JPWO2020191243A5 - - Google Patents

Description

GOVERNMENT SUPPORT This invention was made with Government support under Grants U01AI142756, RM1HG009490, R01EB022376, and R35GM118062 awarded by the National Institutes of Health. The Government has certain rights in this invention.

RELATED APPLICATIONS AND INCORPORATION BY REFERENCE This U.S. provisional application is a continuation of the following applications: U.S. Provisional Application No. 62/820,813, filed March 19, 2019 (Attorney Docket No. B1195.70074US00), U.S. Provisional Application No. 62/858,958, filed June 7, 2019 (Attorney Docket No. B1195.70074US01), U.S. Provisional Application No. 62/858,958, filed August 21, 2019 (Attorney Docket No. B1195.70074US02), No. 889,996 (Attorney Docket No. B1195.70074US02), U.S. Provisional Application No. 62/922,654 filed on August 21, 2019 (Attorney Docket No. B1195.70083US00), U.S. Provisional Application No. 62/913,553 filed on October 10, 2019 (Attorney Docket No. B1195.70074US03), U.S. Provisional Application No. 62/922,654 filed on August 21, 2019 (Attorney Docket No. B1195.70083US00), U.S. Provisional Application No. 62/913,553 filed on October 10, 2019 (Attorney Docket No. B1195.70074US04), U.S. Provisional Application No. 62/922,654 filed on August 21, 2019 (Attorney Docket No. B1195.70083US00), /973,558 (Attorney Docket No. B1195.70083US01), U.S. Provisional Application No. 62/931,195, filed November 5, 2019 (Attorney Docket No. B1195.70074US04), U.S. Provisional Application No. 62/944,231, filed December 5, 2019 (Attorney Docket No. B1195.70074US05), U.S. Provisional Application No. 62/ No. 974,537 (Attorney Docket No. B1195.70083US02), U.S. Provisional Application No. 62/991,069, filed March 17, 2020 (Attorney Docket No. B1195.70074US06), and U.S. Provisional Application No. 62/991,069, filed March 17, 2020 (serial number not available at the time of filing this application) (Attorney Docket No. B1195.70083US03), which are incorporated by reference.

Background of the Invention Pathogenic single-nucleotide mutations contribute, by some estimates, to approximately 50% of human diseases with a genetic component. ⁷ Unfortunately, treatment options for patients with these genetic disorders remain extremely limited, despite decades of gene therapy exploration. ⁸ Perhaps the simplest solution to this therapeutic challenge is the direct correction of single-nucleotide mutations in the patient genome, which may address the underlying cause of the disease and provide lasting benefits. While such a strategy was previously unthinkable, recent improvements in genome editing capabilities brought about by the advent of the CRISRP/Cas system ⁹ now put this therapeutic approach within reach. By simple design of guide RNA (gRNA) sequences containing ∼20 nucleotides complementary to the target DNA sequence, almost any envisioned 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 applications. ¹⁰ This natural diversity of Cas nucleases, together with a growing collection of engineered ^{variants11-14} , provides fertile ground for the development of new genome editing technologies.

Although gene disruption with CRISPR is now a mature technique, high-precision editing of single base pairs in the human genome ^remains a major challenge. 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 encoding the desired edits. ¹⁵ However, existing HDR has extremely low efficiency in most human cell types, particularly in non-dividing cells, and incompatible non-homologous end joining (NHEJ) leads primarily to insertion-deletion (indel) by-products. ¹⁶ Other issues concern the generation of DSBs, which can generate large chromosomal rearrangements and deletions at the target locus, ¹⁷ or activate the p53 axis leading to growth arrest and apoptosis. ^18,19

Several approaches have been explored to address these shortcomings of HDR. For example, repair of single-stranded DNA breaks (nicks) with oligonucleotide donors has been shown to reduce indel formation, but the yield of the desired repair product remains low. ²⁰ Other strategies attempt to bias repair toward HDR over NHEJ using small molecules and biological reagents. ^21-23 However, the efficacy of these methods may be cell type dependent, and perturbation of normal cellular conditions may lead to undesirable and unpredictable effects.

Recently, we, led by Professor David Liu, have developed base editing as a technique to edit targeted nucleotides without creating DSBs or relying on HDR ^{. 4 - 6, 24 - 27} Direct modification of DNA bases by Cas-fused deaminases allows highly efficient C·G → T·A or A·T → G·C base pair conversions in short target windows (~5-7 bases). As a result, base editors have been rapidly adopted by the scientific community. However, the following factors limit their generality for high-precision genome editing: (1) "bystander editing" of non-targeted C or A bases in the target window is observed; (2) a mixture of targeted nucleotide products is observed; (3) the targeted base must be located 15 ± 2 nucleotides upstream of the PAM sequence; and (5) repair of small insertion and deletion mutations is not possible.

Thus, the development of programmable editing elements that can flexibly introduce any desired single nucleotide change and/or incorporate base pair insertions or deletions (e.g., insertions or deletions of 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 alter or modify nucleotide sequences at target sites with high specificity and efficiency would substantially expand the scope and therapeutic potential of CRISPR-based genome editing technologies.

Overview 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 uses a nucleic acid programmable DNA binding protein ("napDNAbp") in cooperation with a polymerase (i.e., in the form of a fusion protein or otherwise provided in trans with napDNAbp) to write new genetic information directly into a specific DNA site, whereas the prime editing system is programmed with a prime editing (PE) guide RNA ("PEgRNA") that specifies both the target site and a template for the synthesis of the desired edit in the form of a replacement DNA strand via an extension (either DNA or RNA) that is engineered onto the guide RNA (e.g., at the 5' or 3' end of the guide RNA, or internally). The replacement strand, which contains 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 encompasses the desired edit). Through DNA repair and/or replication mechanisms, the endogenous strand of the target site is replaced by the newly synthesized replacement strand, which contains the desired edit. In some cases, prime editing may be considered a "search-and-replace" genome editing technique, where prime editing elements as described herein not only search for and locate the desired target site to be edited, but also simultaneously encode a replacement strand containing the desired edit that is incorporated in place of the corresponding target site in the 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 exploited or employed to perform high-precision CRISPR/Cas-based genome editing with high efficiency and genetic flexibility (e.g., as depicted in various embodiments in Figures 1A-1F). TPRT is naturally used by mobile DNA elements such as mammalian non-LTR retrotransposons and bacterial group II ^introns28,29 . Herein, we use Cas protein-reverse transcriptase fusions or related systems that target specific DNA sequences with a guide RNA, generate a single-stranded nick at the target site, and use the nicked DNA as a primer for reverse transcription of a modified reverse transcriptase template incorporated by the guide RNA. However, although the concept originates from prime editors that use reverse transcriptase as a component of the DNA polymerase, the prime editors described herein are not limited to reverse transcriptases and may encompass the use of any DNA polymerase in nature. Indeed, although the application may refer to a prime editor with a "reverse transcriptase" throughout, it is explained herein that reverse transcriptase is the only type of DNA polymerase that can function in prime editing. Thus, wherever "reverse transcriptase" is referred to herein, one skilled in the art will understand that any suitable DNA polymerase may be used in place of reverse transcriptase. Thus, in one aspect, a prime editor may comprise a Cas9 (or equivalent napDNAbp) that is programmed to target a DNA sequence by linking the DNA sequence to a specialized guide RNA (i.e., a PEgRNA) that contains 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 a stretch that codes for a replacement strand of DNA that contains the desired genetic alteration that is used to replace the 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 DNA exposing a 3' hydroxyl group. The exposed 3' hydroxyl group can then be used to prime DNA polymerization of an extension on the PEgRNA that encodes the edit directly into the target site. In various embodiments, the extension - which provides a template for polymerization of the displaced strand containing the edit - can be made from RNA or DNA. In the case of RNA extension, the polymerase of the primed editor can be an RNA-dependent DNA polymerase (such as reverse transcriptase). In the case of DNA extension, the polymerase of the primed editor can be a DNA-dependent DNA polymerase.

The newly synthesized strand (i.e., the replacement DNA strand containing the desired edit) formed by the primed editing factors disclosed herein will be homologous to (i.e., have the same sequence as) the target sequence of the genome, except that it contains the desired nucleotide change (e.g., a single nucleotide change, deletion, or insertion, or a combination thereof). The newly synthesized (or replacement) strand of DNA, sometimes also referred to as a single-stranded DNA flap, competes with the complementary homologous endogenous DNA strand for hybridization, thereby displacing the corresponding endogenous strand. In some embodiments, the system can be combined with the use of an error-prone reverse transcriptase (e.g., provided as a fusion protein with a Cas9 domain or provided in trans with a Cas9 domain). The error-prone reverse transcriptase can introduce modifications during the synthesis of the single-stranded DNA flap. Thus, in some embodiments, an error-prone reverse transcriptase can be utilized to introduce nucleotide changes into the target DNA. Depending on the error-prone reverse transcriptase used with the system, the changes can be random or non-random.

Degradation of the hybridized intermediate (including the single-stranded DNA flap synthesized by the reverse transcriptase hybridized to the endogenous DNA strand) may involve the resulting removal of the displaced flap from the endogenous DNA (e.g., with 5'-end DNA flap endonuclease, FEN1), ligation of the synthesized single-stranded DNA flap to the target DNA, and assimilation of the desired nucleotide change as a result of the cellular DNA repair and/or replication processes. Because templated DNA synthesis confers high precision of single nucleotides for any nucleotide modification, including insertions and deletions, the breadth of this approach is extremely broad and it is foreseeable that it could be used for a myriad of applications in basic science and therapy.

In one aspect, the present 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 performing genome editing by primed reverse transcription to a prime target in the presence of an extended guide RNA.

In some embodiments, the napDNAbp has nickase activity. The napDNAbp can also be a Cas9 protein or a functional equivalent thereof, such as a nuclease-active Cas9, a nuclease-inactive Cas9 (dCas9), or a Cas9 nickase (nCas9).

In some embodiments, the napDNAbp is selected from the group consisting of: Cas9, Cas12e, Cas12d, Cas12a, Cas12b1, Cas13a, Cas12c, and Argonaute, and optionally has nickase activity.

In other embodiments, the fusion protein is capable of binding to a target DNA sequence when complexed with an extended guide RNA.

In yet other embodiments, the target DNA sequence includes a target strand and a complementary non-target strand.

In other embodiments, binding of the complexed fusion protein to the extended guide RNA forms an R-loop. The R-loop may comprise (i) an RNA-DNA hybrid comprising the extended guide RNA and a target strand, and (ii) a complementary non-target strand.

In yet other embodiments, the complementary non-target strand is nicked to form a reverse transcriptase prime sequence with 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 3' end of the guide RNA or at an intramolecular location of the guide RNA. The RNA extension may comprise (i) a reverse transcription template sequence containing the 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-stranded DNA flap that is complementary to an endogenous DNA sequence adjacent to the nick site, the single-stranded DNA flap containing 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-stranded DNA flap hybridizes to the endogenous DNA sequence adjacent to the nick site, thereby incorporating the desired nucleotide change. In still other embodiments, the single-stranded DNA flap has a free 5' end and replaces the endogenous DNA sequence adjacent to the nick site. In some embodiments, the replaced endogenous DNA with the 5' end is excised by the cell.

In various embodiments, cellular repair of the single-stranded DNA flap results in incorporation of the desired nucleotide change, thereby forming the desired product.

In various other embodiments, the desired nucleotide change is incorporated into an editing window between about -4 and +10 of the PAM sequence.

In still other embodiments, the desired nucleotide change is incorporated over an editing window that is between about -5 and +5 at the nick site, or between about -10 and +10 at the nick site, or between about -20 and +20 at the nick site, or between about -30 and +30 at the nick site, or between about -40 and +40 at the nick site, or between about -50 and +50 at the nick site, or between about -60 and +60 at the nick site, or between about -70 and +70 at the nick site, or between about -80 and +80 at the nick site, or between about -90 and +90 at the nick site, or between about -100 and +100 at the nick site, or between about -200 and +200 at the nick site.

In various embodiments, the napDNAbp comprises the 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 NOs: 77-86 (CP-Cas9); SEQ ID NOs: 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 can include 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 yet other embodiments, the reverse transcriptase can include an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to 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. These sequences can be naturally occurring reverse transcriptase sequences, for example from retroviruses or retrotransposons, or the sequences can be recombinant.

In various other embodiments, the fusion proteins disclosed herein may comprise various structural configurations. For example, the fusion protein may comprise the structure _NH2- [napDNAbp]-[reverse transcriptase]-COOH; or _NH2- [reverse transcriptase]-[napDNAbp]-COOH, each of which 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 is at least 80%, 85%, or 90%, or 95%, or 99% identical to any one of the linker amino acid sequences of SEQ ID NOs: 127, 165-176, 446, 453, and 767-769.

In various embodiments, the desired nucleotide change 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 disclosure provides an extended guide RNA comprising a guide RNA and at least one RNA extension. The RNA extension may be located at the 3' end of the guide RNA. In other embodiments, the RNA extension may be located at the 5' end of the guide RNA. In yet other embodiments, the RNA extension may be located at an intramolecular location on the guide RNA. However, preferably, the intramolecular positioning of the extended portion does not block the function of the protospacer.

In various embodiments, a prime editor guide RNA (PEgRNA) is capable of binding to the napDNAbp and directing the napDNAbp to a target DNA sequence. The target DNA sequence may include a target strand and a complementary non-target strand, where the guide RNA hybridizes to the target strand to form an RNA-DNA hybrid and an R-loop.

In various embodiments of the prime editor guide RNA, at least one RNA extension comprises a DNA synthesis template. In various other embodiments, the RNA extension further comprises a reverse transcription primer binding site. In still other embodiments, the RNA extension comprises a linker or spacer that links 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 DNA synthesis template (i.e., with respect to FIG. 27, the editing 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, 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, the reverse transcription primer binding site sequence (i.e., with respect to FIG. 27, 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, 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, any linker or spacer 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 RNA, the reverse transcription template sequence can encode a single-stranded DNA flap that is complementary to the endogenous DNA sequence adjacent to the nick site, the single-stranded DNA flap containing the desired nucleotide change. The single-stranded DNA flap can displace the endogenous single-stranded DNA at the nick site. The displaced endogenous single-stranded DNA at the nick site can have a 5' end and form an endogenous flap that can be excised by the cell. In various embodiments, excision of the 5' end endogenous flap can drive product formation because removal of the 5' end endogenous flap promotes hybridization of the single-stranded 3' DNA flap to the corresponding complementary DNA strand and incorporation or assimilation of the desired nucleotide change carried by the single-stranded 3' DNA flap into the target DNA.

In various embodiments of the extended guide RNA, cellular repair of the single-stranded DNA flap results in incorporation of the desired nucleotide change, thereby forming the desired product.

In some embodiments, the PEG RNA is selected from the group consisting of SEQ ID NOs : 101-104, 131, 181-183, 222-234, 237-244, 277, 324-330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 394, 429-442, 499-505, 641-649, 678-692, 735-73 6, 738, 757-761, 776-777, 2997-3103, 3113-3121, 3305-3455, 3479-3493, 3522-3540, 3549-3556, 3628-3698, 3755-3810, 3874, 3890-3901, 3905-3911, 3913-3929, and 3972-3989, or the nucleotide sequences of SEQ ID NOs : 101-104, 181-183, 223-23 4, 237-244, 277, 324-330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 394, 429-442, 499-505, 641-649, 678-692, 735-736, 757-761, 776-777, 2997-3103, 3113-3121, 3305-3455 , 3479-3493, 3522-3540, 3549-3556, 3628-3698, 3755-3810, 3874, 3890-3901, 3905-3911, 3913-3929, and 3972-3989 .

In another aspect of the present invention, the present invention provides a complex comprising a fusion protein described herein and any of the extended guide RNAs described above.

In yet another aspect of the present invention, the present disclosure provides a complex comprising napDNAbp and an extended guide RNA. napDNAbp can be a Cas9 nickase or can be an amino acid sequence 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 the complex, the extended guide RNA can direct the napDNAbp to the target DNA sequence. In various embodiments, the reverse transcriptase can be provided in trans, i.e., from a source different from the complex itself. For example, the reverse transcriptase can be provided to the same cell that has the complex by introducing a separate vector that individually encodes the reverse transcriptase.

In yet another aspect, the present specification provides a polynucleotide. In certain embodiments, the polynucleotide may encode any of the fusion proteins disclosed herein. In certain other embodiments, the polynucleotide may encode any of the napDNAbps disclosed herein. In still further embodiments, the polynucleotide may encode any of the reverse transcriptases disclosed herein. In still other embodiments, the polynucleotide may encode any of the extended guide RNAs, any of the reverse transcription template sequences, any of the reverse transcription primer sites, or any of the optional linker sequences disclosed herein.

In yet another aspect, the present specification provides a vector comprising a polynucleotide described herein. Thus, in some embodiments, the vector comprises a polynucleotide encoding a fusion protein comprising napDNAbp and reverse transcriptase. In other embodiments, the vector comprises polynucleotides separately encoding napDNAbp and reverse transcriptase. In still other embodiments, the vector may comprise a polynucleotide encoding an extended guide RNA. In various embodiments, the vector may comprise one or more polynucleotides encoding napDNAbp, reverse transcriptase, and extended guide RNA on the same or separate vectors.

In yet another aspect, the present specification provides a cell comprising a fusion protein and an extended guide RNA described herein. The cell can be transformed with a vector comprising the fusion protein, napDNAbp, reverse transcriptase, and the extended guide RNA. These genetic elements can be included on the same vector or on different vectors.

In another aspect, the present disclosure provides pharmaceutical compositions. In some embodiments, the pharmaceutical composition comprises one or more of napDNAbp, a fusion protein, a reverse transcriptase, and an extended guide RNA. In some embodiments, the fusion protein described herein and a pharma- ceutically acceptable excipient. In other embodiments, the pharmaceutical composition comprises any extended guide RNA described herein and a pharma- ceutically acceptable excipient. In yet other embodiments, the pharmaceutical composition comprises any extended guide RNA described herein in combination with any fusion protein described herein and a pharma- ceutically acceptable excipient. In another other embodiment, the pharmaceutical composition comprises any polynucleotide sequence encoding one or more of napDNAbp, a fusion protein, a reverse transcriptase, and an extended guide RNA. In yet 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 napDNAbp, a second pharmaceutical composition may comprise a reverse transcriptase, and a third pharmaceutical composition may comprise an extended guide RNA.

In yet a further aspect, the disclosure provides kits. In one embodiment, the kit includes one or more polynucleotides encoding one or more components including a fusion protein, napDNAbp, reverse transcriptase, and an extended guide RNA. The kit may also include isolated preparations of vectors, cells, and polypeptides including any of the fusion proteins, napDNAbp, or reverse transcriptases disclosed herein.

In another aspect, the disclosure provides methods of using the disclosed compositions of matter.

In one embodiment, the method relates to a method for incorporating a desired nucleotide change into a double-stranded DNA sequence. The method first involves contacting the double-stranded DNA sequence with a complex comprising a fusion protein and an extended guide RNA, the fusion protein comprising napDNAbp and a reverse transcriptase, and the extended guide RNA comprising a reverse transcription template sequence comprising the desired nucleotide change. The method then involves nicking the double-stranded DNA sequence on the non-target strand, thereby generating a free single-stranded DNA having a 3' end. The method then involves hybridizing the 3' end of the free single-stranded 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-stranded DNA flap comprising the desired nucleotide change. The method then involves replacing the endogenous DNA strand adjacent to the cut site with the single-stranded DNA flap, thereby incorporating the desired nucleotide change into the double-stranded DNA sequence.

In another aspect, the disclosure provides a method for introducing one or more changes in a 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 that targets the napDNAbp to the target locus, where the guide RNA comprises a reverse transcriptase (RT) template sequence that comprises at least one desired nucleotide change. The method then involves forming an exposed 3' end in the DNA strand at the target locus, and then priming reverse transcription by hybridizing the exposed 3' end with the RT template sequence. A single-stranded DNA flap that comprises at least one desired nucleotide change based on the RT template sequence is then synthesized or polymerized by reverse transcriptase. Finally, 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 yet another aspect, the disclosure provides a method for introducing one or more changes in a nucleotide sequence of a DNA molecule at a target locus by target-primed reverse transcription, the method comprising: (a) contacting a DNA molecule at the target locus with i) a 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) allowing target-primed reverse transcription of the RT template to generate a single-stranded DNA comprising the desired nucleotide change; and (c) incorporating the desired nucleotide change into the DNA molecule at the target locus through DNA repair and/or replication processes.

In one embodiment, the step of replacing the endogenous DNA strand includes: (i) hybridizing a single-stranded DNA flap to the endogenous DNA strand adjacent to the cleavage site, thereby creating a sequence mismatch; (ii) excising the endogenous DNA strand; and (iii) repairing the mismatch to form a desired product containing the desired nucleotide change in both strands of DNA.

In various embodiments, the desired nucleotide change can be a single nucleotide substitution (e.g., a transition or 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 nucleotide change may be: (1) a G:C base pair to a T:A base pair; (2) a G:C base pair to an A:T base pair; (3) a G:C base pair to a C:G base pair; (4) a T:A base pair to a G:C base pair; (5) a T:A base pair to an A:T base pair; (6) a T:A base pair to a C:G base pair; (7) a C:G base pair to a G:C base pair; (8) a C:G base pair to a T:A base pair; (9) a C:G base pair to an A:T base pair; (10) an A:T base pair to a T:A base pair; (11) an A:T base pair to a G:C base pair; or (12) an A:T base pair to a C:G base pair.

In yet 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 modifies a disease-associated gene. The disease-associated gene may be associated with a monogenic 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; phenylketonuria; severe combined immunodeficiency; sickle cell disease; Smith-Lemli-Opitz syndrome; and Tay-Sachs disease. In other embodiments, the disease-associated gene may 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 a fusion protein having a napDNAbp that is a nuclease-dead Cas9 (dCas9), a Cas9 nickase (nCas9), or a nuclease-active Cas9. In other embodiments, the napDNAbp and reverse transcriptase are not encoded as a single fusion protein, but rather may be provided in separate constructs. Thus, in some embodiments, the reverse transcriptase may be provided in trans to the napDNAbp (rather than as a fusion protein).

In various embodiments involving the methods, the napDNAbp may include the amino acid sequences 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 NOs: 77-86 (CP-Cas9); SEQ ID NOs: 18-25 and 87-88 (SpCas9); and SEQ ID NOs: 62-72 (Cas12). The napDNAbp may also contain an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to any one of the amino acid sequences 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 NOs: 77-86 (CP-Cas9); SEQ ID NOs: 18-25 and 87-88 (SpCas9); and SEQ ID NOs: 62-72 (Cas12).

In various embodiments involving the 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 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 method includes the steps of SEQ ID NOs: 101-104, 131, 181-183, 222-234, 237-244, 277, 324-330 , 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 394, 429-442, 499-505 , 641-649 , 678-692 , 735-736, 738, 757-761, 776-777 , 2997-3103 , 3113-3121, 3305-3455 , 3479- The method may involve the use of a PEgRNA comprising the nucleotide sequence of 3493 , 3522 to 3540 , 3549 to 3556, 3628 to 3698, 3755 to 3810, 3874, 3890 to 3901, 3905 to 3911, 3913 to 3929 , and 3972 to 3989 , 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 method may involve the use of an extended guide RNA comprising an RNA extension at its 3' end, the RNA extension comprising a reverse transcriptase template sequence.

The method may include the use of an extended guide RNA that includes an RNA extension at its 5' end, the RNA extension including a reverse transcription template sequence.

The method may include the use of an extended guide RNA that includes an RNA extension at the intramolecular positioning of the guide RNA, the RNA extension including a reverse transcription template sequence.

The method may include the use of an extended guide RNA 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 understood that the above concepts, and additional concepts discussed below, may be arranged in any suitable combination (although the disclosure is not limited in this respect). Additionally, 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 specification and are included to further demonstrate certain aspects of the disclosure that may 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 single nucleotide changes and/or insertions and/or deletions into a DNA molecule (e.g., a genome) using a fusion protein comprising a reverse transcriptase fused to a Cas9 protein complexed 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 and complexed with a guide RNA (gRNA) binds to a 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 synthesis of a new DNA strand encoding the desired edit. The editing process shown may be referred to as target-primed reverse transcription editing (TRT editing), or "primed editing."

FIG. 1B provides the same diagram as FIG. 1A, except that the prime editor complex is more commonly represented as [napDNAbp]-[P]:PEgRNA or [P]-[napDNAbp]:PEgRNA. In the formula, "P" refers to any polymerase (e.g., reverse transcriptase), "napDNAbp" refers to a nucleic acid programmable DNA binding protein (e.g., SpCas9), "PEgRNA" refers to the prime editing guide RNA, and "]-[" refers to an optional linker. As described elsewhere, e.g., in FIG. 3A-3G, the PEgRNA includes a 5' extension arm that includes 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 includes the primer binding site and the DNA synthesis template) can be DNA or RNA. The specific polymerase contemplated in this configuration will depend on the nature of the DNA synthesis template. For example, if the DNA synthesis template is RNA, the polymerase can be an RNA-dependent DNA polymerase (e.g., reverse transcriptase). When the DNA synthesis template is DNA, the polymerase can be a DNA-dependent DNA polymerase.

FIG. 1C provides a schematic of an exemplary process for introducing single nucleotide changes and/or insertions and/or deletions onto a DNA molecule (e.g., 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 encompass a reverse transcriptase template sequence. The schematic shows how a reverse transcriptase (RT) fused to a Cas9 nickase, in complex with a guide RNA (gRNA), binds to a 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 synthesis of a new DNA strand encoding the desired edit. The editing process shown can be referred to as primed target-primed reverse transcription editing (TRT editing) or equivalently "primed editing".

FIG. 1D provides the same depiction as FIG. 1C, except that the prime editor complex is more generally represented as [napDNAbp]-[P]:PEgRNA or [P]-[napDNAbp]:PEgRNAPEgRNA, where "P" refers to any polymerase (e.g., reverse transcriptase), "napDNAbp" refers to a nucleic acid programmable DNA binding protein (e.g., SpCas9), and "PEgRNA" refers to the prime editing guide RNA, and "]-[" refers to the optional linker. As depicted elsewhere, e.g., in FIGS. 3A-3G, the PEgRNA includes a 3' extension arm that includes a primer binding site and a DNA synthesis template. Although not shown, it is contemplated that the extension arm of the PEgRNA (i.e., including the primer binding site and the DNA synthesis template) can be DNA or RNA. The specific polymerase contemplated in this configuration will depend on the nature of the DNA synthesis template. For example, if the DNA synthesis template is RNA, the polymerase can be an RNA-dependent DNA polymerase (e.g., reverse transcriptase). If the DNA synthesis template is DNA, the polymerase can be a DNA-dependent DNA polymerase. In various embodiments, the PEgRNA can be modified or synthesized to incorporate a DNA-based DNA synthesis template.

IE is a schematic diagram depicting an exemplary process of how a synthesized single strand of DNA (containing a desired nucleotide change) is degraded such that the desired nucleotide change is incorporated into the DNA. As shown, subsequent synthesis of an edited strand (or "mutagenized strand"), equilibration with the endogenous strand, flap cleavage of the endogenous strand, and ligation leads to the incorporation of the DNA edit following degradation of the mismatched DNA duplex through the action of endogenous DNA repair and/or replication processes.

Figure IF is a schematic diagram showing that "opposite strand nicking" can be incorporated into the degradation method of Figure IE to help drive the formation of the desired product versus the restored product. In opposite strand nicking, a second Cas9/gRNA complex is used to introduce a second nick on the strand opposite the first nicked strand. This induces the endogenous cell's 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 diagram of an exemplary process for introducing single nucleotide changes and/or insertions and/or deletions into a DNA molecule (e.g., a genome) at a target locus using a nucleic acid programmable DNA binding protein (napDNAbp) complexed with an extended guide RNA. This process is sometimes referred to as an embodiment of prime editing. The extended guide RNA includes an extension at the 3' or 5' end of the guide RNA, or at some 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 is introduced (e.g., by a nuclease or chemical agent) into one of the strands of the DNA (the R-loop strand, or the PAM-containing strand, or the non-target DNA strand, or the protospacer strand) of the target locus, thereby creating an available 3' end on one of the strands of the target locus. In one embodiment, the nick is created in the strand of DNA corresponding to the R-loop strand, i.e., the strand that is not hybridized with the guide RNA sequence. In step (c), the 3'-end DNA strand interacts with the extended portion of the guide RNA to prime reverse transcription. In some embodiments, the 3'-end DNA strand hybridizes with a specific RT prime sequence on the extended portion of the guide RNA. In step (d), a reverse transcriptase is introduced to synthesize a single strand of DNA from the 3'-end of the primed site to the 3'-end of the guide RNA. This forms a single-stranded DNA flap containing the desired nucleotide change (e.g., a single base change, an insertion, a deletion, or a combination thereof). In step (e), the napDNAbp and the guide RNA are released. Steps (f) and (g) involve the degradation of the single-stranded DNA flap so that the desired nucleotide change is incorporated into the target locus. This process can drive the desired product formation by removing the corresponding 5' endogenous DNA flap that is formed once the 3' single-stranded DNA flap invades and hybridizes with a complementary sequence on the other strand. The process can also drive the formation of a product nicked to the second strand, as illustrated in FIG. 1F. This process may introduce at least one or more of the following genetic alterations: transversions, transitions, deletions, and insertions.

Figure 1H is a schematic diagram depicting the types of genetic changes that can occur in the prime editing process described herein. The types of nucleotide changes that can be achieved by prime editing include deletions (including short and long deletions), single nucleotide changes (including transitions and transversions), and insertions (including short and long).

FIG. 1I is a schematic depicting temporal second strand nicking, exemplified by PE3b (PE3b=PE2 prime editor fusion protein+PEgRNA+guide RNA that nicks the second strand). Temporal second strand nicking is a variant of second strand nicking to facilitate the formation of the desired edited product. The term "temporary" refers to the fact that the second strand nicking to the unedited strand occurs only after the desired edit has been incorporated into the edited strand. This avoids simultaneous nicking on both strands leading to double-stranded DNA breaks.

1J -1K depict variations of prime editing contemplated herein in which napDNAbp (e.g., SpCas9 nickase) is replaced with any programmed nuclease domain, such as a zinc finger nuclease (ZFN) or transcription activator-like effector nuclease (TALEN). Thus, it is contemplated that a suitable nuclease does not necessarily have to be "programmed" by a nucleic acid target molecule (e.g., a guide RNA), but rather may be programmed by defining the specificity of the DNA binding domain, such as the nuclease, among others. Just as with prime editing with napDNAbp moieties, alternative programmed nucleases are preferably modified to cleave only one strand of the target DNA. In other words, the programmed nuclease should preferably function as a nickase. Once a programmed nuclease is selected (e.g., a ZFN or TALEN), additional functionality may be engineered to result in a system that can operate according to a prime editing-like mechanism. For example, the programmable nuclease may be modified by coupling it to an RNA or DNA extension arm (e.g., via a chemical linker), where the extension arm includes a primer binding site (PBS) and a DNA synthesis template. The programmable nuclease may also be coupled to a polymerase (e.g., via a chemical or amino acid linker), but the nature of the polymerase will depend on whether the extension arm is DNA or RNA. In the case of an RNA extension arm, the polymerase may be an RNA-dependent DNA polymerase (e.g., reverse transcriptase). In the case of a DNA extension arm, the polymerase may 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 functions added as fusions with programmable nucleases or in trans to facilitate the overall reaction (e.g., (a) a helicase that unwinds the DNA at the cut site, creating a cut strand with a 3' end that can be used as a primer; (b) a flap endonuclease (e.g., FEN1) that removes the endogenous strand on the cut strand, helping to drive the reaction towards replacing the endogenous strand with the synthesized strand; or (c) an nCas9:gRNA complex that creates a second site nick on the opposite strand, which may also help drive incorporation of the synthetic repair over the preferred cellular repair of the non-edited strand). In a similar manner to priming editing with napDNAbp, such complexes with other programmable nucleases can be used to synthesize and then permanently incorporate a newly synthesized replacement strand of DNA bearing the edit of interest into a target site of DNA.

FIG . 1L depicts anatomical features of a target DNA that may be edited by prime editing in one embodiment. The target DNA includes a "non-target strand" and a "target strand." The target strand is the strand that becomes annealed with the spacer of the PEgRNA of the prime editor complex that recognizes the PAM site (in this case, NGG, which is recognized by standard SpCas9-based prime editors). The target strand may also be referred to as the "non-PAM strand" or "non-edited strand." In contrast, the non-target strand (i.e., the strand containing the protospacer and the PAM sequence of NGG) may also be referred to as the "PAM strand" or "edited strand." In various embodiments, the nick site of the PE complex (e.g., in SpCas9-based PE) will be in the protospacer on the PAM strand. The location of the nick will be a feature of the specific Cas9 forming the PE. For example, with SpCas9-based PE, the nick site is in the phosphodiester bond between bases 3 (position "-3" relative to position 1 of the PAM sequence) and 4 (position "-4" relative to position 1 of the PAM sequence). The nick site in the protospacer forms a free 3' hydroxyl group that complexes with the primer binding site of the extension arm of the PEgRNA as seen in the diagram below, providing a substrate to initiate polymerization of a single strand of DNA encoding 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'→3' direction. Polymerization terminates (e.g., by inclusion of a polymerization termination signal or secondary structure that functions to terminate the polymerization activity of PE) before reaching the gRNA core, producing a single-stranded DNA flap that is extended from the original 3' hydroxyl group of the nicked PAM strand. The DNA synthesis template encodes a single strand of DNA that is homologous to the 5'-terminal single strand of the endogenous DNA immediately following the nick site on the PAM strand and incorporates the desired nucleotide change (e.g., single base substitution, insertion, deletion, inversion). The desired edit position can be at any position following downstream of the nick site on the PAM strand, and can be at positions +1, +2, +3, +4 (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, + 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, + The 3'-terminal single-stranded DNA (containing the edit of interest) may include 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'-terminal single-stranded DNA (containing the edit of interest) replaces the endogenous 5'-terminal single-stranded DNA, DNA repair and replication processes will result in the permanent incorporation of the edit site on the PAM strand and then correction of the mismatch on the non-PAM strand present at the edit site. Thus, the edit will spread to both strands of DNA on the target DNA site. It will be understood that references to the "edited strand" and the "non-edited" strand are intended merely 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' single stranded DNA immediately downstream of the nick site with a synthesized 3' single stranded DNA containing the desired edit. The "non-edited" strand is the counterpart strand to the edited strand, but it also becomes edited (particularly the edit of interest) through repair and/or replication to become complementary to the edited strand.

FIG . 1M depicts the mechanism of prime editing showing the anatomical features of the target DNA, the 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., SpCas9 with an inactivating mutation in the HNH nuclease domain (e.g., H840A) or an activating mutation in the RuvC nuclease domain (D10A)) is complexed with a PEgRNA and a DNA comprising the target DNA to be edited. The PEgRNA comprises a spacer, a gRNA core (also known as a gRNA backbone 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 anywhere within the PEgRNA molecule. As shown, the extension arm is at the 3' end of the PEgRNA. The extension arm contains a primer binding site and a DNA synthesis template (containing both the edit of interest and a homologous region (i.e., the homology arm)) that is homologous in the 3'→5' direction to the single-stranded DNA at the 5' end immediately following the nick site on the PAM strand. As shown, once a nick is introduced, thereby generating 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 primed editor complex. A polymerase (e.g., reverse transcriptase) then polymerizes a strand of DNA from the 3' hydroxyl end to the end of the extension arm. The sequence of the single-stranded DNA is encoded by the DNA synthesis template, which is the portion of the extension arm that is "read" by the polymerase to synthesize new DNA (i.e., excluding the primer binding site). This polymerization effectively extends to the original 3' hydroxyl-terminal sequence at the site of the initial nick. The DNA synthesis template encodes a single strand of DNA that contains not only the desired edit, but also a region of homology to the single strand of endogenous DNA immediately downstream of the nick site on the PAM strand. The encoded 3' single strand of DNA (i.e., the 3' single-stranded DNA flap) then replaces the corresponding homologous endogenous 5' single strand of DNA immediately downstream of the nick site on the PAM strand, forming a DNA intermediate with a 5' single-stranded DNA flap that is removed by the cell (e.g., by a flap endonuclease). The 3' single-stranded DNA flap that anneals to the complement of the endogenous 5' single-stranded DNA flap is ligated to the endogenous strand after the 5' DNA flap is removed. The desired edit in the 3'-terminal single-stranded DNA flap, which has just been annealed and ligated, forms a mismatch with the complementary strand and undergoes DNA repair and/or rounds of replication, thereby permanently incorporating the desired edit on both strands.

Figure 2 shows three Cas complexes (SpCas9, SaCas9, and LbCas12a) that can be used with the prime editors described herein, along with their PAM, gRNA, and DNA cleavage features. The diagram shows the design of the complexes involving SpCas9, SaCas9, and LbCas12a.

Figures 3A-3F show the design of engineered 5' prime editor gRNA (Figure 3A), 3' prime editor gRNA (Figure 3B), and intramolecular extension (Figure 3C). The extended guide RNA (or extended gRNA) may also be referred to herein as PEgRNA or "prime editing guide RNA". Figures 3D and 3E provide additional embodiments of 3' and 5' prime editor gRNA (PEgRNA), respectively. Figure 3F illustrates the interaction between the 3' prime editor guide RNA with the target DNA sequence. The embodiments of Figures 3A-3C illustrate example placements of reverse transcription template sequences (i.e., or more broadly referred to as DNA synthesis templates as indicated, since RT is the only type of polymerase that can be used in the context of a prime editor), primer binding sites, and optional linker sequences, as well as the general placement of spacers and core regions in the extended portions of the 3', 5', and intramolecular versions. The disclosed prime editing process is not limited to these configurations of the extended guide RNA. The embodiment of FIG. 3D provides an exemplary PEgRNA structure contemplated in the present application. The PEgRNA includes three main component components ordered in the 5' to 3' direction: a spacer, a gRNA core, and an extension arm at the 3' end. The extension arm can be further divided into the following structural elements in the 5' to 3' direction: a primer binding site (A), an editing template (B), and a homology arm (C). In addition, the PEgRNA can include an optional 3' end modification region (e1) and an optional 5' end modification region (e2). Furthermore, the PEgRNA can include a transcription termination signal at the 3' end of the PEgRNA (not shown). These structural elements are further defined in the present application. The illustration of the structure of the PEgRNA is not meant to be limiting and encompasses variations in the placement of the elements. For example, the optional sequence modifications (e1) and (e2) can be located within or between any of the other regions shown. It is not limited to being located at the 3' and 5' ends. In some embodiments, the PEgRNA may include secondary RNA structures such as, but not limited to, hairpins, stem loops, toe loops, RNA-binding protein recruitment domains (e.g., MS2 aptamers that recruit and bind MS2cp proteins). For example, such secondary structures may be located within the spacer, gRNA core, or extension arms, particularly within the e1 and/or e2 modified regions. In addition to the secondary RNA structures, the PEgRNA may include (e.g., within the e1 and/or e2 modified regions) a chemical linker or poly(N) linker or tail, where "N" can be any nucleobase. In some embodiments (e.g., as shown in Figure 72(c)), the chemical linker may function to prevent reverse transcription of the sgRNA backbone or core. Additionally, in certain embodiments (see, e.g., Figure 72(c)), the extension arm (3) may be composed of RNA or DNA and/or may include one or more nucleobase analogs (e.g., which may add functionality such as temperature resilience). It is further noted that the orientation of the extension arm (3) may be in the natural 5' to 3' direction or may be synthesized in the opposite direction, 3' to 5' (relative to the orientation of the overall PEgRNA molecule). It is also noted that the skilled artisan will be able to select the appropriate DNA polymerase for use in prime editing depending on the nature of the nucleic acid material of the extension arm (i.e. DNA or RNA). This can be implemented either as a fusion with napDNAbp or provided in trans as a separate part to synthesize a 3' single-stranded DNA flap encoded by the desired template that encompasses the desired edit. For example, if the extension arm is RNA, the DNA polymerase can be a reverse transcriptase or any other suitable RNA-dependent DNA polymerase. However, if the extension arm is DNA, the DNA polymerase can be a DNA-dependent DNA polymerase. In various embodiments, provision of the DNA polymerase can be in trans, such as by use of an RNA-protein recruitment domain (e.g., an MS2 hairpin incorporated onto 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 generally does not form part of the template used by the DNA polymerase (e.g., reverse transcriptase) to encode the resulting 3' single-stranded DNA flap that contains the desired edit. Thus, the designation "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-stranded DNA flap that contains the edit and a region of homology to the 5' endogenous single-stranded DNA flap that is replaced by the 3' single-stranded DNA product of the primed edited DNA synthesis. In some embodiments, the DNA synthesis template includes an "editing template" and a "homologous arm" or one or more homologous arms, for example, before and after the editing template. The editing template can be as small as a single nucleotide substitution, or it can be an insertion or inversion of DNA. In addition, the editing template can also include deletions, which can be engineered by encoding a homology arm containing the desired deletion. In other embodiments, the DNA synthesis template can also include the e2 region or a portion thereof. For example, if the e2 region contains a secondary structure that causes termination of DNA polymerase activity, it is possible that DNA polymerase function will terminate before any part of the e2 region is actually coded on the DNA. It is also possible that some or even all of the e2 region will be coded on the DNA. How much of e2 is actually used as a template will depend on its configuration and whether it disrupts DNA polymerase function.

The embodiment of FIG. 3E provides another PEgRNA structure contemplated in the present application. The PEgRNA comprises three main component elements ordered in the 5' to 3' direction: a spacer, a gRNA core, and an extension arm at the 3' end. The extension arm can be further divided into the following structural elements in the 5' to 3' direction: a primer binding site (A), an editing template (B), and a homology arm (C). In addition, the PEgRNA can comprise an optional 3' end modification region (e1) and an optional 5' end modification region (e2). Furthermore, the PEgRNA can comprise a transcription termination signal at the 3' end of the PEgRNA (not shown). These structural elements are further defined in the present application. The illustration of the structure of the PEgRNA is not meant to be limiting and encompasses variations in the placement of the elements. For example, the optional sequence modifications (e1) and (e2) can be located within or between any of the other regions shown. It is not limited to being located at the 3' and 5' ends. In some embodiments, the PEgRNA may include 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 the MS2cp protein). These secondary structures may be located anywhere on the PEgRNA molecule. For example, such secondary structures may be located within the spacer, gRNA core, or extension arms, particularly within the e1 and/or e2 modified regions. In addition to the secondary RNA structures, the PEgRNA may include a chemical linker or poly(N) linker or tail (e.g., within the e1 and/or e2 modified regions), where "N" can be any nucleobase. In some embodiments (e.g., as shown in Figure 72(c)), the chemical linker may function to prevent reverse transcription of the sgRNA backbone or core. Additionally, in certain embodiments (see, e.g., Figure 72(c)), the extension arm (3) may be comprised of RNA or DNA, and/or may include one or more nucleobase analogs (e.g., which may add functionality such as temperature resilience). It is further noted that the orientation of the extension arm (3) may be in the natural 5' to 3' direction, or may be synthesized in the opposite direction, 3' to 5' (relative to the orientation of the overall PEgRNA molecule). It is also noted that the skilled artisan will be able to select the appropriate DNA polymerase for use in prime editing, depending on the nature of the nucleic acid material of the extension arm (i.e. DNA or RNA). This can be implemented either as a fusion with napDNAbp, or provided in trans as a separate part, to synthesize a 3' single-stranded DNA flap encoded by the desired template that encompasses the desired edit. For example, if the extension arm is RNA, the DNA polymerase can be a reverse transcriptase or any other suitable RNA-dependent DNA polymerase. However, if the extension arm is DNA, the DNA polymerase can be a DNA-dependent DNA polymerase. In various embodiments, provision of the DNA polymerase can be in trans, such as by use of an RNA-protein recruitment domain (e.g., an MS2 hairpin incorporated onto 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 generally does not form part of the template used by the DNA polymerase (e.g., reverse transcriptase) to encode the resulting 3' single-stranded DNA flap that contains the desired edit. Thus, the designation "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-stranded DNA flap that contains the edit and a region of homology to the 5' endogenous single-stranded DNA flap that is replaced by the 3' single-stranded DNA product of the primed edited DNA synthesis. In some embodiments, the DNA synthesis template includes an "editing template" and a "homologous arm" or one or more homologous arms, for example, before and after the editing template. The editing template can be as small as a single nucleotide substitution, or it can be an insertion or inversion of DNA. In addition, the editing template can also include deletions, which can be engineered by encoding a homology arm containing the desired deletion. In other embodiments, the DNA synthesis template can also include the e2 region or a portion thereof. For example, if the e2 region contains a secondary structure that causes termination of DNA polymerase activity, it is possible that DNA polymerase function will terminate before any part of the e2 region is actually coded on the DNA. It is also possible that some or even all of the e2 region will be coded on the DNA. How much of e2 is actually used as a template will depend on its configuration and whether it disrupts DNA polymerase function.

The schematic in FIG. 3F illustrates a typical PEgRNA interaction with a target site in double-stranded DNA and the concomitant generation of a 3′ single-stranded DNA flap containing the desired genetic alteration. The double-stranded DNA is shown with an upper strand in a 3′ to 5′ orientation (i.e., the target strand) and a lower strand in a 5′ to 3′ orientation (i.e., the PAM strand or non-target strand). The upper strand contains 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 targeted by and anneals to the spacer of the PEgRNA. The complementary lower strand is referred to as the “non-target strand” or “PAM strand” or “protospacer strand” because it contains the PAM sequence (e.g., NGG) and the protospacer. Although not shown, the depicted PEgRNA would be complexed with Cas9 or an 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 a DNA/RNA hybrid between the spacer RNA and the complement of the protospacer DNA, inducing the formation of an R-loop on the protospacer. As taught elsewhere in this application, the Cas9 protein (not shown) then induces a nick on the non-target strand as shown. This then leads to the formation of a 3' ssDNA flap region immediately upstream of the nick site, which interacts with the 3' end of the PEgRNA at the primer binding site according to *z*. 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 the reverse transcriptase. Next, the reverse transcriptase (e.g., provided in trans or provided in cis as a fusion protein attached to the Cas9 construct) then polymerizes a single strand of DNA encoded by the DNA synthesis template (comprising the editing template (B) and the homology arm (C)). Polymerization continues toward the 5' end of the extension arm. The polymerized strand of ssDNA forms a ssDNA 3' end flap that invades the endogenous DNA as described elsewhere (e.g., as shown in FIG. 1G), displacing the corresponding endogenous strand (which is removed as a DNA flap at the 5' end of the endogenous DNA) and incorporating the desired nucleotide edit (single nucleotide base pair change, deletion, insertion (including entire genes) by a naturally occurring round of DNA repair/replication.

FIG. 3G illustrates yet another embodiment of the prime editing contemplated in the present application. In particular, the upper schematic illustrates one embodiment of a prime editor (PE), which includes a fusion protein of napDNAbp (e.g., SpCas9) and a polymerase (e.g., reverse transcriptase), which are linked by a linker. PE forms a complex with the PEgRNA by binding to the gRNA core of the PEgRNA. In the embodiment shown, the PEgRNA is equipped with a 3' extension arm, which, starting at the 3' end, includes a primer binding site (PBS) and then a DNA synthesis template. The lower schematic illustrates a variant of the prime editor, referred to as "trans-prime editor (tPE)". In this embodiment, the DNA synthesis template and the PBS are decoupled from the PEgRNA and presented by a separate molecule, referred to as the trans-prime editor RNA template ("tPERT"). It includes an RNA-protein recruitment domain (e.g., an MS2 hairpin). PE itself is further modified to include a fusion to the rPERT recruitment protein ("RP"). This is a protein that specifically recognizes and binds the RNA-protein recruitment domain. In an example where the RNA-protein recruitment domain is an MS2 hairpin, the corresponding rPERT recruitment 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 on 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" tPERT with the appropriate RNA-protein recruitment domain to co-localize with the PE:gRNA complex, thereby providing PBS and DNA synthesis template in trans for use in prime editing as shown in the example illustrated in Figure 3H.

FIG. 3H illustrates the process of trans-prime editing. In this embodiment, the trans-prime editor comprises a "PE2" prime editor (i.e., a fusion of Cas9(H840A) and variant MMLV RT) that is fused to an MS2cp protein (i.e., a type of recruitment protein that recognizes and binds the MS2 aptamer) and 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 non-target strand. The MS2cp protein recruits tPERT in trans by specific interaction with the RNA-protein recruitment domain on the tPERT molecule. 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-stranded DNA flap with a 3' end and containing the desired genetic information encoded by the DNA synthesis template.

Figures 4A-4E demonstrate the TPRT assay (i.e., prime editing assay) in vitro. Figure 4A is a schematic of the fluorescently labeled DNA substrate, extension of the gRNA template by the RT enzyme, and PAGE. Figure 4B shows TPRT (i.e., prime editing) with pre-nicked substrates, dCas9, and 5'-extended gRNAs of different synthetic template lengths. Figure 4C shows the RT reaction with pre-nicked DNA substrates in the absence of Cas9. Figure 4D shows TPRT (i.e., prime editing) on a full-length dsDNA substrate with Cas9(H840A) and a 5'-extended gRNA. Figure 4E shows a 3'-extended gRNA template with a pre-nicked full-length dsDNA substrate. M-MLV RT is present in all reactions.

Figure 5 shows in vitro validations using 5' extended gRNAs with varying synthetic template length. Fluorescently labeled (Cy5) DNA targets were used as substrates and were pre-nicked in this set of experiments. The Cas9 used in these experiments was catalytically inactive Cas9 (dCas9) and the RT used was Superscript III, a commercial RT derived from Moloney Murine Leukemia Virus (M-MLV). dCas9:gRNA complexes were formed from purified components. Fluorescently labeled DNA substrates were then added along with dNTPs and RT enzyme. After 1 hour of incubation at 37°C, reaction products were analyzed by denaturing urea polyacrylamide gel electrophoresis (PAGE). The gel images show an extension of length consistent with the original DNA strand to the reverse transcription template length.

Figure 6 shows in vitro validation results using 5'-extended gRNAs with varying synthetic template lengths, very similar to those shown in Figure 5. However, the DNA substrate was not pre-nicked in this set of experiments. The Cas9 used in these experiments was Cas9 nickase (SpyCas9 H840A mutant) and the RT used was Superscript III, a commercial RT derived from 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 a standard gRNA is used (gRNA_0, lane 3).

Figure 7 demonstrates that 3' extension supports DNA synthesis and does not result in significant Cas9 nickase activity. Pre-nicked substrates (black arrows) are nearly quantitatively converted to RT products when either dCas9 or Cas9 nickase are used (lanes 4 and 5). More than 50% conversion to RT products (red arrows) is observed for the full-length substrate (lane 3). Cas9 nickase (SpyCas9 H840A mutant), catalytically inactive Cas9 (dCas9), and Superscript III, a commercial RT derived from Moloney Murine Leukemia Virus (M-MLV), are used.

Figure 8 demonstrates a dual color experiment that was used to determine if the RT reaction occurs preferentially in cis to the gRNA (bound in the same complex). Two separate experiments were performed for the 5'- and 3'-extended gRNAs. Products were analyzed by PAGE. Product ratios were calculated as (Cy3cis/Cy3trans)/(Cy5trans/Cy5cis).

Figures 9A-9D demonstrate the flap model substrate. Figure 9A shows a dual FP reporter for flap-directed mutagenesis. Figure 9B shows stop codon repair in HEK cells. Figure 9C shows yeast clones that were sequenced after flap repair, and Figure 9D shows the examination of different flap traits in human cells.

FIG. 10 demonstrates prime editing on a plasmid substrate. A dual fluorescent reporter plasmid was constructed for yeast (S. cerevisiae) expression. Expression of this construct in yeast produces only GFP. Prime editing reactions in vitro induce point mutations and transform the parental plasmid or a plasmid nicked in vitro with Cas9(H840A) into yeast. Colonies are visualized by fluorescent imaging. Yeast dual FP plasmid transformants are shown. Transforming the parental plasmid or a plasmid nicked in vitro with Cas9(H840A) results in only green GFP-expressing colonies. Prime editing reactions with 5'- or 3'-extended gRNA produce mixed green and yellow colonies. The latter express both GFP and mCherry. More yellow colonies are observed with the 3'-extended gRNA. A positive control containing no stop codon is also not shown.

Figure 11 shows prime editing on a plasmid substrate similar to the experiment in Figure 10, but instead of incorporating a point mutation in the stop codon, the prime edit incorporates a single nucleotide insertion (left) or deletion (right) that repairs the frameshift mutation and allows synthesis of mCherry downstream. Both experiments used 3' extended gRNAs.

Figure 12 shows the editing products of prime editing on the plasmid substrate characterized by Sanger sequencing. Colonies from the TRT transformation were individually selected and analyzed by Sanger sequencing. Correct editing was observed by sequencing the selected colonies. The green colonies contained the plasmid with the original DNA sequence, whereas the yellow colonies contained the correct mutations designed by the prime editing gRNA. No other point mutations or indels were observed.

Figure 13 illustrates the potential scope of the new prime editing technology and provides a comparison with deaminase-mediated base editing technology.

FIG. 14 shows a schematic of editing in human cells.

FIG. 15 demonstrates the extension of the primer binding site in the gRNA.

FIG. 16 shows gRNAs truncated for adjacent targeting.

Figures 17A-17C are graphs displaying % T→A conversion at the target nucleotide following transfection of the constructs in human embryonic kidney (HEK) cells. Figure 17A shows data presenting the results using an N-terminal fusion of wild-type MLV reverse transcriptase to Cas9 (H840A) nickase (32 amino acid linker). Figure 17B is similar to Figure 17A except for the C-terminal fusion of the RT enzyme. Figure 17C is similar to Figure 17A except that the linker between the MLV RT and Cas9 is 60 amino acids long instead of 32 amino acids.

Figure 18 shows high purity T→A editing at the HEK3 site by high throughput amplicon sequencing. The sequencing analysis output displays the most abundant genotypes in edited cells.

Figure 19 shows the editing efficiency (orange bars) at the targeted nucleotide alongside the indel ratio (blue bars). WT refers to the wild-type MLV RT enzyme. Mutant enzymes (M1-M4) contain the mutations listed to the right. Editing ratios were quantified by high-throughput sequencing of genomic DNA amplicons.

Figure 20 shows the editing efficiency of a target nucleotide when a single-stranded nick is introduced into the complementary DNA strand adjacent to the target nucleotide. Nicking at various distances from the target nucleotide was tested (triangles). Editing efficiency at the target base pair (blue bar) is shown alongside the indel formation rate (orange bar). The "none" example does not contain a guide RNA that nicks the complementary strand. Editing rates were quantified by high-throughput sequencing of genomic DNA amplicons.

FIG. 21 demonstrates processed high-throughput sequencing data showing the desired T→A transversion mutations and the general absence of other major genome editing by-products.

FIG. 22 provides a schematic diagram of an exemplary process for performing targeted mutagenesis on a target locus with an error-prone reverse transcriptase (i.e., prime editing with error-prone RT) using a nucleic acid programmable DNA binding protein (napDNAbp) complexed with an extended guide RNA. This process may also be referred to as an embodiment of prime editing for targeted mutagenesis. The extended guide RNA includes 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 a DNA molecule, and the gRNA guides the napDNAbp to bind to the target locus to be mutagenized. In step (b), a nick is introduced (e.g., by a nuclease or chemical agent) into one of the strands of the DNA at the target locus, thereby creating an available 3' end on one of the strands of the target locus. In one embodiment, 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 extension of the guide RNA to prime reverse transcription. In some embodiments, the 3'-end DNA strand hybridizes with a specific RT prime sequence on the extension of the guide RNA. In step (d), an error-prone reverse transcriptase is introduced, which synthesizes a single strand of mutagenized DNA from the 3'-end of the primed site to the 3'-end of the guide RNA. Exemplary mutations are indicated with an asterisk "*". This forms a single-stranded DNA flap containing the desired mutagenized region. In step (e), the napDNAbp and the guide RNA are released. Steps (f) and (g) involve the degradation of the single-stranded DNA flap (containing the mutagenized region) so that the desired mutagenized region is integrated into the target locus. This process can be driven to the desired product formation by removing the corresponding 5' endogenous DNA flap that is formed once the 3'-single-stranded DNA flap has invaded and hybridized with a complementary sequence on the other strand. The process can also be driven to the formation of a second strand nicked product, as illustrated in Figure IF. Following endogenous DNA repair and/or replication processes, the mutagenized region becomes incorporated into both strands of DNA at the DNA locus.

Figure 23 is a schematic diagram of gRNA design for reducing trinucleotide repeat sequences and the reduction of trinucleotide repeats 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 a CAG triplet, but 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 disease. Pathogenic expansion of trinucleotide repeats can hypothetically be corrected using prime editing. The region upstream of the repeat region can be nicked by an RNA-guided nuclease and then used to prime the synthesis of a new DNA strand containing a healthy number of repeats (this depends on the specific gene and disease). The repeat is followed by a short stretch of homology (red strand) that matches the identity of the sequence adjacent to the other end of the repeat. Invasion of the newly synthesized strand and subsequent replacement of the endogenous DNA with the newly synthesized flap leads to a shortened repeat allele.

Figure 24 is a schematic diagram showing the precise 10-nucleotide deletion in prime editing. Guide RNAs targeting the HEK3 locus were 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.

Figure 25 is a schematic diagram showing gRNA design for peptide tagging genes at endogenous genomic loci and peptide tagging in TPRT genome editing (i.e., prime editing). The FlAsH and ReAsH tagging systems include 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 in cells, tetracysteine motif-containing proteins can be fluorescently labeled with the fluorophore-arsenical probe (see reference: J.Am.Chem.Soc., 2002,124(21),pp6063-6076. DOI:10.1021/ja017687n). The "sortagging" system employs bacterial sortase enzymes to covalently conjugate labeled peptide probes to proteins containing suitable peptide substrates (see reference: Nat. Chem. Biol. 2007 Nov;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 be labeled with a pentafluoro-aromatic substrate (Reference: Nat. Chem. 2016 Feb;8(2):120-8. doi:10.1038/nchem.2413).

Figure 26A shows the precise incorporation of His ₆ and FLAG tags into genomic DNA. Guide RNAs targeting the HEK3 locus were designed with reverse transcription templates encoding either an 18nt His tag insertion or a 24nt FLAG tag insertion. Editing efficiency in transfected HEK cells was assessed using amplicon sequencing. Note that the full 24nt sequence of the FLAG tag is cut off from the frame (sequencing confirmed the full length and precise insertion). FIG. 26B shows a schematic summarizing various applications involving protein/peptide tagging, including (a) solubilizing or insolubilizing proteins, (b) altering or tracking the subcellular localization of proteins, (c) extending protein half-life, (d) facilitating protein purification, and (e) facilitating protein detection.

Figure 27 shows an overview of prime editing by incorporating protective mutations into PRNP that prevent or halt the progression of prion disease. The PEGRNA sequence corresponds to SEQ ID NO: 4082 on the left (i.e., 5' of the sgRNA backbone) and SEQ ID NO: 4083 on the right (i.e., 3' of the sgRNA backbone).

FIG. 28A is a schematic diagram of PE-based insertion of a sequence encoding an RNA motif. FIG. 28B is a non-exhaustive list of some example motifs that could potentially be inserted, and their functions.

Figure 29A is a diagram of a primed editor. Figure 29B shows possible PE-directed modifications to genomic, plasmid, or viral DNA. Figure 29C shows an example scheme for the insertion of a library of peptide loops onto a defined protein (in this case GFP) by a library of PEgRNAs. Figure 29D shows an example of possible programmable deletion of codons or N- or C-terminal truncation of a protein using different PEgRNAs. Deletions would be expected to occur with minimal generation of frameshift mutations.

FIG. 30 shows a possible scheme for the recursive insertion of codons in a continuous evolution system such as PACE.

Figure 31 is a representation of an engineered gRNA, showing the gRNA core, a ∼20 nt spacer that matches the sequence of the target gene, a reverse transcription template with an immunogenic epitope nucleotide sequence, and a primer binding site that matches the sequence of the target gene.

FIG. 32 is a schematic diagram of using prime editing as a means to insert known immunogenic epitopes onto endogenous or exogenous genomic DNA, resulting in the modification of the corresponding protein.

Figure 33 is a schematic diagram showing PEgRNA design for primer binding sequence insertion and primer binding insertion onto genomic DNA with prime editing to determine off-target editing. In this embodiment, prime editing is performed in living cells, tissues, or animal models. As a first step, a suitable PEgRNA is designed. The upper schematic shows an exemplary PEgRNA that can be used in this aspect. The spacer on the PEgRNA (labeled "protospacer") is complementary to one of the strands of the genomic target. The PE:PEgRNA complex (i.e., the PE complex) incorporates a single-stranded 3'-end flap at the nick site, which contains the encoded primer binding sequence and a homologous region (encoded by the homologous arm of the PEgRNA) that is complementary to the region just downstream of the cut site (by red). By flap invasion and DNA repair/replication processes, the synthesized strand becomes incorporated onto the DNA, thereby incorporating a primer binding site. This process can occur not only at the desired genomic target, but also at other genomic sites that may interact with the PEgRNA in an off-target manner (i.e., due to the complementarity of the spacer region to other genomic sites that are not the intended genomic site, the PEgRNA guides the PE complex to other off-target sites). Therefore, primer binding sequences can be incorporated not only at the desired genomic target, but also at other off-target genomic sites on the genome. To detect the insertion of these primer binding sites at both the intended genomic target site and the off-target genomic site, the genomic DNA (after PE) can be isolated, fragmented, and ligated to adapter nucleotides (shown in red). Then, PCR can be performed with PCR oligonucleotides that anneal to the adapter and the inserted primer binding sequence to amplify the on-target and off-target genomic DNA regions where the primer binding sites were inserted by PE. High-throughput sequencing and sequence alignment can then be performed to identify the insertion point of the primer binding sequence inserted by PE at either the on-target site or the off-target site.

FIG. 34 is a schematic diagram showing precise insertion of genes by PE.

Figure 35A is a schematic showing the native insulin signaling pathway and Figure 35B is a schematic showing FKBP12-tagged insulin receptor activation controlled by FK1012.

Figure 36 shows a small molecule monomer. Reference: Bump FK506 mimic (2) ¹⁰⁷ .

Figures 37A-37B show small molecule dimers. References: FK1012 4 ^95,96 ; FK1012 5 ¹⁰⁸ ; FK1012 6 ¹⁰⁷ ; AP1903 7 ¹⁰⁷ ; Cyclosporin A dimer 8 ⁹⁸ ; FK506-Cyclosporin A dimer (FkCsA) 9 ¹⁰⁰ .

Figures 38A-38F provide an overview of prime editing and feasibility studies in vitro and in yeast cells. Figure 38A shows 75,122 known pathogenic human genetic variants in ClinVar (accessed July 2019), categorized by type. Figure 38B shows that the prime editing complex consists of a prime editor (PE) protein containing an RNA-guided DNA nicking domain, e.g., Cas9 nickase, fused to an engineered reverse transcriptase domain and complexed with a prime editing guide RNA (PEgRNA). The PE:PEgRNA complex binds to the target DNA site, enabling a large variety of precise DNA editing at a wide range of DNA positions before and after the protospacer adjacent motif (PAM) of the target site. Figure 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 with the RT template of the PEgRNA, resulting in a newly synthesized DNA strand (3' flap) containing the desired edit. 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 a stably edited DNA. Figure 38D shows an in vitro 5' extended PEgRNA primer extension assay with pre-nicked dsDNA substrates containing a 5' Cy5-labeled PAM strand, dCas9, and a commercially available M-MLV RT variant (RT, Superscript III). dCas9 was complexed with PEgRNA containing RT templates of various lengths and then added to the DNA substrate along with the indicated components. The reactions were incubated at 37°C for 1 hour and then analyzed by denaturing urea PAGE and visualized for Cy5 fluorescence. Figure 38E shows primer extension performed as in Figure 38D using 3'-extended PEgRNA precomplexed with dCas9 or Cas9 H840A nickase and 5' Cy5-labeled dsDNA substrates pre-nicked or unnicked. Figure 38F shows yeast colonies transformed with GFP-mCherry fusion reporter plasmids edited in vitro by PEgRNA, Cas9 nickase, and RT. Plasmids containing nonsense or frameshift mutations between GFP and mCherry were edited with 5'- or 3'-extended PEgRNA that restored mCherry translation by transversion mutation, 1 bp insertion, or 1 bp deletion. GFP and mCherry double positive cells (yellow) reflect successful editing.

Figures 39A-39D show primed editing of genomic DNA in human cells with PE1 and PE2. Figure 39A shows that the PEgRNA contains a spacer sequence, sgRNA backbone, and a 3' extension containing a primer binding site (green) and a reverse transcription (RT) template (purple) containing the base(s) to be edited (red). The primer binding site hybridizes to the PAM-containing DNA strand immediately upstream of the site of nicking. With the exception of the encoded edit, the RT template is homologous to the DNA sequence downstream of the nick. Figure 39B shows incorporation of a T.A to A.T transversion edit at the HEK3 site in HEK293T cells using Cas9 H840A nickase (PE1) fused to wild-type M-MLV reverse transcriptase and PEgRNA of various primer binding site lengths. Figure 39C shows that the use of engineered quintuple mutant M-MLV reverse transcriptase (D200N, L603W, T306K, W313F, T330P) in PE2 substantially improves prime editing transversion efficiency at five genomic sites in HEK293T cells and small insertion and small deletion editing in HEK3. Figure 39D is a comparison of PE2 editing efficiency with various 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.

Figures 40A-40C show that the PE3 and PE3b systems increase prime editing efficiency by nicking the non-edited strand. Figure 40A is an overview of prime editing with PE3. After the 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 mechanisms or DNA replication can degrade the heteroduplex to give either an edited or non-edited product. Nicking the non-edited strand favors repair of that strand, resulting in preferential generation of stable duplex DNA containing the desired edit. Figure 40B shows the effect of complementary strand nicking on prime editing efficiency and indel formation mediated by PE3. "None" refers to the PE2 control, which does not nick the complementary strand. Figure 40C is a comparison of editing efficiency by PE2 (no complementary nick), PE3 (general complementary nick), and PE3b (editing-specific complementary nick). All editing yields reflect the percentage of total sequencing reads that contain the intended edit and no indels among all treated cells without sorting. Values and error bars reflect the mean and s.d. of three independent biological replicates.

Figures 41A-41K show PE3 targeted insertions, deletions, and all 12 types of point mutations at seven endogenous human genomic loci in HEK293T cells. Figure 41A is a graph showing all 12 types of single nucleotide transition and transversion editing at positions +1 to +8 (counting the positioning of the nicks induced by PEgRNA as between positions +1 and -1) of the HEK3 site using a 10 nt RT template. Figure 41B is a graph showing long-range PE3 transversion editing at the HEK3 site using a 34 nt RT template. Figures 41C-41H are graphs showing all 12 types of transition and transversion editing at various positions on the prime editing window for (Figure 41C) RNF2, (Figure 41D) FANCF, (Figure 41E) EMX1, (Figure 41F) RUNX1, (Figure 41G) VEGFA, and (Figure 41H) DNMT1. Figure 41I is a graph showing targeted 1 and 3 bp insertions and 1 and 3 bp deletions by PE3 at seven endogenous genomic loci. Figure 41J is a graph showing targeted precision deletions of 5 to 80 bp at HEK3 target sites. Figure 41K is a graph showing combination editing 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 no indels among all treated cells without sorting. Values and error bars reflect the mean and s.d. of three independent biological replicates.

Figures 42A-42H show a comparison of prime and base editing and off-target editing by Cas9 and PE3 at known Cas9 off-target sites. Figure 42A shows the total C.G to T.A editing efficiency at the same target nucleotide for PE2, PE3, BE2max, and BE4max at endogenous HEK3, FANCF, and EMX1 sites in HEK293T cells. Figure 42B shows the indel frequency from the treatments in Figure 42A. Figure 42C shows the editing efficiency of precise C.G to T.A editing (without bystander editing or indels) for PE2, PE3, BE2max, and BE4max in HEK3, FANCF, and EMX1. In EMX1, precise PE combinatorial editing of all possible combinations of C.G to T.A conversions at the three target nucleotides is also shown. Figure 42D shows the total A.T to G.C editing efficiency for PE2, PE3, ABEdmax, and ABEmax in HEK3 and FANCF. Figure 42E shows the precise A.T to G.C editing efficiency without bystander editing or indels for HEK3 and FANCF. Figure 42F shows the indel frequency from the treatments in Figure 42D. Figure 42G shows the average triplicate editing efficiency (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 observed 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 an sgRNA or each of the four PEgRNAs that recognize the same protospacer. Figure 42H shows the average triplicate on-target and off-target editing efficiency and indel efficiency (below in brackets) for PE2 or PE3 paired with each PEgRNA in (Figure 42G) in HEK293T cells. On-target editing yield reflects the percentage of total sequencing reads that contain intended editing and no indels among all treated cells without sorting. Off-target editing yield reflects off-target locus modifications that are consistent with prime editing. Values and error bars reflect the mean and sd of three independent biological replicates.

Figures 43A-43I show a comparison of prime editing, incorporation and correction of pathogenic transversion, insertion, or deletion mutations, and prime editing and HDR in various human cell lines and primary mouse cortical neurons. Figure 43A is a graph showing incorporation (by a T.A to A.T transversion) and correction (by an A.T to T.A transversion) of a pathogenic E6V mutation in HBB of HEK293T cells. Correction is shown either to wild-type HBB or to HBB containing a silent mutation that blocks the PEgRNA PAM. Figure 43B is a graph showing incorporation (by a 4 bp insertion) and correction (by a 4 bp deletion) of a pathogenic HEXA 1278+TATC allele in HEK293T cells. Correction is shown to either wild-type HEXA or to HEXA containing a silent mutation that blocks the PEgRNA PAM. Figure 43C is a graph showing the incorporation of the protective G127V variant of PRNP in HEK293T cells by G-C to T-A transversion. Figure 43D is a graph showing prime editing in other human cell lines including K562 (leukemia bone marrow cells), U2OS (osteosarcoma cells), and HeLa (cervical cancer cells). Figure 43E is a graph showing the incorporation of a G-C to T-A transversion mutation in DNMT1 in mouse primary cortical neurons using a dual split-intein PE3 lentiviral system, in which the N-terminal half is Cas9(1-573) fused to the N intein and to GFP-KASH via the P2A self-cleaving peptide, and the C-terminal half is the C intein fused to the remainder of PE2. The PE2 halves are expressed from the human synapsin promoter, which is highly specific to mature neurons. Sorted values reflect editing or indels from GFP-positive nuclei, and unsorted values are from all nuclei. Figure 43F is a comparison of HDR editing efficiency mediated by PE3 and Cas9 at endogenous genomic loci in HEK293T cells. Figure 43G is a comparison of HDR editing efficiency mediated by PE3 and Cas9 at endogenous genomic loci in K562, U2OS, and HeLa cells. Figure 43H is a comparison of HDR indel by-product generation mediated by PE3 and Cas9 in HEK293T, K562, U2OS, and HeLa cells. Figure 43I shows targeted insertion of His6 tag (18bp), FLAG epitope tag (24bp), or extended LoxP site (44bp) in HEK293T cells by PE3. All editing yields reflect the percentage of total sequencing reads that contain the intended edit and no indels among all treated cells. Values and error bars reflect the mean and s.d. of three independent biological replicates.

Figures 44A-44G show in vitro prime editing validation studies with fluorescently labeled DNA substrates. Figure 44A shows electrophoretic mobility shift assays with dCas9, 5' extended PEgRNAs, and 5'-Cy5 labeled DNA substrates. PEgRNAs 1 to 5 contain a 15 nt linker sequence between the spacer and PBS (Linker A for PEgRNA 1, Linker B for PEgRNAs 2 to 5), a 5 nt PBS sequence, and 7 nt (PEgRNAs 1 and 2), 8 nt (PEgRNA 3), 15 nt (PEgRNA 4), and 22 nt (PEgRNA 5) RT templates. PEgRNAs are those used in Figures 44E and 44F; complete sequences are listed in Tables 2A-2C. Figure 44B shows in vitro nicking assays of Cas9 H840A with 5' extended and 3' extended PEgRNAs. Figure 44C shows Cas9-mediated indel formation in HEK293T cells in HEK3 with 5'- and 3'-extended PEgRNA. Figure 44D shows an overview of the prime editing in vitro biochemical assay. 5'-Cy5-labeled pre-nicked and unnicked dsDNA substrates were tested. sgRNA, 5'-extended PEgRNA, or 3'-extended PEgRNA were pre-complexed with dCas9 or Cas9 H840A nickase and then combined with dsDNA substrate, M-MLV RT, and dNTPs. Reactions were allowed to proceed for 1 hour at 37°C prior to separation by denaturing urea PAGE and visualization by Cy5 fluorescence. Figure 44E shows that primer extension reactions with 5'-extended PEgRNA, pre-nicked DNA substrate, and dCas9 lead to significant conversion to RT product. Figure 44F shows a primer extension reaction using unnicked DNA substrate and Cas9 H840A nickase with 5'-extended PEgRNA as in Figure 44B. Product yield is greatly reduced compared to pre-nicked substrate. Figure 44G shows that in vitro primer extension reaction using 3'-PEgRNA produces a single clear product by denaturing urea PAGE. The RT product band was excised, eluted from the gel, and then subjected to homopolymeric tailing with terminal transferase (TdT) using either dGTP or dATP. The tailed product was extended with a poly-T or poly-C primer, and the resulting DNA was sequenced. Sanger traces indicate that three nucleotides from the gRNA backbone were reverse transcribed (added to the DNA product as the last 3' nucleotides). Note that in mammalian cell prime editing experiments, PEgRNA backbone insertion is much rarer than in vitro (Figures 56A-56D). This is possibly due to the inability of the tethered reverse transcriptase to access the Cas9-bound guide RNA backbone and/or cellular excision of the mismatched 3' end of the 3' flap containing the PEgRNA backbone sequence.

Figures 45A-45G show cellular repair in yeast of a 3' DNA flap from an in vitro prime editing reaction. Figure 45A shows that a dual fluorescent protein reporter plasmid contains GFP and mCherry open reading frames separated by target sites encoding an in-frame stop codon, a +1 frameshift, or a -1 frameshift. Prime editing reactions were performed in vitro with Cas9 H840A nickase, PEgRNA, dNTPs, and M-MLV reverse transcriptase and then transformed into yeast. Colonies containing unedited plasmids produce GFP but not mCherry. Yeast colonies containing edited plasmids produce both GFP and mCherry as fusion proteins. Figure 45B shows an overlay of GFP and mCherry fluorescence of yeast colonies transformed with reporter plasmids that contain a stop codon between GFP and mCherry (unedited negative control, top) or no stop codon or frameshift between GFP and mCherry (pre-edited positive control, bottom). Figures 45C-45F show visualization of mCherry and GFP fluorescence from yeast colonies transformed with in vitro primed editing reaction products. Figure 45C shows stop codon correction by T.A to A.T transversion using 3'-extended PEgRNA or 5'-extended PEgRNA as shown in Figure 45D. Figure 45E shows +1 frameshift correction of a 1 bp deletion using 3'-extended PEgRNA. Figure 45F shows -1 frameshift correction by a 1 bp insertion using 3'-extended PEgRNA. Figure 45G shows Sanger DNA sequencing traces from plasmids isolated from the GFP-only colony in Figure 45B and the GFP and mCherry double positive colony in Figure 45C.

Figures 46A-46F show correct editing versus indel generation by PE1. Figure 46A shows transversion editing efficiency and indel generation of T.A to A.T by PE1 at the +1 position of HEK3 using a 10 nt RT template and a PBS sequence ranging from 8-17 nt, and a PEgRNA containing the same. Figure 46B shows transversion editing efficiency and indel generation of G.C to T.A by PE1 at the +5 position of EMX1 using a 13 nt RT template and a PBS sequence ranging from 9-17 nt, and a PEgRNA containing the same. Figure 46C shows transversion editing efficiency and indel generation of G.C to T.A by PE1 at the +5 position of FANCF using a 17 nt RT template and a PBS sequence ranging from 8-17 nt, and a PEgRNA containing the same. Figure 46D shows the transversion editing efficiency and indel generation of C.G to A.T by PE1 at the +1 position of RNF2, using a PEgRNA containing an 11 nt RT template and a PBS sequence ranging from 9-17 nt. Figure 46E shows the transversion editing efficiency and indel generation of G.C to T.A by PE1 at the +2 position of HEK4, using a PEgRNA containing a 13 nt RT template and a PBS sequence ranging from 7-15 nt. Figure 46F shows the +1T deletion, +1A insertion, and +1CTT insertion mediated by PE1 at the HEK3 site, using a 13 nt PBS and 10 nt RT template. The sequences of the PEgRNAs are those used in Figure 39C (see Tables 3A-3R). Values and error bars reflect the mean and s.d. of three independent biological replicates.

Figures 47A-47S show evaluation of M-MLV RT variants for prime editing. Figure 47A shows the abbreviations of the prime editor variants used in this figure. Figure 47B shows targeted insertion and deletion editing by PE1 at the HEK3 locus. Figures 47C-47H show a comparison of 18 prime editor constructs containing M-MLV RT variants for their ability to incorporate a +2G.C to C.G transversion edit in HEK3 as shown in Figure 47C, a 24bp FLAG insertion in HEK3 as shown in Figure 47D, a +1C.G to A.T transversion edit in RNF2 as shown in Figure 47E, a +1G.C to C.G transversion edit in EMX1 as shown in Figure 47F, a +2T.A to A.T transversion edit in HBB as shown in Figure 47G, and a +1G.C to C.G transversion edit in FANCF as shown in Figure 47H. Figures 47I-47N show a comparison of four prime editor constructs containing M-MLV variants for their ability to incorporate the edits shown in Figures 47C-47H in a second round of independent experiments. Figures 47O-47S show PE2 editing efficiency at five genomic loci with varying PBS lengths. Figure 47O shows +1T·A to A·T variation in HEK3. Figure 47P shows +5G·C to T·A variation in EMX1. Figure 47Q shows +5G·C to T·A variation in FANCF. Figure 47R shows +1C·G to A·T variation in RNF2. Figure 47S shows +2G·C to T·A variation in HEK4. Values and error bars reflect the mean and s.d. of three independent biological replicates.

Figures 48A-48C show design features of PEgRNA PBS and RT template sequences. Figure 48A shows PE2-mediated +5G·C to T·A transversion editing efficiency in VEGFA of HEK293T cells as a function of RT template length (blue line). Indels (gray line) are plotted for comparison. The sequence below the graph shows the last nucleotide for editing that serves as a template for synthesis by PEgRNA. The G nucleotide (templated by a C on PEgRNA) is highlighted; to maximize prime editing efficiency, RT templates that end with a C should be avoided during PEgRNA design. Figure 48B shows DNMT1 +5G·C to T·A transversion editing and indels as in Figure 48A. Figure 48C shows RUNX1 +5G·C to T·A transversion editing and indels as in Figure 48A. Values and error bars reflect the mean and s.d. of three independent biological replicates.

Figures 49A-49B show the effect 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 using the CellTiter-Glo2.0 assay (Promega) every 24 hours for 3 days post-transfection. Figure 49A shows viability measured by luminescence 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. Figure 49B shows percent editing and indels for PE2, PE2 R110S K103L, Cas9 H840A nickase, or dCas9 with HEK3-targeted PEgRNA plasmids encoding +5G to A edits. Editing efficiency was measured from treated cells at day 3 post-transfection alongside those used to assay viability in Figure 49A. Values and error bars reflect the mean and s.d. of three independent biological replicates.

Figures 50A-50B show PE3-mediated HBB E6V correction and HEXA 1278+TATC correction by various PEgRNAs. Figure 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 mutations. Figure 50B shows a screen of 41 PEgRNAs for correction of the HEXA 1278+TATC allele in HEK293T cells with PE3 or PE3b. PEgRNAs labeled with HEXA correct the pathogenic allele with a shifted 4bp deletion that blocks the PAM and leaves a silent mutation. PEgRNAs labeled with HEXA correct the pathogenic allele back to wild-type. Entries ending in "b" use an editing-specific nicking sgRNA in combination with the PEgRNA (PE3b system). Values and error bars reflect the mean and s.d. of three independent biological replicates.

Figures 51A- 51G show a comparison of PE3 activity in human cell lines and HDR initiated by PE3 and Cas9. Efficiency of generating correct edits (no indels) and indel frequency for HDR initiated by PE3 and Cas9 in HEK293T cells as shown in Figure 51A, K562 cells as shown in Figure 51B, U2OS cells as shown in Figure 51C, and HeLa cells as shown in Figure 51D. Comparison of each bundled edit incorporates identical edits by HDR initiated by PE3 and Cas9. Non-targeting controls are PE3 and PEgRNA targeting a non-targeted locus. Figure 51E shows a control experiment with non-targeting PEgRNA+PE3 and dCas9+sgRNA compared to a wild-type Cas9 HDR experiment. It is confirmed that the common contaminant ssDNA donor HDR template, which spuriously increases the apparent HDR efficiency, does not contribute to the HDR measurements in Figures 51A-51D. Figures 51F -51G show example HEK3 site allele tables from genomic DNA samples isolated from K562 cells after editing with PE3 or Cas9-initiated HDR. Alleles were sequenced by Illumina MiSeq and analyzed by ^CRISPResso2 . The reference HEK3 sequence from this region is at the top. Allele tables are shown for a negative control of non-targeting PEgRNA, a +1CTT insertion in HEK3 with PE3, and a +1CTT insertion in HEK3 with Cas9-initiated HDR. Allele frequencies and corresponding Illumina sequencing read counts are shown for each allele. All alleles observed with a frequency ≥ 0.20% are shown. Values and error bars reflect the mean and sd of three independent biological replicates.

Figures 52A-52D show the distribution of pathogenic insertion, duplication, deletion, and indel lengths on the ClinVar database. The ClinVar variant summary was downloaded from NCBI on July 15, 2019. The lengths of reported insertions, deletions, and duplications were calculated using appropriate identifying information for the reference and alternative alleles, variant start and stop positions, or variant name. Variants that did not report any of the above information were excluded from the analysis. The lengths of reported indels (single variants encompassing 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 alternative alleles.

Figures 53A- 53E show example FACS gating for GFP positive cell sorting. Below are examples of the original batch analysis files outlining the sorting strategy used to generate the HEXA 1278+TATC and HBB E6V HEK293T cell lines. Image data was generated by a Sony LE-MA900 cytometer using Cell Sorter software v.3.0.5. Graphic 1 shows a gating plot of cells not expressing GFP. Graphic 2 shows an example sorting of P2A-GFP expressing cells used to isolate the HBB E6V HEK293T cell line. HEK293T cells were initially gated on populations using FSC-A/BSC-A (gate A) and then sorted for singlets using FSC-A/FSC-H (gate B). Live cells were sorted by gating on DAPI negative cells (gate C). Cells with GFP fluorescence levels above that of negative control cells were sorted using EGFP as the fluorochrome (gate D). Figure 53A shows HEK293T cells (GFP negative). Figure 53B shows a representative plot of FACS gating for cells expressing PE2-P2A-GFP. Figure 53C shows the genotype of HEXA 1278+TATC homozygous HEK293T cells. Figures 53D -53E show the allele table of the HBB E6V homozygous HEK293T cell line.

Figure 54 is a schematic summarizing the PEG RNA cloning procedure.

Figures 55A-55G are schematics of PEgRNA design. Figure 55A shows a simple diagram of PEgRNA with domains labeled (left) and bound to nCas9 at a genomic site (right). Figure 55B shows different types of modifications of PEgRNA expected to increase activity. Figure 55C shows modifications of PEgRNA to increase transcription of longer RNAs through promoter options and 5', 3' processing and termination. Figure 55D shows lengthening of the P1 system. This is an example of backbone modification. Figure 55E shows that incorporation of synthetic modifications on the template region or elsewhere on the PEgRNA can increase activity. Figure 55F shows that designed incorporation of minimal secondary structure on the template can prevent the formation of longer, more inhibitory secondary structures. Figure 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' end of the PEgRNA can enhance RT binding.

Figures 56A-56D show the integration of PEgRNA backbone sequences into the target locus. HTS data was analyzed for PEgRNA backbone sequence insertions as described in Figures 60A-60B. Figure 56A shows the analysis of the EMX1 locus. Shown are the % of total sequencing reads that contain one or more PEgRNA backbone sequence nucleotides on the insertion adjacent to the RT template (left); the percentage of total sequencing reads that contain a PEgRNA backbone sequence insertion of a defined length (center); and the cumulative total percentage of PEgRNA insertions up to encompassing a defined length on the x-axis. Figure 56B shows the same as Figure 56A for FANCF. Figure 56C shows the same as Figure 56A for HEK3. Figure 56D shows the same as Figure 56A for RNF2. Values and error bars reflect the mean and s.d. of three independent biological replicates.

Figures 57A-57I show the effect of PE2, PE2-dRT, and Cas9 H840A nickase on transcriptome-wide RNA abundance. Analysis of ribosomal RNA-depleted cellular RNA isolated from HEK293T cells expressing PE2, PE2-dRT, or Cas9 H840A nickase and PRNP- or HEXA-targeted PEgRNA. RNA corresponding to 14,410 and 14,368 genes were detected in PRNP and HEXA samples, respectively. Figures 57A-57F show volcano plots displaying -log10 FDR-adjusted p-values versus log2 fold change in transcript abundance for each (Aeach) RNA. (Figure 57A) PE2 vs. PE2-dRT with PRNP-targeted PEgRNA, (Figure 57B) PE2 vs. Cas9 H840A with PRNP-targeted PEgRNA, (Figure 57C) PE2-dRT vs. Cas9 H840A with PRNP-targeted PEgRNA, (Figure 57D) PE2 vs. PE2-dRT with HEXA-targeted PEgRNA, (Figure 57E) PE2 vs. Cas9 H840A with HEXA-targeted PEgRNA, (Figure 57F) PE2-dRT vs. Cas9 H840A with HEXA-targeted PEgRNA. Red dots indicate genes that show a ≧2-fold change in relative abundance that is statistically significant (FDR adjusted p<0.05). Figures 57G-57I are Venn diagrams of up- and down-regulated transcripts (≧2-fold change) comparing PRNP and HEXA samples for (Figure 57G) PE2 vs. PE2-dRT, (Figure 57H) PE2 vs. Cas9 H840A, and (Figure 57I) PE2-dRT vs. Cas9 H840A.

Figures 58A-58B show representative FACS gating for neuronal nuclei sorting. Nuclei were sequentially gated based on DyeCycle Ruby signal, FSC/SSC ratio, SSC width/SSC height ratio, and GFP/DyeCycle ratio.

Figures 59A- 59G show the protocol for cloning 3' extended PEgRNA onto a mammalian U6 expression vector by Golden Gate assembly. Figure 59A shows an overview of the cloning. Figure 59B shows "Step 1: Digest pU6-PEgRNA-GG-vector plasmid (component 1)". Figure 59C shows "Steps 2 and 3: Order and anneal oligonucleotide parts (components 2, 3, and 4)". Figure 59D shows "Step 2.b.ii.: sgRNA backbone phosphorylation (not required if oligonucleotides are purchased phosphorylated)". Figure 59E shows "Step 4: PEgRNA assembly". Figure 59F shows "Steps 5 and 6: Transformation of assembled plasmid". Figure 59G shows a diagram summarizing the PEgRNA cloning protocol.

Figures 60A-60B show a Python script for quantifying PEgRNA backbone incorporation. A custom python script was generated to characterize and quantify PEgRNA insertions at the target genomic locus. The script iteratively matches text strings of increasing length taken from the reference sequence (guide RNA backbone sequence) with the sequencing reads in the fastq file 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 backbone sequence. The exact length incorporation and cumulative incorporation up to a specified length were calculated in this manner. To ensure alignment and accurate counting of short slices of sgRNA, the start of the reference sequence encompasses 5 to 6 bases at the 3' end of the new DNA strand synthesized by reverse transcriptase.

Figure 61 is a graph showing the percentage of total sequencing reads with the defined edits for SaCas9(N580A)-MMLV RT HEK3 +6C>A. Values for correct edits and indels are shown.

Figures 62A-62B show the importance of protospacers for efficient incorporation of desired edits at precise positioning by prime editing. Figure 62A is a graph showing the percent of total sequencing reads in which the targeted T-A base pair was converted to A-T for various HEK3 loci. Figure 62B shows the sequence analysis.

Figure 63 is a graph showing SpCas9 PAM variants of PAM editing (N=3). The percentage of total sequencing reads with targeted PAM editing 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.

Figures 64A-64F are schematic diagrams showing the introduction of various site-specific recombinase (SSR) targets onto a genome using PE. Figure 64A provides a general schematic diagram of the insertion of a recombinase target sequence by a primed editor. Figure 64B shows how a single SSR target inserted by PE can be used as a site for genomic incorporation of a DNA donor template. Figure 64C shows how tandem insertion of SSR target sites can be used to delete a portion of a genome. Figure 64D shows how tandem insertion of SSR target sites can be used to invert a portion of a genome. Figure 64E shows how insertion of two SSR target sites at two distal chromosomal regions can result in a chromosomal translocation. Figure 64F shows how insertion of two different SSR target sites on a genome can be used to exchange a cassette from a DNA donor template. See Example 17 for further details.

Figure 65 shows 1) PE-mediated synthesis of an SSR target site on the genome of a human cell and 2) use of that SSR target site to incorporate a DNA donor template containing a GFP expression marker. Once successfully incorporated, the GFP causes the cell to fluoresce. See Example 17 for further details.

Figure 66 illustrates one embodiment of a prime editor provided as two PE half proteins that regenerate the entire prime editor by 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 illustrates the mechanism of intein removal from a polypeptide sequence and reformation of a peptide bond between the N- and C-terminal extein sequences. (a) illustrates the general mechanism of two half proteins, each containing half of an intein sequence. When they come into contact in a cell, they result in a fully functional intein. This then undergoes self-splicing and excision. The excision process results in the formation of a peptide bond between the N-terminal protein half (or "N-extein") and the C-terminal protein half (or "C-extein") to form a whole single polypeptide containing the N-extein and C-extein portions. In various embodiments, the N-extein can correspond to the N-terminal half of a split prime editor fusion protein, and the C-extein can correspond to the C-terminal half of a split prime editor. (b) illustrates the chemical mechanism of intein excision and reformation of the peptide bond linking the N-extein half (half in red) and the C-extein half (half in blue). The excision of a split intein (i.e., the N intein and C intein in a split-intein configuration) can also be referred to as "trans-splicing" since it involves the splicing action of two separate components provided in trans.

Figure 68A demonstrates that delivery of both split-intein halves of SpPE (SEQ ID NO: 762) in a linker maintains activity at the three test loci when co-transfected into HEK293T cells.

Figure 68B demonstrates that delivery of both split-intein halves of SaPE2 (e.g., sequence number 443 and sequence number 450) recapitulates the activity of full-length SaPE2 (sequence number 134) when cotransfected into HEK293T cells .

The residues indicated in quotation marks are the sequence of amino acids 741-743 of SaCa s 9 (the first residues of the C-terminal extein), which are important for the intein trans-splicing reaction. "SMP" are the native residues. We also mutated these to the "CFN" consensus splicing sequence. The consensus sequence has been shown to yield the highest rearrangements, as measured by prime editing percentage.

Figure 68C provides data showing that a variety of the disclosed PE ribonucleoprotein complexes (high concentration PE2, high concentration PE3, and low concentration PE3) can be delivered in this manner.

Figure 69 shows a bacteriophage plaque assay to determine PE efficacy in PANCE. Plaques (dark circles) indicate phages capable of successfully infecting E. coli. Increasing the concentration of L-rhamnose results in increased expression of PE and increased plaque formation. Plaque sequencing revealed the presence of genome edits incorporated by PE.

Figures 70A-70I provide examples of edited target sequences as an illustration of step-by-step instructions for designing PEgRNAs and nicking sgRNAs for prime editing. Figure 70A: Step 1. Define target sequence and edit. Retrieve the sequence of the target DNA region (~200 bp) centered on the location of the desired edit (point mutation, insertion, deletion, or combination thereof). Figure 70B: Step 2. Locate the target PAM. Identify the PAM proximal to the edit location. Take care to look for PAMs on both strands. Although a PAM proximal to the edit location is preferred, it is possible to incorporate an edit using a protospacer and PAM that places the nick ≥ 30 nt from the edit location. Figure 70C: Step 3. Locate the nick site. For each PAM to be considered, identify the corresponding nick site. For Sp Cas9 H840A nickase, cleavage occurs on the PAM-containing strand between the third and fourth bases 5' of the NGG PAM. All edited nucleotides must be 3' to the nick site. Therefore, an appropriate PAM must 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 demonstrate the design of a PEgRNA using only PAM 1. Figure 70D: Step 4. Design the spacer sequence. The protospacer of SpCas9 corresponds to the 20 nucleotides 5' of the NGG PAM on the PAM-containing strand. Efficient Pol III transcription initiation requires that G be the first transcribed nucleotide. If the first nucleotide of the protospacer is G, then the spacer sequence of the PEgRNA is simply the protospacer sequence. If the first nucleotide of the protospacer is not G, then the spacer sequence of the PEgRNA is G, then the protospacer sequence. Figure 70E: Step 5. Design the primer binding site (PBS). Using the starting allele sequence, identify a 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 fourth base 5' of the NGG PAM for SpCas9). 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 containing 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. Regardless of GC content, optimal PBS sequences should be determined empirically. To design a PBS sequence of length p, use the starting allele sequence to take the reverse complement of the first p nucleotides 5' of the nick site on the PAM-containing strand. Figure 70F: Step 6. Design the RT template. The RT template encodes the designed edit and homology to sequences flanking the edit. Optimal RT template length will vary based on the target site. For short-range editing (positions +1 to +6), it is recommended to test short (9 to 12 nt), medium (13 to 16 nt), and long (17 to 20 nt) RT templates. For long-range editing (positions +7 and beyond), it is recommended to use an RT template that extends at least 5 nt (preferably 10 nt or more) beyond the position of editing to allow sufficient 3' DNA flap homology. For long-range editing, several RT templates should be screened to identify functional designs. For larger insertions and deletions (≥ 5 nt), incorporation of more 3' homology (~20 nt or more) on the RT template is recommended. Editing efficiency is typically compromised when the RT template codes for the synthesis of G (corresponding to C in the RT template for PEgRNA) as the last nucleotide on the reverse transcribed DNA product. Because many RT templates support efficient prime editing, avoidance of G as the last synthesized nucleotide is recommended when designing the RT template. To design an RT template sequence of length r, use the desired allele sequence and take the reverse complement of the first r nucleotides 3' of the nick site on the strand that originally contained the PAM. Note that, compared to SNP editing, an insertion or deletion edit using an RT template of the same length will not contain identical homology. Figure 70G: Step 7. Assemble the complete PEgRNA sequence. Concatenate the PEgRNA components in the following order (5' to 3'): spacer, backbone, RT template, and PBS. Figure 70H: Step 8. Design a nicking sgRNA for PE3. Identify PAMs on the non-edited strand upstream and downstream of the edit. The optimal nicking position is highly locus-dependent and should be determined empirically. In general, a nick placed 40 to 90 nucleotides 5' opposite the PEgRNA-induced nick leads to higher editing yields and fewer indels. The nicking sgRNA has a spacer sequence that matches the 20 nt protospacer on the starting allele. If the protospacer does not begin with a G, then have a 5' G added. Figure 70I: Step 9. Design a PE3b nicking sgRNA. If a PAM is present on the complementary strand and its corresponding protospacer overlaps with the sequence targeted for editing, then this edit may 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, not the starting allele. The PE3b system works efficiently when the nucleotide(s) to be edited fall within the seed region (~10 nt adjacent to the PAM) of the nicking sgRNA protospacer. This prevents nicking of the complementary strand until after incorporation of the edited strand, preventing competition between the PEgRNA and the sgRNA for binding to the target DNA. PE3b also avoids the generation of nicks on both strands simultaneously, thus significantly reducing indel formation while maintaining high editing efficiency. The PE3b sgRNA should have a spacer sequence that matches the 20 nt protospacer of the desired allele, with the addition of a 5' G if needed.

Figure 71A shows the nucleotide sequence of the SpCas9 PEgRNA molecule (top), which terminates at the 3' end with "UUU" and does not contain a toe loop element. The lower portion of the figure illustrates the same SpCas9 PEgRNA molecule, but further modified to contain a toe loop element with the sequence 5'-"GAAANNNNN"-3' inserted immediately prior to the "UUU" 3' end. "N" can be any nucleobase.

Figure 71B shows the results of Example 18, demonstrating that the efficiency of prime editing in HEK or EMX cells is increased using a PEgRNA containing a toe loop element, while the percentage of indel formation remains largely unchanged.

Figures 72A-72C illustrate alternative PEgRNA configurations that can be used for prime editing. Figure 72A illustrates the PE2:PEgRNA embodiment of prime editing. This embodiment involves PE2 (a fusion protein comprising Cas9 and reverse transcriptase) complexed with PEgRNA (as also depicted in Figures 1A-1I and/or Figures 3A-3E). In this embodiment, a reverse transcription template is incorporated onto the 3' extension arm of the sgRNA to make the PEgRNA, and the DNA polymerase enzyme is reverse transcriptase (RT) fused directly to Cas9. Figure 72B illustrates the MS2cp-PE2:sgRNA+tPERT embodiment. This embodiment includes a PE2 fusion (Cas9+reverse transcriptase), which 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 sgRNA, which targets the complex to a specific target site on DNA. Then, embodiments involve the introduction of a trans-prime editing RNA template ("tPERT"), which acts in place of the PEgRNA by providing a primer binding site (PBS) and a DNA synthesis template by a separate molecule, i.e., tPERT. It also comprises an MS2 aptamer (stem loop). The MS2cp protein recruits tPERT by binding to the MS2 aptamer of the molecule. Figure 72C illustrates alternative designs of PEgRNA that can be achieved by 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, where the extension arm of the hybrid PEgRNA is DNA instead of RNA. In such embodiments, a DNA-dependent DNA polymerase can be used in place of reverse transcriptase to synthesize a 3' DNA flap containing the desired genetic change formed by prime editing. In another embodiment, the extension arm can be synthesized to include a chemical linker, which prevents the DNA polymerase (e.g., reverse transcriptase) from using the sgRNA backbone or backbone as a template. In yet another embodiment, the extension arm can include a DNA synthesis template that has a reverse orientation relative to the overall orientation of the PEgRNA molecule. For example, as shown for a PEgRNA with an extension attached to the 3' end of the sgRNA backbone and in a 5' to 3' orientation, the DNA synthesis template faces the opposite direction, i.e., 3' to 5'. This embodiment can be advantageous for PEgRNA embodiments with an extension arm positioned at the 3' end of the gRNA. By reversing the orientation of the extension arm, DNA synthesis by the polymerase (e.g., reverse transcriptase) will terminate once it reaches the 5' of the new orientation of the extension arm, and therefore there will be no risk of using the gRNA core as a template.

Figure 73 demonstrates prime editing by tPERT and the MS2 recruitment system (also known as MS2 tagging technology). An sgRNA targeting the prime editor protein (PE2) to the target locus is expressed in combination with tPERT containing a primer binding site (13nt or 17nt PBS), a RT template encoding a His6 tag insert and homology arms, and an MS2 aptamer (located at the 5' or 3' end of the tPERT molecule). Either the prime editor protein (PE2) or a fusion of MS2cp to the N-terminus of PE2 was used. As with the previously developed PE3 system, editing was performed with or without a complementary strand nicking sgRNA (designated on the x-axis as labeled "PE2+nick" or "PE2", respectively). This is also referred to as "second strand nicking" and is defined herein.

Figure 74 demonstrates the MS2 aptamer expression of reverse transcriptase in trans and its recruitment by the MS2 aptamer system. The PEgRNA contains the MS2 RNA aptamer inserted into either one of the two sgRNA backbone hairpins. Wild-type M-MLV reverse transcriptase is expressed as an N- or C-terminal fusion to the MS2 coat protein (MCP). Editing is at the HEK3 site in HEK293T cells.

Figure 75 provides a bar graph comparing the efficiency (i.e., "% of total sequencing reads with a defined edit or indel") of PE2, PE2-trunc, PE3, and PE3-trunc for different target sites in various cell lines. The data show that prime editors containing truncated RT variants were about as efficient as prime editors containing the untruncated RT protein.

Figure 76 demonstrates the editing efficiency of the intein split prime editor of Example 20. HEK239T cells were transfected with plasmids encoding full length PE2 or intein split PE2, PEgRNA, and a nicking guide RNA. The consensus sequence (most amino terminal residue of the C-terminal extein) is indicated. Percent editing at two sites is shown: HEK3 +1 CTT insertion and PRNP +6 G to T. Replicate n=3 independent transfections. See Example 20.

Figure 77 demonstrates the editing efficiency of the intein split-primed editing factor of Example 20. Editing was assessed by delivery of 5E10vg and a small amount of 1E10 nuclear-localized GFP:KASH per half SpPE3 to P0 mice by ICV injection, and by targeted deep sequencing of bulk cortical and GFP+ subpopulations. Editing factors and GFP were packaged into AAV9 with an EFS promoter. Mice were harvested 3 weeks after injection and GFP+ nuclei were isolated by flow cytometry. Individual data points are shown for 1-2 mice per condition analyzed. See Example 20.

Figure 78 demonstrates the editing efficiency of the intein split prime editor of Example 20. Specifically, the figure illustrates the AV split SpPE3 construct used in Example 20. Co-transduction with AAV particles expressing SpPE3-N and SpPE3-C separately recapitulates PE3 activity. Note that the N-terminal genome contains a U6-sgRNA cassette expressing a nicking sgRNA, and the C-terminal genome contains a U6-PEgRNA cassette expressing a PEgRNA. See Example 20.

Figure 79 shows the editing efficiency of certain optimized linkers discussed in Example 21. In particular, the data shows the editing efficiency of the PE2 construct with the current linker (labeled PE2, white box) compared to various versions in which the linker was replaced by the indicated sequence for representative PEgRNAs for transition, transversion, insertion, and deletion edits at the HEK3, EMX1, FANCF, and RNF2 loci. The replacement linkers are referred to as "1xSGGS" (SEQ ID NO:174) , "2xSGGS" (SEQ ID NO:446) , "3xSGGS" (SEQ ID NO:3889) , "1xXTEN" (SEQ ID NO:171) , "No Linker", "1xGly", "1xPro", "1xEAAAK" (SEQ ID NO:3968) , "2xEAAAK" (SEQ ID NO:3969) , and "3xEAAAK" (SEQ ID NO:3970) . Editing efficiency is measured in a bar graph format relative to the "control" editing efficiency of PE2. The linker for PE2 is SGGSSGGSSGSETPGTSESATPESSGGSSGGSS (SEQ ID NO: 127). All edits were made in the context of the PE3 system. That is, this refers to the addition of the PE2 editing construct plus an optimal secondary sgRNA nicking guide. See Example 21.

Taking the average fold potency relative to PE2 produces the graph shown, indicating that use of the 1×XTEN (SEQ ID NO: 171) linker sequence improves editing efficiency by an average of 1.14-fold (n=15). See Example 21.

FIG. 81 illustrates the transcription levels of PEgRNA from different promoters, as described in Example 22.

As illustrated in Example 22, the impact of different types of modifications on the PEgRNA structure on the editing efficiency relative to unmodified PEgRNA.

Figure 83 illustrates a PE experiment that targeted editing of a HEK3 gene. The insertion of a 10 nt insertion at position +1 relative to the nick site was specifically targeted and PE3 was used. See Example 22.

Figure 84A illustrates an exemplary PEgRNA with a spacer, gRNA core, and extension arm (RT template + primer binding site). It is modified at the 3' end of the PEgRNA with a tRNA molecule coupled via a UCU linker. The tRNA includes various post-transcriptional modifications. However, the modifications are not required.

Figure 84B illustrates the structure of a tRNA that can be used to modify the PEgRNA structure. See Example 22. P1 can be variable in length. P1 can be elongated to help prevent RNAse P processing of the PEgRNA-tRNA fusion.

Figure 85 illustrates a PE experiment that targeted editing of the FANCF gene. The G to T conversion at position +5 relative to the nick site was specifically targeted and used the PE3 construct. See Example 22.

Figure 86 illustrates a PE experiment that targeted editing of the HEK3 gene. The insertion of a 71 nt FLAG tag insertion at position +1 relative to the nick site was specifically targeted using the PE3 construct. See Example 22.

Results from screening of N2A cells where pegRNA incorporates 1412Adel, with details of primer binding site (PBS) length and reverse transcriptase (RT) template length shown with and without indels. See Example 23.

Results from screening of N2A cells where pegRNA incorporates 1412Adel, with details of primer binding site (PBS) length and reverse transcriptase (RT) template length shown with and without indels. See Example 23.

Figure 89 illustrates the results of editing at the proxy locus of the β-globin gene in healthy HSCs and in HEK3. Varying concentrations of editing factors versus pegRNA and nicking gRNA. See Example 23.

Definitions : Unless otherwise specified, all technical and scientific terms used in this application have the meanings commonly understood by those skilled in the art to which this invention belongs. The following references provide those skilled in the art with general definitions 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). Unless otherwise specified, the following terms used in this application have the meanings ascribed to them.

Antisense strand In genetics, the "antisense" strand of a segment of double-stranded DNA is considered to be the template strand and runs in a 3'→5' orientation. In contrast, the "sense" strand is the segment of double-stranded DNA that runs 5' to 3' and is complementary to the antisense or template strand of the 3' to 5' DNA. In the case of a DNA segment that codes for a protein, the sense strand is the strand of DNA that has the same sequence as the mRNA that takes the antisense strand as its template during transcription and ends up (typically, but not always) being translated into a protein. Thus, the antisense strand carries the RNA that is then translated into a protein, while the sense strand possesses nearly identical makeup to the mRNA. Note that for each segment of dsDNA, there will likely be two sets of sense and antisense (since sense and antisense are relative viewpoints), depending on which direction one is read from. The gene product or mRNA ultimately refers to either strand of a segment of dsDNA, which is referred to as sense or antisense.

Dual-specific ligands The term "dual-specific ligand" or "dual-specific moiety" as used herein refers to a ligand that binds to two different ligand-binding domains. In some embodiments, the ligand is a small molecule compound or a peptide or polypeptide. In other embodiments, the ligand-binding domain is a "dimerization domain" that can be incorporated onto a protein as a peptide tag. In various embodiments, two proteins, each containing the same or different dimerization domains, can be induced to dimerize by binding of each dimerization domain to a dual-specific ligand. A "dual-specific ligand" as used herein can be equivalently referred to as a "chemical inducer of dimerization" or "CID".

Cas9
The term "Cas9" or "Cas9 nuclease" refers to an RNA-guided nuclease that includes a Cas9 domain or a fragment thereof (e.g., a protein that includes an active or inactive DNA cleavage domain of Cas9 and/or a gRNA binding domain of Cas9). A "Cas9 domain" as used herein is a protein fragment that includes an active or inactive cleavage domain of Cas9 and/or a gRNA binding domain of Cas9. A "Cas9 protein" is a full - length Cas9 protein. Cas9 nuclease is also sometimes referred to as casn1 nuclease or 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 a spacer, a sequence complementary to the preceding mobile element, and a targeting invading nucleic acid. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems, corrective processing of pre-crRNA requires a trans-encoded small RNA (tracrRNA), an endogenous ribonuclease 3 (rnc), and a Cas9 domain. The tracrRNA acts as a guide for ribonuclease 3-aided processing of the pre-crRNA. Cas9/crRNA/tracrRNA then endonucleolytically cleaves linear or circular dsDNA targets that are complementary to the spacer. Target strands that are not complementary to the crRNA are first endonucleolytically cleaved and then 3'-5' exonucleolytically excised. In nature, DNA binding and cleavage typically require both proteins and RNAs. However, single guide RNAs ("sgRNAs", or simply "gNRAs") can be modified to incorporate aspects of both crRNA and tracrRNA into a single RNA species. See, for example, Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna JA, Charpentier E. Science 337:816-821 (2012), the entire contents of which are incorporated herein by reference. Cas9 recognizes short motifs in the CRISPR repeats (PAM or protospacer adjacent motifs) to help distinguish self versus non-self. The Cas9 nuclease sequence and structure are well known to those skilled in the art (see, for example, "Complete genome sequence of an M1 strain of Streptococcus pyogenes." Ferretti et al., JJ, McShan WM, Ajdic DJ, Savic DJ, Savic G., Lyon K., Primeaux C., Sezate S., Suvorov AN, Kenton S., Lai HS, Lin SP, Qian Y., Jia HG, Najar FZ, Ren Q., Zhu H., Song L., White J., Yuan X., Clifton SW, Roe BA, McLaughlin RE, Proc. Natl. Acad. Sci. USA 98:4658-4663 (2001); "CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III." Deltcheva E., Chylinski K., Sharma CM, Gonzales See Jinek M., Chao Y., Pirzada ZA, Eckert MR, 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 JA, 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 a variety of species, including, but not limited to, S. pyogenes and S. thermophilus. Additional suitable Cas9 nucleases and sequences will be apparent to one of skill in the art based on the present disclosure. Such Cas9 nucleases and sequences also 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, the Cas9 nuclease comprises one or more mutations that partially impair or inactivate the DNA cleavage domain.

A nuclease-inactivated Cas9 domain may be interchangeably referred to as a "dCas9" protein (representing a nuclease-inactivated Cas9). Methods for generating a Cas9 domain (or a fragment thereof) with 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, 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, the proteins comprise 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". Cas9 variants share homology with Cas9 or fragments 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 a 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 a wild-type Cas9 (e.g., SpCas9 of SEQ ID NO: 18). In some embodiments, the Cas9 variant comprises a fragment of SEQ ID NO:X 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 a corresponding fragment of a 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. The cDNA is complementary to the RNA template.

Circular permutants As used herein, the term "circular permutant" refers to a protein or polypeptide (e.g., Cas9) that contains a circular permutation, which is a change in the structural conformation of a protein with a change in the order of amino acids that appear in the amino acid sequence of the protein. In other words, a circular permutant is a protein with an altered N-terminus and C-terminus compared to its wild-type counterpart, e.g., the C-terminal half of the wild-type protein becomes a new N-terminal half. Circular permutation (or CP) is essentially a topological rearrangement of a protein primary sequence with its N-terminus and C-terminus connected, often with a peptide linker, while simultaneously splitting the sequence at a different position to create new adjacent N-terminus and C-terminus. The result is a protein structure that may have different connections but often the same or overall similar three-dimensional (3D) shape, possibly including improved or altered characteristics, including reduced susceptibility to proteolysis, improved catalytic activity, altered substrate or ligand binding, and/or improved thermostability. Circularly permuted proteins can occur naturally (e.g., concanavalin A and lectins). In addition, circular permutations can occur as a result of post-translational modifications or can be engineered using recombinant techniques.

Circularly permuted Cas9
The term "circularly permuted Cas9" refers to any Cas9 protein or variant thereof that has been generated as a circular permutation, thereby locally rearranging its N-terminus and C-terminus. Such circularly permuted Cas9 protein ("CP-Cas9"), or variants thereof, retains the ability to bind to DNA when complexed with 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, January 10, 2019, 176:254-267, each of which is incorporated herein by reference. The present disclosure contemplates any previously known or new CP-Cas9 to be used, 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 infection by viruses that have invaded prokaryotes. The snippets of DNA are used by prokaryotic cells to detect and destroy DNA from subsequent attacks by similar viruses, and together with an array of CRISPR-associated proteins (including Cas9 and its homologs) and CRISPR-associated RNAs, effectively constitute 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 the pre-crRNA requires a trans-encoded small RNA (tracrRNA), an endogenous ribonuclease 3 (rnc), and a Cas9 protein. The tracrRNA serves as a guide for ribonuclease 3-assisted processing of the pre-crRNA. Cas9/crRNA/tracrRNA then cleaves linear or circular dsDNA targets that are complementary to the RNA endolytically. Specifically, the target strand that is not complementary to the crRNA is first endolytically cut and then 3'-5' exolytically trimmed. In nature, DNA binding and cleavage typically requires a protein and both RNAs. However, single guide RNAs ("sgRNAs" or simply "gNRAs") can be engineered to incorporate aspects of both crRNA and tracrRNA into the guide RNA of a single RNA species. See, for example, Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna JA, Charpentier E. Science 337:816-821 (2012), the entire contents of which are incorporated herein by reference. Cas9 recognizes short motifs (PAM or protospacer adjacent motifs) on CRISPR repeats to help distinguish self versus non-self. CRISPR biology and 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., JJ, McShan WM, Ajdic DJ, Savic DJ, Savic G., Lyon K., Primeaux C., Sezate S., Suvorov AN, Kenton S., Lai HS, Lin SP, Qian Y., Jia HG, Najar FZ, Ren Q., Zhu H., Song L., White J., Yuan X., Clifton SW, Roe BA, McLaughlin RE, Proc. Natl. Acad. Sci. USA 98:4658-4663 (2001); "CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III." Deltcheva E., Chylinski K., Sharma See CM, Gonzales K., Chao Y., Pirzada ZA, Eckert MR, 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 JA, 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 a variety of species, including, but not limited to, S. pyogenes and S. thermophilus. Additional suitable Cas9 nucleases and sequences will be apparent to one of skill in the art based on the present disclosure. Such Cas9 nucleases and sequences include Cas9 sequences from 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 the pre-crRNA requires a trans-encoded small RNA (tracrRNA), an endogenous ribonuclease 3 (rnc), and a Cas9 protein. The tracrRNA serves as a guide for ribonuclease 3-assisted processing of the pre-crRNA. Cas9/crRNA/tracrRNA then endolytically cleaves linear or circular nucleic acid targets that are complementary to the RNA. Specifically, the target strand that is not complementary to the crRNA is first endolytically cut and then 3'-5' exolytically trimmed. In nature, DNA binding and cleavage typically requires a protein and both RNAs. However, single guide RNAs ("sgRNAs" or simply "gRNAs") can be engineered to incorporate aspects of both the crRNA and tracrRNA into the guide RNA of a single RNA species.

In general, a "CRISPR system" collectively refers to the transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated ("Cas") genes, and includes sequences encoding Cas genes, tracr (transactivating CRISPR) sequences (e.g., tracrRNA or an active partial tracrRNA), tracr mate sequences (which, in the context of an endogenous CRISPR system, encompass "direct repeats" and partial direct repeats processed by tracrRNA), guide sequences (also referred to as "spacers" in the context of an endogenous CRISPR system), or other sequences and transcripts from the 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 the PEgRNA that is utilized by the polymerase of the prime editor as the template strand (containing the desired edit and encoding the 3' single-stranded DNA flap that then replaces the corresponding strand of endogenous DNA at the target site through the mechanism of prime editing). In various embodiments, the DNA synthesis template is shown in Figure 3A (in the context of a PEgRNA containing a 5' extension arm), Figure 3B (in the context of a PEgRNA containing a 3' extension arm), Figure 3C (in the context of an internal extension arm), Figure 3D (in the context of a 3' extension arm), and Figure 3E (in the context of a 5' extension arm). The extension arm encompasses the DNA synthesis template and may be composed of DNA or RNA. In the case of RNA, the polymerase of the prime editor may be an RNA-dependent DNA polymerase (e.g., reverse transcriptase). In the case of DNA, the polymerase of the prime editor may be a DNA-dependent DNA polymerase. In various embodiments (e.g., as depicted in Figures 3D-3E), the DNA synthesis template (4) may comprise the "editing template" and the "homology arm", as well as all or part of the optional 5'-end modification region e2. That is, depending on the nature of the e2 region (e.g., whether it comprises a hairpin, a toe-loop, or a stem/loop secondary structure), the polymerase may encode none, or a part or all of the e2 region. In other words, in the case of a 3' extension arm, the DNA synthesis template (3) may comprise a portion of the extension arm (3) that extends from the 5' end of the primer binding site (PBS) to the 3' end of the gRNA core, which may act as a template for the synthesis of a single strand of DNA by a polymerase (e.g., reverse transcriptase). In the case of a 5' extension arm, the DNA synthesis template (3) may comprise a portion of the extension arm (3) that extends from the 5' end of the PEgRNA molecule to the 3' end of the editing template. Preferably, the DNA synthesis template excludes the primer binding site (PBS) of the PEgRNAs with either the 3' extension arm or the 5' extension arm. Certain embodiments described herein (for example, FIG. 71A) refer to an "RT template" that includes the editing template and the homology arm, i.e., the sequence of the PEgRNA extension arm that 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., Figures 3G and 3H), the primer binding site (PBS) and the DNA synthesis template can be engineered into separate molecules called trans-prime editor RNA template (tPERT).

Dimerization domain The term "dimerization domain" refers to a ligand binding domain that binds to a binding domain of a dual-specific ligand. A "first" dimerization domain binds to a first binding moiety of a dual-specific ligand, and a "second" dimerization domain binds to a second binding moiety of the same dual-specific ligand. When a first dimerization domain is fused (e.g., via PE as discussed herein) to a first protein and a second dimerization domain is fused (e.g., via PE as discussed herein) to a second protein, the first and second proteins dimerize in the presence of the dual-specific ligand, and the dual-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 relative terms that define the linear location of at least two elements located in a nucleic acid molecule (whether single-stranded or double-stranded) oriented in a 5'→3' direction. In particular, if a first element is located anywhere 5' to the second element, the first element is upstream of the second element in the nucleic acid molecule. For example, if the SNP is 5' to the nick site, the SNP is upstream of the Cas9-induced nick site. Conversely, if a first element is located anywhere 3' to the second element, the first element is downstream of the second element in the nucleic acid molecule. For example, if the SNP is 3' to the nick site, the SNP is downstream of the Cas9-induced nick site. The nucleic acid molecule can be DNA (double-stranded or single-stranded), RNA (double-stranded or single-stranded), or a hybrid of DNA and RNA. The analysis is the same for single-stranded nucleic acid molecules and double-stranded molecules, except when it is necessary to select which strand of a double-stranded molecule is considered, where the terms upstream and downstream refer only to a single strand of a nucleic acid molecule. Often, the strand of a double-stranded DNA that can be used to determine the relative positions of at least two elements is the "sense" strand or "coding" strand. In genetics, a "sense" strand is a segment within a double-stranded DNA that extends from 5' to 3' and is complementary to the antisense or template strand of the DNA that extends from 3' to 5'. Thus, as an example, if a SNP nucleobase is 3' to the promoter on the sense or coding strand, the SNP nucleobase is "downstream" of the promoter sequence in genomic DNA (which is double-stranded).

Editing template The term "editing template" refers to the portion of the extension arm that encodes the desired edit on the single-stranded 3' DNA flap that is synthesized by a polymerase, e.g., a DNA-dependent DNA polymerase, an RNA-dependent DNA polymerase (e.g., reverse transcriptase). Certain embodiments described herein (e.g., FIG. 71A) refer to an "RT template" which refers to both the editing template and the homologous arm together, i.e., the sequence of the PEgRNA extension arm that is actually used as a template during DNA synthesis. The term "RT editing template" is also equivalent to the term "DNA synthesis template," where RT editing template reflects the use of a primed editor with a polymerase that is a reverse transcriptase, and DNA synthesis template more broadly reflects the use of a primed editor with any polymerase.

Effective amount The term "effective amount" as used herein refers to an amount of a bioactive agent that is sufficient to induce a desired biological response. For example, in some embodiments, an effective amount of a prime editor (PE) can refer to an amount of the editor that is sufficient to edit a target site nucleotide sequence, such as a genome. In some embodiments, an effective amount of a prime editor (PE) provided herein, such as a fusion protein comprising a nickase Cas9 domain and a reverse transcriptase, can refer to an amount of the fusion protein that is sufficient to induce editing of a target site that is specifically bound and edited by the fusion protein. As will be understood by those skilled in the art, the effective amount of an agent, such as a fusion protein, a nuclease, a hybrid protein, a protein dimer, a complex of a protein (or a protein dimer) and a polynucleotide, or a polynucleotide, can vary depending on various factors, such as, for example, the desired biological response, such as the specific allele, genome, or target site to be edited, the cell or tissue to be targeted, and the agent to be used.

Error-prone reverse transcriptases 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 is naturally occurring or derived from another reverse transcriptase (e.g., wild-type M-MLV reverse transcriptase) that has an error rate less than that of wild-type M-MLV reverse transcriptase. The error rate of wild-type M-MLV reverse transcriptase has been reported to range from 15,000 (higher) to 27,000 (lower) for 1 error. An error rate of 1 in 15,000 corresponds to an error rate of 6.7×10 ⁻⁵ . An error rate of 1 in 27,000 corresponds to 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 a sequence that is more than 1 error in the incorporation of 15,000 nucleobases (6.7×10 ^-5 or higher), for example 1 error in 14,000 nucleobases (7.14×10 ^-5 or higher), 1 error in 13,000 nucleobases or less (7.7×10 ^-5 or higher), 1 error in 12,000 nucleobases or less (7.7×10 ^-5 or higher), 1 error in 11,000 nucleobases or less (9.1×10 ^-5 or higher), 1 error in 10,000 nucleobases or less (1×10 ^-4 or 0.0001 or higher), 1 error in 9,000 nucleobases or less (0.00011 or higher), 1 error in 8,000 nucleobases or less (0.00013 or higher), 1 error in 7,000 nucleobases or less (0.00014 or higher), 1 error in 6,000 nucleobases or less (0.00016 or higher), 1 error in 5,000 nucleobases or less (0.0002 or higher), 1 error in 4,000 nucleobases or less "RTs" refer to those RTs that have an error rate that is 1 error (0.00025 or higher), 1 error in 3,000 nucleobases or less (0.00033 or higher), 1 error in 2,000 nucleobases or less (0.00050 or higher), or 1 error in 1,000 nucleobases or less (0.001 or higher), or 1 error in 500 nucleobases or less (0.002 or higher), or 1 error in 250 nucleobases or less (0.004 or higher).

Extein The term "extein" as used herein refers to a polypeptide sequence that is flanked by an intein and 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, which are ligated together when the intein catalyzes its own excision. An extein is thus a protein analog to an exon found on an mRNA. For example, a polypeptide containing an intein can have the structure extein(N)-intein-extein(C). After excision of the intein and splicing of the two exteins, the resulting structure is extein(N)-extein(C) and a free intein. In various configurations, the exteins can be separate proteins (e.g., half of a Cas9 or PE fusion protein), each fused to a split intein, and excision of the split intein causes the extein sequences to be spliced together.

The term "extension arm" refers to a nucleotide sequence component of the PEgRNA that serves several functions, including a primer binding site and an editing template for reverse transcriptase. In some embodiments, for example in FIG. 3D, the extension arm is located at the 3' end of the guide RNA. In other embodiments, for example in 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 includes the following components in the 5'→3' direction: a homology arm, an editing template, and a primer binding site. Since the polymerization activity of reverse transcriptase is in the 5'→3' direction, the preferred arrangement of the homology arm, editing template, and primer binding site is in the 5'→3' direction such that the reverse transcriptase, once primed by the annealed primer sequence, polymerases the single-stranded DNA using the editing template as the 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 generally comprising two regions: a primer binding site (PBS) and a DNA synthesis template, as illustratively shown in FIG. 3G (top row). The primer binding site binds to a primer sequence formed from the endogenous DNA strand of the target site when it becomes nicked by the prime editor complex, thereby exposing its 3' end on the nicked endogenous strand. As described herein, 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., 3' of the primer sequence), which then provides a substrate for a polymerase that begins polymerizing a single strand of DNA from the exposed 3' end along the length of the DNA synthesis template. The sequence of the single-stranded DNA product is the complement of the DNA synthesis template. Polymerization continues 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 by the polymerase of the primed editor complex into a single-stranded DNA product (i.e., a 3' single-stranded DNA flap containing the desired gene editing information), which replaces the corresponding endogenous DNA strand at the target site immediately downstream of the PE-induced nick site. Without being bound by theory, polymerization of the DNA synthesis template continues to the 5' end of the extension arm until an event known as termination. Polymerization may terminate in a variety of ways, including, but not limited to, (a) reaching the 5' end of the PEgRNA (e.g., in the case of a 5' extension arm, the DNA polymerase simply runs out of template), (b) reaching an RNA secondary structure that cannot be traversed (e.g., a 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 signal on a nucleic acid form such as supercoiled DNA or RNA.

Flap endonucleases (e.g., FEN1)
As used herein, the term "flap endonucleases" refers to enzymes that catalyze the removal of 5' single-stranded 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 described herein may utilize endogenously supplied flap endonucleases or those provided in trans to remove 5' flaps of endogenous DNA formed at the target site during prime editing. Flap endonucleases are known in the art and can be found 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, and Balakrishnan et al., "Flap Endonuclease 1," Annu Rev Biochem, 2013, Vol 82:119-138, each of which is incorporated herein by reference. An exemplary flap endonuclease is FEN1, which can be represented by the following amino acid sequence:

Functional equivalent The term "functional equivalent" refers to a second biomolecule that is functionally equivalent but not necessarily structurally equivalent to a first biomolecule. For example, a "Cas9 equivalent" refers to a protein that has the same or substantially the same function as Cas9, but does not necessarily have the same amino acid sequence. In the context of this disclosure, the specification refers throughout to "protein X, or a functional equivalent thereof." In this context, a "functional equivalent" of protein X embraces any homolog, paralog, fragment, naturally occurring version, modified version, mutated version, or synthetic version that bears the equivalent function of protein X.

Fusion Protein The term "fusion protein" as used herein refers to a hybrid polypeptide that contains protein domains from at least two different proteins. A protein may be located at the amino-terminal (N-terminal) portion of the fusion protein or at the carboxy-terminal (C-terminal) portion of the protein, thus forming an "amino-terminal fusion protein" or a "carboxy-terminal fusion protein", respectively. A protein may contain different domains, such as a nucleic acid binding domain (e.g., the gRNA binding domain of Cas9, which directs the protein to bind to a target site) and a nucleic acid cleavage domain or catalytic domain of a nucleic acid editing protein. Another example includes Cas9 fused with reverse transcriptase or its equivalent. 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 particularly suitable for fusion proteins that include peptide linkers. Methods for expression and purification of recombinant proteins 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, NY (2012) (the contents of which are incorporated herein by reference in their entirety).

Gene of interest (GOI)
The term "gene of interest" or "GOI" refers to a gene that codes for a biomolecule of interest (e.g., a protein or an RNA molecule). The protein of interest may include any intracellular, membrane, or extracellular protein, e.g., nuclear proteins, transcription factors, nuclear membrane transporters, intracellular organelle-associated proteins, membrane receptors, catalytic proteins, and enzymes, therapeutic proteins, membrane proteins, membrane transport proteins, signal transduction proteins, or immunological proteins (e.g., IgG or other antibody proteins), and the like. The gene of interest may also code for 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" refers to a specific type of guide nucleic acid, most commonly associated with the Cas protein of CRISPR-Cas9, that binds to Cas9 and directs the Cas9 protein to a specific sequence in a DNA molecule (including the complementarity of the guide RNA to the protospace sequence). However, the term also embraces equivalent guide nucleic acid molecules that bind to Cas9 equivalents, homologs, orthologs, or paralogs, whether naturally occurring or not (e.g., modified or recombinant), and that otherwise program the Cas9 equivalent to localize to a specific target nucleotide sequence. Cas9 equivalents may also include other napDNAbp from any type of CRISPR system (e.g., type II, type V, type VI), including Cpf1 (type V CRISPR-Cas system), C2c1 (type V CRISPR-Cas system), C2c2 (type VI CRISPR-Cas system), and C2c3 (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 and structures of guide RNAs are provided herein. In addition, methods for designing suitable guide RNA sequences are also provided herein. As used herein, "guide RNA" may also be referred to as "existing guide RNA" to contrast with the modified form of guide RNA called "prime editing guide RNA" (or "PEgRNA") invented for the prime editing methods and compositions disclosed herein.

Guide RNAs or PEgRNAs may contain a variety of structural elements, including, but not limited to:

Spacer sequence - A sequence in the guide RNA or PEgRNA (approximately 20 nt in length) that binds to the protospacer in the target DNA.

gRNA core (or gRNA backbone or backbone sequence) - refers to the sequence within the gRNA responsible for Cas9 binding, this does not include the spacer/targeting sequence used to guide Cas9 to the target DNA.

Extension arm - a single-stranded extension at the 3' or 5' end of the PEGRNA. It contains a primer binding site and a DNA synthesis template sequence that encodes a single-stranded DNA flap containing the desired genetic change by a polymerase (e.g., reverse transcriptase). This is then incorporated onto the endogenous DNA by displacing the corresponding endogenous strand, thereby incorporating the desired genetic change.

Transcription terminator - The guide RNA or PEgRNA may contain a transcription termination sequence at the 3' end of the molecule.

Homologous arm The term "homologous arm" refers to the portion or portions of the extension arm that code for the resulting portion of the reverse transcriptase-encoded single-stranded DNA flap that is incorporated into the target DNA site by displacing the endogenous strand. The portion of the single-stranded DNA flap that is encoded by the homologous arm is complementary to the non-edited strand of the target DNA sequence, facilitating it to displace the endogenous strand and anneal with the single-stranded DNA flap in its place, thereby incorporating the edit. This component is further defined elsewhere. By definition, the homologous arm is part of the DNA synthesis template, since it is encoded by the polymerase of the prime editor described herein.

Host Cell The term "host cell," as used herein, refers to a cell that can host, replicate and express the vectors described herein, including vectors containing a nucleic acid molecule encoding a fusion protein comprising Cas9 or a Cas9 equivalent and reverse transcriptase.

Intein The term "intein" as used herein refers to a self-processing polypeptide domain found in organisms from all domains of life. An intein (intercalating protein) performs a unique self-processing event known as protein splicing, in which it excises itself from a larger precursor polypeptide by cleavage of two peptide bonds, and in the process ligates flanking extein (external protein) sequences by forming new peptide bonds. Since intein genes are found embedded in-frame with other protein-coding genes, this rearrangement occurs post-translationally (or co-translationally). Furthermore, intein-mediated protein splicing is spontaneous; it requires only the folding of the intein domain, not external factors or energy sources. This process is also known as cis protein splicing, in contrast to the natural process of trans protein splicing by "split inteins". Inteins are the protein equivalents of self-splicing RNA introns (see Perler et al., Nucleic Acids Res. 22:1125-1127 (1994)), which catalyze their own excision from precursor proteins and have the concomitant fusion of flanking protein sequences known as exteins (reviewed in Perler et al., Curr. Opin. Chem. Biol. 1:292-299 (1997); Perler, FB 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 the process by which internal regions (inteins) of a precursor protein are excised and flanking regions (exteins) of the protein 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). Intein units contain the necessary components required to catalyze protein splicing and often contain an endonuclease domain responsible for 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). However, the resulting proteins are linked and are not expressed as separate proteins. Protein splicing can also be performed in trans by split inteins expressed on separate polypeptides. These spontaneously combine to form a single intein, which then goes through the protein splicing process to link separate proteins.

Elucidation of the mechanism of protein splicing has led to a number of intein-based applications (Comb, et al., U.S. Patent No. 5,496,714; Comb, et al., U.S. Patent 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. Biomol. 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 includes a ligand-binding domain. Typically, the ligand-binding domain is inserted onto the amino acid sequence of the intein, resulting in the structure intein(N)-ligand-binding domain-intein(C). Typically, the ligand-dependent intein exerts no or only minimal protein splicing activity in the absence of a suitable ligand, and exerts a significant increase in protein splicing activity in the presence of a ligand. In some embodiments, the ligand-dependent intein exerts no observable splicing activity in the absence of a ligand, but exerts splicing activity in the presence of a ligand. In some embodiments, the ligand-dependent intein exerts observable protein splicing activity in the absence of ligand and in the presence of a suitable ligand, a protein splicing activity that is at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 150-fold, at least 200-fold, at least 250-fold, at least 500-fold, at least 1000-fold, at least 1500-fold, at least 2000-fold, at least 2500-fold, at least 5000-fold, at least 10000-fold, at least 20000-fold, at least 25000-fold, at least 50000-fold, at least 100000-fold, at least 500000-fold, or at least 1000000-fold greater than the activity observed in the absence of ligand. In some embodiments, the increase in activity is dose-dependent over at least one order of magnitude, at least two orders of magnitude, at least three orders of magnitude, at least four orders of magnitude, or at least five orders of magnitude, allowing fine-tuning of intein activity by adjusting the concentration of the ligand. Suitable ligand-dependent inteins are known in the art and include those provided below and in published U.S. Patent Application No. US2014/0065711A1; 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 of which are incorporated herein by reference. Exemplary sequences are as follows:

Linker The term "linker" as used herein refers to a molecule that connects two other molecules or moieties. A linker can be an amino acid sequence in the case of a linker that connects two fusion proteins. For example, Cas9 can be fused to reverse transcriptase by an amino acid linker sequence. A linker can also be a nucleotide sequence in the case of linking two nucleotide sequences together. For example, in this case, a conventional guide RNA is linked to the RNA extension of a primed editing guide RNA, which can include a RT template sequence and a RT primer binding site, via a spacer or linker nucleotide sequence. In other embodiments, the linker is an organic molecule, a group, a polymer, or a chemical moiety. In some embodiments, the linker is 5-100 amino acids in length, e.g., 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 its natural state. For example, a nucleic acid or peptide that is naturally present in a living animal is not "isolated," but the same nucleic acid or peptide that is partially or completely separated from its natural state of coexisting materials is "isolated." An isolated nucleic acid or protein may exist in a substantially purified form or may exist in a non-native environment (such as, for example, a host cell).

In some embodiments, the gene of interest is encoded by an isolated nucleic acid. As used herein, the term "isolated" refers to the character of a material as provided herein that has been removed from its original or native environment (e.g., the natural environment if it occurs in nature). Thus, a naturally occurring polynucleotide or protein or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide that has been separated from some or all of the coexisting materials in a natural system by human intervention is isolated. Artificial or modified materials, such as non-naturally occurring nucleic acid constructs, such as the expression constructs and vectors described herein, are also subsequently referred to as isolated. A material does not have to be purified to be isolated. Consequently, a material may be part of a vector and/or part of a composition, and such a vector or composition is still isolated in that it is not part of the environment in which the material is found in nature.

MS2 tagging technology In various embodiments (e.g., as illustrated in the embodiments of Figures 72-73 and Example 19), the term "MS2 tagging technology" refers to the combination of an "RNA-protein interaction domain" (also known as an "RNA-protein recruitment domain or protein") paired with an RNA-protein interaction domain, e.g., an RNA binding protein that specifically recognizes and binds to a particular hairpin structure. These types of systems can be exploited to recruit various functionalities to a prime editor complex that is bound to a target site. MS2 tagging technology is based on the natural interaction of MS2 bacteriophage coat protein ("MCP" or "MS2cp") with a stem-loop or hairpin structure, i.e., an "MS2 hairpin," present on the genome of a phage. In the case of prime editing, MS2 tagging technology involves introducing an MS2 hairpin onto the desired RNA molecule (e.g., PEgRNA or tPERT) involved in prime editing. This then constitutes a specific, interactive 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 the MCP is fused to another protein (e.g., reverse transcriptase or another DNA polymerase), the MS2 hairpin can be used to "recruit" other proteins in trans to the target site occupied by the prime editing complex.

The prime editors described herein may, as an aspect, incorporate any known RNA-protein interaction domain to recruit or "colocalize" a particular function of interest to the prime editor complex. Reviews of other modular domains of RNA-protein interactions 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 is incorporated herein by reference in its entirety. 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 (i.e., referred to as the "MS2 aptamer") is: GCCAACATGAGGATCACCCATGTCTGCAGGGCC (SEQ ID NO: 763).

The amino acid sequence of MCP or MS2cp is: GSASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVATQTVGGEELPVAGWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY (SEQ ID NO: 764).

The MS2 hairpin (or "MS2 aptamer") may also be referred to as a type of "RNA effector recruitment domain" (or equivalently, "RNA binding protein recruitment domain" or simply "recruitment domain") because it is a physical structure (e.g., a hairpin) that is incorporated onto the PEgRNA or tPERT that effectively recruits other effector functions (e.g., RNA binding proteins with various functions, such as DNA polymerases, or other DNA-modifying enzymes) to the so modified PEgRNA or rPERT, thus colocalizing the effector functions in trans to the prime editing machinery. This application is in no way intended to be limited to any particular RNA effector recruitment domain, but may include any available such domain, including the MS2 hairpin. Example 19 and FIG. 72(b) illustrate the use of a prime editor that includes an MS2 aptamer linked to a DNA synthesis domain (i.e., a tPERT molecule) and an MS2cp protein fused to PE2 to induce colocalization of a prime editor complex (MS2cp-PE2:sgRNA complex) bound to a target DNA site with the DNA synthesis domain of the tPERT molecule.

napDNAbp
As used herein, the term "nucleic acid programmed DNA binding protein" or "napDNAbp" (of which Cas9 is an example) refers to a protein that uses RNA:DNA hybridization to target and bind to a specific sequence in a DNA molecule. Each napDNAbp is linked to at least one guide nucleic acid (e.g., a guide RNA) that localizes the napDNAbp to a DNA sequence that includes a DNA strand (i.e., a target strand) that is complementary to the guide nucleic acid or a portion thereof (e.g., a protospacer of the guide RNA). In other words, the guide nucleic acid "programs" the napDNAbp (e.g., Cas9 or equivalent) to localize and bind to the complementary sequence.

Without being bound by theory, the binding mechanism of the napDNAbp-guide RNA complex generally involves the formation of an R-loop in which the napDNAbp induces unwinding of the double-stranded 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 the "non-target strand" that is complementary to the target strand, thereby forming a single-stranded region of the R-loop. In some embodiments, the napDNAbp contains one or more nuclease activities, which then cleave the DNA to eliminate various types of lesions. For example, the napDNAbp may contain a nuclease activity that cleaves the non-target strand at a first location and/or the target strand at a second location. In response to the nuclease activity, the target DNA may be cleaved to form a "double-stranded break" in which both strands are cleaved. In other embodiments, the target DNA may only be cleaved at a single site, i.e., the DNA is "nicked" on one strand. Exemplary napDNAbps with various nuclease activities include "Cas9 nickase" ("nCas9"), and inactivated Cas9 that does not have any nuclease activity ("inactivated Cas9" or "dCas9"). Exemplary sequences for these and other napDNAbps are provided herein.

The term "nickase" refers to Cas9 with one of its two nuclease domains inactivated, allowing the enzyme to cleave only one strand of the target DNA.

Nuclear localization sequence (NLS)
The term "nuclear localization sequence" or "NLS" refers to an amino acid sequence that facilitates the import of a protein into the cell nucleus, for example, by nuclear transport. Nuclear localization sequences are known in the art and will be apparent to those skilled in the art. For example, NLS sequences are described in International PCT Application PCT/EP2000/011690 to Plank et al., filed November 23, 2000, and published May 31, 2001 as WO/2001/038547, the contents of which are incorporated herein by reference for their disclosure of exemplary nuclear localization sequences. In some embodiments, the NLS comprises the amino acid sequence PKKKRKV (SEQ ID NO: 16) or MDSLLMNRRKFLYQFKNVRWAKGRRETYLC (SEQ ID NO: 17).

Nucleic Acid Molecules The term "nucleic acid" as used herein refers to a polymer of nucleotides. The polymer may be any of a variety of nucleotides, including natural nucleosides (i.e., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine), nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C5 bromouridine, C5 fluorouridine, C5 iodouridine, C5 propynyluridine, C5 propynylcytidine, C5 methylcytidine, 7 deazaadenosine, 7 deazaguanosine, 8 oxoadenosine, 8 oxoguanosine, O(6) methylguanine, 4-acetylcytidine, 5-acetylcytidine, 6-acetylcytidine, 7-acetylcytidine, 8 ... , 5-(carboxyhydroxymethyl)uridine, dihydrouridine, methylpseudouridine, 1-methyladenosine, 1-methylguanosine, N6-methyladenosine, 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., phosphorothioate and 5'-N phosphoramidite linkages).

PEgRNA
As used herein, the term "prime edited guide RNA" or "PEgRNA" or "extended guide RNA" refers to a specialized form of guide RNA that has been modified to include one or more additional sequences to practice the prime editing methods and compositions described herein. As described herein, a prime edited guide RNA includes one or more "extended regions" of a nucleic acid sequence. The extended region may include, but is not limited to, a single strand of RNA or DNA. Additionally, the extended region may occur at the 3' end of an existing guide RNA. In other configurations, the extended region may occur at the 5' end of an existing guide RNA. In yet other configurations, the extended region may occur at a region within the molecule at the end of an existing guide RNA (e.g., in the gRNA core region that binds and/or binds to the napDNAbp). The extended region comprises a "DNA synthesis template" that encodes a single-stranded DNA (by the polymerase of the prime editor), which molecule is then (a) designed to be homologous to the endogenous target DNA to be edited, and (b) contains at least one desired nucleotide change (e.g., a transition, transversion, deletion, or insertion) to be introduced or incorporated into the endogenous target DNA. The extended region may also contain 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, an aptamer, a stem loop, a hairpin, a toe loop (e.g., a 3' toe loop), or an RNA-protein recruitment domain (e.g., an MS2 hairpin)). As used herein, a "primer binding site" comprises a sequence that hybridizes to a single-stranded DNA sequence with a 3' end generated from the R-loop nicked DNA.

In one embodiment, the PEgRNA is represented by FIG. 3A, which shows a PEgRNA with a 5' extension arm, a spacer, and a gRNA core. The 5' extension further comprises a reverse transcriptase template in the 5'→3' direction, a primer binding site, and a linker. As shown, the reverse transcriptase template may also be more broadly referred to as a "DNA synthesis template," where the polymerase of the prime editor described herein is not RT but another type of polymerase.

In certain other embodiments, the PEgRNA is represented by FIG. 3B, which shows a PEgRNA with a 3' extension arm, a spacer, and a gRNA core. The 3' extension further comprises a reverse transcriptase template in the 5'→3' direction, a primer binding site. As shown, the reverse transcriptase template may also be more broadly referred to as a "DNA synthesis template," where the polymerase of the prime editor described herein is not RT but another type of polymerase.

In yet another embodiment, the PEgRNA is represented by FIG. 3D, which shows a PEgRNA having a spacer (1), a gRNA core (2), and an extender arm (3) in the 5' to 3' direction. The extender arm (3) is at the 3' end of the PEgRNA. The extender arm (3) further comprises a "primer binding site" (A), an "editing template" (B), and a "homology arm" (C) in the 5' to 3' direction. The extender arm (3) may also comprise optional modification regions at the 3' and 5' ends, which may be the same or different sequences. Additionally, the 3' end of the PEgRNA may comprise a transcription terminator sequence. These sequence elements of the PEgRNA are further described and defined herein.

In yet other embodiments, the PEgRNA is represented by FIG. 3E, which shows a PEgRNA having, in a 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 a 3' to 5' direction, a "primer binding site" (A), an "editing template" (B), and a "homology arm" (C). The extension arm (3) may also comprise optional modification regions at the 3' and 5' ends, which may be the same or different sequences. The 3' end of the PEgRNA may comprise a transcription terminator sequence. These sequence elements of the PEgRNA are further described and defined herein.

PE1
As used herein, "PE1" refers to a PE complex that includes a fusion protein comprising Cas9(H840A) and wild-type MMLV RT having the following structure: [NLS]-[Cas9(H840A)]-[Linker]-[MMLV_RT(wt)]+desired PE gRNA. The PE fusion has the amino acid sequence of SEQ ID NO: 123, which is shown as follows:

PE2
As used herein, "PE2" refers to a PE complex that includes 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)]+desired PE gRNA. The PE fusion has the amino acid sequence of SEQ ID NO: 134, which is set forth as follows:

PE3
As used herein, "PE3" refers to PE2 plus a second strand nicking guide RNA that complexes with PE2 to introduce a nick on the non-edited DNA strand to induce preferential displacement of the edited strand.
PE3b

As used herein, "PE3b" refers to PE3, where a second strand nicking guide RNA is designed for temporal control such that a second strand nick is not introduced until after incorporation of the desired edit. This is accomplished by designing a gRNA with a spacer sequence that matches only the edited strand and not the original allele. Using this strategy, hereafter referred to as PE3b, mismatches between the protospacer and the non-edited allele should not favor nicking by the sgRNA until after the editing event on the PAM strand has occurred.

PE-short
As used herein, " short PE " refers to a PE construct that is fused to a C-terminal truncated reverse transcriptase and has the following amino acid sequence:

Peptide Tags The term "peptide tag" refers to a peptide amino acid sequence that is genetically fused to a protein sequence to confer one or more functions onto the protein, thereby facilitating manipulation of the protein for various purposes such as visualization, purification, solubilization, separation, etc. Peptide tags can include various types of tags classified by purpose or function, which may include "affinity tags" (to facilitate protein purification), "solubilization tags" (to aid in correct folding of the protein), "chromatography tags" (to alter the chromatographic properties of the protein), "epitope tags" (to bind to high affinity antibodies), "fluorescent tags" (to facilitate visualization of the protein in cells or in vitro).

Polymerase The term "polymerase" as used herein refers to an enzyme that synthesizes a nucleotide strand that can be used in conjunction with the prime editor system described herein. The polymerase can be a "template-dependent" polymerase (i.e., a polymerase that 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 that synthesizes a nucleotide strand without the requirement of a template strand). The polymerase can 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 a case, the DNA template molecule can be a PEgRNA and the extension arm comprises a strand of DNA. In such cases, the PEgRNA may be referred to as a chimeric or hybrid PEgRNA, which includes an RNA portion (i.e., a guide RNA component including a spacer and a gRNA core) and a DNA portion (i.e., an extension arm). In various other embodiments, the DNA polymerase may be an "RNA-dependent DNA polymerase" (i.e., according to which the template molecule is a strand of RNA). In such cases, the PEgRNA is RNA, i.e., includes RNA extension. The term "polymerase" may also refer to an enzyme that catalyzes the polymerization of nucleotides (i.e., polymerase activity). Generally, the enzyme will initiate synthesis at the 3' end of a primer annealed to a polynucleotide template sequence (e.g., a primer sequence annealed to a primer binding site of the PEgRNA) and proceed toward the 5' end of the template strand. A "DNA polymerase" catalyzes the polymerization of deoxynucleotides. The term DNA polymerase as used herein with reference to a DNA polymerase includes "functional fragments thereof." A "functional fragment thereof" refers to any portion of a wild-type or mutant DNA polymerase encompassing less than the entire amino acid sequence of the polymerase, which retains the ability to catalyze the polymerization of polynucleotides under at least one set of conditions. Such a functional fragment can exist as a separate entity, or it can be a component 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, which uses napDNAbp, a polymerase (e.g., reverse transcriptase), and a specialized guide RNA that contains a DNA synthesis template to encode the desired new genetic information (or delete genetic information) that is then integrated onto the target DNA sequence. Certain embodiments of prime editing are described in the embodiments of Figures 1A-1H and Figures 72(a)-72(c), among others.

Prime editing represents an entirely new platform for genome editing, a versatile and precise genome editing method that writes new genetic information directly at defined DNA sites, using a nucleic acid programmable DNA binding protein ("napDNAbp") that works in conjunction with a polymerase (i.e., in the form of a fusion protein or otherwise provided in trans with napDNAbp). The prime editing system is programmed by a prime editing (PE) guide RNA ("PEgRNA"), which defines a target site and serves as a template for the synthesis of the desired edit in the form of a replaced DNA strand by an extension (either DNA or RNA) engineered onto the guide RNA (e.g., at the 5' or 3' end of the guide RNA or at an internal portion). The replacement strand, which contains the desired edit (e.g., a single nucleobase substitution), shares the same sequence as (or is homologous to) the endogenous strand (immediately downstream of the nick site) of the target site to be edited (except that it encompasses the desired edit). By DNA repair and/or replication mechanisms, 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 can be considered a "search and replace" genome editing technology, because the prime editors described herein not only search and locate the desired target site to be edited, but also simultaneously encode a replacement strand containing the desired edit that is incorporated in place of the endogenous DNA strand at the corresponding target site. The prime editors of the present disclosure relate, in part, to the discovery that the mechanism of prime target primed reverse transcription (TPRT) or "prime editing" can be exploited or adapted to perform precise CRISPR/Cas-based genome editing with high efficiency and genetic flexibility (e.g., as illustrated in various embodiments of Figures 1A-1F). In nature, TPRT is used by mobile DNA elements, such as mammalian non-LTR retrotransposons and bacterial group II ^introns28,29 . In this application, the inventors have used Cas protein-reverse transcriptase fusions or related systems to target specific DNA sequences with guide RNA, generate single-stranded nicks at the target site, and use the nicked DNA as a primer for reverse transcription of the engineered reverse transcriptase template incorporated into the guide RNA. However, although the concept begins with a prime editor using reverse transcriptase as a DNA polymerase component, the prime editor described herein is not limited to reverse transcriptase and can encompass the use of virtually any DNA polymerase. Indeed, throughout the present application, the prime editor may refer to a prime editor having a "reverse transcriptase", but it is hereby expressly stated that reverse transcriptase is only one type of DNA polymerase that can work in prime editing. Thus, wherever the present application refers to "reverse transcriptase", those skilled in the art should understand that any suitable DNA polymerase can be used in place of reverse transcriptase. Thus, in one aspect, the prime editor may comprise Cas9 (or equivalent napDNAbp), which is programmed to target a DNA sequence by associating it with a specialized guide RNA (i.e., PEgRNA) that contains a spacer sequence that anneals to a complementary protospacer on the target DNA. The specialized guide RNA also contains new genetic information in the form of an extension that codes for a replacement strand of DNA containing the desired genetic alteration, which is used to replace the 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 on one strand of DNA to expose a 3' hydroxyl group. The exposed 3' hydroxyl group can then be used to prime DNA polymerization of the extension that codes for the edit on the PEgRNA directly to the target site. In various embodiments, the extension that provides the template for polymerization of the replacement strand containing the edit can be formed from RNA or DNA. In the case of RNA extension, the polymerase of the prime editor can be an RNA-dependent DNA polymerase (e.g., reverse transcriptase). In the case of DNA extension, the polymerase of the prime editor can be a DNA-dependent DNA polymerase. The newly synthesized strand (i.e., the replacement DNA strand containing the desired edit) formed by the prime editors disclosed herein will be homologous to (i.e., have the same sequence as) the genomic target sequence, except for the inclusion of the 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-stranded DNA flap. It will compete for hybridization with the complementary homologous endogenous DNA strand, thereby displacing the corresponding endogenous strand. In some embodiments, the system may be combined with the use of an error-prone reverse transcriptase enzyme (e.g., provided as a fusion protein with a Cas9 domain or provided in trans to a Cas9 domain). The error-prone reverse transcriptase enzyme may introduce alterations during the synthesis of the single-stranded DNA flap. Thus, in some embodiments, an error-prone reverse transcriptase may be utilized to introduce nucleotide changes into the target DNA. Depending on the error-prone reverse transcriptase used in the system, the changes may be random or non-random. Degradation of the hybridized intermediate (including the single-stranded DNA flap synthesized by the reverse transcriptase hybridized to the endogenous DNA strand) may involve removal of the resulting displaced flap of the endogenous DNA (e.g., by the 5'-end DNA flap endonuclease FEN1), ligation of the synthesized single-stranded 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 any nucleotide modification, including insertions and deletions, the scope of this approach is very broad and can foreseeably be used for a myriad of applications in basic research and therapeutics.

In various embodiments, prime editing operates by contacting a target DNA molecule (in which a nucleotide sequence change is desired to be introduced) with a nucleic acid programmable DNA binding protein (napDNAbp) complexed with a prime editing guide RNA (PEgRNA). With reference to FIG. 1G, the prime editing guide RNA (PEgRNA) includes an extension at the 3' or 5' end of the guide RNA or at an intramolecular positioning of the guide RNA to encode the desired nucleotide change (e.g., a single nucleotide change, an insertion, or a deletion). In step (a), the napDNAbp/extended gRNA complex contacts the DNA molecule, and the extended gRNA guides the napDNAbp to bind to the target locus. In step (b), a nick is introduced (e.g., by a nuclease or chemical agent) into one of the strands of DNA at the target locus, thereby creating an available 3' end on one of the strands at the target locus. In some embodiments, the nick is created on 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." However, the nick can be introduced in either strand. That is, the nick can be introduced in 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 that forms the single-stranded portion of the R-loop that 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 to prime reverse transcription (i.e., "RT primed to prime target"). In some embodiments, the 3' end DNA strand hybridizes to a specific RT prime sequence of the extended portion of the guide RNA, i.e., the "reverse transcriptase prime sequence" or "primer binding site" on the PEgRNA. In step (d), a reverse transcriptase (or other suitable DNA polymerase) is introduced. It synthesizes a single strand of DNA from the 3' end of the primed site toward the 5' end of the primed edited guide RNA. The DNA polymerase (e.g., reverse transcriptase) can be fused to the napDNAbp or alternatively provided in trans to the napDNAbp. This forms a single-stranded DNA flap containing the desired nucleotide change (e.g., a single base change, an insertion, or a deletion, or a combination thereof) that 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) involve degradation of the single-stranded DNA flap, so that the desired nucleotide change becomes incorporated at the target locus. This process can be driven toward the desired product formation by removing the corresponding 5' endogenous DNA flap that forms once the 3' single-stranded DNA flap invades and hybridizes to the endogenous DNA sequence. Without being bound by theory, the cell's endogenous DNA repair and replication processes degrade the mismatched DNA and incorporate the nucleotide change(s) to form the desired altered product. The process can also be driven toward product formation by "second strand nicking" as illustrated in FIG. 1F. This process can introduce at least one or more of the following genetic changes: transversions, transitions, deletions, and insertions.

The term "prime editing element (PE) system" or "prime editing element (PE)" or "PE system" or "PE editing system" refers to a composition involved in the method of genome editing using primed target-primed reverse transcription (TPRT) as described herein, including, but not limited to, napDNAbp, reverse transcriptase, a fusion protein (e.g., comprising napDNAbp and reverse transcriptase), a prime editing guide RNA, and a complex comprising a fusion protein and a prime editing guide RNA, as well as auxiliary elements, such as a second strand nicking component (e.g., second strand sgRNA) and a 5' endogenous DNA flap removal endonuclease (e.g., FEN1) to help drive the prime editing process toward the formation of an edited product.

In the embodiments described thus far, the PEgRNA constitutes a single molecule comprising the guide RNA (which itself comprises a spacer sequence and the gRNA core or backbone) and a 5' or 3' extension arm comprising a primer binding site and a DNA synthesis template (see, for example, FIG. 3D), but the PEgRNA can also take the form of two individual molecules, consisting of a guide RNA and a trans-prime editor RNA template (tPERT), which essentially houses an extension arm (including, inter alia, a primer binding site and a DNA synthesis domain) and an RNA-protein recruitment domain (e.g., an MS2 aptamer or hairpin) on the same molecule, which becomes co-localized or recruited to a modified prime editor complex that includes a tPERT recruitment protein (e.g., an MS2cp protein that binds to the MS2 aptamer). See FIG. 3G and FIG. 3H for an example of a tPERT that can be used for prime editing.

Prime Editor The term "prime editor" refers to a fusion construct described herein that includes napDNAbp (e.g., Cas9 nickase) and reverse transcriptase, capable of performing a prime edit on a target nucleotide sequence in the presence of PEgRNA (or "extended guide RNA"). The term "prime editor" may refer to a fusion protein, or a fusion protein complexed with PEgRNA, and/or a fusion protein further complexed with sgRNA that nicks the second strand. In some embodiments, prime editor may also refer to a complex that includes a fusion protein (reverse transcriptase fused to napDNAbp), PEgRNA, and regular guide RNA that can direct the nicking step to the second site 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 "PBS" refers to a nucleotide sequence located on the PEgRNA as a component of the extension arm (typically at the 3' end of the extension arm) that serves to bind to a primer sequence formed after Cas9 nicking of the target sequence by the prime editor. As detailed elsewhere, when the Cas9 nickase component of the prime editor nicks one of the strands of the target DNA sequence, a 3'-terminal ssDNA flap is formed that anneals to the primer binding site on the PEgRNA and serves as a primer sequence to prime reverse transcription. Figures 27 and 28 show embodiments of primer binding sites located on the 3' and 5' extension arms, respectively.

The term " promoter " is recognized in the art and refers to a nucleic acid molecule having a sequence capable of being recognized by the cellular transcriptional machinery and initiating 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 particular condition. For example, a conditional promoter can be active only in the presence of a particular protein that connects a protein associated with a control element on the promoter to the basic transcriptional machinery, or in the absence of an inhibitory molecule. A subclass of conditionally active promoters are inducible promoters, which 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 those of skill in the art, and the skilled artisan will be able to ascertain a variety of such promoters useful in carrying out the present invention. It is not limited in this respect.

Protospacer: The term "protospacer" as used herein refers to a sequence (~20bp) on DNA adjacent to a 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, to one strand of it, the "target strand" versus the "non-target strand" of the target DNA sequence). For Cas9 to function, it also requires a specific protospacer adjacent motif (PAM), which varies depending on the bacterial species of the Cas9 gene. The most commonly used Cas9 nuclease from S. pyogenes recognizes the PAM sequence NGG on the non-target strand found directly downstream of the target sequence on genomic DNA. Those skilled in the art will appreciate that the state of the art literature sometimes refers to the "protospacer" as the ~20nt target-specific guide sequence on the guide RNA itself, rather than referring to it as a "spacer". Therefore, 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 refers to a gRNA or DNA target.

Protospacer adjacent motif (PAM)
The term "protospacer adjacent sequence" or "PAM" as used herein refers to a DNA sequence of approximately 2-6 base pairs that is the key targeting component of the Cas9 nuclease. Typically, the PAM sequence is on either strand, downstream in the 5' to 3' direction of the site cut by Cas9. The standard PAM sequence (i.e., the PAM sequence associated with Streptococcus pyogenes Cas9 nuclease or SpCas9) is 5'-NGG-3', where "N" is any nucleobase followed by two guanine ("G") nucleobases. Different PAM sequences may 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 sequences.

For example, with reference to the standard SpCas9 amino acid sequence of SEQ ID NO: 18, the PAM sequence can be modified by introducing one or more mutations, including (a) the D1135V, R1335Q, and T1337R "VQR variants" that change the PAM specificity to NGAN or NGNG, (b) the D1135E, R1335Q, and T1337R "EQR variants" that change the PAM specificity to NGAG, and (c) the D1135V, G1218R, R1335E, and T1337R "VRER variants" that change the PAM specificity to NGCG. In addition, the D1135E variant of standard SpCas9 still recognizes NGG, but more selectively than the wild-type SpCas9 protein.

It will also be understood that Cas9 enzymes from different bacterial species (i.e., Cas9 orthologues) may have different 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 thermophilis (StCas9) recognizes NNAGAAW. In yet another example, Cas9 from Treponema denticola (TdCas) recognizes NAAAAC. These are examples and are not meant to be limiting. Furthermore, it will be understood that non-SpCas9 bind to various PAM sequences. This makes them useful when a suitable SpCas9 PAM sequence is not present at the site of the desired target to be cut. Furthermore, non-SpCas9 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. Therefore, it can be packaged into an 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, resulting in the excision, incorporation, 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 names of serine and tyrosine recombinases are derived from the conserved nucleophilic amino acid residues that the recombinase uses to attack DNA and become covalently linked to the DNA during strand exchange. Recombinases have numerous applications, including the generation of gene knockouts/knockins and gene therapy applications. For example, see 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 See, e.g., ΦC31 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: Creating 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 incorporated herein by reference in their entirety). The recombinases provided herein are not meant to be the sole examples of recombinases that may be used in embodiments of the present 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 specificity (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. U.S.A. 2009; 106, 5053-5058, the entire contents of each of which are incorporated herein by reference in their entirety). Other examples of recombinases that are useful in the methods and compositions described herein will be known to those of skill in the art, and it is anticipated that any new recombinases discovered or generated will be capable of being used in different embodiments of the invention. In some embodiments, the catalytic domain of a recombinase is fused to an RNA-programmable nuclease (e.g., dCas9 or a fragment thereof) that inactivates the nuclease, such that the recombinase domain does not contain a nucleic acid binding domain or is incapable of binding to a target nucleic acid (e.g., the recombinase domain is engineered such that it has no specific DNA binding activity). Recombinases lacking DNA binding activity and methods for their modification are known, see 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 of which are incorporated herein by reference in their entirety. For example, serine recombinases of the resolvase-invertase family, such as Tn3 and γδ resolvases and Hin and Gin invertases, have molecular 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). Thus, for example, after isolation of "activated" recombinase mutants that do not require any auxiliary factors (e.g., DNA binding activity), the catalytic domains of these recombinases are amenable to recombination with RNA-programmable nucleases (e.g., dCas9 or fragments thereof) that inactivate the nuclease as described herein (see, e.g., Klippel et al., "Isolation and characterization 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). In addition, many other naturally occurring serine recombinases with N-terminal catalytic domains and C-terminal DNA binding domains are known (e.g., phiC31 integrase, TnpX transposase, IS607 transposase), whose catalytic domains can be selected 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, lambda integrase) are known and can be similarly selected 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 1999;302:27-48). 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 As used herein, the term "recombinase recognition sequence" or, equivalently, "RRS" or "recombinase target sequence" refers to a nucleotide sequence target that is recognized by a recombinase and undergoes strand exchange with another DNA molecule that has an RRS, resulting in excision, incorporation, inversion, or exchange of a DNA fragment between the recombinase recognition sequences.

The terms " recombining " or "recombination" are used in the context of nucleic acid modification (e.g., genome modification) to refer to a process in 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., the inventive recombinase fusion proteins provided herein). Recombination can result in, among other things, an insertion, inversion, excision, or translocation of nucleic acid on or between one or more nucleic acid molecules. Recombinase recognition sequences

Reverse TranscriptaseThe 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 transcriptases have been used primarily to transcribe mRNA into cDNA, which can then be cloned onto 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 possesses 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 of an RNA-DNA hybrid (Perbal, A Practical Guide to Molecular Cloning, New York: Wiley & Sons (1984)). Known viral reverse transcriptases lack the 3'-5' exonuclease activity necessary for proofreading, so transcription errors cannot be corrected by the reverse transcriptase (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 is presented by Berger et al., Biochemistry 22:2365-2372 (1983). Another reverse transcriptase that is extensively used in molecular biology is the reverse transcriptase originating from Moloney murine leukemia virus (M-MLV). See, for example, Gerard, GR, DNA 5:271-279 (1986) and Kotewicz, ML, et al., Gene 35:249-258 (1985). M-MLV reverse transcriptase, which is substantially devoid of RNase H activity, has also been described. See, for example, US Pat. No. 5,244, 797. The present invention contemplates the use of any such reverse transcriptase or variants or mutants thereof.

In addition, the present invention contemplates the use of reverse transcriptases that are error-prone, i.e. may be referred to as error-prone reverse transcriptases, or reverse transcriptases that do not support high fidelity incorporation of nucleotides during polymerization. During synthesis of the single-stranded DNA flap based on the guide RNA and the incorporated RT template, the error-prone reverse transcriptase may introduce one or more nucleotides that are mismatched with the RT template sequence, thereby introducing changes in the nucleotide sequence by error-prone polymerization of the single-stranded DNA flap. These errors introduced during synthesis of the single-stranded DNA flap then become incorporated onto the double-stranded molecule by hybridization to the corresponding endogenous target strand, removal of the displaced endogenous strand, ligation, and then another round of endogenous DNA repair and/or sequencing processes.

Reverse Transcription The term "reverse transcription" as used herein refers to the ability of an enzyme to synthesize a DNA strand (i.e., complementary DNA or cDNA) using RNA as a template. In some embodiments, the reverse transcription can be "error-prone reverse transcription." This refers to the property of certain reverse transcriptase enzymes that their DNA polymerization activity is error-prone.

PACE
The term "phage-assisted continuous evolution (PACE)" as used herein refers to continuous evolution using phages as viral vectors. The general concept of PACE technology is described, for example, in International PCT Application PCT/US2009/056194, filed September 8, 2009, published March 11, 2010 as WO 2010/028347; International PCT Application PCT/US2011/066747, filed December 22, 2011, published June 28, 2012 as WO 2012/088381; U.S. Patent Application, U.S. Patent No. 9,023,594, issued May 5, 2015; International PCT Application PCT/US2015/012022, filed January 20, 2015, published September 11, 2015 as WO 2012/088381; and in International PCT application PCT/US2016/027795, filed April 15, 2016, published as WO 2016/168631, the entire contents of each of which are incorporated herein by reference.

Phage The term "phage", which is used interchangeably with the term "bacteriophage" in this application, refers to a virus that infects bacterial cells. Typically, a phage consists of an outer protein capsid that encapsulates 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 method provided herein are λ (lysogen), T2, T4, T7, T12, R17, M13, MS2, G4, P1, P2, P4, phiX174, N4, Φ6, and Φ29. In one embodiment, 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. The present invention is not limited in this aspect. Exemplary descriptions of additional suitable phages and host cells can be found in Elizabeth Kutter and Alexander Sulakvelidze: Bacteriophages: Biology and Applications. CRC Press; 1st edition (December 2004), ISBN: 0849313368; Martha RJ 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 RJ 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 by reference in their entireties for their disclosure of suitable phages and host cells, as well as methods and protocols for the isolation, culture, and manipulation of such phages.

Proteins, Peptides, and Polypeptides 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 proteins, peptides, or polypeptides 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 group of proteins. One or more of the amino acids in a protein, peptide, or polypeptide may be modified by addition of a chemical entity, such as, for example, 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, and the like. A protein, peptide, or polypeptide may also be a single molecule or 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 through recombinant protein expression and purification, which is particularly suitable for fusion proteins that contain peptide linkers. 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, NY (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 the sequence of an intein (or split intein, as the case may be) is excised from within an amino acid sequence and the exteins of the remaining fragment of the amino acid sequence are ligated by amide bonds to form a continuous amino acid sequence. The term "trans" protein splicing refers to the specific case in which the inteins are split inteins and are located on different proteins.

The degradation of the heteroduplex DNA (i.e., containing one edited and one non-edited strand) formed as a result of second strand nicking prime editing determines the long-term editing outcome. In words, the goal of prime editing is to degrade the heteroduplex DNA (the edited strand paired with the endogenous non-edited strand) formed as a PE intermediate by permanently incorporating the edited strand on the complementary endogenous strand. To help drive the degradation of the heteroduplex DNA in favor of the permanent incorporation of the edited strand on the DNA molecule, the approach of "second strand nicking" can be used in the present application. The concept of "second strand nicking" used in the present application refers to the introduction of a second nick, preferably on the non-edited strand, in a position downstream of the first nick (i.e., the original nick site that provides a free 3' end for use in priming reverse transcriptase on the extended portion of the guide RNA). In some 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-stranded portion of the R-loop) and the second nick is on the target strand. In yet other embodiments, the first nick is on the edited strand and the second nick is on the non-edited strand. The second nick can be located 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 nucleotides or more downstream of the first nick. The second nick can be introduced, in some embodiments, on the non-edited strand between about 5-150 nucleotides, 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 nucleotides away from the site of the PEgRNA-induced nick. 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 directs the cell's endogenous DNA repair and replication processes towards replacing or editing the non-edited strand, thereby permanently incorporating the edited sequence on both strands and dissolving the heteroduplex formed as a result of PE. In some embodiments, the edited strand is a non-target strand and the non-edited strand is a target strand, hi other embodiments, the edited strand is a target strand and the non-edited strand is a non-target strand.

Sense strand In genetics, the "sense" strand is a segment in a double-stranded DNA that runs 5' to 3' and is complementary to the antisense or template strand of the 3' to 5' DNA. In the case of a DNA segment that codes for a protein, the sense strand is the strand of DNA that has the same sequence as the mRNA that takes the antisense strand as its template during transcription and ends up (typically, but not always) being translated into a protein. Thus, the antisense strand carries the RNA that is then translated into a protein, while the sense strand has nearly the same makeup as the mRNA. Note that for each segment of dsDNA, there will likely be two pairs, a sense and an antisense (because sense and antisense are relative perspectives), depending on which direction one is read from. The gene product or mRNA ultimately refers to either strand of a segment of dsDNA, which is referred to as sense or antisense.

In the context of PEgRNA, the first step is the synthesis of a single stranded complementary DNA (i.e., the 3'ssDNA flap to be incorporated) oriented in the 5'→3' direction, but the synthesized strand is off-templated by the PEgRNA extension arm. Whether the 3'ssDNA flap should be considered the sense strand or the antisense strand depends on the direction of transcription, where it is fully accepted that both strands of DNA can serve as templates for transcription (but not simultaneously). Thus, in some embodiments, the 3'ssDNA flap (extending generally in the 5'→3' direction) will serve as the sense strand, since it is the coding strand. In other embodiments, the 3'ssDNA flap (extending generally in the 5'→3' direction) will serve as the antisense strand and thus be a template for transcription.

Spacer Sequence As used herein, the term "spacer sequence" in relation to a guide RNA or PEgRNA refers to a portion of the guide RNA or PEgRNA of about 20 nucleotides that contains a nucleotide sequence complementary to a 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 in the endogenous DNA strand complementary to the protospacer sequence.

The term "subject" as used herein refers to an individual organism, e.g., 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 animal of the order Primates. In some embodiments, the subject is a rodent. In some embodiments, the subject is a sheep, goat, cattle, cat, or dog. In some embodiments, the subject is a vertebrate, amphibian, reptile, fish, insect, fly, or nematode. In some embodiments, the subject is a research animal. In some embodiments, the subject is genetically modified, e.g., a genetically modified non-human subject. The subject may be of either sex and at any stage of development.

Split inteinsInteins are most frequently found as a continuous domain, but some naturally exist in a split form. In this case, the two fragments are expressed as separate polypeptides and must associate before splicing can occur, a process known as protein trans-splicing.

An exemplary split intein is the Ssp DnaE intein, which contains two subunits, DnaE-N and DnaE-C. The two distinct subunits are encoded by separate genes, 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 that can direct the trans-splicing of two separate proteins, each of which contains a fusion with either DnaE-N or DnaE-C.

Additional naturally occurring or engineered split intein sequences are known or may be made from the whole intein sequences described herein or 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 Nostc punctiforme, FEBS Lett, 580: 1853-1858, each of which is incorporated herein by reference. Additional split intein sequences can be found, for example, in WO2013/045632, WO2014/055782, WO2016/069774, and EP2877490, the contents of 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. al., J. Biol. Chem. 275:9091 (2000); Otomo, et al., Biochemistry 38:16040-16044 (1999); Otomo, et al., J. Biomol. NMR 14:105-114 (1999); Scott, et al., Proc. Natl. Acad. Sci. USA 96:13638-13643 (1999)), providing the opportunity to express a protein as two inactive fragments that subsequently undergo ligation to form a functional product, as shown, for example, in Figures 66 and 67 for the formation of a complete PE fusion protein from two separately expressed halves.

The term "target site " refers to a sequence on a nucleic acid molecule that is edited by a prime editor (PE) as disclosed herein. Additionally, a target site refers to a sequence on a nucleic acid molecule to which a complex of a prime editor (PE) and a gRNA binds.

tPERT
See definition for "trans prime editing factor "RNA type (tPERT)."

As used herein, the term " transient second strand nicking" refers to a variant of second strand nicking in which the incorporation of a second nick in the unedited strand occurs only after the desired edit has been incorporated into the edited strand. This avoids simultaneous nicking on both strands, which can lead to double-stranded DNA breaks. The second strand nicking guide RNA is designed for temporal control so that the second strand nick is not introduced until after the desired edit has been incorporated. 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, the mismatch between the protospacer and the unedited allele should prevent nicking by the sgRNA until after the editing event on the PAM strand has occurred.

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., where the PEgRNA is separated into two separate molecules: sgRNA and trans prime editing RNA template (tPERT). The sgRNA serves to target the prime editor (or more generally, the napDNAbp component of the prime editor) to the desired genomic target site, while once tPERT is recruited in trans to the prime editor by the interaction of the binding domains located on the prime editor and on tPERT, tPERT is used by the polymerase (e.g., reverse transcriptase) to write new DNA sequences into the target locus. In one embodiment, the binding domain can include an RNA-protein recruitment moiety, such as the MS2 aptamer located on tPERT and the MS2cp protein fused to the prime editor. The advantage of trans prime editing is that by separating the DNA synthesis template from the guide RNA, a template of potentially longer length can be used.

An embodiment of trans-prime editing is shown in Figures 3G and 3H. Figure 3G shows on the left the composition of a trans-prime editor complex ("RP-PE:gRNA complex"), which includes napDNAbp fused to each of a polymerase (e.g., reverse transcriptase) and a rPERT recruitment protein (e.g., MS2sc), complexed with a guide RNA. Figure 3G further shows a separate tPERT molecule, which includes a DNA synthesis template and an extension arm feature of PEgRNA that includes a primer binding sequence. The tPERT molecule also includes an RNA-protein recruitment domain (which in this case is a stem-loop structure, which can be, for example, an MS2 aptamer). As depicted in the process described in Figure 3H, the RP-PE:gRNA complex binds to and nicks the target DNA sequence. The recruitment protein (RP) then recruits tPERT and colocalizes it to the prime editor complex bound to the DNA target site, allowing the primer binding site to bind to the primer sequence on the nicked strand, and subsequently allowing a polymerase (e.g., RT) to synthesize a single strand of DNA onto the DNA synthesis template, up to 5' of tPERT.

Although tPERT is shown in Figures 3G and 3H as containing a PBS and a DNA synthesis template on the 5' end of the RNA-protein recruitment domain, other configurations of tPERT may also be designed with the PBS and DNA synthesis template located on the 3' end of the RNA-protein recruitment domain. However, tPERT with a 5' extension has the advantage that synthesis of a single strand of DNA will naturally terminate at the 5' end of tPERT, and thus there is no risk of using any part of the RNA-protein recruitment domain as a template during the DNA synthesis step of prime editing.

Trans primed editor RNA template (tPERT)
As used herein, "trans prime editor RNA template (tPERT)" refers to the components used in trans prime editing, a modified version of prime editing works by separating PEgRNA into two separate molecules: guide RNA and tPERT molecules. The tPERT molecule is programmed to colocalize with the prime editor complex at the target DNA site and bring the primer binding site and DNA synthesis template to the prime editor in trans. For example, see FIG. 3G for an embodiment of trans prime editor (tPE) showing a two-component system including (1) RP-PE:gRNA complex and (2) tPERT containing a primer binding site and a DNA synthesis template linked to an RNA-protein recruitment domain. Here, the RP (recruitment protein) component of the RP-PE:gRNA complex recruits tPERT to the target site to be edited, thereby linking the PBS and DNA synthesis template to the prime editor in trans. In other words, tPERT is modified to contain (all or part of) the extension arm of a PEgRNA, which encompasses a primer binding site and a DNA synthesis template.

Transitions As used herein, "transition" refers to the interconversion of purine nucleobases (A⇔G) or pyrimidine nucleobases (C⇔T). This class of interconversion 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 transversions in the same target DNA molecule. These changes involve A⇔G, G⇔A, C⇔T, or T⇔C. In the context of double-stranded DNA with Watson-Crick paired nucleobases, transversion refers 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 transversions, as well as other nucleotide changes, including deletions and insertions, in the same target DNA molecule.

Transversions As used herein, "transversion" refers to the interconversion of purine nucleobases to pyrimidine nucleobases, or vice versa, thus involving the interconversion of dissimilar nucleobases. These changes involve T⇔A, T⇔G, C⇔G, C⇔A, A⇔T, A⇔C, G⇔C, and G⇔T. In the context of double-stranded 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 transversions, as well as other nucleotide changes, including deletions and insertions, in the same target DNA molecule.

Treatment The terms "treatment", "treat" and "treating" refer to a clinical intervention aimed at halting, alleviating, delaying the onset of, or inhibiting the progression of, a disease or disorder as described herein, or one or more symptoms thereof. As used herein, the terms "treatment", "treat" and "treating" refer to a clinical intervention aimed at halting, alleviating, delaying the onset of, or inhibiting the progression of, a disease or disorder as described herein, or one or more symptoms thereof. 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 the onset of symptoms or inhibit the 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, e.g., to prevent or delay their recurrence.

Trinucleotide Repeat Disorders As used herein, "trinucleotide repeat disorders" (or alternatively, "expansion repeat disorders" or "repeat expansion disorders") refer to a set of inherited disorders caused by certain mutations (a trinucleotide repeat in a gene or intron) called "trinucleotide repeat expansions." Trinucleotide repeats were once thought to be common iterations in the genome, but were classified into these disorders in the 1990s. These seemingly "benign" stretches of DNA can sometimes expand and cause disease. Several defining features are common to disorders caused by trinucleotide repeat expansions. First, the mutated repeats show instability in both somatic and germline cells, and more frequently they expand rather than contract with successful transmissions. Second, an early age of onset and increased severity (prognosis) of the phenotype in subsequent generations generally correlate with larger repeat length. Finally, the parental origin of a disease allele can often affect the expectation that paternal inheritance poses a greater risk of spread for many of these disorders.

Triplet expansions are thought to be caused by slippage during DNA replication. Due to the repetitive nature of the DNA sequences in these regions, "loop-out" structures can also form during DNA replication, maintaining complementary base pairing between the parent and daughter strands being synthesized. If the loop-out structure is formed from a 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. These repeat expansions appear to be more common than reductions. In general, the larger the expansions, the more likely they are to cause disease or increase disease severity. This characteristic results in the predictive features seen in trinucleotide repeat disorders. Prediction explains the tendency for an earlier age of onset and an increased severity of symptoms through successive generations of affected families due to these repeat expansions.

Nucleotide repeat disorders may include disorders 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 be used to treat nucleotide repeat disorders, which may include Fragile X Syndrome (FRAXA), Fragile XE MR (FRAXE), Friedreich's Ataxia (FRDA), Myotonic Dystrophy (DM), Spinocerebellar Ataxia Type 8 (SCA8), and Spinocerebellar Ataxia Type 12 (SCA12), among others.

As used herein, the terms " upstream " and "downstream" are relative terms that define the linear location of at least two elements located in a nucleic acid molecule (whether single-stranded or double-stranded) oriented in a 5'→3' direction. In particular, if a first element is located anywhere 5' to the second element, the first element is upstream of the second element in the nucleic acid molecule. For example, if the SNP is located 5' to the nick site, the SNP is upstream of the nick site induced by Cas9. Conversely, if a first element is located anywhere 3' to the second element, the first element is downstream of the second element in the nucleic acid molecule. For example, if the SNP is located 3' to the nick site, the SNP is downstream of the nick site induced by Cas9. The nucleic acid molecule can be DNA (double-stranded or single-stranded), RNA (double-stranded or single-stranded), or a hybrid of DNA and RNA. Unless it is necessary to select which strand of a double-stranded molecule is considered, the terms upstream and downstream refer only to a single strand of a nucleic acid molecule, and the analysis is the same for single-stranded nucleic acid molecules and double-stranded molecules. Often, the strand of a double-stranded DNA that can be used to determine the relative positions of at least two elements is the "sense" strand or the "coding" strand. In genetics, a "sense" strand is a segment within a double-stranded DNA that extends from 5' to 3' and is complementary to the antisense or template strand of the DNA that extends from 3' to 5'. Thus, by way of example, if a SNP nucleobase is 3' to the promoter on the sense or coding strand, the SNP nucleobase is "downstream" of the promoter sequence in genomic DNA (which is double-stranded).

Variant As used herein, the term "variant" should be interpreted as meaning a property that exhibits a pattern of qualities that deviate from those that occur in nature, and for example, a variant Cas9 is a Cas9 that contains one or more changes in amino acid residues compared to the wild-type Cas9 amino acid sequence. The term "variant" encompasses homologous proteins that have 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 the reference sequence and have the same or substantially the same functional activity(ies) as the reference sequence. The term also encompasses mutants, truncations, or domains of the reference sequence that exhibit the same or substantially the same functional activity(ies) 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 can enter a host cell and mutate or replicate within the host cell (and then transfer the replicative form of the vector into another host cell). Exemplary suitable vectors include viral vectors, such as retroviral vectors or bacteriophages and filamentous phages, and conjugative plasmids. Additional suitable vectors will be apparent to one of skill in the art based on the present disclosure.

Wild-type As used herein, the term "wild-type" is a term of art understood by those of skill in the art and means the typical form of an organism, strain, gene, or characteristic as occurring in nature, as distinguished from mutant or variant forms.

5' Endogenous DNA Flap As used herein, the term "endogenous 5' DNA flap" refers to the strand of DNA immediately downstream of the PE-induced nick site on the target DNA. Nicking of the target DNA strand by PE exposes a 3' hydroxyl group upstream of the nick site and a 5' hydroxyl group downstream of the nick site. The endogenous strand terminating in the 3' hydroxyl group is used to prime the DNA polymerase of the prime editor (e.g., the DNA polymerase is a reverse transcriptase). The endogenous strand downstream of the nick site and beginning 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., the "3' replacement DNA flap") encoded by the extension of the PE gRNA.

5' endogenous DNA flap removal As used herein, the term "5' endogenous DNA flap removal" or "5' flap removal" refers to the removal of a 5' endogenous DNA flap formed when a single-stranded DNA flap synthesized by RT competitively invades and hybridizes with endogenous DNA, displacing the endogenous strand in the process. Removing this displaced endogenous strand can drive the reaction to form the desired product containing the desired nucleotide change. The cell's own DNA repair enzymes may catalyze the removal or excision of the 5' endogenous flap (e.g., flap endonucleases such as EXO1 or FEN1). The host cell may also be transformed to express one or more enzymes that catalyze the removal of the 5' endogenous flap, thereby driving the process to form the product (e.g., flap endonucleases). Flap endonucleases are known in the art and can be found 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 is incorporated herein by reference.

3' Replacement DNA Flap As used herein, the term "3' replacement DNA flap", or simply "replacement DNA flap", refers to a strand of DNA synthesized by a prime editor and encoded by the extension arm of a prime editor PEgRNA. More specifically, the 3' replacement DNA flap is encoded by the polymerase template of the PEgRNA. The 3' replacement DNA flap contains the same sequence as the 5' endogenous DNA flap, except that it also contains an edited sequence (e.g., a single nucleotide change). The 3' replacement DNA flap anneals to the target DNA to replace or displace the 5' endogenous DNA flap (which can be excised, for example, by a 5' flap endonuclease such as FEN1 or EXO1), and then ligates the 3' end of the 3' replacement DNA flap to join the exposed 5' hydroxyl end of the endogenous DNA (exposed after excision of the 5' endogenous DNA flap), reforming a phosphodiester bond to incorporate the 3' replacement DNA flap and form a heteroduplex DNA containing one edited strand and one unedited strand. The DNA repair process degrades the heteroduplex and permanently incorporates the edit into the DNA by copying the information of the edited strand to the complementary strand. This degradation process can be further driven to completion by nicking the unedited strand, i.e., via "nicking the second strand" as described herein.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS The adoption of the clustered regularly interspaced short palindromic repeats (CRISPR) system for genome editing has revolutionized life ^sciences1-3 . Although gene disruption using CRISPR is now routine, precise incorporation of single nucleotide edits remains a major challenge, despite being necessary to study or correct a large number of disease-causing mutations. Homologous directed repair (HDR) can achieve such edits, but suffers from low efficiency (often <5%), the requirement for a donor DNA repair template, and the deleterious effects of double-stranded DNA break (DSB) formation. Recently, the laboratory of Professor David Liu and others 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 targeted C·G or A·T base pairs to A·T or G·C ^{, respectively4-6} . Although already widely used by researchers worldwide, current BE can only modify 4 of 12 possible base pair changes and cannot correct small insertions or deletions. Moreover, the target scope of base editing is limited by the editing of non-targeted C or A bases adjacent to the target base ("bystander editing") and by the requirement that the PAM sequence be present 15±2bp from the target base. Therefore, overcoming these shortcomings would greatly expand the basic research and therapeutic applications of genome editing.

This disclosure proposes a new high-precision editing approach that provides many of the benefits of base editing - namely, avoidance of double-strand breaks and donor DNA repair template - while overcoming its major drawbacks. The proposed approach described herein achieves direct incorporation of an edited DNA strand at a site in the target genome using target-primed reverse transcription (TPRT). In the designs discussed herein, a CRISPR guide RNA (gRNA) will be modified to carry a reverse transcriptase (RT) template sequence that encodes a single-stranded DNA containing the desired nucleotide change. The target site DNA nicked by the CRISPR nuclease (Cas9) will act as a primer for reverse transcription of the template sequence on the modified gRNA, allowing for direct incorporation of any desired nucleotide edits.

As a result, the present invention relates in part to the discovery that the mechanism of target-primed reverse transcription (TPRT) or "prime editing" can be exploited or employed to perform high-precision CRISPR/Cas-based genome editing with high efficiency and genetic flexibility (e.g., as depicted in various embodiments in Figures 1A-1F). The inventors herein propose to use a Cas9 protein-reverse transcriptase fusion to target a specific DNA sequence with a modified guide RNA (an "extended guide RNA"), generate a single-stranded nick at the target site, and use the nicked DNA as a primer for a modified reverse transcriptase template incorporated into the extended gRNA. The newly synthesized strand will be homologous to the genomic target sequence except that it contains the desired nucleotide change (e.g., a single nucleotide change, deletion, or insertion, or a combination thereof). The newly synthesized strand of DNA, sometimes referred to as a single-stranded DNA flap, competes for hybridization with the complementary homologous endogenous DNA strand, thereby displacing the corresponding endogenous strand. Degradation of this hybridized intermediate may involve the resulting removal of the displaced flap from the endogenous DNA (e.g., with 5'-end DNA flap endonuclease, FEN1), ligation of the synthesized single-stranded DNA flap to the target DNA, and assimilation of the desired nucleotide change as a result of the cellular DNA repair and/or replication processes. Because templated DNA synthesis confers high precision of single nucleotides, the breadth of this approach is extremely broad, and it is foreseeable that it could be used for myriad applications in basic science and therapy.

[1] napDNAbp
The prime and trans-prime editors described herein can include nucleic acid programmable DNA binding proteins (napDNAbp).

In one aspect, the napDNAbp can be associated or complexed with at least one guide nucleic acid (e.g., guide RNA or PEgRNA). This localizes the napDNAbp to a DNA sequence that includes 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 the guide RNA that 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 the complementary sequence of the protospacer on the DNA.

Any suitable napDNAbp may be used in the prime editing elements 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. Since the rapid development of CRISPR-Cas as a tool for genome editing, there has been a constant development of nomenclature systems used to describe and/or identify CRISPR-Cas enzymes, such as Cas9 and Cas9 orthologues. This application refers to CRISPR-Cas enzymes by nomenclature systems, which may be old and/or new. Those skilled in the art will be able to identify the particular CRISPR-Cas enzymes referenced in this application based on the nomenclature system used, whether it is an old (i.e., "legacy") or new nomenclature system. The CRISPR-Cas nomenclature system is extensively described 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 specific CRISPR-Cas nomenclature system used in any given instance of this application is in no way limiting, and one of skill in the art would 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 their variants may be used in the prime editors described herein:

Without being bound by theory, certain napDNAbp mechanisms of action contemplated herein include forming an R-loop, whereby the napDNAbp induces unwinding of the double-stranded DNA target, thereby separating the strands of the region bound by the napDNAbp. The guide RNA spacer then hybridizes to the "target strand" at the protospacer sequence. This displaces the "non-target strand," which is complementary to the target strand, forming the single-stranded 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 scars. For example, the napDNAbp can include a nuclease activity that cuts the non-target strand in a first position and/or cuts the target strand in a second position. 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 only at a single site, i.e., the DNA is "nicked" one strand. Exemplary napDNAbps with different nuclease activities include "Cas9 nickase" ("nCas9") and inactivated Cas9 with no nuclease activity ("inactivated Cas9" or "dCas9").

The description below of various napDNAbps that may be used with respect to the prime editors of the present disclosure is not meant to be limiting in any way. Prime editors may include standard SpCas9 or any orthologous Cas9 protein or any variant Cas9 protein, which may be known or may be created or evolved by directed evolution or otherwise mutational processes, including any naturally occurring variant, mutant, or otherwise engineered version of Cas9. In various embodiments, the Cas9 or Cas9 variant has nickase activity, i.e., cleaves only one of the strands of the target DNA sequence. In other embodiments, the Cas9 or Cas9 variant has an inactive nuclease, i.e., is an "inactive" Cas9 protein. Other variant Cas9 proteins that may be used are those that have a smaller molecular weight than standard SpCas9 (e.g., for easier delivery) or have a modified or rearranged primary amino acid structure (e.g., a circular permutation format).

The prime editors described herein may also include Cas9 equivalents, including Cas12a (Cpf1) and Cas12b1 proteins, which are the result of convergent evolution. The napDNAbps (e.g., SpCas9, Cas9 variants, or Cas9 equivalents) used herein may also contain various modifications that alter/enhance their PAM specificity. Finally, the present application contemplates any Cas9, Cas9 variant, or Cas9 equivalent having 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 reference SpCas9 standard sequence or a reference Cas9 equivalent (e.g., Cas12a (Cpf1)).

napDNAbp may be a CRISPR (clustered regularly interspaced short palindromic repeats)-associated nuclease. As outlined above, CRISPR is an adaptive immune system that provides protection from mobile genetic elements (viruses, transposable elements, and conjugative plasmids). CRISPR clusters contain a spacer, a sequence complementary to an ancestral mobile element, and a target invading nucleic acid. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems, correct processing of the pre-crRNA requires a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and Cas9 protein. The tracrRNA serves as a guide for ribonuclease 3-assisted processing of the pre-crRNA. Cas9/crRNA/tracrRNA then endolytically cleaves linear or circular dsDNA targets complementary to the spacer. The target strand that is not complementary to the crRNA is first endolytically cut and then 3'-5' exolytically trimmed. In nature, DNA binding and cleavage typically requires a protein and both RNAs. However, single guide RNAs ("sgRNAs" or simply "gRNAs") can be engineered to incorporate aspects of both crRNA and tracrRNA onto a single RNA species. See, for example, Jinek M. et al., Science 337:816-821 (2012), the entire contents of which are incorporated herein by reference.

In some embodiments, the napDNAbp directs cleavage of one or both strands at the location of the target sequence, e.g., on the target sequence and/or on 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 the target sequence. In some embodiments, the vector encodes a napDNAbp that is mutated relative to the corresponding wild-type enzyme such that the mutated napDNAbp lacks the ability to cleave one or both strands of a target polynucleotide containing the target sequence. For example, an aspartate to alanine substitution (D10A) on the RuvC I catalytic domain of Cas9 from S. pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (that cleaves a single strand). Other examples of mutations that render Cas9 a nickase include, without limitation, H840A, N854A, and N863A, with reference to the equivalent amino acid positions in the standard SpCas9 sequence or other Cas9 variants or Cas9 equivalents.

The term "Cas protein" as used herein refers to a full-length Cas protein obtained from nature, a recombinant Cas protein having a sequence that differs from a naturally occurring Cas protein, or any fragment of a Cas protein that still retains all or a significant amount of the essential basic functions required for the methods of the disclosure, namely, (i) possession of nucleic acid-programmed binding of the Cas protein to target DNA and (ii) the ability to nick the target DNA sequence on one strand. The Cas proteins contemplated herein include CRISPR Cas9 proteins, and 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 can include Cas9 equivalents from any Class 2 CRISPR system (e.g., Types 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 term "Cas9" or "Cas9 nuclease" or "Cas9 portion" or "Cas9 domain" encompasses 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 naturally occurring or engineered Cas9. The term Cas9 is not meant to be particularly limiting and may be referred to as "Cas9 or equivalent." Exemplary Cas9 proteins are further described herein and/or in the art and are incorporated herein by reference. This disclosure is not limited with respect to the specific Cas9 used in the prime editor (PE) of the present invention.

The Cas9 nuclease sequence and structure described in this application are well known to those skilled in the art (see, for example, "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 below; however, these specific examples are not meant to be limiting. Primer editors of the present disclosure may use any suitable napDNAbp, including any suitable Cas9 or Cas9 equivalent.

A. Standard wild-type SpCas9
In one embodiment, the primer editor constructs described herein may include the "standard SpCas9" nuclease from S. pyogenes. It is widely used as a tool in genome engineering and is classified as a type II subgroup of enzymes in class 2 CRISPR-Cas systems. The Cas9 protein is a large multi-domain protein that contains two separate nuclease domains. Point mutations to abolish one or both nuclease activities can be introduced onto Cas9, resulting in a nickase Cas9 (nCas9) or an inactive Cas9 (dCas9), respectively. This still retains its ability to bind DNA in a manner programmed by sgRNA. In principle, when fused to another protein or domain, Cas9 or a variant thereof (e.g., nCas9) can target the protein to virtually any DNA sequence simply by co-expression with an appropriate sgRNA. Standard SpCas9 protein as used herein refers to the wild-type protein from Streptococcus pyogenes, which has the following amino acid sequence:

を包含する。 The prime editors described herein may include standard 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 the wild-type Cas9 sequence provided above. These variants may include SpCas9 variants containing one or more mutations, including any of the known mutations reported in SwissProt Accession No. Q99ZW2 (SEQ ID NO: 18) entry. These include:

Includes:

を包含する。 Other wild-type SpCas9 sequences that can be used in the present disclosure include:

Includes:

The prime editors described herein can include any of the above SpCas9 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.

B. Wild-type Cas9 orthologues In other embodiments, the Cas9 protein can be a wild-type Cas9 orthologue from another bacterial species that is different from the standard Cas9 from S. pyogenes. For example, the following Cas9 orthologues can be used in conjunction with the prime editor constructs described herein. In addition, any variant Cas9 orthologue having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to any of the orthologues below can also be used in the present prime editors.

The prime editors described herein may include any of the above Cas9 orthologous 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.

napDNAbp may include any suitable homologs and/or orthologs of 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., mutated, engineered, or otherwise derived from nature) as a nickase, i.e., capable of cleaving only one strand of the target. Additional suitable Cas9 nucleases and sequences will be apparent to those of skill in the art based on this disclosure. Such Cas9 nucleases and sequences include Cas9 sequences from 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, the Cas9 nuclease has an inactive (e.g., inactivated) DNA cleavage domain. That is, 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 provided by any one of the variants in 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 provided by any one of the Cas9 orthologues in the table above.

C. Inactive Cas9 variants
In some embodiments, the prime editor described herein may include an inactive Cas9, such as an inactive SpCas9, which does not have nuclease activity due to one or more mutations that inactivate both nuclease domains of Cas9, namely the RuvC domain (which cleaves the non-protospacer DNA strand) and the HNH domain (which cleaves the protospacer DNA strand). The nuclease inactivation may be due to one or more mutations that result in one or more substitutions and/or deletions in the amino acid sequence of the encoded protein or any variant thereof that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto.

The term "dCas9" as used herein refers to nuclease inactive or dead Cas9 or a functional fragment thereof, and includes any naturally occurring dCas9 from any organism, any naturally occurring dCas9 equivalent or a functional fragment thereof, any dCas9 homolog, ortholog, or paralog from any organism, and any mutant or variant of naturally occurring or engineered dCas9. The term dCas9 is not meant to be particularly limiting and may be referred to as "dCas9 or equivalent." Exemplary dCas9 proteins and methods for making dCas9 proteins are further described herein and/or in the art and are incorporated herein by reference.

In other embodiments, dCas9 corresponds to or includes a Cas9 amino acid sequence having one or more mutations that partially or completely inactivate Cas9 nuclease activity. In other embodiments, Cas9 variants having mutations other than D10A and H840A are provided that may result in complete or partial inactivation of endogenous Cas9 nuclease activity (e.g., nCas9 or dCas9, respectively). Such mutations include, by way of example, other amino acid substitutions at D10 and H820, or other substitutions on the nuclease domains of Cas9 (e.g., substitutions on the HNH nuclease subdomain and/or 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 homologs of Cas9 (e.g., variants of Cas9 from Streptococcus pyogenes (NCBI Reference Sequence: NC_017053.1 (SEQ ID NO: 20)) are provided that 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 that have an amino acid sequence that is shorter or longer than NC_017053.1 (SEQ ID NO:20) by about 5 amino acids, about 10 amino acids, about 15 amino acids, about 20 amino acids, about 25 amino acids, about 30 amino acids, about 40 amino acids, about 50 amino acids, about 75 amino acids, about 100 amino acids, or more.

In one embodiment, the inactive Cas9 can be based on the standard SpCas9 sequence of Q99ZW2 and can have the following sequence, including D10X and H810X substitutions (underlined and bold), where X can be any amino acid, or the variant can be a 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 inactive Cas9 can be based on the standard SpCas9 sequence of Q99ZW2 and can have the following sequence including the D10A and H810A substitutions (underlined and bolded), or can 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.

D. Cas9 nickase variants
In one embodiment, the prime editor described herein comprises Cas9 nickase. The term "Cas9 nickase" or "nCas9" refers to a variant of Cas9 that can introduce single-stranded breaks on double-stranded DNA molecular targets. In some embodiments, Cas9 nickase comprises only a single functional nuclease domain. Wild-type Cas9 (e.g., standard SpCas9) comprises two separate nuclease domains, the RuvC domain (which cleaves non-protospacer DNA strands) and the HNH domain (which cleaves protospacer DNA strands). In one embodiment, Cas9 nickase comprises a mutation in the RuvC domain that inactivates RuvC nuclease activity. For example, mutations of aspartic acid (D)10, histidine (H)983, aspartic acid (D)986, or glutamic acid (E)762 have been reported as loss-of-function mutations in the RuvC nuclease domain and generate functional Cas9 nickases (see, 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 can include D10X, H983X, D986X, or E762X, where X is any amino acid other than the wild-type amino acid. In some embodiments, the nickase can be D10A, or 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 can have one of the following amino acid sequences, or a variant thereof having an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto.

In another embodiment, the Cas9 nickase comprises a mutation in the HNH domain that inactivates HNH nuclease activity. For example, mutation of histidine (H)840 or asparagine (R)863 has been reported as a loss-of-function mutation in the HNH nuclease domain and produces a functional Cas9 nickase (see, 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, the nickase mutation in the HNH domain can include H840X and R863X, where X is any amino acid other than the wild-type amino acid. In one embodiment, the nickase can be H840A or R863A, or a combination thereof.

In various embodiments, the Cas9 nickase may have a mutation in the HNH nuclease domain and may have one of the following amino acid sequences, or a variant thereof having an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto.

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

E. Other Cas9 variants
In addition to inactive Cas9 and Cas9 nickase variants, the Cas9 proteins used herein can also include other "Cas9 variants" that are 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 or mutant Cas9 (e.g., inactive Cas9 or Cas9 nickase) disclosed herein or known in the art, or a fragment Cas9, or a circularly permuted Cas9, or other variant of Cas9. 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 the corresponding wild-type Cas9 (e.g., SEQ ID NO: 18).

In some embodiments, the disclosure may also utilize Cas9 fragments that are fragments of any of the Cas9 proteins disclosed herein that retain their functionality. In some embodiments, the Cas9 fragments are at least 100 amino acids in length. In some embodiments, the fragments are 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 editing element disclosed herein may include one of the Cas9 variants described as follows, or a Cas9 variant thereof 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 reference Cas9 variant.

F. Smaller Cas9 variants
In some embodiments, the prime editor contemplated in the present application comprises a Cas9 protein that is smaller in molecular weight than the standard SpCas9 sequence. In some embodiments, the smaller size of the Cas9 variant can facilitate delivery to cells, for example, by expression vectors, nanoparticles, or other means of delivery. In some embodiments, the smaller size of the Cas9 variant can comprise an enzyme that is classified as a type II enzyme of class 2 CRISPR-Cas system. In some embodiments, the smaller size of the Cas9 variant can comprise an enzyme that is classified as a type V enzyme of class 2 CRISPR-Cas system. In other embodiments, the smaller size of the Cas9 variant can comprise an enzyme that is classified as a type VI enzyme of class 2 CRISPR-Cas system.

The standard SpCas9 protein is 1368 amino acids in length and has a predicted molecular weight of 158 kilodaltons. As used herein, the term "small size Cas9 variant" refers to a Cas9 variant that is at least 1300 amino acids long, or at least 1290 amino acids long, or at least 1280 amino acids long, or at least 1270 amino acids long, or at least 1260 amino acids long, or at least 1250 amino acids long, or at least 1240 amino acids long, or at least 1230 amino acids long, or at least 1220 amino acids long, or at least 1210 amino acids long, or at least 1200 amino acids long, or at least 1190 amino acids long, or at least 1180 amino acids long, or at least 1170 amino acids long, or at least 1160 amino acids long, or at least 1150 amino acids long, or at least 1140 amino acids long, or at least 1130 amino acids long. 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 greater than at least about 400 amino acids and retaining the required function of the Cas9 protein, either naturally occurring, engineered, or otherwise. Cas9 variants may include those categorized as Type II, Type V, or Type VI enzymes of Class 2 CRISPR-Cas systems.

In various embodiments, a prime editor disclosed herein can comprise one of the small size 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 size Cas9 protein.

G. Cas9 equivalents
In some embodiments, the prime editors described herein may include any Cas9 equivalent. The term "Cas9 equivalent" as used herein is a broad term, which includes any napDNAbp protein that provides the same function as Cas9 in the prime editor, even though its primary amino acid sequence and/or its three-dimensional structure may differ and/or may be unrelated from an evolutionary standpoint. Thus, Cas9 equivalents include any Cas9 orthologues, homologues, mutants, or variants described or included in this application that are evolutionarily related, but Cas9 equivalents also include proteins that may have evolved by convergent evolutionary processes to have the same or similar function as Cas9, but do not necessarily have any similarity in amino acid sequence and/or three-dimensional structure. Although Cas9 equivalents may be based on proteins that have developed by convergent evolution, the prime editors described herein include any Cas9 equivalent that would provide the same or similar function as Cas9. For example, where Cas9 refers to a Type II enzyme of a CRISPR-Cas system, a Cas9 equivalent could refer to a Type V or Type VI enzyme of a CRISPR-Cas system.

For example, Cas12e (CasX) is a Cas9 equivalent that reportedly has the same function as Cas9 but evolved by 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 for use in the prime editors described herein. Additionally, any variants or modifications of Cas12e (CasX) are envisioned and are within the scope of the present disclosure.

Cas9 is a bacterial enzyme that has evolved in a wide variety of species. However, Cas9 equivalents contemplated in this application may also be obtained from Archaea, which constitute a distinct domain and kingdom of unicellular prokaryotic microorganisms from Bacteria.

In some embodiments, Cas9 equivalents may refer to Cas12e (CasX) or Cas12d (CasY), as described, for example, in 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 are incorporated herein by reference. Using genome-resolution metagenomics, a number of CRISPR-Cas systems have been identified, including the first reported Cas9 in the archaeal domain of life. This divergent Cas9 protein was found as part of an active CRISPR-Cas system from the little-studied nanoarchaea. In bacteria, two previously unknown systems, CRISPR-Cas12e and CRISPR-Cas12d, have been discovered, which are among the most compact systems discovered to date. In some embodiments, Cas9 refers to Cas12e or a variant of Cas12e. In some embodiments, Cas9 refers to Cas12d or a variant of Cas12d. It should be understood that other RNA-guided DNA binding proteins can be used as nucleic acid programmable DNA binding proteins (napDNAbp) and are within the scope of the present disclosure. See also 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 portion or any Cas portion provided herein.

In various embodiments, 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 with a PAM specificity different from Cas9 is clustered regularly interspaced short palindromic repeats 1 from Prevotella and Francisella (i.e., Cas12a (Cpf1)). Similar to Cas9, Cas12a (Cpf1) is also a class 2 CRISPR effector, but it is a type V subgroup of enzymes rather than a type II subgroup. Cas12a (Cpf1) has been shown to mediate robust DNA interference with characteristics that set it apart from Cas9. Cas12a (Cpf1) is a single RNA-guided endonuclease lacking tracrRNA, which utilizes T-rich protospacer adjacent motifs (TTN, TTTN, or YTN). Moreover, Cpf1 cleaves DNA by staggered DNA double-strand breaks. Of the 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. See, e.g., 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 are incorporated herein by reference.

In still other embodiments, the Cas protein can 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, Csm 6, 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, preferably including 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: Cas9, Cas12a (Cpf1), Cas12e (CasX), Cas12d (CasY), Cas12b1 (C2c1), Cas13a (C2c2), Cas12c (C2c3), GeoCas9, CjCas9, Cas12g, Cas12h, Cas12i, Cas13b, Cas13c, Cas13d, Cas14, Csn2, xCas9, SpCas9-NG, circularly permuted Cas9, or an Argonaute (Ago) domain, or a variant thereof.

Exemplary Cas9 equivalent protein sequences can include:

The prime editors described herein may also include Cas12a(Cpf1)(dCpf1) variants that may be used as DNA-binding protein domains programmable by guide nucleotide sequences. The Cas12a(Cpf1) protein has a RuvC-like endonuclease domain that is similar to the RuvC domain of Cas9, but does not have the HNH endonuclease domain, and the N-terminus of Cas12a(Cpf1) does not have the alpha-helical recognition lobe of Cas9. In Zetsche et al., Cell, 163, 759-771, 2015 (which is incorporated herein by reference), it was shown 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, 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 multi-subunit 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 incorporated herein by reference.

Effectors of two of the systems, Cas12b1 and Cas12c, contain a RuvC-like endonuclease domain related to Cas12a. The third system, Cas13a, contains an effector with two predicated HEPN RNase domains. Unlike CRISPR RNA production by Cas12b1, production of mature CRISPR RNA is tracrRNA-independent. Cas12b1 depends on both CRISPR RNA and tracrRNA for DNA cleavage. Bacterial Cas13a has been shown to possess an intrinsic RNase activity for CRISPR RNA maturation that is separate from its RNA-activated single-stranded RNA degradation activity. These RNase functions are distinct 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 incorporated herein by reference. In vitro biochemical analysis of Leptotrichia shahii Cas13a shows that Cas13a can be guided by a single CRISPR RNA and programmed to cleave ssRNA targets with complementary protospacers. Two conserved catalytic residues on the HEPN domain mediate cleavage. Mutation of the catalytic residues generates a catalytically inactive RNA-binding protein. 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 incorporated herein by reference.

The crystal structure of Alicyclobacillus acidoterrastris Cas12b1 (AacC2c1) has been reported in complex with a chimeric single 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 incorporated herein by reference. A crystal structure has also been reported for Alicyclobacillus acidoterrestris C2c1 bound to target DNA as a ternary complex. 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 incorporated herein by reference. The catalytically competent conformation of AacC2c1 is captured by both the target and non-target DNA strands, which are independently positioned within a single RuvC catalytic pocket. C2c1-mediated cleavage results in staggered seven-nucleotide cuts of the target DNA. Structural comparison between the C2c1 ternary complex and previously identified Cas9 and Cpf1 counterparts demonstrates the diversity of mechanisms employed by the CRISPR-Cas9 system.

In some embodiments, the napDNAbp can be a C2c1, C2c2, or 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 permutation
In various embodiments, the prime editing elements disclosed herein can comprise a circular permutation of Cas9.

The term "circularly permuted Cas9" or "circular permutants" of Cas9 or "CP-Cas9" refers to any Cas9 protein or variant thereof that has been modified to arise or be engineered as a circularly permuted variant. This means that the N-terminus and C-terminus of the Cas9 protein (e.g., wild-type Cas9 protein) are locally 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, January 10, 2019, 176:254-267, each of which is incorporated herein by reference. The present disclosure contemplates any previously known CP-Cas9 or employs 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 equivalents thereof, can be reconstituted as a circularly permuted variant.

In various embodiments, the circular permutation of Cas9 can have the following structure: N-terminus-[original C-terminus]-[optional linker]-[original N-terminus]-C-terminus.

By way of example, the present disclosure contemplates the following circular permutation of the standard S. pyogenes Cas9 (1368 amino acids of UniProtKB-Q99ZW2 (CAS9_STRP1) (numbering is based on amino acid positions 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 permutation of other Cas9 proteins (including other Cas9 orthologs, variants, etc.).

In a specific embodiment, the circular permuant Cas9 has the following structure (based on the 1368 amino acids of S. pyogenes Cas9 (UniProtKB-Q99ZW2 (CAS9_STRP1) (numbering is based on amino acid positions 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 permutation of other Cas9 proteins (including other Cas9 orthologs, variants, etc.).

In yet other embodiments, the circular permuant Cas9 has the following structure (based on the 1368 amino acids of S. pyogenes Cas9 (UniProtKB-Q99ZW2 (CAS9_STRP1) (numbering is based on amino acid positions 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 permutation of other Cas9 proteins (including other Cas9 orthologs, variants, etc.).

In some embodiments, a circular permutation can be formed by linking a C-terminal fragment of Cas9 to an N-terminal fragment of Cas9, either directly or by using a linker, such as an amino acid linker. In some embodiments, the C-terminal fragment can correspond to the C-terminal 95% or more of the amino acids of Cas9 (e.g., about amino acids 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 Cas9 (e.g., any one of SEQ ID NOs:77-86). The N-terminal portion can correspond to the N-terminal 95% or more of the amino acids of Cas9 (e.g., about amino acids 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 Cas9 (e.g., of SEQ ID NO:18).

In some embodiments, a circular permutation can be formed by linking a C-terminal fragment of Cas9 to an N-terminal fragment of Cas9, either directly or by using a linker, such as an amino acid linker. In some embodiments, the C-terminal fragment relocated to the N-terminus includes or corresponds to the C-terminal 30% or less of the amino acids of Cas9 (e.g., amino acids 1012-1368 of SEQ ID NO: 18). In some embodiments, the C-terminal fragment relocated 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 Cas9 (e.g., Cas9 of SEQ ID NO: 18). In some embodiments, the C-terminal fragment relocated to the N-terminus includes or corresponds to the C-terminal 410 or less residues of Cas9 (e.g., Cas9 of SEQ ID NO: 18). In some embodiments, the C-terminal portion relocated 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 Cas9 (e.g., Cas9 of SEQ ID NO: 18). In some embodiments, the C-terminal fragment that is relocated to the N-terminus includes or corresponds to the C-terminal 357, 341, 328, 120, or 69 residues of Cas9 (e.g., Cas9 of SEQ ID NO: 18).

In other embodiments, a circularly permuted Cas9 variant can be defined as a topological rearrangement of the Cas9 primary structure based on S. pyogenes Cas9 of SEQ ID NO: 18: (a) selecting a circularly permuted (CP) site corresponding to an amino acid residue within the Cas9 primary structure, which splits 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 (containing 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 on 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 to the S. pyogenes Cas9 of SEQ ID NO: 18) at the original amino acid residues 181, 199, 230, 270, 310, 1010, 1016, 1023, 1029, 1041, 1247, 1249, or 1282. Thus, once relocated to the N-terminus, the original amino acids 181, 199, 230, 270, 310, 1010, 1016, 1023, 1029, 1041, 1247, 1249, or 1282 will become the new N-terminal amino acids. The nomenclature of these CP-Cas9 proteins can be referred to as Cas9-CP ¹⁸¹ , Cas9-CP ¹⁹⁹ , Cas9-CP ²³⁰ , Cas9-CP ²⁷⁰ , Cas9-CP ³¹⁰ , Cas9-CP ¹⁰¹⁰ , Cas9-CP ¹⁰¹⁶ , Cas9-CP ¹⁰²³ , Cas9-CP ¹⁰²⁹ , Cas9-CP ¹⁰⁴¹ , Cas9-CP ¹²⁴⁷ , Cas9-CP ¹²⁴⁹ , and Cas9-CP ¹²⁸² , respectively. This description is not meant to be limited to making CP variants from SEQ ID NO: 18, and can be implemented to make CP variants either at the CP sites corresponding to these positions or entirely at other CP sites in any Cas9 sequence. This description is in no way meant to be limited to a particular CP site. Virtually any CP site can be used to form a CP-Cas9 variant.

An exemplary CP-Cas9 amino acid sequence based on the Cas9 of SEQ ID NO: 18 is provided below, in which the linker sequence is indicated by underlining and the optional methionine (M) residue is indicated in bold. It should be understood that the present disclosure provides CP-Cas9 sequences that do not include linker sequences or that include different linker sequences. It should be understood that CP-Cas9 sequences can be based on Cas9 sequences other than SEQ ID NO: 18, and any examples provided in this application are not meant to be limiting. An exemplary CP-Cas9 sequence is as follows:

Cas9 circular permutations that may be useful in the prime editing constructs described herein. Exemplary C-terminal fragments of Cas9 based on Cas9 of SEQ ID NO: 18 that can be relocated to the N-terminus of Cas9 are provided below. It should be understood 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:

I. Cas9 variants with altered PAM specificity
Prime editors of the disclosure may also include Cas9 variants with altered PAM specificity. Some aspects of the disclosure provide Cas9 proteins that are active against target sequences that do not include a standard PAM (5'-NGG-3', where N is A, C, G, or T) at their 3' end. In some embodiments, the Cas9 protein is active against target sequences that include a 5'-NGG-3'PAM sequence at their 3' end. In some embodiments, the Cas9 protein is active against target sequences that include a 5'-NNG-3'PAM sequence at their 3' end. In some embodiments, the Cas9 protein is active against target sequences that include a 5'-NNA-3'PAM sequence at their 3' end. In some embodiments, the Cas9 protein is active against target sequences that include a 5'-NNC-3'PAM sequence at their 3' end. In some embodiments, the Cas9 protein is active against a target sequence that comprises a 5'-NNT-3'PAM sequence at its 3' end. In some embodiments, the Cas9 protein is active against a target sequence that comprises a 5'-NGT-3'PAM sequence at its 3' end. In some embodiments, the Cas9 protein is active against a target sequence that comprises a 5'-NGA-3'PAM sequence at its 3' end. In some embodiments, the Cas9 protein is active against a target sequence that comprises a 5'-NGC-3'PAM sequence at its 3' end. In some embodiments, the Cas9 protein is active against a target sequence that comprises a 5'-NAA-3'PAM sequence at its 3' end. In some embodiments, the Cas9 protein is active against a target sequence that comprises a 5'-NAC-3'PAM sequence at its 3' end. In some embodiments, the Cas9 protein is active against a target sequence that comprises a 5'-NAT-3'PAM sequence at its 3' end. In yet other embodiments, the Cas9 protein is active against a target sequence that contains a 5'-NAG-3'PAM sequence at its 3' end.

It should be understood that any of the amino acid mutations described herein from a first amino acid residue (e.g., A) to a second amino acid residue (e.g., T) (e.g., A262T) can also include a mutation from the first amino acid residue to an amino acid residue that is similar to the (e.g., conserved) second amino acid residue. For example, a mutation of an amino acid having a hydrophobic side chain (e.g., alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan) can be a mutation to a second amino acid having a different hydrophobic side chain (e.g., alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan). For example, a mutation from alanine to threonine (e.g., A262T mutation) can also be a mutation from alanine to an amino acid similar in size and chemical properties to threonine, such as serine. As another example, a mutation of an amino acid with a positively charged side chain (e.g., arginine, histidine, or lysine) can be a mutation to a second amino acid with a different positively charged side chain (e.g., arginine, histidine, or lysine). As another example, a mutation of an amino acid with a polar side chain (e.g., serine, threonine, asparagine, or glutamine) can 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: phenylalanine and tyrosine; asparagine and glutamine; methionine and cysteine; aspartic acid and glutamic acid; and arginine and lysine. Those skilled in the art will recognize that such conservative amino acid substitutions will likely have minor effects on protein structure and will likely be well tolerated without impairing function. In some embodiments, any amino acid of the amino acid mutations provided herein from one amino acid to threonine can be an amino acid mutation to serine. In some embodiments, any amino acid of the amino acid mutations provided herein from one amino acid to arginine can be an amino acid mutation to lysine. In some embodiments, any amino acid of the amino acid mutations provided herein from one amino acid to isoleucine can be an amino acid mutation to alanine, valine, methionine, or leucine. In some embodiments, any amino acid of the amino acid mutations provided herein from one amino acid to lysine can be an amino acid mutation to arginine. In some embodiments, any amino acid of the amino acid mutations provided herein from one amino acid to aspartic acid can be an amino acid mutation to glutamic acid or asparagine. In some embodiments, any amino acid of the amino acid mutations provided herein from one amino acid to valine can be an amino acid mutation to alanine, isoleucine, methionine, or leucine. In some embodiments, any amino acid of the amino acid mutations provided herein from one amino acid to glycine can be an amino acid mutation to alanine. However, it should be understood that additional conserved amino acid residues would be recognized by one of skill in the art, and any 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 exerts activity against a target sequence that includes a 5'-NAA-3'PAM sequence at its 3' end. In some embodiments, the combination of mutations is present in any one of the clones listed in Table 1. In some embodiments, the combination of mutations is a conservative mutation of the clones listed in Table 1. In some embodiments, the Cas9 protein comprises a combination of mutations of any one of the Cas9 clones listed in Table 1.

Table 1: NAA PAM clones

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 provided by any one of the variants in 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 provided by any one of the variants in Table 1.

In some embodiments, the Cas9 protein exhibits increased activity against a target sequence that does not include a canonical PAM (5'-NGG-3') at its 3' end, as compared to the Streptococcus pyogenes Cas9 provided by SEQ ID NO: 18. In some embodiments, the Cas9 protein exhibits at least a 5-fold increased activity against a target sequence having a 3' end that is not immediately adjacent to a canonical PAM sequence (5'-NGG-3'), as compared to the activity of the Streptococcus pyogenes Cas9 provided by SEQ ID NO: 18 against the same target sequence. In some embodiments, the Cas9 protein exerts 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 activity against a target sequence that is not immediately adjacent to a standard PAM sequence (5'-NGG-3') compared to the activity of Streptococcus pyogenes provided by SEQ ID NO: 18 against the same target sequence. In some embodiments, the 3' end of the target sequence is immediately adjacent to an AAA, GAA, CAA, or TAA sequence. In some embodiments, the Cas9 protein comprises a combination of mutations that exerts activity against a target sequence that includes a 5'-NAC-3' PAM sequence at its 3' end. In some embodiments, the combination of mutations is present in any one of the clones listed in Table 2. In some embodiments, the combination of mutations is a conservative mutation of the clones listed in Table 2. In some embodiments, the Cas9 protein comprises a combination of mutations of any one of the Cas9 clones listed in Table 2.

Table 2: NAC PAM clones

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 provided by any one of the variants in 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 provided by any one of the variants in Table 2.

In some embodiments, the Cas9 protein exhibits increased activity against a target sequence that does not include a canonical PAM (5'-NGG-3') at its 3' end, as compared to the Streptococcus pyogenes Cas9 provided by SEQ ID NO: 18. In some embodiments, the Cas9 protein exhibits at least 5-fold increased activity against a target sequence having a 3' end that is not immediately adjacent to a canonical PAM sequence (5'-NGG-3'), as compared to the activity of the Streptococcus pyogenes Cas9 provided by SEQ ID NO: 18 against the same target sequence. In some embodiments, the Cas9 protein exerts 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 activity against a target sequence that is not immediately adjacent to a standard PAM sequence (5'-NGG-3') compared to the activity of Streptococcus pyogenes against the same target sequence provided by SEQ ID NO: 18. In some embodiments, the 3' end of the target sequence is immediately adjacent to an AAC, GAC, CAC, or TAC sequence.

In some embodiments, the Cas9 protein comprises a combination of mutations that exerts activity against a target sequence that includes a 5'-NAT-3'PAM sequence at its 3' end. In some embodiments, the combination of mutations is present in any one of the clones listed in Table 3. In some embodiments, the combination of mutations is a conservative mutation of a clone listed in Table 3. In some embodiments, the Cas9 protein comprises a combination of mutations of any one of the Cas9 clones listed in Table 3.

Table 3: NAT PAM clones

The above description of various napDNAbp that may be used in connection with the prime editors of the present disclosure is not meant to be limiting in any way. Prime editors may include standard SpCas9, or any orthologous Cas9 protein, or any variant Cas9 protein, which may be known or created or evolved by directed evolutionary or otherwise mutational processes, including any naturally occurring variant, mutant, or otherwise engineered version of Cas9. In various embodiments, the Cas9 or Cas9 variant has nickase activity, i.e., cleaves only one of the strands of the target DNA sequence. In other embodiments, the Cas9 or Cas9 variant has an inactive nuclease, i.e., is an "inactive" Cas9 protein. Other variant Cas9 proteins that may be used are those that have a smaller molecular weight than standard SpCas9 (e.g., for easier delivery) or have a modified or rearranged primary amino acid structure (e.g., circular permutation format). The prime editors described herein may also include Cas9 equivalents, including Cas12a/Cpf1 and Cas12b proteins that are the result of convergent evolution. The napDNAbps (e.g., SpCas9, Cas9 variants, or Cas9 equivalents) used herein may also contain various modifications that alter/enhance their PAM specificity. Finally, the present application contemplates any Cas9, Cas9 variant, or Cas9 equivalent having 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 reference SpCas9 standard sequence or a reference Cas9 equivalent (e.g., Cas12a/Cpf1).

In a specific embodiment, the Cas9 variant with extended PAM capabilities is SpCas9(H840A)VRQR (SEQ ID NO:87), which has the following amino acid sequence (V, R, Q, R substitutions relative to SpCas9(H840A) of SEQ ID NO:51 are shown in bold and underlined. In addition, the methionine residue of SpCas9(H840) was removed in SpCas9(H840A)VRQR):

In another specific embodiment, the Cas9 variant with extended PAM capabilities is SpCas9(H840A)VRER, which has the following amino acid sequence (V, R, E, R substitutions relative to SpCas9(H840A) of SEQ ID NO:51 are shown in bold and underlined. In addition, the methionine residue of SpCas9(H840) has been removed in SpCas9(H840A)VRER):

In some embodiments, the napDNAbp that functions for non-canonical PAM sequences is an Argonaute protein. One example of such a nucleic acid-programmed DNA-binding protein is the Argonaute protein (NgAgo) from Natronobacterium gregoryi. NgAgo is an endonuclease guided by ssDNA. NgAgo will bind and guide ∼24 nucleotide 5' phosphorylated ssDNA (gDNA) to its target site and create a DNA double-strand break 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 can be targeted. The characterization and use of NgAgo is described in Gao et al., Nat Biotechnol., 2016 Jul;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, napDNAbp is a prokaryotic homolog of an Argonaute protein. Prokaryotic homologs of Argonaute proteins are known and are 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 are incorporated herein by reference. In some embodiments, napDNAbp is a Marinintoga piezophila Argunaute (MpAgo) protein. CRISPR-associated Marinintoga piezophila Argunaute (MpAgo) protein cleaves single-stranded target sequences using a 5'-phosphorylated guide. The 5' guide is used by all known Argonautes. The crystal structure of the MpAgo-RNA complex shows a guide strand binding site that includes residues that block the 5' phosphate interaction. This data suggests the evolution of an Argonaute subclass with noncanonical specificity for 5' hydroxylated guides. See, e.g., Kaya et al., "A bacterial Argonaute with noncanonical guide RNA specificity", Proc Natl Acad Sci U S A. 2016 Apr 12;113(15):4057-62, the entire contents of which are incorporated herein by reference. It should be understood that other argonaute proteins may be used and are within the scope of this disclosure.

Some aspects of the present disclosure provide Cas9 domains with different PAM specificities. Typically, Cas9 proteins, such as Cas9 from S. pyogenes (spCas9), require a standard NGG PAM sequence to bind to a specific nucleic acid region. This may limit the ability to edit a desired base on a genome. In some embodiments, the base editing fusion proteins provided herein may need to be precisely positioned. For example, here, the target base is placed on a 4-base region (e.g., an "editing window") that 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 incorporated herein by reference. Thus, in some embodiments, any of the fusion proteins provided herein may contain a Cas9 domain that can bind to a nucleotide sequence that does not contain a standard (e.g., NGG) PAM sequence. Cas9 domains that bind to non-canonical PAM sequences have been described in the art and will be apparent to one of skill in the art. For example, Cas9 domains that bind to non-canonical PAM sequences are 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 incorporated herein by reference.

For example, napDNAbp domains with altered PAM specificity, e.g., domains having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to wild-type Francisella novicida Cpf1 (D917, E1006, and D1255) (SEQ ID NO: 74) having the following amino acid sequence:

Additional napDNAbp domains with altered PAM specificity, for example, domains with at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the wild-type Geobacillus thermodenitrificans Cas9 (SEQ ID NO: 75) having the following amino acid sequence:

In some embodiments, the nucleic acid programmed DNA binding protein (napDNAbp) is a nucleic acid programmed 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 programmed DNA binding protein is the Argonaute protein (NgAgo) from Natronobacterium gregoryi. NgAgo is an endonuclease guided by ssDNA. NgAgo will bind and guide ∼24 nucleotide 5' phosphorylated ssDNA (gDNA) to its target site and create a DNA double strand break 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 can be targeted. The characterization and use of NgAgo is 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 by SEQ ID NO:76.

The disclosed fusion proteins can include a napDNAbp domain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the wild-type Natronobacterium gregoryi Argonaute (SEQ ID NO:76), which has the following amino acid sequence:

In addition, any available method may be utilized to obtain or construct variant or mutant Cas9 proteins. The term "mutation" as used herein refers to the substitution of a residue on a sequence, e.g., a nucleic acid or amino acid sequence, with another residue, or the deletion or insertion of one or more residues on a sequence. Mutations are typically described herein by identifying the original residue, then the position of the residue on the sequence, and the identity of the newly substituted residue. Various methods for making amino acid substitutions (mutations) provided herein are well known in the art, and are provided, for example, by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)). Mutations may encompass a variety of categories, e.g., single nucleotide polymorphisms, microduplication regions, indels, and inversions, and are in no way meant to be limiting. Mutations may encompass "loss-of-function" mutations, which are the normal consequence of a mutation that reduces or abolishes protein activity. Most loss-of-function mutations are recessive. Because in heterozygotes, the second chromosomal copy carries a fully functional, unmutated version of the gene that codes for the protein, the presence of which compensates for the effect of the mutation. Mutations also encompass "gain-of-function" mutations, which confer an abnormal activity to a protein or cell that is not otherwise present under normal conditions. Many gain-of-function mutations are in regulatory sequences rather than coding regions and therefore can have a number of consequences. For example, a mutation can lead to one or more genes being expressed in the wrong tissues, and these tissues gain a function that they normally lack. By their nature, gain-of-function mutations are usually dominant.

Mutations can be introduced into the reference Cas9 protein using site-directed mutagenesis. Older methods of site-directed mutagenesis known in the art rely on subcloning of the sequence to be mutated onto a vector, such as the M13 bacteriophage vector, that allows the isolation of a single-stranded DNA template. In these methods, a mutagenic primer (i.e., a primer that can anneal to the site to be mutated but has one or more mismatched nucleotides at the site to be mutated) is annealed to the single-stranded template, and then the complement of the template is polymerized starting from the 3' end of the mutagenic primer. The resulting duplex is then transformed into a host bacterium and plaques are screened for the desired mutation. More recently, site-directed mutagenesis has used PCR methodologies. These have the advantage of not requiring a single-stranded template. In addition, methods have been developed that do not require subcloning. When PCR-based site-directed mutagenesis is performed, several issues must be considered. First, in these methods, it is desirable to reduce the number of PCR cycles to prevent the extension of unwanted mutations introduced by the polymerase. Second, selection must be used to reduce the number of unmutated parental molecules remaining in the reaction. Third, extended-length PCR methods are preferred to allow the use of a single PCR primer set. And fourth, due to the non-template-dependent end-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 mutant products generated by PCR.

Mutations can also be introduced by directed evolution processes, such as phage-assisted continuous evolution (PACE) or phage-assisted discontinuous evolution (PANCE). The term "phage-assisted continuous evolution (PACE)" as used herein refers to continuous evolution using phages as viral vectors. The general concept of PACE technology is described, for example, in International PCT application PCT/US2009/056194, filed September 8, 2009, published as WO2010/028347 on March 11, 2010; International PCT application PCT/US2011/066747, filed December 22, 2011, published as WO2012/088381 on June 28, 2012; and International PCT application PCT/US2011/066747, filed December 22, 2011, published as WO2012/088381 on May 5, 2015, in the U.S. US Patent No. 9,023,594, International PCT Application PCT/US2015/012022, filed January 20, 2015, published as WO2015/134121 on September 11, 2015, and International PCT Application PCT/US2016/027795, filed April 15, 2016, published as WO2016/168631 on October 20, 2016, the entire contents of each of which are incorporated herein by reference. Variant Cas9 can also be obtained by phage-assisted non-linear evolution (PANCE). As used herein, this refers to non-linear evolution using phage as a viral vector. PANCE is a simplified technique for rapid in vivo directed evolution that uses serial flask transfer of evolving "selection phage" (SP) containing the gene of interest to be evolved with new E. coli host cells, whereby the gene contained in the SP is continuously evolved while allowing the gene in the host E. coli to be held constant. Serial flask transfer has long served as a widely accessible approach for the laboratory evolution of microorganisms, and more recently, a related approach has been developed for bacteriophage evolution. The PANCE system features lower stringency than the PACE system.

Any of the references set forth above regarding Cas9 or Cas9 equivalents are hereby incorporated by reference in their entirety unless already so claimed.

J. Split napDNAbp domain for split PE delivery
In various embodiments, the prime editors described herein can be delivered to cells as two or more fragments that become assembled into a reconstituted prime editor within the cell (either by passive assembly or by active assembly, such as by using a split intein sequence). In some cases, the self-assembly can be passive, whereby two or more prime editor fragments covalently or non-covalently associate within the cell to reconstitute the prime editor. In other cases, the self-assembly can be catalyzed by a dimerization domain incorporated into each of the fragments. Examples of dimerization domains are described herein. In yet other cases, the self-assembly can be catalyzed by a split intein sequence incorporated into each of the prime editor fragments.

Split PE delivery can be advantageous to address the various size constraints of different delivery approaches. For example, delivery approaches can include viral-based delivery methods, messenger RNA-based delivery methods, or RNP-based delivery (ribonucleoprotein-based delivery). And each of these methods of delivery can be more efficient and/or effective by splitting 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 split prime editor fragments can reassemble in a non-covalent or covalent manner to reform the prime editor. In one embodiment, the prime editor can be split into two or more fragments at one or more split sites. The fragments can be unmodified (other than being split). Once the fragments are delivered to the cell (e.g., by direct delivery of the ribonucleoprotein complex or by nucleic acid delivery, e.g., mRNA delivery or viral vector-based delivery), the fragments can covalently or non-covalently reassemble to reconstitute the prime editor. In another embodiment, the prime editor can be split into two or more fragments at one or more split sites. Each of the fragments can be modified to include a dimerization domain, whereby each formed fragment is coupled to the dimerization domain. Once delivered or expressed in a cell, the dimerization domains of the different fragments associate and bind to each other, bringing the different prime editor fragments together to reform a functional prime editor. In yet another embodiment, the prime editor fragment can be modified to include a split intein. Once delivered or expressed in a cell, the split intein domains of the different fragments associate and bind to each other and then undergo trans-splicing. This results in the excision of the split intein domain from each of the fragments and the concomitant formation of peptide bonds between the fragments, thereby restoring the prime editor.

In one embodiment, the primed editing factor can be delivered using a split-intein approach.

The location of the division site may be between any one or more pairs of residues on the prime editor, as well as on any domain therein, including within the napDNAbp domain, the polymerase domain (e.g., the RT domain), and the linker domain connecting the napDNAbp domain and the polymerase domain.

In one embodiment, illustrated in Figure 66, the prime editor (PE) is cleaved at the cleavage site on the napDNAbp.

In one embodiment, the napDNAbp is a standard SpCas9 polypeptide of SEQ ID NO: 18, as follows:

In some embodiments, SpCas9 is located at a division 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 of the standard SpCas9 of SEQ ID NO:18, or between residues 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 6 It is split into two fragments between any two pairs of residues located anywhere between 0-70, 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.

In some embodiments, the napDNAbp is split into two fragments at a split site located at a pair of residues corresponding to any two pairs 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 the standard SpCas9 of SEQ ID NO:18.

In some embodiments, SpCas9 is expressed at a division 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 of the standard SpCas9 of SEQ ID NO:18, or between residues 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, The fragment is split into two fragments between any two pairs of residues located anywhere between 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. In some embodiments, the split site is located at one or more polypeptide binding sites (i.e., the "split site or split-intein split site") and fused to the split intein, which is then delivered to the cell as a separately encoded fusion protein. Once the split-intein fusion proteins (i.e., the protein halves) are expressed in a cell, the proteins undergo trans-splicing to form the complete or whole PE with the concomitant removal of the linked split-intein sequence.

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). Upon self-association of the N and C inteins in the cell, followed by their self-excision and concomitant formation of a peptide bond between the N-terminal and C-terminal extein portions of the whole prime editor (PE), the N-terminal extein becomes fused to the C-terminal extein to reform the whole prime editor fusion protein including the napDNAbp domain and the polymerase domain (e.g., RT domain).

To take advantage of a split PE delivery strategy using split inteins, the prime editor needs to be split at one or more split sites to generate at least two separate halves of the prime editor, each of which can be rejoined within the cell when each half is fused to a split intein sequence.

In some embodiments, the prime editing factor is split at a single division site. In certain other embodiments, the prime editing factor is split at two division sites, or three division sites, or four division sites, or more.

In a preferred embodiment, the prime editor is split at a single split site to generate two separate halves of the prime editor, each of which can be fused to a split intein sequence.

An exemplary split intein is the Ssp DnaE intein, which contains two subunits, DnaE-N and DnaE-C. The two distinct subunits are encoded by separate genes, 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 that can direct the trans-splicing of two separate proteins, each of which contains a fusion with either DnaE-N or DnaE-C.

Additional naturally occurring or engineered split intein sequences are known in the art or can be made from whole intein sequences described herein or 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 Nostc punctiforme, FEBS Lett, 580: 1853-1858, each of which is incorporated herein by reference. Additional split intein sequences can be found, for example, in WO2013/045632, WO2014/055782, WO2016/069774, and EP2877490, the contents of 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. al., J. Biol. Chem. 275:9091 (2000); Otomo, et al., Biochemistry 38:16040-16044 (1999); Otomo, et al., J. Biomol. NMR 14:105-114 (1999); Scott, et al., Proc. Natl. Acad. Sci. USA 96:13638-13643 (1999)), providing the opportunity to express a protein as two inactive fragments which then undergo ligation to form a functional product, as shown, for example, in Figures 66 and 67 for the formation of a complete PE fusion protein from two separately expressed halves.

In various embodiments described herein, continuous evolution methods (e.g., PACE) can be used to evolve a first portion of a base editor. The first portion can include a single component or domain, such as a Cas9 domain, a deaminase domain, or a UGI domain. The separately evolved component or domain can then be fused to the remaining portion of the base editor in the cell by separately expressing both the evolved portion and the remaining unevolved portion together with the split-intein polypeptide domain. The first portion can more broadly include any first amino acid portion of the base editor that is desired to be evolved using the continuous evolution methods described herein. The second portion, in this embodiment, would refer to the remaining amino acid portion of the base editor that is not evolved using the methods of the present application. The evolved first and second portions of the base editor can each be expressed by the cell together with the split-intein polypeptide domain. The cell's natural protein splicing machinery will reassemble the evolved first portion and the unevolved second portion to form a single fusion protein evolved base editor. The evolved first portion can include either the N- or C-terminal parts of a single fusion protein. In an analogous manner, the use of a second orthogonal trans-splicing intein pair can allow the evolved first portion to include internal parts of a single fusion protein.

Thus, to facilitate the formation of a complete base editor containing evolved and unevolved components in a cell, any of the evolved and unevolved components of the base editors described herein can be expressed with a split intein tag.

The mechanism of 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 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' end of a first gene (e.g., an evolved portion of a base editor). A suitable intein sequence 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 its 3' end to the 5' end of a second gene. For targeting of this gene to certain organelles, 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 many times as desired for expression of multiple proteins in the same cell. In a multi-intein-containing construct, it can 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 such that it can catalyze the excision of the extein from the intein and prevent the ligation of the extein. Mutagenesis of the C-terminal extein junction on Pyrococcus species GB-D DNA polymerase was found to result in an altered splicing element. This induces cleavage of the extein and intein but prevents subsequent ligation of the extein (Xu, M-Q & Perler, F.B. EMBO Journal, 1996, 15, 5146-5153). Mutation of serine 538 to either alanine or glycine induced cleavage but prevented ligation. Due to conservation of amino acids at the C-terminal extein junction to the intein, mutation of equivalent residues in other intein splicing units should also prevent extein ligation. A preferred intein that does not contain an endonuclease domain is the Mycobacterium xenopi GyrA protein (Telenti, et al. J. Bacteriol. 1997, 179, 6378-6382). Others are found in nature or have been artificially generated by removing the endonuclease domain from an intein-containing endonuclease (Chong, et al. J. Biol. Chem. 1997, 272, 15587-15590). In a preferred embodiment, the intein is selected such that it consists of the minimal number of amino acids required to perform the splicing function, such as the intein from 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 Mycobacterium xenopi GyrA protein, or the Saccharomyces cerevisiae VMA intein modified to remove the endonuclease domain (Chong, 1997). Further modification of the intein splicing unit may allow the kinetics of the cleavage reaction to be altered, allowing protein dosage to be controlled by simply modifying the genetic 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 a variety of 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)), which have not been found so far in eukaryotes. Recently, bioinformatics analysis of environmental metagenomic data revealed 26 distinct loci with novel genomic arrangements. In each locus, a conserved enzyme coding region is interrupted by a split intein and an autonomous endonuclease gene is inserted between sections encoding intein subdomains. Of those, five loci have been fully assembled: DNA helicase (gp41-1, gp41-8); inosine-5'-monophosphate dehydrogenase (IMPDH-1); and ribonucleotide reductase catalytic subunits (NrdA-2 and NrdJ-1). This fragmented genetic organization appears to be present primarily 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 a protein trans-splicing reaction. In addition, the Npu DnaE protein splicing reaction is considered robust and high-yielding for 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 a Cysl Ala mutation in the N domain of these inteins was introduced, the initial N to S acyl shift and thus protein splicing was blocked. Unfortunately, the C-terminal cleavage reaction was also almost completely inhibited. The dependence of asparagine cyclization at the C-terminal splice junction on acyl shift at the N-terminal scissile peptide bond appears to be a common, intrinsic property of naturally split DnaE intein alleles (Zettler J. et al. FEBS Letters. 555:909-914 (2009)).

The mechanism of protein splicing typically involves four steps [29-30]: 1) an N-S or N-O acyl shift at the intein N-terminus, which cleaves the upstream peptide bond and forms an ester bond between the N-extein and the side chain of the first amino acid (Cys or Ser) of the intein; 2) transesterification, which repositions the N-extein to the intein C-terminus, which forms a new ester bond and links the N-extein to the side chain of the first amino acid (Cys, Ser, or Thr) of the C-extein; 3) Asn cyclization, which cleaves the peptide bond between the intein and the C-extein; and 4) an S-N or O-N acyl shift, which replaces the ester bond by a peptide bond between the N-extein and the C-extein.

Protein trans-splicing catalyzed by split inteins provides a purely enzymatic method for protein ligation [31]. Split inteins are essentially a contiguous intein (e.g., a mini-intein) that has been split into two pieces, named N-intein and C-intein, respectively. The N-intein and C-intein of a split intein can non-covalently associate to form an active intein and catalyze a splicing reaction in essentially the same way that a contiguous intein does. Split inteins are found in nature and have also been engineered in the laboratory [31-35]. The term "split intein" as used herein refers to any intein in which one or more peptide bond cleavages exist between the N- and C-terminal amino acid sequences, such that the N- and C-terminal sequences are separate molecules that can non-covalently reassociate or reconstitute into an intein that is functional for a trans-splicing reaction. Any catalytically active intein or fragment thereof can be used to derive a split intein for use in the methods of the invention. For example, in one aspect, the split intein can be derived from a eukaryotic intein. In another aspect, the split intein can be derived from a bacterial intein. In another aspect, the split intein can be derived from an archaeal intein. Preferably, the split intein so derived will possess only the amino acid sequences essential for catalyzing the trans-splicing reaction.

As used herein, "N-terminal split intein (In)" refers to any intein sequence that includes an N-terminal amino acid sequence that is functional for a trans-splicing reaction. In therefore also includes sequences that are spliced out when trans-splicing occurs. In can include sequences that are modifications of the N-terminal portion of a naturally occurring intein sequence. For example, In can include additional amino acid residues and/or mutated residues, so long as the inclusion of such additional and/or mutated residues does not render In non-functional in trans-splicing. Preferably, the inclusion of additional and/or mutated residues improves or enhances the trans-splicing activity of In.

As used herein, "C-terminal split intein (Ic)" refers to any intein sequence that includes a C-terminal amino acid sequence that is functional for a trans-splicing reaction. In one aspect, Ic includes a stretch of 4 to 7 amino acid residues, at least 4 of which are from the last β-strand of the intein from which it is derived. Ic therefore also includes a sequence that is spliced out when trans-splicing occurs. Ic can include a sequence that is a modification of the C-terminal portion of a naturally occurring intein sequence. For example, Ic can include additional amino acid residues and/or mutated residues, so long as the inclusion of such additional and/or mutated residues does not render In non-functional in trans-splicing. Preferably, the inclusion of additional and/or mutated residues improves or enhances the trans-splicing activity of Ic.

In some embodiments of the invention, the peptide linked to Ic or In may contain additional chemical moieties including, among others, fluorescent groups, biotin, polyethylene glycol (PEG), amino acid analogs, unnatural amino acids, phosphate groups, glycosyl groups, radioisotope labels, and pharmaceutical molecules. In other embodiments, the peptide linked to Ic may contain one or more chemically reactive groups including, among others, ketones, aldehydes, Cys residues, and Lys residues. When an "intein splicing polypeptide (ISP)" is present, the N intein and C intein of the split intein may non-covalently associate to form an active intein and catalyze the splicing reaction. As used herein, "intein splicing polypeptide (ISP)" refers to the portion of the amino acid sequence of the split intein that remains when Ic, In, or both are removed from the split intein. In some embodiments, In includes an ISP. In other embodiments, Ic includes an ISP. In yet other embodiments, the ISP is a separate peptide that is not covalently linked to either In or Ic.

Split inteins can be generated from consecutive inteins by engineering one or more split sites into the unstructured loop or intervening amino acid sequence between the -12 conserved beta strands found on the mini-intein structure [25-28]. There can be some flexibility in the location of the split site within the region between the beta strands, provided that the creation of the split would not interrupt the intein structure, particularly the structured beta strands, 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 an N intein, another precursor protein consists of a C intein followed by a C extein part, and a trans-splicing reaction (catalyzed together by the N and C inteins) excises the two intein sequences and links the two extein sequences by a peptide bond. Protein trans-splicing, an enzymatic reaction, can work at very low (e.g., micromolar) concentrations of protein and can be carried out under physiological conditions.

[2] Other programmable nucleases In various embodiments described herein, the prime editor comprises a napDNAbp, such as the Cas9 protein. These proteins are "programmable" by becoming complexed with a guide RNA (or a PEgRNA, as the case may be). This guides the Cas9 protein to a target site on DNA that possesses a sequence that is complementary to the spacer portion of the gRNA (or PEgRNA) and also possesses the required PAM sequence. However, in certain embodiments contemplated herein, the napDNAbp can be replaced by a different type of programmable protein, such as a zinc finger nuclease or a transcription activator-like effector nuclease (TALEN).

Figure 1J illustrates such a variation of prime editing contemplated herein, replacing napDNAbp (e.g., SpCas9 nickase) with any programmable nuclease domain, such as a zinc finger nuclease (ZFN) or a transcription activator-like effector nuclease (TALEN). Therefore, it is contemplated that a suitable nuclease does not necessarily need to be "programmed" with a nucleic acid targeting molecule (e.g., a guide RNA), but rather can be programmed by a DNA binding domain, such as by defining the specificity of the nuclease in particular. Just like prime editing with napDNAbp moieties, such alternative programmable nucleases are preferably engineered such that only one strand of the target DNA is cut. In other words, the programmable nuclease should preferably function as a nickase. Once a programmable nuclease is selected (e.g., ZFN or TALEN), additional functionality can then be engineered onto the system to allow it to operate according to a prime editing-like mechanism. For example, a programmable nuclease can be modified by coupling an RNA or DNA extension arm to it (e.g., via a chemical linker), the extension arm including a primer binding site (PBS) and a DNA synthesis template. A programmable nuclease can also be coupled to a polymerase (e.g., via a chemical or amino acid linker). The nature of this will depend on 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 nuclease or added in trans to facilitate the reaction as a whole (e.g., (a) a helicase to unwind DNA at the cut site to create a cut strand with a 3' end that can be used as a primer, (b) FEN1 to help remove the endogenous strand on the cut strand and drive the reaction toward replacement of the endogenous strand with the synthesized strand, or (c) an nCas9:gRNA complex to generate a second site nick on the opposing strand that may help drive incorporation of synthetic repair with favorable cellular repair of the non-edited strand). In a manner analogous to napDNAbp primed editing, such complexes with alternative programmable nucleases may be used to synthesize a newly synthesized replacement strand of DNA with the desired edit and then permanently incorporate it onto the target site of DNA.

Suitable alternative programmable nucleases are well known in the art and can be used in place of the napDNAbp:gRNA complex to construct alternative prime editor systems that can be programmed to selectively bind to target sites in DNA. And, they can be further modified in the manner described above to co-localize the polymerase with the RNA or DNA extension arm containing the primer binding site and the DNA synthesis template to a specific nick site. For example, as represented by FIG. 1J, transcription activator-like effector nucleases (TALENs) can be used in the prime editing methods and compositions described herein as programmable nucleases. TALENs are artificial restriction enzymes generated by fusing a TAL effector DNA binding domain to a DNA cleavage domain. These reagents allow efficient, programmable, and specific DNA cleavage and represent a powerful tool for in situ genome editing. Transcription activator-like effectors (TALEs) can be rapidly engineered to bind to virtually any DNA sequence. The term TALEN as used in this application is broad and includes monomeric TALENs 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 can be referred to as left TALENs and right TALENs. This refers to 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 herein by reference in their entirety. In addition, TALENs have been described in WO2015/027134, US 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. Research, vol. 39, No. 17, e82 (2011), each of which is incorporated herein by reference.

As depicted in Figure 1J, zinc finger nucleases can also be used in place of napDNAbp, such as Cas9 nickases, as alternative programmable nucleases for use in prime editing. Like TALENs, ZFN proteins can be modified such that they function as nickases, i.e., engineering the ZFN such that it cuts only one strand of the target DNA in a manner similar to the napDNAbp used in the prime editors described herein. ZFN proteins have been described extensively in the art, for example, in Carroll et al., "Genome Engineering with Zinc-Finger Nucleases," Genetics, Aug 2011, Vol. 188:773-782; Durai 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 is incorporated herein by reference in its entirety).

[3] Polymerase (e.g., reverse transcriptase)
In various embodiments, the prime editor (PE) system disclosed herein includes a polymerase (e.g., a DNA-dependent DNA polymerase or an RNA-dependent DNA polymerase, such as a reverse transcriptase), or a variant thereof, which can be provided as a fusion protein or in trans with napDNAbp or other programmable nuclease.

Any polymerase may be used in the prime editors disclosed herein. The polymerase may be a wild-type polymerase, a functional fragment, a mutant, a variant, or a truncated variant, and the like. The polymerase may include a wild-type polymerase from a eukaryotic, prokaryotic, archaeal, or viral organism, and/or the polymerase may be modified by processes based on genetic engineering, mutagenesis, directed evolution, and the like. The polymerase may include T7 DNA polymerase, T5 DNA polymerase, T4 DNA polymerase, Klenow fragment DNA polymerase, DNA polymerase III, and the like. The polymerase can also be thermostable and can include Taq, Tne, Tma, Pfu, Tfl, Tth, Stoffel fragment, VENT® and DEEPVENT® DNA polymerase, KOD, Tgo, JDF3, and mutants, variants, and derivatives thereof (U.S. Pat. No. 5,436,149; U.S. Pat. No. 4,889,818; U.S. Pat. No. 4,965,185; U.S. Pat. No. 5,079,352; U.S. Pat. No. 5,614,365; U.S. Pat. No. 5,374,553; U.S. Pat. No. 5,270,179; U.S. Pat. No. 5,047,342; U.S. Pat. No. 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 is incorporated herein by reference. For the synthesis of longer nucleic acid molecules (e.g., nucleic acid molecules longer than about 3-5 Kb in length), at least two DNA polymerases may be used. In certain embodiments, one of the polymerases may substantially lack 3' exonuclease activity, and the other may have 3' exonuclease activity. Such pairings may involve the same or different polymerases. Examples of DNA polymerases that substantially lack 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 polymerases that can be used in the prime editing elements disclosed herein are "template-dependent" polymerases (because the polymerase is intended to rely on a DNA synthesis template to define the sequence of the DNA strand being synthesized during prime editing. As used herein, the term "template DNA molecule" refers to a strand of nucleic acid from which a complementary nucleic acid strand is synthesized by a DNA polymerase, e.g., by a primer extension reaction of a DNA synthesis template of PEGRNA.

The term "template-dependent manner" as used herein is intended to refer to a process involving template-dependent extension of a primer molecule (e.g., DNA synthesis by a DNA polymerase). The term "template-dependent manner" refers to RNA or DNA polynucleotide synthesis in which the sequence of the newly synthesized strand of polynucleotide is dictated by well-known rules of complementary base pairing (see, e.g., 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 by base pairing. It is known that adenine nucleotides can form specific hydrogen bonds ("base pair") with nucleotides that are thymine or uracil. Similarly, it is known that cytosine nucleotides can base pair with guanine nucleotides. Therefore, in the case of prime editing, it can be said that the single strand of DNA synthesized by the polymerase of the prime editor on 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 polymerases. The present disclosure contemplates any wild-type polymerase obtained from any naturally occurring organism or virus, or from commercially available or non-commercial sources. In addition, the polymerases usable in the prime editors of the present disclosure may include any naturally occurring mutant polymerases, engineered mutant polymerases, or other variant polymerases, including truncated variants that retain function. The polymerases usable in the present application 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 present disclosure are template-based polymerases. That is, they synthesize nucleotide sequences in a template-dependent manner.

A polymerase is an enzyme that synthesizes a nucleotide strand that can be used in conjunction with the prime editor system described herein. The polymerase is preferably a "template-dependent" polymerase (i.e., a polymerase that synthesizes a nucleotide strand based on the order of nucleotide bases of a template strand). In certain configurations, the polymerase can also be "template-independent" (i.e., a polymerase that synthesizes a nucleotide strand without the requirement of a template strand). The polymerase can also be further categorized as a "DNA polymerase" or an "RNA polymerase". In various embodiments, the prime editor system includes a DNA polymerase. In various embodiments, the DNA polymerase can be a "DNA-dependent DNA polymerase" (i.e., according to which the template molecule is a strand of DNA). In such cases, the DNA template molecule can be a PEgRNA, and the extension arm comprises a strand of DNA. In such cases, the PEgRNA can also be referred to as a chimeric or hybrid PEgRNA, which includes an RNA portion (i.e., a guide RNA component that includes a spacer and a gRNA core) and a DNA portion (i.e., an extension arm). In various other embodiments, the DNA polymerase can be an "RNA-dependent DNA polymerase" (i.e., according to which the template molecule is a strand of RNA). In such cases, the PEgRNA is RNA, i.e., RNA elongation is involved. The term "polymerase" can also refer to an enzyme that catalyzes the polymerization of nucleotides (i.e., polymerase activity). Generally, the enzyme will initiate synthesis at the 3' end of a primer annealed to a polynucleotide template sequence (e.g., a primer sequence annealed to the primer binding site of the PEgRNA) and proceed toward the 5' end of the template strand. A "DNA polymerase" catalyzes the polymerization of deoxynucleotides. The term DNA polymerase as used herein with reference to a DNA polymerase includes "functional fragments thereof." A "functional fragment thereof" refers to any portion of a wild-type or mutant DNA polymerase encompassing less than the entire amino acid sequence of the polymerase that retains the ability to catalyze the polymerization of a polynucleotide under at least one set of conditions. Such a functional fragment may exist as a separate entity, or it may be a component of a larger polypeptide, such as a fusion protein.

In some embodiments, the polymerase can be from a bacteriophage. Bacteriophage DNA polymerases generally lack 5' to 3' exonuclease activity because this activity is encoded by a separate polypeptide. Examples of suitable DNA polymerases are T4, T7, and Phi29 DNA polymerases. Commercially available enzymes are: T4 (available from many sources, e.g., Epicentre) and T7 (available from many sources, e.g., Epicentre for unmodified and USB for the 3' to 5' exo T7 "Sequenase" DNA polymerase).

In another embodiment, the polymerase is an archaeal polymerase. There are two distinct classes of DNA polymerases that have been identified in archaea: 1. Family B/pol type I (homolog of Pfu from Pyrococcus furiosus) and 2. pol type II (homolog of P. furiosus DP1/DP2 two-subunit polymerase). DNA polymerases from both classes have been shown to naturally lack associated 5' to 3' exonuclease activity and to possess 3' to 5' exonuclease (proofreading) activity. A suitable DNA polymerase (pol I or pol II) may be derived from an archaea that has an optimal growth temperature that is similar to the desired assay temperature.

Thermostable archaeal DNA polymerases have been isolated from Pyrococcus species (furiosus, sp. GB-D, woesii, abysii, horikoshii), Thermococcus species (kodakaraensis KOD1, litoralis, sp. 9 degrees North-7, sp. JDF-3, gorgonarius), Pyrodictium occultum, and Archaeoglobus fulgidus.

The polymerase can also be from a Eubacterium species. There are three classes of Eubacterial DNA polymerases, pol I, II, and III. Enzymes of 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 polymerase represents the major replicative DNA polymerase of the cell and is composed of multiple subunits. The pol III catalytic subunit lacks 5' to 3' exonuclease activity, but in some cases, the 3' to 5' exonuclease activity is located on 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 various 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 present invention further provides for chimeric or non-chimeric DNA polymerases that have been chemically modified according to the methods disclosed in U.S. Patent Nos. 5,677,152, 6,479,264, and 6,183,998, the contents of which are incorporated herein by reference in their entireties.

Additional archaeal 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. Exemplary Reverse Transcriptases
In various embodiments, the prime editors described herein comprise reverse transcriptase as a polymerase. The present disclosure contemplates any wild-type reverse transcriptase obtained from any naturally occurring organism or virus, or from commercially available or non-commercial sources. In addition, the reverse transcriptase usable in the prime editors of the present disclosure can include any naturally occurring mutant RT, engineered mutant RT, or other variant RT, including truncated variants that retain function. RT can also be engineered to contain specific amino acid substitutions, such as those specifically disclosed herein.

Reverse transcriptase is a multifunctional enzyme that typically possesses three enzymatic activities, including RNA- and DNA-dependent DNA polymerization activity, and an RNase H activity that catalyzes the cleavage of RNA in RNA-DNA hybrids. Some mutants of reverse transcriptase have disabled the RNase H portion to prevent unintended damage to mRNA. These enzymes, which synthesize complementary DNA (cDNA) using mRNA as a template, were first identified in RNA viruses. Subsequently, reverse transcriptase was isolated and purified directly from virus particles, cells, or tissues (see, e.g., 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 is incorporated by reference). More recently, mutants and fusion proteins have been created for improved properties, such as thermostability, fidelity, and activity. Any wild-type, variant, and/or mutant form of reverse transcriptase known in the art or that 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 example, the gene can be obtained from a eukaryotic cell infected with a retrovirus, or from a number of plasmids containing either the entire retroviral genome or portions thereof. In addition, messenger RNA-like RNA containing the RT gene can be obtained from a retrovirus. Examples of sources of 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, e.g., 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 is incorporated herein by reference in its entirety.

Wild-type RT
Exemplary enzymes for use with the prime editor disclosed herein may include, but are not limited to, M-MLV reverse transcriptase and RSV reverse transcriptase. Enzymes with reverse transcriptase activity are commercially available. In some embodiments, reverse transcriptase is provided in trans with other components of the prime editor (PE) system. That is, reverse transcriptase is expressed or otherwise provided as an individual component, i.e., not as a fusion protein with napDNAbp.

Those skilled 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 leukosis and sarcoma virus (ASLV) reverse transcriptase, including, but 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 virus associated virus (MAV) reverse transcriptase) may be suitably used in the subject methods and compositions described herein.

An exemplary wild-type RT enzyme is:

Variant and error-prone RT
Reverse transcriptase is essential for the synthesis of a complementary DNA (cDNA) strand from an RNA template. Reverse transcriptase is an enzyme composed of distinct domains that exert different biochemical activities. The enzyme catalyzes the synthesis of DNA from an RNA template as follows: in the presence of an annealed primer, reverse transcriptase binds to the RNA template and initiates the polymerization reaction. The RNA-dependent DNA polymerase activity synthesizes a complementary DNA (cDNA) strand and incorporates dNTPs. The RNase H activity degrades the RNA template in the DNA:RNA complex. Hence, reverse transcriptase contains (a) a binding activity that recognizes and binds RNA/DNA hybrids, (b) an RNA-dependent DNA polymerase activity, and (c) an RNase H activity. In addition, reverse transcriptases are generally considered to have various attributes, including their thermostability, processivity (rate of dNTP incorporation), and fidelity (or error rate). Reverse transcriptase variants contemplated in this application can include any mutation in the reverse transcriptase that impacts or alters 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 enzymatic properties (e.g., thermostability, processivity, or fidelity). Such variants can be available in the public domain, commercially available, or can be made using known methods of mutagenesis, including directed evolution processes (e.g., PACE or PANCE).

In various embodiments, the reverse transcriptase may be a variant reverse transcriptase. As used herein, "variant reverse transcriptase" includes any naturally occurring or engineered variant that contains one or more mutations (including single mutations, inversions, deletions, insertions, and rearrangements) relative to a reference sequence (e.g., a reference wild-type sequence). RT naturally possesses several activities, including RNA-dependent DNA polymerase activity, RNase 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 be incorporated onto the host genome, from which new copies of the RNA can be made by host cell transcription. A variant RT may contain mutations that impact one or more of these activities (either reducing or increasing these activities, or eliminating these activities altogether). In addition, a variant RT may contain one or more mutations that render the RT more or less stable, less prone to aggregation, easier to purify and/or detect, and/or other the modifications of properties or characteristics.

Those skilled 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 leukosis and sarcoma virus (ASLV) reverse transcriptase, including, but 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 suitable for use in the subject methods and compositions described herein.

One way to prepare variant RTs is by genetic modification (e.g., by modifying the DNA sequence of a wild-type reverse transcriptase). Several methods are known in the art that allow random and targeted mutation of DNA sequences (see, e.g., Ausubel et.al. Short Protocols in Molecular Biology (1995) 3.sup.rd Ed. John Wiley & Sons, Inc.). In addition, there are several commercially available kits for site-directed mutagenesis, including both conventional and PCR-based methods. Examples include the QuikChange Site-Directed Mutagenesis Kit (AGILENT®), the Q5® Site-Directed Mutagenesis Kit (NEW ENGLAND BIOLABS®), and the GeneArt™ Site-Directed Mutagenesis System (THERMOFISHER SCIENTIFIC®).

In addition, mutant reverse transcriptases can be generated by insertion mutations or truncations (N-terminal, internal, or C-terminal insertions or truncations) according to methodologies known to those of skill in the art. The term "mutation" as used herein refers to the substitution of a residue on a sequence, e.g., a nucleic acid or amino acid sequence, with another residue, or the deletion or insertion of one or more residues on a sequence. Mutations are typically described herein by identifying the original residue, then the position of the residue on the sequence, and the identity of the newly substituted residue. Various methods for making amino acid substitutions (mutations) provided herein are well known in the art, for example, by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)). Mutations can include various categories, such as single nucleotide polymorphisms, microduplication regions, indels, and inversions, and are not meant to be limiting in any way. Mutations can include "loss-of-function" mutations, which are the normal consequence of a mutation that reduces or abolishes protein activity. Most loss-of-function mutations are recessive. Because in heterozygotes, the second chromosomal copy carries a fully functional, unmutated version of the gene that codes for the protein, the presence of which compensates for the effect of the mutation. Mutations also encompass "gain-of-function" mutations, which confer an abnormal activity to a protein or cell that is not otherwise present under normal conditions. Many gain-of-function mutations are in regulatory sequences rather than coding regions and therefore can have a number of consequences. For example, a mutation can lead to one or more genes being expressed in the wrong tissues, and these tissues gain a function that they normally lack. By their nature, gain-of-function mutations are usually dominant.

Older methods of site-directed mutagenesis known in the art rely on subcloning the sequence to be mutated onto a vector, such as an M13 bacteriophage vector, that allows the isolation of a single-stranded DNA template. In these methods, a mutagenic primer (i.e., a primer that can anneal to the site to be mutated but that has one or more mismatched nucleotides at the site to be mutated) is annealed to the single-stranded template, and then the complement of the template is polymerized starting from the 3' end of the mutagenic primer. The resulting duplex is then transformed into a host bacterium, and plaques are screened for the desired mutation.

More recently, site-directed mutagenesis has used PCR methodologies. These have the advantage of not requiring a single-stranded template. In addition, methods have been developed that do not require subcloning. 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 the extension of unwanted mutations introduced by the polymerase. Second, selection must be used to reduce the number of unmutated parent molecules remaining in the reaction. Third, extended-length PCR methods are preferred to allow the use of a single PCR primer set. And fourth, due to the non-template-dependent end-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 mutant products generated by PCR.

Methods for random mutagenesis exist in the art and will result in a panel of mutants with one or more randomly placed mutations. Such a panel of mutants can then be screened for those that exhibit a desired property, such as increased stability relative to wild-type reverse transcriptase.

An example of a method for random mutagenesis is the so-called "error-prone PCR." As the name implies, the method amplifies a given sequence under conditions where the DNA polymerase does not support high-fidelity incorporation. Conditions that promote error-prone incorporation vary for different DNA polymerases, but one of skill in the art can 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 ions in the buffer. Thus, the use of manganese ions and/or variations in magnesium or manganese ion concentration can be applied to affect the error rate of the polymerase.

In various aspects, the prime editor RT can be an "error-prone" reverse transcriptase variant. Any error-prone reverse transcriptase known and/or available in the art can be used. It will be understood that reverse transcriptases do not naturally possess any proofreading function; therefore, the error rate of reverse transcriptases is generally higher than DNA polymerases that contain proofreading activity. The error rate of any particular reverse transcriptase is a property of the "fidelity" of the enzyme, which represents the accuracy of DNA template-directed polymerization relative to its RNA template. RTs with high fidelity have low error rates. Conversely, RTs with low fidelity have high error rates. The fidelity of M-MLV-based reverse transcriptases is reported to have error rates ranging from 1 error per 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. This is incorporated by reference. Therefore, for purposes of this application, a reverse transcriptase that is considered to be "error-prone" or has "error-prone fidelity" is one that has an error rate that is less than 1 error per 15,000 nucleotides synthesized.

Error-prone reverse transcriptases can also be generated by mutagenesis of the starting RT enzyme (e.g., wild-type M-MLV RT). The method of mutagenesis is not limited and can include directed evolution processes, such as phage-assisted continuous evolution (PACE) or phage-assisted discontinuous evolution (PANCE). The term "phage-assisted continuous evolution (PACE)" as used herein refers to continuous evolution using phages as viral vectors. The general concept of PACE technology is described, for example, in International PCT application PCT/US2009/056194, filed September 8, 2009, published as WO2010/028347 on March 11, 2010; International PCT application PCT/US2011/066747, filed December 22, 2011, published as WO2012/088381 on June 28, 2012; and U.S. application PCT/US2011/066747, filed December 22, 2011, published as WO2012/088381 on May 5, 2015. No. 9,023,594, International PCT Application No. PCT/US2015/012022, filed January 20, 2015, published as WO2015/134121 on September 11, 2015, and International PCT Application No. PCT/US2016/027795, filed April 15, 2016, published as WO2016/168631 on October 20, 2016, the entire contents of each of which are incorporated herein by reference.

Error-prone reverse transcriptases can also be obtained by "phage-assisted discontinuous evolution (PANCE)". As used herein, this refers to discontinuous evolution using phages as viral vectors. PANCE is a simplified technique for rapid in vivo directed evolution, using serial flask transfer of evolving "selection phages" (SPs) containing the genes of interest to be evolved with new E. coli host cells, whereby the genes contained in the SPs are serially evolved while allowing the genes in the host E. coli to be held constant. Serial flask transfer has long served as a widely accessible approach for laboratory evolution of microorganisms, and more recently, a related approach for bacteriophage evolution has been developed. The PANCE system features lower stringency than the PACE system.

Other error-prone reverse transcriptases have been described in the literature. Each of these is contemplated for use in the methods and compositions of the present application. For example, error-prone reverse transcriptases are 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 is incorporated by reference. Additionally, reverse transcriptases, including error-prone reverse transcriptases, can be obtained from commercial suppliers, 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 present disclosure also contemplates reverse transcriptases having mutations in the RNase H domain. As mentioned above, one of the intrinsic properties of reverse transcriptase is RNase H activity, which cleaves the RNA template of an RNA:cDNA hybrid concomitantly with polymerization. RNase H activity may be undesirable for the synthesis of long cDNAs, since the RNA template may be degraded prior to the completion of full-length reverse transcription. RNase H activity may also reduce reverse transcription efficiency, possibly due to its competition with the polymerase activity of the enzyme. Therefore, the present disclosure contemplates any reverse transcriptase variant that contains an altered RNase H activity.

The present disclosure also contemplates reverse transcriptases having mutations in the RNA-dependent DNA polymerase domain. As mentioned above, one of the intrinsic properties of reverse transcriptase is RNA-dependent DNA polymerase activity, which incorporates nucleobases into the nascent cDNA strand encoded by the template RNA strand of an RNA:cDNA hybrid. The RNA-dependent DNA polymerase activity can be increased or decreased to either increase or decrease the processivity of the enzyme (i.e., in terms of its incorporation rate). Thus, the present disclosure contemplates any reverse transcriptase variant that contains modified RNA-dependent DNA polymerase activity such that the processivity of the enzyme is either increased or decreased relative to the unmodified version.

Reverse transcriptase variants with altered thermostability characteristics are also contemplated herein. The ability of reverse transcriptase to tolerate high temperatures is an important aspect of cDNA synthesis. The increased reaction temperature helps to denature RNA with strong secondary structures and/or high GC content, allowing reverse transcriptase to read through the sequence. As a result, reverse transcription at higher temperatures allows full-length cDNA synthesis and higher yields, which may lead to improved generation of 3' flap ssDNA as a result of the prime editing process. Wild-type M-MLV reverse transcriptase typically has an optimum temperature in the range of 37-48°C; however, mutations can be introduced that allow reverse transcription activity at higher temperatures above 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 RT, thermostable RT, and processivity-enhanced RT, can be engineered by a variety of routine strategies, including mutagenesis or evolutionary processes. In some cases, variants can arise by introducing a single mutation. In other cases, variants may require more than one mutation. For variants containing more than one mutation, the effect of a given mutation can be assessed by introducing the identified mutation into the wild-type gene by site-directed mutagenesis in isolation from other mutations carried by the specific mutant. Screening assays of the single mutants thus generated would then allow the determination of the effect of that mutation alone.

The variant RT enzymes used in this application may also include other "RT variants" that are 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 in this application 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 the corresponding wild-type RT (M-MLV reverse transcriptase) (e.g., SEQ ID NO: 89) or any of the reverse transcriptases of SEQ ID NOs: 90-100.

In some embodiments, the present disclosure may also utilize RT fragments that retain their functionality and are fragments of any of the RT proteins disclosed herein. In some embodiments, the RT fragments are at least 100 amino acids in length. In some embodiments, the fragments are 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 may also utilize RT variants in which the N-terminus or C-terminus or both are truncated by a certain number of amino acids, resulting in truncated variants that still retain full polymerase function. In some embodiments, the RT truncated variants have 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 truncation variants have 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 yet other embodiments, the RT truncation variants have truncations of the same or different lengths at the N- and C-terminal ends.

For example, the prime editor disclosed herein may include a truncated version of M-MLV reverse transcriptase. In this embodiment, the reverse transcriptase contains four mutations (D200N, T306K, W313F, T330P; note 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, thus making it potentially useful for applications where delivery of DNA sequences is difficult due to their 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:

In various embodiments, the prime editing factor disclosed herein can include one of the RT variants described herein, or an RT variant thereof 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 reference Cas9 variant.

In still other embodiments, the methods and compositions may utilize DNA polymerases that have been evolved into reverse transcriptases, as described in Effefson et al., "Synthetic evolutionary origin of a proofreading reverse transcriptase," Science, June 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 that also includes napDNAbp. In other words, in some embodiments, the reverse transcriptase is fused to napDNAbp as a fusion protein.

In various embodiments, the variant reverse transcriptase can be engineered from the wild-type M-MLV reverse transcriptase represented by SEQ ID NO:89.

In various embodiments, the primed editing elements described herein (wherein the RT is provided either as a fusion partner or in trans) can include variant RTs that contain 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 at the corresponding amino acid positions in the wild-type M-MLV RT of SEQ ID NO:89 or on another wild-type RT polypeptide sequence.

Provided below are several exemplary reverse transcriptases that may be provided as fusions to napDNAbp proteins or as individual proteins according to various embodiments of the disclosure. Exemplary reverse transcriptases include variants having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the following wild-type or partial enzymes:

In various other embodiments, the primed editing factors described herein (wherein the RT is provided either as a fusion partner or in trans) can include variant RTs that contain 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 at the corresponding amino acid positions in the wild-type M-MLV RT of SEQ ID NO:89 or on another wild-type RT polypeptide sequence, where "X" can be any amino acid.

In various other embodiments, a primed editing factor described herein (wherein RT is provided either as a fusion partner or in trans) can include a variant RT that includes a P51X mutation at the corresponding amino acid position in the wild-type M-MLV RT of SEQ ID NO:89 or on another wild-type RT polypeptide sequence, where "X" can be any amino acid. In some embodiments, X is L.

In various other embodiments, a primed editing factor described herein (wherein RT is provided either as a fusion partner or in trans) can include a variant RT that includes an S67X mutation at the corresponding amino acid position in the wild-type M-MLV RT of SEQ ID NO:89 or on another wild-type RT polypeptide sequence, where "X" can be any amino acid. In some embodiments, X is K.

In various other embodiments, a primed editing factor described herein (wherein RT is provided either as a fusion partner or in trans) can include a variant RT that includes an E69X mutation at the corresponding amino acid position in the wild-type M-MLV RT of SEQ ID NO:89 or on another wild-type RT polypeptide sequence, where "X" can be any amino acid. In some embodiments, X is K.

In various other embodiments, a primed editing factor described herein (wherein RT is provided either as a fusion partner or in trans) can include a variant RT that includes an L139X mutation at the corresponding amino acid position in the wild-type M-MLV RT of SEQ ID NO:89 or on another wild-type RT polypeptide sequence, where "X" can be any amino acid. In some embodiments, X is P.

In various other embodiments, a primed editing factor described herein (wherein RT is provided either as a fusion partner or in trans) can include a variant RT that includes a T197X mutation at the corresponding amino acid position in the wild-type M-MLV RT of SEQ ID NO:89 or on another wild-type RT polypeptide sequence, where "X" can be any amino acid. In some embodiments, X is A.

In various other embodiments, a primed editing factor described herein (wherein RT is provided either as a fusion partner or in trans) can include a variant RT that includes a D200X mutation at the corresponding amino acid position in the wild-type M-MLV RT of SEQ ID NO:89 or on another wild-type RT polypeptide sequence, where "X" can be any amino acid. In some embodiments, X is N.

In various other embodiments, a primed editing factor described herein (wherein RT is provided either as a fusion partner or in trans) can include a variant RT that includes an H204X mutation at the corresponding amino acid position in the wild-type M-MLV RT of SEQ ID NO:89 or on another wild-type RT polypeptide sequence, where "X" can be any amino acid. In some embodiments, X is R.

In various other embodiments, a primed editing factor described herein (wherein RT is provided either as a fusion partner or in trans) can include a variant RT that includes an F209X mutation at the corresponding amino acid position in the wild-type M-MLV RT of SEQ ID NO:89 or on another wild-type RT polypeptide sequence, where "X" can be any amino acid. In some embodiments, X is N.

In various other embodiments, a primed editing factor described herein (wherein RT is provided either as a fusion partner or in trans) can include a variant RT that includes an E302X mutation at the corresponding amino acid position in the wild-type M-MLV RT of SEQ ID NO:89 or on another wild-type RT polypeptide sequence, where "X" can be any amino acid. In some embodiments, X is K.

In various other embodiments, a primed editing factor described herein (wherein RT is provided either as a fusion partner or in trans) can include a variant RT that includes an E302X mutation at the corresponding amino acid position in the wild-type M-MLV RT of SEQ ID NO:89 or on another wild-type RT polypeptide sequence, where "X" can be any amino acid. In some embodiments, X is R.

In various other embodiments, a primed editing factor described herein (wherein RT is provided either as a fusion partner or in trans) can include a variant RT that includes a T306X mutation at the corresponding amino acid position in the wild-type M-MLV RT of SEQ ID NO:89 or on another wild-type RT polypeptide sequence, where "X" can be any amino acid. In some embodiments, X is K.

In various other embodiments, a primed editing factor described herein (wherein RT is provided either as a fusion partner or in trans) can include a variant RT that includes an F309X mutation at the corresponding amino acid position in the wild-type M-MLV RT of SEQ ID NO:89 or on another wild-type RT polypeptide sequence, where "X" can be any amino acid. In some embodiments, X is N.

In various other embodiments, a primed editing factor described herein (wherein RT is provided either as a fusion partner or in trans) can include a variant RT that includes a W313X mutation at the corresponding amino acid position in the wild-type M-MLV RT of SEQ ID NO:89 or on another wild-type RT polypeptide sequence, where "X" can be any amino acid. In some embodiments, X is F.

In various other embodiments, a primed editing factor described herein (wherein RT is provided either as a fusion partner or in trans) can include a variant RT that includes a T330X mutation at the corresponding amino acid position in the wild-type M-MLV RT of SEQ ID NO:89 or on another wild-type RT polypeptide sequence, where "X" can be any amino acid. In some embodiments, X is P.

In various other embodiments, a primed editing factor described herein (wherein RT is provided either as a fusion partner or in trans) can include a variant RT that includes an L345X mutation at the corresponding amino acid position in the wild-type M-MLV RT of SEQ ID NO:89 or on another wild-type RT polypeptide sequence, where "X" can be any amino acid. In some embodiments, X is G.

In various other embodiments, a primed editing factor described herein (wherein RT is provided either as a fusion partner or in trans) can include a variant RT that includes an L435X mutation at the corresponding amino acid position in the wild-type M-MLV RT of SEQ ID NO:89 or on another wild-type RT polypeptide sequence, where "X" can be any amino acid. In some embodiments, X is G.

In various other embodiments, a primed editing factor described herein (wherein RT is provided either as a fusion partner or in trans) can include a variant RT that includes an N454X mutation at the corresponding amino acid position in the wild-type M-MLV RT of SEQ ID NO:89 or on another wild-type RT polypeptide sequence, where "X" can be any amino acid. In some embodiments, X is K.

In various other embodiments, a primed editing factor described herein (wherein RT is provided either as a fusion partner or in trans) can include a variant RT that includes a D524X mutation at the corresponding amino acid position in the wild-type M-MLV RT of SEQ ID NO:89 or on another wild-type RT polypeptide sequence, where "X" can be any amino acid. In some embodiments, X is G.

In various other embodiments, a primed editing factor described herein (wherein RT is provided either as a fusion partner or in trans) can include a variant RT that includes an E562X mutation at the corresponding amino acid position in the wild-type M-MLV RT of SEQ ID NO:89 or on another wild-type RT polypeptide sequence, where "X" can be any amino acid. In some embodiments, X is Q.

In various other embodiments, a primed editing factor described herein (wherein RT is provided either as a fusion partner or in trans) can include a variant RT that includes a D583X mutation at the corresponding amino acid position in the wild-type M-MLV RT of SEQ ID NO:89 or on another wild-type RT polypeptide sequence, where "X" can be any amino acid. In some embodiments, X is N.

In various other embodiments, a primed editing factor described herein (wherein RT is provided either as a fusion partner or in trans) can include a variant RT that includes an H594X mutation at the corresponding amino acid position in the wild-type M-MLV RT of SEQ ID NO:89 or on another wild-type RT polypeptide sequence, where "X" can be any amino acid. In some embodiments, X is Q.

In various other embodiments, a primed editing factor described herein (wherein RT is provided either as a fusion partner or in trans) can include a variant RT that includes an L603X mutation at the corresponding amino acid position in the wild-type M-MLV RT of SEQ ID NO:89 or on another wild-type RT polypeptide sequence, where "X" can be any amino acid. In some embodiments, X is W.

In various other embodiments, a primed editing factor described herein (wherein RT is provided either as a fusion partner or in trans) can include a variant RT that includes an E607X mutation at the corresponding amino acid position in the wild-type M-MLV RT of SEQ ID NO:89 or on another wild-type RT polypeptide sequence, where "X" can be any amino acid. In some embodiments, X is K.

In various other embodiments, a primed editing factor described herein (wherein RT is provided either as a fusion partner or in trans) can include a variant RT that includes a D653X mutation at the corresponding amino acid position in the wild-type M-MLV RT of SEQ ID NO:89 or on another wild-type RT polypeptide sequence, where "X" can be any amino acid. In some embodiments, X is N.

Provided below are several exemplary reverse transcriptases that may be provided as fusions to napDNAbp proteins or as individual proteins according to various embodiments of the disclosure. Exemplary reverse transcriptases include variants having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the following wild-type or partial enzymes:

The Prime Editor (PE) system described herein contemplates any publicly available reverse transcriptases described or disclosed in any of the following U.S. patents, each of which is incorporated by reference in its entirety: U.S. Patent 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 variants thereof that may be made using known methods for incorporating mutations or evolving proteins. The following references describe reverse transcriptases in the art. Each of those disclosures is incorporated by reference in its entirety.

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, REVIEWS 1017 (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 targeted 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. & Zsiros, 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, 859-86 (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 set forth above regarding reverse transcriptase are hereby incorporated by reference in their entirety unless already so claimed.

[4] PE Fusion Protein The prime editor (PE) system described herein contemplates a fusion protein comprising napDNAbp and a polymerase (e.g., a DNA-dependent DNA polymerase or an RNA-dependent DNA polymerase, such as a reverse transcriptase), optionally linked by a linker. The present application contemplates that any suitable napDNAbp and polymerase (e.g., a DNA-dependent DNA polymerase or an RNA-dependent DNA polymerase, such as a reverse transcriptase) are combined into a single fusion protein. Examples of napDNAbp and polymerases (e.g., a DNA-dependent DNA polymerase or an RNA-dependent DNA polymerase, such as a reverse transcriptase), respectively, are defined herein. The present disclosure is in no way meant to be limited to the specific polymerases identified herein, as polymerases are well known in the art and amino acid sequences are readily available.

In various embodiments, the fusion protein may comprise any suitable structural configuration. For example, the fusion protein may comprise, from the N-terminus to the C-terminus, a napDNAbp fused to a polymerase (e.g., a DNA-dependent DNA polymerase or an RNA-dependent DNA polymerase, e.g., a reverse transcriptase). In other embodiments, the fusion protein may comprise, from the N-terminus to the C-terminus, a polymerase (e.g., a reverse transcriptase) fused to the napDNAbp. The fused domains may optionally be linked by a linker, e.g., an amino acid sequence. In other embodiments, the fusion protein may comprise the structure _NH2- [napDNAbp]-[polymerase]-COOH; or _NH2- [polymerase]-[napDNAbp]-COOH, where each "]-[" indicates the presence of an optional linker sequence. In embodiments where the polymerase is a reverse transcriptase, the fusion protein may comprise the structure _NH2- [napDNAbp]-[RT]-COOH; or _NH2- [RT]-[napDNAbp]-COOH, with each of the "]-[" indicating the presence of an optional linker sequence.

An exemplary fusion protein is illustrated in Figure 14, which shows a fusion protein comprising MLV reverse transcriptase ("MLV-RT") fused via a linker sequence to nickase Cas9 ("Cas9(H840A)"). This example is not intended to limit the scope of fusion proteins that may be utilized in the prime editor (PE) systems 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 (i.e., Cas9 nickase) containing the H840A mutation and M-MLV RT wild type, with an N-terminal NLS sequence (19 amino acids) and an amino acid linker (32 amino acids) connecting 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 sequences of PE1 and its individual components are as follows:

In another embodiment, the prime editor fusion protein can have the following amino acid sequence (referred to herein as "PE2"), which includes a Cas9 variant (i.e., Cas9 nickase) containing the H840A mutation and M-MLV RT containing the mutations D200N, T330P, L603W, T306K, and W313F, and an N-terminal NLS sequence (19 amino acids) and an amino acid linker (33 amino acids) that connects 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:

In yet other embodiments, a prime editor fusion protein may have the following amino acid sequence:

In other embodiments, a prime editor fusion protein can be based on SaCas9 or SpCas9 nickase with altered PAM specificity, such as the following exemplary sequences:

In yet other embodiments, a prime editor fusion protein 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 four mutations (D200N, T306K, W313F, T330P; note that the L603W mutation present in PE2 is no longer present due to truncation). The DNA sequence encoding this truncated editor is 522 bp smaller than PE2, thus making it potentially useful for applications where delivery of DNA sequences is difficult due to their size (i.e., adeno-associated virus and lentivirus delivery). This embodiment is referred to as Cas9(H840A)-MMLV-RT(trunc) or "short PE2" or "PE2-trunc" and has the following amino acid sequence:

See Figure 75, which provides a bar graph comparing the efficiency (i.e., "% of total sequencing reads with defined edits or indels") of PE2, PE2-trunc, PE3, and PE3-trunc for different target sites in various cell lines. The data show that prime editors containing truncated RT variants were about as efficient as prime editors containing the untruncated RT protein.

In various embodiments, the prime editor fusion proteins contemplated herein may also include variants of any of the sequences disclosed above 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 prime editor fusion sequences indicated above.

In some embodiments, a linker may be used to link any of the peptides or peptide domains or portions of the invention (e.g., napDNAbp linked or fused to reverse transcriptase).

[5] Linker and other domains
The PE fusion protein may contain various other domains in addition to the napDNAbp (e.g., Cas9 domain) and the polymerase domain (e.g., RT domain). For example, in the case where the napDNAbp is Cas9 and the polymerase is RT, the PE fusion protein may contain one or more linkers linking the Cas9 domain to the RT domain. The linkers may also link other functional domains, such as a nuclear localization sequence (NLS) or FEN1 (or other flap endonucleases), to the PE fusion protein or domains thereof.

Additionally, in embodiments involving trans-prime editing, a linker can be used to link the tPERT recruitment protein to the 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 for separately fusing the polymerase domain and recruitment protein domain to the napDNAbp.

A. Linker
As defined above, the term "linker" as used herein refers to a chemical group or molecule that connects two molecules or moieties, such as the binding domain and cleavage domain of a nuclease. In some embodiments, the linker connects the gRNA binding domain of an RNA-programmable nuclease and the catalytic domain of a polymerase (e.g., reverse transcriptase). In some embodiments, the linker connects dCas9 and reverse transcriptase. Typically, the linker is located between or flanked by two groups, molecules, or other moieties, and is 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, e.g., 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 can be as simple as a covalent bond, or it can be a polymeric linker that is many atoms in length. In some embodiments, the linker is a poly(pol) peptide or based on amino acids. In other embodiments, the linker is not peptide-like. In some embodiments, the linker is a covalent bond (e.g., carbon-carbon bond, disulfide bond, carbon-heteroatom bond, etc.). In some embodiments, the linker is a carbon-nitrogen bond of an amide linkage. In some embodiments, the linker is a cyclic or acyclic, substituted or unsubstituted, branched or unbranched, aliphatic or heteroaliphatic linker. In some embodiments, the linker is polymeric (e.g., polyethylene, polyethylene glycol, polyamide, polyester, etc.). In some embodiments, the linker comprises a monomer, dimer, or polymer of an aminoalkanoic acid. In some embodiments, the linker comprises an aminoalkanoic acid (e.g., glycine, ethanoic acid, alanine, beta-alanine, 3-aminopropanoic acid, 4-aminobutyric acid, 5-pentanoic acid, etc.). In some embodiments, the linker comprises a monomer, dimer, or polymer of aminohexanoic acid (Ahx). In some 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 an amino acid. In some embodiments, the linker comprises a peptide. In some embodiments, the linker comprises an aryl or heteroaryl moiety. In some embodiments, the linker is based on a phenyl ring. The linker may include a functionalized moiety to facilitate attachment of a nucleophile from the peptide to the linker (e.g., thiol, amino). 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, where n is independently an integer between 1 and 30, and X is any amino acid. In some embodiments, the linker comprises the amino acid sequence (GGS)N (SEQ ID NO:176), where 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 SGGSSGGSSGSETPGTSESATPESAGSYPYDVPDYAGSAAPAAKKKKLDGSGSGGSSGGS (SEQ ID NO: 175, 60AA).

In some embodiments, linkers can be used to link any of the peptides or peptide domains or portions of the invention (e.g., napDNAbp linked or fused to reverse transcriptase).

As defined above, the term "linker" as used herein refers to a chemical group or molecule that links two molecules or moieties, such as the binding domain and cleavage domain of a nuclease. In some embodiments, the linker links the gRNA binding domain of an RNA-programmable nuclease and the catalytic domain of a recombinase. In some embodiments, the linker links dCas9 and reverse transcriptase. Typically, the linker is located between or flanked by two groups, molecules, or other moieties and is connected to each one via a covalent bond, thus connecting the two. In some embodiments, the linker is an amino acid or multiple 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, e.g., 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 can be as simple as a covalent bond, or it can be a polymeric linker that is many atoms in length. In some embodiments, the linker is a polypeptide or based on amino acids. In other embodiments, the linker is not peptide-like. In some embodiments, the linker is a covalent bond (e.g., carbon-carbon bond, disulfide bond, carbon-heteroatom bond, etc.). In some embodiments, the linker is a carbon-nitrogen bond of an amide linkage. In some embodiments, the linker is a cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic or heteroaliphatic linker. In some embodiments, the linker is polymeric (e.g., polyethylene, polyethylene glycol, polyamide, polyester, etc.). In some embodiments, the linker comprises a monomer, dimer, or polymer of an aminoalkanoic acid. In some embodiments, the linker comprises an aminoalkanoic acid (e.g., glycine, ethanoic acid, alanine, beta-alanine, 3-aminopropanoic acid, 4-aminobutyric acid, 5-pentanoic acid, etc.). In some embodiments, the linker comprises a monomer, dimer, or polymer of aminohexanoic acid (Ahx). In some 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 an amino acid. In some embodiments, the linker comprises a peptide. In some embodiments, the linker comprises an aryl or heteroaryl moiety. In some embodiments, the linker is based on a phenyl ring. The linker may include a functionalized moiety to facilitate attachment of a nucleophile from the peptide to the linker (e.g., thiol, amino). 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, where n is independently an integer between 1 and 30, and X is any amino acid. In some embodiments, the linker comprises the amino acid sequence (GGS)N (SEQ ID NO: 176), where 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 link the prime editor domains together:
GGS (SEQ ID NO:767);
GGSGGS (SEQ ID NO:768);
GGSGGSGGS (SEQ ID NO:769);
SGGSSGGSSGSETPGTSESATPESSGGSSGGSS (SEQ ID NO: 127);
SGSETPGTSESATPES (SEQ ID NO: 171);
SGGSSGGSSGSETPGTSESATPESAGSYPYDVPDYAGSAAPAAKKKKLDGSGSGGSSGGS (sequence number 175).

B. Nuclear localization sequence (NLS)
In various embodiments, the PE fusion protein may contain one or more nuclear localization sequences (NLS), which aid in promoting the translocation of the protein into the cell nucleus. Such sequences are well known in the art and may be used to , which may include the following examples:

The above NLS examples are not limiting. The PE fusion protein may contain 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 is incorporated herein by reference.

In various embodiments, the prime editing factors and constructs encoding the prime editing factors disclosed herein further comprise one or more, preferably at least two, nuclear localization signals. In some embodiments, the prime editing factor comprises at least two NLSs. In embodiments with at least two NLSs, the NLSs can be the same NLS or they can be different NLSs. In addition, the NLS can be expressed as part of a fusion protein with the remainder of the prime editing factor. In some embodiments, one or more of the NLSs is a bipartite NLS ("bpNLS"). In some embodiments, the fusion proteins of the present disclosure comprise two bipartite NLSs. In some embodiments, the fusion proteins of the present disclosure comprise more than two bipartite NLSs.

The location of the NLS fusion can be at the N-terminus, C-terminus, or within the sequence of the prime editor (e.g., inserted between the encoded napDNAbp component (e.g., Cas9) and the polymerase domain (e.g., reverse transcriptase domain).

The NLS can be any known NLS sequence in the art. The NLS can also be any future discovered NLS for nuclear localization. The NLS can also 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 facilitates the import of a protein into a cell nucleus, for example, by nuclear transport. Nuclear localization sequences are known in the art and will be apparent to those skilled in the art. For example, NLS sequences are described in International PCT Application PCT/EP2000/011690, filed November 23, 2000, by Plank et al., published as WO/2001/038547 on May 31, 2001, the contents of which are incorporated herein by reference. In some embodiments, the 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: 125 ). In other embodiments, the NLS comprises the amino acid sequence NLSKRPAAIKKAGQAKKKK (SEQ ID NO: 136 ), PAAKRVKLD (SEQ ID NO: 192), RQRRNELKRSF (SEQ ID NO: 3934 ), NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 3935 ).

In one aspect of the present disclosure, the prime editor may be modified by one or more nuclear localization signals (NLS), preferably at least two NLSs. In some embodiments, the prime editor is modified by two or more NLSs. The present disclosure contemplates the use of any nuclear localization signal known in the art at the time of this disclosure or any nuclear localization signal identified or otherwise made available by the state of the art at a later time of the present application. Exemplary nuclear localization signals are peptide sequences that direct a protein to the nucleus of the cell in which the sequence is expressed. Nuclear localization signals are predominantly basic and can be located almost anywhere in the amino acid sequence of a protein, generally comprising short sequences of 4 amino acids (Autieri & Agrawal, (1998) J. Biol. Chem. 273:14731-37, incorporated herein by reference) to 8 amino acids, and are typically rich in lysine and arginine residues (Magin et al., (2000) Virology 274:11-16, incorporated herein by reference). Nuclear localization signals often contain proline residues. A variety of nuclear localization signals have been identified and used to effect transport of biological molecules from the cytoplasm to the nucleus of the 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 are incorporated by reference. The transport is currently thought to involve nuclear pore proteins.

Most NLSs can be classified into three general groups: (i) monokaryotic NLSs, exemplified by the SV40 large T antigen NLS (PKKKRKV (SEQ ID NO: 16)); (ii) bikaryotic motifs consisting of two basic domains separated by a variable number of spacer amino acids, exemplified by the Xenopus nucleoplasmin NLS (KRXXXXXXXXXXKKKL (SEQ ID NO: 3936 )); and (iii) non-canonical sequences, such as M9 of the hnRNP A1 protein, influenza virus nucleoprotein NLS, and yeast Gal4 protein NLS (Dingwall and Laskey 1991).

Nuclear localization signals occur at various points along the amino acid sequence of a protein. NLSs have been identified from the N-terminus, C-terminus, and central regions of a protein. Thus, the present disclosure provides prime editors that can be modified with one or more NLSs at the C-terminus, N-terminus, and internal regions of the prime editor. Residues of the longer sequence that do not function as component NLS residues should be selected so as not to interfere, e.g., tonically or sterically, with the nuclear localization signal itself. Thus, while there are no strict limitations on the composition of sequences that include an NLS, such sequences can be functionally limited in length and composition in nature.

The present disclosure contemplates any suitable means for modifying a prime editor to include one or more NLSs. In one aspect, a prime editor can be engineered to express a prime editor protein that is translationally fused to one or more NLSs at its N-terminus or its C-terminus (or both), i.e., to form a prime editor-NLS fusion construct. In other embodiments, a nucleotide sequence encoding a prime editor can be genetically modified to incorporate an open reading frame encoding one or more NLSs in an internal region of the encoded prime editor. In addition, the NLS can include various amino acid linker or spacer regions encoded between the prime editor and an NLS amino acid sequence attached to the N-terminus, C-terminus, or internally, for example, and in the central region of the protein. Thus, the present disclosure also enables nucleotide constructs, vectors, and host cells for expressing fusion proteins comprising a prime editor and one or more NLSs.

The prime editors described herein may also include nuclear localization signals, which are linked to the prime editor via one or more linkers, such as and polymeric, amino acid, nucleic acid, polysaccharide, chemical, or nucleic acid linker elements. Linkers within the contemplated scope of this disclosure are not intended to have any limitation and may be any suitable type of molecule (e.g., polymer, amino acid, polysaccharide, nucleic acid, lipid, or any synthetic chemical linker domain) and may be linked to the prime editor by any suitable strategy that achieves the formation of a bond (e.g., covalent linkage, hydrogen bond) between the prime editor and one or more NLSs.

C. Flap endonucleases (e.g., FEN1)
In various embodiments, the PE fusion protein may comprise one or more flap endonucleases (e.g., FEN1), which refers to enzymes that catalyze the removal of 5' single-stranded 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 described herein may utilize endogenously provided 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 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 is incorporated herein by reference. An exemplary flap endonuclease is FEN1, which can be represented by the following amino acid sequence:

Flap endonucleases can also include any FEN1 variants, mutants, or other flap endonuclease orthologs, homologs, or variants. Non-limiting examples of FEN1 variants are:

In various embodiments, the prime editor fusion proteins contemplated herein may include flap endonuclease variants of any of the sequences disclosed above 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 can be utilized by the present method to facilitate removal of the 5' single-stranded DNA flap include, but are not limited to, (1) trex2, (2) exo1 endonuclease (e.g., Keijzers et al., Biosci Rep. 2015, 35(3):e00206).
Trex2

3' 3 prime repair exonuclease 2 (TREX2) - human

Accession number NM_080701
MSEAPRAETFVFLDLEATGLPSVEPEIAELSLFAVHRSSLENPEHDESGALVLPRVLDKLTLCMCPERPFTAKASEITGLSSEGLARCRKAGFDGAVVRTLQAFLSRQAGPICLVAHNGFDYDFPLLCAELRRLGARLPRDTVCLDTLPALRGLDRAHSHGTRARGRQGYSLGSLFHRYFRAEPSAAHSAEGDVHTLLLIFLHRAAELLAWADEQARGWAHIEPMYLPPDDPSLEA (sequence number 3865).

3' 3 prime repair exonuclease 2 (TREX2) - Mouse

Accession number NM_011907

MSEPPRAETFVFLDLEATGLPNMDPEIAEISLFAVHRSSLENPERDDSGSLVLPRVLDKLTLCMCPERPFTAKASEITGLSSESLMHCGKAGFNGAVVRTLQGFLSRQEGPICLVAHNGFDYDFPLLCTELQRLGAHLPQDTVCLDTLPALRGLDRAHSHGTRAQGRKSYSLASLFHRYFQAEPSAAHSAEGDVHTLLLIFLHRAPELLAWADEQARSWAHIEPMYVPPDGPSLEA (sequence number 3866).

3' 3 prime repair exonuclease 2 (TREX2) - Rat

Accession number NM_001107580

MSEPLRAETFVFLDLEATGLPNMDPEIAEISLFAVHRSSLENPERDDSGSLVLPRVLDKLTLCMCPERPFTAKASEITGLSSEGLMNCRKAAFNDAVVRTLQGFLSRQEGPICLVAHNGFDYDFPLLCTELQRLGAHLPRDTVCLDTLPALRGLDRVHSHGTRAQGRKSYSLASLFHRYFQAEPSAAHSAEGDVNTLLLIFLHRAPELLAWADEQARSWAHIEPMYVPPDGPSLEA (sequence number 3867).

ExoI
Human exonuclease 1 (EXO1) is involved in many different DNA metabolic processes, including DNA mismatch repair (MMR), micro-mediated end-joining, homologous recombination (HR), and replication. Human EXO1 belongs to the family of eukaryotic nucleases Rad2/XPG, which also includes FEN1 and GEN1. The Rad2/XPG family has conserved nuclease domains across species from phages to humans. The EXO1 gene product exhibits both 5' exonuclease and 5' flap activities. In addition, EXO1 is a unique 5' RNase Human EXO1 also contains H activity. Human EXO1 has high affinity for processing double-stranded DNA (dsDNA), nicks, gaps, and pseudo-Y structures, and can resolve Holliday junctions using its inherited flap activity. Human EXO1 is involved in MMR and contains a conserved binding domain that directly interacts with MLH1 and MSH2. EXO1 nucleolytic activity is positively stimulated by PCNA, MutSα (MSH2/MSH6 complex), 14-3-3, MRN, and the 9-1-1 complex.

Exonuclease 1 (EXO1) Accession number NM_003686 (Homo sapiens Exonuclease 1 (EXO1), transcript variant 3) - Isoform A
(Sequence number 3868).

Exonuclease 1 (EXO1) Accession number NM_006027 (Homo sapiens exonuclease 1 (EXO1), transcript variant 3) - isoform B

MGIQGLLQFIKEASEPIHVRKYKGQVVAVDTYCWLHKGAIACAEKLAKGEPTDRYVGFCMKFVNMLLSHGIKPILVFDGCTLPSKKEVERSRRERRQANLLKGKQLLREGKVSEARECFTRSINITHAMAHKVIKAARSQGVDCLVAPYEADAQLAYLNKAGIVQAIITEDSDLLAFGCKKVILKMDQFGNGLEIDQARLGMCRQLGDVFTEEKFRYMCILSGCDYLSSLRGIGLAKACKVLRLANNPDIVKVIKKIGHYLKMNITVPEDYINGFIRANNTFLYQLVFDPIKRKLIPLNAYEDDVDPETLSYAGQYVDDSIALQIALGNKDINTFEQIDDYNPDTAMPAHSRSHSWDDKTCQKSANVSSIWHRNYSPRPESGTVSDAPQLKENPSTVGVERVISTKGLNLPRKSSIVKRPRSAELSED DLLSQYSLSFTKKTKKNSSEGNKSLSFSEVFVPDLVNGPTNKKSVSTPPRTRNKFATFLQRKNEESGAVVVPGTRSRFFCSSDSTDCVSNKVSIQPLDETAVTDKENNLHESEYGDQEGKRLVDTDVARNSSDDIPNNHIPGDHIPDKATVFTDEESYSFESSKFTRTISPPTLGTLRGTLSCFSWSGGLGDFSRTPSPSPSTALQQFRRKSDSPTSLPENNMSDVSQLKSEESSDDESHPLREEACSSQSQESGEFSLQSSNASKLSQCSSKDSDSEESDCNIKLLDSQSDQTSKLRLSHFSKKDTPLRNKVPGLYKSSSADSLSTTKIKPLGPARASGLSKKPASIQKRKHHNAENKPGLQIKLNELWKNFGFKKDSEKLPPCKKPLSPVRDNIQLTPEAEEDIFNKPECGRVQRAIFQ (sequence number 3869).

Exonuclease 1 (EXO1) Accession number NM_001319224 (Homo sapiens exonuclease 1 (EXO1), transcript variant 4) - isoform C

MGIQGLLQFIKEASEPIHVRKYKGQVVAVDTYCWLHKGAIACAEKLAKGEPTDRYVGFCMKFVNMLLSHGIKPILVFDGCTLPSKKEVERSRRERRQANLLKGKQLLREGKVSEARECFTRSINITHAMAHKVIKAARSQGVDCLVAPYEADAQLAYLNKAGIVQAIITEDSDLLAFGCKKVILKMDQFGNGLEIDQARLGMCRQLGDVFTEEKFRYMCILSGCDYLSSLRGIGLAKACKVLRLANNPDIVKVIKKIGHYLKMNITVPEDYINGFIRANNTFLYQLVFDPIKRKLIPLNAYEDDVDPETLSYAGQYVDDSIALQIALGNKDINTFEQIDDYNPDTAMPAHSRSHSWDDKTCQKSANVSSIWHRNYSPRPESGTVSDAPQLKENPSTVGVERVISTKGLNLPRKSSIVKRPRSELSEDD LLSQYSLSFTKKTKKNSSEGNKSLSFSEVFVPDLVNGPTNKKSVSTPPRTRNKFATFLQRKNEESGAVVVPGTRSRFFCSSDSTDCVSNKVSIQPLDETAVTDKENNLHESEYGDQEGKRLVDTDVARNSSDDIPNNHIPGDHIPDKATVFTDEESYSFESSKFTRTISPPTLGTLRGTLSCFSWSGGLGDFSRTPSPSPSTALQQFRRKSDSPTSLPENNMSDVSQLKSEESSDDESHPLREEACSSQSQESGEFSLQSSNASKLSQCSSKDSDSEESDCNIKLLDSQSDQTSKLRLSHFSKKDTPLRNKVPGLYKSSSADSLSTTKIKPLGPARASGLSKKPASIQKRKHHNAENKPGLQIKLNELWKNFGFKKDSEKLPPCKKPLSPVRDNIQLTPEAEEDIFNKPECGRVQRAIFQ (sequence number 3870).

D. Inteins and Split-Inteins
It will be appreciated 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 napDNAbp) or a fusion protein (e.g., a prime editor) into N-terminal and C-terminal halves and deliver them separately, then allow their colocalization to reform the complete protein (or fusion protein, as the case may be) within the cell. The separate halves of the protein or fusion protein may each contain a split intein tag to facilitate reformation of the complete protein or fusion protein by a protein trans-splicing mechanism.

Protein trans-splicing catalyzed by split inteins provides a purely enzymatic method for protein ligation. A split intein is essentially a continuous intein (e.g., a mini-intein) that has been split into two pieces, named N and C inteins, respectively. The N and C inteins of a split intein can non-covalently associate to form an active intein and catalyze a splicing reaction in essentially the same way that a continuous intein does. Split inteins are found in nature and have also been engineered in the laboratory. The term "split intein" as used herein refers to any intein in which one or more peptide bond cleavages exist between the N- and C-terminal amino acid sequences, such that the N- and C-terminal sequences become separate molecules that can non-covalently reassociate or reconstitute into an intein that is functional for a trans-splicing reaction. Any catalytically active intein or fragment thereof can be used to derive a split intein for use in the methods of the invention. For example, in one aspect, the split intein can be derived from a eukaryotic intein. In another aspect, the split intein can be derived from a bacterial intein. In another aspect, the split intein can be derived from an archaeal intein. Preferably, the split intein so derived will possess only the amino acid sequences essential for catalyzing the trans-splicing reaction.

As used herein, "N-terminal split intein (In)" refers to any intein sequence that includes an N-terminal amino acid sequence that is functional for a trans-splicing reaction. In therefore also includes sequences that are spliced out when trans-splicing occurs. In can include sequences that are modifications of the N-terminal portion of a naturally occurring intein sequence. For example, In can include additional amino acid residues and/or mutated residues, so long as the inclusion of such additional and/or mutated residues does not render In non-functional in trans-splicing. Preferably, the inclusion of additional and/or mutated residues improves or enhances the trans-splicing activity of In.

As used herein, "C-terminal split intein (Ic)" refers to any intein sequence that includes a C-terminal amino acid sequence that is functional for a trans-splicing reaction. In one aspect, Ic includes a stretch of 4 to 7 amino acid residues, at least 4 amino acids, that are from the last β-strand of the intein from which it is derived. Ic therefore also includes sequences that are spliced out when trans-splicing occurs. Ic can include sequences that are modifications of the C-terminal portion of a naturally occurring intein sequence. For example, Ic can include additional amino acid residues and/or mutated residues, so long as the inclusion of such additional and/or mutated residues does not render In non-functional in trans-splicing. Preferably, the inclusion of additional and/or mutated residues improves or enhances the trans-splicing activity of Ic.

In some embodiments of the invention, the peptide linked to Ic or In may contain additional chemical moieties including, among others, fluorescent groups, biotin, polyethylene glycol (PEG), amino acid analogs, unnatural amino acids, phosphate groups, glycosyl groups, radioisotope labels, and pharmaceutical molecules. In other embodiments, the peptide linked to Ic may contain one or more chemically reactive groups including, among others, ketones, aldehydes, Cys residues, and Lys residues. When an "intein splicing polypeptide (ISP)" is present, the N intein and C intein of the split intein may non-covalently associate to form an active intein and catalyze the splicing reaction. As used herein, "intein splicing polypeptide (ISP)" refers to the portion of the amino acid sequence of the split intein that remains when Ic, In, or both are removed from the split intein. In some embodiments, In includes an ISP. In other embodiments, Ic includes an ISP. In yet other embodiments, the ISP is a separate peptide that is not covalently linked to either In or Ic.

Split inteins can be generated from a stretch of inteins by engineering one or more split sites into the unstructured loop or intervening amino acid sequence between the -12 conserved beta strands found on the mini-intein structure. There can be some flexibility in the location of the split site within the region between the beta strands, provided that the creation of the split would not interrupt the structure of the intein, particularly the structured beta strands, 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 an N intein, another precursor protein consists of a C intein followed by a C extein part, and a trans-splicing reaction (catalyzed together by the N and C inteins) excises the two intein sequences and links the two extein sequences by a peptide bond. Protein trans-splicing, an enzymatic reaction, can work at very low (e.g., micromolar) concentrations of protein and can be carried out under physiological conditions.

An example sequence is as follows:

Inteins are most frequently found as a continuous domain, but some naturally occur in a split form. In this case, the two fragments are expressed as separate polypeptides and must associate before splicing, known as protein trans-splicing, can occur.

An exemplary split intein is the Ssp DnaE intein, which contains two subunits, DnaE-N and DnaE-C. The two distinct subunits are encoded by separate genes, 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 that can direct the trans-splicing of two separate proteins, each of which contains a fusion with either DnaE-N or DnaE-C.

Additional naturally occurring or engineered split intein sequences are known in the art or can be made from whole intein sequences described herein or 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 Nostc punctiforme, FEBS Lett, 580: 1853-1858, each of which is incorporated herein by reference. Additional split intein sequences can be found, for example, in WO2013/045632, WO2014/055782, WO2016/069774, and EP2877490, the contents of 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. al., J. Biol. Chem. 275:9091 (2000); Otomo, et al., Biochemistry 38:16040-16044 (1999); Otomo, et al., J. Biomol. NMR 14:105-114 (1999); Scott, et al., Proc. Natl. Acad. Sci. USA 96:13638-13643 (1999)), providing the opportunity to express a protein as two inactive fragments that subsequently undergo ligation to form a functional product, as shown, for example, in Figures 66 and 67 for the formation of a complete PE fusion protein from two separately expressed halves.

E. RNA-protein interaction domains
In various embodiments, two separate protein domains (e.g., Cas9 domain and polymerase domain) can be co-localized with each other to form a functional complex (similar to the function of a fusion protein containing two separate protein domains) by using an "RNA-protein recruitment system", e.g., "MS2 tagging technology". Such systems generally tag one protein domain with an "RNA-protein interaction domain" (also known as an "RNA-protein recruitment domain") and the other with an RNA-protein interaction domain, e.g., an "RNA binding protein" that specifically recognizes and binds to a particular hairpin structure. These types of systems can be exploited to co-localize domains of prime editors and to recruit additional functionality to the prime editors, such as UGI domains. In one example, MS2 tagging technology is based on the natural interaction of MS2 bacteriophage coat protein ("MCP" or "MS2cp") with stem-loop or hairpin structures, i.e., "MS2 hairpins", present on the genome of phages. In the case of the MS2 hairpin, it is recognized and bound by the MS2 bacteriophage coat protein (MCP), and therefore, in one exemplary scenario, a deaminase-MS2 fusion can recruit a Cas9-MCP fusion.

Other reviews of 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 is incorporated herein by reference in its entirety. 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: GCCAACATGAGGATCACCCATGTCTGCAGGGCC (SEQ ID NO: 763 ).

The amino acid sequence of MCP or MS2cp is:
GSASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVATQTVGGEELPVAGWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY (sequence number 764 ).

F. UGI domain
In other embodiments, the prime editors described herein may comprise one or more uracil glycosylase inhibitor domains. As used herein, the term "uracil glycosylase inhibitor (UGI)" or "UGI domain" refers to a protein that can inhibit uracil DNA glycosylase base excision repair enzyme. In some embodiments, the UGI domain comprises wild-type UGI or the UGI set forth in SEQ ID NO: 3873. In some embodiments, the UGI proteins provided herein include fragments of UGI and proteins homologous to UGI or UGI fragments. For example, in some embodiments, the UGI domain comprises a fragment of the amino acid sequence set forth in SEQ ID NO: 3873. In some embodiments, the UGI fragment comprises an amino acid sequence comprising 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 set forth in SEQ ID NO: 3873. In some embodiments, 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, a protein comprising UGI or a fragment of UGI or a homolog of UGI or a UGI fragment is referred to as a "UGI variant." 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 wild-type UGI or UGI 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 UGI set forth in SEQ ID NO: 3873. In some embodiments, UGI comprises the following amino acid sequence:

Uracil-DNA glycosylase inhibitors:

＞sp|P14739|UNGI_BPPB2
MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML (SEQ ID NO: 3873).

The prime editing factors described herein may contain more than one UGI domain, which may be separated by one or more linkers as described herein.

G. Additional PE Elements
In some embodiments, the prime editor described herein may comprise an inhibitor of base repair. The term "inhibitor of base repair" or "IBR" refers to a protein that can inhibit the activity of nucleic acid repair enzymes, such as base excision repair enzymes. 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, which can be a small molecule or peptide inhibitor of catalytically inactive glycosylase or catalytically inactive dioxygenase, or oxidase, or a variant thereof. In some embodiments, the IBR is an iBER that can be a TDG inhibitor, an MBD4 inhibitor, or an inhibitor of AlkBH enzyme. In some embodiments, the IBR is an iBER that comprises catalytically inactive TDG or catalytically inactive MBD4. An exemplary catalytically inactive TDG is the N140A mutant of SEQ ID NO: 3872 (human TDG).

Some exemplary glycosylases are provided below. Catalytically inactivated variants of any of these glycosylase domains are iBERs that can be fused to the napDNAbp or polymerase domains of the prime editors provided in this disclosure.

OGG(human)

MPARALLPRRMGHRTLASTPALWASIPCPRSELRLDLVLPSGQSFRWREQSPAHWSGVLADQVWTLTQTEEQLHCTVYRGDKSQASRPTPDELEAVRKYFQLDVTLAQLYHHWGSVDSHFQEVAQKFQGVRLLRQDPIECLFSFICSSNNNIARITGMVERLCQAFGPRLIQLDDVTYHGFPSLQALAGPEVEAHLRKLGLGYRARYVSASARAILEEQGGLAWLQQLRESSYEEAHKALCILPGVGTKVADCICLMALDKPQAVPVDVHMWHIAQRDYSWHPTTSQAKGPSPQTNKELGNFFRSLWGPYAGWAQAVLFSADLRQSRHAQEPPAKRRKGSKGPEG (Sequence number 3937 )

MPG(human)

MVTPALQMKKPKQFCRRMGQKKQRPARAGQPHSSSDAAQAPAEQPHSSSDAAQAPCPRERCLGPPTTPGPYRSIYFSSPKGHLTRLGLEFFDQPAVPLARAFLGQVLVRRLPNGTELRGRIVETEAYLGPEDEAAHSRGGRQTPRNRGMFMKPGTLYVYIIYGMYFCMNISSQGDGACVLLRALEPLEGLETMRQLRSTLRKGTASRVLKDRELCSGPSKLCQALAINKSFDQRDLAQDEAVWLERGPLEPSEPAVVAAARVGVGHAGEWARKPLRFYVRGSPWVSVVDRVAEQDTQA (sequence number 3938 )

MBD4(human)

MGTTGLESLSLGDRGAAPTVTSSERLVPDPPNDLRKEDVAMELERVGEDEEQMMIKRSSECNPLLQEPIASAQFGATAGTECRKSVPCGWERVVKQRLFGKTAGRFDVYFISPQGLKFRSKSSLANYLHKNGETSLKPEDFDFTVLSKRGIKSRYKDCSMALTSHLQNQSNNSNWNLRTRSKCKKDVFMPPSSSSELQESRGLSNFTSTHLLLKEDEGVDDVNFRKVRKPKGKVTILKGIPIKKTKKGCRKSCSGFVQSDSKRESVCNKADAESEPVAQKSQLDRTVCISDAGA CGETLSVTSEENSLVKKKERSLSSGSNFCSEQKTSGIINKFCSAKDSEHNEKYEDTFLESEEIGTKVEVVERKEHLHTDILKRGSEMDNNCSPTRKDFTGEKIFQEDTIPRTQIERRKTSLYFSSKYNKEALSPPRRKAFKKWTPPRSPFNLVQETLFHDPWKLLIATIFLNRTSGKMAIPVLWKFLEKYPSAEVARTADWRDVSELLKPLGLYDLRAKTIVKFSDEYLTKQWKYPIELHGIGKYGNDSYRIFCVNEWKQVHPEDHKLNKYHDWLWENHEKLSLS (sequence number 3871)

TDG(human)

MEAENAGSYSLQQAQAFYTFPFQQLMAEAPNMAVVNEQQMPEEVPAPAPAQEPVQEAPKGRKRKPRTTEPKQPVEPKKPVESKKSGKSAKSKEKQEKITDTFKVKRKVDRFNGVSEAELLTKTLPDILTFNLDIVIIGINPGLMAAYKGHHYPGPGNHFWKCLFMSGLSEVQLNHMDDHTLPGKYGIGFTNMVERTTPGSKDLSSKEFRE GGRILVQKLQKYQPRIAVFNGKCIYEIFSKEVFGVKVKNLEFGLQPHKIPDTETLCYVMPSSSARCAQFPRAQDKVHYYIKLKDLRDQLKGIERNMDVQEVQYTFDLQLAQEDAKKMAVKEEKYDPGYEAAYGGAYGENPCSSEPCGFSSNGLIESVELRGESAFSGIPNGQWMTQSFTDQIPSFSNHCGTQEQEEESHA (sequence number 3872)

In some embodiments, the fusion proteins described herein may contain one or more heterologous protein domains (e.g., about or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more domains in addition to the prime editor component). The fusion protein may contain any additional protein sequences and, optionally, a linker sequence between any two domains. Other exemplary features that may be present are localization sequences, e.g., cytoplasmic localization sequences, export sequences, e.g., nuclear export sequences, or other localization sequences, and sequence tags that are useful for solubilizing, purifying, or detecting the fusion protein.

Examples of protein domains that can be used to fuse a prime editor or a component thereof (e.g., napDNAbp domain, polymerase domain, or 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 blue fluorescent protein (BFP). Prime editors can be fused to genetic sequences encoding proteins or fragments of proteins that bind to DNA molecules or other cellular molecules, including, but not limited to, maltose binding protein (MBP), S-tags, Lex A DNA binding domain (DBD) fusions, GAL4 DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions. Additional domains that can form part of prime editors are described in U.S. Patent Publication No. 2011/0059502, published March 11, 2011, which is incorporated herein by reference in its entirety.

In certain aspects of the disclosure, reporter genes, including but not limited to glutathione-5-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), beta-galactosidase, beta-glucuronidase, luciferase, autofluorescent proteins, including green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and blue fluorescent protein (BFP), can be introduced into cells to encode a gene product that serves as a marker for measuring alteration or modification of expression of the gene product. In certain embodiments of the disclosure, the gene product is luciferase. In further embodiments of the disclosure, 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, Softag (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 one 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 can be temporally controlled by adjusting the residence time, amount, and/or activity of the expressed components of the PE system. For example, the PE described herein can be fused to a protein domain that can modify the intracellular half-life of the PE. In certain embodiments involving two or more vectors (e.g., vector systems in which the components described herein are encoded on two or more separate vectors), the activity of the PE system can be temporally controlled by controlling the timing at which the vectors are delivered. For example, in some embodiments, a vector encoding a nuclease system can deliver the PE preceding a vector encoding a template. In other embodiments, a vector encoding a PEgRNA can deliver the guide preceding a vector encoding a PE system. In some embodiments, the PE system and the vector encoding the PEgRNA are delivered simultaneously. In some embodiments, the vectors delivered simultaneously temporally deliver, for example, the PE, PEgRNA, and/or second strand guide RNA components. In further embodiments, the RNA transcribed from the coding sequence on the vector (e.g., e.g., nuclease transcript) may further comprise at least one element that can modify the intracellular half-life of the RNA and/or regulate 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 increase the stability of the RNA. In some embodiments, the element may decrease 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 comprise a polyadenylation signal (PA). In some embodiments, the element may comprise a cap, e.g., an upstream mRNA or PEgRNA end. In some embodiments, the RNA may be free of PA, such that it undergoes more rapid degradation in the cell after transcription. In some embodiments, the element may comprise at least one AU-rich element (ARE). The ARE may be bound by an ARE-binding protein (ARE-BP) in a manner that is tissue-type, cell-type, timing, cellular localization, and environment dependent. 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 woodchuck hepatitis virus (WHP).

A post-transcriptional regulatory element (WPRE) that generates a tertiary structure to enhance expression from a transcript. In further embodiments, the element is a modified and/or truncated WPRE sequence that can enhance expression from a transcript, e.g., as described 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 can be added to the 3'UTR of the RNA. In some embodiments, the element can be selected from other RNA sequence motifs enriched in either fast or slow decay transcripts.

In some embodiments, a vector encoding a PE or PEgRNA can be self-destructed by cleavage of a target sequence present on the vector by the PE system. Cleavage can prevent continued transcription of the PE or PEgRNA from the vector. Although transcription can occur for any amount of time on a linearized vector, expressed transcripts or proteins that undergo intracellular degradation will have less time to produce off-target effects without a continuous supply from the expression of the encoding vector.

[6] PEgRNA
The prime editing system described herein contemplates the use of any suitable PEgRNA. The inventors have discovered that the mechanism of primed target-primed reverse transcription (TPRT) can be exploited or adapted to perform precise and versatile CRISPR/Cas-based genome editing by using a specially constructed guide RNA that contains a reverse transcription (RT) template sequence that encodes the desired nucleotide change. Since the RT template sequence can be provided as an extension of a standard or conventional guide RNA molecule, the application refers to the specially constructed guide RNA as an "extended guide RNA" or "PEgRNA". The application contemplates any suitable configuration or arrangement of the extended guide RNA.

PEgRNA Architecture FIG. 3A shows one embodiment of an extended guide RNA that can be used in the prime editor system disclosed herein, where the existing guide RNA (in green) includes a ∼20 nt protospacer sequence and a gRNA core region, which binds together with the napDNAbp. In this embodiment, the guide RNA includes an RNA segment extended 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 FIG. 1A-1B, the hybrid of the RT primer binding site with the free 3' end formed after the nick is formed on the non-target strand of the R-loop, thereby priming the reverse transcriptase for DNA polymerization in the 5'-3' direction.

Figure 3B shows another embodiment of an extended guide RNA that can be used in the prime editing system disclosed herein, where the existing guide RNA (in green) includes a ∼20 nt protospacer sequence and a gRNA core, which binds together 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 Figures 1C-1D, the hybrid of the RT primer binding site with the free 3' end formed after the nick is formed on the non-target strand of the R-loop, thereby priming the reverse transcriptase for DNA polymerization in the 5'-3' direction.

Figure 3C shows another embodiment of an extended guide RNA that can be used in the prime editing system disclosed herein, where the existing guide RNA (in green) includes a ∼20 nt protospacer sequence and a gRNA core, which binds together 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 hybridizes to the free 3' end formed after the nick and is formed on the non-target strand of the R-loop, thereby priming the reverse transcriptase for DNA polymerization in the 5'-3' direction.

In one embodiment, the location of the intramolecular RNA extension is not within the protospacer sequence of the guide RNA. In another embodiment, the location of the intramolecular RNA extension is within the gRNA core. In yet another embodiment, the location of the intramolecular RNA extension is anywhere within the guide RNA molecule except within the protospacer sequence or at a position that does not interfere with the protospacer sequence.

In one embodiment, the intramolecular RNA extension is inserted downstream from the 3' end of the protospacer sequence. In another embodiment, the intramolecular 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, an intramolecular RNA extension is inserted into the gRNA, which refers to a portion of the guide RNA that corresponds to or includes the tracrRNA, and which binds to and/or interacts with the Cas9 protein or its equivalent (i.e., a different napDNAbp). Preferably, the insertion of the intramolecular RNA extension does not interfere, or only minimally interferes, with the interaction of the tracrRNA portion with 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 of 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, 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, any 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 some embodiments, encodes a single-stranded DNA molecule that is homologous to the non-target strand (and thus complementary to the corresponding site on the target strand) but contains one or more nucleotide changes. The at 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. At equilibrium, the single-stranded DNA product of the RT template sequence hybridizes to the complementary target strand sequence, thereby displacing the homologous endogenous target strand sequence. The displaced endogenous strand may in some embodiments be referred to as a 5′ endogenous DNA flap species (see, for example, FIG. 1E). This 5′ endogenous DNA flap species may be removed by a 5′ flap endonuclease (for example, FEN1), and the single-stranded DNA product, now hybridized to the endogenous target strand, is ligated to create a mismatch between the endogenous sequence and the newly synthesized strand. The mismatch may be resolved by the cell's intrinsic 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 that is replaced by the 5' flap species and overlaps with the site to be edited.

In various embodiments of the extended guide RNA, the reverse transcription template sequence may encode a single-stranded DNA flap complementary to the endogenous DNA sequence adjacent to the nick site, where the single-stranded DNA flap contains the desired nucleotide change. The single-stranded DNA flap may replace the endogenous single-stranded DNA at the nick site. The endogenous single-stranded DNA replaced at the nick site may have a 5' end and form an endogenous flap, which may be excised by the cell. In various embodiments, excision of the 5' endogenous flap may help drive product formation, where removal of the 5' endogenous flap promotes hybridization of the single-stranded 3' DNA flap with the corresponding complementary DNA strand and incorporation or assimilation of the desired nucleotide change carried by the single-stranded 3' DNA flap into the target DNA.

In various embodiments of the extended guide RNA, cellular repair of the single-stranded DNA flap results in incorporation of the desired nucleotide change, thereby forming the desired product.

In still other embodiments, the desired nucleotide changes are incorporated over an editing window that is between about -5 to +5 at the nick site, or between about -10 to +10 at the nick site, or between about -20 to +20 at the nick site, or between about -30 to +30 at the nick site, or between about -40 to +40 at the nick site, or between about -50 to +50 at the nick site, or between about -60 to +60 at the nick site, or between about -70 to +70 at the nick site, or between about -80 to +80 at the nick site, or between about -90 to +90 at the nick site, or between about -100 to +100 at the nick site, or between about -200 to +200 at the nick site.
In other embodiments, the desired nucleotide changes are from about +1 to +2 from the nick site, or from 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 ~+31, +1~+32, +1~+33, +1~+34, +1~+35, +1~+36, +1~+37, +1~+38, +1~+39, +1~+40, +1~+41, +1~+42, +1~+43, +1~+44, +1~+45, +1~+46, +1~+47, +1~+48, +1~+49, +1~+50, +1~+51, +1~+52, +1~+53, +1~+54, +1~+55, +1~+56, +1~+57, +1~+58, +1~+59, +1~+60, +1~+61, +1~+62, +1~+63, +1~+64, +1~ +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 + The edit window is placed between 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.

In still other embodiments, the desired nucleotide change is between about +1 and +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 +11 5, +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 will be incorporated into the editing window.

In various aspects, the extended guide RNA is a modified version of a guide RNA. The guide RNA may be naturally occurring, expressed from an encoding nucleic acid, or chemically synthesized. Methods for obtaining or otherwise synthesizing a guide RNA that includes a protospacer sequence that interacts with and hybridizes to a target strand at a genomic target site of interest, and for determining the appropriate sequence of the guide RNA, are well known in the art.

In various embodiments, the specific design aspects of the guide RNA sequence will depend on the nucleotide sequence of the genomic target site of interest (i.e., the desired site to be edited) and the type of napDNAbp (e.g., Cas9 protein) present in the prime editing system described herein, among other factors such as the location of the PAM sequence, the percent G/C content in the target sequence, the extent of microhomologous regions, secondary structure, etc.

In general, a guide sequence is any polynucleotide sequence that has sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of a napDNAbp (e.g., Cas9, a Cas9 homolog, or a Cas9 variant) to the target sequence. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence is about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or more, when optimally aligned using a suitable alignment algorithm. Optimal alignment may be determined using any suitable algorithm for aligning sequences, non-limiting examples of which include algorithms based on the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, the Burrows-Wheeler Transform (e.g., 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, the guide sequence is 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, the guide sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12 nucleotides in length, or shorter. The ability of the guide sequence to direct sequence-specific binding of the prime editor (PE) to a target sequence may be assessed by any suitable assay. For example, a prime editor (PE) component (including the guide sequence to be tested) may be provided to a host cell having a corresponding target sequence, such as by transfection with a vector encoding a prime editor (PE) component disclosed herein, followed by assessment of preferential cleavage within the target sequence, such as by a Surveyor assay as described herein. Similarly, cleavage of a target polynucleotide sequence may be assessed in a test tube by providing the target sequence, a prime editor (PE) component (including the guide sequence to be tested), and a control guide sequence that is different from the test guide sequence, and comparing the binding or cleavage rate at the target sequence of the reaction between the test guide sequence and the control guide sequence. Other assays may be performed and will occur to those of skill in the art.

The guide sequence may be selected to target any target sequence. In some embodiments, the target sequence is a sequence in the genome of a cell. Exemplary target sequences include sequences that are unique in the target genome. For example, for S.pyogenes Cas9, a unique target sequence in a genome may include a Cas9 target site of the form MMMMMMMMNNNNNNNNNNNXG G , where NNNNNNNNNNNNXG G ( N is A, G, T, or C; and X can be anything). A unique target sequence in a genome may include a S.pyogenes Cas9 target site of the form MMMMMMMMMNNNNNNNNNNNXG G , where NNNNNNNNNNXG G ( N is A, G, T, or C; and X can be anything). For S. thermophilus CRISPR1Cas9, a unique target sequence in the genome may encompass 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 the genome may encompass a S. thermophilus CRISPR1 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 S. pyogenes Cas9, a unique target sequence in a genome may encompass a Cas9 target site of the form MMMMMMMNNNNNNNNNNNXGGX G , where NNNNNNNNNNNNXGGX G ( N is A, G, T, or C; and X can be anything). A unique target sequence in a genome may encompass a S. pyogenes Cas9 target site of the form MMMMMMMMMNNNNNNNNNNNXGGX G , where NNNNNNNNNNNXGGX G ( 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, the guide sequence is selected to reduce the degree of secondary structure within the guide sequence. The secondary structure may be determined by any suitable polynucleotide folding algorithm. Some programs are based on calculating the minimum Gibbs free energy. One example of such an algorithm is mFold, as described by Zuker and Stiegler (Nucleic Acids Res. 9 (1981), 133-148). Another example folding algorithm is the online web server RNAfold, developed at the Institute for Theoretical Chemistry, University of Vienna, which uses a centroid structure prediction algorithm (see, for example, 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 No. 61/836,080; Broad Reference BI-2013/004A (herein incorporated 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 cells containing the corresponding tracr sequence; and (2) formation of a complex in a target sequence, the complex including a tracr mate sequence hybridized to a tracr sequence. In general, the degree of complementarity is by reference to optimal alignment of the tracr mate sequence and the tracr sequence along the shorter length of the two sequences. Optimal alignment may be determined by any suitable alignment algorithm, and may further comprise secondary structures, such as self-complementarity, within either the tracr sequence or the tracr mate sequence. In some embodiments, the degree of complementarity between the tracr sequence and the tracr mate sequence is about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99% or more, or higher, when optimally aligned along the shorter length of the two. In some embodiments, the tracr sequence is 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, or longer. In some embodiments, the tracr sequence and the tracr mate sequence are contained within a single transcript, such that hybridization between the two produces a transcript with a secondary structure, such as a hairpin. A preferred loop-forming sequence for use in a hairpin structure is 4 nucleotides in length, and most preferably has the sequence GAAA. However, longer or shorter loop sequences may also be used, as may alternative sequences. The sequence preferably includes a nucleotide triplet (e.g., AAA) and an additional nucleotide (e.g., C or G). Examples of loop-forming sequences include CAAA and AAAG. In an embodiment of the present invention, the transcript or transcribed polynucleotide sequence has at least two, or more, hairpins. In a preferred embodiment, the transcript has 2, 3, 4 or 5 hairpins. In further embodiments of the invention, the transcript has at most 5 hairpins. In some embodiments, the single transcript further includes a transcription termination sequence; preferably, this includes a poly-T sequence, such as 6 T nucleotides. Further non-limiting examples of single polynucleotides including a guide sequence, a tracr mate sequence, and a tracr sequence are as follows (listed 5'→3'), where "N" represents the bases of the guide sequence, the first block of lowercase letters represents the tracr block sequence, and the second block of lowercase letters represents the tracr sequence, and the final poly-T sequence represents the transcription terminator: (1) NNNNNNNNGTTTTTGTACTCTCAAGATTTAGAAATAAATCTTGCAGAAGCTACAAAGATAAGGCTTCATGCCGAAATCAACACCCTGTCATTTTATGGCAGGGTGTTTTCGTTATTTAATTTTTT (SEQ ID NO: 216);

(2) NNNNNNNNNNNNNNNNNNgtttttgtactctcaGAAAtgcagaagctacaaagataaggcttcatgccgaaatcaacaccctgtcattttatggcagggtgttttcgttatttaaTTTTTT (SEQ ID NO: 217);

(3)NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaGAAAtgcagaagctacaaagataaggcttcatgccgaaatcaacaccctgtcattttatggcagggtgtTTTTTT (SEQ ID NO: 218);

(4)NNNNNNNNNNNNNNNNNNNNgttttagagctaGAAAtagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcTTTTTT (sequence number 219);

(5) NNNNNNNNNNNNNNNNNNNNgttttagagctaGAAATAGcaagttaaaataaggctagtccgttatcaacttgaaaaagtgTTTTTT (SEQ ID NO: 220); and

(6) NNNNNNNNNNNNNNNNNNNNgttttagagctagAAATAGcaagttaaaataaggctagtccgttatcaTTTTTTTT (sequence number 221).

In some embodiments, sequences (1)-(3) are used in combination with Cas9 from S. thermophilus CRISPR1. In some embodiments, sequences (4)-(6) are used in combination with Cas9 from S. pyogenes. In some embodiments, the tracr sequence is a separate transcript from the transcript that contains the tracr mate sequence.

It will be apparent to one of skill in the art that any fusion protein as disclosed herein comprising a Cas9 domain and a single-stranded DNA binding protein typically requires co-expression of the fusion protein with a guide RNA (e.g., an sgRNA) to target the fusion protein to a target site (e.g., a site containing a point mutation to be edited). As described in more detail elsewhere herein, the guide RNA typically comprises a tracrRNA framework that allows for Cas9 binding and a guide sequence that confers sequence specificity to the Cas9:nucleic acid editing enzyme/domain fusion protein.

In some embodiments, the guide RNA comprises the structure 5'-[guide sequence]-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUU-3' (SEQ ID NO: 222), where the guide sequence comprises a sequence complementary to the target sequence. The guide sequence is typically 20 nucleotides in length. Suitable guide RNA sequences for targeting Cas9:nucleic acid editing enzyme/domain fusion proteins to specific genomic target sites will be apparent to one of skill in the art based on this disclosure. Such suitable guide RNA sequences typically comprise a guide sequence complementary to a nucleic acid sequence within 50 nucleotides upstream or downstream of the target nucleotide to be edited. Some exemplary guide RNA sequences suitable for targeting the provided fusion proteins to specific target sequences are provided herein. Additional guide sequences are known in the art and may be used with the prime editors (PEs) described herein.

In other embodiments, the PEgRNA includes those depicted in Figure 3D.

In yet other embodiments, the PEgRNA may include that depicted in Figure 3E.

Figure 3D provides the structure of an embodiment of a PEgRNA that may be designed according to the methodology contemplated herein and defined in Example 2. The PEgRNA comprises three major components ordered in the 5' to 3' direction: a spacer, a gRNA core, and an extension arm at the 3' end. The extension arm may be further divided in the 5' to 3' direction into the following structural elements: a primer binding site (A), an editing template (B), and a homology arm (C). In addition, the PEgRNA may comprise an optional 3' end modification region (e1) and an optional 5' end modification region (e2). Furthermore, the PEgRNA may also comprise a transcription termination signal at the 3' end of the PEgRNA (not depicted). These structural elements are further defined herein. The depiction of the PEgRNA structure is not intended to be limiting and encompasses variations in the arrangement of the elements. For example, the optional sequence modifiers (e1) and (e2) can be located within or between any of the other regions shown and are not limited to being located at the 3' and 5' ends.

Figure 3E provides another embodiment of a PEgRNA structure that may be designed according to the methodology contemplated herein and defined in Example 2. The PEgRNA comprises three major components ordered in the 5' to 3' direction: a spacer, a gRNA core, and an extension arm at the 3' end. The extension arm may be further divided into the following structural elements in the 5' to 3' direction: a primer binding site (A), an editing template (B), and a homology arm (C). In addition, the PEgRNA may comprise an optional 3' end modification region (e1) and an optional 5' end modification region (e2). Furthermore, the PEgRNA may also comprise a transcription termination signal at the 3' end of the PEgRNA (not depicted). These structural elements are further defined herein. The depiction of the PEgRNA structure is not intended to be limiting and encompasses variations in the arrangement of the elements. For example, the optional sequence modifiers (e1) and (e2) can be located within or between any of the other regions shown and are not limited to being located at the 3' and 5' ends.

PEgRNAs may also include additional design improvements that may modify the properties and/or characteristics of the PEgRNA, thereby improving the efficacy of prime editing. In various embodiments, these improvements may fall into one or more of a number of different categories, including, but not limited to: (1) designs that allow efficient expression of functional PEgRNAs from non-polymerase III (pol III) promoters, which would allow expression of longer PEgRNAs without cumbersome sequence requirements; (2) improvements to the core, Cas9-bound PEgRNA backbone, which may improve efficacy; (3) modifications to the PEgRNA that improve RT processivity, which would allow insertion of longer sequences at the targeted genomic locus; and (4) the addition of RNA motifs to the 5' or 3' end of the PEgRNA, which may improve PEgRNA stability, enhance RT processivity, prevent PEgRNA misfolding, or recruit additional factors important for genome editing.

In one embodiment, PEgRNAs can be designed with pol III promoters to improve expression of longer PEgRNAs 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 expressing short RNAs that are retained in the nucleus. However, pol III is not very processive and cannot express RNAs longer than a few hundred nucleotides in length at the levels required for efficient genome editing. In addition, pol III may stall or terminate at stretches of U's, potentially limiting the sequence diversity that can be inserted using PEgRNAs. Other promoters that recruit polymerase II (such as pCMV) or polymerase I (such as the U1 snRNA promoter) have also been investigated for their ability to express longer sgRNAs. However, these promoters are typically partially transcribed, which results in extra sequence 5' of the spacer in the expressed PEgRNA, which has been shown to result in significantly reduced Cas9:sgRNA activity in a site-dependent manner. In addition, pol III transcribed PEgRNAs can simply terminate at the 6-7 U stretch, whereas pol II or pol I transcribed PEgRNAs will require different termination signals. Often such signals will also result in polyadenylation, which will result in the undesired export of the PEgRNA from the nucleus. Similarly, RNAs expressed from pol II promoters (such as pCMV) are typically 5' capped, which will also result in their export from the nucleus.

Previously, Rinn and coworkers ^screened various expression platforms for the production of sgRNAs tagged with long non-coding RNAs (lncRNAs). These platforms include RNAs expressed from pCMV and terminating in ^an ENE element from human MALAT1 ncRNA, a PAN ENE element from KSHV, or a ³ ' box from ^U1 snRNA. ^Notably , MALAT1 ncRNA and PAN ENE form a triple helix that protects the polyA tail. These constructs may also enhance RNA stability. It is contemplated that these expression systems will also allow the expression of longer PEGRNAs.

In addition, a series of methods have been designed for cleavage of the portion of the pol II promoter that will be transcribed as part of the PEgRNA, and either a self-cleaving ribozyme (such as the ^{hammerhead188} , ^pistol189 , ^hatchet189 , ^hairpin190 , ^VS191 , ^twister192 , or twister ^sister192 ribozyme) or other self-cleaving elements that process the transcribed guide, or a ^hairpin193 that is recognized by Csy4 and also leads to processing of the guide. It is also hypothesized that the incorporation of multiple ENE motifs may lead to improved PEgRNA expression and stability, as previously demonstrated for KSHV PAN RNA ^{and elements185} . It is also predicted that circularizing the PEgRNA into the form of a circular intronic RNA (ciRNA) may also lead to enhanced RNA expression and stability, as well as nuclear localization194.

In various embodiments, the PEgRNA may include various elements above, as exemplified by the following sequences:

Non-limiting example 1 - PEgRNA expression platform consisting of pCMV, Csy4 hairpin, PEgRNA, and MALAT1 ENE
(SEQ ID NO:223)

Non-limiting example 2 - PEgRNA expression platform consisting of pCMV, Csy4 hairpin, PEgRNA, and PAN ENE
(SEQ ID NO:224)

Non-limiting example 3 - PEgRNA expression platform consisting of pCMV, Csy4 hairpin, PEgRNA, and 3xPAN ENE
(SEQ ID NO:225)

Non-limiting Example 4 - PEgRNA Expression Platform Consisting of pCMV, Csy4 Hairpin, PEgRNA, and 3' Box
(SEQ ID NO:226)

Non-limiting Example 5 - PEgRNA Expression Platform Consisting of pU1, Csy4 Hairpin, PEgRNA, and 3' Box
(SEQ ID NO:227).

In various other embodiments, the PEGRNA may be improved by introducing improvements into the backbone or core sequence. This may be done by introducing known improvements.

The core, Cas9-bound PEgRNA backbone could conceivably also be improved to enhance PE activity. Several such approaches have already been demonstrated. As an illustration, the first pairing element (P1) of the backbone contains a GTTTT-AAAAC (SEQ ID NO: 3939) pairing element. Such a stretch of T's has been shown to result in pol III pausing and premature termination of the RNA transcript. Rational mutation of one of the TA pairs in this portion of P1 to a GC pair has been shown to enhance sgRNA activity, suggesting that this approach may also be feasible for ^PEgRNA195 . In addition, increasing the length of P1 has also been shown to enhance sgRNA folding leading to improved ^activity195 , suggesting this as another avenue for improving PEgRNA activity. Examples of improvements to the core may include:

PEgRNA containing a 6 nt extension to P1
GGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGCTCATGAAAATGAGCTAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCCTCTGCCATCAAAGCGTGCTCAGTCTGTTTTTTT (SEQ ID NO: 228)

PEgRNA containing a TA→GC mutation in P1
GGCCCAGACTGAGCACGTGAGTTTGAGAGCTAGAAATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCCTCTGCCATCAAAGCGTGCTCAGTCTGTTTTTTT (SEQ ID NO: 229)

In various other embodiments, PEgRNA may be improved by introducing modifications into the editing template region. As the size of the PEgRNA templated insertion increases, it is more likely to be degraded by endonucleases, undergo spontaneous hydrolysis, or fold into secondary structures that cannot be reverse transcribed by RT or that interfere with the folding of the PEgRNA backbone and subsequent Cas9-RT binding. As a result, modifications to the PEgRNA template may also be required to affect large insertions, such as the insertion of entire genes. Some strategies to do so include the incorporation of modified nucleotides in synthetic or semi-synthetic PEgRNA, which make the ^RNA more resistant to degradation or hydrolysis, or less likely to adopt inhibitory secondary structures.196 Such modifications may include 8-aza-7-deazaguanosine, which will reduce RNA secondary structures in G-rich sequences; locked nucleic acids (LNA), which reduce degradation and enhance certain RNA secondary structures; 2'-O-methyl, 2'-fluoro, or 2'-O-methoxyethoxy modifications, which enhance RNA stability. Such modifications may also be included elsewhere in the PEgRNA to enhance stability and activity. Alternatively, or in addition, the template of the PEgRNA may be designed to encode the desired protein product while also being more likely to adopt a simple secondary structure that cannot be folded by RT. Such a simple structure would act as a thermodynamic sink, which would reduce the likelihood of more complex structures arising that would prevent reverse transcription. Finally, the template may also be split into two separate PEgRNAs. In such a design, PE would be used to initiate transcription and also recruit a separate template RNA to the targeted site, either through an RNA-binding protein fused to Cas9 or an RNA recognition element on the PEgRNA itself (such as the MS2 aptamer). 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 allow for long insertions, not only by preventing misfolding of the PEgRNA upon addition of a long template, but also by not requiring dissociation of Cas9 from the genome to generate long insertions (which may potentially inhibit PE-based long insertions).

In yet other embodiments, the PEgRNA may be improved by introducing additional RNA motifs at the 5' and 3' ends of the PEgRNA, or even at positions between them (e.g., in the gRNA core region or in the spacer). Several such motifs--such as the PAN ENE from KSHV and the ENE from MALAT1--were discussed above as possible means of terminating the expression of longer PEgRNAs from non-pol III promoters. These elements form RNA triple helices that engulf the polyA tails, resulting in their retention in the nucleus. ^184,187 However, by forming complex structures at the 3' end of the PEgRNA that occlude the terminal nucleotides, these structures may also help prevent exonuclease-mediated degradation of the PEgRNA.

Other structural elements inserted at the 3' end may also enhance RNA stability without allowing termination from non-pol III promoters. Such motifs could include hairpins or RNA quadruplexes that would occlude the 3' end, ¹⁹⁷ or self-cleaving ribozymes such as HDV that result in the formation of a 2'-3'-cyclic phosphate at the 3' end, potentially also making the PEgRNA less susceptible to ^exonuclease degradation.198 Inducing the PEgRNA to circularize through incomplete splicing - forming ciRNA - could also increase PEgRNA stability and result in the PEgRNA being retained in the ^nucleus.194

Additional RNA motifs may also improve RT processivity or enhance PEgRNA activity by enhancing RT binding to the DNA-RNA duplex. The addition of native sequences bound by RT in its cognate retroviral genome can enhance RT activity. This may include native primer binding sites (PBSs), polypurine tracts ( ^PPTs ), or kissing loops, which are involved in ^retroviral genome dimerization and transcription initiation.

The addition of dimerization chiefs - such as kissing loops or GNRA tetraloop/tetraloop acceptor pair ²⁰⁰ - at the 5' and 3' ends of the PEgRNA may also result in efficient circularization of the PEgRNA, improving stability. In addition, it is envisioned that the addition of these motifs may allow for physical separation of the PEgRNA spacer and primer, preventing spacer blockage that would otherwise interfere with PE activity. Short 5' or 3' extensions to the PEgRNA that form small toehold hairpins in the spacer region or along the primer binding site may also preferably compete for annealing of intramolecular complementary regions along the length of the PEgRNA (e.g., interactions that may occur between the spacer and primer binding sites). Finally, kissing loops may also be used to recruit other template RNAs to genomic sites and to allow for the switching of RT activity from one RNA to another. As exemplary embodiments of various secondary structures, the PEgRNA depicted in Figures 3D and 3E lists a number of secondary RNA structures (which, upon modification, can be any region of the PEgRNA), including those at the terminal portions of the extended arms (i.e., e1 and e2) as shown.

Examples of improvements include, but are not limited to:

PEGRNA-HDV fusions
GGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCCTCTGCCATCAAAGCGTGCTCAGTCTGGGCCGGCATGGTCCCAGCCTCCTCGCTGGCGCCGGCTGGGCAACATGCTTCGGCATGGCGAATGGGACTTTTTTT (SEQ ID NO: 230)

PEgRNA-MMLV kissing loop
GGTGGGAGACGTCCCACCGGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCCTCTGCCATCAAAGCTTCGACCGTGCTCAGTCTGGTGGGAGACGTCCCACCTTTTTTTT (SEQ ID NO: 231)

PEgRNA-VS ribozyme kissing loop
GAGCAGCATGGCGTCGCTGCTCACGGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCCTCTGCCATCAAAGCTTCGACCGTGCTCAGTCTCCATCAGTTGACACCCTGAGGTTTTTTT (SEQ ID NO: 232)

PEgRNA-GNRA tetraloop/tetraloop receptor
GCAGACCTAAGTGGUGACATATGGTCTGGGCCCAGACTGAGCACGTGAGTTTTAGAGCTAUACGTAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTUACGAAGTGGGACCGAGTCGGTCCTCTGCCATCAAAGCTTCGACCGTGCTCAGTCTGCATGCGATTAGAAATAATCGCATGTTTTTTT (SEQ ID NO: 233)

PEgRNA template switching the secondary RNA-HDV fusion
TCTGCCATCAAAGCTGCGACCGTGCTCAGTCTGGTGGGAGACGTCCCACCGGCCGGCATGGTCCCAGCCTCCTCGCTGGCGCCGGCTGGGCAACATGCTTCGGCATGGCGAATGGGACTTTTTTT (SEQ ID NO: 234)

The PEgRNA backbone can be further improved through directed evolution in a manner similar to how SpCas9 and prime editors (PEs) have been improved. Directed evolution can enhance PEgRNA recognition by Cas9 or evolved Cas9 variants. In addition, different PEgRNA backbone sequences will likely be optimal at different genomic loci, either enhancing PE activity at the site in question, reducing off-target activity, or both. Finally, evolution of PEgRNA backbones with additional RNA motifs will almost always improve the activity of the fused PEgRNA compared to the unevolved fusion RNA. As an illustration, evolution of an allosteric ribozyme composed of a c-di-GMP-I aptamer and a hammerhead ribozyme led to dramatically improved activity, ²⁰² suggesting that evolution will also improve the activity of hammerhead-PEgRNA fusions. In addition, although Cas9 currently does not generally tolerate 5' extensions of sgRNAs, directed evolution will likely generate potent mutations that alleviate this intolerance, allowing additional RNA motifs to be exploited.

The present disclosure contemplates any such manner to further improve the efficacy of the prime editing system disclosed herein.

Since a series of consecutive T's can limit the capacity of the PEgRNA to be transcribed, in various embodiments it may be advantageous to limit the appearance of consecutive sequences of T's from the extension arm. For example, strings of at least 3 consecutive T's, at least 4 consecutive T's, at least 5 consecutive T's, at least 6 consecutive T's, at least 7 consecutive T's, at least 8 consecutive T's, at least 9 consecutive T's, at least 10 consecutive T's, at least 11 consecutive T's, at least 12 consecutive T's, at least 13 consecutive T's, at least 14 consecutive T's, or at least 15 consecutive T's should be avoided when designing the PEgRNA, or at least removed from the final designed sequence. In one embodiment, in addition to avoiding target sites that are rich in consecutive A:T nucleobase pairs, the inclusion of unwanted strings of consecutive T's on the PEgRNA extension arm may be avoided.

Split-PEgRNA Design for Trans-Prime Editing The present disclosure also contemplates trans-prime editing, which refers to a modified version of prime editing that operates by splitting PEgRNA into two separate molecules: guide RNA and tPERT molecules. The tPERT molecule is programmed to co-localize with the prime editor complex at the target DNA site, bringing the primer binding site and DNA synthesis template to the prime editor in trans. For example, see FIG. 3G for an embodiment of a trans-prime editor (tPE). This shows a two-component system that includes (1) a recruitment protein (RP)-PE:gRNA complex and (2) tPERT, which contains a primer binding site and a DNA synthesis template linked to an RNA-protein recruitment domain (e.g., stem-loop or hairpin). The recruitment protein component of the RP-PE:gRNA complex recruits tPERT to the target site to be edited, thereby associating the PBS and DNA synthesis template with the prime editor in trans. In other words, tPERT is engineered to contain (all or part of) the extension arm of PEgRNA, which contains the primer binding site and the DNA synthesis template. One advantage of this approach is that it separates the extension arm of PEgRNA from the guide RNA, thereby minimizing the annealing interaction that tends 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 tPERT to the site of DNA editing, thereby effectively co-localizing all of the functions of the PEgRNA at the site of prime editing. Recruitment can be achieved by incorporating an RNA-protein recruitment domain, such as the MS2 aptamer, into tPERT and fusing the corresponding recruitment protein to a prime editor that can specifically bind to the RNA-protein recruitment domain (e.g., to napDNAbp via a linker, or to the polymerase via a linker linker), thereby recruiting the tPERT molecule to the prime editor complex. As illustrated in the process described in Figure 3H, the RP-PE:gRNA complex binds and nicks the target DNA sequence. The recruitment protein (RP) then recruits tPERT to co-localize with the prime editor complex bound to the DNA target site, thereby allowing the primer binding site located on tPERT to bind to the primer sequence on the nicked strand, and subsequently allowing a polymerase (e.g., RT) to synthesize a single strand of DNA from the 5' end of tPERT to the DNA synthesis template located on tPERT.

Although tPERT is shown in Figures 3G and 3H as containing a PBS and a DNA synthesis template at the 5' end of the RNA-protein recruitment domain, other configurations of tPERT can be designed with the PBS and DNA synthesis template located at the 3' end of the RNA-protein recruitment domain. However, tPERT with a 5' extension has the advantage that synthesis of a single strand of DNA will naturally terminate at the 5' end of tPERT, and therefore there is no risk of using any part of the RNA-protein recruitment domain as a template during the DNA synthesis stage of prime editing.

Methods for Designing PEgRNAs The present disclosure also relates to methods for designing PEgRNAs.

In one aspect of the design, the design approach may take into account the specific application for which prime editing is to be used. For example, as exemplified and discussed herein, prime editing may be used, without limitation, to (a) incorporate mutation-correcting changes into a nucleotide sequence, (b) incorporate protein and RNA tags, (c) incorporate immune epitopes into a protein of interest, (d) incorporate inducible dimerization domains into a protein, (e) incorporate or remove sequences in a biomolecule to alter its activity, (f) incorporate recombinase target sites to direct specific genetic changes, and (g) mutate a target sequence by using error-prone RT. In addition to these methods of generally inserting, altering, or deleting nucleotide sequences at a target site of interest, prime editors may also be used to construct highly programmable libraries, as well as to perform cellular data recording and lineage tracing studies. In these various uses, there may be specific design aspects that apply to the preparation of PEgRNAs that are particularly useful for any given of these applications, as described herein.

A number of considerations can be taken into account when designing a PEgRNA for any particular application or use of prime editing. These are:
(a) a target sequence, i.e., a nucleotide sequence, into which one or more nucleobase modifications are desired to be incorporated by a primed editor;
(b) Positioning of the cut site on the target sequence, i.e., the specific nucleobase position where the primed editor will induce a single-stranded nick to generate a 3'-end RT primer sequence on one side of the nick and a 5'-end endogenous flap on the other side of the nick (which is ultimately removed by FEN1 or its equivalent and replaced by a 3'-ssDNA flap). The cut site is analogous to "positioning of the edit" because it generates a 3'-end RT primer sequence that becomes extended by RT during RNA-dependent DNA polymerization to generate a 3'-ssDNA flap containing the desired edit, which then replaces the 5'-endogenous DNA flap on the target sequence;
(c) available PAM sequences (encompassing canonical SpCas9 PAM sites, as well as non-canonical PAM sites recognized by Cas9 variants and equivalents with extended or different PAM specificities);
(d) the spacing between available PAM sequences on the target sequence and the positioning of the site to be cut;
(e) the specific Cas9, Cas9 variant, or Cas9 equivalent of the prime editing element to be used;
(f) the sequence and length of the primer binding site;
(g) the sequence and length of the editing template;
(h) sequence and length of the homology arms;
(i) spacer sequence and length; and
(j) a core sequence;
These include, but are not limited to:

This disclosure addresses these aspects above.

In one embodiment, an approach is provided herein for designing a suitable PEgRNA and optionally a nicking sgRNA design guide for second site nicking. This embodiment provides a step-by-step set of instructions for designing a PEgRNA and a nicking sgRNA for prime editing, which takes into account one or more of the above considerations. The steps refer to the examples shown in Figures 70A-70I.
1. Define the target sequence and edit. Retrieve the sequence of the target DNA region (~200bp) centered around the location of the desired edit (point mutation, insertion, deletion, or combination thereof). See Figure 70A.
2. Position the target PAM. Identify a PAM proximal to the desired editing position. A PAM can be identified on either strand of DNA proximal to the desired editing position. Although a PAM proximal to the editing position is preferred (i.e., the nick site is less than 30 nt from the editing position, 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 nt from the editing position to the nick site), it is possible to incorporate an edit using a protospacer and PAM that places the nick ≧30 nt from the editing position. See Figure 70B.
3. Position the nick site. For each PAM under consideration, identify the corresponding nick site and on which strand. For Sp Cas9 H840A nickase, cleavage occurs on the PAM-containing strand between the third and fourth bases 5' of the NGG PAM. All edited nucleotides must be 3' to the nick site. Therefore, the appropriate PAM must nick 5' of the targeted edit on the PAM-containing strand. In the example shown below, there are two possible PAMs. For simplicity, the remaining steps demonstrate the design of a PEgRNA using only PAM 1. See Figure 70C.
4. Design the spacer sequence. The protospacer of SpCas9 corresponds to the 20 nucleotides 5' of the NGG PAM on the PAM-containing strand. Efficient Pol III transcription initiation requires that G be the first transcribed nucleotide. If the first nucleotide of the protospacer is a G, then the spacer sequence of the PEgRNA is simply the protospacer sequence. If the first nucleotide of the protospacer is not a G, then the spacer sequence of the PEgRNA is a G followed by the protospacer sequence. See Figure 70D.
5. Design the primer binding site (PBS). Using the starting allele sequence, identify a 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 fourth base 5' of 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 containing .about.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. Regardless of GC content, the optimal PBS sequence should be determined empirically. To design a PBS sequence of length p, use the starting allele sequence to take the reverse complement of the first p nucleotides 5' of the nick site on the PAM-containing strand. See Figure 70E.
6. Design the RT template (or DNA synthesis template). The RT template (or DNA synthesis template, where the polymerase is not a reverse transcriptase) codes for the designed edit and homology to the sequence flanking the edit. In one embodiment, these regions correspond to the DNA synthesis template of FIG. 3D and FIG. 3E, where the DNA synthesis template includes the "edit template" and the "homology arms". The optimal RT template length varies based on the target site. For short-range edits (positions +1 to +6), it is recommended to test short (9 to 12 nt), medium (13 to 16 nt), and long (17 to 20 nt) RT templates. For long-range edits (positions +7 and beyond), it is recommended to use an RT template that extends at least 5 nt (preferably 10 nt or more) from the edit position to allow sufficient 3' DNA flap homology. For long-range edits, several RT templates should be screened to identify a functional design. For larger insertions and deletions (≧5 nt), incorporation of greater 3′ homology (≧20 nt) onto the RT template is recommended. Editing efficiency is typically compromised when the RT template codes for synthesis of a G (corresponding to a C on the RT template for PEG RNA) as the last nucleotide on the reverse transcribed DNA product. Because many RT templates support efficient prime editing, avoidance of G as the last synthesized nucleotide is recommended when designing an RT template. To design an RT template sequence of length r, use the desired allele sequence and take the reverse complement of the first r nucleotides 3′ of the nick site on the strand that originally contained the PAM. Note that compared to SNP editing, insertion or deletion edits using an RT template of the same length will not contain the same homology. See Figure 70F.
7. Assemble the complete PEGRNA sequence. Concatenate the PEGRNA building blocks in the following order (5' to 3'): spacer, backbone, RT template, and PBS. See Figure 70G.
8. Design a nicking sgRNA for PE3. Identify PAMs on the non-edited strand upstream and downstream of the edit. The optimal nicking position is highly locus dependent and should be empirically determined. Generally, a nick placed 40 to 90 nucleotides 5' opposite the PEgRNA-induced nick leads to higher editing yields and fewer indels. The nicking sgRNA has a spacer sequence that matches the 20 nt protospacer on the starting allele. If the protospacer does not begin with a G, have an additional 5' G. See Figure 70H.
9. Design a PE3b nicking sgRNA. If a PAM is present on the complementary strand and its corresponding protospacer overlaps with the sequence targeted for editing, this edit may 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, not the starting allele. The PE3b system works efficiently when the nucleotide(s) to be edited fall within the seed region (~10 nt adjacent to the PAM) of the nicking sgRNA protospacer. This prevents nicking of the complementary strand until after incorporation of the edited strand, preventing competition between the PEgRNA and the sgRNA for binding to the target DNA. PE3b also avoids the generation of nicks on both strands simultaneously, thus significantly reducing indel formation while maintaining high editing efficiency. The PE3b sgRNA should have a spacer sequence that matches the 20 nt protospacer of the desired allele, with the addition of a 5'G if required. See Figure 70I.

The above step-by-step process for designing suitable PEgRNAs and second-site nicking sgRNAs is not meant to be limiting in any way. The present disclosure contemplates variations of the step-by-step process described above that could be derived therefrom by one of skill in the art.

[7] Applications Utilizing Prime Editing In addition to developing the prime editing system described herein as a new "search and replace" genome editing technology that mediates targeted insertions, deletions, and conversions of all 12 possible bases at target loci in human cells without requiring double-stranded DNA breaks or donor DNA templates, the inventors have also contemplated the use of prime editing elements for a wide array of specific applications. For example, as exemplified and discussed in this application, prime editing can be used to (a) incorporate mutation-correcting changes into nucleotide sequences, (b) incorporate protein and RNA tags, (c) incorporate immune epitopes into proteins of interest, (d) incorporate inducible dimerization domains into proteins, (e) incorporate or remove sequences in biomolecules to alter their activity, (f) incorporate recombinase target sites to induce specific genetic changes, and (g) mutate target sequences by using error-prone RT. In addition to these methods of generally inserting, changing or deleting nucleotide sequences at a target site of interest, prime editors can also be used to construct highly programmable libraries and to perform cell data recording and lineage tracing studies. The inventors have also contemplated additional design features of PEgRNA that aim to improve the efficacy of prime editing. Furthermore, the inventors have conceived a method for successfully delivering prime editors using a vector delivery system that involves splitting napDNAbp using an intein domain.

These specific exemplary uses of prime editing are not intended to be limiting in any way. The present application contemplates any use of prime editing that generally involves some form of incorporation, removal, and/or modification of one or more nucleic acid bases at a target site on a nucleotide sequence, e.g., genomic DNA.

For any of the exemplified uses of prime editing, any prime editing factor disclosed herein may be used, 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 (in which a nucleotide sequence change is desired to be introduced) with a nucleic acid programmable DNA binding protein (napDNAbp) complexed with an extended guide RNA. With reference to FIG. 1G, the extended guide RNA includes an extension at the 3' or 5' end of the guide RNA or at the intramolecular positioning of the guide RNA, encoding the desired nucleotide change (e.g., a single nucleotide change, an insertion, or a deletion). In step (a), the napDNAbp/extended gRNA complex contacts the DNA molecule, and the extended gRNA guides the napDNAbp to bind to the target locus. In step (b), a nick is introduced (e.g., by a nuclease or chemical agent) into one of the strands of DNA at the target locus, thereby generating an available 3' end on one of the strands at the target locus. In some embodiments, the nick is generated on the strand of DNA corresponding to the R-loop strand, i.e., the strand that is not hybridized to the guide RNA sequence, i.e., the "non-target strand." However, the nick can be introduced on either strand. That is, the nick can be introduced on the R-loop "target strand" (i.e., the strand that hybridizes to the protospacer sequence of the extended gRNA) or on the "non-target strand" (i.e., the strand that forms the single-stranded portion of the R-loop and 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 to prime reverse transcription (i.e., "RT primed to prime target"). In some embodiments, the 3' end DNA strand hybridizes to a specific RT prime sequence of the extended portion of the guide RNA, i.e., the "reverse transcriptase prime sequence". In step (d), a reverse transcriptase is introduced (as a fusion protein with napDNAbp or in trans), which synthesizes a single strand of DNA from the 3' end of the primed site toward the 5' end of the extended guide RNA. This forms a single-stranded DNA flap containing the desired nucleotide change (e.g., a single base change, an insertion, or a deletion, or a combination thereof) that is otherwise homologous to the endogenous DNA at or adjacent to the nick site. In step (e), the napDNAbp and the guide RNA are released. Steps (f) and (g) involve degradation of the single-stranded DNA flap, so that the desired nucleotide change becomes incorporated at the target locus. This process can be driven toward the formation of the desired product by removing the corresponding 5' endogenous DNA flap that forms once the 3' single-stranded DNA flap invades and hybridizes to the endogenous DNA sequence (e.g., by FEN1 or a similar enzyme provided in trans as a fusion with a primed editing factor, or endogenously provided). Without being bound by theory, the cell's endogenous DNA repair and replication processes degrade the mismatched DNA and incorporate the nucleotide change(s) to form the desired altered product. The process can also be driven towards product formation by "second strand nicking" as illustrated in FIG. 1G or "termporal second strand nicking" as illustrated in FIG. 1I and discussed herein.

The process of prime editing can introduce at least one or more of the following genetic changes: transversions, transitions, deletions, and insertions. In addition, prime editing can be implemented for specific applications. For example, as exemplified and discussed in this application, prime editing can be used to (a) incorporate mutation-correcting changes into nucleotide sequences, (b) incorporate protein and RNA tags, (c) incorporate immune epitopes into proteins of interest, (d) incorporate inducible dimerization domains into proteins, (e) incorporate or remove sequences in biomolecules to alter their activity, (f) incorporate recombinase target sites to direct specific genetic changes, and (g) mutate target sequences by using error-prone RT. In addition to these methods of generally inserting, altering, or deleting nucleotide sequences at target sites of interest, prime editors can also be used to construct highly programmable libraries and to perform cellular data recording and lineage tracing studies. The inventors have also contemplated additional design features of PEgRNA aimed at improving the efficacy of prime editing. Furthermore, the inventors have conceived a method for successfully delivering prime editing factors using a vector delivery system that involves disrupting napDNAbp with an intein domain.

The term "prime editing system" or "prime editing element (PE)" refers to a composition involved in the method of genome editing using primed target-primed reverse transcription (TPRT) described herein, including, but not limited to, napDNAbp, reverse transcriptase, a fusion protein (e.g., comprising a DNAbp and a reverse transcriptase), an extended guide RNA, and a complex comprising a fusion protein and an extended guide RNA, as well as auxiliary elements, such as a second strand nicking component and a 5' endogenous DNA flap removal endonuclease (e.g., FEN1) to help drive the prime editing process toward the formation of an edited product.

In another embodiment, the schematic in FIG. 3F illustrates a typical PEgRNA interaction with a target site in double-stranded DNA and the concomitant production of a 3′ single-stranded DNA flap containing the desired genetic alteration. The double-stranded DNA is shown with the top strand in a 3′ to 5′ orientation and the bottom strand in a 5′ to 3′ orientation. The top strand contains the “protospacer” and PAM sequence and is referred to as the “target strand.” The complementary bottom strand is referred to as the “non-target strand.” Although not shown, the illustrated PEgRNA would complex with Cas9 or an equivalent. As shown in the schematic, the spacer of the PEgRNA anneals to a complementary region on the target strand, referred to as the protospacer, which is located just downstream of the PAM sequence and is approximately 20 nucleotides in length. This interaction forms a DNA/RNA hybrid between the spacer RNA and the protospacer DNA, inducing the formation of an R-loop in the region opposite the protospacer. As taught elsewhere in this application, the Cas9 protein (not shown) then induces a nick on the non-target strand as shown. This then leads to the formation of a 3' ssDNA flap region, which interacts with the 3' end of the PEgRNA at the primer binding site according to *z*. 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 the reverse transcriptase. Next, the reverse transcriptase (e.g., provided in trans or provided in cis as a fusion protein attached to the Cas9 construct) then polymerizes the single strand of DNA encoded by the editing template (B) and the homology arm (C). Polymerization continues toward the 5' end of the extension arm. The polymerized strand of ssDNA forms the ssDNA 3' end flap. It invades the endogenous DNA, displaces the corresponding endogenous strand (which is removed as a 5' DNA flap of the endogenous DNA), and incorporates the desired nucleotide edit (single nucleotide base pair change, deletion, insertion (including entire genes) by naturally occurring rounds of DNA repair/replication), as described elsewhere (e.g., as shown in Figure 1G).

This application of prime editing can be further described in Example 1.

B. Mutagenesis using error-prone RT-primed editing
In various embodiments, a prime editing system (i.e., a prime editing system) may involve the use of an error-prone reverse transcriptase to perform targeted mutagenesis, i.e., to mutate only well-defined stretches of DNA on a genome or other DNA elements of a cell. Figure 22 provides a schematic diagram of an exemplary process for performing targeted mutagenesis by 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 includes an extension at the 3' or 5' end of the guide RNA or at the intramolecular positioning of 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 mutated. In step (b), a nick is introduced (e.g., by a nuclease or chemical agent) into one of the strands of the DNA at the target locus, thereby generating an available 3' end on one of the strands at the target locus. In some embodiments, a nick is generated on the strand of DNA corresponding to the R-loop strand, i.e., the strand that is not hybridized to the guide RNA sequence. In step (c), the 3' end of the DNA strand interacts with the extended portion of the guide RNA to prime reverse transcription. In some embodiments, the 3' end DNA strand hybridizes to a specific RT prime sequence of the extended portion of the guide RNA. In step (d), an error-prone reverse transcriptase is introduced, which synthesizes a mutated single strand of DNA from the 3' end of the primed site toward the 3' end of the guide RNA. Exemplary mutations are indicated by an asterisk "*". This forms a single stranded DNA flap containing the desired mutated region. In step (e), the napDNAbp and the guide RNA are released. Steps (f) and (g) involve degradation of the single stranded DNA flap (containing the mutated region), resulting in the integration of the desired mutated region 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-stranded DNA flap invades and hybridizes to the complementary sequence on the other strand. The process can also be driven towards product formation by second strand nicking as illustrated in Figure 1F. After endogenous DNA repair and/or replication processes, the mutated region becomes incorporated onto both strands of DNA at 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. The term "error-prone" reverse transcriptase as used herein refers to a reverse transcriptase enzyme that is naturally occurring or derived from another reverse transcriptase (e.g., wild-type M-MLV reverse transcriptase) that has an error rate that is less than that of wild-type M-MLV reverse transcriptase. The error rate of wild-type M-MLV reverse transcriptase has been reported to range from one error per 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. Therefore, for purposes of this application, the term "error-prone" refers to a nucleic acid sequence that is more likely to have at least one error per 15,000 nucleobase incorporation (6.7×10 ⁻⁵ or higher), e.g., one error per 14,000 nucleobases (7.14×10 ⁻⁵ or higher), one error per 13,000 nucleobases or fewer (7.7×10 ⁻⁵ or higher), one error per 12,000 nucleobases or fewer (7.7×10 ⁻⁵ or higher), one error per 11,000 nucleobases or fewer (9.1×10 ⁻⁵ or higher), one error per 10,000 nucleobases or fewer (1×10 ^-4 or 0.0001 or higher), 1 error per 9,000 nucleobases or less (0.00011 or higher), 1 error per 8,000 nucleobases or less (0.00013 or higher), 1 error per 7,000 nucleobases or less (0.00014 or higher), 1 error per 6,000 nucleobases or less (0.00016 or higher), 1 error per 5,000 nucleobases or less (0.0002 or higher), 1 error per 4,000 nucleobases or less (0.00016 or higher), 1 error per 5,000 nucleobases or less (0.0002 or higher), 1 error per 4,000 nucleobases or less (0.00016 or higher), 1 error per 6,000 nucleobases or less (0.00016 or higher), 1 error per 5,000 nucleobases or less (0.0002 or higher), 1 error per 4,000 nucleobases or less (0.00016 or higher), 1 error per 8,000 nucleobases or less (0.00016 or higher), 1 error per 9,000 nucleobases or less (0.00016 or higher), 1 error per 10 ... It refers to a RT having an error rate that is greater than 1 error (0.00025 or higher), 1 error per 3,000 nucleobases or fewer (0.00033 or higher), 1 error per 2,000 nucleobases or fewer (0.00050 or higher), or 1 error per 1,000 nucleobases or fewer (0.001 or higher), or 1 error per 500 nucleobases or fewer (0.002 or higher), or 1 error per 250 nucleobases or fewer (0.004 or higher).

For the generation of mutated sequences using prime editing, various mutagenic RTs can be envisaged. 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 herein by reference). In the case of the RT from Bordetella phage (brt), it may also be necessary to deliver an auxiliary protein (bavd) to Cas9 and an additional RNA sequence to the PEgRNA, either additionally or in trans, 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 RT, the template region of the PEgRNA can be enriched for adenosine or AAY codons to enhance diversity.

The amino acid sequence of the mutagenized RT from Bordetella phage is provided below: As with the other RTs disclosed herein, the Brt protein can be fused to the napDNAbp as a fusion protein to form a functional PE.

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

In the case of Brt from Bodetella, the PEgRNA can include additional nucleotide sequences added to the PEgRNA, for example, at the 5' or 3' end. An exemplary sequence is as follows, which is originally from the Bordetella phage genome:

To reduce its length, this PEgRNA addition sequence can be shortened in various ways. For example, the PEgRNA addition 1 sequence can be shortened to the following exemplary alternative addition sequences:

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

In various embodiments involving the use of PE to introduce mutations, special PEgRNA considerations may apply. For example, without being bound by theory, the additional PEgRNA sequences described above may be required to enable efficient mutagenesis by mutagenic RT.

Any mutagenic RT may be used in the prime editors disclosed herein. For example, the error-prone RTs described in the following references may be used, which 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 instrinsic fidelity of retroviral reverse transcriptases," 2009, Viruses, 1(3):1137-1165.

Various error-prone RTs may include, but are not limited to, the following enzymes disclosed in Table 1 of Menendez-Arias et al., as follows (the entire contents of which are incorporated by reference):

C. Use of Prime Editing to Treat Triplet Expansion Disorders
The prime editing system or prime editing (PE) system described herein can be used to reduce trinucleotide repeat mutations (or "triplet expansion disorders") to treat diseases 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 cognitive and sensorimotor functions. The disorders show genetic anticipation (i.e., increasing severity with each generation). DNA expansion or shortening usually occurs meiotically (i.e., during gamete formation or earlier during embryonic development) and often has a sex bias, meaning that some genes are expanded only when inherited from females and others only from males. 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 to be produced with large repeated amino acid sequences. This either disables or alters protein function, often in a dominant-negative manner (eg polyglutamine diseases).

Without being bound by theory, triplet expansion is caused by slippage during DNA replication or DNA repair synthesis. Because the tandem repeats have identical sequences to each other, base pairing between the two DNA strands can occur at multiple points along the sequence. This can lead to the formation of "loop-out" structures during DNA replication or DNA repair synthesis. This can lead to repeated copies of the repeat sequence, extending the number of repeats. Additional mechanisms involving hybrid RNA:DNA intermediates have been proposed. Prime editing can be used to reduce or eliminate these triplet expansion regions by deleting one or more of the offending repeat codon triplets. In one embodiment of this use, FIG. 23 provides a schematic of PEgRNA design for shortening or reducing trinucleotide repeat sequences by prime editing.

Prime editing can be implemented to shorten the triplet expansion region by nicking a region upstream of the triplet repeat region with a prime editor containing a dedicated PEgRNA targeted to the site to be cut. The prime editor then synthesizes a new DNA strand (ssDNA flap) based on the PEgRNA as a template (i.e., its editing template) that encodes a healthy number of triplet repeats (this depends on the specific gene and disease). The newly synthesized ssDNA strand containing the healthy triplet repeat sequence is also synthesized to include a short stretch of homology (i.e., a 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 by the newly synthesized ssDNA flap leads to a shortened repeat allele.

Depending on the specific trinucleotide expansion disorder, the triplet expansion inducing defect may occur in a "trinucleotide repeat expansion protein." Trinucleotide repeat expansion proteins are a diverse set of proteins that are associated with susceptibility to 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). Thus, these disorders are referred to as polyglutamine (polyQ) disorders and include the following diseases: Huntington's disease (HD); spinal-bulbar muscular atrophy (SBMA); spinocerebellar ataxia (SCA types 1, 2, 3, 6, 7, and 17); and dentatorubral-pallidoluysian atrophy (DRPLA). The remaining trinucleotide repeat expansion disorders either do not involve a CAG triplet or the CAG triplet is not in the coding region of the gene and are therefore referred to as non-polyglutamine disorders. Non-polyglutamine disorders include Fragile X syndrome (FRAXA); Fragile XE mental retardation (FRAXE); Friedreich's ataxia (FRDA); Myotonic dystrophy (DM); and Spinocerebellar ataxia (SCA types 8 and 12).

Proteins associated with trinucleotide repeat expansion disorders may be selected based on experimental association of the protein associated with trinucleotide repeat expansion disorders to trinucleotide repeat expansion disorders. 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 with a trinucleotide repeat expansion disorder relative to a population lacking a 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, proteins associated with trinucleotide repeat expansion disorders may be identified by obtaining gene expression profiles of 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 that can be corrected by prime editing include AR (androgen receptor), FMR1 (Fragile X Mental Retardation 1), HTT (huntingtin), DMPK (myotonic dystrophy protein kinase), FXN (frataxin), ATXN2 (ataxin 2), ATN1 (atrophin 1), FEN1 (flap structure-specific endonuclease 1), TNRC6A (trinucleotide repeat-containing 6A), PABPN1 (nuclear polyA binding protein 1), JPH3 (junctophilin 3), MED 15 (Mediator complex subunit 15), ATXN1 (Ataxin 1), ATXN3 (Ataxin 3), TBP (TATA box binding protein), CACNA1A (Voltage-gated calcium channel P/Q type alpha 1A subunit), ATXN80S (ATXN8 opposing 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, folate 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 (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-dependent) gamma), AFF2 (AF4/FMR2 family member 2), THBS1 (thrombospondin 1), TP53 (tumor protein p53), ESR1 (estrogen receptor 1), CGGBP1 (CGG triplet repeat binding protein protein 1), ABT1 (basal transcription activator 1), KLK3 (kallikrein-related peptidase 3), PRNP (prion protein), JUN (jun oncogene), KCNN3 (potassium medium/small conductance calcium-activated channel subfamily N member 3), BAX (BCL2-associated X protein), FRAXA (fragile site, folate-type, rare, fra(X)(q27.3)A (macrothrimi, 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/CTG1), TSC1 (tuberous sclerosis complex 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's disease (pseudoglioma)), ARX (aristaless-associated 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 (intestinal fatty acid binding protein 2), 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 enhanced 2 (S. cerevisiae)), GLA (galactosidase alpha), CBL (Cas-Br-M (murine) ecotropic retroviral transforming sequence), FTH1 (ferritin heavy chain polypeptide 1), IL12RB2 (interleukin-12 receptor beta 2), OTX2 (orthodenticle homeobox A1), IL12RB3 (interleukin-12 receptor beta 2), IL12RB4 (interleukin-12 receptor beta 2), IL12RB5 (interleukin-12 receptor beta 2), IL12RB6 (interleukin-12 receptor beta 2), IL12RB7 (interleukin-12 receptor beta 2), IL12RB8 (interleukin-12 receptor beta 2), IL12RB9 (interleukin-12 receptor beta 2), IL12RB1 (interleukin-12 receptor beta 2), IL12RB2 ...3 (interleukin-12 receptor beta 2), IL12RB4 (interleukin-12 receptor beta 2), IL12RB5 (interleukin-12 receptor beta 2), IL12RB6 (interleukin-12 receptor beta 2), IL12RB7 (interleukin-12 receptor beta 2), IL12RB8 (interleukin-12 receptor beta 2), IL12RB9 (interleukin- box 2), HOXA5 (homeobox A5), POLG2 (polymerase (DNA-dependent) gamma 2 auxiliary 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 editing elements disclosed herein can be used to reduce triplet repeat expansion regions in any of the disease proteins indicated above, including the following polyglutamine triplet expansion disease genes (which indicate the specific locations of the pathogenic repeats that can be removed in whole or in part by prime editing):

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

Prime editing can be implemented to reduce the triplet expansion region using a PEgRNA having an editing template designed to delete at least one codon in the triplet expansion region. In other embodiments, to reach a healthy (i.e., not associated with causing disease) number of triplet repeats, the PEG RNA for use in priming editing therefor has 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 is used to delete 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 the triplet expansion region.

In other embodiments, to reach a healthy (i.e., not associated with causing disease) number of triplet repeats, the PEG RNA for use in priming editing therefor is 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 the triplet expansion region.

In other embodiments, to reach a healthy (i.e., not associated with causing disease) number of triplet repeats, a PEG RNA for use in prime editing therefor is 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 the triplet expansion region.

In other embodiments, to reach a healthy (i.e., not associated with causing disease) number of triplet repeats, a PEgRNA for use in prime editing is 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 the triplet expansion region.

Prime editing can 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, US20011/00165540A1 (genome editing of genes associated with trinucleotide repeat expansion disorders in animals), and US2016/0355796A1 (compositions of the crispr-cas system and methods of use for nucleotide repeat disorders).

In various aspects, the present 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 editing factor fusion comprising napDNAbp and a reverse transcriptase, and (b) a PEgRNA comprising a spacer sequence that targets the trinucleotide repeat expansion region and an extension arm that comprises an editing template that codes for 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 of a defective gene in a cell using prime editing, comprising contacting the cell with a prime editor fusion comprising napDNAbp and a reverse transcriptase, and a PEgRNA comprising a spacer sequence that targets the trinucleotide repeat expansion region and an extension arm that comprises an editing template that encodes for 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 repeats are tetranucleotide, pentanucleotide, or hexanucleotide.

D. Use of Prime Editing for Peptide Tagging
In another aspect, the present disclosure provides a method for using a prime editor described herein to genetically graft one or more peptide tags onto a protein using prime editing. More specifically, the present disclosure provides a method for genetically incorporating one or more peptide tags onto a protein, comprising: contacting a target nucleotide sequence encoding a protein with a prime editor configured to insert a second nucleotide sequence encoding one or more peptide tags therein, resulting in a recombinant nucleotide sequence encoding a fusion protein comprising the protein fused to the protein tag.

In another aspect, 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 a protein with a primed editing element configured to insert therein a second nucleotide sequence encoding one or more peptide tags, resulting in a recombinant nucleotide sequence encoding a fusion protein comprising the protein fused to the protein tag.

In various embodiments, the target nucleotide sequence is a specific gene of interest on 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 signaling 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).

A peptide tag can be any peptide tag or variant thereof that confers one or more functions to a protein, such as for purposes of separation, purification, visualization, solubilization, or detection. Peptide tags can include "affinity tags" (to facilitate protein purification), "solubilization tags" (to aid in proper folding of the protein), "chromatography tags" (to alter the chromatographic properties of the protein), "epitope tags" (to bind high affinity antibodies), and "fluorescent tags" (to facilitate visualization of the protein in cells or in vitro). Examples of peptide tags include, but are not limited to, the following tags:

The peptide tag can also be an affinity tag (for protein isolation and/or purification) (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).

In specific embodiments, peptide tags may include His ⁶ tag, FLAG tag, V5 tag, GCN4 tag, HA tag, Myc tag, FIAsH/ReAsH tag, sortase substrate, pike clamp.

In various embodiments, peptide tags can be used in applications including protein fluorescent labeling, immunoprecipitation, immunoblotting, immunohistochemistry, protein mobilization, inducible protein degrons, and genome-wide screening.

In various other embodiments, the peptide tag may include an intein sequence to incorporate protein self-splicing functionality. The term "intein" as used herein refers to a self-processing polypeptide domain found in organisms from all domains of life. An intein (intercalating protein) performs a unique self-processing event known as protein splicing, in which it excises itself from a larger precursor polypeptide by cleavage of two peptide bonds, ligating flanking extein (external protein) sequences in the process by forming new peptide bonds. Since intein genes are found embedded in-frame within other protein-coding genes, this rearrangement occurs post-translationally (or possibly co-translationally). Furthermore, intein-mediated protein splicing is spontaneous; it requires only the folding of the intein domain, not external factors or energy sources. This process is also known as cis protein splicing, in contrast to the natural process of trans protein splicing by "split inteins". Inteins are the protein equivalents of self-splicing RNA introns (see Perler et al., Nucleic Acids Res. 22:1125-1127 (1994)), which catalyze their own excision from precursor proteins and have the concomitant fusion of 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 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 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 a variety of 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. Recently, bioinformatic analysis of environmental metagenomic data has revealed 26 diverse loci with novel genomic arrangements. At each locus, conserved enzyme-coding regions are interrupted by split inteins, and freestanding endonuclease genes are inserted between sections encoding intein subdomains. Of these, five loci have been fully assembled: DNA helicase (gp41-l, gp41-8); inosine-5'-monophosphate dehydrogenase (IMPDH-1); and ribonucleotide reductase catalytic subunit (NrdA-2 and NrdJ-1). This fragmented gene organization appears to be primarily present in phages (Dassa et al, Nucleic Acids Research. 57:2560-2573 (2009)).

In some embodiments, the prime editors described herein can be used to insert split intein tags onto two different proteins, which when co-expressed cause their intracellular ligation to form a fusion protein. In protein trans-splicing, one precursor protein consists of an N extein part followed by an N intein, and another precursor protein consists of a C intein followed by a C extein part, and the trans-splicing reaction (catalyzed by the N and C inteins together) excises the two intein sequences and links the two extein sequences by a peptide bond. Protein trans-splicing, an enzymatic reaction, can work at very low (e.g., micromolar) concentrations of proteins and can be performed under physiological conditions.

The split intein Npu DnaE was characterized as having the highest rate reported for a protein trans-splicing reaction. In addition, the Npu DnaE protein splicing reaction is considered robust and high-yielding for 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 a Cysl Ala mutation in the N domain of these inteins was introduced, the initial N to S acyl shift and thus protein splicing was blocked. Unfortunately, the C-terminal cleavage reaction was also almost completely inhibited. The dependence of asparagine cyclization at the C-terminal splice junction on acyl shift at the N-terminal scissile peptide bond appears to be a common, intrinsic property of naturally split DnaE intein alleles (Zettler J. et al. FEBS Letters. 555:909-914 (2009)).

Protein trans-splicing catalyzed by split inteins provides a purely enzymatic method for protein ligation. A split intein is essentially a continuous intein (e.g., a mini-intein) that has been split into two pieces, named N and C inteins, respectively. The N and C inteins of a split intein can non-covalently associate to form an active intein and catalyze a splicing reaction in essentially the same way that a continuous intein does. Split inteins are found in nature and have also been engineered in the laboratory. The term "split intein" as used herein refers to any intein in which one or more peptide bond cleavages exist between the N- and C-terminal amino acid sequences, such that the N- and C-terminal sequences become separate molecules that can non-covalently reassociate or reconstitute into an intein that is functional for a trans-splicing reaction. Any catalytically active intein or fragment thereof can be used to derive a split intein for use in the methods of the invention. For example, in one aspect, the split intein can be derived from a eukaryotic intein. In another aspect, the split intein can be derived from a bacterial intein. In another aspect, the split intein can be derived from an archaeal intein. Preferably, the split intein so derived will possess only the amino acid sequences essential for catalyzing the trans-splicing reaction.

Split inteins can be generated from a stretch of inteins by engineering one or more split sites into the unstructured loop or intervening amino acid sequence between the -12 conserved beta strands found on the mini-intein structure. There can be some flexibility in the location of the split site within the region between the beta strands, provided that the creation of the split would not interrupt the structure of the intein, particularly the structured beta strands, to a sufficient degree that protein splicing activity is lost.

The prime editors described herein can incorporate a peptide tag (including an intein) at the C-terminal end of a protein of interest. In other embodiments, a peptide tag (including an intein) can be incorporated at the N-terminal end of a protein of interest. A peptide tag can also be incorporated internally into a protein of interest. The resulting fusion protein produced by the prime editors described herein can have the following structure:

[protein of interest]-[peptide tag];

[peptide tag]-[protein of interest]; or

[N-terminal region of the protein of interest]-[peptide tag]-[C-terminal region of the protein of interest].

The principles of guide RNA design for use in peptide tagging can be applied to peptide tagging throughout. For example, in one embodiment, a PEgRNA structure for peptide tagging can have the following structure: 5'-[spacer sequence]-[gRNA core or backbone]-[extension arm]-3'. The extension arm contains, in the 5' to 3' direction, a homology arm, an editing template (containing a sequence encoding the peptide tag), and a primer binding site. This configuration is illustrated in Figure 3D and Figure 24.

In another embodiment, the PEgRNA structure for peptide tagging can have the following structure: 5'-[extension arm]-[spacer sequence]-[gRNA core or backbone]-3'. The extension arm contains, in the 5' to 3' direction, a homology arm, an editing template (containing a sequence encoding the peptide tag), and a primer binding site. This configuration is illustrated in Figure 3E.

An embodiment of peptide tagging using prime editing is illustrated in Figures 25 and 26 and described in Example 4.

E. Use of Prime Editing to Prevent or Treat Prion Diseases
Prime editing can also be used to prevent or halt the progression of prion diseases by incorporating one or more protective mutations on the prion protein (PRNP) that becomes misfolded during the disease process. 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 a long latency period, characteristic spongiform changes associated with neuronal loss, and failure to induce an inflammatory response.

In humans, prion diseases include Creutzfeldt-Jakob disease (CJD), variant Creutzfeldt-Jakob disease (vCJD), Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia, and kuru. In animals, prion diseases include bovine spongiform encephalopathy (BSE or "mad cow disease"), chronic wasting disease (CWD), scrapie, transmissible mink encephalopathy, feline spongiform encephalopathy, and ungulate spongiform encephalopathy. To prevent or halt the progression of any one of these prion diseases, prime editing can be used to incorporate protective point mutations onto the prion protein.

Classical CJD is a human prion disease. It is a neurodegenerative disorder with characteristic clinical and diagnostic features. The disease is rapidly progressive and invariably fatal. Infection with the disease is usually fatal within one year of disease onset. 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, with an estimated annual incidence in many countries, including the United States, reported to be about one case per million population. The majority of CJD patients usually die within one year of disease onset. CJD is classified as a transmissible spongiform encephalopathy (TSE), along with other prion diseases occurring in humans and animals. In approximately 85% of patients, CJD occurs as an idiopathic disease with no discernible pattern of transmission. A smaller percentage of patients (5 to 15%) develop CJD due to inherited mutations in the prion protein gene. These inherited forms include Gerstmann-Straussler-Scheinker syndrome and fatal familial insomnia. There is currently no known treatment for CJD.

Variant Creutzfeldt-Jakob disease (vCJD) is a prion disease first described in 1996 in the UK. There is now strong scientific evidence that the agent responsible for outbreaks of the prion disease bovine spongiform encephalopathy (BSE or "mad cow" disease) in cattle is the same agent responsible for outbreaks of vCJD in humans. Variant CJD (vCJD) is not the same disease as classical CJD (often simply called CJD). It has different clinical and pathological characteristics than classical CJD. Each disease also has a specific 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 a non-conventionally transmissible agent called a prion. For vCJD, no treatment is currently known.

BSE (bovine spongiform encephalopathy or "mad cow disease") is a progressive neurological disorder in cattle that results from infection with 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 an altered form of a normal protein known as a prion protein. For reasons that are not yet understood, the normal prion protein changes into a pathogenic (harmful) form, which then damages the central nervous system of the cattle. There is growing evidence that there are different strains of BSE: the typical or classical BSE strain, which was responsible for the outbreak in the UK, and two atypical strains (the 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 is found in some areas of North America, including Canada and the United States, Norway, and Korea. It can take more than a year before an infected animal develops symptoms, which can include severe weight loss (wasting), staggering, lethargy, and other neurological 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 is no treatment or vaccine.

The causative agent of TSEs is believed to be prions. The term "prion" refers to an abnormal pathogenic agent that is transmissible and capable of inducing the abnormal folding of certain normal cellular proteins called prion proteins, which are found most abundantly in the brain. The function of these normal prion proteins is not yet fully 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 are always fatal.

The term "prion" as used herein refers to an infectious particle known to cause disease (spongiform encephalopathy) in humans and animals. The term "prion" is a contraction of the words "protein" and "infectious," and the particle is largely, if not exclusively, composed of PRNP ^Sc molecules encoded by the PRNP gene that expresses PRNP ^C , which changes conformation to become PRNP ^Sc . Prions are distinct from bacteria, viruses, and viroids. Known prions include those that infect animals and cause scrapie, a transmissible degenerative disease of the nervous system in sheep and goats, and bovine spongiform encephalopathy (BSE) or mad cow disease, and feline spongiform encephalopathy in cats. The four prion diseases discussed above that are known to affect humans are (1) kuru, (2) Creutzfeldt-Jakob disease (CJD), (3) Gerstmann-Straussler-Scheinker disease (GSS), and (4) fatal familial insomnia (FFI). Prion as used herein includes all forms of prion that cause all or any of these diseases or others in any animal used, particularly humans and domesticated livestock animals.

In general, without being bound by theory, prion diseases are caused by misfolding of prion protein. Such diseases, often referred to as deposition diseases, misfolding of prion protein can be explained as follows: if A is the normally synthesized gene product that performs its intended physiological role in a monomeric or oligomeric state, and A* is the conformationally activated form of A that is capable of undergoing dramatic conformational changes, then B is the conformationally altered state that favors multimeric assembly (i.e., the misfolded form that forms deposits), and _Bn is the multimeric material that is pathogenic and relatively difficult to recycle. In prion diseases, PRNP ^C and PRNP ^Sc correspond to states A and _Bn , where A is largely helical and monomeric, and _Bn is β-rich and multimeric.

It is known that certain mutations in the prion protein may be associated with an increased risk of prion (prior) diseases. Conversely, certain mutations in the prion protein may be naturally protective. 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)) human prion protein is encoded by a 16 kb long gene located on chromosome 20 (4686151-4701588). It contains two exons, exon 2 has an open reading frame, which encodes the 253 amino acid (AA) long PrP protein. Exon 1 is a non-coding exon, which may serve as the transcription start site. Post-translational modification results 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), and the CTF (glycosylphosphatidylinositol [GPI] signal peptide [GPI-SP]) is cleaved by the GPI anchor. The GPI anchor may be involved in PrP protein transport. It may also play a role in the attachment of the prion protein to the outer surface of the cell membrane. Normal PrP consists of a long N-terminal loop (which contains an octapeptide repeat region), two short β-sheets, three α-helices, and a C-terminal region (which contains a GPI anchor). Cleavage of PrP results in a 208AA long glycoprotein that is anchored to the cell membrane.

The amino acid sequence of PRNP (NP_000302.1) is as follows:
MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPVILLISFLIFLIVG (sequence number 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) and is as follows:
(SEQ ID NO:292)

The relative mutation sites for PRNP (NP_000302.1) linked to CJD and FFI have been reported as follows: These mutations can be removed or incorporated using the prime editors disclosed herein.

The mutation sites relative to PRNP (NP_000302.1) (SEQ ID NO: 291) that lead to GSS are reported as follows:

The mutation sites relative to PRNP (NP_000302.1) (SEQ ID NO: 291) that lead to possible protective nature against prion disease are as follows:

Thus, in various embodiments, prime editing can be used to remove PRNP mutations linked to prion disease or to introduce PRNP mutations considered protective against prion disease. For example, prime editing can be used to remove or restore D178N, V180I, T188K, E196K, E196A, E200K, E200G, V203I, R208H, V210I, E211Q, I215V, or M232R mutations in the PRNP protein (relative to PRNP of NP_000302.1) (SEQ ID NO: 291). In other embodiments, prime editing can be used to remove or restore P102L, P105L, A117V, G131V, V176G, H187R, F198S, D202N, Q212P, Q217R, or M232T mutations in the PRNP protein (relative to PRNP of NP_000302.1) (SEQ ID NO: 291). By using prime editing to remove or correct the presence of such mutations in PRNP, the risk of prion disease can be reduced or eliminated.

In other embodiments, prime editing can be used to incorporate protective mutations in PRNP that are linked to a protective effect against one or more prion diseases. For example, prime editing can be used to incorporate G127S, G127V, M129V, D167G, D167N, N171S, E219K, or P238S protective mutations in PRNP (relative to PRNP in NP_000302.1) (SEQ ID NO: 291). In yet other embodiments, the protective mutations can be any alternative amino acid incorporated into G127, G127, M129, D167, D167, N171, E219, or P238 in PRNP (relative to PRNP in NP_000302.1) (SEQ ID NO: 291).

In a specific embodiment, prime editing can be used to incorporate the G127V protective mutation into PRNP, as illustrated in Figure 27 and discussed in Example 5.

In another embodiment, prime editing can be used to incorporate the E219K protective mutation into PRNP.

The PRNP protein and protective mutation sites are conserved in mammals. Therefore, in addition to treating human disease, it could also be used to generate cattle and sheep immune to prion disease, or even help cure wild populations of animals suffering from prion disease. Prime editing has already been used to achieve ∼25% incorporation of the naturally occurring protective allele in human cells, and previous mouse experiments indicate that this level of incorporation is sufficient to induce immunity to most prion diseases. This method is the first and potentially only current way to incorporate this allele with such high efficiency in most cell types. Another possible strategy for treatment is to use prime editing to reduce or eliminate expression of PRNP by incorporating a premature stop codon into the gene.

Using the principles described herein for PEgRNA design, suitable PEgRNAs can be designed to incorporate desired protective mutations or to remove prion disease-associated mutations from PRNP. For example, the following list of PEgRNAs can be used to incorporate the G127V protective allele and the E219K protective allele into human PRNP, and the G127V protective allele into the PRNP of various animals.

F. Use of Prime Editing for RNA Tagging
Prime editing can also be used to manipulate, alter and otherwise modify the sequence of DNA that codes for RNA function by RNA tagging, thus providing a means to indirectly modify RNA structure and function. For example, PE can be used to insert motifs (hereafter RNA motifs) that are functional at the RNA level to tag or otherwise manipulate non-coding RNA or mRNA. These motifs can be used to increase gene expression, decrease gene expression, alter splicing, change post-transcriptional modification, affect the subcellular level location of RNA, enable RNA isolation or determination of intracellular or extracellular location (e.g., using fluorescent RNA aptamers, such as Spinach, Spinach2, Baby Spinach, or Broccoli), recruit endogenous or exogenous proteins or RNA binding factors, introduce sgRNA, or induce RNA processing by either self-cleavage or RNAse (see Figure 28B and Example 6 for further details).

The following RNA tags or motifs can be inserted onto genes of interest using prime editing with appropriate PEgRNA (designed using the guidance provided herein) to affect various properties of the RNA including RNA transport, expression levels, splicing, and detection.

The PEgRNAs in the table above are designed to site-specifically insert the example motifs above into the HEXA gene (defective in Tay-Sachs disease) (e.g., GenBank No. KR710351.1 (SEQ ID NO: 369)). However, this is for illustrative purposes only. The use of prime editing for RNA tagging is not limited to the HEXA gene, and indeed could be any gene.
HEXA mRNA has the following nucleotide sequence:
GTTCGTTGCAACAAATTGATGAGCAATGCTTTTTTATAATGCCAACTTTGTACAAAAAAGTTGGCATGACAAGTTCCAGGCTTTGGTTTTCGCTGCTGCTGGCGGCAGCGTTCGCAGGACGGGCGACGGCCCTCTGGCCCTGGCCTCAGAACTTCCAAACCTCCGACCAGCGCTACGTCCTTTACCCGAACAACTTTCAATTCCAGTACGATGTCAGCTCGGCCGCGCAGCCCGGCTGCTCAGTCCTCGACGAGGCCTTCCAGCGCTATCGTGACCTGCTTTTCGGTTCCGGGTCTTGGCCCCGTCCTTACCTCACAGGGAAACGGCATACACTGGAGAAGAATGTGTTGGTTGTCTCTGTAGTCACACCTGGATGTAACCAGCTTCCTACTTTGGAGTCAGTGGAGAAT TATACCCTGACCATAAATGATGACCAGTGTTTACTCCTCTCTGAGACTGTCTGGGGAGCTCTCCGAGGTCTGGAGACTTTTAGCCAGCTTGTTTGGAAATCTGCTGAGGGCACATTCTTTATCAACAAGACTGAGATTGAGGACTTTCCCCGCTTTCCTCACCGGGGCTTGCTGTTGGATACATCTCGCCATTACCTGCCACTCTCTAGCATCCTGGACACTCTGGATGTCATGGCGTACAATAAATTGAACGTGTTCCACTGGCATCTGGTAGATGATCCTTCCTTCCCATATGAGAGCTTCACTTTTCCAGAGCTCATGAGAAAGGGGTCCTACAACCCTGTCACCCACATCTACACAGCACAGGATGTGAAGGAGGTCATTGAATACGCACGGCTCCGGGGTATCCG TGTGCTTGCAGAGTTTGACACTCCTGGCCACACTTTGTCCTGGGGACCAGGTATCCCTGGATTACTGACTCCTTGCTACCCTGGGTCTGAGCCCTCTGGCACCTTTGGACCAGTGAATCCCAGTCTCAATAATACCTATGAGTTCATGAGCACATTCTTCTTAGAAGTCAGCTCTGTCTTCCCAGATTTTTATCTTCATCTTGGAGGAGATGAGGTTGATTTCACCTGCTGGAAGTCCAACCCAGAGATCCAGGACTTTATGAGGAAGAAAGGCTTCGGTGAGGACTTCAAGCAGCTGGAGTCCTTCTACATCCAGACGCTGCTGGACATCGTCTCTTCTTATGGCAAGGGCTATGTGGTGGCAGGAGGTGTTTGATAATAAAGTAAAGATTCAGCCAGACACAATCA [0043] TACAGGTGTGGCGAGAGGATATTCCAGTGAACTATATGAAGGAGCTGGAACTGGTCACCAAGGCCGGCTTCCGGGCCCTTCTCTCTGCCCCCTGGTACCTGAACCGTATATCCTATGGCCCTGACTGGAAGGATTTCTACGTAGTGGAACCCCTGGCATTTGAAGGTACCCCTGAGCAGAAGGCTCTGGTGATTGGTGGAGAGGCTTGTATGTGGGGAGAATATGTGGACAACACAAACCTGGTCCCCAGGCTCTGGCCCAGAGCAGGGGCTGTTGCCGAAAGGCTGTGGAGCAACAAGTTGACATCTGACCTGACATTTGCCTATGAACGTTTGTCACACTTCCGCTGTGAGTTGCTGAGGCGAGGTGTCCAGGCCCAACCCCTCAATGTAGGCTTCTGTGAGCAGGAGTTTTGAACAGACCTGCCCAACTTTCTTGTACAAAGTTGGCATTATAAGAAAGCATTGCTTATCAATTTGTTGCAACGAAC (SEQ ID NO: 369).

The corresponding HEXA protein has the following amino acid sequence:
(Sequence number 370).

Of note, the resulting RNA motif would be contained within the translational region of the HEXA gene, blocking function of the protein-coding gene. The inserted polyadenylation motif would result in premature transcript termination. This site is merely illustrative of the possible PEgRNAs that could result in the insertion of the RNA motifs listed in the table above within a genomic site that would be transcribed and therefore produce an RNA product.

PEgRNAs for use in PE for RNA tagging can be expressed from a U6 promoter (in this case, for guides containing a protospacer that does not begin with a G, a single guanosine would be added to the 5' end of the PEgRNA and 6-7 thymines would be added to the 3' end) or a polII promoter, such as pCMV (in this case, it may be necessary to remove the naturally transcribed sequence of this promoter from the 5' end of the RNA with 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 will not be inserted on the genome as a result of PE, since it will be 3' of the annealing region). The core PEgRNA backbone is underlined, the homology and annealing regions are italicized, and the insertion sequence is in bold. Note that the inserted sequence is the reverse complement of the example above, as described below, and thus these PEgRNAs will need to be targeted to the coding strand.

Also note that self-cleaving ribozymes other than HDV, in some embodiments, need to be tailored to a given target site; that is, HDV cleaves the encoded transcript immediately 5' to itself, while the cutting site for all other self-cleaving ribozymes is within the ribozyme itself. Thus, the first and last approximately 5-10 nucleotides (and in some cases potentially more than 10) will actually be part of the encoded sequence. As an example, to cleave the sequence 5'NNNNNTCATCCTGATAAACTGCAAA3' (SEQ ID NO:371), where N is any nucleotide, after 5 N's using a hammerhead self-cleaving ribozyme, the following sequence would be inserted, where the underlined sequence forms a non-perfect RNA pairing element:

5'NNNNNCAGTTTGTACGGATGACTGATGAGTCCCAAATAGGACGAAACGCGCTTCGGTGCGTCTCATCCTGATAAACTGCAAA-3' (sequence number 372).

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

(ここで、コアPEgRNA骨格に下線を付し、相同領域およびアニーリング領域は斜字体に、挿入配列は太字にしている)(配列番号373)。

(where the core PEgRNA backbone is underlined, the homology and annealing regions are in italics, and the insert sequence is in bold) (SEQ ID NO:373).

Designing other PEgRNAs for insertion of RNA motifs follows the general principles described herein. However, it is noted that many of the RNA motifs are potentially highly structured. This can make them difficult to reverse transcribe and insert onto the genome. In some RNA sequences, e.g., simple hairpins, both the RNA sequence itself and its complement are structured, however, that is unlikely to be true for the sequences described above. Thus, when inserting these motifs, it would most likely be best for the PEgRNA to code for the reverse complement of these sequences, resulting in insertion onto the genome of the DNA sequence that actually codes for the motif. Similarly, inclusion of a self-cleaving ribozyme in the PEgRNA template region would result in processing and inefficient activity, but inclusion of its reverse complement would not. Therefore, these PEgRNAs would likely have to target the coding strand, although PEgRNAs encoding other types of insertions (e.g., therapeutic modifications) would theoretically be capable of targeting either strand.

Also note that for many of the inserted motifs, the resulting PEgRNA may not be capable of being transcribed from the U6 promoter, necessitating the use of other promoters, such as pCMV. Similarly, longer PEgRNAs may also be less stable. Shorter motifs, such as the ^m6A marker, would not have this problem.

G. Use of Prime Editing to Generate High-Performance Gene Libraries
Prime editing can also be used to generate high performance libraries of genes encoding proteins or RNAs 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 is further described in Example 7.

The generation of variable gene libraries is most commonly accomplished by mutagenic PCR (see Cadwell RC and Joyce GF. PCR Methods Appl. 1992). This method relies on either using reaction conditions that reduce the fidelity of the DNA polymerase or using modified DNA polymerases with higher mutation rates. The biases of these polymerases are then reflected in the library products (e.g., preference for transition mutations over transversions). An inherent limitation of this approach to library construction is the relative inability to affect the size of the gene to be altered. Most DNA polymerases have an extremely low rate of indel mutations (insertions or deletions), and most of these will result in frameshift mutations in protein-coding regions, making it unlikely that library members will pass any downstream selection (see McInerney P, Adams P, and Hadi MZ. Mol Biol Int. 2014).

In addition, PCR and cloning biases can make it difficult to generate a single library of genes of different sizes. These limitations can severely limit the efficacy of directed evolution to enhance existing and 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 standard library generation for mutagenesis. Furthermore, these mutations most commonly occur in regions of the protein that are predicted to form loops versus the hydrophobic core. Therefore, most indels generated using traditional unbiased approaches are likely to be either harmful or ineffective.

Because all libraries have access to only a fraction of the possible mutation space, libraries that can bias mutations towards sites on the protein where they are most likely to be beneficial, such as loop regions, would have a significant advantage over conventional libraries. Finally, while it is possible to generate gene libraries with site-specific indel mutations by multi-step PCR and clone assembly using NNK primers or by DNA shuffling, these libraries cannot undergo additional rounds of "indelgenesis" in continuous evolution. Continuous evolution is a type of directed evolution with minimal user intervention. One such example is PACE (see Esvelt KM, Carlson JC, and Liu DR. Nature. 2011). Since continuous evolution occurs with minimal user intervention, any increase in library diversity during evolution should occur using native replication mechanisms. Thus, in PACE, libraries of genes with inserted or removed codons at specific loci can be generated and screened, but additional rounds of "indelgenesis" are not possible.

It is envisioned that the programmability of prime editing (PE) can be exploited to generate highly sophisticated programmed gene libraries for use in high-throughput screening and directed evolution (see FIG. 29A). PE can insert, change, or remove a defined number of nucleotides from a defined gene locus using information encoded on a prime editing guide RNA (PEgRNA) (see FIG. 29B). This allows the generation of targeted libraries with one or more amino acids inserted or removed from loop regions where the mutation is most likely to result in a change in function, without the background introduction of non-functional frameshift mutations (see FIG. 29C). PE can be used to incorporate a specific set of mutations regardless of the biases inherent in either the DNA polymerase or the sequence to be mutated.

For example, PEs can be used to convert any given target codon to a TGA stop codon in one step, which would be an unlikely occurrence with standard library generation since it would require three consecutive mutations encompassing two transversions. They can also be used to incorporate programmed diversity at a given position, for example by incorporating a codon that codes for any hydrophobic amino acid but not any other at a given site. Furthermore, because of the programmability of PE, multiple PE gRNAs can be utilized to generate multiple different edits at multiple sites simultaneously, enabling the generation of highly programmed libraries (see Figure 29D). In addition, reverse transcriptases with lower fidelity can be used to generate regions of mutagenesis in otherwise immutable libraries (e.g., HIV-I reverse transcriptase or Bordetella phage reverse transcriptase) (see Naorem SS, Hin J, Wang S, Lee WR, Heng X, Miller JF, Guo H. Proc Natl Acad Sci 2017 and Martinez MA, Vartanian JP, Wain-Hobson S. Proc Natl Acad Sci USA 1994).

The possibility of repeated rounds of PE on the same site is also envisioned, for example allowing the insertion of repeated codons at a single site, for example in a loop region. It is also envisioned that all of the approaches described above can be incorporated into continuous evolution, allowing the generation of novel in situ evolution libraries (see FIG. 30). They can also be used to construct these libraries in other cell types where it would otherwise be difficult to assemble large libraries, for example in mammalian cells. The generation of bacterial strains encoding optimized PE 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 PE. In conclusion, library generation with PE would be a highly useful tool in synthetic biology and directed evolution, as well as for high-throughput screening of combinatorial mutants of proteins and RNAs.
Competing approaches

The main way diverse libraries are currently generated is by mutagenic PCR as described above (see Cadwell RC and Joyce GF. PCR Methods Appl. 1992). Insertions or deletions can be introduced by degenerate NNK primers at defined sites during PCR, but introducing such mutations at multiple sites requires multiple rounds of iterative PCR and cloning before a more diverse library can be constructed by mutagenic PCR, making the method slow. An alternative complementary method is DNA shuffling, where fragments of a library of genes generated by DNase treatment are introduced into a primer-free PCR reaction, resulting in annealing of the different fragments to each other and more rapid generation of diverse libraries than mutagenic PCR alone (see Meyer AJ, Ellefson JW, Ellington AD. Curr Protoc Mol Biol. 2014). Although this approach can theoretically generate indel mutations, it more often results in frameshift mutations that disrupt gene function. Furthermore, DNA shuffling requires a high degree of homology between gene fragments.

While both of these methods must be done in vitro and the resulting libraries transformed into cells, libraries generated by PE can be constructed in situ, enabling their use in continuous evolution. Libraries can be constructed in situ by in vivo mutagenesis, and these libraries rely on the host cell machinery and exert a bias against indels. Similarly, traditional cloning methods can be used to generate site-specific mutation profiles, but 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 can be constructed directly within the cell type of interest, versus being cloned into a model organism such as E. coli and then transferred into the cell or organism of interest. Another competing approach to targeted diversification is automated multiplex genome engineering or MAGE, in which multiple single-stranded DNA oligonucleotides can be incorporated into replication forks, resulting 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 A et al. Proc Natl Acad Sci USA. 2016), whereas PE is more highly programmed and is expected to result in fewer off-target effects. In addition, MAGE has not been demonstrated in a wide variety of cell types, including mammalian cells.

In contrast, prime editing is a novel and non-obvious complementary technique for library generation.
Use of PE to construct gene libraries

PE can be used to construct gene libraries in a programmable manner.

In one example, PE can be used in directed evolution experiments to introduce protein variants into gene libraries during continuous evolution experiments using PACE, allowing the iterative accumulation of both point mutations and indels in a manner not possible with conventional approaches.

It has already been shown that PE can site-specifically and programmably insert nucleotides into gene sequences in E. coli. Directed evolution can be used to identify monobodies with improved binding to specific epitopes by engineered two-hybrid protein:protein binding PACE selection. The specific and highly variable loops on these monobodies contribute significantly to affinity and specificity. Improved monobody binding can be rapidly obtained in PACE by altering the length and composition of these loops in a targeted manner. However, altering sequence length is not an established functionality of PACE. Although a library of different loop sizes can be used as a starting point for PACE, subsequent refinement of the length will not occur during PACE selection, preventing access to beneficial synergistic combinations of point and indel mutations.

In various embodiments, PE can be used to improve PACE selection by enabling the in situ generation and evolution of monobodies with various loop lengths. To do so, a PACE E. coli strain can be introduced with an additional PE plasmid, which encodes the PE enzyme and one or more PEgRNAs. Expression of the PE enzyme and PEgRNAs in E. coli will be under the control of small molecules delivered to the PACE lagoon at a rate selected by the experimenter.

In various embodiments, the PEgRNA component will be designed to contain a spacer that directs PE to the site of interest on the selection phage, allowing multiple trinucleotides to be inserted into the target site. This will introduce new PEgRNA binding sites, allowing repeated insertion of one or more codons at the target site.

In parallel, another host E. coli strain could harbor a PEgRNA that would template the removal of one or more codons, allowing the loop size to shrink during evolution. PACE experiments could utilize a mixture of both strains, or alternate between the two to allow for the slow and controlled addition or removal of loop sequences.

In addition to the use of PE and PACE to generate 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 the antibody complementarity determining region loops is critical for their binding function. Furthermore, longer loop lengths have been found to be critical for the development of rare antibodies with broadly protective activity against HIV-1 and other viral infections (see Mascola JR, Haynes BF. Immunol Rev. 2013). Application of the PE described above to antibodies or antibody-derived molecules will allow the generation of antibodies with diverse loop lengths and various loop sequences. In combination with PACE, such an approach will allow enhanced binding by loop geometries not accessible to standard PACE and therefore the evolution of highly functional antibodies.

As a non-limiting example, the following PEgRNA can be used to programmably modify the genome of a bacteriophage used in continuous evolution experiments:

In various embodiments, the use of PE to construct gene libraries can make use of the mutagenic activity of error-prone reverse transcriptases. The use of such mutagenic reverse transcriptases can facilitate the generation of mutagenized programmable libraries due to the lower fidelity of error-prone RT. The term "error-prone" reverse transcriptase as used herein refers to a reverse transcriptase enzyme that is naturally occurring or derived from another reverse transcriptase (e.g., wild-type M-MLV reverse transcriptase) that has an error rate that is less than that of wild-type M-MLV reverse transcriptase. The error rate of wild-type M-MLV reverse transcriptase has been reported to range from 1 error per 15,000 to 27,000 nucleobase incorporation. 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.

Therefore, for purposes of this application, the term "error-prone" refers to a nucleic acid sequence that is more likely to have an error than one error per 15,000 nucleobase incorporation (6.7×10 ⁻⁵ or higher), e.g., one error per 14,000 nucleobases (7.14×10 ⁻⁵ or higher), one error per 13,000 nucleobases or fewer (7.7×10 ⁻⁵ or higher), one error per 12,000 nucleobases or fewer (7.7×10 ⁻⁵ or higher), one error per 11,000 nucleobases or fewer (9.1×10 ⁻⁵ or higher), one error per 10,000 nucleobases or fewer (1×10 ^-4 or 0.0001 or higher), 1 error per 9,000 nucleobases or less (0.00011 or higher), 1 error per 8,000 nucleobases or less (0.00013 or higher), 1 error per 7,000 nucleobases or less (0.00014 or higher), 1 error per 6,000 nucleobases or less (0.00016 or higher), 1 error per 5,000 nucleobases or less (0.0002 or higher), 1 error per 4,000 nucleobases or less (0.00016 or higher), 1 error per 5,000 nucleobases or less (0.0002 or higher), 1 error per 4,000 nucleobases or less (0.00016 or higher), 1 error per 6,000 nucleobases or less (0.00016 or higher), 1 error per 5,000 nucleobases or less (0.0002 or higher), 1 error per 4,000 nucleobases or less (0.00016 or higher), 1 error per 8,000 nucleobases or less (0.00016 or higher), 1 error per 9,000 nucleobases or less (0.00016 or higher), 1 error per 10 ... It refers to a RT having an error rate that is greater than 1 error (0.00025 or higher), 1 error per 3,000 nucleobases or fewer (0.00033 or higher), 1 error per 2,000 nucleobases or fewer (0.00050 or higher), or 1 error per 1,000 nucleobases or fewer (0.001 or higher), or 1 error per 500 nucleobases or fewer (0.002 or higher), or 1 error per 250 nucleobases or fewer (0.004 or higher).

For the generation of highly mutagenized programmable libraries, various mutagenizing RTs can be envisaged. Two such examples are the mutagenizing 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 herein by reference). In the case of the RT from Bordetella phage (brt), it may also be necessary to deliver an auxiliary protein (bavd) to Cas9 and an additional RNA sequence to the PEgRNA, either additionally or in trans, to improve binding of the mutagenizing RT to the target site (see Handa, S., et al. Nucl Acids Res 9711-25 (2018)). When using mutagenic RT, the template region of the PEgRNA can be enriched for adenosine or AAY codons to enhance diversity.

The amino acid sequence of the mutagenized RT from Bordetella phage is provided below: As with the other RTs disclosed herein, the Brt protein can be fused to the napDNAbp as a fusion protein to form a functional PE.

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

In the case of Brt from Bodetella, the PEgRNA can include additional nucleotide sequences added to the PEgRNA, for example, at the 5' or 3' end. An exemplary sequence is as follows, which is originally from the Bordetella phage genome:

To reduce its length, this PEgRNA addition sequence can be shortened in various ways. For example, the PEgRNA addition 1 sequence can be shortened to the following exemplary alternative addition sequences:

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

In various embodiments involving the use of PE to design gene libraries, special PEgRNA considerations may apply. For example, without being bound by theory, the additional PEgRNA sequences described above may be required to enable efficient mutagenesis by mutagenic RT. In another embodiment, repetitive codon insertion using PE may require specific PEgRNA design. For example, to repetitively insert GGG (glycine) codons, as shown above, the entire homology region of the PEgRNA may need to consist of Gs. This would mean that only certain sites would be capable of repetitive insertion. In addition, PAM sequences would not be capable of being blocked by PE.

H. Use of Prime Editing for Insertion of Immune Epitopes
Prime editors can also be used as a means to insert known immunogenic epitopes onto endogenous or exogenous genomic DNA, resulting in the modification of the corresponding protein for therapeutic or biotechnological applications (see Figures 31 and 32). Prior to the present invention of prime editing, such insertions could only be achieved inefficiently and with high indel formation rates from DSBs. Prime editing offers higher efficiency than HDR in general, while solving the problem of high indel formation from insertion editing. This lower indel formation rate presents a major advantage of prime editing compared to HDR as a method for targeted DNA insertion, especially in the described application of inserting immunogenic epitopes. The length of the epitopes ranges from a few bases to several hundred bases. Prime editors are an efficient approach to achieve such targeted insertions in mammalian cells.

The key concept of the present invention is the use of a prime editor to insert nucleotide sequences containing the immunogenic epitopes described above onto endogenous or exogenous genomic DNA for downregulation and/or destruction of their protein products and/or expressing cell types. The nucleotide sequences for immunogenic epitope insertion will be targeted into the gene in a manner that results in a fusion protein between the encoded protein of the target gene and the corresponding protein translation of the inserted immunogenic epitope. The patient's immune system will have been previously trained to recognize these epitopes as a result of prior standard immunization, e.g., from routine vaccinations against tetanus or diphtheria or measles. As a result of the immunogenic nature of the fused epitopes, the patient's immune system will be expected to recognize and disable the prime edited protein (and not just the inserted epitope) and potentially the cells in which it is 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 onto target genomic loci. Previously, homology-directed repair (HDR) was used for this application, requiring a 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 to the cell and is the only method available for inserting large DNA sequences into mammalian cells. However, HDR is hampered by unwanted cellular side effects derived from initiating the 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 work to overcome these drawbacks involves fusing a human Rad51 mutant to the Cas9 D10A nickase (RDN), resulting in a DSB-free HDR system. This features improved HDR product:indel ratios and lower off-target editing, but is still hampered by cell-type dependency and only modest HDR editing efficiency.

The recently developed fusion of Cas9 to a reverse transcriptase coupled with PEgRNA (the "prime editor") represents a novel genome editing technology that offers several advantages over existing genome editing methods, including the ability to incorporate 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. Of note, PE editing is generally achieved with a low rate of unintended indels. Therefore, PE enables targeted insertion-based editing applications that were previously impossible or impractical.

This particular aspect describes a method for using prime editing as a means to insert known immunogenic epitopes onto endogenous or exogenous genomic DNA, resulting in the modification of the corresponding protein for therapeutic or biotechnological applications (see Figures 31 and 32). Prior to the present invention of prime editing, such insertions could only be achieved inefficiently and with high indel formation rates from DSBs. Prime editing offers higher efficiency than HDR in general, while solving the problem of high indel formation from insertion editing. This lower indel formation rate presents a major advantage of prime editing compared to HDR as a method for targeted DNA insertion, especially in the described application of inserting immunogenic epitopes. The length of the epitopes ranges from a few bases to hundreds of bases. Prime editors are an efficient and cleanest approach to achieve such targeted insertions in mammalian cells.

The key concept of this aspect is the use of a prime editor to insert nucleotide sequences containing the immunogenic epitopes described above onto endogenous or exogenous genomic DNA for downregulation and/or destruction of their protein products and/or expressing cell types. The nucleotide sequences for immunogenic epitope insertion will be targeted into the gene in a manner that results in a fusion protein between the encoded protein of the target gene and the corresponding protein translation of the inserted immunogenic epitope. The patient's immune system will have been previously trained to recognize these epitopes as a result of prior standard immunization, e.g., from routine vaccinations against tetanus or diphtheria or measles. As a result of the immunogenic nature of the fused epitopes, the patient's immune system will be expected to recognize and disable the prime edited protein (and not just the inserted epitope) and potentially the cells in which it is expressed.

Fusions to the target gene will be engineered as required to ensure that the inserted epitope protein translation is exposed for immune system recognition. This may include C-terminal fusions of the immunogenic epitope to the target gene, N-terminal fusions of the immunogenic epitope to the target gene, or targeted nucleotide insertions that result in protein translation resulting in the insertion of nucleotides onto the gene such that the immunogenic epitope is encoded in a surface-exposed region of the protein structure.

To facilitate immune system recognition of the target gene, cellular trafficking, protein function, or protein folding, a protein linker encoded as nucleotides inserted between the target gene sequence and the inserted immunogenic epitope nucleotide sequence may need to be engineered as part of the invention. The protein linkers encoded by these inserted nucleotides may include (but are not limited to) XTEN linkers of variable length and sequence or glycine-serine linkers of variable length and sequence. These engineered linkers have been used previously to successfully facilitate protein fusion. Exemplary linkers may include any of those described herein, including the amino acid sequences (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, where n is independently an integer between 1 and 30, and X is any amino acid. In some embodiments, the linker comprises the amino acid sequence (GGS)n (SEQ ID NO: 176), where 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).

A distinguishing feature of this aspect includes the ability to use a previously acquired immune response to a particular amino acid sequence as a means to induce an immune response to an otherwise non-immunogenic protein. Another distinguishing feature is the ability to insert the nucleotide sequences of these immunogenic epitopes in a targeted manner that does not induce high levels of unwanted indels as by-product editing and whose insertion is efficient. This particular application of PE has the ability to combine a cell type specific delivery method (e.g., AAV serotype) to insert an epitope into a cell type against which it is desired to elicit an immune response.

Prime editing as a means of inserting immunogenic epitopes onto pathogenic genes can be used to program a patient's immune system to fight a wide variety of diseases (not limited to cancer in an immuno-oncology strategy). An immediately relevant use of this technology would be as cancer therapeutics, since it could thwart the immune escape mechanisms of tumors by triggering an immune response against relevant oncogenes such as HER2 or growth factors such as EGFR. While such an approach may appear similar to T cell engineering, one novel advancement of this approach is that it can be utilized for many cell types and diseases other than cancer, without the need to generate and introduce engineered T cells into patients.

Using PE, immunogenic epitopes against which most people are already vaccinated (tetanus, whooping cough, diphtheria, measles, mumps, rubella, etc.) are inserted onto exogenous or endogenous disease-driving genes so that the patient's immune system learns to disable the protein.

Diseases that would have potential therapeutic benefit from the aforementioned strategies include those caused by toxic protein aggregation, such as fatal familial insomnia. Other diseases that may benefit include those caused by pathogenic overexpression of otherwise non-toxic endogenous proteins, and those caused by exogenous 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 in some diseases, especially aggressive cancers, or in cases where drug treatments help alleviate disease symptoms until the disease is completely cured, current standard of care medicines may be used in conjunction with prime editing. Below are examples of immune epitopes that can be inserted onto genes by the prime editing elements disclosed herein:

Additional immune epitopes known in the art may also be incorporated. Any of the immune epitopes available from the Immune Epitope Database and Analysis Resource (iedb.org/epitopedetails_v3.php), the contents of which are incorporated herein by reference, may be incorporated by the prime editors disclosed herein.

In some embodiments, immune epitopes that can be incorporated by the prime editors disclosed herein can include any of the following epitopes:

I. Delivery of Prime Editing Factors
In another aspect, the present disclosure allows for the delivery of prime editors in vitro and in vivo using a variety of strategies, including separate vectors using split inteins, as well as direct delivery strategies using ribonucleoprotein complexes (i.e., prime editors complexed with PE gRNA and/or second site gRNA) using techniques such as electroporation, the use of cationic lipid-mediated formulations, and inducible endocytosis methods using receptor ligands fused to ribonucleoprotein complexes. Any such method is contemplated herein.

Overview of Delivery Options In some aspects, the present invention provides methods that include delivering to a host cell a polynucleotide encoding one or more prime editors, such as one or more vectors described herein encoding one or more components of the prime editing system described herein, one or more transcripts thereof, and/or one or more proteins transcribed therefrom. In some aspects, the present invention further provides cells resulting from such methods, and organisms (e.g., animals, plants, or fungi) that contain or result from such cells. In some embodiments, the prime editors described herein are delivered to cells in combination with (and optionally complexed with) a guide sequence. 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 the prime editors to cells in culture or in a host organism. Non-viral vector delivery systems include DNA plasmids, RNA (e.g., transcripts of vectors 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 reviews of gene therapy, 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 See Bihm (eds) (1995); and Yu et al., Gene Therapy 1:13-26 (1994).

Methods for 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 drug-enhanced uptake of DNA. Lipofection is described, for example, in U.S. Pat. Nos. 5,049,386; 4,946,787; and 4,897,355, and lipofection reagents are commercially available (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 to 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 those 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); see U.S. Patent 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 virus-based systems for delivery of nucleic acids takes advantage of the highly evolved processes for targeting viruses to specific cells of the body and transporting 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 can optionally be administered to patients (ex vivo). Traditional virus-based systems can include retroviral, lentiviral, adenoviral, adeno-associated, and herpes simplex virus vectors for gene transfer. Integration into the host genome is possible with retroviral, lentiviral, and adeno-associated viral gene transfer methods, often resulting in long-term expression of the inserted transgene. In addition, high transduction efficiencies have been observed in many different cell types and target tissues.

Viral tropism can be altered by incorporating exogenous envelope proteins, expanding the potential target population of target cells. Lentiviral vectors are retroviral vectors capable of transducing or infecting non-dividing cells and typically produce high viral titers. Thus, the choice of retroviral gene transfer system will depend on the target tissue. Retroviral vectors consist of cis-acting long terminal repeats and have a packaging capacity of up to 6-10 kb of exogenous sequence. The minimal cis-acting LTRs are sufficient for vector replication and packaging, which can then be used to introduce therapeutic genes into target cells to provide permanent transgene expression. Widely used retroviral vectors include those based on murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency 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, adenovirus-based systems may be used. Adenovirus-based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. High titers and expression levels have been obtained with such vectors. The vectors can be produced in large quantities with a relatively simple system. Adeno-associated virus ("AAV") vectors can also be used to transduce target nucleic acids into cells, for example, for in vitro production of nucleic acids and peptides and in vivo and ex vivo gene therapy procedures (see, e.g., West et al., Virology 160:38-47 (1987); U.S. Patent No. 4,797,368; WO 93/24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351 (1994)). The construction of recombinant AAV vectors is described in numerous 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 viral particles capable of infecting host cells. Such cells include 293 cells, which package adenovirus, and ψ2 or PA317 cells, which package retrovirus. Viral vectors used in gene therapy are usually produced by generating cell lines that package nucleic acid vectors into viral particles. The vectors typically contain the minimal viral sequences required for packaging and subsequent incorporation into the host, with other viral sequences being replaced by an expression cassette for the polynucleotide(s) to be expressed. Missing viral functions are typically supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy typically possess only the ITR sequences from the AAV genome required for packaging and incorporation into the host genome. The viral DNA is packaged by a cell line, which contains a helper plasmid that encodes the other AAV genes, namely rep and cap, but lacks the ITR sequences. The cell line can also be infected with adenovirus as a helper. The helper virus facilitates replication of the AAV vector and expression of AAV genes from the helper plasmid, which is not packaged in significant amounts due to the lack of ITR sequences. Contamination with adenovirus can be reduced, for example, by heat treatment, to which adenovirus is more sensitive than AAV. Additional methods for delivery of nucleic acids to cells are known to those of skill in the art. See, for example, US20030087817, which is incorporated herein by reference.

In various embodiments, the PE construct (including split constructs) can be engineered for delivery by one or more rAAV vectors. The rAAV for any of the methods and compositions provided herein can 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). The rAAV can contain a genetic cargo to be delivered to a cell (i.e., a recombinant nucleic acid vector expressing a gene of interest, such as a whole or split PE fusion protein, which is carried into the cell by the rAAV). The rAAV can be chimeric.

Serotype as used herein for rAAV refers to the serotype of the capsid protein 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, AA V6.2, 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 a derivative and pseudotype with a chimeric VP1 protein is rAAV2/5-1VP1u, which has the genome of AAV2, the capsid backbone of AAV5, and the VP1u of AAV1. Other non-limiting examples of derivatives and pseudotypes with chimeric VP1 proteins are rAAV2/5-8VP1u, rAAV2/9-1VP1u, and rAAV2/9-8VP1u.

AAV derivatives/pseudotypes and methods for generating such derivatives/pseudotypes are known in the art (see, e.g., Mol Ther. 2012 Apr;20(4):699-708. doi:10.1038/mt.2011.287. Epub 2012 Jan 24. The AAV vector toolkit: poised at the clinical crossroads. Asokan A1, Schaffer DV, Samulski RJ.). Methods for generating 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 for 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 Nos. US20070015238 and US20120322861, which are incorporated herein by reference; plasmids and kits are available from ATCC and Cell Biolabs, Inc.). For example, a plasmid containing a gene of interest can be combined with one or more helper plasmids containing, for example, rep genes (e.g., encoding Rep78, Rep68, Rep52, and Rep40) and cap genes (encoding VP1, VP2, and VP3, including the modified VP2 region described herein) and transfected into a recombinant cell. As a result, rAAV particles can be packaged and subsequently purified.

A recombinant AAV may comprise a nucleic acid vector, which may contain, 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., an 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). In this application, a heterologous nucleic acid region comprising a sequence encoding a protein or RNA of interest is referred to as a gene of interest.

Any one of the rAAV particles provided herein may have a capsid protein with 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 genes. In some embodiments, the serotype of the backbone of the VP1 capsid protein of the particle is the same as the serotype of the ITRs. In some embodiments, the serotype of the backbone of the VP1 capsid protein of the particle is the same as the serotype of the Rep gene. In some embodiments, the capsid protein of the rAAV particle includes amino acid mutations that result in improved transduction efficiency.

In some embodiments, the nucleic acid vector includes one or more regions that include sequences that facilitate expression of a nucleic acid (e.g., a heterologous nucleic acid), such as expression control sequences operably 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 polyA tails. Any combination of such control sequences is contemplated herein (e.g., promoters and enhancers).

The final AAV construct may incorporate a sequence encoding a PEgRNA. In other embodiments, the AAV construct may incorporate a sequence encoding a second site nicking guide RNA. In yet other embodiments, the AAV construct may incorporate a sequence encoding a second site nicking guide RNA and a sequence encoding a PEgRNA.

In various embodiments, the PEgRNA and the second-site nicking guide RNA can be expressed from a suitable promoter, such as the human U6 (hU6) promoter, the mouse U6 (mU6) promoter, or other suitable promoter. The PEgRNA and the second-site nicking guide RNA can be driven from the same promoter or different promoters.

In some embodiments, the rAAV construct or composition of the present application is administered to the subject enterally. In some embodiments, the rAAV construct or composition of the present application is administered to the subject parenterally. In some embodiments, the rAAV particle or composition of the present application is administered to the subject subcutaneously, intraocularly, intravitreally, subretinal, intravenously (IV), intracerebroventricularly, intramuscularly, intrathecally (IT), intracisternally, intraperitoneally, by inhalation, topically, or by direct injection into one or more cells, tissues, or organs. In some embodiments, the rAAV particle or composition of the present application is administered to the subject by injection into the hepatic artery or portal vein.
Split PE vector-based strategy

In this aspect, the prime editor can be split at the split site and provided as two halves of a whole/complete prime editor. The two halves can be delivered to a cell (e.g., as expressed proteins or on separate expression vectors) and once in contact within the cell, the two halves form a complete prime editor by the self-splicing action of the inteins of each prime editor half. A split intein sequence can be engineered into each of the encoded prime editor halves to facilitate their trans-splicing within the cell and the concomitant restoration of a complete, functional PE.

These split-intein-based methods overcome several barriers to in vivo delivery. For example, the DNA encoding the prime editor is larger than the rAAV packaging limit and therefore requires specialized solutions. One such solution is to formulate the editor fused to a split-intein pair, which are packaged into two separate rAAV particles that reconstitute functional editor proteins when co-delivered to cells. Several other special considerations that account for the unique features of prime editing have been described, including optimization of second-site nicking targets and proper packaging of the prime editor into viral vectors, including lentiviruses and rAAVs.

In this aspect, the prime editor can be split at the split site and provided as two halves of a whole/complete prime editor. The two halves can be delivered to a cell (e.g., as expressed proteins or on separate expression vectors) and once in contact within the cell, the two halves form a complete prime editor by the self-splicing action of the inteins of each prime editor half. A split intein sequence can be engineered into each of the encoded prime editor halves to facilitate their trans-splicing within the cell and the concomitant restoration of a complete, functional PE.

Figure 66 illustrates one embodiment of a prime editor provided as two PE half proteins. These regenerate the entire prime editor by self-splicing 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 that includes an "N intein" at the C-terminal end of the PE N-terminal half of the complete prime editor (i.e., the N-terminal extein). The "N intein" refers to the N-terminal half of the complete fully formed split intein portion. As used herein, the term "PE C-terminal half" refers to the C-terminal half of a complete prime editor that includes a "C intein" at the N-terminal end of the C-terminal half of the complete prime editor (i.e., the C-terminal extein). When the two half proteins, i.e., the PE N-terminal half and the PE C-terminal half, come into contact with each other, e.g., in a cell, the N-intein and the C-intein undergo a simultaneous process of self-excision and 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, including the complete napDNAbp domain (e.g., Cas9 nickase) and the RT domain. Although not shown in the figures, the prime editor may also include additional sequences, including N- and/or C-terminal NLSs and amino acid linker sequences connecting each domain.

In various embodiments, the prime editor can 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 at a "split site". The "split site" refers to the positioning of the insertion of the split intein sequence (i.e., the N intein and the C intein) between two adjacent amino acid residues on the prime editor. More specifically, the "split site" refers to the positioning of the whole prime editor into two separate halves, each half fused to either the N intein or the C intein motif at the split site. The split site can be at any suitable position on the prime editor fusion protein, but preferably, the split site is positioned to allow the formation of two half proteins that are appropriately sized for delivery (e.g., by an expression vector), and the inteins fused to each half protein at the split site end are available to interact sufficiently with each other when one half protein contacts the other half protein in a cell.

In some embodiments, the division site is located on the napDNAbp domain. In other embodiments, the division site is located on the RT domain. In other embodiments, the division site is located on the linker connecting the napDNAbp domain and the RT domain.

In various embodiments, the split site design requires finding sites for splitting and inserting N- and C-terminal inteins that are both structurally acceptable for the purpose of packaging the two halves of the prime editor domain into two different AAV genomes. In addition, intein residues required for trans-splicing can be incorporated by mutating residues at the N-terminus of the C-terminal extein or by inserting residues that would leave an intein "scar".

Exemplary split constructs of split prime editors containing either SpCas9 nickase or SaCas9 nickase are as follows:

In various embodiments, using SpCas9 nickase (SEQ ID NO: 18, 1368 amino acids) as an example, the split can be between any two amino acids between 1 and 1368. However, preferred splits will be located between the central region of the protein, for example, amino acids 50-1250, or 100-1200, or 150-1150, or 200-1100, or 250-1050, or 300-1000, or 350-950, or 400-900, or 450-850, or 500-800, or 550-750, or 600-700 of SEQ ID NO: 18. In certain exemplary embodiments, the split site can be between 740/741, or 801/802, or 1010/1011, or 1041/1042. In other embodiments, the division sites are 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, Between 88/89, 89/90, or 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 1350-13 68, between any two corresponding residues on an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 99.9% sequence identity to SEQ ID NO:18, or between any two corresponding residues on an SpCas9 variant or equivalent of any of the amino acid sequences of SEQ ID NOs:19-88, or between any two corresponding residues on an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 99.9% sequence identity to any of SEQ ID NOs:19-88.

In various embodiments, the split intein sequence can be engineered from the following intein sequences:

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

In various embodiments, split inteins can be used to separately deliver separate portions of a complete PE fusion protein to a cell, which upon expression in the cell become reconstituted into the complete PE fusion protein by trans-splicing.

In some aspects, 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 a 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 the cell;
Including,
As a result of trans-splicing activity that leads to self-excision of the first and second split intein sequences, the N- and C-terminal fragments are reconstituted as PE fusion proteins within the cell.

The division 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 division site is on the napDNAbp domain.

In yet other embodiments, the cleavage site is on the reverse transcriptase or polymerase domain.

In yet other embodiments, the division site is on the linker.

In various embodiments, the disclosure provides a primed editor 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, e.g., a ligand-dependent intein. Typically, the intein is a ligand-dependent intein, which exerts no or minimal protein splicing activity in the absence of a 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.S.N. 14/004,280, published as U.S. 2014/0065711A1, the entire contents of which are incorporated herein by reference. In addition, using a 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 domain is a napDNAbp domain of reduced size compared to the standard SpCas9 domain of SEQ ID NO:18.

The standard SpCas9 protein is 1368 amino acids in length and has a predicted molecular weight of 158 kilodaltons. As used herein, the term "small size Cas9 variant" refers to a Cas9 variant having at least 1300 amino acids, or at least 1290 amino acids, or less than 1280 amino acids, or less than 1270 amino acids, or less than 1260 amino acids, 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. "Cas9 variants" refers to Cas9 variants, either naturally occurring, engineered, or otherwise, that are less than about 400 amino acids long, or less than 1050 amino acids long, or less than 1000 amino acids long, or less than 950 amino acids long, or less than 900 amino acids long, or less than 850 amino acids long, or less than 800 amino acids long, or less than 750 amino acids long, or less than 700 amino acids long, or less than 650 amino acids long, or less than 600 amino acids long, or less than 550 amino acids long, or less than 500 amino acids long, but greater than about 400 amino acids long, and that retain the required function of the Cas9 protein.

In one embodiment, as illustrated in Example 20, the present disclosure encompasses the following split-intein PE constructs, which are split between residues 1024 and 1025 of standard SpCas9 (SEQ ID NO:18) (or these may be referred to as residues 1023 and 1024, respectively, relative to SEQ ID NO:18 minus Met).

First, the amino acid sequence of SEQ ID NO:18 is shown as follows, indicating the location of the split site between residues 1024 ("K") and 1025 ("S"):

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

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

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

As shown in Example 20, the PE2 (based on SpCas9 of SEQ ID NO:18) construct was split into two separate constructs at positions 1023/1024 (relative to Met minus SEQ ID NO:18) as follows:

SpPE2 splitting in the N-terminal half of 1023/1024

SpPE2 cleavage in the C-terminal half of 1023/1024

The present disclosure also contemplates methods of delivering split intein prime editing factors to cells and/or treating cells with split intein prime editing factors.

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 a 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 the cell;
Including,
As a result of trans-splicing activity that causes self-excision of the first and second split intein sequences, the N- and C-terminal fragments are reconstituted intracellularly as a PE fusion protein.

In some embodiments, the N-terminal fragment of the PE fusion protein fused to the 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 to SEQ ID NO:3875.

In other embodiments, the C-terminal fragment of the PE fusion protein fused to the 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 to SEQ ID NO:3876.

In another aspect, the present disclosure provides a method for editing a target DNA sequence in a cell, comprising:
(a) constructing a first expression vector encoding an N-terminal fragment of a 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 the cell;
Including,
As a result of trans-splicing activity that causes self-excision of the first and second split intein sequences, the N- and C-terminal fragments are reconstituted intracellularly as a PE fusion protein.

In some embodiments, the N-terminal fragment of the PE fusion protein fused to the 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 to SEQ ID NO:3875.

In other embodiments, the C-terminal fragment of the PE fusion protein fused to the 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 to SEQ ID NO:3876.
Delivery of PE ribonucleoprotein complexes

In this aspect, the prime editing factor can be delivered by non-viral delivery strategies involving delivery of the prime editing factor complexed with PEgRNA (i.e., PE ribonucleoprotein complexes) by a variety of 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 drug-enhanced uptake of DNA. Lipofection is described, for example, in U.S. Patent Nos. 5,049,386, 4,946,787; and 4,897,355, and lipofection reagents are commercially available (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Feigner, WO91/17424; WO91/16024. Delivery can be to cells (e.g., in vitro or ex vivo administration) or to 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 those 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); see U.S. Patent 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, each of which is incorporated herein by reference, which discuss approaches for non-viral delivery of ribonucleoprotein complexes:

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.

Figure 68C provides data showing that a variety of the disclosed PE ribonucleoprotein complexes (high concentration PE2, high concentration PE3, and low concentration PE3) can be delivered in this manner.
Delivery of PE by mRNA

Another method that can be used to deliver prime editing factors and/or PEgRNA to cells in which prime editing-based genome editing is desired is by using messenger RNA (mRNA) delivery methods and technologies. Examples of mRNA delivery methods and compositions that can be utilized in the present disclosure include, for example, PCT/US2014/028330, US8822663B2, NZ700688A, ES2740248T3, EP2755693A4, EP2755986A4, WO2014152940A1, EP3450553B1, BR112016030852A2, and EP3362461A1, each of which is incorporated herein by reference in their entirety. Additional disclosures incorporated herein 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 vectors encoding prime editors, the use of RNA as a 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 can be translated directly in the cytoplasm into the desired protein (e.g., prime editor fusion protein) and nucleic acid product (e.g., PEgRNA). However, to be more stable (e.g., resistant to cytoplasmic RNA-degrading enzymes), in some embodiments it is necessary to stabilize the mRNA to improve delivery efficiency. Certain delivery vehicles, such as cationic lipids or polymeric delivery vehicles, can also help protect the transfected mRNA from endogenous RNase enzymes, which could otherwise degrade the therapeutic mRNA encoding the desired prime editor fusion protein. In addition, despite the increased stability of modified mRNAs, delivery of mRNAs, especially mRNAs encoding full-length proteins, to cells in vivo in a manner that allows therapeutic levels of protein production remains a challenge.

With some exceptions, intracellular delivery of mRNA is generally more difficult than that of small oligonucleotides, which requires encapsulation into delivery nanoparticles, in part due to the significantly larger size of mRNA molecules (300-5,000 kDa; ~1-15 kb) compared with other types of RNA (small interfering RNA [siRNA], ~14 kDa; antisense oligonucleotides [ASO], 4-10 kDa).

To reach the cytoplasm, mRNA must cross the cell membrane. The cell membrane is a dynamic and formidable barrier to intracellular delivery. It is made up of a lipid bilayer of primarily zwitterionic and negatively charged phospholipids, where the polar heads of the phospholipids face towards the aqueous environment and the hydrophobic tails form a hydrophobic core.

In some embodiments, the mRNA compositions of the present disclosure include an mRNA (encoding a primed editing factor and/or a PEgRNA), a delivery vehicle, and optionally an agent that facilitates contact with and subsequent transfection of a target cell.

In some embodiments, the mRNA may include one or more modifications that confer stability to the mRNA and are involved in the associated aberrant expression of a protein (e.g., compared to a wild-type or native version of the mRNA). One or more modifications of the wild-type that correct a defect may also be included. For example, the nucleic acids of the invention may include modifications of one or both of the 5' untranslated region or the 3' untranslated region. Such modifications may include the inclusion of a sequence encoding the cytomegalovirus (CMV) immediate early 1 (IE1) gene, a polyA tail, a cap 1 structure, or a partial sequence of human growth hormone (hGH). In some embodiments, the mRNA is modified to reduce mRNA immunogenicity.

In one embodiment, the "prime editing factor" mRNA in the composition of the invention may be formulated with a liposomal import vehicle to facilitate delivery to target cells. Contemplated import vehicles may include one or more cationic lipids, non-cationic lipids, and/or PEG-modified lipids. For example, the import vehicle may include at least one of the following cationic lipids: C12-200, DLin-KC2-DMA, DODAP, HGT4003, ICE, HGT5000, or HGT5001. In embodiments, the import vehicle includes cholesterol (chol) and/or a PEG-modified lipid. In some embodiments, the import vehicle includes DMG-PEG2K. In some embodiments, the transfer vehicle has one of the following lipid formulations: C12-200, DOPE, chol, DMG-PEG2K; DODAP, DOPE, cholesterol, DMG-PEG2K; HGT5000, DOPE, chol, DMG-PEG2K, HGT5001, DOPE, chol, DMG-PEG2K.

The present disclosure also provides compositions and methods useful for facilitating transfection of target cells with mRNA molecules encoding one or more PEs. 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 the low density lipoprotein receptor, thus facilitating recognition of the targeting ligand. A vast number of target cells can be selectively 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, neural cells, cardiac cells, adipocytes, vascular smooth muscle includes cells, cardiac myocytes, skeletal muscle cells, beta cells, pituitary cells, synovial lining cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, reticulocytes, leukocytes, granulocytes, and tumor cells. However, it is not limited thereto.

In some embodiments, the mRNA encoding the PE may optionally have chemical or biological modifications, for example, that improve the stability and/or half-life of such mRNA or improve or otherwise facilitate protein production. Upon transfection, the natural mRNA in the compositions of the present disclosure may decay with a half-life between 30 minutes and days. The mRNA in the compositions of the present disclosure may retain at least some ability to be translated, thereby resulting in a functional protein or enzyme. Thus, the present invention provides compositions comprising stabilized mRNA and methods of administering the same. In some embodiments, the activity of the mRNA is prolonged for 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 biweekly basis, or more preferably on a monthly, bimonthly, quarterly, or yearly basis. The extended or prolonged activity of the mRNA of the present invention is directly related to the quantity of protein or enzyme that results from such mRNA. Similarly, the activity of the compositions of the present disclosure may be further prolonged or prolonged by modifications made to improve or enhance translation of the mRNA. Furthermore, the amount of functional protein or enzyme produced by a target cell is a function of the amount of mRNA delivered to the target cell and the stability of such mRNA. To the extent that the stability of the mRNA of the present invention can be improved or enhanced, the half-life, activity of the resulting protein or enzyme, and dosing frequency of the composition can be further extended.

Thus, in some embodiments, the mRNA in the compositions of the present disclosure includes at least one modification that confers increased or enhanced stability to the nucleic acid, including, for example, improved resistance to nuclease digestion in vivo. The terms "modified" and "modified," as such terms relate to the nucleic acids provided herein, preferably include at least one alteration that enhances stability and makes the mRNA more stable (e.g., resistant to nuclease digestion) than a wild-type or naturally occurring version of the mRNA. The terms "stable" and "stability," as such terms relate to the nucleic acids of the present invention, particularly for mRNA, refer to, for example, increased or enhanced resistance to degradation by nucleases (i.e., endonucleases or exonucleases) that would normally degrade such mRNA. Increased stability can include, for example, less susceptibility 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 a longer half-life relative to their naturally occurring unmodified counterparts (e.g., wild-type versions of mRNA). Also contemplated by the terms "modification" and "modified" as such terms relate to the mRNA of the present invention are alterations that improve or enhance translation of the mRNA nucleic acid. For example, they include the inclusion of sequences (e.g., Kozak consensus sequences) that function in the initiation of protein translation. (Kozak, M., Nucleic Acids Res 15(20):8125-48(1987)).

In some embodiments, the mRNAs used in the compositions of the present disclosure have undergone chemical or biological modifications to make them more stable. Exemplary modifications of mRNA include base depletion (e.g., by deleting one nucleotide or substituting another) or modification of the bases, e.g., chemical modification of the bases. As used herein, the phrase "chemical modification" includes modifications that introduce a different chemistry than that found in naturally occurring mRNA, e.g., covalent modifications, e.g., the introduction of modified nucleotides (e.g., inclusion of nucleotide analogs or pendant groups not naturally found in such mRNA molecules).

Other suitable polynucleotide modifications that may be incorporated into the mRNA encoding the PE used in the compositions of the present disclosure include 4'-thio modified bases: 4'-thioadenosine, 4'-thioguanosine, 4'-thiocytidine, 4'-thiouridine, 4'-thio-5-methyl-cytidine, 4'-thiopseudouridine, and 4'-thio-2-thiouridine, pyridin-4-one ribonucleosides, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-5-methyl-cyt ... Thiopseudouridine, 2-thiopseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyluridine, 1-propynylpseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thiouridine, 1-taurinomethyl-4-thiouridine, 5-methyl-uridine, 1-methyl-pseudouridine uridine, 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-thiodihydrouridine, 2-thiodihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thiouridine, 4-methoxy-pseudouridine, 4-methoxy-2-thiopseudouridine , 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thiocytidine, 2-thio-5-methyl-cytidine, 4-thiopseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine doisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methylzebularine, 5-aza-2-thiozebularine, 2-thiozebularine, 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-Threonylcarbamoyladenosine, 2-methylthio-N6-threonylcarbamoyl adenosine, N6,N6-dimethyl adenosine, 7-methyladenine, 2-methylthioadenine, and 2-methoxy-adenine, inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thioguanosine, 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, sN2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thioguanosine, N2-methyl-6-thioguanosine, and N2,N2-dimethyl-6-thioguanosine, and combinations thereof. The term modification also includes, for example, the incorporation of non-nucleotide linkages or modified nucleotides onto the mRNA sequences of the invention (e.g., modification of one or both of the 3' and 5' ends of an mRNA molecule encoding a functional protein or enzyme). Such modifications include adding bases to the mRNA sequence (e.g., inclusion of a polyA tail or a longer polyA tail), altering the 3'UTR or 5'UTR, complexing the mRNA with an agent (e.g., a protein or a complementary nucleic acid molecule), and inclusion of elements that change the structure of the mRNA molecule (e.g., which form secondary structures).

In some embodiments, the mRNA encoding PE includes a 5' cap structure. The 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; then, guanosine triphosphate (GTP) is added to the terminal phosphate by a guanylyltransferase, resulting in a 5'5'5 triphosphate linkage; then, the 7-nitrogen of guanine is 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 contain a 7-methylguanosine linked to the 5' end of the first transcribed nucleotide via a triphosphate bridge, 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 guanylyltransferase. Addition of the cap to the 5' end of the RNA occurs immediately after the initiation of transcription. The terminal nucleoside is typically guanosine and is in the opposite orientation to all other nucleotides, i.e., G(5')ppp(5')GpNpNp.

Additional cap analogs include, but are not limited to, chemical structures selected from the group consisting of m7GpppG, m7GpppA, m7GpppC; unmethylated cap analogs (e.g., GpppG); dimethylated cap analogs (e.g., m2,7GpppG), trimethylated cap analogs (e.g., m2,2,7GpppG), dimethylated symmetric 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. PolyA or polyU tails are thought to stabilize natural messenger and synthetic sense RNA. Thus, in some embodiments, a long polyA or polyU tail can be added to an mRNA molecule, thus making the RNA more stable. PolyA or polyU tails can be added using a variety of art-recognized techniques. For example, a long polyA tail can be added to synthetic or in vitro transcribed RNA using polyA polymerase (Yokoe, et al. Nature Biotechnology. 1996;14:1252-1256). Transcription vectors can also encode long polyA tails. Additionally, polyA tails can be added by transcription directly from PCR products. Poly A can also be ligated to the 3' end of the sense RNA by 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 the polyA or polyU tail can be at least about 10, 50, 100, 200, 300, 400, at least 500 nucleotides. In some embodiments, the polyA tail at the 3' end of the 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, the mRNA includes a 3' polyC tail structure. A suitable polyC tail at the 3' end of the mRNA typically includes 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 can be in addition to or replace the poly-A or poly-U tail.

The mRNA encoding the PE according to the present disclosure may be synthesized according to any of a variety of known methods. For example, the mRNA according to the present invention may be synthesized by 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 inhibitors. Stringent conditions will vary according to the particular application.

In embodiments involving mRNA delivery, the ratio of mRNA encoding the PE fusion protein to PEgRNA can 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. Using prime editing to identify off-target edits in an unbiased manner
Like other genome editing elements, there is some risk that PE may introduce its programmed genetic alteration at unintended sites on the genome, i.e., "off-target" sites. However, currently, there is no method described for detecting off-target editing by prime editing elements. Such a method would allow identification of possible sites of off-target editing using prime editing elements.

The key concept of this aspect is the idea of using prime editing to insert the same adaptor sequences and/or primer binding sites at on-target and off-target sites using the same PEgRNA as a template, enabling rapid identification of genomic off-target modification sites of napDNAbp nucleases or prime editors. This method is distinguished from other techniques for identifying nuclease off-target sites because the adaptor and/or primer binding sequences are inserted in the same event as DNA binding and nicking by napDNAbp, simplifying downstream processing.

Figure 33 illustrates the basic principle of off-target identification. The figure shows a schematic diagram of PEgRNA design for primer binding sequence insertion and primer-bound insertion onto genomic DNA using prime editing to determine off-target editing. In this embodiment, prime editing is performed in a living cell, tissue, or animal model. As a first step, a suitable PEgRNA is designed. The top schematic diagram shows an exemplary PEgRNA that can be used in this aspect. The spacer on the PEgRNA (labeled "protospacer") is complementary to one of the strands of the genomic target. The PE:PEgRNA complex (i.e., the PE complex) incorporates a single-stranded 3' end flap at the nick site, which contains the encoded primer binding sequence and a homologous region (encoded by the homologous arm of the PEgRNA) that is complementary to the region just downstream of the site to be cut (by red). By flap invasion and DNA repair/replication processes, the synthesized strand becomes incorporated onto the DNA, thereby incorporating the primer binding site. This process may occur at the desired genomic target, but also at other genomic sites that may interact with the PEgRNA in an off-target manner (i.e., due to the complementarity of the spacer region to other genomic sites that are not the intended genomic site, the PEgRNA guides the PE complex to other off-target sites). Thus, primer binding sequences may be incorporated not only at the desired genomic target, but also at other off-target genomic sites on the genome. To detect the insertion of these primer binding sites at both the intended genomic target site and the off-target genomic site, the genomic DNA (post-PE) may be isolated, fragmented, and ligated to adapter nucleotides (shown in red). Then, PCR may be performed with PCR oligonucleotides that anneal to the adapter and the inserted primer binding sequence to amplify the on-target and off-target genomic DNA regions where the primer binding sites were inserted by PE. High-throughput sequencing and sequence alignment may then be performed to identify the insertion point of the primer binding sequence inserted by PE at either the on-target site or the off-target site.

Thus, FIG. 33 illustrates one aspect of identifying off-target editing sites when editing in live cells, tissue culture, or animal models. To perform this method, a PEgRNA is generated that has the same spacer as the final desired prime editor (and the same primer binding site sequence as the final desired editor, if prime editing off-targets are being examined), but includes the necessary sequences for incorporating an adapter or primer binding site after reverse transcription by prime editing. In vivo editing using a nuclease fused to the prime editor or RT is performed, and genomic DNA is isolated. The genomic DNA is fragmented by enzymatic or mechanical means, adding different adapters at the sites of DNA fragmentation. PCR is used to amplify from one adapter to the adapter incorporated by the PEgRNA. The resulting product is deep sequenced to identify all modified sites.

In another aspect, evaluation of off-target editing by PE can be performed in vitro. In this aspect, PE is used during in vitro modification of genomic DNA identification of off-target editing sites using in vitro modification of genomic DNA. To perform this method, a purified prime editor fusion protein and a ribonucleoprotein (RNP) of PEgRNA (i.e., a PE complex) is assembled that is configured to incorporate an adapter or primer binding sequence at the target site, but is otherwise identical to the PEgRNA of interest. This RNP (i.e., the PE complex) is incubated with extracted genomic DNA before or after fragmentation of the DNA. After fragmentation, different adapter sequences are ligated to the ends of the fragment DNA. PCR is used to amplify the genomic sites that span the inserted adapter sequence (i.e., inserted by EP) and the adapters ligated to the ends of the fragments. High-throughput sequencing between the adapter 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, as cellular DNA repair will not erase the reverse-transcribed DNA adapters added by the primed editor.

These methods can be used to identify off-target edits for any primed editor or any genome editor that uses a guide RNA to recognize the target site to be cut (most Cas nucleases).

These methods can be applied to all genetic diseases for which genome editing elements are being considered for treatment.

を包含するが、これらに限定されない。 Exemplary adaptor and/or primer binding sequences that may be incorporated by PE are:

These include, but are not limited to:

These adaptor and/or primer binding sequences may also be used in the ligation step following genomic DNA fragmentation as outlined above.

Exemplary PEgRNA designs and their editing target loci illustrating the use of the methods described herein to assess off-target editing are as follows:

K. Use of Prime Editing to Insert Inducible Dimerization Domains
The prime editors described herein can also be used to incorporate a dimerization domain into one or more protein targets. The dimerization domain can facilitate inducible control of the activity associated with dimerization of one or more protein targets through a linking moiety (e.g., a small molecule, peptide, or protein) that binds in a bispecific manner. In various aspects, when the dimerization domains are incorporated on different proteins (e.g., the same type or different proteins), each binds to the same bispecific moiety (e.g., a bispecific small molecule, peptide, or polypeptide having at least two regions that bind separately to the dimerization domain), thereby causing the proteins to dimerize due to their common interaction with the bispecific ligand. In this manner, the bispecific ligand functions as an "inducer" of dimerization of the two proteins. In some cases, the bispecific ligand or compound will have two targeting moieties that are the same. In other embodiments, the bispecific ligand or compound will have a targeting moiety that is different from the other, respectively. A bispecific ligand or compound with the same two targeting moieties will be capable of targeting the same dimerization domain incorporated on different protein targets. A bispecific ligand or compound with different targeting moieties will be capable of targeting different dimerization domains incorporated on different protein targets.

The term "dimerization domain" as used herein refers to a ligand binding domain that binds to a binding portion of a dual-specific ligand. A "first" dimerization domain binds to a first binding portion of a dual-specific ligand, and a "second" dimerization domain binds to a second binding portion of the same dual-specific ligand. When a first dimerization domain is fused to a first protein (e.g., by PE as discussed herein) and a second dimerization domain is fused to a second protein (e.g., by PE as discussed herein), the first and second proteins dimerize in the presence of the dual-specific ligand, and the dual-specific ligand has at least one portion that binds to the first dimerization domain and at least another portion that binds to the second dimerization domain.

The term "dual-specific ligand" or "dual-specific moiety" as used herein refers to a ligand that binds to two different ligand-binding domains. In various embodiments, the bispecific moiety is itself a dimer of two identical or two different chemical moieties, each of which binds tightly and specifically to a dimerization domain. In some embodiments, the ligand is a small molecule compound or a peptide or polypeptide. In other embodiments, the ligand-binding domain is a "dimerization domain" that can be incorporated onto a protein as a peptide tag. In various embodiments, two proteins, each of which contains the same or different dimerization domains, can be induced to dimerize by binding of each dimerization domain to a dual-specific ligand. These molecules can also be referred to as "chemical inducers of dimerization" or CIDs. In addition, dual-specific ligands can be prepared by coupling together (e.g., by standardized chemical linkages) two identical moieties or two different moieties, each of which binds tightly and specifically to a dimerization domain.

In various aspects, the dimerization domains incorporated by the PE can be the same or different.

For example, the dimerization domain can be FKBP12, which has the following amino acid sequence:

In another example, the dimerization domain can be a mutant of FKBP12, termed FKBP12-F36V, a mutant of FKBP12 with an engineered hole that binds to the synthetic bump FK506 mimic (2, Figure 3) ¹⁰⁷ :

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

In various embodiments, the amino acid sequences of these dimerization domains can be altered to optimize binding to the native target or improve binding orthogonality. The nucleic acid sequences of genes encoding small molecule binding proteins can be altered to optimize the efficiency of the PE process, for example, by reducing PEgRNA secondary structure.

Other examples of suitable dimerization domains and corresponding small molecule compounds that bind thereto are provided below. Note that the corresponding small molecule compound can be coupled (e.g., by a chemical linker) to a second small molecule compound (either the same compound or a different compound) to form a dual-specific ligand that can bind to two dimerization domains. In some cases, such as FK506 and cyclosporin A, the respective dimerization (e.g., FK506-FK506 or cyclosporin A-cyclosporin A) reduces or eliminates the immunosuppressive activity of the monomeric compound.

Other examples of naturally occurring bifunctional molecules and their dual target receptors are as follows: Prime editing can be used to incorporate a dual target receptor into a different protein. Once a different protein has been modified by PE to contain a bifunctional molecule receptor, the bifunctional molecule can be introduced, thereby causing dimerization of the proteins modified to contain different dimerization domains. Examples of pairing of (1) biofunctional molecules and (2) their dual target receptors are as follows:

Examples of other bifunctional molecules that can be used in this aspect of prime editing are:

Synstab A:

Paclitaxel:

Discodermolide:

GNE-0011

ARV-825, and

。 dBET1

.

Synstab A, paclitexel, and discodermolide are microtubule stabilizing agents. Therefore, these compounds can be used to dimerize proteins that have been modified with PE to contain microtubule proteins. GNE-0011, ARV-825, and dBET1 contain BRD4- and CRBN-binding motifs. Therefore, these compounds can be used to dimerize proteins that have been modified with PE to contain these targeting domains.

A PEgRNA for incorporating a dimerization domain may comprise the following structure (with reference to FIG. 3D):
5'-[spacer]-[gRNA core]-[extension arm]-3', wherein the extension arm comprises 5'-[homology arm]-[editing template]-[primer binding site]-3'; or
The structure is 5'-[extension arm]-[spacer]-[gRNA core]-3', where the extension arm comprises 5'-[homology arm]-[editing template]-[primer binding site]-3', and in both configurations the "editing template" comprises the nucleotide sequence of the dimerization domain.

In one example, a PEgRNA for insertion of the FKBP12 dimerization domain at the C-terminal end of the human insulin receptor (spacer underlined, gRNA core plain, flap homology bold, FKBP12 insertion italic, annealing region bold italic):

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

を包含するが、これらに限定されない。 The target proteins for incorporating the dimerization domain are not particularly limited; however, it is advantageous that their dimerization in the presence of a dual-specific ligand (once modified by PE) results in some beneficial biological effect, such as signal pathways, reduced immune responsiveness, etc. In various aspects, the target proteins to be dimerized by the PE-dependent incorporation of the dimerization domain can be the same protein or different proteins. Preferably, the proteins, when dimerized, trigger one or more downstream biological cascades, such as signaling cascades, phosphorylation, etc. Exemplary target proteins for which PE can be used to incorporate the dimerization domain are:

These include, but are not limited to:

In one aspect, the prime editing factors described herein can be used to incorporate sequences encoding dimerization domains onto one or more genes encoding target proteins of interest in living cells or patients. This can be referred to as a "prime editing-CID system," where the CID is a bispecific ligand that induces dimerization of target proteins, each fused to a dimerization domain incorporated by PE. This editing alone should have no physiological effect. Upon administration of the bispecific ligand, typically a dimeric small molecule that can simultaneously bind to two dimerization domains, each fused to a copy of the target protein, the bispecific ligand causes dimerization of the target protein. This target protein dimerization event then induces a biological signaling event, such as erythropoiesis or insulin signaling. Described herein is a new method for placing dimerization-induced biological processes, such as receptor signaling, under the control of convenient small molecule drugs (i.e., bispecific ligands) by genomic incorporation of genes encoding small molecule binding proteins (i.e., dimerization domains) via prime editing.

Protein dimerization is a ubiquitous biological process. Of note, homodimerization of many membrane-bound receptors is known to initiate signaling cascades, often with profound biological consequences. Several natural small molecule products approved for use as drugs act as chemical inducers of protein dimerization as part of their mechanism of action92. For example, FK506 tightly binds FKBP12, and the resulting small molecule-protein complex then binds to the phosphatase ^calcineurin , thereby inhibiting certain steps of T cell receptor ^signaling93 . Similarly, cyclosporin A induces dimerization of cyclophilin and calcineurin, and rapamycin induces dimerization of FKBP and ^mTOR93,94 .

In one embodiment, synthetic chemical inducers of dimerization have also been developed, taking advantage of the selective high affinity binding of FK506:FKBP12 and cyclosporin A:cyclophilin small molecule:protein binding interactions. In one example, a two-unit small molecule of FK506, designated FK1012, was shown to achieve signal transduction when the cytoplasmic domain of a signaling receptor was tagged with ^FKBP12.95 Since then, chemical inducers of dimerization (CIDs) have been used to control several signaling ^{pathways.96-103}

Although a useful tool for studying biological processes, one challenge facing synthetic CID for therapeutic applications is the difficulty of introducing FKBP12 or cyclophilin-targeted protein chimeras into patients.

This disclosure brings together two concepts to generate previously inaccessible therapeutic processes. The first concept is prime editing, described herein, which allows precise genome editing, including targeted insertions, in living cells. The second concept is chemically induced dimerization, a powerful tool that has enabled small molecule control of signaling and oligomerization processes in cell culture.

Specific cases have been identified in which chemical control of protein dimerization could have beneficial therapeutic effects.

The insulin receptor is a heterotetrameric transmembrane protein that responds to insulin binding to the extracellular domain by phosphorylation of the cytoplasmic kinase ^domain104 . An engineered chimeric protein consisting of a membrane-localized component, the C-terminal kinase domain of the insulin receptor, and three copies of FKBP12, responds to FK1012 in cell culture and initiates an insulin response99. Similarly, it is predicted that fusion of FKBP12 to the C-terminal end of the kinase domain of the native insulin receptor in patient cells should allow 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 diabetes) or who respond poorly to insulin (e.g., type 2 diabetes).

In addition, erythropoietin stimulates erythrocyte proliferation by binding to the erythropoietin receptor (EpoR) and induces either dimerization or conformational changes in preformed receptor dimers, which result in activation of the Jak/STAT signaling ^cascade105 . It has been demonstrated that FK1012-induced oligomerization of the membrane-anchored cytoplasmic domain of FKBP12-tagged EpoR is sufficient to initiate Jak/STAT signaling cascade signals and promote cell proliferation106. It is expected that fusing FKBP12 to native EpoR by prime editing in patient cells will allow ^FK1012 -induced control of erythrocyte proliferation (erythropoiesis). This system can be used to induce erythrocyte growth in anemic patients. FK1012-inducible EpoR can also be used as an in vivo selection marker for blood cells that have undergone ex vivo manipulation.

In principle, any receptor tyrosine kinase is a potential target for prime editing-CID therapeutics. The table below contains ^a list of all receptor tyrosine kinases in the human genome.

The prime editing-CID system has a number of advantages. One such advantage is that it can replace endogenous ligands, which are typically proteins that present complexities in manufacturing, cost, delivery, production, or storage, with drug-like small molecules. They can be administered orally instead of by IV or injection, can be readily prepared from FDA-approved drugs (or are already drugs themselves), and do not incur specialized production or storage costs typically associated with protein drugs. Another advantage is that the editing should have no physiological effect alone. The amount of target protein dimerization can be controlled by dosing the small molecule CID. Furthermore, target protein dimerization is readily and rapidly reversible by adding a monomeric form of the CID. Yet another advantage is that in cases 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 inducible receptor activation only in specific areas.

If the editing efficiency of prime editing is high enough that two separate editing events can occur at high levels, it may also be possible to tag the two proteins of interest with different small molecule binding domains (e.g., FKBP and cyclophilin) and induce heterodimerization with a small molecule heterodimer (e.g., FK506-cyclosporine A dimer).

Fusion of FKBP12 or other small molecule binding proteins to native proteins is typically achieved by overexpression from plasmids in tissue culture. Subsequent chemically induced dimerization has been demonstrated to induce phenotypic changes in cells bearing the fusion protein.

The following references are cited above in Section G and are incorporated herein by reference:

L. Using Prime Editing to Record Cellular Data
Prime editing factors and the resulting genome modifications can also be used to study and record cellular processes and development. For example, by providing a cell with a first nucleic acid sequence encoding a fusion protein having a nucleic acid programmable DNA binding protein (napDNAbp) and reverse transcriptase, and providing a cell with at least a second nucleic acid sequence encoding a PEgRNA, the prime editing factors described herein can be used to record the presence and duration of a stimulus to a cell. Either the first, second, or both nucleic acid sequences are operably linked to an inducible promoter that is responsive to a cellular stimulus, so that it induces the expression of the fusion protein and/or the PEgRNA, thereby causing modification of a target sequence in the cell.

The prime editors described herein can also be used for cell barcoding and lineage tracing. For example, by barcoding each cell with a unique genomic barcode, the prime editors can help define a cell lineage map by allowing the construction of a lineage tree based on modifications made to one or more target sequences. Starting from progenitor cells, the prime editor system can make it possible to build a cell fate map for a single cell in a whole organism. This can be deciphered by analyzing modifications made to one or more target sequences. A method for tracing the lineage of a cell can include providing a nucleic acid encoding a fusion protein with napDNAbp and reverse transcriptase, and providing at least one second nucleic acid encoding a PEgRNA. A unique cell barcode can be generated using the fusion protein and the PEgRNA to generate one or more modifications to one or more target sequences, thereby allowing the lineage of any cell that develops from the initial cell to be traced using the unique cell barcode. The use of the prime editors for both cell data recording and lineage tracing is further described in Example 13.

Prime editors can perform both lineage tracing and cell signal recording by modifying genomic target sequences or incorporated pre-designed sequences. Prime editors use synthetic fusion proteins containing a Cas9 nickase fragment (including but not limited to the SpCas9 H840A variant) and a reverse transcriptase domain in conjunction with an engineered prime editing guide RNA (PEgRNA). Together, these components target specific genomic sequences or incorporated pre-designed sequences and incorporate predetermined edits. Because the PEgRNA specifies both the target genomic sequence and the editing outcome, highly specific and controlled genome modifications can be achieved simultaneously in the same cell using multiple PEgRNAs. 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 in cells.

The use of prime editing factors for cellular data recording may 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 lifespan. The cellular data recording system may include a fusion protein consisting of napDNAbp (e.g., Cas9 domain) and reverse transcriptase operably linked to a promoter that induces expression of the fusion protein to induce changes by generating targeted and sequence-defined genomic insertions, deletions, or mutations in response to a cellular stimulus or change. In contrast to digital memory devices that store information (e.g., the presence or absence of a stimulus) as one of two distinct states (i.e., "on" or "off"), these cellular data recorders may induce permanent marks on cellular DNA in a manner that reflects both the strength (i.e., amplitude) and duration of one or more stimuli. Thus, in some aspects, the cellular data recording system has the ability to record multiple cellular states simultaneously. Examples include small molecules, proteins, peptides, amino acids, metabolites, inorganic molecules, organometallic molecules, organic molecules, drugs or drug candidates, sugars, lipids, metals, nucleic acids, molecules that arise during activation of endogenous or exogenous signal cascades, light, heat, sound, pressure, mechanical stress, shear stress, or exposure to viruses or other microorganisms. These cellular data recorders can use sequencing technologies (e.g., high-throughput sequencing) to measure readouts (e.g., changes in cellular DNA) and do not rely on large cell populations for both recording the stimulus or readout of the change(s) in cellular DNA induced by the stimulus.

In general, the cell data recorder system provided herein for use in a cell includes a fusion protein consisting of napDNAbp and reverse transcriptase, with the nucleic acid sequence encoding the fusion plasmid operably linked to a promoter (e.g., an inducible promoter or a constitutive promoter). When a stimulus is present or a change in the cell state occurs, the stimulus induces expression of the fusion protein. One or more nucleic acids encoding at least one PEgRNA that associates with the napDNAbp and directs the napDNAbp or the fusion protein to a target sequence are also present in the cell (i.e., the PEgRNA is complementary to the target sequence). The nucleic acid encoding the PEgRNA may also, or alternatively, be operably linked to a promoter (e.g., an inducible promoter or a constitutive promoter). Under the correct stimulus or the correct set of stimuli, both the fusion protein and the PEgRNA are expressed by the cell, and the PEgRNA associates with and directs the fusion protein to the target sequence. This target sequence records the activity of the prime editor, thereby recording the presence of a stimulus or set of stimuli or a change in the cell state. More than one PEgRNA sequence may also be present in a cell, and these additional PEgRNA sequences that may direct the fusion protein to a different target sequence may each be operably linked to a promoter that senses the presence of a different stimulus, allowing complex cellular data recorder systems to be constructed for 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 cellular data recorder system (e.g., the fusion protein and the PEgRNA) may be constitutively expressed by the cell. Exemplary components of the cellular data recorder system for use in the compositions are described herein. Additional suitable combinations of the components provided herein will be apparent to one of skill in the art based on this disclosure and knowledge of the art. Thus, are encompassed within the scope of this disclosure.

Repeated modifications of DNA targets, which can be sequenced by targeted amplicon sequencing and/or RNA sequencing (which is particularly valuable for single-cell recording experiments), can be used to record a number of important biological processes, including activation of signal cascades, metabolic states, and cell differentiation programs. Connecting internal and external cellular signals to sequence modifications on the genome is possible for any signal for which a signal-responsive promoter 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 occurring during the activation of an endogenous or exogenous signal cascade, light, heat, sound, pressure, mechanical stress, shear stress, or a virus or other microorganism, a change in pH, or a change in redox state. In some embodiments, the stimulus is 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 signal molecule occurring during an activated signal cascade (e.g., beta-catenin occurring during an activated Wnt signal cascade). Additional promoters that detect signal molecules can be generated to induce expression of a nucleic acid sequence operably linked to the promoter. For example, promoters that encode endogenous pathways including immune response (IL-2 promoter), cAMP response 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, aryl hydrocarbon receptor, or 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 one of skill in the art based on this disclosure and knowledge in the art. It is within the scope of this disclosure.

Prime editors can also be used to trace cell lineage. To track individual cells, repeated sequence modifications can be used to generate unique cell barcodes. The array of barcodes, their order, and size can all be used to infer cell lineage. For example, the insertion of homologous sequences (i.e., sequences 3' to the positioning of the Cas9 nick), specifically homologous sequences with associated barcodes, appears to be a particularly useful lineage prime editor strategy. These systems can be designed such that sequential rounds of editing result in the insertion of barcodes from the PEgRNA cassette that cannot be modified by other PEgRNA editing events in the same cell. Barcoding systems can utilize multiple barcodes, which can be associated with a given stimulus. The system can preserve many of the target protospacers, but alter the seed sequence, PAM, and downstream adjacent nucleotides. This allows multiple signals to be connected to one editing locus without significant redesign of the PEgRNA to be used. The strategy can allow multiple barcode insertions at a single locus in response to multiple cellular stimuli (either internal or external). It can allow recording of the intensity, duration, and order of as many signals as there are unique barcodes (these can be designed with multiple N nucleotides to generate 4^N possible barcodes; for example, a 5 nt barcode would allow recording of 4^5 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 the prime editors described herein can also be used to control the subcellular localization and modification state of biomolecules such as DNA, RNA, and proteins. Certain biological functions, such as transcriptional control, cellular metabolism, and signal transduction cascades, are carefully orchestrated in specific locations within cells. The ability to transport proteins to these and other specific cellular compartments can provide the opportunity to alter a number of biological processes.

Thus, prime editing can be used to incorporate genetically encoded handles that would allow altered modification states and subcellular transport of biomolecules (e.g., proteins, lipids, sugars, and nucleic acids) via genetically encoded signals. In various embodiments, the target biomolecule of a drug therapy mediated by a prime editor is DNA. For example, DNA can be modified by incorporating a number of DNA sequences that change the accessibility of the target locus. This can lead to either increased or decreased transcription of the desired sequence. In other embodiments, the target biomolecule of a drug therapy mediated by a prime editor is RNA. For example, the activity of the RNA can be modified by changing its cellular localization, interaction partners, structural dynamics, or folding thermodynamics. In yet other embodiments, the target biomolecule of a drug therapy mediated by a prime editor is a protein. Proteins can be modified to impact post-translational modifications, protein motifs can be incorporated to change the subcellular localization of the protein, or proteins can be modified to either create or destroy their ability to exist in protein-protein complexation events.

This application of prime editing can be further described in Example 14.

DNA modification
One target biomolecule for PE-mediated modification is DNA. DNA can be modified to incorporate any number of DNA sequences that change the accessibility of the target locus. Chromatin accessibility controls gene transcription output. Incorporation of a mark to recruit chromatin compacting enzymes should decrease the transcription output of neighboring genes, while incorporation of sequences associated with chromatin opening should make the region more accessible, which in turn should increase transcription. Incorporation of more complex sequence motifs that mimic native regulatory sequences should provide more subtle and biologically sensitive control of different epigenetic reader, writer, or eraser enzymes than currently available dCas9 fusions. Typically, it is a tool that incorporates a large number of possible single-type marks without a specific biological ancestry. As demonstrated in the burgeoning field of 3D genome architecture, incorporation of sequences that would bring two loci into close proximity or bring loci into contact with the nuclear membrane should also alter the transcription output of those loci.

RNA Modifications RNAs can also be modified to alter their activity by changing their cellular localization, interaction partners, structural dynamics, or folding thermodynamics. Incorporation of motifs that would cause translational interruption or frameshifting could alter the abundance of mRNA species by 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 changes in the ratio of protein translation products. This can be used to alter many biological pathways. For example, shifting the balance of mitochondrial versus nuclear DNA repair proteins would alter the resilience of different cancers to chemotherapy agents. Furthermore, RNA can be modified with sequences that allow binding to novel protein targets. A number of RNA aptamers have been developed that bind cellular proteins with high affinity. Incorporation of one of these aptamers can be used either to sequester different RNA species by binding to a protein target that would prevent their translation, biological activity, or to bring the RNA species to a specific subcellular compartment. Biomolecular degradation is another class of localized modification.

For example, RNA methylation is used to control RNA in cells. A consensus motif for methylation can be introduced onto the target RNA coding sequence by PE. RNA can also be modified to include sequences that direct nonsense-mediated decay mechanisms or other nucleic acid metabolic pathways to degrade the target RNA species, which will alter the pool of RNA in the cell. In addition, RNA species can be modified to alter their aggregation state. Sequences to generate RNA tangles can be incorporated onto a single RNA or multiple RNAs of interest, which will make them ineffective substrates for translation or signaling.

Protein Modification Modification of proteins by post-translational modifications (PTMs) also represents an important class of biomolecular manipulations that can be performed by PE. Like RNA species, altering the abundance of proteins in cells is an important capability of PE. Open reading frames can be edited to incorporate stop codons. This will eliminate the full-length product resulting from the edited DNA sequence. Alternatively, peptide motifs can be incorporated that cause the rate of protein degradation to be altered for the target protein. Incorporation of degradation tags onto the gene body can be used to alter the abundance of proteins in cells. Moreover, introduction of small molecule-induced degrons can allow for temporal control of protein degradation. This can have important implications for both research and therapeutics, since researchers can readily assess whether small molecule-mediated therapeutic protein degradation of a given target is a promising therapeutic strategy. Protein motifs can also be incorporated to change the subcellular localization of proteins. Amino acid motifs can be incorporated to preferentially target proteins to any number of subcellular compartments, including the nucleus, mitochondria, cell membrane, peroxisomes, lysosomes, proteasomes, exosomes, and others.

Incorporating or disrupting motifs modified by PTM mechanisms can alter protein post-translational modifications. Phosphorylation, ubiquitination, glycosylation, lipid modifications (e.g., farnesylation, myristoylation, palmitoylation, prenylation, GPI anchors), hydroxylation, methylation, acetylation, crotonylation, sumoylation, disulfide bond formation, side chain bond cleavage events, polypeptide backbone cleavage events (proteolysis), and several other protein PTMs have been identified. These PTMs alter protein function, often by altering subcellular localization. Indeed, kinases often activate downstream signaling cascades through phosphorylation events. Removal of the target phosphosite would prevent signal transduction. The ability to site-specifically excise or incorporate any PTM motif while retaining full-length protein expression would be an important advance for both basic research and therapeutics. PE's sequence incorporation range and targeting window make it well suited to the broad PTM modification space.

Removal of the lipid modification site should prevent transport of the protein to the cell membrane. A major limitation of current therapeutics targeting post-translational modification processes is their specificity. Farnesyltransferase inhibitors are being actively tested for their ability to eliminate KRAS localization to the cell membrane. Unfortunately, blanket inhibition of farnesylation is accompanied by numerous off-target effects that prevent the widespread use of these small molecules. Similarly, specific inhibition of protein kinases by small molecules can be very difficult due to the large size of the human genome and the similarity between various kinases. PE offers a potential solution to this specificity problem because it allows inhibition of target protein modification by excision of the modification site instead of blanket enzyme inhibition. For example, removal of the lipid modification peptide motif on KRAS would be a targeted approach that could be used instead of farnesyltransferase inhibition. This approach is the functional opposite of inhibiting target protein activity by incorporating lipid targeting motifs onto proteins that are not designed to be membrane bound.

PE can also be used to instigate protein-protein complexation events. Proteins often function in complexes to carry out their biological activities. PE can be used to either create or destroy the ability of proteins to reside in these complexes. To eliminate complexation events, amino acid substitutions or insertions on the protein:protein interface can be incorporated to disfavor complexation. SSX18 is a protein component of the BAF complex, an important histone remodeling complex. Mutations in SSX18 drive synovial sarcoma. PE can be used to incorporate side chains that prevent SSX18 from binding to its protein partners in the complex, preventing its oncogenic activity. PE can also be used to remove pathogenic mutations to restore the WT activity of this protein. Alternatively, PE can be used either to keep proteins in their native complexes or to pull them into interactions that are unrelated to their native activity, inhibiting their activity. Forming complexes that maintain one interaction state over another may represent an important therapeutic modality. Altering protein:protein interfaces to decrease the Kd of an interaction will keep the proteins attached to each other longer. Protein complexes can have multiple signaling complexes, such as n-myc, which drives the neuroblastoma signaling cascade in disease but is otherwise involved in healthy transcriptional control in other cells. PE can be used to incorporate mutations that drive n-myc association with healthy interacting partners and decrease its affinity for oncogenic interacting partners.
References cited in Section I
Each of the following references is incorporated herein by reference.

N. Improved design aspects of PEGRNA for prime editing
In other embodiments, the prime editing system may include the use of PEgRNA design and strategies that can improve prime editing efficiency.These strategies seek to overcome some of the issues that exist due to the multi-step process required for prime editing.For example, unfavorable RNA structures that may form on PEgRNA may result in inhibition of DNA editing from PEgRNA to genome locus copying.These limitations may be overcome by redesigning and manipulating PEgRNA components.These redesigns may improve the efficiency of prime editing factors and allow the incorporation of longer insertion sequences on genomes.

Thus, in various embodiments, the PEgRNA design may result in longer PEgRNAs by enabling efficient expression of functional PEgRNAs from non-polymerase III (pol III) promoters. This would avoid the need for laborious sequence requirements. In other embodiments, the core Cas9-binding PEgRNA backbone may be improved to improve the efficacy of the system. In yet other embodiments, modifications may be made to the PEgRNA to improve reverse transcriptase (RT) processivity. This would allow for the insertion of longer sequences at the target genomic locus. In other embodiments, RNA motifs may be added to the 5' and/or 3' ends of the PEgRNA to improve stability, enhance RT processivity, prevent PEgRNA misfolding, 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 may improve the PEgRNA backbone and enhance prime editor efficiency. These designs may be used to improve any PEgRNA recognized by any Cas9 or evolved variants thereof.

This application of prime editing can be further described in Example 15.

The PEgRNA may include additional design improvements that may modify the properties and/or characteristics of the PEgRNA, thereby improving the efficacy of prime editing. In various embodiments, these improvements may fall into one or more of a number of different categories: (1) designs to enable efficient expression of functional PEgRNA from non-polymerase III (pol III) promoters, which would enable expression of longer PEgRNAs without onerous sequence requirements; (2) improvements to the core Cas9-binding PEgRNA backbone, which may improve efficacy; (3) modifications of the PEgRNA to improve RT processivity and enable insertion of longer sequences at the target genomic locus; and (4) addition of RNA motifs to the 5' or 3' end of the PEgRNA that improve PEgRNA stability, enhance RT processivity, prevent PEgRNA misfolding, or recruit additional factors important for genome editing.

In one embodiment, PEgRNAs can be designed with pol III promoters to improve expression of longer lengths of PEgRNAs 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 in the nucleus. However, pol III is not highly processive and is not capable of expressing RNAs longer than several hundred nucleotides in length at the levels required for efficient genome editing. In addition, pol III can stall or terminate at stretches of U, potentially limiting the sequence diversity that can be inserted with PEgRNAs. Other promoters that recruit polymerase II (e.g., pCMV) or polymerase I (e.g., U1 snRNA promoter) have been surveyed for their ability to express longer sgRNAs. However, these promoters are typically partially transcribed, which would result in extra sequence 5' of the spacer on the expressed PEgRNA. This has been shown to result in significantly reduced Cas9:sgRNA activity in a site-dependent manner. In addition, PEgRNA transcribed by pol III may simply terminate with a stretch of 6-7 U's, whereas PEgRNA transcribed from pol II or pol I will require a different termination signal. In many cases, such signals will also result in polyadenylation, which will result in the undesired transport of the PEgRNA from the nucleus. Similarly, RNAs expressed from pol II promoters such as pCMV are typically 5' capped, which will also result in their nuclear export.

Previously, Rinn and colleagues screened various expression platforms for the production of long non-coding RNA (lncRNA) tagged ^sgRNAs . These platforms include RNAs expressed from ^pCMV that terminate with ^an ENE element from human MALAT1 ncRNA, a PAN ENE element from ^KSHV , or a ³ ' box from U1 snRNA. Of note, MALAT1 ncRNA and PAN ENE form triple helices and protect the polyA tail. These constructs may 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 cleavage of the portion of the pol II promoter that will be transcribed as part of the PEgRNA, adding either ^a self-cleaving ribozyme, such as a ^hammerhead , pistol, ^hatchet , ^hairpin , VS, ^twister , or ^{twister sister} ribozyme, or other self-cleaving elements, to process the ^transcribed guide, or a hairpin that is recognized by Csy4 and leads to processing of the guide. It has also been hypothesized that the incorporation of multiple ENE motifs may lead to improved PEgRNA expression and stability, as previously demonstrated for KSHV PAN RNA and elements. ^{It is} also anticipated that circularizing the PEgRNA in the form of a circular intronic RNA (ciRNA) may also lead to enhanced RNA expression and stability as well as nuclear localization.

In various embodiments, the PEgRNA may include various elements of the above, as exemplified by the following sequences:

Non-limiting example 1 - PEgRNA expression platform consisting of pCMV, Csy4 hairpin, PEgRNA, and MALAT1 ENE
(SEQ ID NO:501)

Non-limiting example 2 - PEgRNA expression platform consisting of pCMV, Csy4 hairpin, PEgRNA, and PAN ENE
(SEQ ID NO:502)

Non-limiting example 3 - PEgRNA expression platform consisting of pCMV, Csy4 hairpin, PEgRNA, and 3xPAN ENE
(SEQ ID NO:503)

Non-limiting Example 4 - PEgRNA Expression Platform Consisting of pCMV, Csy4 Hairpin, PEgRNA, and 3' Box
(SEQ ID NO:504)

Non-limiting Example 5 - PEgRNA Expression Platform Consisting of pU1, Csy4 Hairpin, PEgRNA, and 3' Box
(SEQ ID NO:505)

In various other embodiments, the PEGRNA can be improved by introducing improvements to the backbone or core sequence. This can be done by introducing known.

The core Cas9-bound PEgRNA backbone could conceivably be improved to enhance PE activity. Several such approaches have already been demonstrated. For example, the first pairing element (P1) of the backbone contains a GTTTT-AAAAC (SEQ ID NO: 3939) pairing element. Such a run of Ts has been shown to result in pol III pausing and premature termination of the RNA transcript. Rational mutation of one of the TA pairs to a GC pair in this portion of P1 has been shown to enhance sgRNA activity, suggesting that this approach may also be feasible for ^PEgRNA195 . In addition, increasing the length of P1 has also been shown to enhance sgRNA folding, leading to improved ^activity195 , suggesting it as another avenue for improving PEgRNA activity. Example improvements of the core are:

PEgRNA containing a 6 nt extension to P1
GGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGCTCATGAAAATGAGCTAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCCTCTGCCATCAAAGCGTGCTCAGTCTGTTTTTTT (SEQ ID NO: 228)

PEgRNA containing a TA→GC mutation in P1
GGCCCAGACTGAGCACGTGAGTTTGAGAGCTAGAAATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCCTCTGCCATCAAAGCGTGCTCAGTCTGTTTTTTT (SEQ ID NO: 229)
may include.

In various other embodiments, PEgRNA can be improved by introducing modifications to the editing template region.As the size of the PEgRNA-templated insert increases, it is more likely to be degraded by endonucleases, undergo spontaneous hydrolysis, or fold into a secondary structure that is incapable of being reverse transcribed by RT or blocks the folding of the PEgRNA backbone and subsequent Cas9-RT binding.Therefore, it is likely that modifications to the PEgRNA template may be necessary to affect large insertions, such as the insertion of whole genes.Some strategies for doing so include the incorporation of modified nucleotides on synthetic or semi-synthetic PEgRNA, which make the RNA more resistant to degradation or hydrolysis, or less likely to adopt inhibitory secondary ^{structures196} . Such modifications may include 8-aza-7-deazaguanosine, which will reduce RNA secondary structure in G-rich sequences; locked nucleic acid (LNA), which reduces degradation and enhances certain types of RNA secondary structure; 2'-O-methyl, 2'-fluoro, or 2'-O-methoxyethoxy modifications, which enhance RNA stability. Such modifications may also be included elsewhere on the PEgRNA to enhance stability and activity. Alternatively or in addition, the template of the PEgRNA may be designed such that it both encodes the desired protein product and is also more likely to adopt a simple secondary structure capable of being unfolded by RT. Such a simple structure would act as a thermodynamic sink, making it less likely that a more complicated structure would arise that would prevent reverse transcription. Finally, the template may be split into two separate PEgRNAs. In such a design, PE will also be used to initiate transcription and recruit a separate template RNA to the target site by an RNA recognition element on the PEgRNA itself, such as an RNA-binding protein fused to Cas9 or an MS2 aptamer. RT can either directly bind to this separate template RNA or initiate reverse transcription from the original PEgRNA before swapping to the second template. Such an approach can enable long insertions both by preventing misfolding of the PEgRNA upon addition of a long template and by not requiring dissociation of Cas9 from the genome for long insertions to occur, which could potentially inhibit PE-based long insertions.

In still other embodiments, PEgRNAs can be improved by introducing additional RNA motifs into the 5' and 3' ends of the PEgRNA. Some such motifs, such as the PAN ENE from KSHV and the ENE from MALAT1, were discussed above as possible means to terminate the expression of longer PEgRNAs from non-pol III promoters. These elements form RNA triple helices that surround the polyA tail and result in their retention in the nucleus. ^184,187 However, by forming complex structures at the 3' end of the PEgRNA that confine the terminal nucleotides, these structures would likely also help prevent exonuclease-mediated degradation of the PEgRNA.

Other structural elements inserted at the 3' end may also enhance RNA stability, even without enabling termination from a non-pol III promoter. Such motifs could include hairpins or RNA quadruplexes that would trap the 3' end, ¹⁹⁷ or self-cleaving ribozymes such as HDV that would result in the formation of a 2'-3'-cyclic phosphate at the 3' end and potentially make the PEgRNA less likely to be degraded by ^{exonucleases.198} Inducing the PEgRNA to circularize by incomplete splicing to form ciRNA could also increase PEgRNA stability and result in the PEgRNA being retained in the ^nucleus.194

Additional RNA motifs may also improve RT processivity or enhance PEgRNA activity by enhancing RT binding to the DNA-RNA duplex. The addition of native sequences bound by RT on its corresponding retroviral genome can enhance RT activity. This ^may include native primer binding sites (PBSs), polypurine tracts (PPTs), or kissing loops involved in retroviral genome dimerization and initiation ^of transcription.

The addition of kissing loops or dimerization motifs such as GNRA tetraloop/tetraloop receptor pair ²⁰⁰ at the 5' and 3' ends of the PEgRNA can also result in efficient circularization of the PEgRNA, improving stability. In addition, it is envisioned that the addition of these motifs can allow for physical separation of the PEgRNA spacer and primer, preventing spacer entrapment that would interfere with PE activity. Short 5' or 3' extensions of the PEgRNA that form small toehold hairpins in the spacer region can also compete favorably for the annealing region of the PEgRNA that binds to the spacer. Finally, kissing loops can also be used to recruit other template RNAs to genomic sites, allowing swapping of RT activity from one RNA to another. Example improvements are:

PEGRNA-HDV fusions
GGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCCTCTGCCATCAAAGCGTGCTCAGTCTGGGCCGGCATGGTCCCAGCCTCCTCGCTGGCGCCGGCTGGGCAACATGCTTCGGCATGGCGAATGGGACTTTTTTT (SEQ ID NO: 230)

PEgRNA-MMLV kissing loop
GGTGGGAGACGTCCCACCGGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCCTCTGCCATCAAAGCTTCGACCGTGCTCAGTCTGGTGGGAGACGTCCCACCTTTTTTTT (SEQ ID NO: 231)

PEgRNA-VS ribozyme kissing loop
GAGCAGCATGGCGTCGCTGCTCACGGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCCTCTGCCATCAAAGCTTCGACCGTGCTCAGTCTCCATCAGTTGACACCCTGAGGTTTTTTT (SEQ ID NO: 232)

PEgRNA-GNRA tetraloop/tetraloop receptor
GCAGACCTAAGTGGUGACATATGGTCTGGGCCCAGACTGAGCACGTGAGTTTTAGAGCTAUACGTAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTUACGAAGTGGGACCGAGTCGGTCCTCTGCCATCAAAGCTTCGACCGTGCTCAGTCTGCATGCGATTAGAAATAATCGCATGTTTTTTT (SEQ ID NO: 233)

PEgRNA template to switch secondary RNA-HDV fusions
TCTGCCATCAAAGCTGCGACCGTGCTCAGTCTGGTGGGAGACGTCCCACCGGCCGGCATGGTCCCAGCCTCCTCGCTGGCGCCGGCTGGGCAACATGCTTCGGCATGGCGAATGGGACTTTTTTT (SEQ ID NO: 234)
These include, but are not limited to:

The PEgRNA backbone can be further improved by directed evolution in a manner analogous to how SpCas9 and prime editors (PEs) have been improved. Directed evolution can enhance PEgRNA recognition by Cas9 or evolved Cas9 variants. In addition, it is likely that different PEgRNA backbone sequences will be optimal at different genomic loci, either enhancing PE activity at the site, reducing off-target activity, or both. Finally, evolution of the PEgRNA backbone with additional RNA motifs will almost certainly improve the activity of the fusion PEgRNA relative to the unevolved fusion RNA. For example, evolution of an allosteric ribozyme consisting of a c-di-GMP-I aptamer and a hammerhead ribozyme led to dramatically improved activity, ²⁰² suggesting that evolution will also improve the activity of hammerhead-PEgRNA fusions. In addition, although Cas9 currently does not generally tolerate 5' extensions of sgRNAs, directed evolution will likely generate potent mutations that alleviate this intolerance, allowing additional RNA motifs to be exploited.

The present disclosure contemplates any such manner to further improve the efficacy of the prime editing system disclosed herein.

O. Use of Prime Editing with Extended Targeting Range
Prime editing (PE) using Streptococcus pyogenes Cas9 (SpCas9) can efficiently incorporate all single base substitutions, insertions, deletions, and combinations thereof at genomic loci with appropriately placed NGG protospacer adjacent motifs (PAMs) to which SpCas9 can efficiently bind. However, in another aspect, the methods described herein broaden the targeting capabilities of PE by expanding the accessible PAMs, and thus the accessible targetable genomic loci for efficient PE. Prime editing using RNA-guided DNA-binding proteins other than SpCas9 allows for an expanded targetable range of genomic loci by allowing access to different PAMs. In addition, the use of RNA-guided DNA-binding proteins smaller than SpCas9 also allows for more efficient viral delivery. PE with Cas proteins other than SpCas9 or other RNA-guided DNA-binding proteins will allow for highly efficient therapeutic editing that was 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 the edits relative to the NGG PAM, or the overall size of the SpCas9-based construct is prohibitive for cellular expression and/or delivery. Certain disease-relevant loci, such as the huntingtin gene, which have SpCas9's few and poorly positioned NGG PAMs close to the target region, can be easily targeted using a different Cas protein of the PE system, such as SpCas9-VRQR, which recognizes the NGA PAM. Smaller Cas proteins would be used to generate smaller PE constructs that can be more efficiently packaged into AAV vectors, enabling better delivery to target tissues. Figure 61 shows the implementation of prime editing using Staphylococcus aureus CRISPR-Cas as an RNA-guided DNA-binding protein. NT is untreated control.

Figures 62A-62B provide a demonstration of the importance of the protospacer for efficient incorporation of the desired edit at precise positioning by prime editing. This highlights the importance of alternative PAMs and protospacers as a novel feature of this technology. "n.d." in Figure 62A is "not detected."

Figure 63 shows the implementation of PE using SpCas9(H840A)-VRQR and SpCas9(H840A)-VRER as the RNA-guided DNA binding proteins of the prime editor system. SpCas9(H840A)-VRQR napDNAbp is disclosed herein as SEQ ID NO: 87. 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, where the MMLV RT contains D200N, L603W, T330P, T306K, and W313F substitutions relative to wild-type MMLV RT. The SpCas9(H840A)-VRQR-MMLV RT fusion protein is disclosed herein as SEQ ID NO:515, in which the MMLV RT contains D200N, L603W, T330P, T306K, and W313F substitutions relative to wild-type MMLV RT. Seven different loci on the human genome are targeted: four by the SpCas9(H840A)-VRQR-MMLV RT prime editor system and three by the SpCas9(H840A)-VRER-MMLV RT system. The amino acid sequences of the constructs tested are as follows:

As shown in Figure 63, SpCas9(H840A)-VRQR-MMLV RT was active at PAM sites including "AGAG" and "GGAG" and had some editing activity at "GGAT" and "AGAT" PAM sequences. SpCas9(H840A)-VRER-MMLV RT was active at PAM sites including "AGCG" and "GGCG" and had some editing activity at "TGCG".

The data demonstrate that prime editing can be performed using napDNAbp with different PAM specificities, such as the Cas9 variants described herein.

In various embodiments, the napDNAbp (e.g., Cas9) with altered PAM specificity comprises a combination of mutations that exert activity against a target sequence that comprises a 5'-NAA-3'PAM sequence at its 3' end. In some embodiments, the combination of mutations is present in any one of the clones listed in Table 1. In some embodiments, the combination of mutations is a conservative mutation of the clones listed in Table 1. In some embodiments, the Cas9 protein comprises a combination of mutations of any one of the Cas9 clones listed in Table 1.

Table 1: NAA PAM clones

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 provided by any one of the variants in 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 provided by any one of the variants in Table 1.

In some embodiments, the Cas9 protein exhibits increased activity against a target sequence that does not contain a canonical PAM (5'-NGG-3') at its 3' end compared to the Streptococcus pyogenes Cas9 provided by SEQ ID NO: 18. In some embodiments, the Cas9 protein exhibits at least a 5-fold increased activity against a target sequence having a 3' end that is not immediately adjacent to a canonical PAM sequence (5'-NGG-3') compared to the activity of the Streptococcus pyogenes Cas9 provided by SEQ ID NO: 18 against the same target sequence. In some embodiments, the Cas9 protein exerts 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 activity against a target sequence that is not immediately adjacent to a standard PAM sequence (5'-NGG-3') compared to the activity of Streptococcus pyogenes provided by SEQ ID NO: 18 against the same target sequence. In some embodiments, the 3' end of the target sequence is immediately adjacent to an AAA, GAA, CAA, or TAA sequence. In some embodiments, the Cas9 protein comprises a combination of mutations that exerts activity against a target sequence that comprises a 5'-NAC-3' PAM sequence at its 3' end. In some embodiments, the combination of mutations is present in any one of the clones listed in Table 2. In some embodiments, the combination of mutations is a conservative mutation of the clones listed in Table 2. In some embodiments, the Cas9 protein comprises a combination of mutations in any one of the Cas9 clones listed in Table 2.

Table 2: NAC PAM clones

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 provided by any one of the variants in 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 provided by any one of the variants in Table 2.

In some embodiments, the Cas9 protein exhibits increased activity against a target sequence that does not contain a canonical PAM (5'-NGG-3') at its 3' end compared to the Streptococcus pyogenes Cas9 provided by SEQ ID NO: 18. In some embodiments, the Cas9 protein exhibits at least 5-fold increased activity against a target sequence having a 3' end that is not immediately adjacent to a canonical PAM sequence (5'-NGG-3') compared to the activity of the Streptococcus pyogenes Cas9 provided by SEQ ID NO: 18 against the same target sequence. In some embodiments, the Cas9 protein exerts 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 activity against a target sequence that is not immediately adjacent to a canonical PAM sequence (5'-NGG-3') compared to the activity of Streptococcus pyogenes provided by SEQ ID NO: 18 against the same target sequence. In some embodiments, the 3' end of the target sequence is immediately adjacent to an AAC, GAC, CAC, or TAC sequence.

In some embodiments, the Cas9 protein comprises a combination of mutations that exerts activity against a target sequence that comprises a 5'-NAT-3'PAM sequence at its 3' end. In some embodiments, the combination of mutations is present in any one of the clones listed in Table 3. In some embodiments, the combination of mutations is a conservative mutation of the clones listed in Table 3. In some embodiments, the Cas9 protein comprises a combination of mutations of any one of the Cas9 clones listed in Table 3.

Table 3: NAT PAM clones

Any of the above Cas9 variants that exhibit differential PAM specificity compared to standard SpCas9 can be used in the prime editors disclosed herein.

P. Use of prime editing to insert recombinase target sites
In another aspect, prime editing can be used to insert a recombinase site (or "recombinase recognition sequence") into a desired genomic site. The insertion of the recombinase site provides a programmed location to effect site-specific genetic alterations into the genome. Such genetic alterations can include, for example, genomic incorporation of a plasmid, genomic deletion or insertion, chromosomal translocation, and cassette exchange, among other genetic alterations. These exemplary types of genetic alterations are illustrated in Figures 64 B-64F . The incorporated recombinase recognition sequence can then be used to effect site-specific recombination at that site to achieve various recombination outcomes, such as excision, incorporation, inversion, or exchange of DNA fragments. For example, Figure 65 illustrates the incorporation of a recombinase site, which can then be used to incorporate a DNA donor template containing a GFP-expressing marker. Cells containing a GFP expression system incorporated into the recombinase site will fluoresce.

The mechanism of incorporating recombinase sites into a genome is analogous to incorporating other sequences into a genome, such as peptide/protein and RNA tags. A schematic illustrating the incorporation of a recombinase target sequence is shown in Figure 64(a). The process begins by selecting a desired target locus where a recombinase target sequence will be introduced. Next, a primed editor fusion is provided ("RT-Cas9:gRNA"), where "gRNA" refers to a PEgRNA, which may be designed using the principles described herein. The PEgRNA will in various embodiments comprise an architecture corresponding to FIG. 3D (5'-[~20 nt spacer]-[gRNA core]-[extension arm]-3', where the extension arm comprises, in a 3' to 5' direction, a primer binding site ("A"), an editing template ("B"), and a homology arm ("C"). The editing template ("B") will comprise a single strand of PEgRNA encoding a sequence corresponding to a recombinase site, i.e., a complementary single strand of DNA that is either the sense or antisense strand of the recombinase site and that is integrated into the genomic DNA target locus by the primed editing process.

In various aspects, the present disclosure enables the use of PE to introduce recombinase recognition sequences at high-value loci on human or other genomes. These will lead to precise and efficient genome modification after exposure to site-specific recombinase(s) (Figure 64). In various embodiments shown in Figure 64, PE can be used to insert (b) a single SSR target for use as a site of genomic incorporation of a DNA donor template. (c) shows how tandem insertion of SSR target sites can be used to delete a portion of a genome. (d) shows how tandem insertion of SSR target sites can be used to invert a portion of a genome. (e) shows how insertion of two SSR target sites in two distal chromosomal regions can result in a chromosomal translocation. (f) shows how insertion of two different SSR target sites on a genome can be used to exchange a cassette from a DNA donor template. Each of the types of genome modifications envisioned by using PE to insert SSR targets, but this list is also not meant to be limiting.

PE-mediated introduction of recombinase recognition sequences may be particularly useful for the treatment of genetic diseases caused by large-scale genomic defects, such as gene losses, inversions, or duplications, or chromosomal ^{translocations1-7} (Table 6). For example, Williams-Beuren syndrome is a developmental disorder caused by a deletion of 24 on chromosome 721. Currently, no technology exists for efficient and targeted insertion of entire genes in living cells (the potential of PE for such full-length gene insertion is currently being explored but not yet established); however, recombinase-mediated incorporation in PE-inserted targets offers one approach to a permanent cure of this and other diseases. In addition, targeted introduction of recombinase recognition sequences may be highly effective for applications involving the generation of transgenic plants, animal research models, bioproduction cell lines, or other custom eukaryotic cell lines. For example, recombinase-mediated genome reorganization of transgenic plants in PE-specific targets may overcome one of the bottlenecks in generating agricultural crops with improved ^{properties8,9} .

Table 6. Examples of genetic diseases linked to large-scale genome modifications that can be repaired by PE-based integration of recombinase recognition sequences.

Several 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 genome integration rates have also been described for some SSRs. ^22-30 In addition to natural recombinases, programmable recombinases with alternative specificities have been developed. ^31-40 Depending on the desired application, one or more of these recognition sequences can be introduced into the genome at defined locations, such as safe harbor loci, using PE. ^41-43

For example, introduction of a single recombinase recognition sequence into the genome by prime editing would result in integrative recombination with a DNA donor template (Figure 64b). Serine integrase, which operates robustly in human cells, may be particularly well suited for gene ^{integration44,45} .

In addition, depending on the identity and orientation of the targets, introduction of two recombinase recognition sequences can result in an intervening sequence deletion, an intervening sequence inversion, a chromosomal translocation, or a cassette exchange (Figures 64C-64F ). By choosing an endogenous sequence that already closely resembles a recombinase target, the extent of editing required to introduce a perfect recombinase target will be reduced.

Finally, several recombinases have been demonstrated to integrate into human or eukaryotic genomes at natively occurring pseudosites. ^{46-64 PE} editing can be used to modify these loci to enhance integration rates 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 onto eukaryotic genomes using PE. The applications of this are nearly limitless. The genome editing reaction is intended for use with a "prime editor" of a chimeric fusion of CRISPR/Cas9 protein and reverse transcriptase domain, which utilizes a custom prime editing guide RNA (PEgRNA). Extending further, the Cas9 tool and the homology-directed repair (HDR) pathway can also be exploited to introduce recombinase recognition sequences from DNA templates by reducing the indel rate using several ^{techniques65-67} . A proof-of-concept experiment in human cell culture is shown in Figure 65.

表8.ラージセリンインテグラーゼおよびSSR標的配列。

表9.セリンリゾルバーゼおよびSSR標的配列。

表10.チロシンインテグラーゼおよび標的配列。

The next several tables provide a list of exemplary recombinases that can be used in the discussion above with respect to PE-directed incorporation of recombinase recognition sequences and their corresponding recombinase recognition sequences that can be incorporated by PE.
Table 7. Tyrosine recombinases and SSR target sequences.

Table 8. Large serine integrase and SSR target sequences.

Table 9. Serine resolvases and SSR target sequences.

Table 10. Tyrosine integrases and target sequences.

In various other aspects, the disclosure relates to methods of using PE to incorporate one or more recombinase recognition sequences and their use for site-specific recombination.

In some embodiments, site-specific recombination can achieve 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 of a single nucleic acid molecule (e.g., DNA).

In some embodiments, the disclosure provides a method for incorporating a donor DNA template by site-specific recombination, comprising: (a) incorporating a recombinase recognition sequence into a genomic locus by prime editing; (b) contacting the genomic locus with a DNA donor template that also contains a recombinase recognition sequence in the presence of a recombinase.

In another aspect, the disclosure provides a method for deleting a genomic region by site-specific recombination, comprising: (a) incorporating 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 another aspect, the disclosure provides a method for inverting a genomic region by site-specific recombination, comprising: (a) incorporating a pair of recombinase recognition sequences at a genomic locus by prime editing; and (b) contacting the genomic locus with a recombinase, thereby catalyzing inversion of the genomic region between the pair of recombinase recognition sequences.

In yet other aspects, the present disclosure provides a method for inducing a chromosomal translocation between a first genomic site and a second genomic site, comprising: (a) incorporating a first recombinase recognition sequence at the first genomic locus by prime editing; (b) incorporating a second recombinase recognition sequence at the second genomic locus by prime editing; and (c) contacting the first and second genomic loci with a recombinase, thereby catalyzing a chromosomal translocation of the first and second genomic loci.

In other aspects, the disclosure provides a method for inducing cassette exchange between a genomic locus and a donor DNA containing a cassette, comprising: (a) incorporating a first recombinase recognition sequence at a first genomic locus by prime editing; (b) incorporating a second recombinase recognition sequence at a second genomic locus by prime editing; (c) contacting the first and second genomic loci with donor DNA containing a cassette flanked by the first and second recombinase recognition sequences and a recombinase, thereby catalyzing exchange of the flanked genomic locus with the cassette on the DNA donor.

In various embodiments involving the insertion of more than one recombinase recognition sequence onto a genome, the recombinase recognition sequences can be the same or different. In some embodiments, the recombinase recognition sequences are the same. In other embodiments, the 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 can be the RRS of Table 7 that corresponds to the recombinase being used.

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, Int7, Int8, Int9, Int10, Int11, Int12, Int13, L1, peaches, Bxz2, or SV1, as shown in Table 8. In such embodiments, the recombinase recognition sequence can be the RRS of Table 8 corresponding to the recombinase being used.

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, Int7, Int8, Int9, Int10, Int11, Int12, Int13, L1, peaches, Bxz2, or SV1, as shown in Table 8. In such embodiments, the recombinase recognition sequence can be the RRS of Table 8 corresponding to the recombinase being used.

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 can be the RRS of Table 9 that corresponds to the recombinase being used.

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 can be the RRS of Table 10 that corresponds to the recombinase being used.

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 on a cell (e.g., to recombine the genomic DNA of the cell). The cell can be prokaryotic or eukaryotic. The cell, such as a eukaryotic cell, can be in an individual, such as a subject described herein (e.g., a human subject). The methods described herein are useful for genetic modification of cells in vitro and in vivo, e.g., in the context of generating transgenic cells, cell lines, or animals, or in altering the genomic sequence of a cell of a subject, e.g., correcting a genetic defect.

REFERENCES CITED IN SECTION L Each of the following references is cited in Example 17, each of which is incorporated herein by reference.

[8] Methods of Treatment The present disclosure provides methods for the treatment of a subject diagnosed with a disease associated with or caused by a point mutation or other mutation (e.g., deletion, insertion, inversion, duplication, etc.) that can be corrected by the prime editing system provided herein, including, but not limited to, a prion disease (e.g., Example 5 of the present application), a trinucleotide repeat expansion disease (e.g., Example 3 of the present application), or a CDKL5 deficiency disorder (CDD) (e.g., Example 23 of the present application).

Virtually any disease-causing genetic defect can be repaired by using prime editing. This involves the selection of an appropriate prime editor fusion protein, including napDNAbp and a polymerase (e.g., reverse transcriptase), and the design of an appropriate PEgRNA designed to (a) target the appropriate target DNA containing the editing site, and (b) provide a template for synthesis of a single strand of DNA from the 3' end of the nick site that contains the desired edit that displaces and replaces the endogenous strand immediately downstream of the nick site. Prime editing can be used, without limitation, to (a) incorporate mutation-correcting changes into a nucleotide sequence, (b) incorporate protein and RNA tags, (c) incorporate immune epitopes onto a protein of interest, (d) incorporate inducible dimerization domains into a protein, (e) incorporate or remove sequences in a biomolecule to alter its activity, (f) incorporate recombinase target sites to direct specific genetic changes, and (g) mutate a target sequence by using error-prone RT.

Methods of treating disorders involve, as an early step, the design of appropriate PEgRNA and prime editor fusion proteins according to the methods described herein, which encompass a number of considerations that can be taken into account, for example:
(a) a target sequence, i.e., a nucleotide sequence, into which one or more nucleobase modifications are desired to be incorporated by a primed editor;
(b) Positioning of the cut site on the target sequence, i.e., the specific nucleobase position where the primed editing factor will induce a single-stranded nick to generate a 3'-end RT primer sequence on one side of the nick and a 5'-end endogenous flap on the other side of the nick (which is ultimately removed by FEN1 or its equivalent and replaced by a 3'-ssDNA flap). The cut site generates a 3'-end primer sequence that becomes extended by the polymerase of the PE fusion protein (e.g., the RT enzyme) during RNA-dependent DNA polymerization to generate a 3'-ssDNA flap containing the desired edit, which then replaces the 5'-endogenous DNA flap on the target sequence;
(c) available PAM sequences (encompassing canonical SpCas9 PAM sites, as well as non-canonical PAM sites recognized by Cas9 variants and equivalents with extended or different PAM specificities);
(d) the spacing between available PAM sequences on the PAM strand and the positioning of the site to be cut;
(e) the specific Cas9, Cas9 variant, or Cas9 equivalent of the available prime editor to be used (this is dictated in part by the available PAM);
(f) the sequence and length of the primer binding site;
(g) the sequence and length of the editing template;
(h) sequence and length of the homology arms;
(i) spacer sequence and length; and
(j) gRNA core sequence.

A suitable PEgRNA and optionally a nicking sgRNA design guide for second site nicking can be designed by the following exemplarly step-by-step set of instructions, which takes into account one or more of the above considerations. The steps refer to the examples shown in Figures 70A-70I.
1. Define the target sequence and edit. Retrieve the sequence of the target DNA region (~200bp) centered around the location of the desired edit (point mutation, insertion, deletion, or combination thereof). See Figure 70A.
2. Position the target PAM. Identify a PAM proximal to the desired editing position. A PAM can be identified on either strand of DNA proximal to the desired editing position. Although a PAM proximal to the editing position is preferred (i.e., the nick site is less than 30 nt from the editing position, 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 nt from the editing position to the nick site), it is possible to incorporate an edit using a protospacer and PAM that places the nick ≧30 nt from the editing position. See Figure 70B.
3. Position the nick site. For each PAM under consideration, identify the corresponding nick site and on which strand. For Sp Cas9 H840A nickase, cleavage occurs on the PAM-containing strand between the third and fourth bases 5' of the NGG PAM. All edited nucleotides must be 3' to the nick site. Therefore, the appropriate PAM must nick 5' of the targeted edit on the PAM-containing strand. In the example shown below, there are two possible PAMs. For simplicity, the remaining steps demonstrate the design of a PEgRNA using only PAM 1. See Figure 70C.
4. Design the spacer sequence. The protospacer of SpCas9 corresponds to the 20 nucleotides 5' of the NGG PAM on the PAM-containing strand. Efficient Pol III transcription initiation requires that G be the first transcribed nucleotide. If the first nucleotide of the protospacer is a G, then the spacer sequence of the PEgRNA is simply the protospacer sequence. If the first nucleotide of the protospacer is not a G, then the spacer sequence of the PEgRNA is a G followed by the protospacer sequence. See Figure 70D.
5. Design the primer binding site (PBS). Using the starting allele sequence, identify a 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 fourth base 5' of 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 containing .about.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. Regardless of GC content, the optimal PBS sequence should be determined empirically. To design a PBS sequence of length p, use the starting allele sequence to take the reverse complement of the first p nucleotides 5' of the nick site on the PAM-containing strand. See Figure 70E.
6. Design the RT template (or DNA synthesis template). The RT template (or DNA synthesis template, where the polymerase is not a reverse transcriptase) codes for the designed edit and homology to the sequence flanking the edit. In one embodiment, these regions correspond to the DNA synthesis template of FIG. 3D and FIG. 3E, where the DNA synthesis template includes the "edit template" and the "homology arms". The optimal RT template length varies based on the target site. For short-range edits (positions +1 to +6), it is recommended to test short (9 to 12 nt), medium (13 to 16 nt), and long (17 to 20 nt) RT templates. For long-range edits (positions +7 and beyond), it is recommended to use an RT template that extends at least 5 nt (preferably 10 nt or more) from the edit position to allow sufficient 3' DNA flap homology. For long-range edits, several RT templates should be screened to identify a functional design. For larger insertions and deletions (≧5 nt), incorporation of greater 3′ homology (≧20 nt) onto the RT template is recommended. Editing efficiency is typically compromised when the RT template codes for synthesis of a G (corresponding to a C on the RT template for PEG RNA) as the last nucleotide on the reverse transcribed DNA product. Because many RT templates support efficient prime editing, avoidance of G as the last synthesized nucleotide is recommended when designing an RT template. To design an RT template sequence of length r, use the desired allele sequence and take the reverse complement of the first r nucleotides 3′ of the nick site on the strand that originally contained the PAM. Note that compared to SNP editing, insertion or deletion edits using an RT template of the same length will not contain the same homology. See Figure 70F.
7. Assemble the complete PEgRNA sequence. Concatenate the PEgRNA components in the following order (5' to 3'): spacer, backbone, RT template, and PBS. See Figure 70G.
8. Design a nicking sgRNA for PE3. Identify PAMs on the non-edited strand upstream and downstream of the edit. The optimal nicking position is highly locus dependent and should be empirically determined. Generally, a nick placed 40 to 90 nucleotides 5' opposite the PEgRNA-induced nick leads to higher editing yields and fewer indels. The nicking sgRNA has a spacer sequence that matches the 20 nt protospacer on the starting allele. If the protospacer does not begin with a G, have an additional 5' G. See Figure 70H.
9. Design a PE3b nicking sgRNA. If a PAM is present on the complementary strand and its corresponding protospacer overlaps with the sequence targeted for editing, this edit may 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, not the starting allele. The PE3b system works efficiently when the nucleotide(s) to be edited fall within the seed region (~10 nt adjacent to the PAM) of the nicking sgRNA protospacer. This prevents nicking of the complementary strand until after incorporation of the edited strand, preventing competition between the PEgRNA and the sgRNA for binding to the target DNA. PE3b also avoids the generation of nicks on both strands simultaneously, thus significantly reducing indel formation while maintaining high editing efficiency. The PE3b sgRNA should have a spacer sequence that matches the 20 nt protospacer of the desired allele. With the addition of a 5'G if required. See Figure 70I.

The above step-by-step process for designing suitable PEgRNAs and second-site nicking sgRNAs is not meant to be limiting in any way. The present disclosure contemplates variations of the step-by-step process described above, which could be derived therefrom by one of skill in the art.

Once suitable PEgRNA and PE fusion proteins have been selected/designed, they can be administered by a suitable methodology, for example, by vector-based transfection (in which one or more vectors containing DNA encoding the PEgRNA and PE fusion protein are expressed in a cell by transfection with the vector), by direct delivery (e.g., RNP delivery) of the PE fusion protein complexed with the PEgRNA in a delivery format (e.g., lipid particles, nanoparticles), or by an mRNA-based delivery system. Such methods are described by this disclosure in this application and any known method can be utilized.

The PEgRNA and PE fusion protein (or together referred to as a PE complex) can be delivered to a cell in a therapeutically effective amount such that they contact the desired target DNA, thereby allowing the desired edits to be incorporated therein.

Any disease could conceivably be treated by such methods, so long as delivery to the appropriate cells is feasible. One of skill in the art would be able to choose and/or select a PE delivery methodology appropriate for the intended purpose and intended target cells.

For example, in some embodiments, a method is provided that includes administering to a subject having such a disease (e.g., cancer associated with a point mutation as described above) an effective amount of a prime editing system described herein (that corrects the point mutation or introduces an inactivating mutation into the disease-associated gene as mediated by homology-directed repair in the presence of a donor DNA molecule containing the desired genetic alteration). In some embodiments, a method is provided that includes administering to a subject having such a disease (e.g., cancer associated with a point mutation as described above) an effective amount of a prime editing system described herein (that corrects the point mutation or introduces an inactivating mutation into the 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 an inactivating mutation into the disease-associated gene are known to those of skill in the art, and the disclosure is not limited in this respect.

The present disclosure provides methods for the treatment of additional diseases or disorders, for example, diseases or disorders associated with or caused by point mutations 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 the skilled artisan based on the present disclosure. Exemplary suitable diseases and disorders are listed below. It will be understood that the numbering of specific positions or residues in each sequence will depend on the specific protein and numbering scheme used. The numbering may be different, for example, in the precursor of the mature protein and the mature protein itself, and sequence differences from species to species may affect the numbering. The skilled artisan will be able to identify any homologous proteins and the respective residues in the respective encoding nucleic acids by methods well known in the art, for example, by aligning sequences and determining homologous residues. Exemplary suitable diseases and disorders include, without limitation, the following: 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, types 1, 3 and 5; 5-oxoprolinase deficiency; 6-pyruvoyl-tetrahydropterin synthase deficiency; Aarskog syndrome; Aase syndrome; achondroplasia type 2; color vision deficiencies 2 and 7; acquired long QT syndrome; Schinzel type acrocallosal syndrome; acrofemoral head dysplasia; acroostosis deficiency 2 with or without hormone resistance; acroerythrokeratoderma; acrobrachydysplasia; Acth-independent adrenal macronodular hyperplasia 2; activity PI3K-delta syndrome;acute intermittent porphyria;acyl-CoA dehydrogenase family, member 9 deficiency;Adams-Oliver syndrome 5 and 6;adenine phosphoribosyltransferase deficiency;adenylate kinase deficiency;hemolytic anemia due to adenylosuccinate lyase deficiency;nephronophthisis of adolescence;renal-hepatic-pancreatic dysplasia;Meckel syndrome type 7;adrenoleukodystrophy;adult junctional epidermolysis bullosa;epidermolysis bullosa, junctional, focal variant;adult neuronal ceroid lipofuscinosis;adult-onset ataxia with ocular lag;ADULT syndrome;afibrinogenemia and congenital afibrinogenemia;autosomal recessive agammaglobulinemia 2;age-related macular degeneration 3, 6, 11 and 12;Aicardi-Goutier syndrome Alzheimer's syndrome 1, 4, and 5; lupus pernio 1; Alagille syndrome 1 and 2; Alexander disease; alkaptonuria; Allan Herndon-Dudley syndrome; alopecia universalis; Alpers encephalopathy; alpha-1-antitrypsin deficiency; autosomal dominant, autosomal recessive, and X-linked recessive Alport syndrome; Alzheimer's disease, familial, 3, with spastic paraplegia and apraxia; Alzheimer's disease, types 1, 3, and 4; hypocalcifying and hypomature, IIA1 hereditary amelogenesis imperfecta; aminoacylase 1 deficiency; Amish childhood epilepsy syndrome; amyloidogenic transthyretin amyloidosis; transthyretin-related amyloid cardiomyopathy; cardiomyopathy; amyotrophic lateral sclerosis types 1, 6, 15 (with or without frontotemporal dementia), and 22 (with or without frontotemporal dementia) with or without, and type 10; frontotemporal dementia with TDP43 inclusions related to TARDBP; Andermann syndrome; Andersen-Tawil syndrome; congenital long QT syndrome; non-spherolytic anemia due to G6PD deficiency; Angelman syndrome; neonatal onset severe encephalopathy with microcephaly; susceptibility to autism, X-linked 3; hereditary vasculopathy with nephropathy, aneurysms, and muscle spasms; mild serum elevation of angiotensin I converting enzyme; aniridia, cerebellar ataxia, and mental retardation; onychomycosis; antithrombin III deficiency; Antley-Bixler syndrome with genital anomalies and steroidogenesis abnormalities; familial thoracic aortic aneurysms 4, 6, and 9; thoracic aortic aneurysm and aortic dissection; multisystemic smooth muscle dysfunction syndrome 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 congenital, peripheral, X-linked;arthrogryposis-renal dysfunction-cholestasis syndrome;arthrogryposis, renal dysfunction, and cholestasis 2;asparagine synthetase deficiency;neuronal migration abnormalities;ataxia with vitamin E deficiency;sensory, autosomal dominant, ataxia;ataxia-telangiectasia syndrome;hereditary cancer-predisposing syndrome syndrome);atransferrinemia;familial atrial fibrillation 11, 12, 13 and 16;atrial septal defect 2, 4 and 7 (with or without ventricular conduction defect);atrial stop 2;atrioventricular septal defect 4;hereditary ophthalmotrophy;ATR-X syndrome;auriculomandibular syndrome 2;autoimmune disease, multisystem, childhood onset;autoimmune lymphoproliferative syndrome type 1a;autosomal dominant anhidrotic 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 types IA and 1B;autosomal recessive anhidrotic ectodermal dysplasia syndrome;ectodermal dysplasia 11b;hypophidrosis/hair/dentition, autosomal recessive;autosomal recessive hypophosphatemic bone disease;accentre type 3 Feldt-Rieger syndrome;Bainbridge-Lopers syndrome;Banayan-Riley-Rubarka syndrome;PTEN hamartoma syndrome;Baraitser-Winter syndrome 1 and 2;Baracart syndrome;Bardet-Biedl syndrome 1, 11, 16 and 19;Bare lymphocyte syndrome type 2, complementation group E;Prenatal Bartter syndrome type 2;Bartter syndrome type 3, type 3 with hypocalciuria, and type 4;Idiopathic basal ganglia calcification 4;Monethlis;Benign familial hematuria;Benign familial neonatal seizures 1 and 2;Seizures, benign familial neonatal 1, and/or myokymia;Seizures, early childhood epileptic encephalopathy 7;Benign familial neonatal-pediatric seizures;Benign hereditary chorea;Benign scapular periosteal muscular dystrophy with cardiomyopathy;Types A1 and A2 (autosomal dominant) Bernard-Soulier syndrome;Bestrophinopathies (autosomal recessive);Beta Thalassemia;Bethlem myopathy and Bethlem myopathy 2;Vietti crystalline corneal retinal dystrophy;Bile acid synthesis deficiency, congenital 2;Biotinidase deficiency;Birk Barel mental retardation dysmorphic syndrome;Eyelid reduction, ptosis, and entropion;Bloom syndrome;Bielson-Forssmann-Lehmann syndrome;Boucher-Neuhauser syndrome;Papillary disease types A1 and A2;Papillary disease with hypertension;Cerebral small vessel disease with hemorrhage;Branched-chain ketoacid dehydrogenase kinase deficiency;Branched-chain syndromes 2 and 3;Breast cancer, early onset;Breast and ovarian cancer, familial 1, 2, and 4;Corneal fragility syndrome 2;Brody myopathy;Bronchiectasis with or without elevated sweat chloride 3;Brown-Vialet-Van Reale syndrome and Brown-Vialet-Van Reale syndrome Reale 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;Familial candidiasis 2, 5, 6 and 8;Types I and II glycoprotein glycosylation deficiency syndromes;Carbonic anhydrase VA deficiency due to hyperammonemia;Colon cancer;Cardiomyopathy;Long QT syndrome, LQT1 variant;Fatal infantile cardiac encephalomyopathy due to cytochrome c oxidase deficiency;Cardiomyopathy;Danon disease;Hypertrophic cardiomyopathy;Left ventricular noncompaction cardiomyopathy;Carnevale syndrome;Type 1 Carney complex; carnitine acylcarnitine translocase deficiency; carnitine palmitoyltransferase I, II, II (late-onset) and II (pediatric) deficiency; cataract 1, 4, autosomal dominant, autosomal dominant polymorphism, with microcornea, Coppock-like, juvenile, with microcornea and glycosuria, and nuclear diffuse nonprogressive; catecholaminergic polymorphic ventricular tachycardia; caudate regression syndrome; familial Cd8 deficiency; central core disease; central core instability of chromosomes 1, 9 and 16 and immunodeficiency; childhood cerebellar ataxia and cerebellar ataxia with progressive extraocular muscles ataxia, mental retardation, and balance disorder syndrome 2; cerebral amyloid angiopathy related to APP; cerebral autosomal dominant and recessive arteriopathy with subcortical infarctions and leukoencephalopathy; cerebral cavernous malformations 2; cerebro-oculofacial-skeletal syndrome 2; cerebro-oculofacial-skeletal syndrome; cerebro-retinal microangiopathy with calcifications and cysts; cerebro-lipofuscinosis neuronal 2, 6, 7, and 10; Ch＼xc3＼xa9diak-Higashi syndrome; adult Chédiak-Higashi syndrome; Charcot-Marie-Tooth disease types 1B, 2B2, 2C, 2F, 2I, and 2U (axonal ), 1C (demyelinating), dominant intermediate C, recessive intermediate A, 2A2, 4C, 4D, 4H, IF, IVF and X; ventral scapular spinal muscular atrophy; distal spinal muscular atrophy, congenital non-progressive; spinal muscular atrophy, distal, autosomal recessive, 5; CHARGE-related; childhood hypophosphatemia; adult hypophosphatemia; cholecystitis; progressive familial intrahepatic cholestasis 3; intrahepatic cholestasis of pregnancy 3; cholestanol storage disease; cholesterol monooxygenase (side chain truncation) deficiency; Blomstrand chondrodysplasia; perforating chondrodysplasia 1, X-linked recessive and 2 X-linked dominant; CHOPS syndrome; chronic granulomatous disease, autosomal recessive cytochrome b positive, types 1 and 2; Chudhry-McCuller syndrome; Primary ciliary dyskinesia, 7, 11, 15, 20 and 22; citrullinemia type I; citrullinemia types I and II; craniofacial fissure; C-like syndrome; Cockayne syndrome type A; primary coenzyme Q10 deficiency, 1, 4 and 7; Coffin-Siris/intellectual disability; Coffin-Lowry syndrome; Cohen syndrome; cold sweats syndrome 1; Cole-Carpenter syndrome 2; combined cellular and humoral immune deficiency with granulomas; d-2- and l-2-hydroxyglutaric acid complexuria; malonic and methylmalonic acid complexuria; combined oxidative phosphorylation deficiency 1, 3, 4, 12, 15 and 25; partial and total 17-alpha- Combined hydroxylase/17,20-lyase deficiency;common variable immunodeficiency 9;partial deficiency of complement component 4 due to c1 inhibitor dysfunction;complement factor B deficiency;cone monochromacytosis;cone-rod dystrophies 2 and 6;cone-rod dystrophies;hereditary amelogenesis imperfecta;congenital adrenal hyperplasia and congenital adrenal hypoplasia, X-linked;congenital amegakaryocytic thrombocytopenia;congenital aniridia;congenital central hypoventilation;Hirschsprung's disease 3;congenital contractile brachioduodenopathy;congenital contractures, hypotonia, and growth retardation of the limbs and face;congenital disorders of glycosylation types 1B, 1D, 1G, 1H, 1J, 1K, 1N, and 1P type 2, type 2C, type 2J, type 2K, type IIm; types I and II congenital dyserythroid anemia; congenital ectodermal dysplasia of the face; congenital erythropoietic porphyria; type 2 congenital generalized lipodystrophy; congenital heart disease, multiple type 2; congenital heart disease; interrupted aortic arch; congenital lipomatous overgrowth, vascular malformations, and epidermal nevi; non-small cell lung cancer; ovarian neoplasms; cardiac conduction disorders, nonspecific; congenital microvillous atrophy; congenital muscular dystrophies; congenital muscular dystrophies due to partial deficiency of LAMA2; congenital muscular dystrophies - dystroglycanopathy with brain and eye abnormalities, types A2, A7, A8, A11, and A14 type;congenital muscular dystrophy - dystroglycanopathy with mental retardation, types B2, B3, B5 and B15;congenital muscular dystrophy - dystroglycanopathy without mental retardation, type B5;congenital hypertrophy-cerebral syndrome;congenital myasthenic syndrome, acetazolamide responsive;congenital myopathy with fiber type imbalance;congenital ocular coloboma;congenital stationary night blindness, types 1A, 1B, 1C, 1E, 1F and 2A;coproporphyria;planokeratosis 2;corneal dystrophy, Fuchs endothelial 4;endothelial corneal dystrophy type 2;corneal fragility keratoglobus, blue sclera and joint hypermobility;Cornelia de Lange syndrome 1 and 5;autosomal dominant coronary artery disease 2;coronary artery disease;hyperalphalipoproteinemia 2; Cortical dysplasia (combined with other brain anomalies) 5 and 6; occipital cortical malformations; corticosteroid-binding globulin deficiency; corticosterone methyloxidase deficiency type 2; Costello syndrome; Cowden syndrome 1; flat coxa; autosomal dominant cranial dysplasia; craniosynostosis 1 and 4; craniosynostosis and dental anomalies; creatine deficiency, X-linked; Crouzon syndrome; cryptophthalmos syndrome; cryptorchidism, unilateral or bilateral; Cushing synostosis; cutaneous melanoma 1; bone dystrophies with severe pulmonary, gastrointestinal, and urinary anomalies cutis laxa with cyanosis;cyanosis, transient neonatal and atypical nephropathy;cystic fibrosis;cystinuria;cytochrome c oxidase I deficiency;cytochrome c oxidase deficiency;D-2-hydroxyglutaric aciduria 2;segmental Darier's disease;hearing loss with cochlear hypoplasia, microtia, and microdontia (LAMM);hearing loss, autosomal dominant 3a, 4, 12, 13, 15, autosomal dominant nonsyndromic sensorineural 17, 20 and 65;hearing loss, autosomal recessive 1A, 2, 3, 6, 8, 9, 12, 15, 16, 18b, 22, 28, 31, 44, 49 , 63, 77, 86 and 89; hearing loss, cochlear, myopic and intellectual disability, no vestibular disorder, autosomal dominant, X-linked 2; 2-methylbutyryl-CoA dehydrogenase deficiency; 3-hydroxyacyl-CoA dehydrogenase deficiency; alpha-mannosidase deficiency; aromatic-L-amino acid decarboxylase deficiency; bisphosphoglycerate mutase deficiency; butyryl-CoA dehydrogenase deficiency; ferroxidase deficiency; galactokinase deficiency; guanidinoacetate methyltransferase deficiency; hyaluronan Glucosaminidase deficiency;ribose-5-phosphate isomerase deficiency;steroid 11-beta-monooxygenase deficiency;UDP glucose-hexose-1-phosphate uridylyltransferase deficiency;xanthine oxidase deficiency;Dejerine-Sottas disease;Charcot-Marie-Tooth disease, ID and IVF;Dejerine-Sottas syndrome, autosomal dominant;dendritic cell, monocyte, B lymphocyte, and natural killer lymphocyte deficiencies;Desbuquois dysplasia 2;Debuquois syndrome;DFNA 2 nonsyndromic hearing loss;diabetes and diabetes insipidus with optic atrophy and deafness;type 2 diabetes, and insulin-dependent 20;Diamond-Blackfan anemia 1, 5, 8, and 10;diarrhoea 3 (secretory sodium, congenital, symptomatic), and 5 (tufting enteropathy) enteropathy);congenital;dicarboxyaminoaciduria;diffuse palmoplantar keratoderma, Bosnian type;digitrenoencephalic 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;steroidogenesis disorder due to cytochrome p450 oxidoreductase deficiency;distal arthrogryposis type 2B;distal hereditary motor neuronopathy type 2B;distal myopathy Markesbery-Griggs type;distal spinal muscular atrophy, X-linked 3;double lash-lymphoedema syndrome;dominant tropholytic epidermolysis bullosa with absence of skin;dominant hereditary optic atrophy;Donnay-Barrow syndrome;dopamine Beta-hydroxylase deficiency; dopamine receptor d2, brain density reduction; Dowling-Degos disease 4; Doyne honeycomb retinal dystrophy; Malattia leventi 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;X-linked dyskeratosis congenita;Familial dyskinesia with facial myokymia;Plasminogen molecule disorders;Dystonia 2 (torsion, autosomal recessive), 3 (torsion, X-linked), 5 (dopa-responsive), 10, 12, 16, 25, 26 (myoclonic);Benign familial infantile seizures 2;Early infantile epileptic encephalopathy 2, 4, 7, 9, 10, 11, 13 and 14;Atypical Rett syndrome;Early T-cell precursor acute lymphoblastic leukemia disease;ectodermal dysplasia-epidermolysis bullosa syndrome;ectodermal dysplasia-syndactyly syndrome 1;ectopic phakia, isolated autosomal recessive and dominant;ectodactyly, ectodermal dysplasia, and cleft lip and palate syndrome 3;Ehlers-Danlos syndrome type 7 (autosomal recessive), classic, type 2 (progeroid), hydroxylysine deficiency, type 4, type 4 variant, and due to tenascin X deficiency;Eichsfeldt congenital muscular dystrophy;cerebroosteodysplasia;S cone enhancement syndrome;enlarged vestibular aqueduct syndrome;enterokinase deficiency;epidermodysplasia verruciformis;epidermolysis bullosa simplex and limb-girdle muscular dystrophy, mottled pigmentation simple with pyloric atresia, simple, autosomal recessive, and with pyloric atresia;epidermolytic palmoplantar keratoderma;familial febrile convulsions 8;epilepsy, childhood absence 2, 12 (idiopathic generalized, susceptible), 5 (nocturnal frontal type), nocturnal frontal type 1, partial, with variable foci, progressive myoclonia 3, and X-linked, with variable learning and behavioral disorders;epileptic encephalopathy, childhood onset, early infancy, 1, 19, 23, 25, 30, and 32;epiphyseal dysplasia, multiple, with myopia and conductive hearing loss;transient ataxia type 2;transient pain syndrome, familial, 3;Epstein syndrome;Fechtner syndrome;erythropoietic protoporphyria;estrogen resistance;exudation Fabry disease and Fabry disease, cardiac variant; two combined deficiencies of factors H, VII, X, V, and VIII; factor XIII subunit deficiency; familial adenomatous polyposis 1 and 3; familial amyloid nephropathy with urticaria and hearing loss; familial cold urticaria; familial cerebellar vermis agenesis; familial benign pemphigus; familial breast cancer; breast cancer susceptibility 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 glomerular cystic kidney disease;familial infantile myasthenia;familial juvenile gout;familial Mediterranean fever, and familial Mediterranean fever, autosomal dominant;familial polypharyngeal syndrome;familial porphyria cutanea tarda;familial pulmonary capillary hemangiomatosis;familial renal glycosuria;familial renal hypouricemia;familial restrictive cardiomyopathy 1;familial types 1 and 3 hyperlipoproteinemia;Fanconi anemia, complement groups E, I, N and O;Fanconi-Bickel syndrome;Favism susceptibility (Favism, susceptibility to);febrile convulsions, 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-Haber syndrome;focal epilepsy with speech disorder with or without mental retardation;focal segmental glomerulosclerosis 5;forebrain coloboma;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 neuropathic;familial horizontal gaze palsy with progressive scoliosis;generalized dominant dystrophic epidermolysis bullosa;generalized epilepsy with febrile seizures plus type 3, 1, 2;Lennox-Gastaut epileptic encephalopathy;giant axonal neuropathy;Glanzmann thrombosis;glaucoma 1, open angle, e, F and G;glaucoma 3, primary congenital, d;congenital glaucoma, and congenital glaucoma, coloboma;glaucoma, primary open angle, early-onset;glioma susceptibility 1;glucose transporter type 1 deficiency syndrome;glucose-6-phosphate transport deficiency;GLUT1 deficiency syndrome 2;idiopathic generalized epilepsy susceptibility, 12 ;glutamate forminotransferase deficiency;glutaric acidemia IIA and IIB;glutaric aciduria type 1;glutathione synthetase deficiency;glycogen storage diseases types 0 (muscle), II (adult form), IXa2, IXc, 1A; types II, IV, IV (combined hepatic and myopathic), V and VI;Goldman-Fabre syndrome;Gordon syndrome;Gorlin syndrome;holoprosencephaly sequence;holoprosencephaly 7;granulomatous disease, chronic, X-linked, variant;granulosa cell tumor of the ovary;gray platelet syndrome;Griselli syndrome type 3;Grenau corneal dystrophy type I;growth and mental retardation, mandibulofacial dysmorphism, microcephaly, and Cleft palate;growth hormone deficiency with pituitary abnormalities;growth hormone insensitivity with immunodeficiency;GTP cyclohydrolase I deficiency;Hajj-Cheney syndrome;hand-foot-uterine syndrome;hearing impairment;infantile capillary hemangioma;hematological neoplasms;hemochromatosis types 1, 2B, and 3;microvascular complications of diabetes mellitus 7;transferrin serum level quantitative trait locus 2;hemoglobin H disease, nondeficient;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 Cancer syndromes; ataxia-telangiectasia-like disorder; hereditary diffuse gastric cancer; hereditary diffuse leukoencephalopathy with spheroids; hereditary factor II, IX, and VIII deficiency; 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 colon neoplasms; Lynch syndrome I and II; hereditary pancreatitis; chronic pancreatitis susceptibility; hereditary sensory and autonomic neuropathy types IIB and IIA; hereditary sideroblastic anemia; Hermansky-Podlak syndromes 1, 3, 4, and 6; visceral paresis position, 2, 4, and 6, autosomal; heterotaxy, X-linked; histiocytic medullary reticulosis; heterotopia; histiocytosis-lymphadenopathy plus syndrome; holocarboxylase synthase deficiency; holoprosencephaly 2, 3, 7, and 9; Holt-Oram syndrome; homocysteinemia due to MTHFR deficiency, CBS deficiency, and homocysteinuria, pyridoxine responsive; homocysteinuria-megaloblastic anemia, cblE complementation type, due to abnormal cobalamin metabolism; Howel-Evans syndrome; Hurler syndrome; Hutchinson-Gilford syndrome; hydrocephalus; hyperammonemia, type III; hypercholesterolemia hypercholesterolemia, autosomal recessive;hyperconvulsant 2 and hereditary hyperconvulsant 2;hyperferritinemia cataract syndrome;hyperglycinuria;hyperimmunoglobulin D with periodic fever;mevalonic aciduria;hyperimmunoglobulin E syndrome;familial hyperinsulinemic hypoglycemia 3, 4 and 5;hyperinsulinemia-hyperammonemia syndrome;hyperlysinemia;hypermanganemia with dystonia, polycythemia, and cirrhosis;hyperornithinemic-hyperammonemia-homocitrullinuria syndrome;hyperparathyroidism 1 and 2;hyperparathyroidism, neonatal severe;partial pts deficiency, BH4 deficiency, D, non Hyperphenylalaninemia, BH4 deficiency, A due to PKU; hyperphosphatemia with mental retardation syndromes 2, 3, and 4; polychondrodysplasia; hypobetalipoproteinemia, familial, associated with APOB32; hypocalcemia, autosomal dominant 1; hypocalciuric hypercalcemia, familial, types 1 and 3; hypochondroplasia; hypochromic microcytic anemia with iron overload; hypoglycemia with hepatic glycogen synthase deficiency; hypogonadotropic hypogonadism with or without anosmia 11; anhidrotic ectodermal dysplasia with immunodeficiency; X-linked ectodermal dysplasia with hypohidrosis; hypokalemic periodic paralysis 1 and 2 ;hypomagnesemia 1, intestinal;hypomagnesemia, seizures, and mental retardation;hypomedullary leukodystrophy 7;hypoplastic left heart syndrome;atrioventricular septal defect and common atrioventricular junction;hypospadias 1 and 2, X-linked;hypothyroidism, congenital nongoiter, 1;hypotrichosis 8 and 12;hypotrichosis-lymphoedema-vasoectasia syndrome;I blood group system;bullous ichthyosis of Siemens type;exfoliative ichthyosis;ichthyosis of prematurity syndrome;idiopathic basal ganglia calcification 5;idiopathic fibrosing alveolitis, chronic type;dyskeratosis congenita, autosomal dominant, 2 and 5;idiopathic hypercalcemia of infancy;immune dysfunction with T-cell inactivation due to impaired calcium influx 2; Immunodeficiency15,16,19,30,31C,38,40,8 with hyper-IgM, due to defects in CD3-zeta, and with magnesium deficiency, X-linked, Epstein-Barr virus infection, and neoplasia; immunodeficiency-centromeric instability-facial malformation syndrome2; inclusion body myopathy2 and 3; Nonaka myopathy; infantile spasms and paroxysmal chorea, familial; infantile cortical hyperostosis; infantile GM1 gangliosidosis; infantile hypophosphatasia; infantile nephronophthisis; infantile nystagmus, X-linked; infantile Parkinson's disease-dystonia; infertility associated with polyturias and excess DNA; insulin resistance; insulin-resistant diabetes and black table erythroderma; insulin-dependent diabetes mellitus secretory diarrhea syndrome; interstitial nephritis, karyomegalic; intrauterine growth retardation, metaphyseal dysplasia, congenital adrenal hypoplasia, and genital anomalies; iodotyrosine binding deficiency; IRAK4 deficiency; iris angle dysplasia dominant and type 1; brain iron accumulation; ischiopatellar dysplasia; islet cell hyperplasia; isolated 17,20-lyase deficiency; isolated lutropin deficiency; isovaleryl-CoA dehydrogenase deficiency; Jankovic-Rivera syndrome; Jarvell and Lange-Nielsen syndromes 2; Joubert syndromes 1, 6, 7, 9/15 (bigénicent), 14, 16, and 17, and orofaciodigital syndrome xiv; Herlitz's joint syndrome Syndesmotic epidermolysis bullosa;Juvenile GM>1<gangliosidosis;Juvenile polyposis syndrome;Juvenile polyposis/hereditary hemorrhagic telangiectasia syndrome;Juvenile retinitis;Kabuki makeup syndrome;Kalmann syndrome 1, 2 and 6;Delayed puberty;Kanzaki disease;Karak syndrome;Kartagener syndrome;Kenny-Cafe syndrome type 2;Köppen-Lubinsky syndrome;Keratoconus 1;Folliculor keratosis;Palmoplantar pustulosis 1;Kindler syndrome;L-2-hydroxyglutaric aciduria;Larsen syndrome, dominant;Lattice corneal dystrophy type III;Leber amaurosis;Zellweger syndrome;Peroxisome biogenesis disorders;Tu Wellweger syndrome spectrum;Leber congenital amaurosis 11, 12, 13, 16, 4, 7 and 9;Leber optic atrophy;Aminoglycoside-induced hearing loss;Hearing loss, nonsyndromic, sensorineural, mitochondrial;Left ventricular noncompaction 5;Leigh-right axis anomaly;Leigh disease;Mitochondrial short-chain enoyl-CoA hydratase 1 deficiency;Leigh syndrome due to mitochondrial complex I deficiency;Reiner disease;Lelly-Weil chondro-osseous dysplasia;Lethal congenital contracture syndrome 6;Leukocyte adhesion deficiency types I and III;Leukodystrophy, hypomyelination, 11 and 6;Leukoencephalopathy, with ataxia, with brainstem and spinal cord involvement and elevated lactate, with loss of white matter, and progressive , with ovarian failure;white nails;dementia with Lewy bodies;Liechtenstein-Knorr syndrome;Li-Fraumeni syndrome 1;Lig4 syndrome;limb-girdle muscular dystrophy types 1B, 2A, 2B, 2D, C1, C5, C9, C14;congenital muscular dystrophy-dystroglycanopathy with brain and eye abnormalities, types A14 and B14;combined lipase deficiency;lipoproteinosis;lipodystrophy, familial partial, types 2 and 3;liary dysencephaly 1,2 (X-linked), 3,6 (with microcephaly), X-linked;subcortical band heterotopia, X-linked;acute infantile liver failure;Loeys-Dietz syndrome 1,2,3;long QT syndrome 1,2,2/9,2/5,( 2, genetic), 3, 5 and 5, acquired, susceptible;lung cancer;lymphoedema, hereditary, id;lymphoedema, primary, with myelodysplasia;lymphoproliferative syndrome 1, 1 (X-linked) and 2;lysosomal acid lipase deficiency;macrophagia, gigantism, facial dysmorphism syndrome;macular degeneration, vitelliform, adult onset;malignant hyperthermia type 1;malignant lymphoma, non-Hodgkin;malignant melanoma;malignant tumor of the prostate;mandibular dysplasia;mandibular dysplasia with lipodystrophy types A or B, atypical;mandibulofacial dysplasia, Treacher-Collins type, autosomal recessive;mannose-binding protein deficiency;maple syrup urine disease types 1A and 3;Malden-Walker-like syndrome;malf Fan syndrome;Marinesco-Sj＼xc3＼xb6gren syndrome;Martsolf syndrome;Maturity-onset diabetes mellitus, types 1, 2, 11, 3 and 9;May-Hegglin syndrome;MYH9-related disorders;Sebastian syndrome;McCun-Albright syndrome;Somatic adenoma;Sex cord-stromal tumor;Cushing syndrome;Macksick-Kauffman syndrome;McLeod neuroerythrocytosis syndrome;Meckel-Gruber syndrome;Medium-chain acyl-coenzyme A dehydrogenase deficiency;Medulloblastoma;Cerebral leukoencephalopathy with subcortical cysts 1 and 2a;Macrocephaly-congenital marbled cutis;PIK3CA-related overgrowth spectrum;Megalencephaly-polymicrogyria-polydactyly-hydrocephalus syndrome 2;megaloblastic anemia, thiamine-responsive, with diabetes mellitus, and sensorineural hearing loss;Meyer-Gorlin syndrome 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;X-linked mental retardation syndrome 5;mental retardation, maxillary prognathism, 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, stereotypical behavior, epilepsy, and/or brain malformations;mental retardation, syndromic, Claes-Jensen, X-linked;mental retardation, X-linked, nonspecific merosin-deficient congenital muscular dystrophy;metachromatic leukodystrophy juvenile, late infantile, and adult;metachromatic leukodystrophy;metamorphic bone dysplasia;methemoglobinemia types I and 2;methionine adenosyltransferase deficiency, autosomal dominant;methylmalonic acidemia with homocystinuria;methylmalonic aciduria type cblB;methylmalonic aciduria due to methylmalonyl-CoA mutase deficiency;methylmalonic aciduria type mut(0);microcephalic osteodysplasia 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 paraplegia50, autosomal recessive;generalized developmental delay;CNS hypomyelination;cerebral atrophy;microcephaly, normal intelligence and immunodeficiency;microcephaly-capillary malformation syndrome;microcytic anemia;syndromic microphthalmia5,7 and 9;microphthalmia, isolated3,5,6,8, and with coloboma6;microcytosis;migraine, familial hemiplegic;Miller syndrome;minicore myopathy with external ophthalmoplegia;congenital myopathy with core;Mitchell-Riley syndrome;mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthetase deficiency;mitochondrial complexes I, II, III, III (nuclear Mitochondrial DNA depletion syndromes 11, 12 (cardiomyopathic type), 2, 4B (MNGIE type), 8B (MNGIE type); mitochondrial DNA depletion syndromes 3 and 7, hepatocerebral type 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; distal myopathy with pretibial involvement; Mohr-Tranebjaerg syndrome; molybdenum cofactor deficiency, complementation group A; Mowat-Wilson syndrome; mucolipidosis III gamma; mucopolypeptide Saccharidosis type VI, type VI (severe) and type VII;mucopolysaccharidosis, MPS-IH/S, MPS-II, MPS-III-A, MPS-III-B, MPS-III-C, MPS-IV-A, MPS-IV-B;retinitis pigmentosa 73;gangliosidosis GM1 type 1 (with cardiac involvement) 3;multicentric osteolytic nephropathy;multicentric osteolysis, nodular disease and arthropathy;multiple congenital anomalies;atrial septal defect 2;multiple congenital anomalies-hypotonia-seizure syndrome 3;multiple skin and mucosal venous malformations;multiple endocrine neoplasia types 1 and 4;multiple epiphyseal dysplasia type 5 or dominant;multiple intestinal atresia;multiple pterygium syndrome Escobar type;multiple sulfatase deficiency;multiple Synostosis syndrome 3;muscle AMP guanine oxidase deficiency;muscle oculoencephalopathy;muscular dystrophy, congenital, megaconoid type;myasthenia, familial childhood, 1;congenital myasthenic syndrome 11, associated with acetylcholine receptor deficiency;congenital myasthenic syndrome 17, without 2A (slow channel), 4B (fast channel) and tubular assembly;myeloperoxidase deficiency;MYH-associated polyposis;endometrial cancer;myocardial infarction 1;myoclonic dystonia;myoclonic-atonic epilepsy;red rag fiber myoclonic epilepsy;myofibrillar myopathy 1 and ZASP associated;myoglobinuria, acute relapsing, autosomal recessive;myoneurogastroenteric cerebral cerebellar ataxia with progressive external ophthalmoplegia;mitochondrial DNA depletion syndrome 4B, MNGIE type;myopathy, central, 1, congenital, with excess muscle spindles, distal, 1, lactic acidosis, and sideroblastic anemia 1;mitochondrial progressive with congenital cataracts, hearing loss, and developmental delay, and tubular aggregates, 2;myopia 6;myosclerosis, autosomal recessive;myotonia congenita;myotonia congenita, autosomal dominant and recessive;nail-patella syndrome;Nance-Hollan syndrome;microphthalmia 2;Navajo neurohepatopathy;nemaline myopathy 3 and 9;neonatal hypotonia;intellectual disability;seizures;language delay;mental retardation, autosomal dominant 31;citrine deficiency Neonatal intrahepatic cholestasis caused by nephropathy;nephrogenic diabetes insipidus, nephrogenic diabetes insipidus, X-linked;nephrolithiasis/osteoporosis, hypophosphatemic, 2;nephronophthisis 13, 15 and 4;infertility;cerebro-ocular-renal syndrome (nephronophthisis, oculomotor ataxia, and cerebellar abnormalities);nephrotic syndrome, types 3, 5, with or without ocular abnormalities, types 7 and 9;Nester-Greermoprogeria syndrome;Neu-Laxova syndrome 1;neurodegeneration with brain iron accumulation 4 and 6;neuroferritinopathy;neurofibromatosis, types 1 and 2;neurofibrosarcoma;neurohypophyseal diabetes insipidus;neuropathy, hereditary sensory, type IC;neutral 1 amino acid transport disorder;myopathy Neutral lipid storage disease with leukemia;Neutrophil immunodeficiency syndrome;Nicolades-Baraist syndrome;Niemann-Pick disease types C1, C2, A, and C1, adult type;Nonketotic hyperglycinemia;Noonan syndrome 1 and 4, LEOPARD syndrome 1;Noonan-like disorder with or without juvenile myelomonocytic leukemia;Emokalemic periodic paralysis, potassium sensitivity;Norm disease;Epilepsy, deafness, and mental retardation syndrome;Mental retardation, X-linked 102 and syndromic 13;Obesity;Ocular albinism, type I;Oculocutaneous albinism types 1B, 3, and 4;Oculodentodigital dysplasia;Odontohypophosphatasia;Odonto-hair-limb syndrome syndrome); macrostomia; hypodontia-colorectal cancer syndrome; Opitz G/BBB syndrome; optic atrophy 9; orofacial dactyly syndrome; ornithine aminotransferase deficiency; orofacial clefts 11 and 7; lip/palate ectodermal dysplasia syndrome; Olstavic-Lindemann-Solberg syndrome; osteoarthritis with mild chondrodysplasia; osteochondritis dissecans; osteogenesis imperfecta types 12, 5, 7, 8, I, and III, with normal sclera, dominant, recessive perinatal lethal; craniosclerosis striated osteomyelopathy with osteoporosis;autosomal dominant osteopetrosis types 1 and 2, recessive type 4, recessive type 1, recessive type 6;osteoporosis with pseudoglioma;otopalatodactyl syndrome types I and II;ovarian hypoplasia 1;ovarian leukoencephalopathy;congenital pachyonychia types 4 and 2;Paget's disease of bone, familial;Pallister-Hall syndrome;palmoplantar keratoderma, non-epidermolytic, focal or diffuse;pancreatic agenesis and congenital heart disease;Papillon-Lef＼xc3＼xa8vre syndrome;paraganglioma 3;von Oleinburg Congenital anomaly myotonia of Eulenburg;parathyroid carcinoma;Parkinson's disease 14, 15, 19 (early onset), 2, 20 (early onset), 6, (autosomal recessive early onset) and 9;localized albinism;hypoxanthine-guanine phosphoribosyltransferase partial deficiency;pattern dystrophy of the retinal pigment epithelium;PC-K6a;Pelizaeus-Merzbacher disease;Pendred syndrome;peripheral demyelinating neuropathy, central demyelinating;Hirschsprung's disease;permanent neonatal diabetes mellitus;permanent neonatal diabetes mellitus, with neurological features;neonatal insulin-dependent diabetes mellitus;maturity-onset diabetes mellitus of the young, Type 2;Peroxisome biogenesis disorders 14B, 2A, 4A, 5B, 6A, 7A and 7B;Perot syndrome 4;Perry syndrome;Persistent hyperinsulinemic hypoglycemia of the newborn;Familial hyperinsulinism;Phenotype;Phenylketonuria;Pheochromocytoma;Hereditary paraganglioma-pheochromocytoma syndrome;Paraganglioma 1;Intestinal carcinoid tumors;Cowden syndrome 3;Phosphoglycerate dehydrogenase deficiency;Phosphoglycerate kinase 1 deficiency;Photosensitive sulfur deficiency hair dystrophy;Phytanic acid storage disease;Pick's disease;Pearson syndrome;Pigmentary retinal dystrophy;Pigmented nodular adrenal corticopathy aberrant, primary, 1;calcifying epithelioma;Pitt-Hopkins syndrome;pituitary-dependent hypercortisolism;pituitary hormone deficiency, combined types 1, 2, 3, and 4;plasminogen activator inhibitor type 1 deficiency;plasminogen deficiency, type I;platelet-type bleeding disorders 15 and 8;poikiloderma, with hereditary fibrosis, tendon contractures, myopathy, and pulmonary fibrosis;polycystic kidney disease 2, adult and infantile types;polycystic lipomembranous dysplasia with sclerosing leukoencephalopathy;polyglucosan body myopathy 1 with or without immunodeficiency;polymicrogyria, asymmetric, bilateral frontoparietal;polyneuritis, Hearing loss, ataxia, retinitis pigmentosa, and cataract;pontocerebellar hypoplasia type 4;popliteal fold syndrome;porencephaly 2;porokeratosis 8, contagious superficial chemoactive type;porphobilinogen synthase deficiency;porphyria cutanea tarda;spinal 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 heart noncompaction 6;4,left heart noncompaction 10;paroxysmal atrial fibrillation;primary hyperoxaluria, types I and III;primary hypertrophic bone Arthropathy, autosomal recessive 2;primary hypomagnesemia;primary open-angle glaucoma early-onset 1;primary pulmonary hypertension;Primrose syndrome;progressive familial heart block type 1B;progressive familial intrahepatic cholestasis 2 and 3;progressive intrahepatic cholestasis;progressive myoclonic epilepsy with ataxia;progressive pseudorheumatic dysplasia;progressive sclerosing poliodystrophy;prolidase deficiency;proline dehydrogenase deficiency;schizophrenia 4;propazine deficiency, X-linked;propionic acidemia;proprotein convertase 1/3 deficiency;prostate cancer, hereditary, 2;protan deficiency;proteinuria;Finland congenital nephrotic syndrome; Proteus syndrome; breast cancer; pseudoachondroplasia spondyloepiphyseal dysplasia; 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 deficiencies; psoriasis susceptibility 2; PTEN hamartoma syndrome; pulmonary arterial hypertension associated with hereditary hemorrhagic telangiectasia; pulmonary fibrosis and/or bone marrow failure, telomere-related, 1 and and 3; pulmonary hypertension, primary, with hereditary hemorrhagic telangiectasia; 1; purine-nucleoside phosphorylase deficiency; pyruvate carboxylase deficiency; pyruvate dehydrogenase E1-alpha deficiency; erythrocyte pyruvate kinase deficiency; Raine syndrome; rasopathy; recessive dystrophic epidermolysis bullosa; nail disorder, nonsyndromic congenital, 8; Reifenstein syndrome; renal coloboma syndrome; renal dysplasia; renal dysplasia, pigmentary retinal dystrophy, cerebellar ataxia, and skeletal dysplasia; with delayed sensorineural hearing loss or with hemolytic anemia renal tubular acidosis, distal, autosomal recessive; renal tubular acidosis, proximal, with ocular abnormalities and mental retardation; 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's disorder; rhabdoid tumor predisposition syndrome 2; rhegmatogenous retinal detachment, autosomal dominant; rhabdoid chondrodysplasia punctata types 2 and 3; Roberts-SC azoobium limb anomaly syndrome; Robinow-Solauf Sorauf syndrome;Robinow syndrome, autosomal recessive, autosomal recessive with brachypolydactyly;Rothmund-Thomson syndrome;Lapadellino syndrome;RRM2B-related mitochondrial disease;Rubinstein-Taybi syndrome;Salla disease;Sandhoff disease, adult and infantile;Sarcoidosis, early onset;Blau syndrome;Schindler disease, type 1;schizencephaly;schizophrenia 15;cochlear-pelvic dysplasia;schwannomatosis 2;Schwartz-Jampel syndrome type 1;keratosclerosis, autosomal recessive;sclerosteosis;secondary hypothyroidism;Segawa syndrome, autosomal recessive;Senior-Loken syndromes 4 and 5;sensory ataxic neuropathies Qi, dysarthria, and ophthalmoplegia;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 with NK cell positive;severe congenital neutropenia;severe congenital neutropenia 3, autosomal recessive or dominant;severe congenital neutropenia and 6, autosomal recessive;severe myoclonic epilepsy of infancy;generalized epilepsy with febrile convulsions, types 1 and 2;severe X-linked myotubular myopathy;short QT syndrome type 3;short stature with nonspecific skeletal abnormalities;short stature, auditory canal stenosis , mandibular hypoplasia, skeletal abnormalities; short stature, onychodysplasia, facial dysmorphism, and hypotrichosis; primordial dwarfism; short-costothoracic dysplasia 11 or 3 with or without polydactyly; sialidosis types I and II; Silver spastic paraplegia syndrome; nerve conduction velocity slowing, autosomal dominant; Smith-Lemli-Opitz syndrome; Snyder-Robinson syndrome; growth hormone-secreting adenoma; prolactinoma; familial, predisposition to pituitary adenoma; 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 disorder, congenital, 3; spermatogenesis impair, 11, 3, and 8; spherocytosis types 4 and 5; spherocytosis, spherocytosis, 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; spinocarpal synostosis syndrome; spondylopalmal dysplasia, Ehlers-Danlos-like, with immune dysregulation, aggrecan type, with congenital joint dislocation, brachy-hand type, Sedaghatian type, with cone-rod dystrophy, and Kozlowski type; twisted 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, childhood onset;Storm-Olken syndrome;Sturge-Weber syndrome, capillary malformation, congenital, 1;succinyl-CoA acetoacetate transferase deficiency;sucrase-isomaltase deficiency;sudden infant death syndrome;subcutaneous Sulfate oxidase deficiency, isolated;Supravalvular aortic stenosis;Pulmonary surfactant metabolic dysfunction, 2 and 3;Synopsis, proximal, 1b;Senanyi-Lenz syndactyly;Syndactyly type 3;Syndromic X-linked mental retardation 16;Equilateral talipes;Tangier disease;TARP syndrome;Tay-Sachs disease, B1 variant;Gm2 gangliosidosis (adult);Gm2 gangliosidosis (adult onset);Temtamy syndrome;Tenorio syndrome;Epiphyseal dysplasia formation;testosterone 17-beta-dehydrogenase deficiency;atetramelia, autosomal recessive;tetralogy of Fallot;hypoplastic left heart syndrome 2;aneurysm;cardiac and great vascular anomalies;ventricular septal defect 1;Thiel-Behnke corneal dystrophy;thoracic aortic aneurysm and aortic dissection;marfanoid habitus;3M syndrome 2;thrombocytopenia, platelet dysfunction, hemolysis, and globin synthesis imbalance;thrombocytopenia, X-linked;thrombosis, hereditary, due to protein C deficiency, autosomal dominant and recessive;thyroid agenesis;thyroid cancer, follicular;thyroid hormone metabolism disorders;thyroid hormone resistance, generalized, autosomal dominant;thyrotoxic periodic paralysis and thyrotoxic periodic paralysis 2;cytotropin-releasing hormone resistance, generalized;timothy syndrome;tumor necrosis factor receptor-associated periodic fever syndrome (TRAPS);tooth agenesis, selective, 3 and 4;polymorphic ventricular tachycardia (Torsades de pointes);Townes-Brocks branchio-oto-nephroid syndrome;Epidermolysis bullosa transientes of the newborn;Treacher Collins syndrome 1;Lymphocytosis with mental retardation, dwarfism, and pigmentary degeneration of the retina;Trigeminal ganglion dysplasia type I;Trigeminal ganglion syndrome type 3;Trimethylaminuria;Tuberous sclerosis syndrome;Lymphangiomyomatosis;Tuberous sclerosis complex 1 and 2;Tyrosinase-negative oculocutaneous albinism;Tyrosinase-positive oculocutaneous albinism;Tyrosinemia type I;UDP-glucose-4-epimerase deficiency;Ullrich congenital muscular dystrophy;Ulnar-peroneal deficiency with severe limb defects;Upshaw-Schulman syndrome;Urocanic acid hydratase deficiency;Usher syndrome, types 1, 1B, 1D, 1G, 2A, 2C, and 2D;Retinitis pigmentosa 39;UVA-sensitive syndrome;Van der Woude Woude syndrome; Van Maldergem syndrome 2; Henkam lymphangiectasia-lymphedema syndrome 2; variant porphyria; ventricular enlargement with cystic kidney disease; Verheij syndrome; very long-chain acyl-CoA dehydrogenase deficiency; vesicoureteral reflux 8; situs inversus 5, autosomal; abdominal myopathy; vitamin D-dependent rickets, types 1 and 2; vitellogenic dystrophy; von Willebrand disease types 2M and 3; Waardenburg syndrome types 1, 4C, and 2E (with neurological involvement); Klein-Waardenburg syndrome; Walker-Warburg congenital muscular dystrophy; Warburg-Micro syndrome 2 and 4; IgE , hypogammaglobulinemia, infection, and myeloid cellular pools;Weaver syndrome;Weill-Marchesani syndromes 1 and 3;Weill-Marchesani-like syndromes;Weissenbacher-Zweymuller syndrome;Werdnig-Hoffmann disease;Charcot-Marie-Tooth disease;Werner syndrome;WFS1-related disorders;Wiedemann-Steiner syndrome;Wilson's disease;Wolfram-like syndromes, autosomal dominant;Worth disease;van Buchem disease type 2;xeroderma pigmentosum, complementation groups b, D, E, and G;X-linked agammaglobulinemia;X-linked Hereditary motor and sensory neuropathies; X-linked ichthyosis with sterylsulfatase deficiency; X-linked periventricular heterotopic gray matter; Otopalatodactyl syndrome, type I; X-linked severe combined immunodeficiency; Zimmermann-Laband syndrome and Zimmermann-Laband syndrome 2; and Zonular powdery cataract 3.

The target nucleotide sequence may include a target sequence (e.g., a point mutation) associated with a disease, disorder, or condition. The target sequence may include a T to C (or A to G) point mutation associated with a disease, disorder, or condition, where deamination of the mutant C base results in mismatch repair-mediated correction to a sequence not associated with the disease, disorder, or condition. The target sequence may include a G to A (or C to T) point mutation associated with a disease, disorder, or condition, where deamination of the mutant A base results in mismatch repair-mediated correction to a sequence not associated with the disease, disorder, or condition. The target sequence may code for a protein, where the point mutation is in a codon, resulting in a change in the amino acid encoded by the mutant codon compared to the wild-type codon. The target sequence may also be in a splice site, where the point mutation results in an altered splicing of the mRNA transcript compared to the wild-type transcript. In addition, the target may also be in a non-coding sequence of a gene, such as a promoter, where the point mutation results in increased or decreased expression of the gene.

Thus, in some aspects, deamination of mutant C results in a change in the amino acid encoded by the mutant codon, which may in some cases result in the expression of the wild-type amino acid. In other aspects, deamination of mutant A results in a change in the amino acid encoded by the mutant codon, which may in some cases result in the expression of the 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 some embodiments, the contacting step occurs in a subject. In some 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 specific embodiments, the methods involve contacting a retinal cell, a cortical cell, or a cerebellar cell.

The split Cas9 protein or split prime editor delivered using the methods described herein preferably has comparable activity to the original Cas9 protein or prime editor (i.e., the unsplit protein delivered to or expressed by the cell in its entirety). 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 the 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 from which the subject suffers. Any disease or disorder that can be treated and/or prevented using CRISPR/Cas9-based genome editing technology may be treated by the split Cas9 protein or split prime editor described herein. It should be understood that in cases where the nucleotide sequence encoding the split Cas9 protein or prime editor does not further encode a gRNA, a separate nucleic acid vector encoding a gRNA may be administered together with the compositions described herein.

Exemplary suitable diseases, disorders, or conditions include, without limitation, a disease or condition selected from the group consisting of: cystic fibrosis, phenylketonuria, epidermolytic hyperkeratosis (EHK), chronic obstructive pulmonary disease (COPD), Charcot-Marie-Tooth disease type 4J, neuroblastoma (NB), von Willebrand disease (vWD), congenital myotonia, hereditary renal amyloidosis, dilated cardiomyopathy, hereditary lymphedema, familial Alzheimer's disease, prion disease, chronic infantile neurological cutaneous and articular syndrome (CINCA), congenital hearing loss, Niemann-Pick disease type C (NPC) disease, and desmin-related myopathy (DRM). In a specific embodiment, 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 the NPC gene, the DNMT1 gene, the PCSK9 gene, or the TMC1 gene. In some embodiments, the point mutation is a T3182C mutation in NPC, which results in an I1061T amino acid substitution.

In some 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 hearing function. The Y182C amino acid substitution is associated with congenital hearing loss.

In some embodiments, the disease, disorder, or condition is associated with a stop codon, e.g., a point mutation that creates a premature stop codon in the coding region of a gene.

Additional exemplary diseases, disorders, or 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 use a deaminase fusion protein to correct the genetic defect); phenylketonuria - e.g., a phenylalanine to serine mutation (T>C mutation) at position 835 (mouse) or 240 (human) of the phenylalanine hydroxylase gene or at a homologous residue - see, e.g., McDonald et al. al., Genomics. 1997;39:402-405; Bernard-Soulier syndrome (BSS) - e.g., a phenylalanine to serine mutation at position 55 or homologous residues of platelet membrane glycoprotein IX or a cysteine to arginine at residue 24 or homologous residues (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., a leucine to proline mutation at position 160 or 161 (counting the initiator methionine) or homologous residues of 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., a leucine to proline mutation (T>C mutation) at positions 54 or 55 (counting the initiator methionine) or homologous residues in the processed form of _α1 -antitrypsin or at residue 78 or homologous residues in the unprocessed form - see, e.g., Poller et al., Genomics. 1993;17:740-743. see also UNIPROT database accession number P01011; Charcot-Marie-Tooth disease type 4J - e.g., an isoleucine to threonine mutation at position 41 or a homologous residue in FIG4 (T>C mutation) - see, e.g., Lenk et al., PLoS Genetics. 2011;7:e1002104; Neuroblastoma (NB) - e.g., a 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., a 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., See al., Br. J. Haematol. 1992. see also UNIPROT database accession number P04275;82:66-72;myotonia congenita - e.g., a cysteine to arginine mutation (T>C mutation) at position 277 or a homologous residue in the muscle chloride channel gene CLCN1 - see, e.g., Weinberger et al., The J. of Physiology. 2012;590:3449-3464;hereditary renal amyloidosis - e.g., a stop codon to arginine mutation (T>C 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 - see, e.g., Yazaki et al., Kidney Int. 2003;64:11-16;dilated cardiomyopathy (DCM) - e.g., a tryptophan to arginine mutation (T>C mutation) at position 148 or a homologous residue in the FOXD4 gene, see, e.g., Minoretti et.al., Int. J. of Mol. Med. 2007;19:369-372; hereditary lymphedema - e.g., a histidine to arginine mutation (A>G mutation) at position 1035 or a homologous residue in VEGFR3 tyrosine kinase, see e.g., Irrthum et al., Am. J. Hum. Genet. 2000;67:295-301; familial Alzheimer's disease - e.g., an isoleucine to valine mutation (A>G mutation) at position 143 or a homologous residue in presenilin 1, see e.g., Gallo et.al., J. Alzheimer's Disease. 2011;25:425-431; prion disease - e.g., a methionine to valine mutation (A>G mutation) at position 129 or a homologous residue in the prion protein - see e.g., Lewis et.al., J. of General Virology. 2006;87:2443-2449; chronic infantile neurological cutaneous articular syndrome (CINCA) - e.g., a tyrosine to cysteine mutation (A>G mutation) at position 570 or homologous residues in cryopyrin - see, e.g., Fujisawa et.al. Blood. 2007;109:2903-2911; and desmin-related myopathy (DRM) - e.g., an arginine to glycine mutation (A>G mutation) at position 120 or homologous residues in αβ crystallin. See, e.g., Kumar et al., J. Biol. Chem. 1999;274:24137-24141. The entire contents of all references and database entries are incorporated herein by reference.

Trinucleotide repeat expansion diseases Trinucleotide repeat expansions are associated with a number of human diseases, including Huntington's disease, fragile X syndrome, and Friedreich's ataxia. The most common trinucleotide repeat contains a CAG triplet, but GAA triplets (Friedreich's ataxia) and CGG triplets (Fragile X syndrome) also occur. Inheriting a predisposition to an 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.

Following the general mechanism outlined in Figure 1G or Figure 22, a region upstream of the repeat region can be nicked by an RNA-guided nuclease and then used to prime the synthesis of a new DNA strand containing a healthy number of repeats (this depends on the specific gene and disease). The repeat sequence is followed by a short stretch of homology that matches the identity of the sequence adjacent to the other end of the repeat (the red strand). Invasion of the newly synthesized strand by the TPRT system and subsequent replacement of the endogenous DNA by the newly synthesized flap leads to a truncated repeat allele. The term "truncated" 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 can be used to shorten trinucleotide repeat mutations (or "triplet expansion disorders") to treat diseases 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 cognitive and sensorimotor functions. The disorders show genetic anticipation (i.e., increasing severity with each generation). DNA expansion or shortening usually occurs meiotically (i.e., during gamete formation or earlier during embryonic development) and often has a sex bias, meaning that some genes are expanded only when inherited from females and others only from males. 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 to be produced with large repeated amino acid sequences. This either disables or alters protein function, often in a dominant-negative manner (e.g., polyglutamine diseases).

Without being bound by theory, triplet expansion is caused by slippage during DNA replication or DNA repair synthesis. Because the tandem repeats have identical sequences to each other, base pairing between the two DNA strands can occur at multiple points along the sequence. This can lead to the formation of "loop-out" structures during DNA replication or DNA repair synthesis. This can lead to repeated copies of the repeat sequence, extending the number of repeats. Additional mechanisms involving hybrid RNA:DNA intermediates have been proposed. Prime editing can be used to reduce or eliminate these triplet expansion regions by deleting one or more of the offending repeated codon triplets. In one embodiment of this use, FIG. 23 provides a schematic of PEgRNA design for shortening or reducing trinucleotide repeat sequences by prime editing.

Prime editing can be implemented to shorten the triplet expansion region by nicking a region upstream of the triplet repeat region with a prime editor containing a dedicated PEgRNA targeted to the site to be cut. The prime editor then synthesizes a new DNA strand (ssDNA flap) based on the PEgRNA as a template (i.e., its editing template) that encodes a healthy number of triplet repeats (this depends on the specific gene and disease). The newly synthesized ssDNA strand contains the healthy triplet repeat sequence and is also synthesized to include a short stretch of homology (i.e., a 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 by the newly synthesized ssDNA flap leads to a shortened repeat allele.

Depending on the specific trinucleotide expansion disorder, the triplet expansion inducing defect may occur in a "trinucleotide repeat expansion protein." Trinucleotide repeat expansion proteins are a diverse set of proteins that are associated with susceptibility to 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). Thus, these disorders are referred to as polyglutamine (polyQ) disorders and include the following diseases: Huntington's disease (HD); spinal-bulbar muscular atrophy (SBMA); spinocerebellar ataxia (SCA types 1, 2, 3, 6, 7, and 17); and dentatorubral-pallidoluysian atrophy (DRPLA). The remaining trinucleotide repeat expansion disorders either do not involve a CAG triplet or the CAG triplet is not in the coding region of the gene and are therefore referred to as non-polyglutamine disorders. Non-polyglutamine disorders include Fragile X syndrome (FRAXA); Fragile XE mental retardation (FRAXE); Friedreich's ataxia (FRDA); Myotonic dystrophy (DM); and Spinocerebellar ataxia (SCA types 8 and 12).

Proteins associated with trinucleotide repeat expansion disorders may be selected based on experimental association of the protein associated with trinucleotide repeat expansion disorders to trinucleotide repeat expansion disorders. For example, the production rate or circulating concentration of a protein associated with trinucleotide repeat expansion disorders may be elevated or depressed in a population with a trinucleotide repeat expansion disorder relative to a population lacking a trinucleotide repeat expansion disorder. Differences in protein levels may be assessed using proteomic techniques, including but not limited to Western blots, immunohistochemical staining, enzyme-linked immunosorbent assays (ELISAs), and mass spectrometry. Alternatively, proteins associated with trinucleotide repeat expansion disorders may be identified by obtaining gene expression profiles of 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 that can be corrected by prime editing include AR (androgen receptor), FMR1 (Fragile X Mental Retardation 1), HTT (huntingtin), DMPK (myotonic dystrophy protein kinase), FXN (frataxin), ATXN2 (ataxin 2), ATN1 (atrophin 1), FEN1 (flap structure-specific endonuclease 1), TNRC6A (trinucleotide repeat-containing 6A), PABPN1 (nuclear polyA binding protein 1), JPH3 (junctophilin 3), MED 15 (Mediator complex subunit 15), ATXN1 (Ataxin 1), ATXN3 (Ataxin 3), TBP (TATA box binding protein), CACNA1A (Voltage-gated calcium channel P/Q type alpha 1A subunit), ATXN80S (ATXN8 opposing 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, folate 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 (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-dependent) gamma), AFF2 (AF4/FMR2 family member 2), THBS1 (thrombospondin 1), TP53 (tumor protein p53), ESR1 (estrogen receptor 1), CGGBP1 (CGG repeat binding protein 1), ABT1 (basal transcription activator 1), KLK3 (kallikrein-related peptidase 3), PRNP (prion protein), JUN (jun oncogene), KCNN3 (potassium medium/small conductance calcium-activated channel subfamily N member 3), BAX (BCL2-associated X protein), FRA XA (fragile site, folate type, rare, fra(X)(q27.3)A (macrothrimi, 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/CTG1), TSC1 (tubercular 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's disease (pseudoglioma)), ARX (aristaless-associated 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 (intestinal fatty acid binding protein 2), 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 enhanced 2 (S. cerevisiae)), GLA (galactosidase alpha), CBL (Cas-Br-M (murine) ecotropic retroviral transforming sequence), FTH1 (ferritin heavy chain polypeptide 1), IL12RB2 (interleukin-12 receptor beta 2), OTX2 (orthodenticle homeobox A1), IL12RB3 (interleukin-12 receptor beta 2), IL12RB4 (interleukin-12 receptor beta 2), IL12RB5 (interleukin-12 receptor beta 2), IL12RB6 (interleukin-12 receptor beta 2), IL12RB7 (interleukin-12 receptor beta 2), IL12RB8 (interleukin-12 receptor beta 2), IL12RB9 (interleukin-12 receptor beta 2), IL12RB1 (interleukin-12 receptor beta 2), IL12RB2 ...3 (interleukin-12 receptor beta 2), IL12RB4 (interleukin-12 receptor beta 2), IL12RB5 (interleukin-12 receptor beta 2), IL12RB6 (interleukin-12 receptor beta 2), IL12RB7 (interleukin-12 receptor beta 2), IL12RB8 (interleukin-12 receptor beta 2), IL12RB9 (interleukin- box 2), HOXA5 (homeobox A5), POLG2 (polymerase (DNA-dependent) gamma 2 auxiliary 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 specific aspect, the present disclosure provides a TPRT-based method for the treatment of subjects diagnosed with an expanded repeat disorder (also known as a repeat expansion disorder or a trinucleotide repeat disorder). Expanded repeat disorders arise when microsatellite repeats expand beyond a length threshold. Currently, at least 30 genetic diseases are believed to be caused by repeat expansions. Scientific understanding of this diverse group of disorders began in the early 1990s when it was discovered that trinucleotide repeats were involved in the pathogenesis of a number of disorders, including fragile X, spinal-bulbar muscular atrophy, myotonic dystrophy, and Huntington's disease (Nelson et al, "The unstable repeats-3 evolving faces of neurological disease," Neuron, March 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, and synpolydactyly (SPD). This has come to light with the discovery that microsatellite repeat instability is responsible for several major genetic diseases, including glaucoma, hand-foot-genital syndrome (HFGS), cleidocranial dysplasia (CCD), holoprosencephalic deficiency syndrome (HPE), congenital central hypoventilation syndrome (CCHS), ARX nonsyndromic X-linked mental retardation (XLMR), and oculopharyngeal muscular dystrophy (OPMD) (see references). It has been found that instability of microsatellite repeats is a hallmark of these diseases, as repeat expansions can occur in each successive generation, leading to more severe phenotypes in the offspring and earlier ages of onset. Repeat expansions are thought to cause disease through several different mechanisms; the expansions can interfere with cell functioning at the level of the gene, the mRNA transcript, and/or the encoded protein. In some diseases, mutations act through a loss-of-function mechanism by silencing the repeat-containing gene. In others, disease occurs as a result of a gain-of-function mechanism, where either the mRNA transcript or the protein takes on a new abnormal function.

In one embodiment, a method for 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 includes a region encoding 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. An exemplary gRNA design for reducing the trinucleotide repeat sequence and a schematic of trinucleotide repeat reduction with TPRT genome editing are shown in FIG. 23.

Prion diseases Prime editing can also be used to prevent or halt the progression of prion diseases by incorporating one or more protective mutations on the prion protein (PRNP) that becomes misfolded during the course of the 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 a long latency period, characteristic spongiform changes associated with neuronal loss, and failure to induce an inflammatory response.

In humans, prion diseases include Creutzfeldt-Jakob disease (CJD), variant Creutzfeldt-Jakob disease (vCJD), Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia, and kuru. In animals, prion diseases include bovine spongiform encephalopathy (BSE or "mad cow disease"), chronic wasting disease (CWD), scrapie, transmissible mink encephalopathy, feline spongiform encephalopathy, and ungulate spongiform encephalopathy. To prevent or halt the progression of any one of these prion diseases, prime editing can be used to incorporate protective point mutations onto the prion protein.

Classical CJD is a human prion disease. It is a neurodegenerative disorder with characteristic clinical and diagnostic features. The disease is rapidly progressive and invariably fatal. Infection with the disease is usually fatal within one year of disease onset. 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, with an estimated annual incidence in many countries, including the United States, reported to be about one case per million population. The majority of CJD patients usually die within one year of disease onset. CJD is classified as a transmissible spongiform encephalopathy (TSE), along with other prion diseases occurring in humans and animals. In approximately 85% of patients, CJD occurs as an idiopathic disease with no discernible pattern of transmission. A smaller percentage of patients (5 to 15%) develop CJD due to inherited mutations in the prion protein gene. These inherited forms include Gerstmann-Straussler-Scheinker syndrome and fatal familial insomnia. There is currently no known treatment for CJD.

Variant Creutzfeldt-Jakob disease (vCJD) is a prion disease first described in 1996 in the UK. There is now strong scientific evidence that the agent responsible for outbreaks of the prion disease bovine spongiform encephalopathy (BSE or "mad cow" disease) in cattle is the same agent responsible for outbreaks of vCJD in humans. Variant CJD (vCJD) is not the same disease as classical CJD (often simply called CJD). It has different clinical and pathological characteristics than classical CJD. Each disease also has a specific 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 a non-conventionally transmissible agent called a prion. For vCJD, no treatment is currently known.

BSE (bovine spongiform encephalopathy or "mad cow disease") is a progressive neurological disorder in cattle that results from infection with 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 an altered form of a normal protein known as a prion protein. For reasons that are not yet understood, the normal prion protein changes into a pathogenic (harmful) form, which then damages the central nervous system of the cattle. There is growing evidence that there are different strains of BSE: the typical or classical BSE strain, which was responsible for the outbreak in the UK, and two atypical strains (the 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 is found in some areas of North America, including Canada and the United States, Norway, and Korea. It can take more than a year before an infected animal develops symptoms, which can include severe weight loss (wasting), staggering, lethargy, and other neurological 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 is no treatment or vaccine.

The causative agent of TSEs is believed to be prions. The term "prion" refers to an abnormal pathogenic agent that is transmissible and capable of inducing the abnormal folding of certain normal cellular proteins called prion proteins, which are found most abundantly in the brain. The function of these normal prion proteins is not yet fully 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 invariably fatal.

The term "prion" as used herein refers to an infectious particle known to cause disease (spongiform encephalopathy) in humans and animals. The term "prion" is a contraction of the words "protein" and "infectious," and the particle is largely, if not exclusively, composed of PRNP ^Sc molecules encoded by the PRNP gene that expresses PRNP ^C , which changes conformation to become PRNP ^Sc . Prions are distinct from bacteria, viruses, and viroids. Known prions include those that infect animals and cause scrapie, a transmissible degenerative disease of the nervous system in sheep and goats, as well as bovine spongiform encephalopathy (BSE) or mad cow disease and feline spongiform encephalopathy in cats. The four prion diseases discussed above that are known to affect humans are (1) kuru, (2) Creutzfeldt-Jakob disease (CJD), (3) Gerstmann-Straussler-Scheinker disease (GSS), and (4) fatal familial insomnia (FFI). Prion as used herein includes all forms of prion that cause all or any of these diseases or others in any animal used, particularly humans and domesticated livestock animals.

In general, without being bound by theory, prion diseases are caused by misfolding of prion protein. Such diseases, often referred to as deposition diseases, misfolding of prion protein can be explained as follows: if A is the normally synthesized gene product that performs its intended physiological role in a monomeric or oligomeric state, and A* is the conformationally activated form of A that is capable of undergoing dramatic conformational changes, then B is the conformationally altered state that favors multimeric assembly (i.e., the misfolded form that forms deposits), and _Bn is the multimeric material that is pathogenic and relatively difficult to recycle. In prion diseases, PRNP ^C and PRNP ^Sc correspond to states A and _Bn , where A is largely helical and monomeric, and _Bn is β-rich and multimeric.

It is known that certain mutations in the prion protein may be associated with an increased risk of prion (prior) diseases. Conversely, certain mutations in the prion protein may be naturally protective. 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)) human prion protein is encoded by a 16 kb long gene located on chromosome 20 (4686151-4701588). It contains two exons, exon 2 has an open reading frame, which encodes the 253 amino acid (AA) long PrP protein. Exon 1 is a non-coding exon, which may serve as the transcription start site. Post-translational modification results 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), and the CTF (glycosylphosphatidylinositol [GPI] signal peptide [GPI-SP]) is cleaved by the GPI anchor. The GPI anchor may be involved in PrP protein transport. It may also play a role in the attachment of the prion protein to the outer surface of the cell membrane. Normal PrP consists of a long N-terminal loop (which contains an octapeptide repeat region), two short β-sheets, three α-helices, and a C-terminal region (which contains a GPI anchor). Cleavage of PrP results in a 208AA long glycoprotein that is anchored to the cell membrane.

The 253 amino acid sequence of PRNP (NP_000302.1) is as follows:
MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPV (sequence number 291).

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) as follows:
(SEQ ID NO:292)

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

The mutation sites relative to the GSS-linked PRNP (NP_000302.1) (SEQ ID NO: 291) are reported as follows:

The mutation sites relative to PRNP (NP_000302.1) (SEQ ID NO: 291) that are linked to possible protective properties against prion disease are as follows:

Thus, in various embodiments, prime editing can be used to remove PRNP mutations linked to prion disease or to introduce PRNP mutations considered protective against prion disease. For example, prime editing can be used to remove or restore D178N, V180I, T188K, E196K, E196A, E200K, E200G, V203I, R208H, V210I, E211Q, I215V, or M232R mutations in the PRNP protein (relative to PRNP of NP_000302.1) (SEQ ID NO: 291). In other embodiments, prime editing can be used to remove or restore P102L, P105L, A117V, G131V, V176G, H187R, F198S, D202N, Q212P, Q217R, or M232T mutations in the PRNP protein (relative to PRNP of NP_000302.1) (SEQ ID NO: 291). By using prime editing to remove or correct the presence of such mutations in PRNP, the risk of prion disease can be reduced or eliminated.

In other embodiments, prime editing can be used to incorporate protective mutations in PRNP that are linked to a protective effect against one or more prion diseases. For example, prime editing can be used to incorporate G127S, G127V, M129V, D167G, D167N, N171S, E219K, or P238S protective mutations in PRNP (relative to PRNP in NP_000302.1) (SEQ ID NO: 291). In yet other embodiments, the protective mutations can be any alternative amino acid incorporated into G127, G127, M129, D167, D167, N171, E219, or P238 in PRNP (relative to PRNP in NP_000302.1) (SEQ ID NO: 291).

In a specific embodiment, prime editing can be used to incorporate the G127V protective mutation into PRNP, as illustrated in Figure 27 and discussed in Example 5.

In another embodiment, prime editing can be used to incorporate the E219K protective mutation into PRNP.

The PRNP protein and protective mutation sites are conserved in mammals. Therefore, in addition to treating human disease, it could also be used to generate cattle and sheep immune to prion disease, or even help cure wild populations of animals suffering from prion disease. Prime editing has already been used to achieve ∼25% incorporation of the naturally occurring protective allele in human cells, and previous mouse experiments indicate that this level of incorporation is sufficient to induce immunity to most prion diseases. This method is the first and potentially only current way to incorporate this allele with such high efficiency in most cell types. Another possible strategy for treatment is to use prime editing to reduce or eliminate expression of PRNP by incorporating a premature stop codon into the gene.

Using the principles described herein for PEgRNA design, suitable PEgRNAs can be designed to incorporate desired protective mutations or to remove prion disease-associated mutations from PRNP. For example, the below list of PEgRNAs can be used to incorporate the G127V and E219K protective alleles into human PRNP, and the G127V protective allele into 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, including by way of example and not limitation, napDNAbp, reverse transcriptase, fusion proteins (including by way of example napDNAbps and reverse transcriptase), extended guide RNA, and complexes comprising fusion proteins and extended guide RNA, as well as accessory components that serve to drive the prime editing process to form an edited product, such as a second strand nicking component and a 5' endogenous DNA flap removal endonuclease.

The term "pharmaceutical composition" as used herein refers to a composition formulated for pharmaceutical use. In some embodiments, the pharmaceutical composition further comprises a pharma- ceutical acceptable carrier. In some embodiments, the pharmaceutical composition includes additional agents (e.g., for specific delivery, increased half-life, or other therapeutic compounds).

As used herein, the term "pharmaceutical acceptable carrier" means a pharma- ceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, magnesium, calcium, or zinc stearate or stearic acid), or solvent encapsulant involved in carrying or transporting a compound from one site in the body (e.g., a delivery site) to another (e.g., an organ, tissue, or body part). A pharma- ceutically acceptable carrier is "acceptable" in the sense of being compatible with the other ingredients of the formulation but not toxic to the tissue of the subject (e.g., physiologically compatible, sterile, physiological pH, etc.). Some examples of materials which may serve as pharma- ceutically 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 carboxymethylcellulose, methylcellulose, ethylcellulose, microcrystalline cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricants, such as magnesium stearate, sodium lauryl sulfate, and talc; (8) excipients, such as cocoa butter and suppository wax; (9) peanut oil, cottonseed oil, safflower oil, sesame oil, and the like. (10) oils, such as olive oil, corn oil, and soybean oil; (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, 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 buffer solutions; (21) polyesters, polycarbonates, and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids; (23) serum components, such as serum albumin, HDL, and LDL; (22) C2-C12 alcohols, such as ethanol; and (23) other compatible non-toxic substances employed in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, fragrances, preservatives, and antioxidants may also be present in the formulation. Terms such as "excipient," "carrier," "pharmaceutical 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 administration of the pharmaceutical compositions described herein include, without limitation, topical, subcutaneous, transdermal, intradermal, intralesional, intraarticular, intraperitoneal, intravesical, transmucosal, gingival, intradental, intracochlear, transtympanic, intraorgan, epidural, intrathecal, intramuscular, intravenous, intravascular, intraosseous, periocular, intratumoral, intracerebral, and intraventricular administration.

In some embodiments, the pharmaceutical compositions described herein are administered locally to the affected site (e.g., the site of a tumor). In some embodiments, the pharmaceutical compositions described herein are administered to the subject by injection, using a catheter, using a suppository, or using an implant (an implant of a porous, non-porous, or gelatinous material, including membranes such as silastic membranes or fibers).

In other embodiments, the pharmaceutical compositions described herein are 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, a polymeric material may be used (see, e.g., 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 (see above).

In some embodiments, the pharmaceutical composition is formulated according to routine procedures as a composition suitable for intravenous or subcutaneous administration to a subject (e.g., a human). In some embodiments, the pharmaceutical composition for administration by injection is a solution in a sterile isotonic aqueous buffer. If necessary, the medicament may 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 in unit dosage form, either individually or mixed together, for example, as a dry liophilized powder or water-free concentrate in a hermetically sealed container, such as an ampule or sachette indicating the quantity of active agent. If the medicament is to be administered by injection, it may be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. If the pharmaceutical composition is to be administered by injection, an ampoule of sterile water for injection or saline may be provided so that the ingredients may be mixed prior to administration.

Pharmaceutical compositions for systemic administration may be liquids, such as sterile saline, lactated Ringer's solution, or Hank's solution. In addition, pharmaceutical compositions may be in solid form and redissolved or suspended extemporaneously prior to use. Lyophilized forms are also contemplated.

The pharmaceutical composition may be contained within lipid particles or vesicles, such as liposomes or microcrystals, which are also suitable for parenteral administration. The particles may be of any suitable structure, such as unilamellar or plurilamellar, so long as the composition is contained therein. The compound may be encapsulated in "stabilized plasmid-lipid particles" (SPLPs) that contain the fusogenic lipid dioleoylphosphatidylethanolamine (DOPE), low levels (5-10 mol%) of cationic lipids, and stabilized by a polyethylene glycol (PEG) coating (Zhang Y.P. et al., Gene Ther. 1999, 6:1438-47). Positively charged lipids, such as N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium methylsulfate, or "DOTAP", are specifically preferred for such particles and vesicles. Preparation of such lipid particles is well known. See, e.g., U.S. Patent 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 compositions described herein may be administered or packaged, for example, as unit doses. The term "unit dose" as used with respect to the pharmaceutical compositions of the present disclosure refers to physically discrete units suitable for subject administration as a discrete dosage, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect together with the required diluent; i.e., carrier or vehicle.

Furthermore, the pharmaceutical composition may be provided as a pharmaceutical kit comprising (a) a container containing the compound of the invention in lyophilized form, and (b) a second container containing a pharma- ceutically acceptable diluent for injection (e.g., sterile water). The pharma- ceutically acceptable diluent may be used for reconstitution or dilution of the lyophilized compound of the invention. Optionally, notice associated with such container(s) may be in a form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceutical or biological products, said notice reflecting approval by the agency of manufacture, use, or sale for human administration.

In another aspect, an article of manufacture is included that contains materials useful for treating the diseases described above. In some embodiments, the article of manufacture includes 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 the diseases described herein and may have a sterile access port. For example, the container may be an intravenous solution bag or vial with a stopper pierceable by a hypodermic needle. The active agent in the composition is a compound of the present invention. In some embodiments, a label on or associated with the container indicates that the composition is used to treat the disease of choice. The article of manufacture may further include a second container that contains a pharma- ceutically 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, vectors, cells, and delivery
Kits The compositions of the present disclosure can be assembled into a kit. In some embodiments, the kit comprises a nucleic acid vector for the expression of the prime editors described herein. In other embodiments, the kit further comprises a suitable guide nucleotide sequence (e.g., PE gRNA and second site gRNA) or a nucleic acid vector for the expression of such a guide nucleotide sequence (which allows the Cas9 protein or prime editor to target the desired target sequence).

The kits described herein may include one or more containers housing components for carrying out the methods described herein and, optionally, instructions for use. Any of the kits described herein may further include components required for carrying out an assay method. Each component of the kit may be provided in liquid form (e.g., in solution) or in solid form (e.g., as a dry powder), if applicable. In some cases, some of the components may be reconstituted or otherwise processable (e.g., into an active form), for example, by the addition of a suitable solvent or other species (e.g., water), which may or may not be provided with the kit.

In some embodiments, the kit may optionally include instructions and/or promotion for the use of the components provided. As used herein, "instructions" may define the components of instructions and/or promotion, and may typically involve written instructions on or in association with the packaging of the present disclosure. Instructions may also include any oral or electronic instructions in any form (e.g., audiovisual (e.g., videotape, DVD, etc.), internet, and/or web-based communication, etc.) such that a user clearly recognizes that the instructions are relevant to the kit. Written instructions may be in a form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceutical or biological products, which may also reflect approval by the agency of manufacture, use, or sale for animal administration. As used herein, "promoted" includes all methods of business conduct, including educational methods, hospital and other clinical instruction, scientific research, drug discovery or development, academic research, pharmaceutical industry activities (including pharmaceutical sales), and any advertising or other promotional activities (including any form of written, oral, and electronic communication) related to the present disclosure. In addition, the kit may also include other components depending on the particular application as described herein.

The kit may contain one or more of any of the components described herein in one or more containers. The components may be sterilely prepared, packaged in syringes, and shipped frozen. Alternatively, it may be housed in a vial or other container for storage. A second container may also have other components sterilely prepared. Alternatively, the kit may include an active agent that is premixed and shipped in a vial, tube, or other container.

The kit may have various forms, such as a blister pouch, shrink-wrap pouch, vacuum-sealable pouch, sealable thermoformed tray, or similar pouch or tray form, with the accessories loosely packed in a pouch, one or more tubes, containers, boxes, or bags. The kit may be sterilized after the accessories are added, which allows the individual accessories in the container to be opened separately. The kit may be sterilized using any suitable sterilization technique, such as radiation sterilization, heat sterilization, or other sterilization methods known in the art. The kit may also include other components, such as containers, cell culture media, salts, buffers, reagents, syringes, needles, fabrics (such as gauze) for applying or removing disinfectants, disposable gloves, for supporting the agent prior to administration, etc., depending on the particular application. Some aspects of the present disclosure provide kits that include nucleic acid constructs that include nucleotide sequences that encode various components of the prime editing system described herein, including, by way of example and not limitation, napDNAbp, reverse transcriptase, polymerase, fusion proteins (e.g., napDNAbp and reverse transcriptase (or more broadly, polymerase)), 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., gRNAs that nick the second strand) and 5' endogenous DNA flap removal endonucleases that serve to drive the prime editing process to edited product formation). In some embodiments, the nucleotide sequence(s) include a heterologous promoter (or more than a single promoter) that drives expression of the prime editing system components.

Another aspect of the present disclosure provides kits that include one or more nucleic acid constructs encoding various components of the prime editing system described herein, including, by way of example, nucleotide sequences encoding components of the prime editing system capable of modifying a target DNA sequence. In some embodiments, the nucleotide sequences include a heterologous promoter that drives expression of the prime editing system components.

Some aspects of the disclosure provide a kit that includes a nucleic acid construct that includes (a) a nucleotide sequence encoding a napDNAbp (e.g., a Cas9 domain) fused to a reverse transcriptase, and (b) a heterologous promoter driving expression of the sequence of (a).

Cells Cells that may contain any of the compositions described herein include prokaryotic and eukaryotic cells. The methods described herein are used to deliver Cas9 proteins or prime editors into eukaryotic cells (e.g., mammalian cells, such as human cells). In some embodiments, the cells are in vitro (e.g., cultured cells). In some embodiments, the cells are in vivo (e.g., in a subject, such as a human subject). In some embodiments, the cells are ex vivo (e.g., isolated from a subject and may be administered back to the same subject or to 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). Various human cell lines include, but are not limited to, 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, SHSY5Y human neuroblastoma cells (cloned from a myeloma), and Saos-2 (bone cancer) cells. In some embodiments, the rAAV vector is delivered into a human embryonic kidney (HEK) cell (e.g., HEK293 or HEK 293T cell). In some embodiments, the rAAV vector is delivered into a stem cell (e.g., a human stem cell), such as, for example, a pluripotent stem cell (e.g., a human pluripotent stem cell, including a human induced pluripotent stem cell (hiPSC)). Stem cells refer to cells that have the ability to divide indefinitely in culture to give rise to specialized cells. Pluripotent stem cells refer to a type of stem cell that can differentiate into all tissues of an organism, but cannot support the development of a complete organism by itself. Human induced pluripotent stem cells refer to somatic cells (e.g., mature or adult cells) that are reprogrammed into an embryonic stem cell-like state by forcing the expression of genes and factors important for maintaining the characteristics that define embryonic stem cells (see, e.g., Takahashi and Yamanaka, Cell 126(4):663-76, 2006, incorporated herein by reference). Human induced pluripotent stem cells express stem cell markers and are capable of generating cells characteristic of all three germ layers (ectoderm, endoderm, and mesoderm).

Non-limiting examples of additional 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, Hepa1c1c7, 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 the disclosure provide a cell comprising any of the constructs disclosed herein. In some embodiments, the host cell is transiently or non-transiently transfected with one or more vectors described herein. In some embodiments, the cell is transfected as it naturally occurs in the subject. In some embodiments, the transfected cell is taken from the subject. In some embodiments, the cell is derived from a cell taken from the 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 epithelium, BALB/3T3 mouse embryonic fibroblasts, 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, Hepa1c1c7, 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 lines, 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 of skill in the art (see, e.g., American Type Culture Collection (ATCC), Manassus, Va.). In some embodiments, cells transfected with one or more vectors described herein are used to establish new cell lines that contain one or more vector-derived sequences. In some embodiments, cells that have been transiently transfected (such as by transient transfection of one or more vectors or transfection with RNA) with components of the CRISPR system as described herein and modified through the activity of the CRISPR complex are used to establish new cell lines that include cells that contain the modifications but lack any other exogenous sequences. 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 to assess one or more test compounds.

Vectors Some aspects of the disclosure relate to the use of recombinant viral vectors (e.g., adeno-associated viral vectors, adenoviral vectors, or herpes simplex viral vectors) for delivery of the prime editors or components thereof (e.g., split Cas9 proteins or split nucleobase prime editors) described herein into cells. In the case of the split-PE approach, since the full-length Cas9 protein or prime editor exceeds the packaging limit of various viral vectors (e.g., rAAV (~4.9 kb)), the N-terminal portion of the PE fusion protein and the C-terminal portion of the PE fusion are delivered into the same cell by separate recombinant viral vectors (e.g., adeno-associated viral vectors, adenoviral vectors, or herpes simplex viral vectors).

Thus, in one embodiment, the disclosure contemplates vectors capable of delivering split-prime editor fusion proteins or their split components. In some embodiments, compositions for delivering split-Cas9 proteins or split-prime editors into cells (e.g., mammalian cells, human cells) are provided. In some embodiments, compositions of the disclosure include: (i) a first recombinant adeno-associated virus (rAAV) particle comprising a first nucleotide sequence encoding an 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 the C-terminal portion of a Cas9 protein or prime editor. The rAAV particles of the disclosure include a rAAV vector (i.e., a recombinant genome of rAAV) encapsidated with viral capsid proteins.

In some embodiments, the rAAV vector comprises: (1) a heterologous nucleic acid region comprising a first or second nucleotide sequence encoding an N- or C-terminal portion of a split-Cas9 protein or split-prime editor in any form as described herein; (2) one or more nucleotide sequences comprising a sequence (e.g., a promoter) that facilitates expression of the heterologous nucleic acid region; and (3) one or more nucleic acid regions comprising a sequence that facilitates integration of the heterologous nucleic acid region (optionally with one or more nucleic acid regions comprising sequences that facilitate expression) into a cellular genome. In some embodiments, the viral sequence that facilitates integration comprises an inverted terminal repeat (ITR) sequence. In some embodiments, the first or second nucleotide sequence encoding the N- or C-terminal portion of the split-Cas9 protein or split-prime editor is flanked on both sides by ITR sequences. In some embodiments, the nucleic acid vector further comprises a region encoding an AAV Rep protein as described herein, but the region is contained either within or outside the region flanked by the ITRs. The ITR sequences can be derived from any AAV serotype (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) or 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 variants thereof. In specific embodiments, the disclosed rAAV particles are rPHP.B particles, rPHP.eB particles, or rAAV9 particles.

ITR sequences and ITR sequence-containing plasmids are known in the art and commercially available (e.g., products and services available from Vector Biolabs, Philadelphia, PA; Cellbiolabs, San Diego, CA; Agilent Technologies, Santa Clara, Ca; and Addgene, Cambridge, MA; and gene delivery to skeletal muscle results in sustained expression and systemic delivery of therapeutic proteins. Kessler PD, Podsakoff GM, Chen X, McQuiston SA, Colosi PC, Matelis LA, Kurtzman GJ, Byrne BJ. Proc Natl Acad Sci USA. 1996 Nov 26;93(24):14082-7; and Curtis A. Machida. Methods in Molecular Medicine ^TM . Viral Vectors for Gene Therapy Methods and Protocols. 10.1385/1-59259-304-6:201c 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; see U.S. Patent 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 (e.g., a promoter, a transcription terminator, and/or other regulatory elements) that control expression of the heterologous nucleic acid region. In some embodiments, the first and/or second nucleotide sequence is operably linked to one or more (e.g., one, two, three, four, five, or more) transcription terminators. Non-limiting examples of transcription terminators that may be used according to the present disclosure include the bovine growth hormone gene (bGH), human growth hormone gene (hGH), SV40, CW3, φ, or combinations thereof. The efficiency of several transcription terminators has been tested to determine their respective effects on the expression levels of split-Cas9 proteins or split-primed editors. In some embodiments, the transcription terminator used in the present disclosure is a bGH transcription terminator. In some embodiments, the rAAV vector further comprises a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). In some embodiments, the WPRE is a truncated WPRE sequence, such as "W3". In some embodiments, the WPRE is inserted 5' to a transcription terminator. Such sequences, when transcribed, create a tertiary structure that enhances expression, particularly from viral vectors.

In some embodiments, the vectors used herein may encode a PE fusion protein or any of its components (e.g., napDNAbp, linker, or polymerase). In addition, the vectors used herein may encode a PE gRNA, and/or an accessory gRNA for nicking the second strand. The vector 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, e.g., a bacterial cell. In some embodiments, the cell may be a eukaryotic cell, 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 for driving expression in various 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 more effective expression. In another other embodiment, the promoter may be truncated but still retain its function. For example, the promoter may have a normal size or a reduced size suitable for correct packaging of the vector into a virus.

In some embodiments, the promoters that may be used in the prime editor vector may be constitutive, inducible, or tissue-specific. In some embodiments, the promoter may be a constitutive promoter. 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 (EFla) promoter, ubiquitin promoter, actin promoter, tubulin promoter, immunoglobulin promoter, functional fragments thereof, or any combination of the above. 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 EFla promoter. In some embodiments, the promoter may be an inducible promoter. Non-limiting exemplary inducible promoters include those that are 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, for example, the Tet-On® promoter (Clontech). In some embodiments, the promoter may be a tissue-specific promoter. In some embodiments, the tissue-specific promoter is expressed exclusively or primarily 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, Nphsl promoter, OG-2 promoter, SP-B promoter, SYN1 promoter, and WASP promoter.

In some embodiments, the prime editor vector (including, by way of example, any vector encoding a prime editor fusion protein and/or a PE gRNA, and/or an accessory gRNA that nicks the second strand) may include an inducible promoter that begins expression only after delivery to a target cell. Non-limiting exemplary inducible promoters include those that are inducible by heat shock, light, chemicals, peptides, metals, steroids, antibiotics, or alcohol. In some embodiments, the inducible promoter may have a low basal (uninduced) expression level, such as, by way of example, the Tet-On® promoter (Clontech).

In additional embodiments, the prime editor vector (including, by way of example, any vector encoding a prime editor fusion protein and/or a PE gRNA, and/or an accessory gRNA that nicks the second strand) may include a tissue-specific promoter that only begins expression after delivery to a particular tissue. Non-limiting exemplary tissue-specific promoters include B29 promoter, CD14 promoter, CD43 promoter, CD45 promoter, CD68 promoter, desmin promoter, elastase-1 promoter, endocrine promoter, fibronectin promoter, Flt-1 promoter, GFAP promoter, GPIIb promoter, ICAM-2 promoter, INF-β promoter, Mb promoter, Nphsl promoter, OG-2 promoter, SP-B promoter, SYN1 promoter, and WASP promoter.

In some embodiments, the nucleotide sequence encoding the PEgRNA (or any guide RNA 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 using more than one guide RNA, the promoters used to drive expression may be the same or different. In some embodiments, the nucleotides encoding the crRNA of the guide RNA and the nucleotides encoding the tracr RNA of the guide RNA may be provided on the same vector. In some embodiments, the nucleotides encoding the crRNA and the nucleotides 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 a 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 that contains the nucleotide sequence encoding the PE fusion protein. In some embodiments, the expression of the guide RNA and the expression of the PE fusion protein may be driven by their corresponding promoters. In some embodiments, the expression of the guide RNA may be driven by the same promoter that drives the expression of the PE fusion protein. In some embodiments, the transcript of the guide RNA and the PE fusion protein may be contained within a single transcript. For example, the guide RNA may be within the 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, thereby shortening the length of the 3'UTR. In additional embodiments, the guide RNA may be within an intron of the PE fusion protein transcript. In some embodiments, a suitable splicing site may be added to the intron in which the guide RNA is located so that the guide RNA is correctly spliced from the transcript. In some embodiments, expression of the Cas9 protein and guide RNA in close proximity on the same vector may facilitate more efficient formation of CRISPR complexes.

The prime editor vector system may include one vector, or two vectors, or three vectors, or four vectors, or five vectors, or more. In some embodiments, the vector system may include one single vector encoding both the PE fusion protein and the PE gRNA. In other embodiments, the vector system may include two vectors, where one vector encodes the PE fusion protein and the other encodes the PE gRNA. In additional embodiments, the vector system may include three vectors, where a third vector encodes a gRNA that nicks the second strand for use in the methods herein.

In some embodiments, a composition comprising rAAV particles (in any form contemplated herein) further comprises a pharma- ceutically acceptable carrier. In some embodiments, the composition is formulated in a pharmaceutical vehicle suitable for administration to a human or animal subject.

Some examples of materials that may serve as pharma- ceutically 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 carboxymethylcellulose, methylcellulose, ethylcellulose, microcrystalline cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricants, such as magnesium stearate, sodium lauryl sulfate, and talc; (8) excipients, such as cocoa butter and suppository wax; (9) peanut oil, cottonseed oil, safflower oil, sesame oil, and other oils. (10) oils, such as olive oil, corn oil, and soybean oil; (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, 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 buffer solutions; (21) polyesters, polycarbonates, and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids; (23) serum components, such as serum albumin, HDL, and LDL; (22) C2-C12 alcohols, such as ethanol; and (23) other compatible non-toxic substances employed in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, fragrances, preservatives, and antioxidants may also be present in the formulation. Terms such as "excipient," "carrier," "pharmaceutical acceptable carrier" or the like are used interchangeably herein.

Delivery Methods In some aspects, the invention provides methods comprising delivering one or more polynucleotides as described herein, such as one or more vectors encoding one or more transcription products thereof, and/or one or more 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, base editors as described herein are delivered to a cell in combination with (optionally complexed with) a guide sequence.

Exemplary delivery strategies are described elsewhere herein and include vector-based strategies, delivery of PE ribonucleoprotein complexes, and delivery of PE via mRNA methods.

In some embodiments, delivery methods provided include nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, and agents that enhance DNA uptake.

Exemplary nucleic acid delivery methods include lipofection, nucleofection, electroporation, stable genomic integration (e.g., piggybac), microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, and agents that enhance DNA uptake. Lipofection is described, for example, in U.S. Pat. Nos. 5,049,386; 4,946,787; and 4,897,355, and lipofection reagents are commercially available (e.g., Transfectam™, Lipofectin™, and SF cell line 4D-Nucleofector X Kit™ (Lonza)). Cationic and neutral lipids suitable for efficient receptor-recognition lipofection of polynucleotides include those of Feigner, WO91/17424; WO91/16024. Delivery can be to a cell (e.g., in vitro or ex vivo administration) or to a target tissue (e.g., in vivo administration). Delivery may be achieved through the use of an RNP complex.

The preparation of lipid:nucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to those 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); see U.S. Patent 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 delivery methods and vectors provided herein are RNP complexes. RNP delivery of fusion proteins significantly increases 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, greatly reducing 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. Patent No. 9,526,784 issued December 27, 2016; and U.S. Patent No. 9,737,604 issued August 22, 2017, each of which is incorporated herein by reference.

Additional methods for nucleic acid delivery to cells are known to those of skill in the art. See, e.g., US 2003/0087817, incorporated herein by reference.

Other aspects of the disclosure provide methods of delivering a prime editor construct into a cell to form a complete and functional prime editor within the cell. For example, in some embodiments, a cell is contacted with a composition described herein (e.g., a composition comprising an AAV particle containing a nucleotide sequence encoding a split-Cas9 or split-prime editor, or a nucleic acid vector comprising such a nucleotide sequence). In some embodiments, the contacting results in delivery of such a nucleotide sequence into the cell, where an N-terminal portion of the Cas9 protein or prime editor and a C-terminal portion of the Cas9 protein or prime editor are expressed and associated in the cell to form a complete Cas9 protein or complete prime editor.

It should be understood that any rAAV particle, nucleic acid molecule, or composition provided herein may be introduced into a cell in any suitable manner, either stably or transiently. In some embodiments, the disclosed proteins may be transfected into a cell. In some embodiments, a 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 rAAV particle containing a nucleic acid molecule encoding a split protein, or a genome of a virus encoding one or more nucleic acid molecules. Such transduction may be stable or transient. In some embodiments, a cell expressing or containing a split protein may be transduced or transfected with one or more guide RNA sequences, for example, in the delivery of a split Cas9 (e.g., nCas9) protein. In some embodiments, plasmids expressing the disruption proteins may be introduced into cells through electroporation, transient (e.g., lipofection) and stable genomic integration (e.g., piggybac), and viral transduction or other methods known to those of skill in the art.

In some embodiments, the compositions provided herein include lipids and/or polymers. In some embodiments, the lipids and/or polymers are cationic. The preparation of such lipid particles is well known. See, e.g., U.S. Patent 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 comprises a sequence of at least 10, at least 15, or at least 20 contiguous nucleotides complementary to the 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 complementary to the 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 some embodiments, the target nucleotide sequence is in a mammalian (e.g., human) genome.

The compositions of the present disclosure may be administered or packaged, for example, as unit doses. The term "unit dose" as used in reference to pharmaceutical compositions of the present disclosure refers to physically discrete units suitable for subject administration as a discrete dosage, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect together with the required diluent; i.e., carrier or vehicle.

Treating a disease or disorder includes delaying the onset or progression of the disease or reducing the severity of the disease. Treating a disease does not necessarily require a curative outcome.

As used therein, "delaying" the onset of a disease means to defer, hinder, slow, retard, stabilize, and/or postpone the progression of a disease. The delay may be of varying lengths of time, depending on the history of the disease and/or the individual being treated. A method of "delaying" or mitigating the onset of a disease, or a method of delaying the onset of a disease, is a method that reduces the probability of developing one or more symptoms of a disease in a given time frame and/or reduces the severity of symptoms in a given time frame, compared to not using the method. Such comparisons are typically based on clinical studies using a sufficient number of subjects to provide statistically significant results.

"Onset" or "progression" of a disease refers to the first symptoms and/or subsequent progression of the disease. Onset of a disease can be detected and assessed using standard clinical techniques as known in the art. However, onset also refers to progression, which may be undetectable. For purposes of this disclosure, onset or progression refers to the biological course of a condition. "Onset" encompasses occurrence, recurrence, and onset.

As used herein, "onset" or "occurrence" of a disease includes initial onset and/or recurrence. Conventional methods known to those skilled in the art of medicine can be used to administer the isolated polypeptide or pharmaceutical composition to a subject, depending on the type of disease or site of disease to be treated.

Without going into further detail, it is believed that one skilled in the art can utilize the present disclosure to its fullest extent based on the above description. The following specific aspects 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 discussed herein.

example
Example 1. Prime editing (PE) to incorporate precise nucleotide changes into a genome
The goal is to develop a precise genome editing transformation technique and generate the incorporation of single nucleotide changes in mammalian genomes. This technique will allow researchers to study the effects of single nucleotide variations in virtually any mammalian gene, potentially enabling therapeutic intervention to correct pathogenic point mutations in human patients.

The adoption of the clustered regularly interspaced short palindromic repeats (CRISPR) system for genome editing has revolutionized the life sciences. ^{1 - 3} Although gene disruption using CRISPR is now routine, precise incorporation of single-nucleotide edits remains a major challenge, despite being necessary to study or correct a large number of disease-causing mutations. Homologous directed repair (HDR) can achieve such edits but suffers from low efficiency (often <5%), the requirement for a donor DNA repair template, and the 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 targeted 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), the latest BEs can only modify 4 of 12 possible base pair conversions and cannot correct small insertions or deletions. Moreover, the target scope of base editing is limited by the editing of non-targeted C or A bases adjacent to the target base ("bystander editing") and by the requirement that the PAM sequence be present 15±2bp from the target base. Thus, overcoming these shortcomings would greatly broaden the basic research and therapeutic applications of genome editing.

Here, we propose 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 template – while overcoming its major drawbacks. To achieve this ambitious goal, we aim to directly incorporate an edited DNA strand at a site in the target genome using target-primed reverse transcription (TPRT). In the design considered here, a CRISPR guide RNA (gRNA) will be modified to carry a template encoding mutagenic DNA strand synthesis, which is carried out by an attached reverse transcriptase (RT) template sequence. The target site DNA nicked by the CRISPR nuclease (Cas9) will act as a primer for reverse transcription of the template sequence on the modified gRNA, allowing direct incorporation of any desired nucleotide edits.

Section 1
Guide RNA-templated reverse transcription of the mutagenized DNA strand is established. Previous studies have shown that after DNA cleavage but before complex dissociation, Cas9 releases the non-targeted DNA strand to expose a free 3' end. We hypothesize that this DNA strand is accessible to extension by a polymerase enzyme and that gRNAs can be modified to serve as templates for DNA synthesis through extension of their 5' or 3' ends. Preliminary in vitro studies have established that the nicked DNA strand in the Cas9:gRNA-bound complex can indeed prime reverse transcription (RT enzyme in trans) using the bound gRNA as a template. Next, different gRNA linkers, primer binding sites, and synthesis templates are studied in detail to determine optimal design rules in vitro. Different RT enzymes acting in trans or as fusions with Cas9 are then evaluated in vitro. Finally, modified gRNA designs that retain efficient binding and cleavage activity in cells are identified. Successful demonstration experiments to this end will provide the basis for accomplishing mutagenized strand synthesis in cells.

Section 2
Establishing prime editing in human cells. Based on DNA processing and repair mechanisms, we hypothesize that the mutagenized DNA strand (single-stranded flap) can be used to direct specific and efficient editing of targeted nucleotides. In promising preliminary studies, the feasibility of this strategy was established by demonstrating editing in a model plasmid substrate containing the mutagenized flap. Concurrent with objective 1, repair outcomes are further evaluated by systematically varying the length, sequence composition, identity of the targeted nucleotide, and 3' end of the mutagenized flap. Small insertions and deletions of 1-3 nucleotides are also tested. Assembled from objective 1 in parallel with objective 1, Cas9-RT structures encompassing fusion proteins and non-covalent mobilization strategies are evaluated. Cas9-RT structures and extended gRNAs are assayed for cellular editing at multiple target sites in the human genome and then optimized for high efficiency. If successful, this objective will 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 universality of this technology may enable editing of transversion mutations and indels currently uncorrectable by BE. Guided by the results of Aim 1 and Aim 2, we target pathogenic transversion mutations in cultured human cells, including the founder mutation of sickle cell disease in beta globin (requiring an A-T → T-A transversion for correction) and the most prevalent Wilson disease mutation in ATP7B (requiring a G-C → T-A transversion for correction). We also examine the correction of small insertion and deletion mutations, including the three-nucleotide ΔF508 deletion in CFTR that causes cystic fibrosis. If successful, this will lay the foundation for developing powerful therapeutic approaches to address these important human diseases.

The approach goal is to develop genome editing strategies that directly incorporate point mutations at targeted genomic sites. During the technology development phase, efforts to incorporate TPRT functionality into the CRISPR/Cas system will focus on protein and RNA engineering. In vitro assays will be used to carefully explore the function of each step of TPRT and build it from the ground up (Aim 1). A second area of focus will be to evaluate the editing outcomes in mammalian cells using a combination of model substrates and the engineered CRISPR/Cas system (Aim 2). Finally, the application phase will use the technology to correct mutations that are difficult to resolve using other methods of genome editing (Aim 3).

A general editing design is shown in Figure 1A-1B. The Cas9 nickase contains an inactivating mutation to the HNH nuclease domain (Spy Cas9 H840A or N863A) that restricts DNA cleavage to the PAM-containing strand (non-target strand). The guide RNA (gRNA) is modified to contain a template for reverse transcription (design detailed on slide 5). Shown is a 5' extension of the gRNA, but a 3' extension can also be performed. The Cas9 nickase is fused to a reverse transcriptase (RT) enzyme through either 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 end that can prime reverse transcription using the extended gRNA as a template. This DNA synthesis reaction is accomplished by the fused RT enzyme. The gRNA template encodes a DNA sequence that is homologous to the original DNA duplex, except for the nucleotides targeted by the edit. The product of reverse transcription is a single-stranded DNA flap that encodes the desired edit. This flap, which contains a free 3' end, 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 the 5' flap from Okazaki fragments during lagging strand DNA synthesis and removes the 5' flap after strand displacement synthesis that occurs during long-patch base excision repair. Ligation of the nicked DNA produces a mismatched base pair. This intermediate can undergo either restoration to the original base pair or conversion to the desired edited base pair via the mismatch repair (MMR) process. Alternatively, one copy each of restoration and edit can be generated by semi-conservative DNA replication.

1. Establish guide RNA-templated reverse transcription of the mutagenized DNA strand.
Background and Rationale In the proposed genome editing strategy, the non-targeted DNA strand nicked by Cas9 (the PAM-containing strand forming the R-loop) acts as a primer for DNA synthesis. This is hypothesized to occur based on several pieces of biochemical and structural data. Nuclease protection ^{experiments32} , crystallographic ^studies33 , and base editing ^windows4,24 have demonstrated a significant degree of flexibility and disorder for non-targeted strand nucleotides -20 to -10 within the so-called R-loop of the Cas9-bound complex (numbering indicates distance from the 5' of the first PAM nucleotide). Moreover, when complementary ssDNA is added, the PAM-distal portion of the cleaved non-targeted strand can be displaced from the tightly bound ternary ^complex20 . These studies support that the non-targeted strand is highly flexible and accessible to the enzyme and that the 3' end of the PAM-distal fragment is released prior to Cas9 dissociation after nicking. Furthermore, it is hypothesized that the gRNA can be extended to template DNA synthesis. Previous studies have shown that gRNAs for SpCas9, SaCas9, and LbCas12a (formerly Cpf1) tolerate gRNA extension with RNA ^aptamers34 , ligand-induced self-cleaving ^ribozymes35 , and long noncoding ^RNAs.36 This literature establishes precedent for two key features to 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 (Figures 2A-2C).

The designs for modified gRNAs for TRT editing are shown in Figures 3A-3B. DNA synthesis proceeds 5'→3', thus copying the RNA template in the 3'→5' direction. The design for 5' extension contains a linker region, a primer binding site to which 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 vitro experiments have shown that reverse transcription extends 3 nucleotides into the gRNA core of the 3' extended gRNA construct, and in some cases the 3' RNA hairpin of the gRNA core is modified to match the DNA target sequence (modification of the hairpin sequence appears to be well tolerated as long as changes are made that compensate for maintaining the hairpin RNA structure). DNA synthesis proceeds 5'→3' with nucleotides being added to the 3' OH of the growing DNA strand.

Preliminary experiment results
DNA nicked by Cas9 primes reverse transcription of gRNA templates. To assess the accessibility of the nicked non-target DNA strand, an in vitro biochemical assay was performed using Cas9 nuclease from S.pyogenes (SpCas9) and Cy5 fluorescently labeled double-stranded DNA substrates (51 base pairs). First, a series of 5' extension-containing gRNAs of varying lengths on synthetic templates were prepared by in vitro transcription (overall design shown in Figure 2B). Electrophoretic mobility shift assays (EMSAs) using nuclease-inactive Cas9 (dCas9) established that the 5' extended gRNAs maintained binding affinity to the target (data not shown). Next, TPRT activity was tested against pre-nicked Cy5-labeled double-stranded DNA substrates using dCas9, 5' extended gRNAs, and Moloney-Murine Leukemia Virus (M-MLV) reverse transcriptase (Superscript III). After 1 h of incubation at 37°C, products were assessed by denaturing polyacrylamide gel electrophoresis (PAGE) and imaged using Cy5 fluorescence (Figure 4A). Each of the 5'-extended gRNA variants resulted in significant product formation, with the observed DNA product sizes consistent with the length of the extended template (Figure 4B). Importantly, in the absence of dCas9, the pre-nicked substrate was extended to the full 51 bp length of the DNA substrate, strongly suggesting that the complementary DNA strand (not the gRNA) was used as a template for DNA synthesis in the absence of dCas9 (Figure 4C). Of note, the system was designed such that the newly synthesized DNA strand mirrors the product required for editing of the target site (the strand homologous to the single nucleotide change). This result establishes that the 3' end of the non-targeted strand nicked by Cas9:gRNA binding is exposed and that the non-targeted strand is accessible to reverse transcription.

Next, unnicked dsDNA substrates were evaluated using a Cas9(H840A) mutant that nicks the non-target DNA strand. First, to test Cas9(H840A) nickase activity with 5'-extended gRNA, an in vitro cleavage assay was performed as previously ^described37 . Although nicking was impaired compared to standard gRNA, substantial cleavage products were formed (Figure 4D). Importantly, when TPRT reactions were performed with 5'-extended gRNA and Cas9(H840A), RT products were also observed, albeit at lower yields (explainable by reduced nicking activity) (Figure 4D). This result establishes that the 5'-extended gRNA:Cas9(H840A) complex can nick DNA and serve as a template for reverse transcription.

Finally, 3' gRNA extension was evaluated for Cas9(H840A) nicking and TPRT. Compared to 5' extended gRNA, DNA cleavage by 3' extended gRNA was not detectably impaired compared to standard gRNA either. Significantly, 3' extended gRNA templates also supported efficient reverse transcription on both pre-nicked and intact duplex DNA substrates when M-MLV RT was supplied in trans (Figure 4E). Surprisingly, only a single product was observed for the 3' extended template, indicating that reverse transcription terminates at a specific location along the gRNA backbone. Homopolymer tailing of the product with terminal transferase, followed by Klenow extension and Sanger sequencing, revealed that the entire gRNA synthesis template was copied with the addition of the terminal three nucleotides of the gRNA core. In the future, the flap ends will be reprogrammed by modifying the terminal gRNA ^{sequence38,39} . This result demonstrates that the 3'-extended gRNA can serve as an efficient nuclease targeting guide and serve as a template for reverse transcription.

Cas9-TPRT uses nicked DNA and gRNA in cis. Dual-color experiments were used to determine whether the RT reaction occurs preferentially in cis (bound in the same complex) with the gRNA (see Figure 8). Two separate experiments were performed with 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 tube, the gRNA encodes a short RT product and the DNA substrate is labeled with Cy5 (blue). After a short incubation, the complexes are 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 be preferentially formed using gRNA templates that were previously complexed with DNA substrates, indicating that the RT reaction may occur in cis. This result supports the idea that a single Cas9:gRNA complex can target a DNA site and serve as a template for reverse transcription of the mutagenized DNA strand.

Testing TPRT with other Cas systems Experiments similar to those presented in the previous section will be performed using other Cas systems, including Cas9 from S. aureus and Cas12a from L. bacterium (see Figures 2A-2C). If TRPT can also be demonstrated for these Cas variants, it will increase the potential editing scope and the overall chance of success in cells.

Testing TPRT with RT-Cas9 Fusion Proteins First, a series of commercially available or purifiable RT enzymes will be evaluated in trans for TPRT activity. In addition to the already tested RT from M-MLV, RTs from avian myeloblastosis virus (AMV), Geobacillus stearothermophilus group II intron (GsI-IIC) ^41,42 , and Eubacterium rectale group II intron (Eu.re.I2) ^43,44 will be evaluated. Significantly, the latter two RTs perform TPRT in their native biological context. Where relevant, RNAse inactivating mutations and other potentially beneficial RT enzyme modifications will also be tested. Once functional RTs are identified when supplied in trans, each will be evaluated against Cas9 variants as fusion proteins. Both N- and C-terminal fusion orientations will be tested along with various linker lengths and structures. Kinetic time course experiments will be used to determine if TPRT can occur using the RT enzymes in cis. If an RT-Cas9 fusion construct could be constructed that enabled efficient TPRT chemistry, this would greatly increase the likelihood of functional editing in the cellular context.

Cas9 targeting with modified gRNA in cells The modified gRNA candidates developed in the previous sub-aims are evaluated in human cell culture experiments (HEK293) to confirm Cas9 targeting efficiency. Using an established indel formation ^assay45 employing wild-type SpCas9, the modified gRNA is compared side-by-side with standard gRNA across 5 or more sites in the human genome. Genome editing efficiency is characterized by multiplex amplicon sequencing using a laboratory-housed Illumina MiSeq platform. It is expected that the results from this section and the previous section will generate insights that will inform subsequent iterations of design-build-test cycles, where gRNAs can be optimized for both reverse transcription template and efficient Cas9 targeting in cells.

In vitro validation results are shown in Figures 5-7. In vitro experiments demonstrated that the nicked non-target DNA strand is flexible and available to prime DNA synthesis, and that the gRNA extension can serve as a template for reverse transcription (see Figure 5). This series of experiments used 5'-extended gRNAs (designed as shown in Figures 3A-3B) with varying lengths of editing templates (listed on the left). A fluorescently labeled (Cy5) DNA target was used as the substrate, which was pre-nicked in this series of experiments. The Cas9 used in these experiments was a catalytically inactive Cas9 (dCas9), which does not cleave DNA but can still bind efficiently. Superscript III, a commercial RT derived from Moloney Murine Leukemia Virus (M-MLV), was supplied in trans. First, the dCas9:gRNA complex was assembled from purified components. Then, the fluorescently labeled DNA substrate was added together with dNTPs and the RT enzyme. After 1 h of incubation at 37 C, the reaction products were analyzed by denaturing urea-polyacrylamide gel electrophoresis (PAGE). The gel image shows that the original DNA strand was extended to a length consistent with that of the reverse transcription template. Notably, reactions performed in the absence of dCas9 produced a 51-nucleotide-long DNA product, regardless of the gRNA used. This product corresponds to the use of the complementary DNA (not RNA) strand as a template for DNA synthesis (data not shown). Thus, Cas9 binding is required to direct DNA synthesis to the RNA template. This set of in vitro experiments closely parallels the one shown in FIG. 5, except that the DNA substrate was not pre-nicked and Cas9 nickase (SpyCas9 H840A mutant) was used. As shown in the gel, the nickase efficiently cleaves the DNA strand when a standard gRNA is used (gRNA_0, lane 3). Multiple cleavage products are observed, consistent with previous biochemical studies of SpyCas9. 5' extension abolishes nicking activity (lanes 4-8), but some RT product is still observed. Figure 7 shows that 3' extension supports DNA synthesis and does not significantly induce Cas9 nickase activity. Pre-nicked substrates (black arrows) are nearly quantitatively converted to RT products when using either dCas9 or Cas9 nickase (lanes 4 and 5). More than 50% conversion to RT products (red arrows) is observed for all substrates (lane 3). To determine the length and sequence of the RT products, product bands were excised from the gel, extracted, and sequenced. This revealed that RT extended three nucleotides into the 3' terminal hairpin of the gRNA core. Subsequent experiments (not shown) demonstrated that these three nucleotides could be altered to match the target DNA sequence, as long as complementary changes were made that maintained the hairpin RNA structure.

Potential Difficulties and Alternatives
(1) RT does not function as a fusion: molecular crowding and/or unfavorable geometries may hinder polymerase extension by Cas9-fused RT enzyme. First, linker optimization can be tested. Circular permutation variants of Cas9 that can reorient the spatial relationship between the DNA primer, gRNA, and RT enzyme are evaluated. Non-covalent RT recruitment strategies as detailed in objective 2 can be tested. (2) Reduced Cas targeting efficiency by extended gRNA variants: This can be an issue for most 5'-extended gRNAs. Based on structural ^data24 , Cas9 mutants can be designed and screened to identify variants that are more resistant to gRNA extension. In addition, gRNA libraries can be screened in cells for linkers that improve targeting activity.

Significance These preliminary experimental results establish that Cas9 nickase and extended gRNA can initiate target-primed reverse transcription on bound DNA targets using reverse transcriptase supplied in trans. Importantly, Cas9 binding was found to be crucial for product formation. Although perhaps not an absolute requirement for genome editing in cells, further development of a system that incorporates RT enzyme function in cis would significantly increase the chances of success in cell-based applications. Achieving the remaining aspects of this objective would provide a molecular basis for carrying out high-precision genome editing in the context of the human genome.

2. Establishing prime editing in human cells.
Background and Rationale In the proposed strategy, a modified RT-Cas9:gRNA complex introduces a mutagenic 3' DNA flap at a genomic target site. We hypothesize that the mutagenic 3' flap, which contains a ^single mismatch, is incorporated by the DNA repair machinery through equilibrium with the energetically accessible adjacent 5' flap, which is preferentially removed (Figures 1C-1D). DNA replication and repair machinery encounters the 5' ssDNA flap when processing Okazaki fragments46 and during long patch base excision repair (LP-BER) ^.47 The 5' flap is the preferred substrate for the broadly expressed flap endonuclease FEN1, which is recruited to DNA repair sites by the homotrimeric sliding clamp complex ^PCNA.48 PCNA also serves as a scaffold for the simultaneous recruitment of other repair factors, including the DNA ligase ^Lig1.49 Acting as a "toolbelt," PCNA accelerates the sequential flap cleavage and ligation that are essential for processing the millions of Okazaki fragments generated during each cell division. Based on similarities with these natural DNA intermediates, it is hypothesized that the mutagenized strand is incorporated through equilibration with the 5' flap followed by coordinated 5' flap excision and ligation. ^Mismatch repair (MMR) should then occur with equal probability on either strand, leading to editing or restoration (Figures 1C-1D). Alternatively, DNA replication can occur first, leading directly to incorporation of the edit in the newly synthesized daughter strand. Although the highest yield expected from this process is 50%, multiple substrate editing attempts can drive the reaction toward completion due to the irreversibility of edit repair.

Preliminary experiment results
DNA flaps involve site-directed mutagenesis in plasmid model substrates in yeast and HEK cells. To test the proposed editing strategy, we began with model plasmid substrates containing a mutagenized 3' flap that resembles the TPRT product. We created a dual fluorescent protein reporter that encodes a stop codon between GFP and mCherry. The mutagenized flap encodes a correction to the stop codon (Figure 9A), allowing mCherry synthesis. Thus, mutagenesis efficiency can be quantified by the GFP:mCherry ratio. Plasmid substrates were prepared in vitro and introduced into yeast (S. cerevisiae) or human cells (HEK293). Hypermutagenesis was observed in both systems (Figure 9B), and isolated yeast colonies contained either reverted bases, mutated bases, or a mixture of both products (Figure 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 the 3' mutagenized strand.

● Systematic studies on model flap substrates Based on the preliminary experimental results described above, a wider range of flap substrates will be evaluated in HEK cells to infer the principles of efficient editing. The 3'ssDNA flap will be systematically varied to determine the effects of mismatch pairing, the location of the mutagenic nucleotide along the flap, and the identity of the terminal nucleotide (Figure 9D). Single nucleotide insertions and deletions will also be tested. Amplicon sequencing will be used to analyze the editing precision. These results will help inform the design of gRNA reverse transcription templates.

In vitro TPRT on plasmid substrates leads to efficient editing results. The TPRT reaction developed in Objective 1 was used to introduce mutagenesis into plasmid substrates. The reaction was performed on circular DNA plasmid substrates (see Figure 10). This excludes the possibility of DNA strand dissociation as a mechanism for RT extension in previous in vitro experiments. This also allows 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, modified gRNA, MLV RT enzyme, dNTPS]. gRNA extension encodes a mutation for the stop codon. Using the flap strand for repair of the stop codon is expected to produce a plasmid expressing both GFP and mCherry as a fusion protein. Yeast dual FP plasmid transformants are shown in FIG. 10. Transformation of the parental plasmid or the in vitro Cas9(H840A) nicked plasmid results in only green GFP expressing colonies. TRT reactions with 5' extended or 3' extended gRNAs produce a mixture of green and yellow colonies, the latter expressing both GFP and mCherry. More yellow colonies are observed with the 3' extended gRNA. A positive control containing no stop codon is also shown.

These results establish that long double-stranded substrates can undergo TPRT and that TPRT products induce editing in eukaryotic cells.

Another experiment similar to the prime editing experiment above was also performed, but instead of incorporating a point mutation in the stop codon, TRT editing introduces a single nucleotide insertion (left) or deletion (right) that repairs the frameshift mutation and allows synthesis of downstream mCherry (see Figure 11). In both experiments, 3' extended gRNAs were used. Individual colonies from the TRT transformants were selected and analyzed by Sanger sequencing (see Figure 12). Green colonies contained a plasmid with the original DNA sequence, while yellow colonies contained the precise mutation designed by the TRT-edited gRNA. No other point mutations or indels were observed.

Establishing prime editing in HEK cells using RT-Cas9 constructs The optimized constructs from the previous objectives are adapted for mammalian expression and editing at target sites in the human genome. Multiple RT enzymes and fusion constructs are tested, in addition to adjacent targeting with a secondary gRNA (truncated to prevent nicking). Non-covalent RT recruitment is also evaluated using the Sun-Tag ^system52 and the MS2 aptamer ^system53 . Indel formation assays are used to evaluate targeting efficiency with standard gRNA and RT-Cas9 fusions (as above). For each genomic site, the extended gRNA and RT-Cas9 pair are then assayed for single nucleotide editing. Editing outcomes are evaluated with MiSeq.

Initial experiments in HEK cells were performed using Cas9-RT fusions. Editing with intracellularly expressed components requires Cas9(H840A) nickase, reverse transcriptase (expressed as a fusion or supplied in trans), and a modified gRNA with a 3' extension (see Figure 14). Preliminary studies showed that the length of the primer binding site within the gRNA extension is important to increase the efficiency of editing in human cells (see Figure 15).

Optimize the prime editing parameters in HEK cells After identifying the Cas9-RT construct that can perform prime editing in cells, optimize the components and design to achieve high-efficiency editing. Vary the location and nucleotide identity of the encoded point mutation, and the total length of the newly synthesized DNA strand to evaluate the editing range and potential defects. Also evaluate short insertion and deletion mutations. Codon-optimize the protein expression construct. If successful, this will establish efficient prime editing in mammalian cells.

Preliminary experimental results.
In the unlikely event that intramolecular reverse transcription by the fused RT enzyme could not occur, additional gRNAs were designed to bring the RT enzyme to a higher local concentration at the editing locus. These auxiliary guides were truncated at the 5' end (14-15 nt spacer), which has previously been shown to prevent Cas9 cleavage but retain binding (see Figure 16). The HEK3 locus was chosen to explore this strategy.

Potential Difficulties and Alternatives
(1) gRNA degradation in cells: if extended gRNA ends are truncated in cells, synthetic gRNAs incorporating stabilizing secondary structures or with stabilizing modifications can be tested. (2) No editing is observed in human cells: additional strategies are explored, including secondary targeting of RT-Cas9 fusions to adjacent genomic ^sites.54 In addition, potential directed evolution strategies in E. coli or S. cerevisiae can also be explored.

If significant prime editing could be established in experimental cell lines, it would have a direct impact on basic biomedical research by enabling the rapid generation and characterization of large numbers of point mutations in human genes. The generality of the method for base editors and their orthogonal editing windows provide an approach to incorporate many currently inaccessible mutations. Moreover, if prime editing could be optimized for high efficiency and product purity, its potential applicability to correct disease mutations in other human cell types is significant.

3. Achieve site-specific editing of pathogenic mutations in cultured human cells.
Background and rationale.

Many pathogenic mutations cannot be corrected by current base editors due to PAM restriction, i.e., correction of transversion or indel mutations is required. With prime editing, all transitions and transversions can theoretically occur, although insertions and deletions are small. Moreover, the prime editing window (expected to be -3 to +4) in relation to the PAM is different from that of base editors (-18 to -12) (Figure 13). Mendelian diseases that are currently uncorrectable by base editors include: (1) the sickle cell disease Glu6Val founder mutation in hemoglobin beta (requiring an A-T to T-A transversion); (2) the most common Wilson disease variant His1069Gln in ATP7B (requiring a G-C to T-A transversion); and (3) the △Phe508 mutation in CFTR that causes cystic fibrosis (requiring a three nucleotide insertion). Each of these targets contains a PAM appropriately positioned for SpCas9 targeting and prime editing.
Preliminary experimental results.
●T→A editing in HEK3 cells cannot be achieved with modern base editing, but can be achieved with TRPT editing (see Figures 17A-17C).

Figure 17A shows a graph displaying the % T→A conversion at the target nucleotide after transfection of the constructs in human embryonic kidney (HEK) cells. The data presents the results using an N-terminal fusion of wild-type MLV reverse transcriptase with Cas9 (H840A) nickase (32 amino acid linker). Editing efficiency improved dramatically when the length of the primer binding site was extended from 7 nucleotides to 11 or 12 nucleotides. In addition, auxiliary guide A, which is positioned just upstream of the editing locus (see Figure 16), significantly improves editing activity, especially when the length of the primer binding site is shorter. Editing efficiency was quantified by amplicon sequencing using the MiSeq platform. Figure 17B also shows the % T→A conversion at the target nucleotide after transfection of the constructs in human embryonic kidney (HEK) cells, but the data presents the results using a C-terminal fusion of the RT enzyme. Here, auxiliary guide A has not as much of an effect and the overall editing efficiency is higher. FIG. 17C shows data presenting the results of using an N-terminal fusion of wild-type MLV reverse transcriptase with the 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.

●The T→A edit at the HEK3 site by RPT editing shows high purity.
FIG. 18 shows the input of the sequencing analysis by high-throughput amplicon sequencing. The input displays the most abundant genotype of the edited cells. Notably, no major indel products are obtained and the desired point mutation (T→A) is clearly incorporated without bystander editing. The first sequence shows the reference genotype. The top two products are the starting genotype (G or A) containing the endogenous polymorphism. The bottom two products represent the corrected edited genotype.

•MLV RT mutants improve editing.
Mutant reverse transcriptases described in Baranauskas et al. (doi:10.1093/protein/gzs034) were tested as C-terminal fusions with Cas9(H840A) nickase for targeted nucleotide editing in human embryonic kidney (HEK) cells. Cas9-RT editor plasmids were co-transfected with a plasmid encoding a 3' prime editing guide RNA that served as a template for reverse transcription. Editing efficiency at the targeted nucleotide (blue bars) is shown alongside the indel rate (orange bars) in Figure 19. WT refers to wild-type MLV RT enzyme. Mutant enzymes (M1-M4) contain the mutations listed on the right. Editing rates were quantified by high-throughput sequencing of genomic DNA amplicons.

Nicking the complementary strand with a second gRNA improves editing.
This experiment evaluates the editing efficiency of a target nucleotide when a single-stranded nick is introduced into the complementary DNA strand adjacent to the target nucleotide (with the hypothesis that this will 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 a plasmid encoding two guide RNAs, one of which serves as a template for reverse transcription and the other of which targets the complementary DNA strand for nicking. Nicking at various distances from the target nucleotide was tested (orange triangles) (see Figure 20). The editing efficiency at the target base pair (blue bars) is shown alongside the indel rate (orange bars). The "none" example does not contain a guide RNA that nicks the complementary strand. The editing rate was quantified by high-throughput sequencing of genomic DNA amplicons.

Figure 21 shows processed high-throughput sequencing data showing the desired T->A transversion mutation and the overall absence of other major genome editing by-products.

range.
The potential scope for new editing technologies is shown in Figure 13 and compared with deaminase-mediated base editor technologies. Base editors developed to date target a ~15±2 bp region upstream of the PAM. By converting targeted C or A nucleotides to T or G, respectively, base editors developed to date enable all transition mutations (A:T→G:C conversions). However, base editors developed to date cannot incorporate transversion mutations (A→T, A→C, G→T, G→C, T→A, T→G, C→A, C→G). Moreover, if there are multiple targeted nucleotides in the editing window, it may result in unwanted additional edits.

The new prime editing technology can theoretically accommodate any nucleotide and base pair change, potentially even small insertion and deletion edits. For PAM, the prime editing window begins at the site where the DNA is nicked (three bases upstream of the PAM) and ends at a so far undetermined location downstream of the PAM. Notably, this editing window is distinct from that of deaminase base editors. The TPRT system uses a DNA polymerase enzyme to perform the edits, and thus has all of those benefits, including generality, high precision, and fidelity.

Correcting 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 epithelium) are obtained from ATCC, Coriell Biobank, or collaborating Harvard/Broad affiliate laboratories. Editing efficiency is assessed by high-throughput sequencing, and the efficacy of the corrected genotype is tested using phenotypic assays (hemoglobin HPLC, ATP7B immunostaining, and CFTR membrane potential assay).

Characterize off-target editing activity.
Potential off-target editing will be screened using targeted gRNAs paired with wild-type Cas9 with established methods such as GUIDE- ^seq55 and CIRCLE- ^seq.56 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 editing elements (PEs) may require optimization for each target. In this case, gRNA libraries can be tested to identify the best-functioning variants for a particular application. Expression and nuclear localization of RT-Cas fusions can be optimized. Liposomal RNP delivery can be used to limit off-target editing.

Next experiment.
Optimization of gRNA design can be achieved by further exploration of primer binding site length and editing template extension. Testing scope and generality includes various nucleotide conversions, small insertions and deletions, and various editing positions relative to the PAM and multiple sites in the human genome. Optimization of RT components includes exploring mutations that enhance activity in MLV RT (inactivation of Rnase H, increasing primer-template binding affinity, tuning processivity) and mutations in new RT enzymes (group II intron RT, RT of other retroviruses).

Significance.
A myriad of genetic disorders result from single nucleotide changes in individual genes. Developing the genome editing technology described here and applying it to disease-relevant cell types will establish the basis for moving 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 results in a similar disease phenotype. Thus, common genome editing methods could theoretically target such a large number of mutations, providing enormous potential benefits to many of these patients and their families. If proof of principle for these applications can be established in cells, it will establish the basis for studying in animal models of disease.

Advantages High precision: the desired edits are encoded and directed in the nucleic acid sequence. Generality: in theory, any base pair change can be made, including transversion edits as well as small insertions or deletions. There is a different editing window than base editors for the Cas9 protospacer adjacent motif (PAM) sequence. This method captures many of the editing capabilities of homology-directed repair (HDR), but without the major drawbacks of HDR (inefficiency in most cell types, often accompanied by excess unwanted by-products such as indels). The method also lacks double-stranded DNA breaks (DSBs), very few indels, translocations, large deletions, p53 activation, etc.

Example 2 – Error-prone Prime Editing (PE)

The prime editing (PE) system described herein can also be used in conjunction with error-prone reverse transcriptase enzymes to incorporate mutations into the genome.

An embodiment is illustrated in FIG. 22, which is a schematic diagram of an exemplary process for performing error-prone reverse transcriptase targeted mutagenesis at 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 includes an extension at the 3' or 5' end of the guide RNA or at the intramolecular positioning of 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 is introduced (e.g., by a nuclease or chemical agent) into one of the strands of the DNA at the target locus, thereby generating an available 3' end on one of the strands at the target locus. In an embodiment, the nick is generated on the strand of DNA corresponding 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 to prime reverse transcription. In some embodiments, the 3'-end DNA strand hybridizes to a specific RT prime sequence of the extended portion of the guide RNA. In step (d), an error-prone reverse transcriptase is introduced, which synthesizes a mutated single strand of DNA from the 3'-end of the primed site toward the 3'-end of the guide RNA. Exemplary mutations are indicated by an asterisk "*". This forms a single-stranded DNA flap containing the desired mutated region. In step (e), the napDNAbp and the guide RNA are released. Steps (f) and (g) involve the degradation of the single-stranded DNA flap (containing the mutated region), so that the desired mutated region is integrated into the target locus. This process can be driven toward the desired product formation by removing the corresponding 5' endogenous DNA flap that forms once the 3' single-stranded DNA flap invades and hybridizes to a complementary sequence on the other strand. The process can also be driven toward product formation by second strand nicking as illustrated in Figure 1F. After endogenous DNA repair and/or replication processes, the mutated region becomes incorporated onto both strands of DNA at the DNA locus.

Example 3 - Trinucleotide Repeat Truncation by PE The prime editing system or prime editing (PE) system described herein can be used to shorten trinucleotide repeat mutations (or "triplet expansion diseases") to treat diseases such as Huntington's disease and other trinucleotide repeat disorders. Without being bound by theory, triplet expansion is caused by slippage during DNA replication or DNA repair synthesis. Because the tandem repeats have identical sequences to each other, base pairing between the two DNA strands can occur at multiple points along the sequence. This can lead to the formation of "loop-out" structures during DNA replication or DNA repair synthesis. This can lead to repeated copies of the repeat sequence, expanding the number of repeats. An additional mechanism involving a hybrid RNA:DNA intermediate has been proposed. Prime editing can be used to reduce or eliminate these triplet expansion regions by deletion of one or more of the offending repeated codon triplets. In one embodiment of this use, Figure 23 provides a schematic of PEgRNA design for shortening or reducing trinucleotide repeat sequences by prime editing.

Therefore, prime editing may be capable of being used to correct any trinucleotide repeat disorder, including Huntington's disease, Fragile X syndrome, and Friedreich's ataxia.

The most common trinucleotide repeat contains a CAG triplet, but GAA triplets (Friedreich's ataxia) and CGG triplets (Fragile X syndrome) also occur. The CAG triplet codes for glutamine (Q), and therefore, CAG repeats result in polyglutamine tracts in the coding regions of diseased proteins. This specific class of trinucleotide repeat disorders is 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 the expansion or acquiring an already expanded parental allele increases the probability of acquiring the disease. Pathogenic expansions of trinucleotide repeats can be corrected using prime editing.

Prime editing can be implemented to shorten the triplet expansion region by nicking a region upstream of the triplet repeat region with a prime editor containing a dedicated PEgRNA targeted to the site to be cut. The prime editor then synthesizes a new DNA strand (ssDNA flap) based on the PEgRNA as a template (i.e., its editing template) that encodes a healthy number of triplet repeats (this depends on the specific gene and disease). The newly synthesized ssDNA strand containing the healthy triplet repeat sequence is also synthesized to include a short stretch of homology (i.e., a 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 by the newly synthesized ssDNA flap leads to a shortened repeat allele.

Example 4 - Peptide tagging with PE The prime editing system described herein (i.e., the PE system) can also be used to introduce various peptide tags onto protein-coding genes. Such tags can include HEXA histidine tags, FLAG tags, V5 tags, GCN4 tags, HA tags, Myc tags, and others. This approach can be useful for applications such as protein fluorescent labeling, immunoprecipitation, immunoblotting, immunohistochemistry, protein mobilization, inducible protein degrons, and genome-wide screening. An embodiment is illustrated in Figures 25 and 26.

Figure 25 is a schematic diagram showing gRNA design for peptide tagging of genes at endogenous genomic loci and peptide tagging by TPRT genome editing (i.e., prime editing). The FlAsH and ReAsH tagging systems include two parts: (1) a fluorophore-biarsenical probe and (2) a peptide encoded by a gene containing a tetracysteine motif, exemplified by the sequence FLNCCPGCCMEP (SEQ ID NO: 1). When expressed in cells, proteins containing a tetracysteine motif can be fluorescently labeled by the fluorophore-arsenical probe (see ref: J. Am. Chem. Soc., 2002, 124(21), pp 6063-6076. DOI: 10.1021/ja017687n). The "sortagging" system uses bacterial sortase enzymes to covalently conjugate labeled peptide probes to proteins containing suitable peptide substrates (see ref: Nat. Chem. Biol. 2007 Nov;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 used in immunoassays as epitope tags. The pike clamp encodes a peptide sequence (FCPF) (sequence number 622) that can be labeled with a pentafluoro-aromatic substrate (ref: Nat. Chem. 2016 Feb;8(2):120-8.doi:10.1038/nchem.2413).

Figure 26 shows the precise incorporation of His6 and FLAG tags onto genomic DNA. Guide RNAs were designed to target the HEK3 locus with reverse transcription templates encoding either an 18nt His tag insertion or a 24nt FLAG tag insertion. Editing efficiency in transfected HEK cells was assessed using amplicon sequencing. Note that the complete 24nt sequence of the FLAG tag is outside the view frame (sequencing confirmed complete and precise insertion).

Example 5 – Prevention or treatment of prion disease with PE

The present invention may help address the problem of prion disease in humans, livestock, and wildlife. Previously described editing strategies are not efficient and clean enough to incorporate protective mutations or reliably knock down PRNP. Cas9 nuclease and HDR could be used, but would generate a mixture of mostly PRNP indel variants. Some of these are thought to be pathogenic. Moreover, HDR does not work in most types of cells. Prime editing is reliable and efficient in incorporating both types of mutations without generating extra double-stranded DNA breaks or resulting indels.

The present invention describes how to incorporate protective mutations in PRNP that prevent or halt the progression of prion disease. This site is conserved in mammals, so in addition to treating human disease, it can also be used to generate cattle and sheep that are immune to prion disease or even help cure wild populations of animals that suffer from prion disease. Prime editing has already been used to achieve incorporation of ∼25% of the naturally occurring protective allele in human cells, and previous mouse experiments indicate that this level of incorporation is sufficient to induce immunity to most prion diseases. This method is the first and potentially only current way to incorporate this allele in most cell types with such high efficiency. Another possible strategy for treatment is to use prime editing to reduce or eliminate expression of PRNP by incorporating a premature stop codon into the gene. Many researchers predict that doing so will treat the disease.

Three potential therapeutic strategies include prime editing to reduce the expression of PrP. This goal can be achieved by introducing mutations that cause a premature stop codon in PRNP, erase the start codon, mutate or delete an essential amino acid codon, introduce or remove a splice site to generate an aberrant transcript, or alter a regulatory element to reduce transcript levels. Prime editing to erase disease mutations. Many variants of PRNP that lead to an increased probability of suffering from the disease have been described (ncbi.nlm.nih.gov/pmc/articles/PMC6097508/#b154-ndt-14-2067). Each known variant can 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 block 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 the 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 on PRNP that would interfere with prion formation can also be used to protect against or treat prion disease.

The third therapeutic strategy is particularly advantageous because the introduction of a protective variant may confer benefit even when a relatively small number of cells undergo editing. Furthermore, the introduction of a protective variant, particularly one that occurs naturally in the human population, such as G127V, would not be expected to have any adverse consequences, whereas reducing expression of the prion protein as in strategy 1 may have some adverse phenotypes, as has been demonstrated in PRNP knockout mice (ncbi.nlm.nih.gov/pmc/articles/PMC4601510/, ncbi.nlm.nih.gov/pmc/articles/PMC2634447/).

It has previously been demonstrated that mice expressing an approximately 2:1 ratio of wild-type human prion protein:the protective G127V variant of human prion protein (approximately 33% expression of the protective variant) were completely immune to most tested forms of prion disease and were also resistant to variant Creutzfeldt-Jakob disease (vCJD), a human disease transmitted from bovine spongiform encephalopathy (BSE or mad cow disease) ^.91 Mice expressing only the protective G127V variant were completely immune to all tested prion disease challenges, including vCJD.

It is demonstrated herein that the protective G127V mutation can be efficiently introduced into human cells in tissue culture using prime editing (see Figure 27).

Informed by these results, three settings are described in which PRNP editing may be used. One setting, PRNP editing, may be used for prime editing in human patients to prevent or treat prion disease. A second setting, PRNP editing, may be used for prime editing in livestock to prevent the occurrence and spread of prion disease. Livestock, both cattle and sheep, have experienced idiopathic occurrences of prion disease caused by the protein produced by the PRNP gene. In addition to the debilitating and fatal disease suffered by the animals, these cases are also economically devastating, in part due to the care that must be taken to prevent the spread of the highly infectious disease. A single dairy cow in Washington state tested positive for BSE in December 2003, leading to an estimated loss of $2.8-4.2 billion in beef sales the following year (bookstore.ksre.ksu.edu/pubs/MF2678.pdf). The PRNP gene is highly conserved in mammals. Introducing PRNP mutations such as G127V into the germline of livestock may eliminate the occurrence of BSE or scrapie, a prion disease manifestation in sheep. A third set of PRNP edits may be used to prime wildlife to prevent the spread of prion disease in the wild. Currently, cervid populations, including North American deer, elk, and moose, suffer from chronic wasting disease (CWD), a prion disease manifestation caused by PNRP in these species. Incidence 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 Korea. It is not yet known whether the disease is transmissible from these species to humans (cdc.gov/prions/cwd/transmission.html) or livestock. Introducing PRNP mutations such as G127V in the germline of these species may protect them from CWD and reduce the risk of transmission to other species, including humans.

This method can be used to treat Creutzfeldt-Jakob disease (CJD), kuru, Gerstmann-Straussler-Scheinker disease, fatal familial insomnia (FFI), bovine spongiform encephalopathy (BSE; mad cow disease), scrapie (sheep), and chronic wasting disease (CWD; deer, elk, and moose).

The method will need to be combined with delivery methodologies for embryonic or adult neurons, such as microinjection, lipid nanoparticles, or AAV vectors.

Example 6 - RNA tagging and manipulation using PE Described herein are new methods for the insertion of motifs onto gene sequences to tag or otherwise manipulate RNA in mammalian, eukaryotic, and bacterial cells. Although it has been estimated that only 1% of the human genome codes for proteins, virtually all of the genome is transcribed at some level. It remains an open question how many of the resulting non-coding RNAs (ncRNAs) play functional roles, much less what the role of most of these putative RNAs is. "Tagging" these RNA molecules by the insertion of novel RNA coding sequences with useful properties onto genes of interest is a useful method to study the biological function of RNA molecules in cells. It may also be useful to incorporate tags onto mRNAs that code for proteins as a means to perturb and therefore better understand how mRNA modifications can affect cellular functions. For example, polyadenylation, a ubiquitous natural RNA tag, is used by cells to affect the transport of mRNAs into the cytoplasm. Different types of polyadenylation signals result in different transport rates and different mRNA lifetimes and hence different levels at which the encoded protein is expressed.

The common approach to express tagged RNA in cells is to exogenously introduce synthetic constructs, either (i) using transient plasmid transfection, which results in short-term bursts of gene expression, often at supraphysiological levels; or (ii) permanent incorporation of tagged RNA genes (at random sites) into the genome using lentiviral incorporation or transposons, which allow for sustained expression. Both of these approaches are limited by the production of altered expression levels and by the absence of native mechanisms to control gene expression or activity. An alternative strategy is to directly tag the 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. Although this approach allows for the generation of a wide range of endogenously tagged genes, HDR is significantly inefficient and therefore requires significant screening to identify the desired clonal population of successfully tagged cells. Moreover, HDR is typically very inefficient or completely inactive in many cell types, most notably in postmitotic cells. The low efficiency of HDR is further complicated by the generation of unwanted indel products, which can be particularly problematic in the case of RNA genes, as they can lead to the production of RNA whose activity interferes with the function of normal alleles. Finally, to achieve optimal performance, researchers often need to screen various tagging positions on the RNA molecule. Combined, these drawbacks make HDR a less desirable method for the incorporation of tags on RNA.

Prime editing is a new genome editing technology that allows targeted editing of genomic loci by transfer of genetic information from RNA to DNA. With prime editing, RNA genes can be tagged with various components, such as RNA aptamers, ribozymes, or other RNA motifs. Prime editing has the potential to be fast, cheap, and effective in a vast variety of cell types compared to HDR strategies. As such, the described invention represents a novel, useful, and non-obvious tool for interrogating the biology of RNA genes in health and disease. Described herein are novel methods for the insertion of RNA motifs onto gene sequences that tag or otherwise engineer RNA using prime editors (PEs). PEs can site-specifically insert, mutate, and/or delete multiple nucleotides at desired genomic loci that are targetable by the CRISPR/Cas system. PEs consist of a fusion between the Cas9 nuclease domain and the reverse transcriptase domain. They are guided to their genomic targets by engineered PE gRNAs (prime editing guide RNAs) that contain a guide spacer portion for DNA targeting and 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 (hereafter RNA motifs) that are functional at the RNA level to tag or otherwise manipulate non-coding RNAs or mRNAs. These motifs can serve to increase gene expression, decrease gene expression, alter splicing, change post-transcriptional modifications, affect the subcellular localization of RNA, enable isolation of RNA or determination of its intracellular or extracellular localization (e.g., using fluorescent RNA aptamers, e.g., Spinach, Spinach2, Baby Spinach, or Broccoli), recruit endogenous or exogenous proteins or RNA binding factors, introduce sgRNAs, or induce processing of RNA by either self-cleavage or RNAse (see FIG. 28B). Due to the flexibility of prime editing, it is not possible to provide an exhaustive list of RNA motifs that can be incorporated into the genome. Here, a series of examples are presented that broadly illustrate the expected range of RNA motifs incorporated by PE that can be used to tag RNA genes. Making these changes efficiently and fairly cleanly is not currently possible in most types of cells (including many that do not support HDR) using any other reported genome editing method other than PE.

Gene expression can be influenced by encoding 3' untranslated regions (UTRs) that result in nuclear transport or retention or altered mRNA lifetime. For example, the polyA tail from the polyomavirus simian virus 40 (SV40) has additional helper sequences that allow efficient transcription termination and can increase gene expression relative to other 3'UTRs. ^57,58 An example sequence of the SV40 polyA tail:

Post-translational modification signals other than polyadenylation signals can also be encoded by PE. These included signals incorporate N6-methyladenosine, N1-methyladenosine, 5-methylcytosine, and pseudouridine modifications. ⁵⁹ It would be possible to use PE to induce the writing or erasure of these modifications on the RNA transcript by including sequences that are bound by enzymes that write or remove them. This could be used as a tool to study the effects of these markers, to induce cell differentiation, to affect stress responses, or to affect target cells in other ways, since the functions of these markers are yet to be explored.

PEs can encode mutations that affect subcellular localization: for example, incorporation of tRNA-Lys on the mRNA could theoretically result in transport to ^{mitochondria60} , whereas different 3′UTRs could result in nuclear retention or ^transport61 .

example:

The SV40 polyA signal mediates transport.

The U1 snRNA 3 box confers retention.

Determining the subcellular localization of endogenous RNAs can be difficult, requiring the addition of exogenous fluorescently tagged nucleotide probes, as in the case of FISH, or time-consuming and potentially imprecise cell fractionation followed by RNA detection. Coding probes onto endogenous RNAs would eliminate many of these issues. One example would be to code fluorescent RNA aptamers, such as Spinach ⁶² or Broccoli, onto endogenous RNAs, thereby visualizing the presence of the RNA by the addition of a small protofluorophore.

Broccoli aptamer:

PE can insert or remove sequences encoding RNAs recognized by RNA-binding proteins, affecting RNA stability, expression, localization, or modification (see, for example, listed proteins ⁶³ ).

As a viral or cancer defense mechanism, PE can insert sequences encoding sgRNAs into the genome. Similarly, it can be used to insert microRNAs (e.g., pre-microRNAs) to direct the silencing of target genes.

PE can insert sequences that result in processing of the RNA either by itself or by external factors, either for therapeutic purposes or as a tool to study the function of various parts of the RNA. For example, the HDV ribozyme ⁶⁴ , when included on an RNA sequence, results in processing of the RNA immediately 5' of the ribozyme, and the hammerhead ribozyme cleaves preceding the third stem on the ribozyme ^65. Other self-cleaving ribozymes include pistol ⁶⁶ , hatchet ⁶⁶ , hairpin ⁶⁷ , Neuropora Varkud satellite ⁶⁸ , glmS ⁶⁹ , twister ⁷⁰ , and twister sister ^66. These sequences can include wild-type or engineered or evolved versions of the ribozyme. Depending on where the site cut by the ribozyme is located, many of these ribozymes can have different sequences depending on the region of the RNA they are associated with. Sequences that would direct the processing of RNA by external factors, such as sequence-specific RNAses ⁷¹ , RNAses that recognize specific structures ⁷² , such as Dicer ⁷³ and Drosha ⁷⁴ , can also be achieved.

References for Example 6

The following references are incorporated herein by reference in their entirety:

Example 7 - Generation of Gene Libraries by PE Described herein are novel methods for the cellular generation of highly sophisticated libraries of protein- or RNA-encoding genes with defined or variable insertions, deletions, or defined amino acid/nucleotide conversions, and their use in high-throughput screening and directed evolution. References cited in the examples are based on the list of references included at the end of this example.

The generation of variable gene libraries is most commonly achieved by mutagenic PCR. ¹ This method relies on either using reaction conditions that reduce the fidelity of the DNA polymerase or using modified DNA polymerases with higher mutation rates. Thus, the biases of these polymerases are reflected in the library products (e.g., preference for transition mutations over transversions). An inherent limitation of this approach to library construction is the relative inability to affect the size of the genes to be altered. Most DNA polymerases have extremely low indel ^mutation2 (insertion or deletion) rates, and most of these will result in frameshift mutations in protein-coding regions, making it unlikely that library members will pass any downstream selection. In addition, PCR and cloning biases can make it difficult to generate a single library 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 potency are typically associated with insertion and deletion mutations, which are unlikely to occur during standard library generation for mutagenesis. Furthermore, these mutations most commonly occur in regions of the protein that are predicted to form loops versus the hydrophobic core, and therefore most indels generated using traditional unbiased approaches are likely to be either deleterious or ineffective.

Since all libraries have access to only a fraction of the possible mutation space, libraries that can bias mutations towards sites on the protein where they are most likely to be beneficial, such as loop regions, would have a significant advantage over conventional libraries. Finally, although it is possible to generate gene libraries with site-specific indel mutations by multi-step PCR and clone assembly using NNK primers or by DNA shuffling, these libraries cannot undergo additional rounds of continuous evolution "indelgenesis". Continuous evolution is a type of directed evolution with minimal user intervention. One such example is ^PACE3 . Since continuous evolution occurs with minimal user intervention, any increase in library diversity during evolution should occur using native replication mechanisms. Thus, in PACE, libraries of genes with inserted or removed codons at specific loci can be generated and screened, but additional rounds of "indelgenesis" are not possible.

It is envisioned that the programmability of prime editing (PE) can be exploited to generate highly sophisticated programmed gene libraries for use in high-throughput screening and directed evolution (see FIG. 29A). PE can insert, change, or remove a defined number of nucleotides from a defined gene locus using information encoded on a prime editing guide RNA (PEgRNA) (see FIG. 29B). This allows the generation of targeted libraries with one or more amino acids inserted or removed from loop regions where the mutation is most likely to result in a change in function, without the background introduction of non-functional frameshift mutations (see FIG. 29C). PE can be used to incorporate a specific set of mutations regardless of the biases inherent in either the DNA polymerase or the sequence to be mutated.

For example, PEs can be used to convert any given target codon to a TGA stop codon in one step, which would be an unlikely occurrence with standard library generation since it would require three consecutive mutations encompassing two transversions. They can also be used to incorporate programmed diversity at a given position, for example by incorporating a codon that codes for any hydrophobic amino acid but not any other at that site. Furthermore, because of the programmability of PE, multiple PE gRNAs can be utilized to generate multiple different edits at multiple sites simultaneously, enabling the generation of highly programmed libraries (see FIG. 29D). In addition, it is possible to generate regions of mutagenesis in an otherwise unaltered library using reverse transcriptases with lower fidelity (e.g., HIV-I reverse transcriptase ⁴ or Bordetella phage reverse transcriptase ⁵ ).

The possibility of repeated rounds of PE on the same site is also envisioned, for example allowing repeated insertion of a codon at a single site. Finally, it is envisioned that all of the approaches described above can be incorporated into continuous evolution, allowing the generation of novel in situ evolution libraries (see FIG. 30). They can also be used to construct these libraries in other cell types where it would otherwise be difficult to assemble large libraries, for example in mammalian cells. The generation of bacterial strains encoding optimized PE 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 properties of PE. In conclusion, library generation with PE would be a highly useful tool in synthetic biology and directed evolution, as well as for high-throughput screening of combinatorial mutants of proteins and RNAs.

Competing Approaches The primary method by which diverse libraries are currently generated is by mutagenic PCR, as described ^above.1 Although insertions or deletions can be introduced by degenerate NNK primers at defined sites during PCR, introducing such mutations at multiple sites requires multiple rounds of iterative PCR and cloning before a more diverse library can be constructed by mutagenic PCR, making the method slow. An alternative, complementary method is DNA shuffling, in which fragments of a library of genes generated by DNase treatment are introduced into a primer-less PCR reaction, resulting in annealing of the different fragments to each other and more rapid generation of a diverse library than mutagenic PCR alone.6 ^Although this approach can theoretically generate indel mutations, it more often results in frameshift mutations that disrupt gene function. Furthermore, DNA shuffling requires a high degree of homology between gene fragments. Both of these methods must be done in vitro, and the resulting libraries are transformed into cells, whereas libraries generated by PE can be constructed in situ, enabling their use for continuous evolution. Libraries can be constructed in situ by in vivo mutagenesis, and these libraries rely on the host cell machinery and exert a bias against indels. Similarly, traditional cloning methods can be used to generate site-specific mutation profiles, but 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 can be constructed directly within the cell type of interest, versus being cloned into a model organism such as E. coli and then transferred into the cell or organism of interest. Another competing approach to targeted diversification is automated multiplex genome engineering or MAGE, in which multiple single-stranded DNA oligonucleotides can be incorporated into replication ^forks , resulting 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, ^whereas PE is more highly programmed and is expected to result in fewer off-target effects. In addition, 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.
An example of PE in directed evolution for constructing gene libraries

In one example, PE can be used in directed evolution experiments to introduce protein variants into gene libraries during continuous evolution experiments with PACE, allowing the recursive accumulation of both point mutations and indels in a manner not possible with conventional approaches. It has already been shown that PE can site-specifically and programmably insert nucleotides into gene sequences in E. coli. In the directed evolution outlined, modified two-hybrid protein:protein binding PACE selection is proposed to identify monobodies with improved binding to specific epitopes. The specific and highly variable loops on these monobodies contribute significantly to affinity and specificity. Improved monobody binding can be rapidly obtained in PACE by altering the length and composition of these loops in a targeted manner. However, altering sequence length is not an established functionality of PACE. Although a library of various loop sizes can be used as a starting point for PACE, subsequent improvements in length would not occur during PACE selection, preventing access to beneficial synergistic combinations of point and indel mutations. Introducing PE into the PACE selection would allow for the in situ generation and evolution of monobodies with various loop lengths. To do so, it is envisioned the introduction of an additional PE plasmid encoding the PE enzyme and one or more PEgRNAs into the host E. coli strain. Expression of the PE enzyme and PEgRNAs would be under the control of small molecules delivered to the PACE lagoon at rates selected by the experimenter.

In various embodiments, the PEgRNA component will be designed to contain a spacer that directs PE to the site of interest on the selection phage, allowing multiple trinucleotides to be inserted into the target site. This will introduce new PEgRNA binding sites, allowing repeated insertion of one or more codons at the target site.

In parallel, another host E. coli strain could harbor a PEgRNA that templates the removal of one or more codons, allowing the loop size to shrink during evolution. PACE experiments could utilize a mixture of both strains, or alternate between the two to allow for the slow and controlled addition or removal of loop sequences.

It is noted that this technique can also be applied to antibody evolution. The binding principles governing antibodies are very similar to those governing monobodies: the length of antibody complementarity determining region loops is critical for their binding function. Furthermore, 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 the above described PE to antibodies or antibody-derived molecules will allow the generation of antibodies with diverse loop lengths and various loop sequences. In combination with PACE, such an approach will allow enhanced binding by loop geometries not accessible to standard PACE and therefore the evolution of highly functional antibodies.

The experiment will demonstrate the ability to use PE to correct harmful mutations in bacteriophage M3 in phage-assisted discontinuous evolution (PANCE), a necessary first step for using PE in continuous evolution (see Figure 69).

References for Example 7

The following references are incorporated herein by reference in their entirety:

Example 8 – PE-mediated immune epitope insertion
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 onto target genomic loci. Previously, homology-directed repair (HDR) was used for this application, requiring a 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 to the cell and is the only method available for inserting large DNA sequences into mammalian cells. However, HDR is hampered by unwanted cellular side effects derived from initiating the 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 work to overcome these drawbacks involves fusing a human Rad51 mutant to the Cas9 D10A nickase (RDN), resulting in a DSB-free HDR system. This features improved HDR product:indel ratios and lower off-target editing, but is still hampered by cell-type dependency and only modest HDR editing efficiency.

The recently developed fusion of Cas9 to a reverse transcriptase coupled with PEgRNA (the "prime editor") represents a novel genome editing technology that offers several advantages over existing genome editing methods, including the ability to incorporate 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. Of note, PE editing is generally achieved with a low rate of unintended indels. Thus, PE enables targeted insertion-based editing applications that were previously impossible or impractical.

The specific invention describes a method for using prime editing as a means to insert known immunogenic epitopes onto endogenous or exogenous genomic DNA, resulting in the modification of the corresponding protein for therapeutic or biotechnological applications (see Figures 31 and 32). Prior to the invention of prime editing, such insertions could only be achieved inefficiently and with high indel formation rates from DSBs. Prime editing offers higher efficiency than HDR in general, while solving the problem of high indel formation from insertion editing. This lower indel formation rate presents a major advantage of prime editing compared to HDR as a method for targeted DNA insertion, especially in the described application of inserting immunogenic epitopes. The length of the epitopes ranges from a few bases to several hundred bases. Prime editors are an efficient and cleanest technology for achieving such targeted insertions in mammalian cells.

The key concept of the present invention is the use of a prime editing agent to insert nucleotide sequences containing the immunogenic epitopes described above onto endogenous or exogenous genomic DNA for downregulation and/or destruction of their protein products and/or expressing cell types. The nucleotide sequences for immunogenic epitope insertion will be targeted into the gene in a manner that results in a fusion protein between the encoded protein of the target gene and the corresponding protein translation of the inserted immunogenic epitope. The patient's immune system will have been previously trained to recognize these epitopes as a result of prior standard immunization, for example from routine vaccination against tetanus or diphtheria or measles. As a result of the immunogenic nature of the fused epitopes, the patient's immune system will be expected to recognize and disable the prime edited protein (and not just the inserted epitope) and potentially the cells in which it is expressed.

Fusions to the target gene will be engineered as required to ensure that the inserted epitope protein translation is exposed for immune system recognition. This can include C-terminal fusions of the immunogenic epitope to the target gene, N-terminal fusions of the immunogenic epitope to the target gene, or targeted nucleotide insertions that result in protein translation yielding insertion of nucleotides onto the gene such that the immunogenic epitope is encoded in an exposed region on the surface of the protein structure.

To facilitate immune system recognition of the target gene, cellular trafficking, protein function, or protein folding, protein linkers encoded as nucleotides inserted between the target gene sequence and the inserted immunogenic epitope nucleotide sequence may need to be engineered as part of the invention. Protein linkers encoded by these inserted nucleotides may include (but are not limited to) XTEN linkers of variable length and sequence or glycine-serine linkers of variable length and sequence. These engineered linkers have been used previously to successfully facilitate protein fusion.

A distinguishing feature of the present invention includes the ability to use a previously acquired immune response to a particular amino acid sequence as a means to induce an immune response to an otherwise non-immunogenic protein. Another distinguishing feature is the ability to insert the nucleotide sequences of these immunogenic epitopes in a targeted manner that does not induce high levels of unwanted indels as by-product editing and whose insertion is efficient. The invention discussed in this application also has the ability to combine cell type specific delivery methods (e.g., AAV serotypes) to insert epitopes into the cell type against which it is desired to elicit an immune response.

Prime editing as a means of inserting immunogenic epitopes onto pathogenic genes can be used to program a patient's immune system to fight a wide variety of diseases (not limited to cancer in an immuno-oncology strategy). An immediately relevant use of this technology would be as cancer therapeutics, since it could thwart the immune escape mechanisms of tumors by triggering an immune response against relevant oncogenes such as HER2 or growth factors such as EGFR. While such an approach may appear similar to T cell engineering, one novel advancement of this approach is that it can be utilized for many cell types and diseases other than cancer, without the need to generate and introduce engineered T cells into patients.

Using PE, immunogenic epitopes against which most people are already vaccinated (tetanus, whooping cough, diphtheria, measles, mumps, rubella, etc.) are inserted onto exogenous or endogenous disease-driving genes so that the patient's immune system learns to disable the protein.

Diseases that would have potential therapeutic benefit from the aforementioned strategies include those caused by toxic protein aggregation, such as fatal familial insomnia. Other diseases that may benefit include those caused by pathogenic overexpression of otherwise non-toxic endogenous proteins, and those caused by exogenous 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 anticipated that in some diseases, such as particularly aggressive cancers, or in cases where drug therapy helps to alleviate disease symptoms until the disease is completely cured, current standard of care medicine may be used in conjunction with prime editing. Below are examples of immune epitopes that may be inserted by prime editing agents and used to achieve:

Below are examples of additional epitopes that can be incorporated onto target genes for immune epitope tagging:

REFERENCES CITED IN EXAMPLE 8 The following references are incorporated herein by reference in their entirety:

Example 9 - In vivo delivery of PE drugs
Precise genome targeting technologies using the CRISPR/Cas system have been recently explored in a wide range of applications, including gene therapy. A major limitation of the application of Cas9 and Cas9-based genome editing agents in gene therapy is the size of Cas9 (>4kb), which hampers its efficient delivery by recombinant adeno-associated viruses (rAAV). Recently developed fusions of Cas9 to reverse transcriptase ("prime editors") represent a novel genome editing technology that possesses several advantages over existing genome editing methods, including the ability to incorporate any single nucleotide substitution in a site-specific manner and to insert or delete any arbitrarily defined short (<~20) stretches of nucleotides. Thus, this method allows the editing of previously inaccessible human pathogenic variants. Delivery of prime editing reagents may enable the correction of gene sequences that cause human disease or allow the incorporation of disease-preventing gene variants.

The present invention describes methods for delivering prime editors to cells in vitro and in vivo. Prime editors have been developed and characterized only in cultured cells. Known methods cannot deliver prime editors in vivo. The disclosed methods for delivering prime editors by rAAV or preassembled ribonucleoprotein (RNP) complexes will overcome several barriers to in vivo delivery. For example, DNA encoding prime editors is larger than the rAAV packaging limit and therefore requires specialized solutions. One such solution is to formulate the editor fused to a split intein pair, which are packaged into two separate rAAV particles. These reconstitute functional editor proteins when co-delivered to cells. Several other special considerations are described that account for the unique features of prime editing, including optimization of second-site nicking targets and proper packaging of prime editors into viral vectors, including lentiviruses and rAAVs.

A distinctive feature involves using a ribonucleoprotein (RNP) delivery formulation, where the prime editor and nearby nicking targets can be pre-complexed with their specific sgRNA/PEgRNA. This would increase the range of possible targetable sites and allow for greater optimization of editing efficiency relative to current data using DNA delivery. With either an RNP or mRNA delivery formulation, variant Cas proteins can be used, each complexed with their own guide RNA variant. This would also allow for a greater diversity of possible nicking loci. It is therefore anticipated that optimization for greater efficiency can be achieved for any given application. With an RNP, it is anticipated that it will increase editing specificity based on previous RNP reports (Rees et al., 2017). This would reduce off-target prime editing. A potential architecture for splitting the prime editor into two AAV vectors for in vivo or ex vivo delivery is described. Packaging prime editing elements into a binary AAV system requires optimization of design considerations including the division site, reconstitution method (e.g., inteins), and guide expression architecture. Using a mixture of virus and RNPs for delivery of prime editing elements, it is expected that editing will be controlled over time, since RNPs will eventually degrade in vivo, which will stop prime editing after RNPs are no longer supplied.

Prime editor ribonucleoproteins (RNPs), mRNA with prime editor guide RNA, or DNA can be packaged in lipid nanoparticles, rAAV, or lentiviruses and injected, ingested, or inhaled to alter genomic DNA in vivo and ex vivo, including for purposes of establishing animal models of human disease, testing therapeutic and scientific hypotheses in animal models of human disease, and treating human disease.

If suitable means of delivery to the relevant cell types in vivo are developed, primed editors could feasibly be used to correct a large fraction of all genetic diseases (~89% of Clinvar pathogenic human genetic variants). Hematological, retinal, and liver diseases are the most likely first applications due to established delivery systems of other reagents. AAV capsids, other evolved or engineered viral vectors, and lipid nanoparticle formulations will need to be used in combination with the present invention.

In some embodiments, one or more of the prime editor domains (e.g., the napDNAbp domain or the RT domain) can be engineered with an intein sequence.

Example 10 - Use of PE to Identify Off-Target Edits Currently, there are no methods described for detecting off-target editing by prime editors (prime editing itself has not yet been published). These methods would allow researchers to identify possible sites of off-target editing using prime editors, which would be an important consideration if this technology is to be used to treat genetic diseases in patients.

The methods described here may also be useful for identifying off-targets of Cas nucleases. These off-targets have been previously identified using BLESS, Guide-Seq, CIRCLE-Seq, and Digenome-Seq. However, this method has advantages in the sensitivity and simplicity of the process.

The key concept of this aspect is the idea of using prime editing to insert PEgRNA-templated adapter sequences or primer binding sites to enable rapid identification of genomic off-target modification sites for Cas nucleases or prime editors.

No methods are known for identifying prime editing off-target sites in an unbiased manner. This method is distinct 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 downstream processing.

The invention encompasses the identification of off-target editing sites when editing in living cells, in tissue culture or animal models (see FIG. 33). To carry out this method, a PEgRNA is generated that has the same protospacer as the final desired editing element (and the same primer binding site sequence as the final desired editing element if prime editing off-targets are being examined), but includes the necessary sequences for incorporating adapters or primer binding sites after reverse transcription with prime editing. In vivo editing is carried out using a prime editing element or RT fusion nuclease, and genomic DNA is isolated. The genomic DNA is fragmented by enzymatic or mechanical means, and different adapters are added to the sites of DNA fragmentation. PCR is used to amplify from one adapter to the adapter incorporated by the PEgRNA. The resulting product is deep sequenced to identify all modified sites.

The present invention also encompasses the identification of off-target editing sites using in vitro modification of genomic DNA (see FIG. 33). To carry out this method, an RNP is assembled with purified prime editor protein and a PEgRNA that will incorporate an adapter or primer binding sequence, but is otherwise identical to the PEgRNA of interest. This RNP is incubated with extracted genomic DNA before or after DNA fragmentation and attachment of a different adapter to the site of DNA cleavage. PCR is used to amplify from the fragmented site to the adapter incorporated by PE. Deep sequencing identifies the site of modification. This in vitro editing method should enhance the sensitivity of detection, since cellular DNA repair will not erase the reverse transcribed DNA adapter added by the prime editor.

These methods can be used to identify off-target edits for any primed editor or any genome editor that uses a guide RNA to recognize the site of the target cut (most Cas nucleases).

These methods can be applied to all genetic diseases for which genome editing elements are being considered for treatment.

Example 11 - Use of PE to enable chemical-induced dimerization of target proteins in vivo Prime editors described herein can also be used to place dimerization-induced biological processes, such as receptor signaling, under the control of convenient small molecule drugs by genomic incorporation of genes encoding small molecule binding proteins by prime editing, as described herein. Using the prime editors described herein, a genetic sequence encoding a small molecule binding protein can be inserted onto a gene encoding a target protein of interest in a living cell or patient. This editing should have no physiological effect alone. By administration of a small molecule drug, typically a dimeric small molecule that can simultaneously bind to two drug binding protein domains, each fused to a copy of the target protein, the small molecule induces dimerization of the target 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 use programmable nucleases to block, delete, or insert target genes with the concomitant by-products of double-stranded DNA breaks, and base editors to incorporate four transition point mutations at target loci. However, small insertions, small deletions, and eight transversion point mutations, which collectively represent most pathogenic genetic variants, cannot be corrected efficiently and without extra by-products in most cell types. Described herein is a highly versatile and precise genome editing method, prime editing, that uses a catalytically impaired Cas9 fused to an engineered reverse transcriptase that is programmed by an engineered prime editing guide RNA (PEgRNA) that both defines the target site and encodes the desired edit to directly write new genetic information into a defined DNA site. Editing more than 175 variants in human cells has established that prime editing can make targeted insertions, deletions, and point mutations of all 12 possible types, and combinations thereof, efficiently (typically 20-60% in unsorted cells, up to 77%) and with low by-products (typically 1-10%), without requiring double-strand breaks or a donor DNA template. Prime editing has been applied in human cells to correct the primary genetic cause of sickle cell disease (requiring an HBB A.T to T.A transversion) and Tay-Sachs disease (requiring a HEXA tetranucleotide deletion), efficiently reverting the pathogenic genomic allele to wild type in both cases with minimal by-products. To generate human cell lines carrying these pathogenic HBB transversion and HEXA insertion mutations, we also used prime editing to incorporate the G127V mutation (requiring a G-C to T-A transversion) that confers resistance to prion disease into PRNP, and to efficiently insert His6 tags, FLAG epitope tags, and extended LoxP sites into target loci in human cells. Prime editing offers the advantages of efficiency and product purity compared to HDR, and complementary strengths and weaknesses compared to base editing. Consistent with its search-and-replace mechanism, which requires three separate base-pairing events, prime editing is much less prone to off-target DNA modifications than Cas9 at known Cas9 off-target sites. Prime editing substantially expands the scope and capabilities of genome editing and, in principle, can correct ∼89% of known pathogenic human gene variants.

The ability to make virtually any targeted change to the genome of any living cell or organism is a long-standing aspiration of life science. Despite rapid advances in genome editing technology, the majority of the >75,000 known human genetic variants associated with ^disease111 cannot be corrected or incorporated in most therapeutically relevant cells (Figure 38A). Programmable nucleases such as CRISPR-Cas9 create double-stranded DNA breaks (DSBs), which can shut off genes by inducing a mixture of insertions and deletions (indels) at the target ^site112-114 . Nucleases can also be used to delete targeted ^genes115,116 or insert exogenous ^genes117-119 by a homology-independent process. However, double-stranded DNA breaks are also associated with unwanted outcomes including a complex mixture of products, ^{translocations120} , and p53 ^{activation121,122} . Moreover, the majority of pathogenic alleles differ from their nonpathogenic counterparts by small insertions, deletions, or base substitutions, which require significantly more precise editing technologies to correct (Figure 38A). Homologous recombination repair (HDR) stimulated by nuclease-induced ^DSBs123 has been widely used to incorporate a variety of precise DNA changes. However, HDR relies on exogenous donor DNA repair templates, typically generates extra indel by-products from end-joining repair of DSBs, and is inefficient in most therapeutically relevant cell types (T cells and some stem cells are important exceptions) ^124,125 . Enhancing the efficiency and precision of DSB-mediated genome editing remains the focus of promising ^work126-130 , and these challenges necessitate the investigation of alternative precise genome editing strategies.

Although base editing can efficiently incorporate or correct 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 it is currently unable to 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 required to directly correct the most common cause of sickle cell disease (HBB E6V) ^.132 In addition, no DSB-free methods have been reported to make targeted deletions, such as the removal of the 4-base duplication (HEXA 1278+TATC) that causes Tay-Sachs disease, 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 Therefore, although they collectively account for most known pathogenic alleles, targeted transversion point mutations, insertions, and deletions are difficult to incorporate or correct efficiently and without excess by-products in most cell types (Figure 38A).

Described herein is the development of a novel "search and replace" genome editing technology, prime editing, that mediates targeted insertions, deletions, and base conversions from all 12 possible bases at target 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 an extension on the PEgRNA to a target genomic locus. A second generation prime editor (PE2) uses an engineered reverse transcriptase to substantially increase editing efficiency with minimal (typically <2%) indel formation, and a third generation PE3 system adds a second guide RNA to nick the non-edited strand, thereby favoring replacement of the non-edited strand, further increasing editing efficiency to typically about 20-50% in human cells with about 1-10% indel formation. PE3 offers much fewer by-products and higher or similar efficiency compared to HDR initiated by optimized Cas9 nucleases, and offers complementary strengths and weaknesses compared to current generation base editors.

PE3 was applied in genomic loci of 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, incorporation of the G127V mutation into PRNP ¹³⁵ (requiring a G·C to T·A transversion) that confers resistance to prion disease, and targeted insertion of a His6 tag (18 bp), a FLAG epitope tag (24 bp), and an extended LoxP site (44 bp) for Cre-mediated recombination. Prime editing was also successful with various efficiencies in three other human cell lines and in postmitotic primary mouse cortical neurons. Due to the high degree of flexibility in the distance between the initial nick and the positioning of the edit, prime editing is not substantially constrained by the PAM requirement of Cas9 and could in principle target the vast majority of genomic loci. Presumably, due to the requirement of three separate DNA base pairing events for productive prime editing to occur, off-target prime editing is much rarer than off-target Cas9 editing at known Cas9 off-target loci.By enabling precise targeted insertion, deletion, and all 12 possible classes of point mutations at a wide variety of genomic loci without the need for DSB or donor DNA template, prime editing has the potential to advance the study and correction of many genetic variants.

Results Strategy for transferring information from extended guide RNAs to target DNA loci
Cas9 targets DNA using guide RNAs that contain spacer sequences that hybridize to the target DNA site. ^{112-114,136,137} The aim was to engineer guide RNAs to both define DNA targets as in the natural CRISPR system, ^138,139 and also to contain new genetic information that replaces the corresponding DNA nucleotides at the target locus. Direct transfer of genetic information from an extended guide RNA to a defined DNA site, then replacing the original unedited DNA, could in principle provide a general means of incorporating targeted DNA sequence changes into living cells without dependency on DSBs or donor DNA templates. To achieve this direct information transfer, the aim was to prime reverse transcription of genetic information into the target site directly from an extended engineered guide RNA (hereafter referred to as prime editing guide RNA or PEgRNA) using genomic DNA nicked at the target site to expose a 3'-hydroxyl group (Figure 38A).

These initial steps of nicking and reverse transcription, similar to the mechanism used by some natural mobile genetic elements, ¹⁴⁰ result in a branched intermediate with two redundant single-stranded DNA flaps on one strand: a 5' flap containing the unedited DNA sequence and a 3' flap containing the edited sequence copied from the PEgRNA (Figure 38B). To achieve successful editing, this branched intermediate must be degraded, with the edited 3' flap replacing the unedited 5' flap. Because the edited 3' flap can make fewer base pairs with the unedited strand, hybridization of the 5' flap with the unedited strand is likely thermodynamically favored, but the 5' flap is a preferred substrate for structure-specific endonucleases such as ^FEN1,141 which excise the 5' flap generated during lagging strand DNA synthesis and long-patch base excision repair. It was reasoned that preferential 5' flap excision and 3' flap ligation might drive integration of the edited DNA strand, generating a heteroduplex DNA containing one edited and one unedited strand (Figure 38B).

Permanent incorporation of the edit can result from subsequent DNA repair that resolves the mismatch between the two DNA strands in a manner that copies information from the edited strand to the complementary DNA strand (Figure 38C). Based on similar strategies developed to maximize the efficiency of DNA base editing ^{,131-133 it} was hypothesized that nicking the non-edited DNA strand far enough away from the site of the original nick to minimize double-strand break formation could bias DNA repair to preferentially replace the non-edited strand.

Validation of the prime editing step 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 may dissociate from the otherwise stable Cas9:sgRNA:DNA ^complex.143 It was hypothesized that the 3' end of this released strand may be sufficiently accessible to prime DNA polymerization. Guide RNA engineering ^work144-146 and the crystal structure of the Cas9:sgRNA:DNA ^{complex147-149} suggest that the 5' and 3' ends of the sgRNA can be extended without abolishing Cas9:sgRNA activity. PEgRNA was designed by extending the sgRNA to encompass 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 copied directly into the genomic DNA site once the 3' end of the nicked DNA strand is extended onto the RNA template by a polymerase (Figure 38C).

These hypotheses were tested in vitro using purified S. pyogenes Cas9 protein. A series of PEgRNA candidates was constructed by extending sgRNAs at either end with PBS sequences (5 to 6 nucleotides, nt) and RT templates (7 to 22 nt). It was confirmed that 5'-extended PEgRNAs direct Cas9 binding to target DNA, and that both 5'- and 3'-extended PEgRNAs support Cas9-mediated target nicking in vitro and DNA cleavage activity in mammalian cells (Figures 44A-44C). These candidate PEgRNA designs were tested with pre-nicked 5'-Cy5-labeled dsDNA substrates, catalytically inactive Cas9 (dCas9), and a commercially available variant of Moloney murine leukemia virus (M-MLV) reverse transcriptase (Figure 44D). When all components were present, efficient conversion of fluorescently labeled DNA strands to longer DNA products was observed by gel mobility, consistent with reverse transcription along the RT template (Figure 38D, Figure 44D-44E). Products of the desired length were formed with either 5'- or 3'-extended PEgRNA (Figure 38D-38E). Omission of dCas9 led to nick translation products derived from reverse transcriptase-mediated DNA polymerization on the DNA template without PEgRNA information transfer (Figure 38D). No DNA polymerization products were observed when PEgRNA was replaced by a conventional sgRNA, confirming the requirement for the PBS and RT template components of PEgRNA (Figure 38D). These results demonstrate that Cas9-mediated DNA melting exposes single-stranded R-loops that, when nicked, are competent to prime reverse transcription from either 5'- or 3'-extended PEgRNA.

Next, the unnicked dsDNA substrate was tested with Cas9 nickase (H840A mutant), which exclusively nicks the PAM-containing strand. In these reactions, 5' ^- extended PEgRNA generated reverse transcription products inefficiently (Figure 44F), possibly due to impaired Cas9 nickase activity. However, 3'-extended PEgRNA enabled robust Cas9 nicking and efficient reverse transcription (Figure 38E). Despite the potential for reverse transcription to terminate anywhere in the remainder of the PEgRNA in principle, use of 3'-extended PEgRNA generated only a single distinct product. DNA sequencing of the products of reactions with Cas9 nickase, RT, and 3'-extended PEgRNA revealed that the entire RT template sequence was reverse transcribed into the DNA substrate (Figure 44G). These experiments established that 3'-extended PEgRNA could serve as a template for reverse transcription of a new DNA strand while retaining the ability to direct Cas9 nickase activity.

To evaluate the eukaryotic DNA repair outcomes of the 3' flap generated by in vitro PEgRNA-programmed reverse transcription, we performed in vitro DNA nicking and reverse transcription with PEgRNA, Cas9 nickase, and RT on a reporter plasmid substrate, and then transformed the reaction products into yeast (S. cerevisiae) cells (Figure 45A). Encouragingly, when the plasmid was edited in vitro with a 3'-extended PEgRNA encoding a T·A to A·T transversion correcting the premature stop codon, 37% of the yeast transformants expressed both GFP and mCherry proteins (Figure 38F, Figure 45C). Consistent with the results in Figure 38E and Figure 44F, the editing reaction performed in vitro with the 5'-extended PEgRNA yielded fewer GFP and mCherry double-positive colonies (9%) than with the 3'-extended PEgRNA (Figure 38F and Figure 45D). Productive editing was also observed using 3'-extended PEgRNAs that inserted (15% of double-positive transformants) or deleted (29% of double-positive transformants) one nucleotide to correct the frameshift mutation (Figure 38F and Figures 45E-45F). DNA sequencing of edited plasmids recovered from double-positive yeast colonies confirmed that the encoded transversion edit occurred at the desired sequence location (Figure 45G). These results demonstrate that eukaryotic DNA repair can resolve the 3' DNA flap resulting from the prime edit and incorporate precise DNA edits, including transversions, insertions, and deletions.

Design of prime editing factor 1 (PE1)
Encouraged by the in vitro and yeast results, we sought to develop a prime editing system with a minimal number of components capable of editing genomic DNA in mammalian cells. It was hypothesized that a 3'-extended PEgRNA (hereafter simply referred to as PEgRNA, Figure 39A) and a direct fusion of Cas9 H840A to reverse transcriptase via a flexible linker could 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 end of Cas9 H840A nickase and a second plasmid encoding PEgRNA. Initial attempts did not result in detectable T·A to A·T conversion at the HEK3 target locus.

However, extension of the PBS on the PEGRNA to 8-15 bases (Figure 39A) led to detectable T·A to A·T editing at the HEK3 target site (Figure 39B). Primed editor constructs in which the RT was fused to the C-terminus of the Cas9 nickase were more efficient (maximum T·A to A·T conversion of 3.7% with PBS lengths ranging from 8 to 15 nt) compared to N-terminal RT-Cas9 nickase fusions (maximum T·A to A·T conversion of 1.3%) (Figure 39B; unless otherwise specified, all mammalian cell data in this application report values for the entire treated cell population without selection or sorting). These results suggest that wild-type M-MLV RT fused to Cas9 requires a long PBS sequence for genome editing in human cells compared to that required in vitro using a commercially available 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 PE1.

The ability of PE1 to precisely introduce transversion point mutations at four additional genomic target sites defined by the PEgRNA (Figure 39C) was tested. Similar to editing at the HEK3 locus, efficiency at these genomic sites was PBS length dependent, with maximum editing efficiencies ranging from 0.7-5.5% (Figure 39C). Indels from PE1 were low, averaging 0.2 ± 0.1% for the five sites under conditions that maximized editing efficiency at each site (Figure 46A). PE1 was capable of incorporating targeted insertions and deletions at the HEK3 locus, exemplified by a 1-nucleotide deletion (4.0% efficiency), a 1-nucleotide insertion (9.7%), and a 3-nucleotide insertion (17%) (Figure 39C). These results establish the ability of PE1 to directly incorporate targeted transversions, insertions, and deletions without requiring double-stranded DNA breaks or a DNA template.

Design of Prime Editing Factor 2 (PE2)

Although PE1 could incorporate various edits at several loci in HEK293T cells, the editing efficiency was generally low (typically ≦5%) (Figure 39C). It was hypothesized that engineering the reverse transcriptase of PE1 could 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, processivity, and DNA:RNA heteroduplex substrate affinity, as well as inactivate RNase H activity. ^{Nineteen PE1} variants containing various reverse transcriptase mutations were constructed and their prime editing efficiencies were evaluated in human cells.

First, we interrogated ^a series of M-MLV RT variants that had previously emerged from laboratory evolution for their ability to support reverse transcription at elevated temperatures.150 Sequential introduction of three of these amino acid substitutions (D200N, L603W, and T330P) onto M-MLV RT, hereafter referred to as M3, led to an average 6.8-fold increase in transversion and insertion editing efficiency at five genomic loci in HEK293T cells compared to that of PE1 (Figures 47A-47S).

Next, we tested additional reverse transcriptase mutations ¹⁵² that were previously shown to enhance binding to the template:PBS complex, enzymatic processivity, and thermostability in combination with M3. Of the 14 additional mutants analyzed, a variant carrying the M3 mutations plus T306K and W313F substitutions improved editing efficiency by an additional 1.3- to 3.0-fold compared to M3 for six transversion or insertion edits at five genomic sites in human cells (Figures 47A-47S). This quintuple mutant of M-MLV reverse transcriptase (Cas9 H840A-M-MLV RT(D200N L603W T330P T306K W313F)) incorporated into the PE1 architecture is hereafter referred to as PE2.

PE2 incorporated single nucleotide transversion, insertion, and deletion mutations with substantially higher efficiency than PE1 (Figure 39C) and was compatible with the shorter PBS PEgRNA sequence (Figure 39C), consistent with its 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 compared to PE1 (Figure 39C), and in some cases dramatically improved editing yields by up to 46-fold (Figures 47F and 47I). PE2 accomplished targeted insertions and deletions more efficiently than PE1, achieving targeted insertion of a 24-bp FLAG epitope tag at the HEK3 locus with 4.5% efficiency, a 15-fold improvement compared to the efficiency of incorporating this insertion by PE1 (Figure 47D), and mediated a 1-bp deletion in HEK3 with 8.6% efficiency, 2.1-fold higher than that of PE1 (Figure 39C). These results establish PE2 as a more efficient prime editor than PE1.

Optimization of PEgRNA characteristics

The relationship between PEgRNA architecture and prime editing efficiency was systematically explored by PE2 at five genomic loci in HEK293T cells (Figure 39C). In general, prime sites with lower GC content require 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 more extensive GC content support 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) (Figure 39C), consistent with the energetic requirements for hybridization of the nicked DNA strand to the PEgRNA PBS. PBS length or GC content level does not strictly predict prime editing efficiency, and other factors such as the secondary structure of the DNA primer or the PEgRNA extension may also affect editing activity. It is recommended to start with a PBS length of ~13 nt for typical target sequences and to investigate different PBS lengths if the sequence deviates from ~40-60% GC content. When necessary, optimal PBS sequences should be determined empirically.

Next, we investigated the performance determinants of the RT template portion of PEgRNA. PEgRNAs with RT templates ranging in length from 10 to 20 nt were systematically evaluated with PE2 at five genomic target sites (Figure 39D) and with longer RT templates as long as 31 nt at three genomic sites (Figures 48A-48C). Like the PBS length, the RT template length can also be varied to maximize prime editing efficiency, but in general, most RT template lengths ≥ 10 nt support more efficient prime editing (Figure 39D). Because some target sites prefer longer RT templates (> 15 nt) to achieve higher editing efficiency (FANCF, EMX1) and other loci prefer short RT templates (HEK3, HEK4) (Figure 39D), it is recommended that both short and long RT templates be tested when optimizing PEgRNA, starting from ∼10-16 nt.

Importantly, RT templates that placed a C as the nucleotide adjacent to the terminal hairpin of the sgRNA backbone generally resulted in lower editing efficiency compared to other PEgRNAs with RT templates of similar length (Figures 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 may block sgRNA backbone folding by pairing with nucleotide G81, which natively forms a non-canonical base pair with Cas9 Tyr 1356 and sgRNA A68. Because many RT template lengths support primed editing, it is recommended to choose PEgRNAs where the first base of the 3' extension (the last reverse transcribed base of the RT template) is not a C.

Design of the prime editing factor 3 system (PE3 and PE3b)

Although PE2 can transfer genetic information from PEgRNA to the target locus more efficiently than PE1, the manner in which cells degrade the resulting heteroduplex DNA generated by one edited and one unedited strand determines whether the edit is durable. Previous developments in base editing faced similar challenges, since the initial product of cytosine or adenine deamination is heteroduplex DNA containing one edited and one unedited strand. To increase the efficiency of base editing, a nick was introduced into the unedited strand using Cas9 D10A nickase to guide DNA repair to that strand using the edited strand as a template. ^129,130,142 To exploit this principle and enhance the efficiency of prime editing, a similar strategy was tested to nick the unedited strand using Cas9 H840A nickase already present in PE2 and a simple sgRNA to induce preferential replacement of the unedited strand by the cell (Figure 40A). Because the edited DNA strand was also nicked to initiate prime editing, the positioning of the nicks programmed by different sgRNAs was tested on the non-edited strand to minimize the production of double-stranded DNA breaks leading 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 product by 1.5 to 4.2-fold, as much as 55% higher, compared to the PE2 system (Figure 40B). Although the optimal nicking position varied depending on the genomic site, nicks located approximately 40-90 bp 3' of the PAM from the PEgRNA-induced nick (positive distance in Figure 40B) generally resulted in a favorable increase in prime editing efficiency (41% on average) without extra indel formation (6.8% average indels for the sgRNA resulting in the highest editing efficiency at each of the five sites tested) (Figure 40B). As expected, at some sites, placing a non-edited strand nick within 40 bp of the PEgRNA-induced nick led to a large increase in indel formation, up to 22% (Figure 40B), presumably due to the formation of double-stranded breaks from nicking both adjacent strands together. However, at other sites, nicking as close as 14 bp from the PEgRNA-induced nick resulted in only 5% indels (Figure 40B), suggesting that locus-dependent factors control the conversion of the proximal double nick to a double-stranded DNA break. At one tested site (HEK4), even when placed >70 bp away from the PEgRNA-induced nick, complementary strand nicks either provided no benefit or led to indel levels that exceeded the editing efficiency (up to 26%), consistent with the unusual tendency of the edited strand at that site to be nicked or inefficiently ligated by the cells. It is recommended to start with a non-edited strand nick approximately 50 bp from the PEGRNA-mediated nick and to test alternative nick positioning if indel frequency exceeds tolerated levels.

This model of how complementary strand nicking improves prime editing efficiency (Figure 40A) predicted that nicking the non-edited strand only after flap degradation of the edited strand would minimize the presence of simultaneous nicks and subsequently reduce the frequency of double-stranded breaks forming indels. To achieve temporal control of non-edited strand nicking, we designed sgRNAs with spacer sequences that match the edited strand but not the original allele. With this strategy, hereafter referred to as PE3b, mismatches between the spacer and the non-edited allele should not favor nicking by the sgRNA until after an editing event on the PAM strand has occurred. We tested this PE3b approach with five different edits at three genomic sites in HEK293T cells and compared outcomes to those achieved by the PE2 and PE3 systems. In all cases, PE3b was associated with substantially lower levels of indels compared to PE3 (3.5 to 30-fold, with an average of 12-fold lower indels or 0.85%) without any apparent decrease in overall editing efficiency compared to PE3 (Figure 40C). Thus, when editing was on the second protospacer, the PE3b system was able to reduce indels compared to PE2 while still improving editing efficiency, often to levels similar to that of PE3 (Figure 40C).

Together, these findings establish that the PE3 system (Cas9 nickase-optimized reverse transcriptase + PEgRNA + sgRNA) improves editing efficiency by ∼3-fold compared to PE2 (Figures 40B-40C). As expected from the additional nicking activity of PE3, PE3 was accompanied by a broader range of indels than PE2. Use of PE3 is recommended when prioritizing prime editing efficiency. When indel minimization is critical, PE2 offers ∼10-fold lower indel frequency. When it is possible to use a sgRNA that recognizes the incorporated edit and nicks 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 primed editing with PE3, the incorporation of all possible single nucleotide substitutions at positions +1 to +8 (counting the first base 3' of the nick induced by the PEgRNA as position +1) of the HEK3 target site was explored using PE3 and PEgRNA with a 10-nucleotide RT template (Figure 41A). Collectively, these 24 alternative edits covered all four transition mutations and all eight transversion mutations, proceeding with an average of 7.5±1.8% indels, with an editing efficiency (not including indels) of 33±7.9% (ranging between 14% and 48%).

Importantly, long-distance RT templates can also generate efficient prime editing with PE3. For example, PE3 was used with a 34-nt RT template to incorporate point mutations at positions +12, +14, +17, +20, +23, +24, +26, +30, and +33 (12 to 33 bases from the nicks induced by PEgRNA) at the HEK3 locus with an average efficiency of 36±8.7% and 8.6±2.0% indels (Figure 41B). Although editing beyond the +10 position at other loci was not attempted, other RT templates ≥30 nt at three alternative sites also support efficient editing (Figure 48A-C). The feasibility of long RT templates enabled efficient prime editing for dozens of nucleotides from the original nick site. In contrast to other precision genome editing methods, prime editing is not substantially constrained by the availability of nearby PAM sequences, since NGG PAMs on either DNA strand occur on average every ∼8 bp, much less than the maximum distance between edits and PAMs that supports efficient prime editing. ^125,142,154 Given the predicted relationship between RNA secondary structure and prime editing efficiency, it is prudent to test RT templates of different lengths and, if necessary, sequence composition (e.g., synonymous codons) to optimize editing efficiency when designing PEgRNAs for long-range editing.

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 point mutations were tested at six additional genomic target sites (Figures 41C-41H). Overall, indel-free editing efficiency averaged 25 ± 14%, and indel formation averaged 8.3 ± 7.5%. Because the PEgRNA RT template encompassed the PAM sequence, prime editing could induce changes in the PAM sequence. In these cases, higher editing efficiency (average of 39 ± 9.7%) and lower indel generation (average of 5.0 ± 2.9%) were observed (Figures 41A-41K; point mutations at positions +5 or +6). This increase in efficiency of PAM editing and decrease in indel formation may result from the inability of Cas9 nickase to rebind and nick the edited strand prior to repair of the complementary strand. Prime editing supports combinatorial editing without any apparent loss of editing efficiency, so when possible, it is recommended to edit the PAM in addition to other desired changes.

Next, 14 targeted small insertions and 14 targeted small deletions were made with PE3 (Figure 41I) at seven genomic sites. Targeted 1 bp insertions proceeded with an average efficiency of 32 ± 9.8%, and 3 bp insertions were incorporated with an average efficiency of 39 ± 16%. Targeted 1 bp and 3 bp deletions were also efficient, proceeding with average yields of 29 ± 14% and 32 ± 11%, respectively. Indel generation (other than targeted insertions or deletions) averaged 6.8 ± 5.4%. Because insertions and deletions introduced between positions +1 and +6 alter the position or 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 rebind and nick the edited 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 (Figure 41J). Very high editing efficiencies (52 to 78%) were observed for 5, 10, and 15 bp deletions when using the 13 nt PBS and RT templates containing 29, 24, or 19 bp of homology to the target locus, respectively. Using a 26 nt RT template supported larger deletions of 25 bp with an efficiency of 72 ± 4.2%, and a 20 nt RT template enabled an 80 bp deletion with an efficiency of 52 ± 3.8%. These targeted deletions were accompanied by an indel frequency that averaged 11 ± 4.8% (Figure 41J).

Finally, we tested the ability of PE3 to mediate 12 combinations of multiple edits at the same target locus consisting of an insertion and deletion, an insertion and a point mutation, a deletion and a point mutation, or two point mutations at three genomic sites. These combinatorial edits were highly efficient, averaging 55% targeted edits with 6.4% indels (Figure 41K), demonstrating the ability of primed editing to make precise combinations of insertions, deletions, and point mutations at individual target sites with high efficiency and low indel frequency.

Together, the examples in Figures 41A-41K represent 156 distinct transitions, transversions, insertions, deletions, and combinatorial edits at seven human genomic loci. These findings establish the versatility, precision, and targeting flexibility of prime editing.

Prime editing compared to base editing

Current generation cytidine base editors (CBEs) and adenine base editors (ABEs) can incorporate C.G to T.A 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 on the base editing activity window (typically ~5 bp wide) that results in unwanted bystander editing, ^{129,130,142,155} or by the absence of a PAM located approximately 15 ^± 2 nt from the target ^{nucleotide.142,156} It was anticipated that prime editing could be particularly useful for precise incorporation of transition mutations without bystander editing or when the lack of a suitably positioned PAM makes it impossible to favorably position the target nucleotide on the CBE or ABE activity window.

Primed editing and cytosine base editing were compared by editing three genomic loci containing multiple targeted cytidines over a standard base editing window (protospacer positions 4-8, counting the PAM as positions 21-23). Optimized CBE ¹⁵⁷ was used without (BE2max) or with (BE4max) nickase activity, or the related PE2 and PE3 primed editing systems were used. Of the nine total targeted cytosines over the three-site base editing window, BE4max produced 2.2-fold higher average total C.G to T.A conversions than PE3 for bases in the center of the base editing window (protospacer positions 5-7; Figure 42A). Similarly, non-nicking BE2max outperformed PE2 on average by 1.4-fold at these well-positioned bases (Figure 42A). However, for cytosines outside the center of the base editing window, PE3 outperformed BE4max by 2.7-fold and PE2 outperformed BE2max by 2.0-fold (average edits of 40±17% for PE3 vs. 15±18% for BE4max, and 22±11% for PE2 vs. 11±13% for BE2max). Overall, the indel frequency in PE2 was very low (average 0.86±0.47%) and in PE3 it was similar to or modestly higher than that of BE4max (BE4max range: 2.5% to 14%; PE3 range: 2.5% to 21%) (Figure 42B).

When comparing the efficiency of base editing to prime editing for the incorporation 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 over the ~5 bp base editing window (Figure 42C). At these sites, such as EMX1, which contains cytosines at protospacer positions C5, C6, and C7, BE4max generated a small number of products that contained only a single targeted base pair conversion without bystander editing. In contrast, prime editing at this site could be used to selectively incorporate C/G to T/A edits at any position or combination of positions (C5, C6, C7, C5+C6, C6+C7, C5+C7, or C5+C6+C7) (Figure 42C). All precise single- or double-base edits (i.e., edits that do not modify any other nearby bases) were significantly more efficient with PE3 or PE2 than with BE4max or BE2, respectively, while triple C-G to T-A edits were more efficient with BE4max (Figure 42C), reflecting the tendency of base editors to edit all target bases over their activity window. Together, these results demonstrate that cytosine base editors can produce higher levels of editing than PE2 or PE3 at optimally positioned target bases, but primed edits can outperform base edits at non-optimally positioned target bases and can edit with significantly greater precision at multiple editable bases.

A.T to G.C editing by optimized non-nicking ABE (ABEmax ¹⁵² with dCas9 instead of Cas9 nickase, henceforth referred to as ABEdmax) versus PE2 and optimized nicking adenine base editor ABEmax versus PE3 were compared at two genomic loci. At sites containing two targeted adenines in the base editing window (HEK3), ABE was more efficient than PE2 or PE3 for converting A5, while PE3 was more efficient for converting A8, which is at the edge of the ABEmax editing window (Figure 42D). When comparing the efficiency of precision editing where only a single adenine is converted, PE3 outperformed ABEmax at both A5 and A8 (Figure 42E). Overall, ABE produced far fewer indels in HEK3 than prime editors (0.19±0.02% ABEdmax vs. 1.5±0.46% PE2, and 0.53±0.16% ABEmax vs. 11±2.3% PE3, Figure 42F). In FANCF, where only a single A is present in the base editing window, ABE2 and ABEmax outperformed their prime editing counterparts by 1.8-2.9-fold in total target base pair conversion, and virtually all edited products from both base editing and prime editing contained only precise edits (Figures 42D-42E). As in the HEK3 site, ABE produced far fewer indels in the FANCF site (Figure 42F).

Collectively, these results indicate that base editing and prime editing offer complementary strengths and weaknesses for creating targeted transition mutations. In cases where a single target nucleotide falls within the base editing window, or when bystander editing is permitted, current base editors are typically more efficient than prime editors, resulting in fewer indels. Prime editors offer a substantial advantage when multiple cytosines or adenines are present and bystander editing is undesirable, or when the target base is poorly positioned for base editing relative to the available PAMs.

Off-target prime edits

To produce productive editing, prime editing requires a complementary target locus:PEgRNA spacer for the Cas9 domain to bind, a target locus:PEgRNA PBS complementarity for the initiation of reverse transcription primed by PEgRNA, and a target locus:reverse transcriptase product complementarity for flap degradation. It was hypothesized that these three separate DNA hybridization requirements may minimize off-target prime editing compared to other genome editing methods. To test this possibility, HEK293T cells were treated with PE3 or PE2 and a total of 16 PEgRNAs designed to target four on-target genome loci, with four corresponding sgRNAs targeting the same protospacer as Cas9, or with the same 16 PEgRNAs as Cas9. These four target loci were chosen because each has at least four well-characterized off-target sites where Cas9 and the corresponding on-target sgRNA are known to cause substantial off-target DNA modifications in HEK293T ^{cells.118,159} After treatment, the four on-target loci for each on-target spacer and the top four known Cas9 off-target sites were sequenced for a total of 16 off-target sites (Table 1).

Consistent with previous studies, ¹¹⁸ Cas9 and the four targeting sgRNAs modified all 16 of the previously reported off-target loci (Figure 42G). For the HEK3 target locus, the Cas9 off-target modification efficiency at the four off-target sites averaged 16%. Cas9 and sgRNA targeting HEK4 resulted in an average of 60% modification of the four known off-target sites tested. Similarly, the off-target sites of EMX1 and FANCF were modified by Cas9:sgRNA at an average frequency of 48% and 4.3%, respectively (Figure 42G). It was noted that, compared to sgRNA, PEgRNA modified on-target sites with Cas9 nuclease with similar (1- to 1.5-fold lower) efficiency on average, but PEgRNA modified off-target sites with Cas9 nuclease with an average efficiency ~4-fold lower than sgRNA.

Remarkably, PE3 or PE2 with the same 16 tested PEgRNAs containing these four target spacers resulted in significantly less off-target editing (Figure 42H). Of the 16 sites known to undergo off-target editing by Cas9+sgRNA, PE3+PEgRNA or PE2+PEgRNA resulted in detectable off-target prime editing at only 3 of the 16 off-target sites, with only 1 of the 16 showing an off-target editing efficiency of ≥1% (Figure 42H). The average off-target prime editing of 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 (Figure 42H). Of note, at three HEK4 off-target sites where 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. Together, these results suggest that PE3 and PEgRNA induce significantly less off-target DNA editing in human cells than Cas9 and sgRNA targeting the same protospacer.

Reverse transcription of 3'-extended PEgRNA can in principle proceed onto the guide RNA backbone. If the resulting 3' flap is integrated into the target locus despite the lack of complementarity at its 3' end with the unedited DNA strand, the outcome is the insertion of PEgRNA backbone nucleotides, which contributes to the indel frequency. We analyzed sequencing data from 66 PE3-mediated editing experiments at four loci in HEK293T cells and observed PEgRNA backbone insertions at a low frequency, averaging 1.7 ± 1.5% of the total insertion of any number of PEgRNA backbone nucleotides (Figures 56A-56D). We speculate that the inaccessibility of the guide RNA backbone to reverse transcriptase due to Cas9 domain binding and cellular excision during flap degradation of the 3' end of the mismatched 3' flap resulting from PEgRNA backbone reverse transcription minimizes the product of incorporation of PEgRNA backbone nucleotides. Although such events are rare, future work to engineer PEgRNAs or prime editor proteins that minimize PEgRNA backbone incorporation may further reduce indel frequency.

Although deaminases can act in a Cas9-independent manner in some base editors, resulting in low-level but extensive off-target DNA editing in first-generation CBEs (but not ABEs) ^,160-162 and off-target RNA editing in first-generation CBEs and ABEs, ^163-165 newer CBE and ABE variants with engineered deaminases greatly reduce Cas9-independent off-target DNA and RNA editing.163-165 Prime editors lack base-modifying enzymes such as deaminases and therefore do not have the intrinsic ability to modify DNA or RNA bases in ^a Cas9-independent manner.

In principle, the reverse transcriptase domain of a primed editor could process appropriately primed RNA or DNA templates in cells, but it was noted that retrotransposons, such as those of the LINE-1 family, ¹⁶⁶ endogenous retroviruses, ^167,168 and human telomerase all provide active endogenous human reverse transcriptase. Their natural presence in human cells suggests that reverse transcriptase activity is not substantially toxic in itself. Indeed, no PE3-dependent differences in HEK293T cell viability were observed compared to controls expressing dCas9, Cas9 H840A nickase, or PE2 with the R110S+K103L mutations that inactivate reverse transcriptase (PE2-dRT) ¹⁶⁹ (Figures 49A-49B).

Despite the above data and analyses, additional studies are needed to evaluate off-target prime editing in an unbiased genome-wide manner and to characterize the extent to which reverse transcriptase variants of prime editing factors or prime editing intermediates may affect cells.

Prime editing pathogenic transversion, insertion, and deletion mutations in human cells

We tested the ability of PE3 to directly incorporate or correct genetic disease-causing transversions, small insertions, and small deletion mutations in human cells. Sickle cell disease is most commonly caused by an A-T to T-A transversion mutation in HBB, resulting in a Glu6→Val mutation in beta ^globin . Ex vivo treatment of hematopoietic stem cells with Cas9 nuclease and a donor DNA template for HDR, followed by enrichment, transplantation, and engraftment of the edited cells, is a promising potential strategy for the treatment of sickle cell disease.170 However, this approach still generates many indel-containing by-products in addition to the correctly edited HBB ^{allele.170-171} Although base editors generally generate much fewer indels, they are currently unable to make the T-A to A-T transversion mutation required to directly restore the normal sequence of HBB.

Using PE3, we engineered the HBB E6V mutation into HEK293T cells with 44% efficiency and 4.8% indels (Figure 43A). From the mixture of PE3-treated cells, we isolated six HEK293T cell lines that were homozygous (triploid) for the HBB E6V allele (Figures 53A- 53E ), 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 PEgRNA programmed to directly revert the HBB E6V mutation to wild-type HBB. In total, 14 PEgRNA designs were tested. After 3 days, DNA sequencing revealed that all 14 PEgRNAs, when combined with PE3, provided efficient correction of HBB E6V to wild-type HBB with indel levels averaging 2.8±0.70% (>26% wild-type HBB without indels) (Figure 50A). The best PEgRNA provided 52% correction of HBB E6V to wild-type with 2.4% indels (Figure 43A). Introduction of silent mutations that alter the PAM recognized by the PEgRNA modestly improved editing efficiency and product purity to 58% correction with 1.4% indels (Figure 43A). These results establish that prime editing can incorporate and correct pathogenic transversion point mutations in human cell lines with high efficiency and minimal by-products.

Tay-Sachs disease is most often caused by a 4-bp insertion (HEXA 1278+TATC) on the HEXA gene. Using PE3, we integrated this 4-bp insertion into HEK293T cells with 31% efficiency and 0.8% indels (Figure 43B), and isolated two HEK293T cell lines homozygous for the HEXA 1278+TATC allele (Figures 53A - ^53E ). Using these cells, 43 PEgRNAs and three nicking sgRNAs were tested for correction of the pathogenic insertion in HEXA by the PE3 or PE3b system (Figure 50B). Either by complete reversion to the wild-type allele, or by a shifted 4-bp deletion that blocks the PAM and incorporates a silent mutation. Nineteen of the 43 PEgRNAs tested resulted in ≧20% editing. Complete correction of wild-type HEXA by PE3 or PE3b and the best PEgRNA proceeded with similar average efficiency (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) (Figure 43B and Figure 50B). These findings demonstrate the ability of prime editing to make precise small insertions and deletions that incorporate or correct pathogenic alleles in mammalian cells efficiently and with minimal side effects.

Finally, we tested the incorporation of a protective SNP on the gene PRNP, which codes for the human prion protein (PrP). PrP misfolding causes progressive and fatal neurodegenerative prion disease, which can occur spontaneously by inherited dominant mutations on the PRNP gene or by exposure to misfolded ^PrP172 . The naturally occurring PRNP G127V mutant allele confers resistance to prion disease in ^humans138 and ^mice173 . PE3 was used to incorporate 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 3 days of exposure to the most effective PE3 and PEgRNA, DNA sequencing revealed a 53±11% efficiency of incorporating the G127V mutation and an indel level of 1.7±0.7% (Figure 43C). Taken together, these results establish the ability of prime editing to incorporate or correct transversion, insertion, or deletion mutations in human cells that cause or confer resistance to disease efficiently and with minimal by-products.

Prime editing of various human cell lines and primary mouse neurons

Prime editing was then tested for its ability to edit endogenous sites in three additional human cell lines. In K562 (leukemic myeloid) cells, PE3 was used to perform transversion editing of HEK3, EMX1, and FANCF sites, as well as an 18-bp insertion of a 6xHis tag in HEK3. Average editing efficiencies of 15-30% were observed for each of these four PE3-mediated edits, with indels averaging 0.85-2.2% (Figure 43A). In U2OS (osteosarcoma) cells, HEK3 and FANCF transversion mutations, as well as 3-bp insertions and 6xHis tag insertions in HEK3, were incorporated with editing efficiencies of 7.9-22%, which exceeded indel formation by 10-76-fold (Figure 43A). Finally, in HeLa (cervical cancer) cells, 3-bp insertions in HEK3 were performed with 12% average efficiency and 1.3% indels (Figure 43A). Collectively, these data indicate that multiple cell lines other than HEK293T cells support prime editing, although editing efficiency varies by cell type and is generally less efficient than HEK293T cells. Edit:indel ratios remained high in all human cell lines tested.

To determine whether prime editing is possible in postmitotic terminally differentiated primary cells, primary cortical neurons harvested from E18.5 mice were transduced with a binary split PE3 lentiviral delivery system, in which split intein splicing ²⁰³ reconstitutes the PE2 protein from N- and C-terminal halves, each delivered from a separate virus. To restrict editing to postmitotic neurons, the human synapsin promoter ²⁰⁴ , which is highly specific for mature neurons, was used to drive expression of both PE2 protein components. GFP was fused to the N-terminal half of PE2 via a self-cleaving P2A peptide ^205. Nuclei from neurons were isolated 2 weeks after binary viral transduction and either sequenced directly or sorted for GFP expression prior to sequencing. An average prime editing of 7.1 ± 1.2% was observed to incorporate transversions at the DNMT1 locus (Figure 43D), with an average indel of 0.58 ± 0.14% in the sorted nuclei. Cas9 nuclease in the same split intein binary lentiviral system resulted in 31±5.5% indels in sorted cortical neuron nuclei (Figure 43D). These data indicate that postmitotic terminally differentiated primary cells can support prime editing, thus establishing that prime editing does not require cell replication.

Prime editing compared to Cas9-initiated HDR

The performance of PE3 was compared to that of optimized Cas9-initiated ^HDR128,125 in mitotic cell lines that support HDR. ^HEK293T , HeLa, K562, and U2OS cells were treated with Cas9 nuclease, sgRNA, and ssDNA donor oligonucleotide templates designed to incorporate various transversion and insertion edits (Figures 43E-43G and Figures 51A- 51G ). Cas9-initiated HDR successfully incorporated the desired edits in all cases with much higher levels of by-products (predominantly indels) as expected from a treatment that causes double-strand breaks. Using PE3 in HEK293T cells, HBB E6V incorporation and correction proceeded with an average indel of 2.6% and 1.4%, with an average editing efficiency of 42% and 58%, respectively (Figures 43E and 43G). In contrast, the same edits with Cas9 nuclease and HDR templates resulted in average editing efficiencies of 5.2% and 6.7%, with average indel frequencies of 79% and 51% (Figures 43E and 43G). Similarly, PE3 incorporated PRNP G127V with 53% efficiency and 1.7% indels, whereas Cas9-initiated HDR incorporated this mutation with 6.9% efficiency and 53% indels (Figures 43E and 43G). Thus, the edit:indel ratios of HBB E6V incorporation, HBB E6V correction, and PRNP G127V incorporation were, on average, 270-fold higher with PE3 than with Cas9-initiated HDR.

Comparisons between PE3 and HDR in human cell lines other than HEK293T showed similar results, albeit with lower PE3 editing efficiency. For example, in K562 cells, 3bp insertion mediated by PE3 on HEK3 proceeded with 25% efficiency and 2.8% indels compared to 17% editing and 72% indels for Cas9-initiated HDR, a 40-fold edit:indel ratio advantage favoring PE3 (Figures 43F-43G). In U2OS cells, PE3 performed this 3bp insertion with 22% efficiency and 2.2% indels, while Cas9-initiated HDR resulted in 15% editing with 74% indels, a 49-fold lower edit:indel ratio (Figures 43F-43G). In HeLa cells, PE3 achieved this insertion with 12% efficiency and 1.3% indels, a 210-fold difference in edit:indel ratio, compared with 3.0% edits and 69% indels for Cas9-initiated HDR (Figures 43F-43G). Collectively, these data indicated that HDR typically results in lower or similar editing efficiencies and much higher indels than PE3 in the four cell lines tested (Figures 51A- 51G ).

Considerations and future directions

The ability to insert DNA sequences with single nucleotide accuracy is a particularly powerful prime editing capability. For example, PE3 was used to precisely insert a His ₆ tag (18 bp, 65% average efficiency), a FLAG epitope tag (24 bp, 18% average efficiency), and an extended LoxP site that is a native substrate for Cre recombinase (44 bp, 23% average efficiency) into the HEK3 locus in HEK293T cells. The average indels ranged between 3.0% and 5.9% in these examples (Figure 43H). It is envisioned that many biotechnology, synthetic biology, and therapeutic applications will arise from the ability to efficiently and precisely introduce new DNA sequences into desired target sites in living cells.

Collectively, the prime editing experiments described herein incorporated 113 point mutations, including 18 insertions of up to 44 bp, 22 deletions of up to 80 bp, 77 transversions, and 18 combination edits, at 12 endogenous loci in the human and mouse genomes, at locations ranging from 3 bp upstream to 29 bp downstream of the start of the PAM, without creating overt double-stranded DNA breaks. These results establish prime editing as a remarkably versatile genome editing method. Because the vast majority (85-99%) of ClinVar insertions, deletions, indels, and duplications are ≦30 bp (Figures 52A-52D), in principle, prime editing could correct up to ∼89% of the 75,122 currently known pathogenic human genetic variants in ClinVar (transitions, transversions, insertions, deletions, indels, and duplications in Figure 38A), with the additional potential to ameliorate diseases caused by copy number gains or losses.

Importantly, for any desired editing, the flexibility of prime editing offers many possible options for the location of the nick induced by the PEgRNA, the location of the second nick induced by the sgRNA, the PBS length, the RT template length, and which strand is edited first, as demonstrated in this application. In contrast to the more limited options typically available in other precision genome editing methods, ^125,142,154 , this flexibility allows editing efficiency, product purity, DNA specificity, or other parameters to be optimized to suit the needs of a given application. As shown in Figures 50A-50B, in which 14 and 43 PEgRNAs covering a range of prime editing strategies were tested to optimize the correction of pathogenic HBB and HEXA alleles, respectively.

Considerable additional research is needed to further understand and improve prime editing. Additional modifications of the prime editing factor system may be required to extend their compatibility to include other cell types, such as postmitotic cells. Interfacing prime editing with viral and nonviral 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 genetic disease research and treatment. However, by enabling highly precise targeted transitions, transversions, small insertions, and small deletions on mammalian cell genomes without requiring double-strand breaks or HDR, prime editing offers a new "search and replace" capability that substantially expands the scope of genome editing.

Methods <br/> General methods

Unless otherwise noted, DNA amplification was performed by PCR using Phusion U Green Multiplex PCR Master Mix (ThermoFisher Scientific) or Q5 Hot Start High Fidelity 2x Master Mix (New England BioLabs). DNA oligonucleotides encompassing 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 in this application were assembled using the USER cloning method as previously ^described175 . Plasmids expressing sgRNAs were constructed by ligation of annealed oligonucleotides onto BsmBI-digested acceptor vectors. Plasmids expressing PEgRNAs were constructed using custom acceptor plasmids by Gibson assembly or Golden Gate assembly (see supplemental "Golden Gate Assembly" overview). The sequences of sgRNA and PEgRNA constructs used in this application are listed in Tables 2A-2C and 3A-3R. All vectors for mammalian cell experiments were purified using Plasmid Plus miniprep kit (Qiagen) or PureYield plasmid miniprep kit (Promega), which includes an endotoxin removal step. All experiments using live animals were approved by the Broad Institute Animal Care and Use Committee. Wild-type C57BL/6 mice were obtained from Charles River (#027).

In vitro biochemical assays

PEgRNA and sgRNA were in vitro transcribed from PCR amplified templates containing T7 promoter sequences using the HiScribe T7 in vitro transcription kit (New England Biolabs). RNA was purified by denaturing urea PAGE and quality checked by analytical gel prior to use. 5'-Cy5-labeled DNA duplex substrates were annealed with two oligonucleotides (Cy5-AVA024 and AVA025; 1:1.1 ratio) for unnicked substrates or three oligonucleotides (Cy5-AVA023, AVA025, and AVA026; 1:1.1:1.1) for pre-nicked substrates by heating to 95°C for 3 min followed by slow cooling to room temperature (Tables 2A-2C). Cas9 cleavage and reverse transcription reactions were carried out in 1× cleavage buffer ^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 sgRNA or PEgRNA (5 μM final) were preincubated in a 5 μL reaction mixture for 10 min at room temperature prior to the addition of double-stranded DNA substrate (400 nM final) and, when applicable, followed by the addition of undisclosed M-MLV RT variant Superscript III reverse transcriptase (ThermoFisher Scientific). The reaction was carried out at 37°C for 1 h, 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 min. After heat inactivation at 95°C for 10 min, the reaction products were combined with 2× formamide gel loading buffer (90% formamide; 10% glycerol; 0.01% bromophenol blue), denatured at 95°C for 5 min, and separated on a denaturing urea-PAGE gel (15% TBE-urea, 55°C, 200 V). DNA products were visualized by Cy5 fluorescent signal using a Typhoon FLA 7000 biomolecular imager.

Electrophoretic mobility shift assays were performed in 1x binding buffer (1x cleavage buffer + 10 μg/mL heparin) with 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 min incubation at 37 °C, samples were analyzed by native PAGE gel (10% TBE) and imaged for Cy5 fluorescence.

For DNA sequencing of the reverse transcription products, the fluorescent bands were excised from urea-PAGE gels, purified, and then 3'-tailed with terminal deoxyribonuclease (TdT; New England Biolabs) in the presence of dGTP or dATP according to the manufacturer's protocol. The tailed DNA products were diluted 10-fold with binding buffer (40% guanidinium chloride saturated aqueous solution + 60% isopropanol), purified by QIAquick spin columns (Qiagen), and then used as templates for primer extension with Klenow fragment (New England Biolabs) using primers AVA134 (A-tailed products) or AVA135 (G-tailed products) (Tables 2A-2C). The extensions were amplified by 10 cycles of PCR with primers AVA110 and AVA122, and then sequenced by the Sanger method with AVA037 (Tables 2A-2C).

Yeast fluorescent reporter assay

Binary fluorescent reporter plasmids containing an in-frame stop codon, a +1 frameshift, or a -1 frameshift were subjected to in vitro 5'- or 3'-extended PEgRNA primed editing reactions as described above. After 1 h incubation at 37°C, the reactions were diluted with water and the plasmid DNA was precipitated with 0.3 M sodium acetate and 70% ethanol. The resuspended DNA was transformed into S. cerevisiae by electroporation as previously ^described67 and plated on synthetic complete medium without leucine (SC(glucose), L-). GFP and mCherry fluorescent signals were visualized from colonies by a 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 Minimum Essential Medium (EMEM, ATCC), each supplemented with 10% (v/v) fetal bovine serum (Gibco, quality assurance) 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

Grown HEK293T cells were seeded on 48-well poly-D-lysine coated plates (Corning). 16 to 24 hours after seeding, cells were transfected at approximately 60% confluency with 1 μL of Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer's protocol, 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 for 3 days after transfection, after which the medium was removed, cells were washed with 1× PBS solution (Thermo Fisher Scientific), and genomic DNA was extracted by adding 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 to each well of the tissue culture plate. The genomic DNA mixture was incubated at 37°C for 1 to 2 h, followed by a 30-min enzyme inactivation step at 80°C. 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, and 50 ng (1.51 pmol) of 100-nt ssDNA donor template (PAGE-purified; Integrated DNA Technologies) were lipofected per well using 1.4 μL of Lipofectamine 2000 (ThermoFisher). 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 by Illumina MiSeq as previously described with the following ^{modifications129,130} . Briefly, amplification primers containing Illumina forward and reverse adapters (Table 4) were used in a first round PCR (PCR1) to amplify the genomic regions of interest. A 25 μL PCR1 reaction was 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. The PCR reaction was performed as follows: 98°C for 2 min, then 30 cycles [98°C for 10 s, 61°C for 20 s, and 72°C for 30 s], followed by a final 72°C extension for 2 min. A unique Illumina barcoded primer pair was added to each sample in the second PCR reaction (PCR2). Specifically, 25 μL of a given PCR2 reaction contained 0.5 μM of each unique forward and reverse Illumina barcoded primer pair, 1 μL of unpurified PCR1 reaction mixture, and 12.5 μL of Phusion U Green Multiplex PCR 2× master mix. Barcoded PCR2 reactions were run as follows: 98°C for 2 min, then 12 cycles [98°C for 10 s, 61°C for 20 s, and 72°C for 30 s], followed by a final 72°C extension for 2 min. PCR products were analytically assessed by electrophoresis through a 1.5% agarose gel. PCR2 products (pooled by common amplicon) were purified by electrophoresis through a 1.5% agarose gel. Elution was performed with 40 μL of water using a QIAquick gel extraction kit (Qiagen). DNA concentrations were measured by fluorimetric quantification (Qubit, ThermoFisher Scientific) or qPCR (KAPA Library Quantification Kit-Illumina, KAPA Biosystems) and sequenced by an Illumina MiSeq instrument according to the manufacturer's protocol.

Sequencing reads were demultiplexed using MiSeq reporter (Illumina). Alignment of amplicon sequences to reference sequences was performed using CRISPResso2. For quantification of point mutation edits, CRISPResso2 was run in standard ^mode with "discard_indel_reads" on. Editing efficiency was calculated as: (frequency of defined point mutation in non-discarded reads) x (# of non-discarded reads) ÷ total reads. For insertion or deletion edits, CRISPResso2 was run in HDR mode with "discard_indel_reads" on and the desired allele as the expected allele (e flag). Editing yield was calculated as the number of HDR aligned reads divided by total reads. For all edits, indel yield was calculated as the number of discarded reads divided by total reads.

Nucleofection of U2OS, K562, and HeLa cells

Nucleofections were performed with K562, HeLa, and U2OS cells in all experiments. For PE conditions for these cell types, 800 ng prime editor expression plasmid, 200 ng PEgRNA expression plasmid, and 83 ng nicking plasmid were nucleofected on 16-well nucleocuvette strips (Lonza) in a final volume of 20 μL. For HDR conditions for these three cell types, 350 ng nuclease expression plasmid, 150 ng sgRNA expression plasmid, and 200 pmol (6.6 μg) of 100 nt ssDNA donor template (PAGE purified; Integrated DNA Technologies) were nucleofected on 16-well Nucleocuvette strips (Lonza) in a final volume of 20 μL per sample. K562 cells were nucleofected with the SF Cell Line 4D-Nucleofector X Kit (Lonza) at 5 × 10 ⁵ cells per sample (program FF-120) according to the manufacturer's protocol. U2OS cells were nucleofected with the SE Cell Line 4D-Nucleofector X Kit (Lonza) at 3-4 × 10 ⁵ cells per sample (program DN-100) according to the manufacturer's protocol. HeLa cells were nucleofected with the SE Cell Line 4D-Nucleofector X Kit (Lonza) at 2 × 10 ⁵ cells per sample (program CN-114) according to the manufacturer's protocol. Cells were harvested for genomic DNA extraction 72 hours after nucleofection.

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 in 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 transfection was performed as described above. Genomic DNA extraction for PE and BE was performed as described above.

Determining 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 a template for PCR amplification of 16 previously reported Cas9 off-target genomic ^sites118,159 (top four off-target sites in HEK3, EMX1, FANCF, and HEK4 spacers, respectively; primer sequences are listed in Table 4). These genomic DNA samples were identical to those used to quantify on-target PE3 editing activity shown in Figures 41A-41K; PEgRNA and nicking sgRNA sequences are listed in Tables 3A-3R. After PCR amplification of off-target sites, amplicons were sequenced by Illumina MiSeq platform as described above (HTS analysis). To determine the Cas9 nuclease, Cas9 H840A nickase, dCas9, and PE2-dRT on-target and off-target editing activities, 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 the primer sequences in Table 4 and sequenced by Illumina MiSeq.

HTS data analysis was performed using ^{CRISPResso2.178} Editing efficiencies of Cas9 nuclease, Cas9 H840A nickase, and dCas9 were quantified as the percentage of total sequencing reads containing indels. For quantification of PE3 and PE3-dRT off-targets, aligned sequencing reads were inspected for point mutations, insertions, or deletions that were consistent with the expected products of PEgRNA reverse transcription initiated at the Cas9 nick site. Single nucleotide variations occurring at an overall frequency of <0.1% of the total reads in a sample were excluded from analysis. For reads containing single nucleotide variations that both occurred at a frequency ≥0.1% and were partially consistent with the edit encoded by the PEgRNA, a t-test (unpaired, one-sided, α=0.5) was used to determine whether the variant occurred at a significantly higher level compared to samples treated with a PEgRNA containing the same spacer but encoding a different edit. To avoid differences in sequencing errors, comparisons were made between samples sequenced simultaneously by the same MiSeq run. Variants that did not meet the criterion of p-value > 0.05 were excluded. Off-target PE3 editing activity was then calculated as the percentage of total sequenced reads that met the above criterion.

Generation of HEK293T cell lines containing HBB E6V mutations using Cas9-initiated HDR

HEK293T cells were seeded in 48-well plates and transfected at approximately 60% confluency with 1.5 μL Lipofectamine 2000, 300 ng Cas9 D10A nickase plasmid, 100 ng sgRNA plasmid, and 200 ng 100mer ssDNA donor template (Table 5). Three days after transfection, the medium was replaced with fresh medium. Four days after transfection, cells were dissociated with 30 μL TrypLE solution and suspended in 1.5 mL medium. Single cells were isolated into individual wells of two 96-well plates by fluorescence-activated cell sorting (FACS) (Beckman-Coulter Astrios). See Figures 53A-53B for representative FACS sorting examples. Cells were expanded for 14 days prior to genomic DNA sequencing as described above. None of the isolated clonal populations were found to be homozygous for the HBB E6V mutation, so a second round of lipofection-editing, sorting, and outgrowth was repeated in the partially edited cell line to generate a cell line homozygous for the E6V allele.

Generation of HEK293T cell lines containing HBB E6V mutations using PE3

2.5 × 104 HEK293T cells grown in the absence of antibiotics were seeded into 48-well poly-D-lysine-coated plates (Corning). 16 to 24 hours after seeding, cells were transfected at approximately 70% confluency with 1 μL Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer's protocol, as well as 750 ng of PE2-P2A-GFP plasmid, 250 ng of PEgRNA plasmid, and 83 ng of sgRNA plasmid. Three days after 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 performed on a LE-MA900 cell sorter (Sony). Cells were treated with 3 nM DAPI (BioLegend) 15 min prior to sorting. After gating to exclude doublets, DAPI-negative single cells with GFP fluorescence above that of the 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 Figures 53A-53B for a representative FACS sorting example. 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 homozygous for the HBB E6V mutation were identified.

Generation of HEK293T cell lines containing HEXA 1278+TATC insertions using PE3

HEK293T cells containing HEXA 1278+TATC alleles were generated following the protocol described above for the generation of the HBB E6V cell line; PEgRNA and sgRNA sequences are listed in Tables 2A-2C under the subheadings of Figures 43A-43H. 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 containing 50% HEXA 1278+TATC alleles were isolated and two homozygous cell lines containing 100% HEXA 1278+TATC alleles were recovered.

Cell viability assay

HEK293T cells were seeded in 48-well plates and transfected at approximately 70% confluency with 750 ng of editing factor plasmid (PE3, PE3 R110S K103L, Cas9 H840A nickase, or dCas9), 250 ng of HEK3-targeted PEgRNA plasmid, and 1 μL of Lipofectamine 2000 as described above. Cell viability was measured every 24 h for 3 days after transfection using the CellTiter-Glo 2.0 assay (Promega) according to the manufacturer's protocol. Luminescence was measured on 96-well flat-bottom polystyrene microplates (Corning) using an M1000 Pro microplate reader (Tecan) with an integration time of 1 s.

Lentivirus production

Lentiviruses were generated as previously described ^. Rapidly dividing HEK293T cells (ATCC; Manassas, VA, USA) in T-75 flasks were transfected with the lentivirus production helper plasmids pVSV-G and psPAX2 in combination with the modified lentiCRISPR_v2 genome carrying the intein-split PE2 editing element using FuGENE HD (Promega, Madison, WI, USA) according to the manufacturer's instructions. Four split-intein editor constructs were designed: 1) a viral genome encoding a U6-PEgRNA expression cassette and the Npu N intein, a self-cleaving P2A peptide, and the N-terminal portion (1-573) of Cas9 H840A nickase fused to GFP-KASH; 2) a viral genome encoding an Npu C intein fused to the C-terminal remainder of PE2; 3) a viral genome encoding an 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 link the two halves of PE2 or Cas9 together, and the P2A GFP-KASH enables co-translational production of nuclear membrane-localized GFP. After 48 h, the supernatant was collected, centrifuged at 500 g for 5 min to remove cell debris, and filtered using a 0.45 μm filter. The filtered supernatant was concentrated using PEG-it virus precipitation solution (System Biosciences, Palo Alto, CA, USA) according to the manufacturer's instructions. The resulting pellet was resuspended in Opti-MEM (Thermo Fisher Scientific, Waltham, MA, USA) using 1% of the original medium volume. The resuspended pellet was flash frozen and stored at -80°C until use.

Dissection and culture of mouse primary cortical neurons

E18.5 dissociated cortical cultures were harvested from planned pregnant C57BL/6 mice (Charles River). After euthanasia with CO2, fetuses were harvested from pregnant mice and then decapitated. Cortical caps were dissected in ice-cold Hibernate-E supplemented with penicillin/streptomycin (Life Technologies). After rinsing with ice-cold Hibernate-E, tissues were digested with papain/DNase (Worthington/Sigma) for 8 min at 37°C. Tissues were 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 medium was changed twice per week.

Priming and nuclear isolation of primary neurons

At DIV1, 15 μL of lentivirus was added at a 10:10:1 ratio of N-terminal:C-terminal:nicking sgRNA. At DIV14, neuronal nuclei were isolated using 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 of EZ-PREP solution. After 5 min of incubation on ice, EZ-PREP was pipetted onto the surface of the wells to dislodge remaining cells. Samples were centrifuged at 500 g for 5 min and the supernatant was removed. Samples were washed with 200 μL of EZ-PREP and centrifuged at 500 g for 5 min. Samples were resuspended 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 by gentle pipetting and then centrifuged at 500 g for 5 min. The supernatant was removed and nuclei were resuspended in 100 μL NSB and sorted into 100 μL Agencourt DNAdvance lysis buffer using 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-targeted or HEXA-targeted PEgRNA and PE2, PE2-dRT, or Cas9 H840A nickase. 72 hours after transfection, total RNA was harvested from cells using TRIzol reagent (Thermo Fisher) and purified by RNeasy mini kit (Qiagen) including on-column DNaseI treatment. Ribosomes were depleted from total RNA using the rRNA removal protocol of TruSeq Stranded total RNA library prep kit (Illumina) and then washed with RNAClean XP beads (Beckman Coulter). Sequencing libraries were prepared with ribo-depleted RNA by SMARTer PrepX Apollo NGS library prep system (Takara) following the manufacturer's protocol. The resulting libraries were visualized by 2200 TapeStation (Agilent Technologies), normalized using Qubit dsDNA HS assay (Thermo Fisher), and sequenced by NextSeq 550 using a high output v2 flow cell (Illumina) as 75 bp paired-end reads. Fastq files were generated by bcl2fastq2 version 2.20 and trimmed using TrimGalore version 0.6.2 (github.com/FelixKrueger/TrimGalore ) to remove low quality bases, unpaired sequences, and adapter sequences. Trimmed reads were aligned to the Homo sapiens genome assembly GRCh ¹⁴⁸ with custom Cas9 H840A gene entries using RSEM version 1.3.1 ^207. Gene expression levels were normalized using the limma-voom ²⁰⁸ package, and differential expression analysis was performed with batch effect correction. Differentially expressed genes were called with a cutoff of FDR-corrected p-value <0.05 and fold change >2, and results were visualized by R.

ClinVar analysis

The ClinVar variant summary was downloaded from NCBI (accessed on July 15, 2019) and the information contained therein was used for all downstream analyses. The list of all reported variants was filtered by allele ID to remove duplicates and by clinical significance to restrict the analysis to pathogenic variants. To calculate the fraction of pathogenic variants that were insertions, deletions, etc., the list of pathogenic variants was sequentially filtered by variant type. Single nucleotide variants (SNVs) were separated into two categories (transitions and transversions) based on the reported reference and alternative alleles. SNVs that did not report a reference or alternative allele were excluded from the analysis.

The lengths of reported insertions, deletions, and duplications were calculated using appropriate identifying information for the reference/alternate alleles, variant start/stop positions, or variant name. Variants that did not report any of the above information were excluded from the analysis. The lengths of reported indels (single variants encompassing 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 alternative alleles. Frequency distributions of variant lengths were calculated using GraphPad Prism 8.

Data availability

High-throughput sequencing data have been deposited in the NCBI Sequence Read Archive database. Plasmids encoding PE1, PE2/PE3, and the PEgRNA expression vector will be available from Addgene.

Code availability

The script used to quantify PEgRNA backbone insertion is provided in Figures 60A-60B.

Additional information: tables and sequences

Table 1: Activity 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 brackets). % indels are shown at ^the top four previously characterized off-target sites for Cas9, Cas9 H840A nickase (nCas9), and PE2-dRT. sgRNA and PEgRNA sequences can be found in Tables 3A-3R under the headings of Figures 42A-42H. All values are the average of three independent biological replicates.

Tables 2A-2C: Sequences of DNA oligonucleotides, PEgRNA, and sgRNA used for in vitro experiments.

Table 2A: DNA oligonucleotides

Table 2B: 5'-Extended PEG RNA

Table 2C: 3'-Extended PEG RNA

Tables 3A-3R: Sequences of PEgRNA and sgRNA used in mammalian cell experiments. All sequences are shown in 5' to 3' orientation. To construct PEgRNA, the spacer sequence listed below was added to the 5' end of the sgRNA backbone and the 3' extension listed below containing a primer binding site and RT template was added to the 3' end of the sgRNA backbone. The sgRNA backbone sequence is GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 131).

Table 3A: Figures 39A-39D PEgRNA

Table 3B: Figures 40A-40C PEgRNA

Table 3C: Figures 40A-40C sgRNA sequences to be nicked

Table 3D: Figures 41A-41K PEgRNA

Table 3E: sgRNAs for nicking in Figures 41A-41K

Table 3F: Figures 42A-42H PEgRNA

Table 3G: sgRNAs nicked in Figures 42A-42H

Table 3H: Figures 42A-42H base-edited sgRNAs

Table 3I: Figures 42A-42H on-target sgRNAs

Table 3J: Figures 42A-42H On-target PEgRNA

Table 3K: Figures 49A-49B PEgRNA

Table 3L: Figures 47A-74D PEgRNA

Table 3M: Figures 48A-48C PEgRNA

Table 3N: Figures 48A-48C PEgRNA

Table 3O: sgRNA nicking in Figures 48A-48C

Table 3P: Figure 50A-50B PRgRNA

Table 3Q: sgRNA nicking in Figures 50A-50B

Table 3R: Figures 51A- 51G PEgRNA

Table 4: Sequences of primers used for mammalian cell genomic DNA amplification and HTS ¹⁸¹ .

Table 5: Sequences of 100mer single-stranded DNA oligonucleotide donor templates used for HDR experiments and for generation of HBB E6V HEK293T cell lines. Oligonucleotides are 100-103 nt in length with homology arms centered at the site of editing. Oligonucleotides were from Integrated DNA Technologies and were purified by PAGE.

Additional arrays

Sequences of the yeast dual fluorescent reporter plasmids used herein
p425-GFP_stop_mCherry:

pLenti-U6-DNMT1_ニックを入れる_sgRNA:

DNA sequences of mammalian prime editor plasmids and exemplary PEgRNA plasmids

pLenti-U6-DNMT1_Nick_sgRNA:

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

M3-不活性型RT M-MLV RT:

Amino acid sequences of Moloney murine leukemia virus reverse transcriptase (M-MLV RT) variants used herein.
PE1 M-MLV RT:
(SEQ ID NO:739)

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

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

M3-inactive RT M-MLV RT:

REFERENCES FOR EXAMPLE 12 Each of the following references is cited in Example 12, each of which is incorporated herein by reference.

Example 13 - Cellular Data Recording and Lineage Tracing with PE Background Genomic modifications can also be used to study and record cellular processes and development. Linking cellular events such as cell division or signal cascade activation to DNA sequence modifications would store the history of the cell as interpretable DNA sequence changes that would describe whether a particular cellular event occurred. 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. When modifications are made to short-lived protein and RNA molecules, information about cell state and lineage is generally lost. Recording cellular events within a single cell is a powerful way to understand how disease states are initiated, maintained, and altered relative to healthy controls. The ability to explore these questions has relevance to understanding the development of cancer, neurological diseases, and numerous other important problems in human health. Prime editing (PE) provides a system for generating targeted and sequence-defined genomic insertions, deletions, or mutations. Repeated modifications of DNA targets, which can be sequenced by targeted amplicon sequencing and/or RNA sequencing (which is particularly valuable for single-cell recording experiments), can be used to record a number of important biological processes, including activation of signal cascades, metabolic states, and cell differentiation programs. Connecting internal and external cellular signals to sequence modifications on genomes is theoretically possible for any signal for which a signal-responsive promoter exists. It is believed that PE will enable a greatly expanded toolkit for probing the cell lineage and signal history of eukaryotic and prokaryotic cells both in culture and in vivo.

Previous standard

Targeted sequence insertions, deletions, or mutations can be used to study a number of important biological questions, including lineage tracing and recording cellular stimuli. The current toolkit for generating these signatures across genomes is limited. To date, targeted locus mutagenesis has been developed using both DNA nucleases and base editors.

CRISPR/Cas9 nuclease cuts of target sequences generate stochastic sequence changes, producing multiple insertion or deletion (indel) products. The multiple sequence outcomes that arise from Cas9 cuts allow unambiguous determination of the sequences that were cut by the nuclease. The ability to distinguish cut versus uncut sequences has been used in two predominant ways.

First, expression of the Cas9 nuclease and/or its single guide RNA (sgRNA) was connected to a cellular signal. In addition, the occurrence of the signal was recorded based on sequence modification of the Cas9 target genomic locus. 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 of this approach is that multiple target loci are desired for a given sgRNA, because the generation of indels often severely precludes additional mutagenesis of the target locus; in many cases, this means that a pre-engineered target locus is introduced into the cell for editing, instead of direct mutagenesis of the endogenous locus.

Second, Cas9 indels have been used to trace cell lineages. As described herein, the large number of possible indel states generated by Cas9 nuclease activity allows the generation of cell developmental trees. These suggest which cells evolved from each other in time. This approach is a powerful way to understand how cells evolved from each other and has been used to help identify unique cell states and types in developmental time by performing RNA sequencing on selected cell pools. This approach cannot independently report on cell signaling events, their order, and can be biased when reporting on precursor versus terminally differentiated cell states.

Cas9 nuclease-mediated lineage tracing and signal recording is a powerful technique with some important caveats. Signal recording with Cas9 nuclease is often very technically challenging. Cas9 cuts deplete the target locus (once an indel is generated, repeated cuts are difficult), making it difficult to record long-term stimulation at the single-cell level. Although the kinetics of Cas9 cuts can be tuned to allow for longer-term recording of events, the ability to incorporate the sequence, strength, and duration of multiple stimuli remains a formidable technical problem that may not be achievable with this tool. Cas9 lineage tracing experiments are extremely powerful but suffer from minor technical challenges of sequence collapse due to simultaneous Cas9 cuts at the target locus. These lineage tracing experiments require predesigned edits of the target locus, limiting the flexibility of this approach.

DNA base editing has also been used to track cellular signaling events. Due to the low number of outcome states generated by editing events relative to the number of states generated by Cas9 indels, base editing is less suitable for lineage tracing; however, the predefined nature of the sequence modifications made by base editors makes them particularly useful for tracking internal and external cellular stimuli. Either base editors or sgRNA expression described herein can be connected to specific biological or chemical stimuli. Base editing activity has been used to track multiple individual stimuli in both mammalian and bacterial cells. This approach has also been used to track sequential stimuli where the first editing event is required before the second edit can occur.

Although base editing signal recording is an important first step for the field, it has a number of limitations. One such limitation is that base editing depletes its target after editing, limiting the dynamic range of the technique. This means that it is often difficult to use endogenous targets to record events, and one is limited to recording bulk activity instead of activity at the single cell level. An alternative to this would be the introduction of pre-designed repeated recording loci, but this has not been done to date. There is also the issue of two-signal recording. These two-signal recording experiments only report on the presence of a second stimulus after the first; they do not report which stimulus occurred first or how long the stimulus was present. This fundamentally limits the biological understanding that can be gleaned from the experiments.

It has been proposed that PE lineage tracing can both lineage trace and record cell signals by modifying genomic target sequences and incorporating pre-designed sequences. PE uses a synthetic fusion protein containing a Cas9 nickase fragment (often the SpCas9 H840A variant) and a reverse transcriptase (RT) domain in conjunction with an engineered prime editing guide RNA (PEgRNA). Together, these components target specific genomic sequences and incorporate predetermined edits. Because the PEgRNA specifies both the target genomic sequence and the editing outcome, highly specific and controlled genome modifications can be achieved simultaneously using multiple PEgRNAs in the same cell. Accessible genome modifications include all single nucleotide substitutions, small to medium sized sequence insertions, and small to medium sized sequence deletions. The versatility of this genome editing technology should enable temporally coupled signal-specific recording in cells.

The utility of PE lineages and cell signaling recordings

Recording cellular signals can be accomplished in a number of ways. One important first application of this approach is to connect DNA modification events to cell cycle-related signals, such as the expression of cyclins, CDKs, or other proteins specific to aspects of cell life, to generate a cellular clock. A cellular clock allows researchers to understand the sequence of various signals being received and processed by individual cells. A molecular clock would also enable the determination of long-term signals versus short-term bursty signals. Using a prime editing component that can edit only once per cell cycle can also lead to a molecular clock. If editing can proceed only once per cell cycle, without successive DNA modifications (perhaps by not nicking the unedited DNA strand), one can imagine a system in which editing can only be done by a second targeted PEsgRNA in a subsequent cell division. PE is particularly useful as a cellular clock because it can repeatedly insert, delete, or mutate loci in a defined manner, and insertions are particularly handy because repeated, ordered insertions can be made at any target genomic locus.

Another important application of recording cellular signals is the parallel recording of multiple cellular inputs. Linking cellular signaling events to DNA modifications allows for recording whether such signaling events have 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 capable of recording the order, intensity, and duration of signaling events without requiring strict sequence motifs for ordered editing. Indeed, lineage prime editing should be capable of incorporating the cellular counters 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 that pre-exist in the target cells of interest (whether this is in bacteria, mice, rats, monkeys, pigs, humans, zebrafish, C. elegans, etc.). It is also important to note that prime editing recording incorporates barcodes in a guide RNA-dependent manner, and the number of inputs is limited to the number of signals for which there are robust signal-specific guide RNA expression cassettes (which should be very high due to the ability to tether their expression to the activity of RNA Pol II promoters). The number of signals that can be recorded is linearly proportional to the number of PEgRNAs required.

PE can also be used to trace cell lineage. Repetitive sequence modifications can be used to generate unique cellular barcodes to track individual cells. The array of barcodes, their order, and size can all be used to infer cell lineage in a manner that can be complementary to the multiple indel states generated by Cas9 nuclease.

Prime editing methodology for repeated sequence modifications

Several distinct modalities of iterative sequence modification using prime editing (PE) have been envisioned: DNA mutagenesis; sequence deletion; and sequence insertion. Of note, these applications can be used either on pre-existing genomic DNA targets or on pre-designed DNA sequences that researchers introduce into target cells. These techniques of sequential sequence modification are valuable for recording information and for the designed or stochastic modification of target loci in a sequential manner. Serial targeted locus modification can be particularly useful for generating libraries of variants in various hosts.

Repeated sequence mutations can be used to alter either genomic DNA or pre-designed introduced DNA sequences in a repetitive manner to report on cellular signaling events. In this paradigm, mutations incorporated by PE gRNA activity will correspond to the presence of cellular signals. These point mutations can incorporate PAM motifs necessary for sequential editing events, and point mutations corresponding to the presence of specific signals. Because each successive guide RNA will use a new protospacer, this system will require gRNA design prior to use; however, it can be particularly powerful for monitoring individual or small numbers of stimuli of interest. The incorporation of these mutations can be dependent on individual biological stimuli or can be connected to corresponding cellular processes that will mark cellular time. The sequences below correspond to SEQ ID NOs: 743, 744, 744, and 745.

Another similar PE guide RNA intensive method is repeated deletion of target sequences. Removal of individual sequences from the target locus would allow the ability to reconstruct signaling events by loss of DNA motifs. Designing PE gRNAs to delete sequential sequences would enable tracking of sequential signals. This would allow researchers to identify cases where one signal follows another. This would allow researchers to explore which signaling events occur in which order. Such a system has been tested using CAMERA; however, this requires the preselection of specific loci with unique sequence requirements. Since no specific sequence determinants are required, sequential sequence deletion using PE would allow parallel recording of pairwise events in individual cells. This would allow researchers the ability to explore pairwise signaling events in a multiplexed manner in any target cell of interest. The sequences below correspond to SEQ ID NOs: 746, 747, and 748.

Sequence insertion is a third approach to tracking cell signaling events. Some variants of this strategy are less PEGRNA-dependent than mutations or deletions. Several different insertion strategies exist: insertion of a short sequence, insertion of a protospacer, insertion of a protospacer and a barcode, insertion of a novel homologous sequence, and insertion of a homologous sequence with a barcode.

The insertion of short repeat sequences is a way to incrementally increase the size of a target sequence to measure cellular time. In this system, the insertion of repeat sequences of 5 or more nucleotides can cause repeat expansion over either time or the continued presence of a predetermined stimulus. The locus agnostic nature of lineage PE again allows for parallel tracking of multiple unique sequence expansions with respect to separate biological signals. This should allow for measurement of the intensity of multiple biological signals over cellular time in individual cells. The sequences below correspond to SEQ ID NOs: 749, 750, and 751.

Insertion of different short sequences will require few PEgRNAs in a similar fashion to the deletion space. The number of signals to be recorded and the size of the sequences to be inserted will determine the number of PEgRNA combinations required. One challenge of recording multiple sequences in this system will be the differing efficiency of each PEgRNA in inserting its cargo sequence.

Although the insertion of protospacers as indicators of cellular signals is attractive, technical challenges may compromise the efficiency of this approach. Single-PEgRNA systems would be difficult to use because the PEgRNA cassettes are substrates themselves, which would cause insertions onto the PEgRNA that would disrupt the efficiency and fidelity of subsequent edits. This same problem remains for two- or three-PEgRNA systems, because each guide is a substrate for another, allowing the insertion of other sequences into the guide cassette itself, which could lead to inappropriate insertion of the protospacer sequence for the wrong signal. These protospacer insertion systems are also difficult to envision for the inclusion of barcode sequences. A single-PEgRNA barcode system would simply rewrite the barcode used, removing the data stored in the first edit. Again, multiple-guide systems suffer from insertion onto other PEgRNA expression constructs, limiting their usefulness (especially in vivo). The sequences below correspond to SEQ ID NOs: 752, 752, 753, 754, 754, 755, and 756.

Insertion of homologous sequences (i.e., sequences 3' to the position of the Cas9 nick), especially those with associated barcodes, appears to be a particularly useful lineage PE strategy. This system avoids issues associated with protospacer insertion by ensuring that successive rounds of editing result in the insertion of a barcode from the PEgRNA cassette that cannot be altered by other PEgRNA editing events in the same cell. Barcoding systems are useful because 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 allows multiple signals to be connected to one editing locus without significant redesign of the PEgRNA to be used. This strategy would allow multiple barcode insertions at a single locus in response to multiple cellular stimuli (either internal or external). It can allow recording of the intensity, duration, and order of as many signals as there are unique barcodes (these can be designed with multiple N nucleotides to generate 4^N possible barcodes, i.e. a 5nt barcode would allow recording of 4^5 or 1024 unique signals at once). This system can be used both in vitro and in vivo.

References Cited in Example 13 Each of the following references is 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.Jun;570(7759):77-82.doi:10.1038/s41586-019-1184-5.Epub 2019 May 13.

Example 14 - Regulating Biomolecule Activity and/or Localization by PE The subcellular localization and modification state of biomolecules controls their activity. Certain biological functions such as transcriptional control, cellular metabolism, and signaling cascades are all carefully orchestrated in specific locations within the cell. Therefore, modulating the cellular localization and modification state of proteins represents a potential therapeutic strategy for the treatment of diseases. Several existing therapeutics have been developed to alter the localization of target proteins. For example, farnesylation inhibitors are designed to prevent lipid modification and membrane targeting of important oncogenic proteins such as KRAS. Similarly, small molecule-induced ubiquitination of target proteins directs them to the proteasome for degradation. The ability to transport proteins to these and other specific cellular compartments provides an opportunity to alter a number of biological processes. For therapeutic purposes, it is proposed in this application to use prime editing (PE) to incorporate genetically encoded handles to alter the modification state and subcellular trafficking of biomolecules (e.g., proteins, lipids, sugars, and nucleic acids) by genetically encoded signals.

PE is a genome editing technology that allows for the incorporation, deletion, or replacement of short DNA sequences on any genomic locus that can be targeted by the Cas9 enzyme. Using this technology, in principle, one can incorporate or remove important DNA, RNA, or protein coding sequences that alter the activity of these important biomolecules. More specifically, prime editing can be used to incorporate motifs or signals that change the localization or modification properties of biomolecules. Some examples include: alterations of protein amino acid sequences; motifs of post-translational modifications; RNA motifs that change folding or localization; and the incorporation of DNA sequences that change the local chromatin state or architecture of the surrounding DNA.

One target biomolecule for PE-mediated modifications is DNA. DNA can be modified to incorporate any number of DNA sequences that alter the accessibility of the target locus. Chromatin accessibility controls gene transcription output. Incorporation of a mark to recruit chromatin compaction enzymes should decrease the transcription output of neighboring genes, while incorporation of sequences associated with chromatin opening should make the region more accessible and in turn increase transcription. Incorporation of more complex sequence motifs that mimic native regulatory sequences should provide more subtle and biologically sensitive control over different epigenetic reader, writer, or eraser enzymes than currently available dCas9 fusions. Typically, it is a tool that incorporates a large number of possible monotypic marks without a specific biological ancestry. Incorporation of sequences that would bring two loci into close proximity or bring loci into contact with the nuclear membrane should also alter the transcription output of those loci, as demonstrated in the burgeoning field of 3D genome architecture.

Modifications of RNAs can also be made to alter their activity by changing their cellular localization, interaction partners, structural dynamics, or folding thermodynamics. Incorporation of motifs that would cause translational interruption or frameshifting could alter the abundance of mRNA species by 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 changes in the ratio of protein translation products. This can be used to alter many biological pathways. For example, shifting the balance of mitochondrial versus nuclear DNA repair proteins would alter the resilience of different cancers to chemotherapy agents. Furthermore, RNAs can be modified with sequences that allow binding to novel protein targets. A number of RNA aptamers have been developed that bind cellular proteins with high affinity. Incorporation of one of these aptamers can be used either to sequester different RNA species by binding to a protein target that would prevent their translation, biological activity, or to bring the RNA species to a specific subcellular compartment. Biomolecular degradation is another class of localized modification. For example, RNA methylation is used to control RNA in cells. A consensus motif for methylation can be introduced by PE onto the target RNA coding sequence. RNA can also be modified to include sequences that direct nonsense-mediated decay mechanisms or other nucleic acid metabolic pathways to degrade the target RNA species, which would change the pool of RNA in the cell. In addition, RNA species can be modified to alter their aggregation state. Sequences to generate RNA tangles can be incorporated onto the target RNA or RNAs, which would make them ineffective substrates for translation or signaling.

Modification of proteins by post-translational modifications (PTMs) also represents an important class of biomolecular manipulations that can be performed by PE. Like RNA species, altering the abundance of proteins in cells is an important capability of PE. Open reading frames can be edited to incorporate stop codons. This will eliminate the full-length product resulting from the edited DNA sequence. Alternatively, peptide motifs can be incorporated that cause the rate of protein degradation to be altered for the target protein. Incorporation of degradation tags onto the gene body can be used to alter the abundance of proteins in cells. Moreover, introduction of small molecule-induced degrons can allow for temporal control of protein degradation. This can have important implications for both research and therapeutics, since researchers can readily assess whether small molecule-mediated therapeutic protein degradation of a given target is a promising therapeutic strategy. Protein motifs can also be incorporated to change the subcellular localization of proteins. Amino acid motifs can be incorporated to preferentially target proteins to a number of subcellular compartments, including the nucleus, mitochondria, cell membrane, peroxisomes, lysosomes, proteasomes, exosomes, and others.

Incorporating or disrupting motifs modified by PTM mechanisms can alter protein post-translational modifications. Phosphorylation, ubiquitination, glycosylation, lipid modifications (e.g., farnesylation, myristoylation, palmitoylation, prenylation, GPI anchors), hydroxylation, methylation, acetylation, crotonylation, sumoylation, disulfide bond formation, side chain bond cleavage events, polypeptide backbone cleavage events (proteolysis), and several other protein PTMs have been identified. These PTMs alter protein function, often by altering subcellular localization. Indeed, kinases often activate downstream signaling cascades through phosphorylation events. Removal of the target phosphosite would prevent signal transduction. The ability to site-specifically excise or incorporate any PTM motif while retaining full-length protein expression would be an important advance for both basic research and therapeutics. PE's sequence incorporation range and targeting window make it well suited to the broad PTM modification space.

Removal of the lipid modification site should prevent transport of the protein to the cell membrane. A major limitation of current therapeutics targeting post-translational modification processes is their specificity. Farnesyltransferase inhibitors are being actively tested for their ability to eliminate KRAS localization to the cell membrane. Unfortunately, blanket inhibition of farnesylation is accompanied by numerous off-target effects that prevent the widespread use of these small molecules. Similarly, specific inhibition of protein kinases by small molecules can be very difficult due to the large size of the human genome and the similarity between various kinases. PE offers a potential solution to this specificity problem because it allows inhibition of target protein modification by excision of the modification site instead of blanket enzyme inhibition. For example, removal of the lipid modification peptide motif on KRAS would be a targeted approach that could be used instead of farnesyltransferase inhibition. This approach is the functional opposite of inhibiting target protein activity by incorporating lipid targeting motifs onto proteins that are not designed to be membrane bound.

PE can also be used to interrogate protein-protein complexation events. Proteins often function in complexes to carry out their biological activities. PE can be used to either create or destroy the ability of proteins to reside in these complexes. To eliminate complexation events, amino acid substitutions or insertions on the protein:protein interface can be incorporated to disfavor complexation. SSX18 is a protein component of the BAF complex, an important histone remodeling complex. Mutations in SSX18 drive synovial sarcoma. PE can be used to incorporate side chains that prevent SSX18 from binding to its protein partners in the complex to prevent its oncogenic activity. PE can also be used to remove pathogenic mutations to restore WT activity of this protein. Alternatively, PE can be used either to keep proteins in their native complexes or to pull them to engage in interactions that are unrelated to their native activity to inhibit their activity. Forming complexes that maintain one interaction state relative to another may represent an important therapeutic modality. Altering protein:protein interfaces to decrease the Kd of an interaction will keep the proteins attached to each other longer. Protein complexes can have multiple signaling complexes, such as n-myc, which drives the neuroblastoma signaling cascade in disease but is otherwise involved in healthy transcriptional control in other cells. PE can be used to incorporate mutations that drive n-myc association with healthy interacting partners and decrease its affinity for oncogenic interacting partners.

References Cited in Example 14 Each of the following references is 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 September 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 Aug;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 modification of PEgRNAs Described herein are 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 in targeted loci using information encoded in a prime editor guide RNA (PEgRNA). Prime editors (PE) consist of a sequence-programmed DNA binding protein with nuclease activity (Cas9) fused to a reverse transcriptase (RT) enzyme. PE forms a complex with PEgRNA, which contains information for the targeted specific DNA locus in their spacer sequence, as well as information that specifies the desired edit during a modified extension into a standard sgRNA backbone. The PE:PEgRNA complex binds to the programmed target DNA locus and nicks it, allowing hybridization of the nicked DNA strand with a modified primer binding sequence (PBS) of the PEgRNA. The reverse transcriptase domain then copies the editing code information in the RT template portion of the PEgRNA, using the nicked genomic DNA as a primer for DNA polymerization. The subsequent DNA repair process incorporates the newly synthesized edited DNA strand into the genomic locus. Although the versatility of prime editing holds promise as a research tool and potential therapeutic agent, several limitations in efficiency and scope exist due to the multi-step process required for editing. For example, unfavorable RNA structures formed within the PEgRNA may inhibit the copying of DNA edits from the PEgRNA to the genomic locus. One potential way to improve PE technology is through the redesign and modification of essential PEgRNA components. These improvements to the design of the PEgRNA are likely to be required for improved PE efficiency and allow for the incorporation of longer inserted sequences into the genome.

Described herein are a series of PE gRNA designs envisioned to improve PE efficacy. These designs take advantage of a number of previously published approaches to improve sgRNA efficacy and/or stability, as well as utilize a number of novel strategies. These improvements can fall into one or more of a number of different categories:

(1) Longer PEgRNAs. This category concerns improved designs that allow efficient expression of functional PEgRNAs from non-polymerase III (pol III) promoters, which would allow expression of longer PEgRNAs without cumbersome sequence requirements;

(2) Core improvements. This category concerns improvements to the core Cas9-linked PEgRNA scaffold, which may improve efficacy;

(3) RT processivity. This category concerns improvements to PEgRNA that improve RT processivity, allowing the insertion of longer sequences at the target genomic locus; and

(4) Terminal motifs. This category involves the addition of RNA motifs to the 5' and/or 3' ends of the PEgRNA that improve PEgRNA stability, enhance RT processivity, prevent PEgRNA misfolding, or recruit additional factors important for genome editing.

Described herein are a number of potential such PEgRNAs in each category. Several of these designs have been previously described and shown to improve sgRNA activity with Cas9. Also described herein is a platform for the evolution of PEgRNAs for a given sequence target that allows for polishing of the PEgRNA scaffold to enhance PE activity (5). Notably, these designs can also be readily applied to improve the PEgRNAs recognized by any Cas9 or its evolutionary variants.

(1) Longer PEgRNA.

sgRNAs are typically expressed from the U6 snRNA promoter. This promoter recruits pol III to express the associated RNA and is useful for expressing short RNAs that are retained in the nucleus. However, pol III is not very processive and cannot express RNAs longer than a few hundred nucleotides in length at the levels required for efficient genome editing183. In addition, pol III may stall or terminate at the extended ^U , potentially limiting the sequence diversity that can be inserted using PEgRNA. Other promoters that recruit polymerase II (such as pCMV) or polymerase I (such as the U1 snRNA promoter) have also been investigated for their ability to express longer ^sgRNAs183 . However, these promoters are typically partially transcribed, which results in extra sequence 5' of the spacer in the expressed PEgRNA, which has been shown to result in significantly reduced Cas9:sgRNA activity in a site-dependent manner. In addition, pol III transcribed PEgRNAs can simply terminate at the stretch of 6-7 U's, whereas PEgRNAs transcribed from pol II or pol I will require different termination signals. Often such signals will also result in polyadenylation, which will result in the undesired export of the PEgRNA from the nucleus. Similarly, RNAs expressed from pol II promoters (such as pCMV) are typically 5' capped, which will also result in their export from the nucleus.

Previously, Rinn and coworkers ^screened various expression platforms for the production of sgRNAs tagged with long non-coding RNAs (lncRNAs). These platforms include RNAs expressed from ^pCMV and terminating in an ENE element from human MALAT1 ncRNA, a PAN ENE element from KSHV, or a ³ ' box from ^U1 snRNA. Notably, MALAT1 ncRNA and PAN ENE form a triple helix that protects the polyA tail. These constructs are also expected to enhance RNA stability in addition to allowing expression of RNA (see section iv). Using a promoter from ^U1 snRNA was also ^explored to allow expression of these longer sgRNAs. It is expected that these expression systems will also allow expression of longer PEgRNAs. In addition, a series of methods have been designed to cleave the portion of the pol II promoter that will be transcribed as part of the PEgRNA, and either self-cleaving ribozymes (such as ^{hammerhead188} , ^pistol189 , ^hatchet189 , ^hairpin190 , ^VS191 , ^twister192 , or twister ^sister192 ribozymes) or other self-cleaving elements that process the transcribed guide, or ^hairpins193 that are recognized by Csy4 and also lead to processing of the guide. It is also hypothesized that the incorporation of multiple ENE motifs may lead to improved PEgRNA expression and stability, as previously demonstrated for KSHV PAN RNA and ^elements185 . It ^is also predicted that circularizing the PEgRNA into the form of a circular intronic RNA (ciRNA) may also lead to enhanced RNA expression and stability, as well as nuclear localization194.

PEgRNA expression platform consisting of pCMV, Csy4 hairpin, PEgRNA, and MALAT1 ENE
(SEQ ID NO:757)

PEgRNA expression platform consisting of pCMV, Csy4 hairpin, PEgRNA, and PAN ENE
(SEQ ID NO:758)

PEgRNA expression platform consisting of pCMV, Csy4 hairpin, PEgRNA, and 3×PAN ENE
(SEQ ID NO:759)

PEgRNA expression platform consisting of pCMV, Csy4 hairpin, PEgRNA, and 3' box
(SEQ ID NO:760)

PEgRNA expression platform consisting of pU1, Csy4 hairpin, PEgRNA, and 3' box
(SEQ ID NO:761)

(2) Core improvements.

The core, Cas9-bound PEgRNA backbone could conceivably also be improved to enhance PE activity. Several such approaches have already been demonstrated. As an illustration, the first pairing element (P1) of the backbone contains a GTTTT-AAAAC (SEQ ID NO: 3939) pairing element. Such a stretch of T's has been shown to result in pol III pausing and premature termination of the RNA transcript. Rational mutation of one of the TA pairs in this portion of P1 to a GC pair has been shown to enhance sgRNA activity, suggesting that this approach may also be feasible for ^PEgRNA195 . In addition, increasing the length of P1 has also been shown to enhance sgRNA folding leading to improved ^activity195 , suggesting this as another avenue for improving PEgRNA activity. Finally, refinement of the PEgRNA backbone through directed evolution of PEgRNA on a given DNA target may also result in improved activity. This is described in section (v).

PEgRNA containing a 6 nt extension to P1
GGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGCTCATGAAAATGAGCTAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCCTCTGCCATCAAAGCGTGCTCAGTCTGTTTTTTT (SEQ ID NO: 228)

PEgRNA containing a TA→GC mutation in P1
GGCCCAGACTGAGCACGTGAGTTTGAGAGCTAGAAATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCCTCTGCCATCAAAGCGTGCTCAGTCTGTTTTTTT (SEQ ID NO: 229)

(iii) Improved RT processivity through modification of the template region of PEGRNA

As the size of the PEgRNA-templated insertion increases, it is likely to be degraded by endonucleases, undergo spontaneous hydrolysis, or fold into secondary structures that cannot be reverse transcribed by RT or that interfere with the folding of the PEgRNA backbone and subsequent Cas9-RT binding. Consequently, modifications to the PEgRNA template may be necessary to affect large insertions, such as the insertion of entire genes. Some strategies to do so include the incorporation of modified nucleotides within synthetic or semi-synthetic PEgRNAs, which render the RNA more resistant to degradation or hydrolysis or less likely to adopt inhibitory secondary structures. ¹⁹⁶ Such modifications may include 8-aza-7-deazaguanosine, which will reduce RNA secondary structure in G-rich sequences; locked nucleic acids (LNAs), which reduce degradation and enhance certain RNA secondary structures; 2'-O-methyl, 2'-fluoro, or 2'-O-methoxyethoxy modifications, which enhance RNA stability. Such modifications can also be included elsewhere in the PEgRNA to enhance stability and activity. Alternatively or in addition, the template of the PEgRNA can be designed to encode the desired protein product while also being more likely to adopt a simple secondary structure that cannot be folded by RT. Such a simple structure would act as a thermodynamic sink, making it less likely that a more complex structure would arise that would prevent reverse transcription. Finally, the template could also be split into two separate PEgRNAs. In such a design, PE would be used to initiate transcription and also recruit separate template RNAs 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). RT could either directly bind to this separate template RNA or initiate reverse transcription on the original PEgRNA before replacing the second template. Such an approach may allow for long insertions not only by preventing misfolding of the PEgRNA upon addition of long templates, but also by not requiring dissociation of Cas9 from the genome to generate long insertions, which may potentially inhibit PE-based long insertions.

(iv) Incorporation of additional RNA motifs at the 5' or 3' end

PEgRNA design can also be improved through the incorporation of additional motifs at either end of the RNA. Several such motifs - such as the PAN ENE from KSHV and the ENE from MALAT1 - were previously discussed in part (i) as a means by which longer PEgRNAs can be terminated from non-pol ^III promoters184,185. These elements form RNA triple helices that engulf the polyA tail, leading to their retention in the ^{nucleus184,187} . However, by forming a complex structure at the 3' end of the PEgRNA that occludes the terminal nucleotides, these structures could also help prevent exonuclease-mediated degradation of the PEgRNA. Other structural elements inserted at the 3' end may also enhance RNA stability without allowing termination from non-pol III promoters. Such motifs could include hairpins or RNA quadruplexes that would occlude the 3' end, ¹⁹⁷ or self-cleaving ribozymes such as HDV that result in the formation of a 2'- ^3' -cyclic phosphate at the 3' end and potentially also render the PEgRNA less susceptible to exonuclease degradation.198 Inducing circularization of the PEgRNA through incomplete splicing - forming a ciRNA - could also increase PEgRNA stability resulting in PEgRNA being retained in the ^nucleus.194

Additional RNA motifs may also improve RT processivity or enhance PEgRNA activity by enhancing RT binding to the DNA-RNA duplex. The addition of native sequences bound by RT in its cognate retroviral genome can enhance RT activity.199 This may include native primer binding sites (PBS), polypurine tracts ( ^PPT ), or kissing loops, which are involved in retroviral genome dimerization and transcription ^{initiation.199} The addition of dimerization chiefs—such as kissing loops or GNRA tetraloop/ ^tetraloop receptor pairs—at the 5′ and 3′ ends of the PEgRNA may also result in efficient circularization of the PEgRNA, improving stability. In addition, it is envisioned that the addition of these motifs may allow for physical separation of the PEgRNA spacer and primer, preventing spacer occlusion that would otherwise hinder PE activity. A short 5' extension to the PEgRNA, forming a small toehold hairpin in the spacer region, may also preferably compete for the annealing region of the PEgRNA-binding spacer. Finally, kissing loops may also be used to recruit other template RNAs to genomic sites and allow for the swapping of RT activity from one RNA to the other, section iii).

PEGRNA-HDV fusions
GGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCCTCTGCCATCAAAGCGTGCTCAGTCTGGGCCGGCATGGTCCCAGCCTCCTCGCTGGCGCCGGCTGGGCAACATGCTTCGGCATGGCGAATGGGACTTTTTTT (SEQ ID NO: 230)

PEgRNA-MMLV kissing loop
GGTGGGAGACGTCCCACCGGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCCTCTGCCATCAAAGCTTCGACCGTGCTCAGTCTGGTGGGAGACGTCCCACCTTTTTTTT (SEQ ID NO: 231)

PEgRNA-VS ribozyme kissing loop
GAGCAGCATGGCGTCGCTGCTCACGGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCCTCTGCCATCAAAGCTTCGACCGTGCTCAGTCTCCATCAGTTGACACCCTGAGGTTTTTTT (SEQ ID NO: 232)

PEgRNA-GNRA tetraloop/tetraloop receptor
GCAGACCTAAGTGGUGACATATGGTCTGGGCCCAGACTGAGCACGTGAGTTTTAGAGCTAUACGTAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTUACGAAGTGGGACCGAGTCGGTCCTCTGCCATCAAAGCTTCGACCGTGCTCAGTCTGCATGCGATTAGAAATAATCGCATGTTTTTTT (SEQ ID NO: 233)

PEgRNA template to switch secondary RNA-HDV fusions
TCTGCCATCAAAGCTGCGACCGTGCTCAGTCTGGTGGGAGACGTCCCACCGGCCGGCATGGTCCCAGCCTCCTCGCTGGCGCCGGCTGGGCAACATGCTTCGGCATGGCGAATGGGACTTTTTTT (SEQ ID NO: 234)

(v) Evolution of PEGRNA

The PEgRNA backbone can be further improved through directed evolution in a manner similar to how SpCas9 and base editors have been improved. Directed evolution can enhance PEgRNA recognition by Cas9 or evolved Cas9 variants. In addition, different PEgRNA backbone sequences are likely optimal at different genomic loci, either enhancing PE activity at the site of interest, reducing off-target activity, or both. Finally, evolution of PEgRNA backbones with additional RNA motifs will almost always improve the activity of the fused PEgRNA compared to the unevolved fusion RNA. As an illustration, evolution of an allosteric ribozyme composed of a c-di-GMP-I aptamer and a hammerhead ribozyme led to dramatically improved activity, ²⁰² suggesting that evolution will also improve the activity of hammerhead-PEgRNA fusions. In addition, although Cas9 currently does not generally tolerate 5' extensions of sgRNAs, directed evolution could generate mutations that allow for alleviating this intolerance, allowing additional RNA motifs to be exploited.

As described herein, a number of these approaches have already been described for use with Cas9:sgRNA complexes, but no designs have been reported to improve PE gRNA activity. Other strategies for incorporating programmable mutations into genomes include base editing, homology-directed repair (HDR), precise microhomology-mediated end joining (MMEJ), or transposase-mediated editing. However, all of these approaches have significant drawbacks compared to PE. Although current base editors are more efficient than PE, they can only incorporate certain classes of genomic mutations and can result in unwanted additional nucleotide changes at the site of interest. HDR is feasible in only a few cell types and results in a relatively high rate of random insertion and deletion mutations (indels). Precise MMEJ can lead to predictable repair of double-strand breaks, but is largely limited to the incorporation of deletions, is highly site-dependent, and can also have a relatively high rate of unwanted indels. Transposase-mediated editing has only been shown to function in bacteria to date. Thus, improvements to PE represent perhaps the best avenue for therapeutic correction of a broad corpus of genomic mutations.

References cited in Example 15 Each of the following references is cited in Example 15, each of which is incorporated herein by reference.
1. Schechner, D. M., Hacisuleyman, E., Younger, S. T., Rinn, J. L. Nat Methods 664-70 (2015).
2. Brown JA, et al. Nat Struct Mol Biol 633-40(2014).
3. Conrad NA and Steitz JA. EMBO J 1831-41 (2005).
4. Bartlett JS, et al. Proc Natl Acad Sci USA 8852-7(1996).
5. Mitton-Fry RM, DeGregorio SJ, Wang J, Steitz TA, Steitz JA. Science 1244-7 (2010).
6. Forster AC, Symons RH. Cell. 1987.
7. Weinberg Z, Kim PB, Chen TH, Li S, Harris KA, Lunse CE, Breaker RR. Nat. Chem. Biol. 2015.
8. Feldstein PA, Buzayan JM, Bruening G. Gene 1989.
9. Saville BJ, Collins RA. Cell. 1990.
10. Roth A, Weinberg Z, Chen AG, Kim PG, Ames TD, Breaker RR. Nat Chem Biol. 2013.
11. Borchardt EK, 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 MF. Open Biol 170077(2017).
15. Nahar S, et al. Chem Comm 2377-80(2018).

Example 16 - Expanding the targeting range of PE using DNA binding proteins other than SPCAS9
Prime editing (PE) using Streptococcus pyogenes Cas9 (SpCas9) can efficiently incorporate all single base substitutions, insertions, deletions, and combinations thereof at genomic loci with appropriately placed NGG protospacer adjacent motifs (PAMs) to which SpCas9 can efficiently bind. The methods described herein broaden the targeting capabilities of PE by expanding the PAMs accessible to efficient PE, and thus the targetable genomic loci accessible. Prime editing using RNA-guided DNA-binding proteins other than SpCas9 allows for an expanded targetable range of genomic loci by allowing access to different PAMs. In addition, the use of RNA-guided DNA-binding proteins smaller than SpCas9 also allows for more efficient viral delivery. PE with Cas proteins other than SpCas9 or other RNA-guided DNA-binding proteins will allow for highly efficient therapeutic editing that was 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 edits relative to the NGG PAM, or the overall size of the SpCas9-based construct is prohibitive for cellular expression and/or delivery. Certain disease-relevant loci, such as the huntingtin gene, which have SpCas9's few and poorly positioned NGG PAMs close to the target region, can be easily targeted using a different Cas protein of the PE system, such as SpCas9-VRQR, which recognizes the NGA PAM. Smaller Cas proteins would be used to generate smaller PE constructs that can be more efficiently packaged into AAV vectors, enabling better delivery to target tissues. Figure 61 shows the implementation of prime editing using Staphylococcus aureus CRISPR-Cas as an RNA-guided DNA-binding protein. NT is untreated control.

Figures 62A-62B provide a demonstration of the importance of the protospacer for efficient incorporation of the desired edit at precise positioning by prime editing. This highlights the importance of alternative PAMs and protospacers as a novel feature of this technology. "n.d." in Figure 62A is "not detected."

Figure 63 shows the implementation of PE using SpCas9(H840A)-VRQR and SpCas9(H840A)-VRER as the RNA-guided DNA binding proteins of the prime editor system. SpCas9(H840A)-VRQR napDNAbp is disclosed herein as SEQ ID NO: 87. 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, where MMLV RT contains D200N, L603W, T330P, T306K, and W313F substitutions relative to wild-type MMLV RT. The SpCas9(H840A)-VRQR-MMLV RT fusion protein is disclosed herein as SEQ ID NO:515, in which the MMLV RT contains D200N, L603W, T330P, T306K, and W313F substitutions relative to wild-type MMLV RT. Seven different loci on the human genome are targeted: four by the SpCas9(H840A)-VRQR-MMLV RT prime editor system and three by the SpCas9(H840A)-VRER-MMLV RT system. The amino acid sequences of the constructs tested are as follows:

As shown in Figure 63, SpCas9(H840A)-VRQR-MMLV RT was active at PAM sites including "AGAG" and "GGAG" and had some editing activity at "GGAT" and "AGAT" PAM sequences. SpCas9(H840A)-VRER-MMLV RT was active at PAM sites including "AGCG" and "GGCG" and had some editing activity at "TGCG".

The data demonstrate that prime editing can be performed using napDNAbp with different PAM specificities, such as the Cas9 variants described herein.

Example 17 - Introduction of recombinase target sites with PE This example describes a method to address genetic diseases or generate tailored animal or plant models by using prime editing (PE) to introduce recombinase target sites (SSR target sites) into mammalian and other genomes with high specificity and efficiency.

This example describes the use of PE to introduce recombinase recognition sequences at high-value loci on human or other genomes. These will lead to precise and efficient genome modification after exposure to site-specific recombinase(s) (Figure 64). In various embodiments shown in Figure 64, PE can be used to (b) insert a single SSR target for use as a site of genomic incorporation of a DNA donor template. (c) shows how tandem insertion of SSR target sites can be used to delete a portion of a genome. (d) shows how tandem insertion of SSR target sites can be used to invert a portion of a genome. (e) shows how insertion of two SSR target sites at two distal chromosomal regions can result in a chromosomal translocation. (f) shows how insertion of two different SSR target sites on a 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 an SSR target, but this list is also not meant to be limiting.

Many large-scale genomic changes, such as gene insertions, deletions, inversions, or chromosomal translocations, have been associated with genetic diseases. ^1-7 In addition, custom and targeted manipulation of eukaryotic genomes is important for the study of human diseases and for the generation of transgenic ^plants8,9 or other biotechnology products. For example, chromosomal microdeletions can lead to disease, and replacement of these deletions by the insertion of critical DNA elements can lead to permanent amelioration of the disease. In addition, diseases resulting from inversions, gene copy number changes, or chromosomal translocations can be addressed by restoring the previous genetic structure of diseased cells. Alternatively, in plants or other high-value eukaryotes used in industry, the introduction of recombinant DNA or targeted genome rearrangements can lead to improved products, such as crops that require fewer resources or are resistant to pathogens. Current technologies for achieving large-scale genomic changes rely on random or stochastic processes, such as the use of transposons or retroviruses, and other desired genome modifications are achieved only by homologous recombination strategies.

One attractive class of proteins for achieving targeted and efficient genome modification is site-specific recombinases (SSRs). SSRs have a long history of being used as tools for genome modification10-13. SSRs are considered as promising tools for gene therapy because they can induce precise cleavage, strand exchange, and religation of DNA fragments in defined recombination targets14 without relying on endogenous repair of double-strand breaks that can induce ^indels , translocations, other DNA rearrangements, or ^{p53 activation15-18} . Reactions catalyzed by SSRs can result in the direct replacement, insertion, or deletion of target DNA fragments with ^an efficiency that exceeds that of homology-directed repair14,19.

Although SSRs offer many advantages, they have not been widely used because they have strong natural preferences for their corresponding target sequences. The recognition sequences of SSRs are typically ≧20 base pairs and therefore unlikely to occur on the genomes of humans or model organisms. Furthermore, the native substrate preferences of SSRs are not easily altered even by extensive laboratory manipulation or evolution. This limitation is overcome by using ^PE to directly introduce a recombinase target onto the genome or to modify an endogenous genomic sequence that natively resembles the recombinase target. Subsequent exposure of cells to the recombinase protein will allow precise and efficient genome modification guided by the positioning and orientation of the recombinase target(s) (Figure 64).

PE-mediated introduction of recombinase targets may be particularly useful for the treatment of genetic diseases caused by large-scale genomic defects, such as gene losses, inversions, or duplications, or chromosomal ^{translocations1-7} (Table 6). For example, Williams-Beuren syndrome is a developmental disorder caused by a deletion of 24 on chromosome 721. Currently, no technology exists for efficient and targeted insertion of entire genes in living cells (the potential of PE to make such full-length gene insertions is currently being explored but not yet established); however, recombinase-mediated incorporation in PE-inserted targets offers one approach to a permanent cure of this and other diseases. In addition, targeted introduction of recombinase recognition sequences may be highly effective for applications involving the generation of transgenic plants, animal research models, bioproduction cell lines, or other custom eukaryotic cell lines. For example, recombinase-mediated genome reorganization of transgenic plants in PE-specific targets may overcome one of the bottlenecks in generating agricultural crops with improved properties8,9.

Several 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 genome integration rates have also been described for some SSRs. ^22-30 In addition to natural recombinases, programmable recombinases with alternative specificities have been developed. ^31-40 Depending on the desired application, one or more of these recognition sequences can be introduced into the genome at defined locations, e.g., safe harbor loci, using PE. ^41-43

For example, introduction of a single recombinase target on the genome will result in integrative recombination with the DNA donor template (Figure 64B ). Serine integrases, which operate robustly in human cells, may be particularly well suited for gene integration44,45. In addition, depending on the identity and orientation of the targets, introduction of two recombinase targets may result in intervening sequence deletion, intervening sequence inversion, chromosomal translocation, or cassette exchange (Figures 64C - ^64F ). By choosing endogenous sequences that already closely resemble the recombinase target, the extent of editing required to introduce the complete recombinase target will be reduced.

Finally, several recombinases have been demonstrated to integrate into human or eukaryotic genomes at natively occurring pseudosites. ^46-64 PE editing can be used to modify these loci to enhance integration rates at these natural pseudosites, or alternatively to eliminate pseudosites that may serve as unwanted off-target sequences.

表7.チロシンリコンビナーゼおよびSSR標的配列

表8.大セリンインテグラーゼおよびSSR標的配列。

表9.セリンリゾルバーゼおよびSSR標的配列。

表10.チロシンインテグラーゼおよび標的配列。

This report describes a general methodology for introducing recombinase target sequences onto eukaryotic genomes using PE. The applications of this are nearly limitless. The genome editing reaction is intended for use with a "prime editor" of a chimeric fusion of CRISPR/Cas9 protein and reverse transcriptase domain, which utilizes a custom prime editing guide RNA (PEgRNA). Extending further, the Cas9 tool and the homology directed repair (HDR) pathway can also be exploited to introduce recombinase targets from DNA templates by reducing the indel rate using several ^{techniques65-67} . A proof-of-concept experiment in human cell culture is shown in Figure 65.
Table 6. Examples of genetic diseases linked to large-scale genome modifications.

Table 7. Tyrosine recombinases and SSR target sequences

Table 8. Large serine integrase and SSR target sequences.

Table 9. Serine resolvases and SSR target sequences.

Table 10. Tyrosine integrases and target sequences.

References Cited in Example 17 Each of the following references is cited in Example 17, each of which is incorporated herein by reference.

Example 18 - Incorporation of a 3' toe loop in the primer binding site (PBS) improves PEgRNA activity To further improve PE activity, we contemplated adding a toe loop sequence at the 3' end of a PEgRNA with a 3' extension arm. Figure 71A provides an example of a generic SpCas9 PEgRNA (top molecule) with a 3' extension arm. The 3' extension arm then contains the RT template (which contains the desired edit) and a primer binding site (PBS) at the 3' end of the molecule. The molecule terminates with a poly(U) sequence containing three U nucleobases (i.e., 5'-UUU-3').

In contrast, the bottom portion of FIG. 71A shows the same PEgRNA molecule as the top portion of FIG. 71A, but now with a 9 nucleobase sequence of 5'-GAAANNNNN-3' 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 "toe loop" RNA structure, where the 5'-NNNNN-3' sequence of the 9 nucleobase insertion anneals to a complementary sequence in the primer binding site and the 5'-GAAA-3' portion forms a 180° turn. The features of the toe loop sequence depicted in FIG. 71A are not intended to limit or restrict the scope of toe loops that may be used therein. Furthermore, the sequence of the toe loop will depend on the complementary sequence of the primer binding site. However, essentially the toe loop sequence may have a first sequence portion that forms 180° and a second sequence portion that has a sequence complementary to a portion of the primer binding site in various embodiments.

Without being bound by theory, it is believed that the toe loop sequence allows the PEgRNA to use longer and longer primer binding sites than would otherwise be possible. Longer PBS sequences are then believed to improve PE activity. More specifically, the possible function of the toe loop in the PEgRNA is to block the PBS or at least minimize PBS interactions with the spacer. Stable hairpin formation between the PBS and the spacer can lead to an inactive PEgRNA. In the absence of a toe loop, this interaction can require a PBS length restriction. Using a 3' toe loop to block or minimize the interaction between the spacer and the PBS can also lead to improved PE activity.

Example 19 - Prime editing with alternative nucleic acid templates and editor protein constructs Prior to this example, it is described that prime editing requires PEgRNA. Exemplary embodiments describing possible configurations of suitable PEgRNAs for use in prime editing are illustrated in Figure 3A (PEgRNA with 5' extension arm), Figure 3B (PEgRNA with 3' extension arm), Figure 3C (internal extension PEgRNA), Figure 3D (PEgRNA with 3' extension arm and including primer binding site, editing template, homology arm, and optional 3' and 5' modified regions and region designated as DNA synthesis template), and Figure 3E (PEgRNA with 5' extension arm and including primer binding site, editing template, homology arm, and optional 3' and 5' modified regions and region designated as DNA synthesis template). In addition, PEgRNA structure and composition are described in detail in the detailed description and throughout this application.

This example describes additional design variations of PEgRNA, in some cases PEgRNAs that are entirely or partially chemically synthesized outside the cell. These are envisioned to work in conjunction with the prime editing factors of the present 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 additional prime editing factor protein effector components to enhance or extend prime editing. In addition, the use of chemically synthesized PEgRNA may potentially lead to the production of molecules that are more stable and possess desirable features that enhance prime editing efficiency and capacity.

The PEgRNA serves as a nucleic acid template that encodes the desired edited genetic information to be integrated into the target site. In one aspect, the PEgRNA is generated by adding an extension arm to either the 5' or 3' end of the sgRNA (e.g., as shown in the embodiment of Figure 3A, 3B, 3D, or 3E) or by inserting a similar sequence internally onto the sgRNA (e.g., as shown in the embodiment of Figure 3C). The extension arm includes a DNA synthesis template that can encode a ssDNA product by a polymerase (e.g., reverse transcriptase) and that contains the desired edited genetic information. The extension arm includes a primer binding site (PBS) for annealing to the genomic DNA strand nicked by napDNAbp, and a DNA synthesis template for encoding the desired edited DNA strand that becomes integrated into the endogenous DNA target site by displacing the counterpart DNA strand. The PEgRNA can be expressed intracellularly from plasmid DNA or a DNA cassette incorporated into the genome, or they can be made outside the cell by in vitro transcription or by chemical synthesis and then delivered to the cell. Preparation of PEgRNA outside the cell, especially by chemical synthesis, offers the opportunity to substantially modify the PEgRNA. The present invention describes an alternative design of the prime editing template (Figure 72).

(A) A DNA synthesis template expressed as a separate molecule from the guide RNA (i.e., a DNA synthesis template provided in trans to the primed editor complex (napDNAbp + guide RNA).
In various embodiments described herein, prime editing utilizes a single PEgRNA that serves as both a programmable targeting molecule and an editing-encoding molecule. This embodiment is illustrated in Figure 72 A for a PEgRNA with a 3' extension arm. However, in some cases, this may be a disadvantage, especially for more complex PEgRNA molecules, such as those that encode large insertions. These RNAs may contain extensive secondary structures that may interfere with the PEgRNA backbone structure and interaction with Cas9. Alternatively, prime editing may be performed by replacing the PEgRNA with two separate RNA molecules: an sgRNA and a trans-prime editing RNA template (tPERT), as illustrated in Figure 72 B. The sgRNA serves to target Cas9 (or more generally napDNAbp) to the desired genomic target site, and the tPERT is used by a polymerase (e.g., reverse transcriptase) to write new DNA sequences into the target locus.

In general, simple expression of tPERT leads to lower editing efficiency compared to PEgRNA. However, the efficiency of trans-prime editing can be enhanced by the introduction of one or more MS2 RNA aptamers onto the tPERT RNA in conjunction with a fusion of the MS2 coat protein (MS2cp) to the prime editor protein (to make MS2cp-Cas9-RT). This allows the MS2 RNA aptamer to bind to MS2cp, thereby colocalizing tPERT (which contains the DNA synthesis template) to the site of editing by the prime editor complex. To avoid reverse transcription of the aptamer sequence, the MS2 aptamer is preferably placed at the 3' end of tPERT.

While this example utilizes MS2 tagging technology (comprising the MS2 RNA aptamer of tPERT paired with the MS2cp protein fused to the prime editor), other RNA-protein recruitment systems could alternatively be used. The general concept envisioned here is that the DNA synthesis template of tPERT is modified to contain an RNA recruitment secondary structure (e.g., a specialized hairpin such as the MS2 aptamer) such that tPERT can 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. Reviews of other RNA-protein recruitment domains are provided in the art, e.g., 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 is incorporated herein by reference in its entirety. 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. Any of these well-known recruitment systems can be used for trans-prime editing as described herein.

The efficiency of the present tPERT trans-prime editing system was tested. A maximum efficiency of 20% of His6 insertion (18bp) was achieved at the HEK3 site in HEK293T cells. tPERT containing an RT template containing a single 3'MS2 aptamer, a 13nt primer binding site, and 34nt of homology to the insert sequence and locus was used in conjunction with an editor containing MS2cp fused to the N-terminus of PE2. See Figure 73. The trans-prime editing strategy has the potential to address complications associated with the PEgRNA design of the PEgRNA and may be more amenable to longer RT templates to achieve larger insertions, deletions, or edits at greater distances from the prime editor nick site.

(B) Chemically synthesized PEgRNA with RNA and DNA synthesis templates.
Alternative nucleic acid templates can be used on the chemically synthesized PEgRNA (Figure 72C ). For example, the synthetic PEgRNA can be constructed as an RNA/DNA hybrid, with the spacer sequence and sgRNA backbone composed of RNA nucleotides, and the primer binding site and synthesis template (shown as the 3' extension in Figure 72C ) composed of DNA nucleotides. This can allow a DNA-dependent DNA polymerase to be used to prime the editor instead of reverse transcriptase. It can also prevent DNA synthesis templated to the sgRNA backbone sequence. In other designs, a chemical linker composed of non-templated nucleotides or other suitable linker moieties can be used to tether the nucleic acid editing template (composed of RNA or DNA) to the sgRNA backbone. This can prevent continued DNA polymerization of the sgRNA backbone and allow flexibility in the extension to allow more efficient templated synthesis. Finally, the orientation of the nucleic acid synthesis template can be reversed, so that DNA polymerization proceeds in the other direction from the sgRNA backbone versus towards it.

MS2 PEgRNAの配列:

タンパク質配列:

(C) Recruitment of 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 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 a recruitment system such as MS2 RNA aptamer and MS2 coat protein, or other similar recruitment systems known in the art. In this system, the PEgRNA is modified to include the MS2 aptamer in one of the sgRNA backbone 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 for wild-type M-MLV reverse transcriptase (Figure 74) and should be applicable to other RT variants. In addition, other RNA-protein interactions or protein-protein interactions can be used for RT recruitment.
The following sequence applies to Example 19:
tPERT sequence:

MS2 PEGRNA sequence:

Protein sequence:

Example 20. Split Intein Delivery of Prime Editor This example demonstrates that a prime editor can be split using an intein as a means to deliver the prime editor to cells by separate vectors. Each vector encodes a portion of a prime editor fusion protein. This example focuses on a PE fusion protein containing the standard SpCas9 (SEQ ID NO: 18). The split site for other Cas9 proteins can be the same corresponding site or may need to be optimized for each different Cas9 protein. In this 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.

The prime editor (PE) exceeds AAV packaging capacity. Therefore, it was envisioned to split PE by inserting a trans-splicing Npu intein into S. pyogenes Cas9 residue 1024 of SEQ ID NO:18, allowing delivery of the split SpPE as two separate polypeptides, each encoded by one of the binary AAV systems. The split site 1023/1024 was chosen because it (1) allows packaging into two AAVs while allowing space for the guide cassette(s) and minimal control elements, (2) the native serine to cysteine mutation would be relatively conservative, (3) the site is a flexible loop near the edge of Cas9 that is sterically predicted to allow splicing to occur, and (4) Cas9 has been successfully altered by circular permutation in this loop (although not previously at this particular split site).

To determine whether the Npu split prime editors are active, we transfected HEK cells with plasmids encoding the split editors and found that they recapitulated the activity of full-length PE3. We analyzed them by high-throughput sequencing. Furthermore, the three native Npu amino-terminal residues of the C-terminal extein known to splice most efficiently are different from those natively flanking Cas9 residue 1024. Replacement of these Cas9 residues with native Npu residues may alter the activity of the prime editor. We therefore determined whether mutation of the Cas9 native “SEQ” to the Npu intein “CFN” sequence altered the efficiency of prime editing. The “SEQ” residues facilitated prime editing with an efficiency similar to the full-length, suggesting that the intein split PE halves are capable of associating with each other and mediating prime editing. Mutation to “CFN” did not result in any further increase, suggesting that association alone may be sufficient for split PE activity, as we have previously observed with intein split base editors.

Although the associated unspliced editors can be active, steric disturbance of the prime editor resulting from incomplete splicing during the initiation step may affect the editing outcome. Therefore, 1024-CFN was chosen for further studies because it also recapitulates full-length PE3 activity.

Below is the amino acid sequence of the 1023/1024 split.

1023/1024 SpPE2 splitting at the N-terminal half

1023/1024 SpPE2 split at the C-terminal half

Prime editing factors packaged by the binary AAV system
Primed editors split between residues 1023 and 1024 by the Npu trans-splicing intein become packaged into AAV with high titers that compare favorably with those of base editors of similar genome size generated in the same manner.

In vivo prime editing
Split SpPE3 delivered by AAV9 via intracerebroventricular injection into P0 mice mediates prime editing in brain tissue in vivo. A nucleotide substitution at position +5 from G to T (i.e., 5 nucleotide bases downstream of the nick site and on the PAM sequence) results in the incorporation of a mutation encoding Pro>Gln near the N-terminus of DNMT1. This edit demonstrates the feasibility of prime editing in vivo and is not expected to introduce any selective pressure on edited cells ("+5" refers to the +5 position downstream of the nick site). The AAV architectures tested include full-length MMLV RT and a truncated variant lacking the RNAse H domain. The truncated posttranscriptional control element W3 was also evaluated because it has been shown to increase expression from viral cassettes in vivo, but its importance had not been tested in a base editor or prime editor context. It was found that full-length RT outperforms truncated RT and the addition of the W3 sequence improves activity. The increase in editing activity in the presence of W3 indicates that expression of prime editing factors is limiting in vivo and demonstrates the distinct challenge of prime editing in vivo versus cell culture.

References
Oakes, BL, 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. In this example, several variant prime editors were constructed that are all based on PE2. PE2 has the following sequence and structure:

As used herein, "PE2" refers to a PE complex that includes 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)]+desired PE gRNA. The PE fusion has the amino acid sequence of SEQ ID NO: 134, which is set forth as follows:

M-MLV reverse transcriptase (sequence number 139).

The PE2 linker is SGGSSGGSSGSETPGTSESATPESSGGSSGGSS (sequence number 127).

In this experiment, the linker in PE2 was replaced by one of the following alternative linkers (or in one case no linker):

Figure 79 shows the editing efficiency of replacement linker constructs. In particular, the data shows the editing efficiency of the PE2 construct with the current linker (denoted as PE2, white box) compared to various versions with the linker replaced by the indicated sequence for representative PEgRNAs for transition, transversion, insertion, and deletion editing at the HEK3, EMX1, FANCF, and RNF2 loci. The replacement linkers are referred to as "1xSGGS" (SEQ ID NO: 174) , "2xSGGS" (SEQ ID NO: 446) , "3xSGGS" (SEQ ID NO: 3889) , "1xXTEN" (SEQ ID NO: 171) , "No Linker", "1xGly", "1xPro", "1xEAAAK" (SEQ ID NO: 3968) , "2xEAAAK" (SEQ ID NO: 3969) , and "3xEAAAK" (SEQ ID NO: 3970) . Editing efficiency is measured relative to the "control" editing efficiency of PE2 in a bar graph format. The linker for PE2 is SGGSSGGSSGSETPGTSESATPESSGGSSGGSS (SEQ ID NO: 127). All edits were made in the context of the PE3 system, i.e., this refers to the addition of the PE2 editing construct plus an optimal secondary sgRNA nicking guide.

Figure 80 shows that the 1xXTEN (SEQ ID NO: 171) linker provided increased editing efficiency. Taking the average fold potency relative to PE2 produced the graph shown, indicating that use of the 1xXTEN (SEQ ID NO: 171) linker sequence improved editing efficiency by an average of 1.14-fold (n=15).

Example 22. Prime editor guide RNAs with improved activity
In various embodiments, the PEgRNA is likely to be target DNA site and editing dependent. That is, the sequence of the PEgRNA will depend on the target DNA sequence and the specific edit (e.g., deletion, insertion, inversion, substitution) to be introduced therein by prime editing. For example, in some embodiments, when attaching a motif 3' of the primer binding site (PBS) of the PEgRNA, a linker between the PBS and the motif is preferred to prevent steric clashes with the polymerase domain (e.g., reverse transcriptase) of the PE fusion protein. However, the nature of the linker may be different for each site. For example, if the same linker is used for each site, it may spuriously turn a 13nt PBS into a 16nt PBS by fortuitous pairing to the spacer sequence. Similarly, the linker may base pair to the PBS itself, resulting in its entrapment and possibly reducing activity. Therefore, unlike protein-based editing factors such as PE or BE4, a single linker sequence option connecting the two elements may not be effective for each construct and will depend, in part, on the sequence of the target DNA sequence and the desired edit.

Building in part on the information presented in Example 15 (Design and Engineering of PEgRNA), this example constructed various structural modifications made to PEgRNA and then tested their effect on editing function, among other aspects.
Expression of PEgRNA from a non-pol III promoter

Various PEgRNA expression systems were tested for their ability to produce PEgRNA, using the insertion of a 102 nucleotide sequence from FKBP as readout.

Transcription of PEgRNA can be driven by a typical constitutive promoter such as the U6 promoter. Although the U6 promoter is effective in driving transcription of PEgRNA in most cases, the U6 promoter is less effective at driving transcription of longer PEgRNA or U-rich RNAs. U-rich RNA stretches cause premature termination of transcription. This example compares the editing outcomes of guides expressed from the CMV promoter or U1 promoter with the U6 promoter. These promoters require different terminator sequences such as MASC ENE or PAN ENE as provided below. Increased editing was observed with the pCMV/MASC-ENE system. However, these guides resulted in incomplete insertion of sequences, whereas with the U6 promoter, complete insertion was observed at lower levels of editing. See Figure 81. The data suggests the possibility that alternative expression systems may be useful for long insertions.

The nucleotide sequence of the pCMV/MASC-ENE expression system is as follows (5' to 3' orientation) (the names of motifs are in bold immediately preceding the region they refer to):

PEgRNA骨格の改善

Improvement of the PEGRNA backbone

Several structural modifications of the gRNA backbone were also tested. None of these showed a significant increase in editing activity (see Figure 82 at 3.30.13 to 3.30.19 on the X-axis compared 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 tested. Second, it was noted that in HEK cells, transfection was quite efficient, and the amount of guide RNA transfected was in vast excess compared to what was needed (reducing the amount by 4-8 fold had no effect on editing). These improvements may only be seen in other cell types with lower transfection efficiency, or with less effective guides. Many of these changes are precedents for improving sgRNA activity in other cell lines.

末端モチーフが存在しないかまたは一続きのUで終わらない末端モチーフが存在するかどちらかであるところではいつも、転写物は次のHDVリボザイムを用いて終結させられたということに注意せよ:
GGCCGGCAUGGUCCCAGCCUCCUCGCUGGCGCCGGCUGGGCAACAUGCUUCGGCAUGGCGAAUGGGAC [配列番号3990] The sequence of the construct in Figure 82 is as follows:

Note that wherever there was either no terminal motif present or a terminal motif that did not end with a stretch of U's, the transcript was terminated using the following HDV ribozyme:
GGCCGGCAUGGUCCCAGCCUCCUCGCUGGCGCCGGCUGGGCAACAUGCUUCGGCAUGGCGAAUGGGAC [SEQ ID NO: 3990 ]

Additional RNA Motifs Certain motifs, such as the HDV ribozyme 3' of the PEgRNA, or G-quadruplex insertions, P1 extensions, templated hairpins, and tetraloop circles, see Figure 82 for details, which can be introduced onto the PEgRNA to improve its performance.

In particular, this example tested the effect of incorporating a tRNA motif 3' to the primer binding site. This element was chosen because of several possible functions:

(1) The tRNA motif is a highly stable RNA motif and therefore potentially reduces PEgRNA degradation;

(2) MMLV RT uses prolyl-tRNA as a primer to convert the viral genome to DNA during transcription. Therefore, it was thought that the same cap could be bound by RT, improving PE-gRNA binding by PE, RNA stability, and bringing the PBS back into close proximity to the genomic site, potentially improving activity as well.

In these constructs, P1 (see Figure 84) of the tRNA was extended. P1 refers to the first stem/base-pairing element of the tRNA (see Figure 84). This was believed to be necessary to prevent RNAse P-mediated cleavage of the tRNA 5' to P1, which would result in its removal from the PEgRNA.

This design used a prolyl-tRNA (codon CGG) with a short 3 nt linker between the tRNA and PBS and an extended P1. Various tRNA designs were tested and editing efficiency was compared to PEgRNA without a tRNA cap. See comparative data in Figure 83 (illustrating a PE experiment targeted to HEK3 gene editing, specifically targeting the insertion of a 10 nt insertion at position +1 relative to the nick site, using PE3), Figure 85 (illustrating a PE experiment targeted to FANCF gene editing, specifically targeting the G to T conversion at position +5 relative to the nick site, using PE3 construct), and Figure 86 (illustrating a PE experiment targeted to HEK3 gene editing, specifically targeting the insertion of a 71 nt FLAG tag insertion at position +1 relative to the nick site, using PE3 construct). tRNA modified PEgRNA was tested against unmodified PEgRNA control.

UGG/CGG refers to the codons used, the number refers to the length of the P1 extension added, length indicates an 8 nt linker, no specification is a 3 nt linker.

The data suggest that incorporation of tRNA may allow the use of shorter PBSs, which would likely result in additional activity improvements. In the case of RNF2, it is possible/probable that the linker used resulted in improved PBS binding to the spacer and a resulting decrease in activity.

Some sequences used:

HEK3+1 FLAG-tag insertion, prolyl-tRNA{UGG} P1 ext 5nt, linker 3nt

GGCCCAGACUGAGCACGUGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUGGAGGAAGCAGGGCUUCCUUUCCUGCCAUCACUUAUCGUCGUCAUCCUUGUAAUCCGUGCUCAGUCUGUCUGGCGGGGCUCGUUGGUCUAGGGGUAUGAUUCUCGCUUCGGGUGCGAGAGGUCCCGGGUUCAAAUCCCGGACGAGCCCCGCCUUUU[Sequence number 3902]

FANCF+5G→T prolyl-tRNA{CGG} P1 ext 5nt, linker 3nt

GGAAUCCCUUCUGCAGCACCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCGGAAAAGCGAUCAAGGUGCUGCAGAAGGGAUCUGGCGGGGCUCGUUGGUCUAGGGGUAUGAUUCUCGCUUCGGGUGCGAGAGGUCCCGGGUUCAAAUCCCGGACGAGCCCCGCCUUUU[Sequence number 3903]

HEK3++1 10nt insertion, prolyl-tRNA{UGG} P1 ext 5nt, linker 3nt

GGCCCAGACUGAGCACGUGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGGACCGAGUCGGUCCUCUGCCAUCAAAGCUUCGACCGUGCUCAGUCUUCUGCUCGAGGCGGGGCUCGUUGGUCUAGGGGUAUGAUUCUCGCUUCGGGUGCGAGAGGUCCCGGGUUCAAAUCCCGGACGAGCCCCGCCUCGAGCUUUU[Sequence number 3904]

The sequences reported in the data in Figures 85 and 86 are as follows:

Example 23. Use of prime editing to correct CDKL5 and sickle cell disease
Using PE to design a mouse model of CDKL5 harboring the 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 appear in early childhood and include seizures, irregular sleep patterns, gastrointestinal stress, and developmental delay. Some mutations that cause CDD, including 1412delA, cannot be corrected by base editing. However, prime editing has the potential to precisely correct base changes, deletions, and insertions from every base. By focusing on the 1412delA mutation, this example designed and tested a pegRNA that can insert the mutation, with the hope of establishing a mouse neuronal cell line (N2A) that harbors the mutation. This will allow large-scale screening of therapeutic pegRNAs as potential corrections of the mutation. The ultimate goal is to progress to a CDD mouse model carrying the 1412delA mutation with the ability to evaluate therapeutic effects. Current mouse models of CDD do not have a humanized allele. However, optimization of pegRNA is also underway in HEK293T cells. Figures 87 and 88 are results from a pilot screen in N2A cells where pegRNA incorporates 1412Adel, with details on primer binding site (PBS) length and reverse transcriptase (RT) template length (shown with and without the indel).

Use of PE to Treat Sickle Cell Disease (SCA) Sickle cell disease (SCA) is a recessive blood disorder caused by a glutamic acid to valine mutation at position 6 of the β-globin gene. The result is sickle-shaped red blood cells, which are poor oxygen transporters and prone to clumping. The clumping condition can be life-threatening. Previously, the D. Liu lab was able to demonstrate both integration and correction of the SCA locus in HEK293T cells using prime editing by DNA plasmid transfection. Since hematopoietic stem cells (HSCs) are difficult to edit by DNA plasmid transfection, this example tested the PE3 system in HSCs by protein and mRNA nucleofection. Figure 89 shows the results of editing the proxy locus of the β-globin gene in healthy HSCs and HEK3 with varying concentrations of editing factors versus pegRNA and nicking gRNA.

mRNA Nucleofection Protocol:
The protocol was improved by adjusting the ratio of editing factor to guide ([editing factor] to [guide] ratio or editing factor:guide ratio).

The nicking guide protospacer sequence was: CCTTGATACCAACCTGCCCA (SEQ ID NO:3991) .

The pegRNA sequence is:
CATGGTGCACCTGACTCCTGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCAGACTTCTCTTCAGGAGTCAGGTGCACTTT (sequence number 3992) .

1. CD34+ cells were thawed and pre-stimulated with cytokines (SCF, Flt3, and TPO) in X-Vivo15 medium for 24hr. Seeding density 1 million cells/mL.

2. The next day (24 hr later), CD34+ cells (1x105) were aspirated, washed once with PBS (centrifuged at 300g for 10 min at RT), and resuspended in P3 solution (Lonza) (20 ul per condition).

3. In the hood, combine 2ug of SpPE2 mRNA, 1ug of pegRNA and sgRNA combination on a sterile PCR strip on ice. Adjust the volume to 20ul (cells + mRNA&gRNA mix in P3 solution).

4. Pipette 20uL of cell suspension into Lonza4D 16-well strips and electroporate using program DS130.

5. Wait 10 to 15 min after electroporation, add 80uL of X-Vivo10+cytokine medium to the cell suspension, and transfer to pre-warmed X-Vivo10 medium with cytokines.

6. Cells are harvested 72 hours after electroporation to check for cell recovery and also genotype the bulk and sorted CD34+ and CD34+90+ populations.
Wild-type CDKL5 protein (accession number 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 (SEQ ID NO: 3993)

Wild-type CDKL5 protein (accession number 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 EQLPPEMTVARSSVKETSRE 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 (SEQ ID NO: 3994)

Example 24. Use of prime editing to target pathogenic AOL1 alleles for the treatment of non-diabetic chronic kidney disease . This example designs PEgRNAs capable of targeting pathogenic APOL1 alleles for use in prime editing to treat or reduce the likelihood of developing kidney disease.

End-stage kidney disease (ESKD) is a growing problem that now affects over 500,000 individuals in the United States. The cost of caring for patients with ESKD is currently over $40 billion per year. In the United States, subjects of African ancestry are four to five times more likely to develop ESKD than Americans without African ancestry. These facts are reflected in the disparity between the 12-13% of the U.S. population with African ancestry and the 40% of U.S. dialysis patients who are African American. The prevalence of kidney disease risk factors such as obesity and metabolic syndrome suggests that the magnitude of this problem will only increase.

For the vast majority of progressive kidney diseases, there is no specific treatment. Although some types of chronic kidney disease progression can be slowed by blood pressure control with certain medications, nephrologists cannot accurately predict which patients will respond. Moreover, while successful treatment typically slows progression, it does not prevent or halt disease progression.

Recently, it has been determined that certain genetic variants that alter the protein sequence of apolipoprotein L1 (APOL1) are associated with progressive kidney disease. Surprisingly, APOL1 kidney disease variants have a major impact on several different types of kidney disease, including hypertension-related 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 a high-risk APOL1 genotype. African Americans without the high-risk genotype have only a small excess risk compared to Americans of European descent.

Despite evidence that variants in the APOL1 gene cause kidney 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 be common in Africa because they confer protection against the form of trypanosome that causes African sleeping sickness.

There remains a need for treatments for kidney disease in patients with one or more APOL1 risk alleles. These cause significant morbidity and mortality in this and other target populations and have a high economic impact.

This example provides three exemplary PEGRNA design options based on specific exemplary target sequences that can be used for prime editing to correct defective APOL1 alleles.

PEgRNA 1

A PEG RNA was designed for APOL1 allele rs73885319 (p.S342G), which represents the G to A correction in affected individuals. The target sequence is 5-GGAGTCAAGCTCACGGATGTGGCCCCTGTA(G to A)GCTTCTTTCTTGTGCTGGATGTAGTCTACCT-3 (SEQ ID NO: 3995) .

The protospacer (bold above) is AAGCTCACGGAGTGGCCCC (SEQ ID NO: 3996) . The selected PE contains SaCas9(D10A).

The primer binding site may be:

GTGGCCCC (SEQ ID NO:3997)

TGTGGCCCC (SEQ ID NO:3998)

ATGTGGCCCC (SEQ ID NO:3999)

GATGTGGCCCC (SEQ ID NO: 4000)

GGATGTGGCCCC (SEQ ID NO: 4001)

CGGATGTGGCCCC (SEQ ID NO: 4002)

ACGGATGTGGCCCC (SEQ ID NO: 4003)

CACGGATGTGGCCCC (SEQ ID NO:4004)

TCACGGATGTGGCCCC (SEQ ID NO: 4005)

CTCACGGATGTGGCCCC (sequence number 4006) .

The RT template may be:

AAGAAGCTTACA (SEQ ID NO: 4007)

AAAGAAGCTTACA (SEQ ID NO: 4008)

GAAAGAAGCTTACA (SEQ ID NO: 4009)

AGAAAGAAGCTTACA (SEQ ID NO: 4010)

AAGAAAGAAGCTTACA (SEQ ID NO: 4011)

CAAGAAAGAAGCTTACA (SEQ ID NO: 4012)

ACAAGAAAGAAGCTTACA (SEQ ID NO: 4013)

CACAAGAAAGAAGCTTACA (SEQ ID NO: 4014)

GCACAAGAAAGAAGCTTACA (SEQ ID NO: 4015)

AGCACAAGAAAGAAGCTTACA (SEQ ID NO: 4016)

CAGCACAAGAAAGAAGCTTACA (SEQ ID NO: 4017)

CCAGCACAAGAAAGAAGCTTACA (SEQ ID NO: 4018)

TCCAGCACAAGAAAGAAGCTTACA (SEQ ID NO: 4019)

ATCCAGCACAAGAAAGAAGCTTACA (sequence number 4020) .

The nicking template may be GCTTTGATTCGTACACGAGG (SEQ ID NO: 4021) .

PEgRNA 2

Design a PEgRNA for APOL1 allele rs60910145, which represents the G→T correction in affected individuals.

The protospacer is GCTGGAGGAGAAGCTAAACA (SEQ ID NO: 4022) . The selected PE includes SpCas9(D10A)-NG.

The primer binding site may be:

GAAGCTAA (SEQ ID NO: 4023)

AGAAGCTAA (SEQ ID NO: 4024)

GAGAAGCTAA (SEQ ID NO: 4025)

GGAGAAGCTAA (SEQ ID NO: 4026)

AGGAGAAGCTAA (SEQ ID NO: 4027)

GAGGAGAAGCTAA (SEQ ID NO: 4028)

GGAGGAGAAGCTAA (SEQ ID NO: 4029)

TGGAGGAGAAGCTAA (SEQ ID NO: 4030)

CTGGAGGAGAAGCTAA (SEQ ID NO: 4031)

GCTGGAGGAGAAGCTAA (sequence number 4032) .

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

AGAATGT (SEQ ID NO:4033)

GAGAATGT (SEQ ID NO:4034)

TGAGAATGT (SEQ ID NO: 4035)

TTGAGAATGT (SEQ ID NO: 4036)

GTTGAGAATGT (SEQ ID NO: 4037)

TGTTGAGAATGT (SEQ ID NO: 4038)

TTGTTGAGAATGT (SEQ ID NO: 4039)

ATTGTTGAGAATGT (SEQ ID NO: 4040)

TATTGTTGAGAATGT (SEQ ID NO: 4041)

TTATTGTTGAGAATGT (SEQ ID NO: 4042)

ATTATTGTTGAGAATGT (SEQ ID NO: 4043)

AATTATTGTTGAGAATGT (SEQ ID NO: 4044)

TAATTATTGTTGAGAATGT (SEQ ID NO: 4045)

ATAATTATTGTTGAGAATGT (sequence number 4046) .

The template to be nicked may be:

CCTGTGGTCACAGTTCTTGG (SEQ ID NO: 4047)

CCACAGGGCAGGGCAGCCAC (sequence number 4048) .

PEgRNA 3

Design a PEgRNA for APOL1 allele rs71785313, which represents the insertion: ATTCTCAACAA [insertion: TAATTA] TAAGATTC.

The protospacer can be: TCTCAACAATAAGATTCTGC.

PE includes SaKKH-PE2.

The primer binding site may be:

TTCTCAAC (SEQ ID NO: 4051)

ATTCTCAAC (SEQ ID NO: 4052)

CATTCTCAAC (SEQ ID NO: 4053)

ACATTCTCAAC (SEQ ID NO: 4054)

AACATTCTCAAC (SEQ ID NO: 4055)

AAACATTCTCAAC (SEQ ID NO: 4056)

TAAACATTCTCAAC (SEQ ID NO: 4057)

CTAAACATTCTCAAC (SEQ ID NO: 4058)

GCTAAACATTCTCAAC (SEQ ID NO: 4059)

AGCTAAACATTCTCAAC (sequence number 4060) .

The RT template may be:

AATCTTATAATTATT (SEQ ID NO: 4061)

GAATCTTATAATTATT (SEQ ID NO: 4062)

AGAATCTTATAATTATT (SEQ ID NO: 4063)

CAGAATCTTATAATTATT (SEQ ID NO: 4064)

GCAGAATCTTATAATTATT (SEQ ID NO: 4065)

TGCAGAATCTTATAATTATT (SEQ ID NO: 4066)

CTGCAGAATCTTATAATTATT (SEQ ID NO: 4067)

CCTGCAGAATCTTATAATTATT (SEQ ID NO: 4068)

GCCTGCAGAATCTTATAATTATT (SEQ ID NO: 4069)

CGCCTGCAGAATCTTATAATTATT (SEQ ID NO: 4070)

CCGCCTGCAGAATCTTATAATTATT (SEQ ID NO: 4071)

TCCGCCTGCAGAATCTTATAATTATT (SEQ ID NO: 4072)

GTCCGCCTGCAGAATCTTATAATTATT (SEQ ID NO: 4073)

GGTCCGCCTGCAGAATCTTATAATTATT (sequence number 4074) .

The nicking template may be:

CCTGTGGTCACAGTTCTTGG (SEQ ID NO: 4047)

CCACAGGGCAGGGCAGCCAC (sequence number 4048) .
Other References Mentioned Throughout This Disclosure
Each of the following references is incorporated herein by reference in its entirety.

からなる群から選択される低分子の二量体である。
164．プライム編集によって、標的ヌクレオチド配列によってコードされる目当てのタンパク質に免疫エピトープを組み入れる方法であって、方法が:(a)標的ヌクレオチド配列を(i)核酸プログラム型DNA結合タンパク質(napDNAbp)および逆転写酵素を含むプライム編集因子ならびに(ii)機能部分をコードする編集鋳型を含むPEgRNAと接触させること;(b)免疫エピトープをコードする一本鎖DNA配列を重合すること;ならびに(c)DNA修復および/または複製プロセスによって、標的ヌクレオチド配列における対応する内生鎖の代わりに一本鎖DNA配列を組み込むことを含み、方法が、目当てのタンパク質と免疫エピトープとを含む融合タンパク質をコードする組み換え標的ヌクレオチド配列を生ずる。
165．態様164の方法であって、免疫エピトープが:破傷風トキソイド(配列番号396);ジフテリア毒素変異体CRM197(配列番号398);ムンプス免疫エピトープ1(配列番号400);ムンプス免疫エピトープ2(配列番号402);ムンプス免疫エピトープ3(配列番号404);風疹ウイルス(配列番号406);ヘマグルチニン(配列番号408);ノイラミニダーゼ(配列番号410);TAP1(配列番号412);TAP2(配列番号414);クラスI HLAに対するヘマグルチニンエピトープ(配列番号416);クラスI HLAに対するノイラミニダーゼエピトープ(配列番号418);クラスII HLAに対するヘマグルチニンエピトープ(配列番号420);クラスII HLAに対するノイラミニダーゼエピトープ(配列番号422);ヘマグルチニンエピトープH5N1結合クラスIおよびクラスII HLA(配列番号424);ノイラミニダーゼエピトープH5N1結合クラスIおよびクラスII HLA(配列番号426)からなる群から選択される。
166．態様164の方法であって、融合タンパク質がPE1、PE2、またはPE3のアミノ酸配列を含む。
167．態様164の方法であって、napDNAbpがCas9ニッカーゼ(nCas9)である。
168．態様164の方法であって、napDNAbpが配列番号18～88、126、130、137、141、147、153、157、445、460、467、および482～487のアミノ酸配列を含む。
169．態様164の方法であって、ガイドRNAが配列番号222を含む。
170．プライム編集によって、標的ヌクレオチド配列によってコードされる目当てのタンパク質上に低分子二量体化ドメインを組み入れる方法であって、方法が:(a)標的ヌクレオチド配列を(i)核酸プログラム型DNA結合タンパク質(napDNAbp)および逆転写酵素を含むプライム編集因子ならびに(ii)低分子二量体化ドメインをコードする編集鋳型を含むPEgRNAと接触させること;(b)免疫エピトープをコードする一本鎖DNA配列を重合すること;ならびに(c)DNA修復および/または複製プロセスによって、標的ヌクレオチド配列における対応する内生鎖の代わりに一本鎖DNA配列を組み込むことを含み、方法が、目当てのタンパク質と低分子二量体化ドメインとを含む融合タンパク質をコードする組み換え標的ヌクレオチド配列を生ずる。
171．態様170の方法であって、さらに、目当ての第2のタンパク質に対して方法を行うことを含む。
172．態様171の方法であって、目当ての第1のタンパク質および目当ての第2のタンパク質が、前記タンパク質の夫々の二量体化ドメインに結合する低分子の存在下において二量体化する。
173．態様170の方法であって、低分子結合ドメインが配列番号488のFKBP12である。
174．態様170の方法であって、低分子結合ドメインが配列番号489のFKBP12-F36Vである。
175．態様170の方法であって、低分子結合ドメインが配列番号490および493～494のシクロフィリンである。
176．態様170の方法であって、低分子が:

からなる群から選択される低分子の二量体である。
177．態様170の方法であって、融合タンパク質がPE1、PE2、またはPE3のアミノ酸配列を含む。
178．態様170の方法であって、napDNAbpがCas9ニッカーゼ(nCas9)である。
179．態様170の方法であって、napDNAbpが配列番号18～88、126、130、137、141、147、153、157、445、460、467、および482～487のアミノ酸配列を含む。
180．態様170の方法であって、ガイドRNAが配列番号222を含む。
181．プライム編集を用いてタンパク質上にペプチドタグまたはエピトープを組み入れる方法であって:タンパク質をコードする標的ヌクレオチド配列を、ペプチドタグをコードする第2のヌクレオチド配列をそれに挿入するように構成されたプライム編集因子構築物と接触させて、組み換えヌクレオチド配列をもたらすことを含み、その結果、ペプチドタグおよびタンパク質が融合タンパク質として組み換えヌクレオチド配列から発現される。
182．態様181の方法であって、ペプチドタグがタンパク質の精製および/または検出に用いられる。
183．態様181の方法であって、ペプチドタグが、ポリヒスチジン(例えば、HHHHHH（配列番号257）)、FLAG(例えば、DYKDDDDK（配列番号2）)、V5(例えば、GKPIPNPLLGLDST（配列番号3）)、GCN4、HA(例えば、YPYDVPDYA（配列番号5）)、Myc(例えばEQKLISEED（配列番号6）)、GSTなどである。
184．態様181の方法であって、ペプチドタグが、配列番号245-290からなる群から選択されるアミノ酸配列を有する。
185．態様181の方法であって、ペプチドタグがリンカーによってタンパク質に融合される。
186．態様181の方法であって、融合タンパク質が次の構造を有し:[タンパク質]-[ペプチドタグ]または[ペプチドタグ]-[タンパク質]、「]-[」が任意のリンカーを表す。
187．態様181の方法であって、リンカーが配列番号127、165-176、446、453、および767-769のアミノ酸配列を有する。
188．態様181の方法であって、プライム編集因子構築物が、配列番号101～104、181～183、223～234、237～244、277、324～330、332、334、336、338、340、342、344、346、348、350、352、354、356、358、360、362、364、366、368、394、429～442、499～505、641～649、678～692、735～736、757～761、776～777のヌクレオチド配列を含むPEgRNAを含む。
189．態様181の方法であって、PEgRNAがスペーサー、gRNAコア、および伸長アームを含み、スペーサーが標的ヌクレオチド配列に対して相補的であり、伸長アームがペプチドタグをコードする逆転写酵素鋳型を含む。
190．態様181の方法であって、PEgRNAがスペーサー、gRNAコア、および伸長アームを含み、スペーサーが標的ヌクレオチド配列に対して相補的であり、伸長アームがペプチドタグをコードする逆転写酵素鋳型を含む。
191．プライム編集によって標的ヌクレオチド配列によってコードされるPRNP上に1つ以上の防護性変異を組み入れることによって、プリオン病を防止またはその進行を停止させる方法であって、方法が:(a)標的ヌクレオチド配列を(i)核酸プログラム型DNA結合タンパク質(napDNAbp)および逆転写酵素を含むプライム編集因子ならびに(ii)機能部分をコードする編集鋳型を含むPEgRNAと接触させること;(b)防護性変異をコードする一本鎖DNA配列を重合すること;ならびに(c)DNA修復および/または複製プロセスによって、標的ヌクレオチド配列における対応する内生鎖の代わりに一本鎖DNA配列を組み込むことを含み、方法が、ミスフォールディングに対して耐性である防護性変異を含むPRNPをコードする組み換え標的ヌクレオチド配列を生ずる。
192．態様191の方法であって、プリオン病がヒトプリオン病である。
193．態様191の方法であって、プリオン病が動物プリオン病である。
194．態様192の方法であって、プリオン病がクロイツフェルト・ヤコブ病(CJD)、変異型クロイツフェルト・ヤコブ病(vCJD)、ゲルストマン・ストロイスラー・シャインカー症候群、致死性家族性不眠症、またはクールー病である。
195．態様193の方法であって、プリオン病が牛海綿状脳症(BSEまたは「狂牛病」)、慢性消耗病(CWD)、スクレイピー、伝達性ミンク脳症、ネコ海綿状脳症、および有蹄類海綿状脳症である。
196．態様191の方法であって、野生型PRNPアミノ酸配列が配列番号291-292である。
197．態様191の方法であって、方法が、配列番号293-323からなる群から選択される改変されたPRNPアミノ酸配列をもたらし、前記改変されたPRNPタンパク質がミスフォールディングに対して耐性である。
198．態様191の方法であって、融合タンパク質がPE1、PE2、またはPE3のアミノ酸配列を含む。
199．態様191の方法であって、napDNAbpがCas9ニッカーゼ(nCas9)である。
200．態様191の方法であって、napDNAbpが配列番号18～88、126、130、137、141、147、153、157、445、460、467、および482～487のアミノ酸配列を含む。
201．態様191の方法であって、ガイドRNAが配列番号222を含む。
202．プライム編集によって、標的ヌクレオチド配列によってコードされる目当てのRNA上にリボヌクレオチドモチーフまたはタグを組み入れる方法であって、方法が:(a)標的ヌクレオチド配列を(i)核酸プログラム型DNA結合タンパク質(napDNAbp)および逆転写酵素を含むプライム編集因子ならびに(ii)リボヌクレオチドモチーフまたはタグをコードする編集鋳型を含むPEgRNAと接触させること;(b)リボヌクレオチドモチーフまたはタグをコードする一本鎖DNA配列を重合すること;ならびに(c)DNA修復および/または複製プロセスによって、標的ヌクレオチド配列における対応する内生鎖の代わりに一本鎖DNA配列を組み込むことを含み、方法が、リボヌクレオチドモチーフまたはタグを含む目当ての修飾されたRNAをコードする組み換え標的ヌクレオチド配列を生ずる。
203．態様202の方法であって、リボヌクレオチドモチーフまたはタグが検出部分である。
204．態様202の方法であって、リボヌクレオチドモチーフまたはタグが目当てのRNAの発現レベルに影響する。
205．態様202の方法であって、リボヌクレオチドモチーフまたはタグが目当てのRNAの輸送または細胞下レベル位置付けに影響する。
206．態様202の方法であって、リボヌクレオチドモチーフまたはタグが、SV40 1型、SV40 2型、SV40 3型、hGH、BGH、rbGlob、TK、MALAT1 ENE-mascRNA、KSHV PAN ENE、Smbox/U1 snRNAボックス、U1 snRNA 3'ボックス、tRNA-リジン、broccoliアプタマー、spinachアプタマー、mangoアプタマー、HDVリボザイム、およびm6Aからなる群から選択される。
207．態様202の方法であって、PEgRNAが配列番号101～104、181～183、223～234、237～244、277、324～330、332、334、336、338、340、342、344、346、348、350、352、354、356、358、360、362、364、366、368、394、429～442、499～505、641～649、678～692、735～736、757～761、776～777、2997～3103、3113～3121、3305～3455、3479～3493、3522～3540、3549～3556、3628～3698、3755～3810、3874、3890～3901、3905～3911、3913～3929、3972～3989を含む。
208．態様202の方法であって、融合タンパク質がPE1、PE2、またはPE3のアミノ酸配列を含む。
209．態様202の方法であって、napDNAbpがCas9ニッカーゼ(nCas9)である。
210．態様202の方法であって、napDNAbpが配列番号18～88、126、130、137、141、147、153、157、445、460、467、および482～487のアミノ酸配列を含む。
211．プライム編集によって、標的ヌクレオチド配列によってコードされる目当てのタンパク質上の機能部分を組み入れまたは欠失する方法であって、方法が:(a)標的ヌクレオチド配列を(i)核酸プログラム型DNA結合タンパク質(napDNAbp)および逆転写酵素を含むプライム編集因子ならびに(ii)機能部分またはその欠失をコードする編集鋳型を含むPEgRNAと接触させること;(b)機能部分またはその欠失をコードする一本鎖DNA配列を重合すること;ならびに(c)DNA修復および/または複製プロセスによって、標的ヌクレオチド配列における対応する内生鎖の代わりに一本鎖DNA配列を組み込むことを含み、方法が、目当てのタンパク質および機能部分またはその除去を含む改変されたタンパク質をコードする組み換え標的ヌクレオチド配列を生じ、機能部分がタンパク質の修飾状態または局在状態を変改する。
212．態様211の方法であって、機能部分が、目当てのタンパク質のリン酸化、ユビキチン化、グリコシル化、脂質修飾、ヒドロキシル化、メチル化、アセチル化、クロトニル化、SUMO化状態を変改する。
群2．態様213～424
213．融合タンパク質であって、核酸プログラム型DNA結合タンパク質(napDNAbp)およびポリメラーゼを含む。
214．態様213の融合タンパク質であって、融合タンパク質が、プライム編集ガイドRNA(PEgRNA)の存在下においてプライム編集を実行することができる。
215．態様213の融合タンパク質であって、napDNAbpがニッカーゼ活性を有する。
216．態様213の融合タンパク質であって、napDNAbpがCas9タンパク質またはそのバリアントである。
217．態様213の融合タンパク質であって、napDNAbpがヌクレアーゼ活性Cas9、ヌクレアーゼ不活性型Cas9(dCas9)、またはCas9ニッカーゼ(nCas9)である。
218．態様213の融合タンパク質であって、napDNAbpがCas9ニッカーゼ(nCas9)である。
219．態様213の融合タンパク質であって、napDNAbpが:Cas9、Cas12e、Cas12d、Cas12a、Cas12b1、Cas13a、Cas12c、およびアルゴノートからなる群から選択され、任意にニッカーゼ活性を有する。
220．態様213の融合タンパク質であって、融合タンパク質がPEgRNAと複合体化したときに標的DNA配列に結合することができる。
221．態様220の融合タンパク質であって、標的DNA配列が標的鎖および相補的な非標的鎖を含む。
222．態様220の融合タンパク質であって、PEgRNAと複合体化した融合タンパク質の結合がRループを形成する。
223．態様222の融合タンパク質であって、Rループが、(i)PEgRNAと標的鎖とを含むRNA-DNAハイブリッドおよび(ii)相補的な非標的鎖を含む。
224．態様223の融合タンパク質であって、相補的な非標的鎖がニッキングされて、遊離の3’端を有するプライム配列を形成する。
225．態様214の融合タンパク質であって、PEgRNAが(a)ガイドRNAおよび(b)ガイドRNAの5'もしくは3’端またはガイドRNAの分子内の位置付けにおける伸長アームを含む。
226．態様225の融合タンパク質であって、伸長アームが(i)所望のヌクレオチド変化を含むDNA合成鋳型配列および(ii)プライマー結合部位を含む。
227．態様226の融合タンパク質であって、DNA合成鋳型配列が、ニック部位に隣接する内生DNA配列に対して相補的である一本鎖DNAフラップをコードし、一本鎖DNAフラップが所望のヌクレオチド変化を含む。
228．態様225の融合タンパク質であって、伸長アームが、長さが少なくとも5ヌクレオチド、少なくとも6ヌクレオチド、少なくとも7ヌクレオチド、少なくとも8ヌクレオチド、少なくとも9ヌクレオチド、少なくとも10ヌクレオチド、少なくとも11ヌクレオチド、少なくとも12ヌクレオチド、少なくとも13ヌクレオチド、少なくとも14ヌクレオチド、少なくとも15ヌクレオチド、少なくとも16ヌクレオチド、少なくとも17ヌクレオチド、少なくとも18ヌクレオチド、少なくとも19ヌクレオチド、少なくとも20ヌクレオチド、少なくとも21ヌクレオチド、少なくとも22ヌクレオチド、少なくとも23ヌクレオチド、少なくとも24ヌクレオチド、または少なくとも25ヌクレオチドである。
229．態様227の融合タンパク質であって、一本鎖DNAフラップが、ニック部位に隣接する内生DNA配列にハイブリダイズし、それによって所望のヌクレオチド変化を組み入れる。
230．態様227の融合タンパク質であって、一本鎖DNAフラップが、遊離の5'端を有しかつニック部位に隣接する内生DNA配列に取って代わる。
231．態様230の融合タンパク質であって、5'端を有する内生DNA配列が細胞によって切除される。
232．態様230の融合タンパク質であって、一本鎖DNAフラップの細胞性修復が所望のヌクレオチド変化の組み入れをもたらし、それによって所望の産物を形成する。
233．態様226の融合タンパク質であって、所望のヌクレオチド変化がPAM配列の約-4から+10の間もしくはPAM配列の約-10から+20の間もしくはPAM配列の約-20から+40の間もしくはPAM配列の約-30から+100の間である編集窓上に組み入れされるか、または所望のヌクレオチド変化がニック部位の少なくとも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、もしくは100ヌクレオチド下流に組み入れされる。
234．態様213の融合タンパク質であって、napDNAbpが、配列番号18のアミノ酸配列、または配列番号18のアミノ酸配列との少なくとも80％、85％、90％、95％、98％、もしくは99％配列同一性を有するアミノ酸配列を含む。
235．態様213の融合タンパク質であって、napDNAbpが、配列番号18～88、126、130、137、141、147、153、157、445、460、467、および482～487のいずれか1つのアミノ酸配列との少なくとも80％、85％、90％、95％、98％、または99％配列同一性を有するアミノ酸配列を含む。
236．態様213の融合タンパク質であって、ポリメラーゼが逆転写酵素であり、配列番号89～100、105～122、128～129、132、139、143、149、154、159、235、454、471、516、662、700、701～716、739～741、および766のアミノ酸配列のいずれか1つを含む。
237．態様213の融合タンパク質であって、ポリメラーゼが逆転写酵素であり、配列番号89～100、105～122、128～129、132、139、143、149、154、159、235、454、471、516、662、700、701～716、739～741、および766のいずれか1つのアミノ酸配列との少なくとも80％、85％、90％、95％、98％、または99％配列同一性を有するアミノ酸配列を含む。
238．態様213の融合タンパク質であって、ポリメラーゼがレトロウイルスまたはレトロトランスポゾンからの天然に存在する逆転写酵素である。
239．先の態様のいずれか1つの融合タンパク質であって、融合タンパク質が構造NH₂-[napDNAbp]-[ポリメラーゼ]-COOH;またはNH₂-[ポリメラーゼ]-[napDNAbp]-COOHを含み、「]-[」の夫々のものが任意のリンカー配列の存在を指示する。
240．態様239の融合タンパク質であって、リンカー配列が配列番号127、165～176、446、453、および767～769のアミノ酸配列を含む。
241．態様226の融合タンパク質であって、所望のヌクレオチド変化が1ヌクレオチド変化、1つ以上のヌクレオチドの挿入、または1つ以上のヌクレオチドの欠失である。
242．態様241の融合タンパク質であって、挿入または欠失が、少なくとも1、少なくとも2、少なくとも3、少なくとも4、少なくとも5、少なくとも6、少なくとも7、少なくとも8、少なくとも9、少なくとも10、少なくとも11、少なくとも12、少なくとも13、少なくとも14、少なくとも15、少なくとも16、少なくとも17、少なくとも18、少なくとも19、少なくとも20、少なくとも21、少なくとも22、少なくとも23、少なくとも24、少なくとも25、少なくとも26、少なくとも27、少なくとも28、少なくとも29、少なくとも30、少なくとも31、少なくとも32、少なくとも33、少なくとも34、少なくとも35、少なくとも36、少なくとも37、少なくとも38、少なくとも39、少なくとも40、少なくとも41、少なくとも42、少なくとも43、少なくとも44、少なくとも45、少なくとも46、少なくとも47、少なくとも48、少なくとも49、または少なくとも50である。
243．ガイドRNAとDNA合成鋳型を含む少なくとも1つの核酸伸長アームとを含むPEgRNA。
244．態様241のPEgRNAであって、核酸伸長アームがガイドRNAの3'もしくは5'端またはガイドRNAの分子内の位置の位置であり、核酸伸長アームがDNAまたはRNAである。
245．態様242のPEgRNAであって、PEgRNAが、napDNAbpに結合することおよびnapDNAbpを標的DNA配列へと導くことができる。
246．態様245のPEgRNAであって、標的DNA配列が標的鎖および相補的な非標的鎖を含み、ガイドRNAが標的鎖にハイブリダイズしてRNA-DNAハイブリッドおよびRループを形成する。
247．態様243のPEgRNAであって、少なくとも1つの核酸伸長アームが(i)DNA合成鋳型および(ii)プライマー結合部位を含む。
248．態様247のPEgRNAであって、核酸伸長アームが、長さが少なくとも5ヌクレオチド、少なくとも6ヌクレオチド、少なくとも7ヌクレオチド、少なくとも8ヌクレオチド、少なくとも9ヌクレオチド、少なくとも10ヌクレオチド、少なくとも11ヌクレオチド、少なくとも12ヌクレオチド、少なくとも13ヌクレオチド、少なくとも14ヌクレオチド、少なくとも15ヌクレオチド、少なくとも16ヌクレオチド、少なくとも17ヌクレオチド、少なくとも18ヌクレオチド、少なくとも19ヌクレオチド、少なくとも20ヌクレオチド、少なくとも21ヌクレオチド、少なくとも22ヌクレオチド、少なくとも23ヌクレオチド、少なくとも24ヌクレオチド、または少なくとも25ヌクレオチドである。
249．態様247のPEgRNAであって、DNA合成鋳型が、長さが少なくとも3ヌクレオチド、少なくとも4ヌクレオチド、少なくとも5ヌクレオチド、少なくとも6ヌクレオチド、少なくとも7ヌクレオチド、少なくとも8ヌクレオチド、少なくとも9ヌクレオチド、少なくとも10ヌクレオチド、少なくとも11ヌクレオチド、少なくとも12ヌクレオチド、少なくとも13ヌクレオチド、少なくとも14ヌクレオチド、または少なくとも15ヌクレオチドである。
250．態様247のPEgRNAであって、プライマー結合部位が、長さが少なくとも3ヌクレオチド、少なくとも4ヌクレオチド、少なくとも5ヌクレオチド、少なくとも6ヌクレオチド、少なくとも7ヌクレオチド、少なくとも8ヌクレオチド、少なくとも9ヌクレオチド、少なくとも10ヌクレオチド、少なくとも11ヌクレオチド、少なくとも12ヌクレオチド、少なくとも13ヌクレオチド、少なくとも14ヌクレオチド、または少なくとも15ヌクレオチドである。
251．態様243のPEgRNAであって、さらに、リンカー、ステムループ、ヘアピン、トウループ、アプタマー、またはRNA-タンパク質動員ドメインからなる群から選択される少なくとも1つの追加の構造を含む。
252．態様247のPEgRNAであって、DNA合成鋳型が、ニック部位に隣接する内生DNA配列に対して相補的である一本鎖DNAフラップをコードし、一本鎖DNAフラップが所望のヌクレオチド変化を含む。
253．態様252のPEgRNAであって、一本鎖DNAフラップが、ニッキングされた標的DNA配列上の5'端を有する内生一本鎖DNAを押し除け、内生一本鎖DNAがニック部位の下流において直ちに隣接する。
254．態様253のPEgRNAであって、遊離の5'端を有する内生一本鎖DNAが細胞によって切除される。
255．態様253のPEgRNAであって、一本鎖DNAフラップの細胞性修復が所望のヌクレオチド変化の組み入れをもたらし、それによって所望の産物を形成する。
256．態様243のPEgRNAであって、配列番号101～104、181～183、223～234、237～244、277、324～330、332、334、336、338、340、342、344、346、348、350、352、354、356、358、360、362、364、366、368、394、429～442、499～505、641～649、678～692、735～736、757～761、776～777のヌクレオチド配列、または配列番号101～104、181～183、223～234、237～244、277、324～330、332、334、336、338、340、342、344、346、348、350、352、354、356、358、360、362、364、366、368、394、429～442、499～505、641～649、678～692、735～736、757～761、776～777のいずれか1つとの少なくとも85％、または少なくとも90％、または少なくとも95％、または少なくとも98％、または少なくとも99％配列同一性を有するヌクレオチド配列を含む。
257．態様247のPEgRNAであって、DNA合成鋳型が、内生DNA標的と少なくとも80％または85％または90％または95％または99％同一であるヌクレオチド配列を含む。
258．態様247のPEgRNAであって、プライマー結合部位が、カットされたDNAの遊離の3'端とハイブリダイズする。
259．態様251のPEgRNAであって、少なくとも1つの追加の構造がPEgRNAの3'または5'端に位置付けられる。
260．態様213-242のいずれか1つの融合タンパク質とPEgRNAとを含むことを含む複合体。
261．態様260の複合体であって、PEgRNAが、ガイドRNAと、ガイドRNAの3'もしくは5'端またはガイドRNAの分子内の位置における核酸伸長アームとを含む。
262．態様260の複合体であって、PEgRNAが、napDNAbpに結合することおよびnapDNAbpを標的DNA配列へと導くことができる。
263．態様262の複合体であって、標的DNA配列が標的鎖および相補的な非標的鎖を含み、ガイドRNAが標的鎖にハイブリダイズして、RNA-DNAハイブリッドおよびRループを形成する。
264．態様261の複合体であって、少なくとも1つの核酸伸長アームが(i)DNA合成鋳型および(ii)プライマー結合部位を含む。
265．態様260の複合体であって、PEgRNAが、配列番号101～104、181～183、223～234、237～244、277、324～330、332、334、336、338、340、342、344、346、348、350、352、354、356、358、360、362、364、366、368、394、429～442、499～505、641～649、678～692、735～736、757～761、776～777のヌクレオチド配列、または配列番号101～104、181～183、223～234、237～244、277、324～330、332、334、336、338、340、342、344、346、348、350、352、354、356、358、360、362、364、366、368、394、429～442、499～505、641～649、678～692、735～736、757～761、776～777のいずれか1つとの少なくとも85％、もしくは少なくとも90％、もしくは少なくとも95％、もしくは少なくとも98％、もしくは少なくとも99％配列同一性を有するヌクレオチド配列を含む。
266．態様264の複合体であって、DNA合成鋳型が、内生DNA標的と少なくとも80％または85％または90％または95％または99％同一であるヌクレオチド配列を含む。
267．態様264の複合体であって、プライマー結合部位が、カットされたDNAの遊離の3'端とハイブリダイズする。
268．napDNAbpおよびPEgRNAを含むことを含む複合体。
269．態様268の複合体であって、napDNAbpがCas9ニッカーゼである。
270．態様268の複合体であって、napDNAbpが配列番号18のアミノ酸配列を含む。
271．態様268の複合体であって、napDNAbpが、配列番号18～88、126、130、137、141、147、153、157、445、460、467、および482～487のいずれか1つのアミノ酸配列との少なくとも80％、85％、90％、95％、98％、または99％配列同一性を有するアミノ酸配列を含む。
272．態様268の複合体であって、PEgRNAが、ガイドRNAと、ガイドRNAの3'もしくは5'端またはガイドRNAの分子内の位置における核酸伸長アームとを含む。
273．態様268の複合体であって、PEgRNAがnapDNAbpを標的DNA配列へと導くことができる。
274．態様272の複合体であって、標的DNA配列が標的鎖および相補的な非標的鎖を含み、PEgRNAのスペーサー配列が標的鎖にハイブリダイズして、RNA-DNAハイブリッドおよびRループを形成する。
275．態様273の複合体であって、核酸伸長アームが(i)DNA合成鋳型および(ii)プライマー結合部位を含む。
276．態様269の複合体であって、PEgRNAが、配列番号101～104、181～183、223～234、237～244、277、324～330、332、334、336、338、340、342、344、346、348、350、352、354、356、358、360、362、364、366、368、394、429～442、499～505、641～649、678～692、735～736、757～761、776～777、2997～3103、3113～3121、3305～3455、3479～3493、3522～3540、3549～3556、3628～3698、3755～3810、3874、3890～3901、3905～3911、3913～3929、3972～3989のヌクレオチド配列、または配列番号101～104、181～183、223～234、237～244、277、324～330、332、334、336、338、340、342、344、346、348、350、352、354、356、358、360、362、364、366、368、394、429～442、499～505、641～649、678～692、735～736、757～761、776～777のいずれか1つとの少なくとも85％、もしくは少なくとも90％、もしくは少なくとも95％、もしくは少なくとも98％、もしくは少なくとも99％配列同一性を有するヌクレオチド配列を含む。
277．態様276の複合体であって、DNA合成鋳型が、内生DNA標的と少なくとも80％または85％または90％または95％または99％同一であるヌクレオチド配列を含む。
278．態様276の複合体であって、プライマー結合部位が、カットされたDNAの遊離の3'端とハイブリダイズする。
279．態様276の複合体であって、PEgRNAが、さらに、リンカー、ステムループ、ヘアピン、トウループ、アプタマー、またはRNA-タンパク質動員ドメインからなる群から選択される少なくとも1つの追加の構造を含む。
280．態様213～242のいずれかの融合タンパク質をコードするポリヌクレオチド。
281．態様280のポリヌクレオチドを含むベクター。
282．態様213～242のいずれかの融合タンパク質と融合タンパク質のnapDNAbpに結合したPEgRNAとを含む細胞。
283．態様260～279のいずれか1つの複合体を含む細胞。
284．医薬組成物であって:(i)態様213～242のいずれかの融合タンパク質、態様260～279の複合体、態様68のポリヌクレオチド、または態様69のベクター;および(ii)薬学的に許容される賦形剤を含む。
285．医薬組成物であって:(i)態様260～279の複合体、(ii)トランスで提供されるポリメラーゼ;および(iii)薬学的に許容される賦形剤を含む。
286．キットであって:(i)態様213～242のいずれか1つの融合タンパク質をコードする核酸配列;および(ii)(i)の配列の発現を駆動するプロモーターを含む核酸構築物を含む。
287．所望のヌクレオチド変化を二本鎖DNA配列に組み入れるための方法であって、方法が:
(i)融合タンパク質およびPEgRNAを含む複合体と二本鎖DNA配列を接触させ、融合タンパク質がnapDNAbpおよびポリメラーゼを含み、PEgRNAが所望のヌクレオチド変化を含むDNA合成鋳型とプライマー結合部位とを含むこと;
(ii)二本鎖DNA配列にニッキングし、それによって、3'端を有する遊離の一本鎖DNAを生成すること;
(iii)遊離の一本鎖DNAの3'端をプライマー結合部位にハイブリダイズさせ、それによってポリメラーゼをプライムすること;
(iv)プライマー結合部位にハイブリダイズした3'端からDNAの鎖を重合し、それによって、DNA合成鋳型に対して相補的であるかつ所望のヌクレオチド変化を含む一本鎖DNAフラップを生成すること;
(v)カットされた部位に隣接する内生DNA鎖を一本鎖DNAフラップによって置き換え、それによって所望のヌクレオチド変化を二本鎖DNA配列に組み入れること、
を含む。
288．態様287の方法であって、(v)置き換えるステップが:(i)一本鎖DNAフラップをカットされた部位に隣接する内生DNA鎖にハイブリダイズさせて、配列ミスマッチを生出すること;(ii)内生DNA鎖を切り出すこと;および(iii)ミスマッチを修復して、所望のヌクレオチド変化をDNAの両方の鎖に含む所望の産物を形成すること、を含む。
289．態様288の方法であって、所望のヌクレオチド変化が1ヌクレオチド置換、欠失、または挿入である。
290．態様289の方法であって、1ヌクレオチド置換がトランジションまたはトランスバージョンである。
291．態様288の方法であって、所望のヌクレオチド変化が、(1)GからTの置換、(2)GからAの置換、(3)GからCの置換、(4)TからGの置換、(5)TからAの置換、(6)TからCの置換、(7)CからGの置換、(8)CからTの置換、(9)CからAの置換、(10)AからTの置換、(11)AからGの置換、または(12)AからCの置換である。
292．態様288の方法であって、所望のヌクレオチド(nucleoid)変化が、(1)G:C塩基対をT:A塩基対に、(2)G:C塩基対をA:T塩基対に、(3)G:C塩基対をC:G塩基対に、(4)T:A塩基対をG:C塩基対に、(5)T:A塩基対をA:T塩基対に、(6)T:A塩基対をC:G塩基対に、(7)C:G塩基対をG:C塩基対に、(8)C:G塩基対をT:A塩基対に、C:G塩基対をA:T塩基対に、(10)A:T塩基対をT:A塩基対に、(11)A:T塩基対をG:C塩基対に、または(12)A:T塩基対をC:G塩基対に変換する。
293．態様288の方法であって、所望のヌクレオチド変化が、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、または25ヌクレオチドの挿入または欠失である。
294．態様288の方法であって、所望のヌクレオチド変化が疾患関連遺伝子を修正する。
295．態様294の方法であって、疾患関連遺伝子が:アデノシンデアミナーゼ(ADA)欠損症;アルファ-1アンチトリプシン欠損症;嚢胞性線維症;デュシェンヌ型筋ジストロフィー;ガラクトース血症;ヘモクロマトーシス;ハンチントン病;メープルシロップ尿症;マルファン症候群;1型神経線維腫症;先天性爪肥厚症;フェニルケトン尿症;重症複合免疫不全;鎌状赤血球症;スミス・レムリ・オピッツ症候群;トリヌクレオチド反復障害;プリオン病;およびテイ・サックス病からなる群から選択される単一遺伝子障害に関連する。
296．態様294の方法であって、疾患関連遺伝子が:心臓病;高血圧;アルツハイマー病;関節炎;糖尿病;がん;および肥満からなる群から選択される多遺伝子性障害に関連する。
297．態様287の方法であって、napDNAbpが、ヌクレアーゼ不活性型Cas9(dCas9)、Cas9ニッカーゼ(nCas9)、またはヌクレアーゼ活性Cas9である。
298．態様287の方法であって、napDNAbpが、配列番号18のアミノ酸配列、または配列番号18のアミノ酸配列との少なくとも80％、85％、90％、95％、98％、もしくは99％配列同一性を有するアミノ酸配列を含む。
299．態様287の方法であって、napDNAbpが、配列番号18～88、126、130、137、141、147、153、157、445、460、467、および482～487のいずれか1つのアミノ酸配列との少なくとも80％、85％、90％、95％、98％、または99％配列同一性を有するアミノ酸配列を含む。
300．態様287の方法であって、ポリメラーゼが逆転写酵素であり、配列番号89～100、105～122、128～129、132、139、143、149、154、159、235、454、471、516、662、700、701～716、739～741、および766のアミノ酸配列のいずれか1つを含む。
301．態様287の方法であって、ポリメラーゼが逆転写酵素であり、配列番号89～100、105～122、128～129、132、139、143、149、154、159、235、454、471、516、662、700、701～716、739～741、および766のいずれか1つのアミノ酸配列との少なくとも80％、85％、90％、95％、98％、または99％配列同一性を有するアミノ酸配列を含む。
302．態様287の方法であって、PEgRNAが核酸伸長アームをガイドRNAの3'もしくは5'端または分子内の位置付けに含み、伸長アームがDNA合成鋳型配列およびプライマー結合部位を含む。
303．態様302の方法であって、伸長アームが、長さが少なくとも5ヌクレオチド、少なくとも6ヌクレオチド、少なくとも7ヌクレオチド、少なくとも8ヌクレオチド、少なくとも9ヌクレオチド、少なくとも10ヌクレオチド、少なくとも11ヌクレオチド、少なくとも12ヌクレオチド、少なくとも13ヌクレオチド、少なくとも14ヌクレオチド、少なくとも15ヌクレオチド、少なくとも16ヌクレオチド、少なくとも17ヌクレオチド、少なくとも18ヌクレオチド、少なくとも19ヌクレオチド、少なくとも20ヌクレオチド、少なくとも21ヌクレオチド、少なくとも22ヌクレオチド、少なくとも23ヌクレオチド、少なくとも24ヌクレオチド、または少なくとも25ヌクレオチドである。
304．態様287の方法であって、PEgRNAが、配列番号101～104、181～183、223～234、237～244、277、324～330、332、334、336、338、340、342、344、346、348、350、352、354、356、358、360、362、364、366、368、394、429～442、499～505、641～649、678～692、735～736、757～761、776～777からなる群から選択されるヌクレオチド配列を有する。
305．標的座位におけるDNA分子のヌクレオチド配列の1つ以上の変化を導入するための方法であって:
(i)DNA分子を、核酸プログラム型DNA結合タンパク質(napDNAbp)およびnapDNAbpを標的座位へと標的化するPEgRNAと接触させ、PEgRNAが、少なくとも1つの所望のヌクレオチド変化を含む逆転写酵素(RT)鋳型配列とプライマー結合部位とを含むことと;
(ii)標的座位におけるDNA鎖の暴露された3'端を形成すること;
(iii)暴露された3'端をプライマー結合部位にハイブリダイズさせて逆転写をプライムすること;
(iv)逆転写酵素によって、RT鋳型配列に基づく少なくとも1つの所望のヌクレオチド変化を含む一本鎖DNAフラップを合成すること;および
(v)少なくとも1つの所望のヌクレオチド変化を対応する内生DNA上に組み込み、それによって、標的座位においてDNA分子のヌクレオチド配列の1つ以上の変化を導入すること、
を含む。
306．態様305の方法であって、ヌクレオチド配列の1つ以上の変化がトランジションを含む。
307．態様306の方法であって、トランジションが、(a)TからC;(b)AからG;(c)CからT;および(d)GからAからなる群から選択される。
308．態様305の方法であって、ヌクレオチド配列の1つ以上の変化がトランスバージョンを含む。
309．態様308の方法であって、トランスバージョンが、(a)TからA;(b)TからG;(c)CからG;(d)CからA;(e)AからT;(f)AからC;(g)GからC;および(h)GからTからなる群から選択される。
310．態様305の方法であって、ヌクレオチド配列の1つ以上の変化が、(1)G:C塩基対をT:A塩基対に、(2)G:C塩基対をA:T塩基対に、(3)G:C塩基対をC:G塩基対に、(4)T:A塩基対をG:C塩基対に、(5)T:A塩基対をA:T塩基対に、(6)T:A塩基対をC:G塩基対に、(7)C:G塩基対をG:C塩基対に、(8)C:G塩基対をT:A塩基対に、(9)C:G塩基対をA:T塩基対に、(9)A:T塩基対をT:A塩基対に、(11)A:T塩基対をG:C塩基対に、または(12)A:T塩基対をC:G塩基対に変化させることを含む。
311．態様305の方法であって、ヌクレオチド配列の1つ以上の変化が、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、または25ヌクレオチドの挿入または欠失を含む。
312．態様305の方法であって、ヌクレオチド配列の1つ以上の変化が疾患関連遺伝子の修正を含む。
313．態様312の方法であって、疾患関連遺伝子が:アデノシンデアミナーゼ(ADA)欠損症;アルファ-1アンチトリプシン欠損症;嚢胞性線維症;デュシェンヌ型筋ジストロフィー;ガラクトース血症;ヘモクロマトーシス;ハンチントン病;メープルシロップ尿症;マルファン症候群;1型神経線維腫症;先天性爪肥厚症;フェニルケトン尿症;重症複合免疫不全;鎌状赤血球症;スミス・レムリ・オピッツ症候群;トリヌクレオチド反復障害;プリオン病;およびテイ・サックス病からなる群から選択される単一遺伝子障害に関連する。
314．態様312の方法であって、疾患関連遺伝子が:心臓病;高血圧;アルツハイマー病;関節炎;糖尿病;がん;および肥満からなる群から選択される多遺伝子性障害に関連する。
315．態様305の方法であって、napDNAbpがヌクレアーゼ活性Cas9またはそのバリアントである。
316．態様305の方法であって、napDNAbpがヌクレアーゼ不活性型Cas9(dCas9)もしくはCas9ニッカーゼ(nCas9)またはそのバリアントである。
317．態様305の方法であって、napDNAbpが、配列番号2のアミノ酸配列、または配列番号2との少なくとも80％、85％、90％、95％、98％、もしくは99％配列同一性を有するアミノ酸配列を含む。
318．態様305の方法であって、napDNAbpが、配列番号18～88、126、130、137、141、147、153、157、445、460、467、および482～487のいずれか1つのアミノ酸配列との少なくとも80％、85％、90％、95％、98％、または99％配列同一性を有するアミノ酸配列を含む。
319．態様305の方法であって、逆転写酵素がトランスで導入される。
320．態様305の方法であって、napDNAbpが逆転写酵素への融合体を含む。
321．態様305の方法であって、逆転写酵素が配列番号89～100、105～122、128～129、132、139、143、149、154、159、235、454、471、516、662、700、701～716、739～741、および766のアミノ酸配列のいずれか1つを含む。
322．態様305の方法であって、逆転写酵素が、配列番号89～100、105～122、128～129、132、139、143、149、154、159、235、454、471、516、662、700、701～716、739～741、および766のいずれか1つのアミノ酸配列との少なくとも80％、85％、90％、95％、98％、または99％配列同一性を有するアミノ酸配列を含む。
323．態様305の方法であって、標的座位におけるDNA鎖の暴露された3’端を形成するステップが、ヌクレアーゼによってDNA鎖へニックを入れることを含む。
324．態様323の方法であって、ヌクレアーゼがトランスで提供される.
325．態様305の方法であって、標的座位におけるDNA鎖の暴露された3’端を形成するステップが、DNA鎖を化学的薬剤と接触させることを含む。
326．態様305の方法であって、標的座位におけるDNA鎖の暴露された3’端を形成するステップが、複製エラーを導入することを含む。
327．態様305の方法であって、DNA分子をnapDNAbpおよびガイドRNAと接触させるステップがRループを形成する。
328．態様327の方法であって、暴露された3’端が形成されるDNA鎖がRループ上にある。
329．態様315の方法であって、PEgRNAが、逆転写酵素(RT)鋳型配列とプライマー結合部位とを含む伸長アームを含む。
330．態様329の方法であって、伸長アームが、ガイドRNAの3'端、ガイドRNAの5'端、またはガイドRNAの分子内の位置にある。
331．態様305の方法であって、PEgRNAが、さらに、リンカー、ステムループ、ヘアピン、トウループ、アプタマー、またはRNA-タンパク質動員ドメインからなる群から選択される少なくとも1つの追加の構造を含む。
332．態様305の方法であって、PEgRNAがさらに相同アームを含む。
333．態様305の方法であって、RT鋳型配列が、対応する内生DNAに対して相同的である。
334．プライム標的にプライムされた逆転写によって標的座位におけるDNA分子のヌクレオチド配列の1つ以上の変化を導入するための方法であって、方法が:(a)標的座位におけるDNA分子を(i)核酸プログラム型DNA結合タンパク質(napDNAbp)および逆転写酵素を含む融合タンパク質ならびに(ii)所望のヌクレオチド変化を含むRT鋳型を含むガイドRNAと接触させること;(b)RT鋳型のプライム標的にプライムされた逆転写を行なって、所望のヌクレオチド変化を含む一本鎖DNAを生成すること;ならびに(c)DNA修復および/または複製プロセスによって標的座位におけるDNA分子上に所望のヌクレオチド変化を組み込むことを含む。
335．態様334の方法であって、RT鋳型が、ガイドRNAの3'端、ガイドRNAの5'端、またはガイドRNAの分子内の位置付けに位置付けられる。
336．態様334の方法であって、所望のヌクレオチド変化が、トランジション、トランスバージョン、挿入、もしくは欠失、またはそれらのいずれかの組み合わせを含む。
337．態様334の方法であって、所望のヌクレオチド変化が、(a)TからC;(b)AからG;(c)CからT;および(d)GからAからなる群から選択されるトランジションを含む。
338．請求項334の方法であって、所望のヌクレオチド変化が、(a)TからA;(b)TからG;(c)CからG;(d)CからA;(e)AからT;(f)AからC;(g)GからC;および(h)GからTからなる群から選択されるトランスバージョンを含む。
339．請求項334の方法であって、所望のヌクレオチド変化が、(1)G:C塩基対をT:A塩基対に、(2)G:C塩基対をA:T塩基対に、(3)G:C塩基対をC:G塩基対に、(4)T:A塩基対をG:C塩基対に、(5)T:A塩基対をA:T塩基対に、(6)T:A塩基対をC:G塩基対に、(7)C:G塩基対をG:C塩基対に、(8)C:G塩基対をT:A塩基対に、(9)C:G塩基対をA:T塩基対に、(10)A:T塩基対をT:A塩基対に、(11)A:T塩基対をG:C塩基対に、または(12)A:T塩基対をC:G塩基対に変化させることを含む。
340．態様243～259のいずれか1つのPEgRNAをコードするポリヌクレオチド。
341．態様340のポリヌクレオチドを含むベクター。
342．態様341のベクターを含む細胞。
343．態様213の融合タンパク質であって、ポリメラーゼがエラーを起こしやすい逆転写酵素である。
344．プライム標的にプライムされた逆転写によって標的座位におけるDNA分子に変異導入するための方法であって、方法が:(a)標的座位におけるDNA分子を(i)核酸プログラム型DNA結合タンパク質(napDNAbp)およびエラーを起こしやすい逆転写酵素を含む融合タンパク質ならびに(ii)所望のヌクレオチド変化を含むRT鋳型を含むガイドRNAと接触させること;(b)RT鋳型のプライム標的にプライムされた逆転写を行なって、変異導入された一本鎖DNAを生成すること;ならびに(c)DNA修復および/または複製プロセスによって、変異導入された一本鎖DNAを標的座位におけるDNA分子上に組み込むことを含む。
345．いずれかの先行する態様の方法であって、融合タンパク質がPE1、PE2、またはPE3のアミノ酸配列を含む。
346．いずれかの先行する態様の方法であって、napDNAbpがCas9ニッカーゼ(nCas9)である。
347．態様344の方法であって、napDNAbpが配列番号18～88、126、130、137、141、147、153、157、445、460、467、および482～487のアミノ酸配列を含む。
348．態様344の方法であって、ガイドRNAが配列番号222を含む。
349．態様344の方法であって、(b)プライム標的にプライムされた逆転写を行うステップが、ガイドRNA上のプライマー結合部位へのアニーリングによって逆転写をプライムすることができる標的座位における3'端プライマー結合配列を生成することを含む。
350．健康な数の反復トリヌクレオチドを含む健康な配列によって標的DNA分子上のトリヌクレオチド反復拡大変異を置き換えるための方法であって、方法が:(a)標的座位におけるDNA分子を(i)核酸プログラム型DNA結合タンパク質(napDNAbp)およびポリメラーゼを含む融合タンパク質ならびに(ii)置き換え配列を含むDNA合成鋳型とプライマー結合部位とを含むPEgRNAと接触させること;(b)プライム編集を行なって、置き換え配列を含む一本鎖DNAを生成すること;ならびに(c)DNA修復および/または複製プロセスによって、標的座位におけるDNA分子上に一本鎖DNAを組み込むことを含む。
351．態様350の方法であって、融合タンパク質がPE1、PE2、またはPE3のアミノ酸配列を含む。
352．態様350の方法であって、napDNAbpがCas9ニッカーゼ(nCas9)である。
353．態様350の方法であって、napDNAbpが配列番号18～88、126、130、137、141、147、153、157、445、460、467、および482～487のアミノ酸配列を含む。
354．態様350の方法であって、ガイドRNAが配列番号222を含む。
355．態様350の方法であって、(b)プライム編集を行うステップが、ガイドRNA上のプライマー結合部位へのアニーリングによってポリメラーゼをプライムすることができる標的座位における3'端プライマー結合配列を生成することを含む。
356．態様350の方法であって、トリヌクレオチド反復拡大変異がハンチントン病、脆弱X症候群、またはフリードライヒ運動失調症に関連する。
357．態様350の方法であって、トリヌクレオチド反復拡大変異がCAGトリプレットの繰り返し単位を含む。
358．態様350の方法であって、トリヌクレオチド反復拡大変異がGAAトリプレットの繰り返し単位を含む。
359．プライム編集によって標的ヌクレオチド配列によってコードされる目当てのタンパク質上に機能部分を組み入れる方法であって、方法が:(a)標的ヌクレオチド配列を(i)核酸プログラム型DNA結合タンパク質(napDNAbp)およびポリメラーゼを含むプライム編集因子ならびに(ii)機能部分をコードするDNA合成鋳型を含むPEgRNAと接触させること;(b)機能部分をコードする一本鎖DNA配列を重合すること;ならびに(c)DNA修復および/または複製プロセスによって、標的ヌクレオチド配列における対応する内生鎖の代わりに一本鎖DNA配列を組み込むことを含み、方法が、目当てのタンパク質と機能部分とを含む融合タンパク質をコードする組み換え標的ヌクレオチド配列を生ずる。
360．態様359の方法であって、機能部分がペプチドタグである。
361．態様360の方法であって、ペプチドタグがアフィニティータグ、可溶化タグ、クロマトグラフィータグ、エピトープもしくは免疫エピトープタグ、または蛍光タグである。
362．態様360の方法であって、ペプチドタグが:AviTag(配列番号245);Cタグ(配列番号246);カルモジュリンタグ(配列番号247);ポリグルタミン酸タグ(配列番号248);Eタグ(配列番号249);FLAGタグ(配列番号2);HAタグ(配列番号5);Hisタグ(配列番号252～262);Mycタグ(配列番号6);NEタグ(配列番号264);Rho1D4タグ(配列番号265);Sタグ(配列番号266);SBPタグ(配列番号267);Softag-1(配列番号268);Softag-2(配列番号269);Spotタグ(配列番号270);Strepタグ(配列番号271);TCタグ(配列番号272);Tyタグ(配列番号273);V5タグ(配列番号3);VSVタグ(配列番号275);およびXpressタグ(配列番号276)からなる群から選択される。
363．態様360の方法であって、ペプチドタグが:AU1エピトープ(配列番号278);AU5エピトープ(配列番号279);バクテリオファージT7エピトープ(T7タグ)(配列番号280);ブルータングウイルスタグ(Bタグ)(配列番号281);E2エピトープ(配列番号282);ヒスチジンアフィニティータグ(HAT)(配列番号283);HSVエピトープ(配列番号284);ポリアルギニン(Argタグ)(配列番号285);ポリアスパラギン酸(Aspタグ)(配列番号286);ポリフェニルアラニン(Pheタグ)(配列番号287);S1タグ(配列番号288);Sタグ(配列番号266);およびVSV-タグ(配列番号275)からなる群から選択される。
364．態様359の方法であって、機能部分が免疫エピトープである。
365．態様364の方法であって、免疫エピトープが:破傷風トキソイド(配列番号396);ジフテリア毒素変異体CRM197(配列番号398);ムンプス免疫エピトープ1(配列番号400);ムンプス免疫エピトープ2(配列番号402);ムンプス免疫エピトープ3(配列番号404);風疹ウイルス(配列番号406);ヘマグルチニン(配列番号408);ノイラミニダーゼ(配列番号410);TAP1(配列番号412);TAP2(配列番号414);クラスI HLAに対するヘマグルチニンエピトープ(配列番号416);クラスI HLAに対するノイラミニダーゼエピトープ(配列番号418);クラスII HLAに対するヘマグルチニンエピトープ(配列番号420);クラスII HLAに対するノイラミニダーゼエピトープ(配列番号422);ヘマグルチニンエピトープH5N1結合クラスIおよびクラスII HLA(配列番号424);ノイラミニダーゼエピトープH5N1結合クラスIおよびクラスII HLA(配列番号426)からなる群から選択される。
366．態様359の方法であって、機能部分が目当てのタンパク質の局在を変改する。
367．態様359の方法であって、機能部分が分解タグであり、その結果、目当てのタンパク質の分解速度が変改される。
368．態様367の方法であって、分解タグが、タグ付けされたタンパク質の消去をもたらす。
369．態様359の方法であって、機能部分が低分子結合ドメインである。
370．態様359の方法であって、低分子結合ドメインが配列番号488のFKBP12である。
371．態様359の方法であって、低分子結合ドメインが配列番号489のFKBP12-F36Vである。
372．態様359の方法であって、低分子結合ドメインが配列番号490および493～494のシクロフィリンである。
373．態様359の方法であって、低分子結合ドメインが目当ての2つ以上のタンパク質上に組み入れされる。
374．態様373の方法であって、目当ての2つ以上のタンパク質が低分子に接触することによって二量体化し得る。
375．態様369の方法であって、低分子が:

からなる群から選択される低分子の二量体である。
376．プライム編集によって、標的ヌクレオチド配列によってコードされる目当てのタンパク質に免疫エピトープを組み入れる方法であって、方法が:(a)標的ヌクレオチド配列を(i)核酸プログラム型DNA結合タンパク質(napDNAbp)およびポリメラーゼを含むプライム編集因子ならびに(ii)機能部分をコードする編集鋳型を含むPEgRNAと接触させること;(b)免疫エピトープをコードする一本鎖DNA配列を重合すること;ならびに(c)DNA修復および/または複製プロセスによって、標的ヌクレオチド配列における対応する内生鎖の代わりに一本鎖DNA配列を組み込むことを含み、方法が、目当てのタンパク質と免疫エピトープとを含む融合タンパク質をコードする組み換え標的ヌクレオチド配列を生ずる。
377．態様376の方法であって、免疫エピトープが:破傷風トキソイド(配列番号396);ジフテリア毒素変異体CRM197(配列番号398);ムンプス免疫エピトープ1(配列番号400);ムンプス免疫エピトープ2(配列番号402);ムンプス免疫エピトープ3(配列番号404);風疹ウイルス(配列番号406);ヘマグルチニン(配列番号408);ノイラミニダーゼ(配列番号410);TAP1(配列番号412);TAP2(配列番号414);クラスI HLAに対するヘマグルチニンエピトープ(配列番号416);クラスI HLAに対するノイラミニダーゼエピトープ(配列番号418);クラスII HLAに対するヘマグルチニンエピトープ(配列番号420);クラスII HLAに対するノイラミニダーゼエピトープ(配列番号422);ヘマグルチニンエピトープH5N1結合クラスIおよびクラスII HLA(配列番号424);ノイラミニダーゼエピトープH5N1結合クラスIおよびクラスII HLA(配列番号426)からなる群から選択される。
378．態様376の方法であって、融合タンパク質がPE1、PE2、またはPE3のアミノ酸配列を含む。
379．態様376の方法であって、napDNAbpがCas9ニッカーゼ(nCas9)である。
380．態様376の方法であって、napDNAbpが配列番号18～88、126、130、137、141、147、153、157、445、460、467、および482～487のアミノ酸配列を含む。
381．態様376の方法であって、PEgRNAが配列番号101～104、181～183、223～234、237～244、277、324～330、332、334、336、338、340、342、344、346、348、350、352、354、356、358、360、362、364、366、368、394、429～442、499～505、641～649、678～692、735～736、757～761、776～777を含む。
382．プライム編集によって、標的ヌクレオチド配列によってコードされる目当てのタンパク質上に低分子二量体化ドメインを組み入れる方法であって、方法が:(a)標的ヌクレオチド配列を(i)核酸プログラム型DNA結合タンパク質(napDNAbp)およびポリメラーゼを含むプライム編集因子ならびに(ii)低分子二量体化ドメインをコードする編集鋳型を含むPEgRNAと接触させること;(b)免疫エピトープをコードする一本鎖DNA配列を重合すること;ならびに(c)DNA修復および/または複製プロセスによって、標的ヌクレオチド配列における対応する内生鎖の代わりに一本鎖DNA配列を組み込むことを含み、方法が、目当てのタンパク質と低分子二量体化ドメインとを含む融合タンパク質をコードする組み換え標的ヌクレオチド配列を生ずる。
383．態様382の方法であって、さらに、目当ての第2のタンパク質に対して方法を行うことを含む。
384．態様383の方法であって、目当ての第1のタンパク質および目当ての第2のタンパク質が、前記タンパク質の夫々の二量体化ドメインに結合する低分子の存在下において二量体化する。
385．態様382の方法であって、低分子結合ドメインが配列番号488のFKBP12である。
386．態様382の方法であって、低分子結合ドメインが配列番号489のFKBP12-F36Vである。
387．態様382の方法であって、低分子結合ドメインが配列番号490および493～494のシクロフィリンである。
388．態様382の方法であって、低分子が:

からなる群から選択される低分子の二量体である。
389．態様382の方法であって、融合タンパク質がPE1、PE2、またはPE3のアミノ酸配列を含む。
390．態様382の方法であって、napDNAbpがCas9ニッカーゼ(nCas9)である。
391．態様382の方法であって、napDNAbpが配列番号18～88、126、130、137、141、147、153、157、445、460、467、および482～487のアミノ酸配列を含む。
392．態様382の方法であって、PEgRNAが配列番号101～104、181～183、223～234、237～244、277、324～330、332、334、336、338、340、342、344、346、348、350、352、354、356、358、360、362、364、366、368、394、429～442、499～505、641～649、678～692、735～736、757～761、776～777を含む。
393．プライム編集を用いてタンパク質上にペプチドタグまたはエピトープを組み入れる方法であって:タンパク質をコードする標的ヌクレオチド配列を、ペプチドタグをコードする第2のヌクレオチド配列をそれに挿入するように構成されたプライム編集因子構築物と接触させて、組み換えヌクレオチド配列をもたらすことを含み、その結果、ペプチドタグおよびタンパク質が融合タンパク質として組み換えヌクレオチド配列から発現される。
394．態様383の方法であって、ペプチドタグがタンパク質の精製および/または検出に用いられる。
395．態様383の方法であって、ペプチドタグが、ポリヒスチジン(例えば、HHHHHH（配列番号257）)、FLAG(例えば、DYKDDDDK（配列番号2）)、V5(例えば、GKPIPNPLLGLDST（配列番号3）)、GCN4、HA(例えば、YPYDVPDYA（配列番号5）)、Myc(例えばEQKLISEED（配列番号6）)、またはGSTである。
396．態様383の方法であって、ペプチドタグが、配列番号245-290からなる群から選択されるアミノ酸配列を有する。
397．態様383の方法であって、ペプチドタグがリンカーによってタンパク質に融合される。
398．態様383の方法であって、融合タンパク質が次の構造を有し:[タンパク質]-[ペプチドタグ]または[ペプチドタグ]-[タンパク質]、「]-[」が任意のリンカーを表す。
399．態様383の方法であって、リンカーが配列番号127、165～176、446、453、および767～769のアミノ酸配列を有する。
400．態様383の方法であって、プライム編集因子構築物が、配列番号101～104、181～183、223～234、237～244、277、324～330、332、334、336、338、340、342、344、346、348、350、352、354、356、358、360、362、364、366、368、394、429～442、499～505、641～649、678～692、735～736、757～761、776～777のヌクレオチド配列を含むPEgRNAを含む。
401．態様383の方法であって、PEgRNAがスペーサー、gRNAコア、および伸長アームを含み、スペーサーが標的ヌクレオチド配列に対して相補的であり、伸長アームがペプチドタグをコードする逆転写酵素鋳型を含む。
402．態様383の方法であって、PEgRNAがスペーサー、gRNAコア、および伸長アームを含み、スペーサーが標的ヌクレオチド配列に対して相補的であり、伸長アームがペプチドタグをコードする逆転写酵素鋳型を含む。
403．プライム編集によって標的ヌクレオチド配列によってコードされるPRNP上に1つ以上の防護性変異を組み入れることによって、プリオン病を防止またはその進行を停止させる方法であって、方法が:(a)標的ヌクレオチド配列を(i)核酸プログラム型DNA結合タンパク質(napDNAbp)およびポリメラーゼを含むプライム編集因子ならびに(ii)機能部分をコードする編集鋳型を含むPEgRNAと接触させること;(b)防護性変異をコードする一本鎖DNA配列を重合すること;ならびに(c)DNA修復および/または複製プロセスによって、標的ヌクレオチド配列における対応する内生鎖の代わりに一本鎖DNA配列を組み込むことを含み、方法が、ミスフォールディングに対して耐性である防護性変異を含むPRNPをコードする組み換え標的ヌクレオチド配列を生ずる。
404．態様403の方法であって、プリオン病がヒトプリオン病である。
405．態様403の方法であって、プリオン病が動物プリオン病である。
406．態様404の方法であって、プリオン病がクロイツフェルト・ヤコブ病(CJD)、変異型クロイツフェルト・ヤコブ病(vCJD)、ゲルストマン・ストロイスラー・シャインカー症候群、致死性家族性不眠症、またはクールー病である。
407．態様403の方法であって、プリオン病が牛海綿状脳症(BSEまたは「狂牛病」)、慢性消耗病(CWD)、スクレイピー、伝達性ミンク脳症、ネコ海綿状脳症、および有蹄類海綿状脳症である。
408．態様403の方法であって、野生型PRNPアミノ酸配列が配列番号291～292である。
409．態様403の方法であって、方法が、配列番号293～323からなる群から選択される改変されたPRNPアミノ酸配列をもたらし、前記改変されたPRNPタンパク質がミスフォールディングに対して耐性である。
410．態様403の方法であって、融合タンパク質がPE1、PE2、またはPE3のアミノ酸配列を含む。
411．態様403の方法であって、napDNAbpがCas9ニッカーゼ(nCas9)である。
412．態様403の方法であって、napDNAbpが配列番号18～88、126、130、137、141、147、153、157、445、460、467、および482～487のアミノ酸配列を含む。
413．態様403の方法であって、PEgRNAが配列番号101～104、181～183、223～234、237～244、277、324～330、332、334、336、338、340、342、344、346、348、350、352、354、356、358、360、362、364、366、368、394、429～442、499～505、641～649、678～692、735～736、757～761、776～777を含む。
414．プライム編集によって、標的ヌクレオチド配列によってコードされる目当てのRNA上にリボヌクレオチドモチーフまたはタグを組み入れる方法であって、方法が:(a)標的ヌクレオチド配列を(i)核酸プログラム型DNA結合タンパク質(napDNAbp)およびポリメラーゼを含むプライム編集因子ならびに(ii)リボヌクレオチドモチーフまたはタグをコードする編集鋳型を含むPEgRNAと接触させること;(b)リボヌクレオチドモチーフまたはタグをコードする一本鎖DNA配列を重合すること;ならびに(c)DNA修復および/または複製プロセスによって、標的ヌクレオチド配列における対応する内生鎖の代わりに一本鎖DNA配列を組み込むことを含み、方法が、リボヌクレオチドモチーフまたはタグを含む目当ての修飾されたRNAをコードする組み換え標的ヌクレオチド配列を生ずる。
415．態様414の方法であって、リボヌクレオチドモチーフまたはタグが検出部分である。
416．態様414の方法であって、リボヌクレオチドモチーフまたはタグが目当てのRNAの発現レベルに影響する。
417．態様414の方法であって、リボヌクレオチドモチーフまたはタグが目当てのRNAの輸送または細胞下レベル位置付けに影響する。
418．態様414の方法であって、リボヌクレオチドモチーフまたはタグが、SV40 1型、SV40 2型、SV40 3型、hGH、BGH、rbGlob、TK、MALAT1 ENE-mascRNA、KSHV PAN ENE、Smbox/U1 snRNAボックス、U1 snRNA 3’ボックス、tRNA-リジン、broccoliアプタマー、spinachアプタマー、mangoアプタマー、HDVリボザイム、およびm6Aからなる群から選択される。
419．態様414の方法であって、PEgRNAが配列番号101～104、181～183、223～234、237～244、277、324～330、332、334、336、338、340、342、344、346、348、350、352、354、356、358、360、362、364、366、368、394、429～442、499～505、641～649、678～692、735～736、757～761、776～777を含む。
420．態様414の方法であって、融合タンパク質がPE1、PE2、またはPE3のアミノ酸配列を含む。
421．態様414の方法であって、napDNAbpがCas9ニッカーゼ(nCas9)である。
422．態様414の方法であって、napDNAbpが配列番号18～88、126、130、137、141、147、153、157、445、460、467、および482～487のアミノ酸配列を含む。
423．プライム編集によって、標的ヌクレオチド配列によってコードされる目当てのタンパク質上の機能部分を組み入れまたは欠失する方法であって、方法が:(a)標的ヌクレオチド配列を(i)核酸プログラム型DNA結合タンパク質(napDNAbp)およびポリメラーゼを含むプライム編集因子ならびに(ii)機能部分またはその欠失をコードする編集鋳型を含むPEgRNAと接触させること;(b)機能部分またはその欠失をコードする一本鎖DNA配列を重合すること;ならびに(c)DNA修復および/または複製プロセスによって、標的ヌクレオチド配列における対応する内生鎖の代わりに一本鎖DNA配列を組み込むことを含み、方法が、目当てのタンパク質および機能部分またはその除去を含む改変されたタンパク質をコードする組み換え標的ヌクレオチド配列を生じ、機能部分がタンパク質の修飾状態または局在状態を変改する。
424．態様423の方法であって、機能部分が、目当てのタンパク質のリン酸化、ユビキチン化、グリコシル化、脂質修飾、ヒドロキシル化、メチル化、アセチル化、クロトニル化、SUMO化状態を変改する。 Aspects
The following embodiments are within the scope of the present disclosure. Moreover, the present disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the enumerated embodiments are introduced into another embodiment enumerated in this section. For example, any enumerated embodiment that is dependent on another enumerated embodiment may be modified to include one or more limitations found in any other embodiment enumerated in this section that is dependent on the same basic embodiment. Where elements are presented as enumerated in Markush group format as examples, each subgroup of elements is also disclosed, and any element(s) may be removed from the group. In general, when the present disclosure, or aspects of the present disclosure, are viewed as including specific elements and/or features, it should be understood that an embodiment or aspect of the present invention consists of, or consists essentially of, such elements and/or features. It is also noted that the terms "comprising" and "containing" are intended to be open and permit the inclusion of additional elements or steps. When ranges are given, the endpoints are included. Moreover, unless otherwise indicated or otherwise clear from the context and the understanding of one of ordinary skill in the art, values expressed as ranges can assume any specific value, or subrange within the ranges set forth in different embodiments of the invention, up to ten times the unit of the lower limit of the range, unless the context clearly dictates otherwise.
Group 1. Aspects 1-212
1. A fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a reverse transcriptase.
2. A fusion protein of embodiment 1, wherein the fusion protein is capable of performing genome editing by reverse transcription primed to a prime target in the presence of an extended guide RNA.
3. A fusion protein of embodiment 1, wherein napDNAbp has nickase activity.
4. The fusion protein of embodiment 1, wherein napDNAbp is a Cas9 protein or a variant thereof.
5. The fusion protein of embodiment 1, wherein napDNAbp is a nuclease-active Cas9, a nuclease-inactive Cas9 (dCas9), or a Cas9 nickase (nCas9).
6. The fusion protein of embodiment 1, wherein napDNAbp is Cas9 nickase (nCas9).
7. The fusion protein of embodiment 1, wherein napDNAbp is selected from the group consisting of: Cas9, CasX, CasY, Cpf1, C2c1, C2c2, C2C3, and Argonaute, and optionally has nickase activity.
8. A fusion protein of embodiment 1, wherein the fusion protein is capable of binding to a target DNA sequence when complexed with an extended guide RNA.
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 binding of the fusion protein complexed with 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 an extended guide RNA and a target strand, and (ii) a complementary non-target strand.
12. The fusion protein of embodiment 11, wherein the complementary non-target strand is nicked to form a reverse transcriptase prime sequence having a free 3' end.
13. A fusion protein of embodiment 2, wherein the extended guide RNA comprises (a) a guide RNA and (b) an RNA extension at the 5' or 3' end of the guide RNA or at an intramolecular position of the guide RNA.
14. A fusion protein of embodiment 13, wherein the RNA extension comprises (i) a reverse transcription template sequence containing a desired nucleotide change, (ii) a reverse transcription primer binding site, and (iii) optionally a linker sequence.
15. A fusion protein of embodiment 14, wherein the reverse transcription template sequence encodes a single-stranded DNA flap that is complementary to the endogenous DNA sequence adjacent to the nick site, and the single-stranded DNA flap contains 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. A fusion protein of embodiment 15, wherein the single-stranded DNA flap hybridizes to the endogenous DNA sequence adjacent to the nick site, thereby incorporating the desired nucleotide change.
18. A fusion protein of embodiment 15, wherein a single-stranded DNA flap has a free 5' end and replaces the endogenous DNA sequence adjacent to the nick site.
19. A fusion protein of embodiment 18, wherein the endogenous DNA sequence having the 5' end is excised by the cell.
20. A fusion protein of embodiment 18, wherein cellular repair of the single-stranded DNA flap results in incorporation of a desired nucleotide change, thereby forming a desired product.
21. The fusion protein of embodiment 14, wherein the desired nucleotide change is incorporated into an editing window that is between about -4 and +10 of the PAM sequence, or between about -10 and +20 of the PAM sequence, or between about -20 and +40 of the PAM sequence, or between about -30 and +100 of the PAM sequence, or the desired nucleotide change is incorporated into 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 of the nick site. , 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.
22. The fusion protein of embodiment 1, wherein napDNAbp is represented by SEQ ID NO:18or SEQ ID NO:18The amino acid sequence of the present invention is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of
23. The fusion protein of embodiment 1, wherein napDNAbp is represented by SEQ ID NO:18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.The amino acid sequence of any one of the above is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of any one of the above.
24. The fusion protein of embodiment 1, wherein the reverse transcriptase is represented by 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 766The amino acid sequence of any one of the following:
25. The fusion protein of embodiment 1, wherein the reverse transcriptase is selected from the group consisting 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 766The amino acid sequence of any one of the above is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of any one of the above.
26. The fusion protein of embodiment 1, wherein the reverse transcriptase is a naturally occurring reverse transcriptase from a retrovirus or retrotransposon.
27. The fusion protein of any one of the preceding aspects, wherein the fusion protein has the structure NH₂-[napDNAbp]-[reverse transcriptase]-COOH; or NH₂-[reverse transcriptase]-[napDNAbp]-COOH, with each "]-[" indicating the presence of an optional linker sequence.
28. The fusion protein of embodiment 27, wherein the linker sequence is SEQ ID NO:127, 165-176, 446, 453, and 767-769The amino acid sequence of
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 insertion 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. An extended guide RNA of embodiment 1, wherein the RNA extension is at the 3' or 5' end of the guide RNA or at an intramolecular position of the guide RNA.
33. An extended guide RNA of embodiment 31, wherein the extended guide RNA is capable of binding to a napDNAbp and guiding 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, and 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 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-stranded DNA flap that is complementary to the endogenous DNA sequence adjacent to the nick site, and the single-stranded DNA flap contains a desired nucleotide change.
41. The extended guide RNA of embodiment 40, wherein the single-stranded DNA flap displaces the endogenous single-stranded DNA having a 5' end on the nicked target DNA sequence, and the endogenous single-stranded DNA is immediately adjacent to and downstream of the nick site.
42. The extended guide RNA of embodiment 41, wherein the endogenous single-stranded DNA having a free 5' end is excised by the cell.
43. The extended guide RNA of embodiment 41, wherein cellular repair of the single-stranded DNA flap results in incorporation of a desired nucleotide change, thereby forming a desired product.
44. The extended guide RNA of embodiment 31, comprising:394, 429-442, 641-649, 678-692, 2997-3103, 3113-3121, 3305-3455, 3479-3493, 3522-3556, 3628-3698, and 3755-3810or SEQ ID NO:394, 429-442, 641-649, 678-692, 2997-3103, 3113-3121, 3305-3455, 3479-3493, 3522-3556, 3628-3698, and 3755-3810or at least 90% or at least 95% or at least 98% or at least 99% sequence identity with any one of the following:
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 to the 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 to 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 of the guide RNA.
50. A complex of embodiment 48, wherein the extended guide RNA is capable of binding to the napDNAbp and guiding the napDNAbp to a target DNA sequence.
51. A complex of embodiment 50, wherein the target DNA sequence comprises a target strand and a complementary non-target strand, and the guide RNA hybridizes to the target strand to form an RNA-DNA hybrid and an R-loop.
52. A complex of embodiment 49, wherein 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 a nucleotide sequence of SEQ ID NO: 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 to any one of SEQ ID NO: 18-36.
54. A complex of embodiment 52, wherein the reverse transcription template sequence comprises a nucleotide sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity with the endogenous DNA target.
55. A complex of embodiment 52, wherein the reverse transcription primer binding site hybridizes to the free 3' end of the cut DNA.
56. A complex containing napDNAbp and an extended guide RNA.
57. A complex of embodiment 56, wherein napDNAbp is Cas9 nickase.
58. The complex of embodiment 56, wherein napDNAbp is represented by SEQ ID NO:18or SEQ ID NO:18The amino acid sequence has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the
59. The complex of embodiment 57, wherein napDNAbp is represented by SEQ ID NO:18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.The amino acid sequence of any one of the above amino acid sequences has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence of any one of the above amino acid sequences.
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 of the guide RNA.
61. A complex of embodiment 57, wherein the extended guide RNA can guide the napDNAbp to a target DNA sequence.
62. A complex of embodiment 61, wherein the target DNA sequence comprises a target strand and a complementary non-target strand, and 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 is394, 429-442, 641-649, 678-692, 2997-3103, 3113-3121, 3305-3455, 3479-3493, 3522-3556, 3628-3698, and 3755-3810or SEQ ID NO:394, 429-442, 641-649, 678-692, 2997-3103, 3113-3121, 3305-3455, 3479-3493, 3522-3556, 3628-3698, and 3755-3810In one embodiment, the present invention comprises 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 the following:
65. A 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. A complex of embodiment 63, wherein the reverse transcription primer binding site hybridizes to the free 3' end of the cut DNA.
67. The conjugate of embodiment 63, wherein any 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 a fusion protein of any one of embodiments 1 to 30.
69. A vector comprising the polynucleotide of embodiment 68.
70. A cell comprising a fusion protein of any one of embodiments 1 to 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 to 67.
72. A pharmaceutical composition comprising: (i) a fusion protein of any of embodiments 1 to 30, a complex of embodiment 48-67, a polynucleotide of embodiment 68, or a vector of embodiment 69; and (ii) a pharma- ceutically acceptable excipient.
73. A pharmaceutical composition comprising (i) a conjugate of any one of embodiments 48 to 67, (ii) a reverse transcriptase provided in trans; and (iii) a pharma- ceutically acceptable excipient.
74. A kit comprising: (i) a nucleic acid sequence encoding a fusion protein of any one of embodiments 1 to 30; and (ii) a nucleic acid construct comprising a promoter driving expression of the sequence of (i).
75. A method for incorporating a desired nucleotide change into a double-stranded DNA sequence, the method comprising:
(i) contacting a double-stranded DNA sequence with a complex comprising a fusion protein and an extended guide RNA, wherein the fusion protein comprises napDNAbp and a reverse transcriptase, and the extended guide RNA comprises a reverse transcription template sequence containing the desired nucleotide change;
(ii) nicking a double-stranded DNA sequence on the non-target strand, thereby generating a free single-stranded DNA having a 3' end;
(iii) hybridizing the 3' end of the free single-stranded DNA to the reverse transcription template sequence, thereby priming the reverse transcriptase domain;
(iv) polymerizing the DNA strand from the 3' end, thereby generating a single-stranded DNA flap containing the desired nucleotide change;
(v) replacing the endogenous DNA strand adjacent to the cut site with a single-stranded DNA flap, thereby incorporating the desired nucleotide change into the double-stranded DNA sequence;
including.
76. The method of embodiment 75, wherein (v) the replacing step comprises: (i) hybridizing a single-stranded DNA flap to the endogenous DNA strand adjacent to the cut site to create a sequence mismatch; (ii) excising the endogenous DNA strand; and (iii) repairing the mismatch to form a desired product containing 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, deletion, or insertion.
78. The method of embodiment 77, wherein the single nucleotide substitution is a transition or 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 nucleotide change is (1) a G:C base pair to a T:A base pair, (2) a G:C base pair to an A:T base pair, (3) a G:C base pair to a C:G base pair, (4) a T:A base pair to a G:C base pair, (5) a T:A base pair to an A:T base pair, (6) a T:A base pair to a C:G base pair, (7) a C:G base pair to a G:C base pair, (8) a C:G base pair to a T:A base pair, (9) a C:G base pair to an A:T base pair, (10) an A:T base pair to a T:A base pair, (11) an A:T base pair to a G:C base pair, or (12) an A:T base pair to a C:G base pair.
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 single-gene 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; congenital pachyonychia; phenylketonuria; 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-inactive Cas9 (dCas9), a Cas9 nickase (nCas9), or a nuclease-active Cas9.
86. The method of embodiment 76, wherein napDNAbp is SEQ ID NO:18The amino acid sequence of
87. The method of embodiment 76, wherein napDNAbp is selected from the group consisting of SEQ ID NO:18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.The amino acid sequence of any one of the above amino acid sequences has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence of any one of the above amino acid sequences.
88. The method of embodiment 76, wherein the reverse transcriptase is selected from the group consisting 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 766The amino acid sequence of any one of the following:
89. The method of embodiment 76, wherein the reverse transcriptase domain is selected from the group consisting 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 766The amino acid sequence of any one of the above amino acid sequences has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence of any one of the above amino acid sequences.
90. The method of embodiment 76, wherein the extended guide RNA comprises an RNA extension at the 5' or 3' end of the guide RNA or at an intramolecular location, and the RNA extension comprises a 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 is394, 429-442, 641-649, 678-692, 2997-3103, 3113-3121, 3305-3455, 3479-3493, 3522-3556, 3628-3698, and 3755-3810The nucleic acid has a nucleotide sequence selected from the group consisting of:
93. A method for introducing one or more changes in the nucleotide sequence of a DNA molecule at a target locus, comprising:
(i) contacting a DNA molecule with a nucleic acid programmable DNA binding protein (napDNAbp) and a guide RNA that targets the napDNAbp to a target locus, the guide RNA comprising a reverse transcriptase (RT) template sequence containing at least one desired nucleotide change;
(ii) creating an exposed 3' end of the DNA strand at the target locus;
(iii) hybridization of the exposed 3' end to the RT template sequence to prime reverse transcription;
(vi) synthesizing, by reverse transcriptase, a single-stranded DNA flap containing at least one desired nucleotide change based on the RT template sequence; and
(v) incorporating 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;
including.
94. The method of embodiment 93, wherein one or more changes in the nucleotide sequence include 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 one or more changes in the nucleotide sequence include 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 include changing (1) a G:C base pair to a T:A base pair, (2) a G:C base pair to an A:T base pair, (3) a G:C base pair to a C:G base pair, (4) a T:A base pair to a G:C base pair, (5) a T:A base pair to an A:T base pair, (6) a T:A base pair to a C:G base pair, (7) a C:G base pair to a G:C base pair, (8) a C:G base pair to a T:A base pair, (9) a C:G base pair to an A:T base pair, (10) an A:T base pair to a T:A base pair, (11) an A:T base pair to a G:C base pair, or (12) an A:T base pair to a C:G base pair.
99. The method of embodiment 93, wherein the one or more changes in the nucleotide sequence include 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 include a correction of a disease-associated gene.
101. The method of embodiment 100, wherein the disease-associated gene is associated with a single-gene 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; phenylketonuria; 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 napDNAbp is a nuclease-active Cas9 or a variant thereof.
104. The method of embodiment 93, wherein napDNAbp is a nuclease-inactivated Cas9 (dCas9) or Cas9 nickase (nCas9) or a variant thereof.
105. The method of embodiment 93, wherein napDNAbp is SEQ ID NO:18The amino acid sequence of
106. The method of embodiment 93, wherein napDNAbp is selected from the group consisting of SEQ ID NO:18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.The amino acid sequence of any one of the above amino acid sequences has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence of any one of the above.
107. The method of embodiment 93, wherein the reverse transcriptase is introduced in trans.
108. The method of embodiment 93, wherein napDNAbp comprises a fusion to a reverse transcriptase.
109. The method of embodiment 93, wherein the reverse transcriptase is selected from the group consisting 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 766The amino acid sequence of any one of the following:
110. The method of embodiment 93, wherein the reverse transcriptase is selected from the group consisting 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 766The amino acid sequence of any one of the above amino acid sequences has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence of any one of the above amino acid sequences.
111. The method of embodiment 93, wherein the step of forming an exposed 3' end of the DNA strand at the target locus includes nicking the DNA strand with a nuclease.
112. The method of embodiment 111, wherein the nuclease is 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 of the DNA strand at the target locus includes contacting the DNA strand with a chemical agent.
114. The method of embodiment 93, wherein the step of forming an exposed 3' end of the DNA strand at the target locus includes introducing a replication error.
115. The method of embodiment 93, wherein the step of contacting the DNA molecule with napDNAbp and the guide RNA forms an R-loop.
116. The method of embodiment 115, wherein the DNA strand on which the exposed 3' end is formed is on an R loop.
117. The method of embodiment 93, wherein the guide RNA comprises an extended portion comprising a 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 of 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 a nucleotide sequence of a DNA molecule at a target locus by primed target reverse transcription, the method comprising: (a) contacting the DNA molecule at the target locus with (i) a fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a reverse transcriptase, and (ii) a guide RNA comprising an RT template containing a desired nucleotide change; (b) performing primed target reverse transcription of the RT template to generate a single-stranded DNA containing the desired nucleotide change; and (c) incorporating the desired nucleotide change on the DNA molecule at the target locus by DNA repair and/or replication processes.
123. The method of embodiment 122, wherein the RT template is positioned at the 3' end of the guide RNA, the 5' end of the guide RNA, or at an intramolecular position of 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.Aspects122, 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.Aspects122, 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 base pair to a T:A base pair, (2) a G:C base pair to an A:T base pair, (3) a G:C base pair to a C:G base pair, (4) a T:A base pair to a G:C base pair, (5) a T:A base pair to an A:T base pair, (6) a T:A base pair to a C:G base pair, (7) a C:G base pair to a G:C base pair, (8) a C:G base pair to a T:A base pair, (9) a C:G base pair to an A:T base pair, (10) an A:T base pair to a T:A base pair, (11) an A:T base pair to a G:C base pair, or (12) an A:T base pair to a C:G base pair.
128. A polynucleotide encoding an extended guide RNA of any one of embodiments 31 to 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 primed target reverse transcription, the method comprising: (a) contacting the DNA molecule at the target locus with (i) a 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 containing a desired nucleotide change; (b) performing primed target reverse transcription of the RT template to generate a mutated single-stranded DNA; and (c) incorporating the mutated single-stranded DNA onto the DNA molecule at the target locus by DNA repair and/or replication processes.
133. The method of embodiment 132, wherein the fusion protein comprises an amino acid sequence of PE1, PE2, or PE3.
134. The method of embodiment 132, wherein napDNAbp is Cas9 nickase (nCas9).
135. The method of embodiment 132, wherein napDNAbp is selected from SEQ ID NOs: 18 to 135.88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.The amino acid sequence of
136. The method of embodiment 132, wherein the guide RNA comprises sequence number 222.
137. The method of embodiment 132, wherein (b) performing primed reverse transcription on a primed target comprises generating a 3' end primer binding sequence at the target locus that can prime reverse transcription by annealing to a primer binding site on the guide RNA.
138. A method for replacing a trinucleotide repeat expansion mutation on a target DNA molecule with a healthy sequence containing a healthy number of repeated trinucleotides, the method comprising: (a) contacting a DNA molecule at a target locus with (i) a 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 replacement sequence, and introducing said fusion protein; (b) performing primed reverse transcription on the prime target of the RT template to generate a single-stranded DNA comprising the replacement sequence; and (c) incorporating the single-stranded DNA onto the DNA molecule at the target locus by DNA repair and/or replication processes.
139. The method of embodiment 138, wherein the fusion protein comprises an amino acid sequence of PE1, PE2, or PE3.
140. The method of embodiment 138, wherein napDNAbp is Cas9 nickase (nCas9).
141. The method of embodiment 138, wherein napDNAbp is selected from SEQ ID NOs: 18 to 19.88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.The amino acid sequence of
142. The method of embodiment 138, wherein the guide RNA comprises sequence number 222.
143. The method of embodiment 138, wherein (b) performing primed reverse transcription on a primed target comprises generating a 3' end primer binding sequence at the target locus that can prime 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 repeat unit of a CAG triplet.
146. The method of embodiment 138, wherein the trinucleotide repeat expansion mutation comprises a repeat unit of a GAA triplet.
147. A method for incorporating a functional moiety onto a protein of interest encoded by a target nucleotide sequence by prime editing, the method comprising: (a) contacting the target nucleotide sequence with (i) a prime editing factor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a reverse transcriptase and (ii) a PEgRNA comprising an editing template encoding the functional moiety; (b) polymerizing a single-stranded DNA sequence encoding the functional moiety; and (c) incorporating the single-stranded DNA sequence in place of the corresponding endogenous strand in the target nucleotide sequence by DNA repair and/or replication processes, wherein the method produces a recombinant target nucleotide sequence encoding a fusion protein comprising the protein of interest and the functional moiety.
148. The method of embodiment 147, wherein the functional portion is a peptide tag.
149. The method of embodiment 148, wherein the peptide tag is an affinity tag, a solubilization tag, a chromatography tag, an epitope tag, or a fluorescent tag.
150. The method of embodiment 148, wherein the peptide tag is: AviTag (SEQ ID NO: 245); C-tag (SEQ ID NO: 246); calmodulin tag (SEQ ID NO: 247); polyglutamic acid tag (SEQ ID NO: 248); E-tag (SEQ ID NO: 249); FLAG tag (SEQ ID NO: 250);2); HA tag (SEQ ID NO:Five); His tag (SEQ ID NO: 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).
151. The method of embodiment 148, wherein the peptide tags are: 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); polyaspartic acid (Asp tag) (SEQ ID NO: 286); polyphenylalanine (Phe tag) (SEQ ID NO: 287); S1 tag (SEQ ID NO: 288); S tag (SEQ ID NO: 266); and VSVtag(SEQ ID NO:275).
152. The method of embodiment 147, wherein the functional portion is an immune epitope.
153. The method of embodiment 152, wherein the immune epitope is selected from the group consisting of: tetanus toxoid (SEQ ID NO: 396); diphtheria toxin variant CRM197 (SEQ ID NO: 398); mumps immune epitope 1 (SEQ ID NO: 400); mumps immune epitope 2 (SEQ ID NO: 402); mumps immune epitope 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 epitope for class I HLA (SEQ ID NO: 416); neuraminidase epitope for class I HLA (SEQ ID NO: 418); hemagglutinin epitope for class II HLA (SEQ ID NO: 420); neuraminidase epitope for class II HLA (SEQ ID NO: 422); hemagglutinin epitope H5N1 binding class I and class II HLA (SEQ ID NO: 424); neuraminidase epitope H5N1-binding class I and class II HLA (SEQ ID NO: 426).
154. A method of embodiment 147, wherein the functional moiety alters the localization of a target protein.
155. The method of embodiment 147, wherein the functional moiety is a degradation tag, thereby altering the degradation rate of the target protein.
156. The method of embodiment 155, further comprising:comprises an amino acid sequence encoding a degradation tag as disclosed herein..
157. The method of embodiment 147, wherein the functional portion is a small molecule binding domain.
158. The method of embodiment 157, wherein the small molecule binding domain is FKBP12 of sequence number 488.
159. The method of embodiment 157, wherein the small molecule binding domain is FKBP12-F36V of sequence number 489.
160. The method of embodiment 157, wherein the small molecule binding domain is SEQ ID NO: 490 and 493~494 cyclophilin.
161. The method of embodiment 157, wherein small molecule binding domains are incorporated onto two or more proteins of interest.
162. The method of embodiment 161, wherein two or more proteins of interest can be dimerized by contacting them with a small molecule.
163. The method of embodiment 157, wherein the small molecule is:

It is a dimer of a small molecule selected from the group consisting of:
164. A method for incorporating an immune epitope into a protein of interest encoded by a target nucleotide sequence by prime editing, the method comprising: (a) contacting the target nucleotide sequence with (i) a prime editing factor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a reverse transcriptase and (ii) a PEgRNA comprising an editing template encoding a functional moiety; (b) polymerizing a single-stranded DNA sequence encoding the immune epitope; and (c) incorporating the single-stranded DNA sequence in place of the corresponding endogenous strand in the target nucleotide sequence by DNA repair and/or replication processes, the method producing a recombinant target nucleotide sequence encoding a fusion protein comprising the protein of interest and the immune epitope.
165. The method of embodiment 164, wherein the immune epitope is selected from the group consisting of: tetanus toxoid (SEQ ID NO: 396); diphtheria toxin variant CRM197 (SEQ ID NO: 398); mumps immune epitope 1 (SEQ ID NO: 400); mumps immune epitope 2 (SEQ ID NO: 402); mumps immune epitope 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 epitope for class I HLA (SEQ ID NO: 416); neuraminidase epitope for class I HLA (SEQ ID NO: 418); hemagglutinin epitope for class II HLA (SEQ ID NO: 420); neuraminidase epitope for class II HLA (SEQ ID NO: 422); hemagglutinin epitope H5N1 binding class I and class II HLA (SEQ ID NO: 424); selected from the group consisting of neuraminidase epitope H5N1-binding class I and class II HLA (SEQ ID NO: 426).
166. The method of embodiment 164, wherein the fusion protein comprises an amino acid sequence of PE1, PE2, or PE3.
167. The method of embodiment 164, wherein napDNAbp is Cas9 nickase (nCas9).
168. The method of embodiment 164, wherein napDNAbp is selected from SEQ ID NOs: 18 to 19.88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.The amino acid sequence of
169. The method of embodiment 164, wherein the guide RNA comprises sequence number 222.
170. A method for incorporating a small dimerization domain onto a protein of interest encoded by a target nucleotide sequence by prime editing, the method comprising: (a) contacting the target nucleotide sequence with (i) a prime editing factor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a reverse transcriptase, and (ii) a PEgRNA comprising an editing template encoding the small dimerization domain; (b) polymerizing a single-stranded DNA sequence encoding an immune epitope; and (c) incorporating the single-stranded DNA sequence in place of the corresponding endogenous strand in the target nucleotide sequence by DNA repair and/or replication processes, the method producing a recombinant target nucleotide sequence encoding a fusion protein comprising the protein of interest and the small dimerization domain.
171. The method of embodiment 170, further comprising performing the method on a second protein of interest.
172. The method of embodiment 171, wherein a first protein of interest and a second protein of interest dimerize in the presence of a small molecule that binds to the dimerization domains of each of the proteins.
173. The method of embodiment 170, wherein the small molecule binding domain is FKBP12 of sequence number 488.
174. The method of embodiment 170, wherein the small molecule binding domain is FKBP12-F36V of sequence number 489.
175. The method of embodiment 170, wherein the small molecule binding domain is SEQ ID NO: 490 and 493~494 cyclophilin.
176. The method of embodiment 170, wherein the small molecule is:

It is a dimer of a small molecule selected from the group consisting of:
177. The method of embodiment 170, wherein the fusion protein comprises an amino acid sequence of PE1, PE2, or PE3.
178. The method of embodiment 170, wherein napDNAbp is Cas9 nickase (nCas9).
179. The method of embodiment 170, wherein the napDNAbp is selected from SEQ ID NOs: 18 to 19.88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.The amino acid sequence of
180. The method of embodiment 170, wherein the guide RNA comprises sequence number 222.
181. A method for incorporating a peptide tag or epitope onto a protein using prime editing, comprising: contacting a target nucleotide sequence encoding a protein with a prime editor construct configured to insert therein a second nucleotide sequence encoding a peptide tag, resulting 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 a peptide tag is used for purifying and/or detecting the protein.
183. The method of embodiment 181, wherein the peptide tag is a polyhistidine (e.g., HHHHHH(SEQ ID NO: 257)), FLAG (e.g., DYKDDDDK(SEQ ID NO:2)), V5 (for example, GKPIPNPLLGLDST(SEQ ID NO:3)), GCN4, HA (e.g., YPYDVPDYA(SEQ ID NO:5)), Myc (e.g., EQKLISEED(SEQ ID NO:6)), 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 NOs: 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], where "]-[" represents an optional linker.
187. The method of embodiment 181, wherein the linker has the 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 sequence represented by SEQ ID NO:101-104, 181-183, 223-234, 237-244, 277, 324-330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 394, 429-442, 499-505, 641-649, 678-692, 735-736, 757-761, 776-777The pEGRNA comprises a nucleotide sequence of
189. The method of embodiment 181, wherein the PEGRNA comprises a spacer, a gRNA core, and an extension arm, the spacer being complementary to a target nucleotide sequence, and the extension arm comprising a reverse transcriptase template encoding a peptide tag.
190. The method of embodiment 181, wherein the PEGRNA comprises a spacer, a gRNA core, and an extension arm, the spacer being complementary to a target nucleotide sequence, and the extension arm comprising a reverse transcriptase template encoding a peptide tag.
191. A method for preventing or halting the progression of prion disease by incorporating one or more protective mutations into a PRNP encoded by a target nucleotide sequence by prime editing, the method comprising: (a) contacting the target nucleotide sequence with (i) a prime editing factor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a reverse transcriptase, and (ii) a PEgRNA comprising an editing template encoding a functional moiety; (b) polymerizing a single-stranded DNA sequence encoding the protective mutations; and (c) incorporating, by DNA repair and/or replication processes, the single-stranded DNA sequence in place of the corresponding endogenous strand in the target nucleotide sequence, wherein the method produces a recombinant target nucleotide sequence encoding a PRNP containing protective mutations that are 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 wild-type PRNP amino acid sequence is SEQ ID NO: 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, and the modified PRNP protein is resistant to misfolding.
198. The method of embodiment 191, wherein the fusion protein comprises an amino acid sequence of PE1, PE2, or PE3.
199. The method of embodiment 191, wherein the napDNAbp is Cas9 nickase (nCas9).
200. The method of embodiment 191, wherein napDNAbp is selected from SEQ ID NOs: 18 to 19.88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.The amino acid sequence of
201. The method of embodiment 191, wherein the guide RNA comprises sequence number 222.
202. A method for incorporating a ribonucleotide motif or tag onto a desired RNA encoded by a target nucleotide sequence by prime editing, the method comprising: (a) contacting the target nucleotide sequence with (i) a prime editing factor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a reverse transcriptase and (ii) a PEgRNA comprising an editing template encoding the ribonucleotide motif or tag; (b) polymerizing a single-stranded DNA sequence encoding the ribonucleotide motif or tag; and (c) incorporating the single-stranded DNA sequence in place of the corresponding endogenous strand in the target nucleotide sequence by DNA repair and/or replication processes, the method producing a recombinant target nucleotide sequence encoding a modified RNA of interest comprising the ribonucleotide motif or tag.
203. The method of embodiment 202, wherein the ribonucleotide motif or tag is the detection moiety.
204. A 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 transport or subcellular location of the desired RNA.
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 PEG RNA is selected from the group consisting of SEQ ID NO:101-104, 181-183, 223-234, 237-244, 277, 324-330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 394, 429-442, 499-505, 641-649, 678- 692, 735-736, 757-761, 776-777, 2997-3103, 3113-3121, 3305-3455, 3479-3493, 3522-3540, 3549-3556, 3628-3698, 3755-3810, 3874, 3890-3901, 3905-3911, 3913-3929, 3972-3989including.
208. The method of embodiment 202, wherein the fusion protein comprises an amino acid sequence of PE1, PE2, or PE3.
209. The method of embodiment 202, wherein the napDNAbp is Cas9 nickase (nCas9).
210. The method of embodiment 202, wherein napDNAbp is selected from SEQ ID NOs: 18 to 19.88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.The amino acid sequence of
211. A method for inserting or deleting a functional portion on a protein of interest encoded by a target nucleotide sequence by prime editing, the method comprising: (a) contacting the target nucleotide sequence with (i) a prime editing factor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a reverse transcriptase and (ii) a PEGRNA comprising an editing template encoding the functional portion or deletion; (b) polymerizing a single-stranded DNA sequence encoding the functional portion or deletion; and (c) incorporating the single-stranded DNA sequence in place of the corresponding endogenous strand in the target nucleotide sequence by DNA repair and/or replication processes, the method producing a recombinant target nucleotide sequence encoding the protein of interest and an altered protein comprising the functional portion or deletion, and the functional portion altering the modification or localization state of the protein.
212. The method of embodiment 211, wherein the functional portion alters the phosphorylation, ubiquitination, glycosylation, lipid modification, hydroxylation, methylation, acetylation, crotonylation, or sumoylation status of the target protein.
Group 2. Aspects 213-424
213. A fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a polymerase.
214. A fusion protein of embodiment 213, wherein the fusion protein is capable of performing prime editing in the presence of a prime editing guide RNA (PEgRNA).
215. A fusion protein of embodiment 213, wherein napDNAbp has nickase activity.
216. The fusion protein of embodiment 213, wherein napDNAbp is a Cas9 protein or a variant thereof.
217. The fusion protein of embodiment 213, wherein napDNAbp is a nuclease-active Cas9, a nuclease-inactive Cas9 (dCas9), or a Cas9 nickase (nCas9).
218. The fusion protein of embodiment 213, wherein napDNAbp is Cas9 nickase (nCas9).
219. The fusion protein of embodiment 213, wherein napDNAbp is selected from the group consisting of: Cas9, Cas12e, Cas12d, Cas12a, Cas12b1, Cas13a, Cas12c, and Argonaute, and optionally has nickase activity.
220. A fusion protein of embodiment 213, wherein the fusion protein is capable of binding to a target DNA sequence when complexed with PEgRNA.
221. The fusion protein of embodiment 220, wherein the target DNA sequence comprises a target strand and a complementary non-target strand.
222. A fusion protein of embodiment 220, wherein binding of the fusion protein complexed with 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 a target strand and (ii) a complementary non-target strand.
224. A fusion protein of embodiment 223, wherein the complementary non-target strand is nicked to form a prime sequence having a free 3' end.
225. A fusion protein of embodiment 214, wherein the PEgRNA comprises (a) a guide RNA and (b) an extension arm at the 5' or 3' end of the guide RNA or positioned intramolecularly of the guide RNA.
226. A fusion protein of embodiment 225, wherein the extension arm comprises (i) a DNA synthesis template sequence containing 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-stranded DNA flap that is complementary to the endogenous DNA sequence adjacent to the nick site, and the single-stranded DNA flap contains 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. A fusion protein of embodiment 227, wherein a single-stranded DNA flap hybridizes to an endogenous DNA sequence adjacent to the nick site, thereby incorporating a desired nucleotide change.
230. The fusion protein of embodiment 227, wherein a single-stranded DNA flap has a free 5' end and replaces the endogenous DNA sequence adjacent to the nick site.
231. A fusion protein of embodiment 230, wherein an endogenous DNA sequence having a 5' end is excised by the cell.
232. A fusion protein of embodiment 230, wherein cellular repair of the single-stranded DNA flap results in incorporation of a desired nucleotide change, thereby forming a desired product.
233. The fusion protein of embodiment 226, wherein the desired nucleotide change is incorporated into an editing window that is between about -4 and +10 of the PAM sequence, or between about -10 and +20 of the PAM sequence, or between about -20 and +40 of the PAM sequence, or between about -30 and +100 of the PAM sequence, or the desired nucleotide change is incorporated into 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, 100, 101, 102, 103, The sequence is integrated 0, 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.
234. The fusion protein of embodiment 213, wherein napDNAbp is selected from the group consisting of SEQ ID NO:18or SEQ ID NO:18The amino acid sequence includes an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence of
235. The fusion protein of embodiment 213, wherein napDNAbp is selected from the group consisting of SEQ ID NO:18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.The amino acid sequence of any one of the above amino acid sequences has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence of any one of the above amino acid sequences.
236. The fusion protein of embodiment 213, wherein the polymerase is a reverse transcriptase and the sequence represented by 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 766The amino acid sequence of any one of the following:
237. The fusion protein of embodiment 213, wherein the polymerase is a reverse transcriptase and the sequence represented by 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 766The amino acid sequence of any one of the above amino acid sequences has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence of any one of the above amino acid sequences.
238. The fusion protein of embodiment 213, wherein the polymerase is a naturally occurring reverse transcriptase from a retrovirus or retrotransposon.
239. The fusion protein of any one of the preceding aspects, wherein the fusion protein has the structure NH₂-[napDNAbp]-[polymerase]-COOH; or NH₂It contains -[polymerase]-[napDNAbp]-COOH, with each "]-[" indicating the presence of an optional linker sequence.
240. The fusion protein of embodiment 239, wherein the linker sequence is selected from the group consisting of SEQ ID NO:127, 165-176, 446, 453, and 767-769The amino acid sequence of
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 insertion 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. A PEgRNA of embodiment 241, wherein the nucleic acid extension arm is at the 3' or 5' end of the guide RNA or at an intramolecular position of the guide RNA, and the nucleic acid extension arm is DNA or RNA.
245. A 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, and the guide RNA hybridizes to the target strand to form an RNA-DNA hybrid and an R-loop.
247. A PEgRNA of embodiment 243, wherein 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 toe loop, an aptamer, or an RNA-protein recruitment domain.
252. The PEgRNA of embodiment 247, wherein the DNA synthesis template encodes a single-stranded DNA flap that is complementary to the endogenous DNA sequence adjacent to the nick site, and the single-stranded DNA flap contains the desired nucleotide change.
253. A PEGRNA of embodiment 252, wherein the single-stranded DNA flap displaces endogenous single-stranded DNA having a 5' end on the nicked target DNA sequence, and the endogenous single-stranded DNA is immediately adjacent to and downstream of the nick site.
254. A PEGRNA of embodiment 253, wherein the endogenous single-stranded DNA having a free 5' end is excised by the cell.
255. A PEgRNA of embodiment 253, wherein cellular repair of the single-stranded DNA flap results in incorporation of a desired nucleotide change, thereby forming a desired product.
256. The PEG RNA of embodiment 243, comprising:101-104, 181-183, 223-234, 237-244, 277, 324-330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 394, 429-442, 499-505, 641-649, 678-692, 735-736, 757-761, 776-777or SEQ ID NO:101-104, 181-183, 223-234, 237-244, 277, 324-330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 394, 429-442, 499-505, 641-649, 678-692, 735-736, 757-761, 776-777or at least 90%, or at least 95%, or at least 98%, or at least 99% sequence identity with any one of the following:
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. A PEGRNA of embodiment 247, wherein the primer binding site hybridizes to the free 3' end of the cut DNA.
259. A PEgRNA of embodiment 251, wherein at least one additional structure is positioned at the 3' or 5' end of the PEgRNA.
260. A complex comprising any one of the fusion proteins of embodiments 213-242 and PEgRNA.
261. A complex of embodiment 260, 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 of the guide RNA.
262. A complex of embodiment 260, wherein the PEgRNA is capable of binding to the napDNAbp and directing the napDNAbp to a target DNA sequence.
263. A complex of embodiment 262, wherein the target DNA sequence comprises a target strand and a complementary non-target strand, and the guide RNA hybridizes to the target strand to form an RNA-DNA hybrid and an R-loop.
264. A complex of embodiment 261, wherein 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 is selected from the group consisting of SEQ ID NO:101-104, 181-183, 223-234, 237-244, 277, 324-330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 394, 429-442, 499-505, 641-649, 678-692, 735-736, 757-761, 776-777or SEQ ID NO:101-104, 181-183, 223-234, 237-244, 277, 324-330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 394, 429-442, 499-505, 641-649, 678-692, 735-736, 757-761, 776-777In one embodiment, the present invention comprises 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 the following:
266. A 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. A complex of embodiment 264, wherein the primer binding site hybridizes to the free 3' end of the cut DNA.
268. A complex containing napDNAbp and PEgRNA.
269. The complex of embodiment 268, wherein napDNAbp is Cas9 nickase.
270. The complex of embodiment 268, wherein napDNAbp is selected from the group consisting of SEQ ID NO:18The amino acid sequence of
271. The complex of embodiment 268, wherein napDNAbp is selected from the group consisting of SEQ ID NO:18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.The amino acid sequence of any one of the above amino acid sequences has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence of any one of the above amino acid sequences.
272. A 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 of the guide RNA.
273. A complex of embodiment 268, wherein the PEgRNA can guide the napDNAbp to a target DNA sequence.
274. A complex of embodiment 272, wherein the target DNA sequence comprises a target strand and a complementary non-target strand, and the spacer sequence of the PEgRNA hybridizes to the target strand to form an RNA-DNA hybrid and an R-loop.
275. A complex of embodiment 273, wherein the nucleic acid extension arm comprises (i) a DNA synthesis template and (ii) a primer binding site.
276. The conjugate of embodiment 269, wherein the PEGRNA is selected from the group consisting of SEQ ID NO:101-104, 181-183, 223-234, 237-244, 277, 324-330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 394, 429-442, 499-505, 641-649, 678- 692, 735-736, 757-761, 776-777, 2997-3103, 3113-3121, 3305-3455, 3479-3493, 3522-3540, 3549-3556, 3628-3698, 3755-3810, 3874, 3890-3901, 3905-3911, 3913-3929, 3972-3989or SEQ ID NO:101-104, 181-183, 223-234, 237-244, 277, 324-330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 394, 429-442, 499-505, 641-649, 678-692, 735-736, 757-761, 776-777In one embodiment, the present invention comprises 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 the following:
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. A complex of embodiment 276, wherein the primer binding site hybridizes to the 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 toe loop, an aptamer, or an RNA-protein recruitment domain.
280. A polynucleotide encoding a fusion protein of any one of embodiments 213 to 242.
281. A vector comprising a polynucleotide of embodiment 280.
282. A cell comprising a fusion protein of any of embodiments 213 to 242 and a PEgRNA bound to the napDNAbp of the fusion protein.
283. A cell comprising a complex of any one of embodiments 260 to 279.
284. A pharmaceutical composition comprising: (i) a fusion protein of any one of embodiments 213 to 242, a complex of embodiments 260 to 279, a polynucleotide of embodiment 68, or a vector of embodiment 69; and (ii) a pharma- ceutically acceptable excipient.
285. A pharmaceutical composition comprising: (i) a conjugate of any one of embodiments 260 to 279, (ii) a polymerase provided in trans; and (iii) a pharma- ceutically acceptable excipient.
286. A kit comprising: (i) a nucleic acid sequence encoding a fusion protein of any one of embodiments 213-242; and (ii) a nucleic acid construct comprising a promoter driving expression of the sequence of (i).
287. A method for incorporating a desired nucleotide change into a double-stranded DNA sequence, the method comprising:
(i) contacting a double-stranded DNA sequence with a complex comprising a fusion protein and a PEgRNA, wherein the fusion protein comprises napDNAbp and a polymerase, and the PEgRNA comprises a DNA synthesis template containing the desired nucleotide change and a primer binding site;
(ii) nicking the double-stranded DNA sequence, thereby generating a free single-stranded DNA having a 3' end;
(iii) hybridizing the 3' end of the free single-stranded 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-stranded DNA flap that is complementary to the DNA synthesis template and contains the desired nucleotide change;
(v) replacing the endogenous DNA strand adjacent to the cut site with a single-stranded DNA flap, thereby incorporating the desired nucleotide change into the double-stranded DNA sequence;
including.
288. The method of embodiment 287, wherein (v) the replacing step comprises: (i) hybridizing a single-stranded DNA flap to the endogenous DNA strand adjacent to the cut site to create a sequence mismatch; (ii) excising the endogenous DNA strand; and (iii) repairing the mismatch to form a desired product containing 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, deletion, or insertion.
290. The method of embodiment 289, wherein the single nucleotide substitution is a transition or 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 nucleotide change is to convert (1) a G:C base pair to a T:A base pair, (2) a G:C base pair to an A:T base pair, (3) a G:C base pair to a C:G base pair, (4) a T:A base pair to a G:C base pair, (5) a T:A base pair to an A:T base pair, (6) a T:A base pair to a C:G base pair, (7) a C:G base pair to a G:C base pair, (8) a C:G base pair to a T:A base pair, a C:G base pair to an A:T base pair, (10) an A:T base pair to a T:A base pair, (11) an A:T base pair to a G:C base pair, or (12) an A:T base pair to a C:G base pair.
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 single-gene 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; congenital pachyonychia; phenylketonuria; severe combined immunodeficiency; sickle cell disease; Smith-Lemli-Opitz syndrome; trinucleotide repeat disorders; prion diseases; 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 napDNAbp is a nuclease-inactive Cas9 (dCas9), a Cas9 nickase (nCas9), or a nuclease-active Cas9.
298. The method of embodiment 287, wherein the napDNAbp is selected from the group consisting of SEQ ID NO:18or SEQ ID NO:18The amino acid sequence includes an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence of
299. The method of embodiment 287, wherein the napDNAbp is selected from the group consisting of SEQ ID NO:18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.The amino acid sequence of any one of the above amino acid sequences has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence of any one of the above amino acid sequences.
300. The method of embodiment 287, wherein the polymerase is a reverse transcriptase and the sequence represented by 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 766The amino acid sequence of any one of the following:
301. The method of embodiment 287, wherein the polymerase is a reverse transcriptase and the sequence represented by 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 766The amino acid sequence of any one of the above amino acid sequences has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence of any one of the above amino acid sequences.
302. The method of embodiment 287, wherein the PEgRNA comprises a nucleic acid extension arm at the 3' or 5' end of the guide RNA or positioned within the molecule, the extension arm comprising a DNA synthesis template sequence and a 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 PEG RNA is selected from the group consisting of SEQ ID NO:101-104, 181-183, 223-234, 237-244, 277, 324-330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 394, 429-442, 499-505, 641-649, 678-692, 735-736, 757-761, 776-777The nucleic acid has a nucleotide sequence selected from the group consisting of:
305. A method for introducing one or more changes in the nucleotide sequence of a DNA molecule at a target locus, comprising:
(i) contacting a DNA molecule with a nucleic acid programmable DNA binding protein (napDNAbp) and a PEgRNA that targets the napDNAbp to a target locus, the PEgRNA comprising a reverse transcriptase (RT) template sequence containing at least one desired nucleotide change and a primer binding site;
(ii) forming an exposed 3' end of the DNA strand at the target locus;
(iii) hybridizing the exposed 3' end to a primer binding site to prime reverse transcription;
(iv) synthesizing, by reverse transcriptase, a single-stranded DNA flap containing at least one desired nucleotide change based on the RT template sequence; and
(v) incorporating 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;
including.
306. The method of embodiment 305, wherein one or more changes in the nucleotide sequence include 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 one or more changes in the nucleotide sequence include 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 include changing (1) a G:C base pair to a T:A base pair, (2) a G:C base pair to an A:T base pair, (3) a G:C base pair to a C:G base pair, (4) a T:A base pair to a G:C base pair, (5) a T:A base pair to an A:T base pair, (6) a T:A base pair to a C:G base pair, (7) a C:G base pair to a G:C base pair, (8) a C:G base pair to a T:A base pair, (9) a C:G base pair to an A:T base pair, (9) an A:T base pair to a T:A base pair, (11) an A:T base pair to a G:C base pair, or (12) an A:T base pair to a C:G base pair.
311. The method of embodiment 305, wherein the one or more changes in the nucleotide sequence include 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 one or more changes in the nucleotide sequence include a correction of a disease-associated gene.
313. The method of embodiment 312, wherein the disease-associated gene is associated with a single-gene 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; phenylketonuria; severe combined immunodeficiency; sickle cell disease; Smith-Lemli-Opitz syndrome; trinucleotide repeat disorders; prion diseases; 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 napDNAbp is a nuclease-active Cas9 or a variant thereof.
316. The method of embodiment 305, wherein napDNAbp is a nuclease-inactivated 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 to SEQ ID NO:2.
318. The method of embodiment 305, wherein napDNAbp is selected from the group consisting of SEQ ID NO:18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.The amino acid sequence of any one of the above amino acid sequences has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence of any one of the above amino acid sequences.
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 is selected from the group consisting 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 766The amino acid sequence of any one of the following:
322. The method of embodiment 305, wherein the reverse transcriptase is selected from the group consisting 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 766The amino acid sequence of any one of the above amino acid sequences has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence of any one of the above amino acid sequences.
323. The method of embodiment 305, wherein the step of forming an exposed 3' end of the DNA strand at the target locus includes nicking the DNA strand with a nuclease.
324. The method of embodiment 323, wherein the nuclease is provided in trans.
325. In the method of embodiment 305, the step of forming an exposed 3' end of the DNA strand at the target locus includes contacting the DNA strand with a chemical agent.
326. The method of embodiment 305, wherein the step of forming an exposed 3' end of the DNA strand at the target locus includes introducing a replication error.
327. The method of embodiment 305, wherein contacting the DNA molecule with napDNAbp and the guide RNA forms an R-loop.
328. A method of embodiment 327, wherein the DNA strand on which the exposed 3' end is formed is on an R loop.
329. The method of embodiment 315, wherein the PEgRNA comprises an extension arm comprising a reverse transcriptase (RT) template sequence and a 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 of 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 toe loop, 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 a nucleotide sequence of a DNA molecule at a target locus by primed target reverse transcription, the method comprising: (a) contacting the DNA molecule at the target locus with (i) a fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a reverse transcriptase, and (ii) a guide RNA comprising an RT template containing a desired nucleotide change; (b) performing primed target reverse transcription of the RT template to generate a single-stranded DNA containing the desired nucleotide change; and (c) incorporating the desired nucleotide change on the DNA molecule at the target locus by DNA repair and/or replication processes.
335. The method of embodiment 334, wherein the RT template is positioned at the 3' end of the guide RNA, the 5' end of the guide RNA, or at an intramolecular position of 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 claim 334, wherein the desired nucleotide change comprises changing (1) a G:C base pair to a T:A base pair, (2) a G:C base pair to an A:T base pair, (3) a G:C base pair to a C:G base pair, (4) a T:A base pair to a G:C base pair, (5) a T:A base pair to an A:T base pair, (6) a T:A base pair to a C:G base pair, (7) a C:G base pair to a G:C base pair, (8) a C:G base pair to a T:A base pair, (9) a C:G base pair to an A:T base pair, (10) an A:T base pair to a T:A base pair, (11) an A:T base pair to a G:C base pair, or (12) an A:T base pair to a C:G base pair.
340. A polynucleotide encoding a PEgRNA of any one of embodiments 243 to 259.
341. A vector comprising a polynucleotide of embodiment 340.
342. A cell comprising a 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 primed target reverse transcription, the method comprising: (a) contacting the DNA molecule at the target locus with (i) a 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 containing a desired nucleotide change; (b) performing primed target reverse transcription of the RT template to generate a mutated single-stranded DNA; and (c) incorporating the mutated single-stranded DNA onto the DNA molecule at the target locus by DNA repair and/or replication processes.
345. A method of any preceding embodiment, wherein the fusion protein comprises an amino acid sequence of PE1, PE2, or PE3.
346. A method of any preceding embodiment, wherein the napDNAbp is Cas9 nickase (nCas9).
347. The method of embodiment 344, wherein napDNAbp is selected from SEQ ID NOs: 18 to 19.88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.The amino acid sequence of
348. The method of embodiment 344, wherein the guide RNA is SEQ ID NO:222including.
349. The method of embodiment 344, wherein (b) performing primed reverse transcription on a prime target comprises generating a 3' end primer binding sequence at the target locus that can prime reverse transcription by annealing to a primer binding site on the guide RNA.
350. A method for replacing a trinucleotide repeat expansion mutation on a target DNA molecule with a healthy sequence containing a healthy number of repeated trinucleotides, the method comprising: (a) contacting a DNA molecule at a target locus with (i) a fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a polymerase and (ii) a DNA synthesis template comprising the replacement sequence and a PEgRNA comprising a primer binding site; (b) performing prime editing to generate a single-stranded DNA comprising the replacement sequence; and (c) incorporating the single-stranded DNA onto the DNA molecule at the target locus by DNA repair and/or replication processes.
351. The method of embodiment 350, wherein the fusion protein comprises an amino acid sequence of PE1, PE2, or PE3.
352. The method of embodiment 350, wherein napDNAbp is Cas9 nickase (nCas9).
353. The method of embodiment 350, wherein napDNAbp is selected from SEQ ID NOs: 18 to 19.88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.The amino acid sequence of
354. The method of embodiment 350, wherein the guide RNA is222including.
355. The method of embodiment 350, wherein (b) the step of performing prime editing includes generating a 3' end primer binding sequence at the target locus that can prime a polymerase by annealing to a 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 repeat unit of a CAG triplet.
358. The method of embodiment 350, wherein the trinucleotide repeat expansion mutation comprises a repeat unit of a GAA triplet.
359. A method for incorporating a functional moiety onto a protein of interest encoded by a target nucleotide sequence by prime editing, the method comprising: (a) contacting the target nucleotide sequence with (i) a prime editing factor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a polymerase, and (ii) a PEgRNA comprising a DNA synthesis template encoding the functional moiety; (b) polymerizing a single-stranded DNA sequence encoding the functional moiety; and (c) incorporating the single-stranded DNA sequence in place of the corresponding endogenous strand in the target nucleotide sequence by DNA repair and/or replication processes, wherein the method produces a recombinant target nucleotide sequence encoding a fusion protein comprising the protein of interest and the functional moiety.
360. A method of embodiment 359, wherein the functional portion is a peptide tag.
361. The method of embodiment 360, wherein the peptide tag is an affinity tag, a solubilization tag, a chromatography tag, an epitope or immunoepitope tag, or a fluorescent tag.
362. The method of embodiment 360, wherein the peptide tag is: AviTag (SEQ ID NO: 245); C-tag (SEQ ID NO: 246); calmodulin tag (SEQ ID NO: 247); polyglutamic acid tag (SEQ ID NO: 248); E-tag (SEQ ID NO: 249); FLAG tag (SEQ ID NO: 250);2); HA tag (SEQ ID NO:Five); His tag (SEQ ID NO: 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).
363. The method of embodiment 360, wherein the peptide tag is: 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); polyaspartic acid (Asp tag) (SEQ ID NO: 286); polyphenylalanine (Phe tag) (SEQ ID NO: 287); S1 tag (SEQ ID NO: 288); S tag (SEQ ID NO: 266); and VSV-tag(SEQ ID NO:275).
364. The method of embodiment 359, wherein the functional portion is an immune epitope.
365. The method of embodiment 364, wherein the immune epitope is selected from the group consisting of: tetanus toxoid (SEQ ID NO: 396); diphtheria toxin variant CRM197 (SEQ ID NO: 398); mumps immune epitope 1 (SEQ ID NO: 400); mumps immune epitope 2 (SEQ ID NO: 402); mumps immune epitope 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 epitope for class I HLA (SEQ ID NO: 416); neuraminidase epitope for class I HLA (SEQ ID NO: 418); hemagglutinin epitope for class II HLA (SEQ ID NO: 420); neuraminidase epitope for class II HLA (SEQ ID NO: 422); hemagglutinin epitope H5N1 binding class I and class II HLA (SEQ ID NO: 424); neuraminidase epitope H5N1-binding class I and class II HLA (SEQ ID NO: 426).
366. A method of embodiment 359, wherein the functional moiety alters the localization of a protein of interest.
367. The method of embodiment 359, wherein the functional moiety is a degradation tag, thereby altering the degradation rate of the target protein.
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 portion is a small molecule binding domain.
370. The method of embodiment 359, wherein the small molecule binding domain is FKBP12 of sequence number 488.
371. The method of embodiment 359, wherein the small molecule binding domain is FKBP12-F36V of sequence number 489.
372. The method of embodiment 359, wherein the small molecule binding domain is SEQ ID NO: 490 and 493~494 cyclophilin.
373. The method of embodiment 359, wherein small molecule binding domains are incorporated onto two or more proteins of interest.
374. The method of embodiment 373, wherein two or more proteins of interest can be dimerized by contacting them with a small molecule.
375. The method of embodiment 369, wherein the small molecule is:

It is a dimer of a small molecule selected from the group consisting of:
376. A method for incorporating an immune epitope into a protein of interest encoded by a target nucleotide sequence by prime editing, the method comprising: (a) contacting the target nucleotide sequence with (i) a prime editing factor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a polymerase and (ii) a PEgRNA comprising an editing template encoding a functional moiety; (b) polymerizing a single-stranded DNA sequence encoding the immune epitope; and (c) incorporating the single-stranded DNA sequence in place of the corresponding endogenous strand in the target nucleotide sequence by DNA repair and/or replication processes, the method producing a recombinant target nucleotide sequence encoding a fusion protein comprising the protein of interest and the immune epitope.
377. The method of embodiment 376, wherein the immune epitope is selected from the group consisting of: tetanus toxoid (SEQ ID NO: 396); diphtheria toxin variant CRM197 (SEQ ID NO: 398); mumps immune epitope 1 (SEQ ID NO: 400); mumps immune epitope 2 (SEQ ID NO: 402); mumps immune epitope 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 epitope for class I HLA (SEQ ID NO: 416); neuraminidase epitope for class I HLA (SEQ ID NO: 418); hemagglutinin epitope for class II HLA (SEQ ID NO: 420); neuraminidase epitope for class II HLA (SEQ ID NO: 422); hemagglutinin epitope H5N1 binding class I and class II HLA (SEQ ID NO: 424); neuraminidase epitope H5N1-binding class I and class II HLA (SEQ ID NO: 426).
378. The method of embodiment 376, wherein the fusion protein comprises an amino acid sequence of PE1, PE2, or PE3.
379. The method of embodiment 376, wherein napDNAbp is Cas9 nickase (nCas9).
380. The method of embodiment 376, wherein napDNAbp is selected from SEQ ID NOs: 18 to 19.88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.The amino acid sequence of
381. The method of embodiment 376, wherein the PEG RNA is selected from the group consisting of SEQ ID NO:101-104, 181-183, 223-234, 237-244, 277, 324-330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 394, 429-442, 499-505, 641-649, 678-692, 735-736, 757-761, 776-777including.
382. A method for incorporating a small dimerization domain onto a protein of interest encoded by a target nucleotide sequence by prime editing, the method comprising: (a) contacting the target nucleotide sequence with (i) a prime editing factor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a polymerase, and (ii) a PEgRNA comprising an editing template encoding the small dimerization domain; (b) polymerizing a single-stranded DNA sequence encoding an immune epitope; and (c) incorporating the single-stranded DNA sequence in place of the corresponding endogenous strand in the target nucleotide sequence by DNA repair and/or replication processes, wherein the method produces a recombinant target nucleotide sequence encoding a fusion protein comprising the protein of interest and the small dimerization domain.
383. The method of embodiment 382, further comprising performing 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 domains of each of the proteins.
385. The method of embodiment 382, wherein the small molecule binding domain is FKBP12 of sequence number 488.
386. The method of embodiment 382, wherein the small molecule binding domain is FKBP12-F36V of sequence number 489.
387. The method of embodiment 382, wherein the small molecule binding domain is selected from the group consisting of SEQ ID NO: 490 and 493~494 cyclophilin.
388. The method of embodiment 382, wherein the small molecule is:

It is a dimer of a small molecule selected from the group consisting of:
389. The method of embodiment 382, wherein the fusion protein comprises an amino acid sequence of PE1, PE2, or PE3.
390. The method of embodiment 382, wherein napDNAbp is Cas9 nickase (nCas9).
391. The method of embodiment 382, wherein napDNAbp is selected from SEQ ID NOs: 18 to 19.88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.The amino acid sequence of
392. The method of embodiment 382, wherein the PEG RNA is selected from the group consisting of SEQ ID NO:101-104, 181-183, 223-234, 237-244, 277, 324-330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 394, 429-442, 499-505, 641-649, 678-692, 735-736, 757-761, 776-777including.
393. A method for incorporating a peptide tag or epitope onto a protein using prime editing, comprising: contacting a target nucleotide sequence encoding a protein with a prime editor construct configured to insert therein a second nucleotide sequence encoding a peptide tag, resulting 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 a peptide tag is used for purifying and/or detecting the protein.
395. The method of embodiment 383, wherein the peptide tag is a polyhistidine (e.g., HHHHHH(SEQ ID NO: 257)), FLAG (e.g., DYKDDDDK(SEQ ID NO:2)), V5 (for example, GKPIPNPLLGLDST(SEQ ID NO:3)), GCN4, HA (e.g., YPYDVPDYA(SEQ ID NO:5)), Myc (e.g., EQKLISEED(SEQ ID NO:6)), 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 NOs: 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], where "]-[" represents an optional linker.
399. The method of embodiment 383, wherein the linker is SEQ ID NO:127, 165-176, 446, 453, and 767-769It has the amino acid sequence:
400. The method of embodiment 383, wherein the prime editor construct comprises a sequence represented by SEQ ID NO:101-104, 181-183, 223-234, 237-244, 277, 324-330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 394, 429-442, 499-505, 641-649, 678-692, 735-736, 757-761, 776-777The pEGRNA comprises a nucleotide sequence of
401. The method of embodiment 383, wherein the PEGRNA comprises a spacer, a gRNA core, and an extension arm, the spacer being complementary to a target nucleotide sequence, and the extension arm comprising a reverse transcriptase template encoding a peptide tag.
402. The method of embodiment 383, wherein the PEGRNA comprises a spacer, a gRNA core, and an extension arm, the spacer being complementary to a target nucleotide sequence, and the extension arm comprising a reverse transcriptase template encoding a peptide tag.
403. A method for preventing or halting the progression of a prion disease by incorporating one or more protective mutations into a PRNP encoded by a target nucleotide sequence by prime editing, the method comprising: (a) contacting the target nucleotide sequence with (i) a prime editing factor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a polymerase, and (ii) a PEgRNA comprising an editing template encoding a functional moiety; (b) polymerizing a single-stranded DNA sequence encoding the protective mutations; and (c) incorporating, by DNA repair and/or replication processes, the single-stranded DNA sequence in place of the corresponding endogenous strand in the target nucleotide sequence, wherein the method produces a recombinant target nucleotide sequence encoding a PRNP containing protective mutations that are 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 wild-type PRNP amino acid sequence is SEQ ID NO: 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, and the modified PRNP protein is resistant to misfolding.
410. The method of embodiment 403, wherein the fusion protein comprises an amino acid sequence of PE1, PE2, or PE3.
411. The method of embodiment 403, wherein the napDNAbp is Cas9 nickase (nCas9).
412. The method of embodiment 403, wherein napDNAbp is selected from SEQ ID NOs: 18 to 19.88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.The amino acid sequence of
413The method of embodiment 403, wherein the PEG RNA has the sequence represented by SEQ ID NO:101-104, 181-183, 223-234, 237-244, 277, 324-330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 394, 429-442, 499-505, 641-649, 678-692, 735-736, 757-761, 776-777including.
414. A method for incorporating a ribonucleotide motif or tag onto a desired RNA encoded by a target nucleotide sequence by prime editing, the method comprising: (a) contacting the target nucleotide sequence with (i) a prime editing factor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a polymerase and (ii) a PEgRNA comprising an editing template encoding the ribonucleotide motif or tag; (b) polymerizing a single-stranded DNA sequence encoding the ribonucleotide motif or tag; and (c) incorporating the single-stranded DNA sequence in place of the corresponding endogenous strand in the target nucleotide sequence by DNA repair and/or replication processes, wherein the method produces a recombinant target nucleotide sequence encoding a modified RNA of interest that includes the ribonucleotide motif or tag.
415. The method of embodiment 414, wherein the ribonucleotide motif or tag is the 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 transport or subcellular location of the desired RNA.
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 PEG RNA is selected from the group consisting of SEQ ID NO:101-104, 181-183, 223-234, 237-244, 277, 324-330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 394, 429-442, 499-505, 641-649, 678-692, 735-736, 757-761, 776-777including.
420. The method of embodiment 414, wherein the fusion protein comprises an amino acid sequence of PE1, PE2, or PE3.
421. The method of embodiment 414, wherein napDNAbp is Cas9 nickase (nCas9).
422. The method of embodiment 414, wherein napDNAbp is selected from SEQ ID NOs: 18 to 19.88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.The amino acid sequence of
423. A method for introducing or deleting a functional portion on a protein of interest encoded by a target nucleotide sequence by prime editing, the method comprising: (a) contacting the target nucleotide sequence with (i) a prime editing factor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a polymerase and (ii) a PEgRNA comprising an editing template encoding the functional portion or deletion; (b) polymerizing a single-stranded DNA sequence encoding the functional portion or deletion; and (c) incorporating the single-stranded DNA sequence in place of the corresponding endogenous strand in the target nucleotide sequence by DNA repair and/or replication processes, wherein the method produces a recombinant target nucleotide sequence encoding the protein of interest and an altered protein comprising the functional portion or deletion, and wherein the functional portion alters the modification or localization state of the protein.
424. The method of embodiment 423, wherein the functional moiety alters the phosphorylation, ubiquitination, glycosylation, lipid modification, hydroxylation, methylation, acetylation, crotonylation, or sumoylation status 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.

Aspect 2. The fusion protein of aspect 1, wherein the fusion protein is capable of performing genome editing by primed reverse transcription to a primed target in the presence of an extended guide RNA.

Aspect 3. The fusion protein of aspect 1, wherein napDNAbp has nickase activity.

Embodiment 4. The fusion protein of embodiment 1, wherein napDNAbp is a Cas9 protein or a variant thereof.

Embodiment 5. The fusion protein of embodiment 1, wherein 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 napDNAbp is selected from the group consisting of: Cas9, CasX, CasY, Cpf1, C2c1, C2c2, C2C3, and Argonaute, and optionally has nickase activity.

Embodiment 8. The fusion protein of embodiment 1, wherein the fusion protein is capable of binding to a target DNA sequence when complexed with an extended guide RNA.

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 binding of the fusion protein complexed with 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 an extended guide RNA and a target strand, and (ii) a 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 prime 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 3' end of the guide RNA or at an intramolecular location of the guide RNA.

Embodiment 14. The fusion protein of embodiment 13, wherein the RNA extension comprises (i) a reverse transcription template sequence containing 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-stranded DNA flap that is complementary to the endogenous DNA sequence adjacent to the nick site, and the single-stranded DNA flap contains 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-stranded DNA flap hybridizes to the endogenous DNA sequence adjacent to the nick site, thereby incorporating the desired nucleotide change.

Embodiment 18. The fusion protein of embodiment 15, wherein the single-stranded DNA flap has a free 5' end and replaces the endogenous DNA sequence adjacent to the nick site.

Aspect 19. The fusion protein of aspect 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-stranded DNA flap results in incorporation of a desired nucleotide change, thereby forming a desired product.

21. The fusion protein of embodiment 14, wherein the desired nucleotide change is incorporated into an editing window that is between about -4 and +10 of the PAM sequence, or between about -10 and +20 of the PAM sequence, or between about -20 and +40 of the PAM sequence, or between about -30 and +100 of the PAM sequence, or the desired nucleotide change is incorporated into 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 of the nick site. , 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.

Embodiment 22. The fusion protein of embodiment 1, wherein 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 of SEQ ID NO:18.

Embodiment 23. The fusion protein of embodiment 1, wherein napDNAbp comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to any one of the amino acid sequences of SEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.

The fusion protein of embodiment 1, wherein the reverse transcriptase comprises any one of the amino acid sequences in 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 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.

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 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, 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 retrotransposon.

Embodiment 27. The fusion protein of any one of the preceding embodiments, wherein the fusion protein comprises the structure NH2-[napDNAbp]-[reverse transcriptase]-COOH; or NH2-[reverse transcriptase]-[napDNAbp]-COOH, each of the "]-[" indicates the presence of an optional linker sequence.

Aspect 28. The fusion protein of aspect 27, wherein the linker sequence comprises the 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 insertion 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 at the 3' or 5' end of the guide RNA or at an intramolecular position of the guide RNA.

Embodiment 33. The extended guide RNA of embodiment 31, wherein the extended guide RNA is capable of binding to the napDNAbp and guiding 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, and 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 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-stranded DNA flap that is complementary to the endogenous DNA sequence adjacent to the nick site, and the single-stranded DNA flap includes a desired nucleotide change.

Embodiment 41. The extended guide RNA of embodiment 40, wherein the single-stranded DNA flap displaces the endogenous single-stranded DNA having a 5' end on the nicked target DNA sequence, and the endogenous single-stranded DNA is immediately adjacent to and downstream of the nick site.

Aspect 42. The extended guide RNA of aspect 41, wherein the endogenous single-stranded DNA having a free 5' end is excised by the cell.

Embodiment 43. The extended guide RNA of embodiment 41, wherein cellular repair of the single-stranded DNA flap results in incorporation of the desired nucleotide change, thereby forming the desired product.

Aspect 44. 32. The extended guide RNA of embodiment 31, comprising a 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 that has at least 85% or at least 90% or at least 95% or at least 98% or at least 99% sequence identity to 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%, 85%, 90%, 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 to the 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.

Aspect 48. A complex comprising a fusion protein of any one of aspects 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 of the guide RNA.

Embodiment 50. The complex of embodiment 48, wherein the extended guide RNA is capable of binding to the napDNAbp and guiding 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, and 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 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 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, or SEQ ID NOs: 394, 429-442, 641-649, The nucleotide sequence has at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with any one of 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%, 85%, 90%, 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 to the free 3' end of the cut DNA.

Embodiment 56. A complex comprising napDNAbp and an extended guide RNA.

Aspect 57. The complex of aspect 56, wherein napDNAbp is a Cas9 nickase.

Aspect 58. The complex of aspect 56, wherein 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 to SEQ ID NO:18.

Embodiment 59. The complex of embodiment 57, wherein napDNAbp comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any one of the amino acid sequences 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 of 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, and 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 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, or SEQ ID NOs: 394, 429-442, 641-649, The nucleotide sequence has at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with any one of 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%, 85%, 90%, 95%, or 99% identical to the endogenous DNA target.

Embodiment 66. The complex of embodiment 63, wherein the reverse transcription primer binding site hybridizes to the free 3' end of the cut DNA.

Embodiment 67. The conjugate 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.

Aspect 68. A polynucleotide encoding a fusion protein according to any one of aspects 1 to 30.

Aspect 69. A vector comprising the polynucleotide of aspect 68.

Embodiment 70. A cell comprising the fusion protein of any one of embodiments 1 to 30 and an extended guide RNA bound to the napDNAbp of the fusion protein.

Aspect 71. A cell comprising a complex according to any one of aspects 48 to 67.

Aspect 72. A pharmaceutical composition comprising: (i) a fusion protein of any one of aspects 1 to 30, a complex of aspects 48 to 67, a polynucleotide of aspect 68, or a vector of aspect 69; and (ii) a pharma- ceutically acceptable excipient.

Embodiment 73. A pharmaceutical composition comprising: (i) a conjugate of any one of embodiments 48 to 67; (ii) a reverse transcriptase provided in trans; and (iii) a pharma- ceutically acceptable excipient.

Embodiment 74. A kit comprising: (i) a nucleic acid sequence encoding a fusion protein of any one of embodiments 1 to 30; and (ii) a nucleic acid construct comprising a promoter driving expression of the sequence of (i).

Embodiment 75. A method for incorporating a desired nucleotide change into a double-stranded DNA sequence, the method comprising:

(i) contacting a double-stranded DNA sequence with a complex comprising a fusion protein and an extended guide RNA, the fusion protein comprising napDNAbp and a reverse transcriptase, and the extended guide RNA comprising a reverse transcription template sequence comprising the desired nucleotide change;

(ii) nicking a double-stranded DNA sequence on the non-target strand, thereby generating a free single-stranded DNA having a 3' end;

(iii) hybridizing the 3' end of the free single-stranded 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-stranded DNA flap containing the desired nucleotide change;

(v) replacing the endogenous DNA strand adjacent to the cut site with a single-stranded DNA flap, thereby incorporating the desired nucleotide change into the double-stranded DNA sequence;
including.

Embodiment 76. The method of embodiment 75, wherein (v) the replacing step comprises: (i) hybridizing a single-stranded DNA flap to the endogenous DNA strand adjacent to the cut site to generate a sequence mismatch; (ii) excising the endogenous DNA strand; and (iii) repairing the mismatch to form a desired product containing 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, deletion, or 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 is (1) a G:C base pair to a T:A base pair, (2) a G:C base pair to an A:T base pair, (3) a G:C base pair to a C:G base pair, (4) a T:A base pair to a G:C base pair, (5) a T:A base pair to an A:T base pair, (6) a T:A base pair to a C:G base pair, (7) a C:G base pair to a G:C base pair, (8) a C:G base pair to a T:A base pair, (9) a C:G base pair to an A:T base pair, (10) an A:T base pair to a T:A base pair, (11) an A:T base pair to a G:C base pair, or (12) a A:T base pair to a C:G base pair.

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 single-gene 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; phenylketonuria; 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-inactive Cas9 (dCas9), a Cas9 nickase (nCas9), or a nuclease-active Cas9.

Aspect 86. The method of aspect 76, wherein napDNAbp comprises the 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 to any one of the amino acid sequences 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 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 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 to 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, 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' end of the guide RNA or positioned intramolecularly, the RNA extension comprising a 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 a DNA molecule with a nucleic acid programmable DNA binding protein (napDNAbp) and a guide RNA that targets the napDNAbp to a target locus, the guide RNA comprising a reverse transcriptase (RT) template sequence that contains at least one desired nucleotide change;

(ii) creating an exposed 3' end of the DNA strand at the target locus;

(iii) hybridization of the exposed 3' end to the RT template sequence to prime reverse transcription;

(iv) synthesizing a single-stranded DNA flap containing at least one desired nucleotide change based on the RT template sequence by reverse transcriptase; and

(v) incorporating 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;
including.

Embodiment 94. The method of embodiment 93, wherein the one or more changes in the nucleotide sequence include 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 include 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 include changing (1) a G:C base pair to a T:A base pair, (2) a G:C base pair to an A:T base pair, (3) a G:C base pair to a C:G base pair, (4) a T:A base pair to a G:C base pair, (5) a T:A base pair to an A:T base pair, (6) a T:A base pair to a C:G base pair, (7) a C:G base pair to a G:C base pair, (8) a C:G base pair to a T:A base pair, (9) a C:G base pair to an A:T base pair, (10) an A:T base pair to a T:A base pair, (11) an A:T base pair to a G:C base pair, or (12) an A:T base pair to a C:G base pair.

Embodiment 99. The method of embodiment 93, wherein the one or more changes in the nucleotide sequence include 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 include correction of a disease-associated gene.

Embodiment 101. The method of embodiment 100, wherein the disease-associated gene is associated with a single-gene 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; phenylketonuria; 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; hypertension; Alzheimer's disease; arthritis; diabetes; cancer; and obesity.

Embodiment 103. The method of embodiment 93, wherein napDNAbp is a nuclease-active Cas9 or a variant thereof.

Embodiment 104. The method of embodiment 93, wherein napDNAbp is a nuclease-inactivated Cas9 (dCas9) or Cas9 nickase (nCas9) or a variant thereof.

Aspect 105. The method of aspect 93, wherein napDNAbp comprises the 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 to any one of the amino acid sequences 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 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 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 to 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, 701-716, 739-741, and 766.

Embodiment 111. The method of embodiment 93, wherein the step of forming an exposed 3' end of 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 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 of 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 of the DNA strand at the target locus includes introducing a replication error.

Embodiment 115. The method of embodiment 93, wherein contacting the DNA molecule with napDNAbp and the guide RNA forms an R-loop.

Embodiment 116. The method of embodiment 115, wherein the DNA strand on which the exposed 3' end is formed is on an R-loop.

Embodiment 117. The method of embodiment 93, wherein the guide RNA comprises an extended portion that includes a 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 location of 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 a nucleotide sequence of a DNA molecule at a target locus by primed reverse transcription, the method comprising: (a) contacting the DNA molecule at the target locus with (i) a 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) performing primed reverse transcription of the RT template to generate a single-stranded DNA comprising the desired nucleotide change; and (c) incorporating the desired nucleotide change on the DNA molecule at the target locus by DNA repair and/or replication processes.

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 of 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 base pair to a T:A base pair, (2) a G:C base pair to an A:T base pair, (3) a G:C base pair to a C:G base pair, (4) a T:A base pair to a G:C base pair, (5) a T:A base pair to an A:T base pair, (6) a T:A base pair to a C:G base pair, (7) a C:G base pair to a G:C base pair, (8) a C:G base pair to a T:A base pair, (9) a C:G base pair to an A:T base pair, (10) an A:T base pair to a T:A base pair, (11) an A:T base pair to a G:C base pair, or (12) an A:T base pair to a C:G base pair.

Embodiment 128. A polynucleotide encoding an extended guide RNA according to any one of embodiments 31 to 47.

Aspect 129. A vector comprising the polynucleotide of aspect 128.

Aspect 130. A cell comprising the vector of aspect 129.

Embodiment 131. The fusion protein of any one of embodiments 1 to 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 primed reverse transcription, the method comprising: (a) contacting the DNA molecule at the target locus with (i) a 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) performing primed reverse transcription of the RT template to generate a mutated single-stranded DNA; and (c) incorporating the mutated single-stranded DNA onto the DNA molecule at the target locus by DNA repair and/or replication processes.

Embodiment 133. The method of embodiment 132, wherein the fusion protein comprises an amino acid sequence of PE1, PE2, or PE3.

Embodiment 134. The method of embodiment 132, wherein the napDNAbp is 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.

Aspect 136. The method of aspect 132, wherein the guide RNA comprises SEQ ID NO: 222.

Embodiment 137. The method of embodiment 132, wherein (b) performing primed reverse transcription on the primed target comprises generating a 3' end primer binding sequence at the target locus 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 on a target DNA molecule with a healthy sequence containing a healthy number of repeated trinucleotides, the method comprising: (a) contacting a DNA molecule at a target locus with (i) a fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a reverse transcriptase and (ii) a guide RNA comprising an RT template containing a replacement sequence, the fusion protein introducing (intr); (b) performing a primed reverse transcription on the primed target of the RT template to generate a single-stranded DNA containing the replacement sequence; and (c) incorporating the single-stranded DNA on the DNA molecule at the target locus by DNA repair and/or replication processes.

Embodiment 139. The method of embodiment 138, wherein the fusion protein comprises an amino acid sequence of PE1, PE2, or PE3.

Embodiment 140. The method of embodiment 138, wherein the napDNAbp is 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 NO: 222.

Embodiment 143. The method of embodiment 138, wherein (b) performing primed reverse transcription on the primed target comprises generating a 3' end primer binding sequence at the target locus 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 repeat unit of a CAG triplet.

Embodiment 146. The method of embodiment 138, wherein the trinucleotide repeat expansion mutation comprises a repeat unit of a GAA triplet.

Embodiment 147. A method for incorporating a functional moiety on a protein of interest encoded by a target nucleotide sequence by prime editing, the method comprising: (a) contacting the target nucleotide sequence with (i) a prime editing factor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a reverse transcriptase, and (ii) a PEgRNA comprising an editing template encoding the functional moiety; (b) polymerizing a single-stranded DNA sequence encoding the functional moiety; and (c) incorporating the single-stranded DNA sequence in place of the corresponding endogenous strand in the target nucleotide sequence by DNA repair and/or replication processes, the method producing a recombinant target nucleotide sequence encoding a fusion protein comprising the protein of interest and the functional moiety.

Aspect 148. The method of aspect 147, wherein the functional moiety is a peptide tag.

Embodiment 149. The method of embodiment 148, wherein the peptide tag is an affinity tag, a solubilization tag, a chromatography tag, an epitope tag, or a fluorescent 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); Polyglutamic acid 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 NO: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); polyaspartic acid (Asp tag) (SEQ ID NO:286); polyphenylalanine (Phe tag) (SEQ ID NO:287); S1 tag (SEQ ID NO:288); S tag (SEQ ID NO:266); and VSV-G (SEQ ID NO:275).

Aspect 152. The method of aspect 147, wherein the functional moiety is an immune epitope.

Embodiment 153. The method of embodiment 152, wherein the immune epitope is selected from the group consisting of: tetanus toxoid (SEQ ID NO: 396); diphtheria toxin variant CRM197 (SEQ ID NO: 398); mumps immune epitope 1 (SEQ ID NO: 400); mumps immune epitope 2 (SEQ ID NO: 402); mumps immune epitope 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 epitope for class I HLA (SEQ ID NO: 416); neuraminidase epitope for class I HLA (SEQ ID NO: 418); hemagglutinin epitope for class II HLA (SEQ ID NO: 420); neuraminidase epitope for class II HLA (SEQ ID NO: 422); hemagglutinin epitope H5N1 binding class I and class II HLA (SEQ ID NO: 424); Selected from the group consisting of neuraminidase epitope H5N1-binding 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, thereby altering the degradation rate of the protein of interest.

Embodiment 156. The method of embodiment 155, wherein the degradation tag comprises an amino acid sequence encoding a degradation tag 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.

Aspect 159. The method of aspect 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 a cyclophilin of SEQ ID NOs : 490 and 493-494.

Embodiment 161. The method of embodiment 157, wherein the small molecule binding domains are incorporated onto two or more proteins of interest.

Embodiment 162. The method of embodiment 161, wherein two or more proteins of interest can be dimerized by contact 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 the compounds disclosed in embodiment 163 of group 1.

Embodiment 164. A method for incorporating an immune epitope onto a protein of interest encoded by a target nucleotide sequence by prime editing, the method comprising: (a) contacting the target nucleotide sequence with (i) a prime editing factor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a reverse transcriptase, and (ii) a PEgRNA comprising an editing template encoding a functional moiety; (b) polymerizing a single-stranded DNA sequence encoding the immune epitope; and (c) incorporating the single-stranded DNA sequence in place of the corresponding endogenous strand in the target nucleotide sequence by DNA repair and/or replication processes, the method resulting in a recombinant target nucleotide sequence encoding a fusion protein comprising the protein of interest and the immune epitope.

Embodiment 165. The method of embodiment 164, wherein the immune epitope is: tetanus toxoid (SEQ ID NO: 396); diphtheria toxin variant CRM197 (SEQ ID NO: 630); mumps immune epitope 1 (SEQ ID NO: 400); mumps immune epitope 2 (SEQ ID NO: 402); mumps immune epitope 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 epitope for class I HLA (SEQ ID NO: 416); neuraminidase epitope for class I HLA (SEQ ID NO: 418); hemagglutinin epitope for class II HLA (SEQ ID NO: 420); neuraminidase epitope for class II HLA (SEQ ID NO: 422); hemagglutinin epitope H5N1 binding class I and class II HLA (SEQ ID NO: 424); Selected from the group consisting of neuraminidase epitope H5N1-binding class I and class II HLA (SEQ ID NO: 426).

Embodiment 166. The method of embodiment 164, wherein the fusion protein comprises an amino acid sequence of PE1, PE2, or PE3.

Embodiment 167. The method of embodiment 164, wherein the napDNAbp is 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 NO: 222.

Embodiment 170. A method for incorporating a small dimerization domain onto a protein of interest encoded by a target nucleotide sequence by prime editing, the method comprising: (a) contacting the target nucleotide sequence with (i) a prime editing factor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a reverse transcriptase, and (ii) a PEgRNA comprising an editing template encoding the small dimerization domain; (b) polymerizing a single-stranded DNA sequence encoding an immune epitope; and (c) incorporating the single-stranded DNA sequence in place of the corresponding endogenous strand in the target nucleotide sequence by DNA repair and/or replication processes, the method producing a recombinant target nucleotide sequence encoding a fusion protein comprising the protein of interest and the small dimerization domain.

Embodiment 171. The method of embodiment 170, further comprising performing 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 domains of each of the 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 a 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 the compounds disclosed in embodiment 163 of group 1.

Embodiment 177. The method of embodiment 170, wherein the fusion protein comprises an amino acid sequence of PE1, PE2, or PE3.

Embodiment 178. The method of embodiment 170, wherein the napDNAbp is 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 NO: 222.

Embodiment 181. A method of incorporating a peptide tag or epitope onto a protein using prime editing, comprising: contacting a target nucleotide sequence encoding a protein with a prime editor construct configured to insert therein a second nucleotide sequence encoding a peptide tag, resulting 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 purifying and/or detecting the protein.

Aspect 183. The method of aspect 181, wherein the peptide tag is polyhistidine (e.g., HHHHHH) (SEQ ID NOs: 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, or the like.

Embodiment 184. The method of embodiment 181, wherein the peptide tag has an amino acid sequence selected from the group consisting of SEQ ID NOs: 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], where "]-[" represents an optional linker.

Embodiment 187. The method of embodiment 181, wherein the linker has an amino acid sequence of SEQ ID NOs: 127, 165-176, 446, 453, and 767-769.

Embodiment 188. The method of embodiment 181, wherein the prime editor construct comprises a PEgRNA comprising a 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, the spacer being complementary to a target nucleotide sequence, and the extension arm comprises a reverse transcriptase template encoding a peptide tag.

Embodiment 190. The method of embodiment 181, wherein the PEGRNA comprises a spacer, a gRNA core, and an extension arm, the spacer being complementary to a target nucleotide sequence, and the extension arm comprises a reverse transcriptase template encoding a peptide tag.

Embodiment 191. A method for preventing or halting the progression of prion disease by incorporating one or more protective mutations in a PRNP encoded by a target nucleotide sequence by prime editing, the method comprising: (a) contacting the target nucleotide sequence with (i) a prime editing factor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a reverse transcriptase, and (ii) a PEgRNA comprising an editing template encoding a functional moiety; (b) polymerizing a single-stranded DNA sequence encoding the protective mutation; and (c) incorporating the single-stranded DNA sequence in place of the corresponding endogenous strand in the target nucleotide sequence by DNA repair and/or replication processes, the method resulting in a recombinant target nucleotide sequence encoding a PRNP containing a protective mutation that is resistant to misfolding.

Aspect 192. The method of aspect 191, wherein the prion disease is a human prion disease.

Aspect 193. The method of aspect 191, wherein the prion disease is an animal prion disease.

Aspect 194. The method of aspect 192, wherein the prion disease is Creutzfeldt-Jakob disease (CJD), variant Creutzfeldt-Jakob disease (vCJD), Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia, or kuru.

Aspect 195. The method of aspect 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.

Aspect 196. The method of aspect 191, wherein the wild-type 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, and the modified PRNP protein is resistant to misfolding.

Embodiment 198. The method of embodiment 191, wherein the fusion protein comprises an amino acid sequence of PE1, PE2, or PE3.

Embodiment 199. The method of embodiment 191, wherein the napDNAbp is 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 NO: 222.

Embodiment 202. A method for incorporating a ribonucleotide motif or tag on a desired RNA encoded by a target nucleotide sequence by prime editing, the method comprising: (a) contacting the target nucleotide sequence with (i) a prime editing factor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a reverse transcriptase, and (ii) a PEgRNA comprising an editing template encoding the ribonucleotide motif or tag; (b) polymerizing a single-stranded DNA sequence encoding the ribonucleotide motif or tag; and (c) incorporating the single-stranded DNA sequence in place of the corresponding endogenous strand in the target nucleotide sequence by DNA repair and/or replication processes, the method producing a recombinant target nucleotide sequence encoding a modified RNA of interest comprising the ribonucleotide motif or tag.

Embodiment 203. The method of embodiment 202, wherein the ribonucleotide motif or tag is the 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 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 an amino acid sequence of PE1, PE2, or PE3.

Embodiment 209. The method of embodiment 202, wherein the napDNAbp is 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 for inserting or deleting a functional portion on a protein of interest encoded by a target nucleotide sequence by prime editing, the method comprising: (a) contacting the target nucleotide sequence with (i) a prime editing factor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a reverse transcriptase and (ii) a PEgRNA comprising an editing template encoding the functional portion or deletion; (b) polymerizing a single-stranded DNA sequence encoding the functional portion or deletion; and (c) incorporating the single-stranded DNA sequence in place of the corresponding endogenous strand in the target nucleotide sequence by DNA repair and/or replication processes, the method producing a recombinant target nucleotide sequence encoding the protein of interest and an altered protein comprising the functional portion or deletion, the functional portion altering the modification or localization state of the protein.

Embodiment 212. The method of embodiment 211, wherein the functional moiety alters the phosphorylation, ubiquitination, glycosylation, lipid modification, hydroxylation, methylation, acetylation, crotonylation, or sumoylation status 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 performing prime editing in the presence of a prime editing guide RNA (PEgRNA).

Aspect 215. The fusion protein of aspect 213, wherein napDNAbp has nickase activity.

Embodiment 216. The fusion protein of embodiment 213, wherein napDNAbp is a Cas9 protein or a variant thereof.

Embodiment 217. The fusion protein of embodiment 213, wherein 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 napDNAbp is selected from the group consisting of: Cas9, Cas12e, Cas12d, Cas12a, Cas12b1, Cas13a, Cas12c, and Argonaute, and optionally has nickase activity.

Embodiment 220. The fusion protein of embodiment 213, wherein the fusion protein is capable of binding to a target DNA sequence when complexed with the PEGRNA.

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 binding of the fusion protein complexed with 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 a target strand and (ii) a complementary non-target strand.

Embodiment 224. The fusion protein of embodiment 223, wherein the complementary non-target strand is nicked to form a prime 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 3' end of the guide RNA or at an intramolecular position of the guide RNA.

Embodiment 226. The fusion protein of embodiment 225, wherein the extension arm comprises (i) a DNA synthesis template sequence containing 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-stranded DNA flap that is complementary to the endogenous DNA sequence adjacent to the nick site, and the single-stranded DNA flap contains 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-stranded DNA flap hybridizes to the endogenous DNA sequence adjacent to the nick site, thereby incorporating the desired nucleotide change.

Embodiment 230. The fusion protein of embodiment 227, wherein the single-stranded DNA flap has a free 5' end and replaces the endogenous DNA sequence adjacent to the nick site.

Embodiment 231. The fusion protein of embodiment 230, wherein the endogenous DNA sequence having a 5' end is excised by the cell.

Embodiment 232. The fusion protein of embodiment 230, wherein cellular repair of the single-stranded DNA flap results in incorporation of a desired nucleotide change, thereby forming a desired product.

233. The fusion protein of embodiment 226, wherein the desired nucleotide change is incorporated into an editing window that is between about -4 and +10 of the PAM sequence, or between about -10 and +20 of the PAM sequence, or between about -20 and +40 of the PAM sequence, or between about -30 and +100 of the PAM sequence, or the desired nucleotide change is incorporated into 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 of the nick site. , 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.

Embodiment 234 The fusion protein of embodiment 213, 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 to the amino acid sequence of SEQ ID NO: 18.

Embodiment 235. The fusion protein of embodiment 213, wherein napDNAbp comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any one of the amino acid sequences 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 and comprises 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, 701-716, 739-741, and 766.

Embodiment 237. The fusion protein of embodiment 213, wherein the polymerase is a reverse transcriptase and comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to 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, 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 retrotransposon.

Embodiment 239. The fusion protein of any one of the preceding embodiments, wherein the fusion protein comprises the structure NH2-[napDNAbp]-[polymerase]-COOH; or NH2-[polymerase]-[napDNAbp]-COOH, each of the "]-[" indicates the presence of an optional linker sequence.

Embodiment 240. The fusion protein of embodiment 239, wherein the linker sequence comprises the 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 insertion 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 at the 3' or 5' end of the guide RNA or at an intramolecular position of the guide RNA, and the nucleic acid extension arm is DNA or RNA.

Embodiment 245. The PEgRNA of embodiment 242, wherein the PEgRNA is capable of binding to the 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, and 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 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 toe loop, an aptamer, or an RNA-protein recruitment domain.

Embodiment 252. The PEGRNA of embodiment 247, wherein the DNA synthesis template encodes a single-stranded DNA flap that is complementary to the endogenous DNA sequence adjacent to the nick site, and the single-stranded DNA flap contains the desired nucleotide change.

Embodiment 253. The PEGRNA of embodiment 252, wherein the single-stranded DNA flap displaces the endogenous single-stranded DNA having a 5' end on the nicked target DNA sequence, and the endogenous single-stranded DNA is immediately adjacent and downstream of the nick site.

Embodiment 254. The PEGRNA of embodiment 253, wherein the endogenous single-stranded DNA having a free 5' end is excised by the cell.

Embodiment 255. The PEGRNA of embodiment 253, wherein cellular repair of the single-stranded DNA flap results in incorporation of the desired nucleotide change, thereby forming the desired product.

Embodiment 256. The PEG RNA of embodiment 243, comprising 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, or 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 to the free 3' end of the cut DNA.

Embodiment 259. The PEGRNA of embodiment 251, wherein at least one additional structure is located at the 3' or 5' end of the PEGRNA.

Aspect 260. A complex comprising any one of the fusion proteins of aspects 213-242 and PEgRNA.

Embodiment 261. The complex of embodiment 260, 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 of the guide RNA.

Embodiment 262. The complex of embodiment 260, wherein the PEgRNA is capable of binding to the 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, and 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 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 is selected from the group consisting of the nucleotide sequences 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 SEQ ID NOs: 101-104, 181-183, 223-24 4, 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%, 85%, 90%, 95%, or 99% identical to the endogenous DNA target.

Embodiment 267. The complex of embodiment 264, wherein the primer binding site hybridizes to the free 3' end of the cut DNA.

Aspect 268. A complex comprising napDNAbp and PEgRNA.

Embodiment 269. The complex of embodiment 268, wherein napDNAbp is a Cas9 nickase.

Aspect 270. The complex of aspect 268, wherein napDNAbp comprises the amino acid sequence of SEQ ID NO:18.

Embodiment 271. The complex of embodiment 268, wherein napDNAbp comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any one of the amino acid sequences 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 of 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, and 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 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, or SEQ ID NOs: 101-104, 181-183, 223-24 4, 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 to the 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 toe loop, an aptamer, or an RNA-protein recruitment domain.

Aspect 280. A polynucleotide encoding a fusion protein according to any one of aspects 213 to 242.

Aspect 281. A vector comprising the polynucleotide of aspect 280.

Embodiment 282. A cell comprising the fusion protein of any one of embodiments 213 to 242 and a PEgRNA bound to the napDNAbp of the fusion protein.

Embodiment 283. A cell comprising any one of the complexes of embodiments 260-279.

Embodiment 284. A pharmaceutical composition comprising: (i) a fusion protein of any one of embodiments 213 to 242, a complex of embodiments 260 to 279, a polynucleotide of embodiment 68, or a vector of embodiment 69; and (ii) a pharma- ceutically acceptable excipient.

Embodiment 285. A pharmaceutical composition comprising: (i) a conjugate of any one of embodiments 260 to 279; (ii) a polymerase provided in trans; and (iii) a pharma- ceutically acceptable excipient.

Embodiment 286. A kit comprising: (i) a nucleic acid construct comprising a nucleic acid sequence encoding a fusion protein of any one of embodiments 213-242; and (ii) a promoter driving expression of the sequence of (i).

Embodiment 287. A method for incorporating a desired nucleotide change into a double-stranded DNA sequence, the method comprising:

(i) contacting a double-stranded DNA sequence with a complex comprising a fusion protein and a PEgRNA, the fusion protein comprising napDNAbp and a polymerase, and the PEgRNA comprising a DNA synthesis template containing the desired nucleotide change and a primer binding site;

(ii) nicking the double-stranded DNA sequence, thereby generating a free single-stranded DNA having a 3' end;

(iii) hybridizing the 3' end of the free single-stranded 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-stranded DNA flap that is complementary to the DNA synthesis template and contains the desired nucleotide change;

(v) replacing the endogenous DNA strand adjacent to the cut site with a single-stranded DNA flap, thereby incorporating the desired nucleotide change into the double-stranded DNA sequence;
including.

Embodiment 288. The method of embodiment 287, wherein (v) the replacing step comprises: (i) hybridizing a single-stranded DNA flap to the endogenous DNA strand adjacent to the cut site to generate a sequence mismatch; (ii) excising the endogenous DNA strand; and (iii) repairing the mismatch to form a desired product containing 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, deletion, or 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 nucleotide change is (1) a G:C base pair to a T:A base pair, (2) a G:C base pair to an A:T base pair, (3) a G:C base pair to a C:G base pair, (4) a T:A base pair to a G:C base pair, (5) a T:A base pair to an A:T base pair, (6) a T:A base pair to a C:G base pair, (7) a C:G base pair to a G:C base pair, (8) a C:G base pair to a T:A base pair, a C:G base pair to an A:T base pair, (10) an A:T base pair to a T:A base pair, (11) an A:T base pair to a G:C base pair, or (12) a A:T base pair to a C:G base pair.

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 single gene 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; phenylketonuria; severe combined immunodeficiency; sickle cell disease; Smith-Lemli-Opitz syndrome; trinucleotide repeat disorders; prion diseases; 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; hypertension; Alzheimer's disease; arthritis; diabetes; cancer; and obesity.

Embodiment 297. The method of embodiment 287, wherein the napDNAbp is a nuclease-inactive Cas9 (dCas9), a Cas9 nickase (nCas9), or a nuclease-active Cas9.

Embodiment 298. The method of embodiment 287, wherein 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 to 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 to any one of the amino acid sequences 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 and comprises 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, 701-716, 739-741, and 766.

Embodiment 301. The method of embodiment 287, wherein the polymerase is a reverse transcriptase and comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to 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, 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' end of the guide RNA or positioned intramolecularly, the extension arm comprising a DNA synthesis template sequence and a 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, and 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 a DNA molecule with a nucleic acid programmable DNA binding protein (napDNAbp) and a PEgRNA that targets the napDNAbp to a target locus, the PEgRNA comprising a reverse transcriptase (RT) template sequence containing at least one desired nucleotide change and a primer binding site;

(ii) forming an exposed 3' end of the DNA strand at the target locus;

(iii) hybridizing the exposed 3' end to a primer binding site to prime reverse transcription;

(iv) synthesizing a single-stranded DNA flap containing at least one desired nucleotide change based on the RT template sequence by reverse transcriptase; and

(v) incorporating 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;
including.

Embodiment 306. The method of embodiment 305, wherein the one or more changes in the nucleotide sequence include 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 include 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 include changing (1) a G:C base pair to a T:A base pair, (2) a G:C base pair to an A:T base pair, (3) a G:C base pair to a C:G base pair, (4) a T:A base pair to a G:C base pair, (5) a T:A base pair to an A:T base pair, (6) a T:A base pair to a C:G base pair, (7) a C:G base pair to a G:C base pair, (8) a C:G base pair to a T:A base pair, (9) a C:G base pair to an A:T base pair, (9) an A:T base pair to a T:A base pair, (11) an A:T base pair to a G:C base pair, or (12) an A:T base pair to a C:G base pair.

Embodiment 311. The method of embodiment 305, wherein the one or more changes in the nucleotide sequence include 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 include correction of a disease-associated gene.

Embodiment 313. The method of embodiment 312, wherein the disease-associated gene is associated with a single gene 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; phenylketonuria; severe combined immunodeficiency; sickle cell disease; Smith-Lemli-Opitz syndrome; trinucleotide repeat disorders; prion diseases; 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 napDNAbp is a nuclease-active Cas9 or a variant thereof.

Embodiment 316. The method of embodiment 305, wherein the napDNAbp is a nuclease-inactivated Cas9 (dCas9) or Cas9 nickase (nCas9) or a variant thereof.

Embodiment 317. The method of embodiment 305, wherein 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 to 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 to any one of the amino acid sequences 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 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 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 to 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, 701-716, 739-741, and 766.

Embodiment 323. The method of embodiment 305, wherein the step of forming an exposed 3' end of 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 in trans.

Embodiment 325. The method of embodiment 305, wherein the step of forming an exposed 3' end of the DNA strand at the target locus comprises contacting the DNA strand with a chemical agent.

Embodiment 326. The method of embodiment 305, wherein forming an exposed 3' end of the DNA strand at the target locus includes introducing a replication error.

Embodiment 327. The method of embodiment 305, wherein 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 on which the exposed 3' end is formed is on an R-loop.

Embodiment 329. The method of embodiment 315, wherein the PEGRNA comprises an extension arm that comprises a reverse transcriptase (RT) template sequence and a 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 location of 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 toe loop, 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 a nucleotide sequence of a DNA molecule at a target locus by primed reverse transcription, the method comprising: (a) contacting the DNA molecule at the target locus with (i) a 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) performing primed reverse transcription of the RT template to generate a single-stranded DNA comprising the desired nucleotide change; and (c) incorporating the desired nucleotide change on the DNA molecule at the target locus by DNA repair and/or replication processes.

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 of 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 base pair to a T:A base pair, (2) a G:C base pair to an A:T base pair, (3) a G:C base pair to a C:G base pair, (4) a T:A base pair to a G:C base pair, (5) a T:A base pair to an A:T base pair, (6) a T:A base pair to a C:G base pair, (7) a C:G base pair to a G:C base pair, (8) a C:G base pair to a T:A base pair, (9) a C:G base pair to an A:T base pair, (10) an A:T base pair to a T:A base pair, (11) an A:T base pair to a G:C base pair, or (12) an A:T base pair to a C:G base pair.

Embodiment 340. A polynucleotide encoding any one of the PEgRNAs of embodiments 243-259.

Aspect 341. A vector comprising the polynucleotide of aspect 340.

Aspect 342. A cell comprising the vector of aspect 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 primed reverse transcription, the method comprising: (a) contacting the DNA molecule at the target locus with (i) a 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) performing primed reverse transcription of the RT template to generate a mutated single-stranded DNA; and (c) incorporating the mutated single-stranded DNA onto the DNA molecule at the target locus by DNA repair and/or replication processes.

Embodiment 345. The method of any preceding embodiment, wherein the fusion protein comprises an amino acid sequence of PE1, PE2, or PE3.

Embodiment 346. The method of any preceding 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 : 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487 .

Embodiment 348. The method of embodiment 344, wherein the guide RNA comprises SEQ ID NO: 222.

Embodiment 349. The method of embodiment 344, wherein (b) performing primed reverse transcription on the primed target comprises generating a 3' end primer binding sequence at the target locus 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 on a target DNA molecule with a healthy sequence containing a healthy number of repeated trinucleotides, the method comprising: (a) contacting a DNA molecule at a target locus with (i) a fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a polymerase and (ii) a DNA synthesis template comprising the replacement sequence and a PEgRNA comprising a primer binding site; (b) performing prime editing to generate a single-stranded DNA comprising the replacement sequence; and (c) incorporating the single-stranded DNA onto the DNA molecule at the target locus by DNA repair and/or replication processes.

Embodiment 351. The method of embodiment 350, wherein the fusion protein comprises an amino acid sequence of PE1, PE2, or PE3.

Embodiment 352. The method of embodiment 350, wherein the napDNAbp is 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 NO: 222.

Embodiment 355. The method of embodiment 350, wherein (b) the step of performing prime editing comprises generating a 3' end primer binding sequence at the target locus that can prime a polymerase by annealing to a 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 repeat unit of a CAG triplet.

Embodiment 358. The method of embodiment 350, wherein the trinucleotide repeat expansion mutation comprises a repeat unit of a GAA triplet.

Embodiment 359. A method for incorporating a functional moiety on a protein of interest encoded by a target nucleotide sequence by prime editing, the method comprising: (a) contacting the target nucleotide sequence with (i) a prime editing factor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a polymerase, and (ii) a PEgRNA comprising a DNA synthesis template encoding the functional moiety; (b) polymerizing a single-stranded DNA sequence encoding the functional moiety; and (c) incorporating the single-stranded DNA sequence in place of the corresponding endogenous strand in the target nucleotide sequence by DNA repair and/or replication processes, the method resulting in a recombinant target nucleotide sequence encoding a fusion protein comprising the protein of interest and the functional moiety.

Embodiment 360. The method of embodiment 359, wherein the functional moiety is a peptide tag.

Embodiment 361. The method of embodiment 360, wherein the peptide tag is an affinity tag, a solubilization tag, a chromatography tag, an epitope or immunoepitope tag, or a fluorescent 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); Polyglutamic acid 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 NO: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); polyaspartic acid (Asp tag) (SEQ ID NO:286); polyphenylalanine (Phe tag) (SEQ ID NO:287); S1 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 immune epitope.

365. The method of 364, wherein the immune epitope is selected from the group consisting of: tetanus toxoid (SEQ ID NO: 396); diphtheria toxin variant CRM197 (SEQ ID NO: 630); mumps immune epitope 1 (SEQ ID NO: 400); mumps immune epitope 2 (SEQ ID NO: 402); mumps immune epitope 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 epitope for class I HLA (SEQ ID NO: 416); neuraminidase epitope for class I HLA (SEQ ID NO: 418); hemagglutinin epitope for class II HLA (SEQ ID NO: 420); neuraminidase epitope for class II HLA (SEQ ID NO: 422); hemagglutinin epitope H5N1 binding class I and class II HLA (SEQ ID NO: 424); Selected from the group consisting of neuraminidase epitope H5N1-binding 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 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 a cyclophilin of SEQ ID NOs: 490 and 493-494.

Embodiment 373. The method of embodiment 359, wherein the small molecule binding domains are incorporated onto two or more proteins of interest.

Embodiment 374. The method of embodiment 373, wherein two or more proteins of interest can be dimerized by contact 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 the compounds disclosed in embodiment 163 of group 1.

Embodiment 376. A method for incorporating an immune epitope onto a protein of interest encoded by a target nucleotide sequence by prime editing, the method comprising: (a) contacting the target nucleotide sequence with (i) a prime editing factor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a polymerase, and (ii) a PEgRNA comprising an editing template encoding a functional moiety; (b) polymerizing a single-stranded DNA sequence encoding the immune epitope; and (c) incorporating the single-stranded DNA sequence in place of the corresponding endogenous strand in the target nucleotide sequence by DNA repair and/or replication processes, the method resulting in a recombinant target nucleotide sequence encoding a fusion protein comprising the protein of interest and the immune epitope.

377. The method of 376, wherein the immune epitope is selected from the group consisting of: tetanus toxoid (SEQ ID NO: 396); diphtheria toxin variant CRM197 (SEQ ID NO: 630); mumps immune epitope 1 (SEQ ID NO: 400); mumps immune epitope 2 (SEQ ID NO: 402); mumps immune epitope 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 epitope for class I HLA (SEQ ID NO: 416); neuraminidase epitope for class I HLA (SEQ ID NO: 418); hemagglutinin epitope for class II HLA (SEQ ID NO: 420); neuraminidase epitope for class II HLA (SEQ ID NO: 422); hemagglutinin epitope H5N1 binding class I and class II HLA (SEQ ID NO: 424); Selected from the group consisting of neuraminidase epitope H5N1-binding class I and class II HLA (SEQ ID NO: 426).

Embodiment 378. The method of embodiment 376, wherein the fusion protein comprises an amino acid sequence of PE1, PE2, or PE3.

Embodiment 379. The method of embodiment 376, wherein the napDNAbp is 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 for incorporating a small dimerization domain onto a protein of interest encoded by a target nucleotide sequence by prime editing, the method comprising: (a) contacting the target nucleotide sequence with (i) a prime editing factor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a polymerase, and (ii) a PEgRNA comprising an editing template encoding the small dimerization domain; (b) polymerizing a single-stranded DNA sequence encoding an immune epitope; and (c) incorporating the single-stranded DNA sequence in place of the corresponding endogenous strand in the target nucleotide sequence by DNA repair and/or replication processes, the method producing a recombinant target nucleotide sequence encoding a fusion protein comprising the protein of interest and the small dimerization domain.

Embodiment 383. The method of embodiment 382, further comprising performing 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 domains of each of the 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 a 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 the compounds disclosed in embodiment 163 of group 1.

Embodiment 389. The method of embodiment 382, wherein the fusion protein comprises an amino acid sequence of PE1, PE2, or PE3.

Embodiment 390. The method of embodiment 382, wherein the napDNAbp is 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 incorporating a peptide tag or epitope onto a protein using prime editing, comprising: contacting a target nucleotide sequence encoding a protein with a prime editor construct configured to insert therein a second nucleotide sequence encoding a peptide tag, resulting 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 purifying and/or detecting the protein.

Embodiment 395. The method of embodiment 383, wherein the peptide tag is polyhistidine (e.g., HHHHHH) (SEQ ID NOs: 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 NOs: 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], where "]-[" represents an optional linker.

Embodiment 399. The method of embodiment 383, wherein the linker has an amino acid sequence of SEQ ID NOs: 127, 165-176, 446, 453, and 767-769.

Embodiment 400. The method of embodiment 383, wherein the prime editor construct comprises a PEgRNA comprising a 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, the spacer being complementary to a target nucleotide sequence, and the extension arm comprises a reverse transcriptase template encoding a peptide tag.

Embodiment 402. The method of embodiment 383, wherein the PEGRNA comprises a spacer, a gRNA core, and an extension arm, the spacer being complementary to a target nucleotide sequence, and the extension arm comprises a reverse transcriptase template encoding a peptide tag.

Embodiment 403. A method for preventing or halting the progression of prion disease by incorporating one or more protective mutations in a PRNP encoded by a target nucleotide sequence by prime editing, the method comprising: (a) contacting the target nucleotide sequence with (i) a prime editing factor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a polymerase, and (ii) a PEgRNA comprising an editing template encoding a functional moiety; (b) polymerizing a single-stranded DNA sequence encoding the protective mutation; and (c) incorporating the single-stranded DNA sequence in place of the corresponding endogenous strand in the target nucleotide sequence by DNA repair and/or replication processes, the method resulting in a recombinant target nucleotide sequence encoding a PRNP containing a protective mutation that is resistant to misfolding.

Aspect 404. The method of aspect 403, wherein the prion disease is a human prion disease.

Aspect 405. The method of aspect 403, wherein the prion disease is an animal prion disease.

Aspect 406. The method of aspect 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 wild-type 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, and the modified PRNP protein is resistant to misfolding.

Embodiment 410. The method of embodiment 403, wherein the fusion protein comprises an amino acid sequence of PE1, PE2, or PE3.

Embodiment 411. The method of embodiment 403, wherein the napDNAbp is 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 for incorporating a ribonucleotide motif or tag onto a desired RNA encoded by a target nucleotide sequence by prime editing, the method comprising: (a) contacting the target nucleotide sequence with (i) a prime editing factor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a polymerase, and (ii) a PEgRNA comprising an editing template encoding the ribonucleotide motif or tag; (b) polymerizing a single-stranded DNA sequence encoding the ribonucleotide motif or tag; and (c) incorporating the single-stranded DNA sequence in place of the corresponding endogenous strand in the target nucleotide sequence by DNA repair and/or replication processes, the method producing a recombinant target nucleotide sequence encoding a modified RNA of interest comprising the ribonucleotide motif or tag.

Embodiment 415. The method of embodiment 414, wherein the ribonucleotide motif or tag is the 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 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 an amino acid sequence of PE1, PE2, or PE3.

Embodiment 421. The method of embodiment 414, wherein the napDNAbp is 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 for introducing or deleting a functional portion on a protein of interest encoded by a target nucleotide sequence by prime editing, the method comprising: (a) contacting the target nucleotide sequence with (i) a prime editing factor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a polymerase and (ii) a PEgRNA comprising an editing template encoding the functional portion or deletion; (b) polymerizing a single-stranded DNA sequence encoding the functional portion or deletion; and (c) incorporating the single-stranded DNA sequence in place of the corresponding endogenous strand in the target nucleotide sequence by DNA repair and/or replication processes, the method producing a recombinant target nucleotide sequence encoding the protein of interest and an altered protein comprising the functional portion or deletion, the functional portion altering the modification or localization state of the protein.

Embodiment 424. The method of embodiment 423, wherein the functional moiety alters the phosphorylation, ubiquitination, glycosylation, lipid modification, hydroxylation, methylation, acetylation, crotonylation, or sumoylation status of the protein of interest.

Embodiment 425. A fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) domain and a domain comprising RNA-dependent DNA polymerase activity.

Embodiment 426. The fusion protein of embodiment 425, wherein the fusion protein is capable of performing prime editing in the presence of a prime editing guide RNA (PEgRNA) to incorporate a desired nucleotide change into a target sequence.

Embodiment 427. The fusion protein of embodiment 425, wherein the napDNAbp domain has nickase activity.

Embodiment 428. The fusion protein of embodiment 425, wherein the napDNAbp domain is a Cas9 protein or a 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 nickase activity.

Embodiment 432. The fusion protein of embodiment 425, wherein the domain comprising the RNA-dependent DNA polymerase activity is a reverse transcriptase and comprises 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, 701-716, 739-741, and 766.

Embodiment 433. The fusion protein of embodiment 425, wherein the domain comprising RNA-dependent DNA polymerase activity is a reverse transcriptase and comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to 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, 701-716, 739-741, and 766, and optionally, the domain comprising RNA-dependent DNA polymerase is error-prone.

Embodiment 434. The fusion protein of embodiment 425, wherein the domain containing the RNA-dependent DNA polymerase activity is a naturally occurring reverse transcriptase from a retrovirus or retrotransposon.

Embodiment 435. The fusion protein of embodiment 425, wherein the fusion protein is capable of binding to a target DNA sequence when complexed with the PEgRNA.

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 binding of the fusion protein complexed with 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 a target strand and (ii) a complementary non-target strand.

Embodiment 439. The fusion protein of embodiment 437, wherein the target or complementary non-target strand is nicked to form a primed 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 is -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 3' end of the guide RNA or positioned intramolecularly in the guide RNA.

Embodiment 445. The fusion protein of embodiment 443, wherein the extension arm comprises (i) a DNA synthesis template sequence containing 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-stranded DNA flap that is complementary to the endogenous DNA sequence adjacent to the nick site, and the single-stranded DNA flap contains the desired nucleotide change.

Aspect 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-stranded DNA flap hybridizes to an endogenous DNA sequence adjacent to the nick site, thereby incorporating a desired nucleotide change onto the target strand.

Embodiment 449. The fusion protein of embodiment 443, wherein the single-stranded DNA flap has a free 5' end and replaces the endogenous DNA sequence adjacent to the nick site.

Embodiment 450. The fusion protein of embodiment 446, wherein the endogenous DNA sequence having a 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-stranded DNA flap incorporates a desired nucleotide change onto the non-target strand, thereby forming a desired product.

Embodiment 453. The fusion protein of embodiment 449, wherein the desired nucleotide change is incorporated over an editing window that is between about -4 and +10 of the PAM sequence, or between about -10 and +20 of the PAM sequence, or between about -20 and +40 of the PAM sequence, or between about -30 and +100 of the PAM sequence.

Embodiment 454. The fusion protein of embodiment 449, wherein the desired nucleotide change is 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, Incorporated 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.

Embodiment 455 The fusion protein of embodiment 425, 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 to the amino acid sequence of SEQ ID NO: 18.

Embodiment 456. The fusion protein of embodiment 425, wherein napDNAbp comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any one of the amino acid sequences 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 preceding embodiments, wherein the fusion protein comprises the structure NH2-[napDNAbp]-[domain comprising RNA-dependent DNA polymerase activity]-COOH; or NH2-[domain comprising RNA-dependent DNA polymerase activity]-[napDNAbp]-COOH, each of the "]-[" indicates the presence of an optional linker sequence.

Embodiment 458. The fusion protein of embodiment 457, wherein the linker sequence comprises the 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 insertion 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 one of embodiments 425 to 460 and a PEgRNA, wherein the PEgRNA guides 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 of the guide RNA.

Embodiment 463. The complex of embodiment 462, wherein the PEgRNA is capable of binding to the napDNAbp and directing the napDNAbp to a 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, and 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 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 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, or SEQ ID NOs: 101-104, 181-183, 223-24 4, 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%, 85%, 90%, 95%, or 99% identical to the endogenous DNA target.

Embodiment 468. The complex of embodiment 465, wherein the primer binding site hybridizes to the free 3' end of the cut DNA.

Aspect 469. The complex of aspect 461, wherein napDNAbp is a Cas9 nickase.

Embodiment 470. The complex of embodiment 461, wherein 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 to SEQ ID NO:18.

Embodiment 471. The complex of embodiment 461, wherein napDNAbp comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any one of the amino acid sequences 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 of the guide RNA.

Embodiment 473. The complex of embodiment 465, wherein the DNA synthesis template comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the endogenous DNA target.

Embodiment 474. The complex of embodiment 465, wherein the primer binding site hybridizes to the 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 toe loop, an aptamer, or an RNA-protein recruitment domain.

Aspect 476. A polynucleotide encoding a fusion protein of any one of aspects 425 to 461.

Aspect 477. A polynucleotide encoding the PEgRNA of any of the above aspects.

Aspect 478. A vector comprising the polynucleotide of aspect 476, wherein expression of the fusion protein is under the control of a promoter.

Aspect 479. A vector comprising the polynucleotide of aspect 477, wherein expression of the PEG RNA is under the control of a promoter.

Aspect 480. The vector of aspect 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 repeat clusters of T on the extension arm to improve transcription efficiency from the U6 promoter.

Embodiment 483. The vector of embodiment 482, wherein the one or more repeat clusters of T's that are removed include at least 3 T's, at least 4 T's, at least 5 T's, at least 6 T's, at least 7 T's, at least 8 T's, at least 9 T's, at least 10 T's, at least 11 T's, at least 12 T's, at least 13 T's, at least 14 T's, at least 15 T's, at least 16 T's, at least 17 T's, at least 18 T's, at least 19 T's, or at least 20 T's.

Embodiment 484. A cell comprising a fusion protein of any one of embodiments 425 to 460 and a PEgRNA bound to the napDNAbp of the fusion protein.

Aspect 485. A cell comprising any one of the complexes of aspects 461 to 475.

Embodiment 486. A pharmaceutical composition comprising: (i) a fusion protein of any of embodiments 425-460, a complex of embodiments 461-475, a polynucleotide of embodiments 476-477, or a vector of embodiments 478-483; and (ii) a pharma- ceutically acceptable excipient.

Embodiment 487. A pharmaceutical composition comprising: (i) a conjugate of any one of embodiments 461 to 475; (ii) a polymerase provided in trans; and (iii) a pharma- ceutically acceptable excipient.

Embodiment 488. A kit for prime editing comprising: (i) a nucleic acid molecule encoding a fusion protein of any one of embodiments 425 to 460; and (ii) a nucleic acid molecule encoding a PEGRNA capable of directing the fusion protein to a target DNA site, wherein the nucleic acid molecules (i) and (ii) may be contained on a single DNA construct or on separate DNA constructs.

Aspect 489. The kit of aspect 488, wherein the nucleic acid molecule of (i) further comprises a promoter that drives expression of the fusion protein.

Aspect 490. The kit of aspect 488, wherein the nucleic acid molecule of (ii) further comprises a promoter driving expression of the PEgRNA.

Aspect 491. The kit of aspect 490, wherein the promoter is a U6 promoter.

Aspect 492. The kit of aspect 490, wherein the promoter is a CMV promoter.

Embodiment 493. The fusion protein of embodiment 457, wherein the linker sequence comprises the amino acid sequences of SEQ ID NOs: 174 (1xSGGS), 446 (2xSGGS), 3889 (3xSGGS), 171 (1xXTEN), 3968 (1xEAAAK), 3969 (2xEAAAK), and 3970 (3xEAAAK).

Group B. PE Guide and Design Methods Embodiment 1. A PEgRNA comprising a guide RNA and at least one nucleic acid extension arm comprising a DNA synthesis template.

Aspect 2. The PEgRNA of aspect 1, wherein the nucleic acid extension arm is at the 3' or 5' end of the guide RNA or at an intramolecular position of the guide RNA, and the nucleic acid extension arm is DNA or RNA.

Embodiment 3. The PEgRNA of embodiment 1, wherein the PEgRNA is capable of binding to the napDNAbp and directing the napDNAbp to a target DNA sequence.

Aspect 4. The PEgRNA of aspect 3, wherein the target DNA sequence comprises a target strand and a complementary non-target strand.

Aspect 5. The PEgRNA of aspect 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 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, a linker, a stem loop, a hairpin, a toe loop, an aptamer, or an RNA-protein recruitment domain.

Embodiment 11. The PEGRNA of embodiment 1, wherein the DNA synthesis template encodes a single-stranded DNA flap that is complementary to the endogenous DNA sequence adjacent to the nick site, and the single-stranded DNA flap contains the desired nucleotide change.

Embodiment 12. The PEGRNA of embodiment 11, wherein the single-stranded DNA flap displaces the endogenous single-stranded DNA having a 5' end on the nicked target DNA sequence, and the endogenous single-stranded DNA is immediately adjacent to and downstream of the nick site.

Aspect 13. The PEGRNA of aspect 11, wherein the endogenous single-stranded DNA having a free 5' end is excised by the cell.

Embodiment 14. The PEGRNA of embodiment 13, wherein cellular repair of the single-stranded DNA flap results in incorporation of the desired nucleotide change, thereby forming the desired product.

Embodiment 15. The PEG RNA of embodiment 1, comprising a 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 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, 77, 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%, 85%, 90%, 95%, or 99% identical to the endogenous DNA target.

Aspect 17. The PEGRNA of aspect 6, wherein the primer binding site hybridizes to the free 3' end of the cut DNA.

Embodiment 18. The PEGRNA of embodiment 10, wherein 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.

Aspect 20. The PEgRNA of aspect 10, wherein the stem loop comprises a nucleotide sequence selected from the stem loops described herein.

Aspect 21. The PEGRNA of aspect 10, wherein the hairpin comprises a nucleotide sequence selected from the hairpins described herein.

Aspect 22. The PEGRNA of aspect 10, wherein the toe loop comprises a nucleotide sequence selected from the toe loops described herein.

Aspect 23. The PEGRNA of aspect 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 domains described herein.

Embodiment 25. A method for designing a PEgRNA for use in prime editing to incorporate a desired nucleotide change into a target nucleotide sequence, the PEgRNA comprising a spacer, a gRNA core, and an extension arm, the extension arm comprising a primer binding site and a DNA synthesis template, the method comprising:

(i) selecting a desired target editing site on a target nucleotide sequence;

(ii) obtaining context nucleotide sequences upstream and downstream of the target editing site;

(iii) locating a putative protospacer adjacent motif (PAM) site on the context nucleotide sequence that is proximal to the desired target editing site;

(iv) identifying the corresponding nick site for each putative PAM site;

(v) designing the spacer;

(vi) designing the gRNA core;

(vii) designing the extension arm; and

(viii) constructing a complete PEGRNA by concatenating the spacer, gRNA core, and extension arm;
including.

Aspect 26. The method of aspect 25, wherein step (i) of selecting the desired target editing 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, an autoimmune disorder, a neurological disorder, a skin disorder, a respiratory disease, and a cardiac disease.

Embodiment 28. The method of embodiment 25, wherein step (ii) of obtaining context nucleotide sequences upstream and downstream from the target editing site comprises obtaining from 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, Including obtaining about 145-150bp, about 150-155bp, about 155-160bp, about 160-165bp, about 165-170bp, about 170-175bp, about 175-180bp, about 180-185bp, about 185-190bp, about 190-195bp, about 195-200bp, about 200-205bp, about 205-210bp, about 210-215bp, about 215-220bp, about 220-225bp, about 225-230bp, about 230-235bp, about 235-240bp, about 240-245bp, or about 245-250bp.

Embodiment 29. The method of embodiment 28, wherein the desired target editing sites are 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 site is proximal to the desired target editing site.

Embodiment 31. The method of embodiment 25, wherein in step (iii), the putative PAM site has an associated nick site located less than 30 nucleotides from the target editing 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 editing site.

32. The method of claim 25, wherein in step (iii), the putative PAM site is located more than 30 nucleotides from the target editing site, or 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, These include those with associated nick sites located at positions 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 more than 100 nucleotides.

Embodiment 33. The method of embodiment 25, wherein in step (iii), the putative PAM site is associated with one or a corresponding napDNAbp that binds to said PAM site.

Embodiment 34. The method of embodiment 33, wherein the putative PAM sites and their corresponding napDNAbp are selected from any of the following groupings: (a) SpCas9 and NGG of SEQ ID NOs: 18-25 and 87-88; (b) Sp VQR nCas9; and (c) NGAN.

Embodiment 35. The method of embodiment 25, wherein the step (v) of designing the spacers comprises determining a complementary nucleotide sequence of a protospacer sequence associated with each putative PAM.

Embodiment 36. The method of embodiment 25, wherein step (vi) of designing the gRNA core comprises selecting a gRNA core sequence that can bind, in the context of each putative PAM, to a napDNAbp associated with each of the putative PAMs.

Embodiment 37. The method of embodiment 25, wherein step (vii) of designing the extension arm comprises (a) designing a DNA synthesis template containing the desired edit 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, the 3' end of the DNA primer being the first nucleotide upstream of the nick site associated with the PAM site, and (b) designing a complement of the DNA primer, the complement forming the primer binding site.

Aspect 39. The method of aspect 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 when 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 when 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 when the DNA primer contains a GC content greater than about 60%.

Embodiment 43. A method of prime editing comprising contacting a target DNA sequence with a prime editor fusion protein comprising a PEgRNA of any one of embodiments 1 to 24 and a napDNAbp and a domain having RNA-dependent DNA polymerase activity.

Aspect 44. The method of aspect 43, wherein the napDNAbp has nickase activity.

Embodiment 45. The method of embodiment 43, wherein the napDNAbp is a Cas9 protein or a variant thereof.

Embodiment 46. The method of embodiment 43, wherein napDNAbp is a nuclease-active Cas9, a nuclease-inactive Cas9 (dCas9), or a Cas9 nickase (nCas9).

Aspect 47. The method of aspect 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 nickase activity.

Embodiment 49. The method of embodiment 43, wherein the domain comprising RNA-dependent DNA polymerase activity is a reverse transcriptase and comprises 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, 701-716, 739-741, and 766.

Embodiment 50. The method of embodiment 43, wherein the domain comprising the RNA-dependent DNA polymerase activity is a reverse transcriptase and comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to 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, 701-716, 739-741, and 766.

Embodiment 51. The method of embodiment 43, wherein the domain containing the RNA-dependent DNA polymerase activity is a naturally occurring reverse transcriptase from a retrovirus or retrotransposon.

Group C. PE Conjugates Embodiment 1. A conjugate for prime editing comprising:

(i) a fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a domain containing aRNA-dependent DNA polymerase activity; and

(ii) a prime-edited guide RNA (PEgRNA),
including.

Aspect 2. The complex of aspect 1, wherein the fusion protein is capable of performing prime editing in the presence of a prime editing guide RNA (PEgRNA) to incorporate a desired nucleotide change into a target sequence.

Aspect 3. The complex of aspect 11, wherein napDNAbp has nickase activity.

Embodiment 4. The complex of embodiment 1, wherein napDNAbp is a Cas9 protein or a variant thereof.

Embodiment 5. The complex of embodiment 1, wherein napDNAbp is a nuclease-active Cas9, a nuclease-inactive Cas9 (dCas9), or a Cas9 nickase (nCas9).

Aspect 6. The complex of aspect 1, wherein the napDNAbp is a Cas9 nickase (nCas9).

Embodiment 7. The complex of embodiment 1, wherein napDNAbp is selected from the group consisting of: Cas9, Cas12e, Cas12d, Cas12a, Cas12b1, Cas13a, Cas12c, and Argonaute, and optionally has nickase activity.

Embodiment 8. The complex of embodiment 1, wherein the domain comprising the RNA-dependent DNA polymerase activity is a reverse transcriptase and comprises 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, 701-716, 739-741, and 766.

Embodiment 9. The complex of embodiment 1, wherein the domain comprising the RNA-dependent DNA polymerase activity is a reverse transcriptase and comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to 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, 701-716, 739-741, and 766.

Embodiment 10. The complex of embodiment 1, wherein the domain containing the RNA-dependent DNA polymerase activity is a naturally occurring reverse transcriptase from a retrovirus or retrotransposon.

Embodiment 11. The complex of embodiment 1, wherein the fusion protein is capable of binding to a target DNA sequence when complexed with the PEgRNA.

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.

Aspect 13. The complex of aspect 12, wherein the nucleic acid extension arm is at the 3' or 5' end of the guide RNA or at an intramolecular position of the guide RNA, and the nucleic acid extension arm is DNA or RNA.

Embodiment 14. The complex of embodiment 12, wherein the PEgRNA is capable of binding to the 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.

Aspect 17. The complex of aspect 12, wherein 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 toe loop, an aptamer, or an RNA-protein recruitment domain.

Embodiment 22. The complex of embodiment 12, wherein the DNA synthesis template encodes a single-stranded DNA flap that is complementary to the endogenous DNA sequence adjacent to the nick site, and the single-stranded DNA flap contains the desired nucleotide change.

Embodiment 23. The complex of embodiment 22, wherein the single-stranded DNA flap displaces the endogenous single-stranded DNA having a 5' end on the nicked target DNA sequence, and the endogenous single-stranded DNA is immediately adjacent to and downstream of the nick site.

Aspect 24. The complex of aspect 23, wherein endogenous single-stranded DNA having a free 5' end is excised by the cell.

Embodiment 25. The complex of embodiment 23, wherein cellular repair of the single-stranded DNA flap results in incorporation of a desired nucleotide change, thereby forming a desired product.

Embodiment 26 The conjugate of embodiment 12, wherein the PEGRNA is selected from the group consisting of the nucleotide sequences 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 SEQ ID NOs: 101-104, 181-183, 223-24 4, 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%, 85%, 90%, 95%, or 99% identical to the endogenous DNA target.

Embodiment 28. The complex of embodiment 17, wherein the primer binding site hybridizes to the free 3' end of the cut DNA.

Embodiment 29. The complex of embodiment 21, wherein at least one additional structure is located at the 3' or 5' end of the PEGRNA.

Embodiment 30. The conjugate 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.

Aspect 31. The complex of aspect 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.

Aspect 33. The complex of aspect 29, wherein the toe loop comprises a nucleotide sequence selected from the toe loops described herein.

Aspect 34. The complex of aspect 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 a target strand and (ii) a complementary non-target strand.

Embodiment 38. The complex of embodiment 37, wherein the target or complementary non-target strand is nicked to form a primed sequence having a free 3' end.

Embodiment 39. The conjugate 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 conjugate of embodiment 38, wherein the nick site is -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-stranded DNA flap hybridizes to an endogenous DNA sequence adjacent to the nick site, thereby incorporating a desired nucleotide change onto the target strand.

Embodiment 43. The complex of embodiment 22, wherein the single-stranded DNA flap has a free 5' end and replaces the endogenous DNA sequence adjacent to the nick site.

Aspect 44. The complex of aspect 22, wherein the endogenous DNA sequence having a 5' end is excised by the cell.

Aspect 45. The complex of aspect 44, wherein the endogenous DNA sequence having a 5' end is excised by a flap endonuclease.

Embodiment 46. The complex of embodiment 43, wherein cellular repair of the single-stranded DNA flap incorporates a desired nucleotide change onto the non-target strand, thereby forming a desired product.

Embodiment 47. The conjugate of embodiment 46, wherein the desired nucleotide change is incorporated over an editing window that is between about -4 and +10 of the PAM sequence, or between about -10 and +20 of the PAM sequence, or between about -20 and +40 of the PAM sequence, or between about -30 and +100 of the PAM sequence.

EMBODIMENT 48. The complex of embodiment 47, wherein the desired nucleotide change is 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.

Embodiment 49. The conjugate of any one of the preceding embodiments, wherein the fusion protein comprises the structure NH2-[napDNAbp]-[domain containing RNA-dependent DNA polymerase activity]-COOH; or NH2-[domain containing RNA-dependent DNA polymerase activity]-[napDNAbp]-COOH, each of the "]-[" indicates the presence of an optional linker sequence.

Embodiment 50. The conjugate of embodiment 49, wherein the linker sequence comprises the 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 connecting the napDNAbp and the domain comprising the RNA-dependent DNA polymerase activity.

Embodiment 52. The conjugate of embodiment 51, wherein the linker sequence comprises the amino acid sequences of SEQ ID NOs: 174 (1xSGGS), 446 (2xSGGS), 3889 (3xSGGS), 171 (1xXTEN), 3968 (1xEAAAK), 3969 (2xEAAAK), and 3970 (3xEAAAK).
Group D. PE methods for correcting mutations

Embodiment 1. A method for incorporating a desired nucleotide change into a double stranded DNA sequence, the method comprising:
contacting a double-stranded DNA sequence with a complex comprising a fusion protein and a PEgRNA, wherein the fusion protein comprises napDNAbp and a polymerase, and the PEgRNA comprises a DNA synthesis template containing the desired nucleotide change and a primer binding site;

thereby nicking the double-stranded DNA sequence, thereby generating a free single-stranded DNA having a 3' end;

thereby hybridizing the 3' end of the free single-stranded 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-stranded DNA flap that is complementary to the DNA synthesis template and contains the desired nucleotide change;

thereby replacing the endogenous DNA strand adjacent to the cut site with a single-stranded DNA flap, thereby incorporating the desired nucleotide change into the double-stranded DNA sequence;
including.

Embodiment 2. The method of embodiment 1, wherein replacing the endogenous DNA strand comprises: (i) hybridizing a single-stranded DNA flap to the endogenous DNA strand adjacent to the cut site to create a sequence mismatch; (ii) excising the endogenous DNA strand; and (iii) repairing the mismatch to form a desired product containing the desired nucleotide change in both strands of DNA.

Aspect 3. The method of aspect 1, wherein the desired nucleotide change is a single nucleotide substitution, deletion, or insertion.

Aspect 4. The method of aspect 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 is to convert (1) a G:C base pair to a T:A base pair, (2) a G:C base pair to an A:T base pair, (3) a G:C base pair to a C:G base pair, (4) a T:A base pair to a G:C base pair, (5) a T:A base pair to an A:T base pair, (6) a T:A base pair to a C:G base pair, (7) a C:G base pair to a G:C base pair, (8) a C:G base pair to a T:A base pair, a C:G base pair to an A:T base pair, (10) an A:T base pair to a T:A base pair, (11) an A:T base pair to a G:C base pair, or (12) an A:T base pair to a C:G base pair.

Aspect 7. The method of aspect 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 single gene 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; phenylketonuria; severe combined immunodeficiency; sickle cell disease; Smith-Lemli-Opitz syndrome; trinucleotide repeat disorders; prion diseases; 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; hypertension; Alzheimer's disease; arthritis; diabetes; cancer; and obesity.

Embodiment 11. The method of embodiment 1, wherein the napDNAbp is a nuclease-inactive Cas9 (dCas9), a Cas9 nickase (nCas9), or a nuclease-active Cas9.

Aspect 12. The method of aspect 1, wherein napDNAbp comprises the amino acid sequence of SEQ ID NO:18.

Embodiment 13. The method of embodiment 1, wherein napDNAbp comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any one of the amino acid sequences 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 and comprises 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, 701-716, 739-741, and 766.

Embodiment 15. The method of embodiment 1, wherein the polymerase is a reverse transcriptase and comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to 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, 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' end of the guide RNA or positioned intramolecularly, the extension arm comprising a DNA synthesis template sequence and a 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, and 776-777.

Embodiment 19. A method for introducing one or more changes in a 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 that targets the napDNAbp to a target locus, the PEgRNA comprising a reverse transcriptase (RT) template sequence containing at least one desired nucleotide change and a primer binding site;

Thereby creating an exposed 3' end of the DNA strand at the target locus;

thereby priming reverse transcription by hybridizing the exposed 3' end to the primer binding site;

thereby synthesizing, by reverse transcriptase, a single-stranded DNA flap containing at least one desired nucleotide change based on the RT template sequence;

thereby incorporating 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;
including.

Embodiment 20. The method of embodiment 19, wherein the one or more changes in the nucleotide sequence include 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 include 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 include changing (1) a G:C base pair to a T:A base pair, (2) a G:C base pair to an A:T base pair, (3) a G:C base pair to a C:G base pair, (4) a T:A base pair to a G:C base pair, (5) a T:A base pair to an A:T base pair, (6) a T:A base pair to a C:G base pair, (7) a C:G base pair to a G:C base pair, (8) a C:G base pair to a T:A base pair, (9) a C:G base pair to an A:T base pair, (9) an A:T base pair to a T:A base pair, (11) an A:T base pair to a G:C base pair, or (12) an A:T base pair to a C:G base pair.

Embodiment 25. The method of embodiment 19, wherein the one or more changes in the nucleotide sequence include 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 include correction of a disease-associated gene.

Embodiment 27. The method of embodiment 26, wherein the disease-associated gene is associated with a single gene 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; phenylketonuria; severe combined immunodeficiency; sickle cell disease; Smith-Lemli-Opitz syndrome; trinucleotide repeat disorders; prion diseases; 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; hypertension; Alzheimer's disease; arthritis; diabetes; cancer; and obesity.

Embodiment 29. The method of embodiment 19, wherein napDNAbp is a nuclease-active Cas9 or a variant thereof.

Aspect 30. The method of aspect 19, wherein napDNAbp is a nuclease-inactivated Cas9 (dCas9) or Cas9 nickase (nCas9) or a variant thereof.

Aspect 31. The method of aspect 19, wherein napDNAbp comprises the amino acid sequence of SEQ ID NO:18.

Embodiment 32. The method of embodiment 19, wherein napDNAbp comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any one of the amino acid sequences 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 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 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 to 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, 701-716, 739-741, and 766.

Embodiment 37. The method of embodiment 19, wherein the step of forming an exposed 3' end of 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 napDNAbp, is provided as a fusion domain of napDNAbp, or is provided in trans.

Aspect 39. The method of aspect 19, wherein the step of forming an exposed 3' end of the DNA strand at the target locus comprises contacting the DNA strand with a chemical agent.

Aspect 40. The method of aspect 19, wherein the step of forming an exposed 3' end of the DNA strand at the target locus includes introducing a replication error.

Embodiment 41. The method of embodiment 19, wherein contacting the DNA molecule with napDNAbp and the guide RNA forms an R-loop.

Embodiment 42. The method of embodiment 41, wherein the DNA strand on which the exposed 3' end is formed is on an R-loop.

Embodiment 43. The method of embodiment 19, wherein the PEGRNA comprises an extension arm that comprises a reverse transcriptase (RT) template sequence and a 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 of 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 toe loop, an aptamer, or an RNA-protein recruitment domain.

Aspect 46. The method of aspect 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 a nucleotide sequence of a DNA molecule at a target locus by primed reverse transcription, the method comprising: (a) contacting a DNA molecule at a target locus with (i) a fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a reverse transcriptase, and (ii) a guide RNA comprising an RT template containing a desired nucleotide change;

thereby performing primed reverse transcription on the RT template primed target to generate single-stranded DNA containing the desired nucleotide change;

thereby incorporating a desired nucleotide change onto the DNA molecule at the target locus by DNA repair and/or replication processes;
including.

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 of 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 base pair to a T:A base pair, (2) a G:C base pair to an A:T base pair, (3) a G:C base pair to a C:G base pair, (4) a T:A base pair to a G:C base pair, (5) a T:A base pair to an A:T base pair, (6) a T:A base pair to a C:G base pair, (7) a C:G base pair to a G:C base pair, (8) a C:G base pair to a T:A base pair, (9) a C:G base pair to an A:T base pair, (10) an A:T base pair to a T:A base pair, (11) an A:T base pair to a G:C base pair, or (12) an A:T base pair to a C:G base pair.

Embodiment 54. A method for replacing a trinucleotide repeat expansion mutation on a target DNA molecule with a healthy sequence containing a healthy number of repeated trinucleotides, the method comprising: (a) contacting a DNA molecule at a target locus with (i) a fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a polymerase and (ii) a DNA synthesis template comprising the replacement sequence and a PEgRNA comprising a primer binding site; (b) performing prime editing to generate a single-stranded DNA comprising the replacement sequence; and (c) incorporating the single-stranded DNA onto the DNA molecule at the target locus by DNA repair and/or replication processes.

Embodiment 55. The method of embodiment 54, wherein the fusion protein comprises an amino acid sequence of PE1, PE2, or PE3.

Embodiment 56. The method of embodiment 54, wherein the napDNAbp is Cas9 nickase (nCas9).

Embodiment 57. The method of embodiment 54, wherein the napDNAbp comprises the amino acid sequences 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 NO: 222.

Embodiment 59. The method of embodiment 54, wherein (b) the step of performing prime editing comprises generating a 3' end primer binding sequence at the target locus that can prime a polymerase by annealing to a 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 repeat unit of a CAG triplet.

Embodiment 62. The method of embodiment 54, wherein the trinucleotide repeat expansion mutation comprises a repeat unit of a GAA triplet.

63. A method for introducing one or more changes in a nucleotide sequence of a DNA molecule at a target locus by primed target-primed reverse transcription, the method comprising:
(a) contacting a DNA molecule at a target locus with (i) a fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a reverse transcriptase, and (ii) a guide RNA comprising an RT template containing a desired nucleotide change;

thereby performing primed reverse transcription on the RT template primed target to generate single-stranded DNA containing the desired nucleotide change;

thereby incorporating a desired nucleotide change onto the DNA molecule at the target locus by DNA repair and/or replication processes;
including.

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 of 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 base pair to a T:A base pair, (2) a G:C base pair to an A:T base pair, (3) a G:C base pair to a C:G base pair, (4) a T:A base pair to a G:C base pair, (5) a T:A base pair to an A:T base pair, (6) a T:A base pair to a C:G base pair, (7) a C:G base pair to a G:C base pair, (8) a C:G base pair to a T:A base pair, (9) a C:G base pair to an A:T base pair, (10) an A:T base pair to a T:A base pair, (11) an A:T base pair to a G:C base pair, or (12) an A:T base pair to a C:G base pair.

Embodiment 69. A method for preventing or halting the progression of a prion disease by incorporating one or more protective mutations in a PRNP encoded by a target nucleotide sequence by prime editing, the method comprising: (a) contacting the target nucleotide sequence with (i) a prime editing factor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a polymerase, and (ii) a PEgRNA comprising an editing template encoding a functional moiety;

thereby polymerizing a single-stranded DNA sequence that codes for a protective mutation;

thereby incorporating the single-stranded DNA sequence in place of the corresponding endogenous strand at the target nucleotide sequence by DNA repair and/or replication processes;
Includes;

The method produces a recombinant target nucleotide sequence encoding a PRNP that contains a protective mutation that is resistant to misfolding.

Aspect 70. The method of aspect 69, wherein the prion disease is a human prion disease.

Aspect 71. The method of aspect 69, wherein the prion disease is an animal prion disease.

Aspect 72. The method of aspect 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.

Aspect 74. The method of aspect 69, wherein the wild-type 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, and the modified PRNP protein is resistant to misfolding.

Aspect 76. The method of aspect 69, wherein the fusion protein comprises an amino acid sequence of PE1, PE2, or PE3.

Aspect 77. The method of aspect 69, wherein the napDNAbp is Cas9 nickase (nCas9).

Embodiment 78. The method of embodiment 69, wherein the napDNAbp comprises the amino acid sequences 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 for treating a CDKL5 deficiency disorder by correcting a mutation in a cyclin-dependent kinase-like 5 gene (CDKL5) on a target nucleotide sequence by prime editing, the method comprising: (a) contacting the target nucleotide sequence with (i) a prime editing factor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a polymerase, and (ii) a PEgRNA comprising an editing template that corrects the mutation in CDKL5;

Thereby polymerizing a single-stranded DNA sequence that codes for the edit;

thereby incorporating the single-stranded DNA sequence in place of the corresponding endogenous strand at the target nucleotide sequence by DNA repair and/or replication processes;
Includes;

The method produces a recombinant target nucleotide sequence encoding a repaired CDKL5 gene.

Embodiment 81. The method of embodiment D80, wherein the CDKL5 mutation is 1412delA.

Group E. PE Methods for Modifying and/or Mutating Protein Structure/FunctionEmbodiment 1. A method for mutating a DNA molecule at a target locus by prime editing, comprising: (a) contacting the DNA molecule at the target locus with (i) a fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) and an error-prone polymerase (e.g., an error-prone reverse transcriptase) and (ii) a guide RNA comprising an editing template comprising a desired nucleotide change; thereby polymerizing single-stranded DNA templated by the editing template, and incorporating the single-stranded DNA onto the DNA molecule at the target locus by DNA repair and/or replication processes.

Aspect 2. The method of any preceding aspect, wherein the fusion protein comprises an amino acid sequence of PE1, PE2, or PE3.

Aspect 3. The method of any preceding aspect, wherein the napDNAbp is Cas9 nickase (nCas9).

Embodiment 4. The method of embodiment 1, wherein napDNAbp comprises the amino acid sequences of SEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.

Aspect 5. The method of aspect 1, wherein the guide RNA comprises SEQ ID NO: 222.

Embodiment 6. A method for incorporating an immune epitope onto a protein of interest encoded by a target nucleotide sequence by prime editing, the method comprising: (a) contacting the target nucleotide sequence with (i) a prime editing factor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a polymerase, and (ii) a PEgRNA comprising an editing template encoding a functional moiety;

Thereby polymerizing a single-stranded DNA sequence encoding an immune epitope;

thereby incorporating the single-stranded DNA sequence in place of the corresponding endogenous strand at the target nucleotide sequence by DNA repair and/or replication processes;
Includes;

The method produces a recombinant target nucleotide sequence that encodes a fusion protein containing the protein of interest and an immune epitope.

Embodiment 7. The method of embodiment 6, wherein the immune epitope is: tetanus toxoid (SEQ ID NO: 396); diphtheria toxin variant CRM197 (SEQ ID NO: 630); mumps immune epitope 1 (SEQ ID NO: 400); mumps immune epitope 2 (SEQ ID NO: 402); mumps immune epitope 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 epitope for class I HLA (SEQ ID NO: 416); neuraminidase epitope for class I HLA (SEQ ID NO: 418); hemagglutinin epitope for class II HLA (SEQ ID NO: 420); neuraminidase epitope for class II HLA (SEQ ID NO: 422); hemagglutinin epitope H5N1 binding class I and class II HLA (SEQ ID NO: 424); and neuraminidase epitope H5N1-binding class I and class II HLA (SEQ ID NO: 426).

Aspect 8. The method of aspect 6, wherein the fusion protein comprises an amino acid sequence of PE1, PE2, or PE3.

Embodiment 9. The method of embodiment 6, wherein the napDNAbp is Cas9 nickase (nCas9).

Embodiment 10. The method of embodiment 6, wherein napDNAbp comprises the amino acid sequences 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 for incorporating a small dimerization domain onto a protein of interest encoded by a target nucleotide sequence by prime editing, the method comprising: (a) contacting the target nucleotide sequence with (i) a prime editing factor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a polymerase, and (ii) a PEgRNA comprising an editing template encoding the small dimerization domain;

Thereby polymerizing a single-stranded DNA sequence encoding an immune epitope;

thereby incorporating the single-stranded DNA sequence in place of the corresponding endogenous strand at the target nucleotide sequence by DNA repair and/or replication processes;
Includes;

The method produces a modified target nucleotide sequence that encodes a fusion protein comprising the protein of interest and a small dimerization domain.

Aspect 13. The method of aspect 12, further comprising performing 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 domains of each of the proteins.

Aspect 15. The method of aspect 12, wherein the small molecule binding domain is FKBP12 of SEQ ID NO:488.

Aspect 16. The method of aspect 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 a cyclophilin of SEQ ID NOs: 490 and 493-494.

Aspect 18. The method of aspect 12, wherein the small molecule is a dimer of a small molecule described herein.

Aspect 19. The method of aspect 12, wherein the fusion protein comprises an amino acid sequence of PE1, PE2, or PE3.

Aspect 20. The method of aspect 12, wherein the napDNAbp is Cas9 nickase (nCas9).

Embodiment 21. The method of embodiment 12, wherein the napDNAbp comprises the amino acid sequences 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 incorporating a peptide tag or epitope onto a protein using prime editing, comprising: contacting a target nucleotide sequence encoding a protein with a prime editor construct configured to insert therein a second nucleotide sequence encoding a peptide tag, resulting 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 purifying and/or detecting the protein.

Embodiment 25 The method of embodiment 23, wherein the peptide tag is polyhistidine (e.g., HHHHHH , SEQ ID NOs: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 NOs: 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], where "]-[" represents an optional linker.

Embodiment 29. The method of embodiment 23, wherein the linker has an amino acid sequence of SEQ ID NOs: 127, 165-176, 446, 453, and 767-769.

Embodiment 30. The method of embodiment 23, wherein the prime editor construct comprises a PEgRNA comprising a 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, the spacer being complementary to a target nucleotide sequence, and the extension arm comprises a reverse transcriptase template encoding a peptide tag.

Embodiment 32. The method of embodiment 23, wherein the PEGRNA comprises a spacer, a gRNA core, and an extension arm, the spacer being complementary to a target nucleotide sequence, and the extension arm comprises a reverse transcriptase template encoding a peptide tag.

Embodiment 33. A method for inserting or deleting a functional portion on a protein of interest encoded by a target nucleotide sequence by prime editing, the method comprising: (a) contacting the target nucleotide sequence with (i) a prime editing factor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a polymerase and (ii) a PEgRNA comprising an editing template encoding the functional portion or deletion; (b) polymerizing a single-stranded DNA sequence encoding the functional portion or deletion; and (c) incorporating the single-stranded DNA sequence in place of the corresponding endogenous strand in the target nucleotide sequence by DNA repair and/or replication processes, the method producing a recombinant target nucleotide sequence encoding the protein of interest and an altered protein comprising the functional portion or deletion, the functional portion altering the modification or localization state of the protein.

Embodiment 34. The method of embodiment 33, wherein the functional moiety alters the phosphorylation, ubiquitination, glycosylation, lipid modification, hydroxylation, methylation, acetylation, crotonylation, or sumoylation status of the protein of interest.

Group F. PE Delivery Methods and Compositions Embodiment 1. A polypeptide comprising the N-terminal or C-terminal half of a prime editor fusion protein.

Aspect 2. The polypeptide of aspect 1, wherein the prime editor fusion protein comprises a nucleic acid programmable DNA binding protein (napDNAbp) domain and a polymerase domain.

Aspect 3. The polypeptide of aspect 1, wherein the prime editor fusion protein is capable of performing prime editing in the presence of a prime editing guide RNA (PEgRNA).

Aspect 4. The polypeptide of aspect 2, wherein napDNAbp is a Cas9 protein or a variant thereof.

Aspect 5. The polypeptide of aspect 2, wherein napDNAbp is a nuclease having nickase activity.

Aspect 6. The polypeptide of aspect 2, wherein napDNAbp is a nuclease-active Cas9, a nuclease-inactive Cas9 (dCas9), or a Cas9 nickase (nCas9).

Aspect 7. The polypeptide of aspect 2, wherein napDNAbp is selected from the group consisting of: Cas9, Cas12e, Cas12d, Cas12a, Cas12b1, Cas13a, Cas12c, and Argonaute, and optionally has nickase activity.

Aspect 8. The polypeptide of aspect 1, wherein the polypeptide is formed by cleaving a prime editor fusion protein at a cleavage site.

Aspect 9. The polypeptide of aspect 8, wherein the cleavage site is a peptide bond on the napDNAbp domain.

Aspect 10. The polypeptide of aspect 8, wherein the cleavage site is a peptide bond on the polymerase domain.

Embodiment 11. The polypeptide of embodiment 8, wherein the cleavage site is a peptide bond on the linker between the napDNAbp domain and the polymerase domain.

12. The polypeptide of claim 9, wherein the division site is selected from 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 and (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 of SEQ ID NO: 18 (standard SpCas9). 9, 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 residues 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 45 A peptide bond between any two residues between 0-500, 500-600, 600-700, 700-800, 800-900, 900-1000, 1000-1100, 1100-1200, 1200-1300, or 1300-1368, or between any two equivalent amino acid residues of an SpCas9 homolog or equivalent of SEQ ID NO:18.

13. The polypeptide of embodiment 9, wherein the cleavage site is selected from 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 and (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 of SEQ ID NO: 89 (standard reverse transcriptase, M-MLV RT). , 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, 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 the N-terminal half of a prime editor fusion protein.

Embodiment 15. The polypeptide of embodiment 1, wherein the polypeptide is the C-terminal half of a prime editor fusion protein.

Aspect 16. A nucleotide sequence encoding a polypeptide of any one of aspects 1 to 15 and optionally a PEG RNA.

Aspect 17. A viral genome comprising a nucleotide sequence encoding a polypeptide of any one of aspects 1 to 15 and optionally a PEgRNA.

Aspect 18. The viral genome of aspect 17, wherein the nucleotide sequence further comprises a promoter sequence suitable for expressing the polypeptide of any of aspects 1-15.

Aspect 19. The viral genome of aspect 17, wherein the nucleotide sequence further comprises a sequence encoding a PEgRNA.

Embodiment 20. A viral particle comprising a genome comprising a nucleotide sequence encoding a polypeptide of any one of embodiments 1 to 15 and optionally a PEgRNA.

Embodiment 21. The viral particle of embodiment 20, wherein the viral particle is an adenovirus particle, an adeno-associated virus particle, or a lentivirus particle.

Embodiment 22. The viral particle of embodiment 20, wherein the polypeptide encoded by the genome is the N-terminal half of a prime editor fusion protein.

Embodiment 23. The viral particle of embodiment 20, wherein the polypeptide encoded by the genome is the C-terminal half of a prime editor fusion protein.

Aspect 24. A pharmaceutical composition comprising a virus particle according to any one of aspects 20 to 23 and a pharmaceutical excipient.

Aspect 25. A pharmaceutical composition comprising a viral particle of aspect 22 (encoding the N-terminal half) and a pharmaceutical excipient.

Aspect 26. A pharmaceutical composition comprising a viral particle of aspect 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 one of embodiments 1 to 15 and, optionally, a PEgRNA.

Embodiment 28. The ribonucleoprotein (RNP) complex of embodiment 27, wherein the polypeptide encoded by the genome is the 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 the C-terminal half of a prime editor fusion protein.

Aspect 30. A pharmaceutical composition comprising a ribonucleoprotein (RNP) complex of any one of aspects 27 to 29 and a pharmaceutical excipient.

Aspect 31. A pharmaceutical composition comprising a ribonucleoprotein (RNP) complex (encoding the N-terminal half) of aspect 28 and a pharmaceutical excipient.

Aspect 32. A pharmaceutical composition comprising a ribonucleoprotein (RNP) complex (encoding the C-terminal half) of aspect 29 and a pharmaceutical excipient.

Aspect 33. A pharmaceutical composition comprising a first AAV particle and a second AAV particle, the first AAV vector expressing an N-terminal half of a prime editor fusion protein and the second AAV vector expressing a C-terminal half of the prime editor fusion protein, the N-terminal half and the C-terminal half combining in a 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, which targets the reconstituted primed editing element to the target DNA site.

Aspect 35. The pharmaceutical composition of aspect 33, wherein the prime editor fusion protein comprises a nucleic acid programmable DNA binding protein (napDNAbp) domain and a polymerase domain.

Aspect 36. The pharmaceutical composition of aspect 33, wherein the prime editor fusion protein is capable of performing prime editing in the presence of a prime editing guide RNA (PEgRNA).

Aspect 37. The pharmaceutical composition of aspect 35, wherein napDNAbp is a Cas9 protein or a variant thereof.

Aspect 38. The pharmaceutical composition of aspect 35, wherein napDNAbp is a nuclease having nickase activity.

Aspect 39. The pharmaceutical composition of aspect 35, wherein napDNAbp is a nuclease-active Cas9, a nuclease-inactive Cas9 (dCas9), or a Cas9 nickase (nCas9).

Aspect 40. The pharmaceutical composition of aspect 35, wherein the napDNAbp is selected from the group consisting of: Cas9, Cas12e, Cas12d, Cas12a, Cas12b1, Cas13a, Cas12c, and Argonaute, and optionally has nickase activity.

Embodiment 41. The pharmaceutical composition of embodiment 33, wherein the N-terminal and C-terminal halves are formed by cleaving the prime editor fusion protein at a cleavage site.

Aspect 42. The pharmaceutical composition of aspect 41, wherein the cleavage site is a peptide bond on the napDNAbp domain.

Aspect 43. The pharmaceutical composition of aspect 41, wherein the cleavage site is a peptide bond on the polymerase domain.

Aspect 44. The pharmaceutical composition of aspect 41, wherein the cleavage site is a peptide bond on the linker.

Embodiment 45. The pharmaceutical composition of embodiment 41, wherein the cleavage site is selected from 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 and (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 of SEQ ID NO: 18 (standard SpCas9). 9, 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 residues 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 45 A peptide bond between any two residues between 0-500, 500-600, 600-700, 700-800, 800-900, 900-1000, 1000-1100, 1100-1200, 1200-1300, or 1300-1368, 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 cleavage site is selected from 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 and (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 of SEQ ID NO: 89 (standard reverse transcriptase, M-MLV RT). , 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, or between any two equivalent amino acid residues of a reverse transcriptase homolog or equivalent of SEQ ID NO:89.

Aspect 47. The pharmaceutical composition of aspect 33, wherein the N-terminal half of the prime editor fusion protein has an amino acid sequence encoding an N-terminal prime editor fusion protein described herein.

Aspect 48. The pharmaceutical composition of aspect 33, wherein the C-terminal half of the prime editor fusion protein has an amino acid sequence encoding an N-terminal prime editor fusion protein 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 the N-terminal half of the prime editor fusion protein and the second AAV vector expresses the C-terminal half of the prime editor fusion protein, and wherein the N-terminal and C-terminal halves combine in 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, which targets the reconstituted primed editing element to the 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 performing prime editing in the presence of a prime editing guide RNA (PEgRNA).

Embodiment 53. The method of embodiment 51, wherein the napDNAbp is a Cas9 protein or a variant thereof.

Embodiment 54. The method of embodiment 53, wherein napDNAbp is a nuclease having nickase activity.

Embodiment 55. The method of embodiment 53, wherein 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 nickase activity.

Embodiment 57. The method of embodiment 49, wherein the N-terminal and C-terminal halves are formed by cleaving the prime editor fusion protein at a cleavage site.

Embodiment 58. The method of embodiment 57, wherein the cleavage site is a peptide bond on the napDNAbp domain.

Embodiment 59. The method of embodiment 57, wherein the cleavage site is a peptide bond on the polymerase domain.

Embodiment 60. The method of embodiment 57, wherein the cleavage site is a peptide bond on the linker.

Embodiment 61. The method of embodiment 57, wherein the division site is selected from 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 and (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, Between residues 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 residues 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450 A peptide bond between any two residues between 500, 500-600, 600-700, 700-800, 800-900, 900-1000, 1000-1100, 1100-1200, 1200-1300, or 1300-1368, 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 cleavage site is selected from 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 and (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 of SEQ ID NO:89 (standard reverse transcriptase, M-MLV RT). , 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, 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 an N-terminal prime editor fusion protein 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 a C-terminal prime editor fusion protein described herein.

Embodiment 65. The method of embodiment 49, wherein the first AAV particle comprises a recombinant AAV genome that includes a nucleotide sequence encoding a first prime editor component.

Embodiment 66. The method of embodiment 49, wherein the second AAV particle comprises a recombinant AAV genome that includes a nucleotide sequence encoding a second prime editing factor component.

Aspect 67. The method of aspect 49, wherein the transfection step is performed in vivo.

Aspect 68. The method of aspect 49, wherein the transfection step is performed ex vivo.

Aspect 69. The method of aspect 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 single gene 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; phenylketonuria; severe combined immunodeficiency; sickle cell disease; Smith-Lemli-Opitz syndrome; trinucleotide repeat disorders; prion diseases; 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; hypertension; Alzheimer's disease; arthritis; diabetes; cancer; and obesity.

Embodiment 72. The method of embodiment 51, comprising a 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 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 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 to 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, 701-716, 739-741, and 766.

Aspect 76. The method of aspect 73, wherein napDNAbp comprises the 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 to an 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 division site is between 1023 and 1024 of SEQ ID NO: 18, or a corresponding position on 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 for incorporating a ribonucleotide motif or tag on a desired RNA encoded by a target nucleotide sequence by prime editing, the method comprising: (a) contacting the target nucleotide sequence with (i) a prime editing factor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a polymerase, and (ii) a PEgRNA comprising an editing template encoding the ribonucleotide motif or tag; thereby polymerizing a single-stranded DNA sequence encoding the ribonucleotide motif or tag; and incorporating the single-stranded DNA sequence in place of the corresponding endogenous strand in the target nucleotide sequence by DNA repair and/or replication processes, the method resulting in a target nucleotide sequence encoding a modified RNA of interest that includes the ribonucleotide motif or tag.

Embodiment 2. The method of embodiment 1, wherein the ribonucleotide motif or tag is the 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 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 PEG RNA 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).

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

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

Embodiment 9. The method of embodiment 1, wherein napDNAbp comprises the amino acid sequences 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 for constructing a programmed mutant gene library by prime editing, the method comprising:

(a) contacting a library of target nucleotide sequences, each of which comprises one or more target gene loci, with (i) a primed editing factor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a polymerase, and (ii) a PEgRNA comprising an editing template comprising a sequence having at least one genetic change relative to one or more target gene loci;

thereby polymerizing a single-stranded DNA sequence using the editing template as a template; and

incorporating a single stranded DNA sequence in place of one or more target gene loci by DNA repair and/or replication processes, thereby incorporating at least one genetic alteration onto the target gene loci of the target nucleotide sequences of said library;
including.

Aspect 2. The method of aspect 1, wherein the library is a plasmid library.

Aspect 3. The method of aspect 1, wherein the library is a phage library.

Embodiment 4. The method of embodiment 1, wherein one or more target gene loci include a protein-coding region.

Embodiment 5. The method of embodiment 1, wherein one or more target gene loci include regions encoding protein secondary structure motifs.

Aspect 6. The method of aspect 5, wherein the secondary structure motif is an alpha helix.

Aspect 7. The method of aspect 5, wherein the secondary structure motif is a beta sheet.

Embodiment 8. The method of embodiment 1, wherein napDNAbp is a Cas9 protein or a variant thereof.

Aspect 9. The method of aspect 1, wherein napDNAbp is a nuclease having nickase activity.

Embodiment 10. The method of embodiment 1, wherein napDNAbp is a nuclease-active Cas9, a nuclease-inactive Cas9 (dCas9), or a Cas9 nickase (nCas9).

Aspect 11. The method of aspect 1, wherein napDNAbp comprises the amino acid sequence of SEQ ID NO:18.

Embodiment 12. The method of embodiment 1, wherein napDNAbp comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.

Aspect 13. The method of aspect 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 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 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 to 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, 701-716, 739-741, and 766.

Embodiment 16. The method of embodiment 1, wherein at least one genetic change in the editing template is an insertion.

Embodiment 17. The method of embodiment 1, wherein at least one genetic alteration in the editing template is a deletion.

Embodiment 18. The method of embodiment 1, wherein at least one genetic change in the editing template is a substitution.

Aspect 19. The method of aspect 1, wherein at least one genetic change is an insertion of one or more codons.

Aspect 20. The method of aspect 1, wherein at least one genetic alteration is a deletion of one or more codons.

Embodiment 21. The method of embodiment 1, wherein at least one genetic alteration is the insertion of a stop codon.

Embodiment 22. The method of embodiment 1, wherein at least one genetic change is a conversion of a non-stop codon to a stop codon.

Embodiment 23. The method of embodiment 1, wherein the method is performed simultaneously for at least 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or between 10 and 100, or between 100 and 200, or between 200 and 300, or between 300 and 400, or between 400 and 500 target loci for each of the target nucleotide sequences of the library.

Embodiment 24. The method of embodiment 1, wherein the method is performed during PACE or PANCE evolution, and each of the incorporating at least one genetic change into a target locus of a target nucleotide sequence of the library also incorporates a new target sequence.

Group I. PE Methods for Off-Target Detection Embodiment 1. A method for assessing off-target editing by a primed editor, the method comprising:

(a) contacting a target nucleotide sequence having an editing site with (i) a primed editor fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a polymerase, and (ii) a PEgRNA comprising a DNA synthesis template encoding a detectable sequence;

The PEgRNA complexes with the fusion protein and guides the fusion protein to the editing site and, if present, to one or more off-target sites;

The prime editor fusion protein incorporates a detectable sequence at the editing site and, if present, at one or more off-target sites;

(b) determining the nucleotide sequence of the editing site and one or more off-target sites;
including.

Aspect 2. The method of aspect 1, wherein the target nucleotide sequence is a genome.

Aspect 3. The method of aspect 1, wherein the contacting step is in vitro.

Aspect 4. The method of aspect 1, wherein the contacting step is in vivo.

Aspect 5. The method of aspect 1, wherein the editing site is a mutation in a disease-associated gene.

Aspect 6. The method of aspect 5, wherein the mutation is a single base substitution, an insertion, a deletion, or an inversion.

Embodiment 7. The method of embodiment 1, wherein the disease-associated gene is associated with a single-gene 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; phenylketonuria; 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; hypertension; Alzheimer's disease; arthritis; diabetes; cancer; and obesity.

Embodiment 9. The method of embodiment 1, wherein the fusion protein comprises an amino acid 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, or SEQ ID NOs: 101-104, It has an amino acid sequence that has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with 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, and 776-777.

Embodiment 10. The method of embodiment 1, wherein 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 napDNAbp is Cas9 or a variant thereof.

Embodiment 12. The method of embodiment 1, wherein 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 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 to 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 the amino acids of SEQ ID NOs : 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 to 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 napDNAbp is an SpCas9 orthologue.

Embodiment 17. The method of embodiment 1, wherein 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 to any one 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 napDNAbp comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any one of the amino acid sequences 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 RNA-dependent DNA polymerase activity.

Aspect 20. The method of aspect 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 NO:89, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any one of SEQ ID NO:89.

Aspect 22. The method of embodiment 1, wherein the reverse transcriptase is a variant reverse transcriptase and has 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 to 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 23. The method of embodiment 1, wherein the reverse transcriptase comprises 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, 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 to 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, 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 base pair to a T:A base pair, (2) a G:C base pair to an A:T base pair, (3) a G:C base pair to a C:G base pair, (4) a T:A base pair to a G:C base pair, (5) a T:A base pair to an A:T base pair, (6) a T:A base pair to a C:G base pair, (7) a C:G base pair to a G:C base pair, (8) a C:G base pair to a T:A base pair, (9) a C:G base pair to an A:T base pair, (10) an A:T base pair to a T:A base pair, (11) an A:T base pair to a G:C base pair, or (12) an A:T base pair to a C:G base pair.

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 the amplicon using a pair of primers, one primer annealing to the adapter sequence attached to one end of the fragment and another primer annealing to the adapter sequence inserted by the primed edit located on the fragment; and (iv) sequencing the amplicon to determine the location of the edit.

Group J. PE Methods for Cellular Data Recording Embodiment 1. A method for 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 napDNAbp and an RNA-dependent DNA polymerase and (ii) a PEgRNA, wherein expression of the fusion protein and/or the PEgRNA is induced by the occurrence of a cellular event, and expression of the fusion protein and/or the PEgRNA results in prime editing of a target editing site on the genome of the cell to introduce a detectable sequence, and (B) identifying the detectable sequence, thereby identifying the occurrence of the cellular event.

Aspect 2. The method of aspect 1, wherein the prime editing of step (A) simultaneously introduces new target editing sites, such that recording of cellular events can occur repetitively.

Embodiment 3. The method of embodiment 1, wherein the PEgRNA comprises an editing template that encodes a detectable sequence.

Embodiment 4. The method of embodiment 3, wherein the editing template further encodes a new target editing 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.

Aspect 7. The method of aspect 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, 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 base pair to a T:A base pair, (2) a G:C base pair to an A:T base pair, (3) a G:C base pair to a C:G base pair, (4) a T:A base pair to a G:C base pair, (5) a T:A base pair to an A:T base pair, (6) a T:A base pair to a C:G base pair, (7) a C:G base pair to a G:C base pair, (8) a C:G base pair to a T:A base pair, (9) a C:G base pair to an A:T base pair, (10) an A:T base pair to a T:A base pair, (11) an A:T base pair to a G:C base pair, or (12) an A:T base pair to a C:G base pair.

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 repeated insertions 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 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 napDNAbp is Cas9 or a variant thereof.

Embodiment 17. The method of embodiment 1, wherein 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 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 to SEQ ID NO:18.

Embodiment 20. The method of embodiment 1, wherein the napDNAbp is SpCas9 wild type or a variant thereof of an 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, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to 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 napDNAbp is an SpCas9 orthologue.

Embodiment 22 The method of embodiment 1, wherein the napDNAbp is an 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, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.

Aspect 23. The method of aspect 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 NO:89, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any one of SEQ ID NO:89.

Aspect 25. The method of embodiment 23, wherein the reverse transcriptase is a variant reverse transcriptase and has 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 to 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 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 to any one of SEQ ID NOs: 123 and 134 (PE1, PE2).

Embodiment 27. The method of embodiment 1, wherein the one or more constructs encoding the prime editor fusion protein and/or the PEgRNA further comprise one or more promoters that are inducible when a 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 that occurs during activation of an endogenous or exogenous signaling cascade, light, heat, sound, pressure, mechanical stress, shear stress, or a virus or other microorganism, a change in pH, or a change in redox state.

Embodiment 30. A cellular data recording plasmid for recording cellular events using prime editing, comprising:

i. a fusion protein comprising (a) a nucleic acid sequence encoding a nucleic acid programmable DNA binding protein (napDNAbp) and (b) an RNA-dependent DNA polymerase, the 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, the PEgRNA being complementary to a target sequence; and

iii. Replication origin;
Including,

At least one of the promoters is an inducible promoter, and the PEgRNA associates with the napDNAbp under conditions that induce expression of the PEgRNA and expression of the napDNAbp sufficient to incorporate a detectable sequence at the target editing site.

Aspect 31. The cell data recording plasmid of aspect 30, wherein the inducible promoter is induced by a cellular event.

Aspect 32. The cell data recording plasmid of aspect 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 that occurs during activation of an endogenous or exogenous signaling cascade, light, heat, sound, pressure, mechanical stress, shear stress, or a virus or other microorganism, a change in pH, or a change in redox state.

Aspect 34. The cell data recording plasmid of aspect 30, wherein the first and second promoters are the same.

Aspect 35. The cell data recording plasmid of aspect 30, wherein the first and second promoters are different.

Aspect 36. The cell data recording plasmid of aspect 30, wherein at least one inducible promoter is an anhydrotetracycline-inducible promoter, an IPTG-inducible promoter, a rhamnose-inducible promoter, or an arabinose-inducible promoter.

Aspect 37. The cell data recording plasmid of aspect 30, wherein the first or second promoter is a constitutive promoter.

Aspect 38. The cell data recording plasmid of aspect 37, wherein the constitutive promoter is a Lac promoter, a cytomegalovirus (CMV) promoter, a constitutive RNA polymerase III promoter, or a UBC promoter.

Aspect 39. The cell data recording plasmid of aspect 30, wherein the origin of replication comprises a pSC101, pMB1, pBR322, ColE1, or p15A origin of replication sequence.

Aspect 40. The cell data recording plasmid of aspect 30, wherein the PEgRNA comprises an editing template that encodes a detectable sequence.

Aspect 41. The cell data recording plasmid of aspect 40, wherein the editing template further encodes a new target editing 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.

Aspect 44. The cell data recording plasmid of aspect 30, wherein the detectable sequence is a nucleic acid base substitution.

Aspect 45. The cell data recording plasmid of aspect 30, wherein the detectable sequence is a transition mutation.

Aspect 46. The cell data recording plasmid of aspect 30, wherein the detectable sequence is a transversion mutation.

Aspect 47. The cell data recording plasmid of aspect 30, wherein the detectable sequence is a single nucleotide substitution, 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 base pair to a T:A base pair, (2) a G:C base pair to an A:T base pair, (3) a G:C base pair to a C:G base pair, (4) a T:A base pair to a G:C base pair, (5) a T:A base pair to an A:T base pair, (6) a T:A base pair to a C:G base pair, (7) a C:G base pair to a G:C base pair, (8) a C:G base pair to a T:A base pair, (9) a C:G base pair to an A:T base pair, (10) an A:T base pair to a T:A base pair, (11) an A:T base pair to a G:C base pair, or (12) an A:T base pair to a C:G base pair.

Aspect 49. The cell data recording plasmid of aspect 30, wherein the detectable sequence is a barcode sequence.

Aspect 50. The cellular data recording plasmid of aspect 30, wherein the detectable sequence increases in length over time as a result of repeated insertions of the detectable sequence for each occurrence of a cellular event.

Aspect 51. The cell data recording plasmid of aspect 30, wherein the detecting step comprises sequencing the edited target site or an amplicon of the edited target site.

Aspect 52. The cell data recording plasmid of aspect 30, wherein napDNAbp is Cas9, Cas12e, Cas12d, Cas12a, Cas12b1, Cas13a, Cas12c, or Argonaute, or a variant of Cas9, Cas12e, Cas12d, Cas12a, Cas12b1, Cas13a, Cas12c, or Argonaute.

Aspect 53. The cell data recording plasmid of aspect 30, wherein napDNAbp is Cas9 or a variant thereof.

Embodiment 54. The cell data recording plasmid of embodiment 30, wherein napDNAbp is a nuclease-active Cas9, a nuclease-inactive Cas9 (dCas9), or a Cas9 nickase (nCas9).

Aspect 55. The cell data recording plasmid of aspect 30, wherein the napDNAbp is Cas9 nickase (nCas9).

Aspect 56. The cell data recording plasmid of aspect 30, wherein 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 to SEQ ID NO:18.

Embodiment 57. The cell data recording plasmid of embodiment 30, wherein the napDNAbp is an SpCas9 wild type or a variant thereof of an 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, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any of SEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.

Aspect 58. The cell data recording plasmid of aspect 30, wherein napDNAbp is an SpCas9 orthologue.

Embodiment 59 The cell data recording plasmid of embodiment 30, wherein napDNAbp is an 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, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.

Aspect 60. The cell data recording plasmid of aspect 30, wherein the RNA-dependent DNA polymerase is a reverse transcriptase.

Aspect 61. The cell data recording plasmid of aspect 30, wherein the reverse transcriptase is a naturally occurring wild-type reverse transcriptase having an amino acid sequence of any one of SEQ ID NO:89, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any one of SEQ ID NO:89.

Aspect 62. The cell data recording plasmid of embodiment 30, wherein the reverse transcriptase is a variant reverse transcriptase and has 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 to 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.

Aspect 63. The cell data recording plasmid of aspect 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 to any one of SEQ ID NOs: 123 and 134 (PE1, PE2).

Aspect 64. A kit for use with a cell comprising the cell data recorder plasmid of any one of aspects 30-63.

Aspect 65. The kit of aspect 64, wherein the cell is a prokaryotic cell.

Aspect 66. The kit of aspect 64, wherein the cell is a eukaryotic cell.

Aspect 67. A cell comprising a cell data recording plasmid according to any one of aspects 30-63.

Aspect 68. The cell of aspect 67, wherein the cell is a prokaryotic cell.

Aspect 69. The cell of aspect 67, wherein the cell is a eukaryotic cell.

Aspect 70. The cell of aspect 69, wherein the eukaryotic cell is a mammalian cell.

Aspect 71. The cell of aspect 70, wherein the mammalian cell is a human cell.

Embodiment 72. A method for recording a cellular event by prime editing, the method comprising: (A) introducing into a cell a cellular data recording plasmid of any of embodiments 30-63, wherein a fusion protein and/or a PEgRNA is induced by the occurrence of a cellular event, and expression of the fusion protein and/or the PEgRNA results in prime editing of a target editing 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.

Aspect 73. The method of aspect 72, wherein (A) the introducing step is by transfection or electroporation.

Equivalents and Scope In the claims, articles such as "a,""an," and "the" may mean one or more than one, unless otherwise indicated to the contrary or otherwise clear from the context. A claim or description including "or" between one or more members of a group is considered to satisfy that one, more than one, or all of the members of the group are present in, employed in, or otherwise relevant to a given product or process, unless otherwise indicated to the contrary or otherwise clear 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 members of the group are present in, employed in, or otherwise relevant to a given product or process.

Moreover, the present invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the enumerated claims are introduced into another claim. For example, any claim that is dependent on another claim may be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as being enumerated in Markush group format, as an example, each subgroup of elements is also disclosed, and any element or elements may be removed from the group. In general, when the invention, or aspects of the invention, are described as including certain elements and/or features, it should be understood that an embodiment of the invention or an aspect of the invention consists of, or consists essentially of, such elements and/or features. For purposes of brevity, those embodiments have not been specifically recited in haec verba herein. It is also noted that the terms "comprising" and "containing" are intended to be open and permit the inclusion of additional elements or steps. When ranges are given, the endpoints are also included. Moreover, unless otherwise indicated or otherwise clear from the context and the understanding of one of ordinary skill in the art, values expressed as ranges may assume any specific value or subrange within the ranges set forth in different aspects of the invention, up to 10 times the unit of the lower limit of the range, unless the context clearly dictates otherwise.

This application makes reference to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. In the event of a conflict between any of the incorporated references and this specification, this specification shall control. In addition, any particular aspect of the present invention that is within the prior art may be expressly excluded from any one or more of the claims. Because such aspects would be known to those of skill in the art, they may be excluded even if the exclusion is not expressly stated herein. Any particular aspect of the present invention may be excluded from any claim for any reason, whether or not related to the existence of prior art.

Those of ordinary skill in the art will recognize, or be able to ascertain, at most using routine experimentation, many equivalents to the specific embodiments described herein. The scope of the embodiments described herein is not intended to be limited to the above description, but rather is as set forth in the appended claims. 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 invention, as defined in the following claims.

Claims (24)

(i) an N-terminal fragment of a prime editor fusion protein and an N-terminal extein comprising an N-intein, or a polynucleotide encoding an N-terminal extein; and
(ii) a C-terminal fragment of a primed editor fusion protein and a C-terminal extein comprising a C-intein, or a prime editing system comprising a polynucleotide encoding the C-terminal extein,
wherein the N-terminal extein and the C-terminal extein N-intein and C-intein are capable of self excision to join the N-terminal and C-terminal fragments to form a prime editor fusion protein; and said prime editing system, wherein the prime editor fusion protein comprises a nucleic acid-programmed DNA binding protein (napDNAbp) domain and a reverse transcriptase (RT) domain. 2. The prime editing system of claim 1, wherein napDNAbp is selected from the group consisting of Cas9 or variants thereof, Cas12a, Cas12b1, Cas12e, Cas12d, Cas13a, Cas12c, and Argonaute, and/or has nickase activity. 3. The prime editing system of claim 2, wherein Cas9 comprises an amino acid sequence having at least 80% identity with amino acids 2-1368 of SEQ ID NO:18. 4. The prime editing system of Claim 3, wherein Cas9 comprises an H840A amino acid substitution when compared to SEQ ID NO:18. 5. The prime editing system of claim 4, wherein Cas9 further comprises S1025C, E1026F, and/or Q1027N amino acid substitutions when compared to SEQ ID NO:18. RT is avian myoblastosis virus (AMV) RT, Moloney murine leukemia virus (M-MLV) RT, human immunodeficiency virus (HIV) chain a RT, human immunodeficiency virus (HIV) chain b RT, Avian sarcoma and leukemia virus (ASLV) RT, Rous sarcoma virus (RSV) RT, Avian Myeloblastosis virus (AMV) RT, Avian erythroblastosis virus (AEV) helper virus MCAV RT, Avian myelocytoma virus MC29 helper virus MCAV RT, avian reticuloendotheliosis virus (REV-T) helper virus REV-A RT, avian sarcoma virus UR2 helper virus UR2AV RT, avian sarcoma virus Y73 helper virus YAV RT, Rous-associated virus (RAV) RT, myeloblastosis-associated virus (MAV) RT, feline leukemia RT, cauliflower mosaic virus RT, Klebsiella pneumoniae RT, Escheria coli RT, Bacillus subtilis RT, Eubacterium rectale subgroup II intron RT, Geobacillus stearothermophillus RT, or variants thereof A prime editing system according to any one of claims 1 to 5, wherein the prime editing system is (i) Variant M-MLV RT has the following mutations when compared to SEQ ID NO:89: P51X, S67X, E69X, L139X, T197X, D200X, H204X, F209X, E302X, T306X, F309X, W313X, T330X, L345X , L435X, N454X, D524X, E562X, D583X, H594X, L603X, E607X, and D653X, where X is a substitution of any other amino acid; or
(ii) variant M-MLV RT has the following mutations when compared to SEQ ID NO:89: P51L, S67K, E69K, L139P, T197A, D200N, H204R, F209N, E302K, E302R, T306K, F309N, W313F, T330P , L345G, L435G, N454K, D524G, E562Q, D583N, H594Q, L603W, E607K, and D653N; or
(iii) the variant M-MLV RT contains the amino acid substitutions D200N, T330P, and/or L603W when compared to SEQ ID NO:89; or
7. The prime editing system of claim 6, wherein (iv) variant M-MLV RT comprises amino acid substitutions D200N, T306K, W313F, T330P, and L603W when compared to SEQ ID NO:89. the reverse transcriptase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:89 or SEQ ID NO:122; or The prime editing system of any one of claims 1-7, wherein the reverse transcriptase has the sequence of SEQ ID NO:122. 9. The prime editing system of any one of claims 1-8, wherein the reverse transcriptase is a variant of M-MLV RT that is truncated when compared to SEQ ID NO:89. The truncated variant M-MLV RT is
(i) is truncated at the C-terminus and contains the D200N, T306K, W313F, and/or T330P mutations when compared to the amino acid sequence of SEQ ID NO:89; or
10. The prime editing system of claim 9, comprising (ii) the sequence of SEQ ID NO:766. 11. The prime editing system of any one of claims 1-10, wherein the N-intein comprises an amino acid sequence having at least 80% identity with SEQ ID NO:447. 12. The prime editing system of any one of claims 1-11, wherein the C-intein comprises an amino acid sequence having at least 80% identity to SEQ ID NO:452. Claims 1-12, wherein the fusion protein comprises the structure _NH2- [napDNAbp]-[reverse transcriptase]-COOH, wherein each occurrence of "]-[" indicates the presence of an optional linker sequence. A prime editing system according to any one of Claims 1 to 3. (i) the N-terminal fragment of the prime editor fusion protein comprises a portion of the napDNAbp domain and the C-terminal fragment of the prime editor fusion protein comprises a portion of the napDNAbp domain and the RT domain; or
(ii) the N-terminal fragment of the prime editor fusion protein comprises the napDNAbp domain and part of the RT domain, and the C-terminal fragment of the prime editor fusion protein comprises part of the RT domain; or
(iii) the N-terminal fragment of the prime editor fusion protein comprises the napDNAbp domain and part of the linker sequence, and the C-terminal fragment of the prime editor fusion protein comprises part of the linker sequence and the RT domain; or
(iv) the N-terminal fragment of the prime editor fusion protein comprises an amino acid sequence having at least 80% identity with amino acids 2-1024 of SEQ ID NO:18, wherein the C-terminal fragment comprises amino acids 1025-1025 of SEQ ID NO:18; 14. The prime editing system of claim 13, comprising an amino acid sequence having at least 80% identity with 1368. (a) the N-terminal extein comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to the amino acid sequence represented by SEQ ID NO:3875; and
(b) the C-terminal extein comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to the amino acid sequence represented by SEQ ID NO:3876, claims 1- 15. The prime editing system of any one of 14. the napDNAbp has nickase activity, and the prime editing system further comprises a prime editing guide RNA (PEgRNA) or a polynucleotide encoding the PEgRNA;
wherein the PE gRNA binds to a spacer sequence containing a region complementary to the first strand of a double-stranded target DNA sequence, an extension arm containing a DNA synthesis template and a primer binding site in a 5′→3′ orientation, and a napDNAbp. including the core,
wherein the primer binding site comprises a region complementary to a region upstream of the nick site in the second strand of the double-stranded target DNA sequence;
wherein the DNA synthesis template comprises a region complementary to a region downstream of the nick site in the second strand of the double-stranded target DNA sequence and has one or more nucleotide changes compared to the double-stranded target DNA sequence. A prime editing system according to any one of claims 1 to 15, comprising: (i) the spacer, extension arms, and gRNA core are in a single RNA molecule; and/or
(ii) the DNA synthesis template is from 3 nucleotides to 500 nucleotides in length, or the DNA synthesis template is from 8 nucleotides to 31 nucleotides in length; and/or
(iii) the primer binding site is from 8 to 15 nucleotides in length; or the primer binding site is from 8 to 11 nucleotides in length and contains a GC content of greater than about 60%; or the primer binding site is 12 to 13 nucleotides in length and contains a GC content of about 40-60%; or the primer binding site is 14 to 15 nucleotides in length; and contains less than about 40% GC content; or the primer binding site is from 3 to 20 nucleotides in length; or the primer binding site is from 7 to 17 nucleotides in length. 17. The prime editing system of claim 16. a polynucleotide encoding the N-terminal extein is part of the first vector, and
A primed editing system according to any one of claims 1 to 17, wherein the polynucleotide encoding the C-terminal extein is part of a second vector. 19. The primed editing system of claim 18, wherein the first vector and/or the second vector are viral vectors. The viral vector is selected from the group consisting of adenoviral vectors, adeno-associated viral (AAV) vectors, lentiviruses, and herpes simplex viruses; and/or the first vector or the second vector comprises prime editing guide RNA (PEgRNA 20. The primed editing system of claim 19, further comprising a polynucleotide encoding ). A pharmaceutical composition comprising a prime editing system according to any one of claims 1-20. A pharmaceutical composition comprising a first adeno-associated viral (AAV) vector and a second AAV vector,
wherein the first AAV vector encodes an N-terminal extein comprising an N-terminal fragment of the primed editor fusion protein and an N-intein;
wherein the second AAV vector encodes a C-terminal extein comprising a C-terminal fragment of the primed editor fusion protein and a C-intein;
wherein the N-terminal extein and the C-terminal extein N-intein and C-intein are capable of self excision to join the N-terminal and C-terminal fragments to form a prime editor fusion protein; and said pharmaceutical composition, wherein the primed editor fusion protein comprises a nucleic acid-programmed DNA binding protein (napDNAbp) domain and a polymerase domain. 23. The pharmaceutical composition of claim 22, wherein the first AAV vector or the second AAV vector further comprises a polynucleotide encoding prime editing guide RNA (PEgRNA). A method for editing a double-stranded target DNA sequence in a cell, the method comprising:
a first expression vector encoding the N-terminal fragment of the prime editor fusion protein fused to the first division intein sequence; and a second expression vector encoding the C-terminal fragment of the prime editor fusion protein fused to the second division intein sequence including contact with;
wherein the first split intein sequence and the second split intein sequence are capable of self excision to join the N-terminal and C-terminal fragments to form a prime editor fusion protein;
wherein the prime editor fusion protein comprises a nucleic acid-programmed DNA binding protein (napDNAbp) domain with nickase activity and a reverse transcriptase domain, and wherein the first expression vector or the second expression vector comprises the prime editor guide RNA further comprising a polynucleotide encoding (PEgRNA);
where PEgRNA is
(i) a spacer sequence containing a complementary region to the first strand of a double-stranded target DNA sequence;
(ii) an extension arm containing a DNA synthesis template and a primer binding site in a 5' to 3' orientation, where the primer binding site is complementary to the region upstream of the nick site in the second strand of the double-stranded target DNA sequence; and wherein the DNA synthesis template encodes one or more nucleotide changes when compared to the region downstream of the nick site in the second strand of the double-stranded target DNA sequence); and
(iii) contains a gRNA core that interacts with napDNAbp;
Contact here results in:
nicking the second strand of a double-stranded target DNA sequence to form a free 3' end at the nick site;
anneal to the region upstream of the nick site of the second strand of the double-stranded target DNA sequence;
A single strand of DNA encoded by a DNA synthesis template is synthesized from the free 3' end of the second strand of a double-stranded target DNA sequence; and downstream of a nick site in the second strand of a double-stranded target DNA sequence. is replaced with a single strand of DNA, whereby a double-stranded target DNA sequence is edited.

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