
  	(SEQ ID NO: 295)
  E196A	MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPG
  	QGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGG
  	WGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGA
  	VVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQ
  	VYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGANFTE
  	TDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPV
  	(SEQ ID NO: 296)
  E200K	MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPG
  	QGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGG
  	WGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGA
  	VVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQ
  	VYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTK
  	TDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPV
  	(SEQID NO: 297)
  E200G	MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPG
  	QGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGG
  	WGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGA
  	VVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQ
  	VYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTG
  	TDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPV
  	(SEQ ID NO: 298)
  V203I	MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPG
  	QGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGG
  	WGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGA
  	VVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQ
  	VYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTE
  	TDIKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPV
  	(SEQ ID NO: 299)
  R208H	MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPG
  	QGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGG
  	WGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGA
  	VVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQ
  	VYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTE
  	TDVKMMEHVVEQMCITQYERESQAYYQRGSSMVLFSSPPV
  	(SEQ ID NO: 300)
  V210I	MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPG
  	QGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGG
  	WGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGA
  	VVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQ
  	VYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTE
  	TDVKMMERVIEQMCITQYERESQAYYQRGSSMVLFSSPPV
  	(SEQ ID NO: 301)
  E211Q	MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPG
  	QGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGG
  	WGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGA
  	VVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQ
  	VYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTE
  	TDVKMMERVVQQMCITQYERESQAYYQRGSSMVLFSSPPV
  	(SEQ ID NO: 302)
  M232R	MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPG
  	QGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGG
  	WGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGA
  	VVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQ
  	VYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTE
  	TDVKMMERVVEQMCITQYERESQAYYQRGSSRVLFSSPPV
  	(SEQ ID NO: 303)

• Mutation sites relative to PRNP (NP_000302.1) (SEQ ID NO: 291) which are linked to GSS are reported, as follows:

• 	AMINO ACID SEQUENCE OF MUTANT PRNP
  	LINKED TO GSS PRION DISEASE (SEE TABLE 2
  	OF BAGYNSZKY ET AL., 2018) (RELATIVE TO
  MUTATION	SEQ ID NO: 291 OF PRNP NP 000302.1)
  P102L	MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPG
  	QGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGG
  	WGQPHGGGWGQGGGTHSQWNKLSKPKTNMKHMAGAAAAGA
  	VVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQ
  	VYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTE
  	TDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPV
  	(SEQ ID NO: 304)
  P105L	MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPG
  	QGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGG
  	WGQPHGGGWGQGGGTHSQWNKPSKLKTNMKHMAGAAAAGA
  	VVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQ
  	VYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTE
  	TDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPV
  	(SEQ ID NO: 305)
  A117V	MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPG
  	QGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGG
  	WGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAVAGA
  	VVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQ
  	VYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTE
  	TDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPV
  	(SEQ ID NO: 306)
  G131V	MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPG
  	QGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGG
  	WGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGA
  	VVGGLGGYMLVSAMSRPIIHFGSDYEDRYYRENMHRYPNQ
  	VYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTE
  	TDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPV
  	(SEQ ID NO: 307)
  V176G	MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPG
  	QGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGG
  	WGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGA
  	VVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQ
  	VYYRPMDEYSNQNNFGHDCVNITIKQHTVTTTTKGENFTE
  	TDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPV
  	(SEQ ID NO: 308)
  H187R	MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPG
  	QGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGG
  	WGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGA
  	VVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQ
  	VYYRPMDEYSNQNNFVHDCVNITIKQRTVTTTTKGENFTE
  	TDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPV
  	(SEQ ID NO: 309)
  	MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPG
  	QGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGG
  	WGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGA
  	VVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQ
  	VYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTE
  	TDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPV
  	(SEQ ID NO: 291)
  F198S	MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPG
  	QGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGG
  	WGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGA
  	VVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQ
  	VYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENSTE
  	TDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPV
  	(SEQ ID NO: 311)
  D202N	MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPG
  	QGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGG
  	WGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGA
  	VVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQ
  	VYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTE
  	TNVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPV
  	(SEQ ID NO: 312)
  Q212P	MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPG
  	QGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGG
  	WGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGA
  	VVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQ
  	VYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTE
  	TDVKMMERVVEPMCITQYERESQAYYQRGSSMVLFSSPPV
  	(SEQ ID NO: 313)
  Q217R	MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPG
  	QGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGG
  	WGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGA
  	VVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQ
  	VYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTE
  	TDVKMMERVVEQMCITRYERESQAYYQRGSSMVLFSSPPV
  	(SEQ ID NO: 314)
  M232T	MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPG
  	QGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGG
  	WGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGA
  	VVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQ
  	VYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTE
  	TDVKMMERVVEQMCITQYERESQAYYQRGSSTVLFSSPPV
  	(SEQ ID NO: 315)

• Mutation sites relative to PRNP (NP_000302.1) (SEQ ID NO: 291) which are linked to a possible protective nature against prion disease, as follows:

• 	AMINO ACID SEQUENCE OF MUTANT
  	PRNP LINKED TO A PROTECTIVE NATURE
  	AGAINST PRION DISEASE (SEE TABLE 4 OF
  MUTAT-	BAGYNSZKY ET AL., 2018) (RELATIVE
  ION	TO SEQ ID NO: 291 OF PRNP NP_00302.1)
  G1275	MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGS
  	PGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHG
  	GGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGSYM
  	LGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQ
  	NNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERVVEQMCI
  	TQYERESQAYYQRGSSMVLFSSPPV
  	(SEQ ID NO: 316)
  G127V	MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGS
  	PGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHG
  	GGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGVYM
  	LGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQ
  	NNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERVVEQMCI
  	TQYERESQAYYQRGSSMVLFSSPPV
  	(SEQ ID NO: 317)
  M129V	MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGS
  	PGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHG
  	GGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYV
  	LGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQ
  	NNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERVVEQMCI
  	TQYERESQAYYQRGSSMVLFSSPPV
  	(SEQ ID NO: 318)
  D167G	MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGS
  	PGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHG
  	GGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYM
  	LGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMGEYSNQ
  	NNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERVVEQMCI
  	TQYERESQAYYQRGSSMVLFSSPPV
  	(SEQ ID NO: 319)
  D167N	MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGS
  	PGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHG
  	GGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYM
  	LGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMNEYSNQ
  	NNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERVVEQMCI
  	TQYERESQAYYQRGSSMVLFSSPPV
  	(SEQ ID NO: 320)
  N171S	MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGS
  	PGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHG
  	GGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYM
  	LGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSSQ
  	NNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERVVEQMCI
  	TQYERESQAYYQRGSSMVLFSSPPV
  	(SEQ ID NO: 321)
  E219K	MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGS
  	PGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHG
  	GGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYM
  	LGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQ
  	NNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERVVEQMCI
  	TQYKRESQAYYQRGSSMVLFSSPPV
  	(SEQ ID NO: 322)
  P238S	MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGS
  	PGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHG
  	GGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYM
  	LGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQ
  	NNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERVVEQMCI
  	TQYERESQAYYQRGSSMVLFSSSPV
  	(SEQ ID NO: 323)

• Thus, in various embodiments, prime editing may be used to remove a mutation in PRNP that is linked to prion disease or install a mutation in PRNP that is considered to be protective against prion disease. For example, prime editing may be use to remove or restore a D178N, V180I, T188K, E196K, E196A, E200K, E200G, V2031, R208H, V210I, E211Q, I215V, or M232R mutation in the PRNP protein (relative to PRNP of NP_000302.1) (SEQ ID NO: 291). In other embodiments, prime editing may be use to remove or restore a P102L, P105L, A117V, G131V, V176G, H187R, F198S, D202N, Q212P, Q217R, or M232T mutation in the PRNP protein (relative to PRNP of NP_000302.1) (SEQ ID NO: 291). By removing or correcting for the presence of such mutations in PRNP using prime editing, the risk of prion disease may be reduced or eliminated.
• In other embodiments, prime editing may be used to install a protective mutation in PRNP that is linked to a protective effect against one or more prion diseases. For example, prime editing may be used to install a G127S, G127V, M129V, D167G, D167N, N171S, E219K, or P238S protective mutation in PRNP (relative to PRNP of NP_000302.1) (SEQ ID NO: 291). In still other embodiments, the protective mutation may be any alternate amino acid installed at G127, G127, M129, D167, D167, N171, E219, or P238 in PRNP (relative to PRNP of NP_000302.1) (SEQ ID NO: 291).
• In particular embodiments, prime editing may be used to install a G127V protective mutation in PRNP, as illustrated in FIG. 27 and discussed in Example 5.
• In another embodiment, prime editing may be used to install an E219K protective mutation in PRNP.
• The PRNP protein and the protective mutation site are conserved in mammals, so in addition to treating human disease it could also be used to generate cows and sheep that are immune to prion disease, or even help cure wild populations of animals that are suffering from prion disease. Prime editing has already been used to achieve ˜25% installation of a naturally occurring protective allele in human cells, and previous mouse experiments indicate that this level of installation is sufficient to cause immunity from most prion diseases. This method is the first and potentially only current way to install this allele with such high efficiency in most cell types. Another possible strategy for treatment is to use prime editing to reduce or eliminate the expression of PRNP by installing an early stop codon in the gene.
• Using the principles described herein for PEgRNA design, appropriate PEgRNAs may be designed for installing desired protective mutations, or for removing prion disease-associated mutations from PRNP. For example, the below list of PEgRNAs can be used to install the G127V protective allele and the E219K protective allele in human PRNP, as well as the G127V protective allele in PRNP of various animals.
[9] Pharmaceutical Compositions
• Other aspects of the present disclosure relate to pharmaceutical compositions comprising any of the various components of the prime editing system described herein (e.g., including, but not limited to, the napDNAbps, reverse transcriptases, fusion proteins (e.g., comprising napDNAbps and reverse transcriptases), extended guide RNAs, and complexes comprising fusion proteins and extended guide RNAs, as well as accessory elements, such as second strand nicking components and 5′ endogenous DNA flap removal endonucleases for helping to drive the prime editing process towards the edited product formation).
• The term “pharmaceutical composition”, as used herein, refers to a composition formulated for pharmaceutical use. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises additional agents (e.g. for specific delivery, increasing half-life, or other therapeutic compounds).
• As used here, the term “pharmaceutically-acceptable carrier” means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the compound from one site (e.g., the delivery site) of the body, to another site (e.g., organ, tissue or portion of the body). A pharmaceutically acceptable carrier is “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the tissue of the subject (e.g., physiologically compatible, sterile, physiologic pH, etc.). Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; (22) C2-C12 alcohols, such as ethanol; and (23) other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative and antioxidants can also be present in the formulation. The terms such as “excipient”, “carrier”, “pharmaceutically acceptable carrier” or the like are used interchangeably herein.
• In some embodiments, the pharmaceutical composition is formulated for delivery to a subject, e.g., for gene editing. Suitable routes of administrating the pharmaceutical composition described herein include, without limitation: topical, subcutaneous, transdermal, intradermal, intralesional, intraarticular, intraperitoneal, intravesical, transmucosal, gingival, intradental, intracochlear, transtympanic, intraorgan, epidural, intrathecal, intramuscular, intravenous, intravascular, intraosseus, periocular, intratumoral, intracerebral, and intracerebroventricular administration.
• In some embodiments, the pharmaceutical composition described herein is administered locally to a diseased site (e.g., tumor site). In some embodiments, the pharmaceutical composition described herein is administered to a subject by injection, by means of a catheter, by means of a suppository, or by means of an implant, the implant being of a porous, non-porous, or gelatinous material, including a membrane, such as a sialastic membrane, or a fiber.
• In other embodiments, the pharmaceutical composition described herein is delivered in a controlled release system. In one embodiment, a pump may be used (see, e.g., Langer, 1990, Science 249:1527-1533; Sefton, 1989, CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med. 321:574). In another embodiment, polymeric materials can be used. (See, e.g., Medical Applications of Controlled Release (Langer and Wise eds., CRC Press, Boca Raton, Fla., 1974); Controlled Drug Bioavailability, Drug Product Design and Performance (Smolen and Ball eds., Wiley, New York, 1984); Ranger and Peppas, 1983, Macromol. Sci. Rev. Macromol. Chem. 23:61. See also Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71:105). Other controlled release systems are discussed, for example, in Langer, supra.
• In some embodiments, the pharmaceutical composition is formulated in accordance with routine procedures as a composition adapted for intravenous or subcutaneous administration to a subject, e.g., a human. In some embodiments, pharmaceutical composition for administration by injection are solutions in sterile isotonic aqueous buffer. Where necessary, the pharmaceutical can also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the pharmaceutical is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the pharmaceutical composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.
• A pharmaceutical composition for systemic administration may be a liquid, e.g., sterile saline, lactated Ringer's or Hank's solution. In addition, the pharmaceutical composition can be in solid forms and re-dissolved or suspended immediately prior to use. Lyophilized forms are also contemplated.
• The pharmaceutical composition can be contained within a lipid particle or vesicle, such as a liposome or microcrystal, which is also suitable for parenteral administration. The particles can be of any suitable structure, such as unilamellar or plurilamellar, so long as compositions are contained therein. Compounds can be entrapped in “stabilized plasmid-lipid particles” (SPLP) containing the fusogenic lipid dioleoylphosphatidylethanolamine (DOPE), low levels (5-10 mol %) of cationic lipid, and stabilized by a polyethyleneglycol (PEG) coating (Zhang Y. P. et al., Gene Ther. 1999, 6:1438-47). Positively charged lipids such as N-[1-(2,3-dioleoyloxi)propyl]-N,N,N-trimethyl-amoniummethylsulfate, or “DOTAP,” are particularly preferred for such particles and vesicles. The preparation of such lipid particles is well known. See, e.g., U.S. Pat. Nos. 4,880,635; 4,906,477; 4,911,928; 4,917,951; 4,920,016; and 4,921,757; each of which is incorporated herein by reference.
• The pharmaceutical composition described herein may be administered or packaged as a unit dose, for example. The term “unit dose” when used in reference to a pharmaceutical composition of the present disclosure refers to physically discrete units suitable as unitary dosage for the subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; i.e., carrier, or vehicle.
• Further, the pharmaceutical composition can be provided as a pharmaceutical kit comprising (a) a container containing a compound of the invention in lyophilized form and (b) a second container containing a pharmaceutically acceptable diluent (e.g., sterile water) for injection. The pharmaceutically acceptable diluent can be used for reconstitution or dilution of the lyophilized compound of the invention. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
• In another aspect, an article of manufacture containing materials useful for the treatment of the diseases described above is included. In some embodiments, the article of manufacture comprises a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers may be formed from a variety of materials such as glass or plastic. In some embodiments, the container holds a composition that is effective for treating a disease described herein and may have a sterile access port. For example, the container may be an intravenous solution bag or a vial having a stopper pierce-able by a hypodermic injection needle. The active agent in the composition is a compound of the invention. In some embodiments, the label on or associated with the container indicates that the composition is used for treating the disease of choice. The article of manufacture may further comprise a second container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution, or dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. [10] Kits, Cells, Vectors, and Delivery
Kits
• The compositions of the present disclosure may be assembled into kits. In some embodiments, the kit comprises nucleic acid vectors for the expression of the prime editors described herein. In other embodiments, the kit further comprises appropriate guide nucleotide sequences (e.g., PEgRNAs and second-site gRNAs) or nucleic acid vectors for the expression of such guide nucleotide sequences, to target the Cas9 protein or prime editor to the desired target sequence.
• The kit described herein may include one or more containers housing components for performing the methods described herein and optionally instructions for use. Any of the kit described herein may further comprise components needed for performing the assay methods. Each component of the kits, where applicable, may be provided in liquid form (e.g., in solution) or in solid form, (e.g., a dry powder). In certain cases, some of the components may be reconstitutable or otherwise processible (e.g., to an active form), for example, by the addition of a suitable solvent or other species (for example, water), which may or may not be provided with the kit.
• In some embodiments, the kits may optionally include instructions and/or promotion for use of the components provided. As used herein, “instructions” can define a component of instruction and/or promotion, and typically involve written instructions on or associated with packaging of the disclosure. Instructions also can include any oral or electronic instructions provided in any manner such that a user will clearly recognize that the instructions are to be associated with the kit, for example, audiovisual (e.g., videotape, DVD, etc.), Internet, and/or web-based communications, etc. The written instructions may be in a form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, which can also reflect approval by the agency of manufacture, use or sale for animal administration. As used herein, “promoted” includes all methods of doing business including methods of education, hospital and other clinical instruction, scientific inquiry, drug discovery or development, academic research, pharmaceutical industry activity including pharmaceutical sales, and any advertising or other promotional activity including written, oral and electronic communication of any form, associated with the disclosure. Additionally, the kits may include other components depending on the specific application, as described herein.
• The kits may contain any one or more of the components described herein in one or more containers. The components may be prepared sterilely, packaged in a syringe and shipped refrigerated. Alternatively it may be housed in a vial or other container for storage. A second container may have other components prepared sterilely. Alternatively the kits may include the active agents premixed and shipped in a vial, tube, or other container.
• The kits may have a variety of forms, such as a blister pouch, a shrink wrapped pouch, a vacuum sealable pouch, a sealable thermoformed tray, or a similar pouch or tray form, with the accessories loosely packed within the pouch, one or more tubes, containers, a box or a bag. The kits may be sterilized after the accessories are added, thereby allowing the individual accessories in the container to be otherwise unwrapped. The kits can be sterilized using any appropriate sterilization techniques, such as radiation sterilization, heat sterilization, or other sterilization methods known in the art. The kits may also include other components, depending on the specific application, for example, containers, cell media, salts, buffers, reagents, syringes, needles, a fabric, such as gauze, for applying or removing a disinfecting agent, disposable gloves, a support for the agents prior to administration, etc. Some aspects of this disclosure provide kits comprising a nucleic acid construct comprising a nucleotide sequence encoding the various components of the prime editing system described herein (e.g., including, but not limited to, the napDNAbps, reverse transcriptases, polymerases, fusion proteins (e.g., comprising napDNAbps and reverse transcriptases (or more broadly, polymerases), extended guide RNAs, and complexes comprising fusion proteins and extended guide RNAs, as well as accessory elements, such as second strand nicking components (e.g., second strand nicking gRNA) and 5′ endogenous DNA flap removal endonucleases for helping to drive the prime editing process towards the edited product formation). In some embodiments, the nucleotide sequence(s) comprises a heterologous promoter (or more than a single promoter) that drives expression of the prime editing system components.
• Other aspects of this disclosure provide kits comprising one or more nucleic acid constructs encoding the various components of the prime editing system described herein, e.g., the comprising a nucleotide sequence encoding the components of the prime editing system capable of modifying a target DNA sequence. In some embodiments, the nucleotide sequence comprises a heterologous promoter that drives expression of the prime editing system components.
• Some aspects of this disclosure provides kits comprising a nucleic acid construct, comprising (a) a nucleotide sequence encoding a napDNAbp (e.g., a Cas9 domain) fused to a reverse transcriptase and (b) a heterologous promoter that drives expression of the sequence of (a).
Cells
• Cells that may contain any of the compositions described herein include prokaryotic cells and eukaryotic cells. The methods described herein are used to deliver a Cas9 protein or a prime editor into a eukaryotic cell (e.g., a mammalian cell, such as a human cell). In some embodiments, the cell is in vitro (e.g., cultured cell. In some embodiments, the cell is in vivo (e.g., in a subject such as a human subject). In some embodiments, the cell is ex vivo (e.g., isolated from a subject and may be administered back to the same or a different subject).
• Mammalian cells of the present disclosure include human cells, primate cells (e.g., vero cells), rat cells (e.g., GH3 cells, OC23 cells) or mouse cells (e.g., MC3T3 cells). There are a variety of human cell lines, including, without limitation, human embryonic kidney (HEK) cells, HeLa cells, cancer cells from the National Cancer Institute's 60 cancer cell lines (NCI60), DU145 (prostate cancer) cells, Lncap (prostate cancer) cells, MCF-7 (breast cancer) cells, MDA-MB-438 (breast cancer) cells, PC3 (prostate cancer) cells, T47D (breast cancer) cells, THP-1 (acute myeloid leukemia) cells, U87 (glioblastoma) cells, SHSYSY human neuroblastoma cells (cloned from a myeloma) and Saos-2 (bone cancer) cells. In some embodiments, rAAV vectors are delivered into human embryonic kidney (HEK) cells (e.g., HEK 293 or HEK 293T cells). In some embodiments, rAAV vectors are delivered into stem cells (e.g., human stem cells) such as, for example, pluripotent stem cells (e.g., human pluripotent stem cells including human induced pluripotent stem cells (hiPSCs)). A stem cell refers to a cell with the ability to divide for indefinite periods in culture and to give rise to specialized cells. A pluripotent stem cell refers to a type of stem cell that is capable of differentiating into all tissues of an organism, but not alone capable of sustaining full organismal development. A human induced pluripotent stem cell refers to a somatic (e.g., mature or adult) cell that has been reprogrammed to an embryonic stem cell-like state by being forced to express genes and factors important for maintaining the defining properties of embryonic stem cells (see, e.g., Takahashi and Yamanaka, Cell 126 (4): 663-76, 2006, incorporated by reference herein). Human induced pluripotent stem cell cells express stem cell markers and are capable of generating cells characteristic of all three germ layers (ectoderm, endoderm, mesoderm).
• Additional non-limiting examples of cell lines that may be used in accordance with the present disclosure include 293-T, 293-T, 3T3, 4T1, 721, 9L, A-549, A172, A20, A253, A2780, A2780ADR, A2780cis, A431, ALC, B16, B35, BCP-1, BEAS-2B, bEnd.3, BHK-21, BR 293, BxPC3, C2C12, C3H-10T1/2, C6, C6/36, Cal-27, CGR8, CHO, CML T1, CMT, COR-L23, COR-L23/5010, COR-L23/CPR, COR-L23/R23, COS-7, COV-434, CT26, D17, DH82, DU145, DuCaP, E14Tg2a, EL4, EM2, EM3, EMT6/AR1, EMT6/AR10.0, FM3, H1299, H69, HB54, HB55, HCA2, Hepalc1c7, High Five cells, HL-60, HMEC, HT-29, HUVEC, J558L cells, Jurkat, JY cells, K562 cells, KCL22, KG1, Ku812, KYO1, LNCap, Ma-Mel 1, 2, 3 . . . 48, MC-38, MCF-10A, MCF-7, MDA-MB-231, MDA-MB-435, MDA-MB-468, MDCK II, MG63, MONO-MAC 6, MOR/0.2R, MRC5, MTD-1A, MyEnd, NALM-1, NCI-H69/CPR, NCI-H69/LX10, NCI-H69/LX20, NCI-H69/LX4, NIH-3T3, NW-145, OPCN/OPCT Peer, PNT-1A/PNT 2, PTK2, Raji, RBL cells, RenCa, RIN-5F, RMA/RMAS, S2, Saos-2 cells, Sf21, Sf9, SiHa, SKBR3, SKOV-3, T-47D, T2, T84, THP1, U373, U87, U937, VCaP, WM39, WT-49, X63, YAC-1 and YAR cells.
• Some aspects of this disclosure provide cells comprising any of the constructs disclosed herein. In some embodiments, a host cell is transiently or non-transiently transfected with one or more vectors described herein. In some embodiments, a cell is transfected as it naturally occurs in a subject. In some embodiments, a cell that is transfected is taken from a subject. In some embodiments, the cell is derived from cells taken from a subject, such as a cell line. A wide variety of cell lines for tissue culture are known in the art. Examples of cell lines include, but are not limited to, C8161, CCRF-CEM, MOLT, mIMCD-3, NHDF, HeLa-S3, Huh1, Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa, MiaPaCell, Panc1, PC-3, TF1, CTLL-2, C1R, Rat6, CV1, RPTE, A10, T24, J82, A375, ARH-77, Calu1, SW480, SW620, SKOV3, SK-UT, CaCo2, P388D1, SEM-K2, WEHI-231, HB56, TIB55, Jurkat, J45.01, LRMB, Bcl-1, BC-3, IC21, DLD2, Raw264.7, NRK, NRK-52E, MRC5, MEF, Hep G2, HeLa B, HeLa T4, COS, COS-1, COS-6, COS-M6A, BS-C-1 monkey kidney epithelial, BALB/3T3 mouse embryo fibroblast, 3T3 Swiss, 3T3-L1, 132-d5 human fetal fibroblasts; 10.1 mouse fibroblasts, 293-T, 3T3, 721, 9L, A2780, A2780ADR, A2780cis, A 172, A20, A253, A431, A-549, ALC, B16, B35, BCP-1 cells, BEAS-2B, bEnd.3, BHK-21, BR 293. BxPC3. C3H-10T1/2, C6/36, Cal-27, CHO, CHO-7, CHO-IR, CHO-K1, CHO-K2, CHO-T, CHO Dhfr−/−, COR-L23, COR-L23/CPR, COR-L23/5010, COR-L23/R23, COS-7, COV-434, CML T1, CMT, CT26, D17, DH82, DU145, DuCaP, EL4, EM2, EM3, EMT6/AR1, EMT6/AR10.0, FM3, H1299, H69, HB54, HB55, HCA2, HEK-293, HeLa, Hepalc1c7, HL-60, HMEC, HT-29, Jurkat, JY cells, K562 cells, Ku812, KCL22, KG1, KYO1, LNCap, Ma-Mel 1-48, MC-38, MCF-7, MCF-10A, MDA-MB-231, MDA-MB-468, MDA-MB-435, MDCK II, MDCK 11, MOR/0.2R, MONO-MAC 6, MTD-1A, MyEnd, NCI-H69/CPR, NCI-H69/LX10, NCI-H69/LX20, NCI-H69/LX4, NIH-3T3, NALM-1, NW-145, OPCN/OPCT cell lines, Peer, PNT-1A/PNT 2, RenCa, RIN-5F, RMA/RMAS, Saos-2 cells, Sf-9, SkBr3, T2, T-47D, T84, THP1 cell line, U373, U87, U937, VCaP, Vero cells, WM39, WT-49, X63, YAC-1, YAR, and transgenic varieties thereof.
• Cell lines are available from a variety of sources known to those with skill in the art (see, e.g., the American Type Culture Collection (ATCC) (Manassas, Va.)). In some embodiments, a cell transfected with one or more vectors described herein is used to establish a new cell line comprising one or more vector-derived sequences. In some embodiments, a cell transiently transfected with the components of a CRISPR system as described herein (such as by transient transfection of one or more vectors, or transfection with RNA), and modified through the activity of a CRISPR complex, is used to establish a new cell line comprising cells containing the modification but lacking any other exogenous sequence. In some embodiments, cells transiently or non-transiently transfected with one or more vectors described herein, or cell lines derived from such cells are used in assessing one or more test compounds.
Vectors
• Some aspects of the present disclosure relate to using recombinant virus vectors (e.g., adeno-associated virus vectors, adenovirus vectors, or herpes simplex virus vectors) for the delivery of the prime editors or components thereof described herein, e.g., the split Cas9 protein or a split nucleobase prime editors, into a cell. In the case of a split-PE approach, the N-terminal portion of a PE fusion protein and the C-terminal portion of a PE fusion are delivered by separate recombinant virus vectors (e.g., adeno-associated virus vectors, adenovirus vectors, or herpes simplex virus vectors) into the same cell, since the full-length Cas9 protein or prime editors exceeds the packaging limit of various virus vectors, e.g., rAAV (˜4.9 kb).
• Thus, in one embodiment, the disclosure contemplates vectors capable of delivering split prime editor fusion proteins, or split components thereof. In some embodiments, a composition for delivering the split Cas9 protein or split prime editor into a cell (e.g., a mammalian cell, a human cell) is provided. In some embodiments, the composition of the present disclosure comprises: (i) a first recombinant adeno-associated virus (rAAV) particle comprising a first nucleotide sequence encoding a N-terminal portion of a Cas9 protein or prime editor fused at its C-terminus to an intein-N; and (ii) a second recombinant adeno-associated virus (rAAV) particle comprising a second nucleotide sequence encoding an intein-C fused to the N-terminus of a C-terminal portion of the Cas9 protein or prime editor. The rAAV particles of the present disclosure comprise a rAAV vector (i.e., a recombinant genome of the rAAV) encapsidated in the viral capsid proteins.
• In some embodiments, the rAAV vector comprises: (1) a heterologous nucleic acid region comprising the first or second nucleotide sequence encoding the N-terminal portion or C-terminal portion of a split Cas9 protein or a split prime editor in any form as described herein, (2) one or more nucleotide sequences comprising a sequence that facilitates expression of the heterologous nucleic acid region (e.g., a promoter), and (3) one or more nucleic acid regions comprising a sequence that facilitate integration of the heterologous nucleic acid region (optionally with the one or more nucleic acid regions comprising a sequence that facilitates expression) into the genome of a cell. In some embodiments, viral sequences that facilitate integration comprise Inverted Terminal Repeat (ITR) sequences. In some embodiments, the first or second nucleotide sequence encoding the N-terminal portion or C-terminal portion of a split Cas9 protein or a split prime editor is flanked on each side by an ITR sequence. In some embodiments, the nucleic acid vector further comprises a region encoding an AAV Rep protein as described herein, either contained within the region flanked by ITRs or outside the region. The ITR sequences can be derived from any AAV serotype (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) or can be derived from more than one serotype. In some embodiments, the ITR sequences are derived from AAV2 or AAV6.
• Thus, in some embodiments, the rAAV particles disclosed herein comprise at least one rAAV2 particle, rAAV6 particle, rAAV8 particle, rPHP.B particle, rPHP.eB particle, or rAAV9 particle, or a variant thereof. In particular embodiments, the disclosed rAAV particles are rPHP.B particles, rPHP.eB particles, rAAV9 particles.
• ITR sequences and plasmids containing ITR sequences are known in the art and commercially available (see, e.g., products and services available from Vector Biolabs, Philadelphia, Pa.; Cellbiolabs, San Diego, Calif.; Agilent Technologies, Santa Clara, Ca; and Addgene, Cambridge, Mass.; and Gene delivery to skeletal muscle results in sustained expression and systemic delivery of a therapeutic protein. Kessler P D, Podsakoff G M, Chen X, McQuiston S A, Colosi P C, Matelis L A, Kurtzman G J, Byrne B J. Proc Natl Acad Sci USA. 1996 Nov. 26; 93(24):14082-7; and Curtis A. Machida. Methods in Molecular Medicine™. Viral Vectors for Gene Therapy Methods and Protocols. 10.1385/1-59259-304-6:201 © Humana Press Inc. 2003. Chapter 10. Targeted Integration by Adeno-Associated Virus. Matthew D. Weitzman, Samuel M. Young Jr., Toni Cathomen and Richard Jude Samulski; U.S. Pat. Nos. 5,139,941 and 5,962,313, all of which are incorporated herein by reference).
• In some embodiments, the rAAV vector of the present disclosure comprises one or more regulatory elements to control the expression of the heterologous nucleic acid region (e.g., promoters, transcriptional terminators, and/or other regulatory elements). In some embodiments, the first and/or second nucleotide sequence is operably linked to one or more (e.g., 1, 2, 3, 4, 5, or more) transcriptional terminators. Non-limiting examples of transcriptional terminators that may be used in accordance with the present disclosure include transcription terminators of the bovine growth hormone gene (bGH), human growth hormone gene (hGH), SV40, CW3, φ, or combinations thereof. The efficiencies of several transcriptional terminators have been tested to determine their respective effects in the expression level of the split Cas9 protein or the split prime editor. In some embodiments, the transcriptional terminator used in the present disclosure is a bGH transcriptional terminator. In some embodiments, the rAAV vector further comprises a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE). In certain embodiments, the WPRE is a truncated WPRE sequence, such as “W3.” In some embodiments, the WPRE is inserted 5′ of the transcriptional terminator. Such sequences, when transcribed, create a tertiary structure which enhances expression, in particular, from viral vectors.
• In some embodiments, the vectors used herein may encode the PE fusion proteins, or any of the components thereof (e.g., napDNAbp, linkers, or polymerases). In addition, the vectors used herein may encode the PEgRNAs, and/or the accessory gRNA for second strand nicking. The vectors may be capable of driving expression of one or more coding sequences in a cell. In some embodiments, the cell may be a prokaryotic cell, such as, e.g., a bacterial cell. In some embodiments, the cell may be a eukaryotic cell, such as, e.g., a yeast, plant, insect, or mammalian cell. In some embodiments, the eukaryotic cell may be a mammalian cell. In some embodiments, the eukaryotic cell may be a rodent cell. In some embodiments, the eukaryotic cell may be a human cell. Suitable promoters to drive expression in different types of cells are known in the art. In some embodiments, the promoter may be wild-type. In other embodiments, the promoter may be modified for more efficient or efficacious expression. In yet other embodiments, the promoter may be truncated yet retain its function. For example, the promoter may have a normal size or a reduced size that is suitable for proper packaging of the vector into a virus.
• In some embodiments, the promoters that may be used in the prime editor vectors may be constitutive, inducible, or tissue-specific. In some embodiments, the promoters may be a constitutive promoters. Non-limiting exemplary constitutive promoters include cytomegalovirus immediate early promoter (CMV), simian virus (SV40) promoter, adenovirus major late (MLP) promoter, Rous sarcoma virus (RSV) promoter, mouse mammary tumor virus (MMTV) promoter, phosphoglycerate kinase (PGK) promoter, elongation factor-alpha (EF1a) promoter, ubiquitin promoters, actin promoters, tubulin promoters, immunoglobulin promoters, a functional fragment thereof, or a combination of any of the foregoing. In some embodiments, the promoter may be a CMV promoter. In some embodiments, the promoter may be a truncated CMV promoter. In other embodiments, the promoter may be an EF1a promoter. In some embodiments, the promoter may be an inducible promoter. Non-limiting exemplary inducible promoters include those inducible by heat shock, light, chemicals, peptides, metals, steroids, antibiotics, or alcohol. In some embodiments, the inducible promoter may be one that has a low basal (non-induced) expression level, such as, e.g., the Tet-On® promoter (Clontech). In some embodiments, the promoter may be a tissue-specific promoter. In some embodiments, the tissue-specific promoter is exclusively or predominantly expressed in liver tissue. Non-limiting exemplary tissue-specific promoters include B29 promoter, CD14 promoter, CD43 promoter, CD45 promoter, CD68 promoter, desmin promoter, elastase-1 promoter, endoglin promoter, fibronectin promoter, Flt-1 promoter, GFAP promoter, GPIIb promoter, ICAM-2 promoter, INF-β promoter, Mb promoter, Nphs1 promoter, OG-2 promoter, SP-B promoter, SYN1 promoter, and WASP promoter.
• In some embodiments, the prime editor vectors (e.g., including any vectors encoding the prime editor fusion protein and/or the PEgRNAs, and/or the accessory second strand nicking gRNAs) may comprise inducible promoters to start expression only after it is delivered to a target cell. Non-limiting exemplary inducible promoters include those inducible by heat shock, light, chemicals, peptides, metals, steroids, antibiotics, or alcohol. In some embodiments, the inducible promoter may be one that has a low basal (non-induced) expression level, such as, e.g., the Tet-On® promoter (Clontech).
• In additional embodiments, the prime editor vectors (e.g., including any vectors encoding the prime editor fusion protein and/or the PEgRNAs, and/or the accessory second strand nicking gRNAs) may comprise tissue-specific promoters to start expression only after it is delivered into a specific tissue. Non-limiting exemplary tissue-specific promoters include B29 promoter, CD14 promoter, CD43 promoter, CD45 promoter, CD68 promoter, desmin promoter, elastase-1 promoter, endoglin promoter, fibronectin promoter, Flt-1 promoter, GFAP promoter, GPIIb promoter, ICAM-2 promoter, INF-β promoter, Mb promoter, Nphs1 promoter, OG-2 promoter, SP-B promoter, SYN1 promoter, and WASP promoter.
• In some embodiments, the nucleotide sequence encoding the PEgRNA (or any guide RNAs used in connection with prime editing) may be operably linked to at least one transcriptional or translational control sequence. In some embodiments, the nucleotide sequence encoding the guide RNA may be operably linked to at least one promoter. In some embodiments, the promoter may be recognized by RNA polymerase III (Pol III). Non-limiting examples of Pol III promoters include U6, HI and tRNA promoters. In some embodiments, the nucleotide sequence encoding the guide RNA may be operably linked to a mouse or human U6 promoter. In other embodiments, the nucleotide sequence encoding the guide RNA may be operably linked to a mouse or human HI promoter. In some embodiments, the nucleotide sequence encoding the guide RNA may be operably linked to a mouse or human tRNA promoter. In embodiments with more than one guide RNA, the promoters used to drive expression may be the same or different. In some embodiments, the nucleotide encoding the crRNA of the guide RNA and the nucleotide encoding the tracr RNA of the guide RNA may be provided on the same vector. In some embodiments, the nucleotide encoding the crRNA and the nucleotide encoding the tracr RNA may be driven by the same promoter. In some embodiments, the crRNA and tracr RNA may be transcribed into a single transcript. For example, the crRNA and tracr RNA may be processed from the single transcript to form a double-molecule guide RNA. Alternatively, the crRNA and tracr RNA may be transcribed into a single-molecule guide RNA.
• In some embodiments, the nucleotide sequence encoding the guide RNA may be located on the same vector comprising the nucleotide sequence encoding the PE fusion protein. In some embodiments, expression of the guide RNA and of the PE fusion protein may be driven by their corresponding promoters. In some embodiments, expression of the guide RNA may be driven by the same promoter that drives expression of the PE fusion protein. In some embodiments, the guide RNA and the PE fusion protein transcript may be contained within a single transcript. For example, the guide RNA may be within an untranslated region (UTR) of the Cas9 protein transcript. In some embodiments, the guide RNA may be within the 5′ UTR of the PE fusion protein transcript. In other embodiments, the guide RNA may be within the 3′ UTR of the PE fusion protein transcript. In some embodiments, the intracellular half-life of the PE fusion protein transcript may be reduced by containing the guide RNA within its 3′ UTR and thereby shortening the length of its 3′ UTR. In additional embodiments, the guide RNA may be within an intron of the PE fusion protein transcript. In some embodiments, suitable splice sites may be added at the intron within which the guide RNA is located such that the guide RNA is properly spliced out of the transcript. In some embodiments, expression of the Cas9 protein and the guide RNA in close proximity on the same vector may facilitate more efficient formation of the CRISPR complex.
• The prime editor vector system may comprise one vector, or two vectors, or three vectors, or four vectors, or five vector, or more. In some embodiments, the vector system may comprise one single vector, which encodes both the PE fusion protein and PEgRNA. In other embodiments, the vector system may comprise two vectors, wherein one vector encodes the PE fusion protein and the other encodes the PEgRNA. In additional embodiments, the vector system may comprise three vectors, wherein the third vector encodes the second strand nicking gRNA used in the herein methods.
• In some embodiments, the composition comprising the rAAV particle (in any form contemplated herein) further comprises a pharmaceutically acceptable carrier. In some embodiments, the composition is formulated in appropriate pharmaceutical vehicles for administration to human or animal subjects.
• Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; (22) C2-C12 alcohols, such as ethanol; and (23) other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative and antioxidants can also be present in the formulation. The terms such as “excipient”, “carrier”, “pharmaceutically acceptable carrier” or the like are used interchangeably herein.
Delivery Methods
• In some aspects, the invention provides methods comprising delivering one or more polynucleotides, such as or one or more vectors as described herein, one or more transcripts thereof, and/or one or proteins transcribed therefrom, to a host cell. In some aspects, the invention further provides cells produced by such methods, and organisms (such as animals, plants, or fungi) comprising or produced from such cells. In some embodiments, a base editor as described herein in combination with (and optionally complexed with) a guide sequence is delivered to a cell.
• Exemplary delivery strategies are described herein elsewhere, which include vector-based strategies, PE ribonucleoprotein complex delivery, and delivery of PE by mRNA methods.
• In some embodiments, the method of delivery provided comprises nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA.
• Exemplary methods of delivery of nucleic acids include lipofection, nucleofection, electroporation, stable genome integration (e.g., piggybac), microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., Transfectam™, Lipofectin™ and SF Cell Line 4D-Nucleofector X Kit™ (Lonza)). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Feigner, WO 91/17424; WO 91/16024. Delivery may be to cells (e.g. in vitro or ex vivo administration) or target tissues (e.g. in vivo administration). Delivery may be achieved through the use of RNP complexes.
• The preparation of lipid:nucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to one of skill in the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese et al., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res. 52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).
• In other embodiments, the method of delivery and vector provided herein is an RNP complex. RNP delivery of fusion proteins markedly increases the DNA specificity of base editing. RNP delivery of fusion proteins leads to decoupling of on- and off-target DNA editing. RNP delivery ablates off-target editing at non-repetitive sites while maintaining on-target editing comparable to plasmid delivery, and greatly reduces off-target DNA editing even at the highly repetitive VEGFA site 2. See Rees, H. A. et al., Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery, Nat. Commun. 8, 15790 (2017), U.S. Pat. No. 9,526,784, issued Dec. 27, 2016, and U.S. Pat. No. 9,737,604, issued Aug. 22, 2017, each of which is incorporated by reference herein.
• Additional methods for the delivery of nucleic acids to cells are known to those skilled in the art. See, for example, US 2003/0087817, incorporated herein by reference.
• Other aspects of the present disclosure provide methods of delivering the prime editor constructs into a cell to form a complete and functional prime editor within a cell. For example, in some embodiments, a cell is contacted with a composition described herein (e.g., compositions comprising nucleotide sequences encoding the split Cas9 or the split prime editor or AAV particles containing nucleic acid vectors comprising such nucleotide sequences). In some embodiments, the contacting results in the delivery of such nucleotide sequences into a cell, wherein the N-terminal portion of the Cas9 protein or the prime editor and the C-terminal portion of the Cas9 protein or the prime editor are expressed in the cell and are joined to form a complete Cas9 protein or a complete prime editor.
• It should be appreciated that any rAAV particle, nucleic acid molecule or composition provided herein may be introduced into the cell in any suitable way, either stably or transiently. In some embodiments, the disclosed proteins may be transfected into the cell. In some embodiments, the cell may be transduced or transfected with a nucleic acid molecule. For example, a cell may be transduced (e.g., with a virus encoding a split protein), or transfected (e.g., with a plasmid encoding a split protein) with a nucleic acid molecule that encodes a split protein, or an rAAV particle containing a viral genome encoding one or more nucleic acid molecules. Such transduction may be a stable or transient transduction. In some embodiments, cells expressing a split protein or containing a split protein may be transduced or transfected with one or more guide RNA sequences, for example in delivery of a split Cas9 (e.g., nCas9) protein. In some embodiments, a plasmid expressing a split protein may be introduced into cells through electroporation, transient (e.g., lipofection) and stable genome integration (e.g., piggybac) and viral transduction or other methods known to those of skill in the art.
• In certain embodiments, the compositions provided herein comprise a lipid and/or polymer. In certain embodiments, the lipid and/or polymer is cationic. The preparation of such lipid particles is well known. See, e.g. U.S. Pat. Nos. 4,880,635; 4,906,477; 4,911,928; 4,917,951; 4,920,016; 4,921,757; and 9,737,604, each of which is incorporated herein by reference.
• The guide RNA sequence may be 15-100 nucleotides in length and comprise a sequence of at least 10, at least 15, or at least 20 contiguous nucleotides that is complementary to a target nucleotide sequence. The guide RNA may comprise a sequence of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 contiguous nucleotides that is complementary to a target nucleotide sequence. The guide RNA may be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length.
• In some embodiments, the target nucleotide sequence is a DNA sequence in a genome, e.g. a eukaryotic genome. In certain embodiments, the target nucleotide sequence is in a mammalian (e.g. a human) genome.
• The compositions of this disclosure may be administered or packaged as a unit dose, for example. The term “unit dose” when used in reference to a pharmaceutical composition of the present disclosure refers to physically discrete units suitable as unitary dosage for the subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent, i.e., a carrier or vehicle.
• Treatment of a disease or disorder includes delaying the development or progression of the disease, or reducing disease severity. Treating the disease does not necessarily require curative results.
• As used therein, “delaying” the development of a disease means to defer, hinder, slow, retard, stabilize, and/or postpone progression of the disease. This delay can be of varying lengths of time, depending on the history of the disease and/or individuals being treated. A method that “delays” or alleviates the development of a disease, or delays the onset of the disease, is a method that reduces probability of developing one or more symptoms of the disease in a given time frame and/or reduces extent of the symptoms in a given time frame, when compared to not using the method. Such comparisons are typically based on clinical studies, using a number of subjects sufficient to give a statistically significant result.
• “Development” or “progression” of a disease means initial manifestations and/or ensuing progression of the disease. Development of the disease can be detectable and assessed using standard clinical techniques as well known in the art. However, development also refers to progression that may be undetectable. For purpose of this disclosure, development or progression refers to the biological course of the symptoms. “Development” includes occurrence, recurrence, and onset.
• As used herein “onset” or “occurrence” of a disease includes initial onset and/or recurrence. Conventional methods, known to those of ordinary skill in the art of medicine, can be used to administer the isolated polypeptide or pharmaceutical composition to the subject, depending upon the type of disease to be treated or the site of the disease.
• Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present disclosure to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein.
EXAMPLES Example 1. Prime Editing (PE) for Installing Precise Nucleotide Changes in the Genome
• The objective is to develop a transformative genome editing technology for precise and general installation of single nucleotide changes in mammalian genomes. This technology would allow investigators to study the effects of single nucleotide variations in virtually any mammalian gene, and potentially enable therapeutic interventions for correcting pathogenic point mutations in human patients.
• Adoption of the clustered regularly interspaced short palindromic repeat (CRISPR) system for genome editing has revolutionized the life sciences^1-3. Although gene disruption using CRISPR is now routine, the precise installation of single nucleotide edits remains a major challenge, despite being necessary for studying or correcting a large number of disease-causative mutations. Homology directed repair (HDR) is capable of achieving such edits, but suffers from low efficiency (often <5%), a requirement for donor DNA repair templates, and deleterious effects of double-stranded DNA break (DSB) formation. Recently, the Liu laboratory developed base editing, which achieves efficient single nucleotide editing without DSBs. Base editors (BEs) combine the CRISPR system with base-modifying deaminase enzymes to convert target C●G or A●T base pairs to A●T or G●C, respectively^4-6.
• Although already widely used by researchers worldwide (>5,000 Liu lab BE constructs distributed by Addgene), current BEs enable only four of the twelve possible base pair conversions and are unable to correct small insertions or deletions. Moreover, the targeting scope of base editing is limited by the editing of non-target C or A bases adjacent to the target base (“bystander editing”) and by the requirement that a PAM sequence exist 15±2 bp from the target base. Overcoming these limitations would therefore greatly broaden the basic research and therapeutic applications of genome editing.
• Here, it is proposed to develop a new precision editing approach that offers many of the benefits of base editing—namely, avoidance of double strand breaks and donor DNA repair templates—while overcoming its major limitations. To achieve this ambitious goal, it is aimed to directly install edited DNA strands at target genomic sites using target-primed reverse transcription (TPRT). In the design discussed herein, CRISPR guide RNA (gRNA) will be engineered to carry a template encoding mutagenic DNA strand synthesis, to be executed by an associated reverse transcriptase (RT) enzyme. The CRISPR nuclease (Cas9)-nicked target site DNA will serve as the primer for reverse transcription, allowing for direct incorporation of any desired nucleotide edit.
Section 1
• Establish guide RNA-templated reverse transcription of mutagenic DNA strands. Prior studies have shown that, following DNA cleavage but prior to complex dissociation, Cas9 releases the non-target DNA strand to expose a free 3′ terminus. It is hypothesized that this DNA strand is accessible to extension by polymerase enzymes, and that gRNAs can be engineered through extension of their 5′ or 3′ terminus to serve as templates for DNA synthesis. In preliminary in vitro studies, it was established that nicked DNA strands within Cas9:gRNA-bound complexes can indeed prime reverse transcription using the bound gRNA as a template (RT enzyme in trans). Next, different gRNA linkers, primer binding sites, and synthesis templates will be explored to determine optimal design rules in vitro. Then, different RT enzymes, acting in trans or as fusions to Cas9, will be evaluated in vitro. Finally, engineered gRNA designs will be identified that retain efficient binding and cutting activity in cells. Successful demonstration of this aim will provide a foundation for carrying out mutagenic strand synthesis in cells.
Section 2
• Establish prime editing in human cells. Based on DNA processing and repair mechanisms, it is hypothesized that mutagenic DNA strands (single stranded flaps) can be used to direct specific and efficient editing of target nucleotides. In encouraging preliminary studies, feasibility for this strategy was established by demonstrating editing with model plasmid substrates containing mutagenic flaps. Concurrent with Aim 1, repair outcomes will be further evaluated by systematically varying the mutagenic flap's length, sequence composition, target nucleotide identity, and 3′ terminus. Small 1 to 3 nucleotide insertions and deletions will also be tested. In parallel, and building from Aim 1, Cas9-RT architectures will be evaluated, including fusion proteins and non-covalent recruitment strategies. Cas9-RT architectures and extended gRNAs will be assayed for cellular editing at multiple target sites in the human genome, and will then be optimized for high efficiency. If successful, this aim would immediately establish TPRT genome editing (i.e., prime editing) for basic science applications.
Section 3
• Achieve site-specific editing of pathogenic mutations in cultured human cells. The potential generality of this technology could enable editing of transversion mutations and indels that are not currently correctable by BEs. Guided by the results of Aim 1 and Aim 2, pathogenic transversion mutations will be targeted in cultured human cells, including the sickle cell disease founder mutation in beta globin (requires an A●T to T●A transversion to correct) and the most prevalent Wilson's disease mutation in ATP7B (requires a G●C to T●A transversion to correct). The correction of small insertion and deletion mutations will also be examined, including the 3-nucleotide ΔF508 deletion in CFTR that causes cystic fibrosis. If successful, this would lay the foundation for developing powerful therapeutic approaches that address these important human diseases.
Approach
• The objective is to develop a genome editing strategy that directly installs point mutations at targeted genomic sites. In the technology development phase, efforts will focus on protein and RNA engineering to incorporate TPRT functionality into the CRISPR/Cas system. In vitro assays will be used to carefully probe the function of each step of TPRT, building from the ground up (Aim 1). The second focus area will evaluate editing outcomes in mammalian cells using a combination of model substrates and engineered CRISPR/Cas systems (Aim 2). Finally, the application phase will use the technology to correct mutations that have been intractable to genome editing by other methods (Aim 3).
• The general editing design is shown in FIGS. 1A-1B. Cas9 nickases contain inactivating mutations to the HNH nuclease domain (Spy Cas9 H840A or N863A), restricting DNA cleavage to the PAM containing strand (non-target strand). Guide RNAs (gRNAs) are engineered to contain a template for reverse transcription (designs detailed on slide 5). Shown is a 5′ extension of the gRNA, but 3′ extensions can also be implemented. The Cas9 nickase is fused to a reverse transcriptase (RT) enzyme, either through the C-terminus or N-terminus. The gRNA:Cas9-RT complex targets the DNA region of interest and forms an R loop after displacing the non-target strand. Cas9 nicks the non-target DNA strand. Release of the nicked strand exposes a free 3′-OH terminus that is competent to prime reverse transcription using the extended gRNA as a template. This DNA synthesis reaction is carried out by the fused RT enzyme. The gRNA template encodes a DNA sequence that is homologous to the original DNA duplex, with the exception of the nucleotide that is targeted for editing. The product of reverse transcription is a single stranded DNA flap that encodes the desired edit. This flap, which contains a free 3′ terminus, can equilibrate with the adjacent DNA strand, resulting in a 5′ flap species. The latter species is hypothesized to serve as an efficient substrate for FEN1 (flap endonuclease 1), an enzyme that naturally excises 5′ flaps from Okazaki fragments during lagging strand DNA synthesis, and removes 5′ flaps following strand displacement synthesis that occurs during long-patch base excision repair. Ligation of the nicked DNA produces a mismatched base pair. This intermediate could either undergo reversion to the original base pair or conversion to the desired edited base pair via mismatch repair (MMR) processes. Alternatively, semiconservative DNA replication could give rise to one copy each of the reversion and edit.
1. Establish Guide RNA-Templated Reverse Transcription of Mutagenic DNA Strands.
• Background and Rationale
• In the proposed genome editing strategy, the Cas9-nicked non-target DNA strand (PAM-containing strand that forms the R-loop) acts as the primer for DNA synthesis. It is hypothesized that this is possible based on several pieces of biochemical and structural data. Nuclease protection experiments³², crystallographic studies³³, and base editing windows^4,24 have demonstrated a large degree of flexibility and disorder for the non-target strand nucleotides −20 through −10 within the so-called R-loop of the Cas9-bound complex (numbering indicates distance 5′ from first PAM nucleotide). Moreover, the PAM-distal portion of the cleaved non-target strand can be displaced from tightly bound ternary complexes when complementary ssDNA is added in trans²⁰. These studies support that the non-target strand is highly flexible, is accessible to enzymes, and that after nicking, the 3′ terminus of the PAM-distal fragment is released prior to Cas9 dissociation. Furthermore, it is hypothesized that gRNAs can be extended to template DNA synthesis. Prior studies have shown that gRNAs for SpCas9, SaCas9, and LbCas12a (formerly Cpf1) tolerate gRNA extensions with RNA aptamers³⁴, ligand-inducible self-cleaving ribozymes³⁵, and long non-coding RNAs³⁶. This literature establishes precedent for two major features that will be exploited. In assessing this strategy, several CRISPR-Cas systems will be evaluated in conjunction with 5′ and 3′ extended gRNA designs using a combination of in vitro and cellular assays (FIGS. 2A-2C).
• Designs for engineered gRNAs for TRT editing are shown in FIGS. 3A-3B. DNA synthesis proceeds 5′ to 3′, and thus copies the RNA template in the 3′ to 5′ direction. The design for the 5′ extension contains a linker region, a primer binding site where the nicked DNA strand anneals, and a template for DNA synthesis by reverse transcription. The 3′ extended gRNA contains a primer binding site and a reverse transcription template. In some cases, the 3′ RNA hairpin of the gRNA core is modified to match the DNA target sequence, as in vitro experiments showed that reverse transcription extends ˜3 nucleotides into the gRNA core for the 3′ extended gRNA constructs (modification of the hairpin sequence appears well tolerated so long as compensatory changes are made that maintain the hairpin RNA structure). DNA synthesis proceed 5′ to 3′, with nucleotides added to the 3′ OH of the growing DNA strand.
• Preliminary Results
• Cas9 nicked DNA primes reverse transcription of gRNA templates. To evaluate the accessibility of the nicked non-target DNA strand, in vitro biochemical assays were performed using the Cas9 nuclease from S. pyogenes (SpCas9) and Cy5 fluorescently labeled duplex DNA substrates (51 base pairs). First, a series of gRNAs containing 5′ extensions with varying synthesis template lengths were prepared by in vitro transcription (overall design shown in FIG. 2B). Electrophoretic mobility shift assays (EMSA) with nuclease dead Cas9 (dCas9) established that 5′ extended gRNAs maintain target binding affinity (data not shown). Next, TPRT activity was tested on pre-nicked Cy5-labeled duplex DNA substrates using dCas9, 5′-extended gRNAs, and Molony-Murine Leukemia Virus (M-MLV) reverse transcriptase (Superscript III). After 1 hour of incubation at 37° C., products were evaluated by denaturing polyacrylamide gel electrophoresis (PAGE) and imaged using Cy5 fluorescence (FIG. 4A). Each 5′-extended gRNA variant led to significant product formation, with the observed DNA product sizes being consistent with the length of the extension template (FIG. 4B). Importantly, in the absence of dCas9, pre-nicked substrates were extended to the full 51-bp length of the DNA substrate, strongly suggesting that the complementary DNA strand, and not the gRNA, was used as the template for DNA synthesis when dCas9 was not present (FIG. 4C). Of note, the system was designed such that the newly synthesized DNA strand mirrors the product that would be required for target site editing (a homologous strand with a single nucleotide change). This result establishes that Cas9:gRNA binding exposes the nicked non-target strand's 3′ end, and that the non-target strand is accessible to reverse transcription.
• Next, non-nicked dsDNA substrates were evaluated using the Cas9(H840A) mutant, which nicks the non-target DNA strand. First, to test Cas9(H840A) nickase activity with 5′-extended gRNAs, in vitro cleavage assays were performed as previously described³⁷. Although nicking was impaired by comparison to the standard gRNA, appreciable cleavage products were formed (FIG. 4D). Importantly, RT products were also observed when TPRT reactions were carried out with 5′-extended gRNAs and Cas9(H840A), albeit at lower yields that are likely explained by the decreased nicking activity (FIG. 4D). This result establishes that 5′-extended gRNA:Cas9(H840A) complexes can nick DNA and template reverse transcription.
• Finally, 3′ gRNA extensions were evaluated for Cas9(H840A) nicking and TPRT. By comparison to 5′-extended gRNAs, DNA cleavage by 3′-extended gRNAs was not impaired to any detectable extent compared to the standard gRNA. Significantly, 3′-extended gRNA templates also supported efficient reverse transcription with both pre-nicked and intact duplex DNA substrates when M-MLV RT was supplied in trans (FIG. 4E). Surprisingly, only a single product was observed for 3′-extended templates, indicating that reverse transcription terminates at a specific location along the gRNA scaffold. Homopolymer tailing of the product with terminal transferase followed by Klenow extension and Sanger sequencing revealed that the full gRNA synthesis template was copied in addition to the terminal 3 nucleotides of the gRNA core. In the future, the flap terminus will be reprogrammed by modifying the terminal gRNA sequence^38,39. This result demonstrates that 3′-extended gRNAs can serve as efficient nuclease targeting guides and can template reverse transcription.
• Cas9-TPRT uses nicked DNA and gRNA in cis. Dual color experiments were used to determine if the RT reaction preferentially occurs with the gRNA in cis (bound in the same complex) (see FIG. 8 ). Two separate experiments were conducted for 5′-extended and 3′-extended gRNAs. For a given experiment, ternary complexes of dCas9, gRNA, and DNA substrate were formed in separate tubes. In one tube, the gRNA encodes a long RT product and the DNA substrate is labeled with Cy3 (red); in the other, the gRNA encodes a short RT product and the DNA substrate is labeled with Cy5 (blue). After short incubations, the complexes were mixed and then treated with RT enzyme and dNTPs. Products were separated by urea-denaturing PAGE and visualized by fluorescence in the Cy3 and Cy5 channels. Reaction products were found to preferentially form using the gRNA template that was pre-complexed with the DNA substrate, indicating that the RT reaction likely can occur in cis. This results supports that a single Cas9:gRNA complex can target a DNA site and template reverse transcription of a mutagenic DNA strand.
• Testing TPRT with Other Cas Systems
• Similar experiments to those presented in the previous sections will be carried out using other Cas systems, including Cas9 from S. aureus and Cas12a from L. bacterium (see FIGS. 2A-2C). If TRPT can also be demonstrated for these Cas variants, the potential editing scope and likelihood of overall success in cells would increase.
• Testing TPRT with RT-Cas9 Fusion Proteins
• A series of commercially available or purifiable RT enzymes will first be evaluated in trans for TPRT activity. In addition to the already tested RT from M-MLV, the RT from Avian Myeloblastosis Virus (AMV), the Geobacillus stearothermophilus Group II Intron (GsI-IIC)^41,42, and the Eubacterium rectale group II intron (Eu.re.I2)^43,44 will be evaluated. Significantly, the latter two RTs perform TPRT in their natural biological contexts. Where relevant, RNAse inactivating mutations and other potentially beneficial RT enzyme modifications will be tested. Once functional RTs are identified when supplied in trans, each will be evaluated as a fusion protein to Cas9 variants. Both N-terminus and C-terminus fusion orientations will be tested, along with various linker lengths and architectures. Kinetic time course experiments will be used to determine whether TPRT can occur using the RT enzyme in cis. If an RT-Cas9 fusion architecture can be constructed that allows for efficient TPRT chemistry, this will greatly increase the likelihood of functional editing in the context of a cell.
• Cas9 Targeting with Engineered gRNAs in Cells
• Candidate engineered gRNAs developed in the previous sub-aims will be evaluated in human cell culture experiments (HEK293) to confirm Cas9 targeting efficiency. Using established indel formation assays employing wild type SpCas9⁴⁵, engineered gRNAs will be compared side-by-side with standard gRNAs across 5 or more sites in the human genome. Genome editing efficiency will be characterized by amplicon sequencing in multiplex using the Illumina MiSeq platform housed in the laboratory. It is anticipated that results from this and the preceding sections will generate insights that inform subsequent iterations of the design-build-test cycle, where gRNAs can be optimized for both templating reverse transcription and efficient Cas9 targeting in cells.
• Results of in vitro validations are shown in FIGS. 5-7 . In vitro experiments demonstrated that the nicked non-target DNA strand is flexible and available for priming DNA synthesis, and that the gRNA extension can serve as a template for reverse transcription (see FIG. 5 ). This set of experiments used 5′-extended gRNAs (designed as shown in FIGS. 3A-3B) with varying length synthesis templates (listed to the left). Fluorescently labeled (Cy5) DNA targets were used as substrates, and were pre-nicked in this set of experiments. The Cas9 used in these experiments is catalytically dead Cas9 (dCas9), so cannot cut DNA but can still bind efficiently. Superscript III, a commercial RT derived from the Moloney-Murine Leukemia Virus (M-MLV), was supplied in trans. First, dCas9:gRNA complexes were formed from purified components. Then, the fluorescently labeled DNA substrate was added along with dNTPs and the RT enzyme. After 1 hour of incubation at 37 C, the reaction products were analyzed by denaturing urea-polyacrylamide gel electrophoresis (PAGE). The gel image shows extension of the original DNA strand to lengths that are consistent with the length of the reverse transcription template. Of note, reactions carried out in the absence of dCas9 produced DNA products of length 51 nucleotides, regardless of the gRNA used. This product corresponds to use of the complementary DNA strand as the template for DNA synthesis and not the RNA (data not shown). Thus, Cas9 binding is required for directing DNA synthesis to the RNA template. This set of in vitro experiments closely parallels those shown in FIG. 5 , except that the DNA substrate is not pre-nicked, and a Cas9 nickase (SpyCas9 H840A mutant) is used. As shown in the gel, the nickase efficiently cleaves the DNA strand when the standard gRNA is used (gRNA_0, lane 3). Multiple cleavage products are observed, consistent with prior biochemical studies of SpyCas9. The 5′ extension impairs nicking activity (lanes 4-8), but some RT product is still observed. FIG. 7 shows that 3′ extensions support DNA synthesis and do not significantly effect Cas9 nickase activity. Pre-nicked substrates (black arrow) are near-quantitatively converted to RT products when either dCas9 or Cas9 nickase is used (lanes 4 and 5). Greater than 50% conversion to the RT product (red arrow) is observed with full substrates (lane 3). To determine the length and sequence of the RT product, the product band was excised from the gel, extracted, and sequenced. This revealed that RT extended 3 nucleotides into the gRNA core's 3′ terminal hairpin. Subsequent experiments (not shown) demonstrated that these three nucleotides could be changed to match target DNA sequences, so long as complementary changes were made that maintain the hairpin RNA structure.
Potential Difficulties and Alternatives
• (1) RT does not function as a fusion: molecular crowding and/or unfavorable geometries could encumber polymerase extension by Cas9-fused RT enzymes. First, linker optimization can be tested. Circularly permutated variants of Cas9, which could re-orient the spatial relationship between the DNA primer, gRNA, and RT enzyme, will be evaluated. Non-covalent RT recruitment strategies as detailed in Aim 2 can be tested. (2) Decreased Cas targeting efficiency by extended gRNA variants: this is most likely to be an issue for 5′-extended gRNAs. Based on structural data²⁴, Cas9 mutants can be designed and screened to identify variants with greater tolerance to gRNA extension. In addition, gRNA libraries could be screened in cells for linkers that improve targeting activity.
Significance
• These preliminary results establish that Cas9 nickases and extended gRNAs can initiate target-primed reverse transcription on bound DNA targets using a reverse transcriptase supplied in trans. Importantly, Cas9 binding was found to be critically important for product formation. Though perhaps not an absolute requirement for genome editing in cells, further development of the system that incorporates RT enzyme function in cis would significantly increase the likelihood of success in cell-based applications. Achievement of the remaining aspects of this Aim would provide a molecular foundation for carrying out precision genome editing in the context of the human genome.
2. Establish Prime Editing in Human Cells.
• Background and Rationale
• In the proposed strategy, an engineered RT-Cas9:gRNA complex will introduce mutagenic 3′ DNA flaps at genomic target sites. It is hypothesized that mutagenic 3′ flaps containing a single mismatch will be incorporated by the DNA repair machinery through energetically accessible equilibration with adjacent 5′ flaps, which would be preferentially removed (FIGS. 1C-1D). The DNA replication and repair machineries encounter 5′ ssDNA flaps when processing Okazaki fragments⁴⁶ and during long-patch base excision repair (LP-BER)⁴⁷. 5′ flaps are the preferred substrates for the widely expressed flap endonuclease FEN1, which is recruited to DNA repair sites by the homotrimeric sliding clamp complex PCNA⁴⁸. PCNA also serves as a scaffold for simultaneous recruitment of other repair factors including the DNA ligase Lig1⁴⁹. Acting as a ‘toolbelt’, PCNA accelerates serial flap cleavage and ligation, which is essential to processing the millions of Okazaki fragments generated during every cell division^50,51. Based on resemblance to these natural DNA intermediates, it is hypothesized that mutagenic strands would be incorporated through equilibration with 5′ flaps, followed by coordinated 5′ flap excision and ligation. Mismatch repair (MMR) should then occur on either strand with equal probability, leading to editing or reversion (FIGS. 1C-1D). Alternatively, DNA replication could occur first and lead directly to the incorporation of the edit in the newly synthesized daughter strand. While the highest expected yield from this process is 50%, multiple substrate editing attempts could drive the reaction toward completion due to the irreversibility of editing repair.
• Preliminary Result
• DNA flaps induce site-specific mutagenesis in plasmid model substrates in yeast and HEK cells. To test the proposed editing strategy, studies were initiated with model plasmid substrates containing mutagenic 3′ flaps that resemble the product of TPRT. A dual fluorescent protein reporter was created that encodes a stop codon between GFP and mCherry. Mutagenic flaps encode a correction to the stop codon (FIG. 9A), enabling mCherry synthesis. Thus, mutagenesis efficiency can be quantified by GFP:mCherry ratios. Plasmid substrates were prepared in vitro and introduced into yeast (S. cerevisiae) or human cells (HEK293). High frequency mutagenesis was observed in both systems (FIG. 9B), and isolated yeast colonies contained either the reverted base, the mutated base, or a mixture of both products (FIG. 9C). Detection of the latter suggests that plasmid replication occurred prior to MMR in these cases, and further suggests that flap excision and ligation precede MMR. This result establishes the feasibility of DNA editing using 3′ mutagenic strands.
• Systematic Studies with Model Flap Substrates
• Based on the preliminary results described above, a broader spectrum of flap substrates will be evaluated in HEK cells to infer principles of efficient editing. 3′ ssDNA flaps will be systematically varied to determine the influence of mismatch pairings, the location of the mutagenic nucleotide along the flap, and the identity of the terminal nucleotide (FIG. 9D). Single nucleotide insertions and deletions will also be tested. Amplicon sequencing will be used to analyze editing precision. These results will help inform the design of gRNA reverse transcription templates.
• In vitro TPRT on plasmid substrates leads to efficient editing outcomes. TPRT reactions developed in Aim 1 were used to induce mutagenesis within a plasmid substrate. The reaction was carried out on circular DNA plasmid substrates (see FIG. 10 ). This rules out the possibility of DNA strand dissociation as the mechanism for RT extension in the previous in vitro experiments. It also allowed for the testing of DNA repair of flap substrates in cells. A dual-fluorescent reporter plasmid was constructed for yeast (S. cerevisiae) expression. This plasmid encodes GFP (green fluorescent protein) and mCherry (red fluorescent protein) with an intervening stop codon (TGA). Expression of this construct in yeast produces only GFP. The plasmid was used as a substrate for in vitro TRT [Cas9(H840A) nickase, engineered gRNA, MLV RT enzyme, dNTPS]. The gRNA extension encodes a mutation to the stop codon. The flap strand is used for repair of the stop codon and it is anticipated to produce a plasmid that expresses both GFP and mCherry as a fusion protein. Yeast dual-FP plasmid transformants are shown in FIG. 10 . Transforming the parent plasmid or an in vitro Cas9(H840A) nicked plasmid results in only green GFP expressing colonies. TRT reaction with 5′-extended or 3′-extended gRNAs produces a mix of green and yellow colonies. The latter express both GFP and mCherry. More yellow colonies are observed with the 3′-extended gRNA. A positive control that contains no stop codon is shown as well.
• This result establishes that long double stranded substrates can undergo TPRT, and that TPRT products induce editing in eukaryotic cells.
• Another experiment similar to the foregoing prime editing experiment was carried out, but instead of installing a point mutation in the stop codon, TRT editing installs a single nucleotide insertion (left) or deletion (right) that repairs a frameshift mutation and allows for synthesis of downstream mCherry (see FIG. 11 ). Both experiments used 3′ extended gRNAs. Individual colonies from the TRT transformations were selected and analyzed by Sanger sequencing (see FIG. 12 ). Green colonies contained plasmids with the original DNA sequence, while yellow colonies contained the precise mutation designed by the TRT editing gRNA. No other point mutations or indels were observed.
Establish Prime Editing in HEK Cells Using RT-Cas9 Architectures
• The optimized constructs from previous aims will be adapted for mammalian expression and editing at targeted sites in the human genome. Multiple RT enzymes and fusion architectures will be tested, in addition to adjacent targeting with secondary gRNAs (truncated to prevent nicking). Non-covalent RT recruitment will also be evaluated using the Sun-Tag system⁵² and MS2 aptamer system⁵³. Indel formation assays will be used to evaluate targeting efficiency with standard gRNAs and RT-Cas9 fusions (as above). Then, for each genomic site, extended gRNAs and RT-Cas9 pairs will be assayed for single nucleotide editing. Editing outcomes will be evaluated with MiSeq.
• Initial experiments in HEK cells were performed using Cas9-RT fusions. Editing by components expressed within cells requires a Cas9(H840A) nickase, a reverse transcriptase (expressed as a fusion or supplied in trans), and an engineered gRNA with a 3′ extension (see FIG. 14 ). Preliminary studies indicated that the length of the primer binding site within the gRNA extension was important for increasing the efficiency of editing in human cells (see FIG. 15 ).
Optimize Prime Editing Parameters in HEK Cells
• After identifying Cas9-RT architectures that can perform prime editing in cells, the components and design will be optimized to achieve high efficiency editing. The location and nucleotide identity of the encoded point mutation, and the total length of the newly synthesized DNA strand, will be varied to evaluate editing scope and potential limitations. Short insertion and deletion mutations will also be evaluated. Protein expression constructs will be codon optimized. If successful, this would establish efficient prime editing in mammalian cells.
• Preliminary Result. Additional gRNAs were designed to bring the RT enzyme to a higher local concentration at the editing locus, in the event that intramolecular reverse transcription by the fused RT enzyme were not possible. These auxiliary guides are truncated at the 5′ end (14-15 nt spacer), which has previously been shown to prevent Cas9 cutting but retain binding (see FIG. 16 ). The HEK3 locus was chosen to explore this strategy.
Potential Difficulties and Alternatives
• 1) gRNA degradation in cells: if extended gRNA termini are truncated in cells, stabilizing secondary structures could be installed, or synthetic gRNAs with stabilizing modifications could be tested. (2) No observed editing in human cells: additional strategies will be explored, including secondary targeting of RT-Cas9 fusions to adjacent genomic sites⁵⁴. In addition, potential directed evolution strategies in E. coli or S. cerevisiae could be explored.
• Significance
• If prime editing could be established in experimental cell lines, this would have an immediate impact for basic biomedical research by enabling the rapid generation and characterization of a large number of point mutations in human genes. The generality of the method, and its orthogonal editing window with respect to base editors, would provide an approach to installing many currently inaccessible mutations. Moreover, if prime editing could be optimized for high efficiency and product purity, its potential applicability to correcting disease mutations in other human cell types would be significant.
3. Achieve Site-Specific Editing of Pathogenic Mutations in Cultured Human Cells.
• Background and Rationale.
• A large number of pathogenic mutations cannot be corrected by current base editors due to PAM restrictions, or a need for transversion or indel mutation correction. With prime editing, all transitions and transversions are theoretically possible, as may be small insertions and deletions. Moreover, in relation to the PAM, the prime editing window (anticipated −3 to +4) is distinct from that of base editors (−18 to −12) (FIG. 13 ). Mendelian conditions not currently correctable by base editors include: (1) the sickle cell disease Glu6Val founder mutation in hemoglobin beta (requires A●T to T●A transversion); (2) the most common Wilson's disease variant His1069Gln in ATP7B (requires G●C to T●A transversion); and (3) the APhe508 mutation in CFTR that causes cystic fibrosis (requires 3-nucleotide insertion). Each of these targets contains an appropriately positioned PAM for SpCas9 targeting and prime editing.
• Preliminary Results.
• T to A Editing in HEK3 Cells is not Achievable by Current Base Editing but is Achievable by TRPT Editing (See FIGS. 17A-17C).
• FIG. 17A shows a graph displaying the % T to A conversion at the target nucleotide after transfection of components in human embryonic kidney (HEK) cells. This data presents results using an N-terminal fusion of wild type MLV reverse transcriptase to Cas9(H840A) nickase (32-amino acid linker). Editing efficiency was improved dramatically when the length of the primer binding site is extended from 7 nucleotides to 11 or 12 nucleotides. Additionally, the auxiliary guide A, which is positioned just upstream of the editing locus (see FIG. 16 ), significantly improves editing activity, particularly for shorter length primer binding sites. Editing efficiency was quantified by amplicon sequencing using the Illumina MiSeq platform. FIG. 17B also shows % T to A conversion at the target nucleotide after transfection of components in human embryonic kidney (HEK) cells, but this data presents results using a C-terminal fusion of the RT enzyme. Here, the auxiliary guide A does not have as much of an effect, and editing efficiency is overall higher. FIG. 17C shows data presenting results using an N-terminal fusion of wild type MLV reverse transcriptase to Cas9(H840A) nickase similar to that used in FIG. 17A; however the linker between the MLV RT and Cas9 is 60 amino acids long instead of 32 amino acids.
• T to A Editing at HEK3 Site by TRPT Editing Results Displays High Purity.
• FIG. 18 shows the output of sequencing analysis by high-throughput amplicon sequencing. The output displays the most abundant genotypes of edited cells. Of note, no major indel products are obtained, and the desired point mutation (T to A) is cleanly installed without bystander edits. The first sequence shows the reference genotype. The top two products are the starting genotype containing an endogenous polymorphism (G or A). The bottom two products represent the correctly edited genotypes.
• MLV RT Mutants Improve Editing.
• Mutant reverse transcriptases, described in Baranauskas, et al (doi:10.1093/protein/gzs034), were tested as C-terminal fusions to the Cas9(H840A) nickase for target nucleotide editing in human embryonic kidney (HEK) cells. Cas9-RT editor plasmid was co-transfected with a plasmid encoding a 3′-prime editing guide RNA that templates reverse transcription. Editing efficiency at the target nucleotide (blue bars) is shown alongside indel rates (orange bars) in FIG. 19 . WT refers to the wild type MLV RT enzyme. The mutant enzymes (M1 through M4) contain the mutations listed to the right. Editing rates were quantified by high throughput sequencing of genomic DNA amplicons.
• Complementary Strand Nicking with a Second gRNA Improves Editing.
• This experiment evaluates editing efficiency of the target nucleotide when a single strand nick is introduced in the complementary DNA strand in proximity to the target nucleotide, with the hypothesis being that this would direct mismatch repair to preferentially remove the original nucleotide and convert the base pair to the desired edit. The Cas9(H840A)-RT editing construct was co-transfected with two guide RNA encoding plasmids, one of which templates the reverse transcription reaction, while the other targets the complementary DNA strand for nicking. Nicking at various distances from the target nucleotide was tested (orange triangles) (see FIG. 20 ). Editing efficiency at the target base pair (blue bars) is shown alongside the indel formation rate (orange bars). The “none” example does not contain a complementary strand nicking guide RNA. Editing rates were quantified by high throughput sequencing of genomic DNA amplicons.
• FIG. 21 shows processed high throughput sequencing data showing the desired T to A transversion mutation and general absence of other major genome editing byproducts.
• Scope. The potential scope for the new editing technology is shown in FIG. 13 and is compared to deaminase-mediated base editor technologies. Previously developed base editors target a region ˜15±2 bp upstream of the PAM. By converting target C or A nucleotides to T or G, respectively, previously developed base editors enable all transition mutations (A:T to G:C conversions). However, previously developed base editors are unable to install transversion mutations (A to T, A to C, G to T, G to C, T to A, T to G, C to A, C to G). Moreover, if there are multiple target nucleotides in the editing window, additional undesired edits can result.
• The new prime editing technology could theoretically install any nucleotide and base pair conversion, and potentially small insertion and deletion edits as well. With respect to the PAM, prime editing windows start at the site of DNA nicking (3 bases upstream of the PAM) and end at an as-of-yet undetermined position downstream of the PAM. Of note, this editing window is distinct from that of deaminase base editors. Because the TPRT systems performs editing using DNA polymerase enzymes, it potentially has all of their benefits including generality, precision, and fidelity.
Correct Pathogenic Mutations in Patient-Derived Cell Lines.
• Cell lines harboring the relevant mutations (sickle cell disease: CD34+ hematopoietic stem cells; Wilson's disease: cultured fibroblasts; cystic fibrosis: cultured bronchial epithelia) will be obtained from ATCC, the Coriell Biobank, or collaborating Harvard/Broad affiliate laboratories. Editing efficiency will be evaluated by high throughput sequencing, and the efficacy of the corrected genotype will be tested using phenotypic assays (hemoglobin HPLC, ATP7B immunostaining, and CFTR membrane potential assays).
Characterize Off-Target Editing Activity.
• Potential off-target editing will be screened with established methods such as GUIDE-seq⁵⁵ and CIRCLE-seq⁵⁶ using target gRNAs paired with wild type Cas9. If potential off-targets are identified, these loci will be probed in TPRT edited cells to identify true off-target editing events.
Potential Difficulties and Alternatives.
• (1) Low editing efficiency: prime editor (PE)s may require optimization for each target. In this case, gRNA libraries can be tested to identify the highest functioning variants for specific applications. RT-Cas fusion expression and nuclear localization can be optimized. Liposomal RNP delivery could be used to limit off-target editing.
Upcoming Experiments.
• Optimization of gRNA designs can be achieved by further exploration of the primer binding site length and extension of synthesis template. Testing scope and generality will include different nucleotide conversions, small insertions and deletions, as well as, different editing positions with respect to PAM, and multiple sites in the human genome. Optimization of RT component will include exploring mutations in MLV RT to enhance activity (Rnase H inactivation, increase primer-template binding affinity, adjustments to processivity), and new RT enzymes (group II intro RTs, other retroviral RTs).
• Significance.
• Myriad genetic disorders result from single nucleotide changes in individual genes. Developing the genome editing technology described here, and applying it in disease-relevant cell types, would establish a foundation for translation to the clinic. For some diseases, such as Sickle Cell Disease, a single point mutation represents the dominant genotype throughout the population. However, for many other genetic disorders, a large heterogeneity of different point mutations within a single gene is observed throughout the patient population, each of which gives rise to a similar disease phenotype. Therefore, as a general genome editing method that could in theory target a large number of such mutations, this technology could provide enormous potential benefit to many of these patients and their families. If proof of principle for these applications could be established in cells, it would establish the foundation to studies in animal models of disease.
• Advantages
• Precision: the desired edit is encoded directed in nucleic acid sequence. Generality: in theory, could be possible to make any base pair conversion, including transversion edits, as well as small insertions or deletions. There is a distinct editing window from that of base editors with respect to Cas9 protospacer adjacent motif (PAM) sequence. This method achieves many of the editing capabilities of homology-directed repair (HDR), but without the major limitations of HDR (inefficient in most cell types, and is usually accompanied by an excess of undesired byproducts such as indels). Also, it does not make double-stranded DNA breaks (DSBs, so few indels, translocations, large deletions, p53 activation, etc.
Example 2—Error-Prone Prime Editing (PE)
• Prime editing (PE) systems described herein may also be used in conjunction with an error-prone reverse transcriptase enzyme to install mutations in a genome.
• An embodiment is depicted in FIG. 22 , which is a schematic of an exemplary process for conducting targeted mutagenesis with an error-prone reverse transcriptase on a target locus using a nucleic acid programmable DNA binding protein (napDNAbp) complexed with an extended guide RNA. This process may be referred to as an embodiment of prime editing for targeted mutagenesis. The extended guide RNA comprises an extension at the 3′ or 5′ end of the guide RNA, or at an intramolecular location in the guide RNA. In step (a), the napDNAbp/gRNA complex contacts the DNA molecule and the gRNA guides the napDNAbp to bind to the target locus to be mutagenized. In step (b), a nick in one of the strands of DNA of the target locus is introduced (e.g., by a nuclease or chemical agent), thereby creating an available 3′ end in one of the strands of the target locus. In certain embodiments, the nick is created in the strand of DNA that corresponds to the R-loop strand, i.e., the strand that is not hybridized to the guide RNA sequence. In step (c), the 3′ end DNA strand interacts with the extended portion of the guide RNA in order to prime reverse transcription. In certain embodiments, the 3′ ended DNA strand hybridizes to a specific RT priming sequence on the extended portion of the guide RNA. In step (d), an error-prone reverse transcriptase is introduced which synthesizes a mutagenized single strand of DNA from the 3′ end of the primed site towards the 3′ end of the guide RNA. Exemplary mutations are indicated with an asterisk “*”. This forms a single-strand DNA flap comprising the desired mutagenized region. In step (e), the napDNAbp and guide RNA are released. Steps (f) and (g) relate to the resolution of the single strand DNA flap (comprising the mutagenized region) such that the desired mutagenized region becomes incorporated into the target locus. This process can be driven towards the desired product formation by removing the corresponding 5′ endogenous DNA flap that forms once the 3′ single strand DNA flap invades and hybridizes to the complementary sequence on the other strand. The process can also be driven towards product formation with second strand nicking, as exemplified in FIG. 1F. Following endogenous DNA repair and/or replication processes, the mutagenized region becomes incorporated into both strands of DNA of the DNA locus.
Example 3—Trinucleotide Repeat Contraction with PE
• The prime editing system or prime editing (PE) system described herein may be used to contract trinucleotide repeat mutations (or “triplet expansion diseases”) to treating conditions such as Huntington's disease and other trinucleotide repeat disorders. Without wishing to be bound by theory, triplet expansion is caused by slippage during DNA replication or during DNA repair synthesis. Because the tandem repeats have identical sequence to one another, base pairing between two DNA strands can take place at multiple points along the sequence. This may lead to the formation of “loop out” structures during DNA replication or DNA repair synthesis. This may lead to repeated copying of the repeated sequence, expanding the number of repeats. Additional mechanisms involving hybrid RNA:DNA intermediates have been proposed. Prime editing may be used to reduce or eliminate these triplet expansion regions by deletion one or more or the offending repeat codon triplets. In an embodiment of this use, FIG. 23 , provides a schematic of a PEgRNA design for contracting or reducing trinucleotide repeat sequences with prime editing.
• Thus, prime editing may be able to be used to correct any trinucleotide repeat disorder, including, Huntington's disease, Fragile X syndrome, and Friedreich's ataxia.
• The most common trinucleotide repeat contains CAG triplets, though GAA triplets (Friedreich's ataxia) and CGG triplets (Fragile X syndrome) also occur. The CAG triplets code for glutamine (Q), thus, CAG repeats result in polyglutamine tracts in the coding regions of diseased proteins. This particular class of trinucleotide repeat disorders are also called “polyglutamine (PolyQ) diseases.” Other trinucleotide repeats can cause alterations in gene regulation and are referred to as “non-polyglutamine diseases.” Inheriting a predisposition to expansion, or acquiring an already expanded parental allele, increases the likelihood of acquiring the disease. Pathogenic expansions of trinucleotide repeats could be corrected using prime editing.
• Prime editing may be implemented to contract triplet expansion regions by nicking a region upstream of the triplet repeat region with the prime editor comprising a PEgRNA appropriated targeted to the cut site. The prime editor then synthesizes a new DNA strand (ssDNA flap) based on the PEgRNA as a template (i.e., the edit template thereof) that codes for a healthy number of triplet repeats (which depends on the particular gene and disease). The newly synthesized ssDNA strand comprising the healthy triplet repeat sequence also is synthesized to include a short stretch of homology (i.e., the homology arm) that matches the sequence adjacent to the other end of the repeat (red strand). Invasion of the newly synthesized strand, and subsequent replacement of the endogenous DNA with the newly synthesized ssDNA flap, leads to a contracted repeat allele.
Example 4—Peptide Tagging with PE
• The prime editing systems (i.e., PE systems) described herein may also be used to introduce various peptide tags into protein coding genes. Such tags can include HEXAhistidine tags, FLAG-tag, V5-tag, GCN4-tag, HA-tag, Myc-tag and others. This approach may be useful in applications such as protein fluorescent labeling, immunoprecipitation, immunoblotting, immunohistochemistry, protein recruitment, inducible protein degrons, and genome-wide screening. Embodiments are depicted in FIGS. 25 and 26 .
• FIG. 25 is a schematic showing gRNA design for peptide tagging genes at endogenous genomic loci and peptide tagging with TPRT genome editing (i.e., prime editing). The FlAsH and ReAsH tagging systems comprise two parts: (1) a fluorophore-biarsenical probe, and (2) a genetically encoded peptide containing a tetracysteine motif, exemplified by the sequence FLNCCPGCCMEP (SEQ ID NO: 1). When expressed within cells, proteins containing the tetracysteine motif can be fluorescently labeled with fluorophore-arsenic probes (see ref: J. Am. Chem. Soc., 2002, 124 (21), pp 6063-6076. DOI: 10.1021/ja017687n). The “sortagging” system employs bacterial sortase enzymes that covalently conjugate labeled peptide probes to proteins containing suitable peptide substrates (see ref: Nat. Chem. Biol. 2007 November; 3(11):707-8. DOI: 10.1038/nchembio.2007.31). The FLAG-tag (DYKDDDDK (SEQ ID NO: 2)), V5-tag (GKPIPNPLLGLDST (SEQ ID NO: 3)), GCN4-tag (EELLSKNYHLENEVARLKK (SEQ ID NO: 4)), HA-tag (YPYDVPDYA (SEQ ID NO: 5)), and Myc-tag (EQKLISEEDL (SEQ ID NO: 6)) are commonly employed as epitope tags for immunoassays. The pi-clamp encodes a peptide sequence (FCPF) (SEQ ID NO: 622) that can by labeled with a pentafluoro-aromatic substrates (ref: Nat. Chem. 2016 February; 8(2):120-8. doi: 10.1038/nchem.2413).
• FIG. 26 shows precise installation of a His6-tag and a FLAG-tag into genomic DNA. A guide RNA targeting the HEK3 locus was designed with a reverse transcription template that encodes either an 18-nt His-tag insertion or a 24-nt FLAG-tag insertion. Editing efficiency in transfected HEK cells was assessed using amplicon sequencing. Note that the full 24-nt sequence of the FLAG-tag is outside of the viewing frame (sequencing confirmed full and precise insertion).
Example 5—Prevention or Treatment of Prion Disease with PE
• This invention could help address the problem of prion disease in humans, livestock, and wildlife. No previously described editing strategy is efficient and clean enough to install protective mutations or to reliably knock down PRNP. Cas9 nuclease and HDR can be used but will generate mostly a mixture of PRNP indel variants some of which are thought to be pathogenic. Moreover, HDR does not work in most types of cells. Prime editing is reliable and efficient at installing both types of mutations without generating an excess of double-stranded DNA breaks or resulting indels.
• This invention describes how to install a protective mutation in PRNP that prevents or halts the progression of prion disease. This site is conserved in mammals, so in addition to treating human disease it could also be used to generate cows and sheep that are immune to prion disease, or even help cure wild populations of animals that are suffering from prion disease. Prime editing has already been used to achieve ˜25% installation of a naturally occurring protective allele in human cells, and previous mouse experiments indicate that this level of installation is sufficient to cause immunity from most prion diseases. This method is the first and potentially only current way to install this allele with such high efficiency in most cell types. Another possible strategy for treatment is to use prime editing to reduce or eliminate the expression of PRNP by installing an early stop codon in the gene. Many researchers predict that doing so would treat the disease.
• Three potential therapeutic strategies include prime editing to reduce expression of PrP. This goal may be accomplished by the introduction of mutations that cause a premature stop codon in PRNP, eliminate the start codon, mutate or delete essential amino acids codons, introduce or remove splice sites to generate an aberrant transcript, or alter regulatory elements that reduce transcript levels. Prime editing to eliminate disease mutations. Many variants of PRNP have been described (ncbi.nlm.nih.gov/pmc/articles/PMC6097508/#b154-ndt-14-2067) that lead to an increased likelihood of contracting disease. Each known variant could be reversed using prime editing, since prime editing can make all possible types of point mutations, local insertions, and local deletions. Prime editing to introduce one or more protective mutations into PRNP that disrupt prion formation and/or transmission. For example, G127V in the human PRNP gene has been demonstrated to protect against many forms of prion disease (ncbi.nlm.nih.gov/pmc/articles/PMC4486072/). This mutation was later described to interfere with prion formation by preventing formation of stable beta sheets and dimers (ncbi.nlm.nih.gov/pubmed/30181558, ncbi.nlm.nih.gov/pubmed/26906032). In addition to the introduction of single nucleotide polymorphisms, the insertion or deletion of sequences in PRNP that would interfere with prion formation could also be used to protect from or treat prion disease.
• The third therapeutic strategy is particularly advantageous because the introduction of protective variants could confer a benefit even when a relatively small number of cells experience the edit. Furthermore, the introduction of protective variants, especially those naturally occurring in human populations such as G127V, would not be expected to have any detrimental consequences, while reducing expression of prion protein as in strategy 1 could have some detrimental phenotypes, as have been documented in PRNP knock-out mice (ncbi.nlm.nih.gov/pmc/articles/PMC4601510/, ncbi.nlm.nih.gov/pmc/articles/PMC2634447/).
• It has previously been demonstrated that mice expressing a ratio of approximately 2:1 of the wild type human prion protein: the protective G127V variant of the human prion protein (approximately 33% expression of the protective variant) were entirely immune to most tested forms of prion disease and were also resistant to variant Creutzfeldt-Jakob disease (vCJD), the human disease transmitted from bovine spongiform encephalopathy (BSE, or mad cow disease)⁹¹. Mice that only expressed the protective G127V variant were entirely immune to all tested prion disease challenges, including vCJD.
• It is demonstrated herein that the protective G127V mutation can be efficiently installed in human cells in tissue culture using prime editing (see FIG. 27 ).
• Informed by these results, three settings are described in which PRNP editing could be used. One setting PRNP editing can be used is prime editing in human patients to prevent or treat prion disease. A second setting PRNP editing can be used is prime editing in livestock to prevent the occurrence and spread of prion disease. Both cow and sheep livestock have experienced sporadic occurrence of prion disease caused by the protein generated by the PRNP gene. In addition to the debilitating and deadly disease suffered by the animal, these cases are also economically devastating, in part due to the care that must be taken to prevent the spread of the extremely infectious disease. A single dairy cow in the state of Washington tested positive for BSE in December of 2003, which led to a projected loss of 2.8-4.2 billion dollars in beef sales the following year bookstore.ksre.ksu.edu/pubs/MF2678.pdf). The PRNP gene is highly conserved in mammals. Introducing a PRNP mutation such as G127V into the livestock germline could eliminate the occurrence of BSE or scrapie, the manifestation of prion disease in sheep. A third setting PRNP editing can be used is prime editing in wildlife could prevent the spread of wild prion disease. Currently, cervid populations including deer, elk, and moose in North America are suffering from chronic wasting disease (CWD), a manifestation of prion disease caused by PNRP in these species. The occurrence has been reported to be as high as 25% in some populations (cdc.gov/prions/cwd/occurrence.html). CWD has also been reported in Norway, Finland, and South Korea. It is not yet known whether the disease is transmissible from these species to humans (cdc.gov/prions/cwd/transmission.html) or livestock. The introduction of PRNP mutations such as G127V in the germline of these species could protect them from CWD and reduce the risk of transmission to other species including humans.
• This method could be used to treat Creutzfeldt-Jakob Disease (CJD), kuru, Gerstmann-Sträussler-Scheinker disease, fatal familial insomnia (FFI), bovine spongiform encephalopathy (BSE; mad cow disease), scrapie (in sheep), and chronic wasting disease (CWD; in deer, elk, and moose).
• The method would need to be combined with a delivery methodology for embryos or adult neurons, such as microinjection, lipid nanoparticles, or AAV vectors.
Example 6—RNA Tagging and Manipulation Using PE
• A new method for the insertion of motifs into genetic sequences that tag or otherwise manipulate RNA within mammalian, eukaryotic, and bacterial cells is described herein. While it is estimated that only 1% of the human genome encodes proteins, virtually all of the genome is transcribed at some level. It is an open question how much of the resulting non-coding RNA (ncRNA) plays a functional role, let alone what the roles of most of these putative RNAs are. “Tagging” of these RNA molecules via the insertion of a novel RNA-encoding sequence with a useful property into genes of interest is a useful method for studying the biological functions of RNA molecules in cells. It can also be useful install tags onto protein-encoding mRNAs as a means to perturb and thus better understand how mRNA modifications can affect cellular function. For instance, a ubiquitous natural RNA tag—polyadenylation—is used by cells to affect transport of mRNA into the cytoplasm. Different types of polyadenylation signals result in different transport rates and different mRNA lifespans and—thus—differences in the levels to which the encoded protein is expressed.
• A common approach for expressing tagged RNAs within cells is to exogenously introduce a synthetic construct using either (i) transient plasmid transfection that produces a short-term burst of gene expression, often at supraphysiologic levels; or (ii) permanent integration of the tagged RNA gene into the genome (at random sites) using lentiviral integration or transposons, which enables prolonged expression. Both of these approaches are limited by production of altered expression levels, and by the absence of natural mechanisms that regulate the expression or activity of the gene. An alternative strategy is to directly tag a gene of interest at its endogenous locus using homology-directed repair (HDR) of double-stranded DNA breaks induced by Cas9 or other targeted DNA nucleases. While this approach enables the generation of a wide range of endogenously tagged genes, HDR is markedly inefficient and so requires significant screening to identify the desired clonal population of cells that have been successfully tagged. Moreover, HDR is typically very inefficient or entirely inactive in a large number of cell types, most notably in post-mitotic cells. The low efficiency of HDR is further complicated by the generation of undesired indel products, would could be especially problematic in the case of RNA genes as they might lead to the production of an RNA whose activities interfere with the function of normal alleles. Finally, researchers often need to screen various tagging positions within an RNA molecule to achieve optimal performance. Combined, these drawbacks make HDR a less desirable method for installation of tags in RNA.
• Prime editing is a new genome editing technology that enables targeted editing of genomic loci via the transfer of genetic information from RNA to DNA. Using prime editing, RNA genes could be tagged with a variety of components such as RNA aptamers, ribozymes, or other RNA motifs. Prime editing has the potential to be faster, cheaper, and effective in a greater variety of cell types by comparison to HDR strategies. As such, the described invention represents a novel, useful, and non-obvious tool for investigating the biology of RNA genes in health and disease. A new method for the insertion of RNA motifs into genetic sequences that tag or otherwise manipulate RNA using prime editors (PEs) is described herein. PEs are capable of site-specifically inserting, mutating, and/or deleting multiple nucleotides at a desired genomic locus that is targetable by a CRISPR/Cas system. PEs are composed of fusions between Cas9 nuclease domains and reverse transcriptase domains. They are guided to their genomic target by engineered PEgRNAs (prime editing guide RNAs), which contain a guide spacer portion for DNA targeting, as well as a template for reverse transcription that encodes the desired genome edit (see FIG. 28A). It is envisioned that PE can be used to insert motifs that are functional at the RNA level (hereafter RNA motifs) to tag or otherwise manipulate non-coding RNAs or mRNAs. These motifs could serve to increase gene expression, decrease gene expression, alter splicing, change post-transcriptional modification, affect the sub-cellular location of the RNA, enable isolation or determination of the intra- or extra-cellular location of the RNA (using, for instance, fluorescent RNA aptamers such as Spinach, Spinach2, Baby Spinach, or Broccoli), recruit endogenous or exogenous protein or RNA binders, introduce sgRNAs, or induce processing of the RNA, by either self-cleavage or RNAses (see FIG. 28B). Due to the flexibility of prime editing, it is not possible to provide a comprehensive list of RNA motifs that could be installed within the genome. A series of examples are shown here that broadly illustrate the predicted scope of PE-installed RNA motifs that could be used to tag RNA genes. It is currently not possible to efficiently and fairly cleanly make these changes in most types of cells (including the many that do not support HDR) using any other reported genome editing method besides PE.
• Gene expression could be affected by encoding a 3′ untranslated region (UTR) that results in changes in nuclear transport or retention or mRNA lifespan. For instance, the polyA tail from polyomavirus simian virus 40 (SV40) has additional helper sequences that enable efficient transcription termination and can increase gene expression relative to other 3′ UTRs^57,58. Example sequence of SV40 polyA tail:

• SV40 POLYA	AACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGC
  TAIL	AATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCA
  	CTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTA
  	TCTTA (SEQ ID NO: 331)

• Post-translational modification signals, besides polyadenylation signals, could also be encoded by PE. These include signals incorporate N6-methyladenosine, N1-methyladenosine, 5-methylcytosine, and pseudouridine modifications⁵⁹. By using PE to include sequences bound by enzymes that write or remove these modifications within an RNA transcript, it would be possible to induce their writing or erasing. This could be used as a tool to study the effects of these markers, to induce cellular differentiation, affect stress responses, or, given the function of these markers are as yet underexplored, affect targeted cells in other fashions.
• PE could encode mutations that affect subcellular localization. For instance, incorporation of tRNA-Lys within an mRNA can theoretically result in transport to the mitochondria⁶⁰, while various 3′ UTRs can result in nuclear retention or transport⁶¹.
EXAMPLES
• SV40 polyA Signal Results in Transport.

• SV40 POLYA	AACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGC
  TAIL	AATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCA
  	CTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTA
  	TCTTA (SEQ ID NO: 331)

• U1 snRNA 3′ Box Results in Retention.

• U1 SNRNA 3′	TTCATTCAGCAAGTTCAGAGAAATCTGAACTTGCTGGA
  BOX	TTTTTGGAGCAGGGAGATGGAATAGGAGCTTGCTCCGT
  	CCACTCCACGCATCGACCTGGTATTGCAGTACCTCCAG
  	GAACGGTGCACCCACTTTCTGGAGTTTCAAAAGTAGAC
  	TGTACGCTAAGGGTCATATCTTTTTTTGTTTGGTTTGT
  	GTCTTGGTTGGCGTCTTAA
  	(SEQ ID NO: 625)

• Determining the sub-cellular localization of endogenous RNA can be challenging and requires the addition of exogenous, fluorescently-tagged nucleotide probes, as in the case of FISH, or time-consuming and potentially inaccurate cell fractionation followed by RNA detection. Encoding a probe within the endogenous RNA would obviate many of these issues. One example would be to encode a fluorescent RNA aptamer, such as Spinach⁶² or Broccoli within an endogenous RNA, thereby visualizing the presence of that RNA via addition of a small molecule proto-fluorophore.
• Broccoli Aptamer:

• BROCCOLI	GAGACGGTCGGGTCCAGATATTCGTATCTGTCGAGTAGAGT
  APTAMER	GTGGGCTC (SEQ ID NO: 357)

• PE could insert or remove sequences that encode RNA that are recognized by RNA binding proteins, affecting RNA stability, expression, localization, or modification (for instance, see proteins listed⁶³).
• PE could insert sequences that encode sgRNAs within the genome, as a viral or cancer defense mechanism. Similarly, it could be used to insert microRNAs (e.g., pre-microRNAs) to direct silencing of targeted genes.
• PE could insert sequences resulting in processing of the RNA, either by itself, or by external factors, either as a therapy or tool for studying the function of various portions of the RNA. For instance, the HDV ribozyme⁶⁴, when included within an RNA sequence, results in processing of the RNA immediately 5′ of the ribozyme, while the hammerhead ribozyme cleaves prior to the third stem within the ribozyme⁶⁵. Other self-cleaving ribozymes include pistol⁶⁶, hatchet⁶⁶, hairpin⁶⁷, Neuropora Varkud satellite⁶⁸, glmS⁶⁹, twister⁷⁰, and twister sister⁶⁶. These sequences could include wild-type or engineered or evolved versions of ribozymes. The majority of these ribozymes could have different sequences depending on the region of RNA into which they were associated, depending on where the ribozyme cut site is located. Sequences that would direct the processing of the RNA by external factors, such as sequence specific RNAses⁷¹, RNAses that recognize specific structures⁷²—such as Dicer⁷³ and Drosha⁷⁴, could also be achieved.
• HDV Ribozyme:

• HDV	GGCCGGCATGGTCCCAGCCTCCTCGCTGGCGCCGGCTGGGC
  RIBOZYME	AACATGCTTCGGCATGGCGAATGGGAC
  	(SEQ ID NO: 365)

REFERENCES FOR EXAMPLE 6
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Example 7—Generation of Gene Libraries with PE
• A new method for the cellular generation of highly sophisticated libraries of protein- or RNA-coding genes with defined or variable insertions, deletions, or defined amino acid/nucleotide conversions, and their use in high-throughput screening and directed evolution is described herein. The references cited in the Example are based from the list of references included at the end of this Example.
• The generation of variable genetic libraries has most commonly been accomplished through mutagenic PCR¹. This method relies on either using reaction conditions that reduce the fidelity of DNA polymerase, or using modified DNA polymerases with higher mutation rates. As such, biases in these polymerases are reflected in the library product (e.g. a preference for transition mutations versus transversions). An inherent limitation of this approach to library construction is a relative inability to affect the size of the gene being varied. Most DNA polymerases have extremely low rates of indel mutations (insertions or deletions), and most of these will result in frameshift mutations in protein-coding regions, rendering members of the library unlikely to pass any downstream selection. Additionally, biases in PCR and cloning can make it difficult to generate single libraries consisting of genes of different sizes. These limitations can severely limit the efficacy of directed evolution to enhance existing or engineer novel protein functions. In natural evolution, large changes in protein function or efficacy are typically associated with insertion and deletion mutations that are unlikely to occur during canonical library generation for mutagenesis. Furthermore, these mutations most commonly occur in regions of the protein in question that are predicted to form loops, as opposed to the hydrophobic core. Thus, most indels generated using a traditional unbiased approach are likely to either be deleterious or ineffective.
• Libraries that could bias such mutations to the sites within the protein where they would be most likely to be beneficial, i.e. loop regions, would have a significant advantage over traditional libraries given that all libraries access only a fraction of the possible mutation space. Finally, although it is possible to generate genetic libraries with site-specific indel mutations through multistep PCR and clonal assembly using NNK primers or via DNA shuffling, these libraries cannot undergo additional rounds of ‘indelgenesis’ in continual evolution. Continuous evolution is a type of directed evolution with minimal user intervention. One such example is PACE³. Because continuous evolution occurs with minimal user intervention, any increase in library diversity during the evolution must occur using the native replication machinery. As such, although libraries of genes with inserted or removed codons as specific loci can be generated and screened in PACE, additional rounds of ‘indelgenesis’ are not possible.
• It is envisioned that the programmability of prime editing (PE) can be leveraged to generate highly sophisticated, programmed genetic libraries for use in high-throughput screening and directed evolution (see FIG. 29A). PE can insert, change or remove defined numbers of nucleotides from specified genetic loci using information encoded in a prime editing guide RNA (PEgRNA) (see FIG. 29B). This enables the generation of targeted libraries with one or more amino acids inserted or removed from the loop regions wherein mutations are most likely to give rise to changes in function, without background introduction of nonfunctional frameshift mutations (see FIG. 29C). PE can be used to install specific sets of mutations without regard for biases inherent in either DNA polymerase or the sequence being mutated.
• For instance, while converting a CCC codon to a stop codon would be an unlikely occurrence via canonical library generation because it would require three consecutive mutations, including two transversions, PE could be used to convert any given, targeted codon to a TGA stop codon in one step. They could also be used to install programmed diversity at given positions, for instance by incorporating codons encoding any hydrophobic amino acid at a given site, while not encoding any others. Furthermore, because of the programmability of PE, multiple PEgRNAs could be utilized to generate multiple different edits at multiple sites simultaneously, enabling the generation of highly programmed libraries (see FIG. 29D). Additionally, it is possible to use reverse transcriptases with lower fidelity to generate regions of mutagenesis within an otherwise invariable library (such as the HIV-I reverse transcriptase⁴ or Bordetella phage reverse transcriptase⁵).
• The possibility of iterative rounds of PE on the same site is also envisioned, allowing—for instance—the repeated insertion of codons at a single site. Finally, it is envisioned that all of the above described approaches can be incorporated into continual evolution, enabling the generation of novel in situ evolving libraries (see FIG. 30 ). They could also be used to construct these libraries within other cell types where it would otherwise be difficult to assemble large libraries, for instance within mammalian cells. Generation of PE-encoding bacterial strains that have been optimized for directed evolution would be a useful additional tool for the identification of proteins and RNAs with improved or novel functionality. All of these uses of PE are non-obvious due to the novel nature of PEs. In conclusion, library generation via PE would be a highly useful tool in synthetic biology and directed evolution, as well as for high-throughput screening of protein and RNA combinatorial mutants.
Competing Approaches
• The chief method by which diverse libraries are currently generated is by mutagenic PCR¹, described above. Insertions or deletions can be introduced via degenerated NNK primers at defined sites during PCR, although introducing such mutations at multiple sites requires multiple rounds of iterative PCR and cloning before constructing a more diverse library via mutagenic PCR, rendering the method slow. An alternative, complementary method is DNA shuffling, where fragments of a library of genes generated via DNase treatment are introduced into a PCR reaction without primers, resulting in the annealing of different fragments to each other and the rapid generation of more diverse libraries than via mutagenic PCR alone⁶. Although this approach can theoretically generate indel mutations, it more often results in frameshift mutations that destroy gene function. Furthermore, DNA shuffling requires a high degree of homology between gene fragments. Both of these methods must be done in vitro, with the resulting library transformed into cells, while libraries generated by PE can be constructed in situ, enabling their use in continual evolution. While libraries can be constructed in situ through in vivo mutagenesis, these libraries rely on the host cellular machinery and exhibit biases against indels. Similarly, although traditional cloning methods can be used to generate site-specific mutational profiles, they cannot be used in situ and are generally assembled one at a time in vitro before being transformed into cells. The efficiency and broad functionality of PE in both prokaryotic and eukaryotic cell types further suggests that these libraries could be constructed directly in the cell type of interest, as opposed to being cloned into a model organism such as E. coli and then transferred into the cell or organism of interest. Another competing approach for targeted diversification is automated multiplex genome engineering, or MAGE, wherein multiple single-stranded DNA oligonucleotides can be incorporated within replication forks and result in programmable mutations. However, MAGE requires significant modification of the host strain and can lead to a 100-fold increase in off-target or background mutations⁸, whereas PE is more highly programmed and anticipated to result in fewer off-target effects. Additionally, MAGE has not been demonstrated in a wide variety of cell types, including mammalian cells. Prime editing is a novel and non-obvious complementary technique for library generation.
Examples of PE in Directed Evolution to Construct Gene Libraries
• In one example, PE can be used in a directed evolution experiment to introduce protein variants into gene libraries during a continual evolution experiment using PACE, permitting iterative accumulation of both point mutations and indels in a manner not possible via traditional approaches. It has already been shown that PE can site-specifically and programmably insert nucleotides into a genetic sequence in E. coli. In the outlined directed evolution, it is proposed to identify monobodies with improved binding to a specific epitope via a modified two-hybrid protein:protein binding PACE selection. Specific and highly variable loops within these monobodies contribute significantly to affinity and specificity. Improved monobody binding might be obtained rapidly in PACE by varying the length and composition of these loops in a targeted fashion. However, varying sequence length is not an established functionality of PACE. While library of varied loop sizes might be used as a starting point for PACE, no subsequent improvements to length would arise throughout the PACE selection, barring access to beneficial synergistic combinations of point mutations and indel mutations. Introducing PE to the PACE selection would enable the in situ generation and evolution of monobodies with varying loop lengths. To do so, it is envisioned the introduction of an additional PE plasmid to the host E. coli strain, encoding the PE enzyme and one or more PEgRNAs. Expression of PE enzyme and PEgRNAs would be under the control of a small molecule delivered to the PACE lagoon at a rate selected by the experimenter.
• In various embodiments, the PEgRNA components would contain a spacer directing the PE to the site of interest on the selection phage and would be designed such that a multiple of three nucleotides could be inserted at the target site such that a new PEgRNA binding site would be introduced, enabling the iterative insertion of one or more codons at the targeted site.
• In parallel, another host E. coli strain might include PEgRNAs that would template the removal of one or more codons, enabling loop size to shrink during the evolution. A PACE experiment might utilize a mixture of both strains or alternate the two to permit the slow and controlled addition or removal of loop sequences.
• It is noted that this technique can also be applied to the evolution of antibodies. The binding principles governing antibodies are very similar to those governing monobodies: the length of antibody complement-determining region loops is critical to their binding function. Further, longer loop lengths have been found to be critical in the development of rare antibodies with broadly protective activity against HIV-1 and other viral infections⁹. Application of PE as described above to an antibody or antibody-derived molecule would permit the generation of antibodies with diverse loop length and varied loop sequence. In combination with PACE, such an approach would permit enhanced binding through loop geometries not accessible to standard PACE, and thus permit evolution of highly functional antibodies.
• Experiments will show the ability to use PE to correct a deleterious mutation in bacteriophage M3 in phage-assisted non-continuous evolution (PANCE), a necessary first step for using PE in continuous evolution (see FIG. 69 ).
REFERENCES FOR EXAMPLE 7
• The following references are incorporated herein by reference in their entireties.
• 1. Cadwell R C and Joyce G F. PCR Methods Appl. 1992.
• 2. McInerney P, Adams P, and Hadi M Z. Mol Biol Int. 2014.
• 3. Esvelt K M, Carlson J C, and Liu D R. Nature. 2011.
• 4. Naorem S S, Hin J, Wang S, Lee W R, Heng X, Miller J F, Guo H. Proc Natl Acad Sci USA 2017.
• 5. Martinez M A, Vartanian J P, Wain-Hobson S. Proc Natl Acad Sci USA 1994.
• 6. Meyer A J, Ellefson J W, Ellington A D. Curr Protoc Mol Biol. 2014.
• 7. Wang H H, Isaacs F J, Carr P A, Sun Z Z, Xu G, Forest C R, Church G M. Nature. 2009.
• 8. Nyerges A et al. Proc Natl Acad Sci USA. 2016.
• 9. Mascola J R, Haynes B F. Immunol Rev. 2013.
Example 8—Immunoepitope Insertion by PE
• Precise genome targeting technologies using the CRISPR/Cas system have recently been explored in a wide range of applications, including the insertion of engineered DNA sequences into targeted genomic loci. Previously, homology-directed repair (HDR) has been used for this application, requiring an ssDNA donor template and repair initiation by means of a double-stranded DNA break (DSB). This strategy offers the broadest range of possible changes to be made in cells and is the only method available to insert large DNA sequences into mammalian cells. However, HDR is hampered by undesired cellular side effects stemming from its initiating DSB, such as high levels of indel formation, DNA translocations, large deletions, and P53 activation. In addition to these drawbacks, HDR is limited by low efficiency in many cell types (T cells are a notable exception to this observation). Recent efforts to overcome these drawbacks include fusing human Rad51 mutants to a Cas9 D10A nickase (RDN), resulting in a DSB-free HDR system that features improved HDR product:indel ratios and lower off target editing, but is still hampered by cell-type dependencies and only modest HDR editing efficiency.
• Recently developed fusions of Cas9 to reverse transcriptases (“Prime editors”) coupled with PEgRNAs represent a novel genome editing technology that offers a number of advantages over existing genome editing methods, including the ability to install any single nucleotide substitution, and to insert or delete any short stretch of nucleotides (up to at least several dozen bases) in a site-specific manner. Notably, PE edits are achieved with generally low rates of unintended indels. As such, PE enables targeted insertion-based editing applications that have been previously impossible or impractical.
• This particular invention describes a method for using prime editing as a means to insert known immunogenicity epitopes into endogenous or foreign genomic DNA, resulting in modification of the corresponding proteins for therapeutic or biotechnological applications (see FIGS. 31 and 32 ). Prior to the invention of prime editing, such insertions could be achieved only inefficiently and with high rates of indel formation from DSBs. prime editing solves the problem of high indel formation from insertion edits while generally offering higher efficiency than HDR. This lower rate of indel formation presents a major advantage of prime editing over HDR as a method for targeted DNA insertions, especially in the described application of inserting immunogenicity epitopes. The length of epitopes is in a range from few bases to hundreds of bases. Prime editor is the most efficient and cleanest technology to achieve such targeted insertions in mammalian cells.
• The key concept of the invention is the use of prime editors to insert a nucleotide sequence containing previously described immunogenicity epitopes into endogenous or foreign genomic DNA for the downregulation and/or destruction of their protein products and/or expressing cell types. Nucleotide sequences for immunogenic epitope insertion would be targeted to genes in a manner to produce fusion proteins of the targeted gene's coded protein and the inserted immunogenic epitope's corresponding protein translation. Patient's immune systems will have been previously trained to recognize these epitopes as a result of standard prior immunization from routine vaccination against, for example, tetanus or diphtheria or measles. As a result of the immunogenic nature of the fused epitopes, patient's immune systems would be expected to recognize and disable the prime edited protein (not just the inserted epitope) and potentially the cells from which it was expressed.
• Fusions to targeted genes would be engineered as needed to ensure the inserted epitope protein translation is exposed for immune system recognition. This could include targeted nucleotide insertions resulting in protein translations yielding C-terminal fusions of immunogenicity epitopes to targeted genes, N-terminal fusions of immunogenicity epitopes to targeted genes, or the insertion of nucleotides into genes so that immunogenicity epitopes are coded within surfaced-exposed regions of protein structure.
• Protein linkers encoded as nucleotides inserted between the target gene sequence and the inserted immunogenicity epitope nucleotide sequence may need to be engineered as part of this invention to facilitate immune system recognition, cellular trafficking, protein function, or protein folding of the targeted gene. These inserted nucleotide-encoded protein linkers may include (but are not limited to) variable lengths and sequences of the XTEN linker or variable lengths and sequences of Glycine-Serine linkers. These engineered linkers have been previous used to successfully facilitate protein fusions.
• Distinguishing features of this invention include the ability to use previously acquired immune responses to specific amino acid sequences as a means to induce an immune response against otherwise non-immunogenic proteins. Another distinguishing feature is the ability to insert the nucleotide sequences of these immunogenic epitopes in a targets manner that does not induce high levels of unwanted indels as a by-product editing and is efficient in its insertion. The invention discussed herein also has the ability to combine cell type-specific delivery methods (such as AAV serotypes) to insert epitopes in cell types that are of interest to trigger an immune response to.
• Prime editing as a means of inserting immunogenic epitopes into pathogenic genes could be used to program the patient's immune system to fight a wide variety of diseases (not limited to cancer as with immuno-oncology strategies). An immediately relevant use of this technology would be as a cancer therapeutic as it could undermine a tumor's immune escape mechanism by causing an immune response to a relevant oncogene like HER2 or growth factors like EGFR. Such an approach could seem similar to T-cell engineering, but one novel advance of this approach is that it can be utilized in many cell types and for diseases beyond cancer, without needed to generate and introduce engineered T-cells into patients.
• Using PE to insert an immunogenicity epitope which most people are already vaccinated against (tetanus, pertussis, diphtheria, measles, mumps, rubella, etc.) into a foreign or endogenous gene that drives a disease, so the patient's immune system learns to disable that protein.
• Diseases that stand to have a potential therapeutic benefit from the aforementioned strategy include those caused by aggregation of toxic proteins, such as in fatal familial insomnia. Other diseases that could benefit include those caused by pathogenic overexpression of an otherwise nontoxic endogenous protein, and those caused by foreign pathogens.
• Primary therapeutic indications include those mentioned above such as therapeutics for cancer, prion and other neurodegenerative diseases, infectious diseases, and preventative medicine. Secondary therapeutic indications may include preventative care for patients with late-onset genetic diseases. It is expected that current standard of care medicines may be used in conjunction with prime editing for some diseases, like particularly aggressive cancers, or in cases where medications help alleviate disease symptoms until the disease completely cured.
• Below are examples of immunogenic epitopes that can by inserted by prime editing can be used to achieve:

• 		Epitope Amino
  Vaccine	Disease	Acid Sequence	Example Nucleic Acid Sequence (8)
  1	Tetanus toxoid	QYIKANSKFIGITEL	CATGATATAAAAGCAAATTCTAAATTTATAGGT
  		(SEQ ID NO: 396)	ATAACTGAACTA
  			(SEQ ID NO: 397)
  2	Diphtheria	GADDVVDSSKSFVMENFSS	GGCGCCGACGACGTGGTGGACAGCAGCAAGAGC
  	toxin mutant	YHGTKPGYVDSIQKGIQKP	TTCGTGATGGAGAACTTCAGCAGCTACCACGGC
  	CRM197	KSGTQGNYDDDWKEFYSTD	ACCAAGCCCGGCTACGTGGACAGCATCCAGAAG
  		NKYDAAGYSVDNENPLSGK	GGCATCCAGAAGCCCAAGAGCGGCACCCAGGGC
  		AGGVVKVTYPGLTKVLALK	AACTACGACGACGACTGGAAGGAGTTCTACAGC
  		VDNAETIKKELGLSLTEPL	ACCGACAACAAGTACGACGCCGCCGGCTACAGC
  		MEQVGTEEFIKRFGDGASR	GTGGACAACGAGAACCCCCTGAGCGGCAAGGCC
  		VVLSLPFAEGSSSVEYINN	GGCGGCGTGGTGAAGGTGACCTACCCCGGCCTG
  		WEQAKALSVELEINFETRG	ACCAAGGTGCTGGCCCTGAAGGTGGACAACGCC
  		KRGQDAMYEYMAQACAGNR	GAGACCATCAAGAAGGAGCTGGGCCTGAGCCTG
  		VRRSVGSSLSCINLDWDVI	ACCGAGCCCCTGATGGAGCAGGTGGGCACCGAG
  		RDKTKTKIESLKEHGPIKN	GAGTTCATCAAGAGGTTCGGCGACGGCGCCAGC
  		KMSESPNKTVSEEKAKQYL	AGGGTGGTGCTGAGCCTGCCCTTCGCCGAGGGC
  		EEFHQTALEHPELSELKTV	AGCAGCAGCGTGGAGTACATCAACAACTGGGAG
  		TGTNPVFAGANYAAWAVNV	CAGGCCAAGGCCCTGAGCGTGGAGCTGGAGATC
  		AQVIDSETADNLEKTTAAL	AACTTCGAGACCAGGGGCAAGAGGGGCCAGGAC
  		SILPGIGSVMGIADGAVHH	GCCATGTACGAGTACATGGCCCAGGCCTGCGCC
  		NTEEIVAQSIALSSLMVAQ	GGCAACAGGGTGAGGAGGAGCGTGGGCAGCAGC
  		AIPLVGELVDIGFAAYNFV	CTGAGCTGCATCAACCTGGACTGGGACGTGATC
  		ESIINLFQVVHNSYNRPAY	AGGGACAAGACCAAGACCAAGATCGAGAGCCTG
  		SPGHKTQPFLHDGYAVSWN	AAGGAGCACGGCCCCATCAAGAACAAGATGAGC
  		TVEDSIIRTGFQGESGHDI	GAGAGCCCCAACAAGACCGTGAGCGAGGAGAAG
  		KITAENTPLPIAGVLLPTI	GCCAAGCAGTACCTGGAGGAGTTCCACCAGACC
  		PGKLDVNKSKTHISVNGRK	GCCCTGGAGCACCCCGAGCTGAGCGAGCTGAAG
  		IRMRCRAIDGDVTFCRPKS	ACCGTGACCGGCACCAACCCCGTGTTCGCCGGC
  		PVYVGNGVHANLHVAFHRS	GCCAACTACGCCGCCTGGGCCGTGAACGTGGCC
  		SSEKIHSNEISSDSIGVLG	CAGGTGATCGACAGCGAGACCGCCGACAACCTG
  		YQKTVDHTKVNSKLSLFFE	GAGAAGACCACCGCCGCCCTGAGCATCCTGCCC
  		IKS	GGCATCGGCAGCGTGATGGGCATCGCCGACGGC
  		(SEQ ID NO: 630)	GCCGTGCACCACAACACCGAGGAGATCGTGGCC
  			CAGAGCATCGCCCTGAGCAGCCTGATGGTGGCC
  			CAGGCCATCCCCCTGGTGGGCGAGCTGGTGGAC
  			ATCGGCTTCGCCGCCTACAACTTCGTGGAGAGC
  			ATCATCAACCTGTTCCAGGTGGTGCACAACAGC
  			TACAACAGGCCCGCCTACAGCCCCGGCCACAAG
  			ACCCAGCCCTTCCTGCACGACGGCTACGCCGTG
  			AGCTGGAACACCGTGGAGGACAGCATCATCAGG
  			ACCGGCTTCCAGGGCGAGAGCGGCCACGACATC
  			AAGATCACCGCCGAGAACACCCCCCTGCCCATC
  			GCCGGCGTGCTGCTGCCCACCATCCCCGGCAAG
  			CTGGACGTGAACAAGAGCAAGACCCACATCAGC
  			GTGAACGGCAGGAAGATCAGGATGAGGTGCAGG
  			GCCATCGACGGCGACGTGACCTTCTGCAGGCCC
  			AAGAGCCCCGTGTACGTGGGCAACGGCGTGCAC
  			GCCAACCTGCACGTGGCCTTCCACAGGAGCAGC
  			AGCGAGAAGATCCACAGCAACGAGATCAGCAGC
  			GACAGCATCGGCGTGCTGGGCTACCAGAAGACC
  			GTGGACCACACCAAGGTGAACAGCAAGCTGAGC
  			CTGTTCTTCGAGATCAAGAGC
  			(SEQ ID NO: 399)
  3	mumps	GTYRLIPNARANLTA	GGCACCTACAGGCTGATCCCCAACGCCAGGGCC
  		(SEQ ID NO: 400)	AACCTGACCGCC (SEQ ID NO: 401)
  4	mumps	PSKFFTISDSATFAPGPVS	Ccgagcaaattttttaccattagcgatagcgcg
  		NA (SEQ ID NO: 402)	acctttgcgccgggcccggtgagcaacgcg
  		PSKLFIMLDNATFAPGPVV	(SEQ ID NO: 403)
  		NA (SEQ ID NO: 404)	Ccgagcaaactgtttattatgctggataacgcg
  			acctttgcgccgggcccggtggtgaacgcg
  			(SEQ ID NO: 405)
  			Selected examples from
  			Hemagglutinin-neuraminidase (HN)
  			diversity among outbreak strains
  			(table1) Divergence between
  			vaccine strain JL5 and outbreak
  			strains (table2)
  5	Rubella virus	TPPPYQVSCGGESDRASAR	ACCCCCCCCCCCTACCAGGTGAGCTGCGGCGGC
  	(RV)	VIDPAAQS	GAGAGCGACAGGGCCAGCGCCAGGGTGATCGAC
  		(SEQ ID NO: 406)	CCCGCCGCCCAGAGC
  			(SEQ ID NO: 407)
  6	Hemagglutinin	PEYAYKIVKNKKMEDGFLQ	CCCGAGTACGCCTACAAGATCGTGAAGAACAAG
  		GMVDGWYGHHSNEQGSGLM	AAGATGGAGGACGGCTTCCTGCAGGGCATGGTG
  		ENERTLDKANPNNDLCSWS	GACGGCTGGTACGGCCACCACAGCAACGAGCAG
  		DHEASSNNTNQEDLLQRES	GGCAGCGGCCTGATGGAGAACGAGAGGACCCTG
  		RRKKRIGTSTLNQRGNCNT	GACAAGGCCAACCCCAACAACGACCTGTGCAGC
  		KCQTEEARLKREEVSLVKS	TGGAGCGACCACGAGGCCAGCAGCAACAACACC
  		DQCSNGSLQCRANNSTEQV	AACCAGGAGGACCTGCTGCAGAGGGAGAGCAGG
  		D (SEQ ID NO: 408)	AGGAAGAAGAGGATCGGCACCAGCACCCTGAAC
  			CAGAGGGGCAACTGCAACACCAAGTGCCAGACC
  			GAGGAGGCCAGGCTGAAGAGGGAGGAGGTGAGC
  			CTGGTGAAGAGCGACCAGTGCAGCAACGGCAGC
  			CTGCAGTGCAGGGCCAACAACAGCACCGAGCAG
  			GTGGAC (SEQ ID NO: 409)
  7	Neuraminidase	TKSTNSRSGGISGPDNEAP	ACCAAGAGCACCAACAGCAGGAGCGGCGGCATC
  		VGEAPSPYGDNPRPNDGNN	AGCGGCCCCGACAACGAGGCCCCCGTGGGCGAG
  		IRIGSKGYNGIITDTIEES	GCCCCCAGCCCCTACGGCGACAACCCCAGGCCC
  		CSCYPDAKVVKSVELDSTI	AACGACGGCAACAACATCAGGATCGGCAGCAAG
  		WTSGSSPNQKIITIGWDPN	GGCTACAACGGCATCATCACCGACACCATCGAG
  		GWTGTPMSPNGAYGTDGPS	GAGAGCTGCAGCTGCTACCCCGACGCCAAGGTG
  		NGQANQHQAESISAGNSSL	GTGAAGAGCGTGGAGCTGGACAGCACCATCTGG
  		CPIRDNWHGSNRSWSWPDG	ACCAGCGGCAGCAGCCCCAACCAGAAGATCATC
  		AE (SEQ ID NO: 410)	ACCATCGGCTGGGACCCCAACGGCTGGACCGGC
  			ACCCCCATGAGCCCCAACGGCGCCTACGGCACC
  			GACGGCCCCAGCAACGGCCAGGCCAACCAGCAC
  			CAGGCCGAGAGCATCAGCGCCGGCAACAGCAGC
  			CTGTGCCCCATCAGGGACAACTGGCACGGCAGC
  			AACAGGAGCTGGAGCTGGCCCGACGGCGCCGAG
  			(SEQ ID NO: 411)
  8	TAP	EKIVLLLAMMEKIVLLLAK	GAGAAGATCGTGCTGCTGCTGGCCATGATGGAG
  	(transport antigen	CQTPMGAIKAVDGVTNKCP	AAGATCGTGCTGCTGCTGGCCAAGTGCCAGACC
  	presentation) on	YLGSPSF	CCCATGGGCGCCATCAAGGCCGTGGACGGCGTG
  	H5N1 virus	(SEQ ID NO: 412)	ACCAACAAGTGCCCCTACCTGGGCAGCCCCAGC
  	hemagglutinin		TTC (SEQ ID NO: 413)
  9	TAP	IRPCFWVELNPNQKIITIR	ATCAGGCCCTGCTTCTGGGTGGAGCTGAACCCC
  	(transport antigen	PCFWVELICYPDAGEIT	AACCAGAAGATCATCACCATCAGGCCCTGCTTC
  	presentation) on	(SEQ ID NO: 414)	TGGGTGGAGCTGATCTGCTACCCCGACGCCGGC
  	h5n1 virus		GAGATCACC (SEQ ID NO: 415)
  	neuraminidase
  10	hemagglutinin	MEKIVLLLAEKIVLLLAMC	ATGGAGAAGATCGTGCTGCTGCTGGCCGAGAAG
  	epitopes toward	PYLGSPSFKCQTPMGAIKA	ATCGTGCTGCTGCTGGCCATGTGCCCCTACCTG
  	class I HLA	VDGVTNK	GGCAGCCCCAGCTTCAAGTGCCAGACCCCCATG
  		(SEQ ID NO: 416)	GGCGCCATCAAGGCCGTGGACGGCGTGACCAAC
  			AAG (SEQ ID NO: 417)
  11	neuraminidase	NPNQKIITICYPDAGEITI	AACCCCAACCAGAAGATCATCACCATCTGCTAC
  	epitopes toward	RPCFWVELRPCFWVELI	CCCGACGCCGGCGAGATCACCATCAGGCCCTGC
  	class I HLA	(SEQ ID NO: 418)	TTCTGGGTGGAGCTGAGGCCCTGCTTCTGGGTG
  			GAGCTGATC (SEQ ID NO: 419)
  12	hemagglutinin	MVSLVKSDQIGTSTLNQR	ATGGTGAGCCTGGTGAAGAGCGACCAGATCGGC
  	epitopes toward	(SEQ ID NO: 420)	ACCAGCACCCTGAACCAGAGG
  	class II HLA		(SEQ ID NO: 421)
  13	neuraminidase	YNGIITDTI	TACAACGGCATCATCACCGACACCATC
  	epitopes toward	(SEQ ID NO: 422)	(SEQ ID NO: 423)
  	class II HLA
  14	hemagglutinin	MEKIVLLLAEKIVLLLAMM	ATGGAGAAGATCGTGCTGCTGCTGGCCGAGAAG
  	epitope H5N1-	VSLVKSDQCPYLGSPSFIG	ATCGTGCTGCTGCTGGCCATGATGGTGAGCCTG
  	bound class I and	TSTLNQRKCQTPMGAIKAV	GTGAAGAGCGACCAGTGCCCCTACCTGGGCAGC
  	class II HLA	DGVTNK	CCCAGCTTCATCGGCACCAGCACCCTGAACCAG
  		(SEQ ID NO: 424)	AGG (SEQ ID NO: 425)
  15	neuraminidase	NPNQKIITIYNGIITDTIC	AACCCCAACCAGAAGATCATCACCATCTACAAC
  	epitope H5N1-	YPDAGEITIRPCFWVELRP	GGCATCATCACCGACACCATCTGCTACCCCGAC
  	bound class I and	CFWVELI	GCCGGCGAGATCACCATCAGGCCCTGCTTCTGG
  	class II HLA	(SEQ ID NO: 426)	GTGGAGCTGAGGCCCTGCTTCTGGGTGGAGCTG
  			ATC (SEQ ID NO: 427)

• Below are additional examples of epitopes that may be integrated into a target gene for immunoepitope taggin:
REFERENCES CITED IN EXAMPLE 8
• The following references are incorporated by reference in their entireties.
• 1. X. Wen, K. Wen, D. Cao, G. Li, R. W. Jones, J. Li, S. Szu, Y. Hoshino, L. Yuan, Inclusion of a universal tetanus toxoid CD4(+) T cell epitope P2 significantly enhanced the immunogenicity of recombinant rotavirus ΔVP8* subunit parenteral vaccines. Vaccine 32, 4420-4427 (2014).
• 2. G. Ada, D. Isaacs, Carbohydrate-protein conjugate vaccines. Clin Microbiol Infect 9, 79-85 (2003).
• 3. E. Malito, B. Bursulaya, C. Chen, P. L. Surdo, M. Picchianti, E. Balducci, M. Biancucci, A. Brock, F. Berti, M. J. Bottomley, M. Nissum, P. Costantino, R. Rappuoli, G. Spraggon, Structural basis for lack of toxicity of the diphtheria toxin mutant CRM197. Proceedings of the National Academy of Sciences 109, 5229 (2012).
• 4. J. de Wit, M. E. Emmelot, M. C. M. Poelen, J. Lanfermeijer, W. G. H. Han, C. van Els, P. Kaaijk, The Human CD4(+) T Cell Response against Mumps Virus Targets a Broadly Recognized Nucleoprotein Epitope. J Virol 93, (2019).
• 5. M. May, C. A. Rieder, R. J. Rowe, Emergent lineages of mumps virus suggest the need for a polyvalent vaccine. Int J Infect Dis 66, 1-4 (2018).
• 6. M. Ramamurthy, P. Rajendiran, N. Saravanan, S. Sankar, S. Gopalan, B. Nandagopal, Identification of immunogenic B-cell epitope peptides of rubella virus E1 glycoprotein towards development of highly specific immunoassays and/or vaccine. Conference Abstract, (2019).
• 7. U. S. F. Tambunan, F. R. P. Sipahutar, A. A. Parikesit, D. Kerami, Vaccine Design for H5N1 Based on B- and T-cell Epitope Predictions. Bioinform Biol Insights 10, 27-35 (2016).
Example 9—In Vivo Delivery of PE Agents
• Precise genome targeting technologies using the CRISPR/Cas9 system have recently been explored in a wide range of applications, including gene therapy. A major limitation to the application of Cas9 and Cas9-based genome-editing agents in gene therapy is the size of Cas9 (>4 kb), impeding its efficient delivery via recombinant adeno-associated virus (rAAV). Recently-developed fusions of Cas9 to reverse transcriptases (“Prime editors”) represent a novel genome editing technology that possesses a number of advantages over existing genome editing methods, including the ability to install any single nucleotide substitution, and to insert or delete any arbitrarily-defined short (<˜20) stretch of nucleotides in a site-specific manner. As such, this method enables editing of human pathogenic variants that have been intractable to correction previously. The delivery of prime editing reagents could enable correction of genetic sequences that cause human disease, or allow for the installation of disease-preventing gene variants.
• This invention describes methods for delivering prime editors into cells in vitro and in vivo. Prime editors have been developed and characterized solely in cultured cells. No known method can deliver prime editors in vivo. The presently disclosed methods for delivering prime editors via rAAV or pre-assembled ribonucleoprotein (RNP) complexes will overcome several barriers to in vivo delivery. For example, the DNA encoding prime editors is larger than the rAAV packaging limit, and so requires special solutions. One such solution is formulating the editor fused to split intein pairs that are packaged into two separate rAAV particles that, when co-delivered to a cell, reconstitute the functional editor protein. Several other special considerations to account for the unique features of prime editing are described, including the optimization of second-site nicking targets and properly packaging prime editors into virus vectors, including lentiviruses and rAAV.
• Distinguishing features include using ribonucleoprotein (RNP) delivery formulations, prime editors and nearby nicking targets can be pre-complexed with their specific sgRNA/PEgRNA. This will enhance the range of possible targetable sites and allow for greater optimization of editing efficiency relative to current data that has used DNA delivery. Using either RNP or mRNA delivery formulations, variant Cas proteins can be used that each complex with their own guide RNA variant. This will also allow for a greater diversity of potential nicking loci, so it is expected that optimization can be achieved for greater efficiency in any given application. Using RNP, it is expected to increase editing specificity base on previous RNP reports (Rees et al., 2017). This would reduce off-target prime editing. Potential architectures for splitting prime editors into two AAV vectors for delivery in vivo or ex vivo are described. Packaging prime editor into a dual AAV system requires optimization of design considerations including split sites, reconstitution methods (such as inteins), and guide expression architecture. Using a mixture of virus and RNP for delivery of prime editor, it is expected that editing will be controlled over time since RNP eventually degrades in vivo which will stop prime editing after RNP is no longer supplied.
• Prime editor ribonucleoprotein (RNP), mRNA with prime editor guide RNA, or DNA can be packaged into lipid nanoparticles, rAAV, or lentivirus and injected, ingested, or inhaled to alter genomic DNA in vivo and ex vivo, including for the purposes of establishing animal models of human disease, testing therapeutic and scientific hypotheses in animal models of human disease, and treating disease in humans.
• Prime editors could feasibly be used to correct a large fraction of all genetic diseases (˜89% of pathogenic human genetic variants in Clinvar), if suitable means of delivery into relevant cell types in vivo are developed. Blood diseases, retinal diseases, and liver diseases are the most likely first applications due to established delivery systems for other reagents. AAV capsids, other evolved or engineered viral vectors, and lipid nanoparticle formulations would need to be used in combination with this invention.
• In certain embodiments, one or more of the prime editor domains (e.g., the napDNAbp domain or the RT domain) could be engineered with an intein sequence.
Example 10—Use of PE to Identify Off-Target Editing
• There are currently no described methods to detect off-target editing with prime editors (prime editing itself has not been published yet). These methods would allow a researcher to identify potential sites of off-target editing using prime editors, which would be important considerations were this technique used to treat genetic disease in patients.
• Methods described here could also be useful to identify off-targets of Cas nucleases. These off-targets have previously been identified using BLESS, Guide-Seq, CIRCLE-Seq, and Digenome-Seq. However, this method is advantageous in the sensitivity and simplicity of the process.
• The key concept of this aspect is the idea of using prime editing to insert an adapter sequence or primer binding site, templated from a PEgRNA, to enable the rapid identification of genomic off-target modification sites of Cas nucleases or prime editors.
• No method to identify in an unbiased manner prime editing off-target sites is known. This method is distinguished from other techniques that identify nuclease off-target sites because the adapter sequence is inserted in the same event as DNA binding and nicking, simplifying the downstream processing.
• The present invention includes identification of off-target editing sites when editing inside a living cell, in tissue culture or animal models (see FIG. 33 ). To conduct this method, a PEgRNA is generated that has an identical protospacer to the final desired editor (and, if looking at prime editing off-targets, an identical primer-binding site sequence to the final desired editor), but includes the necessary sequences to install an adapter or primer binding site after reverse transcription by prime editing. In vivo editing is conducted using a prime editor or RT-fused nuclease, and isolate genomic DNA. The genomic DNA is fragmented by enzymatic or mechanical means and append a different adapter to sites of DNA fragmentation. PCR is used to amplify from one adapter to the adapter installed via PEgRNA. The resulting product is deep-sequenced to identify all modified sites.
• The invention also includes identification of off-target editing sites using in vitro modification of genomic DNA (see FIG. 33 ). To conduct this method, RNP of purified prime editor protein and a PEgRNA is assembled that will install an adapter or primer binding sequence, but is otherwise the same as the PEgRNA of interest. This RNP is incubated with extracted genomic DNA before or after fragmentation of the DNA and attachment of different adapters to sites of DNA breaks. PCR is used to amplify from fragmented site to the adapter that was installed with PE. Deep sequence to identify sites of modification. This in vitro editing method should enhance the sensitivity of detection, because cellular DNA repair will never eliminate the reverse-transcribed DNA adapter added by the prime editor.
• These methods could be used to identify off-target editing for any prime editor, or any genome editor that uses a guideRNA to recognize a target cut site (most Cas nucleases).
• These methods could be applied to all genetic diseases for which genome editors are considered for use in treatment.
Example 11—Use of PE to Enable Chemical-Induced Dimerization of Target Proteins In Vivo
• The prime editors described herein may also be used to place dimerization-induced biological processes, such as receptor signaling, under control of a convenient small-molecule drug by the genomic integration of genes encoding small-molecule binding proteins with prime editing is described herein. Using the prime editors described herein, the gene sequence encoding a small-molecule binding protein may be inserted within a gene encoding a target protein of interest in a living cell or patient. This edit alone should have no physiological effect. Upon administration of the small-molecule drug, which typically is a dimeric small molecule that can simultaneously bind to two drug-binding protein domains each of which is fused to a copy of the target protein, the small-molecule induces dimerization of the targeted protein. This target protein dimerization event then induces a biological signaling event, such as erythropoiesis or insulin signaling.
Example 12—Prime Editing: Highly Versatile and Precise Search-and-Replace Genome Editing in Human Cells without Double-Stranded DNA Breaks
• Current genome editing methods can disrupt, delete, or insert target genes with accompanying byproducts of double-stranded DNA breaks using programmable nucleases, and install the four transition point mutations at target loci using base editors. Small insertions, small deletions, and the eight transversion point mutations, however, collectively represent most pathogenic genetic variants but cannot be corrected efficiently and without an excess of byproducts in most cell types. Described herein is prime editing, a highly versatile and precise genome editing method that directly writes new genetic information into a specified DNA site using a catalytically impaired Cas9 fused to an engineered reverse transcriptase, programmed with an engineered prime editing guide RNA (PEgRNA) that both specifies the target site and encodes the desired edit. Greater than 175 distinct edits in human cells were performed to establish that prime editing can make targeted insertions, deletions, all 12 possible types of point mutations, and combinations thereof efficiently (typically 20-60%, up to 77% in unsorted cells) and with low byproducts (typically 1-10%), without requiring double-stranded breaks or donor DNA templates. Prime editing was applied in human cells to correct the primary genetic causes of sickle cell disease (requiring an A●T-to-T●A transversion in HBB) and Tay-Sachs disease (requiring a 4-base deletion in HEXA), in both cases efficiently reverting the pathogenic genomic alleles to wild-type with minimal byproducts. Prime editing was also used to create human cell lines with these pathogenic HBB transversion and HEXA insertion mutations, to install the G127V mutation in PRNP that confers resistance to prion disease (requiring a G●C-to-T●A transversion), and to efficiently insert a His6 tag, a FLAG epitope tag, and an extended LoxP site into target loci in human cells. Prime editing offers efficiency and product purity advantages over HDR, and complementary strengths and weaknesses compared to base editing. Consistent with its search-and-replace mechanism, which requires three distinct base-pairing events, prime editing is much less prone to off-target DNA modification at known Cas9 off-target sites than Cas9. Prime editing substantially expands the scope and capabilities of genome editing, and in principle can correct ˜89% of known pathogenic human genetic variants.
• The ability to make virtually any targeted change in the genome of any living cell or organism is a longstanding aspiration of the life sciences. Despite rapid advances in genome editing technologies, the majority of the >75,000 known human genetic variants associated with diseases¹¹¹ cannot be corrected or installed in most therapeutically relevant cells (FIG. 38A). Programmable nucleases such as CRISPR-Cas9 make double-stranded DNA breaks (DSBs) that can disrupt genes by inducing mixtures of insertions and deletions (indels) at target sites^112-114. Nucleases can also be used to delete target genes^115-116, or insert exogenous genes^117-119, through homology-independent processes. Double-stranded DNA breaks, however, are also associated with undesired outcomes including complex mixtures of products, translocations¹²⁰, and p53 activation^121,122. Moreover, the vast majority of pathogenic alleles differ from their non-pathogenic counterparts by small insertions, deletions, or base substitutions that require much more precise editing technologies to correct (FIG. 38A). Homology-directed repair (HDR) stimulated by nuclease-induced DSBs¹²³ has been widely used to install a variety of precise DNA changes. HDR, however, relies on exogenous donor DNA repair templates, typically generates an excess of indel byproducts from end-joining repair of DSBs, and is inefficient in most therapeutically relevant cell types (T cells and some stem cells being important exceptions)^124,125. While enhancing the efficiency and precision of DSB-mediated genome editing remains the focus of promising efforts^126-130, these challenges necessitate the exploration of alternative precision genome editing strategies.
• Base editing can efficiently install or correct the four types of transition mutations (C to T, G to A, A to G, and T to C) without requiring DSBs in a wide variety of cell types and organisms, including mammals^128-131, but cannot currently achieve any of the eight transversion mutations (C to A, C to G, G to C, G to T, A to C, A to T, T to A, and T to G), such as the T●A-to-A●T mutation needed to directly correct the most common cause of sickle cell disease (HBB E6V)¹³². In addition, no DSB-free method has been reported to perform target deletions, such as the removal of the 4-base duplication that causes Tay-Sachs disease (HEXA 1278+TATC)¹³³, or targeted insertions, such as the precise 3-base insertion required to directly correct the most common cause of cystic fibrosis (CFTR ΔF508)¹³⁴. Targeted transversion point mutations, insertions, and deletions thus are difficult to install or correct efficiently and without excess byproducts in most cell types, even though they collectively account for most known pathogenic alleles (FIG. 38A).
• Described herein is the development of prime editing, a new “search-and-replace” genome editing technology that mediates targeted insertions, deletions, and all 12 possible base-to-base conversions at targeted loci in human cells without requiring double-stranded DNA breaks, or donor DNA templates. Prime editors, initially exemplified by PE1, use a reverse transcriptase fused to a programmable nickase and a prime editing extended guide RNA (PEgRNA) to directly copy genetic information from the extension on the PEgRNA into the target genomic locus. A second-generation prime editor (PE2) uses an engineered reverse transcriptase to substantially increase editing efficiencies with minimal (typically <2%) indel formation, while a third-generation PE3 system adds a second guide RNA to nick the non-edited strand, thereby favoring replacement of the non-edited strand and further increasing editing efficiency, typically, to about 20-50% in human cells with about 1-10% indel formation. PE3 offers far fewer byproducts and higher or similar efficiency compared to optimized Cas9 nuclease-initiated HDR, and offers complementary strengths and weaknesses compared to current-generation base editors.
• PE3 was applied at genomic loci in human HEK293T cells to achieve efficient conversion of HBB E6V to wild-type HBB, deletion of the inserted TATC to restore HEXA 1278+TATC to wild-type HEXA, installation in PRNP of the G127V mutation that confers resistance to prion disease¹³⁵ (requiring a G●C-to-T●A transversion), and targeted insertion of a His6 tag (18 bp), FLAG epitope tag (24 bp), and extended LoxP site for Cre-mediated recombination (44 bp). Prime editing was also successful in three other human cell lines, as well as in post-mitotic primary mouse cortical neurons, with varying efficiencies. Due to a high degree of flexibility in the distance between the initial nick and location of the edit, prime editing is not substantially constrained by the PAM requirement of Cas9 and in principle can target the vast majority of genomic loci. Off-target prime editing is much rarer than off-target Cas9 editing at known Cas9 off-target loci, likely due to the requirement of three distinct DNA base pairing events in order for productive prime editing to take place. By enabling precise targeted insertions, deletions, and all 12 possible classes of point mutations at a wide variety of genomic loci without the need for DSBs or donor DNA templates, prime editing has the potential to advance the study and correction of many gene variants.
Results
• Strategy for Transferring Information from an Extended Guide RNA into a Target DNA Locus
• Cas9 targets DNA using a guide RNA containing a spacer sequence that hybridizes to the target DNA site^{112-114,136,137}. The aim was to engineer guide RNAs to both specify the DNA target as in natural CRISPR systems^138,139, and also to contain new genetic information that replaces the corresponding DNA nucleotides at the target locus. The direct transfer of genetic information from an extended guide RNA into a specified DNA site, followed by replacement of the original unedited DNA, in principle could provide a general means of installing targeted DNA sequence changes in living cells, without dependence on DSBs or donor DNA templates. To achieve this direct information transfer, the aim was to use genomic DNA, nicked at the target site to expose a 3′-hydroxyl group, to prime the reverse transcription of the genetic information from an extension on the engineered guide RNA (hereafter referred to as the prime editing guide RNA, or PEgRNA) directly into the target site (FIG. 38A).
• These initial steps of nicking and reverse transcription, which resemble mechanisms used by some natural mobile genetic elements¹⁴⁰, result in a branched intermediate with two redundant single-stranded DNA flaps on one strand: a 5′ flap that contains the unedited DNA sequence, and a 3′ flap that contains the edited sequence copied from the PEgRNA (FIG. 38B). To achieve a successful edit, this branched intermediate must be resolved so that the edited 3′ flap replaces the unedited 5′ flap. While hybridization of the 5′ flap with the unedited strand is likely to be thermodynamically favored since the edited 3′ flap can make fewer base pairs with the unedited strand, 5′ flaps are the preferred substrate for structure-specific endonucleases such as FEN1¹⁴¹, which excises 5′ flaps generated during lagging-strand DNA synthesis and long-patch base excision repair. It was reasoned that preferential 5′ flap excision and 3′ flap ligation could drive the incorporation of the edited DNA strand, creating heteroduplex DNA containing one edited strand and one unedited strand (FIG. 38B).
• Permanent installation of the edit could arise from subsequent DNA repair that resolves the mismatch between the two DNA strands in a manner that copies the information in the edited strand to the complementary DNA strand (FIG. 38C). Based on a similar strategy developed to maximize the efficiency of DNA base editing^131-133, it was envisioned that nicking the non-edited DNA strand, far enough from the site of the initial nick to minimize double-strand break formation, might bias DNA repair to preferentially replace the non-edited strand.
Validation of Prime Editing Steps In Vitro and in Yeast Cells
• Following cleavage of the PAM-containing DNA strand by the RuvC nuclease domain of Cas9, the PAM-distal fragment of this strand can dissociate from otherwise stable Cas9:sgRNA:DNA complexes¹⁴³. It was hypothesized that the 3′ end of this liberated strand might be sufficiently accessible to prime DNA polymerization. Guide RNA engineering efforts^144-146 and crystal structures of Cas9:sg RNA:DNA complexes^147-149 suggest that the 5′ and 3′ termini of the sgRNA can be extended without abolishing Cas9:sgRNA activity. PEgRNAs were designed by extending sgRNAs to include two critical components: a primer binding site (PBS) that allows the 3′ end of the nicked DNA strand to hybridize to the PEgRNA, and a reverse transcriptase (RT) template containing the desired edit that would be directly copied into the genomic DNA site as the 3′ end of the nicked DNA strand is extended across the RNA template by a polymerase (FIG. 38C).
• These hypotheses were tested in vitro using purified S. pyogenes Cas9 protein. A series of PEgRNA candidates were constructed by extending sgRNAs on either terminus with a PBS sequence (5 to 6 nucleotides, nt) and an RT template (7 to 22 nt). It was confirmed that 5′-extended PEgRNAs direct Cas9 binding to target DNA, and that both 5′-extended PEgRNAs and 3′-extended PEgRNAs support Cas9-mediated target nicking in vitro and DNA cleavage activities in mammalian cells (FIGS. 44A-44C). These candidate PEgRNA designs were tested using pre-nicked 5′-Cy5-labeled dsDNA substrates, catalytically dead Cas9 (dCas9), and a commercial variant of Moloney murine leukemia virus (M-MLV) reverse transcriptase (FIG. 44D). When all components were present, efficient conversion of the fluorescently labeled DNA strand into longer DNA products with gel mobilities, consistent with reverse transcription along the RT template, (FIG. 38D, FIGS. 44D-44E) was observed. Products of desired length were formed with either 5′-extended or 3′-extended PEgRNAs (FIGS. 38D-38E). Omission of dCas9 led to nick translation products derived from reverse transcriptase-mediated DNA polymerization on the DNA template, with no PEgRNA information transfer (FIG. 38D). No DNA polymerization products were observed when the PEgRNA was replaced by a conventional sgRNA, confirming the necessity of the PBS and RT template components of the PEgRNA (FIG. 38D). These results demonstrate that Cas9-mediated DNA melting exposes a single-stranded R-loop that, if nicked, is competent to prime reverse transcription from either a 5′-extended or 3′-extended PEgRNA.
• Next, non-nicked dsDNA substrates were tested with a Cas9 nickase (H840A mutant) that exclusively nicks the PAM-containing strand¹¹². In these reactions, 5′-extended PEgRNAs generated reverse transcription products inefficiently, possibly due to impaired Cas9 nickase activity (FIG. 44F). However, 3′-extended PEgRNAs enabled robust Cas9 nicking and efficient reverse transcription (FIG. 38E). The use of 3′-extended PEgRNAs generated only a single apparent product, despite the potential, in principle, for reverse transcription to terminate anywhere within the remainder of the PEgRNA. DNA sequencing of the products of reactions with Cas9 nickase, RT, and 3′-extended PEgRNAs revealed that the complete RT template sequence was reverse transcribed into the DNA substrate (FIG. 44G). These experiments established that 3′-extended PEgRNAs can template the reverse transcription of new DNA strands while retaining the ability to direct Cas9 nickase activity.
• To evaluate the eukaryotic cell DNA repair outcomes of 3′ flaps produced by PEgRNA-programmed reverse transcription in vitro, DNA nicking and reverse transcription using PEgRNAs, Cas9 nickase, and RT in vitro on reporter plasmid substrates were performed, and the reaction products were then transformed into yeast (S. cerevisiae) cells (FIG. 45A). Encouragingly, when plasmids were edited in vitro with 3′-extended PEgRNAs encoding a T●A-to-A●T transversion that corrects the premature stop codon, 37% of yeast transformants expressed both GFP and mCherry proteins (FIG. 38F, FIG. 45C). Consistent with the results in FIG. 38E and FIG. 44F, editing reactions carried out in vitro with 5′-extended PEgRNAs yielded fewer GFP and mCherry double-positive colonies (9%) than those with 3′-extended PEgRNAs (FIG. 38F and FIG. 45D). Productive editing was also observed using 3′-extended PEgRNAs that insert a single nucleotide (15% double-positive transformants) or delete a single nucleotide (29% double-positive transformants) to correct frameshift mutations (FIG. 38F and FIGS. 45E-45F). DNA sequencing of edited plasmids recovered from double-positive yeast colonies confirmed that the encoded transversion edit occurred at the desired sequence position (FIG. 45G). These results demonstrate that DNA repair in eukaryotic cells can resolve 3′ DNA flaps arising from prime editing to incorporate precise DNA edits including transversions, insertions, and deletions.
• Design of Prime Editor 1 (PE1)
• Encouraged by the results in vitro and in yeast, a prime editing system with a minimum number of components capable of editing genomic DNA in mammalian cells was sought for development. It was hypothesized that 3′-extended PEgRNAs (hereafter referred to simply as PEgRNAs, FIG. 39A) and direct fusions of Cas9 H840A to reverse transcriptase via a flexible linker may constitute a functional two-component prime editing system. HEK293T (immortalized human embryonic kidney) cells were transfected with one plasmid encoding a fusion of wild-type M-MLV reverse transcriptase to either terminus of Cas9 H840A nickase as well as a second plasmid encoding a PEgRNA. Initial attempts led to no detectable T●A-to-A●T conversion at the HEK3 target locus.
• Extension of the PBS in the PEgRNA to 8-15 bases (FIG. 39A), however, led to detectable T●A-to-A●T editing at the HEK3 target site (FIG. 39B), with higher efficiencies for prime editor constructs in which the RT was fused to the C-terminus of Cas9 nickase (3.7% maximal T●A-to-A●T conversion with PBS lengths ranging from 8-15 nt) compared to N-terminal RT-Cas9 nickase fusions (1.3% maximal T●A-to-A●T conversion) (FIG. 39B; all mammalian cell data herein reports values for the entire treated cell population, without selection or sorting, unless otherwise specified). These results suggest that wild-type M-MLV RT fused to Cas9 requires longer PBS sequences for genome editing in human cells compared to what is required in vitro using the commercial variant of M-MLV RT supplied in trans. This first-generation wild-type M-MLV reverse transcriptase fused to the C-terminus of Cas9 H840A nickase was designated as PE1.
• The ability of PE1 to precisely introduce transversion point mutations at four additional genomic target sites specified by the PEgRNA (FIG. 39C) was tested. Similar to editing at the HEK3 locus, efficiency at these genomic sites was dependent on PBS length, with maximal editing efficiencies ranging from 0.7-5.5% (FIG. 39C). Indels from PE1 were low, averaging 0.2±0.1% for the five sites under conditions that maximized each site's editing efficiency (FIG. 46A). PE1 was also able to install targeted insertions and deletions, exemplified by a single-nucleotide deletion (4.0% efficiency), a single-nucleotide insertion (9.7%), and a three-nucleotide insertion (17%) at the HEK3 locus (FIG. 39C). These results establish the ability of PE1 to directly install targeted transversions, insertions, and deletions without requiring double-stranded DNA breaks or DNA templates.
• Design of Prime Editor 2 (PE2)
• While PE1 can install a variety of edits at several loci in HEK293T cells, editing efficiencies were generally low (typically ≤5%) (FIG. 39C). It was hypothesized that engineering the reverse transcriptase in PE1 might improve the efficiency of DNA synthesis within the unique conformational constraints of the prime editing complex, resulting in higher genome editing yields. M-MLV RT mutations have been previously reported that increase enzyme thermostability^150,151, processivity¹⁵⁰, and DNA:RNA heteroduplex substrate affinity¹⁵², and that inactivate RNaseH activity¹⁵³. 19 PE1 variants were constructed containing a variety of reverse transcriptase mutations to evaluate their prime editing efficiency in human cells.
• First, a series of M-MLV RT variants that previously emerged from laboratory evolution for their ability to support reverse transcription at elevated temperatures¹⁵⁰ were investigated. Successive introduction of three of these amino acid substitutions (D200N, L603W, and T330P) into M-MLV RT, hereafter referred to as M3, led to a 6.8-fold average increase in transversion and insertion editing efficiency across five genomic loci in HEK293T cells compared to that of PE1 (FIGS. 47A-47S).
• Next, in combination with M3, additional reverse transcriptase mutations that were previously shown to enhance binding to template:PBS complex, enzyme processivity, and thermostability¹⁵² were tested. Among the 14 additional mutants analyzed, a variant with T306K and W313F substitutions, in addition to the M3 mutations, improved editing efficiency an additional 1.3-fold to 3.0-fold compared to M3 for six transversion or insertion edits across five genomic sites in human cells (FIGS. 47A-47S). This pentamutant of M-MLV reverse transcriptase incorporated into the PE1 architecture (Cas9 H840A-M-MLV RT (D200N L603W T330P T306K W313F)) is hereafter referred to as PE2.
• PE2 installs single-nucleotide transversion, insertion, and deletion mutations with substantially higher efficiency than PE1 (FIG. 39C), and is compatible with shorter PBS PEgRNA sequences (FIG. 39C), consistent with an enhanced ability to productively engage transient genomic DNA:PBS complexes. On average, PE2 led to a 1.6- to 5.1-fold improvement in prime editing point mutation efficiency over PE1 (FIG. 39C), and in some cases dramatically improved editing yields up to 46-fold (FIG. 47F and FIG. 47I). PE2 also effected targeted insertions and deletions more efficiently than PE1, achieving the targeted insertion of the 24-bp FLAG epitope tag at the HEK3 locus with 4.5% efficiency, a 15-fold improvement over the efficiency of installing this insertion with PE1 (FIG. 47D), and mediated a 1-bp deletion in HEK3 with 8.6% efficiency, 2.1-fold higher than that of PE1 (FIG. 39C). These results establish PE2 as a more efficient prime editor than PE1.
• Optimization of PEgRNA Features
• The relationship between PEgRNA architecture and prime editing efficiency was systematically probed at five genomic loci in HEK293T cells with PE2 (FIG. 39C). In general, priming sites with lower GC content required longer PBS sequences (EMX1 and RNF2, containing 40% and 30% GC content, respectively, in the first 10 nt upstream of the nick), whereas those with greater GC content supported prime editing with shorter PBS sequences (HEK4 and FANCF, containing 80% and 60% GC content, respectively, in the first 10 nt upstream of the nick) (FIG. 39C), consistent with the energetic requirements for hybridization of the nicked DNA strand to the PEgRNA PBS. No PBS length or GC content level was strictly predictive of prime editing efficiency, and other factors such as secondary structure in the DNA primer or PEgRNA extension may also influence editing activity. It is recommended to start with a PBS length of ˜13 nt for a typical target sequence, and exploring different PBS lengths if the sequence deviates from ˜40-60% GC content. When necessary, optimal PBS sequences should be determined empirically.
• Next, the performance determinants of the RT template portion of the PEgRNA were studied. PEgRNAs with RT templates ranging from 10-20 nt in length were systemically evaluated at five genomic target sites using PE2 (FIG. 39D) and with longer RT templates as long as 31 nt at three genomic sites (FIGS. 48A-48C). As with PBS length, RT template length also could be varied to maximize prime editing efficiency, although in general many RT template lengths ≥10 nt long support more efficient prime editing (FIG. 39D). Since some target sites preferred longer RT templates (>15 nt) to achieve higher editing efficiencies (FANCF, EMX1), while other loci preferred short RT templates (HEK3, HEK4) (FIG. 39D), it is recommend both short and long RT templates be tested when optimizing a PEgRNA, starting with ˜10-16 nt.
• Importantly, RT templates that place a C as the nucleotide adjacent to the terminal hairpin of the sgRNA scaffold generally resulted in lower editing efficiency compared to other PEgRNAs with RT templates of similar length (FIGS. 48A-48C). Based on the structure of sgRNAs bound to Cas9^148,149 it was speculated that the presence of a C as the first nucleotide of the 3′ extension of a canonical sgRNA can disrupt the sgRNA scaffold fold by pairing with G81, a nucleotide that natively forms a pi stack with Tyr 1356 in Cas9 and a non-canonical base pair with sgRNA A68. Since many RT template lengths support prime editing, it is recommended to choose PEgRNAs in which the first base of the 3′ extension (the last reverse-transcribed base of the RT template) is not C.
• Design of Prime Editor 3 Systems (PE3 and PE3b)
• While PE2 can transfer genetic information from the PEgRNA to the target locus more efficiently than PE1, the manner in which the cell resolves the resulting heteroduplex DNA created by one edited strand and one unedited strand determines if the edit is durable. A previous development of base editing faced a similar challenge since the initial product of cytosine or adenine deamination is heteroduplex DNA containing one edited and one non-edited strand. To increase the efficiency of base editing, a Cas9 D10A nickase was used to introduce a nick into the non-edited strand and to direct DNA repair to that strand, using the edited strand as a template^129,130,142. To exploit this principle to enhance prime editing efficiencies, a similar strategy of nicking the non-edited strand using the Cas9 H840A nickase already present in PE2 and a simple sgRNA to induce preferential replacement of the non-edited strand by the cell (FIG. 40A) was tested. Since the edited DNA strand was also nicked to initiate prime editing, a variety of sgRNA-programmed nick locations were tested on the non-edited strand to minimize the production of double-stranded DNA breaks that lead to indels.
• This PE3 strategy was first tested at five genomic sites in HEK293T cells by screening sgRNAs that induce nicks located 14 to 116 bases from the site of the PEgRNA-induced nick, either 5′ or 3′ of the PAM. In four of the five sites tested, nicking the non-edited strand increased the amount of indel-free prime editing products compared to the PE2 system by 1.5- to 4.2-fold, to as high as 55% (FIG. 40B). While the optimal nicking position varied depending on the genomic site, nicks positioned 3′ of the PAM (positive distances in FIG. 40B) approximately 40-90 bp from the PEgRNA-induced nick generally produced favorable increases in prime editing efficiency (averaging 41%) without excess indel formation (6.8% average indels for the sgRNA resulting in the highest editing efficiency for each of the five sites tested) (FIG. 40B). As expected, at some sites, placement of the non-edited strand nick within 40 bp of the PEgRNA-induced nick led to large increases in indel formation up to 22% (FIG. 40B), presumably due to the formation of a double-strand break from nicking both strands close together. At other sites, however, nicking as close as 14 bp away from the PEgRNA-induced nick produced only 5% indels (FIG. 40B), suggesting that locus-dependent factors control conversion of proximal dual nicks into double-strand DNA breaks. At one tested site (HEK4), complementary strand nicks either provided no benefit or led to indel levels that surpassed editing efficiency (up to 26%), even when placed at distances >70 bp from the PEgRNA-induced nick, consistent with an unusual propensity of the edited strand at that site to be nicked by the cell, or to be ligated inefficiently. It is recommend to start with non-edited strand nicks approximately 50 bp from the PEgRNA-mediated nick, and to test alternative nick locations if indel frequencies exceed acceptable levels.
• This model for how complementary strand nicking improved prime editing efficiency (FIG. 40A) predicted that nicking the non-edited strand only after edited strand flap resolution could minimize the presence of concurrent nicks, decreasing the frequency of double-strand breaks that go on to form indels. To achieve temporal control over non-edited strand nicking, sgRNAs with spacer sequences that match the edited strand, but not the original allele, were designed. Using this strategy, referred to hereafter as PE3b, mismatches between the spacer and the unedited allele should disfavor nicking by the sgRNA until after the editing event on the PAM strand takes place. This PE3b approach was tested with five different edits at three genomic sites in HEK293T cells and compared outcomes to those achieved with PE2 and PE3 systems. In all cases, PE3b was associated with substantially lower levels of indels compared to PE3 (3.5- to 30-fold, averaging 12-fold lower indels, or 0.85%), without any evident decrease in overall editing efficiency compared to PE3 (FIG. 40C). Therefore, when the edit lay within a second protospacer, the PE3b system could decrease indels while still improving editing efficiency compared to PE2, often to levels similar to those of PE3 (FIG. 40C).
• Together, these findings established that PE3 systems (Cas9 nickase-optimized reverse transcriptase+PEgRNA+sgRNA) improved editing efficiencies ˜3-fold compared with PE2 (FIGS. 40B-40C). PE3 was accompanied by wider ranges of indels than PE2, as expected given the additional nicking activity of PE3. The use of PE3 is recommended when prioritizing prime editing efficiency. When minimization of indels is critical, PE2 offers ˜10-fold lower indel frequencies. When it is possible to use a sgRNA that recognizes the installed edit to nick the non-edited strand, the PE3b system can achieve PE3-like editing levels while greatly reducing indel formation.
• To demonstrate the targeting scope and versatility of prime editing with PE3, the installation of all possible single nucleotide substitutions across the +1 to +8 positions (counting the first base 3′ of the PEgRNA-induced nick as position +1) of the HEK3 target site using PE3 and PEgRNAs with 10-nucleotide RT templates (FIG. 41A) was explored. Collectively, these 24 distinct edits cover all four transition mutations and all eight transversion mutations, and proceed with editing efficiencies (containing no indels) averaging 33±7.9% (ranging between 14% and 48%), with an average of 7.5±1.8% indels.
• Importantly, long-distance RT templates could also give rise to efficient prime editing with PE3. For example, using PE3 with a 34-nt RT template, point mutations were installed at positions +12, +14, +17, +20, +23, +24, +26, +30, and +33 (12 to 33 bases from the PEgRNA-induced nick) in the HEK3 locus with an average of 36±8.7% efficiency and 8.6±2.0% indels (FIG. 41B). Although edits beyond the +10 position at other loci were not attempted, other RT templates ≥30 nt at three alternative sites also support efficient editing (FIGS. 48A-C). The viability of long RT templates enabled efficient prime editing for dozens of nucleotides from the initial nick site. Since an NGG PAM on either DNA strand occurs on average every ˜8 bp, far less than maximum distances between the edit and the PAM that support efficient prime editing, prime editing is not substantially constrained by the availability of a nearby PAM sequence, in contrast with other precision genome editing methods^125,142,154. Given the presumed relationship between RNA secondary structure and prime editing efficiency, when designing PEgRNAs for long-range edits it is prudent to test RT templates of various lengths and, if necessary, sequence compositions (e.g., synonymous codons) to optimize editing efficiency.
• To further test the scope and limitations of the PE3 system for introducing transition and transversion point mutations, 72 additional edits covering all 12 possible types of point mutations across six additional genomic target sites (FIG. 41C-41H) were tested. Overall, indel-free editing efficiency averaged 25±14%, while indel formation averaged 8.3±7.5%. Since the PEgRNA RT template included the PAM sequence, prime editing could induce changes to the PAM sequence. In these cases, higher editing efficiency (averaging 39±9.7%) and lower indel generation (averaging 5.0±2.9%) were observed (FIGS. 41A-41K, point mutations at positions +5 or +6). This increase in efficiency and decrease in indel formation for PAM edits may arise from the inability of the Cas9 nickase to re-bind and nick the edited strand prior to the repair of the complementary strand. Since prime editing supports combination edits with no apparent loss of editing efficiency, editing the PAM, in addition to other desired changes, when possible, is recommended.
• Next, 14 targeted small insertions and 14 targeted small deletions at seven genomic sites using PE3 (FIG. 41I) were performed. Targeted 1-bp insertions proceeded with an average efficiency of 32±9.8%, while 3-bp insertions were installed with an average efficiency of 39±16%. Targeted 1-bp and 3-bp deletions were also efficient, proceeding with an average yield of 29±14% and 32±11%, respectively. Indel generation (beyond the targeted insertion or deletion) averaged 6.8±5.4%. Since insertions and deletions introduced between positions +1 and +6 alter the position or the structure of the PAM, it was speculated that insertion and deletion edits in this range are typically more efficient due to the inability of Cas9 nickase to re-bind and nick the edited DNA strand prior to repair of the complementary strand, similar to point mutations that edit the PAM.
• PE3 was also tested for its ability to mediate larger precise deletions of 5 bp to 80 bp at the HEK3 site (FIG. 41J). Very high editing efficiencies (52 to 78%) were observed for 5-, 10-, and 15-bp deletions when using a 13-nt PBS and an RT template that contained 29, 24, or 19 bp of homology to the target locus, respectively. Using a 26-nt RT template supported a larger deletion of 25 bp with 72±4.2% efficiency, while a 20-nt RT template enabled an 80-bp deletion with an efficiency of 52±3.8%. These targeted deletions were accompanied by indel frequencies averaging 11±4.8% (FIG. 41J).
• Finally, the ability of PE3 to mediate 12 combinations of multiple edits at the same target locus consisting of insertions and deletions, insertions and point mutations, deletions and point mutations, or two point mutations across three genomic sites was tested. These combination edits were very efficient, averaging 55% of the target edit with 6.4% indels (FIG. 41K), and demonstrating the ability of prime editing to make combinations of precision insertions, deletions, and point mutations at individual target sites with high efficiency and low indel frequencies.
• Together, the examples in FIGS. 41A-41K represent 156 distinct transition, transversion, insertion, deletion, and combination edits across seven human genomic loci. These findings establish the versatility, precision, and targeting flexibility of prime editing.
• Prime Editing Compared with Base Editing
• Current-generation cytidine base editors (CBEs) and adenine base editors (ABEs) can install C●G-to-T●A transition mutations and A●T-to-G●C transition mutations with high efficiency and low indels^129,130,142. The application of base editing can be limited by the presence of multiple cytidine or adenine bases within the base editing activity window (typically ˜5-bp wide), which gives rise to unwanted bystander edits^{129,130,142,155,} or by the absence of a PAM positioned approximately 15±2 nt from the target nucleotide^142,156. It was anticipated that prime editing could be particularly useful for precise installation of transitions mutations without bystander edits, or when the lack of suitably positioned PAMs precludes favorable positioning the target nucleotide within the CBE or ABE activity window.
• Prime editing and cytosine base editing was compared by editing three genomic loci that contain multiple target cytidines in the canonical base editing window (protospacer positions 4-8, counting the PAM as positions 21-23) using optimized CBEs¹⁵⁷ without nickase activity (BE2max) or with nickase activity (BE4max), or using the analogous PE2 and PE3 prime editing systems. Among the nine total target cytosines within the base editing windows of the three sites, BE4max yielded 2.2-fold higher average total C●G-to-T●A conversion than PE3 for bases in the center of the base editing window (protospacer positions 5-7, FIG. 42A). Likewise, non-nicking BE2max outperformed PE2 by 1.4-fold on average at these well-positioned bases (FIG. 42A). However, PE3 outperformed BE4max by 2.7-fold, and PE2 outperformed BE2max by 2.0-fold, for cytosines beyond the center of the base editing window (average editing of 40±17% for PE3 vs. 15±18% for BE4max, and 22±11% for PE2 vs. 11±13% for BE2max). Overall, indel frequencies for PE2 were very low (averaging 0.86±0.47%), and for PE3 were similar to or modestly higher than that of BE4max (BE4max range: 2.5% to 14%; PE3 range: 2.5% to 21%) (FIG. 42B).
• When comparing the efficiency of base editing to prime editing for installation of precise C●G-to-T●A edits (without any bystander editing), the efficiency of prime editing greatly exceeded that of base editing at the above sites, which like most genomic DNA sites, contain multiple cytosines within the ˜5-bp base editing window (FIG. 42C). At these sites, such as EMX1, which contains cytosines at protospacer positions C5, C6, and C7, BE4max generated few products containing only the single target base pair conversion with no bystander edits. In contrast, prime editing at this site could be used to selectively install a C●G-to-T●A edit at any position or combination of positions (C5, C6, C7, C5+C6, C6+C7, C5+C7, or C5+C6+C7) (FIG. 42C). All precise one-base or two-base edits (that is, edits that do not modify any other nearby bases) were much more efficient with PE3 or PE2 than with BE4max or BE2, respectively, while the three-base C●G-to-T●A edit was more efficient with BE4max (FIG. 42C), reflecting the propensity of base editors to edit all target bases within the activity window. Taken together, these results demonstrate that cytosine base editors can result in higher levels of editing at optimally positioned target bases than PE2 or PE3, but prime editing can outperform base editing at non-optimally positioned target bases, and can edit with much higher precision with multiple editable bases.
• A●T-to-G●C editing was compared at two genomic loci by an optimized non-nicking ABE (ABEmax¹⁵² with a dCas9 instead of a Cas9 nickase, hereafter referred to as ABEdmax) versus PE2, and by the optimized nicking adenine base editor ABEmax versus PE3. At a site that contains two target adenines in the base editing window (HEK3), ABEs were more efficient than PE2 or PE3 for conversion of A5, but PE3 was more efficient for conversion of A8, which lies at the edge of the ABEmax editing window (FIG. 42D). When comparing the efficiency of precision edits in which only a single adenine is converted, PE3 outperformed ABEmax at both A5 and A8 (FIG. 42E). Overall, ABEs produced far fewer indels at HEK3 than prime editors (0.19±0.02% for ABEdmax vs. 1.5±0.46% for PE2, and 0.53±0.16% for ABEmax vs. 11±2.3% for PE3, FIG. 42F). At FANCF, in which only a single A is present within the base editing window, ABE2 and ABEmax outperformed their prime editing counterparts in total target base pair conversion by 1.8- to 2.9-fold, with virtually all edited products from both base editing and prime editing containing only the precise edit (FIGS. 42D-42E). As with the HEK3 site, ABEs produced far fewer indels at the FANCF site (FIG. 42F).
• Collectively, these results indicate that base editing and prime editing offer complementary strengths and weaknesses for making targeted transition mutations. For cases in which a single target nucleotide is present within the base editing window, or when bystander edits are acceptable, current base editors are typically more efficient and result in fewer indels than prime editors. When multiple cytosines or adenines are present and bystander edits are undesirable, or when target bases are poorly positioned for base editing relative to available PAMs, prime editors offer substantial advantages.
• Off-Target Prime Editing
• To result in productive editing, prime editing requires target locus:PEgRNA spacer complementary for the Cas9 domain to bind, target locus:PEgRNA PBS complementarity for PEgRNA-primed reverse transcription to initiate, and target locus:reverse transcriptase product complementarity for flap resolution. It was hypothesized that these three distinct DNA hybridization requirements may minimize off-target prime editing compared to that of other genome editing methods. To test this possibility, HEK293T cells were treated with PE3 or PE2 and 16 total PEgRNAs designed to target four on-target genomic loci, with Cas9 and the four corresponding sgRNAs targeting the same protospacers, or with Cas9 and the same 16 PEgRNAs. These four target loci were chosen because each has at least four well-characterized off-target sites for which Cas9 and the corresponding on-target sgRNA in HEK293T cells is known to cause substantial off-target DNA modification^118,159. Following treatment, the four on-target loci and the top four known Cas9 off-target sites for each on-target spacer, were sequenced, for a total of 16 off-target sites (Table 1).
• Consistent with previous studies¹¹⁸, Cas9 and the four target sgRNAs modified all 16 of the previously reported off-target loci (FIG. 42G). Cas9 off-target modification efficiency among the four off-target sites for the HEK3 target locus averaged 16%. Cas9 and the sgRNA targeting HEK4 resulted in an average of 60% modification of the four tested known off-target sites. Likewise, off-target sites for EMX1 and FANCF were modified by Cas9:sgRNA at an average frequency of 48% and 4.3%, respectively (FIG. 42G). It was noted that PEgRNAs with Cas9 nuclease modified on-target sites at similar (1- to 1.5-fold lower) efficiency on average compared to sgRNAs, while PEgRNAs with Cas9 nuclease modified off-target sites at ˜4-fold lower average efficiency than sgRNAs.
• Strikingly, PE3 or PE2 with the same 16 tested PEgRNAs containing these four target spacers resulted in much lower off-target editing (FIG. 42H). Of the 16 sites known to undergo off-target editing by Cas9+sgRNA, PE3+PEgRNAs or PE2+PEgRNAs resulted in detectable off-target prime editing at only 3 of 16 off-target sites, with only 1 of 16 showing off-target editing efficiency ≥1% (FIG. 42H). Average off-target prime editing for the PEgRNAs targeting HEK3, HEK4, EMX1, and FANCF at these 16 known Cas9 off-target sites was <0.1%, <2.2±5.2%, <0.1%, and <0.13±0.11%, respectively (FIG. 42H). Notably, at the HEK4 off-target 3 site that Cas9+PEgRNA1 edits with 97% efficiency, PE2+PEgRNA1 results in only 0.7% off-target editing despite sharing the same spacer sequence, demonstrating how the two additional DNA hybridization events required for prime editing compared to Cas9 editing can greatly reduce off-target editing. Taken together, these results suggest that PE3 and PEgRNAs induce much lower off-target DNA editing in human cells than Cas9 and sgRNAs that target the same protospacers.
• Reverse transcription of 3′-extended PEgRNAs in principle can proceed into the guide RNA scaffold. If the resulting 3′ flap, despite a lack of complementary at its 3′ end with the unedited DNA strand, is incorporated into the target locus, the outcome is insertion of PEgRNA scaffold nucleotides that contributes to indel frequency. We analyzed sequencing data from 66 PE3-mediated editing experiments at four loci in HEK293T cells and observed PEgRNA scaffold insertion at a low frequency, averaging 1.7±1.5% total insertion of any number of PEgRNA scaffold nucleotides (FIGS. 56A-56D). It is speculated that inaccessibility of the guide RNA scaffold to the reverse transcriptase due to Cas9 domain binding, as well as cellular excision during flap resolution of the mismatched 3′ end of the 3′ flap that results from PEgRNA scaffold reverse transcription, minimizes products that incorporate PEgRNA scaffold nucleotides. While such events are rare, future efforts to engineer PEgRNAs or prime editor proteins that minimize PEgRNA scaffold incorporation may further decrease indel frequencies.
• Deaminases in some base editors can act in a Cas9-independent manner, resulting in low-level but widespread off-target DNA editing among first-generation CBEs (but not ABEs)^160-162 and off-target RNA editing among first-generation CBEs and ABEs^163-165, although newer CBE and ABE variants with engineered deaminases greatly reduce Cas9-independent off-target DNA and RNA editing^163-165. Prime editors lack base-modification enzymes such as deaminases, and therefore have no inherent ability to modify DNA or RNA bases in a Cas9-independent manner.
• While the reverse transcriptase domain in prime editors in principle could process properly primed RNA or DNA templates in cells, it was noted that retrotransposons such as those in the LINE-1 family¹⁶⁶, endogenous retroviruses^167,168, and human telomerase all provided active endogenous human reverse transcriptases. Their natural presence in human cells suggests that reverse transcriptase activity itself is not substantially toxic. Indeed, no PE3-dependent differences were observed in HEK293T cell viability compared to that of controls expressing dCas9, Cas9 H840A nickase, or PE2 with R110S+K103L (PE2-dRT) mutations that inactivate the reverse transcriptase¹⁶⁹ (FIGS. 49A-49B).
• The above data and analyses notwithstanding, additional studies are needed to assess off-target prime editing in an unbiased, genome-wide manner, as well as to characterize the extent to which the reverse transcriptase variants in prime editors, or prime editing intermediates, may affect cells.
• Prime Editing Pathogenic Transversion, Insertion, and Deletion Mutations in Human Cells
• The ability of PE3 to directly install or correct in human cells transversion, small insertion, and small deletion mutations that cause genetic diseases, was tested. Sickle cell disease is most commonly caused by an A●T-to-T●A transversion mutation in HBB, resulting in the mutation of Glu6→Val in beta-globin. Treatment of hematopoietic stem cells ex vivo with Cas9 nuclease and a donor DNA template for HDR, followed by enrichment of edited cells, transplantation, and engraftment is a promising potential strategy for the treatment of sickle-cell disease¹⁷⁰. However, this approach still generates many indel-containing byproducts in addition to the correctly edited HBB allele^170-171. While base editors generally produce far fewer indels, they cannot currently make the T●A-to-A●T transversion mutation needed to directly restore the normal sequence of HBB.
• PE3 was used to install the HBB E6V mutation in HEK293T cells with 44% efficiency and 4.8% indels (FIG. 43A. From the mixture of PE3-treated cells, we isolated six HEK293T cell lines that are homozygous (triploid) for the HBB E6V allele (FIGS. 53A-53D), demonstrating the ability of prime editing to generate human cell lines with pathogenic mutations. To correct the HBB E6V allele to wild-type HBB, we treated homozygous HBB E6V HEK293T cells with PE3 and a PEgRNA programmed to directly revert the HBB E6V mutation to wild-type HBB. In total, 14 PEgRNA designs were tested. After three days, DNA sequencing revealed that all 14 PEgRNAs when combined with PE3 gave efficient correction of HBB E6V to wild-type HBB (≥26% wild-type HBB without indels), and indel levels averaging 2.8±0.70% (FIG. 50A). The best PEgRNA resulted in 52% correction of HBB E6V to wild-type with 2.4% indels (FIG. 43A). Introduction of a silent mutation that modifies the PAM recognized by the PEgRNA modestly improved editing efficiency and product purity, to 58% correction with 1.4% indels (FIG. 43A). These results establish that prime editing can install and correct a pathogenic transversion point mutation in a human cell line with high efficiency and minimal byproducts.
• Tay-Sachs disease is most often caused by a 4-bp insertion into the HEXA gene (HEXA 1278+TATC)¹³⁶. PE3 was used to install this 4-bp insertion into HEK293T cells with 31% efficiency and 0.8% indels (FIG. 43B), and isolated two HEK293T cell lines that are homozygous for the HEXA 1278+TATC allele (FIGS. 53A-53D). These cells were used to test 43 PEgRNAs and three nicking sgRNAs with PE3 or PE3b systems for correction of the pathogenic insertion in HEXA (FIG. 50B), either by perfect reversion to the wild-type allele or by a shifted 4-bp deletion that disrupts the PAM and installs a silent mutation. Nineteen of the 43 PEgRNAs tested resulted in ≥20% editing. Perfect correction to wild-type HEXA with PE3 or PE3b and the best PEgRNA proceeded with similar average efficiencies (30% for PE3 vs. 33% for PE3b), but the PE3b system was accompanied by 5.3-fold fewer indel products (1.7% for PE3 vs. 0.32% for PE3b) (FIG. 43B and FIG. 50B). These findings demonstrate the ability of prime editing to make precise small insertions and deletions that install or correct a pathogenic allele in mammalian cells efficiently and with a minimum of byproducts.
• Finally, the installation of a protective SNP into PRNP, the gene encoding the human prion protein (PrP), was tested. PrP misfolding causes progressive and fatal neurodegenerative prion disease that can arise spontaneously, through inherited dominant mutations in the PRNP gene, or through exposure to misfolded PrP¹⁷². A naturally occurring PRNP G127V mutant allele confers resistance to prion disease in humans¹³⁸ and mice¹⁷³. PE3 was used to install G127V into the human PRNP allele in HEK293T cells, which requires a G●C-to-T●A transversion. Four PEgRNAs and three nicking sgRNAs were evaluated with the PE3 system. After three days of exposure to the most effective PE3 and PEgRNA, DNA sequencing revealed 53±11% efficiency of installing the G127V mutation and indel levels of 1.7±0.7% (FIG. 43C). Taken together, these results establish the ability of prime editing in human cells to install or correct transversion, insertion, or deletion mutations that cause or confer resistance to disease efficiently, and with a minimum of byproducts.
• Prime Editing in Various Human Cell Lines and Primary Mouse Neurons
• Next, prime editing was tested for its ability to edit endogenous sites in three additional human cell lines. In K562 (leukemic bone marrow) cells, PE3 was used to perform transversion edits in the HEK3, EMX1, and FANCF sites, as well as the 18-bp insertion of a 6×His tag in HEK3. An average editing efficiency of 15-30% was observed for each of these four PE3-mediated edits, with indels averaging 0.85-2.2% (FIG. 43A). In U2OS (osteosarcoma) cells, transversion mutations in HEK3 and FANCF were installed, as well as a 3-bp insertion and 6×His tag insertion into HEK3, with 7.9-22% editing efficiency that exceeded indel formation 10- to 76 fold (FIG. 43A). Finally, in HeLa (cervical cancer) cells, a 3-bp insertion into HEK3 was performed, with 12% average efficiency and 1.3% indels (FIG. 43A). Collectively, these data indicate that multiple cell lines beyond HEK293T cells support prime editing, although editing efficiencies vary by cell type and are generally less efficient than in HEK293T cells. Editing:indel ratios remained high in all tested human cell lines.
• To determine if prime editing is possible in post-mitotic, terminally differentiated primary cells, primary cortical neurons harvested from E18.5 mice were transduced with a dual split-PE3 lentiviral delivery system in which split-intein splicing²⁰³ reconstitutes PE2 protein from N-terminal and C-terminal halves, each delivered from a separate virus. To restrict editing to post-mitotic neurons, the human synapsin promoter, which is highly specific for mature neurons²⁰⁴, was used to drive expression of both PE2 protein components. GFP was fused through a self-cleaving P2A peptide²⁰⁵ to the N-terminal half of PE2. Nuclei from neurons were isolated two weeks following dual viral transduction and were sequenced directly, or sorted for GFP expression before sequencing. A 7.1±1.2% average prime editing to install a transversion at the DNMT1 locus with 0.58±0.14% average indels in sorted nuclei (FIG. 43D was observed. Cas9 nuclease in the same split-intein dual lentivirus system resulted in 31±5.5% indels among sorted cortical neuron nuclei (FIG. 43D. These data indicate that post-mitotic, terminally differentiated primary cells can support prime editing, and thus establish that prime editing does not require cell replication.
• Prime Editing Compared with Cas9-Initiated HDR
• The performance of PE3 was compared with that of optimized Cas9-initiated HDR^128,125 in mitotic cell lines that support HDR¹²⁸. HEK293T, HeLa, K562 and U2OS cells were treated with Cas9 nuclease, a sgRNA, and an ssDNA donor oligonucleotide template designed to install a variety of transversion and insertion edits (FIGS. 43E-43G, and FIGS. 51A-51F). Cas9-initiated HDR in all cases successfully installed the desired edit, but with far higher levels of byproducts (predominantly indels), as expected from treatments that cause double-stranded breaks. Using PE3 in HEK293T cells, HBB E6V installation and correction proceeded with 42% and 58% average editing efficiency with 2.6% and 1.4% average indels, respectively (FIG. 43E and FIG. 43G). In contrast, the same edits with Cas9 nuclease and an HDR template resulted in 5.2% and 6.7% average editing efficiency, with 79% and 51% average indel frequency (FIG. 43E and FIG. 43G). Similarly, PE3 installed PRNP G127V with 53% efficiency and 1.7% indels, whereas Cas9-initiated HDR installed this mutation with 6.9% efficiency and 53% indels (FIG. 43E and FIG. 43G). Thus, the ratio of editing:indels for HBB E6V installation, HBB E6V correction, and PRNP G127V installation on average was 270-fold higher for PE3 than for Cas9-initiated HDR.
• Comparisons between PE3 and HDR in human cell lines other than HEK293T showed similar results, although with lower PE3 editing efficiencies. For example, in K562 cells, PE3-mediated 3-bp insertion into HEK3 proceeded with 25% efficiency and 2.8% indels, compared with 17% editing and 72% indels for Cas9-initiated HDR, a 40-fold editing:indel ratio advantage favoring PE3 (FIGS. 43F-43G). In U2OS cells, PE3 performed this 3-bp insertion with 22% efficiency and 2.2% indels, while Cas9-initiated HDR resulted in 15% editing with 74% indels, a 49-fold lower editing:indel ratio (FIGS. 43F-43G). In HeLa cells, PE3 made this insertion with 12% efficiency and 1.3% indels, versus 3.0% editing and 69% indels for Cas9-initiated HDR, a 210-fold editing:indel ratio difference (FIGS. 43F-43G). Collectively, these data indicated that HDR typically results in similar or lower editing efficiencies and far higher indels than PE3 in the four cell lines tested (FIGS. 51A-51F).
• Discussion and Future Directions
• The ability to insert DNA sequences with single-nucleotide precision is an especially enabling prime editing capability. For example, PE3 was used to precisely insert into the HEK3 locus in HEK293T cells a His6 tag (18 bp, 65% average efficiency), a FLAG epitope tag (24 bp, 18% average efficiency), and an extended LoxP site (44 bp, 23% average efficiency) that is the native substrate for Cre recombinase. Average indels ranged between 3.0% and 5.9% for these examples (FIG. 43H). Many biotechnological, synthetic biology, and therapeutic applications are envisioned to arise from the ability to efficiently and precisely introduce new DNA sequences into target sites of interest in living cells.
• Collectively, the prime editing experiments described herein installed 18 insertions up to 44 bp, 22 deletions up to 80 bp, 113 point mutations including 77 transversions, and 18 combination edits, across 12 endogenous loci in the human and mouse genomes at locations ranging from 3 bp upstream to 29 bp downstream of the start of a PAM without making explicit double-stranded DNA breaks. These results establish prime editing as a remarkably versatile genome editing method. Because the overwhelming majority (85-99%) of insertions, deletions, indels, and duplications in ClinVar are ≤30 bp (FIGS. 52A-52D), in principle prime editing can correct up to ˜89% of the 75,122 currently known pathogenic human genetic variants in ClinVar (transitions, transversions, insertions, deletions, indels, and duplications in FIG. 38A), with additional potential to ameliorate diseases caused by copy number gain or loss.
• Importantly, for any desired edit the flexibility of prime editing offers many possible choices of PEgRNA-induced nick locations, sgRNA-induced second nick locations, PBS lengths, RT template lengths, and which strand to edit first, as demonstrated extensively herein. This flexibility, which contrasts with more limited options typically available for other precision genome editing methods^125,142,154, allows editing efficiency, product purity, DNA specificity, or other parameters to be optimized to suit the needs of a given application, as shown in FIGS. 50A-50B in which testing 14 and 43 PEgRNAs covering a range of prime editing strategies optimized correction of pathogenic HBB and HEXA alleles, respectively.
• Much additional research is needed to further understand and improve prime editing. Additional modifications of prime editor systems may be required to expand their compatibility to include other cell types, such as post-mitotic cells. Interfacing prime editing with viral and non-viral in vitro and in vivo delivery strategies is needed to fully explore the potential of prime editing to enable a wide range of applications including the study and treatment of genetic diseases. By enabling highly precise targeted transitions, transversions, small insertions, and small deletions in the genomes of mammalian cells without requiring double-stranded breaks or HDR, however, prime editing provides a new “search-and-replace” capability that substantially expands the scope of genome editing.
Methods
• General Methods
• DNA amplification was conducted by PCR using Phusion U Green Multiplex PCR Master Mix (ThermoFisher Scientific) or Q5 Hot Start High-Fidelity 2× Master Mix (New England BioLabs) unless otherwise noted. DNA oligonucleotides, including Cy5-labeled DNA oligonucleotides, dCas9 protein, and Cas9 H840A protein were obtained from Integrated DNA Technologies. Yeast reporter plasmids were derived from previously described plasmids64 and cloned by the Gibson assembly method. All mammalian editor plasmids used herein were assembled using the USER cloning method as previously described¹⁷⁵. Plasmids expressing sgRNAs were constructed by ligation of annealed oligonucleotides into BsmBI-digested acceptor vector. Plasmids expressing PEgRNAs were constructed by Gibson assembly or Golden Gate assembly using a custom acceptor plasmid (see supplemental ‘Golden Gate assembly’ outline). Sequences of sgRNA and PEgRNA constructs used herein are listed in Tables 2A-2C and Tables 3A-3R. All vectors for mammalian cell experiments were purified using Plasmid Plus Midiprep kits (Qiagen) or PureYield plasmid miniprep kits (Promega), which include endotoxin removal steps. All experiments using live animals were approved by the Broad Institute Institutional and Animal Care and Use Committees. Wild-type C57BL/6 mice were obtained from Charles River (#027).
• In Vitro Biochemical Assays
• PEgRNAs and sgRNAs were transcribed in vitro using the HiScribe T7 in vitro transcription kit (New England Biolabs) from PCR-amplified templates containing a T7 promoter sequence. RNA was purified by denaturing urea PAGE and quality-confirmed by an analytical gel prior to use. 5′-Cy5-labeled DNA duplex substrates were annealed using two oligonucleotides (Cy5-AVA024 and AVA025; 1:1.1 ratio) for the non-nicked substrate or three oligonucleotides (Cy5-AVA023, AVA025 and AVA026; 1:1.1:1.1) for the pre-nicked substrate by heating to 95° C. for 3 minutes followed by slowly cooling to room temperature (Tables 2A-2C). Cas9 cleavage and reverse transcription reactions were carried out in 1× cleavage buffer²⁰⁵ supplemented with dNTPs (20 mM HEPES-K, pH 7.5; 100 mM KCl; 5% glycerol; 0.2 mM EDTA, pH 8.0; 3 mM MgCl2; 0.5 mM dNTP mix; 5 mM DTT). dCas9 or Cas9 H840A (5 μM final) and the sgRNA or PEgRNA (5 μM final) were pre-incubated at room temperature in a 5 μL reaction mixture for 10 minutes prior to the addition of duplex DNA substrate (400 nM final), followed by the addition of Superscript III reverse transcriptase (ThermoFisher Scientific), an undisclosed M-MLV RT variant, when applicable. Reactions were carried out at 37° C. for 1 hour, then diluted to a volume of 10 μL with water, treated with 0.2 μL of proteinase K solution (20 mg/mL, ThermoFisher Scientific), and incubated at room temperature for 30 minutes. Following heat inactivation at 95° C. for 10 minutes, reaction products were combined with 2× formamide gel loading buffer (90% formamide; 10% glycerol; 0.01% bromophenol blue), denatured at 95° C. for 5 minutes, and separated by denaturing urea-PAGE gel (15% TBE-urea, 55° C., 200V). DNA products were visualized by Cy5 fluorescence signal using a Typhoon FLA 7000 biomolecular imager.
• Electrophoretic mobility shift assays were carried out in 1× binding buffer (1× cleavage buffer+10 μg/mL heparin) using pre-incubated dCas9:sgRNA or dCas9:PEgRNA complexes (concentration range between 5 nM and 1 μM final) and Cy5-labeled duplex DNA (Cy5-AVA024 and AVA025; 20 nM final). After 15 minutes of incubation at 37° C., the samples were analyzed by native PAGE gel (10% TBE) and imaged for Cy5 fluorescence.
• For DNA sequencing of reverse transcription products, fluorescent bands were excised and purified from urea-PAGE gels, then 3′ tailed with terminal transferase (TdT; New England Biolabs) in the presence of dGTP or dATP according to the manufacturer's protocol. Tailed DNA products were diluted 10-fold with binding buffer (40% saturated aqueous guanidinium chloride+60% isopropanol) and purified by QIAquick spin column (Qiagen), then used as templates for primer extension by Klenow fragment (New England Biolabs) using primer AVA134 (A-tailed products) or AVA135 (G-tailed products) (Tables 2A-2C). Extension were amplified by PCR for 10 cycles using primers AVA110 and AVA122, then sequenced with AVA037 using the Sanger method (Tables 2A-2C).
• Yeast Fluorescent Reporter Assays
• Dual fluorescent reporter plasmids containing an in-frame stop codon, a +1 frameshift, or a −1 frameshift were subjected to 5′-extended PEgRNA or 3′-extended PEgRNA prime editing reactions in vitro as described above. Following incubation at 37° C. for 1 hour, the reactions were diluted with water and plasmid DNA was precipitated with 0.3 M sodium acetate and 70% ethanol. Resuspended DNA was transformed into S. cerevisiae by electroporation as previously described⁶⁷ and plated on synthetic complete media without leucine (SC(glucose), L−). GFP and mCherry fluorescence signals were visualized from colonies with the Typhoon FLA 7000 biomolecular imager.
• General Mammalian Cell Culture Conditions
• HEK293T (ATCC CRL-3216), U2OS (ATTC HTB-96), K562 (CCL-243), and HeLa (CCL-2) cells were purchased from ATCC and cultured and passaged in Dulbecco's Modified Eagle's Medium (DMEM) plus GlutaMAX (ThermoFisher Scientific), McCoy's 5A Medium (Gibco), RPMI Medium 1640 plus GlutaMAX (Gibco), or Eagle's Minimal Essential Medium (EMEM, ATCC), respectively, each supplemented with 10% (v/v) fetal bovine serum (Gibco, qualified) and 1× Penicillin Streptomycin (Corning). All cell types were incubated, maintained, and cultured at 37° C. with 5% CO₂. Cell lines were authenticated by their respective suppliers and tested negative for Mycoplasma.
• HEK293T Tissue Culture Transfection Protocol and Genomic DNA Preparation
• HEK293T cells grown were seeded on 48-well poly-D-lysine coated plates (Corning). 16 to 24 hours post-seeding, cells were transfected at approximately 60% confluency with 1 μL of Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer's protocols and 750 ng of PE plasmid, 250 ng of PEgRNA plasmid, and 83 ng of sgRNA plasmid (for PE3 and PE3b). Unless otherwise stated, cells were cultured 3 days following transfection, after which the media was removed, the cells were washed with 1×PBS solution (Thermo Fisher Scientific), and genomic DNA was extracted by the addition of 150 μL of freshly prepared lysis buffer (10 mM Tris-HCl, pH 7.5; 0.05% SDS; 25 μg/mL Proteinase K (ThermoFisher Scientific)) directly into each well of the tissue culture plate. The genomic DNA mixture was incubated at 37° C. for 1 to 2 hours, followed by an 80° C. enzyme inactivation step for 30 minutes. Primers used for mammalian cell genomic DNA amplification are listed in Table 4. For HDR experiments in HEK293T cells, 231 ng of nuclease-expression plasmid, 69 ng of sgRNA expression plasmid, 50 ng (1.51 pmol) 100-nt ssDNA donor template (PAGE-purified; Integrated DNA Technologies) was lipofected using 1.4 μL Lipofectamine 2000 (ThermoFisher) per well. Genomic DNA from all HDR experiments was purified using the Agencourt DNAdvance Kit (Beckman Coulter), according to the manufacturer's protocol.
• High-Throughput DNA Sequencing of Genomic DNA Samples
• Genomic sites of interest were amplified from genomic DNA samples and sequenced on an Illumina MiSeq as previously described with the following modifications^129,130. Briefly, amplification primers containing Illumina forward and reverse adapters (Table 4) were used for a first round of PCR (PCR 1) amplifying the genomic region of interest. 25 μL PCR 1 reactions were performed with 0.5 μM of each forward and reverse primer, 1 μL of genomic DNA extract and 12.5 μL of Phusion U Green Multiplex PCR Master Mix. PCR reactions were carried out as follows: 98° C. for 2 minutes, then 30 cycles of [98° C. for 10 seconds, 61° C. for 20 seconds, and 72° C. for 30 seconds], followed by a final 72° C. extension for 2 minutes. Unique Illumina barcoding primer pairs were added to each sample in a secondary PCR reaction (PCR 2). Specifically, 25 μL of a given PCR 2 reaction contained 0.5 μM of each unique forward and reverse illumina barcoding primer pair, 1 μL of unpurified PCR 1 reaction mixture, and 12.5 μL of Phusion U Green Multiplex PCR 2× Master Mix. The barcoding PCR 2 reactions were carried out as follows: 98° C. for 2 minutes, then 12 cycles of [98° C. for 10 seconds, 61° C. for 20 seconds, and 72° C. for 30 seconds], followed by a final 72° C. extension for 2 minutes. PCR products were evaluated analytically by electrophoresis in a 1.5% agarose gel. PCR 2 products (pooled by common amplicons) were purified by electrophoresis with a 1.5% agarose gel using a QIAquick Gel Extraction Kit (Qiagen), eluting with 40 μL of water. DNA concentration was measured by fluorometric quantification (Qubit, ThermoFisher Scientific) or qPCR (KAPA Library Quantification Kit-Illumina, KAPA Biosystems) and sequenced on an Illumina MiSeq instrument according to the manufacturer's protocols.
• Sequencing reads were demultiplexed using MiSeq Reporter (Illumina). Alignment of amplicon sequences to a reference sequence was performed using CRISPResso2¹⁷⁸. For quantification of point mutation editing, CRISPResso2 was run in standard mode with “discard_indel_reads” on. Editing efficiency was calculated as: (frequency of specified point mutation in non-discarded reads)×(#of non-discarded reads)÷total reads. For insertion or deletion edits, CRISPResso2 was run in HDR mode using the desired allele as the expected allele (e flag), and with “discard_indel_reads” ON. Editing yield was calculated as the number of HDR aligned reads divided by total reads. For all edits, indel yields were calculated as the number of discarded reads divided by total reads.
• Nucleofection of U2OS, K562, and HeLa Cells
• Nucleofection was performed in all experiments using K562, HeLa, and U2OS cells. For PE conditions in these cell types, 800 ng prime editor-expression plasmid, 200 ng PEgRNA-expression plasmid, and 83 ng nicking plasmid was nucleofected in a final volume of 20 μL in a 16-well nucleocuvette strip (Lonza). For HDR conditions in these three cell types, 350 ng nuclease-expression plasmid, 150 ng sgRNA-expression plasmid and 200 pmol (6.6 μg) 100-nt ssDNA donor template (PAGE-purified; Integrated DNA Technologies) was nucleofected in a final volume of 20 μL per sample in a 16-well Nucleocuvette strip (Lonza). K562 cells were nucleofected using the SF Cell Line 4D-Nucleofector X Kit (Lonza) with 5×10⁵ cells per sample (program FF-120), according to the manufacturers protocol. U2OS cells were nucleofected using the SE Cell Line 4D-Nucleofector X Kit (Lonza) with 3-4×10⁵ cells per sample (program DN-100), according to the manufacturers protocol. HeLa cells were nucleofected using the SE Cell Line 4D-Nucleofector X Kit (Lonza) with 2×10⁵ cells per sample (program CN-114), according to the manufacturers protocol. Cells were harvested 72 hours after nucleofection for genomic DNA extraction.
• Genomic DNA Extraction for HDR Experiments
• Genomic DNA from all HDR comparison experiments in HEK293T, HEK293T HBB E6V, K562, U2OS, and HeLa cells was purified using the Agencourt DNAdvance Kit (Beckman Coulter), according to the manufacturer's protocol.
• Comparison Between PE2, PE3, BE2, BE4max, ABEdmax, and ABEmax
• HEK293T Cells were Seeded on 48-Well Poly-D-Lysine Coated Plates (Corning). After 16 to 24 hours, cells were transfected at approximately 60% confluency. For base editing with CBE or ABE constructs, cells were transfected with 750 ng of base editor plasmid, 250 ng of sgRNA expression plasmid, and 1 μL of Lipofectamine 2000 (Thermo Fisher Scientific). PE transfections were performed as described above. Genomic DNA extraction for PE and BE was performed as described above.
• Determination of PE3 Activity at Known Cas9 Off-Target Sites
• To evaluate PE3 off-target editing activity at known Cas9 off-target sites, genomic DNA extracted from HEK293T cells 3 days after transfection with PE3 was used as template for PCR amplification of 16 previously reported Cas9 off-target genomic sites^118,159 (the top four off-target sites each for the HEK3, EMX1, FANCF, and HEK4 spacers; primer sequences are listed in Table 4). These genomic DNA samples were identical to those used for quantifying on-target PE3 editing activities shown in FIGS. 41A-41K; PEgRNA and nicking sgRNA sequences are listed in Tables 3A-3R. Following PCR amplification of off-target sites, amplicons were sequenced on the Illumina MiSeq platform as described above (HTS analysis). For determining Cas9 nuclease, Cas9 H840A nickase, dCas9, and PE2-dRT on-target and off-target editing activity, HEK293T cells were transfected with 750 ng of editor plasmid (Cas9 nuclease, Cas9 H840A nickase, dCas9, or PE2-dRT), 250 ng of PEgRNA or sgRNA plasmid, and 1 μL of Lipofectamine 2000. Genomic DNA was isolated from cells 3 days after transfection as described above. On-target and off-target genomic loci were amplified by PCR using primer sequences in Table 4 and sequenced on an Illumina MiSeq.
• HTS data analysis was performed using CRISPResso2¹⁷⁸. The editing efficiencies of Cas9 nuclease, Cas9 H840A nickase, and dCas9 were quantified as the percent of total sequencing reads containing indels. For quantification of PE3 and PE3-dRT off-targets, aligned sequencing reads were examined for point mutations, insertions, or deletions that were consistent with the anticipated product of PEgRNA reverse transcription initiated at the Cas9 nick site. Single nucleotide variations occurring at <0.1% overall frequency among total reads within a sample were excluded from analysis. For reads containing single nucleotide variations that both occurred at frequencies ≥0.1% and were partially consistent with the PEgRNA-encoded edit, t-tests (unpaired, one-tailed, α=0.5) were used to determine if the variants occurred at significantly higher levels compared to samples treated with PEgRNAs that contained the same spacer but encoded different edits. To avoid differences in sequencing errors, comparisons were made between samples that were sequenced simultaneously within the same MiSeq run. Variants that did not meet the criteria of p-value >0.05 were excluded. Off-target PE3 editing activity was then calculated as the percentage of total sequencing reads that met the above criteria.
• Generation of a HEK293T Cell Line Containing the HBB E6V Mutation Using Cas9-Initiated HDR
• HEK293T cells were seeded in a 48-well plate and transfected at approximately 60% confluency with 1.5 μL of Lipofectamine 2000, 300 ng of Cas9 D10A nickase plasmid, 100 ng of sgRNA plasmid, and 200 ng of 100-mer ssDNA donor template (Table 5). Three days after transfection, media was exchanged for fresh media. Four days after transfection, cells were dissociated using 30 μL of TrypLE solution and suspended in 1.5 mL of media. Single cells were isolated into individual wells of two 96-well plates by fluorescence-activated cell sorting (FACS) (Beckman-Coulter Astrios). See FIGS. 53A-53B for representative FACS sorting examples. Cells were expanded for 14 days prior to genomic DNA sequencing as described above. Of the isolated clonal populations, none was found to be homozygous for the HBB E6V mutation, so a second round of editing by lipofection, sorting, and outgrowth was repeated in a partially edited cell line to yield a cell line homozygous for the E6V allele.
• Generation of a HEK293T Cell Line Containing the HBB E6V Mutation Using PE3
• 2.5×104 HEK293T cells grown in the absence of antibiotic were seeded on 48-well poly-D-lysine coated plates (Corning). 16 to 24 hours post-seeding, cells were transfected at approximately 70% confluency with 1 μL of Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer's protocols and 750 ng of PE2-P2A-GFP plasmid, 250 ng of PEgRNA plasmid, and 83 ng of sgRNA plasmid. After 3 days post transfection, cells were washed with phosphate-buffered saline (Gibco) and dissociated using TrypLE Express (Gibco). Cells were then diluted with DMEM plus GlutaMax (Thermo Fisher Scientific) supplemented with 10% (v/v) FBS (Gibco) and passed through a 35-μm cell strainer (Corning) prior to sorting. Flow cytometry was carried out on a LE-MA900 cell sorter (Sony). Cells were treated with 3 nM DAPI (BioLegend) 15 minutes prior to sorting. After gating for doublet exclusion, single DAPI-negative cells with GFP fluorescence above that of a GFP-negative control cell population were sorted into 96-well flat-bottom cell culture plates (Corning) filled with pre-chilled DMEM with GlutaMax supplemented with 10% FBS. See FIGS. 53A-53B for representative FACS sorting examples. Cells were cultured for 10 days prior to genomic DNA extraction and characterization by HTS, as described above. A total of six clonal cell lines were identified that are homozygous for the E6V mutation in HBB.
• Generation of a HEK293T Cell Line Containing the HEXA 1278+TATC Insertion Using PE3
• HEK293T cells containing the HEXA 1278+TATC allele were generated following the protocol described above for creation of the HBB E6V cell line; PEgRNA and sgRNA sequences are listed in Tables 2A-2C under the FIGS. 43A-43H subheading. After transfection and sorting, cells were cultured for 10 days prior to genomic DNA extraction and characterization by HTS, as described above. Two heterozygous cell lines were isolated that contained 50% HEXA 1278+TATC alleles, and two homozygous cell lines containing 100% HEXA 1278+TATC alleles were recovered.
• Cell Viability Assays
• HEK293T cells were seeded in 48-well plates and transfected at approximately 70% confluency with 750 ng of editor plasmid (PE3, PE3 R110S K103L, Cas9 H840A nickase, or dCas9), 250 ng of HEK3-targeting PEgRNA plasmid, and 1 μL of Lipofectamine 2000, as described above. Cell viability was measured every 24 hours post-transfection for 3 days using the CellTiter-Glo 2.0 assay (Promega) according to the manufacturer's protocol. Luminescence was measured in 96-well flat-bottomed polystyrene microplates (Corning) using a M1000 Pro microplate reader (Tecan) with a 1-second integration time.
• Lentivirus Production
• Lentivirus was produced as previously described²⁰⁶. T-75 flasks of rapidly dividing HEK293T cells (ATCC; Manassas, Va., USA) were transfected with lentivirus production helper plasmids pVSV-G and psPAX2 in combination with modified lentiCRISPR_v2 genomes carrying intein-split PE2 editor using FuGENE HD (Promega, Madison, Wis., USA) according to the manufacturer's directions. Four split-intein editor constructs were designed: 1) a viral genome encoding a U6-PEgRNA expression cassette and the N-terminal portion (1-573) of Cas9 H840A nickase fused to the Npu N-intein, a self-cleaving P2A peptide, and GFP-KASH; 2) a viral genome encoding the Npu C-intein fused to the C-terminal remainder of PE2; 3) a viral genome encoding the Npu C-intein fused to the C-terminal remainder of Cas9 for the Cas9 control; and 4) a nicking sgRNA for DNMT1. The split-intein mediates trans splicing to join the two halves of PE2 or Cas9, while the P2A GFP-KASH enables co-translational production of a nuclear membrane-localized GFP. After 48 hours, supernatant was collected, centrifuged at 500 g for 5 minutes to remove cellular debris, and filtered using a 0.45 μm filter. Filtered supernatant was concentrated using the PEG-it Virus Precipitation Solution (System Biosciences, Palo Alto, Calif., USA) according to the manufacturer's directions. The resulting pellet was resuspended in Opti-MEM (Thermo Fisher Scientific, Waltham, Mass., USA) using 1% of the original media volume. Resuspended pellet was flash-frozen and stored at −80° C. until use.
• Mouse Primary Cortical Neuron Dissection and Culture
• E18.5 dissociated cortical cultures were harvested from timed-pregnant C57BL/6 mice (Charles River). Embryos were harvested from pregnant mice after euthanasia by CO2 followed by decapitation. Cortical caps were dissected in ice-cold Hibernate-E supplemented with penicillin/streptomycin (Life Technologies). Following a rinse with ice-cold Hibernate-E, tissue was digested at 37° C. for 8 minutes in papain/DNase (Worthington/Sigma). Tissue was triturated in NBActiv4 (BrainBits) supplemented with DNase. Cells were counted and plated in 24-well plates at 100,000 cells per well. Half of the media was changed twice per week.
• Prime Editing in Primary Neurons and Nuclei Isolation
• At DIV 1, 15 μL of lentivirus was added at 10:10:1 ratio of N-terminal:C-terminal:nicking sgRNA. At DIV 14, neuronal nuclei were isolated using the EZ-PREP buffer (Sigma D8938) following the manufacturer's protocol. All steps were performed on ice or at 4° C. Media was removed from dissociated cultures, and cultures were washed with ice-cold PBS. PBS was aspirated and replaced with 200 μL EZ-PREP solution. Following a 5-minute incubation on ice, EZ-PREP was pipetted across the surface of the well to dislodge remaining cells. The sample was centrifuged at 500 g for 5 minutes, and the supernatant removed. Samples were washed with 200 μL EZ-PREP and centrifuged again at 500 g for 5 minutes. Samples were resuspended with gentle pipetting in 200 μL ice-cold Nuclei Suspension Buffer (NSB) consisting of 100 μg/mL BSA and 3.33 μM Vybrant DyeCycle Ruby (Thermo Fisher) in 1×PBS, then centrifuged at 500 g for 5 minutes. The supernatant was removed and nuclei were resuspended in 100 μL NSB and sorted into 100 μL Agencourt DNAdvance lysis buffer using a MoFlo Astrios (Beckman Coulter) at the Broad Institute flow cytometry facility. Genomic DNA was purified according to the manufacturer's Agencourt DNAdvance instructions.
• RNA-Sequencing and Data Analysis
• HEK293T cells were co-transfected with PRNP-targeting or HEXA-targeting PEgRNAs and PE2, PE2-dRT, or Cas9 H840A nickase. 72 hours following transfection, total RNA was harvested from cells using TRIzol reagent (Thermo Fisher) and purified with RNeasy Mini kit (Qiagen) including on-column DNaseI treatment. Ribosomes were depleted from total RNA using the rRNA removal protocol of the TruSeq Stranded Total RNA library prep kit (Illumina) and subsequently washed with RNAClean XP beads (Beckman Coulter). Sequencing libraries were prepared using ribo-depleted RNA on a SMARTer PrepX Apollo NGS library prep system (Takara) following the manufacturer's protocol. Resulting libraries were visualized on a 2200 TapeStation (Agilent Technologies), normalized using a Qubit dsDNA HS assay (Thermo Fisher), and sequenced on a NextSeq 550 using high output v2 flow cell (Illumina) as 75-bp paired-end reads. Fastq files were generated with bc12fastq2 version 2.20 and trimmed using TrimGalore version 0.6.2 (https://github.com/FelixKrueger/TrimGalore) to remove low-quality bases, unpaired sequences, and adaptor sequences. Trimmed reads were aligned to a Homo sapiens genome assembly GRCh¹⁴⁸ with a custom Cas9 H840A gene entry using RSEM version 1.3.1²⁰⁷. The limma-voom²⁰⁸ package was used to normalize gene expression levels and perform differential expression analysis with batch effect correction. Differentially expressed genes were called with FDR-corrected p-value <0.05 and fold-change >2 cutoffs, and results were visualized in R.
• ClinVar Analysis
• The ClinVar variant summary was downloaded from NCBI (accessed Jul. 15, 2019), and the information contained therein was used for all downstream analysis. The list of all reported variants was filtered by allele ID in order to remove duplicates and by clinical significance in order to restrict the analysis to pathogenic variants. The list of pathogenic variants was filtered sequentially by variant type in order to calculate the fraction of pathogenic variants that are insertions, deletions, etc. Single nucleotide variants (SNVs) were separated into two categories (transitions and transversions) based on the reported reference and alternate alleles. SNVs that did not report reference or alternate alleles were excluded from the analysis.
• The lengths of reported insertions, deletions, and duplications were calculated using reference/alternate alleles, variant start/stop positions, or appropriate identifying information in the variant name. Variants that did not report any of the above information were excluded from the analysis. The lengths of reported indels (single variants that include both insertions and deletions relative to the reference genome) were calculated by determining the number of mismatches or gaps in the best pairwise alignment between the reference and alternate alleles. Frequency distributions of variant lengths were calculated using GraphPad Prism 8.
• Data Availability
• High-throughput sequencing data are deposited to the NCBI Sequence Read Archive database. Plasmids encoding PE1, PE2/PE3, and PEgRNA expression vectors will be available from Addgene.
• Code Availability
• The script used to quantify PEgRNA scaffold insertion is provided in FIGS. 60A-60B.
• Supplemental Information: Tables and Sequences
• Table 1: Activities of prime editors, Cas9 nuclease, Cas9 H840A nickase, and PE2-dRT at HEK3, HEK4, EMX1, and FANCF on-target and off-target sites. PE2/PE3 editing is shown as % prime editing alongside % indels (in parentheses). % indels are shown for Cas9, Cas9 H840A nickase (nCas9), and PE2-dRT at the top four previously characterized off-target sites^179,180 sgRNA and PEgRNA sequences can be found in Tables 3A-3R, under the FIGS. 42A-42H heading. All values are the average of three independent biological replicates.

• 	PE pegRNA

  HEK 3 (PE3)

  HEK4 (PE2)

  Site	—	1	2	3	4	—	1	2	3	4
  On-target		44.2	81.2	40.4	45.4		16.2	14.4	9.8	7.9
  		(11.9)	(8.8)	(16.5)	(3.3)		(0.9)	(1.8)	(2.0)	(2.2)
  Off-target 1		(<0.1)	<0.1	<0.1	<0.1		<0.1	0.4	<0.1	0.4
  		(<0.1)	(<0.1)	(<0.1)	(<0.1)		(<0.1)	(<0.1)	(<0.1)	(<0.1)
  Off-target 2		<0.1	<0.1	<0.1	<0.1		<0.1	<0.1	<0.1	<0.1
  		(<0.1)	(<0.1)	(<0.1)	(<0.1)		(<0.1)	(0.1)	(0.1)	(0.1)
  Off-target 3		<0.1	<0.1	<0.1	<0.1		0.2	6.8	18.2	7.9
  		(<0.1)	(<0.1)	(<0.1)	(<0.1)		(0.5)	(1.9)	(0.5)	(3.5)
  Off-target 4		<0.1	<0.1	<0.1	<0.1		<0.1	<0.1	<0.1	<0.1
  		(0.1)	(0.1)	(0.1)	(<0.1)		(<0.1)	(0.1)	(0.2)	(<0.1)

  Cas9 pegRNA

  HEK 3

  HEK4

  Site	sgRNA	1	2	3	4	sgRNA	1	2	3	4
  On-target	91.8	87.5	89.2	89.1	86.8	71.8	68.6	72.8	72.8	70.9
  Off-target 1	17.2	1.9	5.5	5.2	1.8	54.2	39.5	48.4	49.7	49.2
  Off-target 2	38.0	6.5	12.8	11.8	4.7	42.5	19.5	29.4	27.3	30.3
  Off-target 3	8.8	0.6	1.7	1.5	0.5	98.1	96.9	97.3	97.6	97.5
  Off-target 4	0.3	<0.1	<0.1	0.1	<0.1	45.3	16.9	28.0	27.5	29.7

  mCas9 pegRNA

  HEK 3

  HEK4

  Site	—	1	2	3	4	—	1	2	3	4
  Off-target 1	<0.1	<0.1	<0.1	<0.1	<0.1		<0.1	<0.1	<0.1	<0.1
  Off-target 2	<0.1	<0.1	<0.1	<0.1	<0.1		<0.1	<0.1	<0.1	<0.1
  Off-target 3	<0.1	<0.1	<0.1	<0.1	<0.1		0.3	0.5	0.7	0.7
  Off-target 4	<0.1	<0.1	<0.1	<0.1	<0.1		<0.1	0.1	<0.1	<0.1

  PE2-dRT pegRNA

  HEK 3

  HEK4

  Site	—	1	2	3	4	—	1	2	3	4
  Off-target 1	<0.1	<0.1	<0.1	<0.1	<0.1		0.1	0.2	<0.1	<0.1
  Off-target 2	<0.1	<0.1	<0.1	<0.1	<0.1		<0.1	0.1	<0.1	<0.1
  Off-target 3	<0.1	<0.1	<0.1	<0.1	<0.1		<0.1	0.1	1.4	0.9
  Off-target 4	<0.1	<0.1	<0.1	<0.1	<0.1		0.1	0.1	0.1	0.2

  PE pegRNA

  EMX1 (PE3)

  FANCF (PE3)

  Site	—	1	2	3	4	—	1	2	3	4
  On-target		25.6	14.1	35.7	15.4		35.5	32.4	42.8	47.5
  		(3.5)	(2.4)	(3.3)	(2.9)		(9.3)	(16.7)	(13.5)	(12.0)
  Off-target 1		<0.1	<0.1	<0.1	<0.1		<0.1	0.5	<0.1	<0.1
  		(0.1)	(0.1)	(0.1)	(0.1)		(0.1)	(0.1)	(0.1)	(0.1)
  Off-target 2		<0.1	<0.1	<0.1	<0.1		<0.1	<0.1	<0.1	<0.1
  		(<0.1)	(0.1)	(0.1)	(0.1)		(<0.1)	(<0.1)	(0.1)	(<0.1)
  Off-target 3		<0.1	<0.1	<0.1	<0.1		<0.1	<0.1	<0.1	<0.1
  		(0.3)	(0.3)	(0.3)	(0.3)		(<0.1)	(<0.1)	(<0.1)	(<0.1)
  Off-target 4		<0.1	<0.1	<0.1	<0.1		<0.1	<0.1	<0.1	<0.1
  		(0.1)	(0.1)	(0.2)	(0.1)		(<0.1)	(<0.1)	(<0.1)	(<0.1)

  Cas9 pegRNA

  EMX1

  FANCF

  Site	sgRNA	1	2	3	4	sgRNA	1	2	3	4
  On-target	85.6	79.7	70.6	76.6	76.0	78.7	55.9	58.3	51.8	52.0
  Off-target 1	81.1	63.5	48.1	53.0	59.6	12.6	1.9	1.9	1.7	1.7
  Off-target 2	58.3	12.0	6.0	8.2	12.9	1.1	0.2	0.2	0.2	0.1
  Off-target 3	14.8	4.2	3.1	3.6	4.8	2.4	0.2	<0.1	0.2	0.2
  Off-target 4	39.5	1.3	0.9	0.6	1.3	1.0	0.2	0.2	0.2	0.2

  mCas9 pegRNA

  EMX1

  FANCF

  Site	1	2	3	4	—	1	2	3	4	1
  Off-target 1	0.1	0.1	<0.1	0.1		<0.1	<0.1	<0.1	<0.1	0.1
  Off-target 2	0.3	0.1	<0.1	<0.1		<0.1	<0.1	<0.1	<0.1	0.3
  Off-target 3	<0.1	<0.1	<0.1	<0.1		<0.1	<0.1	<0.1	<0.1	<0.1
  Off-target 4	1.5	0.1	0.1	0.4		<0.1	<0.1	<0.1	<0.1	1.5

  PE2-dRT pegRNA

  EMX1

  FANCF

  Site	1	2	3	4	—	1	2	3	4	1
  Off-target 1	0.1	0.1	0.1	0.1		<0.1	<0.1	<0.1	<0.1	0.1
  Off-target 2	<0.1	0.3	<0.1	<0.1		<0.1	<0.1	<0.1	<0.1	<0.1
  Off-target 3	<0.1	<0.1	<0.1	<0.1		<0.1	<0.1	<0.1	<0.1	<0.1
  Off-target 4	0.9	0.1	0.1	0.2		<0.1	<0.1	<0.1	<0.1	0.9

• Tables 2A-2C: Sequences of DNA oligonucleotides, PEgRNAs, and sgRNAs used for in vitro experiments.

• TABLE 2A
  DNA oligonucleotides

  OLIGONUCLEOTIDE	SEQUENCE
  AVA023	5CY5-CCTGGGTCAATCCTTGGGGCCCAGACT
  	GAGCACG (SEQ ID NO: 374)
  AVA024	5CY5-CCTGGGTCAATCCTTGGGGCCCAGACT
  	GAGCACGTGATGGCAGAGGAAAGG
  	(SEQ ID NO: 375)
  AVA025	5PHOS-CCTTTCCTCTGCCATCACGTGCTCAG
  	TCTGGGCCCCAAGGATTGACCCAGG
  	(SEQ ID NO: 376)
  AVA026	5PHOS-TGATGGCAGAGGAAAGG
  	(SEQ ID NO: 377)
  AVA037	GCAGGCTTTAAAGGAACCAATTC
  	(SEQ ID NO: 378)
  AVA110	GCAGGCTTTAAAGGAACCAATTCCCTGGGTCA
  	ATCCTTGGGGC (SEQ ID NO: 379)
  AVA122	CTCTGGAGGATCTAGCGGAG
  	(SEQ ID NO: 380)
  AVA134	CTCTGGAGGATCTAGCGGAGTTTTTTTTTTTT
  	TTTTTTTT (SEQ ID NO: 381)
  AVA135	CTCTGGAGGATCTAGCGGAGCCCCCCCCCCCC
  	CC (SEQ ID NO: 382)

• TABLE 2B
  5′-extended PEgRNAs

  					RT
  			LINKER	PBS	TEMPLATE
  		5′ EXTENSON	LENGTH	LENGTH	LENGTH
  PEGRNA	SPACER SEQUENCE	SEQUENCE	(NT)	(NT)	(NT)
  PEGRNA 1	GGCCCAGACTGAGCAC	GGCTAACCGTGCCATT	15	5	7
  	GTGA	TGATCAGGTCA
  	(SEQ ID NO: 383)	(SEQ ID NO: 429)
  PEGRNA 2	GGCCCAGACTGAGCAC	GGCTAACCGTGCAAAT	15	5	7
  	GTGA	TAACAAACTAA
  	(SEQ ID NO: 384)	(SEQ ID NO: 430)
  PEGRNA 3	GGCCCAGACTGAGAC	GGCCATCTCGTGCAAA	15	5	8
  	GTGA	TTAACAAACTAA
  	(SEQ ID NO: 385)	(SEQ ID NO: 431)
  PEGRNA 4	GGCCCAGACTGAGCAC	GGTCCTCTGCCATCTC	15	5	15
  	GTGA	GTGCAAATTAACAAAC
  	(SEQ ID NO: 386)	TAA
  		(SEQ ID NO: 432)
  PEGRNA 5	GGCCCAGACTGAGCAC	GGCTTCCTTTCCTCTG	15	5	22
  	GTGA	CCATCTCGTGCAAATT
  	(SEQ ID NO: 387)	AACAAACTAA
  		(SEQ ID NO: 433)
  5′-PEGRNA_RT_7_A	GGCCCAGACTGAGCAC	GGCTAACCGTGCCATT	15	5	7
  	GTGA	TGATCAGGTCA
  	(SEQ ID NO: 388)	(SEQ ID NO: 434)
  5′-PEGRNA_RT_7_B	GGCCCAGACTGAGCAC	GGCTAACCGTGCAAAT	15	5	7
  	GTGA	TAACAAACTAA
  	(SEQ ID NO: 389)	(SEQ ID NO: 435)
  5′-PEGRNA_RT_8	GGCCCAGACTGAGCAC	GGCCATCTCGTGCAAA	15	5	8
  	GTGA	TTAACAAACTAA
  	(SEQ ID NO: 390)	(SEQ ID NO: 436)
  5′-PEGRNA_RT_15	GGCCCAGACTGAGCAC	GGTCCTCTGCCATCTC	15	5	15
  	GTGA	GTGCAAATTAACAAAC
  	(SEQ ID NO: 391)	TAA
  		(SEQ ID NO: 437)
  5′-PEGRNA_RT_22	GGCCCAGACTGAGCAC	GGCTTCCTTTCCTCTG	15	5	22
  	GTGA	CCATCTCGTGCAAATT
  	(SEQ ID NO: 392)	AACAAACTAA
  		(SEQ ID NO: 438)

• 3′-extended PEgRNAs

  				RT
  			PBS	TEMPLATE
  		3′ EXTENSION	LENGTH	LENGTH
  PEGRNA	SPACER SEQUENCE	SEQUENCE	(NT)	(NT)
  3′-PEGRNA_10	GGCCCAGACTGAGCAC	TCTGCCATCTCGTGCT		7	10
  	GTGA	C
  	(SEQ ID NO: 506)	(SEQ ID NO: 439)
  3′-PEGRNA_YEAST_TTOA	GGCCCAGACTGAGCAC	TCTGCCATCTCGTGCT		7	10
  	GTGA	C
  	(SEQ ID NO: 507)	(SEQ ID NO: 440)
  3′-PEGRNA_YEAST_+1AINS	GGCCCAGACTGAGCAC	TCTGCCATCATCGTGC		7	11
  	GTGT	C
  	(SEQ ID NO: 508)	(SEQ ID NO: 441)
  3′-PEGRAN_YEAST_+1TDEL	GGCCCAGACTGAGCAC	TCTGCCATCCGTGCTC		7	9
  	GTGA	(SEQ ID NO: 442)
  	(SEQ ID NO: 509)

• Tables 3A-3R: Sequences of PEgRNAs and sgRNAs used in mammalian cell experiments. All sequences are shown in 5′ to 3′ orientation. To construct PEgRNAs, spacer sequences listed below were added to the 5′ end of the sgRNA scaffold and the 3′ extensions listed below containing the primer binding site and RT template were added to the 3′ end of the sgRNA scaffold. The sgRNA scaffold sequence is

• (SEQ ID NO: 131)

  GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAAC

  TTGAAAAAGTGGCACCGAGTCGGTGC.

• TABLE 3A
  FIGS. 39A-39D PEgRNA

  				RT
  			PBS	TEMPLATE
  	SPACER SEQUENCE	3′ EXTENSION	LENGTH	LENGTH
  PEGRNA	(SEQ ID NOS: 2890-2996)	(SEQ ID NOS: 2997-3103)	(NT)	(NT)
  HEK3_2B-C_8	GGCCCAGACTGAGCACGTGA	TCTGCCATCTCGTGCTCA	8	10
  HEK3_2B-C_9	GGCCCAGACTGAGCACGTGA	TCTGCCATCTCGTGCTCAG	9	10
  HEK3_2B-C_10	GGCCCAGACTGAGCACGTGA	TCTGCCATCTCGTGCTCAGT	10	10
  HEK3_2B-C_11	GGCCCAGACTGAGCACGTGA	TCTGCCATCTCGTGCTCAGTC	11	10
  HEK3_2B-C_12	GGCCCAGACTGAGCACGTGA	TCTGCCATCTCGTGCTCAGTCT	12	10
  HEK3_2B-C_13	GGCCCAGACTGAGCACGTGA	TCTGCCATCTCGTGCTCAGTCTG	13	10
  HEK3_2B-C_14	GGCCCAGACTGAGCACGTGA	TCTGCCATCTCGTGCTCAGTCTGG	14	10
  HEK3_2B-C_15	GGCCCAGACTGAGCACGTGA	TCTGCCATCTCGTGCTCAGTCTGGG	15	10
  HEK3_2C_16	GGCCCAGACTGAGCACGTGA	TCTGCCATCTCGTGCTCAGTCTGGGC	16	10
  HEC3_2C_17	GGCCCAGACTGAGCACGTGA	TCTGCCATCTCGTGCTCAGTCTGGGCC	17	10
  EMX1_2C_9	GAGTCCGAGCAGAAGAAGAA	ATGGGAGCACTTCTTCTTCTGC	9	13
  EMX1_2C_10	GAGTCCGAGCAGAAGAAGAA	ATGGGAGCACTTCTTCTTCTGCT	10	13
  EMX1_2C_11	GAGTCCGAGCAGAAGAAGAA	ATGGGAGCACTTCTTCTTCTGCTC	11	13
  EMX1_2C_12	GAGTCCGAGCAGAAGAAGAA	ATGGGAGCACTTCTTCTTCTGCTCG	12	13
  EMX1_2C_13	GAGTCCGAGCAGAAGAAGAA	ATGGGAGCACTTCTTCTTCTGCTCGG	13	13
  EMX1_2C_14	GAGTCCGAGCAGAAGAAGAA	ATGGGAGCACTTCTTCTTCTGCTCGGA	14	13
  EMX1_2C_15	GAGTCCGAGCAGAAGAAGAA	ATGGGAGCACTTCTTCTTCTGCTCGGAC	15	13
  EMX1_2C_16	GAGTCCGAGCAGAAGAAGAA	ATGGGAGCACTTCTTCTTCTGCTCGGACT	16	13
  EMX1_2C_17	GAGTCCGAGCAGAAGAAGAA	ATGGGAGCACTTCTTCTTCTGCTCGGACTC	17	13
  FANCF_2C_8	GGAATCCCTTCTGCAGCACC	GGAAAAGCGATCAAGGTGCTGCAGA	8	17
  FANCF_2C_9	GGAATCCCTTCTGCAGCACC	GGAAAAGCGATCAAGGTGCTGCAGAA	9	17
  FANCF_2C_10	GGAATCCCTTCTGCAGCACC	GGAAAAGCGATCAAGGTGCTGCAGAAG	10	17
  FANCF_2C_11	GGAATCCCTTCTGCAGCACC	GGAAAAGCGATCAAGGTGCTGCAGAAGG	11	17
  FANCF_2C_12	GGAATCCCTTCTGCAGCACC	GGAAAAGCGATCAAGGTGCTGCAGAAGGG	12	17
  FANCF_2C_13	GGAATCCCTTCTGCAGCACC	GGAAAAGCGATCAAGGTGCTGCAGAAGGGA	13	17
  FANCF_2C_14	GGAATCCCTTCTGCAGCACC	GGAAAAGCGATCAAGGTGCTGCAGAAGGGA	14	17
  		T
  FANCF_2C_15	GGAATCCCTTCTGCAGCACC	GGAAAAGCGATCAAGGTGCTGCAGAAGGGA	15	17
  		TT
  FANCF_2C_16	GGAATCCCTTCTGCAGCACC	GGAAAAGCGATCAAGGTGCTGCAGAAGGGA	16	17
  		TTC
  FANCF_2C_17	GGAATCCCTTCTGCAGCACC	GGAAAAGCGATCAAGGTGCTGCAGAAGGGA	17	17
  		TTCC
  RNF2_2C_9	GTCATCTTAGTCATTACCTG	GAACACCTCATGTAATGACT	9	11
  RNF2_2C_10	GTCATCTTAGTCATTACCTG	GAACACCTCATGTAATGACTA	10	11
  RNF2_2C_11	GTCATCTTAGTCATTACCTG	GAACACCTCATGTAATGACTAA	11	11
  RNF2_2C_12	GTCATCTTAGTCATTACCTG	GAACACCTCATGTAATGACTAAG	12	11
  RNF2_2C_13	GTCATCTTAGTCATTACCTG	GAACACCTCATGTAATGACTAAGA	13	11
  RNF2_2C_14	GTCATCTTAGTCATTACCTG	GAACACCTCATGTAATGACTAAGAT	14	11
  RNF2_2C_15	GTCATCTTAGTCATTACCTG	GAACACCTCATGTAATGACTAAGATG	15	11
  RNF2_2C_16	GTCATCTTAGTCATTACCTG	GAACACCTCATGTAATGACTAAGATGA	16	11
  RNF2_2C_17	GTCATCTTAGTCATTACCTG	GAACACCTCATGTAATGACTAAGATGAC	17	11
  HEK4_2C_7	GGCACTGCGGCTGGAGGTGG	GCTTTAACCCCAACCTCCAG	7	13
  HEK4_2C_8	GGCACTGCGGCTGGAGGTGG	GCTTTAACCCCAACCTCCAGC	8	13
  HEK4_2C_9	GGCACTGCGGCTGGAGGTGG	GCTTTAACCCCAACCTCCAGCC	9	13
  HEK4_2C_10	GGCACTGCGGCTGGAGGTGG	GCTTTAACCCCAACCTCCAGCCG	10	13
  HEK4_2C_11	GGCACTGCGGCTGGAGGTGG	GCTTTAACCCCAACCTCCAGCCGC	11	13
  HEK4_2C_12	GGCACTGCGGCTGGAGGTGG	GCTTTAACCCCAACCTCCAGCCGCA	12	13
  HEK4_2C_13	GGCACTGCGGCTGGAGGTGG	GCTTTAACCCCAACCTCCAGCCGCAG	13	13
  HEK4_2C_14	GGCACTGCGGCTGGAGGTGG	GCTTTAACCCCAACCTCCAGCCGCAGT	14	13
  HEK4_2C_15	GGCACTGCGGCTGGAGGTGG	GCTTTAACCCCAACCTCCAGCCGCAGTG	15	13
  HEK3_2C_1TDEL	GGCCCAGACTGAGCACGTGA	TCTGCCATCCGTGCTCAGTCTG	13	10
  HEK3_2C_1AINS	GGCCCAGACTGAGCACGTGA	TCTGCCATCATCGTGCTCAGTCTG	13	10
  HEK3_2C_1CTTINS	GGCCCAGACTGAGCACGTGA	TCTGCCATCAAAGCGTGCTCAGTCTG	13	10
  HEK3_2D_10	GGCCCAGACTGAGCACGTGA	TCTGCCATCTCGTGCTCAGTCTG	13	10
  HEK3_2D_11	GGCCCAGACTGAGCACGTGA	CTCTGCCATCTCGTGCTCAGTCTG	13	11
  HEK3_2D_12	GGCCCAGACTGAGCACGTGA	CCTCTGCCATCTCGTGCTCAGTCTG	13	12
  HEK3_2D_13	GGCCCAGACTGAGCACGTGA	TCCTCTGCCATCTCGTGCTCAGTCTG	13	13
  HEK3_2D_14	GGCCCAGACTGAGCACGTGA	TTCCTCTGCCATCTCGTGCTCAGTCTG	13	14
  HEK3_2D_15	GGCCCAGACTGAGCACGTGA	TTTCCTCTGCCATCTCGTGCTCAGTCTG	13	15
  HEK3_2D_16	GGCCCAGACTGAGCACGTGA	CTTTCCTCTGCCATCTCGTGCTCAGTCTG	13	16
  HEK3_2D_17	GGCCCAGACTGAGCACGTGA	CCTTTCCTCTGCCATCTCGTGCTCAGTCTG	13	17
  HEK3_2D_18	GGCCCAGACTGAGCACGTGA	TCCTTTCCTCTGCCATCTCGTGCTCAGTCT	13	18
  		G
  HEK3_2D_19	GGCCCAGACTGAGCACGTGA	TTCCTTTCCTCTGCCATCTCGTGCTCAGTC	13	19
  		TG
  HEK3_2D_20	GGCCCAGACTGAGCACGTGA	CTTCCTTTCCTCTGCCATCTCGTGCTCAGT	13	20
  		CTG
  EMX1_2D_10	GAGTCCGAGCAGAAGAAGAA	GGAGCCCTTGTTCTTCTGCTCGG	13	10
  EMX1_2D_11	GAGTCCGAGCAGAAGAAGAA	GGGAGCCCTTGTTCTTCTGCTCGG	13	11
  EMX1_2D_12	GAGTCCGAGCAGAAGAAGAA	TGGGAGCCCTTGTTCTTCTGCTCGG	13	12
  EMX1_2D_13	GAGTCCGAGCAGAAGAAGAA	ATGGGAGCCCTTGTTCTTCTGCTCGG	13	13
  EMX1_2D_14	GAGTCCGAGCAGAAGAAGAA	GATGGGAGCCCTTGTTCTTCTGCTCGG	13	14
  EMX1_2D_15	GAGTCCGAGCAGAAGAAGAA	TGATGGGAGCCCTTGTTCTTCTGCTCGG	13	15
  EMX1_2D_16	GAGTCCGAGCAGAAGAAGAA	GTGATGGGAGCCCTTGTTCTTCTGCTCGG	13	16
  EMX1_2D_17	GAGTCCGAGCAGAAGAAGAA	TGTGATGGGAGCCCTTGTTCTTCTGCTCGG	13	17
  EMX1_2D_18	GAGTCCGAGCAGAAGAAGAA	ATGTGATGGGAGCCCTTGTTCTTCTGCTCG	13	18
  		G
  EMX1_2D_19	GAGTCCGAGCAGAAGAAGAA	GATGTGATGGGAGCCCTTGTTCTTCTGCTC	13	19
  		GG
  EMX1_2D_20	GAGTCCGAGCAGAAGAAGAA	TGATGTGATGGGAGCCCTTGTTCTTCTGCT	13	20
  		CGG
  FANCF_2D_10	GGAATCCCTTCTGCAGCACC	CGATCAAGGTGCTGCAGAAGGGA	13	10
  FANCF_2D_11	GGAATCCCTTCTGCAGCACC	GCGATCAAGGTGCTGCAGAAGGGA	13	11
  FANCF_2D_12	GGAATCCCTTCTGCAGCACC	AGCGATCAAGGTGCTGCAGAAGGGA	13	12
  FANCF_2D_13	GGAATCCCTTCTGCAGCACC	AAGCGATCAAGGTGCTGCAGAAGGGA	13	13
  FANCF_2D_14	GGAATCCCTTCTGCAGCACC	AAAGCGATCAAGGTGCTGCAGAAGGGA	13	14
  FANCF_2D_15	GGAATCCCTTCTGCAGCACC	AAAAGCGATCAAGGTGCTGCAGAAGGGA	13	15
  FANCF_2D_16	GGAATCCCTTCTGCAGCACC	GAAAAGCGATCAAGGTGCTGCAGAAGGGA	13	16
  FANCF_2D_17	GGAATCCCTTCTGCAGCACC	GGAAAAGCGATCAAGGTGCTGCAGAAGGGA	13	17
  FANCF_2D_18	GGAATCCCTTCTGCAGCACC	CGGAAAAGCGATCAAGGTGCTGCAGAAGGG	13	18
  		A
  FANCF_2D_19	GGAATCCCTTCTGCAGCACC	TCGGAAAAGCGATCAAGGTGCTGCAGAAGG	13	19
  		GA
  FANCF_2D_20	GGAATCCCTTCTGCAGCACC	CTCGGAAAAGCGATCAAGGTGCTGCAGAAG	13	20
  		GGA
  RNF2_2D_10	GTCATCTTAGTCATTACCTG	AACACCTCATGTAATGACTAAGATG	15	10
  RNF2_2D_11	GTCATCTTAGTCATTACCTG	GAACACCTCATGTAATGACTAAGATG	15	11
  RNF2_2D_12	GTCATCTTAGTCATTACCTG	CGAACACCTCATGTAATGACTAAGATG	15	12
  RNF2_2D_13	GTCATCTTAGTCATTACCTG	ACGAACACCTCATGTAATGACTAAGATG	15	13
  RNF2_2D_14	GTCATCTTAGTCATTACCTG	AACGAACACCTCATGTAATGACTAAGATG	15	14
  RNF2_2D_15	GTCATCTTAGTCATTACCTG	CAACGAACACCTCATGTAATGACTAAGATG	15	15
  RNF2_2D_16	GTCATCTTAGTCATTACCTG	ACAACGAACACCTCATGTAATGACTAAGAT	15	16
  		G
  RNF2_2D_17	GTCATCTTAGTCATTACCTG	TACAACGAACACCTCATGTAATGACTAAGA	15	17
  		TG
  RNF2_2D_18	GTCATCTTAGTCATTACCTG	TTACAACGAACACCTCATGTAATGACTAAG	15	18
  		ATG
  RNF2_2D_19	GTCATCTTAGTCATTACCTG	GTTACAACGAACACCTCATGTAATGACTAA	15	19
  		GATG
  RNF2_2D_20	GTCATCTTAGTCATTACCTG	AGTTACAACGAACACCTCATGTAATGACTA	15	20
  		AGATG
  HEK4_2D_7	GGCACTGCGGCTGGAGGTGG	ACCCCAACCTCCAGCCGC	11	7
  HEK4_2D_8	GGCACTGCGGCTGGAGGTGG	AACCCCAACCTCCAGCCGC	11	8
  HEK4_2D_9	GGCACTGCGGCTGGAGGTGG	TAACCCCAACCTCCAGCCGC	11	9
  HEK4_2D_10	GGCACTGCGGCTGGAGGTGG	TTAACCCCAACCTCCAGCCGC	11	10
  HEK4_2D_11	GGCACTGCGGCTGGAGGTGG	TTTAACCCCAACCTCCAGCCGC	11	11
  HEK4_2D_12	GGCACTGCGGCTGGAGGTGG	CTTTAACCCCAACCTCCAGCCGC	11	12
  HEK4_2D_13	GGCACTGCGGCTGGAGGTGG	GCTTTAACCCCAACCTCCAGCCGC	11	13
  HEK4_2D_14	GGCACTGCGGCTGGAGGTGG	CGCTTTAACCCCAACCTCCAGCCGC	11	14
  HEK4_2D_15	GGCACTGCGGCTGGAGGTGG	CCGCTTTAACCCCAACCTCCAGCCGC	11	15
  HEK4_2D_16	GGCACTGCGGCTGGAGGTGG	TCCGCTTTAACCCCAACCTCCAGCCGC	11	16
  HEK4_2D_17	GGCACTGCGGCTGGAGGTGG	CTCCGCTTTAACCCCAACCTCCAGCCGC	11	17
  HEK4_2D_18	GGCACTGCGGCTGGAGGTGG	CTCCGCTTTAACCCCAACCTCCAGCCGC	11	18
  HEK4_2D_19	GGCACTGCGGCTGGAGGTGG	CTCCGCTTTAACCCCAACCTCCAGCCGC	11	19

• TABLE 3B
  FIGS. 40A-40C PEgRNA

  				RT
  			PBS	TEMPLATE
  	SPACER SEQUENCE
  	3′ EXTENSION	LENGTH	LENGTH
  PEGRNA	(SEQ ID NO: 3104-3112)	(SEQ ID NOS: 3113-3121)	(NT)	(NT)
  RNF2_3B	GTCATCTTAGTCATTACCTG	AACGAACACCTCATGTAATGACTAAGATG		15	14
  EMX1_3B	GAGTCCGAGCAGAAGAAGAA	ATGGGAGCACTTCTTCTTCTGCTCGGAC		15	13
  FANCF_3B	GGAATCCCTTCTGCAGCACC	GGAAAAGCGATCAAGGTGCTGCAGAAGGGATT		15	17
  HE3_3B	GGCCCAGACTGAGCACGTGA	TCTGCCATGACGTGCTCAGTCTG		13	10
  HEK4_3B	GGCACTGCGGCTGGAGGTGG	TTAACCCCAACCTCCAGCC		9	10
  RNF2_3C_4ATOC	GTCATCTTAGTCATTACCTG	AACGAACACCGCAGGTAATGACTAAGATG		15	14
  RNF2_3C_4ATOG	GTCATCTTAGTCATTACCTG	AACGAACACCCCAGGTAATGACTAAGATG		15	14
  FANCF_3C_5GTOT	GGAATCCCTTCTGCAGCACC	GGAAAAGCGATCAAGGTGCTGCAGAAGGGA		13	17
  FANCF_3C_7ATOC	GGAATCCCTTCTGCAGCACC	GGAAAAGCGAGCCAGGTGCTGCAGAAGGGAT		14	17

• TABLE 3C
  FIGS. 40A-40C nicking sgRNA sequences

  NICKING SGRNA	SPACER SEQUENCE	SEQ ID NO:
  RNF2_2B_+41	GTCAACCATTAAGCAAAACAT	3122
  RNF2_2B_+67	GTCTCAGGCTGTGCAGACAAA	3123
  EMX1_2B_−116	GGGGCACAGATGAGAAACTC	3124
  EMX1_2B_−57	GCCGTTTGTACTTTGTCCTC	3125
  EMX1_2B_+14	GCGCCACCGGTTGATGTGAT	3126
  EMX1_2B_+27	GCTTCGTGGCAATGCGCCAC	3127
  EMX1_2B_+53	GACATCGATGTCCTCCCCAT	3128
  EMX1_2B_+80	GTGGTTGCCCACCCTAGTCAT	3129
  FANCF_2B_−78	GCGACTCTCTGCGTACTGAT	3130
  FANCF_2B_−50	GCCCTACTTCCGCTTTCACCT	3131
  FANCF_2B_−27	GGATTCCATGAGGTGCGCGA	3132
  FANCF_2B_−17	GCTGCAGAAGGGATTCCATG	3133
  FANCF_2B_+21	GCTTGAGACCGCCAGAAGCT	3134
  FANCF_2B_+48	GGGGTCCCAGGTGCTGACGT	3135
  HEK3_2B_−108	GCAGAAATAGACTAATTGCA	3136
  HEK3_2B_−38	GGATTGACCCAGGCCAGGGC	3137
  HEK3_2B_+26	GACGCCCTCTGGAGGAAGCA	3138
  HEK3_2B_+37	GCTGTCCTGCGACGCCCTC	3139
  HEK3_2B_+63	GCACATACTAGCCCCTGTCT	3140
  HEK3_2B_+90	GTCAACCAGTATCCCGGTGC	3141
  HEK4_2B_−95	TCCCTTCCTTCCACCCAGCC	3142
  HEK4_2B_−51	CCCTGCCTGTCATCCTGCTT	3143
  HEK4_2B_−26	GCAGTGCCACCGGGGCGCCG	3144
  HEK4_2B_+52	GCGGGGGCTCAGAGAGGGCA	3145
  HEK4_2B_+74	GAGACACACACACAGGCCTGG	3146
  RNF2_2C_+41	GTCAACCATTAAGCAAAACAT	3147
  RNF2_2C_4ATOC_+5	GTGAGTTACAACGAACACCGC	3148
  RNF2_2C_4ATOG_+5	GTGAGTTACAACGAACACCCC	3149
  FANCF_2C_+48	GGGGTCCCAGGTGCTGACGT	3150
  FANCF_2C_5GTOT_+7	GAAGCTCGGAAAAGCGATCA	3151
  FANCF_2C_7ATOC_+7	GAAGCTCGGAAAAGCGAGCC	3152
  HEK3_2C_+90	GTCAACCAGTATCCCGGTGC	3153

• FIGS. 41A-41K PEgRNA

  				RT
  			PBS	TEMPLATE
  	SPACER SEQUENCE
  	3′ EXTENSION	LENGTH	LENGTH
  PEGRNA	(SEQ ID NO: 3154-3304)	(SEQ ID NO: 3305-3455)	(NT)	(NT)
  HEK3_4A_1TTOA	GGCCCAGACTGAGCACGTGA	TCTGCCATCTCGTGCTCAGTCTG		13	10
  HEK3_4A_1TTOC	GGCCCAGACTGAGCACGTGA	TCTGCCATCGCGTGCTCAGTCTG		13	10
  HEK3_4A_1TTOG	GGCCCAGACTGAGCACGTGA	TCTGCCATCCCGTGCTCAGTCTG		13	10
  HEK3_4A_2GTOA	GGCCCAGACTGAGCACGTGA	TCTGCCATTACGTGCTCAGTCTG		13	10
  HEK3_4A_2GTOC	GGCCCAGACTGAGCACGTGA	TCTGCCATGACGTGCTCAGTCTG		13	10
  HEK3_4A_2GTOT	GGCCCAGACTGAGCACGTGA	TCTGCCATAACGTGCTCAGTCTG		13	10
  HEK3_4A_3ATOC	GGCCCAGACTGAGCACGTGA	TCTGCCAGCACGTGCTCAGTCTG		13	10
  HEK3_4A_3ATOG	GGCCCAGACTGAGCACGTGA	TCTGCCACCACGTGCTCAGTCTG		13	10
  HEK3_4A_3ATOT	GGCCCAGACTGAGCACGTGA	TCTGCCAACACGTGCTCAGTCTG		13	10
  HEK3_4A_4TTOA	GGCCCAGACTGAGCACGTGA	TCTGCCTTCACGTGCTCAGTCTG		13	10
  HEK3_4A_4TTOC	GGCCCAGACTGAGCACGTGA	TCTGCCGTCACGTGCTCAGTCTG		13	10
  HEK3_4A_4TTOG	GGCCCAGACTGAGCACGTGA	TCTGCCCTCACGTGCTCAGTCTG		13	10
  HEK3_4A_5GTOA	GGCCCAGACTGAGCACGTGA	TCTGCTATCACGTGCTCAGTCTG		13	10
  HEK3_4A_5GTOC	GGCCCAGACTGAGCACGTGA	TCTGCGATCACGTGCTCAGTCTG		13	10
  HEK3_4A_5GTOT	GGCCCAGACTGAGCACGTGA	TCTGCAATCACGTGCTCAGTCTG		13	10
  HEK3_4A_6GTOA	GGCCCAGACTGAGCACGTGA	TCTGTCATCACGTGCTCAGTCTG		13	10
  HEK3_4A_6GTOC	GGCCCAGACTGAGCACGTGA	TCTGGCATCACGTGCTCAGTCTG		13	10
  HEK3_4A_6GTOT	GGCCCAGACTGAGCACGTGA	TCTGACATCACGTGCTCAGTCTG		13	10
  HEK3_4A_7CTOA	GGCCCAGACTGAGCACGTGA	TCTTCCATCACGTGCTCAGTCTG		13	10
  HEK3_4A_7CTOG	GGCCCAGACTGAGCACGTGA	TCTCCCATCACGTGCTCAGTCTG		13	10
  HEK3_4A_7CTOT	GGCCCAGACTGAGCACGTA	TCTACCATCACGTGCTCAGTCTG		13	10
  HEK3_4A_8ATOC	GGCCCAGACTGAGCACGTGA	TCGGCCATCACGTGCTCAGTCTG		13	10
  HEK3_4A_8ATOG	GGCCCAGACTGAGCACGTGA	TCCGCCATCACGTGCTCAGTCTG		13	10
  HEK3_4A_8ATOT	GGCCCAGACTGAGCACGTGA	TCAGCCATCACGTGCTCAGTCTG		13	10
  HEK3_4B_1TTOA	GGCCCAGACTGAGCACGTGA	TGGAGGAAGCAGGGCTTCCTTTCCTCTGCCATCTCGTG		13	34
  		CTCAGTCTG
  HEK3_4B_12GTOC	GGCCCAGACTGAGCACGTGA	TGGAGGAAGCAGGGCTTCCTTTGCTCTGCCATCACGTG		13	34
  		CTCAGTCTG
  HEK3_4B_14ATOT	GGCCCAGACTGAGCACGTGA	TGGAGGAAGCAGGGCTTCCTATCCTCTGCCATCACGTG		13	34
  		CTCAGTCTG
  HEK3_4B_17GTOC	GGCCCAGACTGAGCACGTGA	TGGAGGAAGCAGGGCTTGCTTTCCTCTGCCATCACGTG		13	34
  		CTCAGTCTG
  HEK3_4B_20GTOC	GGCCCAGACTGAGCACGTGA	TGGAGGAAGCAGGGGTTCCTTTCCTCTGCCATCACGTG		13	34
  		CTCAGTCTG
  HEK3_4B_23CTOG	GGCCCAGACTGAGCACGTGA	TGGAGGAAGCACGGCTTCCTTTCCTCTGCCATCACGTG		13	34
  		CTCAGTCTG
  HEK3_4B_24TTOA	GGCCCAGACTGAGCACGTGA	TGGAGGAAGCTGGGCTTCCTTTCCTCTGCCATCACGTG		13	34
  		CTCAGTCTG
  HEK3_4B_26CTOG	GGCCCAGACTGAGCACGTGA	TGGAGGAACCAGGGCTTCCTTTCCTCTGCCATCACGTG		13	34
  		CTCAGTCTG
  HEK3_4B_30CTOG	GGCCCAGACTGAGCACGTGA	TGGACGAAGCAGGGCTTCCTTTCCTCTGCCATCACGTG		13	34
  		CTCAGTCTG
  HEK3_4B_33CTOG	GGCCCAGACTGAGCACGTGA	TCGAGGAAGCAGGGCTTCCTTTCCTCTGCCATCACGTG		13	34
  		CTCAGTCTG
  RNF2_4C_1CTOA	GTCATCTTAGTCATTACCTG	AACGAACACCTCATGTAATGACTAAGATG		15	14
  RNF2_4C_1CTOG	GTCATCTTAGTCATTACCTG	AACGAACACCTCACGTAATGACTAAGATG	15	14
  RNF2_4C_1CTOT	GTCATCTTAGTCATTACCTG	AACGAACACCTCAAGTAATGACTAAGATG	15	14
  RNF2_4C_2TTOA	GTCATCTTAGTCATTACCTG	AACGAACACCTCTGGTAATGACTAAGATG	15	14
  RNF2_4C_2TTOG	GTCATCTTAGTCATTACCTG	AACGAACACCTCCGGTAATGACTAAGATG	15	14
  RNF2_4C_3GTOC	GTCATCTTAGTCATTACCTG	AACGAACACCTGAGGTAATGACTAAGATG	15	14
  RNF2_4C_4ATOC	GTCATCTTAGTCATTACCTG	AACGAACACCGCAGGTAATGACTAAGATG	15	14
  RNF2_4C_4ATOT	GTCATCTTAGTCATTACCTG	AACGAACACCACAGGTAATGACTAAGATG	15	14
  RNF2_4C_4ATOG	GTCATCTTAGTCATTACCTG	AACGAACACCCCAGGTAATGACTAAGATG	15	14
  RNF2_4C_5GTOT	GTCATCTTAGTCATTACCTG	AACGAACACATCAGGTAATGACTAAGATG	15	14
  RNF2_4C_6GTOA	GTCATCTTAGTCATTACCTG	AACGAACATCTCAGGTAATGACTAAGATG	15	14
  RNF2_4C_7TTOC	GTCATCTTAGTCATTACCTG	AACGAACGCCTCAGGTAATGACTAAGATG	15	14
  FANCF_4D_1ATOG	GGAATCCCTTCTGCAGCACC	GGAAAAGCGATCCAGGCGCTGCAGAAGGGAT	14	17
  FANCF_4D_1ATOT	GGAATCCCTTCTGCAGCACC	GGAAAAGCGATCCAGGAGCTGCAGAAGGGAT	14	17
  FANCF_4D_2CTOA	GGAATCCCTTCTGCAGCACC	GGAAAAGCGATCCAGTTGCTGCAGAAGGGAT	14	17
  FANCF_4D_3CTOG	GGAATCCCTTCTGCAGCACC	GGAAAAGCGATCCACGTGCTGCAGAAGGGAT	14	17
  FANCF_4D_3CTOT	GGAATCCCTTCTGCAGCACC	GGAAAAGCGATCCAAGTGCTGCAGAAGGGAT	14	17
  FANCF_4D_4TTOA	GGAATCCCTTCTGCAGCACC	GGAAAAGCGATCCTGGTGCTGCAGAAGGGAT	14	17
  FANCF_4D_4TTOG	GGAATCCCTTCTGCAGCACC	GGAAAAGCGATCCCGGTGCTGCAGAAGGGAT	14	17
  FANCF_4D_5GTOA	GGAATCCCTTCTGCAGCACC	GGAAAAGCGATCTAGGTGCTGCAGAAGGGAT	14	17
  FANCF_4D_6GTOC	GGAATCCCTTCTGCAGCACC	GGAAAAGCGATGCAGGTGCTGCAGAAGGGAT	14	17
  FANCF_4D_7ATOC	GGAATCCCTTCTGCAGCACC	GGAAAAGCGAGCCAGGTGCTGCAGAAGGGAT	14	17
  FANCF_4D_8TTOC	GGAATCCCTTCTGCAGCACC	GGAAAAGCGGTCCAGGTGCTGCAGAAGGGAT	14	17
  FANCF_4D_10GTOT	GGAATCCCTTCTGCAGCACC	GGAAAAGAGATCCAGGTGCTGCAGAAGGGAT	14	17
  EMX1_4E_2ATOC	GAGTCCGAGCAGAAGAAGAA	GTGATGGGAGCCCTGCTTCTTCTGCTCGGA	14	16
  EMX1_4E_2ATOT	GAGTCCGAGCAGAAGAAGAA	GTGATGGGAGCCCTACTTCTTCTGCTCGGA	14	16
  EMX1_4E_3ATOG	GAGTCCGAGCAGAAGAAGAA	GTGATGGGAGCCCCTCTTCTTCTGCTCGGA	14	16
  EMX1_4E_4GTOC	GAGTCCGAGCAGAAGAAGAA	GTGATGGGAGCCGTTCTTCTTCTGCTCGGA	14	16
  EMX1_4E_5GTOA	GAGTCCGAGCAGAAGAAGAA	GTGATGGGAGCTCTTCTTCTTCTGCTCGGA	14	16
  EMX1_4E_5GTOT	GAGTCCGAGCAGAAGAAGAA	GTGATGGGAGCACTTCTTCTTCTGCTCGGA	14	16
  EMX1_4E_7CTOA	GAGTCCGAGCAGAAGAAGAA	GTGATGGGATCCCTTCTTCTTCTGCTCGGA	14	16
  EMX1_4E_8TTOA	GAGTCCGAGCAGAAGAAGAA	GTGATGGGTGCCCTTCTTCTTCTGCTCGGA	14	16
  EMX1_4E_8TTOC	GAGTCCGAGCAGAAGAAGAA	GTGATGGGGGCCCTTCTTCTTCTGCTCGGA	14	16
  EMX1_4E_8TTOG	GAGTCCGAGCAGAAGAAGAA	GTGATGGGCGCCCTTCTTCTTCTGCTCGGA	14	16
  EMX1_4E_9CTOG	GAGTCCGAGCAGAAGAAGAA	GTGATGGCAGCCCTTCTTCTTCTGCTCGGA	14	16
  EMX1_4E_9CTOT	GAGTCCGAGCAGAAGAAGAA	GTGATGGAAGCCCTTCTTCTTCTGCTCGGA	14	16
  RUNX1_4F_1CTOA	GCATTTTCAGGAGGAAGCGA	TGTCTGAAGCCATCTCTTCCTCCTGAAAAT	15	15
  RUNX1_4F_1CTOG	GCATTTTCAGGAGGAAGCGA	TGTCTGAAGCCATCCCTTCCTCCTGAAAAT	15	15
  RUNX1_4F_1CTOT	GCATTTTCAGGAGGAAGCGA	TGTCTGAAGCCATCACTTCCTCCTGAAAAT	15	15
  RUNX1_4F_2GTOA	GCATTTTCAGGAGGAAGCGA	TGTCTGAAGCCATTGCTTCCTCCTGAAAAT	15	15
  RUNX1_4F_3ATOC	GCATTTTCAGGAGGAAGCGA	TGTCTGAAGCCAGCGCTTCCTCCTGAAAAT	15	15
  RUNX1_4F_3ATOG	GCATTTTCAGGAGGAAGCGA	TGTCTGAAGCCACCGCTTCCTCCTGAAAAT	15	15
  RUNX1_4F_3ATOT	GCATTTTCAGGAGGAAGCGA	TGTCTGAAGCCAACGCTTCCTCCTGAAAAT	15	15
  RUNX1_4F_4TTOA	GCATTTTCAGGAGGAAGCGA	TGTCTGAAGCCTTCGCTTCCTCCTGAAAAT	15	15
  RUNX1_4F_4TTOC	GCATTTTCAGGAGGAAGCGA	TGTCTGAAGCCGTCGCTTCCTCCTGAAAAT	15	15
  RUNX1_4F_4TTOG	GCATTTTCAGGAGGAAGCGA	TGTCTGAAGCCCTCGCTTCCTCCTGAAAAT	15	15
  RUNX1_4F_5GTOT	GCATTTTCAGGAGGAAGCGA	TGTCTGAAGCAATCGCTTCCTCCTGAAAAT	15	15
  RUNX1_4F_6GTOC	GCATTTTCAGGAGGAAGCGA	TGTCTGAAGGCATCGCTTCCTCCTGAAAAT	15	15
  VEGFA_4G_1TTOA	GATGTCTGCAGGCCAGATGA	AATGTGCCATCTGGAGCCCTCTTCTGGCCTGCAGA	13	22
  VEGFA_4G_1TTOC	GATGTCTGCAGGCCAGATGA	AATGTGCCATCTGGAGCCCTCGTCTGGCCTGCAGA	13	22
  VEGFA_4G_1TTOG	GATGTCTGCAGGCCAGATGA	AATGTGCCATCTGGAGCCCTCCTCTGGCCTGCAGA	13	22
  VEGFA_4G_2GTOA	GATGTCTGCAGGCCAGATGA	AATGTGCCATCTGGAGCCCTTATCTGGCCTGCAGA	13	22
  VEGFA_4G_3ATOC	GATGTCTGCAGGCCAGATGA	AATGTGCCATCTGGAGCCCGCATCTGGCCTGCAGA	13	22
  VEGFA_4G_3ATOG	GATGTCTGCAGGCCAGATGA	AATGTGCCATCTGGAGCCCCCATCTGGCCTGCAGA	13	22
  VEGFA_4G_3ATOT	GATGTCTGCAGGCCAGATGA	AATGTGCCATCTGGAGCCCACATCTGGCCTGCAGA	13	22
  VEGFA_4G_5GTOT	GATGTCTGCAGGCCAGATGA	AATGTGCCATCTGGAGCACTCATCTGGCCTGCAGA	13	22
  VEGFA_4G_6GTOC	GATGTCTGCAGGCCAGATGA	AATGTGCCATCTGGAGGCCTCATCTGGCCTGCAGA	13	22
  VEGFA_4G_7CTOA	GATGTCTGCAGGCCAGATGA	AATGTGCCATCTGGATCCCTCATCTGGCCTGCAGA	13	22
  VEGFA_4G_7CTOT	GATGTCTGCAGGCCAGATGA	AATGTGCCATCTGGAACCCTCATCTGGCCTGCAGA	13	22
  VEGFA_4G_9CTOG	GATGTCTGCAGGCCAGATGA	AATGTGCCATCTGCAGCCCTCATCTGGCCTGCAGA	13	22
  DNMT1_4H_1ATOC	GATTCCTGGTGCCAGAAACA	GTCACCCCTGGTTCTGGCACCAGG	13	11
  DNMT1_4H_1ATOG	GATTCCTGGTGCCAGAAACA	GTCACCCCTGCTTCTGGCACCAGG	13	11
  DNMT1_4H_2CTOA	GATTCCTGGTGCCAGAAACA	GTCACCCCTTTTTCTGGCACCAGG	13	11
  DNMT1_4H_2CTOG	GATTCCTGGTGCCAGAAACA	GTCACCCCTCTTTCTGGCACCAGG	13	11
  DNMT1_4H_2CTOT	GATTCCTGGTGCCAGAAACA	GTCACCCCTATTTCTGGCACCAGG	13	11
  DNMT1_4H_3ATOT	GATTCCTGGTGCCAGAAACA	GTCACCCCAGTTTCTGGCACCAGG	13	11
  DNMT1_4H_4GTOA	GATTCCTGGTGCCAGAAACA	GTCACCCTTGTTTCTGGCACCAGG	13	11
  DNMT1_4H_5GTOT	GATTCCTGGTGCCAGAAACA	GTCACCACTGTTTCTGGCACCAGG	13	11
  DNMT1_4H_6GTOC	GATTCCTGGTGCCAGAAACA	GTCACGCCTGTTTCTGGCACCAGG	13	11
  DNMT1_4H_8TTOA	GATTCCTGGTGCCAGAAACA	GCCCTCCCGTCTCCCCTGTTTCTGGCACCAGG	13	19
  DNMT1_4H_8TTOC	GATTCCTGGTGCCAGAAACA	GCCCTCCCGTCGCCCCTGTTTCTGGCACCAGG	13	19
  DNMT1_4H_8TTOG	GATTCCTGGTGCCAGAAACA	GCCCTCCCGTCCCCCCTGTTTCTGGCACCAGG	13	19
  HEK3_4J_DEL1-5	GGCCCAGACTGAGCACGTGA	TGGAGGAAGCAGGGCTTCCTTTCCTCTGCCGTGCTCAG	13	29
  		TCTG
  HEK3_4J_DEL1-10	GGCCCAGACTGAGCACGTGA	TGGAGGAAGCAGGGCTTCCTTTCCCGTGCTCAGTCTG	13	24
  HEK3_4J_DEL1-15	GGCCCAGACTGAGCACGTGA	TGGAGGAAGCAGGGCTTCCCGTGCTCAGTCTG	13	19
  HEK3_4J_DEL1-25	GGCCCAGACTGAGCACGTGA	TGTCCTGCGACGCCCTCTGGAGGAAGCGTGCTCAGTCT	13	26
  		G
  HEK3_4J_DEL1-30	GGCCCAGACTGAGCACGTGA	TGTCCTGCGACGCCCTCTGGACGTGCTCAGTCTG	13	21
  HEK3_4J_DEL1-80	GGCCCAGACTGAGCACGTGA	AGTATCCCGGTGCAGGAGCTCGTGCTCAGTCTG	13	20
  HEK3_4I_1AINS	GGCCCAGACTGAGCACGTGA	TCTGCCATCATCGTGCTCAGTCTG	13	11
  HEK3_4I_CTTINS	GGCCCAGACTGAGCACGTGA	TCTGCCATCAAAGCGTGCTCAGTCTG	13	13
  HEK3_4I_TDEL	GGCCCAGACTGAGCACGTGA	TCTGCCATCCGTGCTCAGTCTG	13	9
  HEK3_4I_1-	GGCCCAGACTGAGCACGTGA	TGGAGGAAGCAGGGCTTCCTTTCCTCTGCCACGTGCTC	13	31
  3TGADEL		AGTCTG
  RNF2_4I_1TINS	GTCATCTTAGTCATTACCTG	AACGAACACCTCAGAGTAATGACTAAGATG	15	15
  RNF2_4I_1GTAINS	GTCATCTTAGTCATTACCTG	AACGAACACCTCAGTACGTAATGACTAAGATG	15	17
  RNF2_4I_4ADEL	GTCATCTTAGTCATTACCTG	AACGAACACCCAGGTAATGACTAAGATG	15	13
  RNF2_4I_3-	GTCATCTTAGTCATTACCTG	AACGAACACAGGTAATGACTAAGATG		15	11
  5GAGDEL
  FANCF_4I_3CINS	GGAATCCCTTCTGCAGCACC	GGAAAAGCGATCCAGGGTGCTGCAGAAGGGAT	14	18
  FANCF_4I_4GATINS	GGAATCCCTTCTGCAGCACC	GGAAAAGCGATCCAATCGGTGCTGCAGAAGGGAT	14	20
  FANCF_4I_6GDEL	GGAATCCCTTCTGCAGCACC	GGAAAAGCGATCAGGTGCTGCAGAAGGGAT	14	16
  FANCF_4I_5-	GGAATCCCTTCTGCAGCACC	GGAAAAGCGAAGGTGCTGCAGAAGGGAT	14	14
  7GGADEL
  EMX1_4I_6TINS	GAGTCCGAGCAGAAGAAGAA	GTGATGGGAGCACCTTCTTCTTCTGCTCGGA	14	17
  EMX1_4I_1TGCINS	GAGTCCGAGCAGAAGAAGAA	GTGATGGGAGCCCTTCGCATTCTTCTGCTCGGA	14	19
  EMX1_4I_5GDEL	GAGTCCGAGCAGAAGAAGAA	GTGATGGGAGCCTTCTTCTTCTGCTCGGA	14	15
  EMX1_4I_4-	GAGTCCGAGCAGAAGAAGAA	GTGATGGGAGTTCTTCTTCTGCTCGGA	14	13
  GGGGDEL
  RUNX1_4I_1CINS	GCATTTTCAGGAGGAAGCGA	TGTCTGAAGCCATCGGCTTCCTCCTGAAAAT	15	16
  RUNX1_4I_1ATGINS	GCATTTTCAGGAGGAAGCGA	TGTCTGAAGCCATCCATCTTCCTCCTGAAAAT	15	18
  RUNX1_4I_2GDEL	GCATTTTCAGGAGGAAGCGA	TGTCTGAAGCCATGCTTCCTCCTGAAAAT	15	14
  RUNX1_4I_2-	GCATTTTCAGGAGGAAGCGA	TGTCTGAAGCCGCTTCCTCCTGAAAAT	15	12
  4GATDEL
  VEGFA_4I_4CINS	GATGTCTGCAGGCCAGATGA	AATGTGCCATCTGGAGCCGCTCATCTGGCCTGCAGA	13	23
  VEGFA_4I_2ACAINS	GATGTCTGCAGGCCAGATGA	AATGTGCCATCTGGAGCCCTTGTCATCTGGCCTGCAGA	13	25
  VEGFA_4I_3ADEL	GATGTCTGCAGGCCAGATGA	AATGTGCCATCTGGAGCCCCATCTGGCCTGCAGA	13	21
  VEGFA_4I_2-	GATGTCTGCAGGCCAGATGA	AATGTGCCATCTGGAGCCATCTGGCCTGCAGA	13	19
  4GAGDEL
  DNMT1_4I_4CINS	GATTCCTGGTGCCAGAAACA	TCCCGTCACCCGCTGTTTCTGGCACCAGG	13	16
  DNMT1_4I_1TCAINS	GATTCCTGGTGCCAGAAACA	TCCCGTCACCCCTGTGATTTCTGGCACCAGG	13	18
  DNMT1_4I_3ADEL	GATTCCTGGTGCCAGAAACA	TCCCGTCACCCCGTTTCTGGCACCAG	13	14
  DNMT1_4I_3-	GATTCCTGGTGCCAGAAACA	TCCCGTCACCGTTTCTGGCACCAGG	13	12
  5AGGDEL
  HEK3_4K_1CTTINS_	GGCCCAGACTGAGCACGTGA	TGGAGGAAGCAGGGCTTCCTTTCCTCTGCATCAAAGCG	13	36
  5GDEL		TGCTCAGTCTG
  HEK3_4K_1CTTINS_	GGCCCAGACTGAGCACGTGA	TGGAGGAAGCAGGGCTTCCTTTCCTCTGCCATGAAAGC	13	37
  2GTOC		GTGCTCAGTCTG
  HEK3_4K_1CTTINS_	GGCCCAGACTGAGCACGTGA	TGGAGGAAGCAGGGCTTCCTTTCCTCTGCGATCCGTGC	13	33
  5GTOC		TCAGTCTG
  HEK3_4K_1CTTINS_	GGCCCAGACTGAGCACGTGA	TGGAGGAAGCAGGGCTTCCTTTCCTCTGACATGACGTG	13	34
  6GTOT		CTCAGTCTG
  RNF2_4K_2AAINS_	GTCATCTTAGTCATTACCTG	AACGAACACCATTGGTAATGACTAAGATG	15	14
  3-4GADEL
  RNF2_4K_1AINS_	GTCATCTTAGTCATTACCTG	AACGAACACGTCAGTGTAATGACTAAGATG	15	15
  5GTOC
  RNF2_4K_1-	GTCATCTTAGTCATTACCTG	AACGAACAACTCGTAATGACTAAGATG	15	12
  2CTDEL_6GTOT
  RNF2_4K_1CTOA_	GTCATCTTAGTCATTACCTG	AACGAACACATCATGTAATGACTAAGATG	15	14
  5GTOT
  FANCF_4K_1TINS_	GGAATCCCTTCTGCAGCACC	GGAAAAGCGATCGGTAGCTGCAGAAGGGAT	14	16
  3-4TGDEL
  FANCF_4K_1TINS_	GGAATCCCTTCTGCAGCACC	GGAAAAGCGATTCAGGTAGCTGCAGAAGGGAT	14	18
  6GTOA
  FANCF_4K_2CDEL_	GGAATCCCTTCTGCAGCACC	GGAAAAGCGATCAAGTGCTGCAGAAGGGAT	14	16
  5GTOT

• TABLE 3E
  FIGS. 41A-41K nicking sgRNA

  		SPACER SEQUENCE
  	NICKING SGRNA	(SEQ ID NOS: 3456-3463)
  	HEK3_4A_+90	GTCAACCAGTATCCCGGTGC
  	HEK3_4B_+90	GTCAACCAGTATCCCGGTGC
  	RNF2_4C_+41	GTCAACCATTAAGCAAAACAT
  	FANCF_4D_+48	GGGGTCCCAGGTGCTGACGT
  	EMX1_4E_+53	GACATCGATGTCCTCCCCAT
  	RUNX1_4F_+38	GATGAAGCACTGTGGGTACGA
  	VEGFA_4G_+57	GATGTACAGAGAGCCCAGGGC
  	DNMT1_4H_+49	GCCCTTCAGCTAAAATAAAGG

• TABLE 3F
  FIGS. 42A-42H PEgRNA

  		3′
  	SPACER	EXTEN-		RT
  	SEQUENCE	SION		TEMP-
  	(SEQ	(SEQ ID	PBS	LATE
  	ID NOS:	NOS:	LENGTH	LENGTH
  PEGRNA	3464-3478)	3479-3493)	(NT)	(NT)
  HEK3_5A_	GGCCCAGACTGA	TCTGTCATCAC	13	10
  C3	GCACGTGA	GTGCTCAGTCTG
  HEK3_5A_	GGCCCAGACTGA	TCTGCTATCACG	13	10
  C4	GCACGTGA	TGCTCAGTCTG
  HEK3_5A_	GGCCCAGACTGA	TCTGCCATTACG	13	10
  C7	GCACGTGA	TGCTCAGTCTG
  FANCF_5A_	GGAATCCCTTCTG	GGAAAAGTGATC	14	17
  C3	CAGCACC	CAGGTGCTGCAG
  		AAGGGAT
  FANCF_5A_	GGAATCCCTTCTG	GGAAAAGCGATT	14	17
  C7	CAGCACC	CAGGTGCTGCAG
  		AAGGGAT
  FANCF_5A_	GGAATCCCTTCTG	GGAAAAGCGATC	14	17
  C8	CAGCACC	TAGGTGCTGCAG
  		AAGGGAT
  EMX1_5A_	GAGTCCGAGCAG	GTGATGGGAGTC	14	16
  C5	AAGAAGAA	CTTCTTCTTCTG
  		CTCGGA
  EMX1_5A_	GAGTCCGAGCAG	GTGATGGGAGCT	14	16
  C6	AAGAAGAA	CTTCTTCTTCTG
  		CTCGGA
  EMX1_5A_	GAGTCCGAGCAG	GTGATGGGAGC	14	16
  C7	AAGAAGAA	CTTTCTTCTTC
  		TGCTCGGA
  EMX1_5C_	GAGTCCGAGCAG	GTGATGGGAGT	14	16
  C5_6	AAGAAGAA	TCTTCTTCTTC
  		TGCTCGGA
  EMX1_5C_	GAGTCCGAGCAG	GTGATGGGAGT	14	16
  C5_7	AAGAAGAA	CTTTCTTCTTC
  		TGCTCGGA
  EMX1_5C_	GAGTCCGAGCAG	GTGATGGGAGC	14	16
  C6_7	AAGAAGAA	TTTTCTTCTTC
  		TGCTCGGA
  EMX1_5C_	GAGTCCGAGCAG	GTGATGGGAGT	14	16
  C5_67	AAGAAGAA	TTTTCTTCTTC
  		TGCTCGGA
  HEK3_5D_	GGCCCAGACTGA	TCTGCCGTCAC	13	10
  A5	GCACGTGA	GTGCTCAGTCT
  		G
  HEK3_5D_	GGCCCAGACTGA	TCTGCCATCGC	13	10
  A8	GCACGTGA	GTGCTCAGTC
  		TG

• TABLE 3G
  FIGS. 42A-42H nicking sgRNA

  	POSSIBLE	POSSIBLE
  	SPACER SEQUENCE	SPACER SEQUENCE
  NICKING	(SEQ ID NOS:	(SEQ ID
  SGRNA	616-618)	NOS: 619-621)
  HEK3_5A-	GTCAACCAGTA	GTCAACCAGTAT
  F_+90	TCCCGGTGC	CCCGGTGC
  FANCF_5A-	GGGGTCCCAGG	GATGTACAGAGA
  F_+48	TGCTGACGT	GCCCAGGGC
  EMX1_5A-	GATGTACAGAG	GGGGTCCCAGGT
  F_+57	AGCCCAGGGC	GCTGACGT

• TABLE 3H
  FIGS. 42A-42H base editing sgRNA

  	BASE EDITING
  	SGRNA	SPACER SEQUENCE
  	HEK3_5A-	GTGCCATCACGTGCTCAGTCT
  	F BE	(SEQ ID NO: 455)
  	FANCF_5A-	GAGCGATCCAGGTGCTGCAGA
  	F_BE	(SEQ ID NO: 456)
  	EMX1_5A-	GGAGCCCTTCTTCTTCTGCT
  	F_BE	(SEQ ID NO: 455)
  	ON-TARGET
  	SGRNA	SPACER SEQUENCE
  	HEK3_5G	GGCCCAGACTGAGCACGTGA
  		(SEQ ID NO: 510)
  	HEK4_5G	GGCACTGCGGCTGGAGGTGG
  		(SEQ ID NO: 511)
  	EMX1_5G	GAGTCCGAGCAGAAGAAGAA
  		(SEQ ID NO: 512)
  	FANCF_5G	GGAATCCCTTCTGCAGCACC
  		(SEQ ID NO: 513)

• TABLE 3I
  FIGS.
  42A-42H on-target sgRNA

• TABLE 3J
  FIGS. 42A-42H on-target PEgRNA

  				RT
  			PBS	TEM-
  	SPACER SEQUENCE	3′ EXTENSION	LENGTH	PLATE
  ON-TARGET	(SEQ ID NO:	(SEQ ID	(NT)	LENGTH
  PEGRNA	663-677)	NO: 678-692)	TH	(NT)

  HEK3_5G-	GGCCCAGACTGAGC	TCTGCCATCTCGTGCTCA	13	10
  H_PEGRNA_1	ACGTGA	GTCTG
  HEK3_5G-	GGCCCAGACTGAGC	TCTGCCATCAAAGCGTG	13	13
  H_PEGRNA_2	ACGTGA	CTCAGTCTG
  HEK3_5G-	GGCCCAGACTGAGC	TCTGCCATCCGTGCTCA	13	9
  H_PEGRNA_3	ACGTGA	GTCTG
  HEK3_5G-	GGCCCAGACTGAGC	TCTGCGATCACGTGCTC	13	10
  H_PEGRNA_4	ACGTGA	AGTCTG
  HEK4_5G-	GGCACTGCGGCTGG	TTAACGCCCACCTCCAG	9	10
  H_PEGRNA_1	AGGTGG	CC
  HEK4_5G-	GGCACTGCGGCTGG	TTAACCCCCCCCTCCAG	9	10
  H_PEGRNA_2	AGGTGG	CC
  HEK4_5G-	GGCACTGCGGCTGG	TTAACCCCTTACACCTCC	9	13
  H_PEGRNA_3	AGGTGG	AGCC
  HEK4_5G-	GGCACTGCGGCTGG	TTAACCCCCCCTCCAGC	9	9
  H_PEGRNA_4	AGGTGG	C
  EMX1_5G-	GAGTCCGAGCAGAA	GTGATGGGAGCACTTCT	14	16
  H_PEGRNA_1	GAAGAA	TCTTCTGCTCGGA
  EMX1_5G-	GAGTCCGAGCAGAA	GTGATGGGAGCCCTGCT	14	16
  H_PEGRNA_2	GAAGAA	TCTTCTGCTCGGA
  EMX1_5G-	GAGTCCGAGCAGAA	GTGATGGGAGCCCTTCG	14	19
  H_PEGRNA_3	GAAGAA	CATTCTTCTGCTCGGA
  EMX1_5G-	GAGTCCGAGCAGAA	GTGATGGGAGTTCTTCTT	14	13
  H_PEGRNA_4	GAAGAA	CTGCTCGGA
  FANCF_5G-	GGAATCCCTTCTGCA	GGAAAAGCGATGCAGGT	14	17
  H_PEGRNA_1	GCACC	GCTGCAGAAGGGAT
  FANCF_5G-	GGAATCCCTTCTGCA	GGAAAAGCGATCCAGGC	14	17
  H_PEGRNA_2	GCACC	GCTGCAGAAGGGAT
  FANCF_5G-	GGAATCCCTTCTGCA	GGAAAAGCGATCCAATC	14	20
  H_PEGRNA_3	GCACC	GGTGCTGCAGAAGGGAT

• TABLE 3K
  FIGS. 49A-49BPEgRNA

  SPACER

  	3′		RT
  	SEQUENCE	EXTENSION		TEM-
  	(SEQ ID	(SEQ ID	PBS	PLATE
  ON-TARGET	NO:	NO:	LENGTH	LENGTH
  PEGRNA	663-677)	678-692)	(NT)	(NT)

  HEK3_5G-	GGCCC	TCTGC	13	10
  H_PEGRNA_	AGACT	CATCT
  1	GAGCA	CGTGC
  	CGTGA	TCAGT
  		CTG
  HEK3_5G-	GGCCC	TCTGC	13	13
  H_PEGRNA_	AGACT	CATCA
  2	GAGCA	AAGCG
  	CGTGA	TGCTC
  		AGTCT
  		G
  HEK3_5G- H_	GGCCC	TCTGC	13	9
  PEGRNA_	AGACT	CATCC
  3	GAGCA	GTGCT
  	CGTGA	CAGTC
  		TG
  HEK3 5G-	GGCCC	TCTGC	13	10
  H PEGRNA_	AGACT	GATCA
  4	GAGCA	CGTGC
  	CGTGA	TCAGT
  		CTG
  HEK4_5G-	GGCAC	TTAAC	9	10
  H PEGRNA_	TGCGG	GCCCA
  1	CTGGA	CCTCC
  	GGTGG	AGCC
  HEK4_5G-	GGCAC	TTAAC	9	10
  H PEGRNA_	TGCGG	CCCCC
  2	CTGGA	CCTCC
  	GGTGG	AGCC
  HEK4_5G-	GGCAC	TTAAC	9	13
  H_PEGRNA_	TGCGG	CCCTT
  _3	CTGGA	ACACC
  	GGTGG	TCCAG
  		CC
  HEK4_5G-	GGCAC	TTAAC	9	9
  H_PEGRNA_	TGCGG	CCCCC
  4	CTGGA	CTCCA
  	GGTGG	GCC
  EMX1_5G-	GAGTC	GTGAT	14	16
  H PEGRNA_1	CGAGC	GGGAG
  	AGAAG	CACTT
  	AAGAA	CTTCT
  		TCTGC
  		TCGGA
  EMX1_5G-	GAGTC	GTGAT	14	16
  H_PEGRNA_	CGAGC	GGGAG
  2	AGAAG	CCCTG
  	AAGAA	CTTCT
  		TCTGC
  		TCGGA
  EMX1_5G-	GAGTC	GTGAT	14	19
  H PEGRNA_	CGAGC	GGGAG
  3	AGAAG	CCCTT
  	AAGAA	CGCAT
  		TCTTC
  		TGCTC
  		GGA
  EMX1_5G-	GAGTC	GTGAT	14	13
  H_PEGRNA_	CGAGC	GGGAG
  4	AGAAG	TTCTT
  	AAGAA	CTTCT
  		GCTCG
  		GA
  FANCF_5G-	GGAAT	GGAAA	14	17
  H_PEGRNA_	CCCTT	AGCGA
  1	CTGCA	TGCAG
  	GCACC	GTGCT
  		GCAGA
  		AGGGA
  		T
  FANCF_5G-	GGAAT	GGAAA	14	17
  H_PEGRNA_	CCCTT	AGCGA
  2	CTGCA	TCCAG
  	GCACC	GCGCT
  		GCAGA
  		AGGGA
  		T
  FANCF_5G-	GGAAT	GGAAA	14	20
  H_PEGRNA_	CCCTT	AGCGA
  3	CTGCA	TCCAA
  	GCACC	TC
  		GG
  		TGCTG
  		CAGAA
  		GGGAT

• TABLE 3L
  FIGS. 47A-74D PEgRNA

  	SPACER	3′
  	SEQUENCE	EXTENSION		RT
  	(SEQ ID	SEQUENCE		TEM-
  	NO:	(SEQ ID	PBS	PLATE
  	3541-	NO:	LENGTH	LENGTH
  PEGRNA	3547)	3549-3556)	(NT)	(NT)

  HEK3_ED4B_1	GGCCC	TCTGC	13	9
  TDEL	AGACT	CATCC
  	GAGCA	GTGCT
  	CGTGA	CAGTC
  		TG
  HEK3_ED4B_1	GGCCC	TCTGC	13	11
  AINS	AGACT	CATCA
  	GAGCA	TCGTG
  	CGTGA	CTCAC
  		TCTG
  HEK3_ED4B_1	GGCCC	TCTGC	13	13
  CTTINS	AGACT	CATCA
  	GAGCA	AAGCG
  	CGTGA	TGCTC
  		AGTCT
  		G
  HEK3_ED4C_2	GGCCC	TCTGC	13	10
  GTOC	AGACT	CATGA
  	GAGCA	CGTGC
  	CGTGA	TCAGT
  		CTG
  HEK3_ED4D_1	GGCCC	TGGAG	13	58
  FLAGINS	AGACT	GAAGC
  	GAGCA	AGGGC
  	CGTGA	TTCCT
  		TTCCT
  		CTGCC
  		ATCAC
  		TTATC
  		GTCGT
  		CATCC
  		TTGTA
  		ATCCG
  		TGCTC
  		AGTCT
  		G
  RNF2_ED4E_1	GTCAT	AACGA	15	14
  CTOA	CTTAG	ACACC
  	TCATT	TCATG
  	ACCTG	TAATG
  		ACTAA
  		GATG
  EMX1_ED4F_1	GAGTC	ATGGG	13	13
  GTOC	CGAGC	AGCCC
  	AGAAG	TTGTT
  	AAGAA	CTTCT
  		GCTCG
  		G
  HBB_EDAG_21	GTAAC	ATCTG	12	14
  TOA	GGCAG	ACTCC
  	ACTTC	TGTGG
  	TCCTC	AGAAG
  		TCTGC
  		C

• TABLE 3M
  FIGS. 48A-48C PEgRNA

  SPACER

  	3′
  	SEQUENCE	EXTENSION		RT
  	(SEQ ID	SEQUENCE		TEM-
  	NO:	(SEQ ID	PBS	PLATE
  	3557-	NO:	LENGTH	LENGTH
  PEGRNA	3627)	3628-3698)	(NT)	(NT)

  VEGFAP_ED	CAGATGA	TGGAGCACTCATCTGGCCTG	13	31
  5A_31		CAGA
  VEGFA_ED	GATGTCTGCAGGC	CCTCTGACAATGTGCCATCT	13	30
  5A_30	CAGATGA	GGAGCACTCATCTGGCCTGC
  		AGA
  VEGFA_ED	GATGTCTGCAGGC	CTCTGACAATGTGCCATCTG	13	29
  5A_29	CAGATGA	GAGCACTCATCTGGCCTGCA
  		GA
  VEGFA_ED	GATGTCTGCAGGC	TCTGACAATGTGCCATCTGG	13	28
  5A_28	CAGATGA	AGCACTCATCTGGCCTGCAG
  		A
  VEGFA_ED	GATGTCTGCAGGC	CTGACAATGTGCCATCTGGA	13	27
  5A_27	CAGATGA	GCACTCATCTGGCCTGCAGA
  VEGFA_ED	GATGTCTGCAGGC	TGACAATGTGCCATCTGGAG	13	26
  5A_26	CAGATGA	CACTCATCTGGCCTGCAGA
  VEGFA_ED	GATGTCTGCAGGC	GACAATGTGCCATCTGGAGC	13	25
  5A_25	CAGATGA	ACTCATCTGGCCTGCAGA
  VEGFA_ED	GATGTCTGCAGGC	ACAATGTGCCATCTGGAGCA	13	24
  5A_24	CAGATGA	CTCATCTGGCCTGCAGA
  VEGFA_ED	GATGTCTGCAGGC	CAATGTGCCATCTGGAGCAC	13	23
  5A_23	CAGATGA	TCATCTGGCCTGCAGA
  VEGFA_ED	GATGTCTGCAGGC	AATGTGCCATCTGGAGCACT	13	22
  5A_22	CAGATGA	CATCTGGCCTGCAGA
  VEGFA_ED	GATGTCTGCAGGC	ATGTGCCATCTGGAGCACTC	13	21
  5A_21	CAGATGA	ATCTGGCCTGCAGA
  VEGFA_ED	GATGTCTGCAGGC	TGTGCCATCTGGAGCACTCA	13	20
  5A_20	CAGATGA	TCTGGCCTGCAGA
  VEGFA_ED	GATGTCTGCAGGC	GTGCCATCTGGAGCACTCAT	13	19
  5A_19	CAGATGA	CTGGCCTGCAGA
  VEGFA_ED	GATGTCTGCAGGC	TGCCATCTGGAGCACTCATC	13	18
  5A_18	CAGATGA	TGGCCTGCAGA
  VEGFA_ED	GATGTCTGCAGGC	GCCATCTGGAGCACTCATCT	13	17
  5A_17	CAGATGA	GGCCTGCAGA
  VEGFA_ED	GATGTCTGCAGGC	CCATCTGGAGCACTCATCTG	13	16
  5A_16	CAGATGA	GCCTGCAGA
  VEGFA_ED	GATGTCTGCAGGC	CATCTGGAGCACTCATCTGG	13	15
  5A_15	CAGATGA	CCTGCAGA
  VEGFA_ED	GATGTCTGCAGGC	ATCTGGAGCACTCATCTGGC	13	14
  5A_14	CAGATGA	CTGCAGA
  VEGFA_ED	GATGTCTGCAGGC	TCTGGAGCACTCATCTGGCC	13	13
  5A_13	CAGATGA	TGCAGA
  VEGFA_ED	GATGTCTGCAGGC	CTGGAGCACTCATCTGGCCT	13	12
  5A_12	CAGATGA	GCAGA
  VEGFA_ED	GATGTCTGCAGGC	TGGAGCACTCATCTGGCCTG	13	11
  5A_11	CAGATGA	CAGA
  VEGFA_ED	GATGTCTGCAGGC	GGAGCACTCATCTGGCCTGC	13	10
  5A_10	CAGATGA	AGA
  VEGFA_ED	GATGTCTGCAGGC	GAGCACTCATCTGGCCTGCA	13	9
  5A_9	CAGATGA	GA
  VEGFA_ED	GATGTCTGCAGGC	AGCACTCATCTGGCCTGCAG	13	8
  5A_8	CAGATGA	A
  DNMT1_ED	GATTCCTGGTGCC	AGGACTAGTTCTGCCCTCCC	13	31
  5B_31	AGAAACA	GTCACCACTGTTTCTGGCAC
  		CAGG
  DNMT1_ED	GATTCCTGGTGCC	GGACTAGTTCTGCCCTCCCG	13	30
  5B_30	AGAAACA	TCACCACTGTTTCTGGCACC
  		AGG
  DNMT1_ED	GATTCCTGGTGCC	GACTAGTTCTGCCCTCCCGT	13	29
  5B_29	AGAAACA	CACCACTGTTTCTGGCACCA
  		GG
  DNMT1_ED	GATTCCTGGTGCC	ACTAGTTCTGCCCTCCCGTC	13	28
  5B_28	AGAAACA	ACCACTGTTTCTGGCACCAG
  		G
  DNMT1_ED	GATTCCTGGTGCC	CTAGTTCTGCCCTCCCGTCA	13	27
  5B_27	AGAAACA	CCACTGTTTCTGGCACCAGG
  DNMT1_ED	GATTCCTGGTGCC	TAGTTCTGCCCTCCCGTCAC	13	26
  5B_26	AGAAACA	CACTGTTTCTGGCACCAGG
  DNMT1_ED	GATTCCTGGTGCC	AGTTCTGCCCTCCCGTCACC	13	25
  5B_25	AGAAACA	ACTGTTTCTGGCACCAGG
  DNMT1_ED	GATTCCTGGTGCC	GTTCTGCCCTCCCGTCACCA	13	24
  5B_24	AGAAACA	CTGTTTCTGGCACCAGG
  DNMT1_ED	GATTCCTGGTGCC	TTCTGCCCTCCCGTCACCAC	13	23
  5B_23	AGAAACA	TGTTTCTGGCACCAGG
  DNMT1_ED	GATTCCTGGTGCC	TCTGCCCTCCCGTCACCACT	13	22
  5B_22	AGAAACA	GTTTCTGGCACCAGG
  DNMT1_ED	GATTCCTGGTGCC	CTGCCCTCCCGTCACCACTG	13	21
  5B_21	AGAAACA	TTTCTGGCACCAGG
  DNMT1_ED	GATTCCTGGTGCC	TGCCCTCCCGTCACCACTGT	13	20
  5B_20	AGAAACA	TTCTGGCACCAGG
  DNMT1_ED	GATTCCTGGTGCC	GCCCTCCCGTCACCACTGTT	13	19
  5B_19	AGAAACA	TCTGGCACCAGG
  DNMT1_ED	GATTCCTGGTGCC	CCCTCCCGTCACCACTGTTT	13	18
  5B_18	AGAAACA	CTGGCACCAGG
  DNMT1_ED	GATTCCTGGTGCC	CCTCCCGTCACCACTGTTTC	13	17
  5B_17	AGAAACA	TGGCACCAGG
  DNMT1_ED	GATTCCTGGTGCC	CTCCCGTCACCACTGTTTCT	13	16
  5B_16	AGAAACA	GGCACCAGG
  DNMT1_ED	GATTCCTGGTGCC	TCCCGTCACCACTGTTTCTG	13	15
  5B_15	AGAAACA	GCACCAGG
  DNMT1_ED	GATTCCTGGTGCC	CCCGTCACCACTGTTTCTGG	13	14
  5B_14	AGAAACA	CACCAGG
  DNMT1_ED	GATTCCTGGTGCC	CCGTCACCACTGTTTCTGGC	13	13
  5B_13	AGAAACA	ACCAGG
  DNMT1_ED	GATTCCTGGTGCC	CGTCACCACTGTTTCTGGCA	13	12
  5B_12	AGAAACA	CCAGG
  DNMT1_ED	GATTCCTGGTGCC	GTCACCACTGTTTCTGGCAC	13	11
  5B_11	AGAAACA	CAGG
  DNMT1_ED	GATTCCTGGTGCC	TCACCACTGTTTCTGGCACC	13	10
  5B_10	AGAAACA	AGG
  DNMT1_ED	GATTCCTGGTGCC	CACCACTGTTTCTGGCACCA	13	9
  5B_9	AGAAACA	GG
  DNMT1_ED	GATTCCTGGTGCC	ACCACTGTTTCTGGCACCAG	13	8
  5B_8	AGAAACA	G
  RUNX1_ED	GCATTTTCAGGAG	AATGACTCAAATATGCTGTC	15	31
  5C_31	GAAGCGA	TGAAGCAATCGCTTCCTCCT
  		GAAAAT
  RUNX1_ED	GCATTTTCAGGAG	ATGACTCAAATATGCTGTCT	15	30
  5C_30	GAAGCGA	GAAGCAATCGCTTCCTCCTG
  		AAAAT
  RUNX1_ED	GCATTTTCAGGAG	TGACTCAAATATGCTGTCTG	15	29
  5C_29	GAAGCGA	AAGCAATCGCTTCCTCCTGA
  		AAAT
  RUNX1_ED	GCATTTTCAGGAG	GACTCAAATATGCTGTCTGA	15	28
  5C_28	GAAGCGA	AGCAATCGCTTCCTCCTGAA
  		AAT
  RUNX1_ED	GCATTTTCAGGAG	ACTCAAATATGCTGTCTGAA	15	27
  5C_27	GAAGCGA	GCAATCGCTTCCTCCTGAAA
  		AT
  RUNX1_ED	GCATTTTCAGGAG	CTCAAATATGCTGTCTGAAG	15	26
  5C_26	GAAGCGA	CAATCGCTTCCTCCTGAAAA
  		T
  RUNX1_ED	GCATTTTCAGGAG	TCAAATATGCTGTCTGAAGC	15	25
  5C_25	GAAGCGA	AATCGCTTCCTCCTGAAAAT
  RUNX1_ED	GCATTTTCAGGAG	CAAATATGCTGTCTGAAGCA	15	24
  5C_24	GAAGCGA	ATCGCTTCCTCCTGAAAAT
  RUNX1_ED	GCATTTTCAGGAG	AAATATGCTGTCTGAAGCAA	15	23
  5C_23	GAAGCGA	TCGCTTCCTCCTGAAAAT
  RUNX1_ED	GCATTTTCAGGAG	AATATGCTGTCTGAAGCAAT	15	22
  5C_22	GAAGCGA	CGCTTCCTCCTGAAAAT
  RUNX1_ED	GCATTTTCAGGAG	ATATGCTGTCTGAAGCAATC	15	21
  5C_21	GAAGCGA	GCTTCCTCCTGAAAAT
  RUNX1_ED	GCATTTTCAGGAG	TATGCTGTCTGAAGCAATCG	15	20
  5C_20	GAAGCGA	CTTCCTCCTGAAAAT
  RUNX1_ED	GCATTTTCAGGAG	ATGCTGTCTGAAGCAATCGC	15	19
  5C_19	GAAGCGA	TTCCTCCTGAAAAT
  RUNX1_ED	GCATTTTCAGGAG	TGCTGTCTGAAGCAATCGCT	15	18
  5C_18	GAAGCGA	TCCTCCTGAAAAT
  RUNX1_ED	GCATTTTCAGGAG	GCTGTCTGAAGCAATCGCTT	15	17
  5C_17	GAAGCGA	CCTCCTGAAAAT
  RUNX1_ED	GCATTTTCAGGAG	CTGTCTGAAGCAATCGCTTC	15	16
  5C_16	GAAGCGA	CTCCTGAAAAT
  RUNX1_ED	GCATTTTCAGGAG	TGTCTGAAGCAATCGCTTCC	15	15
  5C_15	GAAGCGA	TCCTGAAAAT
  RUNX1_ED	GCATTTTCAGGAG	GTCTGAAGCAATCGCTTCCT	15	14
  5C_14	GAAGCGA	CCTGAAAAT
  RUNX1_ED	GCATTTTCAGGAG	TCTGAAGCAATCGCTTCCTC	15	13
  5C_13	GAAGCGA	CTGAAAAT
  RUNX1_ED	GCATTTTCAGGAG	CTGAAGCAATCGCTTCCTCC	15	12
  5C_12	GAAGCGA	TGAAAAT
  RUNX1_ED	GCATTTTCAGGAG	TGAAGCAATCGCTTCCTCCT	15	11
  5C_11	GAAGCGA	GAAAAT
  RUNX1_ED	GCATTTTCAGGAG	GAAGCAATCGCTTCCTCCTG	15	10
  5C_10	GAAGCGA	AAAAT
  RUNX1_ED	GCATTTTCAGGAG	AAGCAATCGCTTCCTCCTGA	15	9
  5C_9	GAAGCGA	AAAT

• TABLE 3N
  FIGS. 48A-48C PEgRNA

  SPACER

  	3′
  	SEQUENCE	EXTENSION		RT
  	(SEQ ID	SEQUENCE		TEM-
  	NO:	(SEQ ID	PBS	PLATE
  	3557-	NO:	LENGTH	LENGTH
  PEGRNA	3627)	3628-3698)	(NT)	(NT)

  HEK3_	GGCCCAGAC	TCTGCTATC	13	10
  ED6_+63	TGAGC	ACGTGCT
  GTOA	ACGTGA	CAGTCTG
  	(SEQ ID	(SEQ ID
  	NO: 393)	NO: 394)

• TABLE 30
  FIGS. 48A-48C nicking sgRNA

  	NICKING
  	SGRNA	SPACER SEQUENCE
  	HEK3_	GCACATACTAGCCCCTGTCT (SEQ
  	ED6_+63	ID NO: 395)

• TABLE 3P
  FIGS. 50A-50B PEgRNA

  SPACER

  	3′
  	SEQUENCE	EXTENSION		RT
  	(SEQ ID	SEQUENCE		TEM-
  	NO:	(SEQ ID	PBS	PLATE
  	3699-	NO:	LENGTH	LENGTH
  PEGRNA	3754	3755-3810)	(NT)	(NT)

  HBB	GTAACGGCAGAC	AGACTTCTCCTCAGGAGTCAGGT	12	14
  3.5	TTCTCCAC	GCAC
  HBB	GCATGGTGCACCT	AGACTTCTCTTCAGGAGTCAGGT	13	14
  3.7	GACTCCTG	GCAC
  HBB	GCATGGTGCACCT	TAACGGCAGACTTCTCCTCAGGA	13	19
  5.2	GACTCCTG	GTCAGGTGCAC
  HBB	GCATGGTGCACCT	ACGGCAGACTTCTCCTCAGGAGT	13	17
  5.3	GACTCCTG	CAGGTGCAC
  HBB	GCATGGTGCACCT	GGCAGACTTCTCCTCAGGAGTCA	13	16
  5.4	GACTCCTG	GGTGCAC
  HBB	GCATGGTGCACCT	GCAGACTTCTCCTCAGGAGTCAG	13	13
  5.5	GACTCCTG	GTGCAC
  HBB	GCATGGTGCACCT	GACTTCTCCTCAGGAGTCAGGTG	13	12
  5.6	GACTCCTG	CAC
  HBB	GCATGGTGCACCT	ACTTCTCCTCAGGAGTCAGGTGC	13	21
  5.7	GACTCCTG	AC
  HBB	GCATGGTGCACCT	TAACGGCAGACTTCTCCTCAGGA	12	19
  5.8	GACTCCTG	GTCAGGTGCA
  HBB	GCATGGTGCACCT	ACGGCAGACTTCTCCTCAGGAGT	12	17
  5.9	GACTCCTG	CAGGTGCA
  HBB	GCATGGTGCACCT	GGCAGACTTCTCCTCAGGAGTCA	12	16
  5.10	GACTCCTG	GGTGCA
  HBB	GCATGGTGCACCT	GCAGACTTCTCCTCAGGAGTCAG	12	13
  5.11	GACTCCTG	GTGCA
  HBB	GCATGGTGCACCT	GACTTCTCCTCAGGAGTCAGGTG	12	12
  5.12	GACTCCTG	CA
  HBB	GCATGGTGCACCT	ACTTCTCCTCAGGAGTCAGGTGC	12	14
  5.13	GACTCCTG	A
  HEX	ATCCTTCCAGTCA	ATATCTTATGGCCCTGACTGGAA	13	14
  AS 1	GGGCCAT
  HEX	ATCCTTCCAGTCA	TATATCTTATGGCCCTGACTGGAA	13	15
  AS 2	GGGCCAT
  HEX	ATCCTTCCAGTCA	GTATATCTTATGGCCCTGACTGGA	13	16
  AS 3	GGGCCAT	A
  HEX	ATCCTTCCAGTCA	ACCGTATATCTTATGGCCCTGACT	13	19
  AS 4	GGGCCAT	GGAA
  HEX	ATCCTTCCAGTCA	AACCGTATATCTTATGGCCCTGAC	13	20
  AS 5	GGGCCAT	TGGAA
  HEX	ATCCTTCCAGTCA	GAACCGTATATCTTATGGCCCTGA	13	21
  AS 6	GGGCCAT	CTGGAA
  HEX	ATCCTTCCAGTCA	TGAACCGTATATCTTATGGCCCTG	13	22
  AS 7	GGGCCAT	ACTGGAA
  HEX	ATCCTTCCAGTCA	ATATCTTATGGCCCTGACT	9	14
  AS 8	GGGCCAT
  HEX	ATCCTTCCAGTCA	TATATCTTATGGCCCTGACT	9	15
  AS 9	GGGCCAT
  HEX	ATCCTTCCAGTCA	GTATATCTTATGGCCCTGACT	9	16
  AS 10	GGGCCAT
  HEX	ATCCTTCCAGTCA	ACCGTATATCTTATGGCCCTGACT	9	19
  AS 11	GGGCCAT
  HEX	ATCCTTCCAGTCA	AACCGTATATCTTATGGCCCTGAC	9	20
  AS 12	GGGCCAT	T
  HEX	ATCCTTCCAGTCA	GAACCGTATATCTTATGGCCCTGA	9	21
  AS 13	GGGCCAT	CT
  HEX	ATCCTTCCAGTCA	TGAACCGTATATCTTATGGCCCTG	9	22
  AS 14	GGGCCAT	ACT
  HEX	ATCCTTCCAGTCA	TGAACCGTATATCTTATGGCCCTG	8	22
  AS 15	GGGCCAT	AC
  HEX	ATCCTTCCAGTCA	TGAACCGTATATCTTATGGCCCTG	10	22
  AS 16	GGGCCAT	ACTG
  HEX	ATCCTTCCAGTCA	TGAACCGTATATCTTATGGCCCTG	11	22
  AS 17	GGGCCAT	ACTGG
  HEX	ATCCTTCCAGTCA	TGAACCGTATATCTTATGGCCCTG	12	22
  AS 18	GGGCCAT	ACTGGA
  HEX	ATCCTTCCAGTCA	TGAACCGTATATCTTATGGCCCTG	13	22
  AS 19	GGGCCAT	ACTGGAA
  HEX	ATCCTTCCAGTCA	TGAACCGTATATCTTATGGCCCTG	14	22
  AS 20	GGGCCAT	ACTGGAAG
  HEX	ATCCTTCCAGTCA	TGAACCGTATATCTTATGGCCCTG	15	22
  AS 21	GGGCCAT	ACTGGAAGG
  HEX	ATCCTTCCAGTCA	ACCTGAACCGTATATCTTATGGCC	9	25
  AS 22	GGGCCAT	CTGACT
  HEX	ATCCTTCCAGTCA	TACCTGAACCGTATATCTTATGGC	9	26
  AS 23	GGGCCAT	CCTGACT
  HEX	ATCCTTCCAGTCA	GTACCTGAACCGTATATCTTATGG	9	27
  AS 24	GGGCCAT	CCCTGACT
  HEX	ATCCTTCCAGTCA	GGTACCTGAACCGTATATCTTATG	9	28
  AS 25	GGGCCAT	GCCCTGACT
  HEX	ATCCTTCCAGTCA	TGGTACCTGAACCGTATATCTTAT	9	29
  AS 26	GGGCCAT	GGCCCTGACT
  HEX	ATCCTTCCAGTCA	ACCTGAACCGTATATCCTATGGCC	13	21
  A 5	GGGCCAT	CTGACTGGAA
  HEX	ATCCTTCCAGTCA	ACCGTATATCCTATGGCCCTGACT	13	15
  A6	GGGCCAT	GGAA
  HEX	ATCCTTCCAGTCA	ACCTGAACCGTATATCCTATGGCC	15	21
  A 7	GGGCCAT	CTGACTGGAAGG
  HEX	ATCCTTCCAGTCA	ACCTGAACCGTATATCCTATGGCC	14	21
  A 8	GGGCCAT	CTGACTGGAAG
  HEX	ATCCTTCCAGTCA	ACCTGAACCGTATATCCTATGGCC	12	21
  A 9	GGGCCAT	CTGACTGGA
  HEX	ATCCTTCCAGTCA	ACCTGAACCGTATATCCTATGGCC	11	21
  A 10	GGGCCAT	CTGACTGG
  HEX	ATCCTTCCAGTCA	ACCTGAACCGTATATCCTATGGCC	10	21
  A 11	GGGCCAT	CTGACTG
  HEX	ATCCTTCCAGTCA	AACCGTATATCCTATGGCCCTGAC	13	16
  A 12	GGGCCAT	TGGAA
  HEX	ATCCTTCCAGTCA	TGAACCGTATATCCTATGGCCCTG	13	18
  A 13	GGGCCAT	ACTGGAA
  HEX	ATCCTTCCAGTCA	TACCTGAACCGTATATCCTATGGC	13	22
  A 14	GGGCCAT	CCTGACTGGAA
  HEX	ATCCTTCCAGTCA	TGGTACCTGAACCGTATATCCTAT	13	25
  A 15	GGGCCAT	GGCCCTGACTGGAA
  HEX	ATCCTTCCAGTCA	GTACCTGAACCGTATATCCTATGG	13	23
  A 16	GGGCCAT	CCCTGACTGGAA
  HEX	ATCCTTCCAGTCA	AACCGTATATCCTATGGCCCTGAC	10	16
  A 17	GGGCCAT	TG
  HEX	ATCCTTCCAGTCA	TGAACCGTATATCCTATGGCCCTG	10	18
  A 18	GGGCCAT	ACTG
  HEX	ATCCTTCCAGTCA	TACCTGAACCGTATATCCTATGGC	10	22
  A 19	GGGCCAT	CCTGACTG
  HEX	ATCCTTCCAGTCA	TGGTACCTGAACCGTATATCCTAT	10	25
  A 20	GGGCCAT	GGCCCTGACTG

• TABLE 3Q
  FIGS. 50A-50B nicking sgRNA

  	NICKING SGRNA	SPACER SEQUENCE
  	HBB_ED7A_+72	GCCTTGATACCAACCTGCCCA
  		(SEQ ID NO: 626)
  	HEXA_ED7B_+60	GCTGGAACTGGTCACCAAGGC
  		(SEQ ID NO: 627)
  	HEXA_ED7B_CORRECT_	GTACCTGAACCGTATATCCTA
  	WT_PE3B	(SEQ ID NO: 628)
  	HEXA_ED7B_CORRECT_	GTACCTGAACCGTATATCTTA
  	SILENT_PE3B	(SEQ ID NO: 629)

• TABLE 3R
  FIGS. 51A-51F PEgRNA

  				RT
  	SPACER

  	3′		TEM-
  	SEQUENCE	EXTENSION	PBS	PLATE
  	(SEQ ID	(SEQ ID NO:	LENGTH	LENGTH
  PEGRNA	NO: 632-640)	641-649)	(NT)	(NT)
  HEK3_	GGCCCAG	TCTGCCA	13	10
  ED8_1	ACTGAGC	TCCCGTG
  TTOG	ACGTGA	CTCAGTC
  		TG
  HEK3_	GGCCCAG	TCTGCCA	13	10
  EDS 3	ACTGAGC	GCACGTG
  ATOC	ACGTGA	CTCAGTC
  		TG
  HEK3_	GGCCCAG	TCTGCCA	13	10
  ED8_3	ACTGAGC	ACACGTG
  ATOT	ACGTGA	CTCAGTC
  		TG
  HEK3_	GGCCCAG	TGGAGGA	13	34
  ED8_3	ACTGAGC	AGCAGGG
  ATOT_5-	ACGTGA	CTTCCTT
  6GGTOTT		TCCTCTG
  		AAAACAC
  		GTGCTCA
  		GTCTG
  HEK3_	GGCCCAG	TCTGCCA	13	10
  ED8_1	ACTGAGC	TCAAAGC
  CTTINS	ACGTGA	GTGCTCA
  		GTCTG
  RNF2_	GTCATCT	AACGAAC	15	14
  ED8_1	TAGTCAT	ACCTCAT
  CTOA	TACCTG	GTAATGA
  		CTAAGAT
  		G
  RNF2_	GTCATCT	AACGAAC	15	14
  ED8_1	TAGTCAT	ACCTCAC
  CTOG	TACCTG	GTAATGA
  		CTAAGAT
  		G
  RNF2_	GTCATCT	AACGAAC	15	17
  ED8_1	TAGTCAT	ACCTCAG
  GTAINS	TACCTG	TACGTAA
  		TGACTAA
  		GATG
  HBB_	GCATGGT	AGACTTC	13	14
  ED8_4A	GCACCTG	TCCACAG
  TOT	ACTCCTG	GAGTCAG
  		GTGCAC

• TABLE 4
  Sequences of primers used for mammalian
  cell genomic DNA amplification and HTS181.

  DESCRIPTION	SEQUENCE (SEQ ID NOS: 3811-3863)
  HEK3 FWD	ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNATGTG
  	GGCTGCCTAGAAAGG
  HEK3 REV	TGGAGTTCAGACGTGTGCTCTTCCGATCTCCCAGCCAAACTT
  	GTCAACC
  RNF2 FWD	ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNACGTC
  	TCATATGCCCCTTGG
  RNF2 REV	TGGAGTTCAGACGTGTGCTCTTCCGATCTACGTAGGAATTTT
  	GGTGGGACA
  HEK4 FWD	ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGAACC
  	CAGGTAGCCAGAGAC
  HEK4 REV	TGGAGTTCAGACGTGTGCTCTTCCGATCTTCCTTTCAACCCG
  	AACGGAG
  EMX1 FWD	ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCAGCT
  	CAGCCTGAGTGTTGA
  EMX1 REV	TGGAGTTCAGACGTGTGCTCTTCCGATCTCTCGTGGGTTTGT
  	GGTTGC
  FANCF FWD	ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCATTG
  	CAGAGAGGCGTATCA
  FANCF REV	TGGAGTTCAGACGTGTGCTCTTCCGATCTGGGGTCCCAGGT
  	GCTGAC
  HBB FWD	ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNAGGGT
  	TGGCCAATCTACTCCC
  HBB REV	TGGAGTTCAGACGTGTGCTCTTCCGATCTGTCTTCTCTGTCT
  	CCACATGCC
  PRNP FWD	ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGTCAG
  	TGGAACAAGCCGAGT
  PRNP REV	TGGAGTTCAGACGTGTGCTCTTCCGATCTACTTGGTTGGGGT
  	AACGGTG
  HEXA FWD	ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCATAC
  	AGGTGTGGCGAGAGG
  HEXA REV	TGGAGTTCAGACGTGTGCTCTTCCGATCTCCAGCCTCCTTTG
  	GTTAGCA
  RUNX1 FWD	ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTCACA
  	AACAAGACAGGGAACTG
  RUNX1 REV	TGGAGTTCAGACGTGTGCTCTTCCGATCTAGATGTAGGGCTA
  	GAGGGGTG
  VEGFA FWD	ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNACTTG
  	GTGCCAAATTCTTCTCC
  VEGFA REV	TGGAGTTCAGACGTGTGCTCTTCCGATCTAAAGAGGGAATG
  	GGCTTTGGA
  DNMT FWD	ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCACAA
  	CAGCTTCATGTCAGCC
  DNMT REV	TGGAGTTCAGACGTGTGCTCTTCCGATCTACGTTAATGTTTC
  	CTGATGGTCC
  HEK3 OFF-TARGET	ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTCCCC
  SITE
  1 FWD	TGTTGACCTGGAGAA
  HEK3 OFF-TARGET	TGGAGTTCAGACGTGTGCTCTTCCGATCTCACTGTACTTGCC
  SITE
  1 REV	CTGACCA
  HEK3 OFF-TARGET	ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTTGGT
  SITE
  2 FWD	GTTGACAGGGAGCAA
  HEK3 OFF-TARGET	TGGAGTTCAGACGTGTGCTCTTCCGATCTCTGAGATGTGGGC
  SITE 2 REV	AGAAGGG
  HEK3 OFF-TARGET	ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTGAGA
  SITE 3 FWD	GGGAACAGAAGGGCT
  HEK3 OFF-TARGET	TGGAGTTCAGACGTGTGCTCTTCCGATCTGTCCAAAGGCCC
  SITE 3 REV	AAGAACCT
  HEK3 OFF-TARGET	ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTCCTA
  SITE 4 FWD	GCACTTTGGAAGGTCG
  HEK3 OFF-TARGET	TGGAGTTCAGACGTGTGCTCTTCCGATCTGCTCATCTTAATCT
  SITE 4 REV	GCTCAGCC
  HEK4 OFF-TARGET	ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGGCAT
  SITE
  1 FWD	GGCTTCTGAGACTCA
  HEK4 OFF-TARGET	TGGAGTTCAGACGTGTGCTCTTCCGATCTGTCTCCCTTGCAC
  SITE
  1 REV	TCCCTGTCTTT
  HEK4 OFF-TARGET	ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTTTGG
  SITE 2 FWD	CAATGGAGGCATTGG
  HEK4 OFF-TARGET	TGGAGTTCAGACGTGTGCTCTTCCGATCTGAAGAGGCTGCC
  SITE
  2 REV	CATGAGAG
  HEK4 OFF-TARGET	ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGGTCT
  SITE 3 FWD	GAGGCTCGAATCCTG
  HEK4 OFF-TARGET	TGGAGTTCAGACGTGTGCTCTTCCGATCTCTGTGGCCTCCAT
  SITE 3 REV	ATCCCTG
  HEK4 OFF-TARGET	ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTTTCC
  SITE 4 FWD	ACCAGAACTCAGCCC
  HEK4 OFF-TARGET	TGGAGTTCAGACGTGTGCTCTTCCGATCTCCTCGGTTCCTCC
  SITE 4 REV	ACAACAC
  EMXI OFF-	ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGTGGG
  TARGET SITE
  1
  FWD	GAGATTTGCATCTGTGGAGG
  EMX1 OFF-	TGGAGTTCAGACGTGTGCTCTTCCGATCTGCTTTTATACCATC
  TARGET SITE
  1	TTGGGGTTACAG
  REV
  EMX1 OFF-	ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCAATG
  TARGET SITE
  2	TGCTTCAACCCATCACGGC
  FWD
  EMX1 OFF-	TGGAGTTCAGACGTGTGCTCTTCCGATCTCCATGAATTTGTG
  TARGET SITE
  2	ATGGATGCAGTCTG
  REV
  EMX1 OFF-	ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGAGAA
  TARGET SITE
  3	GGAGGTGCAGGAGCTAGAC
  FWD
  EMX1 OFF-	TGGAGTTCAGACGTGTGCTCTTCCGATCTCATCCCGACCTTC
  TARGET SITE
  3	ATCCCTCCTGG
  REV
  EMX1 OFF-	ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGTAGT
  TARGET SITE
  4	TCTGACATTCCTCCTGAGGG
  FWD
  EMXI OFF-	TGGAGTTCAGACGTGTGCTCTTCCGATCTTCAAACAAGGTG
  TARGET SITE
  4	CAGATACAGCA
  REV
  FANCF OFF-	ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGCGGG
  TARGET SITE
  1	C AGTGGCGTCTTAGTCG
  FWD
  FANCF OFF-	TGGAGTTCAGACGTGTGCTCTTCCGATCTCCCTGGGTTTGGT
  TARGET SITE
  1	TGGCTGCTC
  REV
  FANCF OFF-	ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCTCCT
  TARGET SITE
  2	TGCCGCCCAGCCGGTC
  FWD
  FANCF OFF-	TGGAGTTCAGACGTGTGCTCTTCCGATCTCACTGGGGAAGA
  TARGET SITE
  2	GGCGAGGACAC
  REV
  FANCF OFF-	ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCCAGT
  TARGET SITE
  3	GTTTCCCATCCCCAACAC
  FWD
  FANCF OFF-	TGGAGTTCAGACGTGTGCTCTTCCGATCTGAATGGATCCCCC
  TARGET SITE
  3	CCTAGAGCTC
  REV
  FANCF OFF-	ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCAGGC
  TARGET SITE
  4	CCACAGGTCCTTCTGGA
  FWD

• TABLE 5
  Sequences of 100-mer single-stranded
  DNA oligonucleotide donor templates
  used in HDR experiments and in the
  creation of the HBB E6V HEK293T cell
  line. Oligonucleotides are 100-103 nt
  in length with homology arms centered
  around the site of the edit. Oligo-
  nucleotides were from Integrated DNA
  Technologies, purified by PAGE.

  	HEK3 +3 A TO T	GCTTCTCCAGCCCTGGCCTG
  		GGTCAATCCTTGGGGCCCAG
  		ACTGAGCACGTGTTGGCAGA
  		GGAAAGGAAGCCCTGCTTCC
  		TCCAGAGGGCGTCGCAGGAC
  		(SEQ ID NO: 717)
  	HEK3 +3 A TOT,	GCTTCTCCAGCCCTGGCCTG
  	+5, 6	GGTCAATCCTTGGGGCCCAG
  	GG TO TT	ACTGAGCACGTGTTTTCAGA
  		GGAAAGGAAGCCCTGCTTCC
  		TCCAGAGGGCGTCGCAGGAC
  		(SEQ ID NO: 718)
  	HEK3+1 TTOG	GCTTCTCCAGCCCTGGCCTG
  		GGTCAATCCTTGGGGCCCAG
  		ACTGAGCACGGGATGGCAGA
  		GGAAAGGAAGCCCTGCTTCC
  		TCCAGAGGGCGTCGCAGGAC
  		(SEQ ID NO: 719)
  	HEK3 +3 A TO C	GCTTCTCCAGCCCTGGCCTG
  		GGTCAATCCTTGGGGCCCAG
  		ACTGAGCACGTGCTGGCAGA
  		GGAAAGGAAGCCCTGCTTCC
  		TCCAGAGGGCGTCGCAGGAC
  		(SEQ ID NO: 720)
  	HEK3 +1 CTT	GCTTCTCCAGCCCTGGCCTG
  	INSERTION	GGTCAATCCTTGGGGCCCAG
  		ACTGAGCACGCTTTGATGGC
  		AGAGGAAAGGAAGCCCTGCT
  		TCCTCCAGAGGGCGTCGCAG
  		GAC (SEQ ID NO: 721)
  	RNF2 +1 CTO A	CCCAGTTTACACGTCTCATA
  		TGCCCCTTGGCAGTCATCTT
  		AGTCATTACATGAGGTGTTC
  		GTTGTAACTCATATAAACTG
  		AGTTCCCATGTTTTGCTTAA
  		(SEQ ID NO: 722)
  	RNF2 +1 CTOG	CCCAGTTTACACGTCTCATA
  		TGCCCCTTGGCAGTCATCTT
  		AGTCATTACGTGAGGTGTTC
  		GTTGTAACTCATATAAACTG
  		AGTTCCCATGTTTTGCTTAA
  		(SEQ ID NO: 723)
  	RNF2 +1 GTA	CAGTTTACACGTCTCATATG
  	INSERTION	CCCCTTGGCAGTCATCTTAG
  		TCATTACGTACTGAGGTGTT
  		CGTTGTAACTCATATAAACT
  		GAGTTCCCATGTTTTGCTTA
  		(SEQ ID NO: 724)
  	HBB E6V	ACTTCATCCACGTTCACCTT
  	INSTALLATION (ALSO	GCCCCACAGGGCAGTAACGG
  	USED FOR CREATION	CAGACTTCTCCACAGGAGTC
  	OF THE HBB E6V	AGATGCACCATGGTGTCTGT
  	HEK293T CELL LINE)	TTGAGGTTGCTAGTGAACAC
  		(SEQ ID NO: 725)
  	HBB E6V	ACTTCATCCACGTTCACCTT
  	CORRECTION	GCCCCACAGGGCAGTAACGG
  	PROTOSPACER A	CAGACTTCTCCTCAGGAGTC
  		AGGTGCACCATGGTGTCTGT
  		TTGAGGTTGCTAGTGAACAC
  		(SEQ ID NO: 726)
  	HBB E6V	GTGTTCACTAGCAACCTCAA
  	CORRECTION	ACAGACACCATGGTGCACCT
  	PROTOSPACER B	GACTCCTGAGGAGAAGTCTG
  		CCGTTACTGCCCTGTGGGGC
  		AAGGTGAACGTGGATGAAGT
  		(SEQ ID NO: 727)
  	HBB E6V	GTGTTCACTAGCAACCTCAA
  	CORRECTION	ACAGACACCATGGTGCACCT
  	PROTOSPACER B.	GACTCCTGATGAGAAGTCTG
  	SILENT PAM	CCGTTACTGCCCTGTGGGGC
  	MUTATION	AAGGTGAACGTGGATGAAGT
  		(SEQ ID NO: 728)
  	PRNP G127V	CACATGGCTGGTGCTGCAGC
  		AGCTGGGGCAGTGGTGGGGG
  		GCCTTGGCGTCTACATGCTG
  		GGAAGTGCCATGAGCAGGCC
  		CATCATACATTTCGGCAGTG
  		(SEQ ID NO: 729)

• Additional Sequences

• Sequences of yeast dual fluorescent reporter plasmids used herein
  p425-GFP_stop_mCherry:
  (SEQ ID NO: 730)
  ATGTCTAAAGGTGAAGAATTATTCACTGGTGTTGTCCCAATTTTGGTTGAATTAGAT
  GGTGATGTTAATGGTCACAAATTTTCTGTCTCCGGTGAAGGTGAAGGTGATGCTAC
  TTACGGTAAATTGACCTTAAAATTTATTTGTACTACTGGTAAATTGCCAGTTCCATG
  GCCAACCTTAGTCACTACTTTCGGTTATGGTGTTCAATGTTTTGCTAGATACCCAGA
  TCATATGAAACAACATGACTTTTTCAAGTCTGCCATGCCAGAAGGTTATGTTCAAG
  AAAGAACTATTTTTTTCAAAGATGACGGTAACTACAAGACCAGAGCTGAAGTCAA
  GTTTGAAGGTGATACCTTAGTTAATAGAATCGAATTAAAAGGTATTGATTTTAAAGA
  AGATGGTAACATTTTAGGTCACAAATTGGAATACAACTATAACTCTCACAATGTTTA
  CATCATGGCTGACAAACAAAAGAATGGTATCAAAGTTAACTTCAAAATTAGACAC
  AACATTGAAGATGGTTCTGTTCAATTAGCTGACCATTATCAACAAAATACTCCAATT
  GGTGATGGTCCAGTCTTGTTACCAGACAACCATTACTTATCCACTCAATCTGCCTTA
  TCCAAAGATCCAAACGAAAAGAGAGACCACATGGTCTTGTTAGAATTTGTTACTG
  CTGCTGGTATTACCCATGGTATGGATGAATTGTACAAAGCTAGCAACCTGGGTCAA
  TCCTTGG GGCCCAGACTGAGCACGTGA TGGCAGAGCACAGGAGACGTCATGGT
  TTCAAAAGGTGAAGAAGATAATATGGCTATTATTAAAGAATTTATGAGATTTAAAGTTCATA
  TGGAAGGTTCAGTTAATGGTCATGAATTTGAAATTGAAGGTGAAGGTGAAGGTAGACCA
  TATGAAGGTACTCAAACTGCTAAATTGAAAGTTACTAAAGGTGGTCCATTACCATTTGCTT
  GGGATATTTTGTCACCACAATTTATGTATGGTTCAAAAGCTTATGTTAAACATCCAGCTGA
  TATTCCAGATTATTTAAAATTGTCATTTCCAGAAGGTTTTAAATGGGAAAGAGTTATGAATT
  TTGAAGATGGTGGTGTTGTTACTGTTACTCAAGATTCATCATTACAAGATGGTGAATTTAT
  TTATAAAGTTAAATTGAGAGGTACTAATTTTCCATCAGATGGTCCAGTTATGCAAAAAAAA
  ACTATGGGTTGGGAAGCTTCATCAGAAAGAATGTATCCAGAAGATGGTGCTTTAAAAGG
  TGAAATTAAACAAAGATTGAAATTAAAAGATGGTGGTCATTATGATGCTGAAGTTAAAACT
  ACTTATAAAGCTAAAAAACCAGTTCAATTACCAGGTGCTTATAATGTTAATATTAAATTGGA
  TATTACTTCACATAATGAAGATTATACTATTGTTGAACAATATGAAAGAGCTGAAGGTAGAC
  ATTCAACTGGTGGTATGGATGAATTATATAAAGGTACCGCTCGAGCAGCTGTGATTGATT
  GAGTCGACTTGGTTGAACACGTTGCCAAGGCTTAAGTGAATTTACTTTAAATCTTG
  CATTTAAATAAATTTTCTTTTTATAGCTTTATGACTTAGTTTCAATTTATATACTATTTT
  AATGACATTTTCGATTCGGATCCACTAGTTCTAGAGCGGCCGCCACCGCGGTGGAG
  CTCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTGCGCGCTTGGCGTAATCATGGTC
  ATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATAGGAG
  CCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGGTAACTCACATT
  AATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTG
  CATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTT
  CCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGT
  ATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCA
  GGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGC
  CGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATC
  GACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGT
  TTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGA
  TACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTG
  TAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAAC
  CCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAAC
  CCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCA
  GAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGG
  CTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCG
  GAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTG
  GTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGA
  TCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAG
  GGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAA
  AATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACC
  AATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAG
  TTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGC
  CCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGC
  AATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCC
  GCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGT
  TAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGT
  CGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGA
  TCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAG
  AAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTC
  TTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAG
  TCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACG
  GGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTT
  CTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAA
  CCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGG
  GTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACAC
  GGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGG
  TTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGG
  GGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGAACGAAGCATCTGTGCTTCA
  TTTTGTAGAACAAAAATGCAACGCGAGAGCGCTAATTTTTCAAACAAAGAATCTG
  AGCTGCATTTTTACAGAACAGAAATGCAACGCGAAAGCGCTATTTTACCAACGAA
  GAATCTGTGCTTCATTTTTGTAAAACAAAAATGCAACGCGAGAGCGCTAATTTTTC
  AAACAAAGAATCTGAGCTGCATTTTTACAGAACAGAAATGCAACGCGAGAGCGCT
  ATTTTACCAACAAAGAATCTATACTTCTTTTTTGTTCTACAAAAATGCATCCCGAGA
  GCGCTATTTTTCTAACAAAGCATCTTAGATTACTTTTTTTCTCCTTTGTGCGCTCTAT
  AATGCAGTCTCTTGATAACTTTTTGCACTGTAGGTCCGTTAAGGTTAGAAGAAGGC
  TACTTTGGTGTCTATTTTCTCTTCCATAAAAAAAGCCTGACTCCACTTCCCGCGTTT
  ACTGATTACTAGCGAAGCTGCGGGTGCATTTTTTCAAGATAAAGGCATCCCCGATT
  ATATTCTATACCGATGTGGATTGCGCATACTTTGTGAACAGAAAGTGATAGCGTTGA
  TGATTCTTCATTGGTCAGAAAATTATGAACGGTTTCTTCTATTTTGTCTCTATATACT
  ACGTATAGGAAATGTTTACATTTTCGTATTGTTTTCGATTCACTCTATGAATAGTTCT
  TACTACAATTTTTTTGTCTAAAGAGTAATACTAGAGATAAACATAAAAAATGTAGAG
  GTCGAGTTTAGATGCAAGTTCAAGGAGCGAAAGGTGGATGGGTAGGTTATATAGG
  GATATAGCACAGAGATATATAGCAAAGAGATACTTTTGAGCAATGTTTGTGGAAGC
  GGTATTCGCAATATTTTAGTAGCTCGTTACAGTCCGGTGCGTTTTTGGTTTTTTGAA
  AGTGCGTCTTCAGAGCGCTTTTGGTTTTCAAAAGCGCTCTGAAGTTCCTATACTTT
  CTAGAGAATAGGAACTTCGGAATAGGAACTTCAAAGCGTTTCCGAAAACGAGCGC
  TTCCGAAAATGCAACGCGAGCTGCGCACATACAGCTCACTGTTCACGTCGCACCT
  ATATCTGCGTGTTGCCTGTATATATATATACATGAGAAGAACGGCATAGTGCGTGTTT
  ATGCTTAAATGCGTACTTATATGCGTCTATTTATGTAGGATGAAAGGTAGTCTAGTAC
  CTCCTGTGATATTATCCCATTCCATGCGGGGTATCGTATGCTTCCTTCAGCACTACCC
  TTTAGCTGTTCTATATGCTGCCACTCCTCAATTGGATTAGTCTCATCCTTCAATGCTA
  TCATTTCCTTTGATATTGGATCATACTAAGAAACCATTATTATCATGACATTAACCTA
  TAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTG
  AAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGA
  TGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCG
  GGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATCG
  ACTACGTCGTAAGGCCGTTTCTGACAGAGTAAAATTCTTGAGGGAACTTTCACCAT
  TATGGGAAATGCTTCAAGAAGGTATTGACTTAAACTCCATCAAATGGTCAGGTCAT
  TGAGTGTTTTTTATTTGTTGTATTTTTTTTTTTTTAGAGAAAATCCTCCAATATCAAA
  TTAGGAATCGTAGTTTCATGATTTTCTGTTACACCTAACTTTTTGTGTGGTGCCCTC
  CTCCTTGTCAATATTAATGTTAAAGTGCAATTCTTTTTCCTTATCACGTTGAGCCATT
  AGTATCAATTTGCTTACCTGTATTCCTTTACTATCCTCCTTTTTCTCCTTCTTGATAA
  ATGTATGTAGATTGCGTATATAGTTTCGTCTACCCTATGAACATATTCCATTTTGTAAT
  TTCGTGTCGTTTCTATTATGAATTTCATTTATAAAGTTTATGTACAAATATCATAAAA
  AAAGAGAATCTTTTTAAGCAAGGATTTTCTTAACTTCTTCGGCGACAGCATCACCG
  ACTTCGGTGGTACTGTTGGAACCACCTAAATCACCAGTTCTGATACCTGCATCCAA
  AACCTTTTTAACTGCATCTTCAATGGCCTTACCTTCTTCAGGCAAGTTCAATGACA
  ATTTCAACATCATTGCAGCAGACAAGATAGTGGCGATAGGGTCAACCTTATTCTTT
  GGCAAATCTGGAGCAGAACCGTGGCATGGTTCGTACAAACCAAATGCGGTGTTCT
  TGTCTGGCAAAGAGGCCAAGGACGCAGATGGCAACAAACCCAAGGAACCTGGGA
  TAACGGAGGCTTCATCGGAGATGATATCACCAAACATGTTGCTGGTGATTATAATAC
  CATTTAGGTGGGTTGGGTTCTTAACTAGGATCATGGCGGCAGAATCAATCAATTGA
  TGTTGAACCTTCAATGTAGGGAATTCGTTCTTGATGGTTTCCTCCACAGTTTTTCTC
  CATAATCTTGAAGAGGCCAAAAGATTAGCTTTATCCAAGGACCAAATAGGCAATGG
  TGGCTCATGTTGTAGGGCCATGAAAGCGGCCATTCTTGTGATTCTTTGCACTTCTG
  GAACGGTGTATTGTTCACTATCCCAAGCGACACCATCACCATCGTCTTCCTTTCTCT
  TACCAAAGTAAATACCTCCCACTAATTCTCTGACAACAACGAAGTCAGTACCTTTA
  GCAAATTGTGGCTTGATTGGAGATAAGTCTAAAAGAGAGTCGGATGCAAAGTTAC
  ATGGTCTTAAGTTGGCGTACAATTGAAGTTCTTTACGGATTTTTAGTAAACCTTGTT
  CAGGTCTAACACTACCGGTACCCCATTTAGGACCAGCCACAGCACCTAACAAAAC
  GGCATCAACCTTCTTGGAGGCTTCCAGCGCCTCATCTGGAAGTGGGACACCTGTA
  GCATCGATAGCAGCACCACCAATTAAATGATTTTCGAAATCGAACTTGACATTGGA
  ACGAACATCAGAAATAGCTTTAAGAACCTTAATGGCTTCGGCTGTGATTTCTTGAC
  CAACGTGGTCACCTGGCAAAACGACGATCTTCTTAGGGGCAGACATAGGGGCAG
  ACATTAGAATGGTATATCCTTGAAATATATATATATATTGCTGAAATGTAAAAGGTAA
  GAAAAGTTAGAAAGTAAGACGATTGCTAACCACCTATTGGAAAAAACAATAGGTC
  CTTAAATAATATTGTCAACTTCAAGTATTGTGATGCAAGCATTTAGTCATGAACGCT
  TCTCTATTCTATATGAAAAGCCGGTTCCGGCCTCTCACCTTTCCTTTTTCTCCCAATT
  TTTCAGTTGAAAAAGGTATATGCGTCAGGCGACCTCTGAAATTAACAAAAAATTTC
  CAGTCATCGAATTTGATTCTGTGCGATAGCGCCCCTGTGTGTTCTCGTTATGTTGAG
  GAAAAAAATAATGGTTGCTAAGAGATTCGAACTCTTGCATCTTACGATACCTGAGT
  ATTCCCACAGTTAACTGCGGTCAAGATATTTCTTGAATCAGGCGCCTTAGACCGCT
  CGGCCAAACAACCAATTACTTGTTGAGAAATAGAGTATAATTATCCTATAAATATAA
  CGTTTTTGAACACACATGAACAAGGAAGTACAGGACAATTGATTTTGAAGAGAAT
  GTGGATTTTGATGTAATTGTTGGGATTCCATTTTTAATAAGGCAATAATATTAGGTAT
  GTGGATATACTAGAAGTTCTCCTCGACCGTCGATATGCGGTGTGAAATACCGCACA
  GATGCGTAAGGAGAAAATACCGCATCAGGAAATTGTAAACGTTAATATTTTGTTAA
  AATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCG
  GCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCA
  GTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGA
  AAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTT
  TTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGA
  TTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAA
  AGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGT
  AACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCGCGCCATTCG
  CCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTAT
  TACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCC
  AGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAGCGCGCGTAATAC
  GACTCACTATAGGGCGAATTGGGTACCGGGCCCCCCCTCGAGGTCGACGGTATCG
  ATAAGCTTGATATCGAATTCCTGCAGCCCGGGGGATCCGTTAGAATCATTTTGAATA
  AAAAACACGCTTTTTCAGTTCGAGTTTATCATTATCAATACTGCCATTTCAAAGAAT
  ACGTAAATAATTAATAGTAGTGATTTTCCTAACTTTATTTAGTCAAAAAATTAGCCTT
  TTAATTCTGCTGTAACCCGTACATGCCCAAAATAGGGGGCGGGTTACACAGAATAT
  ATAACATCGTAGGTGTCTGGGTGAACAGTTTATTCCTGGCATCCACTAAATATAATG
  GAGCCCGCTTTTTAAGCTGGCATCCAGAAAAAAAAAGAATCCCAGCACCAAAATA
  TTGTTTTCTTCACCAACCATCAGTTCATAGGTCCATTCTCTTAGCGCAACTACAGAG
  AACAGGGGCACAAACAGGCAAAAAACGGGCACAACCTCAATGGAGTGATGCAA
  CCTGCCTGGAGTAAATGATGACACAAGGCAATTGACCCACGCATGTATCTATCTCA
  TTTTCTTACACCTTCTATTACCTTCTGCTCTCTCTGATTTGGAAAAAGCTGAAAAAA
  AAGGTTGAAACCAGTTCCCTGAAATTATTCCCCTACTTGACTAATAAGTATATAAAG
  ACGGTAGGTATTGATTGTAATTCTGTAAATCTATTTCTTAAACTTCTTAAATTCTACT
  TTTATAGTTAGTCTTTTTTTTAGTTTTAAAACACCAAGAACTTAGTTTCGAATAAAC
  ACACATAAACAAACAAAGAATTC
  p425-GFP_+1fs_mCherry:
  (SEQ ID NO: 731)
  ATGTCTAAAGGTGAAGAATTATTCACTGGTGTTGTCCCAATTTTGGTTGAATTAGAT
  GGTGATGTTAATGGTCACAAATTTTCTGTCTCCGGTGAAGGTGAAGGTGATGCTAC
  TTACGGTAAATTGACCTTAAAATTTATTTGTACTACTGGTAAATTGCCAGTTCCATG
  GCCAACCTTAGTCACTACTTTCGGTTATGGTGTTCAATGTTTTGCTAGATACCCAGA
  TCATATGAAACAACATGACTTTTTCAAGTCTGCCATGCCAGAAGGTTATGTTCAAG
  AAAGAACTATTTTTTTCAAAGATGACGGTAACTACAAGACCAGAGCTGAAGTCAA
  GTTTGAAGGTGATACCTTAGTTAATAGAATCGAATTAAAAGGTATTGATTTTAAAGA
  AGATGGTAACATTTTAGGTCACAAATTGGAATACAACTATAACTCTCACAATGTTTA
  CATCATGGCTGACAAACAAAAGAATGGTATCAAAGTTAACTTCAAAATTAGACAC
  AACATTGAAGATGGTTCTGTTCAATTAGCTGACCATTATCAACAAAATACTCCAATT
  GGTGATGGTCCAGTCTTGTTACCAGACAACCATTACTTATCCACTCAATCTGCCTTA
  TCCAAAGATCCAAACGAAAAGAGAGACCACATGGTCTTGTTAGAATTTGTTACTG
  CTGCTGGTATTACCCATGGTATGGATGAATTGTACAAAGCTAGCAAACCTGGGTCA
  ATCCTTGG GGCCCAGACTGAGCACGTGA TGGCAGAGCACAGGAGACGTCATGG
  TTTCAAAAGGTGAAGAAGATAATATGGCTATTATTAAAGAATTTATGAGATTTAAAGTTCAT
  ATGGAAGGTTCAGTTAATGGTCATGAATTTGAAATTGAAGGTGAAGGTGAAGGTAGACC
  ATATGAAGGTACTCAAACTGCTAAATTGAAAGTTACTAAAGGTGGTCCATTACCATTTGCT
  TGGGATATTTTGTCACCACAATTTATGTATGGTTCAAAAGCTTATGTTAAACATCCAGCTG
  ATATTCCAGATTATTTAAAATTGTCATTTCCAGAAGGTTTTAAATGGGAAAGAGTTATGAAT
  TTTGAAGATGGTGGTGTTGTTACTGTTACTCAAGATTCATCATTACAAGATGGTGAATTTA
  TT!TATAAAGTTAAATTGAGAGGTACTAATTTTCCATCAGATGGTCCAGTTATGCAAAAAAA
  AACTATGGGTTGGGAAGCTTCATCAGAAAGAATGTATCCAGAAGATGGTGCTTTAAAAG
  GTGAAATTAAACAAAGATTGAAATTAAAAGATGGTGGTCATTATGATGCTGAAGTTAAAAC
  TACTTATAAAGCTAAAAAACCAGTTCAATTACCAGGTGCTTATAATGTTAATATTAAATTGG
  ATATTACTTCACATAATGAAGATTATACTATTGTTGAACAATATGAAAGAGCTGAAGGTAGA
  CATTCAACTGGTGGTATGGATGAATTATATAAAGGTACCGCTCGAGCAGCTGTGATTGAT
  TGAGTCGACTTGGTTGAACACGTTGCCAAGGCTTAAGTGAATTTACTTTAAATCTT
  GCATTTAAATAAATTTTCTTTTTATAGCTTTATGACTTAGTTTCAATTTATATACTATT
  TTAATGACATTTTCGATTCGGATCCACTAGTTCTAGAGCGGCCGCCACCGCGGTGG
  AGCTCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTGCGCGCTTGGCGTAATCATG
  GTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATAGG
  AGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGGTAACTCACA
  TTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCT
  GCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCT
  TCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGG
  TATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCA
  GGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGC
  CGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATC
  GACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGT
  TTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGA
  TACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTG
  TAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAAC
  CCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAAC
  CCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCA
  GAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGG
  CTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCG
  GAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTG
  GTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGA
  TCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAG
  GGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAA
  AATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACC
  AATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAG
  TTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGC
  CCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGC
  AATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCC
  GCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGT
  TAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGT
  CGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGA
  TCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAG
  AAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTC
  TTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAG
  TCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACG
  GGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTT
  CTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAA
  CCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGG
  GTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACAC
  GGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGG
  TTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGG
  GGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGAACGAAGCATCTGTGCTTCA
  TTTTGTAGAACAAAAATGCAACGCGAGAGCGCTAATTTTTCAAACAAAGAATCTG
  AGCTGCATTTTTACAGAACAGAAATGCAACGCGAAAGCGCTATTTTACCAACGAA
  GAATCTGTGCTTCATTTTTGTAAAACAAAAATGCAACGCGAGAGCGCTAATTTTTC
  AAACAAAGAATCTGAGCTGCATTTTTACAGAACAGAAATGCAACGCGAGAGCGCT
  ATTTTACCAACAAAGAATCTATACTTCTTTTTTGTTCTACAAAAATGCATCCCGAGA
  GCGCTATTTTTCTAACAAAGCATCTTAGATTACTTTTTTTCTCCTTTGTGCGCTCTAT
  AATGCAGTCTCTTGATAACTTTTTGCACTGTAGGTCCGTTAAGGTTAGAAGAAGGC
  TACTTTGGTGTCTATTTTCTCTTCCATAAAAAAAGCCTGACTCCACTTCCCGCGTTT
  ACTGATTACTAGCGAAGCTGCGGGTGCATTTTTTCAAGATAAAGGCATCCCCGATT
  ATATTCTATACCGATGTGGATTGCGCATACTTTGTGAACAGAAAGTGATAGCGTTGA
  TGATTCTTCATTGGTCAGAAAATTATGAACGGTTTCTTCTATTTTGTCTCTATATACT
  ACGTATAGGAAATGTTTACATTTTCGTATTGTTTTCGATTCACTCTATGAATAGTTCT
  TACTACAATTTTTTTGTCTAAAGAGTAATACTAGAGATAAACATAAAAAATGTAGAG
  GTCGAGTTTAGATGCAAGTTCAAGGAGCGAAAGGTGGATGGGTAGGTTATATAGG
  GATATAGCACAGAGATATATAGCAAAGAGATACTTTTGAGCAATGTTTGTGGAAGC
  GGTATTCGCAATATTTTAGTAGCTCGTTACAGTCCGGTGCGTTTTTGGTTTTTTGAA
  AGTGCGTCTTCAGAGCGCTTTTGGTTTTCAAAAGCGCTCTGAAGTTCCTATACTTT
  CTAGAGAATAGGAACTTCGGAATAGGAACTTCAAAGCGTTTCCGAAAACGAGCGC
  TTCCGAAAATGCAACGCGAGCTGCGCACATACAGCTCACTGTTCACGTCGCACCT
  ATATCTGCGTGTTGCCTGTATATATATATACATGAGAAGAACGGCATAGTGCGTGTTT
  ATGCTTAAATGCGTACTTATATGCGTCTATTTATGTAGGATGAAAGGTAGTCTAGTAC
  CTCCTGTGATATTATCCCATTCCATGCGGGGTATCGTATGCTTCCTTCAGCACTACCC
  TTTAGCTGTTCTATATGCTGCCACTCCTCAATTGGATTAGTCTCATCCTTCAATGCTA
  TCATTTCCTTTGATATTGGATCATACTAAGAAACCATTATTATCATGACATTAACCTA
  TAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTG
  AAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGA
  TGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCG
  GGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATCG
  ACTACGTCGTAAGGCCGTTTCTGACAGAGTAAAATTCTTGAGGGAACTTTCACCAT
  TATGGGAAATGCTTCAAGAAGGTATTGACTTAAACTCCATCAAATGGTCAGGTCAT
  TGAGTGTTTTTTATTTGTTGTATTTTTTTTTTTTTAGAGAAAATCCTCCAATATCAAA
  TTAGGAATCGTAGTTTCATGATTTTCTGTTACACCTAACTTTTTGTGTGGTGCCCTC
  CTCCTTGTCAATATTAATGTTAAAGTGCAATTCTTTTTCCTTATCACGTTGAGCCATT
  AGTATCAATTTGCTTACCTGTATTCCTTTACTATCCTCCTTTTTCTCCTTCTTGATAA
  ATGTATGTAGATTGCGTATATAGTTTCGTCTACCCTATGAACATATTCCATTTTGTAAT
  TTCGTGTCGTTTCTATTATGAATTTCATTTATAAAGTTTATGTACAAATATCATAAAA
  AAAGAGAATCTTTTTAAGCAAGGATTTTCTTAACTTCTTCGGCGACAGCATCACCG
  ACTTCGGTGGTACTGTTGGAACCACCTAAATCACCAGTTCTGATACCTGCATCCAA
  AACCTTTTTAACTGCATCTTCAATGGCCTTACCTTCTTCAGGCAAGTTCAATGACA
  ATTTCAACATCATTGCAGCAGACAAGATAGTGGCGATAGGGTCAACCTTATTCTTT
  GGCAAATCTGGAGCAGAACCGTGGCATGGTTCGTACAAACCAAATGCGGTGTTCT
  TGTCTGGCAAAGAGGCCAAGGACGCAGATGGCAACAAACCCAAGGAACCTGGGA
  TAACGGAGGCTTCATCGGAGATGATATCACCAAACATGTTGCTGGTGATTATAATAC
  CATTTAGGTGGGTTGGGTTCTTAACTAGGATCATGGCGGCAGAATCAATCAATTGA
  TGTTGAACCTTCAATGTAGGGAATTCGTTCTTGATGGTTTCCTCCACAGTTTTTCTC
  CATAATCTTGAAGAGGCCAAAAGATTAGCTTTATCCAAGGACCAAATAGGCAATGG
  TGGCTCATGTTGTAGGGCCATGAAAGCGGCCATTCTTGTGATTCTTTGCACTTCTG
  GAACGGTGTATTGTTCACTATCCCAAGCGACACCATCACCATCGTCTTCCTTTCTCT
  TACCAAAGTAAATACCTCCCACTAATTCTCTGACAACAACGAAGTCAGTACCTTTA
  GCAAATTGTGGCTTGATTGGAGATAAGTCTAAAAGAGAGTCGGATGCAAAGTTAC
  ATGGTCTTAAGTTGGCGTACAATTGAAGTTCTTTACGGATTTTTAGTAAACCTTGTT
  CAGGTCTAACACTACCGGTACCCCATTTAGGACCAGCCACAGCACCTAACAAAAC
  GGCATCAACCTTCTTGGAGGCTTCCAGCGCCTCATCTGGAAGTGGGACACCTGTA
  GCATCGATAGCAGCACCACCAATTAAATGATTTTCGAAATCGAACTTGACATTGGA
  ACGAACATCAGAAATAGCTTTAAGAACCTTAATGGCTTCGGCTGTGATTTCTTGAC
  CAACGTGGTCACCTGGCAAAACGACGATCTTCTTAGGGGCAGACATAGGGGCAG
  ACATTAGAATGGTATATCCTTGAAATATATATATATATTGCTGAAATGTAAAAGGTAA
  GAAAAGTTAGAAAGTAAGACGATTGCTAACCACCTATTGGAAAAAACAATAGGTC
  CTTAAATAATATTGTCAACTTCAAGTATTGTGATGCAAGCATTTAGTCATGAACGCT
  TCTCTATTCTATATGAAAAGCCGGTTCCGGCCTCTCACCTTTCCTTTTTCTCCCAATT
  TTTCAGTTGAAAAAGGTATATGCGTCAGGCGACCTCTGAAATTAACAAAAAATTTC
  CAGTCATCGAATTTGATTCTGTGCGATAGCGCCCCTGTGTGTTCTCGTTATGTTGAG
  GAAAAAAATAATGGTTGCTAAGAGATTCGAACTCTTGCATCTTACGATACCTGAGT
  ATTCCCACAGTTAACTGCGGTCAAGATATTTCTTGAATCAGGCGCCTTAGACCGCT
  CGGCCAAACAACCAATTACTTGTTGAGAAATAGAGTATAATTATCCTATAAATATAA
  CGTTTTTGAACACACATGAACAAGGAAGTACAGGACAATTGATTTTGAAGAGAAT
  GTGGATTTTGATGTAATTGTTGGGATTCCATTTTTAATAAGGCAATAATATTAGGTAT
  GTGGATATACTAGAAGTTCTCCTCGACCGTCGATATGCGGTGTGAAATACCGCACA
  GATGCGTAAGGAGAAAATACCGCATCAGGAAATTGTAAACGTTAATATTTTGTTAA
  AATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCG
  GCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCA
  GTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGA
  AAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTT
  TTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGA
  TTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAA
  AGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGT
  AACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCGCGCCATTCG
  CCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTAT
  TACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCC
  AGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAGCGCGCGTAATAC
  GACTCACTATAGGGCGAATTGGGTACCGGGCCCCCCCTCGAGGTCGACGGTATCG
  ATAAGCTTGATATCGAATTCCTGCAGCCCGGGGGATCCGTTAGAATCATTTTGAATA
  AAAAACACGCTTTTTCAGTTCGAGTTTATCATTATCAATACTGCCATTTCAAAGAAT
  ACGTAAATAATTAATAGTAGTGATTTTCCTAACTTTATTTAGTCAAAAAATTAGCCTT
  TTAATTCTGCTGTAACCCGTACATGCCCAAAATAGGGGGCGGGTTACACAGAATAT
  ATAACATCGTAGGTGTCTGGGTGAACAGTTTATTCCTGGCATCCACTAAATATAATG
  GAGCCCGCTTTTTAAGCTGGCATCCAGAAAAAAAAAGAATCCCAGCACCAAAATA
  TTGTTTTCTTCACCAACCATCAGTTCATAGGTCCATTCTCTTAGCGCAACTACAGAG
  AACAGGGGCACAAACAGGCAAAAAACGGGCACAACCTCAATGGAGTGATGCAA
  CCTGCCTGGAGTAAATGATGACACAAGGCAATTGACCCACGCATGTATCTATCTCA
  TTTTCTTACACCTTCTATTACCTTCTGCTCTCTCTGATTTGGAAAAAGCTGAAAAAA
  AAGGTTGAAACCAGTTCCCTGAAATTATTCCCCTACTTGACTAATAAGTATATAAAG
  ACGGTAGGTATTGATTGTAATTCTGTAAATCTATTTCTTAAACTTCTTAAATTCTACT
  TTTATAGTTAGTCTTTTTTTTAGTTTTAAAACACCAAGAACTTAGTTTCGAATAAAC
  ACACATAAACAAACAAAGAATTC
  p425_-GFP_−1fs_mCherry:
  (SEQ ID NO: 732)
  ATGTCTAAAGGTGAAGAATTATTCACTGGTGTTGTCCCAATTTTGGTTGAATTAGAT
  GGTGATGTTAATGGTCACAAATTTTCTGTCTCCGGTGAAGGTGAAGGTGATGCTAC
  TTACGGTAAATTGACCTTAAAATTTATTTGTACTACTGGTAAATTGCCAGTTCCATG
  GCCAACCTTAGTCACTACTTTCGGTTATGGTGTTCAATGTTTTGCTAGATACCCAGA
  TCATATGAAACAACATGACTTTTTCAAGTCTGCCATGCCAGAAGGTTATGTTCAAG
  AAAGAACTATTTTTTTCAAAGATGACGGTAACTACAAGACCAGAGCTGAAGTCAA
  GTTTGAAGGTGATACCTTAGTTAATAGAATCGAATTAAAAGGTATTGATTTTAAAGA
  AGATGGTAACATTTTAGGTCACAAATTGGAATACAACTATAACTCTCACAATGTTTA
  CATCATGGCTGACAAACAAAAGAATGGTATCAAAGTTAACTTCAAAATTAGACAC
  AACATTGAAGATGGTTCTGTTCAATTAGCTGACCATTATCAACAAAATACTCCAATT
  GGTGATGGTCCAGTCTTGTTACCAGACAACCATTACTTATCCACTCAATCTGCCTTA
  TCCAAAGATCCAAACGAAAAGAGAGACCACATGGTCTTGTTAGAATTTGTTACTG
  CTGCTGGTATTACCCATGGTATGGATGAATTGTACAAAGCTAGCAAACCTGGGTCA
  ATCCTTGG GGCCCAGACTGAGCACGTGA TGGCAGAGCACAGGACGTCATGGTTT
  CAAAAGGTGAAGAAGATAATATGGCTATTATTAAAGAATTTATGAGATTTAAAGTTCATATG
  GAAGGTTCAGTTAATGGTCATGAATTTGAAATTGAAGGTGAAGGTGAAGGTAGACCATAT
  GAAGGTACTCAAACTGCTAAATTGAAAGTTACTAAAGGTGGTCCATTACCATTTGCTTGG
  GATATTTTGTCACCACAATTTATGTATGGTTCAAAAGCTTATGTTAAACATCCAGCTGATAT
  TCCAGATTATTTAAAATTGTCATTTCCAGAAGGTTTTAAATGGGAAAGAGTTATGAATTTT
  GAAGATGGTGGTGTTGTTACTGTTACTCAAGATTCATCATTACAAGATGGTGAATTTATTT
  ATAAAGTTAAATTGAGAGGTACTAATTTTCCATCAGATGGTCCAGTTATGCAAAAAAAAAC
  TATGGGTTGGGAAGCTTCATCAGAAAGAATGTATCCAGAAGATGGTGCTTTAAAAGGTG
  AAATTAAACAAAGATTGAAATTAAAAGATGGTGGTCATTATGATGCTGAAGTTAAAACTAC
  TTATAAAGCTAAAAAACCAGTTCAATTACCAGGTGCTTATAATGTTAATATTAAATTGGATAT
  TACTTCACATAATGAAGATTATACTATTGTTGAACAATATGAAAGAGCTGAAGGTAGACAT
  TCAACTGGTGGTATGGATGAATTATATAAAGGTACCGCTCGAGCAGCTGTGATTGATTGA
  GTCGACTTGGTTGAACACGTTGCCAAGGCTTAAGTGAATTTACTTTAAATCTTGCA
  TTTAAATAAATTTTCTTTTTATAGCTTTATGACTTAGTTTCAATTTATATACTATTTTA
  ATGACATTTTCGATTCGGATCCACTAGTTCTAGAGCGGCCGCCACCGCGGTGGAGC
  TCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTGCGCGCTTGGCGTAATCATGGTCA
  TAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATAGGAGC
  CGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGGTAACTCACATTA
  ATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGC
  ATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTC
  CGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTAT
  CAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGG
  AAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCG
  CGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGA
  CGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTC
  CCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATAC
  CTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAG
  GTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCC
  CCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCC
  GGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGA
  GCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCT
  ACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGA
  AAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTT
  TTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCC
  TTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGG
  ATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAA
  TGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAA
  TGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTT
  GCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCC
  CAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCA
  ATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCG
  CCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTT
  AATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTC
  GTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGAT
  CCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGA
  AGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTT
  ACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTC
  ATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGG
  ATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCT
  TCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAAC
  CCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGG
  TGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACG
  GAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGT
  TATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGG
  GTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGAACGAAGCATCTGTGCTTCATT
  TTGTAGAACAAAAATGCAACGCGAGAGCGCTAATTTTTCAAACAAAGAATCTGAG
  CTGCATTTTTACAGAACAGAAATGCAACGCGAAAGCGCTATTTTACCAACGAAGA
  ATCTGTGCTTCATTTTTGTAAAACAAAAATGCAACGCGAGAGCGCTAATTTTTCAA
  ACAAAGAATCTGAGCTGCATTTTTACAGAACAGAAATGCAACGCGAGAGCGCTAT
  TTTACCAACAAAGAATCTATACTTCTTTTTTGTTCTACAAAAATGCATCCCGAGAGC
  GCTATTTTTCTAACAAAGCATCTTAGATTACTTTTTTTCTCCTTTGTGCGCTCTATAA
  TGCAGTCTCTTGATAACTTTTTGCACTGTAGGTCCGTTAAGGTTAGAAGAAGGCTA
  CTTTGGTGTCTATTTTCTCTTCCATAAAAAAAGCCTGACTCCACTTCCCGCGTTTAC
  TGATTACTAGCGAAGCTGCGGGTGCATTTTTTCAAGATAAAGGCATCCCCGATTATA
  TTCTATACCGATGTGGATTGCGCATACTTTGTGAACAGAAAGTGATAGCGTTGATG
  ATTCTTCATTGGTCAGAAAATTATGAACGGTTTCTTCTATTTTGTCTCTATATACTAC
  GTATAGGAAATGTTTACATTTTCGTATTGTTTTCGATTCACTCTATGAATAGTTCTTA
  CTACAATTTTTTTGTCTAAAGAGTAATACTAGAGATAAACATAAAAAATGTAGAGG
  TCGAGTTTAGATGCAAGTTCAAGGAGCGAAAGGTGGATGGGTAGGTTATATAGGG
  ATATAGCACAGAGATATATAGCAAAGAGATACTTTTGAGCAATGTTTGTGGAAGCG
  GTATTCGCAATATTTTAGTAGCTCGTTACAGTCCGGTGCGTTTTTGGTTTTTTGAAA
  GTGCGTCTTCAGAGCGCTTTTGGTTTTCAAAAGCGCTCTGAAGTTCCTATACTTTC
  TAGAGAATAGGAACTTCGGAATAGGAACTTCAAAGCGTTTCCGAAAACGAGCGCT
  TCCGAAAATGCAACGCGAGCTGCGCACATACAGCTCACTGTTCACGTCGCACCTA
  TATCTGCGTGTTGCCTGTATATATATATACATGAGAAGAACGGCATAGTGCGTGTTTA
  TGCTTAAATGCGTACTTATATGCGTCTATTTATGTAGGATGAAAGGTAGTCTAGTAC
  CTCCTGTGATATTATCCCATTCCATGCGGGGTATCGTATGCTTCCTTCAGCACTACCC
  TTTAGCTGTTCTATATGCTGCCACTCCTCAATTGGATTAGTCTCATCCTTCAATGCTA
  TCATTTCCTTTGATATTGGATCATACTAAGAAACCATTATTATCATGACATTAACCTA
  TAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTG
  AAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGA
  TGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCG
  GGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATCG
  ACTACGTCGTAAGGCCGTTTCTGACAGAGTAAAATTCTTGAGGGAACTTTCACCAT
  TATGGGAAATGCTTCAAGAAGGTATTGACTTAAACTCCATCAAATGGTCAGGTCAT
  TGAGTGTTTTTTATTTGTTGTATTTTTTTTTTTTTAGAGAAAATCCTCCAATATCAAA
  TTAGGAATCGTAGTTTCATGATTTTCTGTTACACCTAACTTTTTGTGTGGTGCCCTC
  CTCCTTGTCAATATTAATGTTAAAGTGCAATTCTTTTTCCTTATCACGTTGAGCCATT
  AGTATCAATTTGCTTACCTGTATTCCTTTACTATCCTCCTTTTTCTCCTTCTTGATAA
  ATGTATGTAGATTGCGTATATAGTTTCGTCTACCCTATGAACATATTCCATTTTGTAAT
  TTCGTGTCGTTTCTATTATGAATTTCATTTATAAAGTTTATGTACAAATATCATAAAA
  AAAGAGAATCTTTTTAAGCAAGGATTTTCTTAACTTCTTCGGCGACAGCATCACCG
  ACTTCGGTGGTACTGTTGGAACCACCTAAATCACCAGTTCTGATACCTGCATCCAA
  AACCTTTTTAACTGCATCTTCAATGGCCTTACCTTCTTCAGGCAAGTTCAATGACA
  ATTTCAACATCATTGCAGCAGACAAGATAGTGGCGATAGGGTCAACCTTATTCTTT
  GGCAAATCTGGAGCAGAACCGTGGCATGGTTCGTACAAACCAAATGCGGTGTTCT
  TGTCTGGCAAAGAGGCCAAGGACGCAGATGGCAACAAACCCAAGGAACCTGGGA
  TAACGGAGGCTTCATCGGAGATGATATCACCAAACATGTTGCTGGTGATTATAATAC
  CATTTAGGTGGGTTGGGTTCTTAACTAGGATCATGGCGGCAGAATCAATCAATTGA
  TGTTGAACCTTCAATGTAGGGAATTCGTTCTTGATGGTTTCCTCCACAGTTTTTCTC
  CATAATCTTGAAGAGGCCAAAAGATTAGCTTTATCCAAGGACCAAATAGGCAATGG
  TGGCTCATGTTGTAGGGCCATGAAAGCGGCCATTCTTGTGATTCTTTGCACTTCTG
  GAACGGTGTATTGTTCACTATCCCAAGCGACACCATCACCATCGTCTTCCTTTCTCT
  TACCAAAGTAAATACCTCCCACTAATTCTCTGACAACAACGAAGTCAGTACCTTTA
  GCAAATTGTGGCTTGATTGGAGATAAGTCTAAAAGAGAGTCGGATGCAAAGTTAC
  ATGGTCTTAAGTTGGCGTACAATTGAAGTTCTTTACGGATTTTTAGTAAACCTTGTT
  CAGGTCTAACACTACCGGTACCCCATTTAGGACCAGCCACAGCACCTAACAAAAC
  GGCATCAACCTTCTTGGAGGCTTCCAGCGCCTCATCTGGAAGTGGGACACCTGTA
  GCATCGATAGCAGCACCACCAATTAAATGATTTTCGAAATCGAACTTGACATTGGA
  ACGAACATCAGAAATAGCTTTAAGAACCTTAATGGCTTCGGCTGTGATTTCTTGAC
  CAACGTGGTCACCTGGCAAAACGACGATCTTCTTAGGGGCAGACATAGGGGCAG
  ACATTAGAATGGTATATCCTTGAAATATATATATATATTGCTGAAATGTAAAAGGTAA
  GAAAAGTTAGAAAGTAAGACGATTGCTAACCACCTATTGGAAAAAACAATAGGTC
  CTTAAATAATATTGTCAACTTCAAGTATTGTGATGCAAGCATTTAGTCATGAACGCT
  TCTCTATTCTATATGAAAAGCCGGTTCCGGCCTCTCACCTTTCCTTTTTCTCCCAATT
  TTTCAGTTGAAAAAGGTATATGCGTCAGGCGACCTCTGAAATTAACAAAAAATTTC
  CAGTCATCGAATTTGATTCTGTGCGATAGCGCCCCTGTGTGTTCTCGTTATGTTGAG
  GAAAAAAATAATGGTTGCTAAGAGATTCGAACTCTTGCATCTTACGATACCTGAGT
  ATTCCCACAGTTAACTGCGGTCAAGATATTTCTTGAATCAGGCGCCTTAGACCGCT
  CGGCCAAACAACCAATTACTTGTTGAGAAATAGAGTATAATTATCCTATAAATATAA
  CGTTTTTGAACACACATGAACAAGGAAGTACAGGACAATTGATTTTGAAGAGAAT
  GTGGATTTTGATGTAATTGTTGGGATTCCATTTTTAATAAGGCAATAATATTAGGTAT
  GTGGATATACTAGAAGTTCTCCTCGACCGTCGATATGCGGTGTGAAATACCGCACA
  GATGCGTAAGGAGAAAATACCGCATCAGGAAATTGTAAACGTTAATATTTTGTTAA
  AATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCG
  GCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCA
  GTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGA
  AAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTT
  TTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGA
  TTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAA
  AGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGT
  AACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCGCGCCATTCG
  CCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTAT
  TACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCC
  AGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAGCGCGCGTAATAC
  GACTCACTATAGGGCGAATTGGGTACCGGGCCCCCCCTCGAGGTCGACGGTATCG
  ATAAGCTTGATATCGAATTCCTGCAGCCCGGGGGATCCGTTAGAATCATTTTGAATA
  AAAAACACGCTTTTTCAGTTCGAGTTTATCATTATCAATACTGCCATTTCAAAGAAT
  ACGTAAATAATTAATAGTAGTGATTTTCCTAACTTTATTTAGTCAAAAAATTAGCCTT
  TTAATTCTGCTGTAACCCGTACATGCCCAAAATAGGGGGCGGGTTACACAGAATAT
  ATAACATCGTAGGTGTCTGGGTGAACAGTTTATTCCTGGCATCCACTAAATATAATG
  GAGCCCGCTTTTTAAGCTGGCATCCAGAAAAAAAAAGAATCCCAGCACCAAAATA
  TTGTTTTCTTCACCAACCATCAGTTCATAGGTCCATTCTCTTAGCGCAACTACAGAG
  AACAGGGGCACAAACAGGCAAAAAACGGGCACAACCTCAATGGAGTGATGCAA
  CCTGCCTGGAGTAAATGATGACACAAGGCAATTGACCCACGCATGTATCTATCTCA
  TTTTCTTACACCTTCTATTACCTTCTGCTCTCTCTGATTTGGAAAAAGCTGAAAAAA
  AAGGTTGAAACCAGTTCCCTGAAATTATTCCCCTACTTGACTAATAAGTATATAAAG
  ACGGTAGGTATTGATTGTAATTCTGTAAATCTATTTCTTAAACTTCTTAAATTCTACT
  TTTATAGTTAGTCTTTTTTTTAGTTTTAAAACACCAAGAACTTAGTTTCGAATAAAC
  ACACATAAACAAACAAAGAATTC
  KEY:
  GFP open reading frame
  Linker containing stop codon +1 frameshift,
  or −1 frameshift
  mCherry open reading frame
  Plasmid backbone (containing the GPD promoter,
  Leu2 marker, and AmpR)
  Protospacer (underlined)
  PAM (boldfaced)
  DNA sequences of mammalian prime editor
  plasmids and example PEgRNA plasmid
  pCMV-PE1:
  (SEQ ID NO: 733)
  ATGAAACGGACAGCCGACGGAAGCGAGTTCGAGTCACCAAAGAAGAAGCGGAA
  AGTCGACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACTCTGTGGGCTGG
  GCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGC
  AACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGAC
  AGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATA
  CACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATG
  GCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAA
  GAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTG
  GCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACA
  GCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAA
  GTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGT
  GGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAAC
  CCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCA
  AGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATG
  GCCTGTTCGGAAACCTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAG
  CAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACACCTACGA
  CGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTT
  CTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGA
  ACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGACGA
  GCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGA
  GAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATT
  GACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAA
  AAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTG
  CGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGA
  GAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACA
  ACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACGTGGGCCC
  TCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAAC
  CATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAG
  CTTCATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCAACGAGAAGGTGCTG
  CCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGCTGACCAAAG
  TGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGA
  AAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGC
  AGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCTC
  CGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAA
  ATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAG
  ATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCT
  GAAAACCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCG
  GAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGA
  CAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAAC
  AGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCC
  AGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATCT
  GGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGA
  CGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAAAT
  GGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAA
  TGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAAC
  ACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCA
  GAATGGGCGGGATATGTACGTGGACCAGGAACTGGACATCAACCGGCTGTCCGAC
  TACGATGTGGACGCTATCGTGCCTCAGAGCTTTCTGAAGGACGACTCCATCGACA
  ACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCC
  TCCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCC
  AAGCTGATTACCCAGAGAAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGC
  CTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGG
  CAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTACG
  ACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGC
  TGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAA
  CTACCACCACGCCCACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATC
  AAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACG
  ACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCA
  AGTACTTCTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCC
  AACGGCGAGATCCGGAAGCGGCCTCTGATCGAGACAAACGGCGAAACCGGGGAG
  ATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGAGCATGC
  CCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAAAG
  AGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACT
  GGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCT
  GGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAG
  AGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGA
  CTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCT
  GCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCT
  GCCGGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAAC
  TTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATG
  AGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATCG
  AGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAA
  AGTGCTGTCCGCCTACAACAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGA
  GAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGT
  ACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGG
  ACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCT
  GTCTCAGCTGGGAGGTGACTCTGGAGGATCTAGCGGAGGATCCTCTGGCAGCGAGA
  CACCAGGAACAAGCGAGTCAGCAACACCAGAGAGCAGTGGCGGCAGCAGCGGCGGC
  AGCAGC ACCCTAAATATAGAAGATGAGTATCGGCTACATGAGACCTCAAAAGA
  GCCAGATGTTTCTCTAGGGTCCACATGGCTGTCTGATTTTCCTCAGGCCTGG
  GCGGAAACCGGGGGCATGGGACTGGCAGTTCGCCAAGCTCCTCTGATCATA
  CCTCTGAAAGCAACCTCTACCCCCGTGTCCATAAAACAATACCCCATGTCACA
  AGAAGCCAGACTGGGGATCAAGCCCCACATACAGAGACTGTTGGACCAGGG
  AATACTGGTACCCTGCCAGTCCCCCTGGAACACGCCCCTGCTACCCGTTAAG
  AAACCAGGGACTAATGATTATAGGCCTGTCCAGGATCTGAGAGAAGTCAACA
  AGCGGGTGGAAGACATCCACCCCACCGTGCCCAACCCTTACAACCTCTTGAG
  CGGGCTCCCACCGTCCCACCAGTGGTACACTGTGCTTGATTTAAAGGATGCC
  TTTTTCTGCCTGAGACTCCACCCCACCAGTCAGCCTCTCTTCGCCTTTGAGT
  GGAGAGATCCAGAGATGGGAATCTCAGGACAATTGACCTGGACCAGACTCC
  CACAGGGTTTCAAAAACAGTCCCACCCTGTTTGATGAGGCACTGCACAGAGA
  CCTAGCAGACTTCCGGATCCAGCACCCAGACTTGATCCTGCTACAGTACGTG
  GATGACTTACTGCTGGCCGCCACTTCTGAGCTAGACTGCCAACAAGGTACTC
  GGGCCCTGTTACAAACCCTAGGGAACCTCGGGTATCGGGCCTCGGCCAAGA
  AAGCCCAAATTTGCCAGAAACAGGTCAAGTATCTGGGGTATCTTCTAAAAGA
  GGGTCAGAGATGGCTGACTGAGGCCAGAAAAGAGACTGTGATGGGGCAGCC
  TACTCCGAAGACCCCTCGACAACTAAGGGAGTTCCTAGGGACGGCAGGCTT
  CTGTCGCCTCTGGATCCCTGGGTTTGCAGAAATGGCAGCCCCCCTGTACCCT
  CTCACCAAAACGGGGACTCTGTTTAATTGGGGCCCAGACCAACAAAAGGCCT
  ATCAAGAAATCAAGCAAGCTCTTCTAACTGCCCCAGCCCTGGGGTTGCCAGA
  TTTGACTAAGCCCTTTGAACTCTTTGTCGACGAGAAGCAGGGCTACGCCAAA
  GGTGTCCTAACGCAAAAACTGGGACCTTGGCGTCGGCCGGTGGCCTACCTG
  TCCAAAAAGCTAGACCCAGTAGCAGCTGGGTGGCCCCCTTGCCTACGGATGG
  TAGCAGCCATTGCCGTACTGACAAAGGATGCAGGCAAGCTAACCATGGGACA
  GCCACTAGTCATTCTGGCCCCCCATGCAGTAGAGGCACTAGTCAAACAACCC
  CCCGACCGCTGGCTTTCCAACGCCCGGATGACTCACTATCAGGCCTTGCTTT
  TGGACACGGACCGGGTCCAGTTCGGACCGGTGGTAGCCCTGAACCCGGCTA
  CGCTGCTCCCACTGCCTGAGGAAGGGCTGCAACACAACTGCCTTGATATCCT
  GGCCGAAGCCCACGGAACCCGACCCGACCTAACGGACCAGCCGCTCCCAGA
  CGCCGACCACACCTGGTACACGGATGGAAGCAGTCTCTTACAAGAGGGACA
  GCGTAAGGCGGGAGCTGCGGTGACCACCGAGACCGAGGTAATCTGGGCTAA
  AGCCCTGCCAGCCGGGACATCCGCTCAGCGGGCTGAACTGATAGCACTCAC
  CCAGGCCCTAAAGATGGCAGAAGGTAAGAAGCTAAATGTTTATACTGATAGC
  CGTTATGCTTTTGCTACTGCCCATATCCATGGAGAAATATACAGAAGGCGTGG
  GTTGCTCACATCAGAAGGCAAAGAGATCAAAAATAAAGACGAGATCTTGGCC
  CTACTAAAAGCCCTCTTTCTGCCCAAAAGACTTAGCATAATCCATTGTCCAGG
  ACATCAAAAGGGACACAGCGCCGAGGCTAGAGGCAACCGGATGGCTGACCA
  AGCGGCCCGAAAGGCAGCCATCACAGAGACTCCAGACACCTCTACCCTCCT
  CATAGAAAATTCATCACCCTCTGGCGGCTCAAAAAGAACCGCCGACGGCAGC
  GAATTCGAGCCCAAGAAGAAGAGGAAAGTCTAA CCGGTCATCATCACCATCAC
  CATTGAGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATC
  TGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTG
  TCCTTTCCTAATAAAATGAGAAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTA
  TTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATA
  GCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAG
  CTGGGGCTCGATACCGTCGACCTCTAGCTAGAGCTTGGCGTAATCATGGTCATAGC
  TGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGA
  AGCATAAAGTGTAAAGCCTAGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGC
  GTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAAT
  GAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTC
  CTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCT
  CACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGA
  ACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGC
  TGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCA
  AGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTG
  GAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCC
  GCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCT
  CAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTT
  CAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAG
  ACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGG
  TATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAG
  AAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGA
  GTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGT
  TTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATC
  TTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGG
  TCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTT
  TTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAA
  TCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGAC
  TCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCT
  GCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCA
  GCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATC
  CAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTT
  GCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTA
  TGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATG
  TTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGT
  TGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCA
  TGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGA
  GAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATAC
  CGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGG
  CGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCG
  TGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAA
  AAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTT
  GAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCT
  CATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGC
  GCACATTTCCCCGAAAAGTGCCACCTGACGTCGACGGATCGGGAGATCGATCTCC
  CGATCCCCTAGGGTCGACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCC
  AGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATT
  TAAGCTACAACAAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGG
  TTAGGCGTTTTGCGCTGCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGA
  TTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATA
  TGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAAC
  GACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGG
  GACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAG
  TACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAAT
  GGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAG
  TACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATC
  AATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGA
  CGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGT
  AACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCT
  ATATAAGCAGAGCTGGTTTAGTGAACCGTCAGATCCGCTAGAGATCCGCGGCCGCT
  AATACGACTCACTATAGGGAGAGCCGCCACC
  pCMV-PE2:
  (SEQ ID NO: 734)
  ATGAAACGGACAGCCGACGGAAGCGAGTTCGAGTCACCAAAGAAGAAGCGGAA
  AGTCGACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACTCTGTGGGCTGG
  GCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGC
  AACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGAC
  AGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATA
  CACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATG
  GCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAA
  GAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTG
  GCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACA
  GCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAA
  GTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGT
  GGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAAC
  CCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCA
  AGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATG
  GCCTGTTCGGAAACCTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAG
  CAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACACCTACGA
  CGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTT
  CTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGA
  ACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGACGA
  GCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGA
  GAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATT
  GACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAA
  AAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTG
  CGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGA
  GAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACA
  ACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACGTGGGCCC
  TCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAAC
  CATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAG
  CTTCATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCAACGAGAAGGTGCTG
  CCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGCTGACCAAAG
  TGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGA
  AAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGC
  AGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCTC
  CGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAA
  ATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAG
  ATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCT
  GAAAACCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCG
  GAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGA
  CAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAAC
  AGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCC
  AGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATCT
  GGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGA
  CGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAAAT
  GGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAA
  TGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAAC
  ACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCA
  GAATGGGCGGGATATGTACGTGGACCAGGAACTGGACATCAACCGGCTGTCCGAC
  TACGATGTGGACGCTATCGTGCCTCAGAGCTTTCTGAAGGACGACTCCATCGACA
  ACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCC
  TCCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCC
  AAGCTGATTACCCAGAGAAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGC
  CTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGG
  CAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTACG
  ACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGC
  TGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAA
  CTACCACCACGCCCACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATC
  AAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACG
  ACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCA
  AGTACTTCTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCC
  AACGGCGAGATCCGGAAGCGGCCTCTGATCGAGACAAACGGCGAAACCGGGGAG
  ATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGAGCATGC
  CCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAAAG
  AGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACT
  GGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCT
  GGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAG
  AGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGA
  CTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCT
  GCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCT
  GCCGGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAAC
  TTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATG
  AGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATCG
  AGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAA
  AGTGCTGTCCGCCTACAACAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGA
  GAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGT
  ACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGG
  ACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCT
  GTCTCAGCTGGGAGGTGACTCTGGAGGATCTAGCGGAGGATCCTCTGGCAGCGAG
  ACACCAGGAACAAGCGAGTCAGCAACACCAGAGAGCAGTGGCGGCAGCAGCGG
  CGGCAGCAGCACCCTAAATATAGAAGATGAGTATCGGCTACATGAGACCTCAAAA
  GAGCCAGATGTTTCTCTAGGGTCCACATGGCTGTCTGATTTTCCTCAGGCCTGGGC
  GGAAACCGGGGGCATGGGACTGGCAGTTCGCCAAGCTCCTCTGATCATACCTCTG
  AAAGCAACCTCTACCCCCGTGTCCATAAAACAATACCCCATGTCACAAGAAGCCA
  GACTGGGGATCAAGCCCCACATACAGAGACTGTTGGACCAGGGAATACTGGTACC
  CTGCCAGTCCCCCTGGAACACGCCCCTGCTACCCGTTAAGAAACCAGGGACTAAT
  GATTATAGGCCTGTCCAGGATCTGAGAGAAGTCAACAAGCGGGTGGAAGACATCC
  ACCCCACCGTGCCCAACCCTTACAACCTCTTGAGCGGGCTCCCACCGTCCCACCA
  GTGGTACACTGTGCTTGATTTAAAGGATGCCTTTTTCTGCCTGAGACTCCACCCCA
  CCAGTCAGCCTCTCTTCGCCTTTGAGTGGAGAGATCCAGAGATGGGAATCTCAGG
  ACAATTGACCTGGACCAGACTCCCACAGGGTTTCAAAAACAGTCCCACCCTGTTT
  AATGAGGCACTGCACAGAGACCTAGCAGACTTCCGGATCCAGCACCCAGACTTGA
  TCCTGCTACAGTACGTGGATGACTTACTGCTGGCCGCCACTTCTGAGCTAGACTGC
  CAACAAGGTACTCGGGCCCTGTTACAAACCCTAGGGAACCTCGGGTATCGGGCCT
  CGGCCAAGAAAGCCCAAATTTGCCAGAAACAGGTCAAGTATCTGGGGTATCTTCT
  AAAAGAGGGTCAGAGATGGCTGACTGAGGCCAGAAAAGAGACTGTGATGGGGCA
  GCCTACTCCGAAGACCCCTCGACAACTAAGGGAGTTCCTAGGGAAGGCAGGCTTC
  TGTCGCCTCTTCATCCCTGGGTTTGCAGAAATGGCAGCCCCCCTGTACCCTCTCAC
  CAAACCGGGGACTCTGTTTAATTGGGGCCCAGACCAACAAAAGGCCTATCAAGAA
  ATCAAGCAAGCTCTTCTAACTGCCCCAGCCCTGGGGTTGCCAGATTTGACTAAGC
  CCTTTGAACTCTTTGTCGACGAGAAGCAGGGCTACGCCAAAGGTGTCCTAACGCA
  AAAACTGGGACCTTGGCGTCGGCCGGTGGCCTACCTGTCCAAAAAGCTAGACCC
  AGTAGCAGCTGGGTGGCCCCCTTGCCTACGGATGGTAGCAGCCATTGCCGTACTG
  ACAAAGGATGCAGGCAAGCTAACCATGGGACAGCCACTAGTCATTCTGGCCCCCC
  ATGCAGTAGAGGCACTAGTCAAACAACCCCCCGACCGCTGGCTTTCCAACGCCCG
  GATGACTCACTATCAGGCCTTGCTTTTGGACACGGACCGGGTCCAGTTCGGACCG
  GTGGTAGCCCTGAACCCGGCTACGCTGCTCCCACTGCCTGAGGAAGGGCTGCAAC
  ACAACTGCCTTGATATCCTGGCCGAAGCCCACGGAACCCGACCCGACCTAACGGA
  CCAGCCGCTCCCAGACGCCGACCACACCTGGTACACGGATGGAAGCAGTCTCTTA
  CAAGAGGGACAGCGTAAGGCGGGAGCTGCGGTGACCACCGAGACCGAGGTAATC
  TGGGCTAAAGCCCTGCCAGCCGGGACATCCGCTCAGCGGGCTGAACTGATAGCAC
  TCACCCAGGCCCTAAAGATGGCAGAAGGTAAGAAGCTAAATGTTTATACTGATAGC
  CGTTATGCTTTTGCTACTGCCCATATCCATGGAGAAATATACAGAAGGCGTGGGTG
  GCTCACATCAGAAGGCAAAGAGATCAAAAATAAAGACGAGATCTTGGCCCTACTA
  AAAGCCCTCTTTCTGCCCAAAAGACTTAGCATAATCCATTGTCCAGGACATCAAAA
  GGGACACAGCGCCGAGGCTAGAGGCAACCGGATGGCTGACCAAGCGGCCCGAAA
  GGCAGCCATCACAGAGACTCCAGACACCTCTACCCTCCTCATAGAAAATTCATCAC
  CCTCTGGCGGCTCAAAAAGAACCGCCGACGGCAGCGAATTCGAGCCCAAGAAGA
  AGAGGAAAGTCTAACCGGTCATCATCACCATCACCATTGAGTTTAAACCCGCTGAT
  CAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTG
  CCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGAA
  AATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGC
  AGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGG
  TGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCGATACCGTCGAC
  CTCTAGCTAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTA
  TCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTAG
  GGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTT
  CCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGG
  AGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCG
  CTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGG
  TTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAG
  CAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCC
  GCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACC
  CGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCT
  CCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAG
  CGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTC
  GCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTT
  ATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGG
  CAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGA
  GTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCT
  GCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGG
  CAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACG
  CGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACG
  CTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAG
  GATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTAT
  ATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCT
  CAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAA
  CTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGA
  CCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCC
  GAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTG
  CCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCC
  ATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTC
  CGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCG
  GTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATC
  ACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATG
  CTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGC
  GACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAG
  AACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGG
  ATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATC
  TTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAA
  AATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCT
  TCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACAT
  ATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAA
  AAGTGCCACCTGACGTCGACGGATCGGGAGATCGATCTCCCGATCCCCTAGGGTC
  GACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATCTGCTCCCTG
  CTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACAACAAG
  GCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCG
  CTGCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTAT
  TAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTA
  CATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTG
  ACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGT
  CAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCA
  TATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATT
  ATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAG
  TCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGC
  GGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTG
  TTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATT
  GACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTGGT
  TTAGTGAACCGTCAGATCCGCTAGAGATCCGCGGCCGCTAATACGACTCACTATAG
  GGAGAGCCGCCACC
  N-terminal NLS + Cas9 H840A
  Flexible linker
  M-MLV reverse transcriptase + C-terminal NLS
  Plasmid backbone (containing CMV promoter and AmpR)
  pU6-HEK3_PEgRNA CTTins:
  (SEQ ID NO: 735)
  GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGA
  GAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTG
  ACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAAT
  GGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATC
  TTGTGGAAAGGACGAAACACC GGCCCAGACTGAGCACGTGA GTTTTAGAGCTAGA
  AATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAG
  TCGGTCC TCTGCCATCAAAGCGTGCTCAGTCTG TTTTTTTAAGCTTGGGCCGCT
  CGAGGTACCTCTCTACATATGACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAA
  CCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAG
  CATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAA
  AGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCT
  GCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTC
  ATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGC
  TGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCG
  TCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGT
  AACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGT
  GGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAG
  CCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCG
  CTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGG
  ATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAA
  ACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATC
  CTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGG
  TCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTT
  CGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGG
  CTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTC
  CAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTC
  CTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAA
  GTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTG
  GTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAG
  GCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTC
  CGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCA
  CTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAG
  TACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCC
  GGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCA
  TTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATC
  CAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCA
  CCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGA
  ATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGA
  AGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAA
  AATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCG
  CTAGCTGTACAAAAAAGCAGGCTTTAAAGGAACCAATTCAGTCGACTGGATCCGG
  TACCAAGGTCGGGCAGGAA
  U6 Promoter sequence
  Spacer sequence
  sgRNA scaffold
  3′ extension (contains PBS and RT template)
  Backbone (contains AmpR)
  pLenti-hSyn-N-PE2-NpuN-P2A-GFP-KASH_U6-DNMT1-PEgRNA:
  (SEQ ID NO: 736)
  GTCGACGGATCGGGAGATCTCCCGATCCCCTATGGTGCACTCTCAGTACAATCTGCTCT
  GATGCCGCATAGTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTA
  GTGCGCGAGCAAAATTTAAGCTACAACAAGGCAAGGCTTGACCGACAATTGCATGAAG
  AATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCGATGTACGGGCCAGATATACGCG
  TTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGC
  CCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCC
  CAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAG
  GGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTA
  CATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCC
  CGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTA
  CGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGG
  ATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTT
  TGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTG
  ACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGCGCGTTTTGCC
  TGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGG
  GAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCC
  GTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAA
  ATCTCTAGCAGTGGCGCCCGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAGC
  TCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGC
  GACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAGATGGGT
  GCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGCGATGGGAAAAAATTCGGTT
  AAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACATATAGTATGGGCAAGCAGGGAG
  CTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAAAT
  ACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAACTTAGATCATTATATA
  ATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAAGACACCAAGGA
  AGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGACCACCGCACAGCAAGC
  GGCCGCTGATCTTCAGACCTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAATT
  ATATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGA
  GAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCCTTGGGT
  TCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACAGG
  CCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAG
  GCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGA
  ATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCT
  CTGGAAAACTCATTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCT
  CTGGAACAGATTTGGAATCACACGACCTGGATGGAGTGGGACAGAGAAATTAACAATT
  ACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAACCAGCAAGAAAAGAATGA
  ACAAGAATTATTGGAATTAGATAAATGGGCAAGTTTGTGGAATTGGTTTAACATAACAA
  ATTGGCTGTGGTATATAAAATTATTCATAATGATAGTAGGAGGCTTGGTAGGTTTAAGAA
  TAGTTTTTGCTGTACTTTCTATAGTGAATAGAGTTAGGCAGGGATATTCACCATTATCGTT
  TCAGACCCACCTCCCAACCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAGA
  AGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATCGGCACTGCG
  TGCGCCAATTCTGCAGACAAATGGCAGTATTCATCCACAATTTTAAAAGAAAAGGGGG
  GATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAA
  ACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTCGGGTTTATTACAGGGA
  CAGCAGAGATCCAGTTTGGTTAATTAAGGTACCAAGGTCGGGCAGGAAGAGGGCCTAT
  TTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAAT
  TAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAAT
  TTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGT
  AACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACC
  GCGGGCTGGAGCTGTTCGCGCGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGC
  TAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCAAGATGCAAGCGC
  GAACAGCTCCAG TTTTTTTGAATTC AGTGCAAGTGGGTTTTAGGACCAGGATGAGGCG
  GGGTGGGGGTGCCTACCTGACGACCGACCCCGACCCACTGGACAAGCACCCAACCCCCAT
  TCCCCAAATTGCGCATCCCCTATCAGAGAGGGGGAGGGGAAACAGGATGCGGCGAGGCGC
  GTGCGCACTGCCAGCTTCAGCACCGCGGACAGTGCCTTCGCCCCCGCCTGGCGGCGCGC
  GCCACCGCCGCCTCAGCACTGAAGGCGCGCTGACGTCACTCGCCGGTCCCCCGCAAACTC
  CCCTTCCCGGCCACCTTGGTCGCGTCCGCGCCGCCGCCGGCCCAGCCGGACCGCACCAC
  GCGAGGCGCGAGATAGGGGGGCACGGGCGCGACCATCTGCGCTGCGGCGCCGGCGACT
  CAGCGCTGCCTCAGTCTGCGGTGGGCAGCGGAGGAGTCGTGTCGTGCCTGAGAGCGCAG
  ATGAAACGGACAGCCGACGGAAGCGAGTTCGAGT
  CACCAAAGAAGAAGCGGAAAGTCGACAAGAAGTACAGCATCGGCCTGGACATCGGCA
  CCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATT
  CAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCT
  GCTGTTCGACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAA
  GAAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGA
  GATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGA
  AGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGC
  CTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCAC
  CGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGG
  GGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTG
  TTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCA
  GCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGG
  AAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGAAACCTGAT
  TGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGAT
  GCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCC
  CAGATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCA
  TCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGC
  CTCTATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTC
  GTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAAC
  GGCTACGCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCA
  AGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAG
  AGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCC
  ACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAA
  GGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACGTGGGC
  CCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACC
  ATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTC
  ATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGC
  ACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGCTGACCAAAGTGAAATACGT
  GACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGT
  GGACCTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTA
  CTTCAAGAAAATCGAG TGCCTGTCCTACGAGACAGAGATCCTGACAGTGGAGTATGGCCT
  GCTGCCAATCGGCAAGATCGTGGAGAAGAGGATCGAGTGTACCGTGTACTCTGTGGATAAC
  AATGGCAACATCTATACACAGCCCGTGGCACAGTGGCACGATAGGGGAGAGCAGGAGGTGT
  TCGAGTATTGCCTGGAGGACGGCAGCCTGATCAGGGCAACCAAGGACCACAAGTTCATGAC
  AGTGGATGGCCAGATGCTGCCCATCGACGAGATTTTCGAGCGGGAGCTGGACCTGATGAGA
  GTGGATAACCTGCC
  ACGCGTTAAGTCGACAATCAACCTCTGGATTACAAAATTTGT
  GAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTA
  ATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATC
  CTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGT
  GCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCT
  CCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCT
  GCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTT
  GTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGC
  GCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGC
  GGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTC
  GGATCTCCCTTTGGGCCGCCTCCCCGCGTCGACTTTAAGACCAATGACTTACAAGGCA
  GCTGTAGATCTTAGCCACTTTTTAAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACT
  CCCAACGAAGACAAGATCTGCTTTTTGCTTGTACTGGGTCTCTCTGGTTAGACCAGATC
  TGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCT
  TGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGA
  TCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGGGCCCGTTTAAACCCGCT
  GATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTG
  CCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAAT
  TGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACA
  GCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTAT
  GGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCTAGGGGGTATCCCCACGCGCCCTGT
  AGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTG
  CCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCC
  GGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTT
  ACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCG
  CCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACT
  CTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGG
  GATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGC
  GAATTAATTCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAG
  CAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTC
  CCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACC
  ATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTC
  TCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCTGCCTC
  TGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAG
  CTCCCGGGAGCTTGTATATCCATTTTCGGATCTGATCAGCACGTGTTGACAATTAATCAT
  CGGCATAGTATATCGGCATAGTATAATACGACAAGGTGAGGAACTAAACCATGGCCAAG
  TTGACCAGTGCCGTTCCGGTGCTCACCGCGCGCGACGTCGCCGGAGCGGTCGAGTTCT
  GGACCGACCGGCTCGGGTTCTCCCGGGACTTCGTGGAGGACGACTTCGCCGGTGTGGT
  CCGGGACGACGTGACCCTGTTCATCAGCGCGGTCCAGGACCAGGTGGTGCCGGACAA
  CACCCTGGCCTGGGTGTGGGTGCGCGGCCTGGACGAGCTGTACGCCGAGTGGTCGGA
  GGTCGTGTCCACGAACTTCCGGGACGCCTCCGGGCCGGCCATGACCGAGATCGGCGA
  GCAGCCGTGGGGGCGGGAGTTCGCCCTGCGCGACCCGGCCGGCAACTGCGTGCACTT
  CGTGGCCGAGGAGCAGGACTGACACGTGCTACGAGATTTCGATTCCACCGCCGCCTTC
  TATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCG
  CGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTTATAATGG
  TTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTC
  TAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGTATACCGTCGACCTCT
  AGCTAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTC
  ACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAAT
  GAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAAC
  CTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTA
  TTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGG
  CGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATA
  ACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAG
  GCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCG
  ACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCC
  CCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTC
  CGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCA
  GTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCC
  CGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACT
  TATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGG
  TGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTG
  GTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCC
  GGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGC
  GCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCA
  GTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTC
  ACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAA
  ACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCT
  ATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGG
  GCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCC
  AGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGC
  AACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTC
  GCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCT
  CGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGA
  TCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAG
  TAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGT
  CATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAG
  AATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCG
  CCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAAC
  TCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAAC
  TGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGC
  AAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTT
  CCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTT
  GAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGT
  GCCACCTGAC
  U6promoter
  PEgRNA
  hSynpromoter
  N-termPE2
  N-termNpu
  P2A-GFP-KASH
  pLenti-hSyn-C-PE2-NpuC:
  (SEQ ID NO: 737)
  GTCGACGGATCGGGAGATCTCCCGATCCCCTATGGTGCACTCTCAGTACAATCTGCTCT
  GATGCCGCATAGTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTA
  GTGCGCGAGCAAAATTTAAGCTACAACAAGGCAAGGCTTGACCGACAATTGCATGAAG
  AATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCGATGTACGGGCCAGATATACGCG
  TTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGC
  CCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCC
  CAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAG
  GGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTA
  CATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCC
  CGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTA
  CGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGG
  ATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTT
  TGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTG
  ACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGCGCGTTTTGCC
  TGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGG
  GAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCC
  GTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAA
  ATCTCTAGCAGTGGCGCCCGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAGC
  TCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGC
  GACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAGATGGGT
  GCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGCGATGGGAAAAAATTCGGTT
  AAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACATATAGTATGGGCAAGCAGGGAG
  CTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAAAT
  ACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAACTTAGATCATTATATA
  ATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAAGACACCAAGGA
  AGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGACCACCGCACAGCAAGC
  GGCCGCTGATCTTCAGACCTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAATT
  ATATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGA
  GAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCCTTGGGT
  TCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACAGG
  CCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAG
  GCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGA
  ATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCT
  CTGGAAAACTCATTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCT
  CTGGAACAGATTTGGAATCACACGACCTGGATGGAGTGGGACAGAGAAATTAACAATT
  ACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAACCAGCAAGAAAAGAATGA
  ACAAGAATTATTGGAATTAGATAAATGGGCAAGTTTGTGGAATTGGTTTAACATAACAA
  ATTGGCTGTGGTATATAAAATTATTCATAATGATAGTAGGAGGCTTGGTAGGTTTAAGAA
  TAGTTTTTGCTGTACTTTCTATAGTGAATAGAGTTAGGCAGGGATATTCACCATTATCGTT
  TCAGACCCACCTCCCAACCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAGA
  AGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATCGGCACTGCG
  TGCGCCAATTCTGCAGACAAATGGCAGTATTCATCCACAATTTTAAAAGAAAAGGGGG
  GATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAA
  ACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTCGGGTTTATTACAGGGA
  CAGCAGAGATCCAGTTTGGTTAATTAAGGTACCAGTGCAAGTGGGTTTTAGGACCAGG
  ATGAGGCGGGGTGGGGGTGCCTACCTGACGACCGACCCCGACCCACTGGACAAGCAC
  CCAACCCCCATTCCCCAAATTGCGCATCCCCTATCAGAGAGGGGGAGGGGAAACAGGA
  TGCGGCGAGGCGCGTGCGCACTGCCAGCTTCAGCACCGCGGACAGTGCCTTCGCCCC
  CGCCTGGCGGCGCGCGCCACCGCCGCCTCAGCACTGAAGGCGCGCTGACGTCACTCG
  CCGGTCCCCCGCAAACTCCCCTTCCCGGCCACCTTGGTCGCGTCCGCGCCGCCGCCGG
  CCCAGCCGGACCGCACCACGCGAGGCGCGAGATAGGGGGGCACGGGCGCGACCATCT
  GCGCTGCGGCGCCGGCGACTCAGCGCTGCCTCAGTCTGCGGTGGGCAGCGGAGGAGT
  CGTGTCGTGCCTGAGAGCGCAG TCGAGAATCTAGAGCGCTGCCACCATGAAACGGACAGC
  CGACGGAAGCGAGTTCGAGTCACCAAAGAAGAAGCGGAAAGTC ATCAAGATTGCTACACGG
  AAATACCTGGGAAAGCAGAACGTGTACGACATCGGCGTGGAGCGGGATCACAACTTCGCCC
  TGAAGAATGGCTTTATCGCCAGCAAA TGCTTCGACTCCGTGGAAATCTCCGGCGTGGA
  AGATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATTATCAAG
  GACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCGTG
  CTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACC
  TATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATAC
  ACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCA
  GTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAA
  CTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAA
  GCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCC
  GGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAG
  CTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAAATGGCC
  AGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAA
  GCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCC
  CGTGGAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAA
  TGGGCGGGATATGTACGTGGACCAGGAACTGGACATCAACCGGCTGTCCGACTA
  CGATGTGGACCATATCGTGCCTCAGAGCTTTCTGAAGGACGACTCCATCGACAAC
  AAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTC
  CGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAA
  GCTGATTACCCAGAGAAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCT
  GAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCA
  GATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTACGA
  CGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCT
  GGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAAC
  TACCACCACGCCCACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATC
  AAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTAC
  GACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCC
  AAGTACTTCTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGC
  CAACGGCGAGATCCGGAAGCGGCCTCTGATCGAGACAAACGGCGAAACCGGGG
  AGATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGAGCA
  TGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCA
  AAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGG
  ACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTG
  TGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTG
  AAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCC
  ATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCA
  AGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGG
  CCTCTGCCGGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATG
  TGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGG
  ATAATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGAT
  CATCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTG
  GACAAAGTGCTGTCCGCCTACAACAAGCACCGGGATAAGCCCATCAGAGAGCAG
  GCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCT
  TCAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGG
  TGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGA
  TCGACCTGTCTCAGCTGGGAGGTGACTCTGGCGGCTCAAAAAGAACCGCCGACG
  GCAGCGAATTCGAGCCCAAGAAGAAGAGGAAAGTCTAA ACGCGTTAAGTCGACAA
  TCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCC
  TTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATG
  GCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGG
  CCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTG
  GTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCT
  ATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGC
  TGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTG
  CTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGC
  CCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGC
  GTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCGTCGA
  CTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAG
  GGGGGACTGGAAGGGCTAATTCACTCCCAACGAAGACAAGATCTGCTTTTTGCTTGTA
  CTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAA
  CCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTC
  TGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATC
  TCTAGCAGGGCCCGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGC
  CATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACT
  GTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATT
  CTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAG
  GCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGC
  TCTAGGGGGTATCCCCACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGG
  TTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTC
  TTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCT
  CCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGG
  GTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTG
  GAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTAT
  CTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAAT
  GAGCTGATTTAACAAAAATTTAACGCGAATTAATTCTGTGGAATGTGTGTCAGTTAGGG
  TGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATT
  AGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAA
  GCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCC
  CTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATG
  CAGAGGCCGAGGCCGCCTCTGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTT
  TGGAGGCCTAGGCTTTTGCAAAAAGCTCCCGGGAGCTTGTATATCCATTTTCGGATCTG
  ATCAGCACGTGTTGACAATTAATCATCGGCATAGTATATCGGCATAGTATAATACGACAA
  GGTGAGGAACTAAACCATGGCCAAGTTGACCAGTGCCGTTCCGGTGCTCACCGCGCGC
  GACGTCGCCGGAGCGGTCGAGTTCTGGACCGACCGGCTCGGGTTCTCCCGGGACTTCG
  TGGAGGACGACTTCGCCGGTGTGGTCCGGGACGACGTGACCCTGTTCATCAGCGCGGT
  CCAGGACCAGGTGGTGCCGGACAACACCCTGGCCTGGGTGTGGGTGCGCGGCCTGGA
  CGAGCTGTACGCCGAGTGGTCGGAGGTCGTGTCCACGAACTTCCGGGACGCCTCCGG
  GCCGGCCATGACCGAGATCGGCGAGCAGCCGTGGGGGCGGGAGTTCGCCCTGCGCGA
  CCCGGCCGGCAACTGCGTGCACTTCGTGGCCGAGGAGCAGGACTGACACGTGCTACG
  AGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGG
  ACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCC
  AACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACA
  AATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCT
  TATCATGTCTGTATACCGTCGACCTCTAGCTAGAGCTTGGCGTAATCATGGTCATAGCTG
  TTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCAT
  AAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCT
  CACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCA
  ACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGAC
  TCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAA
  TACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCC
  AGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCG
  CCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGAC
  AGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTC
  CGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTT
  TCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGG
  CTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTC
  TTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAG
  GATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAAC
  TACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTT
  CGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGG
  TTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCT
  TTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTT
  GGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTT
  TAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAG
  TGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGT
  CGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATAC
  CGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAG
  GGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTT
  GCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATT
  GCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTC
  CCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCC
  TTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTAT
  GGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGG
  TGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCC
  CGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATT
  GGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTT
  CGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTT
  CTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACA
  CGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTT
  ATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGG
  TTCCGCGCACATTTCCCCGAAAAGTGCCACCTGAC
  hSynpromoter
  C-termNpu
  C-termwtCas9
  pLenti-U6-DNMT1 nicking sgRNA:
  (SEQ ID NO: 738)
  TAACAAATTGGCTGTGGTATATAAAATTATTCATAATGATAGTAGGAGGCTTGGTAGGTT
  TAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATAGAGTTAGGCAGGGATATTCACCAT
  TATCGTTTCAGACCCACCTCCCAACCCCGAGGGGACCCAGAGAGGGCCTATTTCCCATG
  ATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGT
  AAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCA
  GTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTC
  TTGGCTTTATATATCTTGTGGAAAGGACGAAACACC
  TTTTTTAAGCTTGGCGTAACTAGATCTTGAGACAAATGGCAGTATTC
  ATCCACAATTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATA
  GTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACAAAAA
  TTCAAAATTTTCGGGTTTATTACAGGGACAGCAGAGATCCACTTTGGCGCCGGCTCGAG
  GGGGCCCGGGTGCAAAGATGGATAAAGTTTTAAACAGAGAGGAATCTTTGCAGCTAAT
  GGACCTTCTAGGTCTTGAAAGGAGTGGGAATTGGCTCCGGTGCCCGTCAGTGGGCAGA
  GCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGATCCGG
  TGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGC
  CTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCT
  TTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGG
  CCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCA
  GTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCT
  TGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGG
  GCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCT
  CTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTT
  GTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCG
  ACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGC
  CACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCT
  CGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTT
  GCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGG
  ACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTT
  TCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCAC
  CTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTA
  TGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCAC
  TTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAG
  CCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGACGTACGGCCAC
  CATGACCGAGTACAAGCCCACGGTGCGCCTCGCCACCCGCGACGACGTCCCCAGGGC
  CGTACGCACCCTCGCCGCCGCGTTCGCCGACTACCCCGCCACGCGCCACACCGTCGAT
  CCGGACCGCCACATCGAGCGGGTCACCGAGCTGCAAGAACTCTTCCTCACGCGCGTCG
  GGCTCGACATCGGCAAGGTGTGGGTCGCGGACGACGGCGCCGCCGTGGCGGTCTGGA
  CCACGCCGGAGAGCGTCGAAGCGGGGGCGGTGTTCGCCGAGATCGGCCCGCGCATGG
  CCGAGTTGAGCGGTTCCCGGCTGGCCGCGCAGCAACAGATGGAAGGCCTCCTGGCGC
  CGCACCGGCCCAAGGAGCCCGCGTGGTTCCTGGCCACCGTCGGAGTCTCGCCCGACC
  ACCAGGGCAAGGGTCTGGGCAGCGCCGTCGTGCTCCCCGGAGTGGAGGCGGCCGAGC
  GCGCCGGGGTGCCCGCCTTCCTGGAGACCTCCGCGCCCCGCAACCTCCCCTTCTACGA
  GCGGCTCGGCTTCACCGTCACCGCCGACGTCGAGGTGCCCGAAGGACCGCGCACCTG
  GTGCATGACCCGCAAGCCCGGTGCCTGAACGCGTTAAGTCGACAATCAACCTCTGGAT
  TACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGT
  GGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCT
  CCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGC
  AACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGC
  CACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGG
  AACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGA
  CAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTG
  CCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCG
  GACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCG
  CCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCGTCGACTTTAAGACCAAT
  GACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAGGGGGGACTGGAA
  GGGCTAATTCACTCCCAACGAAGACAAGATCTGCTTTTTGCTTGTACTGGGTCTCTCTG
  GTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAG
  CCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTC
  TGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTACGT
  ATAGTAGTTCATGTCATCTTATTATTCAGTATTTATAACTTGCAAAGAAATGAATATCAGA
  GAGTGAGAGGAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCA
  CAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCA
  TCAATGTATCTTATCATGTCTGGCTCTAGCTATCCCGCCCCTAACTCCGCCCATCCCGCC
  CCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTAT
  GCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTT
  TTGGAGGCCTAGGGACGTACCCAATTCGCCCTATAGTGAGTCGTATTACGCGCGCTCAC
  TGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCG
  CCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGAT
  CGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGGACGCGCCCTGTAGCGGCG
  CATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGC
  CCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCC
  CCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACC
  TCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAG
  ACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCA
  AACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCC
  GATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTA
  ACAAAATATTAACGCTTACAATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCC
  CTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTG
  ATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGC
  CCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGT
  GAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGAT
  CTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGA
  GCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAG
  CAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCAC
  AGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACC
  ATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGC
  TAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCG
  GAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGG
  CAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACA
  ATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTC
  CGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATC
  ATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGG
  GAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTG
  ATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAA
  CTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAA
  ATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAG
  GATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCA
  CCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGT
  AACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAG
  GCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTA
  CCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGAT
  AGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCA
  GCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAG
  CGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGG
  AACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCT
  GTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCG
  GAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGG
  CCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCG
  CCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGT
  GAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCC
  GATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGC
  AACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTT
  CCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCT
  ATGACCATGATTACGCCAAGCGCGCAATTAACCCTCACTAAAGGGAACAAAAGCTGGA
  GCTGCAAGCTTAATGTAGTCTTATGCAATACTCTTGTAGTCTTGCAACATGGTAACGATG
  AGTTAGCAACATGCCTTACAAGGAGAGAAAAAGCACCGTGCATGCCGATTGGTGGAA
  GTAAGGTGGTACGATCGTGCCTTATTAGGAAGGCAACAGACGGGTCTGACATGGATTG
  GACGAACCACTGAATTGCCGCATTGCAGAGATATTGTATTTAAGTGCCTAGCTCGATAC
  ATAAACGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGG
  GAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCC
  GTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAA
  ATCTCTAGCAGTGGCGCCCGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAGC
  TCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGC
  GACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAGATGGGT
  GCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGCGATGGGAAAAAATTCGGTT
  AAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACATATAGTATGGGCAAGCAGGGAG
  CTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAAAT
  ACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAACTTAGATCATTATATA
  ATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAAGACACCAAGGA
  AGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGACCACCGCACAGCAAGC
  GGCCGCTGATCTTCAGACCTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAATT
  ATATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGA
  GAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCCTTGGGT
  TCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACAGG
  CCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAG
  GCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGA
  ATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCT
  CTGGAAAACTCATTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCT
  CTGGAACAGATTTGGAATCACACGACCTGGATGGAGTGGGACAGAGAAATTAACAATT
  ACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAACCAGCAAGAAAAGAATGA
  ACAAGAATTATTGGAATTAGATAAATGGGCAAGTTTGTGGAATTGGTTTAACA
  U6promoter
  sgRNA

• Amino acid sequences of Maloney murine leukemia virus reverse transcriptase (M-MLV RT) variants used herein.

• PE1 M-MLV RT:

  (SEQ ID NO: 739)

  TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLI

  IPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPL

  LPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYT

  VLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSP

  TLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ

  TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTP

  KTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKA

  YQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPV

  AYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVE

  ALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEG

  LQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAA

  VTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAF

  ATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPG

  HQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP

  M3 M-MLV RT (D200N, T330P, L603W) (see Baranauskas et al.182):

  (SEQ ID NO: 740)

  TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLI

  IPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPL

  LPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYT

  VLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSP

  TLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ

  TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTP

  KTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKPGTLFNWGPDQQKA

  YQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPV

  AYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVE

  ALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEG

  LQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAA

  VTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAF

  ATAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPKRLSIIHCP

  GHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP

  PE2 M-MLV RT (D200N, T306K, W313F, T330P, L603W):

  (SEQ ID NO: 741)

  TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLI

  IPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPL

  LPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYT

  VLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSP

  TLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ

  TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTP

  KTPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKA

  YQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPV

  AYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVE

  ALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEG

  LQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAA

  VTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAF

  ATAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPG

  HQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP

  M3-deadRT M-MLV RT:

  (SEQ ID NO: 742)

  TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLI

  IPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPL

  LPVKLPGTNDYSPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYT

  VLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSP

  TLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ

  TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTP

  KTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKPGTLFNWGPDQQKA

  YQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPV

  AYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVE

  ALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEG

  LQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAA

  VTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAF

  ATAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPG

  HQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP

REFERENCES FOR EXAMPLE 12
• Each of the following references are cited in Example 12, each of which are incorporated herein by reference.
• 1. Landrum, M. J. et al. ClinVar: public archive of interpretations of clinically relevant variants. Nucleic Acids Res. 44, D862-D868 (2016).
• 2. Jinek, M. et al. A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science 337, 816-821 (2012).
• 3. Cong, L. et al. Multiplex Genome Engineering Using CRISPR/Cas Systems. Science 339, 819-823 (2013).
• 4. Mali, P. et al. RNA-Guided Human Genome Engineering via Cas9. Science 339, 823-826 (2013).
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Example 13—Cell Data Recording and Lineage Tracing by PE Background
• Genome modification can be used to study and record cellular processes and development. Linking cellular events, like cell division or signaling cascade activation, to DNA sequence modifications stores cellular histories as interpretable DNA sequence changes that would describe whether specific cellular events had taken place. DNA editing is necessary for these applications because DNA is faithfully passed from one cell to the next in a way that RNA and proteins are not. Information relating to cellular state and lineage are in general lost when modifications are made to short-lived protein and RNA molecules. Recording cellular events within a single cell is a powerful way to understand how disease states are initiated, maintained, and changed relative to healthy controls. The ability to probe these questions has implications for understanding the development of cancer, neurological disease, and a host of other important problems in human health. prime editing (PE) provides a system for creating targeted and sequence-specified genomic insertions, deletions, or mutations. Repeated modification of a DNA target that can be sequenced by targeted amplicon sequencing and/or RNA sequencing (which is of particular value for single cell recording experiments) can be used to record a host of important biological processes, including activation of signaling cascades, metabolic states, and cellular differentiation programs. Connecting internal and external cellular signals to sequence modifications in the genome is, in theory, possible for any signal for which a signal responsive promoter exists. It is believed that PE enables a greatly expanded toolkit for probing the cellular lineage and the signaling histories of eukaryotic and prokaryotic cells in both cultured conditions and in vivo.
• Previous Standards
• Targeted sequence insertion, deletion, or mutation can be used to study a number of important biological questions including lineage tracing and recording of cellular stimuli. The current toolkit for generating these signatures in the genome is limited. Mutagenesis of target loci has been developed to date using both DNA-nucleases and base editors.
• CRISPR/Cas9 nuclease cutting of target sequences generates stochastic sequence changes to generate a large number of insertion or deletion (indel) products. The large number of sequence outcomes that arise from Cas9 cutting allows for clear determination of sequences that have been cut by the nuclease. The ability to distinguish cut versus non-cut sequences has been used in two predominant ways.
• First, the expression of the Cas9 nuclease and/or its single guide RNA (sgRNA) have been connected to cellular signals. Additionally, whether that signal has occurred based on sequence modifications to the Cas9 targeted genomic locus has been recorded. However, this approach is limited because each signal requires a unique target locus, which makes tracking the relative timing of multiple signals difficult to interpret. Another limitation to this approach is that multiple target loci are desired for a specific sgRNA because generation of an indel often severely hinders additional mutagenesis of the target locus; this often means that pre-engineered target loci are integrated into cells for editing instead of direct mutagenesis of endogenous loci.
• Second, Cas9 indels have been used to track cellular lineages. As described herein, the large number of possible indel states generated by Cas9 nuclease activity allows for the generation of cellular developmental trees that suggest which cells have arisen from one another across time. This approach is a powerful way to understand how cells arise from one another and has been used to help identify unique cell states and types across developmental time by performing RNA sequencing on selected cellular pools. This approach cannot independently report on cellular signaling events, their order, and may have biases when reporting on pre-cursor versus terminally differentiated cell states.
• Cas9 nuclease mediated lineage tracing and signal recording are powerful techniques that come with some important caveats. Signal recording with Cas9 nuclease is often very technically challenging. Cas9 cutting exhausts the target locus (repeated cutting is difficult once an indel is generated) making it difficult to record long term stimuli at the single cell level. The kinetics of Cas9 cutting can be tuned to enable longer term recording events though the ability to integrate the order, intensity, and duration of multiple stimuli remains a very challenging technical problem that may not be achievable with this tool. Cas9 lineage tracing experiments have been incredibly powerful but suffer from minor technical challenges of sequence collapse due to simultaneous Cas9 cuts at a target locus. These lineage-tracing experiments require editing of pre-designed target loci, limiting the flexibility of this approach.
• DNA base editing has also been used to track cellular signaling events. Base editing is not well suited for lineage tracing due to the low number of outcome states generated by an editing event relative to the number of states generated by Cas9 indels; however, the pre-defined nature of the sequence modifications made by a base editor are particularly useful for tracking internal and external cellular stimuli. As described herein, either base editor or sgRNA expression can be connected to a particular biological or chemical stimulus. Base editing activity has been used to track a large number of individual stimuli in both mammalian and bacterial cells. This approach has also been used to track consecutive stimuli where a first editing event was necessary before a second edit could take place.
• Base editing signal recording is an important first step for the field, but it has a number of limitations. One such limitation is that base editing exhausts its target after editing, limiting the dynamic range of the technique. This means that using endogenous targets for recording events is often difficult and limited to recording bulk activities instead of activity at the single cell level. An alternative to this is the introduction of a pre-designed repetitive recording locus though this has not been performed to date. There are also issues with two-signal recording. These two signal-recording experiments only report on the presence of the second stimulus after a first; it does not report which stimulus happened first or how long the stimuli were present. This fundamentally limits the biological understanding gleaned from the experiment.
• It has been proposed that PE lineage tracing can do both lineage tracing and cellular signaling recording by modifying genomic target sequences as well as integrated pre-designed sequences. PE uses a synthetic fusion protein comprising a Cas9 nickase fragment (often the SpCas9 H840A variant) and a reverse transcriptase (RT) domain, along with an engineered prime editing guide RNA (PEgRNA). Together, these components target a specific genomic sequence and install a pre-determined edit. Since the PEgRNA specifies both the target genomic sequence and the editing outcome, highly specific and controlled genome modification can be achieved simultaneously using multiple PEgRNAs within the same cell. Accessible genome modifications include all single nucleotide substitution, small to medium size sequence insertions, and small to medium size sequence deletions. The versatility of this genome editing technology should enable temporally coupled, signal-specific recording within cells.
• Utility of PE Lineage and Cell Signaling Recording
• Recording cellular signaling can be accomplished in a number of ways. One important first application of this approach is to connect DNA modification events to cell cycle associated signals like the expression of cyclins, CDKs, or other proteins specific to phases of the cellular life span one could generate a cellular clock. A cellular clock allows researchers to understand the order of various signals being received and processed by individual cells. A molecular clock would also enable the determination of long-term signaling versus short-term bursts of signaling. Using prime editing components that can only edit once a cell cycle could also lead to a molecular clock. If editing can only proceed once a cell cycle without continued DNA modification (perhaps by not nicking the non-edited DNA strand) one could imagine a system that only can be edited by a second targeting PEsgRNA in a subsequent cell division. PE is particularly useful as a cellular clock as it can repeatedly insert, delete, or mutate loci in a predetermined way with insertions being particularly valuable as repeated, regular insertions can be made at any target genomic locus.
• Another important application related to recording cellular signals is the parallel recording of a large number of cellular inputs. Linking cellular signaling events to DNA modification enables recording of whether such a signaling event has occurred. Similar to Cas9 nuclease-based or base editing-based recording systems, recording of cellular events can be tethered to gRNA or editor expression. Unlike these other approaches, lineage prime editing should be able to record the order, intensity, and duration of signaling events without requiring strict sequence motifs for ordered editing. Indeed, lineage prime editing should be able to integrate the cellular counter described above with signal specific insertions, deletions, or mutations to study the order, intensity, and duration of biological signals. Due to the programmable nature of prime editing, this approach can be achieved at genomic loci pre-existing in the target cell of interest (whether this is in bacteria, mice, rats, monkeys, pigs, humans, zebrafish, C. elegans, etc.). It is important to note as well that prime editing recording installs barcodes in a guide RNA dependent manner, the number of inputs is limited to the number of signals for which there are reliable signal specific guide RNA expression cassettes (which should be very high due to the ability to tether these expressions to the activity of RNA Pol II promoters). The number of recordable signals scales linearly with the number of PEgRNAs needed.
• PE can also be used to trace cellular lineages. Repeated sequence modification can be used to generate unique cellular barcodes to track individual cells. The arrays of barcodes, their order, and size can all be used to infer cellular lineages in a way that can be complementary to the large number of indel states generated by Cas9 nuclease.
• Prime Editing Methodology for Repeated Sequence Modification
• A number of distinct modalities for repeated sequence modification using prime editing (PE) were envisioned: DNA mutagenesis; sequence deletion; and, sequence insertion. Notably, these applications can be used on either pre-existing genomic DNA targets or on pre-designed DNA sequences that researchers integrate into target cells. These techniques of serial sequence modification have value for recording information and for designed or stochastic modification of target loci in a continuous manner. Serial targeted locus modification may be particularly useful for generating libraries of variants in various hosts.
• Repeated sequence mutation can be used to alter either genomic DNA or pre-designed integrated DNA sequences in an iterative manner to report on cellular signaling events. In this paradigm, mutations installed by PE gRNA activity will correspond to the presence of a cellular signal. These point mutations could install PAM motifs necessary for serial editing events, as well as point mutations that correspond to the presence of specific signals. This system would require gRNA design prior to use as each successive guide RNA will use a novel protospacer; however, it could be especially powerful for examining individual or small numbers of stimuli of particular interest. Installation of these mutations could be dependent on individual biological stimuli or could be connected to consistent cellular processes that would mark cellular time. The sequences below correspond to SEQ ID NOs: 743, 744, 744, and 745.

• 	Target genomic	CGTATCGGTAACTGATCCGAT
  	DNA sequence	GGAAAGCCAGTTCAGAACCG
  	Target sequence	CGTATCGGTAACTGATCCGAT
  	post edit #1	GGAATGCCAGGTCAGAACCG
  	(install G and T)
  	Target sequence	CGTATCGGTAACTGATCCGAT
  	post edit #1	GGAATGCCAGGTCAGAACCG
  	(use AGG PAM)
  	Target sequence	CGTATCGGTAACTGATCCGATG
  	post edit #2	GAATGCCAGGTCAAAACOG
  	(install 0 and A)

• Another similar PE guide RNA-intensive method is the repeated deletion of target sequences. Removal of individual sequences from a target locus would allow for the ability to reconstruct signaling events through the loss of DNA motifs. Design of PEgRNAs that delete successive sequences would enable the tracking of consecutive signals. This would allow researchers to identify instances where one signal followed another, which would allow researchers to probe which signaling events happen in which order. Such a system has been tested using CAMERA; however, this required pre-selection of particular loci with unique sequence requirements. Successive sequence deletion using PE would allow for the paralleled recording of pairwise events in individual cells as no specific sequence determinants are required. This would allow researchers the ability to probe pairwise signaling events in a multiplexed manner inside any target cell of interest. The sequences below correspond to SEQ ID NOs: 746, 747, and 748.

• 	Example target	CGTATCGGTAACTGATCCGAT
  	DNA sequence	GGAAAGCCAGTTCAGAACCG
  	Target sequence	CGTATCGGTAACTGATCCGAT
  	post edit #1	GGGCCAGTTCAGAACCG
  	(delete AAA)
  	Target sequence	CGTATCGGTAACTGATCCGAT
  	post edit #2	GGAGTTCAGAACCG
  	(delete GCC)

• Sequence insertion is a third approach for the tracking cellular signaling events. Some variants of this strategy are less PEgRNA-dependent than mutagenesis or deletion. A number of different insertion strategies exist-insertion of short sequences, insertion of protospacers, insertion of a protospacer and a barcode, insertion of novel homology sequences, and insertion of homology sequences with a barcode.
• Insertion of short repetitive sequences is a way to incrementally increase the size of a target sequence to measure the passage of time in a cell. In this system insertion of 5 or more nucleotides of repetitive sequence can cause repeat expansion in connection to either the passage of time or the continued presence of a pre-determined stimulus. The locus-agnostic nature of lineage PE again enables paralleled tracking of multiple unique sequence expansions in connection with discrete biological signals. This should enable measuring of the intensity of multiple biological signals across cellular time in individual cells. The sequences below correspond to SEQ ID NOs: 749, 750, and 751.

• 	Example target	genomic sequence-
  	DNA sequence	CGTATCGTATCGTATCGTATTGG-
  		genomic sequence
  	Target sequence	genomic sequence-
  	post edit #1	CGTATCGTATCGTATCGTATCGCTAtTGG-
  	(install CGTAT)	genomic sequence
  	Target sequence	genomic sequence-
  	post edit #2	CGTATCGTATCGTATCGTATCGTATCGT
  	(install CGTAT)	ATTGG-genomic sequence

• Insertion of different short sequences would require a handful of PEgRNAs in a manner similar to the deletional space. The number of signals being recorded and the size of the inserted sequence would determine the number of combinations of PEgRNAs needed. One challenge with recording multiple sequences in this system would be the different efficiencies of each PEgRNA at inserting its cargo sequence.
• Insertion of protospacers as indicators of cellular signals is enticing though technical challenges may infringe on the efficiency of this approach. Single PEgRNA systems will be challenging to use because PEgRNA cassettes would be substrates for themselves, causing insertions into the PEgRNA that compromise the efficiency and fidelity of successive edits. This same problem persists for two or three PEgRNA systems, as each guide is a substrate for another, enabling insertion of other sequences into the guide cassette itself, which could lead to inappropriate insertion of protospacer sequences in connection with the wrong signal. These protospacer insertion systems are also difficult to imagine with the inclusion of barcode sequences. Single PEgRNA barcode systems would simply write over the barcodes used, removing the data stored in the first edit. Multiple guide systems again suffer from insertions into other PEgRNA expression constructs, limiting its utility (especially in vivo). The sequences below correspond to SEQ ID NOs: 752, 752, 753, 754, 754, 755, and 756.

• Example target DNA	CGTATCGGTAACTGATCCGATGGAAA
  sequence
  Example target DNA  sequence edited with  lineage PE guide  #1	↓

  	gCGTATCGGTAACTGATCCGA-sgRNA Backbone-TTCCATCE-BC1-GGGCCAGGA-
  	GATCAGTTACCGAT

  		HomologyPrimer Binding
  		Reverse ComplementSequence
  		Old Homology Sequence

  Target DNA after	CGTATCGGTAACTGATCTCCTGGCCC-BC1-CGATGGAAA
  editing with lineage PE
  guide #
  1
  Target DNA after  editing with lineage PE  guide #1 edited with  lineage PE guide #2
  	gCGTATCGGTAACTGATCTCC-sgRNA Backbone-GGGCCAGGA-BC1-TTTCCATCG-
  	GATCAGTTACCGAT

  	↓

  Target DNA after

  CGTATCGGTAACTGATCCGATGGAAA-BC2-TCCTGGCCC-BC1-CGATGGAAA

  editing with lineage PE
  guide #
  1 and with
  lineage PE guide #2

• Insertion of homology sequences (i.e., sequences 3′ of the Cas9 nick location), and especially homology sequences with associated barcodes, appear to be particularly useful lineage PE strategies. This system avoids the issues associated with protospacer insertion by ensuring that successive rounds of editing result in the insertion of a barcode from a PEgRNA cassette that cannot be modified by other PEgRNA editing events in the same cell. The barcoding system is valuable as multiple barcodes can be associated with a given stimulus. This system preserves the majority of the target protospacer but alters the seed sequence, PAM, and downstream adjacent nucleotides. This enables multiple signals to be connected to one editing locus without significant re-designing of the PEgRNAs being used. This strategy would enable multiplexed barcode insertion in response to a large number of cellular stimuli (either internal or external) at a single locus. It could enable recording of intensity, duration, and order of as many signals as there exist unique barcodes (which can be designed with multiple N nucleotides to generate 4{circumflex over ( )}N possible barcodes; i.e. a 5-nt barcode would enable recording of 4{circumflex over ( )}5 or 1024 unique signals at once). This system could be used both in vitro and in vivo.
REFERENCES CITED IN EXAMPLE 13
• Each of the following references are incorporated herein by reference.
• 1. Recording development with single cell dynamic lineage tracing. Aaron McKenna, James A. Gagnon.
• 2. Whole-organism lineage tracing by combinatorial and cumulative genome editing.Mckenna et al. Science. 2016 Jul. 29; 353(6298):aaf7907. doi10.1126/science.aaf7907. Epub 2016 May 26.
• 3. Molecular recording of mammalian embryogenesis. Chan et al. 2019. Nature. June; 570(7759):77-82. doi: 10.1038/s41586-019-1184-5. Epub 2019 May 13.
Example 14—Modulating Biomolecule Activity and/or Localization by PE
• The subcellular localization and modification states of biomolecules regulate their activities. Specific biological functions like transcriptional control, cellular metabolism, and signal transduction cascades are all carefully orchestrated in particular locations within the cell. As such, modulating the cellular localization and modification states of proteins represents a potential therapeutic strategy for the treatment of disease. Some existing therapeutics have been developed to alter the localization of target proteins. For example, farnesylation inhibitors are designed to prevent lipidation and membrane targeting of important oncogenic proteins like KRAS. Similarly, small-molecule-induced ubiquitination of target proteins directs them to the proteasome for degradation. The ability to traffic proteins to these and other unique cellular compartments provides an opportunity to alter a number of biological processes. It is proposed herein that the use of prime editing (PE) to install genetically encoded handles for altering the modification state and the subcellular trafficking of biomolecules with a genetically encoded signal (e.g. proteins, lipids, sugars, and nucleic acids) for the purposes of therapeutics.
• PE is a genome editing technology that enables the installation, deletion, or replacement of short DNA sequences into any genomic locus targetable with a Cas9 enzyme. Using this technology, one could in principle install or remove important DNA, RNA, or protein coding sequences that change the activities of these important biomolecules. More specifically, prime editing could be used to install motifs, or signals, that change the localization or modification properties of biomolecules. Some examples include modification to: protein amino acid sequences; motifs for post translational modifications; RNA motifs that change folding or localization; and, installation of DNA sequences that change the local chromatin state or architecture of the surrounding DNA.
• One target biomolecule for PE-mediated modification is DNA. Modifications to DNA could be made to install a number of DNA sequences that change the accessibility of the target locus. Chromatin accessibility controls gene transcriptional output. Installation of marks to recruit chromatin compacting enzymes should decrease the transcriptional output of neighboring genes, while installation of sequences associated with chromatin opening should make regions more accessible and in turn increase transcription. Installation of more complex sequence motifs that mirror native regulatory sequences should provide more nuanced and biologically sensitive control than the currently available dCas9 fusions to different epigenetic reader, writer, or eraser enzymes—tools that typically install large numbers of a single type of mark that may not have a particular biological antecedent. Installation of sequences that will bring two loci into close proximity, or bring loci into contact with the nuclear membrane, should also alter the transcriptional output of those loci as has been demonstrated in the burgeoning field of 3-D genomic architecture.
• Modifications to RNAs can also be made to alter their activity by changing their cellular localization, interacting partners, structural dynamics, or thermodynamics of folding. Installation of motifs that will cause translational pausing or frameshifting could change the abundance of mRNA species through various mRNA processing mechanisms. Modifying consensus splice sequences would also alter the abundance and prevalence of different RNA species. Changing the relative ratio of different splice isoforms would predictably lead to a change in the ratio of protein translation products, and this could be used to alter many biological pathways. For instance, shifting the balance of mitochondrial versus nuclear DNA repair proteins would alter the resilience of different cancers to chemotherapeutic reagents. Furthermore, RNAs could be modified with sequences that enable binding to novel protein targets. A number of RNA aptamers have been developed that bind with high affinity to cellular proteins. Installation of one of these aptamers could be used to either sequester different RNA species through binding to a protein target that will prevent their translation, biological activity, or to bring RNA species to specific subcellular compartments. Biomolecule degradation is another class of localization modification. For example, RNA methylation is used to regulate RNAs within the cell. Consensus motifs for methylation could be introduced into target RNA coding sequences with PE. RNAs could also be modified to include sequences that direct nonsense mediated decay machinery or other nucleic acid metabolism pathways to degrade the target RNA species would change the pool of RNAs in a cell. Additionally, RNA species could be modified to alter their aggregation state. Sequences could be installed on single RNAs of interest or multiple RNAs to generate RNA tangles that would render them ineffective substrates for translation or signaling.
• Modifications to proteins via post-translational modification (PTM) also represent an important class of biomolecule manipulation that can be carried out with PE. As with RNA species, changing the abundance of proteins in a cell is an important capability of PE. Editing can be done to install stop codons in an open reading frame—this will eliminate full-length product from being produced by the edited DNA sequence. Alternatively, peptide motifs can be installed that cause the rate of protein degradation to be altered for a target protein. Installation of degradation tags into a gene body could be used to alter the abundance of a protein in a cell. Moreover, introduction of degrons that are induced by small molecules could enable temporal control over protein degradation. This could have important implications for both research and therapeutics as researchers could readily assess whether small molecule-mediated therapeutic protein degradation of a given target was a viable therapeutic strategy. Protein motifs could also be installed to change the subcellular localization of a protein. Amino acid motifs can be installed to preferentially traffic proteins to a number of subcellular compartments including the nucleus, mitochondria, cell membrane, peroxisome, lysosome, proteasome, exosome, and others.
• Installing or destroying motifs modified by PTM machinery can alter protein post-translational modifications. Phosphorylation, ubiquitylation, glycosylation, lipidation (e.g. farnesylation, myristoylation, palmitoylation, prenylation, GPI anchors), hydroxylation, methylation, acetylation, crotonylation, SUMOylation, disulfide bond formations, side chain bond cleavage events, polypeptide backbone cleavage events (proteolysis), and a number of other protein PTMs have been identified. These PTMs change protein function, often by changing subcellular localization. Indeed, kinases often activate downstream signaling cascades via phosphorylation events. Removal of the target phosphosite would prevent signal transduction. The ability to site-specifically ablate or install any PTM motif while retaining full-length protein expression would be an important advance for both basic research and therapeutics. The sequence installation scope and target window of PE make it well suited for broad PTM modification space.
• Removal of lipidation sites should prevent the trafficking of proteins to cell membranes. A major limitation to current therapeutics that target post-translational modification processes is their specificity. Farnesyl transferase inhibitors have been tested extensively for their ability to eliminate KRAS localization at cell membranes. Unfortunately, global inhibition of farnesylation comes with numerous off target effects that have prevented broad use of these small molecules. Similarly, specific inhibition of protein kinases with small molecules can be very challenging due to the large size of the human genome and similarities between various kinases. PE offers a potential solution to this specificity problem, as it enables inhibition of modification of the target protein by ablation of the modification site instead of global enzyme inhibition. For example, removal of the lapidated peptide motif in KRAS would be a targeted approach that could be used in place of farnesyl transferase inhibition. This approach is the functional inverse of inhibiting a target protein activity by installing a lipid-targeting motif on a protein not designed to be membrane bound.
• PE can also be used to instigate protein-protein complexation events. Proteins often function within complexes to execute their biological activity. PE can be used to either create or destroy the ability of proteins to exist within these complexes. To eliminate complex formation events, amino acid substitutions or insertions along the protein: protein interface could be installed to disfavor complexation. SSX18 is a protein component of the BAF complex, an important histone-remodeling complex. Mutations in SSX18 drive synovial sarcomas. PE could be used to install side chains that prevent SSX18 from binding to its protein partners in the complex to prevent its oncogenic activity. PE could also be used to remove the pathogenic mutations to restore WT activity of this protein. Alternatively, PE could be used to keep proteins within either their native complex or to drag them to participate in interactions that are unrelated to their native activity to inhibit their activity. Forming complexes that maintain one interaction state over another could represent an important therapeutic modality. Altering protein: protein interfaces to decrease the Kd of the interaction would keep those proteins stuck to one another longer. As protein complexes can have multiple signaling complexes, like n-myc driving neuroblastoma signaling cascades in disease but otherwise participating in healthy transcriptional control in other cells. PE could be used to install mutations that drive n-myc association with healthy interactions partners and decrease its affinity for oncogenic interaction partners.
REFERENCES CITED IN EXAMPLE 14
• Each of the following references are incorporated herein by reference.
• 1. Selective Target Protein Degradation via Phthalimide Conjugation. Winter et al. Science. Author manuscript; available in PMC 2016 Jul. 8.
• 2. Reversible disruption of mSWI/SNF (BAF) complexes by the SS18-SSX oncogenic fusion in synovial sarcoma. Kadoch and Crabtree. Cell. 2013 Mar. 28; 153(1):71-85. doi: 10.1016/j.cell.2013.02.036.
• 3. Ribosomal frameshifting and transcriptional slippage: From genetic steganography and cryptography to adventitious use. Atkins et al. Nucleic Acids Research, Volume 44, Issue 15, 6 Sep. 2016, Pages 7007-7078.
• 4. Transcriptional Regulation and its Misregulation in Disease. Lee and Young. Cell. Author manuscript; available in PMC 2014 Mar. 14.
• 5. Protein localization in disease and therapy. Mien-Chie Hung, Wolfgang Link Journal of Cell Science 2011 124: 3381-3392.
• 6. Loss of post-translational modification sites in disease. Li et al. Pac Symp Biocomput. 2010:337-47. PTMD: A Database of Human Disease-associated Post-translational Modifications. Xu et al. Genomics Proteomics Bioinformatics. 2018 August; 16(4):244-251. Epub 2018 Sep. 21.
• 7. Post-transcriptional gene regulation by mRNA modifications. Zhao et al. Nature Reviews Molecular Cell Biology volume 18, pages 31-42 (2017).
Example 15—Design and Engineering of PEgRNAS
• Described herein is a series of PEgRNA designs and strategies that can improve prime editing (PE) efficiency.
• Prime editing (PE) is a genome editing technology that can replace, insert, or remove defined DNA sequences within a targeted genetic locus using information encoded within a prime editing guide RNA (PEgRNA). Prime editors (PEs) consist of a sequence-programmable DNA binding protein with nuclease activity (Cas9) fused to a reverse transcriptase (RT) enzyme. PEs form complexes with PEgRNAs, which contain the information for targeting specific DNA loci within their spacer sequences, as well as information specifying the desired edit in an engineered extension built into a standard sgRNA scaffold. PE:PEgRNA complexes bind and nick the programmed target DNA locus, allowing hybridization of the nicked DNA strand to the engineered primer binding sequence (PBS) of the PEgRNA. The reverse transcriptase domain then copies the edit-encoding information within the RT template portion of the PEgRNA, using the nicked genomic DNA as a primer for DNA polymerization. Subsequent DNA repair processes incorporate the newly synthesized edited DNA strand into the genomic locus. While the versatility of prime editing holds great promise as a research tool and potential therapeutic, several limitations in efficiency and scope exist due to the multi-step process required for editing. For example, unfavorable RNA structures that form within the PEgRNA can inhibit the copying of DNA edits from the PEgRNA to the genomic locus. One potential way to improve PE technology is through redesign and engineering of the critical PEgRNA component. Improvements to the design of these PEgRNAs are likely to be necessary for improved PE efficiency, as well as enable installation of longer inserted sequences into the genome.
• Described herein is a series of PEgRNA designs that are envisioned to improve the efficacy of PE. These designs take advantage of a number of previously published approaches for improving sgRNA efficacy and/or stability, as well as utilize a number of novel strategies. These improvements can belong to one or more of a number of different categories:
• (1) Longer PEgRNAs. This category relations to improved designs that enable efficient expression of functional PEgRNAs from non-polymerase III (pol III) promoters, which would enable the expression of longer PEgRNAs without burdensome sequence requirements;
• (2) Core improvements. This category relates to improvements to the core, Cas9-binding PEgRNA scaffold, which could improve efficacy;
• (3) RT processivity. This category relates to modifications to the PEgRNA that improve RT processivity, enabling the insertion of longer sequences at targeted genomic loci; and
• (4) Termini motifs. This category relates to the addition of RNA motifs to the 5′ and/or 3′ termini of the PEgRNA that improve PEgRNA stability, enhance RT processivity, prevent mis-folding of the PEgRNA, or recruit additional factors important for genome editing.
• Described herein are a number of potential such PEgRNA designs in each category. Several of these designs have been previously described for improving sgRNA activity with Cas9 and are indicated as such. Described herein is also a platform for the evolution of PEgRNAs for given sequence targets that would enable the polishing of the PEgRNA scaffold and enhance PE activity (5). Notably, these designs could also be readily applied to improve PEgRNAs recognized by any Cas9 or evolved variant thereof.
• (1) Longer PEgRNAs.
• sgRNAs are typically expressed from the U6 snRNA promoter. This promoter recruits pol III to express the associated RNA and is useful for expression of short RNAs that are retained within the nucleus. However, pol III is not highly processive and is unable to express RNAs longer than a few hundred nucleotides in length at the levels required for efficient genome editing¹⁸³. Additionally, pol III can stall or terminate at stretches of U's, potentially limiting the sequence diversity that could be inserted using a PEgRNA. Other promoters that recruit polymerase II (such as pCMV) or polymerase I (such as the U1 snRNA promoter) have been examined for their ability to express longer sgRNAs¹⁸³. However, these promoters are typically partially transcribed, which would result in extra sequence 5′ of the spacer in the expressed PEgRNA, which has been shown to result in markedly reduced Cas9:sgRNA activity in a site-dependent manner. Additionally, while pol III-transcribed PEgRNAs can simply terminate in a run of 6-7 U's, PEgRNAs transcribed from pol II or pol I would require a different termination signal. Often such signals also result in polyadenylation, which would result in undesired transport of the PEgRNA from the nucleus. Similarly, RNAs expressed from pol II promoters such as pCMV are typically 5′-capped, also resulting in their nuclear export.
• Previously, Rinn and coworkers screened a variety of expression platforms for the production of long-noncoding RNA-(lncRNA) tagged sgRNAs¹⁸³. These platforms include RNAs expressed from pCMV and that terminate in the ENE element from the MALAT1 ncRNA from humans¹⁸⁴, the PAN ENE element from KSHV¹⁸⁵, or the 3′ box from U1 snRNA¹⁸⁶. Notably, the MALAT1 ncRNA and PAN ENEs form triple helices protecting the polyA-tail^{184, 187}. It is anticipated that, in addition to enabling expression of RNAs, these constructs could also enhance RNA stability (see section iv). Using the promoter from the U1 snRNA to enable expression of these longer sgRNAs¹⁸³ was also explored. It is anticipated that these expression systems will also enable the expression of longer PEgRNAs. In addition, a series of methods have been designed for the cleavage of the portion of the pol II promoter that would be transcribed as part of the PEgRNA, adding either a self-cleaving ribozyme such as the hammerhead¹⁸⁸, pistol¹⁸⁹, hatchet¹⁸⁹, hairpin¹⁹⁰, VS¹⁹¹, twister¹⁹², or twister sister¹⁹² ribozymes, or other self-cleaving elements to process the transcribed guide, or a hairpin that is recognized by Csy4¹⁹³ and also leads to processing of the guide. Also, it is hypothesized that incorporation of multiple ENE motifs could lead to improved PEgRNA expression and stability, as previously demonstrated for the KSHV PAN RNA and element¹⁸⁵. It is also anticipated that circularizing the PEgRNA in the form of a circular intronic RNA (ciRNA) could also lead to enhanced RNA expression and stability, as well as nuclear localization¹⁹⁴.

• PEgRNA expression platform consisting of pCMV,

  Csy4 hairping, the PEgRNA, and MALAT1 ENE

  (SEQ ID NO: 757)

  TAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCAT

  ATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGC

  TGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGT

  TCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGG

  AGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCAT

  ATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGC

  CTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGC

  AGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTT

  TGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATT

  TCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACC

  AAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTG

  ACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAG

  AGCTGGTTTAGTGAACCGTCAGATCGTTCACTGCCGTATAGGCAGGG

  CCCAGACTGAGCACGTGAGTTTTAGAGCTAGAAATAGCAAGTTAAAA

  TAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCC

  TCTGCCATCAAAGCGTGCTCAGTCTGTTTTAGGGTCATGAAGGTTTT

  TCTTTTCCTGAGAAAACAACACGTATTGTTTTCTCAGGTTTTGCTTT

  TTGGCCTTTTTCTAGCTTAAAAAAAAAAAAAGCAAAAGATGCTGGTG

  GTTGGCACTCCTGGTTTCCAGGACGGGGTTCAAATCCCTGCGGCGTC

  TTTGCTTTGACT

  PEgRNA expression platform consisting of pCMV,

  Csy4 hairping, the PEgRNA, and PAN ENE

  (SEQ ID NO: 758)

  TAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCAT

  AGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCG

  GACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAAC

  AGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTG

  CCCAACGCCAATCCCACTTGGCAGTACATCAAGTGTATCATATGCCA

  AGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCA

  TTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACA

  TCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAG

  TACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAG

  TCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATC

  AACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAA

  ATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTGG

  TTTAGTGAACCGTCAGATCGTTCACTGCCGTATAGGCAGGGCCCAGA

  CTGAGCACGTGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGC

  TAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCCTCTGCC

  ATCAAAGCGTGCTCAGTCTGTTTTGTTTTGGCTGGGTTTTTCCTTGT

  TCGCACCGGACACCTCCAGTGACCAGACGGCAAGGTTTTTATCCCAG

  TGTATATTGGAAAAACATGTTATACTTTTGACAATTTAACGTGCCTA

  GAGCTCAAATTAAACTAATACCATAACGTAATGCAACTTACAACATA

  AATAAAGGTCAATGTTTAATCCATAAAAAAAAAAAAAAAAAAA

  PEgRNA expression platform consisting of pCMV,

  Csy4 hairping, the PEgRNA, and 3xPAN ENE

  (SEQ ID NO: 759)

  TAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCAT

  ATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGC

  TGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGT

  TCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGG

  AGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCAT

  ATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGC

  CTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGC

  AGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTT

  TGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATT

  TCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACC

  AAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTG

  ACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAG

  AGCTGGTTTAGTGAACCGTCAGATCGTTCACTGCCGTATAGGCAGGG

  CCCAGACTGAGCACGTGAGTTTTAGAGCTAGAAATAGCAAGTTAAAA

  TAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCC

  TCTGCCATCAAAGCGTGCTCAGTCTGTTTTGTTTTGGCTGGGTTTTT

  CCTTGTTCGCACCGGACACCTCCAGTGACCAGACGGCAAGGTTTTTA

  TCCCAGTGTATATTGGAAAAACATGTTATACTTTTGACAATTTAACG

  TGCCTAGAGCTCAAATTAAACTAATACCATAACGTAATGCAACTTAC

  AACATAAATAAAGGTCAATGTTTAATCCATAAAAAAAAAAAAAAAAA

  AAACACACTGTTTTGGCTGGGTTTTTCCTTGTTCGCACCGGACACCT

  CCAGTGACCAGACGGCAAGGTTTTTATCCCAGTGTATATTGGAAAAA

  CATGTTATACTTTTGACAATTTAACGTGCCTAGAGCTCAAATTAAAC

  TAATACCATAACGTAATGCAACTTACAACATAAATAAAGGTCAATGT

  TTAATCCATAAAAAAAAAAAAAAAAAAATCTCTCTGTTTTGGCTGGG

  TTTTTCCTTGTTCGCACCGGACACCTCCAGTGACCAGACGGCAAGGT

  TTTTATCCCAGTGTATATTGGAAAAACATGTTATACTTTTGACAATT

  TAACGTGCCTAGAGCTCAAATTAAACTAATACCATAACGTAATGCAA

  CTTACAACATAAATAAAGGTCAATGTTTAATCCATAAAAAAAAAAAA

  AAAAAAA

  PEgRNA expression platform consisting of pCMV,

  Csy4 hairping, the PEgRNA, and 3 ′box

  (SEQ ID NO: 760)

  TAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCAT

  ATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGC

  TGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGT

  TCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGG

  AGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCAT

  ATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGC

  CTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGC

  AGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTT

  TGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATT

  TCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACC

  AAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTG

  ACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAG

  AGCTGGTTTAGTGAACCGTCAGATCGTTCACTGCCGTATAGGCAGGG

  CCCAGACTGAGCACGTGAGTTTTAGAGCTAGAAATAGCAAGTTAAAA

  TAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCC

  TCTGCCATCAAAGCGTGCTCAGTCTGTTTGTTTCAAAAGTAGACTGT

  ACGCTAAGGGTCATATCTTTTTTTGTTTGGTTTGTGTCTTGGTTGGC

  GTCTTAAA

  PEgRNA expression platform consisting of pUl,

  Csy4 hairping, the PEgRNA, and 3 ′box

  (SEQ ID NO: 761)

  CTAAGGACCAGCTTCTTTGGGAGAGAACAGACGCAGGGGCGGGAGGG

  AAAAAGGGAGAGGCAGACGTCACTTCCCCTTGGCGGCTCTGGCAGCA

  GATTGGTCGGTTGAGTGGCAGAAAGGCAGACGGGGACTGGGCAAGGC

  ACTGTCGGTGACATCACGGACAGGGCGACTTCTATGTAGATGAGGCA

  GCGCAGAGGCTGCTGCTTCGCCACTTGCTGCTTCACCACGAAGGAGT

  TCCCGTGCCCTGGGAGCGGGTTCAGGACCGCTGATCGGAAGTGAGAA

  TCCCAGCTGTGTGTCAGGGCTGGAAAGGGCTCGGGAGTGCGCGGGGC

  AAGTGACCGTGTGTGTAAAGAGTGAGGCGTATGAGGCTGTGTCGGGG

  CAGAGGCCCAAGATCTCAGTTCACTGCCGTATAGGCAGGGCCCAGAC

  TGAGCACGTGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCT

  AGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCCTCTGCCA

  TCAAAGCGTGCTCAGTCTGTTTCAGCAAGTTCAGAGAAATCTGAACT

  TGCTGGATTTTTGGAGCAGGGAGATGGAATAGGAGCTTGCTCCGTCC

  ACTCCACGCATCGACCTGGTATTGCAGTACCTCCAGGAACGGTGCAC

  CCACTTTCTGGAGTTTCAAAAGTAGACTGTACGCTAAGGGTCATATC

  TTTTTTTGTTTGGTTTGTGTCTTGGTTGGCGTCTTAAA

  (2) Core improvements.

• The core, Cas9-binding PEgRNA scaffold can likely be improved to enhance PE activity. Several such approaches have already been demonstrated. For instance, the first pairing element of the scaffold (P1) contains a GTTTT-AAAAC pairing element. Such runs of Ts have been shown to result in pol III pausing and premature termination of the RNA transcript. Rational mutation of one of the T-A pairs to a G-C pair in this portion of P1 has been shown to enhance sgRNA activity, suggesting this approach would also be feasible for PEgRNAs¹⁹⁵. Additionally, increasing the length of P1 has also been shown to enhance sgRNA folding and lead to improved activity¹⁹⁵, suggesting it as another avenue for the improvement of PEgRNA activity. Finally, it is likely the polishing of the PEgRNA scaffold through directed evolution of PEgRNAs on a given DNA target would also result in improved activity. This is described in section (v).

• PEgRNA containing a 6 nt extension to P1

  (SEQ ID NO: 228)

  GGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGCTCATGAAAATGAGCTA

  GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGA

  GTCGGTCCTCTGCCATCAAAGCGTGCTCAGTCTGTTTTTTT

  PEgRNA containing a T-A to G-C mutation within P1

  (SEQ ID NO: 229)

  GGCCCAGACTGAGCACGTGAGTTTGAGAGCTAGAAATAGCAAGTTTAAAT

  AAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCCTCTG

  CCATCAAAGCGTGCTCAGTCTGTTTTTTT

• (iii) Improvement of RT Processivity Via Modifications to the Template Region of the PEgRNA
• As the size of the insertion templated by the PEgRNA increases, it is more likely to be degraded by endonucleases, undergo spontaneous hydrolysis, or fold into secondary structures unable to be reverse-transcribed by the RT or that disrupt folding of the PEgRNA scaffold and subsequent Cas9-RT binding. Accordingly, it is likely that modification to the template of the PEgRNA might be necessary to affect large insertions, such as the insertion of whole genes. Some strategies to do so include the incorporation of modified nucleotides within a synthetic or semi-synthetic PEgRNA that render the RNA more resistant to degradation or hydrolysis or less likely to adopt inhibitory secondary structures¹⁹⁶. Such modifications could include 8-aza-7-deazaguanosine, which would reduce RNA secondary structure in G-rich sequences; locked-nucleic acids (LNA) that reduce degradation and enhance certain kinds of RNA secondary structure; 2′-O-methyl, 2′-fluoro, or 2′-O-methoxyethoxy modifications that enhance RNA stability. Such modifications could also be included elsewhere in the PEgRNA to enhance stability and activity. Alternatively or additionally, the template of the PEgRNA could be designed such that it both encodes for a desired protein product and is also more likely to adopt simple secondary structures that are able to be unfolded by the RT. Such simple structures would act as a thermodynamic sink, making it less likely that more complicated structures that would prevent reverse transcription would occur. Finally, one could also imagine splitting the template into two, separate PEgRNAs. In such a design, a PE would be used to initiate transcription and also recruit a separate template RNA to the targeted site via an RNA-binding protein fused to Cas9 or an RNA recognition element on the PEgRNA itself such as the MS2 aptamer. The RT could either directly bind to this separate template RNA, or initiate reverse transcription on the original PEgRNA before swapping to the second template. Such an approach could enable long insertions by both preventing mis-folding of the PEgRNA upon addition of the long template and also by not requiring dissociation of Cas9 from the genome for long insertions to occur, which could possibly be inhibiting PE-based long insertions.
• (iv) Installation of Additional RNA Motifs at the 5′ or 3′ Termini
• PEgRNA designs could also be improved via the installation of additional motifs at either end of the terminus of the RNA. Several such motifs—such as the PAN ENE from KSHV and the ENE from MALAT1 were discussed earlier in part (i)^184,185 as possible means to terminate expression of longer PEgRNAs from non-pol III promoters. These elements form RNA triple helices that engulf the polyA tail, resulting in their being retained within the nucleus^184,187. However, by forming complex structures at the 3′ terminus of the PEgRNA that occlude the terminal nucleotide, these structures would also likely help prevent exonuclease-mediated degradation of PEgRNAs. Other structural elements inserted at the 3′ terminus could also enhance RNA stability, albeit without enabling termination from non-pol III promoters. Such motifs could include hairpins or RNA quadruplexes that would occlude the 3′ terminus¹⁹⁷, or self-cleaving ribozymes such as HDV that would result in the formation of a 2′-3′-cyclic phosphate at the 3′ terminus and also potentially render the PEgRNA less likely to be degraded by exonucleases¹⁹⁸. Inducing the PEgRNA to cyclize via incomplete splicing—to form a ciRNA—could also increase PEgRNA stability and result in the PEgRNA being retained within the nucleus¹⁹⁴.
• Additional RNA motifs could also improve RT processivity or enhance PEgRNA activity by enhancing RT binding to the DNA-RNA duplex. Addition of the native sequence bound by the RT in its cognate retroviral genome could enhance RT activity¹⁹⁹. This could include the native primer binding site (PBS), polypurine tract (PPT), or kissing loops involved in retroviral genome dimerization and initiation of transcription¹⁹⁹. Addition of dimerization motifs—such as kissing loops or a GNRA tetraloop/tetraloop receptor pair²⁰⁰—at the 5′ and 3′ termini of the PEgRNA could also result in effective circularization of the PEgRNA, improving stability. Additionally, it is envisioned that addition of these motifs could enable the physical separation of the PEgRNA spacer and primer, prevention occlusion of the spacer which would hinder PE activity. Short 5′ extensions to the PEgRNA that form a small toehold hairpin in the spacer region could also compete favorably against the annealing region of the PEgRNA binding the spacer. Finally, kissing loops could also be used to recruit other template RNAs to the genomic site and enable swapping of RT activity from one RNA to the other (section iii).

• PEgRNA-HDV fusion

  (SEQ ID NO: 230)

  GGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGAAATAGCAAGTTAAAAT

  AAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCCTCTG

  CCATCAAAGCGTGCTCAGTCTGGGCCGGCATGGTCCCAGCCTCCTCGCTG

  GCGCCGGCTGGGCAACATGCTTCGGCATGGCGAATGGGACTTTTTTT

  PEgRNA-MMLV kissing loop

  (SEQ ID NO: 231)

  GGTGGGAGACGTCCCACCGGCCCAGACTGAGCACGTGAGTTTTAGAGCTA

  GAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG

  GACCGAGTCGGTCCTCTGCCATCAAAGCTTCGACCGTGCTCAGTCTGGTG

  GGAGACGTCCCACCTTTTTTT

  PEgRNA-VS ribozyme kissing loop

  (SEQ ID NO: 232)

  GAGCAGCATGGCGTCGCTGCTCACGGCCCAGACTGAGCACGTGAGTTTTA

  GAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

  AAGTGGGACCGAGTCGGTCCTCTGCCATCAAAGCTTCGACCGTGCTCAGT

  CTCCATCAGTTGACACCCTGAGGTTTTTTT

  PEgRNA-GNRA tetraloop/tetraloop receptor

  (SEQ ID NO: 233)

  GCAGACCTAAGTGGUGACATATGGTCTGGGCCCAGACTGAGCACGTGAGT

  TTTAGAGCTAUACGTAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTT

  UACGAAGTGGGACCGAGTCGGTCCTCTGCCATCAAAGCTTCGACCGTGCT

  CAGTCTGCATGCGATTAGAAATAATCGCATGTTTTTTT

  PEgRNA template switching secondary RNA-HDV fusion

  (SEQ ID NO: 234)

  TCTGCCATCAAAGCTGCGACCGTGCTCAGTCTGGTGGGAGACGTCCCACC

  GGCCGGCATGGTCCCAGCCTCCTCGCTGGCGCCGGCTGGGCAACATGCTT

  CGGCATGGCGAATGGGACTTTTTTT

• (v) Evolution of PEgRNAs
• It is likely that the PEgRNA scaffold can be further improved via directed evolution, in an analogous fashion to how SpCas9 and base editors have been improved²⁰¹. Directed evolution could enhance PEgRNA recognition by Cas9 or evolved Cas9 variants. Additionally, it is likely that different PEgRNA scaffold sequences would be optimal at different genomic loci, either enhancing PE activity at the site in question, reducing off-target activities, or both. Finally, evolution of PEgRNA scaffolds to which other RNA motifs have been added would almost certainly improve the activity of the fused PEgRNA relative to the unevolved, fusion RNA. For instance, evolution of allosteric ribozymes composed of c-di-GMP-I aptamers and hammerhead ribozymes led to dramatically improved activity²⁰², suggesting that evolution would improve the activity of hammerhead-PEgRNA fusions as well. In addition, while Cas9 currently does not generally tolerate 5′ extension of the sgRNA, directed evolution will likely generate enabling mutations that mitigate this intolerance, allowing additional RNA motifs to be utilized.
• As described herein, a number of these approaches have already been described for use with Cas9:sgRNA complexes, but no designs for improving PEgRNA activity have been reported. Other strategies for the installation of programmable mutations into the genome include base-editing, homology-directed recombination (HDR), precise microhomology-mediated end-joining (MMEJ), or transposase-mediated editing. However, all of these approaches have significant drawbacks when compared to PEs. Current base editors, while more efficient than existing PEs, can only install certain classes of genomic mutations and can result in additional, undesired nucleotide conversions at the site of interest. HDR is only feasible in a very small minority of cell types and results in comparably high rates of random insertion and deletion mutations (indels). Precise MMEJ can lead to predictable repair of double-strand breaks, but is largely limited to installation of deletions, is very site-dependent, and can also have comparably high rates of undesired indels. Transposase-mediated editing has to date only been shown to function in bacteria. As such improvements to PE represent possibly the best path forward for the therapeutic correction of a wide-swatch of genomic mutations.
REFERENCES CITED IN EXAMPLE 15
• Each of the following references are cited in Example 15, each of which are incorporated herein by reference.
• 1. Schechner, D M, Hacisuleyman E., Younger S T, Rinn J L. Nat Methods 664-70 (2015).
• 2. Brown J A, et al. Nat Struct Mol Biol 633-40 (2014).
• 3. Conrad N A and Steitz J A. EMBO J 1831-41 (2005).
• 4. Bartlett J S, et al. Proc Natl Acad Sci USA 8852-7 (1996).
• 5. Mitton-Fry R M, DeGregorio S J, Wang J, Steitz T A, Steitz J A. Science 1244-7 (2010).
• 6. Forster A C, Symons R H. Cell. 1987.
• 7. Weinberg Z, Kim P B, Chen T H, Li S, Harris K A, Lünse C E, Breaker R R. Nat. Chem. Biol. 2015.
• 8. Feldstein P A, Buzayan J M, Bruening G. Gene 1989.
• 9. Saville B J, Collins R A. Cell. 1990.
• 10. Roth A, Weinberg Z, Chen A G, Kim P G, Ames T D, Breaker R R. Nat Chem Biol. 2013.
• 11. Borchardt E K, et al. RNA 1921-30 (2015).
• 12. Zhang Y, et al. Mol Cell 792-806 (2013).
• 13. Dang Y, et al. Genome Biol 280 (2015).
• 14. Schaefer M, Kapoor U, and Jantsch M F. Open Biol 170077 (2017).
• 15. Nahar S, et al. Chem Comm 2377-80 (2018).
• 16. Gao Y and Zhao Y. J Integr Plant Biol 343-9 (2014).
• 17. Dubois N, Marquet R, Paillart J, Bernacchi S. Front Microbiol 527 (2018).
• 18. Costa M and Michel F. EMBO J 1276-85 (1995).
• 19. Hu J H, et al. Nature 57-63 (2018).
• 20. Furukawa K, Gu H, Breaker R R. Methods Mol Biol 209-20 (2014).
Example 16—Expanding the Targeting Scope of PE Using DNA Binding Proteins Other than SpCas9
• Prime editing (PE) using Streptococcus pyogenes Cas9 (SpCas9) can efficiently install all single base substitutions, insertions, deletions, and combinations thereof at genomic loci where there is a suitably-placed NGG protospacer adjacent motif (PAM) that SpCas9 can efficiently bind. The methods described herein broaden the targeting capability of PE by expanding the accessible PAMs and, therefore, the targetable genomic loci accessible for efficient PE. Prime editors using RNA-guided DNA binding proteins other than SpCas9 enable an expanded targettable scope of genomic loci by allowing access to different PAMs. In addition, use of RNA-guided DNA binding proteins smaller than SpCas9 also allows for more efficient viral delivery. PE with Cas proteins or other RNA-guided DNA binding proteins beyond SpCas9 will allow for high efficiency therapeutic edits that were either inaccessible or inefficient using SpCas9-based PE.
• This is expected to be used in situations where SpCas9-based PE is either inefficient due to non-ideal spacing of an edit to relative to an NGG PAM or the overall size of the SpCas9-based construct is prohibitive for cellular expression and/or delivery. Specific disease-relevant loci such as the Huntingtin gene, which has few and poorly located NGG PAMs for SpCas9 near the target region, can easily be targeted using different Cas proteins in the PE system such as SpCas9-VRQR which recognizes an NGA PAM. Smaller Cas proteins will be used to generate smaller PE constructs that can be packaged into AAV vectors more efficiently, enabling better delivery to target tissues. FIG. 61 shows the reduction to practice of prime editing using Staphylococcus aureus CRISPR-Cas as the RNA-guided DNA binding protein. NT is untreated control.
• FIGS. 62A-62B provide a demonstration of the importance of the protospacer for efficient installation of a desired edit at a precise location with prime editing. This highlights the importance of alternate PAMs and protospacers as novel features of this technology. “n.d.” in FIG. 62A is “not detected.”
• FIG. 63 shows the reduction to practice of PE using SpCas9(H840A)-VRQR and SpCas9(H840A)-VRER as the RNA-guided DNA binding protein in a prime editor system. The SpCas9(H840A)-VRQR napDNAbp is disclosed herein as SEQ ID NO: 87. The SpCas9(H840A)-VRER napDNAbp is disclosed herein as SEQ ID NO: 88. The SpCas9(H840A)-VRER-MMLV RT fusion protein is disclosed herein as SEQ ID NO: 516, wherein the MMLV RT comprises the D200N, L603W, T330P, T306K, and W313F substitutions relative to the wild type MMLV RT. The SpCas9(H840A)-VRQR-MMLV RT fusion protein is disclosed herein as SEQ ID NO: 515, wherein the MMLV RT comprises the D200N, L603W, T330P, T306K, and W313F substitutions relative to the wild type MMLV RT. Seven different loci in the human genome are targeted: 4 with the SpCas9(H840A)-VRQR-MMLV RT prime editor system and 3 with the SpCas9(H840A)-VRER-MMLV RT system. The amino acid sequences of the tested constructs are as follows:

• SACAS9-M-MLV	MKRTADGSEFESPKKKRKVGKRNYILGLDIGITSVGYGIIDYETR
  RT PRIME EDITOR	DVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQRVKK
  	LLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLA
  	KRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLE
  	RLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFID
  	TYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPE
  	ELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQIIEN
  	VFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYH
  	DIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQE
  	EIEQISNLKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKL
  	VPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYG
  	LPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGK
  	ENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHII
  	PRSVSFDNSFNNKVLVKQEEASKKGNRTPFQYLSSSDSKISYETF
  	KKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVD
  	TRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFK
  	KERNKGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMF
  	EEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKK
  	PNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLIN
  	KSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGN
  	YLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVK
  	LSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYE
  	EAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIE
  	VNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNL
  	YEVKSKKHPQIIKKGSGGSSGGSSGSETPGTSESATPESSGGSSG
  	GSSTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGL
  	AVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGIL
  	VPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVP
  	NPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWR
  	DPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDL
  	ILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQ
  	ICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREF
  	LGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEI
  	KQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRR
  	PVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVI
  	LAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVV
  	ALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHT
  	WYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAEL
  	IALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSE
  	GKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRM
  	ADQAARKAAITETPDTSTLLIENSSPSGGSKRTADGSEFEPKKKR
  	KV (SEQ ID NO: 660)
  SPCAS9(H840A)-	MKRTADGSEFESPKKKRKVDKKYSIGLDIGTNSVGWAVITDEY
  VRQR-MALONEY	KVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTAR
  MURINE	RRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKH
  LEUKEMIA VIRUS	ERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLAL
  REVERSE	AHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPIN
  TRANSCRIPTASE	ASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSL
  PRIME EDITOR	GLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADL
  	FLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTL
  	LKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPI
  	LEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAIL
  	RRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTR
  	KSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKH
  	SLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKT
  	NRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLL
  	KIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFD
  	DKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDG
  	FANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSP
  	AIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQ
  	KNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQN
  	GRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSD
  	KNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKA
  	ERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDEND
  	KLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNA
  	VVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATA
  	KYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRD
  	FATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARK
  	KDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
  	TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRK
  	RMLASARELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQ
  	KQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHR
  	DKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRSTKEVL
  	DATLIHQSITGLYETRIDLSQLGGDSGGSSGGSSGSETPGTSESAT
  	PESSGGSSGGSSTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQA
  	WAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKP
  	HIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVN
  	KRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPT
  	SQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDL
  	ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLG
  	YRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPT
  	PKTPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWG
  	PDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVL
  	TQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAG
  	KLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT
  	DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTD
  	QPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPA
  	GTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIY
  	RRRGWLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHS
  	AEARGNRMADQAARKAAITETPDTSTLLIENSSPSGGSKRTADG
  	SEFEPKKKRKV (SEQ ID NO: 661)
  SPCAS9(H840A)-	MKRTADGSEFESPKKKRKVDKKYSIGLDIGTNSVGWAVITDEY
  VRER-MALONEY	KVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTAR
  MURINE	RRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKH
  LEUKEMIA VIRUS	ERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLAL
  REVERSE	AHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPIN
  TRANSCRIPTASE	ASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSL
  PRIME EDITOR	GLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADL
  	FLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTL
  	LKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPI
  	LEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAIL
  	RRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTR
  	KSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKH
  	SLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKT
  	NRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLL
  	KIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFD
  	DKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDG
  	FANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSP
  	AIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQ
  	KNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQN
  	GRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSD
  	KNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKA
  	ERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDEND
  	KLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNA
  	VVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATA
  	KYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRD
  	FATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARK
  	KDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
  	TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRK
  	RMLASARELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQ
  	KQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHR
  	DKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKEYRSTKEVL
  	DATLIHQSITGLYETRIDLSQLGGDSGGSSGGSSGSETPGTSESAT
  	PESSGGSSGGSSTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQA
  	WAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKP
  	HIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVN
  	KRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPT
  	SQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDL
  	ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLG
  	YRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPT
  	PKTPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWG
  	PDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVL
  	TQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAG
  	KLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDT
  	DRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTD
  	QPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPA
  	GTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIY
  	RRRGWLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHS
  	AEARGNRMADQAARKAAITETPDTSTLLIENSSPSGGSKRTADG
  	SEFEPKKKRKV (SEQ ID NO: 662)

• As shown in FIG. 63 , the SpCas9(H840A)-VRQR-MMLV RT was operational at PAM sites that included “AGAG” and “GGAG”, with some editing activity at “GGAT” and “AGAT” PAM sequences. The SpCas9(H840A)-VRER-MMLV RT was operational at PAM sites that included “AGCG” and “GGCG”, with some editing activity at “TGCG.”
• The data demonstrates that prime editing may be conducted using napDNAbps which bear different PAM specificities, such as those Cas9 variant described herein.
Example 17—Introduction of Recombinase Target Sites with PE
• This Example describes a method to address genetic disease or generate tailor-made animal or plant models by using prime editing (PE) to introduce recombinase targets sites (SSR target sites) in mammalian and other genomes with high specificity and efficiency.
• This Example describes use of PE to introduce recombinase recognition sequences at high-value loci in human or other genomes, which, after exposure to site-specific recombinase(s), will direct precise and efficient genomic modifications (FIG. 64 ). In various embodiments show in FIG. 64 , PE may be used to (b) insert a single SSR target for use as a site for genomic integration of a DNA donor template. (c) shows how a tandem insertion of SSR target sites can be used to delete a portion of the genome. (d) shows how a tandem insertion of SSR target sites can be used to invert a portion of the genome. (e) shows how the insertion of two SSR target sites at two distal chromosomal regions can result in chromosomal translocation. (f) shows how the insertion of two different SSR target sites in the genome can be used to exchange a cassette from a DNA donor template. Each of the types of genome modifications are envisioned by using PE to insert SSR targets, but this list also is not meant to be limiting.
• Many large-scale genomic changes, such as gene insertions, deletions, inversions, or chromosomal translocations, are implicated in genetic disease^1-7. In addition, custom and targeted manipulation of eukaryotic genomes is important for research into human disease, as well as generation of transgenic plants^8,9 or other biotechnological products. For example, microdeletions of chromosomes can lead to disease, and replacement of these deletions by insertions of critical DNA elements could lead to a permanent amelioration of disease. In addition, diseases resulting from inversions, gene copy number changes, or chromosomal translocations could be addressed by restoring the previous gene structure in affected cells. Alternatively, in plants or other high value eukaryotic organisms used in industry, introduction of recombinant DNA or targeted genomic rearrangements could lead to improved products, for example crops which require fewer resources or are resistant to pathogens. Current technologies for effecting large-scale genomic changes rely on random or stochastic processes, for example the use of transposons or retroviruses, while other desired genomic modifications have only been achieved by homologous recombination strategies.
• One appealing class of proteins for accomplishing targeted and efficient genomic modification is site-specific recombinases (SSRs). SSRs have a long history of being used as a tool for genomic modification^10-13. SSRs are considered promising tools for gene therapy because they catalyze the precise cleavage, strand exchange, and rejoining of DNA fragments at defined recombination targets¹⁴ without relying on the endogenous repair of double-strand breaks which can induce indels, translocations, other DNA rearrangements, or p53 activation^15-18. The reactions catalyzed by SSRs can result in the direct replacement, insertion, or deletion of target DNA fragments with efficiencies exceeding those of homology-directed repair^14,19.
• Although SSRs offer many advantages, they are not widely used because they have a strong innate preference for their cognate target sequence. The recognition sequences of SSRs are typically ≥20 base pairs and thus unlikely to occur in the genomes of humans or model organisms. Further, the native substrate preferences of SSRs are not easily altered, even with extensive laboratory engineering or evolution²⁰. This limitation is overcome by using PE to directly introduce recombinase targets into the genome, or to modify endogenous genomic sequences which natively resemble recombinase targets. Subsequent exposure of the cell to recombinase protein will permit precise and efficient genomic modification directed by the location and orientation of the recombinase target(s) (FIG. 64 ).
• PE-mediated introduction of recombinase targets could be particularly useful for the treatment of genetic diseases which are caused by large-scale genomic defects, such as gene loss, inversion, or duplication, or chromosomal translocation^1-7 (Table 6). For example, Williams-Beuren syndrome is a developmental disorder caused by a deletion of 24 in chromosome 721. No technology exists currently for the efficient and targeted insertion of multiple entire genes in living cells (the potential of PE to do such a full-length gene insertion is currently being explored but has not yet been established); however, recombinase-mediated integration at a target inserted by PE offers one approach towards a permanent cure for this and other diseases. In addition, targeted introduction of recombinase recognition sequences could be highly enabling for applications including generation of transgenic plants, animal research models, bioproduction cell lines, or other custom eukaryotic cell lines. For example, recombinase-mediated genomic rearrangement in transgenic plants at PE-specific targets could overcome one of the bottlenecks to generating agricultural crops with improved properties8,9.
• A number of SSR family members have been characterized and their target sequences described, including natural and engineered tyrosine recombinases (TABLE 7), large serine integrases (TABLE 8), serine resolvases (TABLE 9), and tyrosine integrases (TABLE 10). Modified target sequences that demonstrate enhanced rates of genomic integration have also been described for several SSRs^22-30. In addition to natural recombinases, programmable recombinases with distinct specificities have been developed^31-40. Using PE, one or more of these recognition sequences could be introduced into the genomic at a specified location, such as a safe harbor locus^41-43, depending on the desired application.
• For example, introduction of a single recombinase target in the genome would result in integrative recombination with a DNA donor template (FIG. 64 b ). Serine integrases, which operate robustly in human cells, may be especially well-suited for gene integration^44,45. Additionally, introduction of two recombinase targets could result in deletion of the intervening sequence, inversion of the intervening sequence, chromosomal translocation, or cassette exchange, depending on the identity and orientation of the targets (FIG. 64 c-f ). By choosing endogenous sequences that already closely resemble recombinase targets, the scope of editing required to introduce the complete recombinase target would be reduced.
• Finally, several recombinases have been demonstrated to integrate into human or eukaryotic genomes at natively occurring pseudosites^46-64. PE editing could be used to modify these loci to enhance rates of integration at these natural pseudosites, or alternatively, to eliminate pseudosites that may serve as unwanted off-target sequences.
• This report describes a general methodology for introducing recombinase target sequences in eukaryotic genomes using PE, the applications of which are nearly limitless. The genome editing reactions are intended for use with “prime editor,” a chimeric fusion of a CRISPR/Cas9 protein and a reverse-transcriptase domain, which utilizes a custom prime editing guide RNA (PEgRNA). By extension, Cas9 tools and homology-directed repair (HDR) pathways may also be exploited to introduce recombinase targets through DNA templates by lowering the rates of indels using several techniques^65-67. A proof-of-concept experiment in human cell culture is shown in FIG. 65 .

• TABLE 6
  Examples of genetic diseases linked to large-scale genomic
  modifications.

  Disease	Source	Cause
  Trisomy 17p	68	Gene duplication
  Charcot-Marie-Tooth	69	Gene duplication
  Smith-Magenis syndrome	70	Gene deletion
  Williams-Beuren	21	Gene deletion
  De la Chapelle syndrome	71	Chromosomal translocation
  Down syndrome (some	72	Chromosomal translocation
  Hemophilia A	73	Gene inversion
  Hunter syndrome	74	Gene inversion

• TABLE 7
  Tyrosine recombinases and SSR target sequences.

  cRecombinase	Source	Target	Name
  Cre	75	ATAACTTCGTATAGCATACATTATACG	loxP
  		AAGTTAT (SEQ ID NO: 517)
  Dre	76	TAACTTTAAATAATGCCAATTATTTAA	rox
  		AGTTA (SEQ ID NO: 518)
  VCre	77	TCAATTTCTGAGAACTGTCATTCTCGG	loxV
  		AAATTGA (SEQ ID NO: 519)
  SCre	77	CTCGTGTCCGATAACTGTAATTATCGG	loxS
  		ACATGAT (SEQ ID NO: 520)
  Flp	78	GAAGTTCCTATTCTCTAGAAAGTATAG	FRT
  		GAACTTC (SEQ ID NO: 521)
  B2	79	GAGTTTCATTAAGGAATAACTAATTCC	loxB
  		CTAATGAAACTC (SEQ ID NO: 522)
  B3	79	GGTTGCTTAAGAATAAGTAATTCTTAA	loxB3
  		GCAACC (SEQ ID NO: 523)
  Kw	80	ACGAAAAATGGTAAGGAATAGACCAT
  		TCCTTACCATTTTTGGT (SEQ ID NO: 524)
  R	81	TTGATGAAAGAATAACGTATTCTTTCA	RS
  		TCAA (SEQ ID NO: 525)
  TD1-40	82	GTGCGTCAAATAATAACGTATTATTTG	TDRS
  		ACACTT (SEQ ID NO: 526)
  Vika	83	AATAGGTCTGAGAACGCCCATTCTCAG	vox
  		ACGTATT (SEQ ID NO: 527)
  Nigri	84	TGAATGTCCTATAATTACACTTATAGG	nox
  		ACATTCA (SEQ ID NO: 528)
  Panto	84	GAAACTTTAAATAATAAGTCTTATTTA	pox
  		AAGTTTC (SEQ ID NO: 259)
  Kd	79	AAACGATATCAGACATTTGTCTGATA	loxK
  		ATGCTTCATTATCAGACAAATGTCTG
  		ATATCGTTT (SEQ ID NO: 530)
  Fre	85	ATATATACGTATATAGACATATATACG	loxH
  		TATATAT (SEQ ID NO: 531)
  CreALSHG	86	ATAACTCTATATAATGTATGCTATATA	loxM7
  		GAGTTAT(SEQ ID NO: 532)
  Tre	87	ACAACATCCTATTACACCCTATATGCC	loxLTR
  		AACATGG (SEQ ID NO: 533)
  Brec1	12	AACCCACTGCTTAAGCCTCAATAAAGC	loxBTR
  		TTGCCTT (SEQ ID NO: 534)
  Cre-R3M3	88	GATACAACGTATATACCTTTCTATACG	loxK2
  		TTGTTTA (SEQ ID NO: 535)

• TABLE 8
  Large serine integrases and SSR target sequences.

  Integrase	Source	Left Target	Right Target

  Bxb1	89	GGTTTGTCTGGTCAACCACC	GGCTTGTCGACGACGGCGG
  		GCGGTCTCAGTGGTGTACGG	TCTCCGTCGTCAGGATCAT
  		TACAAACC (SEQ ID NO: 536)	(SEQ ID NO: 537)
  phiC31	90	GTGCCCCAACTGGGGTAACC	TGCGGGTGCCAGGGCGTGC
  		TTTGAGTTCTCTCAGTTGGG	CCTTGGGCTCCCCGGGCGCG
  		GG (SEQ ID NO: 538)	TACTCC (SEQ ID NO: 539)
  R4	91	TGTTCCCCAAAGCGATACCA	GCATGTTCCCCAAAGCGATA
  		CTTGAAGCAGTGGTACTGCT	CCACTTGAAGCAGTGGTACT
  		TGTGGGTACA (SEQ ID NO:	GCTTGTGGGTACACTCTGCG
  		540)	GGTG (SEQ ID NO: 541)
  phiBT1	92	GGTGCTGGGTTGTTGTCTCT	CAGGTTTTTGACGAAAGTGA
  		GGACAGTGATCCATGGGAA	TCCAGATGATCCAG (SEQ ID
  		ACTACTCAGCACC (SEQ ID	NO: 543)
  		NO: 542)
  MJ1	93	ATTTTAGGTATATGATTTTGT	CAAAGGATCACTGAATCAA
  (phiFC1)		TTATTAGTGTAAATAACACT	AAGTATTGCTCATCCACGCG
  		ATGTACCTAAAAT (SEQ ID	AAA (SEQ ID NO: 545)
  		NO: 544)
  MR11	94	TTTGTGCGGAACTACGAACA	CGAAAATGTATGGAGGCAC
  		GTTCATTAATACGAAGTGTA	TTGTATCAATATAGGATGTA
  		CAAACTTCCATACAA	TACCTTCGAAGACACTT
  		(SEQ ID NO: 546)	(SEQ ID NO: 547)
  TP901-1	95	GAGTTTTTATTTCGTTTATTT	ATGCCAACACAATTAACATC
  		CAATTAAGGTAACTAAAAA	TCAATCAAGGTAAATGCTTT
  		ACTCCTTTTAAGG (SEQ ID	TTGCTTTTTTTGC (SEQ ID
  		NO: 548)	NO: 549)
  A118	96	TTCCTCGTTTTCTCTCGTTGG	TTTCGGATCAAGCTATGAAG
  		AAGAAGAAGAAACGAGAAA	GACGCAAAGAGGGAACTAA
  		(SEQ ID NO: 550)	A (SEQ ID NO: 551)
  U153	97	TTCCTCGTTTTCTCTCGTTGG	TTTCGGATCAAGCTATGAAG
  		ACGGAAACGAATCGAGAAA	GACGCAAAGAGGGAACTAA
  		(SEQ ID NO: 552)	A (SEQ ID NO: 553)
  phiRV1	98	GTAGTGTATCTCACAGGTCC	GAAGGTGTTGGTGCGGGGT
  		ACGGTTGGCCGTGGACTGCT	TGGCCGTGGTCGAGGTGGG
  		GAAGAACATTCC (SEQ ID	GT (SEQ ID NO: 555)
  		NO: 554)
  phi370.1	99	AAAAAAATACAGCGTTTTTC	TTGTAAAGGAGACTGATAA
  		ATGTACAACTATACTAGTTG	TGGCATGTACAACTATACTC
  		TAGTGCCTAAAA (SEQ ID	GTCGGTAAAAAGGCA (SEQ
  		NO: 556)	ID NO: 557)
  TG1	100	TCCAGCCCAACAGTGTTAGT	GATCAGCTCCGCGGGCAAG
  		CTTTGCTCTTACCCAGTTGG	ACCTTTCTCCTTCACGGGGT
  		GCGGGA (SEQ ID NO: 558)	GGAAGGTC (SEQ ID NO: 559)
  WB	101	CTAGTTTTAAAGTTGGTTAT	CGGAAGGTAGCGTCAACGA
  		TAGTTACTGTGATATTTATC	TAGGTGTAACTGTCGTGTTT
  		ACGGTACCCAATAACCAATG	GTAACGGTACTTCCAACAGC
  		AAT (SEQ ID NO: 560)	TGGCGCCGCCAC (SEQ ID
  			NO: 561)
  BL3	102	CAATGAAAAACTAGGCATGT	TTTCCACAGACAACTCACGT
  		AGAAGTTGTTTGT (SEQ ID	GGAGGTAGTCAC (SEQ ID
  		NO: 562)	NO: 563)
  SprA	103	TGTAGTAAGTATCTTAATAT	CACCCATTGTGTTCACAGGA
  		ACAGCTTTATCTGTTTTTTAA	GATACAGCTTTATCTGTACT
  		GATACTTACTACTTT (SEQ ID	GATATTAATGACATGCTG
  		NO: 564)	(SEQ ID NO: 565)
  phiJoe	104	AGTTGTGGCCATGTGTCCAT	ATCTGGATGTGGGTGTCCAT
  		CTGGGGGCAGATGGAGACG	CTGCGGGCAGACGCCGCAG
  		TCGAAGCACGG (SEQ ID	GGGTCACA (SEQ ID NO:
  		NO: 566)	567)
  phiK38	105	CCCTAATACGCAAGTCGATA	GAGCGCCGGATCAGGGAGT
  		ACTCTCCTGGGAGCGTTGAC	GGACGGCCTGGGAGCGCTA
  		AACTTGCGCACCCTGATCTG	CACGCTGTGGCTGCGGTCGG
  		(SEQ ID NO: 569)	TGC (SEQ ID NO: 570)
  Int2	105	GCTCATGTATGTGTCTACGC	GGACGGCGCAGAAGGGGAG
  		GAGATTCTCGCCCGAGAACT	TAGCTCTTCGCCGGACCGTC
  		TCTGCAAGGCACTGCTCTTG	GACATACTGCTCAGCTCGTC
  		GCT (SEQ ID NO: 571)	(SEQ ID NO: 572)
  Int3	105	ATGGATAAAAAAATACAGC	GTTTGTAAAGGAGACTGAT
  		GTTTTTCATGTACAACTATA	AATGGCATGTACAACTATAC
  		CTAGTTGTAGTGCCTAAATA	TCGTCGGTAAAAAGGCATCT
  		ATGCTT (SEQ ID NO: 573)	TAT (SEQ ID NO: 574)
  Int4	105	AAAAATTACAAAGTTTTCAA	TTCCAAAGAGCGCCCAACG
  		CCCTTGATTTGAATTAGCGG	CGACCTGAAATTTGAATAA
  		TCAAATAATTTGTAATTCGT	GACTGCTGCTTGTGTAAAGG
  		TT (SEQ ID NO: 575)	CGATGATT (SEQ ID NO: 576)
  Int7	105	GTGTTATAAACCTGTGTGAG	AGACGAGAAACGTTCCGTC
  		AGTTAAGTTTACATGCCTAA	CGTCTGGGTCAGTTGGGCAA
  		CCTTAACTTTTACGCAGGTT	AGTTGATGACCGGGTCGTCC
  		CAGCTT(SEQ ID NO: 577)	GTT (SEQ ID NO: 578)
  Int8	105	TTAATAAACTATGGAAGTAT	CAATCATCAGATAACTATGG
  		GTACAGTCTTGCAATGTTGA	CGGCACGTGCATTAACCAC
  		GTGAACAAACTTCCATAATA	GGTTGTATCCCGTCTAAAGT
  		AAAT (SEQ ID NO: 579)	ACTCGT (SEQ ID NO: 580)
  Int9	105	GTGGTTGTTTTTGTTGGAAG	TTTATATTGCGAAAAATAAT
  		TGTGTATCAGGTATCTGCAT	TGGCGAACGAGGTAACTGG
  		AGTTATTCCGAACTTCCAAT	ATACCTCATCCGCCAATTAA
  		TA (SEQ ID NO: 581)	AATTTG (SEQ ID NO: 582)
  Int10	105	GGAAAATATAAATAATTTTA	AGCACGCTGATAATCAGCA
  		GTAACCTACATCTCAATCAA	AGACCACCAACATTTCCACC
  		GGATAGTAAAACTCTCACTC	AATGTAAAAGCTTTAACCTT
  		TT (SEQ ID NO: 583)	AGC (SEQ ID NO: 584)
  Int11	105	GTTTATATGTTTACTAATAA	ATGGATTTTGCAGATTCCCA
  		GACGCTCTCAACCCATAAAG	GATGCCCCTACAGAAAGAG
  		TCTTATTAGTAAACATATTT	GTACAAAACATTTATTGGAA
  		CAACT (SEQ ID NO: 585)	TTAATT (SEQ ID NO: 586)
  Int12	105	TTTTTGTATGTTAGTTGTGTC	GTTCGTGGTAACTATGGGTG
  		ACTGGGTAGACCTAAATAGT	GTACAGGTGCCACATTAGTT
  		GACACAACTGCTATTAAAAT	GTACCATTTATGTTTATGTG
  		TTAA (SEQ ID NO: 587)	GTTAAC (SEQ ID NO: 588)
  Int13	105	CAATAACGGTTGTATTTGTA	GCATACATTGTTGTTGTTTT
  		GAACTTGACCAGTTGTTTTA	TCCAGATCCAGTTGGTCCTG
  		GTAACATAAATACAACTCCG	TAAATATAAGCAATCCATGT
  		AATA (SEQ ID NO: 589)	GAGT (SEQ ID NO: 590)
  LI	106	GTTTAGTATCTCGTTATCTCT	TAACTTTTTCGGATCGAGTT
  		CGTTGGAGGGAGAAGAAAC	ATGATGGACGTAAAGAGGG
  		GGGATACCAAAA (SEQ ID	AACAAAGCATCTA (SEQ ID
  		NO: 591)	NO: 592)
  Peaches	107	TAGTTTCCAATGTTACAGGA	CGGTCTCCATCGGGATCTGC
  		ACTGCTGGCAGAATCCAACA	TGATCGAGCAGCATGCCGA
  		CATTGGAAGTCG (SEQ ID	CCA (SEQ ID NO: 594)
  		NO: 593)
  Bxz2	107	TAACCGCAAGTGTACATCCC	CGGTCTCCATCGGGATCTGC
  		TCGGCTGGCCGAGACAAGTA	TGATCGAGCAGCATGCCGA
  		CAGTTGCGACAG (SEQ ID	CCA (SEQ ID NO: 596)
  		NO: 595)
  SV1	108	ATGTGGTCCTTTAGATCCAC	CATCAGGGCGGTCAGGCCG
  		TGACGTGGGTCAGTGTCTCT	TAGATGTGGAAGAAACGGC
  		AAAGGACTCGCG (SEQ ID	AGCACGGCGAGGACG
  		NO: 597)	(SEQ ID NO: 598)

• TABLE 9
  Serine resolvases and SSR target sequences.

  Resolvase	Source	Left Target	Right Target
  Gin	109	CGTTTCCTGTAAACCGAGGT	CGTTTCCTGTAAACCGAGGT
  		TTTGGATAAACA	TTTGGATAATGG (SEQ ID
  		(SEQ ID NO: 599)	NO: 600)
  Cin	110	GAGTTCTCTTAAACCAAGGT	GAGTTCTCTTAAACCAAGGT
  		TTAGGATTGAAA (SEQ ID	ATTGGATAACAG (SEQ ID
  		NO: 601)	NO: 602)
  Hin	111	TGGTTCTTGAAAACCAAGGT	AAATTTTCCTTTTTGGAAGG
  		TTTTGATAAAGC (SEQ ID	TTTTTGATAACCA (SEQ ID
  		NO: 603)	NO: 604)
  Min	112	GCCTTCCCCTAAACCAACGT	GCCTTCCCCCAAACCAAGGT
  		TTTTATGCCGCC	AATCAAGAACGC
  		(SEQ ID NO: 605)	(SEQ ID NO: 606)
  Sin	113	TTGTGAAATTTGGGTACACC	CGTATGATTAGGGTGTATAT
  		CTAATCATACAA (SEQ ID	TAATTT (SEQ ID NO: 608)
  		NO: 607)

• TABLE 10 Tyrosine integrases and target sequences.

  Integrase	Source	attP	attB
  HK022
  	114	CAAATGATTTTATTTTGACTAATAA	GCACTTTAGGTGA
  		TGACCTACTTACATTAATTTACTGAT	AAAAGGTT
  		AATTAAAGAGATTTTAAATATACAA	(SEQ ID NO: 610)
  		CTTATTCACCTAAAGGATGACAAAA
  		(SEQ ID NO: 609)
  P22	115	CTAAGTGGTTTGGGACAAAAATGGG	GCAGCGCATTCGT
  		ACATACAAATCTTTGCATCGGTTTG	AATGCGAAGGTCG
  		CAAGGCTTTGCATGTCTTTCGAAGA	T (SEQ ID NO: 613)
  		TGGGACGTGTGAGCGCAGGTATGAC
  		GTGGTATGTGTTGACTTAAAAGGTA
  		GTTCTTATAATTCGTAATGCGAAGG
  		TCGTAGGTTCGACTCCTATTATCGG
  		CACCAGTTAAATCAAATACTTACGT
  		ATTATTCGTGCCTTCCTTATTTTTAC
  		TGTGGGACATATTTGGGACAGAAGT
  		ACCAAAAA (SEQ ID NO: 612)
  L5	116	GCGATCCCCATCCGCGACGTGCCAA	GAGCGGGCGACG
  		CTAGGTCTCCTCTCGTCGTGAACAA	GGAATCGAACCCG
  		GGCTACCGGGTTGCAACTCCTGTGC	CGTAGCTAGTTTG
  		AACTCTCAGGCTTCAACGCGCTTCT	GAAGA (SEQ ID
  		ACGACCTGCAATTTCTTTCCACTTA	NO: 615)
  		GAGGATGCAGCCGAGAGGGGTAAA
  		AACCTATCTTGACCGGCCCATATGT
  		GGTCGGCAGACACCCATTCTTCCAA
  		ACTAGCTACGCGGGTTCGATTCCCG
  		TCGCCCGCTCCGCTGGTCAGAGGGT
  		GTTTTCGCCCTCTGGCCATTTTTCTT
  		TCCAGGGGTCTGCAACTCTTGTGCG
  		ACTCTTCTGACCTGGGCATACGCGG
  		TTGCAACGCATCCCTGATCTGGCTA
  		CTTTCGATGCTGACAAACGAATAGA
  		GCCCCCCGCCTGCGCGAACAGACG
  		AGGGGCATTCACA (SEQ ID
  		NO: 614)

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Example 18—Incorporation of 3′ Toeloop in the Primer Binding Site (PBS) Improves PEgRNA Activity
• In order to further improve PE activity, the inventors contemplated adding a toeloop sequence at the 3′ end of a PEgRNA having a 3′ extension arm. FIG. 71A provides an example of a generic SpCas9 PEgRNA having a 3′ extension arm (top molecule). The 3′ extension arm, in turn, comprises an RT template (that includes that the desired edit) and a primer binding site (PBS) at the 3′ end of the molecule. The molecule terminates with a poly(U) sequence comprising three U nucleobases (i.e., 5′-UUU-3′).
• By contrast, the bottom portion of FIG. 71A shows the same PEgRNA molecule as the top portion of FIG. 71A, but wherein a 9-nucleobase sequence of 5′-GAAANNNNN-3′ has been inserted between the 3′ end of the primer binding site and the 5′ end of the terminal poly(U) sequence. This structure folds back on itself by 180° to form a “toeloop” RNA structure, wherein the sequences of 5′-NNNNN-3′ of the 9-nucleobase insertion anneals with a complementary sequence in the primer binding site, and wherein the 5′-GAAA-3′ portion forms the 180° turn. The features of the toeloop sequence depicted in FIG. 71A is not intended to limit or narrow the scope of possible toeloops that could be used in its place. Further, the sequence of the toeloop will depend upon the complementary sequence of the primer binding site. Essentially though, the toeloop sequence, in various embodiments, may have a first sequence portion that forms a 180°, and a second sequence portion that has a sequence that is commentary to a portion of the primer binding site.
• Without being bound by theory, the toeloop sequence is thought to enable PEgRNA the use of PEgRNAs with increasingly longer primer binding sites than would otherwise be possible. Longer PBS sequences, in turn, are thought to improve PE activity. PEgRNA More in particular, the likely function of the toeloop is to occlude or at least minimize the PBS from interacting with the spacer. Stable hairpin formation between the PBS and the spacer can lead to an inactive PEgRNA. Without a toeloop, this interaction may require restricting the length of the PBS. Blocking or minimizing the interaction between the spacer and the PBS using a 3′ end toeloop may lead to an improvement in PE activity.
Example 19—Prime Editing with Alternative Nucleic Acid Templates and Editor Protein Constructs
• Prior to this example, prime editing is described as requiring a PEgRNA. Exemplary embodiments describing possible configurations of suitable PEgRNA for use in prime editing are depicted in FIG. 3A (a PEgRNA with a 5′ extension arm), FIG. 3B (a PEgRNA with a 3′ extension arm), FIG. 3C (an internally extended PEgRNA), FIG. 3D (a PEgRNA with a 3′ extension arm, and comprising a primer binding site, edit template, homology arm, and optional 3′ and 5′ modifier regions, and a region indicated as the DNA synthesis template), and FIG. 3E (a PEgRNA with a 5′ extension arm, and comprising a primer binding site, edit template, homology arm, and optional 3′ and 5′ modifier regions, and a region indicated as the DNA synthesis template). In addition, PEgRNA structure and composition are described extensively herein in the Detailed Description and throughout.
• This Example describes additional design variations of PEgRNAs—in some cases, PEgRNAs which are wholly or partially chemically synthesized outside the cell—that are envisioned to work in conjunction with the prime editors of this Specification. Such alternative designs may improve various aspects of prime editing, including the insertion of longer DNA sequences by prime editing, the use of alternative polymerases (i.e., alternatives to reverse transcriptase) that potentially operate with increased efficiency and/or fidelity, and the use or recruitment of alternative and/or addition prime editor protein effector components to enhance or augment prime editing. In addition, the use of chemically synthesized PEgRNAsPEgRNA may potentially lead to the production of molecules that are more stable and possess desirable features that enhance prime editing efficiency and capabilities.
• PEgRNAPEgRNAPEgRNAA PEgRNA serves as the nucleic acid template that encodes the desired edited genetic information that is to be incorporated into a target site. In one aspect, a PEgRNA is created by adding an extension arm to either the 5′ end or 3′ end of an sgRNA (e.g., as shown in the embodiments of FIG. 3A, 3B, 3D, or 3E), or by inserting a similar sequence internally within an sgRNA (e.g., as shown in the embodiment of FIG. 3C), wherein the extension arm comprises a DNA synthesis template that is capable of encoding a ssDNA product by a polymerase (e.g., a reverse transcriptase) and which includes the edited genetic information of interest. The extension arm comprises a primer binding site (PBS) for annealing to the napDNAbp-nicked genomic DNA strand, and a DNA synthesis template to encode t the edited DNA strand of interest, which becomes incorporated into the endogenous DNA target site by replacing the counterpart DNA strand. PEgRNA can be expressed within cells from plasmid DNA or a genomically integrated DNA cassette, or they can be made outside of cells by in vitro transcription or by chemical synthesis and subsequently delivered into cells. Preparation of PEgRNAs outside of cells, particularly by chemical synthesis, offers an opportunity to modify the PEgRNAs substantially. This invention describes alternative designs for prime editing templates (FIG. 72 ).
• (A) DNA Synthesis Template Expressed as a Separate Molecules from Guide RNAs (i.e., DNA Synthesis Template Provided in Trans to the Prime Editor Complex (napDNAbp+Guide RNA).
• In various embodiments described herein, prime editing utilizes a single PEgRNA that serves as both the programmable targeting molecules and the edit-encoding molecule. This embodiment is depicted in FIG. 72(a) with a PEgRNA having a 3′ extension arm. However, in some cases, this could be disadvantageous, particularly for more complex PEgRNA molecules such as those that encode a large insertion. These RNAs could contain extensive secondary structure that interferes with the PEgRNA scaffold structure and interactions with Cas9. Alternatively, prime editing can be carried out by substituting a PEgRNA with two separate RNA molecules: an sgRNA, and a trans prime editing RNA template (tPERT), as depicted in FIG. 72(b). The sgRNA serves to target Cas9 (or more generally, the napDNAbp) to the desired genomic target site, while the tPERT is used by the polymerase (e.g., a reverse transcriptase) to write new DNA sequence into the target locus.
• In general, simple expression of a tPERT leads to lower editing efficiency compared to a PEgRNA. However, the efficiency of trans prime editing can be enhanced by the introduction of one or more MS2 RNA aptamers into the tPERT RNA, along with a fusion of the MS2 coat protein (MS2cp) to the prime editor protein (to make MS2cp-Cas9-RT). This allows for the MS2 RNA aptamer to bind to the MS2cp, thereby co-localizing the tPERT (which comprises the DNA synthesis template) to the site of editing by the prime editor complex. The MS2 aptamer is preferably placed on the 3′ end of the tPERT to avoid reverse transcription of the aptamer sequence.
• Although this example utilizes the MS2 tagging technique (comprising the MS2 RNA aptamer on the tPERT paired with the MS2cp protein fused to the prime editor), other RNA-protein recruitment systems can be used in the alternative. The general concept envisioned here is that the DNA synthesis template of the tPERT is modified to contain an RNA recruitment secondary structure (e.g., a specialized hairpin like the MS2 aptamer) so that the tPERT may be recruited by a modified prime editor fusion protein that further comprises an RNA-binding protein that specifically recognizes and binds to the RNA recruitment second structure on the tPERT molecule. A review of other RNA-protein recruitment domains are described in the art, for example, in Johansson et al., “RNA recognition by the MS2 phage coat protein,” Sem Virol., 1997, Vol. 8(3): 176-185; Delebecque et al., “Organization of intracellular reactions with rationally designed RNA assemblies,” Science, 2011, Vol. 333: 470-474; Mali et al., “Cas9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering,” Nat. Biotechnol., 2013, Vol. 31: 833-838; and Zalatan et al., “Engineering complex synthetic transcriptional programs with CRISPR RNA scaffolds,” Cell, 2015, Vol. 160: 339-350, each of which are incorporated herein by reference in their entireties. Other systems include the PP7 hairpin, which specifically recruits the PCP protein, and the “corn” hairpin, which specifically recruits the Com protein. See Zalatan et al. Any of these well-known recruitment systems may be employed with trans prime editing as described herein.
• The efficiency of the instant tPERT trans prime editing system was tested. Up to 20% efficiency of His6 insertion (18 bp) was achieved at the HEK3 site in HEK293T cells using a tPERT containing a single 3′ MS2 aptamer, a 13-nt primer binding site, and an RT template containing the insertion sequence and 34 nt of homology to the locus, along with an editor containing the MS2cp fused to the N-terminus of PE2. See FIG. 73 . The strategy of trans prime editing has the potential to address complications associated with PEgRNAPEgRNA design, and could be more suited for longer RT templates to achieve larger insertion, deletions, or edits at further distances from the prime editor nick site.
• (B) Chemically Synthesized PEgRNAs with RNA and DNA Synthesis Templates
• Alternative nucleic acid templates can be used within chemically synthesized PEgRNAs (FIG. 72 c ). For example, a synthetic PEgRNA can be constructed as an RNA/DNA hybrid wherein the spacer sequence and sgRNA scaffold is composed of RNA nucleotides and the primer binding site and synthesis template (shown as a 3′ extension in FIG. 72 c ) is composed of DNA nucleotides. This could allow for DNA-dependent DNA polymerases to be used in place of reverse transcriptase within prime editors. It could also prevent the synthesis of DNA that is templated by sgRNA scaffold sequence. In other designs, chemical linkers, composed of non-templating nucleotides or other suitable linker moieties, can be used to tether the nucleic acid edit template (composed of RNA or DNA) to the sgRNA scaffold. This could prevent continued DNA polymerization of the sgRNA scaffold and allow for flexibility in the extension that allows for more efficient templated synthesis. Finally, the directionality of the nucleic acid synthesis template can be inverted such that DNA polymerization proceeds away from the sgRNA scaffold as opposed to toward it.
(C) Recruitment of the DNA Polymerase Expressed in Trans
• In the main embodiment of prime editing, the polymerase (e.g., reverse transcriptase enzyme) is expressed as a fusion to the napDNAbp (e.g., Cas9 nickase). Alternatively, the polymerase (e.g., reverse transcriptase) can be expressed in trans, and its activity can be localized to the editing site using recruitment systems such as the MS2 RNA aptamer and MS2 coat protein, or other similar recruitment system known in the art. In this system, the PEgRNA is modified to include an MS2 aptamer within one of the sgRNA scaffold hairpins, and the polymerase (e.g., reverse transcriptase) is expressed as a fusion protein to MS2cp. The napDNAbp (e.g., Cas9 nickase) is also expressed as an independent polypeptide. This system has been demonstrated with the wild type M-MLV reverse transcriptase (FIG. 74 ), and should be applicable to other RT variants. In addition, other RNA-protein interactions, or protein-protein interactions, could be used for RT recruitment.
• The following sequences are pertinent to Example 19:

• Sequences of tPERTs:
  MS2 aptamer/RT template/PBS/Linker
  5′-MS2_13nt-PBS:
  (SEQ ID NO: 762)
  5′GCCAACATGAGGATCACCCATGTCTGCAGGGCC TGGAGGAAGCAGGGCTTCC
  TTTCCTCTGCCATCAATGATGGTGATGATGGTGCGTGCTCAGTCTG-3′
  5′-MS2_17nt-PBS:
  (SEQ ID NO: 773)
  5′GCCAACATGAGGATCACCCATGTCTGCAGGGCC TGGAGGAAGCAGGGCTTCC
  TTTCCTCTGCCATCAATGATGGTGATGATGGTGCGTGCTCAGTCTGGGCC-3′
  3′-MS2_13nt-PBS:
  (SEQ ID NO: 774)
  5′GGAGGAAGCAGGGCTTCCTTTCCTCTGCCATCAATGATGGTGATGATGGT
  GCGTGCTCAGTCTG AAATTAACAAATCAAGCCAACATGAGGATCACCCATGTCT
  GCAGGGCC-3′
  3′-MS2_17nt-PBS:
  (SEQ ID NO: 775)
  5′GGAGGAAGCAGGGCTTCCTTTCCTCTGCCATCAATGATGGTGATGATGGT
  GCGTGCTCAGTCTGGGCC AAATTAACAAATCAAGCCAACATGAGGATCACCCAT
  GTCTGCAGGGCC-3′
  Sequences of MS2 PEgRNAs:
  Spacer/MS2 aptamer/sgRNA scaffold/RT template/PBS
  HEK3_MS2_1
  (SEQ ID NO: 776)
  5′ GGCCCAGACTGAGCACGTGA GTTTTAGAGCTAG GCCAACATGAGGATCACCCA
  TGTCTGCAGGGCCTAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTG
  GGACCGAGTCGGTCC TCTGCCATCTCGTGCTCAGTCT-3′
  HEK3_MS2_2
  (SEQ ID NO: 777)
  5′ GGCCCAGACTGAGCACGTGA GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGC
  TAGTCCGTTATCAACTTG GCCAACATGAGGATCACCCATGTCTGCAGGGCC AAGTG
  GGACCGAGTCGGTCC TCTGCCATCTCGTGCTCAGTCT-3′
  Protein sequences:
  MS2cp-PE2
  (SEQ ID NO: 778)
  MKRTADGSEFESPKKKRKVGSASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISS
  NSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVATQTVGGEELPVAGWRSYLNMELT
  IPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIYSGGSSGGSSGSETPGTSESATP
  ESSGGSSGGSSDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALL
  FDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKH
  ERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLN
  PDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGL
  FGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLS
  DAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGY
  AGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELH
  AILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVV
  DKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSG
  EQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDK
  DFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSR
  KLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIA
  NLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIE
  EGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSF
  LKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERG
  GLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRK
  DFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEI
  GKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMP
  QVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSP7'VAYSVLVVAKVEK
  GKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRM
  LASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQIS
  EFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRY
  TSTKEVLDATLIHQSITGLYETRIDLSQLGGDSGGSSGGSSGSETPGTSESATPESSGGSSGG
  SSTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVS
  IKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNK
  RVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGIS
  GQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGT
  RALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQ
  LREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGL
  PDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAV
  LTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVA
  LNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKA
  GAAVITETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIY
  RRRGWLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARK
  AAITETPDTSTLLIENSSPSGGSKRTADGSEFEPKKKRKV
  MS2cp-MMLV-RT
  (SEQ ID NO: 779)
  MKRTADGSEFESPKKKRKVGSASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISS
  NSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVATQTVGGEELPVAGWRSYLNMELT
  IPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIYSGGSSGGSSGSETPGTSESATP
  ESSGGSSGGSSTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLII
  PLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRP
  VQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFE
  WRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAAT
  SELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMG
  QPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQ
  ALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPP
  CLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTD
  RVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSL
  LQEGQRKAGAAVITETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFA
  TAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRM
  ADQAARKAAITETPDTSTLLIENSSPSGGSKRTADGSEFEPKKKRKV
  MMLV-RT-MS2cp
  (SEQ ID NO: 780)
  MKRTADGSEFESPKKKRKVTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGM
  GLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPV
  KKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLH
  PTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQ
  YVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLT
  EARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQ
  QKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLD
  PVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTH
  YQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADH
  TWYTDGSSLLQEGQRKAGAAVITETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLN
  VYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKG
  HSAEARGNRMADQAARKAAITETPDTSTLLIENSSPSGGSSGGSSGSETPGTSESATPESS
  GGSSGGSSGSASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCS
  VRQSSAQNRKYTIKVEVPKVATQTVGGEELPVAGWRSYLNMELTIPIFATNSDCELIV
  KAMQGLLKDGNPIPSAIAANSGIYSGGSKRTADGSEFEPKKKRKV

Example 20. Split-Intein Delivery of Prime Editors
• This Example demonstrates that a prime editor may be split using inteins as a means to deliver the prime editor to cells via separate vectors, wherein each vector encodes a portion of the prime editor fusion protein. This Example is focused on a PE fusion protein comprising the canonical SpCas9 (SEQ ID NO: 18). The split sites for other Cas9 proteins may be the corresponding same site, or may need to be optimized for each different Cas9 protein. In the instant Example, the prime editor was split between residues 1023 and 1024 of SpCas9 (SEQ ID NO: 18). This is referred to as the “1023/1024” split site.
• Prime editors (PEs) exceed the AAV packaging capacity. It was therefore contemplated to split the PE by inserting a trans-splicing Npu intein at S. pyogenes Cas9 residue 1024 in SEQ ID NO: 18, allowing delivery of split-SpPEs as two separate polypeptides, each encoded by one of a dual-AAV system. The split site 1023/1024 was chosen because it (1) allows packaging into two AAVs while accommodating space for guide cassette(s) and minimal regulatory elements, (2) mutation of the native Serine to Cysteine would be relatively conservative, (3) the site is a flexible loop near the periphery of Cas9 which sterically is predicted to allow for spicing to occur, and (4) Cas9 has been successfully altered in this loop by circular permutation (but not previously at this specific split site).
• To determine whether Npu-split prime editors are active, HEK cells were transfected with plasmids encoding split-editors, finding they recapitulate activity of full-length PE3 as analyzed by high-throughput sequencing. Furthermore, the three native Npu amino-terminal residues of C-terminal extein are known to splice most efficiently but differ from those natively flanking Cas9 residue 1024. Replacement of these Cas9 residues with the native Npu residues may alter the activity of prime editor. It was therefore determined whether mutation from the Cas9-native “SEQ” towards the Npu intein “CFN” sequence altered efficiency of prime editing. The “SEQ” residues facilitate prime editing with similar efficiency to full-length, suggesting the intein-split PE halves are able to associate and mediate prime editing. No further increase was seen by mutation towards “CFN”, suggesting that association alone may be sufficient for split-PE activity, as we have previously observed with intein-split base editors.
• Although associated, unspliced editors may be active, perturbations of the sterics of the prime editor resulting from incomplete splicing during initiation steps may affect editing outcomes. 1024-CFN was therefore chosen for further studies as it also recapitulates full-length PE3 activity.
• The following are the amino acid sequences of the 1023/1024 split.

• SpPE2 split at 1023/1024 N terminal half

  (SEQ ID NO: 3875)

  MKRTADGSEFESPKKKRKVDKKYSIGLDIGTNSVGWAVITDEYKVPSKKF

  KVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYL

  QEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKY

  PTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVD

  KLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGE

  KKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI

  GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDL

  TLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEK

  MDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPF

  LKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVV

  DKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTE

  GMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISG

  VEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMI

  EERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDF

  LKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPA

  IKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMK

  RIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINR

  LSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNY

  WRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVA

  QILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYH

  SpPE2 split at 1023/1024 C terminal half

  SEQ ID NO: 3876)

  EIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLI

  ARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIME

  RSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGEL

  QKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEII

  EQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGA

  SLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQE

  ARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVN

  KRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFA

  FEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLIL

  LQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYL

  GYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGKAGFCRLFIPGFAE

  MAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELF

  VDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVL

  TKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDR

  VQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADH

  TWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQA

  LKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGWLTSEGKEIKNKDEILA

  LLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTST

Prime Editor Packages in a Dual AAV System
• Prime editors split between residues 1023 and 1024 with Npu trans-splicing intein become packages within AAV with high titer comparable to that of base editors of similar genome size produced in the same manner.
Prime Editing In Vivo
• Split SpPE3 delivered with AAV9 by intracerebroventricular injection to P0 mice mediates prime editing in brain tissue in vivo. A nucleotide substitution at position +5 (i.e., 5 nucleotide bases downstream of nick site and within the PAM sequence) of a G-to-T results in the installation of a Pro>G1n coding mutation near the N terminus of DNMT1. This edit demonstrates the viability of prime editing in vivo and is not thought to introduce any selective pressure on edited cells (with “+5” referring to the +5 position downstream of the nick site). The AAV architectures tested include a full-length MMLV RT as well as a truncated variant lacking the RNAse H domain. The truncated post-transcriptional regulatory element W3 was also assessed as it has been shown to increase expression from viral cassettes in vivo but its importance had not been tested in the context of base editors or prime editors. It was found that full-length RT outperforms truncated RT, and the addition of the W3 sequence improves activity. The increase in editing activity when W3 is present indicates that expression of prime editor is limiting in vivo and demonstrates the distinct challenges of prime editing in vivo versus cell culture.
REFERENCES
• Oakes, B. L., Fellmann, C. et al. CRISPR-Cas9 Circular Permutants as Programmable Scaffolds for Genome Modification. Cell. 2019 Jan. 10; 176(1-2):254-267.e16. doi: 10.1016/j.cell.2018.11.052.
Example 21. Linker Optimization in PE2 Format
• This Example constructed a number of variant prime editors all based on PE2. PE2 has the following sequence and structure:
• As used herein, “PE2” refers to a PE complex comprising a fusion protein comprising Cas9(H840A) and a variant MMLV_RT having the following structure: [NLS]-[Cas9(H840A)]-[linker]-[MMLV_RT(D200N)(T330P)(L603W)(T306K)(W313F)]+a desired PEgRNA, wherein the PE fusion has the amino acid sequence of SEQ ID NO: 134, which is shown as follows:

• (SEQ ID NO: 134)

  MKRTADGSEFESPKKKRKV DKKYSIGLDIGTNSVGWAVITDEYKVPSKKF

  KVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYL

  QEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKY

  PTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVD

  KLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGE

  KKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI

  GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDL

  TLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEK

  MDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPF

  LKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVV

  DKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTE

  GMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISG

  VEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMI

  EERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDF

  LKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPA

  IKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMK

  RIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINR

  LSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNY

  WRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVA

  QILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYH

  HAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKA

  TAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT

  VRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYG

  GFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFL

  EAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKY

  VNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVIL

  ADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTID

  RKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD SGGSSGGSSGSETP

  GTSESATPESSGGSSGGSS TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQ

  AWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRL

  LDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPN

  PYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISG

  QLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAAT

  SELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLT

  EARKETVMGQPTPKTPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPG

  TLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVL

  TQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQP

  LVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPA

  TLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQE

  GQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVY

  TDSRYAFATAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPKRLS

  IIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP SGGS

  KRTADGSEFEPKKKRKV

  KEY:

  NUCLEAR LOCALIZATION SEQUENCE (NLS) TOP: (SEQ ID

  NO: 124), BOTTOM: (SEQ ID NO: 133)

  CAS9(H840A) (SEQ ID NO: 137)

  33-AMINO ACID LINKER (SEQ ID NO: 127)

  M-MLV reverse transcriptase (SEQ ID NO: 139).

  The PE2 linker is SGGSSGGSSGSETPGTSESATPESSGGSSGG

  SS (SEQ ID NO: 127).

• In this experiment, the linker of PE2 was replaced with one of the following substitute linkers (or no linker in one instance):

• Linker Name	Nucleotide Sequence	Amino Acid Sequence
  1x SGGS	TCCGGAGGATCT (SEQ ID NO: 3880)	SGGS (SEQ ID NO: 3887)
  2x SGGS	TCCGGAGGATCTAGCGGAGGCTCC	SGGSSGGS (SEQ ID NO: 3888)
  	(SEQ ID NO: 3881)
  3x SGGS	TCCGGAGGATCTAGCGGAGGCTCCA	SGGSSGGSSGGS (SEQ ID NO:
  	GCGGAGGCAGC (SEQ ID NO: 3882)	3889)
  1x XTEN	TCTGGCTCTGAGACACCTGGCACAAG	SGSETPGTSESATPES (SEQ ID
  	CGAGAGCGCAACACCTGAAAGC	NO: 3890)
  	(SEQ ID NO: 3883)
  No linker
  1x Gly	GGT	G
  1x Pro	CCC	P
  1x	GAAGCAGCTGCTAAA (SEQ ID NO:	EAAAK (SEQ ID NO: 3891)
  EAAAK	3884)
  2x	GAAGCCGCTGCTAAAGAAGCTGCAG	EAAAKEAAAK (SEQ ID NO:
  EAAAK	CTAAG (SEQ ID NO: 3885)	3892)
  3x	GAAGCCGCTGCTAAAGAGGCCGCTG	EAAAKEAAAKEAAAK (SEQ ID
  EAAAK	CTAAAGAAGCTGCAGCTAAG	NO: 3893)
  	(SEQ ID NO: 3886)

• FIG. 79 shows the editing efficiency of the replacement linker constructs. In particular, the data shows the editing efficiency of the PE2 construct with the current linker (noted as PE2—white box) compared to various versions with the linker replaced with a sequence as indicated at the HEK3, EMX1, FANCF, RNF2 loci for representative PEgRNAs for transition, transversion, insertion, and deletion edits. The replacement linkers are referred to as “1×SGGS”, “2×SGGS”, “3×SGGS”, “1×XTEN”, “no linker”, “1× Gly”, “1× Pro”, “1× EAAAK”, “2×EAAAK”, and “3×EAAAK”. The editing efficiency is measured in bar graph format relative to the “control” editing efficiency of PE2. The linker of PE2 is SGGSSGGSSGSETPGTSESATPESSGGSSGGSS (SEQ ID NO: 127). All editing was done in the context of the PE3 system, i.e., which refers the PE2 editing construct plus the addition of the optimal secondary sgRNA nicking guide.
• FIG. 80 . shows that the 1×XTEN linker provided an increase in editing efficiency. Taking the average fold efficacy relative to PE2 yields the graph shown, indicating that use of a 1×XTEN linker sequence improves editing efficiency by 1.14 fold on average (n=15).
Example 22. Prime Editor Guide RNAs with Improved Activities
• In various embodiments, PEgRNA are likely to be target DNA site and edit dependent. That is, the sequence of the PEgRNA will depend upon the target DNA sequence and the particular edit that is being introduced therein by prime editing (e.g., deletion, insertion, inversion, substitution). For instance, in certain embodiments, when attaching a motif 3′ of the primer binding site (PBS) of the PEgRNA, a linker between the PBS and motif is preferred to prevent steric clash with the polymerase domain (e.g., reverse transcriptase) of the PE fusion protein. However, the nature of that linker may be different for each site. For instance, if the same linker for each site were used, it could artificially render a 13 nt PBS a 16 nt PBS by fortuitous pairing to the spacer sequence. Similarly, the linker could basepair to the PBS itself, resulting in its occlusion and potentially reducing activity. So, unlike with protein-based editors, such as PE or BE4, a single linker sequence option connecting two elements may not be effective in each construct but will depend in part on the sequence of the target DNA sequence and the edit of interest.
• Building, in part, on the information presented Example 15 (Design and Engineering of PEgRNAs), this Example constructed and then tested the effects of various structural modifications made to PEgRNA on editing function, among other aspects.
• Expression of PEgRNAs from Non-Pol III Promoters
• A variety of PEgRNA expression systems were tested for their ability to generate PEgRNAs, using insertion of a 102 nucleotide sequence from FKBP as a readout.
• Transcription of PEgRNA can be directed by a typical constitutive promoter, such as U6 promoter. Although the U6 promoter is in most cases effective at directing transcription of PEgRNAs, the U6 promoter is not very effective at directing the transcription of longer PEgRNAs or U-rich RNAs. U-rich RNA stretches of cause premature termination of transcription. This Example compared editing outcomes of guides expressed from the CMV promoter or U1 promoter with the U6 promoter. These promoters require a different terminator sequence, such as MASC ENE or PAN ENE, as provided below. An increase in editing was observed with the pCMV/MASC-ENE system, however these guides resulted in incomplete insertion of the sequence, while, with the U6 promoter, complete insertion was observed at lower levels of editing. See FIG. 81 . The data suggests the likelihood that the alternate expression systems may be useful for long insertions.
• The nucleotide sequence of the pCMV/MASC-ENE expression systems as follows (5′-to-3′ direction) (with the name of the motif in bold immediately preceding the region to which it refers):

• -pCMV promoter-
  TAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAG
  TTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACC
  CCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGAC
  TTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTA
  CATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAAT
  GGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCA
  GTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTAC
  ATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCA
  TTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATG
  TCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGA
  GGTCTATATAAGCAGAGCTGGTTTAGTGAACCGTCAGATC-Csy4 loop-
  GTTCACTGCCGTATAGGCAG-spacer-GGCCCAGACTGAGCACGTGA-scaffold-
  GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA
  AAAGTGGGACCGAGTCGGTCC-template-
  TGGAGGAAGCAGGGCTTCCTTTCCTCTGCCATCA-insert-
  AAATTTCTTTCCATCTTCAAGCATCCCGGTGTAGTGCACCACGCAGGTCTGGCCG
  CGCTTGGGGAAGGTGCGCCCGTCTCCTGGGGAGATGGTTTCCACCTGCACTCC-
  PBS-CGTGCTCAGTCTG-linker-TTT-MASC ENE-
  TAGGGTCATGAAGGTTTTTCTTTTCCTGAGAAAACAACACGTATTGTTTTCTCAGG
  TTTTGCTTTTTGGCCTTTTTCTAGCTTAAAAAAAAAAAAAGCAAAAGATGCTGGT
  GGTTGGCACTCCTGGTTTCCAGGACGGGGTTCAAATCCCTGCGGCGTCTTTGCTTT
  GACT-unrelated plasmid sequence-
  TTTTTTTAAGCTTGGGCCGCTCGAGGTAGCAGC-Ubc promoter-
  GGCCTCCGCGCCGGGTTTTGGCGCCTCCCGCGGGCGCCCCCCTCCTCACGGCGAG
  CGCTGCCACGTCAGACGAAGGGCGCAGGAGCGTTCCTGATCCTTCCGCCCGGAC
  GCTCAGGACAGCGGCCCGCTGCTCATAAGACTCGGCCTTAGAACCCCAGTATCA
  GCAGAAGGACATTTTAGGACGGGACTTGGGTGACTCTAGGGCACTGGTTTTCTTT
  CCAGAGAGCGGAACAGGCGAGGAAAAGTAGTCCCTTCTCGGCGATTCTGCGGAG
  GGATCTCCGTGGGGCGGTGAACGCCGATGATTATATAAGGACGCGCCGGGTGTG
  GCACAGCTAGTTCCGTCGCAGCCGGGATTTGGGTCGCGGTTCTTGTTTGTGGATC
  GCTGTGATCGTCACTTGGTGAGTTGCGGGCTGCTGGGCTGGCCGGGGCTTTCGTG
  GCCGCCGGGCCGCTCGGTGGGACGGAAGCGTGTGGAGAGACCGCCAAGGGCTGT
  AGTCTGGGTCCGCGAGCAAGGTTGCCCTGAACTGGGGGTTGGGGGGAGCGCACA
  AAATGGCGGCTGTTCCCGAGTCTTGAATGGAAGACGCTTGTAAGGCGGGCTGTG
  AGGTCGTTGAAACAAGGTGGGGGGCATGGTGGGCGGCAAGAACCCAAGGTCTTG
  AGGCCTTCGCTAATGCGGGAAAGCTCTTATTCGGGTGAGATGGGCTGGGGCACC
  ATCTGGGGACCCTGACGTGAAGTTTGTCACTGACTGGAGAACTCGGGTTTGTCGT
  CTGGTTGCGGGGGCGGCAGTTATGCGGTGCCGTTGGGCAGTGCACCCGTACCTTT
  GGGAGCGCGCGCCTCGTCGTGTCGTGACGTCACCCGTTCTGTTGGCTTATAATGC
  AGGGTGGGGCCACCTGCCGGTAGGTGTGCGGTAGGCTTTTCTCCGTCGCAGGACG
  CAGGGTTCGGGCCTAGGGTAGGCTCTCCTGAATCGACAGGCGCCGGACCTCTGGT
  GAGGGGAGGGATAAGTGAGGCGTCAGTTTCTTTGGTCGGTTTTATGTACCTATCT
  TCTTAAGTAGCTGAAGCTCCGGTTTTGAACTATGCGCTCGGGGTTGGCGAGTGTG
  TTTTGTGAAGTTTTTTAGGCACCTTTTGAAATGTAATCATTTGGGTCAATATGTAA
  TTTTCAGTGTTAGACTAGTAAATTGTCCGCTAAATTCTGGCCGTTTTTGGCTTTTT
  TGTTAGACAGGATCCCCGGGTACCGGTCGCCACC-Csy4 and NLS-
  ATGGGCTCTTTTACTATGGACCACTACCTGGATATTAGACTGAGACCTGACCCTG
  AGTTCCCACCCGCCCAGCTGATGAGCGTGCTGTTCGGCAAGCTGCACCAGGCCCT
  GGTGGCACAGGGAGGCGACCGGATCGGCGTGAGCTTCCCCGACCTGGATGAGAG
  CAGATCCAGGCTGGGAGAGCGCCTGAGGATCCACGCATCCGCCGACGATCTGCG
  CGCCCTGCTGGCCCGGCCATGGCTGGAGGGCCTGCGCGACCACCTGCAGTTTGG
  AGAGCCAGCAGTGGTGCCACACCCTACCCCATACAGGCAGGTGTCCAGGGTGCA
  GGCAAAGTCTAACCCTGAGCGGCTGCGGAGAAGGCTGATGCGCCGGCACGATCT
  GTCTGAGGAGGAGGCCAGAAAGAGGATCCCCGACACCGTGGCCAGAACACTGG
  ATCTGCCTTTCGTGACCCTGCGGAGCCAGAGCACAGGCCAGCACTTCAGACTGTT
  TATCAGGCACGGCCCACTGCAGGTGACAGCCGAGGAAGGAGGATTCACTTGTTA
  CGGACTGTCTAAAGGAGGATTCGTGCCCTGGTTCAGCAGCCTGAGGCCTCCTAAG
  AAGAAGAGGAAGGTTTAA-SV40 terminator-
  TGATCATAATCAAGCCATATCACATCTGTAGAGGTTTACTTGCTTTAAAAAACCT
  CCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACT
  TGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCAC
  AAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATG
  TATCTTATCATGTCTGGATCTGC.
  Key:
  [pCMV promoter]-binds pol II RNA polymerase
  [Csy4 loop]-bound by Csy4 protein, results in cleavage 3′ of
  the loop. Required because part of [CMV promoter]is
  transcribed, and if this sequence is attached 5′ of the gRNA
  it will lower/eliminate activity (previously known).
  [Spacer sequence]of pegRNA
  [pegRNA scaffold]
  [DNA synthesis template]
  [insertion edit (108 nt from FKBP)]
  [primer binding site]
  [Linker](highly variable)-connects PBS and terminator element
  [MASC ENE transcription terminator]-transcription of this
  element results in termination of transcription; a polyA
  tail is encoded and then sequestered by the ENE element
  [Unimportant sequence]
  [Ubc promoter]-required for expression of the Csy4 protein
  [Csy4 protein and NLS]-required for processing of the 5′ end
  of the guide. Other strategies could also be used that don't
  require expression of a large protein (such as ribozyme-
  mediated cleavage of the spacer), but these would require more
  individual tuning for differentspacer sequences, so we used this.
  [SV40 terminator]-for termination of the Csy4 protein.

Improvements to the PEgRNA Scaffold
• A number of structural modifications to the gRNA scaffold were also tested, none of which showed a significant increase in editing activity (see FIG. 82 at 3.30.13 through 3.30.19 in the X axis, as compare to 3.30). However, this data has two caveats worth noting. First, this guide already worked quite well, and a less effective guide would have been better to test. Second, in HEK cells, transfection is quite efficient, and it was noted that the amount of guide RNA transfected is in large excess compared to what is needed (reducing the amount by ˜4-8 fold has no effect on editing). These improvements might only be seen in other cell types, where transfection efficiency is lower, or with less effective guides. Many of these changes are precedented to improve sgRNA activity in other cell lines.
• The sequences of the constructs of FIG. 82 are as follows:

• HEK3.30 pegRNA sequence:
  [SEQ ID NO: 3873]
  spacer-GGCCCAGACUGAGCACGUGA-scaffold-
  GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACU
  UGAAAAAGUGGCACCGAGUCGGUGC-Templateand PBS-
  UCUGCCAUCAAAGCGUGCUCAGUCUG-Terminal motif-UUUU
  HEK3.30.0 pegRNA sequence:
  [SEQ ID NO: 3874]
  spacer-GGCCCAGACUGAGCACGUGA-scaffold-
  GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACU
  UGAAAAAGUGGCACCGAGUCGGUGC-Template and PBS-
  UCUGCCAUCAAAGCGUGCUCAGUCUG-Terminal motif-UUUUUU
  HEK3.30.1 pegRNA sequence:
  [SEQ ID NO: 3875]
  spacer-GGCCCAGACUGAGCACGUGA-scaffold-
  GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACU
  UGAAAAAGUGGCACCGAGUCGGUGC-Template and PBS-
  UCUGCCAUCAAAGCGUGCUCAGUCUG-Terminal motif-[none]
  HEK3.30.2 pegRNA sequence:
  [SEQ ID NO: 3876]
  spacer-GGCCCAGACUGAGCACGUGA-scaffold-
  GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACU
  UGAAAAAGUGGCACCGAGUCGGUGC-Template and PBS-
  UCUGCCAUCAAAGCGUGCUCAGUCUG-Terminal motif-
  UUUGCUCGAGGCGGAAACGCCUCGAGCUUUU
  HEK3.30.2b pegRNA sequence:
  [SEQ ID NO: 3877]
  spacer-GGCCCAGACUGAGCACGUGA-scaffold-
  GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACU
  UGAAAAAGUGGCACCGAGUCGGUGC-Template and PBS-
  UCUGCCAUCAAAGCGUGCUCAGUCUG-Terminal motif-
  UUUGCUCGAGGCGGAAACGCCUCGAGC
  HEK3.30.3 pegRNA sequence:
  [SEQ ID NO: 3878]
  spacer-GGCCCAGACUGAGCACGUGA-scaffold-
  GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACU
  UGAAAAAGUGGCACCGAGUCGGUGC-Template and PBS-
  UCUGCCAUCAAAGCGUGCUCAGUCUG-Terminal motif-
  UUUGCUCGAGGCGUACGCGAAAGCGUACGCCUCGAGCUUUU
  HEK3.30.3b pegRNA sequence:
  [SEQ ID NO: 3879]
  spacer-GGCCCAGACUGAGCACGUGA-scaffold-
  GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACU
  UGAAAAAGUGGCACCGAGUCGGUGC-Template and PBS-
  UCUGCCAUCAAAGCGUGCUCAGUCUG-Terminal motif-
  UUUGCUCGAGGCGUACGCGAAAGCGUACGCCUCGAGC
  HEK3.30.5 pegRNA sequence:
  [SEQ ID NO: 3880]
  spacer-GGCCCAGACUGAGCACGUGA-scaffold-
  GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACU
  UGAAAAAGUGGCACCGAGUCGGUGC-Template and PBS-
  UCUGCCAUCAAAGCGUGCUCAGUCUG-Terminal motif-
  UUUGCUCGAGGCGUACGCCCGAUGAAAAUCGGGCGUACGCCUCGAGCUUUU
  HEK3.30.5a pegRNA sequence:
  [SEQ ID NO: 3881]
  spacer-GGCCCAGACUGAGCACGUGA-scaffold-
  GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACU
  UGAAAAAGUGGCACCGAGUCGGUGC-Template and PBS-
  UCUGCCAUCAAAGCGUGCUCAGUCUG-Terminal motif-
  UUUUGGGGUUGGGGUUGGGGUUGGGGUUUU
  HEK3.30.5b pegRNA sequence:
  [SEQ ID NO: 3882]
  spacer-GGCCCAGACUGAGCACGUGA-scaffold-
  GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACU
  UGAAAAAGUGGCACCGAGUCGGUGC-Template and PBS-
  UCUGCCAUCAAAGCGUGCUCAGUCUG-Terminal motif-
  UUUGGUGGUGGUGGUUUU
  HEK3.30.13 pegRNA sequence:
  [SEQ ID NO: 3883]
  spacer-GGCCCAGACUGAGCACGUGA-scaffold-
  GUUUUAGAGCUAGCGAAAGCUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUC
  AACUUGAAAAAGUGGGACCGAGUCGGUCC-Template and PBS-
  UCUGCCAUCAAAGCGUGCUCAGUCUG-Terminal motif-UUUU
  HEK3.30.15 pegRNA sequence:
  [SEQ ID NO: 3884]
  spacer-GGCCCAGACUGAGCACGUGA-scaffold-
  GUUUUAGAGCUAGCUCGAAAGAGCUAGCAAGUUAAAAUAAGGCUAGUCCGU
  UAUCAACUUGAAAAAGUGGGACCGAGUCGGUCC-Template and PBS-
  UCUGCCAUCAAAGCGUGCUCAGUCUG-Terminal motif-UUUU
  HEK3.30.15 pegRNA sequence:
  [SEQ ID NO: 3885]
  spacer-GGCCCAGACUGAGCACGUGA-scaffold-
  GUUUUAGAGCUAGCUCAUGAAAAUGAGCUAGCAAGUUAAAAUAAGGCUAGU
  CCGUUAUCAACUUGAAAAAGUGGGACCGAGUCGGUCC-Template and PBS-
  UCUGCCAUCAAAGCGUGCUCAGUCUG-Terminal motif-UUUU
  HEK3.30.16 pegRNA sequence:
  [SEQ ID NO: 3886]
  spacer-GGCCCAGACUGAGCACGUGA-scaffold-
  GUUUUAGAGCUAGCUCAUCCGAAAGGAUGAGCUAGCAAGUUAAAAUAAGGC
  UAGUCCGUUAUCAACUUGAAAAAGUGGGACCGAGUCGGUCC-Template and
  PBS-UCUGCCAUCAAAGCGUGCUCAGUCUG-Terminal motif-UUUU
  HEK3.30.17 pegRNA sequence:
  [SEQ ID NO: 3887]
  spacer-GGCCCAGACUGAGCACGUGA-scaffold-
  GUUUUAGAGCUAGCUCAUCCUGGAAACAGGAUGAGCUAGCAAGUUAAAAUA
  AGGCUAGUCCGUUAUCAACUUGAAAAAGUGGGACCGAGUCGGUCC-Template
  and PBS-UCUGCCAUCAAAGCGUGCUCAGUCUG-Terminal motif-UUUU
  HEK3.30.18 pegRNA sequence:
  [SEQ ID NO: 3888]
  spacer-GGCCCAGACUGAGCACGUGA-scaffold-
  GUUUGAGAGCUAGAAAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACU
  UGAAAAAGUGGGACCGAGUCGGUCC-Template and PBS-
  UCUGCCAUCAAAGCGUGCUCAGUCUG-Terminal motif-UUUU
  HEK3.30.19 pegRNA sequence:
  [SEQ ID NO: 3889]
  spacer-GGCCCAGACUGAGCACGUGA-scaffold-
  GUUUGAGAGCUAGCUCAUGAAAAUGAGCUAGCAAGUUUAAAUAAGGCUAGU
  CCGUUAUCAACUUGAAAAAGUGGGACCGAGUCGGUCC-Template and PBS-
  UCUGCCAUCAAAGCGUGCUCAGUCUG-Terminal motif-UUUU
  HEK3.56 pegRNA sequence:
  [SEQ ID NO: 3890]
  spacer-GGCCCAGACUGAGCACGUGA-scaffold-
  GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACU
  UGAAAAAGUGGCACCGAGUCGGUGC-Template and PBS-
  UCUGCCAUCAAAGCUUCGACCGUGCUCAGUCUG-Terminal motif-UUUU
  HEK3.56.1a pegRNA sequence:
  [SEQ ID NO: 3891]
  spacer-GGCCCAGACUGAGCACGUGA-scaffold-
  GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACU
  UGAAAAAGUGGCACCGAGUCGGUGC-Template and PBS-
  UCUGCCAUCAGGCGAAAGCCUCGUGCUCAGUCUG-Terminal motif-UUUU
  HEK3.56.1b pegRNA sequence:
  [SEQ ID NO: 3892]
  spacer-GGCCCAGACUGAGCACGUGA-scaffold-
  GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACU
  UGAAAAAGUGGCACCGAGUCGGUGC-Template and PBS-
  UCUGCCAUCAGACGAAAGCCUCGUGCUCAGUCUG-Terminal motif-UUUU
  HEK3.56.1c pegRNA sequence:
  [SEQ ID NO: 3892]
  spacer-GGCCCAGACUGAGCACGUGA-scaffold-
  GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACU
  UGAAAAAGUGGCACCGAGUCGGUGC-Template and PBS-
  UCUGCCAUCAGGCGAAAGCCCGUGCUCAGUCUG-Terminal motif-UUUU
  HEK3.56.2a pegRNA sequence:
  [SEQ ID NO: 3893]
  spacer-GGCCCAGACUGAGCACGUGA-scaffold-
  GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACU
  UGAAAAAGUGGCACCGAGUCGGUGC-Template and PBS-
  UCUGCCAUCAGAUGCGAAAGCAUCUCGUGCUCAGUCUG-Terminal motif-UUUU
  HEK3.56.2b pegRNA sequence:
  [SEQ ID NO: 3894]
  spacer-GGCCCAGACUGAGCACGUGA-scaffold-
  GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACU
  UGAAAAAGUGGCACCGAGUCGGUGC-Template and PBS-
  UCUGCCAUCAGAUGCGAAAGCACCUCGUGCUCAGUCUG-Terminal motif-UUUU
  HEK3.56.2c pegRNA sequence:
  [SEQ ID NO: 3895]
  spacer-GGCCCAGACUGAGCACGUGA-scaffold-
  GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACU
  UGAAAAAGUGGCACCGAGUCGGUGC-Template and PBS-
  UCUGCCAUCAGAUGCGAAAGCAUCCGUGCUCAGUCUG-Terminal motif-UUUU
  HEK3.56.3a pegRNA sequence:
  [SEQ ID NO: 3896]
  spacer-GGCCCAGACUGAGCACGUGA-scaffold-
  GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACU
  UGAAAAAGUGGCACCGAGUCGGUGC-Template and PBS-
  UCUGCCAUCAGACAUGCGAAAGCAUGUCUCGUGCUCAGUCUG-Terminal motif-
  UUUU
  HEK3.56.3b pegRNA sequence:
  [SEQ ID NO: 3897]
  spacer-GGCCCAGACUGAGCACGUGA-scaffold-
  GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACU
  UGAAAAAGUGGCACCGAGUCGGUGC-Template and PBS-
  UCUGCCAUCAGACAUGCGAAAGCAGGCCCGUGCUCAGUCUG-Terminal motif-
  UUUU
  HEK3.56.3c pegRNA sequence:
  [SEQ ID NO: 3898]
  spacer-GGCCCAGACUGAGCACGUGA-scaffold-
  GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACU
  UGAAAAAGUGGCACCGAGUCGGUGC-Template and PBS-
  UCUGCCAUCAGACAUGCGAAAGCAUGUCUCGUGCUCAGUCUG-Terminal motif-
  UUUU
  HEK3.56.4a pegRNA sequence:
  [SEQ ID NO: 3899]
  spacer-GGCCCAGACUGAGCACGUGA-scaffold-
  GUUUUAGAGCUAUACGUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACU
  UUACGAAGUGGGACCGAGUCGGUCC-Template and PBS-
  UCUGCCAUCAAAGCUUCGACCGUGCUCAGUCUG-Terminal motif-UUUU
  HEK3.56.4b pegRNA sequence:
  [SEQ ID NO: 3922]
  5′motif-
  GCAGACCUAAGUGGUGACAUAUGGUCUG-spacer-
  GGCCCAGACUGAGCACGUGA-scaffold-
  GUUUUAGAGCUAUACGUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACU
  UUACGAAGUGGGACCGAGUCGGUCC-Template and PBS--Terminal motif-UUUU
  HEK3.56.4c pegRNA sequence:
  [SEQ ID NO: 3900]
  5′motif-
  GCAGACCUAAGUGGUGACAUAUGGUCUG-spacer-
  GGCCCAGACUGAGCACGUGA-scaffold-
  GUUUUAGAGCUAUACGUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACU
  UUACGAAGUGGGACCGAGUCGGUCC-Template and PBS--Terminal motif-UUUU
  HEK3.56.4d pegRNA sequence:
  [SEQ ID NO: 3901]
  5′motif-
  GCAGACCUAAGUGGUGACAUAUGGUCUG-spacer-
  GGCCCAGACUGAGCACGUGA-scaffold-
  GUUUUAGAGCUAUACGUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACU
  UUACGAAGUGGGACCGAGUCGGUCC-Template and PBS--Terminal motif-UUUU

  
  Note that where ever either no terminal motif or a terminal motif that does not end in a run of U's exists, transcript was terminated using the following HDV ribozyme:

• [SEQ ID NO: 3923]

  GGCCGGCAUGGUCCCAGCCUCCUCGCUGGCGCCGGCUGGGCAACAUGCUU

  CGGCAUGGCGAAUGGGAC

Additional RNA Motifs
• See FIG. 82 for details on certain motifs, such as an HDV ribozyme 3′ of the PEgRNA, or G-quadruplex insertion, P1 extensions, template hairpins, and tetraloop circ'd, that may be introduced into a PEgRNA to improve its performance.
• In particular, this Example tested the effect of installing a tRNA motif 3′ of the primer binding site. This element was chosen because of multiple potential functions:
• (1) the tRNA motif is a very stable RNA motif, and so could potentially reduce PEgRNA degradation;
• (2) the MMLV RT uses a prolyl-tRNA as a primer when converting the viral genome into DNA during transcription, so it was suspected the same cap could be bound by the RT, improving binding of the PEgRNA by PE, RNA stability, and bringing the PBS back in closer proximity to the genomic site, potentially also improving activity.
• In these constructs, the P1 of the tRNA (see FIG. 84 ) was extended. P1 refers to the first stem/base-pairing element of the tRNA (see FIG. 84 ). This was believed to be necessary to prevent RNAseP-mediated cleavage of the tRNA 5′ of the P1, which would result in its removal from the PEgRNA.
• In this design a prolyl-tRNA (codon CGG) with an extended P1 and short 3 nt linker between the tRNA and the PBS was used. A variety of tRNA designs were tested and the editing efficiency was tested compared to a PEgRNA having no tRNA cap—see the comparative data in FIG. 83 (depicting a PE experiment that targeted editing of the HEK3 gene, specifically targeting the insertion of a 10 nt insertion at position +1 relative to the nick site and using PE3), FIG. 85 (depicting a PE experiment that targeted editing of the FANCF gene, specifically targeting a G-to-T conversion at position +5 relative to the nick site and using PE3 construct) and FIG. 86 (depicting a PE experiment that targeted editing of the HEK3 gene, specifically targeting the insertion of a 71 nt FLAG tag insertion at position +1 relative to the nick site and using PE3 construct). tRNA-modified PEgRNAs were tested against a non-modified PEgRNA control.
• UGG/CGG refers to the codon used, the number refers to the length of the added P1 extension, long indicates an 8 nt linker, no designation a 3 nt linker.
• The data suggest that the installation of a tRNA may enable use of shorter PBSs, which would likely result in additional activity improvements. In the case of RNF2, it is possible/likely that the linker used resulted in improved PBS binding to the spacer, and the resulting diminishment in activity.
• Some sequences used:

• HEK3 +1 FLAG-tag insertion, proly-tRNA {UGG} P1

  ext

  5 nt, linker 3 nt

  [SEQ ID NO: 3902]

  GGCCCAGACUGAGCACGUGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAU

  AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUGGA

  GGAAGCAGGGCUUCCUUUCCUCUGCCAUCACUUAUCGUCGUCAUCCUUGU

  AAUCCGUGCUCAGUCUGUCUGGCGGGGCUCGUUGGUCUAGGGGUAUGAUU

  CUCGCUUCGGGUGCGAGAGGUCCCGGGUUCAAAUCCCGGACGAGCCCCGC

  CUUUU

  FANCF +5 G to T proly-tRNA {CGG } P1 ext 5 nt,

  linker 3 nt

  [SEQ ID NO: 3903]

  GGAAUCCCUUCUGCAGCACCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAU

  AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCGGAA

  AAGCGAUCAAGGUGCUGCAGAAGGGAUCUGGCGGGGCUCGUUGGUCUAGG

  GGUAUGAUUCUCGCUUCGGGUGCGAGAGGUCCCGGGUUCAAAUCCCGGAC

  GAGCCCCGCCUUUU

  HEK3 ++1 10 nt insertion, proly-tRNA {UGG} P1

  ext

  5 nt, linker 3 nt

  [SEQ ID NO: 3904]

  GGCCCAGACUGAGCACGUGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAU

  AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGGACCGAGUCGGUCCUCUG

  CCAUCAAAGCUUCGACCGUGCUCAGUCUUCUGCUCGAGGCGGGGCUCGUU

  GGUCUAGGGGUAUGAUUCUCGCUUCGGGUGCGAGAGGUCCCGGGUUCAAA

  UCCCGGACGAGCCCCGCCUCGAGCUUUU

• The sequences reported in the data of FIGS. 85 and 86 are as follows:

• FANCF +5 G to T pegRNA sequence: space,

  scaffold template and PBSr-

  [SEQ ID NO: 3905]

  GGAAUCCCUUCUGCAGCACCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAU

  AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCGGAA

  AAGCGAUCAAGGUGCUGCAGAAGGGA-partial CGG tRNA linker

  8-UCUCUCUCUGGUCUAGGGGUAUGAUUCUCGCUUCGGGUGCGAGAGGUC

  CCGGGUUCAAUUUU

  FANCF +5 G to T pegRNA sequence: space,

  scaffold template and PBSr-

  [SEQ ID NO: 3906]

  GGAAUCCCUUCUGCAGCACCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAU

  AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCGGAA

  AAGCGAUCAAGGUGCUGCAGAAGGGA-UGG P1 ext 5 linker 3-U

  CUGGCGGGGCUCGUUGGUCUAGGGGUAUGAUUCUCGCUUUGGGUGCGAGA

  GGUCCCGGGUUCAAAUCCCGGACGAGCCCCGCCUUUU

  FANCF +5 G to T pegRNA sequence: space,

  scaffold template and PBSr-

  [SEQ ID NO: 3907]

  GGAAUCCCUUCUGCAGCACCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAU

  AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCGGAA

  AAGCGAUCAAGGUGCUGCAGAAGGGA-UGG P1 ext 5 linker 8-U

  CUCUCUCGGCGGGGCUCGUUGGUCUAGGGGUAUGAUUCUCGCUUUGGGUG

  CGAGAGGUCCCGGGUUCAAAUCCCGGACGAGCCCCGCCUUUU

  FANCF +5 G to T pegRNA sequence: space,

  scaffold template and PBSr-

  [SEQ ID NO: 3908]

  GGAAUCCCUUCUGCAGCACCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAU

  AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCGGAA

  AAGCGAUCAAGGUGCUGCAGAAGGGA-UGG P1 ext 8 linker 3-U

  CUCGAGGCGGGGCUCGUUGGUCUAGGGGUAUGAUUCUCGCUUUGGGUGCG

  AGAGGUCCCGGGUUCAAAUCCCGGACGAGCCCCGCCUCGUUUU

  FANCF +5 G to T pegRNA sequence: space,

  scaffold template and PBSr-

  [SEQ ID NO: 3909]

  GGAAUCCCUUCUGCAGCACCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAU

  AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCGGAA

  AAGCGAUCAAGGUGCUGCAGAAGGGA-UGG P1 ext 8 linker 8-U

  CUCUCUCCGAGGCGGGGCUCGUUGGUCUAGGGGUAUGAUUCUCGCUUUGG

  GUGCGAGAGGUCCCGGGUUCAAAUCCCGGACGAGCCCCGCCUCGUUUU

  FANCF +5 G to T pegRNA sequence: space,

  scaffold template and PBSr-

  [SEQ ID NO: 3910]

  GGAAUCCCUUCUGCAGCACCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAU

  AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCGGAA

  AAGCGAUCAAGGUGCUGCAGAAGGGA-UGG P1 ext 11 linker 3-

  UCUGCUCGAGGCGGGGCUCGUUGGUCUAGGGGUAUGAUUCUCGCUUUGGG

  UGCGAGAGGUCCCGGGUUCAAAUCCCGGACGAGCCCCGCCUCGAGCUUUU

  FANCF +5 G to T pegRNA sequence: space,

  scaffold template and PBSr-

  [SEQ ID NO: 3911]

  GGAAUCCCUUCUGCAGCACCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAU

  AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCGGAA

  AAGCGAUCAAGGUGCUGCAGAAGGGA-UGG P1 ext 11 linker 8-

  UCUCUCUCGCUCGAGGCGGGGCUCGUUGGUCUAGGGGUAUGAUUCUCGCU

  UUGGGUGCGAGAGGUCCCGGGUUCAAAUCCCGGACGAGCCCCGCCUCGAG

  CUUUU

  HEK3 +1 10 nt insertion pegRNA sequence: space,

  scaffold template and PBSr-

  [SEQ ID NO: 3913

  GGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGAAATAGCAAGTTAAAAT

  AAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTCTG

  CCATCAAAGCTTCGACCGTGCTCAGTCTG-UGG P1 ext 5 linker

  3-UCUGGCGGGGCUCGUUGGUCUAGGGGUAUGAUUCUCGCUUUGGGUGCG

  AGAGGUCCCGGGUUCAAAUCCCGGACGAGCCCCGCCUUUU

  HEK3 +1 FLAG insertion pegRNA sequence: space,

  scaffold template and PBSr-

  [SEQ ID NO: 3914]

  GGCCCAGACUGAGCACGUGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAU

  AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUGGA

  GGAAGCAGGGCUUCCUUUCCUCUGCCAUCACUUAUCGUCGUCAUCCUUGU

  AAUCCGUGCUCAGUCUG-partial CGG tRNA linker 8-UCUCUC

  UCUGGUCUAGGGGUAUGAUUCUCGCUUCGGGUGCGAGAGGUCCCGGGUUC

  AAUUUU

  HEK3 +1 FLAG insertion pegRNA sequence: space,

  scaffold template and PBSr-

  [SEQ ID NO: 3915]

  GGCCCAGACUGAGCACGUGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAU

  AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUGGA

  GGAAGCAGGGCUUCCUUUCCUCUGCCAUCACUUAUCGUCGUCAUCCUUGU

  AAUCCGUGCUCAGUCUG-UGG P1 ext 5 linker 3-UCUGGCGGGG

  CUCGUUGGUCUAGGGGUAUGAUUCUCGCUUUGGGUGCGAGAGGUCCCGGG

  UUCAAAUCCCGGACGAGCCCCGCCUUUU

  HEK3 +1 FLAG insertion pegRNA sequence: space,

  scaffold template and PBSr-

  [SEQ ID NO: 3916]

  GGCCCAGACUGAGCACGUGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAU

  AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUGGA

  GGAAGCAGGGCUUCCUUUCCUCUGCCAUCACUUAUCGUCGUCAUCCUUGU

  AAUCCGUGCUCAGUCUG-UGG P1 ext 5 linker 8-UCUCUCUCGG

  CGGGGCUCGUUGGUCUAGGGGUAUGAUUCUCGCUUUGGGUGCGAGAGGUC

  CCGGGUUCAAAUCCCGGACGAGCCCCGCCUUUU

  HEK3 +1 FLAG insertion pegRNA sequence: space,

  scaffold template and PBSr-

  [SEQ ID NO: 3917]

  GGCCCAGACUGAGCACGUGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAU

  AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUGGA

  GGAAGCAGGGCUUCCUUUCCUCUGCCAUCACUUAUCGUCGUCAUCCUUGU

  AAUCCGUGCUCAGUCUG-UGG P1 ext 8 linker 8-UCUCUCUCCG

  AGGCGGGGCUCGUUGGUCUAGGGGUAUGAUUCUCGCUUUGGGUGCGAGAG

  GUCCCGGGUUCAAAUCCCGGACGAGCCCCGCCUCGUUUU

  HEK3 +1 FLAG insertion pegRNA sequence: space,

  scaffold template and PBSr-

  [SEQ ID NO: 3918]

  GGCCCAGACUGAGCACGUGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAU

  AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUGGA

  GGAAGCAGGGCUUCCUUUCCUCUGCCAUCACUUAUCGUCGUCAUCCUUGU

  AAUCCGUGCUCAGUCUG-UGG P1 ext 11 linker 8-UCUCUCUCG

  CUCGAGGCGGGGCUCGUUGGUCUAGGGGUAUGAUUCUCGCUUUGGGUGCG

  AGAGGUCCCGGGUUCAAAUCCCGGACGAGCCCCGCCUCGAGCUUUU

  HEK3 +1 FLAG insertion pegRNA sequence: space,

  scaffold template and PBSr-

  [SEQ ID NO: 3919]

  GGCCCAGACUGAGCACGUGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAU

  AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUGGA

  GGAAGCAGGGCUUCCUUUCCUCUGCCAUCACUUAUCGUCGUCAUCCUUGU

  AAUCCGUGCUCAGUCUG-UGG P1 ext 14 linker 8-UCUCUCUCG

  GUGCUCGAGGCGGGGCUCGUUGGUCUAGGGGUAUGAUUCUCGCUUUGGGU

  GCGAGAGGUCCCGGGUUCAAAUCCCGGACGAGCCCCGCCUCGAGCACCUU

  UU

  RNF2 +1 C to A pegRNA sequence: space, scaffold

  template and PBSr-

  [SEQ ID NO: 3920]

  GUCAUCUUAGUCAUUACCUGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAU

  AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCGAAC

  ACCUCAUGUAAUGACUAAGAUG-tRNA-Pro {cgg}-5-UCUGGCGGGG

  CUCGUUGGUCUAGGGGUAUGAUUCUCGCUUCGGGUGCGAGAGGUCCCGGG

  UUCAAAUCCCGGACGAGCCCCGCC

  RNF2 +1 C to A pegRNA sequence: space, scaffold

  template and PBSr-

  [SEQ ID NO: 3921]

  GUCAUCUUAGUCAUUACCUGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAU

  AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCGAAC

  ACCUCAUGUAAUGACUAAGAUG-tRNA-Pro {cgg}-8-UCUCGAGGCG

  GGGCUCGUUGGUCUAGGGGUAUGAUUCUCGCUUCGGGUGCGAGAGGUCCC

  GGGUUCAAAUCCCGGACGAGCCCCGCCUCGUUUU

  RNF2 +1 C to A pegRNA sequence: space, scaffold

  template and PBSr-

  [SEQ ID NO: 3922]

  GUCAUCUUAGUCAUUACCUGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAU

  AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCGAAC

  ACCUCAUGUAAUGACUAAGAUG-tRNA-Pro {cgg}-11-UCUGCUCGA

  GGCGGGGCUCGUUGGUCUAGGGGUAUGAUUCUCGCUUCGGGUGCGAGAGG

  UCCCGGGUUCAAAUCCCGGACGAGCCCCGCCUCGAGCUUUU

  RNF2 +1 C to A pegRNA sequence: space, scaffold

  template and PBSr-

  [SEQ ID NO: 3923]

  GUCAUCUUAGUCAUUACCUGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAU

  AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCGAAC

  ACCUCAUGUAAUGACUAAGAUG-tRNA-Pro {Lys}-5-UCUGGCGGGC

  CCGGAUAGCUCAGUCGGUAGAGCAUCAGACUUUUAAUCUGAGGGUCCAGG

  GUUCAAGUCCCUGUUCGGGCCCGCCUUUU

  RNF2 +1 C to A pegRNA sequence: space, scaffold

  template and PBSr-

  [SEQ ID NO: 3924]

  GUCAUCUUAGUCAUUACCUGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAU

  AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCGAAC

  ACCUCAUGUAAUGACUAAGAUG-tRNA-Pro {Lys}-8-UCUCGAGGCG

  GGCCCGGAUAGCUCAGUCGGUAGAGCAUCAGACUUUUAAUCUGAGGGUCC

  AGGGUUCAAGUCCCUGUUCGGGCCCGCCUCGUUUU

  RNF2 +1 C to A pegRNA sequence: space, scaffold

  template and PBSr-

  [SEQ ID NO: 3925]

  GUCAUCUUAGUCAUUACCUGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAU

  AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCGAAC

  ACCUCAUGUAAUGACUAAGAUG-tRNA-Pro {UGG}-8-UCUCGAGGCG

  GGGCUCGUUGGUCUAGGGGUAUGAUUCUCGCUUUGGGUGCGAGAGGUCCC

  GGGUUCAAAUCCCGGACGAGCCCCGCCUCGUUUU

  RNF2 C to A pegRNA sequence: space, scaffold

  template and PBSr-

  [SEQ ID NO: 3926]

  GUCAUCUUAGUCAUUACCUGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAU

  AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCGAAC

  ACCUCAUGUAAUGACUAAGAUG-tRNA-Pro {UGG}-11-UCUGCU

  CGAGGCGGGGCUCGUUGGUCUAGGGGUAUGAUUCUCGCUUUGGGUGCGAG

  AGGUCCCGGGUUCAAAUCCCGGACGAGCCCCGCCUCGAGCUUUU

  RNF2 +1 C to A pegRNA sequence: space, scaffold

  template and PBSr-

  [SEQ ID NO: 3927]

  GUCAUCUUAGUCAUUACCUGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAU

  AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCGAAC

  ACCUCAUGUAAUGACUAAGAUG-tRNA-Pro {UGG}-8-longer

  linker-UCUCUCUCCGAGGCGGGGCUCGUUGGUCUAGGGGUAUGAUUCU

  CGCUUUGGGUGCGAGAGGUCCCGGGUUCAAAUCCCGGACGAGCCCCGCCU

  CGUUUU

  RNF2 +1 C to A pegRNA sequence: space, scaffold

  template and PBSr-

  [SEQ ID NO: 3928]

  GUCAUCUUAGUCAUUACCUGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAU

  AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCGAAC

  ACCUCAUGUAAUGACUAAGAUG-tRNA-Pro {UGG}-11-longer

  linker-UCUCUCUCGCUCGAGGCGGGGCUCGUUGGUCUAGGGGUAUGAU

  UCUCGCUUUGGGUGCGAGAGGUCCCGGGUUCAAAUCCCGGACGAGCCCCG

  CCUCGAGCUUUU

  RNF2 +1 C to A pegRNA sequence: space, scaffold

  template and PBSr-

  [SEQ ID NO: 3929]

  GUCAUCUUAGUCAUUACCUGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAU

  AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCGAAC

  ACCUCAUGUAAUGACUAAGAUG-tRNA-Pro {UGG}-11-longer

  linker-UCUCUCUCGGUGCUCGAGGCGGGGCUCGUUGGUCUAGGGGUAU

  GAUUCUCGCUUUGGGUGCGAGAGGUCCCGGGUUCAAAUCCCGGACGAGCC

  CCGCCUCGAGCACCUUUU

Example 23. Use of Prime Editing to Correct CDKL5 and Sickle Cell Anemia
• Use of PE to Design Mouse Model of CDKL5 with 1412delA Mutation
• CDKL5 Deficiency Disorder (CDD) is a neurodegenerative disease most often caused by spontaneous mutations in the cyclin-dependent kinase-like 5 gene. Symptoms manifest in early childhood and include seizures, irregular sleeping patterns, gastrointestinal stress, and developmental delay. Some mutations which cause CDD, including 1412delA, cannot be corrected with base editing. However, prime editing has the potential to precisely correct all base-to-base changes, deletions, and insertions. With focus on the 1412delA mutation, this Example designed and tested pegRNAs capable of inserting the mutation in hopes to establish a mouse neuronal cell line (N2A) harboring the mutation. This will allow for extensive screening potentially therapeutic pegRNAs that correct the mutation. The ultimate goal is to be able to move into a CDD mouse model with the 1412delA mutation to assess therapeutic effect. No current mouse models of CDD have a humanized allele, however optimization of pegRNAs is underway in HEK293T cells as well. FIGS. 87 and 88 are the results from a pilot screen in N2A cells where the pegRNA installs 1412Adel, with details about the primer binding site (PBS) length and reverse transcriptase (RT) template length. (Shown with and without indels)
Use of PE to Treat Sickle Cell Anemia (SCA)
• Sickle cell anemia (SCA) is a recessive blood disorder caused by a glutamate-to-valine mutation at position 6 in the β-globin gene. The result is sickled red blood cells that are poor oxygen transporters and prone to aggregation. Symptoms of aggregation can be life-threatening. Previously, the D. Liu lab was able to show both the installation and correction of the SCA locus in HEK293T cells using prime editing via DNA plasmid transfection. Since hematopoietic stem cells (HSCs) are difficult to edit via DNA plasmid transfection, this Example tested the PE3 system in HSCs with protein and mRNA nucleofection. FIG. 89 are the results of editing at a proxy locus in the β-globin gene and at HEK3 in healthy HSCs, varying the concentration of editor to pegRNA and nicking gRNA.
• mRNA Nucleofection Protocol:
• The protocol was improved by adjusting the ratio of editor to guides ([editor] to [guide] ratio or editor:guide ratio)
• The nicking guide protospacer sequence was: CCTTGATACCAACCTGCCCA
• The pegRNA sequence was:

• CATGGTGCACCTGACTCCTGGTTTTAGAGCTAGAAATAGCAAGTTAAAAT

  AAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCAGAC

  TTCTCTTCAGGAGTCAGGTGCACTTT

• 1. Thaw CD34+ cells and pre-stimulate in X-Vivo15 media with cytokines (SCF, Flt3 and TPO) for 24 hrs. Seeding density 1 million cells/mL.
• 2. Next day (post 24 hrs), aspirate CD34+ cells (1×105) and wash once with PBS (centrifugation performed at 300 g for 10 mins in RT) and resuspend in P3 solution (Lonza) (20 ul per condition).
• 3. In hood, combine 2 ug of SpPE2 mRNA, 1 ug combined of pegRNA and sgRNA in a sterile PCR strip on ice. Adjust the volume to 20 ul (cells in P3 solution+mRNA &gRNA mix).
• 4. Pipette up cell suspension into 20 uL Lonza4D 16 well strip and electroporate with program DS130.
• 5. Wait for 10 to 15 mins post electroporation and add 80 uL of X-Vivo10+cytokine media to cell suspension and transfer to pre-warmed X-Vivo10 with cytokine media.
• 6. Harvest cells 72 hours after electroporation and check the cell recovery and do genotyping in bulk population and sorted CD34+ and CD34+90+ populations as well.

• 	Wildtype CDKL5 protein (Accession No.
  	NP_001032420) (isoform 1-human)
  	MKIPNIGNVM NKFEILGVVG EGAYGVVLKC RHKETHEIVA
  	IKKFKDSEEN EEVKETTLRE LKMLRTLKQE NIVELKEAFR
  	RRGKLYLVFE YVEKNMLELL EEMPNGVPPE KVKSYIYQLI
  	KAIHWCHKND IVHRDIKPEN LLISHNDVLK LCDFGFARNL
  	SEGNNANYTE YVATRWYRSP ELLLGAPYGK SVDMWSVGCI
  	LGELSDGQPL FPGESEIDQL FTIQKVLGPL PSEQMKLFYS
  	NPRFHGLRFP AVNHPQSLER RYLGILNSVL LDLMKNLLKL
  	DPADRYLTEQ CLNHPTFQTQ RLLDRSPSRS AKRKPYHVES
  	STLSNRNQAG KSTALQSHHR SNSKDIQNLS VGLPRADEGL
  	PANESFLNGN LAGASLSPLH TKTYQASSQP GSTSKDLTNN
  	NIPHLLSPKE AKSKTEFDFN IDPKPSEGPG TKYLKSNSRS
  	QQNRHSFMES SQSKAGTLQP NEKQSRHSYI DTIPQSSRSP
  	SYRTKAKSHG ALSDSKSVSN LSEARAQIAE PSTSRYFPSS
  	CLDLNSPTSP TPTRHSDTRT LLSPSGRNNR NEGTLDSRRT
  	TTRHSKTMEE LKLPEHMDSS HSHSLSAPHE SFSYGLGYTS
  	PFSSQQRPHR HSMYVTRDKV RAKGLDGSLS IGQGMAARAN
  	SLQLLSPQPG EQLPPEMTVA RSSVKETSRE GTSSFHTRQK
  	SEGGVYHDPH SDDGTAPKEN RHLYNDPVPR RVGSFYRVPS
  	PRPDNSFHEN NVSTRVSSLP SESSSGTNHS KRQPAFDPWK
  	SPENISHSEQ LKEKEKQGFF RSMKKKKKKS QTVPNSDSPD
  	LLTLQKSIHS ASTPSSRPKE WRPEKISDLQ TQSQPLKSLR
  	KLLHLSSASN HPASSDPRFQ PLTAQQTKNS FSEIRIHPLS
  	QASGGSSNIR QEPAPKGRPA LQLPDGGCDG RRQRHHSGPQ
  	DRRFMLRTTE QQGEYFCCGD PKKPHTPCVP NRALHRPISS
  	PAPYPVLQVR GTSMCPTLQV RGTDAFSCPT QQSGFSFFVR
  	HVMREALIHR AQVNQAALLT YHENAALTGK
  	Wildtype CDKL5 protein (Accession No.
  	NP_001310218) (isoform 2-human)
  	MKIPNIGNVM NKFEILGVVG EGAYGVVLKC RHKETHEIVA
  	IKKFKDSEEN EEVKETTLRE LKMLRTLKQE NIVELKEAFR
  	RRGKLYLVFE YVEKNMLELL EEMPNGVPPE KVKSYIYQLI
  	KAIHWCHKND IVHRDIKPEN LLISHNDVLK LCDFGFARNL
  	SEGNNANYTE YVATRWYRSP ELLLGAPYGK SVDMWSVGCI
  	LGELSDGQPL FPGESEIDQL FTIQKVLGPL PSEQMKLFYS
  	NPRFHGLRFP AVNHPQSLER RYLGILNSVL LDLMKNLLKL
  	DPADRYLTEQ CLNHPTFQTQ RLLDRSPSRS AKRKPYHVES
  	STLSNRNQAG KSTALQSHHR SNSKDIQNLS VGLPRADEGL
  	PANESFLNGN LAGASLSPLH TKTYQASSQP GSTSKDLTNN
  	NIPHLLSPKE AKSKTEFDFN IDPKPSEGPG TKYLKSNSRS
  	QQNRHSFMES SQSKAGTLQP NEKQSRHSYI DTIPQSSRSP
  	SYRTKAKSHG ALSDSKSVSN LSEARAQIAE PSTSRYFPSS
  	CLDLNSPTSP TPTRHSDTRT LLSPSGRNNR NEGTLDSRRT
  	TTRHSKTMEE LKLPEHMDSS HSHSLSAPHE SFSYGLGYTS
  	PFSSQQRPHR HSMYVTRDKV RAKGLDGSLS IGQGMAARAN
  	SLQLLSPQPG EQLPPEMTVA RSSVKETSRE GTSSFHTRQK
  	SEGGVYHDPH SDDGTAPKEN RHLYNDPVPR RVGSFYRVPS
  	PRPDNSFHEN NVSTRVSSLP SESSSGTNHS KRQPAFDPWK
  	SPENISHSEQ LKEKEKQGFF RSMKKKKKKS QTVPNSDSPD
  	LLTLQKSIHS ASTPSSRPKE WRPEKISDLQ TQSQPLKSLR
  	KLLHLSSASN HPASSDPRFQ PLTAQQTKNS FSEIRIHPLS
  	QASGGSSNIR QEPAPKGRPA LQLPGQMDPG WHVSSVTRSA
  	TEGPSYSEQL GAKSGPNGHP YNRTNRSRMP NLNDLKETAL

Example 24. Use of Prime Editing to Target Pathogenic AOL1 Alleles as Treatment for Non-Diabetic Chronic Kidney Disease
• This Example designed PEgRNAs that are capable of targeting pathogenic APOL1 alleles for use with prime editing to treat or reduce the likelihood of developing a renal disease.
• End-stage kidney failure (ESKD) is a growing problem that now affects over half a million individuals in the United States. The cost of caring for patients with ESKD is currently over 40 billion dollars per year. In the U.S., the likelihood that subjects of African descent will develop ESKD is 4 to 5 times higher than for Americans without African ancestry. These facts are reflected in the disparity between the 12-13% of the U.S. population with African descent and the 40% of U.S. dialysis patients who are African-American. The epidemic of renal disease risk factors, such as obesity and metabolic syndrome, suggests that the magnitude of this problem will only increase.
• There are no specific therapies for the vast majority of progressive kidney diseases. Some types of chronic renal disease progression can be slowed by blood pressure control with specific agents, but nephrologists cannot accurately predict which patients will respond. Moreover, while successful treatment typically slows progression, it neither prevents disease nor halts disease progression.
• Recently it was determined that that specific genetic variants that alter the protein sequence of APOIipoprotein-L1 (APOL1) are associated with progressive kidney disease. Surprisingly, APOL1 kidney disease variants have a major impact on multiple different types of kidney disease including hypertension-associated end-stage renal disease (H-ESRD), focal segmental glomerulosclerosis (FSGS), and HIV-associated nephropathy (HIVAN). Individuals with these variant APOL1 alleles have a 7-30 fold increased risk for kidney disease. Based on the high frequency of these APOL1 risk alleles, more than 3.5 million African Americans likely have the high risk APOL1 genotype. African Americans without the high risk genotype have little excess risk compared with Americans of European ancestry.
• Despite evidence that variants in the APOL1 gene cause renal disease, very little is known about the biology of its product, APOL1, or its role in the kidney. APOL1 has a defined role in resistance to trypanosomes, and the G1 and G2 variants appear to have become common in Africa because they confer protection against the forms of trypanosomes that cause African Sleeping Sickness.
• There still exists a need for therapies for kidney diseases in patients with one or more APOL1 risk alleles, which cause great morbidity and mortality with high economic impact in this and other subject populations.
• This Example provides three exemplary PEgRNA design options based on a specific exemplary target sequence that may be used with prime editing to correct APOL1 defective alleles.
• PEgRNA 1
• Designing PEgRNAs for APOL1 allele rs73885319 (p.S342G). This represents a G→A correction in affected individuals. The target sequence is

• 5-GGAGTCAAGCTCACGGATGTGGCCCCTGTA(G-to-A)GCTTCTTTCT

  TGTGCTGGATGTAGTCTACCT-3.

• The protospacer (bolded above) is AAGCTCACGGATGTGGCCCC. The selected PE comprises a SaCas9(D10A).
• The primer binding sites can be:

• 	GTGGCCCC
  	TGTGGCCCC
  	ATGTGGCCCC
  	GATGTGGCCCC
  	GGATGTGGCCCC
  	CGGATGTGGCCCC
  	ACGGATGTGGCCCC
  	CACGGATGTGGCCCC
  	TCACGGATGTGGCCCC
  	CTCACGGATGTGGCCCC.

• The RT templates can be:

• 	AAGAAGCTTACA
  	AAAGAAGCTTACA
  	GAAAGAAGCTTACA
  	AGAAAGAAGCTTACA
  	AAGAAAGAAGCTTACA
  	CAAGAAAGAAGCTTACA
  	ACAAGAAAGAAGCTTACA
  	CACAAGAAAGAAGCTTACA
  	GCACAAGAAAGAAGCTTACA
  	AGCACAAGAAAGAAGCTTACA
  	CAGCACAAGAAAGAAGCTTACA
  	CCAGCACAAGAAAGAAGCTTACA
  	TCCAGCACAAGAAAGAAGCTTACA
  	ATCCAGCACAAGAAAGAAGCTTACA.

• The nicking template can be GCTTTGATTCGTACACGAGG.
• PEgRNA 2
• Designing PEgRNAs for APOL1 allele rs60910145. This represents a G→T correction in affected individuals.
• The protospacer is GCTGGAGGAGAAGCTAAACA. The selected PE comprises SpCas9(D10A)-NG.
• The primer binding sites can be:

• 	GAAGCTAA
  	AGAAGCTAA
  	GAGAAGCTAA
  	GGAGAAGCTAA
  	AGGAGAAGCTAA
  	GAGGAGAAGCTAA
  	GGAGGAGAAGCTAA
  	TGGAGGAGAAGCTAA
  	CTGGAGGAGAAGCTAA
  	GCTGGAGGAGAAGCTAA.

• The RT template can be (cannot end in C):

• 	AGAATGT
  	GAGAATGT
  	TGAGAATGT
  	TTGAGAATGT
  	GTTGAGAATGT
  	TGTTGAGAATGT
  	TTGTTGAGAATGT
  	ATTGTTGAGAATGT
  	TATTGTTGAGAATGT
  	TTATTGTTGAGAATGT
  	ATTATTGTTGAGAATGT
  	AATTATTGTTGAGAATGT
  	TAATTATTGTTGAGAATGT
  	ATAATTATTGTTGAGAATGT.

• The nicking templates can be:

• 	CCTGTGGTCACAGTTCTTGG
  	CCACAGGGCAGGGCAGCCAC.

• PEgRNA 3
• Designing PEgRNAs for APOL1 allele rs71785313. This represents an insert, as follows: ATTCTCAACAA[insert: TAATTA]TAAGATTC.
• The protospacer can be: TCTCAACAATAAGATTCTGC
• The PE comprises SaKKH-PE2.
• The primer binding site can be:

• 	TTCTCAAC
  	ATTCTCAAC
  	CATTCTCAAC
  	ACATTCTCAAC
  	AACATTCTCAAC
  	AAACATTCTCAAC
  	TAAACATTCTCAAC
  	CTAAACATTCTCAAC
  	GCTAAACATTCTCAAC
  	AGCTAAACATTCTCAAC.

• The RT template can be:

• 	AATCTTATAATTATT
  	GAATCTTATAATTATT
  	AGAATCTTATAATTATT
  	CAGAATCTTATAATTATT
  	GCAGAATCTTATAATTATT
  	TGCAGAATCTTATAATTATT
  	CTGCAGAATCTTATAATTATT
  	CCTGCAGAATCTTATAATTATT
  	GCCTGCAGAATCTTATAATTATT
  	CGCCTGCAGAATCTTATAATTATT
  	CCGCCTGCAGAATCTTATAATTATT
  	TCCGCCTGCAGAATCTTATAATTATT
  	GTCCGCCTGCAGAATCTTATAATTATT
  	GGTCCGCCTGCAGAATCTTATAATTATT.

• The nicking templates can be:

• 	CCTGTGGTCACAGTTCTTGG
  	CCACAGGGCAGGGCAGCCAC.

OTHER REFERENCES MENTIONED THROUGHOUT THIS DISCLOSURE
• The following references are each incorporated herein by reference in their entireties.
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EMBODIMENTS
• The following embodiments are within the scope of the present disclosure. Furthermore, the disclosure encompasses all variations, combinations, and permutations of these embodiments in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed embodiments is introduced into another listed embodiment in this section. For example, any listed embodiment that is dependent on another embodiment can be modified to include one or more limitations found in any other listed embodiment in this section that is dependent on the same base embodiment. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the disclosure, or aspects of the disclosure, is/are referred to as comprising particular elements and/or features, certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements and/or features. It is also noted that the terms “comprising” and “containing” are intended to be open and permits the inclusion of additional elements or steps. Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.
GROUP 1. EMBODIMENTS 1-212
• 1. A fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a reverse transcriptase.
  2. The fusion protein of embodiment 1, wherein the fusion protein is capable of carrying out genome editing by target-primed reverse transcription in the presence of an extended guide RNA.
  3. The fusion protein of embodiment 1, wherein the napDNAbp has a nickase activity.
  4. The fusion protein of embodiment 1, wherein the napDNAbp is a Cas9 protein or variant thereof.
  5. The fusion protein of embodiment 1, wherein the napDNAbp is a nuclease active Cas9, a nuclease inactive Cas9 (dCas9), or a Cas9 nickase (nCas9).
  6. The fusion protein of embodiment 1, wherein the napDNAbp is Cas9 nickase (nCas9).
  7. The fusion protein of embodiment 1, wherein the napDNAbp is selected from the group consisting of: Cas9, CasX, CasY, Cpf1, C2c1, C2c2, C2C3, and Argonaute and optionally has a nickase activity.
  8. The fusion protein of embodiment 1, wherein the fusion protein when complexed with an extended guide RNA is capable of binding to a target DNA sequence.
  9. The fusion protein of embodiment 8, wherein the target DNA sequence comprises a target strand and a complementary non-target strand.
  10. The fusion protein of embodiment 8, wherein the binding of the fusion protein complexed to the extended guide RNA forms an R-loop.
  11. The fusion protein of embodiment 10, wherein the R-loop comprises (i) an RNA-DNA hybrid comprising the extended guide RNA and the target strand, and (ii) the complementary non-target strand.
  12. The fusion protein of embodiment 11, wherein the complementary non-target strand is nicked to form a reverse transcriptase priming sequence having a free 3′ end.
  13. The fusion protein of embodiment 2, wherein the extended guide RNA comprises (a) a guide RNA, and (b) an RNA extension at the 5′ or the 3′ end of the guide RNA, or at an intramolecular location in the guide RNA.
  14. The fusion protein of embodiment 13, wherein the RNA extension comprises (i) a reverse transcription template sequence comprising a desired nucleotide change, (ii) a reverse transcription primer binding site, and (iii) optionally, a linker sequence.
  15. The fusion protein of embodiment 14, wherein the reverse transcription template sequence encodes a single-strand DNA flap that is complementary to an endogenous DNA sequence adjacent to the nick site, wherein the single-strand DNA flap comprises the desired nucleotide change.
  16. The fusion protein of embodiment 13, wherein the RNA extension is at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides, at least 22 nucleotides, at least 23 nucleotides, at least 24 nucleotides, or at least 25 nucleotides in length.
  17. The fusion protein of embodiment 15, wherein the single-strand DNA flap hybridizes to the endogenous DNA sequence adjacent to the nick site, thereby installing the desired nucleotide change.
  18. The fusion protein of embodiment 15, wherein the single-stranded DNA flap displaces the endogenous DNA sequence adjacent to the nick site and which has a free 5′ end. 19. The fusion protein of embodiment 18, wherein the endogenous DNA sequence having the 5′ end is excised by the cell.
  20. The fusion protein of embodiment 18, wherein cellular repair of the single-strand DNA flap results in installation of the desired nucleotide change, thereby forming a desired product.
  21. The fusion protein of embodiment 14, wherein the desired nucleotide change is installed in an editing window that is between about −4 to +10 of the PAM sequence, or between about −10 to +20 of the PAM sequence, or between about −20 to +40 of the PAM sequence, or between about −30 to +100 of the PAM sequence, or wherein the desired nucleotide change is installed at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides downstream of the nick site.
  22. The fusion protein of embodiment 1, wherein the napDNAbp comprises an amino acid sequence of SEQ ID NO: 2, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to the amino acid sequence to SEQ ID NO: 2.
  23. The fusion protein of embodiment 1, wherein the napDNAbp comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 2-10.
  24. The fusion protein of embodiment 1, wherein the reverse transcriptase comprises any one of the amino acid sequences of SEQ ID NO: 11-17.
  25. The fusion protein of embodiment 1, wherein the reverse transcriptase comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 11-17.
  26. The fusion protein of embodiment 1, wherein the reverse transcriptase is a naturally-occurring reverse transcriptase from a retrovirus or a retrotransposon.
  27. The fusion protein of any one of the previous embodiments, wherein the fusion protein comprises the structure NH₂-[napDNAbp]-[reverse transcriptase]-COOH; or NH₂-[reverse transcriptase]-[napDNAbp]-COOH, wherein each instance of “]-[” indicates the presence of an optional linker sequence.
  28. The fusion protein of embodiment 27, wherein the linker sequence comprises an amino acid sequence of SEQ ID NOs: 37-47.
  29. The fusion protein of embodiment 14, wherein the desired nucleotide change is a single nucleotide change, an insertion of one or more nucleotides, or a deletion of one or more nucleotides.
  30. The fusion protein of embodiment 29, wherein the insert or deletion is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, or at least 50.
  31. An extended guide RNA comprising a guide RNA and at least one RNA extension.
  32. The extended guide RNA of embodiment 1, wherein the RNA extension is position at the 3′ or 5′ end of the guide RNA, or at an intramolecular position in the guide RNA.
  33. The extended guide RNA of embodiment 31, wherein the extended guide RNA is capable of binding to a napDNAbp and directing the napDNAbp to a target DNA sequence.
  34. The extended guide RNA of embodiment 33, wherein the target DNA sequence comprises a target strand and a complementary non-target strand, wherein the guide RNA hybridizes to the target strand to form an RNA-DNA hybrid and an R-loop.
  35. The extended guide RNA of embodiment 31, wherein the at least one RNA extension comprises (i) a reverse transcription template sequence, (ii) a reverse transcription primer binding site, and (iii) optionally a linker sequence.
  36. The extended guide RNA of embodiment 35, wherein the RNA extension is at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides, at least 22 nucleotides, at least 23 nucleotides, at least 24 nucleotides, or at least 25 nucleotides in length.
  37. The extended guide RNA of embodiment 35, wherein the reverse transcription template sequence is at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, or at least 15 nucleotides in length.
  38. The extended guide RNA of embodiment 35, wherein the reverse transcription primer binding site sequence is at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, or at least 15 nucleotides in length.
  39. The extended guide RNA of embodiment 35, wherein the optional linker sequence is at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, or at least 15 nucleotides in length.
  40. The extended guide RNA of embodiment 35, wherein the reverse transcription template sequence encodes a single-strand DNA flap that is complementary to an endogenous DNA sequence adjacent to a nick site, wherein the single-strand DNA flap comprises a desired nucleotide change.
  41. The extended guide RNA of embodiment 40, wherein the single-stranded DNA flap displaces an endogenous single-strand DNA having a 5′ end in the target DNA sequence that has been nicked, and wherein the endogenous single-strand DNA is immediately adjacent downstream of the nick site.
  42. The extended guide RNA of embodiment 41, wherein the endogenous single-stranded DNA having the free 5′ end is excised by the cell.
  43. The extended guide RNA of embodiment 41, wherein cellular repair of the single-strand DNA flap results in installation of the desired nucleotide change, thereby forming a desired product.
  44. The extended guide RNA of embodiment 31, comprising the nucleotide sequence of SEQ ID NOs: 18-36, or a nucleotide sequence having at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99% sequence identity with any one of SEQ ID NOs: 18-36.
  45. The extended guide RNA of embodiment 35, wherein the reverse transcription template sequence comprises a nucleotide sequence that is at least 80%, or 85%, or 90%, or 95%, or 99% identical to the endogenous DNA target.
  46. The extended guide RNA of embodiment 35, wherein the reverse transcription primer binding site hybridizes with a free 3′ end of the cut DNA.
  47. The extended guide RNA of embodiment 35, wherein the optional linker sequence is at least 1 nucleotide, or at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 nucleotides in length.
  48. A complex comprising a fusion protein of any one of embodiments 1-30 and an extended guide RNA.
  49. The complex of embodiment 48, wherein the extended guide RNA comprises a guide RNA and an RNA extension at the 3′ or 5′ end of the guide RNA or at an intramolecular position in the guide RNA.
  50. The complex of embodiment 48, wherein the extended guide RNA is capable of binding to a napDNAbp and directing the napDNAbp to a target DNA sequence.
  51. The complex of embodiment 50, wherein the target DNA sequence comprises a target strand and a complementary non-target strand, wherein the guide RNA hybridizes to the target strand to form an RNA-DNA hybrid and an R-loop.
  52. The complex of embodiment 49, wherein the at least one RNA extension comprises (i) a reverse transcription template sequence, (ii) a reverse transcription primer binding site, and (iii) optionally a linker sequence.
  53. The complex of embodiment 48, wherein the extended guide RNA comprises the nucleotide sequence of SEQ ID NOs: 18-36, or a nucleotide sequence having at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99% sequence identity with any one of SEQ ID NOs: 18-36.
  54. The complex of embodiment 52, wherein the reverse transcription template sequence comprises a nucleotide sequence having at least 80%, or 85%, or 90%, or 95%, or 99% sequence identity with the endogenous DNA target.
  55. The complex of embodiment 52, wherein the reverse transcription primer binding site hybridizes with a free 3′ end of the cut DNA.
  56. A complex comprising a napDNAbp and an extended guide RNA.
  57. The complex of embodiment 56, wherein the napDNAbp is a Cas9 nickase.
  58. The complex of embodiment 56, wherein the napDNAbp comprises an amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO: 2.
  59. The complex of embodiment 57, wherein the napDNAbp comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 2-10.
  60. The complex of embodiment 57, wherein the extended guide RNA comprises a guide RNA and an RNA extension at the 3′ or 5′ end of the guide RNA, or at an intramolecular position in the guide RNA.
  61. The complex of embodiment 57, wherein the extended guide RNA is capable of directing the napDNAbp to a target DNA sequence.
  62. The complex of embodiment 61, wherein the target DNA sequence comprises a target strand and a complementary non-target strand, wherein the spacer sequence hybridizes to the target strand to form an RNA-DNA hybrid and an R-loop.
  63. The complex of embodiment 61, wherein the RNA extension comprises (i) a reverse transcription template sequence, (ii) a reverse transcription primer binding site, and (iii) optionally a linker sequence.
  64. The complex of embodiment 57, wherein the extended guide RNA comprises the nucleotide sequence of SEQ ID NOs: 18-36, or a nucleotide sequence having at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99% sequence identity with any one of SEQ ID NOs: 18-36.
  65. The complex of embodiment 63, wherein the reverse transcription template sequence comprises a nucleotide sequence that is at least 80%, or 85%, or 90%, or 95%, or 99% identical to the endogenous DNA target.
  66. The complex of embodiment 63, wherein the reverse transcription primer binding site hybridizes with a free 3′ end of the cut DNA.
  67. The complex of embodiment 63, wherein the optional linker sequence is at least 1 nucleotide, or at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 nucleotides in length.
  68. A polynucleotide encoding the fusion protein of any of embodiments 1-30.
  69. A vector comprising the polynucleotide of embodiment 68.
  70. A cell comprising the fusion protein of any of embodiments 1-30 and an extended guide RNA bound to the napDNAbp of the fusion protein.
  71. A cell comprising a complex of any one of embodiments 48-67.
  72. A pharmaceutical composition comprising: (i) a fusion protein of any of embodiments 1-30, the complex of embodiments 48-67, the polynucleotide of embodiment 68, or the vector of embodiment 69; and (ii) a pharmaceutically acceptable excipient.
  73. A pharmaceutical composition comprising: (i) the complex of embodiments 48-67 (ii) reverse transcriptase provided in trans; and (iii) a pharmaceutically acceptable excipient.
  74. A kit comprising a nucleic acid construct, comprising: (i) a nucleic acid sequencing encoding the fusion protein of any one of embodiments 1-30; and (ii) a promoter that drives expression of the sequence of (i).
  75. A method for installing a desired nucleotide change in a double-stranded DNA sequence, the method comprising:
• (i) contacting the double-stranded DNA sequence with a complex comprising a fusion protein and an extended guide RNA, wherein the fusion protein comprises a napDNAbp and a reverse transcriptase and wherein the extended guide RNA comprises a reverse transcription template sequence comprising the desired nucleotide change;
• (ii) nicking the double-stranded DNA sequence on the non-target strand, thereby generating a free single-strand DNA having a 3′ end;
• (iii) hybridizing the 3′ end of the free single-strand DNA to the reverse transcription template sequence, thereby priming the reverse transcriptase domain;
• (iv) polymerizing a strand of DNA from the 3′ end, thereby generating a single-strand DNA flap comprising the desired nucleotide change;
• (v) replacing an endogenous DNA strand adjacent the cut site with the single-strand DNA flap, thereby installing the desired nucleotide change in the double-stranded DNA sequence.
  76. The method of embodiment 75, wherein the step of (v) replacing comprises: (i) hybridizing the single-strand DNA flap to the endogenous DNA strand adjacent the cut site to create a sequence mismatch; (ii) excising the endogenous DNA strand; and (iii) repairing the mismatch to form the desired product comprising the desired nucleotide change in both strands of DNA.
  77. The method of embodiment 76, wherein the desired nucleotide change is a single nucleotide substitution, a deletion, or an insertion.
  78. The method of embodiment 77, wherein the single nucleotide substitution is a transition or a transversion.
  79. The method of embodiment 76, wherein the desired nucleotide change is (1) a G to T substitution, (2) a G to A substitution, (3) a G to C substitution, (4) a T to G substitution, (5) a T to A substitution, (6) a T to C substitution, (7) a C to G substitution, (8) a C to T substitution, (9) a C to A substitution, (10) an A to T substitution, (11) an A to G substitution, or (12) an A to C substitution.
  80. The method of embodiment 76, wherein the desired nucleoid change converts (1) a G:C basepair to a T:A basepair, (2) a G:C basepair to an A:T basepair, (3) a G:C basepair to C:G basepair, (4) a T:A basepair to a G:C basepair, (5) a T:A basepair to an A:T basepair, (6) a T:A basepair to a C:G basepair, (7) a C:G basepair to a G:C basepair, (8) a C:G basepair to a T:A basepair, (9) a C:G basepair to an A:T basepair, (10) an A:T basepair to a T:A basepair, (11) an A:T basepair to a G:C basepair, or (12) an A:T basepair to a C:G basepair.
  81. The method of embodiment 76, wherein the desired nucleotide change is an insertion or deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides.
  82. The method of embodiment 76, wherein the desired nucleotide change corrects a disease-associated gene.
  83. The method of embodiment 82, wherein the disease-associated gene is associated with a monogenetic disorder selected from the group consisting of: Adenosine Deaminase (ADA) Deficiency; Alpha-1 Antitrypsin Deficiency; Cystic Fibrosis; Duchenne Muscular Dystrophy; Galactosemia; Hemochromatosis; Huntington's Disease; Maple Syrup Urine Disease; Marfan Syndrome; Neurofibromatosis Type 1; Pachyonychia Congenita; Phenylkeotnuria; Severe Combined Immunodeficiency; Sickle Cell Disease; Smith-Lemli-Opitz Syndrome; and Tay-Sachs Disease.
  84. The method of embodiment 82, wherein the disease-associated gene is associated with a polygenic disorder selected from the group consisting of: heart disease; high blood pressure; Alzheimer's disease; arthritis; diabetes; cancer; and obesity.
  85. The method of embodiment 76, wherein the napDNAbp is a nuclease dead Cas9 (dCas9), a Cas9 nickase (nCas9), or a nuclease active Cas9.
  86. The method of embodiment 76, wherein the napDNAbp comprises an amino acid sequence of SEQ ID NO: 2.
  87. The method of embodiment 76, wherein the napDNAbp comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 2-10.
  88. The method of embodiment 76, wherein the reverse transcriptase comprises any one of the amino acid sequences of SEQ ID NO: 11-17.
  89. The method of embodiment 76, wherein the reverse transcriptase domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 11-17.
  90. The method of embodiment 76, wherein the extended guide RNA comprises an RNA extension at the 3′ or 5′ ends or at an intramolecular location in the guide RNA, wherein the RNA extension comprises the reverse transcription template sequence.
  91. The method of embodiment 90, wherein the RNA extension is at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides, at least 22 nucleotides, at least 23 nucleotides, at least 24 nucleotides, or at least 25 nucleotides in length.
  92. The method of embodiment 76, wherein the extended guide RNA has a nucleotide sequence selected from the group consisting of SEQ ID NOs: 18-36.
  93. A method for introducing one or more changes in the nucleotide sequence of a DNA molecule at a target locus, comprising:
• (i) contacting the DNA molecule with a nucleic acid programmable DNA binding protein (napDNAbp) and a guide RNA which targets the napDNAbp to the target locus, wherein the guide RNA comprises a reverse transcriptase (RT) template sequence comprising at least one desired nucleotide change;
• (ii) forming an exposed 3′ end in a DNA strand at the target locus;
• (iii) hybridizing the exposed 3′ end to the RT template sequence to prime reverse transcription;
• (iv) synthesizing a single strand DNA flap comprising the at least one desired nucleotide change based on the RT template sequence by reverse transcriptase;
• (v) and incorporating the at least one desired nucleotide change into the corresponding endogenous DNA, thereby introducing one or more changes in the nucleotide sequence of the DNA molecule at the target locus.
• 94. The method of embodiment 93, wherein the one or more changes in the nucleotide sequence comprises a transition.
  95. The method of embodiment 94, wherein the transition is selected from the group consisting of: (a) T to C; (b) A to G; (c) C to T; and (d) G to A.
  96. The method of embodiment 93, wherein the one or more changes in the nucleotide sequence comprises a transversion.
  97. The method of embodiment 96, wherein the transversion is selected from the group consisting of: (a) T to A; (b) T to G; (c) C to G; (d) C to A; (e) A to T; (f) A to C; (g) G to C; and (h) G to T.
  98. The method of embodiment 93, wherein the one or more changes in the nucleotide sequence comprises changing (1) a G:C basepair to a T:A basepair, (2) a G:C basepair to an A:T basepair, (3) a G:C basepair to C:G basepair, (4) a T:A basepair to a G:C basepair, (5) a T:A basepair to an A:T basepair, (6) a T:A basepair to a C:G basepair, (7) a C:G basepair to a G:C basepair, (8) a C:G basepair to a T:A basepair, (9) a C:G basepair to an A:T basepair, (10) an A:T basepair to a T:A basepair, (11) an A:T basepair to a G:C basepair, or (12) an A:T basepair to a C:G basepair.
  99. The method of embodiment 93, wherein the one or more changes in the nucleotide sequence comprises an insertion or deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides.
  100. The method of embodiment 93, wherein the one or more changes in the nucleotide sequence comprises a correction to a disease-associated gene.
  101. The method of embodiment 100, wherein the disease-associated gene is associated with a monogenetic disorder selected from the group consisting of: Adenosine Deaminase (ADA) Deficiency; Alpha-1 Antitrypsin Deficiency; Cystic Fibrosis; Duchenne Muscular Dystrophy; Galactosemia; Hemochromatosis; Huntington's Disease; Maple Syrup Urine Disease; Marfan Syndrome; Neurofibromatosis Type 1; Pachyonychia Congenita; Phenylkeotnuria; Severe Combined Immunodeficiency; Sickle Cell Disease; Smith-Lemli-Opitz Syndrome; and Tay-Sachs Disease.
  102. The method of embodiment 100, wherein the disease-associated gene is associated with a polygenic disorder selected from the group consisting of: heart disease; high blood pressure; Alzheimer's disease; arthritis; diabetes; cancer; and obesity.
  103. The method of embodiment 93, wherein the napDNAbp is a nuclease active Cas9 or variant thereof.
  104. The method of embodiment 93, wherein the napDNAbp is a nuclease inactive Cas9 (dCas9) or Cas9 nickase (nCas9), or a variant thereof.
  105. The method of embodiment 93, wherein the napDNAbp comprises an amino acid sequence of SEQ ID NO: 2.
  106. The method of embodiment 93, wherein the napDNAbp comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 2-10.
  107. The method of embodiment 93, wherein the reverse transcriptase is introduced in trans.
  108. The method of embodiment 93, wherein the napDNAbp comprises a fusion to a reverse transcriptase.
  109. The method of embodiment 93, wherein the reverse transcriptase comprises any one of the amino acid sequences of SEQ ID NO: 11-17.
  110. The method of embodiment 93, wherein the reverse transcriptase comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 11-17.
  111. The method of embodiment 93, wherein the step of forming an exposed 3′ end in the DNA strand at the target locus comprises nicking the DNA strand with a nuclease.
  112. The method of embodiment 111, wherein the nuclease is the napDNAbp, is provided as a fusion domain of napDNAbp, or is provided in trans.
  113. The method of embodiment 93, wherein the step of forming an exposed 3′ end in the DNA strand at the target locus comprises contacting the DNA strand with a chemical agent.
  114. The method of embodiment 93, wherein the step of forming an exposed 3′ end in the DNA strand at the target locus comprises introducing a replication error.
  115. The method of embodiment 93, wherein the step of contacting the DNA molecule with the napDNAbp and the guide RNA forms an R-loop.
  116. The method of embodiment 115, wherein the DNA strand in which the exposed 3′ end is formed is in the R-loop.
  117. The method of embodiment 93, wherein guide RNA comprises an extended portion that comprises the reverse transcriptase (RT) template sequence.
  118. The method of embodiment 117, wherein the extended portion is at the 3′ end of the guide RNA, the 5′ end of the guide RNA, or at an intramolecular position in the guide RNA.
  119. The method of embodiment 93, wherein the guide RNA further comprises a primer binding site.
  120. The method of embodiment 93, wherein the guide RNA further comprises a spacer sequence.
  121. The method of embodiment 93, wherein the RT template sequence is homologous to the corresponding endogenous DNA.
  122. A method for introducing one or more changes in the nucleotide sequence of a DNA molecule at a target locus by target-primed reverse transcription, the method comprising: (a) contacting the DNA molecule at the target locus with a (i) fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a reverse transcriptase and (ii) a guide RNA comprising an RT template comprising a desired nucleotide change; (b) conducting target-primed reverse transcription of the RT template to generate a single strand DNA comprising the desired nucleotide change; and (c) incorporating the desired nucleotide change into the DNA molecule at the target locus through a DNA repair and/or replication process.
  123. The method of embodiment 122, wherein the RT template is located at the 3′ end of the guide RNA, the 5′ end of the guide RNA, or at an intramolecular location in the guide RNA.
  124. The method of embodiment 122, wherein the desired nucleotide change comprises a transition, a transversion, an insertion, or a deletion, or any combination thereof.
  125. The method of claim 122, wherein the desired nucleotide change comprises a transition selected from the group consisting of: (a) T to C; (b) A to G; (c) C to T; and (d) G to A.
  126. The method of claim 122, wherein the desired nucleotide change comprises a transversion selected from the group consisting of: (a) T to A; (b) T to G; (c) C to G; (d) C to A; (e) A to T; (f) A to C; (g) G to C; and (h) G to T.
  127. The method of embodiment 122, wherein the desired nucleotide change comprises changing (1) a G:C basepair to a T:A basepair, (2) a G:C basepair to an A:T basepair, (3) a G:C basepair to C:G basepair, (4) a T:A basepair to a G:C basepair, (5) a T:A basepair to an A:T basepair, (6) a T:A basepair to a C:G basepair, (7) a C:G basepair to a G:C basepair, (8) a C:G basepair to a T:A basepair, (9) a C:G basepair to an A:T basepair, (10) an A:T basepair to a T:A basepair, (11) an A:T basepair to a G:C basepair, or (12) an A:T basepair to a C:G basepair.
  128. A polynucleotide encoding the extended guide RNA of any one of embodiments 31-47.
  129. A vector comprising the polynucleotide of embodiment 128.
  130. A cell comprising the vector of embodiment 129.
  131. The fusion protein of any of embodiments 1-30, wherein the reverse transcriptase is an error-prone reverse transcriptase.
  132. A method for mutagenizing a DNA molecule at a target locus by target-primed reverse transcription, the method comprising: (a) contacting the DNA molecule at the target locus with a (i) fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) and an error-prone reverse transcriptase and (ii) a guide RNA comprising an RT template comprising a desired nucleotide change; (b) conducting target-primed reverse transcription of the RT template to generate a mutagenized single strand DNA; and (c) incorporating the mutagenized single strand DNA into the DNA molecule at the target locus through a DNA repair and/or replication process.
  133. The method of embodiment 132, wherein the fusion protein comprises the amino acid sequence of PE1, PE2, or PE3.
  134. The method of embodiment 132, wherein the napDNAbp is a Cas9 nickase (nCas9).
  135. The method of embodiment 132, wherein the napDNAbp comprises the amino acid sequence of SEQ ID NOs: 18-25.
  136. The method of embodiment 132, wherein the guide RNA comprises SEQ ID NOs: 222.
  137. The method of embodiment 132, wherein the step of (b) conducting target-primed reverse transcription comprises generating a 3′ end primer binding sequence at the target locus that is capable of priming reverse transcription by annealing to a primer binding site on the guide RNA.
  138. A method for replacing a trinucleotide repeat expansion mutation in a target DNA molecule with a healthy sequence comprising a healthy number of repeat trinucleotides, the method comprising: (a) contacting the DNA molecule at the target locus with a (i) fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a reverse transcriptase and (ii) a guide RNA comprising an RT template comprising the replacement sequence, wherein said fusion protein intr; (b) conducting target-primed reverse transcription of the RT template to generate a single strand DNA comprising the replacement sequence; and (c) incorporating the single strand DNA into the DNA molecule at the target locus through a DNA repair and/or replication process.
  139. The method of embodiment 138, wherein the fusion protein comprises the amino acid sequence of PE1, PE2, or PE3.
  140. The method of embodiment 138, wherein the napDNAbp is a Cas9 nickase (nCas9).
  141. The method of embodiment 138, wherein the napDNAbp comprises the amino acid sequence of SEQ ID NOs: 18-25.
  142. The method of embodiment 138, wherein the guide RNA comprises SEQ ID NOs: 222.
  143. The method of embodiment 138, wherein the step of (b) conducting target-primed reverse transcription comprises generating a 3′ end primer binding sequence at the target locus that is capable of priming reverse transcription by annealing to a primer binding site on the guide RNA.
  144. The method of embodiment 138, wherein the trinucleotide repeat expansion mutation is associated with Huntington's Disease, Fragile X syndrome, or Friedreich's ataxia.
  145. The method of embodiment 138, wherein the trinucleotide repeat expansion mutation comprises a repeating unit of CAG triplets.
  146. The method of embodiment 138, wherein the trinucleotide repeat expansion mutation comprises a repeating unit of GAA triplets.
  147. A method of installing a functional moiety in a protein of interest encoded by a target nucleotide sequence by prime editing, the method comprising: (a) contacting the target nucleotide sequence with a (i) prime editor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a reverse transcriptase and (ii) a PEgRNA comprising an edit template encoding the functional moiety; (b) polymerizing a single strand DNA sequence encoding the functional moiety; and (c) incorporating the single strand DNA sequence in place of a corresponding endogenous strand at the target nucleotide sequence through a DNA repair and/or replication process, wherein the method produces a recombinant target nucleotide sequence that encodes a fusion protein comprising the protein of interest and the functional moiety.
  148. The method of embodiment 147, wherein functional moiety is peptide tag.
  149. The method of embodiment 148, wherein the peptide tag is an affinity tag, solubilization tag, chromatography tag, epitope tag, or a fluorescence tag.
  150. The method of embodiment 148, wherein the peptide tag is selected from the group consisting of: AviTag (SEQ ID NO: 245); C-tag (SEQ ID NO: 246); Calmodulin-tag (SEQ ID NO: 247); polyglutamate tag (SEQ ID NO: 248); E-tag (SEQ ID NO: 249); FLAG-tag (SEQ ID NO: 250); HA-tag (SEQ ID NO: 251); His-tag (SEQ ID NOs: 252-262); Myc-tag (SEQ ID NO: 263); NE-tag (SEQ ID NO: 264); Rho1D4-tag (SEQ ID NO: 265); S-tag (SEQ ID NO: 266); SBP-tag (SEQ ID NO: 267); Softag-1 (SEQ ID NO: 268); Softag-2 (SEQ ID NO: 269); Spot-tag (SEQ ID NO: 270); Strep-tag (SEQ ID NO: 271); TC tag (SEQ ID NO: 272); Ty tag (SEQ ID NO: 273); V5 tag (SEQ ID NO: 274); VSV-tag (SEQ ID NO: 275); and Xpress tag (SEQ ID NO: 276).
  151. The method of embodiment 148, wherein the peptide tag is selected from the group consisting of: AU1 epitope (SEQ ID NO: 278); AU5 epitope (SEQ ID NO: 279); Bacteriophage T7 epitope (T7-tag) (SEQ ID NO: 280); Bluetongue virus tag (B-tag) (SEQ ID NO: 281); E2 epitope (SEQ ID NO: 282); Histidine affinity tag (HAT) (SEQ ID NO: 283); HSV epitope (SEQ ID NO: 284); Polyarginine (Arg-tag) (SEQ ID NO: 285); Polyaspartate (Asp-tag) (SEQ ID NO: 286); Polyphenylalanine (Phe-tag) (SEQ ID NO: 287); S1-tag (SEQ ID NO: 288); S-tag (SEQ ID NO: 289); and VSV-G (SEQ ID NO: 290).
  152. The method of embodiment 147, wherein the functional moiety is an immunoepitope.
  153. The method of embodiment 152, wherein the immunoepitope is selected from the group consisting of: tetanus toxoid (SEQ ID NO: 396); diphtheria toxin mutant CRM197 (SEQ ID NO: 398); mumps immunoepitope 1 (SEQ ID NO: 400); mumps immunoepitope 2 (SEQ ID NO: 402); mumps immunoeptitope 3 (SEQ ID NO: 404); rubella virus (SEQ ID NO: 406); hemagglutinin (SEQ ID NO: 408); neuraminidase (SEQ ID NO: 410); TAP1 (SEQ ID NO: 412); TAP2 (SEQ ID NO: 414); hemagglutinin epitopes toward class I HLA (SEQ ID NO: 416); neuraminidase epitopes toward class I HLA (SEQ ID NO: 418); hemagglutinin epitopes toward class II HLA (SEQ ID NO: 420); neuraminidase epitopes toward class II HLA (SEQ ID NO: 422); hemagglutinin epitope H5N1-bound class I and class II HLA (SEQ ID NO: 424); neuraminidase epitope H5N1-bound class I and class II HLA (SEQ ID NO: 426).
• 154. The method of embodiment 147, wherein the functional moiety alters the localization of the protein of interest.
• 155. The method of embodiment 147, wherein the functional moiety is a degradation tag such that the degradation rate of the protein of interest is altered.
• 156. The method of embodiment 155, wherein the degradation tag.
• 157. The method of embodiment 147, wherein the functional moiety is a small molecule binding domain.
• 158. The method of embodiment 157, wherein the small molecule binding domain is FKBP12 of SEQ ID NO: 488.
• 159. The method of embodiment 157, wherein the small molecule binding domain is FKBP12-F36V of SEQ ID NO: 489.
• 160. The method of embodiment 157, wherein the small molecule binding domain is cyclophilin of SEQ ID NOs: 492-494.
• 161. The method of embodiment 157, wherein the small molecule binding domain is installed in two or more proteins of interest.
• 162. The method of embodiment 161, wherein the two or more proteins of interest may dimerize upon contacting with a small molecule.
• 163. The method of embodiment 157, wherein the small molecule is a dimer of a small molecule selected from the group consisting of:
• 

  
• 164. A method of installing an immunoepitope in a protein of interest encoded by a target nucleotide sequence by prime editing, the method comprising: (a) contacting the target nucleotide sequence with a (i) prime editor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a reverse transcriptase and (ii) a PEgRNA comprising an edit template encoding the functional moiety; (b) polymerizing a single strand DNA sequence encoding the immunoepitope; and (c) incorporating the single strand DNA sequence in place of a corresponding endogenous strand at the target nucleotide sequence through a DNA repair and/or replication process, wherein the method produces a recombinant target nucleotide sequence that encodes a fusion protein comprising the protein of interest and the immunoepitope.
  165. The method of embodiment 164, wherein the immunoepitope is selected from the group consisting of: tetanus toxoid (SEQ ID NO: 396); diphtheria toxin mutant CRM197 (SEQ ID NO: 398); mumps immunoepitope 1 (SEQ ID NO: 400); mumps immunoepitope 2 (SEQ ID NO: 402); mumps immunoeptitope 3 (SEQ ID NO: 404); rubella virus (SEQ ID NO: 406); hemagglutinin (SEQ ID NO: 408); neuraminidase (SEQ ID NO: 410); TAP1 (SEQ ID NO: 412); TAP2 (SEQ ID NO: 414); hemagglutinin epitopes toward class I HLA (SEQ ID NO: 416); neuraminidase epitopes toward class I HLA (SEQ ID NO: 418); hemagglutinin epitopes toward class II HLA (SEQ ID NO: 420); neuraminidase epitopes toward class II HLA (SEQ ID NO: 422); hemagglutinin epitope H5N1-bound class I and class II HLA (SEQ ID NO: 424); neuraminidase epitope H5N1-bound class I and class II HLA (SEQ ID NO: 426).
  166. The method of embodiment 164, wherein the fusion protein comprises the amino acid sequence of PE1, PE2, or PE3.
  167. The method of embodiment 164, wherein the napDNAbp is a Cas9 nickase (nCas9).
  168. The method of embodiment 164, wherein the napDNAbp comprises the amino acid sequence of SEQ ID NOs: 18-25.
  169. The method of embodiment 164, wherein the guide RNA comprises SEQ ID NOs: 222.
• 170. A method of installing a small molecule dimerization domain in a protein of interest encoded by a target nucleotide sequence by prime editing, the method comprising: (a) contacting the target nucleotide sequence with a (i) prime editor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a reverse transcriptase and (ii) a PEgRNA comprising an edit template encoding the small molecule dimerization domain; (b) polymerizing a single strand DNA sequence encoding the immunoepitope; and (c) incorporating the single strand DNA sequence in place of a corresponding endogenous strand at the target nucleotide sequence through a DNA repair and/or replication process, wherein the method produces a recombinant target nucleotide sequence that encodes a fusion protein comprising the protein of interest and the small molecule dimerization domain.
• 171. The method of embodiment 170, further comprising conducting the method on a second protein of interest.
  172. The method of embodiment 171, wherein the first protein of interest and the second protein of interest dimerize in the presence of a small molecule that binds to the dimerization domain on each of said proteins.
  173. The method of embodiment 170, wherein the small molecule binding domain is FKBP12 of SEQ ID NO: 488.
  174. The method of embodiment 170, wherein the small molecule binding domain is FKBP12-F36V of SEQ ID NO: 489.
  175. The method of embodiment 170, wherein the small molecule binding domain is cyclophilin of SEQ ID NOs: 492-494.
  176. The method of embodiment 170, wherein the small molecule is a dimer of a small molecule selected from the group consisting of:
• 

  
• 177. The method of embodiment 170, wherein the fusion protein comprises the amino acid sequence of PE1, PE2, or PE3.
  178. The method of embodiment 170, wherein the napDNAbp is a Cas9 nickase (nCas9).
  179. The method of embodiment 170, wherein the napDNAbp comprises the amino acid sequence of SEQ ID NOs: 18-25.
  180. The method of embodiment 170, wherein the guide RNA comprises SEQ ID NOs: 222.
  181. A method of installing a peptide tag or epitope onto a protein using prime editing, comprising: contacting a target nucleotide sequence encoding the protein with a prime editor construct configured to insert therein a second nucleotide sequence encoding the peptide tag to result in a recombinant nucleotide sequence, such that the peptide tag and the protein are expressed from the recombinant nucleotide sequence as a fusion protein.
  182. The method of embodiment 181, wherein the peptide tag is used for purification and/or detection of the protein.
  183. The method of embodiment 181, wherein the peptide tag is a poly-histidine (e.g., HHHHHH), FLAG (e.g., DYKDDDDK), V5 (e.g., GKPIPNPLLGLDST), GCN4, HA (e.g., YPYDVPDYA), Myc (e.g. EQKLISEED), GST . . . etc.
  184. The method of embodiment 181, wherein the peptide tag has an amino acid sequence selected from the group consisting of SEQ ID NO: 245-290.
  185. The method of embodiment 181, wherein the peptide tag is fused to the protein by a linker.
  186. The method of embodiment 181, wherein the fusion protein has the following structure: [protein]-[peptide tag] or [peptide tag]-[protein], wherein “]-[” represents an optional linker.
  187. The method of embodiment 181, wherein the linker has an amino acid sequence of SEQ ID NO: 127, 165-176, 446, 453, and 767-769.
  188. The method of embodiment 181, wherein the prime editor construct comprises a PEgRNA comprising the nucleotide sequence of SEQ ID NOs: 18-25.
  189. The method of embodiment 181, wherein the PEgRNA comprises a spacer, a gRNA core, and an extension arm, wherein the spacer is complementary to the target nucleotide sequence and the extension arm comprises a reverse transcriptase template that encodes the peptide tag.
  190. The method of embodiment 181, wherein the PEgRNA comprises a spacer, a gRNA core, and an extension arm, wherein the spacer is complementary to the target nucleotide sequence and the extension arm comprises a reverse transcriptase template that encodes the peptide tag.
  191. A method of preventing or halting the progression of a prion disease by installing on or more protective mutations into PRNP encoded by a target nucleotide sequence by prime editing, the method comprising: (a) contacting the target nucleotide sequence with a (i) prime editor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a reverse transcriptase and (ii) a PEgRNA comprising an edit template encoding the functional moiety; (b) polymerizing a single strand DNA sequence encoding the protective mutation; and (c) incorporating the single strand DNA sequence in place of a corresponding endogenous strand at the target nucleotide sequence through a DNA repair and/or replication process, wherein the method produces a recombinant target nucleotide sequence that encodes a PRNP comprising a protective mutation and which is resistant to misfolding.
  192. The method of embodiment 191, wherein the prion disease is a human prion disease.
  193. The method of embodiment 191, wherein the prion disease is an animal prion disease.
  194. The method of embodiment 192, wherein the prion disease is Creutzfeldt-Jakob Disease (CJD), Variant Creutzfeldt-Jakob Disease (vCJD), Gerstmann-Straussler-Scheinker Syndrome, Fatal Familial Insomnia, or Kuru.
  195. The method of embodiment 193, wherein the prion disease is Bovine Spongiform Encephalopathy (BSE or “mad cow disease”), Chronic Wasting Disease (CWD), Scrapie, Transmissible Mink Encephalopathy, Feline Spongiform Encephalopathy, and Ungulate Spongiform Encephalopathy.
  196. The method of embodiment 191, wherein the wildtype PRNP amino acid sequence is SEQ ID NOs: 291-292. 197. The method of embodiment 191, wherein the method results in a modified PRNP amino acid sequence selected from the group consisting of SEQ ID NOs: 293-323, wherein said modified PRNP protein is resistant to misfolding.
  198. The method of embodiment 191, wherein the fusion protein comprises the amino acid sequence of PE1, PE2, or PE3.
  199. The method of embodiment 191, wherein the napDNAbp is a Cas9 nickase (nCas9).
  200. The method of embodiment 191, wherein the napDNAbp comprises the amino acid sequence of SEQ ID NOs: 18-25.
  201. The method of embodiment 191, wherein the guide RNA comprises SEQ ID NOs: 222.
  202. A method of installing a ribonucleotide motif or tag in an RNA of interest encoded by a target nucleotide sequence by prime editing, the method comprising: (a) contacting the target nucleotide sequence with a (i) prime editor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a reverse transcriptase and (ii) a PEgRNA comprising an edit template encoding the ribonucleotide motif or tag; (b) polymerizing a single strand DNA sequence encoding the ribonucleotide motif or tag; and (c) incorporating the single strand DNA sequence in place of a corresponding endogenous strand at the target nucleotide sequence through a DNA repair and/or replication process, wherein the method produces a recombinant target nucleotide sequence that encodes a modified RNA of interest comprising the ribonucleotide motif or tag.
  203. The method of embodiment 202, wherein ribonucleotide motif or tag is a detection moiety.
  204. The method of embodiment 202, wherein the ribonucleotide motif or tag affects the expression level of the RNA of interest.
  205. The method of embodiment 202, wherein the ribonucleotide motif or tag affects the transport or subcellular location of the RNA of interest.
  206. The method of embodiment 202, wherein the ribonucleotide motif or tag is selected from the group consisting of SV40 type 1, SV40 type 2, SV40 type 3, hGH, BGH, rbGlob, TK, MALAT1 ENE-mascRNA, KSHV PAN ENE, Smbox/U1 snRNA box, U1 snRNA 3′ box, tRNA-lysine, broccoli aptamer, spinach aptamer, mango aptamer, HDV ribozyme, and m6A.
  207. The method of embodiment 202, wherein the PEgRNA comprises SEQ ID NOs: 18-25.
  208. The method of embodiment 202, wherein the fusion protein comprises the amino acid sequence of PE1, PE2, or PE3.
  209. The method of embodiment 202, wherein the napDNAbp is a Cas9 nickase (nCas9).
  210. The method of embodiment 202, wherein the napDNAbp comprises the amino acid sequence of SEQ ID NOs: 18-25.
  211. A method of installing or deleting a functional moiety in a protein of interest encoded by a target nucleotide sequence by prime editing, the method comprising: (a) contacting the target nucleotide sequence with a (i) prime editor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a reverse transcriptase and (ii) a PEgRNA comprising an edit template encoding the functional moiety or deletion of same; (b) polymerizing a single strand DNA sequence encoding the functional moiety or deletion of same; and (c) incorporating the single strand DNA sequence in place of a corresponding endogenous strand at the target nucleotide sequence through a DNA repair and/or replication process, wherein the method produces a recombinant target nucleotide sequence that encodes a modified protein comprising the protein of interest and the functional moiety or the removal of same, wherein the functional moiety alters a modification state or localization state of the protein.
  212. The method of embodiment 211, wherein functional moiety alters the phosphorylation, ubiquitylation, glycosylation, lipidation, hydroxylation, methylation, acetylation, crotonylation, SUMOylation state of the protein of interest.
GROUP 2. EMBODIMENTS 213-424
• 213. A fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a polymerase.
  214. The fusion protein of embodiment 213, wherein the fusion protein is capable of carrying out prime editing in the presence of an prime editing guide RNA (PEgRNA).
  215. The fusion protein of embodiment 213, wherein the napDNAbp has a nickase activity.
  216. The fusion protein of embodiment 213, wherein the napDNAbp is a Cas9 protein or variant thereof.
  217. The fusion protein of embodiment 213, wherein the napDNAbp is a nuclease active Cas9, a nuclease inactive Cas9 (dCas9), or a Cas9 nickase (nCas9).
  218. The fusion protein of embodiment 213, wherein the napDNAbp is Cas9 nickase (nCas9).
  219. The fusion protein of embodiment 213, wherein the napDNAbp is selected from the group consisting of: Cas9, Cas12e, Cas12d, Cas12a, Cas12b1, Cas13a, Cas12c, and Argonaute and optionally has a nickase activity.
  220. The fusion protein of embodiment 213, wherein the fusion protein when complexed with a PEgRNA is capable of binding to a target DNA sequence.
  221. The fusion protein of embodiment 220, wherein the target DNA sequence comprises a target strand and a complementary non-target strand.
  222. The fusion protein of embodiment 220, wherein the binding of the fusion protein complexed to the PEgRNA forms an R-loop.
  223. The fusion protein of embodiment 222, wherein the R-loop comprises (i) an RNA-DNA hybrid comprising the PEgRNA and the target strand, and (ii) the complementary non-target strand.
  224. The fusion protein of embodiment 223, wherein the complementary non-target strand is nicked to form a priming sequence having a free 3′ end.
  225. The fusion protein of embodiment 214, wherein the PEgRNA comprises (a) a guide RNA and (b) an extension arm at the 5′ or the 3′ end of the guide RNA, or at an intramolecular location in the guide RNA.
  226. The fusion protein of embodiment 225, wherein the extension arm comprises (i) a DNA synthesis template sequence comprising a desired nucleotide change, and (ii) a primer binding site.
  227. The fusion protein of embodiment 226, wherein the DNA synthesis template sequence encodes a single-strand DNA flap that is complementary to an endogenous DNA sequence adjacent to the nick site, wherein the single-strand DNA flap comprises the desired nucleotide change.
  228. The fusion protein of embodiment 225, wherein the extension arm is at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides, at least 22 nucleotides, at least 23 nucleotides, at least 24 nucleotides, or at least 25 nucleotides in length.
  229. The fusion protein of embodiment 227, wherein the single-strand DNA flap hybridizes to the endogenous DNA sequence adjacent to the nick site, thereby installing the desired nucleotide change.
  230. The fusion protein of embodiment 227, wherein the single-stranded DNA flap displaces the endogenous DNA sequence adjacent to the nick site and which has a free 5′ end.
  231. The fusion protein of embodiment 230, wherein the endogenous DNA sequence having the 5′ end is excised by the cell.
  232. The fusion protein of embodiment 230, wherein cellular repair of the single-strand DNA flap results in installation of the desired nucleotide change, thereby forming a desired product.
  233. The fusion protein of embodiment 226, wherein the desired nucleotide change is installed in an editing window that is between about −4 to +10 of the PAM sequence, or between about −10 to +20 of the PAM sequence, or between about −20 to +40 of the PAM sequence, or between about −30 to +100 of the PAM sequence, or wherein the desired nucleotide change is installed at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides downstream of the nick site.
  234. The fusion protein of embodiment 213, wherein the napDNAbp comprises an amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence to SEQ ID NO: 2.
  235. The fusion protein of embodiment 213, wherein the napDNAbp comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 2-10.
  236. The fusion protein of embodiment 213, wherein the polymerase is a reverse transcriptase comprising any one of the amino acid sequences of SEQ ID NO: 11-17.
  237. The fusion protein of embodiment 213, wherein the polymerase is a reverse transcriptase comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 11-17.
  238. The fusion protein of embodiment 213, wherein the polymerase is a naturally-occurring reverse transcriptase from a retrovirus or a retrotransposon.
  239. The fusion protein of any one of the previous embodiments, wherein the fusion protein comprises the structure NH₂-[napDNAbp]-[polymerase]-COOH; or NH₂-[polymerase]-[napDNAbp]-COOH, wherein each instance of “]-[” indicates the presence of an optional linker sequence.
  240. The fusion protein of embodiment 239, wherein the linker sequence comprises an amino acid sequence of SEQ ID NOs: 37-47.
  241. The fusion protein of embodiment 226, wherein the desired nucleotide change is a single nucleotide change, an insertion of one or more nucleotides, or a deletion of one or more nucleotides.
  242. The fusion protein of embodiment 241, wherein the insert or deletion is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, or at least 50.
  243. A PEgRNA comprising a guide RNA and at least one nucleic acid extension arm comprising a DNA synthesis template.
  244. The PEgRNA of embodiment 241, wherein the nucleic acid extension arm is position at the 3′ or 5′ end of the guide RNA, or at an intramolecular position in the guide RNA, and wherein the nucleic acid extension arm is DNA or RNA.
  245. The PEgRNA of embodiment 242, wherein the PEgRNA is capable of binding to a napDNAbp and directing the napDNAbp to a target DNA sequence.
  246. The PEgRNA of embodiment 245, wherein the target DNA sequence comprises a target strand and a complementary non-target strand, wherein the guide RNA hybridizes to the target strand to form an RNA-DNA hybrid and an R-loop.
  247. The PEgRNA of embodiment 243, wherein the at least one nucleic acid extension arm comprises (i) a DNA synthesis template, and (ii) a primer binding site.
  248. The PEgRNA of embodiment 247, wherein the nucleic acid extension arm is at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides, at least 22 nucleotides, at least 23 nucleotides, at least 24 nucleotides, or at least 25 nucleotides in length.
  249. The PEgRNA of embodiment 247, wherein the DNA synthesis template is at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, or at least 15 nucleotides in length.
  250. The PEgRNA of embodiment 247, wherein the primer binding site is at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, or at least 15 nucleotides in length.
  251. The PEgRNA of embodiment 243, further comprising at least one additional structure selected from the group consisting of a linker, a stem loop, a hairpin, a toeloop, an aptamer, or an RNA-protein recruitment domain.
  252. The PEgRNA of embodiment 247, wherein the DNA synthesis template encodes a single-strand DNA flap that is complementary to an endogenous DNA sequence adjacent to a nick site, wherein the single-strand DNA flap comprises a desired nucleotide change.
  253. The PEgRNA of embodiment 252, wherein the single-stranded DNA flap displaces an endogenous single-strand DNA having a 5′ end in the target DNA sequence that has been nicked, and wherein the endogenous single-strand DNA is immediately adjacent downstream of the nick site.
  254. The PEgRNA of embodiment 253, wherein the endogenous single-stranded DNA having the free 5′ end is excised by the cell.
  255. The PEgRNA of embodiment 253, wherein cellular repair of the single-strand DNA flap results in installation of the desired nucleotide change, thereby forming a desired product.
  256. The PEgRNA of embodiment 243, comprising the nucleotide sequence of SEQ ID NOs: 18-36, or a nucleotide sequence having at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99% sequence identity with any one of SEQ ID NOs: 18-36.
  257. The PEgRNA of embodiment 247, wherein the DNA synthesis template comprises a nucleotide sequence that is at least 80%, or 85%, or 90%, or 95%, or 99% identical to the endogenous DNA target.
  258. The PEgRNA of embodiment 247, wherein the primer binding site hybridizes with a free 3′ end of the cut DNA.
  259. The PEgRNA of embodiment 251, wherein the at least one additional structure is located at the 3′ or 5′ end of the PEgRNA.
  260. A complex comprising a fusion protein of any one of embodiments 213-242 and an PEgRNA.
  261. The complex of embodiment 260, wherein the PEgRNA comprises a guide RNA and an nucleic acid extension arm at the 3′ or 5′ end of the guide RNA or at an intramolecular position in the guide RNA.
  262. The complex of embodiment 260, wherein the PEgRNA is capable of binding to a napDNAbp and directing the napDNAbp to a target DNA sequence.
  263. The complex of embodiment 262, wherein the target DNA sequence comprises a target strand and a complementary non-target strand, wherein the guide RNA hybridizes to the target strand to form an RNA-DNA hybrid and an R-loop.
  264. The complex of embodiment 261, wherein the at least one nucleic acid extension arm comprises (i) a DNA synthesis template, and (ii) a primer binding site.
  265. The complex of embodiment 260, wherein the PEgRNA comprises the nucleotide sequence of SEQ ID NOs: 18-36, or a nucleotide sequence having at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99% sequence identity with any one of SEQ ID NOs: 18-36.
  266. The complex of embodiment 264, wherein the DNA synthesis template comprises a nucleotide sequence that is at least 80%, or 85%, or 90%, or 95%, or 99% identical to the endogenous DNA target.
  267. The complex of embodiment 264, wherein the primer binding site hybridizes with a free 3′ end of the cut DNA.
  268. A complex comprising a napDNAbp and an PEgRNA.
  269. The complex of embodiment 268, wherein the napDNAbp is a Cas9 nickase.
  270. The complex of embodiment 268, wherein the napDNAbp comprises an amino acid sequence of SEQ ID NO: 2.
  271. The complex of embodiment 268, wherein the napDNAbp comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 2-10.
  272. The complex of embodiment 268, wherein the PEgRNA comprises a guide RNA and a nucleic acid extension arm at the 3′ or 5′ end of the guide RNA, or at an intramolecular position in the guide RNA.
  273. The complex of embodiment 268, wherein the PEgRNA is capable of directing the napDNAbp to a target DNA sequence.
  274. The complex of embodiment 272, wherein the target DNA sequence comprises a target strand and a complementary non-target strand, wherein the spacer sequence of the PEgRNA hybridizes to the target strand to form an RNA-DNA hybrid and an R-loop.
  275. The complex of embodiment 273, wherein the nucleic acid extension arm comprises (i) a DNA synthesis template, and (ii) a primer binding site.
  276. The complex of embodiment 269, wherein the PEgRNA comprises the nucleotide sequence of SEQ ID NOs: 18-36, or a nucleotide sequence having at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99% sequence identity with any one of SEQ ID NOs: 18-36.
  277. The complex of embodiment 276, wherein the DNA synthesis template comprises a nucleotide sequence that is at least 80%, or 85%, or 90%, or 95%, or 99% identical to the endogenous DNA target.
  278. The complex of embodiment 276, wherein the primer binding site hybridizes with a free 3′ end of the cut DNA.
  279. The complex of embodiment 276, wherein the PEgRNA further comprises at least one additional structure selected from the group consisting of a linker, a stem loop, a hairpin, a toeloop, an aptamer, or an RNA-protein recruitment domain.
  280. A polynucleotide encoding the fusion protein of any of embodiments 213-242.
  281. A vector comprising the polynucleotide of embodiment 280.
  282. A cell comprising the fusion protein of any of embodiments 213-242 and an PEgRNA bound to the napDNAbp of the fusion protein.
  283. A cell comprising a complex of any one of embodiments 260-279.
  284. A pharmaceutical composition comprising: (i) a fusion protein of any of embodiments 213-242, the complex of embodiments 260-279, the polynucleotide of embodiment 68, or the vector of embodiment 69; and (ii) a pharmaceutically acceptable excipient.
  285. A pharmaceutical composition comprising: (i) the complex of embodiments 260-279 (ii) a polymerase provided in trans; and (iii) a pharmaceutically acceptable excipient.
  286. A kit comprising a nucleic acid construct, comprising: (i) a nucleic acid sequencing encoding the fusion protein of any one of embodiments 213-242; and (ii) a promoter that drives expression of the sequence of (i).
  287. A method for installing a desired nucleotide change in a double-stranded DNA sequence, the method comprising:
• (i) contacting the double-stranded DNA sequence with a complex comprising a fusion protein and a PEgRNA, wherein the fusion protein comprises a napDNAbp and a polymerase and wherein the PEgRNA comprises a DNA synthesis template comprising the desired nucleotide change and a primer binding site;
• (ii) nicking the double-stranded DNA sequence, thereby generating a free single-strand DNA having a 3′ end;
• (iii) hybridizing the 3′ end of the free single-strand DNA to the primer binding site, thereby priming the polymerase;
• (iv) polymerizing a strand of DNA from the 3′ end hybridized to the primer binding site, thereby generating a single-strand DNA flap comprising the desired nucleotide change and which is complementary to the DNA synthesis template;
• (v) replacing an endogenous DNA strand adjacent the cut site with the single-strand DNA flap, thereby installing the desired nucleotide change in the double-stranded DNA sequence.
  288. The method of embodiment 287, wherein the step of (v) replacing comprises: (i) hybridizing the single-strand DNA flap to the endogenous DNA strand adjacent the cut site to create a sequence mismatch; (ii) excising the endogenous DNA strand; and (iii) repairing the mismatch to form the desired product comprising the desired nucleotide change in both strands of DNA.
  289. The method of embodiment 288, wherein the desired nucleotide change is a single nucleotide substitution, a deletion, or an insertion.
  290. The method of embodiment 289, wherein the single nucleotide substitution is a transition or a transversion.
  291. The method of embodiment 288, wherein the desired nucleotide change is (1) a G to T substitution, (2) a G to A substitution, (3) a G to C substitution, (4) a T to G substitution, (5) a T to A substitution, (6) a T to C substitution, (7) a C to G substitution, (8) a C to T substitution, (9) a C to A substitution, (10) an A to T substitution, (11) an A to G substitution, or (12) an A to C substitution.
  292. The method of embodiment 288, wherein the desired nucleoid change converts (1) a G:C basepair to a T:A basepair, (2) a G:C basepair to an A:T basepair, (3) a G:C basepair to C:G basepair, (4) a T:A basepair to a G:C basepair, (5) a T:A basepair to an A:T basepair, (6) a T:A basepair to a C:G basepair, (7) a C:G basepair to a G:C basepair, (8) a C:G basepair to a T:A basepair, a C:G basepair to an A:T basepair, (10) an A:T basepair to a T:A basepair, (11) an A:T basepair to a G:C basepair, or (12) an A:T basepair to a C:G basepair.
  293. The method of embodiment 288, wherein the desired nucleotide change is an insertion or deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides.
  294. The method of embodiment 288, wherein the desired nucleotide change corrects a disease-associated gene.
  295. The method of embodiment 294, wherein the disease-associated gene is associated with a monogenetic disorder selected from the group consisting of: Adenosine Deaminase (ADA) Deficiency; Alpha-1 Antitrypsin Deficiency; Cystic Fibrosis; Duchenne Muscular Dystrophy; Galactosemia; Hemochromatosis; Huntington's Disease; Maple Syrup Urine Disease; Marfan Syndrome; Neurofibromatosis Type 1; Pachyonychia Congenita; Phenylkeotnuria; Severe Combined Immunodeficiency; Sickle Cell Disease; Smith-Lemli-Opitz Syndrome; a trinucleotide repeat disorder; a prion disease; and Tay-Sachs Disease.
  296. The method of embodiment 294, wherein the disease-associated gene is associated with a polygenic disorder selected from the group consisting of: heart disease; high blood pressure; Alzheimer's disease; arthritis; diabetes; cancer; and obesity.
  297. The method of embodiment 287, wherein the napDNAbp is a nuclease dead Cas9 (dCas9), a Cas9 nickase (nCas9), or a nuclease active Cas9.
  298. The method of embodiment 287, wherein the napDNAbp comprises an amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 2.
  299. The method of embodiment 287, wherein the napDNAbp comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 2-10.
  300. The method of embodiment 287, wherein the polymerase is a reverse transcriptase comprising any one of the amino acid sequences of SEQ ID NO: 11-17.
  301. The method of embodiment 287, wherein the polymerase is a reverse transcriptase comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 11-17.
  302. The method of embodiment 287, wherein the PEgRNA comprises a nucleic acid extension arm at the 3′ or 5′ ends or at an intramolecular location in the guide RNA, wherein the extension arm comprises the DNA synthesis template sequence and the primer binding site.
  303. The method of embodiment 302, wherein the extension arm is at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides, at least 22 nucleotides, at least 23 nucleotides, at least 24 nucleotides, or at least 25 nucleotides in length.
  304. The method of embodiment 287, wherein the PEgRNA has a nucleotide sequence selected from the group consisting of SEQ ID NOs: 18-36.
  305. A method for introducing one or more changes in the nucleotide sequence of a DNA molecule at a target locus, comprising:
• (i) contacting the DNA molecule with a nucleic acid programmable DNA binding protein (napDNAbp) and a PEgRNA which targets the napDNAbp to the target locus, wherein the PEgRNA comprises a reverse transcriptase (RT) template sequence comprising at least one desired nucleotide change and a primer binding site;
• (ii) forming an exposed 3′ end in a DNA strand at the target locus;
• (iii) hybridizing the exposed 3′ end to the primer binding site to prime reverse transcription;
• (iv) synthesizing a single strand DNA flap comprising the at least one desired nucleotide change based on the RT template sequence by reverse transcriptase;
• (v) and incorporating the at least one desired nucleotide change into the corresponding endogenous DNA, thereby introducing one or more changes in the nucleotide sequence of the DNA molecule at the target locus.
• 306. The method of embodiment 305, wherein the one or more changes in the nucleotide sequence comprises a transition.
  307. The method of embodiment 306, wherein the transition is selected from the group consisting of: (a) T to C; (b) A to G; (c) C to T; and (d) G to A.
  308. The method of embodiment 305, wherein the one or more changes in the nucleotide sequence comprises a transversion.
  309. The method of embodiment 308, wherein the transversion is selected from the group consisting of: (a) T to A; (b) T to G; (c) C to G; (d) C to A; (e) A to T; (f) A to C; (g) G to C; and (h) G to T.
  310. The method of embodiment 305, wherein the one or more changes in the nucleotide sequence comprises changing (1) a G:C basepair to a T:A basepair, (2) a G:C basepair to an A:T basepair, (3) a G:C basepair to C:G basepair, (4) a T:A basepair to a G:C basepair, (5) a T:A basepair to an A:T basepair, (6) a T:A basepair to a C:G basepair, (7) a C:G basepair to a G:C basepair, (8) a C:G basepair to a T:A basepair, (9) a C:G basepair to an A:T basepair, (9) an A:T basepair to a T:A basepair, (11) an A:T basepair to a G:C basepair, or (12) an A:T basepair to a C:G basepair.
  311. The method of embodiment 305, wherein the one or more changes in the nucleotide sequence comprises an insertion or deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides.
  312. The method of embodiment 305, wherein the one or more changes in the nucleotide sequence comprises a correction to a disease-associated gene.
  313. The method of embodiment 312, wherein the disease-associated gene is associated with a monogenetic disorder selected from the group consisting of: Adenosine Deaminase (ADA) Deficiency; Alpha-1 Antitrypsin Deficiency; Cystic Fibrosis; Duchenne Muscular Dystrophy; Galactosemia; Hemochromatosis; Huntington's Disease; Maple Syrup Urine Disease; Marfan Syndrome; Neurofibromatosis Type 1; Pachyonychia Congenita; Phenylkeotnuria; Severe Combined Immunodeficiency; Sickle Cell Disease; Smith-Lemli-Opitz Syndrome; a trinucleotide repeat disorder; a prion disease; and Tay-Sachs Disease.
  314. The method of embodiment 312, wherein the disease-associated gene is associated with a polygenic disorder selected from the group consisting of: heart disease; high blood pressure; Alzheimer's disease; arthritis; diabetes; cancer; and obesity.
  315. The method of embodiment 305, wherein the napDNAbp is a nuclease active Cas9 or variant thereof.
  316. The method of embodiment 305, wherein the napDNAbp is a nuclease inactive Cas9 (dCas9) or Cas9 nickase (nCas9), or a variant thereof.
  317. The method of embodiment 305, wherein the napDNAbp comprises an amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO: 2.
  318. The method of embodiment 305, wherein the napDNAbp comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 2-10.
  319. The method of embodiment 305, wherein the reverse transcriptase is introduced in trans.
  320. The method of embodiment 305, wherein the napDNAbp comprises a fusion to a reverse transcriptase.
  321. The method of embodiment 305, wherein the reverse transcriptase comprises any one of the amino acid sequences of SEQ ID NO: 11-17.
  322. The method of embodiment 305, wherein the reverse transcriptase comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 11-17.
  323. The method of embodiment 305, wherein the step of forming an exposed 3′ end in the DNA strand at the target locus comprises nicking the DNA strand with a nuclease.
  324. The method of embodiment 323, wherein the nuclease is provided is provided in trans.
  325. The method of embodiment 305, wherein the step of forming an exposed 3′ end in the DNA strand at the target locus comprises contacting the DNA strand with a chemical agent.
  326. The method of embodiment 305, wherein the step of forming an exposed 3′ end in the DNA strand at the target locus comprises introducing a replication error.
  327. The method of embodiment 305, wherein the step of contacting the DNA molecule with the napDNAbp and the guide RNA forms an R-loop.
  328. The method of embodiment 327, wherein the DNA strand in which the exposed 3′ end is formed is in the R-loop.
  329. The method of embodiment 315, wherein the PEgRNA comprises an extension arm that comprises the reverse transcriptase (RT) template sequence and the primer binding site.
  330. The method of embodiment 329, wherein the extension arm is at the 3′ end of the guide RNA, the 5′ end of the guide RNA, or at an intramolecular position in the guide RNA.
  331. The method of embodiment 305, wherein the PEgRNA further comprises at least one additional structure selected from the group consisting of a linker, a stem loop, a hairpin, a toeloop, an aptamer, or an RNA-protein recruitment domain.
  332. The method of embodiment 305, wherein the PEgRNA further comprises a homology arm.
  333. The method of embodiment 305, wherein the RT template sequence is homologous to the corresponding endogenous DNA.
  334. A method for introducing one or more changes in the nucleotide sequence of a DNA molecule at a target locus by target-primed reverse transcription, the method comprising: (a) contacting the DNA molecule at the target locus with a (i) fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a reverse transcriptase and (ii) a guide RNA comprising an RT template comprising a desired nucleotide change; (b) conducting target-primed reverse transcription of the RT template to generate a single strand DNA comprising the desired nucleotide change; and (c) incorporating the desired nucleotide change into the DNA molecule at the target locus through a DNA repair and/or replication process.
  335. The method of embodiment 334, wherein the RT template is located at the 3′ end of the guide RNA, the 5′ end of the guide RNA, or at an intramolecular location in the guide RNA.
  336. The method of embodiment 334, wherein the desired nucleotide change comprises a transition, a transversion, an insertion, or a deletion, or any combination thereof.
  337. The method of embodiment 334, wherein the desired nucleotide change comprises a transition selected from the group consisting of: (a) T to C; (b) A to G; (c) C to T; and (d) G to A.
  338. The method of claim 334, wherein the desired nucleotide change comprises a transversion selected from the group consisting of: (a) T to A; (b) T to G; (c) C to G; (d) C to A; (e) A to T; (f) A to C; (g) G to C; and (h) G to T.
  339. The method of embodiment 334, wherein the desired nucleotide change comprises changing (1) a G:C basepair to a T:A basepair, (2) a G:C basepair to an A:T basepair, (3) a G:C basepair to C:G basepair, (4) a T:A basepair to a G:C basepair, (5) a T:A basepair to an A:T basepair, (6) a T:A basepair to a C:G basepair, (7) a C:G basepair to a G:C basepair, (8) a C:G basepair to a T:A basepair, (9) a C:G basepair to an A:T basepair, (10) an A:T basepair to a T:A basepair, (11) an A:T basepair to a G:C basepair, or (12) an A:T basepair to a C:G basepair.
  340. A polynucleotide encoding the PEgRNA of any one of embodiments 243-259.
  341. A vector comprising the polynucleotide of embodiment 340.
  342. A cell comprising the vector of embodiment 341.
  343. The fusion protein of embodiment 213, wherein the polymerase is an error-prone reverse transcriptase.
  344. A method for mutagenizing a DNA molecule at a target locus by target-primed reverse transcription, the method comprising: (a) contacting the DNA molecule at the target locus with a (i) fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) and an error-prone reverse transcriptase and (ii) a guide RNA comprising an RT template comprising a desired nucleotide change; (b) conducting target-primed reverse transcription of the RT template to generate a mutagenized single strand DNA; and (c) incorporating the mutagenized single strand DNA into the DNA molecule at the target locus through a DNA repair and/or replication process.
  345. The method of any prior embodiment, wherein the fusion protein comprises the amino acid sequence of PE1, PE2, or PE3.
  346. The method of any prior embodiment, wherein the napDNAbp is a Cas9 nickase (nCas9).
  347. The method of embodiment 344, wherein the napDNAbp comprises the amino acid sequence of SEQ ID NOs: 18-25.
  348. The method of embodiment 344, wherein the guide RNA comprises SEQ ID NOs: 26-36.
  349. The method of embodiment 344, wherein the step of (b) conducting target-primed reverse transcription comprises generating a 3′ end primer binding sequence at the target locus that is capable of priming reverse transcription by annealing to a primer binding site on the guide RNA.
  350. A method for replacing a trinucleotide repeat expansion mutation in a target DNA molecule with a healthy sequence comprising a healthy number of repeat trinucleotides, the method comprising: (a) contacting the DNA molecule at the target locus with a (i) fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a polymerase and (ii) a PEgRNA comprising DNA synthesis template comprising the replacement sequence and a primer binding site; (b) conducting prime editing to generate a single strand DNA comprising the replacement sequence; and (c) incorporating the single strand DNA into the DNA molecule at the target locus through a DNA repair and/or replication process.
  351. The method of embodiment 350, wherein the fusion protein comprises the amino acid sequence of PE1, PE2, or PE3.
  352. The method of embodiment 350, wherein the napDNAbp is a Cas9 nickase (nCas9).
  353. The method of embodiment 350, wherein the napDNAbp comprises the amino acid sequence of SEQ ID NOs: 18-25.
  354. The method of embodiment 350, wherein the guide RNA comprises SEQ ID NOs: 26-36.
  355. The method of embodiment 350, wherein the step of (b) conducting prime editing comprises generating a 3′ end primer binding sequence at the target locus that is capable of priming polymerase by annealing to the primer binding site on the guide RNA.
  356. The method of embodiment 350, wherein the trinucleotide repeat expansion mutation is associated with Huntington's Disease, Fragile X syndrome, or Friedreich's ataxia.
  357. The method of embodiment 350, wherein the trinucleotide repeat expansion mutation comprises a repeating unit of CAG triplets.
  358. The method of embodiment 350, wherein the trinucleotide repeat expansion mutation comprises a repeating unit of GAA triplets.
  359. A method of installing a functional moiety in a protein of interest encoded by a target nucleotide sequence by prime editing, the method comprising: (a) contacting the target nucleotide sequence with a (i) prime editor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a polymerase and (ii) a PEgRNA comprising DNA synthesis template encoding the functional moiety; (b) polymerizing a single strand DNA sequence encoding the functional moiety; and (c) incorporating the single strand DNA sequence in place of a corresponding endogenous strand at the target nucleotide sequence through a DNA repair and/or replication process, wherein the method produces a recombinant target nucleotide sequence that encodes a fusion protein comprising the protein of interest and the functional moiety.
  360. The method of embodiment 359, wherein functional moiety is peptide tag.
  361. The method of embodiment 360, wherein the peptide tag is an affinity tag, solubilization tag, chromatography tag, epitope or immunoepitope tag, or a fluorescence tag.
  362. The method of embodiment 360, wherein the peptide tag is selected from the group consisting of: AviTag (SEQ ID NO: 245); C-tag (SEQ ID NO: 246); Calmodulin-tag (SEQ ID NO: 247); polyglutamate tag (SEQ ID NO: 248); E-tag (SEQ ID NO: 249); FLAG-tag (SEQ ID NO: 250); HA-tag (SEQ ID NO: 251); His-tag (SEQ ID NOs: 252-262); Myc-tag (SEQ ID NO: 263); NE-tag (SEQ ID NO: 264); Rho1D4-tag (SEQ ID NO: 265); S-tag (SEQ ID NO: 266); SBP-tag (SEQ ID NO: 267); Softag-1 (SEQ ID NO: 268); Softag-2 (SEQ ID NO: 269); Spot-tag (SEQ ID NO: 270); Strep-tag (SEQ ID NO: 271); TC tag (SEQ ID NO: 272); Ty tag (SEQ ID NO: 273); V5 tag (SEQ ID NO: 274); VSV-tag (SEQ ID NO: 275); and Xpress tag (SEQ ID NO: 276).
  363. The method of embodiment 360, wherein the peptide tag is selected from the group consisting of: AU1 epitope (SEQ ID NO: 278); AU5 epitope (SEQ ID NO: 279); Bacteriophage T7 epitope (T7-tag) (SEQ ID NO: 280); Bluetongue virus tag (B-tag) (SEQ ID NO: 281); E2 epitope (SEQ ID NO: 282); Histidine affinity tag (HAT) (SEQ ID NO: 283); HSV epitope (SEQ ID NO: 284); Polyarginine (Arg-tag) (SEQ ID NO: 285); Polyaspartate (Asp-tag) (SEQ ID NO: 286); Polyphenylalanine (Phe-tag) (SEQ ID NO: 287); 51-tag (SEQ ID NO: 288); S-tag (SEQ ID NO: 289); and VSV-G (SEQ ID NO: 290).
  364. The method of embodiment 359, wherein the functional moiety is an immunoepitope.
  365. The method of embodiment 364, wherein the immunoepitope is selected from the group consisting of: tetanus toxoid (SEQ ID NO: 396); diphtheria toxin mutant CRM197 (SEQ ID NO: 398); mumps immunoepitope 1 (SEQ ID NO: 400); mumps immunoepitope 2 (SEQ ID NO: 402); mumps immunoeptitope 3 (SEQ ID NO: 404); rubella virus (SEQ ID NO: 406); hemagglutinin (SEQ ID NO: 408); neuraminidase (SEQ ID NO: 410); TAP1 (SEQ ID NO: 412); TAP2 (SEQ ID NO: 414); hemagglutinin epitopes toward class I HLA (SEQ ID NO: 416); neuraminidase epitopes toward class I HLA (SEQ ID NO: 418); hemagglutinin epitopes toward class II HLA (SEQ ID NO: 420); neuraminidase epitopes toward class II HLA (SEQ ID NO: 422); hemagglutinin epitope H5N1-bound class I and class II HLA (SEQ ID NO: 424); neuraminidase epitope H5N1-bound class I and class II HLA (SEQ ID NO: 426).
  366. The method of embodiment 359, wherein the functional moiety alters the localization of the protein of interest.
  367. The method of embodiment 359, wherein the functional moiety is a degradation tag such that the degradation rate of the protein of interest is altered.
  368. The method of embodiment 367, wherein the degradation tag results in the elimination of the tagged protein.
  369. The method of embodiment 359, wherein the functional moiety is a small molecule binding domain.
  370. The method of embodiment 359, wherein the small molecule binding domain is FKBP12 of SEQ ID NO: 488.
  371. The method of embodiment 359, wherein the small molecule binding domain is FKBP12-F36V of SEQ ID NO: 489.
  372. The method of embodiment 359, wherein the small molecule binding domain is cyclophilin of SEQ ID NOs: 492-494.
  373. The method of embodiment 359, wherein the small molecule binding domain is installed in two or more proteins of interest.
  374. The method of embodiment 373, wherein the two or more proteins of interest may dimerize upon contacting with a small molecule.
  375. The method of embodiment 369, wherein the small molecule is a dimer of a small molecule selected from the group consisting of:
• 

  
• 376. A method of installing an immunoepitope in a protein of interest encoded by a target nucleotide sequence by prime editing, the method comprising: (a) contacting the target nucleotide sequence with a (i) prime editor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a polymerase and (ii) a PEgRNA comprising an edit template encoding the functional moiety; (b) polymerizing a single strand DNA sequence encoding the immunoepitope; and (c) incorporating the single strand DNA sequence in place of a corresponding endogenous strand at the target nucleotide sequence through a DNA repair and/or replication process, wherein the method produces a recombinant target nucleotide sequence that encodes a fusion protein comprising the protein of interest and the immunoepitope.
  377. The method of embodiment 376, wherein the immunoepitope is selected from the group consisting of: tetanus toxoid (SEQ ID NO: 396); diphtheria toxin mutant CRM197 (SEQ ID NO: 398); mumps immunoepitope 1 (SEQ ID NO: 400); mumps immunoepitope 2 (SEQ ID NO: 402); mumps immunoeptitope 3 (SEQ ID NO: 404); rubella virus (SEQ ID NO: 406); hemagglutinin (SEQ ID NO: 408); neuraminidase (SEQ ID NO: 410); TAP1 (SEQ ID NO: 412); TAP2 (SEQ ID NO: 414); hemagglutinin epitopes toward class I HLA (SEQ ID NO: 416); neuraminidase epitopes toward class I HLA (SEQ ID NO: 418); hemagglutinin epitopes toward class II HLA (SEQ ID NO: 420); neuraminidase epitopes toward class II HLA (SEQ ID NO: 422); hemagglutinin epitope H5N1-bound class I and class II HLA (SEQ ID NO: 424); neuraminidase epitope H5N1-bound class I and class II HLA (SEQ ID NO: 426).
  378. The method of embodiment 376, wherein the fusion protein comprises the amino acid sequence of PE1, PE2, or PE3.
  379. The method of embodiment 376, wherein the napDNAbp is a Cas9 nickase (nCas9).
  380. The method of embodiment 376, wherein the napDNAbp comprises the amino acid sequence of SEQ ID NOs: 18-25.
  381. The method of embodiment 376, wherein the PEgRNA comprises SEQ ID NOs: 26-36.
  382. A method of installing a small molecule dimerization domain in a protein of interest encoded by a target nucleotide sequence by prime editing, the method comprising: (a) contacting the target nucleotide sequence with a (i) prime editor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a polymerase and (ii) a PEgRNA comprising an edit template encoding the small molecule dimerization domain; (b) polymerizing a single strand DNA sequence encoding the immunoepitope; and (c) incorporating the single strand DNA sequence in place of a corresponding endogenous strand at the target nucleotide sequence through a DNA repair and/or replication process, wherein the method produces a recombinant target nucleotide sequence that encodes a fusion protein comprising the protein of interest and the small molecule dimerization domain.
  383. The method of embodiment 382, further comprising conducting the method on a second protein of interest.
  384. The method of embodiment 383, wherein the first protein of interest and the second protein of interest dimerize in the presence of a small molecule that binds to the dimerization domain on each of said proteins.
  385. The method of embodiment 382, wherein the small molecule binding domain is FKBP12 of SEQ ID NO: 488.
  386. The method of embodiment 382, wherein the small molecule binding domain is FKBP12-F36V of SEQ ID NO: 489.
  387. The method of embodiment 382, wherein the small molecule binding domain is cyclophilin of SEQ ID NOs: 492-494.
  388. The method of embodiment 382, wherein the small molecule is a dimer of a small molecule selected from the group consisting of:
• 

  
• 389. The method of embodiment 382, wherein the fusion protein comprises the amino acid sequence of PE1, PE2, or PE3.
  390. The method of embodiment 382, wherein the napDNAbp is a Cas9 nickase (nCas9).
  391. The method of embodiment 382, wherein the napDNAbp comprises the amino acid sequence of SEQ ID NOs: 18-25.
  392. The method of embodiment 382, wherein the PEgRNA comprises SEQ ID NOs: 26-36.
  393. A method of installing a peptide tag or epitope onto a protein using prime editing, comprising: contacting a target nucleotide sequence encoding the protein with a prime editor construct configured to insert therein a second nucleotide sequence encoding the peptide tag to result in a recombinant nucleotide sequence, such that the peptide tag and the protein are expressed from the recombinant nucleotide sequence as a fusion protein.
  394. The method of embodiment 383, wherein the peptide tag is used for purification and/or detection of the protein.
  395. The method of embodiment 383, wherein the peptide tag is a poly-histidine (e.g., HHHHHH), FLAG (e.g., DYKDDDDK), V5 (e.g., GKPIPNPLLGLDST), GCN4, HA (e.g., YPYDVPDYA), Myc (e.g. EQKLISEED), or GST.
  396. The method of embodiment 383, wherein the peptide tag has an amino acid sequence selected from the group consisting of SEQ ID NO: 245-290.
  397. The method of embodiment 383, wherein the peptide tag is fused to the protein by a linker.
  398. The method of embodiment 383, wherein the fusion protein has the following structure: [protein]-[peptide tag] or [peptide tag]-[protein], wherein “]-[” represents an optional linker.
  399. The method of embodiment 383, wherein the linker has an amino acid sequence of SEQ ID NO: 37-47.
  400. The method of embodiment 383, wherein the prime editor construct comprises a PEgRNA comprising the nucleotide sequence of SEQ ID NOs: 18-25.
  401. The method of embodiment 383, wherein the PEgRNA comprises a spacer, a gRNA core, and an extension arm, wherein the spacer is complementary to the target nucleotide sequence and the extension arm comprises a reverse transcriptase template that encodes the peptide tag.
  402. The method of embodiment 383, wherein the PEgRNA comprises a spacer, a gRNA core, and an extension arm, wherein the spacer is complementary to the target nucleotide sequence and the extension arm comprises a reverse transcriptase template that encodes the peptide tag.
  403. A method of preventing or halting the progression of a prion disease by installing on or more protective mutations into PRNP encoded by a target nucleotide sequence by prime editing, the method comprising: (a) contacting the target nucleotide sequence with a (i) prime editor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a polymerase and (ii) a PEgRNA comprising an edit template encoding the functional moiety; (b) polymerizing a single strand DNA sequence encoding the protective mutation; and (c) incorporating the single strand DNA sequence in place of a corresponding endogenous strand at the target nucleotide sequence through a DNA repair and/or replication process, wherein the method produces a recombinant target nucleotide sequence that encodes a PRNP comprising a protective mutation and which is resistant to misfolding.
  404. The method of embodiment 403, wherein the prion disease is a human prion disease.
  405. The method of embodiment 403, wherein the prion disease is an animal prion disease.
  406. The method of embodiment 404, wherein the prion disease is Creutzfeldt-Jakob Disease (CJD), Variant Creutzfeldt-Jakob Disease (vCJD), Gerstmann-Straussler-Scheinker Syndrome, Fatal Familial Insomnia, or Kuru.
  407. The method of embodiment 403, wherein the prion disease is Bovine Spongiform Encephalopathy (BSE or “mad cow disease”), Chronic Wasting Disease (CWD), Scrapie, Transmissible Mink Encephalopathy, Feline Spongiform Encephalopathy, and Ungulate Spongiform Encephalopathy.
  408. The method of embodiment 403, wherein the wildtype PRNP amino acid sequence is SEQ ID NOs: 291-292.
  409. The method of embodiment 403, wherein the method results in a modified PRNP amino acid sequence selected from the group consisting of SEQ ID NOs: 293-323, wherein said modified PRNP protein is resistant to misfolding.
  410. The method of embodiment 403, wherein the fusion protein comprises the amino acid sequence of PE1, PE2, or PE3.
  411. The method of embodiment 403, wherein the napDNAbp is a Cas9 nickase (nCas9).
  412. The method of embodiment 403, wherein the napDNAbp comprises the amino acid sequence of SEQ ID NOs: 18-25.
  413. The method of embodiment 403, wherein the PEgRNA comprises SEQ ID NOs: 26-36.
  414. A method of installing a ribonucleotide motif or tag in an RNA of interest encoded by a target nucleotide sequence by prime editing, the method comprising: (a) contacting the target nucleotide sequence with a (i) prime editor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a polymerase and (ii) a PEgRNA comprising an edit template encoding the ribonucleotide motif or tag; (b) polymerizing a single strand DNA sequence encoding the ribonucleotide motif or tag; and (c) incorporating the single strand DNA sequence in place of a corresponding endogenous strand at the target nucleotide sequence through a DNA repair and/or replication process, wherein the method produces a recombinant target nucleotide sequence that encodes a modified RNA of interest comprising the ribonucleotide motif or tag.
  415. The method of embodiment 414, wherein ribonucleotide motif or tag is a detection moiety.
  416. The method of embodiment 414, wherein the ribonucleotide motif or tag affects the expression level of the RNA of interest.
  417. The method of embodiment 414, wherein the ribonucleotide motif or tag affects the transport or subcellular location of the RNA of interest.
  418. The method of embodiment 414, wherein the ribonucleotide motif or tag is selected from the group consisting of SV40 type 1, SV40 type 2, SV40 type 3, hGH, BGH, rbGlob, TK, MALAT1 ENE-mascRNA, KSHV PAN ENE, Smbox/U1 snRNA box, U1 snRNA 3′ box, tRNA-lysine, broccoli aptamer, spinach aptamer, mango aptamer, HDV ribozyme, and m6A.
  419. The method of embodiment 414, wherein the PEgRNA comprises SEQ ID NOs: 18-25.
  420. The method of embodiment 414, wherein the fusion protein comprises the amino acid sequence of PE1, PE2, or PE3.
  421. The method of embodiment 414, wherein the napDNAbp is a Cas9 nickase (nCas9).
  422. The method of embodiment 414, wherein the napDNAbp comprises the amino acid sequence of SEQ ID NOs: 18-25.
  423. A method of installing or deleting a functional moiety in a protein of interest encoded by a target nucleotide sequence by prime editing, the method comprising: (a) contacting the target nucleotide sequence with a (i) prime editor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a polymerase and (ii) a PEgRNA comprising an edit template encoding the functional moiety or deletion of same; (b) polymerizing a single strand DNA sequence encoding the functional moiety or deletion of same; and (c) incorporating the single strand DNA sequence in place of a corresponding endogenous strand at the target nucleotide sequence through a DNA repair and/or replication process, wherein the method produces a recombinant target nucleotide sequence that encodes a modified protein comprising the protein of interest and the functional moiety or the removal of same, wherein the functional moiety alters a modification state or localization state of the protein.
  424. The method of embodiment 423, wherein functional moiety alters the phosphorylation, ubiquitylation, glycosylation, lipidation, hydroxylation, methylation, acetylation, crotonylation, SUMOylation state of the protein of interest.
Group A. Fusion Proteins, Guides, and Methods
• Embodiment 1. A fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a reverse transcriptase.
• Embodiment 2. The fusion protein of embodiment 1, wherein the fusion protein is capable of carrying out genome editing by target-primed reverse transcription in the presence of an extended guide RNA.
• Embodiment 3. The fusion protein of embodiment 1, wherein the napDNAbp has a nickase activity.
• Embodiment 4. The fusion protein of embodiment 1, wherein the napDNAbp is a Cas9 protein or variant thereof.
• Embodiment 5. The fusion protein of embodiment 1, wherein the napDNAbp is a nuclease active Cas9, a nuclease inactive Cas9 (dCas9), or a Cas9 nickase (nCas9).
• Embodiment 6. The fusion protein of embodiment 1, wherein the napDNAbp is Cas9 nickase (nCas9).
• Embodiment 7. The fusion protein of embodiment 1, wherein the napDNAbp is selected from the group consisting of: Cas9, CasX, CasY, Cpf1, C2c1, C2c2, C2C3, and Argonaute and optionally has a nickase activity.
• Embodiment 8. The fusion protein of embodiment 1, wherein the fusion protein when complexed with an extended guide RNA is capable of binding to a target DNA sequence.
• Embodiment 9. The fusion protein of embodiment 8, wherein the target DNA sequence comprises a target strand and a complementary non-target strand.
• Embodiment 10. The fusion protein of embodiment 8, wherein the binding of the fusion protein complexed to the extended guide RNA forms an R-loop.
• Embodiment 11. The fusion protein of embodiment 10, wherein the R-loop comprises (i) an RNA-DNA hybrid comprising the extended guide RNA and the target strand, and (ii) the complementary non-target strand.
• Embodiment 12. The fusion protein of embodiment 11, wherein the complementary non-target strand is nicked to form a reverse transcriptase priming sequence having a free 3′ end.
• Embodiment 13. The fusion protein of embodiment 2, wherein the extended guide RNA comprises (a) a guide RNA, and (b) an RNA extension at the 5′ or the 3′ end of the guide RNA, or at an intramolecular location in the guide RNA.
• Embodiment 14. The fusion protein of embodiment 13, wherein the RNA extension comprises (i) a reverse transcription template sequence comprising a desired nucleotide change, (ii) a reverse transcription primer binding site, and (iii) optionally, a linker sequence.
• Embodiment 15. The fusion protein of embodiment 14, wherein the reverse transcription template sequence encodes a single-strand DNA flap that is complementary to an endogenous DNA sequence adjacent to the nick site, wherein the single-strand DNA flap comprises the desired nucleotide change.
• Embodiment 16. The fusion protein of embodiment 13, wherein the RNA extension is at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides, at least 22 nucleotides, at least 23 nucleotides, at least 24 nucleotides, or at least 25 nucleotides in length.
• Embodiment 17. The fusion protein of embodiment 15, wherein the single-strand DNA flap hybridizes to the endogenous DNA sequence adjacent to the nick site, thereby installing the desired nucleotide change.
• Embodiment 18. The fusion protein of embodiment 15, wherein the single-stranded DNA flap displaces the endogenous DNA sequence adjacent to the nick site and which has a free 5′ end.
• Embodiment 19. The fusion protein of embodiment 18, wherein the endogenous DNA sequence having the 5′ end is excised by the cell.
• Embodiment 20. The fusion protein of embodiment 18, wherein cellular repair of the single-strand DNA flap results in installation of the desired nucleotide change, thereby forming a desired product.
• Embodiment 21. The fusion protein of embodiment 14, wherein the desired nucleotide change is installed in an editing window that is between about −4 to +10 of the PAM sequence, or between about −10 to +20 of the PAM sequence, or between about −20 to +40 of the PAM sequence, or between about −30 to +100 of the PAM sequence, or wherein the desired nucleotide change is installed at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides downstream of the nick site.
• Embodiment 22. The fusion protein of embodiment 1, wherein the napDNAbp comprises an amino acid sequence of SEQ ID NO: 18, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to the amino acid sequence to SEQ ID NO: 18.
• Embodiment 23. The fusion protein of embodiment 1, wherein the napDNAbp comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.
• [0001] The fusion protein of embodiment 1, wherein the reverse transcriptase comprises any one of the amino acid sequences of SEQ ID NO: 89.
• Embodiment 24. The fusion protein of embodiment 1, wherein the reverse transcriptase comprises any one of the amino acid sequences of SEQ ID NO: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, 662, 700, 701-716, 739-741, and 766.
• [0002] The fusion protein of embodiment 1, wherein the reverse transcriptase comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NO: 89.
• Embodiment 25. The fusion protein of embodiment 1, wherein the reverse transcriptase comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, 662, 700, 701-716, 739-741, and 766.
• Embodiment 26. The fusion protein of embodiment 1, wherein the reverse transcriptase is a naturally-occurring reverse transcriptase from a retrovirus or a retrotransposon.
• Embodiment 27. The fusion protein of any one of the previous embodiments, wherein the fusion protein comprises the structure NH₂-[napDNAbp]-[reverse transcriptase]-COOH; or NH₂-[reverse transcriptase]-[napDNAbp]-COOH, wherein each instance of “]-[” indicates the presence of an optional linker sequence.
• Embodiment 28. The fusion protein of embodiment 27, wherein the linker sequence comprises an amino acid sequence of SEQ ID NOs: 127, 165-176, 446, 453, and 767-769.
• Embodiment 29. The fusion protein of embodiment 14, wherein the desired nucleotide change is a single nucleotide change, an insertion of one or more nucleotides, or a deletion of one or more nucleotides.
• Embodiment 30. The fusion protein of embodiment 29, wherein the insert or deletion is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, or at least 50.
• Embodiment 31. An extended guide RNA comprising a guide RNA and at least one RNA extension.
• Embodiment 32. The extended guide RNA of embodiment 1, wherein the RNA extension is position at the 3′ or 5′ end of the guide RNA, or at an intramolecular position in the guide RNA.
• Embodiment 33. The extended guide RNA of embodiment 31, wherein the extended guide RNA is capable of binding to a napDNAbp and directing the napDNAbp to a target DNA sequence.
• Embodiment 34. The extended guide RNA of embodiment 33, wherein the target DNA sequence comprises a target strand and a complementary non-target strand, wherein the guide RNA hybridizes to the target strand to form an RNA-DNA hybrid and an R-loop.
• Embodiment 35. The extended guide RNA of embodiment 31, wherein the at least one RNA extension comprises (i) a reverse transcription template sequence, (ii) a reverse transcription primer binding site, and (iii) optionally a linker sequence.
• Embodiment 36. The extended guide RNA of embodiment 35, wherein the RNA extension is at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides, at least 22 nucleotides, at least 23 nucleotides, at least 24 nucleotides, or at least 25 nucleotides in length.
• Embodiment 37. The extended guide RNA of embodiment 35, wherein the reverse transcription template sequence is at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, or at least 15 nucleotides in length.
• Embodiment 38. The extended guide RNA of embodiment 35, wherein the reverse transcription primer binding site sequence is at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, or at least 15 nucleotides in length.
• Embodiment 39. The extended guide RNA of embodiment 35, wherein the optional linker sequence is at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, or at least 15 nucleotides in length.
• Embodiment 40. The extended guide RNA of embodiment 35, wherein the reverse transcription template sequence encodes a single-strand DNA flap that is complementary to an endogenous DNA sequence adjacent to a nick site, wherein the single-strand DNA flap comprises a desired nucleotide change.
• Embodiment 41. The extended guide RNA of embodiment 40, wherein the single-stranded DNA flap displaces an endogenous single-strand DNA having a 5′ end in the target DNA sequence that has been nicked, and wherein the endogenous single-strand DNA is immediately adjacent downstream of the nick site.
• Embodiment 42. The extended guide RNA of embodiment 41, wherein the endogenous single-stranded DNA having the free 5′ end is excised by the cell.
• Embodiment 43. The extended guide RNA of embodiment 41, wherein cellular repair of the single-strand DNA flap results in installation of the desired nucleotide change, thereby forming a desired product.
• Embodiment 44. The extended guide RNA of embodiment 31, comprising the nucleotide sequence of SEQ ID NOs: 18-36, or a nucleotide sequence having at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99% sequence identity with any one of SEQ ID NOs: 394, 429-442, 641-649, 678-692, 2997-3103, 3113-3121, 3305-3455, 3479-3493, 3522-3556, 3628-3698, and 3755-3810.
• Embodiment 45. The extended guide RNA of embodiment 35, wherein the reverse transcription template sequence comprises a nucleotide sequence that is at least 80%, or 85%, or 90%, or 95%, or 99% identical to the endogenous DNA target.
• Embodiment 46. The extended guide RNA of embodiment 35, wherein the reverse transcription primer binding site hybridizes with a free 3′ end of the cut DNA.
• Embodiment 47. The extended guide RNA of embodiment 35, wherein the optional linker sequence is at least 1 nucleotide, or at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 nucleotides in length.
• Embodiment 48. A complex comprising a fusion protein of any one of embodiments 1-30 and an extended guide RNA.
• Embodiment 49. The complex of embodiment 48, wherein the extended guide RNA comprises a guide RNA and an RNA extension at the 3′ or 5′ end of the guide RNA or at an intramolecular position in the guide RNA.
• Embodiment 50. The complex of embodiment 48, wherein the extended guide RNA is capable of binding to a napDNAbp and directing the napDNAbp to a target DNA sequence.
• Embodiment 51. The complex of embodiment 50, wherein the target DNA sequence comprises a target strand and a complementary non-target strand, wherein the guide RNA hybridizes to the target strand to form an RNA-DNA hybrid and an R-loop.
• Embodiment 52. The complex of embodiment 49, wherein the at least one RNA extension comprises (i) a reverse transcription template sequence, (ii) a reverse transcription primer binding site, and (iii) optionally a linker sequence.
• Embodiment 53. The complex of embodiment 48, wherein the extended guide RNA comprises the nucleotide sequence of SEQ ID NOs: 394, 429-442, 641-649, 678-692, 2997-3103, 3113-3121, 3305-3455, 3479-3493, 3522-3556, 3628-3698, and 3755-3810, or a nucleotide sequence having at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99% sequence identity with any one of SEQ ID NOs: 394, 429-442, 641-649, 678-692, 2997-3103, 3113-3121, 3305-3455, 3479-3493, 3522-3556, 3628-3698, and 3755-3810.
• Embodiment 54. The complex of embodiment 52, wherein the reverse transcription template sequence comprises a nucleotide sequence having at least 80%, or 85%, or 90%, or 95%, or 99% sequence identity with the endogenous DNA target.
• Embodiment 55. The complex of embodiment 52, wherein the reverse transcription primer binding site hybridizes with a free 3′ end of the cut DNA.
• Embodiment 56. A complex comprising a napDNAbp and an extended guide RNA.
• Embodiment 57. The complex of embodiment 56, wherein the napDNAbp is a Cas9 nickase.
• Embodiment 58. The complex of embodiment 56, wherein the napDNAbp comprises an amino acid sequence of SEQ ID NO: 18, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO: 18.
• Embodiment 59. The complex of embodiment 57, wherein the napDNAbp comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.
• Embodiment 60. The complex of embodiment 57, wherein the extended guide RNA comprises a guide RNA and an RNA extension at the 3′ or 5′ end of the guide RNA, or at an intramolecular position in the guide RNA.
• Embodiment 61. The complex of embodiment 57, wherein the extended guide RNA is capable of directing the napDNAbp to a target DNA sequence.
• Embodiment 62. The complex of embodiment 61, wherein the target DNA sequence comprises a target strand and a complementary non-target strand, wherein the spacer sequence hybridizes to the target strand to form an RNA-DNA hybrid and an R-loop.
• Embodiment 63. The complex of embodiment 61, wherein the RNA extension comprises (i) a reverse transcription template sequence, (ii) a reverse transcription primer binding site, and (iii) optionally a linker sequence.
• Embodiment 64. The complex of embodiment 57, wherein the extended guide RNA comprises the nucleotide sequence of SEQ ID NOs: 394, 429-442, 641-649, 678-692, 2997-3103, 3113-3121, 3305-3455, 3479-3493, 3522-3556, 3628-3698, and 3755-3810, or a nucleotide sequence having at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99% sequence identity with any one of SEQ ID NOs: 394, 429-442, 641-649, 678-692, 2997-3103, 3113-3121, 3305-3455, 3479-3493, 3522-3556, 3628-3698, and 3755-3810.
• Embodiment 65. The complex of embodiment 63, wherein the reverse transcription template sequence comprises a nucleotide sequence that is at least 80%, or 85%, or 90%, or 95%, or 99% identical to the endogenous DNA target.
• Embodiment 66. The complex of embodiment 63, wherein the reverse transcription primer binding site hybridizes with a free 3′ end of the cut DNA.
• Embodiment 67. The complex of embodiment 63, wherein the optional linker sequence is at least 1 nucleotide, or at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 nucleotides in length.
• Embodiment 68. A polynucleotide encoding the fusion protein of any of embodiments 1-30.
• Embodiment 69. A vector comprising the polynucleotide of embodiment 68.
• Embodiment 70. A cell comprising the fusion protein of any of embodiments 1-30 and an extended guide RNA bound to the napDNAbp of the fusion protein.
• Embodiment 71. A cell comprising a complex of any one of embodiments 48-67.
• Embodiment 72. A pharmaceutical composition comprising: (i) a fusion protein of any of embodiments 1-30, the complex of embodiments 48-67, the polynucleotide of embodiment 68, or the vector of embodiment 69; and (ii) a pharmaceutically acceptable excipient.
• Embodiment 73. A pharmaceutical composition comprising: (i) the complex of embodiments 48-67 (ii) reverse transcriptase provided in trans; and (iii) a pharmaceutically acceptable excipient.
• Embodiment 74. A kit comprising a nucleic acid construct, comprising: (i) a nuleic acid sequencing encoding the fusion protein of any one of embodiments 1-30; and (ii) a promoter that drives expression of the sequence of (i).
• Embodiment 75. A method for installing a desired nucleotide change in a double-stranded DNA sequence, the method comprising:
• (i) contacting the double-stranded DNA sequence with a complex comprising a fusion protein and an extended guide RNA, wherein the fusion protein comprises a napDNAbp and a reverse transcriptase and wherein the extended guide RNA comprises a reverse transcription template sequence comprising the desired nucleotide change;
• (ii) nicking the double-stranded DNA sequence on the non-target strand, thereby generating a free single-strand DNA having a 3′ end;
• (iii) hybridizing the 3′ end of the free single-strand DNA to the reverse transcription template sequence, thereby priming the reverse transcriptase domain;
• (iv) polymerizing a strand of DNA from the 3′ end, thereby generating a single-strand DNA flap comprising the desired nucleotide change;
• (v) replacing an endogenous DNA strand adjacent the cut site with the single-strand DNA flap, thereby installing the desired nucleotide change in the double-stranded DNA sequence.
• Embodiment 76. The method of embodiment 75, wherein the step of (v) replacing comprises: (i) hybridizing the single-strand DNA flap to the endogenous DNA strand adjacent the cut site to create a sequence mismatch; (ii) excising the endogenous DNA strand; and (iii) repairing the mismatch to form the desired product comprising the desired nucleotide change in both strands of DNA.
• Embodiment 77. The method of embodiment 76, wherein the desired nucleotide change is a single nucleotide substitution, a deletion, or an insertion.
• Embodiment 78. The method of embodiment 77, wherein the single nucleotide substitution is a transition or a transversion.
• Embodiment 79. The method of embodiment 76, wherein the desired nucleotide change is (1) a G to T substitution, (2) a G to A substitution, (3) a G to C substitution, (4) a T to G substitution, (5) a T to A substitution, (6) a T to C substitution, (7) a C to G substitution, (8) a C to T substitution, (9) a C to A substitution, (10) an A to T substitution, (11) an A to G substitution, or (12) an A to C substitution.
• Embodiment 80. The method of embodiment 76, wherein the desired nucleoid change converts (1) a G:C basepair to a T:A basepair, (2) a G:C basepair to an A:T basepair, (3) a G:C basepair to C:G basepair, (4) a T:A basepair to a G:C basepair, (5) a T:A basepair to an A:T basepair, (6) a T:A basepair to a C:G basepair, (7) a C:G basepair to a G:C basepair, (8) a C:G basepair to a T:A basepair, (9) a C:G basepair to an A:T basepair, (10) an A:T basepair to a T:A basepair, (11) an A:T basepair to a G:C basepair, or (12) an A:T basepair to a C:G basepair.
• Embodiment 81. The method of embodiment 76, wherein the desired nucleotide change is an insertion or deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides.
• Embodiment 82. The method of embodiment 76, wherein the desired nucleotide change corrects a disease-associated gene.
• Embodiment 83. The method of embodiment 82, wherein the disease-associated gene is associated with a monogenetic disorder selected from the group consisting of: Adenosine Deaminase (ADA) Deficiency; Alpha-1 Antitrypsin Deficiency; Cystic Fibrosis; Duchenne Muscular Dystrophy; Galactosemia; Hemochromatosis; Huntington's Disease; Maple Syrup Urine Disease; Marfan Syndrome; Neurofibromatosis Type 1; Pachyonychia Congenita; Phenylkeotnuria; Severe Combined Immunodeficiency; Sickle Cell Disease; Smith-Lemli-Opitz Syndrome; and Tay-Sachs Disease.
• Embodiment 84. The method of embodiment 82, wherein the disease-associated gene is associated with a polygenic disorder selected from the group consisting of: heart disease; high blood pressure; Alzheimer's disease; arthritis; diabetes; cancer; and obesity.
• Embodiment 85. The method of embodiment 76, wherein the napDNAbp is a nuclease dead Cas9 (dCas9), a Cas9 nickase (nCas9), or a nuclease active Cas9.
• Embodiment 86. The method of embodiment 76, wherein the napDNAbp comprises an amino acid sequence of SEQ ID NO: 18.
• Embodiment 87. The method of embodiment 76, wherein the napDNAbp comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.
• Embodiment 88. The method of embodiment 76, wherein the reverse transcriptase comprises any one of the amino acid sequences of SEQ ID NO: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, 662, 700, 701-716, 739-741, and 766.
• Embodiment 89. The method of embodiment 76, wherein the reverse transcriptase domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, 662, 700, 701-716, 739-741, and 766.
• Embodiment 90. The method of embodiment 76, wherein the extended guide RNA comprises an RNA extension at the 3′ or 5′ ends or at an intramolecular location in the guide RNA, wherein the RNA extension comprises the reverse transcription template sequence.
• Embodiment 91. The method of embodiment 90, wherein the RNA extension is at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides, at least 22 nucleotides, at least 23 nucleotides, at least 24 nucleotides, or at least 25 nucleotides in length.
• Embodiment 92. The method of embodiment 76, wherein the extended guide RNA has a nucleotide sequence selected from the group consisting of SEQ ID NOs: 394, 429-442, 641-649, 678-692, 2997-3103, 3113-3121, 3305-3455, 3479-3493, 3522-3556, 3628-3698, and 3755-3810.
• Embodiment 93. A method for introducing one or more changes in the nucleotide sequence of a DNA molecule at a target locus, comprising:
• (i) contacting the DNA molecule with a nucleic acid programmable DNA binding protein (napDNAbp) and a guide RNA which targets the napDNAbp to the target locus, wherein the guide RNA comprises a reverse transcriptase (RT) template sequence comprising at least one desired nucleotide change;
• (ii) forming an exposed 3′ end in a DNA strand at the target locus;
• (iii) hybridizing the exposed 3′ end to the RT template sequence to prime reverse transcription;
• (iv) synthesizing a single strand DNA flap comprising the at least one desired nucleotide change based on the RT template sequence by reverse transcriptase;
• (v) and incorporating the at least one desired nucleotide change into the corresponding endogenous DNA, thereby introducing one or more changes in the nucleotide sequence of the DNA molecule at the target locus.
• Embodiment 94. The method of embodiment 93, wherein the one or more changes in the nucleotide sequence comprises a transition.
• Embodiment 95. The method of embodiment 94, wherein the transition is selected from the group consisting of: (a) T to C; (b) A to G; (c) C to T; and (d) G to A.
• Embodiment 96. The method of embodiment 93, wherein the one or more changes in the nucleotide sequence comprises a transversion.
• Embodiment 97. The method of embodiment 96, wherein the transversion is selected from the group consisting of: (a) T to A; (b) T to G; (c) C to G; (d) C to A; (e) A to T; (f) A to C; (g) G to C; and (h) G to T.
• Embodiment 98. The method of embodiment 93, wherein the one or more changes in the nucleotide sequence comprises changing (1) a G:C basepair to a T:A basepair, (2) a G:C basepair to an A:T basepair, (3) a G:C basepair to C:G basepair, (4) a T:A basepair to a G:C basepair, (5) a T:A basepair to an A:T basepair, (6) a T:A basepair to a C:G basepair, (7) a C:G basepair to a G:C basepair, (8) a C:G basepair to a T:A basepair, (9) a C:G basepair to an A:T basepair, (10) an A:T basepair to a T:A basepair, (11) an A:T basepair to a G:C basepair, or (12) an A:T basepair to a C:G basepair.
• Embodiment 99. The method of embodiment 93, wherein the one or more changes in the nucleotide sequence comprises an insertion or deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides.
• Embodiment 100. The method of embodiment 93, wherein the one or more changes in the nucleotide sequence comprises a correction to a disease-associated gene.
• Embodiment 101. The method of embodiment 100, wherein the disease-associated gene is associated with a monogenetic disorder selected from the group consisting of: Adenosine Deaminase (ADA) Deficiency; Alpha-1 Antitrypsin Deficiency; Cystic Fibrosis; Duchenne Muscular Dystrophy; Galactosemia; Hemochromatosis; Huntington's Disease; Maple Syrup Urine Disease; Marfan Syndrome; Neurofibromatosis Type 1; Pachyonychia Congenita; Phenylkeotnuria; Severe Combined Immunodeficiency; Sickle Cell Disease; Smith-Lemli-Opitz Syndrome; and Tay-Sachs Disease.
• Embodiment 102. The method of embodiment 100, wherein the disease-associated gene is associated with a polygenic disorder selected from the group consisting of: heart disease; high blood pressure; Alzheimer's disease; arthritis; diabetes; cancer; and obesity.
• Embodiment 103. The method of embodiment 93, wherein the napDNAbp is a nuclease active Cas9 or variant thereof.
• Embodiment 104. The method of embodiment 93, wherein the napDNAbp is a nuclease inactive Cas9 (dCas9) or Cas9 nickase (nCas9), or a variant thereof.
• Embodiment 105. The method of embodiment 93, wherein the napDNAbp comprises an amino acid sequence of SEQ ID NO: 18.
• Embodiment 106. The method of embodiment 93, wherein the napDNAbp comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.
• Embodiment 107. The method of embodiment 93, wherein the reverse transcriptase is introduced in trans.
• Embodiment 108. The method of embodiment 93, wherein the napDNAbp comprises a fusion to a reverse transcriptase.
• Embodiment 109. The method of embodiment 93, wherein the reverse transcriptase comprises any one of the amino acid sequences of SEQ ID NO: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, 662, 700, 701-716, 739-741, and 766.
• Embodiment 110. The method of embodiment 93, wherein the reverse transcriptase comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, 662, 700, 701-716, 739-741, and 766.
• Embodiment 111. The method of embodiment 93, wherein the step of forming an exposed 3′ end in the DNA strand at the target locus comprises nicking the DNA strand with a nuclease.
• Embodiment 112. The method of embodiment 111, wherein the nuclease is the napDNAbp, is provided as a fusion domain of napDNAbp, or is provided in trans.
• Embodiment 113. The method of embodiment 93, wherein the step of forming an exposed 3′ end in the DNA strand at the target locus comprises contacting the DNA strand with a chemical agent.
• Embodiment 114. The method of embodiment 93, wherein the step of forming an exposed 3′ end in the DNA strand at the target locus comprises introducing a replication error.
• Embodiment 115. The method of embodiment 93, wherein the step of contacting the DNA molecule with the napDNAbp and the guide RNA forms an R-loop.
• Embodiment 116. The method of embodiment 115, wherein the DNA strand in which the exposed 3′ end is formed is in the R-loop.
• Embodiment 117. The method of embodiment 93, wherein guide RNA comprises an extended portion that comprises the reverse transcriptase (RT) template sequence.
• Embodiment 118. The method of embodiment 117, wherein the extended portion is at the 3′ end of the guide RNA, the 5′ end of the guide RNA, or at an intramolecular position in the guide RNA.
• Embodiment 119. The method of embodiment 93, wherein the guide RNA further comprises a primer binding site.
• Embodiment 120. The method of embodiment 93, wherein the guide RNA further comprises a spacer sequence.
• Embodiment 121. The method of embodiment 93, wherein the RT template sequence is homologous to the corresponding endogenous DNA.
• Embodiment 122. A method for introducing one or more changes in the nucleotide sequence of a DNA molecule at a target locus by target-primed reverse transcription, the method comprising: (a) contacting the DNA molecule at the target locus with a (i) fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a reverse transcriptase and (ii) a guide RNA comprising an RT template comprising a desired nucleotide change; (b) conducting target-primed reverse transcription of the RT template to generate a single strand DNA comprising the desired nucleotide change; and (c) incorporating the desired nucleotide change into the DNA molecule at the target locus through a DNA repair and/or replication process.
• Embodiment 123. The method of embodiment 122, wherein the RT template is located at the 3′ end of the guide RNA, the 5′ end of the guide RNA, or at an intramolecular location in the guide RNA.
• Embodiment 124. The method of embodiment 122, wherein the desired nucleotide change comprises a transition, a transversion, an insertion, or a deletion, or any combination thereof.
• Embodiment 125. The method of claim 122, wherein the desired nucleotide change comprises a transition selected from the group consisting of: (a) T to C; (b) A to G; (c) C to T; and (d) G to A.
• Embodiment 126. The method of claim 122, wherein the desired nucleotide change comprises a transversion selected from the group consisting of: (a) T to A; (b) T to G; (c) C to G; (d) C to A; (e) A to T; (f) A to C; (g) G to C; and (h) G to T.
• Embodiment 127. The method of embodiment 122, wherein the desired nucleotide change comprises changing (1) a G:C basepair to a T:A basepair, (2) a G:C basepair to an A:T basepair, (3) a G:C basepair to C:G basepair, (4) a T:A basepair to a G:C basepair, (5) a T:A basepair to an A:T basepair, (6) a T:A basepair to a C:G basepair, (7) a C:G basepair to a G:C basepair, (8) a C:G basepair to a T:A basepair, (9) a C:G basepair to an A:T basepair, (10) an A:T basepair to a T:A basepair, (11) an A:T basepair to a G:C basepair, or (12) an A:T basepair to a C:G basepair.
• Embodiment 128. A polynucleotide encoding the extended guide RNA of any one of embodiments 31-47.
• Embodiment 129. A vector comprising the polynucleotide of embodiment 128.
• Embodiment 130. A cell comprising the vector of embodiment 129.
• Embodiment 131. The fusion protein of any of embodiments 1-30, wherein the reverse transcriptase is an error-prone reverse transcriptase.
• Embodiment 132. A method for mutagenizing a DNA molecule at a target locus by target-primed reverse transcription, the method comprising: (a) contacting the DNA molecule at the target locus with a (i) fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) and an error-prone reverse transcriptase and (ii) a guide RNA comprising an RT template comprising a desired nucleotide change; (b) conducting target-primed reverse transcription of the RT template to generate a mutagenized single strand DNA; and (c) incorporating the mutagenized single strand DNA into the DNA molecule at the target locus through a DNA repair and/or replication process.
• Embodiment 133. The method of embodiment 132, wherein the fusion protein comprises the amino acid sequence of PE1, PE2, or PE3.
• Embodiment 134. The method of embodiment 132, wherein the napDNAbp is a Cas9 nickase (nCas9).
• Embodiment 135. The method of embodiment 132, wherein the napDNAbp comprises the amino acid sequence of SEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.
• Embodiment 136. The method of embodiment 132, wherein the guide RNA comprises SEQ ID NOs: 222.
• Embodiment 137. The method of embodiment 132, wherein the step of (b) conducting target-primed reverse transcription comprises generating a 3′ end primer binding sequence at the target locus that is capable of priming reverse transcription by annealing to a primer binding site on the guide RNA.
• Embodiment 138. A method for replacing a trinucleotide repeat expansion mutation in a target DNA molecule with a healthy sequence comprising a healthy number of repeat trinucleotides, the method comprising: (a) contacting the DNA molecule at the target locus with a (i) fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a reverse transcriptase and (ii) a guide RNA comprising an RT template comprising the replacement sequence, wherein said fusion protein intr; (b) conducting target-primed reverse transcription of the RT template to generate a single strand DNA comprising the replacement sequence; and (c) incorporating the single strand DNA into the DNA molecule at the target locus through a DNA repair and/or replication process.
• Embodiment 139. The method of embodiment 138, wherein the fusion protein comprises the amino acid sequence of PE1, PE2, or PE3.
• Embodiment 140. The method of embodiment 138, wherein the napDNAbp is a Cas9 nickase (nCas9).
• Embodiment 141. The method of embodiment 138, wherein the napDNAbp comprises the amino acid sequence of SEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.
• Embodiment 142. The method of embodiment 138, wherein the guide RNA comprises SEQ ID NOs: 222.
• Embodiment 143. The method of embodiment 138, wherein the step of (b) conducting target-primed reverse transcription comprises generating a 3′ end primer binding sequence at the target locus that is capable of priming reverse transcription by annealing to a primer binding site on the guide RNA.
• Embodiment 144. The method of embodiment 138, wherein the trinucleotide repeat expansion mutation is associated with Huntington's Disease, Fragile X syndrome, or Friedreich's ataxia.
• Embodiment 145. The method of embodiment 138, wherein the trinucleotide repeat expansion mutation comprises a repeating unit of CAG triplets.
• Embodiment 146. The method of embodiment 138, wherein the trinucleotide repeat expansion mutation comprises a repeating unit of GAA triplets.
• Embodiment 147. A method of installing a functional moiety in a protein of interest encoded by a target nucleotide sequence by prime editing, the method comprising: (a) contacting the target nucleotide sequence with a (i) prime editor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a reverse transcriptase and (ii) a PEgRNA comprising an edit template encoding the functional moiety; (b) polymerizing a single strand DNA sequence encoding the functional moiety; and (c) incorporating the single strand DNA sequence in place of a corresponding endogenous strand at the target nucleotide sequence through a DNA repair and/or replication process, wherein the method produces a recombinant target nucleotide sequence that encodes a fusion protein comprising the protein of interest and the functional moiety.
• Embodiment 148. The method of embodiment 147, wherein functional moiety is peptide tag.
• Embodiment 149. The method of embodiment 148, wherein the peptide tag is an affinity tag, solubilization tag, chromatography tag, epitope tag, or a fluorescence tag.
• Embodiment 150. The method of embodiment 148, wherein the peptide tag is selected from the group consisting of: AviTag (SEQ ID NO: 245); C-tag (SEQ ID NO: 246); Calmodulin-tag (SEQ ID NO: 247); polyglutamate tag (SEQ ID NO: 248); E-tag (SEQ ID NO: 249); FLAG-tag (SEQ ID NO: 2); HA-tag (SEQ ID NO: 5); His-tag (SEQ ID NOs: 252-262); Myc-tag (SEQ ID NO: 6); NE-tag (SEQ ID NO: 264); Rho1D4-tag (SEQ ID NO: 265); S-tag (SEQ ID NO: 266); SBP-tag (SEQ ID NO: 267); Softag-1 (SEQ ID NO: 268); Softag-2 (SEQ ID NO: 269); Spot-tag (SEQ ID NO: 270); Strep-tag (SEQ ID NO: 271); TC tag (SEQ ID NO: 272); Ty tag (SEQ ID NO: 273); V5 tag (SEQ ID NO: 3); VSV-tag (SEQ ID NO: 275); and Xpress tag (SEQ ID NO: 276).
• Embodiment 151. The method of embodiment 148, wherein the peptide tag is selected from the group consisting of: AU1 epitope (SEQ ID NO: 278); AU5 epitope (SEQ ID NO: 279); Bacteriophage T7 epitope (T7-tag) (SEQ ID NO: 280); Bluetongue virus tag (B-tag) (SEQ ID NO: 281); E2 epitope (SEQ ID NO: 282); Histidine affinity tag (HAT) (SEQ ID NO: 283); HSV epitope (SEQ ID NO: 284); Polyarginine (Arg-tag) (SEQ ID NO: 285); Polyaspartate (Asp-tag) (SEQ ID NO: 286); Polyphenylalanine (Phe-tag) (SEQ ID NO: 287); 51-tag (SEQ ID NO: 288); S-tag (SEQ ID NO: 266); and VSV-G (SEQ ID NO: 275).
• Embodiment 152. The method of embodiment 147, wherein the functional moiety is an immunoepitope.
• Embodiment 153. The method of embodiment 152, wherein the immunoepitope is selected from the group consisting of: tetanus toxoid (SEQ ID NO: 396); diphtheria toxin mutant CRM197 (SEQ ID NO: 398); mumps immunoepitope 1 (SEQ ID NO: 400); mumps immunoepitope 2 (SEQ ID NO: 402); mumps immunoeptitope 3 (SEQ ID NO: 404); rubella virus (SEQ ID NO: 406); hemagglutinin (SEQ ID NO: 408); neuraminidase (SEQ ID NO: 410); TAP1 (SEQ ID NO: 412); TAP2 (SEQ ID NO: 414); hemagglutinin epitopes toward class I HLA (SEQ ID NO: 416); neuraminidase epitopes toward class I HLA (SEQ ID NO: 418); hemagglutinin epitopes toward class II HLA (SEQ ID NO: 420); neuraminidase epitopes toward class II HLA (SEQ ID NO: 422); hemagglutinin epitope H5N1-bound class I and class II HLA (SEQ ID NO: 424); neuraminidase epitope H5N1-bound class I and class II HLA (SEQ ID NO: 426).
• Embodiment 154. The method of embodiment 147, wherein the functional moiety alters the localization of the protein of interest.
• Embodiment 155. The method of embodiment 147, wherein the functional moiety is a degradation tag such that the degradation rate of the protein of interest is altered.
• Embodiment 156. The method of embodiment 155, wherein the degradation tag comprises an amino acid sequence encoding the degradation tags as disclosed herein.
• Embodiment 157. The method of embodiment 147, wherein the functional moiety is a small molecule binding domain.
• Embodiment 158. The method of embodiment 157, wherein the small molecule binding domain is FKBP12 of SEQ ID NO: 488.
• Embodiment 159. The method of embodiment 157, wherein the small molecule binding domain is FKBP12-F36V of SEQ ID NO: 489.
• Embodiment 160. The method of embodiment 157, wherein the small molecule binding domain is cyclophilin of SEQ ID NOs: 492-494.
• Embodiment 161. The method of embodiment 157, wherein the small molecule binding domain is installed in two or more proteins of interest.
• Embodiment 162. The method of embodiment 161, wherein the two or more proteins of interest may dimerize upon contacting with a small molecule.
• Embodiment 163. The method of embodiment 157, wherein the small molecule is a dimer of a small molecule selected from the group consisting of those compounds disclosed in Embodiment 163 of Group 1.
• Embodiment 164. A method of installing an immunoepitope in a protein of interest encoded by a target nucleotide sequence by prime editing, the method comprising: (a) contacting the target nucleotide sequence with a (i) prime editor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a reverse transcriptase and (ii) a PEgRNA comprising an edit template encoding the functional moiety; (b) polymerizing a single strand DNA sequence encoding the immunoepitope; and (c) incorporating the single strand DNA sequence in place of a corresponding endogenous strand at the target nucleotide sequence through a DNA repair and/or replication process, wherein the method produces a recombinant target nucleotide sequence that encodes a fusion protein comprising the protein of interest and the immunoepitope.
• Embodiment 165. The method of embodiment 164, wherein the immunoepitope is selected from the group consisting of: tetanus toxoid (SEQ ID NO: 396); diphtheria toxin mutant CRM197 (SEQ ID NO: 630); mumps immunoepitope 1 (SEQ ID NO: 400); mumps immunoepitope 2 (SEQ ID NO: 402); mumps immunoeptitope 3 (SEQ ID NO: 404); rubella virus (SEQ ID NO: 406); hemagglutinin (SEQ ID NO: 408); neuraminidase (SEQ ID NO: 410); TAP1 (SEQ ID NO: 412); TAP2 (SEQ ID NO: 414); hemagglutinin epitopes toward class I HLA (SEQ ID NO: 416); neuraminidase epitopes toward class I HLA (SEQ ID NO: 418); hemagglutinin epitopes toward class II HLA (SEQ ID NO: 420); neuraminidase epitopes toward class II HLA (SEQ ID NO: 422); hemagglutinin epitope H5N1-bound class I and class II HLA (SEQ ID NO: 424); neuraminidase epitope H5N1-bound class I and class II HLA (SEQ ID NO: 426).
• Embodiment 166. The method of embodiment 164, wherein the fusion protein comprises the amino acid sequence of PE1, PE2, or PE3.
• Embodiment 167. The method of embodiment 164, wherein the napDNAbp is a Cas9 nickase (nCas9).
• Embodiment 168. The method of embodiment 164, wherein the napDNAbp comprises the amino acid sequence of SEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.
• Embodiment 169. The method of embodiment 164, wherein the guide RNA comprises SEQ ID NOs: 222.
• Embodiment 170. A method of installing a small molecule dimerization domain in a protein of interest encoded by a target nucleotide sequence by prime editing, the method comprising: (a) contacting the target nucleotide sequence with a (i) prime editor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a reverse transcriptase and (ii) a PEgRNA comprising an edit template encoding the small molecule dimerization domain; (b) polymerizing a single strand DNA sequence encoding the immunoepitope; and (c) incorporating the single strand DNA sequence in place of a corresponding endogenous strand at the target nucleotide sequence through a DNA repair and/or replication process, wherein the method produces a recombinant target nucleotide sequence that encodes a fusion protein comprising the protein of interest and the small molecule dimerization domain.
• Embodiment 171. The method of embodiment 170, further comprising conducting the method on a second protein of interest.
• Embodiment 172. The method of embodiment 171, wherein the first protein of interest and the second protein of interest dimerize in the presence of a small molecule that binds to the dimerization domain on each of said proteins.
• Embodiment 173. The method of embodiment 170, wherein the small molecule binding domain is FKBP12 of SEQ ID NO: 488.
• Embodiment 174. The method of embodiment 170, wherein the small molecule binding domain is FKBP12-F36V of SEQ ID NO: 489.
• Embodiment 175. The method of embodiment 170, wherein the small molecule binding domain is cyclophilin of SEQ ID NOs: 490 and 493-494.
• Embodiment 176. The method of embodiment 170, wherein the small molecule is a dimer of a small molecule selected from the group consisting of those compounds disclosed in Embodiment 163 of Group 1.
• Embodiment 177. The method of embodiment 170, wherein the fusion protein comprises the amino acid sequence of PE1, PE2, or PE3.
• Embodiment 178. The method of embodiment 170, wherein the napDNAbp is a Cas9 nickase (nCas9).
• Embodiment 179. The method of embodiment 170, wherein the napDNAbp comprises the amino acid sequence of SEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.
• Embodiment 180. The method of embodiment 170, wherein the guide RNA comprises SEQ ID NOs: 222.
• Embodiment 181. A method of installing a peptide tag or epitope onto a protein using prime editing, comprising: contacting a target nucleotide sequence encoding the protein with a prime editor construct configured to insert therein a second nucleotide sequence encoding the peptide tag to result in a recombinant nucleotide sequence, such that the peptide tag and the protein are expressed from the recombinant nucleotide sequence as a fusion protein.
• Embodiment 182. The method of embodiment 181, wherein the peptide tag is used for purification and/or detection of the protein.
• Embodiment 183. The method of embodiment 181, wherein the peptide tag is a poly-histidine (e.g., HHHHHH) (SEQ ID NO: 252-262), FLAG (e.g., DYKDDDDK) (SEQ ID NO: 2), V5 (e.g., GKPIPNPLLGLDST) (SEQ ID NO: 3), GCN4, HA (e.g., YPYDVPDYA) (SEQ ID NO: 5), Myc (e.g. EQKLISEED) (SEQ ID NO: 6), GST . . . etc.
• Embodiment 184. The method of embodiment 181, wherein the peptide tag has an amino acid sequence selected from the group consisting of SEQ ID NO: 1-6, 245-249, 252-262, 264-273, 275-276, 281, 278-288, and 622.
• Embodiment 185. The method of embodiment 181, wherein the peptide tag is fused to the protein by a linker.
• Embodiment 186. The method of embodiment 181, wherein the fusion protein has the following structure: [protein]-[peptide tag] or [peptide tag]-[protein], wherein “]-[” represents an optional linker.
• Embodiment 187. The method of embodiment 181, wherein the linker has an amino acid sequence of SEQ ID NO: 127, 165-176, 446, 453, and 767-769.
• Embodiment 188. The method of embodiment 181, wherein the prime editor construct comprises a PEgRNA comprising the nucleotide sequence of SEQ ID NOs: 101-104, 181-183, 223-244, 277, 325-334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 499-505, 735-761, 776-777.
• Embodiment 189. The method of embodiment 181, wherein the PEgRNA comprises a spacer, a gRNA core, and an extension arm, wherein the spacer is complementary to the target nucleotide sequence and the extension arm comprises a reverse transcriptase template that encodes the peptide tag.
• Embodiment 190. The method of embodiment 181, wherein the PEgRNA comprises a spacer, a gRNA core, and an extension arm, wherein the spacer is complementary to the target nucleotide sequence and the extension arm comprises a reverse transcriptase template that encodes the peptide tag.
• Embodiment 191. A method of preventing or halting the progression of a prion disease by installing on or more protective mutations into PRNP encoded by a target nucleotide sequence by prime editing, the method comprising: (a) contacting the target nucleotide sequence with a (i) prime editor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a reverse transcriptase and (ii) a PEgRNA comprising an edit template encoding the functional moiety; (b) polymerizing a single strand DNA sequence encoding the protective mutation; and (c) incorporating the single strand DNA sequence in place of a corresponding endogenous strand at the target nucleotide sequence through a DNA repair and/or replication process, wherein the method produces a recombinant target nucleotide sequence that encodes a PRNP comprising a protective mutation and which is resistant to misfolding.
• Embodiment 192. The method of embodiment 191, wherein the prion disease is a human prion disease.
• Embodiment 193. The method of embodiment 191, wherein the prion disease is an animal prion disease.
• Embodiment 194. The method of embodiment 192, wherein the prion disease is Creutzfeldt-Jakob Disease (CJD), Variant Creutzfeldt-Jakob Disease (vCJD), Gerstmann-Straussler-Scheinker Syndrome, Fatal Familial Insomnia, or Kuru.
• Embodiment 195. The method of embodiment 193, wherein the prion disease is Bovine Spongiform Encephalopathy (BSE or “mad cow disease”), Chronic Wasting Disease (CWD), Scrapie, Transmissible Mink Encephalopathy, Feline Spongiform Encephalopathy, and Ungulate Spongiform Encephalopathy.
• Embodiment 196. The method of embodiment 191, wherein the wildtype PRNP amino acid sequence is SEQ ID NOs: 291-292.
• Embodiment 197. The method of embodiment 191, wherein the method results in a modified PRNP amino acid sequence selected from the group consisting of SEQ ID NOs: 293-309, and 311-323, wherein said modified PRNP protein is resistant to misfolding.
• Embodiment 198. The method of embodiment 191, wherein the fusion protein comprises the amino acid sequence of PE1, PE2, or PE3.
• Embodiment 199. The method of embodiment 191, wherein the napDNAbp is a Cas9 nickase (nCas9).
• Embodiment 200. The method of embodiment 191, wherein the napDNAbp comprises the amino acid sequence of SEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.
• Embodiment 201. The method of embodiment 191, wherein the guide RNA comprises SEQ ID NOs: 222.
• Embodiment 202. A method of installing a ribonucleotide motif or tag in an RNA of interest encoded by a target nucleotide sequence by prime editing, the method comprising: (a) contacting the target nucleotide sequence with a (i) prime editor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a reverse transcriptase and (ii) a PEgRNA comprising an edit template encoding the ribonucleotide motif or tag; (b) polymerizing a single strand DNA sequence encoding the ribonucleotide motif or tag; and (c) incorporating the single strand DNA sequence in place of a corresponding endogenous strand at the target nucleotide sequence through a DNA repair and/or replication process, wherein the method produces a recombinant target nucleotide sequence that encodes a modified RNA of interest comprising the ribonucleotide motif or tag.
• Embodiment 203. The method of embodiment 202, wherein ribonucleotide motif or tag is a detection moiety.
• Embodiment 204. The method of embodiment 202, wherein the ribonucleotide motif or tag affects the expression level of the RNA of interest.
• Embodiment 205. The method of embodiment 202, wherein the ribonucleotide motif or tag affects the transport or subcellular location of the RNA of interest.
• Embodiment 206. The method of embodiment 202, wherein the ribonucleotide motif or tag is selected from the group consisting of SV40 type 1, SV40 type 2, SV40 type 3, hGH, BGH, rbGlob, TK, MALAT1 ENE-mascRNA, KSHV PAN ENE, Smbox/U1 snRNA box, U1 snRNA 3′ box, tRNA-lysine, broccoli aptamer, spinach aptamer, mango aptamer, HDV ribozyme, and m6A.
• Embodiment 207. The method of embodiment 202, wherein the PEgRNA comprises SEQ ID NOs: 101-104, 181-183, 223-244, 277, 325-334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 499-505, 735-761, 776-777.
• Embodiment 208. The method of embodiment 202, wherein the fusion protein comprises the amino acid sequence of PE1, PE2, or PE3.
• Embodiment 209. The method of embodiment 202, wherein the napDNAbp is a Cas9 nickase (nCas9).
• Embodiment 210. The method of embodiment 202, wherein the napDNAbp comprises the amino acid sequence of SEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.
• Embodiment 211. A method of installing or deleting a functional moiety in a protein of interest encoded by a target nucleotide sequence by prime editing, the method comprising: (a) contacting the target nucleotide sequence with a (i) prime editor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a reverse transcriptase and (ii) a PEgRNA comprising an edit template encoding the functional moiety or deletion of same; (b) polymerizing a single strand DNA sequence encoding the functional moiety or deletion of same; and (c) incorporating the single strand DNA sequence in place of a corresponding endogenous strand at the target nucleotide sequence through a DNA repair and/or replication process, wherein the method produces a recombinant target nucleotide sequence that encodes a modified protein comprising the protein of interest and the functional moiety or the removal of same, wherein the functional moiety alters a modification state or localization state of the protein.
• Embodiment 212. The method of embodiment 211, wherein functional moiety alters the phosphorylation, ubiquitylation, glycosylation, lipidation, hydroxylation, methylation, acetylation, crotonylation, SUMOylation state of the protein of interest.
• Embodiment 213. A fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a polymerase.
• Embodiment 214. The fusion protein of embodiment 213, wherein the fusion protein is capable of carrying out prime editing in the presence of an prime editing guide RNA (PEgRNA).
• Embodiment 215. The fusion protein of embodiment 213, wherein the napDNAbp has a nickase activity.
• Embodiment 216. The fusion protein of embodiment 213, wherein the napDNAbp is a Cas9 protein or variant thereof.
• Embodiment 217. The fusion protein of embodiment 213, wherein the napDNAbp is a nuclease active Cas9, a nuclease inactive Cas9 (dCas9), or a Cas9 nickase (nCas9).
• Embodiment 218. The fusion protein of embodiment 213, wherein the napDNAbp is Cas9 nickase (nCas9).
• Embodiment 219. The fusion protein of embodiment 213, wherein the napDNAbp is selected from the group consisting of: Cas9, Cas12e, Cas12d, Cas12a, Cas12b1, Cas13a, Cas12c, and Argonaute and optionally has a nickase activity.
• Embodiment 220. The fusion protein of embodiment 213, wherein the fusion protein when complexed with a PEgRNA is capable of binding to a target DNA sequence.
• Embodiment 221. The fusion protein of embodiment 220, wherein the target DNA sequence comprises a target strand and a complementary non-target strand.
• Embodiment 222. The fusion protein of embodiment 220, wherein the binding of the fusion protein complexed to the PEgRNA forms an R-loop.
• Embodiment 223. The fusion protein of embodiment 222, wherein the R-loop comprises (i) an RNA-DNA hybrid comprising the PEgRNA and the target strand, and (ii) the complementary non-target strand.
• Embodiment 224. The fusion protein of embodiment 223, wherein the complementary non-target strand is nicked to form a priming sequence having a free 3′ end.
• Embodiment 225. The fusion protein of embodiment 214, wherein the PEgRNA comprises (a) a guide RNA and (b) an extension arm at the 5′ or the 3′ end of the guide RNA, or at an intramolecular location in the guide RNA.
• Embodiment 226. The fusion protein of embodiment 225, wherein the extension arm comprises (i) a DNA synthesis template sequence comprising a desired nucleotide change, and (ii) a primer binding site.
• Embodiment 227. The fusion protein of embodiment 226, wherein the DNA synthesis template sequence encodes a single-strand DNA flap that is complementary to an endogenous DNA sequence adjacent to the nick site, wherein the single-strand DNA flap comprises the desired nucleotide change.
• Embodiment 228. The fusion protein of embodiment 225, wherein the extension arm is at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides, at least 22 nucleotides, at least 23 nucleotides, at least 24 nucleotides, or at least 25 nucleotides in length.
• Embodiment 229. The fusion protein of embodiment 227, wherein the single-strand DNA flap hybridizes to the endogenous DNA sequence adjacent to the nick site, thereby installing the desired nucleotide change.
• Embodiment 230. The fusion protein of embodiment 227, wherein the single-stranded DNA flap displaces the endogenous DNA sequence adjacent to the nick site and which has a free 5′ end.
• Embodiment 231. The fusion protein of embodiment 230, wherein the endogenous DNA sequence having the 5′ end is excised by the cell.
• Embodiment 232. The fusion protein of embodiment 230, wherein cellular repair of the single-strand DNA flap results in installation of the desired nucleotide change, thereby forming a desired product.
• Embodiment 233. The fusion protein of embodiment 226, wherein the desired nucleotide change is installed in an editing window that is between about −4 to +10 of the PAM sequence, or between about −10 to +20 of the PAM sequence, or between about −20 to +40 of the PAM sequence, or between about −30 to +100 of the PAM sequence, or wherein the desired nucleotide change is installed at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides downstream of the nick site.
• Embodiment 234. The fusion protein of embodiment 213, wherein the napDNAbp comprises an amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence to SEQ ID NO: 18.
• Embodiment 235. The fusion protein of embodiment 213, wherein the napDNAbp comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.
• Embodiment 236. The fusion protein of embodiment 213, wherein the polymerase is a reverse transcriptase comprising any one of the amino acid sequences of SEQ ID NO: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, 662, 700, 701-716, 739-741, and 766.
• Embodiment 237. The fusion protein of embodiment 213, wherein the polymerase is a reverse transcriptase comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, 662, 700, 701-716, 739-741, and 766.
• Embodiment 238. The fusion protein of embodiment 213, wherein the polymerase is a naturally-occurring reverse transcriptase from a retrovirus or a retrotransposon.
• Embodiment 239. The fusion protein of any one of the previous embodiments, wherein the fusion protein comprises the structure NH₂-[napDNAbp]-[polymerase]-COOH; or NH₂-[polymerase]-[napDNAbp]-COOH, wherein each instance of “]-[” indicates the presence of an optional linker sequence.
• Embodiment 240. The fusion protein of embodiment 239, wherein the linker sequence comprises an amino acid sequence of SEQ ID NOs: 127, 165-176, 446, 453, and 767-769.
• Embodiment 241. The fusion protein of embodiment 226, wherein the desired nucleotide change is a single nucleotide change, an insertion of one or more nucleotides, or a deletion of one or more nucleotides.
• Embodiment 242. The fusion protein of embodiment 241, wherein the insert or deletion is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, or at least 50.
• Embodiment 243. A PEgRNA comprising a guide RNA and at least one nucleic acid extension arm comprising a DNA synthesis template.
• Embodiment 244. The PEgRNA of embodiment 241, wherein the nucleic acid extension arm is position at the 3′ or 5′ end of the guide RNA, or at an intramolecular position in the guide RNA, and wherein the nucleic acid extension arm is DNA or RNA.
• Embodiment 245. The PEgRNA of embodiment 242, wherein the PEgRNA is capable of binding to a napDNAbp and directing the napDNAbp to a target DNA sequence.
• Embodiment 246. The PEgRNA of embodiment 245, wherein the target DNA sequence comprises a target strand and a complementary non-target strand, wherein the guide RNA hybridizes to the target strand to form an RNA-DNA hybrid and an R-loop.
• Embodiment 247. The PEgRNA of embodiment 243, wherein the at least one nucleic acid extension arm comprises (i) a DNA synthesis template, and (ii) a primer binding site.
• Embodiment 248. The PEgRNA of embodiment 247, wherein the nucleic acid extension arm is at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides, at least 22 nucleotides, at least 23 nucleotides, at least 24 nucleotides, or at least 25 nucleotides in length.
• Embodiment 249. The PEgRNA of embodiment 247, wherein the DNA synthesis template is at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, or at least 15 nucleotides in length.
• Embodiment 250. The PEgRNA of embodiment 247, wherein the primer binding site is at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, or at least 15 nucleotides in length.
• Embodiment 251. The PEgRNA of embodiment 243, further comprising at least one additional structure selected from the group consisting of a linker, a stem loop, a hairpin, a toeloop, an aptamer, or an RNA-protein recruitment domain.
• Embodiment 252. The PEgRNA of embodiment 247, wherein the DNA synthesis template encodes a single-strand DNA flap that is complementary to an endogenous DNA sequence adjacent to a nick site, wherein the single-strand DNA flap comprises a desired nucleotide change.
• Embodiment 253. The PEgRNA of embodiment 252, wherein the single-stranded DNA flap displaces an endogenous single-strand DNA having a 5′ end in the target DNA sequence that has been nicked, and wherein the endogenous single-strand DNA is immediately adjacent downstream of the nick site.
• Embodiment 254. The PEgRNA of embodiment 253, wherein the endogenous single-stranded DNA having the free 5′ end is excised by the cell.
• Embodiment 255. The PEgRNA of embodiment 253, wherein cellular repair of the single-strand DNA flap results in installation of the desired nucleotide change, thereby forming a desired product.
• Embodiment 256. The PEgRNA of embodiment 243, comprising the nucleotide sequence of SEQ ID NOs: 101-104, 181-183, 223-244, 277, 325-334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 499-505, 735-761, 776-777, or a nucleotide sequence having at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99% sequence identity with any one of SEQ ID NOs: 101-104, 181-183, 223-244, 277, 325-334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 499-505, 735-761, 776-777.
• Embodiment 257. The PEgRNA of embodiment 247, wherein the DNA synthesis template comprises a nucleotide sequence that is at least 80%, or 85%, or 90%, or 95%, or 99% identical to the endogenous DNA target.
• Embodiment 258. The PEgRNA of embodiment 247, wherein the primer binding site hybridizes with a free 3′ end of the cut DNA.
• Embodiment 259. The PEgRNA of embodiment 251, wherein the at least one additional structure is located at the 3′ or 5′ end of the PEgRNA.
• Embodiment 260. A complex comprising a fusion protein of any one of embodiments 213-242 and an PEgRNA.
• Embodiment 261. The complex of embodiment 260, wherein the PEgRNA comprises a guide RNA and an nucleic acid extension arm at the 3′ or 5′ end of the guide RNA or at an intramolecular position in the guide RNA.
• Embodiment 262. The complex of embodiment 260, wherein the PEgRNA is capable of binding to a napDNAbp and directing the napDNAbp to a target DNA sequence.
• Embodiment 263. The complex of embodiment 262, wherein the target DNA sequence comprises a target strand and a complementary non-target strand, wherein the guide RNA hybridizes to the target strand to form an RNA-DNA hybrid and an R-loop.
• Embodiment 264. The complex of embodiment 261, wherein the at least one nucleic acid extension arm comprises (i) a DNA synthesis template, and (ii) a primer binding site.
• Embodiment 265. The complex of embodiment 260, wherein the PEgRNA comprises the nucleotide sequence of SEQ ID NOs: 101-104, 181-183, 223-244, 277, 325-334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 499-505, 735-761, 776-777, or a nucleotide sequence having at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99% sequence identity with any one of SEQ ID NOs: 101-104, 181-183, 223-244, 277, 325-334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 499-505, 735-761, 776-777.
• Embodiment 266. The complex of embodiment 264, wherein the DNA synthesis template comprises a nucleotide sequence that is at least 80%, or 85%, or 90%, or 95%, or 99% identical to the endogenous DNA target.
• Embodiment 267. The complex of embodiment 264, wherein the primer binding site hybridizes with a free 3′ end of the cut DNA.
• Embodiment 268. A complex comprising a napDNAbp and an PEgRNA.
• Embodiment 269. The complex of embodiment 268, wherein the napDNAbp is a Cas9 nickase.
• Embodiment 270. The complex of embodiment 268, wherein the napDNAbp comprises an amino acid sequence of SEQ ID NO: 18.
• Embodiment 271. The complex of embodiment 268, wherein the napDNAbp comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.
• Embodiment 272. The complex of embodiment 268, wherein the PEgRNA comprises a guide RNA and a nucleic acid extension arm at the 3′ or 5′ end of the guide RNA, or at an intramolecular position in the guide RNA.
• Embodiment 273. The complex of embodiment 268, wherein the PEgRNA is capable of directing the napDNAbp to a target DNA sequence.
• Embodiment 274. The complex of embodiment 272, wherein the target DNA sequence comprises a target strand and a complementary non-target strand, wherein the spacer sequence of the PEgRNA hybridizes to the target strand to form an RNA-DNA hybrid and an R-loop.
• Embodiment 275. The complex of embodiment 273, wherein the nucleic acid extension arm comprises (i) a DNA synthesis template, and (ii) a primer binding site.
• Embodiment 276. The complex of embodiment 269, wherein the PEgRNA comprises the nucleotide sequence of SEQ ID NOs: 101-104, 181-183, 223-244, 277, 325-334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 499-505, 735-761, 776-777, or a nucleotide sequence having at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99% sequence identity with any one of SEQ ID NOs: 101-104, 181-183, 223-244, 277, 325-334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 499-505, 735-761, 776-777.
• Embodiment 277. The complex of embodiment 276, wherein the DNA synthesis template comprises a nucleotide sequence that is at least 80%, or 85%, or 90%, or 95%, or 99% identical to the endogenous DNA target.
• Embodiment 278. The complex of embodiment 276, wherein the primer binding site hybridizes with a free 3′ end of the cut DNA.
• Embodiment 279. The complex of embodiment 276, wherein the PEgRNA further comprises at least one additional structure selected from the group consisting of a linker, a stem loop, a hairpin, a toeloop, an aptamer, or an RNA-protein recruitment domain.
• Embodiment 280. A polynucleotide encoding the fusion protein of any of embodiments 213-242.
• Embodiment 281. A vector comprising the polynucleotide of embodiment 280.
• Embodiment 282. A cell comprising the fusion protein of any of embodiments 213-242 and an PEgRNA bound to the napDNAbp of the fusion protein.
• Embodiment 283. A cell comprising a complex of any one of embodiments 260-279.
• Embodiment 284. A pharmaceutical composition comprising: (i) a fusion protein of any of embodiments 213-242, the complex of embodiments 260-279, the polynucleotide of embodiment 68, or the vector of embodiment 69; and (ii) a pharmaceutically acceptable excipient.
• Embodiment 285. A pharmaceutical composition comprising: (i) the complex of embodiments 260-279 (ii) a polymerase provided in trans; and (iii) a pharmaceutically acceptable excipient.
• Embodiment 286. A kit comprising a nucleic acid construct, comprising: (i) a nuleic acid sequencing encoding the fusion protein of any one of embodiments 213-242; and (ii) a promoter that drives expression of the sequence of (i).
• Embodiment 287. A method for installing a desired nucleotide change in a double-stranded DNA sequence, the method comprising:
• (i) contacting the double-stranded DNA sequence with a complex comprising a fusion protein and a PEgRNA, wherein the fusion protein comprises a napDNAbp and a polymerase and wherein the PEgRNA comprises a DNA synthesis template comprising the desired nucleotide change and a primer binding site;
• (ii) nicking the double-stranded DNA sequence, thereby generating a free single-strand DNA having a 3′ end;
• (iii) hybridizing the 3′ end of the free single-strand DNA to the primer binding site, thereby priming the polymerase;
• (iv) polymerizing a strand of DNA from the 3′ end hybridized to the primer binding site, thereby generating a single-strand DNA flap comprising the desired nucleotide change and which is complementary to the DNA synthesis template;
• (v) replacing an endogenous DNA strand adjacent the cut site with the single-strand DNA flap, thereby installing the desired nucleotide change in the double-stranded DNA sequence.
• Embodiment 288. The method of embodiment 287, wherein the step of (v) replacing comprises: (i) hybridizing the single-strand DNA flap to the endogenous DNA strand adjacent the cut site to create a sequence mismatch; (ii) excising the endogenous DNA strand; and (iii) repairing the mismatch to form the desired product comprising the desired nucleotide change in both strands of DNA.
• Embodiment 289. The method of embodiment 288, wherein the desired nucleotide change is a single nucleotide substitution, a deletion, or an insertion.
• Embodiment 290. The method of embodiment 289, wherein the single nucleotide substitution is a transition or a transversion.
• Embodiment 291. The method of embodiment 288, wherein the desired nucleotide change is (1) a G to T substitution, (2) a G to A substitution, (3) a G to C substitution, (4) a T to G substitution, (5) a T to A substitution, (6) a T to C substitution, (7) a C to G substitution, (8) a C to T substitution, (9) a C to A substitution, (10) an A to T substitution, (11) an A to G substitution, or (12) an A to C substitution.
• Embodiment 292. The method of embodiment 288, wherein the desired nucleoid change converts (1) a G:C basepair to a T:A basepair, (2) a G:C basepair to an A:T basepair, (3) a G:C basepair to C:G basepair, (4) a T:A basepair to a G:C basepair, (5) a T:A basepair to an A:T basepair, (6) a T:A basepair to a C:G basepair, (7) a C:G basepair to a G:C basepair, (8) a C:G basepair to a T:A basepair, a C:G basepair to an A:T basepair, (10) an A:T basepair to a T:A basepair, (11) an A:T basepair to a G:C basepair, or (12) an A:T basepair to a C:G basepair.
• Embodiment 293. The method of embodiment 288, wherein the desired nucleotide change is an insertion or deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides.
• Embodiment 294. The method of embodiment 288, wherein the desired nucleotide change corrects a disease-associated gene.
• Embodiment 295. The method of embodiment 294, wherein the disease-associated gene is associated with a monogenetic disorder selected from the group consisting of: Adenosine Deaminase (ADA) Deficiency; Alpha-1 Antitrypsin Deficiency; Cystic Fibrosis; Duchenne Muscular Dystrophy; Galactosemia; Hemochromatosis; Huntington's Disease; Maple Syrup Urine Disease; Marfan Syndrome; Neurofibromatosis Type 1; Pachyonychia Congenita; Phenylkeotnuria; Severe Combined Immunodeficiency; Sickle Cell Disease; Smith-Lemli-Opitz Syndrome; a trinucleotide repeat disorder; a prion disease; and Tay-Sachs Disease.
• Embodiment 296. The method of embodiment 294, wherein the disease-associated gene is associated with a polygenic disorder selected from the group consisting of: heart disease; high blood pressure; Alzheimer's disease; arthritis; diabetes; cancer; and obesity.
• Embodiment 297. The method of embodiment 287, wherein the napDNAbp is a nuclease dead Cas9 (dCas9), a Cas9 nickase (nCas9), or a nuclease active Cas9.
• Embodiment 298. The method of embodiment 287, wherein the napDNAbp comprises an amino acid sequence of SEQ ID NO: 18, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 18.
• Embodiment 299. The method of embodiment 287, wherein the napDNAbp comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.
• Embodiment 300. The method of embodiment 287, wherein the polymerase is a reverse transcriptase comprising any one of the amino acid sequences of SEQ ID NO: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, 662, 700, 701-716, 739-741, and 766.
• Embodiment 301. The method of embodiment 287, wherein the polymerase is a reverse transcriptase comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, 662, 700, 701-716, 739-741, and 766.
• Embodiment 302. The method of embodiment 287, wherein the PEgRNA comprises a nucleic acid extension arm at the 3′ or 5′ ends or at an intramolecular location in the guide RNA, wherein the extension arm comprises the DNA synthesis template sequence and the primer binding site.
• Embodiment 303. The method of embodiment 302, wherein the extension arm is at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides, at least 22 nucleotides, at least 23 nucleotides, at least 24 nucleotides, or at least 25 nucleotides in length.
• Embodiment 304. The method of embodiment 287, wherein the PEgRNA has a nucleotide sequence selected from the group consisting of SEQ ID NOs: 101-104, 181-183, 223-244, 277, 325-334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 499-505, 735-761, 776-777.
• Embodiment 305. A method for introducing one or more changes in the nucleotide sequence of a DNA molecule at a target locus, comprising:
• (i) contacting the DNA molecule with a nucleic acid programmable DNA binding protein (napDNAbp) and a PEgRNA which targets the napDNAbp to the target locus, wherein the PEgRNA comprises a reverse transcriptase (RT) template sequence comprising at least one desired nucleotide change and a primer binding site;
• (ii) forming an exposed 3′ end in a DNA strand at the target locus;
• (iii) hybridizing the exposed 3′ end to the primer binding site to prime reverse transcription;
• (iv) synthesizing a single strand DNA flap comprising the at least one desired nucleotide change based on the RT template sequence by reverse transcriptase;
• (v) and incorporating the at least one desired nucleotide change into the corresponding endogenous DNA, thereby introducing one or more changes in the nucleotide sequence of the DNA molecule at the target locus.
• Embodiment 306. The method of embodiment 305, wherein the one or more changes in the nucleotide sequence comprises a transition.
• Embodiment 307. The method of embodiment 306, wherein the transition is selected from the group consisting of: (a) T to C; (b) A to G; (c) C to T; and (d) G to A.
• Embodiment 308. The method of embodiment 305, wherein the one or more changes in the nucleotide sequence comprises a transversion.
• Embodiment 309. The method of embodiment 308, wherein the transversion is selected from the group consisting of: (a) T to A; (b) T to G; (c) C to G; (d) C to A; (e) A to T; (f) A to C; (g) G to C; and (h) G to T.
• Embodiment 310. The method of embodiment 305, wherein the one or more changes in the nucleotide sequence comprises changing (1) a G:C basepair to a T:A basepair, (2) a G:C basepair to an A:T basepair, (3) a G:C basepair to C:G basepair, (4) a T:A basepair to a G:C basepair, (5) a T:A basepair to an A:T basepair, (6) a T:A basepair to a C:G basepair, (7) a C:G basepair to a G:C basepair, (8) a C:G basepair to a T:A basepair, (9) a C:G basepair to an A:T basepair, (9) an A:T basepair to a T:A basepair, (11) an A:T basepair to a G:C basepair, or (12) an A:T basepair to a C:G basepair.
• Embodiment 311. The method of embodiment 305, wherein the one or more changes in the nucleotide sequence comprises an insertion or deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides.
• Embodiment 312. The method of embodiment 305, wherein the one or more changes in the nucleotide sequence comprises a correction to a disease-associated gene.
• Embodiment 313. The method of embodiment 312, wherein the disease-associated gene is associated with a monogenetic disorder selected from the group consisting of: Adenosine Deaminase (ADA) Deficiency; Alpha-1 Antitrypsin Deficiency; Cystic Fibrosis; Duchenne Muscular Dystrophy; Galactosemia; Hemochromatosis; Huntington's Disease; Maple Syrup Urine Disease; Marfan Syndrome; Neurofibromatosis Type 1;
• Pachyonychia Congenita; Phenylkeotnuria; Severe Combined Immunodeficiency; Sickle Cell Disease; Smith-Lemli-Opitz Syndrome; a trinucleotide repeat disorder; a prion disease; and Tay-Sachs Disease.
• Embodiment 314. The method of embodiment 312, wherein the disease-associated gene is associated with a polygenic disorder selected from the group consisting of: heart disease; high blood pressure; Alzheimer's disease; arthritis; diabetes; cancer; and obesity.
• Embodiment 315. The method of embodiment 305, wherein the napDNAbp is a nuclease active Cas9 or variant thereof.
• Embodiment 316. The method of embodiment 305, wherein the napDNAbp is a nuclease inactive Cas9 (dCas9) or Cas9 nickase (nCas9), or a variant thereof.
• Embodiment 317. The method of embodiment 305, wherein the napDNAbp comprises an amino acid sequence of SEQ ID NO: 18, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO: 18.
• Embodiment 318. The method of embodiment 305, wherein the napDNAbp comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.
• Embodiment 319. The method of embodiment 305, wherein the reverse transcriptase is introduced in trans.
• Embodiment 320. The method of embodiment 305, wherein the napDNAbp comprises a fusion to a reverse transcriptase.
• Embodiment 321. The method of embodiment 305, wherein the reverse transcriptase comprises any one of the amino acid sequences of SEQ ID NO: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, 662, 700, 701-716, 739-741, and 766.
• Embodiment 322. The method of embodiment 305, wherein the reverse transcriptase comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, 662, 700, 701-716, 739-741, and 766.
• Embodiment 323. The method of embodiment 305, wherein the step of forming an exposed 3′ end in the DNA strand at the target locus comprises nicking the DNA strand with a nuclease.
• Embodiment 324. The method of embodiment 323, wherein the nuclease is provided is provided in trans.
• Embodiment 325. The method of embodiment 305, wherein the step of forming an exposed 3′ end in the DNA strand at the target locus comprises contacting the DNA strand with a chemical agent.
• Embodiment 326. The method of embodiment 305, wherein the step of forming an exposed 3′ end in the DNA strand at the target locus comprises introducing a replication error.
• Embodiment 327. The method of embodiment 305, wherein the step of contacting the DNA molecule with the napDNAbp and the guide RNA forms an R-loop.
• Embodiment 328. The method of embodiment 327, wherein the DNA strand in which the exposed 3′ end is formed is in the R-loop.
• Embodiment 329. The method of embodiment 315, wherein the PEgRNA comprises an extension arm that comprises the reverse transcriptase (RT) template sequence and the primer binding site.
• Embodiment 330. The method of embodiment 329, wherein the extension arm is at the 3′ end of the guide RNA, the 5′ end of the guide RNA, or at an intramolecular position in the guide RNA.
• Embodiment 331. The method of embodiment 305, wherein the PEgRNA further comprises at least one additional structure selected from the group consisting of a linker, a stem loop, a hairpin, a toeloop, an aptamer, or an RNA-protein recruitment domain.
• Embodiment 332. The method of embodiment 305, wherein the PEgRNA further comprises a homology arm.
• Embodiment 333. The method of embodiment 305, wherein the RT template sequence is homologous to the corresponding endogenous DNA.
• Embodiment 334. A method for introducing one or more changes in the nucleotide sequence of a DNA molecule at a target locus by target-primed reverse transcription, the method comprising: (a) contacting the DNA molecule at the target locus with a (i) fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a reverse transcriptase and (ii) a guide RNA comprising an RT template comprising a desired nucleotide change; (b) conducting target-primed reverse transcription of the RT template to generate a single strand DNA comprising the desired nucleotide change; and (c) incorporating the desired nucleotide change into the DNA molecule at the target locus through a DNA repair and/or replication process.
• Embodiment 335. The method of embodiment 334, wherein the RT template is located at the 3′ end of the guide RNA, the 5′ end of the guide RNA, or at an intramolecular location in the guide RNA.
• Embodiment 336. The method of embodiment 334, wherein the desired nucleotide change comprises a transition, a transversion, an insertion, or a deletion, or any combination thereof.
• Embodiment 337. The method of embodiment 334, wherein the desired nucleotide change comprises a transition selected from the group consisting of: (a) T to C; (b) A to G; (c) C to T; and (d) G to A.
• Embodiment 338. The method of claim 334, wherein the desired nucleotide change comprises a transversion selected from the group consisting of: (a) T to A; (b) T to G; (c) C to G; (d) C to A; (e) A to T; (f) A to C; (g) G to C; and (h) G to T.
• Embodiment 339. The method of embodiment 334, wherein the desired nucleotide change comprises changing (1) a G:C basepair to a T:A basepair, (2) a G:C basepair to an A:T basepair, (3) a G:C basepair to C:G basepair, (4) a T:A basepair to a G:C basepair, (5) a T:A basepair to an A:T basepair, (6) a T:A basepair to a C:G basepair, (7) a C:G basepair to a G:C basepair, (8) a C:G basepair to a T:A basepair, (9) a C:G basepair to an A:T basepair, (10) an A:T basepair to a T:A basepair, (11) an A:T basepair to a G:C basepair, or (12) an A:T basepair to a C:G basepair.
• Embodiment 340. A polynucleotide encoding the PEgRNA of any one of embodiments 243-259.
• Embodiment 341. A vector comprising the polynucleotide of embodiment 340.
• Embodiment 342. A cell comprising the vector of embodiment 341.
• Embodiment 343. The fusion protein of embodiment 213, wherein the polymerase is an error-prone reverse transcriptase.
• Embodiment 344. A method for mutagenizing a DNA molecule at a target locus by target-primed reverse transcription, the method comprising: (a) contacting the DNA molecule at the target locus with a (i) fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) and an error-prone reverse transcriptase and (ii) a guide RNA comprising an RT template comprising a desired nucleotide change; (b) conducting target-primed reverse transcription of the RT template to generate a mutagenized single strand DNA; and (c) incorporating the mutagenized single strand DNA into the DNA molecule at the target locus through a DNA repair and/or replication process.
• Embodiment 345. The method of any prior embodiment, wherein the fusion protein comprises the amino acid sequence of PE1, PE2, or PE3.
• Embodiment 346. The method of any prior embodiment, wherein the napDNAbp is a Cas9 nickase (nCas9).
• Embodiment 347. The method of embodiment 344, wherein the napDNAbp comprises the amino acid sequence of SEQ ID NOs: 18-25.
• Embodiment 348. The method of embodiment 344, wherein the guide RNA comprises SEQ ID NOs: 222.
• Embodiment 349. The method of embodiment 344, wherein the step of (b) conducting target-primed reverse transcription comprises generating a 3′ end primer binding sequence at the target locus that is capable of priming reverse transcription by annealing to a primer binding site on the guide RNA.
• Embodiment 350. A method for replacing a trinucleotide repeat expansion mutation in a target DNA molecule with a healthy sequence comprising a healthy number of repeat trinucleotides, the method comprising: (a) contacting the DNA molecule at the target locus with a (i) fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a polymerase and (ii) a PEgRNA comprising DNA synthesis template comprising the replacement sequence and a primer binding site; (b) conducting prime editing to generate a single strand DNA comprising the replacement sequence; and (c) incorporating the single strand DNA into the DNA molecule at the target locus through a DNA repair and/or replication process.
• Embodiment 351. The method of embodiment 350, wherein the fusion protein comprises the amino acid sequence of PE1, PE2, or PE3.
• Embodiment 352. The method of embodiment 350, wherein the napDNAbp is a Cas9 nickase (nCas9).
• Embodiment 353. The method of embodiment 350, wherein the napDNAbp comprises the amino acid sequence of SEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.
• Embodiment 354. The method of embodiment 350, wherein the guide RNA comprises SEQ ID NOs: 222.
• Embodiment 355. The method of embodiment 350, wherein the step of (b) conducting prime editing comprises generating a 3′ end primer binding sequence at the target locus that is capable of priming polymerase by annealing to the primer binding site on the guide RNA.
• Embodiment 356. The method of embodiment 350, wherein the trinucleotide repeat expansion mutation is associated with Huntington's Disease, Fragile X syndrome, or Friedreich's ataxia.
• Embodiment 357. The method of embodiment 350, wherein the trinucleotide repeat expansion mutation comprises a repeating unit of CAG triplets.
• Embodiment 358. The method of embodiment 350, wherein the trinucleotide repeat expansion mutation comprises a repeating unit of GAA triplets.
• Embodiment 359. A method of installing a functional moiety in a protein of interest encoded by a target nucleotide sequence by prime editing, the method comprising: (a) contacting the target nucleotide sequence with a (i) prime editor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a polymerase and (ii) a PEgRNA comprising DNA synthesis template encoding the functional moiety; (b) polymerizing a single strand DNA sequence encoding the functional moiety; and (c) incorporating the single strand DNA sequence in place of a corresponding endogenous strand at the target nucleotide sequence through a DNA repair and/or replication process, wherein the method produces a recombinant target nucleotide sequence that encodes a fusion protein comprising the protein of interest and the functional moiety.
• Embodiment 360. The method of embodiment 359, wherein functional moiety is peptide tag.
• Embodiment 361. The method of embodiment 360, wherein the peptide tag is an affinity tag, solubilization tag, chromatography tag, epitope or immunoepitope tag, or a fluorescence tag.
• Embodiment 362. The method of embodiment 360, wherein the peptide tag is selected from the group consisting of: AviTag (SEQ ID NO: 245); C-tag (SEQ ID NO: 246); Calmodulin-tag (SEQ ID NO: 247); polyglutamate tag (SEQ ID NO: 248); E-tag (SEQ ID NO: 249); FLAG-tag (SEQ ID NO: 2); HA-tag (SEQ ID NO: 5); His-tag (SEQ ID NOs: 252-262); Myc-tag (SEQ ID NO: 6); NE-tag (SEQ ID NO: 264); Rho1D4-tag (SEQ ID NO: 265); S-tag (SEQ ID NO: 266); SBP-tag (SEQ ID NO: 267); Softag-1 (SEQ ID NO: 268); Softag-2 (SEQ ID NO: 269); Spot-tag (SEQ ID NO: 270); Strep-tag (SEQ ID NO: 271); TC tag (SEQ ID NO: 272); Ty tag (SEQ ID NO: 273); V5 tag (SEQ ID NO: 3); VSV-tag (SEQ ID NO: 275); and Xpress tag (SEQ ID NO: 276).
• Embodiment 363. The method of embodiment 360, wherein the peptide tag is selected from the group consisting of: AU1 epitope (SEQ ID NO: 278); AU5 epitope (SEQ ID NO: 279); Bacteriophage T7 epitope (T7-tag) (SEQ ID NO: 280); Bluetongue virus tag (B-tag) (SEQ ID NO: 281); E2 epitope (SEQ ID NO: 282); Histidine affinity tag (HAT) (SEQ ID NO: 283); HSV epitope (SEQ ID NO: 284); Polyarginine (Arg-tag) (SEQ ID NO: 285); Polyaspartate (Asp-tag) (SEQ ID NO: 286); Polyphenylalanine (Phe-tag) (SEQ ID NO: 287); 51-tag (SEQ ID NO: 288); S-tag (SEQ ID NO: 266); and VSV-G (SEQ ID NO: 275).
• Embodiment 364. The method of embodiment 359, wherein the functional moiety is an immunoepitope.
• Embodiment 365. The method of embodiment 364, wherein the immunoepitope is selected from the group consisting of: tetanus toxoid (SEQ ID NO: 396); diphtheria toxin mutant CRM197 (SEQ ID NO: 630); mumps immunoepitope 1 (SEQ ID NO: 400); mumps immunoepitope 2 (SEQ ID NO: 402); mumps immunoeptitope 3 (SEQ ID NO: 404); rubella virus (SEQ ID NO: 406); hemagglutinin (SEQ ID NO: 408); neuraminidase (SEQ ID NO: 410); TAP1 (SEQ ID NO: 412); TAP2 (SEQ ID NO: 414); hemagglutinin epitopes toward class I HLA (SEQ ID NO: 416); neuraminidase epitopes toward class I HLA (SEQ ID NO: 418); hemagglutinin epitopes toward class II HLA (SEQ ID NO: 420); neuraminidase epitopes toward class II HLA (SEQ ID NO: 422); hemagglutinin epitope H5N1-bound class I and class II HLA (SEQ ID NO: 424); neuraminidase epitope H5N1-bound class I and class II HLA (SEQ ID NO: 426).
• Embodiment 366. The method of embodiment 359, wherein the functional moiety alters the localization of the protein of interest.
• Embodiment 367. The method of embodiment 359, wherein the functional moiety is a degradation tag such that the degradation rate of the protein of interest is altered.
• Embodiment 368. The method of embodiment 367, wherein the degradation tag results in the elimination of the tagged protein.
• Embodiment 369. The method of embodiment 359, wherein the functional moiety is a small molecule binding domain.
• Embodiment 370. The method of embodiment 359, wherein the small molecule binding domain is FKBP12 of SEQ ID NO: 488.
• Embodiment 371. The method of embodiment 359, wherein the small molecule binding domain is FKBP12-F36V of SEQ ID NO: 489.
• Embodiment 372. The method of embodiment 359, wherein the small molecule binding domain is cyclophilin of SEQ ID NOs: 490 and 493-494.
• Embodiment 373. The method of embodiment 359, wherein the small molecule binding domain is installed in two or more proteins of interest.
• Embodiment 374. The method of embodiment 373, wherein the two or more proteins of interest may dimerize upon contacting with a small molecule.
• Embodiment 375. The method of embodiment 369, wherein the small molecule is a dimer of a small molecule selected from the group consisting of those compounds disclosed in Embodiment 163 of Group 1.
• Embodiment 376. A method of installing an immunoepitope in a protein of interest encoded by a target nucleotide sequence by prime editing, the method comprising: (a) contacting the target nucleotide sequence with a (i) prime editor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a polymerase and (ii) a PEgRNA comprising an edit template encoding the functional moiety; (b) polymerizing a single strand DNA sequence encoding the immunoepitope; and (c) incorporating the single strand DNA sequence in place of a corresponding endogenous strand at the target nucleotide sequence through a DNA repair and/or replication process, wherein the method produces a recombinant target nucleotide sequence that encodes a fusion protein comprising the protein of interest and the immunoepitope.
• Embodiment 377. The method of embodiment 376, wherein the immunoepitope is selected from the group consisting of: tetanus toxoid (SEQ ID NO: 396); diphtheria toxin mutant CRM197 (SEQ ID NO: 630); mumps immunoepitope 1 (SEQ ID NO: 400); mumps immunoepitope 2 (SEQ ID NO: 402); mumps immunoeptitope 3 (SEQ ID NO: 404); rubella virus (SEQ ID NO: 406); hemagglutinin (SEQ ID NO: 408); neuraminidase (SEQ ID NO: 410); TAP1 (SEQ ID NO: 412); TAP2 (SEQ ID NO: 414); hemagglutinin epitopes toward class I HLA (SEQ ID NO: 416); neuraminidase epitopes toward class I HLA (SEQ ID NO: 418); hemagglutinin epitopes toward class II HLA (SEQ ID NO: 420); neuraminidase epitopes toward class II HLA (SEQ ID NO: 422); hemagglutinin epitope H5N1-bound class I and class II HLA (SEQ ID NO: 424); neuraminidase epitope H5N1-bound class I and class II HLA (SEQ ID NO: 426).
• Embodiment 378. The method of embodiment 376, wherein the fusion protein comprises the amino acid sequence of PE1, PE2, or PE3.
• Embodiment 379. The method of embodiment 376, wherein the napDNAbp is a Cas9 nickase (nCas9).
• Embodiment 380. The method of embodiment 376, wherein the napDNAbp comprises the amino acid sequence of SEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.
• Embodiment 381. The method of embodiment 376, wherein the PEgRNA comprises SEQ ID NOs: 101-104, 181-183, 223-244, 277, 325-334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 499-505, 735-761, 776-777.
• Embodiment 382. A method of installing a small molecule dimerization domain in a protein of interest encoded by a target nucleotide sequence by prime editing, the method comprising: (a) contacting the target nucleotide sequence with a (i) prime editor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a polymerase and (ii) a PEgRNA comprising an edit template encoding the small molecule dimerization domain; (b) polymerizing a single strand DNA sequence encoding the immunoepitope; and (c) incorporating the single strand DNA sequence in place of a corresponding endogenous strand at the target nucleotide sequence through a DNA repair and/or replication process, wherein the method produces a recombinant target nucleotide sequence that encodes a fusion protein comprising the protein of interest and the small molecule dimerization domain.
• Embodiment 383. The method of embodiment 382, further comprising conducting the method on a second protein of interest.
• Embodiment 384. The method of embodiment 383, wherein the first protein of interest and the second protein of interest dimerize in the presence of a small molecule that binds to the dimerization domain on each of said proteins.
• Embodiment 385. The method of embodiment 382, wherein the small molecule binding domain is FKBP12 of SEQ ID NO: 488.
• Embodiment 386. The method of embodiment 382, wherein the small molecule binding domain is FKBP12-F36V of SEQ ID NO: 489.
• Embodiment 387. The method of embodiment 382, wherein the small molecule binding domain is cyclophilin of SEQ ID NOs: 490 and 493-494.
• Embodiment 388. The method of embodiment 382, wherein the small molecule is a dimer of a small molecule selected from the group consisting of those compounds disclosed in Embodiment 163 of Group 1.
• Embodiment 389. The method of embodiment 382, wherein the fusion protein comprises the amino acid sequence of PE1, PE2, or PE3.
• Embodiment 390. The method of embodiment 382, wherein the napDNAbp is a Cas9 nickase (nCas9).
• Embodiment 391. The method of embodiment 382, wherein the napDNAbp comprises the amino acid sequence of SEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.
• Embodiment 392. The method of embodiment 382, wherein the PEgRNA comprises SEQ ID NOs: 101-104, 181-183, 223-244, 277, 325-334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 499-505, 735-761, 776-777.
• Embodiment 393. A method of installing a peptide tag or epitope onto a protein using prime editing, comprising: contacting a target nucleotide sequence encoding the protein with a prime editor construct configured to insert therein a second nucleotide sequence encoding the peptide tag to result in a recombinant nucleotide sequence, such that the peptide tag and the protein are expressed from the recombinant nucleotide sequence as a fusion protein.
• Embodiment 394. The method of embodiment 383, wherein the peptide tag is used for purification and/or detection of the protein.
• Embodiment 395. The method of embodiment 383, wherein the peptide tag is a poly-histidine (e.g., HHHHHH) (SEQ ID NO: 252-262), FLAG (e.g., DYKDDDDK) (SEQ ID NO: 2), V5 (e.g., GKPIPNPLLGLDST) (SEQ ID NO: 3), GCN4, HA (e.g., YPYDVPDYA) (SEQ ID NO: 5), Myc (e.g. EQKLISEED) (SEQ ID NO: 6), or GST.
• Embodiment 396. The method of embodiment 383, wherein the peptide tag has an amino acid sequence selected from the group consisting of SEQ ID NO: 1-6, 245-249, 252-262, 264-273, 275-276, 281, 278-288, and 622.
• Embodiment 397. The method of embodiment 383, wherein the peptide tag is fused to the protein by a linker.
• Embodiment 398. The method of embodiment 383, wherein the fusion protein has the following structure: [protein]-[peptide tag] or [peptide tag]-[protein], wherein “]-[” represents an optional linker.
• Embodiment 399. The method of embodiment 383, wherein the linker has an amino acid sequence of SEQ ID NO: 127, 165-176, 446, 453, and 767-769.
• Embodiment 400. The method of embodiment 383, wherein the prime editor construct comprises a PEgRNA comprising the nucleotide sequence of SEQ ID NOs: 101-104, 181-183, 223-244, 277, 325-334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 499-505, 735-761, 776-777.
• Embodiment 401. The method of embodiment 383, wherein the PEgRNA comprises a spacer, a gRNA core, and an extension arm, wherein the spacer is complementary to the target nucleotide sequence and the extension arm comprises a reverse transcriptase template that encodes the peptide tag.
• Embodiment 402. The method of embodiment 383, wherein the PEgRNA comprises a spacer, a gRNA core, and an extension arm, wherein the spacer is complementary to the target nucleotide sequence and the extension arm comprises a reverse transcriptase template that encodes the peptide tag.
• Embodiment 403. A method of preventing or halting the progression of a prion disease by installing on or more protective mutations into PRNP encoded by a target nucleotide sequence by prime editing, the method comprising: (a) contacting the target nucleotide sequence with a (i) prime editor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a polymerase and (ii) a PEgRNA comprising an edit template encoding the functional moiety; (b) polymerizing a single strand DNA sequence encoding the protective mutation; and (c) incorporating the single strand DNA sequence in place of a corresponding endogenous strand at the target nucleotide sequence through a DNA repair and/or replication process, wherein the method produces a recombinant target nucleotide sequence that encodes a PRNP comprising a protective mutation and which is resistant to misfolding.
• Embodiment 404. The method of embodiment 403, wherein the prion disease is a human prion disease.
• Embodiment 405. The method of embodiment 403, wherein the prion disease is an animal prion disease.
• Embodiment 406. The method of embodiment 404, wherein the prion disease is Creutzfeldt-Jakob Disease (CJD), Variant Creutzfeldt-Jakob Disease (vCJD), Gerstmann-Straussler-Scheinker Syndrome, Fatal Familial Insomnia, or Kuru.
• Embodiment 407. The method of embodiment 403, wherein the prion disease is Bovine Spongiform Encephalopathy (BSE or “mad cow disease”), Chronic Wasting Disease (CWD), Scrapie, Transmissible Mink Encephalopathy, Feline Spongiform Encephalopathy, and Ungulate Spongiform Encephalopathy.
• Embodiment 408. The method of embodiment 403, wherein the wildtype PRNP amino acid sequence is SEQ ID NOs: 291-292.
• Embodiment 409. The method of embodiment 403, wherein the method results in a modified PRNP amino acid sequence selected from the group consisting of SEQ ID NOs: 293-309, 311-323, wherein said modified PRNP protein is resistant to misfolding.
• Embodiment 410. The method of embodiment 403, wherein the fusion protein comprises the amino acid sequence of PE1, PE2, or PE3.
• Embodiment 411. The method of embodiment 403, wherein the napDNAbp is a Cas9 nickase (nCas9).
• Embodiment 412. The method of embodiment 403, wherein the napDNAbp comprises the amino acid sequence of SEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.
• Embodiment 413. The method of embodiment 403, wherein the PEgRNA comprises SEQ ID NOs: 101-104, 181-183, 223-244, 277, 325-334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 499-505, 735-761, 776-777.
• Embodiment 414. A method of installing a ribonucleotide motif or tag in an RNA of interest encoded by a target nucleotide sequence by prime editing, the method comprising: (a) contacting the target nucleotide sequence with a (i) prime editor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a polymerase and (ii) a PEgRNA comprising an edit template encoding the ribonucleotide motif or tag; (b) polymerizing a single strand DNA sequence encoding the ribonucleotide motif or tag; and (c) incorporating the single strand DNA sequence in place of a corresponding endogenous strand at the target nucleotide sequence through a DNA repair and/or replication process, wherein the method produces a recombinant target nucleotide sequence that encodes a modified RNA of interest comprising the ribonucleotide motif or tag.
• Embodiment 415. The method of embodiment 414, wherein ribonucleotide motif or tag is a detection moiety.
• Embodiment 416. The method of embodiment 414, wherein the ribonucleotide motif or tag affects the expression level of the RNA of interest.
• Embodiment 417. The method of embodiment 414, wherein the ribonucleotide motif or tag affects the transport or subcellular location of the RNA of interest.
• Embodiment 418. The method of embodiment 414, wherein the ribonucleotide motif or tag is selected from the group consisting of SV40 type 1, SV40 type 2, SV40 type 3, hGH, BGH, rbGlob, TK, MALAT1 ENE-mascRNA, KSHV PAN ENE, Smbox/U1 snRNA box, U1 snRNA 3′ box, tRNA-lysine, broccoli aptamer, spinach aptamer, mango aptamer, HDV ribozyme, and m6A.
• Embodiment 419. The method of embodiment 414, wherein the PEgRNA comprises SEQ ID NOs: 101-104, 181-183, 223-244, 277, 325-334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 499-505, 735-761, 776-777.
• Embodiment 420. The method of embodiment 414, wherein the fusion protein comprises the amino acid sequence of PE1, PE2, or PE3.
• Embodiment 421. The method of embodiment 414, wherein the napDNAbp is a Cas9 nickase (nCas9).
• Embodiment 422. The method of embodiment 414, wherein the napDNAbp comprises the amino acid sequence of SEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.
• Embodiment 423. A method of installing or deleting a functional moiety in a protein of interest encoded by a target nucleotide sequence by prime editing, the method comprising: (a) contacting the target nucleotide sequence with a (i) prime editor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a polymerase and (ii) a PEgRNA comprising an edit template encoding the functional moiety or deletion of same; (b) polymerizing a single strand DNA sequence encoding the functional moiety or deletion of same; and (c) incorporating the single strand DNA sequence in place of a corresponding endogenous strand at the target nucleotide sequence through a DNA repair and/or replication process, wherein the method produces a recombinant target nucleotide sequence that encodes a modified protein comprising the protein of interest and the functional moiety or the removal of same, wherein the functional moiety alters a modification state or localization state of the protein.
• Embodiment 424. The method of embodiment 423, wherein functional moiety alters the phosphorylation, ubiquitylation, glycosylation, lipidation, hydroxylation, methylation, acetylation, crotonylation, SUMOylation state of the protein of interest.
• Embodiment 425. A fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) domain and a domain comprising an RNA-dependent DNA polymerase activity.
• Embodiment 426. The fusion protein of embodiment 425, wherein the fusion protein is capable of carrying out prime editing in the presence of an prime editing guide RNA (PEgRNA) to install a desired nucleotide change in a target sequence.
• Embodiment 427. The fusion protein of embodiment 425, wherein the napDNAbp domain has a nickase activity.
• Embodiment 428. The fusion protein of embodiment 425, wherein the napDNAbp domain is a Cas9 protein or variant thereof.
• Embodiment 429. The fusion protein of embodiment 425, wherein the napDNAbp domain is a nuclease active Cas9, a nuclease inactive Cas9 (dCas9), or a Cas9 nickase (nCas9).
• Embodiment 430. The fusion protein of embodiment 425, wherein the napDNAbp domain is Cas9 nickase (nCas9).
• Embodiment 431. The fusion protein of embodiment 425, wherein the napDNAbp domain is selected from the group consisting of: Cas9, Cas12e, Cas12d, Cas12a, Cas12b1, Cas13a, Cas12c, and Argonaute and optionally has a nickase activity.
• Embodiment 432. The fusion protein of embodiment 425, wherein the domain comprising an RNA-dependent DNA polymerase activity is a reverse transcriptase comprising any one of the amino acid sequences of SEQ ID NO: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, 662, 700, 701-716, 739-741, and 766.
• Embodiment 433. The fusion protein of embodiment 425, wherein the domain comprising an RNA-dependent DNA polymerase activity is a reverse transcriptase comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, 662, 700, 701-716, 739-741, and 766, and optionally wherein the domain comprising an RNA-dependent DNA polymerase is error-prone.
• Embodiment 434. The fusion protein of embodiment 425, wherein the domain comprising an RNA-dependent DNA polymerase activity is a naturally-occurring reverse transcriptase from a retrovirus or a retrotransposon.
• Embodiment 435. The fusion protein of embodiment 425, wherein the fusion protein when complexed with a PEgRNA is capable of binding to a target DNA sequence.
• Embodiment 436. The fusion protein of embodiment 435, wherein the target DNA sequence comprises a target strand and a complementary non-target strand.
• Embodiment 437. The fusion protein of embodiment 435, wherein the binding of the fusion protein complexed to the PEgRNA forms an R-loop.
• Embodiment 438. The fusion protein of embodiment 437, wherein the R-loop comprises (i) an RNA-DNA hybrid comprising the PEgRNA and the target strand, and (ii) the complementary non-target strand.
• Embodiment 439. The fusion protein of embodiment 437, wherein the target or the complementary non-target strand is nicked to form a priming sequence having a free 3′ end.
• Embodiment 440. The fusion protein of embodiment 439, wherein the nick site is upstream of the PAM sequence on the target strand.
• Embodiment 441. The fusion protein of embodiment 439, wherein the nick site is upstream of the PAM sequence on the non-target strand.
• Embodiment 442. The fusion protein of embodiment 439, wherein the nick site −1, −2, −3, −4, −5, −6, −7, −8, or −9 relative to the 5′ end of the PAM sequence.
• Embodiment 443. The fusion protein of embodiment 426, wherein the PEgRNA comprises a guide RNA and at least one nucleic acid extension arm.
• Embodiment 444. The fusion protein of embodiment 443, wherein the extension arm is at the 5′ or the 3′ end of the guide RNA, or at an intramolecular location in the guide RNA.
• Embodiment 445. The fusion protein of embodiment 443, wherein the extension arm comprises (i) a DNA synthesis template sequence comprising a desired nucleotide change, and (ii) a primer binding site.
• Embodiment 446. The fusion protein of embodiment 445, wherein the DNA synthesis template sequence encodes a single-strand DNA flap that is complementary to an endogenous DNA sequence adjacent to the nick site, wherein the single-strand DNA flap comprises the desired nucleotide change.
• Embodiment 447. The fusion protein of embodiment 443, wherein the extension arm is at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides, at least 22 nucleotides, at least 23 nucleotides, at least 24 nucleotides, at least 25 nucleotides, at least 26 nucleotides, at least 27 nucleotides, at least 28 nucleotides, at least 29 nucleotides, at least 30 nucleotides, at least 31 nucleotides, at least 32 nucleotides, at least 33 nucleotides, at least 34 nucleotides, at least 35 nucleotides, at least 36 nucleotides, at least 37 nucleotides, at least 38 nucleotides, at least 39 nucleotides, at least 40 nucleotides, at least 41 nucleotides, at least 42 nucleotides, at least 43 nucleotides, at least 44 nucleotides, at least 45 nucleotides, at least 46 nucleotides, at least 47 nucleotides, at least 48 nucleotides, at least 49 nucleotides, or at least 50 nucleotides.
• Embodiment 448. The fusion protein of embodiment 443, wherein the single-strand DNA flap hybridizes to the endogenous DNA sequence adjacent to the nick site, thereby installing the desired nucleotide change in the target strand.
• Embodiment 449. The fusion protein of embodiment 443, wherein the single-stranded DNA flap displaces the endogenous DNA sequence adjacent to the nick site and which has a free 5′ end.
• Embodiment 450. The fusion protein of embodiment 446, wherein the endogenous DNA sequence having the 5′ end is excised by the cell.
• Embodiment 451. The fusion protein of embodiment 446, wherein the endogenous DNA sequence having the 5′ end is excised by a flap endonuclease.
• Embodiment 452. The fusion protein of embodiment 448, wherein cellular repair of the single-strand DNA flap incorporates the desired nucleotide change in the non-target strand, thereby forming a desired product.
• Embodiment 453. The fusion protein of embodiment 449, wherein the desired nucleotide change is installed in an editing window that is between about −4 to +10 of the PAM sequence, or between about −10 to +20 of the PAM sequence, or between about −20 to +40 of the PAM sequence, or between about −30 to +100 of the PAM sequence.
• Embodiment 454. The fusion protein of embodiment 449, wherein the desired nucleotide change is installed at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides downstream of the nick site.
• Embodiment 455. The fusion protein of embodiment 425, wherein the napDNAbp comprises an amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence to SEQ ID NO: 18.
• Embodiment 456. The fusion protein of embodiment 425, wherein the napDNAbp comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.
• Embodiment 457. The fusion protein of any one of the previous embodiments, wherein the fusion protein comprises the structure NH₂-[napDNAbp]-[domain comprising an RNA-dependent DNA polymerase activity]-COOH; or NH₂-[domain comprising an RNA-dependent DNA polymerase activity]-[napDNAbp]-COOH, wherein each instance of “]-[” indicates the presence of an optional linker sequence.
• Embodiment 458. The fusion protein of embodiment 457, wherein the linker sequence comprises an amino acid sequence of SEQ ID NOs: 127, 165-176, 446, 453, and 767-769.
• Embodiment 459. The fusion protein of embodiment 425, wherein the desired nucleotide change is a single nucleotide change, an insertion of one or more nucleotides, or a deletion of one or more nucleotides.
• Embodiment 460. The fusion protein of embodiment 459, wherein the insert or deletion is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, or at least 50.
• Embodiment 461. A complex comprising a fusion protein of any of embodiments 425-460 and a PEgRNA, wherein the PEgRNA directs the fusion protein to a target DNA sequence for prime editing.
• Embodiment 462. The complex of embodiment 461, wherein the PEgRNA comprises a guide RNA and a nucleic acid extension arm at the 3′ or 5′ end of the guide RNA or at an intramolecular position in the guide RNA.
• Embodiment 463. The complex of embodiment 462, wherein the PEgRNA is capable of binding to a napDNAbp and directing the napDNAbp to the target DNA sequence.
• Embodiment 464. The complex of embodiment 463, wherein the target DNA sequence comprises a target strand and a complementary non-target strand, wherein the guide RNA hybridizes to the target strand to form an RNA-DNA hybrid and an R-loop.
• Embodiment 465. The complex of embodiment 464, wherein the at least one nucleic acid extension arm comprises (i) a DNA synthesis template, and (ii) a primer binding site.
• Embodiment 466. The complex of embodiment 464, wherein the PEgRNA comprises the nucleotide sequence of SEQ ID NOs: 101-104, 181-183, 223-244, 277, 325-334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 499-505, 735-761, 776-777, or a nucleotide sequence having at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99% sequence identity with any one of SEQ ID NOs: 101-104, 181-183, 223-244, 277, 325-334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 499-505, 735-761, 776-777.
• Embodiment 467. The complex of embodiment 465, wherein the DNA synthesis template comprises a nucleotide sequence that is at least 80%, or 85%, or 90%, or 95%, or 99% identical to the endogenous DNA target.
• Embodiment 468. The complex of embodiment 465, wherein the primer binding site hybridizes with a free 3′ end of the cut DNA.
• Embodiment 469. The complex of embodiment 461, wherein the napDNAbp is a Cas9 nickase.
• Embodiment 470. The complex of embodiment 461, wherein the napDNAbp comprises an amino acid sequence of SEQ ID NO: 18, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO: 18.
• Embodiment 471. The complex of embodiment 461, wherein the napDNAbp comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.
• Embodiment 472. The complex of embodiment 461, wherein the PEgRNA comprises a guide RNA and a nucleic acid extension arm at the 3′ or 5′ end of the guide RNA, or at an intramolecular position in the guide RNA.
• Embodiment 473. The complex of embodiment 465, wherein the DNA synthesis template comprises a nucleotide sequence that is at least 80%, or 85%, or 90%, or 95%, or 99% identical to the endogenous DNA target.
• Embodiment 474. The complex of embodiment 465, wherein the primer binding site hybridizes with a free 3′ end of the cut DNA.
• Embodiment 475. The complex of embodiment 462, wherein the PEgRNA further comprises at least one additional structure selected from the group consisting of a linker, a stem loop, a hairpin, a toeloop, an aptamer, or an RNA-protein recruitment domain.
• Embodiment 476. A polynucleotide encoding the fusion protein of any of embodiments 425-461.
• Embodiment 477. A polynucleotide encoding the PEgRNA of any of the above embodiments.
• Embodiment 478. A vector comprising the polynucleotide of embodiment 476, wherein expression of the fusion protein is under the control of a promoter.
• Embodiment 479. A vector comprising the polynucleotide of embodiment 477, wherein expression of the PEgRNA is under the control of a promoter.
• Embodiment 480. The vector of embodiment 479, wherein the promoter is a U6 promoter.
• Embodiment 481. The vector of embodiment 479, wherein the promoter is a CMV promoter.
• Embodiment 482. The vector of embodiment 480, wherein the PEgRNA is engineered to remove one or more repeating clusters of Ts in the extension arm to improve transcription efficiency by the U6 promoter.
• Embodiment 483. The vector of embodiment 482, wherein the one or more repeating clusters of Ts that is removed comprises at least 3 Ts, at least 4 Ts, at least 5 Ts, at least 6 Ts, at least 7 Ts, at least 8 Ts, at least 9 Ts, at least 10 Ts, at least 11 Ts, at least 12 Ts, at least 13 Ts, at least 14 Ts, at least 15 Ts, at least 16 Ts, at least 17 Ts, at least 18 Ts, at least 19 Ts, or at least 20 Ts.
• Embodiment 484. A cell comprising the fusion protein of any of embodiments 425-460 and an PEgRNA bound to the napDNAbp of the fusion protein.
• Embodiment 485. A cell comprising a complex of any one of embodiments 461-475.
• Embodiment 486. A pharmaceutical composition comprising: (i) a fusion protein of any of embodiments 425-460, the complex of embodiments 461-475, the polynucleotide of embodiments 476-477, or the vector of embodiments 478-483; and (ii) a pharmaceutically acceptable excipient.
• Embodiment 487. A pharmaceutical composition comprising: (i) the complex of embodiments 461-475 (ii) a polymerase provided in trans; and (iii) a pharmaceutically acceptable excipient.
• Embodiment 488. A kit for prime editing comprising: (i) a nucleic acid molecule encoding the fusion protein of any one of embodiments 425-460; and (ii) a nucleic acid molecule encoding a PEgRNA that is capable of directing the fusion protein to a target DNA site, wherein the nucleic acid molecule of (i) and (ii) may be contained within a single DNA construct or separate DNA constructs.
• Embodiment 489. The kit of embodiment 488, wherein the nucleic acid molecule of (i) further comprising a promoter that drives expression of the fusion protein.
• Embodiment 490. The kit of embodiment 488, wherein the nucleic acid molecule of (ii) further comprises a promoter that drives expression of the PEgRNA.
• Embodiment 491. The kit of embodiment 490, wherein the promoter is a U6 promoter.
• Embodiment 492. The kit of embodiment 490, wherein the promoter is a CMV promoter.
• Embodiment 493. The fusion protein of embodiment 457, wherein the linker sequence comprises an amino acid sequence of SEQ ID NOs: 174 (1×SGGS), 3888 (2×SGGS), 3889 (3×SGGS), 3890 (1×XTEN), 3891 (1×EAAAK), 3892 (2×EAAAK), and 3893 (3×EAAAK).
Group B. PE Guides and Methods of Design
• Embodiment 1. A PEgRNA comprising a guide RNA and at least one nucleic acid extension arm comprising a DNA synthesis template.
• Embodiment 2. The PEgRNA of embodiment 1, wherein the nucleic acid extension arm is position at the 3′ or 5′ end of the guide RNA, or at an intramolecular position in the guide RNA, and wherein the nucleic acid extension arm is DNA or RNA.
• Embodiment 3. The PEgRNA of embodiment 1, wherein the PEgRNA is capable of binding to a napDNAbp and directing the napDNAbp to a target DNA sequence.
• Embodiment 4. The PEgRNA of embodiment 3, wherein the target DNA sequence comprises a target strand and a complementary non-target strand.
• Embodiment 5. The PEgRNA of embodiment 3, wherein the guide RNA hybridizes to the target strand to form an RNA-DNA hybrid and an R-loop.
• Embodiment 6. The PEgRNA of embodiment 1, wherein the at least one nucleic acid extension arm further comprises a primer binding site.
• Embodiment 7. The PEgRNA of embodiment 1, wherein the nucleic acid extension arm is at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides, at least 22 nucleotides, at least 23 nucleotides, at least 24 nucleotides, at least 25 nucleotides, at least 26 nucleotides, at least 27 nucleotides, at least 28 nucleotides, at least 29 nucleotides, at least 30 nucleotides, at least 31 nucleotides, at least 32 nucleotides, at least 33 nucleotides, at least 34 nucleotides, at least 35 nucleotides, at least 36 nucleotides, at least 37 nucleotides, at least 38 nucleotides, at least 39 nucleotides, at least 40 nucleotides, at least 41 nucleotides, at least 42 nucleotides, at least 43 nucleotides, at least 44 nucleotides, at least 45 nucleotides, at least 46 nucleotides, at least 47 nucleotides, at least 48 nucleotides, at least 49 nucleotides, or at least 50 nucleotides.
• Embodiment 8. The PEgRNA of embodiment 1, wherein the DNA synthesis template is at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, or at least 15 nucleotides in length.
• Embodiment 9. The PEgRNA of embodiment 6, wherein the primer binding site is at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, or at least 15 nucleotides in length.
• Embodiment 10. The PEgRNA of embodiment 1, further comprising at least one additional structure selected from the group consisting of a tRNA, linker, a stem loop, a hairpin, a toeloop, an aptamer, or an RNA-protein recruitment domain.
• Embodiment 11. The PEgRNA of embodiment 1, wherein the DNA synthesis template encodes a single-strand DNA flap that is complementary to an endogenous DNA sequence adjacent to a nick site, wherein the single-strand DNA flap comprises a desired nucleotide change.
• Embodiment 12. The PEgRNA of embodiment 11, wherein the single-stranded DNA flap displaces an endogenous single-strand DNA having a 5′ end in the target DNA sequence that has been nicked, and wherein the endogenous single-strand DNA is immediately adjacent downstream of the nick site.
• Embodiment 13. The PEgRNA of embodiment 11, wherein the endogenous single-stranded DNA having the free 5′ end is excised by the cell.
• Embodiment 14. The PEgRNA of embodiment 13, wherein cellular repair of the single-strand DNA flap results in installation of the desired nucleotide change, thereby forming a desired product.
• Embodiment 15. The PEgRNA of embodiment 1, comprising the nucleotide sequence of SEQ ID NOs: 101-104, 181-183, 223-244, 277, 325-334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 499-505, 735-761, 776-777, or a nucleotide sequence having at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99% sequence identity with any one of SEQ ID NOs: 101-104, 181-183, 223-244, 277, 325-334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 499-505, 735-761, 776-777.
• Embodiment 16. The PEgRNA of embodiment 1, wherein the DNA synthesis template comprises a nucleotide sequence that is at least 80%, or 85%, or 90%, or 95%, or 99% identical to the endogenous DNA target.
• Embodiment 17. The PEgRNA of embodiment 6, wherein the primer binding site hybridizes with a free 3′ end of the cut DNA.
• Embodiment 18. The PEgRNA of embodiment 10, wherein the at least one additional structure is located at the 3′ or 5′ end of the PEgRNA.
• Embodiment 19. The PEgRNA of embodiment 10, wherein the linker comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 127, 165-176, 446, 453, and 767-769.
• Embodiment 20. The PEgRNA of embodiment 10, wherein the stem loop comprises a nucleotide sequence selected from the stem loops described herein.
• Embodiment 21. The PEgRNA of embodiment 10, wherein the hairpin comprises a nucleotide sequence selected from the hairpins described herein.
• Embodiment 22. The PEgRNA of embodiment 10, wherein the toeloop comprises a nucleotide sequence selected from the toeloops described herein.
• Embodiment 23. The PEgRNA of embodiment 10, wherein the aptamer comprises a nucleotide sequence selected from the aptamers described herein.
• Embodiment 24. The PEgRNA of embodiment 10, wherein the RNA-protein recruitment domain comprises a nucleotide sequence selected from the RNA-protein recruitment domain described herein.
• Embodiment 25. A method for designing a PEgRNA for use in prime editing to install a desired nucleotide change in a target nucleotide sequence, wherein said PEgRNA comprises a spacer, gRNA core, and an extension arm, and wherein said extension arm comprises a primer binding site and a DNA synthesis template, said method comprising:
• (i) selecting a desired target edit site in a target nucleotide sequence;
• (ii) obtaining a context nucleotide sequence upstream and downstream from the target edit site;
• (iii) locating putative protospacer adjacent motif (PAM) sites in the context nucleotide sequence which are proximal to the desired target edit site;
• (iv) identifying the corresponding nick sites for each putative PAM site;
• (v) designing the spacer;
• (vi) designing the gRNA core;
• (vii) designing the extension arm; and
• (viii) constructing the full PEgRNA by concatenating the spacer, gRNA core, and the extension arm.
• Embodiment 26. The method of embodiment 25, wherein the step (i) of selecting the desired target edit site comprises selecting a disease-causing mutation.
• Embodiment 27. The method of embodiment 26, wherein the disease-causing mutation is associated with a disease selected from the group consisting of: cancer, autoimmune disorders, neurological disorders, skin disorders, respiratory diseases, and cardiac diseases.
• Embodiment 28. The method of embodiment 25, wherein the step (ii) of obtaining a context nucleotide sequence upstream and downstream from the target edit site comprises obtaining about 50-55 base pairs (bp), about 55-60 bp, about 60-65 bp, about 65-70 bp, about 70-75 bp, about 75-80 bp, about 80-85 bp, about 85-90 bp, about 90-95 bp, about 95-100 bp, about 100-105 bp, about 105-110 bp, about 110-125 bp, about 125-130 bp, about 130-135 bp, about 135-140 bp, about 140-145 bp, about 145-150 bp, about 150-155 bp, about 155-160 bp, about 160-165 bp, about 165-170 bp, about 170-175 bp, about 175-180 bp, about 180-185 bp, about 185-190 bp, about 190-195 bp, about 195-200 bp, about 200-205 bp, about 205-210 bp, about 210-215 bp, about 215-220 bp, about 220-225 bp, about 225-230 bp, about 230-235 bp, about 235-240 bp, about 240-245 bp, or about 245-250 bp of a region that comprises the desired target edit site.
• Embodiment 29. The method of embodiment 28, wherein the desired target edit site is positioned approximately equidistant from each end of the context nucleotide sequence.
• Embodiment 30. The method of embodiment 25, wherein in step (iii), the putative PAM sites are proximal to the desired target edit site.
• Embodiment 31. The method of embodiment 25, wherein in step (iii), the putative PAM sites comprise those with associated nick sites located at a position less than 30 nucleotides from the target edit site, or less than 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 nucleotides from the target edit site.
• Embodiment 32. The method of embodiment 25, wherein in step (iii), the putative PAM sites comprise those with associated nick sites located at a position more than 30 nucleotides from the target edit site, or more than 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides from the target edit site.
• Embodiment 33. The method of embodiment 25, wherein in step (iii), the putative PAM sites are associated with one or corresponding napDNAbp which bind to said PAM sites.
• Embodiment 34. The method of embodiment 33, the putative PAM sites and their corresponding napDNAbps are selected from any of the following groupings: (a) SpCas9 of SEQ ID NO: 18-25 and 87-88 and NGG; (b) Sp VQR nCas9; and (c) NGAN.
• Embodiment 35. The method of embodiment 25, wherein the step (v) of designing the spacer comprises determining the complement nucleotide sequence of the protospacer sequence associated with each putative PAM.
• Embodiment 36. The method of embodiment 25, wherein the step (vi) of designing gRNA core comprises in the context of each putative PAM, selecting a gRNA core sequence that is capable of binding to a napDNAbp which is associated with each of said putative PAMs.
• Embodiment 37. The method of embodiment 25, wherein the step (vii) of designing the extension arm comprises designing (a) a DNA synthesis template comprising the edit of interest, and (b) a primer binding site.
• Embodiment 38. The method of embodiment 37, wherein designing the primer binding site comprises (a) identifying a DNA primer on the PAM-containing strand of the target nucleotide sequence, wherein the 3′ end of the DNA primer is the first nucleotide upstream of the nick site associated with the PAM site, and (b) designing the complement of the DNA primer, wherein said complement forms the primer binding site.
• Embodiment 39. The method of embodiment 38, wherein the primer binding site is 8 to 15 nucleotides in length.
• Embodiment 40. The method of embodiment 38, wherein the primer binding site is 12-13 nucleotides if the DNA primer contains about 40-60% GC content.
• Embodiment 41. The method of embodiment 38, wherein the primer binding site is 14-15 nucleotides if the DNA primer contains less than about 40% GC content.
• Embodiment 42. The method of embodiment 38, wherein the primer binding site is 8-11 nucleotides if the DNA primer contains greater than about 60% GC content.
• Embodiment 43. A method of prime editing comprising contacting a target DNA sequence with a PEgRNA of any of embodiments 1-24 and a prime editor fusion protein comprising a napDNAbp and a domain having an RNA-dependent DNA polymerase activity.
• Embodiment 44. The method of embodiment 43, wherein the napDNAbp has a nickase activity.
• Embodiment 45. The method of embodiment 43, wherein the napDNAbp is a Cas9 protein or variant thereof.
• Embodiment 46. The method of embodiment 43, wherein the napDNAbp is a nuclease active Cas9, a nuclease inactive Cas9 (dCas9), or a Cas9 nickase (nCas9).
• Embodiment 47. The method of embodiment 43, wherein the napDNAbp is Cas9 nickase (nCas9).
• Embodiment 48. The method of embodiment 43, wherein the napDNAbp is selected from the group consisting of: Cas9, Cas12e, Cas12d, Cas12a, Cas12b1, Cas13a, Cas12c, and Argonaute and optionally has a nickase activity.
• Embodiment 49. The method of embodiment 43, wherein the domain comprising an RNA-dependent DNA polymerase activity is a reverse transcriptase comprising any one of the amino acid sequences of SEQ ID NO: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, 662, 700, 701-716, 739-741, and 766.
• Embodiment 50. The method of embodiment 43, wherein the domain comprising an RNA-dependent DNA polymerase activity is a reverse transcriptase comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, 662, 700, 701-716, 739-741, and 766.
• Embodiment 51. The method of embodiment 43, wherein the domain comprising an RNA-dependent DNA polymerase activity is a naturally-occurring reverse transcriptase from a retrovirus or a retrotransposon.
Group C. PE Complexes
• Embodiment 1. A complex for prime editing comprising:
• (i) fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a domain comprising an RNA-dependent DNA polymerase activity; and
• (ii) a prime editing guide RNA (PEgRNA).
• Embodiment 2. The complex of embodiment 1, wherein the fusion protein is capable of carrying out prime editing in the presence of the prime editing guide RNA (PEgRNA) to install a desired nucleotide change in a target sequence.
• Embodiment 3. The complex of embodiment 11, wherein the napDNAbp has a nickase activity.
• Embodiment 4. The complex of embodiment 1, wherein the napDNAbp is a Cas9 protein or variant thereof.
• Embodiment 5. The complex of embodiment 1, wherein the napDNAbp is a nuclease active Cas9, a nuclease inactive Cas9 (dCas9), or a Cas9 nickase (nCas9).
• Embodiment 6. The complex of embodiment 1, wherein the napDNAbp is Cas9 nickase (nCas9).
• Embodiment 7. The complex of embodiment 1, wherein the napDNAbp is selected from the group consisting of: Cas9, Cas12e, Cas12d, Cas12a, Cas12b1, Cas13a, Cas12c, and Argonaute and optionally has a nickase activity.
• Embodiment 8. The complex of embodiment 1, wherein the domain comprising an RNA-dependent DNA polymerase activity is a reverse transcriptase comprising any one of the amino acid sequences of SEQ ID NO: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, 662, 700, 701-716, 739-741, and 766.
• Embodiment 9. The complex of embodiment 1, wherein the domain comprising an RNA-dependent DNA polymerase activity is a reverse transcriptase comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, 662, 700, 701-716, 739-741, and 766.
• Embodiment 10. The complex of embodiment 1, wherein the domain comprising an RNA-dependent DNA polymerase activity is a naturally-occurring reverse transcriptase from a retrovirus or a retrotransposon.
• Embodiment 11. The complex of embodiment 1, wherein the fusion protein when complexed with a PEgRNA is capable of binding to a target DNA sequence.
• Embodiment 12. The complex of embodiment 1, wherein the PEgRNA comprises a guide RNA and at least one nucleic acid extension arm comprising a DNA synthesis template.
• Embodiment 13. The complex of embodiment 12, wherein the nucleic acid extension arm is position at the 3′ or 5′ end of the guide RNA, or at an intramolecular position in the guide RNA, and wherein the nucleic acid extension arm is DNA or RNA.
• Embodiment 14. The complex of embodiment 12, wherein the PEgRNA is capable of binding to a napDNAbp and directing the napDNAbp to a target DNA sequence.
• Embodiment 15. The complex of embodiment 14, wherein the target DNA sequence comprises a target strand and a complementary non-target strand.
• Embodiment 16. The complex of embodiment 12, wherein the guide RNA hybridizes to the target strand to form an RNA-DNA hybrid and an R-loop.
• Embodiment 17. The complex of embodiment 12, wherein the at least one nucleic acid extension arm further comprises a primer binding site.
• Embodiment 18. The complex of embodiment 12, wherein the nucleic acid extension arm is at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides, at least 22 nucleotides, at least 23 nucleotides, at least 24 nucleotides, at least 25 nucleotides, at least 26 nucleotides, at least 27 nucleotides, at least 28 nucleotides, at least 29 nucleotides, at least 30 nucleotides, at least 31 nucleotides, at least 32 nucleotides, at least 33 nucleotides, at least 34 nucleotides, at least 35 nucleotides, at least 36 nucleotides, at least 37 nucleotides, at least 38 nucleotides, at least 39 nucleotides, at least 40 nucleotides, at least 41 nucleotides, at least 42 nucleotides, at least 43 nucleotides, at least 44 nucleotides, at least 45 nucleotides, at least 46 nucleotides, at least 47 nucleotides, at least 48 nucleotides, at least 49 nucleotides, or at least 50 nucleotides.
• Embodiment 19. The complex of embodiment 12, wherein the DNA synthesis template is at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, or at least 15 nucleotides in length.
• Embodiment 20. The complex of embodiment 17, wherein the primer binding site is at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, or at least 15 nucleotides in length.
• Embodiment 21. The complex of embodiment 12, wherein the PEgRNA further comprises at least one additional structure selected from the group consisting of a linker, a stem loop, a hairpin, a toeloop, an aptamer, or an RNA-protein recruitment domain.
• Embodiment 22. The complex of embodiment 12, wherein the DNA synthesis template encodes a single-strand DNA flap that is complementary to an endogenous DNA sequence adjacent to a nick site, wherein the single-strand DNA flap comprises a desired nucleotide change.
• Embodiment 23. The complex of embodiment 22, wherein the single-stranded DNA flap displaces an endogenous single-strand DNA having a 5′ end in the target DNA sequence that has been nicked, and wherein the endogenous single-strand DNA is immediately adjacent downstream of the nick site.
• Embodiment 24. The complex of embodiment 23, wherein the endogenous single-stranded DNA having the free 5′ end is excised by the cell.
• Embodiment 25. The complex of embodiment 23, wherein cellular repair of the single-strand DNA flap results in installation of the desired nucleotide change, thereby forming a desired product.
• Embodiment 26. The complex of embodiment 12, wherein the PEgRNA comprises the nucleotide sequence of SEQ ID NOs: 18-36, or a nucleotide sequence having at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99% sequence identity with any one of SEQ ID NOs: 101-104, 181-183, 223-244, 277, 325-334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 499-505, 735-761, 776-777.
• Embodiment 27. The complex of embodiment 12, wherein the DNA synthesis template comprises a nucleotide sequence that is at least 80%, or 85%, or 90%, or 95%, or 99% identical to the endogenous DNA target.
• Embodiment 28. The complex of embodiment 17, wherein the primer binding site hybridizes with a free 3′ end of the cut DNA.
• Embodiment 29. The complex of embodiment 21, wherein the at least one additional structure is located at the 3′ or 5′ end of the PEgRNA.
• Embodiment 30. The complex of embodiment 29, wherein the linker comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 127, 165-176, 446, 453, and 767-769.
• Embodiment 31. The complex of embodiment 29, wherein the stem loop comprises a nucleotide sequence selected from the stem loops described herein.
• Embodiment 32. The complex of embodiment 29, wherein the hairpin comprises a nucleotide sequence selected from the hairpins described herein.
• Embodiment 33. The complex of embodiment 29, wherein the toeloop comprises a nucleotide sequence selected from the toeloops described herein.
• Embodiment 34. The complex of embodiment 29, wherein the aptamer comprises a nucleotide sequence selected from the aptamers described herein.
• Embodiment 35. The complex of embodiment 29, wherein the RNA-protein recruitment domain comprises a nucleotide sequence selected from the RNA-protein recruitment domains described herein.
• Embodiment 36. The complex of embodiment 1, wherein the target DNA sequence comprises a target strand and a complementary non-target strand.
• Embodiment 37. The complex of embodiment 36, wherein the R-loop comprises (i) an RNA-DNA hybrid comprising the PEgRNA and the target strand, and (ii) the complementary non-target strand.
• Embodiment 38. The complex of embodiment 37, wherein the target or the complementary non-target strand is nicked to form a priming sequence having a free 3′ end.
• Embodiment 39. The complex of embodiment 38, wherein the nick site is upstream of the PAM sequence on the target strand.
• Embodiment 40. The complex of embodiment 38, wherein the nick site is upstream of the PAM sequence on the non-target strand.
• Embodiment 41. The complex of embodiment 38, wherein the nick site −1, −2, −3, −4, −5, −6, −7, −8, or −9 relative to the 5′ end of the PAM sequence.
• Embodiment 42. The complex of embodiment 22, wherein the single-strand DNA flap hybridizes to the endogenous DNA sequence adjacent to the nick site, thereby installing the desired nucleotide change in the target strand.
• Embodiment 43. The complex of embodiment 22, wherein the single-stranded DNA flap displaces the endogenous DNA sequence adjacent to the nick site and which has a free 5′ end.
• Embodiment 44. The complex of embodiment 22, wherein the endogenous DNA sequence having the 5′ end is excised by the cell.
• Embodiment 45. The complex of embodiment 44, wherein the endogenous DNA sequence having the 5′ end is excised by a flap endonuclease.
• Embodiment 46. The complex of embodiment 43, wherein cellular repair of the single-strand DNA flap incorporates the desired nucleotide change in the non-target strand, thereby forming a desired product.
• Embodiment 47. The complex of embodiment 46, wherein the desired nucleotide change is installed in an editing window that is between about −4 to +10 of the PAM sequence, or between about −10 to +20 of the PAM sequence, or between about −20 to +40 of the PAM sequence, or between about −30 to +100 of the PAM sequence.
• Embodiment 48. The complex of embodiment 47, wherein the desired nucleotide change is installed at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides downstream of the nick site.
• Embodiment 49. The complex of any one of the previous embodiments, wherein the fusion protein comprises the structure NH₂-[napDNAbp]-[domain comprising an RNA-dependent DNA polymerase activity]-COOH; or NH₂-[domain comprising an RNA-dependent DNA polymerase activity]-[napDNAbp]-COOH, wherein each instance of “]-[” indicates the presence of an optional linker sequence.
• Embodiment 50. The complex of embodiment 49, wherein the linker sequence comprises an amino acid sequence of SEQ ID NOs: 127, 165-176, 446, 453, and 767-769.
• Embodiment 51. The complex of embodiment 1, wherein the fusion protein further comprises a linker that joins the napDNAbp and the domain comprising an RNA-dependent DNA polymerase activity.
• Embodiment 52. complex of embodiment 51, wherein the linker sequence comprises an amino acid sequence of SEQ ID NOs. 3887 (1×SGGS), 3888 (2×SGGS), 3889 (3×SGGS), 3890 (1×XTEN), 3891 (1×EAAAK), 3892 (2×EAAAK), and 3893 (3×EAAAK).
Group D. PE Method for Correcting Mutations
• Embodiment 1. A method for installing a desired nucleotide change in a double-stranded DNA sequence, the method comprising: contacting the double-stranded DNA sequence with a complex comprising a fusion protein and a PEgRNA, wherein the fusion protein comprises a napDNAbp and a polymerase, and wherein the PEgRNA comprises a DNA synthesis template comprising the desired nucleotide change and a primer binding site;
• thereby nicking the double-stranded DNA sequence, thereby generating a free single-strand DNA having a 3′ end;
• thereby hybridizing the 3′ end of the free single-strand DNA to the primer binding site, thereby priming the polymerase;
• thereby polymerizing a strand of DNA from the 3′ end hybridized to the primer binding site, thereby generating a single-strand DNA flap comprising the desired nucleotide change and which is complementary to the DNA synthesis template;
• thereby replacing an endogenous DNA strand adjacent the cut site with the single-strand DNA flap, thereby installing the desired nucleotide change in the double-stranded DNA sequence.
• Embodiment 2. The method of embodiment 1, wherein replacing an endogenous DNA strand comprises: (i) hybridizing the single-strand DNA flap to the endogenous DNA strand adjacent the cut site to create a sequence mismatch; (ii) excising the endogenous DNA strand; and (iii) repairing the mismatch to form the desired product comprising the desired nucleotide change in both strands of DNA.
• Embodiment 3. The method of embodiment 1, wherein the desired nucleotide change is a single nucleotide substitution, a deletion, or an insertion.
• Embodiment 4. The method of embodiment 3, wherein the single nucleotide substitution is a transition or a transversion.
• Embodiment 5. The method of embodiment 1, wherein the desired nucleotide change is (1) a G to T substitution, (2) a G to A substitution, (3) a G to C substitution, (4) a T to G substitution, (5) a T to A substitution, (6) a T to C substitution, (7) a C to G substitution, (8) a C to T substitution, (9) a C to A substitution, (10) an A to T substitution, (11) an A to G substitution, or (12) an A to C substitution.
• Embodiment 6. The method of embodiment 1, wherein the desired nucleotide change converts (1) a G:C basepair to a T:A basepair, (2) a G:C basepair to an A:T basepair, (3) a G:C basepair to C:G basepair, (4) a T:A basepair to a G:C basepair, (5) a T:A basepair to an A:T basepair, (6) a T:A basepair to a C:G basepair, (7) a C:G basepair to a G:C basepair, (8) a C:G basepair to a T:A basepair, a C:G basepair to an A:T basepair, (10) an A:T basepair to a T:A basepair, (11) an A:T basepair to a G:C basepair, or (12) an A:T basepair to a C:G basepair.
• Embodiment 7. The method of embodiment 1, wherein the desired nucleotide change is an insertion or deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides.
• Embodiment 8. The method of embodiment 1, wherein the desired nucleotide change corrects a disease-associated gene.
• Embodiment 9. The method of embodiment 8, wherein the disease-associated gene is associated with a monogenetic disorder selected from the group consisting of: Adenosine Deaminase (ADA) Deficiency; Alpha-1 Antitrypsin Deficiency; Cystic Fibrosis; Duchenne Muscular Dystrophy; Galactosemia; Hemochromatosis; Huntington's Disease; Maple Syrup Urine Disease; Marfan Syndrome; Neurofibromatosis Type 1; Pachyonychia Congenita; Phenylkeotnuria; Severe Combined Immunodeficiency; Sickle Cell Disease; Smith-Lemli-Opitz Syndrome; a trinucleotide repeat disorder; a prion disease; and Tay-Sachs Disease.
• Embodiment 10. The method of embodiment 8, wherein the disease-associated gene is associated with a polygenic disorder selected from the group consisting of: heart disease; high blood pressure; Alzheimer's disease; arthritis; diabetes; cancer; and obesity.
• Embodiment 11. The method of embodiment 1, wherein the napDNAbp is a nuclease dead Cas9 (dCas9), a Cas9 nickase (nCas9), or a nuclease active Cas9.
• Embodiment 12. The method of embodiment 1, wherein the napDNAbp comprises an amino acid sequence of SEQ ID NO: 18.
• Embodiment 13. The method of embodiment 1, wherein the napDNAbp comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.
• Embodiment 14. The method of embodiment 1, wherein the polymerase is a reverse transcriptase comprising any one of the amino acid sequences of SEQ ID NO: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, 662, 700, 701-716, 739-741, and 766.
• Embodiment 15. The method of embodiment 1, wherein the polymerase is a reverse transcriptase comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, 662, 700, 701-716, 739-741, and 766.
• Embodiment 16. The method of embodiment 1, wherein the PEgRNA comprises a nucleic acid extension arm at the 3′ or 5′ ends or at an intramolecular location in the guide RNA, wherein the extension arm comprises the DNA synthesis template sequence and the primer binding site.
• Embodiment 17. The method of embodiment 16, wherein the extension arm is at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides, at least 22 nucleotides, at least 23 nucleotides, at least 24 nucleotides, or at least 25 nucleotides in length.
• Embodiment 18. The method of embodiment 1, wherein the PEgRNA has a nucleotide sequence selected from the group consisting of SEQ ID NOs: 101-104, 181-183, 223-244, 277, 325-334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 499-505, 735-761, 776-777.
• Embodiment 19. A method for introducing one or more changes in the nucleotide sequence of a DNA molecule at a target locus, comprising: contacting the DNA molecule with a nucleic acid programmable DNA binding protein (napDNAbp) and a PEgRNA which targets the napDNAbp to the target locus, wherein the PEgRNA comprises a reverse transcriptase (RT) template sequence comprising at least one desired nucleotide change and a primer binding site;
• thereby forming an exposed 3′ end in a DNA strand at the target locus;
• thereby hybridizing the exposed 3′ end to the primer binding site to prime reverse transcription;
• thereby synthesizing a single strand DNA flap comprising the at least one desired nucleotide change based on the RT template sequence by reverse transcriptase;
• thereby incorporating the at least one desired nucleotide change into the corresponding endogenous DNA, thereby introducing one or more changes in the nucleotide sequence of the DNA molecule at the target locus.
• Embodiment 20. The method of embodiment 19, wherein the one or more changes in the nucleotide sequence comprises a transition.
• Embodiment 21. The method of embodiment 19, wherein the transition is selected from the group consisting of: (a) T to C; (b) A to G; (c) C to T; and (d) G to A.
• Embodiment 22. The method of embodiment 19, wherein the one or more changes in the nucleotide sequence comprises a transversion.
• Embodiment 23. The method of embodiment 22, wherein the transversion is selected from the group consisting of: (a) T to A; (b) T to G; (c) C to G; (d) C to A; (e) A to T; (f) A to C; (g) G to C; and (h) G to T.
• Embodiment 24. The method of embodiment 19, wherein the one or more changes in the nucleotide sequence comprises changing (1) a G:C basepair to a T:A basepair, (2) a G:C basepair to an A:T basepair, (3) a G:C basepair to C:G basepair, (4) a T:A basepair to a G:C basepair, (5) a T:A basepair to an A:T basepair, (6) a T:A basepair to a C:G basepair, (7) a C:G basepair to a G:C basepair, (8) a C:G basepair to a T:A basepair, (9) a C:G basepair to an A:T basepair, (9) an A:T basepair to a T:A basepair, (11) an A:T basepair to a G:C basepair, or (12) an A:T basepair to a C:G basepair.
• Embodiment 25. The method of embodiment 19, wherein the one or more changes in the nucleotide sequence comprises an insertion or deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides.
• Embodiment 26. The method of embodiment 19, wherein the one or more changes in the nucleotide sequence comprises a correction to a disease-associated gene.
• Embodiment 27. The method of embodiment 26, wherein the disease-associated gene is associated with a monogenetic disorder selected from the group consisting of: Adenosine Deaminase (ADA) Deficiency; Alpha-1 Antitrypsin Deficiency; Cystic Fibrosis; Duchenne Muscular Dystrophy; Galactosemia; Hemochromatosis; Huntington's Disease; Maple Syrup Urine Disease; Marfan Syndrome; Neurofibromatosis Type 1; Pachyonychia Congenita; Phenylkeotnuria; Severe Combined Immunodeficiency; Sickle Cell Disease; Smith-Lemli-Opitz Syndrome; a trinucleotide repeat disorder; a prion disease; and Tay-Sachs Disease.
• Embodiment 28. The method of embodiment 26, wherein the disease-associated gene is associated with a polygenic disorder selected from the group consisting of: heart disease; high blood pressure; Alzheimer's disease; arthritis; diabetes; cancer; and obesity.
• Embodiment 29. The method of embodiment 19, wherein the napDNAbp is a nuclease active Cas9 or variant thereof.
• Embodiment 30. The method of embodiment 19, wherein the napDNAbp is a nuclease inactive Cas9 (dCas9) or Cas9 nickase (nCas9), or a variant thereof.
• Embodiment 31. The method of embodiment 19, wherein the napDNAbp comprises an amino acid sequence of SEQ ID NO: 18.
• Embodiment 32. The method of embodiment 19, wherein the napDNAbp comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.
• Embodiment 33. The method of embodiment 19, wherein the reverse transcriptase is introduced in trans.
• Embodiment 34. The method of embodiment 19, wherein the napDNAbp comprises a fusion to a reverse transcriptase.
• Embodiment 35. The method of embodiment 19, wherein the reverse transcriptase comprises any one of the amino acid sequences of SEQ ID NO: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, 662, 700, 701-716, 739-741, and 766.
• Embodiment 36. The method of embodiment 19, wherein the reverse transcriptase comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, 662, 700, 701-716, 739-741, and 766.
• Embodiment 37. The method of embodiment 19, wherein the step of forming an exposed 3′ end in the DNA strand at the target locus comprises nicking the DNA strand with a nuclease.
• Embodiment 38. The method of embodiment 37, wherein the nuclease is the napDNAbp, is provided as a fusion domain of napDNAbp, or is provided in trans.
• Embodiment 39. The method of embodiment 19, wherein the step of forming an exposed 3′ end in the DNA strand at the target locus comprises contacting the DNA strand with a chemical agent.
• Embodiment 40. The method of embodiment 19, wherein the step of forming an exposed 3′ end in the DNA strand at the target locus comprises introducing a replication error.
• Embodiment 41. The method of embodiment 19, wherein the step of contacting the DNA molecule with the napDNAbp and the guide RNA forms an R-loop.
• Embodiment 42. The method of embodiment 41, wherein the DNA strand in which the exposed 3′ end is formed is in the R-loop.
• Embodiment 43. The method of embodiment 19, wherein the PEgRNA comprises an extension arm that comprises the reverse transcriptase (RT) template sequence and the primer binding site.
• Embodiment 44. The method of embodiment 43, wherein the extension arm is at the 3′ end of the guide RNA, the 5′ end of the guide RNA, or at an intramolecular position in the guide RNA.
• Embodiment 45. The method of embodiment 19, wherein the PEgRNA further comprises at least one additional structure selected from the group consisting of a linker, a stem loop, a hairpin, a toeloop, an aptamer, or an RNA-protein recruitment domain.
• Embodiment 46. The method of embodiment 19, wherein the PEgRNA further comprises a homology arm.
• Embodiment 47. The method of embodiment 19, wherein the RT template sequence is homologous to the corresponding endogenous DNA.
• Embodiment 48. A method for introducing one or more changes in the nucleotide sequence of a DNA molecule at a target locus by target-primed reverse transcription, the method comprising: (a) contacting the DNA molecule at the target locus with a (i) fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a reverse transcriptase and (ii) a guide RNA comprising an RT template comprising a desired nucleotide change;
• thereby conducting target-primed reverse transcription of the RT template to generate a single strand DNA comprising the desired nucleotide change;
• thereby incorporating the desired nucleotide change into the DNA molecule at the target locus through a DNA repair and/or replication process.
• Embodiment 49. The method of embodiment 48, wherein the RT template is located at the 3′ end of the guide RNA, the 5′ end of the guide RNA, or at an intramolecular location in the guide RNA.
• Embodiment 50. The method of embodiment 48, wherein the desired nucleotide change comprises a transition, a transversion, an insertion, or a deletion, or any combination thereof.
• Embodiment 51. The method of embodiment 48, wherein the desired nucleotide change comprises a transition selected from the group consisting of: (a) T to C; (b) A to G; (c) C to T; and (d) G to A.
• Embodiment 52. The method of embodiment 48, wherein the desired nucleotide change comprises a transversion selected from the group consisting of: (a) T to A; (b) T to G; (c) C to G; (d) C to A; (e) A to T; (f) A to C; (g) G to C; and (h) G to T.
• Embodiment 53. The method of embodiment 48, wherein the desired nucleotide change comprises changing (1) a G:C basepair to a T:A basepair, (2) a G:C basepair to an A:T basepair, (3) a G:C basepair to C:G basepair, (4) a T:A basepair to a G:C basepair, (5) a T:A basepair to an A:T basepair, (6) a T:A basepair to a C:G basepair, (7) a C:G basepair to a G:C basepair, (8) a C:G basepair to a T:A basepair, (9) a C:G basepair to an A:T basepair, (10) an A:T basepair to a T:A basepair, (11) an A:T basepair to a G:C basepair, or (12) an A:T basepair to a C:G basepair.
• Embodiment 54. A method for replacing a trinucleotide repeat expansion mutation in a target DNA molecule with a healthy sequence comprising a healthy number of repeat trinucleotides, the method comprising: (a) contacting the DNA molecule at the target locus with a (i) fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a polymerase and (ii) a PEgRNA comprising DNA synthesis template comprising the replacement sequence and a primer binding site; (b) conducting prime editing to generate a single strand DNA comprising the replacement sequence; and (c) incorporating the single strand DNA into the DNA molecule at the target locus through a DNA repair and/or replication process.
• Embodiment 55. The method of embodiment 54, wherein the fusion protein comprises the amino acid sequence of PE1, PE2, or PE3.
• Embodiment 56. The method of embodiment 54, wherein the napDNAbp is a Cas9 nickase (nCas9).
• Embodiment 57. The method of embodiment 54, wherein the napDNAbp comprises the amino acid sequence of SEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.
• Embodiment 58. The method of embodiment 54, wherein the guide RNA comprises SEQ ID NOs: 222.
• Embodiment 59. The method of embodiment 54, wherein the step of (b) conducting prime editing comprises generating a 3′ end primer binding sequence at the target locus that is capable of priming polymerase by annealing to the primer binding site on the guide RNA.
• Embodiment 60. The method of embodiment 54, wherein the trinucleotide repeat expansion mutation is associated with Huntington's Disease, Fragile X syndrome, or Friedreich's ataxia.
• Embodiment 61. The method of embodiment 54, wherein the trinucleotide repeat expansion mutation comprises a repeating unit of CAG triplets.
• Embodiment 62. The method of embodiment 54, wherein the trinucleotide repeat expansion mutation comprises a repeating unit of GAA triplets.
• Embodiment 63. A method for introducing one or more changes in the nucleotide sequence of a DNA molecule at a target locus by target-primed reverse transcription, the method comprising: (a) contacting the DNA molecule at the target locus with a (i) fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a reverse transcriptase and (ii) a guide RNA comprising an RT template comprising a desired nucleotide change;
• thereby conducting target-primed reverse transcription of the RT template to generate a single strand DNA comprising the desired nucleotide change;
• thereby incorporating the desired nucleotide change into the DNA molecule at the target locus through a DNA repair and/or replication process.
• Embodiment 64. The method of embodiment 63, wherein the RT template is located at the 3′ end of the guide RNA, the 5′ end of the guide RNA, or at an intramolecular location in the guide RNA.
• Embodiment 65. The method of embodiment 63, wherein the desired nucleotide change comprises a transition, a transversion, an insertion, or a deletion, or any combination thereof.
• Embodiment 66. The method of embodiment 63, wherein the desired nucleotide change comprises a transition selected from the group consisting of: (a) T to C; (b) A to G; (c) C to T; and (d) G to A.
• Embodiment 67. The method of embodiment 63, wherein the desired nucleotide change comprises a transversion selected from the group consisting of: (a) T to A; (b) T to G; (c) C to G; (d) C to A; (e) A to T; (f) A to C; (g) G to C; and (h) G to T.
• Embodiment 68. The method of embodiment 63, wherein the desired nucleotide change comprises changing (1) a G:C basepair to a T:A basepair, (2) a G:C basepair to an A:T basepair, (3) a G:C basepair to C:G basepair, (4) a T:A basepair to a G:C basepair, (5) a T:A basepair to an A:T basepair, (6) a T:A basepair to a C:G basepair, (7) a C:G basepair to a G:C basepair, (8) a C:G basepair to a T:A basepair, (9) a C:G basepair to an A:T basepair, (10) an A:T basepair to a T:A basepair, (11) an A:T basepair to a G:C basepair, or (12) an A:T basepair to a C:G basepair.
• Embodiment 69. A method of preventing or halting the progression of a prion disease by installing on or more protective mutations into PRNP encoded by a target nucleotide sequence by prime editing, the method comprising: (a) contacting the target nucleotide sequence with a (i) prime editor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a polymerase and (ii) a PEgRNA comprising an edit template encoding the functional moiety;
• thereby polymerizing a single strand DNA sequence encoding the protective mutation;
• thereby incorporating the single strand DNA sequence in place of a corresponding endogenous strand at the target nucleotide sequence through a DNA repair and/or replication process;
• wherein the method produces a recombinant target nucleotide sequence that encodes a PRNP comprising a protective mutation and which is resistant to misfolding.
• Embodiment 70. The method of embodiment 69, wherein the prion disease is a human prion disease.
• Embodiment 71. The method of embodiment 69, wherein the prion disease is an animal prion disease.
• Embodiment 72. The method of embodiment 69, wherein the prion disease is Creutzfeldt-Jakob Disease (CJD), Variant Creutzfeldt-Jakob Disease (vCJD), Gerstmann-Straussler-Scheinker Syndrome, Fatal Familial Insomnia, or Kuru.
• Embodiment 73. The method of embodiment 69, wherein the prion disease is Bovine Spongiform Encephalopathy (BSE or “mad cow disease”), Chronic Wasting Disease (CWD), Scrapie, Transmissible Mink Encephalopathy, Feline Spongiform Encephalopathy, and Ungulate Spongiform Encephalopathy.
• Embodiment 74. The method of embodiment 69, wherein the wildtype PRNP amino acid sequence is SEQ ID NOs: 291-292.
• Embodiment 75. The method of embodiment 69, wherein the method results in a modified PRNP amino acid sequence selected from the group consisting of SEQ ID NOs: 293-309, 311-323, wherein said modified PRNP protein is resistant to misfolding.
• Embodiment 76. The method of embodiment 69, wherein the fusion protein comprises the amino acid sequence of PE1, PE2, or PE3.
• Embodiment 77. The method of embodiment 69, wherein the napDNAbp is a Cas9 nickase (nCas9).
• Embodiment 78. The method of embodiment 69, wherein the napDNAbp comprises the amino acid sequence of SEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.
• Embodiment 79. The method of embodiment 69, wherein the PEgRNA comprises SEQ ID NOs: 101-104, 181-183, 223-244, 277, 325-334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 499-505, 735-761, 776-777.
• Embodiment 80. A method of treating CDKL5 Deficiency Disorder by correcting a mutation in the cyclin-dependent kinase-like 5 gene (CDKL5) in a target nucleotide sequence by prime editing, the method comprising: (a) contacting the target nucleotide sequence with a (i) prime editor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a polymerase and (ii) a PEgRNA comprising an edit template that corrects the mutation in CDKL5;
• thereby polymerizing a single strand DNA sequence encoding the edit;
• thereby incorporating the single strand DNA sequence in place of a corresponding endogenous strand at the target nucleotide sequence through a DNA repair and/or replication process;
• wherein the method produces a recombinant target nucleotide sequence that encodes a repaired CDKL5 gene.
• Embodiment 81. The method of embodiment D80, wherein the mutation in CDKL5 is 1412delA.
• Group E. PE Methods for Modifying Protein Structure/Function and/or Mutagenesis
• Embodiment 1. A method for mutagenizing a DNA molecule at a target locus by prime editing, the method comprising: (a) contacting the DNA molecule at the target locus with a (i) fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) and an error-prone polymer (e.g., error-prone reverse transcriptase), and (ii) a guide RNA comprising an edit template comprising a desired nucleotide change; thereby polymerizing a single stranded DNA templated from the edit template; and incorporating the single stranded DNA into the DNA molecule at the target locus through a DNA repair and/or replication process.
• Embodiment 2. The method of any prior embodiment, wherein the fusion protein comprises the amino acid sequence of PE1, PE2, or PE3.
• Embodiment 3. The method of any prior embodiment, wherein the napDNAbp is a Cas9 nickase (nCas9).
• Embodiment 4. The method of embodiment 1, wherein the napDNAbp comprises the amino acid sequence of SEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.
• Embodiment 5. The method of embodiment 1, wherein the guide RNA comprises SEQ ID NOs: 222.
• Embodiment 6. A method of installing an immunoepitope in a protein of interest encoded by a target nucleotide sequence by prime editing, the method comprising: (a) contacting the target nucleotide sequence with a (i) prime editor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a polymerase and (ii) a PEgRNA comprising an edit template encoding the functional moiety;
• thereby polymerizing a single strand DNA sequence encoding the immunoepitope;
• thereby incorporating the single strand DNA sequence in place of a corresponding endogenous strand at the target nucleotide sequence through a DNA repair and/or replication process;
• wherein the method produces a recombinant target nucleotide sequence that encodes a fusion protein comprising the protein of interest and the immunoepitope.
• Embodiment 7. The method of embodiment 6, wherein the immunoepitope is selected from the group consisting of: tetanus toxoid (SEQ ID NO: 396); diphtheria toxin mutant CRM197 (SEQ ID NO: 630); mumps immunoepitope 1 (SEQ ID NO: 400); mumps immunoepitope 2 (SEQ ID NO: 402); mumps immunoeptitope 3 (SEQ ID NO: 404); rubella virus (SEQ ID NO: 406); hemagglutinin (SEQ ID NO: 408); neuraminidase (SEQ ID NO: 410); TAP1 (SEQ ID NO: 412); TAP2 (SEQ ID NO: 414); hemagglutinin epitopes toward class I HLA (SEQ ID NO: 416); neuraminidase epitopes toward class I HLA (SEQ ID NO: 418); hemagglutinin epitopes toward class II HLA (SEQ ID NO: 420); neuraminidase epitopes toward class II HLA (SEQ ID NO: 422); hemagglutinin epitope H5N1-bound class I and class II HLA (SEQ ID NO: 424); and neuraminidase epitope H5N1-bound class I and class II HLA (SEQ ID NO: 426).
• Embodiment 8. The method of embodiment 6, wherein the fusion protein comprises the amino acid sequence of PE1, PE2, or PE3.
• Embodiment 9. The method of embodiment 6, wherein the napDNAbp is a Cas9 nickase (nCas9).
• Embodiment 10. The method of embodiment 6, wherein the napDNAbp comprises the amino acid sequence of SEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.
• Embodiment 11. The method of embodiment 6, wherein the PEgRNA comprises SEQ ID NOs: 101-104, 181-183, 223-244, 277, 325-334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 499-505, 735-761, 776-777.
• Embodiment 12. A method of installing a small molecule dimerization domain in a protein of interest encoded by a target nucleotide sequence by prime editing, the method comprising: (a) contacting the target nucleotide sequence with a (i) prime editor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a polymerase, and (ii) a PEgRNA comprising an edit template encoding the small molecule dimerization domain;
• thereby polymerizing a single strand DNA sequence encoding the immunoepitope;
• thereby incorporating the single strand DNA sequence in place of a corresponding endogenous strand at the target nucleotide sequence through a DNA repair and/or replication process;
• wherein the method produces a modified target nucleotide sequence that encodes a fusion protein comprising the protein of interest and the small molecule dimerization domain.
• Embodiment 13. The method of embodiment 12 further comprising conducting the method on a second protein of interest.
• Embodiment 14. The method of embodiment 13, wherein the first protein of interest and the second protein of interest dimerize in the presence of a small molecule that binds to the dimerization domain on each of said proteins.
• Embodiment 15. The method of embodiment 12, wherein the small molecule binding domain is FKBP12 of SEQ ID NO: 488.
• Embodiment 16. The method of embodiment 12, wherein the small molecule binding domain is FKBP12-F36V of SEQ ID NO: 489.
• Embodiment 17. The method of embodiment 12, wherein the small molecule binding domain is cyclophilin of SEQ ID NOs: 490 and 493-494.
• Embodiment 18. The method of embodiment 12, wherein the small molecule is a dimer of a small molecule as described herein.
• Embodiment 19. The method of embodiment 12, wherein the fusion protein comprises the amino acid sequence of PE1, PE2, or PE3.
• Embodiment 20. The method of embodiment 12, wherein the napDNAbp is a Cas9 nickase (nCas9).
• Embodiment 21. The method of embodiment 12, wherein the napDNAbp comprises the amino acid sequence of SEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.
• Embodiment 22. The method of embodiment 12, wherein the PEgRNA comprises SEQ ID NOs: 101-104, 181-183, 223-244, 277, 325-334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 499-505, 735-761, 776-777.
• Embodiment 23. A method of installing a peptide tag or epitope onto a protein using prime editing, comprising: contacting a target nucleotide sequence encoding the protein with a prime editor construct configured to insert therein a second nucleotide sequence encoding the peptide tag to result in a recombinant nucleotide sequence, such that the peptide tag and the protein are expressed from the recombinant nucleotide sequence as a fusion protein.
• Embodiment 24. The method of embodiment 23, wherein the peptide tag is used for purification and/or detection of the protein.
• Embodiment 25. The method of embodiment 23, wherein the peptide tag is a poly-histidine (e.g., HHHHHH) (SEQ ID NO: 252-262), FLAG (e.g., DYKDDDDK) (SEQ ID NO: 2), V5 (e.g., GKPIPNPLLGLDST) (SEQ ID NO: 3), GCN4, HA (e.g., YPYDVPDYA) (SEQ ID NO: 5), Myc (e.g. EQKLISEED) (SEQ ID NO: 6), or GST.
• Embodiment 26. The method of embodiment 23, wherein the peptide tag has an amino acid sequence selected from the group consisting of SEQ ID NO: 1-6, 245-249, 252-262, 264-273, 275-276, 281, 278-288, and 622.
• Embodiment 27. The method of embodiment 23, wherein the peptide tag is fused to the protein by a linker.
• Embodiment 28. The method of embodiment 23, wherein the fusion protein has the following structure: [protein]-[peptide tag] or [peptide tag]-[protein], wherein “]-[” represents an optional linker.
• Embodiment 29. The method of embodiment 23, wherein the linker has an amino acid sequence of SEQ ID NO: 127, 165-176, 446, 453, and 767-769.
• Embodiment 30. The method of embodiment 23, wherein the prime editor construct comprises a PEgRNA comprising the nucleotide sequence of SEQ ID NOs: 101-104, 181-183, 223-244, 277, 325-334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 499-505, 735-761, 776-777.
• Embodiment 31. The method of embodiment 23, wherein the PEgRNA comprises a spacer, a gRNA core, and an extension arm, wherein the spacer is complementary to the target nucleotide sequence and the extension arm comprises a reverse transcriptase template that encodes the peptide tag.
• Embodiment 32. The method of embodiment 23, wherein the PEgRNA comprises a spacer, a gRNA core, and an extension arm, wherein the spacer is complementary to the target nucleotide sequence and the extension arm comprises a reverse transcriptase template that encodes the peptide tag.
• Embodiment 33. A method of installing or deleting a functional moiety in a protein of interest encoded by a target nucleotide sequence by prime editing, the method comprising: (a) contacting the target nucleotide sequence with a (i) prime editor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a polymerase and (ii) a PEgRNA comprising an edit template encoding the functional moiety or deletion of same; (b) polymerizing a single strand DNA sequence encoding the functional moiety or deletion of same; and (c) incorporating the single strand DNA sequence in place of a corresponding endogenous strand at the target nucleotide sequence through a DNA repair and/or replication process, wherein the method produces a recombinant target nucleotide sequence that encodes a modified protein comprising the protein of interest and the functional moiety or the removal of same, wherein the functional moiety alters a modification state or localization state of the protein.
• Embodiment 34. The method of embodiment 33, wherein functional moiety alters the phosphorylation, ubiquitylation, glycosylation, lipidation, hydroxylation, methylation, acetylation, crotonylation, or SUMOylation state of the protein of interest.
Group F. PE Delivery Methods and Compositions
• Embodiment 1. A polypeptide comprising an N-terminal half or a C-terminal half of a prime editor fusion protein.
• Embodiment 2. The polypeptide of embodiment 1, wherein the prime editor fusion protein comprises a nucleic acid programmable DNA binding protein (napDNAbp) domain and a polymerase domain.
• Embodiment 3. The polypeptide of embodiment 1, wherein the prime editor fusion protein is capable of carrying out prime editing in the presence of a prime editing guide RNA (PEgRNA).
• Embodiment 4. The polypeptide of embodiment 2, wherein the napDNAbp is a Cas9 protein or variant thereof.
• Embodiment 5. The polypeptide of embodiment 2, wherein the napDNAbp is a nuclease with nickase activity.
• Embodiment 6. The polypeptide of embodiment 2, wherein the napDNAbp is a nuclease active Cas9, a nuclease inactive Cas9 (dCas9), or a Cas9 nickase (nCas9).
• Embodiment 7. The polypeptide of embodiment 2, wherein the napDNAbp is selected from the group consisting of: Cas9, Cas12e, Cas12d, Cas12a, Cas12b1, Cas13a, Cas12c, and Argonaute and optionally has a nickase activity.
• Embodiment 8. The polypeptide of embodiment 1, wherein the polypeptide is formed by splitting a prime editor fusion protein at a split site.
• Embodiment 9. The polypeptide of embodiment 8, wherein the split site is a peptide bond in the napDNAbp domain.
• Embodiment 10. The polypeptide of embodiment 8, wherein the split site is a peptide bond in the polymerase domain.
• Embodiment 11. The polypeptide of embodiment 8, wherein the split site is a peptide bond in a linker between the napDNAbp domain and the polymerase domain.
• Embodiment 12. The polypeptide of embodiment 9, wherein the split site is in the peptide bond between residues 1 and 2, 2 and 3, 3 and 4, 4 and 5, 5 and 6, 6 and 7, 7 and 8, 8 and 9, 9 and 10, 10 and 11, 11 and 12, 12 and 13, 13 and 14, 14 and 15, 16 and 17, 17 and 18, 18 and 19, 19 and 20, 20 and 21, 21 and 22, 22 and 23, 23 and 24, 24 and 25, 25 and 26, 26 and 27, 27 ad 28, 28 and 29, 29 and 30, 30 and 31, 31 and 32, 32 and 33, 33 and 34, 34 and 35, 35 and 36, 36 and 37, 37 and 38, 38 and 39, 39 and 40, 40 and 41, 41 and 42, 42 and 43, 43 and 44, 44 and 45, 45 and 46, 46 and 47, 47 and 48, 48 and 49, 49 and 50, or between any two residues between residues 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-600, 600-700, 700-800, 800-900, 900-1000, 1000-1100, 1100-1200, 1200-1300, or 1300-1368 of SEQ ID NO: 18 (canonical SpCas9), or between any two equivalent amino acid residues of an SpCas9 homolog or equivalent of SEQ ID NO: 18.
• Embodiment 13. The polypeptide of embodiment 9, wherein the split site is in the peptide bond between residues 1 and 2, 2 and 3, 3 and 4, 4 and 5, 5 and 6, 6 and 7, 7 and 8, 8 and 9, 9 and 10, 10 and 11, 11 and 12, 12 and 13, 13 and 14, 14 and 15, 16 and 17, 17 and 18, 18 and 19, 19 and 20, 20 and 21, 21 and 22, 22 and 23, 23 and 24, 24 and 25, 25 and 26, 26 and 27, 27 ad 28, 28 and 29, 29 and 30, 30 and 31, 31 and 32, 32 and 33, 33 and 34, 34 and 35, 35 and 36, 36 and 37, 37 and 38, 38 and 39, 39 and 40, 40 and 41, 41 and 42, 42 and 43, 43 and 44, 44 and 45, 45 and 46, 46 and 47, 47 and 48, 48 and 49, 49 and 50, or between any two residues between residues 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-600, or 600-667 of SEQ ID NO: 89 (canonical reverse transcriptase, M-MLV RT), or between any two equivalent amino acid residues of a reverse transcriptase homolog or equivalent of SEQ ID NO: 89.
• Embodiment 14. The polypeptide of embodiment 1, wherein the polypeptide is an N-terminal half of a prime editor fusion protein.
• Embodiment 15. The polypeptide of embodiment 1, wherein the polypeptide is a C-terminal half of a prime editor fusion protein.
• Embodiment 16. A nucleotide sequence encoding a polypeptide of any of embodiments 1-15 and optionally a PEgRNA.
• Embodiment 17. A virus genome comprising a nucleotide sequence encoding a polypeptide of any of embodiments 1-15 and optionally a PEgRNA.
• Embodiment 18. The virus genome of embodiment 17, wherein the nucleotide sequence further comprises a promoter sequence suitable for expressing the polypeptide of any of embodiments 1-15.
• Embodiment 19. The virus genome of embodiment 17, wherein the nucleotide sequence further comprises a sequence encoding a PEgRNA.
• Embodiment 20. A virus particle comprising a genome comprising a nucleotide sequence encoding a polypeptide of any of embodiments 1-15 and optionally a PEgRNA.
• Embodiment 21. The virus particle of embodiment 20, wherein the virus particle is an adenovirus particle, an adeno-associated virus particle, or a lentivirus particle.
• Embodiment 22. The virus particle of embodiment 20, wherein the polypeptide encoded by the genome is an N terminal half of a prime editor fusion protein.
• Embodiment 23. The virus particle of embodiment 20, wherein the polypeptide encoded by the genome is a C terminal half of a prime editor fusion protein.
• Embodiment 24. A pharmaceutical composition comprising a virus particle of any of embodiments 20-23 and a pharmaceutical excipient.
• Embodiment 25. A pharmaceutical composition comprising a virus particle of embodiment 22 (encoding the N terminal half) and a pharmaceutical excipient.
• Embodiment 26. A pharmaceutical composition comprising a virus particle of embodiment 23 (encoding the C terminal half) and a pharmaceutical excipient.
• Embodiment 27. A ribonucleoprotein (RNP) complex comprising a nucleotide sequence encoding a polypeptide of any of embodiments 1-15 and optionally a PEgRNA.
• Embodiment 28. The ribonucleoprotein (RNP) complex of embodiment 27, wherein the polypeptide encoded by the genome is an N-terminal half of a prime editor fusion protein.
• Embodiment 29. The ribonucleoprotein (RNP) complex of embodiment 27, wherein the polypeptide encoded by the genome is a C-terminal half of a prime editor fusion protein.
• Embodiment 30. A pharmaceutical composition comprising a ribonucleoprotein (RNP) complex of any of embodiments 27-29 and a pharmaceutical excipient.
• Embodiment 31. A pharmaceutical composition comprising the ribonucleoprotein (RNP) complex of embodiment 28 (encoding the N-terminal half) and a pharmaceutical excipient.
• Embodiment 32. A pharmaceutical composition comprising the ribonucleoprotein (RNP) complex of embodiment 29 (encoding the C-terminal half) and a pharmaceutical excipient.
• Embodiment 33. A pharmaceutical composition comprising a first AAV particle and a second AAV particle, wherein the first AAV vector expresses an N-terminal half of a prime editor fusion protein and the second AAV vector expresses a C-terminal half of a prime editor fusion protein, wherein the N-terminal half and the C-terminal half are combined within the cell to reconstitute the prime editor.
• Embodiment 34. The pharmaceutical composition of embodiment 33, wherein the first or second AAV particle also expresses a PEgRNA that targets the reconstituted prime editor to a target DNA site.
• Embodiment 35. The pharmaceutical composition of embodiment 33, wherein the prime editor fusion protein comprises a nucleic acid programmable DNA binding protein (napDNAbp) domain and a polymerase domain.
• Embodiment 36. The pharmaceutical composition of embodiment 33, wherein the prime editor fusion protein is capable of carrying out prime editing in the presence of a prime editing guide RNA (PEgRNA).
• Embodiment 37. The pharmaceutical composition of embodiment 35, wherein the napDNAbp is a Cas9 protein or variant thereof.
• Embodiment 38. The pharmaceutical composition of embodiment 35, wherein the napDNAbp is a nuclease with a nickase activity.
• Embodiment 39. The pharmaceutical composition of embodiment 35, wherein the napDNAbp is a nuclease active Cas9, a nuclease inactive Cas9 (dCas9), or a Cas9 nickase (nCas9).
• Embodiment 40. The pharmaceutical composition of embodiment 35, wherein the napDNAbp is selected from the group consisting of: Cas9, Cas12e, Cas12d, Cas12a, Cas12b1, Cas13a, Cas12c, and Argonaute and optionally has a nickase activity.
• Embodiment 41. The pharmaceutical composition of embodiment 33, wherein N-terminal and C-terminal halves are formed by splitting the prime editor fusion protein at a split site.
• Embodiment 42. The pharmaceutical composition of embodiment 41, wherein the split site is a peptide bond in a napDNAbp domain.
• Embodiment 43. The pharmaceutical composition of embodiment 41, wherein the split site is a peptide bond in a polymerase domain.
• Embodiment 44. The pharmaceutical composition of embodiment 41, wherein the split site is a peptide bond in a linker.
• Embodiment 45. The pharmaceutical composition of embodiment 41, wherein the split site is in the peptide bond between residues 1 and 2, 2 and 3, 3 and 4, 4 and 5, 5 and 6, 6 and 7, 7 and 8, 8 and 9, 9 and 10, 10 and 11, 11 and 12, 12 and 13, 13 and 14, 14 and 15, 16 and 17, 17 and 18, 18 and 19, 19 and 20, 20 and 21, 21 and 22, 22 and 23, 23 and 24, 24 and 25, 25 and 26, 26 and 27, 27 ad 28, 28 and 29, 29 and 30, 30 and 31, 31 and 32, 32 and 33, 33 and 34, 34 and 35, 35 and 36, 36 and 37, 37 and 38, 38 and 39, 39 and 40, 40 and 41, 41 and 42, 42 and 43, 43 and 44, 44 and 45, 45 and 46, 46 and 47, 47 and 48, 48 and 49, 49 and 50, or between any two residues between residues 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-600, 600-700, 700-800, 800-900, 900-1000, 1000-1100, 1100-1200, 1200-1300, or 1300-1368 of SEQ ID NO: 18 (canonical SpCas9), or between any two equivalent amino acid residues of an SpCas9 homolog or equivalent of SEQ ID NO: 18.
• Embodiment 46. The pharmaceutical composition of embodiment 41, wherein the split site is in the peptide bond between residues 1 and 2, 2 and 3, 3 and 4, 4 and 5, 5 and 6, 6 and 7, 7 and 8, 8 and 9, 9 and 10, 10 and 11, 11 and 12, 12 and 13, 13 and 14, 14 and 15, 16 and 17, 17 and 18, 18 and 19, 19 and 20, 20 and 21, 21 and 22, 22 and 23, 23 and 24, 24 and 25, 25 and 26, 26 and 27, 27 ad 28, 28 and 29, 29 and 30, 30 and 31, 31 and 32, 32 and 33, 33 and 34, 34 and 35, 35 and 36, 36 and 37, 37 and 38, 38 and 39, 39 and 40, 40 and 41, 41 and 42, 42 and 43, 43 and 44, 44 and 45, 45 and 46, 46 and 47, 47 and 48, 48 and 49, 49 and 50, or between any two residues between residues 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-600, or 600-667 of SEQ ID NO: 89 (canonical reverse transcriptase, M-MLV RT), or between any two equivalent amino acid residues of a reverse transcriptase homolog or equivalent of SEQ ID NO: 89.
• Embodiment 47. The pharmaceutical composition of embodiment 33, wherein the N-terminal half of the prime editor fusion protein has an amino acid sequence encoding the N-terminal prime editor fusion proteins as described herein.
• Embodiment 48. The pharmaceutical composition of embodiment 33, wherein the C-terminal half of the prime editor fusion protein has an amino acid sequence encoding the N-terminal prime editor fusion proteins as described herein.
• Embodiment 49. A method of delivering a prime editor fusion protein to a cell comprising transfecting the cell with a first AAV particle and a second AAV particle, wherein the first AAV vector expresses an N-terminal half of a prime editor fusion protein and the second AAV vector expresses a C-terminal half of a prime editor fusion protein, wherein the N-terminal half and the C-terminal half are combined within the cell to reconstitute the prime editor fusion protein.
• Embodiment 50. The method of embodiment 49, wherein the first or second AAV particle also expresses a PEgRNA that targets the reconstituted prime editor to a target DNA site.
• Embodiment 51. The method of embodiment 49, wherein the prime editor fusion protein comprises a nucleic acid programmable DNA binding protein (napDNAbp) domain and a polymerase domain.
• Embodiment 52. The method of embodiment 49, wherein the prime editor fusion protein is capable of carrying out prime editing in the presence of an prime editing guide RNA (PEgRNA).
• Embodiment 53. The method of embodiment 51, wherein the napDNAbp is a Cas9 protein or variant thereof.
• Embodiment 54. The method of embodiment 53, wherein the napDNAbp is a nuclease with nickase activity.
• Embodiment 55. The method of embodiment 53, wherein the napDNAbp is a nuclease active Cas9, a nuclease inactive Cas9 (dCas9), or a Cas9 nickase (nCas9).
• Embodiment 56. The method of embodiment 53, wherein the napDNAbp is selected from the group consisting of: Cas9, Cas12e, Cas12d, Cas12a, Cas12b1, Cas13a, Cas12c, and Argonaute and optionally has a nickase activity.
• Embodiment 57. The method of embodiment 49, wherein N-terminal and C-terminal halves are formed by splitting the prime editor fusion protein at a split site.
• Embodiment 58. The method of embodiment 57, wherein the split site is a peptide bond in a napDNAbp domain.
• Embodiment 59. The method of embodiment 57, wherein the split site is a peptide bond in a polymerase domain.
• Embodiment 60. The method of embodiment 57, wherein the split site is a peptide bond in a linker.
• Embodiment 61. The method of embodiment 57, wherein the split site is in the peptide bond between residues 1 and 2, 2 and 3, 3 and 4, 4 and 5, 5 and 6, 6 and 7, 7 and 8, 8 and 9, 9 and 10, 10 and 11, 11 and 12, 12 and 13, 13 and 14, 14 and 15, 16 and 17, 17 and 18, 18 and 19, 19 and 20, 20 and 21, 21 and 22, 22 and 23, 23 and 24, 24 and 25, 25 and 26, 26 and 27, 27 ad 28, 28 and 29, 29 and 30, 30 and 31, 31 and 32, 32 and 33, 33 and 34, 34 and 35, 35 and 36, 36 and 37, 37 and 38, 38 and 39, 39 and 40, 40 and 41, 41 and 42, 42 and 43, 43 and 44, 44 and 45, 45 and 46, 46 and 47, 47 and 48, 48 and 49, 49 and 50, or between any two residues between residues 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-600, 600-700, 700-800, 800-900, 900-1000, 1000-1100, 1100-1200, 1200-1300, or 1300-1368 of SEQ ID NO: 18 (canonical SpCas9), or between any two equivalent amino acid residues of an SpCas9 homolog or equivalent of SEQ ID NO: 18.
• Embodiment 62. The method of embodiment 57, wherein the split site is in the peptide bond between residues 1 and 2, 2 and 3, 3 and 4, 4 and 5, 5 and 6, 6 and 7, 7 and 8, 8 and 9, 9 and 10, 10 and 11, 11 and 12, 12 and 13, 13 and 14, 14 and 15, 16 and 17, 17 and 18, 18 and 19, 19 and 20, 20 and 21, 21 and 22, 22 and 23, 23 and 24, 24 and 25, 25 and 26, 26 and 27, 27 ad 28, 28 and 29, 29 and 30, 30 and 31, 31 and 32, 32 and 33, 33 and 34, 34 and 35, 35 and 36, 36 and 37, 37 and 38, 38 and 39, 39 and 40, 40 and 41, 41 and 42, 42 and 43, 43 and 44, 44 and 45, 45 and 46, 46 and 47, 47 and 48, 48 and 49, 49 and 50, or between any two residues between residues 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-600, or 600-667 of SEQ ID NO: 89 (canonical reverse transcriptase, M-MLV RT), or between any two equivalent amino acid residues of a reverse transcriptase homolog or equivalent of SEQ ID NO: 89.
• Embodiment 63. The method of embodiment 49, wherein the N-terminal half of the prime editor fusion protein has an amino acid sequence encoding the N-terminal prime editor fusion proteins as described herein.
• Embodiment 64. The method of embodiment 49, wherein the C-terminal half of the prime editor fusion protein has an amino acid sequence encoding the C-terminal prime editor fusion proteins as described herein.
• Embodiment 65. The method of embodiment 49, wherein the first AAV particle comprises a recombinant AAV genome comprising a nucleotide sequence which encodes the first prime editor component.
• Embodiment 66. The method of embodiment 49, wherein the second AAV particle comprises a recombinant AAV genome comprising a nucleotide sequence which encodes the second prime editor component.
• Embodiment 67. The method of embodiment 49, wherein the transfecting step is conducted in vivo.
• Embodiment 68. The method of embodiment 49, wherein the transfecting step is conducted ex vivo.
• Embodiment 69. The method of embodiment 50, wherein the target DNA site is a disease-associated gene.
• Embodiment 70. The method of embodiment 69, wherein the disease-associated gene is associated with a monogenetic disorder selected from the group consisting of: Adenosine Deaminase (ADA) Deficiency; Alpha-1 Antitrypsin Deficiency; Cystic Fibrosis; Duchenne Muscular Dystrophy; Galactosemia; Hemochromatosis; Huntington's Disease; Maple Syrup Urine Disease; Marfan Syndrome; Neurofibromatosis Type 1; Pachyonychia Congenita; Phenylkeotnuria; Severe Combined Immunodeficiency; Sickle Cell Disease; Smith-Lemli-Opitz Syndrome; a trinucleotide repeat disorder; a prion disease; and Tay-Sachs Disease.
• Embodiment 71. The method of embodiment 69, wherein the disease-associated gene is associated with a polygenic disorder selected from the group consisting of: heart disease; high blood pressure; Alzheimer's disease; arthritis; diabetes; cancer; and obesity.
• Embodiment 72. The method of embodiment 51, wherein the programmable DNA binding protein (napDNAbp) domain.
• Embodiment 73. The method of embodiment 51, wherein the polymerase domain is a reverse transcriptase.
• Embodiment 74. The method of embodiment 73, wherein the reverse transcriptase comprises any one of the amino acid sequences of SEQ ID NO: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, 662, 700, 701-716, 739-741, and 766.
• Embodiment 75. The method of embodiment 73, wherein the reverse transcriptase comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, 662, 700, 701-716, 739-741, and 766.
• Embodiment 76. The method of embodiment 73, wherein the napDNAbp comprises an amino acid sequence of SEQ ID NO: 18.
• Embodiment 77. The method of embodiment 73, wherein the napDNAbp comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.
• Embodiment 78. The polypeptide of embodiment 8, wherein the split site is between 1023 and 1024 of SEQ ID NO: 18, or at a corresponding position in an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 99%, or at least 99.5% sequence identity with SEQ ID NO: 18.
Group G. PE Methods for Modifying RNA Structure/Function
• Embodiment 1. A method of installing a ribonucleotide motif or tag in an RNA of interest encoded by a target nucleotide sequence by prime editing, the method comprising: (a) contacting the target nucleotide sequence with a (i) prime editor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a polymerase, and (ii) a PEgRNA comprising an edit template encoding the ribonucleotide motif or tag; thereby polymerizing a single strand DNA sequence encoding the ribonucleotide motif or tag; and incorporating the single strand DNA sequence in place of a corresponding endogenous strand at the target nucleotide sequence through a DNA repair and/or replication process, wherein the method produces a target nucleotide sequence that encodes a modified RNA of interest comprising the ribonucleotide motif or tag.
• Embodiment 2. The method of embodiment 1, wherein ribonucleotide motif or tag is a detection moiety.
• Embodiment 3. The method of embodiment 1, wherein the ribonucleotide motif or tag affects the expression level of the RNA of interest.
• Embodiment 4. The method of embodiment 1, wherein the ribonucleotide motif or tag affects the transport or subcellular location of the RNA of interest.
• Embodiment 5. The method of embodiment 1, wherein the ribonucleotide motif or tag is selected from the group consisting of SV40 type 1, SV40 type 2, SV40 type 3, hGH, BGH, rbGlob, TK, MALAT1 ENE-mascRNA, KSHV PAN ENE, Smbox/U1 snRNA box, U1 snRNA 3′ box, tRNA-lysine, broccoli aptamer, spinach aptamer, mango aptamer, HDV ribozyme, and m6A.
• Embodiment 6. The method of embodiment 1, wherein the PEgRNA comprises SEQ ID NOs: 101-104, 181-183, 223-244, 277, 325-334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 499-505, 735-761, 776-777 (see Table).
• Embodiment 7. The method of embodiment 1, wherein the fusion protein comprises the amino acid sequence of PE1, PE2, or PE3.
• Embodiment 8. The method of embodiment 1, wherein the napDNAbp is a Cas9 nickase (nCas9).
• Embodiment 9. The method of embodiment 1, wherein the napDNAbp comprises the amino acid sequence of SEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.
Group H. PE Methods for Making Gene Libraries
• Embodiment 1. A method of constructing a programmed mutant gene library by prime editing, the method comprising:
• (a) contacting a library of target nucleotide sequences each comprising one or more target genetic loci with a (i) prime editor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a polymerase, and (ii) a PEgRNA comprising an edit template comprising a sequence having at least one genetic change relative to the one or more target genetic loci;
• thereby polymerizing a single strand DNA sequence templated by the edit template; and
• incorporating the single strand DNA sequence in place of the one or more target genetic loci through a DNA repair and/or replication process, thereby incorporating the at least one genetic change into the target genetic loci of the target nucleotide sequences of said library.
• Embodiment 2. The method of embodiment 1, wherein the library is a plasmid library.
• Embodiment 3. The method of embodiment 1, wherein the library is a phage library.
• Embodiment 4. The method of embodiment 1, wherein the one or more target genetic loci comprise a region encoding a protein.
• Embodiment 5. The method of embodiment 1, wherein the one or more target genetic loci comprise a region encoding a secondary structure motif of a protein.
• Embodiment 6. The method of embodiment 5, wherein the secondary structure motif is an alpha helix.
• Embodiment 7. The method of embodiment 5, wherein the secondary structure motif is a beta sheet.
• Embodiment 8. The method of embodiment 1, wherein the napDNAbp is a Cas9 protein or variant thereof.
• Embodiment 9. The method of embodiment 1, wherein the napDNAbp is a nuclease with a nickase activity.
• Embodiment 10. The method of embodiment 1, wherein the napDNAbp is a nuclease active Cas9, a nuclease inactive Cas9 (dCas9), or a Cas9 nickase (nCas9).
• Embodiment 11. The method of embodiment 1, wherein the napDNAbp comprises an amino acid sequence of SEQ ID NO: 18.
• Embodiment 12. The method of embodiment 1, wherein the napDNAbp comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.
• Embodiment 13. The method of embodiment 1, wherein the polymerase domain is a reverse transcriptase.
• Embodiment 14. The method of embodiment 13, wherein the reverse transcriptase comprises any one of the amino acid sequences of SEQ ID NO: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, 662, 700, 701-716, 739-741, and 766.
• Embodiment 15. The method of embodiment 14, wherein the reverse transcriptase comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, 662, 700, 701-716, 739-741, and 766.
• Embodiment 16. The method of embodiment 1, wherein the at least one genetic change in the edit template is an insertion.
• Embodiment 17. The method of embodiment 1, wherein the at least one genetic change in the edit template is a deletion.
• Embodiment 18. The method of embodiment 1, wherein the at least one genetic change in the edit template is substitution.
• Embodiment 19. The method of embodiment 1, wherein the at least one genetic change is an insertion of one or more codons.
• Embodiment 20. The method of embodiment 1, wherein the at least one genetic change is a deletion of one or more codons.
• Embodiment 21. The method of embodiment 1, wherein the at least one genetic change is the insertion of a stop codon.
• Embodiment 22. The method of embodiment 1, wherein the at least one genetic change is the conversion of a non-stop codon to a stop codon.
• Embodiment 23. The method of embodiment 1, wherein the method is conduct simultaneously at least 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or between 10-100, or between 100-200, or between 200-300, or between 300-400, or between 400-500 target genetic loci in each of the target nucleotide sequences of the library.
• Embodiment 24. The method of embodiment 1, wherein the method is conducted during PACE or PANCE evolution, and wherein each instance of incorporating the at least one genetic change into the target genetic loci of the target nucleotide sequences of said library also installs a new target sequence.
Group I. PE Methods for Off-Target Detection
• Embodiment 1. A method of evaluating off-target editing by a prime editor, the method comprising:
• (a) contacting a target nucleotide sequence having an edit site with a (i) prime editor fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a polymerase and (ii) a PEgRNA comprising a DNA synthesis template that encodes a detectable sequence;
• wherein the PEgRNA complexes with the fusion protein and guides said fusion protein to the edit site and, if present, to one or more off-target sites;
• and wherein the prime editor fusion protein installs the detectable sequence at the edit site, and, if present, at the one or more off-target sites;
• (b) determining the nucleotide sequence of the edit site and the one or more off-target sites.
• Embodiment 2. The method of embodiment 1, wherein the target nucleotide sequence is a genome.
• Embodiment 3. The method of embodiment 1, wherein the step of contacting is in vitro.
• Embodiment 4. The method of embodiment 1, wherein the step of contacting is in vivo.
• Embodiment 5. The method of embodiment 1, wherein the edit site is a mutation in a disease-associated gene.
• Embodiment 6. The method of embodiment 5, wherein the mutation is a single base substitution, insertion, deletion, or inversion.
• Embodiment 7. The method of embodiment 1, wherein the disease-associated gene is associated with a monogenetic disorder selected from the group consisting of: Adenosine Deaminase (ADA) Deficiency; Alpha-1 Antitrypsin Deficiency; Cystic Fibrosis; Duchenne Muscular Dystrophy; Galactosemia; Hemochromatosis; Huntington's Disease; Maple Syrup Urine Disease; Marfan Syndrome; Neurofibromatosis Type 1; Pachyonychia Congenita; Phenylkeotnuria; Severe Combined Immunodeficiency; Sickle Cell Disease; Smith-Lemli-Opitz Syndrome; and Tay-Sachs Disease.
• Embodiment 8. The method of embodiment 1, wherein the disease-associated gene is associated with a polygenic disorder selected from the group consisting of: heart disease; high blood pressure; Alzheimer's disease; arthritis; diabetes; cancer; and obesity.
• Embodiment 9. The method of embodiment 1, wherein the fusion protein has an amino acid sequence of SEQ ID NOs: 101-104, 181-183, 223-244, 277, 325-334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 499-505, 735-761, 776-777, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NOs: 101-104, 181-183, 223-244, 277, 325-334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 499-505, 735-761, 776-777.
• Embodiment 10. The method of embodiment 1, wherein the napDNAbp is Cas9, Cas12e, Cas12d, Cas12a, Cas12b1, Cas13a, Cas12c, or Argonaute, or a variant of Cas9, Cas12e, Cas12d, Cas12a, Cas12b1, Cas13a, Cas12c, or Argonaute.
• Embodiment 11. The method of embodiment 1, wherein the napDNAbp is a Cas9 or variant thereof.
• Embodiment 12. The method of embodiment 1, wherein the napDNAbp is a nuclease active Cas9, a nuclease inactive Cas9 (dCas9), or a Cas9 nickase (nCas9).
• Embodiment 13. The method of embodiment 1, wherein the napDNAbp is Cas9 nickase (nCas9).
• Embodiment 14. The method of embodiment 1, wherein the napDNAbp comprises the amino acid sequence of SEQ ID NO: 18, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO: 18.
• Embodiment 15. The method of embodiment 1, wherein the napDNAbp is SpCas9 wild type or a variant thereof of any one of amino acid sequences 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with any of SEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.
• Embodiment 16. The method of embodiment 1, wherein the napDNAbp is an SpCas9 ortholog.
• Embodiment 17. The method of embodiment 1, wherein the napDNAbp is any one of amino acid sequences 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with any of SEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.
• Embodiment 18. The method of embodiment 1, wherein the napDNAbp comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.
• Embodiment 19. The method of embodiment 1, wherein the polymerase comprises an RNA-dependent DNA polymerase activity.
• Embodiment 20. The method of embodiment 1, wherein the polymerase is a reverse transcriptase.
• Embodiment 21. The method of embodiment 1, wherein the reverse transcriptase is a naturally occurring wild type reverse transcriptase having an amino acid sequence of any one of SEQ ID NOs: 89 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with any of SEQ ID NOs: 89.
• Embodiment 22. The method of embodiment 1, wherein the reverse transcriptase is a variant reverse transcriptase having an amino acid sequence of any one of SEQ ID NOs: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, 662, 700, 701-716, 739-741, and 766 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with any of SEQ ID NOs: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, 662, 700, 701-716, 739-741, and 766.
• Embodiment 23. The method of embodiment 1, wherein the reverse transcriptase comprises any one of the amino acid sequences of SEQ ID NO: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, 662, 700, 701-716, 739-741, and 766.
• Embodiment 24. The method of embodiment 1, wherein the reverse transcriptase comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, 662, 700, 701-716, 739-741, and 766.
• Embodiment 25. The method of embodiment 1, wherein the detectable sequence is an insertion of at least 1, or at least 2, or at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or at least 8, or at least 9, or at least 10, or at least 11, or at least 12, or at least 13, or at least 14, or at least 15, or at least 16, or at least 17, or at least 18, or at least 19, or at least 20, or at least 21, or at least 22, or at least 23, or at least 24, or at least 25, or at least 26, or at least 27, or at least 28, or at least 29, or at least 30, or at least 31, or at least 32, or at least 33, or at least 34, or at least 35, or at least 40, or at least 50, or at least 60, or at least 70, or at least 80, or at least 90, or at least 100 nucleobases.
• Embodiment 26. The method of embodiment 1, wherein the detectable sequence is a deletion of at least 1, or at least 2, or at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or at least 8, or at least 9, or at least 10, or at least 11, or at least 12, or at least 13, or at least 14, or at least 15, or at least 16, or at least 17, or at least 18, or at least 19, or at least 20, or at least 21, or at least 22, or at least 23, or at least 24, or at least 25, or at least 26, or at least 27, or at least 28, or at least 29, or at least 30, or at least 31, or at least 32, or at least 33, or at least 34, or at least 35, or at least 40, or at least 50, or at least 60, or at least 70, or at least 80, or at least 90, or at least 100 nucleobases.
• Embodiment 27. The method of embodiment 1, wherein the detectable sequence is a nucleobase substitution.
• Embodiment 28. The method of embodiment 1, wherein the detectable sequence is a transition mutation.
• Embodiment 29. The method of embodiment 1, wherein the detectable sequence is a transversion mutation.
• Embodiment 30. The method of embodiment 1, wherein the detectable sequence is a single nucleotide substitution selected from the group consisting of: (1) a G to T substitution, (2) a G to A substitution, (3) a G to C substitution, (4) a T to G substitution, (5) a T to A substitution, (6) a T to C substitution, (7) a C to G substitution, (8) a C to T substitution, (9) a C to A substitution, (10) an A to T substitution, (11) an A to G substitution, and (12) an A to C substitution.
• Embodiment 31. The method of embodiment 1, wherein the detectable sequence is a single nucleotide substitution that converts (1) a G:C basepair to a T:A basepair, (2) a G:C basepair to an A:T basepair, (3) a G:C basepair to C:G basepair, (4) a T:A basepair to a G:C basepair, (5) a T:A basepair to an A:T basepair, (6) a T:A basepair to a C:G basepair, (7) a C:G basepair to a G:C basepair, (8) a C:G basepair to a T:A basepair, (9) a C:G basepair to an A:T basepair, (10) an A:T basepair to a T:A basepair, (11) an A:T basepair to a G:C basepair, or (12) an A:T basepair to a C:G basepair.
• Embodiment 32. The method of embodiment 1, wherein the detectable sequence is a barcode sequence.
• Embodiment 33. The method of embodiment 1, wherein step (d) of determining the nucleotide sequence at the on-target and off-target sites comprises (i) fragmenting the target nucleotide sequence to form fragments, (ii) attaching adapter sequences to the ends of the fragments, (iii) PCR amplifying an amplicon using a pair of primers wherein one primer anneals to an adapter sequence attached at one end of the fragments, and another primer that anneals to an adapter sequence inserted by prime editing located within the fragment, and (iv) sequencing the amplicon to determine the location of the edit.
Group J. PE Methods for Cell Data Recording
• Embodiment 1. A method of recording a cellular event by prime editing, the method comprising: (A) introducing into a cell one or more constructs encoding (i) a prime editor fusion protein comprising an napDNAbp and an RNA-dependent DNA polymerase and (ii) a PEgRNA, wherein the expression of the fusion protein and/or the PEgRNA is induced by the occurrence of a cellular event, and wherein upon expression of the fusion protein and/or the PEgRNA results in prime editing of a target edit site in the genome of the cell to introduce a detectable sequence, and (B) identifying the detectable sequence, thereby identifying the occurrence of the cellular event.
• Embodiment 2. The method of embodiment 1, wherein the prime editing of step (A) simultaneously introduces a new target edit site such that the recording of the cellular event may occur iteratively.
• Embodiment 3. The method of embodiment 1, wherein the PEgRNA comprises an edit template that encodes the detectable sequence.
• Embodiment 4. The method of embodiment 3, wherein the edit template further encodes a new target edit site.
• Embodiment 5. The method of embodiment 1, wherein the detectable sequence is an insertion of at least 1, or at least 2, or at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or at least 8, or at least 9, or at least 10, or at least 11, or at least 12, or at least 13, or at least 14, or at least 15, or at least 16, or at least 17, or at least 18, or at least 19, or at least 20, or at least 21, or at least 22, or at least 23, or at least 24, or at least 25, or at least 26, or at least 27, or at least 28, or at least 29, or at least 30, or at least 31, or at least 32, or at least 33, or at least 34, or at least 35, or at least 40, or at least 50, or at least 60, or at least 70, or at least 80, or at least 90, or at least 100 nucleobases.
• Embodiment 6. The method of embodiment 1, wherein the detectable sequence is a deletion of at least 1, or at least 2, or at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or at least 8, or at least 9, or at least 10, or at least 11, or at least 12, or at least 13, or at least 14, or at least 15, or at least 16, or at least 17, or at least 18, or at least 19, or at least 20, or at least 21, or at least 22, or at least 23, or at least 24, or at least 25, or at least 26, or at least 27, or at least 28, or at least 29, or at least 30, or at least 31, or at least 32, or at least 33, or at least 34, or at least 35, or at least 40, or at least 50, or at least 60, or at least 70, or at least 80, or at least 90, or at least 100 nucleobases.
• Embodiment 7. The method of embodiment 1, wherein the detectable sequence is a nucleobase substitution.
• Embodiment 8. The method of embodiment 1, wherein the detectable sequence is a transition mutation.
• Embodiment 9. The method of embodiment 1, wherein the detectable sequence is a transversion mutation.
• Embodiment 10. The method of embodiment 1, wherein the detectable sequence is a single nucleotide substitution of wherein the single nucleotide substitution is (1) a G to T substitution, (2) a G to A substitution, (3) a G to C substitution, (4) a T to G substitution, (5) a T to A substitution, (6) a T to C substitution, (7) a C to G substitution, (8) a C to T substitution, (9) a C to A substitution, (10) an A to T substitution, (11) an A to G substitution, or (12) an A to C substitution.
• Embodiment 11. The method of embodiment 1, wherein the detectable sequence is a single nucleotide substitution that converts (1) a G:C basepair to a T:A basepair, (2) a G:C basepair to an A:T basepair, (3) a G:C basepair to C:G basepair, (4) a T:A basepair to a G:C basepair, (5) a T:A basepair to an A:T basepair, (6) a T:A basepair to a C:G basepair, (7) a C:G basepair to a G:C basepair, (8) a C:G basepair to a T:A basepair, (9) a C:G basepair to an A:T basepair, (10) an A:T basepair to a T:A basepair, (11) an A:T basepair to a G:C basepair, or (12) an A:T basepair to a C:G basepair.
• Embodiment 12. The method of embodiment 1, wherein the detectable sequence is a barcode sequence.
• Embodiment 13. The method of embodiment 1, wherein the detectable sequence increases in length over time as a result of iterative insertion of the detectable sequence for each occurrence of the cellular event.
• Embodiment 14. The method of embodiment 1, wherein the detecting step comprises sequencing the edited target site, or an amplicon of the edited target site.
• Embodiment 15. The method of embodiment 1, wherein the napDNAbp is Cas9, Cas12e, Cas12d, Cas12a, Cas12b1, Cas13a, Cas12c, or Argonaute, or a variant of Cas9, Cas12e, Cas12d, Cas12a, Cas12b1, Cas13a, Cas12c, or Argonaute.
• Embodiment 16. The method of embodiment 1, wherein the napDNAbp is a Cas9 or variant thereof.
• Embodiment 17. The method of embodiment 1, wherein the napDNAbp is a nuclease active Cas9, a nuclease inactive Cas9 (dCas9), or a Cas9 nickase (nCas9).
• Embodiment 18. The method of embodiment 1, wherein the napDNAbp is Cas9 nickase (nCas9).
• Embodiment 19. The method of embodiment 1, wherein the napDNAbp comprises the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO: 18.
• Embodiment 20. The method of embodiment 1, wherein the napDNAbp is SpCas9 wild type or a variant thereof of any one of amino acid sequences 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with any of SEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.
• Embodiment 21. The method of embodiment 1, wherein the napDNAbp is an SpCas9 ortholog.
• Embodiment 22. The method of embodiment 1, wherein the napDNAbp is any one of amino acid sequences 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with any of SEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.
• Embodiment 23. The method of embodiment 1, wherein the RNA-dependent DNA polymerase is a reverse transcriptase.
• Embodiment 24. The method of embodiment 23, wherein the reverse transcriptase is a naturally occurring wild type reverse transcriptase having an amino acid sequence of any one of SEQ ID NOs: 89 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with any of SEQ ID NOs: 89.
• Embodiment 25. The method of embodiment 23, wherein the reverse transcriptase is a variant reverse transcriptase having an amino acid sequence of any one of SEQ ID NOs: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, 662, 700, 701-716, 739-741, and 766 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with any of SEQ ID NOs: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, 662, 700, 701-716, 739-741, and 766.
• Embodiment 26. The method of embodiment 1, wherein the fusion protein comprises an amino acid sequence of any one of SEQ ID NOs: 123 and 134 (PE1, PE2), or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with any of SEQ ID NOs:123 and 134 (PE1, PE2).
• Embodiment 27. The method of embodiment 1, wherein the one or more constructs that encode the prime editor fusion protein and/or the PEgRNA further comprises one or more promoters which are inducible when cellular event occurs.
• Embodiment 28. The method of embodiment 1, wherein the cellular event is marked by a stimulus received by the cell.
• Embodiment 29. The method of embodiment 28, wherein the stimulus is a small molecule, a protein, a peptide, an amino acid, a metabolite, an inorganic molecule, an organometallic molecule, an organic molecule, a drug or drug candidate, a sugar, a lipid, a metal, a nucleic acid, a molecule produced during the activation of an endogenous or an exogenous signaling cascade, light, heat, sound, pressure, mechanical stress, shear stress, or a virus or other microorganism, change in pH, or change in oxidation/reduction state.
• Embodiment 30. A cell data recording plasmid for recording a cellular event using prime editing, comprising:
• i. a fusion protein comprising (a) a nucleic acid sequence encoding a nucleic acid programmable DNA binding protein (napDNAbp) and a (b) RNA-dependent DNA polymerase, said fusion protein being operably linked to a first promoter;
• ii. a nucleic acid sequence encoding a prime editor guide RNA (PEgRNA) operably linked to a second promoter, wherein the PEgRNA is complementary to a target sequence; and
• iii. an origin of replication;
• wherein at least one of the promoters is an inducible promoter, and wherein the PEgRNA associates with the napDNAbp under conditions that induce expression of the PEgRNA and expression of the napDNAbp sufficiently to install a detectable sequence at a target edit site.
• Embodiment 31. The cell data recording plasmid of embodiment 30, wherein the inducible promoter is induced by the cellular event.
• Embodiment 32. The cell data recording plasmid of embodiment 30, wherein the cellular event is marked by a stimulus received by the cell.
• Embodiment 33. The cell data recording plasmid of embodiment 32, wherein the stimulus is a small molecule, a protein, a peptide, an amino acid, a metabolite, an inorganic molecule, an organometallic molecule, an organic molecule, a drug or drug candidate, a sugar, a lipid, a metal, a nucleic acid, a molecule produced during the activation of an endogenous or an exogenous signaling cascade, light, heat, sound, pressure, mechanical stress, shear stress, or a virus or other microorganism, change in pH, or change in oxidation/reduction state.
• Embodiment 34. The cell data recording plasmid of embodiment 30, wherein the first and second promoter are the same.
• Embodiment 35. The cell data recording plasmid of embodiment 30, wherein the first and second promoter are different.
• Embodiment 36. The cell data recording plasmid of embodiment 30, wherein the at least one inducible promoter is an anhydrotetracycline-inducible promoter, IPTG-inducible promoter, rhamnose-inducible promoter, or arabinose-inducible promoter.
• Embodiment 37. The cell data recording plasmid of embodiment 30, wherein the first or second promoter is a constitutive promoter.
• Embodiment 38. The cell data recording plasmid of embodiment 37, wherein the constitutive promoter is a Lac promoter, cytomegalovirus (CMV) promoter, a constitutive RNA polymerase III promoter, or a UBC promoter.
• Embodiment 39. The cell data recording plasmid of embodiment 30, wherein the origin of replication comprises a pSC101, pMB1, pBR322, ColE1, or p15A origin of replication sequence.
• Embodiment 40. The cell data recording plasmid of embodiment 30, wherein the PEgRNA comprises an edit template that encodes the detectable sequence.
• Embodiment 41. The cell data recording plasmid of embodiment 40, wherein the edit template further encodes a new target edit site.
• Embodiment 42. The cell data recording plasmid of embodiment 30, wherein the detectable sequence is an insertion of at least 1, or at least 2, or at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or at least 8, or at least 9, or at least 10, or at least 11, or at least 12, or at least 13, or at least 14, or at least 15, or at least 16, or at least 17, or at least 18, or at least 19, or at least 20, or at least 21, or at least 22, or at least 23, or at least 24, or at least 25, or at least 26, or at least 27, or at least 28, or at least 29, or at least 30, or at least 31, or at least 32, or at least 33, or at least 34, or at least 35, or at least 40, or at least 50, or at least 60, or at least 70, or at least 80, or at least 90, or at least 100 nucleobases.
• Embodiment 43. The cell data recording plasmid of embodiment 30, wherein the detectable sequence is a deletion of at least 1, or at least 2, or at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or at least 8, or at least 9, or at least 10, or at least 11, or at least 12, or at least 13, or at least 14, or at least 15, or at least 16, or at least 17, or at least 18, or at least 19, or at least 20, or at least 21, or at least 22, or at least 23, or at least 24, or at least 25, or at least 26, or at least 27, or at least 28, or at least 29, or at least 30, or at least 31, or at least 32, or at least 33, or at least 34, or at least 35, or at least 40, or at least 50, or at least 60, or at least 70, or at least 80, or at least 90, or at least 100 nucleobases.
• Embodiment 44. The cell data recording plasmid of embodiment 30, wherein the detectable sequence is a nucleobase substitution.
• Embodiment 45. The cell data recording plasmid of embodiment 30, wherein the detectable sequence is a transition mutation.
• Embodiment 46. The cell data recording plasmid of embodiment 30, wherein the detectable sequence is a transversion mutation.
• Embodiment 47. The cell data recording plasmid of embodiment 30, wherein the detectable sequence is a single nucleotide substitution of wherein the single nucleotide substitution is (1) a G to T substitution, (2) a G to A substitution, (3) a G to C substitution, (4) a T to G substitution, (5) a T to A substitution, (6) a T to C substitution, (7) a C to G substitution, (8) a C to T substitution, (9) a C to A substitution, (10) an A to T substitution, (11) an A to G substitution, or (12) an A to C substitution.
• Embodiment 48. The cell data recording plasmid of embodiment 30, wherein the detectable sequence is a single nucleotide substitution that converts (1) a G:C basepair to a T:A basepair, (2) a G:C basepair to an A:T basepair, (3) a G:C basepair to C:G basepair, (4) a T:A basepair to a G:C basepair, (5) a T:A basepair to an A:T basepair, (6) a T:A basepair to a C:G basepair, (7) a C:G basepair to a G:C basepair, (8) a C:G basepair to a T:A basepair, (9) a C:G basepair to an A:T basepair, (10) an A:T basepair to a T:A basepair, (11) an A:T basepair to a G:C basepair, or (12) an A:T basepair to a C:G basepair.
• Embodiment 49. The cell data recording plasmid of embodiment 30, wherein the detectable sequence is a barcode sequence.
• Embodiment 50. The cell data recording plasmid of embodiment 30, wherein the detectable sequence increases in length over time as a result of iterative insertion of the detectable sequence for each occurrence of the cellular event.
• Embodiment 51. The cell data recording plasmid of embodiment 30, wherein the detecting step comprises sequencing the edited target site, or an amplicon of the edited target site.
• Embodiment 52. The cell data recording plasmid of embodiment 30, wherein the napDNAbp is Cas9, Cas12e, Cas12d, Cas12a, Cas12b1, Cas13a, Cas12c, or Argonaute, or a variant of Cas9, Cas12e, Cas12d, Cas12a, Cas12b1, Cas13a, Cas12c, or Argonaute.
• Embodiment 53. The cell data recording plasmid of embodiment 30, wherein the napDNAbp is a Cas9 or variant thereof.
• Embodiment 54. The cell data recording plasmid of embodiment 30, wherein the napDNAbp is a nuclease active Cas9, a nuclease inactive Cas9 (dCas9), or a Cas9 nickase (nCas9).
• Embodiment 55. The cell data recording plasmid of embodiment 30, wherein the napDNAbp is Cas9 nickase (nCas9).
• Embodiment 56. The cell data recording plasmid of embodiment 30, wherein the napDNAbp comprises the amino acid sequence of SEQ ID NO: 18, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO: 18.
• Embodiment 57. The cell data recording plasmid of embodiment 30, wherein the napDNAbp is SpCas9 wild type or a variant thereof of any one of amino acid sequences 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with any of SEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.
• Embodiment 58. The cell data recording plasmid of embodiment 30, wherein the napDNAbp is an SpCas9 ortholog.
• Embodiment 59. The cell data recording plasmid of embodiment 30, wherein the napDNAbp is any one of amino acid sequences 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with any of SEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.
• Embodiment 60. The cell data recording plasmid of embodiment 30, wherein the RNA-dependent DNA polymerase is a reverse transcriptase.
• Embodiment 61. The cell data recording plasmid of embodiment 30, wherein the reverse transcriptase is a naturally occurring wild type reverse transcriptase having an amino acid sequence of any one of SEQ ID NOs: 89 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with any of SEQ ID NOs: 89.
• Embodiment 62. The cell data recording plasmid of embodiment 30, wherein the reverse transcriptase is a variant reverse transcriptase having an amino acid sequence of any one of SEQ ID NOs: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, 662, 700, 701-716, 739-741, and 766 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with any of SEQ ID NOs: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, 662, 700, 701-716, 739-741, and 766.
• Embodiment 63. The cell data recording plasmid of embodiment 30, wherein the fusion protein comprises an amino acid sequence of any one of SEQ ID NOs: 123 and 134 (PE1, PE2), or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with any of SEQ ID NOs: 123 and 134 (PE1, PE2).
• Embodiment 64. A kit for use in a cell comprising the cell data recorder plasmid of any one of embodiments 30 63.
• Embodiment 65. The kit of embodiment 64, wherein the cell is a prokaryotic cell.
• Embodiment 66. The kit of embodiment 64, wherein the cell is a eukaryotic cell.
• Embodiment 67. A cell comprising the cell data recording plasmid of any one of embodiments 30-63.
• Embodiment 68. The cell of embodiment 67, wherein the cell is a prokaryotic cell.
• Embodiment 69. The cell of embodiment 67, wherein the cell is a eukaryotic cell.
• Embodiment 70. The cell of embodiment 69, wherein the eukaryotic cell is a mammalian cell.
• Embodiment 71. The cell of embodiment 70, wherein the mammalian cell is a human cell.
• Embodiment 72. A method of recording a cellular event by prime editing, the method comprising: (A) introducing into a cell a cell data recording plasmid of any of embodiments 30-63, wherein the fusion protein and/or the PEgRNA are induced by the occurrence of a cellular event, and wherein expression of the fusion protein and/or the PEgRNA results in prime editing of a target edit site in the genome of the cell to introduce a detectable sequence, and (B) identifying the detectable sequence, thereby identifying the occurrence of the cellular event.
• Embodiment 73. The method of embodiment 72, wherein the step of (A) introducing is by transfection or electroporation.
EQUIVALENTS AND SCOPE
• In the articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Embodiments or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.
• Furthermore, the disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claims that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements and/or features, certain embodiments of the disclosure or aspects of the disclosure consist, or consist essentially of, such elements and/or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein. It is also noted that the terms “comprising” and “containing” are intended to be open and permits the inclusion of additional elements or steps. Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.
• This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, the specification shall control. In addition, any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the embodiments. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the invention can be excluded from any embodiment, for any reason, whether or not related to the existence of prior art.
• Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended embodiments. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following embodiments.

Claims (9)

What is claimed is:

1. A method of installing a ribonucleotide motif or tag in an RNA of interest encoded by a target nucleotide sequence by prime editing, the method comprising: (a) contacting the target nucleotide sequence with a (i) prime editor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a polymerase, and (ii) a PEgRNA comprising an edit template encoding the ribonucleotide motif or tag; thereby polymerizing a single strand DNA sequence encoding the ribonucleotide motif or tag; and incorporating the single strand DNA sequence in place of a corresponding endogenous strand at the target nucleotide sequence through a DNA repair and/or replication process, wherein the method produces a target nucleotide sequence that encodes a modified RNA of interest comprising the ribonucleotide motif or tag.

2. The method of claim 1, wherein ribonucleotide motif or tag is a detection moiety.

3. The method of claim 1, wherein the ribonucleotide motif or tag affects the expression level of the RNA of interest.

4. The method of claim 1, wherein the ribonucleotide motif or tag affects the transport or subcellular location of the RNA of interest.

5. The method of claim 1, wherein the ribonucleotide motif or tag is selected from the group consisting of SV40 type 1, SV40 type 2, SV40 type 3, hGH, BGH, rbGlob, TK, MALAT1 ENE-mascRNA, KSHV PAN ENE, Smbox/U1 snRNA box, U1 snRNA 3′ box, tRNA-lysine, broccoli aptamer, spinach aptamer, mango aptamer, HDV ribozyme, and m6A.

6. The method of claim 1, wherein the PEgRNA comprises SEQ ID NOs: 101-104, 181-183, 223-244, 277, 325-334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 499-505, 735-761, 776-777 (see Table).

7. The method of claim 1, wherein the fusion protein comprises the amino acid sequence of PE1, PE2, or PE3.

8. The method of claim 1, wherein the napDNAbp is a Cas9 nickase (nCas9).

9. The method of claim 1, wherein the napDNAbp comprises the amino acid sequence of SEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.

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