JPWO2020191234A5 - - Google Patents

Description

GOVERNMENT SUPPORT This invention was made with government support under Grants No. 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 incorporated by reference : U.S. Provisional Application No. 62/820,813 (Attorney Docket No. B1195.70074US00) filed March 19, 2019, June 7, 2019; U.S. Provisional Application No. 62/858,958 (Attorney Docket No. B1195.70074US01), filed August 21, 2019, U.S. Provisional Application No. 62/889,996 (Attorney Docket No. B1195.70074US02), August 2019 U.S. Provisional Application No. 62/922,654 (Attorney Docket No. B1195.70083US00) filed on October 21, 2019, U.S. Provisional Application No. 62/913,553 (Attorney Docket No. B1195.70074US03) filed October 10, 2019. U.S. Provisional Application No. 62/973,558 (Attorney Docket No. B1195.70083US01) filed on October 10, U.S. Provisional Application No. 62/931,195 (Attorney Docket No. B1195.70074US04) filed on November 5, 2019; U.S. Provisional Application No. 62/944,231 (Attorney Docket No. B1195.70074US05) filed December 5, 2019; U.S. Provisional Application No. 62/974,537 (Attorney Docket No. B1195.70083US02) filed December 5, 2019 ), U.S. Provisional Application No. 62/991,069 (Attorney Docket No. B1195.70074US06) filed on March 17, 2020, and U.S. Provisional Application No. 62/991,069 (Attorney Docket No. B1195.70074US06) filed on March 17, 2020 (at the time of filing, the serial number is Unavailable) (Agent Docket No. B1195.70083US03), incorporated by reference.

Background of the Invention Pathogenic single nucleotide mutations ^contribute by some estimates to approximately 50% of human diseases that have a genetic component7. Unfortunately, treatment options for patients with these genetic disorders ^remain extremely limited despite decades of gene therapy exploration. Perhaps the simplest solution to this therapeutic challenge is the direct correction of single nucleotide mutations in the patient's genome, which addresses the root cause of the disease and provides lasting benefit. I might. Although such a strategy was previously unthinkable, recent improvements in genome editing performance brought about by the arrival of the CRISRP/Cas system ⁹ now bring this therapeutic approach within reach. By the simple design of guide RNA (gRNA) sequences containing ~20 nucleotides complementary to the target DNA sequence, almost any conceivable genomic site can be specifically accessed by CRISPR-associated (Cas) nucleases ^{. 2} . To date, several monomeric bacterial Cas nuclease systems have been identified and adapted for genome editing applications ^. This natural diversity of Cas nucleases, together with the ever-growing array of engineered variants, ^11-14 provides fertile ground for the development of new genome editing technologies.

Although CRISPR gene disruption is now a mature technique, 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 generate double strand breaks (DSBs) in DNA sequences using a donor DNA repair template that encodes the desired edits. ) have been used for insertion, modification, or exchange at sites15 ^. However, existing HDRs have very low efficiency in most human cell types, specifically in non-dividing cells, and incompatible non-homologous end joining (NHEJ) are mainly used for insert- Deletion (indel) leads to by-products16 ^. Other issues concern the generation of DSBs, which can result in large chromosomal rearrangements and deletions at ^targeted loci17 or activate the p53 axis leading to growth arrest ^and apoptosis18 ^,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 ^low20 . Other strategies use small molecules and biological reagents to attempt to bias repair toward HDR over NHEJ ^{21 - 23} . However, the effectiveness of these methods can depend on the cell type, and perturbation of normal cellular conditions can lead to undesirable and unpredictable effects.

Recently, the present inventors, led by Professor David Liu and colleagues, developed base editing as a technique to edit target nucleotides without creating DSBs or relying on ^HDR4-6,24-27 . Direct modification of DNA bases by Cas-fusion deaminase allows C・G→T・A or A・T→G・C base pair conversion in a short target window (~5-7 bases) with extremely high efficiency. make it possible. As a result, base editors were quickly adopted by the scientific community. However, the following factors limit their generality for precision genome editing: (1) “bystander editing” of non-target C or A bases on the target window is observed; (2) ) a target nucleotide product mixture is observed; (3) the target base must be located 15±2 nucleotides upstream of the PAM sequence; (5) repair of small insertion and deletion mutations is not possible.

It is therefore possible to flexibly introduce any desired single nucleotide changes and/or base pair insertions or deletions (for example, 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 pairs The development of programmed editing elements that can incorporate (insertions or deletions of scope) and therapeutic possibilities.

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 editing factors of the present disclosure utilize a target-primed reverse transcription (TPRT) or "prime editing" mechanism that is highly efficient and highly genetically flexible, such as CRISPR/CRISPR/Cast. In part, the present invention relates in part to the discovery that the present invention can be exploited or employed to perform genome-based editing (eg, as depicted in the various embodiments of FIGS. 1A-1F). TPRT is naturally used by mobile DNA elements such as non-LTR retrotransposons in mammals and group II introns in bacteria28,29 ^. We herein target a specific DNA sequence with a guide RNA, generate a single-stranded nick at the target site, and transfer the nicked DNA to a modified reverse transcriptase template incorporated with the guide RNA. A Cas protein-reverse transcriptase fusion or related system is used as a primer for reverse transcription. However, although this concept began with prime editing agents that use reverse transcriptase as a component of DNA polymerase, the prime editing agents described herein are not limited to reverse transcriptase, and in fact, the use of DNA polymerase may include. Indeed, although this application may refer throughout this application to a prime editing agent having "reverse transcriptase," it is hereby established that reverse transcriptase is the only type of DNA polymerase that can act in prime editing. explained. Thus, wherever "reverse transcriptase" is referred to herein, one of ordinary skill in the art should understand that any suitable DNA polymerase may be used in place of reverse transcriptase. Thus, in one aspect, the prime editing factor targets a DNA sequence by associating the DNA sequence with a specialized guide RNA (i.e., PEgRNA) that contains a spacer sequence that anneals to a complementary protospacer in the target DNA. It may contain Cas9 (or equivalent napDNAbp) that is programmed to do so. The specialized guide RNA also contains new genetic information in the form of an extension encoding a replacement strand of DNA containing the desired genetic modification that is used to replace the corresponding endogenous DNA strand at the target site. do. To transfer information from PEgRNA to target DNA, the prime editing mechanism involves nicking the target site in one strand of the DNA exposing the 3' hydroxyl group. The exposed 3' hydroxyl group can then be used to prime DNA polymerization of the editing-encoding extension onto the PEgRNA directly into the target site. In various embodiments, the extension - which provides a template for polymerization of the replacement strand containing the edit - can be formed from RNA or DNA. In the case of RNA elongation, the prime editing agent polymerase can be an RNA-dependent DNA polymerase (such as reverse transcriptase). In the case of DNA extension, the prime editor polymerase may be a DNA-dependent DNA polymerase.

The newly synthesized strand (i.e., the replacement DNA strand containing the desired edits) formed by the prime editing agents disclosed herein contains the desired nucleotide changes (e.g., single nucleotide changes, deletions). , or insertions, or combinations thereof) will be homologous to (ie, have the same sequence as) the target sequence of the genome. The newly synthesized (or replaced) strand of DNA, sometimes referred to as a single-stranded DNA flap, competes for hybridization with the complementary homologous endogenous DNA strand, thereby Replaces raw chains. In certain embodiments, the system combines 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). can be Error-prone reverse transcriptases can introduce changes during single-stranded DNA flap synthesis. Thus, in certain embodiments, error-prone reverse transcriptase can be utilized to introduce nucleotide changes into target DNA. Depending on the error-prone reverse transcriptase used with the system, changes can be random or non-random.

Degradation of the hybridized intermediate (including the single-stranded DNA flap synthesized by reverse transcriptase that is hybridized with the endogenous DNA strand) causes the resulting endogenous DNA to displace the replaced flap. removal (for example, with the 5'-end DNA flap endonuclease, FEN1), ligation of the synthesized single-stranded DNA flap to the target DNA, and desired nucleotide changes as a result of cellular DNA repair and/or replication processes. can include the assimilation of Because templated DNA synthesis imparts high single-nucleotide precision to any nucleotide modification, including insertions and deletions, the breadth of this approach is extremely broad and has applications in both basic science and therapeutics. It is foreseeable that it could be used for a myriad of applications in the field.

In one aspect, the present specification provides a fusion protein comprising a nucleic acid programmed DNA binding protein (napDNAbp) and a reverse transcriptase. In various embodiments, the fusion protein is capable of performing genome editing by reverse transcription primed to a primed target in the presence of an elongated 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 certain 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 elongated guide RNA.

In still 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 elongated guide RNA forms an R-loop. The R-loop can include (i) an RNA-DNA hybrid comprising an elongated 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 elongated guide RNA comprises (a) a guide RNA and (b) RNA extension at the 5' or 3' end of the guide RNA or at an intramolecular location of the guide RNA. RNA extension can include (i) a reverse transcription template sequence containing the desired nucleotide changes, (ii) a reverse transcription primer binding site, and (iii) an optional linker sequence. In various embodiments, 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, and the single-stranded DNA flap includes the desired nucleotide changes.

In various embodiments, the RNA elongation 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 in length. 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 still other embodiments, single-stranded DNA flaps can hybridize to endogenous DNA sequences adjacent to the nick site, thereby incorporating desired nucleotide changes. In still other embodiments, a single-stranded DNA flap has a free 5' end and replaces the endogenous DNA sequence adjacent to the nick site. In certain embodiments, the displaced endogenous DNA with a 5' end is excised by the cell.

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

In various other embodiments, the desired nucleotide changes are incorporated over an editing window that lies between about -4 and +10 of the PAM sequence.

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

In various embodiments, napDNAbp comprises the amino acid sequence of SEQ ID NO: 18. In various other embodiments, napDNAbp is SEQ ID NO. ); (SpCas9); SEQ ID NO: 77-86 (CP-Cas9); SEQ ID NO: 18-25 and 87-88 (SpCas9); and at least 80% of the amino acid sequence of any one of SEQ ID NO: 62-72 (Cas12) , 85%, 90%, 95%, 98%, or 99% identical.

In other embodiments, the reverse transcriptase of the disclosed fusion proteins and/or compositions is SEQ ID NO: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454 , 471, 516, 662, 700-716, 739-742, and 766. In still other embodiments, the reverse transcriptase is SEQ ID NO: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, 662, 700-716 , 739-742, and 766. These sequences may be naturally occurring reverse transcriptase sequences, for example from retroviruses or retrotransposons, or the sequences may be recombinant.

In various other embodiments, the fusion proteins disclosed herein can include various structural configurations. For example, a fusion protein can include the structure NH ₂ -[napDNAbp]-[reverse transcriptase]-COOH; or NH ₂ -[reverse transcriptase]-[napDNAbp]-COOH, where each of "]-[" Indicates the presence of any linker sequences.

In various embodiments, the linker sequence is the amino acid sequence of SEQ ID NO: 127, 165-176, 446, 453, and 767-769, or the linker amino acid sequence of SEQ ID NO: 127, 165-176, 446, 453, and 767-769. an amino acid sequence that is at least 80%, 85%, or 90%, or 95%, or 99% identical to any one of

In various embodiments, the desired nucleotide change incorporated onto the target DNA can be a single nucleotide change (eg, a transition or transversion), an insertion of one or more nucleotides, or a deletion of one or more nucleotides.

In certain cases, the insertion has a length 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 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 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 in length. , 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 another aspect, the disclosure provides an extended guide RNA comprising a guide RNA and at least one RNA extension. The RNA extension can be located at the 3' end of the guide RNA. In other embodiments, the RNA extension can be located 5' to the guide RNA. In still other embodiments, RNA extension can 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 editing factor guide RNA (PEgRNA) is capable of binding to and directing 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 with the target strand to form an RNA-DNA hybrid and an R-loop.

In various embodiments of the prime editing factor guide RNA, at least one RNA extension comprises a DNA synthesis template. In various other embodiments, the RNA extension further includes a reverse transcription primer binding site. In yet other embodiments, the RNA extension includes a linker or spacer that joins the RNA extension to the guide RNA.

In various embodiments, the RNA elongation 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 in length. 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 obtain.

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 in length. , 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 various embodiments of the elongated guide RNA, the reverse transcribed template sequence can encode a single-stranded DNA flap that is complementary to the endogenous DNA sequence adjacent to the nick site, and the single-stranded DNA flap is Contains nucleotide changes. A single-stranded DNA flap can replace endogenous single-stranded DNA at the nick site. The displaced endogenous single-stranded DNA 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 endogenous flap at the 5' end can drive product formation. This is because removing the endogenous flap at the 5' end reduces the hybridization of the single-stranded 3' DNA flap to the corresponding complementary DNA strand and the desired This is because it facilitates the incorporation or assimilation of nucleotide changes.

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

In certain embodiments, the PEgRNA is SEQ ID NO : 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, 3479-3493, 3522-3540, 3549-3556, 3628-3698, 3755-3810, 3874, 3890-3901, 3905-3911, 3913-3929, and 3972-3989 nucleotide sequence, or 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, and 3972-39 89 nucleotide sequences 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.

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

In yet other aspects of the invention, herein provides complexes comprising napDNAbp and extended guide RNA. napDNAbp can be a Cas9 nickase, or one of the amino acid sequences of SEQ ID NOs: 42-57 (Cas9 nickase) and 65 (AsCas12a nickase), or one of SEQ ID NOs: 42-57 (Cas9 nickase) and 65 (AsCas12a nickase). The amino acid sequence can be at least 80%, 85%, 90%, 95%, 98%, or 99% identical to one amino acid sequence.

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 invention provides polynucleotides. 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 napDNAbp disclosed herein. In still further embodiments, the polynucleotide may encode any of the reverse transcriptases disclosed herein. In still other embodiments, the polynucleotide comprises any of the extended guide RNAs disclosed herein, any of the reverse transcription template sequences, or any of the reverse transcription primer sites, or any of the linker sequences disclosed herein. can be coded.

In yet other aspects, the specification provides vectors comprising the polynucleotides described herein. Therefore, in certain embodiments, the vector comprises a polynucleotide encoding a fusion protein comprising napDNAbp and reverse transcriptase. In other embodiments, the vector comprises polynucleotides that separately encode napDNAbp and reverse transcriptase. In still other embodiments, the vector may include a polynucleotide encoding an extended guide RNA. In various embodiments, a vector can include one or more polynucleotides encoding napDNAbp, reverse transcriptase, and extended guide RNA on the same or separate vectors.

In yet other aspects, the present specification provides cells comprising a fusion protein described herein and an elongated guide RNA. Cells can be transformed with a vector containing the fusion protein, napDNAbp, reverse transcriptase, and extended guide RNA. These genetic elements may be contained on the same vector or on different vectors.

In another aspect, the present specification provides pharmaceutical compositions. In certain embodiments, the pharmaceutical composition comprises one or more of napDNAbp, fusion protein, reverse transcriptase, and extended guide RNA. In certain embodiments, a fusion protein described herein and a pharmaceutically acceptable excipient. In other embodiments, the pharmaceutical composition comprises any extension guide RNA described herein and a pharmaceutically acceptable excipient. In yet other embodiments, the pharmaceutical composition comprises any extension guide RNA described herein in combination with any fusion protein described herein and a pharmaceutically 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, the first pharmaceutical composition may include the fusion protein or napDNAbp, the second pharmaceutical composition may include reverse transcriptase, and the third pharmaceutical composition may include the elongated guide RNA. May contain.

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

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 desired nucleotide changes onto a double-stranded DNA sequence. The method includes first contacting a double-stranded DNA sequence with a complex comprising a fusion protein and an elongated guide RNA, the fusion protein comprising napDNAbp and reverse transcriptase, and the elongated guide RNA , containing the reverse transcription template sequence containing the desired nucleotide changes. The method then involves nicking the double-stranded DNA sequence on the non-target strand, thereby generating free single-stranded DNA with a 3' end. The method then involves hybridizing the 3' end of the free single-stranded DNA to a reverse transcription template sequence, thereby priming the reverse transcriptase domain. The method then involves polymerizing the strands of DNA from the 3' end, thereby producing a single-stranded DNA flap containing the desired nucleotide changes. The method then involves replacing the endogenous DNA strand adjacent to the cut site with a single-stranded DNA flap, thereby incorporating the desired nucleotide change onto the double-stranded DNA sequence.

In other embodiments, the present disclosure provides methods for introducing one or more changes in the nucleotide sequence of a DNA molecule at a target locus, wherein the DNA molecule is combined with a nucleic acid programmed DNA binding protein (napDNAbp). contacting the napDNAbp with a guide RNA that targets the target locus, where the guide RNA includes a reverse transcriptase (RT) template sequence containing at least one desired nucleotide change. The method then primes reverse transcription by forming an exposed 3' end in the DNA strand at the target locus and then hybridizing the exposed 3' end with an RT template sequence. accompanied by something. A single-stranded DNA flap containing at least one desired nucleotide change based on the RT template sequence is then synthesized or polymerized by reverse transcriptase. Finally, 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 other embodiments, the present disclosure provides a method for introducing one or more changes in the nucleotide sequence of a DNA molecule at a target locus by target-primed reverse transcription, the method comprising: A guide comprising: (a) a DNA molecule at the target locus with i) a fusion protein comprising a nucleic acid programmed DNA binding protein (napDNAbp) and a reverse transcriptase; and (ii) an RT template comprising the desired nucleotide changes. (b) causing target-primed reverse transcription of the RT template to generate single-stranded DNA containing the desired nucleotide changes; (c) accessing the target locus through DNA repair and/or replication processes; nucleotide changes into the DNA molecule.

In certain embodiments, replacing the endogenous DNA strand includes: (i) hybridizing a single-stranded DNA flap with the endogenous DNA strand adjacent to the cleavage site, thereby creating a sequence mismatch; (ii) excision of the endogenous DNA strand; and (iii) repair of mismatches to form the desired product containing the desired nucleotide changes 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 changes are (1) G:C base pairs to T:A base pairs, (2) G:C base pairs to A:T base pairs, (3) G:C base pairs. pair into C:G base pair, (4) T:A base pair into G:C base pair, (5) T:A base pair into A:T base pair, (6) T:A base pair. (7) C:G base pair to G:C base pair, (8) C:G base pair to T:A base pair, (9) C:G base pair to A: T base pair, (10) A:T base pair to T:A base pair, (11) A:T base pair to G:C base pair, or (12) A:T base pair to C:G. Can be converted into base pairs.

In still other embodiments, the method introduces the desired nucleotide change that is an insertion. In certain cases, the insertion has a length 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 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 other embodiments, the method introduces the desired nucleotide change that is a deletion. In certain other cases, the deletion is at least 1 in length, 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 various embodiments, the desired nucleotide change modifies a disease-associated gene. Disease-associated genes are: adenosine deaminase (ADA) deficiency; alpha-1 antitrypsin deficiency; cystic fibrosis; Duchenne muscular dystrophy; galactosemia; hemochromatosis; Huntington's disease; maple syrup urine disease; Marfan syndrome; A single gene selected from the group consisting of neurofibromatosis type 1; congenital nail hypertrophy; phenylketonuria; severe combined immunodeficiency; sickle cell disease; Smith-Lemmli-Opitz syndrome; and Tay-Sachs disease May be related to disability. In other embodiments, the disease-associated gene may be associated with a polygenic disorder selected from the group consisting of: heart disease; hypertension; Alzheimer's disease; arthritis; diabetes; cancer; and obesity.

The methods disclosed herein may involve fusion proteins having napDNAbp that is nuclease dead Cas9 (dCas9), Cas9 nickase (nCas9), or nuclease active Cas9. In other embodiments, napDNAbp and reverse transcriptase are not encoded as a single fusion protein, but rather may be provided in separate constructs. Thus, in some embodiments, reverse transcriptase can be provided in trans to napDNAbp (rather than as a fusion protein).

In various embodiments involving the methods, napDNAbp is SEQ ID NO: 26-61, 75-76, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467 and 482-487 (Cas9); ); SEQ ID NO: 77-86 (CP-Cas9); SEQ ID NO: 18-25 and 87-88 (SpCas9); and SEQ ID NO: 62-72 (Cas12). napDNAbp is also SEQ ID NO: 26-61, 75-76, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467 and 482-487 (Cas9); (SpCas9); SEQ ID NO: 77-86 (CP-Cas9); SEQ ID NO: 18-25 and 87-88 (SpCas9); and any one amino acid sequence of SEQ ID NO: 62-72 (Cas12) and at least 80%, 85%, 90%, 95%, 98% , or 99% identical amino acid sequences.

In various embodiments involving the methods, the reverse transcriptase is SEQ ID NO: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, 662, 700 -716, 739-742 and 766. Reverse transcriptase also has 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 at least 80%, 85%, 90%, 95%, 98%, or 99% identical.

The method is as follows : , 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 , 3479-3493 , 3522-3540 , 3549-3556 , 3628-3698, 3755-3810 , 3874, 3890-3901, 3905-3911, 3913-3929, and 3972 ~3989 or 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 include the use of an extended guide RNA that includes an RNA extension at its 3' end, the RNA extension including a reverse transcriptase template sequence.

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

The method can include the use of an elongated guide RNA that includes RNA extension in intramolecular positioning of the guide RNA, where the RNA extension includes a reverse transcription template sequence.

The method has a length 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 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 It may also include the use of an extended guide RNA having one or more RNA extensions that are at least 500 nucleotides.

It should be understood that the above concepts, and the additional concepts discussed below, may be arranged in any suitable combination (although this 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 aspects when considered in conjunction with the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS The following drawings form a part of this specification, and reference may be made to one or more of these drawings in combination with a detailed description of particular embodiments presented herein. are included to further demonstrate certain aspects of the disclosure that may be better understood by.

Figure 1A shows the use of a fusion protein containing reverse transcriptase fused to a Cas9 protein complexed with an elongated guide RNA molecule to create single nucleotide changes and/or insertions and/or deletions in a DNA molecule ( As an example, a schematic diagram of an exemplary process for introduction into a genome is provided. In this embodiment, the guide RNA is extended at the 3' end to include a reverse transcriptase template sequence. Schematic diagram shows how reverse transcriptase (RT) fused to Cas9 nickase and complexed with guide RNA (gRNA) binds to a DNA target site and nicks the PAM-containing DNA strand adjacent to the target nucleotide. shows. The RT enzyme uses the nicked DNA as a primer for DNA synthesis from gRNA, which is used as a template for the synthesis of a new DNA strand encoding the desired edit. The illustrated editing process may be referred to as target-primed reverse transcription editing (TRT editing), or "prime editing."

Figure 1B shows the same diagram as Figure 1A, with the exception that the prime editing factor complex is more commonly represented as [napDNAbp]-[P]:PEgRNA or [P]-[napDNAbp]:PEgRNA. providing. Where "P" refers to any polymerase (e.g. reverse transcriptase), "napDNAbp" refers to a nucleic acid programmed DNA binding protein (e.g. SpCas9), "PEgRNA" refers to prime editing guide RNA, and "] -['' says any linker. As described elsewhere, eg, in Figures 3A-3G, PEgRNA includes a 5' extending arm that includes a primer binding site and a DNA synthesis template. Although not shown, it is contemplated that the extended arm of PEgRNA (ie, it contains the primer binding site and 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 (eg, reverse transcriptase). When the DNA synthesis template is DNA, the polymerase can be a DNA-dependent DNA polymerase.

Figure 1C provides a schematic illustration of an exemplary process for introducing single nucleotide changes and/or insertions and/or deletions onto a DNA molecule (e.g., a genome), as a complex with an elongated guide RNA molecule. A fusion protein containing reverse transcriptase fused to a Cas9 protein is used. In this embodiment, the guide RNA is extended at the 5' end to include the reverse transcriptase template sequence. Schematic diagram shows how reverse transcriptase (RT) fused to Cas9 nickase, as a complex with guide RNA (gRNA), binds to a DNA target site and transfers to a PAM-containing DNA strand adjacent to the target nucleotide. It shows whether to put a nick. The RT enzyme uses the nicked DNA as a primer for DNA synthesis from gRNA, which is used as a template for the synthesis of a new DNA strand encoding the desired edit. The illustrated editing process may be referred to as primed target-primed reverse transcription editing (TRT editing) or equivalently "primed editing."

Figure 1D provides the same depiction as Figure 1C, except that the prime editing factor complex is more commonly 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 programmed DNA binding protein (e.g., SpCas9), and "PEgRNA" refers to a prime editing refers to the guide RNA, and "]-[" refers to the optional linker. As described elsewhere, for example in Figures 3A-3G, PEgRNA includes a 3' extending arm that contains a primer binding site and a DNA synthesis template. Although not shown, it is contemplated that the extended arm of PEgRNA (ie, containing the primer binding site and 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. As an illustration, if the DNA synthesis template is RNA, the polymerase can be an RNA-dependent DNA polymerase (eg, reverse transcriptase). When the DNA synthesis template is DNA, the polymerase can be a DNA-dependent DNA polymerase. In various embodiments, PEgRNA can be modified or synthesized to incorporate DNA-based DNA synthesis templates.

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).

Figure 1G shows the use of a nucleic acid-programmed DNA binding protein (napDNAbp) complexed with an elongated guide RNA to introduce single nucleotide changes into a DNA molecule (e.g., genome) at a target locus, and Figure 3 provides another schematic illustration of an exemplary process for introducing insertions and/or deletions. This process is sometimes referred to as a form of prime editing. An extended guide RNA comprises an extension at the 3' or 5' end of the guide RNA, or at some point within the molecule within 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 into one of the strands of DNA (the R-loop strand, or the PAM-containing strand, or the non-target DNA strand, or the protospacer strand) at the target locus (e.g., by a nuclease or a chemical agent). ), which creates an available 3' end on one strand of the target locus. In certain embodiments, a nick is created in the strand of DNA that corresponds to the R-loop strand, ie, the strand that does not hybridize to the guide RNA sequence. In step (c), the 3' terminal DNA strand interacts with the extended portion of the guide RNA to prime reverse transcription. In some embodiments, the 3' terminal DNA strand hybridizes to a specific RT prime sequence on the extended portion of the guide RNA. In step (d), 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 changes (eg, single base changes, insertions, deletions, or combinations thereof). In step (e) napDNAbp and guide RNA are released. Steps (f) and (g) involve disassembling the single-stranded DNA flap so that the desired nucleotide changes are incorporated into the target locus. This process, once a 3' single-stranded DNA flap enters and hybridizes with a complementary sequence on the other strand, removes the corresponding 5' endogenous DNA flap that is formed, thereby allowing desired product formation. can be promoted. The process can also be driven to form a product with a nick in the second strand, as illustrated in Figure IF. This process may introduce at least one or more of the following genetic changes: transversions, transitions, deletions, and insertions.

FIG. 1H is a schematic diagram depicting the types of genetic changes that can occur during 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 ones). ).

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 second strand nicking into 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.

Figures 1J -1K are contemplated herein to replace napDNAbp (eg, SpCas9 nickase) with any programmed nuclease domain, such as a zinc finger nuclease (ZFN) or a transcription activator-like effector nuclease (TALEN). Draw a variation of the prime editing that will be done. As such, a suitable nuclease need not necessarily be "programmed" by a nucleic acid target molecule (such as a guide RNA), but rather by defining the specificity of the DNA binding domain of the nuclease, among other things. Good things are planned. Just like prime editing with the napDNAbp moiety, the alternative programmed nuclease is preferably modified so that only one strand of the target DNA is cut. In other words, the programmed nuclease should preferably function as a nickase. Once a programmed nuclease is selected (eg, ZFN or TALEN), additional functions may be modified such that it is a system that can operate according to a prime editing-like mechanism. For example, a programmed nuclease may be modified by coupling it to an extended arm of RNA or DNA (eg, via a chemical linker), where the extended arm is a primer binding site (PBS ) and DNA synthesis templates. The programmed nuclease may also be coupled to a polymerase (eg, via a chemical or amino acid linker), although the nature of the polymerase may depend on whether the extending arm is DNA or RNA. Dew. In the case of RNA extension arms, the polymerase can be an RNA-dependent DNA polymerase (eg, reverse transcriptase). In the case of DNA extension arms, the polymerase is a DNA-dependent DNA polymerase (e.g., prokaryotic polymerases including Pol I, Pol II, or Pol III, or Pol a, Pol b, Pol g, Pol d, Pol e, or eukaryotic polymerases including Pol z). The system may also include other functions added as fusions with programmed nucleases or in trans to facilitate the overall reaction (for example, (a) cleaving the DNA Helicase, by unwinding at a site, creates a cleaved strand with a 3' end that can be used as a primer, (b) removes the endogenous strand on the cleaved strand, (c) a flap endonuclease (e.g., FEN1) that helps drive the reaction toward replacement with the nCas9:gRNA complex, which creates a second site nick on the opposite strand (this is may also help promote the uptake of synthetic repair through favorable cellular repair)). In a similar manner to prime editing with napDNAbp, such a complex with a different programmed nuclease synthesizes and then persists the newly synthesized DNA replacement strand with the targeted edit into the target site of DNA. It can be used to incorporate

FIG. 1L depicts, in one embodiment, anatomical features of target DNA that may be edited by prime editing. Target DNA includes a "non-target strand" and a "target strand." The target strand is the strand that becomes annealed to the spacer of the PEgRNA of the prime editor complex, which recognizes the PAM site (in this case the NGG recognized by the standard SpCas9-based prime editor). The target strand may also be referred to as the "non-PAM strand" or the "unedited strand." In contrast, the non-target strand (ie, the strand containing the protospacer and NGG PAM sequences) is sometimes referred to as the "PAM strand" or the "edited strand." In various embodiments, the nick site of the PE complex (eg, in SpCas9-based PE) will be in the protospacer on the PAM strand. The location of the nick would be a feature of the specific Cas9 that forms the PE. For example, in a SpCas9-based PE, the nick sites are between bases 3 (position ``-3'' compared to position 1 of the PAM sequence) and 4 (position ``-4'' compared to position 1 of the PAM sequence). in the phosphodiester bond between. The nick site in the protospacer forms a free 3' hydroxyl group that complexes with the primer binding site on the extended arm of PEgRNA, as seen in the figure below, and the DNA encoding the DNA synthesis template on the extended arm of PEgRNA. provides a substrate for initiating single-chain polymerization. This polymerization reaction is catalyzed in the 5'→3' direction by the polymerase (eg, reverse transcriptase) of the PE fusion protein. Polymerization is terminated before reaching the gRNA core (e.g., by the inclusion of a polymerization termination signal or secondary structure that functions to terminate the polymerization activity of the PE) and is removed from the original 3' hydroxyl group of the nicked PAM strand. Generates a single-stranded DNA flap that is extended. The DNA synthesis template encodes a single-stranded DNA homologous to the 5'-end single strand of endogenous DNA that immediately follows the nick site on the PAM strand and makes the desired nucleotide changes (e.g., single base substitutions). , insertions, deletions, inversions). The position of the desired edit can be any position following the nick site on the PAM strand, including positions +1, +2, +3, +4 (start of PAM site), +5 (start of PAM site), Position 2), +6 (PAM site position 3), +7, +8, +9, +10, +11, +12, +13, +14, +15, +16, +17, +18, +19, +20, +21, +22, +23, +24, +25, +26, +27, +28, +29, +30, +31, +32, +33, +34, +35 , +36, +37, +38, +39, +40, +41, +42, +43, +44, +45, +46, +47, +48, +49, +50, +51, + 52, +53, +54, +55, +56, +57, +58, +59, +60, +61, +62, +63, +64, +65, +66, +67, +68, +69, +70, +71, +72, +73, +74, +75, +76, +77, +78, +79, +80, +81, +82, +83, +84, +85 , +86, +87, +88, +89, +90, +91, +92, +93, +94, +95, +96, +97, +98, +99, +100, +101, + 102, +103, +104, +105, +106, +107, +108, +109, +110, +111, +112, +113, +114, +115, +116, +117, +118, +119, +120, +121, +122, +123, +124, +125, +126, +127, +128, +129, +130, +131, +132, +133, +134, +135 , +136, +137, +138, +139, +140, +141, +142, +143, +144, +145, +146, +147, +148, +149, or +150, or more (relative to a position downstream of the nick site). Once the 3'-end single-stranded DNA (containing the edit of interest) replaces the endogenous 5'-end single-stranded DNA, DNA repair and replication processes lead to permanent incorporation of the editing site on the PAM strand, which then will result in correction of mismatches on non-PAM strands present at the site. In this way, the edit will spread to both strands of DNA on the target DNA site. It will be appreciated that references to "edited strand" and "unedited" strand are merely intended to delineate the strands of DNA involved in the PE mechanism. . The "edited strand" only becomes edited by replacing the 5'-end single-stranded DNA immediately downstream of the nick site with a synthesized 3'-end single-stranded DNA containing the desired edit. It's a chain. The "unedited" strand is the companion strand to the edited strand, but has also been edited (in particular, the editing of interest) to be complementary to the edited strand through repair and/or replication. Become so.

Figure 1M depicts the mechanism of prime editing showing the anatomical features of the target DNA, the prime editing factor complex, and the interaction between PEgRNA and target DNA. First, polymerases (e.g. reverse transcriptase) and napDNAbp (e.g. SpCas9 nickase, e.g. inactivating mutations in the HNH nuclease domain (e.g. H840A) or activating mutations in the RuvC nuclease domain) A prime editing factor comprising a fusion protein with a mutation (SpCas9) with a mutation (D10A) is complexed with PEgRNA and DNA with the target DNA to be edited. PEgRNA includes a spacer, a gRNA core (also known as gRNA backbone or gRNA backbone), which binds to napDNAbp, and an extended arm. The extended arm can be at the 3' end, 5' end, or anywhere within the PEgRNA molecule. As shown, the extended arm is at the 3' end of the PEgRNA. The extending arm contains a primer binding site and a DNA synthesis template (a region homologous to the edit of interest (i.e. , homologous arms)). As shown, once a nick has been introduced, thereby producing a free 3' hydroxyl group immediately upstream of the nick site, the region immediately upstream of the nick site on the PAM strand is termed the "primer binding site." anneal to a complementary sequence at the 3' end of the extended arm, creating a short double-stranded region with an available 3' hydroxyl end for the polymerase of the prime editing factor complex. A matrix is formed. A polymerase (eg, reverse transcriptase) then polymerizes the DNA as a strand from the 3' hydroxyl end to the end of the extending arm. The sequence of single-stranded DNA is encoded by the DNA synthesis template, which is part of the extending arm (ie, eliminates the primer binding site) that is "read" by the polymerase that synthesizes new DNA. This polymerization effectively extends up to the original 3' hydroxyl terminal sequence of the initial nick site. The DNA synthesis template encodes a single strand of DNA that includes not only the desired edit, but also a region homologous to the single strand of endogenous DNA immediately downstream of the nick site on the PAM strand. The encoded single strand of 3'-end DNA (i.e., the 3' single-stranded DNA flap) is then encoded by the corresponding homologous endogenous 5'-end DNA strand immediately downstream of the nick site on the PAM strand. The strands are replaced, forming a DNA intermediate with a single-stranded DNA flap at the 5' end, which is removed by the cell (eg, by a flap endonuclease). The 3'-terminal single-stranded DNA flap, which anneals with the complement of the endogenous 5'-terminal single-stranded DNA flap, is ligated with the endogenous strand after the 5' DNA flap is removed. The desired edit in the single-stranded DNA flap at the 3' end, which has just been annealed and ligated, forms a mismatch with the complementary strand and undergoes DNA repair and/or a series of replications, thereby making the edit on both strands. The desired edits are permanently incorporated.

Figure 2 shows three Cas complexes (SpCas9, SaCas9, and LbCas12a) that can be used in the prime editing factors described herein and their PAM, gRNA, and DNA cleavage features. The figure shows the design of a complex involving SpCas9, SaCas9, and LbCas12a.

Figures 3A-3F show the design of engineered 5' prime editing factor gRNA (Figure 3A), 3' prime editing factor gRNA (Figure 3B), and intramolecular extension (Figure 3C). Elongated guide RNA (or elongated gRNA) may also be referred to herein as PEgRNA or "primed editing guide RNA." Figures 3D and 3E provide additional embodiments of 3' and 5' prime editing factor gRNAs (PEgRNAs), respectively. Figure 3F illustrates the interaction between the 3' prime editor guide RNA and the target DNA sequence. Embodiments of FIGS. 3A-3C represent reverse transcription template sequences in the extended portions of the 3', 5', and intramolecular versions (i.e., or more broadly referred to as DNA synthesis templates as indicated. Because RT Figure 2 illustrates an exemplary arrangement of a primer binding site, and an optional linker sequence, as well as a general arrangement of a spacer and core region (as this is the only type of polymerase that can be used in the context of a prime editing agent), a primer binding site, and an optional linker sequence. The disclosed prime editing process is not limited to these configurations of elongated guide RNA. The embodiment of FIG. 3D provides the structure of an exemplary PEgRNA contemplated herein. PEgRNA contains three main component components ordered in the 5' to 3' direction: a spacer, a gRNA core, and an extended arm at the 3' end. The elongated arm can be further divided in the 5' to 3' direction into the following structural elements: primer binding site (A), editing template (B), and homology arm (C). In addition, PEgRNA can include an optional 3' end modified region (e1) and an optional 5' end modified region (e2). Furthermore, PEgRNA may further include a transcription termination signal at the 3' end of PEgRNA (not shown). These structural elements are further defined in this application. The illustration of the structure of PEgRNA is not meant to be limiting and encompasses variations in the arrangement of the elements. For example, any sequence modifications (e1) and (e2) may be located within or between any of the other regions shown. It is not limited to being located at the 3' and 5' ends. PEgRNA, in certain embodiments, can include secondary RNA structures such as, but not limited to, hairpins, stem loops, toe loops, RNA binding protein recruitment domains (eg, MS2 aptamers that recruit and bind MS2cp protein). For example, such secondary structures may be located within the spacer, gRNA core, or elongated arms, particularly within the e1 and/or e2 modified regions. In addition to the secondary RNA structure, PEgRNAs may include chemical linkers or poly(N) linkers or tails (eg, within the e1 and/or e2 modified regions). Here, "N" can be any nucleobase. In some embodiments (eg, as shown in Figure 72(c)), the chemical linker can function to prevent reverse transcription of the sgRNA backbone or core. Additionally, in certain embodiments (see, e.g., FIG. 72(c)), the extended arm (3) may be composed of RNA or DNA, and/or may include one or more nucleobase analogs (e.g., This may add functionality such as temperature resilience). Additionally, the orientation of the extended arm (3) can be in the native 5' to 3' direction or in the 3' to 5' direction (with respect to the overall PEgRNA molecule orientation) in an opposing orientation. can be synthesized (relatively). It is also noted that one skilled in the art 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 extended arm (i.e. DNA or RNA). . This can be provided in trans, either as a fusion with napDNAbp or as a separate part, to synthesize a 3' single-stranded DNA flap encoded by the desired template that encompasses the desired edits. Can be implemented. For example, if the extending arm is RNA, the DNA polymerase can be reverse transcriptase or any other suitable RNA-dependent DNA polymerase. However, if the extending arm is DNA, the DNA polymerase may be a DNA-dependent DNA polymerase. In various embodiments, the provision of the DNA polymerase can be in trans, for example, an RNA-protein recruitment domain (e.g., an MS2 hairpin incorporated on PEgRNA (e.g., in the e1 or e2 region or elsewhere) and fused to the DNA polymerase). MS2cp protein, which colocalizes DNA polymerase with PEgRNA). The primer binding site generally does not form part of the template used by a DNA polymerase (e.g., reverse transcriptase) to encode the resulting 3' single-stranded DNA flap that encompasses the desired edit. It is also noted that. Therefore, the designation "DNA synthesis template" refers to the desired 3 'Refers to the region or portion of the extended arm (3) that is used as a template by a DNA polymerase to encode a single-stranded DNA flap. In some embodiments, the DNA synthesis template includes an "editing template" and a "homologous arm" or one or more homologous arms, eg, 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, editing templates can also include deletions, which can be manipulated by encoding homology arms containing the desired deletions. In other embodiments, the DNA synthesis template may also include the e2 region or portions thereof. For example, if the e2 region contains secondary structures that cause termination of DNA polymerase activity, then DNA polymerase function would be terminated before any part of the e2 region is actually encoded on the DNA. Is possible. It is also possible that some or even all of the e2 region will be encoded on the DNA. How much of e2 is actually used as template will depend on its composition and whether it disrupts DNA polymerase function.

The embodiment of FIG. 3E provides the structure of another PEgRNA contemplated herein. PEgRNA contains three main component components ordered in the 5' to 3' direction: a spacer, a gRNA core, and an extended arm at the 3' end. The elongated arm can be further divided into the following structural elements in the 5' to 3' direction: primer binding site (A), editing template (B), and homology arm (C). In addition, PEgRNA can include an optional 3' end modified region (e1) and an optional 5' end modified region (e2). Furthermore, PEgRNA may further include a transcription termination signal at the 3' end of PEgRNA (not shown). These structural elements are further defined in this application. The illustration of the structure of PEgRNA is not meant to be limiting and encompasses variations in the arrangement of the elements. For example, any sequence modifications (e1) and (e2) may be located within or between any of the other regions shown. It is not limited to being located at the 3' and 5' ends. PEgRNA, in certain embodiments, can include secondary RNA structures such as, but not limited to, hairpins, stem loops, toe loops, RNA binding protein recruitment domains (eg, MS2 aptamers that recruit and bind MS2cp protein). These secondary structures can be located anywhere on the PEgRNA molecule. For example, such secondary structures may be located within the spacer, gRNA core, or elongated arms, particularly within the e1 and/or e2 modified regions. In addition to secondary RNA structure, PEgRNAs may include chemical linkers or poly(N) linkers or tails (eg, within the e1 and/or e2 modified regions). Here, "N" can be any nucleobase. In some embodiments (eg, as shown in Figure 72(c)), the chemical linker can function to prevent reverse transcription of the sgRNA backbone or core. Additionally, in certain embodiments (see, e.g., FIG. 72(c)), the extended arm (3) may be comprised of RNA or DNA, and/or may include one or more nucleobase analogs (e.g., This may add functionality such as temperature resilience). Additionally, the orientation of the elongated arm (3) can be in the native 5' to 3' direction, or in the 3' to 5' direction (with respect to the overall PEgRNA molecule orientation) in an opposing orientation. (relatively) can be synthesized. It is also noted that one skilled in the art 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 extended arm (i.e. DNA or RNA). . This can be provided in trans, either as a fusion with napDNAbp or as a separate part, to synthesize a 3' single-stranded DNA flap encoded by the desired template that encompasses the desired edits. Can be implemented. For example, if the extending arm is RNA, the DNA polymerase can be reverse transcriptase or any other suitable RNA-dependent DNA polymerase. However, if the extending arm is DNA, the DNA polymerase may be a DNA-dependent DNA polymerase. In various embodiments, the provision of the DNA polymerase can be in trans, e.g., an RNA-protein recruitment domain (e.g., an MS2 hairpin incorporated on PEgRNA (e.g., in the e1 or e2 region or elsewhere) and fused to the DNA polymerase). MS2cp protein, which colocalizes DNA polymerase with PEgRNA). The primer binding site generally does not form part of the template used by a DNA polymerase (e.g., reverse transcriptase) to encode the resulting 3' single-stranded DNA flap that encompasses the desired edit. It is also noted that. Therefore, the designation "DNA synthesis template" refers to the desired 3 'Refers to the region or portion of the extended arm (3) that is used as a template by a DNA polymerase to encode a single-stranded DNA flap. In some embodiments, the DNA synthesis template includes an "editing template" and a "homologous arm" or one or more homologous arms, eg, 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, editing templates can also include deletions, which can be manipulated by encoding homology arms containing the desired deletions. In other embodiments, the DNA synthesis template may also include the e2 region or portions thereof. For example, if the e2 region contains secondary structures that cause termination of DNA polymerase activity, then DNA polymerase function would be terminated before any part of the e2 region is actually encoded on the DNA. Is possible. It is also possible that some or even all of the e2 region will be encoded on the DNA. How much of e2 is actually used as template will depend on its composition and whether it disrupts DNA polymerase function.

The schematic diagram in Figure 3F illustrates a typical PEgRNA interaction with a double-stranded DNA target site and the concomitant generation of a 3' single-stranded DNA flap containing the genetic change of interest. Double-stranded DNA is represented by an upper strand (ie, the target strand) in a 3' to 5' orientation and a lower strand (ie, the PAM strand or non-target strand) in a 5' to 3' orientation. The upper strand contains the complement of the "protospacer" and the complement of the PAM sequence and is referred to as the "target strand." This is because it is the strand that is targeted by and anneals to the spacer of PEgRNA. Because it contains a PAM sequence (eg, NGG) and a protospacer, the complementary lower strand is referred to as the "non-target strand" or "PAM strand" or "protospacer strand." Although not shown, the illustrated PEgRNA will be complexed with the Cas9 or equivalent domain of the prime editor fusion protein. As shown in the schematic, the PEgRNA spacer 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 and induces 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 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, reverse transcriptase (e.g., provided in trans or in cis as a fusion protein attached to a Cas9 construct) is then added to the DNA synthesis template (editing template (B) and homology arm (C)). ) is encoded by a single strand of DNA. Polymerization continues towards the 5' end of the extended arm. The polymerized strands of ssDNA form the ssDNA 3' end flap, which invades the endogenous DNA and displaces the corresponding endogenous strand (as described elsewhere (e.g., as shown in Figure 1G)). This is removed as a DNA flap at the 5' end of the endogenous DNA), performs the desired nucleotide edits (single nucleotide base pair changes, deletions, insertions (encompassing entire genes) in naturally occurring DNA repair/replication rounds. Incorporate by.

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.

Figure 3H illustrates the process of transprime editing. In this embodiment, the transprime editing factor is fused to the MS2cp protein (i.e., a type of recruitment protein that recognizes and binds the MS2 aptamer) and complexed with sgRNA (i.e., standard guide RNA as opposed to PEgRNA). contains the "PE2" prime editing factor (i.e., a fusion of Cas9(H840A) and variant MMLV RT). Transprime editing factors bind to target DNA and nick non-target strands. 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 editing factor, thereby providing PBS and DNA synthesis template functions in trans for use by reverse transcriptase polymerase, with a 3' end and by the DNA synthesis template. A single-stranded DNA flap containing the desired encoded genetic information is synthesized.

Figures 4A-4E demonstrate an in vitro TPRT assay (ie, prime editing assay). Figure 4A is a schematic diagram of fluorescently labeled DNA substrate, elongation of gRNA template with RT enzyme, and PAGE. Figure 4B shows TPRT (ie, prime editing) with pre-nicked substrates, dCas9, and 5' extended gRNAs of different synthetic template lengths. Figure 4C shows RT reactions with pre-nicked DNA substrates in the absence of Cas9. Figure 4D shows TPRT (ie, prime editing) on full-length dsDNA substrates with Cas9(H840A) and 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 lengths. 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). A dCas9:gRNA complex was formed from the purified components. Fluorescently labeled DNA substrate was then added along with dNTPs and RT enzyme. After 1 hour incubation at 37°C, reaction products were analyzed by denaturing urea polyacrylamide gel electrophoresis (PAGE). The gel image shows an extension of the original DNA strand to a length that matches the reverse transcription template length.

Figure 6 shows in vitro validation results using 5' extended gRNAs with varying synthetic template lengths, which are very similar to those shown in Figure 5. However, the DNA substrate is 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, nickase efficiently cuts the DNA strand when 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 whether the RT reaction preferentially occurs in cis on gRNA (bound in the same complex). Two separate experiments were performed with 5' and 3' extended gRNAs. Products were analyzed by PAGE. The product ratio was calculated as (Cy3cis/Cy3trans)/(Cy5trans/Cy5cis).

Figures 9A-9D demonstrate flap model substrates. 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 sequenced after flap repair. Figure 9D shows examination of different flap features in human cells.

Figure 10 demonstrates prime editing on plasmid substrates. A dual fluorescent reporter plasmid was constructed for yeast (S. cerevisiae) expression. Expression of this construct in yeast produces only GFP. An in vitro prime editing reaction induces point mutations and the parental plasmid, or a plasmid nicked in vitro with Cas9(H840A), is transformed into yeast. Colonies are visualized by fluorescence imaging. A yeast double FP plasmid transformant is shown. Transforming the parental plasmid or the Cas9(H840A) nicked plasmid in vitro results in only green GFP expressing colonies. Prime editing reactions with 5' extended gRNA or 3' extended gRNA produce mixed green and yellow colonies. The latter expresses both GFP and mCherry. More yellow colonies are observed with 3' extended gRNA. A positive control that does not contain a 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, prime editing repairs the frameshift mutation and synthesizes downstream mCherry. Incorporate single nucleotide insertions (left) or deletions (right) that allow for. 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. Precise 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 shows a comparison with deaminase-mediated base editing technology.

Figure 14 shows a schematic diagram of editing in human cells.

Figure 15 demonstrates the extension of primer binding sites in gRNA.

Figure 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 output of the sequencing analysis displays the most abundant genotype of the edited cells.

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

Figure 20 shows the editing efficiency of target nucleotides when a single-stranded nick is introduced into the complementary DNA strand in close proximity 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). An example of "none" does not contain a guide RNA that nicks the complementary strand. Editing rates were quantified by high-throughput sequencing of genomic DNA amplicons.

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

Figure 22 shows that targeted mutagenesis is performed on a target locus using a nucleic acid-programmed DNA binding protein (napDNAbp) complexed with an elongated guide RNA and an error-prone reverse transcriptase. Provides a schematic diagram of an exemplary process for performing (i.e., prime editing with error-prone RT). This process is sometimes referred to as a form of prime editing for targeted mutagenesis. An extended guide RNA comprises an extension at the 3' or 5' end of the guide RNA or at some point within the molecule within 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 (eg, 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 at the target locus. Ru. In certain embodiments, a nick is created in the strand of DNA that corresponds to the R-loop strand, ie, the strand that is not hybridized to the guide RNA sequence. In step (c), the 3' terminal DNA strand interacts with the guide RNA extension to prime reverse transcription. In certain embodiments, the 3' terminal DNA strand hybridizes to a specific RT prime sequence on the guide RNA extension. 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 mutagenic region. In step (e) napDNAbp and guide RNA are released. Steps (f) and (g) involve disassembling the single-stranded DNA flap (containing the mutagenic region) so that the desired mutagenic region is incorporated into the target locus. This process, once a 3' single-stranded DNA flap enters and hybridizes with a complementary sequence on the other strand, removes the corresponding 5' endogenous DNA flap that is formed, thereby allowing desired product formation. can be promoted to The process can also be driven to form a product with a nick in the second strand, as illustrated in Figure IF. Following endogenous DNA repair and/or replication processes, the mutagenic region becomes integrated into both strands of DNA at the DNA locus.

Figure 23 is a schematic diagram of gRNA design to reduce trinucleotide repeat sequences and trinucleotide repeat reduction with TPRT genome editing (ie, prime editing). Trinucleotide repeat expansions are associated with numerous human diseases, including Huntington's disease, Fragile X syndrome, and Friedreich's ataxia. The most common trinucleotide repeat contains the CAG triplet, but the GAA triplet (Friedreich's ataxia) and CGG triplet (Fragile X syndrome) also occur. Inheriting a predisposition to expansion or acquiring an already expanded parental allele increases the likelihood of acquiring the disease. Pathogenic expansion of trinucleotide repeats could hypothetically be corrected using prime editing. The region upstream of the repeat region is nicked by an RNA-guided nuclease, and then a new DNA strand containing a healthy number of repeats (this depends on the specific gene and disease) is generated. Can be used to prime synthesis. After the repeat sequence, a short stretch of homology is added (red strand) that matches the identity of the sequence adjacent to the other end of the repeat. Invasion of the newly synthesized strand, followed by replacement of the endogenous DNA with the newly synthesized flap, leads to a reduced repeat allele.

Figure 24 is a schematic diagram showing a precise 10 nucleotide deletion in prime editing. A guide RNA targeted by the HEK3 locus was designed with a reverse transcription template encoding a 10 nucleotide deletion after the nick site. Editing efficiency in transfected HEK cells was assessed using amplicon sequencing.

FIG. 25 is a schematic diagram showing gRNA design for peptide tagging genes at endogenous genomic loci and peptide tagging with TPRT genome editing (ie, prime editing). The FlAsH and ReAsH tagging systems contain two parts: (1) a fluorophore-biarsenical probe, and (2) a genetically encoded peptide containing a tetracysteine motif, with the sequence FLNCCPGCCMEP. (SEQ ID NO: 1). When expressed intracellularly, tetracysteine motif-containing proteins can be fluorescently labeled with fluorophore-arsenic probes (see reference: J.Am.Chem.Soc., 2002, 124(21), pp6063-6076) .DOI:10.1021/ja017687n). The "sortagging" system employs a bacterial sortase enzyme to covalently conjugate a labeled peptide probe to a protein containing a suitable peptide substrate (Reference: Nat.Chem.Biol.2007 Nov;3 (11):707-8. DOI:10.1038/nchembio.2007.31). 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)) is commonly employed as an epitope tag for immunoassays. Pi-clamp encodes a peptide sequence (FCPF (SEQ ID NO: 622) ) that can be labeled with pentafluoro-aromatic substrates (Reference: Nat.Chem.2016 Feb;8(2):120- 8. doi:10.1038/nchem.2413).

Figure 26A shows the correct incorporation of His ₆ and FLAG tags into genomic DNA. Guide RNAs targeting the HEK3 locus were designed with reverse transcription templates encoding either 18nt His tag insertions or 24nt FLAG tag insertions. Editing efficiency in transfected HEK cells was assessed using amplicon sequencing. Note that the full length 24 nt sequence of the FLAG tag is cut out of frame (sequencing confirmed correct insertion of the full length). Figure 26B shows that (a) solubilizing or insolubilizing a protein; (b) altering or tracking the subcellular localization of a protein; (c) extending the half-life of a protein; Figure 2 shows a schematic summarizing various applications involving protein/peptide tagging, including d) facilitating protein purification; and (e) facilitating protein detection.

Figure 27 provides an overview of prime editing by incorporating protective mutations into PRNP that prevent prion disease or halt its progression. The PEgRNA sequences correspond to SEQ ID NO: 4082 (ie, 5' of the sgRNA backbone) on the left and SEQ ID NO: 4083 (ie, 3' of the sgRNA backbone) on the right.

Figure 28A is a schematic diagram of PE-based insertion of sequences encoding RNA motifs. 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 prime editing factors. Figure 29B shows possible modifications to genomic, plasmid, or viral DNA induced by PE. Figure 29C shows an example scheme for insertion of a library of peptide loops onto a defined protein (in this case GFP) with a library of PEgRNAs. Figure 29D shows an example of possible programmable deletion or N- or C-terminal truncation of protein codons 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 an illustration of engineered gRNA. Shown is a gRNA core, a ~20nt 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 using 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 top schematic shows exemplary PEgRNAs that can be used in this aspect. A spacer on 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 is complementary to the encoded primer binding sequence and the region just downstream of the cut site. (in red color) and a homologous region (encoded by the homologous arms of PEgRNA). Through 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 PEgRNA in an off-target manner (i.e., spacers for other genomic sites that are not the intended genomic site). Due to region complementarity, 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 off-target genomic sites elsewhere on the genome. To detect the insertion of these primer binding sites at both intended and off-target genomic sites, genomic DNA (after PE) can be isolated, fragmented, and ligated to adapter nucleotides (indicated in red). It is shown). PCR can then be performed with PCR oligonucleotides that anneal to the adapter and the inserted primer binding sequences to amplify on-target and off-target genomic DNA regions where 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 on-target or off-target sites.

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

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

Figure 36 shows low molecular weight monomers. 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 the 75,122 known pathogenic human gene variants in ClinVar (accessed July 2019), categorized by type. Figure 38B shows that the prime editing complex is guided by an RNA fused to an engineered reverse transcriptase domain, such as a prime containing a DNA nicking domain, such as a Cas9 nickase and complexed with a prime editing guide RNA (PEgRNA). This indicates that it consists of the editing factor (PE) protein. PE:PEgRNA complexes bind to target DNA sites and enable 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 PEgRNA. The reverse transcriptase domain catalyzes primer extension using the PEgRNA RT template, 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 to degrade the heteroduplex DNA. or replication results in stably edited DNA. Figure 38D shows in vitro 5' extended PEgRNA with a pre-nicked dsDNA substrate containing a 5'Cy5 labeled PAM strand, dCas9, and a commercially available M-MLV RT variant (RT, Superscript III). Primer extension assay is shown. dCas9 was complexed with PEgRNA containing RT templates of various lengths and then added to the DNA substrate along with the indicated components. Reactions were incubated for 1 hour at 37°C and then analyzed by denaturing urea PAGE and visualized for Cy5 fluorescence. Figure 38E shows primers performed as in Figure 38D using 3'-extended PEgRNA pre-complexed with dCas9 or Cas9 H840A nickase and 5'Cy5-labeled dsDNA substrate, pre-nicked or unnicked. Shows elongation. Figure 38F shows a yeast colony transformed with a GFP-mCherry fusion reporter plasmid edited in vitro with PEgRNA, Cas9 nickase, and RT. Plasmids containing nonsense or frameshift mutations between GFP and mCherry were edited with 5'- or 3'-extended PEgRNAs that restored mCherry translation by transversion mutations, 1bp insertions, or 1bp deletions. GFP and mCherry double positive cells (yellow) reflect successful editing.

Figures 39A-39D show prime editing of genomic DNA of human cells by PE1 and PE2. Figure 39A shows the 3' PEgRNA contains a spacer sequence, an sgRNA backbone, and a reverse transcription (RT) template (purple) containing a primer binding site (green) and the base(s) to be edited (red). Indicates that it contains elongation. The primer binding site hybridizes to the PAM-containing DNA strand immediately upstream of the site of nicking. With the exception of encoded edits, the RT template is homologous to the DNA sequence downstream of the nick. Figure 39B shows T.A. to A.T. Indicates the inclusion of version editing. Figure 39C shows that the use of an engineered quintuple mutant M-MLV reverse transcriptase (D200N,L603W,T306K,W313F,T330P) to PE2 significantly increases prime editing transversion efficiency at five genomic sites in HEK293T cells. small insertions and small deletion edits. 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 nick the non-edited strand to increase prime editing efficiency. Figure 40A is an overview of prime editing with PE3. After initial synthesis of the edited strand, DNA repair will remove either the newly synthesized strand containing the edit (3' flap excision) or the original genomic DNA strand (5' flap excision). . 5' flap excision leaves behind a DNA heteroduplex containing one edited and one unedited strand. Mismatch repair mechanisms or DNA replication can degrade the heteroduplex to give either edited or unedited products. Nicking the non-edited strand favors repair of that strand, resulting in the preferential production of stable double-stranded DNA containing the desired edits. Figure 40B shows the effect of complementary strand nicking on prime editing efficiency and indel formation mediated by PE3. "None" refers to a PE2 control that does not nick the complementary strand. FIG. 40C is a comparison of editing efficiency with PE2 (no complementary strand nick), PE3 (general complementary strand nick), and PE3b (editing-specific complementary strand nick). All editing yields reflect the percentage of total sequencing reads containing the intended edit and no indels out of all treated cells without sorting. Values and error bars reflect the mean and s.d. of three independent biological replicates.

Figures 41A-41K show targeted insertions, deletions, and point mutations of all 12 types by PE3 at 7 endogenous human genomic loci in HEK293T cells. Figure 41A shows all 12 types of one-nucleotide transitions from position +1 to +8 (counting PEgRNA-induced nick positioning as between positions +1 and -1) of the HEK3 site using a 10nt RT template. and a graph showing transversion editing. Figure 41B is a graph showing long range PE3 transversion editing at the HEK3 site using a 34nt RT template. Figures 41C-41H show all 12 types of transition and transversion edits at various positions on the prime edit window: (Figure 41C) RNF2, (Figure 41D) FANCF, (Figure 41E) EMX1, (Figure 41F) FIG. 41 is a graph showing RUNX1, (FIG. 41G) VEGFA, and (FIG. 41H) DNMT1. FIG. 41I is a graph showing targeted 1 and 3 bp insertions and 1 and 3 bp deletions by PE3 at seven endogenous genomic loci. Figure 41J is a graph showing targeted precision deletions from 5 to 80 bp in the HEK3 target site. FIG. 41K is a graph showing combinatorial 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 containing the intended edit and no indels out of 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 editing 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 frequencies from the treatment of Figure 42A. Figure 42C shows the editing efficiency of precise C.G to T.A. edits (no bystander edits or indels) in HEK3, FANCF, and EMX1 for PE2, PE3, BE2max, and BE4max. In EMX1, precise PE combinatorial editing of all possible combinations of C·G to T·A conversions at the three target nucleotides is also demonstrated. 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 precise A·T to G·C editing efficiency for HEK3 and FANCF without bystander editing or indels. Figure 42F shows the indel frequencies from the treatment of Figure 42D. Figure 42G shows average triplicate editing efficiency (percentage sequencing reads with indels) in HEK293T cells at 4 on-target and 16 known off-target sites for Cas9 nuclease. The 16 off-target sites examined were the top four previously reported off-target sites ^118,159 for each of the four on-target sites. For each on-target site, Cas9 was paired with an sgRNA or with each of the four PEgRNAs that recognize the same protospacer. Figure 42H shows the average triplicate on-target and off-target editing efficiencies and indel efficiencies (in brackets below) 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 containing the intended edit and no indels out of all treated cells without sorting. Off-target editing yield reflects off-target locus modifications 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 the incorporation (by T.A to A.T transversion) and correction (by A.T to T.A transversion) of pathogenic E6V mutations in the HBB of HEK293T cells. Modifications to either wild-type HBB or to HBB containing silent mutations that block PEgRNA PAM are shown. Figure 43B is a graph showing the integration (by 4bp insertion) and correction (by 4bp deletion) of the pathogenic HEXA 1278+TATC allele in HEK293T cells. Modifications to either wild-type HEXA or HEXA containing a silent mutation that blocks PEgRNA PAM are shown. Figure 43C is a graph showing 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). FIG. 43E is a graph showing the incorporation of a G.C to T.A transversion mutation in DNMT1 of mouse primary cortical neurons using a dual split intein PE3 lentiviral system. In this, the N-terminal half was Cas9(1-573) fused to the N-intein and to GFP-KASH via the P2A self-cleaving peptide, and the C-terminal half was fused to the rest of PE2. It is C intein. The two halves of PE2 are expressed from the human synapsin promoter, which is highly specific for mature neurons. Sorted values reflect edits or indels from GFP-positive nuclei, 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 byproduct production mediated by PE3 and Cas9 in HEK293T, K562, U2OS, and HeLa cells. Figure 43I shows targeted insertion of His6 tag (18 bp), FLAG epitope tag (24 bp), or extended LoxP site (44 bp) in HEK293T cells by PE3. All editing yields reflect the percentage of total sequencing reads that contain the intended edit and do not contain indels out of 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 assay with dCas9, 5' extended PEgRNA, and 5'-Cy5 labeled DNA substrate. PEgRNA 1 to 5 have a 15nt linker sequence between the spacer and PBS (linker A for PEgRNA 1, linker B for PEgRNA 2 to 5), a 5nt PBS sequence, and 7nt (PEgRNA 1 and 2), 8nt (PEgRNA 3), Contains 15nt (PEgRNA 4) and 22nt (PEgRNA 5) RT templates. PEgRNA is that used in Figures 44E and 44F; complete sequences are listed in Tables 2A-2C. Figure 44B shows in vitro nicking assay of Cas9 H840A using 5' extended and 3' extended PEgRNA. Figure 44C shows Cas9-mediated indel formation in HEK293T cells in HEK3 with 5' extended and 3' extended PEgRNAs. Figure 44D shows an overview of the prime editing in vitro biochemical assay. 5'-Cy5 labeled prenicked and unnicked dsDNA substrates were tested. sgRNA, 5'-extended PEgRNA, or 3'-extended PEgRNA was 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 using 5'-extended PEgRNA, pre-nicked DNA substrates, and dCas9 lead to significant conversion to RT products. Figure 44F shows a primer extension reaction using unnicked DNA substrate and Cas9 H840A nickase and PEgRNA 5' extended as in Figure 44B. Product yield is greatly reduced compared to pre-nicked substrates. Figure 44G shows by denaturing urea PAGE that in vitro primer extension reactions using 3'-PEgRNA generate a single distinct product. The RT product band was excised, eluted from the gel, and then subjected to homopolymer tailing with terminal transferase (TdT) using either dGTP or dATP. The tailed products were extended with polyT or polyC primers and the resulting DNA was sequenced. The Sanger trace indicates that the 3 nucleotides from the gRNA backbone were reverse transcribed (added to the DNA product as the last 3' nucleotide). Note that in mammalian cell prime editing experiments, PEgRNA backbone insertions are much rarer than in vitro (Figures 56A-56D). Possible explanations include the inability of the tethered reverse transcriptase to access the Cas9-bound guide RNA scaffold and/or cellular excision of the mismatched 3' end of the 3' flap containing the PEgRNA scaffold sequence. Cause.

Figures 45A-45G show cellular repair in yeast of 3' DNA flaps from in vitro prime editing reactions. Figure 45A shows that the dual fluorescent protein reporter plasmid contains GFP and mCherry open reading frames separated by a target site encoding an in-frame stop codon, +1 frameshift, or -1 frameshift. show. 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 the unedited plasmid produce GFP but not mCherry. Yeast colonies containing the edited plasmid will generate both GFP and mCherry as fusion proteins. Figure 45B contains a stop codon between GFP and mCherry (non-edited negative control, top) or no stop codon or frameshift between GFP and mCherry (pre-edited positive control, bottom) ) Shows an overlay of GFP and mCherry fluorescence of yeast colonies transformed by the reporter plasmid. Figures 45C-45F show visualization of mCherry and GFP fluorescence from yeast colonies transformed with in vitro prime editing reaction products. Figure 45C shows stop codon modification 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 with a 1 bp insertion using 3' extended PEgRNA. Figure 45G shows Sanger DNA sequencing traces from plasmids isolated from GFP-only colonies in Figure 45B and GFP and mCherry double positive colonies in Figure 45C.

Figures 46A-46F show correct edits versus indel generation by PE1. Figure 46A shows T·A to A·T transversion editing efficiency and indel generation by PE1 at the +1 position of HEK3, using PEgRNA containing a 10nt RT template and PBS sequences ranging from 8-17nt. Figure 46B shows G·C to T·A transversion editing efficiency and indel generation by PE1 at position +5 of EMX1 using PEgRNA containing a 13nt RT template and PBS sequences ranging from 9-17nt. . Figure 46C shows G·C to T·A transversion editing efficiency and indel generation by PE1 at position +5 of FANCF using PEgRNA containing a 17nt RT template and a PBS sequence ranging from 8-17nt. . Figure 46D shows C·G to A·T transversion editing efficiency and indel generation by PE1 at the +1 position of RNF2 using PEgRNA containing an 11nt RT template and PBS sequences ranging from 9-17nt. . Figure 46E shows the G·C to T·A transversion editing efficiency and indel generation by PE1 at the +2 position of HEK4, using PEgRNA containing a 13nt RT template and a PBS sequence ranging from 7-15nt. Figure 46F shows a +1T deletion, +1A insertion, and +1CTT insertion mediated by PE1 in the HEK3 site, using a 13nt PBS and 10nt RT template. The sequence of PEgRNA is that 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 prime editing factor variants used in this figure. Figure 47B shows targeted insertion and deletion editing by PE1 at the HEK3 locus. Figures 47C-47H show +2G C to C G transversion edit in HEK3 as shown in Figure 47C, 24bp FLAG insertion in HEK3 as shown in Figure 47D, and Figure 47E. +1C G to A T transversion edit in RNF2, +1G C to C G transversion edit in EMX1 as shown in Figure 47F, + in HBB as shown in Figure 47G Containing M-MLV RT variants for their ability to incorporate the 2T A to A T transversion edit and the +1G C to C G transversion edit in FANCF as shown in Figure 47H. A comparison of 18 prime editing factor constructs is shown. 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 various PBS lengths. Figure 47O shows the variation from +1T・A to A・T in HEK3. Figure 47P shows variations from +5G・C to T・A in EMX1. Figure 47Q shows variations from +5G・C to T・A in FANCF. Figure 47R shows variations from +1C・G to A・T in RNF2. Figure 47S shows +2G・C to T・A variations 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 (blue line) in VEGFA of HEK293T cells as a function of RT template length. Indels (gray lines) are plotted for comparison. The sequence below the graph shows the final nucleotide for editing as a template for synthesis with PEgRNA. G nucleotides (templated by C on PEgRNA) are highlighted; to maximize prime editing efficiency, RT templates ending in C should be avoided during PEgRNA design. Figure 48B shows transversion edits and indels from +5G.C to T.A of DNMT1 as shown in Figure 48A. FIG. 48C shows the transversion edit and indel from +5G.C to T.A of RUNX1 as shown in FIG. 48A. Values and error bars reflect the mean and s.d. of three independent biological replicates.

Figures 49A-49B show the effects of PE2, PE2 R110S K103L, Cas9 H840A nickase, and dCas9 on cell viability. HEK293T cells were transfected with plasmids encoding PE2, PE2 R110S K103L, Cas9 H840A nickase, or dCas9 together with HEK3-targeting PEgRNA plasmids. Cell viability was measured every 24 hours for 3 days after transfection using the CellTiter-Glo2.0 assay (Promega). Figure 49A shows viability measured by luminescence 1, 2, or 3 days after 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, along with a HEK3-targeted PEgRNA plasmid encoding a +5G to A edit. Editing efficiency was measured from treated cells on day 3 post-transfection, alongside that 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 and HEXA 1278+TATC modification by various PEgRNAs. Figure 50A shows a screen of 14 PEgRNAs for correction of HBB E6V allele in HEK293T cells by PE3. All PEgRNAs evaluated convert the HBB E6V allele back to wild-type HBB without introducing 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. PEgRNA labeled HEXA corrects the pathogenic allele by a shifted 4bp deletion that blocks PAM and leaves a silent mutation. PEgRNA labeled HEXA corrects the pathogenic allele back to the wild type. Entries ending in "b" use an editing-specific nicking sgRNA in combination with 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 and HDR initiated by PE3 and Cas9 in human cell lines. 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. Efficiency in producing correct edits (no indels) and indel frequencies for HDR initiated. Comparison of each lumped edit incorporates identical edits by HDR initiated by PE3 and Cas9. Non-targeting controls are PEgRNA targeting PE3 and non-target loci. Figure 51E shows control experiments with non-targeted PEgRNA+PE3 and dCas9+sgRNA compared to wild type Cas9 HDR experiments. We confirm that the common contaminant ssDNA donor HDR template, which pseudo-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 by PE3 or Cas9-initiated HDR. Alleles were sequenced by Illumina MiSeq and analyzed by CRISPResso2 ¹⁷⁸ . Reference HEK3 sequences from this region are on the top. Allele tables are shown for the non-targeting PEgRNA negative control, +1CTT insertion in HEK3 with PE3, and +1CTT insertion in HEK3 with Cas9-initiated HDR. Allele frequencies and corresponding Illumina sequencing read counts are shown for each allele. All alleles observed at frequency ≧0.20% are shown. Values and error bars reflect the mean and SD of three independent biological replicates.

Figures 52A-52D show the length distribution of pathogenic insertions, duplications, deletions, and indels on the ClinVar database. ClinVar variant summary downloaded from NCBI on July 15, 2019. The lengths of reported insertions, deletions, and duplications were calculated using appropriate identification of reference and alternative alleles, variant start and stop positions, or variant names. Variants that did not report any of the above information were excluded from the analysis. The reported length of an indel (a single variant encompassing both insertions and deletions relative to the reference genome) is the number of mismatches or gaps in the best pairwise alignment between the reference and alternative alleles. Calculated by determining .

Figures 53A- 53E show examples of FACS gating for GFP positive cell sorting. Below is an example of the original batch analysis file, 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 for cells that do not express GFP. Graphic 2 shows example sorting of P2A-GFP expressing cells used to isolate the HBB E6V HEK293T cell line. HEK293T cells were initially population gated 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 those 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 -5E show allele tables for the HBB E6V homozygous HEK293T cell line.

Figure 54 is a schematic diagram summarizing the PEgRNA cloning procedure.

Figures 55A-55G are schematic diagrams of PEgRNA designs. Figure 55A shows a simple diagram of PEgRNA with labeled domains (left) and bound to nCas9 at a genomic site (right). Figure 55B shows various types of modifications of PEgRNA that are expected to increase activity. Figure 55C shows promoter options and modification of PEgRNA to increase transcription of longer RNAs through 5', 3' processing and termination. Figure 55D shows the elongation 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 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 PEgRNA can enhance RT binding.

Figures 56A-56D show integration of PEgRNA backbone sequences into target loci. HTS data was analyzed for PEgRNA backbone sequence insertions as described in Figures 60A-60B. Figure 56A shows analysis of the EMX1 locus. % of total sequenced reads containing one or more PEgRNA backbone sequence nucleotides on an insert adjacent to the RT template (left); % of total sequenced reads containing a PEgRNA backbone sequence insert of a defined length (middle ); and the cumulative total percentage of PEgRNA insertions encompassing up to the defined length on the X-axis is shown. Figure 56B shows the same thing as Figure 56A for FANCF. Figure 56C shows the same thing as Figure 56A for HEK3. Figure 56D shows the same thing 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 effects 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-targeted or HEXA-targeted PEgRNA. RNA corresponding to 14,410 genes and 14,368 genes were detected in PRNP and HEXA samples, respectively. Figures 57A-57F show volcano plots presenting -log10 FDR-adjusted p-values versus ~fold change in log2 transcript abundance for each (Aeach) RNA. (Figure 57A) PE2 vs. PE2-dRT with PRNP-targeting PEgRNA, (Figure 57B) PE2 vs. Cas9 H840A with PRNP-targeting PEgRNA, (Figure 57C) PE2-dRT vs. Cas9 H840A with PRNP-targeting PEgRNA, (Figure 57D) HEXA Comparing PE2 vs. PE2-dRT with targeting PEgRNA, (FIG. 57E) PE2 vs. Cas9 H840A with HEXA-targeting PEgRNA, (FIG. 57F) PE2-dRT vs. Cas9 H840A with HEXA-targeting PEgRNA. Red dots indicate genes showing a ≧2-fold change in relative abundance that is statistically significant (FDR adjusted p<0.05). Figures 57G-57I are Venn diagrams of upregulated and downregulated transcripts (≥2-fold change), comparing PRNP and HEXA samples to (Figure 57G) PE2 vs. PE2-dRT, (Figure 57H) PE2 vs. Cas9 H840A, and (FIG. 57I) comparing PE2-dRT vs. Cas9 H840A.

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

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

Figures 60A-60B show Python scripts for quantifying PEgRNA backbone incorporation. A custom python script was generated to characterize and quantify PEgRNA insertions at targeted genomic loci. The script iteratively matches increasing lengths of text strings taken from a reference sequence (guide RNA backbone sequence) to sequencing reads in a fastq file and calculates the number of sequencing reads that match the search query. Count. Each sequential text string corresponds to additional nucleotides of the guide RNA backbone sequence. Exact length intake and maximum specified length cumulative intake were calculated in this manner. To ensure alignment and accurate counting of short slices of sgRNA, the 3'-end 5 to 6 bases of the new DNA strand synthesized by reverse transcriptase are included at the start of the reference sequence.

Figure 61 is a graph showing the percentage of total sequencing reads with defined edits for SaCas9(N580A)-MMLV RT HEK3 +6C>A. Correct edit and indel values 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). Percentage of total sequencing reads with targeted PAM editing is shown for SpCas9(H840A)-VRQR-MMLV RT with NGA > NTA and for SpCas9(H840A)-VRER-MMLV RT with NGCG > NTCG. ing. 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 a human cell genome and 2) use of that SSR target site to incorporate a DNA donor template containing a GFP expression marker. Once successfully incorporated, GFP causes cells to fluoresce. See Example 17 for further details.

Figure 66 illustrates one embodiment of a prime editing factor provided as two PE half proteins. These regenerate the entire prime editing factor by the self-splicing action of the split intein halves positioned at the end or beginning of each protein half of the prime editing factor.

Figure 67 illustrates the mechanism of intein removal from a polypeptide sequence and reformation of peptide bonds between N-terminal and C-terminal extein sequences. (a) illustrates the general mechanism of two halves of the protein, each containing half of the intein sequence. When these come into contact within a cell, they result in a fully functional intein. This then undergoes self-splicing and excision. The process of excision results in the formation of a peptide bond between the N-terminal half of the protein (or "N extein") and the C-terminal half of the protein (or "C extein"), resulting in the formation of N- and C-exteins. form a whole single polypeptide containing the in moiety. In various embodiments, the N extein can correspond to the N-terminal half of the split prime editing factor fusion protein and the C extein can correspond to the C-terminal half of the split prime editing factor fusion protein. (b) shows the chemical mechanism of intein excision and reformation of the peptide bond connecting half of the N extein (half colored red) and half of the C extein (half colored blue). Excision of a split intein (ie, 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 shows that delivery of both halves of the split intein of SaPE2 (e.g., SEQ ID NO: 443 and SEQ ID NO: 450) recapitulates the activity of full-length SaPE2 (SEQ ID NO: 134) when co-transfected into HEK293T cells. prove that .

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

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

Figure 69 shows 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 illustrations of step-by-step instructions for designing PEgRNA and nicking sgRNA for prime editing. Figure 70A: Step 1. Define target sequence and edits. Retrieve the sequence of the target DNA region (~200 bp) centered around the location of the desired edit (point mutation, insertion, deletion, or combination thereof). Figure 70B: Step 2. Position the target PAM. Identify PAMs that are proximal to the edit position. Be careful to look for PAM on both strands. Edits can be incorporated using protospacers and PAMs that place nicks ≧30 nt from the edit location, although PAMs that are close to the edit location are preferred. Figure 70C: Step 3. Position the nick site. For each PAM to be considered, the corresponding nick site is identified. For Sp Cas9 H840A nickase, cleavage occurs between the 5' third and fourth bases of the NGG PAM on the PAM-containing strand. All edited nucleotides must be 3' to the nick site. Therefore, a suitable PAM must be nicked 5' of 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 PEgRNA using PAM 1 only. Figure 70D: Step 4. Design the spacer array. The protospacer of Sp Cas9 corresponds to the 5' 20 nucleotides of the NGG PAM on the PAM-containing strand. Efficient Pol III transcription initiation requires G to be the first transcribed nucleotide. If the first nucleotide of the protospacer is G, the spacer sequence of PEgRNA is simply a protospacer sequence. If the first nucleotide of the protospacer is not a G, the spacer sequence of the PEgRNA is G followed by the protospacer sequence. Figure 70E: Step 5. Design the primer binding site (PBS). The starting allele sequence is used to identify DNA primers on the PAM-containing strand. The 3' end of the DNA primer is the nucleotide just upstream of the nick site (ie, the 4th base 5' of the NGG PAM for Sp Cas9). The general design principle for use with PE2 and PE3 is that the PEgRNA primer binding site (PBS) contains 12 to 13 nucleotides of complementarity to the DNA primer and contains ~40-60% GC content. It can be used for arrays. 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, the starting allele sequence is used 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 mold. The RT template encodes the designed edit and homology to the 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 (position +7 and beyond), it is recommended to use RT templates that extend at least 5 nt (preferably 10 nt or more) beyond the location of the edit to allow sufficient 3' DNA flap homology. For long range editing, several RT templates should be screened to identify a functional design. For larger insertions and deletions (≧5nt), incorporation of more 3' homology (~20nt or more) onto the RT template is recommended. Editing efficiency is typically compromised when the RT template encodes the synthesis of a G (corresponding to C in the PEgRNA RT template) as the last nucleotide on the reverse transcribed DNA product. Avoiding G as the last synthesized nucleotide is recommended when designing RT templates, as many RT templates support efficient prime editing. 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 editing using RT templates of the same length will not contain the same 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 unedited strands upstream and downstream of the edit. The optimal nicking position is highly locus dependent and should be determined empirically. Generally, nicks placed 40 to 90 nucleotides 5' across from the nick induced by PEgRNA lead to higher editing yields and fewer indels. The nicking sgRNA has a spacer sequence that matches the 20nt protospacer on the starting allele. If the protospacer does not start with a G, it has an addition of 5'G. Figure 70I: Step 9. Design PE3b nicking sgRNA. If the PAM is present on the complementary strand and its corresponding protospacer overlaps the sequence targeted for editing, then this editing 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 rather than the starting allele. The PE3b system operates 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 and prevents competition between PEgRNA and sgRNA for binding to target DNA. PE3b also avoids simultaneous nick generation on both strands, thus significantly reducing indel formation while maintaining high editing efficiency. The PE3b sgRNA should have a spacer sequence that matches the 20nt protospacer of the desired allele. With addition of 5'G if required.

Figure 71A shows the nucleotide sequence of the SpCas9 PEgRNA molecule (top). It terminates with "UUU" at the 3' end and contains no toe loop element. The lower part 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 before the 'UUU' 3' end. Ru. "N" can be any nucleobase.

FIG. 71B shows the results of Example 18. This demonstrates that the efficiency of prime editing in HEK or EMX cells is increased using PEgRNA containing toe loop elements, but the percentage of indel formation is not significantly changed.

Figures 72A -72C illustrate alternative PEgRNA constructs that can be used for prime editing. Figure 72A illustrates the PE2:PEgRNA aspect of prime editing. This embodiment involves PE2 (a fusion protein comprising Cas9 and reverse transcriptase) complexed with PEgRNA (as also described in Figures 1A-1I and/or Figures 3A-3E). In this embodiment, the reverse transcription template is incorporated onto the 3' extending arm of the sgRNA to create 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 MS2 bacteriophage coat protein (MS2cp) to form an 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 the DNA. Embodiments then involve the introduction of trans-primed editing RNA templates ("tPERT"), which replace PEgRNA by providing the primer binding site (PBS) and DNA synthesis template by separate molecules, i.e., tPERT. It operates. It also contains the MS2 aptamer (stem-loop). The MS2cp protein recruits tPERT by binding to the MS2 aptamer molecule. Figure 72C illustrates an alternative design 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 hybrid RNA/DNA PEgRNA molecules for use in prime editing, where the extended arms of the hybrid PEgRNA are DNA instead of RNA. In such embodiments, a DNA-dependent DNA polymerase can be used in place of reverse transcriptase to synthesize the 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 DNA polymerases (eg, reverse transcriptase) from using the sgRNA scaffold or backbone as a template. In yet another embodiment, the elongated arm can include a DNA synthesis template that has an opposite orientation relative to the overall orientation of the PEgRNA molecule. For example, as shown for PEgRNA with an extension attached to the 3' end of the sgRNA backbone and oriented 5' to 3', the DNA synthesis template is oriented in the opposite direction, i.e. 3' to 5'. . This embodiment may be advantageous for PEgRNA embodiments with an extended arm positioned at the 3' end of the gRNA. By reversing the orientation of the elongated arm, DNA synthesis by the polymerase (e.g. reverse transcriptase) is terminated once it reaches the new orientation 5' of the elongated arm, thus using the gRNA core as a template. There would be no risk.

Figure 73 demonstrates prime editing with tPERT and the MS2 recruitment system (also known as MS2 tagging technology). The sgRNA that targets the prime editing factor protein (PE2) to the target locus contains an RT template encoding a primer binding site (13nt or 17nt PBS), a His6 tag insertion and a homology arm, and an MS2 aptamer (5' or 5' of the tPERT molecule). (located at the 3' end) in combination with tPERT. Either prime editing factor protein (PE2) or a fusion of MS2cp to the N-terminus of PE2 was used. As in the previously developed PE3 system, editing was performed with or without 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 MS2 aptamer expression of reverse transcriptase in trans and its recruitment by the MS2 aptamer system. PEgRNA PEgRNA contains an MS2 RNA aptamer inserted into either one of the two sgRNA backbone hairpins. Wild-type M-MLV reverse transcriptase is expressed as an N-terminal or C-terminal fusion to MS2 coat protein (MCP). The 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 nicking guide RNA. The consensus sequence (most amino-terminal residues of the C-terminal extein) is indicated. Percent editing at two sites is shown: HEK3 +1CTT insertion and PRNP +6G to T. Replicates n = 3 independent transfections. See Example 20.

Figure 77 demonstrates the editing efficiency of the intein split prime editor of Example 20. Editing was assessed by targeted deep sequencing of the bulk cortex and GFP+ subpopulation by delivery of 5E10vg per half of SpPE3 and a small amount of 1E10 of nuclear-localized GFP:KASH to P0 mice by ICV injection. 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 that expresses nicking sgRNA, and the C-terminal genome contains a U6-PEgRNA cassette that expresses PEgRNA. See Example 20.

FIG. 79 shows the editing efficiency of certain optimized linkers discussed in Example 21. In particular, the data show the editing efficiency of the PE2 construct with the current linker (marked as PE2; white box) compared to various versions in which the linker was replaced by the indicated sequence, HEK3, EMX1 , FANCF, and RNF2 loci, representative PEgRNAs undergoing transition, transversion, insertion, and deletion editing are shown. Replacement linkers are “1×SGGS” (SEQ ID NO: 174) , “2×SGGS” (SEQ ID NO: 446) , “3×SGGS” (SEQ ID NO: 3889) , “1×XTEN” (SEQ ID NO: 171) , “No linker ”, “1×Gly”, “1×Pro”, “1×EAAAK” (SEQ ID NO: 3968) , “2×EAAAK” (SEQ ID NO: 3969) , and “3×EAAAK” (SEQ ID NO: 3970) . Editing efficiency is measured in bar graph format relative to the PE2 "control" editing efficiency. The linker for PE2 is SGGSSGGSSGSETPGTSESATPESSGGSSGGSS (SEQ ID NO: 127). All editing was done in the context of the PE3 system. Namely, this refers to the PE2 editing construct plus the addition of 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.

Impact of different types of modifications on PEgRNA structure on editing efficiency relative to unmodified PEgRNA, as illustrated in Example 22.

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

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

Figure 84B illustrates the structure of tRNA that can be used to modify PEgRNA structure. See Example 22. P1 can be variable in length. P1 can be extended to help prevent RNAseP processing of PEgRNA-tRNA fusions.

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

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

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

Results from a screen of N2A cells where pegRNA incorporates 1412Adel and details of primer binding site (PBS) length and reverse transcriptase (RT) template length (shown with and without indel). 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 the concentrations of editing factors versus pegRNA and nicking gRNA. See Example 23.

Definitions Unless defined otherwise, all technical and scientific terms used in this application have the meanings commonly understood by one of ordinary skill 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 defined, 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, running in a 3'→5' direction. In contrast, the "sense" strand is the segment of double-stranded DNA running 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 coding for a protein, the sense strand is the strand of DNA with 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 subsequently 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 “bispecific ligand” or “bispecific moiety” as used herein refers to a ligand that binds to two different ligand binding domains. In certain embodiments, the ligand is a small molecule compound or a peptide or polypeptide. In other embodiments, the ligand binding domain is a "dimerization domain," which can be incorporated onto the 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 bispecific ligand. As used herein, a "bispecific ligand" can equivalently be referred to as a "chemical inducer of dimerization" or "CID."

Cas9
The term "Cas9" or "Cas9 nuclease" refers to an RNA-guided nuclease that contains a Cas9 domain or fragment thereof (e.g., a protein that contains 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. "Cas9 protein" is full length Cas9 protein. Cas9 nuclease is also sometimes referred to as casn1 nuclease or CRISPR ( Clustered Regularly Interspaced Short Palindromic Repeat)-related nuclease . CRISPR is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements, and conjugative plasmids). A CRISPR cluster contains 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 trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and a Cas9 domain. tracrRNA serves as a guide for ribonuclease 3-aided processing of pre-crRNA. Cas9/crRNA/tracrRNA then endonucleolytically cleaves the linear or circular dsDNA target complementary to the spacer. Target strands that are not complementary to the crRNA are first endonucleolytically excised and then 3'-5' exonucleolytically excised. In nature, DNA binding and cleavage typically requires proteins and both RNA. However, a single guide RNA ("sgRNA", or simply "gNRA") can be modified to incorporate aspects of both crRNA and tracrRNA into a single RNA species. See, e.g., 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 helps distinguish between self versus non-self by recognizing short motifs in CRISPR repeats (PAM or protospacer-adjacent motifs). Cas9 nuclease sequences and structures are well known to those skilled in the art (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.USA98:4658-4663(2001);“CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.”Deltcheva E. , Chylinski K., Sharma CM, Gonzales K., Chao Y., Pirzada ZA, Eckert MR, Vogel J., Charpentier E., Nature 471:602-607(2011); Endonuclease in adaptive bacterial immunity.” Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna JA, Charpentier E. Science 337:816-821 (2012) (the entire content of each of which is incorporated herein by reference). (incorporated into the specification)). Cas9 orthologs have been described in various species including, but not limited to, S. pyogenes and S. thermophilus. Additional suitable Cas9 nucleases and sequences will be apparent to those skilled in the art based on this disclosure. Such Cas9 nucleases and sequences are also described 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. Cas9 sequences from the organisms and loci disclosed in (incorporated into). In some embodiments, the Cas9 nuclease contains one or more mutations that partially impair or inactivate the DNA cleavage domain.

A Cas9 domain with nuclease inactivation may be interchangeably referred to as a "dCas9" protein (representing the nuclease "inactive" form of Cas9). Methods for generating Cas9 domains (or fragments thereof) with inactive DNA cleavage domains are known (see, for example, 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 complementary strand to 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 are provided that include fragments of Cas9. For example, in some embodiments, the protein comprises one of two Cas9 domains: (1) the gRNA binding domain of Cas9; or (2) the DNA cleavage domain of Cas9. In some embodiments, a Cas9-comprising protein or fragment thereof is referred to as a "Cas9 variant." Cas9 variants share homology with Cas9 or fragments thereof. For example, the 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 to wild-type Cas9 (e.g., SpCas9 of SEQ ID NO: 18). , 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. In some embodiments, the Cas9 variant is 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, May have 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more amino acid changes. In some embodiments, the Cas9 variant has a fragment thereof that is at least about 70% identical, at least about 80% identical, at least about 90% identical to the corresponding fragment of wild-type Cas9 (e.g., SpCas9 of SEQ ID NO: 18). , 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, Contains a fragment of X Cas9 (eg, gRNA binding domain or DNA cleavage domain). In some embodiments, the fragment is at least 30%, at least 35%, at least 40%, at least 45%, at least 50% of the amino acid length of the corresponding wild-type Cas9 (e.g., SpCas9 of SEQ ID NO: 18). , 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%.

cDNA
The term "cDNA" refers to a strand of DNA copied from an RNA template. cDNA is complementary to the RNA template.

Circular permutant As used herein, the term "circular permutant" refers to a circular permutant that is a change in the structural configuration of a protein that involves a change in the order of the amino acids that occur in the protein's amino acid sequence. Refers to a protein or polypeptide (eg, Cas9) that contains a circular permutation. In other words, a circular permutant is a protein that has an altered N-terminus and C-terminus compared to its wild-type counterpart, eg, the C-terminal half of the wild-type protein has become a new N-terminal half. Circular permutation (or CP) is essentially a morphological rearrangement of a protein's primary sequence, with its N-terminus and C-terminus connected, often with a peptide linker, but at the same time splitting the sequence at a different position. This creates new adjacent N- and C-termini. The result is a protein structure that may have different connections but often the same or overall similar three-dimensional (3D) shape, perhaps with improved or altered features (reduced susceptibility to proteolysis). improved catalytic activity, altered substrate or ligand binding, and/or improved thermal stability). Circularly permuted proteins may occur in nature (eg, concanavalin A and lectins). Additionally, circular substitutions may occur as a result of post-translational modifications or may be modified using recombinant techniques.

Circularly permuted Cas9
The term "circularly permuted Cas9" refers to any Cas9 protein or variant thereof that has arisen as a circular permutant, whereby its N-terminus and C-terminus have been locally rearranged. Such circularly permuted Cas9 proteins (“CP-Cas9”), or variants thereof, retain the ability to bind to DNA when complexed with guide RNA (gRNA). 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 a new CP-Cas9 when the resulting circularly permuted protein is complexed with a guide RNA (gRNA). Use as long as it retains the ability to bind to DNA. 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 a snippet of a previous infection by a virus that invaded prokaryotes. 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. It effectively constitutes a prokaryotic immune defense system. In nature, CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In some types of CRISPR systems (e.g., type II CRISPR systems), correct processing of pre-crRNA requires trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and the Cas9 protein. . tracrRNA serves as a guide for pre-crRNA ribonuclease 3-assisted processing. Cas9/crRNA/tracrRNA then endo-cleaves linear or circular dsDNA targets that are complementary to the RNA. Specifically, target strands that are not complementary to the crRNA are first cut endo-wise and then trimmed 3'-5' exo-wise. In nature, DNA binding and cleavage typically requires proteins and both RNA. However, single guide RNAs ("sgRNAs" or simply "gNRAs") can be engineered to incorporate aspects of both crRNA and tracrRNA into a single RNA species guide RNA. See, e.g., Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna JA, Charpentier E. Science 337:816-821 (2012). The entire contents of this are incorporated herein by reference. Cas9 recognizes short motifs on CRISPR repeats (PAM or protospacer-adjacent motifs) to help distinguish self from non-self. CRISPR biology and Cas9 nuclease sequence and structure are well known to those skilled in the art (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.USA98:4658-4663(2001);“CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. “Deltcheva E., Chylinski K., Sharma 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). (Incorporated herein by reference in its entirety). Cas9 orthologs have been described in various species including, but not limited to, S. pyogenes and S. thermophilus. Additional suitable Cas9 nucleases and sequences will be apparent to those skilled in the art based on this disclosure. Such Cas9 nucleases and sequences include Cas9 nucleases 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 types of CRISPR systems (e.g., type II CRISPR systems), correct processing of pre-crRNA requires trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and the Cas9 protein. . tracrRNA serves as a guide for ribonuclease 3-assisted processing of pre-crRNA. Cas9/crRNA/tracrRNA then endo-cleaves linear or circular nucleic acid targets complementary to the RNA. Specifically, target strands that are not complementary to the crRNA are first cut endo-wise and then trimmed 3'-5' exo-wise. In nature, DNA binding and cleavage typically requires proteins and both RNA. However, single guide RNAs ("sgRNAs" or simply "gRNAs") can be engineered to incorporate aspects of both crRNA and tracrRNA into the guide RNA of a single RNA species.

In general, "CRISPR system" refers collectively to the transcripts and other elements involved in the expression or directing the activity of CRISPR-associated ("Cas") genes, including Cas genes, tracr (transactivation CRISPR) sequences (e.g. tracrRNA or active partial tracrRNA), tracr mate sequences (in the context of endogenous CRISPR systems, encompassing "direct repeats" and partial direct repeats processed by tracrRNA), guide sequences ( (also referred to as a "spacer" in the context of an endogenous CRISPR system), or other sequences from the CRISPR locus and sequences encoding transcripts. 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 a template strand (containing the desired edits) by the polymerase of a prime editing factor, and then through the mechanism of prime editing, the corresponding endogenous Refers to the region or portion of the extended arm of PEgRNA that is utilized as a 3' single-stranded DNA flap (encoding a 3' single-stranded DNA flap that displaces a strand of DNA at the target site). In various embodiments, the DNA synthesis template is configured in the form of 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 a PEgRNA containing an internal extension arm), Figure 3B (in the context of a PEgRNA containing a 3' extension arm), 3D (in the context of the 3′ extended arm), and FIG. 3E (in the context of the 5′ extended arm). The extension arm contains the DNA synthesis template and may be composed of DNA or RNA. In the case of RNA, the prime editor polymerase can be an RNA-dependent DNA polymerase (eg, reverse transcriptase). In the case of DNA, the prime editor polymerase can be a DNA-dependent DNA polymerase. In various embodiments (as depicted in Figures 3D-3E, for example), the DNA synthesis template (4) includes an "editing template" and a "homologous arm" as well as all or part of the optional 5' end modification region e2. May contain parts. That is, depending on the nature of the e2 region (e.g., whether this includes hairpin, toe-loop, or stem/loop secondary structures), the polymerase may encode the e2 region without any or part or all of it may be coded. Stated differently, in the case of the 3' extension arm, the DNA synthesis template (3) may act as a template for the synthesis of a single strand of DNA by a polymerase (e.g. reverse transcriptase). It may include the portion of the extended arm (3) that extends from the 5' end of the primer binding site (PBS) to the 3' end of the gRNA core. In the case of a 5' extending arm, the DNA synthesis template (3) may include the portion of the extending 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 PEgRNAs with either a 3' or 5' extending arm. Certain embodiments described herein (for example, FIG. 71A) provide an "RT template" that includes the editing template and the sequence of the homology arm, i.e., the PEgRNA extension arm that is actually used as a template during DNA synthesis. refers to The term "RT template" is equivalent to the term "DNA synthesis template."

In the case of transprime editing (e.g., Figures 3G and 3H), the primer binding site (PBS) and DNA synthesis template can be engineered into a separate molecule called the transprime editing factor RNA template (tPERT).

Dimerization Domain The term "dimerization domain" refers to a ligand binding domain that binds to the binding domain of a bispecific ligand. The "first" dimerization domain binds to the first binding moiety of the bispecific ligand, and the "second" dimerization domain binds to the second binding moiety of the same bispecific ligand. Join. A first dimerization domain is fused to a first protein (e.g., via a PE as discussed in this application) and a second dimerization domain is fused to a first protein (e.g., via a PE as discussed in this application). when fused to a second protein, the first and second proteins dimerize in the presence of the bispecific ligand, and the bispecific ligand is fused to the first dimerization domain. at least one moiety that binds and at least another moiety that binds to the second dimerization domain.

Downstream As used herein, the terms "upstream" and "downstream" refer to positions in a nucleic acid molecule (whether single-stranded or double-stranded) oriented in the 5'→3' direction. is a relative term that defines the linear position of at least two elements. In particular, a first element is upstream of a second element in the nucleic acid molecule if the first element is positioned somewhere 5' to the second element. For example, if the SNP is 5' to the nick site, the SNP is upstream of the Cas9-induced nick site. Conversely, if the first element is positioned somewhere 3' to the second element, then 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. Nucleic acid molecules can be DNA (double-stranded or single-stranded), RNA (double-stranded or single-stranded), or a hybrid of DNA and RNA. Where the terms upstream and downstream refer only to a single strand of a nucleic acid molecule, the analysis applies to single-stranded and double-stranded molecules, unless one needs to choose which strand of a double-stranded molecule to consider. It's the same. Often, the strand of double-stranded DNA that can be used to determine the relative position of at least two elements is the "sense" or "coding" strand. In genetics, the "sense" strand is a 5' to 3' extending segment within double-stranded DNA that is complementary to the 3' to 5' antisense or template strand of the DNA. Thus, by way of example, a SNP nucleobase is "downstream" of a promoter sequence in genomic DNA (which is double-stranded) if the SNP nucleobase is 3' to a promoter on the sense or coding strand.

Editing template The term "editing template" refers to an extended arm encoding the desired edit on a single-stranded 3' DNA flap synthesized by a polymerase, e.g., DNA-dependent DNA polymerase, RNA-dependent DNA polymerase (e.g., reverse transcriptase). Say the part. Certain embodiments described herein (as an example, FIG. 71A) refer to both the editing template and the homology arm together, i.e., the sequence of the PEgRNA extension arm that is actually used as a template during DNA synthesis. "RT mold". The term "RT editing template" is also equivalent to the term "DNA synthesis template," where the RT editing template reflects the use of a prime editing factor with a polymerase, a reverse transcriptase, and the DNA synthesis template is More broadly reflects the use of prime editing factors with either polymerase.

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

Error-Prone Reverse Transcriptase As used herein, the term "error-prone" reverse transcriptase (or more broadly, any polymerase) refers to naturally occurring or wild-type M-MLV Refers to a reverse transcriptase (or more broadly, any polymerase) that is derived from another reverse transcriptase (eg, wild-type M-MLV reverse transcriptase) that has an error rate less than that of reverse transcriptase. The error rate for wild-type M-MLV reverse transcriptase is reported to range from 15,000 (higher) to 27,000 (lower) per 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 the purposes of this application, the term "error-prone" means an error greater than 1 in 15,000 nucleobases (6.7 x 10 ^-5 or higher), e.g. 1 error in 14,000 nucleobases (7.14 ×10 ^-5 or higher), 1 error in 13,000 nucleobases or 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 (0.00011 or higher) in 9,000 nucleobases or less; 1 error (0.00013 or higher) in 8,000 nucleobases or less; 1 error (0.00013 or higher) in 8,000 nucleobases or less; 1 error in 6,000 nucleobases or less (0.00016 or higher), 1 error in 5,000 nucleobases or less (0.0002 or higher), 4,000 1 error in 3,000 nucleobases or less (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 250 nucleobases. or less, with an error rate of 1 error (0.004 or higher).

Extein The term "extein" as used herein refers to a polypeptide sequence that is flanked by inteins and ligated to another extein during the process of protein splicing to form the mature spliced protein. Typically, an intein is flanked by two extein sequences, which are ligated together when the intein catalyzes its own excision. Exteins are therefore protein analogs to exons found on mRNA. For example, a polypeptide containing an intein can have the structure extein (N)-intein-extein (C). After excision of an intein and splicing of two exteins, the resulting structure is extein (N)-extein (C) and free intein. In various configurations, the exteins can be separate proteins (eg, half of a Cas9 or PE fusion protein) each fused to a split intein, with excision of the split intein causing splicing of the extein sequences together.

Extended arm The term "extended arm" refers to the nucleotide sequence component of PEgRNA that provides several functions, including a primer binding site for reverse transcriptase and an editing template. In some embodiments, for example in FIG. 3D, the extension arm is positioned at the 3' end of the guide RNA. In other embodiments, as an example, in FIG. 3E, the extension arm is positioned at the 5' end of the guide RNA. In some embodiments, extended arms also include homologous arms. In various embodiments, the elongated 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 homologous arm, editing template, and primer binding site means that once the reverse transcriptase is primed by the annealed primer sequence, the editing in the 5'→3' direction so that the template is used as a complementary template strand to polymerases single-stranded DNA. Further details, such as the length of the extension arms, are described elsewhere herein.

The extended arm may also be described as generally containing two regions: a primer binding site (PBS) and a DNA synthesis template, as illustrated in Figure 3G (top row). A primer binding site is formed from the endogenous DNA strand of the target site when it becomes nicked by the prime editing factor complex, thereby exposing the 3' end onto the nicked endogenous strand. Binds to the primer sequence. As described herein, binding of the primer sequence to the primer binding site on the extended arm of PEgRNA creates a duplex region with an exposed 3' end (i.e., 3' of the primer sequence). This, in turn, provides a substrate for the polymerase to begin polymerizing a single strand of DNA along the length of the DNA synthesis template from the exposed 3' end. 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 extending arm) until polymerization is terminated. Thus, the DNA synthesis template represents the portion of the elongated arm that is encoded into a single-stranded DNA product (i.e., a 3' single-stranded DNA flap containing the desired gene-editing information) by the polymerase of the prime editing factor complex; The single-stranded DNA product replaces the corresponding endogenous DNA strand at the target site immediately downstream of the PE-induced nick site. Without wishing to be bound by theory, polymerization of the DNA synthesis template continues to the 5' end of the elongated arm until the event of termination. Polymerization may include, but is not limited to: (a) reaching the 5' end of the PEgRNA (for example, in the case of a 5' extended arm, the DNA polymerase simply runs out of template); (c) replication termination signals, e.g. specific nucleotide sequences that block or inhibit polymerases, or on nucleic acid forms such as supercoiled DNA or RNA; Termination can occur in a variety of ways, including reaching a signal.

flap endonuclease (for example, FEN1)
As used herein, the term "flap endonuclease" refers to an enzyme that catalyzes the removal of a 5' single-stranded DNA flap. These are naturally occurring enzymes that handle the removal of 5' flaps formed during cellular processes, including DNA replication. The prime editing methods described herein utilize an endogenously supplied flap endonuclease, or a flap endonuclease provided in trans to remove the 5' flap of endogenous DNA that is formed at the target site during prime editing. You may use what is provided. Flap endonucleases are known in the art and are described by 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, "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 accept.

Fusion protein The term "fusion protein" as used herein refers to a hybrid polypeptide that includes 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 fusion protein, thus forming an "amino-terminal fusion protein" or a "carboxy-terminal fusion protein," respectively. You may. 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 a catalytic domain of a nucleic acid editing protein. good. Another example includes Cas9 or its equivalent fused to reverse transcriptase. Any of the proteins provided herein may be produced by any method known in the art. For example, the proteins provided herein may be produced through recombinant protein expression and purification, which is particularly suitable for fusion proteins that include peptide linkers. Methods for the expression and purification of recombinant proteins are well known and can be found in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012)). includes those described by )), which is incorporated herein by reference.

Gene of interest (GOI)
The term "gene of interest" or "GOI" refers to a gene encoding a biomolecule of interest (eg, a protein or RNA molecule). The proteins of interest are any intracellular proteins, membrane proteins, or extracellular proteins, such as nuclear proteins, transcription factors, nuclear membrane transporters, intracellular organelle-related proteins, membrane receptors, catalytic proteins, and enzymes. It may include therapeutic proteins, membrane proteins, membrane transport proteins, signal transduction proteins, or immunological proteins (eg, IgG or other antibody proteins), and the like. The genes of interest also include, but are not limited to, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA), antisense RNA, guide RNA, and 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. targeting specific sequences in the DNA molecule, including complementarity with protospace sequences in the guide RNA. However, the term also refers to Cas9 equivalents, homologs, orthologs, or paralogs, whether naturally occurring or non-naturally occurring (e.g., modified or recombinant); Equivalent guide nucleic acid molecules that are otherwise programmed to localize to specific target nucleotide sequences are also accepted. Cas9 equivalents are any of the following, including Cpf1 (V-type CRISPR-Cas system), C2c1 (V-type CRISPR-Cas system), C2c2 (Type VI CRISPR-Cas system) and C2c3 (V-type CRISPR-Cas system). Other napDNAbps from other types of CRISPR systems (eg, type II, type V, type VI) may also be included. Additional Cas equivalents can be found 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. Are listed. Exemplary sequences and structures of guide RNAs are provided herein. Additionally, methods for designing suitable guide RNA sequences are also provided herein. As used herein, "guide RNA" also refers to the modified form referred to as "primed editing guide RNA" (or "PEgRNA") invented for the prime editing methods and compositions disclosed herein. It may also be referred to as "existing guide RNA" in order to contrast with the guide RNA of.

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

Spacer sequence - A sequence in a guide RNA or PEgRNA (having 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, which does not include the spacer/targeting sequence used to guide Cas9 to the target DNA. .

Extended Arm - Single-stranded extension of the 3' or 5' end of PEgRNA. This includes 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 (eg, reverse transcriptase). This is then incorporated onto the endogenous DNA by replacing the corresponding endogenous strand, thereby incorporating the desired genetic change.

Transcription Terminator - A 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 of the extended arm that encodes the portion of the resulting reverse transcriptase-encoded single-stranded DNA flap that is incorporated into the target DNA site by replacing the endogenous strand. Refers to (singular or plural). The portion of the single-stranded DNA flap encoded by the homologous arm is complementary to the unedited strand of the target DNA sequence and is capable of displacing the endogenous strand and annealing with the single-stranded DNA flap at that location. This facilitates editing. This component is further defined elsewhere. By definition, the homology arm is part of the DNA synthesis template because it is encoded by the prime editing factor polymerase described herein.

Host Cell The term "host cell" as used herein includes a vector described herein, such as a nucleic acid molecule encoding a fusion protein comprising Cas9 or a Cas9 equivalent and a reverse transcriptase. Refers to cells that host a vector and can replicate and express it.

Intein The term "intein" as used herein refers to a self-processing polypeptide domain found in organisms from all domains of life. Inteins (intervening proteins) carry out a unique self-processing event known as protein splicing. In this, it excises itself from a larger precursor polypeptide by cleaving two peptide bonds, and in the process ligates the flanking extein (external protein) sequences together by forming a new peptide bond. This rearrangement occurs post-translationally (or co-translationally) since intein genes are found embedded in-frame with other protein-coding genes. Furthermore, protein splicing mediated by inteins is spontaneous; it requires only folding of the intein domain and 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)), and they catalyze their own excision from precursor proteins and with a concomitant fusion of flanking protein sequences known as ins (Perler et al., Curr. Opin. Chem. Biol. 1:292-299(1997); Perler, FBCell 92(1):1-4( 1998); reviewed in Xu et al., EMBO J.15(19):5146-5153 (1996)).

The term "protein splicing" as used in this application refers to a process in which the inner region (intein) of a precursor protein is excised and the flanking regions (extein) of the protein are ligated together to form the mature protein. This natural process has been observed in numerous proteins from both prokaryotes and eukaryotes (Perler, F.B., Xu, M.Q., Paulus, H. Current Opinion in Chemical Biology 1997, 1, 292-299; Perler, F.B. Nucleic Acids Research 1999,27,346-347). The intein unit contains the necessary components needed to catalyze protein splicing and often contains an endonuclease domain 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 combine spontaneously to form a single intein, which then undergoes a protein splicing process to link into separate proteins.

Elucidation of the mechanisms of protein splicing has led to numerous intein-based applications (Comb, et al., U.S. Pat. No. 5,496,714; Comb, et al., U.S. Pat. No. 5,834,247; Camarero and Muir, J. .Amer.Chem.Soc.,121:5597-5598(1999);Chong,et al.,Gene,192:271-281(1997),Chong,et al.,Nucleic Acids Res.,26:5109-5115 (1998);Chong,et al.,J.Biol.Chem.,273:10567-10577(1998);Cotton,et al.J.Am.Chem.Soc.,121:1100-1101(1999);Evans ,et al.,J.Biol.Chem.,274:18359-18363(1999);Evans,et al.,J.Biol.Chem.,274:3923-3926(1999);Evans,et al.,Protein Sci.,7:2256-2264(1998);Evans,et al.,J.Biol.Chem.,275:9091-9094(2000);Iwai and Pluckthun,FEBS Lett.459:166-172(1999); Mathys,et al.,Gene,231:1-13(1999);Mills,et al.,Proc.Natl.Acad.Sci.USA 95:3543-3548(1998);Muir,et al.,Proc.Natl .Acad.Sci.USA 95:6705-6710(1998);Otomo,et al.,Biochemistry 38:16040-16044(1999);Otomo,et al.,J.Biolmol.NMR 14:105-114(1999) ;Scott,et al.,Proc.Natl.Acad.Sci.USA 96:13638-13643(1999);Severinov and Muir,J.Biol.Chem.,273:16205-16209(1998);Shingledecker,et al. ,Gene,207:187-195(1998);Southworth,et al.,EMBO J.17:918-926(1998);Southworth,et al.,Biotechniques,27:110-120(1999);Wood,et al. 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 inteins As used herein, the term "ligand-dependent inteins" refers to inteins that include a ligand binding domain. Typically, the ligand binding domain is inserted onto the amino acid sequence of an intein, resulting in the structure intein (N)-ligand binding domain-intein (C). Typically, ligand-dependent inteins exhibit no or only minimal protein splicing activity in the absence of a suitable ligand, and a significant increase in protein splicing activity in the presence of the ligand. In some embodiments, a ligand-dependent intein does not exhibit observable splicing activity in the absence of ligand, but does exhibit splicing activity in the presence of ligand. In some embodiments, the ligand-dependent intein exhibits an observable protein splicing activity in the absence of ligand that, in the presence of a suitable ligand, is at least 5 times greater than that observed in the absence of ligand, and at least 10 times greater than that observed in the absence of ligand. at least 50 times, at least 100 times, at least 150 times, at least 200 times, at least 250 times, at least 500 times, at least 1000 times, at least 1500 times, at least 2000 times, at least 2500 times, at least 5000 times, at least 10000 times, exerts a protein splicing activity that is at least 20,000 times, at least 25,000 times, at least 50,000 times, at least 100,000 times, at least 500,000 times, or at least 1,000,000 times greater. In some embodiments, the increase in activity is dose-dependent by at least 1 order of magnitude, at least 2 orders of magnitude, at least 3 orders of magnitude, at least 4 orders of magnitude, or at least 5 orders of magnitude, and the increase in intein activity is increased by adjusting the concentration of the ligand. Allows for minor adjustments. Suitable ligand-gated inteins are known in the art, and those provided below and published US Patent Application No. US2014/0065711A1; Mootz et al., “Protein splicng triggered by a small molecule.”J. Am.Chem.Soc.2002;124,9044-9045;Mootz et al., “Conditional protein splicng: a new tool to control protein structure and function in vitro and in vivo.”J.Am.Chem.Soc.2003; 125,10561-10569;Buskirk et al.,Proc.Natl.Acad.Sci.USA. 2004;101,10505-10510);Skretas & Wood,“Regulation of protein activity with small-molecule-controlled inteins.”Protein Sci .2005;14,523-532;Schwartz, et al.,“Post-translational enzyme activation in an animal via optimized conditional protein splicing.”Nat.Chem.Biol.2007;3,50-54;Peck et al.,Chem. Biol. 2011;18(5), 619-630; the entire contents of each are incorporated herein by reference. An example array is:

Linker The term "linker" as used herein refers to a molecule that joins two other molecules or moieties. A linker can be an amino acid sequence in the case of a linker joining two fusion proteins. For example, Cas9 can be fused to reverse transcriptase through an amino acid linker sequence. A linker can also be a nucleotide sequence in the case of joining two nucleotide sequences together. For example, in this case, a conventional guide RNA is linked via a spacer or linker nucleotide sequence to an RNA stretch of prime editing guide RNA, which may include an RT template sequence and an RT primer binding site. In other embodiments, the linker is an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker is 5-100 amino acids in length, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 in length. , 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. Longer or shorter linkers are also contemplated.

" Isolated " means altered or removed from its natural state. For example, a nucleic acid or peptide that occurs naturally in a living animal is not "isolated," but the same nucleic acid or peptide that is partially or completely separated from its natural state in coexisting materials is "single." “separated”. An isolated nucleic acid or protein may exist in substantially purified form or may exist in a non-native environment (eg, a host cell, etc.).

In some embodiments, the gene of interest is encoded by an isolated nucleic acid. As used herein, the term "isolated" refers to a specification that has been removed from its original or native environment (e.g., the natural environment, if naturally occurring). Refers to the characteristics of the material as provided. Therefore, naturally occurring polynucleotides or proteins or polypeptides present in living animals are not isolated, but have been separated from some or all of the coexisting materials in natural systems by human intervention. The same polynucleotide or polypeptide has been isolated. Artificial or modified materials, such as non-naturally occurring nucleic acid constructs, such as the expression constructs and vectors described herein, are accordingly also referred to as isolated. Material does not need to be purified in order to be isolated. Consequently, the material may be part of a vector and/or part of a composition, such that such vector or composition is not yet isolated in that it is not part of the environment in which the material is found in nature. ing.

MS2 Tagging Techniques In various embodiments (e.g., as illustrated in FIGS. 72-73 and the embodiment of Example 19), the term "MS2 tagging techniques" refers to RNA-protein interaction domains, e.g., specific hairpin structures. Refers to a combination of an "RNA-protein interaction domain" (also known as an "RNA-protein recruitment domain or protein") paired with an RNA-binding protein that specifically recognizes and binds. These types of systems can be exploited to recruit various functionalities to the prime editor complex bound to the target site. MS2 tagging technology is based on the natural interaction of the MS2 bacteriophage coat protein ("MCP" or "MS2cp") with a stem-loop or hairpin structure, or "MS2 hairpin," present on the genome of the phage. In the case of prime editing, the MS2 tagging technique involves introducing an MS2 hairpin onto the desired RNA molecule (e.g., PEgRNA or tPERT) involved in prime editing. This in turn constitutes a specific interactable binding target for RNA binding proteins that recognize and bind to that structure. In the case of the MS2 hairpin, it is recognized and bound by the MS2 bacteriophage coat protein (MCP). Then, when MCP is fused to another protein (e.g., reverse transcriptase or other DNA polymerase), the MS2 hairpin "recruits" the other protein in trans to the target site occupied by the prime editing complex. ” can be used to

The prime editors described herein may, in aspect, incorporate any known RNA-protein interaction domain to recruit or "co-localize" the specific function of interest to the prime editor complex. . Reviews of other modular domains of RNA-protein interactions are available in the art, eg, 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 transcription synthetic programs with CRISPR RNA scaffolds,” Cell,2015,Vol.160:339-350( each of which is incorporated herein by reference in its entirety. Other systems also include the PP7 hairpin, which specifically recruits PCP proteins, and the "com" hairpin, which specifically recruits Com proteins. 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 targets and binds to a specific sequence in a DNA molecule. Refers to proteins that use hybridization. Each napDNAbp includes at least one guide nucleic acid ( For example, a guide RNA). In other words, the guide nucleic acid "programs" the napDNAbp (eg, Cas9 or equivalent) to localize and bind to a complementary sequence.

Without being bound by theory, the binding mechanism of the napDNAbp-guide RNA complex is generally that napDNAbp forms an R-loop that induces unwinding of the double-stranded DNA target (thereby causing the strands in the region bound by napDNAbp to (separated) step. The guide RNA protospacer then hybridizes to the "target strand." This replaces the "non-target strand" that is complementary to the target strand, thereby forming the single-stranded region of the R-loop. In some embodiments, the napDNAbp contains one or more nuclease activities and then cutting the DNA eliminates various types of lesions. For example, napDNAbp may contain nuclease activity that cuts the non-target strand at a first location and/or cuts the target strand at a second location. By cutting the target DNA in response to nuclease activity, a "double-strand break" in which both strands are cut can be formed. In other embodiments, the target DNA can only be cut at a single site, ie, the DNA is "nicked" on one strand. Exemplary napDNAbps with various nuclease activities include "Cas9 nickase" ("nCas9") and inactivated Cas9 without any nuclease activity ("inactive Cas9" or "dCas9"). Exemplary sequences for these and other napDNAbps are provided herein.

Nickase The term "nickase" refers to Cas9 with one of the two nuclease domains inactivated. This enzyme is capable of cutting only one strand of 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, eg, 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 the international PCT application PCT/EP2000/011690 of Plank et al., filed on November 23, 2000 and published as WO/2001/038547 on May 31, 2001 ( (This content is incorporated herein by reference for its 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 polymers include natural nucleosides (i.e., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine), nucleoside analogs (for example, 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, 8oxoguanosine, O(6)methylguanine, 4-acetylcytidine, 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. '-fluororibose, ribose, 2'-deoxyribose, 2'-O-methylcytidine, arabinose, and hexose), or modified phosphate groups (e.g., phosphorothioate and 5'-N phosphoramidite linkages). It may be included.

PEgRNA
As used herein, the term "primed editing guide RNA" or "PEgRNA" or "elongated guide RNA" refers to one or more prime editing guide RNAs for practicing the prime editing methods and compositions described herein. Refers to a specialized form of guide RNA that has been modified to include additional sequences. As described herein, the prime editing guide RNA includes one or more "extended regions" of the nucleic acid sequence. The extended region may include, but is not limited to, single-stranded RNA or DNA. Additionally, the extended region may occur at the 3' end of an existing guide RNA. In other arrangements, the extended region may occur at the 5' end of the existing guide RNA. In yet other arrangements, the extended region may occur at some region within the molecule at the end of an existing guide RNA (eg, in the gRNA core region that joins and/or binds to napDNAbp). The elongated region contains a "DNA synthesis template" that encodes (by the prime editing factor polymerase) a single-stranded DNA, which is then (a) homologous to the endogenous target DNA to be edited; and (b) contain at least one desired nucleotide change (eg, a transition, transversion, deletion, or insertion) to be induced or incorporated into the endogenous target DNA. The extended region may also contain other functional sequence elements (such as, but not limited to, "primer binding site" and "spacer or linker" sequences), or other structural elements (including, but not limited to, aptamers, It may also contain a stem-loop, hairpin, toe-loop (eg, 3' toe-loop), or RNA-protein recruitment domain (eg, MS2 hairpin). As used herein, a "primer binding site" includes a sequence that hybridizes to a single-stranded DNA sequence having a 3' end generated from R-loop nicked DNA.

In certain embodiments, the PEgRNA is represented by Figure 3A, which shows a PEgRNA with a 5' extended arm, a spacer, and a gRNA core. The 5' extension further includes a reverse transcriptase template, a primer binding site, and a linker in the 5'→3' direction. As indicated, the reverse transcriptase template can also be referred to more broadly as a "DNA synthesis template," where the polymerase of the prime editing factor described herein is another type of polymerase rather than RT.

In certain other embodiments, the PEgRNA is represented by Figure 3B, which shows a PEgRNA with a 3' extended arm, a spacer, and a gRNA core. The 3' extension further includes a reverse transcriptase template, primer binding site in the 5'→3' direction. As indicated, the reverse transcriptase template can also be referred to more broadly as a "DNA synthesis template," where the polymerase of the prime editing factor described herein is another type of polymerase rather than RT.

In yet other embodiments, the PEgRNA is represented by Figure 3D, which shows the PEgRNA with a spacer (1), a gRNA core (2), and an extended arm (3) in the 5'→3' direction. The extended arm (3) is at the 3' end of PEgRNA. The elongated arm (3) further includes a "primer binding site" (A), an "editing template" (B), and a "homologous arm" (C) in the 5'→3' direction. The extended arm (3) may also contain any modified regions at the 3' and 5' ends, which may be of the same or different sequence. Additionally, the 3' end of PEgRNA may include a transcription terminator sequence. These sequence elements of 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
"PE1" as used in this application refers to a PE complex comprising a fusion protein containing Cas9(H840A) and wild-type MMLV RT with the following structure: [NLS]-[Cas9(H840A)]-[linker] -[MMLV_RT(wt)]+desired PEgRNA. The PE fusion has the amino acid sequence of SEQ ID NO: 123, which is shown below;

PE2
"PE2" as used in this application refers to a PE complex comprising a fusion protein containing Cas9(H840A) and variant MMLV RT with the following structure: [NLS]-[Cas9(H840A)]-[linker]- [MMLV_RT(D200N)(T330P)(L603W)(T306K)(W313F)]+desired PEgRNA. The PE fusion has the amino acid sequence of SEQ ID NO: 134, which is shown below:

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

Short PE (PE-short)
" Short PE " as used herein refers to a PE construct that is fused to a C-terminally truncated reverse transcriptase and has the following amino acid sequence:

Peptide tag 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 on the protein, thereby allowing for various functions such as visualization, purification, solubilization, separation, etc. Easily manipulate proteins for purposes. Peptide tags can encompass various types of tags categorized by purpose or function, such as "affinity tags" (to facilitate protein purification), "solubilization tags" (to facilitate correct folding of proteins), ``chromatographic tags'' (to alter the chromatographic properties of a protein), ``epitope tags'' (for binding to high-affinity antibodies), and ``fluorescent tags'' (to assist in binding to high-affinity antibodies) ) to facilitate visualization of the protein in vitro.

Polymerase The term "polymerase" as used herein refers to an enzyme that synthesizes a nucleotide chain that can be used in connection with the prime editor system described herein. The polymerase can be a "template-dependent" polymerase (ie, a polymerase that synthesizes a nucleotide strand based on the order of the nucleotide bases in a template strand). A polymerase can also be a "template-independent" polymerase (ie, a polymerase that synthesizes a nucleotide strand without the requirement of a template strand). Polymerases can also be further classified as "DNA polymerases" or "RNA polymerases." In various embodiments, the prime editor system includes a DNA polymerase. In various embodiments, the DNA polymerase can be a "DNA-dependent DNA polymerase" (ie, according to which the template molecule is a strand of DNA). In such cases, the DNA template molecule may be PEgRNA and the extending 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 (ie, a guide RNA component that includes a spacer and a gRNA core) and a DNA portion (ie, an extended arm). In various other embodiments, the DNA polymerase can be an "RNA-dependent DNA polymerase" (ie, according to which the template molecule is a strand of RNA). In such cases, PEgRNA is RNA, ie, includes RNA extension. The term "polymerase" can also refer to an enzyme that catalyzes the polymerization of nucleotides (ie, polymerase activity). Generally, the enzyme initiates 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, e.g., PEgRNA) and proceeds toward the 5' end of the template strand. Will. "DNA polymerase" catalyzes the polymerization of deoxynucleotides. The term DNA polymerase, as used herein with reference to DNA polymerase, encompasses "functional fragments thereof.""Functional fragment thereof" means any portion of a wild-type or mutant DNA polymerase that encompasses less than the entire amino acid sequence of the polymerase that retains the ability to catalyze the polymerization of polynucleotides under at least one set of conditions. say. 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.

Prime Editing The term "prime editing" as used herein refers to a novel approach for gene editing, in which napDNAbp, a polymerase (e.g. reverse transcriptase), and the desired new genetic information are then incorporated onto the target DNA sequence. A specialized guide RNA is used that contains the DNA synthesis template to encode (or delete genetic information). Certain embodiments of prime editing are described in the embodiments of FIGS. 1A-1H and 72(a)-72(c), among other figures.

Prime editing represents a completely new platform for genome editing, a versatile and precise genome editing method that directly writes new genetic information into defined DNA sites, using 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 the target site and which is located on the guide RNA (e.g., at the 5' or 3' end of the guide RNA or internally). (in part) serves as a template for the synthesis of the desired edit in the form of a DNA strand replaced by an engineered extension (either DNA or RNA). The replacement strand containing 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. ) (with the exception that it includes the desired edits). Through DNA repair and/or replication machinery, the endogenous strand downstream of the nick site is replaced by a newly synthesized replacement strand containing the desired edit. In some cases, prime editing can be considered a "search and replace" genome editing technology. This is because the prime editing factors described herein not only locate and locate the desired target site to be edited, but also simultaneously generate the desired edit to be incorporated in place of the endogenous DNA strand of the corresponding target site. This is because it encodes the contained replacement strand. The prime editing factors of the present disclosure provide, in part, that the target-primed reverse transcription (TPRT) or "prime editing" mechanism is based on precision CRISPR/Cas with high efficiency and genetic flexibility. Relating to the discovery that it can be exploited or adapted to perform genome editing (eg, as illustrated in the various embodiments of FIGS. 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, we use Cas protein-reverse transcriptase fusions or related systems to target specific DNA sequences by guide RNA, generate single-stranded nicks at the target site, and incorporate them into the guide RNA. The nicked DNA was used as a primer for reverse transcription of the engineered reverse transcriptase template. However, although the concept begins with prime editing agents that use reverse transcriptase as the DNA polymerase component, the prime editing agents described herein are not limited to reverse transcriptase, and may include the use of virtually any DNA polymerase. can be included. Indeed, although this application may refer to a prime editing agent with "reverse transcriptase" throughout, it is hereby acknowledged that reverse transcriptase is only one type of DNA polymerase that can act in prime editing. be made explicit. Therefore, wherever this specification refers to "reverse transcriptase," one of ordinary skill in the art should understand that any suitable DNA polymerase can be used in place of reverse transcriptase. Therefore, in one aspect, the prime editing factor may include Cas9 (or equivalent napDNAbp), which is a specialized guide RNA containing a spacer sequence that anneals to a complementary protospacer on the target DNA (i.e., PEgRNA ) is programmed to target a DNA sequence. The specialized guide RNA also contains new genetic information in the form of an extension encoding a replacement strand of DNA containing the desired genetic modification, which replaces the corresponding endogenous DNA strand at the target site. used for To transfer information from PEgRNA to target DNA, the prime editing mechanism 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 editing encoding extension on 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 elongation, the prime editor polymerase can be an RNA-dependent DNA polymerase (eg, reverse transcriptase). In the case of DNA elongation, the prime editor polymerase can be a DNA-dependent DNA polymerase. The newly synthesized strand formed by the prime editing agents disclosed herein (i.e., the replacement DNA strand containing the desired edit) contains the desired nucleotide change (e.g., a single nucleotide change, deletion, or insertion). , or a combination thereof) will be homologous to (ie, have the same sequence as) the genomic target sequence. The newly synthesized (or replaced) strand of DNA can also be referred to as a single-stranded DNA flap. This will compete for hybridization with the complementary homologous endogenous DNA strand, thereby displacing the corresponding endogenous strand. In certain embodiments, the system may be combined with the use of error-prone reverse transcriptase enzymes (eg, provided as a fusion protein with or in trans to a Cas9 domain). The error-prone reverse transcriptase enzyme can introduce alterations during the synthesis of single-stranded DNA flaps. Therefore, in certain embodiments, error-prone reverse transcriptase can be utilized to introduce nucleotide changes to target DNA. Depending on the error-prone reverse transcriptase used in the system, changes can be random or non-random. Degradation of hybridized intermediates (including single-stranded DNA flaps synthesized by reverse transcriptase hybridized to endogenous DNA strands) results in removal of the displaced flap of endogenous DNA (e.g., 5'-end DNA flap endonuclease FEN1), ligation of the synthesized single-stranded DNA flap to the target DNA, and assimilation of desired nucleotide changes as a result of cellular DNA repair and/or replication processes. . Because template-directed DNA synthesis offers single nucleotide precision for any nucleotide modification, including insertions and deletions, the scope of this approach is very broad and foreseeably has countless applications in basic research and therapeutics. It can be used for.

In various embodiments, prime editing involves the use of a nucleic acid programmed DNA binding protein (napDNAbp) complexed with a prime editing guide RNA (PEgRNA) and a target DNA molecule (for which it is desired to introduce a nucleotide sequence change). ) is activated by touching. Referring to Figure 1G, a prime editing guide RNA (PEgRNA) contains an extension at the 3' or 5' end of the guide RNA or at an intramolecular position of the guide RNA to accommodate the desired nucleotide change (e.g., single nucleotide change, insertion , or deletion). In step (a), the napDNAbp/extended gRNA complex contacts the DNA molecule and the extended gRNA guides the napDNAbp to bind to 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 at the target locus, thereby creating an available 3' end on one of the strands at the target locus. . In certain embodiments, the nick is created on the strand of DNA that corresponds to the R-loop strand, ie, the strand that is not hybridized to the guide RNA sequence, ie, the "non-target strand." However, nicks can be introduced into either of the strands. That is, the nick is either the R-loop "target strand" (i.e., the strand that hybridizes to the protospacer of the elongated gRNA) or the "non-target strand" (i.e., one of the R-loops that is complementary to the target strand). (the chain forming the chain part). 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., 'primed RT ”). In certain embodiments, the 3' end DNA strand hybridizes to a specific RT prime sequence of the extended portion of the guide RNA, ie, a "reverse transcriptase prime sequence" or "primer binding site" on the PEgRNA. In step (d), reverse transcriptase (or other suitable DNA polymerase) is introduced. This synthesizes a single strand of DNA from the 3' end of the primed site towards the 5' end of the prime editing guide RNA. A DNA polymerase (eg, reverse transcriptase) can be fused to napDNAbp or alternatively provided in trans to napDNAbp. This is a single strand containing the desired nucleotide changes (e.g., single base changes, insertions, or deletions, or combinations thereof) that are otherwise homologous to the endogenous DNA at or adjacent to the nick site. Form a DNA flap. In step (e) napDNAbp and guide RNA are released. Steps (f) and (g) involve disassembly of the single-stranded DNA flap so that the desired nucleotide change is incorporated at the target locus. This process can be driven toward 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 mismatched DNA and incorporate nucleotide change(s) to form the desired modified product. The process can also be driven towards product formation by "second strand nicking" as illustrated in FIG. 1F. This process may introduce at least one or more of the following genetic changes: transversions, transitions, deletions, and insertions.

The term "primed editing element (PE) system" or "primed editing factor (PE)" or "PE system" or "PE editing system" refers to the primed reverse transcription ( TPRT) and includes napDNAbp, reverse transcriptase, a fusion protein (e.g., including napDNAbp and reverse transcriptase), prime editing guide RNA, and fusion protein and prime editing guide RNA. complex, as well as auxiliary elements, such as a second strand nicking component (e.g., second strand sgRNA) and 5' endogenous DNA to help drive the prime editing process toward edited product formation. Including, but not limited to, flap removal endonucleases (eg, FEN1).

In the embodiments described so far, the PEgRNA comprises a guide RNA (which itself includes a spacer sequence and a gRNA core or backbone) and a 5' or 3' extending arm that contains a primer binding site and a DNA synthesis template. PEgRNA can also take the form of two individual molecules, consisting of a guide RNA and a transprime editing factor RNA template (tPERT). This essentially accommodates an elongated arm (including, inter alia, a primer binding site and a DNA synthesis domain) and an RNA-protein recruitment domain (eg, an MS2 aptamer or hairpin) on the same molecule. It becomes colocalized or recruited to a modified prime editor complex that includes tPERT recruitment proteins (eg, MS2cp protein that binds the MS2 aptamer). See Figures 3G and 3H for examples of tPERT that can be used for prime editing.

Prime editing factor The term "primed editing factor" refers to a fusion construct described herein comprising napDNAbp (e.g., Cas9 nickase) and reverse transcriptase in the presence of PEgRNA (or "elongated guide RNA"). It is possible to perform prime editing on a target nucleotide sequence with The term "prime editor" may refer to a fusion protein, or a fusion protein complexed with a PEgRNA, and/or a fusion protein further complexed with an sgRNA that nicks the second strand. In some embodiments, the prime editing factor is also a fusion protein (reverse transcriptase fused to napDNAbp) capable of directing the nicking step to the second site of the unedited strand as described herein. ), PEgRNA, and a regular guide RNA. 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 the nucleotide sequence located on PEgRNA as a component of the elongating arm (typically at the 3' end of the elongating arm) and that is Refers to the nucleotide sequence that helps bind to the primer sequence formed after Cas9 nicking of the target sequence. As detailed elsewhere, when the Cas9 nickase component of the prime editing factor nicks one strand of the target DNA sequence, a 3'-terminal ssDNA flap is formed, which is connected to the primer on the PEgRNA. It acts as a primer sequence that anneals to the binding site and primes reverse transcription. Figures 27 and 28 show embodiments of primer binding sites located on the 3' and 5' extension arms, respectively.

Promoter The term "promoter" is art-recognized 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 either 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 specific conditions. It can be. For example, a conditional promoter may be active only in the presence of a specific protein that connects the protein associated with regulatory elements 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 that require the presence of small molecule "inducers" 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 skilled in the art, and one of skill in the art will be able to ascertain a variety of such promoters that are 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 an important 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 the "N" is followed by two guanine ("G") nucleobases Any nucleobase. Different PAM sequences may be associated with different Cas9 nucleases or equivalent proteins from different organisms. In addition, any given Cas9 nuclease, such as SpCas9, can be modified so that the nuclease recognizes alternative PAM sequences, altering the PAM specificity of the nuclease.

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 appreciated that Cas9 enzymes (ie, Cas9 orthologs) from different bacterial species may have varying PAM specificities. For example, Cas9 from Staphylococcus aureus (SaCas9) recognizes NGRRT or NGRRN. In addition, Cas9 from Neisseria meningitis (NmCas) recognizes NNNNGATT. In another example, Cas9 from Streptococcus thermophilis (StCas9) recognizes NNAGAAW. In yet another example, Cas9 (TdCas) from Treponema denticola recognizes NAAAAC. These are examples and are not meant to be limiting. Additionally, it will be appreciated that non-SpCas9 binds to a variety of 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-SpCas9s may have other characteristics that make them more useful than SpCas9s. For example, Cas9 from Staphylococcus aureus (SaCas9) is approximately 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, which involves excision, incorporation, inversion, or exchange of DNA fragments between recombinase recognition sequences (e.g. translocation). Recombinases can be classified into two distinct families: serine recombinases (eg resolvases and invertases) and tyrosine recombinases (eg 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. includes. Examples of tyrosine recombinases include, without limitation, Cre, FLP, R, Lambda, HK101, HK022, and pSAM2. The name serine and tyrosine recombinases is derived from the conserved nucleophilic amino acid residues that recombinases use to attack DNA and become covalently linked to DNA during strand exchange. Recombinases have numerous applications including generating gene knockouts/knockins and gene therapy applications. For example, 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 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: Cre-ating a new biological era.”J.Zhejiang Univ.Sci.B.2012;13(7):511-24;Karpenshif and Bernstein,“From yeast to mammals:recent advances in Genetic control of homologous recombination.” DNA Repair (Amst). 2012;1;11(10):781-8, the entire contents of each of which are incorporated herein by reference in their entirety. The recombinases provided in this application are not meant to be the exclusive examples of recombinases that can be used in embodiments of the invention. The methods and compositions of the invention can be extended by mining databases for new orthogonal recombinases or by 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 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 skilled in the art, and any new recombinases that are discovered or generated may be used in different aspects of the invention. expected to be capable. In some embodiments, the catalytic domain of the recombinase is fused to an RNA-programmable nuclease (e.g., dCas9 or a fragment thereof) that has been nuclease-inactivated, such that the recombinase domain does not include a nucleic acid binding domain or or is incapable of binding the target nucleic acid (eg, the recombinase domain is engineered such that it does not have specific DNA binding activity). Recombinases lacking DNA-binding activity and methods for modifying them are known and are described in 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;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 is incorporated herein by reference in its entirety. . For example, serine recombinases of the resolvase-invertase group, such as Tn3 and γδ resolvases and Hin and Gin invertases, have molecular structures with autonomous catalytic and DNA-binding domains (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). Therefore, for example, after isolation of "activated" recombinase variants that do not require any auxiliary factors (e.g. DNA binding activity), the catalytic domains of these recombinases can be used as nucleases as described herein. amenable to being recombined with a programmable nuclease (e.g., dCas9 or a fragment thereof) by inactivated RNA (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;See Akopian et al., “Chimeric recombinases with designed DNA sequence recognition.” Proc Natl Acad Sci USA.2003;100:8688-8691).In addition, it has an N-terminal catalytic domain and a C-terminal DNA binding domain. Many other naturally occurring serine recombinases are known (e.g., PhiC31 integrase, TnpX transposase, IS607 transposase), and their catalytic domains can be used as programmable site-specific recombinases as described herein. (see, e.g., Smith et al., “Diversity in the serine recombinases.” Mol Microbiol. 2002;44:299-307, the entire contents of which are incorporated by reference). Similarly, the core catalytic domains of tyrosine recombinases (e.g., Cre, λ integrase) are known and can be similarly selected to engineer programmable site-specific recombinases as described herein (e.g. As Guo et al., “Structure of Cre recombinase complexed with DNA in a site-specific recombination synapse.”Nature.1997;389:40-46;Hartung et al., “Cre mutants with altered DNA binding properties.”J Biol Chem 1998;273:22884-22891;Shaikh et al.,“Chimeras of the Flp and Cre recombinases:Tests of the mode of cleavage by Flp and Cre.J Mol Biol.2000;302:27-48;Rongrong et al .,“Effect of deletion mutation on the recombination activity of Cre recombinase.”Acta Biochim Pol.2005;52:541-544;Kilbride et al.,“Determinants of product topology in a hybrid Cre-Tn3 resolvase site-specific recombination system .”J Mol Biol.2006;355:185-195;Warren et al.,“A chimeric cre recombinase with regulated directionality.”Proc Natl Acad Sci USA.2008 105:18278-18283;Van Duyne,“Teaching Cre to follow directions.”Proc Natl Acad Sci USA.2009 Jan 6;106(1):4-5;Numrych et al.,“A comparison of the effects of single-base and triple-base changes in the integrase arm-type binding sites on the site-specific recombination of bacteriophage λ.”Nucleic Acids Res.1990;18:3953-3959;Tirumalai et al.,“The recognition of core-type DNA sites by λ integrase.”J Mol Biol.1998;279: 513-527;Aihara et al.,“A conformational switch controls the DNA cleavage activity of λ integrase.”Mol Cell.2003;12:187-198;Biswas et al.,“A structural basis for allosteric control of DNA recombination by λ integrase.”Nature.2005;435:1059-1066; and Warren et al., “Mutations in the amino-terminal domain of λ-integrase have differential effects on integrative and excisive recombination.”Mol Microbiol.2005;55:1104 -1112 (the entire contents of each of which 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. To tell. This results in excision, incorporation, inversion, or exchange of DNA fragments between recombinase recognition sequences.

Recombine or Recombinant The term "recombinant" or "recombinant" in the context of nucleic acid modification (e.g. genome modification) means that two or more nucleic acid molecules or two or more regions of a single nucleic acid molecule are combined with a recombinase protein (e.g. used to refer to processes that are modified by the action of the recombinase fusion proteins of the invention provided herein. Recombination can result in inter alia insertions, inversions, excisions, or translocations of nucleic acids, eg, on or between one or more nucleic acid molecules. Recombinase recognition sequence

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 (ie, complementary DNA or cDNA) using RNA as a template. In some embodiments, reverse transcription can be "error-prone reverse transcription." This refers to the property of certain reverse transcriptases 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", used interchangeably with the term "bacteriophage" herein, refers to a virus that infects bacterial cells. Typically, phages consist of an outer protein capsid that encloses 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 skilled in the art, and non-limiting examples of phages that are useful for carrying out the PACE methods provided herein include λ (lysogen), T2, T4, T7, T12, They are R17, M13, MS2, G4, P1, P2, P4, PhiX174, N4, Φ6, and Φ29. In certain embodiments, the phage utilized in the invention is M13. Additional suitable phages and host cells will be apparent to those skilled in the art. The invention is not limited in this aspect. Illustrative 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 RJClokie 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 RJClokie and Andrew M.Kropinski:Bacteriophages:Methods and Protocols,Volume 2 See: Molecular and Applied Aspects (Methods in Molecular Biology) Humana Press; 1st edition (December 2008), ISBN: 1603275649; all of which describe suitable phages and host cells, and the isolation, culture, and manipulation of such phages. (incorporated herein by reference in its entirety for disclosure of methods and protocols for).

Protein, Peptide, and Polypeptide The terms "protein,""peptide," and "polypeptide" are used interchangeably herein and refer to a polymer of amino acid residues linked together by peptide (amide) bonds. refers to The term refers to a protein, peptide, or polypeptide of any size, structure, or function. Typically a protein, peptide or polypeptide will be at least 3 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, for example, by 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. It may also be modified by the addition of chemical entities such as. A protein, peptide, or polypeptide may also be a single molecule or a multimolecular complex. A protein, peptide, or polypeptide may be just a fragment of a naturally occurring protein or peptide. The 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 include peptide linkers. Methods for the expression and purification of recombinant proteins are well known and can be found in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012)). includes those described by )), which is incorporated herein by reference.

Protein splicing The term "protein splicing" as used in this application refers to the sequence of an intein (or split intein, as the case may be) being excised from within the amino acid sequence, and the remaining fragments of the amino acid sequence extein being ligated together by amide bonds. refers to the process of forming a specific amino acid sequence. The term "trans" protein splicing refers to the particular case where the inteins are split inteins and are located on different proteins.

The degradation of the heteroduplex DNA (i.e., containing one edited and one unedited 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 permanently incorporate the edited strand onto the complementary endogenous strand, thereby reducing the heteroduplex DNA (endogenous non-edited strand) formed as an intermediate to PE. (edited strands paired with). The approach of "second strand nicking" can be used herein to help drive degradation of heteroduplex DNA in favor of permanent incorporation of the edited strand onto the DNA molecule. The concept of "second strand nicking" as used in this application refers to the initial nick (i.e., the initial nick) that provides a free 3' end for use in priming reverse transcriptase on the extended portion of the guide RNA. Refers to the introduction of a second nick, preferably on the non-edited strand, in a position downstream of the nick site (nick site). In certain embodiments, the first nick and the second nick are on opposite strands. In other embodiments, the first nick and the second nick are on opposite strands. In yet another embodiment, the first nick is on the non-target strand (ie, the strand that forms the single-stranded portion of the R-loop) and the second nick is on the target strand. In still other embodiments, the first nick is on the edited strand and the second nick is on the non-edited strand. The second nick is 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. The second nick is, in some embodiments, between about 5-150 nucleotides, or between about 5-140, or between about 5-130 nucleotides from the site of the PEgRNA-induced nick on the non-edited strand, 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 about 5 -60, or about 5-50, or about 5-40, or about 5-30, or about 5-20, or about 5-10. In one embodiment, the second nick is introduced between 14-116 nucleotides away from the PEgRNA-induced nick. Without being bound by theory, the second nick directs the cell's endogenous DNA repair and replication processes towards replacing or editing the unedited strand, thereby leaving the edited sequence on both strands. Permanently incorporates and degrades heteroduplexes formed as a result of PE. In some embodiments, the edited strand is the non-target strand and the non-edited strand is the target strand. In other embodiments, the edited strand is the target strand and the non-edited strand is the non-target strand.

Sense Strand In genetics, the "sense" strand is a segment within double-stranded DNA that extends 5' to 3' and is complementary to the antisense or template strand of DNA that extends 3' to 5'. be. In the case of a DNA segment that encodes a protein, the sense strand has the same sequence as the mRNA that takes the antisense strand as its template during transcription and eventually (typically, but not always) undergoes translation to become a protein. It is a strand of DNA. Thus, the antisense strand carries the RNA that is later translated into protein, while the sense strand has nearly the same makeup as the mRNA. Note that for each segment of dsDNA, there will probably be two sets of sense and antisense (as sense and antisense are relative to the viewpoint), depending on which direction one is read. The gene product or mRNA ultimately indicates that either strand of one segment of dsDNA 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 that is to be incorporated) that is oriented in the 5'→3' direction, while the synthetic strand is attached to the PEgRNA extension arm. comes out of the mold. As to whether the 3'ssDNA flap should be considered the sense or antisense strand, it is well accepted that both strands of DNA can serve as templates for transcription (but not simultaneously). Where it is depends on the direction of transcription. 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) may serve as the antisense strand and thus be the template for transcription.

Spacer sequence As used herein, the term "spacer sequence" in relation to guide RNA or PEgRNA refers to a guide RNA of approximately 20 nucleotides containing a nucleotide sequence complementary to a protospacer sequence in a target DNA sequence. Or refers to the PEgRNA part. The spacer sequence anneals with the protospacer sequence to form an ssRNA/ssDNA hybrid structure at the target site and a corresponding R-loop ssDNA structure of the endogenous DNA strand that is complementary to the protospacer sequence.

The subject term "subject" as used herein refers to an individual organism, such as 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 Primate. 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, such as a genetically modified non-human subject. The subject may be of either sex and at any stage of development.

Split Inteins Inteins are most often found as continuous domains, but some exist in naturally split forms. In this case, the two fragments are expressed as separate polypeptides and must come together before splicing can occur, so-called protein trans-splicing.

An exemplary fission intein is the Ssp DnaE intein, which contains two subunits, DnaE-N and DnaE-C. The two different 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 fission intein of Synechocytis sp. PCC6803 that can direct the trans-splicing of two separate proteins, each containing a fusion with either DnaE-N or DnaE-C.

Additional naturally occurring or engineered split intein sequences are known or can be made from whole intein sequences described herein or available in the art. Examples of split intein sequences are 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 in, for example, 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.

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

tPERT
See the definition of "trans prime editing factor 'RNA type (tPERT)'".

Temporary Second Strand Nicking As used herein, the term "temporary second strand nicking" means that the incorporation of a second nick in the unedited strand causes the desired edit to occur. Refers to a variant of nicking the second strand that occurs only after it has been incorporated into the edited strand. This avoids simultaneous nicks on both strands that could lead to double-stranded DNA breaks. The guide RNA that nicks the second strand is designed for temporal control so that the second strand nick is not introduced until after the desired edit has been incorporated. This is accomplished by designing gRNAs with spacer sequences that match only the edited strand, but not the original allele. Using this strategy, mismatches 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 split PEgRNA, i.e., where PEgRNA is divided into two separate molecules: sgRNA and trans Isolated into prime editing RNA template (tPERT). While the sgRNA acts to target the prime editing factor (or more generally, targets the napDNAbp component of the prime editing factor) to the desired genomic target site, once tPERT is on the prime editing factor and tPERT Once recruited in trans to the prime editing factor by interaction of the overlying binding domain, tPERT is used by a polymerase (eg, reverse transcriptase) to write a new DNA sequence into the target locus. In one embodiment, the binding domain can include an RNA-protein recruitment moiety, such as the MS2cp protein fused to the MS2 aptamer and prime editing factor positioned on tPERT. The advantage of trans prime editing is that by separating the DNA synthesis template from the guide RNA, potentially longer template lengths can be used.

Aspects of trans prime editing are shown in Figures 3G and 3H. Figure 3G shows on the left the composition of the trans prime editing factor complex (“RP-PE:gRNA complex”), which is composed of polymerases (e.g. reverse transcriptase) and rPERT recruitment proteins (e.g. MS2sc) and is complexed with guide RNA. Figure 3G further shows a separate tPERT molecule containing an extended arm feature of PEgRNA that includes the DNA synthesis template and primer binding sequences. The tPERT molecule also includes an RNA-protein recruitment domain, which in this case is a stem-loop structure and can be, for example, an MS2 aptamer. As depicted in the process described in Figure 3H, the RP-PE:gRNA complex binds and nicks the target DNA sequence. Recruitment protein (RP) then recruits tPERT to colocalize to the prime editor complex bound to the DNA target site, thereby directing the primer binding site to the primer sequence on the nicked strand. Then, a polymerase (eg, RT) can synthesize a single strand of DNA up to 5' of tPERT to the DNA synthesis template.

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

Trans prime editing factor RNA template (tPERT)
As used herein, "trans prime editing factor RNA template (tPERT)" refers to the component used for trans prime editing, a modified version of prime editing that combines PEgRNA into two separate molecules: the guide It works by separating into RNA and tPERT molecules. The tPERT molecule is programmed to colocalize with the prime editing factor complex at the target DNA site, bringing the primer binding site and DNA synthesis template in trans to the prime editing factor. For example, the trans prime editing factor ( tPE), see Figure 3G. Here, the RP (recruiting 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 in trans to the prime editing factor. Stated differently, tPERT has been modified to contain (all or part of) an extended arm of PEgRNA, which includes a primer binding site and a DNA synthesis template.

Transitions As used herein, "transition" refers to the interconversion of purine nucleobases (A⇔G) or the interconversion of pyrimidine nucleobases (C⇔T). This class of interconversions involves similarly shaped nucleobases. 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 pairs of nucleobases, transversion involves the following base pair exchanges: A:T⇔G:C, G:G⇔A:T, C:G⇔T:A. , or refer to 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.

Transversion As used herein, "transversion" refers to the interconversion of purine nucleobases to pyrimidine nucleobases, or vice versa, thus involving the interconversion of nucleobases that are dissimilar in shape. These changes involve T⇔A, T⇔G, C⇔G, C⇔A, A⇔T, A⇔C, G⇔C, and G⇔T. In the context of double-stranded DNA with Watson-Crick pairs of nucleobases, transversion refers 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.

The treatment terms "treatment,""treating," and "treating" refer to halting, alleviating, delaying the onset of a disease or disorder, or one or more symptoms thereof, as described herein; or clinical interventions aimed at inhibiting its progression. As used herein, the terms "treatment,""treating," and "treating" mean to prevent the onset of a disease or disorder, or one or more symptoms thereof, as described herein. , refers to clinical interventions aimed at alleviating, delaying, or inhibiting its progression. In some embodiments, treatment may be administered after one or more symptoms have developed and/or after the disease has been diagnosed. In other embodiments, treatment may be administered in the absence of symptoms, eg, to prevent or delay the onset of symptoms, or to inhibit the onset or progression of a disease. For example, treatment may be administered to susceptible individuals prior to the onset of symptoms (eg, 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, eg, to prevent or delay their recurrence.

Trinucleotide repeat disorders As used herein, "trinucleotide repeat disorders" (or alternatively, "expanded repeat disorders" or "repeat expansion disorders") refer to certain mutations (certain trinucleotide repeat disorders). Refers to a group of genetic disorders caused by "trinucleotide repeat expansions," in which nucleotide repeats are found in certain genes or introns. Trinucleotide repeats, once thought to be commonplace iterations in the genome, 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, mutant repeats exhibit both somatic and germline instability, and more often they expand rather than shrink with successful transmissions. Second, younger age of onset and increased severity of the phenotype in subsequent generations (expected) generally correlates with larger repeat length. Finally, the parental origin of a disease allele can often influence the expectation that paternal inheritance carries a greater risk of spread for many of these disorders.

Triplet expansion is thought to be caused by slippage during DNA replication. Due to the repetitive nature of the DNA sequence in these regions, "loop-out" structures are created during DNA replication while maintaining complementary base pairing between the parent and daughter strands being synthesized. may be formed. If a loop-out structure is formed from sequences on the daughter strand, this will result in an increased number of repeats. However, if a loop-out structure is formed on the parent strand, a reduction in the number of repeats occurs. Expansion of these iterations appears to be more common than reduction. Generally, the larger the spread, the more likely they are to cause disease or increase the severity of the disease. This property results in the expected features seen in trinucleotide repeat disorders. The prediction explains the trend towards younger age of onset and increasing severity of symptoms over successive generations of affected families due to the expansion of these repeats.

Nucleotide repeat disorders may include disorders in which triplet repeats occur in non-coding regions (ie, non-coding trinucleotide repeat disorders) or disorders in which triplet repeats occur in coding regions.

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.

Upstream As used herein, the terms "upstream" and "downstream" refer to positions in a nucleic acid molecule (whether single-stranded or double-stranded) oriented in the 5'→3' direction. is a relative term that defines the linear position of at least two elements. In particular, a first element is upstream of a second element in the nucleic acid molecule if the first element is positioned somewhere 5' to the second element. For example, if the SNP is 5' to the nick site, the SNP is upstream of the Cas9-induced nick site. Conversely, if the first element is positioned somewhere 3' to the second element, then 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. Nucleic acid molecules can be DNA (double-stranded or single-stranded), RNA (double-stranded or single-stranded), or a hybrid of DNA and RNA. Where the terms upstream and downstream refer only to a single strand of a nucleic acid molecule, the analysis applies to single-stranded and double-stranded molecules, unless one needs to choose which strand of a double-stranded molecule to consider. The strand of double-stranded DNA that is often the same and can be used to determine the relative position of at least two elements is the "sense" or "coding" strand. In genetics, the "sense" strand is a 5' to 3' extending segment within double-stranded DNA that is complementary to the 3' to 5' antisense or template strand of the DNA. Thus, by way of example, a SNP nucleobase is "downstream" of a promoter sequence in genomic DNA (which is double-stranded) if the SNP nucleobase is 3' to a promoter on the sense or coding strand.

Variants As used herein, the term "variant" shall be taken to mean exhibiting qualities that have a pattern that deviates from those that occur in nature, including, by way of example, the 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" refers to a sequence that has a percent identity of at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 99% with a reference sequence and is the same as or or encompasses homologous proteins having substantially the same functional activity(s). The term also encompasses mutants, truncations, or domains of the reference sequence that exhibit the same or substantially the same functional activity(s) 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 mutate or replicate within the host cell). refers to a nucleic acid that is capable of transferring its replicative form 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 those skilled in the art based on this disclosure.

Wild Type As used herein, the term "wild type" is a terminology understood by those skilled in the art that refers to wild type as it occurs in nature, as distinguished from mutant or variant forms. means the typical form of an organism, strain, gene, or characteristic.

5' Endogenous DNA Flap The term "endogenous 5' DNA flap" as used herein refers to the strand of DNA located immediately downstream of the PE-induced nick site on the target DNA. Nicking of the target DNA strand with PE exposes the 3' hydroxyl group upstream of the nick site and the 5' hydroxyl group downstream of the nick site. The endogenous chain ending with a 3' hydroxyl group is used to prime the prime editing factor DNA polymerase (eg, the DNA polymerase is reverse transcriptase). The endogenous strand that begins downstream of the nick site and with an exposed 5' hydroxyl group is referred to as the "5' endogenous DNA flap" and is ultimately removed, leading to the newly synthesized DNA encoded by the extension of PEgRNA. replaced by the replaced strand (i.e., the "3' replaced DNA flap").

5' Endogenous DNA Flap Removal As used herein, the term "5' endogenous DNA flap removal" or "5' flap removal" refers to the competitive invasion of single-stranded DNA flaps synthesized by RT. refers to the removal of the 5' endogenous DNA flap that is formed when the strand hybridizes with endogenous DNA, displacing the endogenous strand in the process. Removal of this displaced endogenous strand may 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 (eg, a flap endonuclease, 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 of forming the product (e.g., flap end nuclease). Flap endonucleases are known in the art and are described in Patel et al., “Flap endonucleases pass 5'-flaps through a flexible arch using a disorder-thread-order mechanism to confer specificity for free 5'-ends,” Nucleic Acids Research, 2012, 40(10): 4507-4519 and Tsutakawa et al., “Human flap endonuclease structures, DNA double-base flipping, and a unified understanding of the FEN1 superfamily,” Cell, 2011, 145(2): 198-211, each of which is incorporated herein by reference.

3' Replacement DNA Flap As used herein, the term "3' replacement DNA flap", or simply "replacement DNA flap", is synthesized by the prime editing factor and encoded by the extending arm of the prime editing factor PEgRNA. Refers to a strand of DNA. More specifically, the 3' replacement DNA flap is encoded by a polymerase template of PEgRNA. The 3' replacement DNA flap contains the same sequence as the 5' endogenous DNA flap, except that it also contains edited sequences (eg, single nucleotide changes). The 3' displacement DNA flap anneals with the target DNA and displaces or displaces the 5' endogenous DNA flap (which can be excised, e.g., by a 5' flap endonuclease such as FEN1 or EXO1). , the 3' end of the 3' replacement DNA flap is then ligated to join the exposed 5' hydroxyl end of the endogenous DNA (exposed after excision of the 5' endogenous DNA flap), thereby binding the phosphodiester The bonds are reformed to incorporate the 3'-displaced DNA flap, forming a heteroduplex DNA containing one edited and one unedited strand. The DNA repair process permanently incorporates the edit into the DNA by breaking down the heteroduplex and copying the information from the edited strand to the complementary strand. This degradation process can be further driven to completion by nicking the unedited strand, i.e. via "second strand nicking" as described herein. .

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS The adoption of the clustered regularly spaced short palindromic repeat (CRISPR) system for genome editing has ^{revolutionized} the life sciences. Although gene disruption using CRISPR is now routine, the precise incorporation of single nucleotide edits is necessary to study or correct the large number of disease-causing mutations. This remains a major challenge. Homologous recombination repair (HDR) is capable of achieving 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. I'm worried about the effects. Recently, the laboratory of Professor David Liu and colleagues developed base editing, which achieves efficient single nucleotide editing without DSBs. Base editors (BEs) combine the CRISPR system with base-modifying deaminase enzymes to convert target C·G or A·T base pairs to A·T or G·C, respectively ^{4 - 6} . Although already widely used by researchers worldwide, current BEs can only make four of the possible 12 base pair transformations and cannot correct small insertions or deletions. Moreover, the target range 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 located 15 ± 2 bp from the target base. limited by. Therefore, overcoming these drawbacks would greatly expand the basic research and therapeutic applications of genome editing.

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

Consequently, the present invention provides that target-primed reverse transcription (TPRT) or "prime editing" mechanisms enable high-precision CRISPR/Cas-based genome editing with high efficiency and genetic flexibility. Relating in part to the discovery that the present invention can be exploited or employed to perform (as depicted in the various embodiments of FIGS. 1A-1F, for example). We herein target a specific DNA sequence with a modified guide RNA (“elongated guide RNA”), generate a single-stranded nick at the target site, and incorporate it into the elongated gRNA. proposed the use of a Cas9 protein-reverse transcriptase fusion, using nicked DNA as a primer for modified reverse transcriptase templates. The newly synthesized strand will be homologous to the target sequence of the genome except for including the desired nucleotide changes (eg, single nucleotide changes, deletions, or insertions, or combinations thereof). The newly synthesized strand of DNA, sometimes referred to as a single-stranded DNA flap, competes for hybridization with a complementary homologous endogenous DNA strand, thereby displacing the corresponding endogenous strand. Replace. Degradation of this hybridized intermediate involves removal (e.g., with the 5' end DNA flap endonuclease, FEN1) of the superseded flap of the resulting endogenous DNA, resulting in the synthesis of a single strand. It may involve ligation of a DNA flap to target DNA and assimilation of desired nucleotide changes as a result of cellular DNA repair and/or replication processes. Because templated DNA synthesis affords high precision of single nucleotides, it is foreseeable that the breadth of this approach is extremely broad and can be used for countless applications in basic science and therapeutics.

[1]napDNAbp
The prime editing factors and transprime editing factors described herein can include nucleic acid programmed 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 can be used in the prime editing factors described herein. In various embodiments, napDNAbp can 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 orthologs. This application refers to CRISPR-Cas enzymes by a nomenclature system that may be old and/or new. One of ordinary skill in the art will be able to identify the particular CRISPR-Cas enzyme referenced in this application based on the nomenclature system used, whether it is an old (i.e. "legacy") or new nomenclature system. There will be. The CRISPR-Cas nomenclature system is thoroughly 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 this document are incorporated by reference into this application. The specific CRISPR-Cas nomenclature system used in any given case of this application is in no way limiting, and one of ordinary skill in the art will be able to identify which CRISPR-Cas enzyme is being referred to.

For example, the following Type II, Type V, and Type VI class 2 CRISPR-Cas enzymes have the following art-recognized old (ie, legacy) and new names: Each of these enzymes and/or their variants can be used in the prime editing factors 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).

Prime editing factors described herein may also include Cas9 equivalents, including Cas12a (Cpf1) and Cas12b1 proteins that are the result of convergent evolution. The napDNAbp (eg, SpCas9, Cas9 variants, or Cas9 equivalents) used in this application may also contain various modifications that alter/enhance their PAM specificity. Finally, the present application provides 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%, Any having 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)) Cas9, Cas9 variants, or Cas9 equivalents are contemplated.

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 conjugatively transmissible plasmids). A CRISPR cluster contains a spacer, a sequence complementary to the 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 pre-crRNA requires trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and Cas9 protein. tracrRNA serves as a guide for ribonuclease 3-assisted processing of pre-crRNA. Cas9/crRNA/tracrRNA then endo-cleaves the linear or circular dsDNA target complementary to the spacer. Target strands that are not complementary to the crRNA are first cut endo- then trimmed 3'-5' exo. In nature, DNA binding and cleavage typically requires proteins and both RNA. 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, e.g., Jinek M. et al., Science 337:816-821 (2012), the entire contents of which are incorporated herein by reference.

In some embodiments, the napDNAbp leads to cleavage of one or both strands at the location of the target sequence, eg, on the target sequence and/or on the complement of the target sequence. In some embodiments, napDNAbp is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, Leading to cleavage of one or both strands within 200, 500, or more base pairs. In some embodiments, the vector comprises a napDNAbp that has been mutated relative to a corresponding wild-type enzyme such that the mutated napDNAbp lacks the ability to cleave one or both strands of a target polynucleotide containing the target sequence. code. 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 (which cleaves single strands). Other examples of mutations that make Cas9 a nickase include, without limitation, H840A, N854A, and N863A, with reference to the standard SpCas9 sequence or equivalent amino acid positions in other Cas9 variants or Cas9 equivalents. .

As used herein, the term "Cas protein" refers to a full-length Cas protein obtained from nature, a recombinant Cas protein having a sequence different from a naturally occurring Cas protein, or all or a significant amount required for the methods of the present disclosure. of any of the Cas proteins that still retains the essential basic functions of (i) the possession of nucleic acid-programmed binding of the Cas protein to the target DNA and (ii) the ability to nick the target DNA sequence on one strand. Say fragment. Cas proteins contemplated herein include naturally occurring or or non-naturally occurring (e.g., engineered or recombinant) and may include Cas9 equivalents from any class 2 CRISPR system (e.g., type II, V, VI), Cas12a ( Cpf1), Cas12e(CasX), Cas12b1(C2c1), Cas12b2, Cas12c(C2c3), C2c4, C2c8, C2c5, C2c10, C2c9 Cas13a(C2c2), Cas13d, Cas13c(C2c7), Cas13b(C2c6), and Cas13b do. Further Cas equivalents can be found 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. These contents are incorporated into this application by reference.

The term "Cas9" or "Cas9 nuclease" or "Cas9 moiety" or "Cas9 domain" refers to any naturally occurring Cas9 from any organism, any naturally occurring Cas9 equivalent or its functional Encompasses fragments, any Cas9 homologs, orthologs, or paralogs from any organism, and any naturally occurring or engineered mutations or variants of 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 to the specific Cas9 used in the prime editing element (PE) of the invention.

The Cas9 nuclease sequences and structures described herein are well known to those skilled in the art (e.g., “Complete genome sequence of an M1 strain of Streptococcus pyogenes.” Ferretti et al., J.J., McShan W.M., Ajdic D.J., Savic D.J. ,Savic G.,Lyon K.,Primeaux C.,Sezate S.,Suvorov A.N.,Kenton S.,Lai H.S.,Lin S.P.,Qian Y.,Jia H.G.,Najar F.Z.,Ren Q.,Zhu H.,Song L .,White J.,Yuan X.,Clifton S.W.,Roe B.A.,McLaughlin R.E.,Proc.Natl.Acad.Sci.U.S.A.98:4658-4663(2001);“CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.”Deltcheva E., Chylinski K., Sharma C.M., Gonzales K., Chao Y., Pirzada Z.A., Eckert M.R., Vogel J., Charpentier E., Nature 471:602-607(2011); and “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.”Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J.A., Charpentier E.Science 337:816-821(2012) (Contents of each of these (Incorporated herein by reference in its entirety).

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 "canonical SpCas9" nuclease from S. pyogenes. It is widely used as a tool for genome engineering and is classified as a type II subgroup of enzymes in class 2 CRISPR-Cas systems. The Cas9 protein is a large multidomain protein that contains two separate nuclease domains. Point mutations can be introduced on Cas9 to abolish one or both nuclease activities, resulting in nickase Cas9 (nCas9) or inactive Cas9 (dCas9), respectively. It still retains its ability to bind DNA in the manner programmed by the sgRNA. In principle, when fused to another protein or domain, Cas9 or a variant thereof (e.g. nCas9) could bind the protein to virtually any DNA sequence by simply co-expressing it with an appropriate sgRNA. and can be targeted. Standard SpCas9 protein as used in this application refers to the wild type protein from Streptococcus pyogenes having the following amino acid sequence:

を包含する。 The prime editing factors described herein are at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence with standard SpCas9, or the wild type Cas9 sequence provided above. Any variant thereof with identity may be included. These variants may include SpCas9 variants containing one or more mutations, including any known mutations reported in SwissProt Accession No. Q99ZW2 (SEQ ID NO: 18) entry. these are:

includes.

を包含する。 Other wild-type SpCas9 sequences that can be used in this disclosure are:

includes.

The prime editing factors described herein are any of the SpCas9 sequences above, or any thereof having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto. Variants of this may be included.

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

The prime editing factors described herein are any of the above Cas9 ortholog sequences, or those having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto. Any variant may be included.

napDNAbp may include any suitable homolog and/or ortholog of a naturally occurring enzyme 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 as a nickase (eg, mutagenized, recombinantly engineered, or otherwise obtained from nature). That is, only a single strand of the target can be cleaved. Additional suitable Cas9 nucleases and sequences will be apparent to those skilled in the art based on this disclosure. Such Cas9 nucleases and sequences include Cas9 nucleases 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 has an inactive (eg, inactivated) DNA cleavage domain. In other words, 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 the Cas9 protein provided by any one of the variants in Table 3. In some embodiments, the Cas9 protein has at least 85%, at least 90%, at least 92%, at least 95%, at least 96% the amino acid sequence of the Cas9 protein provided by any one of the Cas9 orthologs in the table above. , at least 97%, at least 98%, at least 99%, or at least 99.5% identical.

C. Inactive Cas9 variant
In certain embodiments, the prime editing factors described herein can include an inactive form of Cas9, such as an inactive form of SpCas9. It consists of 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). Due to this, it has no nuclease activity. Nuclease inactivation occurs when the amino acid sequence of the encoded protein, or any variant thereof, has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to it. It may be due to one or more mutations resulting in one or more substitutions and/or deletions.

The term "dCas9" as used herein refers to nuclease inactive Cas9 or nuclease dead Cas9 or a functional fragment thereof, including any naturally occurring dCas9 from any organism, any any naturally occurring dCas9 equivalent or functional fragment thereof, any dCas9 homolog, ortholog, or paralog from any organism, and any naturally occurring or engineered dCas9 variant or Subsuming variants. 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 in this application and/or in the art, and are incorporated herein by reference.

In other embodiments, the dCas9 corresponds to or includes a Cas9 amino acid sequence that has one or more mutations that inactivate Cas9 nuclease activity, in part or in its entirety. In other embodiments, Cas9 variants with mutations other than D10A and H840A are provided, which can result in complete or partial inactivation of endogenous Cas9 nuclease activity (eg, nCas9 or dCas9, respectively). Such mutations may include, for example, other amino acid substitutions in D10 and H820, or other substitutions on the nuclease domain of Cas9 (e.g., substitutions on the HNH nuclease subdomain and/or RuvC1 subdomain) of Cas9 from Streptococcus pyogenes. (NCBI reference sequence: NC_017053.1) is included by reference. 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, which are similar to the NCBI Reference Sequence: NC_017053.1 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 be. In some embodiments, a variant of dCas9 (e.g., a variant of NCBI reference sequence: NC_017053.1 (SEQ ID NO: 20)) is provided that has about 5 amino acids, about 10 amino acids, about having an amino acid sequence shorter or longer by 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 canonical SpCas9 sequence of Q99ZW2 and have the following sequence including the 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 may be based on the standard SpCas9 sequence of Q99ZW2 and may have the following sequence, including the D10A and H810A substitutions (underlined and bold), or at least 80%, at least 85%, It can be a variant of SEQ ID NO: 41 having at least 90%, at least 95%, or at least 99% sequence identity thereto.

D. Cas9 nickase variant
In one embodiment, the prime editing factor described herein comprises a Cas9 nickase. The term "Cas9 nickase" or "nCas9" refers to a variant of Cas9 that is capable of introducing a single-strand break on a double-stranded DNA molecule target. In some embodiments, the Cas9 nickase contains only a single functional nuclease domain. Wild-type Cas9 (e.g. standard SpCas9) contains two separate nuclease domains: the RuvC domain (which cleaves the non-protospacer DNA strand) and the HNH domain (which cleaves the protospacer DNA strand) . In one embodiment, the Cas9 nickase comprises a mutation in the RuvC domain that inactivates RuvC nuclease activity. For example, mutations in aspartate (D)10, histidine (H)983, aspartate (D)986, or glutamate (E)762 have been reported as loss-of-function mutations in the RuvC nuclease domain and generation of a functional Cas9 nickase. (eg, 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). Therefore, nickase mutations in the RuvC domain may include D10X, H983X, D986X, or E762X, where X is any amino acid other than the wild type amino acid. In certain embodiments, the nickase can be D10A, or (of) H983A, or D986A, or E762A, or a combination thereof.

In various embodiments, the Cas9 nickase can have a mutation in the RuvC nuclease domain and has one of the following amino acid sequences, or at least 80%, at least 85%, at least 90%, at least 95%, or at least 99 It may have a variant thereof that has an amino acid sequence with which it has % sequence identity.

In another embodiment, the Cas9 nickase comprises a mutation in the HNH domain that inactivates HNH nuclease activity. For example, mutations in histidine (H)840 or asparagine (R)863 have been reported as loss-of-function mutations in the HNH nuclease domain and generation of a functional Cas9 nickase (e.g., Nishimasu et al., “Crystal structure of Cas9 in complex with guide RNA and target DNA,” Cell 156(5), 935-949, which is incorporated herein by reference). Therefore, nickase mutations in the HNH domain can include H840X and R863X, where X is any amino acid other than the wild type amino acid. In certain embodiments, the nickase can be H840A or R863A or a combination thereof.

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

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

E. Other Cas9 variants
In addition to inactive Cas9 and Cas9 nickase variants, Cas9 proteins used herein may include any wild-type Cas9 or mutant Cas9 (e.g., inactive Cas9) disclosed herein or known in the art. at least about 70% identical, at least about 80% identical, at least about 90% identical to any reference Cas9 protein, including Cas9 or Cas9 nickase), or fragment Cas9, or circularly substituted Cas9, or other variants of Cas9; Other "Cas9 Variants" that are 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 may also be included. In some embodiments, the Cas9 variant is 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, It may have 46, 47, 48, 49, 50, or more amino acid changes compared to the 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 to the corresponding fragment of wild-type Cas9. % 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% are the same. 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%.

In some embodiments, the present disclosure may also utilize Cas9 fragments that retain their functionality, which are fragments of any of the Cas9 proteins disclosed herein. In some embodiments, the Cas9 fragment is at least 100 amino acids in length. In some embodiments, the fragment is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, or at least 1300 amino acids.

In various embodiments, the prime editing factors disclosed herein are at least about 70% identical, at least about 80% identical, at least about That Cas9 is 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 May contain variants.

F. Smaller size Cas9 variant
In some embodiments, prime editing agents contemplated herein include Cas9 proteins that are smaller in molecular weight than the standard SpCas9 sequence. In some embodiments, smaller sized Cas9 variants may facilitate delivery to cells, eg, by expression vectors, nanoparticles, or other means of delivery. In certain embodiments, smaller sized Cas9 variants may include enzymes classified as type II enzymes of class 2 CRISPR-Cas systems. In some embodiments, smaller sized Cas9 variants may include enzymes classified as type V enzymes of class 2 CRISPR-Cas systems. In other embodiments, smaller sized Cas9 variants may include enzymes classified as type VI enzymes of class 2 CRISPR-Cas systems.

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" means at least less than 1300 amino acids, or at least less than 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, 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 retains the required function of the Cas9 protein. Refers to Cas9 variants that are either naturally occurring, engineered, or otherwise. Cas9 variants can include those classified as type II, type V, or type VI enzymes of class 2 CRISPR-Cas systems.

In various embodiments, the prime editing factors disclosed herein are at least about 70% identical to one of the small size Cas9 variants described as follows, or to any reference small size Cas9 protein; 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 It may include a Cas9 variant thereof having 99.9% identity.

G. Cas9 equivalent
In some embodiments, the prime editing factors described herein may include any Cas9 equivalent. The term "Cas9 equivalent" as used in this application is a broad term, which means that its primary amino acid sequence and/or its three-dimensional structure may differ and/or be unrelated from an evolutionary standpoint. , subsumes any napDNAbp protein that serves the same function as Cas9 in this prime editing factor. Therefore, while Cas9 equivalents include any evolutionarily related Cas9 orthologs, homologs, mutants, or variants described or encompassed herein, Cas9 equivalents do not have the same or similar functionality as Cas9. It also encompasses proteins that may have evolved by a convergent evolutionary process to have a molecule, 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 arisen by convergent evolution, the prime editing factors described here encompass any Cas9 equivalent that would provide the same or similar functionality as Cas9. do. For example, if Cas9 refers to the type II enzyme of the CRISPR-Cas system, the Cas9 equivalent can refer to the type V or type VI enzyme of the CRISPR-Cas system.

For example, Cas12e (CasX) is a Cas9 equivalent that reportedly has the same function as Cas9 but evolved by convergent evolution. Therefore, 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 herein referred to as It is contemplated for use in the described prime editing factors. Additionally, any variants or modifications of Cas12e (CasX) are envisioned and within the scope of this 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, a Cas9 equivalent can refer to Cas12e (CasX) or Cas12d (CasY). These are 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 this are incorporated herein by reference. Using genome-resolved metagenomics, a number of CRISPR-Cas systems were identified, including the first reported Cas9 in the archaeal domain of life. This divergent Cas9 protein was discovered as part of an active CRISPR-Cas system from a poorly studied nanoarchaea. In bacteria, two previously unknown systems were discovered: CRISPR-Cas12e and CRISPR-Cas12d. These are among the most compact systems ever discovered. 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 programmed DNA binding proteins (napDNAbp) and are within the scope of this disclosure. See also Liu et al., “CasX enzymes comprise 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 is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, naturally occurring Cas12e (CasX) or Cas12d (CasY) protein. Includes amino acid sequences that are at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical. In some embodiments, napDNAbp is a naturally occurring Cas12e (CasX) or Cas12d (CasY) protein. According to some embodiments, NAPDNABP is at least 85 %, at least 85 %, at least 90 %, at least 92 %, at least 93 %, at least 94 %, at least 94 %, at least 93 %, at least 92 %, at least 92 %, at least 92 %, at least 92 %, at least 92 %, at least 92 %, at least 92 %, at least 92 %, at least 92 %, at least 92 %, at least 92 %, at least 92 % Includes amino acid sequences that are 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical.

In various embodiments, the nucleic acid programmed 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-programmed DNA-binding protein with a PAM specificity different from Cas9 is clustered regularly interspaced short palindromic repeat 1 from Prevotella and Francisella (ie, 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 distinct features from Cas9. Cas12a (Cpf1) is a single RNA-guided endonuclease lacking tracrRNA, which utilizes a T-rich protospacer flanking motif (TTN, TTTN, or YTN). Moreover, Cpf1 cleaves DNA by alternating DNA double-strand breaks. Among 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 previously described. For example, Yamano et al., “Crystal structure of Cpf1 in complex with guide RNA and target DNA.” Cell (165) 2016, p. 949-962; the entire contents of which are incorporated herein by reference.

In still other embodiments, the Cas protein may include any CRISPR-associated protein, including Cas12a, Cas12b1, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also as Csn1 and Csx12). (known), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17 , Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, homologues thereof, or modified versions thereof, preferably with nickase mutations. (eg, a mutation corresponding to the D10A mutation of the wild-type Cas9 polypeptide of SEQ ID NO: 18).

In various other embodiments, 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 Argonaute (Ago) domain, or a variant thereof.

Exemplary Cas9 equivalent protein sequences may include:

The prime editing factors described herein can also include Cas12a (Cpf1) (dCpf1) variants that can be used as DNA binding protein domains programmable by guide nucleotide sequences. Cas12a (Cpf1) protein has a RuvC-like endonuclease domain that is similar to the RuvC domain of Cas9, but does not have an HNH endonuclease domain, and the N-terminus of Cas12a (Cpf1) has a recognition lobe of the alpha-helical system of Cas9. I don't have it. In Zetsche et al., Cell, 163, 759-771, 2015 (which is incorporated herein by reference), the RuvC-like domain of Cas12a (Cpf1) is responsible for cleaving both DNA strands, and the RuvC-like domain It was shown that activation 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, whereas 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 this are incorporated herein by reference.

Two of the system's effectors, Cas12b1 and Cas12c, contain RuvC-like endonuclease domains related to Cas12a. A third system, Cas13a, contains an effector with two predicated HEPN RNase domains. Unlike CRISPR RNA production by Cas12b1, mature CRISPR RNA production is tracrRNA-independent. Cas12b1 relies 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 the single-stranded RNA degradation activity activated by its RNA. 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 this 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 catalytic residues produces 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 this are incorporated herein by reference.

The crystal structure of Alicyclobaccillus acidoterrastris Cas12b1 (AacC2c1) in complex with a chimeric single molecule guide RNA (sgRNA) has been reported. See, for example, 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 this are incorporated herein by reference. The crystal structure has also been reported as 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 this are incorporated herein by reference. The catalytically competent conformation of AacC2c1 is captured by both target and non-target DNA strands independently positioned within a single RuvC catalytic pocket. Cleavage mediated by C2c1 results in a staggered 7-nucleotide cleavage of the target DNA. Structural comparisons between the C2c1 tripartite complex and previously identified Cas9 and Cpf1 counterparts demonstrate the diversity of mechanisms used by the CRISPR-Cas9 system.

In some embodiments, napDNAbp can be a C2c1, C2c2, or C2c3 protein. In some embodiments, napDNAbp is a C2c1 protein. In some embodiments, napDNAbp is a Cas13a protein. In some embodiments, napDNAbp is a Cas12c protein. In some embodiments, napDNAbp is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, naturally occurring Cas12b1 (C2c1), Cas13a (C2c2), or Cas12c (C2c3) protein. Includes amino acid sequences that are at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical. In some embodiments, napDNAbp is a naturally occurring Cas12b1 (C2c1), Cas13a (C2c2), or Cas12c (C2c3) protein.

H. Cas9 circular permutant
In various embodiments, the prime editing factors disclosed herein can include circular permutations of Cas9.

The term "circularly permuted Cas9" or "circularly permuted variant" of Cas9 or "CP-Cas9" refers to any Cas9 protein or variant thereof that has been modified to occur or be engineered as a circularly permuted variant. . This means that the N-terminus and C-terminus of the Cas9 protein (eg, wild-type Cas9 protein) are locally rearranged. Such circularly permuted Cas9 proteins or variants thereof retain the ability to bind DNA when complexed with guide RNA (gRNA). 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, See 2019,176:254-267. Each is incorporated herein by reference. This disclosure contemplates either the previously known CP-Cas9 or the new Uses CP-Cas9.

Any variant, ortholog, or any of the Cas9 proteins described herein, including naturally occurring Cas9, or equivalents thereof, can be reconstituted as a circular permutant variant.

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

As an example, this disclosure contemplates the following circular permutations of standard S. pyogenes Cas9 (1368 amino acids of UniProtKB-Q99ZW2 (CAS9_STRP1) (numbering based on amino acid position of 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 permutations 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 (numbering is based on the amino acid position of SEQ ID NO: 18) of S. pyogenes Cas9 (UniProtKB-Q99ZW2 (CAS9_STRP1)) :
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 permutations 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 (numbering is based on the amino acid position of SEQ ID NO: 18) of S. pyogenes Cas9 (UniProtKB-Q99ZW2 (CAS9_STRP1)) :
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 permutations of other Cas9 proteins (including other Cas9 orthologs, variants, etc.).

In some embodiments, circular permutations 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 comprises the C-terminal 95% or more of the amino acids (e.g., about amino acids 1300-1368) of Cas9, or the C-terminal 90% of the amino acids of Cas9 (e.g., any one of SEQ ID NO: 77-86). %, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%, or more. The N-terminal portion is at least 95% of the N-terminus of Cas9 (e.g., about amino acids 1-1300), or 90%, 85%, 80%, 75%, 70% of the N-terminus of Cas9 (e.g., SEQ ID NO: 18). , 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%, or more.

In some embodiments, circular permutations 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 that is relocated to the N-terminus includes or corresponds to the C-terminal 30% or less of the amino acids of Cas9 (eg, amino acids 1012-1368 of SEQ ID NO: 18). In some embodiments, the C-terminal fragment that is relocated to the N-terminus is the C-terminal 30%, 29%, 28%, 27%, 26%, 25 of the amino acids of Cas9 (e.g., Cas9 of SEQ ID NO: 18). %, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, Includes or corresponds to 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%. In some embodiments, the C-terminal fragment that is relocated to the N-terminus includes or corresponds to the C-terminal 410 residues or less of Cas9 (eg, Cas9 of SEQ ID NO: 18). In some embodiments, the C-terminal portion that is relocated to the N-terminus is the C-terminus 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, Includes or corresponds to 70, 60, 50, 40, 30, 20, or 10 residues. In some embodiments, the C-terminal fragment that is relocated to the N-terminus includes or comprises the C-terminal 357, 341, 328, 120, or 69 residues of Cas9 (e.g., Cas9 of SEQ ID NO: 18). corresponds to

In other embodiments, circular permutant Cas9 variants may be defined as topological rearrangements of the Cas9 primary structure based on the following method, which is based on S. pyogenes Cas9 of SEQ ID NO: 18: (a) of the Cas9 primary structure. (b) selecting circular permutation (CP) sites corresponding to internal amino acid residues that cut the original protein into two halves: an N-terminal region and a C-terminal region; (b) preceding the original N-terminal region; Modifying the Cas9 protein sequence (e.g., by genetic engineering techniques) by moving the original C-terminal region (containing the CP-site amino acid) such that the Cas9 protein now begins with the CP-site amino acid residue to form a new N-terminus. 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 includes the original amino acid residues 181, 199, 230, 270, 310, 1010, 1016, 1023, 1029, 1041, 1247, 1249 (relative to S. pyogenes Cas9 of SEQ ID NO: 18). , or may be located at 1282. Therefore, once relocated to the N-terminus, the original amino acid 181, 199, 230, 270, 310, 1010, 1016, 1023, 1029, 1041, 1247, 1249, or 1282 becomes the new N-terminal amino acid. Will. The naming system for these CP-Cas9 proteins is Cas9-CP ¹⁸¹ , Cas9-CP ¹⁹⁹ , Cas9-CP ²³⁰ , Cas9-CP ²⁷⁰ , Cas9-CP ³¹⁰ , Cas9-CP ¹⁰¹⁰ , Cas9-CP ¹⁰¹⁶ , Cas9-CP, respectively. ¹⁰²³ , Cas9- ^CP1029 , Cas9- ^CP1041 , Cas9- ^CP1247 , Cas9- ^CP1249 , and Cas9- ^CP1282 . This description is not meant to be limited to making CP variants from SEQ ID NO: 18, but in any Cas9 sequence, either at the CP sites corresponding to these positions or at entirely other CP sites. or can be implemented to create CP variants. This description is in no way meant to limit specific CP sites. Virtually any CP site can be used to form a CP-Cas9 variant.

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

Cas9 circular permutants that may be useful in the prime editing constructs described herein. An exemplary C-terminal fragment of Cas9 based on Cas9 of SEQ ID NO: 18 that can be relocated to the N-terminus of Cas9 is provided below. It should be understood that such C-terminal fragments of Cas9 are exemplary and not meant to be limiting. These exemplary CP-Cas9 fragments have the following sequences:

I. Cas9 variants with altered PAM specificity
Prime editing agents of the present disclosure can also include Cas9 variants with altered PAM specificity. Some aspects of the present disclosure apply to target sequences that do not contain a standard PAM (5'-NGG-3', where N is A, C, G, or T) at its 3' end. The present invention provides a Cas9 protein that exhibits activity. In some embodiments, the Cas9 protein exerts activity against a target sequence that includes a 5'-NGG-3'PAM sequence at its 3' end. In some embodiments, the Cas9 protein exerts activity against a target sequence that includes a 5'-NNG-3'PAM sequence at its 3' end. In some embodiments, the Cas9 protein exerts activity against a target sequence that includes a 5'-NNA-3'PAM sequence at its 3' end. In some embodiments, the Cas9 protein exerts activity against a target sequence that includes a 5'-NNC-3'PAM sequence at its 3' end. In some embodiments, the Cas9 protein exerts activity against a target sequence that includes a 5'-NNT-3'PAM sequence at its 3' end. In some embodiments, the Cas9 protein exerts activity against a target sequence that includes a 5'-NGT-3'PAM sequence at its 3' end. In some embodiments, the Cas9 protein exerts activity against a target sequence that includes a 5'-NGA-3'PAM sequence at its 3' end. In some embodiments, the Cas9 protein exerts activity against a target sequence that includes a 5'-NGC-3'PAM sequence at its 3' end. In some embodiments, the Cas9 protein exerts activity against a target sequence that includes a 5'-NAA-3'PAM sequence at its 3' end. In some embodiments, the Cas9 protein exerts activity against a target sequence that includes a 5'-NAC-3'PAM sequence at its 3' end. In some embodiments, the Cas9 protein exerts activity against a target sequence that includes a 5'-NAT-3'PAM sequence at its 3' end. In yet other embodiments, the Cas9 protein exerts activity against a target sequence that includes 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 exhibit 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 are conservative mutations of the listed clones of Table 1. In some embodiments, the Cas9 protein comprises a combination of mutations in 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 the Cas9 protein provided by any one of the variants in Table 1. In some embodiments, the Cas9 protein has at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least the amino acid sequence of the Cas9 protein provided by any one of the variants in Table 1. Comprising amino acid sequences that are 97%, at least 98%, at least 99%, or at least 99.5% identical.

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 the Cas9 protein provided by any one of the variants in Table 2. In some embodiments, the Cas9 protein has at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least the amino acid sequence of the Cas9 protein provided by any one of the variants in Table 2. Comprising amino acid sequences that are 97%, at least 98%, at least 99%, or at least 99.5% identical.

In some embodiments, the Cas9 protein is directed 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. exerts increased activity. In some embodiments, the Cas9 protein is provided by SEQ ID NO: 18 for a target sequence with a 3' end that is not directly adjacent to the canonical PAM sequence (5'-NGG-3') for the same target sequence. Streptococcus pyogenes Cas9 exhibits at least a 5-fold increased activity compared to that of Streptococcus pyogenes Cas9. In some embodiments, the Cas9 protein is directed against a target sequence that is not directly adjacent to the canonical PAM sequence (5'-NGG-3'), while the Cas9 protein is directed against a target sequence of Streptococcus pyogenes provided by SEQ ID NO: 18 against the same target sequence. at least 10 times, at least 50 times, at least 100 times, at least 500 times, at least 1,000 times, at least 5,000 times, at least 10,000 times, at least 50,000 times, at least 100,000 times, at least 500,000 times, or at least 1,000,000 times greater than the activity Demonstrates a certain level of activity. In some embodiments, the 3' end of the target sequence is directly adjacent to an AAC, GAC, CAC, or TAC sequence.

In some embodiments, the Cas9 protein comprises a combination of mutations that exhibit activity 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 are conservative mutations of the listed clones of Table 3. In some embodiments, the Cas9 protein comprises a combination of mutations in any one of the Cas9 clones listed in Table 3.

Table 3: NAT PAM clone

The above description of various napDNAbps that may be used with respect to the prime editing factors of the present disclosure is not meant to be limiting in any way. The prime editing factor is the standard SpCas9, or any orthologous Cas9 protein, or any variant that is known or may be created or evolved by directed evolutionary or otherwise mutational processes. A Cas9 protein may be included, including any naturally occurring variants, mutants, or otherwise engineered versions of Cas9. In various embodiments, Cas9 or a Cas9 variant has nickase activity, ie, cleaves only one (of) strands of the target DNA sequence. In other embodiments, the Cas9 or Cas9 variant has an inactive nuclease, ie, is an "inactive" Cas9 protein. Other variant Cas9 proteins that may be used have lower molecular weights than standard SpCas9 (e.g., for easier delivery) or modified or rearranged primary amino acid structures (e.g., circular permutant format). It is something that you have. The prime editing factors described herein can also include Cas9 equivalents, including Cas12a/Cpf1 and Cas12b proteins that are the result of convergent evolution. The napDNAbp (eg, SpCas9, Cas9 variant, or Cas9 equivalent) used in this application may also contain various modifications that alter/enhance their PAM specificity. Finally, the present application provides 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%, Any having 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) Cas9, Cas9 variants, or Cas9 equivalents are contemplated.

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

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

In some embodiments, the napDNAbp that functions with non-canonical PAM sequences is an Argonaute protein. One example of such a nucleic acid programmed DNA binding protein is Argonaute protein (NgAgo) from Natronobacterium gregoryi. NgAgo is an ssDNA-guided endonuclease. NgAgo will bind to ~24 nucleotides of 5'-phosphorylated ssDNA (gDNA) and guide it to its target site, creating 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 nuclease-inactive NgAgo (dNgAgo) can greatly expand the bases that can be targeted. Characterization and use of NgAgo can be found 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 these is incorporated herein by reference.

In some embodiments, napDNAbp is a prokaryotic homologue of Argonaute protein. Prokaryotic homologs of Argonaute proteins are known, for example 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 this are incorporated herein by reference. In some embodiments, napDNAbp is Marinitoga piezophila Argunaute (MpAgo) protein. The CRISPR-associated Marinitoga piezophila Argunaute (MpAgo) protein uses a 5'-phosphorylated guide to cleave single-stranded target sequences. The 5' guide is used by all known Argonauts. The crystal structure of the MpAgo-RNA complex shows a guide strand binding site that contains residues that block 5' phosphate interactions. This data suggests the evolution of an argonaute subclass with non-canonical specificity for 5'-hydroxylated guides. See, eg, 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 can be used and are within the scope of this disclosure.

Some aspects of this disclosure provide Cas9 domains with different PAM specificities. Typically, Cas9 proteins, such as Cas9 from S. pyogenes (spCas9), require the standard NGG PAM sequence to bind to a specific nucleic acid region. This can limit the ability to edit desired bases on the genome. In some embodiments, the base editing fusion proteins provided herein may require precise positioning. For example, here the target base is placed on a four base region (eg, an "editing window"), which is approximately 15 bases upstream of the PAM. See Komor, A.C., et al., “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage” Nature 533, 420-424 (2016). The entire contents of this 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 canonical (eg, NGG) PAM sequence. Cas9 domains that bind non-canonical PAM sequences have been described in the art and will be apparent to those skilled in the art. For example, Cas9 domains that bind non-canonical PAM sequences have been described by 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); each of which is incorporated herein by reference in its entirety.

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:

an additional napDNAbp domain with modified PAM specificity, such as at least 80%, at least 85%, at least 90%, at least 95%, or Domains with at least 99% sequence identity:

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, napDNAbp is an argonaute protein. One example of such a nucleic acid programmed DNA binding protein is Argonaute protein (NgAgo) from Natronobacterium gregoryi. NgAgo is an ssDNA-guided endonuclease. NgAgo will bind to ~24 nucleotides of 5'-phosphorylated ssDNA (gDNA) and guide it to its target site, creating 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 nuclease-inactive NgAgo (dNgAgo) can greatly expand the bases that can be targeted. Characterization and use of NgAgo are described by 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 these is incorporated herein by reference. The sequence of Natronobacterium gregoryi Argonaute is provided by SEQ ID NO: 76.

The disclosed fusion proteins have a napDNAbp having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with wild type Natronobacterium gregoryi Argonaute (SEQ ID NO: 76) having the following amino acid sequence: May contain domains:

Additionally, any available method can 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, such as a nucleic acid or amino acid sequence, by 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 the amino acid substitutions (mutations) provided herein are well known in the art and are described, for example, in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)). Mutations may encompass various categories, such as single nucleotide polymorphisms, microduplicated regions, indels, and inversions, and are not meant to be limiting in any way. Mutations can include "loss-of-function" mutations that are the normal result of mutations that reduce or abolish protein activity. Most loss-of-function mutations are recessive. This is because in a heterozygote, the second chromosome copy carries an unmutated version of the gene encoding a fully functional protein, and this presence compensates for the effects of the mutation. Mutations also encompass "gain-of-function" mutations. This confers on a protein or cell an abnormal activity 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, mutations can lead to one or more genes being expressed in the wrong tissues, and these tissues acquire functions that they normally lack. Because of their nature, gain-of-function mutations are usually dominant.

Mutations can be introduced onto a reference Cas9 protein using site-directed mutagenesis. Older methods of site-directed mutagenesis known in the art rely on subcloning the sequence to be mutated onto a vector such as the M13 bacteriophage vector, which allows isolation of a single-stranded DNA template. In these methods, a mutagenic primer (i.e., a primer capable of annealing to the site to be mutated, but with one or more mismatched nucleotides at the site to be mutated) is annealed to a single-stranded template; The complement of the template is then polymerized starting from the 3' end of the mutagenic primer. The resulting duplex is then transformed into host bacteria and plaques are screened for the desired mutations. More recently, site-directed mutagenesis has used PCR methodology. These have the advantage of not requiring single-stranded templates. Additionally, 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 expansion 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, because of the nontemplate-dependent end extension activity of some thermostable polymerases, blunt-end ligation of PCR-generated mutant products is often preceded by an end-polishing step. It is necessary to incorporate this into the procedure.

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 e.g. international PCT application PCT/US2009/056194 filed September 8, 2009, published as WO2010/028347 on March 11, 2010; WO2012/088381 filed June 28, 2012. International PCT Application PCT/US2011/066747, filed December 22, 2011; U.S. Application U.S. Patent No. 9,023,594, filed May 5, 2015; published September 11, 2015 as WO2015/134121; International PCT Application PCT/US2015/012022, filed on January 20, 2015, and International PCT Application PCT/US2016/027795, filed on April 15, 2016, published as WO2016/168631 on October 20, 2016. Are listed. The entire contents of each of these are incorporated herein by reference. Variant Cas9 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, in which serial flask planting of evolving "selection phages" (SPs) containing the genes of interest to be evolved by new E. coli host cells Using splicing, the genes contained in the SP evolve continuously while allowing the genes in the host E. coli to remain constant. Serial flask transfer has long served as a widely accessible approach for the 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.

Any of the references noted above regarding Cas9 or Cas9 equivalents are herein incorporated by reference in their entirety, unless already so claimed.

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

Split PE delivery may be advantageous to address 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 made more efficient and/or effective by dividing the prime editing factor into smaller pieces. Once inside the cell, the smaller pieces can assemble into a functional prime editor. Depending on the means of disruption, the split prime editing factor fragments can be reassembled in a non-covalent or covalent manner to reform the prime editing factor. In one embodiment, the prime editing element can be split into two or more fragments at one or more cleavage sites. A fragment can be unmodified (other than split). Once the fragments are delivered to the cell (e.g., by direct delivery of ribonucleoprotein complexes or by nucleic acid delivery, e.g. mRNA delivery or viral vector-based delivery), the fragments can be covalently or non-covalently linked. can reassemble to reconstitute prime editing factors. In another embodiment, the prime editing element 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 fragment formed is coupled to a dimerization domain. Once delivered or expressed into a cell, the dimerization domains of the different fragments associate and bind to each other, bringing the different prime editing factor fragments together to re-form a functional prime editing factor. do. In yet another embodiment, the prime editing factor fragment can be modified to include a splitting intein. Once delivered or expressed into a cell, the divided intein domains of 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 editing factor.

In one embodiment, prime editing factors can be delivered using a split intein approach.

The location of the splitting site is between any one or more pairs of residues on the prime editing factor, as well as within the linker domain that connects the napDNAbp domain, the polymerase domain (e.g., RT domain), the napDNAbp domain, and the polymerase domain. It can be located on any domain therein.

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

In certain embodiments, napDNAbp is a standard SpCas9 polypeptide of SEQ ID NO: 18 as follows:

In certain embodiments, the SpCas9 comprises 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 at the cleavage site located between 8 and 9, or 9 and 10, or at residues 1 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 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, Between any two pairs of residues located anywhere between 1100-1200, 1200-1300, or 1300-1368, it is split into two fragments.

In certain embodiments, napDNAbp is at positions 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80 of the canonical SpCas9 of SEQ ID NO: 18. ～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 into two fragments at a cleavage site located at a pair of residues corresponding to any two pairs of residues located anywhere between 1300 and 1368.

In certain embodiments, the SpCas9 comprises 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 at the cleavage site located between 8 and 9, or 9 and 10, or at residues 1 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 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, It is split into two fragments between any two pairs of residues located anywhere between 1100-1200, 1200-1300, or 1300-1368. In certain embodiments, the fission site is located at one or more polypeptide binding sites (i.e., "split site or fission intein fission site"), fused to the fission intein, and then released into the cell as a separately encoded fusion protein. delivered to. Once the split intein fusion proteins (i.e., the protein halves) are expressed in a cell, the proteins undergo trans-splicing to form a complete or whole PE with concomitant removal of the linked split intein sequences. Form.

For example, as shown in Figure 66, an N-terminal extein can be fused to a first split intein (e.g., N intein) and a C-terminal extein can be fused to a second split intein (e.g., C intein). can be done. By self-association of N- and C-inteins within the cell, then their self-excision and concomitant formation of a peptide bond between the N-terminal and C-terminal extein moieties of the whole prime editing element (PE). The N-terminal extein becomes fused to the C-terminal extein to reform an entire prime editor fusion protein containing a napDNAbp domain and a polymerase domain (eg, RT domain).

To benefit from a split PE delivery strategy using split inteins, the prime editing factor must be cleaved at one or more split sites to generate at least two separate halves of the prime editing factor. do. Each of these can be religated intracellularly if each half is fused to a split intein sequence.

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

In a preferred embodiment, the prime editing factor is split at a single cleavage site to generate two separate halves of the prime editing factor. Each of these can be fused to a split intein sequence.

An exemplary fission intein is the Ssp DnaE intein, which contains two subunits, DnaE-N and DnaE-C. The two different 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 fission intein of Synechocytis sp. PCC6803 that can direct the trans-splicing of two separate proteins, each containing 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 are 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 in, for example, 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 (eg, PACE) can be used to evolve the first portion of the base editor. The first portion may include a single component or domain, such as a Cas9 domain, a deaminase domain, or a UGI domain. The separately evolved components or domains are then fused to the rest of the base editor within the cell by expressing both the evolved portion and the remaining unevolved portion together with the split intein polypeptide domain. obtain. The first portion may be broader and include the first amino acid portion of any base editor 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 cannot be evolved using the methods of the present application. The evolved first portion and second portion of the base editor can each be expressed by the cell with a split intein polypeptide domain. The cell's natural protein splicing machinery will reassemble the evolved first part and the unevolved second part to form a single fusion protein evolved base editor. The first portion evolved can include either the N- or C-terminal part of a single fusion protein. In a related fashion, the use of a second orthogonal trans-splicing intein pair may allow the evolved first portion to contain the 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), 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 (eg, an evolved portion of a base editor). Suitable intein sequences may be selected from any of the proteins known to contain protein splicing elements. A database containing all known inteins can be found on the World Wide Web (Perler, F.B. Nucleic Acids Research, 1999, 27, 346-347). The intein sequence is fused at the 3' end to the 5' end of the 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 multi-intein-containing constructs, it may be useful to use intein elements from different sources. A transcription termination sequence must be inserted after the last gene sequence to be expressed. In one embodiment, an engineered intein splicing unit is designed such that it can catalyze excision of extein from intein and prevent ligation of extein. Mutagenesis of the C-terminal extein junction on the Pyrococcus sp. GB-D DNA polymerase was found to result in altered splicing elements. This induces cleavage of exteins and inteins, but prevents subsequent ligation of exteins (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 the conservation of amino acids at the C-terminal extein junction to inteins, 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 created artificially by removing the endonuclease domain from intein-containing endonucleases (Chong, et al. J. Biol. Chem. 1997, 272, 15587 -15590). In a preferred embodiment, the intein is selected such that it consists of the minimum number of amino acids required to perform the splicing function, such as the intein from the Mycobacterium xenopi GyrA protein (Telenti, A., et al. al., J. Bacteriol. 1997, 179, 6378-6382). In an alternative embodiment, an intein without endonuclease activity is selected, such as the intein from the Mycobacterium xenopi GyrA protein, or the Saccharaomyces 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. Fission inteins have been identified in diverse cyanobacteria and archaea (Caspi et al,Mol Microbiol.50:1569-1577(2003);Choi J.et al,J Mol Biol.556:1093-1106(2006 .);Dassa B.et al,Biochemistry.46:322-330(2007.);Liu X. and Yang J.,J Biol Chem.275:26315-26318(2003);Wu H.et al.

Proc Natl Acad Sci USA. ￡5:9226-9231 (1998.); and Zettler J. et al, FEBS Letters. 553:909-914 (2009)), so far it has not been found in eukaryotes. Recently, bioinformatics analysis of environmental metagenomic data revealed 26 distinct loci with novel genomic locations. At each locus, the conserved enzyme-coding region is interrupted by a split intein, and an independent endonuclease gene is inserted between sections encoding intein subdomains. Among them, five loci were fully assembled: DNA helicase (gp41-1, gp41-8); inosine-5'-monophosphate dehydrogenase (IMPDH-1); and ribonucleotide reductase catalytic subunit (NrdA -2 and NrdJ-1). This disjointed 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 protein splicing mechanism typically has four steps [29-30]: 1) N-S or N-O acyl shift at the intein N-terminus; This cleaves the upstream peptide bond and forms an ester bond between the N-extein and the side chain of the intein's first amino acid (Cys or Ser); 2) positions the N-extein to the intein C-terminus; Transesterification to change. Form a new ester bond and link the N extein to the side chain of the first amino acid (Cys, Ser, or Thr) of the C extein; 3) Break the peptide bond between the intein and the C extein Asn cyclization; and 4) S-N or O-N acyl shift replacing the ester bond by a peptide bond between the N and C exteins.

Protein trans-splicing catalyzed by split inteins provides a purely enzymatic method for protein ligation [31]. A split intein is essentially a stretch of intein (eg, a mini-intein) that has been split into two pieces, named N-intein and C-intein, respectively. The N and C inteins of a split intein non-covalently associate to form an active intein and can catalyze splicing reactions in essentially the same way that a continuous intein does. Split inteins are found in nature and have also been manipulated in the laboratory [31-35]. As used herein, the term "split intein" refers to any intein in which one or more peptide bond cleavages exist between the N-terminal and C-terminal amino acid sequences, such that the N-terminal and C-terminal sequences are distinct. molecules, which can non-covalently reassemble or reconstitute into inteins that are functional for trans-splicing reactions. 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, a fission intein can be derived from a eukaryotic intein. In another aspect, the fission intein can be derived from a bacterial intein. In another aspect, the fission 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, an "N-terminally 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 naturally occurring intein sequences. For example, an In may contain additional amino acid residues and/or mutated residues, so long as the inclusion of such additional and/or mutated residues does not render the In non-functional in trans-splicing. Preferably, the inclusion of additional and/or mutated residues improves or enhances the trans-splicing activity of the 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 includes fluorescent groups, biotin, polyethylene glycol (PEG), amino acid analogs, unnatural amino acids, phosphate groups, glycosyl groups, radioisotope labels, among others. , and additional chemical moieties including pharmaceutical molecules. In other embodiments, the peptide linked to Ic may contain one or more chemically reactive groups including ketones, aldehydes, Cys residues, and Lys residues, among others. When an "intein splicing polypeptide (ISP)" is present, the N and C inteins of the split intein can non-covalently associate to form the active intein and catalyze the splicing reaction. As used herein, "intein splicing polypeptide (ISP)" refers to the portion of the amino acid sequence of a splitting intein that remains when Ic, In, or both are removed from the splitting intein. In certain embodiments, In includes ISP. In another embodiment, Ic comprises ISP. In yet another embodiment, the ISP is a separate peptide that is not covalently linked to either In or Ic.

Split inteins are created by engineering one or more splitting sites in unstructured loops or intervening amino acid sequences between the 12 conserved beta strands found on the structure of mini-inteins. [25-28]. Some of the location of the cleavage site within the inter-beta strand region, provided that the generation of cleavages will not interrupt the structure of the intein, particularly the structured beta strand, to a sufficient degree that protein splicing activity is lost. flexibility can exist.

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 the trans-splicing reaction (together N and C intein) excises the two intein sequences and connects the two extein sequences by a peptide bond. Protein trans-splicing, an enzymatic reaction, can work with very low (eg, micromolar) concentrations of protein and can be carried out under physiological conditions.

[2] Other Programmable Nucleases In various embodiments described herein, the prime editing factor comprises a napDNAbp, such as a Cas9 protein. These proteins are "programmable" by virtue of their becoming complexed with a guide RNA (or PEgRNA, as the case may be). This guides the Cas9 protein to a target site on the 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 envisioned herein, 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 shows such a variation of prime editing as contemplated herein in which napDNAbp (e.g. SpCas9 nickase) is replaced by some programmable nuclease domain, e.g. zinc finger nuclease (ZFN) or transcription activator-like effector nuclease (TALEN). Illustrated. As such, it is understood that a suitable nuclease does not necessarily need to be "programmed" by a nucleic acid targeting molecule (e.g. guide RNA), but rather can be programmed by a DNA binding domain, e.g. planned. Just like prime editing with the napDNAbp moiety, such alternative programmable nucleases are preferably modified 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 (eg, 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 (eg, via a chemical linker), where the extension arm contains a primer binding site (PBS) and a DNA synthesis template. Programmable nucleases can also be coupled to polymerases (eg, via chemical or amino acid linkers). The nature of this will depend on whether the extended arm is DNA or RNA. In the case of RNA extension arms, the polymerase can be an RNA-dependent DNA polymerase (eg, reverse transcriptase). In the case of DNA extending arms, the polymerase can be a DNA-dependent DNA polymerase (e.g., a prokaryotic polymerase including Pol I, Pol II, or Pol III, or Pol a, Pol b, Pol g, Pol d, Pol e, or eukaryotic polymerases including Pol z). The system may also include other functionality added as a fusion to the programmable nuclease or added in trans to facilitate the whole reaction (e.g., (a) cut site (b) a helicase to unwind the DNA and create a cut strand with a 3' end that can be used as a primer; FEN1 to drive the reaction towards replacement of the endogenous strand by (c) a second on the opposing strand that may help drive the incorporation of synthetic repair by favoring cellular repair of the non-edited strand. nCas9:gRNA complex to generate site nicks). In a manner analogous to prime editing by napDNAbp, such a complex with a differently programmable nuclease synthesizes a newly synthesized replacement strand of DNA with the desired edit and then permanently places it on the target site of DNA. can be used to incorporate into

Suitable alternative programmable nucleases are well known in the art, and these can be used to construct alternative prime editor systems that can be programmed to selectively bind to target sites in DNA. :Can be used instead of gRNA complex. And these can be further modified in the manner described above to co-localize the polymerase and the RNA or DNA extension arm containing the primer binding site and DNA synthesis template to a particular nick site. For example, as represented by FIG. 1J, transcription activator-like effector nucleases (TALENs) can be used as programmable nucleases in the prime editing methods and compositions described herein. TALENs are artificial restriction enzymes produced by fusing a TAL effector DNA binding domain to a DNA cutting domain. These reagents enable efficient, programmable, and specific DNA cleavage and represent powerful tools for in situ genome editing. Transcription activator-like effectors (TALEs) can be rapidly engineered to bind to virtually any DNA sequence. As used herein, the term TALEN is broad and includes monomeric TALENs that are capable of cleaving 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 cut DNA at the same site. TALENs that work together can be referred to as a left TALEN and a right TALEN. This refers to the handedness of DNA. U.S.Ser.No.12/965,590;U.S.Ser.No.13/426,991(U.S.Pat.No.8,450,471);U.S.Ser.No.13/427,040(U.S.Pat.No.8,440,431);U.S.Ser.No.13/ 427,137 (U.S. Pat. No. 8,440,432); and U.S. Ser. No. 13/738,381. All of which are incorporated by reference into this application in their entirety. In addition, TALEN is 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. al.,TAL Effectors:Customizable Proteins for DNA Targeting,Science,vol.333,pp.1843-1846(2011),Cade et al.,“Highly efficient generation of heritable zebrafish gene mutations using homo- and heterodimeric TALENs”,Nucleic Acids Research, vol. 40, pp. 8001-8010 (2012), and Cermak et al., “Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting”, Nucleic Acids Research, vol. 39, No. 17, e82 (2011). Each of these is incorporated herein by reference.

As depicted in Figure 1J, zinc finger nucleases can also be used in place of napDNAbp, such as Cas9 nickase, as alternative programmable nucleases for use in prime editing. Like TALENs, ZFN proteins can be modified so that they function as nickases. That is, the ZFN is engineered such that it cuts only one strand of the target DNA, in a manner similar to the napDNAbp used in the prime editing factors described herein. ZFN proteins have been described in the art, for example, by 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, 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 editing element (PE) systems disclosed herein include a polymerase (e.g., a DNA-dependent DNA polymerase or an RNA-dependent DNA polymerase, e.g., reverse transcriptase) or a variant thereof, which It can be provided as a fusion protein with napDNAbp or other programmable nucleases or in trans.

Any polymerase can be used in the prime editing agents disclosed herein. Polymerases can be wild-type polymerases, functional fragments, mutants, variants, or truncated variants, and the like. The polymerase may include wild-type polymerases from eukaryotic, prokaryotic, archaeal, or viral organisms, and/or the polymerase may be modified by processes based on genetic engineering, mutagenesis, directed evolution. Polymerases can include T7 DNA polymerase, T5 DNA polymerase, T4 DNA polymerase, Klenow fragment DNA polymerase, DNA polymerase III, and the like. Polymerases can also be thermostable, such as Taq, Tne, Tma, Pfu, Tfl, Tth, Stoffel fragments, VENT® and DEEPVENT® DNA polymerases, KOD, Tgo, JDF3, and variants thereof. , variants, and derivatives (US Pat. No. 5,436,149; US Pat. No. 4,889,818; US Pat. No. 4,965,185; US Pat. No. 5,079,352; US Pat. No. 5,614,365; US Pat. No. 5,374,553; US Pat. No. 5,270,179). No.; U.S. Patent No. 5,047,342; U.S. Patent 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). (incorporated into the specification)). At least two DNA polymerases can be used to synthesize longer nucleic acid molecules (eg, nucleic acid molecules greater than about 3-5 Kb in length). In certain embodiments, one of the polymerases may be substantially devoid of 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 are 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 agents disclosed herein are "template-dependent" polymerases (because during prime editing, the polymerase is used to define the sequence of the DNA strand being synthesized). As used herein, the term "template DNA molecule" refers to the term "template DNA molecule" from which a complementary nucleic acid strand is synthesized by a DNA polymerase, e.g., by a primer extension reaction of a PEgRNA DNA synthesis template. refers to a chain of nucleic acid.

As used herein, the term "template-dependent manner" is intended to refer to a process that involves template-dependent extension of a primer molecule (eg, DNA synthesis by a DNA polymerase). The term "template-dependent mode" refers to polynucleotide synthesis of RNA or DNA in which the sequence of the newly synthesized strand of polynucleotide is described by the well-known rules of complementary base pairing (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, the single strand of DNA synthesized by the polymerase of the prime editing factor against the DNA synthesis template is said to be "complementary" to the sequence of the DNA synthesis template. It can be said.

A. Exemplary polymerases
In various embodiments, the prime editing agent described herein comprises a polymerase. This disclosure contemplates any wild-type polymerase obtained from any naturally occurring organism or virus, or obtained from commercial or non-commercial sources. In addition, polymerases that can be used in the prime editing agents of the present disclosure can include any naturally occurring, engineered, or other variant polymerases that retain function. Includes all variants. Polymerases that can be used herein can also be engineered to contain certain amino acid substitutions, such as those specifically disclosed herein. In certain preferred embodiments, the polymerases that can be used in the prime editing agents of the present disclosure are template-based polymerases. That is, they synthesize nucleotide sequences in a template-dependent manner.

Polymerases are enzymes that synthesize nucleotide chains that can be used in conjunction with the prime editor systems described herein. The polymerase is preferably a "template-dependent" polymerase (ie, a polymerase that synthesizes a nucleotide strand based on the order of the nucleotide bases of a template strand). In certain configurations, a polymerase can also be "template-independent" (ie, a polymerase that synthesizes a nucleotide strand without the requirement of a template strand). Polymerases can also be further classified as "DNA polymerases" or "RNA polymerases." In various embodiments, the prime editor system includes a DNA polymerase. In various embodiments, the DNA polymerase can be a "DNA-dependent DNA polymerase" (ie, according to which the template molecule is a strand of DNA). In such cases, the DNA template molecule may be PEgRNA and the extending arm comprises a strand of DNA. In such cases, the PEgRNA may also be referred to as a chimeric or hybrid PEgRNA, which includes an RNA portion (ie, a guide RNA component that includes a spacer and a gRNA core) and a DNA portion (ie, an extended arm). In various other embodiments, the DNA polymerase can be an "RNA-dependent DNA polymerase" (ie, according to which the template molecule is a strand of RNA). In such cases, PEgRNA is RNA, ie, includes RNA extension. The term "polymerase" can also refer to an enzyme that catalyzes the polymerization of nucleotides (ie, polymerase activity). Generally, the enzyme initiates 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, e.g., PEgRNA) and towards the 5' end of the template strand. will proceed. "DNA polymerase" catalyzes the polymerization of deoxynucleotides. The term DNA polymerase, as used herein with reference to DNA polymerase, includes "functional fragments thereof." A "functional fragment thereof" means any portion of a wild-type or mutant DNA polymerase that encompasses less than the entire amino acid sequence of the polymerase that retains the ability to catalyze the polymerization of polynucleotides under at least one set of conditions. To tell. 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. This is 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 3' to 5' exo T7 "Sequenase" DNA polymerase) available from ).

In other embodiments, 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 the relevant 5' to 3' exonuclease activity and to possess 3' to 5' exonuclease (proofreading) activity. . Suitable DNA polymerases (pol I or pol II) may be derived from archaea, which have optimal growth temperatures similar to the desired assay temperature.

Thermostable archaeal DNA polymerases include 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 may also be from Eubacterium sp. There are three classes of Eubacterium 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 exert 3' to 5' exonuclease activity. Pol II DNA polymerase naturally lacks 5' to 3' exonuclease activity, but does exert 3' to 5' exonuclease activity. Pol III DNA polymerase represents the cell's major replicative DNA polymerase and is composed of multiple subunits. Although the pol III catalytic subunit lacks 5' to 3' exonuclease activity, in some cases 3' to 5' exonuclease activity is mapped onto 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 are isolated from various thermophilic eubacteria, including Thermus species and Thermotoga maritima, such as Thermus aquaticus (Taq), Thermus thermophilus (Tth), and Thermotoga maritima (Tma UlTma). can be done.

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 enables chimeric or non-chimeric DNA polymerases that have been chemically modified according to the methods disclosed in US Pat. Nos. 5,677,152, 6,479,264, and 6,183,998. These contents are incorporated herein by reference in their entirety.

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 transcriptase
In various embodiments, the prime editing factors described herein include reverse transcriptase as the polymerase. This disclosure contemplates any wild-type reverse transcriptase obtained from any naturally occurring organism or virus or from commercial or non-commercial sources. In addition, reverse transcriptases that can be used in the prime editing agents of the present disclosure can include any naturally occurring, engineered, or other variant RT that retains functionality. Includes shortened variants. RTs can also be engineered to contain certain amino acid substitutions, such as those specifically disclosed herein.

Reverse transcriptase is a multifunctional enzyme, typically having three enzymatic activities, including RNA- and DNA-dependent DNA polymerization activities, and RNaseH activity, which catalyzes the cleavage of RNA in RNA-DNA hybrids. Some variants of reverse transcriptase have the RNaseH moiety disabled to prevent unintended damage to the mRNA. These enzymes, which synthesize complementary DNA (cDNA) using mRNA as a template, were first identified in RNA viruses. Later, reverse transcriptase was isolated and purified directly from virus particles, cells, or tissues (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 generated in search of improved properties such as thermostability, fidelity, and activity. Any wild-type, variant, and/or mutant forms of reverse transcriptase that are known in the art or that can be made using methods known in the art are contemplated in this application.

The reverse transcriptase (RT) gene (or the genetic information it contains) can be obtained from a number of different sources. For example, genes can be obtained from eukaryotic cells infected with a retrovirus or from any number of plasmids containing either the entire retroviral genome or a portion thereof. In addition, messenger RNA-like RNA containing RT genes can be obtained from retroviruses. Examples of sources of RT are 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); yeasts, including Saccharomyces; Neurospora, Drosophila; primates; and rodents. For example, Weiss, et al., U.S. Patent 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 editing factors 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 certain embodiments, reverse transcriptase is provided in trans to other components of the prime editing element (PE) system. That is, the reverse transcriptase is expressed or otherwise provided as an individual component, ie, not as a fusion protein with napDNAbp.

Those skilled in the art will appreciate that wild-type reverse transcriptases include (Moloney murine leukemia virus (M-MLV); human immunodeficiency virus (HIV) reverse transcriptase; and avian leukemia-sarcoma virus (ASLV) reverse transcriptase; These include, but are 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 myelocytoma disease 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 enzymes, including but not limited to Rous-associated virus (RAV) reverse transcriptase, and myeloblastosis virus-associated virus (MAV) reverse transcriptase), in the subject methods and compositions described herein. It will be appreciated that it can be used suitably.

Exemplary wild type RT enzymes are:

Variants and error-prone RTs
Reverse transcriptase is essential for synthesizing complementary DNA (cDNA) strands from RNA templates. 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 annealed primers, reverse transcriptase binds to the RNA template and initiates the polymerization reaction. RNA-dependent DNA polymerase activity synthesizes complementary DNA (cDNA) strands and incorporates dNTPs. RNase H activity degrades RNA templates in DNA:RNA complexes. Therefore, 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, throughput (rate of dNTP incorporation), and fidelity (or error rate). Reverse transcriptase variants contemplated herein may have different 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) that impacts or alters any one or more of the reverse transcriptase enzymes. Such variants may be available in the public domain, commercially available in the art, or may be created using known methods of mutagenesis, including directed evolution processes (e.g., PACE or PANCE). .

In various embodiments, the reverse transcriptase can be a variant reverse transcriptase. As used herein, a "variant reverse transcriptase" refers to one or more mutations (single mutations, inversions, deletions, insertions, and rearrangements) relative to a reference sequence (e.g., reference wild-type sequence). including any naturally occurring or genetically engineered variant. RT naturally possesses several activities, including RNA-dependent DNA polymerase activity, ribonuclease H activity, and DNA-dependent DNA polymerase activity. Collectively, these activities enable the enzyme to convert single-stranded RNA into double-stranded cDNA. For retroviruses and retrotransposons, this cDNA can then be incorporated onto the host genome, from which new RNA copies can be made by host cell transcription. Variant RTs may contain mutations that impact one or more of these activities, either reducing or increasing these activities, or eliminating them altogether. In addition, a variant RT may contain one or more mutations that make the RT more or less stable, less prone to aggregation, easier to purify and/or detect, and/or other the alterations in properties or characteristics. may include.

Those skilled in the art will appreciate that other reverse transcriptases, including Moloney murine leukemia virus (M-MLV); human immunodeficiency virus (HIV) reverse transcriptase, and avian leukemia-sarcoma virus (ASLV) reverse transcriptase, These include, but are 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) are the subject matter described herein. It will be appreciated that the present invention may be suitably used in methods and compositions of the invention.

One way to prepare variant RTs is by genetic modification (eg, by modifying the DNA sequence of wild-type reverse transcriptase). A number of methods are known in the art that allow for random and targeted mutations of DNA sequences (e.g., Ausubel et.al.Short Protocols in Molecular Biology (1995)3.sup.rd Ed.John Wiley & (See Sons, Inc.). In addition, there are a number of commercially available kits for site-directed mutagenesis, including both conventional and PCR-based methods. Examples are 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, variant reverse transcriptases can be generated by insertional mutagenesis or truncation (N-terminal, internal, or C-terminal insertions or truncation) according to methodologies known to those skilled in the art. The term "mutation" as used herein refers to the substitution of a residue on a sequence, such as a nucleic acid or amino acid sequence, by 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 the amino acid substitutions (mutations) provided herein are well known in the art and are described, for example, in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)). Mutations may encompass various categories, such as single nucleotide polymorphisms, microduplicated regions, indels, and inversions, and are not meant to be limiting in any way. Mutations can include "loss-of-function" mutations that are the normal result of mutations that reduce or abolish protein activity. Most loss-of-function mutations are recessive. This is because in a heterozygote, the second chromosome copy carries an unmutated version of the gene encoding a fully functional protein, and this presence compensates for the effects of the mutation. Mutations also encompass "gain-of-function" mutations. This confers on a protein or cell an abnormal activity 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, mutations can lead to one or more genes being expressed in the wrong tissues, and these tissues acquire functions that they normally lack. Because of their nature, gain-of-function mutations are usually dominant.

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

More recently, site-directed mutagenesis has used PCR methodology. These have the advantage of not requiring single-stranded templates. Additionally, 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 expansion 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, because of the non-template-dependent end extension activity of some thermostable polymerases, blunt-end ligation of PCR-generated mutant products is often preceded by an end-polishing step. It is necessary to incorporate this into the procedure.

Methods of random mutagenesis exist in the art and these will result in a panel of variants with one or more randomly located mutations. Such a panel of variants 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 mutation introduction is the so-called "error-prone PCR method". As the name implies, the method amplifies a given sequence under conditions where the DNA polymerase does not support high-fidelity integration. Conditions that promote error-prone integration will vary for different DNA polymerases, but one skilled 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 concentrations can be applied to affect the error rate of the polymerase.

In various aspects, the prime editing factor RT can be an "error-prone" reverse transcriptase variant. Any error-prone reverse transcriptase known and/or available in the art may be used. It will be appreciated that reverse transcriptase does not naturally have any proofreading function; therefore, the error rate of reverse transcriptase is generally higher than that of DNA polymerases that contain proofreading activity. . The error rate of any particular reverse transcriptase is a property of the enzyme's "fidelity," which represents the accuracy of polymerization directed by its DNA template 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 reverse transcriptase based on M-MLV 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 the purposes of this application, a reverse transcriptase that is considered "error-prone" or has "error-prone fidelity" has a have an error rate of less than one error.

Error-prone reverse transcriptases can also be generated by mutagenesis of the starting RT enzyme (eg, wild-type M-MLV RT). The method of mutagenesis is not limited and may include directed evolution processes, such as phage-assisted continuous evolution (PACE) or phage-assisted 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 e.g. international PCT application PCT/US2009/056194 filed September 8, 2009, published as WO2010/028347 on March 11, 2010; WO2012/088381 filed June 28, 2012. International PCT Application PCT/US2011/066747 filed December 22, 2011; U.S. Application U.S. Patent No. 9,023,594 filed May 5, 2015; published September 11, 2015 as WO2015/134121; International PCT Application PCT/US2015/012022, filed on January 20, 2015, and International PCT Application PCT/US2016/027795, filed on April 15, 2016, published as WO2016/168631 on October 20, 2016. Are listed. The entire contents of each of these 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, in which serial flask planting of evolving "selection phages" (SPs) containing the genes of interest to be evolved by new E. coli host cells Using splicing, the genes contained in the SP evolve continuously while allowing the genes in the host E. coli to remain constant. Serial flask transfer has long served as a widely accessible approach for the 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 include Bebenek et al., “Error-prone Polymerization by HIV-1 Reverse Transcriptase,” J Biol Chem, 1993, Vol. 268:10324-10334 and Sebastian-Martin et al., Described in “Transcriptional inaccuracy threshold attenuates differences in RNA-dependent DNA synthesis fidelity between retroviral reverse transcriptases,” Scientific Reports, 2018, Vol. 8:627. Each of these 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, all from NEW ENGLAND BIOLABS®. Transcriptase, WarmStart® Reverse Transcriptase, and M-MuLV Reverse Transcriptase, or AMV Reverse Transcriptase XL, SMARTScribe Reverse Transcriptase, GPR Ultra Pure MMLV, all from TAKARA BIO USA, INC. (formerly CLONTECH) Includes reverse transcriptase.

The present disclosure also contemplates reverse transcriptases having mutations in the RNaseH domain. As mentioned above, one of the inherent properties of reverse transcriptase is its RNase H activity, which cleaves the RNA template of RNA:cDNA hybrids simultaneously with polymerization. RNase H activity may be undesirable for the synthesis of long cDNAs because the RNA template may be degraded before full-length reverse transcription is complete. RNase H activity can also reduce reverse transcription efficiency, possibly due to its competition with the enzyme's polymerase activity. Therefore, this disclosure contemplates any reverse transcriptase variants that include altered RNaseH activity.

This disclosure also contemplates reverse transcriptases having mutations in the RNA-dependent DNA polymerase domain. As mentioned above, one of the inherent properties of reverse transcriptase is RNA-dependent DNA polymerase activity. This incorporates the nucleobase into the nascent cDNA strand encoded by the template RNA strand of the RNA:cDNA hybrid. RNA-dependent DNA polymerase activity can be increased or decreased to either increase or decrease the processivity of the enzyme (ie, in terms of its rate of incorporation). Therefore, the present disclosure discloses modified RNA-dependent DNA polymerase activity such that the processivity of the enzyme is either increased or decreased relative to the unmodified version. Any reverse transcriptase variant containing is contemplated.

Reverse transcriptase variants with altered thermostability characteristics are also contemplated herein. The ability of reverse transcriptase to withstand high temperatures is an important aspect of cDNA synthesis. Elevated reaction temperature helps denature RNA with strong secondary structure 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 can lead to improved generation of 3' flap ssDNA as a result of the prime editing process. Wild-type M-MLV reverse transcriptase typically has a temperature optimum in the range 37-48 °C; however, 49 °C, 50 °C, 51 °C, 52 °C, 53 °C, 54 °C, 55 °C, 56 °C reverse transcription activity at higher temperatures above 48°C, including 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, and higher. Forgiving mutations can be introduced.

Variant reverse transcriptases contemplated herein, including error-prone RTs, thermostable RTs, throughput-enhancing RTs, can be engineered by a variety of routine strategies, including mutagenesis or evolutionary processes. In some cases, variants can be generated by introducing a single mutation. In other cases, a variant may require more than one mutation. In mutants containing more than one mutation, the effects of a given mutation can be isolated from other mutations carried by a particular mutant and identified on the wild-type gene by site-directed mutagenesis. This can be evaluated by the introduction of mutations. Screening assays for the single mutants thus generated will then allow determination of the effect of that mutation alone.

Variant RT enzymes as used in this application refer to any reference including any wild-type RT or mutant RT or fragment RT or other variants of RT disclosed or contemplated in this application or known in the art. 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 to RT protein Other "RT variants" that are at least about 99.5% identical, or at least about 99.9% identical may also be included.

In some embodiments, the RT variant is 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 the reference RT. It is possible. In some embodiments, 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 to the corresponding fragment of the reference RT. , at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical, an RT variant comprises a fragment of a reference RT. In some embodiments, the fragment is at least 30% of the amino acid length of either the corresponding wild type RT (M-MLV reverse transcriptase) (e.g., SEQ ID NO: 89) or the reverse transcriptase of SEQ ID NO: 90-100. , 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%.

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 fragment is at least 100 amino acids in length. In some embodiments, the fragment is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, or up to 600, or more amino acids in length.

In yet other embodiments, the present disclosure may also utilize RT variants in which the N-terminus or the C-terminus, or both, are truncated by a certain number of amino acids, which is a truncated variant that still retains sufficient polymerase function. bring about. In some embodiments, the RT shortening variant 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 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 acid truncations at the N-terminal end of the protein. In other embodiments, the RT shortening variant 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 30, 40, 50, 60, 70, 80, 90, A truncation of 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 amino acids at the C-terminal end of the protein. In still other embodiments, the RT truncation variants have truncations at the N-terminal and C-terminal ends that are the same or different lengths.

For example, the prime editing factors disclosed herein can include truncated versions 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 truncation). The DNA sequence encoding this truncated editing factor is 522 bp smaller than PE2, thus making it a potential for applications where delivery of the DNA sequence is difficult due to its size (i.e., adeno-associated virus and lentivirus delivery). Make useful. This embodiment is called MMLV-RT(trunc) and has the following amino acid sequence:

In various embodiments, the prime editing factors disclosed herein are at least about 70% identical, at least about 80% identical, at least about That RT that is 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 May contain variants.

In still other embodiments, the methods and compositions are as described in Effefson et al., “Synthetic evolutionary origin of a proofreading reverse transcriptase,” Science, June 24, 2016, Vol. 352:1590-1593. DNA polymerases evolved into reverse transcriptases can be used. The contents of which are incorporated into this application by reference.

In certain other embodiments, reverse transcriptase is provided as a component of a fusion protein that also includes napDNAbp. In other words, in some embodiments, reverse transcriptase is fused to napDNAbp as a fusion protein.

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

In various embodiments, the prime editing factors described herein (wherein the RT is provided either as a fusion partner or in trans) can include variant RTs that include one or more of the following mutations: SEQ ID NO: P51L, S67K, E69K, L139P, T197A, D200N, H204R, F209N, E302K, E302R, T306K, F309N, W313F in the wild-type M-MLV RT of 89 or at the corresponding amino acid position on another wild-type RT polypeptide sequence , T330P, L345G, L435G, N454K, D524G, E562Q, D583N, H594Q, L603W, E607K, or D653N.

Some exemplary reverse transcriptases that can be fused to napDNAbp proteins or provided as individual proteins according to various aspects of this disclosure are provided below. 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 enzymes 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, the prime editing factors described herein (wherein the RT is provided either as a fusion partner or in trans) are primed in the wild-type M-MLV RT of SEQ ID NO: 89 or in another wild-type Variant RTs may be included that include a P51X mutation at the corresponding amino acid position on the RT polypeptide sequence, where "X" can be any amino acid. In certain embodiments, X is L.

In various other embodiments, the prime editing factors described herein (wherein the RT is provided either as a fusion partner or in trans) are primed in the wild-type M-MLV RT of SEQ ID NO: 89 or in another wild-type Variant RTs containing the S67X mutation at the corresponding amino acid position on the RT polypeptide sequence may be included, where "X" can be any amino acid. In certain 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, the prime editing factors described herein (wherein the RT is provided either as a fusion partner or in trans) are primed in the wild-type M-MLV RT of SEQ ID NO: 89 or in another wild-type Variant RTs containing the L139X mutation at the corresponding amino acid position on the RT polypeptide sequence can be included, where "X" can be any amino acid. In certain embodiments, X is P.

In various other embodiments, the prime editing factors described herein (wherein the RT is provided either as a fusion partner or in trans) are primed in the wild-type M-MLV RT of SEQ ID NO: 89 or in another wild-type Variant RTs can be included that include a T197X mutation at the corresponding amino acid position on the RT polypeptide sequence, where "X" can be any amino acid. In certain 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, the prime editing factors described herein (wherein the RT is provided either as a fusion partner or in trans) are primed in the wild-type M-MLV RT of SEQ ID NO: 89 or in another wild-type Variant RTs can be included that include a H204X mutation at the corresponding amino acid position on the RT polypeptide sequence, where "X" can be any amino acid. In certain embodiments, X is R.

In various other embodiments, the prime editing factors described herein (wherein the RT is provided either as a fusion partner or in trans) are primed in the wild-type M-MLV RT of SEQ ID NO: 89 or in another wild-type Variant RTs can be included that include a F209X mutation at the corresponding amino acid position on the RT polypeptide sequence, where "X" can be any amino acid. In certain 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, the prime editing factors described herein (wherein the RT is provided either as a fusion partner or in trans) are primed in the wild-type M-MLV RT of SEQ ID NO: 89 or in another wild-type Variant RTs can be included that include the E302X mutation at the corresponding amino acid position on the RT polypeptide sequence, where "X" can be any amino acid. In certain embodiments, X is R.

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

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

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

In various other embodiments, the prime editing factors described herein (wherein the RT is provided either as a fusion partner or in trans) are primed in the wild-type M-MLV RT of SEQ ID NO: 89 or in another wild-type Variant RTs can be included that include a T330X mutation at the corresponding amino acid position on the RT polypeptide sequence, where "X" can be any amino acid. In certain 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, the prime editing factors described herein (wherein the RT is provided either as a fusion partner or in trans) are primed in the wild-type M-MLV RT of SEQ ID NO: 89 or in another wild-type Variant RTs can be included that include the L435X mutation at the corresponding amino acid position on the RT polypeptide sequence, where "X" can be any amino acid. In certain 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, the prime editing factors described herein (wherein the RT is provided either as a fusion partner or in trans) are primed in the wild-type M-MLV RT of SEQ ID NO: 89 or in another wild-type Variant RTs can be included that include a D524X mutation at the corresponding amino acid position on the RT polypeptide sequence, where "X" can be any amino acid. In certain 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, the prime editing factors described herein (wherein the RT is provided either as a fusion partner or in trans) are primed in the wild-type M-MLV RT of SEQ ID NO: 89 or in another wild-type Variant RTs can be included that include a D583X mutation at the corresponding amino acid position on the RT polypeptide sequence, where "X" can be any amino acid. In certain embodiments, X is N.

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

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

In various other embodiments, the prime editing factors described herein (wherein the RT is provided either as a fusion partner or in trans) are primed in the wild-type M-MLV RT of SEQ ID NO: 89 or in another wild-type Variant RTs can be included that include the E607X mutation at the corresponding amino acid position on the RT polypeptide sequence, where "X" can be any amino acid. In certain 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 editing factor (PE) system described herein is described in the following U.S. patents (each of which is incorporated by reference in their entirety): U.S. Patent No. 10,202,658; No. 10,189,831; No. 10,150,955; No. 9,783,791; No. 9,580,698; No. 9,534,201; and any variants thereof that can be made using known methods for evolving. The following references describe reverse transcriptases in the art. Each of those disclosures is incorporated by reference into this application 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, REVIEWS1017(2001).

Baranauskas,A.et al.Generation and characterization of new highly thermostable and processive M-MuLV reverse transcriptase variants.Protein Eng Des Sel 25,657-668(2012).

Zimmerly, S., Guo, H., Perlman, P. S. & Lambowltz, A. M. Group II intron mobility occurs by target DNA-primed reverse transcription. Cell 82, 545-554 (1995).

Feng, Q., Moran, J.V., Kazazian, H.H. & Boeke, J.D.Human L1 retrotransposon encodes a conserved endonuclease required for retrotransposition.Cell 87,905-916(1996).

Berkhout, B., Jebbink, M. & 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 noted above relating to reverse transcriptase are herein incorporated by reference in their entirety, unless already so claimed.

[4] PE fusion protein The prime editing element (PE) system described herein consists of napDNAbp and a polymerase (e.g., DNA-dependent DNA polymerase or RNA-dependent DNA polymerase, e.g. reverse transcriptase), optionally linked by a linker. ) are contemplated. This application contemplates that any suitable napDNAbp and polymerase (eg, DNA-dependent DNA polymerase or RNA-dependent DNA polymerase, eg, reverse transcriptase) are combined as a single fusion protein. Examples of napDNAbp and polymerases (eg, DNA-dependent DNA polymerases or RNA-dependent DNA polymerases, eg, reverse transcriptase) are each defined in this application. This disclosure is in no way meant to be limited to the particular polymerases identified in this application, as polymerases are well known in the art and amino acid sequences are readily available.

In various embodiments, the fusion protein can include any suitable structural configuration. For example, a fusion protein can include napDNAbp fused to a polymerase (eg, a DNA-dependent DNA polymerase or an RNA-dependent DNA polymerase, eg, reverse transcriptase) in an N-terminal to C-terminal direction. In other embodiments, the fusion protein may include a polymerase (eg, reverse transcriptase) fused to napDNAbp in an N-terminal to C-terminal direction. The fused domains may optionally be joined by a linker, such as an amino acid sequence. In other embodiments, the fusion protein may include the structure _NH2- [napDNAbp]-[polymerase]-COOH; or _NH2- [polymerase]-[napDNAbp]-COOH, where each occurrence of "]-[" is optional. Indicates the presence of a linker sequence. In embodiments where the polymerase is reverse transcriptase, the fusion protein may include the structure NH ₂ -[napDNAbp]-[RT]-COOH; or NH ₂ -[RT]-[napDNAbp]-COOH, where "]-[" Each one indicates the presence of an optional linker sequence.

Exemplary fusion proteins are illustrated in FIG. 14. This shows a fusion protein comprising MLV reverse transcriptase ("MLV-RT") fused to nickase Cas9 ("Cas9(H840A)") via a linker sequence. This example is not intended to limit the range of fusion proteins that can be utilized in the prime editor (PE) system described herein.

In various embodiments, the prime editing factor fusion protein can have the following amino acid sequence (referred to herein as "PE1"), which includes Cas9 variants containing the H840A mutation (i.e., Cas9 nickase) and M-MLV RT wild type, 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 sequence of PE1 and its individual components is as follows:

In another aspect, the prime editing factor fusion protein can have the following amino acid sequence (referred to herein as "PE2"), which includes the Cas9 variant containing the H840A mutation (i.e., Cas9 nickase) as well as the mutation D200N, Contains an M-MLV RT containing T330P, L603W, T306K, and W313F with 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. do. 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:

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

In other embodiments, prime editing factor fusion proteins can be based on SaCas9 or SpCas9 nickases with modified PAM specificity, such as the following exemplary sequences:

In yet other embodiments, prime editing factor fusion proteins contemplated herein may include a Cas9 nickase (eg, 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 editing factor is 522 bp smaller than PE2, thus making it potentially useful for applications where delivery of the DNA sequence is difficult due to its size (i.e., adeno-associated virus and lentiviral delivery). Make it. This embodiment is referred to as Cas9(H840A)-MMLV-RT(trunc) or "PE2-short" or "PE2-trunc" and has the following amino acid sequence:

See Figure 75. This is 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. I will provide a. The data show that prime editors containing truncated RT variants were approximately as efficient as prime editors containing non-truncated RT proteins.

In various embodiments, the prime editing factor fusion proteins contemplated herein are at least about 70% identical, at least about 80% identical, at least about 80% identical to PE1, PE2, or any of the prime editing factor fusion sequences indicated above. Amino acids that are 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 Variants of any of the sequences disclosed above may also be included.

In certain embodiments, a linker can be used to join any of the peptides or peptide domains or portions of the invention (eg, napDNAbp linked or fused to reverse transcriptase).

[5] Linkers and other domains
PE fusion proteins can contain various other domains besides napDNAbp (eg, Cas9 domain) and polymerase domain (eg, RT domain). For example, in the case where napDNAbp is Cas9 and the polymerase is RT, the PE fusion protein can include one or more linkers that join the Cas9 domain with the RT domain. Linkers may also connect other functional domains, such as the nuclear localization sequence (NLS) or FEN1 (or other flap endonucleases) to the PE fusion protein or domain thereof.

Additionally, in embodiments involving transprime editing, a linker can be used to join the tPERT recruitment protein to the prime editing factor, eg, between the tPERt recruitment protein and napDNAbp. For example, see Figure 3G for an exemplary schematic of a transprime editing element (tPE) that includes a linker to separately fuse the polymerase domain and recruitment protein domain to napDNAbp.

A. linker
As defined above, the term "linker" as used herein refers to a chemical group or molecule that joins two molecules or moieties, such as a binding domain and a 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 (eg, reverse transcriptase). In some embodiments, a linker connects dCas9 and reverse transcriptase. Typically, a 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 (eg, 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 in length. , 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. Longer or shorter linkers are also contemplated.

The linker can be as simple as a covalent bond, or it can be a polymeric linker many atoms in length. In certain embodiments, the linker is a poly(pol) peptide or based on amino acids. In other embodiments, the linker is not peptidic. In certain embodiments, the linker is a covalent bond (eg, a carbon-carbon bond, a disulfide bond, a carbon-heteroatom bond, etc.). In certain embodiments, the linker is a carbon-nitrogen bond of an amide linkage. In certain embodiments, the linker is a cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic or heteroaliphatic linker. In certain embodiments, the linker is polymeric (eg, polyethylene, polyethylene glycol, polyamide, polyester, etc.). In certain embodiments, the linker comprises an aminoalkanoic acid monomer, dimer, or polymer. In certain embodiments, the linker comprises an aminoalkanoic acid (eg, glycine, ethanoic acid, alanine, beta-alanine, 3-aminopropanoic acid, 4-aminobutyric acid, 5-pentanoic acid, etc.). In certain embodiments, the linker comprises a monomer, dimer, or polymer of aminohexanoic acid (Ahx). In certain embodiments, the linker is based on a carbocyclic moiety (eg, cyclopentane, cyclohexane). In other embodiments, the linker includes a polyethylene glycol moiety (PEG). In other embodiments, the linker includes amino acids. In certain embodiments, the linker comprises a peptide. In certain embodiments, the linker includes an aryl or heteroaryl moiety. In certain embodiments, the linker is based on a phenyl ring. The linker may include a functionalized moiety (eg, thiol, amino) to facilitate attachment of a nucleophile from the peptide to the linker. Any electrophile can 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 certain embodiments, a linker can be used to join any of the peptides or peptide domains or moieties of the invention (eg, napDNAbp linked or fused to reverse transcriptase).

As defined above, the term "linker" as used herein refers to a chemical group or molecule that joins two molecules or moieties, such as a binding domain and a cleavage domain of a nuclease. In some embodiments, the linker connects the gRNA binding domain of the RNA programmable nuclease and the catalytic domain of the recombinase. In some embodiments, a linker joins dCas9 and reverse transcriptase. Typically, a 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 (eg, a peptide or protein). In some embodiments, the linker is an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker is between 5 and 100 amino acids in length, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 in length. , 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. Longer or shorter linkers are also contemplated.

The linker can be as simple as a covalent bond, or it can be a polymeric linker many atoms in length. In certain embodiments, the linker is polypeptide or based on amino acids. In other embodiments, the linker is not peptidic. In certain embodiments, the linker is a covalent bond (eg, a carbon-carbon bond, a disulfide bond, a carbon-heteroatom bond, etc.). In certain embodiments, the linker is a carbon-nitrogen bond of an amide linkage. In certain embodiments, the linker is a cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic or heteroaliphatic linker. In certain embodiments, the linker is polymeric (eg, polyethylene, polyethylene glycol, polyamide, polyester, etc.). In certain embodiments, the linker comprises an aminoalkanoic acid monomer, dimer, or polymer. In certain embodiments, the linker comprises an aminoalkanoic acid (eg, glycine, ethanoic acid, alanine, beta-alanine, 3-aminopropanoic acid, 4-aminobutyric acid, 5-pentanoic acid, etc.). In certain embodiments, the linker comprises a monomer, dimer, or polymer of aminohexanoic acid (Ahx). In certain embodiments, the linker is based on a carbocyclic moiety (eg, cyclopentane, cyclohexane). In other embodiments, the linker includes a polyethylene glycol moiety (PEG). In other embodiments, the linker includes amino acids. In certain embodiments, the linker comprises a peptide. In certain embodiments, the linker includes an aryl or heteroaryl moiety. In certain embodiments, the linker is based on a phenyl ring. The linker may include a functionalized moiety (eg, thiol, amino) to facilitate attachment of a nucleophile from the peptide to the linker. Any electrophile can 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 It is an 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 SGGSGGSSGSETPGTSESATPESSGGSSGGS (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 can include one or more nuclear localization sequences (NLS), which help facilitate translocation of the protein into the cell nucleus. Such sequences are well known in the art and may include the following examples:

The above NLS example is not limiting. PE fusion proteins can include any known NLS sequence, including 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 these is incorporated herein by reference.

In various embodiments, the prime editing factors and constructs encoding prime editing factors disclosed herein further comprise one or more, preferably at least two, nuclear localization signals. In certain 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. Additionally, NLS can be expressed as part of a fusion protein with the rest of the prime editing factor. In some embodiments, one or more of the NLSs is a bibaric NLS (“bpNLS”). In certain embodiments, the fusion proteins of the present disclosure include two bisegmented NLSs. In some embodiments, the fusion proteins of the present disclosure include more than two bisegmented NLSs.

The positioning of the NLS fusion can be at the N-terminus, C-terminus, or within the sequence of the prime editing factor (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. NLS could also be any NLS discovered in the future for nuclear localization. The NLS can also be any naturally occurring NLS or any non-naturally occurring NLS (eg, an NLS with one or more desired mutations).

The term "nuclear localization sequence" or "NLS" refers to an amino acid sequence that facilitates import of proteins into the nucleus of a cell, eg, 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 Plank et al.'s international PCT application PCT/EP2000/011690, filed November 23, 2000, published as WO/2001/038547 on May 31, 2001. The contents of which are incorporated into this application by reference. In some embodiments, the NLS comprises the amino acid sequence PKKKRKV (SEQ ID NO: 16), MDSLLNMNRKFLYQFKNVRWAKGRRETYLC (SEQ ID NO: 17), KRTADGSEFESPKKKRKV (SEQ ID NO: 3864), or KRTADGSEFEPKKKRKV (SEQ ID NO: 125 ). In other embodiments, the NLS comprises the amino acid sequences NLSKRPAIKKAGQAKKKK (SEQ ID NO : 136 ), PAAKRVKLD (SEQ ID NO: 192), RQRRNELKRSF (SEQ ID NO: 3934 ), NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO : 3935 ).

In one aspect of the disclosure, the prime editing factor may be modified with one or more nuclear localization signals (NLS), preferably at least two NLSs. In certain embodiments, the prime editing factor is modified with two or more NLSs. The present disclosure describes 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 later state of the art at the time of this application. Contemplate the use of signals. A typical nuclear localization signal is a peptide sequence that directs the protein to the nucleus of the cell where the sequence is expressed. Nuclear localization signals are predominantly basic and can be located almost anywhere in a protein's amino acid sequence, typically consisting of four amino acids (Autieri & Agrawal, (1998) J. Biol. Chem. 273:14731- 37. (incorporated herein by reference) and is typically rich in lysine and arginine residues (Magin et al., (2000) Virology 274:11-16. ). Nuclear localization signals often contain proline residues. A variety of nuclear localization signals have been identified and used to accomplish the transport of biological molecules from the cytoplasm to the nucleus of the cell. See, for example, Tinland et al., (1992) Proc. Natl. Acad. Sci. U.S.A. 89:7442-46; Moede et al., (1999) FEBS Lett. 461:229-34. This is incorporated by reference. Translocation is currently thought to involve nuclear pore proteins.

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

Nuclear localization signals appear at various points on a protein's amino acid sequence. NLSs have been identified from the N-terminus, C-terminus, and central region of the protein. Therefore, the present disclosure provides prime editing factors that can be modified by one or more NLSs at the C-terminus, N-terminus, and internal regions of the prime editing factor. Residues of the longer sequence should be selected that do not function as component NLS residues, eg, tonically or sterically, so as not to interfere with the nuclear localization signal itself. Thus, although there are no strict limitations on the composition of sequences comprising an NLS, such sequences may in fact be functionally limited in length and composition.

This disclosure contemplates any suitable means for modifying the prime editing factor to include one or more NLSs. In one aspect, the prime editing factor is configured to express a prime editing factor protein that is translationally fused at its N-terminus or its C-terminus (or both) to one or more NLSs, i.e., prime editing factor-NLS Can be engineered to form fusion constructs. In other embodiments, the nucleotide sequence encoding the prime editing factor can be genetically modified to incorporate reading frames encoding one or more NLSs into regions internal to the encoded prime editing factor. In addition, NLS encodes various amino acid linkers or spacer regions between the prime editing factor and the NLS amino acid sequence attached at the N-terminus, at the C-terminus, or internally, e.g., and in the central region of the protein. can be included. Therefore, the present disclosure also enables nucleotide constructs, vectors, and host cells for expressing fusion proteins that include a prime editing factor and one or more NLSs.

The prime editing factors described herein may also include nuclear localization signals, which are linked via one or more linkers, e.g., and polymeric, amino acid, nucleic acid, polysaccharide, chemical, or nucleic acid linker elements. linked to the prime editing factor. Linkers within the contemplated scope of this disclosure are not intended to have any limitations and may include any suitable type of molecule (e.g., polymer, amino acid, polysaccharide, nucleic acid, lipid, or any synthetic chemical (e.g., covalent linkage, hydrogen bonding) between the prime editing factor and one or more NLSs. can be connected to

C. flap endonuclease (e.g. FEN1)
In various embodiments, the PE fusion protein can include one or more flap endonucleases (eg, FEN1), which refers to enzymes that catalyze the removal of the 5' single-stranded DNA flap. These are naturally occurring enzymes that process the removal of the 5' flap that is formed during cellular processes including DNA replication. The prime editing method described herein uses an endogenously supplied flap endonuclease or trans to remove the 5' flap of endogenous DNA that forms at the target site during prime editing. You can take advantage of what is provided. Flap endonucleases are known in the art and are described in Patel et al., “Flap endonucleases pass 5'-flaps through a flexible arch using a disorder-thread-order mechanism to confer specificity for free 5'-ends,” Nucleic Acids Research, 2012, 40(10): 4507-4519 and Tsutakawa et al., “Human flap endonuclease structures, DNA double-base flipping, and a unified understanding of the FEN1 superfamily,” Cell, 2011, 145(2): 198-211, each of which is incorporated herein by reference. An exemplary flap endonuclease is FEN1, which can be represented by the following amino acid sequence:

Flap endonucleases may also include any FEN1 variants, mutants, or other flap endonuclease orthologs, homologs, or variants. Examples of non-limiting FEN1 variants are:

In various embodiments, the prime editing factor fusion proteins contemplated herein 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 to any of the above sequences. % 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 sequences disclosed above. Flap endonuclease variants may be included.

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 (SEQ ID NO:3 865).

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

Accession number NM_011907

MSEPPRAETFVFLDLEATGLPNMDPEIAEISLFAVHRSSLENPERDDSGSLVLPRVLDKLTLCMCPERPFTAKASEITGLSSESLMHCGKAGFNGAVVRTLQGFLSRQEGPICLVAHNGFDYDFPLLCTELQRLGAHLPQDTVCLDTLPALRGLDRAHSHGTRAQGRKSYSLASLFHRYFQAEPSAAHSAEGDVHTLLLIFLHRAPELLAWADEQARSWAHIEPMYVPPDGPSLEA (SEQ ID NO: 3866).

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

Accession number NM_001107580

MSEPLRAETFVFLDLEATGLPNMDPEIAEISLFAVHRSSLENPERDDSGSLVLPRVLDKLTLCMCPERPFTAKASEITGLSSEGLMNCRKAAFNDAVVRTLQGFLSRQEGPICLVAHNGFDYDFPLLCTELQRLGAHLPRDTVCLDTLPALRGLDRVHSHGTRAQGRKSYSLASLFHRYFQAEPSAAHSAEGDVNTLLLIFLHRAPELLAWADEQARSWAHIEPMYVPPDGPSLEA( SEQ ID NO: 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 a conserved nuclease domain across species from phages to humans. EXO1 The gene product exhibits both 5' exonuclease and 5' flap activities. In addition, EXO1 also contains unique 5' RNase H activity. It has a high affinity for processing gap, pseudo-Y structures and can use its inherited flap activity to disassemble Holliday contacts. Human EXO1 is involved in MMR and interacts directly with MLH1 and MSH2 Contains a conserved binding domain. EXO1 nucleolytic activity is positively stimulated by PCNA, MutSα (MSH2/MSH6 complex), 14-3-3, MRN, and 9-1-1 complex.

Exonuclease 1 (EXO1) Accession Number NM_003686 (Homo sapiens exonuclease 1 (EXO1), transcript variant 3) - Isoform A
(SEQ ID NO: 3868).

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

MGIS FRYMCILSGCDYLSSLRGIGLAKACKVLRLANNPDIVKVIKKIGHYLKMNITVPEDYINGFIRANNTFLYQLVFDPIKRKLIPLNAYEDDVDPETLSYAGQYVDDSIALQIALGNKDINTFEQIDDYNPDTAMPAHSRSHSWDDKTCQKSANVSSIWHRNYSPRPESGTVSDAPQLKENPSTVGVERVISTKGLNLPRKSSIVKRPRSAELSEDDLLSQYSLSFTKKTKKNS SEGNKSLSFSEVFVPDLVNGPTNKKSVSTPPRTRNKFATFLQRKNEESGAVVVPGTRSRFFCSSDSTDCVSNKVSIQPLDETAVTDKENNLHESEYGDQEGKRLVDTDVARNSSDDIPNNHIPGDHIPDKATVFTDEESYSFESSKFTRTISPPTLGTLRSCFSWSGGLGDFSRTPSPSPSTALQQFRRKSDSPTSLPENNMSDVSQLKSEESSDDESHPLREESQSQ ESGEFSLQSNASKLSQCSSKDSDSEESDCNIKLLDSQSDQTSKLRLSHFSKKDTPLRNKVPGLYKSSSADSLSTTKIKPLGPARASGLSKKPASIQKRKHHNAENKPGLQIKLNELWKNFGFKKDSEKLPPCKKPLSPVRDNIQLTPEAEEDIFNKPECGRVQRAIFQ (SEQ ID NO: 3869).

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

MGIS FRYMCILSGCDYLSSLRGIGLAKACKVLRLANNPDIVKVIKKIGHYLKMNITVPEDYINGFIRANNTFLYQLVFDPIKRKLIPLNAYEDDVDPETLSYAGQYVDDSIALQIALGNKDINTFEQIDDYNPDTAMPAHSRSHSWDDKTCQKSANVSSIWHRNYSPRPESGTVSDAPQLKENPSTVGVERVISTKGLNLPRKSSIVKRPRSELSEDDLLSQYSLSFTKKTKKNS SEGNKSLSFSEVFVPDLVNGPTNKKSVSTPPRTRNKFATFLQRKNEESGAVVVPGTRSRFFCSSDSTDCVSNKVSIQPLDETAVTDKENNLHESEYGDQEGKRLVDTDVARNSSDDIPNNHIPGDHIPDKATVFTDEESYSFESSKFTRTISPPTLGTLRSCFSWSGGLGDFSRTPSPSPSTALQQFRRKSDSPTSLPENNMSDVSQLKSEESSDDESHPLREESQSQ ESGEFSLQSSNASKLSQCSSKDSDSEESDCNIKLLDSQSDQTSKLRLSHFSKKDTPLRNKVPGLYKSSSADSLSTTKIKPLGPARASGLSKKPASIQKRKHHNAENKPGLQIKLNELWKNFGFKKDSEKLPPCKKPLSPVRDNIQLTPEAEEDIFNKPECGRVQRAIFQ (SEQ ID NO: 3870).

D. Inteins and split inteins
In some embodiments (e.g., delivery of prime editing factor in vivo using AAV particles), a polypeptide (e.g., deaminase or napDNAbp) or fusion protein (e.g., prime editing factor) is added to the N-terminal half and the C-terminal half. It is understood that it may be advantageous to deliver them separately and then allow their co-localization to re-form the complete protein (or fusion protein, as the case may be) within the cell. will be done. 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 the mechanism of protein trans-splicing.

Protein trans-splicing catalyzed by split inteins provides a purely enzymatic method for protein ligation. A split intein is essentially a continuous intein (eg, a mini-intein) that has been split into two pieces named N-intein and C-intein, respectively. The N and C inteins of a split intein can non-covalently associate to form an active intein and catalyze splicing reactions in essentially the same way that a continuous intein does. Split inteins are found in nature and have also been manipulated in the laboratory. As used herein, the term "split intein" refers to any intein in which one or more peptide bond cleavages exist between the N-terminal and C-terminal amino acid sequences, such that the N-terminal and C-terminal sequences are distinct. molecules, which can non-covalently reassemble or reconstitute into inteins that are functional for trans-splicing reactions. 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, a fission intein can be derived from a eukaryotic intein. In another aspect, the fission intein can be derived from a bacterial intein. In another aspect, the fission 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, a "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, the Ic comprises a stretch of 4 to 7 amino acid residues, at least 4 amino acids, 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 may include a sequence that is a modification of the C-terminal portion of a naturally occurring intein sequence. For example, Ic may include additional amino acid residues and/or mutated residues, so long as the inclusion of such additional and/or mutated residues does not render the In non-functional in trans-splicing. Preferably, the inclusion of 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 includes fluorescent groups, biotin, polyethylene glycol (PEG), amino acid analogs, unnatural amino acids, phosphate groups, glycosyl groups, radioisotope labels, among others. , and additional chemical moieties including pharmaceutical molecules. In other embodiments, the peptide linked to Ic may contain one or more chemically reactive groups including ketones, aldehydes, Cys residues, and Lys residues, among others. When an "intein splicing polypeptide (ISP)" is present, the N and C inteins of the split intein can non-covalently associate to form the active intein and catalyze the splicing reaction. As used herein, "intein splicing polypeptide (ISP)" refers to the portion of the amino acid sequence of a splitting intein that remains when Ic, In, or both are removed from the splitting intein. In certain embodiments, In includes ISP. In another embodiment, Ic comprises ISP. In yet another embodiment, the ISP is a separate peptide that is not covalently linked to either In or Ic.

Split inteins are created by engineering one or more splitting sites in unstructured loops or intervening amino acid sequences between the 12 conserved beta strands found on the structure of mini-inteins. can be generated from. Some of the location of the cleavage site within the inter-beta strand region, provided that the generation of cleavages will not interrupt the structure of the intein, particularly the structured beta strand, to a sufficient degree that protein splicing activity is lost. flexibility can exist.

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 the trans-splicing reaction (together N and C intein) excises the two intein sequences and connects the two extein sequences by a peptide bond. Protein trans-splicing, an enzymatic reaction, can work with very low (eg, micromolar) concentrations of protein and can be carried out under physiological conditions.

An example array is:

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 fission intein is the Ssp DnaE intein, which contains two subunits, DnaE-N and DnaE-C. The two different 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 fission intein of Synechocytis sp. PCC6803 that can direct the trans-splicing of two separate proteins, each containing 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 the entire 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.

Additionally, protein splicing in trans has been described in vivo and in vitro (Shingledecker, et al.,[0076]Gene 207:187(1998), Southworth, et al., EMBO J.17:918(1998). ;Mills,et al.,Proc.Natl.Acad.Sci.USA,95:3543-3548(1998);Lew,et al.,J.Biol.Chem.,273:15887-15890(1998);Wu, et al.,Biochim.Biophys.Acta 35732:1(1998b),Yamazaki,et al.,J.Am.Chem.Soc.120:5591(1998),Evans,et al.,J.Biol.Chem.275 :9091(2000);Otomo,et al.,Biochemistry 38:16040-16044(1999);Otomo,et al.,J.Biolmol.NMR 14:105-114(1999);Scott,et al.,Proc. Natl. Acad. Sci. USA 96:13638-13643 (1999)), provides an opportunity to express a protein as two inactive fragments that subsequently undergo ligation to form a functional product. . For example, as shown in Figures 66 and 67 for the formation of a complete PE fusion protein from two separately expressed halves.

E. RNA-protein interaction domain
In various embodiments, two separate protein domains (e.g., a Cas9 domain and a polymerase domain) are assembled into a functional complex (two can be co-localized with each other to form (similar to the function of fusion proteins containing separate protein domains). Such systems generally define one protein domain by an "RNA-protein interaction domain" (also known as an "RNA-protein recruitment domain") and another by an RNA-protein interaction domain, e.g. a particular hairpin. The structure is tagged with an "RNA-binding protein" that specifically recognizes and binds to it. These types of systems can be exploited to colocalize domains of prime editing factors and to recruit additional functionality to prime editing factors, such as UGI domains. In one example, MS2 tagging technology is based on the natural interaction of the MS2 bacteriophage coat protein ("MCP" or "MS2cp") with a stem-loop or hairpin structure, or "MS2 hairpin," present on the phage's genome. based on. In the case of the MS2 hairpin, it is recognized and bound by the MS2 bacteriophage coat protein (MCP). Therefore, in one exemplary scenario, a deaminase-MS2 fusion may recruit a Cas9-MCP fusion.

Reviews of other modular RNA-protein interaction domains are available in the art, for example, by 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 transcription synthetic programs with CRISPR RNA scaffolds,” Cell,2015,Vol.160:339-350. There is. Each of these is incorporated herein by reference in its entirety. Other systems include the PP7 hairpin, which specifically recruits PCP proteins, and the "com" hairpin, which specifically recruits Com proteins. See Zalatan et al.

The nucleotide sequence of the MS2 hairpin (or equivalently referred to as "MS2 aptamer") is: GCCAACATGAGGATCACCCATGTCTGCAGGGCC (SEQ ID NO : 763 ).

The amino acid sequence of MCP or MS2cp is:
GSASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVATQTVGGEELPVAGWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY (SEQ ID NO : 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 inhibitor:

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

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

G. Additional PE elements
In certain embodiments, the prime editing factors described herein can include inhibitors of base repair. The term "inhibitor of base repair" or "IBR" refers to a protein that can inhibit the activity of a nucleic acid repair enzyme, such as a base excision repair enzyme. In some embodiments, the IBR is an inhibitor of OGG base excision repair. In some embodiments, the IBR is an inhibitor of base excision repair (“iBER”). Exemplary inhibitors of base excision repair include inhibitors of APE1, Endo III, Endo IV, Endo V, Endo VIII, Fpg, hOGG1, hNEIL1, T7 EndoI, T4PDG, UDG, hSMUG1, and hAAG. In some embodiments, the IBR is an inhibitor of Endo V or hAAG. In some embodiments, the IBR is an iBER that can be a small molecule or peptide inhibitor of a catalytically inactive glycosylase or a catalytically inactive dioxygenase, or an 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 the AlkBH enzyme. In some embodiments, the IBR is an iBER that includes catalytically inactive TDG or catalytically inactive MBD4. An exemplary catalytically inactive TDG is the N140A variant 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 editing factors provided in this disclosure.

OGG (human)

MPARALLPRRMGHRTLASTPALWASIPCPRSELRLDLVLPSGQSFRWREQSPAHWSGVLADQVWTLTQTEEQLHCTVYRGDKSQASRPTPDELEAVRKYFQLDVTLAQLYHHWGSVDSHFQEVAQKFQGVRLLRQDPIECLFSFICSSNNNIARITGMVERLCQAFGPRLIQLDDVTYHGFPSLQALAGPEVEAHLRKLGLGYRARYVSASARAILEEQGG LAWLQQLRESSYEEAHKALCILPGVGTKVADCICLMALDKPQAVPVDVHMWHIAQRDYSWHPTTSQAKGPSPQTNKELGNFFRSLWGPYAGWAQAVLFSADLRQSRHAQEPPAKRRKGSKGPEG (SEQ ID NO: 3937 )

MPG (human)

MVTPALQMKKPKQFCRRMGQKKQRPARAGQPHSSSDAAQAPAEQPHSSSDAAQAPCPRERCLGPPTPGPYRSIYFSSPKGHLTRLGLEFFDQPAVPLARAFLGQVLVRRLPNGTELRGRIVETEAYLGPEDEAAHSRGGRQTPRNRGMFMKPGTLYVYIIYGMYFCMNISSQGDGACVLLRALEPLEGLETMRQLRSTLRKGTASRVLKDRELCSGPSKLCQALAIN KSFDQRDLAQDEAVWLERGPLEPSEPAVVAAARVGVGHAGEWARKPLRFYVRGSPWVSVVDRVAEQDTQA (SEQ ID NO: 3938 )

MBD4(human)

MGTTGLESLSLGDRGAAPTVTSSERLVPDPPNDLRKEDVAMELERVGEDEEQMMIKRSSECNPLLQEPIASAQFGATAGTECRKSVPCGWERVVKQRLFGKTAGRFDVYFISPQGLKFRSKSSLANYLHKNGETSLKPEDFDFTVLSKRGIKSRYKDCSMAALTSHLQNQSNNSNWNLRTRSKCKKDVFMPPSSSSELQESRGLSNFTSTHLLLKEDEGVDDVN FRKVRKPKGKVTILKGIPIKKTKKGCRKSCSGFVQSDSKRESVCNKADAESEPVAQKSQLDRTVCISDAGACGETLSVTSEENSLVKKKERSLSSGSNFCSEQKTSGIINKFCSAKDSEHNEKYEDTFLESEEIGTKVEVVERKEHLHTDILKRGSEMDNNCSPTRKDFTGEKIFQEDTIPRTQIERRKTSLYFSSKYNKEALSPPRRKAFKWTPPRSPFNLVQKKWTPPRSPFNLVQ ETLFHDPWKLLIATIFLNRTSGKMAIPVLWKFLEKYPSAEVARTADWRDVSELLKPLGLYDLRAKTIVKFSDEYLTKQWKYPIELHGIGKYGNDSYRIFCVNEWKQVHPEDHKLNKYHDWLWENHEKLSLS (SEQ ID NO: 3871)

TDG (human)

MEAENAGSYSLQQAQAFYTFPFQQLMAEAPNMAVVNEQQMPEEVPAPAPAQEPVQEAPKGRKRKPRTTEPKQPVEPKKPVESKKSGKSAKSKEFREGGRI LVQKLQKYQPRIAVFNGKCIYEIFSKEVFGVKVKNLEFGLQPHKIPDTETLCYVMPSSSARCAQFPRAQDKVHYYIKLKDLRDQLKGIERNMDVQEVQYTFDLQLAQEDAKKMAVKEEKYDPGYEAAYGGAYGENPCSSEPCGFSSNGLIESVELRGESAFSGIPNGQWMTQSFTDQIPSFSNHCGTQEQEEESHA (Sequence number 3872 )

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

Examples of protein domains that can be used to fuse prime editing factors or components thereof (eg, napDNAbp domains, polymerase domains, or NLS domains) include, without limitation, epitope tags, and reporter gene sequences. Non-limiting examples of epitope tags include histidine (His) tag, V5 tag, FLAG tag, influenza hemagglutinin (HA) tag, Myc tag, VSV-G tag, and thioredoxin (Trx) tag. Examples of reporter genes are glutathione-5-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), beta-galactosidase, beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed , autofluorescent proteins including, but not limited to, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and blue fluorescent protein (BFP). Prime editing factors can be fused to genetic sequences encoding proteins or fragments of proteins that bind to DNA molecules or bind to other cellular molecules, such as maltose binding protein (MBP), S-tag, Lex A DNA-binding domain (DBD). fusions, GAL4 DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions. Additional domains that may form part of the prime editing factor are described in US 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 biotin carboxylase carrier protein (BCCP) tag, myc tag, calmodulin tag, FLAG tag, hemagglutinin (HA) tag, polyhistidine tag, also referred to as histidine tag or His tag, maltose binding protein (MBP) tag, nus tag, glutathione-S-transferase (GST) tag, green fluorescent protein (GFP) tag, thioredoxin tag, S tag, Softag (e.g. Softag 1, Softag 3), strep tag, biotin ligase tag , FlAsH tag, V5 tag, and SBP tag. Additional suitable sequences will be apparent to those skilled in the art. In some embodiments, the fusion protein includes 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 alter the intracellular half-life of PE. In certain embodiments involving two or more vectors (e.g., vector systems in which components described herein are encoded on two or more separate vectors), the activity of the PE system depends on whether the vector is delivered or not. can be temporally controlled by controlling the timing of For example, in some embodiments, a vector encoding a nuclease system can deliver PE prior to a vector encoding a template. In other embodiments, a vector encoding PEgRNA can deliver a guide prior to a vector encoding a PE system. In some embodiments, the PE system and the vector encoding PEgRNA are delivered simultaneously. In certain embodiments, co-delivered vectors temporally deliver, eg, PE, PEgRNA, and/or second strand guide RNA components. In a further embodiment, the RNA transcribed from the coding sequence on the vector (e.g., a nuclease transcript) further comprises at least one element capable of altering the intracellular half-life of the RNA and/or modulating translational control. may be included. In some embodiments, the half-life of RNA can be increased. In some embodiments, the half-life of RNA can be decreased. In some embodiments, the element may be capable of increasing RNA stability. In some embodiments, the element may be capable of decreasing RNA stability. In some embodiments, the element can be within the 3'UTR of the RNA. In some embodiments, the element may include a polyadenylation signal (PA). In some embodiments, the element may include a cap, eg, an upstream mRNA or PEgRNA end. In some embodiments, the RNA can be free of PA, so that it undergoes more rapid degradation within the cell after transcription. In some embodiments, the element can include at least one AU-rich element (ARE). AREs can be bound by ARE-binding proteins (ARE-BPs) 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 can include 50 to 150 nucleotides in length. In some embodiments, the ARE can include at least one copy of the sequence AUUUA. In some embodiments, at least one ARE can be added to the 3'UTR of the RNA. In some embodiments, the element can be woodchuck hepatitis virus (WHP).

Post-transcriptional regulatory elements (WPREs) that generate tertiary structure to enhance expression from transcripts. In a further embodiment, the element is a modified and/or truncated WPRE sequence capable of enhancing expression from the transcript, e.g. Zufferey et al., J Virol, 73(4): 2886-92 (1999) and Flajolet et al., J Virol, 72(7): 6175-80 (1998). In some embodiments, the WPRE or equivalent may be added to the 3'UTR of the RNA. In some embodiments, elements may be selected from other RNA sequence motifs enriched in either fast or slow decaying transcripts.

In some embodiments, vectors encoding PE or PEgRNA can be self-destructed by cleavage of target sequences present on the vector by the PE system. Cleavage may prevent continued transcription of PE or PEgRNA from the vector. Although transcription can occur for any amount of time on a linearized vector, the expressed transcript or protein that undergoes intracellular degradation will be off-target without continuous supply from the expression of the encoding vector. It will have less time to take effect.

[6] PEgRNA
The prime editing system described herein contemplates the use of any suitable PEgRNA. By the use of specially constructed guide RNAs containing reverse transcription (RT) template sequences encoding the desired nucleotide changes, the inventors have demonstrated that the mechanism of target-primed reverse transcription (TPRT) can be precisely and We have discovered that it can be utilized or adapted to perform general-purpose CRISPR/Cas-based genome editing. Since the RT template sequence can be provided as an extension of a standard or conventional guide RNA molecule, this application refers to this specially constructed guide RNA as "extended guide RNA" or "PEgRNA." This application contemplates any suitable configuration or arrangement for the elongated guide RNA.

PEgRNA architecture Figure 3A shows one embodiment of an elongated guide RNA that can be used in the prime editor system disclosed herein, but the existing guide RNA (green area) has a ~20nt protospacer sequence and a gRNA core. region and binds with napDNAbp. In this embodiment, the guide RNA includes an extended RNA segment at the 5' end, ie, 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 Figures 1A-1B, a hybrid of the RT primer binding site with the free 3' end formed after nicking is formed on the non-target strand of the R-loop, thereby allowing DNA in the 5'-3' direction. Reverse transcriptase is primed for polymerization.

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 position of intramolecular RNA extension is not in the protospacer sequence of the guide RNA. In another embodiment, the location of intramolecular RNA extension is in the gRNA core. In yet another embodiment, the location of 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, an intramolecular RNA extension is inserted downstream from the 3' end of the protospacer sequence. In another embodiment, the intramolecular RNA extension comprises 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 the 3' end of the protospacer sequence. 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.

In other embodiments, the intramolecular RNA extension is inserted into gRNA, which refers to a portion of the guide RNA that corresponds to or includes tracrRNA, which is linked to the Cas9 protein or its equivalent (i.e., a different napDNAbp). bind to and/or interact with. Preferably, the insertion of an intramolecular RNA extension does not or only minimally interferes with the interaction of the tracrRNA portion with napDNAbp.

The length of RNA extension can be any useful length. In various embodiments, the RNA elongation 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 in length. 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.

The RT template sequence can also be of any suitable length. For example, the RT template sequence has a length of 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 The nucleotides can be 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 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 in length, 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 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 in length. 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.

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 has one or more nucleotide changes. include. 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 Figure 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 with 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 can be removed by a 5' flap endonuclease (e.g., FEN1) and the single-stranded DNA product, now hybridized to the endogenous target strand, is ligated to A mismatch is created between the strand and the newly synthesized strand. Mismatches may be resolved by cell-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 replaces the 5' flap species and overlaps the site to be edited.

In various embodiments of the elongated guide RNA, the reverse transcription template sequence may encode a single-stranded DNA flap that is complementary to the endogenous DNA sequence adjacent to the nick site, where the single-stranded DNA flap is Contains desired nucleotide changes. A single-stranded DNA flap may replace endogenous single-stranded DNA at the nick site. The endogenous single-stranded DNA displaced at the nick site has a 5' end and can form an endogenous flap, which can be excised by the cell. In various embodiments, removing the 5' end endogenous flap reduces hybridization of the single stranded 3' DNA flap with the corresponding complementary DNA strand and the desired nucleotide changes that the single stranded 3' DNA flap has. Excision of the 5' endogenous flap may help drive product formation, where it promotes incorporation or assimilation into the target DNA.

In various embodiments of the elongated guide RNA, cellular repair of the single-stranded DNA flap results in the incorporation of the desired nucleotide changes, 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 +2 from the nick site. +20, +1~+25, +1~+30, +1~+35, +1~+40, +1~+45, +1~+50, +1~+55, +1~+100 , +1 to +105, +1 to +110, +1 to +115, +1 to +120, +1 to +125, +1 to +130, +1 to +135, +1 to +140, + 1~+145, +1~+150, +1~+155, +1~+160, +1~+165, +1~+170, +1~+175, +1~+180, +1~ It is incorporated on the editing window which is +185, +1 to +190, +1 to +195, or +1 to +200.

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, the guide sequence is any polynucleotide sequence that has sufficient complementarity with the target polynucleotide sequence to hybridize with the target sequence and that napDNAbp (e.g., Cas9, Cas9 homolog, or Cas9 variant) It is directed toward sequence-specific binding to a target sequence. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence is about 50%, 60%, 75% when optimally aligned using a suitable alignment algorithm. %, 80%, 85%, 90%, 95%, 97.5%, 99% or higher. Optimal alignment may be determined using any suitable algorithm for aligning arrays, non-limiting examples of which algorithms include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, the Burrows-Wheeler Transform (e.g. , Burrows Wheeler Aligner), ClustalW, Clustal (available at sourceforge.net). In some embodiments, the guide sequence has a length of 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 or longer.

In some embodiments, the guide sequence is less than or shorter than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12 nucleotides in length. The ability of a guide sequence to direct sequence-specific binding of a prime editing element (PE) to a target sequence may be assessed by any suitable assay. For example, a prime editing element (PE) component (including a guide sequence to be tested) can be prepared by transfection with a vector encoding a prime editing element (PE) component disclosed herein. Subsequent provision of the corresponding target sequence to a host cell may be performed, such as by assessment of preferential cleavage within the target sequence, such as by Surveyor assay as described herein. Similarly, cleavage of a target polynucleotide sequence in vitro provides the target sequence, a component of the prime editing element (PE) (including the guide sequence to be tested), and a regulatory guide sequence distinct from the test guide sequence. The reaction between the test guide sequence and the control guide sequence may be evaluated by comparing the rate of binding or cleavage at the target sequence. Other assays may be performed and will occur to those skilled in the art.

The guide sequence may be selected to target any target sequence. In some embodiments, the target sequence is a sequence within the genome of the cell. Exemplary target sequences include sequences that are unique in the target genome. For example, for S.pyogenes Cas9, a unique target sequence in the genome may encompass a Cas9 target site of the form MMMMMMMMNNNNNNNNNNNNXG G , where N is A, G, T, or C. ; and X can be anything). A unique target sequence in the genome may encompass an S.pyogenes Cas9 target site of the form MMMMMMMMMNNNNNNNNNNNXG G , where NNNNNNNNNNNXG ( N is A, G, T, or C; and X is 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 include a S. thermophilus CRISPR1 Cas9 target site of the form MMMMMMMMMMNNNNNNNNNNXXAGAAW (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 the genome may encompass a Cas9 target site of the form MMMMMMMMNNNNNNNNNNNNXGGX G , where NNNNNNNNNNNNNXGGX G( N is A, G, T, or C; and X can be anything). A unique target sequence in the genome may encompass an S.pyogenes Cas9 target site of the form MMMMMMMMMNNNNNNNNN ). In each of these sequences, "M" may be A, G, T, or C and need not be taken into account in identifying the sequence as unique.

In some embodiments, the guide sequence is selected to reduce the degree of secondary structure within the guide sequence. Secondary structure may be determined by any suitable polynucleotide folding algorithm. Some programs are based on calculating the 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 RNA fold developed at the Institute for Theoretical Chemistry at the University of Vienna, which uses a centroid structure prediction algorithm (for example , A. R. Gruber et al., 2008, Cell 106(1):23-24; and P. A. Carr and GM Church, 2009, Nature Biotechnology 27(12):1151-62). Additional algorithms may be found in US Application No. 61/836,080; Broad Reference BI-2013/004A) (incorporated herein by reference).

Generally, a tracr mate sequence induces one or more of: (1) excision of the guide sequence flanked by the tracr mate sequence in cells containing the corresponding tracr sequence; and (2) formation of a complex at the target sequence. To facilitate this, the complex includes a tracr mate sequence hybridized to the tracr sequence. Generally, the degree of complementarity is by reference to the optimal alignment of the tracr mate sequences and the tracr sequences along the shorter length of the two sequences. Optimal alignment may be determined by any suitable alignment algorithm and may also constitute secondary structure, such as self-complementarity, within the sequence of either the tracr or tracr mate sequences. In some embodiments, the degree of complementarity between the tracr sequence and the tracr mate sequence is about 25%, 30%, 40%, 50% when optimally aligned along their two shorter lengths. %, 60%, 70%, 80%, 90%, 95%, 97.5%, 99% or higher. In some embodiments, the tracr sequence has a length of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50 or more nucleotides or longer. In some embodiments, a tracr sequence and a 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. Preferred loop-forming sequences for use in hairpin structures are 4 nucleotides in length and most preferably have the sequence GAAA. However, longer or shorter loop sequences may also be used as may alternative sequences. The sequence preferably includes a nucleotide triplet (eg, AAA) and an additional nucleotide (eg, C or G). Examples of loop-forming sequences include CAAA and AAAG. In embodiments of the invention, the transcript or transcribed polynucleotide sequence has at least two or more hairpins. In one preferred embodiment, the transcript has 2, 3, 4 or 5 hairpins. In a further embodiment of the invention, the transcript has at most 5 hairpins. In some embodiments, the single transcript further includes a transcription termination sequence; preferably it includes a polyT sequence, eg, 6 T nucleotides. Further non-limiting examples of single polynucleotides that include a guide sequence, a tracr mate sequence, and a tracr sequence are as follows (listed 5'→3'), where "N" is a base of the guide sequence. , the lowercase letters in the first block represent the tracr block sequence, and the lowercase letters in the second block represent the tracr sequence, and the last polyT sequence represents the transcription terminator: (1) NNNNNNNNGTTTTTGTACTCTCAAGATTTAGAAATAAATCTTGCAGAAGCTACAAAGATAAGGCTTCATGCCGAAATCAACACCCTGTCATTTTATGGCAGGGTGTTTTCGTTATTTAATTTTTT (SEQ ID NO: 216 );

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

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

(4) NNNNNNNNNNNNNNNNNNNNgttttagagctaGAAAtagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcTTTTTT (SEQ ID NO: 219);

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

(6) NNNNNNNNNNNNNNNNNNNNgttttagagctagAAATAGcaagttaaaataaggctagtccgttatcaTTTTTTTT (SEQ ID NO: 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 includes the tracr mate sequence.

Any of the fusion proteins as disclosed herein comprising a Cas9 domain and a single-stranded DNA binding protein can be targeted to a target site (e.g., a site containing a point mutation to be edited). It will be clear to those skilled in the art that it is typically necessary to co-express the fusion protein together with a guide RNA (eg, sgRNA). As described in more detail elsewhere herein, the guide RNA typically includes a tracrRNA framework that allows for Cas9 binding and a sequence-specific Cas9:nucleic acid editing enzyme/domain fusion. and a guide sequence attached to the 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 those depicted in Figure 3E.

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

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

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

In one embodiment, PEgRNAs can be designed with a pol III promoter to improve expression of longer length PEgRNAs with larger extended arms. sgRNAs are typically expressed from the U6 snRNA promoter. This promoter recruits pol III to express 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 at the levels required for efficient genome editing. Additionally, pol III stalls or terminates at stretches of U's, 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 sgRNAs. However, these promoters are typically partially transcribed, resulting in an extra sequence 5' of the spacer in the expressed PEgRNA, which is significantly reduced in a site-dependent manner. Cas9:sgRNA activity has been shown to result in increased Cas9:sgRNA activity. In addition, pol III-transcribed PEgRNAs can easily terminate at 6-7 extended U's, whereas PEgRNAs transcribed from pol II or pol I will require different termination signals. Frequently, such signals will also result in polyadenylation, leading to unwanted export of PEgRNA from the nucleus. Similarly, RNAs expressed from pol II promoters (such as pCMV) are typically 5' capped, which also results in their nuclear export.

Previously, Rinn and co-workers 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 the ENE element ¹⁸⁴ from human MALAT1 ncRNA, the PAN ENE element ¹⁸⁵ from KSHV, or the 3' box ¹⁸⁶ from U1 snRNA. Remarkably, 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 expression of longer PEgRNAs.

In addition, a series of methods have been designed for the cleavage of the portion of the pol II promoter that would be transcribed as part of PEgRNA, including self-cleaving ribozymes (Hammerhead ¹⁸⁸ , Pistol ¹⁸⁹ , Hatchet ¹⁸⁹ , Hairpin ¹⁹⁰ , VS ¹⁹¹ , Twister ¹⁹² , or Twister Sister ¹⁹² ribozymes) or other self-cleaving elements that process the transcribed guide, or a hairpin ¹⁹³ that is recognized by Csy4 and also leads to guide processing. has been done. 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 elements. It is also anticipated that circularizing PEgRNA into the form of circular intronic RNA (ciRNA) may also lead to enhanced RNA expression and stability, as well as nuclear localization ¹⁹⁴ .

In various embodiments, PEgRNA may include various elements above, as exemplified by the sequences below.

Non-limiting example 1 - PEgRNA expression platform consisting of pCMV, Csy4 hairpin, PEgRNA, and MALAT1 ENE
(Sequence number 223)

Non-limiting example 2 - PEgRNA expression platform consisting of pCMV, Csy4 hairpin, PEgRNA, and PAN ENE
(Sequence number 224)

Non-limiting example 3 - PEgRNA expression platform consisting of pCMV, Csy4 hairpin, PEgRNA, and 3×PAN ENE
(Sequence number 225)

Non-limiting example 4 - PEgRNA expression platform consisting of pCMV, Csy4 hairpin, PEgRNA, and 3' box
(Sequence number 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, PEgRNAs may be improved by introducing improvements to the backbone or core sequence. This can be done by introducing what is known.

The core, Cas9-bound PEgRNA backbone could probably also be improved to enhance PE activity. Several such approaches have already been demonstrated. Illustratively, the first pairing element (P1) of the scaffold contains a GTTTT-AAAAC (SEQ ID NO: 3939) pairing element. Such T(s) elongation has been shown to result in pol III pausing and premature termination of RNA transcripts. Rational mutation of one of the TA pairs in this P1 region to a GC pair was shown to enhance sgRNA activity, suggesting that this approach may also be feasible for PEgRNA. ¹⁹⁵ . In addition, increasing the length of P1 was also shown to enhance sgRNA folding leading to improved activity, ¹⁹⁵ which has been suggested as another avenue for improving PEgRNA activity. Ru. Examples of improvements to the core may include:

PEgRNA containing a 6nt 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, PEgRNAs may be improved by introducing modifications to the editing template region. As the size of the PEgRNA-templated insert increases, it is degraded by endonucleases, undergoes spontaneous hydrolysis, or folds into secondary structures (which cannot be reverse transcribed by RT). , or interfere with PEgRNA backbone folding and subsequent Cas9-RT binding). Consequently, modifications to the PEgRNA template may also be necessary to affect large insertions, such as whole gene insertions. Some strategies to do so include the incorporation of modified nucleotides within synthetic or semi-synthetic PEgRNAs (making the RNA more resistant to degradation or hydrolysis or adopting inhibitory secondary structures). ¹⁹⁶ . Such modifications will reduce RNA secondary structure in G-rich sequences, 8-aza-7-deazaguanosine; locked nucleic acids (LNA), which reduce degradation and enhance certain RNA secondary structures; 2'-O-methyl, 2'-fluoro, or 2'-O-methoxyethoxy modifications may be included that enhance RNA stability. Such modifications may also be included elsewhere in the PEgRNA to enhance stability and activity. Alternatively, or in addition, PEgRNA templates can be designed such that while encoding the desired protein product, they are also more likely to adopt simple secondary structures that cannot be folded by RT. Such a simple structure would act as a thermodynamic sink, but this reduces the likelihood of more complex structures that would prevent reverse transcription. Ultimately, it is also possible to split the template into two separate PEgRNAs. In such designs, PE can initiate transcription and recruit separate template RNAs to targeted sites via RNA binding proteins fused to Cas9 or RNA recognition elements on PEgRNA itself (such as the MS2 aptamer). It may also be used to The RT can either bind directly to this separate template RNA or initiate reverse transcription on the original PEgRNA before swapping to the second template. Such an approach not only prevents PEgRNA misfolding upon addition of long templates, but also prevents dissociation of Cas9 from the genome (possibly inhibiting PE-based long inserts) to generate long inserts. This may also allow long insertions.

In yet other embodiments, the PEgRNA introduces additional RNA motifs at the 5' and 3' ends of the PEgRNA, even at positions therebetween (e.g., in the gRNA core region or in the spacer). It may be improved by doing so. Several such motifs--such as PAN ENE from KSHV and ENE from MALAT1--were discussed above as means by which expression of longer PEgRNAs from non-pol III promoters could be terminated. These elements form an RNA triple helix that engulfs the polyA tails, resulting in their retention within the nucleus ^. However, by forming complex structures at the 3' end of PEgRNA that occlude the terminal nucleotides, these structures may also help prevent exonuclease-mediated degradation of 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 processing capacity or enhance PEgRNA activity by enhancing RT binding to DNA-RNA duplexes. Addition of native sequences bound by RT in its cognate retroviral genome can enhance RT activity ¹⁹⁹ . This may include the native primer binding site (PBS), polypurine tract (PPT), or kissing loop, which are involved in retroviral genome dimerization and transcription initiation ¹⁹⁹ .

Addition of dimerizing chiefs - such as kissing loops or GNRA tetraloop/tetraloop receptor pair ²⁰⁰ - at the 5' and 3' ends of PEgRNA can also result in efficient circularization of PEgRNA, stabilizing it. sex is improved. 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 impede PE activity. Short 5' or 3' extensions into the PEgRNA that form small toehold hairpins in the spacer region or along the primer binding site are also preferably included in intramolecular complementary regions along the length of the PEgRNA. Annealing (eg, the interaction that may occur between a spacer and a primer binding site) may also be competed for. Finally, kissing loops can also be used to recruit other template RNAs to genomic sites and to allow swapping of RT activity from one RNA to another. As an exemplary embodiment of the various secondary structures, the PEgRNA depicted in Figures 3D and 3E contains numerous secondary RNAs, including those in the terminal portions of the extended arms (i.e., e1 and e2) as shown. List the structures (which can be modified to become any region of PEgRNA).

Examples of improvements include, but are not limited to:

PEgRNA-HDV fusion
GGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCCTCTGCCATCAAAGCGTGCTCAGTCTGGGCCGGCATGGTCCCAGCCTCCTCGCTGGCGCCGGCTGGGCAACATGCTTCGGCATGGCGAATGGGACTTTTTTT (SEQ ID NO: 230)

PEgRNA-MMLV kissing loop
GGTGGGAGACGTCCCACCGGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCCTCTGCCATCAAAGCTTCGACCGTGCTCAGTCTGGTGGGAGACGTCCCACCTTTTTT (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 templates for switching secondary RNA-HDV fusions
TCTGCCATCAAAGCTGCGACCGTGCTCAGTCTGGTGGGAGACGTCCCACCGGCCGGCATGGTCCCAGCCTCCTCGCTGGCGCCGGCTGGGCAACATGCTTCGGCATGGCGAATGGGACTTTTTTT (SEQ ID NO: 234)

PEgRNA scaffolds can be further improved through directed evolution in a manner similar to how SpCas9 and prime editing elements (PEs) 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 to enhance PE activity at the site in question, reduce off-target activity, or both. Probably either. Ultimately, evolution of the PEgRNA backbone with the addition of other RNA motifs will almost always improve the activity of the fused PEgRNA compared to the unevolved fusion RNA. As an illustration, ^the evolution of allosteric ribozymes composed of c-di-GMP-I aptamers and hammerhead ribozymes has led to dramatically improved activity202, but this is because evolution of hammerhead-PEgRNA fusions has led to dramatically improved activity. suggesting that activity would also be improved. In addition, although Cas9 currently does not generally tolerate 5' extensions of sgRNAs, directed evolution could conceivably generate potent mutations that alleviate this intolerance, creating additional RNA motifs. will be allowed to be used.

This disclosure contemplates any such approach to further improve the efficacy of the prime editing system disclosed herein.

In various embodiments, it may be advantageous to limit the appearance of a contiguous sequence of T's from an extended arm, as a contiguous series of T's can limit the capacity for PEgRNA to be transcribed. For example, 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 , a string of at least 10 consecutive Ts, at least 11 consecutive Ts, at least 12 consecutive Ts, at least 13 consecutive Ts, at least 14 consecutive Ts, or at least 15 consecutive Ts, design PEgRNA. or at least be 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 undesired strings of consecutive T's on the PEgRNA extended arms may be avoided.

Split PEgRNA Design for Transprime Editing This disclosure also contemplates transprime editing. This refers to a modified version of prime editing that operates by separating PEgRNA into two separate molecules: the guide RNA and the tPERT molecule. The tPERT molecule is programmed to colocalize with the prime editor complex at the target DNA site, bringing the primer binding site and DNA synthesis template in trans to the prime editor. For example, see Figure 3G for an embodiment of transprime editing element (tPE). It consists of tPERT, which contains (1) a recruitment protein (RP)-PE:gRNA complex and (2) a primer binding site and a DNA synthesis template linked to an RNA-protein recruitment domain (e.g., a stem-loop or hairpin). A two-component system containing: The recruitment protein component of the RP-PE:gRNA complex recruits tPERT to the target site to be edited, thereby associating PBS and DNA synthesis template in trans with the prime editing factor. Stated differently, tPERT is engineered to contain (all or part of) an extended arm of PEgRNA that includes a primer binding site and a DNA synthesis template. One advantage of this approach is that it separates the extended arm of the PEgRNA from the guide RNA, thereby minimizing annealing interactions that tend to occur between the PBS of the extended arm and the spacer sequence of the guide RNA. .

A key feature of transprime editing is the ability of transprime editing factors to recruit tPERT to the site of DNA editing, thereby effectively colocalizing all of the PEgRNA functions at the site of prime editing. Recruitment involves incorporating an RNA-protein recruitment domain, such as the MS2 aptamer, into tPERT and the corresponding recruitment protein to a prime editing factor that can specifically bind to the RNA-protein recruitment domain (e.g., napDNAbp via a linker). or via a linker to a polymerase), thereby recruiting the tPERT molecule to the prime editor complex. The RP-PE:gRNA complex binds and nicks the target DNA sequence, as illustrated in the process described in Figure 3H. The recruitment protein (RP) then recruits tPERT to co-localize with the prime editing factor complex bound to the DNA target site, thereby causing the primer binding site located on tPERT to become on the nicked strand. and then allow a polymerase (eg, RT) to synthesize a single strand of DNA from the 5' end of tPERT to a DNA synthesis template positioned on tPERT.

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

PEgRNA Design Methods The present disclosure also relates to methods for designing PEgRNA.

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 illustrated and discussed herein, prime editing can be used, without limitation, to (a) incorporate changes in a nucleotide sequence that correct mutations, (b) incorporate protein and RNA tags, and (c) target immune epitopes. (d) incorporating an inducible dimerization domain into the protein; (e) incorporating or removing sequences to alter the activity of the biomolecule; (f) directing specific genetic changes. and (g) mutagenesis of the target sequence by using error-prone RT. In addition to these methods of generally inserting, changing, or deleting nucleotide sequences at the target site of interest, prime editing factors can be used to construct highly programmable libraries, as well as for cell data recording and lineage tracing. It can also be used to conduct research. For these various uses, as described herein, there may be specific design aspects that apply to the preparation of PEgRNA that are particularly useful for any given one of these applications.

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 to design suitable PEgRNAs and optionally a nicking sgRNA design guide for second site nicking. This embodiment provides a step-by-step set of instructions for designing PEgRNAs and nicking sgRNAs for prime editing, which takes into account one or more of the above considerations. The steps refer to the example shown in Figures 70A-70I.
1. Define target sequence and edits. Retrieve the sequence of the target DNA region (~200 bp) centered around the location of the desired edit (point mutation, insertion, deletion, or combination thereof). See Figure 70A.
2. Position the target PAM. Identify the PAM proximal to the desired editing location. PAMs can be identified on either strand of DNA proximal to the desired editing location. PAMs that are close to the edit position are preferred (i.e., the nick site is less than 30 nt from the edit position; or 29 nt, 28 nt, 27 nt, 26 nt, 25 nt, 24 nt, 23 nt, 22 nt, 21 nt, 20 nt, Protospacer that places a nick ≧30nt from the editing position And it is possible to incorporate editing using PAM. See Figure 70B.
3． Position the nick site. For each PAM considered, identify the corresponding nick site and on which strand. For the Sp Cas9 H840A nickase, cleavage occurs on the PAM-containing strand between the 5' third and fourth bases of the NGG PAM. All edited nucleotides must be 3' to the nick site. Therefore, a suitable PAM must be nicked 5' of 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 PEgRNA using PAM 1 only. See Figure 70C.
Four. Design the spacer array. The protospacer of Sp Cas9 corresponds to the 5' 20 nucleotides of NGG PAM on the PAM-containing strand. Efficient Pol III transcription initiation requires G to be the first nucleotide transcribed. If the first nucleotide of the protospacer is G, the spacer sequence of PEgRNA is simply a protospacer sequence. If the first nucleotide of the protospacer is not a G, the spacer sequence of the PEgRNA is G followed by the protospacer sequence. See Figure 70D.
Five. Design the primer binding site (PBS). The starting allele sequence is used to identify DNA primers on the PAM-containing strand. The 3' end of the DNA primer is the nucleotide just upstream of the nick site (ie, the 4th 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 is combined with a sequence containing ~40-60% GC content. can be used. 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, the starting allele sequence is used 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) encodes the designed edit and homology to the sequences flanking the edit. In one embodiment, these regions correspond to the DNA synthesis template of Figures 3D and 3E, where the DNA synthesis template includes an "editing template" and a "homologous arm." 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 (position +7 and beyond), it is recommended to use RT templates that extend at least 5 nt (preferably 10 nt or more) from the editing position 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 (≧5nt), incorporation of more 3' homology (~20nt or more) onto the RT template is recommended. Editing efficiency is typically compromised when the RT template encodes the synthesis of a G (corresponding to C on the RT template of PEgRNA) as the last nucleotide on the reverse transcribed DNA product. Avoiding G as the last synthesized nucleotide is recommended when designing RT templates, as many RT templates support efficient prime editing. 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 editing using RT templates 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 unedited strands upstream and downstream of the edit. The optimal nicking position is highly locus dependent and should be determined empirically. Generally, nicks placed 40 to 90 nucleotides 5' across from the nick induced by PEgRNA lead to higher editing yields and fewer indels. The nicking sgRNA has a spacer sequence that matches the 20nt protospacer on the starting allele. If the protospacer does not begin with a G, it has an addition of 5'G. See Figure 70H.
9. Design PE3b nicking sgRNA. If the PAM is present on the complementary strand and its corresponding protospacer overlaps the sequence targeted for editing, this editing 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 rather than the starting allele. The PE3b system operates efficiently when the edited nucleotide(s) falls within the seed region of the nicking sgRNA protospacer (~10 nt adjacent to the PAM). This prevents nicking of the complementary strand until after incorporation of the edited strand and prevents competition between PEgRNA and sgRNA for binding to target DNA. PE3b also avoids simultaneous nick generation on both strands, thus significantly reducing indel formation while maintaining high editing efficiency. The PE3b sgRNA should have a spacer sequence that matches the 20nt protospacer of the desired allele. With addition of 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. This disclosure contemplates variations on the step-by-step process described above, which may be derived therefrom by those skilled in the art.

[7] Applying targeted insertions, deletions, and base conversions that utilize prime editing to require double-stranded DNA breaks or donor DNA templates at the target locus in human cells In addition to the development of the prime editing system described herein as a new "search-and-replace" genome editing technology mediated by I planned it again. For example, as illustrated and discussed herein, prime editing involves (a) incorporating changes to the nucleotide sequence that correct for mutations, (b) incorporating protein and RNA tags, (c) incorporating immune epitopes into the protein of interest, (d) incorporating inducible dimerization domains into proteins; (e) incorporating or removing sequences to alter the activity of the biomolecule; (f) recombinase target sites to direct specific genetic changes. and (g) mutagenesis of a target sequence by using error-prone RT. In addition to these methods of generally inserting, changing, or deleting nucleotide sequences at the target site of interest, prime editing factors can be used to construct highly programmable libraries, as well as for cell data recording and lineage tracing. It can also be used to conduct research. The inventors also contemplated additional design features of PEgRNA aimed at improving the efficacy of prime editing. Still further, the inventors have conceived a method for successfully delivering prime editing factors using a vector delivery system that involves splitting napDNAbp using an intein domain.

These particular exemplary uses of prime editing are not intended to be limiting in any way. This application contemplates any use of prime editing, which generally involves some form of incorporation, removal, and/or modification of one or more nucleobases at a target site on a nucleotide sequence, such as genomic DNA.

For any of the illustrated uses of prime editing, any of the prime editing factors 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") is a nucleic acid program that complexes a target DNA molecule (into which it is desired to introduce a nucleotide sequence change) with an elongated guide RNA. It operates by contacting the type DNA binding protein (napDNAbp). Referring to Figure 1G, the elongated guide RNA includes an extension at the 3' or 5' end of the guide RNA or at an intramolecular position of the guide RNA and includes a desired nucleotide change (e.g., a single nucleotide change, an insertion, or (deletion). In step (a), the napDNAbp/elongated gRNA complex contacts the DNA molecule, and the elongated 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 at the target locus, thereby creating an available 3' end on one of the strands at the target locus. . In certain embodiments, the nick is created on the strand of DNA that corresponds to the R-loop strand, ie, the strand that is not hybridized to the guide RNA sequence, ie, the "non-target strand." However, nicks can be introduced into either of the strands. That is, the nick forms either the R-loop "target strand" (i.e., the strand that hybridizes to the protospacer sequence of the elongated gRNA) or the "non-target strand" (i.e., the single-stranded portion of the R-loop and the target strand (which is complementary to the 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. "primed RT to prime target"). ). In certain embodiments, the 3' end DNA strand hybridizes to a specific RT prime sequence of the extended portion of the guide RNA, ie, a "reverse transcriptase prime sequence." In step (d), reverse transcriptase is introduced (either as a fusion protein with napDNAbp or in trans), which instructs the DNA from the 3' end of the primed site towards the 5' end of the extended guide RNA. Synthesize the main chain. This is a single strand containing the desired nucleotide changes (e.g., single base changes, insertions, or deletions, or combinations thereof) that is otherwise homologous to the endogenous DNA at or adjacent to the nick site. Strands form DNA flaps. In step (e) napDNAbp and guide RNA are released. Steps (f) and (g) involve disassembly of the single-stranded DNA flap so that the desired nucleotide change is incorporated at 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 endogenous DNA sequence ( For example, by FEN1 or a similar enzyme provided in trans, as a fusion with a prime editing factor, or endogenously). Without being bound by theory, the cell's endogenous DNA repair and replication processes degrade mismatched DNA and incorporate nucleotide change(s) to form the desired modified product. The process can also be driven towards product formation by "second strand nicking" as illustrated in FIG. 1G or "temporal second strand nicking" as illustrated in FIG. 1I and discussed herein. .

The process of prime editing may introduce at least one or more of the following genetic changes: transversions, transitions, deletions, and insertions. Additionally, prime editing may be implemented for specific applications. For example, as illustrated and discussed herein, prime editing involves (a) incorporating changes to the nucleotide sequence that correct for mutations, (b) incorporating protein and RNA tags, (c) incorporating immune epitopes into the protein of interest, (d) incorporating inducible dimerization domains into proteins; (e) incorporating or removing sequences to modify the biomolecule's activity; (f) recombinase target sites to direct specific genetic changes. and (g) mutagenesis of a target sequence by using error-prone RT. In addition to these methods of generally inserting, changing, or deleting nucleotide sequences at the target site of interest, prime editing factors can be used to construct highly programmable libraries, as well as for cell data recording and lineage tracing. It can also be used to conduct research. The inventors also contemplated additional design features of PEgRNA aimed at improving the efficacy of prime editing. Still further, the inventors have conceived a method for successfully delivering prime editing factors using a vector delivery system that involves splitting napDNAbp using an intein domain.

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

In another embodiment, the schematic diagram in Figure 3F depicts a typical PEgRNA interaction with a double-stranded DNA target site and the concomitant production of a 3' single-stranded DNA flap containing the genetic change of interest. Illustrated. Double-stranded DNA is shown with the upper strand in the 3' to 5' orientation and the lower strand in the 5' to 3' orientation. The upper strand contains the "protospacer" and PAM sequences and is referred to as the "target strand." The complementary lower strand is referred to as the "non-target strand." Although not shown, the illustrated PEgRNA will be complexed with Cas9 or equivalent. As shown in the schematic diagram, the PEgRNA spacer anneals to a complementary region on the target strand called the protospacer, which is positioned 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 protospacer DNA and induces the formation of an R-loop in the region facing 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 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. Reverse transcriptase (e.g., provided in trans or in cis as a fusion protein attached to a Cas9 construct) is then encoded by the editing template (B) and the homology arm (C). Polymerize a single strand of DNA. Polymerization continues towards the 5' end of the extended arm. The polymerized strands of ssDNA form the ssDNA 3' end flap. This invades the endogenous DNA and displaces the corresponding endogenous strand (which is removed as a 5′ DNA flap of the endogenous DNA), as described elsewhere (e.g., as shown in Figure 1G). , the desired nucleotide edits (single nucleotide base pair changes, deletions, insertions (including entire genes) are incorporated by naturally occurring rounds of DNA repair/replication.

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

B. Mutation introduction using prime editing with error-prone RT
In various embodiments, the prime editing system (i.e., prime editing system) is used to perform targeted mutagenesis, i.e., to mutate only well-defined stretches of DNA on other DNA elements of the genome or cell. may include the use of error-prone reverse transcriptase enzymes. Figure 22 provides a schematic diagram of an exemplary process for introducing targeted mutagenesis by error-prone reverse transcriptase on the target locus, complexed with an elongated guide RNA. Nucleic acid programmed DNA binding protein (napDNAbp) is used. This process can be referred to as a form of prime editing for targeted mutagenesis. An extended guide RNA includes an extension at the 3' or 5' end of the guide RNA or at a position within the molecule 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 creating an available 3' end on one of the strands at the target locus. . In certain embodiments, the nick is created on the strand of DNA that corresponds to the R-loop strand, ie, 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 certain embodiments, the 3' end DNA strand hybridizes to a specific RT prime sequence of the extended portion of the guide RNA. In step (d), 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 mutagenized region. In step (e) napDNAbp and guide RNA are released. Steps (f) and (g) involve disassembling the single-stranded DNA flap (containing the mutagenized region) so that the desired mutagenized region is integrated into the target locus. This process directs desired product formation by removing the corresponding 5' endogenous DNA flap that forms once a 3' single-stranded DNA flap invades and hybridizes to complementary sequences on the other strand. can be driven to. The process can also be driven towards product formation by second strand nicking as illustrated in Figure IF. After endogenous DNA repair and/or replication processes, the mutagenized region becomes integrated on 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. As used herein, the term "error-prone" reverse transcriptase refers to a naturally occurring or another reverse transcriptase (e.g., a wild-type Refers to reverse transcriptase enzyme derived from 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 integrations. See Boutabout et al. (2001) “DNA synthesis fidelity by the reverse transcriptase of the yeast retrotransposon Ty1,” Nucleic Acids Res 29(11):2217-2222. This is incorporated herein by reference. Therefore, for the purposes of this application, the term "error-prone" refers to 1 error per 15,000 nucleobase integrations (6.7 × 10 ^-5 or higher), e.g., 1 error per 14,000 nucleobases (7.14 × 10 ^{- 5} or higher), 1 error per 13,000 nucleobases or fewer (7.7 x 10 ^-5 or higher), 1 error per 12,000 nucleobases or fewer (7.7 x 10 ^-5 or higher), 11,000 nucleobases or 1 error per 10,000 nucleobases or ^0.0001 or higher; 1 error per 10,000 ^nucleobases or 0.0001 or higher; 1 error per 9,000 nucleobases or 0.0001 or higher 1 error per 8,000 nucleobases or fewer (0.00013 or higher), 1 error per 7,000 nucleobases or fewer (0.00014 or higher), 1 error per 6,000 nucleobases or fewer (0.00016 or higher) ), 1 error in 5,000 nucleobases or fewer (0.0002 or higher), 1 error in 4,000 nucleobases or fewer (0.00025 or higher), 1 error in 3,000 nucleobases or fewer (0.00033 or higher), 1 error 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 RT with an error rate that is greater than 1 error per 250 nucleobases or fewer (0.004 or higher).

Various mutagenic RTs can be envisioned for the generation of mutagenized sequences using prime editing. Two such examples are Bordetella phage (Handa, S., et al. Nucl Acids Res 9711-25 (2018), which is incorporated herein by reference) and Legionella pneumophila (Arambula, D., et al. Proc. See Natl Acad Sci USA 8212-7 (2013), which is incorporated by reference). In the case of RT from Bordetella phage (brt), an auxiliary protein (bavd) is added to Cas9 and an additional RNA sequence is added to PEgRNA, either in trans or to improve binding of the mutagenic RT to the target site. may also need to be delivered (see Handa, S., et al. Nucl Acids Res 9711-25 (2018)). When using mutagenic RT, the PEgRNA template region can be enriched with adenosine or AAY codons to enhance diversity.

The amino acid sequence of mutagenic RT from Bordetella phage is provided below. Like other RTs disclosed herein, the Brt protein can be fused to 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 ancillary protein (Bavd). Supplementary proteins can be fused or provided in trans to the PE fusion protein. The amino acid sequence of Bavd auxiliary protein is provided as follows:

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

To reduce length, this PEgRNA additional sequence can be reduced in a variety of ways. For example, a PEgRNA addition sequence can be reduced to the following exemplary alternative addition sequences:

In other embodiments, PEgRNA additional sequences can also be mutated. For example, a PEgRNA add1 sequence can be mutated to the following exemplary alternative add sequences:

In various embodiments regarding 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. BiolChem, 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. (the entire content of this reference is incorporated by reference): (Incorporated):

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 tandem repeats have identical sequences to each other, base pairing between the two DNA strands can occur at multiple points on 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 repeating sequences, 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 harmful repeat codon triplets. In some embodiments of this use, Figure 23 provides a schematic representation of PEgRNA design to shorten or reduce trinucleotide repeat sequences by prime editing.

Prime editing can be implemented to shorten the triplet expansion region by nicking the region upstream of the triplet repeat region with a prime editing agent containing a dedicated PEgRNA targeted to the site to be cut. . The prime editing factor then creates a new DNA strand (ssDNA flap) based on PEgRNA as a template (i.e., its editing template) encoding a healthy number of triplet repeats (this depends on the specific gene and disease). Synthesize. A newly synthesized ssDNA strand containing a healthy triplet repeat sequence is also synthesized to include a short stretch of homology (i.e. homology arm) that matches the sequence flanking the other end of the repeat (shown in red). chain). Invasion of the newly synthesized strand and subsequent replacement of the endogenous DNA by the newly synthesized ssDNA flap leads to a truncated repeat allele.

Depending on the specific trinucleotide expansion disorder, defect-inducing triplet expansions can occur in "trinucleotide repeat expansion proteins." Trinucleotide repeat expansion proteins are a diverse set of proteins associated with susceptibility to developing trinucleotide repeat expansion disorders, presence of trinucleotide repeat expansion disorders, severity of trinucleotide repeat expansion disorders, or any combination thereof. be. 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 encodes the amino acid glutamine (Q). These disorders are therefore referred to as polyglutamine (polyQ) disorders and include: Huntington's disease (HD); spinobulbar muscular atrophy (SBMA); spinocerebellar ataxia (SCA1, 2, 3, types 6, 7, and 17); and dentate nucleus pallidum Louisian atrophy (DRPLA). The remaining trinucleotide repeat expansion disorders either do not involve the CAG triplet or the CAG triplet is not in the coding region of the gene and are therefore referred to as non-polyglutamine disorders. Non-polyglutamine disorders include fragile including.

Proteins associated with trinucleotide repeat expansion disorders may be selected based on the experimental association of proteins 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 increased or decreased in a population with a trinucleotide repeat expansion disorder relative to a population lacking the trinucleotide repeat expansion disorder. Differences in protein levels can 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 can be identified by obtaining gene expression profiles of protein-coding genes, using DNA microarray analysis, serial analysis of gene expression (SAGE), and quantitative real-time polymerase analysis. Genomic techniques are used, including but not limited to 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), MED15 (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 opposite strand (non-protein coding)), PPP2R2B (protein phosphatase 2 regulatory subunit B beta), ATXN7 (ataxin 7), TNRC6B (trinucleotide repeat containing 6B), TNRC6C (trinucleotide repeat containing 6C), CELF3 (CUGBP Elav-like family member 3), MAB21L1 (mab-21-like 1 (C. elegans)), MSH2 (mutS homolog 2 colon cancer non-polyposis 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 (transcriptional 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 (oncoprotein p53), ESR1 ( estrogen receptor 1), CGGBP1 (CGG triplet repeat binding protein 1), ABT1 (basic transcriptional 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-related ), KBTBD10 (containing kelch repeats and BTB(POZ) domain 10), MBNL1 (muscleblind-like (Drosophila)), RAD51 (RAD51 homolog (RecA homolog, E. coli) (S. cerevisiae)), NCOA3 (nuclear receptor core activator 3), ERDA1 (elongated repeat domain CAG/CTG1), TSC1 (tuberous sclerosis 1), COMP (cartilage oligomeric matrix protein), GCLC (glutamate-cysteine ligase catalytic subunit), RRAD (associated with diabetes). Ras related), MSH3 (mutS homolog 3 (E. coli)), DRD2 (dopamine receptor D2), CD44 (CD44 molecule (Indian blood type)), CTCF (CCCTC binding factor (zinc finger protein)), CCND1 ( cyclin D1), CLSPN (claspin homologue (Xenopus laevis)), MEF2A (myocyte enhancer factor 2A), PTPRU (protein tyrosine phosphatase receptor type U), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), TRIM22 (trisegmental type motif containing 22), WT1 (Wilms tumor 1), AHR (aryl hydrocarbon receptor), GPX1 (glutathione peroxidase 1), TPMT (thiopurine S-methyltransferase), NDP (Norie disease (pseudoglioma)), ARX (aristaless related homeobox), MUS81 (MUS81 endonuclease homolog (S. cerevisiae)), TYR (tyrosinase (oculocutaneous albinism IA)), EGR1 (early proliferative 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 14kDa (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 increase 2 (S.cerevisiae)), GLA (galactosidase alpha), CBL (Cas-Br-M (murine) homotropic retroviral transformation sequence), FTH1 (ferritin heavy chain polypeptide 1), IL12RB2 (interleukin-12 receptor beta 2), OTX2 (orthodenticle homeobox 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 (midbrain astrocyte-derived neurotrophic factor), TMEM158 (transmembrane protein 158 (gene/pseudogene)), and ENSG00000078687.

The prime editing factors disclosed herein can be used to reduce triplet repeat expansion regions of any of the disease proteins indicated above, including the following polyglutamine triplet expansion disease genes (these are , indicating specific locations of pathogenic repeats that can be removed in whole or in part by prime editing):

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

Prime editing can be implemented to reduce a triplet expansion region using PEgRNA with an editing template designed to delete at least one codon of the triplet expansion region. In other embodiments, to reach a healthy (i.e., not associated with disease producing) number of triplet repeats, the PEgRNA for use in prime editing is at least 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20 , or 21, or 22, or 23, or 24, or 25, or 26, or 27, or 28, or 29, or 30, or 31, or 32, or 33, or 34, or 35, or 36, or 37, or 38, or 39, or 40, or 41, or 42, or 43, or 44, or 45, or 46, or 47, or 48, or 49, or 50, or 51, or 52, or 53; or 54, or 55, or 56, or 57, or 58, or 59, or 60, or 61, or 62, or 63, or 64, or 65, or 66, or 67, or 68, or 69, or 70 , or 71, or 72, or 73, or 74, or 75, or 76, or 77, or 78, or 79, or 80, or 81, or 82, or 83, or 84, or 85, or 86, or Triplet expansion region with 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 used to delete from.

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 disease producing) number of triplet repeats, the PEgRNA for use in prime editing is at least 1, or 2, or 3, or for deletion of 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. used for.

In other embodiments, to reach a healthy (i.e., not associated with disease producing) number of triplet repeats, the PEgRNA for use in prime editing is at least 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or more codons are used to delete from the triplet expansion region.

Prime editing can be performed on any triplet expansion region, such as Budworth et al., “A Brief History of Triplet Repeat Diseases,” Methods Mol Biol, 2013, 1010:3-17, US20011/00165540A1 (animal trinucleotide repeat expansion disorders). (genome editing of genes associated with), US2016/0355796A1 (compositions of the crispr-cas system and methods of use for nucleotide repeat disorders);

In various embodiments, the present disclosure provides prime editing constructs suitable for use in cells having trinucleotide repeat expansions in defective genes, comprising: (a) a prime editing factor fusion comprising napDNAbp and reverse transcriptase; (b) comprises a PEgRNA comprising a spacer sequence that targets the trinucleotide repeat expansion region and an extended arm containing an editing template encoding removal of the trinucleotide repeat expansion region.

In various other embodiments, the present disclosure provides methods for deleting all or a portion of a trinucleotide repeat expansion region of a defective gene in a cell using prime editing, wherein a prime comprising napDNAbp and reverse transcriptase is used. contacting a cell with a PEgRNA comprising an editing factor fusion and an extended arm comprising a spacer sequence targeting a trinucleotide repeat expansion region and an editing template encoding 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, it is a tetranucleotide repeat, a pentanucleotide repeat, or a hexanucleotide repeat.

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 other aspects, the disclosure provides methods for making fusion proteins comprising a peptide of interest and one or more peptide tags, the method comprising: combining a target nucleotide sequence encoding a protein with one or more peptide tags; contacting a second nucleotide sequence encoding a tag with a prime editing agent configured to insert therein a recombinant nucleotide sequence encoding a fusion protein comprising a protein fused to the protein tag. .

In various embodiments, the target nucleotide sequence is a particular gene of interest on genomic DNA. The gene of interest may encode a protein of interest (eg, a receptor, enzyme, therapeutic protein, membrane protein, transport protein, signal transduction protein, or immunological protein, etc.). The gene of interest may also encode RNA molecules, including messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA), antisense RNA, guide RNA, microRNA ( miRNA), small interfering RNA (siRNA), and cell-free RNA (cfRNA).

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

Peptide tags can also be affinity tags (Kimple et al., “Overview of Affinity Tags for Protein Purification,” Curr Protoc Protein Sci, 2013, 73: Unit-9.9 Table 9.9.1, which is incorporated herein by reference).

In specific embodiments, peptide tags can include ^His6 tags, FLAG tags, V5 tags, GCN4 tags, HA tags, Myc tags, FIAsH/ReAsH tags, sortase substrates, pike clamps.

In various embodiments, peptide tags can be used for 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 self-processing polypeptide domains found in organisms from all domains of life. Inteins (intervening proteins) carry out a unique self-processing event known as protein splicing. In this, it excises itself from the larger precursor polypeptide by cleavage of two peptide bonds, and in the process ligates the flanking extein (external protein) sequences by the formation of 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, protein splicing mediated by inteins is spontaneous; it requires only folding of the intein domain and 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)), and they catalyze their own excision from precursor proteins and (Perler et al., Curr. Opin. Chem. Biol. 1:292-299(1997); Perler, F.B. Cell 92(1):1-4( 1998); reviewed in 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), 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. Fission inteins have been identified in diverse cyanobacteria and archaea (Caspi et al,Mol Microbiol.50:1569-1577(2003);Choi J.et al,J Mol Biol.556:1093-1106(2006 .);Dassa B.et al,Biochemistry.46:322-330(2007.);Liu X. and Yang J.,J Biol Chem.275:26315-26318(2003);Wu H.et al.Proc Natl Acad Sci USA. £5:9226-9231 (1998.); and Zettler J. et al, FEBS Letters. Not served. Recently, bioinformatic analysis of environmental metagenomic data has revealed 26 different genetic loci with novel genomic locations. At each locus, the conserved enzyme-coding region is interrupted by a split intein, and a freestanding endonuclease gene is inserted between sections encoding intein subdomains. Among them, five loci were 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 disjointed genetic organization appears to be present primarily in phages (Dassa et al, Nucleic Acids Research. 57:2560-2573 (2009)).

In certain embodiments, the prime editing factors described herein can be used to insert split intein tags onto two different proteins, causing their intracellular ligation when coexpressed to create a fusion protein. Form. 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 the trans-splicing reaction (together N and C intein) excises the two intein sequences and connects the two extein sequences by a peptide bond. Protein trans-splicing, an enzymatic reaction, can work with very low (eg, micromolar) concentrations of protein and can be carried out under physiological conditions.

The splitting intein Npu DnaE was characterized as having the highest rate reported for protein trans-splicing reactions. 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 6 M urea (Zettler J. et al, FEBS Letters.553 :909-914(2009);Iwai I. et al,FEBS Letters 550:1853-1858(2006)). As expected, when the 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 an acyl shift at the N-terminal scissile peptide bond appears to be a common and inherent 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 (eg, a mini-intein) that has been split into two pieces named N-intein and C-intein, respectively. The N and C inteins of a split intein non-covalently associate to form an active intein and can catalyze splicing reactions in essentially the same way that a continuous intein does. Split inteins are found in nature and have also been manipulated in the laboratory. As used herein, the term "split intein" refers to any intein in which one or more peptide bond cleavages exist between the N-terminal and C-terminal amino acid sequences, such that the N-terminal and C-terminal sequences are distinct. molecules, which can non-covalently reassemble or reconstitute into inteins that are functional for trans-splicing reactions. 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, a fission intein can be derived from a eukaryotic intein. In another aspect, the fission intein can be derived from a bacterial intein. In another aspect, the fission 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 are created by engineering one or more splitting sites in unstructured loops or intervening amino acid sequences between the 12 conserved beta strands found on the structure of mini-inteins. can be generated from. Some of the location of the cleavage site within the inter-beta strand region, provided that the generation of cleavages will not interrupt the structure of the intein, particularly the structured beta strand, to a sufficient degree that protein splicing activity is lost. flexibility can exist.

The prime editors described herein can incorporate peptide tags (including inteins) at the C-terminal end of the protein of interest. In other embodiments, peptide tags (including inteins) can be incorporated at the N-terminal end of the protein of interest. Peptide tags can also be incorporated inside the protein of interest. The resulting fusion protein produced by the prime editing factors described herein can have the following structure:

[Target protein]-[peptide tag];

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

[Protein of interest - N-terminal region] - [Peptide tag] - [Protein of interest - C-terminal region].

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

Embodiments of peptide tagging using prime editing are illustrated in FIGS. 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 onto the prion protein (PRNP), which becomes misfolded during the disease process. obtain. 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 disease. 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.

Classic CJD is a human prion disease. It is a neurodegenerative disorder with distinctive clinical and diagnostic features. The disease is rapidly progressive and always fatal. Infection with this disease usually results in death within one year of the onset of the disease. CJD is a rapidly progressive, permanently fatal neurodegenerative disorder believed to be caused by an abnormal isoform of a cellular glycoprotein known as prion protein. CJD occurs worldwide, with an estimated annual incidence of approximately 1 case per million population in many countries, including the United States. The majority of CJD patients usually die within a year from the onset of the disease. CJD, along with other prion diseases that occur in humans and animals, is classified as a transmissible spongiform encephalopathy (TSE). In approximately 85% of patients, CJD occurs as an idiopathic disease without a recognizable pattern of transmission. A smaller proportion 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. For CJD, no treatment is currently known.

Variant Creutzfeldt-Jakob disease (vCJD) is a prion disease first described in the United Kingdom in 1996. 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 classic CJD (often referred to simply as CJD). It has different clinical and pathological features from classic CJD. Each disease also has a specific genetic profile of prion protein genes. Both disorders are persistently fatal brain diseases with unusually long incubation periods measured in years and are caused by unconventional transmitters called prions. 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 unusually transmittable substance called a prion. The nature of communicable substances is not well understood. Currently, the most accepted theory is that the substance is a modified form of a normal protein known as prion protein. For reasons not yet understood, the normal prion protein transforms into a pathogenic (harmful) form, which then damages the central nervous system of cattle. There is growing evidence that there are different strains of BSE: the typical or classic BSE strain responsible for the outbreak in the UK and two atypical strains (strains H and L). For BSE, no treatment is currently known.

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, Norway, and South Korea, including Canada and the United States. It can take more than a year before infected animals develop symptoms, and these can include severe weight loss (wasting), staggering, lethargy, and other neurological symptoms. CWD can affect animals of all ages, and some infected animals can eventually die without developing disease. CWD is fatal to animals and there is no treatment or vaccine.

The causative agent of TSE is believed to be a prion. The term "prion" refers to an abnormal pathogenic substance that is transmissible and capable of inducing abnormal folding of certain normal cellular proteins called prion proteins, which are most abundantly found in the brain. The function of these normal prion proteins is still not completely understood. Abnormal folding of the prion protein leads to brain damage and characteristic signs and symptoms of the disease. Prion diseases are usually rapidly progressive and always fatal.

The term "prion" as used herein refers to an infectious particle known to cause disease in humans and animals (spongiform encephalopathies). The term "prion" is a contraction of the words "protein" and "infection," and the particles are primarily, if not exclusively, infected by the PRNP gene expressing PRNP ^C , which changes conformation to become PRNP ^Sc . Consists of encoded PRNP ^Sc molecules. Prions are distinct from bacteria, viruses, and viroids. Known prions include those that infect animals and cause scrapie, a contagious 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 known and discussed to affect humans are (1) Kuru disease, (2) Creutzfeldt-Jakob disease (CJD), and (3) Gerstmann-Streussler-Scheinker disease (GSS). , and (4) fatal familial insomnia (FFI). Prions, as used herein, encompass all forms of prions that cause these diseases and/or others in any animal used, specifically humans and domesticated livestock.

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

It is known that certain mutations in the prion protein may be associated with an increased risk of prion disease. Conversely, certain mutations of 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 into this application 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:
MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERV VEQMCITQYERESQAYYQRGSSMVLFSSPPVILLISFLIFLIVG (SEQ ID NO: 291).

The amino acid sequence of PRNP (NP_000302.1) is encoded by the following nucleotide sequence (NCBI Ref.Seq No.NM_000311.5, “homo sapiens prion protein (PRNP), transcript variant 1, mRNA), and is as follows. :
(Sequence number 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 editing factors disclosed herein.

The mutation site compared to PRNP (NP_000302.1) (SEQ ID NO: 291) leading to GSS is reported as follows:

The mutation sites compared to PRNP (NP_000302.1) (SEQ ID NO: 291) leading to possible protective nature against prion diseases are as follows:

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

In other embodiments, prime editing can be used to incorporate protective mutations in PRNP that are linked to protective effects 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 still other embodiments, the protective mutation can be any alternative amino acid incorporated at G127, G127, M129, D167, D167, N171, E219, or P238 of PRNP (for PRNP of NP_000302.1 relative) (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 diseases, it could be used to generate cattle and sheep that are immune to prion diseases, or even to help cure wild populations of animals suffering from prion diseases. It can also be used. Prime editing has already been used to achieve ~25% incorporation of naturally occurring protective alleles in human cells, and previous mouse experiments have shown that this level of incorporation is sufficient to induce immunity against most prion diseases. It indicates that. This method is the first and potentially the 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 list below of PEgRNA can be used to incorporate the G127V and E219K protective alleles into human PRNPs, and the G127V protective allele into PRNPs of various animals.

F. Using prime editing for RNA tagging
Prime editing can also be used to manipulate, alter, and otherwise modify the sequences of DNA encoding RNA functions by RNA tagging, and in this way indirectly alter RNA structure and function. provide the means to do so. For example, PE can be used to insert motifs (hereinafter RNA motifs) that are functional at the RNA level for tagging or otherwise manipulating non-coding RNA or mRNA. These motifs may increase gene expression, decrease gene expression, alter splicing, alter post-transcriptional modifications, affect subcellular localization of RNA, or affect RNA isolation or allow determination of intracellular or extracellular localization (e.g., using fluorescent RNA aptamers such as Spinach, Spinach2, Baby Spinach, or Broccoli), recruit endogenous or exogenous protein or RNA binding factors, It can be used to introduce sgRNA or induce processing of the RNA either by self-cleavage or by RNAse (see Figure 28B and Example 6 for further details).

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

The PEgRNAs in the table above are designed to site-specifically insert examples of the 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, but can indeed be any gene.
HEXA mRNA has the following nucleotide sequence:
GTTCGTTGCAACAAATTGATGAGCAATGCTTTTTATAATGCCAACTTTGTACAAAAAAGTTGGCATGACAAGTTCCAGGCTTTGGTTTTCGCTGCTGCTGGCGGCAGCGTTCGCAGGACGGGCGACGGCCCTCTGGCCCTGGCCTCAGAACTTCCAAACCTCCGACCAGCGCTACGTCCTTTACCCGAACAACTTTCAATTCCAGTACGATGTCAGCTCGGCCGCGCAGCCCGGCTGCTCAGTCCTCGACGAGGCCTTCCA GCGCTATCGTGACCTGCTTTTCGGTTCCGGGTCTTGGCCCCGTCCTTACCTCACAGGGAAACGGCATACACTGGAGAAGAATGTGTTGGTTGTCTCTGTAGTCACACCTGGATGTAACCAGCTTCCTACTTTGGAGTCAGTGGAGAATTATACCTGACCATAAATGATGACCAGTGTTTACTCCTCTCTGAGACTGTCTGGGGAGCTCTCCGAGGTCTGGAGACTTTTAGCCAGCTTGTTTGGAAATCTGCTGAGGGC ACATTCTTTATCAACAAGACTGAGATTGAGGACTTTCCCCGCTTTCCTCACCGGGGCTTGCTGTTGGATACATCTCGCCATTACCTGCCACTCTCTAGCATCCTGGACACTCTGGATGTCATGGCGTACAATAAATTGAACGTGTTCCACTGGCATCTGGTAGATGATCCTTCCTTCCCATATGAGAGCTTCACTTTTCCAGAGCTCATGAGAAAGGGGTCCTACAACCCTGTCACCCACATCTACACAGCACAGGATGTGAAGGAGG TCATTGAATACGCACGGCTCCGGGGTATCCGTGTGCTTGCAGAGTTTGACACTCCTGGCCACACTTTGTCCTGGGGACCAGGTATCCCTGGATTACTGACTCCTTGCTACCCTGGGTCTGAGCCCTCTGGCACCTTTGGACCAGTGAATCCCAGTCTCAATAATACCTATGAGTTCATGAGCACATTCTTCTTAGAAGTCAGCTCTGTCTTCCCAGATTTTTATCTTCATCTTGGAGGAGATGAGGTTGATTTCACCTGCT GGAAGTCCAACCCAGAGATCCAGGACTTTATGAGGAAGAAAGGCTTCGGTGAGGACTTCAAGCAGCTGGAGTCCTTCTACATCCAGACGCTGCTGGACATCGTCTCTTCTTATGGCAAGGGCTATGTGGTGTGGCAGGAGGTGTTTGATAATAAAGTAAAGATTCAGCCAGACACAATCATACAGGTGTGGCGAGAGGATATTCCAGTGAACTATATGAAGGAGCTGGAACTGGTCACCAAGGCCGGCTTCCGGGCCCTTC TCTCTGCCCCCTGGTACCTGAACCGTATATCCTATGGCCCTGACTGGAAGGATTTCTACGTAGTGGAACCCCTGGCATTTGAAGGTACCCCTGAGCAGAAGGCTCTGGTGATTGGTGGAGAGGCTTGTATGTGGGGAGAATATGTGGACAACAACAACCTGGTCCCAGGCTCTGGCCCAGAGCAGGGGCTGTTGCCGAAAGGCTGTGGAGCAACAAGTTGACATCTGACCTGACATTTGCCTATGAACGTTTGTCACACTTC CGCTGTGAGTTGCTGAGGCGAGGTGTCCAGGCCCAACCCCTCAATGTAGGCTTCTGTGAGCAGGAG TTTGAACAGACCTGCCCAACTTTCTTGTACAAAGTTGGCATTATAAGAAAGCATTGCTTATCAATTTGTTGCAACGAAC (SEQ ID NO: 369).

The corresponding HEXA protein has the following amino acid sequence:
(SEQ ID NO: 370).

Of note, the resulting RNA motif will be included within the coding region of the HEXA gene and will block the function of the protein-coding gene. The inserted polyadenylation motif will result in premature transcript termination. This site is merely illustrative of potential PEgRNAs that could result in the insertion of the RNA motifs listed in the table above within the genomic site that would be transcribed and thus give rise to an RNA product.

PEgRNA for use in PE for RNA tagging is a guide that encompasses the U6 promoter (in this case, a protospacer that does not start with a G), a single guanosine is added to the 5' end of the PEgRNA, and 6 ~7 thymines would be added to the 3' end) or the polII promoter, e.g. pCMV (in this case, the naturally transcribed sequence of this promoter from the 5' end of the RNA by a self-cleaving element or Csy4 motif) may need to be removed and a termination motif added to the 3' end of the RNA that does not result in export of the RNA from the nucleus. For example, the 3' box motif (note that since it will be 3' of the annealing region, this motif will not be inserted onto the genome as a result of PE). The core PEgRNA backbone is underlined, homology and annealing regions are italicized, and inserted sequences are bolded. Note that, as described below, the inserted sequences are the reverse complement of the above example, so these PEgRNAs will need to be targeted to the coding strand.

Also note that self-cleaving ribozymes other than HDV require, in some embodiments, to be tailored to a given target site; It cuts immediately 5', but all other self-cleaving ribozyme cut sites are within the ribozyme itself. Thus, approximately the first and last 5 to 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, using a hammerhead self-cleaving ribozyme after 5 N's, the following sequence is inserted: Will. Here, the underlined sequences form a non-perfect RNA pairing element.

5'NNNNNCAGTTTGTACGGATGACTGATGAGTCCCAAATAGGACGAAACGCGCTTCGGTGCGTCTCATCCTGATAAACTGCAAA-3' (SEQ ID NO: 372).

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

(Here, the core PEgRNA backbone is underlined, homology and annealing regions are in italics, and inserted sequences are 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, such as simple hairpins, both the RNA sequence itself and its complement are structured, however, this is unlikely to be true for the sequences noted above. . Therefore, when inserting these motifs, it is best to most likely ensure that the PEgRNA encodes the reverse complement of these sequences, resulting in the insertion of the DNA sequence actually encoding the motif onto the genome. Dew. Similarly, inclusion of a self-cleaving ribozyme into the PEgRNA template region will result in processing and inefficient activity, but inclusion of its reverse complement will not. Therefore, these PEgRNAs would likely have to target the coding strand, whereas PEgRNAs encoding other types of insertions (e.g. therapeutic modifications) could theoretically target either strand. There will be targeting capabilities.

Also note that for many of the inserted motifs, the resulting PEgRNA may not be capable of being transcribed from the U6 promoter, requiring 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. Using prime editing for the generation of high-performance gene libraries
Prime editing can also be used to generate high-performance libraries of protein- or RNA-encoding genes with defined or variable insertions, deletions, or defined amino acid/nucleotide conversions, allowing for high-throughput screening. and their use in directed evolution described herein. This application of prime editing can be further described in Example 7.

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 that have higher mutation rates. As such, these polymerase biases are reflected in the library product (eg, preference for transition mutations over transversions). An inherent limitation of this approach to library construction is the relative inability to influence the size of the genes being altered. Most DNA polymerases have extremely low rates of indel mutations (insertions or deletions), and most of these result in frameshift mutations in protein coding regions, making it difficult for library members to pass through 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 consisting of genes of different sizes. These limitations can severely limit the efficacy of directed evolution to enhance existing protein functions and engineer new protein functions. In natural evolution, large changes in protein function or potency are typically associated with insertion and deletion mutations that are unlikely to occur during standard library generation for mutagenesis. . Moreover, these mutations most commonly occur in regions of the protein in question that are predicted to form loops relative to the hydrophobic core. Therefore, most indels generated using traditional unbiased approaches are likely to be either harmful or invalid.

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 performing programmed gene libraries for use in high-throughput screening and directed evolution (Fig. (see 29A). PE can insert, change, or remove a defined number of nucleotides from a defined genetic locus using information encoded on the prime editing guide RNA (PEgRNA) (see Figure 29B). This is a targeted method that has one or more amino acids inserted or removed from the loop region where the mutation is most likely to result in a change in function, without the background introduction of non-functional frameshift mutations. (See Figure 29C). PE can be used to incorporate specific sets of mutations regardless of biases inherent in either the DNA polymerase or the sequence to be mutated.

For example, converting a CCC codon to a stop codon would be an unlikely occurrence by standard library generation, since it would require three consecutive mutations encompassing two transversions. PE can be used to convert any given target codon into a TGA stop codon in one step. They can also be used to incorporate programmed diversity into a given position, for example, by incorporating at a given site a codon that encodes for one hydrophobic amino acid but not for any other. It can be done. Moreover, because of the programmability of PE, multiple PEgRNAs can be utilized to generate multiple different edits at multiple sites simultaneously, allowing the generation of highly programmed libraries (see Figure 29D). ). In addition, it is possible to generate regions of mutagenesis in otherwise invariant libraries using reverse transcriptases with lower fidelity (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 to the same site is also envisaged, allowing repeated insertion of codons at a single site, for example in loop regions. It is also envisioned that all of the approaches described above can be incorporated into sequential evolution, allowing the generation of new in situ evolution libraries (see Figure 30). They can also be used to construct these libraries in other cell types where it would otherwise be difficult to assemble large libraries, such as in mammalian cells. Generation of bacterial strains encoding optimized PEs 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 by PE will be a highly useful tool in synthetic biology and directed evolution, as well as for high-throughput screening of combinatorial variants of proteins and RNA.
competing approaches

The main way that diverse libraries are currently generated is by mutagenic PCR as described above (see Cadwell RC and Joyce GF. PCR Methods Appl. 1992). Although insertions or deletions can be introduced by degenerate NNK primers at defined sites during PCR, introducing such mutations at multiple sites is important before a more diverse library is constructed by mutagenic PCR. Requires multiple rounds of iterative PCR and cloning, 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-free PCR reaction, resulting in annealing of the different fragments to each other and a faster response than mutagenic PCR alone. (See Meyer AJ, Ellefson JW, Ellington AD. Curr Protoc Mol Biol. 2014). Although this approach could theoretically generate indel mutations, it more often results in frameshift mutations that disrupt gene function. Moreover, DNA shuffling requires a high degree of homology between gene fragments.

While both of these methods have to be done in vitro and the resulting libraries are transformed into cells, libraries generated by PE can be constructed in situ, making their use for continuous evolution enable. Libraries can be constructed in situ by in vivo mutagenesis, and these libraries rely on host cellular machinery to exert a bias against indels. Similarly, while traditional cloning methods can be used to generate site-specific mutation profiles, they cannot be used in situ and are generally grown in vitro before being transformed into cells. Assembled one at a time. The efficiency and broad functionality of PEs in both prokaryotic and eukaryotic cell types further emphasizes their ability to be cloned into model organisms such as E. coli and then transferred into cells or organisms of interest. We suggest that libraries can be constructed directly within the cell type of interest. Another competing approach to targeted diversification is automated multiplex genome engineering or MAGE, in which multiple single-stranded DNA oligonucleotides can be integrated into a replication fork, resulting in programmable mutations ^. However, while 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), PE is more is expected to result in fewer off-target effects. Additionally, 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 point mutations and indels to be introduced in a manner not possible with traditional approaches. Allows repeated accumulation of both.

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

In various embodiments, PE can be used to improve PACE selection by allowing in situ generation and evolution of monobodies with varying loop lengths. To do so, the PACE E. coli strain can be introduced with an additional PE plasmid, which encodes the PE enzyme and one or more PEgRNAs. Expression of PE enzyme and PEgRNA 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 contain a spacer that directs the PE to the site of interest on the selection phage and will be designed such that multiple 3 nucleotides can be inserted into the target site. As a result, new PEgRNA binding sites will be introduced, allowing repeated insertion of one or more codons at the target site.

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

In addition to using PE and PACE to generate monobody libraries, this technique can also be applied to antibody evolution using PE and PACE. The binding principles governing antibodies are very similar to those governing monobodies: the length of antibody complementarity determining region loops is critical to their binding function. Additionally, 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 (Mascola JR, Haynes BF. Immunol Rev. (see .2013). Application of PE as described above to antibodies or antibody-derived molecules will allow the generation of antibodies with a variety of loop lengths and a variety of loop sequences. In combination with PACE, such an approach would allow enhanced binding due to loop geometries not accessible to standard PACE, and therefore the evolution of highly functional antibodies.

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

In various embodiments, the use of PE to construct gene libraries may make use of the error-prone mutagenic activity of reverse transcriptase. The use of such mutagenic reverse transcriptases may facilitate the generation of mutagenized programmable libraries due to the lower fidelity of error-prone RTs. As used herein, the term "error-prone" reverse transcriptase refers to a naturally occurring or another reverse transcriptase (e.g., wild-type A reverse transcriptase enzyme derived from M-MLV reverse transcriptase. The error rate for wild-type M-MLV reverse transcriptase has been reported to range from 1 error per 15,000 to 27,000 nucleobase integrations. See Boutabout et al. (2001) “DNA synthesis fidelity by the reverse transcriptase of the yeast retrotransposon Ty1,” Nucleic Acids Res 29(11):2217-2222. This is incorporated herein by reference.

Therefore, for the purposes of this application, the term "error-prone" means 1 error per 15,000 nucleobase integrations (6.7 × 10 ^-5 or higher), e.g., 1 error per 14,000 nucleobases (7.14 × 10 ^-5 or higher), 1 error per 13,000 nucleobases or fewer (7.7 x 10 ^-5 or higher), 1 error per 12,000 nucleobases or fewer (7.7 x 10 ^-5 or higher), 11,000 nucleobases or 1 error per 10,000 nucleobases or lower (9.1 × 10 ^-5 or higher), 1 error per 10,000 nucleobases or lower (1 × 10 ^-4 or 0.0001 or higher), 1 error per 9,000 nucleobases or lower (0.00011) or higher), 1 error per 8,000 nucleobases or lower (0.00013 or higher), 1 error per 7,000 nucleobases or lower (0.00014 or higher), 1 error per 6,000 nucleobases or lower (0.00016 or higher), higher), 1 error per 5,000 nucleobases or fewer (0.0002 or higher), 1 error per 4,000 nucleobases or fewer (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 has an error rate that is greater than 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 mutagenic RT from Bordetella phage is provided below. Like other RTs disclosed herein, the Brt protein can be fused to 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 ancillary protein (Bavd). Supplementary proteins can be fused or provided in trans to the PE fusion protein. The amino acid sequence of Bavd auxiliary protein is provided as follows:

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

To reduce length, this PEgRNA additional sequence can be reduced in a variety of ways. For example, a PEgRNA addition sequence can be reduced 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 a GGG (glycine) codon, 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, the PAM sequence 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 to incorporate previously described immunogenic epitopes onto endogenous or exogenous genomic DNA for downregulation and/or destruction of their protein products and/or expressing cell types. The use of a prime editing agent to insert the containing nucleotide sequence. The nucleotide sequence for immunogenic epitope insertion is targeted to 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. Will. The patient's immune system will have previously been trained to recognize these epitopes as a result of previous standard immunization, for example from routine vaccination against tetanus or diphtheria or measles. As a result of the immunogenic nature of the epitope being fused, the patient's immune system recognizes and recognizes the prime-edited protein (not just the inserted epitope) and potentially the cells in which it is expressed. It is expected that it will be disabled.

Precise genome targeting technology using the CRISPR/Cas system has recently been investigated in a wide range of applications, including the insertion of engineered DNA sequences onto target genomic loci. Previously, homologous recombination repair (HDR) was used for this application, requiring an ssDNA donor template and initiation of repair by means of double-stranded DNA breaks (DSB). This strategy offers the widest range of possible changes to be made to cells and is the only method available for inserting large DNA sequences into mammalian cells. However, HDR is hampered by undesired cellular side effects derived from initiating its DSBs, 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 human Rad51 mutants to Cas9 D10A nickase (RDN), resulting in a DSB-free HDR system. It features an improved HDR product:indel ratio and lower off-target editing, but is still hampered by cell type dependence and only modest HDR editing efficiency.

A recently developed fusion of Cas9 to reverse transcriptase coupled to PEgRNA (the "prime editing factor") represents a novel genome editing technology that offers several advantages compared to existing genome editing methods. Includes the ability to incorporate any single nucleotide substitutions and insert or delete any short stretches of nucleotides (up to at least several dozen bases) in a site-specific manner. Of note, PE editing is generally achieved with low unintended indel rates. PE therefore enables targeted insertion-based editing applications that were previously impossible or impractical.

This specific aspect describes a method for using prime editing as a means to insert known immunogenic epitopes onto endogenous or exogenous genomic DNA, 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 rates of indel formation from DSBs. Prime editing generally offers higher efficiency than HDR 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. Epitope lengths range from a few bases to several hundred bases. Prime editing factors are the efficient and cleanest approach to achieve such targeted insertions in mammalian cells.

The key concept of this aspect is that nucleotide sequences containing the previously described immunogenic epitopes can be synthesized into endogenous or exogenous proteins for downregulation and/or destruction of their protein products and/or expressing cell types. The use of prime editing factors to insert onto genomic DNA. The nucleotide sequence for immunogenic epitope insertion is targeted to 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. Will. The patient's immune system will have previously been trained to recognize these epitopes as a result of previous standard immunization, for example from routine vaccination against tetanus or diphtheria or measles. As a result of the immunogenic nature of the epitope being fused, the patient's immune system recognizes and recognizes the prime-edited protein (not just the inserted epitope) and potentially the cells in which it is expressed. It is expected that it will be disabled.

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

A protein linker encoded as a nucleotide inserted between a target gene sequence and an inserted immunogenic epitope nucleotide sequence to facilitate immune system recognition, cellular trafficking, protein function, or protein folding of the target gene. may need to be manipulated as part of the invention. The protein linkers encoded by these inserted nucleotides can 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 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 1 and 30. is an integer between 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 SGGSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 172). In some embodiments, the linker comprises the amino acid sequence SGGSGGSGGS (SEQ ID NO: 173). In some embodiments, the linker comprises the amino acid sequence SGGS (SEQ ID NO: 174).

Distinguishing features of this aspect include the ability to use previously acquired immune responses against specific amino acid sequences as a means to induce immune responses against otherwise non-immunogenic proteins. 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 editing byproducts and where the insertion is efficient. This particular application of PE has the ability to combine cell type-specific delivery methods (eg, AAV serotypes) to insert epitopes into the cell type against which it is aimed 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 immuno-oncology strategies). can be used. An immediate relevant use of this technology would be as a cancer therapeutic. This is because it can thwart the tumor's immune escape mechanism by triggering an immune response against the relevant oncogenes, such as HER2, or growth factors, such as EGFR. Although such an approach may appear similar to T cell engineering, one novel advance in this approach is that it can be used to generate engineered T cells and to introduce them into patients, allowing them to be used in many cell types. This means that it can be used to treat diseases other than cancer.

PE is used to insert an immunogenic epitope (such as tetanus, pertussis, diphtheria, measles, mumps, rubella) against which most people are already vaccinated onto an exogenous or endogenous gene that drives the disease; Therefore, 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 the therapeutics of cancer, prions, and other neurodegenerative diseases, infectious diseases, and preventive medicine. Secondary therapeutic indications may include preventive care of patients with late-onset genetic diseases. In some diseases, especially aggressive cancers, or in cases where drug therapy helps alleviate disease symptoms until the disease is completely cured, the current standard of care medicine is used in conjunction with prime editing. It is expected that this may occur. Below are examples of immune epitopes that can be inserted onto genes by the prime editing factors 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, can be incorporated by the prime editing factors disclosed herein. obtain.

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

I. Delivery of prime editing factors
In another aspect, the present disclosure enables the delivery of prime editing factors in vitro and in vivo using a variety of strategies, including separate vectors using split inteins, and ribonucleoprotein complexes using techniques such as electroporation. the use of cationic lipid-mediated formulations, and receptor ligands fused to ribonucleoprotein complexes (i.e., PEgRNA and/or prime editing factors complexed with second-site gRNAs) It includes a direct delivery strategy called endocytosis. Any such method is contemplated herein.

Overview of Delivery Options In some aspects, the invention provides polynucleotides encoding one or more prime editing factors, e.g., polynucleotides encoding one or more components of the prime editing systems described herein. , one or more transcripts thereof, and/or one or more proteins transcribed therefrom, into a host cell. In some aspects, the invention further provides cells produced by such methods, and organisms (eg, animals, plants, or fungi) that contain or arise from such cells. In some embodiments, the prime editing factors described herein are delivered to cells in combination with (optionally conjugated to) 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 prime editing factors to cells in culture or in a host organism. Non-viral vector delivery systems include DNA plasmids, RNA (eg, transcripts of the vectors described herein), naked nucleic acids, and nucleic acids complexed with delivery vehicles such as liposomes. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to cells. 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 Bihm(eds) (1995); and Yu et al., Gene See Therapy 1:13-26 (1994).

Methods of non-viral delivery of nucleic acids include lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycations or lipid:nucleic acid conjugates, naked DNA, artificial virions, and DNA agents. Includes enhanced uptake. Lipofection has been described, for example, in US Pat. Cationic and neutral lipids suitable for efficient receptor recognition lipofection of polynucleotides include those of Feigner, WO91/17424; WO91/16024. Delivery can be to cells (eg, in vitro or ex vivo administration) or target tissues (eg, in vivo administration).

Preparation of lipid:nucleic acid complexes including targeted liposomes, such as immunolipid complexes, is well known to those skilled in the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese et al., Cancer Gene Ther. .2:291-297(1995);Behr et al.,Bioconjugate Chem.5:382-389(1994);Remy et al.,Bioconjugate Chem.5:647-654(1994);Gao et al.,Gene Therapy 2:710-722(1995);Ahmad et al., Cancer Res.52:4817-4820(1992);U.S. Pat. 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 highly evolved processes for targeting viruses to specific cells of the body and transporting viral payloads to the nucleus. Viral vectors can be administered directly to a patient (in vivo), or they can be used to treat cells in vitro and the modified cells optionally administered to a patient (ex vivo). Conventional 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 by retroviral, lentiviral, and adeno-associated viral gene transfer methods, often resulting in long-term expression of the inserted transgene. In addition, high transduction efficiency has been observed in many different cell types and target tissues.

Viral tropism can be modified by incorporating exogenous envelope proteins, expanding the possible target population of target cells. Lentiviral vectors are retroviral vectors that are capable of transducing or infecting non-dividing cells and typically produce high viral titers. Therefore, 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 sequences. Minimal cis-acting LTRs are sufficient for vector replication and packaging, and these are then used to incorporate therapeutic genes into target cells to provide permanent transgene expression. Commonly used retroviral vectors include those based on murine leukemia virus (MuLV), gibbon leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (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); see 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. This vector can be produced in large quantities by a relatively simple system. Adeno-associated virus (“AAV”) vectors can also be used to transduce cells with target nucleic acids, e.g., for in vitro production of nucleic acids and peptides, and for in vivo and ex vivo gene therapy procedures (see, e.g., West et al. al., Virology 160:38-47 (1987); US Patent No. 4,797,368; WO93/24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351 (1994) ). Construction of recombinant AAV vectors is described in US Pat. No. 5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985); 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 that can infect host cells. Such cells include 293 cells, which package adenoviruses, and ψ2 cells or PA317 cells, which package retroviruses. Viral vectors used in gene therapy are typically produced by generating cell lines that package nucleic acid vectors into viral particles. The vector typically contains the minimal viral sequences required for packaging and subsequent incorporation into the host, with other viral sequences carried by the expression cassette of the polynucleotide(s) to be expressed. Replaced. The missing viral functions are typically supplied in trans by the packaging cell line. For example, AAV vectors used for 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 encoding the other AAV genes, rep and cap, but lacking ITR sequences. Cell lines can also be infected with adenovirus as a helper. Helper viruses facilitate replication of AAV vectors and expression of AAV genes from helper plasmids. The helper plasmid is not packaged in significant quantities due to the lack of ITR sequences. Contamination by adenoviruses can be reduced, for example, by heat treatment, to which adenoviruses are more sensitive than AAV. Additional methods for delivery of nucleic acids to cells are known to those skilled in the art. See, for example, US20030087817, which is incorporated herein by reference.

In various embodiments, PE constructs (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 may contain the gene cargo to be delivered to the cell (ie, a recombinant nucleic acid vector expressing the gene of interest, such as a whole or split PE fusion protein, which is brought into the cell by the rAAV). rAAV can be chimeric.

As used herein, serotype of 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, AAV6.2, AAV2i8, AAV-HSC15/17, AAVM41, AAV9.45, AAV6(Y445F/Y731F), AAV2.5T, Includes 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 having a chimeric VP1 protein is rAAV2/5-1VP1u, which has the genome of AAV2, the capsid backbone of AAV5, and VP1u of AAV1. Other non-limiting examples of derivatives and pseudotypes having chimeric VP1 proteins are rAAV2/5-8VP1u, rAAV2/9-1VP1u, and rAAV2/9-8VP1u.

AAV derivatives/pseudotypes and methods for producing such derivatives/pseudotypes are known in the art (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.See Asokan A1, Schaffer DV, Samulski RJ.). Methods for generating and using pseudotyped rAAV vectors are known in the art (e.g., Duan et al., J. Virol., 75:7662-7671, 2001; Halbert et al., J. Virol. , 74:1524-1532, 2000; Zolotukhin et al., Methods, 28:158-167, 2002; and Auricchio et al., Hum. Molec. Genet., 10: 3075-3081, 2001).

Methods of making or packaging rAAV particles are known in the art, and reagents are commercially available (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 US Patent Publication Nos. US20070015238 and US20120322861, which are incorporated herein by reference; plasmids and kits available from ATCC and Cell Biolabs, Inc.). For example, a plasmid containing the genes of interest may contain, for example, the rep genes (e.g., encoding Rep78, Rep68, Rep52, and Rep40) and the cap genes (e.g., VP1, VP2, including the modified VP2 regions described herein). and VP3) and transfected into recombinant cells. As a result, rAAV particles can be packaged and subsequently purified.

A recombinant AAV can include a nucleic acid vector, which at a minimum includes: (a) one or more heterologous nucleic acid regions containing sequences encoding a protein or polypeptide of interest or an RNA of interest (e.g., siRNA or microRNA); (b) one or more regions comprising inverted terminal repeat (ITR) sequences (e.g., wild-type or engineered ITR sequences) flanking one or more nucleic acid regions (e.g., a heterologous nucleic acid region); may include. In this application, a heterologous nucleic acid region containing a sequence encoding a protein of interest or an 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 ITR and/or Rep genes. In some embodiments, the VP1 capsid protein backbone serotype of the particle is the same as the ITR serotype. 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 contains 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 the nucleic acid (eg, 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 (eg, 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, PEgRNA and second site nicking guide RNA can be expressed from a suitable promoter, such as the human U6 (hU6) promoter, mouse U6 (mU6) promoter, or other suitable promoter. PEgRNA and 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 enterally to the subject. In some embodiments, the rAAV construct or composition of the present application is administered parenterally to the subject. In some embodiments, rAAV particles or compositions of the present invention are administered to a subject subcutaneously, intraocularly, intravitreally, subretally, intravenously (IV), intracerebroventricularly, intramuscularly, intrathecally. (IT), intracisternally, intraperitoneally, by inhalation, locally, or by direct injection into one or more cells, tissues, or organs. In some embodiments, rAAV particles or compositions herein are administered to a subject by injection into the hepatic artery or portal vein.
Strategy based on split PE vectors

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 the cell (e.g., as an expressed protein or on a separate expression vector), and once in contact within the cell, the two halves are freed from the self-splicing action of the intein of each prime editing factor half. to form a complete prime editing factor. Split intein sequences can be engineered into each half of the encoded prime editing factor to facilitate their trans-splicing within the cell and the concomitant restoration of a fully functional PE.

These split intein-based methods overcome several barriers to in vivo delivery. For example, the DNA encoding prime editing factors is larger than rAAV packaging limits and therefore requires special solutions. One such solution is to formulate editing factors fused to divisible intein pairs, which are then packaged into two separate rAAV particles that when co-delivered to cells form functional editing factors. Reconstitute proteins. Several other special considerations are described that explain the unique features of prime editing, including optimization of second-site nicking targets and proper packaging of prime editing factors into viral vectors, including lentiviruses and rAAV. It includes doing.

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 the cell (e.g., as an expressed protein or on a separate expression vector), and once in contact within the cell, the two halves are freed from the self-splicing action of the intein of each prime editing factor half. to form a complete prime editing factor. Split intein sequences can be engineered into each half of the encoded prime editing factor to facilitate their trans-splicing within the cell and the concomitant restoration of a fully functional PE.

Figure 66 illustrates one embodiment of a prime editing factor provided as two PE half proteins. These regenerate the entire prime editing factor by the self-splicing action of the split intein halves positioned at the end or beginning of each protein half of the prime editing factor. As used herein, the term "PE N-terminal half" refers to the N-terminal half of an intact prime editing factor that contains an "N-intein" at the C-terminal end of the PE N-terminal half of the intact prime editing factor (i.e. N-terminal extein). "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 an intact prime editing element that contains a "C intein" at the N-terminal end of the C-terminal half of the intact prime editing element (i.e., the C-terminal Extain). When the two halves of the protein, namely the PE N-terminal half and the PE C-terminal half, come into contact with each other, e.g. within a cell, the N-intein and the C-intein self-excise and release the C-terminal end of the PE N-terminal half and the PE C-terminal end. A simultaneous process with the formation of a peptide bond between the N-terminal ends of the halves re-forms the complete prime editor protein containing the complete napDNAbp domain (e.g. Cas9 nickase) and RT domain. Although not shown in the figures, the prime editor may also contain additional sequences, including an N-terminal and/or C-terminal NLS and an amino acid linker sequence connecting each domain.

In various embodiments, the prime editing factor is engineered into two half-proteins (i.e., a PE N-terminal half and a PE C-terminal half) by "splitting" the whole prime editing factor (as) at a "splitting site." can be done. "Split site" refers to the positioning of the insertion of a split intein sequence (ie, N-intein and C-intein) between two adjacent amino acid residues on the prime editing element. More specifically, the "split site" refers to the positioning of the entire prime editing element into two separate halves, each half fused to either an N-intein or a C-intein motif at the split site. Although the splitting site can be in any suitable location on the prime editing factor fusion protein, preferably the splitting site is in a position that allows the formation of two half-proteins of appropriate size for delivery (e.g., by an expression vector). The inteins that are positioned and fused to each half protein at the end of the division site are available to fully interact with each other when one half protein contacts the other half protein within the cell.

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

In various embodiments, the split site design includes sites for splitting and inserting N- and C-terminal inteins that both structurally permit the purpose of packaging the two half prime editor domains into two different AAV genomes. We request you to find out. In addition, intein residues required for trans-splicing can be incorporated by mutating residues at the N-terminus of the C-terminal extein or inserting residues that will leave a "scar" of the intein.

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 are between the central regions of the protein, for example amino acids 50-1250, or 100-1200, or 150-1150, or 200-1100, or 250-1050, or 300-1000 of SEQ ID NO: 18; or 350-950, or 400-900, or 450-850, or 500-800, or 550-750, or 600-700. In certain exemplary embodiments, the cleavage site can be between 740/741, or 801/802, or 1010/1011, or 1041/1042. In other embodiments, the split sites are 1/2, 2/3, 3/4, 4/5, 5/6, 6/7, 7/8, 8 relative to SpCas9 of SEQ ID NO: 18. /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 , between 81/82, 82/83, 83/84, 84/85, 85/86, 86/87, 87/88, 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, Adjacent residues between 850-900, 900-950, 950-1000, 1000-1050, 1050-1100, 1100-1150, 1150-1200, 1200-1250, 1250-1300, 1300-1350, and 1350-1368 Any two correspondences on the amino acid sequence that have at least 80%, 85%, 90%, 95%, 98%, 99%, or 99.9% sequence identity with SEQ ID NO: 18 between any two pairs of groups. or at least 80%, 85%, 90%, 95%, 98% between residues that , 99%, or 99.9% sequence identity between any two corresponding residues of the amino acid sequences.

In various embodiments, a split intein sequence can be engineered from the next intein sequence.

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 embodiments, the present disclosure provides a method of delivering a PE fusion protein to a cell:
(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 the trans-splicing activity that causes self-excision of the first and second split intein sequences, the N-terminal and C-terminal fragments are reconstituted as a PE fusion protein 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 still other embodiments, the cleavage site is on the reverse transcriptase or polymerase domain.

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

In various embodiments, the present disclosure provides prime editing factors that include napDNAbp (e.g., a Cas9 domain) and a reverse transcriptase, wherein one or both of napDNAbp and/or reverse transcriptase is an intein, e.g., a ligand-dependent intein. including. Typically, inteins are ligand-dependent inteins, which exert protein splicing activity in the absence of ligands (e.g., small molecules such as 4-hydroxytamoxifen, peptides, proteins, polynucleotides, amino acids, and nucleotides). No or minimal performance. Ligand-dependent inteins are known and include those described in US patent application U.S.S.N.14/004,280, published as U.S.2014/0065711A1. The entire contents of this document are incorporated by reference into this application. 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 invention encompasses the following split intein PE construct. These are split between residues 1024 and 1025 of standard SpCas9 (SEQ ID NO: 18) (or they can be referred to as residues 1023 and 1024, respectively, relative to Met minus SEQ ID NO: 18). ).

First, the amino acid sequence of SEQ ID NO: 18 is shown as follows. Indicating the location of the split site between 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 (which is based on the SpCas9 of SEQ ID NO: 18) construct was constructed with two separate molecules at positions 1023/1024 (relative to Met minus SEQ ID NO: 18) as follows: Split into constructs:

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

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

The present disclosure also contemplates methods of delivering a dividing intein prime editing factor to a cell and/or treating a cell with a dividing intein prime editing factor.

In some embodiments, the present disclosure provides methods of delivering PE fusion proteins to cells:
(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 the trans-splicing activity that causes self-excision of the first and second split intein sequences, the N-terminal and C-terminal fragments are reconstituted intracellularly as a PE fusion protein.

In certain embodiments, the N-terminal fragment of the PE fusion protein fused to the first split intein sequence is SEQ ID NO: 3875, or at least 80%, at least 85%, at least 90%, at least 95%, at least SEQ ID NO: 3875. Amino acid sequences having 98%, or at least 99.9% sequence identity.

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 at least 80%, at least 85%, at least 90%, at least 95% with SEQ ID NO: 3876, Amino acid sequences having at least 98%, or at least 99.9% sequence identity.

In other embodiments, the disclosure provides a method of editing a target DNA sequence in a cell:
(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-terminal 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 at least 80%, at least 85%, at least 90%, at least 95% with SEQ ID NO: 3876, Amino acid sequences having at least 98%, or at least 99.9% sequence identity.
Delivery of PE ribonucleoprotein complexes

In this aspect, prime editing agents can be delivered using non-viral methods that involve the delivery of prime editing agents (i.e., PE ribonucleoprotein complexes) complexed with PEgRNA by various methods including electroporation and lipid nanoparticles. delivery strategy. Methods of non-viral delivery of nucleic acids include lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycations or lipid:nucleic acid conjugates, naked DNA, artificial virions, and DNA agents. Includes enhanced uptake. Lipofection is described in, for example, US Pat. Cationic and neutral lipids suitable for efficient receptor recognition lipofection of polynucleotides include those of Feigner, WO91/17424; WO91/16024. Delivery can be to cells (eg, in vitro or ex vivo administration) or target tissues (eg, 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 that discuss approaches for non-viral delivery of ribonucleoprotein complexes. Each of these is incorporated herein by reference.

Chen,Sean,et al.“Highly efficient mouse genome editing by CRISPR ribonucleoprotein electroporation of zygotes.”Journal of Biological Chemistry(2016):jbc-M116.PubMed

Zuris, John A., et al. “Cationic lipid-mediated efficient delivery of proteins enables 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 various disclosed PE ribonucleoprotein complexes (high concentration of PE2, high concentration of PE3, and low concentration of 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, BR11201603085 2A2, and EP3362461A1. Each of these is incorporated herein by reference in its entirety. Additional disclosure can be found in Kowalski et al., “Delivering the Messenger: Advances in Technologies for Therapeutic mRNA Delivery,” Mol Therap., 2019; 27(4):710-728, which is incorporated herein by reference.

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, this is due to the significantly larger size of mRNA molecules (300–5,000 kDa, ~1-15kb).

mRNA must cross the cell membrane to reach the cytoplasm. 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 prime editing factor and/or a PEgRNA), a transport vehicle, and optionally a target cell to facilitate contact and subsequent transfection. Contains drugs.

In some embodiments, the mRNA includes one or more modifications that confer stability to the mRNA (e.g., compared to a wild-type or native version of the mRNA) and are involved in associated aberrant expression of the protein. It is possible. One or more modifications of the wild type that correct the defect may also be included. For example, a nucleic acid 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 sequences encoding the cytomegalovirus (CMV) immediate early 1 (IE1) gene, the poly A tail, the cap 1 structure, or a subsequence 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 compositions of the invention can be formulated with a liposomal transfer 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 can 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 comprises cholesterol (chol) and/or PEG-modified lipids. In some embodiments, the import vehicle comprises DMG-PEG2K. In certain embodiments, the import vehicle has the following lipid formulation: C12-200, DOPE, chol, DMG-PEG2K; DODAP, DOPE, cholesterol, DMG-PEG2K; HGT5000, DOPE, chol, DMG-PEG2K, HGT5001, DOPE , chol, one of DMG-PEG2K.

The present disclosure also provides compositions and methods useful for facilitating transfection of target cells with mRNA molecules encoding one or more PEs. For example, the compositions and methods of the 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 cell expresses a 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, nerve cells, cardiac cells, adipocytes, vascular smooth muscle cells. (Includes) cells, cardiomyocytes, skeletal muscle cells, beta cells, pituitary cells, synovial surface cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, reticulocytes, leukocytes, granulocytes, and tumors. Contains cells. However, it is not limited to these.

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

Thus, in some embodiments, the mRNA in the compositions of the present disclosure comprises at least one modification that confers increased or enhanced stability to the nucleic acid, including, for example, improved resistance to nuclease digestion in vivo. As used in this application, the terms "modified" and "altered", as such terms relate to the nucleic acids provided herein, preferably enhance stability and make the mRNA more stable than the wild-type or naturally occurring version of the mRNA. Includes at least one modification that renders it stable (eg, resistant to nuclease digestion). As used in this application, the terms "stable" and "stability" refer to the nucleic acids of the invention, particularly when such terms refer to mRNA, e.g. Refers to increased or enhanced resistance to degradation. Increased stability may 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, whereby the target cell, Increase or enhance the retention of such mRNA in the tissue, subject, and/or cytoplasm. The stabilized mRNA molecules provided herein demonstrate long half-lives relative to their naturally occurring, unmodified counterparts (eg, the wild-type version of the mRNA). Also, when such terms relate to the mRNA of the present invention, the terms "modification" and "altered" contemplate modifications that improve or enhance translation of the mRNA nucleic acid. For example, this includes the inclusion of sequences that function in the initiation of protein translation (eg, Kozak consensus sequences). (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 modification to make them more stable. Exemplary modifications of mRNA include base depletion (eg, by deleting one nucleotide or substituting another) or base modification, eg, chemical modification of the base. As used herein, the phrase "chemical modification" refers to modifications that introduce chemistry different from that found in naturally occurring mRNA, such as covalent modifications, such as the introduction of modified nucleotides (e.g., nucleotide analogs, or (inclusion of pendant groups not found in nature).

Other suitable polynucleotide modifications that may be incorporated into the mRNA encoding 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'-thio2-thiouridine, pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio- 5-aza-uridine, 2-thiouridine, 4-thiopseudouridine, 2-thiopseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl Uridine, 1-propynylpseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thiouridine, 1-taurinomethyl-4-thiouridine, 5-methyl-uridine, 1-methyl-pseudouridine , 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydro Uridine, 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- Pseudisocytidine, 2-Thiocytidine, 2-thio-5-methyl-cytidine, 4-Thiopseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudo Isocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methylzebularine, 5-aza-2-thiosebularine, 2-thiosebularine, 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-glycinylcarbamoyl Adenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine, N6,N6-dimethyladenosine, 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, Not limited to these. The term modification also encompasses, for example, the incorporation of non-nucleotide linkages or modified nucleotides onto the mRNA sequences of the invention (e.g., at the 3' and 5' ends of an mRNA molecule encoding a functional protein or enzyme). one or both qualifications). Such modifications may 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, or converting the mRNA into a drug (e.g., protein or complementary nucleic acid). molecule) and the inclusion of elements that alter the structure of the mRNA molecule (eg, which form secondary structure).

In some embodiments, the mRNA encoding PE includes a 5' cap structure. The 5' cap is typically added as follows: first, RNA terminal phosphatases remove one of the terminal phosphate groups from the 5' nucleotide, leaving two terminal phosphates; Phosphate (GTP) is added to the terminal phosphate by guanylyltransferase, creating a 5'5'5 triphosphate linkage; then the 7-nitrogen of the guanine is methylated by 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. The naturally occurring cap structure contains a 7-methylguanosine linked to the 5' end of the first transcribed nucleotide via a triphosphate bridge, m7G(5')ppp(5')N resulting in a dinucleotide cap, where N is any nucleoside. In vivo, the cap is added enzymatically. The cap is added in the nucleus, catalyzed by the enzyme guanylyltransferase. The 5' of the RNA Addition of a cap to the terminal end occurs immediately after the initiation of transcription. The terminal nucleoside is typically a guanosine and is oriented in the opposite direction to all other nucleotides, i.e. G(5')ppp(5')GpNpNp It is.

Additional cap analogs include 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 symmetrical cap analogs (e.g. m7Gpppm7G), or anti-reverse cap analogs (e.g. ARCA; m7,2'OmeGpppG, m72'dGpppG, m7,3'OmeGpppG, m7,3'dGpppG, and their tetraphosphate derivatives) including, but not limited to, chemical structures selected from the group (e.g., Jemielity, J. et al., “Novel 'anti-reverse'cap analogs with superior translational properties”, RNA, 9:1108-1122( (see 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 polyC tail can be in addition to or replace the polyA or polyU tail.

mRNA encoding PE according to the present disclosure can be synthesized according to any of a variety of known methods. For example, mRNA according to the invention can be synthesized by in vitro transcription (IVT). Briefly, IVT typically comprises 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 a suitable RNA polymerase ( For example, T3, T7, or SP6 RNA polymerase), DNAse I, pyrophosphatase, and/or RNAse inhibitors. The exact 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 primed edits to identify off-target edits in an unbiased manner
Like other genome editing agents, there is some risk that PE may introduce its programmed genetic changes at unintended or "off-target" sites on the genome. However, currently there is no described method for detecting off-target edits due to prime editing factors. Such methods would allow identification of potential sites of off-target editing using prime editing factors.

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 is a schematic diagram showing PEgRNA design for primer binding sequence insertion and primer binding insertion onto genomic DNA using 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 top schematic shows exemplary PEgRNAs that can be used in this aspect. A spacer on PEgRNA (labeled "protospacer") is complementary to one of the strands of the genomic target. The PE:PEgRNA complex (i.e., PE complex) incorporates a single-stranded 3' end flap at the nick site, which is complementary to the encoded primer binding sequence and the region just downstream of the site to be cut. (in red color) and a homologous region (encoded by the homologous arms of PEgRNA). Through flap invasion and DNA repair/replication processes, the synthesized strand becomes incorporated onto the DNA, thereby incorporating a primer binding site. This process may occur at the desired genomic target, but may also occur at other genomic sites that may interact with PEgRNA in an off-target manner (i.e., to other genomic sites that are not the intended genomic site). Due to the complementarity of the spacer region, 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 off-target genomic sites elsewhere on the genome. To detect the insertion of these primer binding sites at both intended and off-target genomic sites, genomic DNA (post-PE) can be isolated, fragmented, and ligated to adapter nucleotides (indicated in red). It is shown). PCR can then be performed with PCR oligonucleotides that anneal to the adapter and the inserted primer binding sequences to amplify on-target and off-target genomic DNA regions where 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 on-target or off-target sites.

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, purified prime editing factor fusion proteins and PEgRNA ribonucleoproteins (RNPs) (ie, PE complexes) are assembled. It 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 (ie, PE complex) is incubated with extracted genomic DNA before or after DNA fragmentation. After fragmentation, different adapter sequences are ligated to the ends of the fragment DNA. PCR is used to amplify the genomic site spanning the inserted adapter sequence (ie, inserted by EP) and the adapter ligated to the end of the fragment. High-throughput sequencing between 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 since cellular DNA repair will not erase the reverse transcribed DNA adapters added by the prime editing factor.

These methods are used to identify off-target edits for either prime editing factors or for any genome editing factors (most Cas nucleases) that use a guide RNA to recognize the cut site of the target. can be used.

These methods can be applied to all genetic diseases for which genome editing agents are considered for use in treatment.

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

including but not limited to.

These adapter and/or primer binding sequences can also be used in the ligation step after genomic DNA fragmentation as outlined above.

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

K. Use of prime editing for insertion of inducible dimerization domains
The prime editing factors described herein can also be used to incorporate dimerization domains into one or more protein targets. A dimerization domain is an inducible domain of activity associated with dimerization of one or more protein targets via a linking moiety (e.g., a small molecule, peptide, or protein) that binds in a bispecific manner. Control can be facilitated. In various aspects, the dimerization domains can each be incorporated into the same bispecific moiety (e.g., separately into the dimerization domains) when incorporated onto different proteins (e.g., of the same type or different proteins). bispecific small molecules, peptides, or polypeptides that have at least two regions that bind, thereby forming a dimer of proteins by their common interaction with the bispecific ligand. cause In this manner, the dual-specific ligand functions as an "inducer" of the dimerization of two proteins. In some cases, a bispecific ligand or compound will have two targeting moieties that are the same. In other embodiments, the bispecific ligands or compounds will each have a targeting moiety that is different from the other. 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. Dual specific ligands or compounds 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 the binding moiety of a bispecific ligand. The "first" dimerization domain binds to the first binding moiety of the bispecific ligand, and the "second" dimerization domain binds to the second binding moiety of the same bispecific ligand. Join. The first dimerization domain is fused to the first protein (e.g., by PE as discussed in this application) and the second dimerization domain (e.g., by PE as discussed in this application) is fused to the first protein. When fused to a second protein, the first and second proteins dimerize in the presence of a dual-specific ligand, the dual-specific ligand having at least one protein that binds to the first dimerization domain. one portion and at least another portion that binds to the second dimerization domain.

The term "bispecific ligand" or "bispecific 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 moiety specifically and tightly binding to a dimerization domain. In certain 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 bispecific ligand. These molecules may also be referred to as "chemical inducers of dimerization" or CIDs. In addition, bispecific ligands can be prepared by coupling two identical moieties together (e.g., by standardized chemical linkages) or two different moieties together, where each moiety has two Binds tightly and specifically to the merization 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. It has the following amino acid sequence:

In another example, the dimerization domain can be a mutant of FKBP12 designated FKBP12-F36V, a mutant of FKBP12 with an engineered hole that binds to a 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 modified to optimize binding to the native target or improve binding orthogonality. The nucleic acid sequences of genes encoding small molecule binding proteins can be modified to optimize the efficiency of the PE process, eg, by reducing PEgRNA secondary structure.

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

Other examples of naturally occurring bifunctional molecules and their dual target receptors are: Prime editing can be used to incorporate dual target receptors into different proteins. Once different proteins have been modified by PE to contain bifunctional molecule receptors, bifunctional molecules can be introduced, thereby allowing two of the proteins modified to contain different dimerization domains. Causes quantification. Examples of pairings of (1) biofunctional molecules and (2) their dual target receptors are:

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

Synstab A:

Paclitaxel:

Disco del Molido:

GNE-0011

ARV-825, and

。 dBET1

.

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

PEgRNA for incorporating a dimerization domain can include the following structure (with reference to Figure 3D):
5'-[spacer]-[gRNA core]-[extending arm]-3', where the extending arm comprises 5'-[homologous arm]-[editing template]-[primer binding site]-3'; or
5'-[extended arm]-[spacer]-[gRNA core]-3', the extended arm comprises 5'-[homologous arm]-[editing template]-[primer binding site]-3'; In either configuration, the "editing template" includes the nucleotide sequence of the dimerization domain.

In one example, 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 italicized, annealed) Areas are 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。 Target proteins for incorporating dimerization domains are not particularly limited; however, their dimerization in the presence of dual-specific ligands (once modified by PE) may have some advantageous biological effects. Advantageously, effects such as signaling pathways, decreased immune responsiveness, etc. are produced. In various aspects, the target proteins to be dimerized by PE-dependent incorporation of dimerization domains can be the same protein or different proteins. Preferably, the protein, when dimerized, triggers one or more downstream biological cascades, such as signal transduction cascades, phosphorylation, etc. Exemplary target proteins for which PE can be used to incorporate dimerization domains are:

including but not limited to.

In one aspect, the prime editing factors described herein are used to incorporate sequences encoding dimerization domains onto one or more genes encoding target proteins of interest in living cells or patients. obtain. This can be referred to as the "prime editing-CID system," in which CID is a dual-specificity ligand that supports dimerization of the target protein, each fused to a dimerization domain incorporated by PE. was induced. This editing alone should have no physiological effect. By administration of the bispecific ligand, which is 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 protein dimerization. This targeted protein dimerization event then induces a biological signaling event, such as erythropoiesis or insulin signaling. Genomic incorporation of genes encoding small molecule binding proteins (i.e., dimerization domains) by prime editing allows biological processes induced by dimerization, such as receptor signaling, to be integrated into convenient small molecule drugs (i.e. , dual-specific ligands) are described herein.

Protein dimerization is a ubiquitous biological process. Of note, homodimerization of many membrane-bound receptors is known to initiate signal cascades, often with profound biological consequences. A number of natural small molecule products approved for use as drugs act as chemical inducers of protein dimerization as part of their mechanism of action ⁹² . For example, FK506 tightly binds to FKBP12, and the resulting small molecule-protein complex then binds the phosphatase calcineurin, thereby inhibiting certain steps in T cell receptor signaling ⁹³ . Similarly, cyclosporine A induces dimerization of cyclophilin and calcineurin, and rapamycin induces dimerization of FKBP and ^mTOR93,94 .

In one embodiment, chemical inducers of synthesis of dimerization are also developed, taking advantage of the selective high affinity binding of FK506:FKBP12 and cyclosporin A:cyclophilin small molecule:protein binding interactions. There is. In one example, a small molecule consisting of two units of FK506, designated FK1012, was shown to accomplish signal transduction when the cytoplasmic domain of the signal receptor was tagged with FKBP12 ^. Since then, chemical inducers of dimerization (CID) have been used to control a number of 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-target protein chimeras into patients.

The present disclosure brings two concepts together to create a previously inaccessible therapeutic process. The first concept is prime editing, described herein, which allows precise genome editing involving 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.

Certain cases have been identified where chemical control of protein dimerization could have a beneficial therapeutic effect.

The insulin receptor is ^a heterotetrameric transmembrane protein that responds to insulin binding to its extracellular domain by phosphorylation of its cytoplasmic kinase domain. 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 response. Similarly, it is expected 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 may replace or supplement insulin use in patients who cannot make insulin (eg, type 1 diabetes) or are weakly responsive to insulin (eg, type 2 diabetes).

In addition, erythropoietin stimulates erythrocyte proliferation by binding to the erythropoietin receptor (EpoR), inducing either dimerization or conformational changes in preformed receptor dimers, which leading to activation of the Jak/STAT signal cascade ¹⁰⁵ . It was demonstrated that FK1012-induced oligomerization of the membrane-anchored cytoplasmic domain of FKBP12-tagged EpoR is sufficient to initiate the Jak/STAT signal cascade and promote cell proliferation. There are ¹⁰⁶ . It is anticipated that fusing FKBP12 to native EpoR by prime editing in patient cells will allow FK1012-induced control of red blood cell proliferation (erythropoiesis). This system can be used to induce red blood cell growth in anemic patients. FK1012-induced EpoR can also be used as an in vivo selectable marker for blood cells that have undergone ex vivo manipulation.

In principle, any receptor tyrosine kinase is a promising target for prime editing-CID therapeutics. The table below contains a list of all receptor tyrosine kinases on the human genome110 ^.

The Prime Edit-CID system has a number of advantages. One such advantage is that it allows drug-like small molecules to replace endogenous ligands, which are typically proteins that present complications in manufacturing, cost, delivery, production, or storage. They can be administered orally instead of being administered by IV or injection, are effortlessly prepared from FDA-approved drugs (or are themselves already drugs), and require specialized production or storage costs typically associated with protein drugs. do not invite Another advantage is that editing alone should have no physiological effect. The amount of target protein dimerization can be controlled by dosing small molecule CID. Furthermore, target protein dimerization is easily and rapidly reversible by adding the monomeric form of CID. Yet another advantage is that when 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 localized tissues or organs, 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).

Fusions of FKBP12 or other small molecule binding proteins to native proteins are commonly accomplished by overexpression from plasmids in tissue culture. Subsequent chemical-induced dimerization has been demonstrated to induce phenotypic changes in cells producing the fusion protein.

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

L. Using prime editing for cellular data recording
Prime editing factors and the resulting genome modifications can also be used to study and document cellular processes and development. For example, providing a cell with a first nucleic acid sequence encoding a fusion protein having a nucleic acid programmed DNA binding protein (napDNAbp) and a reverse transcriptase; and providing the cell with at least a second nucleic acid sequence encoding PEgRNA. Alternatively, the prime editing factors described herein can be used to record the presence and duration of stimulation to cells. either the first, second, or both nucleic acid sequences are operably linked to an inducible promoter that is responsive to cellular stimulation so that it induces expression of the fusion protein and/or PEgRNA; thereby causing modification of the target sequence within the cell.

The prime editing factors described herein can also be used for cell barcoding and lineage tracing. For example, by barcoding each cell with a unique genomic barcode, prime editing factors reveal cell lineage maps by allowing the construction of a phylogenetic tree based on the modifications made to one or more target sequences. can help you do that. Starting from progenitor cells, the prime editor system can make it possible to build cell fate maps for single cells in whole organisms. This can be deciphered by analyzing the modifications made to one or more target sequences. A method for tracing the lineage of a cell includes providing a nucleic acid encoding a fusion protein with napDNAbp and reverse transcriptase, and providing at least one second nucleic acid encoding PEgRNA. It is possible. A unique cellular barcode can be generated using a fusion protein and PEgRNA to generate one or more modifications in one or more target sequences, thereby allowing the differentiation of any cell lineage ( linage) can be traced using unique cell barcodes. The use of prime editing factors for both cell data recording and lineage tracing is further described in Example 13.

Prime editing factors can perform both lineage tracing and cell signal recording by modifying genomic target sequences or incorporated pre-designed sequences. Prime editing factors 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 to incorporate pre-determined edits. Because PEgRNAs define both the target genome sequence and the editing outcome, highly specific and controlled genome modifications can be achieved simultaneously within the same cell using multiple PEgRNAs. Accessible genomic modifications include all single nucleotide substitutions, small to moderate sized sequence insertions, and small to moderate sized deletions. The versatility of this genome editing technology may enable temporally coupled signal-specific recording within cells.

The use of prime editing factors for cell data recording refers to the composition (e.g., nucleic acids), cell , systems, kits, and methods. The cellular data recording system induces the expression of fusion proteins to induce changes by producing targeted and sequenced genomic insertions, deletions, or mutations in response to cellular stimuli or changes. A fusion protein consisting of napDNAbp (eg, a Cas9 domain) and a reverse transcriptase operably linked to a promoter that promotes the use of a protein can be included. 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 Permanent marks can be induced on cellular DNA in a manner that reflects both the strength (i.e. amplitude) and duration of one or more stimuli. Therefore, in some aspects, a cellular data recording system has the ability to record multiple cellular states simultaneously. For example, during activation of small molecules, proteins, peptides, amino acids, metabolites, inorganic molecules, organometallic molecules, organic molecules, drugs or drug candidates, sugars, lipids, metals, nucleic acids, endogenous or exogenous signal cascades. exposure to molecules, light, heat, sound, pressure, mechanical stress, shear stress, or viruses or other microorganisms caused by microorganisms. These cellular data recorders may use sequencing technology (e.g., high-throughput sequencing) to measure readouts (e.g., changes in cellular DNA) and record stimulation or stimulus-induced changes in cellular DNA. Both readout(s) are independent of large cell populations.

Generally, the cellular data recorder systems provided herein for use in cells include a fusion protein consisting of napDNAbp and reverse transcriptase, and the nucleic acid sequence encoding the fusion plasmid is linked to a promoter (e.g., an inducible promoter). or a constitutive promoter). When a stimulus is present or a change in cellular state occurs, the stimulus induces expression of the fusion protein. Also present within the cell are one or more nucleic acids encoding at least one PEgRNA that associates with napDNAbp and directs napDNAbp or the fusion protein to the target sequence (ie, the PEgRNA is complementary to the target sequence). A nucleic acid encoding PEgRNA can also, or alternatively, be operably linked to a promoter (eg, an inducible promoter or a constitutive promoter). Under the correct stimulus or set of stimuli, both the fusion protein and PEgRNA are expressed by the cell, and the PEgRNA associates with the fusion protein and directs it to the target sequence. This targeting sequence records the activity of the prime editing factor, thereby recording the presence of a stimulus or set of stimuli or a change in cellular state. More than one PEgRNA sequence may also be present in the cell, and each of these additional PEgRNA sequences can direct the fusion protein to a different target sequence, each operable to a promoter that senses the presence of a different stimulus. can be coupled, 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 (eg, fusion protein and PEgRNA) can be constitutively expressed by the cell. Exemplary components of a cellular data recorder system for use in the compositions are described herein. Additional suitable combinations of the components provided herein will be apparent to those skilled in the art based on this disclosure and knowledge in the art. Therefore, it is within the scope of this disclosure.

Repetitive 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), may influence activation of signal cascades, metabolic status, and cell differentiation. The program can be used to record a number of important biological processes. Connecting internal and external cellular signals to sequence modifications on the genome is possible for any signal for which signal-responsive promoters are present. In some embodiments, the promoter is a promoter suitable for use in prokaryotic systems (ie, a bacterial promoter). In some embodiments, the promoter is a promoter suitable for use in eukaryotic systems (ie, a eukaryotic promoter). In some embodiments, the promoter is a promoter suitable for use in mammalian (eg, human) systems (ie, a mammalian promoter). In some embodiments, the promoter is induced by stimulation (ie, an inducible promoter). In some embodiments, the stimulus is a small molecule, protein, peptide, amino acid, metabolite, inorganic molecule, organometallic molecule, organic molecule, drug or drug candidate, sugar, lipid, metal, nucleic acid, endogenous or exogenous molecules, light, heat, sound, pressure, mechanical stress, shear stress, or viruses or other microorganisms, changes in pH, or changes in redox conditions that occur during activation of a signal cascade. 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 that occurs during an activated signal cascade (eg, beta-catenin that occurs during an activated Wnt signal cascade). Additional promoters that detect signal molecules can be generated to direct expression of nucleic acid sequences operably linked to the promoter. For example, immune response (IL-2 promoter), cAMP response element (CREB), NFκB signal, interferon response, P53 (DNA damage), Sox2, TGF-β signal (SMAD), Erk (e.g. activated Ras/Raf /Mek/Erk cascade), PI3K/AKT (e.g., from the activated Ras/PI3K/Akt cascade), heat shock, Notch signaling, Oct4, aryl hydrocarbon receptor, or endogenous transcription factors, including AP-1 transcription factors. It is a promoter that records the target pathway. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is a promoter listed in Table 3. Additional suitable promoters for use in both prokaryotic and eukaryotic systems will be apparent to those skilled in the art based on this disclosure and knowledge in the art. Within the scope of this disclosure.

Prime editing factors can also be used to trace cell lineages. 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, insertion of a homologous sequence (ie, a sequence 3' of the positioning of the Cas9 nick), specifically a homologous sequence with an associated barcode, appears to be a particularly useful lineage prime editor strategy. These systems can be designed such that successive rounds of editing result in the insertion of a barcode from the PEgRNA cassette that cannot be modified by other PEgRNA editing events in the same cell. A barcoding system may utilize multiple barcodes, which may be associated with a given stimulus. This system can preserve much of the target protospacer, but can modify the seed sequence, PAM, and downstream flanking nucleotides. This allows multiple signals to be connected to one editing locus without significant redesign of the PEgRNA to be used. The strategy may allow multiplexed barcode insertion at a single locus in response to multiple cellular stimuli (either internal or external). It can enable the recording of the intensity, duration, and order of as many signals as there are unique barcodes (these are designed with multiple N nucleotides to generate 4^N possible barcodes). For example, 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.

M. Using prime editing to modulate biomolecule activity
The use of prime editing factors described herein can also be used to control the subcellular localization and modification status of biomolecules such as DNA, RNA, and proteins. Specific 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 unique cellular compartments may provide opportunities for modifying a number of biological processes.

Therefore, prime editing involves the use of gene-encoded handles that would allow altered modification states of biomolecules (e.g. proteins, lipids, sugars, and nucleic acids) and their transport at the subcellular level by gene-encoded signals. can be used to incorporate. In various embodiments, the target biomolecule for drug therapy mediated by prime editing factors is DNA. For example, DNA can be modified by incorporating any 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 for drug therapy mediated by a prime editing factor is RNA. For example, the activity of an RNA can be modified by changing its cellular localization, interaction partners, structural dynamics, or thermodynamics of folding. In yet other embodiments, the target biomolecule for drug therapy mediated by a prime editing factor is a protein. Proteins may be modified to impact post-translational modifications, protein motifs may be incorporated to alter the subcellular localization of the protein, or proteins may be present in protein-protein complexing events. can be modified to either create or destroy abilities.

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

DNA modification
One target biomolecule for PE-mediated modification is DNA. Modifications to the DNA can be made to incorporate any number of DNA sequences that change the accessibility of the target locus. Chromatin accessibility controls gene transcriptional output. Incorporation of marks to recruit chromatin-compacting enzymes should reduce the transcriptional output of neighboring genes, whereas incorporation of sequences associated with chromatin opening makes regions more accessible and, in turn, increases transcription. It should be increased. 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. It is. Typically, it is a tool that incorporates a large number of possible single types of marks without specific biological ancestry. As has been demonstrated in the burgeoning field of 3D genome architecture, the incorporation of sequences that would bring two loci into close proximity or bring loci into contact with the nuclear envelope also It should alter the transcriptional output of the locus.

RNA Modifications Modifications of RNAs can also be made to alter their activity by changing their cellular localization, interaction partners, structural dynamics, or thermodynamics of folding. Incorporation of motifs that would cause translation interruption or frameshifting can alter the abundance of mRNA species through various mRNA processing mechanisms. Altering the consensus splice sequence will also alter the abundance and prevalence of different RNA species. Altering the relative ratios of different splice isoforms will predictably lead to changes in the ratio of protein translation products. This can be used to modify many biological pathways. For example, shifting the balance of mitochondrial versus nuclear DNA repair proteins would alter the resilience of different cancers to chemotherapy reagents. Furthermore, RNA can be modified with sequences that allow binding to new protein targets. A number of RNA aptamers have been developed that bind with high affinity to cellular proteins. Incorporation of one of these aptamers can be used to sequester different RNA species by binding to protein targets that would prevent their translation, biological activity, or bring RNA species to specific subcellular level compartments. can be used for either purpose. Biomolecular degradation is another class of localization modification.

For example, RNA methylation is used to control RNA within cells. A consensus motif of 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 target RNA species, which would alter the pool of RNA within the cell. . Additionally, 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 signals.

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 within cells is an important ability of PE. Edits can be made to incorporate stop codons into the open reading frame. This will eliminate the full length product resulting from the edited DNA sequence. Alternatively, peptide motifs can be incorporated that cause the rate of proteolysis to be altered for the target protein. Incorporation of degradation tags onto gene bodies can be used to alter the abundance of proteins within cells. Moreover, introduction of degrons induced by small molecules may allow temporal control of protein degradation. This may have important implications for both research and therapeutics. This is because researchers can easily assess whether small molecule-mediated therapeutic proteolysis of a given target is a promising therapeutic strategy. Protein motifs can also be incorporated to alter the subcellular localization of the protein. Amino acid motifs can be incorporated to preferentially transport proteins to any number of subcellular level compartments, including the nucleus, mitochondria, cell membranes, peroxisomes, lysosomes, proteasomes, exosomes, and others.

Incorporating or destroying motifs modified by the PTM machinery can alter protein post-translational modifications. Phosphorylation, ubiquitination, glycosylation, lipid modification (e.g. farnesylation, myristoylation, palmitoylation, prenylation, GPI anchor), hydroxylation, methylation, acetylation, crotonylation, SUMOylation, disulfide bond formation, side chains Bond cleavage events, polypeptide backbone cleavage events (proteolysis), and a number of other protein PTMs have been identified. These PTMs alter protein function, often by altering subcellular localization. Indeed, kinases activate downstream signal cascades, often through phosphorylation events. Removal of the target phosphosite will prevent signal transduction. The ability to site-specifically excise or incorporate either PTM motif while preserving 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 for a wide range of PTM modification spaces.

Removal of lipid modification sites should prevent protein transport to the cell membrane. A major limitation of current therapeutics targeting post-translational modification processes is their specificity. Farnesyltransferase inhibitors have been extensively tested for their ability to eliminate KRAS localization to the cell membrane. Unfortunately, total inhibition of farnesylation is associated with a number of off-target effects, which 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 similarities between various kinases. PE offers a possible solution to this specificity problem. This is because it allows inhibition of target protein modification by excision of the modification site instead of total enzyme inhibition. For example, removal of the lipid-modified 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 on proteins that are not designed to bind to membranes.

PE can also be used to investigate protein-protein conjugation events. Proteins often function within complexes to carry out their biological activities. PE can be used to either create or destroy the ability of proteins to reside within these complexes. To eliminate complexation events, amino acid substitutions or insertions on the protein:protein interface can be engineered 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 partner in the complex, thus preventing its oncogenic activity. PE can also be used to remove pathogenic mutations and restore WT activity of the protein. Alternatively, PE inhibits the activity of proteins in order to keep them in their native complexes or by pulling them to engage in interactions that are independent of their native activity. can be used for either purpose. Forming complexes that maintain one interaction state relative to another may represent an important therapeutic modality. Modifying the protein:protein interface to reduce the Kd of interaction will keep those proteins stuck together longer. Protein complexes can have multiple signaling complexes, such as n-myc, which drives the neuroblastoma signal 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 interaction partners and reduce its affinity for oncogenic interaction 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, prime editing systems can include the use of PEgRNA designs 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 can form on PEgRNA can result in inhibition of DNA editing being copied from PEgRNA to genomic loci. These limitations can be overcome by redesign and engineering of PEgRNA components. These redesigns can improve prime editing element efficiency and allow the incorporation of longer insert sequences onto the genome.

Thus, in various embodiments, PEgRNA designs may result in longer PEgRNAs by allowing efficient expression of functional PEgRNAs from non-polymerase III (pol III) promoters. This would avoid the need for onerous alignment requirements. In other embodiments, the core Cas9-bound PEgRNA backbone can be modified to improve the efficacy of the system. In yet other embodiments, modifications can be made to PEgRNA to improve reverse transcriptase (RT) processability. This would allow insertion of longer sequences at the target genomic locus. In other embodiments, the RNA motif is 5' and/or may be added to the 3' end. In yet another embodiment, a platform is provided for the evolution of PEgRNA for a given sequence target that can improve the PEgRNA backbone and enhance prime editor efficiency. These designs can be used to improve any PEgRNA recognized by any Cas9 or its evolved variants.

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

PEgRNA may include additional design improvements that may modify the properties and/or characteristics of PEgRNA, thereby improving the efficacy of prime editing. In various embodiments, these improvements may belong to one or more of a number of different categories: (1) non-polymerase III (pol III) promoters that would allow expression of longer PEgRNAs without difficult sequence requirements; (2) improvement of the core Cas9-bound PEgRNA backbone that may improve potency; (3) improve RT processing ability and (4) modification of PEgRNA that allows insertion of longer sequences; and (4) modification of PEgRNA that improves PEgRNA stability, enhances RT processing capacity, prevents PEgRNA misfolding, or recruits additional factors important for genome editing. Including, but not limited to, the addition of RNA motifs to the 5' or 3' ends.

In one embodiment, PEgRNAs with pol III promoters can be designed to improve expression of longer length PEgRNAs with larger extended arms. sgRNAs are typically expressed from the U6 snRNA promoter. This promoter recruits pol III to express the associated RNA and is useful for the 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 a few hundred nucleotides in length at the levels required for efficient genome editing. In addition, pol III may stall or terminate in stretches of U, potentially limiting the sequence diversity that can be inserted using PEgRNA. Other promoters that recruit polymerase II (eg, pCMV) or polymerase I (eg, the U1 snRNA promoter) have been tested for their ability to express longer sgRNAs. However, these promoters are typically partially transcribed, which will result in an extra sequence 5' of the spacer on the PEgRNA that is expressed. This has been shown to result in significantly reduced Cas9:sgRNA activity in a site-dependent manner. In addition, PEgRNAs transcribed by pol III may simply terminate with a stretch of 6-7 U, whereas PEgRNAs transcribed from pol II or pol I will require different termination signals. In many cases, such signals will also result in polyadenylation, which will result in unwanted export of 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 RNA expressed from pCMV that terminates in the ENE element ¹⁸⁴ from human MALAT1 ncRNA, the PAN ENE element ¹⁸⁵ from KSHV, or the 3' box ¹⁸⁶ from U1 snRNA. Of note, MALAT1 ncRNA and PAN ENE form a triple helix and shield the polyA tail184,187 ^. These constructs may also enhance RNA stability. It is contemplated that these expression systems will also allow expression of longer PEgRNAs.

In addition, a series of methods have been designed for cleavage of the portion of the pol II promoter that would be transcribed as part of PEgRNA, and a self-cleaving ribozyme, e.g. hammerhead ¹⁸⁸ , has been designed to process the transcribed guide. , Pistol ¹⁸⁹ , Hatchet ¹⁸⁹ , Hairpin ¹⁹⁰ , VS ¹⁹¹ , Twister ¹⁹² , or Twister Sister ¹⁹² ribozyme, or other self-cleaving elements, or recognized by Csy4 ¹⁹³ , and have added either a hairpin leading to the 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 PEgRNA in the form of 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 above, as exemplified by the sequences below.

Non-limiting example 1 - PEgRNA expression platform consisting of pCMV, Csy4 hairpin, PEgRNA, and MALAT1 ENE
(Sequence number 501)

Non-limiting example 2 - PEgRNA expression platform consisting of pCMV, Csy4 hairpin, PEgRNA, and PAN ENE
(Sequence number 502)

Non-limiting example 3 - PEgRNA expression platform consisting of pCMV, Csy4 hairpin, PEgRNA, and 3×PAN ENE
(Sequence number 503)

Non-limiting example 4 - PEgRNA expression platform consisting of pCMV, Csy4 hairpin, PEgRNA, and 3' box
(Sequence number 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, PEgRNAs can be improved by introducing improvements to the backbone or core sequence. This can be done by introducing known information.

The core Cas9-bound PEgRNA backbone could conceivably be improved to enhance PE activity. Some such approaches have already been demonstrated. For example, the first pairing element (P1) of the scaffold contains a GTTTT-AAAAC (SEQ ID NO: 3939) pairing element. Such stretches of T have been shown to result in pol III disruption and premature termination of RNA transcripts. Rational mutation of one of the TA pairs to a GC pair in this part of P1 has been shown to enhance sgRNA activity, suggesting that this approach may also be feasible for PEgRNA. There are ¹⁹⁵ . In addition, increasing the length of P1 has also been shown to enhance sgRNA ^folding and lead to improved activity, suggesting it as another avenue for improving PEgRNA activity. ing. Improvements to the core example:

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

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

In various other embodiments, PEgRNA can be improved by introducing modifications of the editing template region. As the size of the PEgRNA-templated insert increases, it may be degraded by endonucleases, undergo spontaneous hydrolysis, or lack the ability to be reverse transcribed by RT or folding of the PEgRNA backbone and subsequent Cas9 -It is more likely that it folds into a secondary structure that blocks RT binding. Therefore, it is likely that modification of the PEgRNA template may be necessary to affect large insertions, such as insertions of entire genes. Some strategies to do so include the incorporation of modified nucleotides on synthetic or semi-synthetic PEgRNAs that make the RNA more resistant to degradation or hydrolysis, or that create inhibitory secondary structures. ¹⁹⁶ . Such modifications would reduce RNA secondary structure in G-rich sequences such as 8-aza-7-deazaguanosine; locking nucleic acids (LNAs) that reduce degradation and enhance certain types of RNA secondary structure. ;2'-O-methyl, 2'-fluoro, or 2'-O-methoxyethoxy modifications that enhance RNA stability may be included. Such modifications may also be included elsewhere on PEgRNA to enhance stability and activity. Alternatively or additionally, it is both more likely to encode the desired protein product and adopt a simple secondary structure that is capable of being unfolded by RT. In this way, PEgRNA templates can be designed. Such a simple structure would act as a thermodynamic sink, making it less likely that more complex structures would occur that would prevent reverse transcription. Finally, the template can be split into two separate PEgRNAs. In such designs, PE is also used to initiate transcription and recruit a separate template RNA to the target site by an RNA recognition element on PEgRNA itself, such as an RNA binding protein fused to Cas9 or the MS2 aptamer. It will be done. RT can either bind directly to this separate template RNA or can initiate reverse transcription from the original PEgRNA before swapping to the second template. Such an approach prevents PEgRNA misfolding due to the addition of long templates, and also does not require dissociation of Cas9 from the genome, which could potentially inhibit PE-based long insertions, for long insertions to occur. Both may allow long insertions.

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

Other structural elements inserted at the 3' end may also enhance RNA stability, even without allowing termination from non-pol III promoters. Such a motif would result in the formation of a hairpin or RNA quadruplex ¹⁹⁷ that would trap the 3' end, or a 2'-3'-cyclic phosphate at the 3' end, and potentially also allow PEgRNA to be catalyzed by an exonuclease. Self-cleaving ribozymes ¹⁹⁸ such as HDV may be included, which would make them less likely to be degraded. Inducing PEgRNA to circularize through incomplete splicing to form ciRNA may also increase PEgRNA stability and result in PEgRNA being retained in the nucleus ¹⁹⁴ .

Additional RNA motifs may also improve RT processing capacity or enhance PEgRNA activity by enhancing RT binding to DNA-RNA duplexes. Addition of native sequences bound by RT on its corresponding retroviral genome can enhance RT activity ¹⁹⁹ . This may include the native primer binding site (PBS), the polypurine tract (PPT), or the kissing loop involved in retroviral genome dimerization and initiation of transcription ¹⁹⁹ .

Addition of dimerization motifs such as kissing loops or GNRA tetraloop/tetraloop receptor pair ²⁰⁰ at the 5′ and 3′ ends of PEgRNA may also result in effective circularization of PEgRNA, improving stability. In addition, it is envisioned that the addition of these motifs may enable physical separation of the PEgRNA spacer and primer, preventing entrapment of the spacer that would interfere with PE activity. Short 5' or 3' stretches of PEgRNA that form small toehold hairpins in the spacer region may also compete favorably for the annealing region of PEgRNA binding 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. Improvements to the example:

PEgRNA-HDV fusion
GGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCCTCTGCCATCAAAGCGTGCTCAGTCTGGGCCGGCATGGTCCCAGCCTCCTCGCTGGCGCCGGCTGGGCAACATGCTTCGGCATGGCGAATGGGACTTTTTTT (SEQ ID NO: 230)

PEgRNA-MMLV kissing loop
GGTGGGAGACGTCCCACCGGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCCTCTGCCATCAAAGCTTCGACCGTGCTCAGTCTGGTGGGAGACGTCCCACCTTTTTT (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 templates to switch secondary RNA-HDV fusions
TCTGCCATCAAAGCTGCGACCGTGCTCAGTCTGGTGGGAGACGTCCCACCGGCCGGCATGGTCCCAGCCTCCTCGCTGGCGCCGGCTGGGCAACATGCTTCGGCATGGCGAATGGGACTTTTTTT (SEQ ID NO: 234)
including but not limited to.

PEgRNA scaffolds can be further improved by directed evolution in a manner analogous to how SpCas9 and prime editing elements (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. be. Finally, evolution of the PEgRNA backbone with the addition of other RNA motifs will almost certainly improve the activity of the fused PEgRNA relative to the unevolved fusion RNA. For example, evolution of allosteric ribozymes consisting of c-di-GMP-I aptamers and hammerhead ribozymes has led to dramatically improved activity202, suggesting that evolution will also improve the activity ^of hammerhead-PEgRNA fusions. suggested. In addition, although Cas9 currently does not generally tolerate 5' extensions of sgRNAs, directed evolution could conceivably generate potent mutations that alleviate this intolerance, creating additional RNA motifs. will be allowed to be used.

This disclosure contemplates any such approach to further improve the effectiveness of the prime editing system disclosed herein.

O. Using prime editing with expanded targeting scope
Prime editing (PE) using Streptococcus pyogenes Cas9 (SpCas9) removes all single base substitutions, insertions, and Deletions, and combinations thereof, can be efficiently incorporated and incorporated. However, in another aspect, the methods described herein enhance the targeting ability of PE by expanding the accessible PAM and thus the accessible targetable genomic loci for efficient PE. Make it broader. Prime editing factors using DNA-binding proteins guided by RNAs other than SpCas9 enable an expanded targetable range of genomic loci by allowing access to different PAMs. In addition, the use of smaller RNA-guided DNA binding proteins than SpCas9 also allows more efficient virus delivery. PE by DNA binding proteins guided by Cas proteins or other RNAs other than SpCas9 may not allow highly efficient therapeutic editing that was either inaccessible or inefficient using SpCas9-based PE. Probably.

This may be due to the inefficiency of SpCas9-based PE due to non-ideal spacing of edits relative to the NGG PAM, or the overall size of SpCas9-based constructs may affect cellular expression and/or It is anticipated that it will be used in situations where delivery is either prohibitive. Certain disease-relevant loci, such as the huntingtin gene, which has a small number and poorly positioned NGG PAM of SpCas9 near its target region, are distinct from PE systems such as SpCas9-VRQR, which recognizes the NGA PAM. Can be easily targeted using Cas proteins. Smaller Cas proteins will be used to generate smaller PE constructs that can be more efficiently packaged into AAV vectors, allowing 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 the untreated control.

Figures 62A-62B provide demonstration of the importance of protospacers for efficient incorporation of desired edits in precise positioning by prime editing. This highlights the importance of alternative PAM and protospacers as novel features of this technology. "n.d." in FIG. 62A is "not detected."

Figure 63 shows the implementation of PE using SpCas9(H840A)-VRQR and SpCas9(H840A)-VRER as RNA-guided DNA binding proteins of the prime editing factor 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, and 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, and 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 sequence of the tested construct is 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, napDNAbp with modified PAM specificity (e.g., Cas9) carries a combination of mutations at its 3' end that is active against a target sequence containing a 5'-NAA-3' PAM sequence. include. 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 are conservative mutations of the listed clones of Table 1. In some embodiments, the Cas9 protein comprises a combination of mutations in 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 the Cas9 protein provided by any one of the variants in Table 1. In some embodiments, the Cas9 protein has at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least the amino acid sequence of a Cas9 protein provided by any one of the variants in Table 1. Comprising amino acid sequences that are 97%, at least 98%, at least 99%, or at least 99.5% identical.

In some embodiments, the Cas9 protein has an increased target sequence that does not contain a canonical PAM (5'-NGG-3') at its 3' end compared to Streptococcus pyogenes Cas9 provided by SEQ ID NO: 18. Demonstrates a certain level of activity. In some embodiments, the Cas9 protein has a canonical PAM sequence (5'-NGG-3') that is at least 5-fold increased compared to the activity of Streptococcus pyogenes Cas9 provided by SEQ ID NO: 18 against the same target sequence. It exerts activity against target sequences with a 3' end that is not directly adjacent to the target sequence. In some embodiments, the Cas9 protein has at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1,000-fold, as compared to the activity of Streptococcus pyogenes provided by SEQ ID NO: 18 against the same target sequence. 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 target sequences that are not directly adjacent to the canonical PAM sequence (5'-NGG-3') demonstrate. In some embodiments, the 3' end of the target sequence is directly adjacent to an AAA, GAA, CAA, or TAA sequence. In some embodiments, the Cas9 protein comprises a combination of mutations that exert 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 are conservative mutations of the listed clones of 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 has an increased target sequence that does not contain a canonical PAM (5'-NGG-3') at its 3' end compared to Streptococcus pyogenes Cas9 provided by SEQ ID NO: 18. Demonstrates a certain level of activity. In some embodiments, the Cas9 protein has a canonical PAM sequence (5'-NGG-3') that is at least 5-fold increased compared to the activity of Streptococcus pyogenes Cas9 provided by SEQ ID NO: 18 against the same target sequence. It exerts activity against target sequences with a 3' end that is not directly adjacent to the target sequence. In some embodiments, the Cas9 protein has at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1,000-fold, as compared to the activity of Streptococcus pyogenes provided by SEQ ID NO: 18 against the same target sequence. to a target sequence that is not directly adjacent to a canonical PAM sequence (5'-NGG-3') that is increased 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 Demonstrate activity. In some embodiments, the 3' end of the target sequence is directly adjacent to an AAC, GAC, CAC, or TAC sequence.

In some embodiments, the Cas9 protein contains a combination of mutations that exert 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 are conservative mutations of the listed clones of Table 3. In some embodiments, the Cas9 protein comprises a combination of mutations in any one of the Cas9 clones listed in Table 3.

Table 3: NAT PAM clone

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") at a desired genomic site. Insertion of recombinase sites provides programmed positioning for accomplishing site-specific genetic changes in the genome. Such genetic changes can include, for example, plasmid genomic integration, genomic deletions or insertions, chromosomal translocations, and cassette exchanges, among other genetic changes. These exemplary types of genetic changes are illustrated in Figures 64B -64F . The incorporated recombinase recognition sequences can then be used to perform 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 expression marker. Cells containing the GFP expression system incorporated into the recombinase site will fluoresce.

The mechanism for incorporating recombinase sites into the genome is analogous to incorporating other sequences into the genome, such as peptide/protein and RNA tags. A schematic diagram illustrating the incorporation of recombinase target sequences is shown in Figure 64A . The process begins by selecting the desired target locus into which the recombinase target sequence will be introduced. A prime editing factor fusion is then provided ("RT-Cas9:gRNA"). Here, "gRNA" refers to PEgRNA, which can be designed using the principles described herein. The PEgRNA will in various embodiments contain an architecture corresponding to Figure 3D (5'-[~20nt spacer]-[gRNA core]-[extended arm]-3', with the extended arm varying from 3' to 5'). ' orientation includes a primer binding site ('A'), an editing template ('B'), and a homology arm ('C'). The editing template ('B') contains a sequence corresponding to the recombinase site, i.e. It will contain single-stranded RNA of PEgRNA that is either the sense or antisense strand of the recombinase site and encodes complementary single-stranded DNA that is incorporated into the genomic DNA target locus by the prime 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 (Tables ^1-7). 6). For example, Williams-Beuren syndrome is a developmental disorder caused by a 24 deletion on chromosome 721. Currently, no technology exists for efficient and targeted insertion of multiple entire genes in living cells (the potential of PE for such full-length gene insertions is currently being investigated but not yet established). ); However, recombinase-mediated uptake at targets inserted by PE offers one approach to permanent cure of this and other diseases. In addition, targeted introduction of recombinase recognition sequences can be highly capable for applications including the generation of transgenic plants, animal research models, bioproduction cell lines, or other custom eukaryotic cell lines. For example, recombinase-mediated genome rearrangement of transgenic plants in PE-specific targets ^may overcome one of the bottlenecks in generating agricultural crops with improved properties.

Table 6. Examples of genetic diseases linked to large-scale genome modifications that can be repaired by PE-based incorporation of recombinase recognition sequences.

Several SSR family members have been characterized and their recombinase recognition sequences have been described, including natural and engineered tyrosine recombinases (Table 7), large serine integrases (Table 8), and serine resolvases (Table 9). , and tyrosine integrase (Table 10). Modified target sequences demonstrating enhanced genomic uptake rates have also been described for several ^SSRs22-30 . In addition to natural recombinases, programmable recombinases with distinct specificities have been developed ^31-40 . Depending on the desired application, using PE one or more of these recognition sequences can be introduced onto the genome at defined positions, such as safe harbor loci ^{41 - 43} .

For example, introduction of a single recombinase recognition sequence on the genome by prime editing will result in integral recombination with the DNA donor template (Figure 64b). Serine integrases that operate robustly in human cells may be particularly well suited for gene transfer44,45 ^.

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, some recombinases have been demonstrated to integrate into human or eukaryotic genomes at natively occurring ^{pseudosites46-64} . PE editing alters these loci to enhance incorporation rates at these natural pseudosites, or alternatively to eliminate pseudosites that can serve as unwanted off-target sequences. It can be used for.

This disclosure describes a general methodology for introducing recombinase target sequences onto eukaryotic genomes using PE. The applications of this are nearly endless. The genome editing reaction is intended for use with a "prime editing factor", a chimeric fusion of a CRISPR/Cas9 protein and a reverse transcriptase domain, which utilizes a custom prime editing guide RNA (PEgRNA). By further extension, the Cas9 tool and homologous recombination repair (HDR) pathway can also be exploited to introduce recombinase recognition sequences from DNA templates by reducing the indel rate using ^several techniques. ^~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 following several tables are cited above with respect to PE-guided incorporation of recombinase recognition sequences and provide a list of exemplary recombinases that may be used and their corresponding recombinase recognition sequences that may be incorporated by PE. .
Table 7. Tyrosine recombinase and SSR target sequences.

Table 8. Large serine integrase and SSR target sequences.

Table 9. Serine resolvase and SSR target sequences.

Table 10. Tyrosine integrase 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 accomplish various recombination outcomes, such as excision, incorporation, 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 (eg, 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 (eg, DNA).

In some embodiments, the present disclosure provides methods for incorporating a donor DNA template by site-specific recombination: (a) incorporating a recombinase recognition sequence into a genomic locus by prime editing; (b) incorporating a recombinase recognition sequence into a genomic locus by prime editing; in the presence of a DNA donor template that also includes a recombinase recognition sequence.

In other aspects, the disclosure provides methods for deleting a genomic region by site-specific recombination: (a) incorporating a pair of recombinase recognition sequences at a genomic locus by prime editing; (b) genomic contacting the locus with a recombinase, thereby catalyzing deletion of the genomic region between the pair of recombinase recognition sequences.

In yet other aspects, the present disclosure provides methods for inverting genomic regions by site-specific recombination: (a) incorporating a pair of recombinase recognition sequences at a genomic locus by prime editing; (b) It involves contacting a genomic locus with a recombinase, thereby catalyzing the inversion of the genomic region between a 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 embodiments, the present disclosure provides a method for inducing cassette exchange between a genomic locus and donor DNA comprising a cassette, comprising: (a) directing a first recombinase recognition to a first genomic locus by prime editing; (b) incorporating a second recombinase recognition sequence at a second genomic locus by prime editing; (c) donor DNA comprising a cassette flanked by first and second recombinase recognition sequences; and contacting the first and second genomic loci with a recombinase, thereby catalyzing the exchange of the flanked genomic loci with a cassette on the DNA donor.

In various embodiments involving the insertion of more than one recombinase recognition sequence on the genome, the recombinase recognition sequences can be the same or different. In some embodiments, the recombinase recognition sequences are the same. In other embodiments, the recombinase recognition sequences are different.

In various embodiments, the recombinase is 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 corresponding to the recombinase in use.

In various other embodiments, the recombinase is a large serine recombinase, such as Bxb1, PhiC31, R4, phiBT1, MJ1, MR11, TP901-1, A118, V153, phiRV1, phi370.1, TG1, WB, BL3, SprA, phiJoe, It can be 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 in use.

In still other embodiments, the recombinase is a serine recombinase, such as Bxb1, PhiC31, R4, phiBT1, MJ1, MR11, TP901-1, A118, V153, phiRV1, phi370.1, TG1, WB, BL3, SprA, phiJoe, phiK38, It can be 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 in use.

In other embodiments, the recombinase can be a serine resolvase, such as Gin, Cin, Hin, Min, or Sin, as shown in Table 9. In such embodiments, the recombinase recognition sequence can be the RRS of Table 9 corresponding to the recombinase in use.

In various other embodiments, the recombinase can be a tyrosine integrase, such as HK022, P22, or L5, as shown in Table 10. In such embodiments, the recombinase recognition sequence can be the RRS of Table 10 corresponding to the recombinase in use.

In some embodiments, any of the methods for site-specific recombination by PE can be performed in vivo or in vitro. In some embodiments, any method for site-specific recombination is performed on the cell (eg, recombining the genomic DNA of the cell). Cells may be prokaryotic or eukaryotic. Cells, such as eukaryotic cells, can be in individuals such as the subjects described herein (eg, human subjects). The methods described herein are useful for genetic modification of cells in vitro and in vivo, e.g., in the context of the generation of transgenic cells, cell lines, or animals, or for alteration of the genomic sequence of a cell of interest, e.g. Useful in correcting defects.

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, in which one or more nucleobase modifications are desired to be incorporated by a prime editing agent;
(b) Positioning of the site to be cut on the target sequence, i.e., the prime editing factor induces a single-stranded nick, with the 3' end RT primer sequence on one side of the nick and the 5' end on the other side of the nick. A specific nucleobase position that will give rise to an endogenous flap at the end, which will ultimately be removed by FEN1 or its equivalent and replaced by a 3'ssDNA flap. The cut site generates a 3' end primer sequence, which becomes extended by the polymerase of the PE fusion protein (e.g. RT enzyme) during RNA-dependent DNA polymerization to contain the desired edits. Generating a 3'ssDNA flap, which then replaces the 5' endogenous DNA flap on the target sequence;
(c) available PAM sequences (including standard SpCas9 PAM sites and non-canonical PAM sites recognized by Cas9 variants and equivalents with extended or different PAM specificities);
(d) the distance between the available PAM sequence 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 editing factor to be used (this is described in part by the available PAM);
(f) sequence and length of the primer binding site;
(g) sequence and length of the editing template;
(h) Sequence and length of homologous 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. This takes into account one or more of the above considerations. The steps refer to the example shown in Figures 70A-70I.
1. Define target sequence and edits. 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). See Figure 70A.
2. Position the target PAM. Identify the PAM proximal to the desired editing location. PAMs can be identified on either strand of DNA proximal to the desired editing location. PAMs that are close to the edit position are preferred (i.e., the nick site is less than 30 nt from the edit position; or 29 nt, 28 nt, 27 nt, 26 nt, 25 nt, 24 nt, 23 nt, 22 nt, 21 nt, 20 nt, Protospacer that places a nick ≧30nt from the editing position And it is possible to incorporate editing using PAM. See Figure 70B.
3． Position the nick site. For each PAM considered, identify the corresponding nick site and on which strand. For Sp Cas9 H840A nickase, cleavage occurs on the PAM-containing strand between the 5' third and fourth bases of the NGG PAM. All edited nucleotides must be present 3' to the nick site. Therefore, a suitable PAM must be nicked 5' of 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 PEgRNA using PAM 1 only. See Figure 70C.
Four. Design the spacer array. The protospacer of Sp Cas9 corresponds to the 5' 20 nucleotides of the NGG PAM on the PAM-containing strand. Efficient Pol III transcription initiation requires G to be the first nucleotide transcribed. If the first nucleotide of the protospacer is G, the spacer sequence of PEgRNA is simply a protospacer sequence. If the first nucleotide of the protospacer is not a G, the spacer sequence of the PEgRNA is G followed by the protospacer sequence. See Figure 70D.
Five. Design the primer binding site (PBS). The starting allele sequence is used to identify DNA primers on the PAM-containing strand. The 3' end of the DNA primer is the nucleotide just upstream of the nick site (ie, the 4th 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 is combined with a sequence containing ~40-60% GC content. can be used. 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, the starting allele sequence is used 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) encodes the designed edit and homology to the sequences flanking the edit. In one embodiment, these regions correspond to the DNA synthesis template of Figures 3D and 3E, where the DNA synthesis template includes an "editing template" and a "homologous arm." 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 (position +7 and beyond), it is recommended to use RT templates that extend at least 5 nt (preferably 10 nt or more) from the editing position 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 (≧5nt), incorporation of more 3' homology (~20nt or more) onto the RT template is recommended. Editing efficiency is typically compromised when the RT template encodes the synthesis of a G (corresponding to C on the RT template of PEgRNA) as the last nucleotide on the reverse transcribed DNA product. Avoiding G as the last synthesized nucleotide is recommended when designing RT templates, as many RT templates support efficient prime editing. 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 editing using RT templates 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 unedited strands upstream and downstream of the edit. The optimal nicking position is highly locus dependent and should be determined empirically. Generally, nicks placed 40 to 90 nucleotides 5' across from the nick induced by PEgRNA lead to higher editing yields and fewer indels. The nicking sgRNA has a spacer sequence that matches the 20nt protospacer on the starting allele. If the protospacer does not begin with a G, it has an addition of 5'G. See Figure 70H.
9. Design PE3b nicking sgRNA. If the PAM is present on the complementary strand and its corresponding protospacer overlaps the sequence targeted for editing, then this editing 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 rather than the starting allele. The PE3b system operates 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 and prevents competition between PEgRNA and sgRNA for binding to target DNA. PE3b also avoids simultaneous nick generation on both strands, thus significantly reducing indel formation while maintaining high editing efficiency. PE3b sgRNA should have a spacer sequence that matches the 20nt protospacer of the desired allele. With addition of 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. This disclosure contemplates variations on the step-by-step process described above, which may be derived therefrom by those skilled in the art.

Once suitable PEgRNA and PE fusion proteins have been selected/designed, they can be prepared by suitable methodologies, such as vector-based transfection, in which PEgRNA and one or more vectors containing DNA encoding the PE fusion protein), by direct delivery of the PE fusion protein complexed with PEgRNA (e.g., RNP delivery) in a delivery format (e.g., lipid particles, nanoparticles), or by direct delivery of the PE fusion protein (e.g., RNP delivery), or by mRNA can be administered by a delivery system based on Such methods are described herein by the present disclosure, and any known method may be utilized.

PEgRNA and PE fusion protein (or together referred to as PE conjugate) are delivered to cells in therapeutically effective amounts by contacting the desired target DNA such that the desired edits are incorporated into it. can be delivered.

Any disease could conceptually be treated by such methods, so long as delivery to the appropriate cells is feasible. Those skilled in the art will be able to choose and/or select PE delivery methodologies to suit the intended purpose and intended target cells.

For example, in some embodiments, an effective amount of a prime editing system described herein (desired Correcting point mutations or introducing inactivating mutations into disease-associated genes, as mediated by homology-directed repair in the presence of a donor DNA molecule containing the genetic alteration) A method is provided comprising administering. In some embodiments, an effective amount of a prime editing system as described herein (which has a point mutation) is administered to a subject having such a disease (e.g., a cancer associated with a point mutation as described above). or introducing an inactivating mutation into a disease-associated gene). In some embodiments, the disease is a proliferative disease. In some embodiments, the disease is a genetic disease. In some embodiments, the disease is a neoplastic disease. In some embodiments, the disease is a metabolic disease. In some embodiments, the disease is a lysosomal storage disease. Other diseases that can be treated by correcting point mutations or introducing inactivating mutations into disease-associated genes are known to those skilled in the art, and the present 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 delay, 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;Lymphangioleiomyomatosis;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 (eg, a point mutation) associated with a disease, disorder, or disease. The target sequence may contain a T to C (or A to G) point mutation that is associated with a disease, disorder, or illness, and deamination of the mutant C base leads to a sequence that is not associated with the disease, disorder, or disease. resulting in correction mediated by mismatch repair. The target sequence may contain a G to A (or C to T) point mutation that is associated with a disease, disorder, or illness, and deamination of the mutant A base leads to a sequence that is not associated with the disease, disorder, or illness. resulting in correction mediated by mismatch repair. The target sequence may encode a protein, and where a point mutation is in a codon, it results in a change in the amino acid encoded by the mutant codon compared to the wild-type codon. Target sequences can also be at splice sites, with point mutations resulting in altered splicing of the mRNA transcript compared to the wild-type transcript. In addition, targets can also be in non-coding sequences of genes, such as promoters, where point mutations result in increased or decreased expression of the gene.

Therefore, in some aspects, deamination of variant C results in a change in the amino acid encoded by the variant codon, which in some cases may result in expression of the wild-type amino acid. In other aspects, deamination of variant A results in a change in the amino acid encoded by the variant codon, which in some cases may result in expression of the wild-type amino acid.

Methods described herein that involve contacting cells with compositions or rAAV particles can occur in vitro, ex vivo, or in vivo. In some embodiments, the contacting step occurs to the subject. In certain embodiments, the subject has been diagnosed with a disease, disorder, or disease.

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 retinal cells, cortical cells, or cerebellar cells.

The split Cas9 protein or split prime editing factor delivered using the methods described herein is preferably the original Cas9 protein or prime editing factor (i.e., delivered to or expressed by the cell whole). It has an activity comparable to that of undivided proteins (undivided proteins). For example, the split Cas9 protein or split prime editor has at least 50% of the activity of the original Cas9 protein or prime editor (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%). In some embodiments, the split Cas9 protein or split prime editing factor is greater than that of the original Cas9 protein or prime editing factor (e.g., 2x, 5x, 10x, 100x, 1000x, or more) active.

The compositions described herein can be administered to a subject in need thereof in a therapeutically effective amount to treat and/or prevent a disease or disorder afflicting the subject. Any disease or disorder that can be treated and/or prevented using CRISPR/Cas9-based genome editing technology can be treated by the split Cas9 proteins or split prime editing factors described herein. If the nucleotide sequence encoding the split Cas9 protein or prime editing factor does not further encode a gRNA, this means that a separate nucleic acid vector encoding the gRNA can be administered with the compositions described herein. It should be understood.

Illustrative and preferred diseases, disorders, or diseases include, without limitation: cystic fibrosis, phenylketonuria, epidermal 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 neuropathy Includes diseases or disorders selected from the group consisting of skin and joint syndrome (CINCA), congenital deafness, Niemann-Pick type C (NPC) disease, and desmin-related myopathy (DRM). In a specific embodiment, the disease or illness 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, DNMT1 gene, PCSK9 gene, or TMC1 gene. In certain embodiments, the point mutation is the 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 disease is associated with a stop codon, eg, a point mutation that produces a premature stop codon within 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 Disease 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 the CAG triplet, but the GAA triplet (Friedreich's ataxia) and CGG triplet (Fragile X syndrome) also occur. Inheriting a predisposition to expansion or acquiring an allele from an already expanded parent increases the probability of acquiring the disease. Pathogenic expansion of trinucleotide repeats could hypothetically be corrected using prime editing.

According to the general mechanism outlined in Figure 1G or Figure 22, the region upstream of the repeat region can be nicked by an RNA-guided nuclease, and then the healthy repeat number (which is determined by the specific gene and (depending on the disease) can be used to prime the synthesis of new DNA strands containing After the repeat sequence, a short stretch of homology is added that matches the identity of the sequence flanking the other end of the repeat (red strand). Invasion of the newly synthesized strand by the TPRT system and subsequent replacement of the endogenous DNA by the newly synthesized flap leads to a truncated repeat allele. The term "truncated" refers to a shortening in the length of a nucleotide repeat region, thereby resulting in repair of the trinucleotide repeat region.

The prime editing system or prime editing (PE) system described herein truncates trinucleotide repeat mutations (or "triplet expansion diseases") to treat diseases such as Huntington's disease and other trinucleotide repeat disorders. It can be used to Trinucleotide repeat expansion disorders are complex, progressive disorders that involve developmental neurobiology and often affect cognitive and sensorimotor functions. The disorder exhibits a genetic facilitation phenomenon (ie, increasing severity with each generation). DNA elongation or shortening usually occurs meiotically (i.e., during the time of gametogenesis or early during embryonic development) and often has a sex bias, with some genes being isolated from females. This means that only when inherited, others only grow from males. In humans, trinucleotide repeat expansion disorders can cause gene silencing at either the transcriptional or translational level, essentially knocking out gene function. Alternatively, trinucleotide repeat expansion disorders may result in modified proteins produced with large repeat 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 tandem repeats have identical sequences to each other, base pairing between the two DNA strands can occur at multiple points on 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 repeating sequences, extending the number of repeats. Additional mechanisms involving hybrid RNA:DNA intermediates have been proposed. Prime editing can be used to reduce or eliminate regions of these triplet expansions by deleting one or more of the harmful repeat codon triplets. In some embodiments of this use, Figure 23 provides a schematic representation of PEgRNA design to shorten or reduce trinucleotide repeat sequences by prime editing.

Prime editing can be implemented to shorten the triplet expansion region by nicking the region upstream of the triplet repeat region with prime editing factors containing appropriate PEgRNA targeted to the site to be cut. . The prime editing factor then creates a new DNA strand (ssDNA flap) based on PEgRNA as a template (i.e., its editing template) encoding a healthy number of triplet repeats (this depends on the specific gene and disease). Synthesize. The newly synthesized ssDNA strand contains a healthy triplet repeat sequence and is also synthesized to include a short stretch of homology (i.e. homology arm) that matches the sequence flanking the other end of the repeat (shown in red). chain). Invasion of the newly synthesized strand and subsequent replacement of the endogenous DNA by the newly synthesized ssDNA flap leads to a truncated repeat allele.

Depending on the specific trinucleotide expansion disorder, defect-inducing triplet expansions can occur in "trinucleotide repeat expansion proteins." Trinucleotide repeat expansion proteins are a diverse set of proteins associated with susceptibility to developing trinucleotide repeat expansion disorders, presence of trinucleotide repeat expansion disorders, severity of trinucleotide repeat expansion disorders, or any combination thereof. be. 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 encodes the amino acid glutamine (Q). These disorders are therefore referred to as polyglutamine (polyQ) disorders and include: Huntington's disease (HD); spinobulbar muscular atrophy (SBMA); spinocerebellar ataxia (SCA 1, 2, 3). , types 6, 7, and 17); and dentate nucleus pallidum Louisian atrophy (DRPLA). The remaining trinucleotide repeat expansion disorders either do not involve the CAG triplet or the CAG triplet is not in the coding region of the gene and are therefore referred to as non-polyglutamine disorders. Non-polyglutamine disorders include fragile including.

Proteins associated with trinucleotide repeat expansion disorders may be selected based on the experimental association of proteins 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 increased or decreased in a population with a trinucleotide repeat expansion disorder relative to a population lacking the trinucleotide repeat expansion disorder. Differences in protein levels can 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 can be identified by obtaining gene expression profiles of protein-coding genes, using DNA microarray analysis, serial analysis of gene expression (SAGE), and quantitative real-time polymerase analysis. Genomic techniques are used, including but not limited to 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), MED15 (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 opposite strand (non-protein coding)), PPP2R2B (protein phosphatase 2 regulatory subunit B beta), ATXN7 (ataxin 7), TNRC6B (trinucleotide repeat containing 6B), TNRC6C (trinucleotide repeat containing 6C), CELF3 (CUGBP Elav-like family member 3), MAB21L1 (mab-21-like 1 (C. elegans)), MSH2 (mutS homolog 2 colon cancer non-polyposis 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 (transcriptional 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 (oncoprotein p53), ESR1 ( estrogen receptor 1), CGGBP1 (CGG triplet repeat binding protein 1), ABT1 (basic transcriptional 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-related )), KBTBD10 (containing kelch repeats and BTB (POZ) domain 10), MBNL1 (muscleblind-like (Drosophila)), RAD51 (RAD51 homolog (RecA homolog, E. coli) (S. cerevisiae)), NCOA3 (nuclear receptor coactivator 3), ERDA1 (elongated repeat domain CAG/CTG1), TSC1 (tuberous sclerosis 1), COMP (cartilage oligomeric matrix protein), GCLC (glutamate-cysteine ligase catalytic subunit), RRAD (associated with diabetes) (Ras-related), MSH3 (mutS homolog 3 (E.coli)), DRD2 (dopamine receptor D2), CD44 (CD44 molecule (Indian blood type)), CTCF (CCCTC binding factor (zinc finger protein)), CCND1 (cyclin D1), CLSPN (claspin homologue (Xenopus laevis)), MEF2A (myocyte enhancer factor 2A), PTPRU (protein tyrosine phosphatase receptor type U), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), TRIM22 ( Contains three-node motif22), WT1 (Wilms tumor 1), AHR (aryl hydrocarbon receptor), GPX1 (glutathione peroxidase 1), TPMT (thiopurine S-methyltransferase), NDP (Norie disease (pseudoglioma)) , ARX (aristaless-related homeobox), MUS81 (MUS81 endonuclease homolog (S. cerevisiae)), TYR (tyrosinase (oculocutaneous albinism IA)), EGR1 (early proliferative response 1), UNG (uracil-DNA glycosylase) , NUMBL (numb homologue (Drosophila)-like), FABP2 (intestinal fatty acid binding protein 2), EN2 (engrailed homeobox 2), CRYGC (crystallin gamma C), SRP14 (signal recognition particle 14kDa (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 augmentation 2 (S.cerevisiae)), GLA (galactosidase alpha) , CBL (Cas-Br-M (murine) homotropic retroviral transformation sequence), FTH1 (ferritin heavy chain polypeptide 1), IL12RB2 (interleukin 12 receptor beta 2), OTX2 (orthodenticle homeobox 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 (midbrain astrocyte-derived neurotrophic factor), TMEM158 (transmembrane protein 158 (gene/pseudogene)), and ENSG00000078687.

In a specific aspect, the present disclosure provides TPRT-based methods for the treatment of subjects diagnosed with expanded repeat disorder (also known as repeat expanded disorder or trinucleotide repeat disorder). Expanded repeat disorders occur when microsatellite repeats expand beyond a length threshold. At least 30 genetic diseases are currently thought to be caused by repeat expansions. Scientific understanding of this diverse group of disorders began in the early 1990s when trinucleotide repeats were identified in fragile X, spinobulbar muscular atrophy, myotonic dystrophy, and Huntington's disease (Nelson et al, “The unstable repeats Haw River syndrome, Jacobsen syndrome, Denticulopallidoid Luysian atrophy (DRPLA), Machado-Joseph disease, synpolydactyly (SPD II), hand-foot-genital syndrome (HFGS), cleidocranial dysplasia (CCD), holoprosencephalic defect Several major hereditary disorders, including Congenital Central Hypoventilation Syndrome (HPE), Congenital Central Hypoventilation Syndrome (CCHS), ARX Nonsyndromic X-Linked Mental Retardation (XLMR), and Oculopharyngeal Muscular Dystrophy (OPMD) The instability of microsatellite repeats has been brought to light with the discovery that microsatellite repeat instability could occur in each subsequent generation, leading to a more severe phenotype in offspring and a younger age of onset. It has been found that repeat expansions are a hallmark of these diseases.Repeat expansions are thought to cause disease through several different mechanisms; that is, expansions , and/or may interfere with cell functioning at the level of the encoded protein. In some diseases, mutations act through a loss-of-function mechanism by silencing repeat-containing genes. In others, diseases arise as a result of gain-of-function mechanisms in which either mRNA transcripts or proteins take on new abnormal functions.

In one embodiment, a method of treating trinucleotide repeat disorders is depicted in FIG. 23. Generally, the approach involves combining an elongated gRNA containing a region encoding the desired and healthy replacement trinucleotide repeat sequence with the intention of replacing the endogenous diseased trinucleotide repeat sequence through the mechanism of a prime editing process. involves using TPRT genome editing (i.e., prime editing). An exemplary gRNA design to reduce trinucleotide repeat sequences and a schematic diagram of trinucleotide repeat reduction with TPRT genome editing is shown in FIG. 23.

Prion Disease Prime editing can also be used to prevent prion diseases or halt their progression by incorporating one or more protective mutations onto the prion protein (PRNP), which becomes misfolded during the disease process. can be used. 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-Sträussler-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 the United Kingdom in 1996. 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 classic CJD (often referred to simply as CJD). It has different clinical and pathological features from classic CJD. Each disease also has a specific genetic profile of prion protein genes. Both disorders are persistently fatal brain diseases with unusually long incubation periods measured in years and are caused by unconventional transmitters called prions. 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 unusually transmittable substance called a prion. The nature of communicable substances is not well understood. Currently, the most accepted theory is that the substance is a modified form of a normal protein known as prion protein. For reasons not yet understood, the normal prion protein transforms into a pathogenic (harmful) form, which then damages the central nervous system of cattle. There is growing evidence that there are different strains of BSE: the typical or classic BSE strain responsible for the outbreak in the UK and two atypical strains (strains H and L). For BSE, no treatment is currently known.

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, Norway, and South Korea, including Canada and the United States. It can take more than a year before infected animals develop symptoms, and these can include severe weight loss (wasting), staggering, lethargy, and other neurological symptoms. CWD can affect animals of all ages, and some infected animals can eventually die without developing disease. CWD is fatal to animals and there is no treatment or vaccine.

The causative agent of TSE is believed to be a prion. The term "prion" refers to an abnormal pathogenic substance that is transmissible and capable of inducing abnormal folding of certain normal cellular proteins called prion proteins, which are most abundantly found in the brain. The function of these normal prion proteins is still not completely understood. Abnormal folding of the prion protein leads to brain damage and characteristic signs and symptoms of the disease. Prion diseases are usually rapidly progressive and always fatal.

The term "prion" as used herein refers to an infectious particle known to cause disease in humans and animals (spongiform encephalopathies). The term "prion" is a contraction of the words "protein" and "infection," and the particles are primarily, if not exclusively, infected by the PRNP gene expressing PRNP ^C , which changes conformation to become PRNP ^Sc . Consists of encoded PRNP ^Sc molecules. Prions are distinct from bacteria, viruses, and viroids. Known prions include those that infect animals and cause scrapie, a contagious 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 known and discussed to affect humans are (1) Kuru disease, (2) Creutzfeldt-Jakob disease (CJD), and (3) Gerstmann-Streussler-Scheinker disease (GSS). , and (4) fatal familial insomnia (FFI). Prions, as used herein, encompass all forms of prions that cause these diseases and/or others in any animal used, specifically humans and domesticated livestock.

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

It is known that certain mutations in the prion protein may be associated with an increased risk of prion disease. Conversely, certain mutations of 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 into this application 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 can serve as a transcription initiation site. Post-translational modification results in the removal of the first 22AA N-terminal fragment (NTF) and the last 23AA C-terminal fragment (CTF). NTF is cleaved after PrP transport to the endoplasmic reticulum (ER), and CTF (glycosylphosphatidylinositol [GPI] signal peptide [GPI-SP]) is cleaved by the GPI anchor. GPI anchors may be involved in PrP protein transport. It may also play a role in attaching 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:
MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERV VEQMCITQYERESQAYYQRGSSMVLFSSPPV (SEQ ID NO: 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), and the following It is as follows:
(Sequence number 292)

The relative mutation sites for 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 editing factors disclosed herein.

The mutation sites relative to 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:

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

In other embodiments, prime editing can be used to incorporate protective mutations in PRNP that are linked to protective effects 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 still other embodiments, the protective mutation can be any alternative amino acid incorporated at G127, G127, M129, D167, D167, N171, E219, or P238 of PRNP (for PRNP of NP_000302.1 relative) (SEQ ID NO: 291).

In a specific embodiment, prime editing can be used to incorporate the G127V protective mutation into PRNP, as illustrated in FIG. 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 diseases, it could be used to generate cattle and sheep that are immune to prion diseases, or even to help cure wild populations of animals suffering from prion diseases. It can also be used. Prime editing has already been used to achieve ~25% incorporation of naturally occurring protective alleles in human cells, and previous mouse experiments have shown that this level of incorporation is sufficient to induce immunity against most prion diseases. It indicates that. This method is the first and potentially the 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 list below of PEgRNA can be used to incorporate the G127V and E219K protective alleles into human PRNPs, and the G127V protective allele into PRNPs of various animals.

[9] Pharmaceutical compositions Other aspects of the present disclosure include pharmaceutical compositions that contain any of the various components of the prime editing system described herein, including, but not limited to, napDNAbp, reverse transcriptase, fusion proteins ( Examples include napDNAbps and reverse transcriptase), an extended guide RNA, and a complex comprising a fusion protein and extended guide RNA, as well as a component that nicks the second strand and the 5' endogenous DNA. The present invention relates to pharmaceutical compositions containing adjuncts that help drive the prime editing process to the formation of edited products, such as flap removal endonucleases.

The term "pharmaceutical composition" as used herein refers to a composition formulated for pharmaceutical use. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition includes additional agents (eg, for specific delivery, half-life enhancement, or other therapeutic compounds).

As used herein, the term "pharmaceutically acceptable carrier" refers to liquid or solid fillers, diluents, excipients, manufacturing aids such as lubricants, talc, magnesium, calcium, or stearin. zinc acid or stearic acid), or solvent-encapsulated materials involved in transporting or transporting the compound from one site of the body (e.g., the site of delivery) to another (e.g., an organ, tissue, or body part). means a pharmaceutically acceptable material, composition, or vehicle. A pharmaceutically acceptable carrier is one that is compatible with the other ingredients of the formulation but not injurious to the target tissue (e.g., a physiologically compatible, sterile, "acceptable" in the sense of pH, etc.). Some examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) ) Cellulose and its derivatives, such as sodium carboxymethylcellulose, methylcellulose, ethylcellulose, microcrystalline cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) magnesium stearate, lauryl (8) Excipients, such as cocoa butter and suppository wax; (9) Lubricant agents, such as sodium sulfate, and talc; (9) Excipients, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil. (10) Glycols, such as propylene glycol; (11) Polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol (PEG); (12) Esters, such as ethyl oleate and ethyl laurate; (13) ) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ) ethyl alcohol; (20) pH buffer solutions; (21) polyesters, polycarbonates, and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids; (23) 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, mold release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservatives, and antioxidants may also be present in the formulation. Terms such as "excipient", "carrier", "pharmaceutically acceptable carrier" or the like are used interchangeably herein.

In some embodiments, the pharmaceutical composition is formulated for delivery to a subject, eg, for gene editing. Suitable routes for administering the pharmaceutical compositions described herein include, but are not limited to: topical, subcutaneous, transdermal, intradermal, intralesional, intraarticular, intraperitoneal, intravesical, intravesical. Mucosal, gingival, intradental, intracochlear, transtympanic, intraorgan, epidural, intrathecal, intramuscular, intravenous, intravascular, intraosseous, periocular, intratumor, intracerebral, and cerebral. Indoor administration.

In some embodiments, the pharmaceutical compositions described herein are administered locally to an affected site (eg, a tumor site). In some embodiments, the pharmaceutical compositions described herein are administered by injection, using a catheter, using a suppository, or using an implant (porous material, non-porous material, or gelatin material (silastic membrane)). (including membranes or fibers such as

In other embodiments, the pharmaceutical compositions described herein are delivered in a controlled release system. In one embodiment, a pump may be used (see, for example, Langer, 1990, Science 249:1527-1533; Sefton, 1989, CRC Crit.Ref.Biomed.Eng.14:201; Buchwald et al., 1980, Surgery 88:507; see Saudek et al., 1989, N. Engl. J. Med. 321:574). In another embodiment, polymeric materials may be used. (For example, 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, See 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 also 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 (eg, a human). In some embodiments, pharmaceutical compositions for administration by injection are solutions in sterile isotonic aqueous buffer. Where necessary, the drug may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. In general, the ingredients are either individually or mixed together in a dry liophilized powder or waterless concentrate in a sealed container such as an ampoule or sachette indicating the amount of active agent. Supplied in unit dosage form as If the drug is to be administered by injection, it may be dispensed in an infusion bottle containing sterile pharmaceutical grade water or saline. When the pharmaceutical composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

Pharmaceutical compositions for systemic administration may be liquids, such as sterile saline, lactated Ringer's solution or Hank's solution. Additionally, the pharmaceutical compositions are in solid form and can be reconstituted or suspended extemporaneously prior to use. Lyophilized forms are also contemplated.

Pharmaceutical compositions 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 contains the fusogenic lipid dioleoylphosphatidylethanolamine (DOPE), low levels (5-10 mol%) of cationic lipids, and is stabilized by a polyethylene glycol (PEG) coating. plasmid-lipid particles (SPLP) (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 methyl sulfate, or "DOTAP", are effective for such particles and vesicles. Specifically preferred. The preparation of such lipid particles is well known. See, by way of example, U.S. Pat. No. 4,880,635;

The pharmaceutical compositions described herein may be administered or packaged as unit doses, for example. The term "unit dose" as used in reference to the pharmaceutical compositions of the present disclosure refers to physically discrete units suitable for a subject as a unitary dosage and containing a predetermined amount of active material. Each unit is calculated to produce the desired therapeutic effect in association with the required diluent; ie, carrier or vehicle.

Additionally, the pharmaceutical composition comprises (a) a container containing a compound of the invention in lyophilized form, and (b) a container containing a pharmaceutically acceptable diluent for injection (eg, sterile water). It may be provided as a pharmaceutical kit containing two containers. Pharmaceutically acceptable diluents may be used for reconstitution or dilution of lyophilized compounds of the invention. Optionally, the notice associated with such container(s) may be in a form prescribed by a governmental agency that regulates the manufacture, use, or sale of pharmaceutical or biological products; reflects an agency's approval of manufacture, use, or sale for.

In another aspect, articles of manufacture containing materials useful for treating the diseases described above are included. 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 container may be formed from a variety of materials such as glass or plastic. In some embodiments, the container retains a composition effective to treat a disease 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 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 containing a pharmaceutically acceptable buffer, such as phosphate buffered saline, Ringer's solution, or dextrose solution. It further encompasses other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. It's okay.

[10] Kits, vectors, cells, and delivery
Kits Compositions of the present disclosure can be assembled into kits. In some embodiments, the kit includes a nucleic acid vector for expression of a prime editing factor described herein. In other embodiments, the kit further provides for expression of suitable guide nucleotide sequences (e.g., PEgRNA and second site gRNA), or such guide nucleotide sequences (which direct the Cas9 protein or prime editing factor to the desired target sequence). Contains nucleic acid vectors for.

The kits described herein may include one or more containers housing the components for carrying out the methods described herein and optionally instructions for use. Any of the kits described herein may further include components needed to carry out the assay method. Each component of the kit may be provided in liquid form (eg, in solution) or solid form (eg, dry powder), where applicable. In some cases, some of the components are reconstituted or otherwise reconstituted, 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. (eg, into an active form).

In some embodiments, the kit may optionally include instructions and/or promotion for the use of the provided components. As used herein, "instructions" may define instructions and/or promotional components, typically written on or in connection with the packaging of the present disclosure. May be accompanied by instructions. The instructions should also be clearly printed so that the user clearly knows that the instructions pertain to the kit (e.g., audiovisual (e.g., videotape, DVD, etc.), Internet, and/or web-based). communication, etc.), any form of oral or electronic instruction may be included. The written instructions may be in the form prescribed by a governmental agency that regulates the manufacture, use, or sale of pharmaceutical or biological products; It can also reflect approval. As used herein, "facilitated" means 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 all forms of written, oral, and electronic communications) . In addition, the kit may also include other components depending on the particular use as described herein.

A 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 placed in a vial or other container for storage. The second container may also contain other sterilely prepared components. Alternatively, the kit may include the active agents premixed and shipped in a vial, tube, or other container.

The kit includes a blister pouch, a shrink-wrapped pouch, a vacuum-sealable pouch, a sealable thermoformable tray, along with accessories loosely packed in a pouch, one or more tubes, containers, boxes, or bags. or similar pouch or tray configurations. The kit may be sterilized after the accessories are added, so that individual accessories in the container can be opened differently. 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, depending on the specific application, such as containers, cell culture media, salts, buffers, reagents, syringes, needles, fabrics (such as gauze) for applying or removing the sterilant, and disposable Gloves, materials to support the agent prior to administration, etc. may also be included. Some aspects of the present disclosure describe various components of the prime editing system described herein, including, but not limited to, napDNAbp, reverse transcriptase, polymerase, fusion proteins (for example, napDNAbp and reverse a transcriptase (or more broadly, a polymerase)), an extended guide RNA, and a complex comprising a fusion protein and the extended guide RNA, as well as components that nick the second strand (e.g. nucleotide sequence encoding a gRNA that nicks the second strand) and accessory elements such as a 5' endogenous DNA flap removal endonuclease (which helps drive the prime editing process to the formation of the edited product). A kit comprising a nucleic acid construct comprising the method is provided. In some embodiments, the nucleotide sequence(s) comprises a heterologous promoter (or more than a single promoter) that drives expression of the prime editing system components.

Other aspects of the present disclosure provide for nucleotide sequences encoding various components of the prime editing factor system described herein (e.g., components of the prime editing factor system capable of modifying a target DNA sequence). A kit is provided that includes one or more nucleic acid constructs encoding a nucleotide sequence comprising: In some embodiments, the nucleotide sequence includes a heterologous promoter that drives expression of the prime editor system components.

Some aspects of the present disclosure include (a) a nucleotide sequence encoding napDNAbp (e.g., a Cas9 domain) fused to a reverse transcriptase, and (b) a heterologous promoter driving expression of the sequence of (a). Kits containing the nucleic acid constructs are provided.

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). do. Various human cell lines include, but are not limited to, human embryonic kidney (HEK) cells, HeLa cells, cancer cells from the National Cancer Institute 60 Cancer Cell Lines (NCI60), DU145 (prostate ) 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 bone marrow) U87 (glioblastoma) cells, SHSY5Y human neuroblastoma cells (cloned from myeloma), and Saos-2 (bone cancer) cells. In some embodiments, the rAAV vector is delivered into human embryonic kidney (HEK) cells (eg, HEK293 or HEK 293T cells). In some embodiments, rAAV vectors are derived from stem cells (e.g., human pluripotent stem cells, including, e.g., human induced pluripotent stem cells (hiPSCs)). stem cells). Stem cells refer to cells that have the ability to divide indefinitely in culture and give rise to specialized cells. Pluripotent stem cells refer to a type of stem cell that is capable of differentiating into all tissues of an organism, but is not capable of supporting the development of a complete organism on its own. Human induced pluripotent stem cells are somatic cells (e.g., mature (mature or adult 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 cellular characteristics 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 this disclosure provide cells that include 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 when naturally present in the subject. In some embodiments, the cells to be transfected are taken from a subject. In some embodiments, the cells are derived from cells taken from the subject, such as cell lines. 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 fibers Blast, 293-T, 3T3, 721, 9L, A2780, A2780ADR, A2780cis, A 172, A20, A253, A431, A-549, ALC, B16, B35, BCP-1 cell, 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 line, Peer, PNT-1A/PNT 2, RenCa, RIN-5F, RMA/RMAS, Saos-2 cells, Sf-9, SkBr3, T2, T-47D, T84, Includes THP1 cell lines, U373, U87, U937, VCaP, Vero cells, WM39, WT-49, X63, YAC-1, YAR, and transgenic strains thereof.

Cell lines are available from a variety of sources known to those skilled in the art (see, for example, the 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 containing one or more vector-derived sequences. In some embodiments, the patient is transiently transfected (such as by transient transfection of one or more vectors, or by transfection with RNA) with components of a CRISPR system as described herein. , cells modified through the activity of the CRISPR complex are used to establish new cell lines containing cells containing the modification but lacking any of the other foreign 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 assayed for one or more test compounds. used for.

Vectors Some aspects of the present disclosure provide recombinant vectors for the delivery of a prime editing agent described herein or a component thereof (eg, a splitting Cas9 protein or a splitting nucleobase prime editing factor) into a cell. Concerning the use of viral vectors (eg, adeno-associated virus vectors, adenovirus vectors, or herpes simplex virus vectors). In the case of the split-PE approach, the N-terminal portion of the PE fusion protein and the PE fusion, as full-length Cas9 proteins or prime editing factors exceed the packaging limits of various viral vectors (e.g., rAAV (~4.9 kb)). The C-terminal portion of is delivered into the same cell by a separate recombinant viral vector (eg, an adeno-associated viral vector, an adenoviral vector, or a herpes simplex virus vector).

Thus, in one aspect, the present disclosure contemplates vectors capable of delivering mitotic prime editing factor fusion proteins or their mitotic components. In some embodiments, compositions for delivering a mitotic Cas9 protein or mitotic prime editing factor into a cell (eg, a mammalian cell, a human cell) are provided. In some embodiments, the compositions of the present disclosure include: (i) a first nucleotide sequence encoding the N-terminal portion of a Cas9 protein or prime editing factor fused at its C-terminus to intein-N; 1 a recombinant adeno-associated virus (rAAV) particle; and (ii) a second recombinant adeno-associated virus particle comprising a second nucleotide sequence encoding intein-C fused to the N-terminus of the protein of Cas9 or the C-terminal portion of the prime editing factor. Virus (rAAV) particles. The rAAV particles of the present disclosure include an rAAV vector (ie, a recombinant genome of rAAV) encapsidated with a viral capsid protein.

In some embodiments, the rAAV vector comprises: (1) a first or a second nucleotide sequence; (2) one or more nucleotide sequences comprising a sequence (e.g., a promoter) that facilitates expression of the heterologous nucleic acid region; and (3) a heterologous nucleic acid region (optionally, one or more nucleic acid regions comprising a sequence that facilitates integration into the genome of a cell. In some embodiments, the viral sequences that facilitate integration include inverted terminal repeat (ITR) sequences. In some embodiments, the first or second nucleotide sequence encoding the N-terminal or C-terminal portion of the splitting Cas9 protein or splitting 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. Ru. The ITR sequences may 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. obtain. In some embodiments, the ITR sequence is derived from AAV2 or AAV6.

Thus, in some embodiments, the rAAV particles disclosed herein include at least one rAAV2 particle, rAAV6 particle, rAAV8 particle, rPHP.B particle, rPHP.eB particle, or rAAV9 particle, or a variant thereof. In specific embodiments, the disclosed rAAV particles are rPHP.B particles, rPHP.eB particles, rAAV9 particles.

ITR sequences and ITR sequence-containing plasmids are known in the art and are commercially available (e.g., Vector Biolabs, Philadelphia, PA; Cellbiolabs, San Diego, CA; Agilent Technologies, Santa Clara, CA; and Addgene, Products and services available from 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- 59259-304-6:201cHumana Press Inc.2003.Chapter 10.Targeted Integration by Adeno-Associated Virus.Matthew D. Weitzman, Samuel M. Young Jr., Toni Cathomen and Richard Jude Samulski; U.S. Patent Nos. 5,139,941 and 5,962,313 (all of which are incorporated herein by reference).

In some embodiments, the rAAV vectors of the present disclosure include one or more regulatory elements (eg, promoters, transcription terminators, and/or other regulatory elements) that control expression of the heterologous nucleic acid region. In some embodiments, the first and/or second nucleotide sequences are operably linked to one or more (eg, 1, 2, 3, 4, 5, or more) transcription terminators. ing. Non-limiting examples of transcription terminators that may be used in accordance with this disclosure include transcription terminators for the bovine growth hormone gene (bGH), human growth hormone gene (hGH), SV40, CW3, φ, or combinations thereof. The efficiency of several transcription terminators was tested to determine their respective effects on the expression levels of mitotic Cas9 proteins or mitotic prime editors. In some embodiments, the transcription terminator used in this disclosure is a bGH transcription terminator. In some embodiments, the rAAV vector further comprises a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). In certain embodiments, the WPRE is a truncated WPRE sequence, such as "W3." In some embodiments, the WPRE is inserted 5' of the 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 (eg, napDNAbp, linker, or polymerase). In addition, the vectors used herein may encode PEgRNA and/or accessory gRNA for nicking the second strand. The vector may be capable of driving the expression of one or more coding sequences in a cell. In some embodiments, the cell may be a prokaryotic cell, such as a bacterial cell. In some embodiments, the cell may be a eukaryotic cell, such as, for example, 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 that drive expression in various types of cells are known in the art. In some embodiments, the promoter may be wild type. In other embodiments, promoters may be modified for more efficient or 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 the virus.

In some embodiments, promoters that may be used in prime editing factor vectors may be constitutive, inducible, or tissue-specific. In some embodiments, the promoter may be a constitutive promoter. Non-limiting example constitutive promoters include the cytomegalovirus immediate early promoter (CMV), the simian virus (SV40) promoter, the adenovirus major late (MLP) promoter, the Rous sarcoma virus (RSV) promoter, the 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 of the above. Includes combinations. 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 the EFla promoter. In some embodiments, the promoter may be an inducible promoter. Non-limiting examples of inducible promoters include those inducible by heat shock, light, chemicals, peptides, metals, steroids, antibiotics, or alcohol. In some embodiments, an inducible promoter may have a low basal (non-induced) expression level, such as 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 example 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, Includes 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 editing factor vector (including, by way of example, any vector encoding a prime editing factor fusion protein and/or PEgRNA and/or an accessory gRNA that nicks the second strand) The vector may contain an inducible promoter that initiates expression only after delivery to the vector. Non-limiting examples of inducible promoters include those inducible by heat shock, light, chemicals, peptides, metals, steroids, antibiotics, or alcohol. In some embodiments, an inducible promoter may have a low basal (non-induced) expression level, such as the Tet-On® promoter (Clontech).

In additional embodiments, the prime editing factor vector (including, by way of example, any vector encoding a prime editing factor fusion protein and/or a PEgRNA and/or an accessory gRNA that nicks the second strand) It may also contain a tissue-specific promoter that initiates expression only after delivery to the tissue. Non-limiting example 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, Includes 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 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 crRNA and nucleotides encoding tracr RNA may be driven by the same promoter. In some embodiments, crRNA and tracr RNA may be transcribed into a single transcript. For example, crRNA and tracr RNA may be processed from a single transcript to form a double-molecule guide RNA. Alternatively, crRNA and tracr RNA may be transcribed into single molecule guide RNAs.

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 editing factor 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 PEgRNA. In other embodiments, the vector system may include two vectors, where one vector encodes the PE fusion protein and the other encodes PEgRNA. In additional embodiments, the vector system may include three vectors, where the 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 pharmaceutically 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 can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) ) Cellulose and its derivatives, such as sodium carboxymethylcellulose, methylcellulose, ethylcellulose, microcrystalline cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) magnesium stearate, lauryl (8) Excipients, such as cocoa butter and suppository wax; (9) Lubricant agents, such as sodium sulfate, and talc; (9) Excipients, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil. (10) Glycols, such as propylene glycol; (11) Polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol (PEG); (12) Esters, such as ethyl oleate and ethyl laurate; (13) ) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ) ethyl alcohol; (20) pH buffer solutions; (21) polyesters, polycarbonates, and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids; (23) 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, mold release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservatives, and antioxidants may also be present in the formulation. Terms such as "excipient", "carrier", "pharmaceutically acceptable carrier" or the like are used interchangeably herein.

Methods of Delivery In some aspects, the invention provides vectors as described herein, such as one or more vectors encoding one or more transcripts thereof, and/or one or more proteins transcribed therefrom. A method is provided that includes delivering one or more polynucleotides to a host cell. In some aspects, the invention further provides cells produced by such methods, and organisms (such as animals, plants, or fungi) that include or are produced from such cells. In some embodiments, base editors as described herein are delivered to cells in combination with (optionally conjugated to) a guide sequence.

Exemplary delivery strategies are described elsewhere herein and include vector-based strategies, delivery of PE ribonucleoprotein complexes, and delivery of PE by 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, polycations or lipid:nucleic acids. Includes conjugates, naked DNA, artificial virions, and agents that enhance DNA uptake. 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 (for example, Transfectam(TM), Lipofectin(TM)). , and SF cell line 4D-Nucleofector X Kit™ (Lonza)). Suitable cationic and neutral lipids for efficient receptor recognition lipofection of polynucleotides include the lipids of Feigner, WO91/17424; WO91/16024. Delivery can be to cells (eg, in vitro or ex vivo administration) or to target tissues (eg, in vivo administration). Delivery may be achieved through the use of RNP complexes.

The preparation of lipid:nucleic acid complexes including targeted liposomes, such as immunolipid complexes, is well known to those skilled in the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese et al., Cancer Gene Ther.2:291-297(1995);Behr et al.,Bioconjugate Chem.5:382-389(1994);Remy et al.,Bioconjugate Chem.5:647-654(1994);Gao et al., Gene Therapy 2:710-722(1995);Ahmad et al., Cancer Res.52:4817-4820(1992);U.S. Pat. 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 the DNA specificity of base editing. RNP delivery of fusion proteins leads to decoupling of on- and off-target DNA editing. RNP delivery ablates off-target editing at non-repetitive sites while maintaining on-target editing comparable to plasmid delivery, greatly reducing off-target DNA editing even at highly repetitive VEGFA site 2. reduce 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 US Pat. 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 skilled in the art. See, eg, US 2003/0087817 (incorporated herein by reference).

Other aspects of the present disclosure provide methods of delivering a prime editing factor construct into a cell to form a complete and functional prime editing factor within the cell. For example, in some embodiments, the cells contain AAV particles containing a composition described herein (e.g., a nucleotide sequence encoding a mitotic Cas9 or mitotic prime editing factor, or a nucleic acid vector comprising such a nucleotide sequence). (composition containing). In some embodiments, the contacting results in delivery of such nucleotide sequences into the cell, wherein the N-terminal portion of the Cas9 protein or prime editing factor and the C-terminal portion of the Cas9 protein or prime editing factor are Expressed in cells and combined to form the complete Cas9 protein or the complete prime editor.

It is understood that any rAAV particle, nucleic acid molecule, or composition provided herein may be introduced into cells in any suitable manner, either stably or transiently. It should be. In some embodiments, the disclosed proteins may be transfected into cells. In some embodiments, cells may be transduced or transfected with a nucleic acid molecule. For example, a cell may be transduced with a nucleic acid molecule encoding a fission protein, or an rAAV particle containing the genome of a virus encoding one or more nucleic acid molecules (e.g., with a virus encoding a fission protein). or may be transfected (eg, with a plasmid encoding a fission protein). Such transduction may be stable transduction or transient transduction. In some embodiments, cells expressing or containing a fission protein are transduced or transfected with one or more guide RNA sequences, e.g., in the delivery of a fission Cas9 (e.g., nCas9) protein. Good too. In some embodiments, plasmids expressing split proteins are produced by electroporation, transient (e.g., lipofection) and stable genomic incorporation (e.g., piggybac), and viral transduction or other methods known to those skilled in the art. It may also be introduced into cells through other methods described.

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 includes a sequence of at least 10, at least 15, or at least 20 contiguous nucleotides complementary to the target nucleotide sequence. The guide RNA is complementary to the target nucleotide sequence. , 35, 36, 37, 38, 39, or 40 contiguous nucleotides. Guide RNAs were 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 in length. , 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides.

In some embodiments, the target nucleotide sequence is a DNA sequence in a genome (eg, a eukaryotic genome). In certain embodiments, the target nucleotide sequence is in the mammalian (eg, human) genome.

Compositions of the present disclosure may be administered or packaged as unit doses, for example. The term "unit dose" as used in reference to the pharmaceutical compositions of the present disclosure refers to physically discrete units suitable for a subject as a unitary dosage and containing a predetermined amount of active material. Each unit is calculated to produce the desired therapeutic effect in association with the required diluent; ie, carrier or vehicle.

Treatment of 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 result.

As used there, "delaying" the onset of a disease means deferring, hindering, slowing, retarding, stabilizing, and / or means to postpone (postpone). This delay may be of varying length of time depending on the medical history and/or the individual being treated. A method of "delaying" or mitigating the onset of a disease, or of delaying the onset of a disease, increases the probability of developing one or more symptoms of the disease in a given time frame compared to not using the method. and/or reduce the severity of symptoms over a given time frame. Such comparisons are typically based on clinical studies using a sufficiently large number of subjects to yield statistically significant results.

"Onset" or "progression" of a disease refers to the first signs and/or subsequent progression of the disease. The onset of 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. "Development" includes occurrence, recurrence, and onset.

As used herein, "onset" or "occurrence" of a disease includes initial onset and/or recurrence. Conventional methods known to those skilled in the art of medicine may be used to administer the isolated polypeptide or pharmaceutical composition to a subject, depending on the type of disease or site of the 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 the genome
The goal is to develop transformative technologies for precise genome editing and to generate incorporation of single nucleotide changes in mammalian genomes. This technology will allow researchers to study the effects of single nucleotide variations in virtually any mammalian gene, allowing therapeutic interventions to correct pathogenic point mutations in human patients. would potentially make it possible.

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.

It is now proposed to develop a new precision editing approach that provides many of the benefits of base editing - namely, avoidance of double-strand breaks and donor DNA repair templates - while overcoming its major drawbacks. To achieve this ambitious goal, we aim to use target primed reverse transcription (TPRT) to directly incorporate edited DNA strands at targeted genomic sites. In the designs discussed herein, a CRISPR guide RNA (gRNA) can be modified to carry a template encoding mutagenic DNA strand synthesis carried out by an associated reverse transcriptase (RT) template sequence. Probably. Target site DNA nicked by CRISPR nuclease (Cas9) will serve as a primer for reverse transcription of the template sequence on the modified gRNA, allowing direct incorporation of any desired nucleotide edits. .

Section 1
Establish guide RNA-templated reverse transcription of the mutagenic DNA strand. Previous studies have shown that after DNA cleavage but before complex dissociation, Cas9 releases the non-target DNA strand, exposing the free 3' end. Assume that this DNA strand is accessible for 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 established that the nicked DNA strand within the Cas9:gRNA binding complex can indeed prime reverse transcription (RT enzyme in trans), which uses the bound gRNA as a template. . Next, various gRNA linkers, primer binding sites, and synthetic templates are studied in detail to determine optimal design rules in vitro. Various RT enzymes acting in trans or as fusions with Cas9 are then evaluated in vitro. Ultimately, modified gRNA designs that retain efficient binding and cleavage activity in cells are identified. Successful demonstration experiments to this end provide the basis for carrying out mutagenic chain synthesis in cells.

Section 2
Establishing prime editing in human cells. Based on DNA processing and repair mechanisms, we hypothesize that mutagenic DNA strands (single-stranded flaps) can be used to direct specific and efficient editing of target nucleotides. In promising preliminary studies, the feasibility of this strategy was established by demonstrating editing with a model plasmid substrate containing a mutagenic flap. Concurrent with objective 1, repair outcomes will be further evaluated by systematically varying the length, sequence composition, target nucleotide identity, and 3' end of the mutagenic flap. Small insertions and deletions of 1-3 nucleotides are also tested. Evaluate Cas9-RT constructs assembled from Aim 1 in parallel with Aim 1 and encompassing fusion proteins and non-covalent recruitment strategies. The Cas9-RT construct and extended gRNA 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
Achieving site-specific editing of pathogenic mutations in cultured human cells. The potential ubiquity of this technology may enable the editing of transversion mutations and indels that are currently uncorrectable by BE. Guided by the results of Aim 1 and Aim 2, we identified the founder mutation in beta-globin of sickle cell disease (correction requires an A.T→T.A transversion) and the most prevalent Wilson disease. targeting pathogenic transversion mutations in cultured human cells containing mutations in ATP7B (requiring G·C→T·A transversion for correction). Correction of small insertion and deletion mutations, including the 3-nucleotide ΔF508 deletion in CFTR that causes cystic fibrosis, will also be examined. If successful, this will lay the foundation for developing powerful therapeutic approaches to address these important human diseases.

The goal of the approach is to develop genome editing strategies that directly incorporate point mutations at targeted genomic sites. During the technology development stage, efforts to incorporate TPRT functionality into CRISPR/Cas systems are focused on protein and RNA engineering. Use in vitro assays to carefully explore the function of each step of TPRT and assemble it from the ground up (Aim 1). The second focus area evaluates editing outcomes in mammalian cells using a combination of model substrates and a modified CRISPR/Cas system (Aim 2). Finally, the application phase uses the technology to correct mutations that are difficult to resolve with other methods of genome editing (Objective 3).

Typical editorial designs are shown in Figures 1A-1B. Cas9 nickase contains an inactivating mutation (Spy Cas9 H840A or N863A) to the HNH nuclease domain that limits DNA cleavage to the PAM-containing strand (non-target strand). Modify the guide RNA (gRNA) to contain the template for reverse transcription (design detailed on slide 5). Although 5' extension of gRNA is shown, 3' extension can also be practiced. Cas9 nickase is fused with reverse transcriptase (RT) enzyme through either the C-terminus or the N-terminus. The gRNA:Cas9-RT complex targets the DNA region of interest and forms an R-loop after replacing the non-target strand. Cas9 nicks non-target DNA strands. Release of the nicked strand exposes a free 3'-OH end, which is capable of priming reverse transcription using the extended gRNA as a template. This DNA synthesis reaction is carried out by a fused RT enzyme. The gRNA template encodes a DNA sequence homologous to the original DNA duplex, except for the nucleotides targeted by the edit. The reverse transcription product is a single-stranded DNA flap encoding the desired edit. This flap, containing a free 3' end, can equilibrate with adjacent DNA strands, yielding the 5' flap species. The latter species is hypothesized to serve as an efficient substrate for FEN1 (flap endonuclease 1), which naturally excises the 5' flap from the Okazaki fragment during lagging strand DNA synthesis. The 5' flap is removed after strand displacement synthesis that occurs during long-patch base excision repair. Ligation of nicked DNA produces mismatched base pairs. This intermediate can undergo either restoration to the original base pair or conversion to the desired edited base pair via a mismatch repair (MMR) process. Alternatively, semi-conservative DNA replication can produce one restored and one edited copy.

1. Establish guide RNA-templated reverse transcription of the mutagenic DNA strand.
Background and Rationale In the proposed genome editing strategy, the non-target DNA strand (PAM-containing strand forming an R-loop) nicked by Cas9 acts as a primer for DNA synthesis. It is hypothesized that this may occur based on several pieces of biochemical and structural data. Nuclease protection experiments, ³² crystallographic studies, ³³ and base editing ^windows4,24 have revealed a significant degree of flexibility and disorder for non-target strand nucleotides -20 to -10 within the so-called R-loop of the Cas9 binding complex. demonstrated (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-target strand ^can be dislodged from the tightly bound ternary complex. These studies support that the non-target strand is highly flexible and accessible to the enzyme and that after nicking, the 3' end of the PAM distal fragment is released prior to Cas9 dissociation. be done. Furthermore, it is assumed that the gRNA can be extended until template DNA synthesis. Previous studies have shown that gRNAs for SpCas9, SaCas9 ^, and LbCas12a (formerly Cpf1) are permissive for gRNA elongation with RNA aptamers, ³⁴ ligand-induced self-cleaving ribozymes, ³⁵ and long noncoding RNAs. Shown. This literature establishes a precedent for two main features that are exploited. In assessing this strategy, several CRISPR-Cas systems are evaluated in conjunction with 5' and 3' extended gRNA designs using a combination of in vitro and cellular assays (Figures 2A-2C). .

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 showed that reverse transcription extends ~3 nucleotides into the gRNA core of 3'-extended gRNA constructs, and in some cases the 3' RNA hairpin of the gRNA core Modify the DNA to match the target sequence (as long as changes are made that compensate for the maintenance of the hairpin RNA structure, modifications of the hairpin sequence appear to be well tolerated). DNA synthesis proceeds 5'→3' with nucleotides being added to the 3'OH of the growing DNA strand.

Preliminary experiment results
The Cas9-nicked DNA primes reverse transcription of the gRNA template. To assess the accessibility of nicked non-target DNA strands, in vitro biochemical assays were performed using Cas9 nuclease from S. pyogenes (SpCas9) and Cy5 fluorescently labeled double-stranded DNA substrate (51 Base pairs) were used. First, a series of 5' extension-containing gRNAs with varying synthetic template lengths were prepared by in vitro transcription (the overall design is shown in Figure 2B). Electrophoretic mobility shift assay (EMSA) using nuclease-inactive Cas9 (dCas9) established that 5'-extended gRNA maintains binding affinity to target (data not shown). TPRT activity was then determined using dCas9, 5'-extended gRNA, and Moloney-murine leukemia virus (M-MLV) reverse transcriptase (Superscript III) on a pre-nicked Cy5-labeled duplex. Tested against DNA substrate. After 1 h incubation at 37 °C, products were evaluated by denaturing polyacrylamide gel electrophoresis (PAGE) and imaged using Cy5 fluorescence (Figure 4A). Each 5'-extended gRNA variant resulted in significant product formation, and the observed DNA product size was consistent with the length of the extended template (Figure 4B). Importantly, in the absence of dCas9, the pre-nicked substrate was elongated to the full length of the DNA substrate, 51 bp, which is because the complementary DNA strand (not gRNA) Strongly suggests that it was used as a template for DNA synthesis (Figure 4C). Of note, the system was designed so that the newly synthesized DNA strand mirrors the product required for editing the target site (a strand homologous to a single nucleotide change). This result establishes that Cas9:gRNA binding exposes the 3' end of the nicked non-target strand and that the non-target strand is accessible to reverse transcription.

The unnicked dsDNA substrate was then evaluated using the Cas9(H840A) mutant, which nicks the non-target DNA strand. First, to test Cas9(H840A) nickase activity using 5′-extended gRNA, in vitro cleavage assays were performed as previously described ³⁷ . Although nicking was impaired compared to standard gRNA, substantial cleavage products were formed (Figure 4D). Importantly, when the TPRT reaction was carried out with 5′-extended gRNA and Cas9(H840A), RT products were also observed, albeit at a lower yield (which can be explained by the decreased 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. When compared to 5'-extended gRNA, DNA cleavage by 3'-extended gRNA was not detectably impaired in any case compared to standard gRNA. Significantly, 3'-extended gRNA templates were also efficient on both pre-nicked and intact duplex DNA substrates when M-MLV RT was supplied in trans. reverse transcription was supported (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, flap ends will be reprogrammed by modifying the terminal gRNA sequences38,39 ^. This result demonstrates that 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. Two-color experiments were used to determine whether the RT reaction preferentially occurs in cis (bound in the same complex) with gRNA (see Figure 8). Two separate experiments were performed with 5' extended gRNA and 3' extended gRNA. 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) has been done. 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 Cy3 and Cy5 channels. Reaction products were found to be preferentially formed using gRNA templates previously complexed with DNA substrates, indicating that the RT reaction may occur in cis. This result supports 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 are performed using other Cas systems, including Cas9 from S. aureus and Cas12a from L. bacterium ( (see Figures 2A-2C). If TRPT could also be demonstrated for these Cas variants, the potential editing scope and overall chances of success in cells would increase.

Testing TPRT with RT-Cas9 fusion proteins First, a series of commercially available or purified RT enzymes are evaluated in trans for TPRT activity. In addition to the already tested RTs from M-MLV, avian myeloblastosis virus (AMV), Geobacillus stearothermophilus group II intron (GsI-IIC) ^41,42 and Eubacterium rectale group II intron (Eu.re.I2 ) Rate RT from ^43,44 . Significantly, the latter two RTs perform TPRT in their natural biological context. Where relevant, RNAse inactivating mutations and other potentially beneficial RT enzyme modifications will also be tested. Once functional RTs are identified when delivered in trans, each is evaluated as a fusion protein against the Cas9 variant. Both N-terminal and C-terminal fusion orientations are tested along with various linker lengths and structures. Kinetic time course experiments are used to determine whether TPRT can occur using the RT enzyme in cis. If RT-Cas9 fusion constructs could be constructed that allow efficient TPRT chemistry, this would greatly increase the potential for functional editing in a 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. Modified gRNAs are compared side-by-side with standard gRNAs across ^five or more sites in the human genome using an established indel formation assay employing wild-type SpCas9. Genome editing efficiency is characterized by multiplex amplicon sequencing using the Illumina MiSeq platform housed in the laboratory. It is anticipated that the results from this and preceding sections will generate insights that will inform subsequent iterations of the design-build-test cycle, in which gRNAs are reverse transcribed in cells. templating and efficient Cas9 targeting.

The in vitro verification results are shown in Figures 5 to 7. In vitro experiments demonstrated that nicked non-target DNA strands are flexible and available to prime DNA synthesis and that 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). Fluorescently labeled (Cy5) DNA targets were used as substrates and were previously nicked in this series of experiments. The Cas9 used in these experiments is a catalytically inactive form of Cas9 (dCas9), so it cannot cut 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 constructed from purified components. Fluorescently labeled DNA substrates were then added together with dNTPs and RT enzyme. After 1 hour incubation at 37C, reaction products were analyzed by denaturing urea-polyacrylamide gel electrophoresis (PAGE). The gel image shows that the original DNA strand has been extended to a length that matches the length of the reverse transcription template. Remarkably, reactions carried out in the absence of dCas9 produced DNA products 51 nucleotides long, regardless of the gRNA used. This product corresponds to the use of a 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 series of in vitro experiments is exactly similar to that shown in Figure 5, except that the DNA substrate is not pre-nicked and a Cas9 nickase (SpyCas9 H840A mutant) is used ( closely parallels). As shown in the gel, nickase efficiently cleaves DNA strands when using standard gRNA (gRNA_0, lane 3). Multiple cleavage products are observed, consistent with previous biochemical studies of SpyCas9. Although 5' extension impairs nicking activity (lanes 4-8), some RT products are still observed. Figure 7 shows that 3' extension supports DNA synthesis and does not significantly generate Cas9 nickase activity. Pre-nicked substrates (black arrows) are almost quantitatively converted to RT products when using either dCas9 or Cas9 nickase (lanes 4 and 5). More than 50% conversion to RT product (red arrow) is observed for all substrates (lane 3). To determine the length and sequence of the RT product, the product band was excised from the gel, extracted and sequenced. This revealed that RT extended 3 nucleotides into the 3' terminal hairpin of the gRNA core. Subsequent experiments (not shown) demonstrated that these three nucleotides could be changed 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 can prevent polymerase extension by the Cas9 fusion RT enzyme. First, linker optimization can be tested. We evaluate circular substitution variants of Cas9 that can reorient the spatial relationship between DNA primers, gRNA, and RT enzymes. Non-covalent RT mobilization strategies as detailed in Aim 2 can be tested. (2) Reduced Cas targeting efficiency by extended gRNA variants: This can be a problem with most 5' extended gRNAs. Based on structural ^data24 , Cas9 mutants can be designed and screened to identify variants that are more resistant to gRNA elongation. Additionally, gRNA libraries can be screened in cells for linkers that improve targeting activity.

Significance These preliminary experimental results demonstrate that Cas9 nickase and elongated gRNA can initiate target-primed reverse transcription on bound DNA targets using reverse transcriptase supplied in trans. Establish. Importantly, Cas9 binding was found to be critical for product formation. Although probably not an absolute requirement for genome editing in cells, further development of systems that incorporate RT enzyme function in cis will significantly increase the likelihood of success in cell-based applications. Achieving the remaining aspects of this objective will provide the molecular basis for performing 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 target site in the genome. We hypothesize that the mutagenic 3' flap containing a single mismatch is incorporated by the DNA repair machinery through equilibrium with the energetically more available adjacent 5' flap, which is preferentially removed (Fig. 1C-1D). The 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 a preferred substrate for the widely 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). Acting as a "toolbelt", ^PCNA accelerates the sequential flap cleavage and ligation that is essential for processing the millions of Okazaki fragments generated during each cell division50,51. Based on these similarities to natural DNA intermediates, it is hypothesized that the mutagenic strand is incorporated through a combination of equilibration with the 5' flap, followed by 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 editing 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 work with a model plasmid substrate containing a mutagenic 3' flap that resembles the TPRT product. We created a dual fluorescent protein reporter that encodes a stop codon between GFP and mCherry. The mutagenic flap encodes a modification to the stop codon (Figure 9A), allowing mCherry synthesis. Therefore, 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), with isolated yeast colonies containing either the reverted base, the mutant base, or a mixture of both products ( Figure 9C). The latter detection suggests that plasmid replication occurred prior to MMR in these cases, further suggesting that flap excision and ligation precede MMR. This result establishes the feasibility of DNA editing using the 3' mutagenic strand.

●Systematic studies on model Wrap substrates Based on the preliminary experimental results described above, a broader range of flap substrates will be evaluated in HEK cells in order to infer the principles of efficient editing. Systematically vary the 3'ssDNA flap to determine the effects of mismatch pairing, the location of the mutagenic nucleotides along the flap, and the identity of the terminal nucleotides (Figure 9D). Single nucleotide insertions and deletions are also tested. Use amplicon sequencing to analyze the editing with high precision. These results help inform the design of gRNA reverse transcription templates.

In vitro TPRT on plasmid substrates leads to efficient editing outcomes. The TPRT reaction developed in Aim 1 was used to introduce mutagenesis into the plasmid substrate. Reactions were carried out on circular DNA plasmid substrates (see Figure 10). This excludes the possibility of DNA strand dissociation as a mechanism for RT elongation in the previous in vitro experiments. This also allows DNA repair of the flap substrate in the cell. 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 TRT [Cas9(H840A) nickase, modified gRNA, MLV RT enzyme, dNTPS] in vitro. gRNA extension encodes a mutation to the stop codon. Using the flap strand for stop codon repair would be expected to produce a plasmid expressing both GFP and mCherry as a fusion protein. A yeast double FP plasmid transformant is shown in FIG. Transformation of the parental plasmid or the Cas9(H840A) nicked plasmid in vitro results in only green GFP-expressing colonies. TRT reactions with 5' extended gRNA or 3' extended gRNA produce a mixture of green and yellow colonies. The latter expresses both GFP and mCherry. More yellow colonies are observed with 3' extended gRNA. A positive control that does not contain a stop codon is also shown.

This result establishes that long double-stranded substrates can undergo TPRT and that TPRT products induce editing in eukaryotic cells.

We also performed another experiment similar to the prime editing experiment described above, but instead of incorporating a point mutation in the stop codon, TRT editing repaired the frameshift mutation and enabled synthesis of downstream mCherry. Incorporate nucleotide insertions (left) or deletions (right) (see Figure 11). Both experiments used 3' extended gRNA. Individual colonies from TRT transformants were selected and analyzed by Sanger sequencing (see Figure 12). Green colonies contained the plasmid with the original DNA sequence, while yellow colonies contained the exact mutations designed by the TRT-edited gRNA. No other point mutations or indels were observed.

Establishing Prime Editing in HEK Cells Using the RT-Cas9 Construct The optimized construct from the previous purpose is adapted for mammalian expression and editing at targeted sites in the human genome. Multiple RT enzymes and fusion constructs are tested in addition to flanking targeting with a secondary gRNA (truncated to prevent nicking). Non-covalent RT recruitment is also assessed using the Sun-Tag system ⁵² and the MS2 aptamer system ⁵³ . Indel formation assays are used to assess targeting efficiency with standard gRNA and RT-Cas9 fusions (as above). For each genomic site, the extended gRNA and RT-Cas9 pairs are then assayed for single nucleotide editing. Evaluate the editing results with MiSeq.

Initial experiments in HEK cells were performed using the Cas9-RT fusion. Editing by 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 in increasing the efficiency of editing in human cells (see Figure 15).

Optimizing Prime Editing Parameters in HEK Cells After identifying a Cas9-RT construct capable of performing prime editing in cells, the components and design are optimized to achieve high efficiency editing. The location and nucleotide identity of the encoded point mutations and the total length of the newly synthesized DNA strand are varied to assess the extent of editing and potential drawbacks. Short insertion and deletion mutations are also evaluated. Codon-optimize protein expression constructs. 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 provide a higher local concentration of the RT enzyme at the edited locus. These auxiliary guides are truncated at the 5' end (14-15 nt spacer), which has previously been shown to prevent Cas9 cleavage but preserve 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 that incorporate stabilizing secondary structures or have stabilizing modifications can be tested. (2) No editing observed in human cells: explore additional strategies, including secondary targeting of RT-Cas9 fusions to adjacent genomic sites ^. Additionally, 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, this would have direct implications for basic biomedical research by allowing the rapid generation and characterization of large numbers of point mutations in human genes. will have an impact. The generalization of the method for base editors and their orthogonal editing windows provides an approach that incorporates many mutations that are currently inaccessible. Moreover, if prime editing can be optimized for high efficiency and product purity, its potential applicability to correcting disease mutations in other human cell types is significant.

3． Achieving site-specific editing of pathogenic mutations in cultured human cells.
Background and rationale.

Many pathogenic mutations cannot be corrected by modern base editing agents due to limitations of PAM, ie, correction of transversion or indel mutations is required. With prime editing, all transitions and transversions can theoretically occur, albeit with minor insertions and deletions. Moreover, in relation to PAM, the prime editing window (predicted from -3 to +4) is different from the base editing factor (-18 to -12) (Figure 13). Mendelian diseases that are currently uncorrectable by base editors include: (1) the sickle cell Glu6Val founder mutation in hemoglobin beta (requiring A.T→T.A transversion); (2) the most common Wilson disease variant His1069Gln in ATP7B (requiring G・C→T・A transversion); and (3) the △Phe508 mutation in CFTR (requiring a 3-nucleotide insertion) that causes cystic fibrosis. required). 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 following transfection of the components in human embryonic kidney (HEK) cells. This data presents the results of 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 to 11 or 12 nucleotides. In addition, an auxiliary guide A located just upstream of the editing locus (see Figure 16) significantly improves editing activity, particularly 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; this data is the result of using a C-terminal fusion of the RT enzyme. present. Here, auxiliary guide A does not have as much of an effect, and the overall editing efficiency is higher. Figure 17C shows data presenting the results of using an N-terminal fusion of wild-type MLV reverse transcriptase with Cas9(H840A) nickase similar to that used in Figure 17A; however, MLV RT and Cas9 The linker between is 60 amino acids long instead of 32 amino acids.

●T→A editing at HEK3 site according to RPT editing results shows high purity.
Figure 18 shows input for sequencing analysis by high-throughput amplicon sequencing. The input displays the most abundant genotype of the edited cells. Remarkably, no major indel product is obtained and the desired point mutation (T→A) is clearly incorporated without bystander editing. The first sequence represents 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 and edited genotypes.

●MLV RT mutants improve editing.
The mutant reverse transcriptase described in Baranauskas et al. (doi:10.1093/protein/gzs034) was tested as a C-terminal fusion with Cas9 (H840A) nickase for targeted nucleotide editing in human embryonic kidney (HEK) cells. did. The Cas9-RT editing factor plasmid was co-transfected with a plasmid encoding a 3' prime editing guide RNA that served as a template for reverse transcription. In Figure 19, the editing efficiency at the target nucleotide (blue bar) is shown alongside the indel rate (orange bar). WT refers to wild type MLV RT enzyme. The 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 in close proximity to the target nucleotide, directing mismatch repair to preferentially replace the original nucleotide. ) and convert the base pairs to the desired edit). The Cas9(H840A)-RT editing construct was co-incorporated with a plasmid encoding two guide RNAs, one of which serves as the template for the reverse transcription reaction and the other that targets the complementary DNA strand for nicking. transfected. Nicking at various distances from the target nucleotide was tested (orange triangle) (see Figure 20). Editing efficiency at the target base pair (blue bar) is shown alongside the indel rate (orange bar). An example of "none" does not contain a guide RNA that nicks the complementary strand. Editing rates were quantified by high-throughput sequencing of genomic DNA amplicons.

Figure 21 shows processed high-throughput sequencing data demonstrating the desired T→A transversion mutation and the overall absence of other major genome editing byproducts.

range.
The potential range for the new editing technology is shown in Figure 13 and compared to deaminase-mediated base editor technology. Base editing factors developed to date target an ~15±2 bp region upstream of PAM. By converting target C or A nucleotides to T or G, respectively, the base editors developed so far allow for all transition mutations (A:T→G:C conversions). However, the base editing factors developed so far are transversion mutations (A→T, A→C, G→T, G→C, T→A, T→G, C→A, C→G). cannot be incorporated. Moreover, multiple target nucleotides in the editing window may result in undesirable additional editing.

The new prime editing technology could theoretically incorporate any nucleotide and base pair changes, and potentially small insertion and deletion edits. For PAM, the prime editing window begins at the site of nicking the DNA (three bases upstream of PAM) and ends at a so far undetermined position downstream of PAM. Notably, this editing window is different from deaminase base editing factors. The TPRT system has all of its benefits, including generality, high precision, and fidelity, since it uses a DNA polymerase enzyme to perform the editing.

Correcting pathogenic mutations in patient-derived cell lines.
Cell lines harboring relevant mutations (sickle cell disease: CD34+ hematopoietic stem cells; Wilson disease: cultured fibroblasts; cystic fibrosis: cultured bronchial epithelium) were collected from ATCC, Coriell Biobank, or the Harvard/Broad collaboration. Obtained from collaborating Harvard/Broad affiliate laboratories. Editing efficiency will be assessed by high-throughput sequencing and efficacy of the corrected genotypes will be tested using phenotypic assays (hemoglobin HPLC, ATP7B immunostaining, and CFTR membrane potential assay).

Characterizing off-target editing activity.
Screen potential off-target edits with established methods such as GUIDE-seq ⁵⁵ and CIRCLE-seq ⁵⁶ using target gRNA paired with wild-type Cas9. If potential off-targets are identified, these loci are probed from TPRT-edited cells to identify true off-target editing events.

Potential difficulties and alternatives.
(1) Low editing efficiency: Prime editing factors (PEs) may require optimization for each target. In this case, gRNA libraries can be tested to identify the best performing 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.
Myriad 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 translation into 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, large heterogeneity of different point mutations within a single gene is observed across patient populations, each of which produces a similar disease phenotype. Therefore, while common genome editing methods could theoretically target such large numbers of mutations, this technology could offer enormous potential benefits to many of these patients and their families. If proof of principle for these applications could be established in cells, it would establish the basis for studies in animal models of disease.

Advantages High precision: the desired edits are encoded and directed into 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 the error-prone reverse transcriptase enzyme to incorporate mutations onto the genome.

One embodiment is illustrated in FIG. 22. This is a schematic illustration of an exemplary process for error-prone reverse transcriptase-mediated targeted mutagenesis of a target locus, with nucleic acid-programmed DNA binding complexed with an elongated guide RNA. A protein (napDNAbp) is used. This process can be referred to as a form of prime editing for targeted mutagenesis. An extended guide RNA includes an extension at the 3' or 5' end of the guide RNA or at a position within the molecule 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 creating an available 3' end on one of the strands at the target locus. . In certain embodiments, the nick is created on the strand of DNA that corresponds to the R-loop strand, ie, 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 certain 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 mutagenized region. In step (e) napDNAbp and guide RNA are released. Steps (f) and (g) involve disassembly of the single-stranded DNA flap (containing the mutagenized region) so that the desired mutagenized region becomes integrated at the target locus. This process directs desired product formation by removing the corresponding 5' endogenous DNA flap that forms once a 3' single-stranded DNA flap invades and hybridizes to complementary sequences on the other strand. can be driven to. The process can also be driven towards product formation by second strand nicking as illustrated in Figure IF. After endogenous DNA repair and/or replication processes, the mutagenized region becomes integrated on 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 shorten trinucleotide repeat mutations (or "triplet expansion diseases") to treat Huntington's disease and other trinucleotide repeat mutations. It can be used to treat diseases such as repetitive disorders. Without being bound by theory, triplet expansion is caused by slippage during DNA replication or DNA repair synthesis. Because tandem repeats have identical sequences to each other, base pairing between the two DNA strands can occur at multiple points on 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 repeating sequences, 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 some embodiments of this use, Figure 23 provides a schematic representation of PEgRNA design to shorten or reduce trinucleotide repeat sequences by prime editing.

Therefore, prime editing may have the potential to be used to correct any trinucleotide repeat disorder, including Huntington's disease, Fragile X syndrome, and Friedreich's ataxia.

The most common trinucleotide repeat contains the CAG triplet, but the GAA triplet (Friedreich's ataxia) and CGG triplet (Fragile X syndrome) also occur. The CAG triplet encodes glutamine (Q) and therefore the CAG repeats give rise to polyglutamine tracts in the coding regions of diseased proteins. This specific class of trinucleotide repeat disorders is also referred to as "polyglutamine (polyQ) diseases." Other trinucleotide repeats can cause alterations in gene regulation and are referred to as "non-polyglutamine diseases." Inheriting a predisposition to expansion or acquiring an allele from an already expanded parent increases the probability of acquiring the disease. Pathogenic expansion of trinucleotide repeats can be corrected using prime editing.

Prime editing can be implemented to shorten the triplet expansion region by nicking the region upstream of the triplet repeat region with a prime editing agent containing a dedicated PEgRNA targeted to the site to be cut. . The prime editing factor then creates a new DNA strand (ssDNA flap) based on PEgRNA as a template (i.e., its editing template) encoding a healthy number of triplet repeats (this depends on the specific gene and disease). Synthesize. A newly synthesized ssDNA strand containing a healthy triplet repeat sequence is also synthesized to include a short stretch of homology (i.e. homology arm) that matches the sequence flanking the other end of the repeat (shown in red). chain). Invasion of the newly synthesized strand and subsequent replacement of the endogenous DNA by the newly synthesized ssDNA flap leads to a truncated 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.

FIG. 25 is a schematic diagram showing gRNA design for peptide tagging genes at endogenous genomic loci and peptide tagging by TPRT genome editing (ie, prime editing). The FlAsH and ReAsH tagging systems include two parts: (1) a fluorophore-biarsenic probe and (2) a peptide encoded by a gene containing a tetracysteine motif exemplified by the sequence FLNCCPGCCMEP (SEQ ID NO: 1). It is. When expressed intracellularly, proteins containing tetracysteine motifs can be fluorescently labeled by fluorophore-arsenic probes (ref: J.Am.Chem.Soc., 2002, 124(21), pp 6063-6076 .DOI:10.1021/ja017687n). The "Sortagging" system uses a bacterial sortase enzyme to covalently conjugate a labeled peptide probe to a protein containing a suitable peptide substrate (ref:Nat.Chem.Biol.2007 Nov;3(11 ):707-8.DOI:10.1038/nchembio.2007.31). 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)) is commonly used in immunoassays as an epitope tag. Pyclamp encodes a peptide sequence (FCPF) (SEQ ID NO: 622) that can be labeled with pentafluoro-aromatic substrates (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 out of the view frame (sequencing confirmed complete and precise insertion).

Example 5 – Prevention or treatment of prion disease with PE

The present invention can help address the problem of prion diseases in humans, livestock, and wildlife. The previously described editing strategies are not efficient and clean enough to incorporate protective mutations or reliably knock down PRNP. Cas9 nuclease and HDR can be used, but will mostly generate a mixture of PRNP indel variants. Some of these are considered pathogenic. Furthermore, HDR does not work in most cell types. Prime editing is robust 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 diseases. This site is conserved in mammals, so in addition to treating human disease, it could produce cows and sheep immune to prion diseases or even cure wild populations of animals suffering from prion diseases. It can also be used to help make things happen. Prime editing has already been used to achieve ~25% incorporation of naturally occurring protective alleles in human cells, and previous mouse experiments have shown that this level of incorporation induces immunity against most prion diseases. It indicates that it is sufficient. This method is the first and possibly the 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 PrP expression. The goal may be to cause a premature stop codon in PRNP, eliminate a start codon, mutate or delete an essential amino acid codon, introduce or remove a splice site to generate an aberrant transcript, or or may be accomplished by introducing mutations that alter regulatory elements to reduce transcript levels. Prime editing to eliminate disease mutations. A number of variants of PRNP have been described that lead to an increased likelihood of contracting the disease (ncbi.nlm.nih.gov/pmc/articles/PMC6097508/#b154-ndt-14-2067). Since prime editing can make all possible types of point mutations, local insertions, and local deletions, each known variant can be reversed using prime editing. Prime editing to introduce one or more protective mutations into PRNP that block prion formation and/or transmission. For example, the human PRNP gene G127V 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 introducing single nucleotide polymorphisms, insertions or deletions of sequences on PRNP that would interfere with prion formation can also be used to protect against or treat prion diseases.

The third therapeutic strategy is particularly advantageous because the introduction of a protective variant can confer benefit even when a relatively small number of cells undergo editing. Furthermore, the introduction of protective variants, particularly those naturally present in the human population such as G127V, would not be expected to have any adverse consequences, as has been demonstrated in PRNP knockout mice. As per strategy 1, reducing prion protein expression may have some disadvantageous phenotypes (ncbi.nlm.nih.gov/pmc/articles/PMC4601510/, ncbi.nlm.nih. gov/pmc/articles/PMC2634447/).

Mice expressing an approximately 2:1 ratio of the protective G127V variant of human prion protein (approximately 33% expression of the protective variant) wild-type human prion protein: It was previously demonstrated that they were completely immune and also resistant to variant Creutzfeldt-Jakob disease (vCJD), a human disease transmitted from bovine spongiform encephalopathy (BSE or mad cow disease). There are ⁹¹ . Mice expressing only the protective G127V variant were completely immune to all prion disease challenges tested, including vCJD.

It is demonstrated herein that the protective G127V mutation can be efficiently incorporated into human cells in tissue culture using prime editing (see Figure 27).

Informed by these results, three settings in which PRNP editing can be used are described. PRNP editing in one setting can be used for prime editing in human patients to prevent or treat prion diseases. PRNP editing in the second setting can be used for prime editing in livestock to prevent the initiation and spread of prion diseases. Livestock, both cattle and sheep, have experienced idiopathic outbreaks of prion disease caused by the protein produced by the PRNP gene. In addition to the debilitating and fatal diseases that animals suffer from, these cases are also economically devastating. In part, this is 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 of 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 a PRNP mutation such as G127V into the livestock germline can eliminate the occurrence of BSE or scrapie, a manifestation of prion disease in sheep. PRNP editing in the third setting can be used to prime edit wildlife and prevent the spread of prion diseases in the wild. Currently, North American cervid populations, including deer, elk, and moose, suffer from chronic wasting disease (CWD), a manifestation of prion disease caused by PNRP in these species. Occurrence has been reported to be as high as 25% in some populations (cdc.gov/prions/cwd/occurrence.html). CWD has also been reported in Norway, Finland, and South Korea. It is still unknown whether the disease is transmissible from these species to humans (cdc.gov/prions/cwd/transmission.html) or livestock. Introduction of 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 is used to treat Creutzfeldt-Jakob disease (CJD), kuru disease, Gerstmann-Straussler-Scheinker disease, fatal familial insomnia (FFI), bovine spongiform encephalopathy (BSE; mad cow disease), and scrapie (sheep). , and chronic wasting disease (CWD; deer, elk, and moose).

The method will require being 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 A new method for the insertion of motifs onto gene sequences to tag or otherwise manipulate RNA in mammalian, eukaryotic, and bacterial cells is described herein. As stated in. Although it is estimated that only 1% of the human genome encodes proteins, virtually all of the genome is transcribed at some level. How many of the resulting non-coding RNAs (ncRNAs) play a functional role, much less what the role of most of these putative RNAs is, is an open question. "Tagging" these RNA molecules by inserting new RNA coding sequences with useful properties onto genes of interest is a useful method for studying the biological functions of RNA molecules in cells. It may also be useful to incorporate tags onto protein-encoding mRNAs as a means to perturb and therefore better understand how mRNA modifications can affect cellular function. For example, polyadenylation of the ubiquitous natural RNA tag is used by cells to influence export of mRNA to the cytoplasm. Different types of polyadenylation signals result in different transport rates and different mRNA lifetimes, and therefore different levels at which the encoded protein is expressed.

A common approach to expressing tagged RNA in cells is to exogenously introduce synthetic constructs, which (i) produce short-term bursts of gene expression that are often supraphysiological; or (ii) permanent incorporation of tagged RNA genes onto the genome (at random sites) using lentiviral incorporation or transposons to allow prolonged expression. or Both of these approaches are limited by the production of altered expression levels and by the absence of natural mechanisms controlling gene expression or activity. An alternative strategy is to tag the gene of interest directly at its endogenous locus using homologous recombination repair (HDR) of double-stranded DNA breaks induced by Cas9 or other targeted DNA nucleases. It is to be. Although this approach allows the generation of a wide range of endogenously tagged genes, HDR is markedly inefficient and therefore difficult to identify the desired clonal population of successfully tagged cells. requires meaningful screening of Moreover, HDR is typically very inefficient or completely inactive in many cell types, most notably post-mitotic 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. This is because they can lead to the production of RNA whose activity interferes with the function of the normal allele. 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 incorporation of tags on RNA.

Prime editing is an emerging genome editing technology that enables targeted editing of genomic loci by transferring genetic information from RNA to DNA. Using 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 wide variety of cell types compared to HDR strategies. As such, the described invention represents a new, useful and non-obvious tool for interrogating the biology of RNA genes in health and disease. Described herein is a new method for insertion of RNA motifs onto gene sequences that tags or otherwise manipulates RNA using prime editing elements (PE). PE can site-specifically insert, mutate, and/or delete multiple nucleotides at desired genomic loci that can be targeted by the CRISPR/Cas system. PE consists of a fusion between a Cas9 nuclease domain and a reverse transcriptase domain. They are guided to their genomic targets by an engineered PEgRNA (prime editing guide RNA) containing a guide spacer moiety for DNA targeting and a reverse transcription template encoding the desired genome editing (Fig. (see 28A). It is envisioned that PE can be used to tag or otherwise manipulate non-coding RNA or mRNA by inserting motifs that are functional at the RNA level (hereinafter RNA motifs). These motifs may increase gene expression, decrease gene expression, alter splicing, alter post-transcriptional modifications, affect subcellular localization of RNA, or affect RNA isolation or enable 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 protein or RNA binding factors; , can serve to introduce sgRNA or induce processing of the RNA by either self-cleavage or RNAse (see Figure 28B). Due to the flexibility of prime editing, it is not possible to provide an exhaustive list of RNA motifs that can be incorporated onto the genome. Here, a series of examples are provided that broadly illustrate the predicted range of RNA motifs incorporated by PE that can be used to tag RNA genes. With any other reported genome editing method other than PE, it is difficult to make these changes efficiently and fairly cleanly in most cell types (including many that do not support HDR). This is not currently possible.

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 modifications59 ^. Using PE, it would be possible to induce the writing or erasing of these modifications on RNA transcripts by including sequences that are bound by enzymes that write or remove them. This may be useful for studying the effects of these markers, for inducing cell differentiation, for influencing stress responses, or for other modalities since the function of these markers is still unexplored. can be used as a tool to influence target cells.

PE can encode mutations that affect subcellular localization. For example, incorporation of tRNA-Lys on mRNA could theoretically result in export to the mitochondria ⁶⁰ , but various 3'UTRs could result in nuclear retention or export ⁶¹ .

example:

SV40 polyA signal results in transport.

U1 snRNA 3 box provides retention.

Determining the subcellular localization of endogenous RNA can be difficult and requires the addition of exogenous fluorescently tagged nucleotide probes, as in the case of FISH, or the time-consuming and potentially inaccurate cellular Require fractionation and then RNA detection. Coding probes on endogenous RNA would eliminate many of these issues. One example would be to encode a fluorescent RNA aptamer, such as Spinach ⁶² or Broccoli, on endogenous RNA, thereby visualizing the presence of the RNA by the addition of a small protofluorophore.

Broccoli aptamer:

PEs can insert or remove sequences encoding RNAs that are recognized by RNA binding proteins, affecting RNA stability, expression, localization, or modification (see, eg, 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 the processing of RNA either by itself or by external factors, either therapeutically 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 prior to 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 may include wild type or engineered or evolved versions of ribozymes. 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 with which they are associated. Sequences that will guide the processing of the RNA by external factors, such as sequence-specific RNAses ⁷¹ , RNAses ⁷² that recognize specific structures, 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 Cellular generation of highly high-performance libraries of protein- or RNA-encoding genes with defined or variable insertions, deletions, or defined amino acid/nucleotide conversions. New methods for and their use for high-throughput screening and directed evolution are described herein. References cited in the example are based on the list of references included at the end of this example.

Generation of variable gene libraries is most commonly accomplished by mutagenic PCR1 ^. This method relies on either using reaction conditions that reduce the fidelity of the DNA polymerase or using modified DNA polymerases that have higher mutation rates. As such, these polymerase biases are reflected in the library product (eg, preference for transition mutations over transversions). An inherent limitation of this approach to library construction is the relative inability to influence the size of the genes being altered. Most DNA polymerases have extremely low rates of indel ^mutations2 (insertions or deletions), and most of these result in frameshift mutations in protein-coding regions, making it difficult for library members to pass through any downstream selection. would make it improbable. In addition, PCR and cloning biases can make it difficult to generate a single library consisting of genes of different sizes. These limitations can severely limit the efficacy of directed evolution to enhance existing protein functions or engineer new protein functions. In natural evolution, large changes in protein function or efficacy are typically associated with insertion and deletion mutations, which are unlikely to occur during standard library generation for mutagenesis. . Moreover, these mutations most commonly occur in regions of the protein in question that are predicted to form loops relative to the hydrophobic core. Therefore, most indels generated using traditional unbiased approaches are likely to be either harmful or invalid.

Because all libraries access only a fraction of the possible mutation space, libraries that can bias mutations toward sites on the protein where they are most likely to be beneficial, such as loop regions, , would have significant advantages compared to traditional 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 be No additional rounds of "Genesis" can pass. 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 the native replication machinery. Therefore, in PACE, libraries of genes with codons inserted or removed as specific loci can be generated and screened, but additional rounds of "indergenesis" are not possible.

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

For example, converting a CCC codon to a stop codon would be an unlikely occurrence by standard library generation, since it would require three consecutive mutations encompassing two transversions. PE can be used to convert any given target codon into a TGA stop codon in one step. They can also be used to incorporate programmed diversity into a given position, for example, by incorporating at a given site a codon that encodes for one hydrophobic amino acid but not for any other. It can be done. Moreover, because of the programmability of PE, multiple PEgRNAs can be utilized to generate multiple different edits at multiple sites simultaneously, allowing the generation of highly programmed libraries (see Figure 29D). ). In addition, it is possible to generate regions of mutagenesis in otherwise invariant libraries using reverse transcriptases with lower fidelity (e.g., HIV-I reverse transcriptase ⁴ or Bordetella phage reverse transcriptase). Enzyme ⁵ ).

The possibility of repeated rounds of PE to the same site is also envisioned, allowing for example repeated insertions of codons at a single site. Finally, it is envisaged that all of the approaches described above can be incorporated into sequential evolution, allowing the generation of new in situ evolution libraries (see Figure 30). They can also be used to construct these libraries in other cell types where it would otherwise be difficult to assemble large libraries, such as in mammalian cells. Generation of bacterial strains encoding optimized PEs 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 by PE will be a highly useful tool in synthetic biology and directed evolution, as well as for high-throughput screening of combinatorial variants of proteins and RNA.

Competing Approaches The main way that diverse libraries are currently generated ^is by mutagenic PCR as described above1. Although insertions or deletions can be introduced by degenerate NNK primers at defined sites during PCR, introducing such mutations at multiple sites is important before a more diverse library is constructed by mutagenic PCR. Requires multiple rounds of iterative PCR and cloning, making the method slow. An alternative and complementary method is DNA shuffling, in which 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 a faster response than mutagenic PCR alone. It also brings with it the rapid generation of diverse libraries6 ^. Although this approach could theoretically generate indel mutations, it more often results in frameshift mutations that disrupt gene function. Moreover, DNA shuffling requires a high degree of homology between gene fragments. While both of these methods have to be done in vitro and the resulting libraries are transformed into cells, libraries generated by PE can be constructed in situ, making their use for continuous evolution enable. Libraries can be constructed in situ by in vivo mutagenesis, and these libraries rely on host cellular machinery to exert a bias against indels. Similarly, while traditional cloning methods can be used to generate site-specific mutation profiles, they cannot be used in situ and are generally grown in vitro before being transformed into cells. Assembled one at a time. The efficiency and broad functionality of PEs in both prokaryotic and eukaryotic cell types further emphasizes their ability to be cloned into model organisms such as E. coli and then transferred into cells or organisms of interest. We suggest that libraries can be constructed directly within the cell type of interest. Another competing approach to targeted diversification is automated multiplex genome engineering or MAGE, in which multiple single-stranded DNA oligonucleotides can be integrated into a replication fork, resulting in programmable mutations ^. However, MAGE requires significant modification of the host strain and can lead to off-target or 100-fold increases in background mutations8, ^whereas PE is more highly programmed and may result in fewer off-target effects. is expected. Additionally, MAGE has not been demonstrated in a wide variety of cell types, including mammalian cells. Prime editing is a novel and non-trivial complementary technique for library generation.
Example of PE in directed evolution to build gene libraries

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 point mutations and indels to be introduced in a manner not possible with traditional approaches. Allows repeated accumulation of both. It has previously been shown that PE can site-specifically and programmably insert nucleotides onto E. coli gene sequences. Directed evolution outlined proposes to identify monobodies with improved binding to specific epitopes by engineered two-hybrid protein:protein binding PACE selection. Specific and highly variable loops on these monobodies contribute significantly to affinity and specificity. Improved monobody binding can be rapidly obtained in PACE by varying the length and composition of these loops in a targeted manner. However, varying sequence length is not an established functionality of PACE. Although libraries of various loop sizes can be used as a starting point for PACE, subsequent improvements in length do not occur during PACE selection, preventing access to beneficial synergistic combinations of point and indel mutations. Will. Introducing PE into PACE selection will allow in situ generation and evolution of monobodies with various loop lengths. To do so, it is envisaged the introduction of an additional PE plasmid encoding the PE enzyme and one or more PEgRNAs into the host E. coli strain. Expression of PE enzyme and PEgRNA 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 contain a spacer that directs the PE to the site of interest on the selection phage and will be designed such that multiple 3 nucleotides can be inserted into the target site. As a result, new PEgRNA binding sites will be introduced, allowing repeated insertion of one or more codons at the target site.

In parallel, another host E. coli strain may contain PEgRNA that templates the removal of one or more codons, allowing the loop size to shrink during evolution. PACE experiments can utilize a mixture of both strains or alternate between the two to allow 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 to 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 infections ^. Application of PE as described above to antibodies or antibody-derived molecules will allow the generation of antibodies with a variety of loop lengths and a variety of loop sequences. In combination with PACE, such an approach would allow enhanced binding due to loop geometries not accessible to standard PACE, and therefore the evolution of highly functional antibodies.

Experiments will demonstrate the ability to use PE to correct deleterious mutations of bacteriophage M3 in phage-assisted discontinuous evolution (PANCE). This is a necessary first step in using PE for continuous evolution (see Figure 69).

References for example 7

The following references are incorporated herein by reference in their entirety:

Example 8 – Immune epitope insertion by PE
Precise genome targeting technology using the CRISPR/Cas system has recently been investigated in a wide range of applications, including the insertion of engineered DNA sequences onto target genomic loci. Previously, homologous recombination repair (HDR) was used for this application, requiring an ssDNA donor template and initiation of repair by means of double-stranded DNA breaks (DSB). This strategy offers the widest range of possible changes to be made to cells and is the only method available for inserting large DNA sequences into mammalian cells. However, HDR is hampered by undesired cellular side effects derived from initiating its DSBs, 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 human Rad51 mutants to Cas9 D10A nickase (RDN), resulting in a DSB-free HDR system. It features an improved HDR product:indel ratio and lower off-target editing, but is still hampered by cell type dependence and only modest HDR editing efficiency.

A recently developed fusion of Cas9 to reverse transcriptase coupled to PEgRNA (the "prime editing factor") represents a novel genome editing technology that offers several advantages compared to existing genome editing methods. Includes the ability to incorporate any single nucleotide substitutions and insert or delete any short stretches of nucleotides (up to at least several dozen bases) in a site-specific manner. Of note, PE editing is generally achieved with low unintended indel rates. PE therefore enables targeted insertion-based editing applications that were previously impossible or impractical.

Specifically, the present invention describes a method for using prime editing as a means to insert known immunogenic epitopes onto endogenous or exogenous genomic DNA, 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 rates of indel formation from DSBs. Prime editing generally offers higher efficiency than HDR while solving the problem of high indel formation from insert 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. Epitope lengths range from a few bases to several hundred bases. Prime editing factors are the efficient and cleanest technology to achieve such targeted insertions in mammalian cells.

The key concept of the present invention is to use endogenous or exogenous nucleotide sequences containing the immunogenic epitopes described above for the downregulation and/or destruction of their protein products and/or expressing cell types. The use of prime editing factors to insert onto genomic DNA. The nucleotide sequence for immunogenic epitope insertion is targeted to 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. Will. The patient's immune system will have previously been trained to recognize these epitopes as a result of previous standard immunization, for example from routine vaccination against tetanus or diphtheria or measles. As a result of the immunogenic nature of the epitope being fused, the patient's immune system recognizes and recognizes the prime-edited protein (not just the inserted epitope) and potentially the cells in which it is expressed. It is expected that it will be disabled.

Fusions to the target gene will be manipulated as required to ensure that the inserted epitope protein translation is exposed for immune system recognition. This may result in a C-terminal fusion of an immunogenic epitope to a target gene, an N-terminal fusion of an immunogenic epitope to a target gene, or a targeted nucleotide that results in protein translation resulting in the insertion of a nucleotide onto a gene. Insertions may be involved such that immunogenic epitopes are encoded in exposed regions of the surface of the protein structure.

A protein linker encoded as a nucleotide inserted between a target gene sequence and an inserted immunogenic epitope nucleotide sequence to facilitate immune system recognition, cellular trafficking, protein function, or protein folding of the target gene. may need to be manipulated as part of the invention. The protein linkers encoded by these inserted nucleotides can 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 distinctive feature of the 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 against 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 editing byproducts and where the insertion is efficient. The invention discussed herein also has the ability to combine cell type-specific delivery methods (eg, AAV serotypes) to insert epitopes into the cell type against which it is aimed 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 immuno-oncology strategies). can be used. An immediate relevant use of this technology would be as a cancer therapeutic. This is because it can thwart the tumor's immune escape mechanism by triggering an immune response against the relevant oncogenes, such as HER2, or growth factors, such as EGFR. Although such an approach may appear similar to T cell engineering, one novel advance in this approach is that it can be used to generate engineered T cells and to introduce them into patients, allowing them to be used in many cell types. This means that it can be used to treat diseases other than cancer.

PE is used to insert an immunogenic epitope (such as tetanus, pertussis, diphtheria, measles, mumps, rubella) against which most people are already vaccinated onto an exogenous or endogenous gene that drives the disease; Therefore, the patient's immune system learns to disable the protein.

Diseases likely to 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 the therapeutics of cancer, prions, and other neurodegenerative diseases, infectious diseases, and preventive medicine. Secondary therapeutic indications may include preventive care of patients with late-onset genetic diseases. In some diseases, particularly aggressive cancers, or in cases where drug therapy can help alleviate disease symptoms until the disease is completely cured, current standard of care medicine is used in conjunction with prime editing. It is expected that this may occur. Below are examples of immune epitopes that can be inserted by prime editing factors and can be used to achieve:

Below are additional examples of 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 editing factors to cells in vitro and in vivo. Prime editing factors have only been developed and characterized in cultured cells. Known methods cannot deliver prime editing factors in vivo. The disclosed methods for delivering prime editing factors by rAAV or preassembled ribonucleoprotein (RNP) complexes will overcome several barriers to in vivo delivery. For example, the DNA encoding prime editing factors is larger than rAAV packaging limits and therefore requires special solutions. One such solution is to formulate editing factors fused to split intein pairs, which are then packaged into two separate rAAV particles. These reconstitute functional editor proteins when co-delivered to cells. Several other special considerations are described that explain the unique features of prime editing, optimization of second site nicking targets and proper packaging of prime editing factors into viral vectors including lentiviruses and rAAV. It includes things.

Distinguishing features include the use of ribonucleoprotein (RNP) delivery formulations, where prime editing factors and nearby nicking targets can be pre-complexed with their specific sgRNA/PEgRNA. This will enhance the range of possible targetable sites and allow for significant optimization of editing efficiency relative to current data using DNA delivery. Variant Cas proteins, each complexed with their own guide RNA variant, can be used using either RNP or mRNA delivery formulations. This would also allow for greater diversity of possible nicking loci. It is therefore anticipated that optimization for greater efficiency can be achieved for any given application. Using RNP, it is expected to increase editing specificity based on previous RNP reports (Rees et al., 2017). This will reduce off-target prime edits. A potential architecture for splitting the prime editing factor into two AAV vectors for in vivo or ex vivo delivery is described. Packaging prime editing factors into a dual AAV system requires optimization of design considerations including the division site, reconstitution method (eg, intein), and guiding expression architecture. Using a mixture of viruses and RNPs for the delivery of prime editing factors, it is anticipated that editing will be controlled over time. This is because RNPs will eventually degrade in vivo, which will stop prime editing after RNPs are no longer supplied.

To modify genomic DNA in vivo and ex vivo, prime editing factor ribonucleoproteins (RNPs), mRNA along with prime editing factor guide RNA, or DNA can be packaged, injected, or ingested into lipid nanoparticles, rAAV, or lentiviruses. , or can be inhaled. Includes purposes of establishing animal models of human diseases, testing therapeutic and scientific hypotheses in animal models of human diseases, and treating human diseases.

If suitable means of delivery to the relevant cell types in vivo are developed, prime editing factors could feasibly be used to treat a large fraction of all genetic diseases (~89 of the pathogenic human genetic variants of Clinvar). %). Blood diseases, retinal diseases, and liver diseases are the most likely first applications due to the 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 certain embodiments, one or more of the prime editing factor domains (eg, napDNAbp domain or RT domain) can be engineered with an intein sequence.

Example 10 - Using PE to identify off-target edits Currently, there is no described method for detecting off-target edits by prime editing factors (prime edits themselves have not yet been published). These methods will allow researchers to identify potential sites of off-target editing using prime editing factors. This would be an important consideration if this technology were 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 in this aspect is to use prime editing to insert PEgRNA-templated adapter sequences or primer binding sites to allow rapid identification of genomic off-target modification sites for Cas nucleases or prime editing factors. This is the idea.

There are no known methods to identify prime editing off-target sites in an unbiased manner. This method is distinguishable from other techniques for identifying 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 Figure 33). To perform this method, you need to have a protospacer identical to the final desired editing agent (and a primer binding site sequence identical to the final desired editing agent when examining prime editing off-targets); , PEgRNA containing the necessary sequences to incorporate adapter or primer binding sites is generated after reverse transcription by prime editing. In vivo editing is performed using prime editing factors or RT fusion nucleases to isolate genomic DNA. Genomic DNA is fragmented by enzymatic or mechanical means and different adapters are added at the sites of DNA fragmentation. PCR is used to amplify from one adapter to the adapter incorporated by PEgRNA. The resulting product is deep sequenced to identify all modified sites.

The invention also encompasses the identification of off-target editing sites using in vitro modification of genomic DNA (see Figure 33). To perform 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 fragmentation of the DNA and attachment of different adapters to the site of DNA cleavage. PCR is used to amplify from the fragmented site to the adapter incorporated by PE. Deep sequencing to identify sites of modification. This in vitro editing method should enhance the sensitivity of detection since cellular DNA repair will not erase the reverse transcribed DNA adapters added by the prime editing factor.

These methods are used to identify off-target edits for either prime editing factors or for any genome editing factors (most Cas nucleases) that use a guide RNA to recognize the cut site of the target. can be used.

These methods can be applied to all genetic diseases for which genome editing agents are considered for use in treatment.

Example 11 - Use of PE to enable chemical-induced dimerization of target proteins in vivo Genomic incorporation can also be used to bring biological processes induced by dimerization, such as receptor signaling, under the control of convenient small molecule drugs, as described herein. Using the prime editing factors described herein, gene sequences encoding small molecule binding proteins can be inserted onto genes encoding target proteins of interest in living cells or patients. This editing alone should have no physiological effect. 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, causes the small molecule to dimerize the target protein. induce. This targeted 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 the life sciences. Despite rapid advances in genome editing technology, many of the >75,000 known human gene 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 block genes by inducing mixtures of insertions and deletions (indels) at target sites ^{112 - 114} . Nucleases can also be used to delete target ^genes115,116 or insert exogenous ^genes117-119 by homology-independent processes. However, double-stranded DNA breaks are also associated with undesirable outcomes, including complex mixtures of products, translocations, ¹²⁰ and ^p53 activation. Moreover, the majority of pathogenic alleles differ from their non-pathogenic 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 DSBs ¹²³ 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 byproducts 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 genome editing mediated by DSBs remains the focus of promising work ^{, 126-130} and these challenges require investigation of alternative precision genome editing strategies.

Base editing produces four types of transition mutations (C to T, G to A, A to G, and T to C) in a wide variety of cell types and organisms, including mammals, without requiring DSBs. ^128-131 Currently, 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), e.g., the T A to A T mutation required to directly correct the most common cause of sickle cell disease (HBB E6V) cannot be achieved. ¹³² . In addition, targeted deletions, e.g. removal of the four base duplication (HEXA 1278+TATC) that causes Tay-Sachs disease (HEXA 1278+TATC) ¹³³ , or targeted insertions, e.g. the most common cause of cystic fibrosis (CFTR ΔF508) No DSB-free method has been reported to perform the precise three-base insertions ¹³⁴ required for direct modification. Therefore, targeted transversion point mutations, insertions, and deletions are efficient and redundant in most cell types, even though they collectively account for most known pathogenic alleles. difficult to incorporate or modify without harmful by-products (Figure 38A).

A new “search” 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. The development of a 'replacement' genome editing technology, prime editing, is described herein. Prime editing factors, originally exemplified by PE1, use a reverse transcriptase fused to a programmable nickase and a prime editing extended guide RNA (PEgRNA) to transfer genetic information from an extension on PEgRNA to a target genome. Copy directly to the locus. The second generation prime editing factor (PE2) uses an engineered reverse transcriptase to substantially increase editing efficiency with minimal (typically <2%) indel formation, and the third generation PE3 system A second guide RNA is added to nick the non-edited strand, thereby favoring its displacement, increasing the editing efficiency to typically about 20-50% in human cells by about 1-10% indel formation. Increase further. PE3 offers much fewer byproducts and higher or similar efficiency compared to optimized Cas9 nuclease-initiated HDR, and complementary strengths and weaknesses compared to current generation base editors. Make an offer.

Application of PE3 at the genomic locus of human HEK293T cells for efficient conversion of HBB E6V to wild-type HBB, deletion of inserted TATC to restore HEXA 1278+TATC to wild-type HEXA, and resistance to prion disease. Incorporation of the G127V mutation into PRNP that confers ¹³⁵ (requiring G·C to T·A transversion), as well as a His6 tag (18bp), a FLAG epitope tag (24bp), and for recombination mediated by Cre. Targeted insertion of an extended LoxP site (44 bp) was achieved. Prime editing was also successful with varying 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 editing position, prime editing is not substantially constrained by the PAM requirements of Cas9 and could in principle target the majority of genomic loci. . Off-target prime editing is much rarer than off-target Cas9 editing at known Cas9 off-target loci, likely due to the requirement of three distinct DNA base pairing events for productive prime editing to occur. It is. By allowing precise targeted insertions, deletions, and point mutations in all 12 possible classes at a wide variety of genomic loci without the need for DSBs or donor DNA templates, prime editing can It has the potential to advance variant research and modification.

Results Strategies for importing information from elongated guide RNA onto target DNA loci
Cas9 ^targets DNA using a guide RNA containing a spacer sequence that hybridizes to the target DNA site. The aim was to engineer the guide RNA both to define the DNA target as in the natural CRISPR system, ^138,139 and also to contain new genetic information that replaces the corresponding DNA nucleotide at the target locus. . Direct transfer of genetic information from an elongated guide RNA to a defined DNA site, then replacing the original unedited DNA, could, in principle, result in targeted transfer without dependence on DSBs or donor DNA templates. This provides a general means of incorporating modified DNA sequence changes into living cells. To achieve this direct information transfer, the aim is to use engineered guide RNA (hereinafter prime edited guide RNA or PEgRNA) directly from the elongation to the target site (Figure 38A).

These initial steps of nicking and reverse transcription, similar to the mechanisms used by some naturally occurring mobile genetic ^elements140 , produced a branched intermediate with two redundant single-stranded DNA flaps on one strand. Result: a 5' flap containing unedited DNA sequences and a 3' flap containing edited sequences copied from PEgRNA (Figure 38B). To achieve successful editing, this branched intermediate must be resolved such that the edited 3' flap replaces the unedited 5' flap. Since the edited 3' flap can make fewer base pairs with the unedited strand, hybridization of the 5' flap with the unedited strand is likely to be thermodynamically favorable, whereas the 5' Flap is a preferred substrate for structure-specific endonucleases such as FEN1 ¹⁴¹ . This excises the 5' flap generated during lagging strand DNA synthesis and long patch base excision repair. That preferential 5' flap excision and 3' flap ligation can drive the incorporation of edited DNA strands, producing heteroduplex DNA containing one edited strand and one unedited strand. was inferred (Figure 38B).

Permanent incorporation of an edit can occur 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 nicks into the unedited DNA strand at a sufficient distance from the site of the original nick to minimize double-strand break formation. It was hypothesized that the inclusion of a molecule 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 were constructed by extending the sgRNA at either end with a PBS sequence (5 to 6 nucleotides, nt) and an RT template (7 to 22 nt). that 5'-extended PEgRNA directs Cas9 binding to target DNA, and that both 5'-extended and 3'-extended PEgRNA exhibit Cas9-mediated target nicking in vitro and DNA cleavage activity in mammalian cells. It was confirmed that it supported the following (Figures 44A to 44C). These candidate PEgRNA designs were developed using a pre-nicked 5'-Cy5-labeled dsDNA substrate, 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 (Figures 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 conventional sgRNA, confirming the need for the PBS and RT template components of PEgRNA (Figure 38D). These results demonstrate that Cas9-mediated DNA melting exposes single-stranded R-loops, which, when nicked, prime reverse transcription from either 5'- or 3'-extended PEgRNAs. Demonstrates that he or she is capable of doing so.

The unnicked dsDNA substrate ^was then tested with a Cas9 nickase (H840A mutant) that exclusively nicks PAM-containing strands. In these reactions, the 5'-extended PEgRNA produced reverse transcripts 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 in principle for reverse transcription to terminate anywhere on the remainder of the PEgRNA, the use of 3'-extended PEgRNA produced only a single apparent product. DNA sequencing of the products of the reaction with Cas9 nickase, RT, and 3' extended PEgRNA revealed that the complete RT template sequence was reverse transcribed into the DNA substrate (Figure 44G). These experiments established that 3'-extended PEgRNA can serve as a template for reverse transcription of new DNA strands while retaining the ability to direct Cas9 nickase activity.

DNA nicking and reverse transcription using in vitro PEgRNA, Cas9 nickase, and RT on reporter plasmid substrates to evaluate eukaryotic DNA repair outcomes of 3' flaps produced by in vitro PEgRNA-programmed reverse transcription. and the reaction products were then transformed into yeast (S. cerevisiae) cells (Figure 45A). Encouragingly, 37% of yeast transformants Both GFP and mCherry proteins were expressed (Figure 38F, Figure 45C). Consistent with the results in Figures 38E and 44F, the editing reaction performed in vitro with 5'-extended PEgRNA resulted in fewer GFP and mCherry double-positive colonies (9) than with 3'-extended PEgRNA. %) (Figures 38F and 45D). Productive editing involves inserting one nucleotide (15% of double-positive transformants) or deleting one nucleotide (29% of double-positive transformants) to correct the frameshift mutation. 'Also observed with elongated PEgRNA (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 position (Figure 45G). These results demonstrate that eukaryotic DNA repair can disassemble the 3' DNA flap generated from prime editing and incorporate precision DNA editing encompassing transversions, insertions, and deletions. There is.

Design of Prime Editing Factor 1 (PE1)
Prompted 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. A direct fusion of Cas9 H840A to the 3'-extended PEgRNA (hereafter simply referred to as PEgRNA, Figure 39A) and reverse transcriptase via a flexible linker is a functional two-component prime editing It was hypothesized that a system could be constructed. HEK293T (immortalized human embryonic kidney) cells were infected 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. transfected. Initial attempts did not result in a detectable T·A to A·T conversion at the HEK3 target locus.

However, extension of PBS on PEgRNA to bases 8-15 (FIG. 39A) led to detectable T·A to A·T editing at the HEK3 target site (FIG. 39B). In prime editing factor constructs in which RT was fused to the C-terminus of Cas9 nickase (3.7% maximal T·A to A·T conversion, depending on PBS length ranging from 8 to 15 nt), the N-terminal RT-Cas9 nickase fusion (maximal T A to A T conversion of 1.3%) (Figure 39B; unless otherwise specified, all mammalian cell data in this application refers to selection or (Reporting values for the entire treated cell population without sorting). These results demonstrate that wild-type M-MLV RT fused to Cas9 improves genome editing in human cells compared to that required in vitro using commercially available variants of M-MLV RT delivered in trans. suggests that long PBS sequences are required for This first generation wild-type M-MLV reverse transcriptase fused to the C-terminus of Cas9 H840A nickase was designated as PE1.

The ability of PE1 to precisely introduce transversion point mutations at four additional genomic target sites defined by PEgRNA (Figure 39C) was tested. Similar to editing at the HEK3 locus, efficiency at these genomic sites was dependent on PBS length, with maximum editing efficiency 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 the editing efficiency of each site (Figure 46A). PE1 is capable of incorporating targeted insertions and deletions at the HEK3 locus, exemplified by 1-nucleotide deletions (4.0% efficiency), 1-nucleotide insertions (9.7%), and 3-nucleotide insertions (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 DNA templates.

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 ¹⁵⁰ M-MLV RT variants that had previously emerged from laboratory evolution for their ability to support reverse transcription at elevated temperatures. Sequential introduction of three of these amino acid substitutions (D200N, L603W, and T330P) onto M-MLV RT, hereafter referred to as M3, resulted in 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 template:PBS complexes, enzymatic processability, and thermostability in combination with M3. Among the 14 additional variants analyzed, the variant with T306K and W313F substitutions in addition to the M3 mutation showed higher editing efficiency compared to M3 for 6 transversion or insertion edits at 5 genomic sites in human cells. was improved by 1.3 to 3.0 times compared to the addition (Figures 47A to 47S). This quintuple mutant of M-MLV reverse transcriptase (Cas9 H840A-M-MLV RT(D200N L603W T330P T306K W313F)) integrated into the PE1 architecture is referred to as PE2 from here on.

PE2 incorporates single-nucleotide transversion, insertion, and deletion mutations with substantially higher efficiency than PE1 (Figure 39C), is compatible with shorter PBS PEgRNA sequences (Figure 39C), and Consistent with an enhanced ability to productively engage genomic DNA:PBS complexes. On average, PE2 led to a 1.6- to 5.1-fold improvement in prime edit point mutation efficiency compared to PE1 (Figure 39C), and in some cases dramatically improved editing yield by up to 46-fold (Figure 47F). and Figure 47I). PE2 accomplished targeted insertions and deletions more efficiently than PE1, targeting a 24bp FLAG epitope tag at the HEK3 locus with an efficiency of 4.5%, 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 an efficiency of 8.6%, 2.1-fold higher than that of PE1 (Figure 39C). These results establish PE2 as a more efficient prime editing factor than PE1.

Optimization of PEgRNA characteristics

The relationship between PEgRNA architecture and prime editing efficiency was systematically explored with 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, which contain 40% and 30% GC content in the first 10 nt upstream of the nick, respectively), but with more extensive Those with GC content favor prime editing with shorter PBS sequences (HEK4 and FANCF, which contain 80% and 60% GC content in the first 10 nt upstream of the nick, respectively) (Figure 39C), and PEgRNA nicking against PBS. consistent with the energetic requirements for hybridization of the hybridized DNA strands. PBS length or GC content level does not strictly predict prime editing efficiency, and other factors such as secondary structure of DNA primers or PEgRNA extension may also influence editing activity. It is recommended to start with a PBS length of ˜13 nt for typical target sequences and explore different PBS lengths if the sequence deviates from ˜40-60% GC content. When necessary, the optimal PBS sequence 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 generated using PE2 at five genomic target sites (Figure 39D) and at three genomic sites with longer RT templates as long as 31 nt (Figures 48A to 48D). 48C) Systematically evaluated. Like PBS length, RT template length can also be varied to maximize prime editing efficiency, but in general, many RT template lengths ≧10 nt length favor more efficient prime editing. (Figure 39D). Some target sites prefer longer RT templates (>15 nt) to achieve higher editing efficiency (FANCF, EMX1), while other loci prefer shorter RT templates (HEK3, HEK4) (Figure 39D ), starting from ~10-16 nt, it is recommended that both short and long RT templates be tested when optimizing PEgRNA.

Importantly, RT templates placing C as a 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 (Figure 48A-48C). Based on the structure of sgRNA bound to ^Cas9148,149 , the presence of C as the first nucleotide of the canonical sgRNA 3' extension leads to the pi-stacking with Tyr 1356 of Cas9 and the non-canonical base pairing with sgRNA A68. It was speculated that sgRNA backbone folding could be blocked by pairing with nucleotide G81, which natively forms . Since many RT template lengths support prime 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 prime editing factor 3 systems (PE3 and PE3b)

Although PE2 may transfer genetic information from PEgRNA to target loci more efficiently than PE1, the manner in which cells degrade the resulting heteroduplex DNA produced by one edited strand and one unedited strand determines whether the edit is durable. Previous developments in base editing faced similar challenges since the original product of cytosine or adenine deamination is heteroduplex DNA containing one edited and one unedited strand. To increase the efficiency of base editing, Cas9 D10A nickase was used to introduce a nick into the unedited strand and direct DNA repair to that strand using the edited strand as a template ^. To take advantage of this principle and enhance prime editing efficiency, we used the Cas9 H840A nickase already present in PE2 and a simple sgRNA to induce preferential replacement of the unedited strand by cells. A similar strategy of nicking (Figure 40A) was tested. Since the edited DNA strand was also nicked to initiate prime editing, the nick positioning programmed by various sgRNAs was tested on the unedited strand to minimize the production of double-stranded DNA breaks leading to indels. It became.

We first applied this PE3 strategy to five genomic sites in HEK293T cells by screening for nick-inducing sgRNAs located 14 to 116 bases from the site of the nick induced by PEgRNA either 5' or 3' of the PAM. It was tested in At 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 times, or as much as 55% higher, compared to the PE2 system. (Figure 40B). Although the optimal nicking position varied depending on the genomic site, a nick located approximately 40-90 bp 3' of the PAM from the PEgRNA-induced nick (positive distance in Figure 40B) generally A favorable increase in prime editing efficiency (41% on average) occurred without extra indel formation (an average indel of 6.8% for the sgRNA yielding the highest editing efficiency at each of the five sites tested) (Fig. 40B). As expected, at some sites, placing the 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). Possibly due to the formation of a double-strand break 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), indicating that locus-dependent factors move from the proximal binary nick to the double-stranded DNA. It was suggested that the conversion to cleavage could be controlled. In one tested site (HEK4), complementary strand nicks provided no benefit or resulted in indel levels exceeding editing efficiency (up to 26 %), consistent with an unusual tendency for the edited strand at that site to be nicked or ligated inefficiently by the cell. It is recommended to start with an unedited strand nick approximately 50 bp from the PEgRNA-mediated nick and to test alternative nick positions if the indel frequency exceeds acceptable levels.

This model of how complementary strand nicking improves prime editing efficiency (Figure 40A) is that nicking the non-edited strand only after flap disassembly of the edited strand is due to the presence of simultaneous nicks. We predicted that this would reduce the number of double-strand breaks that subsequently form indels. To achieve temporal control of non-edited strand nicking, we designed sgRNAs with spacer sequences that matched the edited strand rather than the original allele. Using 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 the editing event on the PAM strand has occurred. This PE3b approach was tested with five different edits at three genomic sites in HEK293T cells, and outcomes were compared 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; on average 12 times lower indels or 0.85%) (Figure 40C). Thus, when editing was on a second protospacer, the PE3b system reduced indels while still improving editing efficiency compared to PE2, often to a level similar to that of PE3. (Figure 40C).

Together, these findings established that the PE3 system (Cas9 nickase-optimized reverse transcriptase + PEgRNA + sgRNA) improves editing efficiency ~3-fold compared to PE2 (Figures 40B-40C ). As expected from the additional nicking activity of PE3, PE3 was accompanied by a wider range of indels than PE2. Use of PE3 is recommended when prime editing efficiency is a priority. When indel minimization is critical, PE2 offers ~10 times lower indel frequencies. When it is possible to use sgRNAs that recognize incorporated edits and nick into the non-edited strand, the PE3b system can achieve PE3-like editing levels while greatly reducing indel formation.

To demonstrate the targeting scope and versatility of prime editing with PE3, at positions +1 to +8 of the HEK3 target site (counting the first base 3' of the PEgRNA-induced nick as position +1). Incorporation of all possible single nucleotide substitutions was investigated using PE3 and PEgRNA with a 10 nucleotide RT template (Figure 41A). Collectively, these 24 distinct edits cover all 4 transition mutations and all 8 transversion mutations, with an average of 33 ± 7.9% (ranging between 14% and 48%). ) editing efficiency (containing no indels), proceeding with an average of 7.5 ± 1.8% indels.

Importantly, long-range RT templates can also result in efficient prime editing by PE3. For example, using PE3 with a 34nt RT template, point mutations were made at positions +12, +14, +17, +20, +23, +24, +26, +30, and +33 of the HEK3 locus (induced by PEgRNA). (12 to 33 bases from the nick) with an average efficiency of 36±8.7% and indels of 8.6±2.0% (Figure 41B). Other RT templates ≧30 nt at three alternative sites also support efficient editing, although editing beyond position +10 at other loci was not attempted (Figures 48A-C). The feasibility of long RT templates enabled efficient prime editing of dozens of nucleotides from the original nick site. In contrast to other precision genome editing methods, NGG PAMs on either DNA strand occur on average every ~8 bp, much less than the maximum distance between edits and PAMs that favors efficient prime editing. In general, prime editing is not substantially constrained by ^the availability of nearby PAM sequences125,142,154. Given the presumed relationship between RNA secondary structure and prime editing efficiency, when designing PEgRNAs for long-range editing, a variety of lengths and, if necessary, sequence compositions (e.g., synonymous codons) are recommended. It is prudent to test RT templates to optimize editing efficiency.

To further test the scope and limitations of the PE3 system for introducing transition and transversion point mutations, we made 72 additional edits covering all 12 possible point mutations at 6 additional genomic target sites. (Figures 41C to 41H). Overall, indel-free editing efficiency averaged 25±14% and indel formation averaged 8.3±7.5%. Since the PEgRNA RT template contained the PAM sequence, prime editing could induce changes in the PAM sequence. In these cases, higher editing efficiency (on average 39 ± 9.7%) and lower indel generation (on average 5.0 ± 2.9%) was observed (Figures 41A-41K. Positions +5 or +6 point mutations in ). This increase in the efficiency of PAM editing and decrease in indel formation may result from the inability of Cas9 nickase to recombine and nick the edited strand prior to repair of the complementary strand. Prime editing supports combinatorial editing without obvious loss of editing efficiency, so it is recommended to edit PAM in addition to other desired changes when possible.

Next, 14 targeted small insertions and 14 targeted small deletions were performed using PE3 (Figure 41I) at 7 genomic sites. Targeted 1bp insertions proceeded with an average efficiency of 32±9.8%, and 3bp insertions were incorporated with an average efficiency of 39±16%. Targeted 1bp and 3bp 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%. Insertions and deletions introduced between positions +1 and +6 alter the position or structure of PAM, so that Cas9 nickase precedes repair of the complementary strand, analogous to point mutations that edit PAM. It has been surmised that this range of insertion and deletion editing is typically more efficient due to the inability to recombine and nick the strands that have been removed.

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 insertions and deletions at three genomic sites, insertions and point mutations, deletions and point mutations, or multiple edits at the same target locus consisting of two point mutations. did. These combinatorial edits are highly efficient, averaging 55% target editing with 6.4% indels (Figure 41K), allowing combinations of precise insertions, deletions, and point mutations to be highly efficient at individual target sites. The ability of prime editing to be done with efficiency and low indel frequency was demonstrated.

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) incorporate and incorporate C, G to T, A and A, T to G, C transition mutations with high efficiency and low indels. Get ^129,130,142 . The application of base editing is limited by the presence of multiple cytidine or adenine bases on the base editing active window (typically ~5 bp wide), which results in ^unwanted bystander editing, or approximately 15 ± 2 nt from the target nucleotide. ^142,156 may be limited by the absence of PAM positions. Prime editing is useful for the precise incorporation of transition mutations without bystander editing or when the lack of a suitably positioned PAM makes it impossible to position the target nucleotide favorably on the CBE or ABE activity window. It was anticipated that it could be particularly useful.

Prime editing and cytosine base editing by editing three genomic loci that contain multiple target cytidines on the standard base editing window (protospacer positions 4-8; counting PAM as positions 21-23). compared. Optimized CBE ¹⁵⁷ without (BE2max) or with (BE4max) nickase activity was used, or the related PE2 and PE3 prime editing systems were used. Among the nine total target cytosines on the three-site base editing window, BE4max has a 2.2-fold higher average total C/G to T/A conversion than PE3 at the central base of the base editing window (protocol). Spacer positions 5 to 7. Generated for Figure 42A). Similarly, non-nicking BE2max outperformed PE2 by an average of 1.4 times at these well-positioned bases (Figure 42A). However, for cytosines outside the center of the base editing window, PE3 outperformed BE4max by 2.7 times and PE2 outperformed BE2max by 2.0 times (40 ± 17% for PE3 vs. 15 ± 18% for BE4max, and 22 ± 11% for PE2). mean edit of 11 ± 13% of BE2max). Overall, the indel frequencies in PE2 were very low (0.86 ± 0.47% on average), and in PE3 they were similar to or modestly higher than those in BE4max (BE4max range: 2.5% to 14%). ; PE3 range: 2.5% to 21%) (Figure 42B).

When we compare the efficiency of base editing with prime editing for the incorporation of precise C, G to T, and A edits (without any bystander editing), we find that the efficiency of prime editing greatly outweighs that of base editing at the upper sites. exceeded. These, like most genomic DNA sites, contain multiple cytosines over a ˜5 bp base editing window (FIG. 42C). At these sites, such as EMX1, which contains cytosines at protospacer positions C5, C6, and C7, BE4max generated a small number of products containing only a single target base pair conversion without bystander editing. In contrast, prime editing at this site involves editing C-G to T-A at any position or combination of positions (C5, C6, C7, C5+C6, C6+C7, C5+C7, or C5 +C6+C7) (Figure 42C). All precise one- or two-base edits (i.e., edits that did not alter any other neighboring bases) were significantly more efficient for PE3 or PE2 than for BE4max or BE2, respectively, but for the three-base C. G to T·A editing was more efficient in BE4max (FIG. 42C), reflecting the tendency of base editors to edit all target bases on the activity window. Taken together, these results indicate that cytosine base editors may result in higher levels of editing than PE2 or PE3 at optimally positioned target bases, whereas prime editing may result in base editing at non-optimally positioned target bases. It has been demonstrated that multiple editable bases can be edited with considerably high accuracy.

Optimized non-nicking ABE (ABEmax ¹⁵² with dCas9 instead of Cas9 nickase, hereafter referred to as ABEdmax) vs. PE2 and optimized nicking adenine base editor ABEmax vs. A.T to G.C with PE3. editing was compared at two genomic loci. At a site containing two target adenines on the base editing window (HEK3), ABE was more efficient than PE2 or PE3 in converting A5, whereas PE3 was more efficient in converting A8 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 in both A5 and A8 (Figure 42E). Overall, ABE generated much fewer indels in HEK3 than prime editing factors (0.19 ± 0.02% of ABEdmax vs. 1.5 ± 0.46% of PE2, and 0.53 ± 0.16% of ABEmax vs. 11 ± 2.3% of PE3). , Figure 42F). In FANCF, where only a single A is present on the base-editing window, ABE2 and ABEmax outperform their prime-edited counterparts by a factor of 1.8 to 2.9 in total target base-pair conversions, resulting in substantial reductions from both base-editing and prime-editing. Generally all edited products contained only precise edits (Figures 42D-42E). Like the HEK3 site, ABE produced much fewer indels at 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 lies on the base editing window, or when bystander editing is allowed, current base editors are typically more efficient than prime editors and eliminate small numbers of indels. bring. Prime editing factors offer substantial advantages 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 PAM. do.

Off-target prime editing

To result in productive editing, prime editing requires a complementary target locus for the Cas9 domain to bind: PEgRNA spacer, a target locus for reverse transcription to initiate primed by PEgRNA: PEgRNA PBS complementarity, and target locus for flap degradation: Requires reverse transcriptase product complementation. It was hypothesized that these three distinct DNA hybridization requirements may minimize off-target prime editing compared to those of other genome editing methods. To test this possibility, HEK293T cells were incubated with PEgRNAs targeting the same protospacer as Cas9 with PE3 or PE2 and a total of 16 PEgRNAs designed to target 4 on-target genomic loci. or with the same 16 PEgRNAs as Cas9. These four target loci each have at least four well-characterized off-target sites where Cas9 and the corresponding on-target sgRNAs are known to cause substantial off-target DNA modifications in ^HEK293T cells. was selected. After treatment, the four on-target loci and the top four known Cas9 off-target sites for each on-target spacer were sequenced for a total of 16 off-target sites (Table 1).

Consistent with previous studies, ^Cas9 and the four targeted sgRNAs modified all 16 of the previously reported off-target loci (Figure 42G). For the HEK3 target locus, Cas9 off-target modification efficiency at the four off-target sites was 16% on average. sgRNAs targeting Cas9 and HEK4 resulted in an average of 60% modification of the four known off-target sites tested. Similarly, off-target sites of EMX1 and FANCF were modified by Cas9:sgRNA with an average frequency of 48% and 4.3%, respectively (Figure 42G). Compared to sgRNA, PEgRNA with Cas9 nuclease modifies on-target sites with similar (1 to 1.5-fold lower) efficiency on average, but PEgRNA with Cas9 nuclease modifies off-target sites with an average ~4-fold lower efficiency than sgRNA. It was noted that modification is done with efficiency.

Remarkably, PE3 or PE2 with the same 16 tested PEgRNAs containing these four target spacers resulted in significantly lower 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; Only one of them showed off-target editing efficiency ≧1% (Figure 42H). The average off-target prime editing of PEgRNA 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, respectively. It was ±0.11% (Figure 42H). Of note, at HEK4 off-target 3 sites where Cas9+PEgRNA1 edits with 97% efficiency, PE2+PEgRNA1 results in only 0.7% off-target editing despite sharing the same spacer sequence, compared to Cas9 editing. We demonstrate how the two additional DNA hybridization events required for prime 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 the 3'-extended PEgRNA could in principle proceed onto the guide RNA backbone. If the resulting 3' flap is incorporated 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 increases the indel frequency. Contribute to We analyzed sequencing data from 66 PE3-mediated editing experiments at four loci in HEK293T cells and found that an average of 1.7 ± 1.5% total insertion of PEgRNA backbone nucleotides in any number of low At high frequencies, PEgRNA backbone insertion was observed (Figures 56A-56D). Inaccessibility of the guide RNA backbone to reverse transcriptase due to Cas9 domain binding and cellular excision during flap degradation at the 3' end of the 3' flap mismatch resulting from PEgRNA backbone reverse transcription It is speculated that this will minimize products that incorporate PEgRNA backbone nucleotides. Although such events are rare, future work to engineer PEgRNA or prime editor proteins that minimize PEgRNA backbone integration could further reduce indel frequency.

Deaminases can act in a Cas9-independent manner for some base editors, causing low-level but widespread off-target DNA editing in first-generation CBE (but not ABE) ^160-162 as well as off-target RNA editing. Although in one generation CBE and ^ABE163-165 newer CBE ^and ABE variants with engineered deaminases greatly reduce Cas9-independent off-target DNA and RNA editing163-165. Prime editors lack base modifying enzymes such as deaminases and therefore have no intrinsic ability to modify DNA or RNA bases in a Cas9-independent manner.

In principle, the reverse transcriptase domain of primed editing factors could process appropriately primed RNA or DNA templates intracellularly, but retrotransposons, such as the LINE-1 family ¹⁶⁶ , endogenous retroviruses ^167,168 , and humans It was noted that all telomerase versions provided active endogenous human reverse transcriptase. Their natural presence in human cells suggests that reverse transcriptase activity itself is not substantially toxic. Indeed, PE3-dependent differences in HEK293T cell viability compared to those of controls expressing PE2 with an R110S+K103L mutation (PE2-dRT) that inactivates dCas9, Cas9 H840A nickase, or reverse transcriptase. was not observed169 ⁽ Figures 49A-49B).

Despite the above data and analysis, it is difficult to assess off-target prime editing in an unbiased, genome-wide manner and to determine the extent to which reverse transcriptase variants of prime editing factors or intermediates can affect cells. Additional studies are needed to characterize it.

Prime editing pathogenic transversion, insertion, and deletion mutations in human cells

The ability of PE3 to directly incorporate or correct transversions, small insertions, and small deletion mutations that cause genetic diseases into human cells was tested. 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. Treatment of ex vivo hematopoietic stem cells with Cas9 nuclease and donor DNA template for HDR, followed by enrichment, transplantation, and colonization of the edited cells is a promising potential strategy for the treatment of sickle cell disease. There are ¹⁷⁰ . However, this approach ^still generates many indel-containing byproducts in addition to correctly edited HBB alleles. Although base editors generally produce much fewer indels, they currently cannot make the T·A to A·T transversion mutation required to directly restore the normal sequence of HBB. do not have.

PE3 was used to incorporate the HBB E6V mutation into HEK293T cells with 44% efficiency and 4.8% indels (Figure 43A). From a mixture of PE3-treated cells, we isolated six HEK293T cell lines that were homozygous (triploid) for the HBB E6V allele (Figures 53A to 53E ) and showed that prime-edited human demonstrated the ability to generate cell lines. 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 showed that when all 14 PEgRNAs were combined with PE3, efficient correction of HBB E6V to wild-type HBB (≧26% wild-type HBB without indels) and an average of 2.8 It was revealed that the indel level was ±0.70% (Figure 50A). The best PEgRNA resulted in a 52% correction of HBB E6V to wild type with 2.4% indels (Figure 43A). Introduction of silent mutations that modify the PAM recognized by 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 side products.

Tay-Sachs disease is most often caused by a 4bp insertion onto the HEXA gene (HEXA 1278+TATC) ¹³⁶ . Using PE3, we incorporated this 4bp 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 (Figure 43B). 53A- 53E ). Using these cells, 43 PEgRNAs and 3 nicking sgRNAs were tested for correction of HEXA pathogenic insertions by PE3 or PE3b systems (Figure 50B). Either by a complete reversion to the wild type allele or by a shifted 4 bp deletion that blocks PAM and incorporates a silent mutation. 19 of the 43 PEgRNAs tested resulted in ≧20% editing. Complete modification 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 times fewer indel products. (1.7% of PE3 vs. 0.32% of 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 products.

Finally, the incorporation of a protective SNP onto the gene PRNP encoding human prion protein (PrP) was tested. PrP misfolding causes a progressive and fatal neurodegenerative prion disease, which can arise spontaneously by inherited dominant mutations on the PRNP gene or by exposure to misfolded PrP ¹⁷² . The naturally occurring PRNP G127V mutant allele confers resistance to prion disease in ^humans138 and ^mice173 . G127V was incorporated into the human PRNP allele in HEK293T cells using PE3. This requires a transversion of T.A from G.C. Four PEgRNAs and three nicking sgRNAs were evaluated by 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). Together, these results establish the ability of prime editing to incorporate or correct transversion, insertion, or deletion mutations in human cells. They cause or confer resistance to disease efficiently and with minimal side 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 (leukemia bone marrow) cells, PE3 was used to perform transversion editing of HEK3, EMX1, and FANCF sites, as well as 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, transversion mutations in HEK3 and FANCF, as well as 3bp insertions and 6xHis tag insertions in HEK3, were incorporated with editing efficiencies of 7.9-22%, exceeding indel formation by 10- to 76-fold (Figure 43A ). Finally, in HeLa (cervical cancer) cells, a 3bp insertion into HEK3 was 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, but editing efficiency varies by cell type and is generally less efficient than HEK293T cells. do not have. 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 HDR initiated by Cas9

The performance of PE3 was compared to that ^{of an optimized Cas9-initiated HDR 128,125 in} a mitotic cell line that supports 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 byproducts (predominantly indels) as expected from treatments that cause double-strand breaks. Using PE3 in HEK293T cells, HBB E6V integration and correction proceeded with average indels of 2.6% and 1.4% with average editing efficiencies of 42% and 58%, respectively (Figures 43E and 43G). In contrast, the same editing with Cas9 nuclease and HDR template 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). Therefore, the edit:indel ratios for HBB E6V integration, HBB E6V modification, and PRNP G127V integration were on average 270-fold higher in PE3 than in Cas9-initiated HDR.

Comparison 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, PE3-mediated 3bp insertion onto HEK3 favors PE3 with 25% efficiency and 2.8% indels compared to 17% editing of HDR and 72% indels initiated by Cas9. 40x editing: proceeded by the advantage of indel rate (Figures 43F-43G). In U2OS cells, PE3 performed this 3bp insertion with 22% efficiency and 2.2% indels, and Cas9-initiated HDR resulted in 15% editing with 74% indels, a 49-fold lower edit:indel ratio (Figure 43F-43G). In HeLa cells, PE3 carries out this insertion with 12% efficiency and 1.3% indels, with a 210-fold difference in edit:indel ratio, compared to 3.0% editing and 69% indels of HDR initiated by Cas9. (Figures 43F to 43G). Collectively, these data indicated that HDR typically resulted in lower or similar editing efficiency and much higher indels than PE3 in the four cell lines tested (Figure 51A- 51G ).

Discussion and future directions

The ability to insert DNA sequences with single nucleotide precision is a particularly potent prime editing ability. For example, PE3 was used to inject a His ₆ tag (18 bp, 65% average efficiency), a FLAG epitope tag (24 bp, 18% average efficiency), and an extended LoxP, a native substrate of Cre recombinase, into the HEK3 locus in HEK293T cells. The site (44 bp, 23% average efficiency) was precisely inserted. Average indels ranged between 3.0% and 5.9% in these examples (Figure 43H). Many biotechnology, synthetic biology, and therapeutic applications are envisioned to 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 generated 113 point mutations encompassing 18 insertions of up to 44 bp, 22 deletions of up to 80 bp, 77 transversions, and 18 combinatorial edits. , integrated without creating overt double-stranded DNA breaks at 12 endogenous loci in the human and mouse genomes, at positions ranging from 3 bp upstream to 29 bp downstream of the start of PAM. These results establish prime editing as an extremely versatile genome editing method. Since the vast majority (85-99%) of ClinVar insertions, deletions, indels, and duplications are ≦30bp (Figures 52A-52D), prime editing could in principle reduce the current known size of ClinVar's 75,122 It can correct up to ~89% of pathogenic human gene variants (transitions, transversions, insertions, deletions, indels, and duplications in Figure 38A) and provides additional potential for ameliorating diseases caused by copy number gain or loss. It has potential.

Importantly, for any desired edit, the flexibility of prime editing depends on the positioning of the nick induced by PEgRNA, the positioning of the second nick induced by sgRNA, the PBS length, the RT template length. , and many possible choices of which strand is edited first, as will be extensively demonstrated in this application. This flexibility, in contrast to the more limited options typically available with other precision genome editing methods, ^125,142,154 allows for the flexibility of editing efficiency, product purity, DNA specificity, or other parameters for a given application. allow it to be optimized to suit its needs. As shown in Figures 50A-50B, testing 14 and 43 PEgRNAs covering a range of prime editing strategies optimized the correction of pathogenic HBB and HEXA alleles, respectively. did.

Considerable additional research is needed to further understand and improve prime editing. Additional modifications of prime editing factor systems may be required to extend their suitability to include other cell types such as post-mitotic cells. To fully explore the potential of prime editing, interfacing prime editing with viral and non-viral in vitro and in vivo delivery strategies will enable a wide range of applications encompassing genetic disease research and treatment. required. However, by enabling highly precise targeted transitions, transversions, small insertions, and small deletions on the genome of mammalian cells without requiring double-strand breaks or HDR, prime editing provides new "search and replace" capabilities that substantially expand the scope of genome editing.

Method <br/> General method

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 2× Master Mix (New England BioLabs). DNA oligonucleotides containing Cy5-labeled DNA oligonucleotides, dCas9 protein, and Cas9 H840A protein were obtained from Integrated DNA Technologies. The yeast reporter plasmid was derived from previously described plasmid ⁶⁴ and was cloned by the Gibson assembly method. All mammalian editing factor plasmids used in this application were assembled using the USER cloning method as previously described175 ^. Plasmids expressing sgRNA were constructed by ligation of annealed oligonucleotides onto the BsmBI-digested acceptor vector. Plasmids expressing PEgRNA were constructed using custom acceptor plasmids by Gibson assembly or Golden Gate assembly (see "Golden Gate Assembly" overview in the supplement). The sequences of the sgRNA and PEgRNA constructs used in this application are listed in Tables 2A-2C and Tables 3A-3R. All vectors for mammalian cell experiments were purified using the Plasmid Plus miniprep kit (Qiagen) or the PureYield plasmid miniprep kit (Promega). These include 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 transcribed in vitro 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 prepared using two oligonucleotides (Cy5-AVA024 and AVA025; 1:1.1 ratio) for unnicked substrates or three oligonucleotides (Cy5-AVA024 and AVA025; 1:1.1 ratio) for pre-nicked substrates. -AVA023, AVA025, and AVA026;1:1.1:1.1) were annealed by heating to 95°C for 3 minutes, 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 ( ^20mM HEPES-K, pH 7.5; 100mM KCl; 5% glycerol; 0.2mM EDTA, pH 8.0; 3mM MgCl2; 0.5mM dNTP mix; 5mM DTT). Add dCas9 or Cas9 H840A (5 μM final) and sgRNA or PEgRNA (5 μM final) to double-stranded DNA substrate (400 nM final), when applicable, then undisclosed M-MLV RT variant Superscript III reverse transcriptase (ThermoFisher Scientific) was pre-incubated at room temperature in 5 μL reaction mixture for 10 minutes. Reactions were carried out for 1 hour at 37°C, 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 for 30 minutes at room temperature. . After heat inactivation at 95 °C for 10 min, the reaction product was combined with 2x formamide gel loading buffer (90% formamide; 10% glycerol; 0.01% bromophenol blue) and denatured at 95 °C for 5 min. , separated by denaturing urea-PAGE gel (15% TBE-urea, 55°C, 200V). DNA products were visualized by Cy5 fluorescence signal using a Typhoon FLA 7000 biomolecule imager.

Electrophoretic mobility shift assays were performed using preincubated dCas9:sgRNA or dCas9:PEgRNA complexes (concentration range between 5 nM and 1 μM final) and Cy5-labeled double-stranded DNA (Cy5-AVA024 and AVA025; final 20 nM). ) in 1× binding buffer (1× cleavage buffer + 10 μg/mL heparin). After 15 minutes of incubation at 37°C, samples were analyzed by native PAGE gel (10% TBE) and imaged for Cy5 fluorescence.

For DNA sequencing of the reverse transcripts, fluorescent bands were excised and purified from urea-PAGE gels and then 3' transduced by terminal transferase (TdT; New England Biolabs) in the presence of dGTP or dATP according to the manufacturer's protocol. I tailed it. The tailed DNA product was diluted 10 times with binding buffer (40% saturated aqueous guanidinium chloride + 60% isopropanol), purified by QIAquick spin columns (Qiagen), and then purified with primers AVA134 (A-tailed product) or AVA135. (G-tailed product) was used as template for primer extension with the Klenow fragment (New England Biolabs) (Tables 2A-2C). The extension was amplified by 10 cycles of PCR using primers AVA110 and AVA122 and then sequenced using the Sanger method with AVA037 (Tables 2A-2C).

Yeast fluorescent reporter assay

Dual fluorescent reporter plasmids containing an in-frame stop codon, +1 frameshift, or -1 frameshift were added to in vitro 5'-extended PEgRNA or 3'-extended PEgRNA as described above. Submitted to Prime Edit Reaction. After 1 hour incubation at 37°C, the reaction was diluted with water and the plasmid DNA was precipitated with 0.3M sodium acetate and 70% ethanol. The resuspended DNA was transformed into S. cerevisiae by electroporation as previously described ⁶⁷ 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.

Common 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 each supplemented with 10% (v/v) fetal bovine serum ( Dulbecco's Modified Eagle's Medium (DMEM) plus GlutaMAX (ThermoFisher Scientific), McCoy's 5A Medium (Gibco), RPMI Medium 1640 plus GlutaMAX (Gibco), or Eagle supplemented with Gibco, Quality Assurance) and 1× Penicillin-Streptomycin (Corning), respectively. Cultured and passaged with minimal essential medium (EMEM, ATCC). All cell types were incubated, maintained, and cultured at 37 °C with 5% _CO2 . Cell lines were certified by their respective suppliers and tested negative for mycoplasma.

HEK293T tissue culture transfection protocol and genomic DNA preparation

Grown HEK293T cells were seeded onto 48-well poly D-lysine coated plates (Corning). 16 to 24 hours after seeding, cells were incubated 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 ( PE3 and PE3b). Unless otherwise stated, cells were cultured for 3 days after transfection, after which the medium was removed, cells were washed with 1x PBS solution (Thermo Fisher Scientific), and genomic DNA was transferred directly to tissue culture. Extraction was performed 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)) to each well of the plate. The genomic DNA mixture was incubated at 37°C for 1 to 2 hours, followed by a 30 minute 80°C enzyme inactivation step. Primers used for mammalian cell genomic DNA amplification are listed in Table 4. For HDR experiments in HEK293T cells, 231 ng of nuclease expression plasmid, 69 ng of sgRNA expression plasmid, 50 ng (1.51 pmol) of 100 nt ssDNA donor template (PAGE purified; Integrated DNA Technologies) were mixed with 1.4 μL of Lipofectamine 2000 (ThermoFisher ) was used for lipofection per well. Genomic DNA from all HDR experiments was purified using the Agencourt DNAdvance kit (Beckman Coulter) according to the manufacturer's protocol.

High-throughput DNA sequencing of genomic DNA samples

Genomic sites of interest were amplified from genomic DNA samples and ^sequenced by Illumina MiSeq as previously described with the following modifications. Briefly, amplification primers containing Illumina forward and reverse adapters (Table 4) were used in the first round of PCR (PCR1) to amplify the genomic region 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. PCR reactions were 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], then a final 72°C extension for 2 min. . A unique Illumina barcoded primer pair was added to each sample in the secondary PCR reaction (PCR2). Specifically, 25 μL of a given PCR2 reaction contains 0.5 μM of each unique forward and reverse illumina barcoded primer pair, 1 μL of unpurified PCR1 reaction mix, and 12.5 μL of Phusion U Green Multiplex PCR 2× Contains a master mix. Barcoding PCR2 reactions were performed 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], then a final cycle of 2 min. Extended at 72℃. PCR products were evaluated analytically by electrophoresis on a 1.5% agarose gel. PCR2 products (pooled by common amplicon) were purified by electrophoresis on a 1.5% agarose gel. Elution was performed with 40 μL of water using the QIAquick gel extraction kit (Qiagen). DNA concentration was measured by fluorometric 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

Nucleofection was performed using K562, HeLa, and U2OS cells in all experiments. For PE conditions for these cell types, 800 ng prime editing factor 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 added in a final volume of 20 μL per sample. Nucleofection was performed on 16-well Nucleocuvette strips (Lonza). K562 cells were nucleofected using 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 using 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 using 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 of PE and BE was performed as described above.

Determination of PE3 activity at known Cas9 off-target sites

To assess PE3 off-target editing activity at known Cas9 off-target sites, genomic DNA extracted from HEK293T cells 3 days after transfection with PE3 was subjected to PCR for 16 previously reported Cas9 off-target genomic sites. ^118,159 (top four off-target sites of HEK3, EMX1, FANCF, and HEK4 spacers, respectively; primer sequences are listed in Table 4) used as templates for amplification. 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. There is. After PCR amplification of off-target sites, amplicons were sequenced by the Illumina MiSeq platform as described above (HTS analysis). To determine Cas9 nuclease, Cas9 H840A nickase, dCas9, and PE2-dRT on-target and off-target editing activities, HEK293T cells were incubated with 750 ng of editing factor plasmid (Cas9 nuclease, Cas9 H840A nickase, dCas9, or PE2-dRT); Transfection was performed with 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 ^{CRISPResso2178} . The editing efficiency of Cas9 nuclease, Cas9 H840A nickase, and dCas9 was quantified as the percentage of total sequencing reads containing indels. For PE3 and PE3-dRT off-target quantification, aligned sequencing reads were inspected for point mutations, insertions, or deletions consistent with the expected product 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 the sample were excluded from the analysis. For reads containing a single nucleotide variation that both occurs at a frequency ≧0.1% and is partially consistent with the edit encoded by PEgRNA, a t-test (unpaired, one-tailed, α=0.5) was used to determine whether the same We determined whether the variants occurred at significantly higher levels compared to samples treated with PEgRNAs containing spacers but encoding different edits. Comparisons were made between samples sequenced simultaneously by the same MiSeq run to avoid differences in sequencing errors. Variants that did not meet the criteria of p-value > 0.05 were excluded. Off-target PE3 editing activity was then calculated as the percentage of total sequencing reads that met the above criteria.

Generation of HEK293T cell line containing HBB E6V mutation 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-based 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 line containing HBB E6V mutation using PE3

2.5 x 104 HEK293T cells grown in the absence of antibiotics were seeded in 48-well poly-D-lysine coated plates (Corning). 16 to 24 hours after seeding, cells were incubated at approximately 70% confluency with 1 μL of 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 PEgRNA plasmid. transfected with the sgRNA plasmid. Three days after transfection, cells were washed with phosphate buffered saline (Gibco) and dissociated using TrypLE Express (Gibco). Cells were then diluted in 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 with a LE-MA900 cell sorter (Sony). Cells were treated with 3nM DAPI (BioLegend) for 15 minutes prior to sorting. After gating for doublet removal, select DAPI-negative single cells with GFP fluorescence above that of the GFP-negative control cell population in 96-well flat-bottomed cells filled with pre-chilled DMEM with GlutaMax supplemented with 10% FBS. Sorted onto culture plates (Corning). See Figures 53A-53B for representative FACS sorting examples. Cells were cultured for 10 days prior to genomic DNA extraction and HTS characterization as described above. A total of six clonal cell lines homozygous for the E6V mutation in HBB were identified.

Generation of HEK293T cell lines containing HEXA 1278+TATC insertions using PE3

HEK293T cells containing the HEXA 1278+TATC allele were generated following the protocol described above for the generation of HBB E6V cell lines; PEgRNA and sgRNA sequences are shown in Tables 2A-2C under the subheadings of Figures 43A-43H. listed. After transfection and sorting, cells were cultured for 10 days prior to genomic DNA extraction and HTS characterization as described above. Two heterozygous cell lines containing 50% HEXA 1278+TATC allele were isolated and two homozygous cell lines containing 100% HEXA 1278+TATC allele were recovered.

Cell viability assay

HEK293T cells were seeded in 48-well plates at approximately 70% confluence with 750 ng of editing factor plasmid (PE3, PE3 R110S K103L, Cas9 H840A nickase, or dCas9), 250 ng of HEK3-targeting PEgRNA plasmid, and 1 μL of Lipofectamine 2000. Transfection was performed as described above. Cell viability was measured every 24 hours for 3 days after transfection. The CellTiter-Glo 2.0 assay (Promega) was used 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 second.

lentivirus production

Lentivirus was generated as previously described206 ^. Rapidly dividing HEK293T cells (ATCC; Manassas, VA, USA) in T-75 flasks were infected with lentivirus-produced helper plasmids pVSV-G and psPAX2 in combination with a modified lentiCRISPR_v2 genome carrying the intein-splitting PE2 editing factor. transfected. FuGENE HD (Promega, Madison, WI, USA) was used according to the manufacturer's instructions. We designed four split intein editing factor constructs: 1) a U6-PEgRNA expression cassette with Npu N intein, a self-cleaving P2A peptide, and the N-terminal part of Cas9 H840A nickase (1-573) fused to GFP-KASH; 2) a viral genome encoding the Npu C intein fused to the C-terminal remainder of PE2; 3) a viral genome encoding the Npu C intein fused to the C-terminal remainder of Cas9 for a Cas9 control; and 4) DNMT1 nicking sgRNA. The mitotic intein mediates trans-splicing to join the two halves of PE2 or Cas9, and P2A GFP-KASH allows co-translational production of nuclear envelope-localized GFP. After 48 hours, the supernatant was collected, centrifuged at 500g for 5 minutes 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 pellets were snap frozen and stored at -80°C until use.

Mouse primary cortical neuron dissection and culture

E18.5 dissociated cortical cultures were harvested from planned pregnancy C57BL/6 mice (Charles River). Fetuses were harvested from pregnant mice after CO2 euthanasia 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 minutes at 37°C. Tissues were triturated in NBActiv4 (BrainBits) supplemented with DNase. Cells were counted and placed in 24-well plates at 100,000 cells per well. Half of the medium was changed twice per week.

Prime editing of primary neurons and nuclear isolation

At DIV1, 15 μL of lentivirus was added with a 10:10:1 ratio of N-terminus:C-terminus: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. The medium was removed from the dissociated cultures and the cultures were washed with ice-cold PBS. PBS was aspirated and replaced by 200 μL of EZ-PREP solution. After 5 minutes of incubation on ice, EZ-PREP was pipetted onto the surface of the wells to remove remaining cells. Samples were centrifuged at 500g for 5 minutes and the supernatant was removed. Samples were washed with 200 μL of EZ-PREP and centrifuged at 500 g for 5 minutes. Samples were resuspended in 200 μL ice-cold nuclear 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 incubated at 500 g Centrifuged for minutes. The supernatant was removed and the nuclei were resuspended in 100 μL of NSB and sorted into 100 μL of Agencourt DNAdvance lysis buffer using a MoFlo Astrios (Beckman Coulter) at the Broad Institute flow cytometry facility. Genomic DNA was purified according to the manufacturer's Agencourt DNAdvance instructions.

RNA sequencing and data analysis

HEK293T cells were co-transfected with PRNP-targeted or HEXA-targeted PEgRNA and PE2, PE2-dRT, or Cas9 H840A nickase. Seventy-two hours after transfection, total RNA was harvested from cells using TRIzol reagent (Thermo Fisher) and purified by the RNeasy mini kit (Qiagen), which includes on-column DNaseI treatment. Ribosomes were depleted from total RNA using the rRNA removal protocol of the TruSeq Stranded Total RNA Library Prep Kit (Illumina) and then washed with RNAClean XP beads (Beckman Coulter). Sequencing libraries were prepared using ribo-depleted RNA with the SMARTer PrepX Apollo NGS Library Prep System (Takara) following the manufacturer's protocol. The resulting libraries were visualized by a 2200 TapeStation (Agilent Technologies), normalized using the Qubit dsDNA HS assay (Thermo Fisher), and sequenced by NextSeq 550 using a high output v2 flow cell (Illumina) as a 75bp paired-end read. did. Fastq files were generated by bcl2fastq2 version 2.20 and trimmed using TrimGalore version 0.6.2 (g ithub.com/FelixKrueger/TrimGalore) to remove low quality bases, unpaired sequences, and adapter sequences. The trimmed reads were aligned to the Homo sapiens genome assembly GRCh ¹⁴⁸ by custom Cas9 H840A gene entry 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 July 15, 2019) and the information it contains 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 limit the analysis to pathogenic variants. To calculate the fraction of pathogenic variants that are 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.

Lengths of reported insertions, deletions, and duplications were calculated using appropriate identification of reference/alternative alleles, variant start/stop positions, or variant names. Variants that did not report any of the above information were excluded from the analysis. The reported length of indels (single variants encompassing both insertions and deletions relative to the reference genome) reflects the number of mismatches or gaps in the best pairwise alignment between the reference and alternative alleles. Calculated by determining. Frequency distributions of variant lengths were calculated using GraphPad Prism 8.

Data availability

High-throughput sequencing data has been deposited in the NCBI Sequence Read Archive database. Plasmids encoding PE1, PE2/PE3, and PEgRNA expression vectors will be available from Addgene.

Code usage

The script used to quantify PEgRNA backbone insertion is provided in Figures 60A-60B.

Additional information: Tables and arrays

Table 1: Activities of prime editing factors, Cas9 nuclease, Cas9 H840A nickase, and PE2-dRT at HEK3, HEK4, EMX1, and FANCF on- and off-target sites. PE2/PE3 edits are shown as % prime edits alongside % indels (in parentheses). % indels ^are shown in the top four previously characterized off-target sites for Cas9, Cas9 H840A nickase (nCas9), and PE2-dRT. The 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 PEgRNA

Table 2C: 3'-extended PEgRNA

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, add the spacer sequence listed below to the 5' end of the sgRNA backbone and add the 3' extension listed below containing the primer binding site and RT template to the 3' end of the sgRNA backbone. Added at the end. 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 Nicked sgRNA sequences

Table 3D: Figures 41A-41K PEgRNA

Table 3E: Figures 41A to 41K nicking sgRNAs

Table 3F: Figures 42A-42H PEgRNA

Table 3G: Figures 42A to 42H Nicking sgRNAs

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: Figures 48A-48C Nicking sgRNAs

Table 3P: Figures 50A-50B PRgRNA

Table 3Q: Figure 50A-50B Nicking sgRNA

Table 3R: Figures 51A-51 G PEgRNA

Table 4: Sequences of primers used for mammalian cell genomic DNA amplification and HTS ¹⁸¹ .

Table 5: Sequence of 100mer single-stranded DNA oligonucleotide donor template used for HDR experiments and generation of HBB E6V HEK293T cell line. The oligonucleotides are 100-103 nt in length and have homologous arms centered around the site of editing. Oligonucleotides were from Integrated DNA Technologies and purified by PAGE.

additional array

Sequence of yeast dual fluorescent reporter plasmid 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:
(Sequence number 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 - Cell data recording and lineage tracing with PE Background Genomic modification can also be used to study and document cellular processes and development. Linking cellular events such as cell division or signal cascade activation to DNA sequence modifications stores the cell's history as interpretable DNA sequence changes, and these can be linked to the occurrence of specific cellular events. I will describe what. 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 proteins and RNA molecules, information about cellular state and lineage is generally lost. Recording cellular events within single cells is a powerful way to understand how disease states initiate, maintain, and change relative to healthy controls. . The ability to explore these questions has relevance to understanding the occurrence 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), influence activation of signal cascades, metabolic state, and cellular differentiation programs. It can be used to record a number of important biological processes involved. Connecting internal and external cellular signals to sequence modifications on the genome is theoretically possible for any signal for which signal-responsive promoters are present. It is believed that PE enables a greatly expanded toolkit for probing the cell lineage and signal history of eukaryotic and prokaryotic cells both in culture conditions and in vivo.

standard ahead

Targeted sequence insertions, deletions, or mutations can be used to study a number of important biological questions, including lineage tracing and recording cellular stimulation. Current toolkits for generating these signatures on the genome are limited. Targeted locus mutagenesis has been developed to date using both DNA nucleases and base editing agents.

CRISPR/Cas9 nuclease cuts of target sequences generate stochastic sequence changes, producing multiple insertion or deletion (indel) products. The multiple sequence outcomes generated from Cas9 cuts allow unambiguous determination of the sequences cut by the nuclease. The ability to distinguish cut versus uncut sequences was used in two dominant 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 were used to trace cell lineage. As described herein, the large number of possible indel states generated by Cas9 nuclease activity allows for the generation of a cell developmental dendrogram. These suggest which cells evolved from each other in time. This approach is a powerful way to understand how cells develop from each other, and by performing RNA sequencing on selected cell pools, unique cell states and types can be identified in developmental time. used to aid in identification. This approach cannot independently report on cell signaling events, their order, and can have bias when reporting on progenitor 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 difficult. 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 events to be recorded over longer periods of time, the ability to incorporate multiple stimulus orders, intensities, and durations remains a very difficult technical problem. , this may not be achievable with this tool. Although Cas9 lineage tracing experiments are tremendously powerful, they suffer from the minor technical challenge of sequence disruption due to simultaneous Cas9 cuts at target loci. These lineage tracing experiments require editing of predesigned target loci, 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 not well suited for lineage tracing; however, sequences created by base editors The predetermined nature of the modification is particularly useful for tracking internal and external cellular stimuli. Either the base editors or sgRNA expression described herein can be coupled to specific biological or chemical stimuli. Base editing activity has been used to track a large number of individual stimuli in both mammalian and bacterial cells. This approach was also used to track sequential stimuli where a first editing event is required before a second editing 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 the target after editing, limiting the dynamic range of the technique. This means that using endogenous targets to record events is often difficult and limited to recording bulk activity instead of activity at the single cell level. . An alternative to this is the introduction of pre-designed repeat recording loci, but this has not been done to date. There is also the issue of 2-signal recording. These two-signal recording experiments only report on the presence of the second stimulus after the first; it does not report which stimulus occurred first or how long the stimulus was present. This fundamentally limits the biological understanding that can be gleaned from experiments.

It has been proposed that by modifying the genomic target sequence and incorporated pre-designed sequences, PE lineage tracing can be both lineage tracing and cell signal recording. 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 PEgRNAs define both the target genome sequence and the editing outcome, highly specific and controlled genome modifications can be achieved simultaneously using multiple PEgRNAs within the same cell. Accessible genomic 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 within cells.

Utility of PE lineage and cell signal recording

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 lifespan, allowing cells to Can generate a clock. Cellular clocks allow researchers to understand the order of various signals being received and processed by individual cells. Molecular clocks would also allow determination of long-term signals versus short-term bursts. The use of prime editing components that can edit only once per cell cycle can also lead to a molecular clock. If editing can proceed only once in the cell cycle without successive DNA modifications (perhaps by not nicking the non-edited DNA strand), it can only be edited by a second targeting PEsgRNA in subsequent cell divisions. I can imagine a system. PE is particularly useful as a cellular clock, and insertions are particularly useful because it can repeatedly insert, delete, or mutate loci in a defined manner. This is because repeated regular insertions can be made at any target genomic locus.

Another important application for recording cellular signals is the parallel recording of multiple cellular inputs. Linking cellular signaling events to DNA modifications makes it possible to record 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 editing factor expression. Unlike these other approaches, lineage prime editing should have the ability to record the order, intensity, and duration of signal events without requiring strict sequence motifs for ordered editing. It is. Indeed, lineage prime editing incorporates the cellular counters described above with signal-specific insertions, deletions, or mutations to study the order, intensity, and duration of biological signals. He must have the ability. Due to the programmable nature of prime editing, this approach can be achieved at pre-existing genomic loci in the desired target cells (such as in bacteria, mice, rats, monkeys, pigs, humans, zebrafish, C. elegans, etc.). ). It is also important to note that prime editing records incorporate barcodes in a guide RNA-dependent manner, and the number of inputs is limited to the number of signals for which there is a robust signal-specific guide RNA expression cassette. (This should be very high due to their ability to tether their expression to the activity of the RNA Pol II promoter). The number of recordable signals is linearly proportional to the number of PEgRNA required.

PE can also be used to trace cell lineages. Iterative sequence modification can be used to generate unique cell barcodes for tracking 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 large number of indel states generated by Cas9 nuclease.

Prime editing methodology for repetitive sequence modification

A number of different modalities of repetitive sequence modification using prime editing (PE) have been envisioned: DNA mutagenesis; sequence deletion; and sequence insertion. Of note, these applications can be used either against pre-existing genomic DNA targets or against pre-designed DNA sequences that researchers introduce into target cells. These techniques of sequential sequence modification are valuable for recording information in a continuous manner and for designed or stochastic modification of target loci. Sequential targeted locus modification may be particularly useful for generating libraries of variants in different hosts.

Repetitive sequence variation can be used to modify either genomic DNA or pre-designed incorporated DNA sequences in a recursive manner to report on cellular signaling events. In this paradigm, mutations incorporated by PE gRNA activity would correspond to the presence of cellular signals. These point mutations can incorporate and incorporate the PAM motif required for sequential editing events, and point mutations that correspond to the presence of specific signals. Since each sequential guide RNA will use a novel protospacer, this system will require gRNA design prior to use; however, it specifically examines individual or small numbers of stimuli of interest. It can be particularly powerful for Incorporation of these mutations may be dependent on individual biological stimuli or may be connected to coordinating cellular processes that would mark cellular time. The sequences below correspond to SEQ ID NOs: 743, 744, 744, and 745.

Another similar PE-guided RNA-intensive method is repeat deletion of the target sequence. Removal of individual sequences from the target locus will allow the ability to reconstruct the signaling event by loss of the DNA motif. Designing PEgRNAs that delete sequential sequences will allow continuous signal tracking. This will allow researchers to identify cases in which one signal follows another. This will allow researchers to explore which signal events occur in which order. Such a system has been tested using CAMERA; however, this requires prior selection of specific loci with unique sequence requirements. Since no specific sequence determinants are required, sequential sequence deletion using PE will allow parallel recording of pairwise events in individual cells. This will allow researchers the ability to probe pairwise signaling events in a multiplexed manner within 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 dependent on PEgRNA than mutagenesis or deletion. There are a number of different insertion strategies: insertion of short sequences, insertion of protospacers, insertion of protospacers and barcodes, insertion of novel homologous sequences, and insertion of homologous sequences with barcodes.

Insertion of short repeat sequences is a way to incrementally increase the size of a target sequence to measure cellular time course. In this system, insertion of repeat sequences of 5 or more nucleotides can cause repeat expansion either over time or with the continued presence of a predetermined stimulus. The loci-agnostic nature of phylogenetic PE once again allows parallel tracking of multiple unique sequence stretches for separate biological signals. This should allow 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 a different short sequence will require fewer PEgRNAs in a similar manner in the deletion space. The number of signals to be recorded and the size of the inserted sequences will determine the number of PEgRNA combinations required. One challenge of recording multiple sequences in this system would be the different efficiency of each PEgRNA in inserting its cargo sequence.

Although protospacer insertion as an indicator of cellular signals is attractive, technical challenges can compromise the efficiency of this approach. A single PEgRNA system would be difficult to use because the PEgRNA cassettes are themselves substrates and would cause insertion onto the PEgRNA, compromising the efficiency and fidelity of sequential editing. This same problem remains for 2 or 3 PEgRNA systems. This is 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 with respect to 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 and remove the data stored in the first compilation. Again, the multiple guide system suffers from insertion onto other PEgRNA expression constructs, limiting its utility (especially in vivo). The sequences below correspond to SEQ ID NOs: 752, 752, 753, 754, 754, 755, and 756.

Insertion of homologous sequences (ie, sequences 3' of the positioning of the Cas9 nick), especially homologous sequences with associated barcodes, appears to be a particularly useful phylogenetic PE strategy. This system addresses issues related to protospacer insertion by ensuring that sequential rounds of editing result in barcode insertions from PEgRNA cassettes that cannot be modified by other PEgRNA editing events in the same cell. Avoid. Barcoding systems are useful because multiple barcodes can be associated with a given stimulus. This system preserves much of the target protospacer but alters the seed sequence, PAM, and downstream flanking nucleotides. This allows multiple signals to be connected to one editing locus without significant redesign of the PEgRNA to be used. This strategy will allow multiplexed barcode insertion at a single locus in response to multiple cellular stimuli (either internal or external). It can enable recording of the intensity, duration, and order of as many signals as there are unique barcodes (these are 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 - Modulating biomolecule activity and/or localization by PE The subcellular localization and modification status of biomolecules controls their activity. Specific biological functions such as transcriptional control, cellular metabolism, and signal transduction cascades are all carefully orchestrated in specific locations within the cell. Modulating the cellular localization and modification status of proteins therefore 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 unique cellular compartments provides opportunities for modifying a number of biological processes. To incorporate genetically encoded handles to alter the modification status and subcellular level transport of biomolecules (e.g. proteins, lipids, sugars, and nucleic acids) by genetically encoded signals for therapeutic purposes. In this application, it is proposed to use prime editing (PE).

PE is a genome editing technology that allows the insertion, deletion, or replacement of short DNA sequences onto any genomic locus that can be targeted by the Cas9 enzyme. This technology could, in principle, be used to incorporate or remove important DNA, RNA, or protein coding sequences that would alter the activity of these important biomolecules. More specifically, prime editing can be used to incorporate motifs or signals that alter the localization or modification properties of a biomolecule. Some examples include: modifications of protein amino acid sequences; post-translational modification motifs; RNA motifs that alter folding or localization; and the incorporation of DNA sequences that alter 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 compacting 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 RNA can also be made to alter their activity by changing their cellular localization, interaction partners, structural dynamics, or thermodynamics of folding. Incorporation of motifs that would cause translation interruption or frameshifting can alter the abundance of mRNA species by various mRNA processing mechanisms. Altering the consensus splice sequence will also alter the abundance and prevalence of different RNA species. Altering the relative ratios of different splice isoforms will predictably lead to changes in the ratio of protein translation products. This can be used to modify many biological pathways. For example, shifting the balance of mitochondrial versus nuclear DNA repair proteins would alter the resilience of different cancers to chemotherapy reagents. Furthermore, RNA can be modified with sequences that allow binding to new protein targets. A number of RNA aptamers have been developed that bind with high affinity to cellular proteins. Incorporation of one of these aptamers can be used to sequester different RNA species by binding to protein targets that would prevent their translation, biological activity, or bring RNA species to specific subcellular level compartments. can be used for either purpose. Biomolecular degradation is another class of localization modification. For example, RNA methylation is used to control RNA within cells. A consensus motif of 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 target RNA species, which would alter the pool of RNA within the cell. . Additionally, 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 signals.

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 destroying motifs modified by the PTM machinery can alter protein post-translational modifications. Phosphorylation, ubiquitination, glycosylation, lipid modification (e.g. farnesylation, myristoylation, palmitoylation, prenylation, GPI anchor), hydroxylation, methylation, acetylation, crotonylation, SUMOylation, disulfide bond formation, side chains Bond cleavage events, polypeptide backbone cleavage events (proteolysis), and a number of other protein PTMs have been identified. These PTMs alter protein function, often by altering subcellular localization. Indeed, kinases activate downstream signal cascades, often through phosphorylation events. Removal of the target phosphosite will prevent signal transduction. The ability to site-specifically excise or incorporate either 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 for a wide range of PTM modification spaces.

Removal of lipid modification sites should prevent protein transport to the cell membrane. A major limitation of current therapeutics targeting post-translational modification processes is their specificity. Farnesyltransferase inhibitors have been extensively tested for their ability to eliminate KRAS localization to the cell membrane. Unfortunately, total inhibition of farnesylation is associated with a number of off-target effects, which 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 similarities between various kinases. PE offers a possible solution to this specificity problem. This is because it allows inhibition of target protein modification by excision of the modification site instead of total enzyme inhibition. For example, removal of the lipid-modified 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 on proteins that are not designed to be membrane bound.

PE can also be used to interrogate protein-protein conjugation events. Proteins often function within complexes to carry out their biological activities. PE can be used to either create or destroy the ability of proteins to reside within these complexes. To eliminate complexation events, amino acid substitutions or insertions on the protein:protein interface can be engineered 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 partner in the complex, thus preventing its oncogenic activity. PE can also be used to remove pathogenic mutations and restore WT activity of the protein. Alternatively, PE inhibits the activity of proteins in order to keep them in their native complexes or by pulling them to engage in interactions that are independent of their native activity. can be used for either purpose. Forming complexes that maintain one interaction state relative to another may represent an important therapeutic modality. Modifying the protein:protein interface to reduce the Kd of interaction will keep those proteins stuck together longer. Protein complexes can have multiple signaling complexes, such as n-myc, which drives the neuroblastoma signal 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 interaction partners and reduce its affinity for oncogenic interaction 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 volume18, pages31-42(2017).

Example 15 - PEgRNAS Design and Modification Described herein is a series of PEgRNA designs and strategies that can improve prime editing (PE) efficiency.

Prime editing (PE) uses information encoded within a prime editing factor guide RNA (PEgRNA) to replace, insert, or remove defined DNA sequences within a targeted genetic locus. This is the genome editing technology that can be used. Prime editing factors (PEs) consist of sequence-programmed DNA-binding proteins with nuclease activity (Cas9) fused to reverse transcriptase (RT) enzymes. PE forms a complex with PEgRNA, which contains information for targeted specific DNA loci within their spacer sequences, as well as modifications provided into the standard sgRNA backbone for desired editing during extension. Contains information that identifies the person. The PE:PEgRNA complex binds and nicks the programmed target DNA locus, allowing hybridization of the nicked DNA strand with the PEgRNA's modified primer binding sequence (PBS). . The reverse transcriptase domain then uses the nicked genomic DNA as a primer for DNA polymerization to copy the editing code information within the RT template portion of PEgRNA. 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, several limitations in efficiency and scope exist due to the multi-step process required for editing. For example, unfavorable RNA structures formed within PEgRNA can inhibit DNA editing copying from PEgRNA to genomic loci. One potential way to improve PE technology is through redesign and modification of essential PEgRNA components. Improvements to the design of these PEgRNAs are likely required for improved PE efficiency and are likely to allow the incorporation of longer inserted sequences into the genome.

Described herein is a series of PEgRNA designs envisioned to improve the efficacy of PE. These designs take advantage of numerous previously published approaches and utilize a number of novel strategies to improve sgRNA efficacy and/or stability. These improvements may belong to one or more of a number of different categories:

(1) Longer PEgRNA. This category includes improved designs that allow efficient expression of functional PEgRNAs from non-polymerase III (pol III) promoters, which allow expression of longer PEgRNAs without cumbersome sequence requirements. concerning);

(2) Core improvement. This category concerns improvements to the core Cas9-bound PEgRNA backbone, which may improve efficacy;

(3) RT processing ability. This category relates to improvements in PEgRNA that improve RT throughput, allowing insertion of longer sequences at targeted genomic loci; and

(4) Terminal motif. This category pertains to the addition of RNA motifs to the 5' and/or 3' ends of PEgRNAs that may improve PEgRNA stability, enhance RT processing capacity, prevent misfolding of PEgRNAs, or Mobilize additional factors important for editing.

Described herein are a number of potential such PEgRNAs in each category. Several of these designs have been previously described and demonstrated as such to improve sgRNA activity with Cas9. Also described herein is a platform for the evolution of PEgRNA for a given sequence target that allows polishing of the PEgRNA backbone to enhance PE activity (5). . Of note, these designs can also be easily applied to improve PEgRNA recognized by any Cas9 or its evolved variants.

(1) Longer PEgRNA.

sgRNAs are typically expressed from the U6 snRNA promoter. This promoter recruits pol III to express 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 at the levels required for efficient genome editing ^. In addition, pol III stalls or terminates 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 sgRNAs ^. However, these promoters are typically partially transcribed, resulting in an extra sequence 5' of the spacer in the expressed PEgRNA, which is significantly reduced in a site-dependent manner. Cas9:sgRNA activity has been shown to result in increased Cas9:sgRNA activity. In addition, pol III-transcribed PEgRNAs can easily terminate at 6-7 extended U's, whereas PEgRNAs transcribed from pol II or pol I will require different termination signals. Frequently, such signals will also result in polyadenylation, leading to unwanted export of PEgRNA from the nucleus. Similarly, RNAs expressed from pol II promoters (such as pCMV) are typically 5' capped, which also results in their nuclear export.

Previously, Rinn and colleagues 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 the ENE element ¹⁸⁴ from human MALAT1 ncRNA, the PAN ENE element ¹⁸⁵ from KSHV, or the 3' box ¹⁸⁶ from U1 snRNA. Remarkably, MALAT1 ncRNA and PAN ENE form ^a triple helix that protects the polyA tail. It is anticipated that these constructs, in addition to allowing expression of RNA, may also enhance RNA stability (see section iv). We also ^explored that using the promoter from U1 snRNA allows expression of these longer sgRNAs. It is anticipated that these expression systems will also allow expression of longer PEgRNAs. In addition, a series of methods have been designed for cleavage of the portion of the pol II promoter that would be transcribed as part of the PEgRNA, using self-cleaving ribozymes (Hammerhead ¹⁸⁸ , Pistol ¹⁸⁹ , Hatchet ¹⁸⁹ , Hairpin ¹⁹⁰ , VS ¹⁹¹ , Twister ¹⁹² , or Twister Sister ¹⁹² ribozymes) or other self-cleaving elements that process the transcribed guide, or a hairpin ¹⁹³ that is recognized by Csy4 and also leads to guide processing. has been done. 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 elements. It is also anticipated that circularizing PEgRNA into the form of circular intronic RNA (ciRNA) may also lead to enhanced RNA expression and stability, as well as nuclear localization ¹⁹⁴ .

PEgRNA expression platform consisting of pCMV, Csy4 hairpin, PEgRNA, and MALAT1 ENE
(Sequence number 757)

PEgRNA expression platform consisting of pCMV, Csy4 hairpin, PEgRNA, and PAN ENE
(Sequence number 758)

PEgRNA expression platform consisting of pCMV, Csy4 hairpin, PEgRNA, and 3×PAN ENE
(Sequence number 759)

PEgRNA expression platform consisting of pCMV, Csy4 hairpin, PEgRNA, and 3' box
(Sequence number 760)

PEgRNA expression platform consisting of pU1, Csy4 hairpin, PEgRNA, and 3' box
(Sequence number 761)

(2) Core improvements.

The core, Cas9-bound PEgRNA backbone could probably also be improved to enhance PE activity. Several such approaches have already been demonstrated. Illustratively, the first pairing element (P1) of the scaffold contains a GTTTT-AAAAC (SEQ ID NO: 3939) pairing element. Such T(s) elongation has been shown to result in pol III pausing and premature termination of RNA transcripts. Rational mutation of one of the TA pairs in this P1 region to a GC pair was shown to enhance sgRNA activity, suggesting that this approach may also be feasible for PEgRNA. ¹⁹⁵ . In addition, increasing the length of P1 was also shown to enhance sgRNA folding leading to improved activity, ¹⁹⁵ which has been suggested as another avenue for improving PEgRNA activity. Ru. Ultimately, 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 6nt extension to P1
GGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGCTCATGAAAATGAGCTAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCCTCTGCCATCAAAGCGTGCTCAGTCTGTTTTTTT (SEQ ID NO: 228)

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

(iii) Improvement of RT processing ability through modification of PEgRNA template region

As the size of the PEgRNA-templated insert increases, it is degraded by endonucleases, undergoes spontaneous hydrolysis, or folds into secondary structures (which cannot be reverse transcribed by RT). , or interfere with PEgRNA backbone folding and subsequent Cas9-RT binding). Consequently, modifications to the PEgRNA template may also be necessary to affect large insertions, such as whole gene insertions. Some strategies to do so include the incorporation of modified nucleotides within synthetic or semi-synthetic PEgRNAs (making the RNA more resistant to degradation or hydrolysis or adopting inhibitory secondary structures). ¹⁹⁶ . Such modifications will reduce RNA secondary structure in G-rich sequences, 8-aza-7-deazaguanosine; locked nucleic acids (LNA), which reduce degradation and enhance certain RNA secondary structures; 2'-O-methyl, 2'-fluoro, or 2'-O-methoxyethoxy modifications may be included that enhance RNA stability. Such modifications may also be included elsewhere in the PEgRNA to enhance stability and activity. Alternatively, or in addition, PEgRNA templates can be designed such that while encoding the desired protein product, they are also more likely to adopt simple secondary structures that cannot be folded by RT. Such a simple structure would act as a thermodynamic sink, but this reduces the likelihood of more complex structures that would prevent reverse transcription. Ultimately, it is also possible to split the template into two separate PEgRNAs. In such designs, PE can initiate transcription and recruit separate template RNAs to targeted sites via RNA binding proteins fused to Cas9 or RNA recognition elements on PEgRNA itself (such as the MS2 aptamer). It may also be used to The RT can either bind directly to this separate template RNA or initiate reverse transcription on the original PEgRNA before replacing it with the second template. Such an approach not only prevents PEgRNA misfolding upon addition of long templates, but also prevents dissociation of Cas9 from the genome (possibly inhibiting PE-based long inserts) to generate long inserts. This may also allow 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 PAN ENE from KSHV and ENE from MALAT1, were previously ^discussed in part (i) as means by which expression of longer PEgRNAs from non-pol III promoters could be terminated. These elements form an RNA triple helix that engulfs the polyA tails, resulting in their retention within the nucleus ^. However, by forming complex structures at the 3' end of PEgRNA that occlude the terminal nucleotides, these structures may also help prevent exonuclease-mediated degradation of PEgRNA. Other structural elements inserted at the 3' end may also enhance RNA stability without allowing termination from non-pol III promoters. Such motifs result in the formation of a hairpin or RNA quadruplex ¹⁹⁷ that would occlude the 3' end, or a 2'-3'-cyclic phosphate at the 3' end, and also potentially render PEgRNA susceptible to degradation by exonucleases. self-cleaving ^ribozymes such as HDV, which also reduce the likelihood of Inducing PEgRNA to circularize through defective splicing - forming ciRNA - can also increase PEgRNA stability and result in PEgRNA retention 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 fusion
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 templates to switch secondary RNA-HDV fusions
TCTGCCATCAAAGCTGCGACCGTGCTCAGTCTGGTGGGAGACGTCCCACCGGCCGGCATGGTCCCAGCCTCCTCGCTGGCGCCGGCTGGGCAACATGCTTCGGCATGGCGAATGGGACTTTTTTT (SEQ ID NO: 234)

(v) Evolution of PEgRNA

PEgRNA scaffolds 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 to enhance PE activity at the site in question, reduce off-target activity, or both. Probably either. Ultimately, evolution of the PEgRNA backbone with the addition of other RNA motifs will almost always improve the activity of the fused PEgRNA compared to the unevolved fusion RNA. As an illustration, ^the evolution of allosteric ribozymes composed of c-di-GMP-I aptamers and hammerhead ribozymes has led to dramatically improved activity202, but this is because evolution of hammerhead-PEgRNA fusions has led to dramatically improved activity. suggesting that activity would also be improved. Additionally, although currently Cas9 is generally intolerant of sgRNA 5' extensions, directed evolution may generate mutations that allow this intolerance to be alleviated, and additional RNA motifs may become available. There must be a sex.

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 PEgRNA activity. Other strategies for incorporating programmable mutations into the genome also include base editing, homologous recombination repair (HDR), precise microhomology-mediated end joining (MMEJ), or transposase-mediated editing. However, all of these approaches have significant drawbacks when compared to PE. Although modern base editors are more efficient than current PEs, they can only incorporate certain classes of genomic mutations and may result in additional unwanted nucleotide changes at the site of interest. HDR is only feasible for a small number of 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 incorporation of deletions, is highly site-dependent, and also has a relatively high proportion of undesired indels. obtain. Transposase-mediated editing has to date only been shown to function in bacteria. As such, improvements to PE represent perhaps the best path to therapeutic correction of a broad sample 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, DM, Hacisuleyman E., Younger ST, Rinn JL. 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 – Extending the targeting scope of PE using DNA binding proteins other than SPCAS9
Prime editing (PE) using Streptococcus pyogenes Cas9 (SpCas9) removes all single base substitutions, insertions, and deletions at genomic loci with well-placed NGG protospacer-adjacent motifs (PAMs) to which SpCas9 can bind efficiently. errors, and combinations thereof can be efficiently incorporated and incorporated. The methods described herein broaden the targeting capabilities of PE by expanding the PAMs and thus the targetable genomic loci accessible to efficient PE. Prime editing factors using DNA-binding proteins guided by RNAs other than SpCas9 enable an expanded targetable range of genomic loci by allowing access to different PAMs. In addition, the use of smaller RNA-guided DNA binding proteins than SpCas9 also allows more efficient virus delivery. PE with DNA-binding proteins guided by Cas proteins other than SpCas9 or other RNAs may not allow highly efficient therapeutic editing that was either inaccessible or inefficient using SpCas9-based PEs. Probably.

This may be due to the inefficiency of SpCas9-based PE due to non-ideal spacing of edits relative to the NGG PAM, or the overall size of SpCas9-based constructs may affect cellular expression and/or It is anticipated that it will be used in situations where delivery is either prohibitive. Certain disease-relevent loci, such as the huntingtin gene, which has a small number and poorly positioned NGG PAM of SpCas9 near its target region, are distinct from PE systems such as SpCas9-VRQR, which recognizes the NGA PAM. Can be easily targeted using Cas proteins. Smaller Cas proteins will be used to generate smaller PE constructs that can be more efficiently packaged into AAV vectors, allowing 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 the untreated control.

Figures 62A-62B provide demonstration of the importance of protospacers for efficient incorporation of desired edits in precise positioning by prime editing. This highlights the importance of alternative PAM and protospacers as novel features of this technology. "n.d." in FIG. 62A is "not detected."

Figure 63 shows the implementation of PE using SpCas9(H840A)-VRQR and SpCas9(H840A)-VRER as RNA-guided DNA binding proteins of the prime editing factor 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, and 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, and 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 sequence of the tested construct is as follows:

As shown in Figure 63, SpCas9(H840A)-VRQR-MMLV RT is agonistic at PAM sites encompassing "AGAG" and "GGAG" and imparts some editing activity to "GGAT" and "AGAT" PAM sites. had in the array. SpCas9(H840A)-VRER-MMLV RT was agonistic 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 shows how to introduce recombinase target sites (SSR target sites) into mammalian and other genomes with high specificity and efficiency by using prime editing (PE). Methods for addressing disease or generating tailored animal or plant models are described.

This example describes the use of PE to introduce recombinase recognition sequences into 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 FIG. 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 the genome. (d) shows how tandem insertion of SSR target sites can be used to invert a portion of the 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 the genome can be used to exchange cassettes from the DNA donor template. Each type of genome modification is envisioned by using PE to insert an SSR target, but this list is also not meant to be limiting.

Many large-scale genomic alterations, 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 the generation of transgenic plants8,9 or other biotechnological products. For example, chromosomal microdeletions can lead to disease, and replacement of these deletions by 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 genomic rearrangements could lead to improved products, e.g. requiring fewer resources or reducing pathogens. can lead to crops that are resistant to Current technologies for achieving large-scale genomic changes rely on random or stochastic processes, such as the use of transposons or retroviruses; other desired genome modifications have been achieved solely by homologous recombination strategies.

One attractive class of proteins for achieving targeted and efficient genome modification is site-specific recombinases (SSRs). SSR has a long history of being used as a tool for genome modification ^{10 - 13} . SSR is considered as a promising tool for gene therapy. ^15-18 This is because they allow precise recombination at defined recombination targets without relying on indels, translocations, other DNA rearrangements, or endogenous repair of double-strand breaks that can induce p53 activation. ^This is because it can induce cleavage, strand exchange, and religation of DNA fragments14. Reactions catalyzed by SSR can result in direct replacement, insertion, or deletion of target DNA fragments with efficiencies that exceed those of homologous recombination repair ^.

Although SSRs offer many advantages, they have not been widely used because they have a strong natural preference for their corresponding target sequences. SSR recognition sequences are typically ≧20 base pairs and are therefore unlikely to occur on the genome of humans or model organisms. Furthermore, the native substrate preference of SSRs is ^not easily altered, even by extensive laboratory manipulation or evolution. This limitation is overcome by using PE to introduce recombinase targets directly onto the genome or to modify endogenous genomic sequences that natively resemble recombinase targets. Subsequent exposure of cells to recombinase proteins 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 24 deletion on chromosome 721. Currently, no technology exists for the efficient and targeted insertion of multiple entire genes in living cells (the potential of PE for such full-length gene insertions is currently being investigated but not yet established). ); However, recombinase-mediated uptake at targets inserted by PE offers one approach to permanent cure of this and other diseases. In addition, targeted introduction of recombinase recognition sequences can be highly capable for applications including the generation of transgenic plants, animal research models, bioproduction cell lines, or other custom eukaryotic cell lines. For example, recombinase-mediated genome rearrangement of transgenic plants in PE-specific targets may overcome one of the bottlenecks in generating agricultural crops with improved properties.

A number of SSR family members have been characterized and their target sequences have been described, including natural and engineered tyrosine recombinases (Table 7), large serine integrases (Table 8), serine resolvases (Table 9), and tyrosine integrase (Table 10). Modified target sequences demonstrating enhanced genomic uptake rates have also been described for several ^SSRs22-30 . In addition to natural recombinases, programmable recombinases with distinct specificities have been developed ^31-40 . Depending on the desired application, using PE one or more of these recognition sequences can be introduced onto the genome at defined positions, such as safe harbor loci ^{41 - 43} .

For example, introduction of a single recombinase target on the genome will result in integral recombination with the DNA donor template (Figure 64 B ). Serine integrases that operate robustly in human cells may be particularly well suited for gene transfer44,45 ^. Additionally, depending on the identity and orientation of the targets, introduction of two recombinase targets can result in deletion of the intervening sequence, inversion of the intervening sequence, chromosomal translocation, or cassette exchange (Figures 64 C to 64F ) . By choosing an endogenous sequence that already closely resembles the recombinase target, the extent of editing required to introduce the complete recombinase target will be reduced.

Finally, some recombinases have been demonstrated to integrate into human or eukaryotic genomes at natively occurring ^{pseudosites46-64} . PE editing alters these loci in order to enhance incorporation rates at these natural pseudosites, or alternatively to eliminate pseudosites that can serve as unwanted off-target sequences. It can be used for

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 endless. The genome editing reaction is intended for use with a "prime editing factor", a chimeric fusion of a CRISPR/Cas9 protein and a reverse transcriptase domain, which utilizes a custom prime editing guide RNA (PEgRNA). By further extension, Cas9 tools and the homologous recombination repair (HDR) pathway can also be exploited to introduce recombinase targets from DNA templates by reducing the indel rate using ^several techniques. . 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 modification.

Table 7. Tyrosine recombinase and SSR target sequences

Table 8. Large serine integrase and SSR target sequences.

Table 9. Serine resolvase and SSR target sequences.

Table 10. Tyrosine integrase 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 It was planned to add a toe loop sequence. Figure 71A provides an example (top molecule) of a generic SpCas9 PEgRNA with a 3' extended arm. The 3' extending arm then contains the RT template (which contains the desired edits) 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 (ie, 5'-UUU-3').

In contrast, the lower part of Figure 71A shows the same PEgRNA molecule as the upper part of Figure 71A, but now the 9 nucleobase sequence of 5'-GAAANNNNN-3' is located at the 3' end of the primer binding site and at the terminal poly( U) It is inserted between the 5' end of the sequence. This structure folds itself backwards by 180° to form a "toe-loop" RNA structure, where the 5'-NNNNN-3' sequence of the 9 nucleobase insert anneals to the complementary sequence in the primer binding site. The 5'-GAAA-3' portion forms a 180° turn. The toe loop arrangement features depicted in FIG. 71A are not intended to limit or limit the range of toe loops that may be used in that location. 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 in various embodiments have a first sequence portion forming a 180° and a second sequence portion having a complementary sequence to a portion of the primer binding site. good.

Without being bound by theory, it is believed that the toe loop sequence allows PEgRNA to use PEgRNAs with increasingly longer primer binding sites than would otherwise be possible. It will be done. Next, longer PBS sequences are thought to improve PE activity. More specifically for PEgRNA, a possible function of the toe loop is to occlude the PBS or at least minimize the PBS interacting with the spacer. Stable hairpin formation between PBS and spacer can lead to inactive PEgRNA. Without the toe loop, this interaction could require length limitations of the PBS. Blocking or minimizing the interaction between the spacer and PBS using the 3'-terminal toe loop may also lead to improved PE activity.

Example 19 - Prime Editing with Alternative Nucleic Acid Templates and Editing Factor 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 shown in Figure 3A (PEgRNA with 5' extended arm), Figure 3B (PEgRNA with 3' extended arm), Figure 3C ( Internally extended PEgRNA), Figure 3D (with 3' extended arm, primer binding site, editing template, homology arm, and any 3' and 5' modified regions and region designated as DNA synthesis template). PEgRNA), and Figure 3E (having a 5' extended arm and containing a primer binding site, editing template, homology arm, and any 3' and 5' modification regions and a region designated as a DNA synthesis template). PEgRNA). In addition, PEgRNA structure and composition are extensively described in the detailed description and elsewhere in this application.

This example describes additional design variations of PEgRNA, in some cases PEgRNA that is chemically synthesized in whole or in part outside the cell. These are envisioned to work in conjunction with the prime editing factors herein. Such alternative designs could improve various aspects of prime editing, including the insertion of longer DNA sequences by prime editing, and potentially the use of alternative polymerases operating with increased efficiency and/or fidelity (i.e., inversion). the use of alternative and/or additional prime editing factor protein effector components to enhance or amplify prime editing. In addition, the use of chemically synthesized PEgRNA can potentially lead to the production of molecules that are more stable and possess desirable features that enhance prime editing efficiency and capacity.

The P EgRNA serves as a nucleic acid template encoding the desired edited genetic information to be incorporated into the target site. In one aspect, the PEgRNA is prepared by adding an extended arm to either the 5' or 3' end of the sgRNA (e.g., as shown in the embodiments of Figures 3A, 3B, 3D, or 3E) or similar generated by inserting the sequence internally on the sgRNA (eg, as shown in the embodiment of Figure 3C). The extension arm contains a DNA synthesis template, which can encode an ssDNA product by a polymerase (eg, reverse transcriptase), and which contains the edited genetic information of interest. The extended arm contains a primer binding site (PBS) for annealing to the genomic DNA strand nicked by napDNAbp and the intended edited DNA to become incorporated into the endogenous DNA target site by displacing the counterpart DNA strand. and a DNA synthesis template for encoding the strand. PEgRNAs can be expressed intracellularly from plasmid DNA or genomically incorporated DNA cassettes, or they can be made extracellularly by in vitro transcription or by chemical synthesis and then delivered to the cell. The preparation of PEgRNA extracellularly, especially by chemical synthesis, offers the opportunity to substantially modify PEgRNA. The present invention describes an alternative design of prime editing templates (Figure 72).

(A) DNA synthesis template expressed as a separate molecule from the guide RNA (i.e., DNA synthesis template provided in trans to the prime editing factor 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 72A for PEgRNA with a 3' extended arm. However, in some cases this may be a disadvantage, especially for more complex PEgRNA molecules, such as those encoding large insertions. These RNAs can contain extensive secondary structure, which can interfere with PEgRNA backbone structure and interaction with Cas9. Alternatively, prime editing can be performed by replacing PEgRNA by two separate RNA molecules: sgRNA and transprime editing RNA template (tPERT) as illustrated in Figure 72B . sgRNA serves to target Cas9 (or napDNAbp more generally) to a desired genomic target site, and tPERT is used by a polymerase (e.g. reverse transcriptase) to write a new DNA sequence into the target locus. used.

In general, simple expression of tPERT leads to lower editing efficiency compared to PEgRNA. However, the efficiency of transprime editing is limited by the addition of one or more MS2 RNA aptamers onto tPERT RNA in conjunction with a fusion of MS2 coat protein (MS2cp) to the prime editing factor protein (to create MS2cp-Cas9-RT). can be enhanced by introduction. 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.

Although this example utilizes MS2 tagging technology (including a tPERT MS2 RNA aptamer paired with an MS2cp protein fused to a prime editing factor), other RNA-protein recruitment systems can alternatively be used. The general concept envisioned here is that the DNA synthesis template for tPERT is modified to contain an RNA recruitment secondary structure (e.g., a specialized hairpin such as the MS2 aptamer), such that tPERT It can be recruited by a modified prime editor fusion protein that further includes an RNA binding protein that specifically recognizes and binds RNA recruitment second structures on the molecule. Reviews of other RNA-protein recruitment domains are available in the art, for example, by Johansson et al., “RNA recognition by the MS2 phage coat protein,” Sem Virol., 1997, Vol. 8(3):176-185; Delebecque et al. 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 these is incorporated herein by reference in its entirety. Other systems include the PP7 hairpin, which specifically recruits PCP proteins, and the "com" hairpin, which specifically recruits Com proteins. See Zalatan et al. Any of these well-known recruitment systems can be used for the transprime editing described herein.

The efficiency of the present tPERT transprime editing system was tested. Up to 20% efficiency of His6 insertion (18 bp) was achieved at the HEK3 site in HEK293T cells. tPERT containing a single 3'MS2 aptamer, a 13nt primer binding site, and an RT template containing 34nt of homology to the insert sequence and the locus was combined with an editing element containing MS2cp fused to the N-terminus of PE2. Used together. See Figure 73. The transprime editing strategy has the potential to address the complications associated with PEgRNA design of PEgRNAs and is more convenient for achieving larger insertions, deletions, or edits at greater distances from the prime editor nick site. May be more suitable for long RT templates.

(B) Chemically synthesized PEgRNA with RNA and DNA synthesis templates
Alternative nucleic acid templates can be used on chemically synthesized PEgRNA (Figure 72C ). For example, synthetic PEgRNA can be constructed as an RNA/DNA hybrid, with the spacer sequence and sgRNA backbone consisting of RNA nucleotides, and the primer binding site and synthetic template (shown as a 3' extension in Figure 72C ) consisting of DNA nucleotides. This could allow a DNA-dependent DNA polymerase to be used on the prime editing factor instead of reverse transcriptase. It may also prevent synthesis of DNA templated by the sgRNA backbone sequence. In other designs, chemical linkers consisting of non-templated nucleotides or other suitable linker moieties can be used to tether the nucleic acid editing template (consisting of RNA or DNA) to the sgRNA backbone. This may prevent sequential DNA polymerization of the sgRNA backbone and allow extension flexibility allowing more efficient templated synthesis. Finally, the directionality of the nucleic acid synthesis template can be reversed so that DNA polymerization proceeds in the opposite direction away from the sgRNA backbone as opposed to towards it.

MS2 PEgRNAの配列:

タンパク質配列:

(C) Mobilization of DNA Polymerase Expressed in Trans In the main embodiment of prime editing, the polymerase (eg, reverse transcriptase enzyme) is expressed as a fusion to napDNAbp (eg, Cas9 nickase). Alternatively, the polymerase (e.g., reverse transcriptase) can be expressed in trans and its activity is linked to mobilization systems such as the MS2 RNA aptamer and MS2 coat protein, or other similar mobilization systems known in the art. can be used to localize to the editing site. In this system, PEgRNA is modified to include the MS2 aptamer in one of the sgRNA backbone hairpins, and the polymerase (eg, reverse transcriptase) is expressed as a fusion protein to MS2cp. napDNAbp (eg, 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. Additionally, other RNA-protein or protein-protein interactions can be used for RT recruitment.
The following array applies to Example 19:
Array of tPERT:

Sequence of MS2 PEgRNA:

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.

Prime editing factor (PE) exceeds AAV packaging capacity. Thus, we split the PE by inserting a trans-splicing Npu intein into S. pyogenes Cas9 residue 1024 of SEQ ID NO: 18, resulting in its creation as two separate polypeptides, each encoded by one of the dualistic AAV systems. It was contemplated to allow delivery of split SpPE. It (1) allows packaging into two AAVs while accommodating space for guide cassette(s) and minimal control elements; (2) the native serine to cysteine mutation is relatively likely to be conservative, (3) the site is a flexible loop near the margin of Cas9 that is sterically predicted to allow splicing to occur, and (4) Cas9 (previously Split site 1023/1024 was chosen because it has been successfully modified by circular substitution in this loop (although not at this particular split site).

To determine whether the Npu mitotic prime editor is activating, we transfected HEK cells with a plasmid encoding the mitotic editor and found that they recapitulated the activity of full-length PE3. Analyzed by high-throughput sequencing. Moreover, the three native Npu amino-terminal residues of the C-terminal extein, which are known to splice most efficiently, are distinct from those that natively flank 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 alters the efficiency of prime editing. The "SEQ" residue facilitates prime editing with an efficiency similar to the full-length one, suggesting that the halves of the intein split PE are capable of associating with each other and mediating prime editing. No further increase was seen upon mutation to 'CFN', suggesting that association alone may be sufficient for fission PE activity, as we previously observed with intein fission 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 in the N-terminal half

1023/1024 SpPE2 splitting in the C-terminal half

Prime editing factors packaged by dual AAV systems
Prime editors split between residues 1023 and 1024 by the Npu trans-splicing intein become packaged within AAV and are similar to those of similar genome-sized base editors generated in the same manner. It has a comparable high potency.

In vivo prime editing
Split SpPE3 delivered by AAV9 by intracerebroventricular injection into P0 mice mediates prime editing in brain tissue in vivo. A nucleotide substitution at position +5 from G to T (ie, 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 editing demonstrates the feasibility of prime editing in vivo and is not expected to introduce any selective pressure on the edited cells ('+5' refers to the +5 position downstream of the nick site). The AAV architecture tested includes the full-length MMLV RT and a truncated variant lacking the RNAse H domain. The truncated post-transcriptional control element W3 was also evaluated. This is because, although it has been shown to increase expression from viral cassettes in vivo, its significance was not tested in the base editor or prime editor context. It was found that full-length RT was superior to truncated RT and that addition of the W3 sequence improved activity. The increased editing activity in the presence of W3 indicates that the expression of prime editing factors is limited in vivo and demonstrates the distinct challenges of in vivo prime editing 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 This example constructed a number of variant prime editing factors, all based on PE2. PE2 has the following sequence and structure:

As used herein, "PE2" refers to a PE complex comprising a fusion protein containing Cas9(H840A) and variant MMLV RT with the following structure: [NLS]-[Cas9(H840A)]-[Linker]- [MMLV_RT(D200N)(T330P)(L603W)(T306K)(W313F)]+desired PEgRNA. The PE fusion has the amino acid sequence of SEQ ID NO: 134, which is shown below:

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

The PE2 linker is SGGSSGGSSGSETPGTSESATPESSGGSSGGSS (SEQ ID NO: 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 compare the editing efficiency of the PE2 construct (marked as PE2; white box) with the current linker to various versions with the linker replaced by the indicated sequences, HEK3, EMX1 Representative PEgRNAs for transition, transversion, insertion, and deletion editing at the , FANCF, and RNF2 loci are shown. Replacement linkers are “1×SGGS” (SEQ ID NO: 174) , “2×SGGS” (SEQ ID NO: 446) , “3×SGGS” (SEQ ID NO: 3889) , “1×XTEN” (SEQ ID NO: 171) , “No linker ”, “1×Gly”, “1×Pro”, “1×EAAAK” (SEQ ID NO: 3968) , “2×EAAAK” (SEQ ID NO: 3969) , and “3×EAAAK” (SEQ ID NO: 3970) . Editing efficiency is measured relative to the PE2 "control" editing efficiency in bar graph format. The linker for PE2 is SGGSSGGSSGSETPGTSESATPESSGGSSGGSS (SEQ ID NO: 127). All editing was done in the context of the PE3 system. Namely, this refers to the PE2 editing construct plus the addition of 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 yields the graph shown, indicating that use of the 1×XTEN (SEQ ID NO: 171) linker sequence improves editing efficiency by a factor of 1.14 on average. giving instructions (n=15).

Example 22. Prime editing factor guide RNA with improved activity
In various embodiments, PEgRNA is likely to be target DNA site and editing dependent. Thus, the sequence of the PEgRNA will depend on the target DNA sequence and the specific edit (eg, deletion, insertion, inversion, substitution) being introduced into it by prime editing. For example, in some embodiments, when attaching a motif 3' of the primer binding site (PBS) of PEgRNA, the PBS and the motif are A linker between is preferred. However, the nature of the linker may be different for each site. For example, if the same linker is used at each site, it can pseudo-morph a 13nt PBS into a 16nt PBS by fortuitous pairing to a spacer sequence. Similarly, the linker can base pair to PBS itself, resulting in its confinement and potentially 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, in part due to the target DNA sequence and the desired editing will depend on the array.

Building in part on the information presented in Example 15 (PEgRNA Design and Manipulation), this example builds on the various structural modifications made to PEgRNA and then introduces the editing functionality, among other aspects. The effect of it on was tested.
Expression of PEgRNA from non-pol III promoters

Various PEgRNA expression systems were tested for their ability to produce PEgRNA. A 102 nucleotide sequence insert from FKBP was used as the readout.

Transcription of PEgRNA can be guided by typical constitutive promoters such as the U6 promoter. Although the U6 promoter is effective in directing the transcription of PEgRNA in most cases, the U6 promoter is less effective in directing the transcription of longer PEgRNAs or U-rich RNAs. U-rich RNA stretches cause premature termination of transcription. This example compared the editing outcomes of guides expressed from the CMV promoter or the U1 promoter to the U6 promoter. These promoters require different terminator sequences such as MASC ENE or PAN ENE provided below. Increased editing was observed in the pCMV/MASC-ENE system. However, these guides resulted in incomplete insertion of sequences, whereas in the U6 promoter, complete insertion was observed at lower levels of editing. See Figure 81. The data suggest 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' direction) (motif names are in bold immediately preceding the region to which they refer):

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 works reasonably well, and it would have been better if a less effective guide was tested. Second, it was noted that in HEK cells, transfection was relatively efficient, and the amount of guide RNA transfected was in large excess compared to that required (the amount , ~4-8x reduction has 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 serve as 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 either a terminal motif was absent or a terminal motif that did not end with a series of U was present, the transcript was terminated using the following HDV ribozyme:
GGCCGGCAUGGUCCCAGCCUCCUCGCUGGCGCCGGCUGGGCAACAUGCUUCGGCAUGGCGAAUGGGAC [SEQ ID NO : 3990 ]

Additional RNA Motifs See Figure 82 for details of certain motifs, such as HDV ribozyme 3' of PEgRNA, or G-quadruplex insertions, P1 extensions, template hairpins, and tetraloop circles. These can be introduced onto PEgRNA to improve its performance.

In particular, this example tested the effect of incorporating a tRNA motif 3' of the primer binding site. This element was chosen because of its multiple possible functions:

(1) The tRNA motif is a very stable RNA motif and therefore could potentially reduce PEgRNA degradation;

(2) MMLV RT uses prolyl-tRNA as a primer when converting the viral genome into DNA during transcription. Therefore, the same cap can be attached by RT, improving binding of PEgRNA by PE, RNA stability, and bringing the PBS back into close proximity to the genomic site, potentially also improving activity. I thought it might be.

In these constructs, tRNA P1 (see Figure 84) 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 RNAseP-mediated cleavage of the 5' tRNA of P1, which would result in its removal from PEgRNA.

This design used prolyl-tRNA (codon CGG) with a short 3nt linker between the tRNA and PBS and an extended P1. Various tRNA designs were tested and editing efficiency was tested compared to PEgRNA without a tRNA cap. Figure 83 (Illustrating PE experiments targeting editing of the HEK3 gene, using PE3 to specifically target the insertion of a 10 nt insertion at position +1 relative to the nick site), Figure 85 (FANCF Figure 86 illustrates PE experiments targeting gene editing (specifically targeting the G to T conversion at position +5 relative to the nick site, using the PE3 construct), and Figure 86 (HEK3 gene Figure 1 illustrates a PE experiment targeting the editing of (using the PE3 construct) specifically targeting the insertion of a 71nt FLAG tag insertion at position +1 relative to the nick site (see comparative data). tRNA modified PEgRNA was tested against unmodified PEgRNA control.

UGG/CGG refers to the codon used, the number refers to the length of the added P1 extension, length indicates an 8nt linker, and unspecified is a 3nt linker.

Data suggest that incorporation of tRNA may allow the use of shorter PBS. This 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 reduction in activity.

Some arrays used:

HEK3+1 FLAG-tag insertion, prolyl-tRNA{UGG} P1 ext 5nt, linker 3nt

GGCCCAGACUGAGCACGUGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUGGAGGAAGCAGGGCUUCCUUUCCUCUGCCAUCACUUAUCGUCGUCAUCCUUGUAAUCCGUGCUCAGUCUGUCUGGCGGGGCUCGUUGGUCUAGGGGUAUGAUUCCGCUUCGGGUGCGAGAGGUCCCGGGUUCAAAUCCCGGAC GAGCCCCGCCUUUU [SEQ ID NO: 3902]

FANCF+5G→T prolyl-tRNA{CGG} P1 ext 5nt, linker 3nt

GGAAUCCCUUCUGCAGCACCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCGGAAAAGCGAUCAAGGUGCUGCAGAAGGGAUCUGGCGGGGCUCGUUGGUCUAGGGGUAUGAUUCUCGCUUCGGGUGCGAGAGGUCCCGGGUUCAAAUCCCGGACGAGCCCCGCCUUUU [SEQ ID NO: 3903]

HEK3++1 10nt insertion, prolyl-tRNA{UGG} P1 ext 5nt, linker 3nt

GGCCCAGACUGAGCACGUGAGUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGGACCGAGUCGGUCCUCUGCCAUCAAAGCUUCGACCGUGCUCAGUCUUCUGCUCGAGGCGGGGCUCGUUGGUCUAGGGGUAUGAUUCUCGCUUCGGGUGCGAGAGGUCCCGGGUUCAAAUCCCGGACGAGCCCCGCCUCGAGCUUUU [SEQ ID NO: 390 4]

The sequences reported for the data in Figures 85 and 86 are as follows:

Example 23. Using prime editing to correct CDKL5 and sickle cell disease
Use of PE to design a mouse model of CDKL5 with 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 all bases. By focusing on the 1412delA mutation, this example designed and tested a pegRNA capable of inserting the mutation, with the hope of establishing a mouse neuronal cell line (N2A) harboring the mutation. This would allow large-scale screening of therapeutic pegRNAs as potential mutation correctors. The ultimate goal is the ability to develop mouse models of CDD carrying the 1412delA mutation to assess therapeutic efficacy. Current mouse models of CDD do not have humanized alleles. 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 (with and without indel). It is shown).

Use of PE to Treat Sickle Cell Anemia (SCA) Sickle cell anemia (SCA) is a recessive blood disorder caused by a glutamate to valine mutation in position 6 of the β-globin gene. The result is sickle cells, which are poor oxygen transporters and prone to clumping. Symptoms of aggregation can be life-threatening. Previously, the D. Liu lab had the ability to demonstrate both the 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 is the result of editing at the proxy locus of the β-globin gene and HEK3 in healthy HSCs, varying the concentration of editing factors versus pegRNA and nicking gRNA.

mRNA nucleofection protocol:
The protocol was improved by adjusting the edit factor to guide ratio ([edit factor] to [guide] ratio or edit factor: guide ratio).

The nicking guide protospacer sequence was: CCTTGATACCAACCTGCCCA (SEQ ID NO: 3991) .

The pegRNA sequence is:
CATGGTGCACCTGACTCCTGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCAGACTTCTCTTCAGGAGTCAGGTGCACTTT (SEQ ID NO: 3992) .

1. CD34+ cells are thawed and primed with cytokines (SCF, Flt3, and TPO) in X-Vivo15 medium for 24 hr. Seeding density 1 million cells/mL.

2. The next day (after 24 hr), CD34+ cells (1 x 105) were aspirated, washed once with PBS (centrifuged at 300 g for 10 min at RT) and resuspended in P3 solution (Lonza). (20ul 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).

Four. Pipette the cell suspension into 20 uL Lonza4D 16-well strips and electroporate with program DS130.

Five. Wait 10-15 min after electroporation, add 80 uL of X-Vivo10+ cytokine medium to the cell suspension and transfer into pre-warmed X-Vivo10 medium with cytokines.

6. Cells are harvested 72 hours after electroporation to check cell recovery and also genotype for 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 SVDMWSVGC I LGELSDGQPL FPGESEIDQL FTIQKVLGPL PSEQMKLFYS NPRFHGLRFP AVNHPQSLER RYLGILNSVL LDLMKNLLKL DPADRYLTEQ CLNHPTFQTQ RLLDRSPSRS AKRKPYHVES STLSNRNQAG KSTALQSHHR SNSKDIQNLS VGLPRADEGL PANESFLNGN LAGASLSPLH TKTYQASSQP GST SKDLTNN 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 RHLY NDPVPR 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 SVDMWSVGC I 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 LS EARAQIAE PSTSRYFPSS CLDLNSPTSP TPTRHSDTRT LLSPSGRNNR NEGTLDSRRT TTRHSKTMEE LKLPEHMDSS HSHSLSAPHE SFSYGLGYTS PFSSQQRPHR HSMYVTRDKV RAKGLDGSLS IGQGMAARAN SLQLLSPQPG EQLPPEMTVARSSVKETSRE GTSSFHTRQK SEGGVYHDPH SDDGTAPKEN RHLYNDPVPR RV GSFYRVPS 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 Treatment of Non-Diabetic Chronic Kidney Disease This example uses prime editing to treat kidney disease or reduce the likelihood of developing it. designed a PEgRNA capable of targeting the pathogenic APOL1 allele for use in.

End-stage renal disease (ESKD) is now a growing problem affecting more than 500,000 individuals in the United States. The cost of caring for patients with ESKD currently exceeds $40 billion per year. In the United States, subjects of African descent are four to five times more likely to develop ESKD than Americans without African descent. 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.

There is no specific treatment for the majority of progressive kidney diseases. Although the progression of some types of chronic kidney disease can be slowed by blood pressure control with certain drugs, nephrologists cannot accurately predict which patients will respond. Moreover, although successful treatment typically slows progression, it neither prevents the disease nor halts disease progression.

Recently, it was determined that certain genetic variants that alter the protein sequence of apolipoprotein L1 (APOL1) are associated with progressive kidney disease. Remarkably, APOL1 kidney disease variants are associated with multiple kidney diseases including hypertension-associated end-stage renal disease (H-ESRD), focal segmental glomerulosclerosis (FSGS), and HIV-associated nephropathy (HIVAN). It has major impact on different types. Individuals with these variant APOL1 alleles have a 7- to 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 high-risk APOL1 genotypes. African Americans without high-risk genotypes have only a small extra risk compared to Americans of European ancestry.

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. This is because they confer protection against the form of trypanosomes that causes African sleeping sickness.

There remains a need for treatments for kidney disease in patients with one or more APOL1 risk alleles. They cause significant morbidity and mortality in this and other target populations and have 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 APOL1 defective alleles.

PEgRNA 1

Design PEgRNA for APOL1 allele rs73885319 (p.S342G). This represents a G→A correction in diseased individuals. The target sequence is 5-GGAGTCAAGCTCACGGATGTGGCCCCTGTA (G to A)GCTTCTTTCTTGTGCTGGATGTAGTCTACCT-3 (SEQ ID NO: 3995) .

The protospacer (in bold above) is AAGCTCACGGATGTGGCCCC (SEQ ID NO: 3996) . Selected PEs include SaCas9(D10A).

Primer binding sites 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) .

RT templates 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 (SEQ ID NO: 4020) .

The nicking template may be GCTTTGATTCGTACACGAGG (SEQ ID NO: 4021) .

PEgRNA 2

Design PEgRNA for APOL1 allele rs60910145. This represents a G→T correction in diseased 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 (SEQ ID NO: 4032) .

The RT template may be (does not 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 (SEQ ID NO: 4046) .

The nicking mold may be:

CCTGTGGTCACAGTTCTTGG (SEQ ID NO: 4047)

CCACAGGGCAGGGGCAGCCAC (SEQ ID NO: 4048) .

PEgRNA 3

Design PEgRNA for APOL1 allele rs71785313. This represents the insertion as follows: ATTCTCAACAA[insert:TAATTA]TAAGATTC (SEQ ID NO: 4049) .

The protospacer can be: TCTCAACAATAAGATTCTGC (SEQ ID NO: 4050) .

PE includes SaKKH-PE2.

Primer binding sites 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) .

RT templates 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)

CCTGGAGAATCTTTATAATTATT (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 (SEQ ID NO: 4074) .

The nicking mold may be:

CCTGTGGTCACAGTTCTTGG (SEQ ID NO: 4047)

CCACAGGGCAGGGGCAGCCAC (SEQ ID NO: 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18のアミノ酸配列、または配列番号18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。 aspect
The following aspects are within the scope of this disclosure. Moreover, this disclosure discloses that all limitations, elements, clauses, and descriptive terms from one or more of the enumerated embodiments are introduced into another embodiment enumerated in this section. encompassing variations, combinations, and permutations of. For example, any enumerated embodiment that is subordinate to another enumerated embodiment includes one or more limitations found in any other enumerated embodiment in this section that is subordinate to the same base embodiment. It can be modified as follows. When elements are presented as enumerated in Markush group format, as an example, each subgroup of the elements is also disclosed, and any element(s) may be removed from the group. In general, when the disclosure, or an aspect of the disclosure, is viewed as including specific elements and/or features, an embodiment of the invention or an aspect of the invention consists of such elements and/or features; or should be understood to consist essentially of them. It is also noted that the terms "comprising" and "containing" are intended to be open, allowing the inclusion of additional elements or steps. When a range is given, endpoints are also included. Moreover, unless otherwise indicated or otherwise clear from the context and understanding of those skilled in the art, a value expressed as a range may refer to any specific value or value stated in different aspects of the invention. Subranges within the specified ranges may be envisioned up to 10 times the units of the lower limit of the range unless the context clearly dictates otherwise.
Group 1. Aspects 1 to 212
1. A fusion protein that includes a nucleic acid programmed DNA binding protein (napDNAbp) and a reverse transcriptase.
2. The fusion protein of embodiment 1, wherein the fusion protein is capable of performing genome editing by primed target-primed reverse transcription in the presence of an elongated guide RNA.
3． The fusion protein of embodiment 1, wherein napDNAbp has nickase activity.
Four. The fusion protein of embodiment 1, wherein napDNAbp is a Cas9 protein or a variant thereof.
Five. The fusion protein of embodiment 1, wherein napDNAbp is nuclease-active Cas9, nuclease-inactive Cas9 (dCas9), or 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. The fusion protein of embodiment 1 is capable of binding to a target DNA sequence when the fusion protein is complexed with an elongated 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 with a free 3' end.
13. The fusion protein of embodiment 2, wherein the extended guide RNA comprises (a) a guide RNA and (b) an RNA extension at the 5' or 3' end of the guide RNA or at an intramolecular location of the guide RNA.
14. The fusion protein of embodiment 13, wherein the RNA extension comprises (i) a reverse transcription template sequence containing the desired nucleotide changes, (ii) a reverse transcription primer binding site, and (iii) an optional linker sequence.
15. The fusion protein of embodiment 14, wherein the reverse transcription template sequence encodes a single-stranded DNA flap that is complementary to an endogenous DNA sequence adjacent to the nick site, and wherein the single-stranded DNA flap carries a desired nucleotide change. including.
16. The fusion protein of embodiment 13, wherein the RNA elongation 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 in length. 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 It is.
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.
18. The fusion protein of embodiment 15, wherein the single-stranded DNA flap replaces the endogenous DNA sequence with a free 5' end and 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.
twenty one. The fusion protein of embodiment 14, wherein the desired nucleotide change 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 The desired nucleotide change is incorporated on an editing window that is between approximately -30 and +100 of the PAM sequence, or 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, Incorporates 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides downstream.
twenty two. The fusion protein of embodiment 1, wherein napDNAbp is SEQ ID NO.18Amino acid sequence of or SEQ ID NO.18an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of
twenty three. The fusion protein of embodiment 1, wherein napDNAbp is SEQ ID NO.18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to any one amino acid sequence of
twenty four. The fusion protein of embodiment 1, wherein the reverse transcriptase is 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:
twenty five. The fusion protein of embodiment 1, wherein the reverse transcriptase is 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 766an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to any one amino acid sequence of
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 embodiments, 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 767-769Contains the 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. The elongated guide RNA of embodiment 1, wherein the RNA extension is at the 3' or 5' end of the guide RNA or at a position within the molecule of the guide RNA.
33. The elongated guide RNA of embodiment 31, wherein the elongated guide RNA is capable of binding napDNAbp and directing napDNAbp to a target DNA sequence.
34. The elongated guide RNA of embodiment 33, wherein the target DNA sequence includes 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 the at least one RNA extension comprises (i) a reverse transcription template sequence, (ii) a reverse transcription primer binding site, and (iii) an optional linker sequence.
36. The elongated guide RNA of embodiment 35, wherein the RNA elongation 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 in length. , 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.
37. The elongated 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.
38. The elongated 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 in length. , 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.
39. The elongated 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.
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 elongated guide RNA of embodiment 40, 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 nicked. Immediately adjacent downstream of the site.
42. The elongated guide RNA of embodiment 41, wherein the endogenous single-stranded DNA with a free 5' end is excised by the cell.
43. The elongated guide RNA of embodiment 41, wherein cellular repair of the single-stranded DNA flap results in the incorporation of the desired nucleotide change, thereby forming the desired product.
44. The elongated guide RNA of embodiment 31, comprising SEQ ID NO:394, 429-442, 641-649, 678-692, 2997-3103, 3113-3121, 3305-3455, 3479-3493, 3522-3556, 3628-3698, and 3755-3810nucleotide sequence, or SEQ ID NO.394, 429-442, 641-649, 678-692, 2997-3103, 3113-3121, 3305-3455, 3479-3493, 3522-3556, 3628-3698, and 3755-3810nucleotide sequences 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.
45. The elongated 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 elongated guide RNA of embodiment 35, wherein the reverse transcription primer binding site hybridizes to the free 3' end of the cut DNA.
47． The elongated guide RNA of embodiment 35, wherein any linker sequence is at least 1 nucleotide in length, 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.
48. A complex comprising the fusion protein of any one of embodiments 1 to 30 and an elongated guide RNA.
49. Embodiment 48, wherein the elongated guide RNA comprises a guide RNA and an RNA extension at the 3' or 5' end of the guide RNA or at a position within the molecule of the guide RNA.
50. The complex of embodiment 48, wherein the elongated guide RNA is capable of binding napDNAbp and directing napDNAbp to a target DNA sequence.
51. The complex of embodiment 50, wherein the target DNA sequence comprises a target strand and a complementary non-target strand, and the guide RNA hybridizes to the target strand to form an RNA-DNA hybrid and an R-loop.
52. Embodiment 49, wherein the at least one RNA extension comprises (i) a reverse transcription template sequence, (ii) a reverse transcription primer binding site, and (iii) an optional linker sequence.
53. Embodiment 48, wherein the elongated guide RNA is at least 85%, or at least 90%, or at least 95% with the nucleotide sequence of SEQ ID NO: 18-36, or with any one of SEQ ID NO: 18-36. , or nucleotide sequences having at least 98%, or at least 99% sequence identity.
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. The complex of embodiment 52, wherein the reverse transcription primer binding site hybridizes to the free 3' end of the cut DNA.
56. complex containing napDNAbp and elongated guide RNA.
57. The conjugate of embodiment 56, wherein napDNAbp is Cas9 nickase.
58. The complex of embodiment 56, wherein napDNAbp is SEQ ID NO.18Amino acid sequence of or SEQ ID NO.18an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with.
59. Embodiment 57, wherein napDNAbp is SEQ ID NO:18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with any one amino acid sequence of
60. Embodiment 57, wherein the elongated guide RNA comprises a guide RNA and an RNA extension at the 3' or 5' end of the guide RNA or at a position within the molecule of the guide RNA.
61. Embodiment 57. The complex of embodiment 57, wherein the elongated guide RNA is capable of directing napDNAbp to a target DNA sequence.
62. The complex of embodiment 61, wherein the target DNA sequence comprises a target strand and a complementary non-target strand, 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 elongated guide RNA has SEQ ID NO.394, 429-442, 641-649, 678-692, 2997-3103, 3113-3121, 3305-3455, 3479-3493, 3522-3556, 3628-3698, and 3755-3810nucleotide sequence, or SEQ ID NO.394, 429-442, 641-649, 678-692, 2997-3103, 3113-3121, 3305-3455, 3479-3493, 3522-3556, 3628-3698, and 3755-3810nucleotide sequences 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. The conjugate of embodiment 63, wherein the reverse transcription template sequence comprises a nucleotide sequence that is at least 80% or 85% or 90% or 95% or 99% identical to the endogenous DNA target.
66. The complex of embodiment 63, wherein the reverse transcription primer binding site hybridizes to the free 3' end of the cut DNA.
67. The conjugate of embodiment 63, wherein any linker sequence is at least 1 nucleotide in length, 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.
68. A polynucleotide encoding the fusion protein of any of aspects 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 the complex of any one of embodiments 48-67.
72. A medicament comprising: (i) the fusion protein of any of embodiments 1 to 30, the conjugate of embodiments 48 to 67, the polynucleotide of embodiment 68, or the vector of embodiment 69; and (ii) a pharmaceutically acceptable excipient. Composition.
73. A pharmaceutical composition comprising (i) a conjugate of embodiments 48-67, (ii) a reverse transcriptase provided in trans; and (iii) a pharmaceutically acceptable excipient.
74. A kit comprising: (i) a nucleic acid sequence encoding the 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 desired nucleotide changes into a double-stranded DNA sequence, the method comprising:
(i) Reverse transcription by contacting a double-stranded DNA sequence with a complex containing a fusion protein and an elongated guide RNA, where the fusion protein contains napDNAbp and reverse transcriptase, and the elongated guide RNA contains the desired nucleotide change. Contains a template sequence;
(ii) nicking a double-stranded DNA sequence on the non-target strand, thereby generating free single-stranded DNA with a 3' end;
(iii) hybridizing the 3′ end of the free single-stranded DNA to a reverse transcription template sequence, thereby priming the reverse transcriptase domain;
(iv) polymerizing the strands of DNA from the 3' end, thereby producing a single-stranded DNA flap containing the desired nucleotide changes;
(v) replacing the endogenous DNA strand adjacent to the cut site by a single-stranded DNA flap, thereby incorporating the desired nucleotide change into the double-stranded DNA sequence;
including.
76. 75. The method of embodiment 75, wherein (v) the step of replacing: (i) hybridizing the single-stranded DNA flap to an 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 the desired product containing the desired nucleotide changes in both strands of the DNA.
77. 77. The method of embodiment 76, wherein the desired nucleotide change is a single nucleotide substitution, deletion, or insertion.
78. 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. 76. 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) G:C base pair to C:G base pair, (4) T:A base pair to G:C base pair, (5) T:A base pair to A:T base pair, (6) T:A base pair becomes C:G base pair, (7) C:G base pair becomes G:C base pair, (8) C:G base pair becomes T:A base pair, (9) C: G base pair to A:T base pair, (10) A:T base pair to T:A base pair, (11) A:T base pair to G:C base pair, or (12) A:T. Converts a 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. 77. The method of embodiment 76, wherein the desired nucleotide change modifies a disease-associated gene.
83. The method of embodiment 82, wherein the disease-associated gene is: adenosine deaminase (ADA) deficiency; alpha-1 antitrypsin deficiency; cystic fibrosis; Duchenne muscular dystrophy; galactosemia; hemochromatosis; Huntington's disease; maple Consists of syrup urine disease; Marfan syndrome; type 1 neurofibromatosis; congenital nail hyperplasia; phenylketonuria; severe combined immunodeficiency; sickle cell disease; Smith-Lemli-Opitz syndrome; and Tay-Sachs disease. associated with a single gene disorder selected from the group.
84. 83. The method of embodiment 82, 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.
85. 77. The method of embodiment 76, wherein the napDNAbp is nuclease-inactive Cas9 (dCas9), Cas9 nickase (nCas9), or nuclease-active Cas9.
86． The method of embodiment 76, wherein napDNAbp is SEQ ID NO.18Contains the amino acid sequence of
87. 76. The method of embodiment 76, wherein napDNAbp is SEQ ID NO.18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with any one amino acid sequence of
88. 76. The method of embodiment 76, wherein the reverse transcriptase is 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 766Contains any one of the following amino acid sequences.
89. 76. The method of embodiment 76, wherein the reverse transcriptase domain is 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 766an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with any one amino acid sequence of
90. 77. The method of embodiment 76, wherein the elongated guide RNA comprises 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. 90. The method of embodiment 90, wherein the RNA elongation 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 in length. , 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 be.
92. 76. The method of embodiment 76, wherein the elongated guide RNA has SEQ ID NO.394, 429-442, 641-649, 678-692, 2997-3103, 3113-3121, 3305-3455, 3479-3493, 3522-3556, 3628-3698, and 3755-3810having 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, the method comprising:
(i) a reverse transcriptase (RT) template in which the DNA molecule is contacted with a nucleic acid programmed DNA binding protein (napDNAbp) and a guide RNA that targets napDNAbp to the target locus, the guide RNA containing at least one desired nucleotide change; Contains an array;
(ii) creating an exposed 3' end of the DNA strand at the target locus;
(iii) hybridizing 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 onto the corresponding endogenous DNA, thereby introducing one or more changes in the nucleotide sequence of the DNA molecule at the target locus;
including.
94． 94. The method of embodiment 93, wherein the one or more changes in the nucleotide sequence comprises a transition.
95. 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． 94. The method of embodiment 93, wherein the one or more changes in the nucleotide sequence comprises a transversion.
97. 96. The method of aspect 96, wherein the transversion is: (a) T to A; (b) T to G; (c) C to G; (d) C to A; (e) A to T; (f) (g) G to C; and (h) G to T.
98. 94. The method of embodiment 93, wherein the one or more changes in the nucleotide sequence include: (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) G:C base pair to C:G base pair, (4) T:A base pair to G:C base pair, (5) T:A base pair to A:T base pair, (6 )T:A base pair to C:G base pair, (7)C:G base pair to G:C base pair, (8)C:G base pair to T:A base pair, (9)C :G base pair to A:T base pair, (10) A:T base pair to T:A base pair, (11) A:T base pair to G:C base pair, or (12) A: It involves changing a T base pair to a C:G base pair.
99． 94. The method of embodiment 93, wherein the one or more changes in the nucleotide sequence are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, Contains insertions or deletions of 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides.
100. 94. The method of embodiment 93, wherein the one or more changes in the nucleotide sequence comprises 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. 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.
103. 94. The method of embodiment 93, wherein napDNAbp is nuclease active Cas9 or a variant thereof.
104. 94. The method of embodiment 93, wherein napDNAbp is nuclease-inactive Cas9 (dCas9) or Cas9 nickase (nCas9) or a variant thereof.
105. The method of embodiment 93, wherein napDNAbp is SEQ ID NO.18Contains the 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 482-487an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with any one amino acid sequence of
107. 94. The method of embodiment 93, wherein the reverse transcriptase is introduced in trans.
108. 94. The method of embodiment 93, wherein napDNAbp comprises a fusion to reverse transcriptase.
109. The method of embodiment 93, wherein the reverse transcriptase is 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 766Contains any one of the following amino acid sequences.
110. 94. The method of embodiment 93, wherein the reverse transcriptase is 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 766an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with any one amino acid sequence of
111. 94. 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.
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. 94. The method of embodiment 93, wherein forming an exposed 3' end of the DNA strand at the target locus comprises introducing a replication error.
115. 94. The method of embodiment 93, wherein the step of contacting the DNA molecule with napDNAbp and guide RNA forms an R-loop.
116. Embodiment 115. The method of embodiment 115, wherein the DNA strand from which the exposed 3' end is formed is on the R-loop.
117. 94. The method of embodiment 93, wherein the guide RNA comprises an extended portion that includes a reverse transcriptase (RT) template sequence.
118. The method of embodiment 117, wherein the elongated portion is at the 3' end of the guide RNA, at the 5' end of the guide RNA, or at a position within the molecule of the guide RNA.
119. 94. 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. 94. The method of embodiment 93, wherein the RT template sequence is homologous to the corresponding endogenous DNA.
122. A method for introducing one or more changes in the nucleotide sequence of a DNA molecule at a target locus by primed reverse transcription to a primed target, the method comprising: (a) converting the DNA molecule at the target locus to (i) a nucleic acid program; (b) reverse transcription primed to the primed target of the RT template. (c) incorporating the desired nucleotide changes onto the DNA molecule at the target locus by DNA repair and/or replication processes.
one two three. The method of embodiment 122, wherein the RT template is located at the 3' end of the guide RNA, at the 5' end of the guide RNA, or at an intramolecular location of the guide RNA.
124. Embodiment 122 The method of embodiment 122, wherein the desired nucleotide change comprises a transition, transversion, insertion, or deletion, or any combination thereof.
125.aspect122, wherein the desired nucleotide change is 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. include.
126．aspect122, 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. Embodiment 122, 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 T:A base pair; :C base pair becomes C:G base pair, (4) T:A base pair becomes G:C base pair, (5) T:A base pair becomes A:T base pair, (6) T:A base pair to C:G base pair, (7) C:G base pair to G:C base pair, (8) C:G base pair to T:A base pair, (9) C:G base pair. into an A:T base pair, (10) an A:T base pair into a T:A base pair, (11) an A:T base pair into a G:C base pair, or (12) an A:T base pair. This includes changing to a C:G base pair.
128． A polynucleotide encoding the elongated guide RNA of any one of embodiments 31-47.
129. A vector comprising the polynucleotide of embodiment 128.
130. A cell comprising the vector of embodiment 129.
131. The fusion protein of any of embodiments 1 to 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. 133. The method of embodiment 132, wherein the fusion protein comprises the amino acid sequence of PE1, PE2, or PE3.
134. 133. The method of embodiment 132, wherein napDNAbp is Cas9 nickase (nCas9).
135. The method of embodiment 132, wherein napDNAbp is SEQ ID NO: 18 to88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487The amino acid sequence of
136. 133. The method of embodiment 132, wherein the guide RNA comprises SEQ ID NO: 222.
137. Embodiment 132, wherein the step of (b) performing primed reverse transcription on a primed target comprises a 3' end primer at the target locus capable of priming reverse transcription by annealing to a primer binding site on a guide RNA; Including generating associative arrays.
138. A method for replacing a trinucleotide repeat expansion mutation on a target DNA molecule by a healthy sequence containing a healthy number of repeat trinucleotides, the method comprising: (a) converting the DNA molecule at the target locus to (i) a nucleic acid programmed contacting a fusion protein comprising a DNA binding protein (napDNAbp) and a reverse transcriptase and (ii) a guide RNA comprising an RT template comprising a replacement sequence, which said fusion protein introduces; (b) priming the RT template to the primed target; (c) incorporating the single-stranded DNA onto the DNA molecule at the target locus by a DNA repair and/or replication process; .
139. 139. The method of embodiment 138, wherein the fusion protein comprises the amino acid sequence of PE1, PE2, or PE3.
140. Embodiment 138, wherein the napDNAbp is Cas9 nickase (nCas9).
141. Embodiment 138. The method of embodiment 138, wherein napDNAbp is SEQ ID NO: 18 to88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487Contains the amino acid sequence of
142. 139. The method of embodiment 138, wherein the guide RNA comprises SEQ ID NO: 222.
143. Embodiment 138, wherein (b) performing primed reverse transcription on a primed target comprises a 3' end primer at the target locus capable of priming reverse transcription by annealing to a primer binding site on a guide RNA; Including generating associative arrays.
144. 139. 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. Embodiment 138, wherein the trinucleotide repeat expansion comprises a CAG triplet repeat unit.
146. 139. The method of embodiment 138, wherein the trinucleotide repeat expansion comprises a GAA triplet repeat unit.
147. A method of incorporating a functional moiety onto a protein of interest encoded by a target nucleotide sequence by prime editing, the method comprising: (a) converting the target nucleotide sequence to (i) nucleic acid programmed DNA binding protein (napDNAbp) and reverse transcriptase; (b) polymerizing the single-stranded DNA sequence encoding the functional moiety; and (c) DNA repair and/or or incorporating, by a replication process, a single-stranded DNA sequence in place of the corresponding endogenous strand in the target nucleotide sequence, wherein the method generates a recombinant target nucleotide sequence encoding a fusion protein comprising the protein of interest and the functional moiety. arise.
148. Embodiment 147. The method of embodiment 147, wherein the functional moiety is a peptide tag.
149. Embodiment 148, wherein the peptide tag is an affinity tag, solubilization tag, chromatography tag, epitope tag, or fluorescent tag.
150． 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(Sequence number2);HA tag(sequence numberFive);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. Embodiment 148, 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 VSVtag(Sequence number 275) is selected from the group consisting of
152. Embodiment 147. The method of embodiment 147, wherein the functional moiety is an immune epitope.
153. Embodiment 152, wherein the immune epitope is: 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); Class I HLA 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. Embodiment 147. The method of embodiment 147, wherein the functional moiety is a degradation tag, so that the degradation rate of the protein of interest is altered.
156. 155. The method of aspect 155, wherein the decomposition tagcomprises an amino acid sequence encoding a degradation tag as disclosed herein..
157. Embodiment 147. The method of embodiment 147, wherein the functional moiety is a small molecule binding domain.
158. The method of embodiment 157, wherein the small molecule binding domain is FKBP12 of SEQ ID NO: 488.
159. The method of embodiment 157, wherein the small molecule binding domain is FKBP12-F36V of SEQ ID NO: 489.
160． Embodiment 157, wherein the small molecule binding domain is SEQ ID NO: 49.0 and 493~494 cyclophilin.
161. Embodiment 157. The method of embodiment 157, wherein the small molecule binding domain is incorporated onto two or more proteins of interest.
162. 162. The method of embodiment 161, wherein the two or more proteins of interest can be dimerized by contacting the small molecule.
163. The method of embodiment 157, wherein the small molecule is:

It is a low molecular weight dimer 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. 164. The method of embodiment 164, wherein the immune epitope is: 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); Class I HLA hemagglutinin epitope for class I HLA (SEQ ID NO: 416); neuraminidase epitope for class I 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).
166. Embodiment 164. The method of embodiment 164, wherein the fusion protein comprises the amino acid sequence of PE1, PE2, or PE3.
167. Embodiment 164. The method of embodiment 164, wherein napDNAbp is Cas9 nickase (nCas9).
168. The method of embodiment 164, wherein napDNAbp is SEQ ID NO: 18 to88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487Contains the amino acid sequence of
169. 165. The method of embodiment 164, wherein the guide RNA comprises SEQ ID NO: 222.
170. A method of incorporating small molecule dimerization domains onto a protein of interest encoded by a target nucleotide sequence by prime editing, the method comprising: (a) converting the target nucleotide sequence into (i) a nucleic acid programmed DNA binding protein ( (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 a DNA repair and/or replication process, the method comprising: A recombinant target nucleotide sequence is generated that encodes a fusion protein comprising an integration domain.
171. Embodiment 170. The method of embodiment 170, further comprising performing the method on a second protein of interest.
172. 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 a respective dimerization domain of the proteins.
173. 170. The method of embodiment 170, wherein the small molecule binding domain is FKBP12 of SEQ ID NO: 488.
174. The method of embodiment 170, wherein the small molecule binding domain is FKBP12-F36V of sequence number 489.
175. Embodiment 170, wherein the small molecule binding domain is SEQ ID NO: 49.0 and 493~494 cyclophilins.
176. Embodiment 170, wherein the small molecule is:

It is a low molecular weight dimer selected from the group consisting of:
177. Embodiment 170, wherein the fusion protein comprises the amino acid sequence of PE1, PE2, or PE3.
178. Embodiment 170, wherein the napDNAbp is Cas9 nickase (nCas9).
179. The method of embodiment 170, wherein napDNAbp is SEQ ID NO: 18 to88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487Contains the amino acid sequence of
180. Embodiment 170, wherein the guide RNA comprises SEQ ID NO: 222.
181. A method of incorporating a peptide tag or epitope onto a protein using prime editing, comprising: a prime editing agent configured to insert into a target nucleotide sequence encoding a protein a second nucleotide sequence encoding a peptide tag; contacting the recombinant nucleotide sequence with a construct so that the peptide tag and protein are expressed from the recombinant nucleotide sequence as a fusion protein.
182. 182. The method of embodiment 181, wherein the peptide tag is used for protein purification and/or detection.
183. 182. The method of embodiment 181, wherein the peptide tag is polyhistidine (e.g., HHHHHH(Sequence number 257)), FLAG (for example, DYKDDDDK(Sequence number 2)), V5 (for example, GKPIPNPLLGLLDST(Sequence number 3)), GCN4, HA (e.g., YPYDVPDYA(Sequence number 5)), Myc (e.g. EQKLISEED(Sequence number 6)), GST, etc.
184. 182. 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. 182. 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 NOs: 127, 165-176, 446, 453, and 767-769.
188. 182. The method of embodiment 181, wherein the prime editing factor construct has 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, 776-777PEgRNA containing the nucleotide sequence of
189. 182. The method of embodiment 181, wherein the PEgRNA comprises a spacer, a gRNA core, and an extended arm, the spacer being complementary to a target nucleotide sequence, and the extended arm comprising a reverse transcriptase template encoding a peptide tag.
190. 182. The method of embodiment 181, wherein the PEgRNA comprises a spacer, a gRNA core, and an extended arm, the spacer being complementary to a target nucleotide sequence, and the extended arm comprising a reverse transcriptase template encoding a peptide tag.
191. A method of preventing or halting the progression of a prion disease by incorporating one or more protective mutations on a PRNP encoded by a target nucleotide sequence by prime editing, the method comprising: (a) (i) a prime editing factor comprising a nucleic acid programmed DNA binding protein (napDNAbp) and a reverse transcriptase; and (ii) a PEgRNA comprising an editing template encoding a functional moiety; (b) a prime editing factor comprising a nucleic acid programmed DNA binding protein (napDNAbp) and a reverse transcriptase; polymerizing the single-stranded DNA sequence; and (c) incorporating the single-stranded DNA sequence in place of the corresponding endogenous strand in the target nucleotide sequence by a DNA repair and/or replication process, the method comprising: A recombinant target nucleotide sequence is generated that encodes a PRNP containing a protective mutation that is resistant to.
192. 192. The method of embodiment 191, wherein the prion disease is a human prion disease.
193. 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. 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. It is encephalopathy.
196． The method of embodiment 191, wherein the wild type PRNP amino acid sequence is SEQ ID NO: 291-292.
197. Embodiment 191, wherein the method results in a modified PRNP amino acid sequence selected from the group consisting of SEQ ID NOs: 293-323, wherein the modified PRNP protein is resistant to misfolding.
198． 192. The method of embodiment 191, wherein the fusion protein comprises the amino acid sequence of PE1, PE2, or PE3.
199. 192. The method of embodiment 191, wherein napDNAbp is Cas9 nickase (nCas9).
200. The method of embodiment 191, wherein napDNAbp is SEQ ID NO: 18 to88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487Contains the amino acid sequence of
201. 192. The method of embodiment 191, wherein the guide RNA comprises SEQ ID NO: 222.
202. A method of incorporating a ribonucleotide motif or tag onto an RNA of interest encoded by a target nucleotide sequence by prime editing, the method comprising: (a) converting the target nucleotide sequence into (i) a nucleic acid programmed DNA binding protein (napDNAbp); and a prime editing agent comprising a reverse transcriptase and (ii) an editing template encoding a ribonucleotide motif or tag; (b) polymerizing a single-stranded DNA sequence encoding a ribonucleotide motif or tag. and (c) incorporating a single-stranded DNA sequence in place of the corresponding endogenous strand in the target nucleotide sequence by a DNA repair and/or replication process, the method comprising: A recombinant target nucleotide sequence is generated that encodes a modified RNA.
203． Embodiment 202. The method of embodiment 202, wherein the ribonucleotide motif or tag is the detection moiety.
204. The method of embodiment 202, wherein the ribonucleotide motif or tag affects the expression level of the RNA of interest.
205. Embodiment 202 The method of embodiment 202, wherein the ribonucleotide motif or tag affects the trafficking or subcellular localization of the RNA of interest.
206. Embodiment 202, wherein the ribonucleotide motif or tag is SV40 type 1, SV40 type 2, SV40 type 3, hGH, BGH, rbGlob, TK, MALAT1 ENE-mascRNA, KSHV PAN ENE, Smbox/U1 snRNA box, selected from the group consisting of U1 snRNA 3' box, tRNA-lysine, broccoli aptamer, spinach aptamer, mango aptamer, HDV ribozyme, and m6A.
207. The method of embodiment 202, wherein the PEgRNA is 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. Embodiment 202, wherein the napDNAbp is Cas9 nickase (nCas9).
210. The method of embodiment 202, wherein napDNAbp is SEQ ID NO: 18 to88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487Contains the amino acid sequence of
211． A method of incorporating or deleting a functional moiety on a protein of interest encoded by a target nucleotide sequence by prime editing, the method comprising: (a) converting the target nucleotide sequence into (i) a nucleic acid programmed DNA binding protein (napDNAbp); ) and a prime editing factor comprising reverse transcriptase and (ii) a PEgRNA comprising an editing template encoding the functional moiety or its deletion; (b) a single-stranded DNA sequence encoding the functional moiety or its deletion; and (c) incorporating a single-stranded DNA sequence in place of the corresponding endogenous strand in the target nucleotide sequence by a DNA repair and/or replication process, the method comprising or a deletion thereof, resulting in a recombinant target nucleotide sequence encoding a modified protein, wherein the functional moiety alters the modification state or localization state of the protein.
212． Embodiment 211. 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.
Group 2. Aspects 213-424
213． A fusion protein that includes a nucleic acid programmed DNA binding protein (napDNAbp) and a polymerase.
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).
215. The 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 nuclease-active Cas9, nuclease-inactive Cas9 (dCas9), or 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. The 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. The fusion protein of embodiment 220, wherein the binding of the fusion protein complexed to PEgRNA forms an R-loop.
223. The fusion protein of embodiment 222, wherein the R-loop comprises (i) an RNA-DNA hybrid comprising PEgRNA and a target strand and (ii) a complementary non-target strand.
224． The fusion protein of embodiment 223, wherein the complementary non-target strand is nicked to form a prime sequence with 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. The fusion protein of embodiment 225, wherein the extended arm comprises (i) a DNA synthesis template sequence containing a desired nucleotide change and (ii) a primer binding site.
227． Embodiment 226. The fusion protein of embodiment 226, wherein the DNA synthesis template sequence encodes a single-stranded DNA flap that is complementary to an endogenous DNA sequence adjacent to the nick site, the single-stranded DNA flap containing a desired nucleotide change. including.
228. Embodiment 225, wherein the elongated 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 in length. 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 It is.
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.
230. The fusion protein of embodiment 227, wherein the single-stranded DNA flap replaces the endogenous DNA sequence with a free 5' end and adjacent to the nick site.
231. The fusion protein of embodiment 230, wherein the 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 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 The desired nucleotide change is incorporated on an editing window that is between approximately -30 and +100 of the PAM sequence, or 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, Incorporates 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 SEQ ID NO:18Amino acid sequence of or SEQ ID NO.18an 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 SEQ ID NO:18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with any one amino acid sequence of
236. The fusion protein of embodiment 213, wherein the polymerase is reverse transcriptase, and wherein 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 766Contains any one of the following amino acid sequences.
237. The fusion protein of embodiment 213, wherein the polymerase is reverse transcriptase, and wherein 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 766an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with any one amino acid sequence of
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 embodiments, wherein the fusion protein has the structure NH₂-[napDNAbp]-[polymerase]-COOH; or NH₂-[polymerase]-[napDNAbp]-COOH, with each occurrence of "]-[" 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 767-769Contains the 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. PEgRNA comprising a guide RNA and at least one nucleic acid extension arm comprising a DNA synthesis template.
244． The PEgRNA of embodiment 241, wherein the nucleic acid extension arm is located at the 3' or 5' end of the guide RNA or a position within the molecule of the guide RNA, and the nucleic acid extension arm is DNA or RNA.
245. The PEgRNA of embodiment 242, wherein the PEgRNA is capable of binding napDNAbp and directing 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. 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.
248． The PEgRNA of embodiment 247, wherein the nucleic acid extended 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 in length. 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 It is.
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 in length. nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, or at least 15 nucleotides.
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 in length. nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, or at least 15 nucleotides.
251． The PEgRNA of embodiment 243 further comprises at least one additional structure selected from the group consisting of a linker, stem-loop, hairpin, toe-loop, aptamer, or 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 an endogenous DNA sequence adjacent to the nick site, and the single-stranded DNA flap includes 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． The PEgRNA of embodiment 253, wherein the endogenous single-stranded DNA with a free 5' end is excised by the cell.
255. Embodiment 253 PEgRNA, wherein cellular repair of the single-stranded DNA flap results in the incorporation of the desired nucleotide change, thereby forming the desired product.
256． PEgRNA of embodiment 243, comprising 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, 776-777nucleotide sequence, or 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, 776-777nucleotide sequences 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.
257. The PEgRNA of embodiment 247, wherein the DNA synthesis template comprises a nucleotide sequence that is at least 80% or 85% or 90% or 95% or 99% identical to the endogenous DNA target.
258. The PEgRNA of embodiment 247, wherein the primer binding site hybridizes to the free 3' end of the cut DNA.
259． The PEgRNA of embodiment 251, wherein the at least one additional structure is located at the 3' or 5' end of the PEgRNA.
260． A complex comprising the fusion protein of any one of embodiments 213-242 and PEgRNA.
261． 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 a position within the molecule of the guide RNA.
262. Embodiment 260, wherein the PEgRNA is capable of binding napDNAbp and directing napDNAbp to a target DNA sequence.
263． 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. 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.
265. Embodiment 260, wherein the PEgRNA is 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, 776-777nucleotide sequence, or 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:
266． The conjugate 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． Embodiment 264. The 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 SEQ ID NO.18Contains the amino acid sequence of
271． Embodiment 268, wherein napDNAbp is SEQ ID NO:18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with any one amino acid sequence of
272． 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 a position within the molecule of the guide RNA.
273． Embodiment 268. The complex of embodiment 268, wherein the PEgRNA is capable of directing napDNAbp to a target DNA sequence.
274. The complex of embodiment 272, wherein the target DNA sequence comprises a target strand and a complementary non-target strand, and the spacer sequence of PEgRNA hybridizes to the target strand to form an RNA-DNA hybrid and an R-loop.
275． The complex of embodiment 273, wherein the nucleic acid extension arm comprises (i) a DNA synthesis template and (ii) a primer binding site.
276． Embodiment 269, wherein the PEgRNA is 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, 3972-3989nucleotide sequence, or 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, 776-777nucleotide sequences 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 conjugate of embodiment 276, wherein the DNA synthesis template comprises a nucleotide sequence that is at least 80% or 85% or 90% or 95% or 99% identical to the endogenous DNA target.
278. The complex of embodiment 276, wherein the primer binding site hybridizes to the free 3' end of the cut DNA.
279. The conjugate 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 the fusion protein of any of embodiments 213-242.
281. A vector comprising the polynucleotide of embodiment 280.
282． A cell comprising the fusion protein of any of embodiments 213-242 and PEgRNA bound to napDNAbp of the fusion protein.
283． A cell comprising the complex of any one of embodiments 260-279.
284. A pharmaceutical composition comprising: (i) a fusion protein of any of embodiments 213-242, a conjugate of embodiments 260-279, a polynucleotide of embodiment 68, or a vector of embodiment 69; and (ii) a pharmaceutically acceptable Contains excipients.
285. A pharmaceutical composition comprising: (i) a conjugate of embodiments 260-279, (ii) a polymerase provided in trans; and (iii) a pharmaceutically 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 desired nucleotide changes into a double-stranded DNA sequence, the method comprising:
(i) contacting the double-stranded DNA sequence with a complex comprising a fusion protein and PEgRNA, the fusion protein comprising napDNAbp and a polymerase, and the PEgRNA comprising a DNA synthesis template containing the desired nucleotide changes and a primer binding site;
(ii) nicking the double-stranded DNA sequence, thereby generating free single-stranded DNA with 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 producing a single-stranded DNA flap that is complementary to the DNA synthesis template and contains the desired nucleotide changes; ;
(v) replacing the endogenous DNA strand adjacent to the cut site by a single-stranded DNA flap, thereby incorporating the desired nucleotide change into the double-stranded DNA sequence;
including.
288. 287. The method of embodiment 287, wherein (v) the replacing step comprises: (i) hybridizing the single-stranded DNA flap to an 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 the desired product containing the desired nucleotide changes in both strands of the DNA.
289. Embodiment 288, wherein the desired nucleotide change is a single nucleotide substitution, deletion, or insertion.
290. Embodiment 289. The method of embodiment 289, wherein the single nucleotide substitution is a transition or transversion.
291． 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) Substitution of A from T, (6) Substitution of C from T, (7) Substitution of G from C, (8) Substitution of T from C, (9) Substitution of A from C, (10) Substitution of A (11) A to G substitution, or (12) A to C substitution.
292. Embodiment 288, 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) G:C base pair to C:G base pair, (4) T:A base pair to G:C base pair, (5) T:A base pair to A:T base pair, (6) T:A base pair becomes C:G base pair, (7) C:G base pair becomes G:C base pair, (8) C:G base pair becomes T:A base pair, C:G base pair. into an A:T base pair, (10) an A:T base pair into a T:A base pair, (11) an A:T base pair into a G:C base pair, or (12) an A:T base pair. Convert to C:G base pair.
293. Embodiment 288, wherein the desired nucleotide change is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, An insertion or deletion of 19, 20, 21, 22, 23, 24, or 25 nucleotides.
294. Embodiment 288. The method of embodiment 288, wherein the desired nucleotide change modifies a disease-associated gene.
295. Embodiment 294, wherein the disease-associated gene is: adenosine deaminase (ADA) deficiency; alpha-1 antitrypsin deficiency; cystic fibrosis; Duchenne muscular dystrophy; galactosemia; hemochromatosis; Huntington's disease; maple Syrupuria; Marfan syndrome; type 1 neurofibromatosis; congenital nail hypertrophy; phenylketonuria; severe combined immunodeficiency; sickle cell disease; Smith-Lemli-Opitz syndrome; trinucleotide repeat disorder; prion disease associated with a single gene disorder selected from the group consisting of; and Tay-Sachs disease.
296. The method of embodiment 294, wherein the disease-associated gene is associated with a polygenic disorder selected from the group consisting of: heart disease; high blood pressure; Alzheimer's disease; arthritis; diabetes; cancer; and obesity.
297. The method of embodiment 287, wherein the napDNAbp is a nuclease-inactive Cas9 (dCas9), a Cas9 nickase (nCas9), or a nuclease-active Cas9.
298. 287. The method of embodiment 287, wherein napDNAbp is SEQ ID NO.18Amino acid sequence of or SEQ ID NO.18an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence of
299． 287. The method of embodiment 287, wherein napDNAbp is SEQ ID NO.18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487.an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with any one amino acid sequence of
300. 287. The method of embodiment 287, wherein the polymerase is reverse transcriptase, and wherein 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 766Contains any one of the following amino acid sequences.
301. 287. The method of embodiment 287, wherein the polymerase is reverse transcriptase, and wherein 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 766an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with any one amino acid sequence of
302. Embodiment 287, wherein the PEgRNA comprises a nucleic acid extension arm at the 3' or 5' end of the guide RNA or at an intramolecular location, the extension arm comprising a DNA synthesis template sequence and a primer binding site.
303. Embodiment 302, wherein the extended 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 in length. , 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 be.
304. Embodiment 287 The method of embodiment 287, wherein the PEgRNA has 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, 776-777having 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, the method comprising:
(i) contacting the DNA molecule with a nucleic acid programmed DNA binding protein (napDNAbp) and a PEgRNA that targets the napDNAbp to the target locus, the PEgRNA forming a reverse transcriptase (RT) template containing at least one desired nucleotide change; comprising a sequence 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 onto the corresponding endogenous DNA, thereby introducing one or more changes in the nucleotide sequence of the DNA molecule at the target locus;
including.
306. Embodiment 305. The method of embodiment 305, wherein the one or more changes in the nucleotide sequence comprises a transition.
307. 306. 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． Embodiment 305. The method of embodiment 305, wherein the one or more changes in the nucleotide sequence comprises a transversion.
309． Embodiment 308, wherein the transversion is: (a) T to A; (b) T to G; (c) C to G; (d) C to A; (e) A to T; (f) (g) G to C; and (h) G to T.
310． Embodiment 305, wherein the one or more changes in the nucleotide sequence include: (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) G:C base pair to C:G base pair, (4) T:A base pair to G:C base pair, (5) T:A base pair to A:T base pair, (6 )T:A base pair to C:G base pair, (7)C:G base pair to G:C base pair, (8)C:G base pair to T:A base pair, (9)C :G base pair to A:T base pair, (9) A:T base pair to T:A base pair, (11) A:T base pair to G:C base pair, or (12) A: It involves changing a T base pair to a C:G base pair.
311. Embodiment 305, wherein the one or more changes in the nucleotide sequence are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, Contains insertions or deletions of 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: adenosine deaminase (ADA) deficiency; alpha-1 antitrypsin deficiency; cystic fibrosis; Duchenne muscular dystrophy; galactosemia; hemochromatosis; Huntington's disease; maple Syrupuria; Marfan syndrome; type 1 neurofibromatosis; congenital nail hypertrophy; phenylketonuria; severe combined immunodeficiency; sickle cell disease; Smith-Lemli-Opitz syndrome; trinucleotide repeat disorder; prion disease associated with a single gene disorder selected from the group consisting of; and Tay-Sachs disease.
314. Embodiment 312. The method of embodiment 312, 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.
315. Embodiment 305, wherein the napDNAbp is nuclease-active Cas9 or a variant thereof.
316． Embodiment 305, wherein the napDNAbp is nuclease-inactive Cas9 (dCas9) or Cas9 nickase (nCas9) or a variant thereof.
317. Embodiment 305, wherein napDNAbp is SEQ ID NO.18Amino acid sequence of or SEQ ID NO.18an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with.
318. Embodiment 305, wherein napDNAbp is SEQ ID NO.18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with any one amino acid sequence of
319． Embodiment 305. The method of embodiment 305, wherein the reverse transcriptase is introduced in trans.
320. Embodiment 305, wherein the napDNAbp comprises a fusion to reverse transcriptase.
321． Embodiment 305, wherein the reverse transcriptase is 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． Embodiment 305, wherein the reverse transcriptase is 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 766an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with any one amino acid sequence of
323． Embodiment 305. 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.
324． Embodiment 323. The method of embodiment 323, wherein the nuclease is provided in trans.
325. Embodiment 305. The method of embodiment 305, wherein forming an exposed 3' end of the DNA strand at the target locus comprises contacting the DNA strand with a chemical agent.
326. Embodiment 305. The method of embodiment 305, wherein forming an exposed 3' end of the DNA strand at the target locus comprises introducing a replication error.
327． Embodiment 305. The method of embodiment 305, wherein the step of contacting the DNA molecule with napDNAbp and guide RNA forms an R-loop.
328. The method of embodiment 327, wherein the DNA strand from which the exposed 3' end is formed is on the R-loop.
329. Embodiment 315. The method of embodiment 315, wherein the PEgRNA comprises an extended arm that includes a reverse transcriptase (RT) template sequence and a primer binding site.
330． 329. The method of embodiment 329, wherein the elongated arm is at the 3' end of the guide RNA, the 5' end of the guide RNA, or an intramolecular position of the guide RNA.
331. Embodiment 305, wherein the PEgRNA further comprises at least one additional structure selected from the group consisting of a linker, stem-loop, hairpin, toe-loop, aptamer, or RNA-protein recruitment domain.
332. Embodiment 305, wherein the PEgRNA further comprises a homology arm.
333. Embodiment 305. The method of embodiment 305, wherein the RT template sequence is homologous to the corresponding endogenous DNA.
334. A method for introducing one or more changes in the nucleotide sequence of a DNA molecule at a target locus by primed reverse transcription to a primed target, the method comprising: (a) converting the DNA molecule at the target locus to (i) a nucleic acid program; (b) reverse transcription primed to the primed target of the RT template. (c) incorporating the desired nucleotide changes onto the DNA molecule at the target locus by DNA repair and/or replication processes.
335. Embodiment 334. The method of embodiment 334, wherein the RT template is located at the 3' end of the guide RNA, at the 5' end of the guide RNA, or at an intramolecular location of the guide RNA.
336． Embodiment 334. The method of embodiment 334, wherein the desired nucleotide change comprises a transition, transversion, insertion, or deletion, or any combination thereof.
337. Embodiment 334, wherein the desired nucleotide change is 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. including.
338. 335. The method of claim 334, wherein the desired nucleotide change is (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． 335. The method of claim 334, 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) G:C base pair to C:G base pair, (4) T:A base pair to G:C base pair, (5) T:A base pair to A:T base pair, (6) T: A base pair becomes C:G base pair, (7) C:G base pair becomes G:C base pair, (8) C:G base pair becomes T:A base pair, (9) C:G base pair. pair to A:T base pair, (10) A:T base pair to T:A base pair, (11) A:T base pair to G:C base pair, or (12) A:T base pair. It involves changing to a C:G base pair.
340. A polynucleotide encoding the PEgRNA of any one of embodiments 243-259.
341. A vector comprising the polynucleotide of embodiment 340.
342． A cell comprising the vector of embodiment 341.
343． The fusion protein of embodiment 213, wherein the polymerase is an error-prone reverse transcriptase.
344. A method for mutagenizing a DNA molecule at a target locus by 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． The method of any preceding embodiment, wherein the fusion protein comprises the 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 482-487Contains the amino acid sequence of
348. Embodiment 344 The method of embodiment 344, wherein the guide RNA is SEQ ID NO.222including.
349. Embodiment 344 The method of embodiment 344, wherein the step of (b) performing primed reverse transcription on a primed target comprises a 3' end primer at the target locus capable of priming reverse transcription by annealing to a primer binding site on a guide RNA. Including generating associative arrays.
350. A method for replacing a trinucleotide repeat expansion mutation on a target DNA molecule by a healthy sequence containing a healthy number of repeat trinucleotides, the method comprising: (a) converting the DNA molecule at the target locus to (i) a nucleic acid programmed contacting a fusion protein containing a DNA binding protein (napDNAbp) and a polymerase; and (ii) a DNA synthesis template containing a replacement sequence and a PEgRNA containing a primer binding site; producing single-stranded DNA; and (c) incorporating single-stranded DNA onto a DNA molecule at a target locus by a DNA repair and/or replication process.
351． Embodiment 350, wherein the fusion protein comprises the amino acid sequence of PE1, PE2, or PE3.
352. Embodiment 350, wherein the napDNAbp is Cas9 nickase (nCas9).
353． The method of embodiment 350, wherein napDNAbp is SEQ ID NO: 18 to88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487Contains the amino acid sequence of
354． Embodiment 350, wherein the guide RNA is SEQ ID NO.222including.
355. Embodiment 350, wherein (b) performing prime editing generates a 3' end primer binding sequence at the target locus that is capable of priming a polymerase by annealing to a primer binding site on the guide RNA. include.
356. Embodiment 350, wherein the trinucleotide repeat expansion mutation is associated with Huntington's disease, Fragile X syndrome, or Friedreich's ataxia.
357. Embodiment 350, wherein the trinucleotide repeat expansion comprises a CAG triplet repeat unit.
358. The method of embodiment 350, wherein the trinucleotide repeat expansion mutation comprises a repeat unit of a GAA triplet.
359． A method of incorporating a functional moiety onto a protein of interest encoded by a target nucleotide sequence by prime editing, the method comprising: (a) converting the target nucleotide sequence to (i) a nucleic acid programmed DNA binding protein (napDNAbp) and a polymerase; contacting PEgRNA containing a prime editing factor and (ii) a DNA synthesis template encoding a functional moiety; (b) polymerizing a single-stranded DNA sequence encoding a functional moiety; and (c) DNA repair and/or incorporating a single-stranded DNA sequence in place of the corresponding endogenous strand in the target nucleotide sequence by a replication process, the method yielding a recombinant target nucleotide sequence encoding a fusion protein comprising the protein of interest and the functional moiety. .
360. Embodiment 359. The method of embodiment 359, wherein the functional moiety is a peptide tag.
361. 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. 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.2);HA tag(Sequence numberFive);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． 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(Sequence number 275) is selected from the group consisting of
364. Embodiment 359. The method of embodiment 359, wherein the functional moiety is an immune epitope.
365. Embodiment 364, wherein the immune epitope is: 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); Class I HLA hemagglutinin epitope for class I HLA (SEQ ID NO: 416); neuraminidase epitope for class I 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. Embodiment 359. The method of embodiment 359, wherein the functional moiety alters localization of the protein of interest.
367． Embodiment 359. The method of embodiment 359, wherein the functional moiety is a degradation tag, so that the degradation rate of the protein of interest is altered.
368. Embodiment 367. The method of embodiment 367, wherein the degradation tag results in elimination of the tagged protein.
369． Embodiment 359. The method of embodiment 359, wherein the functional moiety is a small molecule binding domain.
370． Embodiment 359. The method of embodiment 359, wherein the small molecule binding domain is FKBP12 of SEQ ID NO: 488.
371． Embodiment 359. The method of embodiment 359, wherein the small molecule binding domain is FKBP12-F36V of SEQ ID NO: 489.
372． Embodiment 359, wherein the small molecule binding domain is SEQ ID NO: 49.0 and 493~494 cyclophilins.
373. Embodiment 359. The method of embodiment 359, wherein the small molecule binding domain is incorporated onto two or more proteins of interest.
374. Embodiment 373. The method of embodiment 373, wherein the two or more proteins of interest can be dimerized by contacting the small molecule.
375． The method of embodiment 369, wherein the small molecule is:

It is a low molecular weight dimer selected from the group consisting of:
376． A method of incorporating an immune epitope into a protein of interest encoded by a target nucleotide sequence by prime editing, the method comprising: (a) converting the target nucleotide sequence to (i) a nucleic acid programmed DNA binding protein (napDNAbp) and a polymerase; contacting a PEgRNA containing a prime editing factor and (ii) an editing template encoding a functional moiety; (b) polymerizing a single-stranded DNA sequence encoding an immune epitope; and (c) DNA repair and/or replication. The process involves incorporating a single-stranded DNA sequence in place of the corresponding endogenous strand in the target nucleotide sequence, and the method results in 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: 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); Class I HLA hemagglutinin epitope for class I HLA (SEQ ID NO: 416); neuraminidase epitope for class I 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. Embodiment 376. The method of embodiment 376, wherein the fusion protein comprises the amino acid sequence of PE1, PE2, or PE3.
379． Embodiment 376, wherein the napDNAbp is Cas9 nickase (nCas9).
380. The method of embodiment 376, wherein napDNAbp is SEQ ID NO: 18 to88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487Contains the amino acid sequence of
381. 376. The method of embodiment 376, wherein the PEgRNA is 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 of incorporating small molecule dimerization domains onto a protein of interest encoded by a target nucleotide sequence by prime editing, the method comprising: (a) converting the target nucleotide sequence into (i) a nucleic acid programmed DNA binding protein ( (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 a DNA repair and/or replication process, the method comprising: small molecule dimerization with the protein of interest; A recombinant target nucleotide sequence is generated that encodes a fusion protein comprising a domain.
383. The method of embodiment 382, further comprising performing the method on a second protein of interest.
384． Embodiment 383. 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 a respective dimerization domain of the proteins.
385. The method of embodiment 382, wherein the small molecule binding domain is FKBP12 of SEQ ID NO: 488.
386. The method of embodiment 382, wherein the small molecule binding domain is FKBP12-F36V of SEQ ID NO: 489.
387． Embodiment 382, wherein the small molecule binding domain is SEQ ID NO: 49.0 and 493~494 cyclophilins.
388． Embodiment 382 The method of embodiment 382, wherein the small molecule is:

It is a low molecular weight dimer selected from the group consisting of:
389． Embodiment 382, wherein the fusion protein comprises the amino acid sequence of PE1, PE2, or PE3.
390. Embodiment 382, wherein the napDNAbp is Cas9 nickase (nCas9).
391． The method of embodiment 382, wherein napDNAbp is SEQ ID NO: 18 to88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487Contains the amino acid sequence of
392. Embodiment 382 The method of embodiment 382, wherein the PEgRNA is 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 of incorporating a peptide tag or epitope onto a protein using prime editing, comprising: a prime editing agent configured to insert into a target nucleotide sequence encoding a protein a second nucleotide sequence encoding a peptide tag; contacting the recombinant nucleotide sequence with a construct so that the peptide tag and protein are expressed from the recombinant nucleotide sequence as a fusion protein.
394． Embodiment 383. The method of embodiment 383, wherein the peptide tag is used for protein purification and/or detection.
395． Embodiment 383. The method of embodiment 383, wherein the peptide tag is polyhistidine (e.g., HHHHHH(Sequence number 257)), FLAG (for example, DYKDDDDK(Sequence number 2)), V5 (for example, GKPIPNPLLGLLDST(Sequence number 3)), GCN4, HA (e.g., YPYDVPDYA(Sequence number 5)), Myc (e.g. EQKLISEED(Sequence number 6)), or GST.
396. 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． 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 an amino acid sequence of
400. Embodiment 383, wherein the prime editing factor construct comprises 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, 776-777PEgRNA containing the nucleotide sequence of
401． Embodiment 383, wherein the PEgRNA comprises a spacer, a gRNA core, and an extended arm, the spacer being complementary to a target nucleotide sequence, and the extended arm comprising a reverse transcriptase template encoding a peptide tag.
402． Embodiment 383, wherein the PEgRNA comprises a spacer, a gRNA core, and an extended arm, the spacer being complementary to a target nucleotide sequence, and the extended arm comprising a reverse transcriptase template encoding a peptide tag.
403． A method of preventing or halting the progression of a prion disease by incorporating one or more protective mutations on a PRNP encoded by a target nucleotide sequence by prime editing, the method comprising: (a) (i) contacting with a prime editing factor comprising a nucleic acid programmed DNA binding protein (napDNAbp) and a polymerase and (ii) a PEgRNA comprising an editing template encoding a functional moiety; (b) a single strand encoding a protective mutation; polymerizing the DNA sequence; and (c) incorporating the single-stranded DNA sequence in place of the corresponding endogenous strand in the target nucleotide sequence by a DNA repair and/or replication process, wherein the method is resistant to misfolding. A recombinant target nucleotide sequence is generated that encodes a PRNP containing a protective mutation that is resistant.
404. Embodiment 403. 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. Embodiment 404, wherein the prion disease is Creutzfeldt-Jakob disease (CJD), variant Creutzfeldt-Jakob disease (vCJD), Gerstmann-Strausler-Scheinker syndrome, fatal familial insomnia, or Kuru disease. It is.
407． 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. It is encephalopathy.
408. The method of embodiment 403, wherein the wild type PRNP amino acid sequence is SEQ ID NO: 291-292.
409. Embodiment 403, wherein the method results in a modified PRNP amino acid sequence selected from the group consisting of SEQ ID NOs: 293-323, wherein the modified PRNP protein is resistant to misfolding.
410. Embodiment 403, wherein the fusion protein comprises the amino acid sequence of PE1, PE2, or PE3.
411. Embodiment 403, wherein the napDNAbp is Cas9 nickase (nCas9).
412． The method of embodiment 403, wherein napDNAbp is SEQ ID NO: 18 to88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487Contains the amino acid sequence of
413．． Embodiment 403, wherein the PEgRNA is 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 of incorporating a ribonucleotide motif or tag onto an RNA of interest encoded by a target nucleotide sequence by prime editing, the method comprising: (a) converting the target nucleotide sequence into (i) a nucleic acid programmed DNA binding protein (napDNAbp); and a prime editing agent comprising a polymerase; and (ii) contacting PEgRNA comprising an editing template encoding a ribonucleotide motif or tag; (b) polymerizing a single-stranded DNA sequence encoding a ribonucleotide motif or tag; and (c) incorporating a single-stranded DNA sequence in place of the corresponding endogenous strand in the target nucleotide sequence by a DNA repair and/or replication process, the method comprising A recombinant target nucleotide sequence is generated that encodes the desired RNA.
415． Embodiment 414. The method of embodiment 414, wherein the ribonucleotide motif or tag is the detection moiety.
416． Embodiment 414. The method of embodiment 414, wherein the ribonucleotide motif or tag affects the expression level of the RNA of interest.
417． Embodiment 414. The method of embodiment 414, wherein the ribonucleotide motif or tag affects trafficking or subcellular localization of the RNA of interest.
418. The method of embodiment 414, wherein the ribonucleotide motif or tag is selected from the group consisting of SV40 type 1, SV40 type 2, SV40 type 3, hGH, BGH, rbGlob, TK, MALAT1 ENE-mascRNA, KSHV PAN ENE, Smbox/U1 snRNA box, U1 snRNA 3' box, tRNA-lysine, broccoli aptamer, spinach aptamer, mango aptamer, HDV ribozyme, and m6A.
419． 414. The method of embodiment 414, wherein the PEgRNA is 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. Embodiment 414. The method of embodiment 414, wherein the fusion protein comprises the amino acid sequence of PE1, PE2, or PE3.
421. Embodiment 414. The method of embodiment 414, wherein napDNAbp is Cas9 nickase (nCas9).
422. The method of embodiment 414, wherein napDNAbp is SEQ ID NO: 18 to88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487Contains the amino acid sequence of
423． A method of incorporating or deleting a functional moiety on a protein of interest encoded by a target nucleotide sequence by prime editing, the method comprising: (a) converting the target nucleotide sequence into (i) a nucleic acid programmed DNA binding protein (napDNAbp); ) and a prime editing factor comprising a polymerase and (ii) PEgRNA comprising an editing template encoding the functional moiety or its deletion; (b) polymerizing the single-stranded DNA sequence encoding the functional moiety or its deletion; and (c) incorporating a single-stranded DNA sequence in place of the corresponding endogenous strand in the target nucleotide sequence by a DNA repair and/or replication process, the method comprising: A recombinant target nucleotide sequence is generated that encodes a modified protein containing a deletion, the functional moiety altering the modification state or localization state of the protein.
424． Embodiment 423. 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 that includes a nucleic acid programmed DNA binding protein (napDNAbp) and a reverse transcriptase.

Aspect 2. The fusion protein of embodiment 1, wherein the fusion protein is capable of performing genome editing by primed target-primed reverse transcription in the presence of an elongated guide RNA.

Aspect 3. The fusion protein of embodiment 1, wherein napDNAbp has nickase activity.

Aspect 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).

Aspect 6. The fusion protein of embodiment 1, wherein napDNAbp is Cas9 nickase (nCas9).

Aspect 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.

Aspect 8. The fusion protein of embodiment 1 is capable of binding to a target DNA sequence when the fusion protein is complexed with an elongated guide RNA.

Aspect 9. The fusion protein of embodiment 8, wherein the target DNA sequence comprises a target strand and a complementary non-target strand.

Aspect 10. In the fusion protein of embodiment 8, the binding of the fusion protein complexed with the elongated guide RNA forms an R-loop.

Aspect 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.

Aspect 12. The fusion protein of embodiment 11, wherein the complementary non-target strand is nicked to form a reverse transcriptase prime sequence with a free 3' end.

Aspect 13. The fusion protein of embodiment 2, wherein the elongated 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.

Aspect 14. The fusion protein of embodiment 13, wherein the RNA extension comprises (i) a reverse transcription template sequence containing the desired nucleotide changes, (ii) a reverse transcription primer binding site, and (iii) an optional linker sequence.

Aspect 15. The fusion protein of embodiment 14, wherein the reverse transcription template sequence encodes a single-stranded DNA flap that is complementary to an endogenous DNA sequence adjacent to the nick site, and wherein the single-stranded DNA flap carries a desired nucleotide change. including.

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.

Aspect 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.

Aspect 18. The fusion protein of embodiment 15, wherein the single-stranded DNA flap replaces the endogenous DNA sequence with a free 5' end and adjacent to the nick site.

Aspect 19. The fusion protein of embodiment 18, wherein the endogenous DNA sequence having a 5' end is excised by the cell.

Mode 20. The fusion protein of embodiment 18, wherein cellular repair of the single-stranded DNA flap results in the incorporation of the desired nucleotide change, thereby forming the desired product.

Aspect 21. The fusion protein of embodiment 14, wherein the desired nucleotide change 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 The desired nucleotide change is incorporated on an editing window that is between approximately -30 and +100 of the PAM sequence, or 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, Incorporates 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides downstream.

Aspect 22. The fusion protein of embodiment 1, wherein napDNAbp is the amino acid sequence of SEQ ID NO: 18, or an amino acid that is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 18. Contains arrays.

Aspect 23. The fusion protein of embodiment 1, 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. Comprising amino acid sequences that are at least 80%, 85%, 90%, 95%, 98%, or 99% identical.

The fusion protein of embodiment 1, wherein the reverse transcriptase comprises any one of the amino acid sequences of SEQ ID NO: 89.

Aspect 24. The fusion protein of embodiment 1, wherein the reverse transcriptase is SEQ ID NO: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, 662, 700, 701-716, 739-741, and 766.

The fusion protein of embodiment 1, wherein the reverse transcriptase has an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to any one amino acid sequence of SEQ ID NO: 89. including.

Aspect 25. The fusion protein of embodiment 1, wherein the reverse transcriptase is SEQ ID NO: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, 662, 700, 701-716, 739-741, and 766.

Aspect 26. The fusion protein of embodiment 1, wherein the reverse transcriptase is a naturally occurring reverse transcriptase from a retrovirus or retrotransposon.

Aspect 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 indicates the presence of an arbitrary linker sequence.

Aspect 28. The fusion protein of embodiment 27, wherein the linker sequence comprises the amino acid sequences of SEQ ID NOs: 127, 165-176, 446, 453, and 767-769.

Aspect 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.

Aspect 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.

Aspect 31. An extended guide RNA comprising a guide RNA and at least one RNA extension.

Aspect 32. The elongated guide RNA of embodiment 1, wherein the RNA extension is at the 3' or 5' end of the guide RNA or at a position within the molecule of the guide RNA.

Aspect 33. The elongated guide RNA of embodiment 31, wherein the elongated guide RNA is capable of binding napDNAbp and directing napDNAbp to a target DNA sequence.

Aspect 34. The elongated 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.

Aspect 35. The extended guide RNA of embodiment 31, wherein the at least one RNA extension comprises (i) a reverse transcription template sequence, (ii) a reverse transcription primer binding site, and (iii) an optional linker sequence.

Aspect 36. The elongated guide RNA of embodiment 35, wherein the RNA elongation 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 in length. , 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.

Aspect 37. The elongated 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.

Aspect 38. The elongated 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 in length. , 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.

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.

Aspect 40. The elongated guide RNA of embodiment 35, wherein the reverse transcription template sequence encodes a single-stranded DNA flap that is complementary to an endogenous DNA sequence adjacent to the nick site, and wherein the single-stranded DNA flap is desired. Contains nucleotide changes.

Aspect 41. The elongated guide RNA of embodiment 40, 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 nicked. Immediately adjacent downstream of the site.

Aspect 42. The elongated guide RNA of embodiment 41, wherein the endogenous single-stranded DNA with a free 5' end is excised by the cell.

Aspect 43. The elongated guide RNA of embodiment 41, wherein cellular repair of the single-stranded DNA flap results in the incorporation of the desired nucleotide change, thereby forming the desired product.

Aspect 44. The elongated guide RNA of embodiment 31, comprising SEQ ID NOs : 394, 429-442, 641-649, 678-692, 2997-3103, 3113-3121, 3305-3455, 3479-3493, 3522-3556, 3628- 3698, and the nucleotide sequence of 3755-3810 , or SEQ ID NO: 394, 429-442, 641-649, 678-692, 2997-3103, 3113-3121, 3305-3455, 3479-3493, 3522-3556, 3628-3698 , and any one of 3755-3810.

Aspect 45. The elongated 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.

Aspect 46. The elongated guide RNA of embodiment 35, wherein the reverse transcription primer binding site hybridizes to the free 3' end of the cut DNA.

Aspect 47. The elongated guide RNA of embodiment 35, wherein any linker sequence is at least 1 nucleotide in length, 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.

Aspect 48. A complex comprising the fusion protein of any one of embodiments 1 to 30 and an elongated guide RNA.

Aspect 49. Embodiment 48, wherein the elongated guide RNA comprises a guide RNA and an RNA extension at the 3' or 5' end of the guide RNA or at a position within the molecule of the guide RNA.

Aspect 50. The complex of embodiment 48, wherein the elongated guide RNA is capable of binding napDNAbp and directing napDNAbp to a target DNA sequence.

Aspect 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.

Aspect 52. Embodiment 49, wherein the at least one RNA extension comprises (i) a reverse transcription template sequence, (ii) a reverse transcription primer binding site, and (iii) an optional linker sequence.

Aspect 53. The complex of embodiment 48, wherein the elongated guide RNA is SEQ ID NO: 394, 429-442, 641-649, 678-692, 2997-3103, 3113-3121, 3305-3455, 3479-3493, 3522- Nucleotide sequences of 3556, 3628-3698, and 3755-3810, or SEQ ID NO: 394, 429-442, 641-649, 678-692, 2997-3103, 3113-3121, 3305-3455, 3479-3493, 3522-3556 , 3628-3698, and 3755-3810.

Aspect 54. The conjugate of embodiment 52, wherein the reverse transcription template sequence comprises a nucleotide sequence having at least 80% or 85% or 90% or 95% or 99% sequence identity with the endogenous DNA target.

Aspect 55. 53. The complex of embodiment 52, wherein the reverse transcription primer binding site hybridizes to the free 3' end of the cut DNA.

Aspect 56. complex containing napDNAbp and elongated guide RNA.

Aspect 57. The conjugate of embodiment 56, wherein napDNAbp is Cas9 nickase.

Aspect 58. The conjugate of embodiment 56, wherein napDNAbp has the amino acid sequence of SEQ ID NO: 18, or an amino acid having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO: 18. Contains arrays.

Aspect 59. The complex of embodiment 57, 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. amino acid sequences having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity.

Aspect 60. Embodiment 57, wherein the elongated guide RNA comprises a guide RNA and an RNA extension at the 3' or 5' end of the guide RNA or at a position within the molecule of the guide RNA.

Aspect 61. Embodiment 57. The complex of embodiment 57, wherein the elongated guide RNA is capable of directing napDNAbp to a target DNA sequence.

Aspect 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.

Aspect 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) an optional linker sequence.

Aspect 64. The complex of embodiment 57, wherein the elongated guide RNA is SEQ ID NO: 394, 429-442, 641-649, 678-692, 2997-3103, 3113-3121, 3305-3455, 3479-3493, 3522- Nucleotide sequences of 3556, 3628-3698, and 3755-3810, or SEQ ID NO: 394, 429-442, 641-649, 678-692, 2997-3103, 3113-3121, 3305-3455, 3479-3493, 3522-3556 , 3628-3698, and 3755-3810.

Aspect 65. The conjugate 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.

Aspect 66. The complex of embodiment 63, wherein the reverse transcription primer binding site hybridizes to the free 3' end of the cut DNA.

Aspect 67. The conjugate of embodiment 63, wherein any linker sequence is at least 1 nucleotide in length, 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.

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 embodiment 68.

Mode 70. A cell comprising the fusion protein of any of embodiments 1 to 30 and an elongated guide RNA bound to napDNAbp of the fusion protein.

Aspect 71. A cell comprising the complex of any one of embodiments 48-67.

Mode 72. A pharmaceutical composition comprising: (i) a fusion protein of any of embodiments 1 to 30, a conjugate of embodiments 48 to 67, a polynucleotide of embodiment 68, or a vector of embodiment 69; and excipients.

Aspect 73. A pharmaceutical composition comprising: (i) a conjugate of embodiments 48-67, (ii) a reverse transcriptase provided in trans; and (iii) a pharmaceutically acceptable excipient.

Aspect 74. A kit comprising: (i) a nucleic acid sequence encoding the 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).

Aspect 75. A method for incorporating desired nucleotide changes into a double-stranded DNA sequence, the method comprising:

(i) Reverse transcription by contacting a double-stranded DNA sequence with a complex containing a fusion protein and an elongated guide RNA, where the fusion protein contains napDNAbp and reverse transcriptase, and the elongated guide RNA contains the desired nucleotide change. Contains a template sequence;

(ii) nicking a double-stranded DNA sequence on the non-target strand, thereby generating free single-stranded DNA with a 3' end;

(iii) hybridizing the 3′ end of the free single-stranded DNA to a reverse transcription template sequence, thereby priming the reverse transcriptase domain;

(iv) polymerizing the strands of DNA from the 3' end, thereby producing a single-stranded DNA flap containing the desired nucleotide changes;

(v) replacing the endogenous DNA strand adjacent to the cut site by a single-stranded DNA flap, thereby incorporating the desired nucleotide change into the double-stranded DNA sequence;
including.

Aspect 76. 75. The method of embodiment 75, wherein (v) the step of replacing: (i) hybridizing the single-stranded DNA flap to an 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 the desired product containing the desired nucleotide changes in both strands of the DNA.

Aspect 77. 77. The method of embodiment 76, wherein the desired nucleotide change is a single nucleotide substitution, deletion, or insertion.

Aspect 78. 78. The method of embodiment 77, wherein the single nucleotide substitution is a transition or transversion.

Aspect 79. 76. 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) Substitution of A from T, (6) Substitution of C from T, (7) Substitution of G from C, (8) Substitution of T from C, (9) Substitution of A from C, (10) Substitution of A T substitution, (11) A to G substitution, or (12) A to C substitution.

Mode 80. 76. 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) G:C base pair to C:G base pair, (4) T:A base pair to G:C base pair, (5) T:A base pair to A:T base pair, (6) T:A base pair becomes C:G base pair, (7) C:G base pair becomes G:C base pair, (8) C:G base pair becomes T:A base pair, (9) C: G base pair to A:T base pair, (10) A:T base pair to T:A base pair, (11) A:T base pair to G:C base pair, or (12) A:T. Converts base pairs to C:G base pairs.

Aspect 81. 77. The method of embodiment 76, wherein the desired nucleotide change is 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 nucleotide insertions or deletions.

Mode 82. 77. The method of embodiment 76, wherein the desired nucleotide change modifies a disease-associated gene.

Aspect 83. The method of embodiment 82, wherein the disease-associated gene is: adenosine deaminase (ADA) deficiency; alpha-1 antitrypsin deficiency; cystic fibrosis; Duchenne muscular dystrophy; galactosemia; hemochromatosis; Huntington's disease; maple Consists of syrup urine disease; Marfan syndrome; type 1 neurofibromatosis; congenital nail hyperplasia; phenylketonuria; severe combined immunodeficiency; sickle cell disease; Smith-Lemli-Opitz syndrome; and Tay-Sachs disease. associated with a single gene disorder selected from the group.

Mode 84. 83. The method of embodiment 82, 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.

Mode 85. 77. The method of embodiment 76, wherein the napDNAbp is nuclease-inactive Cas9 (dCas9), Cas9 nickase (nCas9), or nuclease-active Cas9.

Mode 86. 77. The method of embodiment 76, wherein napDNAbp comprises the amino acid sequence of SEQ ID NO: 18.

Mode 87. 76. The method of embodiment 76, wherein napDNAbp has 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. Comprising amino acid sequences with at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity.

Mode 88. The method of embodiment 76, wherein the reverse transcriptase is SEQ ID NO: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, 662, 700 , 701-716, 739-741, and 766.

Mode 89. The method of embodiment 76, wherein the reverse transcriptase domain is 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 90. 77. The method of embodiment 76, wherein the elongated guide RNA comprises an RNA extension at the 3' or 5' end of the guide RNA or at an intramolecular position, and the RNA extension comprises a reverse transcription template sequence.

Aspect 91. 90. The method of embodiment 90, wherein the RNA elongation 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 in length. , 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 be.

Aspect 92. The method of embodiment 76, wherein the elongated guide RNA is SEQ ID NO: 394, 429-442, 641-649, 678-692, 2997-3103, 3113-3121, 3305-3455, 3479-3493, 3522-3556. , 3628-3698, and 3755-3810.

Aspect 93. A method for introducing one or more changes in the nucleotide sequence of a DNA molecule at a target locus, the method comprising:

(i) a reverse transcriptase (RT) template in which the DNA molecule is contacted with a nucleic acid programmed DNA binding protein (napDNAbp) and a guide RNA that targets napDNAbp to the target locus, the guide RNA containing at least one desired nucleotide change; Contains an array;

(ii) forming an exposed 3' end of the DNA strand at the target locus;

(iii) hybridizing the exposed 3′ end to the RT template sequence 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 onto the corresponding endogenous DNA, thereby introducing one or more changes in the nucleotide sequence of the DNA molecule at the target locus;
including.

Aspect 94. 94. The method of embodiment 93, wherein the one or more changes in the nucleotide sequence comprises a transition.

Aspect 95. 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.

Aspect 96. 94. The method of embodiment 93, wherein the one or more changes in the nucleotide sequence comprises a transversion.

Aspect 97. 96. The method of aspect 96, wherein the transversion is: (a) T to A; (b) T to G; (c) C to G; (d) C to A; (e) A to T; (f) (g) G to C; and (h) G to T.

Aspect 98. 94. The method of embodiment 93, wherein the one or more changes in the nucleotide sequence include: (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) G:C base pair to C:G base pair, (4) T:A base pair to G:C base pair, (5) T:A base pair to A:T base pair, (6 )T:A base pair to C:G base pair, (7)C:G base pair to G:C base pair, (8)C:G base pair to T:A base pair, (9)C :G base pair to A:T base pair, (10) A:T base pair to T:A base pair, (11) A:T base pair to G:C base pair, or (12) A: It involves changing a T base pair to a C:G base pair.

Aspect 99. 94. The method of embodiment 93, wherein the one or more changes in the nucleotide sequence are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, Contains insertions or deletions of 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides.

Mode 100. 94. The method of embodiment 93, wherein the one or more changes in the nucleotide sequence comprises correction of a disease-associated gene.

Aspect 101. Embodiment 100, wherein the disease-associated gene is: adenosine deaminase (ADA) deficiency; alpha-1 antitrypsin deficiency; cystic fibrosis; Duchenne muscular dystrophy; galactosemia; hemochromatosis; Huntington's disease; maple Consists of syrup urine disease; Marfan syndrome; type 1 neurofibromatosis; congenital nail hyperplasia; phenylketonuria; severe combined immunodeficiency; sickle cell disease; Smith-Lemli-Opitz syndrome; and Tay-Sachs disease. associated with a single gene disorder selected from the group.

Aspect 102. 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.

Aspect 103. 94. The method of embodiment 93, wherein napDNAbp is nuclease active Cas9 or a variant thereof.

Aspect 104. 94. The method of embodiment 93, wherein napDNAbp is nuclease-inactive Cas9 (dCas9) or Cas9 nickase (nCas9) or a variant thereof.

Aspect 105. 94. The method of embodiment 93, wherein napDNAbp comprises the amino acid sequence of SEQ ID NO: 18.

Aspect 106. Embodiment 93, wherein napDNAbp has 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. Comprising amino acid sequences with at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity.

Aspect 107. 94. The method of embodiment 93, wherein the reverse transcriptase is introduced in trans.

Aspect 108. 94. The method of embodiment 93, wherein napDNAbp comprises a fusion to reverse transcriptase.

Aspect 109. The method of embodiment 93, wherein the reverse transcriptase is f SEQ ID NO: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, 662, 700, 701-716, 739-741, and 766.

Aspect 110. Embodiment 93, wherein the reverse transcriptase is SEQ ID NO: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, 662, 700 , 701-716, 739-741, and 766.

Aspect 111. 94. 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.

Aspect 112. Embodiment 111, wherein the nuclease is napDNAbp, is provided as a fusion domain of napDNAbp, or is provided in trans.

Aspect 113. 94. The method of embodiment 93, wherein forming an exposed 3' end of the DNA strand at the target locus comprises contacting the DNA strand with a chemical agent.

Aspect 114. 94. The method of embodiment 93, wherein forming an exposed 3' end of the DNA strand at the target locus comprises introducing a replication error.

Aspect 115. 94. The method of embodiment 93, wherein the step of contacting the DNA molecule with napDNAbp and guide RNA forms an R-loop.

Aspect 116. Embodiment 115. The method of embodiment 115, wherein the DNA strand from which the exposed 3' end is formed is on the R-loop.

Aspect 117. 94. The method of embodiment 93, wherein the guide RNA comprises an extended portion that includes a reverse transcriptase (RT) template sequence.

Aspect 118. The method of embodiment 117, wherein the elongated portion is at the 3' end of the guide RNA, at the 5' end of the guide RNA, or at a position within the molecule of the guide RNA.

Aspect 119. 94. The method of embodiment 93, wherein the guide RNA further comprises a primer binding site.

Aspect 120. 94. The method of embodiment 93, wherein the guide RNA further comprises a spacer sequence.

Aspect 121. 94. The method of embodiment 93, wherein the RT template sequence is homologous to the corresponding endogenous DNA.

Aspect 122. A method for introducing one or more changes in the nucleotide sequence of a DNA molecule at a target locus by primed reverse transcription to a primed target, the method comprising: (a) converting the DNA molecule at the target locus into (i) a nucleic acid molecule; contacting a fusion protein comprising a programmed DNA binding protein (napDNAbp) and a reverse transcriptase and (ii) a guide RNA comprising an RT template containing the desired nucleotide changes; (b) a reverse primed target of the RT template; (c) incorporating the desired nucleotide changes onto the DNA molecule at the target locus by DNA repair and/or replication processes.

Aspect 123. The method of embodiment 122, wherein the RT template is located at the 3' end of the guide RNA, at the 5' end of the guide RNA, or at an intramolecular location of the guide RNA.

Aspect 124. Embodiment 122. The method of embodiment 122, wherein the desired nucleotide change comprises a transition, transversion, insertion, or deletion, or any combination thereof.

Aspect 125. 123. The method of claim 122, wherein the desired nucleotide change is selected from the group consisting of: (a) T to C; (b) A to G; (c) C to T; and (d) G to A. Contains transitions.

Aspect 126. 123. The method of claim 122, wherein the desired nucleotide change is (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.

Aspect 127. Embodiment 122, 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 T:A base pair; :C base pair becomes C:G base pair, (4) T:A base pair becomes G:C base pair, (5) T:A base pair becomes A:T base pair, (6) T:A base pair to C:G base pair, (7) C:G base pair to G:C base pair, (8) C:G base pair to T:A base pair, (9) C:G base pair. into an A:T base pair, (10) an A:T base pair into a T:A base pair, (11) an A:T base pair into a G:C base pair, or (12) an A:T base pair. This includes changing to a C:G base pair.

Aspect 128. A polynucleotide encoding the elongated guide RNA of any one of embodiments 31-47.

Aspect 129. A vector comprising the polynucleotide of embodiment 128.

Aspect 130. A cell comprising the vector of embodiment 129.

Aspect 131. The fusion protein of any of embodiments 1 to 30, wherein the reverse transcriptase is an error-prone reverse transcriptase.

Aspect 132. A method for mutagenizing a DNA molecule at a target locus by primed reverse transcription, the method comprising: (a) converting the DNA molecule at the target locus to (i) a nucleic acid programmed DNA binding protein (napDNAbp); contacting a fusion protein comprising an error-prone reverse transcriptase and (ii) a guide RNA comprising an RT template comprising the desired nucleotide changes; (b) performing primed reverse transcription on a primed target of the RT template; producing mutagenized single-stranded DNA; and (c) incorporating the mutagenized single-stranded DNA onto a DNA molecule at a target locus by a DNA repair and/or replication process.

Aspect 133. 133. The method of embodiment 132, wherein the fusion protein comprises the amino acid sequence of PE1, PE2, or PE3.

Aspect 134. 133. The method of embodiment 132, wherein napDNAbp is Cas9 nickase (nCas9).

Aspect 135. Embodiment 132, 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.

Aspect 136. 133. The method of embodiment 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.

Aspect 138. A method for replacing a trinucleotide repeat expansion mutation on a target DNA molecule by a healthy sequence containing a healthy number of repeat trinucleotides, the method comprising: (a) converting the DNA molecule at the target locus to (i) a nucleic acid programmed contacting a fusion protein comprising a 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 (intr); (b) priming the RT template; performing target-primed reverse transcription to generate single-stranded DNA containing 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. Including.

Embodiment 139. The method of embodiment 138, wherein the fusion protein comprises an amino acid sequence of PE1, PE2, or PE3.

Aspect 140. Embodiment 138, wherein the napDNAbp is Cas9 nickase (nCas9).

Aspect 141. Embodiment 138, 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.

Aspect 142. 139. The method of embodiment 138, wherein the guide RNA comprises SEQ ID NO: 222.

Aspect 143. Embodiment 138, wherein (b) performing primed reverse transcription on a primed target comprises a 3' end primer at the target locus capable of priming reverse transcription by annealing to a primer binding site on a guide RNA; Including generating associative arrays.

Aspect 144. 139. The method of embodiment 138, wherein the trinucleotide repeat expansion mutation is associated with Huntington's disease, Fragile X syndrome, or Friedreich's ataxia.

Aspect 145. 139. The method of embodiment 138, wherein the trinucleotide repeat expansion comprises a CAG triplet repeat unit.

Aspect 146. 139. The method of embodiment 138, wherein the trinucleotide repeat expansion comprises a GAA triplet repeat unit.

Aspect 147. A method of incorporating a functional moiety onto a protein of interest encoded by a target nucleotide sequence by prime editing, the method comprising: (a) converting the target nucleotide sequence to (i) nucleic acid programmed DNA binding protein (napDNAbp) and reverse transcription; contacting a prime editing factor comprising an enzyme and (ii) a PEgRNA comprising an editing template encoding a functional moiety; (b) polymerizing a single-stranded DNA sequence encoding a functional moiety; and (c) DNA repair and and/or incorporating a single-stranded DNA sequence in place of the corresponding endogenous strand in the target nucleotide sequence by a replication process, the method comprising a recombinant target nucleotide sequence encoding a fusion protein comprising the protein of interest and a functional moiety. will occur.

Aspect 148. Embodiment 147. The method of embodiment 147, wherein the functional moiety is a peptide tag.

Aspect 149. Embodiment 148, wherein the peptide tag is an affinity tag, solubilization tag, chromatography tag, epitope tag, or fluorescent tag.

Mode 150. 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: 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).

Aspect 151. Embodiment 148, 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-G (SEQ ID NO: 275). selected.

Aspect 152. Embodiment 147. The method of embodiment 147, wherein the functional moiety is an immune epitope.

Aspect 153. Embodiment 152, wherein the immune epitope is: 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); Class I HLA hemagglutinin epitope for class I HLA (SEQ ID NO: 416); neuraminidase epitope for class I 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).

Aspect 154. Embodiment 147. The method of embodiment 147, wherein the functional moiety alters localization of the protein of interest.

Aspect 155. Embodiment 147. The method of embodiment 147, wherein the functional moiety is a degradation tag, so that the degradation rate of the protein of interest is altered.

Aspect 156. Embodiment 155. The method of embodiment 155, wherein the degradation tag comprises an amino acid sequence encoding a degradation tag as disclosed herein.

Aspect 157. Embodiment 147. 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 embodiment 157, wherein the small molecule binding domain is FKBP12-F36V of SEQ ID NO: 489.

Mode 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.

Aspect 163. Embodiment 157. 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.

Aspect 164. A method of incorporating an immune epitope onto a protein of interest encoded by a target nucleotide sequence by prime editing, the method comprising: (a) converting the target nucleotide sequence to (i) nucleic acid programmed DNA binding protein (napDNAbp) and reverse transcription; contacting a prime editing factor comprising an enzyme and (ii) PEgRNA comprising an editing template encoding a functional moiety; (b) polymerizing a single-stranded DNA sequence encoding an immune epitope; and (c) DNA repair and and/or incorporating a single-stranded DNA sequence in place of the corresponding endogenous strand in the target nucleotide sequence by a replication process, wherein the method comprises recombinant target nucleotide sequences encoding a fusion protein comprising the protein of interest and the immune epitope. will occur.

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).

Aspect 166. Embodiment 164. The method of embodiment 164, wherein the fusion protein comprises the amino acid sequence of PE1, PE2, or PE3.

Aspect 167. Embodiment 164. The method of embodiment 164, wherein napDNAbp is Cas9 nickase (nCas9).

Mode 168. Embodiment 164, 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.

Mode 169. 165. The method of embodiment 164, wherein the guide RNA comprises SEQ ID NO: 222.

Mode 170. A method of incorporating a small molecule dimerization domain onto a protein of interest encoded by a target nucleotide sequence by prime editing, the method comprising: (a) converting the target nucleotide sequence into (i) a nucleic acid programmed DNA binding protein ( (b) polymerizing a single-stranded DNA sequence encoding an immune epitope; and (c) incorporating a single-stranded DNA sequence in place of the corresponding endogenous strand in the target nucleotide sequence by a DNA repair and/or replication process, the method comprising: A recombinant target nucleotide sequence is generated that encodes a fusion protein comprising an integration domain.

Embodiment 171. The method of embodiment 170, further comprising performing the method on a second protein of interest.

Aspect 172. Embodiment 171 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 a respective dimerization domain of the proteins.

Embodiment 173. The method of embodiment 170, wherein the small molecule binding domain is FKBP12 of SEQ ID NO:488.

Aspect 174. Embodiment 170, wherein the small molecule binding domain is FKBP12-F36V of SEQ ID NO: 489.

Aspect 175. Embodiment 170, wherein the small molecule binding domain is a cyclophilin of SEQ ID NOs: 490 and 493-494.

Aspect 176. Embodiment 170. 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.

Aspect 177. Embodiment 170, wherein the fusion protein comprises the amino acid sequence of PE1, PE2, or PE3.

Aspect 178. Embodiment 170, wherein the napDNAbp is Cas9 nickase (nCas9).

Aspect 179. Embodiment 170, 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.

Mode 180. Embodiment 170, wherein the guide RNA comprises SEQ ID NO: 222.

Mode 181. A method of incorporating a peptide tag or epitope onto a protein using prime editing, comprising: a prime editing agent configured to insert into a target nucleotide sequence encoding a protein a second nucleotide sequence encoding a peptide tag; contacting the recombinant nucleotide sequence with a construct so that the peptide tag and 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. 182. The method of embodiment 181, wherein the peptide tag is polyhistidine (e.g., HHHHHH) (SEQ ID NO: 252-262), FLAG (e.g., DYKDDDDK) (SEQ ID NO: 2), V5 (e.g., GKPIPNPLLGLLDST) (SEQ ID NO: 3). , GCN4, HA (eg, YPYDVPDYA) (SEQ ID NO: 5), Myc (eg, EQKLISEED) (SEQ ID NO: 6), GST, and the like.

Mode 184. 182. The method of embodiment 181, wherein the peptide tag is an amino acid selected from the group consisting of SEQ ID NOs: 1-6, 245-249, 252-262, 264-273, 275-276, 281, 278-288, and 622. It has an array.

Mode 185. 182. 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.

Mode 187. The method of embodiment 181, wherein the linker has the 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.

Mode 190. 182. The method of embodiment 181, wherein the PEgRNA comprises a spacer, a gRNA core, and an extended arm, the spacer being complementary to a target nucleotide sequence, and the extended arm comprising a reverse transcriptase template encoding a peptide tag.

Aspect 191. A method of preventing or halting the progression of a prion disease by incorporating one or more protective mutations on a PRNP encoded by a target nucleotide sequence by prime editing, the method comprising: (a) (i) a prime editing factor comprising a nucleic acid programmed DNA binding protein (napDNAbp) and a reverse transcriptase; and (ii) a PEgRNA comprising an editing template encoding a functional moiety; (b) a prime editing factor comprising a nucleic acid programmed DNA binding protein (napDNAbp) and a reverse transcriptase; polymerizing the single-stranded DNA sequence; and (c) incorporating the single-stranded DNA sequence in place of the corresponding endogenous strand in the target nucleotide sequence by a DNA repair and/or replication process, the method comprising: A recombinant target nucleotide sequence is generated that encodes a PRNP containing a protective mutation that is resistant to.

Mode 192. 192. The method of embodiment 191, wherein the prion disease is a human prion disease.

Aspect 193. Embodiment 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.

Mode 195. 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 sponges. It is a shape encephalopathy.

Aspect 196. The method of embodiment 191, wherein the wild type PRNP amino acid sequence is SEQ ID NO: 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.

Mode 198. 192. The method of embodiment 191, wherein the fusion protein comprises the amino acid sequence of PE1, PE2, or PE3.

Mode 199. 192. The method of embodiment 191, wherein napDNAbp is Cas9 nickase (nCas9).

Mode 200. The method of embodiment 191, 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.

Mode 201. 192. 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.

Mode 204. Embodiment 202. The method of embodiment 202, wherein the ribonucleotide motif or tag affects the expression level of the RNA of interest.

Mode 205. Embodiment 202 The method of embodiment 202, wherein the ribonucleotide motif or tag affects the trafficking or subcellular localization 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.

Mode 207. Embodiment 202, wherein the PEgRNA is SEQ ID NO: 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.

Mode 208. Embodiment 202, wherein the fusion protein comprises the amino acid sequence of PE1, PE2, or PE3.

Mode 209. Embodiment 202, wherein the napDNAbp is Cas9 nickase (nCas9).

Mode 210. Embodiment 202, 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.

Mode 211. A method of incorporating or deleting a functional moiety on a protein of interest encoded by a target nucleotide sequence by prime editing, the method comprising: (a) converting the target nucleotide sequence into (i) a nucleic acid programmed DNA binding protein (napDNAbp); ) and a prime editing factor comprising reverse transcriptase and (ii) a PEgRNA comprising an editing template encoding the functional moiety or its deletion; (b) a single-stranded DNA sequence encoding the functional moiety or its deletion; and (c) incorporating a single-stranded DNA sequence in place of the corresponding endogenous strand in the target nucleotide sequence by a DNA repair and/or replication process, the method comprising or a deletion thereof, resulting in a recombinant target nucleotide sequence encoding a modified protein, wherein the functional moiety alters the modification state 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.

Mode 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).

Mode 215. The fusion protein of embodiment 213, wherein napDNAbp has nickase activity.

Mode 216. The fusion protein of embodiment 213, wherein napDNAbp is a Cas9 protein or a variant thereof.

Mode 217. The fusion protein of embodiment 213, wherein napDNAbp is nuclease-active Cas9, nuclease-inactive Cas9 (dCas9), or Cas9 nickase (nCas9).

Mode 218. The fusion protein of embodiment 213, wherein napDNAbp is Cas9 nickase (nCas9).

Mode 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.

Mode 220. The fusion protein of embodiment 213, wherein the fusion protein is capable of binding to a target DNA sequence when complexed with PEgRNA.

Mode 221. The fusion protein of embodiment 220, wherein the target DNA sequence comprises a target strand and a complementary non-target strand.

Mode 222. The fusion protein of embodiment 220, wherein the binding of the fusion protein complexed to 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.

Mode 224. The fusion protein of embodiment 223, wherein the complementary non-target strand is nicked to form a prime sequence with a free 3' end.

Mode 225. The fusion protein of embodiment 214, wherein the PEgRNA comprises (a) a guide RNA and (b) an extended arm at the 5' or 3' end of the guide RNA or at an intramolecular location of the guide RNA.

Mode 226. The fusion protein of embodiment 225, wherein the extended 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.

Mode 228. Embodiment 225, wherein the elongated 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 in length. 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 It is.

Mode 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.

Mode 230. The fusion protein of embodiment 227, wherein the single-stranded DNA flap replaces the endogenous DNA sequence with a free 5' end and adjacent to the nick site.

Aspect 231. The fusion protein of embodiment 230, wherein the endogenous DNA sequence having a 5' end is excised by the cell.

Mode 232. The fusion protein of embodiment 230, wherein cellular repair of the single-stranded DNA flap results in the incorporation of the desired nucleotide change, thereby forming the desired product.

Aspect 233. The fusion protein of embodiment 226, wherein the desired nucleotide change 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 The desired nucleotide change is incorporated on an editing window that is between approximately -30 and +100 of the PAM sequence, or 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, Incorporates 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides downstream.

Aspect 234. The fusion protein of embodiment 213, wherein napDNAbp is the amino acid sequence of SEQ ID NO: 18 , or at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 18. It contains an amino acid sequence having the following.

Mode 235. The fusion protein of embodiment 213, 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. amino acid sequences having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity.

Aspect 236. The fusion protein of embodiment 213, wherein the polymerase is reverse transcriptase, 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 237. The fusion protein of embodiment 213, wherein the polymerase is reverse transcriptase, 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. including.

Mode 238. The fusion protein of embodiment 213, wherein the polymerase is a naturally occurring reverse transcriptase from a retrovirus or retrotransposon.

Mode 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 which indicates the presence of an optional linker sequence.

Mode 240. The fusion protein of embodiment 239, wherein the linker sequence comprises the amino acid sequences of SEQ ID NOs: 127, 165-176, 446, 453, and 767-769.

Aspect 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.

Mode 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.

Aspect 243. PEgRNA comprising a guide RNA and at least one nucleic acid extension arm comprising a DNA synthesis template.

Mode 244. The PEgRNA of embodiment 241, wherein the nucleic acid extension arm is located at the 3' or 5' end of the guide RNA or a position within the molecule of the guide RNA, and the nucleic acid extension arm is DNA or RNA.

Mode 245. The PEgRNA of embodiment 242, wherein the PEgRNA is capable of binding napDNAbp and directing napDNAbp to a target DNA sequence.

Mode 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.

Aspect 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.

Mode 248. The PEgRNA of embodiment 247, wherein the nucleic acid extended 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 in length. 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 It is.

Mode 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 in length. nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, or at least 15 nucleotides.

Mode 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 in length. nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, or at least 15 nucleotides.

Mode 251. The PEgRNA of embodiment 243 further comprises at least one additional structure selected from the group consisting of a linker, stem-loop, hairpin, toe-loop, aptamer, or RNA-protein recruitment domain.

Mode 252. The PEgRNA of embodiment 247, wherein the DNA synthesis template encodes a single-stranded DNA flap that is complementary to an endogenous DNA sequence adjacent to the nick site, and the single-stranded DNA flap includes the desired nucleotide change. .

Aspect 253. The 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 located downstream of the nick site. Immediately adjacent.

Mode 254. The PEgRNA of embodiment 253, wherein the endogenous single-stranded DNA with a free 5' end is excised by the cell.

Mode 255. Embodiment 253 PEgRNA, wherein cellular repair of the single-stranded DNA flap results in the incorporation of the desired nucleotide change, thereby forming the desired product.

Aspect 256. PEgRNA of embodiment 243, comprising 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 nucleotide sequence, or SEQ ID NO: 101-104, 181-183, 223-244, 277, 325-334, 336, At least one of the following nucleotide sequences having 85% or at least 90% or at least 95% or at least 98% or at least 99% sequence identity.

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.

Mode 258. The PEgRNA of embodiment 247, wherein the primer binding site hybridizes to the free 3' end of the cut DNA.

Mode 259. The PEgRNA of embodiment 251, wherein the at least one additional structure is located at the 3' or 5' end of the PEgRNA.

Mode 260. A complex comprising the fusion protein of any one of embodiments 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.

Mode 262. Embodiment 260, wherein the PEgRNA is capable of binding napDNAbp and directing napDNAbp to a target DNA sequence.

Mode 263. 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.

Mode 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.

Mode 265. The complex of embodiment 260, wherein the PEgRNA is SEQ ID NO: 101-104, 181-183, 223-244, 277, 325-334, 336, 338, 340, 342, 344, 346, 348, 350, 352, Nucleotide sequences of 354, 356, 358, 360, 362, 364, 366, 368, 499-505, 735-761, 776-777, or SEQ ID NO: 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 nucleotide sequences having at least 85% or at least 90% or at least 95% or at least 98% or at least 99% sequence identity with one.

Mode 266. The conjugate 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.

Mode 267. Embodiment 264. The complex of embodiment 264, wherein the primer binding site hybridizes to the free 3' end of the cut DNA.

Mode 268. complex containing napDNAbp and PEgRNA.

Mode 269. Embodiment 268, wherein napDNAbp is Cas9 nickase.

Mode 270. Embodiment 268, wherein the napDNAbp comprises the amino acid sequence of SEQ ID NO: 18.

Mode 271. Embodiment 268, 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. amino acid sequences having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity.

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.

Aspect 273. Embodiment 268. The complex of embodiment 268, wherein the PEgRNA is capable of directing napDNAbp to a target DNA sequence.

Mode 274. Embodiment 272, wherein the target DNA sequence comprises a target strand and a complementary non-target strand, and the PEgRNA spacer sequence hybridizes to the target strand to form an RNA-DNA hybrid and an R-loop.

Mode 275. The complex of embodiment 273, wherein the nucleic acid extension arm comprises (i) a DNA synthesis template and (ii) a primer binding site.

Mode 276. The complex of embodiment 269, wherein the PEgRNA is SEQ ID NO: 101-104, 181-183, 223-244, 277, 325-334, 336, 338, 340, 342, 344, 346, 348, 350, 352, Nucleotide sequences of 354, 356, 358, 360, 362, 364, 366, 368, 499-505, 735-761, 776-777, or SEQ ID NO: 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 nucleotide sequences having at least 85% or at least 90% or at least 95% or at least 98% or at least 99% sequence identity with one.

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.

Mode 278. The complex of embodiment 276, wherein the primer binding site hybridizes to the free 3' end of the cut DNA.

Mode 279. The conjugate 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.

Mode 280. A polynucleotide encoding the fusion protein of any of embodiments 213-242.

Mode 281. A vector comprising the polynucleotide of embodiment 280.

Mode 282. A cell comprising the fusion protein of any of embodiments 213-242 and PEgRNA bound to napDNAbp of the fusion protein.

Mode 283. A cell comprising the complex of any one 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.

Mode 285. A pharmaceutical composition comprising: (i) a conjugate of embodiments 260-279, (ii) a polymerase provided in trans; and (iii) a pharmaceutically acceptable excipient.

Mode 286. A kit comprising: (i) a nucleic acid sequence encoding the fusion protein of any one of embodiments 213 to 242; and (ii) a nucleic acid construct comprising a promoter driving expression of the sequence of (i). include.

Mode 287. A method for incorporating desired nucleotide changes into a double-stranded DNA sequence, the method comprising:

(i) contacting the double-stranded DNA sequence with a complex comprising a fusion protein and PEgRNA, the fusion protein comprising napDNAbp and a polymerase, and the PEgRNA comprising a DNA synthesis template containing the desired nucleotide changes and a primer binding site;

(ii) nicking the double-stranded DNA sequence, thereby generating free single-stranded DNA with 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 the strand of DNA from the 3' end hybridized to the primer binding site, thereby producing a single-stranded DNA flap that is complementary to the DNA synthesis template and contains the desired nucleotide changes; ;

(v) replacing the endogenous DNA strand adjacent to the cut site by a single-stranded DNA flap, thereby incorporating the desired nucleotide change into the double-stranded DNA sequence;
including.

Mode 288. Embodiment 287, wherein (v) the replacing step comprises: (i) hybridizing the single-stranded DNA flap to an 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 the desired product containing the desired nucleotide changes in both strands of the DNA.

Mode 289. Embodiment 288, wherein the desired nucleotide change is a single nucleotide substitution, deletion, or insertion.

Mode 290. Embodiment 289. The method of embodiment 289, wherein the single nucleotide substitution is a transition or transversion.

Mode 291. 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) Substitution of A from T, (6) Substitution of C from T, (7) Substitution of G from C, (8) Substitution of T from C, (9) Substitution of A from C, (10) Substitution of A substitution of T, (11) substitution of A to G, or (12) substitution of A to C.

Mode 292. 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 T:A base pair; :C base pair becomes C:G base pair, (4) T:A base pair becomes G:C base pair, (5) T:A base pair becomes A:T base pair, (6) T:A base pair to C:G base pair, (7) C:G base pair to G:C base pair, (8) C:G base pair to T:A base pair, C:G base pair to A: T base pair, (10) A:T base pair to T:A base pair, (11) A:T base pair to G:C base pair, or (12) A:T base pair to C:G. Convert to base pairs.

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.

Mode 294. Embodiment 288. The method of embodiment 288, wherein the desired nucleotide change modifies a disease-associated gene.

Mode 295. Embodiment 294, wherein the disease-associated gene is: adenosine deaminase (ADA) deficiency; alpha-1 antitrypsin deficiency; cystic fibrosis; Duchenne muscular dystrophy; galactosemia; hemochromatosis; Huntington's disease; maple Syrupuria; Marfan syndrome; type 1 neurofibromatosis; congenital nail hypertrophy; phenylketonuria; severe combined immunodeficiency; sickle cell disease; Smith-Lemli-Opitz syndrome; trinucleotide repeat disorder; prion disease associated with a single gene disorder selected from the group consisting of; and Tay-Sachs disease.

Mode 296. 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.

Mode 297. Embodiment 287, wherein the napDNAbp is nuclease-inactive Cas9 (dCas9), Cas9 nickase (nCas9), or nuclease-active Cas9.

Mode 298. Embodiment 287, wherein napDNAbp has the amino acid sequence of SEQ ID NO: 18, or at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 18. Contains an amino acid sequence with

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.

Mode 300. The method of embodiment 287, wherein the polymerase is a reverse transcriptase, SEQ ID NOs: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, Contains any one of the amino acid sequences 662, 700, 701-716, 739-741, and 766.

Aspect 301. The method of embodiment 287, wherein the polymerase is a reverse transcriptase, and SEQ ID NOs: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, An amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with any one amino acid sequence of 662, 700, 701-716, 739-741, and 766. include.

Aspect 302. Embodiment 287, wherein the PEgRNA comprises a nucleic acid extension arm at the 3' or 5' end of the guide RNA or at an intramolecular location, the extension arm comprising a DNA synthesis template sequence and a primer binding site.

Aspect 303. Embodiment 302, wherein the extended 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 in length. , 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 be.

Aspect 304. Embodiment 287, wherein the PEgRNA is SEQ ID NO: 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.

Aspect 305. A method for introducing one or more changes in the nucleotide sequence of a DNA molecule at a target locus, the method comprising:

(i) contacting the DNA molecule with a nucleic acid programmed DNA binding protein (napDNAbp) and a PEgRNA that targets napDNAbp to the target locus, and the PEgRNA contains 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 onto the corresponding endogenous DNA, thereby introducing one or more changes in the nucleotide sequence of the DNA molecule at the target locus;
including.

Aspect 306. Embodiment 305. The method of embodiment 305, wherein the one or more changes in the nucleotide sequence comprises a transition.

Aspect 307. 306. 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.

Aspect 308. Embodiment 305, wherein the one or more changes in the nucleotide sequence comprises a transversion.

Aspect 309. Embodiment 308, wherein the transversion is: (a) T to A; (b) T to G; (c) C to G; (d) C to A; (e) A to T; (f) (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.

Aspect 311. Embodiment 305, wherein the one or more changes in the nucleotide sequence are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, Contains insertions or deletions of 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides.

Aspect 312. Embodiment 305. The method of embodiment 305, wherein the one or more changes in the nucleotide sequence comprises correction of a disease-associated gene.

Aspect 313. The method of embodiment 312, wherein the disease-associated gene is: adenosine deaminase (ADA) deficiency; alpha-1 antitrypsin deficiency; cystic fibrosis; Duchenne muscular dystrophy; galactosemia; hemochromatosis; Huntington's disease; maple Syrupuria; Marfan syndrome; type 1 neurofibromatosis; congenital nail hypertrophy; phenylketonuria; severe combined immunodeficiency; sickle cell disease; Smith-Lemli-Opitz syndrome; trinucleotide repeat disorder; prion disease associated with a single gene disorder selected from the group consisting of; and Tay-Sachs disease.

Aspect 314. Embodiment 312. The method of embodiment 312, 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.

Aspect 315. Embodiment 305, wherein the napDNAbp is nuclease-active Cas9 or a variant thereof.

Aspect 316. Embodiment 305, wherein the napDNAbp is nuclease-inactive Cas9 (dCas9) or Cas9 nickase (nCas9) or a variant thereof.

Aspect 317. Embodiment 305, wherein napDNAbp is the amino acid sequence of SEQ ID NO: 18, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO: 18. including.

Aspect 318. Embodiment 305, wherein napDNAbp has 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. Comprising amino acid sequences with at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity.

Aspect 319. Embodiment 305. The method of embodiment 305, wherein the reverse transcriptase is introduced in trans.

Mode 320. Embodiment 305, wherein the napDNAbp comprises a fusion to reverse transcriptase.

Aspect 321. Embodiment 305, wherein the reverse transcriptase is SEQ ID NO: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, 662, 700 , 701-716, 739-741, and 766.

Aspect 322. Embodiment 305, wherein the reverse transcriptase is SEQ ID NO: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, 662, 700 , 701-716, 739-741, and 766.

Aspect 323. Embodiment 305. 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.

Aspect 324. Embodiment 323. The method of embodiment 323, wherein the nuclease is provided and 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.

Aspect 326. Embodiment 305. The method of embodiment 305, wherein forming an exposed 3' end of the DNA strand at the target locus comprises introducing a replication error.

Aspect 327. Embodiment 305. The method of embodiment 305, wherein the step of contacting the DNA molecule with napDNAbp and guide RNA forms an R-loop.

Aspect 328. Embodiment 327. The method of embodiment 327, wherein the DNA strand from which the exposed 3' end is formed is on the R-loop.

Aspect 329. Embodiment 315. The method of embodiment 315, wherein the PEgRNA comprises an extended arm that includes a reverse transcriptase (RT) template sequence and a primer binding site.

Aspect 330. 329. The method of embodiment 329, wherein the elongated arm is at the 3' end of the guide RNA, the 5' end of the guide RNA, or an intramolecular position of the guide RNA.

Aspect 331. Embodiment 305, wherein the PEgRNA further comprises at least one additional structure selected from the group consisting of a linker, stem-loop, hairpin, toe-loop, aptamer, or RNA-protein recruitment domain.

Aspect 332. Embodiment 305, wherein the PEgRNA further comprises a homology arm.

Aspect 333. Embodiment 305. The method of embodiment 305, wherein the RT template sequence is homologous to the corresponding endogenous DNA.

Aspect 334. A method for introducing one or more changes in the nucleotide sequence of a DNA molecule at a target locus by primed reverse transcription to a primed target, the method comprising: (a) converting the DNA molecule at the target locus into (i) a nucleic acid molecule; contacting a fusion protein comprising a programmed DNA binding protein (napDNAbp) and a reverse transcriptase and (ii) a guide RNA comprising an RT template containing the desired nucleotide changes; (b) a reverse primed target of the RT template; (c) incorporating the desired nucleotide changes onto the DNA molecule at the target locus by DNA repair and/or replication processes.

Aspect 335. Embodiment 334. The method of embodiment 334, wherein the RT template is located at the 3' end of the guide RNA, at the 5' end of the guide RNA, or at an intramolecular location of the guide RNA.

Aspect 336. Embodiment 334. The method of embodiment 334, wherein the desired nucleotide change comprises a transition, transversion, insertion, or deletion, or any combination thereof.

Aspect 337. Embodiment 334, wherein the desired nucleotide change is 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. including.

Aspect 338. 335. The method of claim 334, wherein the desired nucleotide change is (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.

Aspect 339. 334. The method of embodiment 334, 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 T:A base pair; :C base pair becomes C:G base pair, (4) T:A base pair becomes G:C base pair, (5) T:A base pair becomes A:T base pair, (6) T:A base pair to C:G base pair, (7) C:G base pair to G:C base pair, (8) C:G base pair to T:A base pair, (9) C:G base pair. into an A:T base pair, (10) an A:T base pair into a T:A base pair, (11) an A:T base pair into a G:C base pair, or (12) an A:T base pair. This includes changing to a C:G base pair.

Embodiment 340. A polynucleotide encoding a PEgRNA according to any one of embodiments 243 to 259.

Aspect 341. A vector comprising the polynucleotide of aspect 340.

Aspect 342. A cell comprising the vector of embodiment 341.

Aspect 343. The fusion protein of embodiment 213, wherein the polymerase is an error-prone reverse transcriptase.

Aspect 344. A method for mutagenizing a DNA molecule at a target locus by primed reverse transcription, the method comprising: (a) converting the DNA molecule at the target locus to (i) a nucleic acid programmed DNA binding protein (napDNAbp); contacting a fusion protein comprising an error-prone reverse transcriptase and (ii) a guide RNA comprising an RT template comprising the desired nucleotide changes; (b) performing primed reverse transcription on a primed target of the RT template; producing mutagenized single-stranded DNA; and (c) incorporating the mutagenized single-stranded DNA onto a DNA molecule at a target locus by a DNA repair and/or replication process.

Aspect 345. The method of any preceding embodiment, wherein the fusion protein comprises the amino acid sequence of PE1, PE2, or PE3.

Aspect 346. The method of any preceding embodiment, wherein napDNAbp is Cas9 nickase (nCas9).

Aspect 347. Embodiment 344, 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 .

Aspect 348. Embodiment 344. The method of embodiment 344, wherein the guide RNA comprises SEQ ID NO: 222.

Aspect 349. Embodiment 344 The method of embodiment 344, wherein the step of (b) performing primed reverse transcription on a primed target comprises a 3' end primer at the target locus capable of priming reverse transcription by annealing to a primer binding site on a guide RNA. Including generating associative arrays.

Mode 350. A method for replacing a trinucleotide repeat expansion mutation on a target DNA molecule by a healthy sequence containing a healthy number of repeat trinucleotides, the method comprising: (a) converting the DNA molecule at the target locus to (i) a nucleic acid programmed contacting a fusion protein containing a DNA binding protein (napDNAbp) and a polymerase; and (ii) a DNA synthesis template containing a replacement sequence and a PEgRNA containing a primer binding site; producing single-stranded DNA; and (c) incorporating single-stranded DNA onto a DNA molecule at a target locus by a DNA repair and/or replication process.

Aspect 351. Embodiment 350, wherein the fusion protein comprises the amino acid sequence of PE1, PE2, or PE3.

Aspect 352. Embodiment 350, wherein the napDNAbp is Cas9 nickase (nCas9).

Aspect 353. Embodiment 350, 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 354. The method of embodiment 350, wherein the guide RNA comprises SEQ ID NO: 222.

Aspect 355. Embodiment 350, wherein (b) performing prime editing generates a 3' end primer binding sequence at the target locus that is capable of priming a polymerase by annealing to a primer binding site on the guide RNA. include.

Aspect 356. Embodiment 350, wherein the trinucleotide repeat expansion mutation is associated with Huntington's disease, Fragile X syndrome, or Friedreich's ataxia.

Aspect 357. Embodiment 350, wherein the trinucleotide repeat expansion comprises a CAG triplet repeat unit.

Aspect 358. Embodiment 350, wherein the trinucleotide repeat expansion comprises a GAA triplet repeat unit.

Aspect 359. A method of incorporating a functional moiety onto a protein of interest encoded by a target nucleotide sequence by prime editing, the method comprising: (a) converting the target nucleotide sequence to (i) a nucleic acid programmed DNA binding protein (napDNAbp) and a polymerase; (b) polymerizing the single-stranded DNA sequence encoding the functional moiety; and (c) DNA repair and/or or incorporating, by a replication process, a single-stranded DNA sequence in place of the corresponding endogenous strand in the target nucleotide sequence, wherein the method generates a recombinant target nucleotide sequence encoding a fusion protein comprising the protein of interest and the functional moiety. arise.

Mode 360. Embodiment 359. The method of embodiment 359, wherein the functional moiety is a peptide tag.

Aspect 361. 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 NOs:252-262); Myc-tag (SEQ ID NO:6); NE-tag (SEQ ID NO:264); Rho1D4-tag (SEQ ID NO:265); S-tag (SEQ ID NO:266); SBP-tag (SEQ ID NO:267); Softag-1 (SEQ ID NO:268); Softag-2 (SEQ ID NO:269); Spot-tag (SEQ ID NO:270); Strep-tag (SEQ ID NO:271); TC-tag (SEQ ID NO:272); Ty-tag (SEQ ID NO:273); V5-tag (SEQ ID NO:3); VSV-tag (SEQ ID NO:275); and Xpress-tag (SEQ ID NO:276).

Aspect 363. 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-G (SEQ ID NO: 275). selected.

Embodiment 364. The method of embodiment 359, wherein the functional moiety is an immune epitope.

Mode 365. Embodiment 364, 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); Class I HLA hemagglutinin epitope for class I HLA (SEQ ID NO: 416); neuraminidase epitope for class I 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).

Aspect 366. Embodiment 359. The method of embodiment 359, wherein the functional moiety alters localization of the protein of interest.

Aspect 367. Embodiment 359. The method of embodiment 359, wherein the functional moiety is a degradation tag, so that the degradation rate of the protein of interest is altered.

Mode 368. Embodiment 367. The method of embodiment 367, wherein the degradation tag results in elimination of the tagged protein.

Aspect 369. Embodiment 359. The method of embodiment 359, wherein the functional moiety is a small molecule binding domain.

Mode 370. Embodiment 359. The method of embodiment 359, wherein the small molecule binding domain is FKBP12 of SEQ ID NO: 488.

Aspect 371. Embodiment 359. The method of embodiment 359, wherein the small molecule binding domain is FKBP12-F36V of SEQ ID NO: 489.

Aspect 372. The method of embodiment 359, wherein the small molecule binding domain is a cyclophilin of SEQ ID NOs: 490 and 493-494.

Aspect 373. Embodiment 359. The method of embodiment 359, wherein the small molecule binding domain is incorporated onto two or more proteins of interest.

Aspect 374. Embodiment 373. The method of embodiment 373, wherein the two or more proteins of interest can be dimerized by contacting with a small molecule.

Embodiment 375. The method of embodiment 369, wherein the small molecule is a dimer of a small molecule selected from the group consisting of the compounds disclosed in embodiment 163 of group 1.

Aspect 376. A method of incorporating an immune epitope onto a protein of interest encoded by a target nucleotide sequence by prime editing, the method comprising: (a) converting the target nucleotide sequence to (i) a nucleic acid programmed DNA binding protein (napDNAbp) and a polymerase; (b) polymerizing a single-stranded DNA sequence encoding an immune epitope; and (c) DNA repair and/or incorporating, by a replication process, a single-stranded DNA sequence in place of the corresponding endogenous strand in the target nucleotide sequence, the method yielding a recombinant target nucleotide sequence encoding a fusion protein comprising the protein of interest and the immune epitope. .

Aspect 377. Embodiment 376, 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); Class I HLA hemagglutinin epitope for class I HLA (SEQ ID NO: 416); neuraminidase epitope for class I 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).

Aspect 378. Embodiment 376. The method of embodiment 376, wherein the fusion protein comprises the amino acid sequence of PE1, PE2, or PE3.

Aspect 379. Embodiment 376, wherein the napDNAbp is Cas9 nickase (nCas9).

Mode 380. Embodiment 376, 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.

Aspect 381. 376. The method of embodiment 376, wherein the PEgRNA is , 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.

Aspect 383. Embodiment 382. The method of embodiment 382, further comprising performing the method on a second protein of interest.

Aspect 384. Embodiment 383. 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 a respective dimerization domain of the proteins.

Aspect 385. The method of embodiment 382, wherein the small molecule binding domain is FKBP12 of SEQ ID NO: 488.

Aspect 386. The method of embodiment 382, wherein the small molecule binding domain is FKBP12-F36V of SEQ ID NO: 489.

Aspect 387. The method of embodiment 382, wherein the small molecule binding domain is a cyclophilin of SEQ ID NOs: 490 and 493-494.

Mode 388. Embodiment 382. 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.

Aspect 389. Embodiment 382, wherein the fusion protein comprises the amino acid sequence of PE1, PE2, or PE3.

Aspect 390. 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.

Aspect 392. Embodiment 382, wherein the PEgRNA is SEQ ID NO: 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.

Aspect 393. A method of incorporating a peptide tag or epitope onto a protein using prime editing, comprising: a prime editing agent configured to insert into a target nucleotide sequence encoding a protein a second nucleotide sequence encoding a peptide tag; contacting the recombinant nucleotide sequence with a construct so that the peptide tag and protein are expressed from the recombinant nucleotide sequence as a fusion protein.

Aspect 394. Embodiment 383. The method of embodiment 383, wherein the peptide tag is used for protein purification and/or detection.

Aspect 395. Embodiment 383, wherein the peptide tag is polyhistidine (e.g., HHHHHH) (SEQ ID NO: 252-262), FLAG (e.g., DYKDDDDK) (SEQ ID NO: 2), V5 (e.g., GKPIPNPLLGLLDST) (SEQ ID NO: 3) , GCN4, HA (eg, YPYDVPDYA) (SEQ ID NO: 5), Myc (eg, EQKLISEED) (SEQ ID NO: 6), or GST.

Aspect 396. Embodiment 383, wherein the peptide tag is an amino acid selected from the group consisting of SEQ ID NOs: 1-6, 245-249, 252-262, 264-273, 275-276, 281, 278-288, and 622. It has an array.

Aspect 397. Embodiment 383. The method of embodiment 383, wherein the peptide tag is fused to the protein by a linker.

Aspect 398. 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.

Mode 400. Embodiment 383, wherein the prime editing factor construct comprises SEQ ID NOs: 101-104, 181-183, 223-244, 277, 325-334, 336, 338, 340, 342, 344, 346, 348, 350, Contains PEgRNA containing nucleotide sequences 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.

Aspect 402. Embodiment 383, wherein the PEgRNA comprises a spacer, a gRNA core, and an extended arm, the spacer being complementary to a target nucleotide sequence, and the extended arm comprising a reverse transcriptase template encoding a peptide tag.

Aspect 403. A method of preventing or halting the progression of a prion disease by incorporating one or more protective mutations on a PRNP encoded by a target nucleotide sequence by prime editing, the method comprising: (a) (i) contacting with a prime editing factor comprising a nucleic acid programmed DNA binding protein (napDNAbp) and a polymerase and (ii) a PEgRNA comprising an editing template encoding a functional moiety; (b) a single strand encoding a protective mutation; polymerizing the DNA sequence; and (c) incorporating the single-stranded DNA sequence in place of the corresponding endogenous strand in the target nucleotide sequence by a DNA repair and/or replication process, wherein the method is resistant to misfolding. A recombinant target nucleotide sequence is generated that encodes a PRNP containing a protective mutation that is resistant.

Aspect 404. Embodiment 403. The method of embodiment 403, wherein the prion disease is a human prion disease.

Aspect 405. Embodiment 403. The method of embodiment 403, wherein the prion disease is an animal prion disease.

Aspect 406. Embodiment 404, wherein the prion disease is Creutzfeldt-Jakob disease (CJD), variant Creutzfeldt-Jakob disease (vCJD), Gerstmann-Strausler-Scheinker syndrome, fatal familial insomnia, or Kuru disease. It is.

Aspect 407. 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. It is encephalopathy.

Aspect 408. The method of embodiment 403, wherein the wild type PRNP amino acid sequence is SEQ ID NO: 291-292.

Aspect 409. 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 wherein the modified PRNP protein is resistant to misfolding. be.

Aspect 410. Embodiment 403, wherein the fusion protein comprises the amino acid sequence of PE1, PE2, or PE3.

Aspect 411. Embodiment 403, wherein the napDNAbp is Cas9 nickase (nCas9).

Aspect 412. Embodiment 403, 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.

Aspect 413. Embodiment 403, wherein the PEgRNA is SEQ ID NO. , 356, 358, 360, 362, 364, 366, 368, 499-505, 735-761, 776-777.

Aspect 414. A method of incorporating a ribonucleotide motif or tag onto an RNA of interest encoded by a target nucleotide sequence by prime editing, the method comprising: (a) converting the target nucleotide sequence into (i) a nucleic acid programmed DNA binding protein (napDNAbp); and a prime editing agent comprising a polymerase; and (ii) contacting PEgRNA comprising an editing template encoding a ribonucleotide motif or tag; (b) polymerizing a single-stranded DNA sequence encoding a ribonucleotide motif or tag; and (c) incorporating a single-stranded DNA sequence in place of the corresponding endogenous strand in the target nucleotide sequence by a DNA repair and/or replication process, the method comprising A recombinant target nucleotide sequence is generated that encodes the desired RNA.

Aspect 415. Embodiment 414. The method of embodiment 414, wherein the ribonucleotide motif or tag is the detection moiety.

Aspect 416. Embodiment 414. The method of embodiment 414, wherein the ribonucleotide motif or tag affects the expression level of the RNA of interest.

Aspect 417. Embodiment 414. The method of embodiment 414, wherein the ribonucleotide motif or tag affects trafficking or subcellular localization of the RNA of interest.

Aspect 418. Embodiment 414, wherein the ribonucleotide motif or tag is SV40 type 1, SV40 type 2, SV40 type 3, hGH, BGH, rbGlob, TK, MALAT1 ENE-mascRNA, KSHV PAN ENE, Smbox/U1 snRNA box, selected from the group consisting of 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.

Aspect 420. Embodiment 414. The method of embodiment 414, wherein the fusion protein comprises the amino acid sequence of PE1, PE2, or PE3.

Aspect 421. Embodiment 414. The method of embodiment 414, wherein napDNAbp is Cas9 nickase (nCas9).

Aspect 422. Embodiment 414, 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 423. A method for inserting or deleting 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 an editing template encoding the functional moiety or deletion; (b) polymerizing a single-stranded DNA sequence encoding the functional moiety 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 moiety or deletion, the functional moiety altering the modification or localization state of the protein.

Aspect 424. Embodiment 423. 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.

Aspect 425. A fusion protein comprising a nucleic acid programmed DNA binding protein (napDNAbp) domain and a domain containing RNA-dependent DNA polymerase activity.

Aspect 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 desired nucleotide changes onto the target sequence.

Aspect 427. The fusion protein of embodiment 425, wherein the napDNAbp domain has nickase activity.

Aspect 428. The fusion protein of embodiment 425, wherein the napDNAbp domain is a Cas9 protein or a variant thereof.

Aspect 429. The fusion protein of embodiment 425, wherein the napDNAbp domain is nuclease-active Cas9, nuclease-inactive Cas9 (dCas9), or Cas9 nickase (nCas9).

Aspect 430. The fusion protein of embodiment 425, wherein the napDNAbp domain is Cas9 nickase (nCas9).

Aspect 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.

Aspect 432. The fusion protein of embodiment 425, wherein the domain comprising RNA-dependent DNA polymerase activity is reverse transcriptase, and 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 433. The fusion protein of embodiment 425, wherein the domain comprising RNA-dependent DNA polymerase activity is reverse transcriptase, and 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. Domains containing amino acid sequences with % sequence identity and optionally containing an RNA-dependent DNA polymerase are error-prone.

Aspect 434. The fusion protein of embodiment 425, wherein the domain comprising RNA-dependent DNA polymerase activity is a naturally occurring reverse transcriptase from a retrovirus or retrotransposon.

Aspect 435. Embodiment 425. The fusion protein of embodiment 425, wherein the fusion protein is capable of binding to a target DNA sequence when complexed with PEgRNA.

Aspect 436. Embodiment 435. The fusion protein of embodiment 435, wherein the target DNA sequence comprises a target strand and a complementary non-target strand.

Aspect 437. The fusion protein of embodiment 435, wherein the binding of the fusion protein complexed to PEgRNA forms an R-loop.

Aspect 438. The fusion protein of embodiment 437, wherein the R-loop comprises (i) an RNA-DNA hybrid comprising PEgRNA and a target strand and (ii) a complementary non-target strand.

Aspect 439. The fusion protein of embodiment 437, wherein the target or complementary non-target strand is nicked to form a prime sequence with a free 3' end.

Aspect 440. The fusion protein of embodiment 439, wherein the nick site is upstream of the PAM sequence on the target strand.

Aspect 441. The fusion protein of embodiment 439, wherein the nick site is upstream of the PAM sequence on the non-target strand.

Aspect 442. The fusion protein of embodiment 439, wherein the nick site is -1, -2, -3, -4, -5, -6, -7, -8, or - relative to the 5' end of the PAM sequence. It is 9.

Aspect 443. The fusion protein of embodiment 426, wherein the PEgRNA comprises a guide RNA and at least one nucleic acid extension arm.

Aspect 444. The fusion protein of embodiment 443, wherein the extended arm is located at the 5' or 3' end of the guide RNA or within the molecule of the guide RNA.

Aspect 445. The fusion protein of embodiment 443, wherein the extended arm comprises (i) a DNA synthesis template sequence containing a desired nucleotide change and (ii) a primer binding site.

Aspect 446. Embodiment 445. The fusion protein of embodiment 445, wherein the DNA synthesis template sequence encodes a single-stranded DNA flap that is complementary to an endogenous DNA sequence adjacent to the nick site, and wherein the single-stranded DNA flap comprises a desired nucleotide change. including.

Aspect 447. Embodiment 443, wherein the elongated arm comprises 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.

Aspect 448. The fusion protein of embodiment 443, wherein the single-stranded DNA flap hybridizes to the endogenous DNA sequence adjacent to the nick site, thereby incorporating the desired nucleotide change on 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.

Mode 450. The fusion protein of embodiment 446, wherein the endogenous DNA sequence having a 5' end is excised by the cell.

Aspect 451. The fusion protein of embodiment 446, wherein the endogenous DNA sequence having a 5' end is excised by a flap endonuclease.

Aspect 452. The fusion protein of embodiment 448, wherein cellular repair of the single-stranded DNA flap incorporates the desired nucleotide change onto the non-target strand, thereby forming the desired product.

Aspect 453. 449. The fusion protein of embodiment 449, wherein the desired nucleotide change 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 approximately -30 and +100 of the PAM array.

Aspect 454. 449. 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 of the nick site. , 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.

Aspect 455. The fusion protein of embodiment 425, wherein napDNAbp is the amino acid sequence of SEQ ID NO: 18 , or at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 18. It contains an amino acid sequence having the following.

Aspect 456. The fusion protein of embodiment 425, 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. amino acid sequences having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity.

Aspect 457. The fusion protein of any one of the preceding embodiments, wherein the fusion protein has the structure NH2-[napDNAbp]-[domain comprising RNA-dependent DNA polymerase activity]-COOH; or NH2-[domain comprising RNA-dependent DNA polymerase activity]-COOH; The domain ]-[napDNAbp]-COOH, with each "]-[" indicating the presence of an optional linker sequence.

Aspect 458. The fusion protein of embodiment 457, wherein the linker sequence comprises the amino acid sequences of SEQ ID NOs: 127, 165-176, 446, 453, and 767-769.

Aspect 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.

Mode 460. 459. 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.

Aspect 461. A complex comprising the fusion protein of any of embodiments 425-460 and PEgRNA, wherein the PEgRNA directs the fusion protein to a target DNA sequence for prime editing.

Embodiment 462. The complex of embodiment 461, wherein the PEgRNA comprises a guide RNA and a nucleic acid extension arm at the 3' or 5' end of the guide RNA or at an intramolecular position of the guide RNA.

Aspect 463. Embodiment 462, wherein the PEgRNA is capable of binding napDNAbp and directing napDNAbp to a target DNA sequence.

Aspect 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.

Aspect 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.

Aspect 466. The complex of embodiment 464, wherein the PEgRNA is SEQ ID NO: 101-104, 181-183, 223-244, 277, 325-334, 336, 338, 340, 342, 344, 346, 348, 350, 352, Nucleotide sequences of 354, 356, 358, 360, 362, 364, 366, 368, 499-505, 735-761, 776-777, or SEQ ID NO: 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 nucleotide sequences having at least 85% or at least 90% or at least 95% or at least 98% or at least 99% sequence identity with one.

Aspect 467. The conjugate of embodiment 465, wherein the DNA synthesis template comprises a nucleotide sequence that is at least 80% or 85% or 90% or 95% or 99% identical to the endogenous DNA target.

Mode 468. Embodiment 465. 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 embodiment 461, wherein napDNAbp is Cas9 nickase.

Mode 470. The conjugate of embodiment 461, wherein napDNAbp has the amino acid sequence of SEQ ID NO: 18, or an amino acid having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO: 18. Contains arrays.

Aspect 471. Embodiment 461, 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. amino acid sequences having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity.

Aspect 472. 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 a position within the molecule of the guide RNA.

Aspect 473. The conjugate of embodiment 465, wherein the DNA synthesis template comprises a nucleotide sequence that is at least 80% or 85% or 90% or 95% or 99% identical to the endogenous DNA target.

Aspect 474. Embodiment 465. The complex of embodiment 465, wherein the primer binding site hybridizes to the free 3' end of the cut DNA.

Aspect 475. The conjugate of embodiment 462, wherein the PEgRNA further comprises at least one additional structure selected from the group consisting of a linker, stem-loop, hairpin, toe-loop, aptamer, or RNA-protein recruitment domain.

Aspect 476. A polynucleotide encoding the fusion protein of any of embodiments 425-461.

Aspect 477. A polynucleotide encoding PEgRNA of any of the above embodiments.

Aspect 478. Embodiment 476 A vector comprising the polynucleotide of embodiment 476, wherein expression of the fusion protein is under the control of a promoter.

Aspect 479. A vector comprising the polynucleotide of embodiment 477, wherein expression of PEgRNA is under the control of a promoter.

Mode 480. The vector of embodiment 479, wherein the promoter is the U6 promoter.

Aspect 481. The vector of embodiment 479, wherein the promoter is a CMV promoter.

Aspect 482. Embodiment 480, wherein the PEgRNA is engineered to remove one or more repeat clusters of T on the extended arm to improve transcription efficiency by the U6 promoter.

Aspect 483. The vector of embodiment 482, wherein the cluster of one or more repeats of T to be removed comprises at least 3 Ts, at least 4 Ts, at least 5 Ts, at least 6 Ts, at least 7 Ts, at least 8 Ts. T, at least 9 T, at least 10 T, at least 11 T, at least 12 T, at least 13 T, at least 14 T, at least 15 T, at least 16 T, at least 17 T, at least 18 T, at least 19 T, or at least 20 T.

Aspect 484. A cell comprising the fusion protein of any of embodiments 425-460 and PEgRNA bound to napDNAbp of the fusion protein.

Aspect 485. A cell comprising the complex of any one of embodiments 461-475.

Aspect 486. A pharmaceutical composition comprising: (i) a fusion protein of any of embodiments 425-460, a conjugate of embodiments 461-475, a polynucleotide of embodiments 476-477, or a vector of embodiments 478-483; and (ii) a pharmaceutical composition. Contains legally acceptable excipients.

Aspect 487. A pharmaceutical composition comprising: (i) a conjugate of embodiments 461-475, (ii) a polymerase provided in trans; and (iii) a pharmaceutically acceptable excipient.

Aspect 488. A kit for prime editing, comprising: (i) a nucleic acid molecule encoding a fusion protein of any one of embodiments 425-460; and (ii) encoding a PEgRNA capable of directing the fusion protein to a target DNA site. The nucleic acid molecules of (i) and (ii) may be contained on a single DNA construct or on separate DNA constructs.

Aspect 489. The kit of embodiment 488, wherein the nucleic acid molecule of (i) further comprises a promoter that drives expression of the fusion protein.

Aspect 490. The kit of embodiment 488, wherein the nucleic acid molecule of (ii) further comprises a promoter that drives expression of PEgRNA.

Aspect 491. The kit of aspect 490, wherein the promoter is a U6 promoter.

Aspect 492. The kit of embodiment 490, wherein the promoter is a CMV promoter.

Aspect 493. The fusion protein of embodiment 457, wherein the linker sequence is SEQ ID NO: 174 (1×SGGS), 446 (2×SGGS), 3889 (3×SGGS), 171 (1×XTEN), 3968 (1×EAAAK), 3969 (2×EAAAK), and 3970 (3×EAAAK) amino acid sequences.

Group B. PE guide and design method aspect 1. PEgRNA comprising a guide RNA and at least one nucleic acid extension arm comprising a DNA synthesis template.

Aspect 2. PEgRNA of embodiment 1, wherein the nucleic acid extension arm is at the 3' or 5' end of the guide RNA or at a position within the molecule of the guide RNA, and the nucleic acid extension arm is DNA or RNA.

Aspect 3. PEgRNA of embodiment 1, wherein the PEgRNA is capable of binding to napDNAbp and directing 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. PEgRNA of embodiment 3, wherein the guide RNA hybridizes to the target strand to form an RNA-DNA hybrid and an R-loop.

Aspect 6. The PEgRNA of embodiment 1, wherein at least one nucleic acid extension arm further comprises a primer binding site.

Aspect 7. PEgRNA of embodiment 1, wherein the nucleic acid extending arm comprises 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.

Aspect 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 in length. nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, or at least 15 nucleotides.

Aspect 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 in length. nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, or at least 15 nucleotides.

Aspect 10. The PEgRNA of embodiment 1 further comprises 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.

Aspect 11. The PEgRNA of embodiment 1, wherein the DNA synthesis template encodes a single-stranded DNA flap that is complementary to an endogenous DNA sequence adjacent to the nick site, and the single-stranded DNA flap contains a desired nucleotide change. .

Aspect 12. PEgRNA of embodiment 11, 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 located downstream of the nicked site. Immediately adjacent.

Aspect 13. PEgRNA of embodiment 11, wherein the endogenous single-stranded DNA with a free 5' end is excised by the cell.

Aspect 14. PEgRNA of embodiment 13, wherein cellular repair of the single-stranded DNA flap results in the incorporation of the desired nucleotide change, thereby forming the desired product.

Aspect 15. PEgRNA of embodiment 1, comprising 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 nucleotide sequence, or SEQ ID NO: 101-104, 181-183, 223-244, 277, 325-334, 336, At least one of the following nucleotide sequences having 85% or at least 90% or at least 95% or at least 98% or at least 99% sequence identity.

Aspect 16. The PEgRNA of embodiment 1, wherein the DNA synthesis template comprises a nucleotide sequence that is at least 80% or 85% or 90% or 95% or 99% identical to the endogenous DNA target.

Aspect 17. PEgRNA of embodiment 6, wherein the primer binding site hybridizes to the free 3' end of the cut DNA.

Aspect 18. The PEgRNA of embodiment 10, wherein the at least one additional structure is located at the 3' or 5' end of the PEgRNA.

Aspect 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.

Mode 20. The PEgRNA of embodiment 10, wherein the stem-loop comprises a nucleotide sequence selected from the stem-loops described herein.

Aspect 21. The PEgRNA of embodiment 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 embodiment 10, wherein the aptamer comprises a nucleotide sequence selected from the aptamers described herein.

Aspect 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.

Aspect 25. A method for designing PEgRNA for use in prime editing to incorporate a desired nucleotide change onto a target nucleotide sequence, the PEgRNA comprising a spacer, a gRNA core, and an extended arm, the extended arm comprising: comprising a primer binding site and a DNA synthesis template, the method comprising:

(i) selecting the desired target editing site on the target nucleotide sequence;

(ii) obtaining the context nucleotide sequences upstream and downstream of the target editing site;

(iii) positioning 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 spacers;

(vi) designing the gRNA core;

(vii) designing an extension arm; and

(viii) constructing a complete PEgRNA by concatenating a spacer, a gRNA core, and an extended 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.

Aspect 27. Embodiment 26. The method of embodiment 26, wherein the disease-causing mutation is associated with a disease selected from the group consisting of: cancer, autoimmune disorders, neurological disorders, skin disorders, respiratory disorders, and heart disorders.

Aspect 28. Embodiment 25, wherein step (ii) of obtaining upstream and downstream context nucleotide sequences from the target editing site comprises about 50-55 base pairs (bp), about 55-60 bp of the region containing the desired target editing site. , about 60-65bp, about 65-70bp, about 70-75bp, about 75-80bp, about 80-85bp, about 85-90bp, about 90-95bp, about 95-100bp, about 100-105bp, about 105-110bp , about 110-125bp, about 125-130bp, about 130-135bp, about 135-140bp, about 140-145bp, 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.

Aspect 29. Embodiment 28. The method of embodiment 28, wherein the desired target editing site is located approximately equidistant from each end of the context nucleotide sequence.

Aspect 30. Embodiment 25. The method of embodiment 25, wherein in step (iii) the putative PAM site is proximal to the desired target editing site.

Aspect 31. The method of embodiment 25, wherein in step (iii) the putative PAM site is less than 30 nucleotides from the target editing site, or 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 related nicks located at less than 2 nucleotide positions Including those with parts.

Aspect 32. The method of embodiment 25, wherein in step (iii) the putative PAM site is more than 30 nucleotides from the target editing site, or 31, 32, 33, 34, 35, 36, 37, 38, 39 from the target editing site. , 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 with an associated nick site located at more than 100 nucleotide positions.

Aspect 33. 26. 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.

Aspect 35. Embodiment 25. The method of embodiment 25, wherein step (v) of designing a spacer comprises determining the complementary nucleotide sequence of the protospacer sequence associated with each putative PAM.

Aspect 36. Embodiment 25, wherein the step (vi) of designing a gRNA core comprises, in the context of each putative PAM, selecting a gRNA core sequence capable of binding to the napDNAbp associated with each of said putative PAMs. .

Aspect 37. Embodiment 25. The method of embodiment 25, wherein step (vii) of designing the extension arm comprises designing (a) a DNA synthesis template containing the desired edit and (b) a primer binding site.

Aspect 38. 38. The method of aspect 37, wherein designing a primer binding site comprises: (a) identifying a DNA primer on a PAM-containing strand of the target nucleotide sequence, wherein the 3' end of the DNA primer has a nick site associated with the PAM site; and (b) designing a complement of the DNA primer, said complement forming a primer binding site.

Aspect 39. 39. The method of embodiment 38, wherein the primer binding site is 8 to 15 nucleotides in length.

Aspect 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.

Aspect 42. The method of embodiment 38, wherein the DNA primer contains a GC content of greater than about 60%, wherein the primer binding site is 8-11 nucleotides.

Aspect 43. A method of prime editing comprising contacting a target DNA sequence with a PEgRNA of any of embodiments 1-24 and a prime editing factor fusion protein comprising napDNAbp and a domain having RNA-dependent DNA polymerase activity.

Aspect 44. 44. The method of embodiment 43, wherein the napDNAbp has nickase activity.

Aspect 45. 44. The method of embodiment 43, wherein napDNAbp is a Cas9 protein or a variant thereof.

Aspect 46. 44. The method of embodiment 43, wherein the napDNAbp is nuclease-active Cas9, nuclease-inactive Cas9 (dCas9), or Cas9 nickase (nCas9).

Aspect 47. Embodiment 43. The method of embodiment 43, wherein napDNAbp is Cas9 nickase (nCas9).

Aspect 48. 44. 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.

Aspect 49. 44. The method of embodiment 43, wherein the domain comprising RNA-dependent DNA polymerase activity is reverse transcriptase, and 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 50. 44. The method of embodiment 43, wherein the domain comprising RNA-dependent DNA polymerase activity is reverse transcriptase, and SEQ ID NOs: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, At least 80%, 85%, 90%, 95%, 98%, or 99% with any one amino acid sequence of 235, 454, 471, 516, 662, 700, 701-716, 739-741, and 766 Contains amino acid sequences that have sequence identity.

Aspect 51. 44. The method of embodiment 43, wherein the domain comprising RNA-dependent DNA polymerase activity is a naturally occurring reverse transcriptase from a retrovirus or retrotransposon.

Group C. PE composite embodiment 1. A complex for prime editing:

(i) a fusion protein comprising a nucleic acid programmed DNA binding protein (napDNAbp) and a domain containing aRNA-dependent DNA polymerase activity; and

(ii) prime editing guide RNA (PEgRNA),
including.

Aspect 2. The complex of embodiment 1, wherein the fusion protein is capable of performing prime editing in the presence of a prime editing guide RNA (PEgRNA) to incorporate desired nucleotide changes onto the target sequence.

Aspect 3. The complex of embodiment 11, wherein napDNAbp has nickase activity.

Embodiment 4. The complex of embodiment 1, wherein napDNAbp is a Cas9 protein or a variant thereof.

Aspect 5. The complex of embodiment 1, wherein napDNAbp is nuclease-active Cas9, nuclease-inactive Cas9 (dCas9), or Cas9 nickase (nCas9).

Aspect 6. The complex of embodiment 1, wherein napDNAbp is Cas9 nickase (nCas9).

Aspect 7. The complex of embodiment 1, wherein the napDNAbp is selected from the group consisting of: Cas9, Cas12e, Cas12d, Cas12a, Cas12b1, Cas13a, Cas12c, and Argonaute, and optionally has nickase activity.

Aspect 8. The complex of embodiment 1, wherein the domain containing RNA-dependent DNA polymerase activity is reverse transcriptase, and 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 9. The complex of embodiment 1, wherein the domain containing RNA-dependent DNA polymerase activity is reverse transcriptase, and 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. % sequence identity.

Aspect 10. The complex of embodiment 1, wherein the domain comprising RNA-dependent DNA polymerase activity is a naturally occurring reverse transcriptase from a retrovirus or retrotransposon.

Aspect 11. The complex of embodiment 1, wherein the fusion protein is capable of binding to a target DNA sequence when complexed with PEgRNA.

Aspect 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 embodiment 12, wherein the nucleic acid extension arm is at the 3' or 5' end of the guide RNA or at a position within the molecule of the guide RNA, and the nucleic acid extension arm is DNA or RNA.

Aspect 14. The complex of embodiment 12, wherein the PEgRNA is capable of binding napDNAbp and directing napDNAbp to a target DNA sequence.

Aspect 15. Embodiment 14. The conjugate of embodiment 14, wherein the target DNA sequence comprises a target strand and a complementary non-target strand.

Aspect 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 conjugate of embodiment 12, wherein at least one nucleic acid extension arm further comprises a primer binding site.

Aspect 18. The conjugate of embodiment 12, wherein the nucleic acid extending arm comprises 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 .

Aspect 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.

Mode 20. 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.

Aspect 21. The conjugate 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.

Aspect 22. The complex of embodiment 12, wherein the DNA synthesis template encodes a single-stranded DNA flap that is complementary to an endogenous DNA sequence adjacent to the nick site, and the single-stranded DNA flap makes a desired nucleotide change. include.

Aspect 23. The complex of embodiment 22, 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 disposed downstream of the nick site. Immediately adjacent.

Aspect 24. The complex of embodiment 23, wherein the endogenous single-stranded DNA with a free 5' end is excised by the cell.

Aspect 25. The complex of embodiment 23, wherein cellular repair of the single-stranded DNA flap results in the incorporation of a desired nucleotide change, thereby forming a desired product.

Aspect 26. The complex of embodiment 12, wherein PEgRNA is SEQ ID NO : 101-104, 181-183, 223-244, 277, 325-334, 336, 338, 340, 342, 344, 346, 348, 350, 352, Nucleotide sequences of 354, 356, 358, 360, 362, 364, 366, 368, 499-505, 735-761, 776-777, or SEQ ID NO: 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 nucleotide sequences having at least 85% or at least 90% or at least 95% or at least 98% or at least 99% sequence identity with one.

Aspect 27. The conjugate of embodiment 12, wherein the DNA synthesis template comprises a nucleotide sequence that is at least 80% or 85% or 90% or 95% or 99% identical to the endogenous DNA target.

Aspect 28. The complex of embodiment 17, wherein the primer binding site hybridizes to the free 3' end of the cut DNA.

Aspect 29. The conjugate of embodiment 21, wherein the at least one additional structure is located at the 3' or 5' end of the PEgRNA.

Aspect 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 conjugate of embodiment 29, wherein the stem-loop comprises a nucleotide sequence selected from the stem-loops described herein.

Aspect 32. The conjugate 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.

Aspect 35. The conjugate of embodiment 29, wherein the RNA-protein recruitment domain comprises a nucleotide sequence selected from the RNA-protein recruitment domains described herein.

Aspect 36. The complex of embodiment 1, wherein the target DNA sequence comprises a target strand and a complementary non-target strand.

Aspect 37. The complex of embodiment 36, wherein the R-loop comprises (i) an RNA-DNA hybrid comprising PEgRNA and a target strand and (ii) a complementary non-target strand.

Aspect 38. The conjugate of embodiment 37, wherein the target or complementary non-target strand is nicked to form a prime sequence with a free 3' end.

Aspect 39. The conjugate of embodiment 38, wherein the nick site is upstream of the PAM sequence on the target strand.

Aspect 40. The conjugate of embodiment 38, wherein the nick site is upstream of the PAM sequence on the non-target strand.

Aspect 41. The complex of embodiment 38, wherein the nick site is -1, -2, -3, -4, -5, -6, -7, -8, or - relative to the 5' end of the PAM sequence. It is 9.

Aspect 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.

Aspect 43. The complex of embodiment 22, wherein the single-stranded DNA flap replaces the endogenous DNA sequence having a free 5' end and adjacent to the nick site.

Aspect 44. The complex of embodiment 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.

Aspect 47. The conjugate of embodiment 46, wherein the desired nucleotide change 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 approximately -30 and +100 of the PAM array.

Aspect 48. The conjugate 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 of the nick site. , 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.

Aspect 49. The complex of any one of the preceding embodiments, wherein the fusion protein has the structure NH2-[napDNAbp]-[domain comprising RNA-dependent DNA polymerase activity]-COOH; or NH2-[domain comprising RNA-dependent DNA polymerase activity]-COOH; The domain ]-[napDNAbp]-COOH, with each "]-[" indicating the presence of an optional linker sequence.

Aspect 50. The conjugate of embodiment 49, wherein the linker sequence comprises the amino acid sequences of SEQ ID NOs: 127, 165-176, 446, 453, and 767-769.

Aspect 51. The conjugate of embodiment 1, wherein the fusion protein further comprises a linker connecting napDNAbp and a domain containing RNA-dependent DNA polymerase activity.

Aspect 52. The complex of embodiment 51, wherein the linker sequence is SEQ ID NO: 174 (1×SGGS), 446 (2×SGGS), 3889 (3×SGGS), 171 (1×XTEN), 3968 (1×EAAAK), 3969 (2×EAAAK), and 3970 (3×EAAAK) amino acid sequences.
Group D. PE method to correct mutations

Aspect 1. A method for incorporating desired nucleotide changes into a double-stranded DNA sequence, the method comprising:
contacting the double-stranded DNA sequence with a complex comprising the fusion protein and 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;

thereby nicking a double-stranded DNA sequence, thereby producing free single-stranded DNA with 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 producing a single-stranded DNA flap that is complementary to the DNA synthesis template and contains the desired nucleotide changes. ;

thereby replacing the endogenous DNA strand adjacent to the cut site by a single-stranded DNA flap, thereby incorporating the desired nucleotide change into the double-stranded DNA sequence;
including.

Aspect 2. The method of embodiment 1, wherein the endogenous DNA strand is replaced by: (i) hybridizing a single-stranded DNA flap to the endogenous DNA strand adjacent to the cut site, creating a sequence mismatch; (ii) excising the endogenous DNA strand; and (iii) repairing the mismatch to form the desired product containing the desired nucleotide changes in both strands of the DNA.

Aspect 3. The method of embodiment 1, wherein the desired nucleotide change is a single nucleotide substitution, deletion, or insertion.

Aspect 4. The method of embodiment 3, wherein the single nucleotide substitution is a transition or transversion.

Aspect 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) Substitution of A from T, (6) Substitution of C from T, (7) Substitution of G from C, (8) Substitution of T from C, (9) Substitution of A from C, (10) Substitution of A (11) A to G substitution, or (12) A to C substitution.

Aspect 6. The method of embodiment 1, 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 becomes C:G base pair, (4) T:A base pair becomes G:C base pair, (5) T:A base pair becomes A:T base pair, (6) T:A base pair to C:G base pair, (7) C:G base pair to G:C base pair, (8) C:G base pair to T:A base pair, C:G base pair to A: T base pair, (10) A:T base pair to T:A base pair, (11) A:T base pair to G:C base pair, or (12) A:T base pair to C:G. Convert to base pairs.

Aspect 7. The method of embodiment 1, wherein the desired nucleotide change is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, An insertion or deletion of 19, 20, 21, 22, 23, 24, or 25 nucleotides.

Aspect 8. Embodiment 1. The method of embodiment 1, wherein the desired nucleotide change modifies a disease-associated gene.

Aspect 9. The method of embodiment 8, wherein the disease-related gene is: adenosine deaminase (ADA) deficiency; alpha-1 antitrypsin deficiency; cystic fibrosis; Duchenne muscular dystrophy; galactosemia; hemochromatosis; Huntington's disease; maple Syrupuria; Marfan syndrome; neurofibromatosis type 1; congenital nail hyperplasia; phenylketonuria; severe combined immunodeficiency; sickle cell disease; Smith-Lemli-Opitz syndrome; trinucleotide repeat disorder; prion disease associated with a single gene disorder selected from the group consisting of; 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.

Aspect 11. The method of embodiment 1, wherein napDNAbp is nuclease-inactive Cas9 (dCas9), Cas9 nickase (nCas9), or nuclease-active Cas9.

Aspect 12. The method of embodiment 1, wherein napDNAbp comprises the amino acid sequence of SEQ ID NO: 18.

Aspect 13. The method of embodiment 1, wherein napDNAbp has 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. Comprising amino acid sequences with at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity.

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.

Aspect 15. The method of embodiment 1, wherein the polymerase is reverse transcriptase, SEQ ID NOs: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, An amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with any one amino acid sequence of 662, 700, 701-716, 739-741, and 766. include.

Aspect 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 at an intramolecular location, the extension arm comprising a DNA synthesis template sequence and a primer binding site.

Aspect 17. 17. The method of embodiment 16, wherein the extended 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 in length. , 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 be.

Aspect 18. The method of embodiment 1, wherein the PEgRNA is SEQ ID NO: 101-104, 181-183, 223-244, 277, 325-334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354 , 356, 358, 360, 362, 364, 366, 368, 499-505, 735-761, 776-777.

Embodiment 19. A method for introducing one or more changes in 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 onto the corresponding endogenous DNA, thereby introducing one or more changes in the nucleotide sequence of the DNA molecule at the target locus;
including.

Mode 20. Embodiment 19. The method of embodiment 19, wherein the one or more changes in the nucleotide sequence comprises a transition.

Embodiment 21. The method of embodiment 19, wherein the transition is selected from the group consisting of: (a) T to C; (b) A to G; (c) C to T; and (d) G to A.

Embodiment 22. The method of embodiment 19, wherein the one or more changes in the nucleotide sequence include a transversion.

Aspect 23. 23. The method of embodiment 22, wherein the transversion is (a) T to A; (b) T to G; (c) C to G; (d) C to A; (e) A to T; (f) (g) G to C; and (h) G to T.

Aspect 24. The method of embodiment 19, wherein the one or more changes in the nucleotide sequence include (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) G:C base pair to C:G base pair, (4) T:A base pair to G:C base pair, (5) T:A base pair to A:T base pair, (6 )T:A base pair to C:G base pair, (7)C:G base pair to G:C base pair, (8)C:G base pair to T:A base pair, (9)C :G base pair to A:T base pair, (9) A:T base pair to T:A base pair, (11) A:T base pair to G:C base pair, or (12) A: It involves changing a T base pair to a C:G base pair.

Aspect 25. The method of embodiment 19, wherein the one or more changes in the nucleotide sequence are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, Contains insertions or deletions of 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides.

Aspect 26. Embodiment 19. The method of embodiment 19, wherein the one or more changes in the nucleotide sequence comprises correction of a disease-associated gene.

Aspect 27. The method of embodiment 26, wherein the disease-associated gene is: adenosine deaminase (ADA) deficiency; alpha-1 antitrypsin deficiency; cystic fibrosis; Duchenne muscular dystrophy; galactosemia; hemochromatosis; Huntington's disease; maple Syrupuria; Marfan syndrome; type 1 neurofibromatosis; congenital nail hypertrophy; phenylketonuria; severe combined immunodeficiency; sickle cell disease; Smith-Lemli-Opitz syndrome; trinucleotide repeat disorder; prion disease associated with a single gene disorder selected from the group consisting of; and Tay-Sachs disease.

Aspect 28. Embodiment 26. 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.

Aspect 29. Embodiment 19. The method of embodiment 19, wherein napDNAbp is nuclease active Cas9 or a variant thereof.

Aspect 30. The method of embodiment 19, wherein napDNAbp is nuclease-inactive Cas9 (dCas9) or Cas9 nickase (nCas9) or a variant thereof.

Aspect 31. 19. The method of embodiment 19, wherein napDNAbp comprises the amino acid sequence of SEQ ID NO: 18.

Aspect 32. The method of embodiment 19, wherein napDNAbp has 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. Comprising amino acid sequences with at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity.

Aspect 33. Embodiment 19. The method of embodiment 19, wherein the reverse transcriptase is introduced in trans.

Aspect 34. Embodiment 19. The method of embodiment 19, wherein napDNAbp comprises a fusion to reverse transcriptase.

Aspect 35. The method of embodiment 19, wherein the reverse transcriptase is SEQ ID NO: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, 662, 700 , 701-716, 739-741, and 766.

Aspect 36. The method of embodiment 19, wherein the reverse transcriptase is SEQ ID NO: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, 662, 700 , 701-716, 739-741, and 766.

Aspect 37. Embodiment 19. 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.

Aspect 38. Embodiment 37. 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. Embodiment 19. The method of embodiment 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. Embodiment 19. The method of embodiment 19, wherein forming an exposed 3' end of the DNA strand at the target locus comprises introducing a replication error.

Aspect 41. Embodiment 19. The method of embodiment 19, wherein the step of contacting the DNA molecule with napDNAbp and guide RNA forms an R-loop.

Aspect 42. The method of embodiment 41, wherein the DNA strand from which the exposed 3' end is formed is on the R-loop.

Aspect 43. Embodiment 19. The method of embodiment 19, wherein the PEgRNA comprises an extended arm that includes a reverse transcriptase (RT) template sequence and a primer binding site.

Aspect 44. 44. The method of embodiment 43, wherein the elongated arm is at the 3' end of the guide RNA, at the 5' end of the guide RNA, or at a position within the molecule of the guide RNA.

Aspect 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. Embodiment 19. The method of embodiment 19, wherein the PEgRNA further comprises a homology arm.

Aspect 47. The method of embodiment 19, wherein the RT template sequence is homologous to the corresponding endogenous DNA.

Aspect 48. A method for introducing one or more changes in the nucleotide sequence of a DNA molecule at a target locus by primed reverse transcription to a primed target, the method comprising: (a) converting the DNA molecule at the target locus into (i) a nucleic acid molecule; contacting a fusion protein comprising a programmed DNA binding protein (napDNAbp) and a reverse transcriptase and (ii) a guide RNA comprising an RT template comprising the desired nucleotide changes;

thereby performing primed reverse transcription on the primed target of the RT template to generate single-stranded DNA containing the desired nucleotide changes;

thereby incorporating desired nucleotide changes on the DNA molecule at the target locus by DNA repair and/or replication processes;
including.

Aspect 49. 49. The method of embodiment 48, wherein the RT template is located at the 3' end of the guide RNA, at the 5' end of the guide RNA, or at an intramolecular position of the guide RNA.

Aspect 50. 49. The method of embodiment 48, wherein the desired nucleotide change comprises a transition, transversion, insertion, or 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.

Aspect 52. Embodiment 48, wherein the desired nucleotide change is (a) T to A; (b) T to G; (c) C to G; (d) C to A; (e) A to T; (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.

Aspect 54. A method for replacing a trinucleotide repeat expansion mutation on a target DNA molecule by a healthy sequence containing a healthy number of repeat trinucleotides, the method comprising: (a) converting the DNA molecule at the target locus to (i) a nucleic acid programmed contacting a fusion protein containing a DNA binding protein (napDNAbp) and a polymerase; and (ii) a DNA synthesis template containing a replacement sequence and a PEgRNA containing a primer binding site; producing single-stranded DNA; and (c) incorporating single-stranded DNA onto a DNA molecule at a target locus by a DNA repair and/or replication process.

Aspect 55. 55. The method of embodiment 54, wherein the fusion protein comprises the amino acid sequence of PE1, PE2, or PE3.

Aspect 56. 55. The method of embodiment 54, wherein napDNAbp is Cas9 nickase (nCas9).

Aspect 57. 55. The method of embodiment 54, 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 58. 55. The method of embodiment 54, wherein the guide RNA comprises SEQ ID NO: 222.

Aspect 59. 55. The method of embodiment 54, wherein (b) performing prime editing generates a 3' end primer binding sequence at the target locus that is capable of priming a polymerase by annealing to a primer binding site on the guide RNA. include.

Aspect 60. 55. The method of embodiment 54, wherein the trinucleotide repeat expansion mutation is associated with Huntington's disease, Fragile X syndrome, or Friedreich's ataxia.

Aspect 61. 55. The method of embodiment 54, wherein the trinucleotide repeat expansion comprises a CAG triplet repeat unit.

Aspect 62. 55. The method of embodiment 54, wherein the trinucleotide repeat expansion comprises a GAA triplet repeat unit.

Aspect 63. A method for introducing one or more changes in the nucleotide sequence of a DNA molecule at a target locus by primed reverse transcription to a primed target, the method comprising:
(a) contacting a DNA molecule at a target locus with a guide RNA comprising (i) a fusion protein comprising a nucleic acid programmed DNA binding protein (napDNAbp) and a reverse transcriptase and (ii) an RT template comprising the desired nucleotide changes;

thereby performing primed reverse transcription on the primed target of the RT template to generate single-stranded DNA containing the desired nucleotide changes;

thereby incorporating desired nucleotide changes on the DNA molecule at the target locus by DNA repair and/or replication processes;
including.

Aspect 64. 64. The method of embodiment 63, wherein the RT template is located at the 3' end of the guide RNA, at the 5' end of the guide RNA, or at an intramolecular position of the guide RNA.

Aspect 65. 64. The method of embodiment 63, wherein the desired nucleotide change comprises a transition, transversion, insertion, or deletion, or any combination thereof.

Aspect 66. 64. The method of embodiment 63, wherein the desired nucleotide change is selected from the group consisting of: (a) T to C; (b) A to G; (c) C to T; and (d) G to A. including.

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.

Aspect 68. Embodiment 63, 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 T:A base pair; :C base pair becomes C:G base pair, (4) T:A base pair becomes G:C base pair, (5) T:A base pair becomes A:T base pair, (6) T:A base pair to C:G base pair, (7) C:G base pair to G:C base pair, (8) C:G base pair to T:A base pair, (9) C:G base pair. into an A:T base pair, (10) an A:T base pair into a T:A base pair, (11) an A:T base pair into a G:C base pair, or (12) an A:T base pair. This includes changing to a C:G base pair.

Aspect 69. A method of preventing or halting the progression of a prion disease by incorporating one or more protective mutations on a PRNP encoded by a target nucleotide sequence by prime editing, the method comprising: (a) contacting (i) a prime editing factor comprising a nucleic acid programmed 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 a protective mutation;

thereby incorporating, by DNA repair and/or replication processes, a single-stranded DNA sequence in place of the corresponding endogenous strand in the target nucleotide sequence;
including;

The method produces a recombinant target nucleotide sequence encoding a PRNP containing a protective mutation that is resistant to misfolding.

Mode 70. 69. The method of embodiment 69, wherein the prion disease is a human prion disease.

Aspect 71. 69. The method of embodiment 69, wherein the prion disease is an animal prion disease.

Aspect 72. 69. The method of embodiment 69, wherein the prion disease is Creutzfeldt-Jakob disease (CJD), variant Creutzfeldt-Jakob disease (vCJD), Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia, or Kuru disease. It is.

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. 69. The method of embodiment 69, wherein the wild-type PRNP amino acid sequence is SEQ ID NO: 291-292.

Aspect 75. 69. 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 wherein the modified PRNP protein is resistant to misfolding. be.

Aspect 76. 69. The method of embodiment 69, wherein the fusion protein comprises the amino acid sequence of PE1, PE2, or PE3.

Aspect 77. 69. The method of embodiment 69, wherein 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.

Aspect 79. The method of embodiment 69, wherein the PEgRNA is SEQ ID NO: 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 encoding the edit;

thereby incorporating, by DNA repair and/or replication processes, a single-stranded DNA sequence in place of the corresponding endogenous strand in the target nucleotide sequence;
including;

The method produces a recombinant target nucleotide sequence encoding a repaired CDKL5 gene.

Aspect 81. The method of embodiment D80, wherein the CDKL5 mutation is 1412delA.

Group E. PE method embodiment 1 for modifying protein structure/function and/or introducing mutations . A method of mutagenizing a DNA molecule at a target locus by prime editing, the method comprising: (a) converting the DNA molecule at the target locus to (i) a nucleic acid programmed DNA binding protein (napDNAbp) and an error-prone polymerase (polymer); (e.g., an error-prone reverse transcriptase) and (ii) a guide RNA containing an editing template containing the desired nucleotide changes; thereby making the single-stranded DNA templated with the editing template It involves incorporating single-stranded DNA onto a DNA molecule at a target locus by polymerizing and by DNA repair and/or replication processes.

Aspect 2. The method of any preceding embodiment, wherein the fusion protein comprises the amino acid sequence of PE1, PE2, or PE3.

Aspect 3. The method of any preceding embodiment, wherein 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 embodiment 1, wherein the guide RNA comprises SEQ ID NO: 222.

Aspect 6. A method of incorporating an immune epitope onto a protein of interest encoded by a target nucleotide sequence by prime editing, the method comprising: (a) converting the target nucleotide sequence to (i) a nucleic acid programmed DNA binding protein (napDNAbp) and a polymerase; (ii) a PEgRNA comprising an editing template encoding a functional moiety;

thereby polymerizing a single-stranded DNA sequence encoding an immune epitope;

thereby incorporating, by DNA repair and/or replication processes, a single-stranded DNA sequence in place of the corresponding endogenous strand in the target nucleotide sequence;
including;

The method produces a recombinant target nucleotide sequence that encodes a fusion protein that includes the protein of interest and the immune epitope.

Aspect 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); Class I HLA hemagglutinin epitope for class I HLA (SEQ ID NO: 416); neuraminidase epitope for class I 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. Embodiment 6. The method of embodiment 6, wherein the fusion protein comprises the amino acid sequence of PE1, PE2, or PE3.

Aspect 9. The method of embodiment 6, wherein napDNAbp is Cas9 nickase (nCas9).

Aspect 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.

Aspect 11. The method of embodiment 6, wherein the PEgRNA is SEQ ID NO: 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, by DNA repair and/or replication processes, a single-stranded DNA sequence in place of the corresponding endogenous strand in the target nucleotide sequence;
including;

The method produces a modified target nucleotide sequence that encodes a fusion protein that includes the protein of interest and a small dimerization domain.

Aspect 13. The method of embodiment 12, further comprising performing the method on a second protein of interest.

Aspect 14. Embodiment 13. 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 a respective dimerization domain of the protein.

Aspect 15. The method of embodiment 12, wherein the small molecule binding domain is FKBP12 of SEQ ID NO: 488.

Aspect 16. The method of embodiment 12, wherein the small molecule binding domain is FKBP12-F36V of SEQ ID NO: 489.

Aspect 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 embodiment 12, wherein the small molecule is a dimer of a small molecule as described herein.

Aspect 19. The method of embodiment 12, wherein the fusion protein comprises the amino acid sequence of PE1, PE2, or PE3.

Mode 20. The method of embodiment 12, wherein napDNAbp is Cas9 nickase (nCas9).

Aspect 21. The method of embodiment 12, 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 22. The method of embodiment 12, wherein the PEgRNA is SEQ ID NO: 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.

Aspect 23. A method of incorporating a peptide tag or epitope onto a protein using prime editing, comprising: a prime editing agent configured to insert into a target nucleotide sequence encoding a protein a second nucleotide sequence encoding a peptide tag; contacting the recombinant nucleotide sequence with a construct so that the peptide tag and protein are expressed from the recombinant nucleotide sequence as a fusion protein.

Aspect 24. Embodiment 23. The method of embodiment 23, wherein the peptide tag is used for protein purification and/or detection.

Aspect 25. Embodiment 23, wherein the peptide tag is polyhistidine (eg, HHHHHH, SEQ ID NO: 252-262), FLAG (eg, DYKDDDDK, SEQ ID NO: 2), V5 (eg, GKPIPNPLLGLLDST, SEQ ID NO: 3), GCN4, HA (eg, YPYDVPDYA, SEQ ID NO: 5), Myc (eg, EQKLISEED, SEQ ID NO: 6), or GST.

Aspect 26. 24. The method of embodiment 23, wherein the peptide tag is an amino acid selected from the group consisting of SEQ ID NOs: 1-6, 245-249, 252-262, 264-273, 275-276, 281, 278-288, and 622. It has an array.

Aspect 27. Embodiment 23. The method of embodiment 23, wherein the peptide tag is fused to the protein by a linker.

Aspect 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.

Aspect 29. The method of embodiment 23, wherein the linker has the amino acid sequence of SEQ ID NOs: 127, 165-176, 446, 453, and 767-769.

Aspect 30. 24. The method of embodiment 23, wherein the prime editing factor construct comprises SEQ ID NOs: 101-104, 181-183, 223-244, 277, 325-334, 336, 338, 340, 342, 344, 346, 348, 350, Contains PEgRNA containing the nucleotide sequences 352, 354, 356, 358, 360, 362, 364, 366, 368, 499-505, 735-761, 776-777.

Aspect 31. Embodiment 23. The method of embodiment 23, wherein the PEgRNA comprises a spacer, a gRNA core, and an extended arm, the spacer being complementary to a target nucleotide sequence, and the extended arm comprising a reverse transcriptase template encoding a peptide tag.

Aspect 32. Embodiment 23. The method of embodiment 23, wherein the PEgRNA comprises a spacer, a gRNA core, and an extended arm, the spacer being complementary to a target nucleotide sequence, and the extended arm comprising a reverse transcriptase template encoding a peptide tag.

Aspect 33. A method of incorporating or deleting a functional moiety on a protein of interest encoded by a target nucleotide sequence by prime editing, the method comprising: (a) converting the target nucleotide sequence into (i) a nucleic acid programmed DNA binding protein (napDNAbp); ) and a prime editing factor comprising a polymerase and (ii) PEgRNA comprising an editing template encoding the functional moiety or its deletion; (b) polymerizing the single-stranded DNA sequence encoding the functional moiety or its deletion; and (c) incorporating a single-stranded DNA sequence in place of the corresponding endogenous strand in the target nucleotide sequence by a DNA repair and/or replication process, the method comprising: A recombinant target nucleotide sequence is generated that encodes a modified protein containing a deletion, the functional moiety altering the modification state or localization state of the protein.

Aspect 34. Embodiment 33. 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 Method and Composition Embodiment 1. A polypeptide comprising the N-terminal half or the C-terminal half of a prime editing factor fusion protein.

Aspect 2. The polypeptide of embodiment 1, wherein the prime editing factor fusion protein comprises a nucleic acid programmed DNA binding protein (napDNAbp) domain and a polymerase domain.

Aspect 3. The polypeptide of embodiment 1, wherein the prime editing factor 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 embodiment 2, wherein napDNAbp is a nuclease having nickase activity.

Aspect 6. The polypeptide of embodiment 2, wherein napDNAbp is nuclease-active Cas9, nuclease-inactive Cas9 (dCas9), or Cas9 nickase (nCas9).

Aspect 7. The polypeptide of embodiment 2, wherein the napDNAbp is selected from the group consisting of: Cas9, Cas12e, Cas12d, Cas12a, Cas12b1, Cas13a, Cas12c, and Argonaute, and optionally has nickase activity.

Aspect 8. The polypeptide of embodiment 1, wherein the polypeptide is formed by splitting a prime editing factor 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 embodiment 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.

Aspect 12. The polypeptide of embodiment 9, wherein the cleavage site is residues 1 and 2, 2 and 3, 3 and 4, 4 and 5, 5 and 6, 6 and 7, 7 of SEQ ID NO: 18 (standard SpCas9). 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, 39 and 40, 40 and 41, 41 and 42, 42 and 43, 43 and 44, 44 and 45, between 45 and 46, 46 and 47, 47 and 48, 48 and 49, 49 and 50, or residues 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350 Between ~400, 400-450, 450-500, 500-600, 600-700, 700-800, 800-900, 900-1000, 1000-1100, 1100-1200, 1200-1300, or 1300-1368 A peptide bond between any two residues or between any two equivalent amino acid residues of the SpCas9 homologue of SEQ ID NO: 18 or equivalent.

Aspect 13. The polypeptide of embodiment 9, wherein the cleavage site comprises 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, 39 and 40, 40 and 41, 41 and 42, 42 and 43, 43 and between 44, 44 and 45, 45 and 46, 46 and 47, 47 and 48, 48 and 49, 49 and 50, or residues 50 to 100, 100 to 150, 150 to 200, 200 to 250, 250 to between any two residues between 300, 300-350, 350-400, 400-450, 450-500, 500-600, or 600-667, or a reverse transcriptase homolog or equivalent of SEQ ID NO: 89 is a peptide bond between any two equivalent amino acid residues.

Aspect 14. The polypeptide of embodiment 1, wherein the polypeptide is the N-terminal half of a prime editing factor 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 the polypeptide of any of embodiments 1 to 15 and optionally PEgRNA.

Aspect 17. A viral genome comprising a nucleotide sequence encoding a polypeptide of any of embodiments 1 to 15 and optionally 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 to 15.

Aspect 19. The viral genome of embodiment 17, wherein the nucleotide sequence further comprises a sequence encoding PEgRNA.

Mode 20. A virus particle comprising a genome comprising a nucleotide sequence encoding a polypeptide of any of embodiments 1 to 15 and optionally PEgRNA.

Aspect 21. The virus particle of embodiment 20, wherein the virus particle is an adenovirus particle, an adeno-associated virus particle, or a lentivirus particle.

Aspect 22. The viral particle of embodiment 20, wherein the polypeptide encoded by the genome is the N-terminal half of a prime editing factor fusion protein.

Aspect 23. The viral particle of embodiment 20, wherein the polypeptide encoded by the genome is the C-terminal half of a prime editing factor fusion protein.

Aspect 24. A pharmaceutical composition comprising the virus particles of any of embodiments 20-23 and a pharmaceutical excipient.

Aspect 25. A pharmaceutical composition comprising the virus particle of embodiment 22 (encoding the N-terminal half) and a pharmaceutical excipient.

Aspect 26. A pharmaceutical composition comprising the virus particle of embodiment 23 (encoding the C-terminal half) and a pharmaceutical excipient.

Aspect 27. A ribonucleoprotein (RNP) complex comprising a nucleotide sequence encoding a polypeptide of any of embodiments 1 to 15 and optionally PEgRNA.

Aspect 28. The ribonucleoprotein (RNP) complex of embodiment 27, wherein the polypeptide encoded by the genome is the N-terminal half of a prime editing factor 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 according to any of embodiments 27-29 and a pharmaceutical excipient.

Aspect 31. A pharmaceutical composition comprising a ribonucleoprotein (RNP) complex of embodiment 28 (encoding the N-terminal half) 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 the N-terminal half of a prime editing factor fusion protein, and the second AAV vector expressing the prime editing factor fusion protein. The C-terminal half of the protein is expressed, and the N-terminal and C-terminal halves are combined within the cell to reconstitute the prime editing factor.

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. Embodiment 33. The pharmaceutical composition of embodiment 33, wherein the prime editing factor fusion protein comprises a nucleic acid programmed DNA binding protein (napDNAbp) domain and a polymerase domain.

Aspect 36. Embodiment 33. The pharmaceutical composition of embodiment 33, wherein the prime editing factor fusion protein is capable of performing prime editing in the presence of a prime editing guide RNA (PEgRNA).

Aspect 37. The pharmaceutical composition of embodiment 35, wherein napDNAbp is a Cas9 protein or a variant thereof.

Aspect 38. Embodiment 35. The pharmaceutical composition of embodiment 35, wherein napDNAbp is a nuclease with nickase activity.

Aspect 39. The pharmaceutical composition of embodiment 35, wherein napDNAbp is nuclease-active Cas9, nuclease-inactive Cas9 (dCas9), or Cas9 nickase (nCas9).

Aspect 40. The pharmaceutical composition of embodiment 35, wherein the napDNAbp is selected from the group consisting of: Cas9, Cas12e, Cas12d, Cas12a, Cas12b1, Cas13a, Cas12c, and Argonaute, and optionally has nickase activity.

Aspect 41. Embodiment 33. The pharmaceutical composition of embodiment 33, wherein the N-terminal and C-terminal halves are formed by splitting the prime editing factor fusion protein at a cleavage site.

Aspect 42. The pharmaceutical composition of embodiment 41, wherein the cleavage site is a peptide bond on the napDNAbp domain.

Aspect 43. 42. The pharmaceutical composition of embodiment 41, wherein the cleavage site is a peptide bond on the polymerase domain.

Aspect 44. Embodiment 41. The pharmaceutical composition of embodiment 41, wherein the cleavage site is a peptide bond on the linker.

Aspect 45. 42. The pharmaceutical composition of embodiment 41, wherein the cleavage site comprises residues 1 and 2, 2 and 3, 3 and 4, 4 and 5, 5 and 6, 6 and 7 of SEQ ID NO: 18 (standard SpCas9); 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, 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, Between 350-400, 400-450, 450-500, 500-600, 600-700, 700-800, 800-900, 900-1000, 1000-1100, 1100-1200, 1200-1300, or 1300-1368 or between any two equivalent amino acid residues of the SpCas9 homologue of SEQ ID NO: 18 or equivalent.

Aspect 46. The pharmaceutical composition of embodiment 41, wherein the cleavage site comprises residues 1 and 2, 2 and 3, 3 and 4, 4 and 5, 5 of SEQ ID NO: 89 (standard reverse transcriptase, M-MLV RT). 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, 39 and 40, 40 and 41, 41 and 42, 42 and 43, between 43 and 44, 44 and 45, 45 and 46, 46 and 47, 47 and 48, 48 and 49, 49 and 50, or residues 50-100, 100-150, 150-200, 200-250, 250 Between any two residues between ~300, 300-350, 350-400, 400-450, 450-500, 500-600, or 600-667, or a reverse transcriptase homolog or equivalent of SEQ ID NO: 89 is a peptide bond between any two equivalent amino acid residues of a substance.

Aspect 47. The pharmaceutical composition of embodiment 33, wherein the N-terminal half of the prime editing factor fusion protein has an amino acid sequence encoding an N-terminal prime editing factor fusion protein as described herein.

Aspect 48. The pharmaceutical composition of embodiment 33, wherein the C-terminal half of the prime editing factor fusion protein has an amino acid sequence encoding an N-terminal prime editing factor fusion protein as described herein.

Embodiment 49. A method of delivering a prime editor fusion protein to a cell, comprising transfecting the cell with a first AAV particle and a second AAV particle, wherein the first AAV vector expresses 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.

Aspect 50. Embodiment 49. The method of embodiment 49, wherein the first or second AAV particle also expresses PEgRNA, which targets the reconstituted prime editing factor to the target DNA site.

Aspect 51. 49. The method of embodiment 49, wherein the prime editing factor fusion protein comprises a nucleic acid programmed DNA binding protein (napDNAbp) domain and a polymerase domain.

Aspect 52. Embodiment 49. The method of embodiment 49, wherein the prime editing factor fusion protein is capable of performing prime editing in the presence of a prime editing guide RNA (PEgRNA).

Aspect 53. 52. The method of embodiment 51, wherein napDNAbp is a Cas9 protein or a variant thereof.

Embodiment 54. The method of embodiment 53, wherein napDNAbp is a nuclease having nickase activity.

Aspect 55. 54. The method of embodiment 53, wherein the napDNAbp is nuclease-active Cas9, nuclease-inactive Cas9 (dCas9), or Cas9 nickase (nCas9).

Aspect 56. 54. 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.

Aspect 57. 49. The method of embodiment 49, wherein the N-terminal and C-terminal halves are formed by splitting the prime editing factor fusion protein at a cleavage site.

Aspect 58. 58. The method of embodiment 57, wherein the cleavage site is a peptide bond on the napDNAbp domain.

Aspect 59. 58. The method of embodiment 57, wherein the cleavage site is a peptide bond on the polymerase domain.

Aspect 60. Embodiment 57. The method of embodiment 57, wherein the cleavage site is a peptide bond on the linker.

Aspect 61. 58. The method of embodiment 57, wherein the cleavage site comprises 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, 39 and 40, 40 and 41, 41 and 42, 42 and 43, 43 and 44, 44 and 45, 45 and between 46, 46 and 47, 47 and 48, 48 and 49, 49 and 50, or residues 50 to 100, 100 to 150, 150 to 200, 200 to 250, 250 to 300, 300 to 350, 350 to Any between 400, 400-450, 450-500, 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 the SpCas9 homolog of SEQ ID NO: 18 or equivalent.

Aspect 62. 58. The method of embodiment 57, wherein the cleavage site comprises residues 1 and 2, 2 and 3, 3 and 4, 4 and 5, 5 and 6 of SEQ ID NO: 89 (standard reverse transcriptase, M-MLV RT). , 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, 39 and 40, 40 and 41, 41 and 42, 42 and 43, 43 and between 44, 44 and 45, 45 and 46, 46 and 47, 47 and 48, 48 and 49, 49 and 50, or 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 a reverse transcriptase homologue of SEQ ID NO: 89 or an equivalent. A peptide bond between any two equivalent amino acid residues.

Aspect 63. 49. The method of embodiment 49, wherein the N-terminal half of the prime editing factor fusion protein has an amino acid sequence encoding an N-terminal prime editing factor fusion protein as described herein.

Aspect 64. 49. The method of embodiment 49, wherein the C-terminal half of the prime editing factor fusion protein has an amino acid sequence encoding a C-terminal prime editing factor fusion protein as described herein.

Aspect 65. 49. The method of embodiment 49, wherein the first AAV particle comprises a recombinant AAV genome comprising a nucleotide sequence encoding a first prime editing element component.

Aspect 66. 49. The method of embodiment 49, wherein the second AAV particle comprises a recombinant AAV genome comprising a nucleotide sequence encoding a second prime editing element component.

Aspect 67. Embodiment 49. The method of embodiment 49, wherein the transfection step is performed in vivo.

Aspect 68. Embodiment 49. The method of embodiment 49, wherein the transfection step is performed ex vivo.

Aspect 69. 50. The method of embodiment 50, wherein the target DNA site is a disease-related gene.

Mode 70. 69. The method of embodiment 69, wherein the disease-associated gene is: adenosine deaminase (ADA) deficiency; alpha-1 antitrypsin deficiency; cystic fibrosis; Duchenne muscular dystrophy; galactosemia; hemochromatosis; Huntington's disease; maple Syrupuria; Marfan syndrome; type 1 neurofibromatosis; congenital nail hypertrophy; phenylketonuria; severe combined immunodeficiency; sickle cell disease; Smith-Lemli-Opitz syndrome; trinucleotide repeat disorder; prion disease associated with a single gene disorder selected from the group consisting of; and Tay-Sachs disease.

Aspect 71. 69. 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.

Aspect 72. 52. The method of embodiment 51, wherein the programmable DNA binding protein (napDNAbp) domain.

Aspect 73. 52. 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.

Aspect 75. The method of embodiment 73, wherein the reverse transcriptase is SEQ ID NO: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, 662, 700 , 701-716, 739-741, and 766.

Aspect 76. 74. The method of embodiment 73, wherein napDNAbp comprises the amino acid sequence of SEQ ID NO: 18.

Aspect 77. 74. The method of embodiment 73, wherein napDNAbp has 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. Comprising amino acid sequences with at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity.
Aspect 78. The polypeptide of embodiment 8, wherein the splitting site is between 1023 and 1024 of SEQ ID NO: 18, or at least 85%, at least 90%, at least 95%, at least 99%, or at least 99.5% sequence with SEQ ID NO: 18. Corresponding positions on amino acid sequences that have identity.

Group G. PE method embodiment 1 for modifying RNA structure/function . A method of incorporating a ribonucleotide motif or tag onto an RNA of interest encoded by a target nucleotide sequence by prime editing, the method comprising: (a) converting the target nucleotide sequence into (i) a nucleic acid programmed DNA binding protein (napDNAbp); and a prime editing agent comprising a polymerase and (ii) PEgRNA comprising an editing template encoding a ribonucleotide motif or tag; thereby polymerizing the single-stranded DNA sequence encoding the ribonucleotide motif or tag; and incorporating a single-stranded DNA sequence in place of the corresponding endogenous strand in the target nucleotide sequence by a DNA repair and/or replication process, the method comprising generate a target nucleotide sequence encoding the target nucleotide sequence.

Aspect 2. The method of embodiment 1, wherein the ribonucleotide motif or tag is the detection moiety.

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

Aspect 4. The method of embodiment 1, wherein the ribonucleotide motif or tag affects the trafficking or subcellular localization of the RNA of interest.

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

Aspect 6. The method of embodiment 1, wherein the PEgRNA is SEQ ID NO: 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 embodiment 1, wherein the fusion protein comprises the amino acid sequence of PE1, PE2, or PE3.

Aspect 8. The method of embodiment 1, wherein napDNAbp is Cas9 nickase (nCas9).

Aspect 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 method mode 1 for creating a gene library . A method of constructing a programmed mutant gene library by prime editing, the method comprising:

(a) a library of target nucleotide sequences each comprising one or more target gene loci; (i) a prime editing factor comprising a nucleic acid programmed DNA binding protein (napDNAbp) and a polymerase; and (ii) one or more target gene loci. contacting PEgRNA comprising an editing template comprising a sequence having at least one genetic change relative to;

thereby polymerizing a single-stranded DNA sequence templated with the editing template; and

Incorporating a single-stranded DNA sequence in place of one or more target genetic loci by a DNA repair and/or replication process, thereby incorporating at least one genetic change onto the target genetic locus of the target nucleotide sequences of said library. ,
including.

Aspect 2. The method of embodiment 1, wherein the library is a plasmid library.

Aspect 3. The method of embodiment 1, wherein the library is a phage library.

Aspect 4. Embodiment 1. The method of embodiment 1, wherein the one or more target gene loci comprises a protein-coding region.

Aspect 5. Embodiment 1. The method of embodiment 1, wherein the one or more target gene loci comprises a region encoding a protein secondary structure motif.

Aspect 6. Embodiment 5. The method of embodiment 5, wherein the secondary structure motif is an alpha helix.

Aspect 7. The method of embodiment 5, wherein the secondary structure motif is a beta sheet.

Aspect 8. The method of embodiment 1, wherein napDNAbp is a Cas9 protein or a variant thereof.

Aspect 9. The method of embodiment 1, wherein napDNAbp is a nuclease with nickase activity.

Aspect 10. The method of embodiment 1, wherein napDNAbp is nuclease-active Cas9, nuclease-inactive Cas9 (dCas9), or Cas9 nickase (nCas9).

Aspect 11. The method of embodiment 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 embodiment 1, wherein the polymerase domain is a reverse transcriptase.

Aspect 14. The method of embodiment 13, wherein the reverse transcriptase is SEQ ID NO: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, 662, 700 , 701-716, 739-741, and 766.

Aspect 15. The method of embodiment 14, wherein the reverse transcriptase is SEQ ID NO: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, 662, 700 , 701-716, 739-741, and 766.

Aspect 16. The method of embodiment 1, wherein at least one genetic change in the editing template is an insertion.

Aspect 17. The method of embodiment 1, wherein at least one genetic change in the editing template is a deletion.

Aspect 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.

Mode 20. The method of embodiment 1, wherein the at least one genetic change is a deletion of one or more codons.

Aspect 21. The method of embodiment 1, wherein the at least one genetic change is the insertion of a stop codon.

Embodiment 22. The method of embodiment 1, wherein at least one genetic change is a conversion of a non-stop codon to a stop codon.

Aspect 23. The method of embodiment 1, wherein the method simultaneously comprises 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 Each of the target nucleotide sequences of the library is performed for between 200, or between 200 and 300, or between 300 and 400, or between 400 and 500 target gene loci.

Aspect 24. The method of embodiment 1, wherein the method is performed during PACE or PANCE evolution, and each of the steps of incorporating at least one genetic change into a target locus of a target nucleotide sequence of said library introduces a new target sequence. Also incorporate.

Group I. PE method aspect 1 for off-target detection . A method for evaluating off-target editing by prime editing factors, the method comprising:

(a) A target nucleotide sequence containing an editing site is combined with (i) a prime editing factor fusion protein containing a nucleic acid programmed DNA binding protein (napDNAbp) and a polymerase and (ii) a PEgRNA containing a DNA synthesis template encoding a detectable sequence. bring into contact;

PEgRNA complexed with a fusion protein and guiding said fusion protein to the editing site and, if present, to one or more off-target sites;

the prime editing factor 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 embodiment 1, wherein the target nucleotide sequence is a genome.

Aspect 3. The method of embodiment 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 embodiment 1, wherein the editing site is a mutation in a disease-related gene.

Aspect 6. Embodiment 5. The method of embodiment 5, wherein the mutation is a single base substitution, insertion, deletion, or inversion.

Aspect 7. The method of embodiment 1, wherein the disease-associated gene is: adenosine deaminase (ADA) deficiency; alpha-1 antitrypsin deficiency; cystic fibrosis; Duchenne muscular dystrophy; galactosemia; hemochromatosis; Huntington's disease; maple Consisting of syrup urine disease; Marfan syndrome; type 1 neurofibromatosis; congenital nail hyperplasia; phenylketonuria; severe combined immunodeficiency; sickle cell disease; Smith-Lemli-Opitz syndrome; and Tay-Sachs disease. associated with a single gene disorder selected from the group.

Aspect 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.

Aspect 9. The method of embodiment 1, wherein the fusion protein comprises SEQ ID NOs: 101-104, 181-183, 223-244, 277, 325-334, 336, 338, 340, 342, 344, 346, 348, 350, 352, Amino acid sequence of 354, 356, 358, 360, 362, 364, 366, 368, 499-505, 735-761, 776-777, or SEQ ID NO: 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 and at least Having amino acid sequences that have 80%, 85%, 90%, 95%, 98%, or 99% sequence identity.

Aspect 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. .

Aspect 11. The method of embodiment 1, wherein napDNAbp is Cas9 or a variant thereof.

Aspect 12. The method of embodiment 1, wherein napDNAbp is nuclease-active Cas9, nuclease-inactive Cas9 (dCas9), or Cas9 nickase (nCas9).

Aspect 13. The method of embodiment 1, wherein napDNAbp is Cas9 nickase (nCas9).

Aspect 14. The method of embodiment 1, wherein napDNAbp is the amino acid sequence of SEQ ID NO: 18, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO: 18. including.

Aspect 15. The method of embodiment 1, wherein napDNAbp is SpCas9 wild type or any one of SEQ ID NOs : 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487. or at least 80%, 85%, 90%, 95 with any of SEQ ID NOs : 18-88 , 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487. %, 98%, or 99% sequence identity.

Aspect 16. The method of embodiment 1, wherein napDNAbp is an SpCas9 ortholog.

Aspect 17. The method of embodiment 1, wherein napDNAbp is any one amino acid of SEQ ID NO : 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487, or at least 80%, 85%, 90%, 95%, 98% with any of SEQ ID NO: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487; or an amino acid sequence with 99% sequence identity.

Aspect 18. The method of embodiment 1, wherein napDNAbp has 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. Comprising amino acid sequences with at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity.

Aspect 19. The method of embodiment 1, wherein the polymerase comprises RNA-dependent DNA polymerase activity.

Mode 20. The method of embodiment 1, wherein the polymerase is 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 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 SEQ ID NO: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, At least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with any of 159, 235, 454, 471, 516, 662, 700, 701-716, 739-741, and 766 It has an amino acid sequence.

Aspect 23. The method of embodiment 1, wherein the reverse transcriptase is SEQ ID NO: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, 662, 700. , 701-716, 739-741, and 766.

Embodiment 24. The method of embodiment 1, wherein the reverse transcriptase comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity 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 25. The method of embodiment 1, wherein the detectable sequence is 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 The insertion is 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.

Aspect 27. The method of embodiment 1, wherein the detectable sequence is a nucleobase substitution.

Aspect 28. The method of embodiment 1, wherein the detectable sequence is a transition mutation.

Aspect 29. The method of embodiment 1, wherein the detectable sequence is a transversion mutation.

Aspect 30. The method of embodiment 1, wherein the detectable sequence is a single nucleotide substitution: (1) G to T substitution, (2) G to A substitution, (3) G to C substitution, (4) T Substitution of G from (5) Substitution of A from T, (6) Substitution of C from T, (7) Substitution of G from C, (8) Substitution of T from C, (9) Substitution of A from C, selected from the group consisting of (10) A to T substitution, (11) A to G substitution, and (12) A to C substitution.

Aspect 31. The method of embodiment 1, wherein the detectable sequence is a single nucleotide substitution, which converts (1) a G:C base pair into a T:A base pair, and (2) a G:C base pair into an A:T base pair. (3) G:C base pair to C:G base pair, (4) T:A base pair to G:C base pair, (5) T:A base pair to A:T base pair. , (6) T:A base pair to C:G base pair, (7) C:G base pair to G:C base pair, (8) C:G base pair to T:A base pair, ( 9) C:G base pair to A:T base pair, (10) A:T base pair to T:A base pair, (11) A:T base pair to G:C base pair, or (12) )Convert A:T base pair to C:G base pair.

Aspect 32. The method of embodiment 1, wherein the detectable sequence is a barcode sequence.

Aspect 33. The method of embodiment 1, wherein step (d) of determining the nucleotide sequences at the on-target and off-target sites comprises: (i) fragmenting the target nucleotide sequence to form fragments; (ii) adding an adapter sequence to the fragments; (iii) PCR amplify the amplicon using a pair of primers, one primer annealing to an adapter sequence attached to one end of the fragment, and another primer that positions it on the fragment. (iv) sequencing the amplicon to determine the location of the edit.

Group J. PE method mode 1 for recording cell data . 1. A method for recording cellular events by prime editing, the method comprising: (A) injecting into a cell one encoding a prime editing factor fusion protein comprising (i) napDNAbp and an RNA-dependent DNA polymerase, and (ii) PEgRNA. one or more constructs are introduced, expression of the fusion protein and/or PEgRNA is induced by the occurrence of a cellular event, and expression of the fusion protein and/or PEgRNA primes the targeted editing site on the genome of the cell. (B) identifying the detectable sequence and thereby identifying the occurrence of the cellular event.

Aspect 2. The method of embodiment 1, wherein the prime editing of step (A) simultaneously introduces new target editing sites, so that recording of cellular events can occur repeatedly.

Aspect 3. The method of embodiment 1, wherein the PEgRNA comprises an editing template encoding a detectable sequence.

Aspect 4. The method of embodiment 3, wherein the editing template further encodes a new target editing site.

Aspect 5. The method of embodiment 1, wherein the detectable sequence is 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 The insertion is 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 embodiment 1, wherein the detectable sequence is a nucleobase substitution.

Aspect 8. The method of embodiment 1, wherein the detectable sequence is a transition mutation.

Aspect 9. The method of embodiment 1, wherein the detectable sequence is a transversion mutation.

Aspect 10. The method of embodiment 1, wherein the detectable sequence is a single nucleotide substitution, wherein the single nucleotide substitutions include (1) a G to T substitution, (2) a G to A substitution, and (3) a G to C substitution. (4) Substitution from T to G, (5) Substitution from T to A, (6) Substitution from T to C, (7) Substitution from C to G, (8) Substitution from C to T, (9 ) C to A substitution, (10) A to T substitution, (11) A to G substitution, or (12) A to C substitution.

Aspect 11. The method of embodiment 1, wherein the detectable sequence is a single nucleotide substitution, which converts (1) a G:C base pair into a T:A base pair, and (2) a G:C base pair into an A:T base pair. (3) G:C base pair to C:G base pair, (4) T:A base pair to G:C base pair, (5) T:A base pair to A:T base pair. , (6) T:A base pair to C:G base pair, (7) C:G base pair to G:C base pair, (8) C:G base pair to T:A base pair, ( 9) C:G base pair to A:T base pair, (10) A:T base pair to T:A base pair, (11) A:T base pair to G:C base pair, or (12) )Convert A:T base pair to C:G base pair.

Aspect 12. The method of embodiment 1, wherein the detectable sequence is a barcode sequence.

Aspect 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 a cellular event.

Aspect 14. The method of embodiment 1, wherein the detecting step comprises sequencing the edited target site or an amplicon of the edited target site.

Aspect 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. be.

Aspect 16. The method of embodiment 1, wherein napDNAbp is Cas9 or a variant thereof.

Aspect 17. The method of embodiment 1, wherein napDNAbp is nuclease-active Cas9, nuclease-inactive Cas9 (dCas9), or Cas9 nickase (nCas9).

Aspect 18. The method of embodiment 1, wherein napDNAbp is Cas9 nickase (nCas9).

Aspect 19. The method of embodiment 1, wherein napDNAbp is the amino acid sequence of SEQ ID NO: 18 , or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO: 18. including.

Mode 20. The method of embodiment 1, wherein napDNAbp is SpCas9 wild type or any one of SEQ ID NOs : 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487. or at least 80%, 85%, 90% with any of SEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487; A variant thereof with an amino acid sequence that has 95%, 98%, or 99% sequence identity.

Aspect 21. The method of embodiment 1, wherein napDNAbp is an SpCas9 ortholog.

Aspect 22. The method of embodiment 1, wherein napDNAbp has 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 at least 80%, 85%, 90%, 95%, 98% with any of SEQ ID NO: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487 , or an amino acid sequence with 99% sequence identity.

Aspect 23. The method of embodiment 1, wherein the RNA-dependent DNA polymerase is reverse transcriptase.

Aspect 24. 24. The method of embodiment 23, wherein the reverse transcriptase is a naturally occurring wild-type reverse transcriptase, and the amino acid sequence of any one of SEQ ID NO: 89, or at least 80%, 85%, Having amino acid sequences with 90%, 95%, 98%, or 99% sequence identity.

Aspect 25. The method of embodiment 23, wherein the reverse transcriptase is a variant reverse transcriptase, and 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 SEQ ID NO: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, At least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with any of 159, 235, 454, 471, 516, 662, 700, 701-716, 739-741, and 766 It has an amino acid sequence.

Aspect 26. The method of embodiment 1, wherein the fusion protein has an amino acid sequence of any one of SEQ ID NO: 123 and 134 (PE1, PE2), or at least 85 %, 90%, 95%, 98%, or 99% sequence identity.

Aspect 27. The method of embodiment 1, wherein the one or more constructs encoding the prime editing factor fusion protein and/or PEgRNA further comprise one or more promoters that are inducible when a cellular event occurs.

Aspect 28. The method of embodiment 1, wherein the cellular event is marked by a stimulus received by the cell.

Aspect 29. 29. The method of embodiment 28, wherein the stimulus is a small molecule, protein, peptide, amino acid, metabolite, inorganic molecule, organometallic molecule, organic molecule, drug or drug candidate, sugar, lipid, metal, nucleic acid, endogenous or exogenous agent. molecules, light, heat, sound, pressure, mechanical stress, shear stress, or viruses or other microorganisms, changes in pH, or changes in redox conditions that occur during the activation of a biological signal cascade.

Aspect 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 programmed DNA binding protein (napDNAbp) and (b) an RNA-dependent DNA polymerase, said fusion protein being operably linked to a first promoter;

ii. a nucleic acid sequence encoding a prime editing factor guide RNA (PEgRNA) operably linked to a second promoter, where the PEgRNA is complementary to the target sequence; and

iii. origin of replication;
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. Embodiment 30. The cellular data recording plasmid of embodiment 30, wherein the inducible promoter is induced by a cellular event.

Aspect 32. The cellular data recording plasmid of embodiment 30, wherein cellular events are marked by stimuli received by the cell.

Aspect 33. The cell data recording plasmid of embodiment 32, wherein the stimulus is a small molecule, protein, peptide, amino acid, metabolite, inorganic molecule, organometallic molecule, organic molecule, drug or drug candidate, sugar, lipid, metal, nucleic acid, molecules, light, heat, sound, pressure, mechanical stress, shear stress, or viruses or other microorganisms, changes in pH, or changes in redox conditions that occur during activation of biological or extrinsic signal cascades. .

Aspect 34. The cell data recording plasmid of embodiment 30, wherein the first and second promoters are the same.

Aspect 35. Embodiment 30. The cell data recording plasmid of embodiment 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 embodiment 30, wherein the first or second promoter is a constitutive promoter.

Aspect 38. The cell data recording plasmid of embodiment 37, wherein the constitutive promoter is the Lac promoter, the cytomegalovirus (CMV) promoter, the constitutive RNA polymerase III promoter, or the UBC promoter.

Aspect 39. The cell data recording plasmid of embodiment 30, wherein the origin of replication comprises a pSC101, pMB1, pBR322, ColE1, or p15A origin of replication sequence.

Aspect 40. The cell data recording plasmid of embodiment 30, wherein the PEgRNA comprises an editing template encoding a detectable sequence.

Aspect 41. The cell data recording plasmid of aspect 40, wherein the editing template further encodes a new target editing site.

Aspect 42. The cell data recording plasmid of embodiment 30, wherein the detectable sequence is 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 nucleobase insertions.

Aspect 43. The cell data recording plasmid of embodiment 30, wherein the detectable sequence is 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 The deletion is 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 embodiment 30, wherein the detectable sequence is a nucleobase substitution.

Aspect 45. The cell data recording plasmid of embodiment 30, wherein the detectable sequence is a transition mutation.

Aspect 46. The cell data recording plasmid of embodiment 30, wherein the detectable sequence is a transversion mutation.

Aspect 47. The cell data recording plasmid of embodiment 30, wherein the detectable sequence is a single nucleotide substitution, from which the single nucleotide substitution is: (1) a G to T substitution, (2) a G to A substitution, (3) a G to A substitution; ) G to C substitution, (4) T to G substitution, (5) T to A substitution, (6) T to C substitution, (7) C to G substitution, (8) C to T substitution. (9) C to A substitution, (10) A to T substitution, (11) A to G substitution, or (12) A to C substitution.

Aspect 48. The cell data recording plasmid of embodiment 30, wherein the detectable sequence is a single nucleotide substitution, (1) a G:C base pair becomes a T:A base pair, and (2) a G:C base pair becomes an A base pair. :T base pair, (3) G:C base pair to C:G base pair, (4) T:A base pair to G:C base pair, (5) T:A base pair to A:T. (6) T:A base pair to C:G base pair, (7) C:G base pair to G:C base pair, (8) C:G base pair to T:A base pair. (9) C:G base pair to A:T base pair, (10) A:T base pair to T:A base pair, (11) A:T base pair to G:C base pair, or (12) convert A:T base pairs to C:G base pairs.

Aspect 49. The cell data recording plasmid of embodiment 30, wherein the detectable sequence is a barcode sequence.

Aspect 50. The cellular data recording plasmid of embodiment 30, wherein the detectable sequence increases in length over time as a result of repeated insertion of the detectable sequence for each occurrence of a cellular event.

Aspect 51. The cell data recording plasmid of embodiment 30, wherein the detecting step comprises sequencing the edited target site or an amplicon of the edited target site.

Aspect 52. The cell data recording plasmid of embodiment 30, wherein napDNAbp is Cas9, Cas12e, Cas12d, Cas12a, Cas12b1, Cas13a, Cas12c, or Argonaute, or Cas9, Cas12e, Cas12d, Cas12a, Cas12b1, Cas13a, Cas12c, or Argonaute. This is a variant of

Aspect 53. The cell data recording plasmid of embodiment 30, wherein napDNAbp is Cas9 or a variant thereof.

Aspect 54. The cell data recording plasmid of embodiment 30, wherein napDNAbp is nuclease-active Cas9, nuclease-inactive Cas9 (dCas9), or Cas9 nickase (nCas9).

Aspect 55. The cell data recording plasmid of embodiment 30, wherein napDNAbp is Cas9 nickase (nCas9).

Aspect 56. The cell data recording plasmid of embodiment 30, wherein napDNAbp has the amino acid sequence of SEQ ID NO: 18, or at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO: 18. Contains an amino acid sequence with

Aspect 57. The cell data recording plasmid of embodiment 30, wherein napDNAbp is SpCas9 wild type or SEQ ID NO : 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487. at least 80%, 85% with any one amino acid sequence , or with any of SEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487; A variant thereof with an amino acid sequence that has 90%, 95%, 98%, or 99% sequence identity.

Aspect 58. The cell data recording plasmid of embodiment 30, wherein napDNAbp is an SpCas9 ortholog.

Aspect 59. The cell data recording plasmid of embodiment 30, wherein napDNAbp is any one of SEQ ID NOs : 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487. or at least 80%, 85%, 90%, 95% with any of the amino acid sequences , or SEQ ID NOs: 18-88, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487. , 98%, or 99% sequence identity.

Aspect 60. The cell data recording plasmid of embodiment 30, wherein the RNA-dependent DNA polymerase is reverse transcriptase.

Aspect 61. Embodiment 30 The cell data recording plasmid of embodiment 30, wherein the reverse transcriptase is a naturally occurring wild-type reverse transcriptase, the amino acid sequence of any one of SEQ ID NO: 89, or at least 80% with any one of SEQ ID NO: 89; Having amino acid sequences with 85%, 90%, 95%, 98%, or 99% sequence identity.

Aspect 62. The cell data recording plasmid of embodiment 30, wherein the reverse transcriptase is a variant reverse transcriptase, SEQ ID NOs: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, Any one amino acid sequence of 454, 471, 516, 662, 700, 701-716, 739-741, and 766, or SEQ ID NO: 89-100, 105-122, 128-129, 132, 139, 143, 149 , 154, 159, 235, 454, 471, 516, 662, 700, 701-716, 739-741, and 766 at least 80%, 85%, 90%, 95%, 98%, or 99% have amino acid sequences with sequence identity.

Aspect 63. The cell data recording plasmid of embodiment 30, wherein the fusion protein has an amino acid sequence of any one of SEQ ID NOs: 123 and 134 (PE1, PE2), or at least 80% with any one of SEQ ID NOs: 123 and 134 (PE1, PE2). , 85%, 90%, 95%, 98%, or 99% sequence identity.

Aspect 64. A kit for use in a cell comprising the cell data recorder plasmid of any one of embodiments 30-63.

Aspect 65. Embodiment 64. The kit of embodiment 64, wherein the cell is a prokaryotic cell.

Aspect 66. 65. The kit of embodiment 64, wherein the cell is a eukaryotic cell.

Aspect 67. A cell comprising the cell data recording plasmid of any one of embodiments 30-63.

Aspect 68. Embodiment 67. The cell of embodiment 67, wherein the cell is a prokaryotic cell.

Aspect 69. The cell of embodiment 67, wherein the cell is a eukaryotic cell.

Mode 70. 69. The cell of embodiment 69, wherein the eukaryotic cell is a mammalian cell.

Aspect 71. Embodiment 70, wherein the mammalian cell is a human cell.

Aspect 72. A method for recording a cellular event by prime editing, the method comprising: (A) introducing the cellular data recording plasmid of any of aspects 30 to 63 into a cell, the fusion protein and/or PEgRNA recording a cellular event; (B) that expression of the fusion protein and/or PEgRNA results in prime editing of the targeted editing site on the cell's genome to introduce the detectable sequence; and (B) identify the detectable sequence. and thereby identifying the occurrence of cellular events.

Aspect 73. 73. The method of embodiment 72, wherein the step of (A) introducing is by transfection or electroporation.

In equivalents and scope claims, articles such as "a,""an," and "the" may mean one or more than one, unless there is an indication to the contrary or otherwise. Unless it is clear from the context. A claim or statement that includes "or" between one or more members of a group means that one, more than one, or all of the members of the group are present in a given product or process. , adopted by, or otherwise related to, is considered to satisfy, unless there is an indication to the contrary or otherwise is not clear from the context. The invention encompasses embodiments in which exactly one member of the group is present in, employed in, or otherwise pertains to a given product or process. The invention encompasses embodiments in which more than one or all of the members of the group are present in, employed in, or otherwise relate 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 references 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 incorporated reference and this specification, the present specification will control. In addition, any particular embodiment of the invention that belongs to the prior art may be specifically excluded from any one or more of the claims. Since such aspects will be known to those skilled in the art, they may be excluded even if the exclusion is not explicitly stated herein. Any particular aspect of the invention may be excluded from any claim for any reason, whether or not related to the existence of prior art.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. The scope of the embodiments described herein is not intended to be limited to the above description, but rather is as expressed in the appended claims. Those skilled in the art will appreciate that various changes and modifications may be made to this description without departing from the spirit or scope of the invention, as defined in the following claims.

Claims (21)

A system for prime editing, comprising:
Prime editors, including nucleic acid-programmed DNA binding proteins (napDNAbp) and reverse transcriptase, and
Prime Editing Guide RNA (PEgRNA)
including
Here, the prime editor complexed with PEgRNA is
binding to a target DNA sequence comprising a target strand and a complementary non-target strand;
forming (i) an RNA-DNA hybrid comprising the PEgRNA and the target strand and (ii) an R-loop comprising the complementary non-target strand; and
capable of nicking the complementary non-target strand to form a reverse transcriptase prime sequence with a free 3'end;
wherein the PEgRNA comprises (a) a guide RNA and an RNA extension comprising (b) (i) a reverse transcription template sequence containing one or more desired nucleotide changes and (ii) a reverse transcription primer binding site,
wherein the reverse transcription primer binding site is capable of hybridizing with the free 3' end of the non-target strand of the nicked target DNA sequence. napDNAbp is
(i) a Cas9 protein or a functional equivalent thereof;
(ii) is a Cas9 nickase;
(iii) selected from the group consisting of Cas9, CasX, CasY, Cpf1, C2c1, C2c2, C2C3, and Argonaute and having nickase activity;
(iv) is selected from the group consisting of Cas9, Cas12a, and Cas12b1 and has nickase activity; or
(v) having H840A, N854A, or N863A with reference to the canonical SpCas9 sequence;
The system of claim 1. reverse transcriptase is
(i) a reverse transcriptase from a retrovirus or retrotransposon, or
(ii) Moloney murine leukemia virus reverse transcriptase (M-MLV RT) or variant M-MLV RT
3. A system according to claim 1 or 2, wherein: (i) the variant M-MLV RT has the following amino acid substitutions:
P51X, S67X, E69X, L139X, T197X, D200X, H204X, F209X, E302X, T306X, F309X, W313X, T330X, L345X, L435X, N454X, D524X, E562X, D583X, H594X, E607X, E607X, for SEQ ID NO:89 , and D653X, where X is any amino acid substitution,
including one or more of
(ii) the variant M-MLV RT has the following amino acid mutations:
P51L, S67K, E69K, L139P, T197A, D200N, H204R, F209N, E302K, E302R, T306K, F309N, W313F, T330P, L345G, L435G, N454K, D524G, E562Q, D583WQN, L609WQ, H59 for SEQ ID NO:89 , E607K, and D653N
including one or more of
(iii) the variant M-MLV RT comprises D200N, T330P, and/or L603W amino acid substitutions relative to SEQ ID NO:89;
(iv) the variant M-MLV RT comprises amino acid substitutions D200N, T306K, W313F, T330P, and L603W relative to SEQ ID NO:89;
(v) 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;
(vi) the reverse transcriptase has the sequence of SEQ ID NO: 122, or
(vii) variant M-MLV RT is truncated at the C-terminus and contains mutations D200N, T306K, W313F, and/or T330P relative to the amino acid sequence of SEQ ID NO:89;
4. The system of claim 3. 5. The system of claim 4, wherein the truncated variant M-MLV RT comprises the sequence of SEQ ID NO:766. The system of any one of claims 1-5, wherein napDNAbp and reverse transcriptase are connected to form a fusion protein. The fusion protein comprises the structure NH2-[napDNAbp]-[reverse transcriptase]-COOH or NH2-[reverse transcriptase]-[napDNAbp]-COOH, where each of the "]-[" 7. The system of claim 6, which indicates the presence of any peptide linker. 8. The system of claim 6 or 7, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO:134 or an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:134. RNA elongation 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 in length , 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. A system according to any one of clauses. (i) the one or more desired nucleotide changes are within an editing window that is between about -4 and +10 of the PAM sequence, and/or
(ii) the one or more desired nucleotide changes are one or more single base nucleotide changes, deletions of one or more nucleotides, insertions of one or more nucleotides, or combinations thereof, and/or ,
(iii) the one or more desired nucleotide changes are at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 at the nick site , 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42 , 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67 , 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92 , 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides downstream of
A system according to any one of claims 1-9. insertion of one or more nucleotides or deletion of one or more nucleotides in length 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 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 11. The system of claim 10, which is 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, or at least 50 nucleotides. the reverse transcription template sequence is
(i) contains a nucleotide sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the target DNA sequence, and/or
(ii) the system of any one of claims 1-11 encoding a replacement strand that is homologous to the target DNA sequence downstream of the nick, with the exception of the desired nucleotide edit; The system of any one of claims 1-12, wherein the primer binding site is 3-20 nucleotides in length. The system of any one of claims 1-13, wherein the primer binding site is 7-17 nucleotides in length. 15. The system of any one of claims 1-14, wherein the guide RNA and RNA elongation are within a single molecule. 16. The system of any one of claims 1-15, wherein RNA elongation comprises, in the 5' to 3' direction, a reverse transcription template and a primer binding site. One or more polynucleotides encoding the system of any one of claims 1-16. 18. One or more vectors comprising one or more polynucleotides of claim 17. A cell comprising the system of any one of claims 1-16, one or more polynucleotides of claim 17, or one or more vectors of claim 18. (i) a system according to any one of claims 1 to 16, one or more polynucleotides according to claim 17, or one or more vectors according to claim 18, and
(ii) a pharmaceutical composition comprising a pharmaceutically acceptable excipient; A system according to any one of claims 1 to 16, one or more polynucleotides according to claim 17, one or more vectors according to claim 18, claim 19, for use in medicine or the pharmaceutical composition of claim 20.

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