Methods and compositions for editing nucleotide sequences

Abstract

The present disclosure provides a novel prime editer guide RNA for prime editing, a construct for prime editing, and a method of using the same. Additionally, the present disclosure provides a composition and a method for performing prime editing of a target DNA molecule (e.g., a genome) that enables the incorporation of nucleotide changes and / or targeted mutagenic agents (e.g., insertions or deletions). Nucleotide changes may include single-nucleotide changes (e.g., any transition or any conversion), the insertion of one or more nucleotides, or the deletion of one or more nucleotides. More particularly, the present disclosure provides a fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a polymerase (e.g., a reverse transcriptase) that is guided to a specific DNA sequence by a prime editer RNA (PEgRNA). Prime editer guide RNA comprises an extension arm that provides a DNA synthesis template sequence encoding a single-stranded DNA flap that is homologous to the endogenous DNA sequence but contains one or more desired nucleotide changes and is incorporated into a target DNA molecule after synthesis by a polymerase (e.g., reverse transcriptionase).

Description

METHODS AND COMPOSITIONS FOR EDITING NUCLEOTIDE SEQUENCESGOVERNMENT SUPPORT

[0001] This invention was made with government support under grant numbers U01AI142756, RM1HG009490, R01EB022376, and R35GM118062 awarded by the National Institutes of Health. The government has certain rights in the invention. RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

[0002] This U.S. Provisional Application refers to and incorporates by reference the following applications, namely, 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 (Attorney Docket No. B1195.70074US01), filed June 7, 2019, U.S. Provisional Application No. 62 / 889,996 (Attorney Docket No. B1195.70074US02), filed August 21, 2019, U.S. Provisional Application No.62 / 922,654, filed August 21, 2019 (Attorney Docket No. B1195.70083US00), U.S. Provisional Application No.62 / 913,553 (Attorney Docket No. B1195.70074US03), filed October 10, 2019, U.S. Provisional Application No.62 / 973,558 (Attorney Docket No.B1195.70083US01), filed October 10, 2019, U.S. Provisional Application No.62 / 931,195 (Attorney Docket No. B1195.70074US04), filed November 5, 2019, U.S. ProvisionalApplication No.62 / 944,231 (Attorney Docket No. B1195.70074US05), filed December 5, 2019, U.S. Provisional Application No.62 / 974,537 (Attorney Docket No. B1195.70083US02), filed December 5, 2019, U.S. Provisional Application No.62 / 991,069 (Attorney Docket No.B1195.70074US06), filed March 17, 2020, and U.S. Provisional Application No. (serial number not available as of this filing) (Attorney Docket No. B1195.70083US03), filed March 17, 2020.SEQUENCE LISTING INCORPORATION BY REFERENCE

[0003] Pursuant to 37 CFR § 1.52(e), this Specification includes a Sequence Listing submitted concurrently herewith on a compact disc (2 copies). 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,” which was created on March 19, 2020, and is 371.109 MB in size. By this statement, the Sequence Listing constitutes a part of the instant Specification. The compact disc contains no other files.BACKGROUND OF THE INVENTION

[0004] Pathogenic single nucleotide mutations contribute to approximately 67% of human diseases for which there is a genetic component7. Unfortunately, treatment options for patients with these genetic disorders remain extremely limited, despite decades of gene therapy exploration8. Perhaps one of the most straightforward solutions to this therapeutic challenge is direct correction of single nucleotide mutations in the patients’ genomes, which would address the root cause of disease and would likely provide lasting benefit. Although such a strategy was previously unthinkable, recent improvements in genome editing capabilities brought about by the advent of the CRISRP / Cas system9have now brought this therapeutic approach within reach. By straightforward design of a guide RNA (gRNA) sequence that contains ~20 nucleotides complementary to the target DNA sequence, nearly any conceivable genomic site can be specifically accessed by CRISPR associated (Cas) nucleases1,2. To date, several monomeric bacterial Cas nuclease systems have been identified and adapted for genome editingapplications10. This natural diversity of Cas nucleases, along with a growing collection of engineered variants11–14, offers fertile ground for developing new genome editing technologies.

[0005] While gene disruption with CRISPR is now a mature technique, precision editing of single base pairs in the human genome remains a major challenge3. Homology directed repair (HDR) has long been used in human cells and other organisms to insert, correct, or exchange DNA sequences at sites of double strand breaks (DSBs) using donor DNA repair templates that encode the desired edits15. However, traditional HDR has very low efficiency in most human cell types, particularly in non-dividing cells, and competing non-homologous end joining (NHEJ) leads predominantly to insertion-deletion (indel) by-products16. Other issues relate to the generation of DSBs, which can give rise to large chromosomal rearrangements and deletions at target loci17, or activate the p53 axis leading to growth arrest and apoptosis18,19.

[0006] Several approaches have been explored to address the drawbacks of HDR. For example, repair of single-stranded DNA breaks (nicks) with oligonucleotide donors has been shown to reduce indel formation, but yields of desired repair products remain low20. Other strategies attempt to bias repair toward HDR over NHEJ using small molecule and biologic reagents21–23. However, the effectiveness of these methods is cell-type dependent, and perturbation of the normal cell state could lead to undesirable and unforeseeable effects.

[0007] Recently, Liu et al. developed base editing as a technology that edits target nucleotides without creating DSBs or relying on HDR4–6,24–27. Direct modification of DNA bases by Cas- fused deaminases allows for C•G to T•A, or A•T to G•C, base pair conversions in a short target window (~5-7 bases) with high efficiency. As a result, base editors have been rapidly adopted by the scientific community. However, several factors may limit their generality for precision genome editing.

[0008] Therefore, the development of programmable editors that are capable of introducing any desired single or multiple nucleotide change, which could install nucleotide insertions or deletions (e.g., at least 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more base pair insertions or deletions), and / or which could alter or modify the nucleotide sequence at a target site with high specificity and efficiency would substantially expand the scope and therapeutic potential of genome editing technologies based on CRISPR.SUMMARY OF THE INVENTION

[0009] The present invention disclosed new compositions (e.g., new PEgRNA and PE complexes comprising same) and methods for using prime editing (PE) to repair therapeutic targets, e.g., those targets identified in the ClinVar database, using PEgRNA designed using a specialized algorithm that is described herein. Thus, in one aspect, the present application discloses an algorithm for predicting on a large-scale the sequences for PEgRNA that may be used to repair therapeutic targets (e.g., those included in the ClinVar database). In addition, the present application discloses predicted sequences for therapeutic PEgRNAs designed and which can be designed using the disclosed algorithm and which may be used with prime editing to repair therapeutic targets.

[0010] The herein disclosed algorithm and the predicted PEgRNA sequences relate in general to prime editing. Thus, this disclosure also provides a description for the various components and aspects of prime editing, including suitable napDNAbp (e.g., Cas9 nickase) and a polymerase (e.g., a reverse transcriptase), as well as other suitable components (e.g., linkers, NLS) and PE fusion proteins, that may be used with the therapeutic PEgRNA disclosed herein.

[0011] As disclosed herein, prime editing is a versatile and precise genome editing method that directly writes new genetic information into a specified DNA site using a nucleic acid programmable DNA binding protein (“napDNAbp”) working in association with a polymerase(i.e., in the form of a fusion protein or otherwise provided in trans with the napDNAbp), wherein the prime editing system is programmed with a prime editing (PE) guide RNA (“PEgRNA”) that both specifies the target site and templates the synthesis of the desired edit in the form of a replacement DNA strand by way of an extension (either DNA or RNA) engineered onto a guide RNA (e.g., at the 5ʹ or 3ʹ end, or at an internal portion of a guide RNA). The replacement strand containing the desired edit (e.g., a single nucleobase substitution) shares the same sequence as the endogenous strand of the target site to be edited (with the exception that it includes the desired edit). Through DNA repair and / or replication machinery, the endogenous strand of the target site is replaced by the newly synthesized replacement strand containing the desired edit. In some cases, prime editing may be thought of as a“search-and-replace” genome editing technology since the prime editors, as described herein, not only search and locate the desired target site to be edited, but at the same time, encode a replacement strand containing a desired edit which is installed in place of the corresponding target site endogenous DNA strand. The prime editors of the present disclosure relate, in part, to the discovery that the mechanism of target-primed reverse transcription (TPRT) or“prime editing” can be leveraged or adapted for conducting precision CRISPR / Cas-based genome editing with high efficiency and genetic flexibility (e.g., as depicted in various embodiments of FIGs.1A-1F). TPRT is naturally used by mobile DNA elements, such as mammalian non-LTR retrotransposons and bacterial Group II introns28,29. The inventors have herein used Cas protein-reverse transcriptase fusions or related systems to target a specific DNA sequence with a guide RNA, generate a single strand nick at the target site, and use the nicked DNA as a primer for reverse transcription of an engineered reverse transcriptase template that is integrated with the guide RNA. However, while the concept begins with prime editors that use reverse trancriptases as the DNA polymerase component, the prime editors described herein are not limited to reverse transcriptases but may include the use of virtually and DNA polymerase. Indeed, while the application throughout may refer to prime editors with“reverse transcriptases,” it is set forth here that reverse transcriptases are only one type of DNA polymerase that may work with prime editing. Thus, where ever the specification mentions“reverse transcriptases,” the person having ordinary skill in the art should appreciate that any suitable DNA polymerase may be used in place of the reverse transcriptase. Thus, in one aspect, the prime editors may comprise Cas9 (or an equivalent napDNAbp) which isprogrammed to target a DNA sequence by associating it with a specialized guide RNA (i.e.,PEgRNA) containing a spacer sequence that anneals to a complementary protospacer in the target DNA. The specialized guide RNA also contains new genetic information in the form of an extension that encodes a replacement strand of DNA containing a desired genetic alteration which is used to replace a corresponding endogenous DNA strand at the target site. To transfer information from the PEgRNA to the target DNA, the mechanism of prime editing involves nicking the target site in one strand of the DNA to expose a 3′-hydroxyl group. The exposed 3′- hydroxyl group can then be used to prime the DNA polymerization of the edit-encoding extension on PEgRNA directly into the target site. In various embodiments, the extension— which provides the template for polymerization of the replacement strand containing the edit— can be formed from RNA or DNA. In the case of an RNA extension, the polymerase of the prime editor can be an RNA-dependent DNA polymerase (such as, a reverse transcriptase). In the case of a DNA extension, the polymerase of the prime editor may be a DNA-dependent DNA polymerase.

[0012] The newly synthesized strand (i.e., the replacement DNA strand containing the desired edit) that is formed by the herein disclosed prime editors would be homologous to the genomic target sequence (i.e., have the same sequence as) except for the inclusion of a desired nucleotide change (e.g., a single nucleotide change, a deletion, or an insertion, or a combination thereof). The newly synthesized (or replacement) strand of DNA may also be referred to as a single strand DNA flap, which would compete for hybridization with the complementary homologous endogenous DNA strand, thereby displacing the corresponding endogenous strand. In certain embodiments, the system can be combined with the use of an error-prone reverse transcriptase enzyme (e.g., provided as a fusion protein with the Cas9 domain, or provided in trans to the Cas9 domain). The error-prone reverse transcriptase enzyme can introduce alterations during synthesis of the single strand DNA flap. Thus, in certain embodiments, error-prone reverse transcriptase can be utilized to introduce nucleotide changes to the target DNA. Depending on the error-prone reverse transcriptase that is used with the system, the changes can be random or non-random.

[0013] Resolution of the hybridized intermediate (comprising the single strand DNA flap synthesized by the reverse transcriptase hybridized to the endogenous DNA strand) can include removal of the resulting displaced flap of endogenous DNA (e.g., with a 5ʹ end DNA flap endonuclease, FEN1), ligation of the synthesized single strand DNA flap to the target DNA, andassimilation of the desired nucleotide change as a result of cellular DNA repair and / or replication processes. Because templated DNA synthesis offers single nucleotide precision for the modification of any nucleotide, including insertions and deletions, the scope of this approach is very broad and could foreseeably be used for myriad applications in basic science and therapeutics.Algorithm and methods of designing therapeutic PEgRNA

[0014] In one aspect, the present disclosure relates to a novel algorithm for designing therapeutic PEgRNA, in particular, on a large-scale as opposed to a one-off PEgRNA design exercise.

[0015] Accordingly, some aspects relate to a computerized method for determining a sequence of a prime editor guide RNA (PEgRNA). The method includes using at least one computer hardware processor to access data indicative of an input allele, an output allele, and a fusion protein comprising a nucleic acid programmable DNA binding protein and a polymerase (e.g., a reverse transcriptase). The method includes determining the PEgRNA sequence based on the input allele, the output allele, and the fusion protein, wherein the PEgRNA sequence is designed to be associated with the fusion protein to change the input allele to the output allele, including determining for the PEgRNA sequence one or more of the following features: a spacer complementary to a target nucleotide sequence in the input allele (i.e., the spacer, as defined in FIG.27); a gRNA backbone for interacting with the fusion protein (i.e., the gRNA core as defined in FIG.27); and an extension (i.e., the extension arm as shown in FIG.27) comprising one or more of: a DNA synthesis template(as shown in FIG.27) comprising a desired nucleotide change to change the input allele to the output allele; primer binding site (i.e., the primer binding site as shown in FIG.27). The PEgRNA may also comprise a 3’ termination signal that terminates transcription from a 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 FIG.27) may include stem-loop sequences, which may increase the stability of the PEgRNA.

[0016] In some examples, the method includes determining the spacer and the extension, and determining the spacer is at the 5′ end of the PEgRNA , and the extension is at a 3′ end of the PEgRNA structure.

[0017] In some examples, the method includes determining the spacer and the extension, and determining the spacer is at the 5′ end of the PEgRNA , and the extension is 3′ to the spacer.

[0018] In some examples, accessing data indicative of the input allele and the output allele comprises accessing a database comprising a set of input alleles and associated output alleles. Accessing the database can include accessing a ClinVar database of the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / clinvar / ) comprising a plurality of entries, each entry comprising an input allele from the set of input alleles and an output allele from the set of output alleles (e.g., wild-type or alleles with the desired activity). Determining the PEgRNA sequence can include determining one or more PEgRNA sequences for each input allele and associated output allele in the set.

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

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

[0021] In some examples, changing the input allele to the output allele includes a single nucleotide change, an insertion of one or more nucleotides, a deletion of one or more nucleotides, or a combination thereof.

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

[0023] In some examples, the method can include: determining a set of initial candidate protospacers based on the input allele and the fusion protein, wherein each initial candidate protospacer comprises 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 comprise an incompatible nick position; removing the determined one or more initial candidate protospacers from the set to generate a set of remaining candidate protospacers; and wherein determining the PEgRNA structure comprises determining a plurality of PEgRNA structures, wherein each of the PEgRNA structure comprises a different spacer determined based on a corresponding protospacer from the set of remaining candidate protospacers.

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

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

[0026] In some examples, the DNA synthesis template (e.g., RT template sequence) encodes a single-strand DNA flap that is complementary to an endogenous DNA sequence adjacent to a nick site, wherein the single-strand DNA flap comprises the desired nucleotide change. The single-strand DNA flap can hybridize to the endogenous DNA sequence adjacent to the nick site, thereby installing the desired nucleotide change. The single-stranded DNA flap can displace the endogenous DNA sequence adjacent to the nick site. Cellular repair of the single-strand DNA flap can result in installation of the desired nucleotide change, thereby forming a desired product.

[0027] In some examples, the fusion protein when complexed with the PEgRNA is capable of binding to a target DNA sequence. The target DNA sequence can include a target strand at which the change occurs and a complementary non-target strand.

[0028] In some examples, 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 encoded thereon instructions which, when executed, cause the at least one processor to perform the computerized methods for determining the PEgRNA structure.

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

[0031] In another aspect, the present disclosure provide therapeutic PEgRNA that have been designed using the herein disclosed algorithm, as represented by FIG.27 and FIG.28.

[0032] For example, the PEgRNA that may be used in the herein disclosure are exemplified in FIG.27. This figure provides the structure of an embodiment of a PEgRNA contemplated herein and which may be designed in accordance with the methodology defined in Example 2. The PEgRNA comprises three main component elements ordered in the 5′ to 3′ direction, namely: a spacer, a gRNA core, and an extension arm at the 3′ end. The extension arm may further be divided into the following structural elements in the 5′ to 3′ direction, namely: a primer binding site (A), an edit template (B), and a homology arm (C). In addition, the PEgRNA may comprise an optional 3′ end modifier region (e1) and an optional 5′ end modifier region (e2). Still further, the PEgRNA may comprise a transcriptional termination signal at the 3′ end of the PEgRNA (not depicted). These structural elements are further defined herein. The depiction of the structure of the PEgRNA is not meant to be limiting and embraces variations in the arrangement of the elements. For example, the optional sequence modifiers (e1) and (e2) could be positioned within or between any of the other regions shown, and not limited to beinglocated at the 3′ and 5′ ends. The PEgRNA shown in FIG.27 can be designed by the herein disclosed algorithm.

[0033] In another example, FIG.28 provides the structure of another embodiment of a PEgRNA contemplated herein and which may be designed in accordance with the methodology defined in Example 2. The PEgRNA comprises three main component elements ordered in the 5′ to 3′ direction, namely: a spacer, a gRNA core, and an extension arm at the 3′ end. The extension arm may further be divided into the following structural elements in the 5′ to 3′ direction, namely: a primer binding site (A), an edit template (B), and a homology arm (C). In addition, the PEgRNA may comprise an optional 3′ end modifier region (e1) and an optional 5′ end modifier region (e2). Still further, the PEgRNA may comprise a transcriptional termination signal on the 3′ end of the PEgRNA (not depicted). These structural elements are further defined herein. The depiction of the structure of the PEgRNA is not meant to be limiting and embraces variations in the arrangement of the elements. For example, the optional sequence modifiers (e1) and (e2) could be positioned within or between any of the other regions shown, and not limited to being located at the 3′ and 5′ ends. The PEgRNA shown in FIG.27 can be designed by the herein disclosed algorithm.

[0034] In various embodiments, the disclosure provides therapeutic PEgRNA of SEQ ID NOs: 1-135514 and 813085-880462 designed using the herein disclosed algorithm against ClinVar database entries.

[0035] In various other embodiments, exemplary PEgRNA designed against the ClinVar database using the herein disclosed algorithm are included in the Sequence Listing, which forms a part of this specification. The Sequence Listing includes complete PEgRNA sequences of SEQ ID NOs: 1-135514 and 813085-880462. Each of these complete PEgRNA are each comprised 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 PEgRNA comprises a gRNA core, for example, as defined by SEQ ID NOs: 1361579-1361580. The extension arms of SEQ ID NOs: 271029– 406542 and 947841-1015218 are further each comprised of a primer binding site (SEQ ID NOs.: 406543– 542056 and 1015219-1082596), an edit template (SEQ ID NOs.: 542057– 677570 and 1082597-1149974), and a homology arm (SEQ ID NOs.: 677571– 813084 and 1149975-1217352). The PEgRNA optionally may comprise a 5′ end modifier region and / or a 3′ end modifier region. The PEgRNA may also comprise a reverse transcription terminationsignal (e.g., SEQ ID NOs: 1361560-1361566) at the 3′ of the PEgRNA. The application embraces the design and use of all of these sequences.

[0036] In various embodiments, the prime editor guide RNA comprises (a) a guide RNA and (b) an RNA extension at the 5´ or the 3´ end of the guide RNA, or at an intramolecular location in the guide RNA, examples of which are depicted in Figs.3A-C. The RNA extension can comprise (i) a DNA synthesis template comprising a desired nucleotide change, (ii) a reverse transcription primer binding site, and (iii) optionally, a linker sequence. In variousembodiments, the DNA synthesis template encodes a single-strand DNA flap that iscomplementary to an endogenous DNA sequence adjacent to the nick site, wherein the single- stranded DNA flap comprises 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 certain 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 with any one of SEQ ID NOs: 1361548-1361581.

[0039] In some embodiments, the prime editor guide RNA (PEgRNA ) comprises a variant of a nucleotide sequence of SEQ ID NOs: 1361548-1361581, comprising at least one mutation as compared to the nucleotide sequence of SEQ ID NOs: 1361548-1361581. In someembodiments, the variant comprises more than 1 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more) mutation as compared to the nucleotide sequence of SEQ ID NOs: 1361548-1361581.

[0040] In another aspect, the present disclosure provides an prime editor guide RNA comprising a guide RNA and at least one RNA extension (i.e., extension arm, per FIG.27). The RNA extension is positioned at the 3´ end of the guide RNA. In other embodiments, the RNA extension is positioned at the 5´ of the guide RNA. In still other embodiments, the RNA extension is positioned at an intramolecular position within the guide RNA, preferably, theintramolecular positioning of the extended portion does not disrupt the functioning of the protospacer.

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

[0042] In various embodiments of the prime editor guide RNA, the at least one RNA extension comprises a DNA synthesis template. In various other embodiment, the RNA extension further comprises a reverse transcription primer binding site. In still 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 extension can be at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides, at least 22 nucleotides, at least 23 nucleotides, at least 24 nucleotides, at least 25 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides, at least 80 nucleotides, at least 90 nucleotides, at least 100 nucleotides, at least 150 nucleotides, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, or at least 500 nucleotides in length.

[0044] In other embodiments, the DNA synthesis template (i.e., the edit template, per FIG.27) 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, wherein the reverse transcription primer binding site sequence (i.e., the primer binding site, per FIG.27) is at least 3 nucleotides, at least 4nucleotides, 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, the optional 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 PEgRNA disclosed herein may be complexed with a prime editor fusion protein.

[0048] In one aspect, the specification 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 carrying out 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 and optionally has nickase activity.

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

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

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

[0053] In still other embodiments, the complementary non-target strand is nicked to form a reverse transcriptase priming sequence having a free 3´ end.

[0054] In still other embodiments, the single-strand DNA flap hybridizes to the endogenous DNA sequence adjacent to the nick site, thereby installing the desired nucleotide change. In still other embodiments, the single-stranded DNA flap displaces the endogenous DNA sequence adjacent to the nick site and which has a free 5' end. In some embodiments, the displaced endogenous DNA having the 5´ end is excised by the cell.

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

[0056] In various other embodiments, the desired nucleotide change is installed in an editing window that is between about -4 to +10 of the PAM sequence.

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

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

[0059] In other embodiments, the reverse transcriptase of the discloses fusion proteins and / or compositions may comprise any one of the amino acid sequences of SEQ ID NO: 1361485- 1361514, and 1361597-1361598. In still other embodiments, the reverse transcriptase may comprise an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 1361485-1361514, and 1361597-1361598.These sequences may be naturally occurring reverse transcriptase sequences, e.g., from a retrovirus or a retrotransposon, or the sequences may be non-naturally occurring or engineered.

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

[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 this at least 80%, 85%, or 90%, or 95%, or 99% identical to any one of the linker amino acid sequence of SEQ ID NOs: 1361520-1361530, 1361585, and 1361603.

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

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

[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 RNAs, the DNA synthesis template (i.e., the edit template, per FIG.27) may encode a single-strand DNA flap that is complementary to an endogenous DNA sequence adjacent to a nick site, wherein the single-strand DNA flap comprises a desired nucleotide change. The single-stranded DNA flap may displace an endogenous single-strand DNA at the nick site. The displaced endogenous single-strand DNA at the nick site can have a 5´ end and form an endogenous flap, which can be excised by the cell. In various embodiments, excision of the 5´ end endogenous flap can help drive product formation since removing the 5´ end endogenous flap encourages hybridization of the single-strand 3´ DNA flap to the corresponding complementary DNA strand, and the incorporation or assimilation of the desired nucleotide change carried by the single-strand 3´ DNA flap into the target DNA.

[0066] In various embodiments of the prime editor guide RNAs, the cellular repair of the single- strand DNA flap results in installation of the desired nucleotide change, thereby forming a desired product.

[0067] In yet another aspect of the invention, the specification provides for complexes comprising a fusion protein described herein and any prime editor guide RNA (PEgRNA ) described above.

[0068] In still other aspects of the invention, the specification provides a complex comprising a napDNAbp (e.g., Cas9) and an prime editor guide RNA. The napDNAbp can be a Cas9 nickase (e.g., spCas9), or can be an amino acid sequence of SEQ ID NO: 1361421, or an amino acid sequence that is 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 any one of SEQ ID NOs: 1361421-1361484, and 1361593-1361596.

[0069] In various embodiments involving a complex, the prime editor guide RNA is capable of directing the napDNAbp to a target DNA sequence. In various embodiments, a reverse transcriptase may be provided in trans, i.e., provided from a different source than the complex itself. For example, a reverse transcriptase could be provided to the same cell having the complex by introducing a separate vector separately encoding the reverse transcriptase.

[0070] In yet another aspect, the specification provides pharmaceutical compositions (e.g., fusion proteins described herein, PEgRNA 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 an prime editor guide RNA. In some embodiments, the fusion protein described herein and a pharmaceutically acceptable excipient. In other embodiments, the pharmaceutical compositions comprise any extend guide RNA described herein and a pharmaceutically acceptable excipient. In still other embodiments, the pharmaceutical compositions comprise any extend guide RNA described herein in combination with any fusion protein described herein and a pharmaceutically acceptable excipient. In yet other embodiments, the pharmaceutical compositions comprise any polynucleotide sequence encoding one or more of a napDNAbp, a fusion protein, a reverse transcriptase, and an prime editor guide RNA. In stillother embodiments, the various components disclosed herein may be separated into one or more pharmaceutical compositions. For example, a first pharmaceutical composition may comprise a fusion protein or a napDNAbp, a second pharmaceutical compositions may comprise a reverse transcriptase, and a third pharmaceutical composition may comprise an prime editor guide RNA.

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

[0072] In yet another aspect, the present disclosure provides for methods of using the disclosed PEgRNA compositions of matter.

[0073] In one embodiment, the methods relate to a method for installing a desired nucleotide change in a double-stranded DNA using the PEgRNA disclosed herein. The method first comprises contacting the double-stranded DNA sequence with a complex comprising a fusion protein and a prime editor guide RNA as described herein, wherein the fusion protein comprises a napDNAbp and a reverse transcriptase, and wherein the prime editor guide RNA comprises a DNA synthesis template comprising the desired nucleotide change. The napDNAbp nicks the double-stranded DNA sequence on the non-target strand, thereby generating a free single-strand DNA having a 3´ end. Subsequent to nicking, the 3´ end of the free single-strand DNA hybridizes to the DNA synthesis template, thereby priming the reverse transcriptase domain. Reverse transcriptase then facilitates DNA polymerization from the 3´ end, thereby generating a single-strand DNA flap comprising the desired nucleotide change. The single-strand DNA flap then, replaces the endogenous DNA strand adjacent the cut site, thereby installing the desired nucleotide change in the double-stranded DNA sequence.

[0074] In other embodiments, the disclosure provides for a method for introducing one or more changes in the nucleotide sequence of a DNA molecule at a target locus, comprising contacting the DNA molecule with a nucleic acid programmable DNA binding protein (napDNAbp) and a guide RNA which targets the napDNAbp to the target locus, wherein the guide RNA comprises a reverse transcriptase (RT) template sequence comprising at least one desired nucleotide change. The napDNAbp exposes a 3´ end in a DNA strand at the target locus which hybridizes to theDNA synthesis template (e.g., RT template sequence) to prime reverse transcription. Next, a single strand DNA flap comprising the at least one desired nucleotide change based on the DNA synthesis template (e.g., RT template sequence) is synthesized or polymerized by reverse transcriptase. Lastly, the at least one desired nucleotide change is incorporated into the corresponding endogenous DNA, thereby introducing one or more changes in the nucleotide sequence of the DNA molecule at the target locus.

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

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

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

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

[0079] In various embodiments involving methods, the reverse transcriptase may comprise any one of the amino acid sequences of SEQ ID NO: 1361485-1361514, and 1361597-1361598. The reverse transcriptase may also comprise an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 1361485- 1361514, and 1361597-1361598.

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

[0081] The methods may comprise the use of prime editor guide RNAs that comprise an RNA extension at the 3' end, wherein the RNA extension comprises the DNA synthesis template, for example the PEgRNA show in Fig.3B (with the following components as described from 5' to 3': spacer; gRNA core; reverse transcription template; primer binding site) has an extension arm comprising, from 5′ to 3′, a reverse transcription template and a primer binding site.

[0082] The methods may comprise the use of prime editor guide RNAs that comprise an RNA extension at the 5' end, wherein the RNA extension comprises the DNA synthesis template, for example the PEgRNA show in Fig.3A (with the following components as described from 5' to 3': reverse transcription template; primer binding site; linker; spacer; gRNA core) has an extension arm comprising, from 5′ to 3′, a reverse transcription template, primer binding site, and a 5-20 nucleotide long linker.

[0083] The methods may comprise the use of prime editor guide RNAs that comprise an RNA extension at an intramolecular location in the guide RNA, wherein the RNA extension comprises the DNA synthesis template.

[0084] The methods may comprise 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 appreciated that the foregoing concepts, and additional concepts discussed below, may be arranged in any suitable combination, as the present disclosure is not limited in this respect. Further, other advantages and novel features of the present disclosure will becomeapparent from the following detailed description of various non-limiting embodiments when considered in conjunction with the accompanying figures.BRIEF DESCRIPTION OF THE DRAWINGS

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

[0087] FIG.1A.1 provides a schematic of an exemplary process for introducing a single nucleotide change, insertion, and / or deletion into a DNA molecule (e.g., a genome) using a fusion protein comprising a reverse transcriptase fused to a napDNAbp (e.g., Cas9) protein in complex with a prime editor guide RNA. In this embodiment, the guide RNA is extended at the 3' end to include a DNA synthesis template. The schematic shows how a reverse transcriptase (RT) fused to a Cas9 nickase, in a complex with a guide RNA (gRNA), binds the DNA target site and nicks the PAM-containing DNA strand adjacent to the target nucleotide. The RT template uses the nicked DNA as a primer for DNA synthesis from the gRNA, which is used as a template for the synthesis of a new DNA strand that encodes the desired edit. The editing process shown may be referred to as target-primed reverse transcription editing (prime editing). FIG.1A.2 provides the same representation as in FIG.1A.1, except that the prime editor complex is represented more generally as [napDNAbp]-[P]:PEgRNAPEgRNA or [P]- [napDNAbp]:PEgRNAPEgRNA, wherein“P” refers to any polymerase (e.g., a reverse transcriptase),“napDNAbp” refers to a nucleic acid programmable DNA binding protein (e.g., SpCas9), and“PEgRNAPEgRNA” refers to a prime editing guide RNA, and“]-[“ refers to an optional linker. As described elsewhere, e.g., FIGs.3A-3G, the PEgRNAPEgRNA comprises an 5ʹ extension arm comprising a primer binding site and a DNA synthesis template. Although not shown, it is contemplated that the extension arm of the PEgRNAPEgRNA (i.e., which comprises a primer binding site and a DNA synthesis template) can be DNA or RNA. The particular polymerase contemplated in this configuration will depend upon the nature of the DNA synthesis template. For instance, if the DNA synthesis template is RNA, then the polymerase case be an RNA-dependent DNA polymerase (e.g., reverse transcriptase). If the DNA synthesis template is DNA, then the polymerase can be a DNA-dependent DNA polymerase. In variousembodiments, the PEgRNA can be engineered or synthesized to incorporate a DNA-based DNA synthesis template.

[0088] FIG.1B.1 provides a schematic of an exemplary process for introducing a single nucleotide change, insertion, and / or deletion into a DNA molecule (e.g., a genome) using a fusion protein comprising a reverse transcriptase fused to a napDNAbp (e.g., Cas9) in complex with an prime editor guide RNA. In this embodiment, the guide RNA is extended at the 5' end to include a DNA synthesis template. The schematic shows how a reverse transcriptase (RT) fused to a Cas9 nickase, in a complex with a guide RNA (gRNA), binds the DNA target site and nicks the PAM-containing DNA strand adjacent to the target nucleotide. The canonical PAM sequence is 5′-NGG-3′, but different PAM sequences can be associated with different Cas9 proteins or equivalent proteins from different organisms. In addition, any given Cas9 nuclease, e.g., SpCas9, may be modified to alter the PAM specificity of the protein to recognize alternative PAM sequence. The RT enzyme uses the nicked DNA as a primer for DNA synthesis from the gRNA, which is used as a template for the synthesis of a new DNA strand that encodes the desired edit. The editing process shown may be referred to as target-primed reverse transcription editing (TPRT editor or prime editor). FIG.1B.2 provides the same representation as in FIG. 1B.1, except that the prime editor complex is represented more generally as [napDNAbp]- [P]:PEgRNAPEgRNA or [P]-[napDNAbp]:PEgRNAPEgRNA, wherein“P” refers to any polymerase (e.g., a reverse transcriptase),“napDNAbp” refers to a nucleic acid programmable DNA binding protein (e.g., SpCas9), and“PEgRNAPEgRNA” refers to a prime editing guide RNA, refers to an optional linker. As described elsewhere, e.g., FIGs.3A-3G, the PEgRNAPEgRNA comprises an 3ʹ extension arm comprising a primer binding site and a DNA synthesis template. Although not shown, it is contemplated that the extension arm of the PEgRNAPEgRNA (i.e., which comprises a primer binding site and a DNA synthesis template) can be DNA or RNA. The particular polymerase contemplated in this configuration will depend upon the nature of the DNA synthesis template. For instance, if the DNA synthesis template is RNA, then the polymerase case be an RNA-dependent DNA polymerase (e.g., reverse transcriptase). If the DNA synthesis template is DNA, then the polymerase can be a DNA- dependent DNA polymerase.

[0089] FIG.1C is a schematic depicting an exemplary process of how the synthesized single strand of DNA (which comprises the desired nucleotide change) becomes resolved such that thedesired nucleotide change, insertion, and / or deletion is incorporated into the DNA. As shown, following synthesis of the edited strand (or“mutagenic strand”), equilibration with the endogenous strand, flap cleavage of the endogenous strand, and ligation leads to incorporation of the DNA edit after resolution of the mismatched DNA duplex through the action of endogenous DNA repair and / or replication processes.

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

[0091] FIG.1E provides another schematic 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) of a target locus using a nucleic acid programmable DNA binding protein (napDNAbp) complexed with an prime editor guide RNA (e.g., prime editing). The prime editor guide RNA comprises an extension at the 3' or 5' end of the guide RNA, or at an intramolecular location in the guide RNA. In step (a), the napDNAbp / gRNA complex contacts the DNA molecule, and the gRNA guides the napDNAbp to bind to the target locus. In step (b), a nick in one of the strands of DNA (the R-loop strand, or the PAM-containing strand, or the non-target DNA strand, or the protospacer strand) of the target locus is introduced (e.g., by a nuclease or chemical agent), thereby creating an available 3' end in one of the strands of the target locus. In certain embodiments, the nick is created in the strand of DNA that corresponds to the R-loop strand, i.e., the strand that is not hybridized to the guide RNA sequence. In step (c), the 3' end DNA strand interacts with the extended portion of the guide RNA in order to prime reverse transcription. In some embodiments, the 3' end 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 which synthesizes a single strand of DNA from the 3' end of the primed site towards the 3' end of the guide RNA. This forms a single-strand DNA flap comprising the desired nucleotide change (e.g., 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) relate to the resolution of the single strand DNA flap such that the desirednucleotide change becomes incorporated into the target locus. This process can be driven towards the desired product formation by removing the corresponding 5′ endogenous DNA flap that forms once the 3' single strand DNA flap invades and hybridizes to the complementary sequence on the other strand. The process can also be driven towards product formation with second strand nicking, as exemplified in FIG.1D. This process may introduce at least one or more of the following genetic changes: transversions, transitions, deletions, and insertions.

[0092] FIG.1F is a schematic depicting the types of genetic changes that are possible with the target-primed reverse transcription editing (prime editing) processes described herein. The types of nucleotide changes achievable by prime editing include deletions (including short and long deletions), single and / or multiple nucleotide changes, and insertions (including short and long insertions).

[0093] FIG.1G is a schematic depicting an example of temporal second strand nicking exemplified by a prime editor complex. Temporal second strand nicking is a variant of second strand nicking in order to facilitate the formation of the desired edited product. The term “temporal” refers to the fact that the second-strand nick to the unedited strand occurs only after the desired edit is installed in the edited strand. This avoids concurrent nicks on both strands that could lead to double-stranded DNA breaks.

[0094] FIG.1H depicts a variation of prime editing contemplated herein that replaces the napDNAbp (e.g., SpCas9 nickase) with any programmable nuclease domain, such as zinc finger nucleases (ZFN) or transcription activator-like effector nucleases (TALEN). As such, it is contemplated that suitable nucleases do not necessarily need to be“programmed” by a nucleic acid targeting molecule (such as a guide RNA), but rather, may be programmed by defining the specificity of a DNA-binding domain, such as and in particular, a nuclease. Just as in prime editing with napDNAbp moieties, it is preferable that such alternative programmable nucleases be modified such that only one strand of a target DNA is cut. In other words, the programmable nucleases should function as nickases, preferably. Once a programmable nuclease is selected (e.g., a ZFN or a TALEN), then additional functionalities may be engineered into the system to allow it to operate in accordance with a prime editing-like mechanism. For example, the programmable nucleases may be modified by coupling (e.g., via a chemical linker) an RNA or DNA extension arm thereto, wherein the extension arm comprises a primer binding site (PBS) and a DNA synthesis template. The programmable nuclease may also be coupled (e.g., via achemical or amino acid linker) to a polymerase, the nature of which will depend upon whether the extension arm is DNA or RNA. In the case of an RNA extension arm, the polymerase can be an RNA-dependent DNA polymerase (e.g., reverse transcriptase). In the case of a DNA extension arm, the polymerase can be a DNA-dependent DNA polymerase (e.g., a prokaryotic polymerase, including Pol I, Pol II, or Pol III, or a eukaryotic polymerase, including Pol a, Pol b, Pol g, Pol d, Pol e, or Pol z). The system may also include other functionalities added as fusions to the programmable nucleases, or added in trans to facilitate the reaction as a whole (e.g., (a) a helicase to unwind the DNA at the cut site to make the cut strand with the 3’ end available as a primer, (b) a FEN1 to help remove the endogenous strand on the cut strand to drive the reaction towards replacement of the endogenous strand with the synthesized strand, or (c) a nCas9:gRNA complex to create a second site nick on the opposite strand, which may help drive the integration of the synthesize repair through favored cellular repair of the non-edited strand). In an analogous manner to prime editing with a napDNAbp, such a complex with an otherwise programmable nuclease could be used to synthesize and then install a newly synthesized replacement strand of DNA carrying an edit of interest permanently into a target site of DNA.

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

[0096] FIG.1J depicts the mechanism of prime editing showing the anatomical features of the target DNA, prime editor complex, and the interaction between the PEgRNA and the target DNA. First, a prime editor comprising a fusion protein having a polymerase (e.g., reverse transcriptase) and a napDNAbp (e.g., SpCas9 nickase, e.g., a SpCas9 having a deactivating mutation in an HNH nuclease domain (e.g., H840A) or a deactivating mutation in a RuvC nuclease domain (D10A)) is complexed with a PEgRNA and DNA having a target DNA to be edited. The PEgRNA comprises a spacer, gRNA core (aka gRNA scaffold or gRNA backbone) (which binds to the napDNAbp), and an extension arm. The extension arm can be at the 3’ end, the 5’ end, or somewhere within the PEgRNA molecule. As shown, the extension arm is at the 3’ end of the PEgRNA. The extension arm comprises in the 3’ to 5’ direction a primer binding site and a DNA synthesis template (comprising both an edit of interest and regions of homology (i.e., homology arms) that are homologous with the 5’ ended single stranded DNA immediately following the nick site on the PAM strand. As shown, once the nick is introduced thereby producing a free 3’ hydroxyl group immediately upstream of the nick site, the regionimmediately upstream of the nick site on the PAM strand anneals to a complementary sequence at the 3’ end of the extension arm referred to as the“primer binding site,” creating a short double-stranded region with an available 3’ hydroxyl end, which forms a substrate for the polymerase of the prime editor complex. The polymerase (e.g., reverse transcriptase) then polymerase as strand of DNA from the 3’ hydroxyl end to the end of the extension arm. The sequence of the single stranded DNA is coded for by the DNA synthesis template, which is the portion of the extension arm (i.e., excluding the primer binding site) that is“read” by the polymerase to synthesize new DNA. This polymerization effectively extends the sequence of the original 3’ hydroxyl end of the initial nick site. The DNA synthesis template encodes a single strand of DNA that comprises not only the desired edit, but also regions that are homologous to the endogenous single strand of DNA immediately downstream of the nick site on the PAM strand. Next, the encoded 3’ ended single strand of DNA (i.e., the 3’ single strand DNA flap) displaces the corresponding homologous endogenous 5’-ended single strand of DNAimmediately downstream of the nick site on the PAM strand, forming a DNA intermediate having a 5’-ended single strand DNA flap, which is removed by the cell (e.g., by a flap endonuclease). The 3’-ended single strand DNA flap, which anneals to the complement of the endogenous 5’-ended single strand DNA flap, is ligated to the endogenous strand after the 5’DNA flap is removed. The desired edit in the 3’ ended single strand DNA flap, now annealed and ligate, forms a mismatch with the complement strand, which undergoes DNA repair and / or a round of replication, thereby permanently installing the desired edit on both strands.

[0097] FIG.2 shows three Cas complexes that will be tested and their PAM, gRNA, and DNA cleavage features. The figure shows designs for complexes involving SpCas9, SaCas9, and LbCas12a.

[0098] FIGs.3A-3C show designs for engineered 5' extended gRNA (FIG.3A), 3' extended gRNA (FIG.3B), and an intramolecular extension (FIG.3C), each of which may be used for prime editing. The embodiments depict exemplary arrangements of the DNA synthesis template, the primer binding site, and an optional linker sequence in the extended portions of the 3', 5', and intramolecular extended gRNAs, as well as the arrangement of the protospacer and core regions. The disclosed TPRT process is not limited to these configurations of prime editor guide RNAs.

[0099] FIGs.4A-4E demonstrate in vitro TPRT assays. FIG.4A is a schematic of fluorescently labeled DNA substrate gRNA templated extension by an RT enzyme, polyacrylamide gel electrophoresis (PAGE) assay of the reverse transcriptase products. FIG.4B shows TPRT with pre-nicked substrates, dCas9, and 5′-extended gRNAs of differing edit template length. FIG.4C shows the RT reaction with pre-nicked DNA substrates in the absence of Cas9. FIG.4D shows TPRT on full dsDNA substrates with Cas9 (H840A) and 5′-extended gRNAs. FIG.4E shows a 3′-extended gRNA template with pre-nicked and full dsDNA substrates. All reactions are with M-MLV RT.

[0100] FIG.5 shows in vitro validations using 5′-extended gRNAs with varying length edit templates. Fluorescently labeled (Cy5) DNA targets were used as substrates and were pre-nicked in this set of experiments. The Cas9 used in these experiments is catalytically dead Cas9(dCas9), and the RT used is Superscript III, a commercially available RT derived from Moloney- Murine Leukemia Virus (M-MLV). dCas9:gRNA complexes were formed from purified components. Then, the fluorescently labeled DNA substrate was added along with dNTPs and the RT enzyme. After 1 hour of incubation at 37 ºC, the reaction products were analyzed by denaturing urea-polyacrylamide gel electrophoresis (PAGE). The gel image shows extension of the original DNA strand to lengths that are consistent with the length of the reverse transcription template.

[0101] FIG.6 shows in vitro validations using 5'-extended gRNAs with varying length edit templates, which closely parallels those shown in FIG.5. However, the DNA substrates are not pre-nicked in this set of experiments. The Cas9 used in these experiments is a Cas9 nickase (SpyCas9 H840A mutant), and the RT used is Superscript III, a commercially available RT derived from the Moloney-Murine Leukemia Virus (M-MLV). The reaction products were analyzed by denaturing urea-polyacrylamide gel electrophoresis (PAGE). As shown in the gel, the nickase efficiently cleaves the DNA strand when the gRNA is used (gRNA_0, lane 3).

[0102] FIG.7 demonstrates that 3' extensions support DNA synthesis and do not significantly affect Cas9 nickase activity. Pre-nicked substrates (black arrow) are near-quantitatively converted to RT products when either dCas9 or Cas9 nickase is used (lanes 4 and 5). Greater than 50% conversion to the RT product (red arrow) is observed with full substrates (lane 3). Cas9 nickase (SpyCas9 H840A mutant), catalytically dead Cas9 (dCas9), and Superscript III, a commercially available RT derived from the Moloney-Murine Leukemia Virus (M-MLV) were used.

[0103] FIG.8 demonstrates dual color experiments that were used to determine if the RT reaction preferentially occurs with the gRNA in cis (bound in the same complex). Two separate experiments were conducted for 5′-extended and 3′-extended gRNAs. Products were analyzed by PAGE. Product ratio calculated as (Cy3cis / Cy3trans) / (Cy5trans / Cy5cis).

[0104] FIGs.9A-9D demonstrates a flap model substrate. FIG.9A shows a dual-FP reporter for flap-directed mutagenesis. FIG.9B shows stop codon repair in HEK cells. FIG.9C shows sequenced yeast clones after flap repair. FIG.9D shows testing of different flap features in human cells.

[0105] FIG.10 demonstrates prime editing on plasmid substrates. A dual-fluorescent reporter plasmid was constructed for yeast (S. cerevisiae) expression. Expression of this construct in yeast produces only GFP. The in vitro TRT reaction introduces a point mutation, and transforms the parent plasmid or an in vitro Cas9(H840A) nicked plasmid into yeast. The colonies are visualized by fluorescence imaging. Yeast dual-FP plasmid transformants are shown.Transforming the parent plasmid or an in vitro Cas9 (H840A) nicked plasmid results in only green GFP expressing colonies. The TRT reaction with 5′-extended or 3′-extended gRNAs produces a mix of green and yellow colonies. The latter express both GFP and mCherry. Moreyellow colonies are observed with the 3′-extended gRNA. A positive control that contains no stop codon is shown as well.

[0106] FIG.11 shows prime editing on plasmid substrates similar to the experiment in FIG.10, but instead of installing a point mutation in the stop codon, prime editing installs a single nucleotide insertion (left) or deletion (right) that repairs a frameshift mutation and allows for synthesis of downstream mCherry. Both experiments used 3′ extended gRNAs.

[0107] FIG.12 shows editing products of prime editing on plasmid substrates, characterized by Sanger sequencing. Individually colonies from the TRT transformations were selected and analyzed by Sanger sequencing. Precise edits were observed by sequencing select colonies. Green colonies contained plasmids with the original DNA sequence, while yellow colonies contained the precise mutation designed by the prime editing gRNA. No other point mutations or indels were observed.

[0108] FIG.13 shows the potential scope for the new prime editing technology is shown and compared to deaminase-mediated base editor technologies.

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

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

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

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

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

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

[0115] FIG.20 shows editing efficiency of the target nucleotide when a single strand nick is introduced in the complementary DNA strand in proximity to the target nucleotide. Nicking at various distances from the target nucleotide was tested (orange triangles). Editing efficiency at the target base pair (blue bars) is shown alongside the indel formation rate (orange bars). The “none” example does not contain a complementary strand nicking guide RNA. Editing rates were quantified by high throughput sequencing of genomic DNA amplicons.

[0116] FIG.21 demonstrates processed high throughput sequencing data showing the desired T to A transversion mutation and general absence of other major genome editing byproducts.

[0117] FIG.22 provides a schematic of an exemplary process for conducting targeted mutagenesis with an error-prone reverse transcriptase on a target locus using a nucleic acid programmable DNA binding protein (napDNAbp) complexed with a prime editor guide RNA. This process may be referred to as an embodiment of prime editing for targeted mutagenesis. The prime editor guide RNA comprises an extension at the 3' or 5' end of the guide RNA, or at an intramolecular location in the guide RNA. In step (a), the napDNAbp / gRNA complex contacts the DNA molecule and the gRNA guides the napDNAbp to bind to the target locus to be mutagenized. In step (b), a nick in one of the strands of DNA of the target locus is introduced (e.g., by a nuclease or chemical agent), thereby creating an available 3' end in one of the strands of the target locus. In certain embodiments, the nick is created in the strand of DNA that corresponds to the R-loop strand, i.e., the strand that is not hybridized to the guide RNA sequence. In step (c), the 3' end DNA strand interacts with the extended portion of the guide RNA in order to prime reverse transcription. In some embodiments, the 3' ended 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 mutagenized single strand of DNA from the 3' end of the primed site towards the 3' end of the guide RNA.Exemplary mutations are indicated with an asterisk“*”. This forms a single-strand DNA flap comprising the desired mutagenized region. In step (e), the napDNAbp and guide RNA are released. Steps (f) and (g) relate to the resolution of the single strand DNA flap (comprising the mutagenized region) such that the desired mutagenized region becomes incorporated into the target locus. This process can be driven towards the desired product formation by removing the corresponding 5′ endogenous DNA flap that forms once the 3' single strand DNA flap invades and hybridizes to the complementary sequence on the other strand. The process can also bedriven towards product formation with second strand nicking, as exemplified in FIG.1D.Following endogenous DNA repair and / or replication processes, the mutagenized region becomes incorporated into both strands of DNA of the DNA locus.

[0118] FIG.23 is a schematic of gRNA design for contracting trinucleotide repeat sequences and trinucleotide repeat contraction with TPRT genome editing. Trinucleotide repeat expansion is associated with a number of human diseases, including Huntington’s Disease, Fragile X syndrome, and Friedreich’s ataxia. The most common trinucleotide repeat contains CAG triplets, though GAA triplets (Friedreich’s ataxia) and CGG triplets (Fragile X syndrome) also occur. Inheriting a predisposition to expansion, or acquiring an already expanded parental allele, increases the likelihood of acquiring the disease. Pathogenic expansions of trinucleotide repeats could hypothetically be corrected using prime editing. A region upstream of the repeat region can be nicked by an RNA-guided nuclease, then used to prime synthesis of a new DNA strand that contains a healthy number of repeats (which depends on the particular gene and disease). After the repeat sequence, a short stretch of homology is added that matches the identity of the sequence adjacent to the other end of the repeat (red strand). Invasion of the newly synthesized strand, and subsequent replacement of the endogenous DNA with the newly synthesized flap, leads to a contracted repeat allele.

[0119] FIG.24 is a schematic showing precise 10-nucleotide deletion with prime editing. A guide RNA targeting the HEK3 locus was designed with a reverse transcription template that encodes a 10-nucleotide deletion after the nick site. Editing efficiency in transfected HEK cells was assessed using amplicon sequencing.

[0120] FIG.25 is a schematic showing gRNA design for peptide tagging genes at endogenous genomic loci and peptide tagging with 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, exemplified by the sequence FLNCCPGCCMEP (SEQ ID NO: 1361586). When expressed within cells, proteins containing the tetracysteine motif can be fluorescently labeled with fluorophore-arsenic probes (see ref: J. Am. Chem.Soc., 2002, 124 (21), pp 6063–6076. DOI: 10.1021 / ja017687n). The“sortagging” system employs bacterial sortase enzymes that covalently conjugate labeled peptide probes to proteins containing suitable peptide substrates (see ref: Nat. Chem. Biol.2007 Nov;3(11):707-8.DOI: 10.1038 / nchembio.2007.31). The FLAG-tag (DYKDDDDK (SEQ ID NO: 1361587)), V5-tag (GKPIPNPLLGLDST (SEQ ID NO: 1361588)), GCN4-tag (EELLSKNYHLENEVARLKK (SEQ ID NO: 1361589)), HA-tag (YPYDVPDYA (SEQ ID NO: 1361590)), and Myc-tag (EQKLISEEDL (SEQ ID NO: 1361591)) are commonly employed as epitope tags for immunoassays. The pi-clamp encodes a peptide sequence (FCPF (SEQ ID NO: 1361592)) that can by labeled with a pentafluoro-aromatic substrates (ref: Nat. Chem.2016 Feb;8(2):120-8. doi: 10.1038 / nchem.2413).

[0121] FIG.26 shows precise installation of a His6-tag and a FLAG-tag into genomic DNA. A guide RNA targeting the HEK3 locus was designed with a reverse transcription template that encodes either an 18-nt His-tag insertion or a 24-nt FLAG-tag insertion. Editing efficiency in transfected HEK cells was assessed using amplicon sequencing. Note that the full 24-nt sequence of the FLAG-tag is outside of the viewing frame (sequencing confirmed full and precise insertion).

[0122] FIG.27 provides the structure of an embodiment of a PEgRNA contemplated herein and which may be designed in accordance with the methodology defined in Example 2. The PEgRNA comprises three main component elements ordered in the 5′ to 3′ direction, namely: a spacer, a gRNA core, and an extension arm at the 3′ end. The extension arm may further be divided into the following structural elements in the 5′ to 3′ direction, namely: a primer binding site (A), an edit template (B), and a homology arm (C). In addition, the PEgRNA may comprise an optional 3′ end modifier region (e1) and an optional 5′ end modifier region (e2). Still further, the PEgRNA may comprise a transcriptional termination signal at the 3′ end of the PEgRNA (not depicted). These structural elements are further defined herein. The depiction of the structure of the PEgRNA is not meant to be limiting and embraces variations in the arrangement of the elements. For example, the optional sequence modifiers (e1) and (e2) could be positioned within or between any of the other regions shown, and not limited to being located at the 3′ and 5′ ends. The PEgRNAPEgRNA could comprise, in certain embodiments, secondary RNA structure, such as, but not limited to, hairpins, stem / loops, toe loops, RNA-binding protein recruitment domains (e.g., the MS2 aptamer which recruits and binds to the MS2cp protein). For instance, such secondary structures could be position within the spacer, the gRNA core, or the extension arm, and in particular, within the e1 and / or e2 modifier regions. In addition to secondary RNA structures, the PEgRNAPEgRNAs could comprise (e.g., within the e1 and / or e2 modifier regions) a chemical linker or a poly(N) linker or tail, where“N” can be any nucleobase.In some embodiments (e.g., as shown in FIG.72(c)), the chemical linker may function to prevent reverse transcription of the sgRNA scaffold or core. In addition, in certain embodiments (e.g., see FIG.72(c)), the extension arm (3) could be comprised of RNA or DNA, and / or could include one or more nucleobase analogs (e.g., which might add functionality, such as temperature resilience). Still further, the orientation of the extension arm (3) can be in the natural 5ʹ-to-3ʹ direction, or synthesized in the opposite orientation in the 3ʹ-to-5ʹ direction (relative to the orientation of the PEgRNAPEgRNA molecule overall). It is also noted that one of ordinary skill in the art will be able to select an appropriate DNA polymerase, depending on the nature of the nucleic acid materials of the extension arm (i.e., DNA or RNA), for use in prime editing that may be implemented either as a fusion with the napDNAbp or as provided in trans as a separate moiety to synthesize the desired template-encoded 3ʹ single-strand DNA flap that includes the desired edit. For example, if the extension arm is RNA, then the DNA polymerase could be a reverse transcriptase or any other suitable RNA-dependent DNA polymerase. However, if the extension arm is DNA, then the DNA polymerase could be a DNA-dependent DNA polymerase. In various embodiments, provision of the DNA polymerase could be in trans, e.g., through the use of an RNA-protein recruitment domain (e.g., an MS2 hairpin installed on thePEgRNAPEgRNA (e.g., in the e1 or e2 region, or elsewhere and an MS2cp protein fused to the DNA polymerase, thereby co-localizing the DNA polymerase to the PEgRNAPEgRNA). It is also noted that the primer binding site does not generally form a part of the template that is used by the DNA polymerase (e.g., reverse transcriptase) to encode the resulting 3ʹ single-strand DNA flap that includes the desired edit. Thus, the designation of the“DNA synthesis template” refers to the region or portion of the extension arm (3) that is used as a template by the DNA polymerase to encode the desired 3ʹ single-strand DNA flap containing the edit. In some embodiments, the DNA synthesis template includes the“edit template” and the“homology arm”. In other embodiments, the DNA synthesis template may also include the e2 region or a portion thereof. For instance, if the e2 region comprises a secondary structure that causes termination of DNA polymerase activity, then it is possible that DNA polymerase function will be terminated before any portion of the e2 region is actual encoded into DNA. It is also possible that some or even all of the e2 region will be encoded into DNA. How much of e2 is actually used as a template will depend on its constitution and whether that constitution interrupts DNApolymerase function.

[0123] FIG.28 provides the structure of another embodiment of a PEgRNA contemplated herein and which may be designed in accordance with the methodology defined in Example 2. The PEgRNA comprises three main component elements ordered in the 5′ to 3′ direction, namely: a spacer, a gRNA core, and an extension arm at the 3′ end. The extension arm may further be divided into the following structural elements in the 5′ to 3′ direction, namely: a primer binding site (A), an edit template (B), and a homology arm (C). In addition, the PEgRNA may comprise an optional 3′ end modifier region (e1) and an optional 5′ end modifier region (e2). Still further, the PEgRNA may comprise a transcriptional termination signal on the 3′ end of the PEgRNA (not depicted). These structural elements are further defined herein. The depiction of the structure of the PEgRNA is not meant to be limiting and embraces variations in the arrangement of the elements. For example, the optional sequence modifiers (e1) and (e2) could be positioned within or between any of the other regions shown, and not limited to being located at the 3′ and 5′ ends. The PEgRNAPEgRNA could comprise, in certain embodiments, secondary RNA structures, such as, but not limited to, hairpins, stem / loops, toeloops, RNA- binding protein recruitment domains (e.g., the MS2 aptamer which recruits and binds to the MS2cp protein). These secondary structures could be positioned anywhere in thePEgRNAPEgRNA molecule. For instance, such secondary structures could be position within the spacer, the gRNA core, or the extension arm, and in particular, within the e1 and / or e2 modifier regions. In addition to secondary RNA structures, the PEgRNAPEgRNAs could comprise (e.g., within the e1 and / or e2 modifier regions) a chemical linker or a poly(N) linker or tail, where“N” can be any nucleobase. In some embodiments (e.g., as shown in FIG.27), the chemical linker may function to prevent reverse transcription of the sgRNA scaffold or core. In addition, in certain embodiments (e.g., see FIG.28), the extension arm (3) could be comprised of RNA or DNA, and / or could include one or more nucleobase analogs (e.g., which might add functionality, such as temperature resilience). Still further, the orientation of the extension arm (3) can be in the natural 5ʹ-to-3ʹ direction, or synthesized in the opposite orientation in the 3ʹ-to- 5ʹ direction (relative to the orientation of the PEgRNAPEgRNA molecule overall). It is also noted that one of ordinary skill in the art will be able to select an appropriate DNA polymerase, depending on the nature of the nucleic acid materials of the extension arm (i.e., DNA or RNA), for use in prime editing that may be implemented either as a fusion with the napDNAbp or as provided in trans as a separate moiety to synthesize the desired template-encoded 3ʹ single-strand DNA flap that includes the desired edit. For example, if the extension arm is RNA, then the DNA polymerase could be a reverse transcriptase or any other suitable RNA-dependent DNA polymerase. However, if the extension arm is DNA, then the DNA polymerase could be a DNA- dependent DNA polymerase. In various embodiments, provision of the DNA polymerase could be in trans, e.g., through the use of an RNA-protein recruitment domain (e.g., an MS2 hairpin installed on the PEgRNAPEgRNA (e.g., in the e1 or e2 region, or elsewhere and an MS2cp protein fused to the DNA polymerase, thereby co-localizing the DNA polymerase to thePEgRNAPEgRNA). It is also noted that the primer binding site does not generally form a part of the template that is used by the DNA polymerase (e.g., reverse transcriptase) to encode the resulting 3ʹ single-strand DNA flap that includes the desired edit. Thus, the designation of the “DNA synthesis template” refers to the region or portion of the extension arm (3) that is used as a template by the DNA polymerase to encode the desired 3ʹ single-strand DNA flap containing the edit. In some embodiments, the DNA synthesis template includes the“edit template” and the “homology arm”. In other embodiments, the DNA synthesis template may also include the e2 region or a portion thereof. For instance, if the e2 region comprises a secondary structure that causes termination of DNA polymerase activity, then it is possible that DNA polymerase function will be terminated before any portion of the e2 region is actual encoded into DNA. It is also possible that some or even all of the e2 region will be encoded into DNA. How much of e2 is actually used as a template will depend on its constitution and whether that constitution interrupts DNA polymerase function.

[0124] FIG.29 is a schematic depicting the interaction of a typical PEgRNA with a target site of a double stranded DNA and the concomitant production of a 3ʹ single stranded DNA flap containing the genetic change of interest. The double strand DNA is shown with the top strand (i.e., the target strand) in the 3ʹ to 5ʹ orientation and the lower strand (i.e., the PAM strand or non-target strand) in the 5ʹ to 3ʹ direction. The top strand comprises the complement of the “protospacer” and the complement of the PAM sequence and is referred to as the“target strand.”” because it is the strand that is target by and anneals to the spacer of the PEgRNA. The complementary lower strand is referred to as the“non-target strand.”” or the“PAM strand” or the“protospacer strand” since it contains the PAM sequence (e.g., NGG) and the protospacer. Although not shown, the PEgRNA depicted would be complexed with a Cas9 or equivalent. domain of a prime editor fusion protein. As shown in the schematic, the spacer of the PEgRNAanneals to the complementary region of the protospacer on the target strand, which is referred to as the protospacer, which is located just downstream of the PAM sequence is approximately 20 nucleotides in length.. This interaction forms as DNA / RNA hybrid between the spacer RNA and the complement of the protospacer DNA, and induces the formation of an R loop in the region opposite the protospacer. As taught elsewhere herein, the Cas9 protein (not shown) then induces a nick in the non-target strand, as shown. This then leads to the formation of the 3ʹ ssDNA flap region immediately upstream of the nick site which, in accordance with *z*, interacts with the 3ʹ end of the PEgRNA at the primer binding site. The 3ʹ end of the ssDNA flap (i.e., the reverse transcriptase primer sequence) anneals to the primer binding site (A) on the PEgRNA, thereby priming reverse transcriptase. Next, reverse transcriptase (e.g., provided in trans or provided cis as a fusion protein, attached to the Cas9 construct) then polymerizes a single strand of DNA which is coded for by the DNA synthesis template (including the edit template (B) and homology arm (C).)). The polymerization continues towards the 5ʹ end of the extension arm.The polymerized strand of ssDNA forms a ssDNA 3′ end flap which, as describe elsewhere (e.g., as shown in FIG.1E), invades the endogenous DNA, displacing thecorresponding endogenous strand (which is removed as a 5′ DNA flap of endogenous DNA), and installing the desired nucleotide edit (single nucleotide base pair change, deletions, insertions (including whole genes) through naturally occurring DNA repair / replication rounds.

[0125] FIG.30 assists in understanding the disclosure of the PEgRNA of the Sequence Listing. The figures shows two exemplary PEgRNA sequences (SEQ ID NO: 135529 (top) and SEQ ID NO: 135880 (bottom)) and how the various disclosed sequence subsets map thereon. 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), edit template (SEQ ID NO: 677585), and the homology arm (SEQ ID NO: 813099). For SEQ ID NO: 135880,corresponding sequences are spacer (SEQ ID NO: 880463), extension arm (SEQ ID NO:947841), primer binding site (SEQ ID NO: 1015219), edit template (SEQ ID NO:1082597), and the homology arm (SEQ ID NO: 1149975).

[0126] FIG.31 is a flow chart showing an exemplary high level computerized method 3100 for determining an extended gRNA structure, according to some embodiments of the disclosure. At step 3102, a computing device (e.g., the computing device 3400 described in conjunction with FIG.34) accesses data indicative of an input allele, an output allele, and a fusion protein thatincludes 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, and it should be appreciated that such data can be accessed using one or more steps without departing from the spirit of the techniques described herein. Accessing data can include receiving data, storing data, accessing a database, and / or the like.

[0127] FIG.32 is a flow chart showing an exemplary computerized method 3200 for determining the components of an extended gRNA structure, including the components of the extension, according to some embodiments. It should be appreciated that FIG.32 is intended to be illustrative, and therefore, techniques used to determine the extended gRNA can include more, or fewer, steps than those shown in FIG.32.

[0128] FIG.33 is a flow chart showing an exemplary computerized method 3300 for determining sets 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., a ClinVar database, which is accessible at www.ncbi.nlm.nih.gov / clinvar / ) that includes a set of mutation entries that each include an input allele representing the mutation and an output allelerepresenting the corrected wild-type sequence.

[0129] FIG.34 is an illustrative implementation of a computer system 3400 that may be used to perform any of the aspects of the techniques and embodiments disclosed herein. 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 writing data to and reading data from the memory 3420 and the non-volatile storage device 3430 in any suitable manner, as the aspects of the invention described herein are not limited in this respect.

[0130] FIG.35A is a schematic of PE-based insertion of sequences encoding RNA motifs in connection with Example 3.

[0131] FIG.35B is a list (not exhaustive) of some example motifs that could potentially be inserted, and their functions, in connection with Example 3.

[0132] FIG.36 provides a bar graph comparing the efficiency (i.e.,of total sequencing reads with the specified edit or indels”) of PE2, PE2-trunc, PE3, and PE3-trunc over different target sites in various cell lines. The data shows that the prime editors comprising the truncated RT variants were about as efficient as the prime editors comprising the non-truncated RT proteins.

[0133] FIG.37A shows the nucleotide sequence of a SpCas9 PEgRNA molecule (top) which terminates at the 3ʹ end in a“UGU” and does not contain a toe loop element. The lower portion of the figure depicts the same SpCas9 PEgRNA molecule but is further modified to contain a toe loop element having the sequence 5ʹ-“GAAANNNNN”-3ʹ inserted immediately before the “UUU” 3ʹ end. The“N” can be any nucleobase.

[0134] FIG.37B shows the results of Example 4, which demonstrates that the efficiency of prime editing in HEK cells or EMX cells is increased using PEgRNA containing toe loop elements, wherease the percent of indel formation is largely unchanged.

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

[0136] FIG.39 depicts the mechanism of intein removal from a polypeptide sequence and the reformation of a peptide bond between the N-terminal and the C-terminal extein sequences. (a) depicts the general mechanism of two half proteins each containing half of an intein sequence, which when in contact within a cell result in a fully-functional intein which then undergoes self- spicing and excision. The process of excision results in the formation of a peptide bond between the N-terminal protein half (or the“N extein”) and the C-terminal protein half (or the“C extein”) to form a whole, single polypeptide comprising the N extein and the C extein portions. In various embodiments, the N extein may correspond to the N-terminal half of a split prime editor fusion protein and the C extein may correspond to the C-terminal half of a split prime editor. (b) shows a chemical mechanics of intein excision and the reformation of a peptide bond that joins the N extein half (the red-colored half) and the C extein half (the blue-colored half). Excision of the split inteins (i.e., the N intein and the C intein in the split intein configuration) may also be referred to as“trans splicing” as it involves the splicing action of two separate components provided in trans. DEFINITIONSAntisense strand

[0137] In genetics, the“antisense” strand of a segment within double-stranded DNA is the template strand, and which is considered to run in the 3' to 5' orientation. By contrast, the “sense” strand is the segment within double-stranded DNA that runs from 5' to 3', and which iscomplementary to the antisense strand of DNA, or template strand, which runs from 3' to 5'. In the case of a DNA segment that encodes a protein, the sense strand is the strand of DNA that has the same sequence as the mRNA, which takes the antisense strand as its template during transcription, and eventually undergoes (typically, not always) translation into a protein. The antisense strand is thus responsible for the RNA that is later translated to protein, while the sense strand possesses a nearly identical makeup to that of the mRNA. Note that for each segment of dsDNA, there will possibly be two sets of sense and antisense, depending on which direction one reads (since sense and antisense is relative to perspective). It is ultimately the gene product, or mRNA, that dictates which strand of one segment of dsDNA is referred to as sense or antisense. Cas9

[0138] The term“Cas9” or“Cas9 nuclease” refers to an RNA-guided nuclease comprising a Cas9 domain, or a fragment thereof (e.g., a protein comprising an active or inactive DNA cleavage domain of Cas9, and / or the gRNA binding domain of Cas9). A“Cas9 domain” as used herein, is a protein fragment comprising an active or inactive cleavage domain of Cas9 and / or the gRNA binding domain of Cas9. A“Cas9 protein” is a full length Cas9 protein. A Cas9 nuclease is also referred to sometimes as a casn1 nuclease or a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)-associated nuclease. CRISPR is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements, and conjugative plasmids). CRISPR clusters contain spacers, sequences complementary to antecedent mobile elements, and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems correct processing of pre-crRNA requires a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc) and a Cas9 domain. The tracrRNA serves as a guide for ribonuclease 3-aided processing of pre- crRNA. Subsequently, Cas9 / crRNA / tracrRNA endonucleolytically cleaves linear or circular dsDNA target complementary to the spacer. The target strand not complementary to crRNA is first cut endonucleolytically, then trimmed 3′-5′ exonucleolytically. In nature, DNA-binding and cleavage typically requires protein and both RNAs. However, single guide RNAs (“sgRNA”, or simply“gNRA”) can be engineered so as to incorporate aspects of both the crRNA and tracrRNA into a single RNA species. See, e.g., Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J.A., Charpentier E. Science 337:816-821(2012), the entire contents of which are hereby incorporated by reference. Cas9 recognizes a short motif in the CRISPR repeat sequences (thePAM or protospacer adjacent motif) to help distinguish self versus non-self. Cas9 nuclease sequences and structures are well known to those of skill in the art (see, e.g.,“Complete genome sequence of an M1 strain of Streptococcus pyogenes.” Ferretti et al., J.J., McShan W.M., Ajdic D.J., Savic D.J., Savic G., Lyon K., Primeaux C., Sezate S., Suvorov A.N., Kenton S., Lai H.S., Lin S.P., Qian Y., Jia H.G., Najar F.Z., Ren Q., Zhu H., Song L., White J., Yuan X., Clifton S.W., Roe B.A., McLaughlin R.E., Proc. Natl. Acad. Sci. U.S.A.98:4658-4663(2001);“CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.” Deltcheva E., Chylinski K., Sharma C.M., Gonzales K., Chao Y., Pirzada Z.A., Eckert M.R., Vogel J., Charpentier E., Nature 471:602-607(2011); and“A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.” Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J.A., Charpentier E. Science 337:816-821(2012), the entire contents of each of which are incorporated herein by reference). Cas9 orthologs have been described in various species, including, but not limited to, S. pyogenes and S. thermophilus. Additional suitable Cas9 nucleases and sequences will be apparent to those of skill in the art based on this disclosure, and such Cas9 nucleases and sequences include Cas9 sequences from the organisms and loci disclosed in Chylinski, Rhun, and Charpentier,“The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems” (2013) RNA Biology 10:5, 726-737; the entire contents of which are incorporated herein by reference. In some embodiments, a Cas9 nuclease comprises one or more mutations that partially impair or inactivate the DNA cleavage domain.

[0139] A nuclease-inactivated Cas9 domain may interchangeably be referred to as a“dCas9” protein (for nuclease-“dead” Cas9). Methods for generating a Cas9 domain (or a fragment thereof) having an inactive DNA cleavage domain are known (see, e.g., Jinek et al., Science. 337:816-821(2012); Qi et al.,“Repurposing CRISPR as an RNA-Guided Platform for Sequence- Specific Control of Gene Expression” (2013) Cell.28;152(5):1173-83, the entire contents of each of which are incorporated herein by reference). For example, the DNA cleavage domain of Cas9 is known to include two subdomains, the HNH nuclease subdomain and the RuvC1 subdomain. The HNH subdomain cleaves the strand complementary to the gRNA, whereas the RuvC1 subdomain cleaves the non-complementary strand. Mutations within these subdomains can silence the nuclease activity of Cas9. For example, the mutations D10A and H840A completely inactivate the nuclease activity of S. pyogenes Cas9 (Jinek et al., Science.337:816- 821(2012); Qi et al., Cell.28;152(5):1173-83 (2013)). In some embodiments, proteinscomprising fragments of Cas9 are provided. For example, in some embodiments, a protein comprises one of two Cas9 domains: (1) the gRNA binding domain of Cas9; or (2) the DNA cleavage domain of Cas9. In some embodiments, proteins comprising Cas9 or fragments thereof are referred to as“Cas9 variants.” A Cas9 variant shares homology to Cas9, or a fragment thereof. For example, a Cas9 variant is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, at least about 99.8% identical, or at least about 99.9% identical to wild type Cas9 (e.g., SpCas9 of SEQ ID NO: 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 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 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).cDNA

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

[0141] As used herein, the term“circular permutant” refers to a protein or polypeptide (e.g., a Cas9) comprising a circular permutation, which is change in the protein’s structuralconfiguration involving a change in order of amino acids appearing in the protein’s amino acid sequence. In other words, circular permutants are proteins that have altered N- and C-termini ascompared to a wild-type counterpart, e.g., the wild-type C-terminal half of a protein becomes the new N-terminal half. Circular permutation (or CP) is essentially the topological rearrangement of a protein’s primary sequence, connecting its N- and C-terminus, often with a peptide linker, while concurrently splitting its sequence at a different position to create new, adjacent N- and C- termini. The result is a protein structure with different connectivity, but which often can have the same overall similar three-dimensional (3D) shape, and possibly include improved or altered characteristics, including, reduced proteolytic susceptibility, improved catalytic activity, altered substrate or ligand binding, and / or improved thermostability. Circular permutant proteins can occur in nature (e.g., concanavalin A and lectin). In addition, circular permutation can occur as a result of posttranslational modifications or may be engineered using recombinant techniques. Circularly permuted Cas9

[0142] The term“circularly permuted Cas9” refers to any Cas9 protein, or variant thereof, that occurs as a circular permutant, whereby its N- and C-termini have been reconfigured though rearrangement of the protein’s primary sequence. Such circularly permuted Cas9 proteins (“CP- Cas9”), or variants thereof, retain the ability to bind DNA when complexed with a guide RNA (gRNA). See, Oakes et al.,“Protein Engineering of Cas9 for enhanced function,” Methods Enzymol, 2014, 546: 491–511 and Oakes et al.,“CRISPR-Cas9 Circular Permutants asProgrammable Scaffolds for Genome Modification,” Cell, January 10, 2019, 176: 254-267, each of are incorporated herein by reference. The instant disclosure contemplates any previously known CP-Cas9 or use a new CP-Cas9 so long as the resulting circularly permuted protein retains the ability to bind DNA when complexed with a guide RNA (gRNA). Exemplary CP- Cas9 proteins are SEQ ID NOs: 1361475-1361484.DNA synthesis template

[0143] As used herein, the term“DNA synthesis template” refers to the region or portion of the extension arm of a PEgRNA that is utilized as a template strand by a polymerase of a prime editor to encode a 3ʹ single-strand DNA flap that contains the desired edit and which then, through the mechanism of prime editing, replaces the corresponding endogenous strand of DNA at the target site. In various embodiments, the DNA synthesis template is shown in FIG.3A (in the context of a PEgRNA comprising a 5ʹ extension arm), FIG.3B (in the context of a PEgRNA comprising a 3ʹ extension arm), FIG.3C (in the context of an internal extension arm), FIG.3D (in the context of a 3ʹ extension arm), and FIG.3E (in the context of a 5ʹ extension arm). Theextension arm, including the DNA synthesis template, may be comprised of DNA or RNA. In the case of RNA, the polymerase of the prime editor can be an RNA-dependent DNApolymerase (e.g., a reverse transcriptase). In the case of DNA, the polymerase of the prime editor can be a DNA-dependent DNA polymerase. In various embodiments (e.g., as depicted in FIGs.3D-3E), the DNA synthesis template (4) may comprise the“edit template” and the “homology arm”, and all or a portion of the optional 5’ end modifier region, e2. That is, depending on the nature of the e2 region (e.g., whether it includes a hairpin, toe loop, or stem / loop secondary structure), the polymerase may encode none, some, or all of the e2 region, as well. Said another way, in the case of a 3ʹ extension arm, the DNA synthesis template (3) can include the portion of the extension arm (3) that spans from the 5ʹ end of the primer binding site (PBS) to 3ʹ end of the gRNA core that may operate as a template for the synthesis of a single- strand of DNA by a polymerase (e.g., a reverse transcriptase). In the case of a 5ʹ extension arm, the DNA synthesis template (3) can include the portion of the extension arm (3) that spans from the 5ʹ end of the PEgRNA molecule to the 3ʹ end of the edit template. Preferably, the DNA synthesis template excludes the primer binding site (PBS) of PEgRNAs either having a 3ʹ extension arm or a 5ʹ extension arm. Certain embodiments described here (e.g., FIG.71A) refer to an“an RT template,” which is inclusive of the edit template and the homology arm, i.e., the sequence of the PEgRNA extension arm which is actually used as a template during DNA synthesis. The term“RT template” is equivalent to the term“DNA synthesis template.”Downstream

[0144] As used herein, the terms“upstream” and“downstream” are terms of relativity that define the linear position of at least two elements located in a nucleic acid molecule (whether single or double-stranded) that is orientated in a 5ʹ-to-3ʹ direction. In particular, a first element is upstream of a second element in a nucleic acid molecule where the first element is positioned somewhere that is 5’ to the second element. For example, a SNP is upstream of a Cas9-induced nick site if the SNP is on the 5’ side of the nick site. Conversely, a first element is downstream of a second element in a nucleic acid molecule where the first element is positioned somewhere that is 3’ to the second element. For example, a SNP is downstream of a Cas9-induced nick site if the SNP is on the 3’ side of the nick site. The nucleic acid molecule can be a DNA (double or single stranded). RNA (double or single stranded), or a hybrid of DNA and RNA. The analysis is the same for single strand nucleic acid molecule and a double strand molecule since the termsupstream and downstream are in reference to only a single strand of a nucleic acid molecule, except that one needs to select which strand of the double stranded molecule is being considered. Often, the strand of a double stranded DNA which can be used to determine the positional relativity of at least two elements is the“sense” or“coding” strand. In genetics, a“sense” strand is the segment within double-stranded DNA that runs from 5' to 3', and which is complementary to the antisense strand of DNA, or template strand, which runs from 3' to 5'. Thus, as an example, a SNP nucleobase is“downstream” of a promoter sequence in a genomic DNA (which is double-stranded) if the SNP nucleobase is on the 3' side of the promoter on the sense or coding strand.CRISPR

[0145] CRISPR is a family of DNA sequences (i.e., CRISPR clusters) in bacteria and archaea that represent snippets of prior infections by a virus that has invaded the prokaryote. The snippets of DNA are used by the prokaryotic cell to detect and destroy DNA from subsequent attacks by similar viruses and effectively compose, along with an array of CRISPR-associated proteins (including Cas9 and homologs thereof) and CRISPR-associated RNA, a prokaryotic immune defense system. In nature, CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In certain types of CRISPR systems (e.g., type II CRISPR systems), correct processing of pre-crRNA requires a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc) and a Cas9 protein. The tracrRNA serves as a guide for ribonuclease 3-aided processing of pre-crRNA. Subsequently, Cas9 / crRNA / tracrRNA endonucleolytically cleaves linear or circular dsDNA target complementary to the RNA. Specifically, the target strand not complementary to crRNA is first cut endonucleolytically, then trimmed 3′-5′ exonucleolytically. In nature, DNA-binding and cleavage typically requires protein and both RNAs. However, single guide RNAs (“sgRNA”, or simply“gNRA”) can be engineered so as to incorporate aspects of both the crRNA and tracrRNA into a single RNA species– the guide RNA. See, e.g., Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J.A., Charpentier E. Science 337:816- 821(2012), the entire contents of which is hereby incorporated by reference. Cas9 recognizes a short motif in the CRISPR repeat sequences (the PAM or protospacer adjacent motif) to help distinguish self versus non-self. CRISPR biology, as well as Cas9 nuclease sequences and structures are well known to those of skill in the art (see, e.g.,“Complete genome sequence of an M1 strain of Streptococcus pyogenes.” Ferretti et al., J.J., McShan W.M., Ajdic D.J., Savic D.J.,Savic G., Lyon K., Primeaux C., Sezate S., Suvorov A.N., Kenton S., Lai H.S., Lin S.P., Qian Y., Jia H.G., Najar F.Z., Ren Q., Zhu H., Song L., White J., Yuan X., Clifton S.W., Roe B.A., McLaughlin R.E., Proc. Natl. Acad. Sci. U.S.A.98:4658-4663(2001);“CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.” Deltcheva E., Chylinski K., Sharma C.M., Gonzales K., Chao Y., Pirzada Z.A., Eckert M.R., Vogel J., Charpentier E., Nature 471:602-607(2011); and“A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.” Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J.A., Charpentier E. Science 337:816-821(2012), the entire contents of each of which are incorporated herein by reference). Cas9 orthologs have been described in various species, including, but not limited to, S. pyogenes and S. thermophilus. Additional suitable Cas9 nucleases and sequences will be apparent to those of skill in the art based on this disclosure, and such Cas9 nucleases and sequences include Cas9 sequences from the organisms and loci disclosed in Chylinski, Rhun, and Charpentier,“The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems” (2013) RNA Biology 10:5, 726-737; the entire contents of which are incorporated herein by reference.

[0146] The term“Cas9” or“Cas9 nuclease” refers to an RNA-guided nuclease comprising a Cas9 domain, or a fragment thereof (e.g., a protein comprising an active or inactive DNA cleavage domain of Cas9, and / or the gRNA binding domain of Cas9). A“Cas9 domain” as used herein, is a protein fragment comprising an active or inactive cleavage domain of Cas9 and / or the gRNA binding domain of Cas9. A“Cas9 protein” is a full length Cas9 protein. A Cas9 nuclease is also referred to sometimes as a casn1 nuclease or a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)-associated nuclease. CRISPR is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements, and conjugative plasmids). CRISPR clusters contain spacers, sequences complementary to antecedent mobile elements, and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems correct processing of pre-crRNA requires a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc) and a Cas9 domain. The tracrRNA serves as a guide for ribonuclease 3-aided processing of pre- crRNA. Subsequently, Cas9 / crRNA / tracrRNA endonucleolytically cleaves linear or circular dsDNA target complementary to the spacer. The target strand not complementary to crRNA is first cut endonucleolytically, then trimmed 3′-5′ exonucleolytically. In nature, DNA-binding andcleavage typically requires protein and both RNAs. However, single guide RNAs (“sgRNA”, or simply“gNRA”) can be engineered so as to incorporate aspects of both the crRNA and tracrRNA into a single RNA species. See, e.g., Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J.A., Charpentier E. Science 337:816-821(2012), the entire contents of which are hereby incorporated by reference. Cas9 recognizes a short motif in the CRISPR repeat sequences (the PAM or protospacer adjacent motif) to help distinguish self versus non-self. Cas9 nuclease sequences and structures are well known to those of skill in the art (see, e.g.,“Complete genome sequence of an M1 strain of Streptococcus pyogenes.” Ferretti et al., J.J., McShan W.M., Ajdic D.J., Savic D.J., Savic G., Lyon K., Primeaux C., Sezate S., Suvorov A.N., Kenton S., Lai H.S., Lin S.P., Qian Y., Jia H.G., Najar F.Z., Ren Q., Zhu H., Song L., White J., Yuan X., Clifton S.W., Roe B.A., McLaughlin R.E., Proc. Natl. Acad. Sci. U.S.A.98:4658-4663(2001);“CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.” Deltcheva E., Chylinski K., Sharma C.M., Gonzales K., Chao Y., Pirzada Z.A., Eckert M.R., Vogel J., Charpentier E., Nature 471:602-607(2011); and“A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.” Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J.A., Charpentier E. Science 337:816-821(2012), the entire contents of each of which are incorporated herein by reference). Cas9 orthologs have been described in various species, including, but not limited to, S. pyogenes and S. thermophilus. Additional suitable Cas9 nucleases and sequences will be apparent to those of skill in the art based on this disclosure, and such Cas9 nucleases and sequences include Cas9 sequences from the organisms and loci disclosed in Chylinski, Rhun, and Charpentier,“The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems” (2013) RNA Biology 10:5, 726-737; the entire contents of which are incorporated herein by reference. In some embodiments, a Cas9 nuclease comprises one or more mutations that partially impair or inactivate the DNA cleavage domain.

[0147] A nuclease-inactivated Cas9 domain may interchangeably be referred to as a“dCas9” protein (for nuclease-“dead” Cas9). Methods for generating a Cas9 domain (or a fragment thereof) having an inactive DNA cleavage domain are known (see, e.g., Jinek et al., Science. 337:816-821(2012); Qi et al.,“Repurposing CRISPR as an RNA-Guided Platform for Sequence- Specific Control of Gene Expression” (2013) Cell.28;152(5):1173-83, the entire contents of each of which are incorporated herein by reference). For example, the DNA cleavage domain of Cas9 is known to include two subdomains, the HNH nuclease subdomain and the RuvC1subdomain. The HNH subdomain cleaves the strand complementary to the gRNA, whereas the RuvC1 subdomain cleaves the non-complementary strand. Mutations within these subdomains can silence the nuclease activity of Cas9. For example, the mutations D10A and H840A completely inactivate the nuclease activity of S. pyogenes Cas9 (Jinek et al., Science.337:816- 821(2012); Qi et al., Cell.28;152(5):1173-83 (2013)). In some embodiments, proteins comprising fragments of Cas9 are provided. For example, in some embodiments, a protein comprises one of two Cas9 domains: (1) the gRNA binding domain of Cas9; or (2) the DNA cleavage domain of Cas9. In some embodiments, proteins comprising Cas9 or fragments thereof are referred to as“Cas9 variants.” A Cas9 variant shares homology to Cas9, or a fragment thereof. For example, a Cas9 variant is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, at least about 99.8% identical, or at least about 99.9% identical to wild type Cas9 (e.g., SpCas9 of SEQ ID NO: 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 wild type Cas9 (e.g., SpCas9 of SEQ ID NO: 1361421). In some embodiments, the Cas9 variant comprises a fragment of SEQ ID NO: 1361421 (e.g., a gRNA binding domain or a DNA-cleavage domain), such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of wild type Cas9 (e.g., SpCas9 of SEQ ID NO: 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).Edit template

[0148] The term“edit template” refers to a portion of the extension arm that encodes the desired edit in the single strand 3ʹ DNA flap that is synthesized by the polymerase, e.g., a DNA-dependent DNA polymerase, RNA-dependent DNA polymerase (e.g., a reverse transcriptase). Certain embodiments described here (e.g, FIG.71A) refer to“an RT template,” which refers to both the edit template and the homology arm together, i.e., the sequence of the PEgRNA extension arm which is actually used as a template during DNA synthesis. The term“RT edit template” is also equivalent to the term“DNA synthesis template,” but wherein the RT edit template reflects the use of a prime editor having a polymerase that is a reverse transcriptase, and wherein the DNA synthesis template reflects more broadly the use of a prime editor having any polymerase.Error-prone

[0149] As used herein, the term“error-prone” reverse transcriptase (or more broadly, any polymerase) refers to a reverse transcriptase (or more broadly, any polymerase) that occurs naturally or which has been derived from another reverse transcriptase (e.g., a wild type M-MLV reverse transcriptase) which has an error rate that is less than the error rate of wild type M-MLV reverse transcriptase. The error rate of wild type M-MLV reverse transcriptase is reported to be in the range of one error in 15,000 (higher) to 27,000 (lower). An error rate of 1 in 15,000 corresponds with an error rate of 6.7 x 10-5. An error rate of 1 in 27,000 corresponds with an error rate of 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 those RT that have an error rate that is greater than one error in 15,000 nucleobase incorporation (6.7 x 10-5or higher), e.g., 1 error in 14,000 nucleobases (7.14 x 10-5or higher), 1 error in 13,000 nucleobases or fewer (7.7 x 10-5or higher), 1 error in 12,000 nucleobases or fewer (7.7 x 10-5or higher), 1 error in 11,000 nucleobases or fewer (9.1 x 10-5or higher), 1 error in 10,000 nucleobases or fewer (1 x 10-4or 0.0001 or higher), 1 error in 9,000 nucleobases or fewer (0.00011 or higher), 1 error in 8,000 nucleobases or fewer (0.00013 or higher) 1 error in 7,000 nucleobases or fewer (0.00014 or higher), 1 error in 6,000 nucleobases or fewer (0.00016 or higher), 1 error in 5,000 nucleobases or fewer (0.0002 or higher), 1 error in 4,000 nucleobases or fewer (0.00025 or higher), 1 error in 3,000 nucleobases or fewer (0.00033 or higher), 1 error in 2,000 nucleobase or fewer (0.00050 or higher), or 1 error in 1,000 nucleobases or fewer (0.001 or higher), or 1 error in 500 nucleobases or fewer (0.002 or higher), or 1 error in 250 nucleobases or fewer (0.004 or higher).Extension arm

[0150] The term“extension arm” refers to a nucleotide sequence component of a PEgRNA which provides several functions, including a primer binding site and an edit template for reverse transcriptase. In some embodiments, e.g., FIG.3D, the extension arm is located at the 3ʹ end of the guide RNA. In other embodiments, e.g., FIG.3E, the extension arm is located at the 5ʹ 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 5ʹ to 3ʹ direction: the homology arm, the edit template, and the primer binding site. Sincepolymerization activity of the reverse transcriptase is in the 5ʹ to 3ʹ direction, the preferred arrangement of the homology arm, edit template, and primer binding site is in the 5ʹ to 3ʹ direction such that the reverse transcriptase, once primed by an annealed primer sequence, polymerases a single strand of DNA using the edit template as a complementary template strand. Further details, such as the length of the extension arm, are described elsewhere herein.

[0151] The extension arm may also be described as comprising generally two regions: a primer binding site (PBS) and a DNA synthesis template, as shown in FIG.3G (top), for instance. The primer binding site binds to the primer sequence that is formed from the endogenous DNA strand of the target site when it becomes nicked by the prime editor complex, thereby exposing a 3ʹ end on the endogenous nicked strand. As explained herein, the binding of the primer sequence to the primer binding site on the extension arm of the PEgRNA creates a duplex region with an exposed 3ʹ end (i.e., the 3ʹ of the primer sequence), which then provides a substrate for a polymerase to begin polymerizing a single strand of DNA from the exposed 3ʹ end along the length of the DNA synthesis template. The sequence of the single strand DNA product is the complement of the DNA synthesis template. Polymerization continues towards the 5ʹ of the DNA synthesis template (or extension arm) until polymerization terminates. Thus, the DNA synthesis template represents the portion of the extension arm that is encoded into a single strand DNA product (i.e., the 3ʹ single strand DNA flap containing the desired genetic edit information) by the polymerase of the prime editor complex and which ultimately replaces the corresponding endogenous DNA strand of the target site that sits immediate downstream of the PE-induced nick site. Without being bound by theory, polymerization of the DNA synthesis template continues towards the 5ʹ end of the extension arm until a termination event. Polymerization may terminate in a variety of ways, including, but not limited to (a) reaching a 5ʹ terminus of the PEgRNA (e.g.,in the case of the 5ʹ extension arm wherein the DNA polymerase simply runs out of template), (b) reaching an impassable RNA secondary structure (e.g., hairpin or stem / loop), or (c) reaching a replication termination signal, e.g., a specific nucleotide sequence that blocks or inhibits the polymerase, or a nucleic acid topological signal, such as, supercoiled DNA or RNA. Effective amount

[0152] The term“effective amount,” as used herein, refers to an amount of a biologically active agent that is sufficient to elicit a desired biological response. For example, in someembodiments, an effective amount of a prime editor may refer to the amount of the editor that is sufficient to edit a target site nucleotide sequence, e.g., a genome. In some embodiments, an effective amount of a prime editor provided herein, e.g., of a fusion protein comprising a nickase Cas9 domain and a reverse transcriptase may refer to the amount of the fusion protein that is sufficient to induce editing of a target site specifically bound and edited by the fusion protein. As will be appreciated by the skilled artisan, the effective amount of an agent, e.g., a fusion protein, a nuclease, a hybrid protein, a protein dimer, a complex of a protein (or protein dimer) and a polynucleotide, or a polynucleotide, may vary depending on various factors as, for example, on the desired biological response, e.g., on the specific allele, genome, or target site to be edited, on the cell or tissue being targeted, and on the agent being used.Functional equivalent

[0153] The term“functional equivalent” refers to a second biomolecule that is equivalent in function, but not necessarily equivalent in structure to a first biomolecule. For example, a“Cas9 equivalent” refers to a protein that has the same or substantially the same functions as Cas9, but not necessarily the same amino acid sequence. In the context of the disclosure, the specification refers throughout to“a protein X, or a functional equivalent thereof.” In this context, a “functional equivalent” of protein X embraces any homolog, paralog, fragment, naturally occurring, engineered, mutated, or synthetic version of protein X which bears an equivalent function.Fusion protein

[0154] The term“fusion protein” as used herein refers to a hybrid polypeptide which comprises protein domains from at least two different proteins. One protein may be located at the amino- terminal (N-terminal) portion of the fusion protein or at the carboxy-terminal (C-terminal) protein thus forming an“amino-terminal fusion protein” or a“carboxy-terminal fusion protein,”respectively. A protein may comprise different domains, for example, a nucleic acid binding domain (e.g., the gRNA binding domain of Cas9 that directs the binding of the protein to a target site) and a nucleic acid cleavage domain or a catalytic domain of a nucleic-acid editing protein. Another example includes a Cas9 or equivalent thereof to a reverse transcriptase. Any of the proteins provided herein may be produced by any method known in the art. For example, the proteins provided herein may be produced via recombinant protein expression and purification, which is especially suited for fusion proteins comprising a peptide linker. Methods for recombinant protein expression and purification are well known, and include those described by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)), the entire contents of which areincorporated herein by reference.Gene Product

[0155] The term“gene product,” as used herein, refers to any product encoded by a nucleic acid sequence. Accordingly, a gene product may, for example, be a primary transcript, a mature transcript, a processed transcript, or a protein or peptide encoded by a transcript. Examples for gene products, accordingly, include mRNAs, rRNAs, tRNAs, hairpin RNAs, microRNAs (miRNAs), shRNAs, siRNAs, and peptides and proteins, for example, reporter proteins or therapeutic proteins.Gene of interest (GOI)

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

[0157] As used herein, the term“guide RNA” is a particular type of guide nucleic acid which is mostly commonly associated with a Cas protein of a CRISPR-Cas9 and which associates with Cas9, directing the Cas9 protein to a specific sequence in a DNA molecule that includes complementarity to protospace sequence of the guide RNA. As described elsewhere, the PEgRNA are a subcategory of guide RNA which further comprise an extension arm on the 3’ or 5’ end of the guide that enables the molecule to be used with the prime editors disclosed herein. The term“guide RNA” also embraces the equivalent guide nucleic acid molecules that associate with Cas9 equivalents, homologs, orthologs, or paralogs, whether naturally occurring or non- naturally occurring (e.g., engineered or recombinant), and which otherwise program the Cas9 equivalent to localize to a specific target nucleotide sequence. The Cas9 equivalents may include other napDNAbp from any type of CRISPR system (e.g., type II, V, VI), including Cpf1 (a type-V CRISPR-Cas systems), C2c1 (a type V CRISPR-Cas system), C2c2 (a type VI CRISPR-Cas system) and C2c3 (a type V CRISPR-Cas system). Further Cas-equivalents are described in Makarova et al.,“C2c2 is a single-component programmable RNA-guided RNA- targeting CRISPR effector,” Science 2016; 353(6299), the contents of which are incorporated herein by reference. Exemplary sequences are and structures of guide RNAs are provided herein. In addition, methods for designing appropriate guide RNA sequences are provided herein. As used herein, the“guide RNA” may also be referred to as a“traditional guide RNA” to contrast it with the modified forms of guide RNA termed“prime editor guide RNAs” (or “PEgRNA”) which have been invented for the prime editing methods and composition disclosed herein.

[0158] Guide RNAs or PEgRNA may comprise various structural elements that include, but are not limited to:

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

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

[0161] Extension arm– refers to the extended portion of the guide RNA at either the 5′ or the 3′ end comprising the homology arm, edit template, and primer binding site. This component is further defined elsewhere.

[0162] Homology arm– refers to a portion(s) of the extension arm that encodes a portion of the resulting reverse transcriptase-encoded single strand DNA flap that is to be integrated into the target DNA site by replacing the endogenous strand. The portion of the single strand DNA flap encoded by the homology arm is complementary to the non-edited strand of the target DNA sequence, which facilitates the displacement of the endogenous strand and annealing of the single strand DNA flap in its place, thereby installing the edit. This component is further defined elsewhere.

[0163] Edit template– refers to a portion of the extension arm that encodes the desired edit in the single strand DNA flap that is synthesized by reverse transcriptase. This component is further defined elsewhere.

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

[0165] Transcription terminator– the guide RNA or PEgRNA may comprise a transcriptional termination sequence at the 3′ of the molecule. Typically transcription terminator sequences (e.g., SEQ ID NOs: 1361560-1361565) are about 70 to about 125 nucleotides in length, but short and longer transcription terminator sequences are contemplated and any known in the art may be used.Flap endonuclease (e.g., FEN1)

[0166] As used herein, the term“flap endonuclease” refers to an enzyme that catalyzes the removal of 5′ single strand DNA flaps. These are naturally occurring enzymes that process the removal of 5′ flaps formed during cellular processes, including DNA replication. The prime editing methods herein described may utilize endogenously supplied flap endonucleases or those provided in trans to remove the 5′ flap of endogenous DNA formed at the target site during prime editing. Flap endonucleases are known in the art and can be found described in Patel et al.,“Flap endonucleases pass 5′-flaps through a flexible arch using a disorder-thread-order mechanism to confer specificity for free 5′-ends,” Nucleic Acids Research, 2012, 40(10): 4507- 4519 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 are incorporated herein by reference). An exemplary flap endonuclease is FEN1, which can be represented by the following amino acid sequence:Fusion protein

[0167] The term“fusion protein” as used herein refers to a hybrid polypeptide which comprises protein domains from at least two different proteins. One protein may be located at the amino- terminal (N-terminal) portion of the fusion protein or at the carboxy-terminal (C-terminal) protein thus forming an“amino-terminal fusion protein” or a“carboxy-terminal fusion protein,” respectively. A protein may comprise different domains, for example, a nucleic acid binding domain (e.g., the gRNA binding domain of Cas9 that directs the binding of the protein to a target site) and a nucleic acid cleavage domain (e.g., Cas9 nickase, napDNAbp) or a catalytic domain of a nucleic-acid editing protein (e.g., RT domain). Another example includes a napDNAbp (e.g., Cas9) or equivalent thereof fused to a reverse transcriptase. Any of the proteins provided herein may be produced by any method known in the art. For example, the proteins provided herein may be produced via recombinant protein expression and purification, which is especially suited for fusion proteins comprising a peptide linker. Methods for recombinant protein expression and purification are well known, and include those described by Green andSambrook, Molecular Cloning: A Laboratory Manual (4thed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)), the entire contents of which are incorporated herein by reference.Homology arm

[0168] The term“homology arm” refers to a portion of the extension arm that includes a sequence of the resulting reverse transcriptase-encoded single strand DNA flap that is to be integrated into the target DNA site by replacing the endogenous strand. The portion of the single strand DNA flap encoded by the homology arm is complementary to the non-edited strand of thetarget DNA sequence, which facilitates the displacement of the endogenous strand and annealing of the single strand DNA flap in its place, thereby installing the edit. This component is further defined elsewhere.Host cell

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

[0170] "Isolated" means altered or removed from the natural state. For example, a nucleic 20 acid or a peptide naturally present in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0171] In some embodiments, a gene of interest is encoded by an isolated nucleic acid. As used herein, the term“isolated,” refers to the characteristic of a material as provided herein being removed from its original or native environment (e.g., the natural environment if it is naturally occurring). Therefore, a naturally-occurring polynucleotide or protein or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated by human intervention from some or all of the coexisting materials in the natural system, is isolated. An artificial or engineered material, for example, a non-naturally occurring nucleic acid construct, such as the expression constructs and vectors described herein, are, accordingly, also referred to as isolated. A material does not have to be purified in order to be isolated. Accordingly, a material may be part of a vector and / or part of a composition, and still be isolated in that such vector or composition is not part of the environment in which the material is found in nature. napDNAbp

[0172] As used herein, the term“nucleic acid programmable DNA binding protein” or “napDNAbp,” of which Cas9 is an example, refers to proteins which use RNA:DNAhybridization to target and bind to specific sequences in a DNA molecule. Each napDNAbp is associated with at least one guide nucleic acid (e.g., guide RNA), which localizes the napDNAbp to a DNA sequence that comprises a DNA strand (i.e., a target strand) that is complementary tothe guide nucleic acid, or a portion thereof (e.g., the protospacer of a guide RNA). In other words, the guide nucleic-acid“programs” the napDNAbp (e.g., Cas9 or equivalent) to localize and bind to a complementary sequence.

[0173] Without being bound by theory, the binding mechanism of a napDNAbp– guide RNA complex, in general, includes the step of forming an R-loop whereby the napDNAbp induces the unwinding of a double-strand DNA target, thereby separating the strands in the region bound by the napDNAbp. The guide RNA protospacer then hybridizes to the“target strand.” This displaces a“non-target strand” that is complementary to the target strand, which forms the single strand region of the R-loop. In some embodiments, the napDNAbp includes one or more nuclease activities, which then cut the DNA leaving various types of lesions. For example, the napDNAbp may comprises a nuclease activity that cuts the non-target strand at a first location, and / or cuts the target strand at a second location. Depending on the nuclease activity, the target DNA can be cut to form a“double-stranded break” whereby both strands are cut. In other embodiments, the target DNA can be cut at only a single site, i.e., the DNA is“nicked” on one strand. Exemplary napDNAbp with different nuclease activities include“Cas9 nickase”(“nCas9”) and a deactivated Cas9 having no nuclease activities (“dead Cas9” or“dCas9”).Exemplary sequences for these and other napDNAbp are provided herein.Linker

[0174] The term“linker,” as used herein, refers to a molecule linking two other molecules or moieties. Linkers are well known in the art and can comprise any suitable combination of nucleic acids or amino acids to facilitate the proper function of the structures they join. The linker can be a series of amino acids. The linker can be an amino acid sequence in the case of a linker joining two fusion proteins. For example, a napDNAbp (e.g., Cas9) can be fused to a reverse transcriptase by an amino acid linker sequence. The linker can also be a nucleotide sequence in the case of joining two nucleotide sequences together. For example, in the instant case, the traditional guide RNA is linked via a spacer or linker nucleotide sequence to the RNA extension of an prime editor guide RNA which may comprise a DNA synthesis template (e.g., RT template sequence) and an Primer binding site. In other embodiments, the linker is an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker is 5- 100 amino acids in length, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30-35, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-150, or 150-200 amino acids in length. In some embodiments, the linker is 5-100 nucleotides in length, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30-35, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-150, 150-200, 200-300, 300-500, 500-1000, 1000-2000, or 2000-5000 nucleotides. Longer or shorter linkers are also contemplated.Nickase

[0175] The term“nickase” refers to a napDNAbp (e.g., Cas9) with one of the two nuclease domains inactivated. This enzyme is capable of cleaving only one strand of a target DNA.Nuclear localization sequence (NLS)

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

[0177] The term“nucleic acid,” as used herein, refers to a polymer (i.e., multiple, more than one, (e.g., 2, 3, 4, etc.) of nucleotides. The polymer may include natural nucleosides (i.e., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine), nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo- pyrimidine, 3-methyl adenosine, 5-methylcytidine, C5 bromouridine, C5 fluorouridine, C5 iodouridine, C5 propynyl uridine, C5 propynyl cytidine, C5 methylcytidine, 7 deazaadenosine, 7 deazaguanosine, 8 oxoadenosine, 8 oxoguanosine, O(6) methylguanine, 4-acetylcytidine, 5- (carboxyhydroxymethyl)uridine, dihydrouridine, methylpseudouridine, 1-methyl adenosine, 1- methyl guanosine, N6-methyl adenosine, and 2-thiocytidine), chemically modified bases, biologically modified bases (e.g., methylated bases), intercalated bases, modified sugars (e.g., 2′- fluororibose, ribose, 2′-deoxyribose, 2´-O-methylcytidine, arabinose, and hexose), or modified phosphate groups (e.g., phosphorothioates and 5′-N phosphoramidite linkages).Nucleobase

[0178] As used herein, the term“nucleobase,” also known as“nitrogenous base” or often simply“base,” are nitrogen-containing biological compounds that form nucleosides, which in turn are components of nucleotides, with all of these monomers constituting the basic building blocks of nucleic acids. The ability of nucleobases to form base pairs and to stack one upon another leads directly to long-chain helical structures such as ribonucleic acid (RNA) and deoxyribonucleic acid (DNA).

[0179] Five nucleobases, which are adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U), can be referred to as primary or canonical. They function as the fundamental units of the genetic code, with the bases A, G, C, and T being 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 adenosine and guanosine nucleobases, alternate nucleobases can include hypoxanthine, xanthine, or 7- methylguanine, which correspond with the alternate nucleosides of inosine, xanthosine, and 7- methylguanosine, respectively. In addition, for example, cytosine, thymine, or uridine nucleobases, alternate nucleobases can include 5,6dihydrouracil, 5-methylcytosine, or 5- hydroxymethylcytosine, which correspond with the alternate nucleosides of dihydrouridine, 5- methylcytidine, and 5-hydroxymethylcytidine, respectively. Nucleobases may also include nucleobase analogues, for which a vast number are known in the art. Typically the analogue nucleobases confer, among other things, different base pairing and base stacking properties. Examples include universal bases, which can pair with all four canonical bases, and phosphate- sugar backbone analogues such as PNA, which affect the properties of the chain (PNA can even form a triple helix). Nucleic acid analogues are also called“xeno nucleic acid” and represent one of the main pillars of xenobiology, the design of new-to-nature forms of life based on alternative biochemistries. Artificial nucleic acids include peptide nucleic acid (PNA), morpholino and locked nucleic acid (LNA), as well as glycol nucleic acid (GNA) and threose nucleic acid (TNA). Each of these is distinguished from naturally occurring DNA or RNA by changes to the backbone of the molecule. Example analogues are (e.g., 2-aminoadenosine, 2- thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C5 bromouridine, C5 fluorouridine, C5 iodouridine, C5 propynyl uridine, C5 propynyl cytidine, C5 methylcytidine, 7 deazaadenosine, 7 deazaguanosine, 8 oxoadenosine, 8 oxoguanosine, O(6)methylguanine, 4-acetylcytidine, 5-(carboxyhydroxymethyl)uridine, dihydrouridine, methylpseudouridine, 1-methyl adenosine, 1-methyl guanosine, N6-methyl adenosine, and 2- thiocytidine), chemically modified bases, biologically modified bases (e.g., methylated bases), intercalated bases, modified sugars (e.g., 2′-fluororibose, ribose, 2′-deoxyribose, 2´-O- methylcytidine, arabinose, and hexose), or modified phosphate groups (e.g., phosphorothioates and 5′-N phosphoramidite linkages).PEgRNA

[0180] As used herein, the terms“prime editor guide RNA” or“PEgRNA” or“extended guide RNA” refers to a specialized form of a guide RNA that has been modified to include one or more additional sequences for use in the prime editing methods, compositions, and systems described herein. As described herein, the prime editor guide RNA comprise one or more “extended regions” of nucleic acid sequence. The extended regions may comprise, but are not limited to, single-stranded RNA. Further, the extended regions may occur at the 3' end of a traditional guide RNA. In other arrangements, the extended regions may occur at the 5' end of a traditional guide RNA. In still other arrangements, the extended region may occur at an intramolecular region, rather than one of the ends, of the traditional guide RNA, for example, in the gRNA core region which associates and / or binds to the napDNAbp. The extended region comprises a“reverse transcriptase template sequence” which is single-stranded RNA molecule which encodes a single-stranded complementary DNA (cDNA) which, in turn, has been designed to be (a) homologous to the endogenous target DNA to be edited, and (b) which comprises at least one desired nucleotide change (e.g., transition, transversion, deletion, insertion, or combination thereof) to be introduced or integrated into the endogenous target DNA. The extended region may also comprise other functional sequence elements, such as, but not limited to, a“primer binding site” and / or a“spacer or linker” sequence. As used herein the“primer binding site” comprises a sequence that hybridizes to a single-strand DNA sequence having a 3' end generated from the nicked DNA of the R-loop and which comprises a primer for reverse transcriptase.

[0181] In some embodiments, the PEgRNA are represented by FIG.3A, which shows a PEgRNA having a 5′ extension arm, a spacer, and a gRNA core. The 5′ extension further comprises in the 5′ to 3′ direction a reverse transcriptase template, a primer binding site, and a linker.

[0182] In some embodiments, the PEgRNA are represented by FIG.3B, which shows a PEgRNA having a 3′ extension arm, a spacer, and a gRNA core. The 3′ extension further comprises in the 5′ to 3′ direction a reverse transcriptase template and a primer binding site.

[0183] In still other embodiments, the PEgRNA are represented by FIG.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), a“edit template” (B), and a “homology arm” (C). The extension arm (3) may also comprise an optional modifier region at the 3′ and 5′ ends, which may be the same sequences or different sequences. In addition, the 3′ end of the PEgRNA may comprise a transcriptional terminator sequence. These sequence elements of the PEgRNA are further described and defined herein. In addition, the specification discloses exemplary PEgRNA , which have been designed in accordance with the methods disclosed herein, in the accompanying Sequence Listing.

[0184] In still other embodiments, the PEgRNA are represented by FIG.28, which shows a PEgRNA having in the 5′ to 3′ direction an extension arm (3), a spacer (1), and a gRNA core (2). The extension arm (3) is at the 5′ end of the PEgRNA . The extension arm (3) further comprises in the 3′ to 5′ direction a“primer binding site” (A), an“edit template” (B), and a “homology arm” (C). The extension arm (3) may also comprise an optional modifier region at the 3′ and 5′ ends, which may be the same sequences or different sequences. The PEgRNA may also comprise a transcriptional terminator sequence at the 3′ end. These sequence elements of the PEgRNA are further described and defined herein.Peptide tag

[0185] The term“peptide tag” refers to a peptide amino acid sequence that is genetically fused to a protein sequence to impart one or more functions onto the proteins that facilitate the manipulation of the protein for various purposes, such as, visualization, identification, localization, purification, solubilization, separation, etc. Peptide tags can include various types of tags categorized by purpose or function, which may include“affinity tags” (to facilitate protein purification),“solubilization tags” (to assist in proper folding of proteins),“chromatography tags” (to alter chromatographic properties of proteins),“epitope tags” (to bind to high affinity antibodies),“fluorescence tags” (to facilitate visualization of proteins in a cell or in vitro).PE1

[0186] As used herein,“PE1” refers to a PE complex comprising a fusion protein comprising Cas9(H840A) and a wild type MMLV RT having the following structure: [NLS]- [Cas9(H840A)]-[linker]-[MMLV_RT(wt)] + a desired PEgRNA, wherein the PE fusion has the amino acid sequence of SEQ ID NO: 1361515, which is shown as follows;PE2

[0187] As used herein,“PE2” refers to a PE complex comprising a fusion protein comprising Cas9(H840A) and a variant MMLV RT having the following structure: [NLS]-[Cas9(H840A)]-[linker]-[MMLV_RT(D200N)(T330P)(L603W)(T306K)(W313F)] + a desired PEgRNA, wherein the PE fusion has the amino acid sequence of SEQ ID NO: 1361516, which is shown as follows:PE3

[0188] As used herein,“PE3” refers to PE2 plus a second-strand nicking guide RNA that complexes with the PE2 and introduces a nick in the non-edited DNA strand in order to induce preferential replacement of the edited strand.PE3b

[0189] As used herein,“PE3b” refers to PE3 but wherein the second-strand nicking guide RNA is designed for temporal control such that the second strand nick is not introduced until after the installation of the desired edit. This is achieved by designing a gRNA with a spacer sequence that matches only the edited strand, but not the original allele. Using this strategy, referred to hereafter as PE3b, mismatches between the protospacer and the unedited allele should disfavor nicking by the sgRNA until after the editing event on the PAM strand takes place.PE-short

[0190] As used herein,“PE-short” refers to a PE construct that is fused to a C-terminally truncated reverse transcriptase, and has the following amino acid sequence:M-MLV TRUNCATED reverse transcriptase(SEQ ID NO: 1361597)Percent identity

[0191] The“percent identity,”“sequence identity,”“% identity,” or“% sequence identity” (as they may be interchangeably used herein) of sequences (e.g., nucleic acid or amino acid) refers to a quantitative measurement of the similarity between two sequences (e.g., nucleic acid or amino acid). The percent identity of genomic DNA sequence, intron and exon sequence, and amino acid sequence between humans and other species varies by species type, with chimpanzee having the highest percent identity with humans of all species in each category. Percent identity can be determined using the algorithms 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 algorithms is 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 the protein molecules of interest. Where gaps exist between two sequences, Gapped BLAST can be utilized 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 a percent identity is stated or recited, or a range thereof (e.g., at least, more than, between, etc.), unless otherwise specified, the endpoints shall be inclusive and the range (e.g., at least 70% identity) shall include all ranges within the cited 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 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 (e.g., tenths of a percent (i.e., 0.1%), hundredths of a percent (i.e., 0.01%), etc.).Prime editor

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

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

[0194] The terms“protein,”“peptide,” and“polypeptide” are used interchangeably herein and refer to a polymer of amino acid residues linked together by peptide (amide) bonds. The terms refer to a protein, peptide, or polypeptide of any size, structure, or function. Typically, a protein, peptide, or polypeptide will be at least three amino acids long. A protein, peptide, or polypeptide may refer to an individual protein or a collection of proteins. One or more of the amino acids in a protein, peptide, or polypeptide may be modified, for example, by the addition of a chemical entity, such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc. A protein, peptide, or polypeptide may also be a single molecule or may be a multi-molecular complex. A protein, peptide, or polypeptide may be just a fragment of a naturally occurring protein or peptide. A protein, peptide, or polypeptide may be naturally occurring, recombinant, or synthetic, or any combination thereof. Any of the proteins provided herein may be produced by any method known in the art. For example, the proteins provided herein may be produced via recombinant protein expression and purification, which is especially suited for fusion proteins comprising a peptide linker. Methods for recombinant proteinexpression and purification are well known, and include those described by Green andSambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)), the entire contents of which are incorporated herein by reference.Operably linked

[0195] The term“operably linked,” as may be used herein, refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence (e.g., transgene) resulting in expression of the heterologous nucleic acid sequence (e.g., transgene). For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked nucleic acid sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.Promoter

[0196] The term“promoter” is art-recognized and refers to a nucleic acid molecule with a sequence recognized by the cellular transcription machinery and able to initiate transcription of a downstream gene. A promoter can be constitutively active, meaning that the promoter is always active in a given cellular context, or conditionally active, meaning that the promoter is only active in the presence of a specific condition. For example, a conditional promoter may only be active in the presence of a specific protein that connects a protein associated with a regulatory element in the promoter to the basic transcriptional machinery, or only in the absence of an inhibitory molecule. A subclass of conditionally active promoters are inducible promoters that require the presence of a small molecule“inducer” for activity. Examples of inducible promoters include, but are not limited to, arabinose-inducible promoters, Tet-on promoters, and tamoxifen-inducible promoters. A variety of constitutive, conditional, and inducible promoters are well known to the skilled artisan, and the skilled artisan will be able to ascertain a variety of such promoters useful in carrying out the instant invention, which is not limited in this respect. Protospacer adjacent motif (PAM)

[0197] As used herein, the term“protospacer adjacent sequence” or“PAM” refers to an approximately 2-6 base pair DNA sequence that is an important targeting component of a Cas9nuclease. Typically, the PAM sequence is on either strand and is downstream in the 5' to 3' direction of Cas9 cut site. The canonical PAM sequence (i.e., the PAM sequence that is associated with the Cas9 nuclease of Streptococcus pyogenes or SpCas9) is 5ʹ-NGG-3ʹ, wherein “N” is any nucleobase followed by two guanine (“G”) nucleobases. Different PAM sequences can be associated with different Cas9 nucleases or equivalent proteins from different organisms, for example, 5ʹ-NG-3ʹ, wherein“N” is any nucleobase followed by one guanine (“G”) nucleobases, or 5ʹ-KKH-3ʹ, wherein two lysine (“K”) are followed by one histidine (“H”). In addition, any given Cas9 nuclease, e.g., SpCas9, may be modified to alter the PAM specificity of the nuclease such that the nuclease recognizes alternative PAM sequence.

[0198] For example, with reference to the canonical 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) D1135V, R1335Q, and T1337R“the VQR variant”, which alters the PAM specificity to NGAN or NGNG, (b) D1135E, R1335Q, and T1337R“the EQR variant”, which alters the PAM specificity to NGAG, and (c) D1135V, G1218R, R1335E, and T1337R“the VRER variant”, which alters the PAM specificity to NGCG. In addition, the D1135E variant of canonical SpCas9 still recognizes NGG, but it is more selective compared to the wild type SpCas9 protein.

[0199] It will also be appreciated that Cas9 enzymes from different bacterial species (i.e., Cas9 orthologs) can have varying PAM specificities. For example, Cas9 from Staphylococcus aureus (SaCas9) recognizes NGRRT or NGRRN. In addition, Cas9 from Neisseria meningitis (NmCas) recognizes NNNNGATT. In another example, Cas9 from Streptococcus thermophilis (StCas9) recognizes NNAGAAW. In still another example, Cas9 from Treponema denticola (TdCas) recognizes NAAAAC. These examples are not meant to be limiting. It will be further appreciated that non-SpCas9s bind a variety of PAM sequences, which makes them useful when no suitable SpCas9 PAM sequence is present at the desired target cut site. Furthermore, non- SpCas9s may have other characteristics that make them more useful than SpCas9. For example, Cas9 from Staphylococcus aureus (SaCas9) is about 1 kilobase smaller than SpCas9, so it can be packaged into adeno-associated virus (AAV). Further reference may be made to Shah et al., “Protospacer recognition motifs: mixed identities and functional diversity,” RNA Biology, 10(5): 891-899 (which is incorporated herein by reference).Protospacer

[0200] As used herein, the term“protospacer” refers to the sequence (~20 bp) in DNA adjacent to the PAM (protospacer adjacent motif) sequence which 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, one strand thereof, i.e, the“target strand” versus the “non-target strand” of the target sequence). In order for Cas9 to function it also requires a specific protospacer adjacent motif (PAM) that varies depending on the bacterial species of the Cas9 gene. The most commonly used Cas9 nuclease, derived from S. pyogenes, recognizes a PAM sequence of NGG that is found directly downstream of the target sequence in the genomic DNA, on the non-target strand. The skilled person will appreciate that the literature in the state of the art sometimes refers to the“protospacer” as the ~20-nt target-specific guide sequence on the guide RNA itself, rather than referring to it as a“spacer.” Thus, in some cases, the term “protospacer” as used herein may be used interchangeably with the term“spacer.” The context of the description surrounding the appearance of either“protospacer” or“spacer” will help inform the reader as to whether the term is refine to the gRNA or the DNA target. Both usages of these terms are acceptable since the state of the art uses both terms in each of these ways. Reverse transcriptase

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

[0202] In addition, the invention contemplates the use of reverse transcriptases which are error- prone, i.e., which may be referred to as error-prone reverse transcriptases or reversetranscriptases which do not support high fidelity incorporation of nucleotides during polymerization. During synthesis of the single-strand DNA flap based on the RT template integrated with the guide RNA, the error-prone reverse transcriptase can introduce one or more nucleotides which are mismatched with the DNA synthesis template (e.g., RT template sequence), thereby introducing changes to the nucleotide sequence through erroneous polymerization of the single-strand DNA flap. These errors introduced during synthesis of the single strand DNA flap then become integrated into the double strand molecule through hybridization to the corresponding endogenous target strand, removal of the endogenous displaced strand, ligation, and then through one more rounds of endogenous DNA repair and / or replication.Reverse transcription

[0203] As used herein, the term“reverse transcription” indicates the capability of enzyme to synthesize DNA strand (that is, complementary DNA or cDNA) using RNA as a template. In some embodiments, the reverse transcription can be“error-prone reverse transcription,” which refers to the properties of certain reverse transcriptase enzymes which are error-prone in their DNA polymerization activity.Sense strand

[0204] In genetics, a“sense” strand is the segment within double-stranded DNA that runs from 5' to 3', and which is complementary to the antisense strand of DNA, or template strand, which runs from 3' to 5'. In the case of a DNA segment that encodes a protein, the sense strand is the strand of DNA that has the same sequence as the mRNA, which takes the antisense strand as its template during transcription, and eventually undergoes (typically, not always) translation into a protein. The antisense strand is thus responsible for the RNA that is later translated to protein,while the sense strand possesses a nearly identical makeup to that of the mRNA. Note that for each segment of dsDNA, there will possibly be two sets of sense and antisense, depending on which direction one reads (since sense and antisense is relative to perspective). It is ultimately the gene product, or mRNA, that dictates which strand of one segment of dsDNA is referred to as sense or antisense.

[0205] In the context of a PEgRNA, the first step is the synthesis of a single-strandcomplementary DNA (i.e., the 3ʹ ssDNA flap, which becomes incorporated) oriented in the 5ʹ to 3ʹ direction which is templated off of the PEgRNA extension arm. Whether the 3ʹ ssDNA flap should be regarded as a sense or antisense strand depends on the direction of transcription since it well accepted that both strands of DNA may serve as a template for transcription (but not at the same time). Thus, in some embodiments, the 3ʹ ssDNA flap (which overall runs in the 5ʹ to 3ʹ direction) will serve as the sense strand because it is the coding strand. In other embodiments, the 3ʹ ssDNA flap (which overall runs in the 5ʹ to 3ʹ direction) will serve as the antisense strand and thus, the template for transcription.Second strand nicking

[0206] As used herein, the 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 the free 3' end for use in priming of the reverse transcriptase on the extended portion of the guide RNA). In some embodiments, the first nick and the second nick are on opposite strands. In other embodiments, the first nick and the second nick are on opposite strands. In yet another embodiment, the first nick is on the non-target strand (i.e., the strand that forms the single strand portion of the R- loop), and the second nick is on the target strand. The second nick is positioned at least 5 nucleotides downstream of the first nick, or at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 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 towards replacement of the unedited strand. In some embodiments, the edited strand is the non-target strand and the unedited strand is the target strand. In other embodiments, the edited strand is the target strand, and the unedited strand is the non-target strand.Spacer sequence

[0207] As used herein, the term“spacer sequence” in connection with a guide RNA or aPEgRNA refers to the 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 which contains a nucleotide sequence that is complementary to the protospacer sequence in the target DNA sequence. The spacer sequence anneals to the protospacer sequence to form a ssRNA / ssDNA hybrid structure at the target site and a corresponding R loop ssDNA structure of the endogenous DNA strand that is complementary to the protospacer sequence.Subject

[0208] The term“subject,” as used herein, refers to an individual organism, for example, an individual mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human mammal. In some embodiments, the subject is a non-human primate. In some embodiments, the subject is a rodent. In some embodiments, the subject is a sheep, a goat, a cattle, a cat, or a dog. In some embodiments, the subject is a vertebrate, an amphibian, a reptile, a fish, an insect, a fly, or a nematode. In some embodiments, the subject is a research animal. In some embodiments, the subject is genetically engineered, e.g., a genetically engineered non-human subject. The subject may be of either sex and at any stage ofdevelopment.Target site

[0209] The term“target site” refers to a sequence within a nucleic acid molecule that is edited by a prime editor disclosed herein. The target site further refers to the sequence within a nucleic acid molecule to which a complex of the base editor and gRNA binds.Temporal second-strand nicking

[0210] As used herein, the term“temporal second-strand nicking” refers to a variant of second strand nicking whereby the installation of the second nick in the unedited strand occurs only after the desired edit is installed in the edited strand. This avoids concurrent nicks on both strands that could lead to double-stranded DNA breaks. The second-strand nicking guide RNA is designed for temporal control such that the second strand nick is not introduced until after the installation of the desired edit. This is achieved by designing a gRNA with a spacer sequence that matches only the edited strand, but not the original allele. Using this strategy, mismatches between theprotospacer and the unedited allele should disfavor nicking by the sgRNA until after the editing event on the PAM strand takes place.tPERT

[0211] See definition for“trans prime editor RNA template (tPERT).”Temporal second-strand nicking

[0212] As used herein, the term“temporal second-strand nicking” refers to a variant of second strand nicking whereby the installation of the second nick in the unedited strand occurs only after the desired edit is installed in the edited strand. This avoids concurrent nicks on both strands that could lead to double-stranded DNA breaks. The second-strand nicking guide RNA is designed for temporal control such that the second strand nick is not introduced until after the installation of the desired edit. This is achieved by designing a gRNA with a spacer sequence that matches only the edited strand, but not the original allele. Using this strategy, mismatches between the protospacer and the unedited allele should disfavor nicking by the sgRNA until after the editing event on the PAM strand takes place.Trans prime editing

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

[0214] An embodiment of trans prime editing is shown in FIGs.3G and 3H. FIG.3G shows the composition of the trans prime editor complex on the left (“RP-PE:gRNA complex), which comprises an napDNAbp fused to each of a polymerase (e.g., a reverse transcriptase) and a rPERT recruiting protein (e.g., MS2sc), and which is complexed with a guide RNA. FIG.3G further shows a separate tPERT molecule, which comprises the extension arm features of aPEgRNA, including the DNA synthesis template and the primer binding sequence. The tPERT molecule also includes an RNA-protein recruitment domain (which, in this case, is a stem loop structure and can be, for example, MS2 aptamer). As depicted in the process described in FIG. 3H, the RP-PE:gRNA complex binds to and nicks the target DNA sequence. Then, the recruiting protein (RP) recruits a tPERT to co-localize to the prime editor complex bound to the DNA target site, thereby allowing the primer binding site to bind to the primer sequence on the nicked strand, and subsequently, allowing the polymerase (e.g., RT) to synthesize a single strand of DNA against the DNA synthesis template up through the 5ʹ of the tPERT.

[0215] While the tPERT is shown in FIG.3G and FIG.3H as comprising the PBS and DNA synthesis template on the 5ʹ end of the RNA-protein recruitment domain, the tPERT in other configurations may be designed with the PBS and DNA synthesis template located on the 3ʹ end of the RNA-protein recruitment domain. However, the tPERT with the 5’ extension has the advantage that synthesis of the single strand of DNA will naturally terminate at the 5’ end of the tPERT and thus, does not risk using any portion of the RNA-protein recruitment domain as a template during the DNA synthesis stage of prime editing.Trans prime editor RNA template (tPERT)

[0216] As used herein, a“trans prime editor RNA template (tPERT)” refers to a component used in trans prime editing, a modified version of prime editing which operates by separating the PEgRNA into two distinct molecules: a guide RNA and a tPERT molecule. The tPERT molecule is programmed to co-localize with the prime editor complex at a target DNA site, bringing the primer binding site and the DNA synthesis template to the prime editor in trans. For example, see FIG.3G for an embodiment of a trans prime editor (tPE) which shows a two- component system comprising (1) an RP-PE:gRNA complex and (2) a tPERT that includes the primer binding site and the DNA synthesis template joined to an RNA-protein recruitment domain, wherein the RP (recruiting protein) component of the RP-PE:gRNA complex recruits the tPERT to a target site to be edited, thereby associating the PBS and DNA synthesis template with the prime editor in trans. Said another way, the tPERT is engineered to contain (all or part of) the extension arm of a PEgRNA, which includes the primer binding site and the DNA synthesis template.Transitions

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

[0218] As used herein,“transversions” refer to the interchange of purine nucleobases for pyrimidine nucleobases, or in the reverse and thus, involve the interchange of nucleobases with dissimilar shape. These changes involve T↔ A, T↔ G, C↔ G, C↔ A, A↔ T, A↔ C, G↔ C, and G↔ T. In the context of a double-strand DNA with Watson-Crick paired nucleobases, transversions refer to the following base pair exchanges: T:A↔ A:T, T:A↔ G:C, C:G↔ G:C, C:G↔ A:T, A:T↔ T:A, A:T↔ C:G, G:C↔ C:G, and G:C↔ T:A. The compositions and methods disclosed herein are capable of inducing one or more transversions in a target DNA molecule. The compositions and methods disclosed herein are also capable of inducing both transitions and transversion in the same target DNA molecule, as well as other nucleotide changes, including deletions and insertions.Treatment

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

[0220] As used herein, a“trinucleotide repeat disorder” (or alternatively,“expansion repeat disorder” or“repeat expansion disorder”) refers to a set of genetic disorders which are cause by “trinucleotide repeat expansion,” which is a kind of mutation where a certain trinucleotide repeats in certain genes or introns. Trinucleotide repeats were once thought to be commonplace iterations in the genome, but the 1990s clarified these disorders. These apparently‘benign’ stretches of DNA can sometimes expand and cause disease. Several defining features are shared amongst disorders caused by trinucleotide repeat expansions. First, the mutant repeats show both somatic and germline instability and, more frequently, they expand rather than contract in successive transmissions. Secondly, an earlier age of onset and increasing severity of phenotype in subsequent generations (anticipation) generally are correlated with larger repeat length.Finally, the parental origin of the disease allele can often influence anticipation, with paternal transmissions carrying a greater risk of expansion for many of these disorders.

[0221] Triplet expansion is thought to be caused by slippage during DNA replication. Due to the repetitive nature of the DNA sequence in these regions 'loop out' structures may form during DNA replication while maintaining complementary base pairing between the parent strand and daughter strand being synthesized. If the loop out structure is formed from sequence on the daughter strand this will result in an increase in the number of repeats. However, if the loop out structure is formed on the parent strand a decrease in the number of repeats occurs. It appears that expansion of these repeats is more common than reduction. Generally the larger the expansion the more likely they are to cause disease or increase the severity of disease. This property results in the characteristic of anticipation seen in trinucleotide repeat disorders.Anticipation describes the tendency of age of onset to decrease and severity of symptoms to increase through successive generations of an affected family due to the expansion of these repeats.

[0222] Nucleotide repeat disorders may include those in which the triplet repeat occurs in a non- coding region (i.e., a non-coding trinucleotide repeat disorder) or in a coding region

[0223] The prime editor (PE) system described herein may use to treat nucleotide repeat disorders, which may include fragile X syndrome (FRAXA), fragile XE MR (FRAXE),Freidreich ataxia (FRDA), myotonic dystrophy (DM), spinocerebellar ataxia type 8 (SCA8), and spinocerebellar ataxia type 12 (SCA12), among others.Prime editing or“prime editing (PE)”

[0224] 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 which is exemplified in the embodiments of FIG.1A-1J. TPRT refers to “target-primed reverse transcription” because the 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 (for which a change in the nucleotide sequence is desired to be introduced) with a nucleic acid programmable DNA binding protein (napDNAbp) complexed with an prime editor guide RNA. In reference to FIG.1E, the prime editor guide RNA comprises an extension at the 3' or 5' end of the guide RNA, or at an intramolecular location in the guide RNA and encodes the desired nucleotide change (e.g., single nucleotide change, insertion, or deletion). In step (a), the napDNAbp / extended gRNA complex contacts the DNA molecule and the extended gRNA guides the napDNAbp to bind to a target locus. In step (b), a nick in one of the strands of DNA of the target locus is introduced (e.g., by a nuclease or chemical agent), thereby creating an available 3' end in one of the strands of the target locus. In some embodiments, the nick is created in the strand of DNA that corresponds to the R-loop strand, i.e., the strand that is not hybridized to the guide RNA sequence, i.e., the“non-target strand.” The nick, however, could be introduced in either of the strands. That is, the nick could be introduced into the“target strand” (i.e., the strand that hybridized to the spacer of the extended gRNA) or the“non-target strand” (i.e, the strand forming the single-stranded portion of the R-loop and which is complementary to the target strand). In step (c), the 3' end of the DNA strand (formed by the nick) interacts with the extended portion of the guide RNA in order to prime reversetranscription (i.e,“target-primed RT”). In some embodiments, the 3' end DNA strand hybridizes to a specific primer binding site on the extended portion of the guide RNA, i.e, the“reversetranscriptase priming sequence.” In step (d), a reverse transcriptase is introduced which synthesizes a single strand of DNA from the 3' end of the primed site towards the 3' end of the prime editor guide RNA. This forms a single-strand DNA flap comprising the desired nucleotide change (e.g., the single base change, insertion, or deletion, or a combination thereof) and which is otherwise homologous to the endogenous DNA at or adjacent to the nick site. In step (e), the napDNAbp and guide RNA are released. Steps (f) and (g) relate to the resolution of the single strand DNA flap such that the desired nucleotide change becomes incorporated into the target locus. This process can be driven towards the desired product formation by removing the corresponding 5' endogenous DNA flap that forms once the 3' single strand DNA flap invades and hybridizes to the endogenous DNA sequence. Without being bound by theory, the cells endogenous DNA repair and replication processes resolves the mismatched DNA to incorporate the nucleotide change(s) to form the desired altered product. The process can also be driven towards product formation with“second strand nicking,” as exemplified in FIG.1D. This process may introduce at least one or more of the following genetic changes: transversions, transitions, deletions, and insertions.

[0225] The term“prime editor (PE) system” or“prime editor” or“PE system” or“PE editing system” refers the compositions involved in the method of genome editing using target-primed reverse transcription (TPRT) describe herein, including, but not limited to the napDNAbps, reverse transcriptases, fusion proteins (e.g., comprising napDNAbps and reverse transcriptases), prime editor guide RNAs, and complexes comprising fusion proteins and prime editor guide RNAs, as well as accessory elements, such as second strand nicking components and 5' endogenous DNA flap removal endonucleases for helping to drive the prime editing process towards the edited product formation.Upstream

[0226] As used herein, the terms“upstream” and“downstream” are terms of relativity that define the linear position of at least two elements located in a nucleic acid molecule (whether single or double-stranded) that is orientated in a 5ʹ-to-3ʹ direction. In particular, a first element is upstream of a second element in a nucleic acid molecule where the first element is positioned somewhere that is 5’ to the second element. For example, a SNP is upstream of a Cas9-induced nick site if the SNP is on the 5’ side of the nick site. Conversely, a first element is downstream of a second element in a nucleic acid molecule where the first element is positioned somewherethat is 3’ to the second element. For example, a SNP is downstream of a Cas9-induced nick site if the SNP is on the 3’ side of the nick site. The nucleic acid molecule can be a DNA (double or single stranded). RNA (double or single stranded), or a hybrid of DNA and RNA. The analysis is the same for single strand nucleic acid molecule and a double strand molecule since the terms upstream and downstream are in reference to only a single strand of a nucleic acid molecule, except that one needs to select which strand of the double stranded molecule is being considered. Often, the strand of a double stranded DNA which can be used to determine the positional relativity of at least two elements is the“sense” or“coding” strand. In genetics, a“sense” strand is the segment within double-stranded DNA that runs from 5' to 3', and which is complementary to the antisense strand of DNA, or template strand, which runs from 3' to 5'. Thus, as an example, a SNP nucleobase is“downstream” of a promoter sequence in a genomic DNA (which is double-stranded) if the SNP nucleobase is on the 3' side of the promoter on the sense or coding strand.Variant

[0227] As used herein the term“variant” should be taken to mean the exhibition of qualities that have a pattern that deviates from what occurs in nature, e.g., a variant Cas9 is a Cas9 comprising one or more changes in amino acid residues as compared to a wild type Cas9 amino acid sequence. The term“variant” encompasses homologous proteins having at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 99% percent identity with a reference sequence and having the same or substantially the same functional activity or activities as the reference sequence. The term also encompasses mutants, truncations, or domains of a reference sequence, and which display the same or substantially the same functional activity or activities as the reference sequence.Vector

[0228] The term“vector,” as used herein, refers to a nucleic acid that can be modified to encode a gene of interest and that is able to enter into a host cell, mutate and replicate within the host cell, and then transfer a replicated form of the vector into another host cell. Exemplary suitable vectors include viral vectors, such as retroviral vectors or bacteriophages and filamentous phage, and conjugative plasmids. Additional suitable vectors will be apparent to those of skill in the art based on the instant disclosure.Wild Type

[0229] As used herein the term“wild type” is a term of the art understood by skilled persons and means the typical form of an organism, strain, gene or characteristic as it occurs in nature as distinguished from mutant or variant forms.5' endogenous DNA flap removal

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

[0231] As used herein, the term“5' endogenous DNA flap” refers to the strand of DNA situated immediately downstream of the PE-induced nick site in the target DNA. The nicking of the target DNA strand by PE exposes a 3' hydroxyl group on the upstream side of the nick site and a 5' hydroxyl group on the downstream side of the nick site. The endogenous strand ending in the 3' hydroxyl group is used to prime the DNA polymerase of the prime editor (e.g., wherein the DNA polymerase is a reverse transcriptase). The endogenous strand on the downstream side of the nick site and which begins with the exposed 5' hydroxyl group is referred to as the“5' endogenous DNA flap” and is ultimately removed and replaced by the newly synthesized replacement strand (i.e.,“3' replacement DNA flap”) the encoded by the extension of the PEgRNA.3' replacement DNA flap

[0232] As used herein, the term“3' replacement DNA flap” or simply,“replacement DNA flap,” refers to the strand of DNA that is synthesized by the prime editor and which is encoded by the extension arm of the prime editor PEgRNA. More in particular, the 3' replacement DNA flap is encoded by the polymerase template of the PEgRNA. The 3' replacement DNA flap comprises the same sequence as the 5' endogenous DNA flap except that it also contains the edited sequence (e.g., single nucleotide change). The 3' replacement DNA flap anneals to the target DNA, displacing or replacing the 5' endogenous DNA flap (which can be excised, for example, by a 5' flap endonuclease, such as FEN1 or EXO1) and then is ligated to join the 3' end of the 3' replacement DNA flap to the exposed 5' hydoxyl end of endogenous DNA (exposed after excision of the 5' endogenous DNA flap, thereby reforming a phosophodiester bond and installing the 3' replacement DNA flap to form a heteroduplex DNA containing one edited strand and one unedited strand. DNA repair processes resolve the heteroduplex by copying the information in the edited strand to the complementary strand permanently installs the edit in to the DNA. This resolution process can be driven further to completion by nicking the unedited strand, i.e., by way of“second-strand nicking,” as described herein.DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0233] The present invention disclosed new compositions (e.g., new PEgRNA and PE complexes comprising same) and methods for using prime editing (PE) to repair therapeutic targets, e.g., those targets identified in the ClinVar database, using PEgRNA designed using a specialized algorithm that is described herein. Thus, present application discloses an algorithm for predicting on a large-scale the sequences for PEgRNA that may be used to repair therapeutic targets (e.g., those included in the ClinVar database). In addition, the present application discloses predicted sequences for therapeutic PEgRNA designed using the disclosed algorithm and which may be used with prime editing to repair therapeutic targets.

[0234] The herein disclosed algorithm and the predicted PEgRNA sequences relate in general to prime editing. Thus, this disclosure also provides a description for the various components and aspects of prime editing, including suitable napDNAbp (e.g., Cas9 nickase) and reverse transcriptases, as well as other suitable components (e.g., linkers, NLS) and PE fusion proteins, that may be used with the therapeutic PEgRNA disclosed herein.

[0235] Adoption of the clustered regularly interspaced short palindromic repeat (CRISPR) system for genome editing has revolutionized the life sciences1–3. Although gene disruption using CRISPR is now routine, the precise installation of single nucleotide edits remains a major challenge, despite being necessary for studying or correcting a large number of disease-causative mutations. Homology directed repair (HDR) is capable of achieving such edits, but suffers from low efficiency (often <5%), a requirement for donor DNA repair templates, and deleterious effects of double-stranded DNA break (DSB) formation. Recently, Prof. David Liu et al.’s laboratory developed base editing, which achieves efficient single nucleotide editing without DSBs. Base editors (BEs) combine the CRISPR system with base-modifying deaminase enzymes to convert target C•G or A•T base pairs to A•T or G•C, respectively4–6. Although already widely used by researchers worldwide, current BEs enable only four of the twelve possible base pair conversions and are unable to correct small insertions or deletions. Moreover, the targeting scope of base editing is limited by the editing of non-target C or A bases adjacent to the target base (“bystander editing”) and by the requirement that a PAM sequence exist 15±2 bp from the target base. Overcoming these limitations would therefore greatly broaden the basic research and therapeutic applications of genome editing.

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

[0237] Accordingly, the present invention relates in part to the discovery that the mechanism of target-primed reverse transcription (TPRT) can be leveraged or adapted for conducting precision CRISPR / Cas-based genome editing with high efficiency and genetic flexibility (e.g., as depicted in various embodiments of FIGs.1A-1G). The inventors have proposed herein to use napDNAbp-polymerase fusions (e.g., Cas9 nickase fused to a reverse transcriptase) to target a specific DNA sequence with a modified guide RNA (“an extended guide RNA” or PEgRNA),generate a single strand nick at the target site, and use the nicked DNA as a primer for synthesis of DNA by a polymerase (e.g., reverse transcriptase) based on a DNA synthesis template that is a component of the PEgRNA. The newly synthesized strand would be homologous to the genomic target sequence except for the inclusion of a desired nucleotide change (e.g., a single nucleotide change, a deletion, or an insertion, or a combination thereof). The newly synthesize strand of DNA may be referred to as a single strand DNA flap, which would compete for hybridization with the complementary homologous endogenous DNA strand, thereby displacing the corresponding endogenous strand. Resolution of this hybridized intermediate can include removal of the resulting displaced flap of endogenous DNA (e.g., with a 5' end DNA flap endonuclease, FEN1), ligation of the synthesized single strand DNA flap to the target DNA, and assimilation of the desired nucleotide change as a result of cellular DNA repair and / or replication processes. Because templated DNA synthesis offers single nucleotide precision, the scope of this approach is very broad and could foreseeably be used for myriad applications in basic science and therapeutics.I. Therapeutic PEgRNA s

[0238] The prime editor (PE) system described herein contemplates the use of any suitable prime editor guide RNA or PEgRNA . The inventors have discovered that the mechanism of target- primed reverse transcription (TPRT) can be leveraged or adapted for conducting precision and versatile CRISPR / Cas-based genome editing through the use of a specially configured guide RNA comprising a DNA synthesis template that codes for the desired nucleotide change by a polymerase (e.g., reverse transcriptase). The application refers to this specially configured guide RNA as a“prime editor guide RNA” (or PEgRNA) since the DNA synthesis template can be provided as an extension of a standard or traditional guide RNA molecule. The application contemplates any suitable configuration or arrangement for the prime editor guide RNA.

[0239] In various embodiments, the disclosure provides therapeutic PEgRNA of SEQ ID NOs: 1-135514 and 813085-880462 designed using the herein disclosed algorithm against ClinVar database entries.

[0240] In various other embodiments, exemplary PEgRNA designed against the ClinVar database using the herein disclosed algorithm are included in the Sequence Listing, which forms a part of this specification. The Sequence Listing includes complete PEgRNA sequences of SEQ ID NOs: 1-135514 and 813085-880462. Each of these complete PEgRNA are each comprisedof 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 PEgRNA comprises a gRNA core, for example, as defined by SEQ ID NOs: 1361579-1361580. The extension arms of SEQ ID NOs: 271029– 406542 and 947841-1015218 are further each comprised of a primer binding site (SEQ ID NOs.: 406543– 542056 and 1015219-1082596), an edit template (SEQ ID NOs.: 542057– 677570 and 1082597-1149974), and a homology arm (SEQ ID NOs.: 677571– 813084 and 1149975-1217352). The PEgRNA optionally may comprise a 5′ end modifier region and / or a 3′ end modifier region. The PEgRNA may also comprise a reverse transcription termination signal (e.g., SEQ ID NOs: 1361560-1361566) at the 3′ of the PEgRNA. The application embraces the design and use of all of these sequences.

[0241] FIG.3A shows one embodiment of a prime editor guide RNA (referred to as either a “PEgRNA” or an“extended gRNA”) usable in the prime editor (PE) system disclosed herein whereby a traditional guide RNA (the green portion) includes a spacer and a gRNA core region, which binds with the napDNAbp. In this embodiment, the guide RNA includes an extended RNA segment at the 5' end, i.e., a 5' extension. In this embodiment, the 5'extension includes a DNA synthesis template, a primer binding site, and an optional 5-20 nucleotide linker sequence. As shown in FIG.1A, the Primer binding site hydrides to the free 3′ end that is formed after a nick is formed in the non-target strand of the R-loop, thereby priming the polymerase (e.g., reverse transcriptase) for DNA polymerization in the 5' to 3' direction.

[0242] FIG.3B shows another embodiment of a prime editor guide RNA usable in the prime editor (PE) system disclosed herein whereby a traditional guide RNA (the green portion) includes a ~20 nt spacer and a gRNA core, which binds with the napDNAbp. In thisembodiment, the guide RNA includes an extended RNA segment at the 3' end, i.e., a 3' extension. In this embodiment, the 3'extension includes a DNA synthesis template, and a primer binding site. As shown in FIG.1B, the primer binding site hydrides to the free 3' end that is formed after a nick is formed in the non-target strand of the R-loop, thereby priming the polymerase for DNA polymerization in the 5' to 3' direction.

[0243] FIG.3C shows another embodiment of an extend guide RNA usable in the prime editor (PE) system disclosed herein whereby a traditional guide RNA (the green portion) includes a ~20 nt spacer and a gRNA core, which binds with the napDNAbp. In this embodiment, the guide RNA includes an extended RNA segment at an intermolecular position within the gRNA core,i.e., an intramolecular extension. In this embodiment, the intramolecular extension includes a DNA synthesis template, and a primer binding site. The primer binding site hybridizes to the free 3' end that is formed after a nick is formed in the non-target strand of the R-loop, thereby priming the polymerase 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 still another embodiment, the position of the intramolecular RNA extension is anywhere within the guide RNA molecule except within the spacer, or at a position which disrupts 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, at least 25 nucleotides downstream of the 3' end of the spacer.

[0246] In other embodiments, the intramolecular RNA extension is inserted into the gRNA, which refers to the portion of the guide RNA corresponding or comprising the tracrRNA, which binds and / or interacts with the Cas9 protein or equivalent thereof (i.e, a different napDNAbp). Preferably the insertion of the intramolecular RNA extension does not disrupt or minimally disrupts the interaction between the tracrRNA portion and the napDNAbp.

[0247] The length of the RNA extension can be any useful length. In various embodiments, the RNA extension is at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides, at least 22 nucleotides, at least 23 nucleotides, at least 24 nucleotides, at least 25 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides, at least 80 nucleotides, at least 90nucleotides, at least 100 nucleotides, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, or at least 500 nucleotides in length.

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

[0250] In other embodiments, the optional 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] The DNA synthesis template (e.g., RT template sequence), In some embodiments, encodes a single-stranded DNA molecule which is homologous to the non-target strand (andthus, complementary to the corresponding site of the target strand) but includes one or more nucleotide changes. The nucleotide change may include one or more single-base nucleotide changes, one or more deletions, one or more insertions, and combinations thereof.

[0252] As depicted in FIG.1E, the synthesized single-stranded DNA product of the DNA synthesis template (e.g., 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., RT template sequence) hybridizes in equilibrium with thecomplementary target strand sequence, thereby displacing the homologous endogenous target strand sequence. The displaced endogenous strand may be referred to in some embodiments as a 5' endogenous DNA flap species (e.g., see FIG.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 to the endogenous target strand, may be ligated, thereby creating a mismatch between the endogenous sequence and the newly synthesized strand. The mismatch may be resolved by the cell’s innate DNA repair and / or replication processes.

[0253] In various embodiments, the nucleotide sequence of the DNA synthesis template (e.g., RT template sequence) corresponds to the nucleotide sequence of the non-target strand which becomes displaced as the 5' flap species and which overlaps with the site to be edited.

[0254] In various embodiments of the prime editor guide RNAs, the DNA synthesis template may encode a single-strand DNA flap that is complementary to an endogenous DNA sequence adjacent to a nick site, wherein the single-strand DNA flap comprises a desired nucleotide change. The single-stranded DNA flap may displace an endogenous single-strand DNA at the nick site. The displaced endogenous single-strand DNA at the nick site can have a 5´ end and form an endogenous flap, which can be excised by the cell. In various embodiments, excision of the 5´ end endogenous flap can help drive product formation since removing the 5´ end endogenous flap encourages hybridization of the single-strand 3´ DNA flap to the corresponding complementary DNA strand, and the incorporation or assimilation of the desired nucleotide change carried by the single-strand 3´ DNA flap into the target DNA.

[0255] In various embodiments of the prime editor guide RNAs, the cellular repair of the single- strand DNA flap results in installation of the desired nucleotide change, thereby forming a desired product.

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

[0257] In various aspects, the prime editor guide RNAs are modified versions of a guide RNA. Guide RNAs maybe naturally occurring, expressed from an encoding nucleic acid, or synthesized chemically. Methods are well known in the art for obtaining or otherwise synthesizing guide RNAs and for determining the appropriate sequence of the guide RNA, including the spacer which interacts and hybridizes with the target strand of a genomic target site of interest.

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

[0259] In general, a guide sequence is any polynucleotide sequence having sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of a napDNAbp (e.g., a Cas9, Cas9 homolog, or Cas9 variant) to the target sequence. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence, when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non-limiting example of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g., the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net). In some embodiments, aguide sequence is about or more than 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, a guide sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides in length. The ability of a guide sequence to direct sequence-specific binding of a base editor to a target sequence may be assessed by any suitable assay. For example, the components of a base editor, including the guide sequence to be tested, may be provided to a host cell having the corresponding target sequence, such as by transfection with vectors encoding the components of a base editor disclosed herein, followed by an assessment of preferential cleavage within the target sequence, such as by Surveyor assay as described herein. Similarly, cleavage of a target polynucleotide sequence may be evaluated in a test tube by providing the target sequence, components of a base editor, including the guide sequence to be tested and a control guide sequence different from the test guide sequence, and comparing binding or rate of cleavage at the target sequence between the test and control guide sequence reactions. Other assays are possible, and will occur to those skilled in the art.

[0261] A guide sequence may be selected to target any target sequence. In some embodiments, the target sequence is a sequence within a genome of a cell. Exemplary target sequences include those that are unique in the target genome. For example, for the S. pyogenes Cas9, a unique target sequence in a genome may include a Cas9 target site of the formMMMMMMMMNNNNNNNNNNNNXGG (SEQ ID NO: 1361548) where NNNNNNNNNNNNXGG (SEQ ID NO: 1361549) (N is A, G, T, or C; and X can be anything) has a single occurrence in the genome. A unique target sequence in a genome may include an S. pyogenes Cas9 target site of the form MMMMMMMMMNNNNNNNNNNNXGG (SEQ ID NO: 1361550) where NNNNNNNNNNNXGG (SEQ ID NO: 1361551) (N is A, G, T, or C; and X can be anything) has a single occurrence in the genome. For the S. thermophilus CRISPR1Cas9, a unique target sequence in a genome may include a Cas9 target site of the form MMMMMMMMNNNNNNNNNNNNXXAGAAW (SEQ ID NO: 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 a single occurrence in the genome. A unique target sequence in a genome may include an S. thermophilus CRISPR 1 Cas9 target site of the formMMMMMMMMMNNNNNNNNNNNXXAGAAW (SEQ ID NO: 1361554) whereNNNNNNNNNNNXXAGAAW (SEQ ID NO: 1361555) (N is A, G, T, or C; X can be anything; and W is A or T) has a single occurrence in the genome. For the S. pyogenes Cas9, a unique targetsequence in a genome may include a Cas9 target site of the formMMMMMMMMNNNNNNNNNNNNXGGXG (SEQ ID NO: 1361556) where NNNNNNNNNNNNXGGXG (SEQ ID NO: 1361557) (N is A, G, T, or C; and X can be anything) has a single occurrence in the genome. A unique target sequence in a genome may include an S. pyogenes Cas9 target site of the form MMMMMMMMMNNNNNNNNNNNXGGXG (SEQ ID NO: 1361558) whereNNNNNNNNNNNXGGXG (SEQ ID NO: 1361559) (N is A, G, T, or C; and X can be anything) has a single occurrence in the genome. In each of these sequences“M” may be A, G, T, or C, and need not be considered in identifying a sequence as unique.

[0262] In some embodiments, a guide sequence is selected to reduce the degree of secondary structure within the guide sequence. Secondary structure may be determined by any suitable polynucleotide folding algorithm. Some programs are based on calculating the minimal Gibbs free energy. An example of one such algorithm is mFold, as described by Zuker and Stiegler (Nucleic Acids Res.9 (1981), 133-148). Another example folding algorithm is the online webserver RNA fold, developed at Institute for Theoretical Chemistry at the University of Vienna, using the centroid structure prediction algorithm (see e.g. A. R. Gruber et al., 2008, Cell 106(1): 23-24; and PA Carr and GM Church, 2009, Nature Biotechnology 27(12): 1151-62). Further algorithms may be found in U.S. application Ser. No.61 / 836,080; Broad Reference BI- 2013 / 004A); incorporated herein by reference.

[0263] In general, a tracr mate sequence includes any sequence that has sufficientcomplementarity with a tracr sequence to promote one or more of: (1) excision of a guide sequence flanked by tracr mate sequences in a cell containing the corresponding tracr sequence; and (2) formation of a complex at a target sequence, wherein the complex comprises the tracr mate sequence hybridized to the tracr sequence. In general, degree of complementarity is with reference to the optimal alignment of the tracr mate sequence and tracr sequence, along the length of the shorter of the two sequences. Optimal alignment may be determined by any suitable alignment algorithm, and may further account for secondary structures, such as self- complementarity within either the tracr sequence or tracr mate sequence. In some embodiments, the degree of complementarity between the tracr sequence and tracr mate sequence along the length of the shorter of the two when optimally aligned is about or more than about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99%, or higher. In some embodiments, the tracr sequence is about or more than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25,30, 40, 50, or more nucleotides in length. In some embodiments, the tracr sequence and tracr mate sequence are contained within a single transcript, such that hybridization between the two produces a transcript having a secondary structure, such as a hairpin. Preferred loop forming sequences for use in hairpin structures are four nucleotides in length, and most preferably have the sequence GAAA. However, longer or shorter loop sequences may be used, as may alternative sequences. The sequences preferably include a nucleotide triplet (for example, AAA), and an additional nucleotide (for example C or G). Examples of loop forming sequences include CAAA and AAAG. In an embodiment of the invention, the transcript or transcribed polynucleotide sequence has at least two or more hairpins. In preferred embodiments, the transcript has two, three, four or five hairpins. In a further embodiment of the invention, the transcript has at most five hairpins. In some embodiments, the single transcript further includes a transcription termination sequence; preferably this is a polyT sequence, for example six T nucleotides. Further non-limiting examples of single polynucleotides comprising a guide sequence, a tracr mate sequence, and a tracr sequence are as follows (listed 5′ to 3′), where“N” represents a base of a guide sequence, the first block of lower case letters represent the tracr mate sequence, and the second block of lower case letters represent the tracr sequence, and the final poly-T sequence represents the transcription terminator: (1)NNNNNNNNgtttttgtactctcaagatttaGAAAtaaatcttgcagaagctacaaagataaggc ttcatgccgaaatcaacaccctgtcattttatggcagggtgttttcgttatttaaTTTTTT (SEQ ID NO: 1361560); (2)NNNNNNNNNNNNNNNNNNgtttttgtactctcaGAAAtgcagaagctacaaagataaggcttca tgccgaaatcaacaccctgtcattttatggcagggtgttttcgttatttaaTTTTTT (SEQ ID NO: 1361561); (3)NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaGAAAtgcagaagctacaaagataaggctt catgccgaaatcaacaccctgtcattttatggcagggtgtTTTTT (SEQ ID NO:1361562); (4)NNNNNNNNNNNNNNNNNNNNgttttagagctaGAAAtagcaagttaaaataaggctagtccgtt atcaacttgaaaaagtggcaccgagtcggtgcTTTTTT (SEQ ID NO: 1361563); (5) NNNNNNNNNNNNNNNNNNNNgttttagagctaGAAATAGcaagttaaaataaggctagtccgtt atcaacttgaaaaagtgTTTTTTT (SEQ ID NO: 1361564 and (6)NNNNNNNNNNNNNNNNNNNNgttttagagctagAAATAGcaagttaaaataaggctagtccgttatcaTTTTTTTT (SEQ ID NO: 1361565). In some embodiments, sequences (1) to (3) are used in combination with Cas9 from S. thermophilus CRISPR1. In some embodiments, sequences (4) to (6) are used in combination with Cas9 from S. pyogenes. In some embodiments, the tracr sequence is a separate transcript from a transcript comprising the tracr mate sequence.

[0264] It will be apparent to those of skill in the art that in order to target any of the fusion proteins comprising a Cas9 domain and a single-stranded DNA binding protein, as disclosed herein, to a target site, e.g., a site comprising a point mutation to be edited, it is typically necessary to co-express the fusion protein together with a guide RNA, e.g., an sgRNA. As explained in more detail elsewhere herein, a guide RNA typically comprises a tracrRNA framework allowing for Cas9 binding, and a guide sequence, which confers sequence specificity to the Cas9:nucleic acid editing enzyme / domain fusion protein.

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

[0266] In other embodiments, PEgRNA may include those depicted by the structure shown in FIG.27, which comprises a guide RNA and a 3′ extension arm.

[0267] FIG.27 provides the structure of an embodiment of a PEgRNA contemplated herein and which may be designed in accordance with the methodology defined in Example 2. The PEgRNA comprises three main component elements ordered in the 5′ to 3′ direction, namely: a spacer, a gRNA core, and an extension arm at the 3′ end. The extension arm may further be divided into the following structural elements in the 5′ to 3′ direction, namely: a primer binding site (A), an edit template (B), and a homology arm (C). In addition, the PEgRNA may comprisean optional 3′ end modifier region (e1) and an optional 5′ end modifier region (e2). Still further, the PEgRNA may comprise a transcriptional termination signal at the 3′ end of the PEgRNA (not depicted). These structural elements are further defined herein. The depiction of the structure of the PEgRNA is not meant to be limiting and embraces variations in the arrangement of the elements. For example, the optional sequence modifiers (e1) and (e2) could be positioned within or between any of the other regions shown, and not limited to being located at the 3′ and 5′ ends.

[0268] In still other embodiments, PEgRNA may include those depicted by the structure shown in FIG.28, which comprises a guide RNA and a 5′ extension arm.

[0269] FIG.28 provides the structure of another embodiment of a PEgRNA contemplated herein and which may be designed in accordance with the methodology defined in Example 2. The PEgRNA comprises three main component elements ordered in the 5′ to 3′ direction, namely: a spacer, a gRNA core, and an extension arm at the 3′ end. The extension arm may further be divided into the following structural elements in the 5′ to 3′ direction, namely: a primer binding site (A) (SEQ ID NOs: 406543-542056 and 1015219-1082596), an edit 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 modifier region (e1) and an optional 5′ end modifier region (e2). Still further, the PEgRNA may comprise a transcriptional termination signal on the 3′ end of the PEgRNA (not depicted). These structural elements are further defined herein. The depiction of the structure of the PEgRNA is not meant to be limiting and embraces variations in the arrangement of the elements. For example, the optional sequence modifiers (e1) and (e2) could be positioned within or between any of the other regions shown, and not limited to being located at the 3′ and 5′ ends.

[0270] The PEgRNA may also include additional design improvements that may modify the properties and / or characteristics of PEgRNA thereby improving the efficacy of prime editing. In various embodiments, these improvements may belong to one or more of a number of different categories, including but not limited to: (1) designs to enable efficient expression of functional PEgRNA from non-polymerase III (pol III) promoters, which would enable the expression of longer PEgRNA without burdensome sequence requirements; (2) improvements to the core, Cas9-binding PEgRNA scaffold, which could improve efficacy; (3) modifications to the PEgRNA to improve RT processivity, enabling the insertion of longer sequences at targetedgenomic loci; and (4) addition of RNA motifs to the 5′ or 3′ termini of the PEgRNA that improve PEgRNA stability, enhance RT processivity, prevent misfolding of the PEgRNA , or recruit additional factors important for genome editing.

[0271] In one embodiment, PEgRNA could be designed with polIII promoters to improve the expression of longer-length PEgRNA with larger extension arms. sgRNAs are typically expressed from the U6 snRNA promoter. This promoter recruits pol III to express the associated RNA and is useful for expression of short RNAs that are retained within the nucleus. However, pol III is not highly processive and is unable to express RNAs longer than a few hundred nucleotides in length at the levels required for efficient genome editing. Additionally, pol III can stall or terminate at stretches of U’s, potentially limiting the sequence diversity that could be inserted using a PEgRNA . Other promoters that recruit polymerase II (such as pCMV) or polymerase I (such as the U1 snRNA promoter) have been examined for their ability to express longer sgRNAs. However, these promoters are typically partially transcribed, which would result in extra sequence 5′ of the spacer in the expressed PEgRNA , which has been shown to result in markedly reduced Cas9:sgRNA activity in a site-dependent manner. Additionally, while pol III- transcribed PEgRNA can simply terminate in a run of 6-7 U’s, PEgRNA transcribed from pol II or pol I would require a different termination signal. Often such signals also result inpolyadenylation, which would result in undesired transport of the PEgRNA from the nucleus. Similarly, RNAs expressed from pol II promoters such as pCMV are typically 5′-capped, also resulting in their nuclear export.

[0272] Previously, Rinn and coworkers screened a variety of expression platforms for the production of long-noncoding RNA- (lncRNA) tagged sgRNAs183. These platforms include RNAs expressed from pCMV and that terminate in the ENE element from the MALAT1 ncRNA from humans184, the PAN ENE element from KSHV185, or the 3′ box from U1 snRNA186.Notably, the MALAT1 ncRNA and PAN ENEs form triple helices protecting the polyA-tail184,187. These constructs could also enhance RNA stability. It is contemplated that these expression systems will also enable the expression of longer PEgRNA .

[0273] In addition, a series of methods have been designed for the cleavage of the portion of the pol II promoter that would be transcribed as part of the PEgRNA , adding either a self-cleaving ribozyme such as the hammerhead188, pistol189, hatchet189, hairpin190, VS191, twister192, or twister sister192ribozymes, or other self-cleaving elements to process the transcribed guide, or a hairpinthat is recognized by Csy4193and also leads to processing of the guide. Also, it is hypothesized that incorporation of multiple ENE motifs could lead to improved PEgRNA expression and stability, as previously demonstrated for the KSHV PAN RNA and element185. It is also anticipated that circularizing the PEgRNA in the form of a circular intronic RNA (ciRNA) could also lead to enhanced RNA expression and stability, as well as nuclear localization194.

[0274] In various embodiments, the PEgRNA may include various above elements, as exemplified by the following sequence.

[0275] Non-limiting example 1 - PEgRNA expression platform consisting of pCMV, Csy4 hairpin, the PEgRNA , and MALAT1 ENE( Q )

[0276] Non-limiting example 2 - PEgRNA expression platform consisting of pCMV, Csy4 hairing, the PEgRNA , and PAN ENE36 568)

[0277] Non-limiting example 3 - PEgRNA expression platform consisting of pCMV, Csy4 hairing, the PEgRNA , and 3xPAN ENECC (S Q O: 36 569)

[0278] Non-limiting example 4 - PEgRNA expression platform consisting of pCMV, Csy4 hairing, the PEgRNA , and 3′ boxT A A T C A C C T T CT

[0279] Non-limiting example 5 - PEgRNA expression platform consisting of pU1, Csy4 hairpin, the PEgRNA , and 3′ box

[0280] In various other embodiments, the PEgRNA may be improved by introducing improvements to the scaffold or core sequences. This can be done by introducing known

[0281] The core, Cas9-binding PEgRNA scaffold can likely be improved to enhance PE activity. Several such approaches have already been demonstrated. For instance, the first pairing element of the scaffold (P1) contains a GTTTT-AAAAC pairing element. Such runs of Ts have been shown to result in pol III pausing and premature termination of the RNA transcript.Rational mutation of one of the T-A pairs to a G-C pair in this portion of P1 has been shown to enhance sgRNA activity, suggesting this approach would also be feasible for PEgRNA195. Additionally, increasing the length of P1 has also been shown to enhance sgRNA folding and lead to improved activity195, suggesting it as another avenue for the improvement of PEgRNA activity. Example improvements to the core can include:

[0282] PEgRNA containing a 6 nt extension to P1

[0283] PEgRNA containing a T-A to G-C mutation within P1

[0284] In various other embodiments, the PEgRNA may be improved by introducing modifications to the edit template region. As the size of the insertion templated by the PEgRNA increases, it is more likely to be degraded by endonucleases, undergo spontaneous hydrolysis, or fold into secondary structures unable to be reverse-transcribed by the RT or that disrupt folding of the PEgRNA scaffold and subsequent Cas9-RT binding. Accordingly, it is likely thatmodification to the template of the PEgRNA might be necessary to affect large insertions, such as the insertion of whole genes. Some strategies to do so include the incorporation of modified nucleotides within a synthetic or semi-synthetic PEgRNA that render the RNA more resistant to degradation or hydrolysis or less likely to adopt inhibitory secondary structures196. Such modifications could include 8-aza-7-deazaguanosine, which would reduce RNA secondary structure in G-rich sequences; locked-nucleic acids (LNA) that reduce degradation and enhance certain kinds of RNA secondary structure; 2’-O-methyl, 2’-fluoro, or 2’-O-methoxyethoxy modifications that enhance RNA stability. Such modifications could also be included elsewhere in the PEgRNA to enhance stability and activity. Alternatively or additionally, the template of the PEgRNA could be designed such that it both encodes for a desired protein product and is also more likely to adopt simple secondary structures that are able to be unfolded by the RT. Such simple structures would act as a thermodynamic sink, making it less likely that more complicated structures that would prevent reverse transcription would occur. Finally, one could also split the template into two, separate PEgRNA . In such a design, a PE would be used to initiate transcription and also recruit a separate template RNA to the targeted site via an RNA- binding protein fused to Cas9 or an RNA recognition element on the PEgRNA itself such as the MS2 aptamer. The RT could either directly bind to this separate template RNA, or initiate reverse transcription on the original PEgRNA before swapping to the second template. Such an approach could enable long insertions by both preventing misfolding of the PEgRNA upon addition of the long template and also by not requiring dissociation of Cas9 from the genome for long insertions to occur, which could possibly be inhibiting PE-based long insertions.(iv) Installation of additional RNA motifs at the 5′ or 3′ termini

[0285] In still other embodiments, the PEgRNA may be improved by introducing additional RNA motifs at the 5′ and 3′ termini of the PEgRNA . Several such motifs - such as the PAN ENE from KSHV and the ENE from MALAT1 were discussed above as possible means to terminate expression of longer PEgRNA from non-pol III promoters. These elements form RNA triple helices that engulf the polyA tail, resulting in their being retained within the nucleus184,187. However, by forming complex structures at the 3′ terminus of the PEgRNA that occlude the terminal nucleotide, these structures would also likely help prevent exonuclease- mediated degradation of PEgRNA.

[0286] Other structural elements inserted at the 3′ terminus could also enhance RNA stability, albeit without enabling termination from non-pol III promoters. Such motifs could include hairpins or RNA quadruplexes that would occlude the 3′ terminus197, or self-cleaving ribozymes such as HDV that would result in the formation of a 2’-3′-cyclic phosphate at the 3′ terminus and also potentially render the PEgRNA less likely to be degraded by exonucleases198. Inducing the PEgRNA to cyclize via incomplete splicing - to form a ciRNA - could also increase PEgRNA stability and result in the PEgRNA being retained within the nucleus194.

[0287] Additional RNA motifs could also improve RT processivity or enhance PEgRNA activity by enhancing RT binding to the DNA-RNA duplex. Addition of the native sequence bound by the RT in its cognate retroviral genome could enhance RT activity199. This could include the native primer binding site (PBS), polypurine tract (PPT), or kissing loops involved in retroviral genome dimerization and initiation of transcription199.

[0288] Addition of dimerization motifs - such as kissing loops or a GNRA tetraloop / tetraloop receptor pair200- at the 5′ and 3′ termini of the PEgRNA could also result in effective circularization of the PEgRNA , improving stability. Additionally, it is envisioned that addition of these motifs could enable the physical separation of the PEgRNA spacer and primer, prevention occlusion of the spacer which would hinder PE activity. Short 5′ extensions to the PEgRNA that form a small toehold hairpin in the spacer region could also compete favorably against the annealing region of the PEgRNA binding the spacer. Finally, kissing loops could also be used to recruit other template RNAs to the genomic site and enable swapping of RT activity from one RNA to the other. Example improvements include, but are not limited to:

[0289] PEgRNA -HDV fusion

[0290] PEgRNA -MMLV kissing loop

[0291] PEgRNA -VS ribozyme kissing loop

[0292] PEgRNA -GNRA tetraloop / tetraloop receptor( Q )

[0293] PEgRNA template switching secondary RNA-HDV fusion

[0294] PEgRNA scaffold could be further improved via directed evolution, in an analogous fashion to how SpCas9 and base editors have been improved. Directed evolution could enhance PEgRNA recognition by Cas9 or evolved Cas9 variants. Additionally, it is likely that different PEgRNA scaffold sequences would be optimal at different genomic loci, either enhancing PE activity at the site in question, reducing off-target activities, or both. Finally, evolution of PEgRNA scaffolds to which other RNA motifs have been added would almost certainly improve the activity of the fused PEgRNA relative to the unevolved, fusion RNA. For instance, evolution of allosteric ribozymes composed of c-di-GMP-I aptamers and hammerhead ribozymes led to dramatically improved activity202, suggesting that evolution would improve the activity of hammerhead-PEgRNA fusions as well. In addition, while Cas9 currently does not generally tolerate 5′ extension of the sgRNA, directed evolution will likely generate enabling mutations that mitigate this intolerance, allowing additional RNA motifs to be utilized.

[0295] The present disclosure contemplates any such ways to further improve the efficacy of the prime editing systems disclosed here.II. Algorithm and method to design therapeutic PEgRNA

[0296] As described herein, the inventors discovered and appreciated that prime editing using PEgRNA can be used to install a wide variety of nucleotide changes, including insertions (of any length, including whole genes or protein coding regions), deletions (of any length), and the correct pathogenic mutations. However, techniques do not yet exist to determine and / or predict PEgRNA structures, including specifying the various components of the PEgRNA , such as the spacer, gRNA core, and extension arm (and components of the extension as described herein). The inventors have developed computerized techniques for determining PEgRNA , 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 a PAM motif and the relative position of the prime editors nick). The difference between the input allele and the output allele represents the desired edit (e.g., a single nucleotide change, insertion, deletion, and / or the like). The determined structures can be created and used to perform base editing to change the input allele to the output allele, as described further herein.

[0297] FIG.31 is a flow chart showing an exemplary high level computerized method 3100 for determining an extended gRNA structure, according to some embodiments. At step 3102, a computing device (e.g., the computing device 3400 described in conjunction with FIG.34) accesses data indicative of an input allele, an output allele, and a fusion protein that includes 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 and it should be appreciated that such data can be accessed using one or more steps without departing from the spirit of the techniques described herein. Accessing data can include receiving data, storing data, accessing a database, and / or the like.

[0298] At step 3104, the computing device determines the extended gRNA structure based on the input allele, the output allele, and the fusion protein accessed in step 3102. The extended gRNA structure is designed to be associated with the fusion protein to change the input allele to the output allele. The fusion protein, when it is complexed with the extended gRNA, is capable of binding to a target DNA sequence that includes a target strand at which the change occurs and a complementary non-target strand. As described herein, the input allele can represent a pathogenic DNA mutation, and the output allele can represent a corrected DNA sequence.

[0299] Changing the input allele to the output allele can include 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 achieve the output allele. In particular, exemplary classes of edits that can be induced by a single PEgRNA include single nucleotide substitutions, insertions from 1 nt up to approximately 40 nt, deletions from 1 nt up to approximately 30 nt, and a combination thereof. For example, prime editing can support changes of these types from spacer position -3 (e.g., immediately 3′ of the nick) to spacer position +27 (e.g., 30 nt 3′ of the nick in the input allele). Other positions can also be used. For example, edits at spacer position -4 can be performed using the SpCas9 system with prime editing (e.g., which can be caused by occasional RuvCcleavage between spacer positions -5 and -4). The type of change, the number of nt changes, and / or the position of the change can be configurable parameter(s) that the computerized techniques can use to determine extended gRNA structures.

[0300] As discussed in conjunction with FIGS.3A-3B and FIGS.27-28, an extended gRNA can include various components such as a spacer for the extended gRNA that is complementary to a target nucleotide sequence in the input allele, a gRNA backbone for interacting with the fusion protein, and an extension. Referring further to step 3104, the computing device determines one or more of the spacer, the gRNA backbone, and the extension. In some embodiments, while the techniques can include determining any combination of the 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, fixed, etc.), and therefore may not be determined as part of step 3104.

[0301] As described herein, the gRNA extension can include various components. For example, as shown in FIGS.3A-3B and 27-28, the extension can include one or more of an RT template (which includes an RT edit template and a homology arm), an Primer binding site, an RT termination signal, an optional 5′ end modifier region, and an optional 3′ end modifier region. FIG.32 is a flow chart showing an exemplary computerized method 3200 for determining the components of an extended gRNA structure, including the components of the extension, according to some embodiments. It should be appreciated that FIG.32 is intended to be illustrative, and therefore techniques used to determine the extended gRNA can include more, or fewer, steps than those shown in FIG.32.

[0302] At step 3202, the computing device determines the 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 computing device determines an initial set of protospacers and filters out protospacers whose associated nick positions are incompatible with prime editing to the output allele to generate a set of remaining candidate protospacers. For example, the computing device may determine that a protospacer is incompatible because the nick is on the 3′ side of the desired edit on the strand. As another example, the computing device may determine that the distance between the nick and the desired edit is too large (e.g., greater than a user-defined threshold, for example 30 nt, 35 nt, etc.).

[0303] At step 3204, the computing device selects a protospacer from the set of determined protospacers. At step 3206, the computing device determines a spacer and an edit template sequence using the protospacer sequence of the input allele, the position of the nick, and the sequence of the desired edit. The spacer can include a nucleotide sequence of approximately 20 nucleotides.

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

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

[0306] The method 3200 proceeds to step 3216, and the computing device determines whether there are more sets of parameters. If yes, the method proceeds to step 3210 and the computing device selects another set of parameters. If no, the method proceeds to step 3218 and the computing device determines whether there are more protospacers. If yes, the method proceedsback to step 3204 and 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 (e.g., RT template sequence) of the extension includes a desired nucleotide change to change the input allele to the output allele, and includes the RT edit template (e.g., determined in step 3206) and the homology arm (e.g., determined at step 3212). As also described herein, the DNA synthesis template (e.g., RT template sequence) encodes a single-strand DNA flap that is complementary to an endogenous DNA sequence adjacent to the nick site. The single-strand DNA flap comprises the desired nucleotide change (e.g., a single nucleotide change, one or more nucleotide insertions, one or more nucleotide deletions, and / or the like). In some base editing deployments, the single-strand DNA flap can hybridize to the endogenous DNA sequence that is adjacent to the nick site to install the desired nucleotide change. In some base editing deployments, the single-stranded DNA flap displaces the endogenous DNA sequence that is adjacent to the nick site. Cellular repair of the single-strand DNA flap can result in installation of the desired nucleotide change to form the desired output allele product. The DNA synthesis template (e.g., RT template sequence) can have a variable number of nucleotides, and can range from approximately 7 nucleotides to 34 nucleotides.

[0308] While not shown in FIG.32, the computing device can be configured to determine other components of the extended gRNA. For example, in some embodiments the computing device is configured to determine an RT termination signal adjacent to the RT template. In some embodiments, the computing device can be configured to determine a first modifier adjacent to the RT termination signal. In some embodiments, the computing device is configured to determine a second modifier adjacent to the Primer binding site.

[0309] The extended gRNA components can be arranged in different configurations, such as those shown in FIGS.3A-3B and FIGS.27-28. For example, referring to FIG.3A, the extension is at the 5′ end of the extended gRNA structure, the spacer is 3′ to the extension and is 5′ to the gRNA core. As another example, referring to FIG.3B, the spacer is at a 5′ end of the extended gRNA structure (and is 5′ to the gRNA core), and the extension is at a 3′ end of the extended gRNA structure (and is 3′ to the gRNA core).

[0310] In some embodiments, the computing device accesses a database that includes a set of input alleles and associated output alleles. For example, the computing device can access adatabase provided by ClinVar that includes hundreds of thousands of mutations, each of which includes an allele representing a pathogenic mutation and an allele representing the corrected wild-type sequence. The techniques can be used to determine one or more extended gRNA structures for each database entry. FIG.33 is a flow chart showing an exemplary computerized method 3300 for determining sets 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., a ClinVar database) that includes a set of mutation entries that each include an input allele representing the mutation and an output allele representing the corrected wild-type sequence.

[0311] At step 3304, the computing device accesses a set of one or more fusion proteins. In some embodiments, the techniques can include generating sets 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 computing device can be configured to access data indicative of the plurality of fusion proteins, and can create a set of extended gRNA structures for each fusion protein (e.g., a Cas9-NG protein and an SpCas9 protein) as described herein.

[0312] At step 3306, the computing device selects a fusion protein from the set of fusion proteins. At step 3308, the computing device selects a mutation entry from the set of entries in the database. The computing device can be configured, for example, to iterate through each entry in the database and create a set of extended gRNA structures for the entry (e.g., one set for a particular fusion protein, and / or multiple sets for each of a plurality of fusion proteins). In some embodiments, the computing device can be configured to generate extended gRNA structures for a subset of entries in the database, such as a pre-configured set, a set of mutations with a highest significance (e.g., those with known therapeutic benefits), 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 can be configured to determine which entries in the database are compatible for prime editing using the selected fusion protein from step 3304, and to select entries that are compatible with the selected fusion protein in step 3308.

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

[0314] In some embodiments, the techniques can design PEgRNA with gRNA extensions that contain non-complementary sequences, such as non-complementary sequences that are 5′ of the homology arm, 3′ of the primer binding site, or both. For example, non-complementary sequences can be designed to form a kissing loop interaction, to act as a protecting hairpin for RNA stability, and / or the like.

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

[0316] An exemplary implementation of the computerized techniques described herein for determining extended gRNA structures is as follows:# Python 3# b_design_PEgRNA .pyfrom __future__ import divisionimport _configimport sys, os, fnmatch, datetime, subprocesssys.path.append(' / home / unix / maxwshen / ')import numpy as npfrom collections import defaultdictfrom mylib import util, compbioimport pandas as pd # Default paramsinp_dir = _config.OUT_PLACE + 'a_annotate / 'NAME = util.get_fn(__file__)out_dir = _config.OUT_PLACE + NAME + ' / 'util.ensure_dir_exists(out_dir) SPLIT = None # Hyperparametersgrna_nick_pos = 17grna_len = 20 max_dist_nick_to_edit = 20 primer_binding_len = 13homology_arm_len = 13 grna_hairpin ='GTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTG AAAAAGTGGCACCGAGTCGGTGC' (SEQ ID NO: 1361579)terminator = 'TTTTTTGTTTT' (SEQ ID NO: 1361581) castypes = {'SpCas9 (NGG)': 'NGG','SpCas9-NG (NG)': 'NG',} assert grna_nick_pos < grna_lenassert primer_binding_len < grna_nick_pos ### Find gRNAs##iupac_nt = {'A': list('A'),'C': list('C'),'G': list('G'),'T': list('T'),'Y': list('CT'),'R': list('AG'),'W': list('AT'),'S': list('GC'),'K': list('TG'),'M': list('AC'),'D': list('AGT'),'V': list('ACG'),'H': list('ACT'),'B': list('CGT'),'N': list('ACGT'),}def match(template, dna):if len(dna) != len(template):return Falsefor char, t in zip(dna, template):if char not in iupac_nt[t]:return Falsereturn Truedef pam_match(seq, grna_pos1, pam):flag, stats = None, dict()cand_pam = seq[grna_pos1 + grna_len : grna_pos1 + grna_len + len(pam)]if match(pam, cand_pam):flag = Truestats['Designed gRNA (NGG orientation)'] = seq[grna_pos1 : grna_pos1 + grna_len]stats['PAM'] = cand_pamstats['gRNA pos1 within sequence'] = grna_pos1return flag, stats def find_grnas(seq, alt_start, alt_len, ref_allele, path_idx, orient):min_grna_pos1 = alt_start - grna_nick_pos - max_dist_nick_to_editmax_grna_pos1 = alt_start - grna_nick_pos '''gRNA nick site must be on the 5' side of the edit. Limit up to 10 nt away and consider gRNAs on both strands''' all_grnas = defaultdict(list)for grna_pos1 in range(min_grna_pos1, max_grna_pos1 + 1): for castype in castypes:pam = castypes[castype]flag, grna_details = pam_match(seq, grna_pos1, pam) if flag:for key in grna_details:all_grnas[key].append(grna_details[key])all_grnas['Cas type'].append(castype) grna = grna_details['Designed gRNA (NGG orientation)'] grna_pos1 = grna_details['gRNA pos1 within sequence'] primer_binding = grna[grna_nick_pos - primer_binding_len : grna_nick_pos]edit_template = seq[grna_pos1 + grna_nick_pos : path_idx] + ref_allelehomology_arm = seq[path_idx + alt_len : path_idx + alt_len + homology_arm_len] grna_extension = primer_binding + edit_template + homology_armall_grnas['Designed primerbinder'].append(primer_binding)all_grnas['Designed edittemplate'].append(edit_template)all_grnas['Designed homology arm'].append(homology_arm) all_grnas['Designed gRNAextension'].append(grna_extension)all_grnas['Designed orientation'].append(orient) all_grnas['Designed gRNA full (NGGorientation)'].append(grna + grna_hairpin +compbio.reverse_complement(grna_extension) + terminator) return all_grnas #####def process_row(row):'''Find gRNAs in sequence at a single row.''' dis_seq = row['Sequence - alternate']ref_seq = row['Sequence - reference']path_start = row['buffer_length_bp'] # [, ) intervalalt_len = len(row['AlternateAllele'])path_end = path_start + alt_len fwd_grnas = find_grnas(dis_seq,path_start,alt_len,row['ReferenceAllele'],path_start,'+') rev_grnas = find_grnas(compbio.reverse_complement(dis_seq),len(dis_seq) - path_end,alt_len,compbio.reverse_complement(row['ReferenceAllele']), path_start,'-') fwd_df = pd.DataFrame(fwd_grnas)rev_df = pd.DataFrame(rev_grnas)df = fwd_df.append(rev_df, ignore_index = True)for col in row.index:df[col] = row[col] return df def process_df():df = pd.read_csv(inp_dir + f'clinvar_{SPLIT}.csv', index_col = 0) mdf = pd.DataFrame()timer = util.Timer(total = len(df))for idx, row in df.iterrows():d = process_row(row)mdf = mdf.append(d, ignore_index = True)timer.update() mdf.to_csv(out_dir + f'clinvar_{SPLIT}.csv')return ### qsub##def gen_qsubs():# Generate qsub shell scripts and commands for easyparallelizationprint('Generating qsub scripts...')qsubs_dir = _config.QSUBS_DIR + NAME + ' / 'util.ensure_dir_exists(qsubs_dir)qsub_commands = [] num_scripts = 0for idx in range(0, 60):command = 'python %s.py %s' % (NAME, idx)script_id = NAME.split('_')[0] # Write shell scriptssh_fn = qsubs_dir + 'q_%s_%s.sh' % (script_id, idx)with open(sh_fn, 'w') as f:f.write('#! / bin / bash\n%s\n' % (command))num_scripts += 1# Write qsub commandsqsub_commands.append('qsub -V -lh_rt=12:00:00,h_vmem=1G,os=RedHat7 -wd %s %s &' %(_config.SRC_DIR, sh_fn)) # Save commandscommands_fn = qsubs_dir + '_commands.sh'with open(commands_fn, 'w') as f:f.write('\n'.join(qsub_commands)) subprocess.check_output('chmod +x %s' % (commands_fn), shell = True) print('Wrote %s shell scripts to %s' % (num_scripts,qsubs_dir))return ### Main##@util.time_decdef main(argv):print(NAME) # Function callsglobal SPLITSPLIT = int(argv[0]) process_df() return if __name__ == '__main__':if len(sys.argv) > 1:main(sys.argv[1:])else:gen_qsubs()

[0317] The exemplary sequence listings submitted herewith were generated using the techniques described herein using the ClinVar database for the input alleles and corresponding output alleles. The entries in the ClinVar database were first filtered to germline mutations annotated as pathogenic or likely pathogenic. For these examples, Cas9-NG and SpCas9 were used to identify compatible mutations. Of the filtered mutations, approximately 72,020 unique ClinVar mutations were identified as compatible with prime editing with Cas9-NG, and approximately63,496 unique ClinVar mutations were identified as compatible with prime editing with SpCas9 with an NGG PAM. It should be appreciated that other and / or additional mutations could be correctable if using a prime editor containing a different Cas9 variant with different PAM compatibility.

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

[0319] In various other embodiments, the algorith was used to design PEgRNA against the ClinVar database using the herein disclosed algorithm are included in the Sequence Listing, which forms a part of this specification. The Sequence Listing includes complete PEgRNA sequences of SEQ ID NOs: 1-135514 and 813085-880462. Each of these complete PEgRNA are each comprised 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 PEgRNA comprises a gRNA core, for example, as defined by SEQ ID NOs: 1361579-1361580. The extension arms of SEQ ID NOs: 271029– 406542 and 947841-1015218 are further each comprised of a primer binding site (SEQ ID NOs.: 406543– 542056 and 1015219-1082596), an edit template (SEQ ID NOs.: 542057– 677570 and 1082597-1149974), and a homology arm (SEQ ID NOs.: 677571– 813084 and 1149975-1217352). The PEgRNA optionally may comprise a 5′ end modifier region and / or a 3′ end modifier region. The PEgRNA may also comprise a reverse transcription termination signal (e.g., SEQ ID NOs: 1361560-1361566) at the 3′ of the PEgRNA. The application embraces the design and use of all of these sequences.

[0320] The mutations were classified into four classes of clinical significance using minor allele frequency, number of submitters, whether or not submitters conflicted in their interpretations, and whether or not the mutation was reviewed by an expert panel. Among the 63,496 SpCas9- compatible mutations: 4,627 mutations were identified at the most significant level (four);13,943 mutations were identified at significance levels three or four; and 44,385 mutations were identified at significance levels two, three, or four.

[0321] The provided sequence listings enumerate a single PEgRNA per unique mutation, selected as the PEgRNA with the shortest distance between the nick and the edit. The PEgRNA were designed with homology arm length of 13 nt, a primer binding site length of 13 nt, a gRNA nick position at 17 nt, and a gRNA length of 20 nt. Protospacers with nick sites farther than 20 ntto the edit were disregarded. The gRNA backbone sequence used wasGTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 1361579). The terminator sequence used was TTTTTTGTTTT (SEQ ID NO: 1361581).

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

[0323] An illustrative implementation of a computer system 3400 that may be used to perform any of the aspects of the techniques and embodiments 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 writing data to and reading data from the memory 3420 and the non-volatile storage device 3430 in any suitable manner, as the aspects of the invention described herein are not limited in this respect. To perform functionality and / or techniques described herein, the processor 3410 may execute one or more instructions stored in one or more computer-readable storage media (e.g., the memory 3420, storage media, etc.), which may serve as non-transitory computer-readable storage media storing instructions for execution by the processor 3410.

[0324] In connection with techniques described herein, code used to, for example, to determine extended gRNA structures 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 techniques for planning an exercise as described herein. Any other software, programs or instructions described herein may also be stored and executed by computer system 3400. It will be appreciated that computer code may be applied to any aspects of methods and techniques described herein. For example, computer code may be applied to interact with an operating system to determine extended gRNA structures through conventional operating system processes.

[0325] The various methods or processes outlined herein may be coded as software that is executable on one or more processors that employ any one of a variety of operating systems or platforms. Additionally, such software may be written using any of numerous suitableprogramming languages and / or programming or scripting tools, and also may be compiled as executable machine language code or intermediate code that is executed on a virtual machine or a suitable framework.

[0326] In this respect, various inventive concepts may be embodied as at least one non-transitory computer readable storage medium (e.g., a computer memory, one or more floppy discs, compact discs, optical discs, magnetic tapes, flash memories, circuit configurations in FieldProgrammable Gate Arrays or other semiconductor devices, etc.) encoded with one or more programs that, when executed on one or more computers or other processors, implement the various embodiments of the present invention. The non-transitory computer-readable medium or media may be transportable, such that the program or programs stored thereon may be loaded onto any computer resource to implement 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 can be employed to program a computer or other processor to implement various aspects ofembodiments as discussed above. Additionally, it should be appreciated that according to one aspect, one or more computer programs that when executed perform methods of the present invention need not reside on a single computer or processor, but may be distributed in a modular fashion among different computers or processors to implement 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] Also, data structures may be stored in non-transitory computer-readable storage media in any suitable form. Data structures may have fields that are related through location in the data structure. Such relationships may likewise be achieved by assigning storage for the fields with locations in a non-transitory computer-readable medium that convey relationship between the fields. However, any suitable mechanism may be used to establish relationships amonginformation in fields of a data structure, including through the use of pointers, tags or other mechanisms that establish relationships among data elements.

[0330] Various inventive concepts may be embodied as one or more methods, of which examples have been provided. The acts performed as part of a method may be ordered in any suitable way. Accordingly, embodiments may be constructed in which acts are performed in an order different than illustrated, which may include performing some acts simultaneously, even though shown as sequential acts in illustrative embodiments.III. Prime editors for use with therapeutic PEgRNA

[0331] The therapeutic PEgRNA designed in accordance with the herein disclosed algorithm can be used to conduct prime editing when in complex with a prime editor. Prime editors comprise a napDNAbp fused with a polymerase (e.g., a reverse transcriptase) (or one which is provided in trans), optionally where the two domains are joined by linkers and further may comprise one or more NLS. These aspects are further described, as follows.A. napDNAbp

[0332] The prime editors described herein may comprise a nucleic acid programmable DNA binding protein (napDNAbp).

[0333] In one aspect, a napDNAbp can be associated with or complexed with at least one guide nucleic acid (e.g., guide RNA or a PEgRNA), which localizes the napDNAbp to a DNA sequence that comprises a DNA strand (i.e., a target strand) that is complementary to the guide nucleic acid, or a portion thereof (e.g., the spacer of a guide RNA which anneals to the protospacer of the DNA target). In other words, the guide nucleic-acid“programs” the napDNAbp (e.g., Cas9 or equivalent) to localize and bind to complementary sequence of the protospacer in the DNA.

[0334] Any suitable napDNAbp may be used in the prime editors described herein. In various embodiments, the napDNAbp may be any Class 2 CRISPR-Cas system, including any type II, type V, or type VI CRISPR-Cas enzyme. Given the rapid development of CRISPR-Cas as a tool for genome editing, there have been constant developments in the nomenclature used to describe and / or identify CRISPR-Cas enzymes, such as Cas9 and Cas9 orthologs. This application references CRISPR-Cas enzymes with nomenclature that may be old and / or new. The skilled person will be able to identify the specific CRISPR-Cas enzyme being referenced in this Application based on the nomenclature that is used, whether it is old (i.e.,“legacy”) or newnomenclature. CRISPR-Cas nomenclature is extensively discussed in Makarova et al., “Classification and Nomenclature of CRISPR-Cas Systems: Where from Here?,” The CRISPR Journal, Vol.1. No.5, 2018, the entire contents of which are incorporated herein by reference. The particular CRISPR-Cas nomenclature used in any given instance in this Application is not limiting in any way and the skilled person will be able to identify which CRISPR-Cas enzyme is being referenced.

[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 variants thereof, may be used with the prime editors described herein:*See Makarova et al., The CRISPR Journal, Vol. 1, No. 5, 2018.

[0336] Without being bound by theory, the mechanism of action of certain napDNAbp contemplated herein includes the step of forming an R-loop whereby the napDNAbp induces the unwinding of a double-strand DNA target, thereby separating the strands in the region bound by the napDNAbp. The guide RNA spacer then hybridizes to the“target strand” at the protospacer sequence. This displaces a“non-target strand” that is complementary to the target strand, which forms the single strand region of the R-loop. In some embodiments, the napDNAbp includes one or more nuclease activities, which then cut the DNA leaving various types of lesions. Forexample, the napDNAbp may comprises a nuclease activity that cuts the non-target strand at a first location, and / or cuts the target strand at a second location. Depending on the nuclease activity, the target DNA can be cut to form a“double-stranded break” whereby both strands are cut. In other embodiments, the target DNA can be cut at only a single site, i.e., the DNA is “nicked” on one strand. Exemplary napDNAbp with different nuclease activities include“Cas9 nickase” (“nCas9”) and a deactivated Cas9 having no nuclease activities (“dead Cas9” or “dCas9”).

[0337] The below description of various napDNAbps which can be used in connection with the presently disclose prime editors is not meant to be limiting in any way. The prime editors may comprise the canonical SpCas9, or any ortholog Cas9 protein, or any variant Cas9 protein— including any naturally occurring variant, mutant, or otherwise engineered version of Cas9—that is known or which can be made or evolved through a directed evolutionary or otherwise mutagenic process. In various embodiments, the Cas9 or Cas9 variants have a nickase activity, i.e., only cleave of strand of the target DNA sequence. In other embodiments, the Cas9 or Cas9 variants have inactive nucleases, i.e., are“dead” Cas9 proteins. Other variant Cas9 proteins that may be used are those having a smaller molecular weight than the canonical SpCas9 (e.g., for easier delivery) or having modified or rearranged primary amino acid structure (e.g., the circular permutant formats).

[0338] The prime editors described herein may also comprise Cas9 equivalents, including Cas12a (Cpf1) and Cas12b1 proteins which are the result of convergent evolution. The napDNAbps used herein (e.g., SpCas9, Cas9 variant, or Cas9 equivalents) may also may also contain various modifications that alter / enhance their PAM specificities. Lastly, the application contemplates any Cas9, Cas9 variant, or Cas9 equivalent which has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.9% sequence identity to a reference Cas9 sequence, such as a references SpCas9 canonical sequence or a reference Cas9 equivalent (e.g., Cas12a (Cpf1)).

[0339] The napDNAbp can be a CRISPR (clustered regularly interspaced short palindromic repeat)-associated nuclease. As outlined above, CRISPR is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements and conjugative plasmids). CRISPR clusters contain spacers, sequences complementary toantecedent mobile elements, and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems correct processing of pre-crRNA requires a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc) and a Cas9 protein. The tracrRNA serves as a guide for ribonuclease 3-aided processing of pre- crRNA. Subsequently, Cas9 / crRNA / tracrRNA endonucleolytically cleaves linear or circular dsDNA target complementary to the spacer. The target strand not complementary to crRNA is first cut endonucleolytically, then trimmed 3´-5′ exonucleolytically. In nature, DNA-binding and cleavage typically requires protein and both RNAs. However, single guide RNAs (“sgRNA”, or simply“gRNA”) can be engineered so as to incorporate aspects of both the crRNA and tracrRNA into a single RNA species. See, e.g., Jinek M. et al., Science 337:816-821(2012), the entire contents of which is hereby incorporated by reference.

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

[0341] As used herein, the term“Cas protein” refers to a full-length Cas protein obtained from nature, a recombinant Cas protein having a sequences that differs from a naturally occurring Cas protein, or any fragment of a Cas protein that nevertheless retains all or a significant amount of the requisite basic functions needed for the disclosed methods, i.e., (i) possession of nucleic-acid programmable binding of the Cas protein to a target DNA, and (ii) ability to nick the target DNA sequence on one strand. The Cas proteins contemplated herein embrace CRISPR Cas 9 proteins, as well as Cas9 equivalents, variants (e.g., Cas9 nickase (nCas9) or nuclease inactiveCas9 (dCas9)) homologs, orthologs, or paralogs, whether naturally occurring or non-naturally occurring (e.g., engineered or recombinant), and may include a Cas9 equivalent from any Class 2 CRISPR system (e.g., type II, V, VI), including Cas12a (Cpf1), Cas12e (CasX), Cas12b1 (C2c1), Cas12b2, Cas12c (C2c3), C2c4, C2c8, C2c5, C2c10, C2c9 Cas13a (C2c2), Cas13d, Cas13c (C2c7), Cas13b (C2c6), and Cas13b. Further Cas-equivalents are described inMakarova et al.,“C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector,” Science 2016; 353(6299) and Makarova et al.,“Classification andNomenclature 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 terms“Cas9” or“Cas9 nuclease” or“Cas9 moiety” or“Cas9 domain” embrace any naturally occurring Cas9 from any organism, any naturally-occurring Cas9 equivalent or functional fragment thereof, any Cas9 homolog, ortholog, or paralog from any organism, and any mutant or variant of a Cas9, naturally-occurring or engineered. The term Cas9 is not meant to be particularly limiting and may be referred to as a“Cas9 or equivalent.” Exemplary Cas9 proteins are further described herein and / or are described in the art and are incorporated herein by reference. The present disclosure is unlimited with regard to the particular Cas9 that is employed in the prime editor (PE) of the invention.

[0343] As noted herein, Cas9 nuclease sequences and structures are well known to those of skill in the art (see, e.g.,“Complete genome sequence of an M1 strain of Streptococcus pyogenes.” Ferretti et al., J.J., McShan W.M., Ajdic D.J., Savic D.J., Savic G., Lyon K., Primeaux C., Sezate S., Suvorov A.N., Kenton S., Lai H.S., Lin S.P., Qian Y., Jia H.G., Najar F.Z., Ren Q., Zhu H., Song L., White J., Yuan X., Clifton S.W., Roe B.A., McLaughlin R.E., Proc. Natl. Acad. Sci. U.S.A.98:4658-4663(2001);“CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.” Deltcheva E., Chylinski K., Sharma C.M., Gonzales K., Chao Y., Pirzada Z.A., Eckert M.R., Vogel J., Charpentier E., Nature 471:602-607(2011); and“A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.” Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J.A., Charpentier E. Science 337:816-821(2012), the entire contents of each of which are incorporated herein by reference).

[0344] Examples of Cas9 and Cas9 equivalents are provided as follows; however, these specific examples are not meant to be limiting. The primer editor of the present disclosure may use any suitable napDNAbp, including any suitable Cas9 or Cas9 equivalent.(i) Wild type canonical SpCas9

[0345] In one embodiment, the primer editor constructs described herein may comprise the “canonical SpCas9” nuclease from S. pyogenes, which has been widely used as a tool for genome engineering and is categorized as the type II subgroup of enzymes of the Class 2 CRISPR-Cas systems. This Cas9 protein is a large, multi-domain protein containing two distinct nuclease domains. Point mutations can be introduced into Cas9 to abolish one or both nuclease activities, resulting in a nickase Cas9 (nCas9) or dead Cas9 (dCas9), respectively, that still retains its ability to bind DNA in a sgRNA-programmed manner. In principle, when fused to another protein or domain, Cas9 or variant thereof (e.g., nCas9) can target that protein to virtually any DNA sequence simply by co-expression with an appropriate sgRNA. As used herein, the canonical SpCas9 protein refers to the wild type protein from Streptococcus pyogenes having the following amino acid sequence:

[0346] The prime editors described herein may include canonical SpCas9, or any variant thereof having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with a wild type Cas9 sequence provided above. These variants may include SpCas9 variants containing one or more mutations, including any known mutation reported with the SwissProt Accession No. Q99ZW2 entry, which include:

[0347] Other wild type SpCas9 sequences that may be used in the present disclosure, include:

[0348] The prime editors described herein may include any of the above SpCas9 sequences, or any variant thereof having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto.(ii) Wild type Cas9 orthologs

[0349] In other embodiments, the Cas9 protein can be a wild type Cas9 ortholog from another bacterial species different from the canonical Cas9 from S. pyogenes. For example, the following Cas9 orthologs can be used in connection with the prime editor constructs described inthis specification. In addition, any variant Cas9 orthologs having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to any of the below orthologs may also be used with the present prime editors.

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

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

[0352] In some embodiments, the prime editors described herein may include a dead Cas9, e.g., dead SpCas9, which has no nuclease activity due to one or more mutations that inactive both nuclease domains of Cas9, namely the RuvC domain (which cleaves the non-protospacer DNA strand) and HNH domain (which cleaves the protospacer DNA strand). The nuclease inactivation may be due to one or mutations that result in one or more substitutions and / or deletions in the amino acid sequence of the encoded protein, or any variants thereof having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto.

[0353] As used herein, the term“dCas9” refers to a nuclease-inactive Cas9 or nuclease-dead Cas9, or a functional fragment thereof, and embraces any naturally occurring dCas9 from any organism, any naturally-occurring dCas9 equivalent or functional fragment thereof, any dCas9 homolog, ortholog, or paralog from any organism, and any mutant or variant of a dCas9, naturally-occurring or engineered. The term dCas9 is not meant to be particularly limiting and may be referred to as a“dCas9 or equivalent.” Exemplary dCas9 proteins and method for making dCas9 proteins are further described herein and / or are described in the art and are incorporated herein by reference.

[0354] In other embodiments, dCas9 corresponds to, or comprises in part or in whole, a Cas9 amino acid sequence having one or more mutations that inactivate the Cas9 nuclease activity. In other embodiments, Cas9 variants having mutations other than D10A and H840A are provided which may result in the full or partial inactivate of the endogenous Cas9 nuclease activity (e.g., nCas9 or dCas9, respectively). Such mutations, by way of example, include other amino acid substitutions at D10 and H820, or other substitutions within the nuclease domains of Cas9 (e.g., substitutions in the HNH nuclease subdomain and / or the RuvC1 subdomain) with reference to a wild type sequence such as Cas9 from Streptococcus pyogenes (NCBI Reference Sequence: NC_017053.1 (SEQ ID NO: 1361424)). In some embodiments, variants or homologues of Cas9 (e.g., variants of Cas9 from Streptococcus pyogenes (NCBI Reference Sequence: NC_017053.1 (SEQ ID NO: 1361424))) are provided which are at least about 70% identical, at least about 80%identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to NCBI Reference Sequence: NC_017053.1 (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 which are shorter, or longer thanNC_017053.1 (SEQ ID NO: 1361424) by about 5 amino acids, by about 10 amino acids, by about 15 amino acids, by about 20 amino acids, by about 25 amino acids, by about 30 amino acids, by about 40 amino acids, by about 50 amino acids, by about 75 amino acids, by about 100 amino acids or more.

[0355] In one embodiment, the dead Cas9 may be based on the canonical SpCas9 sequence of Q99ZW2 and may have the following sequence, which comprises a D10A and an H810A substitutions (underlined and bolded), or a variant of SEQ ID NO: 1361444 having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto:(iv) Cas9 nickase variant

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

[0357] In various embodiments, the Cas9 nickase can having a mutation in the RuvC nuclease domain and have one of the following amino acid sequences, or a variant thereof having an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto.

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

[0359] In various embodiments, the Cas9 nickase can have a mutation in the HNH nuclease domain and have one of the following amino acid sequences, or a variant thereof, having an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto.(v) Other Cas9 variants

[0360] Besides dead Cas9 and Cas9 nickase variants, the Cas9 proteins used herein may also include other“Cas9 variants” having at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to any reference Cas9 protein, including any wild type Cas9, or mutant Cas9 (e.g., a dead Cas9 or Cas9 nickase), or fragment Cas9, or circular permutant Cas9, or other variant of Cas9 disclosed herein or known in the art. In some embodiments, a Cas9 variant may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acid changes compared to a reference Cas9. In some embodiments, the Cas9 variant comprises a fragment of a reference Cas9 (e.g., a gRNA binding domain or a DNA-cleavage domain), such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of wild type Cas9. In some embodiments, the fragment is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid length of a corresponding wild type Cas9 (e.g., SEQ ID NO: 1361421).

[0361] In some embodiments, the disclosure also may utilize Cas9 fragments which retain their functionality and which are fragments of any herein disclosed Cas9 protein. In someembodiments, the Cas9 fragment is at least 100 amino acids in length. In some embodiments, the fragment is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, or at least 1300 amino acids in length.

[0362] In various embodiments, the prime editors disclosed herein may comprise one of the Cas9 variants described as follows, or a Cas9 variant thereof having at least about 70% identical, at least about 80% identical, at least about 90% identical, at least ...

Claims

CLAIMS What is claimed is:

1. A guide RNA comprising a spacer, a gRNA core, and an extension arm, wherein the guide RNA comprises a sequence selected from the group consisting of SEQ ID NOs: 1- 135514, or a sequence having at least 90% sequence identity with any of SEQ ID NOs: 1- 135514.

2. A guide RNA comprising a spacer, a gRNA core, and an extension arm, wherein the spacer comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 135515– 271028, or a spacer having a nucleotide sequence having at least 90% sequence identity with any of SEQ ID NOs: 135515– 271028.

3. A guide RNA comprising a spacer, a gRNA core, and an extension arm, wherein the extension arm has a nucleotide sequence selected from the group consisting of SEQ ID NOs: 271029– 406542, or an extension arm having a nucleotide sequence having at least 90% sequence identity with any of SEQ ID NOs: 271029– 406542.

4. A guide RNA comprising a spacer, a gRNA core, and an extension arm, wherein the extension arm comprises (i) a primer binding site, (ii) an edit template, and (iii) a homology arm.

5. A guide RNA comprising a spacer, a gRNA core, and an extension arm, wherein the extension arm comprises an primer binding site having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 406543– 542056, or a primer binding site having a nucleotide sequence that is at least 90% sequence identical to any of SEQ ID NOs: 406543– 542056.

6. A guide RNA comprising a spacer, a gRNA core, and an extension arm, wherein the extension arm comprises an edit template comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 542057– 677570, or an edit template havinga nucleotide sequence that is at least 90% identical to any of SEQ ID NOs: 542057– 677570.

7. A guide RNA comprising a spacer, a gRNA core, and an extension arm, wherein the extension arm comprises a homology arm having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 677571– 813084, or a homology arm having a nucleotide sequence that is at least 90% identical to any of SEQ ID NOs: 677571– 813084.

8. A guide RNA comprising:(i) a spacer having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 135515– 271028, or a spacer having a nucleotide sequence having at least 90% sequence identity with any of SEQ ID NOs: 135515– 271028, and (ii) an extension arm selected from the group consisting of SEQ ID NOs: 271029– 406542, or an extension arm having a nucleotide sequence having least 90% sequence identity with SEQ ID NOs: 271029– 406542.

9. A guide RNA comprising:(i) a spacer having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 135515– 271028, or a spacer having a nucleotide sequence that is at least 90% identical to any of SEQ ID NOs: 135515– 271028, and (ii) a primer binding site selected from the group consisting of SEQ ID NOs: 406543 – 542056, or a primer binding site having a nucleotide sequence that is at least 90% identical to any of SEQ ID NOs: 406543– 542056.

10. A guide RNA comprising:(i) a spacer having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 135515– 271028, or a spacer having a nucleotide sequence having at least 90% sequence identity with any of SEQ ID NOs: 135515– 271028, and (ii) an edit template having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 542057– 677570, or an edit template having a nucleotide sequence that is at least 90% identical to any of SEQ ID NOs: 542057– 677570.

11. A guide RNA comprising:(i) a spacer having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 135515– 271028, or a spacer having a nucleotide sequence that is at least 90% identical to any of SEQ ID NOs: 135515– 271028, and (ii) a homology arm having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 677571– 813084, or a spacer having a nucleotide sequence that is at least 90% identical to any of SEQ ID NOs: 677571– 813084.

12. The guide RNA of any of the above claims further comprising an termination signal of SEQ ID NO: 813086, or a termination signal having at least 90% sequence identity with SEQ ID NO: 813086.

13. The guide RNA of any of the above claims further comprising a 5′ end modifier region comprising a hairpin sequence, a stem / loop sequence, or a toeloop sequence.

14. The guide RNA of any of the above claims further comprising a 3′ end modifier region comprising a hairpin sequence, a stem / loop sequence, or a toeloop sequence.

15. The guide RNA of any of the above claims further comprising a gRNA core comprising SEQ ID NO: 813085, or a gRNA core having at least 90% sequence identity with SEQ ID NO: 813085.

16. The guide RNA of any of the above claims, wherein the guide RNA is capable of binding to a napDNAbp suitable for prime editing and directing the napDNAbp to a target DNA sequence.

17. The guide RNA of claim 16, wherein the target nucleic acid sequence comprises a target strand (or PAM strand) and a complementary non-target strand (or non-PAM strand), wherein the spacer of the guide RNA hybridizes to the complementary non-target strand (non-PAM strand) to form an RNA-DNA hybrid and an R-loop.

18. The guide RNA of any of the above claims, wherein the primer binding site is between approximately 8 and approximately 20 nucleotides in length.

19. The guide RNA of any of the above claims, wherein the primer binding site is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length.

20. The guide RNA of any of the above claims, wherein the primer binding site is 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 18nucleotides, at least 19 nucleotides, or at least 20 nucleotides in length.

21. The guide RNA of any of the above claims, wherein the homology arm is complementary to a strand of the target DNA.

22. The guide RNA of any of the above claims, wherein the extension arm is betweenapproximately 7 and approximately 500 nucleotides in length.

23. The guide RNA of any of the above claims, wherein the extension arm is at least 7nucleotides, 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 18nucleotides, 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 29nucleotides, 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 40nucleotides, or at least 100 nucleotides in length.

24. The guide RNA of any of the above claims, wherein the edit template is at least 1nucleotides, 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 20nucleotides, 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 31nucleotides, 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.

25. The guide RNA of any of the above claims, wherein the homology arm is at least 1nucleotides, 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 20nucleotides, 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.

26. The guide RNA of any of the above claims, wherein the edit template and homology arm can be used by a reverse transcriptase as a template sequence for the synthesis of a corresponding single-strand DNA flap having a 3′ end, wherein the DNA flap is complementary to a strand of the endogenous target DNA sequence adjacent to a nick site, and wherein the single-strand DNA flap comprises a nucleotide change encoded by the edit template.

27. The guide RNA of claim 26, wherein the single-strand DNA flap displaces anendogenous single-strand DNA having a 5´ end in the target DNA sequence that has been nicked.

28. The guide RNA of claim 27, wherein the endogenous single-strand DNA having the free 5´ end is excised by the cell.

29. The guide RNA of claim 27, whereby cellular repair of the single-strand DNA flapresults in installation of the nucleotide change, thereby forming a desired product.

30. The guide RNA of claim 29, wherein the desired nucleotide change is an insertion.

31. The guide RNA of claim 30, wherein in the insertion 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 21nucleotides, 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 32nucleotides, 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.

32. The guide RNA of claim 30, wherein the insertion is a sequence encoding a polypeptide.

33. A prime editing complex comprising a napDNAbp, a reverse transcriptase, and any one of the guide RNAs of claims 1-32.

34. The prime editing complex of claim 33, wherein the napDNAbp and the reversetranscriptase are formed as a fusion protein.

35. The prime editing complex of claim 33, wherein the napDNAbp is a Cas9.

36. The prime editing complex of claim 35, wherein the Cas9 is selected from the group consisting of Cas9 nickases or variants thereof.

37. The prime edting complex of claim 35, wherein the Cas9 has an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-135514.

38. The prime editing complex of claim 34, wherein the fusion protein has an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-135514.

39. The prime editing complex of claim 34, wherein the fusion protein comprises a linker joining the napDNAbp and reverse transcriptase.

40. The prime editing complex of claim 39, wherein the linker has an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-135514.

41. One or more polynucleotides encoding the prime editing complex of any of claims 33-40.

42. A vector comprising the polynucleotide of claim 41 and one or more promoters that drive the expression of the guide RNA and the fusion protein of the prime editing complex.

43. A cell comprising the a vector of claim 42.

44. A cell comprising a prime editing complex of any of claims 33-40.

45. A pharmaceutical composition comprising: (i) a guide RNA of any of claims 1-32, a prime editing complex of claims 33-40, a polynucleotide of claim 41, or a vector of claim 42; and (ii) a pharmaceutically acceptable excipient.

46. A method for installing a nucleotide change in a nucleic acid sequence, the methodcomprising: contacting the nucleic acid sequence with a complex comprising a fusion protein and a guide RNA of any of claims 1-32 or any of claims 56-81, wherein the fusion protein comprises a napDNAbp and a polymerase, and wherein the guide RNA comprises a spacer, gRNA core, and an extension arm that comprises an edit template encoding a nucleotide change; thereby(i) nicking the double-stranded DNA sequence on the target strand (or the PAMstrand), and generating a free single-strand DNA having a 3′ end;(ii) hybridizing the 3′ end of the free single-strand DNA to the guide RNA at the primer binding site, thereby priming the polymerase;(iii) polymerizing a strand of DNA from the 3′ end, thereby generating a single-strand DNA flap comprising the nucleotide change; and(iv) replacing the endogenous DNA strand immediately adjacent downstream of the cut site on the target strand (or PAM strand) with the single-strand DNA flap,thereby installing the desired nucleotide change in the double-stranded DNA sequence.

47. The method of claim 46, wherein the nucleotide change is a single nucleotidesubstitution, a deletion, an insertion, or a combination thereof.

48. The method of claim 46, wherein the single nucleotide substitution is a transition or a transversion.

49. The method of claim 46, wherein the nucleotide change is (1) a G to T substitution, (2) a G to A substitution, (3) a G to C substitution, (4) a T to G substitution, (5) a T to A substitution, (6) a T to C substitution, (7) a C to G substitution, (8) a C to T substitution, (9) a C to A substitution, (10) an A to T substitution, (11) an A to G substitution, or (12) an A to C substitution.

50. The method of claim 46, wherein the nucleoid change converts (1) a G:C basepair to a T:A basepair, (2) a G:C basepair to an A:T basepair, (3) a G:C basepair to C:G basepair, (4) a T:A basepair to a G:C basepair, (5) a T:A basepair to an A:T basepair, (6) a T:A basepair to a C:G basepair, (7) a C:G basepair to a G:C basepair, (8) a C:G basepair to a T:A basepair, (9) a C:G basepair to an A:T basepair, (10) an A:T basepair to a T:A basepair, (11) an A:T basepair to a G:C basepair, or (12) an A:T basepair to a C:G basepair.

51. The method of claim 46, wherein the nucleotide change is an insertion or deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides.

52. The method of claim 46, wherein the nucleotide change is an insertion of a polypeptide- encoding sequence.

53. The method of claim 46, wherein the nucleotide change corrects a disease-associated gene.

54. The method of claim 46, wherein the disease-associated gene is associated with amonogentic disorder selected from the group consisting of: Adenosine Deaminase (ADA)Deficiency; Alpha-1 Antitrypsin Deficiency; Cystic Fibrosis; Duchenne Muscular Dystrophy; Galactosemia; Hemochromatosis; Huntington’s Disease; Maple Syrup Urine Disease; Marfan Syndrome; Neurofibromatosis Type 1; Pachyonychia Congenita;Phenylkeotnuria; Severe Combined Immunodeficiency; Sickle Cell Disease; Smith- Lemli-Opitz Syndrome; and Tay-Sachs Disease.

55. The method of claim 46, wherein the disease-associated gene is associated with apolygenic disorder selected from the group consisting of: cardiac disease; high blood pressure; neurological disease; autoimmune disorder, arthritis; diabetes; cancer; and obesity.

56. A guide RNA for use in prime editing to correct a disease allele at an edit site in a target DNA sequence to form a healthy allele, said guide comprising a spacer, a gRNA core, and an extension arm, wherein the spacer is capable of binding to a ~20 nucleotide region within SEQ ID NOs: 1217353-1289387 or the complement strand thereof.

57. A guide RNA comprising a spacer, gRNA core, and an extension arm, wherein theextension arm comprises a DNA synthesis template and a primer binding site effective to conduct prime editing.

58. The guide RNA of claim 56, wherein the edit site in any of the nucleotide sequences of SEQ ID NOs: 1217353-1289387 begins at position 201 in the 5' to 3' orientation.

59. A guide RNA for prime editing comprising a spacer, a gRNA core, and an extension arm, wherein the extension arm comprises a primer binding site and a DNA synthesis template.

60. The guide RNA of claim 59, wherein the primer binding site has a nucleotide sequence selected from the group consisting of SEQ ID NOs: 406543– 542056 (primer binding site), or a nucleotide sequence that has at least 90% sequence identity with any of SEQ ID NOs: 406543– 542056.

61. The guide RNA of claim 59, wherein the DNA synthesis template comprises a nucleotide sequence of SEQ ID NOs: 542057– 677570 (edit template), or a nucleotide sequence that has at least 90% sequence identity with any of SEQ ID NOs: 542057– 677570.

62. The guide RNA of claim 59, wherein the DNA synthesis template comprises a nucleotide sequence of SEQ ID NOs: 677571– 813084 (homology arm), or a nucleotide sequence that has at least 90% sequence identity with any of SEQ ID NOs: 677571– 813084.

63. The guide RNA of claim 59, wherein the DNA synthesis template comprises an edit template and a homology arm, wherein the edit template comprises a nucleotide sequence of SEQ ID NOs: 542057– 677570, and the homology arm comprises a nucleotide sequence of SEQ ID NOs: 677571– 813084.

64. The guide RNA of any of claims 56-63 further comprising an termination signal of SEQ ID NO: 813086, or a termination signal having at least 90% sequence identity with SEQ ID NO: 813086.

65. The guide RNA of any of claims 56-64 further comprising a 5′ end modifier regioncomprising a hairpin sequence, stem / loop sequence, or a toeloop sequence.

66. The guide RNA of any of claims 56-65 further comprising a 3′ end modifier regioncomprising a hairpin sequence, stem / loop sequence, or a toeloop sequence.

67. The guide RNA of any of claims 56-66, further comprising a gRNA core comprising SEQ ID NO: 813085, or a gRNA core having at least 90% sequence identity with SEQ ID NO: 813085.

68. The guide RNA of any of claims 56-67, wherein the guide RNA is capable of binding to a napDNAbp suitable for prime editing and directing the napDNAbp to a target DNA sequence.

69. The guide RNA of claim 68, wherein the target nucleic acid sequence comprises a target strand (or PAM or edit strand) and a complementary non-target strand (or non-PAM or non-edit strand) wherein the spacer of the guide RNA hybridizes to the non-PAM strand to form an RNA-DNA hybrid and an R-loop.

70. The guide RNA of any of claims 56-69, wherein the primer binding site is between approximately 8 and approximately 20 nucleotides in length.

71. The guide RNA of any of claims 56-70, wherein the primer binding site is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length.

72. The guide RNA of any of claims 56-71, wherein the extension arm is at least 7nucleotides, 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 18nucleotides, 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 29nucleotides, 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 40nucleotides, at least 100 nucleotides in length.

73. The guide RNA of any of claims 56-72, wherein the primer binding site is at least 7nucleotides, 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 18nucleotides, at least 19 nucleotides, or at least 20 nucleotides in length.

74. The guide RNA of any of claims 56-73, wherein the DNA synthesis template is at least 1 nucleotides, 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 20nucleotides, 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 31nucleotides, at least 32 nucleotides, at least 33 nucleotides, at least 34 nucleotides, at least 35 nucleotides, at least 36 nucleotides, at least 37 nucleotides, at least 38 nucleotides, at least 39 nucleotides, at least 40 nucleotides, at least 100 nucleotides in length.

75. The guide RNA of any of claims 56-74, wherein the DNA synthesis template can be used by an RNA-dependent DNA polymerase (e.g., reverse transcriptase) as a template for the synthesis of a corresponding single-strand DNA flap having a 3′ end, wherein the DNA flap is complementary to a strand of the endogenous target DNA sequence adjacent to a nick site, and wherein the single-strand DNA flap comprises a desired nucleotide change encoded by the DNA synthesis template.

76. The guide RNA of claim 75, wherein the single-strand DNA flap displaces anendogenous single-strand DNA having a 5´ end in the target DNA sequence that has been nicked.

77. The guide RNA of claim 76, wherein the endogenous single-strand DNA having the free 5´ end is excised by the cell.

78. The guide RNA of claim 77, whereby cellular repair of the single-strand DNA flapresults in installation of the nucleotide change, thereby forming an edited DNA product.

79. The guide RNA of claim 78, wherein the nucleotide change is an insertion.

80. The guide RNA of claim 79, wherein in the insertion 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 21nucleotides, 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 32nucleotides, at least 33 nucleotides, at least 34 nucleotides, at least 35 nucleotides, at least36 nucleotides, at least 37 nucleotides, at least 38 nucleotides, at least 39 nucleotides, at least 40 nucleotides, at least 100 nucleotides in length.

81. The guide RNA of claim 79, wherein the insertion is a sequence encoding a polypeptide.

82. A prime editing complex comprising a napDNAbp, an RNA-dependent DNApolymerase, and any one of the guide RNA of claims 56-81.

83. The prime editing complex of claim 82, wherein the napDNAbp and the RNA-dependent DNA polymerase are formed as a fusion protein.

84. The prime editing complex of claim 82, wherein the napDNAbp is a Cas9.

85. The prime editing complex of claim 84, wherein the Cas9 is a Cas9 nickase or variant thereof.

86. The prime editing complex of claim 84, wherein the Cas9 has an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-135514.

87. The prime editing complex of claim 83, wherein the fusion protein has an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-135514.

88. The prime editing complex of claim 83, wherein the fusion protein comprises a linker joining the napDNAbp and RNA-dependent DNA polymerase.

89. The prime editing complex of claim 88, wherein the linker has an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-135514.

90. One or more polynucleotides encoding the prime editing complex of any of claims 82-89.

91. A vector comprising the polynucleotide of claim 90 and one or more promoters that drive the expression of the guide RNA and the fusion protein of the prime editing complex.

92. A cell comprising the vector of claim 91.

93. A cell comprising a prime editing complex of any of claims 82-89.

94. A pharmaceutical composition comprising: (i) a guide RNA of any of claims 56-81, a prime editing complex of claims 82-89, a polynucleotide of claim 90, or a vector of claim 91; and (ii) a pharmaceutically acceptable excipient.

95. A method for installing a nucleotide change in a nucleic acid sequence, the methodcomprising: contacting the nucleic acid sequence with a complex comprising a fusion protein and a guide RNA of any of claims 56-81, wherein the fusion protein comprises a napDNAbp and an RNA-dependent DNA polymerase, wherein the guide RNA comprises a spacer, gRNA core, and an extension arm that comprises a DNA synthesis template and primer binding site, said DNA synthesis template encoding a nucleotide change, and wherein the spacer is capable of annealing to the non-PAM strand proximal to an available PAM and protospacer; thereby(i) nicking the double-stranded DNA sequence on the PAM strand, therebygenerating a free single-strand DNA having a 3′ end;(ii) hybridizing the 3′ end of the free single-strand DNA to the guide RNA at the primer binding site, thereby priming the RNA-dependent DNA polymerase;(iii) polymerizing a strand of DNA from the 3′ end of DNA, coding from the DNA synthesis template, thereby generating a single-strand DNA flap extended from the 3′ end of the DNA, wherein the flap comprises the nucleotide change;(iv) replacing an endogenous DNA strand adjacent immediately downstream of the cut site on the PAM strand with the single-strand DNA flap, thereby installing the nucleotide change in the double-stranded DNA sequence.

96. The method of claim 95, wherein when step (v) is completed within a cell, the cell repairs the non-edited strand through cellular DNA repair and / or replication.

97. The method of claim 95, wherein the nucleotide change is a single nucleotidesubstitution, a deletion, an insertion, or a combination thereof.

98. The method of claim 97, wherein the single nucleotide substitution is a transition or a transversion.

99. The method of claim 97, wherein the single nucleotide substitution is (1) a G to T substitution, (2) a G to A substitution, (3) a G to C substitution, (4) a T to G substitution, (5) a T to A substitution, (6) a T to C substitution, (7) a C to G substitution, (8) a C to T substitution, (9) a C to A substitution, (10) an A to T substitution, (11) an A to G substitution, or (12) an A to C substitution.

100. The method of claim 97, wherein the single nucleotide substitution converts (1) a G:C basepair to a T:A basepair, (2) a G:C basepair to an A:T basepair, (3) a G:C basepair to C:G basepair, (4) a T:A basepair to a G:C basepair, (5) a T:A basepair to an A:T basepair, (6) a T:A basepair to a C:G basepair, (7) a C:G basepair to a G:C basepair, (8) a C:G basepair to a T:A basepair, (9) a C:G basepair to an A:T basepair, (10) an A:T basepair to a T:A basepair, (11) an A:T basepair to a G:C basepair, or (12) an A:T basepair to a C:G basepair.

101. The method of claim 97, wherein the nucleotide change is an insertion or deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides.

102. The method of claim 97, wherein the nucleotide change is an insertion of a polypeptide- encoding sequence.

103. The method of claim 97, wherein the nucleotide change corrects a disease-associated gene.

104. The method of claim 103 wherein the disease-associated gene is associated with amonogenetic disorder selected from the group consisting of: Adenosine Deaminase (ADA) Deficiency; Alpha-1 Antitrypsin Deficiency; Cystic Fibrosis; DuchenneMuscular Dystrophy; Galactosemia; Hemochromatosis; Huntington’s Disease; Maple Syrup Urine Disease; Marfan Syndrome; Neurofibromatosis Type 1; Pachyonychia Congenita; Phenylkeotnuria; Severe Combined Immunodeficiency; Sickle Cell Disease; Smith-Lemli-Opitz Syndrome; and Tay-Sachs Disease.

105. The method of claim 103, wherein the disease-associated gene is associated with a polygenic disorder selected from the group consisting of: heart disease; high blood pressure; Alzheimer’s disease; arthritis; diabetes; cancer; and obesity.

106. A guide RNA for use in prime editing to alter the nucleotide sequence of a target DNA molecule with an insertion, deletion, inversion, substitution, or combination thereof to produce a corresponding edited DNA molecule, wherein: (i) the guide RNA is capable of forming a complex with a fusion protein comprising a napDNAbp and a domain comprising an RNA-dependent DNA polymerase activity; (ii) the guide RNA comprises (a) a spacer that is capable of annealing to the non- PAM strand proximal to an available PAM and protospacer on the PAM strand on the target DNA molecule, and (b) a gRNA core; (iii) the guide RNA further comprises an extension arm at the 5ʹ or 3ʹ end of the guide RNA; (iv) the extension arm comprises (a) a primer binding site and (b) a DNA synthesis template, wherein the DNA synthesis template codes for a single-strand DNA flap that includes an edit to be integrated in place of the endogenous strand immediately downstream of the cut site on the PAM strand; (v) the target DNA molecule is selected from the group consisting of SEQ ID NOs:SEQ ID NOs: 1217353-1289387; and (vi) the corresponding edited DNA molecule is selected from the group consisting of SEQ ID NOs: 1289388-1361420.

107. The guide RNA of claim 106, wherein the target DNA molecule is a Clinvar variant sequence.

108. The guide RNA of claim 106, wherein the napDNAbp is Cas9, Cas12e, Cas12d, Cas12a, Cas12b1, Cas13a, Cas12c, or Argonaute, or a variant of Cas9, Cas12e, Cas12d, Cas12a, Cas12b1, Cas13a, Cas12c, or Argonaute.

109. The guide RNA of claim 106, wherein the napDNAbp domain comprises nickase activity.

110. The guide RNA of claim 106, wherein the napDNAbp is a Cas9 or variant thereof.

111. The guide RNA of claim 106, wherein the napDNAbp is a nuclease active Cas9, a nuclease inactive Cas9 (dCas9), or a Cas9 nickase (nCas9).

112. The guide RNA of claim 106, wherein the napDNAbp is Cas9 nickase (nCas9).

113. The guide RNA of claim 106, wherein the napDNAbp comprises the amino acid 114. The guide RNA of claim 106, wherein the napDNAbp is SpCas9 wild type or a variant thereof of any one of amino acid sequences 1361421-1361428, or an amino acid sequence having at least 80% sequence identity with any of SEQ ID NOs: 1361421-1361428.

115. The guide RNA of claim 106, wherein the napDNAbp is an SpCas9 ortholog of any one of amino acid sequences 1361429-1361442, or an amino acid sequence having at least 80% sequence identity with any of SEQ ID NOs: 1361429-1361442.

116. The guide RNA of claim 106, wherein the napDNAbp is any one of amino acid sequences 1361421-1361484, or an amino acid sequence having at least 80% sequence identity with any of SEQ ID NOs: 1361421-1361484.

117. The guide RNA of claim 106, wherein the domain comprising an RNA-dependent DNA polymerase activity is a reverse transcriptase.

118. The guide RNA of claim 117, wherein the reverse transcriptase is a naturally occurring wild type reverse transcriptase having an amino acid sequence of any one of SEQ ID NOs: 1361485-1361496, or an amino acid sequence having at least 80% sequence identity with any of SEQ ID NOs: 1361485-1361496.

119. The guide RNA of claim 117, wherein the reverse transcriptase is a variant reverse transcriptase having an amino acid sequence of any one of SEQ ID NOs: 1361497-1361514, or an amino acid sequence having at least 80% sequence identity with any of SEQ ID NOs:1361497-1361514.

120. The guide RNA of claim 106, wherein the fusion protein comprises an amino acid sequence of any one of SEQ ID NOs: 1361515-1361519, or an amino acid sequence having at least 80% sequence identity with any of SEQ ID NOs: 1361515-1361519.

121. The guide RNA of claim 106, wherein the fusion protein comprises an amino acid sequence of SEQ ID NO: 1361515 (PE1) or 1361516 (PE2), or an amino acid sequence having at least 80% sequence identity with any of SEQ ID NOs: 1361515 or 1361516.

122. The guide RNA of claim 106, wherein the available PAM sequence is a function of the napDNAbp used in step (i).

123. The guide RNA of claim 106, wherein the available PAM sequence is selected from the group consisting of: (a) 5'-NGG-3' (the canonical PAM sequence), (b) 5'-NNG-3', (c) 5'-NNA- 3', (d) 5'-NNC-3', (e) 5'-NNT-3', (f) 5'-NGT-3', (g) 5'-NGA-3', (h) 5'-NGC-3', (i) 5'-NAA-3', (j) 5'-NAC-3', (k) 5'-NAG-3', and (l) 5'-NAT-3', the selection of which is a function of the choice of napDNAbp.

124. The guide RNA of claim 106, wherein the edit site in any of the nucleotide sequences of SEQ ID NOs: 1217353-1289387 of step (v) begins at position 201 in the 5' to 3' orientation.

125. The guide RNA of claim 106, wherein the nucleotide change is a nucleotide substitution, a deletion, an insertion, or a combination thereof.

126. The guide RNA of claim 106, wherein the nucleotide substitution is a transition or a transversion.

127. The guide RNA of claim 106, wherein the single nucleotide substitution is (1) a G to T substitution, (2) a G to A substitution, (3) a G to C substitution, (4) a T to G substitution, (5) a T to A substitution, (6) a T to C substitution, (7) a C to G substitution, (8) a C to T substitution, (9)a C to A substitution, (10) an A to T substitution, (11) an A to G substitution, or (12) an A to C substitution.

128. The guide RNA of claim 106, wherein the single nucleotide substitution converts (1) a G:C basepair to a T:A basepair, (2) a G:C basepair to an A:T basepair, (3) a G:C basepair to C:G basepair, (4) a T:A basepair to a G:C basepair, (5) a T:A basepair to an A:T basepair, (6) a T:A basepair to a C:G basepair, (7) a C:G basepair to a G:C basepair, (8) a C:G basepair to a T:A basepair, (9) a C:G basepair to an A:T basepair, (10) an A:T basepair to a T:A basepair, (11) an A:T basepair to a G:C basepair, or (12) an A:T basepair to a C:G basepair.

129. The guide RNA of claim 106, wherein the desired nucleotide change is an insertion or deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides.

130. The guide RNA of claim 106, wherein the nucleotide change is an insertion of a polypeptide-encoding sequence.

131. The guide RNA of claim 106, wherein the nucleotide change corrects a disease- associated gene.

132. The guide RNA of claim 131, wherein the disease-associated gene is associated with a monogenetic disorder selected from the group consisting of: Adenosine Deaminase (ADA) Deficiency; Alpha-1 Antitrypsin Deficiency; Cystic Fibrosis; Duchenne Muscular Dystrophy; Galactosemia; Hemochromatosis; Huntington’s Disease; Maple Syrup Urine Disease; Marfan Syndrome; Neurofibromatosis Type 1; Pachyonychia Congenita; Phenylkeotnuria; Severe Combined Immunodeficiency; Sickle Cell Disease; Smith-Lemli-Opitz Syndrome; and Tay- Sachs Disease.

133. The guide RNA of claim 131, wherein the disease-associated gene is associated with a polygenic disorder selected from the group consisting of: heart disease; high blood pressure; Alzheimer’s disease; arthritis; diabetes; cancer; and obesity.

134. A method for installing a nucleotide change in a nucleic acid sequence, the method comprising: contacting the nucleic acid sequence with a complex comprising a fusion protein and a guide RNA of any of claims 56-81.

135. The method of claim 134, wherein the fusion protein comprises a napDNAbp and an RNA-dependent DNA polymerase.

136. The method of claim 134, wherein the guide RNA comprises a spacer, gRNA core, and an extension arm that comprises a DNA synthesis template and primer binding site.

137. The method of claim 136, wherein the DNA synthesis template encodes a nucleotide change.

138. The method of any of claims 134-137, wherein the guide RNA is capable of binding to a napDNAbp suitable for prime editing and directing the napDNAbp to a target DNA sequence.

139. The method of claim 138, wherein the target nucleic acid sequence comprises a target strand (or PAM or edit strand) and a complementary non-target strand (or non-PAM or non-edit strand) wherein the spacer of the guide RNA hybridizes to the non-PAM strand to form an RNA- DNA hybrid and an R-loop.

140. The method of claim 136, wherein the primer binding site is between approximately 8 and approximately 20 nucleotides in length.

141. The method of claim 136, wherein the primer binding site is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length.

142. The method of claim 136, wherein the extension arm is 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 28nucleotides, at least 29 nucleotides, at least 30 nucleotides, at least 31 nucleotides, at least 32 nucleotides, at least 33 nucleotides, at least 34 nucleotides, at least 35 nucleotides, at least 36 nucleotides, at least 37 nucleotides, at least 38 nucleotides, at least 39 nucleotides, at least 40 nucleotides, at least 100 nucleotides in length.

143. The method of claim 136, wherein the primer binding site is 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, or at least 20 nucleotides in length.

144. The method of claim 136, wherein the DNA synthesis template is at least 1 nucleotides, 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, at least 100 nucleotides in length.

145. The method of claim 136, wherein the DNA synthesis template can be used by an RNA- dependent DNA polymerase (e.g., reverse transcriptase) as a template for the synthesis of a corresponding single-strand DNA flap having a 3′ end, wherein the DNA flap is complementary to a strand of the endogenous target DNA sequence adjacent to a nick site, and wherein the single-strand DNA flap comprises a desired nucleotide change encoded by the DNA synthesis template.

146. The method of claim 145, wherein the single-strand DNA flap displaces an endogenous single-strand DNA having a 5´ end in the target DNA sequence that has been nicked.

147. The method of claim 146, wherein the endogenous single-strand DNA having the free 5´ end is excised by the cell.

148. The method of claim 145, whereby cellular repair of the single-strand DNA flap results in installation of the nucleotide change, thereby forming an edited DNA product.

149. The method of claim 148, wherein the nucleotide change is an insertion.

150. The method of claim 149, wherein in the insertion 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, at least 100 nucleotides in length.

151. The method of claim 149, wherein the insertion is a sequence encoding a polypeptide.