Methods and compositions for editing nucleotide sequences

Abstract

The present disclosure provides compositions and methods for prime editing of target DNA molecules (e.g., genomes) that enables incorporation of nucleotide changes and / or targeted mutagenesis. Nucleotide changes can include single nucleotide changes (e.g., any transition or any transversion), one or more nucleotide insertions, or one or more nucleotide deletions. More specifically, the present disclosure provides fusion proteins comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a polymerase (e.g., a reverse transcriptase) that are guided to a specific DNA sequence by a modified guide RNA (termed a PEgRNA). The PEgRNA (relative to a standard guide RNA) has been altered to comprise an extension portion that provides a DNA synthesis template sequence encoding a single-stranded DNA flap that is homologous to a strand of the targeted endogenous DNA sequence to be edited but comprises a desired nucleotide change(s) and is incorporated into the target DNA molecule after synthesis by a polymerase (e.g., a reverse transcriptase). Also disclosed herein are various methods of utilizing prime editing, including treating trinucleotide repeat contraction diseases, installing targeted peptide tags, treating prion diseases by installing protective mutations, manipulating RNA coding genes to install RNA tags for controlling RNA function and expression, using prime editing to construct complex gene libraries, using prime editing to insert immunogenic epitopes into proteins, using prime editing to insert inducible dimerization domains into protein targets, and delivery methods, among others.

Description

[0001] Government Support

[0002] This application was made with government support under Grant Numbers U01 AI142756, RM1 HG009490, R01 EB022376, and R35 GM118062 awarded by the National Institutes of Health. The government has certain rights in the application.

[0003] Related Applications and Incorporation by Reference

[0004] This U.S. provisional application is related to and incorporates by reference the following applications, i.e., U.S. Provisional Application No. 62 / 820,813 (Attorney Docket No. B1195.70074US00), filed March 19, 2019, U.S. Provisional Application No. 62 / 858,958 (Attorney Docket No. B1195.70074US01), filed June 7, 2019, U.S. Provisional Application No. 62 / 889,996 (Attorney Docket No. B1195.70074US02), filed August 21, 2019, U.S. Provisional Application No. 62 / 922,654 (Attorney Docket No. B1195.70083US00), filed August 21, 2019, U.S. Provisional Application No. 62 / 889,996, 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 October 10, 2019, U.S. Provisional Application No. 62 / 931,195 (Attorney Docket No. B1195.70074US04), filed 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 March 17, 2020, and U.S. Provisional Application No. (no serial number obtained at time of filing of this application) (Attorney Docket No. B1195.70083US03), filed March 17, 2020. BACKGROUND

[0006] According to certain estimates, pathogenic single nucleotide mutations cause about 50% of human diseases with a genetic component 7 Unfortunately, despite decades of gene therapy exploration, treatment options for these patients with genetic diseases remain very limited8 Perhaps the most parsimonious solution to this therapeutic challenge is to directly correct the single nucleotide mutation in the patient's genome, which would address the root cause of the disease and potentially provide a durable benefit. Although this strategy was previously unimaginable, the advent of the CRISPR / Cas system 9 has now brought genome editing capabilities within reach. By directly designing a guide RNA (gRNA) sequence comprising ~20 nucleotides complementary to the target DNA sequence, a CRISPR-associated (Cas) nuclease can be specifically directed to nearly any conceivable genomic locus 1，2 To date, several monomeric bacterial Cas nuclease systems have been identified and adapted for genome editing applications 10 This natural diversity of Cas nucleases, coupled with an increasing number of engineered variants 11-14 has provided fertile ground for the development of new genome editing technologies.

[0007] While gene disruption using CRISPR is now a mature technology, precise editing of single base pairs in the human genome remains a major challenge 3 Homology-directed repair (HDR) has long been used to repair templates in human cells and other organisms using a donor DNA encoding the desired edit to insert, correct, or exchange DNA sequences at double-strand break (DSB) locations. However, traditional HDR is very inefficient in most human cell types, especially in non-dividing cells, and competitive non-homologous end joining (NHEJ) predominantly leads to indel byproducts 16 Other issues are associated with the generation of DSBs, which can lead to large chromosomal rearrangements and deletions at the target locus 17 , or activation of the p53 axis, leading to growth arrest and apoptosis 18，19 .

[0008] Several approaches have been explored to address these shortcomings of HDR. For example, it has been demonstrated that utilizing oligonucleotide donors to repair single-stranded DNA breaks (nicks) reduces indel formation, but the yield of the desired repair product is still low 20 Other strategies attempt to bias repair towards HDR over NHEJ using small molecules and biological reagents 21-23 However, the effectiveness of these approaches can depend on cell type, and perturbing normal cellular states can lead to undesirable and unpredictable effects.

[0009] Recently, the inventors led by Professor David Liu developed base editing as a technology to edit target nucleotides without forming DSBs or relying on HDR 4-6，24-27Direct modification of DNA bases by Cas fusion deaminases can convert C.G to T.A, or A.T to G.C, within a short target window (about 5-7 bases) with very high efficiency. Thus, base editors have rapidly been adopted by the scientific community. However, the following factors limit their universality in precise genome editing: (1) "bystander editing" of non-target C or A bases within the target window is observed; (2) a mixture of target nucleotide products is observed; (3) the target base must be located 15+2 nucleotides upstream of the PAM sequence; and (5) repair of small insertion and deletion mutations is not possible.

[0010] Accordingly, developing the ability to flexibly introduce any desired single nucleotide change and / or the ability to install base pair insertions or deletions (e.g., at least 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more base pair insertions or deletions) and / or the ability to change or modify nucleotide sequences at a target site with high specificity and high efficiency would greatly expand the scope and therapeutic potential of CRISPR-based genome editing technologies. SUMMARY

[0012] A novel genome editing platform, termed "prime editing," is described. Prime editing is a universal and precise method of genome editing that directly writes new genetic information to a specified DNA site using a nucleic acid programmable DNA binding protein ("napDNAbp") in conjunction with a polymerase (i.e., in the form of a fusion protein or otherwise provided in trans to the napDNAbp), where the prime editing system is programmed with a prime editing (PE) guide RNA ("PEgRNA") that both specifies the target site and provides a template for the synthesis of a replacement DNA strand engineered into the guide RNA (e.g., at the 5' or 3' end or in an internal portion of the guide RNA) for the desired edit. The replacement strand containing the desired edit (e.g., a single nucleobase substitution) shares the same sequence as the endogenous strand of the target site to be edited (except that it includes the desired edit). Through DNA repair and / or replication mechanisms, the endogenous strand of the target site is replaced with the newly synthesized replacement strand containing the desired edit. In certain instances, prime editing can be considered a "search and replace" genome editing technology, as the prime editing described herein not only searches for and locates the desired target site to be edited, but also simultaneously encodes the replacement strand containing the desired edit, which gets installed in place of the corresponding target site endogenous DNA strand.

[0013] The guide editor portion of the present disclosure relates, in part, to the discovery that CRISPR / Cas-based precise genome editing can be performed with or adapted to the mechanism of target-primed reverse transcription (TPRT) or "prime editing," with high efficiency and genetic plasticity (as depicted in different embodiments of FIGS. 1A-1F Mammalian non-LTR retrotransposons and bacterial group II introns naturally use TPRT 28，29 The present inventors herein use Cas protein-reverse transcriptase fusions or related systems, with guide RNAs to target a specific DNA sequence, make a single-strand nick at the target site, and use the nicked DNA as a primer for reverse transcription of an engineered reverse transcriptase template that integrates with the guide RNA. However, while this concept starts with a prime editor using a reverse transcriptase as the DNA polymerase component, the prime editors described herein are not limited to reverse transcriptases, but can include DNA polymerases that use DNA in fact. Indeed, while the present application refers throughout to prime editors with "reverse transcriptases," it is presented herein that reverse transcriptases are only one type of DNA polymerase that can work with prime editing. Thus, wherever the specification refers to "reverse transcriptases," one of ordinary skill in the art will understand that any suitable DNA polymerase can be used in place of a reverse transcriptase. Thus, in one aspect, a prime editor can comprise a Cas9 (or equivalent napDNAbp) that is programmed to target a DNA sequence by associating it with a specialized guide RNA (i.e., a PEgRNA) that comprises a spacer sequence that anneals to a complementary protospacer in the target DNA. The specialized guide RNA also comprises in extended form new genetic information that encodes a DNA replacement strand comprising a desired genetic change for replacing a corresponding endogenous DNA strand at the target site. To transfer information from the PEgRNA to the target DNA, the prime editing mechanism includes making a nick in one strand of the DNA at the target site to expose a 3'-hydroxyl. The exposed 3'-hydroxyl can then be used to directly extend the DNA in the target site primed on the PEgRNA that encodes the edit. In different embodiments, the extension, which provides a template for the polymerization of the replacement strand containing the edit, can be formed from RNA or DNA. In the case of RNA extension, the polymerase of the prime editor can be an RNA-dependent DNA polymerase (e.g., a reverse transcriptase). In the case of DNA extension, the polymerase of the prime editor can be a DNA-dependent DNA polymerase.

[0014] The newly synthesized strand formed by the guide editor disclosed herein (i.e., the substituted DNA strand containing the desired edit) is homologous to (i.e., has the same sequence as) the genomic target sequence, in addition to containing the desired nucleotide change (e.g., single nucleotide change, deletion, or insertion, or a combination thereof). The newly synthesized (or substituted) DNA strand can also be referred to as a single-stranded DNA flap, which can compete for hybridization with the complementary homologous endogenous DNA strand, thereby displacing the corresponding endogenous strand. In certain embodiments, the system can be used in combination with an error-prone reverse transcriptase (e.g., provided as a fusion protein with the Cas9 domain, or provided in trans with the Cas9 domain). The error-prone reverse transcriptase can introduce changes during the synthesis of the single-stranded DNA flap. Thus, in certain embodiments, the error-prone reverse transcriptase can be utilized to introduce nucleotide changes into the target DNA. Depending on the error-prone reverse transcriptase used with the system, the changes can be random or non-random.

[0015] Disassembly of the hybrid intermediate (including the single-stranded DNA flap synthesized by the reverse transcriptase hybridized to the endogenous DNA strand) can include removal of the resulting endogenous DNA substitution flap (e.g., using a 5' end DNA flap endonuclease, FENl), ligation of the synthesized single-stranded DNA flap to the target DNA, and assimilation of the desired nucleotide change due to cellular DNA repair and / or replication processes. Because DNA synthesis provided by the template provides single nucleotide precision for any nucleotide modification (including insertions and deletions), the scope of this approach is very broad, and numerous applications in basic science and therapeutics are envisioned.

[0016] In an aspect, the present specification provides a fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a reverse transcriptase. In various embodiments, the fusion protein is capable of genome editing by target primed reverse transcription in the presence of an extended guide RNA.

[0017] In certain 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).

[0018] In certain embodiments, the napDNAbp is selected from the group consisting of: Cas9, Casl2e, Casl2d, Casl2a, Casl2bl, Casl3a, Casl2c, and an Argonaute protein, and optionally has nickase activity.

[0019] In other embodiments, the fusion protein, when complexed with an extended guide RNA, is capable of binding to a target DNA sequence.

[0020] In other embodiments, the target DNA sequence comprises a target strand and a complementary non-target strand.

[0021] In other embodiments, binding of the fusion protein complexed with the extended guide RNA forms an R-loop. The R-loop can comprise (i) an RNA-DNA hybrid comprising the extended guide RNA and the target strand, and (ii) the complementary non-target strand.

[0022] In other embodiments, the complementary non-target strand is nicked to form a reverse transcriptase priming sequence having a free 3' end.

[0023] In different embodiments, the extended guide RNA comprises (a) a guide RNA and (b) an RNA extension at a 5' or 3' end of the guide RNA or in an intramolecular position of the guide RNA. The RNA extension can comprise (i) a reverse transcription template sequence comprising a desired nucleotide alteration, (ii) a reverse transcription primer binding site, and (iii) an optional linker sequence. In different embodiments, the reverse transcription template sequence can encode a single-stranded DNA flap that is complementary to an endogenous DNA sequence adjacent to a nick site, wherein the single-stranded DNA flap comprises a desired nucleotide alteration.

[0024] In different embodiments, the RNA extension is at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides, at least 22 nucleotides, at least 23 nucleotides, at least 24 nucleotides, or at least 25 nucleotides in length.

[0025] In other embodiments, the single-stranded DNA flap can hybridize to an endogenous DNA sequence adjacent to a nick site, thereby installing a desired nucleotide alteration. In other embodiments, the single-stranded DNA flap displaces an endogenous DNA sequence adjacent to a nick site and having a free 5' end. In certain embodiments, the displaced endogenous DNA having a 5' end is excised by the cell.

[0026] In different embodiments, cellular repair of the single-stranded DNA flap results in installation of a desired nucleotide alteration, thereby forming a desired product.

[0027] In different other embodiments, the desired nucleotide alteration is installed in an editing window between about -4 to +10 of a PAM sequence.

[0028] In other embodiments, the desired nucleotide change is installed in an editing window between about -5 to +5 of the nick site, or between about -10 to +10 of the nick site, or between about -20 to +20 of the nick site, or between about -30 to +30 of the nick site, or between about -40 to +40 of the nick site, or between about -50 to +50 of the nick site, or between about -60 to +60 of the nick site, or between about -70 to +70 of the nick site, or between about -80 to +80 of the nick site, or between about -90 to +90 of the nick site, or between about -100 to +100 of the nick site, or between about -200 to +200 of the nick site.

[0029] In different embodiments, the napDNAbp comprises the amino acid sequence of SEQ ID NO: 18. In different other embodiments, the napDNAbp comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 26-39, 42-61, 75-76, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487 (Cas9); (SpCas9); SEQ ID NOs: 77-86 (CP-Cas9); SEQ ID NOs: 18-25 and 87-88 (SpCas9); and SEQ ID NOs: 62-72 (Cas12).

[0030] In other embodiments, the reverse transcriptase of the disclosed fusion proteins and / or compositions can comprise the amino acid sequence of any one of SEQ ID NOs: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, 662, 700-716, 739-742, and 766. In other embodiments, the reverse transcriptase can comprise an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, 662, 700-716, 739-742, and 766. These sequences can be naturally occurring reverse transcriptase sequences, as from a retrovirus or a retrotransposon, which sequences can be recombinant.

[0031] In different other embodiments, the fusion proteins disclosed herein can comprise various structural configurations. For example, the fusion protein can comprise the structure NH2- [napDNAbp]-[reverse transcriptase]-COOH; or NH2-[reverse transcriptase]-[napDNAbp]-COOH, wherein each "-[" case indicates the presence of an optional linker sequence.

[0032] In different embodiments, the linker sequence comprises the amino acid sequence of SEQ ID NO: 127, 165-176, 446, 453, and 767-769, or an amino acid sequence that is at least 80%, 85%, or 90%, or 95%, or 99% identical to any one of the linker amino acid sequences of SEQ ID NO: 127, 165-176, 446, 453, and 767-769.

[0033] In different embodiments, the desired nucleotide change(s) incorporated into the target DNA can be a single nucleotide change (e.g., a transition or a transversion), an insertion of one or more nucleotides, or a deletion of one or more nucleotides.

[0034] In certain instances, the insertion is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, or at least 500 nucleotides in length.

[0035] In certain other instances, the deletion is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, or at least 500 nucleotides in length.

[0036] 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 at the 5' end of the guide RNA. In other embodiments, the RNA extension can be located at an intramolecular position of the guide RNA, however, preferably the intramolecular positioning of the extension portion does not disrupt the function of the protospacer.

[0037] In different embodiments, the extended guide RNA is capable of binding to a napDNAbp and directing the napDNAbp to a target DNA sequence. The target DNA sequence can comprise a target strand and a complementary non-target strand, wherein the guide RNA hybridizes to the target strand to form an RNA-DNA hybrid and an R-loop.

[0038] In different embodiments of the extended guide RNA, the at least one RNA extension can comprise a reverse transcription template sequence. In different other embodiments, the RNA extension can further comprise a reverse transcription primer binding site. In still further embodiments, the RNA extension can comprise a linker or spacer sequence that links the RNA extension to the guide RNA.

[0039] In different embodiments, the length of the RNA extension can be at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides, at least 22 nucleotides, at least 23 nucleotides, at least 24 nucleotides, at least 25 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides, at least 80 nucleotides, at least 90 nucleotides, at least 100 nucleotides, at least 150 nucleotides, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, or at least 500 nucleotides.

[0040] In other embodiments, the reverse transcription template sequence is at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides, at least 80 nucleotides, at least 90 nucleotides, at least 100 nucleotides, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, or at least 500 nucleotides in length.

[0041] In other embodiments, the length of the reverse transcription primer binding site sequence is at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides, at least 80 nucleotides, at least 90 nucleotides, at least 100 nucleotides, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, or at least 500 nucleotides.

[0042] In other embodiments, the length of the optional linker or spacer sequence is at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides, at least 80 nucleotides, at least 90 nucleotides, at least 100 nucleotides, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, or at least 500 nucleotides.

[0043] In different embodiments of the extended guide RNA, the reverse transcription template sequence can encode a single-stranded DNA flap that is complementary to an endogenous DNA sequence adjacent to the nick site, where the single-stranded DNA flap comprises the desired nucleotide change. The single-stranded DNA flap can displace the endogenous single-stranded DNA at the nick site. The endogenous single-stranded DNA displaced at the nick site can have a 5' end and form an endogenous flap that can be excised by the cell. In different embodiments, excision of the 5' end endogenous flap can help drive product formation, as removal of the 5' end endogenous flap facilitates hybridization of the single-stranded 3' DNA flap to the corresponding complementary DNA strand, as well as incorporation or assimilation of the desired nucleotide change carried by the single-stranded 3' DNA flap into the target DNA.

[0044] In different embodiments of the extended guide RNA, cellular repair of the single-stranded DNA flap results in installation of the desired nucleotide change, thereby forming the desired product.

[0045] In certain embodiments, a PEgRNA comprises the nucleotide sequence of: SEQ ID NOs: 101-104, 131, 181-183, 222-234, 237-244, 277, 324-330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 394, 429-442, 499-505, 641-649, 678-692, 735-736, 738, 757-761, 776-777, 2997-3103, 3113-3121, 3305-3455, 3479-3493, 3522-3540, 3549-3556, 3628-3698, 3755-3810, 3874, 3890-3901, 3905-3911, 3913-3929, and 3972-3989, or a nucleotide sequence having at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99% sequence identity to any one of the sequences of: SEQ ID NOs: 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 3972-3989.

[0046] In yet another aspect of the application, the present specification provides a complex comprising a fusion protein described herein and any of the extended guide RNAs described above.

[0047] In other aspects of the application, the present description provides a complex comprising a napDNAbp and an extended guide RNA. The napDNAbp can be a Cas9 nickase, or can be an amino acid sequence of SEQ ID NOs: 42-57 (Cas9 nickases) and 65 (AsCasl2a nickase), or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to any one of the amino acid sequences of SEQ ID NOs: 42-57 (Cas9 nickases) and 65 (AsCasl2a nickase).

[0048] In different embodiments involving the complex, the extended guide RNA is capable of directing the napDNAbp to a target DNA sequence. In different embodiments, the reverse transcriptase can be provided in trans, i.e., provided from a source different from the complex itself. For example, the reverse transcriptase can be provided to the same cell as the complex by introducing a separate vector encoding the reverse transcriptase alone.

[0049] In other aspects, the present description provides polynucleotides. In certain embodiments, the polynucleotides can encode any of the fusion proteins disclosed herein. In certain other embodiments, the polynucleotides can encode any of the napDNAbps disclosed herein. In still further embodiments, the polynucleotides can encode any of the reverse transcriptases disclosed herein. In other embodiments, the polynucleotides can encode any of the extended guide RNAs, any of the reverse transcription template sequences, or any of the reverse transcription primer sites, or any of the optional linker sequences disclosed herein.

[0050] In other aspects, the present description provides vectors comprising the polynucleotides described herein. Thus, in certain embodiments, the vectors comprise a polynucleotide for encoding a fusion protein comprising a napDNAbp and a reverse transcriptase. In other embodiments, the vectors comprise polynucleotides encoding a napDNAbp and a reverse transcriptase, respectively. In other embodiments, the vectors can comprise a polynucleotide encoding an extended guide RNA. In different embodiments, the vectors can comprise one or more polynucleotides encoding a napDNAbp, a reverse transcriptase, and an extended guide RNA, on the same or different vectors.

[0051] In other aspects, the present description provides cells comprising a fusion protein as described herein and an extended guide RNA. The cells can be transformed with vectors comprising the fusion protein, the napDNAbp, the reverse transcriptase, and the extended guide RNA. These genetic elements can be comprised on the same vector or on different vectors.

[0052] In another aspect, the present description provides pharmaceutical compositions. In certain embodiments, the pharmaceutical compositions comprise one or more of a napDNAbp, a fusion protein, a reverse transcriptase, and an extended guide RNA. In certain embodiments, the fusion proteins described herein and a pharmaceutically acceptable excipient. In other embodiments, the pharmaceutical compositions comprise any of the extended guide RNAs described herein and a pharmaceutically acceptable excipient. In other embodiments, the pharmaceutical compositions comprise any of the extended guide RNAs described herein in combination with any of the fusion proteins described herein and a pharmaceutically acceptable excipient. In other embodiments, the pharmaceutical compositions comprise any polynucleotide sequence encoding one or more of a napDNAbp, a fusion protein, a reverse transcriptase, and an extended guide RNA. In other embodiments, the various components disclosed herein can be isolated into one or more pharmaceutical compositions. For example, a first pharmaceutical composition can comprise a fusion protein or a napDNAbp, a second pharmaceutical composition can comprise a reverse transcriptase, and a third pharmaceutical composition can comprise an extended guide RNA.

[0053] In yet another aspect, the present disclosure provides kits. In one embodiment, the kits comprise one or more polynucleotides encoding one or more components, including a fusion protein, a napDNAbp, a reverse transcriptase, and an extended guide RNA. The kits can also comprise vectors, cells, and isolated polypeptide preparations, including any of the fusion proteins, napDNAbps, or reverse transcriptases disclosed herein.

[0054] In yet another aspect, the present disclosure provides methods of using the disclosed compositions of matter.

[0055] In one embodiment, the method involves a method of installing a desired nucleotide change in a double-stranded DNA sequence. The method first involves contacting the double-stranded DNA sequence with a complex comprising a fusion protein and an extended guide RNA, wherein the fusion protein comprises a napDNAbp and a reverse transcriptase, wherein the extended guide RNA comprises a reverse transcription template sequence comprising the desired nucleotide change. Next, the method involves making a nick in the double-stranded DNA sequence on the non-target strand, thereby generating a free single-stranded DNA with a 3’ end. The method then involves hybridizing the 3’ end of the free single-stranded DNA to the reverse transcription template sequence, thereby priming the reverse transcriptase domain. Then, the method involves polymerizing a strand of DNA from the 3’ end, thereby generating a single-stranded DNA flap comprising the desired nucleotide change. The method then involves displacing the endogenous DNA strand adjacent to the cleavage site with the single-stranded DNA flap, thereby installing the desired nucleotide change in the double-stranded DNA sequence.

[0056] In other embodiments, the present disclosure provides methods for introducing one or more changes in a nucleotide sequence of a DNA molecule at a target locus, comprising contacting the DNA molecule with a nucleic acid programmable DNA binding protein (napDNAbp) and a guide RNA that targets the napDNAbp to the target locus, wherein the guide RNA comprises a reverse transcriptase (RT) template sequence comprising at least one desired nucleotide change. Next, the method involves forming an exposed 3' end in a DNA strand at the target locus, followed by hybridizing the exposed 3' end to the RT template sequence to prime reverse transcription. Next, a single-stranded DNA flap comprising the at least one desired nucleotide change is synthesized or polymerized by the reverse transcriptase based on the RT template sequence. Finally, the at least one desired nucleotide change is incorporated into the corresponding endogenous DNA, thereby introducing one or more changes in the nucleotide sequence of the DNA molecule at the target locus.

[0057] In other embodiments, the present disclosure provides methods for introducing one or more changes in a nucleotide sequence of a DNA molecule at a target locus by target primed reverse transcription, the method comprising: (a) contacting a DNA molecule at the target locus with: (i) a fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a reverse transcriptase, and (ii) a guide RNA comprising an RT template comprising a desired nucleotide change; (b) performing target primed reverse transcription on the RT template to generate a single-stranded DNA comprising the desired nucleotide change; and (c) incorporating the desired nucleotide change into the DNA molecule at the target locus by DNA repair and / or replication processes.

[0058] In certain embodiments, the step of displacing the endogenous DNA strand comprises: (i) hybridizing the single-stranded DNA flap to the endogenous DNA strand proximal to the cleavage site to create a sequence mismatch; (ii) excising the endogenous DNA strand; and (iii) repairing the mismatch to form the desired product comprising the desired nucleotide change in both DNA strands.

[0059] In different 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) a A to T substitution, (11) a A to G substitution, or (12) a A to C substitution.

[0060] In other embodiments, the desired nucleotide change can convert (1) a G:C base pair to a T:A base pair, (2) a G:C base pair to an A:T base pair, (3) a G:C base pair to a C:G base pair, (4) a T:A base pair to a G:C base pair, (5) a T:A base pair to an A:T base pair, (6) a T:A base pair to a C:G base pair, (7) a C:G base pair to a G:C base pair, (8) a C:G base pair to a T:A base pair, (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.

[0061] In other embodiments, the method introduces a desired nucleotide change that is an insertion. In certain instances, the insertion is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, or at least 500 nucleotides in length.

[0062] In other embodiments, the method introduces a desired nucleotide change that is a deletion. In certain other instances, the deletion is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, or at least 500 nucleotides in length.

[0063] In various embodiments, the desired nucleotide change corrects a disease-associated gene. The disease-associated gene can be associated with a monogenetic disorder selected from the group consisting of adenosine deaminase (ADA) deficiency; alpha- 1 antitrypsin deficiency; cystic fibrosis; Duchenne muscular dystrophy; galactosemia; hemochromatosis; Huntington's disease; maple syrup urine disease; Marfan syndrome; neurofibromatosis type 1; pachyonychia congenita; phenylketonuria; severe combined immunodeficiency; sickle cell disease; Smith-Lemli-Opitz syndrome; and Tay-Sachs disease. In other embodiments, the disease-associated gene can be associated with a polygenic disorder selected from the group consisting of heart disease; hypertension; Alzheimer's disease; arthritis; diabetes; cancer; and obesity.

[0064] The methods disclosed herein can involve a fusion protein having a napDNAbp that is a nuclease dead Cas9 (dCas9), a Cas9 nickase (nCas9), or a nuclease active Cas9. In other embodiments, the napDNAbp and reverse transcriptase are not encoded as a single fusion protein, but can be provided in separate constructs. Thus, in some embodiments, the reverse transcriptase can be provided in trans relative to the napDNAbp (rather than by way of a fusion protein).

[0065] In various embodiments involving methods, the napDNAbp can comprise the amino acid sequence of SEQ ID NOs: 26-61, 75-76, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487 (Cas9); (SpCas9); SEQ ID NOs: 77-86 (CP-Cas9); SEQ ID NOs: 18-25 and 87-88 (SpCas9); and SEQ ID NOs: 62-72 (Cas12). The napDNAbp can also comprise an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 26-61, 75-76, 126, 130, 137, 141, 147, 153, 157, 445, 460, 467, and 482-487 (Cas9); (SpCas9); SEQ ID NOs: 77-86 (CP-Cas9); SEQ ID NOs: 18-25 and 87-88 (SpCas9); and SEQ ID NOs: 62-72 (Cas12).

[0066] In various embodiments involving a method, the reverse transcriptase can comprise the amino acid sequence of SEQ ID NOs: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, 662, 700-716, 739-742, and 766. The reverse transcriptase can also comprise an amino acid sequence that is at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 99% identical to any one of the amino acid sequences of SEQ ID NOs: 89-100, 105-122, 128-129, 132, 139, 143, 149, 154, 159, 235, 454, 471, 516, 662, 700-716, 739-742, and 766.

[0067] The method can involve using a PEgRNA comprising a nucleotide sequence of SEQ ID NOs: 101-104, 131, 181-183, 222-234, 237-244, 277, 324-330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 394, 429-442, 499-505, 641-649, 678-692, 735-736, 738, 757-761, 776-777, 2997-3103, 3113-3121, 3305-3455, 3479-3493, 3522-3540, 3549-3556, 3628-3698, 3755-3810, 3874, 3890-3901, 3905-3911, 3913-3929, and 3972-3989, or a nucleotide sequence having at least 80% sequence identity thereto, or at least 85% sequence identity thereto, or at least 90% sequence identity thereto, or at least 95% sequence identity thereto, or at least 99% sequence identity thereto. The method can comprise using an extended guide RNA comprising an RNA extension at the 3’ end, wherein the RNA extension comprises a reverse transcription template sequence.

[0068] The method can comprise using an extended guide RNA comprising an RNA extension at the 5’ end, wherein the RNA extension comprises a reverse transcription template sequence.

[0069] The method can comprise using an extended guide RNA comprising an RNA extension at an intra-molecular position of the guide RNA, wherein the RNA extension comprises a reverse transcription template sequence.

[0070] The method may include using an extended guide RNA having one or more RNA extensions having 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.

[0071] It should be understood that the foregoing concepts and other concepts discussed below can be arranged in any suitable combination, as this disclosure is not limited in this respect. Furthermore, other advantages and novel features of this disclosure will become apparent when considered in conjunction with the accompanying drawings, based on the following detailed description of various non-limiting embodiments. DETAILED DESCRIPTION

[0073] The following figures form part of this specification and are included to further illustrate certain aspects of this disclosure. A better understanding of this disclosure can be achieved by referring to one or more of these figures in conjunction with the detailed description of the specific embodiments presented herein.

[0074] FIG. 1A A schematic diagram is provided illustrating an exemplary process for introducing single nucleotide alterations and / or insertions and / or deletions into DNA molecules (e.g., genomes) using a guide RNA extended from a fusion protein complex containing a reverse transcriptase fused to a Cas9 protein. In this embodiment, the guide RNA is extended at its 3′ end to contain a reverse transcriptase template sequence. The schematic diagram illustrates how the complex formed by the reverse transcriptase (RT) fused to the Cas9 nicking enzyme and the guide RNA (gRNA) binds to a DNA target site and creates a nick in a PAM-containing DNA strand adjacent to the target nucleotide. The RT enzyme uses the nicked DNA as a primer for DNA synthesis from the gRNA, which serves as a template for synthesizing a new DNA strand encoding the desired edit. The editing process illustrated may be referred to as target-initiated reverse transcription editing (TRT editing) or equivalently, “guided editing.”

[0075] FIG. 1B Provide with FIG. 1A The same diagram is used, except that the guide editor complex is more generally represented as [napDNAbp]-[P]:PEgRNA or [P]-[napDNAbp]:PEgRNA, where "P" refers to any polymerase (such as reverse transcriptase), "napDNAbp" refers to a nucleic acid programmable DNA-binding protein (such as SpCas9), and "PEgRNA" refers to the guide editing RNA, and "]-[" refers to an optional adapter. As described elsewhere, for example FIGS. 3A-3GAs shown, the PEgRNA comprises a 5' extension arm comprising a primer binding site and a DNA synthesis template. While not shown, it is contemplated that the extension arm of the PEgRNA (i.e., that comprises the primer binding site and the DNA synthesis template) can be DNA or RNA. The particular polymerase contemplated in this configuration depends on the nature of the DNA synthesis template. For example, if the DNA synthesis template is RNA, then the polymerase can be an RNA-dependent DNA polymerase (e.g., a reverse transcriptase). If the DNA synthesis template is DNA, then the polymerase can be a DNA-dependent DNA polymerase.

[0076] FIG. 1C A schematic of an exemplary process for introducing a single nucleotide change and / or insertion and / or deletion into a DNA molecule (e.g., a genome) using a fusion protein complexed extension of a guide RNA molecule comprising a reverse transcriptase fused to a Cas9 protein. In this embodiment, the guide RNA is extended at the 5' end to comprise a reverse transcriptase template sequence. This schematic shows how a complex of a reverse transcriptase (RT) fused to a Cas9 nickase binds to a DNA target site and makes a nick in the PAM-containing DNA strand adjacent to the target nucleotide. The RT enzyme uses the nicked DNA as a primer for DNA synthesis from the gRNA, which serves as a template for synthesizing a new DNA strand encoding the desired edit. The edit process shown can be referred to as target primed reverse transcriptional editing (TRT editing) or equivalently "prime editing."

[0077] FIG. 1D A schematic of the same as FIG. 1D the same except that the prime editor complex is more generally represented as [napDNAbp]-[P]:PEgRNA or [P]-[napDNAbp]:PEgRNA, where "P" refers to any polymerase (e.g., a reverse transcriptase), "napDNAbp" refers to a nucleic acid programmable DNA binding protein (e.g., SpCas9), "PEgRNA" refers to a prime editing guide RNA, and "-[" refers to an optional linker. As described elsewhere, for example FIGS. 3A-3G As shown, the PEgRNA comprises a 3' extension arm comprising a primer binding site and a DNA synthesis template. While not shown, it is contemplated that the extension arm of the PEgRNA (i.e., that comprises the primer binding site and the DNA synthesis template) can be DNA or RNA. The particular polymerase contemplated in this configuration depends on the nature of the DNA synthesis template. For example, if the DNA synthesis template is RNA, then the polymerase can be an RNA-dependent DNA polymerase (e.g., a reverse transcriptase). If the DNA synthesis template is DNA, then the polymerase can be a DNA-dependent DNA polymerase. In various embodiments, the PEgRNA can be engineered or synthesized to incorporate a DNA-based DNA synthesis template.

[0078] FIG. 1E is a schematic diagram depicting an exemplary process by which a synthesized DNA single strand, which contains a desired nucleotide change, is resolved to incorporate the desired nucleotide change into the DNA. As shown, the synthetic edit strand (or "mutagenic strand") is then synthesized, balanced with the endogenous strand, and cleavage and ligation of the flap of the endogenous strand results in resolution of the mismatched DNA duplex by action of endogenous DNA repair and / or replication processes to incorporate the DNA edit.

[0079] FIG. 1F is a schematic diagram showing a resolution method that can incorporate a "reverse strand nick" to aid in driving formation of the desired product over a reversion product. In reverse strand nicking, a second Cas9 / gRNA complex is used to introduce a second nick on the strand opposite the initial nicked strand. This induces endogenous cellular DNA repair and / or replication processes to preferentially displace the non-edit strand (i.e., the strand containing the second nick site). FIG. 1E

[0080] FIG. 1G ​Another schematic of an exemplary process for introducing a single nucleotide change and / or an insertion and / or a deletion into a DNA molecule (e.g., a genome) using a nucleic acid programmable DNA binding protein (napDNAbp) complexed extended guide RNA is provided. This process can be considered an embodiment of prime editing. The extended guide RNA comprises an extension at the 3' or 5' end of the guide RNA or in a position within the molecule of the guide RNA. In step (a), a napDNAbp / gRNA complex contacts the DNA molecule, and the gRNA directs the napDNAbp to bind to the target locus. In step (b), one of the DNA strands at the target locus (the R-loop strand, or the PAM-containing strand, or the non-target DNA strand, or the protospacer strand) is introduced with a nick (e.g., by a nuclease or a chemical agent), thereby creating an available 3' end in one of the strands at the target locus. In certain embodiments, the nick is created in the DNA strand corresponding to the R-loop strand, i.e., the strand that is not hybridized to the guide RNA sequence. In step (c), the 3' end DNA strand interacts with the extended portion of the guide RNA to prime reverse transcription. In certain embodiments, the 3' end DNA strand hybridizes to a specific RT priming sequence on the extended portion of the guide RNA. In step (d), a reverse transcriptase is introduced, which synthesizes a single-stranded DNA from the 3' end of the priming site to the 3' end of the guide RNA. This forms a single-stranded DNA flap comprising the desired nucleotide change (e.g., a single base change, an insertion, or a deletion, or a combination thereof). In step (e), the napDNAbp and the guide RNA are released. Steps (f) and (g) involve resolution of the single-stranded DNA flap, such that the desired nucleotide change is incorporated into the target locus. This process can be driven towards forming the desired product by removal of the corresponding 5' endogenous DNA flap, which forms once the 3' single-stranded DNA flap invades and hybridizes to the complementary sequence of the other strand. This process can also be driven towards forming the product with a second strand nick, as shown. This process introduces at least one or more of the following genetic changes: transversions, transitions, deletions, and insertions. FIG. 1F

[0081] FIG. 1H is a schematic depicting the types of genetic changes possible with 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), inversions, and insertions (including short and long insertions).

[0082] FIG. 1I ​Figure 2 is a schematic depicting a temporal second strand nicking by PE3b (PE3b = PE2 guide editor fusion protein + PE gRNA + second strand nicking guide RNA) example. Temporal second strand nicking is a variant of second strand nicking to facilitate formation of a desired edit product. The "temporal" term refers to the fact that the second strand nick of the non-editing strand only occurs after the desired edit is installed to the editing strand. This avoids the two strands having nicks at the same time leading to a double stranded DNA break.

[0083] FIGS. 1J-1KVariants of the guided editing contemplated herein are depicted that utilize any programmable nuclease domain such as a zinc finger nuclease (ZFN) or a transcription activator-like effector nuclease (TALEN) in place of a napDNAbp (e.g., SpCas9 nickase). Thus, it is contemplated that a suitable nuclease does not necessarily need to be "programmed" by a nucleic acid targeting molecule (e.g., a guide RNA), but rather can be programmed by defining the specificity of the DNA binding domain, e.g., of the nuclease in particular. Just as with guided editing utilizing a napDNAbp moiety, it is preferred that such alternative programmable nucleases are modified so as to only cleave one target DNA strand. In other words, the programmable nuclease should preferably function as a nickase. Once a programmable nuclease (e.g., a ZFN or TALEN) is selected, additional functionality can be engineered into the system to make it function in a guided editing-like mechanism. For example, the programmable nuclease can be modified by coupling (e.g., by a chemical linker) to an RNA or DNA extension arm, where the extension arm comprises a primer binding site (PBS) and a DNA synthesis template. The programmable nuclease can also be coupled (e.g., by a chemical or amino acid linker) to a polymerase, the nature of which depends on whether the extension arm is DNA or RNA. In the case of an RNA extension arm, the polymerase can be an RNA-dependent DNA polymerase (e.g., a reverse transcriptase). In the case of a DNA extension arm, the polymerase can be a DNA-dependent DNA polymerase (e.g., a prokaryotic polymerase, including Pol I, Pol II, or Pol III, or a eukaryotic polymerase, including Pola, Polb, Polg, Pold, Pole, or Polz). The system can also include additional functionality added as a fusion to the programmable nuclease or added in trans to facilitate the overall reaction (e.g., (a) a helicase unwinds the DNA at the cleavage site to form a cleaved strand with an available 3' end to serve as a primer, (b) a flap endonuclease (e.g., FEN1) helps remove the endogenous strand on the cleaved strand to drive the reaction toward replacement of the endogenous strand with the synthesized strand, or (c) a nCas9:gRNA complex forms a second site nick on the opposite strand, which can help drive integration of the synthesized repair by favored cellular repair of the non-edited strand). In a manner analogous to guided editing utilizing a napDNAbp, such a complex with an otherwise programmable nuclease can be used to synthesize and then permanently install a new synthesized DNA replacement strand carrying the edit of interest into the target site of the DNA.

[0084] FIG. 1LPAM sequence) can be referred to as the "PAM strand" or "editing strand." In different embodiments, the nicking site of the PE complex is located in the protospacer of the PAM strand (e.g., with SpCas9-based PE). The positioning of the nick will be characteristic of the particular Cas9 forming the PE. For example, with SpCas9-based PE, the nicking site is in the phosphodiester bond between base 3 (the "-3" position relative to position 1 of the PAM sequence) and base 4 (the "-4" position relative to position 1 of the PAM sequence). The nicking site in the protospacer forms a free 3' hydroxyl, as shown in the figure below, which complexes with the primer binding site of the PEgRNA extension arm and provides a substrate to initiate single-stranded DNA encoded polymerization by the DNA synthesis template of the PEgRNA extension arm. This polymerization reaction is catalyzed by the polymerase (e.g., reverse transcriptase) of the PE fusion protein in the 5' to 3' direction. The polymerization is terminated before reaching the gRNA core (e.g., by inclusion of a polymerization termination signal or secondary structure that acts to terminate the polymerization activity of the PE), resulting in a single-stranded DNA flap extending from the original 3' hydroxyl of the nicked PAM strand. The DNA synthesis template encodes a single-stranded DNA that is homologous to the endogenous 5' end DNA single strand immediately adjacent to the nicking site of the PAM strand and incorporates the desired nucleotide change (e.g., single base substitution, insertion, deletion, inversion).The position of the desired edit can be any position downstream of the nick site on the PAM strand, can include position +1, +2, +3, +4 (the start of the PAM site), +5 (position 2 of the PAM site), +6 (position 3 of the PAM site), +7, +8, +9, +10, +11, +12, +13, +14, +15, +16, +17, +18, +19, +20, +21, +22, +23, +24, +25, +26, +27, +28, +29, +30, +31, +32, +33, +34, +35, +36, +37, +38, +39, +40, +41, +42, +43, +44, +45, +46, +47, +48, +49, +50, +51, +52, +53, +54, +55, +56, +57, +58, +59, +60, +61, +62, +63, +64, +65, +66, +67, +68, +69, +70, +71, +72, +73, +74, +75, +76, +77, +78, +79, +80, +81, +82, +83, +84, +85, +86, +87, +88, +89, +90, +91, +92, +93, +94, +95, +96, +97, +98, +99, +100, +101, +102, +103, +104, +105, +106, +107, +108, +109, +110, +111, +112, +113, +114, +115, +116, +117, +118, +119, +120, +121, +122, +123, +124, +125, +126, +127, +128, +129, +130, +131, +132, +133, +134, +135, +136, +137, +138, +139, +140, +141, +142, +143, +144, +145, +146, +147, +148, +149, or +150 or more (relative to the downstream position of the nick site). Once the 3' end single-stranded DNA (containing the edit of interest) displaces the endogenous 5' end single-stranded DNA, the DNA repair and replication processes will result in permanent installation of the edit at the edit position on the PAM strand, which then corrects the mismatch present on the non-PAM strand at the edit position. In this way, the edit extends to both DNA strands at the target DNA position. It should be understood that reference to "edit strand" and "non-edit" are intended to merely depict the DNA strands involved in the PE mechanism. The "edit strand" is the strand that is first edited by displacing the 5' end single-stranded DNA immediately downstream of the nick site with the synthesized 3' end single-stranded DNA containing the desired edit. The "non-edit" strand is the strand that pairs with the edit strand, but which itself is also edited to be complementary to the edit strand, particularly the edit of interest, through repair and / or replication.

[0085] FIG. 1M The prime editing mechanism is described that shows the anatomy of the target DNA, guides the editor complex, and the interaction between the PEgRNA and the target DNA. First, a prime editor comprising a polymerase (e.g., reverse transcriptase) and a napDNAbp (e.g., SpCas9 nickase, e.g., SpCas9 with an inactivating mutation in the HNH nuclease domain (e.g., H840A) or with an inactivating mutation in the RuvC nuclease domain (D10A)) is complexed with a PEgRNA and a DNA with a target DNA to be edited. The PEgRNA comprises a spacer, a gRNA core (a.k.a. gRNA scaffold or gRNA backbone) that binds to the napDNAbp, and an extension arm. The extension arm can be at the 3' end of the PEgRNA molecule, the 5' end of the PEgRNA molecule, or somewhere within the PEgRNA molecule. As shown, the extension arm is at the 3' end of the PEgRNA. The extension arm comprises, in the 3' to 5' direction, a primer binding site and a DNA synthesis template (including a homologous region of interest for editing (i.e., a homology arm) that is homologous to the immediately PAM strand single-stranded DNA immediately 5' of the nick site. As shown, once the nick is introduced, thereby creating a free 3' hydroxyl immediately upstream of the nick site, the region immediately upstream of the nick site on the PAM strand anneals to the complementary sequence at the 3' end of the extension arm known as the primer binding site, creating a short double-stranded region with an available 3' hydroxyl end, which forms a substrate for the polymerase of the prime editor complex. The polymerase (e.g., reverse transcriptase) then polymerizes a DNA strand from the 3' hydroxyl end to the end of the extension arm. The sequence of the single-stranded DNA is encoded by the DNA synthesis template, which is the portion of the extension arm (i.e., not including the primer binding site) that is "read" by the polymerase to synthesize the new DNA. This polymerization effectively extends the sequence of the original 3' hydroxyl end of the initial nick site. The DNA synthesis template encodes single-stranded DNA that not only contains the desired edit, but also contains a region that is homologous to the endogenous single-stranded DNA immediately downstream of the nick site on the PAM strand. Next, the encoded 3' end single-stranded DNA (i.e., 3' single-stranded DNA flap) displaces the corresponding homologous endogenous 5' end DNA single-strand immediately downstream of the nick site on the PAM strand, forming a DNA intermediate with a 5' end single-stranded DNA flap that is removed by the cell (e.g., by a flap endonuclease). The 3' end single-stranded DNA flap, which anneals to the complementary sequence of the endogenous 5' end single-stranded DNA flap, is ligated to the endogenous strand upon removal of the 5' DNA flap. The desired edit in the 3' end single-stranded DNA flap, now annealed and ligated, forms a mismatch with the complementary strand, which undergoes a round of DNA repair and / or replication, thereby permanently installing the desired edit on both strands.

[0086] FIG. 2Three Cas complexes (SpCas9, SaCas9, and LbCasl2a) and their PAMs, gRNAs, and DNA cleavage characteristics that can be used with the prime editors described herein are shown. This figure shows the design of complexes involving SpCas9, SaCas9, and LbCasl2a.

[0087] FIGS. 3A-3F Designs of engineered 5' prime editor gRNAs FIG. 3A , 3' prime editor gRNAs FIG. 3B , and intramolecular extensions FIG. 3C are shown. Extended guide RNAs (or extended gRNAs) are also referred to herein as PEgRNAs or "prime editing guide RNAs." FIG. 3D and FIG. 3E provide additional embodiments of 3' and 5' prime editor gRNAs (PEgRNAs), respectively. FIG. 3F Interactions between 3' end prime editor guide RNAs and target DNA sequences are shown. FIGS. 3A-3C Embodiments of the prime editor template sequence (i.e., or more broadly referred to as a DNA synthesis template, as shown, since the RT is simply a class of polymerases that can be used in the context of the prime editor), primer binding site, and exemplary arrangement of optional linker sequences in the 3', 5', and intramolecular versions of the extension, as well as the general arrangement of the spacer and core regions are depicted. The disclosed prime editing process is not limited to these configurations of the extended guide RNA. FIG. 3Dembodiments, the PEgRNA can comprise a secondary RNA structure, such as, but not limited to, a hairpin, stem / loop, toe loop, RNA binding protein recruiting domain (e.g., MS2 aptamer that recruits and binds the MS2 cp protein). For example, such secondary structure can be within the spacer, gRNA core, or extension arm, particularly within the el and / or e2 modifier regions. In addition to secondary RNA structures, the PEgRNA can also comprise (e.g., within the el and / or e2 modifier regions) a chemical linker or a poly(N) linker or tail, where "N" can be any nucleobase. In some embodiments (e.g., as shown in FIG. 72(c)), the chemical linker can serve to prevent reverse transcription of the sgRNA scaffold or core. Furthermore, in certain embodiments (e.g., see FIG. 72(c)), the extension arm (3) can be composed of RNA or DNA, and / or can include one or more nucleobase analogs (e.g., which can add functionality, such as temperature elasticity). Still further, the orientation of the extension arm (3) can be the natural 5' to 3' direction, or synthesized in the opposite orientation (3' to 5', relative to the orientation of the entire PEgRNA molecule). It should also be noted that one of ordinary skill in the art will be able to select an appropriate DNA polymerase for use in the directed editing, which can be implemented as a fusion with the napDNAbp or as a separate moiety in trans, to synthesize the 3' single-stranded DNA flap containing the desired template encoded for the desired edit, depending on the nature of the nucleic acid material (i.e., DNA or RNA) of the extension arm. For example, if the extension arm is RNA, the DNA polymerase can be a reverse transcriptase or any other suitable RNA-dependent DNA polymerase. However, if the extension arm is DNA, the DNA polymerase can be a DNA-dependent DNA polymerase.In different embodiments, the DNA polymerase can be provided in trans, for example by using an RNA-protein recruitment domain (e.g., MS2 hairpins installed on the PEgRNA (e.g., in the el or e2 region or elsewhere, and MS2 cp proteins fused to the DNA polymerase, thereby co-localizing the DNA polymerase to the PEgRNA). It should also be noted that the primer binding site does not typically form part of the template for the DNA polymerase (e.g., reverse transcriptase) to encode the resulting 3' single-stranded DNA flap containing the desired edit. Thus, the designation "DNA synthesis template" refers to the region or portion of the extended arm (3) that is used by the DNA polymerase as a template to encode the desired 3' single-stranded DNA flap containing the edit and the region that is homologous to the 5' endogenous single-stranded DNA flap that is replaced by the 3' single-stranded DNA strand product of the guided edit DNA synthesis. In some embodiments, the DNA synthesis template includes the "edit template" and a "homology arm," or one or more homology arms, for example, before and after the edit template. The edit template can be as small as a single nucleotide substitution, or it can be an insertion or a DNA inversion. In addition, the edit template can also include a deletion, which can be engineered by encoding homology arms that contain the desired deletion. In other embodiments, the DNA synthesis template can also include the e2 region or a portion thereof. For example, if the e2 region contains a secondary structure that results in termination of DNA polymerase activity, it is possible that the function of the DNA polymerase terminates before any portion of the e2 region is actually encoded into the DNA. It is also possible that a portion or even the entire e2 region is encoded into the DNA. How much of e2 is actually used as a template will depend on its composition and whether that composition disrupts the function of the DNA polymerase.

[0088] FIG. 3Eembodiments, the PEgRNA can comprise secondary RNA structures such as, but not limited to, hairpins, stem / loops, toe loops, RNA binding protein recruiting domains (e.g., MS2 aptamer that recruits and binds the MS2 cp protein). These secondary structures can be located anywhere in the PEgRNA molecule. For example, such secondary structures can be located within the spacer, gRNA core, or extension arm, particularly within the el and / or e2 modification regions. In addition to secondary RNA structures, the PEgRNA can also comprise (e.g., within the el and / or e2 modification regions) a chemical linker or a poly(N) linker or tail, where “N” can be any nucleobase. In some embodiments (e.g., as shown in FIG. 72(c)), the chemical linker can serve to prevent reverse transcription of the sgRNA scaffold or core. Furthermore, in certain embodiments (e.g., see FIG. 72(c)), the extension arm (3) can be composed of RNA or DNA, and / or can include one or more nucleobase analogs (e.g., which can add functionality such as temperature elasticity). Still further, the orientation of the extension arm (3) can be the natural 5’ to 3’ direction, or synthesized in the opposite orientation (3’ to 5’, relative to the orientation of the entire PEgRNA molecule). It should also be noted that one of ordinary skill in the art will be able to select an appropriate DNA polymerase for the properties of the nucleic acid material (i.e., DNA or RNA) of the extension arm, for the guided editing, which can be provided as a fusion with the napDNAbp or as a separate moiety in trans to synthesize the 3’ single-stranded DNA flap containing the desired template encoded for the desired edit. For example, if the extension arm is RNA, the DNA polymerase can be a reverse transcriptase or any other suitable RNA-dependent DNA polymerase. However, if the extension arm is DNA, the DNA polymerase can be a DNA-dependent DNA polymerase.In different embodiments, the provision of the DNA polymerase can be in trans, for example by using an RNA-protein recruitment domain (e.g., MS2 hairpins installed on the PEgRNA (e.g., in the el or e2 region or elsewhere, and MS2 cp proteins fused to the DNA polymerase, thereby co-localizing the DNA polymerase to the PEgRNA). It should also be noted that the primer binding site does not typically form part of the template for the DNA polymerase (e.g., reverse transcriptase) to encode the resulting 3' single-stranded DNA flap containing the desired edit. Thus, the designation "DNA synthesis template" refers to the region or portion of the extended arm (3) that is used by the DNA polymerase as a template to encode the desired 3' single-stranded DNA flap containing the edit and the region homologous to the 5' endogenous single-stranded DNA flap that is replaced by the 3' single-stranded DNA strand product of the guided edit DNA synthesis. In some embodiments, the DNA synthesis template includes the "edit template" and the "homology arm," or one or more homology arms, for example, before and after the edit template. The edit template can be as small as a single nucleotide substitution, or it can be an insertion or a DNA inversion. In addition, the edit template can also include a deletion, which can be engineered by encoding homology arms containing the desired deletion. In other embodiments, the DNA synthesis template can also include the e2 region or a portion thereof. For example, if the e2 region contains a secondary structure that results in termination of DNA polymerase activity, it is possible that the function of the DNA polymerase is terminated before any portion of the e2 region is actually encoded into the DNA. It is also possible that a portion or even the entire e2 region is encoded into the DNA. How much of e2 is actually used as a template will depend on its composition and whether that composition interrupts the function of the DNA polymerase.

[0089] FIG. 3FFigure 1. Schematic depicting the interaction of a typical PEgRNA with a target site of double-stranded DNA and the concomitant production of a 3' single-stranded DNA flap containing a genetic change of interest. The double-stranded DNA is shown as the top strand (i.e., the target strand) in the 3' to 5' direction and the lower strand (i.e., the PAM strand or non-target strand) in the 5' to 3' direction. The top strand contains the complement of the "protospacer" and the complement of the PAM sequence and is referred to as the "target strand" because it is the strand to which the spacer of the PEgRNA is targeted and anneals. The complementary lower strand is referred to as the "non-target strand" or "PAM strand" or "protospacer strand" because it contains the PAM sequence (e.g., NGG) and the protospacer. Although not shown, the depicted PEgRNA would be complexed with the Cas9 or equivalent domain of the prime editor fusion protein. As shown in the schematic, the spacer of the PEgRNA anneals to the complementary region of the protospacer sequence of the target strand. This interaction forms a DNA / RNA hybrid between the spacer RNA and the complementary sequence of the protospacer DNA and induces the formation of an R-loop in the protospacer. As taught elsewhere herein, the Cas9 protein (not shown) then induces a nick in the non-target strand, as shown. This then results in the formation of a 3' ssDNA flap region immediately upstream of the nick site, which interacts with the 3' end of the PEgRNA's 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. In turn, the reverse transcriptase (e.g., provided in trans or as a fusion protein in cis, linked to the Cas9 construct) polymerizes the single-stranded DNA encoded by the DNA synthesis template (comprising the edit template (B) and the homology arm (C)). Polymerization continues to the 5' end of the extension arm. The polymerized strand of ssDNA forms a ssDNA 3' end flap, which invades the endogenous DNA, displaces the corresponding endogenous strand (which is removed as a 5' end DNA flap of the endogenous DNA), and installs the desired nucleotide edit (single nucleotide base pair change, deletion, insertion (including entire genes)) through the naturally occurring DNA repair / reproduction cycle. FIG. 1G

[0090] FIG. 3G ​This paper depicts another embodiment of the guided editing anticipated in this paper. Specifically, the top schematic depicts one embodiment of the guided editor (PE), which comprises a fusion protein of napDNAbp (e.g., SpCas9) and polymerase (e.g., reverse transcriptase) linked by a adapter. The PE forms a complex with the PEgRNA by binding to the gRNA core of the PEgRNA. In the illustrated embodiment, the PEgRNA is equipped with a 3′ extension arm containing a primer binding site (PBS) starting from the 3′ end, followed by the DNA synthesis template. The bottom schematic depicts a variant of the guided editor, referred to as the “trans-guided editor (tPE)”. In this embodiment, the DNA synthesis template and PBS are separated from the PEgRNA and presented on a separate molecule, referred to as the trans-guided editor RNA template (“tPERT”), which contains an RNA-protein recruitment domain (e.g., MS2 hairpin). The PE itself is further modified to include a fusion with an rPERT recruitment protein (“RP”), which is a protein that specifically recognizes and binds to the RNA-protein recruitment domain. In the example where the RNA protein recruitment domain is an MS2 hairpin, the corresponding rPERT recruiting protein could be MS2cp from the MS2 tagging system. The MS2 tagging system is based on the natural interaction between the MS2 phage coat protein (“MCP” or “MS2cp”) and stem-loop or hairpin structures present in the phage genome, i.e., “MS2 hairpins” or “MS2 aptamers”. In the case of trans-guided 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 trans-form PBS and DNA synthesis templates used in guided editing, such as... FIG. 3H The example shown.

[0091] FIG. 3H The process of trans-guided editing is described. In this embodiment, the trans-guided editor comprises a “PE2” guide editor (i.e., a fusion of Gas9(H840A) and the variant MMLV RT) fused to the MS2cp protein (i.e., a class of recruitment proteins that recognize and bind to the MS2 aptamer) and complexed with sgRNA (i.e., a standard guide RNA opposite to PEgRNA). The trans-guided editor binds to the target DNA and creates a nick in the non-target strand. The MS2cp protein trans-recruits tPERT through a specific interaction with the RNA protein recruitment domain of the tPERT molecule. tPERT co-localizes with the trans-guided editor, thereby trans-providing PBS and a DNA synthesis template for reverse transcriptase polymerase to synthesize a single-stranded DNA lobe having a 3′ end and containing the desired genetic information encoded by the DNA synthesis template.

[0092] FIGS. 4A-4EIn vitro TPRT assay (i.e., prime editing assay) showing the fluorescently labeled DNA substrate gRNA templated extension by RT enzyme. FIG. 4A Schematic of PAGE showing fluorescently labeled DNA substrate gRNA templated extension by RT enzyme. FIG. 4B TPRT (i.e., prime editing) with pre-nicked substrate, dGas9, and 5'-extended gRNAs of different synthetic template lengths. FIG. 4C RT reaction with pre-nicked DNA substrate in the absence of Gas9. FIG. 4D TPRT (i.e., prime editing) on intact dsDNA substrate with Cas9 (H840A) and 5'-extended gRNAs. FIG. 4E 3' extended gRNA templates in the context of pre-nicked and intact dsDNA substrates. All reactions utilized M-MLV RT.

[0093] FIG. 5 In vitro validation using 5'-extended gRNAs with different lengths of synthetic templates. A fluorescently labeled (Cy5) DNA target was used as the substrate and was pre-nicked in this set of experiments. The Gas9 used in these experiments was catalytically dead Cas9 (dCas9) and the RT used was commercial RT derived from Moloney murine leukemia virus (M-MLV), Superscript III. The dCas9:gRNA complex was formed from purified components. The fluorescently labeled DNA substrate was then added with dNTPs and the RT enzyme. After incubation at 37°C for 1 hour, the reaction products were analyzed by denaturing urea-polyacrylamide gel electrophoresis (PAGE). The gel image shows that the original DNA strand was extended to a length consistent with the length of the reverse transcription template.

[0094] FIG. 6 In vitro validation using 5'-extended gRNAs with different lengths of synthetic templates, closely paralleling those shown in FIG. 5 However, in this set of experiments, the DNA substrate was not pre-nicked. The Gas9 used in these experiments was Cas9 nickase (SpyCas9 H840A mutant) and the RT used was commercial RT derived from Moloney murine leukemia virus (M-MLV), Superscript III. The reaction products were analyzed by denaturing urea-polyacrylamide gel electrophoresis (PAGE). As shown in the gel, the nickase was able to efficiently cleave the DNA strand when a standard gRNA was used (gRNA_0, lane 3).

[0095] FIG. 7The 3' extension supports DNA synthesis and does not significantly affect Cas9 nickase activity. When using dGas9 or Cas9 nickase, substrates that are pre-nicked (black arrows) are almost quantitatively converted to RT products (lanes 4 and 5). With intact substrates, over 50% conversion to RT products is observed (red arrows) (lane 3). Commercial RT, Superscript III, derived from Moloney murine leukemia virus (M-MLV) was used with Cas9 nickase (SpyCas9 H840A mutant), catalytically dead Cas9 (dCas9).

[0096] FIG. 8 A two-color experiment to determine if RT reactions preferentially occur in cis with gRNA (bound in the same complex) is shown. Two independent experiments were performed with 5'-extended and 3'-extended gRNAs. Products were analyzed by PAGE. Product ratios were calculated as (Cy3 cis / Cy3 trans) / (Cy5 trans / Cy5 cis).

[0097] FIGS. 9A-9D A flap model substrate is shown. FIG. 9A A dual FP reporter for flap-directed mutagenesis is shown. FIG. 9B Termination codon repair in HEK cells is shown. FIG. 9C Yeast colonies sequenced after flap repair are shown. FIG. 9D Testing of different flap features in human cells is shown.

[0098] FIG. 10 Prime editing on a plasmid substrate is shown. A dual fluorescent reporter plasmid was constructed for expression in yeast (S. cerevisiae). Expression of this construct in yeast only produces GFP. In vitro prime editing reactions introduce point mutations and the parental plasmid or in vitro Cas9 (H840A) nicked plasmid is transformed into yeast. Clones are visualized by fluorescent imaging. Yeast dual FP plasmid transformants are shown. Transforming the parental plasmid or the in vitro Cas9 (H840A) nicked plasmid only produces green GFP expressing colonies. Prime editing reactions using 5'-extended or 3'-extended gRNAs produce a mixture of green and yellow colonies. The latter express both GFP and mCherry. More yellow colonies are observed with 3' extended gRNAs. A positive control without a stop codon is also shown.

[0099] FIG. 11 Prime editing on a plasmid substrate similar to the experiment in FIG. 10 Prime editing on a plasmid substrate similar to the experiment in

[0100] FIG. 12 Edit products of prime editing on plasmid substrates characterized by Sanger sequencing are shown. Individual colonies from TRT transformation were selected and analyzed by Sanger sequencing. Precise edits were observed by sequencing selected colonies. Green colonies contain plasmids with original DNA sequence, while yellow colonies contain precise mutations designed by prime editing gRNAs. No other point mutations or indels were observed.

[0101] FIG. 13 Potential scope of new prime editing technology is shown and compared to deaminase-mediated base editor technology.

[0102] FIG. 14 Schematic showing editing in human cells.

[0103] FIG. 15 Extension of primer binding site in gRNA is shown.

[0104] FIG. 16 Truncated gRNA for proximity targeting is shown.

[0105] FIGS. 17A-17C Graph showing % T to A conversion at the target nucleotide after component transfection in human embryonic kidney (HEK) cells. FIG. 17A Data showing results using N-terminal fusion of wild type MLV reverse transcriptase with Cas9 (H840A) nickase (32 amino acid linker). FIG. 17B Similar to FIG. 17A except for C-terminal fusion of RT enzyme. FIG. 17C Similar to FIG. 17A except the linker between MLVRT and Cas9 is 60 amino acids long instead of 32 amino acids.

[0106] FIG. 18 High purity T to A editing at HEK3 position by high throughput amplicon sequencing is shown. Output of sequencing analysis shows the most abundant genotype of edited cells.

[0107] FIG. 19 Editing efficiency (blue bars) and indel rate (orange bars) at the target nucleotide are shown. WT refers to wild type MLV RT enzyme. Mutant enzymes (M1 to M4) contain mutations listed on the right. Editing rate was quantified by high throughput sequencing of genomic DNA amplicons.

[0108] FIG. 20Editing efficiency of target nucleotides when a single-strand nick is introduced adjacent to the target nucleotide in the complementary DNA strand is shown. Nick generation at different distances from the target nucleotide (triangles) was tested. Editing efficiency of the target base pair (blue bars) is shown along with indel formation rate (orange bars). "None" examples do not contain a complementary strand nick generation guide RNA. Editing rates were quantified by high-throughput sequencing of genomic DNA amplicons.

[0109] FIG. 21 Processed high-throughput sequencing data is shown, demonstrating the expected T to A transversion mutation and the general absence of other major genome editing byproducts.

[0110] FIG. 22 A schematic of an exemplary process for targeted mutagenesis at a target locus using a nucleic acid programmable DNA binding protein (napDNAbp) complexed with an extended guide RNA, utilizing an error-prone reverse transcriptase (i.e., prime editing with error-prone RT) is provided. This process can be referred to as an embodiment of prime editing for targeted mutagenesis. The extended guide RNA comprises an extension at the 3' or 5' end of the guide RNA or at an intramolecular position of the guide RNA. In step (a), the napDNAbp / gRNA complex is contacted with a DNA molecule, and the gRNA directs the napDNAbp to bind to the target locus to be mutagenized. In step (b), a nick is introduced in one of the DNA strands at the target locus (e.g., by a nuclease or chemical agent), thereby creating an available 3' end in one of the strands at the target locus. In certain embodiments, the nick is created in the DNA strand corresponding to the R-loop strand, i.e., the strand that is not hybridized to the guide RNA sequence. In step (c), the 3' end DNA strand interacts with the extended portion of the guide RNA to prime reverse transcription. In certain embodiments, the 3' end DNA strand hybridizes to a specific RT priming sequence on the extended portion of the guide RNA. In step (d), an error-prone reverse transcriptase is introduced, which synthesizes a mutagenized single-stranded DNA from the 3' end of the priming site to the 3' end of the guide RNA. Exemplary mutations are indicated with an asterisk "*". This forms a single-stranded DNA flap comprising the desired mutagenized region. In step (e), the napDNAbp and guide RNA are released. Steps (f) and (g) involve resolution of the single-stranded DNA flap (comprising the mutagenized region), such that the desired mutagenized region is integrated into the target locus. This process can be driven toward the desired product formation by removal of the corresponding 5' endogenous DNA flap, which is formed once the 3' single-stranded DNA flap invades and hybridizes to the complementary sequence on the other strand. This process can also be driven toward product formation with a second strand nicking event, as exemplified. Following endogenous DNA repair and / or replication processes, the mutagenized region is incorporated into both DNA strands of the DNA locus. FIG. 1F

[0111] FIG. 23 ​is a schematic of gRNA design for reducing trinucleotide repeat sequences and reducing trinucleotide repeat sequences using TPRT genome editing (i.e., prime editing). Trinucleotide repeat expansions are associated with many human diseases, including Huntington’s disease, fragile X syndrome, and Friedreich’s ataxia. The most common trinucleotide repeats contain CAG triplets, but GAA triplets (Friedreich’s ataxia) and CGG triplets (fragile X syndrome) also exist. Inheritance of an expanded predisposition or parent allele that has been expanded increases the likelihood of disease. Hypothetically, prime editing can be used to correct the pathogenic expansion of trinucleotide repeats. The region upstream of the repeat region can be nicked by an RNA-guided nuclease, then used to prime synthesis of a new DNA strand containing a healthy number of repeats (dependent on the specific gene and disease). Following the repeat sequence, a short homology segment is added that matches the identity of the sequence on the other end of the adjacent repeat sequence (red strand). Invasion of the newly synthesized strand, and subsequent replacement of the endogenous DNA with the newly synthesized flap, results in a reduced repeat allele.

[0112] FIG. 24 is a schematic showing a precise 10 nucleotide deletion obtained using prime editing. The guide RNA designed to target the HEK3 locus has a reverse transcription template that encodes a 10 nucleotide deletion after the nicking site. Amplicon sequencing was used to assess editing efficiency of transfected HEK cells.

[0113] FIG. 25is a schematic showing gRNA design and utilization of TPRT genome editing (i.e., prime editing) of a peptide-tagged gene at an endogenous genomic locus. The FlAsH and ReAsH tagging systems include two parts: (1) a fluorophore-biarsenical probe, and (2) a gene-encoded peptide containing a four-cysteine motif, such as the sequence FLNCCPGCCMEP (SEQ ID NO: 1). When expressed in cells, proteins containing a four-cysteine motif can be fluorescently labeled with the fluorophore-biarsenical probe (see reference: J. Am. Chem. Soc, 2002, 124(21), pp6063-6076. DOI: 10.1021 / ja017687n). The “sortagging” system employs a bacterial sortase that covalently conjugates a labeled peptide probe to a protein containing a suitable peptide substrate (see reference: Nat. Chem. Biol. 2007 Nov;3(11):707-8. DOI: 10.1038 / nchembio.2007.31). The FLAG tag (DYKDDDDK (SEQ ID NO: 2)), V5 tag (GKPIPNPLLGLDST (SEQ ID NO: 3)), GCN4 tag (EELLSKNYHLENEVARLKK (SEQ ID NO: 4)), HA tag (YPYDVPDYA (SEQ ID NO: 4) ID NO: 5)), and Myc-tag (EQKLISEEDL (SEQ ID NO: 6)) are commonly used as epitope tags for immunoassays. The p-clamp encoded peptide sequence (FCPF (SEQ ID NO: 622)) can be labeled with a pentafluorinated aromatic substrate (reference: Nat. Chem. 2016 Feb;8(2):120-8. doi: 10.1038 / nchem.2413).

[0114] FIG. 26A shows the precise installation of His6-tag and FLAG-tag into genomic DNA. Guide RNAs targeting the HEK3 locus were designed with reverse transcription templates that encode either an 18-nt His-tag insertion or a 24-nt FLAG-tag insertion. Amplicon sequencing was used to assess editing efficiency in transfected HEK cells. Note that the full 24-nt sequence of the FLAG-tag is out of the viewing frame (sequencing confirms full and precise insertion). FIG. 26B shows a schematic outlining various applications involving protein / peptide tagging, including (a) making proteins soluble or insoluble, (b) altering or tracking cellular localization of proteins, (c) extending protein half-life, (d) facilitating protein purification, and (e) facilitating protein detection.

[0115] FIG. 27 A summary of prime editing by installing a protective mutation in PRNP that prevents or halts prion disease progression. The PEgRNA sequence corresponds to SEQ ID NO:4082 (i.e., 5' of sgRNA scaffold) on the left and SEQ ID NO:4083 (i.e., 3' of sgRNA scaffold) on the right

[0116] FIG. 28A A schematic of PE-based insertion of a sequence encoding an RNA motif. FIG. 28B A list (non-exhaustive) of some example motifs that can be inserted and their functions.

[0117] FIG. 29A A depiction of a prime editor. FIG. 29B A possible modification of a PE-directed genome, plasmid, or viral DNA is shown. FIG. 29C An example protocol for inserting a library of peptide loops into a prescribed protein (in this case GFP) by a library of PEgRNAs is shown. FIG. 29D An example of a possible programmable deletion of a codon or N- or C-terminal truncation of a protein using different PEgRNAs is shown. Deletions are expected to occur with minimal production of frameshift mutations.

[0118] FIG. 30 A possible protocol for repeated insertion of codons in a continuous evolution system such as PACE is shown.

[0119] FIG. 31 A diagram showing an engineered gRNA with a gRNA core, a ~20 nt spacer matching the targeted gene sequence, a reverse transcription template with an immunogenic epitope nucleotide sequence, and a primer binding site matching the targeted gene sequence.

[0120] FIG. 32 A schematic showing the use of prime editing as a means to insert known immunogenic epitopes into endogenous or exogenous genomic DNA, resulting in modification of the corresponding protein.

[0121] FIG. 33FIG. 1 is a schematic diagram showing the use of prime editing to determine off-target editing for PEgRNA design for primer binding sequence insertion and primer binding insertion into genomic DNA. In this embodiment, prime editing is performed within a living cell, tissue, or animal model. In the first step, a suitable PEgRNA is designed. The upper schematic shows an exemplary PEgRNA that can be used in this regard. The spacer region in the PEgRNA (labeled "protospacer") is complementary to one of the strands of the genomic target. The PE:PEgRNA complex (i.e., the PE complex) installs a single-stranded 3' end flap at the nick site, which contains the encoded primer binding sequence and a homology region (encoded by the homology arm of the PEgRNA) that is complementary to the region just downstream of the cleavage site (in red). Through flap invasion and DNA repair / repl ication processes, the synthesized strand is incorporated into the DNA, thereby installing the primer binding site. This process can occur at the desired genomic target, but also at other genomic sites that can interact with the PEgRNA in an off-target manner (i.e., the PEgRNA directs the PE complex to other off-target sites due to complementarity of the spacer region to other genomic sites of the non-desired genomic site). Thus, primer binding sequences can be installed not only at the desired genomic target, but also at off-target genomic sites elsewhere in the genome. To detect the insertion of these primer binding sites at the intended genomic target site and at off-target genomic sites, genomic DNA (post-PE) can be isolated, fragmented, and ligated to adaptor nucleotides (shown in red). PCR can then be performed with PCR oligonucleotides that anneal to the adaptors and the inserted primer binding sequences to amplify the on-target and off-target genomic DNA regions where the PE inserted primer binding sites. High-throughput sequencing and sequence alignment can then be performed to identify the insertion points of the PE inserted primer binding sequences at the on-target sites or off-target sites.

[0122] FIG. 34 FIG. 2 is a schematic diagram showing precise insertion of a gene with PE.

[0123] FIG. 35A FIG. 3 is a schematic diagram showing the natural insulin signaling pathway. FIG. 35B FIG. 4 is a schematic diagram showing FKBP12-tagged insulin receptor activation controlled by FK1012.

[0124] FIG. 36 FIG. 5 shows a small molecule monomer. Reference: bumped FK506 mimetic (2) 107 .

[0125] FIGS. 37A-37B FIG. 6 shows a small molecule dimer. Reference: FK10124 95，96 ; FK10125 108 ; FK10126107 ; AP19037 107 ; cyclosporin A dimer 8 98 ; FK506-cyclosporin A dimer (FkCsA) 9 100 .

[0126] FIGS. 38A-38F Overview of in vitro and yeast cell guide-editing and feasibility studies are provided. FIG. 38A Show 75,122 known human pathogenic genetic variants in ClinVar, categorized by type (accessed July 2019). FIG. 38B Show a guide-editing complex composed of a guide-editing (PE) protein comprising an RNA-guided DNA nicking domain, such as a Cas9 nickase, fused to an engineered reverse transcriptase domain, complexed with a guide-editing guide RNA (PEgRNA). The PE:PEgRNA complex binds to a target DNA location and effects a wide variety of precise DNA edits at various DNA sites before or after the protospacer adjacent motif (PAM) of the target site. FIG. 38C Show that upon DNA target binding, the PE:PEgRNA complex nicks the DNA strand containing the PAM. The resulting free 3’ end hybridizes to the primer binding site of the PEgRNA. The reverse transcriptase domain catalyzes primer extension using the RT template of the PEgRNA, resulting in a newly synthesized DNA strand (3’ flap) containing the desired edit. Equilibration between the edited 3’ flap and the non-edited 5’ flap containing the original DNA, followed by cell 5’ flap cleavage and ligation, and DNA repair or replication to resolve the heteroduplex DNA, results in stably edited DNA. FIG. 38D Show an in vitro 5’-extension PEgRNA primer extension assay utilizing a pre- nicked dsDNA substrate containing a 5’-Cy5-labeled PAM strand, dCas9, and a commercial M MLV RT variant (RT, Superscript III). dGas9 is complexed with PEgRNAs containing RT templates of varying lengths, then added to the DNA substrate with the indicated components. Reactions are incubated at 37°C for 1 hour, then analyzed by urea denaturing PAGE and visualized for Cy5 fluorescence. FIG. 38E Show 3’-extension PEgRNAs pre-complexed with dGas9 or Cas9 H840A nickase and pre-nicked or un-nicked 5’-Cy5-labeled dsDNA substrates as in FIG. 38D Perform primer extension assays. FIG. 38FYeast colonies transformed with GFP-mCherry fusion reporter plasmid edited in vitro with PEgRNA, Gas9 nickase, and RT. Plasmids containing nonsense or frameshift mutations between GFP and mCherry were edited with 5'-extended or 3'-extended PEgRNAs that restored mCherry translation through transversion mutations, 1-bp insertions, or 1-bp deletions. GFP and mCherry double positive cells (yellow) reflect successful editing.

[0127] FIGS. 39A-39D Directed editing of genomic DNA in human cells by PE1 and PE2. FIG. 39A PEgRNA contains a spacer sequence, an sgRNA scaffold, and a 3' extension containing a primer binding site (green) and a reverse transcription (RT) template (purple) that contains the edited base (red). The primer binding site hybridizes to the PAM-containing DNA strand immediately upstream of the nicking site. The RT template is homologous to the DNA sequence downstream of the nick, in addition to encoding the edit. FIG. 39B HEK3 site T·A to A·T transversion editing installation in HEK293T cells using Gas9 H840A nickase fused to wild-type M-MLV reverse transcriptase (PE1) and PEgRNAs with different primer binding site lengths. FIG. 39C Using an engineered five-mutant M-MLV reverse transcriptase (D200N, L603W, T306K, W313F, T330P) in PE2 significantly improves directed editing transversion efficiency at 5 genomic sites in HEK293T cells, as well as small insertion and small deletion edits at HEK3. FIG. 39D is a comparison of PE2 editing efficiency at 5 genomic sites in HEK293T cells with different RT template lengths. Values and error bars reflect mean and standard deviation of 3 independent biological replicates.

[0128] FIGS. 40A-40C PE3 and PE3b systems nick the non-edited strand to increase directed editing efficiency. FIG. 40A is an overview of directed editing by PE3. After initial synthesis of the edited strand, DNA repair will remove either the newly synthesized strand containing the edit (3' flap excision) or the original genomic DNA strand (5' flap excision). 5' flap excision leaves a DNA heteroduplex containing one edited and one non-edited strand. Mismatch repair mechanisms or DNA replication can resolve the heteroduplex to provide either edited or non-edited product. Nicking the non-edited strand favors repair of that strand, preferentially generating stable duplex DNA containing the desired edit. FIG. 40B is a demonstration of the effect of complementary strand nicking on PE3-mediated directed editing efficiency and indel formation. "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 (edit-specific complementary strand nick). All editing yields reflect the percentage of total sequencing reads containing the intended edit but not an indel among all treated cells without sorting. Values and error bars reflect the mean and standard deviation of 3 independent biological replicates.

[0129] FIGS. 41A-41K shows targeted insertion, deletion, and all 12 types of point mutations with PE3 at 7 endogenous human genomic loci in HEK293T cells. FIG. 41A shows all 12 types of single nucleotide conversion and transversion edits from site +1 to site +8 (position of PE gRNA-induced nick calculated to be between site +1 and site -1) at HEK3 position using a 10-nt RT template. FIG. 41B shows remote PE3 transversion edits at HEK3 position using a 34-nt RT template. FIGS. 41C-41H shows all 12 types of conversion and transversion edits at different sites in the guide-editing window of FIG. 41C RNF2, FIG. 41D FANCF, FIG. 41E EMX1, FIG. 41F RUNX1, FIG. 41G VEGFA, and FIG. 41H DNMT1. FIG. 41I shows 1 and 3 bp insertions and 1 and 3 bp deletions targeted at 7 endogenous genomic loci with PE3. FIG. 41J is a graph showing targeted precise deletions of 5 to 80 bp at HEK3 target loci. FIG. 41K is a graph showing combinatorial edits of insertions and deletions, insertions and point mutations, deletions and point mutations, and double point mutations at 3 endogenous genomic loci. All editing yields reflect the percentage of total sequencing reads containing the intended edit but not an indel among treated cells without sorting. Values and error bars reflect the mean and standard deviation of 3 independent biological replicates.

[0130] FIGS. 42A-42H shows a comparison of guide editing and base editing by Cas9 and PE3, and off-target editing at known Cas9 off-target sites. FIG. 42A shows total C·G to T·A editing efficiency at the same target nucleotides of PE2, PE3, BE2max, and BE4max at endogenous HEK3, FANCF, and EMX1 positions of HEK293T cells. FIG. 42B shows indel frequencies from the treatments in Figure 42. FIG. 42CEdit efficiency (without bystander edits or indels) of precise C·G to T·A edits at PE2, PE3, BE2max, and BE4max in HEK3, FANCF, and EMX1. For EMX1, also shown is precise PE combinatorial editing of all possible combinations of C·G to T·A conversions at 3 targeted nucleotides. FIG. 42D Total A·T to G·C edit efficiency of PE2, PE3, ABEdmax, and ABEmax at HEK3 and FANCF. FIG. 42E Precise A·T to G·C edit efficiency without bystander edits or indels at HEK3 and FANCF. FIG. 42F Indel frequencies from processing in Figure 42. FIG. 42G One-triplicate averaged edit efficiency (percentage of sequencing reads with indels) of Cas9 nuclease at 4 on-target sites and 16 known off-target sites in HEK293T cells. The 16 off-target sites detected are the top 4 previously reported off-target sites for each of the 4 on-target sites 118，159 . For each on-target site, Cas9 was paired with either an sgRNA or each of the 4 PEgRNAs recognizing the same protospacer. FIG. 42H One-triplicate averaged on-target and off-target edit efficiency and indel efficiency (in parentheses below) of PE2 or PE3 paired with each PEgRNA in ( FIG. 42G ). On-target edit yield reflects the percentage of total sequencing reads in all treated cells that contain the intended edit and do not contain indels, without sorting. Off-target edit yield reflects modification of off-target loci consistent with guided editing. Values and error bars reflect mean and standard deviation of three independent biological replicates.

[0131] FIGS. 43A-43I Guided editing, installation, and correction of pathogenic transversions, insertions, or deletion mutations, and comparison of guided editing and HDR in different human cell lines and primary mouse cortical neurons. FIG. 43A is a plot showing installation (by T·A to A·T transversion) and correction (by A·T to T·A transversion) of the pathogenic E6V mutation in HBB of HEK293T cells. Shown is correction of wild-type HBB or HBB containing a silent mutation that disrupts the PAM of the PEgRNA. FIG. 43B is a plot showing installation (by 4-bp insertion) and correction (by 4-bp deletion) of the pathogenic HEXA 1278+TATC allele in HEK293T cells. Shown is correction of wild-type HEXA or HEXA containing a silent mutation that disrupts the PAM of the PEgRNA. FIG. 43CFigure 1 is a graph showing installation of a protective G127V variant in PRNP in HEK293T cells by G-C to T-A transversion. FIG. 43D Figure 2 is a graph showing prime editing in other human cell lines including K562 (leukemic myeloid cells), U20S (osteosarcoma cells), and HeLa (cervical cancer cells). FIG. 43E Figure 3 is a graph showing installation of G-C to T-A transversion mutations in DNMT1 in mouse primary cortical neurons using a dual split-intein PE3 lentivirus system, where the N-terminal half is an N-intein fused to Cas9 (1-573) and fused to GFP-KASH via a P2A self-cleaving peptide, and the C-terminal half is a C-intein fused to the rest of PE2. PE2 halves are expressed from a human synapsin promoter that is highly specific to mature neurons. Sorted values reflect editing or indels from GFP-positive nuclei, while unsorted values are from all nuclei. FIG. 43F Figure 4 is a comparison of PE3 and Cas9-mediated HDR editing efficiency at endogenous genomic loci in HEK293T cells. FIG. 43G Figure 5 is a comparison of PE3 and Cas9-mediated HDR editing efficiency at endogenous genomic loci in K562, U20S, and HeLa cells. FIG. 43H Figure 6 is a comparison of PE3 and Cas9-mediated HDR indel byproduct generation in HEK293T, K562, U20S, and HeLa cells. FIG. 43I Figure 7 shows insertion of a His6 tag (18 bp), a FLAG epitope tag (24 bp), or an extended LoxP site (44 bp) in HEK293T cells targeted by PE3. All editing yields reflect the percentage of total sequencing reads in all treated cells that contain the intended edit but do not contain an indel. Values and error bars reflect the mean and standard deviation of 3 independent biological replicates.

[0132] FIGS. 44A-44G Figure 8 shows in vitro prime editing validation studies with fluorescently labeled DNA substrates. FIG. 44A Figure 9 shows a mobility shift assay with dCas9, 5'-extended PEgRNAs, and 5'-Cy5-labeled DNA substrates. PEgRNAs 1-5 contain a 15-nt linker sequence between the spacer and PBS (Linker A for PEgRNA 1, Linker B for PEgRNAs 2-5), a 5-nt PBS sequence, and a RT template of 7 nt (PEgRNAs 1 and 2), 8 nt (PEgRNA 3), 15 nt (PEgRNA 4), and 22 nt (PEgRNA 5). PEgRNAs are FIG. 44E and those used in 44F Figures 10A-10C.FIG. 44B In vitro nicking assay with Cas9 H840A using 5'-extended and 3'-extended PEgRNA is shown. FIG. 44C Cas9-mediated indel formation at HEK3 in HEK293T cells using 5'-extended and 3'-extended PEgRNA is shown. FIG. 44D Overview of in vitro biochemical assay for prime editing is shown. 5'-Cy5 labeled pre-nicked and non-nicked dsDNA substrates were tested. sgRNA, 5'-extended PEgRNA, or 3'-extended PEgRNA were pre-complexed with dCas9 or Cas9 H840A nickase, then combined with dsDNA substrate, M-MLV RT, and dNTPs. Reactions were allowed to proceed for 1 hour at 37°C, then separated by urea denaturing PAGE and visualized by Cy5 fluorescence. FIG. 44E Primer extension reactions using 5'-extended PEgRNA, pre-nicked DNA substrate, and dCas9 resulted in significant conversion to RT product is shown. FIG. 44F Primer extension reactions using 5'-extended PEgRNA and non-nicked DNA substrate, and Cas9 H840A nickase as in FIG. 44B Primer extension reactions using 5'-extended PEgRNA and non-nicked DNA substrate, and Cas9 H840A nickase as in FIG. 44G In vitro primer extension reactions using 3'-PEgRNA produced a single apparent product by urea denaturing PAGE. The RT product band was excised from the gel, eluted, then tailed with a homopolymer using terminal transferase (TdT) using either dGTP or dATP. The tailed product was extended by a poly-T or poly-C primer, and the resulting DNA was sequenced. Sanger traces indicate that the three nucleotides derived from the gRNA scaffold were reverse transcribed (added to the DNA product as the last 3' nucleotide). Note that PEgRNA scaffold insertion is much more rare in mammalian cell prime editing experiments than in vitro FIGS. 56A-56D ), likely due to cellular excision of the mismatched 3' end of the Cas9-bound guide RNA scaffold and / or 3' flap containing PEgRNA scaffold sequence.

[0133] FIGS. 45A-45G Cellular repair of 3' DNA flap from in vitro prime editing reactions in yeast is shown. FIG. 45ADual fluorescent protein reporter plasmids are shown containing GFP and mCherry open reading frames separated by a target site encoding an in-frame stop codon, +1 frameshift or -1 frameshift. Guide editing reactions are performed in vitro using Cas9 H840A nickase, PEgRNA, dNTPs and M-MLV reverse transcriptase, then transformed into yeast. Colonies containing non-edited plasmids produce GFP but not mCherry. Yeast colonies containing edited plasmids produce GFP and mCherry as a fusion protein. FIG. 45B GFP and mCherry fluorescence overlay of yeast colonies transformed with reporter plasmids containing a stop codon between GFP and mCherry (non-edited negative control, top panel), or no stop codon or frameshift between GFP and mCherry (pre-edited positive control, bottom panel) are shown. FIGS. 45C-45F Visualization of mCherry and GFP fluorescence from yeast colonies transformed with the product of in vitro guide editing reactions are shown. FIG. 45C Stop codon correction by T·A to A·T transversion using 3'-extended PEgRNA or using 5'-extended PEgRNA is shown, as FIG. 45D indicated. FIG. 45E +1 frameshift correction by 1-bp deletion using 3'-extended PEgRNA is shown. FIG. 45F -1 frameshift correction by 1-bp insertion using 3'-extended PEgRNA is shown. FIG. 45G Sanger DNA sequencing traces of plasmids isolated from only GFP colonies from FIG. 45B and GFP and mCherry double positive colonies from FIG. 45C are shown.

[0134] FIGS. 46A-46F Correct editing versus indel generation with PE1 is shown. FIG. 46A T·A to A·T transversion editing efficiency and indel generation by PE1 at site +1 in HEK3 using PEgRNAs containing 10-nt RT templates and PBS sequences ranging from 8-17 nt is shown. FIG. 46B G·C to T·A transversion editing efficiency and indel generation by PE1 at site +5 in EMX1 using PEgRNAs containing 13-nt RT templates and PBS sequences ranging from 9-17 nt is shown. FIG. 46C G·C to T·A transversion editing efficiency and indel generation by PE1 at site +5 in FANCF using PEgRNAs containing 17-nt RT templates and PBS sequences ranging from 8-17 nt is shown. FIG. 46DC to A·T transversion editing efficiency and indel generation by PE1 at site +1 of RNF2 using PEgRNAs containing 11-nt RT templates and PBS sequences ranging from 9-17 nt. FIG. 46E G to T·A transversion editing efficiency and indel generation by PE1 at site +2 of HEK4 using PEgRNAs containing 13-nt RT templates and PBS sequences ranging from 7-15 nt. FIG. 46F +1 T deletion, +1 A insertion, and +1 CTT insertion at HEK3 position mediated by PE1 using 13-nt PBS and 10-nt RT templates. The sequence of the PEgRNA is FIG. 39C those used in FIG. 47A-47R. Values and error bars reflect mean and standard deviation of three independent biological replicates.

[0135] FIGS. 47A-47S Evaluation of M-MLV RT variants for prime editing. FIG. 47A Abbreviations for prime editor variants used in this figure. FIG. 47B Targeted insertion and deletion editing at the HEK3 locus with PE1. FIGS. 47C-47H Comparison of 18 prime editor constructs containing M-MLV RT variants for their ability to install +2 G·C to C·G transversion edits at HEK3, as FIG. 47C Comparison of 18 prime editor constructs containing M-MLV RT variants for their ability to install 24-bp FLAG insertions at HEK3, as FIG. 47D Comparison of 18 prime editor constructs containing M-MLV RT variants for their ability to install +1 C·G to A·T transversion edits at RNF2, as FIG. 47E Comparison of 18 prime editor constructs containing M-MLV RT variants for their ability to install +1 G·C to C·G transversion edits at EMX1, as FIG. 47F Comparison of 18 prime editor constructs containing M-MLV RT variants for their ability to install +2 T·A to A·T transversion edits at HBB, as FIG. 47G Comparison of 18 prime editor constructs containing M-MLV RT variants for their ability to install +1 G·C to C·G transversion edits at FANCF, as FIG. 47H Comparison of 18 prime editor constructs containing M-MLV RT variants for their ability to install +1 G·C to C·G transversion edits at FANCF, as FIGS. 47I-47N Comparison of 4 prime editor constructs containing M-MLV variants for their ability to install FIGS. 47C-47H Comparison of 4 prime editor constructs containing M-MLV variants for their ability to install FIG. 47P +5 G·C to T·A alteration at EMX1. FIG. 47Q +5 G·C to T·A alteration at FANCF. FIG. 47R +1 C·G to A·T alteration at RNF2. FIG. 47SThe variation from +2G·C to T·A at HEK4 is shown. Values ​​and error bars reflect the mean and standard deviation of three independent biological replicates.

[0136] FIGS. 48A-48C The design features of the PEgRNA PBS and RT template sequences are shown. FIG. 48A The efficiency of PE2-mediated +5G·C to T·A transversion editing at VEGFA in HEK293T cells (blue line) as a function of RT template length is shown. Indels (gray lines) are plotted for comparison. The sequence below the figure shows the last template-providing nucleotide synthesized by PEgRNA. G nucleotides (with C as the template in PEgRNA) are highlighted; C-terminated RT templates should be avoided during PEgRNA design to maximize guided editing efficiency. FIG. 48B Showing the +5G·C to T·A transpose edit and indel of DNMT, as follows FIG. 48A Same as in China. FIG. 48C Showing the +5G·C to T·A transpose edit and indel of RUNX1, as follows: FIG. 48A The values ​​and error bars reflect the mean and standard deviation of three independent biological replicates.

[0137] FIGS. 49A-49B The effects of PE2, PE2 R110S K103L, Cas9 H840A nickase, and dCas9 on cell viability are illustrated. HEK293T cells were transfected using plasmids encoding PE2, PE2 R110S K103L, Cas9 H840A nickase, or dCas9, as well as a PEgRNA plasmid targeting HEK3. Cell viability was measured every 24 hours for 3 days post-transfection using the CellTiter-G1o2.0 assay (Promega). FIG. 49A Viability is shown as measured by luminescence at 1, 2, or 3 days post-transfection. Values ​​and error bars reflect the mean and SEM of three independent biological replicates, each replicate being technically performed in triplicate. FIG. 49B The percentage of edits and indels of PE2, PE2 R110S K103L, Cas9 H840A nickase, or dCas9 along with PEgRNA plasmids encoding +5G to A editing targeting HEK3 are shown. Measurements were taken on day 3 post-transfection of treated cells along with... FIG. 49A The editing efficiency of those cells used to measure viability. The values ​​and error bars reflect the mean and standard deviation of three independent biological replicates.

[0138] FIGS. 50A-50B The images show PE3-mediated HBB E6V correction and HEXA 1278+TATC correction mediated by different PEgRNAs. FIG. 50AScreening 14 PEgRNAs to correct the HBB E6V allele in HEK293T cells using PE3 is shown. All PEgRNAs evaluated converted the HBB E6V allele back to wild-type HBB without introducing any silent PAM mutations. FIG. 50B Screening 41 PEgRNAs to correct the HEXA 1278+TATC allele in HEK293T cells using PE3 or PE3b is shown. Those PEgRNAs labeled HEXA correct the pathogenic allele by moving a 4bp deletion that would disrupt the PAM and leave a silent mutation. Those PEgRNAs labeled HEXA correct the pathogenic allele back to wild-type. Entries ending in “b” use an edit-specific nicking generating sgRNA in combination with a PEgRNA (PE3b system). Values and error bars reflect the mean and standard deviation of three independent biological replicates.

[0139] FIGS. 51A-51G PE3 activity in human cell lines and comparison of PE3 and Cas9-initiated HDR is shown. PE3-initiated HDR in HEK293T cells is shown in FIG. 51A PE3-initiated HDR in K562 cells is shown in FIG. 51B PE3-initiated HDR in U20S cells is shown in FIG. 51C PE3-initiated HDR in HeLa cells is shown in FIG. 51D PE3 and Cas9-initiated HDR generate efficiency of correct edits (no indels) and indel frequencies. Each bracketed edit comparison utilizes PE3 and Cas9-initiated HDR to install the same edit. Non-targeting controls are PE3 and PEgRNAs targeting non-target loci. FIG. 51E Control experiments with non-targeting PEgRNAs + PE3 and with dCas9 + sgRNAs demonstrate that ssDNA donor HDR templates (a common contaminant that artificially boosts apparent HDR efficiency) do not contribute to FIGS. 51A-51D HDR measurements in FIGS. 51F-51G Example HEK3 site allele tables for genomic DNA samples isolated from K562 cells after editing with PE3 or with Cas9-initiated HDR is shown. Alleles were sequenced with Illumina MiSeq and analyzed with CRISPResso2 178Reference HEK3 sequences from this region are at the top. Allele tables are shown for non-targeting PEgRNA negative control, +1 CTT insertion at HEK3 using PE3, and +1 CTT insertion at HEK3 using Cas9-initiated HDR. Allele frequencies and corresponding Illumina sequencing read counts are shown for each allele. All alleles with observed frequencies > 0.20% are shown. Values and error bars reflect mean and standard deviation of three independent biological replicates.

[0140] FIGS. 52A-52D Distribution of pathogenic insertions, duplications, deletions, and indels by length in the ClinVar database is shown. ClinVar variant summaries were downloaded from NCBI on July 15, 2019. Lengths of reported insertions, deletions, and duplications were calculated using appropriate identifying information in reference and alternate alleles, variant start and end positions, or variant names. Variants for which none of the above information was reported were excluded from the analysis. Lengths of reported indels (including individual variants for insertions and deletions relative to the reference genome) were calculated by determining the number of mismatches or gaps in the best pairing alignment between the reference and alternate alleles.

[0141] FIGS. 53A-53E An example of FACS gating for GFP-positive cell sorting is shown. The following is an example of an original bulk analysis file outlining the sorting strategy used to generate the HEXA 1278+TATC and HBB E6V HEK293T cell lines. Image data was generated on a Sony LE-MAP900 cytometer using Cell Sorter software v.3.0.5. Figure 1 shows a gating map for cells that do not express GFP. FIG. 2 An example of P2A-GFP expressing cells for isolation of the HBB E6V HEK293T cell line is shown. HEK293T cells were initially gated on the population using FSC-A / BSC-A (gate A), then singlets were sorted using FSC-A / FSC-H (gate B). Live cells were sorted by gating on DAPI-negative cells (gate C). Cells with a higher level of GFP fluorescence than negative control cells were sorted using EGFP as a fluorescent dye (gate D). FIG. 53A HEK293T cells (GFP-negative) are shown. FIG. 53B A representative plot of FACS gating for cells expressing PE2-P2A-GFP is shown. FIG. 53C Genotypes of HEK293T cell HEXA 1278+TATC homozygotes are shown. FIGS. 53D-53E Allele tables for HEK293T cell line HBB E6V homozygotes are shown.

[0142] FIG. 54is a schematic summarizing the PEgRNA cloning procedure.

[0143] FIGS. 55A-55G is a schematic of PEgRNA design. FIG. 55A shows a simplified diagram of PEgRNA, with domains labeled (left) and bound to nCas9 at a genomic site (right). FIG. 55B shows various types of modifications to PEgRNA that are expected to increase activity. FIG. 55C shows modifications to PEgRNA to increase transcription of longer RNAs by promoter selection and 5', 3' processing and termination. FIG. 55D shows elongation of the P1 system, an example of scaffold modification. FIG. 55E shows that incorporation of synthetic modifications within the template region or elsewhere in the PEgRNA can increase activity. FIG. 55F shows that incorporation of minimal secondary structure within the design template can prevent formation of longer, more inhibitory secondary structures. FIG. 55G shows a split PEgRNA with a second template sequence anchored by a 3' end (left) RNA element of the PEgRNA. Incorporation of elements at the 5' or 3' end of the PEgRNA can enhance RT binding.

[0144] FIGS. 56A-56D shows incorporation of PEgRNA scaffold sequences into the target locus. As described in FIGS. 60A-60B , HTS data was analyzed for PEgRNA scaffold sequence insertion. FIG. 56A shows analysis for the EMX1 locus. Total sequencing reads containing one or more PEgRNA scaffold sequence nucleotides in the insertion adjacent to the RT template (left); total sequencing reads containing PEgRNA scaffold sequence insertions of the indicated lengths (middle); and cumulative total percentage of PEgRNA insertions up to and including the indicated lengths on the X axis. FIG. 56B shows the same as FIG. 56A except for FANCF. FIG. 56C shows the same as FIG. 56A except for HEK3. FIG. 56D shows the same as FIG. 56A except for RNF2. Values and error bars reflect mean and standard deviation of three independent biological replicates.

[0145] FIG. 57A to 57IPE2, PE2-dRT, and Cas9 H840A nickase effects on whole transcriptome RNA abundance. Cell RNA isolated from HEK293T cells expressing PE2, PE2-dRT, or Cas9 H840A nickase and PRNP-targeting or HEXA-targeting PEgRNAs was analyzed, with ribosomal RNA removed. RNA corresponding to 14,410 genes and 14,368 genes was detected in PRNP and HEXA samples, respectively. FIG. 57A to 57F Volcano plots showing -log10 FDR-adjusted p-value vs. log2 fold change of Aeach RNA transcript abundance comparing FIG. 57A ) PE2 vs. PE2-dRT in the context of PRNP-targeting PEgRNAs, FIG. 57B ) PE2 vs. Cas9 H840A in the context of PRNP-targeting PEgRNAs, FIG. 57C ) PE2-dRT vs. Cas9 H840A in the context of PRNP-targeting PEgRNAs, FIG. 57D ) PE2 vs. PE2-dRT in the context of HEXA-targeting PEgRNAs, FIG. 57E ) PE2 vs. Cas9 H840A in the context of HEXA-targeting PEgRNAs, FIG. 57F ) PE2-dRT vs. Cas9 H840A in the context of HEXA-targeting PEgRNAs. Red dots indicate genes with a change in relative abundance > 2-fold and statistically significant (FDR-adjusted p < 0.05). FIG. 57G to 57I Venn diagrams of up- and down-regulated transcripts (> 2-fold change) comparing PRNP and HEXA samples, FIG. 57G ) PE2 vs. PE2-dRT, FIG. 57H ) PE2 vs. Cas9 H840A, and FIG. 57I ) PE2-dRT vs. Cas9 H840A.

[0146] FIG. 58A-58B Representative FACS gating for neuronal nuclei sorting is shown. Nuclei were sequentially gated based on DyeCycle Ruby signal, FSC / SSC ratio, SSC width / SSC height ratio, and GFP / DyeCycle ratio.

[0147] FIG. 59A to 59G A scheme for cloning 3 '-extended PEgRNAs into mammalian U6 expression vectors by Golden Gate assembly is shown. FIG. 59A A cloning overview is shown. FIG. 59B "Step 1: Digest pU6-PEgRNA-GG-Vector Plasmid (Component 1)" is shown. FIG. 59C“Step 2 and 3: Ordering and annealing of oligonucleotides parts (components 2, 3 and 4)” is shown. FIG. 59D “Step 2.b.ii.: sgRNA scaffold phosphorylation (not needed if phosphorylated oligos are purchased)” is shown. FIG. 59E “Step 4: PEgRNA assembly” is shown. FIG. 59F “Step 5 and 6: Transformation of assembled plasmid” is shown. FIG. 59G A figure summarizing the PEgRNA cloning protocol is shown.

[0148] FIG. 60A to 60B A Python script for quantifying PEgRNA scaffold integration is shown. A custom python script was generated to characterize and quantify PEgRNA insertion at the target genomic locus. The script iteratively matches length-increased text strings harvested from the reference sequence (guide RNA scaffold sequence) to sequencing reads in a fastq file and counts the number of sequencing reads that match the search query. Each consecutive text string corresponds to an additional nucleotide of the guide RNA scaffold sequence. In this way, an exact length integral and cumulative integral to a specified length are calculated. At the start of the reference sequence, 5 to 6 bases comprising the 3’ end of the newly synthesized DNA strand by reverse transcriptase are included to ensure alignment and accurate counting of sgRNA short fragments.

[0149] FIG. 61 is a graph showing the percentage of total sequencing reads with the indicated edits for SaCas9(N580A)-MMLV RT HEK3+6 C>A. Values for correct edits and indels are shown.

[0150] FIG. 62A to 62B The importance of protospacers for efficient installation of the desired edit at the precise location with prime editing is shown. FIG. 62A is a graph showing the percentage of total sequencing reads with target A·T base pairs converted to T·A for each HEK3 locus. FIG. 62B is a graph showing the same sequence analysis.

[0151] FIG. 63 is a graph showing SpCas9 PAM variants (N=3) in PAM editing. The percentage of total sequencing reads with targeted PAM edits is shown for SpCas9(H840A)-VRQR-MMLV RT, where NGA > NTA, and for SpCas9(H840A)-VRER-MMLV RT, where NGCG > NTCG. The PEgRNA primer binding site (PBS) length, RT template (RT) length, and PE system used are listed.

[0152] FIG. 64A-64Fis a schematic showing the introduction of individual site-specific recombinase (SSR) targets into the genome using PE. FIG. 64A A general schematic showing the insertion of a recombinase target sequence by a prime editor is provided. FIG. 64B A site of genomic integration is shown using a single SSR target inserted by PE as a DNA donor template. FIG. 64C How a portion of the genome can be deleted using tandem insertion of SSR target sites is shown. FIG. 64D How a portion of the genome can be inverted using tandem insertion of SSR target sites is shown. FIG. 64E How insertion of two SSR target sites in two distal chromosomal regions can result in a chromosomal translocation is shown. FIG. 64F How a cassette from a DNA donor template can be exchanged using insertion of two different SSR target sites in the genome is shown. See Example 17 for more details.

[0153] FIG. 65 1) PE-mediated synthesis of an SSR target site in the genome of a human cell and 2) integration of a DNA donor template containing a GFP expression marker using this SSR target site is shown. Once successfully integrated, GFP causes the cell to fluoresce. See Example 17 for more details.

[0154] FIG. 66 One embodiment of a prime editor provided as two PE half proteins that are regenerated into a complete prime editor by the action of self-splicing of a split intein half located at the end or beginning of each prime editor half protein is depicted.

[0155] Figure 67 depicts the mechanism of removing an intein and reforming a peptide bond between N-terminal and C-terminal extein sequences from a polypeptide sequence. (a) Describes the general mechanism of two half proteins, each comprising one half of an intein sequence, that when brought into contact within a cell produce a fully functional intein that then undergoes self-splicing and excision. The excision process results in the formation of a peptide bond between the N-terminal protein half (or “N-extein”) and the C-terminal protein half (or “C-extein”) to form a single polypeptide comprising the N-extein and C-extein portions. In different embodiments, the N-extein can correspond to the N-terminal half of a split prime editor fusion protein, and the C-extein can correspond to the C-terminal half of a split prime editor. (b) Shows the chemical mechanism of intein excision and reforming a peptide bond linking the N-extein half (red half) and the C-extein half (blue half). Excision of a split intein (i.e., the N-intein and C-intein in a split intein construct) can also be referred to as “trans-splicing” because it involves the splicing action of two separate components provided in trans.

[0156] FIG. 68ADelivery of the two split intein halves of SpPE (SEQ ID NO: 762) at the linker was shown to retain activity at three tested loci when co-transfected into HEK293T cells.

[0157] FIG. 68B Delivery of the two split inteins of SaPE2 (e.g., SEQ ID NO: 443 and SEQ ID NO: 450) was shown to recapitulate the activity of full-length SaPE2 (SEQ ID NO: 134) when co-transfected into HEK293T cells. Residues indicated in quotes are the sequence of amino acids 741-743 in SaCas9 (first residues of the C-terminal extein), which are important for the intein trans-splicing reaction. "SMP" is the native residue, which we also mutated to the "CFN" consensus splicing sequence. The consensus sequence was shown to produce the highest reconstitution as measured by percent guide editing.

[0158] FIG. 68C Data is provided showing that various disclosed PE ribonucleoprotein complexes (high concentration PE2, high concentration PE3, and low concentration PE3) can be delivered in this manner.

[0159] FIG. 69 A phage plaque assay is shown to determine PE effectiveness in PANCE. Plaque (black circles) indicates that phage was able to successfully infect E. coli. Increasing concentrations of L-rhamnose resulted in increased PE expression and increased plaque formation. Sequencing of the plaques revealed the presence of genome edits installed by the PE.

[0160] FIG. 70A to 70I An example of editing a target sequence is provided as a graphical illustration of the step-by-step instructions for designing PE gRNAs and nicking sgRNAs for prime editing. FIG. 70A Step 1, Define target sequence and edit. Retrieve the sequence of the target DNA region (about 200 bp) centered around the location of the desired edit (point mutation, insertion, deletion, or combination thereof). FIG. 70B Step 2, Locate target PAM. Identify a PAM close to the edit location. Be sure to look for PAMs on both strands. While PAMs close to the edit location are preferred, PAM installation edits can be used that place the nick >30 nt away from the edit location. FIG. 70C: Step 3, locate the nick site. For each PAM under consideration, identify the corresponding nick site. For Sp Cas9 H840A nickase, cleavage occurs between the 3rd and 4th bases 5' of the NGG PAM on the PAM-containing strand. All edited nucleotides must be present 3' of the nick site, so the appropriate PAM must place the nick on the PAM-containing strand 5' of the target edit. In the example shown below, there are two possible PAMs. For simplicity, the remaining steps will show PEgRNA design using only PAM1. FIG. 70D : Step 4, design the spacer sequence. The protospacer of Sp Cas9 corresponds to the 20 nucleotides 5' of the NGG PAM on the PAM-containing strand. Efficient Pol III transcription initiation requires a G as the first transcribed nucleotide. If the first nucleotide of the protospacer is a G, then the spacer sequence of the PEgRNA is the protospacer. If the first nucleotide of the protospacer is not a G, then the spacer sequence of the PEgRNA is a G followed by the protospacer. FIG. 70E : Step 5, design the primer binding site (PBS). Using the starting allele sequence, identify the DNA primer on the PAM-containing strand. The 3' end of the DNA primer is exactly the nucleotide upstream of the nick site (i.e., 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 complementary to the DNA primer can be used for sequences containing approximately 40-60% GC content. For sequences with lower 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. The optimal PBS sequence should be determined empirically, regardless of GC content. To design a PBS sequence of length p, use Initiation the reverse complement of the first p nucleotides 5' of the nick site in the PAM-containing strand. FIG. 70F: Step 6, design the RT template. The RT template encodes the edit of interest and homology to the sequence adjacent to the edit. The optimal RT template length varies by target site. For short-range edits (positions +1 to +6), it is recommended to test short (9 to 12 nt), medium (13 to 16 nt), and long (17 to 20 nt) RT templates. For long-range edits (+7 position and above), it is recommended to use an RT template that extends at least 5 nt (preferably 10 nt or more) past the edit position to allow sufficient 3' flap homology. For long-range edits, several RT templates should be screened to identify a functional design. For larger insertions and deletions (>5 nt), it is recommended to incorporate larger 3' homology (~20 nt or more) into the RT template. When the RT template encodes a G as the last nucleotide synthesized in the reverse-transcribed DNA product (corresponding to a C in the RT template of the PEgRNA), it often impairs editing efficiency. As many RT templates support efficient prime editing, it is recommended to avoid a G as the final synthesized nucleotide when designing the RT template. To design an RT template sequence of length r, use Desired the allele sequence and take the reverse complement of the first r nucleotides 3' of the cut site in the original PAM-containing strand. Note that, in contrast to SNP editing, insertions or deletion edits using the same length of RT template will not contain the same homology. FIG. 70G : Step 7, assemble the complete PEgRNA sequence. Concatenate the PEgRNA components in the following order (5' to 3'): spacer, scaffold, RT template, and PBS. FIG. 70H : Step 8, design a nicking sgRNA for PE3. Identify PAMs on the non-edited strand upstream and downstream of the edit. The optimal nicking sites are highly dependent on the locus and should be determined empirically. Generally, a nick 5' of the site opposite the PEgRNA-induced nick results in higher editing yields and fewer indels. The nicking sgRNA has a spacer sequence that matches the 20-nt protospacer in the allele, with a 5'-G added if the protospacer does not begin with a G. Initiation FIG. 70I ​: Step 9, Design PE3b nicking gRNAs. If the PAM exists in the complementary strand and its corresponding protospacer overlaps the sequence targeted for editing, this edit can be a candidate for the PE3b system. In the PE3b system, the spacer sequence of the nicking gRNA matches the sequence of the desired edited allele, but not the sequence of the starting allele. The PE3b system works effectively when the edited nucleotide falls within the seed region (about 10 nt adjacent to the PAM) of the nicking gRNA protospacer. This prevents a nick on the complementary strand before the installation of the edited strand, thus preventing competition between the PEgRNA and the sgRNA for binding to the target DNA. PE3b also avoids the generation of simultaneous nicks on both strands, thus significantly reducing indel formation while maintaining high editing efficiency. PE3b sgRNAs should have a spacer sequence that matches the 20-nt protospacer in the allele, with a 5' G added if needed. Desired

[0161] FIG. 71A The nucleotide sequence of a SpCas9 PEgRNA molecule is shown (upper portion), which terminates at the 3' end with "UUU" and does not contain a toe-loop element. The lower portion of this figure depicts the same SpCas9 PEgRNA molecule, but further modified to contain a toe-loop element with the sequence 5'- "GAAANNNNN" -3' inserted just prior to the 3' end of "UUU". "N" can be any nucleobase.

[0162] FIG. 71B Results of Example 18 are shown, which indicate that the use of PEgRNAs containing a toe-loop element improves the prime editing efficiency in HEK cells or EMX cells, with little change in the percentage of indel formation.

[0163] FIG. 72A-72C Alternative PEgRNA configurations that can be used in prime editing are depicted. FIG. 72A A PE2:PEgRNA embodiment of prime editing is depicted. This embodiment involves PE2 (a fusion protein comprising Cas9 and a reverse transcriptase) complexed with a PEgRNA (also as described in FIG. 1A-1I and / or FIG. 3A-3E ). In this embodiment, the template for reverse transcription is incorporated into a 3' extension arm on the sgRNA to make the PEgRNA, and the DNA polymerase is a reverse transcriptase (RT) fused directly to Cas9. FIG. 72B ​Depiction of MS2cp-PE2:sgRNA + tPERT implementation. This implementation includes a PE2 fusion (Cas9 + reverse transcriptase) that is further fused with MS2 bacteriophage coat protein (MS2cp) to form a MS2cp-PE2 fusion protein. To implement prime editing, the MS2cp-PE2 fusion protein is complexed with a sgRNA that targets the complex to a specific target site in DNA. This implementation then involves the introduction of a trans prime editing RNA template ("tPERT") that operates in place of a PEgRNA by providing a primer binding site (PBS) and a DNA synthesis template on a separate molecule (i.e., tPERT) that is also equipped with MS2 aptamer (stem loop). The MS2cp protein recruits the tPERT by binding to the MS2 aptamer of the molecule. FIG. 72C Depiction of alternative design of PEgRNA that can be implemented by known methods of chemical synthesis of nucleic acid molecules. For example, chemical synthesis can be used to synthesize a hybrid RNA / DNA PEgRNA molecule for use in prime editing, where the extension arm of the hybrid PEgRNA is DNA rather than RNA. In such an implementation, a DNA-dependent DNA polymerase can be used in place of a reverse transcriptase to synthesize the 3' DNA flap that contains the desired genetic change formed by prime editing. In another implementation, the extension arm can be synthesized to contain a chemical linker that prevents a DNA polymerase (e.g., reverse transcriptase) from using the sgRNA scaffold or backbone as a template. In yet another implementation, the extension arm can contain a DNA synthesis template that has an opposite orientation relative to the overall orientation of the PEgRNA molecule. For example, as shown for a PEgRNA in a 5' to 3' orientation and with an extension linked to the 3' end of the sgRNA scaffold, the DNA synthesis template is in the opposite reverse orientation, i.e., 3' to 5' direction. This implementation can be advantageous for PEgRNA implementations that have an extension arm located at the 3' end of the gRNA. By reversing the orientation of the extension arm, DNA synthesis by a polymerase (e.g., reverse transcriptase) will terminate upon reaching the 5' end of the newly oriented extension arm, thus there is no risk of using the gRNA core as a template.

[0164] FIG. 73Display of prime editing with tPERT and MS2 recruitment system (aka MS2-tagging technology). sgRNA targeting the guide editor protein (PE2) to the target locus is expressed in combination with tPERT, which comprises a primer binding site (13-nt or 17-nt PBS), a RT template encoding a His6 tag insertion and homology arms, and an MS2 aptamer (located at the 5' or 3' end of the tPERT molecule). The guide editor protein (PE2) or a fusion of MS2cp to the N-terminus of PE2 is used. Editing is performed with or without a complementary strand nicking generating sgRNA, as in the previously developed PE3 system (designated as the label "PE2+nick" or "PE2" on the x-axis, respectively). This is also referred to and defined herein as "second strand nicking generation".

[0165] FIG. 74 MS2 aptamer to reverse transcriptase is expressed in trans and its utilization with the MS2 aptamer system recruitment. PEgRNA PEgRNA comprises an MS2 RNA aptamer inserted into one of the two sgRNA scaffold hairpins. Wild-type M-MLV reverse transcriptase is expressed as an N-terminal or C-terminal fusion to MS2 coat protein (MCP). Editing is at the HEK3 position in HEK293T cells.

[0166] FIG. 75 Bar graphs comparing the efficiency of PE2, PE2-truncated, PE3, and PE3-truncated at different target sites in different cell lines (i.e., "% of total sequencing reads with the indicated edit or indel"). The data show that guide editors comprising truncated RT variants are about as efficient as guide editors comprising non-truncated RT proteins.

[0167] FIG. 76 Display of editing efficiency of the intein-split guide 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 (the majority of the amino-terminal residues of the C-terminal extein peptide) is shown. Editing percentages are shown at two positions: HEK3+1 CTT insertion and PRNP+6 G to T. Repeats n=3 independent transfections. See Example 20.

[0168] FIG. 77Editing efficiency of the intein-splitting prime editor of Example 20 is shown. Editing was assessed by bulk-targeted deep sequencing in P0 mice delivered with 5E10 vg / SpPE3 half and a small amount of 1E10 nuclear-localized GFP:KASH via ICV injection. The editor and GFP were packaged in AAV9 with an EFS promoter. Mice were harvested 3 weeks post-injection and GFP+ nuclei were isolated by flow cytometry. Individual data points are shown, 1-2 mice were analyzed per condition. See Example 20.

[0169] FIG. 78 Editing efficiency of the intein-splitting prime editor of Example 20 is shown. Specifically, this figure depicts the AAV split-SpPE3 construct used in Example 20. PE3 activity is recapitulated by co-transduction of AAV particles expressing SpPE3-N and SpPE3-C, respectively. Note that the N-terminal genome contains a U6-sgRNA cassette expressing a nicking sgRNA, and the C-terminal genome contains a U6-PEgRNA cassette expressing a PEgRNA. See Example 20.

[0170] FIG. 79 Editing efficiency of certain optimized linkers as discussed in Example 21 is shown. In particular, the data show editing efficiency of PE2 constructs with the current linker (labeled PE2 - white box) compared to various versions with linkers replaced with sequences as shown for transversion, transversion, insertion, and deletion edits at the HEK3, EMX1, FANCF, RNF2 loci for a representative PEgRNA. The replacement linkers are called “lxSGGS” (SEQ ID NO: 174), “2xSGGS” (SEQ ID NO: 446), “3xSGGS” (SEQ ID NO: 3889), “lxXTEN” (SEQ ID NO: 171), “no linker”, “lxGly”, “lxPro”, “lx EAAAK” (SEQ ID NO: 3968), “2xEAAAK” (SEQ ID NO: 3969), and “3xEAAAK” (SEQ ID NO: 3970). Editing efficiency is measured as a bar graph relative to the “control” editing efficiency of PE2. The linker of PE2 is SGGSSGGSSGSETPGTSESATPESSGGSSGGSS (SEQ ID NO: 127). All edits were done in the context of the PE3 system, i.e., PE2 editing construct plus addition of optimal secondary sgRNA nicking guides. See Example 21.

[0171] FIG. 80. Taking the average fold potency relative to PE2 results in the graph shown, indicating that using a 1x XTEN (SEQ ID NO: 171) linker sequence increases the editing efficiency by an average of 1.14 fold (n=15). See Example 21.

[0172] FIG. 81 Depiction of transcription levels of PEgRNAs from different promoters, as described in Example 22.

[0173] FIG. 82 Depiction of the effect of different types of modifications to PEgRNA structure on editing efficiency relative to unmodified PEgRNA, as shown in Example 22.

[0174] FIG. 83 Depiction of PE experiments targeting editing of the HEK3 gene, specifically targeting an insertion of a 10 nt insertion at position +1 relative to the nick site and using PE3. See Example 22.

[0175] FIG. 84A Depiction of an exemplary PEgRNA with a spacer, gRNA core, and extended arm (RT template + primer binding site) modified at the 3’ end of the PEgRNA with a tRNA molecule coupled by a UCU linker. The tRNA includes various post-transcriptional modifications. However, the modifications are not essential.

[0176] FIG. 84B Depiction of tRNA structures that can be used to modify PEgRNA structure. See Example 22. The length of P1 can be variable. P1 can be extended to help prevent RNAse P processing of the PEgRNA-tRNA fusion.

[0177] FIG. 85 Depiction of PE experiments targeting editing of the FANCF gene, specifically targeting a G to T conversion at position +5 relative to the nick site and using the PE3 construct. See Example 22.

[0178] FIG. 86 Depiction of PE experiments targeting editing of the HEK3 gene, specifically targeting an insertion of a 71 nt FLAG tag insertion at position +1 relative to the nick site and using the PE3 construct. See Example 22.

[0179] FIG. 87 is a result from a screen in N2A cells where the pegRNA was installed with a 1412A del with details on primer binding site (PBS) length and reverse transcriptase (RT) template length (shown with and without indel). See Example 23.

[0180] FIG. 88Results from a screen in N2A cells with pegRNAs installed with 1412AdeI with details on primer binding site (PBS) length and reverse transcriptase (RT) template length (shown with and without indels). See Example 23.

[0181] FIG. 89 Depiction of results of editing at proxy loci in the beta-globin gene and at HEK3 in healthy HSCs, varying concentrations of editor to pegRNA and nicking gRNA. See Example 23.

[0182] Definitions

[0183] Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this application belongs. The following references provide one of ordinary skill with a general definition of many of the terms used in this application: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

[0184] Antisense strand

[0185] In genetics, the "antisense" strand of a segment within double-stranded DNA is the template strand, and is considered to be extended in the 3' to 5' direction. In contrast, the "sense" strand of a segment within double-stranded DNA is extended 5' to 3', and is complementary to the antisense or template strand (which is extended 3' to 5'). In the case of a DNA segment that encodes a protein, the sense strand is the DNA strand with the same sequence as the mRNA, which is templated by the antisense strand during transcription, and eventually (not always) undergoes translation into protein. Thus, the antisense strand is responsible for the RNA that is subsequently translated into protein, while the sense strand has nearly the same composition as the mRNA. Note that for each segment of dsDNA, there can be two sets of sense and antisense, depending on the direction of reading (since sense and antisense are relative to the point of view). Ultimately, which strand of a segment of dsDNA is designated sense or antisense is a matter of the gene product or mRNA.

[0186] Bispecific ligand

[0187] As used herein, the term "bispecific ligand" or "bispecific moiety" 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 domains are "dimerization domains" that can be installed as peptide tags onto proteins. In different embodiments, two proteins each comprising the same or different dimerization domains can be induced to dimerize through binding of each dimerization domain to a bispecific ligand. As used herein, "bispecific ligand" can be equally referred to as "dimerization chemical inducer" or "CID".

[0188] Cas9

[0189] The term "Cas9" or "Cas9 nuclease" refers to a protein comprising a Cas9 domain or fragment thereof (e.g., a protein comprising the active or inactive DNA cleavage domain of Cas9, and / or the gRNA binding domain of Cas9). As used herein, a "Cas9 domain" is a protein fragment comprising the active or inactive cleavage domain of Cas9 and / or the gRNA binding domain of Cas9. A "Cas9 protein" is a full-length Cas9 protein. Cas9 nucleases are sometimes also referred to as casnl nucleases or CRISPR (clustered regularly interspaced short palindromic repeats)-associated nucleases. CRISPR is an adaptive immune system that can provide protection against mobile genetic elements (viruses, transposable elements, and conjugative plasmids). CRISPR clusters contain spacer regions, sequences complementary to preceding mobile elements, and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems, proper processing of pre-crRNA requires a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and a Cas9 domain. The tracrRNA acts as a guide for ribonuclease 3 to assist in processing pre-crRNA. Subsequently, Cas9 / crRNA / tracrRNA cleaves linear or circular dsDNA targets complementary to the spacer regions in an endonucleolytic manner. The target strand not complementary to the crRNA is first cleaved in an endonucleolytic manner, then trimmed in a 3'-5' exonucleolytic manner. In fact, DNA binding and cleavage generally require the protein and both RNAs. However, a single guide RNA ("sgRNA", or simply "gNRA") can be engineered to incorporate aspects of both the crRNA and tracrRNA into a single RNA species. See, e.g., Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J.A., Charpentier E. Science 337:816-821 (2012), the entire contents of which are incorporated herein by reference. Cas9 recognizes a short motif in the CRISPR repeat sequence (PAM or protospacer adjacent motif) to help distinguish self from non-self.Cas9 nuclease sequences and structures are well known to those of skill in the art (see, e.g., "Complete genome sequence of an Ml strain of Streptococcus pyogenes." Ferretti et al., J.J., McShan W.M., Ajdic D.J., Savic D.J., Savic G., Lyon K., Primeaux C., Sezate S., Suvorov A.N., Kenton S., Lai H.S., Lin S.P., Qian Y., Jia H.G., Najar F.Z., Ren Q., Zhu H., Song L., White J., Yuan X., Clifton S.W., Roe B.A., McLaughlin R.E., Proc. Natl. Acad. Sci. U.S.A. 98:4658-4663 (2001); "CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III." Deltcheva E., Chylinski K., Sharma C.M., Gonzales K., Chao Y., Pirzada Z.A., Eckert M.R., Vogel J., Charpentier E., Nature 471 :602-607 (2011); and "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity." Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J.A., Charpentier E. Science 337:816-821 (2012), the entire contents of each of which are incorporated herein by reference). Cas9 orthologs have been described in a variety of species, including, but not limited to, S. pyogenes and S. thermophilus.Based on the present disclosure, other suitable Cas9 nucleases and sequences will be apparent to those skilled in the art, and such Cas9 nucleases and sequences include Cas9 sequences from organisms and loci disclosed in Chylinski, Rhun, and Charpentier, "The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems" (2013) RNA Biology 10:5, 726-737 (the entire contents of which are incorporated herein by reference). In some embodiments, the Cas9 nuclease comprises one or more mutations that partially impair or inactivate the DNA cleavage domain.

[0190] A nuclease-inactivated Cas9 domain is interchangeably referred to as a "dCas9" protein (for nuclease- "dead" Cas9). Methods for producing Cas9 domains (or fragments thereof) with inactive DNA cleavage domains are known (see, e.g., Jinek et al., Science. 337:816-821 (2012); Qi et al., "Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression" (2013) Cell. 28; 152(5): 1173-83, the entire contents of each of which are incorporated herein by reference). For example, the DNA cleavage domain of Cas9 includes two subdomains, an HNH nuclease subdomain and a RuvC1 subdomain. The HNH subdomain cleaves the strand complementary to the gRNA, while 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, a protein comprising a fragment of Cas9 is provided. 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 protein comprising Cas9 or a fragment thereof is referred to as a "Cas9 variant." A Cas9 variant has homology to Cas9 or a fragment thereof. For example, a Cas9 variant is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, at least about 99.8% identical, or at least about 99.9% identical to wild-type Cas9 (e.g., SpCas9 of SEQ ID NO: 18). In some embodiments, a Cas9 variant can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more amino acid changes compared to wild-type Cas9 (e.g., SpCas9 of SEQ ID NO: 18).In some embodiments, a Cas9 variant comprises a fragment of SEQ ID NO: 18 Cas9 (e.g., a gRNA binding domain or a DNA cleavage domain) such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of wild-type Cas9 (e.g., SpCas9 of SEQ ID NO: 18). In some embodiments, the fragment is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid length of the corresponding wild-type Cas9 (e.g., SpCas9 of SEQ ID NO: 18).

[0191] cDNA

[0192] The term "cDNA" refers to a DNA strand copied from an RNA template. The cDNA is complementary to the RNA template.

[0193] Circular permutant

[0194] As used herein, the term "circularly permuted" refers to a protein or polypeptide (e.g., Cas9) comprising a circular permutation, which is a change in the order of amino acids occurring in the protein amino acid sequence that results in a change in the N- and C-termini of the protein. In other words, a circularly permuted protein has an altered N- and C-termini compared to the wild-type counterpart, such as the wild-type C-terminal half of the protein becomes the new N-terminal half. Circular permutations (or CPs) are essentially topological rearrangements of the primary sequence of a protein, typically using a peptide linker to connect its N- and C-termini, while splitting its sequence at different positions to form new adjacent N- and C-termini. The result is a protein structure with different connectivity, but often can have the same overall similar three-dimensional (3D) shape, and can include improved or altered characteristics, including reduced proteolytic susceptibility, increased catalytic activity, altered substrate or ligand binding, and / or increased thermal stability. Circularly permuted proteins can exist in nature (e.g., concanavalin A and lectins). In addition, circular permutations can arise as a result of post-translational modifications, or can be engineered using recombinant techniques.

[0195] Circularly permutated Cas9

[0196] The term "circularly permuted Cas9" refers to any Cas9 protein or variant thereof that has existed as a circularly permuted, whereby its N- and C-termini have been partially rearranged. Such circularly permuted Cas9 proteins ("CP-Cas9") or variants thereof retain the ability to bind DNA when complexed with a guide RNA (gRNA). See Oakes et al., "Protein Engineering of Cas9 for enhanced function," Methods Enzymol, 2014, 546:491-511 and Oakes et al., "CRISPR-Cas9 Circular Permutants as Programmable Scaffolds for Genome Modification," Cell, January 10, 2019, 176:254-267, each incorporated herein by reference. The present disclosure contemplates any previously known CP-Cas9 or use of a new CP-Cas9, so long as the resulting circularly permuted protein retains the ability to bind DNA when complexed with a guide RNA (gRNA). Exemplary CP-Cas9 proteins are SEQ ID NOs: 77-86.

[0197] CRISPR

[0198] CRISPR is a family of DNA sequences (i.e., CRISPR clusters) in bacteria and archaea that represent fragments of previously infecting viruses that have invaded the prokaryote. Prokaryotic cells use the DNA fragments to detect and destroy DNA from similar viruses in subsequent attacks and effectively constitute a prokaryotic immune defense system with a series of CRISPR-associated proteins, including Cas9 and its homologs, and CRISPR-associated RNAs. In fact, CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In certain types of CRISPR systems (e.g., Type II CRISPR systems), proper processing of pre-crRNA requires a small RNA encoded in trans (tracrRNA), an endogenous ribonuclease 3 (rnc), and a Cas9 protein. The tracrRNA acts as a guide for ribonuclease 3 to assist in processing the pre-crRNA. Subsequently, Cas9 / crRNA / tracrRNA cleaves linear or circular dsDNA targets complementary to the RNA in an endonucleolytic manner. Specifically, the target strand not complementary to the crRNA is first cleaved in an endonucleolytic manner, and then trimmed in a 3'-5' exonucleolytic manner. In fact, DNA binding and cleavage generally require the protein and both RNAs. However, a single guide RNA ("sgRNA", or simply "gNRA") can be engineered to incorporate aspects of both the crRNA and the tracrRNA into a single RNA species - the guide RNA. See, e.g., Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J. A., Charpentier E. Science 337:816-821 (2012), the entire contents of which are incorporated herein by reference. Cas9 recognizes a short motif in the CRISPR repeat sequence (PAM or protospacer adjacent motif) to help distinguish self from non-self.CRISPR biology as well as Cas9 nuclease sequences and structures are well known to those of skill in the art (see, e.g., "Complete genome sequence of an Ml strain of Streptococcus pyogenes." Ferretti et al., J.J., McShan W.M., Ajdic D.J., Savic D.J., Savic G., Lyon K., Primeaux C., Sezate S., Suvorov A.N., Kenton S., Lai H.S., Lin S.P., Qian Y., Jia H.G., Najar F.Z., Ren Q., Zhu H., Song L., White J., Yuan X., Clifton S.W., Roe B.A., McLaughlin R.E., Proc. Natl. Acad. Sci. U.S.A. 98:4658-4663 (2001); "CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III." Deltcheva E., Chylinski K., Sharma C.M., Gonzales K., Chao Y., Pirzada Z.A., Eckert M.R., Vogel J., Charpentier E., Nature 471 :602-607 (2011); and "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity." Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J.A., Charpentier E. Science 337:816-821 (2012), the entire contents of which are incorporated herein by reference). Cas9 orthologs have been described in different species, including but not limited to S. pyogenes and S. thermophilus.Other suitable Cas9 nucleases and sequences will be apparent to those skilled in the art based on the present disclosure, and such Cas9 nucleases and sequences include those from Chylinski, Rhun, and Charpentier, "The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems" (2013) RNA Biology 10:5, 726-737, the entirety of which is incorporated herein by reference.

[0199] In certain types of CRISPR systems (e.g., type II CRISPR systems), proper processing of pre-crRNA requires a trans-encoded small RNA (tracrRNA), an endogenous ribonuclease 3 (rnc), and a Cas9 protein. The tracrRNA acts as a guide for ribonuclease 3 to assist in processing the pre-crRNA. Subsequently, Cas9 / crRNA / tracrRNA cleaves linear or circular nucleic acid targets that are complementary to the RNA in an endonucleolytic manner. Specifically, it first cleaves the target strand that is not complementary to the crRNA in an endonucleolytic manner, and then trims in a 3'-5' exonucleolytic manner. In fact, DNA binding and cleavage generally requires the protein and both RNAs. However, a single guide RNA ("sgRNA", or simply "gRNA") can be engineered to incorporate both the crRNA and tracrRNA embodiments into a single RNA species - the guide RNA.

[0200] Generally, a "CRISPR system" collectively refers to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated ("Cas") genes, including sequences encoding Gas genes, tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or active portions of tracrRNA), tracr-mate sequences (including "direct repeats" and portions of direct repeats processed by tracrRNA in the context of endogenous CRISPR systems), guide sequences (also referred to as "spacers" in the context of endogenous CRISPR systems), or other sequences and transcripts from CRISPR loci. The tracrRNA of the system is complementary (fully or partially) to the tracr-mate sequence present on the guide RNA.

[0201] DNA synthesis template

[0202] As used herein, the term "DNA synthesis template" refers to the region or portion of the extension arm of a PEgRNA that is used by the polymerase of the prime editor as a template strand to encode a 3' single-stranded DNA flap that contains the desired edit, which is then substituted for the corresponding endogenous DNA strand at the target site by the prime editing mechanism. In different embodiments, the DNA synthesis template is shown in FIG. 3A (in the context of a PEgRNA comprising a 5' extension arm), FIG. 3B (in the context of a PEgRNA comprising a 3' extension arm), FIG. 3C (in the context of an internal extension arm), FIG. 3D (in the context of a 3' extension arm), and FIG. 3E (in the context of a 5' extension arm). The extension arm (including the DNA synthesis template) can be composed of DNA or RNA. In the case of RNA, the polymerase of the prime editor can be an RNA-dependent DNA polymerase (e.g., a reverse transcriptase). In the case of DNA, the polymerase of the prime editor can be a DNA-dependent DNA polymerase. In different embodiments (e.g., as shown in FIG. 3D-3E ), the DNA synthesis template (4) can comprise all or part of the "editing template" and "homology arm," as well as the optional 5' end modification region e2. That is, depending on the nature of the e2 region (e.g., whether it includes a hairpin, toe-loop, or stem / loop secondary structure), the polymerase can also encode none, some, or the entire e2 region. In other words, in the case of a 3' extension arm, the DNA synthesis template (3) can include the portion of the extension arm (3) spanning the 5' end of the primer binding site (PBS) to the 3' end of the gRNA core, which can operate as a template for the polymerase (e.g., reverse transcriptase) to synthesize a DNA single strand. In the case of a 5' extension arm, the DNA synthesis template (3) can include the portion of the extension arm (3) spanning the 5' end of the PEgRNA molecule to the 3' end of the editing template. Preferably, the DNA synthesis template does not include the primer binding site (PBS) of the PEgRNA with either a 3' extension arm or a 5' extension arm. Certain embodiments described herein (e.g., FIG. 71A ) refer to the "RT template," which includes the editing template and homology arm, i.e., the sequence of the PEgRNA extension arm that is actually used as a template during DNA synthesis. The term "RT template" is equivalent to the term "DNA synthesis template."

[0203] In the context of trans prime editing (e.g., FIG. 3G and FIG. 3H ), the primer binding site (PBS) and the DNA synthesis template can be engineered as separate molecules, referred to as a trans prime editor RNA template (tPERT).

[0204] Dimerization domain

[0205] The term "dimerization domain" refers to a ligand binding domain that binds to a binding moiety of a bispecific ligand. A "first" dimerization domain binds to a first binding moiety of a bispecific ligand, and a "second" dimerization domain binds to a second binding moiety of the same bispecific ligand. When the first dimerization domain is fused to a first protein (e.g., by a PE, as discussed herein) and the second dimerization domain is fused to a second protein (e.g., by a PE, as discussed herein), the first and second proteins dimerize in the presence of the bispecific ligand, wherein the bispecific ligand has at least one moiety that binds to the first dimerization domain and at least another moiety that binds to the second dimerization domain.

[0206] Downstream

[0207] As used herein, the terms "upstream" and "downstream" are relative terms that define the linear position of at least two elements in a nucleic acid molecule (whether single-stranded or double-stranded) oriented in a 5' to 3' direction. Specifically, a first element is upstream of a second element in a nucleic acid molecule, where the first element is located somewhere 5' of the second element. For example, if a SNP is located 5' of a nick site, the SNP is upstream of the Cas9-induced nick site. Conversely, a first element is downstream of a second element in a nucleic acid molecule, where the first element is located somewhere 3' of the second element. For example, if a SNP is located 3' of a nick site, the SNP is downstream of the Cas9-induced nick site. A nucleic acid molecule can be DNA (double-stranded or single-stranded), RNA (double-stranded or single-stranded), or a hybrid of DNA and RNA. Analysis of single-stranded nucleic acid molecules and double-stranded molecules is the same because the terms upstream and downstream refer only to the single strand of the nucleic acid molecule, except that consideration is given to which strand of a double-stranded molecule is selected. Generally, 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 the segment of double-stranded DNA that extends from 5' to 3' and is complementary to the antisense or template strand of DNA that extends from 3' to 5'. Thus, for example, if a SNP nucleobase is 3' of a promoter on the sense or coding strand, the SNP nucleobase is "downstream" of the promoter sequence in genomic DNA (double-stranded).

[0208] Editing template

[0209] The term "editing template" refers to a portion of an extension arm that encodes a desired edit in a single-stranded 3' DNA flap synthesized by a polymerase, e.g., a DNA-dependent DNA polymerase, an RNA-dependent DNA polymerase (e.g., a reverse transcriptase). Certain embodiments described herein (e.g., FIG. 71A) refers to the "RT template" which refers to both the edit template and the homology arm, i.e., the sequence of the PEgRNA extension arm that is actually used as a template during DNA synthesis. The term "RT edit template" is also equivalent to the term "DNA synthesis template" but where the RT edit template reflects the use of a prime editor with a polymerase that is a reverse transcriptase, where the DNA synthesis template more broadly reflects the use of a prime editor with any polymerase.

[0210] Effective amount

[0211] As used herein, the term "effective amount" refers to the amount of a biologically active agent sufficient to elicit a desired biological response. For example, in some embodiments, an effective amount of a prime editor (PE) can refer to the amount of an editor sufficient to edit a nucleotide sequence (e.g., a genome) at a target site. In some embodiments, an effective amount of a prime editor (PE) provided herein, e.g., an effective amount of a fusion protein comprising a nickase Cas9 domain and a reverse transcriptase, can refer to the amount of the fusion protein sufficient to cause the fusion protein to specifically bind and edit an edit at a target site. One of skill in the art will appreciate that an effective amount of an agent, e.g., a fusion protein, a nuclease, a hybrid protein, a protein dimer, a complex of a protein (or protein dimer) and a polynucleotide, or a polynucleotide, can vary depending on various factors, e.g., the desired biological response, e.g., a particular allele, genome, or target site to be edited, the cell or tissue being targeted, and the agent being used.

[0212] Error-prone reverse transcriptase

[0213] As used herein, the term "error-prone" reverse transcriptase (or more broadly, any polymerase) refers to a reverse transcriptase (or more broadly, any polymerase) that naturally occurs or is derived from another reverse transcriptase (e.g., wild-type M-MLV reverse transcriptase) that has an error rate that is less than the error rate of the wild-type M-MLV reverse transcriptase. The error rate of wild-type M-MLV reverse transcriptase has been reported to range from one error in 15,000 (higher) to one error in 27,000 (lower). An error rate of one in 15,000 corresponds to an error rate of 6.7 x 10 -5 An error rate of one in 27,000 corresponds to an error rate of 3.7 x 10 -5error rate. 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” refers to those RTs having an error rate greater than one error in 15,000 nucleobase incorporations (6.7 x 10 -5 or higher), 1 error in 14,000 nucleobases (7.14 x 10 -5 or higher), 1 error in 13,000 or fewer nucleobases (7.7 x 10 -5 or higher), 1 error in 12,000 or fewer nucleobases (7.7 x 10 -5 or higher), 1 error in 11,000 or fewer nucleobases (9.1 x 10 -5 or higher), 1 error in 10,000 or fewer nucleobases (1 x 10 -4 or higher), 1 error in 9,000 or fewer nucleobases (0.00011 or higher), 1 error in 8,000 or fewer nucleobases (0.00013 or higher), 1 error in 7,000 or fewer nucleobases (0.00014 or higher), 1 error in 6,000 or fewer nucleobases (0.00016 or higher), 1 error in 5,000 or fewer nucleobases (0.0002 or higher), 1 error in 4,000 or fewer nucleobases (0.00025 or higher), 1 error in 3,000 or fewer nucleobases (0.00033 or higher), 1 error in 2,000 or fewer nucleobases (0.00050 or higher), or 1 error in 1,000 or fewer nucleobases (0.001 or higher), or 1 error in 500 or fewer nucleobases (0.002 or higher), or 1 error in 250 or fewer nucleobases (0.004 or higher).

[0214] Epitope

[0215] As used herein, the term "extein" refers to a polypeptide sequence flanked by inteins and linked to another extein in the process of protein splicing to form a mature spliced protein. Typically, an intein is flanked on both sides by two extein sequences that are linked together when the intein catalyzes its own excision. Thus, an extein is a protein analog of an exon present in mRNA. For example, a polypeptide comprising an intein can have the structure extein(N)-intein-extein(C). After excision of the intein and splicing of the two exteins, the resulting structure is extein(N)-extein(C) and a free intein. In different configurations, the exteins can be separate proteins (e.g., halves of a Cas9 or PE fusion protein), each fused to a split intein, where excision of the split intein results in splicing of the extein sequences together.

[0216] Extension arm

[0217] The term "extension arm" refers to a nucleotide sequence component of a PEgRNA that provides multiple functions, including a primer binding site and an editing template for a reverse transcriptase. In some embodiments, for example FIG. 3D , the extension arm is located at the 3' end of the guide RNA. In other embodiments, for example FIG. 3E , the extension arm is located at the 5' end of the guide RNA. In some embodiments, the extension arm further comprises a homology arm. In different embodiments, the extension arm comprises, in the 5' to 3' direction, the following components: a homology arm, an editing template, and a primer binding site. Because the polymerization activity of a reverse transcriptase is in the 5' to 3' direction, the preferred arrangement of the homology arm, editing template, and primer binding site in the 5' to 3' direction is such that the reverse transcriptase, once primed by the annealed primer sequence, uses the editing template as the complementary template strand to polymerize a DNA single strand. More details are described elsewhere herein, for example the length of the extension arm.

[0218] The extension arm can also be described as generally comprising two regions: a primer binding site (PBS) and a DNA synthesis template, for example, as FIG. 3G(upper panel) is shown. The primer binding site binds a primer sequence that is formed from the endogenous DNA strand of the target site as it is nicked by the prime editor complex, thereby exposing the 3' end on the endogenous nicked strand. As explained herein, the binding of the primer sequence to the primer binding site on the PEgRNA extension arm creates a duplex region with an exposed 3' end (i.e., 3' of the primer sequence) that then provides a substrate for a polymerase to begin polymerizing a single strand of DNA from the exposed 3' end along the length of the DNA synthesis template. The sequence of the single-stranded DNA product is the complement of the DNA synthesis template. Polymerization continues in the 5' direction toward the DNA synthesis template (or extension arm) until polymerization terminates. Thus, the DNA synthesis template represents a portion of the extension arm that is encoded by the polymerase of the prime editor complex into a single-stranded DNA product (i.e., a 3' single-stranded DNA flap containing the desired genetic edit information) and ultimately replaces the corresponding endogenous DNA strand of the target site located immediately downstream of the PE-induced nick site. Without being bound by theory, the DNA synthesis template continues to polymerize toward the 5' end of the extension arm until a termination event. Polymerization can terminate in a variety of ways, including but not limited to (a) reaching the 5' end of the PEgRNA (as in the case of the 5' extension arm, where the DNA polymerase simply runs out of template), (b) reaching an insurmountable RNA secondary structure (e.g., a hairpin or stem loop), or (c) reaching a replication termination signal, such as a specific nucleotide sequence that blocks or inhibits the polymerase, or a nucleic acid topological signal, such as supercoiled DNA or RNA.

[0219] Flap endonuclease (e.g., FEN1)

[0220] As used herein, the term "flap endonuclease" refers to an enzyme that catalyzes the removal of 5' single-stranded DNA flaps. These are naturally occurring enzymes that process cellular processes, including the removal of 5' flaps formed during DNA replication. The prime editing methods described herein can utilize endogenously provided flap endonucleases or those provided in trans to remove the 5' flap of endogenous DNA formed at the target site during prime editing. Flap endonucleases are known in the art and described in Patel et al., "Flap endonucleases pass 5'-flaps through a flexible arch using a disorder-thread-order mechanism to confer specificity for free 5'-ends," Nucleic Acids Research, 2012, 40(10): 4507-4519, Tsutakawa et al., "Human flap endonuclease structures, DNA double-base flipping, and a unified understanding of the FEN1 superfamily," Cell, 2011, 145(2): 198-211, and Balakrishnan et al., "Flap Endonuclease 1," Annu Rev Biochem, 2013, Vol 82: 119-138 (each incorporated herein by reference). An exemplary flap endonuclease is FEN1, which can be represented by the following amino acid sequence:

[0221]

[0222] Functional equivalent

[0223] The term "functional equivalent" refers to a second biological molecule that is functionally equivalent to a first biological molecule but not necessarily structurally equivalent. For example, a "Cas9 equivalent" refers to a protein that has the same or substantially the same function as Cas9 but not necessarily the same amino acid sequence. In the context of the present disclosure, the specification refers to "protein X or a functional equivalent thereof" throughout. In this context, a "functional equivalent" of protein X includes any homolog, paralog, fragment, naturally occurring, engineered, mutated, or synthetic version of protein X that has equivalent function.

[0224] Fusion protein

[0225] As used herein, the term "fusion protein" refers to a hybrid polypeptide comprising protein domains from at least two different proteins. One protein can be located in the amino-terminal (N-terminal) portion or the carboxy-terminal (C-terminal) protein of the fusion protein, forming an "amino-terminal fusion protein" or a "carboxy-terminal fusion protein," respectively. A protein can comprise different domains, for example, a nucleic acid binding domain (e.g., a gRNA binding domain of Cas9 that directs the protein to bind to a target site) and a nucleic acid cleavage domain or catalytic domain of a nucleic acid editing protein. Another example includes Cas9 or its equivalent to a reverse transcriptase. Any of the proteins provided herein can be produced by any method known in the art. For example, the proteins provided herein can be produced by recombinant protein expression and purification, which is particularly suitable for fusion proteins comprising a peptide linker. Methods for recombinant protein expression and purification are well known, including those described in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4thed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)), which is incorporated by reference herein in its entirety. th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)), which is incorporated by reference herein in its entirety.

[0226] Gene of interest (GOI)

[0227] The term "gene of interest" or "GOI" refers to a gene that encodes a biomolecule of interest (e.g., a protein or an RNA molecule). Proteins of interest can include any intracellular protein, membrane protein, or extracellular protein, such as a nuclear protein, a transcription factor, a nuclear membrane transport protein, an intracellular organelle-associated protein, a membrane receptor, a catalytic protein and enzyme, a therapeutic protein, a membrane protein, a membrane transport protein, a signal transduction protein, or an immune protein (e.g., an IgG or other antibody protein), and the like. Genes of interest can also encode RNA molecules, including but not limited to messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA), antisense RNA, guide RNA, microRNA (miRNA), small interfering RNA (siRNA), and cell-free RNA (cfRNA).

[0228] Guide RNA ("gRNA")

[0229] As used herein, the term "guide RNA" is a particular type of guide nucleic acid that is typically associated with the Gas protein of CRISPR-Cas9 and associates with Cas9 to direct the Cas9 protein to a particular sequence in a DNA molecule that includes complementarity to the protospacer of the guide RNA. However, the term also includes equivalent guide nucleic acid molecules that associate with Cas9 equivalents, homologs, orthologs, or paralogs, whether naturally occurring or non-naturally occurring (e.g., engineered or recombinant), and otherwise program the Cas9 equivalent to localize to a particular target nucleotide sequence. Cas9 equivalents can include other napDNAbps from any type of CRISPR system (e.g., Type II, V, VI), including Cpfl (Type V CRISPR-Cas system), C2cl (Type V CRISPR-Cas system), C2c2 (Type VI CRISPR-Cas system), and C2c3 (Type V CRISPR-Cas system). Other Gas equivalents are described in Makarova et al., "C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector," Science 2016; 353(6299), the contents of which are incorporated herein by reference. Exemplary sequences and structures of guide RNAs are provided herein. In addition, methods for designing suitable guide RNA sequences are provided herein. As used herein, "guide RNA" is also referred to as "traditional guide RNA" to contrast it with a modified form of guide RNA known as a "prime editing guide RNA" (or "PEgRNA"), which has been invented for the prime editing methods and compositions disclosed herein.

[0230] A guide RNA or PEgRNA can comprise various structural elements, including but not limited to:

[0231] Protospacer sequence - the sequence in a guide RNA or PEgRNA (of about 20 nt in length) that binds to a protospacer in a target DNA.

[0232] gRNA core (or gRNA scaffold or backbone sequence) - refers to the sequence within a gRNA that is responsible for Cas9 binding, which does not include the 20 bp protospacer / targeting sequence that is used to direct Cas9 to a target DNA.

[0233] Extension arm - a single-stranded extension at the 3' end or 5' end of a PEgRNA that contains a primer binding site and a DNA synthesis template sequence that encodes a single-stranded DNA flap containing a genetic change of interest, which is then integrated into endogenous DNA by displacing the corresponding endogenous strand, thereby installing the desired genetic change.

[0234] Transcriptional terminator - a guide RNA or PEgRNA can contain a transcriptional termination sequence at the 3' end of the molecule.

[0235] Homology arm

[0236] The term "homology arm" refers to the portion of the extension arm that encodes the portion of the single-stranded DNA flap that will be integrated into the target DNA location by displacing the endogenous strand. The portion of the single-stranded DNA flap encoded by the homology arm is complementary to the non-edited strand of the target DNA sequence, which facilitates displacement of the endogenous strand and in-place annealing of the single-stranded DNA flap, thereby installing the edit. This component is further defined elsewhere. The homology arm is part of the DNA synthesis template because, by definition, it is encoded by the polymerase of the prime editor described herein.

[0237] Host cell

[0238] As used herein, the term "host cell" refers to a cell that can contain, replicate, and express a vector described herein, e.g., a vector comprising a nucleic acid molecule encoding a fusion protein comprising a Cas9 or Cas9 equivalent and a reverse transcriptase.

[0239] Intein

[0240] As used herein, the term "intein" refers to an autoprocessing polypeptide domain found in organisms from all domains of life. Inteins (intermediates proteins) perform a unique autoprocessing event known as protein splicing, in which it excises itself from a larger precursor polypeptide by cleavage of two peptide bonds and in the process links flanking extein (external protein) sequences by formation of new peptide bonds. This rearrangement occurs post-translationally (or possibly co-translationally), as intein genes are found embedded in-frame within other protein-coding genes. Furthermore, intein-mediated protein splicing is spontaneous; it requires no external factors or energy source, only the folding of the intein domain. This process is also known as cis protein splicing, as opposed to the natural process of trans protein splicing with "split-inteins." Inteins are the protein equivalent of self-splicing RNA introns (see, Perler et al., Nucleic Acids Res. 22: 1125-1127 (1994)), which catalyze their own excision from a precursor protein, accompanied by fusion of flanking protein sequences known as exteins (for review, see Perler et al., Curr. Opin. Chem. Biol. 1 : 292-299 (1997); Perler, F. B. Cell 92(1): 1-4 (1998); Xu et al., EMBO J. 15(19): 5146-5153 (1996)).

[0241] As used herein, the term "protein splicing" refers to the process in which an internal region of a precursor protein (intein) is excised and the flanking regions of the protein (extein) are joined to form a mature protein. This natural process has been observed in many proteins from both prokaryotes and eukaryotes (Perler, F. B., Xu, M. Q., Paulus, H. Current Opinion in Chemical Biology 1997, 1, 292-299; Perler, F. B. Nucleic Acids Research 1999, 27, 346-347). Intein units contain the necessary components required to catalyze protein splicing and often contain endonuclease domains involved in intein migration (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 protein is linked and not expressed as separate proteins. Protein splicing can also occur in trans, with split inteins expressed on separate polypeptides spontaneously combining to form a single intein, which then undergoes the protein splicing process to link to the separate proteins.

[0242] The elucidation of the protein splicing mechanism has led to a number of intein-based applications (Comb, et al., U.S. Patent No. 5,496,714; Comb, et al., U.S. Patent No. 5,834,247; Camarero and Muir, J. Amer. Chem. Soc, 121:5597-5598 (1999); Chong, et al., Gene, 192:271-281 (1997), Chong, et al., Nucleic Acids Res., 26:5109-5115 (1998); Chong, et al., J. Biol. Chem., 273:10567-10577 (1998); Cotton, et al. J. Am. Chem. Soc, 121:1100-1101 (1999); Evans, et al., J. Biol. Chem., 274:18359-18363 (1999); Evans, et al., J. Biol. Chem., 274:3923-3926 (1999); Evans, et al., Protein Sci., 7:2256-2264 (1998); Evans, et al., J. Biol. Chem., 275:9091-9094 (2000); Iwai and Pluckthun, FEBS Lett. 459:166-172 (1999); Mathys, et al., Gene, 231:1-13 (1999); Mills, et al., Proc. Natl. Acad. Sci. USA 95:3543-3548 (1998); Muir, et al., Proc. Natl. Acad. Sci. USA 95:6705-6710 (1998); Otomo, et al., Biochemistry 38:16040-16044 (1999); Otomo, et al., J. Biomol. NMR 14:105-114 (1999); Scott, et al., Proc. Natl. Acad. Sci. USA 96:13638-13643 (1999); Severinov and Muir, J. Biol. Chem., 273:16205-16209 (1998); Shingledecker, et al., Gene, 207:187-195 (1998); Southworth, et al., EMBO J. 17:918-926 (1998); Southworth, et al., Biotechniques, 27:110-120 (1999); Wood, et al., Nat. Biotechnol., 17:889-892 (1999); Wu, et al., Proc. Natl. Acad. Sci. USA 95:9226-9231 (1998a); Wu, et al., Biochim Biophys Acta 1387:422-432 (1998b); Xu, et al., Proc. Natl. Acad. Sci. USA 96:388-393 (1999); Yamazaki, et al., J. Am. Chem. Soc, 120:5591-5592 (1998)). Each of these references is incorporated herein by reference.

[0243] Ligand-dependent intein

[0244] As used herein, the term "ligand-dependent intein" refers to an intein that comprises a ligand binding domain. Typically, the ligand binding domain is inserted into the amino acid sequence of the intein, forming the structure: Intein(N)-ligand binding domain-intein(C). Typically, the ligand-dependent intein shows no or only minimal protein splicing activity in the absence of a suitable ligand, while the protein splicing activity is significantly increased in the presence of the ligand. In some embodiments, the ligand-dependent intein shows no observable splicing activity in the absence of the ligand, but does show splicing activity in the presence of the ligand. In some embodiments, the ligand-dependent intein shows observable protein splicing activity in the absence of the ligand, and the protein splicing activity in the presence of a suitable ligand is at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 150-fold, at least 200-fold, at least 250-fold, at least 500-fold, at least 1000-fold, at least 1500-fold, at least 2000-fold, at least 2500-fold, at least 5000-fold, at least 10000-fold, at least 20000-fold, at least 25000-fold, at least 50000-fold, at least 100000-fold, at least 500000-fold, or at least 1000000-fold greater than the activity observed in the absence of the ligand. In some embodiments, the increase in activity is dose-dependent over at least 1 order of magnitude, at least 2 orders of magnitude, at least 3 orders of magnitude, at least 4 orders of magnitude, or at least 5 orders of magnitude, allowing for fine-tuning of the intein activity by adjusting the ligand concentration.Suitable ligand-dependent inteins are known in the art and include those provided below and described in published U.S. Patent Application U.S. 2014 / 0065711 Al; Mootz et al., "Protein splicing triggered by a small molecule." J. Am. Chem. Soc. 2002; 124, 9044-9045; Mootz et al., "Conditional protein splicing: a new tool to control protein structure and function in vitro and in vivo." J. Am. Chem. Soc. 2003; 125, 10561-10569; Buskirk et al., Proc. Natl. Acad. Sci. USA. 2004; 101, 10505-10510); Skretas & Wood, "Regulation of protein activity with small-molecule-controlled inteins." Protein Sci. 2005; 14, 523-532; Schwartz, et al., "Post-translational enzyme activation in an animal via optimized conditional protein splicing." Nat. Chem. Biol. 2007; 3, 50-54; Peck et al., Chem. Biol. 2011; 18(5), 619-630; the entire contents of each of which are hereby incorporated by reference. Exemplary sequences are as follows:

[0245]

[0246]

[0247] Linker

[0248] As used herein, the term "linker" refers to a molecule that connects two other molecules or moieties. In the case of a linker connecting two fusion proteins, the linker can be an amino acid sequence. For example, Cas9 can be fused to a reverse transcriptase through an amino acid linker sequence. In the case of linking two nucleotide sequences together, the linker can also be a nucleotide sequence. For example, in the current case, a traditional guide RNA is linked through a spacer or linker nucleotide sequence to the RNA extension of a prime editing guide RNA that can comprise 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 to 100 amino acids in length, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30-35, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-150, or 150-200 amino acids in length. Longer or shorter linkers are also contemplated.

[0249] Isolated

[0250] "Isolated" means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of the natural state is "isolated." An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0251] In some embodiments, a gene of interest is encoded by an isolated nucleic acid. As used herein, the term "isolated" refers to the property of a material, as provided herein, that is removed from its original or natural environment, e.g., the natural environment if it is naturally occurring. Thus, a naturally occurring polynucleotide or protein or polypeptide that is present in a living animal is not isolated, but the same polynucleotide or polypeptide separated from some or all of the coexisting materials of the natural system by the hand of man is isolated. Thus, artificial or engineered materials, e.g., non-naturally occurring nucleic acid constructs, such as the expression constructs and vectors described herein, are also referred to as isolated. The material need not be purified to be isolated. Thus, the material can be part of a vector and / or part of a composition, and still be isolated in that such vector or composition is not part of the environment in which the material is found in nature.

[0252] MS2 tagging technology

[0253] In various embodiments (e.g., as depicted in the embodiments of FIGS. 72-73 and Example 19), the term “MS2 tagging technology” refers to the combination of an “RNA-protein interaction domain” (a.k.a. “RNA-protein recruiting domain or protein”) paired with an RNA binding protein that specifically recognizes and binds to the RNA-protein interaction domain (e.g., a particular hairpin structure). These types of systems can be utilized to recruit various functionalities to a prime editor complex bound to a target site. MS2 tagging technology is based on the natural interaction of the MS2 bacteriophage coat protein (“MCP” or “MS2 cp”) with a stem loop or hairpin structure (i.e., “MS2 hairpin”) present in the bacteriophage genome. In the case of prime editing, MS2 tagging technology involves introducing an MS2 hairpin into a desired RNA molecule involved in prime editing (e.g., a PEgRNA or tPERT), which then constitutes a specific interactable binding target for an RNA binding protein that recognizes and binds to the structure. In the case of an MS2 hairpin, it is recognized and bound by the MS2 bacteriophage coat protein (MCP). And, if the MCP is fused to another protein (e.g., a reverse transcriptase or other DNA polymerase), the MS2 hairpin can be used to “recruit” the other protein in trans to a target site occupied by a prime editing complex.

[0254] In one aspect, the guide editors described herein can incorporate any known RNA-protein interaction domain to recruit or "co-localize" a particular functionality of interest to the guide editor complex. A review of other modular RNA-protein interaction domains has been described in the art, for example, Johansson et al., "RNA recognition by the MS2 phage coat protein," Sem Virol., 1997, Vol. 8(3): 176-185; Delebecque et al., "Organization of intracellular reactions with rationally designed RNA assemblies," Science, 2011, Vol. 333: 470-474; Mali et al., "Cas9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering," Nat. Biotechnol., 2013, Vol. 31: 833-838; and Zalatan et al., "Engineering complex synthetic transcriptional programs with CRISPR RNA scaffolds," Cell, 2015, Vol. 160: 339-350, each incorporated herein by reference in their entirety. Other systems include PP7 hairpins, which specifically recruit PCP protein, and "com" hairpins, which specifically recruit Com protein. See Zalatan et al.

[0255] The nucleotide sequence of the MS2 hairpin (or equivalently referred to as "MS2 aptamer") is:

[0256] GCCAACATGAGGATCACCCATGTCTGCAGGGCC (SEQ ID NO: 763).

[0257] The amino acid sequence of MCP or MS2cp is:

[0258] GSASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVATQTVGGEELPVAGWRSYLNMELTIPFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY (SEQ ID NO: 764).

[0259] MS2 hairp in (or "MS2 aptamer") can also be referred to as a class of "RNA effector recruiting domains" (or equivalently as "RNA binding protein recruiting domains" or simply "recruiting domains") because it is a physical structure (e.g., a hairpin) installed into a PEgRNA or tPERT that effectively recruits other effector functions (e.g., RNA binding proteins with different functions such as DNA polymerases or other DNA modifying enzymes) to the so-modified PEgRNA or rPERT, thereby colocalizing the effector functions in trans to the prime editing machinery. The present application is not intended to be limited in any way to any particular RNA effector recruiting domain, and can include any such domain available, including the MS2 hairpin. Example 19 and FIG. 72(b) depict the use of an MS2 aptamer linked to a DNA synthesis domain (i.e., a tPERT molecule) and a prime editor comprising an MS2cp protein fused to a PE2 to cause colocalization of the prime editor complex (MS2cp-PE2:sgRNA complex) bound to the target DNA location and the DNA synthesis domain of the tPERT molecule to complete.

[0260] napDNAbp

[0261] As used herein, the term "nucleic acid programmable DNA binding protein" or "napDNAbp" (with Cas9 as an example) refers to a protein that uses RNA:DNA hybridization to target and bind to a specific sequence in a DNA molecule. Each napDNAbp associates with at least one guide nucleic acid (e.g., a guide RNA), which localizes the napDNAbp to a DNA sequence comprising a DNA strand (i.e., a target strand) that is complementary to the guide nucleic acid or portion thereof (e.g., protospacer of a guide RNA). In other words, the guide nucleic acid "programs" the napDNAbp (e.g., Cas9 or equivalent) to locate and bind to a complementary sequence.

[0262] Without being bound by theory, the binding mechanism of napDNAbp-guide RNA complexes generally includes a step of forming an R-loop, whereby the napDNAbp induces the unwinding of the double-stranded DNA target, separating the strands in the region bound by the napDNAbp. The guide RNA protospacer then hybridizes to the "target strand." This displaces the "non-target strand," which is complementary to the target strand, forming a single-stranded region of the R-loop. In some embodiments, the napDNAbp includes one or more nuclease activities, which then cleave the DNA, leaving various types of lesions. For example, the napDNAbp can comprise a nuclease activity that cleaves the non-target strand at a first location and / or cleaves the target strand at a second location. Depending on the nuclease activity, the target DNA can be cleaved to form a "double-stranded break," cleaving both strands. In other embodiments, the target DNA can be cleaved only at a single location, i.e., the DNA has a "nick" on one strand. Exemplary napDNAbps with different nuclease activities include "Cas9 nickases" ("nCas9") and inactive Cas9s without nuclease activity ("dead Cas9" or "dCas9"). Exemplary sequences of these and other napDNAbps are provided herein.

[0263] Nicking enzyme

[0264] The term "nickase" refers to a Cas9 protein with one of the two nuclease domains inactivated. as 9. This enzyme can only cleave one strand of the target DNA.

[0265] Nuclear localization sequence (NLS)

[0266] The term "nuclear localization sequence" or "NLS" refers to an amino acid sequence that imports into the nucleus as by nuclear transport facilitating proteins. Nuclear localization sequences are known in the art and will be apparent to the skilled person. For example, the disclosure of exemplary nuclear localization sequences of International PCT Application No. PCT / EP2000 / 011690, filed November 23, 2000, published as WO / 2001 / 038547 on May 31, 2001, is incorporated herein by reference. In some embodiments, the NLS comprises the amino acid sequence PKKKRKV (SEQ ID NO: 16) or MDSLLMNRRKFLYQFKNVRWAKGRRETYLC (SEQ ID NO: 17).

[0267] Nucleic acid molecule

[0268] As used herein, the term "nucleic acid" refers to a polymer of nucleotides. The polymer can include natural nucleosides (i.e., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine), nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C5 bromouridine, C5 fluoro uridine, C5 iodo uridine, C5 propynyl uridine, C5 propynyl cytidine, C5 methyl cytidine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6) methyl guanine, 4-acetylcytidine, 5-(carboxyhydroxylmethyl) uridine, dihydrouridine, methylpseudo-uridine, 1-methyladenosine, 1-methylguanosine, N6-methyladenosine, and 2-thiocytidine), chemically modified bases, biologically modified bases (e.g., methylated bases), intervening bases, modified sugars (e.g., 2'-fluoro-ribose, ribose, 2'-deoxyribose, 2'-O-methylcytidine, arabinose, and hexose), or modified phosphate groups (e.g., phosphorothioates and 5' N phosphoramidite linkages).

[0269] PEgRNA

[0270] As used herein, the term "prime editing guide RNA" or "PEgRNA" or "extended guide RNA" refers to a particular form of guide RNA that has been modified to include one or more additional sequences for carrying out the prime editing methods and compositions described herein. As described herein, a prime editing guide RNA comprises one or more "extension regions" of nucleic acid sequence. The extension region can include, but is not limited to, single-stranded RNA or DNA. Further, the extension region can be present at the 3' end of the traditional guide RNA. In other configurations, the extension region can be present at the 5' end of the traditional guide RNA. In other configurations, the extension region can be present at an intramolecular region of the traditional guide RNA, e.g., in the gRNA core region that associates and / or binds with the napDNAbp. The extension region comprises a "DNA synthesis template" that encodes (by the polymerase of the prime editor) a single-stranded DNA that is in turn designed to be (a) homologous to the endogenous target DNA to be edited, and (b) which comprises at least one desired nucleotide change (e.g., transition, transversion, deletion, or insertion) to be introduced or integrated into the endogenous target DNA. The extension region can further comprise other functional sequence elements, such as but not limited to "primer binding sites" and "spacer or linker" sequences, or additional structural elements, such as but not limited to aptamers, stem loops, hairpins, toe loops (e.g., 3' toe loops), or RNA-protein recruitment domains (e.g., MS2 hairpins). As used herein, a "primer binding site" comprises a sequence that hybridizes to a single-stranded DNA sequence with a 3' end that is generated from nicked DNA of the R-loop.

[0271] In certain embodiments, the PEgRNA is derived from FIG. 3APEgRNA having a 5' extension arm, a spacer, and a gRNA core. The 5' extension further includes, in the 5' to 3' direction, a reverse transcriptase template, a primer binding site, and a linker. As shown, the reverse transcriptase template can also be more broadly referred to as a "DNA synthesis template," where the polymerase of the prime editor described herein is not an RT, but another type of polymerase.

[0272] In certain other embodiments, the PEgRNA is comprised of FIG. 3B PEgRNA having a 5' extension arm, a spacer, and a gRNA core. The 5' extension further includes, in the 5' to 3' direction, a reverse transcriptase template, a primer binding site, and a linker. As shown, the reverse transcriptase template can also be more broadly referred to as a "DNA synthesis template," where the polymerase of the prime editor described herein is not an RT, but another type of polymerase.

[0273] In other embodiments, the PEgRNA is comprised of FIG. 3D PEgRNA having a 5' extension arm, a spacer, and a gRNA core. The 5' extension further includes, in the 5' to 3' direction, a reverse transcriptase template, a primer binding site, and a linker. As shown, the reverse transcriptase template can also be more broadly referred to as a "DNA synthesis template," where the polymerase of the prime editor described herein is not an RT, but another type of polymerase.

[0274] In other embodiments, the PEgRNA is comprised of FIG. 3E PEgRNA having a 5' extension arm, a spacer, and a gRNA core. The 5' extension further includes, in the 5' to 3' direction, a reverse transcriptase template, a primer binding site, and a linker. As shown, the reverse transcriptase template can also be more broadly referred to as a "DNA synthesis template," where the polymerase of the prime editor described herein is not an RT, but another type of polymerase.

[0275] PE1

[0276] As used herein, "PE1" refers to a PE complex comprising a fusion protein comprising Cas9(H840A) and wild type MMLV RT: [NLS]-[Cas9(H840A)]-[linker]-[MMLV_RT(wt)] + desired PEgRNA, where the PE fusion has the amino acid sequence of SEQ ID NO: 123, as shown below;

[0277]

[0278]

[0279] Explanation:

[0280] Nuclear localization sequence (NLS) Start: (SEQ ID NO: 124), End: (SEQ ID NO: 133)

[0281] CAS9(H840A) (SEQ ID NO: 126)

[0282] 33-amino acid linker (SEQ ID NO: 127)

[0283] M-MLV Reverse Transcriptase (SEQ ID NO: 128).

[0284] PE2

[0285] As used herein, "PE2" refers to a PE complex comprising a fusion protein comprising Cas9(H840A) and variant MMLV RT: (NLS)-[(Cas9(H840A)]-[linker]-[MMLV_RT(D200N)(T330P)(L603W)(T306K)(W313F)] + desired PEgRNA, where the PE fusion has the amino acid sequence of SEQ ID NO: 134, as shown below;

[0286]

[0287]

[0288] Explanation:

[0289] Nuclear localization sequence (NLS) Start: (SEQ ID NO: 124), End: (SEQ ID NO: 133)

[0290] CAS9(H840A) (SEQ ID NO: 137)

[0291] 33-amino acid linker(SEQ ID NO: 127)

[0292] M-MLV reverse transcriptase (SEQ ID NO: 139).

[0293] PE3

[0294] As used herein, "PE3" refers to PE2 plus a second strand nicking generating guide RNA that complexes with PE2 and introduces a nick in the non-edited DNA strand to cause preferential displacement of the edited strand.

[0295] PE3b

[0296] As used herein, "PE3b" refers to PE3 but where the second strand nicking generating guide RNA is designed for temporal control such that the second strand nick is not introduced until after the desired edit is installed. This is achieved by designing the gRNA to have a spacer sequence that only matches the edited strand and not the original allele. Using this strategy (hereafter referred to as PE3b), the mismatch between the original spacer and the non-edited allele should be unfavorable for nicking by the sgRNA until after the editing event on the PAM strand has occurred.

[0297] PE-Short

[0298] As used herein, "PE-short" refers to a PE construct fused to a C-terminally truncated reverse transcriptase, having the following amino acid sequence:

[0299]

[0300]

[0301] Explanation:

[0302] Nuclear localization sequence (NLS) Start: (SEQ ID NO: 124), End: (SEQ ID NO: 133)

[0303] CAS9 (H840A) (SEQ ID NO: 157)

[0304] 33-amino acid linker 1 (SEQ ID NO: 127)

[0305] M-MLV truncated reverse transcriptase

[0306] (SEQ ID NO: 766)

[0307] Peptide tag

[0308] 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 to the protein that aid in manipulating the protein for different purposes, such as visualization, purification, solubilization, and isolation, among others. Peptide tags can include different types of tags that are classified by purpose or function, which can include "affinity tags" (facilitate protein purification), "solubilization tags" (assist in proper protein folding), "chromatography tags" (alter the chromatographic properties of the protein), "epitope tags" (bind to high affinity antibodies), "fluorescent tags" (facilitate protein visualization in cells or in vitro).

[0309] Polymerase

[0310] As used herein, the term "polymerase" refers to an enzyme that synthesizes a nucleotide chain and can be used in conjunction with the prime editor system described herein. The polymerase can be a "template-dependent" polymerase (i.e., a polymerase that synthesizes a nucleotide chain based on the sequence of nucleotide bases of a template strand). The polymerase can also be a "template-independent" polymerase (i.e., a polymerase that synthesizes a nucleotide chain without a template strand). The polymerase can be further classified as a "DNA polymerase" or an "RNA polymerase." In different embodiments, the prime editor system comprises a DNA polymerase. In different embodiments, the DNA polymerase can be a "DNA-dependent DNA polymerase" (i.e., whereby the template molecule is a DNA strand). In this case, the DNA template molecule can be a PEgRNA, where the extension arm comprises a DNA strand. In this case, the PEgRNA can be referred to as a chimeric or hybrid PEgRNA, which comprises an RNA portion (i.e., the guide RNA component, including the spacer and gRNA core) and a DNA portion (i.e., the extension arm). In different other embodiments, the DNA polymerase can be an "RNA-dependent DNA polymerase" (i.e., whereby the template molecule is an RNA strand). In this case, the PEgRNA is RNA, i.e., includes an RNA extension. The term "polymerase" can also refer to an enzyme that catalyzes the polymerization of nucleotides (i.e., polymerase activity). Typically, the enzyme will start synthesis at the 3'-end of a primer (e.g., a primer sequence that anneals to the primer binding site of a PEgRNA) that is annealed to a polynucleotide template sequence, and will proceed towards the 5' end of the template strand. A "DNA polymerase" catalyzes the polymerization of deoxynucleotides. As used herein with respect to DNA polymerases, the term DNA polymerase includes "functional fragments thereof." By "functional fragments thereof" is meant any portion of a wild-type or mutant DNA polymerase that comprises less than the complete amino acid sequence of the polymerase and retains the ability to catalyze the polymerization of polynucleotides under at least one set of conditions. Such functional fragments can exist as separate entities, or it can be a component of a larger polypeptide (e.g., a fusion protein).

[0311] Prime editing

[0312] As used herein, the term "prime editing" refers to a new method of gene editing using napDNAbps, a polymerase (e.g., a reverse transcriptase), and a specialized guide RNA that includes a DNA synthesis template for encoding a desired new genetic information (or deleting genetic information) that is then incorporated into a target DNA sequence. FIG. 1A to 1H Certain embodiments of prime editing are described in particular in the embodiments of FIGS. 72(a) to 72(c).

[0313] Prime editing represents a new genome editing platform for a universal and precise genome editing method that uses a nucleic acid programmable DNA binding protein ("napDNAbp") running with a polymerase (i.e., in the form of a fusion protein or otherwise provided in trans to the napDNAbp) to write new genetic information to a prescribed DNA site, where the prime editing system is programmed with a prime editing (PE) guide RNA ("PEgRNA") that both specifies the target site and provides a template for synthesizing a desired edit in the form of a replacement DNA strand through an extension (DNA or RNA) engineered to the guide RNA (e.g., at the 5' or 3' end or internal portion of the guide RNA). The replacement strand containing the desired edit (e.g., a single nucleobase substitution) shares the same (or homologous) sequence (except that it includes the desired edit) with the endogenous strand of the target site to be edited (immediately downstream of the nick site). Through DNA repair and / or replication mechanisms, the endogenous strand downstream of the nick site is replaced with the newly synthesized replacement strand containing the desired edit. In certain instances, prime editing can be considered a "search-and-replace" genome editing technique because the prime editors described herein not only search for and locate the desired target site to be edited, but also simultaneously encode the replacement strand containing the desired edit that is installed in place of the corresponding target site endogenous DNA strand. The prime editors of the present disclosure are based in part on the discovery that the mechanism of target primed reverse transcription (TPRT) or "prime editing" can be harnessed or adapted for CRISPR / Cas-based precise genome editing with high efficiency and genetic plasticity (e.g., as depicted in different embodiments of FIG. 1). FIG. 1A to 1F TPRT is naturally used by mobile DNA elements, such as mammalian non-LTR retrotransposons and bacterial group II introns 28，29The present inventors herein use Cas protein-reverse transcriptase fusions or related systems to target a specific DNA sequence with a guide RNA, create a single-stranded nick at the target site, and use the nicked DNA as a primer to perform reverse transcription of an engineered reverse transcriptase template that integrates with the guide RNA. However, while the concept starts with a prime editor using a reverse transcriptase as the DNA polymerase component, the prime editors described herein are not limited to reverse transcriptases, but can include the use of virtually any DNA polymerase. In fact, while the present application can refer to a prime editor with a "reverse transcriptase" throughout, it is presented herein that a reverse transcriptase is just one type of DNA polymerase that can function with a prime editor. Thus, wherever the specification refers to a "reverse transcriptase," one of ordinary skill in the art will understand that any suitable DNA polymerase can be used in place of a reverse transcriptase. Thus, in one aspect, a prime editor can comprise a Cas9 (or equivalent napDNAbp) that is programmed to target a DNA sequence by associating it with a specialized guide RNA (i.e., a PEgRNA) that comprises a spacer sequence that anneals to a protospacer in the target DNA that is complementary. The specialized guide RNA also comprises new genetic information in the form of an extension that encodes a replacement strand of DNA comprising a desired genetic change that is used to replace a corresponding endogenous DNA strand at the target site. To transfer the information from the PEgRNA to the target DNA, the prime editing mechanism involves making a nick in one strand of the DNA at the target site to expose a 3'-hydroxyl. The exposed 3'-hydroxyl can then be used to prime DNA polymerization on the PEgRNA that encodes the extension of the edit in the target site. In different 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 extension, the polymerase of the prime editor can be an RNA-dependent DNA polymerase (e.g., a reverse transcriptase). In the case of DNA extension, the polymerase of the prime editor can be a DNA-dependent DNA polymerase. The newly synthesized strand (i.e., the replacement DNA strand containing the desired edit) formed by the prime editor disclosed herein will be homologous (i.e., have the same sequence) to the genomic target sequence except for comprising the desired nucleotide change (e.g., a single nucleotide change, a deletion, or an insertion, or a combination thereof). The newly synthesized (or replacement) DNA strand is also referred to as a single-stranded DNA flap, which will compete for hybridization with the complementary homologous endogenous DNA strand, thereby replacing the corresponding endogenous strand. In certain embodiments, the system can be combined with the use of an error-prone reverse transcriptase (e.g., provided as a fusion protein with the Cas9 domain, or provided in trans with the Cas9 domain). The error-prone reverse transcriptase can introduce changes during the synthesis of the single-stranded DNA flap. Thus, in certain embodiments, the error-prone reverse transcriptase can be utilized to introduce nucleotide changes into the target DNA. Depending on the error-prone reverse transcriptase used with the system, the changes can be random or non-random.Disassembly of the hybridized intermediates (including the single-stranded DNA flap synthesized by reverse transcriptase hybridized to an endogenous DNA strand) can include removal of the displaced flap of endogenous DNA (e.g., using 5' end DNA flap endonuclease, FENl), ligation of the synthesized single-stranded DNA flap to the target DNA, and assimilation of the desired nucleotide changes due to cellular DNA repair and / or replication processes. Because DNA synthesis to provide a template provides single nucleotide precision for any nucleotide modification (including insertions and deletions), the scope of this approach is very broad and foreseeably can be used for myriad applications in basic science and therapeutics.

[0314] In different embodiments, prime editing operates by contacting a target DNA molecule to which a change in nucleotide sequence is desired with a nucleic acid programmable DNA binding protein (napDNAbp) complexed with a prime editing guide RNA (PEgRNA). See FIG. 1GAs shown, the prime editing guide RNA (PEgRNA) comprises an extension at the 3' or 5' end of the guide RNA or an intramolecular position of the guide RNA and encodes a 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) in one of the DNA strands of the target locus, thereby creating an accessible 3' end in one of the strands of the target locus. In certain embodiments, the nick is created in the DNA strand corresponding to the R-loop strand (i.e., the strand that is not hybridized to the guide RNA sequence, i.e., the "non-target strand"). However, the nick can be introduced in either strand. In other words, the nick can be introduced into the R loop "target strand" (i.e., the strand that is hybridized to the protospacer of the extended gRNA) or the "non-target strand" (i.e., the strand that forms the single-stranded portion of the R loop, which is complementary to the target strand). In step (c), the 3' end of the DNA strand (created by the nick) interacts with the extended portion of the guide RNA to prime reverse transcription (i.e., "target-primed RT"). In certain embodiments, the 3' end DNA strand hybridizes to a specific RT priming sequence on the extended portion of the guide RNA, i.e., the "reverse transcriptase priming sequence" or "primer binding site" on the PEgRNA. In step (d), a reverse transcriptase (or other suitable DNA polymerase) is introduced, which synthesizes a single-stranded DNA from the 3' end of the priming site towards the 5' end of the prime editing guide RNA. The DNA polymerase (e.g., reverse transcriptase) can be fused to the napDNAbp, or alternatively, can be provided in trans to the napDNAbp. This forms a single-stranded DNA flap, which comprises the desired nucleotide change (e.g., single base change, insertion, or deletion, or combinations thereof), and is otherwise homologous to the endogenous DNA at or near the nick site. In step (e), the napDNAbp and guide RNA are released. Steps (f) and (g) involve resolution of the single-stranded DNA flap, such that the desired nucleotide change is incorporated into the target locus. Without being bound by theory, cellular endogenous DNA repair and replication processes resolve the mismatched DNA to incorporate the nucleotide change to form the desired altered product. The product formation can also be driven by "second strand nicking" as FIG. 1F shown. This process can introduce at least one or more of the following genetic changes: transversions, transitions, deletions, and insertions.

[0315] The term "prime editor (PE) system" or "prime editor (PE)" or "PE system" or "PE editing system" refers to compositions involved in the method of genome editing using target primed reverse transcription (TPRT) described herein, including but not limited to napDNAbps, reverse transcriptases, fusion proteins (e.g., comprising a napDNAbp and a reverse transcriptase), prime editing guide RNAs, and complexes comprising a fusion protein and a prime editing guide RNA, as well as ancillary elements, such as second strand nicking producing components (e.g., second strand sgRNAs) and 5' endogenous DNA flap removing endonucleases (e.g., FEN1), for aiding in the formation of driving the prime editing process toward an editing product.

[0316] While in the embodiments described thus far, the PE gRNA constitutes a single molecule comprising a guide RNA (which itself comprises a spacer sequence and a gRNA core or scaffold) and a 5' or 3' extension arm containing a primer binding site and a DNA synthesis template (see, e.g., FIG. 3D , the PE gRNA can also take the form of two separate molecules consisting of a guide RNA and a trans- prime editor RNA template (tPERT) that essentially houses the extension arm (including, in particular, the primer binding site and the DNA synthesis domain) and the RNA-protein recruiting domain (e.g., MS2 aptamer or hairpin) in the same molecule, which co-localize or recruit to a modified prime editor complex comprising a tPERT recruiting protein (e.g., MS2 cp protein, which binds to the MS2 aptamer). See FIG. 3G and FIG. 3H , as examples of tPERTs that can be used with prime editing.

[0317] Prime editor

[0318] The term "prime editor" refers to a fusion construct described herein comprising a napDNAbp (e.g., Cas9 nickase) and a reverse transcriptase, and capable of prime editing a target nucleotide sequence in the presence of a PE gRNA (or "extended guide RNA"). The term "prime editor" can refer to a fusion protein or a fusion protein complexed with a PE gRNA and / or further complexed with a second strand nicking producing sgRNA. In some embodiments, a prime editor can also refer to a complex comprising a fusion protein (a reverse transcriptase fused to a napDNAbp), a PE gRNA, and a conventional guide RNA capable of directing a second site nicking producing step of the non-edited strand, as described herein. In other embodiments, the reverse transcriptase component of the "prime editor" can be provided in trans.

[0319] Primer binding site

[0320] The term "primer binding site" or "PBS" refers to a nucleotide sequence located on a PEgRNA as an extension arm component (typically at the 3' end of the extension arm) and used to bind to a primer sequence formed following a Cas9 nick of a target sequence by a prime editor. As detailed elsewhere, when the Cas9 nickase component of a prime editor makes a nick in one strand of a target DNA sequence, a 3' ended ssDNA flap is formed, which acts as a primer sequence to anneal with the primer binding site on the PEgRNA to prime reverse transcription. FIG. 27 FIGS. 28 show embodiments of primer binding sites located on 3' and 5' extension arms, respectively.

[0321] Promoter

[0322] The term "promoter" is art-recognized and refers to a nucleic acid molecule having a sequence that is recognized by the cellular transcription machinery and is capable of initiating the transcription of a downstream gene. The promoter can be constitutively active, meaning that the promoter is always active in a given cellular environment, or conditionally active, meaning that the promoter is only active in the presence of a particular condition. For example, a conditionally active promoter can only be active in the presence of a particular protein that links the protein in the promoter to the basal transcription machinery, or only in the absence of an inhibitory molecule. One subclass of conditionally active promoters are inducible promoters, which require the presence of a small molecule "inducer" to be active. Examples of inducible promoters include, but are not limited to, arabinose inducible promoters, Tet-on promoters, and tamoxifen inducible promoters. Various constitutive, conditional, and inducible promoters are well known to those of skill in the art, and one of skill in the art would be able to determine a variety of such promoters for use in practicing the present application, which is not limited in this respect.

[0323] Protospacer

[0324] As used herein, the term "protospacer" refers to the sequence (about 20 bp) in DNA that is 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 complementary sequence of the protospacer on the target DNA (specifically, one strand, the "target strand" of the target DNA sequence, as opposed to the "non-target strand"). A specific protospacer adjacent motif (PAM) is also required for Cas9 to function, and this motif varies with the bacterial species of the Cas9 gene. The most commonly used Cas9 nuclease derived from S. pyogenes recognizes a PAM sequence of NGG on the non-target strand, which is immediately downstream of the genomic DNA target sequence. The skilled artisan will appreciate that the literature in the art sometimes refers to the "protospacer" as the approximately 20-nt target-specific guide sequence on the guide RNA itself, rather than as the "spacer." Thus, in some instances, the term "protospacer" as used herein can be used interchangeably with the term "spacer." The context of the specification in which "protospacer" or "spacer" appears will help inform the reader as to whether the term refers to the gRNA or to the DNA target.

[0325] Protospacer adjacent motif (PAM)

[0326] As used herein, the term "protospacer adjacent motif" or "PAM" refers to a DNA sequence of about 2-6 base pairs that is an important targeting component of Cas9 nucleases. Typically, the PAM sequence is on either strand and is located downstream in the 5' to 3' direction of the Cas9 cleavage site. The canonical PAM sequence (i.e., the PAM sequence associated with the Cas9 nuclease of S. pyogenes or SpCas9) is 5'-NGG-3', where "N" is any nucleobase, followed by two guanine ("G") nucleobases. Different PAM sequences can be associated with different Cas9 nucleases or equivalent proteins from different organisms. In addition, any given Cas9 nuclease, such as SpCas9, can be modified to change the PAM specificity of the nuclease, such that the nuclease recognizes alternative PAM sequences.

[0327] For example, with reference to the canonical SpCas9 amino acid sequence of SEQ ID NO: 18, the PAM sequence can be modified by introducing one or more mutations including (a) D1135V, R1335Q, and T1337R, the“VQR variant,” which changes the PAM specificity to NGAN or NGNG, (b) D1135E, R1335Q, and T1337R, the“EQR variant,” which changes the PAM specificity to NGAG, and (c) D1135V, G1218R, R1335E, and T1337R, the“VRR variant,” which changes the PAM specificity to NGCG. In addition, the D1135E variant of canonical SpCas9 can still recognize NGG, but it is more selective than the wild-type SpCas9 protein.

[0328] It is also understood that Cas9 enzymes from different bacterial species (i.e., Cas9 orthologs) can have different PAM specificities. For example, Cas9 from Staphylococcus aureus (SaCas9) recognizes NGRRT or NGRRN. In addition, Cas9 from Neisseria meningitis (NmCas) recognizes NNNNGATT. In another example, Cas9 from Streptococcus thermopbilis (StCas9) recognizes NNAGAAW. In another example, Cas9 from Treponema denticola (TdCas) recognizes NAAAAC. These are examples and are not meant to be limiting. It is further understood that non-SpCas9 bind to a variety of PAM sequences, which makes them useful when a suitable SpCas9 PAM sequence is not present at the desired target cleavage site. In addition, non-SpCas9 can have other features that can make them more useful than SpCas9. For example, Cas9 from Staphylococcus aureus (SaCas9) is about 1 kb smaller than SpCas9, so it can be packaged into adeno-associated virus (AAV). Further reference can be made to Shah et al.,“Protospacer recognition motifs: mixed identities and functional diversity,” RNA Biology, 10(5): 891-899 (incorporated herein by reference).

[0329] Recombinase

[0330] As used herein, the term "recombinase" refers to a site-specific enzyme that mediates DNA recombination between recombinase recognition sequences resulting in excision, integration, inversion, or exchange (e.g., translocation) of a DNA fragment between recombinase recognition sequences. Recombinases can be divided into two different families: serine recombinases (e.g., resolvases and invertases) and tyrosine recombinases (e.g., integrases). Examples of serine recombinases include, but are not limited to, Hin, Gin, Tn3, p-6, CinH, ParA, γδ, Bxb1, TP901, TG1, R4, MR11, A118, U153, and gp29. Examples of tyrosine recombinases include, but are not limited to, Cre, FLP, R, Lambda, HK101, HK022, and pSAM2. The names of the serine and tyrosine recombinases are derived from the conserved nucleophilic amino acid residues that the recombinases use to attack DNA and covalently link to DNA during strand exchange. Recombinases have many applications, including creating gene knockouts / knockins and gene therapy applications. See, e.g., Brown et al.,“Serine recombinases as tools for genome engineering.” Methods. 2011; 53(4): 372-9; Hirano et al.,“Site-specific recombinases as tools for heterologous gene integration.” Appl. Microbiol. Biotechnol. 2011; 92(2): 227-39; Chavez and Calos,“Therapeutic applications of the ΦC31 integrase system.” Curr. Gene Ther. 2011; 11(5): 375-81; Turan and Bode,“Site-specific recombinases: from tag-and-target-to tag-and-exchange-based genomic modifications.” FASEB J. 2011; 25(12): 4088-107; Venken and Bellen,“Genome-wide manipulations of Drosophila melanogaster with transposons, Flp recombinase, and 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(6): 479-91; and Zhang et al.,“Cre-ating a new biological era: conditional gene manipulation in plants.” Plant Cell Rep. 2012; 31(6): 971-89.2012; 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 by reference herein in their entirety. The recombinases provided herein are not meant to be exclusive examples of recombinases that can be used in embodiments of the application. The methods and compositions of the application can be extended by mining databases of new orthogonal recombinases or designing synthetic recombinases with defined DNA specificity (see, e.g., Groth et al., "Phage integrases: biology and applications." J. Mol. Biol. 2004; 335, 667-678; Gordley et al., "Synthesis of programmable integrases." Proc. Natl. Acad. Sci. USA. 2009; 106, 5053-5058; the entire contents of each of which are incorporated by reference herein in their entirety). Other examples of recombinases that can be used in the methods and compositions described herein are known to those of skill in the art, and any new recombinases discovered or generated are contemplated to be useful in different embodiments of the application. In some embodiments, the catalytic domain of the recombinase is fused to a nuclease-inactivated RNA programmable nuclease (e.g., dCas9 or a fragment thereof) such that the recombinase domain does not comprise a nucleic acid binding domain or is unable to bind to a target nucleic acid (e.g., the recombinase domain is engineered such that it does not have specific DNA binding activity). Recombinases lacking DNA binding activity and methods of engineering the same are known, including those described in Klippel et al., "Isolation and characterisation of unusual gin mutants." EMBO J. 1988; 7: 3983-3989: Burke et al., "Activating mutations of Tn3 resolvase marking interfaces important in recombination catalysis and its regulation. Mol Microbiol. 2004; 51 : 937-948; Olorunniji et al., "A novel Gin recombinase with a single-stranded DNA binding activity." Nucleic Acids Res. 2008; 36: 6577-6586; the entire contents of each of which are incorporated by reference herein in their entirety., “Synapsis and catalysis by activated Tn3 resolvasemutants.” Nucleic Acids Res. 2008; 36: 7181-7191; Rowland et al., “Regulatory mutations in Sin recombinase support a structure-based model of the synaptosome.” Mol Microbiol. 2009; 74: 282-298; Akopian et al., “Chimeric recombinases with designed DNA sequence recognition.” Proc Natl Acad Sci USA. 2003; 100: 8688-8691; Gordley et al., “Evolution of programmable zinc finger-recombinases with activity in human cells. J Mol Biol. 2007; 367: 802-813; Gordley et al., “Synthesis of programmable integrases.” Proc Natl Acad Sci USA. 2009; 106: 5053-5058; Arnold et al., “Mutants of Tn3 resolvase which do not require accessory binding sites for recombination activity.” EMBO J. 1999; 18: 1407-1414; Gaj et al., “Structure-guided reprogramming of serine recombinase DNA sequence specificity.” Proc Natl Acad Sci USA. 2011; 108(2): 498-503; and Proudfoot et al., “Zinc finger recombinases with adaptable DNA sequence specificity.” PLoS One.2011; 6(4):e19537; the entire contents of each of which are incorporated by reference. For example, the serine recombinases in the resolvase- invertase group (e.g., Tn3 and γδ resolvases with Hin and Gin invertases) have a modular structure containing autonomous catalytic and DNA binding domains (see, e.g., Grindley et al.,“Mechanism of site-specific recombination.” Ann Rev Biochem. 2006; 75:567-605, the entire contents of which are incorporated by reference). Thus, as described herein, the catalytic domains of these recombinases are amenable to recombination with nuclease-inactivated RNA-programmable nucleases (e.g., dCas9 or fragments thereof), for example, following isolation of“activated” recombinase mutants that do not require any cofactors (e.g., DNA binding activity) for activity (see, e.g., Klippel et al.,“Isolation and characterisation of unusual gin mutants.” EMBO J. 1988; 7:3983-3989; Burke et al.,“Activating mutations of Tn3 resolvase marking interfaces important in recombination catalysis and its regulation. Mol Microbiol. 2004; 51:937-948; Olorunniji et al.,“Synapsis and catalysis by activated Tn3 resolvase mutants.” Nucleic Acids Res. 2008; 36:7181-7191; Rowland et al.,“Regulatory mutations in Sin recombinase support a structure-based model of the synaptosome.” Mol Microbiol. 2009; 74:282-298; Akopian et al.,“Chimeric recombinases with designed DNA sequence recognition.” Proc Natl Acad Sci USA. 2009; 106:690-695; the entire contents of each of which are incorporated by reference).2003; 100: 8688-8691). In addition, many other natural serine recombinases with N-terminal catalytic domains and C-terminal DNA binding domains are known (e.g., phiC31 integrase, TnpX transposase, IS607 transposase), and their catalytic domains can be co-opted to engineer programmable site-specific recombinases as described herein (see, e.g., Smith et al., "Diversity in the serine recombinases." Mol Microbiol. 2002; 44: 299-307, the entire contents of which are incorporated by reference). Similarly, the core catalytic domains of tyrosine recombinases (e.g., Cre, lambda integrase) are known and can be similarly co-opted to engineer programmable site-specific recombinases as described herein (see, e.g., Guo et al., "Structure of Cre recombinase complexed with DNA in a site-specific recombination synapse." Nature. 1997; 389: 40-46; Hartung et al., "Cre mutants with altered DNA binding properties." J Biol Chem 1998; 273: 22884-22891; Shaikh et al., "Chimeras of the Flp and Cre recombinases: Tests of the mode of cleavage by Flp and Cre. J Mol Biol. 2000; 302: 27-48; Rongrong et al., "Effect of deletion mutation on the recombination activity of Cre recombinase." Acta Biochim Pol. 2005; 52: 541-544; Kilbride et al., "Determinants of product topology in a hybrid Cre-Tn3 resolvase site-specific recombination system." J Mol Biol. 2006; 355: 185-195; Warren et al., "A chimeric cre recombinase with regulated directionality.Proc Natl Acad Sci USA. 2008 105: 18278-18283; Van Duyne, "Teaching Cre to follow directions." Proc Natl Acad Sci USA. 2009 Jan 6; 106(1): 4-5; Numrych et al., "A comparison of the effects of single-base and triple-base changes in the integrase arm-type binding sites on the site-specific recombination of bacteriophage lambda." Nucleic Acids Res. 1990; 18: 3953-3959; Tirumalai et al., "The recognition of core-type DNA sites by lambda integrase." J Mol Biol. 1998; 279: 513-527; Aihara et al., "A conformational switch controls the DNA cleavage activity of lambda integrase." Mol Cell. 2003; 12: 187-198; Biswas et al., "A structural basis for allosteric control of DNA recombination by lambda integrase." Nature. 2005; 435: 1059-1066; and Warren et al., "Mutations in the amino-terminal domain of lambda-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.

[0331] Recombinase recognition sequence

[0332] 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 having a RRS, resulting in excision, integration, inversion, or exchange of a DNA fragment between recombinase recognition sequences.

[0333] Recombination

[0334] In the context of nucleic acid modification (e.g., genome modification), the term "recombination" is used to refer to the process by which two or more nucleic acid molecules, or two or more regions of a single nucleic acid molecule, are modified by the action of a recombinase protein (e.g., a recombinase fusion protein of the application provided herein). Recombination can result in, inter alia, insertion, inversion, excision, or translocation of nucleic acid, e.g., within or between recombinase recognition sequences of one or more nucleic acid molecules.

[0335] Reverse transcriptase

[0336] The term "reverse transcriptase" describes a class of polymerases characterized as RNA-dependent DNA polymerases. All known reverse transcriptases require a primer to synthesize a DNA transcript from an RNA template. Historically, reverse transcriptases have been used primarily to transcribe mRNA into eDNA, which can then be cloned into a vector for further manipulation. The avian myoblastosis virus (AMV) reverse transcriptase was the first RNA-dependent DNA polymerase to be widely used (Verma, Biochem. Biophys. Acta 473: 1 (1977)). The enzyme has 5' to 3' RNA-directed DNA polymerase activity, 5' to 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)). Reverse transcriptases cannot correct errors in transcription because known viral reverse transcriptases lack the 3' to 5' exonuclease activity necessary for proofreading (Saunders and Saunders, Microbial Genetics Applied to Biotechnology, London: Croom Helm (1987)). Detailed studies of AMV reverse transcriptase activity and its associated RNase H activity are provided by Berger et al., Biochemistry 22: 2365-2372 (1983). Another reverse transcriptase widely used in molecular biology is the reverse transcriptase derived from the Moloney murine leukemia virus (M-MLV). See, e.g., Gerard, G.R., DNA 5: 271-279 (1986) and Kotewicz, M.L, et al., Gene 35: 249-258 (1985). M-MLV reverse transcriptases that are essentially lacking in RNase H activity have also been described. See, e.g., U.S. Patent No. 5,244,797. The present invention contemplates the use of any such reverse transcriptases, or variants or mutants thereof.

[0337] Further, the present application contemplates the use of error-prone reverse transcriptases, i.e., that can be referred to as error-prone reverse transcriptases or reverse transcriptases that do not support high fidelity nucleotide incorporation during the polymerization process. During the RT template-based single-stranded DNA flap synthesis process integrated with a guide RNA, the error-prone reverse transcriptase can introduce one or more nucleotides that are mismatched to the RT template sequence, thereby introducing a change to the nucleotide sequence through error polymerization of the single-stranded DNA flap. These errors introduced during the single-stranded DNA flap synthesis process are then integrated into a double-stranded molecule as follows: hybridization to the corresponding endogenous target strand, removal of the endogenous displacement strand, ligation, and then again through a round of endogenous DNA repair and / or sequencing process.

[0338] Reverse transcription

[0339] As used herein, the term “reverse transcription” refers to the ability of an enzyme to synthesize a DNA strand (i.e., complementary DNA or cDNA) using RNA as a template. In some embodiments, reverse transcription can be “error-prone reverse transcription,” which refers to the property of certain reverse transcriptases to be error-prone in their DNA polymerization activity.

[0340] PACE

[0341] As used herein, the term “phage-assisted continuous evolution (PACE)” refers to continuous evolution employing bacteriophages as viral vectors. The general concept of PACE technology has been described in, for example, International PCT Application PCT / US2009 / 056194, filed September 8, 2009, published as WO 2010 / 028347 on March 11, 2010; International PCT Application PCT / US2011 / 066747, filed December 22, 2011, published as WO 2012 / 088381 on June 28, 2012; U.S. Application, U.S. Patent No. 9,023,594, issued May 5, 2015; International PCT Application PCT / US2015 / 012022, filed January 20, 2015, published as WO 2015 / 134121 on September 11, 2015; and International PCT Application PCT / US2015 / 012022, filed January 20, 2015, published as WO 2016 / 168631 on October 20, 2016, each of which is incorporated herein by reference in its entirety.

[0342] Phage

[0343] As used herein, the term "bacteriophage," which can be used interchangeably with the term "phage," refers to a virus that infects bacterial cells. Typically, a phage consists of an outer protein coat that encloses genetic material. The genetic material can be ssRNA, dsRNA, ssDNA, or dsDNA in linear or circular form. Phages and phage vectors are well known to those skilled in the art, and non-limiting examples of phages that can be used to practice the PACE methods provided herein are lambda (lysogeny), T2, T4, T7, T12, R17, M13, MS2, G4, PI, P2, P4, PhiX174, N4, Φ6, and Φ29. In certain embodiments, the phage used in the present application is M13. Other suitable phages and host cells will be apparent to those skilled in the art, and the present application is not limited in this respect. For exemplary descriptions of other suitable phages and host cells, see Elizabeth Kutter and Alexander Sulakvelidze: Bacteriophages: Biology and Applications. CRC Press; 1st edition (December 2004), ISBN: 0849313368; Martha R.J. Clokie and Andrew M. Kropinski: Bacteriophages: Methods and Protocols, Volume 1: Isolation, Characterization, and Interactions (Methods in Molecular Biology) Humana Press; 1st edition (December, 2008), ISBN: 1588296822; Martha R.J. Clokie and Andrew M. Kropinski: Bacteriophages: Methods and Protocols, Volume 2: Molecular and Applied Aspects (Methods in Molecular Biology) Humana Press; 1st edition (December 2008), ISBN: 1603275649; all of which are incorporated herein by reference in their entirety for disclosure of suitable phages and host cells and methods and protocols for isolating, culturing, and manipulating such phages).

[0344] Proteins, peptides, and polypeptides

[0345] The terms "protein," "peptide," and "polypeptide" are used interchangeably herein to refer to a polymer of amino acid residues linked together by peptide (amide) bonds. These terms refer to proteins, peptides, or polypeptides of whatever size, structure, or function. Typically, a protein, peptide, or polypeptide is at least 3 amino acids in length. A protein, peptide, or polypeptide can refer to a single protein or a collection of proteins. One or more amino acids in a protein, peptide, or polypeptide can be modified, e.g., by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnyl group, an isofarnyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc. A protein, peptide, or polypeptide can also be a single molecule or can be a multimeric complex. A protein, peptide, or polypeptide can be just a fragment of a naturally occurring protein or peptide. A protein, peptide, or polypeptide can be naturally occurring, recombinant, or synthetic, or any combination thereof. Any protein provided herein can be produced by any method known in the art. For example, a protein provided herein can be produced by recombinant protein expression and purification, which is particularly suitable for fusion proteins comprising peptide linkers. Methods of recombinant protein expression and purification are well known, including those described in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4thed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)), the entire contents of which are incorporated herein by reference.

[0346] Protein splicing

[0347] As used herein, the term "protein splicing" refers to the process of excising a sequence, an intein (or split intein, as the case can be), from within an amino acid sequence, and the remaining fragments of the amino acid sequence exteins are joined by amide bonds to form a continuous amino acid sequence. The term "trans" protein splicing refers to the specific case where the inteins are split inteins and they are located on different proteins.

[0348] Second strand nicking

[0349] Disassembly of heteroduplex DNA formed as a result of prime editing (i.e., comprising one edited strand and one non-edited strand) determines the long-term editing outcome. In other words, the goal of prime editing is to disassemble the heteroduplex DNA formed as an intermediate in PE (the edited strand paired with the endogenous non-edited strand) by permanently integrating the edited strand into the complementary endogenous strand. Methods of “second strand nicking” can be used herein to help drive disassembly of the heteroduplex DNA to favor permanent integration of the edited strand into the DNA molecule. As used herein, the concept of “second strand nicking” refers to the introduction of a second nick at a position downstream of the first nick (i.e., the initial nick site that provides a free 3’ end for priming the reverse transcriptase on the extension portion of the guide RNA), preferably on the non-edited strand. In certain embodiments, the first and second nicks are on opposite strands. In other embodiments, the first and second nicks are on opposite strands. In yet another embodiment, the first nick is on the non-target strand (i.e., the strand that forms the single-stranded portion of the R-loop), and the second nick is on the target strand. In other embodiments, the first nick is on the edited strand, and the second nick is on the non-edited strand. The second nick can be at least 5 nucleotides downstream of the first nick, or at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 or more nucleotides downstream of the first nick. In certain embodiments, a second nick can be introduced on the non-edited strand between about 5 and 150 nucleotides away from the PE gRNA-induced nick site, or between about 5 and 140, or between about 5 and 130, or between about 5 and 120, or between about 5 and 110, or between about 5 and 100, or between about 5 and 90, or between about 5 and 80, or between about 5 and 70, or between about 5 and 60, or between about 5 and 50, or between about 5 and 40, or between about 5 and 30, or between about 5 and 20, or between about 5 and 10 nucleotides away from the PE gRNA-induced nick. In one embodiment, a second nick is introduced between 14 and 116 nucleotides away from the PE gRNA-induced nick. Without being bound by theory, the second nick induces the cell’s endogenous DNA repair and replication processes to shift or edit toward the non-edited strand, thereby permanently installing the edited sequence on both strands and disassembling the heteroduplex 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.

[0350] Sense strand

[0351] In genetics, the "sense" strand is the segment of double-stranded DNA that extends from 5' to 3' and is complementary to the antisense or template strand of DNA that extends from 3' to 5'. In the case of a DNA segment that encodes a protein, the sense strand is the DNA strand that has the same sequence as the mRNA, which is templated from the antisense strand during transcription and eventually undergoes (usually, not always) translation into a protein. Thus, the antisense strand is responsible for generating the RNA that is later translated into a protein, while the sense strand has nearly the same composition as the mRNA. Note that for each segment of dsDNA, there can be two sets of sense and antisense, depending on the direction of reading (since sense and antisense are relative to the point of view). The final designation of which strand of a dsDNA segment is called sense or antisense is the gene product or mRNA.

[0352] In the case of a PEgRNA, the first step is the synthesis of a single-stranded complementary DNA (i.e., the incorporated 3' ssDNA flap) oriented in the 5' to 3' direction that leaves the PEgRNA extension arm as a template. Whether the 3' ssDNA flap should be considered the sense or antisense strand depends on the direction of transcription, since it is recognized that both DNA strands can serve as templates for transcription (but not simultaneously). Thus, in some embodiments, the 3' ssDNA flap (which extends generally in the 5' to 3' direction) will serve as the sense strand, since it is the coding strand. In other embodiments, the 3' ssDNA flap (which extends generally in the 5' to 3' direction) will serve as the antisense strand, and thus as the transcription template.

[0353] Spacer sequence

[0354] As used herein, the term "spacer sequence" in relation to a guide RNA or PEgRNA refers to a portion of about 20 nucleotides of the guide RNA or PEgRNA that comprises a nucleotide sequence that is complementary to a protospacer in a target DNA sequence. The spacer sequence anneals to the protospacer to form a ssRNA / ssDNA hybrid structure at the target site and a corresponding R-loop ssDNA structure of the endogenous DNA strand that is complementary to the protospacer.

[0355] Subject

[0356] As used herein, the term "subject" 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 primate. In some embodiments, the subject is a rodent. In some embodiments, the subject is a sheep, goat, cow, 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 engineered, e.g., a genetically engineered non-human subject. The subject can be of any gender and at any stage of development.

[0357] Split intein

[0358] Although inteins are most often found as contiguous domains, some exist in a naturally split form. In this case, the two fragments are expressed as separate polypeptides and must associate prior to splicing, a process known as protein trans-splicing.

[0359] An exemplary split intein is the Ssp DnaE intein, which comprises two subunits, DnaE-N and DnaE-C. The two distinct subunits are encoded by different genes, dnaE-n and dnaE-c, which encode the DnaE-N and DnaE-C subunits, respectively. DnaE is a naturally occurring split intein in Synechocytis sp. PCC6803 and is capable of directing the trans-splicing of two different proteins, each comprising a fusion to either DnaE-N or DnaE-C.

[0360] Other naturally occurring or engineered split intein sequences are known in the art or can be prepared from the complete intein sequences described herein or those available in the art. Examples of split intein sequences can be found in Stevens et al.,“A promiscuous split intein with expanded protein engineering applications,” PNAS, 2017, Vol. 114:8538-8543; Iwai et al.,“Highly efficient protein trans-splicing by a naturally split DnaE intein from Nostc punctiforme, FEBS Lett, 580:1853-1858, each of which is incorporated herein by reference. Other 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.

[0361] In addition, trans-splicing has been described in vivo and in vitro (Shingledecker, et al., Gene 207:187 (1998), Southworth, et al., EMBO J. 17:918 (1998); Mills, et al., Proc. Natl. Acad. Sci. USA, 95:3543-3548 (1998); Lew, et al., J. Biol. Chem., 273:15887-15890 (1998); Wu, et al., Biochim. Biophys. Acta 35732:1 (1998b), Yamazaki, et al., J. Am. Chem. Soc 120:5591 (1998), Evans, et al., J. Biol. Chem. 275:9091 (2000); Otomo, et al., Biochemistry 38:16040-16044 (1999); Otomo, et al., J. Biolmol. NMR 14:105-114 (1999); Scott, et al., Proc. Natl. Acad. Sci. USA 96:13638-13643 (1999)), and provides an opportunity to express proteins from two inactive fragments that subsequently undergo ligation to form a functional product, for example, as FIG. 66 and FIG. 67, regarding formation of a complete PE fusion protein from two separately expressed halves.

[0362] Target site

[0363] The term“target site” refers to a sequence within a nucleic acid molecule that is edited by a prime editor (PE) disclosed herein. The target site also refers to the sequence within a nucleic acid molecule that is bound by a complex of a prime editor (PE) and a gRNA.

[0364] tPERT

[0365] See the definition of“trans prime editor RNA template (tPERT)” for additional information.

[0366] Temporal second strand nicking

[0367] As used herein, the term“temporally second strand nicking” refers to a variant of second strand nicking whereby installation of a second nick in the unedited strand only occurs after installation of the desired edit in the edited strand. This avoids the simultaneous occurrence of nicks on both strands, which can lead to a double-stranded DNA break. Temporally controlling second strand nicking is achieved by designing the gRNA with a spacer sequence that only matches the edited strand, but not the original allele. Using this strategy, the mismatch between the original spacer and the unedited allele should be disfavored from being nicked by the sgRNA until after the editing event on the PAM strand has occurred.

[0368] Trans prime editing

[0369] As used herein, the term“trans prime editing” refers to a modified form of prime editing that utilizes a split PEgRNA, i.e., where the PEgRNA is split into two separate molecules: a sgRNA and a trans prime editor RNA template (tPERT). The sgRNA is used to target (or more generally, target the napDNAbp component of the prime editor to) the desired genomic target site, while the tPERT is used by the polymerase (e.g., reverse transcriptase) to write a new DNA sequence to the target locus once the tPERT is recruited in trans to the prime editor through the interaction of binding domains located on the prime editor and the tPERT. In one embodiment, the binding domains can include an RNA-protein recruiting moiety, such as an MS2 aptamer located on the tPERT and an MS2 cp protein fused to the prime editor. An advantage of trans prime editing is that by separating the DNA synthesis template from the guide RNA, longer length templates can potentially be used.

[0370] Embodiments of trans prime editing are shown in FIG. 3G and FIG. 3H .FIG. 3G The left side shows the components of the trans-guided editor complex (“RP-PE:gRNA complex”), which comprises a napDNAbp fused to each of a polymerase (e.g., reverse transcriptase) and an rPERT recruiting protein (e.g., MS2sc), and complexed with a guide RNA. FIG. 3G Also shown is a separate tPERT molecule, which comprises the extension arm features of a PEgRNA, including a DNA synthesis template and a primer binding sequence. The tPERT molecule also includes an RNA-protein recruiting domain (in this case, a stem loop structure, which can be, for example, an MS2 aptamer). As FIG. 3H As shown by the process described in the middle, the RP-PE:gRNA complex binds to the target DNA sequence and makes a nick in the target DNA sequence. The recruiting protein (RP) then recruits a tPERT to co-localize to the guide editor complex bound to the DNA target site, such that the primer binding site binds to the primer sequence on the nicked strand, and subsequently, a polymerase (e.g., RT) is allowed to synthesize a DNA single strand against the DNA synthesis template up to the 5’ of the tPERT.

[0371] While FIG. 3G and FIG. 3H While the tPERT shown in the middle comprises a PBS and a DNA synthesis template at the 5’ end of the RNA-protein recruiting domain, tPERTs in other configurations can be designed to have a PBS and a DNA synthesis template located at the 3’ end of the RNA-protein recruiting domain. However, the advantage of tPERTs with 5’ extensions is that the synthesis of the DNA single strand will naturally terminate at the 5’ end of the tPERT, so there is no risk of using any portion of the RNA-protein recruiting domain as a template during the DNA synthesis phase of the prime editing.

[0372] Trans prime editor RNA template (tPERT)

[0373] As used herein, “trans-guided editor RNA template (tPERT)” refers to a component used for trans-guided editing, which is a modified version of prime editing that is run by separating the PEgRNA into two different molecules (a guide RNA and a tPERT molecule). The tPERT molecule is programmed to co-localize with the prime editor complex at the target DNA site, thereby bringing the primer binding site and DNA synthesis template to the prime editor in trans. See, e.g., FIG. 3G, embodiments for trans -prime editor (tPE) showing a two-component system comprising (1) RP-PE:gRNA complex and (2) tPERT comprising a primer binding site and a DNA synthesis template linked to a RNA-protein recruitment domain, wherein the RP (recruiting protein) component of the RP-PE:gRNA complex recruits the tPERT to the target site to be edited, thereby associating the PBS and the DNA synthesis template in trans with the prime editor. In other words, the tPERT is engineered to comprise an extension arm (in whole or in part) of the PEgRNA, which includes the primer binding site and the DNA synthesis template.

[0374] Transition

[0375] As used herein, “transversion” refers to the interchange of a purine nucleobase or an interchange of a pyrimidine nucleobase Such interchanges involve nucleobases of similar shape. 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 a transversion and a transition in the same target DNA molecule. These changes involve or In the case of double-stranded DNA with Watson-Crick-paired nucleobases, a transversion refers to the following base pair exchange: or 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 a transversion and a transition in the same target DNA molecule, as well as other nucleotide changes, including deletions and insertions.

[0376] Transversion

[0377] As used herein, “transversion” refers to the interchange of a purine nucleobase and In the case of double-stranded DNA with Watson-Crick-paired nucleobases, a transversion refers to the following base pair exchange: and 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 a transversion and a transition in the same target DNA molecule, as well as other nucleotide changes, including deletions and insertions.

[0378] Treatment

[0379] The term "treatment" refers to clinical intervention designed to alter, arrest, delay the onset of, or inhibit the progress of a disease or disorder, or one or more symptoms thereof as described herein. The term "treatment" as used herein refers to clinical intervention designed to alter, arrest, delay the onset of, or inhibit the progress of a disease or disorder, or one or more symptoms thereof as described herein. In some embodiments, treatment can be post-symptom onset and / or post-diagnosis of the disease. In other embodiments, treatment can be in the absence of symptoms, e.g., to prevent or delay onset or progression of symptoms or of the disease. For example, treatment can be of a susceptible individual prior to the onset of symptoms (e.g., in light of a medical history and / or in light of a genetic or other predisposition). Treatment can also continue after symptoms have resolved, e.g., to prevent or delay their recurrence.

[0380] Trinucleotide repeat disorder

[0381] As used herein, a "trinucleotide repeat disorder" (or, alternatively, an "expanding repeat disorder" or "repeat expansion disorder") refers to a group of genetic disorders caused by "trinucleotide repeat expansions," which are a mutation of certain trinucleotide repeats in certain genes or introns. Trinucleotide repeats were once thought to be common repeats in the genome, but these disorders were clarified in the 1990s. These apparently "benign" stretches of DNA sometimes expand and cause disease. Disorders caused by trinucleotide repeat expansions share several defining features. First, the mutated repeats show both somatic and germline instability, and more commonly, they expand rather than contract upon successive transmission. Second, earlier age of onset and increasing severity of phenotype in later generations (expected) are often associated with larger repeat lengths. Finally, parental origin of the disease allele often influences expectations, with paternal transmission having a greater risk of expansion for many of these disorders.

[0382] Trinucleotide expansions are thought to arise from slippage during the process of DNA replication. Due to the repetitive nature of the DNA sequence in these regions, it is possible to form a "loop out" structure during DNA replication while maintaining complementary base pairing between the parent strand and the daughter strand being synthesized. If the loop out structure is formed from the sequence on the daughter strand, this results in an increase in the number of repeats. However, if the loop out structure is formed on the parent strand, a decrease in the number of repeats occurs. These expansions of repeats appear to be more common than contractions. Generally, the more expansions, the more likely they are to cause disease or increase the severity of disease. This property leads to the observed features in trinucleotide repeat disorders. Expectations describe a trend of decreasing age of onset and increasing severity of symptoms through successive generations of affected families due to expansion of these repeats.

[0383] Nucleotide repeat disorders can include those in which triplets repeats are present in non-coding regions (i.e., non-coding trinucleotide repeat disorders) or in coding regions.

[0384] The prime editor (PE) systems described herein can be used to treat nucleotide repeat disorders, which can 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.

[0385] Upstream

[0386] As used herein, the terms “upstream” and “downstream” are relative terms that define the linear position of at least two elements in a nucleic acid molecule (whether single-stranded or double-stranded) oriented in the 5’ to 3’ direction. In particular, in a nucleic acid molecule in which a first element is located somewhere 5’ of a second element, the first element is upstream of the second element. For example, if a SNP is located 5’ of a nick site, the SNP is upstream of the Cas9-induced nick site. Conversely, in a nucleic acid molecule in which a first element is located somewhere 3’ of a second element, the first element is downstream of the second element. For example, if a SNP is located 3’ of a nick site, the SNP is downstream of the Cas9-induced nick site. A nucleic acid molecule can be DNA (double-stranded or single-stranded), RNA (double-stranded or single-stranded), or a hybrid of DNA and RNA. Analysis of single-stranded nucleic acid molecules and double-stranded molecules is the same because the terms upstream and downstream refer only to the single strand of a nucleic acid molecule, it is just a matter of choosing which strand of a double-stranded molecule to consider. Generally, 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 the segment of double-stranded DNA that extends from 5’ to 3’, which is complementary to the antisense or template strand of DNA (which extends from 3’ to 5’). Thus, for example, if a SNP nucleobase is 3’ of a promoter on the sense or coding strand, the SNP nucleobase is “downstream” of the promoter sequence in genomic DNA (which is double-stranded).

[0387] Variant

[0388] As used herein, the term "variant" is understood to refer to a characteristic that exhibits a pattern that deviates from patterns that occur in nature, e.g., a variant Cas9 is a Cas9 that comprises one or more amino acid residue changes as compared to a wild-type Cas9 amino acid sequence. The term "variant" includes homologous proteins that have at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 99% percent identity to a reference sequence and have the same or substantially the same functional activity as the reference sequence. The term also includes mutants, truncations, or domains of a reference sequence and display the same or substantially the same functional activity(s) as the reference sequence.

[0389] Vector

[0390] As used herein, the term "vector" refers to a nucleic acid that can be modified to encode a gene of interest and is capable of entering a host cell, where it can undergo mutation and replication, and then transfer the replicated form of the vector into another host cell. Exemplary suitable vectors include viral vectors, such as retroviral vectors or bacteriophages and filamentous phages, as well as conjugative plasmids. Other suitable vectors will be apparent to those of skill in the art based on the present disclosure.

[0391] Wild type

[0392] As used herein, the term "wild-type" is a term understood by the skilled artisan and means an organism, strain, gene, or feature in its typical form as it exists in nature, as opposed to a mutant or variant form.

[0393] 5' endogenous DNA flap

[0394] As used herein, the term "5' endogenous DNA flap" refers to the strand of DNA located immediately downstream of the PE-induced nick site in the target DNA. The generation of a nick in the target DNA strand by the PE exposes a 3' hydroxyl on the upstream side of the nick site and a 5' hydroxyl on the downstream side of the nick site. The endogenous strand ending in a 3' hydroxyl is used to prime the DNA polymerase of the prime editor (e.g., where the DNA polymerase is a reverse transcriptase). The endogenous strand on the downstream side of the nick site and beginning with the exposed 5' hydroxyl is referred to as the "5' endogenous DNA flap" and is ultimately removed and replaced by a newly synthesized replacement strand (i.e., a "3' replacement DNA flap") encoded by the extension of the PEgRNA.

[0395] 5' endogenous DNA flap removal

[0396] As used herein, the term "5' endogenous DNA flap removal" or "5' flap removal" refers to the removal of the 5' endogenous DNA flap that forms when a RT-synthesized single-stranded DNA flap invades and hybridizes to an endogenous DNA, displacing the endogenous strand in the process. Removal of this endogenous displaced strand can drive the reaction toward the formation of the desired product comprising the desired nucleotide change. Cellular DNA repair enzymes can catalyze the removal or excision of the 5' endogenous flap (e.g., flap endonucleases such as EXOl or FENl). Host cells can also be transformed to express one or more enzymes that catalyze the removal of the 5' endogenous flap, driving the process toward product formation (e.g., flap endonucleases). Flap endonucleases are known in the art and can be found and 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 FEN 1 superfamily," Cell, 2011, 145(2): 198-211, each of which is incorporated herein by reference.

[0397] 3' replacement DNA flap

[0398] As used herein, the term "3' replacement DNA flap" or simply "replacement DNA flap" refers to a strand of DNA synthesized by a prime editor and encoded by the extension arm of a prime editor PEgRNA. More specifically, the 3' replacement DNA flap is encoded by the polymerase template of the PEgRNA. The 3' replacement DNA flap contains the same sequence as the 5' endogenous DNA flap except that it also contains an edit sequence (e.g., a single nucleotide change). The 3' replacement DNA flap anneals to the target DNA, displaces or replaces the 5' endogenous DNA flap (e.g., can be excised by a 5' flap endonuclease such as FEN1 or EXOl), and then ligates to join the 3' end of the 3' replacement DNA flap to the exposed 5' hydroxyl end of the endogenous DNA (exposed upon excision of the 5' endogenous DNA flap, thereby reforming a phosphodiester bond and installing the 3' replacement DNA flap to form a heteroduplex DNA that contains one edited strand and one unedited strand. DNA repair processes resolve the heteroduplex by copying the information in the edited strand to the complementary strand, thereby permanently installing the edit into the DNA. This resolution process can be further driven by nicking the unedited strand, i.e., by "second strand nicking," as described herein.

[0399] Detailed description of certain embodiments

[0400] Genome editing with the clustered regularly interspaced short palindromic repeat (CRISPR) system has revolutionized the life sciences 1-3 . While gene disruption using CRISPR is now routine, the precise installation of single nucleotide edits remains a major challenge, despite being necessary for research or correction of a large number of pathogenic mutations. Homology directed repair (HDR) enables such edits, but suffers from low efficiency (typically <5%), the need for a donor DNA repair template, and the deleterious effects of double strand DNA break (DSB) formation. Recently, the laboratories of Prof. David Liu and others have developed base editing, enabling highly efficient single nucleotide editing without the need for a DSB. Base editors (BEs) combine the CRISPR system with a base-modifying deaminase to convert a target C·G or A·T base pair to an A·T or G·C 4-6 , respectively. While widely used by researchers around the world, current BEs only enable 4 of the 12 possible base pair conversions, and are unable to correct small insertions or deletions. Furthermore, the targeting scope of base editing is limited by editing of non-target C or A bases adjacent to the target base ("bystander editing") and by the requirement that a PAM sequence be present 15+2 bp from the target base. Thus, overcoming these limitations would greatly expand the basic research and therapeutic applications of genome editing.

[0401] The present disclosure presents a new method of precise editing that offers many of the benefits of base editing - i.e., avoidance of double-strand breaks and donor DNA repair templates - while overcoming its major limitations. The method presented herein uses target primed reverse transcription (TPRT) to directly install an edited DNA strand at a target genomic site. In the design discussed herein, a CRISPR guide RNA (gRNA) is engineered to carry a reverse transcriptase (RT) template sequence that encodes a single-stranded DNA containing the desired nucleotide change. The target site DNA nicked by the CRISPR nuclease (Cas9) will serve as a primer for reverse transcription of the template sequence on the modified gRNA, allowing direct incorporation of any desired nucleotide edit.

[0402] Accordingly, the present invention relates, in part, to the discovery that CRISPR / Cas-based precise genome editing can be performed with high efficiency and genetic plasticity (e.g., as depicted in different embodiments of FIG. 1A-1F The present inventors herein propose the use of Cas protein-reverse transcriptase fusions to target a specific DNA sequence with a modified guide RNA ("extended guide RNA"), create a single-stranded nick at the target site, and use the nicked DNA as a reverse transcription primer for an engineered reverse transcriptase template that has been integrated into the extended guide RNA. In addition to containing the desired nucleotide change (e.g., single nucleotide change, deletion, or insertion, or a combination thereof), the newly synthesized strand will be homologous to the genomic target sequence. The newly synthesized DNA strand can be referred to as a single-stranded DNA flap that will compete for hybridization with the complementary homologous endogenous DNA strand, thereby displacing the corresponding endogenous strand. Resolution of this hybrid intermediate can include removal of the displaced flap of endogenous DNA that results therefrom (e.g., using a 5' end DNA flap endonuclease, FEN1), ligation of the synthesized single-stranded DNA flap to the target DNA, and assimilation of the desired nucleotide change due to cellular DNA repair and / or replication processes. Because DNA synthesis provides single nucleotide precision, the scope of this method is very broad and foreseeably useful for countless applications in basic science and therapeutics.

[0403] [1] napDNAbp

[0404] The prime editors and trans- prime editors described herein can comprise a nucleic acid programmable DNA binding protein (napDNAbp).

[0405] In one aspect, a napDNAbp can be bound or complexed with at least one guide nucleic acid (e.g., a guide RNA or a PEgRNA) that localizes the napDNAbp to a DNA sequence comprising a DNA strand (i.e., a target strand) that is complementary to the protospacer of the DNA target (e.g., the spacer of a guide RNA that anneals to the protospacer of a DNA target). In other words, the guide nucleic acid “programs” the napDNAbp (e.g., Cas9 or an equivalent) to locate and bind to the complement of the protospacer in DNA.

[0406] Any suitable napDNAbp can be used in the prime editors described herein. In different embodiments, the napDNAbp can be any Class 2 CRISPR-Cas system, including any Type II, Type V, or Type VI CRISPR-Cas enzyme. Given the rapid development of CRISPR-Cas as a genome editing tool, the nomenclature used to describe and / or identify CRISPR-Cas enzymes has been continually evolving, e.g., Cas9 and Cas9 orthologs. This application refers to both old and / or new CRISPR-Cas enzyme nomenclature. One of skill in the art will be able to determine the particular CRISPR-Cas enzyme referenced in this application based on the nomenclature used (whether it is old (i.e., “legacy”) nomenclature or new nomenclature). CRISPR-Cas nomenclature is discussed extensively in Makarova et al., “Classification and Nomenclature of CRISPR-Cas Systems: Wherefrom Here?,” The CRISPR Journal, Vol. 1. No. 5, 2018, which is incorporated by reference herein in its entirety. The particular CRISPR-Cas nomenclature used in any given example of this application is not limiting in any way, and one of skill in the art will be able to determine which CRISPR-Cas enzyme is being referenced.

[0407] For example, the following Type II, Type V, and Type VI Class 2 CRISPR-Cas enzymes have the following art-recognized old (i.e., legacy) and new names. Each of these enzymes and / or variants thereof can be used with the prime editors described herein:

[0408]

[0409] See Makarova et al., The CRISPR Journal, Vol. 1, No. 5, 2018

[0410] Without being bound by theory, the mechanism of action of certain napDNAbps contemplated herein includes a step of forming an R-loop, whereby the napDNAbp induces unwinding of a double-stranded DNA target, separating the strands in the region bound by the napDNAbp. The guide RNA spacer then hybridizes to the "target strand" at the protospacer. This displaces the "non-target strand" that is complementary to the target strand, which forms a single-stranded region of the R-loop. In some embodiments, the napDNAbp includes one or more nuclease activities that cleave DNA, leaving different types of lesions. For example, the napDNAbp can comprise a nuclease activity that cleaves the non-target strand at a first location and / or cleaves the target strand at a second location. Depending on the nuclease activity, the target DNA can be cleaved to form a "double-stranded break," cleaving both strands. In other embodiments, the target DNA can be cleaved only at a single site, i.e., "nicking" the DNA on one strand. Exemplary napDNAbps with different nuclease activities include "Cas9 nickases" ("nCas9") and inactive Cas9s without nuclease activity ("dead Cas9" or "dCas9").

[0411] The following description of various napDNAbps that can be used in conjunction with the presently disclosed prime editors is not meant to be limiting in any way. The prime editors can include classic SpCas9, or any orthologous Cas9 protein, or any variant Cas9 protein - including any naturally occurring Cas9 variant, mutant, or other engineered version - that is known or can be made or evolved through directed evolution or other mutagenesis processes. In different embodiments, the Cas9 or Cas9 variant has nickase activity, i.e., cleaves only one strand of the target DNA sequence. In other embodiments, the Cas9 or Cas9 variant has inactive nuclease, i.e., a "dead" Cas9 protein. Other variant Cas9 proteins that can be used are those that have a smaller molecular weight than classic SpCas9 (e.g., for easier delivery) or have a modified or rearranged primary amino acid structure (e.g., in a circularly permuted form).

[0412] The guide editors described herein can also comprise Cas9 equivalents, including Casl2a (Cpf1) and Casl2b1 proteins, which are the result of convergent evolution. The napDNAbps used herein (e.g., SpCas9, Cas9 variants, or Cas9 equivalents) can also comprise various modifications that alter / enhance their PAM specificity. Finally, the present application contemplates any Cas9, Cas9 variant, or Cas9 equivalent having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.9% sequence identity to a reference Cas9 sequence (e.g., a reference SpCas9 canonical sequence or a reference Cas9 equivalent (e.g., Casl2a (Cpf1))).

[0413] The napDNAbp can be a CRISPR (clustered regularly interspaced short palindromic repeat)-associated nuclease. As noted above, CRISPR is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements, and conjugative plasmids). CRISPR clusters contain spacer regions, sequences complementary to preceding mobile elements, and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems, proper processing of pre-crRNA requires a trans-encoded small RNA (tracrRNA), an endogenous ribonuclease 3 (rnc), and a Cas9 protein. The tracrRNA acts as a guide for ribonuclease 3 to assist in processing the pre-crRNA. Subsequently, Cas9 / crRNA / tracrRNA cleaves linear or circular dsDNA targets that are complementary to the spacer regions in an endonucleolytic fashion. The target strand that is not complementary to the crRNA is first cleaved in an endonucleolytic fashion, and then trimmed 3’-5’ in an exonucleolytic fashion. In fact, DNA binding and cleavage generally requires the protein and both RNAs. However, single- stranded guide RNAs (“sgRNAs,” or simply “gRNAs”) can be engineered to incorporate aspects of both the crRNA and the tracrRNA into a single RNA species. See, e.g., Jinek M. et al., Science 337:816-821 (2012), which is incorporated by reference herein in its entirety.

[0414] In some embodiments, the napDNAbp directs cleavage of one or both strands at a target sequence location (e.g., within the target sequence and / or within the complement of the target sequence). In some embodiments, the napDNAbp directs cleavage of one or both strands from within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs of the first or last nucleotide of the target sequence. In some embodiments, the vector encodes a napDNAbp that is mutated relative to the corresponding wild-type enzyme such that the mutated napDNAbp lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence. For example, an aspartate-to-alanine substitution (D10A) in the RuvC I catalytic domain of Cas9 from Streptococcus pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (cleaves a single strand). Other examples of mutations that render Cas9 a nickase include, but are not limited to, H840A, N854A, and N863A, with reference to the equivalent amino acid positions in the canonical SpCas9 sequence or other Cas9 variants or Cas9 equivalents.

[0415] 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 any fragment of a Cas protein that retains all or a substantial portion of the essential basic functions required for the disclosed methods, i.e., (i) having nucleic acid programmable binding of the Cas protein to a target DNA, and (ii) the ability to make a nick in a target DNA sequence on one strand. Cas proteins contemplated herein include CRISPR Cas9 proteins, as well as Cas9 equivalents, variants (e.g., Cas9 nickases (nCas9) or nuclease-inactive Cas9 (dCas9)) homologs, orthologs, or paralogs, whether naturally occurring or non-naturally occurring (e.g., engineered or recombinant), and can include Cas9 equivalents from any Class 2 CRISPR system (e.g., Type II, V, VI), including Casl2a (Cpfl), Casl2e (CasX), Casl2bl (C2cl), Casl2b2, Casl2c (C2c3), C2c4, C2c8, C2c5, C2clO, C2c9 Casl3a (C2c2), Casl3d, Casl3c (C2c7), Casl3b (C2c6), and Casl3b. Other Cas equivalents are described in Makarova et al.,“C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector,” Science 2016; 353(6299) and Makarova et al.,“Classification and Nomenclature of CRISPR-Cas Systems: Where from Here?” The CRISPR Journal, Vol. 1. No. 5, 2018, the contents of which are incorporated herein by reference.

[0416] The term“Cas9” or“Cas9 nuclease” or“Cas9 moiety” or“Cas9 domain” includes any naturally occurring Cas9 from any organism, any naturally occurring Cas9 equivalent or functional fragment thereof, any Cas9 homolog, ortholog, or paralog from any organism, as well as any naturally occurring or engineered mutant or variant of Cas9. The term Cas9 is not meant to be particularly limiting and can be referred to as“Cas9 or equivalent.” Exemplary Cas9 proteins are further described herein and / or described in the art and incorporated herein by reference. The present disclosure is not limited as to the particular Cas9 used in the prime editors (PEs) of the invention.

[0417] As described herein, Cas9 nuclease sequences and structures are well known to those of skill in the art (see, e.g., "Complete genome sequence of an Ml strain of Streptococcus pyogenes." Ferretti et al., J.J., McShan W.M., Ajdic D.J., Savic D.J., Savic G., Lyon K., Primeaux C., Sezate S., Suvorov A.N., Kenton S., Lai H.S., Lin S.P., Qian Y., Jia H.G., Najar F.Z., Ren Q., Zhu H., Song L., White J., Yuan X., Clifton S.W., Roe B.A., McLaughlin R.E., Proc. Natl. Acad. Sci. U.S.A. 98:4658-4663 (2001); "CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III." Deltcheva E., Chylinski K., Sharma C.M., Gonzales K., Chao Y., Pirzada Z.A., Eckert M.R., Vogel J., Charpentier E., Nature 471 :602-607 (2011); and "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity." Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J.A., Charpentier E. Science 337:816-821 (2012), the entire contents of each of which are incorporated herein by reference).

[0418] Examples of Cas9 and Cas9 equivalents are provided below; however, these specific examples are not meant to be limiting. The guide editors of the present disclosure can use any suitable napDNAbp, including any suitable Cas9 or Cas9 equivalent.

[0419] A. Wild type canonical SpCas9

[0420] In one embodiment, the guide editor constructs described herein can comprise the“canonical SpCas9” nuclease from Streptococcus pyogenes, which has been widely used as a tool for genome engineering and classified as a type II sub-group enzyme of the class 2 CRISPR-Cas system. This Cas9 protein is a large multidomain protein that comprises two distinct nuclease domains. Point mutations can be introduced into Cas9 to abolish one or both nuclease activities, resulting in either a nickase Cas9 (nCas9) that still retains the ability to bind DNA in a programmable manner with an sgRNA or a dead Cas9 (dCas9). In principle, Cas9 or its variants (e.g., nCas9) can target a protein to almost any DNA sequence when fused to another protein or domain by co-expression with the appropriate sgRNA. As used herein, canonical SpCas9 protein refers to the wild-type protein from Streptococcus pyogenes having the following amino acid sequence:

[0421]

[0422]

[0423]

[0424] The guide editors described herein can include canonical SpCas9, or any variant thereof having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the wild-type Cas9 sequence provided above. These variants can include SpCas9 variants containing one or more mutations, including any known mutations reported in the SwissProt accession number Q99ZW2 (SEQ ID NO: 18) entry, which includes:

[0425]

[0426] Other wild-type SpCas9 sequences that can be used in the present disclosure include:

[0427]

[0428]

[0429]

[0430]

[0431]

[0432]

[0433]

[0434]

[0435]

[0436]

[0437] The guide editors described herein can include any of the above SpCas9 sequences, or any variant thereof having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto.

[0438] B. Wild type Cas9 ortholog

[0439] In other embodiments, the Cas9 protein can be a wild-type Cas9 ortholog from another bacterial species that is different from the canonical Cas9 from S. pyogenes. For example, the following Cas9 orthologs can be used in conjunction with the guide editor constructs described in this specification. In addition, any variant Cas9 ortholog having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to any of the following orthologs can also be used with the guide editors described herein.

[0440]

[0441]

[0442]

[0443]

[0444]

[0445]

[0446]

[0447]

[0448] The guide editors described herein can include any of the above Cas9 ortholog sequences, or any variant thereof having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto.

[0449] The napDNAbp can include any suitable homolog and / or ortholog or naturally occurring enzyme, for example, Cas9, Cas9 homologs and / or orthologs have been described in different species including, but not limited to, Streptococcus pyogenes and Streptococcus thermophilus. Preferably, the Cas moiety is configured (e.g., mutagenized, recombineering, or otherwise obtained from nature) to be a nickase, i.e., capable of cutting only a single strand of a target doublet, and sequences are apparent to the skilled artisan based on the present disclosure, such Cas9 nucleases and sequences include Cas9 sequences from organisms and loci disclosed in Chylinski, Rhun, and Charpentier, "The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems" (2013) RNA Biology 10:5, 726-737 (the entire contents of which are incorporated herein by reference). In some embodiments, the Cas9 nuclease has an inactive (e.g., inactivated) DNA cleavage domain, i.e., the Cas9 is a nickase. In some embodiments, the Cas9 protein comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of a Cas9 protein provided by any one of the variants of Table 3. In some embodiments, the Cas9 protein comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the amino acid sequence of a Cas9 protein provided by any one of the Cas9 orthologs in the above table.

[0450] C. Dead Cas9 variant

[0451] In certain embodiments, the prime editors described herein can include a dead Cas9, for example, a dead SpCas9, which has no nuclease activity due to one or more mutations that inactivate both nuclease domains of Cas9, i.e., the RuvC domain (which cleaves the non-protospacer DNA strand) and the HNH domain (which cleaves the protospacer DNA strand). The nuclease inactivation can be due to one or more substitutions and / or deletions in the amino acid sequence of the encoded protein or any variant having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto.

[0452] As used herein, the term "dCas9" refers to a nuclease inactive Cas9 or nuclease dead Cas9, or a functional fragment thereof, and includes any naturally occurring dCas9, any naturally occurring dCas9 equivalent, or a functional fragment thereof, from any organism, any dCas9 homolog, ortholog, or paralog from any organism, as well as any mutant or variant of a naturally occurring or engineered dCas9. The term dCas9 does not imply a particular limitation and can be referred to as "dCas9 or equivalent." Exemplary dCas9 proteins and methods for making dCas9 proteins are further described herein and / or are described in the art and incorporated herein by reference.

[0453] In other embodiments, dCas9 corresponds to or comprises a portion or the entire Cas9 amino acid sequence having one or more mutations that inactivate Cas9 nuclease activity. In other embodiments, Cas9 variants are provided having mutations other than D10A and H840A that can result in complete or partial inactivation of endogenous Cas9 nuclease (e.g., nCas9 or dCas9, respectively) activity. For example, with reference to a wild-type sequence such as Cas9 from Streptococcus pyogenes (NCBI Reference Sequence: NC_017053.1), such mutations include other amino acid substitutions at D10 and H820 of Cas9, or other substitutions within the nuclease domain (e.g., substitutions in the HNH nuclease subdomain and / or RuvC1 subdomain). In some embodiments, variants or homologs of Cas9 are provided (e.g., variants of Cas9 from Streptococcus pyogenes (NCBI Reference Sequence: NC_017053.1 (SEQ ID NO:20)) that are at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to NCBI Reference Sequence: NC_017053.1. In some embodiments, variants of Cas9 are provided (e.g., variants of NCBI Reference Sequence: NC_017053.1 (SEQ ID NO:20)) that have an amino acid sequence that is about 5 amino acids, about 10 amino acids, about 15 amino acids, about 20 amino acids, about 25 amino acids, about 30 amino acids, about 40 amino acids, about 50 amino acids, about 75 amino acids, about 100 amino acids or more shorter or longer than NC_017053.1 (SEQ ID NO:20).

[0454] In one embodiment, the dead Cas9 can be based on the canonical SpCas9 sequence of Q99ZW2 and can have the following sequence comprising D10X and H810X, where X can be any amino acid, a substitution (underlined and bolded), or a variant that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 40.

[0455] In one embodiment, the dead Cas9 can be based on the canonical SpCas9 sequence of Q99ZW2 and can have the following sequence comprising D10A and H810A substitutions (underlined and bolded), or a variant that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 41.

[0456]

[0457]

[0458]

[0459] D. Cas9 nickase variants

[0460] In an embodiment, the guide editors described herein comprise a Cas9 nickase. The term "Cas9 nickase" refers to a Cas9 variant that is capable of introducing a single-strand break in a target double-stranded DNA molecule. In some embodiments, a Cas9 nickase comprises only a single functional nuclease domain. Wild-type Cas9 (e.g., canonical SpCas9) comprises two independent nuclease domains, i.e., a RuvC domain (cleaves the non-protospacer DNA strand) and an HNH domain (cleaves the protospacer DNA strand). In an embodiment, a Cas9 nickase comprises a mutation in the RuvC domain that inactivates the RuvC nuclease activity. For example, mutations in aspartate (D) 10, histidine (H) 983, aspartate (D) 986, or glutamate (E) 762 have been reported as loss-of-function mutations of the RuvC nuclease domain and the creation of a functional Cas9 nickase (e.g., Nishimasu et al., "Crystal structure of Cas9 in complex with guide RNA and target DNA," Cell 156(5), 935-949, incorporated herein by reference). Thus, a nickase mutation in the RuvC domain can include D10X, H983X, D986X, or E762X, where X is any amino acid other than the wild-type amino acid. In certain embodiments, the nickase can be D10A, H983A, or D986A, or E762A, or a combination thereof.

[0461] In different embodiments, the Cas9 nickase can have a mutation in the RuvC nuclease domain and have one of the following amino acid sequences or a variant of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto.

[0462]

[0463]

[0464]

[0465]

[0466]

[0467]

[0468]

[0469] 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 of the HNH nuclease domain and creation of a functional Cas9 nickase (e.g., Nishimasu et al., "Crystal structure of Cas9 in complex with guide RNA and target DNA," Cell 156(5), 935-949, which is incorporated herein by reference). Thus, nickase mutations in the HNH domain 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.

[0470] In different embodiments, the Cas9 nickase can have a mutation in the HNH nuclease domain and have one of the following amino acid sequences or a variant of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto.

[0471]

[0472]

[0473]

[0474]

[0475] In some embodiments, the N-terminal methionine is removed from the Cas9 nickase or from any of the Cas9 variants, orthologs, or equivalents disclosed or contemplated herein. For example, a methionine-reduced Cas9 nickase includes the following sequence or a variant of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto.

[0476]

[0477]

[0478]

[0479] E. Other Cas9 variants

[0480] In addition to dead Cas9 and Cas9 nickase variants, Cas9 proteins used herein can also include other "Cas9 variants" having at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to any reference Cas9 protein, including any wild-type Cas9 or mutant Cas9 (e.g., inactivated Cas9 or Cas9 nickase), or Cas9 fragment, or circularly permuted Cas9, or other variant of Cas9 disclosed herein or known in the art. In some embodiments, a Cas9 variant can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acid changes compared to a reference Cas9. In some embodiments, a Cas9 variant includes a fragment of a reference Cas9 (e.g., gRNA binding domain or DNA cleavage domain) such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of a wild-type Cas9. In some embodiments, the fragment is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% in amino acid length to the corresponding wild-type Cas9 (e.g., SEQ ID NO: 18).

[0481] In some embodiments, the disclosure can also utilize Cas9 fragments that retain functionality and are fragments of any Cas9 protein disclosed herein. In some embodiments, a Cas9 fragment is at least 100 amino acids in length. In some embodiments, the fragment is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, or at least 1300 amino acids in length.

[0482] In various embodiments, the prime editors disclosed herein can comprise one of the Cas9 variants described below or a Cas9 variant that is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to any of the reference Cas9 variants.

[0483] F. Small Cas9 variants

[0484] In some embodiments, the prime editors contemplated herein can include Cas9 proteins having a molecular weight that is less than that of a canonical SpCas9 sequence. In some embodiments, the small Cas9 variants can facilitate delivery to a cell, for example, by an expression vector, a nanoparticle, or other delivery means. In certain embodiments, the small Cas9 variants can include enzymes classified as Type II enzymes of the Class 2 CRISPR-Cas system. In some embodiments, the small Cas9 variants can include enzymes classified as Type V enzymes of the Class 2 CRISPR-Cas system. In other embodiments, the small Cas9 variants can include enzymes classified as Type VI enzymes of the Class 2 CRISPR-Cas system.

[0485] The length of the canonical SpCas9 protein is 1368 amino acids and has a predicted molecular weight of 158 kilodaltons. As used herein, the term "miniature Cas9 variant" refers to any Cas9 variant - naturally occurring, engineered, or otherwise - that is less than at least 1300 amino acids, or at least less than 1290 amino acids, or 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 at least more than about 400 amino acids and retains the desired Cas9 protein function. Cas9 variants can include those classified as Type II, Type V, or Type VI enzymes of the Class 2 CRISPR-Cas system.

[0486] In different embodiments, the guided editors disclosed herein can comprise a miniature Cas9 variant as described below or a Cas9 variant that is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to any of the reference miniature Cas9 proteins.

[0487]

[0488]

[0489]

[0490]

[0491]

[0492] G. Cas9 equivalents

[0493] In some embodiments, the prime editors described herein can include any Cas9 equivalent. As used herein, the term "Cas9 equivalent" is a broad term and includes any napDNAbp protein that functions the same as Cas9 in the present prime editors, although its amino acid primary sequence and / or its three-dimensional structure can be different and / or unrelated from an evolutionary perspective. Thus, while a Cas9 equivalent includes any Cas9 ortholog, homolog, mutant, or variant described or encompassed herein, a Cas9 equivalent also includes proteins that can have evolved through a process of convergent evolution to have the same or similar function as Cas9, but they do not necessarily have any similarity in amino acid sequence and / or three-dimensional structure. The prime editors described herein include any Cas9 equivalent that will provide the same or similar function as Cas9, although the Cas9 equivalent can be based on a protein that arose through convergent evolution. For example, if Cas9 refers to a type II enzyme of the CRISPR-Cas system, then a Cas9 equivalent can refer to a type V or VI enzyme of the CRISPR-Cas system.

[0494] For example, Gasl2e (CasX) is a Cas9 equivalent that is reported to have the same function as Cas9 but evolved through convergent evolution. Thus, the Gasl2e (CasX) protein described in Liu et al., "CasX enzymes comprises a distinct family of RNA-guided genome editors," Nature, 2019, Vol. 566: 218-223 is contemplated for use with the prime editors described herein. Furthermore, any variant or modification of Gasl2e (CasX) is contemplated and within the scope of the present disclosure.

[0495] Cas9 is a bacterial enzyme that evolved in a very large number of species. However, the Cas9 equivalents contemplated herein can also be obtained from archaea, which constitute a domain and kingdom of single-celled p...

Claims

1. A system for prime editing, comprising: (i) a prime editor comprising a nucleic acid programmable DNA binding protein and a reverse transcriptase, wherein the nucleic acid programmable DNA binding protein is a Cas protein, and wherein the Cas protein has nickase activity, and (ii) an extended guide RNA, wherein the prime editor complexed with the extended guide RNA is capable of: binding to a target DNA sequence comprising a target strand and a complementary non-target strand, forming an R-loop comprising (i) an RNA-DNA hybrid comprising the extended guide RNA and the target strand, and (ii) the complementary non-target strand, and nicking the complementary non-target strand to form a free 3’ end; wherein the extended guide RNA comprises (a) a guide RNA and (b) an RNA extension comprising (i) a reverse transcription template sequence comprising one or more desired nucleotide changes and (ii) a reverse transcription primer binding site, and wherein the reverse transcription primer binding site is capable of hybridizing to the free 3’ end of the complementary non-target strand of the nicked target DNA sequence.

2. The system of claim 1, wherein the nucleic acid programmable DNA binding protein is a Cas9 nickase.

3. The system of claim 1, wherein the nucleic acid programmable DNA binding protein is selected from the group consisting of: Cas9, Cas12e, Cas12a, Cas12b1, Cas13a, and Cas12c nickases.

4. The system of claim 1, wherein the nucleic acid programmable DNA binding protein is a Cas9 nickase that has an H840A amino acid substitution relative to the SpCas9 sequence of SEQ ID NO:

18.

5. The system of claim 1, 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, at least 25 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides, at least 80 nucleotides, at least 90 nucleotides, at least 100 nucleotides, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, or at least 500 nucleotides in length.

6. The system of claim 1, wherein the one or more desired nucleotide changes are in an editing window between -4 to +10 of a PAM sequence.

7. The system of claim 1, wherein the one or more desired nucleotide changes comprise one or more nucleotide substitutions, one or more nucleotide deletions, one or more nucleotide insertions, or a combination thereof.

8. The system of claim 7, wherein each of the one or more nucleotide insertions or one or more nucleotide deletions 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, or at least 15 nucleotides in length.

9. The system of claim 1, wherein the one or more desired nucleotide changes are at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 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, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, at least 68, at least 69, at least 70, at least 71, at least 72, at least 73, at least 74, at least 75, at least 76, at least 77, at least 78, at least 79, at least 80, at least 81, at least 82, at least 83, at least 84, at least 85, at least 86, at least 87, at least 88, at least 89, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, at least 99, or at least 100 nucleotides downstream of the nick.

10. The system of claim 1, wherein the reverse transcription template sequence comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the target DNA sequence.

11. The system of claim 1, wherein the reverse transcription template sequence is homologous to the target DNA sequence downstream of the nick except for the one or more desired nucleotide changes.

12. The system of any one of claims 1-11, wherein the reverse transcriptase is a reverse transcriptase from a retrovirus or a retrotransposon.

13. The system of any one of claims 1-11, wherein the reverse transcriptase is a Moloney-Murine Leukemia Virus reverse transcriptase (M-MLV RT).

14. The system of any one of claims 1-11, wherein the reverse transcriptase consists of the sequence of SEQ ID NO:

89.

15. The system of any one of claims 1-11, wherein the reverse transcriptase consists of the sequence of SEQ ID NO:

122.

16. The system of claim 11, wherein the M-MLV RT consists of the sequence of SEQ ID NO:

766.

17. The system of any one of claims 1-11, wherein the nucleic acid programmable DNA binding protein and the reverse transcriptase are linked to form a fusion protein.

18. The system of claim 17, wherein the fusion protein comprises the structure NH2- [nucleic acid programmable DNA binding protein]-[reverse transcriptase]-COOH, or NH2- [reverse transcriptase]-[nucleic acid programmable DNA binding protein]-COOH, wherein each instance of “-” indicates the presence of an optional peptide linker.

19. The system of claim 17, wherein the fusion protein consists of the amino acid sequence of SEQ ID NO:

134.

20. One or more polynucleotides encoding the nucleic acid programmable DNA binding protein, reverse transcriptase, and extended guide RNA of the system of any one of claims 1-19.

21. One or more vectors comprising the one or more polynucleotides of claim 20.

22. A cell comprising the system of any one of claims 1-19, the one or more polynucleotides of claim 20, or the one or more vectors of claim 21.

23. A pharmaceutical composition comprising (i) the system of any one of claims 1-19, the one or more polynucleotides of claim 20, the one or more vectors of claim 21, or the cell of claim 22, and (ii) a pharmaceutically acceptable excipient.

24. Use of the system of any one of claims 1-19, the one or more polynucleotides of claim 20, the one or more vectors of claim 21, the cell of claim 22, or the pharmaceutical composition of claim 23 in the manufacture of a medicament.

Images