Editing Methods and compositions for editing nucleotide sequences

Abstract

The present disclosure provides novel prime editor guide RNAs for prime editing, constructs for prime editing, and methods for using the constructs. Additionally, the present disclosure provides compositions and methods for performing prime editing of a target DNA molecule (e.g., a genome) that allow for the incorporation of nucleotide changes and / or targeted mutagenesis (e.g., insertions or deletions). Nucleotide changes can include single nucleotide changes (e.g., any transition or any transversion), the insertion of one or more nucleotides, or the deletion of one or more nucleotides. In particular, the present disclosure provides a fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a polymerase (e.g., reverse transcriptase), which is guided to a specific DNA sequence by a prime editor RNA (PEgRNA). The prime editor guide RNA comprises an extension arm that provides a DNA synthesis template sequence that encodes a single-stranded DNA flap that is homologous to the endogenous DNA sequence but contains one or more desired nucleotide changes, and that is incorporated into the target DNA molecule after synthesis by a polymerase (e.g., reverse transcriptase).

Description

[Technical Field]

[0001] Government support This invention was made with government support under Grant Nos. U01AI142756, RM1HG009490, R01EB022376, and R35GM118062 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0002] Related Applications and Incorporation by Reference This U.S. provisional application is a continuation of the following applications: U.S. Provisional Application No. 62 / 820,813, filed March 19, 2019 (Attorney Docket No. B1195.70074US00), U.S. Provisional Application No. 62 / 858,958, filed June 7, 2019 (Attorney Docket No. B1195.70074US01), U.S. Provisional Application No. 62 / 889,996, filed August 21, 2019 (Attorney Docket No. U.S. Provisional Application No. 62 / 922,654, filed August 21, 2019 (Attorney Docket No. B1195.70074US02), U.S. Provisional Application No. 62 / 922,654, filed August 21, 2019 (Attorney Docket No. B1195.70083US00), U.S. Provisional Application No. 62 / 913,553, filed October 10, 2019 (Attorney Docket No. B1195.70074US03), U.S. Provisional Application No. 62 / 973,558, filed October 10, 2019 No. (Attorney Docket No. B1195.70083US01), U.S. Provisional Application No. 62 / 931,195, filed November 5, 2019 (Attorney Docket No. B1195.70074US04), U.S. Provisional Application No. 62 / 944,231, filed December 5, 2019 (Attorney Docket No. B1195.70074US05), U.S. Provisional Application No. 62 / 974, filed December 5, 2019 No. 537, filed March 17, 2020 (Attorney Docket No. B1195.70083US02), U.S. Provisional Application No. 62 / 991,069, filed March 17, 2020 (Attorney Docket No. B1195.70074US06), and U.S. Provisional Application No. (serial number not available at the time of filing this) filed March 17, 2020 (Attorney Docket No. B1195.70083US03), which are incorporated by reference.

[0003] Incorporation by Reference of Sequence Listing Pursuant to 37 CFR § 1.52(e), this specification contains a sequence listing on a compact disc (two copies) filed concurrently herewith. As required by 37 CFR § 1.52(e)(5), applicant expressly incorporates by reference all of the information and material located on the compact disc in the file designated "B119570083WO00-SEQ.txt," created on March 19, 2020, and 371.109 MB in size. The sequence listing is hereby made a part of this specification. The compact disc contains no other files. [Background technology]

[0004] Background of the Invention Pathogenic single-nucleotide mutations contribute to approximately 67% of human diseases with a genetic component 7 Unfortunately, treatment options for patients with these genetic disorders remain extremely limited, despite decades of gene therapy research. 8 Perhaps one of the simplest solutions to this therapeutic challenge is the direct correction of single-nucleotide mutations in the patient's genome, which would address the underlying cause of the disease and may provide lasting benefit. Such a strategy has never been considered before, but the CRISRP / Cas system has proven to be a promising tool. 9 Recent improvements in genome editing capabilities brought about by the advent of CRISPR now put this therapeutic approach within reach. By simple design of guide RNA (gRNA) sequences containing ~20 nucleotides complementary to the target DNA sequence, almost any conceivable genomic site can be specifically accessed by CRISPR-associated (Cas) nucleases. 1,2 To date, several monomeric bacterial Cas nuclease systems have been identified and adapted for genome editing applications. 10 This natural diversity of Cas nucleases, along with a growing collection of engineered variants, 11~14 , providing fertile ground for developing new genome editing technologies.

[0005] Although CRISPR gene disruption is now a mature technique, precisely editing single base pairs in the human genome remains a major challenge. 3 Homology-directed repair (HDR) has long been used in human cells and other organisms to insert, correct, or exchange DNA sequences at sites of double-strand breaks (DSBs) using a donor DNA repair template that encodes the desired edit. 15 However, existing HDR has extremely low efficiency in most human cell types, particularly in non-dividing cells, and incompatible non-homologous end joining (NHEJ) leads primarily to insertion-deletion (indel) by-products. 16 Another issue concerns the generation of DSBs, which can result in large chromosomal rearrangements and deletions at the target locus. 17 or activate the p53 axis, which leads to growth arrest and apoptosis 18,19 .

[0006] Several approaches have been explored to address the shortcomings of HDR. For example, repair of single-strand DNA breaks (nicks) with oligonucleotide donors has been shown to reduce indel formation, but the yield of the desired repair product remains low. 20 Other strategies have attempted to bias repair toward HDR over NHEJ using small molecules and biological reagents. 21~23 However, the effectiveness of these methods is cell type dependent, and perturbation of normal cellular conditions can lead to unwanted and unpredictable effects.

[0007] Recently, Liu et al. developed base editing, a technique for editing targeted nucleotides without creating DSBs or relying on HDR. 4~6,24~27Direct modification of DNA bases by Cas-fused deaminases allows for highly efficient C·G → T·A or A·T → G·C base pair conversions within short target windows (~5–7 bases). As a result, base editors have been rapidly adopted by the scientific community. However, several factors may limit their generality for high-precision genome editing.

[0008] Thus, the development of programmable editing elements capable of introducing any desired single or multiple nucleotide changes that can incorporate nucleotide insertions or deletions (e.g., insertions or deletions of at least 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more base pairs) and / or alter or modify the nucleotide sequence at a target site with high specificity and efficiency would substantially expand the scope and therapeutic potential of CRISPR-based genome editing technology. Summary of the Invention

[0009] SUMMARY OF THE INVENTION The present invention discloses novel compositions (e.g., novel PEgRNAs and PE complexes comprising the same) and methods for using prime editing (PE) to repair therapeutic targets (e.g., those targets identified in the ClinVar database) using PEgRNAs designed using specialized algorithms described herein. Thus, in one aspect, the present application discloses algorithms for large-scale prediction of PEgRNA sequences that may be used to repair therapeutic targets (e.g., therapeutic targets contained in the ClinVar database). Additionally, the present application discloses designed therapeutic PEgRNAs and therapeutic PEgRNAs that can be designed using the disclosed algorithms, and predicted sequences of therapeutic PEgRNAs that may be used to repair therapeutic targets with prime editing.

[0010] The algorithms and predicted PEgRNA sequences disclosed herein generally relate to prime editing. Thus, the present disclosure also provides a description of various components and aspects of prime editing, including suitable napDNAbps (e.g., Cas9 nickase) and polymerases (e.g., reverse transcriptase), as well as other suitable components (e.g., linkers, NLSs) and PE fusion proteins, that may be used with the therapeutic PEgRNAs disclosed herein.

[0011] As disclosed herein, prime editing is a versatile and precise genome editing method that uses a nucleic acid-programmed DNA-binding protein ("napDNAbp") in conjunction with a polymerase (i.e., in the form of a fusion protein or otherwise provided in trans with the napDNAbp) to write new genetic information directly into a specific DNA site; the prime editing system is programmed with a prime editing (PE) guide RNA ("PEgRNA") that specifies both the target site and a template for synthesis of the desired edit in the form of a replacement DNA strand via an extension (either DNA or RNA) engineered onto the guide RNA (e.g., at the 5' or 3' end of the guide RNA, or internally). The replacement strand, containing the desired edit (e.g., a single nucleobase substitution), shares the same sequence as the endogenous strand at the target site to be edited (except that it encompasses the desired edit). Through DNA repair and / or replication mechanisms, the endogenous strand at the target site is replaced by the newly synthesized replacement strand containing the desired edit. In some cases, prime editing may be considered a "search-and-replace" genome editing technique, where a prime editor as described herein not only searches for and locates the desired target site to be edited, but also simultaneously encodes a replacement strand containing the desired edit that is incorporated in place of the corresponding target site in the endogenous DNA strand. The prime editors of the present disclosure relate, in part, to the discovery that the mechanism of target-primed reverse transcription (TPRT) or "prime editing" can be exploited or employed to perform high-precision CRISPR / Cas-based genome editing with high efficiency and genetic flexibility (e.g., as depicted in various embodiments in Figures 1A-1F). TPRT is naturally used by mobile DNA elements, such as mammalian non-LTR retrotransposons and bacterial group II introns. 28,29Herein, we use a Cas protein-reverse transcriptase fusion or related system that targets a specific DNA sequence with a guide RNA, generates a single-stranded nick at the target site, and uses the nicked DNA as a primer for reverse transcription of an engineered reverse transcriptase template incorporated with the guide RNA. However, although the concept originates with prime editors that use reverse transcriptase as a DNA polymerase component, the prime editors described herein are not limited to reverse transcriptase and may in fact encompass the use of any DNA polymerase. Indeed, although the application sometimes refers to prime editors with "reverse transcriptase" throughout, it is explained herein that reverse transcriptase is the only type of DNA polymerase that can function in prime editing. Thus, wherever "reverse transcriptase" is referred to herein, those skilled in the art will understand that any suitable DNA polymerase may be used in place of reverse transcriptase. Thus, in one aspect, a prime editor may comprise a Cas9 (or equivalent napDNAbp) programmed to target a DNA sequence by linking it to a specialized guide RNA (i.e., a PEgRNA) containing a spacer sequence that anneals to a complementary protospacer in the target DNA. The specialized guide RNA also contains new genetic information in the form of an extension that encodes a replacement strand of DNA containing the desired genetic alteration that is used to replace the corresponding endogenous DNA strand at the target site. To transfer information from the PEgRNA to the target DNA, the mechanism of prime editing involves nicking the target site in one strand of DNA, exposing a 3' hydroxyl group. The exposed 3' hydroxyl group can then be used to prime DNA polymerization of the edit-encoding extension on the PEgRNA directly into the target site. In various embodiments, the extension—which provides a template for polymerization of the replacement strand containing the edit—can be formed from RNA or DNA. In the case of RNA elongation, the polymerase of the prime editor can be an RNA-dependent DNA polymerase (such as a reverse transcriptase).In the case of DNA elongation, the polymerase of the prime editor may be a DNA-dependent DNA polymerase.

[0012] The newly synthesized strand (i.e., the replacement DNA strand containing the desired edit) formed by the primed editors disclosed herein will be homologous to (i.e., have the same sequence as) the target sequence in the genome, except for the desired nucleotide change (e.g., a single nucleotide change, deletion, or insertion, or a combination thereof). The newly synthesized (or replacement) strand of DNA, sometimes referred to as a single-stranded DNA flap, competes with the complementary homologous endogenous DNA strand for hybridization, thereby displacing the corresponding endogenous strand. In some embodiments, the system can be combined with the use of an error-prone reverse transcriptase (e.g., provided as a fusion protein with a Cas9 domain or provided in trans with the Cas9 domain). The error-prone reverse transcriptase can introduce modifications during single-stranded DNA flap synthesis. Thus, in some embodiments, an error-prone reverse transcriptase can be utilized to introduce nucleotide changes into the target DNA. Depending on the error-prone reverse transcriptase used with the system, the modifications can be random or non-random.

[0013] Degradation of the hybridized intermediate (including the single-stranded DNA flap synthesized by the reverse transcriptase hybridized to the endogenous DNA strand) can involve the resulting removal of the displaced flap from the endogenous DNA (e.g., with the 5'-terminal DNA flap endonuclease, FEN1), ligation of the synthesized single-stranded DNA flap to the target DNA, and assimilation of the desired nucleotide change as a result of cellular DNA repair and / or replication processes. Because templated DNA synthesis confers single-nucleotide precision on any nucleotide modification, including insertions and deletions, the breadth of this approach is extremely broad, and it is foreseeable that it could be used for countless applications in basic science and therapeutics.

[0014] Algorithms and methods for designing therapeutic PEGRNAs In one aspect, the present disclosure relates to novel algorithms for designing therapeutic PEG RNAs, particularly on a large scale, as opposed to one-off PEG RNA design exercises.

[0015] Accordingly, some aspects relate to computational methods for determining the sequence of a prime editor guide RNA (PEgRNA), the methods comprising using at least one computer hardware processor to access data representing an input allele, an output allele, and a fusion protein comprising a nucleic acid-programmable DNA-binding protein and a polymerase (e.g., reverse transcriptase). The method includes determining a PEgRNA sequence based on an input allele, an output allele, and a fusion protein, where the PEgRNA sequence is designed to associate with a fusion protein that changes the input allele to the output allele, and includes determining one or more of the following features for the PEgRNA sequence: a spacer complementary to the target nucleotide sequence in the input allele (i.e., a spacer as defined in FIG. 27); a gRNA backbone for interacting with the fusion protein (i.e., a gRNA core as defined in FIG. 27); and an extension (i.e., an extension arm as shown in FIG. 27) comprising one or more of the following: a DNA synthesis template (as shown in FIG. 27) containing the desired nucleotide change that changes the input allele to the output allele; a primer binding site (i.e., a primer binding site as shown in FIG. 27). The PEgRNA may also include a 3' termination signal that terminates transcription from the promoter. In addition, the PEgRNA may include a first modifier at the 5' end of the extension arm and a second modifier at the 3' end of the extension arm. Such sequences (shown as "e1" and "e2" in Figure 27) may include stem-loop sequences that may increase the stability of the PEGRNA.

[0016] In some instances, the method includes determining a spacer and an extension, and determining that the spacer is at the 5' end of the PEGRNA and the extension is at the 3' end of the PEGRNA structure.

[0017] In some examples, the method includes determining a spacer and an extension, and determining that the spacer is at the 5' end of the PEGRNA and the extension is at the 3' end relative to the spacer.

[0018] In some examples, accessing data indicating input alleles and output alleles includes accessing a database containing a set of input alleles and associated output alleles. Accessing the database may include accessing the National Center for Biotechnology Information's Clinvar database (www.ncbi.nlm.nih.gov / clinvar / ), which contains a plurality of entries, each entry containing an input allele from the set of input alleles and an output allele (e.g., a wild-type or an allele with a desired activity) from the set of output alleles. Determining the PEG RNA sequence may include determining, for one or more PEG RNA sequences, each input allele and associated output allele in the set.

[0019] In some instances, accessing data indicative of the fusion protein includes determining the fusion protein from a plurality of fusion proteins.

[0020] In some instances, the fusion protein comprises a Cas9 protein. The fusion protein may include a Cas9-NG protein, a Cas9-NGG protein, a saCas9-KKH protein, or a SpCas9 protein.

[0021] In some examples, changing the input allele to the output allele involves changing a single nucleotide, inserting one or more nucleotides, deleting one or more nucleotides, or a combination thereof.

[0022] In some embodiments, the method includes determining a spacer, wherein the spacer comprises a nucleotide sequence between 1 and 40 nucleotides. In some embodiments, the method includes determining a spacer, wherein the spacer comprises a nucleotide sequence between 5 and 35 nucleotides. In some embodiments, the method includes determining a spacer, wherein the spacer comprises a nucleotide sequence between 10 and 30 nucleotides. In some embodiments, the method includes determining a spacer, wherein the spacer comprises a nucleotide sequence between 15 and 25 nucleotides. In some embodiments, the method includes determining a spacer, wherein the spacer comprises a nucleotide sequence of approximately 20 nucleotides. The method can include determining a spacer based on the position of the change in the corresponding protospacer nucleotide sequence. The change can be incorporated in an editing window that is between about protospacer position -15 and protospacer position +39. The change can be incorporated in an editing window that is between about protospacer position -10 and protospacer position +34. Changes can be incorporated into an editing window that is between about protospacer position -5 and protospacer position +29. Changes can be incorporated into an editing window that is between about protospacer position -1 and protospacer position +27.

[0023] In some examples, the method may include: determining a set of initial candidate protospacers based on an input allele and a fusion protein (wherein each initial candidate protospacer includes a PAM of the fusion protein in the input allele); determining one or more initial candidate protospacers from the set of initial candidate protospacers (each including a mismatched nick position); removing one or more of the determined initial candidate protospacers from the set to generate a set of remaining candidate protospacers; wherein determining a PEG RNA structure includes determining a plurality of PEG RNA structures, each of which includes a different spacer determined based on a corresponding protospacer from the remaining set of candidate protospacers.

[0024] In some examples, the method includes determining an extension and DNA synthesis template (e.g., an RT template sequence), wherein the DNA synthesis template (e.g., the RT template sequence) comprises approximately 1 nucleotide to 40 nucleotides. In some examples, the method includes determining an extension and DNA synthesis template (e.g., an RT template sequence), wherein the DNA synthesis template (e.g., the RT template sequence) comprises approximately 3 nucleotides to 38 nucleotides. In some examples, the method includes determining an extension and DNA synthesis template (e.g., an RT template sequence), wherein the DNA synthesis template (e.g., the RT template sequence) comprises approximately 5 nucleotides to 36 nucleotides. In some examples, the method includes determining an extension and DNA synthesis template (e.g., an RT template sequence), wherein the DNA synthesis template (e.g., the RT template sequence) comprises approximately 7 nucleotides to 34 nucleotides.

[0025] In some examples, determining the PEGRNA includes determining a spacer based on the input allele and / or fusion protein, and determining a DNA synthesis template (e.g., an RT template sequence) based on the spacer.

[0026] In some instances, the DNA synthesis template (e.g., an RT template sequence) encodes a single-stranded DNA flap complementary to the endogenous DNA sequence adjacent to the nick site, where the single-stranded DNA flap contains a desired nucleotide change. The single-stranded DNA flap hybridizes with the endogenous DNA sequence adjacent to the nick site, thereby incorporating the desired nucleotide change. The single-stranded DNA flap can replace the endogenous DNA sequence adjacent to the nick site. Cell repair of the single-stranded DNA flap results in the incorporation of the desired nucleotide change, thereby forming the desired product.

[0027] In some instances, the fusion protein, when complexed with PEGRNA, is capable of binding to a target DNA sequence, which may include the target strand where the alteration occurs and the complementary non-target strand.

[0028] In some instances, the input allele comprises a pathogenic DNA mutation and the output allele comprises a corrected DNA sequence.

[0029] Some embodiments relate to a system including at least one processor and at least one computer-readable storage medium having instructions encoded thereon that, when executed, cause the at least one processor to perform a computational method for determining a PEG RNA structure.

[0030] Some embodiments relate to at least one computer-readable storage medium having encoded thereon instructions that, when executed, cause at least one processor to perform a computational method for determining the sequence of a PEG RNA. Some embodiments relate to methods of base editing using a PEGRNA determined according to a computational method for determining a PEGRNA.

[0031] Therapeutic PEgRNA In another aspect, the present disclosure provides therapeutic PEG RNAs designed using the algorithms disclosed herein as represented by Figures 27 and 28.

[0032] For example, a PEgRNA that may be used in the present disclosure is illustrated in Figure 27. This figure provides the structure of one embodiment of a PEgRNA that may be designed according to the methodology contemplated herein and defined in Example 2. The PEgRNA comprises three major component elements ordered in the 5' to 3' direction: a spacer, a gRNA core, and an extension arm at the 3' end. The extension arm may be further divided in the 5' to 3' direction into the following structural elements: a primer binding site (A), an editing template (B), and a homology arm (C). In addition, the PEgRNA may comprise an optional 3'-end modifier region (e1) and an optional 5'-end modifier region (e2). The PEgRNA may also comprise a transcription termination signal at the 3' end of the PEgRNA (not depicted). These structural elements are further defined herein. The depiction of the PEgRNA structure is not intended to be limiting and encompasses variations in the arrangement of elements. For example, any sequence modifiers (e1) and (e2) can be located within or between any other regions shown and are not limited to being located at the 3' and 5' ends. The PEG RNA shown in Figure 27 can be designed by the algorithm disclosed herein.

[0033] In another example, Figure 28 provides the structure of another embodiment of a PEgRNA that may be designed according to the methodology contemplated herein and defined in Example 2. The PEgRNA comprises three major components ordered in the 5' to 3' direction: a spacer, a gRNA core, and an extension arm at the 3' end. The extension arm may be further divided into the following structural elements in the 5' to 3' direction: a primer binding site (A), an editing template (B), and a homology arm (C). In addition, the PEgRNA may comprise an optional 3'-end modification region (e1) and an optional 5'-end modification region (e2). Furthermore, the PEgRNA may comprise a transcription termination signal at the 3' end of the PEgRNA (not depicted). These structural elements are further defined herein. The depiction of the PEgRNA structure is not intended to be limiting and encompasses variations in the arrangement of elements. For example, any sequence modifiers (e1) and (e2) can be located within or between any other regions shown and are not limited to being located at the 3' and 5' ends. The PEG RNA shown in Figure 27 can be designed by the algorithm disclosed herein.

[0034] In various embodiments, the present disclosure provides therapeutic PEGRNAs of SEQ ID NOs: 1-135514 and 813085-880462 that were designed using the algorithms disclosed herein against Clinver database entries.

[0035] In various other embodiments, exemplary PEG-gRNAs designed using the algorithms disclosed herein against the Clinver database are included in the Sequence Listing, which forms a part of the present specification. The Sequence Listing includes the complete PEG-gRNA sequences set forth in SEQ ID NOS: 1-135514 and 813085-880462. Each of these complete PEG-gRNAs is composed of a respective spacer (SEQ ID NOS: 135515-271028 and 880463-947840) and extension arm (SEQ ID NOS: 271029-406542 and 947841-1015218). In addition, each PEG-gRNA includes a gRNA core, e.g., as defined by SEQ ID NOS: 1361579-1361580. The extension arms of SEQ ID NOs: 271029-406542 and 947841-1015218 further comprise respective primer binding sites (SEQ ID NOs: 406543-542056 and 1015219-1082596), editing templates (SEQ ID NOs: 542057-677570 and 1082597-1149974), and homology arms (SEQ ID NOs: 677571-813084 and 1149975-1217352). The PEG RNA may optionally contain a 5'-end modified region and / or a 3'-end modified region. The PEG RNA may also contain a reverse transcription termination signal (e.g., SEQ ID NOs: 1361560-1361566) at the 3' end of the PEG RNA. This application covers all designs and uses of these sequences.

[0036] In various embodiments, a prime editor guide RNA comprises (a) a guide RNA and (b) an RNA extension at the 5' or 3' end of the guide RNA, or at an intramolecular location within the guide RNA (examples of which are depicted in Figures 3A-C). The RNA extension may comprise (i) a DNA synthesis template containing the desired nucleotide change, (ii) a reverse transcription primer binding site, and (iii) optionally, a linker sequence. In various embodiments, the DNA synthesis template encodes a single-stranded DNA flap complementary to the endogenous DNA sequence adjacent to the nick site, where the single-stranded DNA flap contains the desired nucleotide change.

[0037] In various embodiments, the RNA extension arm is at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides, at least 22 nucleotides, at least 23 nucleotides, at least 24 nucleotides, or at least 25 nucleotides in length.

[0038] In some embodiments, the prime editor guide RNA comprises the nucleotide sequence of SEQ ID NOs: 1361548-1361581, or a nucleotide sequence having at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 1361548-1361581.

[0039] In some embodiments, the prime editor guide RNA (PEgRNA) comprises a variant of the nucleotide sequence of SEQ ID NOs: 1361548-1361581 that comprises at least one mutation compared to the nucleotide sequence of SEQ ID NOs: 1361548-1361581. In some embodiments, the variant comprises more than one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more) mutations compared to the nucleotide sequence of SEQ ID NOs: 1361548-1361581.

[0040] In another aspect, the present disclosure provides a prime editor guide RNA comprising a guide RNA and at least one RNA extension (i.e., with reference to Figure 27, an extension arm). The RNA extension is positioned at the 3' end of the guide RNA. In other embodiments, the RNA extension is positioned at the 5' end of the guide RNA. In yet other embodiments, the RNA extension is positioned at an intramolecular position within the guide RNA, preferably such that the intramolecular positioning of the extended portion does not interfere with the function of the protospacer.

[0041] In various embodiments, a prime editor guide RNA (PEgRNA) is capable of binding to the napDNAbp and directing the napDNAbp to a target DNA sequence, which may include a target strand and a complementary non-target strand, where the guide RNA hybridizes to the target strand to form an RNA-DNA hybrid and an R-loop.

[0042] In various embodiments of the prime editor guide RNA, at least one RNA extension comprises a DNA synthesis template. In various other embodiments, the RNA extension further comprises a reverse transcription primer binding site. In yet other embodiments, the RNA extension comprises a linker or spacer that joins the RNA extension to the guide RNA.

[0043] In various embodiments, the RNA extensions can be at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides, at least 22 nucleotides, at least 23 nucleotides, at least 24 nucleotides, at least 25 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides, at least 80 nucleotides, at least 90 nucleotides, at least 100 nucleotides, at least 150 nucleotides, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, or at least 500 nucleotides in length.

[0044] In other embodiments, the DNA synthesis template (i.e., with respect to Figure 27, the editing template) is at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides, at least 80 nucleotides, at least 90 nucleotides, at least 100 nucleotides, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, or at least 500 nucleotides in length.

[0045] In still other embodiments, the reverse transcription primer binding site sequence (i.e., with respect to Figure 27, the primer binding site) is at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides, at least 80 nucleotides, at least 90 nucleotides, at least 100 nucleotides, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, or at least 500 nucleotides in length.

[0046] In other embodiments, any linker or spacer is at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides, at least 80 nucleotides, at least 90 nucleotides, at least 100 nucleotides, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, or at least 500 nucleotides in length.

[0047] The designed PEGRNAs disclosed herein may be complexed with a prime editor fusion protein.

[0048] In one aspect, the present disclosure provides a primer editor fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a reverse transcriptase. In various embodiments, the fusion protein is capable of performing genome editing by target-primed reverse transcription in the presence of a prime editor guide RNA (PEgRNA).

[0049] In some embodiments, the napDNAbp is selected from the group consisting of Cas9, CasX, CasY, Cpf1, C2c1, C2c2, C2C3, and Argonaute, optionally having nickase activity.

[0050] In other embodiments, the fusion protein is capable of binding to a target DNA sequence (e.g., genomic DNA) when complexed with a primed editor guide RNA as described herein.

[0051] In yet other embodiments, the target DNA sequence comprises a target strand and a complementary non-target strand.

[0052] In other embodiments, binding of the fusion protein complexed to the prime editor guide RNA results in the formation of an R-loop, which may comprise (i) an RNA-DNA hybrid comprising the prime editor guide RNA and a target strand, and (ii) a complementary non-target strand.

[0053] In yet another embodiment, the complementary non-target strand is nicked to create a reverse transcriptase priming sequence with a free 3' end.

[0054] In still other embodiments, the single-stranded DNA flap hybridizes to the endogenous DNA sequence adjacent to the nick site, thereby incorporating the desired nucleotide change. In still other embodiments, the single-stranded DNA flap replaces the endogenous DNA sequence adjacent to the nick site and with a free 5' end. In some embodiments, the replaced endogenous DNA with a 5' end is excised by the cell.

[0055] In various embodiments, cellular repair of the single-stranded DNA flap results in the incorporation of the desired nucleotide change, thereby forming the desired product.

[0056] In various other embodiments, the desired nucleotide changes are incorporated into an editing window that is between about -4 and +10 of the PAM sequence.

[0057] In still other embodiments, the desired nucleotide change is incorporated into an editing window that is between about -5 to +5 nucleotides from the nick site, or between about -10 to +10 from the nick site, or between about -20 to +20 from the nick site, or between about -30 to +30 from the nick site, or between about -40 to +40 from the nick site, or between about -50 to +50 from the nick site, or between about -60 to +60 from the nick site, or between about -70 to +70 from the nick site, or between about -80 to +80 from the nick site, or between about -90 to +90 from the nick site, or between about -100 to +100 from the nick site, or between about -200 to +200 from the nick site.

[0058] In various embodiments, the napDNAbp comprises the amino acid sequence of SEQ ID NO: 1361421. In various other embodiments, the napDNAbp comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 1361421-1361484 and 1361593-1361596.

[0059] In other embodiments, the reverse transcriptase, fusion protein, and / or composition of the disclosure may comprise any one of the amino acid sequences set forth in SEQ ID NOs: 1361485-1361514 and 1361597-1361598. In still other embodiments, the reverse transcriptase may comprise an amino acid sequence at least 80%, 85%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 1361485-1361514 and 1361597-1361598. These sequences may be naturally occurring reverse transcriptase sequences (e.g., from a retrovirus or retrotransposon), or the sequences may be non-naturally occurring or modified.

[0060] In various other embodiments, the fusion proteins disclosed herein may comprise various structural configurations. For example, a fusion protein may comprise the structure NH2-[napDNAbp]-[reverse transcriptase]-COOH; or NH2-[reverse transcriptase]-[napDNAbp]-COOH, where each occurrence of " ]-[ " indicates the presence of an optional linker sequence.

[0061] In various embodiments, the linker sequence comprises an amino acid sequence of SEQ ID NOs: 1361520-1361530, 1361585, and 1361603, or an amino acid sequence that is at least 80%, 85%, or 90%, or 95%, or 99% identical to any one of the linker amino acid sequences of SEQ ID NOs: 1361520-1361530, 1361585, and 1361603.

[0062] In various embodiments, the desired nucleotide change incorporated into the target DNA can be a single nucleotide change (e.g., a transition or transversion), an insertion of one or more nucleotides, a deletion of one or more nucleotides, or a combination thereof.

[0063] In some cases, the insertion is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, or at least 500 in length.

[0064] In certain other cases, the deletion is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, or at least 500 nucleotides in length.

[0065] In various embodiments of the prime editor guide RNA, the DNA synthesis template (i.e., with reference to Figure 27, the editing template) may encode a single-stranded DNA flap complementary to the endogenous DNA sequence adjacent to the nick site, where the single-stranded DNA flap contains the desired nucleotide change. The single-stranded DNA flap may replace the endogenous single-stranded DNA at the nick site. The endogenous single-stranded DNA replaced at the nick site may have a 5' end, forming an endogenous flap, which can be excised by the cell. In various embodiments, excision of the 5' endogenous flap helps drive product formation, as removal of the 5' endogenous flap facilitates hybridization of the single-stranded 3' DNA flap with the corresponding complementary DNA strand and incorporation or assimilation of the desired nucleotide change carried by the single-stranded 3' DNA flap into the target DNA.

[0066] In various embodiments of the primed editor guide RNA, cellular repair of the single-stranded DNA flap results in the incorporation of the desired nucleotide change, thereby forming the desired product.

[0067] In another aspect of the invention, the description provides a complex comprising a fusion protein described herein and any of the prime editor guide RNAs (PEgRNAs) described above.

[0068] In yet another aspect of the present invention, the present disclosure provides a complex comprising a napDNAbp (e.g., Cas9) and a prime editor guide RNA. The napDNAbp may be a Cas9 nickase (e.g., spCas9) or may be an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 1361421 or any one of SEQ ID NOs: 1361421-1361484 and 1361593-1361596.

[0069] In various embodiments involving the complex, a prime editor guide RNA can direct the napDNAbp to the target DNA sequence. In various embodiments, the reverse transcriptase can be provided in trans, i.e., from a source different from the complex itself. For example, the reverse transcriptase can be provided to the same cell containing the complex by introducing a separate vector encoding the reverse transcriptase.

[0070] In another aspect, the present disclosure provides pharmaceutical compositions (e.g., fusion proteins described herein, PEGRNAs of SEQ ID NOS: 1-135,514). In some embodiments, the pharmaceutical compositions comprise one or more of a napDNAbp, a fusion protein, a reverse transcriptase, and a prime editor guide RNA. In some embodiments, the fusion protein described herein and a pharmaceutically acceptable excipient. In other embodiments, the pharmaceutical composition comprises any extended guide RNA described herein and a pharmaceutically acceptable excipient. In yet other embodiments, the pharmaceutical composition comprises any extended guide RNA described herein in combination with any fusion protein described herein and a pharmaceutically acceptable excipient. In yet another embodiment, the pharmaceutical composition comprises any polynucleotide sequence encoding one or more of a napDNAbp, a fusion protein, a reverse transcriptase, and a prime editor guide RNA. In still other embodiments, the various components disclosed herein may be separated into one or more pharmaceutical compositions. For example, a first pharmaceutical composition may comprise a fusion protein or napDNAbp, a second pharmaceutical composition may comprise a reverse transcriptase, and a third pharmaceutical composition may comprise a prime editor guide RNA.

[0071] In yet a further aspect, the present disclosure provides kits. In one embodiment, the kit includes one or more polynucleotides encoding one or more components, including a fusion protein, a napDNAbp, a reverse transcriptase, and a prime editor guide RNA (e.g., any of SEQ ID NOS: 1-135514 or 813085-880462). The kit may also include isolated preparations of vectors, cells, and polypeptides, including any of the fusion proteins, napDNAbp, or reverse transcriptases disclosed herein.

[0072] In another aspect, the present disclosure provides methods of using the disclosed PEG RNA compositions of matter.

[0073] In one embodiment, a method is provided for incorporating a desired nucleotide change into double-stranded DNA using the PEGRNA disclosed herein. The method first involves contacting a double-stranded DNA sequence with a complex comprising a fusion protein as described herein and a prime editor guide RNA, wherein the fusion protein comprises a napDNAbp and a reverse transcriptase, and wherein the prime editor guide RNA comprises a DNA synthesis template containing the desired nucleotide change. The napDNAbp nicks the double-stranded DNA sequence on the non-target strand, thereby generating a free single-stranded DNA having a 3' end. Following nicking, the 3' end of the free single-stranded DNA hybridizes to the DNA synthesis template, thereby priming the reverse transcriptase domain. The reverse transcriptase then proceeds with DNA polymerization from the 3' end, thereby generating a single-stranded DNA flap containing the desired nucleotide change. The single-stranded DNA flap then displaces the endogenous DNA strand adjacent to the cleavage site, thereby incorporating the desired nucleotide change into the double-stranded DNA sequence.

[0074] In another aspect, the present disclosure provides a method for introducing one or more changes into the nucleotide sequence of a DNA molecule at a target locus, comprising contacting the DNA molecule with a nucleic acid programmable DNA-binding protein (napDNAbp) and a guide RNA that targets the napDNAbp to the target locus, wherein the guide RNA comprises a reverse transcriptase (RT) template sequence containing at least one desired nucleotide change. The napDNAbp exposes the 3' end of a DNA strand at the target locus, which primes reverse transcription by hybridizing with a DNA synthesis template (e.g., an RT template sequence). Next, a single-stranded DNA flap containing at least one desired nucleotide change is synthesized or polymerized by reverse transcriptase based on the DNA synthesis template (e.g., an RT template sequence). Finally, the at least one desired nucleotide change is incorporated into the corresponding endogenous DNA, thereby introducing one or more changes into the nucleotide sequence of the DNA molecule at the target locus.

[0075] In yet another aspect, the present disclosure provides a method for introducing one or more changes in the nucleotide sequence of a DNA molecule at a target locus by target-primed reverse transcription, the method comprising contacting the DNA molecule at the target locus with (i) a fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a reverse transcriptase, and (ii) a guide RNA comprising an RT template comprising a desired nucleotide change (e.g., any of SEQ ID NOS: 1-135514 or 813085-880462); the contacting allows target-primed reverse transcription of the RT template to generate single-stranded DNA comprising the desired nucleotide change and facilitates incorporation of the desired nucleotide change into the DNA molecule at the target locus through DNA repair and / or replication processes.

[0076] In some embodiments, the step of replacing the endogenous DNA strand comprises: (i) hybridizing a single-stranded DNA flap to the endogenous DNA strand adjacent to the cleavage site, thereby creating a sequence mismatch; (ii) excising the endogenous DNA strand; and (iii) repairing the mismatch to form a desired product containing the desired nucleotide changes in both strands of DNA.

[0077] The methods disclosed herein may involve a fusion protein having a napDNAbp that is a nuclease-dead Cas9 (dCas9), a Cas9 nickase (nCas9), or a nuclease-active Cas9. In other embodiments, the napDNAbp and reverse transcriptase are not encoded as a single fusion protein, but rather can be provided in separate constructs. Thus, in some embodiments, the reverse transcriptase can be provided in trans to the napDNAbp (rather than as a fusion protein).

[0078] In various embodiments involving the methods, the napDNAbp may comprise the amino acid sequence of SEQ ID NO: 1361421 (Cas9). The napDNAbp may also comprise an amino acid sequence at least 80%, 85%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NO: 1361421.

[0079] In various embodiments involving the methods, the reverse transcriptase may comprise any one of the amino acid sequences set forth in SEQ ID NOs: 1361485-1361514 and 1361597-1361598. The reverse transcriptase may also comprise an amino acid sequence at least 80%, 85%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 1361485-1361514 and 1361597-1361598.

[0080] The method may involve the use of an extended RNA having the nucleotide sequence of SEQ ID NOs: 271029-406542 and 947841-1015218, or a nucleotide sequence having at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 99% sequence identity thereto.

[0081] The method may include the use of a primed editor guide RNA that comprises an RNA extension at its 3' end, where the RNA extension comprises a DNA synthesis template, for example, the PEG RNA shown in Figure 3B (with the following components as written from 5' to 3': spacer; gRNA core; reverse transcription template; primer binding site), which has an extension arm that comprises, from 5' to 3', a reverse transcription template and a primer binding site.

[0082] The method may include the use of a primed editor guide RNA that comprises an RNA extension at its 5' end, where the RNA extension comprises a DNA synthesis template, for example, the PEG RNA shown in Figure 3A (with the following components as listed from 5' to 3': reverse transcription template; primer binding site; linker; spacer; gRNA core), which has an extension arm that comprises, from 5' to 3', a reverse transcription template, a primer binding site, and a 5-20 nucleotide long linker.

[0083] The method may include the use of a primed editor guide RNA that comprises an RNA extension at an intramolecular location in the guide RNA, where the RNA extension comprises a DNA synthesis template.

[0084] The methods may involve the use of prime editor guide RNAs having one or more RNA extensions that are at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, or at least 500 nucleotides in length.

[0085] It should be understood that the above concepts, and additional concepts discussed below, may be arranged in any suitable combination (although the disclosure is not limited in this respect). Furthermore, other advantages and novel features of the present disclosure will become apparent from the following detailed description of various non-limiting embodiments when considered in conjunction with the accompanying figures. [Brief explanation of the drawings]

[0086] Brief description of the drawings The following drawings form part of the present specification and are included to further demonstrate certain aspects of the disclosure that may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0087] [Figure 1A-1] Figure 1A.1 provides a schematic of an exemplary process for introducing single nucleotide changes, insertions, and / or deletions into a DNA molecule (e.g., a genome) using a fusion protein comprising a reverse transcriptase fused to a napDNAbp (e.g., Cas9) protein complexed with a primed editor guide RNA. In this embodiment, the guide RNA is extended at its 3' end to encompass a DNA synthesis template. The schematic shows how reverse transcriptase (RT) fused to a Cas9 nickase and complexed with a guide RNA (gRNA) binds to a DNA target site and nicks the PAM-containing DNA strand adjacent to the target nucleotide. The RT template uses the nicked DNA as a primer for DNA synthesis from the gRNA, which is used as a template for synthesis of a new DNA strand encoding the desired edit. The editing process shown may be referred to as target-primed reverse transcription editing (primed editing). [Figure 1A-2]Figure 1A.2 provides the same depiction as Figure 1A.1, except that the prime editor complex is more generally represented as [napDNAbp]-[P]:PEgRNAPEgRNA or [P]-[napDNAbp]:PEgRNAPEgRNA, where "P" refers to any polymerase (e.g., reverse transcriptase), "napDNAbp" refers to a nucleic acid-programmed DNA-binding protein (e.g., SpCas9), and "PEgRNAPEgRNA" refers to the prime editing guide RNA, and " ]-[ ]" refers to the optional linker. As depicted elsewhere, e.g., in Figures 3A-3G, PEgRNAPEgRNA includes a 5' extension arm that includes a primer binding site and a DNA synthesis template. Although not shown, it is contemplated that the extension arm of PEgRNAPEgRNA (i.e., including the primer binding site and DNA synthesis template) can be DNA or RNA. The specific polymerase contemplated in this configuration will depend on the nature of the DNA synthesis template. For example, if the DNA synthesis template is RNA, the polymerase can be an RNA-dependent DNA polymerase (e.g., reverse transcriptase). If the DNA synthesis template is DNA, the polymerase can be a DNA-dependent DNA polymerase. In various embodiments, the PEG RNA can be modified or synthesized to incorporate a DNA-based DNA synthesis template.

[0088] [Figure 1B-1]Figure 1B.1 provides a schematic of an exemplary process for introducing single-nucleotide changes, insertions, and / or deletions into a DNA molecule (e.g., a genome) using a fusion protein comprising a reverse transcriptase fused to a napDNAbp (e.g., Cas9) protein complexed with a prime editor guide RNA. In this embodiment, the guide RNA is extended at its 5' end to encompass a DNA synthesis template. The schematic shows how a reverse transcriptase (RT) fused to a Cas9 nickase and complexed with a guide RNA (gRNA) binds to a DNA target site and nicks the PAM-containing DNA strand adjacent to the target nucleotide. The standard PAM sequence is 5'-NGG-3', but different PAM sequences may be associated with different Cas9 proteins or equivalent proteins from different organisms. Additionally, a given Cas9 nuclease, e.g., SpCas9, may be modified to alter the PAM specificity of the protein to recognize alternative PAM sequences. The RT enzyme uses the nicked DNA as a primer for DNA synthesis from the gRNA, which is used as a template for synthesis of a new DNA strand encoding the desired edit. The editing process described is sometimes referred to as target-primed reverse transcription editing (TPRT editing or prime editing). [Figure 1B-2]Figure 1B.2 provides the same depiction as Figure 1B.1, except that the prime editor complex is more generally represented as [napDNAbp]-[P]:PEgRNAPEgRNA or [P]-[napDNAbp]:PEgRNAPEgRNA, where "P" refers to any polymerase (e.g., reverse transcriptase), "napDNAbp" refers to a nucleic acid-programmed DNA-binding protein (e.g., SpCas9), and "PEgRNAPEgRNA" refers to the prime editing guide RNA, and " ]-[ ]" refers to the optional linker. As depicted elsewhere, e.g., in Figures 3A-3G, PEgRNAPEgRNA includes a 3' extension arm that includes a primer binding site and a DNA synthesis template. Although not shown, it is contemplated that the extension arm of PEgRNAPEgRNA (i.e., including the primer binding site and DNA synthesis template) can be DNA or RNA. The specific polymerase contemplated in this configuration will depend on the nature of the DNA synthesis template. For example, if the DNA synthesis template is RNA, the polymerase can be an RNA-dependent DNA polymerase (e.g., reverse transcriptase). If the DNA synthesis template is DNA, the polymerase can be a DNA-dependent DNA polymerase.

[0089] [Figure 1C] 1C is a schematic depicting an exemplary process of how a synthesized single strand of DNA (containing a desired nucleotide change) is degraded so that the desired nucleotide change, insertion, and / or deletion is incorporated into the DNA. As shown, subsequent synthesis of an edited strand (or "mutagenized strand"), equilibration with the endogenous strand, flap cleavage of the endogenous strand, and ligation leads to the incorporation of the DNA edit after degradation of the mismatched DNA duplex through the action of endogenous DNA repair and / or replication processes.

[0090] [Figure 1D]Figure 1D is a schematic diagram showing that "opposite strand nicking" can be incorporated into the degradation method of Figure 1C to help drive the formation of the desired product versus the restored product. In opposite strand nicking, a second napDNAbp / gRNA complex (e.g., a Cas9 / gRNA complex) is used to introduce a second nick on the strand opposite the originally nicked strand. This directs the endogenous cellular DNA repair and / or replication processes to preferentially replace the unedited strand (i.e., the strand containing the second nick site).

[0091] [Figure 1E]FIG. 1E provides another schematic diagram of an exemplary process for introducing at least one nucleotide change (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more), insertion, and / or deletion into a DNA molecule (e.g., a genome) at a target locus using a nucleic acid programmable DNA-binding protein (napDNAbp) complexed with a prime editor guide RNA (e.g., prime edit). The prime editor guide RNA includes an extension at the 3' or 5' end of the guide RNA or at an intramolecular location within the guide RNA. In step (a), the napDNAbp / gRNA complex contacts the DNA molecule, and the gRNA guides the napDNAbp to bind to the target locus. In step (b), a nick is introduced (e.g., by a nuclease or chemical agent) into one strand of DNA (the R-loop strand, or the PAM-containing strand, or the non-target DNA strand, or the protospacer strand) at the target locus, thereby creating an available 3' end on one of the strands at the target locus. In some embodiments, a nick is created in the strand of DNA corresponding to the R-loop strand, i.e., the strand that is not hybridized to the guide RNA sequence. In step (c), the 3'-terminal DNA strand interacts with the extended portion of the guide RNA to prime reverse transcription. In some embodiments, the 3'-terminal DNA strand hybridizes to a specific primer-binding site on the extended portion of the guide RNA. In step (d), a reverse transcriptase is introduced to synthesize a single strand of DNA from the 3' end of the primed site to the 3' end of the guide RNA. This forms a single-stranded DNA flap containing the desired nucleotide change (e.g., single or multiple base change(s), insertion(s), deletion(s), or a combination thereof). In step (e), the napDNAbp and guide RNA are released. Steps (f) and (g) involve degradation of the single-stranded DNA flap so that the desired nucleotide change is incorporated into the target locus. This process can drive the formation of the desired product by removing the corresponding 5′ endogenous DNA flap that is formed once the 3′ single-stranded DNA flap invades and hybridizes with the complementary sequence on the other strand.The process can also be driven to form a nicked product in the second strand, as illustrated in Figure ID. This process may introduce at least one or more of the following genetic alterations: transversions, transitions, deletions, and insertions.

[0092] [Figure 1F] 1F is a schematic diagram depicting the types of genetic changes that can occur in the target-primed reverse transcription editing (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 and / or multiple nucleotide changes, and insertions (including short and long insertions).

[0093] [Figure 1G] Figure 1G is a schematic diagram depicting an example of temporal second-strand nicking, exemplified by a primed editor complex. Temporal second-strand nicking is a variant of second-strand nicking to facilitate the formation of the desired edited product. The term "temporary" refers to the fact that second-strand nicking of the unedited strand occurs only after the desired edit has been incorporated into the edited strand. This avoids simultaneous nicking on both strands, which can lead to double-stranded DNA breaks.

[0094] [Figure 1H]Figure 1H depicts a variation of prime editing contemplated herein in which napDNAbp (e.g., SpCas9 nickase) is replaced with any programmable nuclease domain, such as a zinc finger nuclease (ZFN) or transcription activator-like effector nuclease (TALEN). Therefore, it is contemplated that a suitable nuclease need not necessarily be "programmed" by a nucleic acid target molecule (e.g., a guide RNA), but rather may be programmed by defining the specificity of the DNA-binding domain, such as a nuclease. Just as with prime editing with napDNAbp moieties, alternative programmable nucleases are preferably modified to cleave only one strand of the target DNA. In other words, the programmable nuclease should preferably function as a nickase. Once a programmable nuclease is selected (e.g., a ZFN or TALEN), additional functionality may be engineered to result in a system capable of operating according to a prime editing-like mechanism. For example, a programmable nuclease may be modified by coupling it to an RNA or DNA extension arm (e.g., via a chemical linker), where the extension arm includes a primer binding site (PBS) and a DNA synthesis template. A programmable nuclease may also be coupled to a polymerase (e.g., via a chemical or amino acid linker), although the nature of the polymerase will depend on whether the extension arm is DNA or RNA. In the case of an RNA extension arm, the polymerase may be an RNA-dependent DNA polymerase (e.g., reverse transcriptase). In the case of a DNA extension arm, the polymerase may be a DNA-dependent DNA polymerase (e.g., a prokaryotic polymerase including Pol I, Pol II, or Pol III, or a eukaryotic polymerase including Pol a, Pol b, Pol g, Pol d, Pol e, or Pol z).The system may also include other functions added as fusions with the programmable nuclease or added in trans to facilitate the overall reaction (e.g., (a) a helicase that unwinds the DNA at the cut site, creating a cut strand with a 3' end that can be used as a primer; (b) FEN1, which removes the endogenous strand on the cut strand, helping to drive the reaction toward replacement of the endogenous strand with the synthesized strand; or (c) the nCas9:gRNA complex that creates a second-site nick on the opposite strand (which may also help drive incorporation of synthetic repair over preferred cellular repair of the non-edited strand). In a manner similar to priming editing with napDNAbp, such complexes with other programmable nucleases can be used to synthesize and then permanently incorporate a newly synthesized replacement strand of DNA bearing the edit of interest into a target site in DNA.

[0095] [Figure 1I]Figure 1I depicts anatomical features of target DNA that may be edited by prime editing in one embodiment. The target DNA includes a "non-target strand" and a "target strand." The target strand is the strand that becomes annealed with the spacer of the PE gRNA of the prime editor complex that recognizes the PAM site (in this case, NGG, recognized by standard SpCas9-based prime editors). The target strand may also be referred to as the "non-PAM strand" or "non-edited strand." In contrast, the non-target strand (i.e., the strand containing the protospacer and NGG PAM sequence) may also be referred to as the "PAM strand" or "edited strand." In various embodiments, the nick site of the PE complex (e.g., in SpCas9-based PE) will be in the protospacer on the PAM strand. The location of the nick will be a characteristic of the specific Cas9 forming the PE. For example, in the case of SpCas9-based PE, the nick site is located in the phosphodiester bond between bases 3 (position "-3" relative to position 1 of the PAM sequence) and 4 (position "-4" relative to position 1 of the PAM sequence). The nick site in the protospacer forms a free 3' hydroxyl group that complexes with the primer-binding site of the extension arm of the PE-gRNA, as shown in the diagram below, providing a substrate for initiating polymerization of a single strand of DNA encoding the DNA synthesis template for the extension arm of the PE-gRNA. This polymerization reaction is catalyzed by the polymerase (e.g., reverse transcriptase) of the PE fusion protein in the 5'→3' direction. Polymerization terminates (e.g., by the inclusion of a polymerization termination signal or secondary structure that functions to terminate PE polymerization activity) before reaching the gRNA core, producing a single-stranded DNA flap that is extended from the original 3' hydroxyl group of the nicked PAM strand. The DNA synthesis template encodes a single-stranded DNA that is homologous to the 5'-terminal single strand of the endogenous DNA immediately following the nick site on the PAM strand, and incorporates the desired nucleotide change (e.g., single-base substitution, insertion, deletion, inversion).The desired editing position can be at any position downstream of the nick site on the PAM strand, including positions +1, +2, +3, +4 (start of the PAM site), +5 (position 2 of the PAM site), +6 (position 3 of the PAM site), +7, +8, +9, +10, +11, +12, +13, +14, +15, +16, +17, +18, +19, +20, +21, +22, +23, +24, +25, +26, +27, +28, +29, +30, +31, +32, +33, +34, +35, +36, +37, +38, +39, +40, +41, +42, +43, +44, +45, +46, +47, +48, ​​+49, +50, +51, +52, +53, +54, +55, +56, +57, +58, +59, +60, +61, +62, +63, +64, +65, +66, +67, +68, +69, +70, +71, +72, +73, +74, +75, +76, +77, +78, +79, +80, +81, +82, +83, +84, +85, +86, +87, +88, +89, +90, +91, +92, +93, +94, +95, +96, +97, +98, +99, +100, +101, +102, +103, +104, +105, +106, +107, +108, +109, +110, +111, +112, +113, +114, + 20, +21, +22, +23, +24, +25, +26, +27, +28, +29, +30, +31, +32, +33, +34, +35, +36, +37, +38, +39, +40, +41, +42, +43, +44, +45, +46, +47, +48, ​​+49, +50, +51, +52, +53, +54, +55 , +56, +57, +58, +59, +60, +61, +62, +63, +64, +65, +66, +67, +68, +69, +70, +71, +72, +73, +74, +75, +76, +77, +78, +79, +80, +81, +82, +83, +84, +85, +86, +87, +88, +89, +90, +91, +92, +93, +94, +95, +96, +97, +98, +99, +100, +101, +102, +103, +104, +105, +106, +107, +108, +109, +110, +111, +112, +113, +114, +115, +116, +117, +118, +119, +120, + The nick site may include 121, +122, +123, +124, +125, +126, +127, +128, +129, +130, +131, +132, +133, +134, +135, +136, +137, +138, +139, +140, +141, +142, +143, +144, +145, +146, +147, +148, +149, or +150 or more (relative to the downstream position of the nick site). Once the 3'-terminal single-stranded DNA (containing the target edit) replaces the endogenous 5'-terminal single-stranded DNA, DNA repair and replication processes will result in permanent incorporation of the edited site on the PAM strand and then correction of the mismatch on the non-PAM strand present at the edit site. In this way, the edit will spread to both strands of DNA at the target DNA site. It will be understood that references to "edited strand" and "non-edited" strand are merely intended to delineate the strand of DNA involved in the PE mechanism.The "edited strand" is the strand that first becomes edited by replacing the 5'-single-stranded DNA immediately downstream of the nick site with a synthesized 3'-single-stranded DNA containing the desired edit. The "non-edited" strand, which is the counterpart to the edited strand, also becomes edited (particularly with the edit of interest) through repair and / or replication to become complementary to the edited strand.

[0096] [Figure 1J]Figure 1J depicts the mechanism of prime editing, showing the anatomical features of the target DNA, the prime editor complex, and the interaction between the PEgRNA and the target DNA. First, a prime editor, including a fusion protein with a polymerase (e.g., reverse transcriptase) and a napDNAbp (e.g., SpCas9 nickase, e.g., SpCas9 with an inactivating mutation in the HNH nuclease domain (e.g., H840A) or an activating mutation in the RuvC nuclease domain (D10A)), is complexed with a PEgRNA and a DNA containing the target DNA to be edited. The PEgRNA includes a spacer, a gRNA core (also known as the gRNA backbone or gRNA backbone), which binds to the napDNAbp, and an extension arm. The extension arm can be at the 3' end, 5' end, or anywhere within the PEgRNA molecule. As shown, the extension arm is at the 3' end of the PEgRNA. The extender arm contains a primer binding site and a DNA synthesis template (containing both the edit of interest and a homologous region (i.e., the homologous arm)) that is homologous in the 3' to 5' direction to the single-stranded DNA at the 5' end immediately following the nick site on the PAM strand. As shown, once a nick is introduced, thereby generating a free 3' hydroxyl group immediately upstream of the nick site, the region immediately upstream of the nick site on the PAM strand anneals to a complementary sequence at the 3' end of the extender arm, termed the "primer binding site," creating a short double-stranded region with an available 3' hydroxyl end, which forms a substrate for the polymerase of the primed editor complex. The polymerase (e.g., reverse transcriptase) then polymerizes a strand of DNA from the 3' hydroxyl end to the end of the extender arm. The sequence of the single-stranded DNA is encoded by the DNA synthesis template, and it is the portion of the extender arm (i.e., excluding the primer binding site) that is "read" by the polymerase to synthesize new DNA. This polymerization effectively extends to the sequence originally 3' hydroxyl-terminus of the initial nick site. The DNA synthesis template encodes a single strand of DNA containing not only the desired edit, but also a region of homology to the single strand of endogenous DNA immediately downstream of the nick site on the PAM strand.The encoded 3'-terminal single-stranded DNA (i.e., the 3'-single-stranded DNA flap) then displaces the corresponding homologous endogenous 5'-terminal single-stranded DNA just downstream of the nick site on the PAM strand, forming a DNA intermediate with a 5'-terminal single-stranded DNA flap, which is removed by the cell (e.g., by a flap endonuclease). The 3'-terminal single-stranded DNA flap that anneals to the complement of the endogenous 5'-terminal single-stranded DNA flap is ligated to the endogenous strand after the 5'-DNA flap is removed. The desired edit in the 3'-terminal single-stranded DNA flap, which just annealed and ligated, forms a mismatch with the complementary strand and undergoes DNA repair and / or rounds of replication, thereby permanently incorporating the desired edit on both strands.

[0097] [Figure 2] Figure 2 shows the three Cas complexes that will be tested, along with their PAM, gRNA, and DNA cleavage features. The figure shows schematic diagrams for complexes involving SpCas9, SaCas9, and LbCas12a.

[0098] [Figure 3A] Figures 3A-3C show schematic diagrams for modified 5'-extended gRNAs (Figure 3A), 3'-extended gRNAs (Figure 3B), and intramolecularly extended gRNAs (Figure 3C), each of which may be used for prime editing. The embodiments depict exemplary arrangements of the DNA synthesis template, primer binding site, and optional linker sequence, as well as the arrangement of the protospacer and core regions, in the extended portions of the 3', 5', and intramolecularly extended gRNAs. The disclosed TPRT process is not limited to these configurations of the prime editor guide RNA. [Figure 3B-C]Figures 3B and 3C show schematic diagrams for modified 3'-extended gRNAs (Figure 3B) and intramolecular extensions (Figure 3C), each of which may be used for prime editing. The embodiments depict exemplary arrangements of the DNA synthesis template, primer binding site, and optional linker sequence, as well as the arrangement of the protospacer and core regions, in the extended portions of the 3', 5', and intramolecularly extended gRNAs. The disclosed TPRT process is not limited to these configurations of the prime editor guide RNA.

[0099] [Figure 4A] Figures 4A-4E demonstrate an in vitro TPRT assay. Figure 4A is a schematic diagram of a fluorescently labeled DNA substrate, extension of a gRNA template by the RT enzyme, and polyacrylamide gel electrophoresis (PAGE) assay of the reverse transcriptase product. [Figure 4B-C] Figure 4B shows TPRT with pre-nicked substrates, dCas9, and 5'-extended gRNAs of different editing template lengths. Figure 4C shows RT reactions with pre-nicked DNA substrates in the absence of Cas9. [Figure 4D-E] Figure 4D shows TPRT on a full-length dsDNA substrate with Cas9(H840A) and a 5'-extended gRNA. Figure 4E shows a 3'-extended gRNA template with a pre-nicked full-length dsDNA substrate. M-MLV RT is present in all reactions.

[0100] [Figure 5]Figure 5 shows in vitro validation results using 5'-extended gRNAs with varying editing template lengths. A fluorescently labeled (Cy5) DNA target was used as the substrate and was pre-nicked in this set of experiments. The Cas9 used in these experiments was catalytically inactive Cas9 (dCas9), and the RT used was Superscript III, a commercially available RT derived from Moloney murine leukemia virus (M-MLV). The dCas9:gRNA complex was formed from purified components. The fluorescently labeled DNA substrate was then added along with dNTPs and the RT enzyme. After a 1-hour incubation at 37°C, the reaction products were analyzed by denaturing urea-polyacrylamide gel electrophoresis (PAGE). The gel image shows an extension of the original DNA strand to a length consistent with the reverse transcription template length.

[0101] [Figure 6] Figure 6 shows in vitro validation results using 5'-extended gRNAs with varying editing template lengths, similar to those shown in Figure 5. However, the DNA substrate was not pre-nicked in this set of experiments. The Cas9 used in these experiments was Cas9 nickase (SpyCas9 H840A mutant), and the RT used was Superscript III, a commercially available RT derived from Moloney murine leukemia virus (M-MLV). Reaction products were analyzed by denaturing urea-polyacrylamide gel electrophoresis (PAGE). As shown on the gel, the nickase efficiently cleaves the DNA strand when gRNA is used (gRNA_0, lane 3).

[0102] [Figure 7]Figure 7 demonstrates that 3' extension supports DNA synthesis and does not significantly affect Cas9 nickase activity. Pre-nicked substrates (black arrows) are nearly quantitatively converted to RT products when either dCas9 or Cas9 nickase is used (lanes 4 and 5). Greater than 50% conversion to RT products (red arrows) is observed for the full-length substrate (lane 3). Cas9 nickase (SpyCas9 H840A mutant), catalytically inactive Cas9 (dCas9), and Superscript III (a commercially available RT derived from Moloney murine leukemia virus (M-MLV)) were used.

[0103] [Figure 8] Figure 8 demonstrates a dual-color experiment used to determine whether the RT reaction occurs preferentially in cis with the gRNA (bound in the same complex). Two separate experiments were performed for the 5'-extended gRNA and the 3'-extended gRNA. Products were analyzed by PAGE. Product ratios were calculated as (Cy3cis / Cy3trans) / (Cy5trans / Cy5cis).

[0104] [Figure 9A-B] Figures 9A-9D demonstrate the flap model substrate. Figure 9A shows a dual FP reporter for flap-directed mutagenesis. Figure 9B shows stop codon repair in HEK cells. [Figure 9C-D] Figure 9C shows yeast clones sequenced after flap repair, and Figure 9D shows examination of different flap features in human cells.

[0105] [Figure 10]Figure 10 demonstrates prime editing on a plasmid substrate. A dual-fluorescent reporter plasmid was constructed for yeast (S. cerevisiae) expression. Expression of this construct in yeast produces only GFP. An in vitro TRT reaction induces point mutations, and the parental plasmid or a plasmid in vitro nicked with Cas9(H840A) is transformed into yeast. Colonies are visualized by fluorescent imaging. Yeast dual-FP plasmid transformants are shown. Transformation of the parental plasmid or a plasmid in vitro nicked with Cas9(H840A) results in only green GFP-expressing colonies. TRT reactions with 5'- or 3'-extended gRNAs produce mixed green and yellow colonies, the latter expressing both GFP and mCherry. More yellow colonies are observed with the 3'-extended gRNA. A positive control containing no stop codon is also not shown.

[0106] [Figure 11] Figure 11 shows prime editing on a plasmid substrate similar to the experiment in Figure 10, but instead of incorporating a point mutation in the stop codon, the prime edit incorporates a single nucleotide insertion (left) or deletion (right) that repairs the frameshift mutation and allows synthesis of mCherry downstream. Both experiments used 3'-extended gRNAs.

[0107] [Figure 12] Figure 12 shows the editing products of prime editing on the plasmid substrate, characterized by Sanger sequencing. Colonies from the TRT transformation were individually selected and analyzed by Sanger sequencing. Correct editing was observed by sequencing selected colonies. Green colonies contained the plasmid with the original DNA sequence, while yellow colonies contained the correct mutation designed by the prime-editing gRNA. No other point mutations or indels were observed.

[0108] [Figure 13]Figure 13 illustrates the potential scope of the new prime-editing technology and provides a comparison with deaminase-mediated base editing techniques.

[0109] [Figure 14] FIG. 14 shows a schematic diagram of editing in human cells.

[0110] [Figure 15] Figure 15 demonstrates the extension of the primer binding site in the gRNA.

[0111] [Figure 16] Figure 16 shows gRNAs truncated for adjacent targeting.

[0112] [Figures 17A-C] Figures 17A-17C are graphs displaying % T→A conversion at the target nucleotide after transfection of the constructs in human embryonic kidney (HEK) cells. Figure 17A shows data presenting the results using an N-terminal fusion of wild-type MLV reverse transcriptase to the Cas9 (H840A) nickase (32 amino acid linker). Figure 17B is similar to Figure 17A except for the C-terminal fusion of the RT enzyme. Figure 17C is similar to Figure 17A, except that the linker between the MLV RT and Cas9 is 60 amino acids long instead of 32 amino acids.

[0113] [Figure 18] Figure 18 shows high-purity T→A editing at the HEK3 site by high-throughput amplicon sequencing. The sequencing analysis output displays the most abundant genotypes in edited cells.

[0114] [Figure 19] Figure 19 shows the editing efficiency at the target nucleotide (left bar of each pair of bars) alongside the indel rate (right bar of each pair of bars). WT refers to the wild-type MLV RT enzyme. Mutant enzymes (M1-M4) contain the mutations listed to the right. Editing rates were quantified by high-throughput sequencing of genomic DNA amplicons.

[0115] [Figure 20] Figure 20 shows the editing efficiency of a target nucleotide when a single-stranded nick is introduced into the complementary DNA strand adjacent to the target nucleotide. Nicking at various distances from the target nucleotide was tested (orange triangle). The editing efficiency at the target base pair (blue bar) is shown alongside the indel formation rate (orange bar). The "none" example does not contain a guide RNA that nicks the complementary strand. The editing rate was quantified by high-throughput sequencing of genomic DNA amplicons.

[0116] [Figure 21] Figure 21 demonstrates the processed high-throughput sequencing data showing the desired T→A transversion mutation and the general absence of other major genome editing by-products.

[0117] [Figure 22]Figure 22 provides a schematic diagram of an exemplary process for performing targeted mutagenesis at a target locus using a nucleic acid-programmed DNA-binding protein (napDNAbp) complexed with a prime editor guide RNA and an error-prone reverse transcriptase. This process is sometimes referred to as a prime editing embodiment for targeted mutagenesis. The prime editor guide RNA involves extension at the 3' or 5' end of the guide RNA, or at an intramolecular location within the guide RNA. In step (a), the napDNAbp / gRNA complex contacts a DNA molecule, and the gRNA guides the napDNAbp to bind to the target locus to be mutagenized. In step (b), a nick is introduced (e.g., by a nuclease or chemical agent) into one strand of DNA at the target locus, thereby creating an available 3' end on one strand of the target locus. In one embodiment, the nick is created in the strand of DNA corresponding to the R-loop strand, i.e., the strand that is not hybridized to the guide RNA sequence. In step (c), the 3'-terminal DNA strand interacts with the extended portion of the guide RNA to prime reverse transcription. In some embodiments, the 3'-terminal DNA strand hybridizes to a specific primer-binding site on the extended portion of the guide RNA. In step (d), an error-prone reverse transcriptase is introduced, which synthesizes a single strand of mutagenized DNA from the 3' end of the primed site to the 3' end of the guide RNA. Exemplary mutations are indicated with an asterisk (*). This forms a single-stranded DNA flap containing the desired mutagenized region. In step (e), the napDNAbp and guide RNA are released. Steps (f) and (g) involve degradation of the single-stranded DNA flap (containing the mutagenized region) so that the desired mutagenized region is integrated into the target locus. This process can be driven to the desired product formation by removing the corresponding 5' endogenous DNA flap formed once the 3'-terminal single-stranded DNA flap invades and hybridizes with a complementary sequence on the other strand. The process can also be driven to form a second-strand nicked product, as illustrated in Figure ID.Following endogenous DNA repair and / or replication processes, the mutagenic region becomes incorporated into both strands of DNA at the DNA locus.

[0118] [Figure 23] Figure 23 shows a schematic diagram of gRNA design for reducing trinucleotide repeat sequences and trinucleotide repeat reduction with TPRT genome editing. Trinucleotide repeat expansions are associated with a number of human diseases, including Huntington's disease, fragile X syndrome, and Friedreich's ataxia. The most common trinucleotide repeat contains a CAG triplet, but GAA triplets (Friedreich's ataxia) and CGG triplets (Fragile X syndrome) also occur. Inheriting a predisposition to expansion or acquiring an already expanded parental allele increases the likelihood of acquiring disease. Pathogenic trinucleotide repeat expansions could hypothetically be corrected using prime editing. A region upstream of the repeat region could be nicked by an RNA-guided nuclease and then used to prime the synthesis of a new DNA strand containing a healthy number of repeats (depending on the specific gene and disease). The repeat sequence is followed by a short stretch of homology (red strand) that matches the identity of the sequence adjacent to the other end of the repeat. Invasion of the newly synthesized strand, followed by displacement of the endogenous DNA with the newly synthesized flap, leads to a shortened repeat allele.

[0119] [Figure 24] Figure 24 is a schematic diagram showing the precise 10-nucleotide deletion in prime editing. Guide RNAs targeting the HEK3 locus were designed with a reverse transcription template encoding a 10-nucleotide deletion after the nick site. Editing efficiency in transfected HEK cells was assessed using amplicon sequencing.

[0120] [Figure 25]Figure 25 is a schematic diagram showing gRNA design for peptide tagging of genes at endogenous genomic loci and peptide tagging in TPRT genome editing. The FlAsH and ReAsH tagging systems comprise two parts: (1) a fluorophore-biarsenical probe, and (2) a genetically encoded peptide containing a tetracysteine ​​motif, as exemplified by the sequence FLNCCPGCCMEP (SEQ ID NO: 1361586). When expressed in cells, tetracysteine ​​motif-containing proteins can be fluorescently labeled with the fluorophore-arsenical probe (see reference: J. Am. Chem. Soc., 2002, 124 (21), pp. 6063-6076. DOI: 10.1021 / ja017687n). The "sortagging" system employs bacterial sortase enzymes to covalently conjugate labeled peptide probes to proteins containing suitable peptide substrates (see reference: Nat. Chem. Biol. 2007 Nov;3(11):707-8. DOI: 10.1038 / nchembio.2007.31). FLAG tags (DYKDDDDK (SEQ ID NO: 1361587)), V5 tags (GKPIPNPLLGLDST (SEQ ID NO: 1361588)), GCN4 tags (EELLSKNYHLENEVARLKK (SEQ ID NO: 1361589)), HA tags (YPYDVPDYA (SEQ ID NO: 1361590)), and Myc tags (EQKLISEEDL (SEQ ID NO: 1361591)) are commonly employed as epitope tags for immunoassays. Pi-clamp encodes a peptide sequence (FCPF (SEQ ID NO: 1361592)) that can be labeled with pentafluoro-aromatic substrates (Reference: Nat. Chem. 2016 Feb;8(2):120-8. doi:10.1038 / nchem.2413).

[0121] [Figure 26]Figure 26 shows the precise incorporation of His6 and FLAG tags into genomic DNA. Guide RNAs targeting the HEK3 locus were designed with reverse transcription templates encoding either an 18nt His tag insertion or a 24nt FLAG tag insertion. Editing efficiency in transfected HEK cells was assessed using amplicon sequencing. Note that the full 24nt sequence of the FLAG tag is truncated (sequencing confirmed the full length and precise insertion).

[0122] [Figure 27]Figure 27 provides the structure of one embodiment of a PEgRNA that may be designed according to the methodology contemplated herein and defined in Example 2. The PEgRNA comprises three major components ordered in the 5' to 3' direction: a spacer, a gRNA core, and an extension arm at the 3' end. The extension arm may be further divided into the following structural elements in the 5' to 3' direction: a primer binding site (A), an editing template (B), and a homology arm (C). In addition, the PEgRNA may comprise an optional 3'-end modification region (e1) and an optional 5'-end modification region (e2). Furthermore, the PEgRNA may also comprise a transcription termination signal at the 3' end of the PEgRNA (not depicted). These structural elements are further defined herein. The depiction of the PEgRNA structure is not intended to be limiting and encompasses variations in the arrangement of elements. For example, any sequence modifiers (e1) and (e2) can be positioned within or between any of the other regions shown and are not limited to being positioned at the 3' and 5' ends. The PEgRNA shown in Figure 27 can be designed by the algorithm disclosed herein. In certain embodiments, the PEgRNA PEgRNA can include secondary RNA structures such as, but not limited to, hairpins, stem / loops, toe-loops, and RNA-binding protein recruitment domains (e.g., the MS2 aptamer, which recruits and binds to the MS2cp protein). Illustratively, such secondary structures can be located within the spacer, gRNA core, or extension arms, particularly within the modification regions of e1 and / or e2. In addition to secondary RNA structures, the PEgRNA PEgRNA can include chemical linkers, or poly(N) linkers or tails (where "N" can be any nucleobase) (e.g., within the modification regions of e1 and / or e2). In some embodiments (e.g., as shown in Figure 72(c)), the chemical linker may function to prevent reverse transcription of the sgRNA backbone or core.Additionally, in certain embodiments (see, e.g., Figure 72(c)), extension arm (3) may be composed of RNA or DNA and / or may include one or more nucleobase analogs (which may, e.g., confer functionality such as temperature resilience). Furthermore, the orientation of extension arm (3) may be the natural 5'→3' orientation, or may be synthesized in a reverse 3'→5' orientation (relative to the orientation of the entire PEgRNAPEgRNA molecule). It is also noted that one skilled in the art will be able to select an appropriate DNA polymerase for use in prime editing depending on the nature of the nucleic acid material of the extension arm (i.e., DNA or RNA). Prime editing here may be practiced either as a fusion with napDNAbp or provided in trans as a separate moiety to synthesize a desired template-encoded 3' single-stranded DNA flap encompassing the desired edit. For example, if the extension arm is RNA, the DNA polymerase may be a reverse transcriptase or any other suitable RNA-dependent DNA polymerase. However, if the extension arm is DNA, the DNA polymerase can be a DNA-dependent DNA polymerase. In various embodiments, the DNA polymerase can be provided in trans, for example, through the use of an RNA-protein recruitment domain (for example, an MS2 hairpin incorporated onto the PEgRNAPEgRNA (for example, in the e1 or e2 region, or elsewhere)) and the use of an MS2cp protein fused to the DNA polymerase (thereby co-localizing the DNA polymerase with the PEgRNAPEgRNA). Note also that the primer binding site generally does not form part of the template used by the DNA polymerase (for example, reverse transcriptase) (encoding the resulting 3' single-stranded DNA flap (containing the desired edit)). Thus, the label "DNA synthesis template" refers to the region or portion of the extension arm (3) used by the DNA polymerase as a template (encoding the desired 3' single-stranded DNA flap containing the edit). In some embodiments, the DNA synthesis template includes an "editing template" and a "homology arm."In other embodiments, the DNA synthesis template may also include the e2 region or a portion thereof. By way of illustration, if the e2 region contains a secondary structure that causes termination of DNA polymerase activity, it is possible that DNA polymerase function will be 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, will be encoded into the DNA. How much of the e2 region is actually used as a template will depend on its configuration and whether that configuration interferes with DNA polymerase function.

[0123] [Figure 28]Figure 28 provides the structure of another embodiment of a PEgRNA that may be constructed according to the methodology contemplated herein and defined in Example 2. The PEgRNA comprises three major components ordered in the 5' to 3' direction: a spacer, a gRNA core, and an extension arm at the 3' end. The extension arm may be further divided into the following structural elements in the 5' to 3' direction: a primer binding site (A), an editing template (B), and a homology arm (C). In addition, the PEgRNA may comprise an optional 3'-end modification region (e1) and an optional 5'-end modification region (e2). Furthermore, the PEgRNA may comprise a transcription termination signal on the 3' end of the PEgRNA (not depicted). These structural elements are further defined herein. The depiction of the PEgRNA structure is not intended to be limiting and encompasses variations in the arrangement of elements. For example, any sequence modifiers (e1) and (e2) can be located within or between any of the other regions shown and are not limited to being located at the 3' and 5' ends. In certain embodiments, a PEgRNA PEgRNA can include secondary RNA structures, such as, but not limited to, a hairpin, a stem / loop, a toe-loop, or an RNA-binding protein recruitment domain (e.g., an MS2 aptamer that recruits and binds to the MS2cp protein). These secondary structures can be located anywhere within the PEgRNA PEgRNA molecule. Illustratively, such secondary structures can be located within the spacer, the gRNA core, or the extension arm, particularly within the modification regions of e1 and / or e2. In addition to secondary RNA structures, a PEgRNA PEgRNA can include a chemical linker, or a poly(N) linker or tail (where "N" can be any nucleobase) (e.g., within the modification regions of e1 and / or e2). In some embodiments (e.g., as shown in Figure 27), the chemical linker may function to prevent reverse transcription of the sgRNA backbone or core. Additionally, in certain embodiments (see, e.g., Figure 28), the extension arm (3) may be composed of RNA or DNA and / or may include one or more nucleobase analogs (e.g., which may provide added functionality such as temperature resistance).Furthermore, the orientation of the extension arm (3) can be the natural 5'→3' direction, or it can be synthesized in the reverse 3'→5' direction (relative to the orientation of the entire PEgRNAPEgRNA molecule). It should also be noted that one skilled in the art would be able to select an appropriate DNA polymerase for use in prime editing depending on the nature of the nucleic acid material of the extension arm (i.e., DNA or RNA). Prime editing may be performed here either as a fusion with napDNAbp or provided in trans as a separate moiety to synthesize a 3' single-stranded DNA flap encoded in the desired template that encompasses the desired edit. For example, if the extension arm is RNA, the DNA polymerase can be a reverse transcriptase or any other suitable RNA-dependent DNA polymerase. However, if the extension arm is DNA, the DNA polymerase can be a DNA-dependent DNA polymerase. In various embodiments, the DNA polymerase can be provided in trans, for example, through the use of an RNA-protein recruitment domain (for example, an MS2 hairpin incorporated onto the PEgRNAPEgRNA (for example, in the e1 or e2 region, or elsewhere)) and the use of an MS2cp protein fused to the DNA polymerase (thereby co-localizing the DNA polymerase with the PEgRNAPEgRNA). Note also that the primer binding site generally does not form part of the template used by the DNA polymerase (for example, reverse transcriptase) (encoding the resulting 3' single-stranded DNA flap (containing the desired edit)). Thus, the label "DNA synthesis template" refers to the region or portion of the extension arm (3) used by the DNA polymerase as a template (encoding the desired 3' single-stranded DNA flap containing the edit). In some embodiments, the DNA synthesis template includes an "editing template" and a "homology arm." In other embodiments, the DNA synthesis template may also include the e2 region or a portion thereof. For example, if the e2 region contains a secondary structure that causes termination of DNA polymerase activity, it is possible that DNA polymerase function will be terminated before any part of the e2 region is actually encoded into the DNA.It is also possible that some portion, or even all, of the e2 region will be encoded into the DNA. How much of e2 is actually used as a template will depend on its organization and whether it interferes with DNA polymerase function.

[0124] [Figure 29]Figure 29 is a schematic depicting the interaction of an exemplary PEG RNA with a target site in double-stranded DNA and the concomitant production of a 3' single-stranded DNA flap containing the genetic alteration of interest. The double-stranded DNA is shown with the top strand (i.e., the target strand) in a 3' to 5' orientation and the bottom strand (i.e., the PAM strand or non-target strand) in a 5' to 3' orientation. 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 targeted by and anneals to the spacer of the PEG RNA. The complementary bottom strand contains the PAM sequence (e.g., NGG) and the protospacer and is therefore referred to as the "non-target strand" or "PAM strand" or "protospacer strand." Although not shown, the depicted PEG RNA is complexed with Cas9 or the equivalent domain of a prime editor fusion protein. As shown in the schematic, the spacer of the PEgRNA anneals to the complementary region of the protospacer on the target strand, referred to as the protospacer, located just downstream of the PAM sequence and approximately 20 nucleotides in length. This interaction forms a DNA / RNA hybrid between the spacer RNA and the complement of the protospacer DNA, inducing the formation of an R-loop in the region opposite the protospacer. As taught elsewhere herein, the Cas9 protein (not shown) then induces a nick in the non-target strand, as shown. This then leads to the formation of a 3' ssDNA flap region immediately upstream of the nick site, which interacts with the 3' end of the PEgRNAat primer binding site according to *z*. The 3' end of the ssDNA flap (i.e., the reverse transcriptase primer sequence) anneals to the primer binding site (A) on the PEgRNA, thereby priming the reverse transcriptase. Next, a reverse transcriptase (e.g., provided in trans or provided in cis as a fusion protein and attached to the Cas9 construct) then polymerizes the single strand of DNA encoded by the DNA synthesis template (including the editing 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, displacing the corresponding endogenous strand (which is removed as a 5' DNA flap from the endogenous DNA), as described elsewhere (e.g., as shown in Figure 1E), and incorporating the desired nucleotide edit (single nucleotide base pair change, deletion, or insertion (including the entire genome) through naturally occurring DNA repair / replication processes.

[0125] [Figure 30-1] Figure 30 aids in understanding the disclosed PEGRNAs in the Sequence Listing. The figure shows two exemplary PEGRNA sequences (SEQ ID NO:135529 (top row) and SEQ ID NO:135880 (bottom row)) and how various disclosed sequence subsets map onto them. For SEQ ID NO:135529, the corresponding sequences are: spacer (SEQ ID NO:271043), extension arm (SEQ ID NO:406557), primer binding site (SEQ ID NO:542071), editing template (SEQ ID NO:677585), and homology arm (SEQ ID NO:813099). For SEQ ID NO:135880, the corresponding sequences are: spacer (SEQ ID NO:880463), extension arm (SEQ ID NO:947841), primer binding site (SEQ ID NO:1015219), editing template (SEQ ID NO:1082597), and homology arm (SEQ ID NO:1149975). [Figure 30-2] Figure 30 aids in understanding the disclosed PEGRNAs in the sequence listing. The figure shows how the various disclosed sequence subsets are mapped onto it. For SEQ ID NO: 135529, the corresponding sequences are the spacer (SEQ ID NO: 271043), extension arm (SEQ ID NO: 406557), primer binding site (SEQ ID NO: 542071), editing template (SEQ ID NO: 677585), and homology arm (SEQ ID NO: 813099). For SEQ ID NO: 135880, the corresponding sequences are the spacer (SEQ ID NO: 880463), extension arm (SEQ ID NO: 947841), primer binding site (SEQ ID NO: 1015219), editing template (SEQ ID NO: 1082597), and homology arm (SEQ ID NO: 1149975).

[0126] [Figure 31] Figure 31 is a flowchart illustrating an exemplary high-level computational method 3100 for determining an extended gRNA structure according to some embodiments of the present disclosure. At step 3102, a computational device (e.g., computational device 3400 described in conjunction with Figure 34) accesses data indicating an input allele, an output allele, and a fusion protein comprising a nucleic acid-programmable DNA-binding protein and a reverse transcriptase. While step 3102 describes accessing all three of the input allele, output allele, and fusion protein in one step, this is for illustrative purposes only, and it should be understood that such data may be accessed using one or more steps without departing from the spirit of the techniques described herein. Accessing the data may include receiving data, storing data, accessing a database, and / or the like.

[0127] [Figure 32] Figure 32 is a flow chart illustrating an exemplary computational method 3200 for determining components of an extended gRNA structure (including extension components) according to some embodiments. It should be understood that Figure 32 is intended for illustration purposes, and thus the technique used to determine the extended gRNA may include more or fewer steps than those shown in Figure 32.

[0128] [Figure 33]33 is a flowchart illustrating an exemplary computational method 3300 for determining a set of extended gRNA structures for each mutation entry in a database, according to some embodiments. In step 3302, a computational device accesses a database (e.g., the Clinvar database, accessible at www.ncbi.nlm.nih.gov / clinvar / ) containing a set of mutation entries, each of which includes an input allele representing a mutation and an output allele representing a corrected wild-type sequence.

[0129] [Figure 34] 34 is an illustrative implementation of a computer system 3400 that may be used to implement any of the techniques and aspects of the embodiments disclosed herein. The computer system 3400 may include one or more processors 3410, as well as one or more non-transitory computer-readable storage media (e.g., memory 3420 and one or more non-volatile storage media 3430), and a display 3440. The processor 3410 may control the writing of data to and reading data from the memory 3420 and non-volatile storage 3430 in any suitable manner, although aspects of the invention described herein are not limited in this respect.

[0130] [Figure 35A] FIG. 35A is a schematic diagram of PE-based insertion of a sequence encoding an RNA motif related to Example 3.

[0131] [Figure 35B] Figure 35B is a non-exhaustive list of some example motifs that can potentially be inserted, and their functions, related to Example 3.

[0132] [Figure 36]Figure 36 provides a bar graph comparing the efficiency (i.e., "% of total sequencing reads in which edits or indels were identified") of PE2, PE2-trunc, PE3, and PE3-trunc against various target sites in various cell lines. The data show that prime editors containing truncated RT variants were nearly as efficient as prime editors containing the non-truncated RT protein.

[0133] [Figure 37A] Figure 37A shows the nucleotide sequence of an SpCas9 PEGRNA molecule that terminates at the 3' end in "UGU" and does not contain a toe loop element (top row). The bottom of the figure depicts the same SpCas9 PEGRNA molecule, but further modified to contain a toe loop element with the sequence 5'-"GAAANNNNN"-3' inserted just before the "UUU" 3' end. "N" can be any nucleobase.

[0134] [Figure 37B] Figure 37B shows the results of Example 4 demonstrating that the efficiency of prime editing in HEK or EMX cells is increased using toe-loop element-containing PEGRNAs, while the percentage of indel formation does not change significantly.

[0135] [Figure 38] Figure 38 depicts one embodiment of a prime editor provided as halves of two PE proteins that regenerate the entire prime editor through the self-splicing action of split-intein halves located at the end or beginning of each half protein of the prime editor.

[0136] [Figure 39A]Figure 39 depicts the mechanism of intein removal from a polypeptide sequence and the reformation of a peptide bond between the N-terminal and C-terminal extein sequences. (a) depicts the general mechanism in which two protein halves (each containing half of the intein sequence) come into contact within a cell, resulting in a fully functional intein, which 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"), forming a single, integrated polypeptide comprising the N-extein and C-extein portions. In various embodiments, the N-extein may correspond to the N-terminal half of a split-prime editor fusion protein, and the C-extein may correspond to the C-terminal half of a split-prime editor. [Figure 39B] Figure 39 depicts the mechanism of intein removal from a polypeptide sequence and the reformation of the peptide bond between the N- and C-terminal extein sequences. (b) shows the chemical dynamics of intein excision and the reformation of the peptide bond joining the N-extein half (red half) and the C-extein half (blue half). Split-intein excision (i.e., the N- and C-inteins in a split-intein configuration) is also sometimes referred to as "trans-splicing," as it involves the splicing of two separate components provided in trans.

[0137] definition antisense strand In genetics, the "antisense" strand of a segment of double-stranded DNA is considered to be the template strand, running in a 3' to 5' direction. In contrast, the "sense" strand is the 5' to 3' running segment of double-stranded DNA that is complementary to the antisense or template strand of the 3' to 5' running DNA. In the case of a DNA segment encoding a protein, the sense strand is the strand of DNA with the same sequence as the mRNA that takes the antisense strand as its template during transcription and ultimately (typically, but not always) is translated into the protein. Thus, the antisense strand carries the RNA that is subsequently translated into protein, while the sense strand possesses nearly identical makeup to the mRNA. Note that for each segment of dsDNA, there will likely be two pairs: sense and antisense (since sense and antisense are relative perspectives), depending on the direction from which one is read. The gene product or mRNA ultimately refers to either strand of a segment of dsDNA, which may be designated as sense or antisense.

[0138] Cas9 The term "Cas9" or "Cas9 nuclease" refers to an RNA-guided nuclease comprising a Cas9 domain or a fragment thereof (e.g., a protein comprising an active or inactive DNA cleavage domain of Cas9 and / or a gRNA-binding domain of Cas9). A "Cas9 domain," as used herein, is a protein fragment comprising an active or inactive cleavage domain of Cas9 and / or a gRNA-binding domain of Cas9. A "Cas9 protein" is a full-length Cas9 protein. Cas9 nuclease is also sometimes referred to as casn1 nuclease or CRISPR (clustered regularly interspaced short palindromic repeats (CRISPR)). C Lustered R regularly I Interspaced S hort P alindromic RCRISPR is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements, and conjugative plasmids). CRISPR clusters contain a spacer, a sequence complementary to the preceding mobile element, and a targeting invading nucleic acid. The CRISPR cluster is transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems, corrective processing of the pre-crRNA requires a trans-encoded small RNA (tracrRNA), an endogenous ribonuclease 3 (rnc), and a Cas9 domain. The tracrRNA acts as a guide for ribonuclease 3-aided processing of the pre-crRNA. Subsequently, the Cas9 / crRNA / tracrRNA endonucleolytically cleaves linear or circular dsDNA targets complementary to the spacer. The target strand that is not complementary to the crRNA is first cleaved endonucleolytically, followed by 3'-5' exonucleolytic excision. In nature, DNA binding and cleavage typically require both a protein and an RNA. However, single guide RNAs ("sgRNAs," or simply "gRNAs") can be engineered to incorporate aspects of both the crRNA and tracrRNA into a single RNA species. See, for example, Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna JA, Charpentier E. Science 337:816-821 (2012), the entire contents of which are incorporated herein by reference. Cas9 recognizes a short motif in the CRISPR repeat (the PAM or protospacer adjacent motif) to help distinguish self from non-self.Cas9 nuclease sequences and structures are well known to those skilled in the art (e.g., "Complete genome sequence of an M1 strain of Streptococcus pyogenes." Ferretti et al., JJ, McShan WM, Ajdic DJ, Savic DJ, Savic G., Lyon K., Primeaux C., Sezate S., Suvorov AN, Kenton S., Lai HS, Lin SP, Qian Y.,Jia HG,Najar FZ,Ren Q.,Zhu H.,Song L.,White J.,Yuan X.,Clifton SW,Roe BA,McLaughlin RE,Proc.Natl.Acad.Sci.USA98:4658-4663(2001);"CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III."Deltcheva E., Chylinski K., Sharma See CM, Gonzales K., Chao Y., Pirzada ZA, Eckert MR, Vogel J., Charpentier E., Nature 471:602-607 (2011); and "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity." Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna JA, Charpentier E. Science 337:816-821 (2012), the entire contents of each of which are incorporated herein by reference. Cas9 orthologs have been described in a variety of species, including, but not limited to, S. pyogenes and S. thermophilus. Additional suitable Cas9 nucleases and sequences will be apparent to those of skill in the art based on the present disclosure.Such Cas9 nucleases and sequences also include Cas9 sequences from the organisms and loci disclosed in Chylinski, Rhun, and Charpentier, "The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems" (2013) RNA Biology 10:5, 726-737, the entire contents of which are incorporated herein by reference. In some embodiments, the Cas9 nuclease comprises one or more mutations that partially impair or inactivate the DNA cleavage domain.

[0139] A Cas9 domain with inactivated nucleases may be interchangeably referred to as a "dCas9" protein (representing a nuclease-inactivated Cas9). Methods for generating a Cas9 domain (or a fragment thereof) with an inactive DNA cleavage domain are known (see, e.g., Jinek et al., Science. 337:816-821 (2012); Qi et al., "Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression" (2013) Cell. 28;152(5):1173-83 (the entire contents of each of which are incorporated herein by reference). For example, the DNA cleavage domain of Cas9 is known to contain 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, proteins comprising fragments of Cas9 are 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, proteins comprising Cas9 or fragments thereof are referred to as "Cas9 variants." Cas9 variants share homology with Cas9 or fragments thereof.For example, a Cas9 variant is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, at least about 99.8% identical, or at least about 99.9% identical to a wild-type Cas9 (e.g., SpCas9 of SEQ ID NO: 1361421). In some embodiments, the Cas9 variant may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more amino acid changes compared to a wild-type Cas9 (e.g., SpCas9 of SEQ ID NO: 1361421). In some embodiments, the Cas9 variant comprises a fragment of SEQ ID NO:X Cas9 (e.g., a gRNA binding domain or a DNA cleavage domain), such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of a wild-type Cas9 (e.g., SpCas9 of SEQ ID NO: 1361421). In some embodiments, the fragment is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid length of a corresponding wild-type Cas9 (e.g., SpCas9 of SEQ ID NO: 1361421).

[0140] cDNA The term "cDNA" refers to a strand of DNA copied from an RNA template. The cDNA is complementary to the RNA template.

[0141] circular permutants As used herein, the term "circular permutant" refers to a protein or polypeptide (e.g., Cas9) containing a circular permutation, which is a change in the structural configuration of a protein accompanied by a change in the order of amino acids appearing in the protein's amino acid sequence. In other words, a circular permutant is a protein with altered N- and C-termini compared to its wild-type counterpart, e.g., the C-terminal half of the wild-type protein becomes a new N-terminal half. A circular permutation (or CP) is essentially a topological rearrangement of a protein's primary sequence, with its N- and C-termini connected, often with a peptide linker, while simultaneously splitting the sequence at a different location to create new adjacent N- and C-termini. The result is a protein structure that may have different connections but often the same or an overall similar three-dimensional (3D) shape, potentially including improved or altered characteristics, including reduced susceptibility to proteolysis, improved catalytic activity, altered substrate or ligand binding, and / or improved thermostability. Circularly permuted proteins can occur naturally (e.g., concanavalin A and lectins). In addition, circular permutations can occur as a result of post-translational modifications or can be engineered using recombinant techniques.

[0142] Circularly permuted Cas9 The term "circularly permuted Cas9" refers to any Cas9 protein or variant thereof that is generated as a circular permutation, whereby its N- and C-termini are rearranged through rearrangement of the protein's primary sequence. Such circularly permuted Cas9 proteins ("CP-Cas9"), or variants thereof, retain the ability to bind to DNA when complexed with a guide RNA (gRNA). See Oakes et al., "Protein Engineering of Cas9 for enhanced function," Methods Enzymol, 2014, 546:491-511, and Oakes et al., "CRISPR-Cas9 Circular Permutants as Programmable Scaffolds for Genome Modification," Cell, January 10, 2019, 176:254-267, each of which is incorporated herein by reference. The present disclosure contemplates the use of any previously known CP-Cas9 or new CP-Cas9, so long as the resulting circularly permuted protein retains the ability to bind to DNA when complexed with a guide RNA (gRNA). Exemplary CP-Cas9 proteins are SEQ ID NOS: 1361475-1361484.

[0143] DNA synthesis template As used herein, the term "DNA synthesis template" refers to the region or portion of the extension arm of a PEgRNA that is utilized by the polymerase of a prime editor as the template strand (encoding a 3' single-stranded DNA flap that contains the desired edit and then replaces the corresponding strand of endogenous DNA at the target site through the mechanism of prime editing). In various embodiments, DNA synthesis templates are shown in Figure 3A (in the context of a PEgRNA comprising a 5' extension arm), Figure 3B (in the context of a PEgRNA comprising a 3' extension arm), Figure 3C (in the context of an internal extension arm), Figure 3D (in the context of a 3' extension arm), and Figure 3E (in the context of a 5' extension arm). The extension arm encompasses the DNA synthesis template and may be composed of DNA or RNA. In the case of RNA, the polymerase of the prime editor can be an RNA-dependent DNA polymerase (e.g., reverse transcriptase). In the case of DNA, the polymerase of the prime editor can be a DNA-dependent DNA polymerase. In various embodiments (e.g., as depicted in Figures 3D-3E), the DNA synthesis template (4) can include all or part of the "editing template" and "homology arm," as well as any 5'-terminal 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 encode none, part, or all of the e2 region. In other words, in the case of a 3' extension arm, the DNA synthesis template (3) can include a portion of the extension arm (3) extending from the 5' end of the primer binding site (PBS) to the 3' end of the gRNA core, which can serve as a template for synthesis of a single strand of DNA by a polymerase (e.g., reverse transcriptase). In the case of a 5' extension arm, the DNA synthesis template (3) can include a portion of the extension arm (3) extending from the 5' end of the PEGRNA molecule to the 3' end of the editing template. Preferably, the DNA synthesis template excludes the primer binding site (PBS) of PEGRNAs with either a 3' extension arm or a 5' extension arm.Certain embodiments described herein (e.g., Figure 71A) refer to an "RT template" that encompasses the editing template and homology arms, i.e., the sequences of the PEGRNA extension arms that are actually used as templates during DNA synthesis. The term "RT template" is equivalent to the term "DNA synthesis template."

[0144] downstream As used herein, the terms "upstream" and "downstream" are relative terms that define the linear positions of at least two elements located in a nucleic acid molecule (whether single-stranded or double-stranded) oriented in a 5' to 3' direction. In particular, if a first element is located anywhere 5' relative to the second element, the first element is upstream of the second element in the nucleic acid molecule. For example, if a SNP is located 5' to the nick site, the SNP is upstream of the Cas9-induced nick site. Conversely, if a first element is located anywhere 3' relative to the second element, the first element is downstream of the second element in the nucleic acid molecule. For example, if a SNP is located 3' to the nick site, the SNP is downstream of the Cas9-induced nick site. The nucleic acid molecule can be DNA (double-stranded or single-stranded), RNA (double-stranded or single-stranded), or a hybrid of DNA and RNA. The terms upstream and downstream refer only to a single strand of a nucleic acid molecule, except when it is necessary to select which strand of a double-stranded molecule is considered, and the analysis is the same for single-stranded nucleic acid molecules and double-stranded molecules. Often, the strand of double-stranded DNA that can be used to determine the relative positions of at least two elements is the "sense" strand or "coding" strand. In genetics, the "sense" strand is the segment within 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 located 3' from the promoter on the sense strand or coding strand, the SNP nucleobase is "downstream" of the promoter sequence in genomic DNA (which is double-stranded).

[0145] CRISPR CRISPR is a family of DNA sequences (i.e., CRISPR clusters) in bacteria and archaea that represent snippets of a previous viral infection that has invaded prokaryotes. These DNA fragments are used by prokaryotic cells to detect and destroy DNA from subsequent attacks by similar viruses, effectively constituting the prokaryotic immune defense system along with an array of CRISPR-associated proteins (including Cas9 and its homologs) and CRISPR-associated RNAs. In nature, CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In some types of CRISPR systems (e.g., type II CRISPR systems), corrective processing of the pre-crRNA requires a trans-encoded small RNA (tracrRNA), an endogenous ribonuclease 3 (rnc), and a Cas9 domain. The tracrRNA acts as a guide for ribonuclease 3-assisted processing of the pre-crRNA. Cas9 / crRNA / tracrRNA then endonucleolytically cleaves the linear or circular dsDNA target that is complementary to the RNA. Specifically, the target strand that is not complementary to the crRNA is first endonucleolytically cleaved, and then 3'-5' exonucleolytically excised. In nature, DNA binding and cleavage typically require both protein and RNA. However, single guide RNA ("sgRNA", or simply "gNRA") can be modified to incorporate both crRNA and tracrRNA aspects into a single RNA species. For example, Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna JA, Charpentier E. Science 337:816-821 (2012) (the entire contents of which are incorporated herein by reference). Cas9 helps distinguish self from non-self by recognizing a short motif in the CRISPR repeats (PAM or protospacer adjacent motif).The biology of CRISPR and the sequence and structure of Cas9 nuclease are well known to those skilled in the art (e.g., "Complete genome sequence of an M1 strain of Streptococcus pyogenes." Ferretti et al., JJ, McShan WM, Ajdic DJ, Savic DJ, Savic G., Lyon K., Primeaux C., Sezate S., Suvorov AN, Kenton S., Lai HS,Lin SP,Qian Y.,Jia HG,Najar FZ,Ren Q.,Zhu H.,Song L.,White J.,Yuan X.,Clifton SW,Roe BA,McLaughlin RE,Proc.Natl.Acad.Sci.USA98:4658-4663(2001);"CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III."Deltcheva See Jinek M., 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. Additional suitable Cas9 nucleases and sequences will be apparent to those of skill in the art based on the present disclosure.Such Cas9 nucleases and sequences also include Cas9 sequences from the organisms and loci disclosed in Chylinski, Rhun, and Charpentier, "The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems" (2013) RNA Biology 10:5, 726-737, the entire contents of which are incorporated herein by reference.

[0146] The term "Cas9" or "Cas9 nuclease" refers to an RNA-guided nuclease (e.g., a protein comprising the Cas9 DNA cleavage domain and / or the Cas9 gRNA binding domain, whether active or inactive) that comprises a Cas9 domain or a fragment thereof. A "Cas9 domain," as used herein, is a protein fragment comprising the Cas9 cleavage domain and / or the Cas9 gRNA binding domain, whether active or inactive. A "Cas9 protein" is a full-length Cas9 protein. Cas9 nuclease is also sometimes referred to as casn1 nuclease or CRISPR (clustered regularly interspaced short palindromic repeats (CRISPR)). C Lustered R regularly I Interspaced S hort P alindromic RCRISPR is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements, and conjugative plasmids). CRISPR clusters contain a spacer, a sequence complementary to the preceding mobile element, and a targeting invading nucleic acid. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems, corrective processing of the pre-crRNA requires a trans-encoded small RNA (tracrRNA), an endogenous ribonuclease 3 (rnc), and a Cas9 domain. The tracrRNA acts as a guide for ribonuclease 3-assisted processing of the pre-crRNA. Subsequently, the Cas9 / crRNA / tracrRNA endonucleolytically cleaves linear or circular dsDNA targets complementary to the spacer. The target strand that is not complementary to the crRNA is first endonucleolytically cleaved, followed by 3'-5' exonucleolytic excision. In nature, DNA binding and cleavage typically require both a protein and an RNA. However, single guide RNAs ("sgRNAs," or simply "gRNAs") can be engineered to incorporate aspects of both the crRNA and tracrRNA into a single RNA species. See, for example, Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna JA, Charpentier E. Science 337:816-821 (2012), the entire contents of which are incorporated herein by reference. Cas9 recognizes a short motif in the CRISPR repeat (the PAM or protospacer adjacent motif) to help distinguish self from non-self.The sequence and structure of Cas9 nuclease are well known to those skilled in the art (e.g., "Complete genome sequence of an M1 strain of Streptococcus pyogenes." Ferretti et al., JJ, McShan WM, Ajdic DJ, Savic DJ, Savic G., Lyon K., Primeaux C., Sezate S., Suvorov AN, Kenton S., Lai HS, Lin SP, Qian Y.,Jia HG,Najar FZ,Ren Q.,Zhu H.,Song L.,White J.,Yuan X.,Clifton SW,Roe BA,McLaughlin RE,Proc.Natl.Acad.Sci.USA98:4658-4663(2001);"CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III."Deltcheva E., Chylinski K., Sharma See CM, Gonzales K., Chao Y., Pirzada ZA, Eckert MR, Vogel J., Charpentier E., Nature 471:602-607 (2011); and "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity." Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna JA, Charpentier E. Science 337:816-821 (2012), the entire contents of each of which are incorporated herein by reference. Cas9 orthologs have been described in a variety of species, including, but not limited to, S. pyogenes and S. thermophilus. Additional suitable Cas9 nucleases and sequences will be apparent to those of skill in the art based on the present disclosure.Such Cas9 nucleases and sequences also include Cas9 sequences from the organisms and loci disclosed in Chylinski, Rhun, and Charpentier, "The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems" (2013) RNA Biology 10:5, 726-737, the entire contents of which are incorporated herein by reference. In some embodiments, the Cas9 nuclease comprises one or more mutations that partially impair or inactivate the DNA cleavage domain.

[0147] A Cas9 domain with inactivated nucleases may be interchangeably referred to as a "dCas9" protein (representing a nuclease-inactivated Cas9). Methods for generating a Cas9 domain (or a fragment thereof) with an inactive DNA cleavage domain are known (see, e.g., Jinek et al., Science. 337:816-821 (2012); Qi et al., "Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression" (2013) Cell. 28;152(5):1173-83 (the entire contents of each of which are incorporated herein by reference). For example, the DNA cleavage domain of Cas9 is known to contain 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, proteins comprising fragments of Cas9 are 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, proteins comprising Cas9 or fragments thereof are referred to as "Cas9 variants." Cas9 variants share homology with Cas9 or fragments thereof.For example, a Cas9 variant is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, at least about 99.8% identical, or at least about 99.9% identical to a wild-type Cas9 (e.g., SpCas9 of SEQ ID NO: 1361421). In some embodiments, the Cas9 variant may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more amino acid changes compared to a wild-type Cas9 (e.g., SpCas9 of SEQ ID NO: 1361421). In some embodiments, the Cas9 variant comprises a fragment of SEQ ID NO:X Cas9 (e.g., a gRNA binding domain or a DNA cleavage domain), such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of a wild-type Cas9 (e.g., SpCas9 of SEQ ID NO: 1361421). In some embodiments, the fragment is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid length of a corresponding wild-type Cas9 (e.g., SpCas9 of SEQ ID NO: 1361421).

[0148] Editing Template The term "editing template" refers to the portion of the extension arm that encodes the desired edit in the 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., Figure 71A) refer to an "RT template," which refers to both the editing template and the homologous arm together, i.e., the sequence of the PEGRNA extension arm that is actually used as a template during DNA synthesis. The term "RT editing template" is also equivalent to the term "DNA synthesis template," except that an RT editing template reflects the use of a primed editor with a polymerase that is a reverse transcriptase, while a DNA synthesis template more broadly reflects the use of a primed editor with either polymerase.

[0149] error-prone As used herein, the term "error-prone" reverse transcriptase (or, more broadly, any polymerase) refers to a reverse transcriptase (or, more broadly, any polymerase) that is naturally occurring or derived from another reverse transcriptase (e.g., wild-type M-MLV reverse transcriptase) that has an error rate less than that of wild-type M-MLV reverse transcriptase. The error rate of wild-type M-MLV reverse transcriptase has been reported to range from 15,000 (higher) to 27,000 (lower) errors per 1 error. An error rate of 1 in 15,000 is 6.7 x 10 -5 The error rate of 1 in 27,000 is 3.7 x 10 -5 See Boutabout et al. (2001) "DNA synthesis fidelity by the reverse transcriptase of the yeast retrotransposon Ty1," Nucleic Acids Res 29(11):2217-2222, which is incorporated herein by reference. Thus, for purposes of this application, the term "error-prone" refers to a rate of more than 1 error (6.7 x 10) in the incorporation of 15,000 nucleobases. -5 or higher), e.g., 1 error in 14,000 nucleobases (7.14 × 10 -5or higher), 1 error (7.7 × 10) in 13,000 nucleobases or less -5 or higher), 1 error (7.7 × 10) in 12,000 nucleobases or less -5 or higher), 1 error in 11,000 nucleobases or less (9.1 × 10 -5 or higher), 1 error (1 × 10) in 10,000 nucleobases or less -4 or 0.0001 or higher), 1 error in 9,000 nucleobases or less (0.00011 or higher), 1 error in 8,000 nucleobases or less (0.00013 or higher), 1 error in 7,000 nucleobases or less (0.00014 or higher), 1 error in 6,000 nucleobases or less (0.00016 or higher), 1 error in 5,000 nucleobases or less (0.0002 or higher), 1 error in 4,000 nucleobases or less "RTs" refer to those RTs that have an error rate that is one error in 3,000 or fewer nucleic acid bases (0.00025 or higher), one error in 3,000 or fewer nucleic acid bases (0.00033 or higher), one error in 2,000 or fewer nucleic acid bases (0.00050 or higher), or one error in 1,000 or fewer nucleic acid bases (0.001 or higher), or one error in 500 or fewer nucleic acid bases (0.002 or higher), or one error in 250 or fewer nucleic acid bases (0.004 or higher).

[0150] Extension arm The term "extension arm" refers to a nucleotide sequence component of a PEG RNA that serves several functions, including a primer binding site and an editing template for reverse transcriptase. In some embodiments, e.g., in FIG. 3D, the extension arm is located at the 3' end of the guide RNA. In other embodiments, e.g., in FIG. 3E, the extension arm is located at the 5' end of the guide RNA. In some embodiments, the extension arm also includes a homology arm. In various embodiments, the extension arm comprises the following components in a 5' to 3' direction: a homology arm, an editing template, and a primer binding site. Because the polymerization activity of reverse transcriptase is in a 5' to 3' direction, the preferred arrangement of the homology arm, editing template, and primer binding site is in a 5' to 3' direction such that the reverse transcriptase, once primed by the annealed primer sequence, polymerizes single-stranded DNA using the editing template as the complementary template strand. Further details, such as the length of the extension arm, are described elsewhere herein.

[0151] The extension arm may also be described as generally comprising two regions: a primer binding site (PBS) and a DNA synthesis template, as illustrated in Figure 3G (top row). The primer binding site binds to a primer sequence formed from the endogenous DNA strand of the target site when it becomes nicked by a prime editor complex, thereby exposing its 3' end on the nicked endogenous strand. As described herein, binding of the primer sequence to the primer binding site on the extension arm of the PEGRNA creates a double-stranded region with an exposed 3' end (i.e., 3' of the primer sequence), which then provides a substrate for a polymerase that begins polymerizing single strands of DNA from the exposed 3' end along the length of the DNA synthesis template. The sequence of the single-stranded DNA product is the complement of the DNA synthesis template. Polymerization continues 5' of the DNA synthesis template (or extension arm) until polymerization terminates. Thus, the DNA synthesis template represents the portion of the extension arm that is encoded by the polymerase of the primed editor complex into a single-stranded DNA product (i.e., a 3' single-stranded DNA flap containing the desired gene editing information), which replaces the corresponding endogenous DNA strand at the target site immediately downstream of the PE-induced nick site. Without being bound by theory, polymerization of the DNA synthesis template continues toward the 5' end of the extension arm until a termination event occurs. Polymerization can terminate in a variety of ways, including, but not limited to, (a) reaching the 5' end of the PE-gRNA (e.g., in the case of a 5' extension arm, the DNA polymerase simply runs out of template), (b) reaching an impassable RNA secondary structure (e.g., a hairpin or stem / loop), or (c) reaching a replication termination signal, e.g., a specific nucleotide sequence that blocks or inhibits the polymerase, or a signal on a nucleic acid form such as supercoiled DNA or RNA.

[0152] Effective dose The term "effective amount," as used herein, refers to an 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 may refer to the amount of the editor sufficient to edit a target site nucleotide sequence (e.g., a genome). In some embodiments, an effective amount of a prime editor provided herein (e.g., of a fusion protein comprising a nickase-Cas9 domain and a reverse transcriptase) may refer to the amount of the fusion protein sufficient to induce editing of a target site specifically bound and edited by the fusion protein. Those skilled in the art will appreciate that the effective amount of an agent, e.g., a fusion protein, a nuclease, a hybrid protein, a protein dimer, a complex of a protein (or protein dimer) and a polynucleotide, or a polynucleotide, may vary depending on various factors, such as the desired biological response, e.g., the particular allele, genome, or target site to be edited, the cell or tissue being targeted, and the agent being used.

[0153] functional equivalent The term "functional equivalent" refers to a second biomolecule that is functionally equivalent, but not necessarily structurally equivalent, to a first biomolecule. For example, a "Cas9 equivalent" refers to a protein that has the same or substantially the same function as Cas9, but does not necessarily have the same amino acid sequence. In the context of this disclosure, the specification refers throughout to "protein X, or a functional equivalent thereof." In this context, a "functional equivalent" of protein X embraces any homolog, paralog, fragment, naturally occurring version, modified version, mutated version, or synthetic version that bears the equivalent function of protein X.

[0154] fusion proteins The term "fusion protein," as used herein, refers to a hybrid polypeptide comprising protein domains from at least two different proteins. A protein may be located at the amino-terminal (N-terminal) portion of the fusion protein or at the carboxy-terminal (C-terminal) portion of the protein, thereby forming an "amino-terminal fusion protein" or a "carboxy-terminal fusion protein," respectively. A protein may contain different domains, such as a nucleic acid-binding domain (e.g., the gRNA-binding domain of Cas9, which directs the protein to bind to a target site) and a nucleic acid-cleavage domain or catalytic domain of a nucleic acid-editing protein. Another example includes Cas9 fused to a reverse transcriptase or its equivalent. Any of the proteins provided herein may be produced by any method known in the art. For example, the proteins provided herein may be produced via recombinant protein expression and purification, which is particularly suitable for fusion proteins containing peptide linkers. Methods for recombinant protein expression and purification are well known and include those described by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012) (the entire contents of which are incorporated herein by reference).

[0155] gene product The term "gene product" as used herein refers to any product encoded by a nucleic acid sequence. Consequently, a gene product may be, for example, a primary transcript, a mature transcript, a processed transcript, or a protein or peptide encoded by a transcript. Consequently, examples of gene products include mRNA, rRNA, tRNA, hairpin RNA, microRNA (miRNA), shRNA, siRNA, and peptides and proteins, such as reporter proteins or therapeutic proteins.

[0156] Gene of interest (GOI) The term "gene of interest" or "GOI" refers to a gene encoding a biomolecule of interest (e.g., a protein or an RNA molecule). The protein of interest may include any intracellular protein, membrane protein, or extracellular protein, such as a nuclear protein, a transcription factor, a nuclear membrane transporter, an intracellular organelle-associated protein, a membrane receptor, a catalytic protein, and an enzyme, a therapeutic protein, a membrane protein, a membrane transport protein, a signal transduction protein, or an immunological protein (e.g., an IgG or other antibody protein), etc. The gene of interest may also encode an RNA molecule, including, but not limited to, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA), antisense RNA, guide RNA, microRNA (miRNA), small interfering RNA (siRNA), and cell-free RNA (cfRNA).

[0157] Guide RNA ("gRNA") As used herein, the term "guide RNA" refers to a specific type of guide nucleic acid, most commonly associated with the Cas protein of CRISPR-Cas9, that binds to Cas9 and directs the Cas9 protein to a specific sequence in a DNA molecule (including complementarity with the protospace sequence of the guide RNA). As described elsewhere, PEGRNA is a subcategory of guide RNA that further comprises an extension arm on the 3' or 5' end of the guide that allows the molecule to be used with the primed editors disclosed herein. The term "guide RNA" also encompasses equivalent guide nucleic acid molecules, whether naturally occurring or non-naturally occurring (e.g., engineered or recombinant), that bind to a Cas9 equivalent, homolog, ortholog, or paralog and otherwise program the Cas9 equivalent to localize to a specific target nucleotide sequence. Cas9 equivalents may also include other napDNAbp from any type of CRISPR system (e.g., type II, type V, type VI), including Cpf1 (type V CRISPR-Cas system), C2c1 (type V CRISPR-Cas system), C2c2 (type VI CRISPR-Cas system), and C2c3 (type V CRISPR-Cas system). Additional Cas equivalents are described in Makarova et al., "C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector," Science 2016;353(6299), the contents of which are incorporated herein by reference. Exemplary sequences and structures of guide RNAs are provided herein. Additionally, methods for designing appropriate guide RNA sequences are provided herein. As used herein, "guide RNA" may also be referred to as "existing guide RNA" to contrast with the modified form of guide RNA called "prime editor guide RNA" (or "PEgRNA") that has been invented for the prime editing methods and compositions disclosed herein.

[0158] Guide RNAs or PEGRNAs may contain a variety of structural elements, including but not limited to:

[0159] Spacer sequence - a sequence in the guide RNA or PEGRNA (having a length of about 10 to about 40 (e.g., about 10, about 15, about 20, about 25, about 30) nucleotides) that binds to a protospacer (as defined herein below) in the target DNA.

[0160] gRNA core (or gRNA backbone or backbone sequence) - refers to the sequence within the gRNA responsible for napDNAbp (e.g., Cas9) binding, this does not include the spacer / targeting sequence used to guide the napDNAbp (e.g., Cas9) to the target DNA.

[0161] Extension Arm - refers to the portion of the guide RNA extended at either the 5' or 3' end, including the homology arm, editing template, and primer binding site. This component is further defined elsewhere.

[0162] Homologous arm refers to the portion or portions of the extension arm that encode the resulting reverse transcriptase-encoded single-stranded DNA flap (which is incorporated into the target DNA site by displacing the endogenous strand). The portion of the single-stranded DNA flap encoded by the homologous arm is complementary to the non-edited strand of the target DNA sequence, facilitating its displacement of the endogenous strand and annealing with the single-stranded DNA flap in its place, thereby incorporating the edit. This component is further defined elsewhere.

[0163] Editing template—refers to the portion of the extension arm that encodes the desired edit in the single-stranded DNA flap synthesized by reverse transcriptase. This component is further defined elsewhere.

[0164] Primer binding site - refers to the portion of the extension arm that anneals to the primer sequence formed from the target DNA strand after Cas9-mediated nickase action thereon. This component is further defined elsewhere.

[0165] Transcription terminator-guide RNAs or PEG RNAs may include a transcription termination sequence 3' of the molecule. Typically, transcription terminator sequences (e.g., SEQ ID NOS: 1361560-1361565) are about 70 to about 125 nucleotides in length, although shorter and longer transcription terminator sequences are contemplated and any known in the art may be used.

[0166] Flap endonucleases (e.g., FEN1) 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 the removal of 5' flaps formed during cellular processes, including DNA replication. The prime editing methods described herein may utilize endogenously supplied flap endonucleases or those provided in trans to remove 5' flaps of endogenous DNA formed at the target site during prime editing. Flap endonucleases are known in the art and can be found in Patel et al., "Flap endonucleases pass 5'-flaps through a flexible arch using a disorder-thread-order mechanism to confer specificity for free 5'-ends," Nucleic Acids Research, 2012, 40(10):4507-4519 and Tsutakawa et al., "Human flap endonuclease structures, DNA double-base flipping, and a unified understanding of the FEN1 superfamily," Cell, 2011, 145(2):198-211, and Balakrishnan et al., "Flap Endonuclease 1," Annu Rev Biochem, 2013, Vol 82:119-138, each of which is incorporated herein by reference. An exemplary flap endonuclease is FEN1, which can be represented by the following amino acid sequence: [Table 1]

[0167] fusion proteins The term "fusion protein," as used herein, refers to a hybrid polypeptide comprising protein domains from at least two different proteins. A protein may be located at the amino-terminal (N-terminal) portion or at the carboxy-terminal (C-terminal) portion of the fusion protein, thereby forming an "amino-terminal fusion protein" or a "carboxy-terminal fusion protein," respectively. The protein may comprise different domains, such as a nucleic acid-binding domain (e.g., the gRNA-binding domain of Cas9, which directs the protein to bind to a target site) and a nucleic acid-cleavage domain (e.g., Cas9 nickase, napDNAbp) or catalytic domain of a nucleic acid-editing protein (e.g., an RT domain). Another example includes napDNAbp (e.g., Cas9) fused to a reverse transcriptase enzyme, or its equivalent. Any of the proteins provided herein may be produced by any method known in the art. For example, the proteins provided herein may be produced via recombinant protein expression and purification, which is particularly suitable for fusion proteins containing peptide linkers. Methods for expression and purification of recombinant proteins are well known and are described in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4 th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012), the entire contents of which are incorporated herein by reference.

[0168] Homologous arms The term "homologous arm" refers to the portion of the extension arm that contains the resulting sequence of the reverse transcriptase-encoded single-stranded DNA flap (which is incorporated into the target DNA site by displacing the endogenous strand). The portion of the single-stranded DNA flap encoded by the homologous arm is complementary to the unedited strand of the target DNA sequence, facilitating its replacement with the endogenous strand and annealing with the single-stranded DNA flap in its place, thereby incorporating the edit. This component is further defined elsewhere.

[0169] host cell The term "host cell," as used herein, refers to a cell that can host, replicate, and express a vector described herein, e.g., a vector containing a nucleic acid molecule encoding a fusion protein comprising napDNAbp or a napDNAbp equivalent (e.g., Cas9 or equivalent) and a reverse transcriptase.

[0170] isolated "Isolated" means altered or removed from its natural state. For example, a nucleic acid or peptide that is naturally present in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from its natural state from coexisting materials is "isolated." An isolated nucleic acid or protein can exist in a substantially purified form or can exist in a non-native environment (e.g., a host cell).

[0171] In some embodiments, the gene of interest is encoded by an isolated nucleic acid. As used herein, the term "isolated" refers to the characteristic of a material as provided herein, which is removed from its original or native environment (e.g., the natural environment if it occurs in nature). Thus, a naturally occurring polynucleotide or protein or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide separated from some or all of the coexisting materials in a natural system by human intervention is isolated. Artificial or modified materials, such as non-naturally occurring nucleic acid constructs, such as the expression constructs and vectors described herein, are also consequently referred to as isolated. A material does not need to be purified to be isolated. Consequently, a material may be part of a vector and / or part of a composition, and such a vector or composition is still isolated in that it is not part of the environment in which the material is found in nature.

[0172] napDNAbp As used herein, the term "nucleic acid-programmed DNA-binding protein" or "napDNAbp" (of which Cas9 is an example) refers to a protein that uses RNA:DNA hybridization to target and bind to specific sequences in a DNA molecule. Each napDNAbp is linked to at least one guide nucleic acid (e.g., a guide RNA) that localizes the napDNAbp to a DNA sequence that includes a DNA strand (i.e., a target strand) that is complementary to the guide nucleic acid or a portion thereof (e.g., a protospacer of the guide RNA). In other words, the guide nucleic acid "programs" the napDNAbp (e.g., Cas9 or equivalent) to localize and bind to the complementary sequence.

[0173] Without being bound by theory, the binding mechanism of the napDNAbp-guide RNA complex generally involves the napDNAbp forming an R-loop that induces unwinding of the double-stranded DNA target (thereby separating the strands in the region bound by the napDNAbp). The guide RNA protospacer then hybridizes to the "target strand." This displaces the "non-target strand" that is complementary to the target strand, thereby forming the single-stranded region of the R-loop. In some embodiments, the napDNAbp contains one or more nuclease activities, which then cleave the DNA to eliminate various types of lesions. For example, the napDNAbp may contain nuclease activities that cleave the non-target strand at a first location and / or the target strand at a second location. In response to the nuclease activity, the target DNA can be cleaved to form a "double-stranded break" in which both strands are cleaved. In other embodiments, the target DNA can be cleaved at only a single site, i.e., the DNA is "nicked" on one strand. Exemplary napDNAbps with various nuclease activities include "Cas9 nickase" ("nCas9") and inactivated Cas9 that does not have any nuclease activity ("inactivated Cas9" or "dCas9"). Exemplary sequences for these and other napDNAbps are provided herein.

[0174] Linker The term "linker," as used herein, refers to a molecule that connects two other molecules or moieties. Linkers are well known in the art and can contain any suitable combination of nucleic acids or amino acids to facilitate the correct function of the structures they link. A linker can be a series of amino acids. A linker can be an amino acid sequence, in the case of a linker that connects two fusion proteins. For example, napDNAbp (e.g., Cas9) can be fused to reverse transcriptase via an amino acid linker sequence. A linker can also be a nucleotide sequence, in the case of connecting two nucleotide sequences together. For example, in a simple case, an existing guide RNA is linked via a spacer or linker nucleotide sequence to a DNA synthesis template (e.g., an RT template sequence) and the RNA extension of a prime editor guide RNA, which may include a 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. In some embodiments, the linker is 5 to 100 nucleotides 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, 150-200, 200-300, 300-500, 500-1000, 1000-2000, or 2000-5000 nucleotides in length. Longer or shorter linkers are also contemplated.

[0175] Nickase The term "nickase" refers to a napDNAbp (e.g., Cas9) that has one of its two nuclease domains inactivated, allowing the enzyme to cleave only one strand of the target DNA.

[0176] Nuclear localization sequence (NLS) The term "nuclear localization sequence" or "NLS" refers to an amino acid sequence that facilitates the import of a protein into the cell nucleus, for example, by nuclear transport. Nuclear localization sequences are known in the art and will be apparent to those skilled in the art. For example, NLS sequences are described in International PCT Application PCT / EP2000 / 011690 to Plank et al., filed November 23, 2000, and published May 31, 2001 as WO2001 / 038547 (the contents of which are incorporated herein by reference for their disclosure of exemplary nuclear localization sequences). In some embodiments, the NLS comprises the amino acid sequence PKKKRKV (SEQ ID NO: 1361531) or MDSLLMNRRKFLYQFKNVRWAKGRRETYLC (SEQ ID NO: 1361533).

[0177] nucleic acid molecule The term "nucleic acid," as used herein, refers to a polymer (i.e., a plurality of, more than one, (e.g., 2, 3, 4, etc.) nucleotides). A polymer can be any of naturally occurring nucleosides (i.e., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine), nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C5 bromouridine, C5 fluorouridine, C5 iodouridine, C5 propynyluridine, C5 propynylcytidine, C5 methylcytidine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)methylguanine, 4-acetylcytidine, and the like). , 5-(carboxyhydroxymethyl)uridine, dihydrouridine, methylpseudouridine, 1-methyladenosine, 1-methylguanosine, N6-methyladenosine, and 2-thiocytidine), chemically modified bases, biologically modified bases (e.g., methylated bases), intercalated bases, modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, 2'-O-methylcytidine, arabinose, and hexose), or modified phosphate groups (e.g., phosphorothioate and 5'-N phosphoramidite linkages).

[0178] Nucleic acid bases As used herein, the term "nucleobase," also known as a "nitrogenous base" or often simply a "base," is a nitrogen-containing biological compound that forms the building blocks of nucleosides, which in turn form nucleotides, all of which constitute the basic building blocks of nucleic acids. The ability of nucleobases to form base pairs and the ability of one nucleobase to stack with another leads directly to long-chain helical structures such as ribonucleic acid (RNA) and deoxyribonucleic acid (DNA).

[0179] The five nucleobases, adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U), may be referred to as primary or standard. They serve as the basic units of the genetic code; the bases A, G, C, and T are found in DNA, while A, G, C, and U are found in RNA. Thymine and uracil are identical, except that T includes a methyl group that U lacks. DNA and RNA may also contain modified nucleobases. For example, for the nucleobases adenosine and guanosine, alternative nucleobases may include hypoxanthine, xanthine, or 7-methylguanosine, which correspond to the alternative nucleosides of inosine, xanthosine, and 7-methylguanosine, respectively. In addition, for example, alternative nucleobases to cytosine, thymine, or uridine can include 5,6-dihydrouracil, 5-methylcytosine, or 5-hydroxymethylcytosine, which correspond to alternative nucleosides of dihydrouridine, 5-methylcytidine, and 5-hydroxymethylcytidine, respectively. Nucleobases can also include nucleobase analogs, numerous of which are known in the art. Typically, analog nucleobases have, among other things, different base-pairing and base-stacking properties. Examples include universal bases (which can pair with all four standard bases) and phosphate-sugar backbone analogs such as PNAs (which can also form triple helices) that affect chain properties. Nucleic acid analogs, also known as "xenonucleic acids," represent one of the major pillars of xenobiology, which designs new-to-natural forms of life based on alternative biochemistries. Artificial nucleic acids include peptide nucleic acids (PNAs), morpholinos and locked nucleic acids (LNAs), as well as glycol nucleic acids (GNAs) and threose nucleic acids (TNAs), each of which is distinguished from naturally occurring DNA or RNA by changes to the backbone of the molecule.Examples of analogs include (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C5 bromouridine, C5 fluorouridine, C5 iodouridine, C5 propynyluridine, C5 propynylcytidine, C5 methylcytidine, 7 deazaadenosine, 7 deazaguanosine, 8 oxoadenosine, 8 oxoguanosine, O(6) methylguanine, 4-acetylcytidine, 5-(carboxyhydroxymethyl)uridine, dihydrouridine, methyl pseudouridine, 1-methyladenosine, 1-methylguanosine, N6-methyladenosine, and 2-thiocytidine), chemically modified bases, biologically modified bases (e.g., methylated bases), intercalated bases, modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, 2'-O-methylcytidine, arabinose, and hexose), or modified phosphate groups (e.g., phosphorothioate and 5'-N phosphoramidite linkages).

[0180] PEG-RNA As used herein, the terms "prime editor guide RNA" or "PEgRNA" or "extended guide RNA" refer to specialized forms of guide RNA that have been modified to include one or more additional sequences for use in the prime editing methods, compositions, and systems described herein. As described herein, a prime editor guide RNA comprises one or more "extended regions" of nucleic acid sequence. The extended region may include, but is not limited to, a single-stranded RNA. Furthermore, the extended region may occur at the 3' end of an existing guide RNA. In other configurations, the extended region may occur at the 5' end of an existing guide RNA. In yet other configurations, the extended region may occur at a region within the molecule (e.g., in the gRNA core region that attaches and / or binds to the napDNAbp) rather than at one of the ends of the existing guide RNA. The extended region comprises a "reverse transcriptase template sequence," a single-stranded RNA molecule that encodes a single-stranded complementary DNA (cDNA), which in turn (a) is designed to be homologous to the endogenous target DNA to be edited and (b) contains at least one desired nucleotide change (e.g., a transition, transversion, deletion, insertion, or a combination thereof) to be introduced or incorporated into the endogenous target DNA. The extended region may also contain other functional sequence elements, such as, but not limited to, a "primer binding site" and / or a "spacer or linker" sequence. As used herein, a "primer binding site" includes a sequence that hybridizes to a single-stranded DNA sequence having a 3' end generated from the R-loop nicked DNA and that contains a primer for reverse transcriptase.

[0181] In some embodiments, the PEGRNA is represented by Figure 3A, which shows a PEGRNA with a 5' extension arm, a spacer, and a gRNA core. The 5' extension further comprises, in the 5' to 3' direction, a reverse transcriptase template, a primer binding site, and a linker.

[0182] In some embodiments, the PEGRNA is represented by Figure 3B, which shows a PEGRNA with a 3' extension arm, a spacer, and a gRNA core. The 3' extension further comprises a reverse transcriptase template and a primer binding site in the 5' to 3' direction.

[0183] In yet another embodiment, the PEgRNA is represented by Figure 27, which shows a PEgRNA having, in the 5' to 3' direction, a spacer (1), a gRNA core (2), and an extension arm (3). The extension arm (3) is at the 3' end of the PEgRNA. The extension arm (3) further comprises, in the 5' to 3' direction, a "primer binding site" (A), an "editing template" (B), and a "homology arm" (C). The extension arm (3) may also comprise optional modification regions at the 3' and 5' ends, which may be the same or different sequences. In addition, the 3' end of the PEgRNA may comprise a transcription terminator sequence. These sequence elements of the PEgRNA are further described and defined herein. Additionally, the present specification discloses exemplary PEgRNAs designed according to the methods disclosed herein in the accompanying sequence listing.

[0184] In yet another embodiment, the PEgRNA is represented by Figure 28, which shows a PEgRNA having an extension arm (3), a spacer (1), and a gRNA core (2) in the 5' to 3' direction. The extension arm (3) is at the 5' end of the PEgRNA. The extension arm (3) further comprises a "primer binding site" (A), an "editing template" (B), and a "homology arm" (C) in the 3' to 5' direction. The extension arm (3) may also comprise optional modification regions at the 3' and 5' ends, which may be the same or different sequences. The PEgRNA may also comprise a transcription terminator sequence at the 3' end. These sequence elements of the PEgRNA are further described and defined herein.

[0185] Peptide tags The term "peptide tag" refers to a peptide amino acid sequence that is genetically fused to a protein sequence to confer one or more functions onto the protein, thereby facilitating manipulation of the protein for various purposes such as visualization, identification, localization, purification, solubilization, isolation, etc. Peptide tags can include various types of tags classified by purpose or function, which may include "affinity tags" (to facilitate protein purification), "solubilization tags" (to assist in correct folding of the protein), "chromatography tags" (to alter the chromatographic properties of the protein), "epitope tags" (to bind to high-affinity antibodies), and "fluorescent tags" (to facilitate visualization of the protein in cells or in vitro).

[0186] PE1 As used herein, "PE1" refers to a PE complex comprising a fusion protein comprising Cas9(H840A) and wild-type MMLV RT and having the following structure: [NLS]-[Cas9(H840A)]-[linker]-[MMLV_RT(wt)]+desired PE gRNA, wherein the PE fusion has the amino acid sequence of SEQ ID NO: 1361515, said sequence being as shown below; [ka]

[0187] PE2 As used herein, "PE2" refers to a PE complex comprising a fusion protein comprising Cas9(H840A) and a variant MMLV RT and having the following structure: [NLS]-[Cas9(H840A)]-[linker]-[MMLV_RT(D200N)(T330P)(L603W)(T306K)(W313F)]+a desired PE gRNA, wherein the PE fusion has the amino acid sequence of SEQ ID NO: 1361516, said sequence being as shown below: [ka]

[0188] PE3 As used herein, "PE3" refers to PE2 plus a second-strand-nicking guide RNA that complexes with PE2 and introduces a nick in the non-edited DNA strand to induce preferential displacement of the edited strand.

[0189] PE3b As used herein, "PE3b" refers to PE3, where the guide RNA that nicks the second strand is designed for temporal control so that the second strand nick is not introduced until after the desired edit has been incorporated. This is achieved by designing a gRNA with a spacer sequence that matches only the edited strand but not the original allele. Using this strategy, hereafter referred to as PE3b, the mismatch between the protospacer and the unedited allele should prevent nicking by the sgRNA until after the editing event on the PAM strand has occurred.

[0190] Short PE (PE-short) As used herein, "short PE" refers to a PE construct fused to a C-terminally truncated reverse transcriptase and having the following amino acid sequence: [ka]

[0191] Percent Identity "Percent identity," "sequence identity," "% identity," or "% sequence identity" (which may be used interchangeably herein) of a sequence (e.g., nucleic acid or amino acid) refers to a quantitative measure of similarity between two sequences (e.g., nucleic acid or amino acid). The percent identity of genomic DNA sequences, intron and exon sequences, and amino acid sequences between humans and other species varies by species type, with chimpanzees having the highest percent identity with humans of all species in each category. Percent identity may be determined using the algorithm of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified as in Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm has been incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul et al., J. Mol. Biol. 215:403-10, 1990. BLAST protein searches can be performed with the XBLAST program, score=50, word length=3, to obtain amino acid sequences homologous to a protein molecule of interest. When gaps exist between the two sequences, Gapped BLAST can be used as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.When percent identities, or ranges thereof (e.g., at least, more than, between, etc.) are specified or recited, unless otherwise specified, the endpoints are to be included, and ranges (e.g., at least 70% identity) include all ranges within the recited range (e.g., 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 "identity" should encompass 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 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% identity), and all increments thereof (for example, one-tenth of a percent (i.e., 0.1%), one-hundredth of a percent (i.e., 0.01%), etc.).

[0192] Prime editing factor The term "prime editor" refers to a fusion construct described herein comprising a napDNAbp (e.g., Cas9 nickase) and a reverse transcriptase, which is capable of performing prime editing on a target nucleotide sequence in the presence of a PEgRNA (or "extended guide RNA"). The term "prime editor" may also refer to a fusion protein or a fusion protein complexed with a PEgRNA. In some embodiments, a prime editor may also refer to a complex comprising a fusion protein (reverse transcriptase fused to a napDNAbp), a PEgRNA, and a regular guide RNA, which is capable of directing a nicking step to a second site in the non-edited strand as described herein. In some embodiments, the reverse transcriptase component of a "primer editor" is provided in trans.

[0193] Primer binding site The term "primer binding site" or "PBS" refers to a nucleotide sequence located on the PEgRNA as a component of the extension arm (typically at the 3' end of the extension arm) that serves to bind to a primer sequence formed after napDNAbp (e.g., Cas9) nicking of the target sequence by a prime editor. As detailed elsewhere, when the Cas9 nickase component of the prime editor nicks one of the strands of the target DNA sequence, a 3'-terminal ssDNA flap is formed, which anneals to the primer binding site on the PEgRNA and serves as a primer sequence to prime reverse transcription. Figures 27 and 28 show embodiments of primer binding sites located on the 3' and 5' extension arms, respectively.

[0194] Proteins, peptides, and polypeptides The terms "protein," "peptide," and "polypeptide" are used interchangeably herein and refer to a polymer of amino acid residues linked together by peptide (amide) bonds. The terms refer to proteins, peptides, or polypeptides of any size, structure, or function. Typically, a protein, peptide, or polypeptide will be at least three amino acids in length. A protein, peptide, or polypeptide may refer to an individual protein or a group of proteins. One or more of the amino acids in a protein, peptide, or polypeptide may be modified by the addition of a chemical entity, such as, for example, a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification. A protein, peptide, or polypeptide may also be a single molecule or a multimolecular complex. A protein, peptide, or polypeptide may be merely a fragment of a naturally occurring protein or peptide. A protein, peptide, or polypeptide may be naturally occurring, recombinant, synthetic, or any combination thereof. Any of the proteins provided herein may be produced by any method known in the art. For example, the proteins provided herein may be produced through recombinant protein expression and purification, which is particularly suitable for fusion proteins containing peptide linkers. Methods for recombinant protein expression and purification are well known and include those described by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012) (the entire contents of which are incorporated herein by reference).

[0195] Operably linked The term "operably linked," as used herein, refers to a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence (e.g., a transgene) that results in expression of the heterologous nucleic acid sequence (e.g., a transgene). For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. Illustratively, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence. Generally, operably linked nucleic acid sequences are contiguous, as is necessary to join two protein-encoding regions in the same reading frame.

[0196] promoter The term "promoter" is art-recognized and refers to a nucleic acid molecule having a sequence recognized by a cell's transcriptional machinery and capable of initiating transcription of a downstream gene. Promoters can be constitutively active (meaning the promoter is always active under a given cellular context) or conditionally active (meaning the promoter is only active in the presence of specific conditions). For example, a conditional promoter may be active only in the presence of a specific protein that connects proteins associated with regulatory elements in the promoter to the basal transcriptional machinery, or in the absence of an inhibitory molecule. A subclass of conditionally active promoters is inducible promoters, which require the presence of a small molecule "inducer" for activity. Examples of inducible promoters include, but are not limited to, arabinose-inducible promoters, Tet-on promoters, and tamoxifen-inducible promoters. Constitutive, conditional, and inducible promoters are well known to those skilled in the art, and one of skill in the art would be able to ascertain a variety of such promoters useful in carrying out the present invention. The present invention is not limited in this respect.

[0197] Protospacer adjacent motif (PAM) As used herein, the term "protospacer adjacent sequence" or "PAM" refers to an approximately 2-6 base pair DNA sequence that is the key targeting component of the Cas9 nuclease. Typically, the PAM sequence is on either strand and is downstream of the Cas9 cleavage site in the 5' to 3' direction. The standard PAM sequence (i.e., the PAM sequence associated with Streptococcus pyogenes Cas9 nuclease or SpCas9) is 5'-NGG-3', where "N" is any nucleobase and is followed by two guanine ("G") nucleobases. Various PAM sequences may be associated with different Cas9 nucleases or equivalent proteins from different organisms, such as 5'-NG-3' (where "N" is any nucleobase and is followed by one guanine ("G") nucleobase) or 5'-KKH-3' (where two lysines ("K") followed by one histidine ("H"). Additionally, a given Cas9 nuclease (e.g., SpCas9) may be modified to alter the PAM specificity of the nuclease so that it recognizes alternative PAM sequences.

[0198] For example, referring to the standard SpCas9 amino acid sequence SEQ ID NO: 1361421 (SpCas9 M1 QQ99ZW2 wild-type), the PAM sequence can be modified by introducing one or more mutations, including (a) the D1135V, R1335Q, and T1337R "VQR variant" (which alters PAM specificity to NGAN or NGNG), (b) the D1135E, R1335Q, and T1337R "EQR variant" (which alters PAM specificity to NGAG), and (c) the D1135V, G1218R, R1335E, and T1337R "VRER variant" (which alters PAM specificity to NGCG). In addition, the D1135E variant of standard SpCas9 still recognizes NGG, but is more selective than the wild-type SpCas9 protein.

[0199] It will also be understood that Cas9 enzymes from different bacterial species (i.e., Cas9 orthologs) can have varying PAM specificities. For example, Cas9 from Staphylococcus aureus (SaCas9) recognizes NGRRT or NGRRN. Additionally, Cas9 from Neisseria meningitis (NmCas) recognizes NNNNGATT. In another example, Cas9 from Streptococcus thermophilis (StCas9) recognizes NNAGAAW. In yet another example, Cas9 from Treponema denticola (TdCas) recognizes NAAAAC. These examples are not intended to be limiting. It will further be understood that non-SpCas9s also bind to a variety of PAM sequences, thereby becoming useful when a suitable SpCas9 PAM sequence is not present at the desired targeted cleavage site. Furthermore, non-SpCas9s may also have other characteristics that make them more useful than SpCas9s. For example, Cas9 from Staphylococcus aureus (SaCas9) is about 1 kilobase smaller than SpCas9 so that it can be packaged into adeno-associated virus (AAV). See also Shah et al., "Protospacer recognition motifs: mixed identities and functional diversity," RNA Biology, 10(5):891-899, which is incorporated herein by reference.

[0200] Protospacer As used herein, the term "protospacer" refers to a sequence (~20 bp) in DNA adjacent to a PAM (protospacer adjacent motif) sequence that has the same sequence as the spacer sequence of the guide RNA. The guide RNA anneals to the complement of the protospacer sequence on the target DNA (specifically, to one strand of it, i.e., the "target strand" versus the "non-target strand" of the target sequence). For Cas9 to function, a specific protospacer adjacent motif (PAM) is also required, which varies depending on the bacterial species of the Cas9 gene. The most commonly used Cas9 nuclease, derived from S. pyogenes, recognizes the NGG PAM sequence found on the non-target strand, immediately downstream of the target sequence in genomic DNA. Those skilled in the art will understand that state-of-the-art literature sometimes refers to the "protospacer" as the ~20 nt targeting-specific guide sequence of the guide RNA itself, rather than referring to it as a "spacer." Thus, in some cases, the term "protospacer" may be used interchangeably with the term "spacer" as used herein. The written context surrounding the appearance of either "protospacer" or "spacer" will help inform the reader as to whether the term refers to a gRNA or a DNA target. Use of both of these terms is acceptable where the state of the art uses both terms in each of these ways.

[0201] reverse transcriptase The term "reverse transcriptase" describes a class of polymerases characterized as RNA-dependent DNA polymerases. All known reverse transcriptases require a primer to synthesize DNA transcripts from an RNA template. Historically, reverse transcriptases have primarily been used to transcribe mRNA into cDNA, which can then be cloned into vectors for further manipulation. Avian myeloblastosis virus (AMV) reverse transcriptase was the first widely used RNA-dependent DNA polymerase (Verma, Biochim. Biophys. Acta 473:1 (1977)). The enzyme possesses 5'-3' RNA-directed DNA polymerase activity, 5'-3' DNA-specific DNA polymerase activity, and RNase H activity. RNase H is a processive 5' and 3' ribonuclease specific for the RNA strand for RNA-DNA hybrids (Perbal, A Practical Guide to Molecular Cloning, New York: Wiley & Sons (1984)). Because known viral reverse transcriptases lack the 3'-5' exonuclease activity required for proofreading, errors in transcription cannot be corrected by reverse transcriptase (Saunders and Saunders, Microbial Genetics Applied to Biotechnology, London: Croom Helm (1987)). A detailed study of the activity of AMV reverse transcriptase and its associated RNase H activity was presented by Berger et al., Biochemistry 22:2365-2372 (1983). Another reverse transcriptase widely used in molecular biology is the reverse transcriptase derived from Moloney murine leukemia virus (M-MLV). See, e.g., Gerard, GR, DNA 5:271-279 (1986) and Kotewicz, ML, et al., Gene 35:249-258 (1985). M-MLV reverse transcriptases that are substantially devoid of RNase H activity have been described. See, e.g., U.S. Patent No. 5,244,797.The present invention contemplates the use of any such reverse transcriptase, or variants or mutants thereof.

[0202] In addition, the present invention also contemplates the use of reverse transcriptases that are error-prone, i.e., may be referred to as error-prone reverse transcriptases, or reverse transcriptases that do not support the incorporation of high-fidelity nucleotides during polymerization.During the synthesis of a single-stranded DNA flap based on the RT template incorporated into the guide RNA, the error-prone reverse transcriptase may introduce one or more nucleotides that are mismatched with the DNA synthesis template (for example, the RT template sequence), thereby introducing changes into the nucleotide sequence through the incorrect polymerization of the single-stranded DNA flap.These errors introduced during the synthesis of the single-stranded DNA flap are then incorporated into the double-stranded molecule through hybridization with the corresponding endogenous target strand, removal of the displaced endogenous strand, ligation, and then one more round of endogenous DNA repair and / or replication.

[0203] Reverse transcription 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 error-prone nature of certain reverse transcriptases in their DNA polymerization activity.

[0204] Sense strand In genetics, a "sense" strand is a 5' to 3' segment of double-stranded DNA that is complementary to the 3' to 5' antisense or template strand of DNA. In the case of a DNA segment encoding a protein, the sense strand is the strand of DNA with the same sequence as the mRNA that takes the antisense strand as its template during transcription and ultimately (typically, but not always) is translated into the protein. Thus, the antisense strand carries the RNA that is subsequently translated into protein, while the sense strand possesses nearly identical makeup to the mRNA. Note that for each segment of dsDNA, there will likely be two pairs: sense and antisense (because sense and antisense are relative perspectives), depending on which direction one is read from. The gene product or mRNA ultimately represents either strand of a segment of dsDNA, which may be designated as sense or antisense.

[0205] In the context of PEGRNA, the first step is the synthesis of a single-stranded complementary DNA (i.e., the 3' ssDNA flap to be incorporated) oriented in the 5' to 3' direction, but the synthesized strand is off-templated for the PEGRNA extension arm. Whether the 3' ssDNA flap should be considered the sense strand or the antisense strand depends on the direction of transcription, where it is fully accepted that both strands of DNA can serve as templates for transcription (but not simultaneously). Thus, in some embodiments, the 3' ssDNA flap (extending generally in the 5' to 3' direction) will serve as the sense strand, since it is the coding strand. In other embodiments, the 3' ssDNA flap (extending generally in the 5' to 3' direction) will serve as the antisense strand and thus will be a transcription template.

[0206] Nicking the second strand As used herein, this concept refers to the introduction of a second nick at a location downstream of the first nick (i.e., the initial nick site that provides a free 3' end for use in priming reverse transcriptase on the extended portion of the guide RNA). In some embodiments, the first nick and the second nick are on opposite strands. In other embodiments, the first nick and the second nick are on opposite strands. In 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. The second nick is located at least 5 nucleotides downstream of the first nick, or at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more nucleotides downstream of the first nick. Without being bound by theory, the second nick induces the cell's endogenous DNA repair and replication processes to replace the unedited strand. In some embodiments, the edited strand is a non-target strand, and the unedited strand is a target strand. In other embodiments, the edited strand is a target strand, and the unedited strand is a non-target strand.

[0207] Spacer sequence As used herein, the term "spacer sequence" in relation to a guide RNA or PEgRNA refers to a portion of the guide RNA or PEgRNA of about 10 to about 40 (e.g., about 10, about 15, about 20, about 25, about 30) nucleotides that contains a nucleotide sequence complementary to a protospacer sequence in the target DNA sequence. The spacer sequence anneals to the protospacer sequence, forming an ssRNA / ssDNA hybrid structure at the target site and a corresponding R-loop ssDNA structure in the endogenous DNA strand complementary to the protospacer sequence.

[0208] subject The term "subject," as used herein, refers to an individual organism, e.g., an individual mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human mammal. In some embodiments, the subject is a non-human animal of the order Primates. In some embodiments, the subject is a rodent. In some embodiments, the subject is a sheep, goat, cattle, cat, or dog. In some embodiments, the subject is a vertebrate, amphibian, reptile, fish, insect, fly, or nematode. In some embodiments, the subject is a research animal. In some embodiments, the subject is genetically modified, e.g., a genetically modified non-human subject. The subject may be of either sex and at any stage of development.

[0209] target site The term "target site" refers to a sequence within a nucleic acid molecule that is edited by a primed editor disclosed herein. Target site also refers to a sequence within a nucleic acid molecule to which a complex of a base editor and a gRNA binds.

[0210] Temporary second strand nicking As used herein, the term "transient second-strand nicking" refers to a variant of second-strand nicking in which the incorporation of a second nick in the unedited strand occurs only after the desired edit has been incorporated into the edited strand. This avoids simultaneous nicking on both strands, which can lead to double-stranded DNA breaks. The second-strand nicking guide RNA is designed for temporal control so that the second-strand nick is not introduced until after the desired edit has been incorporated. This is achieved by designing a gRNA with a spacer sequence that matches only the edited strand but not the original allele. Using this strategy, the mismatch between the protospacer and the unedited allele should prevent nicking by the sgRNA until after the editing event on the PAM strand has occurred.

[0211] tPERT See definition for "trans-primed editor RNA template (tPERT)."

[0212] Temporary second strand nicking As used herein, the term "transient second-strand nicking" refers to a variant of second-strand nicking in which the incorporation of a second nick in the unedited strand occurs only after the desired edit has been incorporated into the edited strand. This avoids simultaneous nicking on both strands, which can lead to double-stranded DNA breaks. The second-strand nicking guide RNA is designed for temporal control so that the second-strand nick is not introduced until after the desired edit has been incorporated. This is achieved by designing a gRNA with a spacer sequence that matches only the edited strand but not the original allele. Using this strategy, the mismatch between the protospacer and the unedited allele should prevent nicking by the sgRNA until after the editing event on the PAM strand has occurred.

[0213] Trans PrimeEdit As used herein, the term "trans prime editing" refers to a modified form of prime editing that utilizes a split PEG RNA, i.e., in which the PEG RNA is separated into two separate molecules: an sgRNA and a trans prime editing RNA template (tPERT). The sgRNA serves to target the prime editor (or more generally, the napDNAbp component of the prime editor) to a desired genomic target site, while once tPERT is recruited in trans to the prime editor through the interaction of binding domains located on the prime editor and tPERT, tPERT is used by a polymerase (e.g., reverse transcriptase) to write new DNA sequences into the target locus. In one embodiment, the binding domain may include an RNA-protein recruitment moiety, such as the MS2 aptamer located on tPERT and the MS2cp protein fused to the prime editor. An advantage of trans prime editing is that by separating the DNA synthesis template from the guide RNA, longer templates can potentially be used.

[0214] An embodiment of trans-prime editing is shown in Figures 3G and 3H. Figure 3G shows, on the left, the composition of a trans-prime editor complex ("RP-PE:gRNA complex"), which includes napDNAbp fused to each of a polymerase (e.g., reverse transcriptase) and an rPERT recruitment protein (e.g., MS2sc), complexed with a guide RNA. Figure 3G further shows a separate tPERT molecule, including a DNA synthesis template and an extended arm feature of PE-gRNA encompassing a primer-binding sequence. The tPERT molecule also includes an RNA-protein recruitment domain (which, in this case, is a stem-loop structure and may be, for example, an MS2 aptamer). As depicted in the process in Figure 3H, the RP-PE:gRNA complex binds to and nicks the target DNA sequence. The recruitment protein (RP) then recruits and colocalizes tPERT to the primed editor complex bound to the DNA target site, allowing the primer-binding site to bind to the primer sequence on the nicked strand, and subsequently allowing a polymerase (e.g., RT) to synthesize a single strand of DNA onto the DNA synthesis template, up to 5' of tPERT.

[0215] Although tPERT is shown in Figures 3G and 3H as containing a PBS and a DNA synthesis template on the 5' end of the RNA-protein recruitment domain, other configurations of tPERT may also be designed with the PBS and DNA synthesis template located on the 3' end of the RNA-protein recruitment domain. However, tPERT with a 5' extension has the advantage that synthesis of a single strand of DNA will naturally terminate at the 5' end of tPERT, thus eliminating the risk of using any part of the RNA-protein recruitment domain as a template during the DNA synthesis step of prime editing.

[0216] Trans-primed editor RNA template (tPERT) As used herein, "trans prime editor RNA template (tPERT)" refers to the components used in trans prime editing. This modified version of prime editing operates by separating the PE-gRNA into two separate molecules: a guide RNA and a tPERT molecule. The tPERT molecule is programmed to colocalize with the prime editor complex at the target DNA site and bring the primer binding site and DNA synthesis template to the prime editor in trans. For example, see Figure 3G for an embodiment of a trans prime editor (tPE) showing a two-component system including (1) an RP-PE:gRNA complex and (2) tPERT, which contains a primer binding site and a DNA synthesis template linked to an RNA-protein recruitment domain. Here, the RP (recruitment protein) component of the RP-PE:gRNA complex recruits tPERT to the target site to be edited, thereby linking the PE and DNA synthesis template to the prime editor in trans. In other words, tPERT is modified to contain (all or part of) the extension arm of a PEG RNA, which encompasses a primer binding site and a DNA synthesis template.

[0217] Transitions As used herein, "transition" refers to the interconversion of purine nucleobases (A⇔G) or pyrimidine nucleobases (C⇔T). This class of interconversion involves nucleobases of similar shape. The compositions and methods disclosed herein are capable of inducing one or more transitions in a target DNA molecule. The compositions and methods disclosed herein are also capable of inducing both a transition and a transversion in the same target DNA molecule. These changes involve A⇔G, G⇔A, C⇔T, or T⇔C. In the context of double-stranded DNA with Watson-Crick paired nucleobases, a transversion refers to the following base pair exchanges: A:T⇔G:C, G:G⇔A:T, C:G⇔T:A, or T:A⇔C:G. The compositions and methods disclosed herein are capable of inducing one or more transitions in a target DNA molecule. The compositions and methods disclosed herein are also capable of inducing both transitions and transversions, as well as other nucleotide changes, including deletions and insertions, in the same target DNA molecule.

[0218] Transversion As used herein, "transversion" refers to the interconversion of a purine nucleobase to a pyrimidine nucleobase, or vice versa, and thus involves the interconversion of dissimilar nucleobases. These changes involve T⇔A, T⇔G, C⇔G, C⇔A, A⇔T, A⇔C, G⇔C, and G⇔T. In the context of double-stranded DNA with Watson-Crick paired nucleobases, transversion refers to the following base pair exchanges: T:A⇔A:T, T:A⇔G:C, C:G⇔G:C, C:G⇔A:T, A:T⇔T:A, A:T⇔C:G, G:C⇔C:G, and G:C⇔T:A. The compositions and methods disclosed herein are capable of inducing one or more transversions in a target DNA molecule. The compositions and methods disclosed herein are also capable of inducing both transitions and transversions, as well as other nucleotide changes, including deletions and insertions, in the same target DNA molecule.

[0219] treatment The terms "treatment," "treat," and "treating" refer to a clinical intervention aimed at halting, alleviating, delaying the onset of, or inhibiting the progression of, a disease or disorder as described herein, or one or more symptoms thereof. As used herein, the terms "treatment," "treat," and "treating" refer to a clinical intervention aimed at halting, alleviating, delaying the onset of, or inhibiting the progression of, a disease or disorder as described herein, or one or more symptoms thereof. In some embodiments, treatment may be administered after one or more symptoms have developed and / or after a disease has been diagnosed. In other embodiments, treatment may be administered in the absence of symptoms, e.g., to prevent or delay the onset of symptoms or inhibit the onset or progression of a disease. For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and / or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, e.g., to prevent or delay their recurrence.

[0220] Trinucleotide repeat disorders As used herein, "trinucleotide repeat disorders" (or alternatively, "expansion repeat disorders" or "repeat expansion disorders") refer to a group of genetic disorders caused by a type of mutation (a trinucleotide repeat in a gene or intron) known as a "trinucleotide repeat expansion." Trinucleotide repeats were once thought to be commonplace iterations in the genome, but in the 1990s these disorders were classified. These seemingly "benign" stretches of DNA can sometimes expand and cause disease. Several defining features are common to disorders caused by trinucleotide repeat expansions. First, the mutant repeats exhibit instability in both somatic cells and the germline, and more frequently they expand rather than contract with successive transmission. Second, an earlier age of onset and increased severity (prognosis) of the phenotype in subsequent generations generally correlate with larger repeat length. Finally, the parental origin of a disease allele can often affect the expectation that paternal inheritance carries a greater risk of spread for many of these disorders.

[0221] Triplet expansions are thought to be caused by slippage during DNA replication. Due to the repetitive nature of DNA sequences in these regions, "loop-out" structures can also form during DNA replication, maintaining complementary base pairing between the parent and daughter strands being synthesized. If a loop-out structure is formed from a sequence on the daughter strand, this will result in an increase in the number of repeats. However, if a loop-out structure is formed on the parent strand, a decrease in the number of repeats occurs. These repeat expansions appear to be more common than reductions. In general, the larger the expansions, the more likely they are to cause disease or increase disease severity. This characteristic leads to the predictive features seen in trinucleotide repeat disorders. Prediction explains the tendency for an earlier age of onset and increasing symptom severity through successive generations of affected families due to these repeat expansions.

[0222] Nucleotide repeat disorders may encompass disorders in which the triplet repeat occurs in non-coding regions (i.e., non-coding trinucleotide repeat disorders) or disorders in which the triplet repeat occurs in coding regions.

[0223] The prime editor (PE) system described herein may be used to treat nucleotide repeat disorders, which may include Fragile X Syndrome (FRAXA), Fragile XE MR (FRAXE), Friedreich's Ataxia (FRDA), Myotonic Dystrophy (DM), Spinocerebellar Ataxia Type 8 (SCA8), and Spinocerebellar Ataxia Type 12 (SCA12), among others.

[0224] Prime Editing or "Prime Editing (PE)" As used herein, the term "prime editing" or "prime editing (PE)" refers to a novel approach for gene editing using napDNAbps and specialized guide RNAs as described in the present application and illustrated in the embodiments of Figures 1A-1J. TPRT refers to "target-primed reverse transcription," where a target DNA molecule is used, in one embodiment, to prime the synthesis of a strand of DNA by reverse transcriptase (or another polymerase). In various embodiments, prime editing operates by contacting a target DNA molecule (in which a nucleotide sequence change to be introduced is desired) with a nucleic acid-programmed DNA-binding protein (napDNAbp) complexed with a prime editor guide RNA. Referring to Figure 1E, the prime editor guide RNA contains an extension at the 3' or 5' end of the guide RNA, or elsewhere intramolecularly within the guide RNA, that encodes the desired nucleotide change (e.g., a single nucleotide change, insertion, or deletion). In step (a), the napDNAbp / extended gRNA complex contacts a DNA molecule, and the extended gRNA guides the napDNAbp to bind to the target locus. In step (b), a nick is introduced (e.g., by a nuclease or chemical agent) into one of the strands of DNA at the target locus, thereby creating an available 3' end on that strand. In some embodiments, the nick is created in the strand of DNA corresponding to the R-loop strand, i.e., the strand that does not hybridize with the guide RNA sequence, i.e., the "non-target strand." However, the nick cannot be introduced into either strand. That is, the nick can be introduced into the "target strand" (i.e., the strand that hybridizes with the spacer of the extended gRNA) or the "non-target strand" (i.e., the strand that forms the single-stranded portion of the R-loop and is complementary to the target strand). In step (c), the 3' end of the DNA strand (formed by the nick) interacts with the extended portion of the guide RNA to prime reverse transcription (i.e., "target-primed RT").In some embodiments, the 3'-terminal DNA strand hybridizes to a specific primer-binding site on the extended portion of the guide RNA, i.e., the "reverse transcriptase priming sequence." In step (d), reverse transcriptase is introduced to synthesize a single strand of DNA from the 3' end of the primed site to the 3' end of the primed editor guide RNA. This forms a single-stranded DNA flap that contains the desired nucleotide change (e.g., a single-base change, insertion, or deletion, or a combination thereof) (or is otherwise homologous to the endogenous DNA at or adjacent to the nick site). In step (e), the napDNAbp and guide RNA are released. Steps (f) and (g) involve the disassembly of the single-stranded DNA flap so that the desired nucleotide change is incorporated into the target locus. This process can drive desired product formation by removing the corresponding 5' endogenous DNA flap that is formed once the 3' single-stranded DNA flap has invaded and hybridized to the endogenous DNA sequence. Without being bound by theory, the cell's endogenous DNA repair and replication processes incorporate the nucleotide change(s) by degrading the mismatched DNA, resulting in the formation of the desired altered product. The process may also drive the formation of a "second-strand nicking" product, as illustrated in Figure ID. This process may introduce at least one or more of the following genetic changes: transversions, transitions, deletions, and insertions.

[0225] The terms "prime editor (PE) system" or "prime editor" or "PE system" or "PE editing system" refer to compositions involved in the methods of genome editing using target-primed reverse transcription (TPRT) described herein, including, but not limited to, napDNAbp, reverse transcriptase, fusion proteins (e.g., comprising napDNAbps and reverse transcriptase), prime editor guide RNA, and complexes comprising fusion proteins and prime editor guide RNA, as well as accessory elements (such as second-strand nicking components and 5' endogenous DNA flap removal endonuclease) that help drive the prime editing process toward edited product formation.

[0226] Upstream As used herein, the terms "upstream" and "downstream" are relative terms that define the linear positions of at least two elements located in a nucleic acid molecule (whether single-stranded or double-stranded) oriented in a 5' to 3' direction. In particular, if a first element is located anywhere 5' relative to the second element, the first element is upstream of the second element in the nucleic acid molecule. For example, if a SNP is located 5' to the nick site, the SNP is upstream of the Cas9-induced nick site. Conversely, if a first element is located anywhere 3' relative to the second element, the first element is downstream of the second element in the nucleic acid molecule. For example, if a SNP is located 3' to the nick site, the SNP is downstream of the Cas9-induced nick site. The nucleic acid molecule can be DNA (double-stranded or single-stranded), RNA (double-stranded or single-stranded), or a hybrid of DNA and RNA. Unless it is necessary to select which strand of a double-stranded molecule is considered, the terms upstream and downstream refer only to a single strand of a nucleic acid molecule, and the analysis is the same for single-stranded nucleic acid molecules and double-stranded molecules. Often, the strand of double-stranded DNA that can be used to determine the relative positions of at least two elements is the "sense" strand or "coding" strand. In genetics, a "sense" strand is a segment within 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' to the promoter on the sense or coding strand, the SNP nucleobase is "downstream" of the promoter sequence in genomic DNA (which is double-stranded).

[0227] variant As used herein, the term "variant" should be interpreted as meaning something that exhibits qualities with a pattern that deviates from those found in nature; for example, a variant Cas9 is a Cas9 that contains one or more changes in amino acid residues compared to the wild-type Cas9 amino acid sequence. The term "variant" encompasses homologous proteins that have at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 99% percent identity with a reference sequence and have the same or substantially the same functional activity(ies) as the reference sequence. The term also encompasses mutants, truncations, or domains of the reference sequence that exhibit the same or substantially the same functional activity(ies) as the reference sequence.

[0228] vector The term "vector," as used herein, refers to a nucleic acid that can be modified to encode a gene of interest and that can enter a host cell and mutate or replicate within the host cell (and then transfer the replicative form of the vector into another host cell). Exemplary suitable vectors include viral vectors, such as retroviral vectors or bacteriophages and filamentous phages, and conjugative plasmids. Additional suitable vectors will be apparent to those of skill in the art based on the present disclosure.

[0229] Wild type As used herein, the term "wild-type" is a term of art understood by those skilled in the art and means the typical form of an organism, strain, gene, or characteristic as occurring in nature, as distinguished from mutant or variant forms.

[0230] 5' endogenous DNA flap removal As used herein, the term "5' endogenous DNA flap removal" or "5' flap removal" refers to the removal of a 5' endogenous DNA flap formed when a single-stranded DNA flap synthesized by RT competitively invades and hybridizes with endogenous DNA, displacing the endogenous strand in the process. Removal of this displaced endogenous strand can drive a reaction to form a desired product containing the desired nucleotide change. The cell's own DNA repair enzymes may catalyze the removal or excision of the 5' endogenous flap (e.g., flap endonucleases such as EXO1 or FEN1). Host cells may also be transformed to express one or more enzymes that catalyze the removal of the 5' endogenous flap, thereby driving the process to form the product (e.g., flap endonucleases). Flap endonucleases are known in the art and can be found in Patel et al., "Flap endonucleases pass 5'-flaps through a flexible arch using a disorder-thread-order mechanism to confer specificity for free 5'-ends," Nucleic Acids Research, 2012, 40(10):4507-4519 and Tsutakawa et al., "Human flap endonuclease structures, DNA double-base flipping, and a unified understanding of the FEN1 superfamily," Cell, 2011, 145(2):198-211, each of which is incorporated herein by reference.

[0231] 5' endogenous DNA flap 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. Nicking of the target DNA strand by PE exposes a 3' hydroxyl group upstream of the nick site and a 5' hydroxyl group downstream of the nick site. The endogenous strand terminating in the 3' hydroxyl group is used to prime the DNA polymerase of the prime editor (e.g., the DNA polymerase here is reverse transcriptase). The endogenous strand downstream of the nick site and beginning with the exposed 5' hydroxyl group, referred to as the "5' endogenous DNA flap," is eventually removed and replaced by a newly synthesized replacement strand (i.e., a "3' replacement DNA flap") encoded by extension of the PE-gRNA.

[0232] 3' replacement DNA flap As used herein, the term "3' replacement DNA flap," or simply "replacement DNA flap," refers to the strand of DNA synthesized by a prime editor and encoded by the extension arm of the prime editor PEgRNA. More specifically, the 3' replacement DNA flap is encoded by the polymerase template of the PEgRNA. The 3' replacement DNA flap contains the same sequence as the 5' endogenous DNA flap, except that it also contains an edited sequence (e.g., a single nucleotide change). The 3' replacement DNA flap anneals to the target DNA, replacing or displacing the 5' endogenous DNA flap (which can be excised, for example, by a 5' flap endonuclease such as FEN1 or EXO1), and is then ligated to join the 3' end of the 3' replacement DNA flap to the exposed 5' hydroxyl end of the endogenous DNA (exposed after excision of the 5' endogenous DNA flap), thereby reforming a phosphodiester bond to incorporate the 3' replacement DNA flap and form a heteroduplex DNA containing one edited strand and one unedited strand. The DNA repair process degrades the heteroduplex and copies the information in the edited strand to the complementary strand, thereby permanently incorporating the edit into the DNA. This degradation process can be further driven to completion by nicking the unedited strand, i.e., via "second-strand nicking" as described herein. DETAILED DESCRIPTION OF THE INVENTION

[0233] DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS The present invention discloses methods for using novel compositions (e.g., novel PEgRNAs and PE complexes comprising the same) and prime editing (PE) to repair therapeutic targets (e.g., those targets identified in the Clinver database) using PEgRNAs designed using specialized algorithms described herein. Thus, the present application discloses algorithms for large-scale prediction of sequences for PEgRNAs that may be used to repair therapeutic targets (e.g., those contained in the Clinver database). Additionally, the present application discloses predicted sequences for therapeutic PEgRNAs (which may be used with prime editing to repair therapeutic targets) designed using the disclosed algorithms.

[0234] The algorithms and predicted PEgRNA sequences disclosed herein generally relate to prime editing. Thus, the present disclosure also provides a description of various components and aspects of prime editing, including suitable napDNAbp (e.g., Cas9 nickase) and reverse transcriptase, as well as other suitable components (e.g., linkers, NLSs) and PE fusion proteins, that may be used with the therapeutic PEgRNAs disclosed herein.

[0235] The adoption of the clustered regularly interspaced short palindromic repeats (CRISPR) system for genome editing has revolutionized the life sciences. 1~3While gene disruption using CRISPR is now routine, the precise integration of single-nucleotide edits remains a major challenge, despite being necessary to study or correct numerous disease-causing mutations. Homologous recombination repair (HDR) can achieve such edits but suffers from low efficiency (often <5%), the requirement for a donor DNA repair template, and the deleterious effects of double-strand DNA break (DSB) formation. Recently, Professor David Liu's laboratory developed base editing, which achieves efficient single-nucleotide edits without DSBs. Base editors (BEs) combine the CRISPR system with base-modifying deaminase enzymes to convert targeted C·G or A·T base pairs to A·T or G·C, respectively. 4~6 Although already widely used by researchers worldwide, current BEs can only correct four of 12 possible base pair changes and are unable to correct small insertions or deletions. Furthermore, the target range of base editing is limited by the editing of non-targeted C or A bases adjacent to the target base ("bystander editing") and by the requirement that a PAM sequence be present 15 ± 2 bp from the target base. Therefore, overcoming these drawbacks would significantly expand the basic research and therapeutic applications of genome editing.

[0236] This disclosure proposes a new high-precision editing approach that offers many of the benefits of base editing—namely, avoidance of double-strand breaks and a donor DNA repair template—while overcoming its major drawbacks. The proposed approach described herein achieves direct integration of an edited DNA strand at a target genomic site using target-primed reverse transcription (TPRT). In the design discussed herein, a CRISPR guide RNA (gRNA) will be modified to carry a reverse transcriptase (RT) template sequence that encodes a single-stranded DNA containing the desired nucleotide change. The target site DNA nicked by the CRISPR nuclease (Cas9) will serve as a primer for reverse transcription of the template sequence on the modified gRNA, allowing for the direct integration of any desired nucleotide edits.

[0237] Consequently, the present invention relates, in part, to the discovery that the mechanism of target-primed reverse transcription (TPRT) or "prime editing" can be exploited or employed to perform highly efficient and genetically flexible, high-precision CRISPR / Cas-based genome editing (e.g., as depicted in various embodiments in Figures 1A-1G). The inventors herein propose using a napDNAbp-polymerase fusion (e.g., Cas9 nickase fused to reverse transcriptase) to target a specific DNA sequence with a modified guide RNA (an "extended guide RNA" or PEgRNA), generate a single-stranded nick at the target site, and use the nicked DNA as a primer for DNA synthesis by a polymerase (e.g., reverse transcriptase) based on a DNA synthesis template that is a component of the PEgRNA. The newly synthesized strand will be homologous to the genomic target sequence except for the inclusion of the desired nucleotide change (e.g., a single-nucleotide change, deletion, or insertion, or a combination thereof). The newly synthesized strand of DNA, sometimes referred to as a single-stranded DNA flap, competes with the complementary homologous endogenous DNA strand for hybridization, thereby displacing the corresponding endogenous strand. Degradation of this hybridized intermediate can involve the resulting removal of the displaced flap from the endogenous DNA (e.g., by 5'-end DNA flap endonuclease, FEN1), ligation of the synthesized single-stranded DNA flap to the target DNA, and assimilation of the desired nucleotide change as a result of cellular DNA repair and / or replication processes. Because templated DNA synthesis offers high precision down to a single nucleotide, the breadth of this approach is extremely broad, and it is foreseeable that it could be used for countless applications in basic science and therapeutics.

[0238] I. Therapeutic PEgRNA The prime editor (PE) system described herein contemplates the use of any suitable prime editor guide RNA or PEgRNA. The inventors have discovered that through the use of a specially designed guide RNA containing a DNA synthesis template that encodes the desired nucleotide change via a polymerase (e.g., reverse transcriptase), the mechanism of target-primed reverse transcription (TPRT) can be exploited or employed to perform highly accurate and versatile CRISPR / Cas-based genome editing. The present application refers to this specially designed guide RNA as a "prime editor guide RNA" (or PEgRNA) because the DNA synthesis template can be provided as an extension of a standard or pre-existing guide RNA molecule. The present application contemplates any suitable configuration or arrangement for the prime editor guide RNA.

[0239] In various embodiments, the present disclosure provides therapeutic PEGRNAs of SEQ ID NOs: 1-135514 and 813085-880462 that were designed using the algorithms disclosed herein against Clinver database entries.

[0240] In various other embodiments, exemplary PEG-gRNAs designed against the Clinver database using the algorithms disclosed herein are included in the Sequence Listing, which forms a part of this specification. The Sequence Listing includes the complete PEG-gRNA sequences set forth in SEQ ID NOS: 1-135514 and 813085-880462. Each of these complete PEG-gRNAs is composed of a spacer (SEQ ID NOS: 135515-271028 and 880463-947840) and an extension arm (SEQ ID NOS: 271029-406542 and 947841-1015218), respectively. In addition, each PEG-gRNA also includes a gRNA core, e.g., as defined by SEQ ID NOS: 1361579-1361580. The extension arms of SEQ ID NOs: 271029-406542 and 947841-1015218 further comprise a primer binding site (SEQ ID NOs: 406543-542056 and 1015219-1082596), an editing template (SEQ ID NOs: 542057-677570 and 1082597-1149974), and a homology arm (SEQ ID NOs: 677571-813084 and 1149975-1217352), respectively. The PEG RNA may optionally contain a 5'-end modified region and / or a 3'-end modified region. The PEG RNA may also contain a reverse transcription termination signal (e.g., SEQ ID NOs: 1361560-1361566) at the 3' end of the PEG RNA. This application covers all designs and uses of these sequences.

[0241] Figure 3A shows one embodiment of a prime editor guide RNA (referred to as either a "PE gRNA" or an "extended gRNA") that can be used in the prime editor (PE) system disclosed herein, where the existing guide RNA (shown in green) includes a spacer and a gRNA core region and binds to the nap DNAbp. In this embodiment, the guide RNA includes an RNA segment extended at its 5' end, i.e., a 5' extension. In this embodiment, the 5' extension includes a DNA synthesis template, a primer binding site, and an optional 5- to 20-nucleotide linker sequence. As shown in Figure 1A, a hybrid of the primer binding site with the free 3' end formed after the nick forms with the non-target strand of the R-loop, thereby priming a polymerase (e.g., reverse transcriptase) for DNA polymerization in the 5' to 3' direction.

[0242] Figure 3B shows another embodiment of a prime editor guide RNA that can be used in the prime editor (PE) system disclosed herein, where the existing guide RNA (shown in green) includes a ∼20 nt spacer and a gRNA core, and binds together with the nap DNAbp. In this embodiment, the guide RNA includes an RNA segment extended at its 3' end, i.e., a 3' extension. In this embodiment, the 3' extension includes a DNA synthesis template and a primer binding site. As shown in Figure 1B, the hybrid of the primer binding site with the free 3' end formed after the nick forms on the non-target strand of the R-loop, thereby priming a polymerase (e.g., reverse transcriptase) for DNA polymerization in the 5'→3' direction.

[0243] Figure 3C shows another embodiment of an extended guide RNA that can be used in the prime editor (PE) system disclosed herein, where the existing guide RNA (green) includes a ~20 nt spacer and gRNA core, which binds together with the nap DNAbp. In this embodiment, the guide RNA includes an extended RNA segment at an intermolecular position within the gRNA core, i.e., an intramolecular extension. In this embodiment, the intramolecular extension includes a DNA synthesis template and a primer binding site. The primer binding site hybridizes to the free 3' end formed after the nick and is formed on the non-target strand of the R-loop, thereby priming a polymerase (e.g., reverse transcriptase) for DNA polymerization in the 5'-3' direction.

[0244] In one embodiment, the position of the intramolecular RNA extension is in the spacer of the guide RNA.In another embodiment, the position of the intramolecular RNA extension is in the gRNA core.In yet another embodiment, the position of the intramolecular RNA extension is anywhere in the guide RNA molecule except in the spacer, or at a position that does not interfere with the spacer.

[0245] In one embodiment, the intramolecular RNA extension is inserted downstream from the 3' end of the spacer. In another embodiment, the intramolecular RNA extension is inserted at least 1 nucleotide, at least 2 nucleotides, at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides, at least 22 nucleotides, at least 23 nucleotides, at least 24 nucleotides, or at least 25 nucleotides downstream of the 3' end of the spacer.

[0246] In other embodiments, an intramolecular RNA extension is inserted into the gRNA, which refers to a portion of the guide RNA that corresponds to or includes the tracrRNA, which binds to and / or interacts with the Cas9 protein or its equivalent (i.e., a different napDNAbp). Preferably, the insertion of the intramolecular RNA extension does not interfere with, or only minimally interferes with, the interaction of the tracrRNA portion with the napDNAbp.

[0247] The length of RNA extension can be any useful length.In various embodiments, the length of 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.

[0248] The DNA synthesis template (e.g., RT template sequence) can also be any suitable length. For example, the DNA synthesis template (e.g., RT template sequence) can be at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides, at least 80 nucleotides, at least 90 nucleotides, at least 100 nucleotides, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, or at least 500 nucleotides in length.

[0249] In still other embodiments, the reverse transcription primer binding site sequence is at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides, at least 80 nucleotides, at least 90 nucleotides, or at least 100 nucleotides in length.

[0250] In other embodiments, any linker or spacer is at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides, at least 80 nucleotides, at least 90 nucleotides, at least 100 nucleotides, at least 200 nucleotides, at least 300 nucleotides, or at least 400 nucleotides in length.

[0251] A DNA synthesis template (e.g., an RT template sequence) in some embodiments encodes a single-stranded DNA molecule that is homologous to the non-target strand (and thus complementary to the corresponding site on the target strand) but contains one or more nucleotide changes, which may include one or more single-base nucleotide changes, one or more deletions, one or more insertions, and combinations thereof.

[0252] As depicted in Figure 1E, the synthesized single-stranded DNA product of the DNA synthesis template (e.g., the RT template sequence) is homologous to the non-target strand and contains one or more nucleotide changes. The single-stranded DNA product of the DNA synthesis template (e.g., the RT template sequence) hybridizes with the complementary target strand sequence at equilibrium, thereby displacing the homologous endogenous target strand sequence. In some embodiments, the displaced endogenous strand may be referred to as a 5' endogenous DNA flap species (see, e.g., Figure 1C). This 5' endogenous DNA flap species can be removed by a 5' flap endonuclease (e.g., FEN1), and the single-stranded DNA product, now hybridized with the endogenous target strand, is ligated to create a mismatch between the endogenous sequence and the newly synthesized strand. The mismatch may be resolved by the cell's inherent DNA repair and / or replication processes.

[0253] In various embodiments, the nucleotide sequence of the DNA synthesis template (eg, the RT template sequence) corresponds to the nucleotide sequence of the non-target strand that is replaced by the 5' flap species and overlaps with the site to be edited.

[0254] In various embodiments of the prime editor guide RNA, the DNA synthesis template may encode a single-stranded DNA flap complementary to the endogenous DNA sequence adjacent to the nick site, where the single-stranded DNA flap contains the desired nucleotide change. The single-stranded DNA flap may replace the endogenous single-stranded DNA at the nick site. The endogenous single-stranded DNA replaced at the nick site may have a 5' end, forming an endogenous flap that can be excised by the cell. In various embodiments, excision of the 5' end endogenous flap can help promote product formation, as removing the 5' end endogenous flap promotes hybridization of the single-stranded 3' DNA flap with the corresponding complementary DNA strand and incorporation or assimilation of the desired nucleotide change carried by the single-stranded 3' DNA flap into the target DNA.

[0255] In various embodiments of the primed editor guide RNA, cellular repair of the single-stranded DNA flap results in the incorporation of the desired nucleotide change, thereby forming the desired product.

[0256] In still other embodiments, the desired nucleotide change is incorporated into an editing window that is a nick site between about -5 and +5, or a nick site between about -10 and +10, or a nick site between about -20 and +20, or a nick site between about -30 and +30, or a nick site between about -40 and +40, or a nick site between about -50 and +50, or a nick site between about -60 and +60, or a nick site between about -70 and +70, or a nick site between about -80 and +80, or a nick site between about -90 and +90, or a nick site between about -100 and +100, or a nick site between about -200 and +200.

[0257] In various aspects, prime editor guide RNA is the modified version of guide RNA.Guide RNA can be naturally occurring, can be expressed from encoding nucleic acid, or can be chemically synthesized.The method for obtaining or otherwise synthesizing guide RNA, which comprises the spacer that interacts and hybridizes with the target strand of the target site of genome of interest, and the method for determining the appropriate sequence of guide RNA are well known in the art.

[0258] In various embodiments, the specific design aspects of the guide RNA sequence will depend on the nucleotide sequence of the genomic target site of interest (i.e., the desired site to be edited), among other factors such as the location of the PAM sequence, the percent G / C content in the target sequence, the extent of microhomologous regions, secondary structure, etc., and the type of napDNAbp (e.g., Cas9 protein) present in the prime editor (PE) system described herein.

[0259] Generally, a guide sequence is any polynucleotide sequence that has sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of a napDNAbp (e.g., a Cas9, a Cas9 homolog, or a Cas9 variant) to the target sequence. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence is about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more when optimally aligned using a suitable alignment algorithm. Optimal alignment may be determined using any suitable algorithm for aligning sequences, including, but not limited to, algorithms based on the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, the Burrows-Wheeler Transform (e.g., the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies), ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net). In some embodiments, the guide sequence is about 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, 35, 40, 45, 50, 75 or more nucleotides in length.

[0260] In some embodiments, the guide sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12 nucleotides in length, or shorter. The ability of a guide sequence to direct sequence-specific binding of a base editor to a target sequence may be assessed by any suitable assay. For example, base editor components (including the guide sequence to be tested) may be provided to a host cell having the corresponding target sequence, such as by transfection with a vector encoding the base editor components disclosed herein, followed by assessment of preferential cleavage within the target sequence, such as by a Surveyor assay as described herein. Similarly, cleavage of a target polynucleotide sequence may be assessed in a test tube by providing the target sequence, the base editor components (including the guide sequence to be tested), and a control guide sequence that differs from the test guide sequence, and comparing the binding or cleavage rate at the target sequence of the reaction between the test guide sequence and the control guide sequence. Other assays may also be performed and will occur to those of skill in the art.

[0261] The guide sequence may be selected to target any target sequence. In some embodiments, the target sequence is a sequence within the genome of a cell. Exemplary target sequences include sequences that are unique in the target genome. For example, for S. pyogenes Cas9, a unique target sequence in the genome may include a Cas9 target site of the form MMMMMMMMNNNNNNNNNNNNNXGG (SEQ ID NO: 1361548), where NNNNNNNNNNNNXGG (SEQ ID NO: 1361549) (N is A, G, T, or C; and X can be anything) has only one occurrence in the genome. A unique target sequence in the genome may include an S. pyogenes Cas9 target site of the form MMMMMMMMMMNNNNNNNNNNNXGG (SEQ ID NO: 1361550), where NNNNNNNNNNNXGG (SEQ ID NO: 1361551) (N is A, G, T, or C; and X can be anything) has only one occurrence in the genome. For S. thermophilus CRISPR1Cas9, a unique target sequence in the genome may include a Cas9 target site of the form MMMMMMMMNNNNNNNNNNNNXXAGAAW (SEQ ID NO: 1361552), where NNNNNNNNNNNNXXAGAAW (SEQ ID NO: 1361553) (N is A, G, T, or C; X can be anything; and W is A or T) has only one occurrence in the genome. A unique target sequence in the genome may encompass a S. thermophilus CRISPR1 Cas9 target site of the form MMMMMMMMMNNNNNNNNNNNXXAGAAW (SEQ ID NO: 1361554), where NNNNNNNNNNNXXAGAAW (SEQ ID NO: 1361555) (N is A, G, T, or C; X is anything; and W is A or T) has only one occurrence in the genome.For S. pyogenes Cas9, a unique target sequence in a genome may include a Cas9 target site of the form MMMMMMMMNNNNNNNNNNNXGGXG (SEQ ID NO: 1361556), where NNNNNNNNNNNNXGGXG (SEQ ID NO: 1361557) (N is A, G, T, or C; and X can be anything) has only one occurrence in the genome. A unique target sequence in a genome may include an S. pyogenes Cas9 target site of the form MMMMMMMMMMNNNNNNNNNNNXGGXG (SEQ ID NO: 1361558), where NNNNNNNNNNNXGGXG (SEQ ID NO: 1361559) (N is A, G, T, or C; and X can be anything) has only one occurrence in the genome. In each of these sequences, "M" may be A, G, T, or C and need not be considered when identifying a sequence as unique.

[0262] In some embodiments, the guide sequence is selected to reduce the degree of secondary structure within the guide sequence. The secondary structure may be determined by any suitable polynucleotide folding algorithm. Some programs are based on calculating the minimum Gibbs free energy. One example of such an algorithm is mFold, as described by Zuker and Stiegler (Nucleic Acids Res. 9 (1981), 133-148). Another example folding algorithm is the online web server RNAfold, developed at the Institute for Theoretical Chemistry of the University of Vienna, which uses a centroid structure prediction algorithm (see, for example, A.R. Gruber et al., 2008, Cell 106(1):23-24; and P.A. Carr and G.M. Church, 2009, Nature Biotechnology 27(12):1151-62). Further algorithms may be found in US Application No. 61 / 836,080; Broad Reference BI-2013 / 004A) (hereby incorporated by reference).

[0263] Generally, a tracr mate sequence encompasses any sequence that has sufficient complementarity with a tracr sequence to promote one or more of the following: (1) excision of the guide sequence flanked by the tracr mate sequence in cells containing the corresponding tracr sequence; and (2) formation of a complex with the target sequence, wherein the complex comprises the tracr mate sequence hybridized with the tracr sequence. Generally, the degree of complementarity refers to optimal alignment of the tracr mate sequence and the tracr sequence along the shorter length of the two sequences. Optimal alignment may be determined by any suitable alignment algorithm and may further allow for the formation of secondary structure, such as self-complementarity, within either the tracr sequence or the tracr mate sequence. In some embodiments, the degree of complementarity between a tracr sequence and a tracr mate sequence, when optimally aligned along the shorter length of the two sequences, is about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99%, or greater. In some embodiments, the tracr sequence is about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or more nucleotides in length. In some embodiments, the tracr sequence and the tracr mate sequence are contained within a single transcript such that hybridization between the two produces a transcript with a secondary structure, such as a hairpin. A preferred loop-forming sequence for use in a hairpin structure is 4 nucleotides in length and most preferably has the sequence GAAA. However, longer or shorter loop sequences may also be used, as may alternative sequences. The sequence preferably includes a nucleotide triplet (e.g., AAA) and an additional nucleotide (e.g., C or G). Examples of loop-forming sequences include CAAA and AAAG. In embodiments of the present invention, a transcript or transcribed polynucleotide sequence has at least two or more hairpins. In a preferred embodiment, the transcript has 2, 3, 4 or 5 hairpins.In further embodiments of the invention, the transcript has at most five hairpins. In some embodiments, the single transcript further comprises a transcription termination sequence; preferably, this comprises a poly-T sequence, for example, six T nucleotides.Further non-limiting examples of single polynucleotides comprising a guide sequence, a tracr mate sequence, and a tracr sequence are as follows (listed 5' to 3'), where "N" represents the bases of the guide sequence, the first block of lowercase letters represents the tracr block sequence, and the second block of lowercase letters represents the tracr sequence, and the final poly-T sequence represents the transcription terminator: (1) NNNNNNNNgtttttgtactctcaagatttaGAAAtaaatcttgcagaagct acaaagataaggcttcatgccgaaatcaacaccctgtcattttatggcagggtgttttcgttatttaaTTTTTT (SEQ ID NO: 1361560); (2) NNNNNNNNNNNNNNNNNNgtttttgtactctcaGAAAtgcagaagctacaaagataaggcttcatgccgaaatcaacaccctgtcattttatggcagggtgttttcgttatttaaTTTTTT (SEQ ID NO: 1361561 );(3)NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaGAAAtgcagaagctacaaagataaggcttcatgccgaaatcaacaccctgtcattttatggcagggtgtTTT TT (SEQ ID NO: 1361562); (4)NNNNNNNNNNNNNNNNNNNNgttttagagctaGAAAtagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcgg (5) NNNNNNNNNNNNNNNNNNNNgttttagagctaGAAATAGcaagttaaaataaggctagtccgttatcaacttgaaaaagtgTTTTTTT (SEQ ID NO: 1361564); and (6) NNNNNNNNNNNNNNNNNNNNgttttagagctagAAATAGcaagttaaaataaggctagtccgttatcaTTTTTTTT (SEQ ID NO: 1361565). In some embodiments, sequences (1)-(3) are used in combination with Cas9 from S. thermophilus CRISPR1.In some embodiments, sequences (4)-(6) are used in combination with Cas9 from S. pyogenes. In some embodiments, the tracr sequence is a separate transcript from the transcript containing the tracr mate sequence.

[0264] It will be apparent to one of skill in the art that any of the fusion proteins as disclosed herein comprising a Cas9 domain and a single-stranded DNA-binding protein typically require co-expression of a guide RNA (e.g., an sgRNA) to target the fusion protein to a target site (e.g., a site containing a point mutation to be edited). As described in more detail elsewhere herein, the guide RNA typically comprises a tracrRNA framework that allows for Cas9 binding and a guide sequence that confers sequence specificity to the Cas9:nucleic acid editing enzyme / domain fusion protein.

[0265] In some embodiments, the guide RNA comprises the structure 5'-[guide sequence]-guuuuagagcuagaaauagcaaguuaaaauaaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuuu-3' (SEQ ID NO: 1361566), where the guide sequence comprises a sequence complementary to the target sequence. Guide sequences are typically 20 nucleotides in length. Suitable guide RNA sequences for targeting Cas9:nucleic acid editing enzyme / domain fusion proteins to specific genomic target sites will be apparent to those of skill in the art based on this disclosure. Such suitable guide RNA sequences typically comprise a guide sequence complementary to a nucleic acid sequence within 50 nucleotides upstream or downstream of the target nucleotide to be edited. Some exemplary guide RNA sequences suitable for targeting the provided fusion proteins to specific target sequences are provided herein. Additional guide sequences are known in the art and can be used with the base editors described herein.

[0266] In other embodiments, the PEGRNA may include those depicted by the structure shown in Figure 27, including the guide RNA and 3' extension arm.

[0267] Figure 27 provides the structure of one embodiment of a PEgRNA that may be designed according to the methodology contemplated herein and defined in Example 2. The PEgRNA comprises three major components ordered in the 5' to 3' direction: a spacer, a gRNA core, and an extension arm at the 3' end. The extension arm may be further divided into the following structural elements in the 5' to 3' direction: a primer binding site (A), an editing template (B), and a homology arm (C). In addition, the PEgRNA may comprise an optional 3'-end modification region (e1) and an optional 5'-end modification region (e2). Furthermore, the PEgRNA may also comprise a transcription termination signal at the 3' end of the PEgRNA (not depicted). These structural elements are further defined herein. The depiction of the PEgRNA structure is not intended to be limiting and encompasses variations in the arrangement of elements. For example, any sequence modifiers (e1) and (e2) may be located within or between any of the other regions shown and are not limited to being located at the 3' and 5' ends.

[0268] In still other embodiments, the PEGRNA may include those depicted by the structure shown in Figure 28, which includes a guide RNA and a 5' extension arm.

[0269] Figure 28 provides the structure of another embodiment of a PEGRNA that may be designed according to the methodology contemplated herein and defined in Example 2. The PEGRNA comprises three major components ordered in the 5' to 3' direction: a spacer, a gRNA core, and an extension arm at the 3' end. The extension arm may be further divided in the 5' to 3' direction into the following structural elements: a primer binding site (A) (SEQ ID NOS: 406543-542056 and 1015219-1082596), an editing template (B) (SEQ ID NOS: 542057-677570 and 1082597-1149974), and a homology arm (C) (SEQ ID NOS: 677571-813084 and 1149975-1217352). In addition, the PEGRNA may comprise an optional 3'-end modification region (e1) and an optional 5'-end modification region (e2). Furthermore, the PEGRNA may also include a transcription termination signal at the 3' end of the PEGRNA (not depicted). These structural elements are further defined herein. The depiction of the PEGRNA structure is not intended to be limiting and encompasses variations in the placement of elements. For example, any of the sequence modifiers (e1) and (e2) may be located within or between any of the other regions shown and are not limited to being located at the 3' and 5' ends.

[0270] PEgRNAs may also incorporate additional design improvements that may modify the properties and / or characteristics of the PEgRNA, thereby improving the efficacy of prime editing. In various embodiments, these improvements may fall into one or more of several different categories, including, but not limited to: (1) designs that enable efficient expression of functional PEgRNAs from non-polymerase III (pol III) promoters (which may allow expression of longer PEgRNAs without cumbersome sequence requirements); (2) improvements to the core, Cas9-binding PEgRNA backbone (which may improve efficacy); (3) modifications to the PEgRNA that improve RT processivity (which may allow insertion of longer sequences at targeted genomic loci); and (4) the addition of RNA motifs to the 5' or 3' end of the PEgRNA (which may improve PEgRNA stability, enhance RT processivity, prevent PEgRNA misfolding, or recruit additional factors important for genome editing).

[0271] In one embodiment, PEGRNAs can be designed with a pol III promoter to improve expression of longer PEGRNAs with larger extension arms. sgRNAs are typically expressed from the U6 snRNA promoter. This promoter recruits pol III to express the associated RNA and is useful for expressing short RNAs that are retained in the nucleus. However, pol III has limited processivity and cannot express RNAs longer than several hundred nucleotides at the levels required for efficient genome editing. In addition, pol III may stall or terminate at stretches of U's, potentially limiting the sequence diversity that can be inserted using PEGRNAs. Other promoters that recruit polymerase II (e.g., pCMV) or polymerase I (e.g., the U1 snRNA promoter) have also been investigated for their ability to express longer sgRNAs. However, these promoters are typically partially transcribed, resulting in extra sequence 5' of the spacer in the expressed PEGRNA, which has been shown to result in significantly reduced Cas9:sgRNA activity in a site-dependent manner. Additionally, Pol III-transcribed PEG RNAs can simply terminate at the six- to seven-U stretch, whereas PEG RNAs transcribed from Pol II or Pol I will require different termination signals. Often, such signals also result in polyadenylation, which will result in the undesired export of PEG RNAs from the nucleus. Similarly, RNAs expressed from Pol II promoters (such as pCMV) are typically 5'-capped, which also results in their nuclear export.

[0272] Previously, Rinn and colleagues screened various expression platforms for the production of long non-coding RNA (lncRNA)-tagged sgRNAs. 183 These platforms were expressed from pCMV and contained the ENE element from human MALAT1 ncRNA. 184 , PAN ENE element from KSHV 185, or the 3' box from U1 snRNA 186 Notably, the MALAT1 ncRNA and the PAN ENE form a triple helix that protects the poly(A) tail. 184,187 These constructs may also enhance RNA stability. It is contemplated that these expression systems will also allow for the expression of longer PEG RNAs.

[0273] In addition, a series of methods are designed for cleavage of the portion of the pol II promoter that will be transcribed as part of the PEG RNA, using a self-cleaving ribozyme (hammerhead 188 ,pistol 189 , Hatchet 189 ,hairpin 190 , VS 191 , Twister 192 , or Twister Sister 192 ribozymes) or other self-cleaving elements that process the transcribed guide, or hairpins that are recognized by Csy4 and also lead to guide processing. 193 In addition, the incorporation of multiple ENE motifs has been demonstrated for KSHV PAN RNA and elements. 185 It is also hypothesized that circularizing PEG RNA into a circular intronic RNA (ciRNA) form may also lead to enhanced RNA expression and stability, as well as nuclear localization. 194 .

[0274] In various embodiments, the PEGRNA may include various elements above, as exemplified by the following sequences:

[0275] Non-limiting Example 1 - PEGRNA Expression Platform Consisting of pCMV, Csy4 Hairpin, PEGRNA, and MALAT1 ENE (query number 1361567)

[0276] Non-limiting Example 2—PEG-RNA Expression Platform Consisting of pCMV, Csy4 Hairpin, PEG-RNA, and PAN ENE (query number 1361568)

[0277] Non-limiting Example 3 - PEGRNA Expression Platform Consisting of pCMV, Csy4 Hairpin, PEGRNA, and 3xPAN ENE

[0278] Non-limiting Example 4 - A PEgRNA expression platform consisting of pCMV, Csy4 hairpin, PEgRNA, and 3' box TAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTGGTTTAGTGAACCGTCAGATCGTTCACTGCCGTATAGGCAGGGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCCTCTGCCATCAAAGCGTGCTCAGTCTGTTTGTTTCAAAAGTAGACTGTACGCTAAGGGTCATATCTTTTTTTGTTTGGTTTGTGTCTTGGTTGGCGTCTTAAA (SEQ ID NO: 1361570)

[0279] Non-limiting Example 5 - PEG-RNA Expression Platform Consisting of pU1, Csy4 Hairpin, PEG-RNA, and 3' Box (SEQ ID NO: 1361571)

[0280] In various other embodiments, the PEG RNA may be improved by introducing improvements into the backbone or core sequence, which may be done by introducing known improvements.

[0281] The core, Cas9-bound PEGRNA backbone could conceivably also be improved to enhance PE activity. Several such approaches have already been demonstrated. Illustratively, the first pairing element (P1) of the backbone contains a GTTTT-AAAAC pairing element. Such a stretch of T's has been shown to result in pol III pausing and premature termination of the RNA transcript. Rational mutation of one of the TA pairs in this P1 region to a GC pair has been shown to enhance sgRNA activity, suggesting that this approach may also be feasible for PEGRNA. 195 In addition, increasing the length of P1 has also been shown to enhance sgRNA folding, leading to improved activity. 195 This is suggested as another avenue for improving PEG RNA activity. Examples of improvements to the core may include:

[0282] PEGRNA containing a 6-nt extension to P1 GGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGCTCATGAAAATGAGCTAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCCTCTGCCATCAAAGCGTGCTCAGTCTGTTTTTTT (SEQ ID NO: 1361572)

[0283] PEgRNA containing a TA→GC mutation in P1 GGCCCAGACTGAGCACGTGAGTTTGAGAGCTAGAAATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCCTCTGCCATCAAAGCGTGCTCAGTCTGTTTTTTT (SEQ ID NO: 1361573)

[0284] In various other embodiments, PEGRNAs may be improved by introducing modifications into the editing template region. As the size of the PEGRNA-templated insert increases, it is more likely to be degraded by endonucleases, undergo spontaneous hydrolysis, or fold into a secondary structure (which cannot be reverse transcribed by RT or which interferes with the folding of the PEGRNA backbone and subsequent Cas9-RT binding). Consequently, modifications to the PEGRNA template may be necessary to affect large insertions, such as the insertion of entire genes. Some strategies for doing so include incorporating modified nucleotides into synthetic or semi-synthetic PEGRNAs (making the RNA more resistant to degradation or hydrolysis, or less likely to adopt inhibitory secondary structures). 196Such modifications may include 8-aza-7-deazaguanosine, which will reduce RNA secondary structure in G-rich sequences; locked nucleic acids (LNAs), which reduce degradation and enhance certain RNA secondary structures; and 2'-O-methyl, 2'-fluoro, or 2'-O-methoxyethoxy modifications, which enhance RNA stability. Such modifications may also be included elsewhere in the PEgRNA to enhance stability and activity. Alternatively, or in addition, the PEgRNA template may be designed to encode the desired protein product while also increasing the likelihood of adopting a simple secondary structure that cannot be folded by RT. Such a simple structure would act as a thermodynamic sink, reducing the likelihood of more complex structures that would prevent reverse transcription. Finally, the template may also be split into two separate PEgRNAs. In such a design, PE would be used to initiate transcription and also recruit a separate template RNA to the targeted site, either via an RNA-binding protein fused to Cas9 or an RNA recognition element (such as the MS2 aptamer) on the PEgRNA itself. RT could either directly bind to this separate template RNA or initiate reverse transcription on the original PEgRNA before swapping to the second template. Such an approach would not only prevent misfolding of the PEgRNA upon addition of a long template, but also enable long insertions by not requiring dissociation of Cas9 from the genome to generate long insertions (which may potentially inhibit PE-based long insertions).

[0285] (iv) Incorporation of additional RNA motifs at the 5' or 3' ends In yet other embodiments, the PEG RNA may be improved by introducing additional RNA motifs at the 5' or 3' end of the PEG RNA. Several such motifs—such as the PAN ENE from KSHV and the ENE from MALAT1—were discussed above as a means of potentially terminating expression of longer PEG RNAs from non-pol III promoters. These elements form an RNA triple helix that engulfs the polyA tail, resulting in their retention in the nucleus. 184,187 However, by forming a complex structure at the 3' end of the PEGRNA that occludes the terminal nucleotide, these structures may also serve to prevent exonuclease-mediated degradation of the PEGRNA.

[0286] Other structural elements inserted at the 3' end may also enhance RNA stability without allowing termination from non-pol III promoters. Such motifs include hairpins or RNA quadruplexes that would occlude the 3' end. 197 or a self-cleaving ribozyme such as HDV, which results in the formation of a 2'-3'-cyclic phosphate at the 3' end and potentially also makes the PEG RNA less susceptible to exonucleolytic degradation. 198 Inducing PEgRNA to circularize through incomplete splicing—forming ciRNA—may also increase PEgRNA stability, resulting in PEgRNA retention in the nucleus. 194 .

[0287] Additional RNA motifs may also improve RT processivity or enhance PEGRNA activity by enhancing RT binding to the DNA-RNA duplex. Addition of native sequences bound by RT in its cognate retroviral genome can enhance RT activity. 199 This may encompass a native primer binding site (PBS), polypurine tract (PPT), or kissing loop, which are involved in retroviral genome dimerization and transcription initiation. 199 .

[0288] Dimerization chief-kissing loop or GNRA tetraloop / tetraloop receptor pair 200 The addition of motifs such as (e.g., (I)-(I)-(I)) at the 5' and 3' ends of the PEgRNA can also result in efficient circularization of the PEgRNA, improving its stability. In addition, it is conceivable that the addition of these motifs can allow for physical separation of the PEgRNA spacer and primer, preventing spacer blockage that would otherwise interfere with PE activity. A short 5' extension to the PEgRNA, forming a small toehold hairpin in the spacer region, can also preferably compete for the annealing region of the PEgRNA-binding spacer. Finally, kissing loops can also be used to recruit other template RNAs to genomic sites and to allow RT activity to be switched from one RNA to another. Examples of improvements include, but are not limited to, the following:

[0289] PEGRNA-HDV fusions GGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCCTCTGCCATCAAAGCGTGCTCAGTCTGGGCCGGCATGGTCCCAGCCTCCTCGCTGGCGCCGGCTGGGCAACATGCTTCGGCATGGCGAATGGGACTTTTTTT (SEQ ID NO: 1361574)

[0290] PEGRNA-MMLV kissing loop GGTGGGAGACGTCCCACCGGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCCTCTGCCATCAAAGCTTCGACCGTGCTCAGTCTGGTGGGAGACGTCCCACCTTTTTT (SEQ ID NO: 1361575)

[0291] PEGRNA-VS ribozyme kissing loop GAGCAGCATGGCGTCGCTGCTCACGGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCCTCTGCCATCAAAGCTTCGACCGTGCTCAGTCTCCATCAGTTGACACCCTGAGGTTTTTTT (SEQ ID NO: 1361576)

[0292] PEG-RNA-GNRA tetraloop / tetraloop receptor GCAGACCTAAGTGGUGACATATGGTCTGGGCCCAGACTGAGCACGTGAGTTTTAGAGCTAUACGTAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTUACGAAGTGGGACCGAGTCGGTCCTCTGCCATCAAAGCTTCGACCGTGCTCAGTCTGCATGCGATTAGAAATAATCGCATGTTTTTTT (SEQ ID NO: 1361577)

[0293] PEG RNA template switching secondary RNA-HDV fusion TCTGCCATCAAAGCTGCGACCGTGCTCAGTCTGGTGGGAGACGTCCCACCGGCCGGCATGGTCCCAGCCTCCTCGCTGGCGCCGGCTGGGCAACATGCTTCGGCATGGCGAATGGGACTTTTTTT (SEQ ID NO: 1361578)

[0294] The PEgRNA backbone can be further improved through directed evolution, in a manner similar to how SpCas9 and base editors have been improved. Directed evolution can enhance PEgRNA recognition by Cas9 or evolved Cas9 variants. In addition, different PEgRNA backbone sequences are likely optimal at different genomic loci, either enhancing PE activity at the site of interest, reducing off-target activity, or both. Finally, evolution of the PEgRNA backbone with additional RNA motifs will almost always improve the activity of the fused PEgRNA compared to the unevolved fusion RNA. As an illustration, evolution of an allosteric ribozyme composed of a c-di-GMP-I aptamer and a hammerhead ribozyme led to dramatically improved activity. 202 , suggesting that evolution may also improve the activity of hammerhead-PEG RNA fusions. Additionally, although Cas9 currently does not generally tolerate 5' extensions of sgRNAs, directed evolution may generate mutations that allow for alleviation of this intolerance, potentially allowing additional RNA motifs to be utilized.

[0295] The present disclosure contemplates any such manner of further improving the effectiveness of the prime editing system disclosed herein.

[0296] II. Algorithms and methods for designing therapeutic PEG RNAs As described herein, the inventors have discovered and understood that prime editing using PEgRNAs can be used to incorporate a wide variety of nucleotide changes (including insertions (of any length, including entire genes or protein-coding regions), deletions (of any length)) and correct pathogenic mutations. However, no techniques yet exist for determining and / or predicting PEgRNA structures, including identifying various components of PEgRNAs, such as spacers, gRNA cores, and extension arms (and the component of extension as described herein). The inventors have developed a computational technique for determining PEgRNAs, including determining extended gRNA structures. Each extended gRNA structure can be determined based on an input allele (e.g., representing a pathogenic mutation), an output allele (e.g., representing a corrected wild-type sequence), and a fusion protein (e.g., a CRISPR system for prime editing, including the relative positions of the PAM motif and the nick of the prime editor). The difference between the input allele and the output allele represents the desired edit (e.g., a single nucleotide change, an insertion, a deletion, and / or the like). The determined structure can be created and used to perform base edits that change the input allele to the output allele, as further described herein.

[0297] Figure 31 is a flowchart illustrating an exemplary high-level computational method 3100 for determining an extended gRNA structure, according to some embodiments. In step 3102, a computational device (e.g., computational device 3400 described in conjunction with Figure 34) accesses data indicating input alleles, output alleles, and fusion proteins comprising a nucleic acid-programmable DNA-binding protein and a reverse transcriptase. While step 3102 is described as accessing all three of the input alleles, output alleles, and fusion proteins in one step, it should be understood that this is for illustrative purposes only and that such data may be accessed using one or more steps without departing from the spirit of the techniques described herein. Accessing the data may include receiving data, storing data, accessing a database, and / or the like.

[0298] In step 3104, the computational device determines an extended gRNA structure based on the input allele, output allele, and fusion protein accessed in step 3102. The extended gRNA structure is designed to be associated with a fusion protein that alters the input allele to the output allele. When complexed with the extended gRNA, the fusion protein is capable of binding to the target DNA sequence, including the altered target strand, and the complementary non-target strand. As described herein, the input allele may represent a pathogenic DNA mutation, and the output allele may represent a corrected DNA sequence.

[0299] Changing the input allele to the output allele can involve a single nucleotide change, an insertion of one or more nucleotides, a deletion of one or more nucleotides, and / or any other change designed to obtain an output allele. In particular, exemplary classes of edits that can be induced by a single PEG RNA include single nucleotide substitutions, insertions of 1 nt up to approximately 40 nt, deletions of 1 nt up to approximately 30 nt, and combinations thereof. For example, prime editing can support these types of changes from spacer position -3 (e.g., immediately 3' from the nick) to spacer position +27 (e.g., 30 nt 3' from the nick in the input allele). Other positions can also be used. For example, editing at spacer position -4 can be performed using an SpCas9 system with prime editing (e.g., this can be caused by accidental RuvC cleavage between spacer positions -5 and -4). The type of alteration, the number of nt changes, and / or the location of the alteration may be configurable parameter(s) that computational techniques may use to determine the extended gRNA structure.

[0300] As discussed in conjunction with Figures 3A-3B and 27-28, the extended gRNA may include various components, such as a spacer for the extended gRNA complementary to the target nucleotide sequence in the input allele, a gRNA backbone for interacting with the fusion protein, and an extension. Further referring to step 3104, the computational device determines one or more of the spacer, gRNA backbone, and extension. While in some embodiments, the technique may involve determining any combination of spacer, gRNA backbone, and / or extension, in some embodiments, one or more of such components and / or aspects of such components are known (e.g., predetermined, pre-specified, determined, etc.) and therefore may not be determined as part of step 3104.

[0301] As described herein, gRNA extension can include various components. For example, as shown in Figures 3A-3B and 27-28, extension can include one or more of an RT template (including an RT editing template and homology arms), a primer binding site, an RT termination signal, an optional 5'-end modification region, and an optional 3'-end modification region. Figure 32 is a flowchart illustrating an exemplary computational method 3200 for determining components of an extended gRNA structure (including components of extension), according to some embodiments. It should be understood that Figure 32 is intended to be illustrative, and thus the technique used to determine an extended gRNA can include more or fewer steps than those shown in Figure 32.

[0302] In step 3202, the computational device determines a set of protospacers that are compatible with the PAM motif of the selected CRISPR system in the input allele on both strands. In some embodiments, the computational device first determines a set of protospacers and removes protospacers whose associated nick positions are incompatible with priming edits to the output allele to generate a set of remaining candidate protospacers. For example, the computational device may determine that a protospacer is incompatible because the nick is 3' to the desired edit on the strand. As another example, the computational device may determine that the spacing between the nick and the desired edit is too large (e.g., greater than a user-defined threshold, e.g., 30 nt, 35 nt, etc.).

[0303] In step 3204, the computing device selects a protospacer from the set of determined protospacers. In step 3206, the computing device determines a spacer and editing template sequence using the protospacer sequence of the input allele, the location of the nick, and the sequence of the desired edit. The spacer may comprise a nucleotide sequence of approximately 20 nucleotides.

[0304] In step 3208, the computing device selects one or more sets of parameters, where each set of parameters includes a primer binding site length value (which may vary in nt, e.g., from approximately 8 nt to 17 nt), a homology arm length value (which may vary in nt, e.g., from approximately 2 nt to 33 nt), and a gRNA backbone sequence. For example, the gRNA backbone sequence can be GTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 1361579), GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 1361580), and / or other gRNA backbone sequences, such as gRNA backbone sequences that retain wild-type RNA secondary structure.

[0305] In step 3210, the computing device selects the set of parameters determined in step 3208. In step 3212, the computing device uses the selected set of parameters to determine the homology arms, primer binding site sequence, and gRNA backbone. In step 3214, the computing device then forms a PEgRNA sequence by concatenating the spacer, gRNA backbone, and PEgRNA extension arm (which includes the homology arms and editing template). In addition, the extension arm may include a terminator signal, which is a sequence that triggers termination of reverse transcription. Such a terminator sequence may include, for example, TTTTTGTTTT (SEQ ID NO: 1361581). In some embodiments, the PEgRNA extension arm may be considered to include a termination signal. In other embodiments, the PEgRNA extension arm may be considered to exclude the termination signal, but instead the extension arm is attached to the termination signal as an element outside the extension arm.

[0306] The method 3200 proceeds to step 3216, where the computing device determines whether there are more sets of parameters. If yes, the method proceeds to step 3210, where the computing device selects another set of parameters. If no, the method proceeds to step 3218, where the computing device determines whether there are more protospacers. If yes, the method returns to step 3204 where the computing device selects another protospacer from the set of protospacers. If no, the method proceeds to step 3220 and ends.

[0307] As described herein, the DNA synthesis template for extension (e.g., an RT template sequence) includes the desired nucleotide change that changes the input allele to the output allele, and includes an RT editing template (e.g., determined in step 3206) and a homology arm (e.g., determined in step 3212). Also as described herein, the DNA synthesis template (e.g., an RT template sequence) encodes a single-stranded DNA flap that is complementary to the endogenous DNA sequence adjacent to the nick site. The single-stranded DNA flap includes the desired nucleotide change (e.g., a single nucleotide change, an insertion of one or more nucleotides, a deletion of one or more nucleotides, and / or the like). In some base editing deployments, the single-stranded DNA flap can hybridize with the endogenous DNA sequence adjacent to the nick site that incorporates the desired nucleotide change. In some base editing deployments, the single-stranded DNA flap replaces the endogenous DNA sequence adjacent to the nick site. Cellular repair of the single-stranded DNA flap can result in incorporation of the desired nucleotide change to form the desired output allele product. The DNA synthesis template (e.g., the RT template sequence) can have a variable number of nucleotides, ranging from approximately 7 nucleotides to 34 nucleotides.

[0308] Although not shown in Figure 32, the computational device can be configured to determine other components of the extended gRNA. For example, in some embodiments, the computational device is configured to determine an RT stop signal adjacent to the RT template. In some embodiments, the computational device can be configured to determine a first modifier adjacent to the RT stop signal. In some embodiments, the computational device is configured to determine a second modifier adjacent to the primer binding site.

[0309] The extended gRNA components can be arranged in various configurations, such as those shown in Figures 3A-3B and 27-28. For example, with reference to Figure 3A, the extension is at the 5' end of the extended gRNA structure, and the spacer is 3' to the extension and 5' to the gRNA core. As another example, with reference to Figure 3B, the spacer is at the 5' end of the extended gRNA structure (and is 5' to the gRNA core), and the extension is at the 3' end of the extended gRNA structure (and is 3' to the gRNA core).

[0310] In some embodiments, the computing device accesses a database containing a set of input alleles and associated output alleles. For example, the computing device may access a database provided by Clinver that contains hundreds of thousands of mutations, each of which includes an allele representing a pathogenic mutation and an allele representing a corrected wild-type sequence. Techniques may be used to determine one or more extended gRNA structures for each database entry. Figure 33 is a flowchart illustrating an exemplary computer-implemented method 3300 for determining a set of extended gRNA structures for each mutation entry in a database, according to some embodiments. At step 3302, the computing device accesses a database (e.g., the Clinver database) containing a set of mutation entries, each of which includes an input allele representing a mutation and an output allele representing a corrected wild-type sequence.

[0311] At step 3304, the computational device accesses a set of one or more fusion proteins. In some embodiments, the technique may involve generating a set of extended gRNA structures for a single fusion protein and / or for a combination of different fusion proteins (e.g., for different Cas9 proteins). The computational device may be configured to access data representing multiple fusion proteins and may create a set of extended gRNA structures for each fusion protein (e.g., Cas9-NG protein and SpCas9 protein) as described herein.

[0312] In step 3306, the computing device selects a fusion protein from the set of fusion proteins. In step 3308, the computing device selects a mutant entry from the set of entries in the database. The computing device may be configured, for example, to iterate through each entry in the database to create a set of extended gRNA structures for that entry (e.g., one set for a specific fusion protein and / or multiple sets for each of a plurality of fusion proteins). In some embodiments, the computing device may be configured to generate extended gRNA structures for a subset (e.g., a predetermined set) of the set of entries in the database, a set of most significant mutations (e.g., those with known therapeutic benefit), and / or the like. In some embodiments, if the database includes entries that are not compatible with some fusion proteins for prime editing, the computing device may be configured to use the fusion protein selected from step 3304 to determine which entries in the database may be compatible with prime editing, and select entries that are compatible with the fusion protein selected in step 3308.

[0313] At step 3310, the computing device determines a set of one or more extended gRNA structures using techniques described herein. The method proceeds from step 3310 to step 3312, where the computing device determines whether there are any additional entries in the database. If yes, the computing device returns to step 3308 to select another entry. If no, the computing device proceeds to step 3314 to determine whether there are more fusion proteins. If yes, the computing device returns to step 3306 to select another fusion protein. If no, the computing device proceeds to step 3316 to end method 3300.

[0314] In some embodiments, techniques can be used to design PE gRNAs with gRNA extensions that contain non-complementary sequences (such as non-complementary sequences 5' to the homologous arms, 3' to the primer binding site, or both). For example, the non-complementary sequences can be designed to form kissing loop interactions and / or the like that act as protective hairpins for RNA stability.

[0315] In some embodiments, PEGRNAs may be designed using a strategy to prioritize among multiple design candidates. For example, techniques may be designed to avoid PEGRNA extensions in which the 5'-most nucleotide is a cytosine (e.g., due to blocking native nucleotide-protein interactions in the sgRNA:Cas9 complex). As another example, techniques may use RNA secondary structure prediction tools to select preferred PBS lengths, flap lengths, and / or the like based on other parameters of the extended gRNA (such as protospacer, desired editing, and / or the like).

[0316] An exemplary implementation of the computational techniques described herein for determining extended gRNA structures is as follows:

number

number

number

number

number

number

[0317] The exemplary sequence listings submitted herewith were generated using the techniques described herein, using the Climbor database for input alleles and corresponding output alleles. Entries in the Climbor database were first filtered for germline mutations annotated as pathogenic or pathogenic-like. For these examples, Cas9-NG and SpCas9 were used to identify compatible mutations. Of the filtered mutations, approximately 72,020 unique Climbor mutations were identified as compatible with prime editing by Cas9-NG, and approximately 63,496 unique Climbor mutations were identified as compatible with prime editing by SpCas9 with an NGG PAM. It should be understood that other and / or additional mutations may be correctable using prime editors containing different Cas9 variants with different PAM compatibility.

[0318] In various embodiments, an algorithm was used to design therapeutic PEGRNAs of SEQ ID NOs: 1-135514 and 813085-880462, which were designed using the algorithm disclosed herein against Clinver database entries.

[0319] In various other embodiments, an algorithm was used to design PEG RNAs against the Clinber database; the algorithm used is disclosed herein and included in the Sequence Listing, which forms a part of this specification. The Sequence Listing includes the complete PEG RNA sequences set forth in SEQ ID NOS: 1-135514 and 813085-880462. Each of these full-length PEG RNAs is composed of a spacer (SEQ ID NOS: 135515-271028 and 880463-947840) and an extension arm (SEQ ID NOS: 271029-406542 and 947841-1015218). In addition, each PEG RNA contains a gRNA core, e.g., as defined by SEQ ID NOS: 1361579-1361580. The extension arms of SEQ ID NOs: 271029-406542 and 947841-1015218 further comprise a primer binding site (SEQ ID NOs: 406543-542056 and 1015219-1082596), an editing template (SEQ ID NOs: 542057-677570 and 1082597-1149974), and a homology arm (SEQ ID NOs: 677571-813084 and 1149975-1217352). The PEG RNA may optionally contain a 5'-end modified region and / or a 3'-end modified region. The PEG RNA may also contain a reverse transcription termination signal (e.g., SEQ ID NOs: 1361560-1361566) at the 3' end of the PEG RNA. This application covers all designs and uses of these sequences.

[0320] Mutations were classified into four classes of clinical significance using the frequency of minor alleles, the number of patients registered, whether there were any patients with discrepancies in their interpretation, and whether the mutation was reviewed by an expert. Of the 63,496 mutations compatible with SpCas9, 4,627 were identified at the most significant level (4); 13,943 were identified at significance levels 3 or 4; and 44,385 were identified at significance levels 2, 3, or 4.

[0321] The provided sequence listing lists a single PEG RNA per unique mutation, selected as the PEG RNA with the shortest distance between the nick and the edit. PEG RNAs were designed with a 13-nt homology arm length, a 13-nt primer-binding site length, a gRNA nick position at 17 nt, and a 20-nt gRNA length. Protospacers in which the nick site was further than 20 nt from the edit were ignored. The gRNA backbone sequence used was GTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 1361579). The terminator sequence used was TTTTTGTTTT (SEQ ID NO: 1361581).

[0322] As described herein, the exemplary sequence listings provided are not intended to be limiting. It should be understood that variations on the PEG RNA designs provided can encompass variations described herein, including altering the gRNA backbone sequence, primer binding site length, flap length, and / or the like.

[0323] An embodiment of a computer system 3400 that may be used to implement any of the aspects and embodiments of the techniques disclosed herein is shown in FIG. 34. The computer system 3400 may include one or more processors 3410 and one or more non-transitory computer-readable storage media (e.g., memory 3420 and one or more non-volatile storage media 3430) and a display 3440. The processor 3410 may control the writing of data to and reading of data from the memory 3420 and non-volatile storage 3430 in any suitable manner, although the aspects of the invention described herein are not limited in this respect. To implement the functionality and / or techniques described herein, the processor 3410 may execute one or more instructions stored on one or more computer-readable storage media (e.g., memory 3420, storage media, etc.) (which may act as non-transitory computer-readable storage media that store instructions for execution by the processor 3410).

[0324] Code used in connection with the techniques described herein, for example, code used to determine extended gRNA structure, may be stored on one or more computer-readable storage media of computer system 3400. Processor 3410 may execute any such code to provide any technique for orchestrating exercises as described herein. Any other software, programs, or instructions described herein may also be stored and executed by computer system 3400. It will be understood that computer code may be applied to any aspect of the methods and techniques described herein. For example, computer code may be applied to interact with an operating system to determine extended gRNA structure through conventional operating system processing.

[0325] The various methods or processes outlined herein may be coded as software executable on one or more processors employing any one of a variety of operating systems or platforms. In addition, such software may be written using any of a number of suitable programming languages ​​and / or programming or scripting tools, and may also be compiled as executable machine code or intermediate code that runs on a virtual machine or suitable framework.

[0326] In this regard, various inventive concepts may be embodied as at least one non-transitory computer-readable storage medium (e.g., computer memory, one or more floppy disks, compact disks, optical disks, magnetic tapes, flash memory, circuitry in a field programmable gate array or other semiconductor device, etc.) encoded with one or more programs that, when executed on one or more computers or other processors, practice various aspects of the present invention. The non-transitory computer-readable medium(s) may be transportable such that the program or programs stored thereon may be loaded onto any computer resource that practices various aspects of the present invention as discussed above.

[0327] The terms "program," "software," and / or "application" are used herein in a generic sense to refer to any type of computer code or set of computer-executable instructions that may be employed to program a computer or other processor to practice various aspects of the present invention as discussed above. Additionally, it should also be understood that, according to one aspect, one or more computer programs that, when executed, perform the methods of the present invention need not reside on a single computer or processor, but may be distributed in a modular manner among various computers or processors that practice various aspects of the present invention.

[0328] Computer-executable instructions may be in many forms, such as program modules, executed by one or more computers or other devices. Generally, program modules include routines, programs, objects, components, data structures, etc. that perform particular tasks or implement particular abstract data types. Typically, the functionality of the program modules may be combined or distributed as desired in various embodiments.

[0329] The data structure may also be stored in any suitable form of non-transitory computer-readable storage medium. The data structure may have fields that are related through their locations in the data structure. Such relationships may also be achieved by assigning storage to fields with locations in the non-transitory computer-readable medium that convey the relationships between the fields. However, any suitable mechanism may be used to establish relationships between information in the fields of the data structure, for example, through the use of pointers, tags, or other mechanisms that establish relationships between data elements.

[0330] Various inventive concepts may be embodied as one or more methods, examples of which are provided. The acts performed as part of a method may be ordered in any suitable manner. Consequently, embodiments may be constructed in which acts are performed in an order different from that described, and may include performing some acts simultaneously even though the illustrative embodiments show acts as sequential.

[0331] III. Prime editing factors for use with therapeutic PEG RNA Therapeutic PEG RNAs designed according to the algorithms disclosed herein may be used to perform prime editing when complexed with a prime editor. The prime editor comprises a napDNAbp (or one provided in trans) fused to a polymerase (e.g., reverse transcriptase), optionally with the two domains connected by a linker, and may further comprise one or more NLSs. These aspects are further described below.

[0332] A. napDNAbp Primed editing factors described herein may include nucleic acid programmable DNA binding proteins (napDNAbp).

[0333] In one aspect, the napDNAbp can be associated with 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 that includes the DNA strand complementary to the guide nucleic acid (i.e., the target strand) or a portion thereof (e.g., the spacer of the guide RNA that anneals to the protospacer of the DNA target). In other words, the guide nucleic acid "programs" the napDNAbp (e.g., Cas9 or equivalent) to localize and bind to the complementary sequence of the protospacer in the DNA.

[0334] Any suitable napDNAbp may be used in the prime editing elements described herein. In various embodiments, the napDNAbp may be any Class 2 CRISPR-Cas system, including any Type II, Type V, or Type VI CRISPR-Cas enzyme. While CRISPR-Cas is rapidly developing as a tool for genome editing, the nomenclature used to describe and / or identify CRISPR-Cas enzymes, such as Cas9 and Cas9 orthologs, is also evolving. This application refers to CRISPR-Cas enzymes by their nomenclature, whether old (i.e., "legacy") or new. Those skilled in the art will be able to identify the specific CRISPR-Cas enzymes referred to in this application based on the nomenclature used, whether old (i.e., "legacy") or new. CRISPR-Cas nomenclature is discussed extensively in Makarova et al., "Classification and Nomenclature of CRISPR-Cas Systems: Where from Here?," The CRISPR Journal, Vol. 1, No. 5, 2018, the entire contents of which are incorporated herein by reference. The specific CRISPR-Cas nomenclature used in any given example in this application is not intended to be limiting in any way, and one of skill in the art would be able to identify which CRISPR-Cas enzyme is being referred to.

[0335] For example, the following Type II, Type V, and Type VI Class 2 CRISPR-Cas enzymes have the following art-recognized old (i.e., legacy) and new names. Each of these enzymes and / or their variants may be used with the prime editors described herein: [Table 2]

[0336] Without being bound by theory, one mechanism of action of napDNAbp contemplated herein involves the napDNAbp forming an R-loop that induces unwinding of the double-stranded DNA target (thereby separating the strands in the region bound by the napDNAbp). The guide RNA spacer then hybridizes to the "target strand" at the protospacer sequence. This displaces the "non-target strand" that is complementary to the target strand, thereby forming the single-stranded region of the R-loop. In some embodiments, the napDNAbp contains one or more nuclease activities, which then cleave the DNA to eliminate various types of lesions. For example, the napDNAbp may contain nuclease activities that cleave the non-target strand at a first location and / or the target strand at a second location. In response to the nuclease activity, the target DNA may be cleaved to form a "double-stranded break" in which both strands are cleaved. In other embodiments, the target DNA may be cleaved at only a single site, i.e., the DNA is "nicked" on one strand. Exemplary napDNAbps with various nuclease activities include "Cas9 nickase" ("nCas9") and inactivated Cas9 that does not have any nuclease activity ("inactivated Cas9" or "dCas9").

[0337] The description below of various napDNAbps that can be used in conjunction with the prime editors of the present disclosure is not intended to be limiting in any way. The prime editor may include standard SpCas9, or any orthologous Cas9 protein, or any variant Cas9 protein—including any naturally occurring variant, mutant, or otherwise modified version of Cas9—whether known or created or evolved through directed evolution or other mutagenesis processes. In various 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 an inactive nuclease activity, i.e., is a "dead" Cas9 protein. Other variant Cas9 proteins that may be used are those with a smaller molecular weight than standard SpCas9 (e.g., for easier delivery) or those with modified or rearranged primary amino acid structures (e.g., circularly permuted forms).

[0338] Prime editing factors described herein may also include Cas9 equivalents, including Cas12a (Cpf1) and Cas12b1 proteins, which are the result of convergent evolution. As used herein, napDNAbp (e.g., SpCas9, Cas9 variants, or Cas9 equivalents) may also contain various modifications that alter / enhance their PAM specificity. Finally, the present application contemplates any Cas9, Cas9 variant, or Cas9 equivalent having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.9% sequence identity with a reference Cas9 sequence (such as a reference SpCas9 standard sequence or a reference Cas9 equivalent (e.g., Cas12a (Cpf1))).

[0339] napDNAbp may be a CRISPR (clustered regularly interspaced short palindromic repeats)-associated nuclease. CRISPR is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements, and conjugative plasmids). CRISPR clusters contain a spacer, a sequence complementary to the preceding mobile element, and a targeting invading nucleic acid. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems, corrective processing of pre-crRNA requires a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and a Cas9 domain. The tracrRNA acts as a guide for ribonuclease 3-aided processing of the pre-crRNA. Subsequently, the Cas9 / crRNA / tracrRNA endonucleolytically cleaves linear or circular dsDNA targets complementary to the spacer. The target strand that is not complementary to the crRNA is first cleaved endonucleolytically, and then exonucleolytically cleaved 3'-5'. In nature, DNA binding and cleavage typically require both protein and RNA. However, single guide RNAs ("sgRNAs," or simply "gRNAs") can be engineered to incorporate both crRNA and tracrRNA aspects into a single RNA species. For example, see Jinek M. et al., Science 337:816-821 (2012), the entire contents of which are incorporated herein by reference.

[0340] In some embodiments, the napDNAbp directs cleavage of one or both strands at the location of the target sequence, such as within the target sequence and / or within the complement of the target sequence. In some embodiments, the napDNAbp directs cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500 or more base pairs from the first or last nucleotide of the target sequence. In some embodiments, the vector encodes a napDNAbp that is mutated with respect to the corresponding wild-type enzyme such that the mutant napDNAbp lacks the ability to cleave one or both strands of a target polynucleotide containing the target sequence. For example, an aspartate to alanine substitution (D10A) in the RuvC I catalytic domain of Cas9 from S. pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (cleaves a single strand). Other examples of mutations that result in a Cas9 nickase include, without limitation, H840A, N854A, and N863A, with reference to the equivalent amino acid positions in the standard SpCas9 sequence, or other Cas9 variants or Cas9 equivalents.

[0341] As used herein, the term "Cas protein" refers to a full-length Cas protein obtained from nature, a recombinant Cas protein having a sequence that differs from a naturally occurring Cas protein, or any fragment of a Cas protein that still retains all or a substantial portion of the essential basic functions required for the disclosed methods, namely, (i) the Cas protein's possession of nucleic acid-programmable binding to target DNA, and (ii) the ability to nick a target DNA sequence on one strand. Cas proteins contemplated herein encompass CRISPR Cas9 proteins, as well as Cas9 equivalents, variants (e.g., Cas9 nickase (nCas9) or nuclease-inactive Cas9 (dCas9)), homologs, orthologs, or paralogs, whether naturally occurring or non-naturally occurring (e.g., modified or recombinant), including any Class 2 Cas protein, including Cas12a (Cpf1), Cas12e (CasX), Cas12b1 (C2c1), Cas12b2, Cas12c (C2c3), C2c4, C2c8, C2c5, C2c10, C2c9, Cas13a (C2c2), Cas13d, Cas13c (C2c7), Cas13b (C2c6), and Cas13b. Cas9 equivalents from CRISPR systems (e.g., Type II, Type V, Type VI) may be included. Additional 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.

[0342] The term "Cas9" or "Cas9 nuclease" or "Cas9 portion" or "Cas9 domain" encompasses any naturally occurring Cas9 from any organism, any naturally occurring Cas9 equivalent or functional fragment thereof, any Cas9 homolog, ortholog, or paralog from any organism, and any mutant or variant of naturally occurring or modified Cas9. The term Cas9 is not intended to be specifically limiting and may also be referred to as "Cas9 or equivalent." Exemplary Cas9 proteins are further described herein and / or in the art and are incorporated herein by reference. The present disclosure is not limited with respect to the particular Cas9 employed in the prime editors (PEs) of the present invention.

[0343] As mentioned herein, the sequence and structure of the Cas9 nuclease is well known to those skilled in the art (e.g., "Complete genome sequence of an M1 strain of Streptococcus pyogenes." Ferretti et al., JJ, McShan WM, Ajdic DJ, Savic DJ, Savic G., Lyon K., Primeaux C., Sezate S., Suvorov AN, Kenton S., Lai HS,Lin SP,Qian Y.,Jia HG,Najar FZ,Ren Q.,Zhu H.,Song L.,White J.,Yuan X.,Clifton SW,Roe BA,McLaughlin RE,Proc.Natl.Acad.Sci.USA98:4658-4663(2001);"CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III."Deltcheva E., Chylinski K., Sharma CM, Gonzales K., Chao Y., Pirzada ZA, Eckert MR, Vogel J., Charpentier E., Nature 471:602-607(2011); and "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity." Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna JA, Charpentier E. Science 337:816-821(2012) (the entire contents of each of which are incorporated herein by reference).

[0344] Examples of Cas9 and Cas9 equivalents are provided below; however, these specific examples are not intended to be limiting. Primer editors of the present disclosure may use any suitable napDNAbp, including any suitable Cas9 or Cas9 equivalent.

[0345] (i) Wild type standard SpCas9 In one embodiment, the primer editor constructs described herein may comprise the "standard SpCas9" nuclease from S. pyogenes, which has been widely used as a tool for genome engineering and is classified as a Type II subgroup enzyme, known as Class 2 CRISPR-Cas systems. The Cas9 protein is a large, multidomain protein containing two distinct nuclease domains. Point mutations can be introduced into Cas9 to disable one or both nuclease activities, resulting in nickase Cas9 (nCas9) or inactive Cas9 (dCas9), each of which still retains its ability to bind DNA in an sgRNA-programmed manner. In principle, when fused to another protein or domain, Cas9 or a variant thereof (e.g., nCas9) can be directed to virtually any DNA sequence simply by coexpression with the appropriate sgRNA. As used herein, a standard SpCas9 protein refers to the wild-type protein from Streptococcus pyogenes, which has the following amino acid sequence: [Table 3-1] [Table 3-2]

[0346] Prime editors described herein can include standard SpCas9 or any variant thereof having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the wild-type Cas9 sequence provided above. These variants can include SpCas9 variants containing one or more mutations, including any of the known mutations reported in SwissProt accession entry Q99ZW2, including the following: [Table 4]

[0347] Other wild-type SpCas9 sequences that may be used in the present disclosure include: [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5] [Table 5-6]

[0348] The prime editors described herein can include any of the above SpCas9 sequences or any variants thereof having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto.

[0349] (ii) Wild-type Cas9 ortholog In other embodiments, the Cas9 protein can be a wild-type Cas9 ortholog from another bacterial species that differs from the standard Cas9 from S. pyogenes. For example, the following Cas9 orthologs can be used in conjunction with the prime editor constructs described herein. In addition, any variant Cas9 ortholog having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with any of the orthologs below can also be used with the present prime editors. [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5]

[0350] The prime editors described herein may include any of the above Cas9 orthologous sequences, or any variants thereof having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto.

[0351] The napDNAbp may include any suitable homolog and / or ortholog, such as Cas9, or a naturally occurring enzyme. Cas9 homologs and / or orthologs have been described in various species, including, but not limited to, S. pyogenes and S. thermophilus. Preferably, the Cas moiety is configured (e.g., mutagenized, recombinantly modified, or otherwise naturally occurring) as a nickase (i.e., capable of cleaving only one strand of a target duplex). Suitable Cas9 nucleases and sequences will be apparent to those of skill in the art based on this disclosure, and include Cas9 sequences from the organisms and loci disclosed in Chylinski, Rhun, and Charpentier, "The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems" (2013) RNA Biology 10:5, 726-737, the entire contents of which are incorporated herein by reference. In some embodiments, the Cas9 nuclease has an inactive (e.g., inactivated) DNA-cleavage domain, i.e., Cas9 is a nickase. In some embodiments, the Cas9 protein comprises an amino acid sequence at least 80% identical to the amino acid sequence of a Cas9 protein as provided by any one of the variants in Table 3. In some embodiments, the Cas9 protein comprises an amino acid sequence at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the amino acid sequence of a Cas9 protein as provided by any one of the Cas9 orthologs in the table above.

[0352] (iii) Inactive (Dead) Cas9 variants In some embodiments, the prime editors described herein can include inactive Cas9, e.g., inactive SpCas9, which lacks any nuclease activity due to one or more mutations that render both Cas9 nuclease domains inactive: the RuvC domain (which cleaves the non-protospacer DNA strand) and the HNH domain (which cleaves the protospacer DNA strand). Nuclease inactivation can also result from one or more mutations that result in one or more substitutions and / or deletions in the amino acid sequence of the encoded protein or any variant thereof that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto.

[0353] As used herein, the term "dCas9" refers to nuclease-inactive Cas9 or nuclease-inactive Cas9, or a functional fragment thereof, and encompasses any naturally occurring dCas9 from any organism, any naturally occurring dCas9 equivalent or functional fragment thereof, any dCas9 homolog, ortholog, or paralog from any organism, and any mutant or variant of naturally occurring or modified dCas9. The term dCas9 is not intended to be specifically limiting and may also be referred to as "dCas9 or equivalent." Exemplary dCas9 proteins and methods for producing dCas9 proteins are further described herein and / or in the art and are incorporated herein by reference.

[0354] In other embodiments, dCas9 corresponds to or includes part or all of a Cas9 amino acid sequence with one or more mutations that inactivate Cas9 nuclease activity. In other embodiments, Cas9 variants are provided with mutations other than D10A and H840A that result in completely or partially inactivated endogenous Cas9 nuclease activity (e.g., nCas9 or dCas9, respectively). Such mutations include, by way of example, other amino acid substitutions at D10 and H820, or other substitutions within the nuclease domain of Cas9 (e.g., substitutions in the HNH nuclease subdomain and / or the RuvC1 subdomain), with reference to a wild-type sequence, such as Cas9 from Streptococcus pyogenes (NCBI Reference Sequence: NC_017053.1 (SEQ ID NO: 1361424)). In some embodiments, provided are variants or homologs of Cas9 (e.g., a variant of Cas9 from Streptococcus pyogenes (NCBI Reference Sequence: NC_017053.1 (SEQ ID NO: 1361424))) 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 (SEQ ID NO: 1361424). In some embodiments, variants of dCas9 (e.g., variants of NCBI Reference Sequence: NC_017053.1 (SEQ ID NO: 1361424)) are provided having amino acid sequences that are about 5, 10, 15, 20, 25, 30, 40, 50, 75, or 100 or more amino acids shorter or longer than NC_017053.1 (SEQ ID NO: 1361424).

[0355] In one embodiment, the inactive Cas9 may be based on the standard SpCas9 sequence of Q99ZW2 and may have the following sequence containing the D10A and H810A substitutions (underlined and bold), or a variant thereof having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 1361444: [Table 7]

[0356] (iv) Cas9 nickase variants In one embodiment, the prime editor described herein comprises a Cas9 nickase. The term "Cas9 nickase" in "nCas9" refers to a variant of Cas9 capable of introducing a single-strand break into a double-stranded DNA molecule target. In some embodiments, the Cas9 nickase comprises only a single functional nuclease domain. Wild-type Cas9 (e.g., standard SpCas9) comprises two separate nuclease domains: a RuvC domain (which cleaves the non-protospacer DNA strand) and an HNH domain (which cleaves the protospacer DNA strand). In one embodiment, the Cas9 nickase comprises a mutation in the RuvC domain that inactivates RuvC nuclease activity. For example, mutations at aspartate (D) 10, histidine (H) 983, aspartate (D) 986, or glutamate (E) 762 have been reported as loss-of-function mutations in the RuvC nuclease domain and the creation of functional Cas9 nickases (see, e.g., Nishimasu et al., "Crystal structure of Cas9 in complex with guide RNA and target DNA," Cell 156(5), 935-949, which is incorporated herein by reference). Thus, nickase mutations in the RuvC domain can include D10X, H983X, D986X, or E762X, where X is any amino acid other than the wild-type amino acid. In some embodiments, the nickase can be D10A, of H983A, or D986A, or E762A, or a combination thereof.

[0357] In various embodiments, the Cas9 nickase may have a mutation in the RuvC nuclease domain and have one of the following amino acid sequences, or a variant thereof having an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto: [Table 8-1] [Table 8-2] [Table 8-3] [Table 8-4]

[0358] In another embodiment, the Cas9 nickase contains a mutation in the HNH domain that inactivates HNH nuclease activity. For example, mutations at histidine (H)840 or asparagine (R)863 have been reported as loss-of-function mutations in the HNH nuclease domain and create functional Cas9 nickases (see, e.g., Nishimasu et al., "Crystal structure of Cas9 in complex with guide RNA and target DNA," Cell 156(5), 935-949, which is incorporated herein by reference). Thus, nickase mutations in the HNH domain can include H840X and R863X, where X is any amino acid other than the wild-type amino acid. In some embodiments, the nickase can be H840A or R863A, or a combination thereof.

[0359] In various embodiments, the Cas9 nickase may have a mutation in the HNH nuclease domain and may have one of the following amino acid sequences, or a variant thereof having an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto: [Table 9-1] [Table 9-2]

[0360] (v) Other Cas9 variants In addition to inactive Cas9 and Cas9 nickase variants, Cas9 proteins as used herein can also include other "Cas9 ...

Claims

1. A guide RNA comprising a spacer sequence, a gRNA core, and an extension arm, wherein the extension arm comprises (i) a primer binding site, (ii) a reverse transcriptase (RT) editing template, and (iii) a homology arm; wherein the spacer sequence comprises 10 to 40 nucleotides containing a nucleotide sequence complementary to a protospacer sequence in the target strand of the target DNA sequence; Here the gRNA core is capable of binding to the Cas9 nickase, a nucleic acid programmed DNA binding protein (napDNAbp); wherein the extension arm can serve as a template for reverse transcription synthesis of a newly synthesized single strand of DNA and contains a homology arm, an RT editing template, and a primer binding site in the 5'→3' direction; wherein the primer binding site is 8-20 nucleotides in length and is capable of annealing to the 3'-terminal single-stranded DNA (ssDNA) flap that is formed after the napDNAbp nicks the non-target strand of the target DNA sequence and serves as a primer sequence to prime the reverse transcription of the DNA single strand; wherein the RT editing template encodes a desired edit in a single strand of DNA; and wherein the homology arms encode a portion of a single strand of DNA that is complementary to a target strand of a target DNA sequence.

2. 2. The guide RNA of claim 1, wherein the protospacer sequence comprises 20 nucleotides.

3. 3. The guide RNA of claim 1 or 2, wherein the extension arm is between 10 and 500 nucleotides in length.

4. 3. The guide RNA of Claim 1 or 2, wherein the extension arm is at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides, at least 22 nucleotides, at least 23 nucleotides, at least 24 nucleotides, at least 25 nucleotides, at least 26 nucleotides, at least 27 nucleotides, at least 28 nucleotides, at least 29 nucleotides, at least 30 nucleotides, at least 31 nucleotides, at least 32 nucleotides, at least 33 nucleotides, at least 34 nucleotides, at least 35 nucleotides, at least 36 nucleotides, at least 37 nucleotides, at least 38 nucleotides, at least 39 nucleotides, at least 40 nucleotides, or at least 100 nucleotides in length.

5. 5. The guide RNA of any one of claims 1 to 4, wherein the extension arm is a 5' extension arm.

6. 5. The guide RNA of any one of claims 1 to 4, wherein the extension arm is a 3' extension arm.

7. The guide RNA of any one of claims 1 to 6, wherein the RT editing template is at least 1 nucleotide, at least 2 nucleotides, at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides, at least 22 nucleotides, at least 23 nucleotides, at least 24 nucleotides, at least 25 nucleotides, at least 26 nucleotides, at least 27 nucleotides, at least 28 nucleotides, at least 29 nucleotides, at least 30 nucleotides, at least 31 nucleotides, at least 32 nucleotides, at least 33 nucleotides, at least 34 nucleotides, at least 35 nucleotides, at least 36 nucleotides, at least 37 nucleotides, at least 38 nucleotides, at least 39 nucleotides, at least 40 nucleotides, or at least 100 nucleotides in length.

8. The guide RNA of any one of claims 1 to 7, wherein the desired edit comprises one or more single base nucleotide changes, one or more deletions, one or more insertions, or a combination thereof.

9. 9. The guide RNA of any one of claims 1 to 8, wherein the homology arms are at least 1 nucleotide, at least 2 nucleotides, at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides, at least 22 nucleotides, at least 23 nucleotides, at least 24 nucleotides, at least 25 nucleotides, at least 26 nucleotides, at least 27 nucleotides, at least 28 nucleotides, at least 29 nucleotides, or at least 30 nucleotides in length.

10. The guide RNA of any one of claims 1 to 9, further comprising a 5' end modification region comprising a hairpin sequence, a stem / loop sequence, or a toe-loop sequence.

11. The guide RNA of any one of claims 1 to 10, further comprising a 3' end modification region comprising a hairpin sequence, a stem / loop sequence, or a toe-loop sequence.

12. The guide RNA of any one of claims 1 to 11, wherein the spacer sequence comprises a nucleotide sequence that is at least 90% identical to any one of SEQ ID NOs: 135515-271028 or 880463-947840.

13. The guide RNA of any one of claims 1 to 11, wherein the spacer sequence comprises a nucleotide sequence of any one of SEQ ID NOs: 135515 to 271028 or 880463 to 947840.

14. The guide RNA of any one of claims 1 to 13, wherein the gRNA core comprises the nucleotide sequence of SEQ ID NO: 1361579 or 1361580.

15. The guide RNA of any one of claims 1 to 14, wherein the primer binding site comprises a nucleotide sequence at least 90% identical to any one of SEQ ID NOs: 406543-542056 or 1015219-1082596.

16. The guide RNA of any one of claims 1 to 14, wherein the primer binding site comprises a nucleotide sequence of any one of SEQ ID NOs: 406543-542056 or 1015219-1082596.

17. The guide RNA of any one of claims 1 to 16, wherein the RT editing template comprises a nucleotide sequence that is at least 90% identical to any one of SEQ ID NOs: 542057-677570 or 1082597-1149974.

18. The guide RNA of any one of claims 1 to 16, wherein the RT editing template comprises a nucleotide sequence of any one of SEQ ID NOs: 542057-677570 or 1082597-1149974.

19. The guide RNA of any one of claims 1 to 18, wherein the homology arms comprise a nucleotide sequence at least 90% identical to any one of SEQ ID NOs: 677571-813084 or 1149975-1217352.

20. The guide RNA of any one of claims 1 to 18, wherein the homology arms comprise a nucleotide sequence of any one of SEQ ID NOs: 677571-813084 or 1149975-1217352.

21. 21. The guide RNA of any one of claims 1 to 20, wherein the extension arm comprises a nucleotide sequence at least 90% identical to any one of SEQ ID NOs: 271029-406542 or 947841-1015218.

22. 21. The guide RNA of any one of claims 1 to 20, wherein the extension arm comprises a nucleotide sequence of any one of SEQ ID NOs: 271029-406542 or 947841-1015218.

23. 23. The guide RNA of any one of claims 1 to 22, comprising a sequence at least 90% identical to any one of SEQ ID NOs: 1 to 135514 or 813085 to 880462.

24. 23. The guide RNA of any one of claims 1 to 22, comprising any one of SEQ ID NOs: 1 to 135514 or 813085 to 880462.

25. A polynucleotide encoding a guide RNA according to any one of claims 1 to 24.

26. A vector comprising the polynucleotide of claim 25.

27. 27. A pharmaceutical composition comprising: (a) a guide RNA according to any one of claims 1 to 24, a polynucleotide according to claim 25, or a vector according to claim 26; and (b) a pharma- ceutically acceptable excipient.

28. A prime editing complex comprising napDNAbp, a Cas9 nickase, a reverse transcriptase, and a guide RNA described in any one of claims 1 to 24.

29. The prime editing complex of claim 28, wherein the napDNAbp and the reverse transcriptase are formed as a fusion protein.

30. 30. One or more polynucleotides encoding the prime editing complex of claim 28 or 29.

31. 31. One or more vectors comprising one or more polynucleotides of claim 30.

32. A pharmaceutical composition comprising a prime editing complex described in claim 28 or 29, one or more polynucleotides described in claim 30, or one or more vectors described in claim 31, and a pharma- ceutically acceptable excipient.

33. 25. A composition comprising the guide RNA of any one of claims 1 to 24 and one or more polynucleotides encoding primed editors comprising a Cas9 nickase, napDNAbp, and a reverse transcriptase.

34. 27. A composition comprising the polynucleotide of Claim 25 or the vector of Claim 26 and one or more polynucleotides encoding primed editors comprising a Cas9 nickase, napDNAbp, and a reverse transcriptase.

35. The composition of Claim 33 or 34, wherein the prime editing factor is a fusion protein.

36. A guide RNA described in any one of claims 1 to 24, a polynucleotide described in claim 25, a vector described in claim 26, a pharmaceutical composition described in claim 27 or 32, a prime editing complex described in claim 28 or 29, one or more polynucleotides described in claim 30, one or more vectors described in claim 31, or a composition described in any one of claims 33 to 35, for use as a pharmaceutical.

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