CA3203876A1

CA3203876A1 - Prime editor variants, constructs, and methods for enhancing prime editing efficiency and precision

Info

Publication number: CA3203876A1
Application number: CA3203876A
Authority: CA
Inventors: David R. Liu; Peter J. Chen; Brittany ADAMSON; Jeffrey HUSSMANN
Original assignee: Harvard College; University of California; Princeton University; Broad Institute Inc
Current assignee: Harvard College; University of California; Princeton University; Broad Institute Inc
Priority date: 2021-01-11
Filing date: 2022-01-11
Publication date: 2022-07-14
Also published as: JP2024503437A; WO2022150790A3; EP4274894A2; AU2022206476A1; WO2022150790A2

Abstract

The present disclosure provides compositions and methods for prime editing with improved editing efficiency and/or reduced indel formation by inhibiting the DNA mismatch repair path way while conducting prime editing of a target site. Accordingly, the present disclosure provides a method for editing a nucleic acid molecule by prime editing that involves contacting a nucleic acid molecule with a prime editor, a pegRNA, and an inhibitor of the DNA mismatch repair pathway, thereby installing one or more modifications to the nucleic acid molecule at a target site with increased editing efficiency and/or lower indel formation. The present disclosure further provides polynucleotides for editing a DNA target site by prime editing comprising a nucleic acid sequence encoding a napDNAbp, a polymerase, and an inhibitor of the DNA mismatch repair pathway, wherein the napDNAbp and polymerase is capable in the presence of a pegRNA of installing one or more modifications in the DNA target site with increased editing efficiency and/or lower indel formation. The disclosure further provides, vectors, cells, and kits comprising the compositions and polynucleotides of the disclosure. The present disclosure also provides compositions and methods for prime editing with improved editing efficiency and/or reduced indel formation with modified prime editor fusion proteins. The disclosure further provides, vectors, cells, and kits comprising the compositions and polynucleotides of the disclosure.

Description

DEMANDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.

NOTE : Pour les tomes additionels, veuillez contacter le Bureau canadien des brevets JUMBO APPLICATIONS/PATENTS
THIS SECTION OF THE APPLICATION/PATENT CONTAINS MORE THAN ONE
VOLUME

NOTE: For additional volumes, please contact the Canadian Patent Office NOM DU FICHIER / FILE NAME:
NOTE POUR LE TOME / VOLUME NOTE:

PRIME EDITOR VARIANTS, CONSTRUCTS, AND METHODS FOR ENHANCING
PRIME EDITING EFFICIENCY AND PRECISION
GOVERNMENT SUPPORT
[1] This invention was made with government support under Grant Nos.
AI142756, All 50551, HG009490, EB022376, EB031172, GM118062, CA072720, GM138167, U01AI142756, RM1HG009490, R01EB022376, and R35GM118062 awarded by the National Institutes of Health, and Grant No. EIR0011-17-2-0049 awarded by the Department of Defense. The government has certain rights in the invention.
RELATED APPLICATIONS

[2] This application claims the priority under 35 U.S.C. 119(e) to U.S.
Provisional Application U.S.S.N. 63/255,897, filed October 14, 2021, U.S. Provisional Application U.S.S.N.
63/231,230, filed August 9, 2021, U.S. Provisional Application U.S.S.N.
63/194,913, filed May 28, 2021, U.S. Provisional Application U.S.S.N. 63/194,865, filed May 28, 2021, U.S.
Provisional Application U.S.S.N. 63/176,202, filed April 16, 2021, and U.S.
Provisional Application U.S.S.N. 63/136,194, filed January 11, 2021, each of which is incorporated herein by reference.
INCORPORATION BY REFERENCE
131 In addition, this application refers to and incorporates by reference the entire contents of each of the following patent applications directed to prime editing previously filed by one or more of the present inventors: U.S. Provisional Application U.S.S.N.
62/820,813, filed March 19, 2019; U.S. Provisional Application U.S.S.N. 62/858,958, filed June 7, 2019; U.S. Provisional Application U.S.S.N. 62/889,996, filed August 21, 2019; U.S. Provisional Application U.S.S.N.
62/922,654, filed August 21, 2019; U.S. Provisional Application U.S.S.N.
62/913,553, filed October 10, 2019; U.S. Provisional Application U.S.S.N. 62/973,558, filed October 10, 2019;
U.S. Provisional Application U.S.S.N. 62/931,195, filed November 5, 2019; U.S.
Provisional Application U.S.S.N. 62/944,231, filed December 5, 2019; U.S. Provisional Application U.S.S.N. 62/974,537, filed December 5, 2019; U.S. Provisional Application U.S.S.N.

62/991,069, filed March 17, 2020; U.S. Provisional Application U.S.S.N.
63/100,548, filed March 17, 2020; International PCT Application No. PCT/US2020/023721, filed March 19, 2020;
International PCT Application No. PCT/US2020/023553, filed March 19, 2020;
International PCT Application No. PCT/US2020/023583, filed March 19, 2020; International PCT

Application No. PCT/US2020/023730, filed March 19, 2020; International PCT
Application No.
PCT/US2020/023713, filed March 19, 2020; International PCT Application No.
PCT/US2020/023712, filed March 19, 2020; International PCT Application No.
PCT/US2020/023727, filed March 19, 2020; International PCT Application No.
PCT/US2020/023724, filed March 19, 2020; International PCT Application No.
PCT/US2020/023725, filed March 19, 2020; International PCT Application No.
PCT/US2020/023728, filed March 19, 2020; International PCT Application No.
PCT/US2020/023732, filed March 19, 2020; and International PCT Application No.

PCT/US2020/023723, filed March 19, 2020.
BACKGROUND OF THE INVENTION
[4] The recent development of prime editing enables the insertion, deletion, and/or replacement of genomic DNA sequences without requiring error-prone double-strand DNA
breaks. See Anzalone et at,"Search-and-replace genome editing without double-strand breaks or donor DNA," Nature, 2019, Vol.576, pp. 149-157, the contents of which are incorporated herein by reference. Prime editing uses an engineered Cas9 nickase-reverse transcriptase fusion protein (e.g., PE1 or PE2) paired with an engineered prime editing guide RNA (pegRNA) that not only directs Cas9 to a target genomic site, but also which encodes the information for installing the desired edit. Without wishing to be bound by any particular theory, prime editing proceeds through a presumed multi-step editing process: 1) the Cas9 domain binds and nicks the target genomic DNA site, which is specified by the pegRNA's spacer sequence; 2) the reverse transcriptase domain uses the nicked genomic DNA as a primer to initiate the synthesis of an edited DNA strand using an engineered extension on the pegRNA as a template for reverse transcription-this generates a single-stranded 3' flap containing the edited DNA sequence; 3) cellular DNA repair resolves the 3' flap intermediate by the displacement of a 5' flap species that occurs via invasion by the edited 3' flap, excision of the 5' flap containing the original DNA

sequence, and ligation of the new 3' flap to incorporate the edited DNA
strand, forming a heteroduplex of one edited and one unedited strand; and 4) cellular DNA repair replaces the unedited strand within the heteroduplex using the edited strand as a template for repair, completing the editing process.
151 Since 2019, prime editing has been applied to introduce genetic changes in a wide variety of cells and/or organisms. Given its rapid adoption, prime editing represents a powerful tool for genomic editing. Despite its versatility and wide-scale use, prime editing efficiency can vary widely across different edit classes, target loci, and cell types (Anzalone etal., 2019). Thus, modifications to prime editing systems which result in increasing the specificity and/or efficiency of the prime editing process would significantly help advance the art. In particular, modifications that facilitate more efficient incorporation of the edited DNA
strand synthesized by the prime editor into the target genomic site are desirable. It is also desirable to reduce the frequency of indel byproducts that can form as a result of prime editing. Such further modifications to prime editing would advance the art.
SUMMARY OF THE INVENTION
161 In one aspect, the present disclosure relates to the observation that the efficiency and/or specificity of prime editing is impacted by a cell's own DNA mismatch repair (MMR) DNA
repair pathway. MMR is a multi-factor pathway that is involved in correcting basepair mismatches and insertion/deletion mispairs generated during DNA replication and recombination. As described herein, the inventors developed a novel genetic screening method¨referred to in one embodiment as "pooled CRISPRi screen for prime editing outcomes"¨which led to the identification of various genetic determinates, including MMR, as affecting the efficiency and/or specificity of prime editing. Accordingly, in one aspect, the present disclosure provides novel prime editing systems comprising a means for inhibiting and/or evade the effects of MMR, thereby increasing the efficiency and/or specificity of prime editing.
In one embodiment, the disclosure provides a prime editing system that comprises an MMR-inhibiting protein, such as, but not limited to, a dominant negative variant of an MMR protein, such as a dominant negative MLI-11 protein (i.e., "MLIIIdn"). In another embodiment, the prime editing system comprises the installation of one or more silent mutations nearby an intended edit,

3/699 thereby allowing the intended edit from evading MMR recognition, even in the absence of an 1M/1R-inhibiting protein, such as an MtHldn. In another aspect, the disclosure provides a novel genetic screen for identifying genetic determinants, such as MMR, that impact the efficiency and/or specificity of prime editing. In still further aspects, the disclosure provides nucleic acid constructs encoding the improved prime editing systems described herein. The disclosure in other aspects also provides vectors (e.g., AAV or lentivirus vectors) comprising nucleic acids encoding the improved prime editing system described herein. In still other aspects, the disclosure provides cells comprising the improved prime editing systems described herein. The disclosure also provides in other aspects the components of the genetic screens, including nucleic acid and/or vector constructs, guide .RNA, pegRNAs, cells (e.g., CRISPRi cells), and other reagents and/or materials for conducting the herein disclosed genetic screens.
In still other aspects, the disclosure provides compositions and kits, e.g., pharmaceutical compositions, comprising the improved prime editing system described herein and which are capable of being administered to a cell, tissue, or organism by any suitable means, such as by gene therapy, mRNA delivery, virus-like particle delivery, or ribonucleoprotein (RNP) delivery. In yet another aspect, the present disclosure provides methods of using the improved prime editing system to install one or more edits in a target nucleic acid molecule, e.g., a genomic locus. In still another aspect, the present disclosure provides methods of treating a disease or disorder using the improved prime editing system to correct or otherwise repair one or more genetic changes (e.g., a single nucleotide polymorphism) in a target nucleic acid molecule, e.g., a genomic locus comprising one or more disease-causing mutations.
171 Thus, in various aspects, the present disclosure describes an improved and modified approach to prime editing that comprises inhibiting the DNA mismatch repair (MMR) system during prime editing. The inventors have surprisingly found that the editing efficiency of prime editing may be significantly increased (e.g., at least a 2-fold increase, at least a 3-fold increase, at least a 4-fold increase, at least a 5-fold increase, at least a 6-fold increase, at least a 7-fold increase, at least an 8-fold increase, at least a 9-fold increase, at least a 10-fold increase, or more) when one or more functions of the DNA mismatch repair (MMR) system are inhibited, blocked, or otherwise inactivated during prime editing (such as using the MLHldn inhibitor of MMR). In addition, the inventors have surprisingly found that the frequency of indel formation resulting from prime editing may be significantly decreased (e.g., about a 2-fold decrease, about a 3-fold

4/699 decrease, about a 4-fold decrease, about a 5-fold decrease, about a 6-fold decrease, about a 7-fold decrease, about a 8-fold decrease, about a 9-fold decrease, or about a 10-fold decrease or lower) when one or more functions of the DNA mismatch repair (MMR) system are inhibited, blocked, or otherwise inactivated during prime editing.
E81 The present disclosure also describes in other embodiments an improved and modified approach to prime editing that comprises evading the DNA mismatch repair (MMR) system during prime editing. The inventors have surprisingly found that the editing efficiency of prime editing may be significantly increased (e.g., at least 1.5-fold, at least 2.0-fold, at least 2.5-fold, at least 3.0-fold, at least 3.5-fold, at least 4.0-fold, at least 4.5-fold, at least 5.0-fold, at least 5.5-fold, at least 6.0-fold, at least 6.5-fold, at least 7.0-fold, at least 7.5-fold, at least 8.0-fold, at least 8.5-fold, at least 9.0-fold, at least 9.5-fold, at least 10.0-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, at least 20-fold, at least 21-fold, at least 22-fold, at least 23-fold, at least 24-fold, at least 25-fold, at least 26-fold, at least 27-fold, at least 28-fold, at least 29-fold, at least 30-fold, at least 31-fold, at least 32-fold, at least 33-fold, at least 34-fold, at least 35-fold, at least 36-fold, at least 37-fold, at least 38-fold, at least 39-fold, at least 40-fold, at least 41-fold, at least 42-fold, at least 43-fold, at least 44-fold, at least 45-fold, at least 46-fold, at least 47-fold, at least 48-fold, at least 49-fold, at least 50-fold, at least 51-fold, at least 52-fold, at least 53-fold, at least 54-fold, at least 55-fold, at least 56-fold, at least 57-fold, at least 58-fold, at least 59-fold, at least 60-fold, at least 61-fold, at least 62-fold, at least 63-fold, at least 64-fold, at least 65-fold, at least 66-fold, at least 67-fold, at least 68-fold, at least 69-fold, at least 70-fold, at least 71-fold, at least 72-fold, at least 73-fold, at least 74-fold, or at least 75-fold increased) when one or more silent mutations are installed nearby a desired site for installing a genetic change by prime editing, in the presence or absence of an inhibitor of IMMR.
In addition, the inventors have surprisingly found that the frequency of indel formation resulting from prime editing may be significantly decreased (e.g., at least 1.5-fold, at least 2.0-fold, at least 2.5-fold, at least 3.0-fold, at least 3.5-fold, at least 4.0-fold, at least 4.5-fold, at least 5.0-fold, at least 5.5-fold, at least 6.0-fold, at least 6.5-fold, at least 7.0-fold, at least 7.5-fold, at least 8.0-fold, at least 8.5-fold, at least 9.0-fold, at least 9.5-fold, at least 10.0-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, at least 20-fold, at least 21-fold, at least 22-fold, at least 23-

5/699 fold, at least 24-fold, at least 25-fold, at least 26-fold, at least 27-fold, at least 28-fold, at least 29-fold, at least 30-fold, at least 31-fold, at least 32-fold, at least 33-fold, at least 34-fold, at least 35-fold, at least 36-fold, at least 37-fold, at least 38-fold, at least 39-fold, at least 40-fold, at least 41-fold, at least 42-fold, at least 43-fold, at least 44-fold, at least 45-fold, at least 46-fold, at least 47-fold, at least 48-fold, at least 49-fold, at least 50-fold, at least 51-fold, at least 52-fold, at least 53-fold, at least 54-fold, at least 55-fold, at least 56-fold, at least 57-fold, at least 58-fold, at least 59-fold, at least 60-fold, at least 61-fold, at least 62-fold, at least 63-fold, at least 64-fold, at least 65-fold, at least 66-fold, at least 67-fold, at least 68-fold, at least 69-fold, at least 70-fold, at least 71-fold, at least 72-fold, at least 73-fold, at least 74-fold, or at least 75-fold decreased) when one or more silent mutations are installed nearby a desired site for installing a genetic change by prime editing, in the presence or absence of an inhibitor of MMR.
[9] In some embodiments, the disclosure describes an improved prime editing system referred to herein as "PE4," which includes PE2 plus an MLH1 dominant negative protein (e.g., wild-type MLH1 with amino acids 754-756 truncated as described further herein). In certain embodiments, the MLHIdn is expressed in trans in a cell comprising the PE2 fusion protein.
The MLFIldn and the PE2 may be provided together or separate, e.g., by delivery on separate plasmids, separate vectors (e.g., AAV or lentivirus vectors), separate vector-like particles, separate ribonucleoprotein complexes (RNPs), or by delivery on the same plasmids, same vectors (e.g., AAV or lentivirus vectors), same vector-like particles, same ribonucleoprotein complexes (RNPs). In other embodiments, the MLHldn may be fused to PE2 or otherwise associated with, coupled, or joined to PE2 such that they are co-delivered.
11.01 In other embodiments, the disclosure describes an improved prime editing system referred to as "PIES," which includes IPE3 (which is PE2 plus a second-strand nicking guide RNA) plus an MLH1 dominant negative protein (e.g., wild-type MLH1 with amino acids 754-756 truncated as described further herein). In certain embodiments, the MLHldn is expressed in trans in a cell comprising the PE3 prime editor. The MLHIdn and the PE3 may be provide together or separate, e.g., by delivery on separate plasmids, separate vectors (e.g., AAV or lentivirus vectors), separate vector-like particles, separate ribonucleoprotein complexes (RNPs), or by delivery on the same plasmid, same vector (e.g., AAV or lentivirus vectors), same vector-like particles, same ribonucleoprotein complexes (RNPs). In other embodiments, the MLHldn

6/699 may be fused to PE3 or otherwise associated with, coupled, or joined to PE3 such that they are co-delivered.
[111 In other aspects, the present disclosure describes an optimized PE2 prime editor architecture referred to herein as "PEmax." PEmax is a modified form of PE2 which comprises modified reverse transcriptase codon usage, SpCas9 mutations, NLS sequences, and is described in FIG. 54B. Specifically, PEmax refers to a PE complex comprising a fusion protein comprising Cas9 (R221K N394K H840A) and a variant MMLV RT pentamutant (D200N
T306K W313F T330P L603W) having the following structure: [bipartite NLS]-[Cas9(R221K)(N394K)(H840A)MlinkerHIVIMLV_RT(D200N)(T330P)(1,603W)]-1bipartite NLSHNLS] + a desired PEgRNA, wherein the PE fusion has the amino acid sequence of SEQ
ID NO: 99, which is shown as follows:
MKRTADGSEFESPKKKRKVDICKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNT

RLIYLALAHMEICFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDA
KAILSARLSKSRKLENLIAQLPGEKKAGLFGNLIALSLGLTPNFKSNFDLAEDAKLQ
LSKDTYDDDILDNILAQIGIDQYADILFLAAKNLSDAILLSDILRVNTEITKAPLSASMIK
RYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPI
LEKMDGTEELLVKLKREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKD
NREKIEKILTFRIPYYVGPLA.RGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFI
ERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKA
IVDLLFKTNRK'VTVKQLKEDYFKKIECIFIDSVEISGVE:DRFNASLGTYH:DLLKIIKDIK
DFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGW
GRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLLHDDSLTFKEDIQKAQVSGQ
GDSLHEIHANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQK

LDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYW
RQLLNAKLITQRKF.DN.LTKAERGGLSE.LDKAGFIKRQLVETRQITKHVAQILDSRM
NTKYDENDKLIREVKVITLKSKLVSDITIKDFQFYKVREINNYHHAHDAYLNAVVGT
ALIKKYPKLESEFVYGD'YKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITL
ANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSNIPQVNIVICKTEVQTGGFSKE
SILPKRNSDKLIARKICDWDPKKYGGFDSPTVAYSVINVAKVEKGKSKKLKSVICEL
LGITIMERSSFEKNPMFLEAKGYKEVKXDLILKLPIKYSLFELENGRKRMLASAGEL
QKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIEEQISEFS
KRVILADANLDKVLSAYNKHRDKPIREQAENHHLFTLTNLGAPAAFKNIFDTTIDRK
RYTSTKEVLDATLIHQSITGLYETRIDLSQLGG.DSGGSSGGSKRTA.DGSEFESPKKK
RKVSGGSSGGSTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAP
LIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTND
YRPVQDLREVNKRVEDIEIPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPL
FAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQIIPDLILLQYVDDLL

7/699 LAAISELDCQQGTRALLQTLGNLGYRA.SAKKAQICQKQVKYLGYLLKEGQRWLTEAR
KETVMGQPTPKTPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKA
YQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDP
VAAGWPPCLRMVAAIAVLIKDAGKLTMGQPINILAPHA'VEALVKQPPDRWLSNARMI
HYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDA
DHTWYTDGSSLLQEGQRKAGAAVETETEVIWAKALPAGISAQRAELIALTQALKMAEG
KKLNVYTDSRYAFATAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPKRLSBHCP
GHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSPSGGSKRTADGSEFESPKK
KRKVGSGPAAKRVKID (SEQ ID NO: 99) KEY:
BIPARTITE SV40 NUCLEAR LOCALIZATION SEQUENCE (NLS) TOP: (SEQ ID NO:
101), BOTTOM: (SEQ ED NO: 140) CAS9(R221K N39K I1840A) (SEQ ID NO: 104) SGGSX2-BIPARTITE SV4ONLS-SGGSX2 LINKER (SEQ ID NO: 105) M-MLV reverse transcriptase (D200N T306K W313F T330P L603W) (SEQ ID NO: 98) Other linker sequence (SEQ ID NO: 122) Other linker sequence (SEQ ID NO: 106) c-Myc NLS PAAKRVKLD (SEQ ID NO: 135) [121 In some embodiments, the PE4 may be modified to substitute the PE2 fusion protein with PEmax. In such cases, the modified prime editing system may be referred to as "PE4max."
[I.3] In some embodiments, the PE5 may be modified to substitute the PE3 prime editor with PEmax. In such cases, the modified prime editing system may be referred to as "PE5max" and includes a second stranding nicking guide RNA.
[141 The inventors developed prime editing which enables the insertion, deletion, and/or replacement of genomic DNA sequences without requiring error-prone double-strand DNA
breaks. The present disclosure now provides an improved method of prime editing involving the blocking, inhibiting, evading, or inactivation of the MMR pathway (e.g., by inhibiting, blocking, or inactivating an MMR pathway protein, including MUM ) during prime editing, whereby doing so surprisingly results in increased editing efficiency and reduced indel formation. As used herein, "during" prime editing can embrace any suitable sequence of events, such that the prime editing step can be applied before, at the same time, or after the step of blocking, inhibiting, evading, or inactivating the MMR pathway (e.g., by targeting the inhibition of MLH1).

8/699 11.51 In various aspects and without wishing to be bound by any particular theory, prime editing uses an engineered Cas9 nickase¨reverse transcriptase fusion protein (e.g., PE1 or PE2) paired with an engineered prime editing guide RNA (pegRNA) that both directs Cas9 to the target genomic site and encodes the information for installing the desired edit. Prime editing proceeds through a multi-step editing process: 1) the Cas9 domain binds and nicks the target genomic DNA site, which is specified by the pegRNA's spacer sequence; 2) the reverse transcriptase domain uses the nicked genomic DNA as a primer to initiate the synthesis of an edited DNA strand using an engineered extension on the pegRNA as a template for reverse transcription¨this generates a single-stranded 3' flap containing the edited DNA sequence; 3) cellular DNA repair resolves the 3' flap intermediate by the displacement of a 5' flap species that occurs via invasion by the edited 3' flap, excision of the 5' flap containing the original DNA
sequence, and ligation of the new 3' flap to incorporate the edited DNA
strand, forming a heteroduplex of one edited and one unedited strand; and 4) cellular DNA repair replaces the unedited strand within the heteroduplex using the edited strand as a template for repair, completing the editing process.
1161 Efficient incorporation of the desired edit requires that the newly synthesized 3' flap contains a portion of sequence that is homologous to the genomic DNA site.
This homology enables the edited 3' flap to compete with the endogenous DNA strand (the corresponding 5' flap) for incorporation into the DNA duplex. Because the edited 3' flap will contain less sequence homology than the endogenous 5' flap, the competition is expected to favor the 5' flap strand. Thus, a potential limiting factor in the efficiency of prime editing may be the failure of the 3' flap, which contains the edit, to effectively invade and displace the 5' flap strand.
Moreover, successful 3' flap invasion and removal of the 5' flap only incorporates the edit on one strand of the double-stranded DNA genome. Permanent installation of the edit requires cellular DNA repair to replace the unedited complementary DNA strand using the edited strand as a template. While the cell can be made to favor replacement of the unedited strand over the edited strand (step 4 above) by the introduction of a nick in the unedited strand adjacent to the edit using a secondary sgRNA (i.e., the PE3 system), this process still relies on a second stage of DNA repair.
1171 This disclosure describes a modified approach to prime editing that comprises additionally inhibiting, blocking, or otherwise inactivating the DNA mismatch repair (MMR)

9/699 system. In certain embodiments, the DNA mismatch repair (MMR) system can be inhibited, blocked, or otherwise inactivating one or more proteins of the MMR system, including, but not limited to MLH1, PMS2 (or Mud, alpha), PMS1 (or MutL beta), MLH3 (or Mud., gamma), MutS alpha (MSH2-MSH6), MutS beta (MSH2-MSH3), MSH2, MSH6, PCNA, RFC, EX01, POLS, and PCNA. The disclosure contemplates any suitable means by which to inhibit, block, or otherwise inactivate the DNA mismatch repair (MMR) system, including, but not limited to inactivating one or more critical proteins of the MMR system at the genetic level, e.g., by introducing one or more mutations in the genes encoding a protein of the MMR
system, e.g., MLH1, PMS2 (or MutL alpha), PMS1 (or MutL beta), MLH3 (or MutL gamma), MutS
alpha (MSH2-MSH6), MutS beta (MSH2-MSH3), MSH2, MSH6, PCNA, RFC, EX01, POLO, and PCNA.
[181 Thus, in one aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome) using prime editing while blocking, inhibiting, evading, or otherwise inactivating the DNA mismatch repair (MMR) system.
[191 In another aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome) using prime editing while blocking, inhibiting, evading, or otherwise inactivating a protein of the MMR system, e.g., MUD, PMS2 (or MutL alpha), PMS1 (or Mud, beta), MLH3 (or MutL gamma), MutS alpha (MSH2-MSH6), MutS beta (MSH2-MSH3), MSH2, MSH6, PCNA, RFC, EX01, POLO, and PCNA.
1201 In still another aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome) using prime editing while blocking, inhibiting, evading, or otherwise inactivating MLH1 or variant thereof 1211 In another aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome) using prime editing while blocking, inhibiting, evading, or otherwise inactivating PMS2 (or MutL alpha) or variant thereof.
1221 In yet another aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome) using prime editing while blocking, inhibiting, evading, or otherwise inactivating PMS1 (or Mud, beta) or variant thereof [231 in still another aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome) using prime editing while blocking, inhibiting, evading, or otherwise inactivating MLH3 (or MutL gamma) or variant thereof.

10/699 1241 In another aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome) using prime editing while blocking, inhibiting, evading, or otherwise inactivating MutS alpha (MSH2-MSH6) or variant thereof.
[251 In still another aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome) using prime editing while blocking, inhibiting, evading, or otherwise inactivating MSH2 or variant thereof.
[261 In another aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome) using prime editing while blocking, inhibiting, evading, or otherwise inactivating MSH6 or variant thereof.
1271 In yet another aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome) using prime editing while blocking, inhibiting, evading, or otherwise inactivating PCNA or variant thereof.
[281 In still another aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome) using prime editing while blocking, inhibiting, evading, or otherwise inactivating RFC or variant thereof.
[291 In another aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome) using prime editing while blocking, inhibiting, evading, or otherwise inactivating EX01 or variant thereof.
1301 In yet another aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome) using prime editing while blocking, inhibiting, evading, or otherwise inactivating POLS or variant thereof.
[311 Thus, in one aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome) using prime editing while blocking, inhibiting, evading, or otherwise inactivating the DNA mismatch repair (MMR) system.
1321 In another aspect, the disclosure provides a method for evading MMR by installing one or more silent mutations nearby an intended edit, resulting in the evading of MMR and thereby improving editing efficiency of prime editing. In various embodiments, the number of silent mutations installed can be one, or two, or three, or four, or five, or six, or seven, or eight, or nine, or ten, or eleven, or twelve, or thirteen, or fourteen, or fifteen, or sixteen, or seventeen, or eighteen, or nineteen, or twenty or more. The one more silent mutations may be located upstream or downstream (or a combination if multiple silent mutations are involved) of the

11/699 intended edit site, on the same or opposite strand of DNA as the intended edit site (or a combination if multiple silent mutations are involved). The silent mutations may be located upstream or downstream of the intended edit by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or more nucleotide positions away from the intended edit. In various embodiments, the method of evading by silent mutation installation results in a significant increase in editing efficiency of prime editing (e.g., at least 1.5-fold, at least 2.0-fold, at least 2.5-fold, at least 3.0-fold, at least 3.5-fold, at least 4.0-fold, at least 4.5-fold, at least 5.0-fold, at least 5.5-fold, at least 6.0-fold, at least 6.5-fold, at least 7.0-fold, at least 7.5-fold, at least 8.0-fold, at least 8.5-fold, at least 9.0-fold, at least 9.5-fold, at least 10.0-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, at least 20-fold, at least 21-fold, at least 22-fold, at least 23-fold, at least 24-fold, at least 25-fold, at least 26-fold, at least 27-fold, at least 28-fold, at least 29-fold, at least 30-fold, at least 31-fold, at least 32-fold, at least 33-fold, at least 34-fold, at least 35-fold, at least 36-fold, at least 37-fold, at least 38-fold, at least 39-fold, at least 40-fold, at least 41-fold, at least 42-fold, at least 43-fold, at least 44-fold, at least 45-fold, at least 46-fold, at least 47-fold, at least 48-fold, at least 49-fold, at least 50-fold, at least 51-fold, at least 52-fold, at least 53-fold, at least 54-fold, at least 55-fold, at least 56-fold, at least 57-fold, at least 58-fold, at least 59-fold, at least 60-fold, at least 61-fold, at least 62-fold, at least 63-fold, at least 64-fold, at least 65-fold, at least 66-fold, at least 67-fold, at least 68-fold, at least 69-fold, at least 70-fold, at least 71-fold, at least 72-fold, at least 73-fold, at least 74-fold, or at least 75-fold increased) when one or more silent mutations are installed nearby a desired site for installing a genetic change by prime editing, in the presence or absence of an inhibitor of MIVIR. In various embodiments, the method of evading MMR by silent mutation installation results in a significant decrease in the frequency of indel formation of prime editing (e.g., at least 1.5-fold, at least 2.0-fold, at least 2.5-fold, at least 3.0-fold, at least 3.5-fold, at least 4.0-fold, at least 4.5-fold, at least 5.0-fold, at least 5.5-fold, at least 6.0-fold, at least 6.5-fold, at least 7.0-fold, at least 7.5-fold, at least 8.0-fold, at least 8.5-fold, at least 9.0-fold, at least 9.5-fold, at least 10.0-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, at least 20-fold, at least 21-fold, at least 22-fold, at least 23-fold, at least 24-fold, at least 25-fold, at least 26-fold, at least 27-fold, at least 28-fold, at least 29-

12/699 fold, at least 30-fold, at least 31-fold, at least 32-fold, at least 33-fold, at least 34-fold, at least 35-fold, at least 36-fold, at least 37-fold, at least 38-fold, at least 39-fold, at least 40-fold, at least 41-fold, at least 42-fold, at least 43-fold, at least 44-fold, at least 45-fold, at least 46-fold, at least 47-fold, at least 48-fold, at least 49-fold, at least 50-fold, at least 51-fold, at least 52-fold, at least 53-fold, at least 54-fold, at least 55-fold, at least 56-fold, at least 57-fold, at least 58-fold, at least 59-fold, at least 60-fold, at least 61-fold, at least 62-fold, at least 63-fold, at least 64-fold, at least 65-fold, at least 66-fold, at least 67-fold, at least 68-fold, at least 69-fold, at least 70-fold, at least 71-fold, at least 72-fold, at least 73-fold, at least 74-fold, or at least 75-fold decrease) when one or more silent mutations are installed nearby a desired site for installing a genetic change by prime editing, in the presence or absence of an inhibitor of MMR.
[331 In another aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome), comprising contacting a target nucleotide molecule with a prime editor and an inhibitor of the MMR system, e.g., an inhibitor of one or more of MLH1, PMS2 (or MutL alpha), PMS1 (or MutL beta), MLH3 (or MutL gamma), MutS alpha (MSH2-MSH6), MutS beta (MSH2-MSH3), MSH2, MSH6, PCNA, RFC, EX01, POLS, or PCNA. In various embodiments, the inhibitor can be a small molecule inhibitor. In other embodiments, the inhibitor can be an antibody, e.g., a neutralizing antibody. In still other embodiments, the inhibitor can be a variant of an MMR protein (e.g., a variant encoded by a dominant negative mutant of the gene encoding the MMR protein that adversely affects the function or expression of the normal wild type MMR protein, also referred to herein as a "dominant negative mutant,"
"dominant negative variant," or a "dominant negative protein," e.g., a "dominant negative MMR
protein"). In some embodiments, the inhibitor is a dominant negative variant of an MMR protein that inhibits the activity of a wild type MMR protein. For example, the inhibitor can be an MLH1 protein variant (e.g., a dominant negative mutant) of one or more of IMLH1, PMS2 (or MutL
alpha), PMS1 (or MutL beta), MLH3 (or MutL gamma), MutS alpha (MSH2-MSH6), MutS beta (MSH2-MSH3), MSH2, MSH6, PCNA, RFC, EX01, POLE, or PCNA, e.g., a dominant negative mutant of MLH1. In still other embodiments, the inhibitor can be targeted at the level of transcription, e.g., an siRNA or other nucleic acid agent that knocks down the level of a transcript encoding MLH1, IPMS2 (or 1MutL alpha), PMS1 (or Mutt. beta), IMLH3 (or MutL
gamma), MutS alpha (MSH2-MSH6), MutS beta (MSH2-MSH3), MSH2, MSH6, PCNA, RFC, EX01, POLS, or PCNA. In yet other embodiments, the step of "contacting a target nucleotide

13/699 molecule with a prime editor" can include (i) delivering directly to a cell an effective amount of a prime editor fusion protein (e.g., PE1 or PE2) complexed with a lipid delivery system; (ii) delivery to a cell an mRNA or delivery complex comprising an mRNA that encodes a prime editor fusion protein and/or a suitable pegRNA; and (iii) a DNA vector (e.g., an AAV or lentivirus vector, plasmid, or other nucleic acid delivery vector) that encodes a prime editor fusion protein and/or a suitable pegRNA on one or more DNA vectors.
[341 In still another aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome) using prime editing while blocking, inhibiting, or otherwise inactivating MLH1 or variant thereof. In another aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome), comprising contacting a target nucleotide molecule with a prime editor and an inhibitor of MLH1. In various embodiments, the inhibitor can be a small molecule inhibitor. In other embodiments, the inhibitor can be an anti-MLH1 antibody, e.g., a neutralizing antibody that inactivates MLH1. In still other embodiments, the inhibitor can be a dominant negative mutant of MLH I. In still other embodiments, the inhibitor can be targeted at the level of transcription of MLH1, e.g., an siRNA or other nucleic acid agent that knocks down the level of a transcript encoding MLH1. In yet other embodiments, the step of "contacting a target nucleotide molecule with a prime editor" can include (i) delivering directly to a cell an effective amount of a prime editor fusion protein (e.g., PEI or PE2) complexed with a lipid delivery system; (ii) delivery to a cell an mRNA
or delivery complex comprising an mRNA that encodes a prime editor fusion protein and/or a suitable pegRNA; and (iii) a DNA vector (e.g., an AAV or lentivirus vector, plasmid, or other nucleic acid delivery vector) that encodes a prime editor fusion protein and/or a suitable pegRNA on one or more DNA vectors.
[351 In still another aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome) using prime editing while blocking, inhibiting, or otherwise inactivating PMS2 (or Mud- alpha) or variant thereof In another aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome), comprising contacting a target nucleotide molecule with a prime editor and an inhibitor of PMS2 (or MutL alpha). In various embodiments, the inhibitor can be a small molecule inhibitor. In other embodiments, the inhibitor can be an anti-PMS2 (or MutL alpha) antibody, e.g., a neutralizing antibody that inactivates PMS2 (or MutL alpha). In still other embodiments, the inhibitor can be a dominant

14/699 negative mutant of PMS2 (or MutL alpha). In still other embodiments, the inhibitor can be targeted at the level of transcription of PMS2 (or MutL alpha), e.g., an siRNA
or other nucleic acid agent that knocks down the level of a transcript encoding ML PMS2 (or MutL alpha). In yet other embodiments, the step of "contacting a target nucleotide molecule with a prime editor" can include (i) delivering directly to a cell an effective amount of a prime editor fusion protein (e.g., PEI or PE2) complexed with a lipid delivery system; (ii) delivery to a cell a mRNA or delivery complex comprising an mRNA that encodes a prime editor fusion protein and/or a suitable pegRNA; and (iii) a DNA vector (e.g., an AAV or lentivina vector, plasmid, or other nucleic acid delivery vector) that encodes a prime editor fusion protein and/or a suitable pegRNA on one or more DNA vectors.
[361 In still another aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome) using prime editing while blocking, inhibiting, or otherwise inactivating PM S1 (or MutL beta) or variant thereof. In another aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome), comprising contacting a target nucleotide molecule with a prime editor and an inhibitor of PMS1 (or MutL beta). in various embodiments, the inhibitor can be a small molecule inhibitor. In other embodiments, the inhibitor can be an anti-PMS1 (or MutL beta) antibody, e.g., a neutralizing antibody that inactivates PMS1 (or MutL beta). In still other embodiments, the inhibitor can be a dominant negative mutant of PMS1 (or MutL beta). In still other embodiments, the inhibitor can be targeted at the level of transcription of PMS1 (or MutL beta), e.g., an siRNA
or other nucleic acid agent that knocks down the level of a transcript encoding PMS1 (or MutL
beta). In yet other embodiments, the step of "contacting a target nucleotide molecule with a prime editor" can include (i) delivering directly to a cell an effective amount of a prime editor fusion protein (e.g., PEI or PE2) complexed with a lipid delivery system; (ii) delivery to a cell a mRNA or delivery complex comprising an mRNA that encodes a prime editor fusion protein and/or a suitable pegRNA; and (iii) a DNA vector (e.g., an AAV or lentivina vector, plasmid, or other nucleic acid delivery vector) that encodes a prime editor fusion protein and/or a suitable pegRNA on one or more DNA vectors.
[371 lEn still another aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome) using prime editing while blocking, inhibiting, or otherwise inactivating MLH3 (or MutL gamma) or variant thereof. In another aspect, the present disclosure

15/699 provides a method for editing a nucleotide molecule (e.g., a genome), comprising contacting a target nucleotide molecule with a prime editor and an inhibitor of MLH3 (or MutL gamma). In various embodiments, the inhibitor can be a small molecule inhibitor. In other embodiments, the inhibitor can be an anti-MLH3 (or MutL gamma) antibody, e.g., a neutralizing antibody that inactivates MLH3 (or MutL gamma). In still other embodiments, the inhibitor can be a dominant negative mutant of MLH3 (or MutL gamma). In still other embodiments, the inhibitor can be targeted at the level of transcription of P MLH3 (or MutL gamma), e.g., an siRNA or other nucleic acid agent that knocks down the level of a transcript encoding MLH3 (or MutL gamma).
In yet other embodiments, the step of "contacting a target nucleotide molecule with a prime editor" can include (i) delivering directly to a cell an effective amount of a prime editor fusion protein (e.g., PEI or PE2) complexed with a lipid delivery system; (ii) delivery to a cell a mRNA
or delivery complex comprising an mRNA that encodes a prime editor fusion protein and/or a suitable peg,RNA; and (iii) a DNA vector (e.g., an AAV or lentivirus vector, plasmid, or other nucleic acid delivery vector) that encodes a prime editor fusion protein and/or a suitable pegRNA
on one or more DNA vectors.
[381 In still another aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome) using prime editing while blocking, inhibiting, or otherwise inactivating MutS alpha (MSH2-MSH6) or variant thereof. In another aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome), comprising contacting a target nucleotide molecule with a prime editor and an inhibitor of MutS alpha (MSH2-MSH6). In various embodiments, the inhibitor can be a small molecule inhibitor. In other embodiments, the inhibitor can be an anti-MutS alpha (MSH2-MSH6) antibody, e.g., a neutralizing antibody that inactivates MutS alpha (MSH2-MSH6). In still other embodiments, the inhibitor can be a dominant negative mutant of MutS alpha (MSH2-MSH6). In still other embodiments, the inhibitor can be targeted at the level of transcription of MutS alpha (MSH2-MSH6), e.g., an siRNA or other nucleic acid agent that knocks down the level of a transcript encoding MutS alpha (MSH2-MSH6). In yet other embodiments, the step of "contacting a target nucleotide molecule with a prime editor" can include (i) delivering directly to a cell an effective amount of a prime editor fusion protein (e.g., PE1 or PE2) complexed with a lipid delivery system; (ii) delivery to a cell a mRNA or delivery complex comprising an mRNA
that encodes a prime editor fusion protein and/or a suitable pegRNA; and (iii) a DNA vector (e.g., an AAV or

16/699 lentivirus vector, plasmid, or other nucleic acid delivery vector) that encodes a prime editor fusion protein and/or a suitable pegRNA on one or more DNA vectors.
1391 In still another aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome) using prime editing while blocking, inhibiting, or otherwise inactivating MSH2 or variant thereof. In another aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome), comprising contacting a target nucleotide molecule with a prime editor and an inhibitor of MSH2. In various embodiments, the inhibitor can be a small molecule inhibitor. In other embodiments, the inhibitor can be an anti- MSH2 antibody, e.g., a neutralizing antibody that inactivates MSH2. In still other embodiments, the inhibitor can be a dominant negative mutant of MSH2. In still other embodiments, the inhibitor can be targeted at the level of transcription of MSH2, e.g., an siRNA or other nucleic acid agent that knocks down the level of a transcript encoding MSH2. In yet other embodiments, the step of "contacting a target nucleotide molecule with a prime editor" can include (1) delivering directly to a cell an effective amount of a prime editor fusion protein (e.g., PEI or PE2) complexed with a lipid delivery system; (ii) delivery to a cell a mRNA or delivery complex comprising an mRNA
that encodes a prime editor fusion protein and/or a suitable pegRNA; and (iii) a DNA vector (e.g., an AAV or lentivirus vector, plasmid, or other nucleic acid delivery vector) that encodes a prime editor fusion protein and/or a suitable pegRNA on one or more DNA
vectors.
1401 In still another aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome) using prime editing while blocking, inhibiting, or otherwise inactivating MSH6 or variant thereof. In another aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome), comprising contacting a target nucleotide molecule with a prime editor and an inhibitor of MSH6. In various embodiments, the inhibitor can be a small molecule inhibitor. In other embodiments, the inhibitor can be an anti-MSH6 antibody, e.g., a neutralizing antibody that inactivates MSH6. In still other embodiments, the inhibitor can be a dominant negative mutant of MSH6. In still other embodiments, the inhibitor can be targeted at the level of transcription of MSH6, e.g., an siRNA or other nucleic acid agent that knocks down the level of a transcript encoding MSH6. In yet other embodiments, the step of "contacting a target nucleotide molecule with a prime editor" can include (i) delivering directly to a cell an effective amount of a prime editor fusion protein (e.g., PEI. or PE2) complexed with a lipid delivery system; (ii) delivery to a cell a mRNA or delivery complex comprising an mRNA

17/699 that encodes a prime editor fusion protein and/or a suitable pegRNA; and (iii) a DNA vector (e.g., an AAV or lentivirus vector, plasmid, or other nucleic acid delivery vector) that encodes a prime editor fusion protein and/or a suitable pegRNA on one or more DNA
vectors.
[411 In still another aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome) using prime editing while blocking, inhibiting, or otherwise inactivating PCNA or variant thereof. In another aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome), comprising contacting a target nucleotide molecule with a prime editor and an inhibitor of PCNA. In various embodiments, the inhibitor can be a small molecule inhibitor. In other embodiments, the inhibitor can be an anti- PCNA
antibody, e.g., a neutralizing antibody that inactivates PCNA. In still other embodiments, the inhibitor can be a dominant negative mutant of PCNA. In still other embodiments, the inhibitor can be targeted at the level of transcription of PCNA, e.g., an siRNA or other nucleic acid agent that knocks down the level of a transcript encoding PCNA. In yet other embodiments, the step of "contacting a target nucleotide molecule with a prime editor" can include (i) delivering directly to a cell an effective amount of a prime editor fusion protein (e.g., PEI or PE2) complexed with a lipid delivery system; (ii) delivery to a cell a mRNA or delivery complex comprising an mRNA
that encodes a prime editor fusion protein and/or a suitable pegRNA; and (iii) a DNA vector (e.g., an AAV or lentivirus vector, plasmid, or other nucleic acid delivery vector) that encodes a prime editor fusion protein and/or a suitable pegRNA on one or more DNA
vectors.
[421 In still another aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome) using prime editing while blocking, inhibiting, or otherwise inactivating RFC or variant thereof. In another aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome), comprising contacting a target nucleotide molecule with a prime editor and an inhibitor of RFC. In various embodiments, the inhibitor can be a small molecule inhibitor. In other embodiments, the inhibitor can be an anti-RFC antibody, e.g., a neutralizing antibody that inactivates RFC. In still other embodiments, the inhibitor can be a dominant negative mutant of RFC. In still other embodiments, the inhibitor can be targeted at the level of transcription of RFC, e.g., an siRNA or other nucleic acid agent that knocks down the level of a transcript encoding RFC. In yet other embodiments, the step of "contacting a target nucleotide molecule with a prime editor" can include (i) delivering directly to a cell an effective amount of a prime editor fusion protein (e.g., PE1 or PE2) complexed with a lipid delivery

18/699 system; (ii) delivery to a cell a mRNA or delivery complex comprising an mRNA
that encodes a prime editor fusion protein and/or a suitable pegRNA; and (iii) a DNA vector (e.g., an AAV or lentivirus vector, plasmid, or other nucleic acid delivery vector) that encodes a prime editor fusion protein and/or a suitable pegRNA on one or more DNA vectors.
1431 In still another aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome) using prime editing while blocking, inhibiting, or otherwise inactivating EX01 or variant thereof. In another aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome), comprising contacting a target nucleotide molecule with a prime editor and an inhibitor of EX01. In various embodiments, the inhibitor can be a small molecule inhibitor. In other embodiments, the inhibitor can be an anti-EX01 antibody, e.g., a neutralizing antibody that inactivates EX01. In still other embodiments, the inhibitor can be a dominant negative mutant of EX01. In still other embodiments, the inhibitor can be targeted at the level of transcription of EX01, e.g., an siRNA or other nucleic acid agent that knocks down the level of a transcript encoding EX01. In yet other embodiments, the step of "contacting a target nucleotide molecule with a prime editor" can include (i) delivering directly to a cell an effective amount of a prime editor fusion protein (e.g., PE1 or PE2) complexed with a lipid delivery system; (ii) delivery to a cell a mRNA or delivery complex comprising an mRNA
that encodes a prime editor fusion protein and/or a suitable pegRNA; and (iii) a DNA vector (e.g., an AAV or lentivirus vector, plasmid, or other nucleic acid delivery vector) that encodes a prime editor fusion protein and/or a suitable pegRNA on one or more DNA
vectors.
1441 In still another aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome) using prime editing while blocking, inhibiting, or otherwise inactivating POLS or variant thereof In another aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome), comprising contacting a target nucleotide molecule with a prime editor and an inhibitor of POLS. In various embodiments, the inhibitor can be a small molecule inhibitor. In other embodiments, the inhibitor can be an anti-POLS
antibody, e.g., a neutralizing antibody that inactivates POLS. In still other embodiments, the inhibitor can be a dominant negative mutant of POLS. In still other embodiments, the inhibitor can be targeted at the level of transcription of POLO, e.g., an si RNA or other nucleic acid agent that knocks down the level of a transcript encoding POLO. In yet other embodiments, the step of "contacting a target nucleotide molecule with a prime editor" can include (i) delivering directly

19/699 to a cell an effective amount of a prime editor fusion protein (e.g., PE1 or PE2) complexed with a lipid delivery system; (ii) delivery to a cell a mRNA or delivery complex comprising an mRNA
that encodes a prime editor fusion protein and/or a suitable pegRNA; and (iii) a DNA vector (e.g., an AAV or lentivirus vector, plasmid, or other nucleic acid delivery vector) that encodes a prime editor fusion protein and/or a suitable pegRNA on one or more DNA
vectors.
[451 In one aspect, the present disclosure provides methods for editing a nucleic acid molecule by prime editing. In some embodiments, the method comprises contacting a nucleic acid molecule with a prime editor, a pegRNA, and an inhibitor of the DNA mismatch repair pathway, thereby installing one or more modifications to the nucleic acid molecule at a target site.
[461 The method may increase the efficiency of prime editing and/or decrease the frequency of indel formation. In some embodiments, the prime editing efficiency is increased by at least 1.5-fold, at least 2.0-fold, at least 2.5-fold, at least 3.0-fold, at least 3.5-fold, at least 4.0-fold, at least 4.5-fold, at least 5.0-fold, at least 5.5-fold, at least 6.0-fold, at least 6.5-fold, at least 7.0-fold, at least 7.5-fold, at least 8.0-fold, at least 8.5-fold, at least 9.0-fold, at least 9.5-fold, or at least 10.0-fold in the presence of the inhibitor of the DNA mismatch repair pathway. In some embodiments, the frequency of indel formation is decreased by at least 1.5-fold, at least 2.0-fold, at least 2.5-fold, at least 3.0-fold, at least 3.5-fold, at least 4.0-fold, at least 4.5-fold, at least 5.0-fold, at least 5.5-fold, at least 6.0-fold, at least 6.5-fold, at least 7.0-fold, at least 7.5-fold, at least 8.0-fold, at least 8.5-fold, at least 9.0-fold, at least 9.5-fold, or at least 10.0-fold in the presence of the inhibitor of the DNA mismatch repair pathway.
[471 In some embodiments, the inhibitor of the DNA mismatch repair pathway inhibits one or more proteins of the DNA mismatch repair pathway. In some embodiments, the one or more proteins is selected from the group consisting of MLH1, PMS2 (or MutL alpha), PMS1 (or MutL
beta), MLH3 (or MutL gamma), MutS alpha (MSH2-MS116), MutS beta (MSH2-MSH3), MSH2, MSH6, PCNA, RFC, EX01, POL8, and PCNA. In certain embodiments, the one or more proteins is MLH.1. In some embodiments, MLH1 comprises an amino acid sequence of SEQ ID
NO: 204, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to and including 100% sequence identity with SEQ ID NO: 204.
[481 The inhibitor utilized in the method may be an antibody, a small molecule, a small interfering RNA (siRNA), a small non-coding microRNA, or a dominant negative variant of an

20/699 MMR protein that inhibits the activity of a wild type MMR protein (e.g., a dominant negative variant of MLH1). In certain embodiments, the inhibitor is an antibody that inhibits the activity of one or more proteins of the DNA mismatch repair pathway. In some embodiments, the inhibitor is a small molecule that inhibits the activity of one or more proteins of the DNA
mismatch repair pathway. In certain embodiments, the inhibitor is a small interfering RNA
(siRNA) or a small non-coding microRNA that inhibits the activity of one or more proteins of the DNA mismatch repair pathway. In some embodiments, the inhibitor is a dominant negative variant of MLH1 that inhibits MLH1.
1491 In some embodiments, the dominant negative variant is (a) MLH1 E34A (SEQ
ID NO:
222), (b) MLH1 A756 (SEQ ID NO: 208), (c) MLH1 A754-756 (SEQ ID NO: 209), (d) E34A M54-756 (SEQ ID NO: 210), (e) MLH1 1-335 (SEQ ID NO: 211), (f) MLH1 1-335 E34A (SEQ ID NO: 212), (g) MLH1 1-335 NLSsv40 (SEQ ID NO: 213), (h) MLH1 501-(SEQ ID NO: 215), (i) MLH I 501-753 (SEQ ID NO: 216), (j) MUD 461-753 (SEQ ID
NO:
218), or (k) NLSsv4 MLH1 501-753 (SEQ ID NO: 223), or a polypeptide comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to and including 100%
sequence identity with any of SEQ ID NOs: 208-213, 215, 216, 218, 222, or 223.
1501 The prime editors utilized in the methods of the present disclosure may comprise multiple components. In some embodiments, the prime editor comprises a napDNAbp and a polymerase.
In some embodiments, the napDNAbp is a nuclease active Cas9 domain, a nuclease inactive Cas9 domain, or a Cas9 nickase domain or variant thereof. In certain embodiments, the napDNAbp is selected from the group consisting of: Cas9, Cas12e, Cas12d, Cas12a, Cas12b1, Cas13a, Cas12c, Cas12b2, Cas13a, Cas12c, Cas12d, Casi2e, Cas12h, Cas12i, Cas12g, Cas12f (Cas14), Casl2f1, Cas12j (Cas(Io), and Argonaute and optionally has a nickase activity. In certain embodiments, the napDNAbp comprises an amino acid sequence of any one of SEQ
ID NOs: 2, 4-67, or 99 (PEmax) or an amino acid sequence having at least an 80%, 85%, 90%, 95%, or 99%
sequence identity with any one of SEQ NOs: 2,4-67, or 99 (PEmax). In certain embodiments, the napDNAbp comprises an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 37 (e.g., the napDNAbp of PEI and PE2) or an amino acid sequence having at least an 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 2. In some embodiments, the polymerase is a DNA-dependent DNA polymerase or an RNA-dependent DNA polymerase. In some embodiments, the

21/699 polymerase is a reverse transcriptase. In certain embodiments, the reverse transcriptase comprises an amino acid sequence of any one of SEQ ID NOs: 69-98 or an amino acid sequence having at least an 80%, 85%, 90%, 95%, or 99% sequence identity with any one of SEQ ID NOs:
69-98.
1511 The napDNAbp and the polymerase of the prime editor may be joined together to form a fusion protein. In some embodiments, the napDNAbp and the polymerase of the prime editor are joined by a linker to form a fusion protein. In certain embodiments, the linker comprises an amino acid sequence of any one of SEQ ID NOs: 102, or 118-131, or an amino acid sequence having at least an 80%, 85%, 90%, 95%, or 99% sequence identity with any one of SEQ ID NOs:
102, or 118-131. In some embodiments, the linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 38, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length.
1521 The components used in the method (e.g., the prime editor, the pegRNA, and/or the inhibitor of the DNA mismatch repair pathway) may be encoded on a DNA vector.
In some embodiments, the prime editor, the pegRNA, and the inhibitor of the DNA
mismatch repair pathway are encoded on one or more DNA vectors. In certain embodiments, the one or more DNA vectors comprise AAV or lentivirus DNA vectors. In some embodiments, the AAV vector is serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
1531 The prime editors utilized in the presently disclosed methods may also be further joined to additional components. In some embodiments, the prime editor as a fusion protein is further joined by a second linker to the inhibitor of the DNA mismatch repair pathway.
In certain embodiments, the second linker is a self-hydrolyzing linker. In certain embodiments, the second linker comprises an amino acid sequence of any one of SEQ ID NOs: 102, 118-131, or 233-236, or an amino acid sequence having at least an 80%, 85%, 90%, 95%, or 99%
sequence identity with any one of SEQ ID NOs: 102, 118-131, or 233-236. In some embodiments, the second linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46,47, 48, 49, or 50 amino acids in length.
[541 lEn some embodiments, the one or more modifications to the nucleic acid molecule installed at the target site comprise one or more transitions, one or more transversions, one or more insertions, one or more deletions, or one more inversions. In certain embodiments, the one

22/699 or more transitions are selected from the group consisting of: (a) T to C; (b) A to G; (c) C to T;
and (d) G to A. In certain embodiments, the one or more transversions are selected from the group consisting of: (a) T to A; (b) T to G; (c) C to G; (d) C to A; (e) A to T; (f) A to C; (g) G to C; and (h) G to T. In certain embodiments, the one or more modifications comprises changing (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 a 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. In some embodiments, the one or more modifications comprises 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.
[55] The methods of the present disclosure may be used for making corrections to one or more disease-associated genes. In some embodiments, the one or more modifications comprises a correction to a disease-associated gene. In certain embodiments, 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.
In certain embodiments, the disease-associated gene is associated with a monogenic disorder selected from the group consisting of: Adenosine Deaminase (ADA) Deficiency; Alpha-1 Antitrypsin Deficiency; Cystic Fibrosis; Duchenne Muscular Dystrophy; Galactosemia;
Hemochromatosis;
Huntington's Disease; Maple Syrup Urine Disease; Madan Syndrome;
Neurofibromatosis Type 1; Pachyonychia Congenita; Phenylketonuria; Severe Combined Immunodeficiency;
Sickle Cell Disease; Smith-Lemli-Opitz Syndrome; a trinucleotide repeat disorder; a prion disease; and Tay-Sachs Disease.
[561 In another aspect, the present disclosure provides compositions for editing a nucleic acid molecule by prime editing. In some embodiments, the composition comprises a prime editor, a pegRNA, and an inhibitor of the DNA mismatch repair pathway, wherein the composition is capable of installing one or more modifications to the nucleic acid molecule at a target site.
[571 The composition may increase the efficiency of prime editing and/or decrease the frequency of indel formation. In some embodiments, the prime editing efficiency is increased by at least 1.5-fold, at least 2.0-fold, at least 2.5-fold, at least 3.0-fold, at least 3.5-fold, at least 4.0-fold, at least 4.5-fold, at least 5.0-fold, at least 5.5-fold, at least 6.0-fold, at least 6.5-fold, at least

23/699 7.0-fold, at least 7.5-fold, at least 8.0-fold, at least 8.5-fold, at least 9.0-fold, at least 9.5-fold, or at least 10.0-fold in the presence of the inhibitor of the DNA mismatch repair pathway. In some embodiments, the frequency of indel formation is decreased by at least 1.5-fold, at least 2.0-fold, at least 2.5-fold, at least 3.0-fold, at least 3.5-fold, at least 4.0-fold, at least 4.5-fold, at least 5.0-fold, at least 5.5-fold, at least 6.0-fold, at least 6.5-fold, at least 7.0-fold, at least 7.5-fold, at least 8.0-fold, at least 8.5-fold, at least 9.0-fold, at least 9.5-fold, or at least 10.0-fold in the presence of the inhibitor of the DNA mismatch repair pathway.
1581 In some embodiments, the inhibitor of the DNA mismatch repair pathway inhibits one or more proteins of the DNA mismatch repair pathway. In some embodiments, the one or more proteins is selected from the group consisting of MLH1, PMS2 (or MutL alpha), PMS1 (or MutL
beta), MLH3 (or MutL gamma), MutS alpha (MSH2-MSH6), MutS beta (MSH2-MSH3), MSH2, MSH6, PCNA, RFC, EX01, POLo, and PCNA. In certain embodiments, the one or more proteins is MLH1. In some embodiments, MLH1 comprises an amino acid sequence of SEQ ID
NO: 204, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to and including 100% sequence identity with SEQ ID NO: 204.
1591 The inhibitor utilized in the composition may be an antibody, a small molecule, a small interfering RNA (siRNA), a small non-coding microRNA, or a dominant negative variant of an MMR protein that inhibits the activity of a wild type MMR protein (e.g., a dominant negative variant of MLH1). In certain embodiments, the inhibitor is an antibody that inhibits the activity of one or more proteins of the DNA mismatch repair pathway. In some embodiments, the inhibitor is a small molecule that inhibits the activity of one or more proteins of the DNA
mismatch repair pathway. In certain embodiments, the inhibitor is a small interfering RNA
(siRNA) or a small non-coding microRNA that inhibits the activity of one or more proteins of the DNA mismatch repair pathway. In some embodiments, the inhibitor is a dominant negative variant of MLH1 that inhibits MLH1.
1601 In some embodiments, the dominant negative variant is (a) MLH1 E34A (SEQ
ID NO:
222), (b) MLH1 A756 (SEQ ID NO: 208), (c) MLH1 A754-756 (SEQ ID NO: 209), (d) E34A A754-756 (SEQ ID NO: 210), (e) MLLE 1-335 (SEQ ID NO: 211), (0 MLH1 1-335 E34A (SEQ ID NO: 212), (g) MLH1 1-335 NLSsys (SEQ ID NO: 213), (h) MLH1 501-(SEQ ID NO: 215), (i)MLH1 501-753 (SEQ ID NO: 216), (j) MLH1 461-753 (SEQ ED
NO:

24/699 218), or (k) NLSsv4 MUD 501-753 (SEQ ID NO: 223), or a polypeptide comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to and including 100%
sequence identity with any of SEQ ID NOs: 208-213, 215, 216, 218, 222, or 223.
1611 The prime editors utilized in the compositions of the present disclosure comprise multiple components. In some embodiments, the prime editor comprises a napDNAbp and a polymerase.
In some embodiments, the napDNAbp is a nuclease active Cas9 domain, a nuclease inactive Cas9 domain, or a Cas9 nickase domain or variant thereof. In certain embodiments, the napDNAbp is selected from the group consisting of: Cas9, Cas12e, Cas12d, Cas12a, Cas12b1, Cas13a, Cas12c, Cas12b2, Cas13a, Cas12c, Cas12d, Cas12e, Cas12h, Cas12i, Cas12g, Casl 2f (Cas14), Casl2f1, Cas12j (Cas(I)), and Argonaute and optionally has a nickase activity. In certain embodiments, the napDNAbp comprises an amino acid sequence of any one of SEQ
ID NOs: 2, 4-67, or 99 (PEmax) or an amino acid sequence having at least an 80%, 85%, 90%, 95%, or 99%
sequence identity with any one of SEQ ID NOs: 2, 4-67, or 99 (PEmax). In certain embodiments, the napDNAbp comprises an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 37 (i.e., the napDNAbp of PEI and PE2) or an amino acid sequence having at least an 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 2 or SEQ ID NO: 37. In some embodiments, the polymerase is a DNA-dependent DNA polymerase or an RNA-dependent DNA
polymerase. In some embodiments, the polymerase is a reverse transcriptase. In certain embodiments, the reverse transcriptase comprises an amino acid sequence of any one of SEQ ID
NOs: 69-98 or an amino acid sequence having at least an 80%, 85%, 90%, 95%, or 99% sequence identity with any one of SEQ ID NOs: 69-98.
1621 The napDNAbp and the polymerase of the prime editor may be joined together to form a fusion protein. In some embodiments, the napDNAbp and the polymerase of the prime editor are joined by a linker to form a fusion protein. In certain embodiments, the linker comprises an amino acid sequence of any one of SEQ ID NOs: 102, 118-131, or an amino acid sequence having at least an 80%, 85%, 90%, 95%, or 99% sequence identity with any one of SEQ ID NOs:
102, 118-131. In some embodiments, the linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 38, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length.

25/699 [631 The components used in the compositions disclosed herein (e.g., the prime editor, the pegRNA, and/or the inhibitor of the DNA mismatch repair pathway) may be encoded on a DNA
vector. In some embodiments, the prime editor, the pegRNA, and the inhibitor of the DNA
mismatch repair pathway are encoded on one or more DNA vectors. In certain embodiments, the one or more DNA vectors comprise AAV or lentivirus DNA vectors. In some embodiments, the AAV vector is serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
[641 The prime editors utilized in the presently disclosed compositions may also be further joined to additional components. In some embodiments, the prime editor as a fusion protein is further joined by a second linker to the inhibitor of the DNA mismatch repair pathway. In certain embodiments, the second linker is a self-hydrolyzing linker. In certain embodiments, the second linker comprises an amino acid sequence of any one of SEQ ID NOs: 102, 118-131, or 233-236, or an amino acid sequence having at least an 80%, 85%, 90%, 95%, or 99%
sequence identity with any one of SEQ ID NOs: 102, 118-131, or 233-236. In some embodiments, the second linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length.
[651 In some embodiments, the one or more modifications to the nucleic acid molecule installed at the target site comprise one or more transitions, one or more transversions, one or more insertions, one or more deletions, or one more inversions. In certain embodiments, the one or more transitions are selected from the group consisting of: (a) T to C; (b) A to G; (c) C to T;
and (d) G to A. In certain embodiments, the one or more transversions are selected from the group consisting of: (a) T to A; (b) T to G; (c) C to G; (d) C to A; (e) A to T; (f) A to C; (g) G to C; and (h) G to T. In certain embodiments, the one or more modifications comprises changing (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 a 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. In some embodiments, the one or more modifications comprises 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.

26/699 [661 The compositions of the present disclosure may be used for making corrections to one or more disease-associated genes. In some embodiments, the one or more modifications comprises a correction to a disease-associated gene. In certain embodiments, 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.
In certain embodiments, the disease-associated gene is associated with a monogenic disorder selected from the group consisting of: Adenosine Deaminase (ADA) Deficiency; Alpha-1 Antitrypsin Deficiency; Cystic Fibrosis; Duchenne Muscular Dystrophy; Galactosemia;
Hemochromatosis;
Huntington's Disease; Maple Syrup Urine Disease; Marfan Syndrome;
Neurofibromatosis Type 1; Pachyonychia Congenita; Phenylketonuria; Severe Combined Immunodeficiency;
Sickle Cell Disease; Smith-Lemli-Opitz Syndrome; a trinucleotide repeat disorder; a prion disease; and Tay-Sachs Disease.
[671 In another aspect, this disclosure provides polynucleotides for editing a DNA target site by prime editing. In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding a napDNAbp, a polymerase, and an inhibitor of the DNA mismatch repair pathway, wherein the napDNAbp and polymerase is capable in the presence of a pegRNA of installing one or more modifications in the DNA target site.
[681 The polynucleotide may increase the efficiency of prime editing and/or decrease the frequency of indel formation. In some embodiments, the prime editing efficiency is increased by at least 1.5-fold, at least 2.0-fold, at least 2.5-fold, at least 3.0-fold, at least 3.5-fold, at least 4.0-fold, at least 4.5-fold, at least 5.0-fold, at least 5.5-fold, at least 6.0-fold, at least 6.5-fold, at least 7.0-fold, at least 7.5-fold, at least 8.0-fold, at least 8.5-fold, at least 9.0-fold, at least 9.5-fold, or at least 10.0-fold in the presence of the inhibitor of the DNA mismatch repair pathway. In some embodiments, the frequency of indel formation is decreased by at least 1.5-fold, at least 2.0-fold, at least 2.5-fold, at least 3.0-fold, at least 3.5-fold, at least 4.0-fold, at least 4.5-fold, at least 5.0-fold, at least 5.5-fold, at least 6.0-fold, at least 6.5-fold, at least 7.0-fold, at least 7.5-fold, at least 8.0-fold, at least 8.5-fold, at least 9.0-fold, at least 9.5-fold, or at least 10.0-fold in the presence of the inhibitor of the DNA mismatch repair pathway.
[691 In some embodiments, the inhibitor of the DNA mismatch repair pathway inhibits one or more proteins of the DNA mismatch repair pathway. In some embodiments, the one or more proteins is selected from the group consisting of MLH1, PMS2 (or MutL alpha), PMS1 (or MutL

27/699 beta), MUD (or MutL gamma), MutS alpha (MSI12-MSH6), MutS beta (MSH2-MSH3), MSH2, MSH6, PCNA, RFC, EX01, POL8, and PCNA. In certain embodiments, the one or more proteins is MLF11. In some embodiments, MLH1 comprises an amino acid sequence of SEQ ID
NO: 204, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to and including 100% sequence identity with SEQ ID NO: 204.
[701 The inhibitor utilized in the polynucleotide may be an antibody, a small molecule, a small interfering RNA (siRNA), a small non-coding microRNA, or a dominant negative variant of an MIVIR protein that inhibits the activity of a wild type MMR protein (e.g., a dominant negative variant of (MLLE). In certain embodiments, the inhibitor is an antibody that inhibits the activity of one or more proteins of the DNA mismatch repair pathway. In some embodiments, the inhibitor is a small molecule that inhibits the activity of one or more proteins of the DNA
mismatch repair pathway. In certain embodiments, the inhibitor is a small interfering RNA
(siRNA) or a small non-coding microRNA that inhibits the activity of one or more proteins of the DNA mismatch repair pathway. in some embodiments, the inhibitor is a dominant negative variant of MLH1 that inhibits MLH1.
1711 In some embodiments, the dominant negative variant is (a) MLH1 E34A (SEQ
ID NO:
222), (b) MLH1 A756 (SEQ ID NO: 208), (c) MLH1 M54-756 (SEQ ID NO: 209), (d) E34A A754-756 (SEQ ID NO: 210), (e) MLH1 1-335 (SEQ ID NO: 211), (f) MLH1 1-E34A (SEQ ID NO: 212), (g) MLH1 1-335 NLSsv" (SEQ ID NO: 213), (h) MLH1 501-(SEQ ID NO: 215), (i) MLH1 501-753 (SEQ ID NO: 216), (j) MLH1 461-753 (SEQ ID
NO:
218), or (k)NLSsm MLH1 501-753 (SEQ ID NO: 223), or a polypeptide comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to and including 100%
sequence identity with any of SEQ ID NOs: 208-213, 215, 216, 218, 222, or 223.
1721 The prime editors utilized in the polynucleotides of the present disclosure comprise multiple components (e.g., a napDNAbp and a polymerase). In some embodiments, the napDNAbp is a nuclease active Cas9 domain, a nuclease inactive Cas9 domain, or a Cas9 nickase domain or variant thereof. In certain embodiments, the napDNAbp is selected from the group consisting of: Cas9, Cas12e, Cas12d, Casi2a, Cas12b1, Cas13a, Cas12c, Cas12b2, Cas13a, Cas12c, Cas12d, Cas12e, Cas12h, Cas12i, Cas12g, Cas12f (Cas14), Casl2f1, Cas12j

28/699 (Case), and Argonaute and optionally has a nickase activity. In certain embodiments, the napDNAbp comprises an amino acid sequence of any one of SEQ ID NOs: 2, 4-67, or 99 (PEmax) or an amino acid sequence having at least an 80%, 85%, 90%, 95%, or 99% sequence identity with any one of SEQ ID NOs: 2,4-67, or 99 (PEmax). In certain embodiments, the napDNAbp comprises an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 37 (i.e., the napDNAbp of PE1 and PE2) or an amino acid sequence having at least an 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 2 or SEQ ID NO: 37. In some embodiments, the polymerase is a DNA-dependent DNA polymerase or an RNA-dependent DNA
polymerase. In some embodiments, the polymerase is a reverse transcriptase. In certain embodiments, the reverse transcriptase comprises an amino acid sequence of any one of SEQ TD
NOs: 69-98 or an amino acid sequence having at least an 80%, 85%, 90%, 95%, or 99% sequence identity with any one of SEQ ID NOs: 69-98.
[731 The napDNAbp and the polymerase of the prime editor may be joined together to form a fusion protein. In some embodiments, the napDNAbp and the polymerase of the prime editor are joined by a linker to form a fusion protein. In certain embodiments, the linker comprises an amino acid sequence of any one of SEQ ID NOs: 102, 118-131, or an amino acid sequence having at least an 80%, 85%, 90%, 95%, or 99% sequence identity with any one of SEQ ID NOs:
102, 118-131. In some embodiments, the linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 38, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length.
[741 The polynucleotides disclosed herein may comprise vectors. In some embodiments, the polynucleotide is a DNA vector. In certain embodiments, the DNA vector is an AAV or lentivirus DNA vector. In some embodiments, the AAV vector is serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
[751 The prime editors encoded by the presently disclosed polynucleotides may also be further joined to additional components. In some embodiments, the prime editor as a fusion protein is further joined by a second linker to the inhibitor of the DNA mismatch repair pathway. In certain embodiments, the second linker comprises a self-hydrolyzing linker. In certain embodiments, the second linker comprises an amino acid sequence of any one of SEQ ID NOs: 102, 118-131, or 233-236, or an amino acid sequence having at least an 80%, 85%, 90%, 95%, or 99% sequence identity with any one of SEQ ID NOs: 102, 118-131, or 233-236. In some embodiments, the

29/699 second linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length.
[761 In some embodiments, the one or more modifications to the nucleic acid molecule installed at the target site comprise one or more transitions, one or more transversions, one or more insertions, one or more deletions, or one more inversions. In certain embodiments, the one or more transitions are selected from the group consisting of: (a) T to C; (b) A to G; (c) C to T;
and (d) G to A. In certain embodiments, the one or more transversions are selected from the group consisting of: (a) T to A; (b) T to G; (c) C to G; (d) C to A; (e) A to T; (f) A to C; (g) G to C; and (h) G to T. In certain embodiments, the one or more modifications comprises changing (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 a 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. In some embodiments, the one or more modifications comprises 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.
1771 The polynucleotides of the present disclosure may be used for making corrections to one or more disease-associated genes. In some embodiments, the one or more modifications comprises a correction to a disease-associated gene. In certain embodiments, 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.
In certain embodiments, the disease-associated gene is associated with a monogenic disorder selected from the group consisting of: Adenosine Deaminase (ADA) Deficiency;
Alpha-1 Antitrypsin Deficiency; Cystic Fibrosis; Duchenne Muscular Dystrophy;
Galactosemia;
Hemochromatosis; Huntington's Disease; Maple Syrup Urine Disease; Marfan Syndrome;
Neurofibromatosis Type 1; Pachyonychia Congenita; Phenylketonuria; Severe Combined Immunodeficiency; Sickle Cell Disease; Smith-Lemli-Opitz Syndrome; a trinucleotide repeat disorder; a prion disease; and Tay-Sachs Disease.
[781 In another aspect, the present disclosure provides cells. In some embodiments, the cell comprises any of the polynucleotides described herein.

30/699 1791 In another aspect, the present disclosure provides pharmaceutical compositions. In some embodiments, the pharmaceutical composition comprises any of the compositions disclosed herein. In some embodiments, the pharmaceutical composition comprises any of the compositions disclosed herein and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises any of the polynucleotides disclosed herein. In some embodiments, the pharmaceutical composition comprises any of the polynucleotides disclosed herein and a pharmaceutically acceptable excipient.
1801 In another aspect, the present disclosure provides kits. In some embodiments, the kit comprises any of the compositions disclosed herein, a pharmaceutical excipient, and instructions for editing a DNA target site by prime editing. In some embodiments, the kit comprises any of the polynucleotides disclosed herein, a pharmaceutical excipient, and instructions for editing a DNA target site by prime editing.
1811 The present disclosure also provides methods and pegRNAs for prime editing whereby correction by the MMR pathway of the alterations introduced into a target nucleic acid molecule is evaded, without the need to provide an inhibitor of the MMR pathway.
Surprisingly, pegRNAs designed with consecutive nucleotide mismatches compared to the endogenous sequence of a target site on a target nucleic acid, for example, pegRNAs that have three or more consecutive mismatching nucleotides, can evade correction by the MMR pathway, resulting in an increase in prime editing efficiency and a decrease in the frequency of indel formation compared to the introduction of a single nucleotide mismatch using prime editing. In addition, insertions or deletions of consecutive nucleotides at the target site of the target nucleic acid, for example, insertions or deletions greater than 10 nucleotides in length, introduced by prime editing also evade correction by the MMR pathway, resulting in an increase in prime editing efficiency and a decrease in the frequency of indel formation compared to the introduction of an insertion or deletion of less than 10 nucleotides in length using prime editing.
1821 Thus, in another aspect, the present disclosure provides methods for editing a nucleic acid molecule by prime editing comprising contacting a nucleic acid molecule with a prime editor (e.g., PE2, PE3, or any of the other prime editors described herein) and a pegRNA with a DNA
synthesis template on its extension arm comprising three or more consecutive nucleotide mismatches relative to the endogenous sequence of a target site on the nucleic acid molecule. In some embodiments, at least one of the consecutive nucleotide mismatches results in an alteration

31/699 in the amino acid sequence of a protein expressed from the nucleic acid molecule, while at least one of the remaining nucleotide mismatches is a silent mutation. The silent mutations may be in coding regions of the target nucleic acid molecule (i.e., in a part of a gene that encodes a protein), or the silent mutations may be in non-coding regions of the target nucleic acid molecule. In some embodiments, when the silent mutations are in a coding region, the silent mutations introduce into the nucleic acid molecule one or more alternate codons encoding the same amino acid as the unedited nucleic acid molecule. In some embodiments, when the silent mutations are in a non-coding region, the silent mutations are present in a region of the nucleic acid molecule that does not influence splicing, gene regulation, RNA lifetime, or other biological properties of the target site on the nucleic acid molecule.
[831 Any number of consecutive nucleotide mismatches compared to the sequence of the target site can be designed in the DNA synthesis template of a pegRNA to achieve the benefits of evading correction by the IMMR pathway, and thereby increase prime editing efficiency and/or reduce indel formation. In some embodiments, the DNA synthesis template comprises at least three consecutive nucleotide mismatches compared to the sequence of the target site. In some embodiments, the DNA synthesis template of the extension arm on the pegRNA
comprises 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 consecutive nucleotide mismatches relative to the endogenous sequence of a target site in the nucleic acid molecule edited by prime editing. In some embodiments, the DNA synthesis template of the extension arm on the pegRNA comprises three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more consecutive nucleotide mismatches relative to the endogenous sequence of a target site on the nucleic acid molecule. In certain embodiments, the use of three or more consecutive nucleotide mismatches results in an increase in prime editing efficiency by at least 1.5-fold, at least 2.0-fold, at least 2.5-fold, at least 3.0-fold, at least 3.5-fold, at least 4.0-fold, at least 4.5-fold, at least 5.0-fold, at least 5.5-fold, at least 6.0-fold, at least 6.5-fold, at least 7.0-fold, at least 7.5-fold, at least 8.0-fold, at least 8.5-fold, at least 9.0-fold, at least 9.5-fold, or at least 10.0-fold relative to a method using a pegRNA comprising a DNA synthesis template comprising only one consecutive nucleotide mismatch relative to tbe endogenous sequence of a target site on the nucleic acid molecule. In certain embodiments, the use of three or more consecutive nucleotide mismatches results in a decrease in the frequency of indel formation by at least 1.5-fold, at least 2.0-fold, at least 2.5-fold, at least 3.0-fold, at least

32/699 3.5-fold, at least 4.0-fold, at least 4.5-fold, at least 5.0-fold, at least 5.5-fold, at least 6.0-fold, at least 6.5-fold, at least 7.0-fold, at least 7.5-fold, at least 8.0-fold, at least 8.5-fold, at least 9.0-fold, at least 9.5-fold, or at least 10.0-fold relative to a method using a pegRNA comprising a DNA synthesis template comprising only one consecutive nucleotide mismatch relative to the endogenous sequence of a target site on the nucleic acid molecule.
[841 In another aspect, the present disclosure provides methods for editing a nucleic acid molecule by prime editing comprising contacting a nucleic acid molecule with a prime editor (e.g., PE2, PE3, or any of the other prime editors described herein) and a pegRNA with a DNA
synthesis template on its extension arm comprising an insertion or deletion of 10 or more contiguous nucleotides relative to the endogenous sequence of a target site on the nucleic acid molecule. In some embodiments, the DNA synthesis template of a pegRNA can be designed to introduce insertions or deletions greater than 3 nucleotides to avoid or reduce the impact of mismatch correction by the cellular MMR. pathway, thereby improving prime editing efficiency.
In some embodiments, the DNA synthesis template of the pegRNA is designed to introduce one or more insertions and/or deletions of 3, 4, 5, 6, 7, 8, 9, 10, or more contiguous nucleotides to avoid or reduce the impact of mismatch correction by the cellular MlvIR
pathway, thereby improving prime editing efficiency. In some embodiments, insertions or deletions of any length greater than 10 contiguous nucleotides can be used to achieve the benefits of evading correction by the MMR pathway. In some embodiments, the DNA synthesis template comprises an insertion of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, or 25 contiguous nucleotides relative to the endogenous sequence of a target site on a nucleic acid molecule edited by prime editing. In some embodiments, the DNA synthesis template comprises a deletion of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 contiguous nucleotides relative to the endogenous sequence of a target site on a nucleic acid molecule edited by prime editing. In some embodiments, the DNA synthesis template comprises an insertion of 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, or 40 contiguous nucleotides relative to the endogenous sequence of a target site on a nucleic acid molecule edited by prime editing. In some embodiments, the DNA synthesis template comprises a deletion of 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, or 40 contiguous nucleotides relative to the endogenous sequence of a target site on a nucleic acid molecule edited by prime

33/699 editing. In some embodiments, the DNA synthesis template comprises an insertion or deletion of 11 or more contiguous nucleotides, 12 or more contiguous nucleotides, 13 or more contiguous nucleotides, 14 or more contiguous nucleotides, 15 or more contiguous nucleotides, 16 or more contiguous nucleotides, 17 or more contiguous nucleotides, 18 or more contiguous nucleotides, 19 or more contiguous nucleotides, 20 or more contiguous nucleotides, 21 or more contiguous nucleotides, 22 or more contiguous nucleotides, 23 or more contiguous nucleotides, 24 or more contiguous nucleotides, or 25 or more contiguous nucleotides relative to a target site on a nucleic acid molecule. In certain embodiments, the DNA synthesis template comprises an insertion or deletion of 15 or more contiguous nucleotides relative to the endogenous sequence of a target site on the nucleic acid molecule.
[851 In some embodiments, prime editing with a pegRNA designed to introduce an insertion and/or deletion of multiple contiguous nucleotides, for example, three or more contiguous nucleotides, relative to the endogenous sequence of a target site results in an increase in prime editing efficiency compared to prime editing with a corresponding control pegRNA (e.g., a control pegRNA that does not introduce an insertion or deletion of three or more contiguous nucleotides) by at least 1.5-fold, at least 2.0-fold, at least 2.5-fold, at least 3.0-fold, at least 3.5-fold, at least 4.0-fold, at least 4.5-fold, at least 5.0-fold, at least 5.5-fold, at least 6.0-fold, at least 6.5-fold, at least 7.0-fold, at least 7.5-fold, at least 8.0-fold, at least 8.5-fold, at least 9.0-fold, at least 9.5-fold, or at least 10.0-fold. In some embodiments, prime editing with a pegRNA
designed to introduce an insertion or deletion of 3, 4, 5, 6, 7, 8, 9, 10, or more contiguous nucleotides relative to the endogenous sequence of a target site results in an increase in prime editing efficiency relative to prime editing with a corresponding control pegRNA (e.g., a control pegRNA that does not introduce insertion or deletion of the three or more contiguous nucleotides) by at least 1.5-fold, at least 2.0-fold, at least 2.5-fold, at least 3.0-fold, at least 3.5-fold, at least 4.0-fold, at least 4.5-fold, at least 5.0-fold, at least 5.5-fold, at least 6.0-fold, at least 6.5-fold, at least 7.0-fold, at least 7.5-fold, at least 8.0-fold, at least 8.5-fold, at least 9.0-fold, at least 9.5-fold, or at least 10.0-fold. In some embodiments, making an insertion or deletion of 10 or more contiguous nucleotides results in an increase in prime editing efficiency by at least 1.5-fold, at least 2.0-fold, at least 2.5-fold, at least 3.0-fold, at least 3.5-fold, at least 4.0-fold, at least 4.5-fold, at least 5.0-fold, at least 5.5-fold, at least 6.0-fold, at least 6.5-fold, at least 7.0-fold, at least 7.5-fold, at least 8.0-fold, at least 8.5-fold, at least 9.0-fold, at least 9.5-fold, or at least 10.0-

34/699 fold relative to a method using a pegRNA comprising a DNA synthesis template comprising an insertion or deletion of fewer than 10 nucleotides relative to the endogenous sequence of a target site on the nucleic acid molecule. In some embodiments, making an insertion or deletion of 10 or more nucleotides results in a decrease in the frequency of indel formation by at least 1.5-fold, at least 2.0-fold, at least 2.5-fold, at least 3.0-fold, at least 3.5-fold, at least 4.0-fold, at least 4.5-fold, at least 5.0-fold, at least 5.5-fold, at least 6.0-fold, at least 6.5-fold, at least 7.0-fold, at least 7.5-fold, at least 8.0-fold, at least 8.5-fold, at least 9.0-fold, at least 9.5-fold, or at least 10.0-fold relative to a method using a pegRNA comprising a DNA synthesis template comprising an insertion or deletion of fewer than 10 nucleotides relative to the endogenous sequence of a target site on the nucleic acid molecule.
[861 In another aspect, the present disclosure also provides pegRNAs useful for editing a nucleic acid molecule by prime editing while evading correction by the MMR
pathway of the alterations introduced into the nucleic acid molecule, thereby increasing prime editing efficiency and/or reducing indel formation. In some embodiments, the extension arm of the pegRNAs provided by the present disclosure comprise three or more consecutive nucleotide mismatches relative to the endogenous sequence of a target site on the nucleic acid molecule. In some embodiments, at least one of the three consecutive nucleotide mismatches relative to the endogenous sequence of the target site is a silent mutation. In some embodiments, at least one of the consecutive nucleotide mismatches results in an alteration in the amino acid sequence of a protein expressed from the target nucleic acid molecule, while at least one of the remaining nucleotide mismatches is a silent mutation. The silent mutations may be in coding regions of the target nucleic acid molecule (i.e. ,in a part of a gene that encodes a protein), or the silent mutations may be in non-coding regions of the target nucleic acid molecule. In some embodiments, when the silent mutations are in a coding region, the silent mutations introduce into the nucleic acid molecule one or more alternate codons encoding the same amino acid as the unedited nucleic acid molecule. In some embodiments, when the silent mutations are in a non-coding region, the silent mutations are present in a region of the nucleic acid molecule that does not influence splicing, gene regulation, RNA lifetime, or other biological properties of the target site on the nucleic acid molecule.
[871 Any number of consecutive nucleotide mismatches of three or more can be incorporated into the extension arm of the pegRNAs described herein to achieve the benefits of evading

35/699 correction by the MMR pathway. In some embodiments, the DNA synthesis template of the extension arm of the pegRNA comprises 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 consecutive nucleotide mismatches relative to the endogenous sequence of a target site on the nucleic acid molecule edited by prime editing. In some embodiments, the DNA synthesis template of the extension arm of the pegRNA
comprises at least three consecutive nucleotide mismatches relative to the endogenous sequence of a target site on the nucleic acid molecule edited by prime editing. In some embodiments, the DNA
synthesis template of the extension arm of the pegRNA comprises 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive nucleotide mismatches relative to the endogenous sequence of a target site on the nucleic acid molecule edited by prime editing. In some embodiments, the DNA synthesis template of the extension arm of the pegRNA
comprises 3, 4, 5, 6, 7, 8, 9, or 10 consecutive nucleotide mismatches relative to the endogenous sequence of a target site on the nucleic acid molecule edited by prime editing. In some embodiments, the DNA
synthesis template of the extension arm on the pegRNA comprises three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more consecutive nucleotide mismatches relative to the endogenous sequence of a target site on the nucleic acid molecule. In certain embodiments, the presence of three or more consecutive nucleotide mismatches on the extension arm of the pegRNA results in an increase in prime editing efficiency by at least 1.5-fold, at least 2.0-fold, at least 2.5-fold, at least 3.0-fold, at least 3.5-fold, at least 4.0-fold, at least 4.5-fold, at least 5.0-fold, at least 5.5-fold, at least 6.0-fold, at least 6.5-fold, at least 7.0-fold, at least 7.5-fold, at least 8.0-fold, at least 8.5-fold, at least 9.0-fold, at least 9.5-fold, or at least 10.0-fold relative to a pegRNA comprising a DNA
synthesis template comprising only one consecutive nucleotide mismatch relative to the endogenous sequence of a target site on the nucleic acid molecule. In certain embodiments, the use of three or more consecutive nucleotide mismatches results in a decrease in the frequency of indel formation by at least 1.5-fold, at least 2.0-fold, at least 2.5-fold, at least 3.0-fold, at least 3.5-fold, at least 4.0-fold, at least 4.5-fold, at least 5.0-fold, at least 5.5-fold, at least 6.0-fold, at least 6.5-fold, at least 7.0-fold, at least 7.5-fold, at least 8.0-fold, at least 8.5-fold, at least 9.0-fold, at least 9.5-fold, or at least 10.0-fold relative to a pegRNA comprising a DNA synthesis template comprising only one consecutive nucleotide mismatch relative to the endogenous sequence of a target site on the nucleic acid molecule.

36/699 [881 In another aspect, the present disclosure provides a prime editor system for site specific genome modification comprising (a) a prime editor comprising (i) a nucleic acid programmable DNA binding protein (napDNAbp) and (ii) a DNA polymerase, and (b) an inhibitor of the DNA
mismatch repair pathway. In some embodiments, the inhibitor of the DNA
mismatch repair pathway inhibits one or more proteins of the DNA mismatch repair pathway (e.g., MLH1, PMS2 (or MutL alpha), PMS1 (or MutL beta), MLH3 (or MutL gamma), MutS alpha (MSH2-MSH6), MutS beta (MSH2-MSH3), MSH2, MSH6, PCNA, RFC, EX01, POLE, and/or PCNA). In some embodiments, the one or more proteins is MLH1. In certain embodiments, the MLH1 comprises an amino acid sequence of SEQ ID NO: 204, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to and including 100% sequence identity with SEQ ID NO: 204.
[891 Any inhibitor of the DNA mismatch repair pathway may be used in the systems described herein. In some embodiments, the inhibitor is an antibody that inhibits the activity of one or more proteins of the DNA mismatch repair pathway. In some embodiments, the inhibitor is a small molecule that inhibits the activity of one or more proteins of the DNA
mismatch repair pathway.
In some embodiments, the inhibitor is a small interfering RNA (siRNA) or a small non-coding microRNA that inhibits the activity of one or more proteins of the DNA
mismatch repair pathway. In some embodiments, the inhibitor is a dominant negative variant of an MMR protein that inhibits the activity of a wild type MMR protein (e.g., a dominant negative variant of MLH1 that inhibits MLH1).
[901 In certain embodiments, the dominant negative variant used in the systems of the present disclosure is (a) MLH1 E34A (SEQ ID NO: 222), (b) MLH1 A756 (SEQ ID NO: 208), (c) MLH1 A754-756 (SEQ ID NO: 209), (d) MLH1 E34A A754-756 (SEQ ID NO: 210), (e) 1-335 (SEQ ID NO: 211), (f) MLH1 1-335 E34A (SEQ ED NO: 212), (g) MLH1 1-335 NLSSV40 (SEQ ID NO: 213), (h) MLH1 501-756 (SEQ ID NO: 215), (i) MLH1 501-753 (SEQ
ID NO: 216), (j) MLH1 461-753 (SEQ ID NO: 218), or (k) NLSSV40 MLIT1 501-753 (SEQ ID
NO: 223), or a polypeptide comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to and including 100% sequence identity with any of SEQ ID .N0s:
208-213, 215, 216, 218, 222, or 223. The present disclosure also contemplates methods for performing prime editing on a nucleic acid molecule in a cell in which MMR
activity is knocked

37/699 out entirely (e.g., by knocking down one or more genes involved in the MMR
pathway in the genome of the cell). Such methods provide the benefits of inhibiting MMR
(e.g., improved editing efficiency and decreased indel formation) without the need to provide an inhibitor of MMR. Thus, in another aspect, the present disclosure provides methods for editing a nucleic acid molecule by prime editing comprising: contacting a nucleic acid molecule with a prime editor and a pegRNA, thereby installing one or more modifications to the nucleic acid molecule at a target site, wherein the nucleic acid molecule is in a cell comprising a knockout of one or more genes involved in the DNA mismatch repair (MMR) pathway. In some embodiments, the method further comprises contacting the nucleic acid molecule with a second strand nicking gRNA. In certain embodiments, the prime editing efficiency is increased by at least 1.5-fold, at least 2.0-fold, at least 2.5-fold, at least 3.0-fold, at least 3.5-fold, at least 4.0-fold, at least 4.5-fold, at least 5.0-fold, at least 5.5-fold, at least 6.0-fold, at least 6.5-fold, at least 7.0-fold, at least 7.5-fold, at least 8.0-fold, at least 8.5-fold, at least 9.0-fold, at least 9.5-fold, or at least 10.0-fold relative to a method performed in a cell that does not comprise a knockout of one or more genes involved in MMR. In certain embodiments, the frequency of indel formation is decreased by at least I.5-fold, at least 2.0-fold, at least 2.5-fold, at least 3.0-fold, at least 3.5-fold, at least 4.0-fold, at least 4.5-fold, at least 5.0-fold, at least 5.5-fold, at least 6.0-fold, at least 6.5-fold, at least 7.0-fold, at least 7.5-fold, at least 8.0-fold, at least 8.5-fold, at least 9.0-fold, at least 9.5-fold, or at least 10.0-fold relative to a method performed in a cell that does not comprise a knockout of one or more genes involved in MMR. In some embodiments, the one or more genes involved in MMR is selected from the group consisting of genes encoding the proteins MLH1, PMS2 (or MutL alpha), PMS1 (or MutL beta), MLH3 (or MutL gamma), MutS alpha (MSH2-MSH6), MutS beta (MSH2-MSH3), MSH2, MSH6, PCNA, RFC, EX01, POL6, and PCNA. In certain embodiments, the one or more genes is the gene encoding MLH1 (e.g., comprising an amino acid sequence of SEQ ID NO: 204, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to and including 100% sequence identity with SEQ ID NO: 204).
[911 In another aspect, the present disclosure provides methods for editing a nucleic acid molecule by prime editing comprising: contacting a nucleic acid molecule with a prime editor, a pegRNA, and an inhibitor of p53, thereby installing one or more modifications to the nucleic

38/699 acid molecule at a target site. In some embodiments, the method further comprises contacting the nucleic acid molecule with a second strand nicking gRNA.
1921 In some embodiments, the prime editing efficiency is increased by at least 1.5-fold, at least 2.0-fold, at least 2.5-fold, at least 3.0-fold, at least 3.5-fold, at least 4.0-fold, at least 4.5-fold, at least 5.0-fold, at least 5.5-fold, at least 6.0-fold, at least 6.5-fold, at least 7.0-fold, at least 7.5-fold, at least 8.0-fold, at least 8.5-fold, at least 9.0-fold, at least 9.5-fold, at least 10.0-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, at least 20-fold, at least 21-fold, at least 22-fold, at least 23-fold, at least 24-fold, at least 25-fold, at least 26-fold, at least 27-fold, at least 28-fold, at least 29-fold, at least 30-fold, at least 31-fold, at least 32-fold, at least 33-fold, at least 34-fold, at least 35-fold, at least 36-fold, at least 37-fold, at least 38-fold, at least 39-fold, at least 40-fold, at least 41-fold, at least 42-fold, at least 43-fold, at least 44-fold, at least 45-fold, at least 46-fold, at least 47-fold, at least 48-fold, at least 49-fold, at least 50-fold, at least 51-fold, at least 52-fold, at least 53-fold, at least 54-fold, at least 55-fold, at least 56-fold, at least 57-fold, at least 58-fold, at least 59-fold, at least 60-fold, at least 61-fold, at least 62-fold, at least 63-fold, at least 64-fold, at least 65-fold, at least 66-fold, at least 67-fold, at least 68-fold, at least 69-fold, at least 70-fold, at least 71-fold, at least 72-fold, at least 73-fold, at least 74-fold, or at least 75-fold, in the presence of the inhibitor of p53. In some embodiments, the frequency of indel formation is decreased by at least 1.5-fold, at least 2.0-fold, at least 2.5-fold, at least 3.0-fold, at least 3.5-fold, at least 4.0-fold, at least 4.5-fold, at least 5.0-fold, at least 5.5-fold, at least 6.0-fold, at least 6.5-fold, at least 7.0-fold, at least 7.5-fold, at least 8.0-fold, at least 8.5-fold, at least 9.0-fold, at least 9.5-fold, at least 10.0-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, at least 20-fold, at least 21-fold, at least 22-fold, at least 23-fold, at least 24-fold, at least 25-fold, at least 26-fold, at least 27-fold, at least 28-fold, at least 29-fold, at least 30-fold, at least 31-fold, at least 32-fold, at least 33-fold, at least 34-fold, at least 35-fold, at least 36-fold, at least 37-fold, at least 38-fold, at least 39-fold, at least 40-fold, at least 41-fold, at least 42-fold, at least 43-fold, at least 44-fold, at least 45-fold, at least 46-fold, at least 47-fold, at least 48-fold, at least 49-fold, at least 50-fold, at least 51-fold, at least 52-fold, at least 53-fold, at least 54-fold, at least 55-fold, at least 56-fold, at least 57-fold, at least 58-fold, at least 59-fold, at least 60-fold, at least 61-fold, at least 62-fold, at least 63-fold, at

39/699 least 64-fold, at least 65-fold, at least 66-fold, at least 67-fold, at least 68-fold, at least 69-fold, at least 70-fold, at least 71-fold, at least 72-fold, at least 73-fold, at least 74-fold, or at least 75-fold, in the presence of the inhibitor of p53.
1931 In some embodiments, the inhibitor of p53 is a protein. In certain embodiments, the inhibitor of p53 is the protein 153. In some embodiments, the inhibitor of p53 is an antibody that inhibits the activity of p53. In some embodiments, the inhibitor of p53 is a small molecule that inhibits the activity of p53. In some embodiments, the inhibitor of p53 is a small interfering RNA (siRNA) or a small non-coding microRNA that inhibits the activity of p53.
194I In another aspect, the present disclosure describes improved prime editor fusion proteins, including PEmax of SEQ ID NO: 99. The disclosure also contemplates fusion proteins having an amino acid sequence with a sequence identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least up to 100% with SEQ
ID NO: 99.
1951 The inventors have surprisingly found that the editing efficiency of prime editing may be significantly increased (e.g., 2-fold increase, 3-fold increase, 4-fold increase, 5-fold increase, 6-fold increase, 7-fold increase, 8-fold increase, 9-fold increase, or 10-fold increase or more) when one or more components of the canonical prime editor fusion protein (i.e., PE2) are modified.
Modifications may include a modified amino acid sequence of one or more components (e.g., a Cas9 component, a reverse transcriptase component, or a linker).
[961 In other aspects, the present disclosure also provides compositions and pharmaceutical compositions comprising PEmax, methods of prime editing using PEmax, polynucleotides and vectors encoding PEmax, and kits and cells comprising PEmax.
[971 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 become apparent from the following detailed description of various non-limiting embodiments when considered in conjunction with the accompanying figures.

40/699 BRIEF DESCRIPTION OF THE DRAWINGS
[98] 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.
[991 FIG. 1 provides a schematic showing that prime editing enables guide RNA-templated genomic manipulations. DNA prime editing intermediates capable of being repaired by cellular factors are shown in boxes.
[100] FIG. 2 provides a schematic for a DNA repair CRISPRi screen for prime editing outcomes.
11011 FIGs. 3A-3C show optimization of prime editing efficiency at the target site. FIG. 3A
provides a schematic for the optimization process. FIG. 3B shows percent reads with a specified modification at a target site in HeLa cells. FIG. 3C shows percent reads with a specified modification at a target site with blasticidin selection in HeLa cells.
[102] IFIGs. 4A-4B show a prime editing CRISPRi screen with a DNA repair library. FIG. 4A
provides a schematic of the screening process. FIG. 4B shows percent reads with a specified modification in bulk editing of post-screen HeLa cells.
[103] FIGs. 5A-5B show that the CRISPRi screen reveals that DNA mismatch repair limits prime editing efficiency. Knockdown of mismatch repair proteins (MSH2, MS116, PMS2, and MLH1) improves the efficiency of PE2 by 3-fold and PE3 by 2-fold.
[104] FIGS. 6A-6C show that siRNA knockdown of MMR improves prime editing in HEK293T cells. Editing results at multiple endogenous loci validate the findings of the CRISPRi screen.
[105] :FIGS. 7A-7B show that complete MMR knockout dramatically enhances prime editing.
In the absence of MMR, PE2 editing efficiency is shown to match PE3 editing efficiency.
[106] FIG. 8A provides a schematic for the mechanism of mismatch repair (MMR).
In the first step, MSH2:MSH6 (MutSa) binds the mismatch and recruits MLH1:PMS2 (MutLa). The DNA
nick signals to MMR which strand to repair. In the second step, MutLa indiscriminately incises the nicked strand 5' and 3' of the mismatch. In the third step, EX01 excises the mismatch from MutLa-generated nicks. In the fourth step, POLE resynthesizes the excised strand, followed by LIG1 ligation.

41/699 [107] FIG. 8B provides yet another schematic for the mechanism of mismatch repair, MMR., in eukaryotic cells. The left side of the schematic depicts 5' MMR. (A) The MutS
homolog proteins (MSH, purple) MutSa (MSH2-MSH6), or MutSI3 (MSH2-MSH3) recognize and bind a mismatch. RPA bound to single-strand DNA prevents EX01 from accessing and degrading DNA. (B) In the sliding clamp model, MutSa/r3 at a mismatch binds ATP and undergoes nucleotide switch activation, becoming a sliding clamp that diffuses along the DNA. Multiple MSH clamps are loaded at a single mismatch. The interaction of EX01 with MSH
sliding clamps overcomes the RPA bather and activates EX01 for 5' to 3' excision from the 5' nick.
MutL homolog proteins (MLH) (MutLa is ScM1h1-Pms1 or HsMLH1-PMS2) bind ATP and may interact with MSH sliding clamps, though MLH is not absolutely required in vitro for 5' MMR. In other models, MSH remains at the mismatch to authorize excision or can load multiple MLH clamps onto the DNA in the vicinity of the mismatch (not shown). (C) In the sliding clamp model, the EXOi/MSHI complex dissociates after excising several hundred nucleotides. Iterative rounds of MSH-EX01 excision create an excision tract coated with RPA that extends from the 5' nick to just beyond the mismatch. MLH may limit excision by modulating the number of MSH
clamps on DNA. (D) RFC (not shown) loads PCNA clamps with specific orientation at 3' termini of strand breaks or gaps, and PCNA facilitates high-fidelity DNA synthesis by Pol 8 or e. (E) DNA ligase I seals the nick. The right side of the schematic depicts 3' MMR.
(A) MSH
recognizes a mismatch. (B) In the sliding clamp model, ATP-dependent binding and nucleotide switching creates MSH sliding clamps that diffuse from the mismatch. The interaction of ATP-bound MLH heterodimers with MSH sliding clamps and PCNA oriented with respect to 3' termini activates MLH strand-specific nicking. Alternatively, ATP-activated MSH may remain at the mismatch to load MLH and activate nicking (not shown). (C) Excision is EX01-dependent or -independent, leading to an RPA-coated excision track. An EX01-independent IPol 8 strand-displacement pathway is not shown. (D) Pol 6 or a with the aid of PCNA
completes gap filling.
(E) DNA ligase I seals the nick.
[108] FIGs. 9A-9C provide a schematic of mismatch repair of PE2 intermediates.
MMR
inhibition provides additional time for flap ligation, removing the strand discrimination signal for repair of the heteroduplex.

42/699 [109] FIG. 10 shows that expression of dominant-negative MLH1 mutants boosts efficiency. MLH1 dominant-negative mutants improve PE2 efficiency by 2- to 4-fold. RNF2 +3 G to C is not responsive to MMR-inhibition.
[110] FIGs. 11A-118 show the effect of MUil mutants on PE3. MLH1 mutants reduce PE3 indels by half.
[111] IFIGs. 12A-12B show that MLH1 mutant improvements translate to other sites. FIG. 12A
shows that PE2 editing efficiency increases with MLH1 mutants, and only RNF2 +3 G to C is resistant to MMR-inhibition. FIG. 12B shows that MLH1 mutants reduce the occurrence of indels by half.
[112] FIG. 13 provides a schematic showing mismatch repair of PE3 intermediates.
[113] FIG. 14 provides a schematic showing that mismatch repair differentially resolves PE3 intermediates. Mismatch repair is required for the one edit-favored intermediate.
[114] FIG. 15A-1511 show screening of MLH1 mutants for smaller size and improved activity.
FIG. 15A shows that MLH1 A754-756 most strongly promotes PE2 editing (hereafter named MLHldn). MLH1 N-terminal domain approaches the effectiveness of MU-Ildn (hereafter named MLHldn'TD). MLH1 dominant negative mutants may function by saturating binding of MutS.
FIG. 15B shows that the MLH1 N-terminal domain + NLS approaches the activity of MLH1neg.
FIG. 15C shows that MLHldn fusion to PE by a self-cleavable P2A linker (PE-2A-MLH1dn) can improve prime editing efficiency. FIGs. 15D-15F show that MMR KD
phenocopies MLH1neg expression. FIGs. 15G-151I show that the efficiency of PE2 and PE3 is equal in the absence of MMR, suggesting that the complementary nick only serves to bias MMR.
[115] FIG. 16 shows that MLHidn reduces indels for PE3. Silent pegRNA is pegRNA that does not encode an edit or produce a mismatch. MLHldn only reduces PE3 indels if a mismatch is generated.
[116] FIG. 17 show that mismatch repair of PE heteroduplexes produces a diffuse indel pattern. Indel distribution is broad for PE3 for these edits, but inhibiting MMR with IVILHldn narrows that distribution. This suggests that MMR makes incisions after mismatch recognition that contribute to the indels generated by PE3.
[117] FIG. 18 shows mismatch repair of PE3 intermediates.
[118] FIGs. 19A-19B show that MMR excision of the target locus generates indels in PE3.

43/699 [119] FIGs. 20A-20B show that MMR knockdown or knockout has no effect on RNF2 -F3 G to C. This suggests that the RNF2 site is not repaired by MMR or the resulting C:C mismatch is not repaired by MMR.
[120] FIGs. 21A-21C show that other substitution edits at RNF2 can be improved with MLHldn.
[121] FIGs. 22A-22B show that MLHldn improves substitution edits at other sites, including HEK3. MLHidn strongly enhances PE2 editing and lowers PE3 indels.
[122] FIGs. 23A-23D show that MLHldn improves substitution edits at other sites, including FANCF. MLHldn strongly enhances PE2 editing and lowers PE3 indels.
[123] FIGs. 24A-24B show that PE improvement by MHLldn is mismatch dependent.
MLHIdn increases PE2 editing by 2-fold on average in HEK293T cells. FIG. 24A
shows that G
to C edits (C:C mismatches) are unaffected by MMR in HEK293T cells. This suggests that G to C edits have a higher baseline efficiency than other substitutions. FIG. 24B
shows a substantial increase in the ratio of edit:indel purity from MLH ldn used with PE3, which is also mismatch dependent.
[1241 FIGs. 25A-25D show that MLHldn also improves the efficiency of small insertion and deletion edits. MMR is known to repair insertions and deletions <15 nucleotides in length.
[125] FIGs. 26A-26B show that MLHldn reduced pegRNA scaffold integration.
Scaffold integration events at these sites occur through a double-strand break (DSB) intermediate.
[126] FIG. 27 shows that MLHidn does not promote substantial PE off-target editing. Small increases in off-target (OT) editing were observed at the HEK4 off-target site 3.
[127] FIGs. 28A-28B show that MLHldn does not induce detectable microsatellite instability at biomarker loci. MMR inhibition is known to cause shortening of homopolymer microsatellite regions.
[128] FIG. 29 shows that MLHldn offers a method to increase prime editing efficiency at sites without good ngRNAs, such as HEK4 [129] FIG. 30 shows that MLHldn improves PE at disease sites.
[130] FIG. 31 shows that MLHIdn enhances installation of the protective APOE
Christchurch allele in mouse astrocytes. A 50% boost in editing efficiency and a large reduction in indels is shown.

44/699 [131] FIG. 32 shows that HEK293T cells are MMR-compromised. The MLH1 promoter is hypermethylated in HEF293T, resulting in lower MUD. expression.
[132] FIGs. 33A-33B show that MLH1 dn enhances prime editing in HeLa cells.
FIG. 33A
shows prime editing with PE2. FIG. 338 shows prime editing with PE3.
[133] FIGs. 34A-34B show that MLHldn enhances prime editing in HeLa cells.
FIG. 34A
shows editing of PRNP +6 G to T. FIG. 34B shows editing of APOE +6 G to T and +10 C to A.
[134] FIGs. 35A-35B show that MtHldn has a larger effect in MMR competent cell lines like HeLa.
[135] FIGs. 36A-36D show that MLI-Ildn improvements synergize with stabilized pegRNAs.
[136] FIGs. 37A-37B show that contiguous substitutions are useful as another strategy for evading MMR.
[137] FIG. 38 shows that MMR does not efficiently repair 3 or more contiguous substitutions.
Contiguous substitutions therefore offer a method for circumventing MMR and boosting PE
efficiency.
[138] FIGs. 39A-39C show that MLH1neg improves PE in HeLa cells.
[139] FIGs. 40A-40G show that pooled Repair-seq CRISPRi screens reveal genetic determinants of substitution on prime editing outcomes. FIG. 40A shows that prime editing with the PE2 system is mediated by the PE2 enzyme (Streptococcus pyogenes Cas9 (SpCas9) H840A
nickase fused to a reverse transcriptase) and a prime editing guide RNA
(pegRNA). The PE3 system uses an additional single guide RNA (sgRNA) to nick the non-edited strand and yield higher editing efficiency. PBS, primer binding site. RT template, reverse transcription template.
FIG. 40B provides an overview of prime editing Repair-seq CRISPRi screens. A
library of CRISPRi sgRNAs and a pre-validated prime edit site are transduced into CRISPRi cell lines and transfected with prime editors targeting the edit site. CRISPRi sgRNA
identities and prime edited sites are amplified together from genomic DNA and paired-end sequenced together to link each genetic perturbation with editing outcome. SaCas9, Staphylococcus aureus Cas9.
FIG. 40C
shows the effect of each CRISPRi sgRNA on the percentage of sequencing reads reporting the intended CrC-to-C=G prime edit at the targeted edit site in pooled CRISPRi screens. Each value depicts all sequencing reads carrying the same CR1SPRi sgRNA. FIG. 401) shows the effect of CRISPRi sgRNAs on editing efficiency in all screen conditions. Black dots represent individual non-targeting sgRNAs, black lines show the mean of all non-targeting sgRNAs, and gray

45/699 shading represents kernel density estimates of the distributions of all sgRNAs. FIGs. 40E-40G
show comparisons of gene-level effects of CRISPRi targeting on the intended GC-to-C=G prime edit across different screen conditions. (FIG. 40E) K562 PE2 vs. HeLa PE2.
(FIG. 40F) K562 PE3+50 vs. HeLa PE3+50. (FIG. 40G) 1K562 PE2 vs. K562 PE3+50. The effect of each gene is calculated as the average log2 fold change in frequency from non-targeting sgRNAs for the two most extreme sgRNAs targeting the gene. Plotted quantities are the mean of n=2 independent biological replicates for each cell type, with bars showing the range of values spanned by the replicates. Black dots represent 20 random sets of three non-targeting sgRNAs.
11401 FIGs. 41A-41J show genetic modulators of unintended prime editing outcomes. FIGs.
41A-41D show representative examples of four categories of unintended prime editing outcomes observed in CRISPRi screens. In each panel, the black bar depicts the sequence of an editing outcome, the blue bar depicts genomic sequence around the targeted editing site, and the orange bar depicts the pegRNA sequence. Blue and orange lines between the editing outcome and the genome or pegRNA depict local alignments between the outcome sequence and the relevant reference sequence. Mismatches in alignments are marked by X's, and insertions are marked by downward dimples. The location of the programmed edit is marked by a grey box.
Red and cyan rectangles on the genome mark SaCas9 protospacers and PAMs, and black vertical lines mark the locations of SaCas9 nick sites. Orange, beige, grey, and red rectangles on the pegRNA mark the primer binding site (PBS), reverse transcription template (RTT), scaffold, and spacer, respectively. FIGs. 41E-41F provide a summary of editing outcome categories observed in PE2 screens (FIG. 41E) and in PE3+50 screens (FIG. 41F) in K562 cells. Plotted quantities are the mean SD of all sgRNAs for each indicated gene (60 non-targeting sgRNAs, three sgRNAs per targeted gene), averaged across n=2 independent biological replicates. FIGs.
41G-4111 show a comparison of the effects of knockdown of all genes targeted in CRISPRi screens on the frequency of joining of reverse transcribed sequence at unintended locations (FIG. 41G) or the frequency of deletions (FIG. 4111) from PE3+50. The effect of each gene is calculated as the average 1og2 fold change in frequency from non-targeting sgRNAs for the two most extreme sgRNAs targeting the gene. Plotted quantities are the mean of n=2 independent biological replicates for each cell type, with bars showing the range of values spanned by the replicates.
Black dots represent 20 random sets of three non-targeting sgRNAs. FIG. 411 shows the frequency of deletion as a function of genomic position relative to programmed PE3+50 nicks

46/699 (dashed vertical lines) in K562 screen replicate 1 across all reads for indicated sets of CRISPRi sgRNAs (black line: 60 non-targeting sgRNAs; orange and green lines: three sgRNAs targeting each of MSH2, MSH6, MLH1, and PMS2) (top). Log2 fold change in frequency of deletion as a function of genomic position from MSH2, MSH6, IMLHI1, and PMS2 sgRNAs compared to non-targeting sgRNAs (bottom). FIG. 41 J shows the effect of gene knockdowns on the fraction of all observed deletions that remove sequence at least 25-nt outside of programmed PE3+50 nicks in K562 screens. Each dot represents all reads for all sgRNAs targeting each gene. Black dots represent 20 sets of three random non-targeting sgRNAs.
11411 FIGs. 42A-42D show a model for mismatch repair of prime editing intermediates. FIG.
42A shows a model for DNA mismatch repair (MMR) of PE2 intermediates. MMR.
excises and replaces the nicked strand during repair of the prime editor-generated heteroduplex substrate.
Infrequent ligation of the nick before MMR recognition deprives the strand discrimination signal for MMR, resulting in un-biased resolution of the heteroduplex. FIG. 42B shows a model for MMR of PE3 intermediates. PE3 installs an additional nick on the non-edited strand that can direct MMR to replace the non-edited strand. Ligation of the edited strand nick leaves only the complementary-strand nick to signal repair by MMR, resulting in the desired prime editing outcome. FIG. 42C shows prime editing efficiencies of PE2 and PE3 prime editors at endogenous sites (HEK3, EMX1, and MINX]) in HEK293T cells pre-treated with knockdown siRNAs against MSH2, MSH6, MLH1, or PMS2 transcripts. Cells were pre-transfected with siRNAs 3 days prior to transfection with prime editor components and siRNAs.
Genomic DNA
was harvested 3 days following transfection with prime editors and additional siRNA, then sequenced. Bars represent the mean of n=3 independent biological replicates.
FIG. 42D shows prime editing efficiencies in HAP! AMSH2 and HAP! AMLH1 cells (mean of n=3 independent biological replicates). A, gene knockout.
11421 F1Gs. 43A-43F show that engineered dominant negative MMR proteins (dominant negative variants of MSH2, MSH6, PMS2, and MLII1) enhance prime editing. FIG.
43A shows editing improvement at HEK2, ENLY1, and MINX] sites by co-expression of PE2 in trans with human MMR proteins or dominant negative variants in HEK293T cells. MMR
proteins include MSH2, MSH6, PM S2, and MLH1. Dominant negative variants are designated as MSH2 K675R, MSH6 K1 140R, PMS2 E41A, PMS2 E705K, MLH1 E34A, and MLH1 A756. All values from n =3 independent biological replicates are shown. FIG. 43B shows functional annotation of the

47/699 756-aa human MLH1 protein, including an ATPase domain, MSH2 interaction domain, NLS
domain, PMS2 dimerization domain, and an endonuclease domain. FIG. 43C shows editing enhancement of MLIT1 variants co-expressed with PE2 in HEK293T cells at HIM, ENIX1, and Rti7VX/ sites. Red boxes indicate mutations that inactivate MLH1 ATPase or endonuclease function. MLHldn, MLH1 A754-756. MLH1NTD¨NLS, codon-optimized MLH1 1-335¨
NLSSV40. All values from n=3 independent biological replicates are shown. FIG.
43D shows a comparison of the top three dominant negative MLH1 variants at additional prime edits. All values from n=3 independent biological replicates are shown. FIG. 43E shows prime editing with PE2 and MLHldn in trans, PE2 and MLH1NTD¨NLS in trans, and PE2¨P2A¨MLH1dn (human codon optimized) in HEK293T cells. Bars represent the mean of n = 3 independent biological replicates. FIG. 43F compares the structure of PE2, PE3, PE4, and PE5. In particular, the PE4 editing system consists of a prime editor enzyme (nickase Cas9-RT
fusion), MLHldn, and pegRNA. The PE5 editing system consists of a prime editor enzyme, MLHldn, pegRNA, and second-strand nicking sgRNA. FIG. 43G shows editing efficiencies of PE2, PE3, PE4, and PE5 systems in HEK293T cells. Bars represent the mean of n = 3 independent biological replicates).
[143] FIGs. 44A-44G show the characterization of PE4 and PE5 across diverse prime editing classes and cell types. FIG. 44A provides a summary of prime editing enhancement by PE4 and PE5 compared to PE2 and PE3 for 84 single-base substitution edits (seven for each substitution type) across seven endogenous sites in HEK293T cells. The grand mean SD of all individual values of n = 3 independent biological replicates are shown. FIG. 44B shows installation of single base mutations at the FANCF locus with PE2, PE3, PE4, and PE5 in HEK293T cells. Bars represent the mean of n =3 independent biological replicates. FIG. 44C shows that PE4 improves the 1- and 3-bp insertion and deletion prime edits compared to PE2 in HEK293T cells.
Bars represent the mean of n = 3 independent biological replicates. FIG. 44D
shows PE4 editing enhancement over PE2 across 33 different insertion and deletion prime edits.
Bars represent the mean of all individual values of n=3 independent biological replicates. FIGs.
44E-44F provide a summary of PE2 and PE4 editing efficiencies for 35 different substitutions of 1 to 5 contiguous bases at five endogenous sites in HEK293T cells. Seven pegRNAs were tested for each number of contiguous bases altered. The mean SD of all individual values of n = 3 independent biological replicates are shown. FIGs. 44G-441I show that installation of additional silent or

48/699 benign mutations near the intended edit can increase editing efficiency by generating a heteroduplex substrate that evades MMR. The PAM sequence (NGG) for each target is underlined. The amino acid sequence of the targeted gene is centered above each DNA codon.
Values represent the mean SD of n=3 independent biological replicates. FIG.
441 shows a comparison of prime editing enhancement in different cell types. PE4 and PE5 systems enhance prime editing to a greater extent in MMR deficient cells (MMR-) than in MMR
proficient cells (MMR+). The same set of 30 pegRNAs encoding single-base substitution edits were tested in HEK293T and HeLa cells. K562 and U2OS cells were edited with 10 pegRNAs that are a direct subset of the 30 pegRNAs tested in HEK293T and HeLa cells. The mean SD of all individual values of sets of n = 3 independent biological replicates are shown. P values were calculated using the Mann-Whitney U test. FIG. 44J shows prime editing with PE2, PE3, PE4, and PE5 in HeLa, K562, and U2OS cells. Bars represent the mean of n =3 independent biological replicates).
[144] FIGs. 45A-4511 show the effect of dominant negative MLH1 on prime editing product purity and off-targeting. FIG. 45A shows that edit-encoding pegRNAs program a base change within the nascent 3' DNA flap and generate a heteroduplex following flap interconversion. Non-editing pegRNAs template a 3' DNA flap with perfect complementarity to the genomic target site. FIG. 45B shows the frequency of indels from PE3 or PE5 with four edit-encoding pegRNAs that program single base mutations or four non-editing pegRNAs. Short horizontal bars indicate the mean of all individual values of sets of n = 3 independent biological replicates.
FIG. 45C shows the ratio of indel frequency from PE5 over PE3 with 4 edit-encoding pegRNAs that program single base mutations or four edit-encoding pegRNAs that program single base mutations or four non-editing pegRNAs. Short horizontal bars indicate the mean of all individual values of sets of n =3 independent biological replicates. FIG. 45D shows distribution of deletions at genomic target DNA formed by PE3 and PE5 using 12 substitution-encoding pegRNAs at endogenous DNMT1 and RNF2 loci in HEK293T cells. Dotted lines indicate position of pegRNA- and sgRNA-directed nicks. Data represent the mean SD of n=3 independent biological replicates. FIG. 45E shows PE5/PE3 ratio of frequency of deletions that remove sequence greater than 25-nt outside of pegRNA- and sgRNA-directed nicks in HEK293T
cells. Each dot represents one of 84 total pegRNAs that program substitution edits at a combined seven loci (mean of n=3 independent biological replicates). FIG. 45F shows PE5/PE3 ratio of

49/699 frequency of editing outcomes with unintended pegRNA scaffold sequence incorporation or unintended flap rejoining in HEK293T cells. Each dot represents one of 84 total pegRNAs that program substitution edits at a combined seven loci (mean of n = 3 independent biological replicates). FIG. 45G shows off-target prime editing by IPE2 and PE4 in HEK293T cells. Bars represent the mean of n =3 independent biological replicates). FIG. 45H shows high-throughput sequencing analysis of 17 sensitive microsatellite repeat loci used for clinical diagnosis of MMR
deficiency. HAP1 and HeLa cells are MMR-proficient, and HCT116 cells have impaired MMR.
HAP1 AMSH2 cells underwent 60 cell divisions following MSH2 knockout. HeLa cells were transiently transfected with PE2 or PE4 components and incubated for 3 days before sequencing.
wt, wild-type. All values from n=2 independent biological replicates are shown.
[145] FIGs. 46A-46F show that PEmax architecture with PE4 and PE5 editing systems enhances editing at disease-relevant gene targets and cell types. FIG 46A
shows a schematic of PE2 and PEmax editor architectures. bpNLSSV40, bipartite SV40 NLS nuclear localization signal. MMLV RT, Moloney Murine Leukemia Virus reverse transcriptase pentamutant; codon opt., human codon-optimized. FIG. 46B shows that engineered pegRNAs (epegRNAs) contain a 3' RNA structural motif that improve prime editing performance. FIG. 46C shows prime editing efficiencies of PE4 and PE5 combined with PEmax architectures and epegRNAs.
Seven single-base substitution edits targeting different loci were tested in HeLa and HEK293T cells. Fold changes indicate the average of fold increases from each edit tested. The meand-SD of all individual values of n=3 independent biological replicates are shown. FIG. 46D
shows prime editing at therapeutically-relevant sites in wild-type HeLa and HEK293T cells.
The HBB locus is edited at the E6 codon commonly mutated in patients with sickle cell disease (E6V). The CDKL5 edit is at a site for which the c.1412delA mutation causes CDKL5 deficiency disorder.
epegRNAs were used for editing the HBB, PRNP, and CDKL5 loci. Bars represent the mean of n=3 independent biological replicates. FIG. 46E shows correction of CDKL5 c.1412delA via an A=T insertion and a silent G=C-to-AT edit in iPSCs derived from a patient heterozygous for the allele. Editing efficiencies indicate the percentage of sequencing reads with c.1412delA
correction out of editable alleles that carry the mutation. lndel frequencies reflect all sequencing reads that contain any indels. Bars represent the mean of n=3 independent biological replicates.
FIG. 46F shows prime editing in primary human T cells. Bars represent the mean of n=3 independent biological replicates from different healthy T cell donors.

50/699 [146] FIGs. 47A-47J show the design and results of Repair-seq screens for substitution prime editing outcomes. FIG. 47A shows optimization of a Staphylococcus aureus (Sa)-pegRNA for installation of a G=C-to-C=G edit within a lentivirally integrated HBB
sequence using SaPE2 in HEK293T cells. PBS, primer-binding site. Data represent the mean of n =3 independent biological replicates. FIG. 47B shows the design of the prime editing Repair-seq lentiviral vector (pPC1000). In Repair-seq screens, a 453-bp region containing CRISPRi sgRNA
sequence and prime editing outcome is amplified from genomic DNA for paired-end Illumina sequencing. The CRISPRi sgRNA is sequenced with a 44-nt Illumina forward read (R1), and the prime edited site (including +50 and ¨50 nick sites) is sequenced with a 263-nt Illumina reverse read (R2). Black triangles indicate positions of SaPE2-induced nicks programmed by Sa-pegRNA
and Sa-sgRNAs. Sizes of all vector components are to scale. FIG. 47C shows a schematic of PE2, PE3+50, and PE3-50 prime editing configurations with SaPE2 protein (SaCas9 N580A fused to an engineered MMLV RT). FIG. 47D shows validation of intended CrC-to-C,G
editing at the lentivirally-integrated Repair-seq edit site in HeLa cells expressing dCas9¨BFP¨KRAB cells.
Bars represent the mean of n =2 independent biological replicates. FIG. 47E
shows prime editing at the Repair-seq edit site with and without blasticidin selection in HeLa cells expressing dCas9¨BFP¨KRAB. SaPE2¨P2A¨BlastR prime editor was used for all conditions.
Bars represent the mean of n =2. FIG. 47F shows functional annotation classes of the genes targeted by the pooled CRISPRi sgRNA library used in Repair-seq screens. FIGs. 47G-47J
show that the knockdown of MSH2, MSH6, MLH1, and PMS2 increases the frequency of the intended +6 GC-to-C=G prime edit in all Repair-seq screens. Dots represent reads from individual CRISPRi sgRNAs.
[147] FIGs. 48A-48I show the genetic modulators of unintended prime editing outcomes. FIG.
48A shows an overview of PE3-50 outcomes in HeLa CRISPRi screens. TP53BP1 knockdown dramatically reduces formation of all unintended editing outcomes. FIG. 48B
shows additional details of PE2 outcomes in K562 CRISPRi screens, supplementing FIG. 41H. FIG.
48C shows additional details of PE3+50 outcomes in K562 CRISPRi screens, supplementing information in FIG. 41G. FIGs. 48D-48I show comparisons of effects of gene knockdown on frequencies of indicated outcome categories in indicated screen conditions. Platted quantities are the mean of the 1og2 fold changes from non-targeting sgRNAs for the two most extreme sgRNAs per gene, averaged over n =2 independent biological replicates per condition. Error bars mark the range of

51/699 values spanned by the replicates. Black dots represent 20 random sets of three non-targeting sgRNAs. FIG. 48D shows that MSH2, MLH1, and PMS2 knockdown produce larger fold changes in installation of additional edits than in intended edits in K562 PE2 screens. FIG. 48E
shows unintended joining of reverse transcribed sequence in PE2 screens in K562 and HeLa cells are most increased by knockdown of Fanconi anemia genes (red) as well as a set of RAD51 homologs and other genes involved in homologous recombination (blue). FIG.
48:F shows deletions in in PE2 screens in K562 and HeLa cells are most increased by a set of RAD51 homologs and other genes involved in homologous recombination (blue). FIG. 48G
shows that in addition to MSH2, MLH1, and PMS2, HLTF knockdown produces larger fold changes in installation of additional edits than in intended edits in K562 PE3+50 screens. FIG. 48111 shows that tandem duplications in HeLa and K562 PE3+50 screens are most decreased by knockdown of FOLD and RFC subunits. FIG. 481 shows deletions in HeLa PE3+50 and PE3-50 screens have dramatically divergent genetic regulators, highlighting differences in the processing of the different overhang configurations.
1148] FIGs. 49A-49F show validation of prime editing Repair-seq screen results. FIGs. 49A-49B show alignment of Sa-pegRNAs, their templated 3' DNA flaps following SaPE2 reverse transcription, and the genomic target sequence (top). Compared to the Sa-pegRNA used in Repair-seq screens (FIG. 49A), an Sa-pegRNA with recoded scaffold sequence (FIG. 49B) templates an extended 3' DNA flap with reduced homology with genomic target sequence. The recoded Sa-pegRNA contains 2 base pair changes that preserve base pairing interactions within the scaffold. Reverse transcription of the Sa-pegRNA scaffold can generate a misextended 3' flap that is incorporated into the genome. Vertical lines depict base pairing. X's depict mismatches between the misextended reverse-transcribed 3' flap and genomic sequence.
FIGs. 49A-49B also show frequencies of editing outcome categories observed at the screen edit site from arrayed PE
and PE3+50 experiments in HeLa CRISPRi cells (bottom). Prime editing with the Sa-pegRNA
used in siteRepair-seq screens (FIG. 49A) or a recoded Sa-pegRNA (FIG. 49B) results in different frequencies of installation of unintended edits from nearly-matched scaffold. Plotted quantities are the meand.,SD of n=4 independent biological replicates, for each cell line containing MSH2 or non-targeting CR1SPRi sgRNAs. FIG. 49C shows the mechanism of DNA
mismatch repair in humans. FIG. 49D shows mismatch repair of a prime editing heteroduplex intermediate could install additional non-programmed nicks from MutLa endonuclease activity.

52/699 Excision from these non-programmed nicks and subsequent repair of the resulting intermediates may contribute to larger and more frequent indel byproducts observed from MMR
activity. FIG.
49E shows the knockdown efficiency of siRNA treatment relative to a non-targeting siRNA
control in HEK293T cells. Cells were transfected with siRNAs, incubated for 3 days, transfected with PE2, pegRNAs, and the same siRNAs, then incubated for another 3 days before relative RNA abundances were assayed by RT-qPCR. NT, non-targeting. Data represent the mean of n=3 independent biological replicates. Each dot represents the mean of n = 3 technical replicates.
FIG. 49F shows editing in HEK293T cells co-transfected with prime editor components and siRNAs. Cells were not pre-treated with siRNAs before transfection with prime editor. Bars represent the mean of n =3 independent biological replicates.
[149] FIGs. 50A-50H show the development and characterization of dominant negative MMR
proteins that enhance prime editing. FIG. 50A shows the prime editing efficiencies from MMR
proteins or dominant negative variants expressed in trans with or fused directly to PE2 in HEK293T cells. 32aa linker, (SGGS)x2¨XTEN¨(SGGS)x2 (SEQ ID NO: 125) (SGGSSGGSSGSETPGTSESATPES SGGSSGGS (SEQ ID NO: 125) or structurally, [SGGS}-[SGGS]-[SGSETPGTSESATPESMSGGSMSGGS] (SEQ ID NO: 125)). codon opt., human codon optimized. Data within the same graph originate from experiments performed at the same time. Data represent the mean SD of n =3 independent biological replicates.
FIG. 50B shows titration of MLI-11 dn plasmid and PE2 plasmid transfection doses in HEK293T
cells. Maximum plasmid amounts tested were 200 ng PE2 and 100 ng MLHIdn. Data represent the mean SD of n =3 independent biological replicates. FIG. 50C shows prime editing with MLHldn co-expression in MMR-deficient HCT116 cells that contain a biallelic deletion in MLH1. Bars represent the mean of 3 replicates. FIG. 50D shows a comparison of prime editing with human MLHidn (human codon-optimized) or mouse MLH1 dn (mouse codon optimized) in human HEK293T cells. Bars represent the mean of n = 3 independent biological replicates. FIG. 50E
shows a comparison of prime editing with human MLITIdn (human codon optimized) or mouse MLHldn (mouse codon optimized) in mouse N2A cells. Bars represent the mean of n = 3 replicates. FIG. 50F shows that MLIII knockout in clonal HeLa cell lines enhances prime editing efficiency to a greater extent than ML111 co-expression in clonal wild-type HeLa cells. d, knockout. Bars represent the mean of n = 3 or 4 independent biological replicates. FIG. 50G
shows editing at the IFANCF locus with PE3b and IPE5b (complementary-strand nick that is

53/699 specific for the edited sequence) in HEK293T cells. PE5b, PE3b editing system with MLHldn co-expression. Bars represent the mean of n = 3 independent biological replicates. FIG. 50H
shows editing at the I1EK2 locus with complementary-strand nicks in HEK293T
cells. "None"
indicates the lack of a nick, which denotes a PE2 or PE4 editing strategy.
Bars represent the mean of n = 3 independent biological replicates.
[150] FIGs. 51A-51J show the characterization of PE4 and PE5 across diverse prime edit classes and cell types. FIG. 51A shows a comparison of PE2, PE3, PE4, and PE5 for 84 single-base substitution prime edits across seven endogenous sites in HEK293T cells.
Bars represent the mean of n =3 independent biological replicates). FIG. 51B provides a summary of PE4 enhancement in editing efficiency over PE2 for 84 single-base substitution edits across seven endogenous sites in HEK293T cells. PE4/PE2 fold improvements may be lower for PAM edits due to the high basal editing efficiency for PAM edits or the high representation of G=C-to-C=G
edits (five out of 15 in this category). Data represent the mean SID of n =
3 independent biological replicates. FIG. 51C shows the efficiencies of single-base substitution prime edits that alter the PAM (+5 G and +6 G bases) of prime editing target protospacers in HEK293T cells.
Four G=C-to-A=T, five G=C-to-C=G, and six G=C-to-T=A PAM edits across a combined seven endogenous sites are shown. The mean of all individual values of n =3 independent biological replicates are shown. FIG. 51D shows the effect of siRNA knockdown of MMR
genes on G=C-to-C=G editing at the RNF2 locus in HEK293T cells. Bars represent the mean of n =3 independent biological replicates. FIG. 51E shows the effect of MMR gene knockout on G=C-to-C=G editing at the RNF2 locus in HAP! cells. A, gene knockout. Bars represent the mean of n =
3 independent biological replicates. FIG. 51F shows prime editing at the integrated screen edit site with CRISPRi knockdown in HeLa CRISPRi cells. PE2 indicates editing with SaPE2 protein and Sa-pegRNA. PE3+50 indicates editing with SaPE2 protein, Sa-pegRNA, and Sa-sgRNA that programs a +50 complementary-strand nick. Bars represent the mean of n = 5 independent biological replicates. FIG. 51G provides a summary of PE5 enhancement in editing efficiency over PE3 for 84 single-base substitution edits in HEK293T cells. The grand mean SD of all individual values of n = 3 independent biological replicates are shown. FIG.
5111 shows PE4 enhancement in editing efficiency over PE2 across a range of insertion and deletion prime edit lengths in HEK293T cells. A total of 33 different prime edits at a combined three endogenous loci are shown. The mean of all individual values of n =3 independent biological replicates are

54/699 shown. FIG. 511 shows that PE5 improves editing efficiency and reduces indel byproducts compared to PE3 across small insertion and deletion prime edits in HEK293T
cells. FIG. 51J
shows PE2 and PE4 editing efficiencies at 33 different insertion and deletion prime edits across a combined three endogenous loci. Bars represent the mean of all individual values of n=3 independent biological replicates.
[151] FEGs. 52A-52C show characterization of PE4 and PE5 systems and improved prime editing efficiency with additional silent mutations. FIG. 52A shows substitutions of contiguous bases with PE2 and PE4 in HEK293T cells. The top sequence indicates the original, unedited genomic sequence. Numbers denote the position of the edited nucleotide relative to the PE2 nick site. Nucleotides within the SpCas9 PAM sequence (NGG) are underlined.
Sequences of the intended edited product are shown below, with edited nucleotides marked in red. Bars represent the mean of n =3 independent biological replicates. FIG. 52B shows that installation of additional silent mutations can increase prime editing efficiency by evading MMR. PE4/PE2 fold-change in editing frequency reflects the extent to which MMR activity impedes the indicated prime edit. Edited nucleotides that make the indicated coding mutation are marked in red, and edited nucleotides that make silent mutations are marked in green.
Data represent the mean:ESD of n=3 independent biological replicates. FIG. 52C shows installation of 22 single-base substitution prime edits across seven endogenous sites in HeLa cells with PE2, PE3, PE4, and PE5. Bars represent the mean of n=3 independent biological replicates.
[152] FIGs. 53A-53G show the effect of dominant negative MLH1 on prime editing product purity and off-targeting. FIG. 53A shows the frequency of indels in HEK293T
cells treated with pegRNAs, nicking sgRNAs, and PE2 enzyme, R'F-impaired PE2 (PE2--dRT), or nickase Cas9 (SpCas9 H840A), with and without MLHldn. Non-editing pegRNAs encode a 3' DNA
flap with perfect homology to the genomic target. Bars represent the mean of n =3 independent biological replicates. FIG. 53B shows the distribution of deletion outcomes from PE3 and PE5 at endogenous loci in HEK293T cells. 12 different pegRNAs that program single-base substitutions were tested at each indicated locus. Dotted lines indicate position of pegRNA-and sgRNA-directed nicks. Data represent the mean a: SD of n = 3 independent biological replicates. FIG.
53C shows the distribution of deletion outcomes from PE3 and PE5 with an edit-encoding and non-editing pegRNA in HEK293T cells. The non-editing pegRNA templates a 3' DNA
flap with perfect complementarity to the genomic target sequence. Data represent the mean SD of n =3

55/699 independent biological replicates. FIG. 53D shows the frequency of all prime editing outcomes with unintended pegRNA scaffold sequence incorporation or unintended flap rejoining in 1-1EK293T cells. 12 pegRNAs each programming a different single-base substitution were tested at each of the seven indicated loci. Each dot represents an individual pegRNA
at the indicated loci (mean of n = 3 independent biological replicates). FIG. 53E shows the off-target prime editing by PE2 and PE4 in :HEK293T cells (mean of n =3 independent biological replicates).
FIG. 53F shows the distribution and cumulative distribution of microsatellite repeat lengths in the indicated cell types and treatments. HAP1 and HeLa cells are MN/TR-proficient, and HCT116 cells have impaired MMR. HAP! AMSH2 cells underwent 60 cell divisions following knockout of MSH2. HeLa cells were transiently transfected with PE2 or PE4 components and grown for a following 3 days before sequencing. wt, wild-type. All values from n =2 independent biological replicates are shown. FIG. 53G shows prime editing at the on-target locus in HeLa cells transfected with PE2 or PE4 components. Bars represent the mean of n =2 independent biological replicates. Microsatellite lengths were assayed from genomic DNA
taken from these PE2 and PE4-treated HeLa cells.
[153] FIGs. 54A-54F show that use of PEmax architecture with PE4 and PE5 editing systems enhances editing at disease-relevant gene targets and cell types. FIG. 54A
shows a schematic of PE2 and PEmax editor architectures. bpNLSsw , bipartite SV40 NLS. MMLV RT, Moloney Murine Leukemia Virus reverse transuiptase pentamutant. GS codon, Genscript human codon optimized. FIG. 548 shows engineered pegRNAs (epegRNAs) containing a 3' RNA
structural motif that improve prime editing performance. FIG. 54C shows prime editing efficiencies of PE4 and PE5 combined with PEmax architectures and epegRNAs. Seven single-base substitution edits targeting different loci were tested in HeLa and HEK293T cells. Fold changes indicate the average of fold increases from each edit tested. The mean SD of all individual values of n =3 independent biological replicates are shown. FIG. 54D shows prime editing at therapeutically-relevant sites in wild-type HeLa and HEK293T cells. The HBB locus is edited at the E6 codon commonly mutated in patients with sickle cell disease (E6V). The CDKL5 edit is at a site for which the c.1412delA mutation causes CDKL5 deficiency disorder. epegRNAs were used for editing the HBB, PRNP, and CDKL5 loci. Bars represent the mean of n =3 independent biological replicates. FIG. 54E shows the correction of CDKL5 c.1412delA via an A=T insertion and a silent GC-to-A=T edit in iPSCs derived from a patient heterozygous for the allele. Editing

56/699 efficiencies indicate the percentage of sequencing reads with c.1412delA
correction out of editable alleles that carry the mutation. Indel frequencies reflect all sequencing reads that contain any indels. Bars represent the mean of n = 3 independent biological replicates. FIG. 54F shows prime editing in primary T cells. Bars represent the mean of n = 3 independent biological replicates from different healthy T cell donors.
[154] FIGs. 55A-55B show the development of PEmax and application of PE4 and PE5 to primary cell types. FIGs. 55A-55B show screening of prime editor variants to maximize editing efficiency in HeLa cells. All prime editor architectures carry a Cas9 H840A
mutation to prevent nicking of the complementary DNA strand at the target protospacer. *NLSSV40 contains a 1-aa deletion outside the PKKKRKV (SEQ ID NO: 132) NLSSV40 consensus sequence. All individual values of n =3 independent biological replicates are shown.
[1551 FIGs. 56A-56G show development of PEmax and application of PE4 and PE5 to primary cell types. MG. 56A shows a screen of prime editor variants for improved editing efficiency with the PE3 system in HeLa cells. All prime editor architectures carry a SpCas9 H840A
mutation to prevent nicking of the complementary DNA strand at the target protospacer.
NLSSV40 indicates the bipartite SV40 NLS. *NLSSV40 contains a 1-aa deletion outside the PKKKRKV (SEQ ID NO: 132) NLSSV40 consensus sequence. All individual values of n=3 independent biological replicates are shown. FIG. 56B shows the architecture of the original PE2 editor (Anzalone et al., 2019), PE2* (Liu et al., 2021), CMP-PE-V1 (Park et al., 2021), and prime editor variants developed in this work (PEmax, CMP-PEmax). HN1, HIVIGN1;
H1G, histone H1 central globular domain; codon opt., human codon optimized. FIG.
56C shows that PEmax outperforms other prime editor architectures with the PE3 system in HeLa cells. Bars represent the mean of n=3 independent biological replicates. FIG. 56D shows fold-change in editing efficiency of prime editor architectures compared to PE2 with the PE3 system in HeLa cells. The mean SD of all individual values of n=3 independent biological replicates are shown.
FIG. 56E shows the intended editing and indel frequencies from PE4, PE4max (PE4 editing system with PEmax architecture), PE5, and PE5max (PE5 editing system with PEmax architecture) in HeLa and HEK293T cells. Seven substitution prime edits targeting different endogenous loci were tested for each condition. The mean SD of all individual values of n =3 independent biological replicates are shown. FIG. 56F shows the correction of c.1412delA via an .A=T insertion and a GC-to-A=T edit in iPSCs derived from a patient

57/699 heterozygous for the disease allele. Editing efficiencies indicate the percentage of sequencing reads with c.1412delA correction out of editable alleles that early the mutation. Indel frequencies reflect all sequencing reads that contain any indels that do not map to the c.1412delA allele or wild-type sequence. 1 pg of IPE2 mItNA was used in all conditions shown. Bars represent the mean of n = 3 independent biological replicates. FIG. 56G shows prime editing in primary T
cells. Bars represent the mean of n = 3 independent replicates from different T cell donors.
11561 FIGs. 57A-57B show that the recoded pegRNA scaffold reduces unintended outcomes from scaffold sequence incorporation. FIG. 57A shows an alignment of the prime editing Repair-seq target site and SaPE2-generated 3' DNA flaps templated by (top) the Sa-pegRNA
used in Repair-seq screens, or (bottom) an Sa-pegRNA with a recoded scaffold sequence. 3' flap sequences are aligned with the templated region of the Sa-pegRNA shown above (RT template or scaffold). Red indicates position of the intended +6 CrC to C=G edit programmed by both Sa-pegRNAs. Blue indicates positions at which the genomic target sequence does not align with the 3' flap sequence templated by the Sa-pegRNA scaffold. Unintended edits from incorporation of a 3' flap containing a reverse transcribed Sa-pegRNA scaffold sequence may occur at these blue-indicated nucleotides. FIG. 57B shows a summary of editing outcome categories observed in PE2 and PE3+50 experiments in HeLa CRISPRi cells. Screen pegRNA indicates the Sa-pegRNA
used in prime editing Repair-seq screens. Sa-pegRNA with recoded scaffold (sequence shown in FIG. 54A) avoids sequence homology with the Repair-seq edit site. Plotted quantities are the mean SD of one CRISPRi sgRNA for each indicated target (MSH2 and non-targeting), averaged across n =4 independent biological replicates.
[157] FIG. 58 shows a comparison of PE3max (PE3 editing system with PEmax protein) and PE3 (PE3 editing system with PE2 protein) in HeLa cells (mean of n = 3 independent biological replicates).
1158] FIG. 59 shows that PE improvement with MLHldn depends on prime edit size. MMR
most efficiently repairs substitutions and insertion and deletion errors of fewer than or equal to approximately 13 bp in length.
11.591 FIG. 60 shows that PEA and epegRNAs enable prime editing with a single pegRNA
integrant.
[1601 FIG. 61 shows that PE5 improves installation of the protective Christchurch allele in an APOE4 mouse astrocyte model.

58/699 [161] FIGs. 62A-62C show that inhibiting p53 enhances the efficiency and precision of PE3 prime editing. This is particularly true when the nicking sgRNA makes a nick upstream (- side) of the pegRNA-directed nick. Each point on the graphs represents an individual CRISPRi gene knockdown in the Repair-seq screens. The axes depict 1og2 fold changes compared to control.
Knocking down TP53BP1 (p53 gene) increases intended editing (x-axis) and decreases three types of unintended editing (y-axes), including joining of the reverse transcribed sequence at unintended locations (FIG. 62A), unintended deletions (FIG. 62B), and unintended tandem duplications (FIG. 62C).
[162] FIG. 63 shows that a p53 inhibitor (i53) can enhance the efficiency and precision of PE3 prime editing. This is particularly true when the nicking sgRNA makes a nick upstream (- side) of the pegRNA-directed nick. Only the EMX1 site uses a nick on the "-"
side.FIG. 64 represents various aspects of the disclosure, including the use of CRISPRi screens to reveal cellular genes---including mismatch repair genes¨having an impact on prime editing outcomes, the use of engineered MLH1 of the mismatch repair (MMR) pathway to enhance the efficiency and precision of prime editing, and the demonstration that improved prime editing systems described herein (e.g., PE4 and PE5 systems, and PEmax editor) were shown to exhibit the same beneficial effects in many cell types.
[163] FIG. 64 shows that CRISPRi screens reveal cellular determinants of prime genome editing, that engineered MLHI protein enhances prime editing efficiency and precision, and that improved prime editing systems were characterized across edit and cell types.
1164.1 FIG. 65 provides a schematic showing the optimization of PE2 protein.
[165] FIG. 66 shows the fold change in the frequency of the intended edit using PE2 and various other PE constructs in HEK293T cells (low plasmid dose) at a range of gene targets (HEK3, EMX1, RNF2, FANCF, FUNX , DNA/17'1, YEGFA, HEK4, PRNP, APOE, CXCR4, HEK3).
[166] FIG. 67 shows the fold change in the frequency of the intended edit using PE3 and various prime editor constructs in HeLa cells at a range of gene targets (HEK3, FANCF, RUNX1, VEGFA).
[167] FIG. 68 shows a comparison of prime editing in I-1E1(293T vs. lieLa editing using various PE constructs.
[168] FIG. 69 shows NLS architecture optimization of PE3 in HeLa cells.

59/699 [169] FIG. 70 provides a schematic showing the final PEmax construct, which corresponds to SEQ ID NO: 99.
[170] FIG. 71 shows that PEmax increases indels in addition to the intended edit.
DEFINITIONS
[171] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton etal., Dictionary of Microbiology and Molecular Biology (2nd ed.
1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988);
The Glossary of Genetics, 5th Ed., R. Rieger etal. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them unless specified otherwise.
Cas9 [172] 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 casnl 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 rib onuclease 3 (rric), and a Cas9 domain. The tracrRNA serves as a guide for ribonuclease 3-aided processing of pre-crRNA. Subsequently, Cas9/crRNA/tracrRNA endonucleolytically cleaves a 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

60/699 cleavage typically requires protein and both RNAs. However, single guide RNAs ("sgRNA", or simply "gNRA") can be engineered to incorporate aspects of both the crRNA and tracrRNA into a single RNA species. See, e.g., Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J.A., Charpentier E. Science 337:816-821(2012), the entire contents of which are 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 MI strain of Streptococcus pyogenes." Ferretti etal., 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 HG., 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., IDoudna 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.
[173] 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 etal., "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

61/699 subdomain. The IINH 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 Di OA
and 11840A
completely inactivate the nuclease activity of S. pyogenes Cas9 (jinek etal., Science. 337:816-821(2012); Qi etal., 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: 2). 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: 2). In some embodiments, the Cas9 variant comprises a fragment of SEQ ID NO: 2 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: 2). 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: 2).
Circular perm utant [174] As used herein, the term "circular permutant" refers to a protein or polypeptide (e.g., a Cas9) comprising a circular permutation, which is a change in the protein's structural configuration involving a change in the order of amino acids appearing in the protein's amino

62/699 acid sequence. In other words, circular perrnutants are proteins that have altered N- and C-termini as compared 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 11751 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 topically rearranged.
Such circularly permuted Cas9 proteins ("CP-Cas9"), or variants thereof, retain the ability to bind DNA when complexed with a guide RNA (gRNA). See, Oakes etal., "Protein Engineering of Cas9 for enhanced function," Methods Enzymol, 2014, 546: 491-511 and Oakes et al., "CRISPR-Cas9 Circular Permutants as Programmable Scaffolds for Genome Modification,"
Cell, January 10, 2019, 176: 254-267, each of which are incorporated herein by reference. The instant disclosure contemplates any previously known CP-Cas9 or use of a new CP-Cas9 so long as the resulting circularly permuted protein retains the ability to bind DNA
when complexed with a guide RNA (gRNA). Exemplary CP-Cas9 proteins are SEQ ID NOs: 54-63.
CRISPR
11761 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 have 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

63/699 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 (me) and a Cas9 protein. The tracrRNA serves as a guide for ribonuclease 3-aided processing of pre-crRNA. Subsequently, Cas9/crRNA/tracrRNA endonucleolytically cleaves a 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. pyagenes 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 11 CRISPR-Cas immunity systems" (2013) RNA
Biology 10:5, 726-737; the entire contents of which are incorporated herein by reference.
11771 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),

64/699 and a Cas9 protein. The tracrRNA serves as a guide for ribonuclease 3-aided processing of pre-crRNA. Subsequently, Cas9/crRNA/tracrRNA endonucleolytically cleaves a linear or circular nucleic acid 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 "gRNA") can be engineered to incorporate embodiments of both the crRNA and tracrRNA into a single RNA species¨the guide RNA.
11781 In general, a "CRISPR system" refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated ("Cm") genes, including sequences encoding a Cas gene, a tracr (trans-activating CRISPR) sequence (e.g.
tracrRNA or an active partial tracrRNA), a tracr mate sequence (encompassing a "direct repeat"
and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system), a guide sequence (also referred to as a "spacer" in the context of an endogenous CRISPR
system), or other sequences and transcripts from a CRISPR locus. The tracrRNA
of the system is complementary (fully or partially) to the tracr mate sequence present on the guide RNA.
DNA synthesis template [179] 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 3' 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. The extension 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 DNA polymerase (e.g., a reverse transcriptase). In the case of DNA, the polymerase of the prime editor can be a DNA-dependent DNA polymerase. In various embodiments the DNA
synthesis template 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, toeloop, 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 3' extension arm, the DNA synthesis template can include the portion of the extension arm that spans from the 5' end of the primer binding site (PBS) to 3' 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

65/699 ftanscriptase). In the case of a 5' extension arm, the DNA synthesis template can include the portion of the extension arm that spans from the 5' end of the PEgRNA molecule to the 3' end of the edit template. In some embodiments, the DNA synthesis template excludes the primer binding site (PBS) of PEgRNAs either having a 3' extension arm or a 5' extension arm. Certain embodiments described here 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." In certain embodiments, an RT template may be used to refer to a template polynucleotide for reverse transcription, e.g., in a prime editing system, complex, or method using a prime editor having a polymerase that is a reverse transcriptase. In some embodiments, a DNA synthesis template may be used to refer to a template polynucleotide for DNA
polymerization, e.g., RNA-dependent DNA polymerization or DNA-dependent DNA
polymerization, e.g., in a prime editing system, complex, or method using a prime editor having a polymerase that is an RNA-dependent DNA polymerase or a DNA-dependent DNA
polymerase.
[180] In some embodiments, the DNA synthesis template is a single-stranded portion of the PEgRNA that is 5' of the PBS and comprises a region of complementarity to the PAM strand (i.e., the non-target strand or the edit strand), and comprises one or more nucleotide edits compared to the endogenous sequence of the double stranded target DNA. In some embodiments, the DNA synthesis template is complementary or substantially complementary to a sequence on the non-target strand that is downstream of a nick site, except for one or more non-complementary nucleotides at the intended nucleotide edit positions. In some embodiments, the DNA synthesis template is complementary or substantially complementary to a sequence on the non-target strand that is immediately downstream (i.e., directly downstream) of a nick site, except for one or more non-complementary nucleotides at the intended nucleotide edit positions.
In some embodiments, one or more of the non-complementary nucleotides at the intended nucleotide edit positions are immediately downstream of a nick site. In some embodiments, the DNA synthesis template comprises one or more nucleotide edits relative to the double-stranded target DNA sequence. In some embodiments, the DNA synthesis template comprises one or more nucleotide edits relative to the non-target strand of the double-stranded target DNA
sequence. For each PEgRNA described herein, a nick site is characteristic of the particular

66/699 napDNAbp to which the gRNA core of the PEgRNA associates, and is characteristic of the particular PAM required for recognition and function of the napDNAbp. For example, for a PEgRNA that comprises a gRNA core that associates with a SpCas9, the nick site in the phosphocliester 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).
In some embodiments, the DNA synthesis template and the primer binding site are immediately adjacent to each other. The terms "nucleotide edit", "nucleotide change", "desired nucleotide change", and "desired nucleotide edit" are used interchangeably to refer to a specific nucleotide edit, e.g., a specific deletion of one or more nucleotides, a specific insertion of one or more nucleotides, a specific substitution (or multiple substitutions) of one or more nucleotides, or a combination thereof, at a specific position in a DNA synthesis template of a PEgRNA to be incorporated in a target DNA sequence. In some embodiments, the DNA synthesis template comprises more than one nucleotide edit relative to the double-stranded target DNA sequence. In such embodiments, each nucleotide edit is a specific nucleotide edit at a specific position in the DNA synthesis template, each nucleotide edit is at a different specific position relative to any of the other nucleotide edits in the DNA synthesis template, and each nucleotide edit is independently selected from a specific deletion of one or more nucleotides, a specific insertion of one or more nucleotides, a specific substitution (or multiple substitutions) of one or more nucleotides, or a combination thereof. A nucleotide edit may refer to the edit on the DNA
synthesis template as compared to the sequence on the target strand of the target gene, or a nucleotide edit may refer to the edit encoded by the DNA synthesis template on the newly synthesized single stranded DNA
that replaces the endogenous target DNA sequence on the non-target strand.
Dominant Negative Variant [ 1811 The terms "dominant negative variant" and "dominant negative mutant"
refer to genes or gene products (e.g., proteins) that comprise a mutation that results in the gene product acting antagonistically to the wild-type gene product (i.e., inhibiting its activity). Dominant negative mutations generally result in an altered molecular function (often inactive).
For example, the present disclosure provides dominant negative variants of MMR proteins that inhibit the activity of wild-type MMR proteins (e.g., the dominant negative MLH1 proteins described herein).

67/699 Edit template [182] The term "edit template" refers to a portion of the extension arm that encodes the desired edit in the single strand 3' 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 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.
Extension arm [183] The term "extension arm" refers to a nucleotide sequence component of a PEgRNA
which comprises a primer binding site and a DNA synthesis template (e.g., an edit template and a homology arm) for a polymerase (e.g., a reverse transcriptase). In some embodiments, the extension arm is located at the 3' end of the guide RNA. In other embodiments, the extension arm is located at the 5' end of the guide RNA. In some embodiments, the extension arm comprises a DNA synthesis template and a primer binding site. In some embodiments, the extension arm comprises the following components in a 5' to 3' direction: the DNA synthesis template and the primer binding site. In some embodiments, the extension arm also includes a homology arm. In various embodiments, the extension arm comprises the following components in a 5' to 3' direction: the homology arm, the edit template, and the primer binding site. Since polymerization activity of the reverse transcriptase is in the 5' to 3' direction, the preferred arrangement of the homology arm, edit template, and primer binding site is in the 5' to 3' direction such that the reverse transcriptase, once primed by an annealed primer sequence, polymerizes a single strand of DNA using the edit template as a complementary template strand.
[184] The extension arm may also be described as comprising generally two regions: a primer binding site (PBS) and a DNA synthesis template, 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 3' 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 3' end (i.e., the 3'

68/699 of the primer sequence), which then provides a substrate for a polymerase to begin polymerizing a single strand of DNA from the exposed 3' end along the length of the DNA
synthesis template.
The sequence of the single strand DNA product is the complement of the DNA
synthesis template. Polymerization continues towards the 5' of the DNA synthesis template (or extension arm) until polymerization terminates. Thus, the DNA synthesis template represents the portion of the extension arm that is encoded into a single strand DNA product (i.e., the 3' 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 immediately downstream of the PE-induced nick site. Without being bound by theory, polymerization of the DNA synthesis template continues towards the 5' 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 5' terminus of the PEgRNA (e.g., in the case of the 5' 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.
Fusion protein 11851 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 Laboraloiy Manual (4th ed., Cold Spring Harbor

69/699 Laboratory Press, Cold Spring Harbor, N.Y. (2012)), the entire contents of which are incorporated herein by reference.
Guide RNA ("gRNA") [186] 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 the protospacer sequence of the guide RNA. However, this term 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 Cpfl (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 editing guide RNAs" (or "PEgRNAs").
[187] Guide RNAs or PEgRNAs may comprise various structural elements that include, but are not limited to:
11881 Spacer sequence ¨ the sequence in the guide RNA or PEgRNA (having about 20 nts in length) which binds to the protospacer in the target DNA.
[189] gRNA core (or gRNA scaffold or backbone sequence) - refers to the sequence within the gRNA that is responsible for Cas9 binding, it does not include the 20 bp spacer/targeting sequence that is used to guide Cas9 to target DNA. In some embodiments, the gRNA core or scaffold comprises a sequence that comprises one or more nucleotide alterations compared to a naturally occurring CRISPR-Cas guide RNA scaffold, for example, a Cas9 guide RNA scaffold.
In some embodiments, the sequence of the gRNA core is designed to comprise minimal or no sequence homology to the endogenous sequence of the target nucleic acid at the target site,

70/699 thereby reducing unintended edits. For example, in some embodiments, one or more base pairs in the second stem loop of a Cas9 gRNA core may be "flipped" (e.g., the G-U base pair and the U-A base pair as exemplified in Fig. 49A) to reduce unintended edits. In some embodiments, the gRNA core comprises no more than 1%, 5%, 10%, 15%, 20%, 25%, or 30% sequence homology to the sequence of the double stranded target DNA that flanks 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides upstream or downstream of the position of the one or more nucleotide edits [1901 Extension arm - a single strand extension at the 3' end or the 5' end of the PEgRNA
which comprises a primer binding site and a DNA synthesis template sequence that encodes via a polymerase (e.g., a reverse transcriptase) a single stranded DNA flap containing the genetic change of interest, which then integrates into the endogenous DNA by replacing the corresponding endogenous strand, thereby installing the desired genetic change.
[191] Transcription terminator - the guide RNA or PEgRNA may comprise a transcriptional termination sequence at the 3' of the molecule. in some embodiments, the PEgRNA comprises a transcriptional termination sequence between the DNA synthesis template and the gRNA core.
Homology [192] The terms "homologous," "homology," or "percent homology" as used herein refer to the degree of sequence identity between an amino acid or polynucleotide sequence and a corresponding reference sequence. "Homology" can refer to polymeric sequences, e.g., polypeptide or DNA sequences that are similar. Homology can mean, for example, nucleic acid sequences with at least about: 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
identity. In other embodiments, a "homologous sequence" of nucleic acid sequences may exhibit 93%, 95%, or 98% sequence identity to the reference nucleic acid sequence. For example, a "region of homology to a genomic region" can be a region of DNA that has a similar sequence to a given genomic region in the genome. A region of homology can be of any length that is sufficient to promote binding of a spacer or protospacer sequence to the genomic region. For example, the region of homology can comprise at least 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, or more bases in length such

71/699 that the region of homology has sufficient homology to undergo binding with the corresponding genomic region.
[193] When a percentage of sequence homology or identity is specified, in the context of two nucleic acid sequences or two polypeptide sequences, the percentage of homology or identity generally refers to the alignment of two or more sequences across a portion of their length when compared and aligned for maximum correspondence. When a position in the compared sequence can be occupied by the same base or amino acid, then the molecules can be homologous at that position. Unless stated otherwise, sequence homology or identity is assessed over the specified length of the nucleic acid, polypeptide, or portion thereof. In some embodiments, the homology or identity is assessed over a functional portion or a specified portion of the length.
[194] Alignment of sequences for assessment of sequence homology can be conducted by algorithms known in the art, such as the Basic Local Alignment Search Tool (BLAST) algorithm, which is described in Altschul eta!, J. Mot Biol. 215:403- 410, 1990. A publicly available, intemet interface, for performing BLAST analyses is accessible through the National Center for Biotechnology Information. Additional known algorithms include those published in:
Smith & Waterman, "Comparison of Biosequences", Adv. App!. Math. 2:482, 1981;
Needleman & Wunsch, "A general method applicable to the search for similarities in the amino acid sequence of two proteins" J. MoL Biol. 48:443, 1970; Pearson & Lipman "Improved tools for biological sequence comparison", Proc. Natl. Acad. Sci. USA 85:2444, 1988; or by automated implementation of these or similar algorithms. Global alignment programs may also be used to align similar sequences of roughly equal size. Examples of global alignment programs include NEEDLE (available at www.ebi.ac.uk/Too1s/psa/emboss_needle0 which is part of the EMBOSS
package (Rice P etal., Trends Genet., 2000; 16: 276-277), and the GGSEARCH
program fasta.bioch.virginia.edu/fasta_www2/, which is part of the FASTA package (Pearson W and Lipman D, 1988, Proc. Natl. Acad. ScL USA, 85: 2444-2448). Both of these programs are based on the Needleman-Wunsch algorithm, which is used to find the optimum alignment (including gaps) of two sequences along their entire length. A detailed discussion of sequence analysis can also be found in Unit 19.3 of Ausubel eta! ("Current Protocols in Molecular Biology" John Wiley & Sons Inc, 1994-1998, Chapter 15, 1998).
[195] A skilled person understands that amino acid (or nucleotide) positions may be determined in homologous sequences based on alignment. For example, "H840" in a reference Cas9

72/699 sequence may correspond to 11839, or another corresponding position in a Cas9 homolog when aligned to the reference Cas9 sequence.
Host cell 11961 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 an MLH1 variant and a fusion protein comprising a Cas9 or Cas9 equivalent and a reverse transcriptase.
Inhibit 1197] As used herein the term "inhibiting," "inhibit," or "inhibition" in the context of proteins and enzymes, for example, in the context of enzymes involved in the DNA
mismatch repair pathway, refers to a reduction in the activity of the protein or enzyme. In some embodiments, the term refers to a reduction of the level of enzyme activity, e.g., the activity of one or more enzymes in the DNA mismatch repair pathway, to a level that is statistically significantly lower than an initial level, which may, for example, be a baseline level of enzyme activity. In some embodiments, the term refers to a reduction of the level of enzyme activity, e.g., the activity of one or more enzymes in the DNA mismatch repair pathway, to a level that is less than 75%, less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.1%, less than 0.01%, less than 0.001%, or less than 0.0001% of an initial level, which may, for example, be a baseline level of enzyme activity.
Linker [198] The term "linker," as used herein, refers to a molecule linking two other molecules or moieties. The linker can be an amino acid sequence in the case of a linker joining two fusion proteins. For example, a 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 a prime editing guide RNA which may comprise a RT template sequence and an RT primer binding site. In other embodiments, the linker is an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker is 5-100 amino acids in length, 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,

73/699 80-90, 90-100, 100-150, or 150-200 amino acids in length. Longer or shorter linkers are also contemplated. In certain embodiments, the linker is a self-hydrolyzing linker (e.g., a 2A self-cleaving peptide as described further herein). Self-hydrolyzing linkers such as 2A self-cleaving peptides are capable of inducing ribosomal skipping during protein translation, resulting in the ribosome failing to make a peptide bond between two genes, or gene fragments.

[199] The term "MLH1" refers to a gene encoding MLH1 (or MutL Homolog 1), a DNA
mismatch repair enzyme. The protein encoded by this gene can heterodimerize with mismatch repair endonuclease PMS2 to form MutL alpha (MutLa), part of the DNA mismatch repair system. MLHI mediates protein-protein interactions during mismatch recognition, strand discrimination, and strand removal. In mismatch repair, the heterodimer MSH2:MSH6 (MutSa) forms and binds the mismatch. MLH1 then forms a heterodimer with PMS2 (MutLa) and binds the MSH2:MSH6 heterodimer. The MutLa heterodimer then incises the nicked strand 5' and 3' of the mismatch, followed by excision of the mismatch from MutLa-generated nicks by EX01.
Finally, POLS resynthesizes the excised strand, followed by LIG1 [200] An exemplary amino acid sequence of MLH1 is human isoform 1, P40692-1:
>sp1P406921MLH1JILIMAN DNA mismatch repair protein Mlhl OS=Homo sapiens 0X-GN=MLH1 PE=1 SV=1:
MSFVAGVIRRLDETVVNRIAAGEVIQRPANAIKEMIENCLDAKSTSIQVIVKEGGLICLIQIQDNGTGIRKEDL
DIVCERPITSKLQSFEDLASIS'FYGFRGEALASISHVAHVTITIKTADGKCAYRASYSDGKLKAPPKPCAGN
QGTQFTVEDLFYNIATRRKALKNPSEEYGKILEVVGRY SVHNAGISFS VKKQGETVADVR'FLPNASTVDNIR
SIFGNAVSRELIEIGCEDKTLAFKMNGYISNANYSVICKCIFLLFINHRLVESTSLRKAIETVYAKYLPKNTHPF
LYLSLEISPQNVDVNVIIPTICHEVHFLHEESILERVQQMESKLLGSNSSRMYFTQTLLPGLAGPSGEMVKST
TSLTSSSTSGSSDKVYAHQMVRTDSREQKLDAFLQPLSICPLSSQPQAIVTEDKTDISSGRARQQDEEMLELP
APAEVAAICNQSLEGDITICGTSEMSEKRGPTSSNPRICRHREDSDVEMVEDDSRKEMTAACTPRRRIINLTSV
LSWEEINEQGHEVLREIVILIINHSFVGCVNPQWALAQHQTKLYLLNTTKLSEELFYQILIYDFANFGVLRLS
EPAPLFDLAMLALDSPESGWTEEDGPKEGLAEYIVEFLICKKAEMLADYF'SLEIDEEGNLIGLPLUDNYVPPL
EGLPIFILRLATEVNWDEEKECFESLSKECAMFYSIRKQYISEESTLSGQQSEVPGSIPNSWKWTVEHIVYKA
LRSHILPPKIIF'rEDGNILQLANLPDLYICVFERC (SEQ ID NO: 204), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to and including 100% sequence identity with SEQ ID NO: 204.
[201] Another exemplary amino acid sequence of MLH1 is human isoform 2, P40692-(wherein amino acids 1-241 of isoform 1 are missing): >spIP40692-21MLH1_HUMAN
lllsofonn 2 of DNA mismatch repair protein Mlhl OS=Homo sapiens 0X=9606 GN=MLH1:

74/699 MNGY I SNANYSVKKCIFT.LFINITRINESTSLIZ K AIETVYA AYLPKNTE PF SLEISPQNVDVNVI-I
PTKITE
VHFLHEESILERVQQHIESKLLG SNS SRMYFTQTLLPGLAGPSGEM VK S TTSLTSSSTSGSSDKVYAHQMVR
TDSREQKLDAFLQPISKPL S SQPQAWTEDK.TDI SSGRARQQDEEMLEIPAP AEVAAKNQSLEGDTTKGTSE
MSEKRGPTSSNPRKRHRED SDVEMVEDDSRKEMTAACTPRRRIINLTSVL SLQEEINEQGHEVLREMLHNH
SFVGCVNPQWALAQHQTKLYLLNTFKLSEELFYQILIYDFANFGVLRLSEPAPLFDLAMLALDSPESGWTE
EDGPKEGLAEYIVEFLKKKAEML ADYFSLEIDEEGNLIGLPILIDNY VPPLEGLPIFILRLATEVNWDEEKEC
FESLSKECAMFYSIRKQYISEESTLSGQQSEVPGSIPNSWKWIVEHIVYKAIRSHILPPICHF1 __ EUGNILQL
AN
LPDLYKVFERC (SEQ ID NO: 205), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to and including 1000/0 sequence identity with SEQ ID NO: 205.
12021 Another exemplary amino acid sequence of MLH1 is human isoform 3, P40692-3 (where amino acids 1-101 (MSFVAGVIRR...ASISTYGFRG (SEQ ID NO: 206) is replaced with MAF): >spIP40692-2[MLH1 HUMAN Isoform 2 of DNA mismatch repair protein Mlhl OS=Homo sapiens OX=9606 GN=MLH1:
MAFEALASISHVAHVTITTKTADGKCAYRASYSDGKLKAPPKPCAGNQGTQITVEDLFYNIATRRKALKNP
SEEYGKILEVVGRYSVHNAGI SF SVKKQGETVADVRTLPNASTVDNIRSIFGNAVSRELIEIGCEDKTL AFKM
NGYISNANYSVICKCIFLLFINIIRLVESTSLRKAIETVYAAYLPKNTHPFLYLSLEISPQNVDVNVIIPTKIIEVH
FLIMPSILERVQQIIIESKLLGSNSSRMYFTQTLLPGLAGPSGEMVKSTTSLTSSSTSGSSDKVYAHQMVRTD
SREQICLDAFLQPLSKPLSSQPQAIVTEDKTDISSGRARQQDEEMLELPAPAEVAAICNQSLEGDTTKOTSEMS
EKROPTSSNPRKRHREDSDVEMVEDDSRKEMTAACTPRRRIINLTSVLSLQEEINEQGHEVLREMLIINHSF

GPKEGLAEYIVEFLKKKAEMLADYFSLEIDEEGNLIGLPLLIDNYVPPLEGLPIFII,RLATEVNWDEEKECFES
LSKECAMFYSIRKQYISEESTLSGQQSEVPGSIPNSWKWTVEHIVYKAIRSHILPPKIIFTEDGNILQLANLPD
LYKVFERC (SEQ ID NO: 207), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to and including 100% sequence identity with SEQ ID NO: 207.
[203] The present disclosure contemplates targeting MLH1 and/or MMR pathway components that interact with MLHI, including any wildtype or naturally occurring variant of MLHI, including any amino acid sequence having at least 70%, or 75%, or 80%, or 85%, or 90%, or 95%, or 99% or more sequence identity with any of SEQ ID NOs: 204, 208-213, 215, 216, 218, 222, or 223, or nucleic acid molecules encoding any MLH1 or variant of MLH1 (e.g., a dominant negative mutant of MLHI as described herein), for inhibiting, blocking, or otherwise inactivating the wild type MLH1 function in the MMR pathway, and consequently, inhibiting, blocking, or otherwise inactivating the MMR pathway, e.g., during genome editing with a prime editor.

75/699 [204] In some embodiments, inactivation of the MMR pathway involves an inhibitor that disrupts, blocks, interferes with, or otherwise inactivates the wild type function of the MLH1 protein. In some embodiments, inactivation of the MMR pathway involves a mutant of the MLH1 protein, for example, contacting a target cell with a MLH1 mutant protein or expressing in a target cell an MLH1 mutant nucleic acid that encodes an MLH1 mutant protein. In some embodiments, the MLH1 mutant protein interferes with, and thereby inactivates, the function of a wild type MLH1 protein in the MMR pathway. In some embodiments, the MLH1 mutant is a dominant negative mutant. In some embodiments, the MLH mutant protein is capable of binding to an MLH1-interacting protein, for example, MutS.
[205] Without being bound by theory, MLH1 dominant negative mutants function by saturating binding of MutS, thereby blocking MutS-wild type MLH1 binding and interfering with the function of the wild type MLH1 protein in the MMR pathway.
[206] In various embodiments, the dominant negative IMLHI can include, for example, MLH1 E34A, which is based on SEQ ID NO: 222 and having the following amino acid sequence (underline and bolded to show the E34A mutation):
MSFVAGVIRRLDETVVNRIAAGEVIQRPANAIICAMIENCLDAKSTSIQVIVKEGGLKLIQIQDNGTGIRKEDL
DIVCERFTTSKLQSFEDLASISTYGFRGEALASISHVAHVTITTKTADGKCAYRASYSDGKLICAPPKPCAGN
QGTQITVEDLFYNIATRRKALKNPSEEYGKILEVVGRYSVHNAGISFSVKICQGETVADVRTLPNASTVDNIR
SIFGNAVSRELIEIGCEDKTLAFKMNGYISNANYSVKKCIFLLFINHRLVESTSLRICAIETVYAAYLPKNTHPF

TSLTSSSTSGSSDKVYAHQMVRTDSREQKLDAFLQPLSKPLSSQPQAIVTEDKTDISSGRARQQDEEMLELP
APAEVAAKNQSLEGDTIKGTSEMSEKRGPTSSNPRKRHREDSDVEMVEDDSRICEMTAACT'PRRREINLTSV
LSLQEEINEQGIIEVLREMLHNHSFVGCVNPQWALAQHQTKLYLLNITKLSEELFYQILIYDFANFGVLRLS
EPAPLFDLAINALALDSPESGWTEEDGPKEGLAEYIVEFLKKKAEMLADYFSLEIDEEGNLIGLPLLIDNYVPPL
EGLPIFILRL ATE VNWDEEXECFESLSKECAMFY SIRKQYISEESTLSGQQSEVPGSIPNSWKWTVEHIVYKA
IASHILPPICHFTEDGNILQLANLPDLYKVFERC (SEQ ID NO: 222), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 900/o, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to and including 100% sequence identity with SEQ ID NO: 222.
12071 In various other embodiments, the dominant negative MLH1 can include, for example, MLH1 A756, which is based on SEQ ID NO: 208 and having the following amino acid sequence (underline and bolded to show the A756 mutation at the C terminus of the sequence):
MSFVAGVIRRLDETVVNRIAAGEVIQRPANAMEMIENCLDAKSTSIQVIVKEGGLICLIQIQDNOTGIRKEDL
DIVCERFITSKLQSFEDLASISTYGFRGEALASISHVAHVTITTICTADGKCAYRASYSDGKLKAPPICPCAGN

SUFGNAVSRELIEIGCEDICILAFKNINGYISNANYSVKKCIFLLFINIIRLVESTSLIMMETVYAAYLPKNTIIPF
LYLSLEISPQNVDVNVIIPTKREVHFLIIEESILERVQQMESKLLGSNSSRMYFTQTLLPGLAGPSGEMVKST

76/699 TSLTSSSTSGSSDKVYAHQMVRTDSREQKID AFLQPLSKPLSSQPQAIVT.EDKTDISSGRARQQDEEML ELY
APAEVAAKNQSLEGDITKGTSEMSEKRGPTSSNPRICRHREDSDVEMVEDDSRKEMTAACTPRRRIINLTSV
LSLQEEINEQGHEVLREML:HNHSFVGCVNPQWALAQHQTKLYLLNTTKLSEELFYQILIYDFANFG'VLRLS
EPAPLFDLAMLALDSPESGWTEEDGPKEGLAEYIVEFLKICKAEMLADYFSLEIDMGNLIGLPLLIDNYVPPL
EGLI:VITAL ATEVNVVDEFICECFESLSKECAMFYSIRKQYISEESTLSGQQSEVPGSIPNSWKWTVEHIVYKA.
LRSHILPPKHF1EDGNILQLANLPDLYKVFERH(SEQ ID NO: 208), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to and including 100% sequence identity with SEQ ID NO: 208 (wherein the [-] indicates deleted amino acid residue(s) relative to the parent or wildtype sequence).
12081 In still other embodiments, the dominant negative MLH1 can include, for example, MLH1 A754-A756, which is based on SEQ ID NO: 209 and having the following amino acid sequence (underline and bolded to show the A754-A756 mutation at the C
terminus of the sequence):
MSFVAGVIRRLDETVVNRIAAGEVIQRPANAIKEMIENCIDAKSTSIQVIVKEGGLICLIQIQDNGTGIIRKEDL
DIVCERFITSKLQSFE.DLASISTYGFRGEALASISHVAHVTITTKTADGXCAYRASYSDGKLKAPPICPCAGN
QGTQITVEDLEYNIATRRKALKNPSEEYGKILEVVGRYSVHNAGISFSVICKQGETVADVRTLPNASTVDNIR
SIFGNAVSRELIEIGC.EDKTLAFKMNGYISNANYS'VKKCIFLLFINHRLVESTSLRKAIETVY AAYLPKNTHPF
LYLSLEISPQNV.DVNVHPTKHEVHFLHEESILERVQQHIESKLLGSNSSRMYFTQTLLPGLAGPSG.EMVKST
TSLTSSSTSGSSDKVYAHQMVR.TDSREQKLDAFLQPLSKPLSSQPQAIVTEDKTDISSGRARQQDEEMLELP
APAEVAAKNQSLEG.DTT.KGTSEMSEKRGPTSSNPRKRHREDSDVEMV.EDDSRKEMTAACT.PRRRIINLTSV
LSLQEEINEQGHEVLREMLHNHSFVGCVNPQWALAQHQT.KLYLLN'TTKLSEELFYQILIYDFANFGVLRLS
EPAPLFDLAMLALDSPESGWTEEDGPICEGLAEYIVEFLKKKAEMLADYFSLEIDEEGNLIGLPLLIDNYVPPL
EGLPIFILRLATEVNWDEEKECFESLSICECAMFYS1RKQYISEESTLSGQQSEVPGSIPNSWK.WTVEHIVYKA
LRSHILPPKHFTEDGNILQLANLPDLYKVFF - (SEQ ID NO: 209), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to and including 100% sequence identity with SEQ ID NO: 209 (wherein the [- - -] indicates deleted amino acid residue(s) relative to the parent or wildtype sequence).
12091 In yet other embodiments, the dominant negative MLH1 can include, for example, MUD
E34A A754-A756, which is based on SEQ ID NO: 210 and having the following amino acid sequence (underline and bolded to show the E34A and A754-A756 mutations):
MSFVAGVIRRLDETVVNRIAAGE'VIQRPANAIKAMIENCLDAKSTSIQVIVKEGGLKLIQIQDNGTGIRICEDL
DIVCMFTTSKLQSFEDLASISTYGFRGEALASISHVAHV'ITITKTADGKCAYRASYSDGKLKAPPKF'CAGN
QGTQITVEDLFYNIATRRKALKNPSEEYGKILEVVGRYSVHNAGISFSVKKQGETVADVRTLPNASTVDNIR
SIFGNAVSRELIEIGCEDKTLAFKMNGYISNANYSVKKCIFLLFINHRLVESTSLRKAIETVYAAYLPKNTHPF
LYLSLEISPQNVDVNVIIPTKHEVHFLHEESILERVQQHTESKLLGSNSSRMYFTQTLLPGLAGPSGEMVKST
TSLTSSSTSGSSDKVYAHQMVR'FDSRF,QKLDAFLQPLSKPLSSQPQAIVTEDKTDISSGRAR.QQDEEMLELP
APAEVAAICNQSLEGDTIXGTSEMSEKRGPTSSNPRKRHREDSDVE'MVEDDSRKEMTAACTPRRMINLTSV
LSLQEEINEQGHEVLREMLHNHSFVGCVNPQWALAQHQTKLYLLNTTKLSEELFYQILIYDFANFGVLRLS
EPAP.LFDLAMLALDSPESGWTEEDGPKEGLAEYIVEFLKKKAEMLADYFSLEIDEEGNLIGLPLLTDNYVPPL

77/699 EGLPIF II,RI, ATE VNWDEEK
ECFESLSKECAMFYS1RKQYISEESTLSGQQSEVPGSIPNSWKWTVEHIVYKA.
IRSHILPPIGIFTEDGNILQLANLPDLYKVF[- - -] (SEQ ID NO: 210), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to and including 100% sequence identity with SEQ ID NO: 210.
12101 In certain embodiments, the dominant negative MIAll can include, for example, :MLH1 1-335, which is based on SEQ ID NO: 211 and having the following amino acid sequence (contains amino acids 1-335 of SEQ NO: 204):
MSFVAGVIRRLDETVVN.R1AAGEVIQRPANAIKEMIENCLDAKSTS1QVIVKEGGLKLIQIQDNGTGIRKEDL
DIVCERFTTSKLQSPEDLASISTYGFR.GEALASISHVAHVTITTKTADGKCAYRASYSDGKLKAPPKPCAGN

SIFGNAVSRELIEIGCEDKTLAFKMNGYISNANYSVICKCIFLLFINHRLVESTSLRICAIETVYAAYLPKNTHPF
LYLSLEISPQNVDVNVHPTKHEVHFLHEES1LERVQQHIESKLL (SEQ ID NO: 211), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to and including 100% sequence identity with SEQ 1E) NO: 211.
[211] In other embodiments, the dominant negative MLH1 can include, for example, MLH1 1-335 E34A, which is based on SEQ 113 NO: 212 and having the following amino acid sequence (contains amino acids 1-335 of SEQ NO: 204 and a E34A mutation relative to SEQ
ID NO:
204):
MSFVAGVIRRLDETVVNRIAAGEVIQRPANAIKAMIENCLDAKSTSIQVIVICEGGLKLIQIQDNGTOIRICEDL
DIVCERFITSKLQSFEDLASISTYGFRGEALASISHVAHVTITIKTADGKCAYRASYSDGKIKAPPICPC.AGN
QGTQITVEDLFYNIA.TRRKALKNPSEEYGKILEVVGRYSVIINAGISFSVKICQGETVAD'VRTI,PNA.STVDNIR

SIFGNAVSRELIEIGCEDKTLAFKMNGYISNANYS'VKICCIFILFINFIRINESTSIRKAIETVYAAYLPKNTHPF
LYI-SLEISPQNVDVNVHPTIMEVHFIEFESILERVQQHIESKLL (SEQ ID NO: 212), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to and including 100% sequence identity with SEQ ID NO: 212.
[212] In still other embodiments, the dominant negative MLH1 can include, for example, MLH1 1-335 NLSs'm (or referred to as MLH1CITINT0, which is based on SEQ ID NO:
204 and having the following amino acid sequence (contains amino acids 1-335 of SEQ
NO: 204 and an NLS sequence of SV40):
MSFVAGVIRRLDETVVNRIAAGEVIQRPANAIKENIIENCLDAKSTSIQVIVKEGGLKLIQIQDNGTGIRKEDL
DIVCERFTTSKLQSFEDLASISTYGFRGEALASISHVAHVIITTK'FADGKCAYRASYSDGKLICAPPKPCAGN
QGTQITVEDLFYNIATRRKALKNPSEEYGICILEVVGRYSVHNAGISFSVKKQGETVADVRTLPNASTVDNIR

78/699 SIFGNAVSRELIEIGCEDKTI.AFKMNGYISNANYSVKKCIFLLFINIIRINESTSLRKATETVYAAYLPKNTHPF
LYLSLEISPQNVDVNVI-IPTKHEVHFIBEESILERVQQHIESKLLPIKKKRKV (SEQ NO: 213), with the underlined and bolded portion referring to the NLS of SV40), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to and including 100% sequence identity with SEQ ID NO: 213.
[2131 In still other embodiments, the dominant negative MLH1 can include, for example, MLH I 1-335 NLSaliernate (which is based on SEQ ID NO: 204 and having the following amino acid sequence (contains amino acids 1-335 of SEQ NO: 204 and an alternate NLS
sequence)):

DIVCERFITSICLQSFEDLASISTYGFRGEALASISHVAHVIIITKTADGKCAYRASYSDGKLICAPPKPCAGN
QGTQITVEDLFYNIATRRICALKNPSEEYGICILEVVGRYSVIINAGISFSVKKQGETVAD'VRTLPNASTVDNIR
SIFGNAVSRELIEIGCEDKTLAFKMNGYISNANYSVKKCIFLLFINIIRLVESTSLRKAIETVYAAYLPICN'TIIPF

LYLSLEISPQNVDVNVHPTKHEVHFLBFFSILERVQQHIESKLL-[alternate NLS sequence] (SEQ ID
NO: 214)-[alternate NLS sequence], or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to and including 100% sequence identity with SEQ ID NO: 214. The alternate NLS sequence can be any suitable NLS sequence, including but not limited to:
Description Sequence SEQ ID NO:
NLS MKRTADGSEFESPKKKRKV SEQ ID NO: 101 NLS MDSLLMNRRKFLYQFICNVRWAKGRRETYLC SEQ ID NO: 1 NLS of nucleoplasmin A VKIIPAATKKAGQAKKKKID SEQ ID NO: 133 NLS of EGL-13 MSRRRICANPTKLSENAKKLAICEVEN SEQ ID NO: 134 NLS of c-MYC PAAKRVKLD SEQ ID NO: 135 NLS of TUS-protein KLKIKRPVK SEQ ID NO: 136 NLS of polyoma large T-Ag VSRKRPRP SEQ
ID NO: 137 NLS of Hepatitis r) virus antigen EGAPPAKRAR SEQ ID NO: 138 NLS of murine p53 PPQPKKKPLDGE SEQ ID NO: 139 NLS of PEI and PE2 SGGSKRTADGSEFhPKKKRKV SEQ ID NO: 103 In some embodiments, an NLS sequence is appended to the N-terminus of a protein and begins with a methionine ("M"). In other embodiments, an NLS sequence may be appended at the C-terminus of a protein, or between multiple domains of a fusion protein, and does not begin with a methionine (i.e., the M in, for example, SEQ ID NOs: 101, 1, and 134 is not included in the NLS
when it is appended at the C-terminus or between two domains in a fusion protein).

79/699 [214] In still other embodiments, the dominant negative MLII1 can include, for example, MLH1 501-756, which corresponds to a C-terminal fragment of SEQ ID NO: 204 that corresponds to amino acids 501-756 of SEQ ID NO: 204:
INLTSVLSLQEEINEQGHEVLRENILHNHSFVGCVNPQWALAQHQTKLYLLNTTICI,SF,ELFYQII,IYDFANFG
VLRLSEPAPLFDLAMLALDSPESGWTEEDGPKF,GIAEYIVEFLKKICAEMLADYFSLEIDEEGNLIGLPLIADN
YVPPLEGLPIFILRIATEVNWDEFICECFESLSKECAMFYSIRKQYISEESTI,SGQQSEVPGSIPNSWKWTVEHI
VYKALRSHILPPKHFTF,DGNR,QLANI,PDLYK.VFF,RC (SEQ ID NO: 215), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to and including 100% sequence identity with SEQ NO: 215.
[215] in still other embodiments, the dominant negative MLH1 can include, for example, MLH1 501-753, which corresponds to a C-terminal fragment of SEQ ID NO: 204 that corresponds to amino acids 501-753 of SEQ ID NO: 204:
INLTSVLSWEEINEQGHEVLREMIENHSFVGCVNPQWALAQHQTKLYLLNTTKLSEELFYQ11,IYDFANFG
VLRLSEPAPLFDLAMLAIDSPESGWTEEDGPKEGLAEYIVEFLICKKAEMLADYFSLEIDEEGNLIGLPILIDN
YVPPLEGLPIFTLRIATEVNWDEEKECI. __ ESL SKECAMFYSERKQYISEES71, VYKALRSHILPPKIIFFEDGNILQLANLPDLYK.VFF - (SEQ ID NO: 216), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to and including 100% sequence identity with SEQ ID NO: 216.
[216] In still other embodiments, the dominant negative MLEI1 can include, for example, MLH1 461-756, which is a C-terminal fragment of SEQ ID NO: 204 that corresponds to amino acids 461-756 of SEQ ID NO: 204:
KRGPTSSNPRKRHREDSDVFNIVEDDSRKEMTAACTPRRRIINLTSVISI.,QEEINF-QGBEVLREMIRNHSFV
GCVNPQWALAQHQTKLYLLNITKI,SEELFYQ11,1YDFANFGVLRLSEPAPLFDLAMLALDSPESGWTF.EDG
PKEGLAEYIVEFLKKKAEMLADYFSLEIDEEGNLIGLPI.LIDNYVPPLEGLPIFILRLATEVNWDEEKECFESL
SKECANIFYSIRKQYISEESTI,SGQQSEVPGSIPNSWKWTVEHIVYKAIRSHILPPKHFIEDGNILQI,ANI,PDL, YKVFERC (SEQ ID NO: 217), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to and including 100% sequence identity with SEQ B3 NO: 217.
[217] In various embodiments, the dominant negative MLH1 can. include, for example. MLII1 461-753, which is a C-terminal fragment of SEQ ID NO: 204 that corresponds to amino acids 461-753 of SEQ ID NO: 204:

80/699 KRGPTSSNPRICRHREDSDVFIvIVEDDSRKEMTAACTPR RRIINLTS SLQEEINEQGHEVI,REMLIINHSFV
GCVNPQWALAQHQTKLYI,I,NITKLSEET,FYQ11.1YDFANFGVLRI,SF,PAPLFDLAMLAI,DSPESGWTEEDG

PKEGLAEYI'VEFLKKKAEMLADYFSLEIDEEGNLIGLPLUDNYVPPLEGLPIPTIALATEVNWDEXKECFESI, SKECANIFYSIRKQYISEESTLSGQQSE'VPGSIPNSWKWTVEHTVYKALRSHILPPKHF _________________ I EDGNILQLANI.PDI, YKVF[- - -] (SEQ ID NO: 218), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to and including 100% sequence identity with SEQ ID NO: 218.
In various other embodiments, the dominant negative MLH1 can include, for example, MLH1 461-753, which is a C-terminal fragment of SEQ ID NO: 204 that corresponds to amino acids 461-753 of SEQ ID NO: 204, and which further comprises an N-terminalNLS, e.g., NLSsv :
[NLSI-KRGPTSSNPRKRHREDSDVEMVEDDSRKEMTAACIPRRRIINLTSVLSLQEEINEQGHEVLREMLHNHSFV
GCVNPQWALAQHQTKLYLLNTTKLSEELFYQILIYDFANFOVLRLSEPAPLFDLAMLALDSPESGWTEEDG
PKEGLAEYIVEFLKKKAEMLADYFSLEIDEEGNLIGLPLLIDNYVPPLEGLPIFILRLATEVNWDEEKECFESL

YKVF[- - -1 (SEQ ID NO: 218), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to and including 100% sequence identity with SEQ ID NO: 218. The NLS sequence can be any suitable NLS
sequence, including but not limited to SEQ ID NOs: 1, 101, 103, 133-139.
napDNAbp 12181 As used herein, the term "nucleic acid programmable DNA binding protein"
or "napDNAbp," of which Cas9 is an example, refer to proteins that use RNA:DNA
hybridization to target and bind to specific sequences in a DNA molecule. Each napDNAbp is associated with at least one guide nucleic acid (e.g., vide RNA), 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 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.
[219] 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 spacer sequence then hybridizes to the "target strand." This displaces a "non-target strand" that is complementary to the target strand, which forms the single

81/699 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.
Nickase [220] The term "nickase," as used herein, may refer to a Cas9 with one of the two nuclease domains inactivated. This enzyme is capable of cleaving only one strand of a target DNA. As used herein, a "nickase" may refer to a napDNAbp (e.g., a Cas protein) which is capable of cleaving only one of the two complementary strands of a double-stranded target DNA sequence, thereby generating a nick in that strand. In some embodiments, the nickase cleaves a non-target strand of a double stranded target DNA sequence. In some embodiments, the nickase comprises an amino acid sequence with one or more mutations in a catalytic domain of a canonical napDNAbp (e.g., a Cas protein), wherein the one or more mutations reduces or abolishes nuclease activity of the catalytic domain. In certain embodiments, the napDNAbp is a Cas9 nickase, a Cas12a nickase, or a Cas12b1 nickase. In some embodiments, the nickase is a Cas9 that comprises one or more mutations in a RuvC-like domain relative to a wild type Cas9 sequence or to an equivalent amino acid position in other Cas9 variants or Cas9 equivalents. In some embodiments, the nickase is a Cas9 that comprises one or more mutations in an HNH-like domain relative to a wild type Cas9 sequence or to an equivalent amino acid position in other Cas9 variants or Cas9 equivalents. In some embodiments, the nickase is a Cas9 that comprises an aspartate-to-alanine substitution (D10A) in the RuvC I catalytic domain of Cas9 relative to a canonical Cas9 sequence or to an equivalent amino acid position in other Cas9 variants or Cas9 equivalents. In some embodiments, the nickase is a Cas9 that comprises a H840A, N854A, and/or N863A mutation relative to a canonical Cas9 sequence, or to an equivalent amino acid position in other Cas9 variants or Cas9 equivalents. In some embodiments, the term "Cas9 nickase" refers to a Cas9 with one of the two nuclease domains inactivated.
This enzyme is

82/699 capable of cleaving only one strand of a target DNA. In some embodiments, the nickase is a Cas protein that is not a Cas9 nickase.
12211 In some embodiments, the napDNAbp of the prime editing complex comprises an endonuclease having nucleic acid programmable DNA binding ability. In some embodiments, the napDNAbp comprises an active endonuclease capable of cleaving both strands of a double stranded target DNA. In some embodiments, the napDNAbp is a nuclease active endonuclease, e.g., a nuclease active Cas protein, that can cleave both strands of a double stranded target DNA
by generating a nick on each strand. For example, a nuclease active Cas protein can generate a cleavage (a nick) on each strand of a double stranded target DNA. In some embodiments, the two nicks on both strands are staggered nicks, for example, generated by a napDNAbp comprising a Cas12a or Cas12b1. In some embodiments, the two nicks on both strands are at the same genomic position, for example, generated by a napDNAbp comprising a nuclease active Cas9. In some embodiments, the napDNAbp comprises an endonuclease that is a nickase.
For example, in some embodiments, the napDNAbp comprises an endonuclease comprising one or more mutations that reduce nuclease activity of the endonuclease, rendering it a nickase. In some embodiments, the napDNAbp comprises an inactive endonuclease. For example, in some embodiments, the napDNAbp comprises an endonuclease comprising one or more mutations that abolish the nuclease activity. In various embodiments, the napDNAbp is a Cas9 protein or variant thereof. The napDNAbp can also be a nuclease active Cas9, a nuclease inactive Cas9 (dCas9), or a Cas9 nickase (nCas9). In a preferred embodiment, the napDNAbp is Cas9 nickase (nCas9) that nicks only a single strand. In certain embodiments, the napDNAbp is a Cas9 nickase, a Cas12a nickase, or a Cas12b1 nickase. In some embodiments, the napDNAbp can be selected from the group consisting of: Cas9, Cas12e, Cas12d, Cas12a, Cas12b1, Cas12b2, Cas13a, Cas12c, Cas12d, Cas12e, Cas12h, Cas12i, Cas12g, Casl2f (Cas14), Casl2f1, Cas12j (Case), and Argonaute, and optionally has a nickase activity such that only one strand is cut. In some embodiments, the napDNAbp is selected from Cas9, Cas12e, Casl 2d, Casl 2a, Cas12b1, Cas12b2, Cas13a, Cas12c, Cas12d, Cas12e, Cas12h, Cas12i, Cas12g, Cas12f (Cas14), Casl2f1, Casi2j (Case), and Argonaute , and optionally has a nickase activity such that one DNA strand is cut preferentially to the other DNA strand.

83/699 Nick site [222] The terms "cleavage site," "nick site," and "cut site" as used interchangeably herein in the context of prime editing, refer to a specific position in between two nucleotides or two base pairs in the double-stranded target DNA sequence. lin some embodiments, the position of a nick site is determined relative to the position of a specific PAM sequence. In some embodiments, the nick site is the particular position where a nick will occur when the double stranded target DNA
is contacted with a napDNAbp, e.g., a nickase such as a Cas nickase, that recognizes a specific PAM sequence. For each PEgRNA described herein, a nick site is characteristic of the particular napDNAbp to which the gRNA core of the PEgRNA associates with, and is characteristic of the particular PAM required for recognition and function of the napDNAbp. For example, for a PEgRNA that comprises a gRNA core that associates with a SpCas9, 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 l of the PAM sequence).
[223] In some embodiments, a nick site is in a target strand of the double-stranded target DNA
sequence. In some embodiments, a nick site is in a non-target strand of the double-stranded target DNA sequence. In some embodiments, the nick site is in a protospacer sequence. In some embodiments, the nick site is adjacent to a protospacer sequence. In some embodiments, a nick site is downstream of a region, e.g., on a non-target strand, that is complementary to a primer binding site of a PEgRNA. In some embodiments, a nick site is downstream of a region, e.g., on a non-target strand, that binds to a primer binding site of a PEgRNA. In some embodiments, a nick site is immediately downstream of a region, e.g., on a non-target strand, that is complementary to a primer binding site of a PEgRNA. In some embodiments, the nick site is upstream of a specific PAM sequence on the non-target strand of the double stranded target DNA, wherein the PAM sequence is specific for recognition by a napDNAbp that associates with the gRNA core of a PEgRNA. In some embodiments, the nick site is downstream of a specific PAM sequence on the non-target strand of the double stranded target DNA. wherein the PAM sequence is specific for recognition by a napDNAbp that associates with the gRNA core of a PEgRNA. In some embodiments, the nick site is 3 nucleotides upstream of the PAM sequence, and the PAM sequence is recognized by a Streptococcus pyogenes Cas9 nickase, a P.
lavamentivorans Cas9 nickase, a C diphtheriae Cas9 nickase, a N. cinerea Cas9, a S. aureus Cas9, or a N. lari Cas9 nickase. In some embodiments, the nick site is 3 nucleotides upstream of

84/699 the PAM sequence, and the PAM sequence is recognized by a Cas9 nickase, wherein the Cas9 nickase comprises a nuclease active HNH domain and a nuclease inactive RuvC
domain. In some embodiments, the nick site is 2 base pairs upstream of the PAM sequence, and the PAM
sequence is recognized by a S'. thermophilus Cas9 nickase.
Nucleic acid molecule [2241 The term "nucleic acid," as used herein, refers to a polymer 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, 0(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'-0-methylcytidine, arabinose, and hexose), or modified phosphate groups (e.g., phosphorothioates and 5'N phosphoramidite linkages).

12251 As used herein, "p53" refers to tumor protein 53. Among other functions, p53 plays a role in DNA damage and repair, specifically in its role in regulation of the cell cycle, apoptosis, and genomic stability. P53 can activate DNA repair proteins when DNA has been damaged. P53 may also arrest cell growth by holding the cell cycle at the Gl/S regulation point on DNA damage recognition, thereby providing DNA repair proteins more time to fix the DNA
damage before allowing the cell to continue the cell cycle. Thus, in some embodiments of the methods described herein, p53 is inhibited (e.g., by the p53 inhibitor protein "i53," or another p53 inhibitor) to increase the efficiency of prime editing.
PEgRNA
12261 As used herein, the terms "prime editing guide RNA" or "PEgRNA" or "extended guide RNA" refer to a specialized form of a guide RNA that has been modified to include one or more additional sequences for implementing the prime editing methods and compositions described

85/699 herein. As described herein, the prime editing 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 or DNA. 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 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 "DNA
synthesis template" which encodes (by the polymerase of the prime editor) a single-stranded DNA which, in turn, has been designed to be (a) homologous with the endogenous target DNA
to be edited, and (b) which comprises at least one desired nucleotide change (e.g., a transition, a transversion, a deletion, or an insertion) 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 a "spacer or linker" sequence, or other structural elements, such as, but not limited to aptamers, stem loops, hairpins, toe loops (e.g., a 3' toeloop), or an RNA-protein recruitment domain (e.g., MS2 hairpin). 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.
1227] In certain embodiments, the PEgRNAs have 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. The reverse transcriptase template may also be referred to more broadly as the "DNA synthesis template" where the polymerase of a prime editor described herein is not an RT, but another type of polymerase.
[228] In certain other embodiments, the PEgRNAs have 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. The reverse transcriptase template may also be referred to more broadly as the "DNA synthesis template" where the polymerase of a prime editor described herein is not an RT, but another type of polymerase.
[229] In still other embodiments, the PEgRNAs have 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 "homology arm," an "edit template," and a "primer binding site." In certain embodiments, a PEgRNA
comprises from 5' to

86/699 3', a space, a DNA synthesis template, and a primer binding site. 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 PEgRNAs are further described and defined herein.
[230] In still other embodiments, the PEgRNAs have 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 "homology arm," an "edit template," and a "primer binding site." 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 PEgRNAs may also comprise a transcriptional terminator sequence at the 3' end. These sequence elements of the PEgRNAs are further described and defined herein.

[231] As used herein, "PEI" refers to a prime editor system 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: 100, which is shown as follows;
MKRTADGSEP __ SPKKKRK'VDKKYSIGLDIGTNSVGWAVITDE YKVPSKICFICVLGNTDRHSIKKNLIGA
LLFDSGETAEATRLKRTARRRYTRRKNRIC'YLQUFSNEMAKVDDSFFIIRLEESFLVEEDICKHERHPI
FGNI'VDEVAYHEICYPTIYILLRKKL'VDSTDKADLRLIYLALAHMIKFRGHELIEGDLNPDNSDVDKLFI
QLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSN
FDIAEDAKLQLSKRTYDDDLDNLLAQIGDQYADLFLAAKNISDAILLSDILRVNTEITKAPLSASMIKR
YDEIIIIQDLTLLKALVRQQLPEKYKEIFFDQSICNGYAGYIDGGASQEEFYKIIKPILEKMDGTEELLV
KLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYP.FLKDNREICIEICILTFRIPYYVGPLARGNS
RFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKRSLLYEYFTVYNELTKV
KYVTEGMRKPAFLSGEQKKAIVDLLFKTNRICVTVKQLKE D YFICKIECFDSVEISGVEDRFNASLGTY
HDLLKIIKDKDFLDNEENEDIL.EDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLICRRRYTGWGR
LSRICLINGIRDKQSGICTIL.DFLKSDGFANRNFMQLIEIDDSLTFICEDIQ.KAQVSGQGDSLHERIANLAG

KEHPVENTQLQNEKLYL Y YLQN GRDMYVDQELDINRLSD YDVDAIVPQSFLKDDSIDNKVLTRSDKN
RGKSDNVPSEEVVKKIVIKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGEIKRQLVETR(NTK

GGFDSPTVAYSVLVVAKVEKGKS1CKLKSVICELLGITIMERSSFEKNP1DFLEAKGYKEVICKDLIII:LP
KYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQIIKII Y
LDELIEQISEFSKRVILADANLDICVLSAYN KIIRDKPIREQAEN MILFILTNLGAPAAFKYFDTTIDRICR
YTSTKEVLDATLILIQSITGL 'YETRIDLSQLGGDSGGSSGGSSGSETPGTSESATPESSGGSSGGSSILME
DEYRLIIE'ISKEPDVS'LGSTWLSDI;PQAWAETGGMGLAVRQAPLHPLKATSTPVSIKQYPMSQEARLGIKPIHQRL
L
DQGILVPCQSPWNTPLLPVKKPG'ThDYRPVQDLREVNKRVEDIHPTVPNPYNLLS'GLPPSHQWYTfrIDLKDAFFC

LRLHPTSQPLFAFEWRDPEI4GB`GQLTIVTRLPQGFICAISPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATS

87/699 ELDCQQGTRA LLQTLGNLGYRASAKKAQICQKQVICYLGYLLKEGQRWLTEARKETIMGQPTPKTPRQLREFLGT
AGFCRLWTPGFA.EMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELF'VDEKQGYAKGV
LTQICLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGICLTMGQPLVILAPHAVEALVKQPPDRWIõSN
A RMTHYQALLLDTDRVOFGPVVA LNPA TLLPLPEEGLQHNCLDILA
EAHGTRPDLTDQPLPDADH7'WYT'DGSSL
LQEGQRKAGAAPTTETEVIWAKALPAGTSAQRAELIALTQALKAMEGICKLNVY7'DSRYA
FATAHIHGEIYRRRGLL
TSEGKEIKNKDEJLALLKALFLPKRLSHHCPGHQKGHSAEARGNRMADQAARKAAITET.'PDTSTLLIENSSPSGGS

ICRTADGSEFE.PKKKIIKV (SEQ 113 NO: 100) KEY:
NUCLEAR. LOCALIZATION SEOUENCE (NLS) TOP:(SEQ ID NO: 101), BOTTOM: (SEQ ID NO:
103) CAS9(11840A) (SEQ ID NO: 37) (SEQ ID NO: 37 corresponds identically to SEQ ID
NO: 2, except with an H840A
substitution) 33-AMINO ACID LINKER (SEQ. ID NO: 102) M-IVILV reverse transcriptase (SEQ ID NO: 81).
[232] Alternatively, PE1 may also refer to the prime editor fusion protein of SEQ m NO: 100, i.e., without the pegRNA complexed thereto. PEI may be complexed with a pegRNA
during operation and/or use in prime editing.

[233] As used herein, "PE2" refers to a prime editing system comprising a fusion protein comprising Cas9(H840A) and a variant MNILV RT having the following structure:
[NLS]-[Cas9(H840AW[1inker]-[MMLV_RT(D200N)(7330P)(L603W)(T306K)(W313F)] + a desired PEgRNA, wherein the PE fusion has the amino acid sequence of SEQ ID NO: 107, which is shown as follows:
MKRTADGSEFESPKICKRKVDICKYSIGLDIGTNSVGWAVITDEYKVPSICKFICVLGNTDRHSIKKNLIGA
LLFDSGEMEATRLICRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERIIPI
FGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAIIMIKFRGHFLIEGDLNPDNSDVDKLFI
QLVQTYNQLFEENPINASGVDAKMISARLSKSRRLENLIAQLPGEKICNGLFGNLIALSLGLTPNFKSN

YDEMIQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEICMDGTEELLV
ICLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLICDNREKIEKILTFRIPYYVGPIARGNS
RFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEICVLPKILSLLYEYFTVYNELTKV
KYVTEGMRKPAFISGEQICKAIVDLLFKTNRKVTVKQLKEDYFICKIECFDSVEISGVEDRFNASLGTY
HDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGR
ISRICLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLIIEFIIANLAG
SPAIKKGILQTVICVVDELVKVMGRHKPENIVIEMARENQTTQKGQICNSRERMKRIEEGIKELGSQIL
KEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFIKDDSIDNICVLTRSDKN
RGKSDNVPSEEVVICICMKNYWRQLLNAICLITQRKFDNLTICAERGGLSELDICAGFIKRQLVETRQITK
EIVAQILDSRMNTKYDENDKLIREVKVITLICSICINSDFRICDFQFYKVREINNYEIHAHDAYLNAVVGTA
LIKKYPICLESEFVYGDYKVYDVRKMIAKSEQEIGKATAICYFFYSNIMNFFKTEITLANGEIRKRPLIET
NGETGEIVWDKGRDFATVRKVISMPQVNIVICKTEVQTGGFSKESILPKRNSDKLIARKICDWDPKKY
GGFDSPTVAYSVLVVAICVEKG.KSKKLKSVKELLGMMERSSFEKNPIDFLEAKGYKEVKKDLIIKLP
KYSLFELENGRKRMIASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKFIY
LDEHEQISEFSKR.VILADANLDKVISAYNKFIRDICPIREQAENIIRLFTLTNLGAPAAFKYFDITIDRKR

88/699 YTSTKEVLDATLIHQSITGLYETRIDLSQLGGDSGGSSGGSSGSETPGTSESATPESSGGSSGGSSTLN/E
DEYRLHETSKEP DLSIESTWLSDFPQA WA ETGGMGLAVRQAPLI IP LKA TSTPVSIKQY PMSQ EA
RLGIKPHIQRLL
DQGILVPCQSPW ATTP LLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSG L PPSHQ WYTYLDLKDA
FFC
LRLHPTSQ PLFAFEWRDPEMGISGQLTYPTRLPQGFKNSPTLFNEALHRDLA DFRIQHPDLILLQYVDDLLLAA
TSE
LDCQQGTRALLQTLENLGYRASAKKAQICQKQVKYLGYLLKEGORWLTEA RKETYMGQPTPKTPRQLREFLGKA
GFCRLFIPGFA.EMAAPLYPL.TKPGTLFNWGPDQQKAYQEIKQALLTAPA LGLPDLTKPFELFVDEKQGYAKGVLT

QKLGP WRRPVAY LSKKLDPVAAGWPPCIRMVA AIAVLTKDAG KLTMGQ P LVI LA PHAVEALVKQ
PPDRWLSNAR
MTHYQA LLIDTDRVQFGPVVA LNPATLLPL PEEGLQ HNCI.DILA EA HGTRPDLIDQPLPDA
DIITWYTDGSSLLQ
EGQRKAGAA VITETEVIWA KALPAGTSA ORA EUALTOALKMA EGKKL NVIITDSRYA FA TA H IHGEIY
RRRGWL TS' EGKEIKNKDE LALL KALFLPKRISIILICPGIIQKGHSAEA RGNRMADQAARKAAITETPDTSTL
LIEMSSPSGGSKR
TADGSEFEPKKKRKV (SEQ ID NO: 107) KEY:
NUCLEAR LOCALIZATION SEQUENCE (NLS) TOP:(SEQ ID NO: 101), BOTTOM: (SEQ ID NO:
103) CAS9(11840A) (SEQ ID NO: 37) 33-AMINO ACID LINKER (SEO ID NO: 102) M-M1.1" reverse transcriptase (SEQ ID NO: 98).
12341 Alternatively, PE2 may also refer to the prime editor fusion protein of SEQ ID NO: 107, i.e., without the pegRNA complexed thereto. PE2 may be complexed with a pegRNA
during operation and/or use in prime editing.

[235] A.s 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 [236] 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.

12371 As used herein, "PE4" refers to a system comprising PE2 plus an MLH1 dominant negative protein (ix., wild-type ML:Hl. with amino acids 754-756 truncated as described further herein) expressed in trans. In some embodiments, PE4 refers to a fusion protein comprising PE2 and an MLII1 dominant negative protein joined via an optional linker.

89/699

90 PCT/US2022/012054 [238] As used herein, "PE5" refers to a system comprising PE3 plus an MLH1 dominant negative protein (i.e., wild-type MLH1 with amino acids 754-756 deleted as described further herein, which may be referred to as "M_LH1 A754-756" or "MLHIdn") expressed in trans. In some embodiments, PE5 refers to a fusion protein comprising PE3 and an MLH1 dominant negative protein joined via an optional linker.
PEmax [239] As used herein, "PEmax" (see FIG. 54B) refers to a PE complex comprising a fusion protein comprising Cas9(R221K N394K H840A) and a variant MMLV RT pentamutant (13200N
T306K W313F T330P L603W) having the following structure: [bipartite NLS]-[Cas9(12221K)(N394K)(H840A)Mlinker]-[MMLV_RT(D200N)(T330P)(L603W)Hbipartite NLSHNLS] + a desired PEgRNA, wherein the PE fusion has the amino acid sequence of SEQ
ID NO: 99.
PE4max [240] As used herein, "PE4max" refers to PE4 but wherein the PE2 component is substituted with PEmax.
PE5max [241] As used herein, "PE5max" refers to PE5 but wherein the PE2 component of PE3 is substituted with PEmax.
PE-short [2421 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:
NIKRTADGSEFESPKKKRKVDKKYSIGLDIG'TNSVGWAVITDEYKVPSKKFKVLGNTDRIISIKKNLIGA
LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEEFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPI

QLVQTYNQLFEENPINASGVDAICAIL,SARLSKSRRLENLIAQLPGEICKNGLFGNL1ALSLGLTPNFKSN

YDEIIRQDLTLLKAL'VRQQLPEICYKEIFIFDQS1CNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLV
ICLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDF'YPFLKDNREKIEKILTFRIPYITVGPLARGNS
RFAWMTRKSEET1TPWNFEEV'VDKGASAQSFIERMTNFDKNLPNEKVLPICHSLLYEYFTVYNELTKV
KYVTEGMRKPAFLSGEQKKANDLLFKTNRKVTVKQLICEDYFICICIECFDS'VEISGVEDRFNASLGTY
IIDLLICIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDICVMKQLKRRRYTGWGR
LSRKLINGIRDKQSGICTILDFLKSDGFANRNFMQLIHDDSLTFICEDIQICAQVSGQGDSLHEHIANLAG
SPAIKKGILQTVICVVDELVKVMGRHICPENIVIEMARENQTTQKG'QICNSRERMKRIEEGIKELGSQ1L
ICERPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSD'YDVDAIVPQSFLICDDSIDNKVLTRSDKN
RGKSDN'VPSEEVVIOCMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAG1FIKRQLVETRQITIC

HVAQILDSRMNTKVDENDKLIREVICVITI,KSKINSDFRKDFQVYKVRETNNYHHAHDAYLNAVVGTA
LIKKATKLESEFVYGDYKVYDVRKNIIAKSEQEIGKATAKYFFYSNEVINFFKTEITLANGEIRKRPLIET
NGETGEIVWDKGRDFA.TVRKVLSMPQVNIVKKTEVQTGGFSKESELPKRNSDIKILIARKKDWDPKKY

KYSLFELENGRKIR1VHASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQI.FVEQHMIY
LDEITEQISEFSKRVILADANIDKVISAYNKFIRDICPIREQAENITHLFTITNLGAPAAFKYFDTTIDRKR
YTST.KEVLDATLIHQSITGLYETRIDLSQLGGDSGGSSGGSSGSETPGTSESATPESSGGSSGGSSTLN/E
DEYR1JTh7.'SKEPDVSLGS7W1SDFPQAWAETGGMGLAVRQAPLHPIXATSTPVSIKQYP.MSQEARLGIKPHIQRL
L
DQGILVPCQSPWN7'PLLPVKKPGTND.YRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFC
LRLHPTSQPLFAFEWRDPEMCHSGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIOHPDLILLOYVDDLLLAATSE
LDCQQGTRALLQTLGNLGYRASAKKAQK'QKQVKYLGYLIXEGQRWLTF,ARKETBYGQPTPKTPROLREFLGKA
GFCRLFIPGFAEMAAPLYPLTKPGTI,F'NWGPDQQKAYOEIKQALL7:4PALGLPDLTKPFELFVDEKQGYAKGVLT

QKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVIIAPHAVEALVKQPPDRWLSNAR
MTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDNSRLINSGGSKRTADGSEFEPKKKRK.V (SEQ
ID NO: 117) KEY:
NUCLEAR LOCALIZATION SEQUENCE (NLS) TOP:(SEQ ID NO: 101), BOTTOM: (SEQ ID NO:
103) CAS9(H840A) (SEQ ID NO: 37) 33-AM1NO ACID LINKER 1 (SEQ ID NO: 102) M-MLV TRUNCATED reverse tremscriptase (SEQ ID NO: 80) Polymerase [243] As used herein, the term "polymerase" refers to an enzyme that synthesizes a nucleotide strand and that may be used in connection with the prime editor systems described herein. The polymerase can be a "template-dependent" polymerase (i.e., a polymerase that synthesizes a nucleotide strand based on the order of nucleotide bases of a template strand). The polymerase can also be a "template-independent" polymerase (i.e., a polymerase that synthesizes a nucleotide strand without the requirement of a template strand). A polymerase may also be further categorized as a "DNA polymerase" or an "RNA polymerase." In various embodiments, the prime editor system comprises a DNA polymerase. In various embodiments, the DNA
polymerase can be a "DNA-dependent DNA polymerase" (i.e., whereby the template molecule is a strand of DNA). In such cases, the DNA template molecule can be a PEgRNA, wherein the extension arm comprises a strand of DNA. In such cases, the PEgRNA may be referred to as a chimeric or hybrid PEgRNA which comprises an RNA portion (i.e., the guide RNA
components, including the spacer and the gRNA core) and a DNA portion (i.e., the extension arm). In various other embodiments, the DNA polymerase can be an "RNA-dependent DNA polymerase"
(i.e., whereby the template molecule is a strand of RNA). In such cases, the PEgRNA
is RNA, i.e., including an RN.A extension. The term "polymerase" may also refer to an enzyme that catalyzes the polymerization of nucleotide (i.e., the polymerase activity). Generally, the enzyme will

91/699 initiate synthesis at the 3'-end of a primer annealed to a polynucleotide template sequence (e.g., such as a primer sequence annealed to the primer binding site of a PEgRNA) and will proceed toward the 5' end of the template strand. A "DNA polymerase" catalyzes the polymerization of deoxynucleotides. As used herein in reference to a DNA polymerase, the term DNA polymerase includes a "functional fragment thereof." A "functional fragment thereof' refers to any portion of a wild-type or mutant DNA polymerase that encompasses less than the entire amino acid sequence of the polymerase and which retains the ability, under at least one set of conditions, to catalyze the polymerization of a polynucleotide. Such a functional fragment may exist as a separate entity, or it may be a constituent of a larger polypeptide, such as a fusion protein.
Prime editing [244] As used herein, the term "prime editing" refers to an approach for gene editing using napDNAbps, a polymerase (e.g., a reverse transcriptase), and specialized guide RNAs that include a DNA synthesis template for encoding desired new genetic information (or deleting genetic information) that is then incorporated into a target DNA sequence.
Certain embodiments of prime editing are described in the embodiments of FIG. 1. Classical prime editing is described in the inventors' publication of Anzalone, A. V. et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149-157(2019), which is incorporated herein by reference in its entirety.
12451 Prime editing represents a platform for genome editing that 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 5' or 3' 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 (or is homologous to) sequence as the endogenous strand (immediately downstream of the nick site) 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 downstream of the nick site is replaced by the newly synthesized replacement strand containing the desired edit. In

92/699 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 mechanism of target-primed reverse transcription (TPRT), which can be engineered for conducting precision CRISPR/Cas-based genome editing with high efficiency and genetic flexibility. TPRT is used by mobile DNA
elements, such as mammalian non-LIR retrotransposons and bacterial Group II
introns. 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 transcriptase as the DNA polymerase component, the prime editors described herein are not limited to reverse transcriptases but may include the use of virtually any 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, wherever the specification mentions a "reverse transcriptase," the person having ordinary skill in the art should appreciate that any suitable DNA polyinerase may be used in place of the reverse transcriptase.
Thus, in one aspect, the prime editors may comprise Cas9 (or an equivalent napDNAbp) which is programmed 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-

93/699 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. 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. 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 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 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.
[246] 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 a prime editing guide RNA
(PEgRNA). In various embodiments, the prime editing guide RNA (PEgRNA) 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

94/699 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, 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 R-loop "target strand" (i.e., the strand hybridized to the protospacer of the extended gRNA) or the "non-target strand" (i.e., the strand 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 reverse transcription (i.e., "target-primed RT"). In certain embodiments, the 3' end DNA strand hybridizes to a specific RT priming sequence on the extended portion of the guide RNA, i.e., the "reverse transcriptase priming sequence" or "primer binding site" on the PEgRNA. In step (d), a reverse transcriptase (or other suitable DNA polymerase) is introduced which synthesizes a single strand of DNA from the 3' end of the primed site towards the 5' end of the prime editing guide RNA. The DNA polymerase (e.g., reverse transcriptase) can be fused to the napDNAbp or alternatively can be provided in trans to the napDNAbp. This forms a single-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."
This process may introduce at least one or more of the following genetic changes:
transversions, transitions, deletions, and insertions.
[247] The term "prime editor (PE) system" or "prime editor (PE)" 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

95/699 napDNAbps, reverse transcriptases (or another DNA polymerase), fusion proteins (e.g., comprising napDNAbps and reverse transcriptases or comprising napDNAbps and DNA
polymerases), prime editing guide RNAs, and complexes comprising fusion proteins and prime editing guide RNAs, as well as accessory elements, such as second strand nicking components (e.g., second strand sgRNAs) and 5' endogenous DNA flap removal endonucleases (e.g., FEN I) for helping to drive the prime editing process towards the edited product formation.
[2481 Although in the embodiments described thus far the PEgRNA constitutes a single molecule comprising a guide RNA (which itself comprises a spacer sequence and a gRNA core or scaffold) and a 5' or 3' extension arm comprising the primer binding site and a DNA synthesis template, the PEgRNA may also take the form of two individual molecules comprised of a guide RNA and a trans prime editor RNA template (tPERT), which essentially houses the extension arm (including, in particular, the primer binding site and the DNA synthesis domain) and an RNA-protein recruitment domain (e.g., MS2 aptamer or hairpin) in the same molecule which becomes co-localized or recruited to a modified prime editor complex that comprises a tPERT
recruiting protein (e.g., MS2cp protein, which binds to the MS2 aptamer).
Prime editor [249] The term "prime editor" refers to 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 guide RNA"). The term "prime editor" may refer to the fusion protein or to the fusion protein complexed with a PEgRNA, and/or further complexed with a second-strand nicking sgRNA. 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, a prime editor (e.g., PE1, PE2, or PE3) may be provided as a system along with an inhibitor of the DNA mismatch repair pathway, such as a dominant negative MLH I protein. In various embodiments, the inhibitor of the DNA mismatch repair pathway, such as a dominant negative MLH1 protein, may be provided in trans to the prime editor. In other embodiments, the inhibitor of the DNA
mismatch repair pathway, such as a dominant negative MLH1 protein, may be complexed to the prime editor, e.g., coupled through a linker to the prime editor fusion protein.

96/699 Primer binding site 1250] The term "primer binding site" or "the PBS" refers to the portion of a PEgRNA as a component of the extension arm (for example, at the 3' end of the extension arm) . The term "primer binding site" refers to a single-stranded portion of the PEgRNA as a component of the extension arm that comprises a region of complementarity to a sequence on the non-target strand.
In some embodiments, the primer binding site is complementary to a region upstream of a nick site in a non-target strand. In some embodiments, the primer binding site is complementary to a region immediately upstream of a nick site in the non-target strand. In some embodiments, the primer binding site is capable of binding to the primer sequence that is formed after nicking (e.g., by a nickase component of a prime editor, for example, a Cas9 nikcase) of the target sequence by the prime editor. When a prime editor nicks one strand of the target DNA
sequence (e.g., by a Cas nickase component of the prime editor), 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. In some embodiments, the PBS is complementary to, or substantially complementary to, and can anneal to, a free 3' end on the non-target strand of the double stranded target DNA at the nick site. In some embodiments, the PBS annealed to the free 3' end on the non-target strand can initiate target-primed DNA synthesis.
Protospacer 12511 As used herein, the term "protospacer" refers to the sequence (-20 bp) in DNA adjacent to the PAM (protospacer adjacent motif) sequence. The protospacer shares the same sequence as the spacer sequence of the guide RNA. The guide RNA anneals to the complement of the protospacer sequence on the target DNA (specifically, one strand thereof, i.e., the "target strand"
versus the "non-target strand" of the target DNA sequence). In some embodiments, in order for a Cas nickase component of the prime editor to function it also requires a specific protospacer adjacent motif (PAM) which varies depending on the Cas protein component itself, e.g., the type of Cas protein. For example, 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

97/699 context of the description surrounding the appearance of either "protospacer"
or "spacer" will help inform the reader as to whether the term is in reference to the ,gRNA or the DNA target.
Protospacer adjacent motif (PAM) 12521 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 Cas9 nuclease. Typically, the PAM sequence is on either strand, and is downstream in the 5' to 3' direction of the 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 5'-NGG-3' 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.
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.
1.2531 For example, with reference to the canonical SpCas9 amino acid sequence is SEQ ED
NO: 2, 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 D1 135E variant of canonical SpCas9 still recognizes NGG, but it is more selective compared to the wild type SpCas9 protein.
12541 lIt 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 (T
dCas) recognizes NAAAAC. These are examples and 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 ldlobase smaller than SpCas9, so it can be packaged into adeno-associated virus (AAV). Further reference may be made to Shah et al.,

98/699 "Protospacer recognition motifs: mixed identities and functional diversity,"
RNA Biology, 10(5):
891-899 (which is incorporated herein by reference).
Reverse transcriptase [255] 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 IH 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 proofreading (Saunders and Saunders, Microbial Genetics Applied to Biotechnology, London:
Croom Helm (1987)). A detailed study of the activity of A.MV reverse transcriptase and its associated RNase H activity has been presented by Berger etal., Biochemistry 22:2365-2372 (1983). Another reverse 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., etal., 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.
[256] In addition, the invention contemplates the use of reverse transcriptases that are error-prone, i.e., that may be referred to as error-prone reverse transcriptases or reverse transcriptases that do not support high fidelity incorporation of nucleotides during polymerization. During synthesis of the single-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 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

99/699 strand molecule through hybridization to the corresponding endogenous target strand, removal of the endogenous displaced strand, ligation, and then through one more round of endogenous DNA
repair and/or sequencing processes.
Reverse transcription 12571 As used herein, the term "reverse transcription" indicates the capability of an enzyme to synthesize a 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.
Protein, peptide, and polypeptide [2581 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, finctionalization, 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 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 are incorporated herein by reference.

100/699 Silent mutation [259] As used herein, the term "silent mutation" refers to a mutation in a nucleic acid molecule that does not have an effect on the phenotype of the nucleic acid molecule, or the protein it produces if it encodes a protein. Silent mutations can be present in coding regions of a nucleic acid (i.e., segments of a gene that encode for a protein), or they can be present in non-coding regions of a nucleic acid. A silent mutation in a nucleic acid sequence, e.g., in a target DNA
sequence or in a DNA synthesis template sequence to be installed in the target sequence, may be a nucleotide alteration that does not result in expression or function of the amino acid sequence encoded by the nucleic acid sequence, or other functional features of the target nucleic acid sequence. When silent mutations are present in a coding region, they may be synonymous mutations. Synonymous mutations refer to substitutions of one base for another in a gene such that the corresponding amino acid residue of the protein produced by the gene is not modified.
This is due to the redundancy of the genetic code, allowing for multiple different codons to encode for the same amino acid in a particular organism. When a silent mutation is in a non-coding region or a junction of a coding region and a non-coding region (e.g., an intron/exon junction), it may be in a region that does not impact any biological properties of the nucleic acid molecule (e.g., splicing, gene regulation, RNA lifetime, etc.). Silent mutations may be useful, for example, for increasing the length of an edit made to a target nucleotide sequence using prime editing to evade correction of the edit by the MA4R pathway as described herein. In certain embodiments, the number of silent mutations installed may be one, or two, or three, or four, or five, or six, or seven, or eight, or nine, or ten, or more. In certain other embodiments involving at least two silent mutations, the silent mutations may be installed within one, or two, or three, or four, or five, or six, or seven, or eight, or nine, or ten, or 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides from the intended edit site.
Spacer sequence [2601 As used herein, the term "spacer sequence" in connection with a guide RNA or a PEgRNA refers to the portion of the guide RNA or PEgRNA of about 20 nucleotides (e.g., 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides) which contains a nucleotide sequence that shares the same sequence as the protospacer sequence in the target DNA sequence. The spacer sequence anneals to the complement of the protospacer sequence to form a ssRNAJssDNA
hybrid structure at the target site and a corresponding R loop ssDNA structure of the endogenous DNA strand.

101/699 Target site 1261] The term "target site" refers to a sequence within a nucleic acid molecule to be edited by a prime editor (PE) disclosed herein. The target site may refer to the endogenous sequence within the nucleic acid molecule to be edited, e.g., endogenous genomic sequence of a target genome, which is identical to the sequence of the DNA synthesis template except for the one or more nucleotide edits to be installed present on the DNA synthesis template (and except that the DNA
synthesis template contains Uracil instead of Thymine), or the corresponding endogenous sequence on the non-target strand that is complementary to the DNA synthesis template except for one or more mismatches at the position of the one or more nucleotide edits to be installed present on the DNA synthesis template. The target site may also further refer to the sequence within a nucleic acid molecule to which a complex of the prime editor (PE) and gRNA binds.
Variant [262] 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 [263] 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.

102/699 DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS
12641 The present disclosure provides compositions and methods for prime editing with improved editing efficiency and/or reduced indel formation by inhibiting the DNA mismatch repair pathway while conducting prime editing of a target site. The inventors have surprisingly found that the editing efficiency of prime editing may be significantly increased (e.g., 2-fold increase, 3-fold increase, 4-fold increase, 5-fold increase, 6-fold increase, 7-fold increase, 8-fold increase, 9-fold increase, 10-fold increase, 11-fold increase, 12-fold increase,13-fold increase, 14-fold increase, 15-fold increase, 16-fold increase, 17-fold increase, 18-fold increase, 19-fold increase, 20-fold increase, 21-fold increase, 22-fold increase, 23-fold increase, 24-fold increase, 26-fold increase, 27-fold increase,28-fold increase, 29-fold increase, 30-fold increase, 31-fold increase, 32-fold increase, 33-fold increase, 34-fold increase, 35-fold increase, 36-fold increase, 37-fold increase, 38-fold increase, 39-fold increase, 40-fold increase ,41-fold increase, 42-fold increase, 43-fold increase, 44-fold increase, 45-fold increase, 46-fold increase, 47-fold increase, 48-fold increase, 49-fold increase, 50-fold increase, 51-fold increase, 52-fold increase, 53-fold increase, 54-fold increase, 55-fold increase, 56-fold increase, 57-fold increase, 58-fold increase, 59-fold increase, 60-fold increase, 61-fold increase, 62-fold increase, 63-fold increase, 64-fold increase, 65-fold increase, 66-fold increase, 67-fold increase, 68-fold increase, 69-fold increase, 70-fold increase, 71-fold increase, 72-fold increase, 73-fold increase, 74-fold increase, 75-fold increase, 76-fold increase, 77-fold increase, 78-fold increase, 79-fold increase, 80-fold increase, 81-fold increase, 82-fold increase, 83-fold increase, 84-fold increase, 85-fold increase, 86-fold increase, 87-fold increase, 88-fold increase, 89-fold increase, 90-fold increase, 91-fold increase, 92-fold increase, 93-fold increase, 94-fold increase, 95-fold increase, 96-fold increase, 97-fold increase, 98-fold increase, 99-fold increase, 100-fold increase or more) when one or more functions of the DNA mismatch repair (MMR) system are inhibited, blocked, or otherwise inactivated during prime editing. In addition, the inventors have surprisingly found that the frequency of indel formation resulting from prime editing may be significantly decreased (e.g., 2-fold decrease, 3-fold decrease, 4-fold decrease, 5-fold decrease, 6-fold decrease, 7-fold decrease, 8-fold decrease, 9-fold decrease, or 10-fold decrease or lower) when one or more functions of the IDN.A mismatch repair (MMR) system are inhibited, blocked, or otherwise inactivated during prime editing.

103/699 [2651 The disclosure relates to the surprising finding that the efficiency and/or specificity of prime editing is impacted by a cell's own DNA mismatch repair (MAIR) DNA
repair pathway.
As described herein (e.g., in Example 1), the inventors developed a novel genetic screening method---referred to in one embodiment as "pooled CRISPRi screen for prime editing outcomes"¨which led to the identification of various genetic determinates, including MMR, as affecting the efficiency and/or specificity of prime editing. Accordingly, in one aspect, the present disclosure provides novel prime editing systems comprising a means for inhibiting and/or evade the effects of MMR, thereby increasing the efficiency and/or specificity of prime editing.
In one embodiment, the disclosure provides a prime editing system that comprises an MMR-inhibiting protein, such as, but not limited to, a dominant negative MMR
protein, such as a dominant negative MLH1 protein (i.e., "MLHldn"). In another embodiment, the prime editing system comprises the installation of one or more silent mutations nearby an intended edit, thereby allowing the intended edit from evading MMR recognition, even in the absence of an MMR-inhibiting protein, such as an MLHldn. In another aspect, the disclosure provides a novel genetic screen for identifying genetic determinants, such as MMR, that impact the efficiency and/or specificity of prime editing. In still further aspects, the disclosure provides nucleic acid constructs encoding the improved prime editing systems described herein. The disclosure in other aspects also provides vectors (e.g., AAV or lentivirus vectors) comprising nucleic acids encoding the improved prime editing system described herein. In still other aspects, the disclosure provides cells comprising the improved prime editing systems described herein. The disclosure also provides in other aspects the components of the genetic screens, including nucleic acid and/or vector constructs, guide RNA, pegRNAs, cells (e.g., CRISPRi cells), and other reagents and/or materials for conducting the herein disclosed genetic screens.
In still other aspects, the disclosure provides compositions and kits, e.g., pharmaceutical compositions, comprising the improved prime editing system described herein and which are capable of being administered to a cell, tissue, or organism by any suitable means, such as by gene therapy, mRNA delivery, virus-like particle delivery, or ribonucleoprotein (RNP) delivery. In yet another aspect, the present disclosure provides methods of using the improved prime editing system to install one or more edits in a target nucleic acid molecule, e.g., a genomic locus. In still another aspect, the present disclosure provides methods of treating a disease or disorder using the improved prime editing system to correct or otherwise repair one or more genetic changes (e.g., a

104/699 single polymorphism) in a target nucleic acid molecule, e.g., a genomic locus comprising one or more disease-causing mutations.
[266] In one embodiment, the MLI-11 protein is inhibited, blocked, or otherwise inactivated. In other embodiments, other proteins of the MMR system are inhibited, blocked, or otherwise inactivated, including, but not limited to, PMS2 (or MutL alpha), PMS1 (or MutL beta), MLH3 (or MutL gamma), MutS alpha (MSH2-MSH6), MutS beta (MSH2-MSH3), MSH2, MSH6, PCNA, RFC, EX01, POLS, and PCNA. The inhibition may involve inhibiting the protein with an inhibitor (e.g., antibody or small molecule inhibitor or a dominant negative variant of the protein which disrupts, blocks, or otherwise inactivates the function of the protein, e.g., a dominant negative form of MLII1). The inhibition may also involve any other suitable means, such as by protein degradation (e.g., PROTAC-based degradation of MLH1), transcript-level inhibition (e.g., siRNA transcript degradation / gene silencing or microRNA-based inhibition of translation of the MLH1 transcript), or at the genetic level (i.e., installing a mutation in the MLHI gene (or regulatory regions) which inactivates or reduces the expression of the MLHI
gene, or which installs a mutation which inactivates, blocks, or minimizes that activity of the encoded MLH1 product). In addition, the disclosure contemplates that the prime editor (e.g., delivered as a fusion protein comprising a napDNAbp and a polymerase, such as a Cas9 nickase fused to a reverse transcriptase) may be administered together with any inhibitor of the DNA
mismatch repair pathway.
[267] Accordingly, the present disclosure provides a method for editing a nucleic acid molecule by prime editing that involves contacting a nucleic acid molecule with a prime editor, a pegRNA, and an inhibitor of the DNA mismatch repair pathway, thereby installing one or more modifications to the nucleic acid molecule at a target site with increased editing efficiency and/or lower indel formation. The present disclosure further provides polynucleotides for editing a DNA target site by prime editing comprising a nucleic acid sequence encoding a napDNAbp, a polymerase, and an inhibitor of the DNA mismatch repair pathway, wherein the napDNAbp and polymerase is capable in the presence of a pegRNA of installing one or more modifications in the DNA target site with increased editing efficiency and/or lower indel formation. The disclosure further provides, vectors, cells, and kits comprising the compositions and polynucleotides of the disclosure, as well as methods of making such vectors, cells, and kits, as well as methods for delivery such compositions, polynucleotides, vectors, cells and kits to cells

105/699 in vitro, ex vivo (e.g., during cell-based therapy which modify cells outside of the body), and in vivo.
MMR pathway 12681 As noted above, the present disclosure relates to the observation that the efficiency and/or specificity of prime editing is impacted by a cell's own DNA mismatch repair (MMR) DNA
repair pathway. DNA mismatch repair (MMR) is a highly conserved biological pathway that plays a key role in maintaining genomic stability (e.g., see FIG. 8A and 8B).
Escherichia colt MutS and Mutl, and their eukaryotic homologs, MutSa and MutLa, respectively, are key players in MMR-associated genome maintenance. In various aspects, the disclosure contemplates any suitable means by which to inhibit, block, or otherwise inactivate the DNA
mismatch repair (MMR) system, including, but not limited to inactivating one or more critical proteins of the MMR. system at the genetic level, e.g., by introducing one or more mutations in the gene(s) encoding a protein of the MMR system. Such proteins include, but are not limited to MLH1, PMS2 (or MutL alpha), PMS1 (or MutL beta), MLH3 (or MutL gamma), MutS alpha (MSH2-MSH6), MutS beta (MSH2-MSI-13), MSH2, MSH6, PCNA, RFC, EX01, POLO, and PCNA.
The nucleotide and amino acid sequences of such naturally occurring proteins and variants thereof are known in the art. Exemplary sequences are provided herein. The present disclosure embraces using any inhibitor, blocking agent, knockdown strategy, or other means of inactivating any known protein involved in MMR ("MMR protein"), including any wild type or naturally occurring variant of such MMR protein, and any engineered variant (including single or multiple amino acid substitutions, deletions, insertions, rearrangements, or fusions) of such MMR protein, so long as the inhibiting, blocking, or otherwise inactivation of one or more of said M:MR
proteins or variants thereof result in the inhibition, blockage, or inactivation of the MMR
pathway. The inhibiting, blocking, or inactivation of any one or more MMR
proteins or variants may be by any suitable means applied at the genetic level (e.g., in the gene encoding the one or more MMR proteins, such as introducing a mutation that inactivates the MMR
protein or variant thereof), transcriptional level (e.g., by transcript knockdown), translational level (e.g., by blocking translation of one or more MMR proteins from their cognate transcripts), or at the protein level (e.g., administering of an inhibitor (e.g., small molecule, antibody, dominant negative protein variant) or by targeted protein degradation (e.g., PROTAC-based degradation).

106/699 [269] In one aspect, the present disclosure provides an improved method of prime editing comprising additionally inhibiting the DNA mismatch repair ('MMR) system during prime editing by inhibiting, blocking, or otherwise inactivating MLH1 or a variant thereof.
[270] Without being bound by theory, MLH1 is a key MMR protein that heterodirrierizes with PMS2 to form MutL alpha, a component of the post-replicative DNA mismatch repair system (MMR). DNA repair is initiated by MutS alpha (MSH2-MSH6) or MutS beta (MSH2-MSH3) binding to a dsDNA mismatch, then MutL alpha is recruited to the heteroduplex.
Assembly of the MutL-MutS-heteroduplex ternary complex in presence of RFC and PCNA is sufficient to activate endonuclease activity of PMS2. It introduces single-strand breaks near the mismatch and thus generates new entry points for the exonuclease EX01 to degrade the strand containing the mismatch. DNA methylation would prevent cleavage and therefore assure that only the newly mutated DNA strand is going to be corrected. MutL alpha (MLH1-PMS2) interacts physically with the clamp loader subunits of DNA polymerase III, suggesting that it may play a role to recruit the DNA polymerase Ill to the site of the MMR. Also implicated in DNA
damage signaling, a process which induces cell cycle arrest and can lead to apoptosis in case of major DNA damages. MLH1 also heterodimerizes with MLH3 to form MutL gamma which plays a role in meiosis.
12711 The "canonical" human MLH1 amino acid sequence is represented by SEQ ID
NO: 204.
1272] MLH1 also may include other human isoforms, including P40692-2 (SEQ ID
NO: 205), which differs from the canonical sequence in that residues 1-241 of the canonical sequence are missing.
[273] MLH1 also may include a third known isoform known as P40692-3 (SEQ ID
NO: 207), which differs from the canonical sequence in that residues 1-101 (of MSFVAG'VIRR...ASISTYGFRG (SEQ ID NO: 206)) are replaced with MAF.
MMR inhibitors and methods of IVIIVIR inhibition [274] The present disclosure provides a method for editing a nucleic acid molecule by prime editing that involves contacting a nucleic acid molecule with a prime editor, a pegRNA, and an inhibitor of the DNA mismatch repair pathway, thereby installing one or more modifications to the nucleic acid molecule at a target site with increased editing efficiency and/or lower indel formation. Thus, the present disclosure contemplates any suitable means to inhibit MMR. In one embodiment, the disclosure embraces administering an effective amount of an inhibitor of the

107/699 MMR pathway. In various embodiments, the MMR pathway may be inhibited by inhibiting, blocking, or inactivating any one or more MMR proteins or variants at the genetic level (e.g., in the gene encoding the one or more MMR proteins, such as introducing a mutation that inactivates the MMR protein or variant thereof), transcriptional level (e.g., by transcript knockdown), translational level (e.g., by blocking translation of one or more MMR proteins from their cognate transcripts), or at the protein level (e.g., application of an inhibitor (e.g., small molecule, antibody, dominant negative protein partner) or by targeted protein degradation (e.g., PROTAC-based degradation). The present disclosure also contemplates methods of prime editing which are designed to install modifications to a nucleic acid molecule that evade correction by the MMR pathway, without the need to provide an MMR inhibitor.
12751 The inventors developed prime editing which enables the insertion, deletion, or replacement of genomic DNA sequences without requiring error-prone double-strand DNA
breaks. The present disclosure now provides an improved method of prime editing involving the blocking, inhibiting, or inactivation of the MMR pathway (e.g., by inhibiting, blocking, or inactivating an MMR pathway protein, including MLH1) during prime editing, whereby doing so surprisingly results in increased editing efficiency and reduced indel formation. As used herein, "during" prime editing can embrace any suitable sequence of events, such that the prime editing step can be applied before, at the same time, or after the step of blocking, inhibiting, or inactivating the MMR pathway (e.g., by targeting the inhibition of MLH1).
12761 Prime editing uses an engineered Cas9 nickase¨reverse transcriptase fusion protein (e.g., PE1 or PE2) paired with an engineered prime editing guide RNA (pegRNA) that both directs Cas9 to the target genomic site and encodes the information for installing the desired edit. Prime editing proceeds through a multi-step editing process: 1) the Cas9 domain binds and nicks the target genomic DNA site, which is specified by the pegRNA's spacer sequence;
2) the reverse transcriptase domain uses the nicked genomic DNA as a primer to initiate the synthesis of an edited DNA strand using an engineered extension on the pegRNA as a template for reverse transcription¨this generates a single-stranded 3' flap containing the edited DNA sequence; 3) cellular DNA repair resolves the 3' flap intermediate by the displacement of a 5' flap species that occurs via invasion by the edited 3' flap, excision of the 5' flap containing the original DNA
sequence, and ligation of the new 3' flap to incorporate the edited DNA
strand, forming a heteroduplex of one edited and one unedited strand; and 4) cellular DNA repair replaces the

108/699 unedited strand within the heteroduplex using the edited strand as a template for repair, completing the editing process.
[277] Efficient incorporation of the desired edit requires that the newly synthesized 3' flap contains a portion of sequence that is homologous to the genomic DNA site.
This homology enables the edited 3' flap to compete with the endogenous DNA strand (the corresponding 5' flap) for incorporation into the DNA duplex. Because the edited 3' flap will contain less sequence homology than the endogenous 5' flap, the competition is expected to favor the 5' flap strand. Thus, a potential limiting factor in the efficiency of prime editing may be the failure of the 3' flap, which contains the edit, to effectively invade and displace the 5' flap strand.
Moreover, successful 3' flap invasion and removal of the 5' flap only incorporates the edit on one strand of the double-stranded DNA genome. Permanent installation of the edit requires cellular DNA repair to replace the unedited complementary DNA strand using the edited strand as a template. While the cell can be made to favor replacement of the unedited strand over the edited strand (step 4 above) by the introduction of a nick in the unedited strand adjacent to the edit using a secondary sgRNA (the PE3 system), this process still relies on a second stage of DNA repair.
[278] This disclosure describes a modified approach to prime editing that comprises additionally inhibiting, blocking, or otherwise inactivating the DNA mismatch repair (MMR) system. In some embodiments, an MMR inhibitor is provided to the target nucleic acid along with other components of a prime editing system, for example, an exogenous MMR
inhibitor such as an siRNA can be provided to a cell comprising the target nucleic acid.
In some embodiments, a prime editing system component, e.g., a pegRNA, is designed to install modifications in the target nucleic acid which evade the MMR system, without the need to provide an inhibitor. In certain embodiments, the DNA mismatch repair (MMR) system can be inhibited, blocked, or otherwise inactivating one or more proteins of the MMR
system, including, but not limited to MLH1, PMS2 (or Muth alpha), PMSI (or MutL beta), MLH3 (or MutL gamma), MutS alpha (MSH2-MSH6), MutS beta (MSH2-MSH3), MSH2, MSH6, PCNA, RFC, EXO I, PODS, and PCNA. The disclosure contemplates any suitable means by which to inhibit, block, or otherwise inactivate the DNA mismatch repair (MMR) system, including, but not limited to inactivating one or more critical proteins of the MMR system at the genetic level, e.g., by introducing one or more mutations in the genes encoding a protein of the MMR system,

109/699 e.g., MLH1, PMS2 (or MutL alpha), PMSI (or MutL beta), MLII3 (or MutL gamma), MutS
alpha (MSH2-MSH6), MutS beta (MSH2-MSH3), MSH2, MSH6, PCNA, RFC, EX0I, POL8, and PCNA.
[279] Thus, in one aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome) using prime editing while blocking, inhibiting, or otherwise inactivating the DNA mismatch repair (MMR) system.
[2801 In another aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome) using prime editing while blocking, inhibiting, or otherwise inactivating a protein of the MMR system, e.g., MLH1, PMS2 (or MutL alpha), PMS1 (or MutL
beta), MI.J13 (or MutL gamma), MutS alpha (MSH2-MSH6), MutS beta (MSH2-MSH3), MSH2, MSH6, PCNA, RFC, EX01, POLO, and PCNA.
[281] In one aspect, the present disclosure provides an improved method of prime editing comprising additionally inhibiting the DNA mismatch repair (MMR) system during prime editing by inhibiting, blocking, or otherwise inactivating MLH1 or a variant thereof. Without being bound by theory, MLH1 is a key MMR protein that heterodimerizes with PMS2 to form MutL alpha, a component of the post-replicative DNA mismatch repair system (MMR). DNA
repair is initiated by MutS alpha (MSH2-MSH6) or MutS beta (MSFI2-MSH3) binding to a dsDNA mismatch, then MutL alpha is recruited to the heteroduplex. Assembly of the MutL-MutS-heteroduplex ternary complex in presence of RFC and PCNA is sufficient to activate endonuclease activity of PMS2. It introduces single-strand breaks near the mismatch and thus generates new entry points for the exonuclease EX01 to degrade the strand containing the mismatch. DNA methylation would prevent cleavage and therefore assure that only the newly mutated DNA strand is going to be corrected. MutL alpha (MLH1-PMS2) interacts physically with the clamp loader subunits of DNA polymerase III, suggesting that it may play a role to recruit the DNA polymerase III to the site of the MMR. Also implicated in DNA
damage signaling, a process which induces cell cycle arrest and can lead to apoptosis in case of major DNA damages. MLH1 also heterodimerizes with MLH3 to form MutL gamma which plays a role in meiosis. The "canonical" human MLH1 amino acid sequence is represented by SEQ ID
NO: 204

110/699 [282] MLH1 also may include other human isoforms, including P40692-2 (SEQ ID
NO: 205), which differs from the canonical sequence in that residues 1-241 of the canonical sequence are missing.
[283] MLH1 also may include a third known isoform known as P40692-3 (SEQ ID
NO: 207), which differs from the canonical sequence in that residues 1-101 (of MSFVAGVIRR...ASISTYGFRG (SEQ ID NO: 206)) are replaced with MAF.
[284] The disclosure contemplates that any of the following MLH1 proteins may be inhibited by an inhibitor, or otherwise blocked or inactivated in order to inhibit the MMR pathway during prime editing. In addition, such exemplary proteins may also be used to engineer or otherwise make a dominant negative variant that may be used as a type of inhibitor when administered in an effective amount which blocks, inactivates, or inhibits the MMR. Without being bound by theory, it is believed that MLH1 dominant negative mutants can saturate binding of MutS.
Exemplary MLH1 proteins include the following amino acid sequences, or amino acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to 100% sequence identity with any of the following sequences:
Description Sequence SEQ
ID NO:
MLH1 MSFVA.GVIRRLDETWNRIAAGEVIQRPANAIKEMIENCLDAKSTSIQVIVKEGG 204 Homo sapiens LKLIQIQDNGTORKEDLDIVCERFTTSKLQSFEDLASISTYGFR.GEALASTSIIVAH
SwissProt VTITTKTADGKCAYRASYSDGKLICAPPKPCAGNQGTQITVEDLFYNIATRRICAL
Accession No. KNPSEEYGKILEVVGRYSVHNAGISFSVKK.QGETVADVRTLPNASTVDNIRSIFG
P40692 NAVSRELIEIGCEDKTLAFKMNGYISNANYSVKKCIFIA,FINHRLVE.STSLRKAM
Wild type TVYAAYL.PKNTHPFLYLSLEISPQNVDVNVHPTICHEVHFLHEESILERVQQIITES
ICLLGSNSSRMYFTQTLLPGLAGPSGEMVKSITSLTSSSTSGSSDKVYAIIQMVR.T
DSREQKLDAFLQPLSKPLSSQPQAIVTEDKTDISSGRARQQDEEMLELPAPAEVA
AKNQSLEGDTTK.GTSEMSEKR.GPTSSNPRKRHREDSDVEMVEDDSRICEMTAAC
TPRRRITNLTS'VLSLQEEINE,QGHEVLREMLHNHSFVGCVNPQWALAQHQTKLY
LLNITKLSEELFYQUAYDFANFGVLRLSEPAPLFDLAMLALDSPESGWTEEDGP
ICEGLAEYIVEF.LKKKAEMLADYFSLEIDEEGNLIGLPLLIDNYVPPLEGLPIFILRL
ATEVNWDEEKECFESLSKECAMFYSIRKQYISEESTLSGQQSE'VPGSIPNS'WKWT
VENIVYKALRSHILPPKHF thOGNILQLANLPDLYKVFERC

Mus muscu/us GLKLIQIQDNGTGIRICEDLDIVCERFTTSKLQWEDLASISTYGFRGEALASISIIV
SwissProt AFIVTITIKTADGKCAYRASYSDGICLQAPPICPCAGNQGTLITVEDLFYNIITRRK
Accession No. ALKNPSEEYGKILEVVGRYSLFINSGISFSVKKQGETVSDVRTLPNATTVDNIRSIF
Q9JK91 GNAVSRELIEVGCEDKTLAFKMNGYISNANYSVKKCIFLLFINHRLVE.SAALRK
Wild type AIETVYAAYLPICNTHPFLYLSLEISPQNVDVNVHPTICHEVHFLHEESILQRVQQH
IESKLLGSNSSRMYFTQTLLPGLAGPSGEAARPTTGVASSSTSGSGDKVYAYQM
VRTDSRF,QICL.DAFLQPVSSLGPSQPQDPAPVRGARTEGSPERATREDEEMLALP
APAEAAAESENLMESLMETSDAAQKAAPTSSPGSSRKRHREDSDVEMVEN A S
GKEMTAA.CYPRRRIINLTSVLSLQEEISERCHETLREMLRNHSFVGCVNPQWAL

111/699 AQHQTKLYLLNTTICLSEELF'YQILIYDFANFGVLRLSEPAPLFDLAMLALDSPES
G'WTEDDGPKEGLAEYIVEFLKKICAEMLADYFSVEIDEEGNLIGLPLLIDSYVPPL

STSICPWKWIVEHITYKAF'RSHILPPKHFTE'DGNVLQLANLPDLYKVFERC
=
MLHi Rattus GLKLIQIQDNGTGIRKEDLD1VCERFTTSKLQ EFEDLAMISTYGFRGEALASISHV
norvegicus AHVTITTKTADGKCAYRASYSDGICLQAPPKPCAGNQGTLITVEDLFYNIrFRKK
SwissProt ALKNPSEEYGKILEVVGRYSIHNSGISFSVKICQGETVSDVRTLPNATTVDNJRSIF
Accession No. GNAVSRELIEVGCEDKTLAFKMNGYISNANYSVKKCIFLLFINHRLVESAALKK

Wild type ESKLLGSNSSRMYFTQTLLPGLAGPSGEAVKSTTGIASSSTSGSGDKVHAYQMV
WIDSRDQKLDAFMQPVSRRLPSQPQDPVPGNRTEGSPEKAMQKDOEISELPAPM
EAAADSASLERESVIGASEVVAPQRHPSSPGSSRKRHPEDSDVEMMENDSRICEM
TAACYPRRRIINLTSVLSLQEEINDRGHETLREMLRNHTFVGC'VNPQWALAQHQ
TKLYLLNITKLSEELFYQILIYDFANFGVLRLPEPAPLFDFAMLALDSPESGWTE
EDGPKEGLAEYIVEFLKKKAKMLAD'YFS'VEIDEEGNLIGLPLLIDSYVPPLEGLPI

WKWTVEHIIYKAFRSHLLPPKHFTEDGNVLQLANLPDLCKVFERC

Bos taurus GLICLIQIQDNGTOIRKEDLEIVCERFTTSKLQSFEDLAHISTYGFRGEALASISHV
SwissProt AHVTITIXTADGKCAYRAHYSDGKLKAPPICPCAGNQGTQUVEDLFYNISTRRIC
Accession No. ALKNPSFEYGKILEVVGRYAVHNSGIGFSVKKQGETVADVRTI,PNATTVDNIRS
F IMPGO IFGNAVSRELIEVECEDK.TLAFKMNGYISNANYSVKKCIFILFINHRINESASLRK
Wild type ATETVYAAYLPKSTHPFIXLSLEISPQNVDVNVHPTKHEVHFLHEDSILERLQQHI
ESRLLGSNASRTYFTQTLLPGLPGPSGEAVKSTASVTSSSTAGSGDRVYAHQMV
R.TDCREQKLDAFLQPVSKALSSQPQAVVPEHRTDA.SSSGTRQQDEEMLELPAPA
AVAAKSQALEDDATMRAADLAEICRGPSSSPENPRKRPRF,DSDVEMVEDASRKE
MTA.ACTPRRRIENLTSVISLQEEINERGBETLREMLENHSFVGCVNPQWALA.QH
QTKLYLLN'TTRLSEELFYQIIõVYDFANFGVLRLSF..PAFLFDLAMLALDSPESGWT

PIFILRLATEVNWDFEKECFESISKECAMFYSIRKQYVSAE:STLSGQQSEVPGST
ANPWKWTVEHVIYKAFRSHLLPPKHFTEDGNILQLANLPDLYKVFFIZC
[2851 The methods and compositions described herein utilize MLH1 mutants or truncated variants. In some embodiments, the mutants and truncated variants of th.e human MLIII wild-type protein are utilized.
[286] in one aspect, a truncated variant of human. MLH1 is provided by this disclosure. In some embodiments, amino acids 754-756 of the wild-type human MLH1 protein are truncated (6.754-756, hereinafter referred to as MLHldn). In some embodiments, a truncated variant of human MLHI comprising only the N-terminal domain (amino acids 1-335) is provided (hereinafter referred to as MLH1 dem. In various embodiments, the following MLH1 variants are provided in this disclosure:
Description Sequence SEQ
ID NO:

LKLIQIQDNGTGIRKEDLDIVCERFTISICLQSFEDLASISTYGFRGEALASISHVAH
VTITTKTADGKCAYRASYSDGKLICAPPKPCAGNQGTQIT'VEDLFYNIATRRICAL
KNPSEEYGKELEVVGRYSVIINAGISFSVICKQGFIVADVRTLPNASTVDNIRSIFG
NAVSRELIEIGCEDKTLAFKMNGYISNANYSVKICCIFLLFINHRLVESTSLRKAIE

112/699 TVYAAYLPKNTHPFLYL SLEISPQNVD VNVHPTICEIEVHFLHEESILER VQQHIES
ICLLGSNS SRIvIYFTQTLLPGL AGP SGEM VKSTI' SLT S S ST SGS SDK VYAHQMVRT
DSREQICLDAFLQPL SKPLSSQPQAIVTEDKTDISSGRARQQDEEMLELPAPAEVA
AKNQSLEGDITICGTSEMSEKRGPTSSNPRKRHREDSDVEMVEDDSRICEMTAAC
TPRRRIINLTS'VLSLQEEINEQGHEVLREMLHNHSFVGCVNPQWALAQHQTKLY
LLNITKLSEELFYQILIYDFANFGVLRLSEPAPLFDLAMLALDSPESGWiEEDGP
ICEGLAE'YIVEFLICKICAEMLADYFSLEIDEMNLIGLPLUDNYVPPLEGLP1FILRL
ATEVNWDEEICECFESLSICECAMFYSTRKQYISEESTLSGQQSEVPGSIPNSWKWT
VEHIVYKALRSHILPPICHF IEDGNILQLANLPDLYKVFERC
.MLH I E756 MSEVAGVIRRLDETVVNRIAAGEVIQRPANATICEIViTENCLDAKSTSIQVIVKEGG 208 LKLIQIQDNGTGIRKEDLDIVCERFTTSICLQSFEDLASISTYGFRGEALASISHVAH
VTITTKTADGKCAYRASYSDGKLKAP.PKPCAGNQGTQITVEDLFYNIATRRKAL
KNPSEEYGICTLE'VVGRYS'VHNAGISFSVKKQGETVADVRTLPNASTVDNIRSIFG
NAVSRELIEIGCEDKTLAFKMNGYISNANYSVKKCIFLLFINHRL'VESTSLRKAIE
TVYAAYLPICNTHPFLYLSLEISPQNVDVNVHPTKHEVHFLHEESILERVQQHIES
KLLGSNSSRIvIYFTQTLLPGLAGPSGEMVKSTTSLTSSSTSGSSDKVYAHQMVRT
DSREQICLDAFLQPLSKPLSSQPQAIVTEDKTDISSGRARQQDEEMLELPAPAEVA
AKNQSLEGDTTICGTSENLSEKRGPTSSNPRKRHREDSD'VEMVEDDSRKEMTAAC
TPRRRTINLTSVLSLQEETN.EQGHEVLREMLHNHSFVCICVNPQWALAQHQTKLY
LLNTTKLSEELFYQILIYDFANFG'VLRLSEPAPLFDLAMLALDSPESGWTEEDGP
KEGLAEYIVEFLKKKAEMLADYFSLEIDEEGNLIGLPLLIDNYVPPLEGLPEFILRL
ATE VNWDEEKECFESLSKECAMFY SIRICQYISEESILSGQQSEVPGSIPNSWKWT
VEHIVYKALRSHILPPKHFTEDGNILQLANLPDLYKVFERH
MLH I A754- MSFNAGVIRRLDETVVNRIAA.GEVIQRPANAIICEMIENCLDAKSTSIQVIVICEGG 209 VTITTKTADGKCAYRASYSDGKLKAPPKPCAGNQGTQITVEDLFYNIATRRKAL
KNPSEEYGKTLEVVGRYSVHNAGISFSVKKQGETVADVRTLPNASTVDNIRSTFG
NAVSRELIEIGCEDKTLAFICMNGYISNANYSVKK CIFLLFINHRLVESTSLRICAIE
TVYAAYLPKNTHPFLYI,SLEISPQNVDVNVIIPTKHEVHFLHFISILERVQQHIES
KLLGSNSSRMYFTQTLLPGLAGPSGEMVKSTTSLTSSSTSGSSDKVYAHQMVRT
DSREQKLDAFLQPI,SKPLSSQPQAIVTEDKTDISSGRARQQDEEMLELPAPAEVA
AICNQSLEGD111(GTS:a4SEICRGPTSSNPRKRHREDSDVEMVEDDSRKEMTAAC

LLNT.TKLSERT FYQILTYDFANFGVLRLSEPAPLFDLAMLALDSPESGWTEEDGP
KEGLAEYIVEFLKKKAEMLADYFSLEIDEEGNLIGLPLLIDNYVPPLEGLPIFILRL
ATEVNWDEEICECFESLSKECAMFYSIRKQYISEESTLSGQQSEVPGSIPNSWKWT

VTITTKTADGKCAYRASYSDGKLKAPPKPCAGNQGTQTTVEDLFYNIATRRKAL
KNPSEEYGKILEVVGRYSVHNAGISFSVKKQGETVADVRTLPNASTVDNIRSIFG
NAVSRELIEIGCEDKTLAFKMNGYISNANYSVKKCIFLLFINHRLVESTSLRKAIE
TVYAAYLPKNTHPFLYLSLEISPQNVDVNVIIPTICHEVHFLITEESILERVQQHEES
KLLGSNSSRMYFTQTLLPGLAGPSGEMVKSTTSLTSSSTSGSSDKVYAHQMVRT
DSREQKLDAFLQPLSKPLSSQPQAIVTEDK.TDISSGRARQQDEEMLELPAPAEVA
AKNQSLEGDTTKGTSEMSEKRGPTSSNPRKRHREDSDVEMVEDDSRICEMTA.AC
TPRRRIINLTSVLSLQEEINEQGHEVLREMLHNHSFVGCVNPQWALAQHQTKLY
LLNITKLSJEtLFYQILIYDFANFGVLRLSEPAPLFDLAMLALDSPESGWTEEDGP
KEGLAEYIVEFLKICKAEMLADYFSLEIDEEGNLIGLPLLIDNYVPPLEGLPIFILRI, ATEVNWDEFJCECFESLSKECAMFYSIRKQYISEESTT.,SGQQSEVPGSIPNSWKWT

LKLIQIQDNGTGIRKEDLDIVCERFITSKLQSFEDLASISTYGFRGEALASISHVAH
VITITKTADGKCAYRASYSDGKLKAPPKPCAGNQGTQITVEDLEYNIATRRKAL
KNPSEEYGKILEVVGRYSVHNAGISFSVKKQGETVADVRTLPNASTVDNIRSIFG

113/699 NAVSRELffiIGCEDKTLAFKMNGYISNANYS'VKKCIFLLFINHRLVESTSLRKAIE
TVYAAYLPKNTHPFLYL SLEISPQNVDVNVHPTKEIEVHFLHEESILER VQQHIES
KLL
MLH I. 1-335 MSFVAGVIRRLDETWNRIAAGEVIQRPANAIKA.MIENCLDAKSTSIQVIVKEGG 212 E34A LKLIQIQDNGTGIRKEDLDIVCERFITSICLQSFEDLA SISTYGFRGEALA.SISHVAH
VTITIXTADGKCAYRA SYSD GKLKAPPKPCAGNQGTQITVEDLFYNIATRRKAL
KNPSFXYGKILEVVGRYSVHNAGISFSVKKQGETVADVR.TLPNASTVDNIRSIFG
NA.VSRELIEIGCEDKTLAFKMNGYISNANYSVKKCIFLLFINHRLVESTSLRKAIE
TVY AAYLPKNTHPFLYL SLEISPQNVDVNVHPTKHEVHFLHEE SILERVQQHIES
KLL

Nie ssvo LKLIQIQDNGTG1RKEDLDIVCERFTTSKLQSFEDLASISTYGFRGEALASISHVAH
V=KTADGKCAYRASYSDGKLKAPPKPCAGNQGTQITVEDLFYNIA'FRRKAL

NAVSRELIEIGCEDKTLAFKIANGYISNANYSVKKCIFLLFINIIRLVESTSLRKAIE
TVY AAYLPKNTHPFLYL SLEISPQNVDVNVHPTKFIEVHFLHEE SILERVQQHIE S
KLLPICKKRICV

KLSEELFYQ1L1YDFANFGVLRLSEPAPLFDLAMLALDSPESGW rhE,DGPKEGLA
EYIVEFLKKKAEIVILADYFSLEIDEEGNLIGLPLLIDNYVPPLEGLPIFILRLATEVN
WDEEKECFESLSKECAMFYSIRKQYISEESTLSGQQSEVPGSIPNSWKWTVEHIV
YKALRSHILPPKHFTEDGNILQLANLPDLYKVFERC
MLH 1 501-753 INLTSVLSLQEEINEQGHEVLREMLHNHSFVGCVNPQWALAQHQTKLYLLN'rr 216 KLSFXLFYQIIIYDFANFGVLRLSEPAPLFDL AMLALDSPESGW11 ,DGPKEGLA

YKALRSHILPPKHFTEDGNILQL ANLPDLYKVF [- - -1 QGHEVLREMLHNHSFV GCVNPQWALAQHQTKLYLLNTTKL SEELFYQILIYDF
ANFG VLRLSEPAPLFDL AIVILALD SPE SGWTEEDGPKEGLAEYIVEFLKKKAEML
ADYFSLEIDEEGNLIGLPLLIDNYVPPLEGLPIFILRLATEVNWDEEKECFESLSKE
CAIVIFYSIRKQYISEESTLSGQQSEVPGSIPNSWKWTVEHIVYKALRSHILPPIGIFT
MGNILQLANLPDLYKVFERC

QGHEVLREMLIINHSFVGCVNPQWALAQHQ1KLYLLNITKLSEEL.FYQILIYDF
ANFGVIAL SEPAPLFDLAMLALD SPE SGWTEEDGPKEGLAEYIVFYIICKKAEML
ADYFSLEIDEEGNLIGLPLLIDNYVPPLEGLPIFILRLATEVNWDEEKECFESLSKE
CAMFYSIRKQYISEESTLSGQQSEVPGSIPNSVVKWTVEHIVYKALRSHILPPKHFT

NLS8v" MLH1 P KKKRKV IN LTS VL SLQEE1NEQGHEVL REMLHNHSFVGCVNPQWALAQHQT 223 DGYKEGLAFYIVEFLICKKAEMLADYFSLEIDEEGNLIGLPLLIDNYVPPLEGLPIFI
1_,RL ATEVNVVDEEKECTESLSICECAMFYSIRKQYISEESTLSGQQSEVPGSWNSW
K WTVEHIVYKALRSHILPPKHFTEDGNILQLANLPDLYKVFL- -NL Ss V4 MLH1 PKKICRKVKRGPTSSNPRKRHREDSD'VEMVEDD SRKEMTAACTPRRRIINLTSV 224 461-753 L SLQEEINEQGHE'VLREMLHNHSPVGCVNPQWALAQHQTKLYLLNTTKL SEEL
FYQILIYDFANFGVLRLSEPAPLFDLAMLALDSPESGWTEEDGPKEGLAEYIVEF
LK KKAEML ADYFSLEIDEEGNLIGLPLLIDNYVPPLEGLPIFILRLATEVNWDEEK
ECFESLSKECAMFY SIRKQYISEESTLS GQQSEVPGSIPNSWKWTVEHT VYK ALR
SHILPPICHFTEDGNILQLANLPDLYKVPF -

114/699 [287] In another aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome) using prime editing while blocking, inhibiting, or otherwise inactivating PMS2 (or Mut', alpha) or variant thereof.
[288] In yet another aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome) using prime editing while blocking, inhibiting, or otherwise inactivating PMS1 (or MutL beta) or variant thereof.
[289] In still another aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome) using prime editing while blocking, inhibiting, or otherwise inactivating MLH3 (or MutL gamma) or variant thereof.
[290] In another aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome) using prime editing while blocking, inhibiting, or otherwise inactivating MutS alpha (MSH2-MSH6) or variant thereof.
[291] In still another aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome) using prime editing while blocking, inhibiting, or otherwise inactivating MSH2 or variant thereof.
[2921 In another aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome) using prime editing while blocking, inhibiting, or otherwise inactivating MSH6 or variant thereof.
[293] In yet another aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome) using prime editing while blocking, inhibiting, or otherwise inactivating PCNA or variant thereof.
[294] In still another aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome) using prime editing while blocking, inhibiting, or otherwise inactivating RFC or variant thereof.
[295] In another aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome) using prime editing while blocking, inhibiting, or otherwise inactivating EXOlor variant thereof.
[296] In yet another aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome) using prime editing while blocking, inhibiting, or otherwise inactivating POL8 or variant thereof.

115/699 [297] Exemplary amino acid sequences for these MMR. proteins (PMS2 (or MutL
alpha), PMS1 (or MutL beta), MLH3 (or MutL gamma), MutS alpha (MSH2-MSH6), MutS beta (MSH2-MSH3), MS1712, MSH6, PCNA, RFC, EX01, POLE, and PCNA) are as follows:
Description Sequence SEQ
ID
NO:
PMS2 MERAESSSTEPAKAIKPIDRKSVHQ1CSGQV'VLSLSTAVKELVENSLDAGATNIDL 225 Homo sapiens KLKDYGVDLIEVSDNGCGVEE.ENFEGLTLKHHTSKIQEFADLTQVETFGFRGEAL
SwissProt SSLCALSDVTISTCHASAKVGTRLMFDHNGKIIQKTPYPRPRGTTVSVQQLFSTLP
Accession No. VRHKEFQRNIKKEYAKMVQVLHAYCIISAGIR VSCTNQLGQGKRQPVVCTGGSP
P54278 &MEN IGS VFGQKQLQSLIPFVQLPPSDSVCEEY GLSCSDALHNLFYISGFISQCTHG
Wild type VGRSSTDRQFFFINRRPCDPAK VCRLVNE'VYHMYNRHQYPFVVLNISVDSECVDI
NVTPDKRQILLQEEKLLLAVLKTSLIGMFDSDVNICLNVSQQPLLDVEGNLIK.MH
AADLP.X.PMVEKQDQSPSLRTGELICKDVSISRLREAFSLRHTTENKPHSPKTPEPR
RSPLCiQKRGMLSSSTSGAISDKGVLRF'QKEAVSSSHGPSDPIDRAEVEICDSGHGS
TSVDSEGFSIPDTGSHCSSEY AASSPGDRGSQEHVDSQEKAPKTDDSFSD VDCHS
NQEDTGCKFRVLPQPINLATPNTICRFICKE'EILSSSDICQICL VNTQDMSASQVDVA
VKINKK'VVPLDFSMSSLAKRIKQLHHEAQQSEGEQNYRKFRAKICPGENQAAED
ELRKEISKTMFAEMEIIGQFNLGFITTKLNEDIFIVDQHATDEKYNFEMLQQHTVL
QGQRLIAPQTLNLTAVNEAVLIENLEIFRICNGFDIWIDENAPVTERAICLISLPTSICN
WTFGPQDVDELIFMLSDSPGVMCRPSRVKQMFASRACRKSVIVIIGTALNTSEMK
ICLITHMGEMDHPWNCPHGRPTMRHIANLGVISQN

Homo sapiens NGEGIKA VD APVMAMKYYTSKINSHEDLENLTTY GFRGEALGSICCIAEV.LITTR
SwissProt TAADNFSTQYVLDGSGHILSQKPSHLGQGITVTALRLFKNLPVRKQFYSTAKKC
Accession No. KDEIKKIQDLLMSFGILKPDLR1VFVHNKAV1WQKSRVSDHKMALMSVLGTAVM

Wild type HHYNLKCLICESTRLYPVFFIXIDVPTADVDVNLTPDKSQVLLQNKESVLIALENL
MT.TCYGPLPSTNSYENNKTDVSAADIVLSKTAETDVLFNKVESSGKNYSNVDTS
VIPFQNDMHNDESGICNTDDCLNHQISIGDFGYGHCSSEISNIDICNTICNAFQDISMS
NVSWENSQTEYSKTCFISSVICH.TQSENGNKDHIDESGENEEEAGLENSSEISADE

DNKSGK VTAYDLLSNRVIKKPMSASALFVQDHRPQFLIENPKTSLEDATLQIEEL
WKTLSEEEICLKYEEKAIICDLERYNSQMKRAIEQESQMSLKDGRKKIKPTSAWN

DLEEICDEPCLIHNLRFPDAWLMTSKTEVMLLNPYRVEEALLFKRLLE'NHKLPAEP
LEKPIMLTESLFNGSHYLDVLYKMTADDQRY SGSTYLSDPRLTANGFKIKLIPGV
SITENYLEIEGMANCLPFYGVADLICEILNAILNRNAKEVYECRPRKVISYLEGEAV
RLSRQLPMYLSKEDIQDILYRIVIKHQFGNEIKECVTIGRPITHHLTYLPETT
MIKCLSVEVQAKIRSGLAISSLGQCVEELALNSIDAEAKCVAVRVNMETF'QVQVI 227 Homo sapiens DNGFGMGSDDVEKVGNRYFTSKCHSVQDLENPRFYGFRGEALANIADMASAVE
SwissProt ISSKKNRTMKTFVKLFQSGKALKACEADVTRASAGTTVTVYNLFYQUVRRKC
Accession No. MDPRLF..FEKVRQRIEALSLMHPSISFSLRNDVSGSMVLQLPKTKDVCSRFCQIYGL
Q9UHC I GKSQICLREISFICYKEFELSGYISSF..AHYNKNMQFLFVNKRLVIATKLIIKLIDFLLR
Wild type KESIICKPKNGPTSRQMNSSLRHRSTPELYGlYVINVQCQFCEYDVCMEPAKTLIE
FQNWDTLLFCIQEGVIC.MFLKQEKLFVELSGEDIKEFSEDNGFSLFDATLQKRVTS
DERSN.FQEACNNILDSYEMFNLQSICA'VKRKTTAENVNTQSSRDSEATRKNTNDA
FLYIYESGGPGHSKMTEPSLQNKDSSCSESKMLEQETIVASEAGENEKHKKSFLE
HSSLENPCGTSLEM.FLSPFQTPCHFEESGQDLEIWKESTTVNGMAANILKNNRION
QPKRFKDATEVGCQPLPFATILWGVHSAQT.EKEKKKESSNCGRRNVFSYGRVKL
CSTGFITHVVQNE.K.TKSTETEHSFKNYV.RPG.PTRAQETFGNRTRHSVETPDIKDL
ASTLSKESGQLPNKKNCRTNISYGLENEPTATYTMFSAFQEGSKKSQIDCILSDTS

116/699 PSFPWYRHVSNDSRKTDKLIGFSKPIVRKKL SLSSQLGSLEKFKRQYGK VENPLD
TEVEESNGVTTNLSLQ'VEF'DILLKDKNRLENSDVCKITTMEHSDSDSSCQPASHIL
NSEKFPFSKDEDCLEQQMPSLRESPMTLKELSLFNRKPLDLEKSSESLASKLSRLK
GSERETQTMGIVIMSRFNELPNSDSSRKDSKLCSVLTQDFCMLFNNKHEKTENGVI
PTSDSATQDN SFNKNSKTHSNSNTIENCVISETPL VLPYNNSK VTGKDSDVLIRAS
EQQIGSLDSPSGMLMNPVEDATGDQNGICFQSEESKARACSETEESNTCCSDWQR
HIDVALGRMVYVNKMTGLSTFIAPTEDIQAACTKDLTIVAVDVVLENGSQYRC
QPFRSDLVLPFLPRARAERTVMRQDNRDTVDDTVSSESLQSLFSEWDNPVFARYP
EVAVDVSSGQAESLAVKIHNILYPYRFTKGMMISMQVLQQVDNKFIACLMSTKT
EENGEAGGNLLVLVDQHAAHERIRLEQUIDSYEKQQAQGSGRKKLLSSTLIPPLE
ITVTEEQRRLLWCYHICNLEDLGLEFVFPDTSDSLVLVGKVPLCFVEREANELRRG
RSTVTKSIVEEFIREQLELLQTTGGIQGTLPLTVQKVLASQACHGAIKFNDGLSLQ
ESCRLIEALSSCQLPFQCAHGRPSIVILPLADIDHLEQEKQIKPNLTKLRKMAQAWR
LFGKAECDTRQSLQQSMPPCEPP

Homo sapiens REVFKTQGVIKYMGPAGAKNLQSV'VLSKMNFESFVKDLILLVRQYRVEVYKNRA
SwissProt GNKASICENDWYLAYKASPGNLSQFEDILFGNNDMSAS1GVVGVKMSAVDGQRQ
Accession No. VGVGYVDS1QRKLGLCEF.PDNDQFSNLEALLIQIGPKECVLPGGETAGDMGKLRQ

Wild type SAVIKFLELLSDDSNFGQFELTTFDFSQY.MKLDIAAVRALNLFQGSVEDTrGSQSL
AALLNKCKTPQGQRLVNQWIKQPLMDICNRIEERLNLVEAFVEDAELRQTLQEDL
LRRFPDLNRLAKKFQRQAANLQDCYRLYQGINQLPNVIQALEKHEGKHQKLLLA
VFVTPLIDLRSDFSKFQEMIETTLDIMDQVENHEFLVKPSFDPNLSELREIMNDLE
KK.MQSTLISAARDLGLDPGKQIKLDSSAQFGYYFRVTCKEEKVLRNNKNFSTVDI
QKNGVKFI'NSKLTSLNEEYTKNKTEYEEAQDALVKEIVNISSGYVEPMQTLNDVL
AQLDAVVSFAHVSNGAPVPY'VRPAJLEKGQGRIILKASRHACVEVQDEIAFIPND
VYFEKDKQMFHIITGPNMGGKSTYIRQTGVIVLMAQIGCFVPCESAEVSIVDCILA
RVGAGDSQLKGVSTFMAEMLETASILRSATKDSLIIIDELGRGTSTYDGFGLAWAI
SEYIATKIGAFCMFATHFHELTALANQIPTVNNLHVTALITEETLTMLYQVKKGV
CDQSFGIHVAELANFPKHVIECAKQKALELEEFQYIGESQGYDIMEPAAKKCYLE
REQGEKIIQEFLSKVKQMPFTEMSEENITIKLKQLKAEVIAKNNSFVNEIISRIKVTI' MSH6 MSRQSTLYSFFPKSPALSDANK.A.SARASREGGRAAAAPGASPSPGGDAAWSEAG 229 Homo sapiens PGPRPLAR SASPPKAKNLN GGLRRSVAP AAPTSCDFSPGDLVWAKMEGYPWWP
SwissProt CLVYNHPFDG'TFIREKGKSVRVFIVQFFDDSPTRGWVSKRLLKPYTGSKSKEAQK
Accession No. GGHFYSAKPEILRAMQRADEALNKDKIKRLEL AVCDEPSEPEEEEEMEVG1TYV

Wild typo GSSDEISSGVGDSESEGLNSPVKVARKRKRMVTGNGSLKRKSSRKETPSATKQAT
SISSETFCNTLRAFSAPQNSESQAHVSGGGDDSSRPTV'VVYHETLEWLICEEKRRDEH
RRRPDHPDFDASTLYVPEDFLNSCTPGMRKWWQIKSQNFDLVICYKVGKFTELY
HMDAL1GVSELGLVFMKGNWAHSGFPEIAF'GRYSDSLVQKGYKVAR'VEQTETPE
MMEARCRKMAHISKYDRVVRREICRIITKGTQTYSVLEGDPSENYSKYLLSLKEK
EEDSSGHTRAYGVCF'VDTSLGKFFIGQFSDDRHCSRFRTLVAHYPPVQVLFEKGN
LSKETICTILKSSLSCSLQEGLIPGSQF'WDASKTLRTLLEEEYFREKLSDGIGVMLPQ
VLICGMTSESDSIGLTPGEKSELALSALGGCVFYLKKCLIDQELLSMANFEEYIPLD
SDTVSTTRSGAIFTKAYQRMVLDAVTLNNLEIFLNGTNGSTEGTLLERVDTCHTP
FGKRLLKQWLCAPLCNHYAINDRLDAIEDLMV'VPDKISEVVELLKKLPDLERLLS
KIHNVGSPLKSQNHPDSRAIMYEETTYSKKKIIDFLSALEGFKVMCKIIGIMEEVA
DGFXSKILKQVISLQTKNPEGRFPDLTVELNRWDTAFDHEKARKTGLITPKAGFD
SDYDQALADIRENEQSLLEYLEKQRNRIGCRTIVYWGIGRNRYQLEIPENFITRNL
PEEYELKSTKKGCKRYWTKTIEKKLANLINAEMRRDVSLKDCMRRLFYNFDKNY
KDWQSAVECIAVLDVLLCLANYSRGGDGPMCRPVILLPEDTPPFLELKGSRHPCI
TKTFFGDDFIPNDILIGCEEEEQE'NGKAYCVLVTGPNMGGKSTLMRQAGLLAVM
AQMGCYVPAEVCRLTPIDRVFTRLGASDRIMSGESTFFVELSETASILMHATAHS
LVLVDELGRGTATFDGTAIANAVVKELAETIKCRTLFSTHYHSLVEDYSQNVAV

117/699 RLGHMACMVENECEDPSQETITFLYKFIKGACPKSYGFNAARLANLPEEVIQKGH
RICAREFEKMNQSLRLFREVCLASERSTVDAEAVHKLLTLIKEL
PCNA. MFEARLVQGSILICICVLEALKDLINEACWDISSSGVNLQSMDSSHVSLVQLTIASE 230 Homo sapiens GFDTYRCDRNLAMGVNLTSMSKILKCAGNEDITTLRAEDNADTLALVFEAPNQE
SwissProt KVSDYEMKLMDLDVEQLGIPEQEYSCVVKMPSGEFARICRDLSHIGDAVVISCA
Accession No. KDGVKFSASGELGNGNIKLSQTSNVDICEEEAVTIEMNEPVQLTFALRYLNFFTKA

Wild type Homo sapiens QKQPSKKKRHYDSDSESEETLQVKNAKKPPEKLPVSSKPGKISRQDPVTYISETDE
SwissProt EDDFMCKKAASKSKENGRSTNSILLGTSNMKKNEENTKTIC.NKPLSPIKLTPTSVL
Accession No. DYFGTGSVQRSNI(KMVASKRKELSQN1DESGLNDEAIAICQLQLDEDAELERQL

Wild type AQVSDERKSYSPRICQSKYESSKESQQHSKSSADKIGEVSSPICASSKLAINIKRKEE
SSYKEIEPVASKRKENAIKLKGETKTPKKTKSSP.AKKESVSPEDSEKKRTNYQAY
RSYLNREGPKALGSKEIPKGAENCLEGLIFVITGVLESIERDEAKSLIERYGGKVTG
NVSKKTNYLVMGRDSGQSKSDKAAALGTIUIDEDGLLNLIRTMPGICKSICYEIAV
ETEMKKESKLERIPQKNVQGKRKISPSKKESESKK.SRPTSKRDSLAKTIKKEIDV
FWKSLDFKEQVAEETSGDSKARNLADDSSENKVENLLWVDKYKPTSLKTIIGQQ
GDQSCANKLLRWLRNVVQKSSSEDKKHAAICFGKFSGKDDGSSFKAALLSGPPGV
GK1TTASLVMELGYSYVF1.,NASDTRSKSSIKAIVAESLNNTSIKGFYSNGAASS
VSTKHALIMDEVDGMAGNEDRGGIQELIGLIKHTKIPIICMCNDRNHPKIRSLVHY

CARSKALTYDQAKADSHRAKICDIKMGPFDVARKVFAAGEETAHMSLVDKSDLF
FFIDYSIAPLFVQENYINVKPVAAGGDMKKHI,MLLSRAADSICDGDLVDSQIRSK
QNWSLIPAQAIYASVLPGFLMRGYMTQFPTFPSWLGKHSSTGKHDRIVQDLALH
MSLRTYSSKRTVNMDYLSLIRDALVQPLTSQGVDGVQDVVALMD'TYYLMKFD
FENTMEISSWGGKPSPFSKLDPKVICAAFTRAYNKEAHLTPYSLQAIICASRHSTSPS
LDSEYNEELNF,DDSQSDEKDQDAIETDAMEKKKTKSSKPSKPEKDKEPRICGKGK
SSICK
EX01 MGIQGLLQFIICEASEPIHVRKYKGQVVAVDTYCWLHKGAIACAEKLAICGEF'TDR 156 Homo sapiens YVGFCMKFVNIvILLSHGIKPILVFDCrCTLPSKKEVERSRRERRQANLLKGKQLLR
SwissProt EGKVSEARECFIRSINITHAMAHKVIKAARSQGVDCLVAPYEADAQLAYLNKAG
Accession No. IVQAIITEDSDLLAFGCKKVILKMDQFGNGLEIDQARLGMCRQLGDVFTEEICFRY

Wild type GFIRANNTFLYQLVFDPEKRKLIPLNAYEDDVDPETLSYAGQYVDDSIALQIALGN

VSDAPQLKENPSTVGVERVISTKGLNLPRKSSIVKRPRSAELSEDDLLSQYSLSFT
KKTKICNSSEGNKSLSFSEVFVPDLVNGPTNKKSVSTPPRTRNKFATFLQRKNEES
GAVVVF'GTRSRFFCSSDSTDCVSNKVSIQPLDETAVTDKENNLFIESEYGDQEGICR
LVDTDVARNSSDDIPNNHIPGDHIPDKATWIDEESYSFESSKFTRTISPPTLGTLR
SCFSWSGGLGDFSRTPSPSPSTALQQFRRKSDSPTSLPENN. MSDVSQLKSEESSDD
ESHPLREEACSSQSQESGEFSLQSSNASKLSQCSSKDSDSEESDCNIKLLDSQSDQT
SKLRLSHFSICICDTPLRNICVPGLYKSSSADSLSTTKIKPLGPARASGLSKKPASIQK
RKIIHNAENKPGLQIKLNELWKNFGFKKDSEKLPPCKKPLSPVRDNIQLTPEAEED

Homo sapiens EEEELQS VLEGVADGQVPPSAIDPRWLRPTPPALDPQMPLIFQQLEIDHYVGPAQ
SwissProt PVPGGPPPSRGSVPVLRAFG'VTDEGFSVCCHIHGFAPYFYTPAPPGFGPEHlvIGDL
Accession No. QRELNLAISRDSRGGRELTGPAVLAVELCSRESMFGYHGHGPSPFLRITVALPRLV

Wild type RLKEXATQCQLEADVLWSD VVSHPPEGPWQR1APLRVLSFDIECAGRKGIFPEPE
RDPVIQICSLGLRWGEPEPFLRLALTLRPCAPILGAKVQSYEKEEDLLQAWSTFIRI
MDPDVITGYNIQNFDLPYLISRAQTLKVQTFPFLGRVAGLCSNIRDSSFQSKQTGR
RDTIKVVSMVGRVQMDMLQVLLREYKLRSYTLNAVSFHFLGEQKEDVQHSIIID

118/699 LQNGNDQTRRRLA VY CLKDAYLPLRLLERLMVLVNA'VEMAR VTGVPLS YLL SR

FSSLYPSIMMAHNLC'Y1TLLRPGTAQKLGLTEDQFIRTPTGDEFVKTSVRKGLLP
QILENLLSARKRAKAELAKETDPLRRQVLDGRQLALKVSANSVYGFTGAQVGKL

SSVAEAMALGREAADWVSGHFPSP1RLEF'EKVYFPYLLISKKRYAGLLFSSRPDA
HDRMDCKGLEAVRRDNCPL VANLVFASLRRLLIDRDPEGAVAHAQDVISDLLCN
RIDISQLVITKELTRAASDYAGKQAHVELAERMRKRDPGSAPSLGDRVPYVIISAA
KGVAAYMKSEDPLFVLEHSLPIDTQYYLEQQLAKPLLRIFEPILGEGRAEA'VLLR
GDHTRCKTVLTGKVGGLLAFAKRRNCCIGCRTVLSHQGAVCEFCQPRESELYQK
EVSHLNALEERFSRLWTQCQRCQGSLHEDVICTSRDCPIFYMRICKVRKDLEDQE
QLLRRFGPPGPEAW
12981 Thus, in one aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome) using prime editing while blocking, inhibiting, or otherwise inactivating the DNA mismatch repair (MMR) system.
[299] In another aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome), comprising contacting a target nucleotide molecule with a prime editor and an inhibitor of the MMR system, e.g., an inhibitor of one or more of MLH1, PMS2 (or MutL alpha), PMS1 (or MutL beta), MLH3 (or Mud, gamma), MutS alpha (MSH2-MSH6), MutS beta (MSH2-MSH3), MSH2, MSH6, PCNA, RFC, EX01, POLS, or PCNA. In various embodiments, the inhibitor can be a small molecule inhibitor. In other embodiments, the inhibitor can be an antibody, e.g., a neutralizing antibody. In still other embodiments, the inhibitor can be a dominant negative mutant of one or more of MLHI, PMS2 (or MutL alpha), PMS1 (or MutL beta), MLH3 (or MutL gamma), MutS alpha (MSH2-MSH6), MutS beta (MSH2-MSH3), MSH2, MSH6, PCNA, RFC, EX01, POLS, or PCNA, e.g., a dominant negative mutant of MLH1. In still other embodiments, the inhibitor can be targeted at the level of transcription, e.g., an siRNA or other nucleic acid agent that knocks down the level of a transcript encoding MLH1, PMS2 (or MutL alpha), PMS I (or MutL beta), MLFL3 (or MutL
gamma), MutS alpha (MSH2-MSH6), MutS beta (MSH2-MSH3), MSH2, MSH6, PCNA, RFC, EX01, POLE, or PCNA. In yet other embodiments, the step of "contacting a target nucleotide molecule with a prime editor" can include (i) delivering directly to a cell an effective amount of a prime editor fusion protein (e.g., PE1 or PE2) complexed with a lipid delivery system; (ii) delivery to a cell a mRNA or delivery complex comprising an inRNA that encodes a prime editor fusion protein and/or a suitable pegRNA; and (iii) a DNA vector (e.g., an AAV
or lentivirus

119/699 vector, plasmid, or other nucleic acid delivery vector) that encodes a prime editor fusion protein and/or a suitable pegRNA on one or more DNA vectors.
[300] In still another aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome) using prime editing while blocking, inhibiting, or otherwise inactivating MLH1 or variant thereof. In another aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome), comprising contacting a target nucleotide molecule with a prime editor and an inhibitor of MLH1. In various embodiments, the inhibitor can be a small molecule inhibitor. In other embodiments, the inhibitor can be an anti-MLH1 antibody, e.g., a neutralizing antibody that inactivates MLH1. In still other embodiments, the inhibitor can be a dominant negative mutant of MLH1. In still other embodiments, the inhibitor can be targeted at the level of transcription of MLH1, e.g., an siRNA or other nucleic acid agent that knocks down the level of a transcript encoding MLH1. In yet other embodiments, the step of "contacting a target nucleotide molecule with a prime editor" can include (i) delivering directly to a cell an effective amount of a prime editor fusion protein (e.g., PE1 or PE2) complexed with a lipid delivery system; (ii) delivery to a cell a MRNA or delivery complex comprising an mRNA that encodes a prime editor fusion protein and/or a suitable pegRNA; and (iii) a DNA vector (e.g., an AA.V or lentivirus vector, plasmid, or other nucleic acid delivery vector) that encodes a prime editor fusion protein and/or a suitable pegRNA on one or more DNA vectors.
[301] In still another aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome) using prime editing while blocking, inhibiting, or otherwise inactivating PMS2 (or MutL alpha) or variant thereof. In another aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome), comprising contacting a target nucleotide molecule with a prime editor and an inhibitor of PMS2 (or MutL alpha). In various embodiments, the inhibitor can be a small molecule inhibitor. In other embodiments, the inhibitor can be an anti-PMS2 (or MutL alpha) antibody, e.g., a neutralizing antibody that inactivates PMS2 (or MutL alpha). In still other embodiments, the inhibitor can be a dominant negative mutant of PMS2 (or MutL alpha). In still other embodiments, the inhibitor can be targeted at the level of transcription of PMS2 (or MutL alpha), e.g., an siRNA
or other nucleic acid agent that knocks down the level of a transcript encoding ML PMS2 (or MutL alpha). In yet other embodiments, the step of "contacting a target nucleotide molecule with a prime editor" can

120/699 include (i) delivering directly to a cell an effective amount of a prime editor fusion protein (e.g., PEI or PE2) cornplexed with a lipid delivery system; (ii) delivery to a cell a mRNA or delivery complex comprising an mRNA that encodes a prime editor fusion protein and/or a suitable pegRNA; and (iii) a DNA vector (e.g., an AAV or lentivirus vector, plasmid, or other nucleic acid delivery vector) that encodes a prime editor fusion protein and/or a suitable pegRNA on one or more DNA vectors.
[302] In still another aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome) using prime editing while blocking, inhibiting, or otherwise inactivating PMS1 (or MutL beta) or variant thereof. In another aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome), comprising contacting a target nucleotide molecule with a prime editor and an inhibitor of PMS1 (or MutL beta). In various embodiments, the inhibitor can be a small molecule inhibitor. In other embodiments, the inhibitor can be an anti-PMS1 (or MutL beta) antibody, e.g., a neutralizing antibody that inactivates PMS1 (or MutL beta). In still other embodiments, the inhibitor can be a dominant negative mutant of PMS1 (or MutL beta). In still other embodiments, the inhibitor can be targeted at the level of transcription of PMS1 (or MutL beta), e.g., an siRNA
or other nucleic acid agent that knocks down the level of a transcript encoding PMS1 (or MutL
beta). In yet other embodiments, the step of "contacting a target nucleotide molecule with a prime editor" can include (i) delivering directly to a cell an effective amount of a prime editor fusion protein (e.g., PEI or PE2) complexed with a lipid delivery system; (ii) delivery to a cell a mRNA or delivery complex comprising an mRNA that encodes a prime editor fusion protein and/or a suitable pegRNA; and (iii) a DNA vector (e.g., an AAV or lentivinis vector, plasmid, or other nucleic acid delivery vector) that encodes a prime editor fusion protein and/or a suitable pegRNA on one or more DNA vectors.
[303] In still another aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome) using prime editing while blocking, inhibiting, or otherwise inactivating MLH3 (or MutL gamma) or variant thereof. In another aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome), comprising contacting a target nucleotide molecule with a prime editor and an inhibitor of MLH3 (or MutL gamma). In various embodiments, the inhibitor can be a small molecule inhibitor. In other embodiments, the inhibitor can be an anti-MLH3 (or MutL gamma) antibody, e.g., a neutralizing antibody that

121/699 inactivates MLH3 (or MutL gamma). In still other embodiments, the inhibitor can be a dominant negative mutant of MLH3 (or MutL gamma). In still other embodiments, the inhibitor can be targeted at the level of transcription of P MLH3 (or Mud, gamma), e.g., an siRNA or other nucleic acid agent that knocks down the level of a transcript encoding MLH3 (or MutL gamma).
In yet other embodiments, the step of "contacting a target nucleotide molecule with a prime editor" can include (i) delivering directly to a cell an effective amount of a prime editor fusion protein (e.g., PE1 or PE2) complexed with a lipid delivery system; (ii) delivery to a cell a mRNA
or delivery complex comprising an mRNA that encodes a prime editor fusion protein and/or a suitable pegRNA; and (iii) a DNA vector (e.g., an AAV or lentivirus vector, plasmid, or other nucleic acid delivery vector) that encodes a prime editor fusion protein and/or a suitable pegRNA
on one or more DNA vectors.
[304] In still another aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome) using prime editing while blocking, inhibiting, or otherwise inactivating MutS alpha (MSH2-MSH6) or variant thereof. In another aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome), comprising contacting a target nucleotide molecule with a prime editor and an inhibitor of MutS alpha (MSH2-MSH6). In various embodiments, the inhibitor can be a small molecule inhibitor. In other embodiments, the inhibitor can be an anti-MutS alpha (MSH2-MSH6) antibody, e.g., a neutralizing antibody that inactivates MutS alpha (MSH2-MSH6). In still other embodiments, the inhibitor can be a dominant negative mutant of MutS alpha (MSH2-MSH6). In still other embodiments, the inhibitor can be targeted at the level of transcription of MutS alpha (MSH2-MSH6), e.g., an siRNA or other nucleic acid agent that knocks down the level of a transcript encoding MutS alpha (MSH2-MSH6). In yet other embodiments, the step of "contacting a target nucleotide molecule with a prime editor" can include (i) delivering directly to a cell an effective amount of a prime editor fusion protein (e.g., PE1 or PE2) complexed with a lipid delivery system; (ii) delivery to a cell a mRNA or delivery complex comprising an mRNA
that encodes a prime editor fusion protein and/or a suitable pegRNA; and (iii) a DNA vector (e.g., an AAV or lentivirus vector, plasmid, or other nucleic acid delivery vector) that encodes a prime editor fusion protein and/or a suitable pegRNA on one or more DNA vectors.
[305] In still another aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome) using prime editing while blocking, inhibiting, or otherwise

122/699 inactivating MSH2 or variant thereof. In another aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome), comprising contacting a target nucleotide molecule with a prime editor and an inhibitor of MSH2. In various embodiments, the inhibitor can be a small molecule inhibitor. In other embodiments, the inhibitor can be an anti- MSH2 antibody, e.g., a neutralizing antibody that inactivates MSH2. In still other embodiments, the inhibitor can be a dominant negative mutant of MSH2. In still other embodiments, the inhibitor can be targeted at the level of transcription of MSH2, e.g., an siRNA or other nucleic acid agent that knocks down the level of a transcript encoding MSH2. In yet other embodiments, the step of "contacting a target nucleotide molecule with a prime editor" can include (i) delivering directly to a cell an effective amount of a prime editor fusion protein (e.g., PE1 or PE2) complexed with a lipid delivery system; (ii) delivery to a cell a mRNA or delivery complex comprising an mRNA
that encodes a prime editor fusion protein and/or a suitable pegRNA; and (iii) a DNA vector (e.g., an AAV or lentivirus vector, plasmid, or other nucleic acid delivery vector) that encodes a prime editor fusion protein and/or a suitable pegRNA on one or more DNA
vectors.
[306] In still another aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome) using prime editing while blocking, inhibiting, or otherwise inactivating MSH6 or variant thereof. In another aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome), comprising contacting a target nucleotide molecule with a prime editor and an inhibitor of MSH6. In various embodiments, the inhibitor can be a small molecule inhibitor. In other embodiments, the inhibitor can be an anti- MSH6 antibody, e.g., a neutralizing antibody that inactivates MSH6. In still other embodiments, the inhibitor can be a dominant negative mutant of MSH6. In still other embodiments, the inhibitor can be targeted at the level of transcription of MSH6, e.g., an siRNA or other nucleic acid agent that knocks down the level of a transcript encoding MSH6. In yet other embodiments, the step of "contacting a target nucleotide molecule with a prime editor" can include (i) delivering directly to a cell an effective amount of a prime editor fusion protein (e.g., PEI or PE2) complexed with a lipid delivery system; (ii) delivery to a cell a mRNA or delivery complex comprising an mRNA
that encodes a prime editor fusion protein and/or a suitable pegRNA; and (iii) a DNA vector (e.g., an AAV or lentivirus vector, plasmid, or other nucleic acid delivery vector) that encodes a prime editor fusion protein and/or a suitable pegRNA on one or more DNA
vectors.

123/699 [307] In still another aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome) using prime editing while blocking, inhibiting, or otherwise inactivating PCNA or variant thereof. In another aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome), comprising contacting a target nucleotide molecule with a prime editor and an inhibitor of PCNA. In various embodiments, the inhibitor can be a small molecule inhibitor. In other embodiments, the inhibitor can be an anti- PCNA
antibody, e.g., a neutralizing antibody that inactivates PCNA. In still other embodiments, the inhibitor can be a dominant negative mutant of PCNA. In still other embodiments, the inhibitor can be targeted at the level of transcription of PCNA, e.g., an siRNA or other nucleic acid agent that knocks down the level of a transcript encoding PCNA. In yet other embodiments, the step of "contacting a target nucleotide molecule with a prime editor" can include (i) delivering directly to a cell an effective amount of a prime editor fusion protein (e.g., PE1 or PE2) complexed with a lipid delivery system; (ii) delivery to a cell a mRNA or delivery complex comprising an mRNA
that encodes a prime editor fusion protein and/or a suitable pegRNA; and (iii) a DNA vector (e.g., an AAV or lentivirus vector, plasmid, or other nucleic acid delivery vector) that encodes a prime editor fusion protein and/or a suitable pegRNA on one or more DNA
vectors.
[308] In still another aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome) using prime editing while blocking, inhibiting, or otherwise inactivating RFC or variant thereof. In another aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome), comprising contacting a target nucleotide molecule with a prime editor and an inhibitor of RFC. In various embodiments, the inhibitor can be a small molecule inhibitor. In other embodiments, the inhibitor can be an anti-RFC antibody, e.g., a neutralizing antibody that inactivates RFC. In still other embodiments, the inhibitor can be a dominant negative mutant of IRFC. In still other embodiments, the inhibitor can be targeted at the level of transcription of RFC, e.g., an siRNA or other nucleic acid agent that knocks down the level of a transcript encoding RFC. In yet other embodiments, the step of "contacting a target nucleotide molecule with a prime editor" can include (i) delivering directly to a cell an effective amount of a prime editor fusion protein (e.g., PE1 or PE2) complexed with a lipid delivery system; (ii) delivery to a cell a mRNA or delivery complex comprising an mRNA
that encodes a prime editor fusion protein and/or a suitable pegRNA; and (iii) a DNA vector (e.g., an AAV or

124/699 lentivirus vector, plasmid, or other nucleic acid delivery vector) that encodes a prime editor fusion protein and/or a suitable pegRNA on one or more DNA vectors.
[309] In still another aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome) using prime editing while blocking, inhibiting, or otherwise inactivating EXO I or variant thereof. In another aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome), comprising contacting a target nucleotide molecule with a prime editor and an inhibitor of EX01. In various embodiments, the inhibitor can be a small molecule inhibitor. In other embodiments, the inhibitor can be an anti- EX01 antibody, e.g., a neutralizing antibody that inactivates EX01. In still other embodiments, the inhibitor can be a dominant negative mutant of EX01. In still other embodiments, the inhibitor can be targeted at the level of transcription of EX01, e.g., an siRNA or other nucleic acid agent that knocks down the level of a transcript encoding EX0I. In yet other embodiments, the step of "contacting a target nucleotide molecule with a prime editor" can include (1) delivering directly to a cell an effective amount of a prime editor fusion protein (e.g., PEI or PE2) complexed with a lipid delivery system; (ii) delivery to a cell a mRNA or delivery complex comprising an mRNA
that encodes a prime editor fusion protein and/or a suitable pegRNA; and (iii) a DNA vector (e.g., an AAV or lentivirus vector, plasmid, or other nucleic acid delivery vector) that encodes a prime editor fusion protein and/or a suitable pegRNA on one or more DNA
vectors.
[310] In still another aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome) using prime editing while blocking, inhibiting, or otherwise inactivating POLS or variant thereof. In another aspect, the present disclosure provides a method for editing a nucleotide molecule (e.g., a genome), comprising contacting a target nucleotide molecule with a prime editor and an inhibitor of POLO. In various embodiments, the inhibitor can be a small molecule inhibitor. In other embodiments, the inhibitor can be an anti- POLO
antibody, e.g., a neutralizing antibody that inactivates POLS. In still other embodiments, the inhibitor can be a dominant negative mutant of POLO. In still other embodiments, the inhibitor can be targeted at the level of transcription of POLO, e.g., an siRNA or other nucleic acid agent that knocks down the level of a transcript encoding POLO. In yet other embodiments, the step of "contacting a target nucleotide molecule with a prime editor" can include (i) delivering directly to a cell an effective amount of a prime editor fusion protein (e.g., PEI. or PE2) complexed with a lipid delivery system; (ii) delivery to a cell a mRNA or delivery complex comprising an mRNA

125/699 that encodes a prime editor fusion protein and/or a suitable pegRNA; and (iii) a DNA vector (e.g., an AAV or lentivirus vector, plasmid, or other nucleic acid delivery vector) that encodes a prime editor fusion protein and/or a suitable pegRNA on one or more DNA
vectors.
13111 In still other aspects, the present disclosure provides methods for prime editing whereby correction by the MMR pathway of the alterations introduced into a target nucleic acid molecule is evaded, without the need to provide an inhibitor of the MMR pathway.
Surprisingly, pegRNAs designed with consecutive nucleotide mismatches compared to a target site on the target nucleic acid, for example, pegRNAs that have three or more consecutive mismatching nucleotides, can evade correction by the MMR pathway, resulting in an increase in prime editing efficiency and/or a decrease in the frequency of indel formation compared to the introduction of a single nucleotide mismatch using prime editing. In addition, insertions and deletions of multiple consecutive nucleotides, for example, three or more contiguous nucleotides, or 10 or more contiguous nucleotides in length introduced by prime editing may also evade correction by the MMR pathway, resulting in an increase in prime editing efficiency and/or a decrease in the frequency of indel formation compared to prime editing with a corresponding control pegRNA
(e.g., a control pegRNA that does not introduce insertion or deletion of three or more contiguous nucleotides). In some embodiments, prime editing that introduces insertion or deletion of 10 or more contiguous nucleotides results in an increase in prime editing efficiency and/or a decrease in indel frequency compared to the introduction of an insertion or deletion of less than 10 nucleotides in length using prime editing.
[312] Thus, in one aspect, the present disclosure provides methods for editing a nucleic acid molecule by prime editing comprising contacting a nucleic acid molecule with a prime editor and a pegRNA comprising a DNA synthesis template on its extension arm comprising three or more consecutive nucleotide mismatches relative to a target site on the nucleic acid molecule. In some embodiments, the pegRNA comprises a DNA synthesis template comprising one or more nucleotide edits compared to the endogenous sequence of the nucleic acid molecule (e.g., a double stranded target DNA) to be edited, wherein the one or more nucleotide edits comprises (i) an intended change is an insertion, deletion, or substitution of x consecutive nucleotides that corrects a mutation (e.g. a disease associated mutation) in the nucleic acid molecule, and (ii) an insertion, deletion, or substitution of y consecutive nucleotides directly adjacent to the x nucleotides, wherein (x+y) is an integer no less than 3. In some embodiments, the insertion,

126/699 deletion, or substitution of the y consecutive nucleotides is a silent mutation. In some embodiments, the insertion, deletion, or substitution of the y consecutive nucleotides is a benign mutation. The silent mutations may be present in coding regions of the target nucleic acid molecule or in non-coding regions of the target nucleic acid molecule. When the silent mutations are present in a coding region, they introduce into the nucleic acid molecule one or more alternate codons encoding the same amino acid as the unedited nucleic acid molecule.
Alternatively, when the silent mutations are in a non-coding region or a junction of a coding region and a non-coding region (e.g., an intron/exon junction), the silent mutations may be present in a region of the nucleic acid molecule that does not influence splicing, gene regulation, RNA lifetime, or other biological properties of the target site on the nucleic acid molecule. A.
benign mutation may refer to a nucleotide alteration or amino acid alteration that alters the amino acid sequence of the protein or polypeptide encoded by the target nucleic acid sequence, but does not impair or substantively impair expression and/or function of the protein or polypeptide. In some embodiments, x is an integer between 1 and 50. In some embodiments, y is an integer between 1 and 50. In some embodiments, y is an integer no less than 1. In some embodiments, the inclusion of the silent mutation(s) increases the efficiency, reduces unintended indel frequency, and/or improves editing outcome purity by prime editing. As used herein, the term "prime editing outcome purity" may refer to the ratio of intended edit to unintended indels that result from prime editing. In some embodiments, the inclusion of the silent mutation(s) increases the efficiency, reduces unintended indel frequency, and/or improves editing outcome purity by prime editing by at least 1.5-fold, at least 2.0 fold, at least 2.5-fold, at least 3.0-fold, at least 3.5-fold, at least 4.0-fold, at least 4.5-fold, at least 5.0-fold, at least 5.5-fold, at least 6.0-fold, at least 6.5-fold, at least 7.0-fold, at least 7.5-fold, at least 8.0-fold, at least 8.5-fold, at least 9.0-fold, at least 9.5-fold, at least 10.0 fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, at least 20-fold, at least 21-fold, at least 22-fold, at least 23-fold, at least 24-fold, at least 25-fold, at least 26-fold, at least 27-fold, at least 28-fold, at least 29-fold, at least 30-fold, at least 31-fold, at least 32-fold, at least 33-fold, at least 34-fold, at least 35-fold, at least 36-fold, at least 37-fold, at least 38-fold, at least 39-fold, at least 40-fold, at least 41-fold, at least 42-fold, at least 43-fold, at least 44-fold, at least 45-fold, at least 46-fold, at least 47-fold, at least 48-fold, at least 49-fold, at least 50-fold, at least 51-fold, at least 52-fold, at least 53-fold, at least 54-fold, at least 55-fold, at least 56-fold,

127/699 at least 57-fold, at least 58-fold, at least 59-fold, at least 60-fold, at least 61-fold, at least 62-fold, at least 63-fold, at least 64-fold, at least 65-fold, at least 66-fold,at least 67-fold, at least 68-fold, at least 69-fold, at least 70-fold, at least 71-fold, at least 72-fold, at least 73-fold, at least 74-fold, or at least 75-fold compared to prime editing with a control pegRNA that does not include the silent mutation(s), e.g., a control pegRNA that only includes the insertion, deletion, or substitution of the x consecutive nucleotides and not the insertion, deletion, or substitution of the y consecutive nucleotides.
13131 In some embodiments, at least one of the three or more consecutive nucleotide mismatches results in an alteration in the amino acid sequence of a protein expressed from the nucleic acid molecule. In some embodiments, more than one of the consecutive nucleotide mismatches results in an alteration in the amino acid sequence of a protein expressed from the nucleic acid molecule. In some embodiments, at least one of the nucleotide mismatches are silent mutations that do not result in an alteration in the amino acid sequence of a protein expressed from the nucleic acid molecule. The silent mutations may be present in coding regions of the target nucleic acid molecule or in non-coding regions of the target nucleic acid molecule. When the silent mutations are present in a coding region, they introduce into the nucleic acid molecule one or more alternate codons encoding the same amino acid as the unedited nucleic acid molecule. Alternatively, when the silent mutations are in a non-coding region, the silent mutations may be present in a region of the nucleic acid molecule that does not influence splicing, gene regulation, RNA lifetime, or other biological properties of the target site on the nucleic acid molecule.
[314] Any number of consecutive nucleotide mismatches of three or more can be used to achieve the benefits of evading correction by the MMR pathway. In some embodiments, the DNA synthesis template of the extension arm on the pegRNA comprises 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive nucleotide mismatches relative to the endogenous sequence of a target site in the nucleic acid molecule edited by prime editing. In some embodiments, the DNA synthesis template of the extension arm on the pegRNA
comprises 3, 4, or 5 consecutive nucleotide mismatches relative to the endogenous sequence of a target site in the nucleic acid molecule edited by prime editing. In some embodiments, the DNA
synthesis template of the extension arm on the pegRNA comprises 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive nucleotide mismatches relative to the endogenous sequence

128/699 of a target site in the nucleic acid molecule edited by prime editing. In some embodiments, the DNA synthesis template of the extension arm on the pegRNA comprises three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more consecutive nucleotide mismatches relative to a target site on the nucleic acid molecule.
[315] In another aspect, the present disclosure provides methods for editing a nucleic acid molecule by prime editing comprising contacting a nucleic acid molecule with a prime editor and a pegRNA comprising a DNA synthesis template on its extension arm comprising an insertion or deletion of 10 or more nucleotides relative to a target site on the nucleic acid molecule.
Insertions and deletions of 10 or more nucleotides in length evade correction by the MMR
pathway when introduced by prime editing and thus can benefit from the inhibition of the MMR.
pathway without the need to provide an inhibitor of MMR. Insertions and deletions of any length greater than 10 nucleotides can be used to achieve the benefits of evading correction by the MMR pathway. lin some embodiments, the DNA synthesis template comprises an insertion or deletion of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides relative to the endogenous sequence at a target site of the nucleic acid molecule edited by prime editing. In some embodiments, the DNA synthesis template comprises an insertion or deletion of 11 or more nucleotides, 12 or more nucleotides, 13 or more nucleotides, 14 or more nucleotides, 15 or more nucleotides, 16 or more nucleotides, 17 or more nucleotides, 18 or more nucleotides, 19 or more nucleotides, 20 or more nucleotides, 21 or more nucleotides, 22 or more nucleotides, 23 or more nucleotides, 24 or more nucleotides, or 25 or more nucleotides relative to a target site on a nucleic acid molecule. In certain embodiments, the DNA synthesis template comprises an insertion or deletion of 15 or more nucleotides relative to a target site on the nucleic acid molecule.
[316] The present disclosure provides compositions and methods for prime editing with improved editing efficiency and/or reduced indel formation by inhibiting the DNA mismatch repair pathway while conducting prime editing of a target site. Accordingly, the present disclosure provides a method for editing a nucleic acid molecule by prime editing that involves contacting a nucleic acid molecule with a prime editor, a pegRNA, and an inhibitor of the DNA
mismatch repair pathway, thereby installing one or more modifications to the nucleic acid molecule at a target site with increased editing efficiency and/or lower indel formation. The present disclosure further provides polynucleotides for editing a DNA target site by prime

129/699 editing comprising a nucleic acid sequence encoding a napDNAbp, a polymerase, and an inhibitor of the DNA mismatch repair pathway, wherein the napDNAbp and polymerase is capable in the presence of a pegRNA of installing one or more modifications in the DNA target site with increased editing efficiency and/or lower indel formation. Thus, the methods and compositions described herein utilize prime editors, which may comprise a nucleic acid programmable DNA binding protein (napDNAbp).
Prime editors: napDNAbp domain [31 7] In one aspect, a napDNAbp of the prime editors described herein can be associated with or complexed with at least one vide 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.
[318] Any suitable napDNAbp may be used in the prime editors utilized in the methods and compositions described herein. In various embodiments, the napDNAbp may be any Class 2 CRISPR-Cas system, including any type H, 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 new nomenclature. 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.
[319] For example, the following type 11, 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,

130/699 and/or variants thereof, may be used with the prime editors utilized in the methods and compositions described herein:
Legacy nomenclature J Cu ne u eu datti re4 type II CRISPR-Cas enzymes Cas9 type V CRISPR-Cas enzymes Cpfl Cas12a CasX Cas 12e __ C2c 1 Cas12b1 Cas 12b2 _____________________________ same C2c3 Cas 12c CasY Cas12d C2c4 same C2c8 same C2c5 same C2c10 same C2c9 same type 1,7 CRISPR-Cas enzymes C2c2 Cas 13a Cas 13d same C2c7 Cas 13c C2c6 Cas13b * See Makarova et aL, The CRISPR Journal, Vol. 1, No. 5, 2018 [320] 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 a region that is complementary to 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. 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").
[321] The below description of various napDNAbps which can be used in connection with the prime editors utilized in the presently disclosed methods and compositions is not meant to be

131/699 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 that can be made or evolved through a directed evolutionary or otherwise rnutagenic process. In various embodiments, the Cas9 or Cas9 variants have a nickase activity, i.e., only cleave one 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).
[322] The prime editors utilized in the methods and compositions described herein may also comprise Cas9 equivalents, including Cas12a (Cpfl) and Cas12b1 proteins which are the result of convergent evolution. The napDNAbps used herein (e.g., SpCas9, Cas9 variant, or Cas9 equivalents) 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 reference SpCas9 canonical sequence or a reference Cas9 equivalent (e.g., Cas12a (Cpfl)).
[323] 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 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 protein. The tracrRNA serves as a guide for ribonuclease 3-aided processing of pre-crRNA. Subsequently, Cas9/crRNA/tracrRNA endonucleolytically cleaves a 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

132/699 DEMANDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.

NOTE : Pour les tomes additionels, veuillez contacter le Bureau canadien des brevets JUMBO APPLICATIONS/PATENTS
THIS SECTION OF THE APPLICATION/PATENT CONTAINS MORE THAN ONE
VOLUME

NOTE: For additional volumes, please contact the Canadian Patent Office NOM DU FICHIER / FILE NAME:
NOTE POUR LE TOME / VOLUME NOTE:

Claims

PCT/U520221012054What is claimed is:
I. A rnethod for editing a nucleic acid rnolecule by prime editing comprising:
contacting a nucleic acid molecule with a prime editor, a pegRNA, and an inhibitor of the DNA mismatch repair pathway, thereby installing one or more modifications to the nucleic acid molecule at a target site.
2. A method for editing a double stranded target DNA sequence, comprising:
contacting the double stranded target DNA with (i) a prime editor, (ii) a prime editing guide RNA
(pegRNA), and (iii) an inhibitor of a DNA mismatch repair pathway, wherein the prime editor comprises a nucleic acid programmable DNA binding protein (n.apDNAbp) and a DNA polymerase, wherein the pegRNA comprises a spacer sequence, a gRNA core, and an extension arm comprising a DNA synthesis template and a primer binding site (PBS), wherein the spacer sequence comprises a region of complementarity to a target strand of the double stranded target DNA sequence, wherein the gRNA core associates with the nap.DNAbp, wherein the DNA synthesis template comprises a region of complementarity to the non-target strand of the double-stranded target DNA. sequence and one or more nucleotide edits compared to the target strand double-stranded target DNA sequence;
wherein the primer binding site comprises a region of complementarity to a non-target strand of the double-stranded target DNA sequence, wherein the contacting installs the one or more nucleotide edits in the double stranded target =DNA, thereby editing the double stranded target DNA
3. The method of claim 2, wherein the PBS comprises a region of complementarity to a region upstream of a nick site in the non-target strand of the target DNA sequence, wherein the nick site is characteristic of the napDNAbp.
4. The method of claim 1, wherein the method further cornprises contacting the nucleic acid molecule with a second strand nicking gRNA.
5. The rnethod of claim 1, wherein the prime editing efficiency is increased by at least 1.5-fold, at least 2.0 fold, at least 2.5-fold, at least 3.0-fold, at least 3.5-fold, at least 4.0-fold, at least 4.5-fold, at least 5.0-fold, at least 5.5-fold, at least 6.0-fold, at least 6.5-fold, at least 7.0-fold, at least 7.5-fold, at least 8.0-fold, at least 8.5-fold, at least 9.0-fold, at least 9.5-fold, at least 10.0 fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, at least 20-fold, at least 21-fold, at least 22-fold, at least 23-fold, at least 24-fold, at least 25-fold, at least 26-fold, at least 27-fold, at least 28-fold, at least 29-fold, at least 30-fold, at least 31-fold, at least 32-fold, at least 33-fold, at least 34-fold, at least 35-fold, at least 36-fold, at least 37-fold, at least 38-fold, at least 39-fold, at least 40-fold, at least 41.-fold, at least 42-fold, at least 43-fold, at least 44-fold, at least 45-fold, at least 46-fold, at least 47-fold, at least 48-fold, at least 49-fold, at least 50-fold, at least 51-fold, at least 52-fold, at least 53-fold, at least 54-fold, at least 55-fold, at least 56-fold, at least 57-fold, at least 58-fold, at least 59-fold, at least 60-fold, at least 61-fold, at least 62-fold, at least 63-fold, at least 64-fold, at least 65-fold, at least 66-fold, at least 67-fold, at least 68-fold, at least 69-fold, at least 70-fold, at least 71-fold, at least 72-fold, at least 73-fold, at least 74-fold, or at least 75-fold, in the presence of the inhibitor of the DNA mismatch repair pathway compared to prime editing efficiency with the prime editor and the pegRNA in the absence of the inhibitor of the DNA
mismatch repair pathway.
6. The rnethod of claim 1, wherein the frequency of indel form.ation is decreased by at least 1.5-fold, at least 2.0 fold, at least 2.5-fold, at least 3.0-fold, at least 3.5-fold, at least 4.0-fold, at least 4.5-fold, at least 5.0-fold, at least 5.5-fold, at least 6.0-fold, at least 6.5-fold, at least 7.0-fold, at least 7.5-fold, at least 8.0-fold, at least 8.5-fold, at least 9.0-fold, at least 9.5-fold, at 1 east 10 . 0-fol d, at least I 1 -fol d, at 1 east 12-fol d, at 1 east 13-fol d, at 1 east 14-fol d, at 1 east 15-fold, at least 16-fold, at least 17-fold, at least 18-fo1d, at least 19-fold, at least 20-fold, at least 21-fold, at least 22-fold, at least 23-fold, at least 24-fold, at least 25-fold, at least 26-fo1d, at least 27-fold, at least 28-fold, at least 29-fold, at least 30-fold, at least 31-fold, at least 32-fold, at least 33-fold, at least 34-fold, at least 35-fold, at least 36-fold, at least 37-fold, at least 38-fold, at least 39-fold, at least 40-fold, at least 41-fold, at least 42-fold, at least 43-fold, at least 44-fold, at least 45-fold, at least 46-fold, at least 47-fold, at least 48-fold, at least 49-fold, at least 50-fold, at least 51-fold, at least 52-fold, at least 53-fold, at least 54-fold, at least 55-fold, at least 56-fold, at least 57-fold, at least 58-fold, at least 59-fo1d, at least 60-fold, at least 61-fo1d, at least 62-fold, at least 63-fold, at least 64-fo1d, at least 65-fold, at least 66-fold, at least 67-fold, at least 68-fold, at least 69-fold, at least 70-fold, at least 71-fold, at least 72-fold, at least 73-fold, at least 74-fold, or at least 75-fold, in the presence of the inhibitor of the DNA mismatch repair pathway compared to the frequency of indel formation with the prime editor and the pegRNA in the absence of the inhibitor of the DNA mismatch repair pathway.
7. 7. The method of claim 1, wherein the purity of editing outcome is increased by at least 1.5-fold, at least 2.0-fold, at least 2.5-fold, at least 3.0-fold, at least 3.5-fold, at least 4.0-fold, at least 4.5-fold, at least 5.0-fold, at least 5.5-fold, at least 6.0-fold, at least 6.5-fo1d, at least 7.0-fold, at least 7.5-fold, at least 8.0-fold, at least 8.5-fold, at least 9.0-fold, at least 9.5-fold, at least 10.0-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least =14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, at least 20-fold, at least 21-fold, at least 22-fold, at least 23-fold, at least 24-fold, at least 25-fold, at least 26-fold, at least 27-fold, at least 28-fold, at least 29-fold, at least 30-fold, at least 31-fold, at least 32-fold, at least 33-fold, at least 34-fold, at least 35-fold, at least 36-fold, at least 37-fo1d, at least 38-fold, at least 39-fold, at least 40-fold, at least 41-fold, at least 42-fold, at least 43-fold, at least 44-fold, at least 45-fold, at least 46-fold, at least 47-fold, at least 48-fold, at least 49-fold, at least 50-fold, at least 51-fo1d, at least 52-fold, at least 53-fold, at least 54-fold, at least 55-fold, at least 56-fold, at least 57-fold, at least 58-fold, at least 59-fold, at least 60-fold, at least 61-fold, at least 62-fold, at least 63-fold, at least 64-fold, at least 65-fold, at least 66-fold, at least 67-fold, at least 68-fold, at least 69-fold, at least 70-fold, at least 71-fold, at least 72-fo1d, at least 73-fold, at least 74-fold, or at least 75-fold, in the presence of the inhibitor of the DNA mismatch repair pathway compared to the purity of editing outcome with the prime editor and the pegRNA in the absence of the inhibitor of the DNA mismatch repair pathway, wherein the purity of editing outcome is measured by the ratio of intended edit/unintended indels.
8. The rnethod of claim 1, wherein the inhibitor of the DNA mismatch repair pathway inhibits the expression or function of one or more proteins of the DNA misrnatch repair pathway (MMR proteins).
9. The method of claim 8, wherein the one or more MMR proteins is selected from the group consisting of MLH1, PMS2 (or MutL alpha), PMS I (or MutL beta), MLH3 (or Mud, gamma), MutS alpha (MSH2-MSH6), MutS beta (MSH2-MSH3), MSH2, MSH6, PCNA, RFC, EX01, POL5, and PCNA.
10. The rnethod of elairn 8, wherein the one or more MMR proteins is MLH1.
11. The rnethod of claim 10, wherein ML111 cornprises an arnino acid sequence of SEQ ID NO:
204, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to and including 100% sequence identity with SEQ ID NO: 204.
12. The method of claim 8, wherein the inhibitor is an antibody that inhibits the activity of one or more proteins of the DNA mismatch repair pathway.
13. The method of claim 8, wherein the inhibitor is a small molecule that inhibits the activity of the one or more proteins of the DNA mismatch repair pathway.
14. The method of claim 8, wherein the inhibitor is a small interfering RNA.
(siRNA.) or a small non-coding rnicroRNA that inhibits the activity of the one or more proteins of the DNA
mismatch repair pathway.
15. The method of claim 8, wherein the inhibitor is a dominant negative variant of an MMR
protein that inhibits the activity of a wild type MMR protein.
16. The method of claim 8, wherein the inhibitor is a dominant negative variant of MLH1 that inhibits the activity of MLH1.
17. The method of claim 16, wherein the dorninant negative variant of MLH1 comprises one or more amino acid substitutions, insertions, and/or deletions in an ATPase domain compared to a wild type MLIT1 protein as set forth in SEQ ID NO: 204, wherein the one or more amino acid alterations impairs or abolishes ATPase activity of the dominant negative variant of MLIT1.
18. The method of claim 16, wherein the dominant negative variant of MLH1 comprises one or more amino acid substitutions, insertions, and/or deletions in an endonuclease domain compared to a wild type MLH1 protein as set forth in SEQ ID NO: 204, wherein the one or more amino acid alterations impairs or abolishes endonuclease activity of the dominant negative variant of MLH1.
19. The method of claim 16, wherein the dominant negative variant of MiLH1 is truncated at the C terminus compared to a wild type MLI-11 protein as set forth in SEQ ID NO:
204.
20. The method of claim 16, wherein the dominant negative variant of MUT] is truncated at the N terminus compared to a wild type ML.H1 protein as set forth in SEQ ID NO:
204.
21. The method of any one of claims 16-20, wherein the dominant negative variant of M:LH1 further comprises a nuclear localization signal (NLS) at the N terminus and/or a NTS at the C
terminus.
22. The method of claim 16, wherein the dominant negative variant is (a) MLH1 E34A (SEQ
NO: 222), (b) MLH1 A756 (SEQ ID NO: 208), (c) MLH1 A754-756 (SEQ ID NO: 209), (d) MLITI E34A A754-756 (SEQ ID NO: 210), (e) MLI-11 1-335 (SEQ ID NO: 211), (0 1-335 E34A (SEQ ID NO: 212), (g) MLH1 1-335 NLSs114 (SEQ ID NO: 213), (h) 501-756 (SEQ ID NO: 215), (i) MLIT1 501-753 (SEQ ID NO: 216), (j) MLH1 461-753 (SEQ
ID NO: 218), or (k) NL,Ssv4O MLH1 501-753 (SEQ ID NO: 223), or a polypeptide comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to and including 100% sequence identity with any of SEQ ID NOs: 208-213, 215, 216, 218, 222, or 223.
23. The method of claim 16, wherein the dominant negative variant (a) comprises a E34A amino acid substitution; (b) a deletion of amino acid 756; (c) a deletion of amino acids 754-756; (d) a E34A arnino acid substitution and a deletion of amino acids 754-756; (e) a deletion of amino acids 336-756; (f) a E34A amino acid substitution and a deletion of amino acids 336-756; (g) a deletion of amino acids 1-500; (h) a deletion of amino acids 1-500 and a deletion of amino acids 754-756; or (i) a deletion of amino acids =I-460 and a deletion of amino acids 754-756, optionally wherein the dominant negative variant further comprises an NLS
comprising the sequence KRTADGSEFESPKKKIKKV at the C terminus and/or at the N
terminus.
24. The method of claim 8, wherein the inhibitor is a dominant negative variant of PMS2 that inhibits the activity of PMS2, optionally wherein the dominant negative variant comprises one or more amino acid substitutions, insertions, and/or deletions compared to a wild type PMS2 protein, optionally wherein the dominant negative variant comprises (a) a substitution, (b) a deletion of amino acids 2-607, (c) a deletion of arnino acids 2-635, (d) a deletion of amino acids 1-635, (e) a E41A substitution, and/or (f) a deletion after amino acid 134 compared to a wild type PMS2 protein.
25. The method of claim 8, wherein the inhibitor is a dominant negative variant of MSH6 that inhibits the activity of MSH6, optionally wherein the dominant negative variant comprises one or more amino acid substitutions, insertions, and/or deletions compared to a wild type MSH6 protein, optionally wherein the dominant negative variant comprises (a) a substitution, and/or (b) a deletion of amino acids 2-361 compared to a wild type MSH6 protein.
26. The method of claim 8, wherein the inhibitor comprises CDKN IA.
27. The method of claim I, wherein the prime editor comprises a napDNAbp and a polymerase
28. The method of claim 27, wherein the napDNAbp is a nuclease active Cas9 domain, a nuclease inactive Cas9 domain, or a Cas9 nickase domain or variant thereof.
29. The method of claim 27, wherein the napDNAbp is a Cas9 nickase comprising one or more amino acid substitutions in the HNH domain, optionally wherein the one or more a.mino acid substitutions comprises H840X, N854X, and/or N863X, wherein X is any amino acid except for the original amino acid, optionally wherein the one or more amino acid substitutions comprises H840A, N854A, and/or N863A.
30. The method of claim 27, wherein the napDNAbp is selected from the group consisting of:
Cas9, Cas12e, Casi2d, Cas12a, Cas12b1, Cas13a, Cas I2c, Cas12b2, Cas13a, Casi2c, Cas12d, Cas12e, Casi2h, Casi2i, Cas.12g, Casl2f (Cas14), Cas12f1, Cas.12j (Cas(1:0), and Argonaute and optionally has a nickase activity.
31. The method of claim 27, wherein the napDNAbp comprises an amino acid sequence of any one of SEQ ID NOs: 2, 4-67, or 104, or an amino acid sequence having at least an 80%, 85%, 90%, 95%, or 99% sequence identity with any one of SEQ I NOs: 2,4-67, or 104.
32. The method of claim 27, wherein the napDNAbp comprises an amino acid sequence of SEQ
ID NO: 2 or SEQ ID NO: 37 (i.e., the napDNAbp of PE1 and PE2) or an amino acid sequence having at least an 80%, 85%, 90%, 95%, or 99% sequence identity with S:EQ ID
NO: 2 or SEQ ID NO: 37.
33. The rnethod of claim 27, wherein the polyrnerase is a DNA-dependent DNA
polymerase or an RNA-dependent DNA polymerase.
34. The method of claim 27, wherein the polymerase is a reverse transcriptase.
35. The method of claim 34, wherein the reverse transcriptase is a retroviral reverse transcriptase, optionally wherein the reverse transcriptase is a Moloney Murine Leukemia virus reverse transcriptase (MMLV-RT), optionally wherein the MMLV-RT
comprises one or more amino acid substitutions selected from D200N, T306K, W313F, T330P, and compared to a wild type MMLV-RT.
36. The method of claim 34, wherein the reverse transcriptase comprises an amino acid sequence of any one of SEQ ID NOs: 69-98 or an amino acid sequence having at least an 80%, 85%, 90%, 95%, or 99% sequence identity with any one of SEQ ID NOs: 69-98, optionally wherein the reverse transcriptase cotnprises an arnino acid sequence of SEQ ID
NO: 105.
37. The rnethod of claim 27, wherein the napDNAbp and the polymerase of the prime editor are joined to form a fusion protein, optionally wherein the napDNAbp and the polymerase are joined by a linker.
38. The method of claim 37, wherein the linker comprises an amino acid sequence of any one of SEQ ID NOs: '102 or .118-131, or an amino acid sequence having at least an 80%, 85%, 90%, 95%, or 99% sequence identity with any one of SEQ TT) NOs: 102 or 118-131.
39. The method of claim 37, wherein the linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 38, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length.
40. The method of claim 37, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 99 or SEQ ID NO: 107, or an amino acid sequence having at least an 80%, 85%, 90%, 95%, or 99% sequence identity with any one of SEQ ID NO: 99 or SEQ
ID NO:
107.
41. The method of claim 1, wherein the priine editor, the pegRNA, and the inhibitor of the DNA
mismatch repair pathway are encoded on one or more DNA vectors.
42. The method of clai rn 41, wherein the one or more DNA. vectors comprise AAV or lentivirus DNA vectors.
43. The method of claim 42, wherein the AAV vector is serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
44. The method of any one of claims 27-36, wherein the prime editor and the inhibitor of the DNA mism.atch repair pathway are not covalently linked.
45. The method of any one of claims 27-36, wherein the napDNAbp, the polymerase, or the prime editor as a fusion protein is further joined by a second linker to the inhibitor of the DNA mismatch repair pathway.
46. The method of claim 45, wherein the second linker comprises a self-hydrolyzing linker, optionally wherein the second linker i.s a T2A linker or a P2A. linker.
47. The rnethod of claim 45, wherein the second linker comprises an arnino acid sequence of any one of SEQ ID NOs: 102, 118-131, or 233-236, or an amino acid sequence having at least an 80%, 85%, 90%, 95%, or 99% sequence identity with any one of SEQ ID NOs: 102, 131, or 233-236.
48. The method of claim 45, wherein the second linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, =16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length.
49. The rnethod of claim 1, wherein the one or more modifications to the nucleic acid molecule installed at the target site comprises one or more transitions, one or more transversions, one or more insertions, one or more deletions, one more inversions, or any combination thereof, and optionally are less than 15 bp.
50. The method of clairn 49, wherein the one or inore transitions are selected from the group consisting of: (a) T to C; (b) A to G; (c) C to T; and (d) G to A.
51. The method of claim 49, wherein the one or more transversions are selected from the group consisting of: (a) T to A; (b) 17 to G; (c) C to G; (d) C to A; (e) A. to T;
(t) A to C; (g) G to C;
and (h) G to T.
52. The method of claim 1, wherein the one or more modifications comprises changing (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 a 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.
53. The method of claim 1, wherein the one or more modifications comprises 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, optionally wherein the one or more edits comprises an insertion or deletion of 1-15 nucleotides.
54. The rnethod claim 1, wherein the one or more modifications comprises a correction to a mutation associated with a disease in a disease-associated gene.
55. The method of claim 54, 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.
56. The method of claim 54, wherein the disease-associated gene is associated with a rnonogenic disorder selected from the group consisting of: Adenosine Deaminase (ADA) Deficiency;
Alpha-1 Anti trypsin Deficiency; Cystic Fibrosis; Duchenne Muscular Dystrophy;

Galactosemia; Hemochromatosis; Huntington's Disease; Maple Syrup Urine Disease; Marfan Syndrome; Neurotibromatosis Type 1; Pachyonychia Congenita; Phenylketonuria;
Severe Combined Immunodeficiency; Sickle Cell Disease; Smith-Lemli-Opitz Syndrome; a trinucleotide repeat disorder; a prion disease; and Tay-Sachs Disease.
57. The method of any one of claims 1-56, wherein the gRNA core comprises minirnal sequence homology to the sequence of the target site, optionally wherein the gRNA core comprises no more than 1%, 5%, 10%, 15%, 20%, 25%, or 30% sequence homology to the sequence of the double stranded target DNA that flanks 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides upstream or downstream of the position of the one or more nucleotide edits.
58. A composition for editing a nucleic acid molecule by prime editing comprising a prime editor, a pegRNA, and an inhibitor of the DNA mismatch repair pathway, wherein the composition is capable of installing one or more modifications to the nucleic acid molecule at a target site.
59. The composition of claim 58, wherein the composition further comprises a second strand nicking gRNA.
60. The composition of claim 58, wherein the prime editing efficiency is increased by at least 1.5-fold, at least 2.0 fold, at least 2.5-fold, at least 3.0-fold, at least 3.5-fold, at least 4.0-fold, at least 4.5-fold, at least 5.0-fold, at least 5.5-fold, at least 6.0-fold, at least 6.5-fold, at least 7.0-fo1d, at least 7.5-fold, at least 8.0-fold, at least 8.5-fold, at least 9.0-fold, at least 9.5-fold, at least 10.0 fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, at least 20-fold, at least 21-fold, at least 22-fold, at least 23-fold, at least 24-fold, at least 25-fold, at least 26-fold, at least 27-fold, at least 28-fold, at least 29-fold, at least 30-fold, at least 3 I-fold, at least 32-fold, at least 33-fold, at least 34-fold, at least 35-fold, at least 36-fold, at least 37-fold, at least 38-fold, at least 39-fold, at least 40-fold, at least 41-fold, at least 42-fold, at least 43-fold, at least 44-fold, at least 45-fold, at least 46-fold, at least 47-fold, at least 48-fold, at least 49-fold, at least 50-fold, at least 51-fold, at least 52-fold, at least 53-fold, at least 54-fold, at least 55-fold, at least 56-fold, at least 57-fold, at least 58-fold, at least 59-fold, at least 60-fold, at least 61-fold, at least 62-fold, at least 63-fold, at least 64-fold, at least 65-fold, at least 66-fold, at least 67-fold, at least 68-fold, at least 69-fold, at least 70-fold, at least 71-fold, at least 72-fold, at least 73-fold, at least 74-fold, or at least 75-fold in the presence of the inhibitor of the DNA mismatch repair pathway compared to prime editing efficiency with the prime editor and the pegRNA in the absence of the inhibitor of the DNA
mismatch repair pathway.
61. The composition of claim 58, wherein the frequency of indel formation is decreased by at least 1.5-fold, at least 2.0 fold, at least 2.5-fold, at least 3.0-fold, at least 3.5-fold, at least 4.0-fold, at least 4.5-fold, at least 5.0-fold, at least 5.5-fold, at least 6.0-fold, at least 6.5-fold, at least 7.0-fold, at least 7.5-fold, at least 8.0-fold, at least 8.5-fold, at least 9.0-fold, at least 9.5-fold, at least 10.0 fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, at least 20-fold, at least 21-fold, at least 22-fold, at least 23-fold, at least 24-fold, at least 25-fold, at least 26-fold, at least 27-fold, at least 28-fold, at least 29-fold, at least 30-fo1d, at least 31-fold, at least 32-fold, at least 33-fold, at least 34-fold, at least 35-fo1d, at least 36-fold, at least 37-fold, at least 38-fold, at least 39-fold, at least 40-fold, at least 41-fold, at least 42-fold, at least 43-fold, at least 44-fold, at least 45-fold, at least 46-fold, at least 47-fold, at least 48-fold, at least 49-fold, at least 50-fold, at least 51-fold, at least 52-fold, at least 53-fold, at least 54-fold, at least 55-fold, at least 56-fold, at least 57-fold, at least 58-fold, at least 59-fold, at least 60-fold, at least 61-fold, at least 62-fold, at least 63-fold, at least 64-fold, at least 65-fold, at least 66-fold, at least 67-fold, at least 68-fold, at least 69-fold, at least 70-fold, at least 71-fold, at least 72-fold, at least 73-fold, at least 74-fold, or at least 75-fold in the presence of the inhibitor of the DNA mismatch repair pathway compared to the frequency of indel formation with the prime editor and the pegRNA in the absence of the inhibitor of the DNA mismatch repair pathway.
62. The composition of claim 58, wherein the purity of editing outcome is increased by at least 1.5-fold, at least 2.0-fold, at least 2.5-fold, at least 3.0-fold, at least 3.5-fold, at least 4.0-fold, at least 4.5-fold, at least 5.0-fold, at least 5.5-fold, at least 6.0-fold, at least 6.5-fold, at least 7.0-fold, at least 7.5-fold, at least 8.0-fold, at least 8.5-fold, at least 9.0-fold, at least 9.5-fold, at least 10.0-fold, at least 11-fold, at least 12-fo1d, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, at least 20-fold, at least 21-fold, at least 22-fold, at least =23-fo1d, at least 24-fold, at least 25-fold, at least 26-fold, at least 27-fold, at least 28-fold, at least 29-fold, at least 30-fold, at least 31-fold, at least 32-fold, at least 33-fold, at least 34-fold, at least 35-fold, at least 36-fold, at least 37-fold, at least 38-fold, at least 39-fold, at least 40-fold, at least 41-fold, at least 42-fold, at least 43-fold, at least 44-fold, at least 45-fold, at least 46-fold, at least 47-fold, at least 48-fold, at least 49-fold, at least 50-fold, at least 51-fold, at least 52-fold, at least 53-fold, at least 54-fold, at least 55-fold, at least 56-fold, at least 57-fold, at least 58-fold, at least 59-fold, at least 60-fold, at least 61-fold, at least 62-fold, at least 63-fold, at least 64-fold, at least 65-fold, at least 66-fold, at least 67-fold, at least 68-fold, at least 69-fold, at least 70-fold, at least 71-fold, at least 72-fold, at least 73-fold, at least 74-fold, or at least 75-fold, in the presence of the inhibitor of the DNA mismatch repair pathway compared to the purity of editing outcome with the prime editor and the pegRN A in the absence of the inhibitor of the DNA mismatch repair pathway, wherein the purity of editing outcome is measured by the ratio of intended edit/unintended indels.
63. The composition of claim 58, wherein the inhibitor of the DNA mismatch repair pathway inhibits the expression of one or more proteins of the DNA mismatch repair pathway (MMR
proteins).
64. The composition of claim 63, wherein the one or more MMR proteins is selected from the group consisting of MLH1, PMS2 (or MutL alpha), PMS1 (or MutL beta), MLH3 (or MutL

gamma), MutS alpha (MSH2-MSI-16), MutS beta (MSH2-MSH3), MSH2, MSH6, PCNA, RFC, EX01, POLS, and PCNA.
65. The composition of claim 63, wherein the one or more MMR proteins is MLH1.
66. The composition of claim 65, wherein MLH I comprises an amino acid sequence of SEQ ID
NO: 204, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to and including =100% sequence identity with SEQ ID NO: 204.
67. The composition of claim 63, wherein the inhibitor is an antibody that inhibits the activity of one or more proteins of the DNA mismatch repair pathway.
68. The composition of claim 63, wherein the inhibitor is a small molecule that inhibits the activity of the one or more proteins of the DNA mismatch repair pathway.
69. The composition of claim 63, wherein the inhibitor is a small interfering RNA (siRNA) or a small non-coding microRNA that inhibits the activity of the one or more proteins of the DNA
mismatch repair pathway.
70. The composition of claim 63, wherein the inhibitor i s a dominant negative variant of an MMR protein that inhibits the activity of a wild type MMR protein.
71. The composition of claim 63, wherein the inhibitor is a dominant negative valiant of MLH1 that inhibits the activity of MLHI.
72. The composition of claim 71, wherein the dominant negative valiant of ML111 comprises one or more amino acid substitutions, insertions, and/or deletions in an ATPase domain compared to a wild type MLH1 protein as set forth in SEQ JD NO: 204, wherein the one or more amino acid alterations impairs or abolishes ATPase activity of the dominant negative variant of MLH1.
73. The composition of claim 71, wherein the dominant negative valiant of MLH1 comprises one or more amino acid substitutions, insertions, and/or deletions in an endonuclease domain compared to a wild type MLH1 protein as set forth in SEQ ID NO: 204, wherein the one or rnore amino acid alterations impairs or abolishes endonuclease activity of the dominant negative variant of MLI11.
74. The composition of claim 71, wherein the dominant negative variant of MLH1 is truncated at the C terminus compared to a wild type MLH=l protein as set forth in SEQ ID
NO: 204.
75. The composition of claim 71, wherein the dominant negative variant of :MLH:1 is truncated at the N terminus compared to a wild type MLHI protein as set forth in SEQ ID NO:
204.
76. The composition of any one of claims 71-75, wherein the dominant negative variant of MLH1 further comprises a nuclear localization signal (NLS) at the N terminus and/or a NLS
at the C terminus.
77. The composition of claim 71, wherein the dominant negative variant is (a) (SEQ ID NO: 222), (b) IvILH1 A756 (SEQ ID NO: 208), (c) IVILH1 A754-756 (SEQ
ID NO:
209), (d) MLH1 E34A A754-756 (SEQ ID NO: 210), (e) MLH1 1-335 (S:EQ ID NO:
211), (I) MLH1 1-335 E34A (SEQ ID NO: 212), (g) MLH1 1-335 NLSw4 (SEQ ID NO: 213), (h) ML:H1 501-756 (SEQ ID NO: 215), (i) ML:H1 501-753 (SEQ ID=NO: 216), (j) MLH1 753 (SEQ ID NO: 218), or (k) NLS8v4 MLH1 501-753 (SEQ ID NO: 223), or a polypeptide comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to and including 100% sequence identity with any of SEQ ID NOs: 208-213, 215, 216, 218, 222, or 223.
78. The composition of claim 71, wherein the dominant negative variant (a) comprises a E34A
amino acid substitution; (b) a deletion of amino acid 756; (c) a deletion of amino acids 754-756; (d) a E34A amino acid substitution and a deletion of amino acids 754-756;
(e) a deletion of amino acids 336-756; (I) a E34A amino acid substitution and a deletion of amino acids 336-756; (g) a deletion of amino acids 1-500; (h) a deletion of amino acids 1-500 and a deletion of amino acids 754-756; or (i) a deletion of amino acids 1-460 and a deletion of arnino acids 754-756, optionally wherein the dominant negative variant further comprises an NES cornprising the sequence KRTADGSEFESPKKKRKV at the C terminus and/or at the N
terminus.
79. The composition of claim 73, wherein the inhibitor is a dominant negative variant of PMS2 that inhibits the activity of PMS2, optionally wherein the dominant negative variant comprises one or more amino acid substitutions, insertions, and/or deletions compared to a wild type PMS2 protein, optionally wherein the dominant negative variant comprises (a) a E705K substitution, (b) a deletion of amino acids 2-607, (c) a deletion of amino acids 2-635, (d) a deletion of amino acids 1-635, (e) a E41A substitution, and/or (f) a deletion after amino acid 134 compared to a wild type PMS2 protein.
80. The composition of claim 73, wherein the inhibitor is a dominant negative variant of MST-16 that inhibits the activity of MSH6, optionally wherein the dominant negative variant comprises one or more amino acid substitutions, insertions, and/or deletions compared to a wild type MSH6 protein, optionally wherein the dominant negative variant comprises (a) a K114OR substitution, and/or (b) a deletion of amino acids 2-361 compared to a wild type MSH6 protein.
81. The composition of claim 73, wherein the inhibitor comprises CDKN1A.
82. The composition of claim 58, wherein the prime editor comprises a napDNAbp and a polymerase.
83. The composition of claim 82, wherein the napDNAbp is a nuclease active Cas9 dornain, a nuclease inactive Cas9 domain, or a Cas9 nickase domain or variant thereof.
84. The composition of claim 82, wherein the napDNAbp is a Cas9 nickase comprising one or more amino acid substitutions in the HMI domain, optionally wherein the one or more amino acid substitutions comprises H840X, N854X, and/or N863X, wherein X is any amino acid except for the original amino acid, optionally wherein the one or more amino acid substitutions comprises H840A, N854A, and/or N83A.
85. The composition of claim 84, wherein the napDNAbp is selected from the group consisting of: Cas9, Cas12e, Cas12d, Cas12a, Cas12b1, Cas13a, Cas12c, ArgonauteCas12b2, Cas13a, Cas12c, Cas12d, Cas12e, Cas12h, Cas12i, Cas12g, Cas12f (Cas14), Casl 2f1., Casi 2j (Cas(1)), and Argonaute and optionally has a nickase activity.
86. The composition of claim 84, wherein the napDNAbp comprises an amino acid sequence of any one of SEQ ID NOs: 2, 4-67, or PEmax or an amino acid sequence having at least an 80%, 85%, 90%, 95%, or 99% sequence identity with any one of SEQ ID NOs: 2, 4-67, or I 04.
87. The composition of claim 84, wherein the napDNAbp comprises an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 37 (i.e., the napDNAbp of PE1 and PE2) or an amino acid sequence having at least an 800A, 85%, 90%, 95%, or 99% sequence identity with SEQ ID
NO: 2 or SEQ ID NO: 37.
88. The composition of claim 84, wherein the polymerase is a DNA-dependent DNA
polymerase or an RNA-dependent DNA polymerase.
89. The composition of claim 84, wherein the polymerase is a reverse transcriptase.
90. The composition of claim 89, wherein the reverse transcriptase is a retroviral reverse transcriptase, optionally wherein the reverse transcriptase is a Moloney Murine Leukemia virus reverse transcriptase (MMLV-RT), optionally wherein the MMLV-RT
comprises one or more amino acid substitutions selected from D200N, T306K, W313F, T330P, and compared to a wild type MMLV-RT.
91. The composition of claim 89, wherein the reverse transcriptase comprises an amino acid sequence of any one of SEQ ID NOs: 69-98 or an amino acid sequence having at least an 80%, 85%, 90%, 95%, or 99% sequence identity with any one of SEQ ID NOs: 69-98, optionally wherein the reverse transcriptase comprises an amino acid sequence of SEQ ID
NO: 105.
92. The composition of claim 84, wherein the napDNAbp and the polymerase of the prime editor a.re joined to form a fusion protein, optionally wherein the napDNAbp and the polymerase are joined by a linker.
93. The composition of claim 92, wherein the linker cornprises an amino acid sequence of any one of SEQ ID NOs: 102, 118-131, or an amino acid sequence having at least an 80%, 85%, 90%, 95%, or 99% sequence identity with any one of SEQ ID NOs: 102, 118-131.
94. The composition of claim 92, wherein the linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, =19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length.
95. The composition of claim 92, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 99 or SEQ ID NO: 107, or an amino acid sequence having at least an 80%, 85%, 90%, 95%, or 99% sequence identity with any one of SEQ ID NO: 99 or SEQ
ID NO:
107.
96. The composition of claim 58, wherein the prime editor, the pegRNA, and the inhibitor of the DNA mismatch repair pathway are encoded on one or more DNA vectors.
97. The composition of claim 96, wherein the one or more DNA vectors comprise AAV or lentivirus DNA vectors.
98. The composition of claim 97, wherein the AAV vector is serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
99. The composition of any one of claims 84-91, wherein the prime editor and the inhibitor of the DNA mismatch repair pathway are not covalently linked.
100. The composition of any one of claims 84-91, wherein the prime editor as a fusion protein is further joined by a second linker to the inhibitor of the DNA. mismatch repair pathway.
101. The composition of claim 100, wherein the second linker comprises a self-hydrolyzing linker, optionally wherein the second linker is a T2A linker or a P2A linker.
102. The composition of claim 100, wherein the second linker comprises an amino acid sequence of any one of SEQ ID NOs: 102, 118-131, or 233-236, or an amino acid sequence having at least an 80%, 85%, 90%, 95%, or 99% sequence identity with any one of SEQ. ID
NOs: 102, 118-131, 233-236.
103. The composition of claim 100, wherein the second linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 I, 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, or 50 amino acids in length.
104. The composition of claim 58, wherein the one or more modifications to the nucleic acid molecule installed at the target site comprises one or more transitions, one or more transversions, one or more insertions, one or more deletions, one more inversions, or any combination thereof, and optionally are less than 15 bp.
105. The composition of claim 104, wherein the one or more transitions are selected from the group consisting of: (a) T to C; (b) A to G; (c) C to T; and (d) G to A.
106. The composition of claim 104, wherein the one or more transversions are selected from the group consisting of: (a) T to A; (b) T to G; (c) C to G; (c1) C to A; (e) A to T; (f) A to C;
(g) G to C; and (h) G to T.
107. The composition of claim 58, wherein the one or more modifications comprises changing (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 a 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.
108. The composition of claim 58, wherein the one or more modifications comprises 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, optionally wherein the one or more edits comprises an insertion or deletion of 1-15 nucleotides.
109. The composition claim 58, wherein the one or more modifications comprises a correction to a mutation associated with a disease in a disease-associated gene.
110. The composition of claim 109, 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.
111. The composition of claim 109, wherein the disease-associated gene is associated with a monogenic disorder selected from the group consisting of: Adenosine :Deaminase (ADA) Deficiency; Alpha- l Antitrypsin Deficiency; Cystic Fibrosis; Duchenne Muscular Dystrophy; Galactosemia; Hemochromatosis; H:untington's Disease; Maple Syrup Urine Disease; Marfan Syndrome; Neurofibromatosis Type 1; Pachyonychia Congenita;
Phenylketonuria; Severe Combined immunodeficiency; Sickle Cell Disease; Smith-Lemli-Opitz Syndrome; a trinucleotide repeat disorder; a prion disease; and Tay-Sachs Disease.
112. The composition of any one of claims 58-111, wherein the gRNA core comprises minimal sequence hornology to the sequence of the target site, optionally wherein the gRNA
core comprises no more than 1%, 5%, 10%, 15%, 20%, 25%, or 30% sequence honiology to the sequence of the double stranded target DNA that flanks 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides upstream or downstream of the position of the one or more nucleotide edits.
113. A polynucleotide for editing a DNA target site by prime editimr comprising a nucleic acid sequence encoding a napDNAbp, a polymerase, and an inhibitor of the DNA
mismatch repair pathway, wherein the napDNAbp and polymerase is capable in the presence of a pegRNA of installing one or more modifications in the DNA target site.
114. The polynucleotide of claim 113, wherein the polynucleotide further comprises a nucleic acid sequence encoding a second strand nicking gRNA.
115. The polynucleotide of claim 113, wherein the prime editing efficiency is increased by at least 1.5-fold, at least 2.0 fold, at least 2.5-fold, at least 3.0-fold, at least 3.5-fold, at least 4.0-fold, at least 4.5-fold, at least 5.0-fold, at least 5.5-fold, at least 6.0-fold, at least 6.5-fold, at least 7.0-fold, at least 7.5-fold, at least 8.0-fold, at least 8.5-fold, at least 9.0-fold, at least 9.5-fold, at least 10.0 fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-thld, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, at least 20-fold, at least 21-fold, at least 22-fold, at least 23-fold, at least 24-fold, at least 25-fold, at least 26-fold, at least 27-fold, at least 28-fold, at least 29-fold, at least 30-fold, at least 31-fold, at least 32-fold, at least 33-fold, at least 34-fold, at least 35-fold, at least 36-fold, at least 37-fold, at least 38-fold, at least 39-fold, at least 40-fold, at least 41-fold, at least 42-fo1d, at least 43-fold, at least 44-fold, at least 45-fold, at least 46-fold, at least 47-fold, at least 48-fold, at least 49-fold, at least 50-fold, at least 51-fold, at least 52-fold, at least 53-fold, at least 54-fold, at least 55-fold, at least 56-fold, at least 57-fold, at least 58-fold, at least 59-fold, at least 60-fold, at least 61-fold, at least 62-fold, at least 63-fold, at least 64-fold, at least 65-fold, at least 66-fold, at least 67-fold, at least 68-fold, at least 69-fold, at least 70-fold, at least 71-fold, at least 72-fold, at least 73-fold, at least 74-fold, or at least 75-fold, in the presence of the inhibitor of the DNA mismatch repair pathway.
116. The polynucleotide of claim 113, wherein the frequency of indel formation is decreased by at least 1.5-fold, at least 2.0 fold, at least 2.5-fold, at least 3.0-fold, at least 3.5-fold, at least 4.0-fold, at least 4.5-fold, at least 5.0-fold, at least 5.5-fold, at least 6.0-fold, at least 6.5-fold, at least 7.0-fold, at least 7.5-fold, at least 8.0-fold, at least 8.5-fold, at least 9.0-fold, at least 9.5-fold, at least 10.0 fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fo1d, at least 18-fold, at least 19-fold, at least 20-fold, at least 21-fold, at least 22-fold, at least 23-fold, at least 24-fold, at least 25-fo1d, at least 26-fold, at least 27-fold, at least 28-fold, at least 29-fold, at least 30-fold, at least 31-fold, at least 32-fold, at least 33-fold, at least 34-fold, at least 35-fold, at least 36-fold, at least 37-fold, at least 38-fold, at least 39-fo1d, at least 40-fold, at least 41-fold, at least 42-fold, at least 43-fold, at least 44-fold, at least 45-fold, at least 46-fold, at least 47-fold, at least 48-fold, at least 49-fold, at least 50-fold, at least 51-fold, at least 52-fold, at least 53-fold, at least 54-fold, at least 55-fold, at least 56-fold, at least 57-fold, at least 58-fold, at least 59-fold, at least 60-fold, at least 61-fold, at least 62-fo1d, at least 63-fold, at least 64-fold, at least 65-fo1d, at least 66-fold, at least 67-fold, at least 68-fold, at least 69-fold, at least 70-fold, at least 71-fold, at least 72-fold, at least 73-fold, at least 74-fold, or at least 75-fold, in the presence of the inhibitor of the DNA mismatch repair pathway.
117. The polynucleotide of claim 113, wherein the inhibitor of the DNA
mismatch repair pathway inhibits one or more proteins of the DNA mismatch repair pathway.
118. The polynucleotide of claim 117, wherein the one or rnore proteins is selected from the group consisting of MLH1, PMS2 (or MutL alpha), PMS1 (or MutL beta), MLH3 (or MutL
gamrna), MutS alpha (MSH2-MS116), MutS beta (MSH2-MSH3), MSH2, MSH6, PCNA, RFC, EX01, POL8, and PCNA.
119. The polynucleotide of claim 117, wherein the one or more proteins is MLH.l .
120. The polynucleotide of claim 119, wherein MLH1 comprises an amino acid sequence of SEQ ID NO: 204, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to and including 100% sequence identity with SEQ ID NO: 204.
121. The polynucleotide of claim 113, wherein the inhibitor is an antibody that inhibits the activity of one or more proteins of the DNA mismatch repair pathway.
122. The polynucleotide of claim 113, wherein the inhibitor is a small interfering RNA
(siRNA) or a small non-coding inicro:RNA that inhibits the activity of one or more proteins of the DNA mismatch repair pathway.
123. The polynucleotide of claim 113, wherein the inhibitor is a dominant negative variant of an MMR protein that inhibits the activity of a wild type :MMR protein.
124. The polynucleotide of claim 113, wherein the inhibitor is a dominant negative variant of 1VILH1 that inhibits M11-11.
125. The polynucleotide of claim 124, wherein the dominant negative variant is (a) MLH1 E34A (SEQ ID NO: 222), (b) MLH I 6,756 (SEQ ID NO: 208), (c) MLFII A754-756 (SEQ
ID NO: 209), (d) :MLH:1 E34A A754-756 (SEQ ID NO: 210), (e) MLH1 1-335 (SEQ ID
NO:
211), (f) MLH1 1-335 E34A (SEQ ID NO: 212), (g) MLH1 1-335 NLSsv40 (SEQ ID NO:

213), (h) :MLH:1 501-756 (SEQ ID NO: 215), (i) :MLH:1 501-753 (SEQ ID NO:
216), (j) MLF11 461-753 (SEQ ID NO: 218), or (k) NLS5v4 MLH1 501-753 (SEQ ID NO: 223), or a polypeptide comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to and including 100% sequence identity with any of SEQ ID NOs: 208-213, 215, 216, 218, 222, or 223.
126. The polynucleotide of claim 113, wherein the napDNAbp is a nuclease active Cas9 domain, a nuclease inactive Cas9 domain, or a Cas9 nickase domain or variant thereof.
127. The polynucleotide of claim 113, wherein the napDNAbp is selected from the group consisting of: Cas9, Cas12e, Cas12d, Cas12a, Cas12b1, Cas13a, Cas12c, ArgonauteCas12b2, Cas13a, Cas12c, Cas12d, Cas12e, Cas12h, Cas12i, Cas12g, Casl2f (Cas14), Casl2f1, Cas12j (Cas(1)), and Argonaute and optionally has a nickase activity.
128. The polynucleotide of claim 113, wherein the napDNAbp cornprises an amino acid sequence of any one of SEQ ID NOs: 2, 4-67, or 104 or an amino acid sequence having at least an 80%, 85%, 90%, 95%, or 99% sequence identity with any one of SEQ ID
NOs: 2,4-67, or 104.
129. The polynucleotide of claim 113, wherein the napDNAbp comprises an amino acid sequence of SEQ ID NO: 2 (i.e., the napDNAbp of PE1 and PE2) or an amino acid sequence having at least an 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID
NO: 2.
130. The polynucleotide of claim 113, wherein the polymerase is a :DNA-dependent DNA
polymerase or an RNA-dependent DNA polymerase.
131. The polynucleotide of claim 113, wherein the polymerase is a reverse transcriptase.
132. The polynucleotide of claim 131, wherein the reverse transcriptase comprises an amino acid sequence of any one of SEQ ID NOs: 69-98 or an arnino acid sequence having at least an 80%, 85%, 90%, 95%, or 99% sequence identity with any one of SEQ ID NOs: 69-98.
133. The polynucleotide of claim 113, wherein the napDNAbp and the polymerase of the prime editor are joined by a linker to form a fusion protein.
134. The polynucleotide of clairn 133, wherein the linker comprises an arnino acid sequence of any one of SEQ ID NOs: 102 or 118-131, or an amino acid sequence having at least an 80%, 85%, 90%, 95%, or 99% sequence identity with any one of SEQ ID NOs: 102 or 118-131.
135. The polynucleotide of claim 133, wherein the linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, l4, 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, 4 l, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length.
136. The polynucleotide of clairn 113, wherein the polynucleotide is a DNA
vector.
137. The polynucleotide of clairn 136, wherein the :DNA vector is an AAV or lentivirus DNA
vector.
138. The polynucleotide of clairn 137, wherein the AAV vector is serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
139. The polynucleotide of claim 133, wherein the prime editor as a fusion protein is further joined by a second linker to the inhibitor of the DNA mismatch repair pathway.
140. The polynucleotide of claim 139, wherein the second linker comprises a self-hydrolyzing linker.
141. The polynucleotide of claim 139, wherein the second linker comprises an amino acid sequence of any one of SEQ ID NOs: 102, 118-131, or 233-236, or an amino acid sequence having at least an 80%, 85%, 90%, 95%, or 99% sequence identity with any one of SEQ ID NOs:
102, 118-131, or 233-236.
142. The polynucleotide of claim 139, wherein the second linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length.
143. The polynucleotide of claim 113, wherein the one or more modifications to the nucleic acid molecule installed at the target site comprises one or more transitions, one or more transversions, one or more insertions, one or more deletions, or one more inversions.
144. The polynucleotide of claim 143, wherein the one or rnore transitions are selected from the group consisting of: (a) T to C; (b) A to G; (c) C to T; and (d) G to A.
145. The polynucleotide of claim 143, wherein the one or more transversions are selected from the group consisting of: (a) T to A; (b) T to G; (c) C to G; (d) C to A; (e) A
to T; (f) A to C; (g) G to C; and (h) G to T.
146. A polynucleotide of claim 113, wherein the one or more modifications comprises changing (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 a C:G= basepair, (4) a T:A basepair to a G:C basepair, (5) a T: A
hasepair 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:6 basepair to an A :T basepair, (1 0) 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.
147. A polynucleotide of claim 113, wherein the one or more modifications comprises 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.
148. The polynucleotide of claim 113, wherein the one or more modifications comprises a correction to a disease-associated gene.
149. The polynucleotide of claim 148, 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.
150. The polynucleotide of claim 148, wherein the disease-associated gene is associated with a monogenic disorder selected from the group consisting of: Adenosine Deaminase (ADA) Deficiency; Alpha-1 Antitiypsin :Deficiency; Cystic Fibrosis; Duchenne Muscular Dystrophy;
Galactosemia; Hemochromatosis; Huntington's Disease; Maple Syrup Urine Disease; Marfan Syndrome; Neurofibromatosis Type 1; :Pachyonychia Congenita; Phenylketonuria;
Severe Combined Immunodeficiency; Sickle Cell Disease; Smith-Lemli-Opitz Syndrome; a trinucleotide repeat disorder; a pion disease; and Tay-Sachs Disease.
151. A cell comprising a polynucleotide of any of claims 113-150, optionally wherein the cell is a mammalian cell, a non-human primate cell, or a human cell.
152. A pharmaceutical composition comprising the composition of any of claims 41-80 or the polynucleotide of any of claims 113-151, or the cell of claim 151, and a pharmaceutical excipient.
153. A kit comprising the composition of any of claims 58-112 or the polynucleotide of any of claims 113-150, a pharmaceutical excipient, and instructions for editing a DNA
target site by pri me editing.
154. A composition comprising a first nucleic acid sequence encoding a nucleic acid programmable DNA binding protein (napDNAbp), a second nucleic acid sequence encoding a pot ymerase, and a third nucleic acid sequence encoding an inhibitor of the DNA mismatch repair pathway.
155. The composition of claim 154, wherein the composition further comprises a prime editing guide RNA (pegRNA) or a nucleic acid sequence encoding the pegRNA, wherein the pegRNA comprises a spacer sequence, a gRNA core, and an extension arm comprising a DNA
synthesis template and a primer binding site (PBS), wherein the spacer sequence comprises a region of complementarity to a target strand of a double stranded target DNA
sequence, wherein the gRNA core associates with the napDNAbp, wherein the DNA synthesis template comprises a region of complementarity to the non-target strand of the double-stranded target DNA sequence and one or more nucleotide edits compared to the target strand double-stranded target DNA
sequence, and wherein the primer binding site comprises a region of complementarity to a non-target strand of the double-stranded target :DNA sequence.
156. The composition of claim 154, wherein the first and the second nucleic acid sequences are on a single polynucleotide.
157. The composition of claim 154, wherein the first, the second, and the third nucleic acid sequences are on a single polynucleotide.
158. The composition of claim 157, wherein the first and the second nucleic acid sequences are connected to encode a napDNAbp-DNA polymerase fusion protein.
159. The composition of claim 158, wherein the first and the second nucleic acid sequences are connected to encode a napDNAbp-DNA polymerase fusion protein, wherein the third nucleic acid sequence is connected to the first or the second nucleic acide sequence via a linker nucleic acid sequence, optionally wherein the linker nucleic acid sequence encodes a peptide linker, optionally wherein the peptide linker is a self-hydrolyzing linker, optionally wherein the self-hydrolyzing linker is a T2A linker or a P2A linker, optionally wherein the self-hydrolyzing linker comprises an amino acid sequence of any one of SEQ. 1T3 NOs: 102, 118-131, or 233-236, or an amino acid sequence having at least an 80%, 85%, 90%, 95%, or 99%
sequence identity with any one of SEQ ID NOs: 102, 118-131, or 233-236.
160. The composition of claim 156 or 157, wherein the single polynucleotide is part of a DNA
vector.
161. The composition of claim 156 or 157, wherein the single polynucleotide is part of an mRNA sequence.
162. The composition of claim 160, wherein the DNA vector is an AAV or lentivirus DNA
vector, optionally wherein the DNA vector further comprises a promoter.
163. The composition of claim 161, wherein the mRNA sequence further comprises a promoter.
164. A method for editing a nucleic acid molecule by prime editing comprising:
contacting a nucleic acid molecule with a prime editor and a prime txliting guide RNA
(pegRNA), wherein the prime editor comprises a nucleic acid programmable DNA binding protein and a DNA
polymerase, wherein the pegRNA a spacer sequence, a gRNA core, and an extension arm comprising a DNA synthesis template and a primer binding site (PBS), wherein the DNA
synthesis template comprises three or more consecutive nucleotide mismatches relative to the endogenous sequence of a target site on the nucleic acid molecule, wherein the three or more consecutive nucleotide mismatches comprise (i) an insertion, deletion, or substitution of x consecutive nucleotides that corrects a mutation (e.g., a disease associated mutation) and (ii) an insertion, deletion, or substitution of y consecutive nucleotides directly adjacent to the x consecutive nucleotides, wherein the insertion, deletion, or substitution of y consecutive nucleotides is a silent mutation, wherein (x y) is an integer no less than 3, wherein y is an integer no less than 1, and wherein inclusion of the silent mutation(s) increases the efficiency, reduces unintended indel frequency, and/or improves editing outcome purity by prime editing.
165. The method of claim 164, wherein at least one of the three or more consecutive nucleotide mismatches results in an alteration in the amino acid sequence of a protein expressed from the nucleic acid molecule, and wherein at least one of the remaining three or more consecutive nucleotide mismatches are silent mutations.
166. The method of claim 165, wherein the silent mutations are in a coding region of the target nucleic acid molecule.
167. The method of claim 166, wherein the silent mutations introduce into the nucleic acid molecule one or more alternate codons encoding the same amino acid as the unedited nucleic acid molecule.
168. The method of claim 165, wherein the silent mutations are in a non-coding region of the target nucleic acid molecule.
169. The method of claim 168, wherein the silent rnutations do not influence splicing, gene regulation, RNA lifetime, or other biological properties of the target site on the nucleic acid molecule.
170. The method of any one of claims 164-169, wherein the DNA synthesis template of the pegRN=A comprises four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more consecutive nucleotide mismatches relative to the endogenous sequence of a target site on the nucleic acid molecule.
171. The method of any one of clairns 164-170, wherein the three or more consecutive nucleotide mismatches evade correction by the DNA mismatch repair pathway.
172. The m.ethod of any one of claims 164-171, wherein the prime editing efficiency is increased by at least 1.5-fold, at least 2.0 fold, at least 2.5-fold, at least 3.0-fold, at least 3.5-fold, at least 4.0-fold, at least 4.5-fold, at least 5.0-fold, at least 5.5-fold, at least 6.0-fold, at least 6.5-fold, at least 7.0-fold, at least 7.5-fold, at least 8.0-fold, at least 8.5-fold, at least 9.0-fold, at least 9.5-fold, at least 10.0 fold, at least 11-fold, at least 12-fold, at least 13-fo1d, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, at least 20-fold, at least 21-fold, at least 22-fold, at least 23-fold, at least 24-fold, at least 25-fold, at least 26-fold, at least 27-fold, at least 28-fold, at least 29-fold, at least 30-fold, at least 31-fold, at least 32-fold, at least 33-fold, at least 34-fold, at least 35-fold, at least 36-fold, at least 37-fold, at least 38-fold, at least 39-fold, at least 40-fold, at least 41-fold, at least 42-fold, at least 43-fold, at least 44-fold, at least 45-fold, at least 46-fold, at least 47-fold, at least 48-fold, at least 49-fold, at least 50-fold, at least 51-fold, at least 52-fold, at least 53-fold, at least 54-fold, at least 55-fold, at least 56-fold, at least 57-fold, at least 58-fold, at least 59-fold, at least 60-fold, at least 61-fold, at least 62-fold, at least 63-fold, at least 64-fold, at least 65-fold, at least 66-fold, at least 67-fold, at least 68-fold, at least 69-fold, at least 70-fold, at least 71-fold, at least 72-fold, at least 73-fold, at least 74-fold, or at least 75-fold relative to a method using a pegRNA. comprising a DNA synthesis template comprising only one consecutive nucleotide mismatch relative to the endogenous sequence of a target site on the nucleic acid molecule.
173. The method of any one of claims 164-171, wherein the frequency of indel formation is decreased by at least 1.5-fold, at least 2.0 fold, at least 2.5-fold, at least 3.0-fold, at least 3.5-fold, at least 4.0-fold, at least 4.5-fold, at least 5.0-fold, at least 5.5-fold, at least 6.0-fold, at least 6.5-fold, at least 7.0-fold, at least 7.5-fold, at least 8.0-fold, at least 8.5-fold, at least 9.0-fold, at least 9.5-fold, at least 10.0 fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at 1 east l 9-fo1d, at least 20-fold, at least 21-fold, at least 22-fold, at least 23-fold, at least 24-fold, at least 25-fold, at least 26-fold, at least 27-fold, at least 28-fold, at least 29-fold, at least 30-fold, at least 31-fold, at least 32-fb1d, at least 33-fo1d, at least 34-fold, at least 35-fold, at least 36-fold, at least 37-fold, at least 38-fold, at least 39-fold, at least 40-fold, at least 41-fold, at least 42-fold, at least 43-fold, at least 44-fold, at least 45-fold, at least 46-fold, at least 47-fold, at least 48-fold, at least 49-fold, at least 50-fold, at least 51-fold, at least 52-fold, at least 53-fold, at least 54-fold, at least 55-fold, at least 56-fold, at least 57-fold, at least 58-fold, at least 59-fold, at least 60-fold, at least 61-fold, at least 62-fold, at least 63-fold, at least 64-fold, at least 65-fold, at least 66-fold, at least 67-fold, at least 68-fold, at least 69-fold, at least 70-fold, at least 71-fold, at least 72-fold, at least 73-fold, at least 74-fold, or at least 75-fold relative to a method using a pcgRNA comprising a DNA synthesis template comprising only onc consecutive nucleotide mismatch relative to the endogenous sequence of a target site on the nucleic acid molecule.
174. A method for editing a nucleic acid molecule by prime editing comprising:
contacting a nucleic acid molecule with a prime editor and a pegRNA, wherein the extension arm of the pegRNA comprises a DNA synthesis template comprising an insertion or deletion of 10 or more nucleotides relative to the endogenous sequence of a target site on the nucleic acid molecule.
175. The method of claim 174, wherein the DNA synthesis template comprises an insertion or deletion of 11 or more nucleotides, 12 or rnore nucleotides, 13 or more nucleotides, 14 or more nucleotides, 15 or more nucleotides, 16 or more nucleotides, 17 or more nucleotides, 18 or more nucleotides, 19 or more nucleotides, 20 or more nucleotides, 21 or more nucleotides, 22 or more nucleotides, 23 or more nucleotides, 24 or more nucleotides, or 25 or more nucleotides relative to the endogenous sequence of a target site on the nucleic acid molecule.
176. The method of claim 175, wherein the DNA synthesis template comprises an insertion or deletion of 15 or more nucleotides relative to the endogenous sequence of a target site on the nucleic acid molecule.
177. The method of any one of claims 174-176, wherein the insertion or deletion of 10 or rnore nucleotides relative to the endogenous sequence of the target site on the nucleic acid molecule evades correction by the DNA mismatch repair pathway.
178. The method of any one of claims 174-176, wherein the prime editing efficiency is increased by at least 1.5-fold, at least 2.0 fold, at least 2.5-fold, at least 3.0-fold, at least 3.5-fold, at least 4.0-fold, at least 4.5-fo1d, at least 5.0-fold, at least 5.5-fold, at least 6.0-fold, at least 6.5-fold, at least 7.0-fold, at least 7.5-fold, at least 8.0-fold, at least 8.5-fold, at least 9.0-fold, at least 9.5-fold, or at least 10.0-fold relative to a method using a pegRNA comprising a DNA synthesis template comprising an insertion or deletion of fewer than 10 nucleotides relative to the endogenous sequence of a target site on the nucleic acid molecule.
179. The method of claim 174-176, wherein the frequency of indel formation is decreased by at least 1.5-fold, at least 2.0 fold, at least 2.5-fold, at least 3.0-fold, at least 3.5-fold, at least 4.0-fold, at least 4.5-fold, at least 5.0-fold, at least 5.5-fold, at least 6.0-fold, at least 6.5-fold, at least 7.0-fold, at least 7.5-fold, at least 8.0-fold, at least 8.5-fold, at least 9.0-fold, at least 9.5-fold, at least 10.0 fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, at least 20-fold, at least 21-fo1d, at least 22-fo1d, at least 23-fold, at least 24-fold, at least 25-fo1d, at least 26-fold, at least 27-fold, at least 28-fold, at least 29-fold, at least 30-fold, at least 31-fold, at least 32-fold, at least 33-fold, at least 34-fold, at least 35-fold, at least 36-fold, at least 37-fo1d, at least 38-fold, at least 39-fold, at least 40-fold, at least 41-fold, at least 42-fold, at least 43-fold, at least 44-fold, at least 45-fold, at least 46-fold, at least 47-fold, at least 48-fold, at least 49-fold, at least 50-fold, at least 51-fold, at least 52-fold, at least 53-fold, at least 54-fold, at least 55-fold, at least 56-fold, at least 57-fold, at least 58-fold, at least 59-fold, at least 60-fold, at least 61-fold, at least 62-fold, at least 63-fold, at least 64-fold, at =least 65-fold, at least 66-fold, at least 67-fold, at least 68-fold, at least 69-fold, at least 70-fold, at least 71-fold, at least 72-fold, at least 73-fold, at least 74-fold, or at least 75-fold relative to a rnethod using a pegRNA comprising a DNA synthesis template comprising an insertion or deletion of fewer than 10 nucleotides relative to the endogenous sequence of a target site on the nucleic acid molecule.
180. A prime editing guide RNA (pegRNA) for editing a nucleic acid molecule by prime editing, wherein the pegRNA comprises a spacer sequence, a gRNA core, and an extension arm comprising a DNA synthesis template and a primer binding site (PBS), wherein the DNA
synthesis template comprising three or more consecutive nucleotide mismatches relative to the endogenous sequence of a target site on the nucleic acid molecule, wherein the three or more consecutive nucleotide mismatches cornprise (i) an insertion, deletion, or substitution of x nucleotides that corrects a mutation (e.g. a disease associated mutation) and (ii) an insertion, deletion, or substitution of y nucleotides directly adjacent to the x nucleotides, wherein the insertion, deletion, or substitution of y nucleotides is a silent mutation, wherein (x-l-y) is an integer no less than 3, wherein y is an integer no less than 1, and wherein inclusion of the silent mutation(s) increases the efficiency, reduces unintended indel frequency, and/or improves editing outcome purity by prime editing.
181. The pegRNA of claim 180, wherein at least one of the three or more consecutive nucleotide mismatches results in an alteration in the amino acid sequence of a protein expressed from the nucleic acid molecule, and wherein at least one of the remaining three or more consecutive nucleotide mismatches are silent mutations.
182. The pegRNA of claim 181, wherein the silent mutations are in a coding region of the target nucleic acid molecule.
183. The pegRNA of claim 182, wherein the silent mutations introduce into the nucleic acid molecule one or more alternate codons encoding the same amino acid as the unedited nucleic acid molecule.
184. The pegRNA of claim 181, wherein the silent mutations are in a non-coding region of the target nucleic acid molecule.
185. The pegRNA of claim 183, wherein the silent mutations are in a region of the nucleic acid molecule that does not influence splicing, gene regulation, RNA lifetime, or other biological properties of the target site on the nucleic acid molecule.
186. The pegRNA of any one of claims 180-185, wherein the extension arm of the pegRNA
comprises four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more consecutive nucleotide mismatches relative to the endogenous sequence of a target site on the nucleic acid molecule.
187. The pegRNA of any one of claims 180-186, wherein the three or more consecutive nucleotide mismatches evade the DNA mismatch repair pathway.
188. The pegRNA of any one of claims 180-186, wherein use of the pegRNA in prime editing results in the prime editing efficiency being increased by at least 1.5-fold, at least 2.0 fold, at least 2.5-fold, at least 3.0-fold, at least 3.5-fold, at least 4.0-fold, at least 4.5-fold, at least 5.0-fold, at least 5.5-fold, at least 6.0-fo1d, at least 6.5-fold, at least 7.0-fold, at least 7.5-fold, at least 8.0-fo1d, at least 8.5-fold, at least 9.0-fold, at least 9.5-fold, at least 10.0 fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, at least 20-fold, at least 21-fold, at least 22-fold, at least 23-fold, at least 24-fold, at least 25-fold, at least 26-fold, at least 27-fold, at least 28-fold, at least 29-fold, at least 30-fold, at least 31-fold, at least 32-fold, at least 33-fold, at least 34-fold, at least 35-fold, at least 36-fold, at least 37-fold, at least 38-fold, at least 39-fold, at least 40-fold, at least 41-fold, at least 42-fold, at least 43-fold, at least 44-fold, at least 45-fold, at least 46-fold, at least 47-fold, at least 48-fold, at least 49-fold, at least 50-fold, at least 51-fold, at least 52-fold, at least 53-fold, at least 54-fold, at least 55-fold, at least 56-fold, at least 57-fold, at least 58-fold, at least 59-fold, at least 60-fold, at least 61-fold, at least 62-fold, at least 63-fold, at least 64-fold, at least 65-fold, at least 66-fold, at least 67-fold, at least 68-fold, at least 69-fold, at least 70-fold, at least 71-fold, at least 72-fold, at least 73-fold, at least 74-fold, (m-at least 75-fold relative to a pegRNA comprising a DNA synthesis template comprising only one consecutive nucleotide mismatch relative to the endogenous sequence of a target site on the nucleic acid molecule.
189. The pegRNA of any one of claims 180-186, wherein use of the pegRNA in prime editing results in the frequency of indel formation being decreased by at least 1.5-fold, at least 2.0 fold, at least 2.5-fold, at least 3.0-fold, at least 3.5-fold, at least 4.0-fold, at least 4.5-fold, at least 5.0-fold, at least 5.5-fold, at least 6.0-fold, at least 6.5-fold, at least 7.0-fold, at least 7.5-fold, at least 8.0-fo1d, at least 8.5-fold, at least 9.0-fold, at least 9.5-fold, or at least 10.0-fold relative to a pegRNA comprising a :DNA synthesis template comprising only one consecutive nucleotide mismatch relative to the endogenous sequence of a target site On the nucleic acid molecule.
190. A prime editor system comprising the pegRNA of any one of claims 180-189 and a prime editor, wherein the prime editor comprises a napDNAbp and a polymerase.
191. The prime editor system of claim 190, further comprising an inhibitor of DNA mismatch repair pathway.
192. A prime editor comprising a fusion protein comprising (i) a nucleic acid programmable DNA binding protein (napDNAbp) and (ii) a .DNA polymerase, wherein the napDNAbp is a Cas9 nickase (nCas9) cornprising a R221K amino acid substitution, a N39K amino acid substitution, and an amino acid substitution that inactivates HNH domain nuclease activity, or corresponding amino acid substitutions thereof, relative to a wild type Cas9 as set forth in SEQ
ID NO: 2.
193. The prime editor of claim 192, wherein the nCas9 comprises a R221K, a N39K, and a H840A amino acid substitution compared to a wild type Cas9 as set forth in SEQ
ID NO: 2.
194. The prime editor of claim 193, wherein the nCas9 and the DNA polymerase are connected by a linker, optionally wherein the linker comprises the sequence of SEQ ID NO: X5, optionally wherein the prime editor thrther comprises a SV40 NLS at the N
terminus, optionally wherein the prime editor further comprises a SV40 NTS and/or a c-Myc NIS at the C terminus.
195. The prime editor of claim 192 comprising the amino acid sequence of SEQ
IT) NO: 99 or an amino acid sequence have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity with SEQ ID NO: 99.
196. A prime editor systern comprising the prime editor of any one of claims 192-195 and an inhibitor of DNA mismatch repair pathway.
197. A prime editor system comprising the prime editor of any one of claims 192-195 and a prime editing guide RNA (pegRNA).
198. A polynucleotide encoding the prime editor of any of claims 192-195.
199. The polynucleotide of claim 198, wherein the polynucleotide is DNA.
200. The polynucleotide of claim 198, wherein the polynucleotide is mRNA.
201. A vector comprising the polynucleotide of claim 198, optionally wherein expression of the fusion protein is under the control of a promoter, optionally wherein the promoter is a U6 promoter.
202. A prime editor systern for site specific genome modification, comprising a (a) prime editor comprising (i) a nucleic acid programmable DNA binding protein (napDNAbp) and (ii) a DNA polyrnerase, and (b) an inhibitor of the DNA mismatch repair pathway.
203. The prime editor system of claim 202, wherein the inhibitor of the DNA
mismatch repair pathway inhibits one or more proteins of the DNA mismatch repair pathway.
204. The prime editor system of claim 203, wherein the one or more proteins is selected from the group consisting of MLH1, PMS2 (or MutL alpha), PMS1 (or MutL beta), MLH3 (or MutL
gamma), MutS alpha (MSH2-MSH6), MutS beta (MSH2-MSH3), MSH2, MSH6, FCNA, RFC, EXOI, POL8, and PCNA.
205. The prime editor system of claim 203, wherein the one or more proteins is MiL,H1.
206. The prime editor system of claim 205, wherein the MLH1 comprises an amino acid sequence of SEQ ID NO: 204, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to and including 100% sequence identity with SEQ ID NO. 204.
207. The prime editor system of claim 202, wherein the inhibitor is an antibody that inhibits the activity of one or more proteins of the DNA mismatch repair pathway.
208. The prime editor system of claim 202, wherein the inhibitor is a small molecule that inhibits the activity of one or more proteins of the DNA mismatch repair pathway.
209. The prime editor system of claim 202, wherein the inhibitor is a small interfering RNA
(siRNA) or a small non-coding microRNA that inhibits the activity of one or more proteins of the :DNA mismatch repair pathway.
210. The prime editor system of claim 202, wherein the inhibitor is a dominant negative variant of an MIVIR protein that inhibits the activity of a wild type MMR
protein.
211. The prime editor system of claim 202, wherein the inhibitor is a dominant negative variant of MLH1 that inhibits MLH1.
212. The prim.e editor system of claim 202, wherein the dominant negative variant is (a) MLH1 E34A (SEQ ID NO: 222), (b) MLH1 A756 (SEQ ID NO: 208), (c) MLH1 A754-756 (SEQ ID NO: 209), (d) MUM E34A A754-756 (SEQ ID NO: 210), (e) MLH1 1-335 (SEQ
ID
NO: 211), (f) MLH1 1-335 E34A (S:EQ ID NO: 212), (g) MLH1 1-335 NLS8v4O (SEQ
ID NO:
213), (h) MLH1 501-756 (SEQ ED NO: 215), (i) MLH1 501-753 (SEQ II) NO: 216), (j) MLH1 461-753 (SEQ ID NO: 218), or (k) NL.Ssy4O MLH1 501-753 (SEQ ID NO: 223), or a polypeptide comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to and including 100% sequence identity with any of SEQ ID NOs: 208-213, 215, 216, 218, 222, or 223.
213. The prime editor system of claim 202, wherein the nap:DNAbp is a nuclease active Cas9 domain, a nuclease inactive Cas9 domain, or a Cas9 nickase domain or variant thereof.
214. The prime editor systeni of claiin 202, wherein the napDNAbp is selected from the group consisting of: Cas9, Cas12e, Casi2d, Cas12a, Cas12b1, Cas13a, Cas12c, ArgonauteCas12b2, Cas13a, Cas12c, Cas12d, Cas12e, Cas12h, Cas12i, Cas12g, Cas12f (Cas14), Casl2f1, Cas12j (Cast,), and Argonaute and optionally has a nickase activity.
215. The prime editor system of claim 202, wherein the napDNAbp comprises an amino acid sequence of any one of SEQ ID NOs: 2, 4-67, 104 or an amino acid sequence having at least an 80%, 85%, 90%, 95%, or 99% sequence identity with any one of SEQ ID NOs: 2, 4-67, 104.
216. The prime editor system of claim 202, wherein the nap:DNAbp comprises an amino acid sequence of SEQ ID NO: 2 (i.e., the napDNAbp of PE1 and PE2) or an amino acid sequence having at least an 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID
NO: 2.
217. The prime editor system of claim 202, wherein the polymerase is a :DNA-dependent DNA
polymerase or an RNA-dependent DNA polymerase.
218. The prime editor system of claim 202, wherein the polymerase is a reverse transcriptase.
219. The prime editor system of claim 218, wherein the reverse transcriptase comprises an amino acid sequence of any one of SEQ ID NOs: 69-98 or an amino acid sequence having at least an 80%, 85%, 90%, 95%, or 99% sequence identity with any one of SEQ ID
NOs: 69-98.
220. The prime editor system of claim 202, wherein the napDNAbp and the polymerase of the prime editor are joined by a linker to form a fusion protein, and optionally wherein the inhibitor is joined by a second linker to either the napDNAbp or the polymerase.
221. The prime editor system of claim 220, wherein the linker and/or second linker comprises an amino acid sequence of any one of SEQ ID NOs: 102 or 118-131, or an amino acid sequence having at least an 80%, 85%, 90%, 95%, or 99% sequence identity with any one of SEQ ID NOs:
102 or 118-131.
222. The prime editor system of claim 220, wherein the linker and/or second linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 38, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length.
223. The prime editor system of claim 202, wherein the prime editor is PE1 of SEQ ID NO:
100 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or up to 100% sequence identity with SEQ ID NO: 100.
224. The prime editor system of claim 202, wherein the prime editor is PE2 of SEQ ID NO:
107 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or up to 100% sequence identity with SEQ ID NO: 107.
225. The prime editor system of claim 202, wherein the prime editor is PE1 of SEQ ID NO:
100 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or up to 100% sequence identity with SEQ ID NO: 100, and the inhibitor is a dominant negative variant of MLH1 that inhibits MLH I .
226. The prime editor system of claim 225, wherein the dominant negative variant of MLH1 is (a) MLII1 E34A (SEQ ID NO: 222), (b) ML/I1 A756 (SEQ ID NO: 208), (c) MLII1 (SEQ ID NO: 209), (d) MLH1 E34A A754-756 (SEQ ID NO: 210), (e) MLH1 1-335 (SEQ
ID
=NO: 211), (1) MLH1 1-335 E34A (SEQ ID NO: 212), (g) MLIII 1-335 NI,Ssv4O (SEQ
ID NO:

213), (h) MLHI 501-756 (SEQ ID NO: 215), (i) MLH1 501-753 (SEQ ID NO: 216), (j) MLH1 461-753 (SEQ ID NO: 218), or (k) N'LSsv4O MLH1 501-753 (SEQ ID NO: 223), or a polypeptide comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to and including 100% sequence identity with any of SEQ ID NOs: 208-213, 215, 216, 218, 222, or 223.The prime editor system of claim 145, wherein the prime editor is PE2 of SEQ ID NO: 107 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%õ at least 99%, or up to 100% sequence identity with SEQ ID NO: 107 and the inhibitor is a dominant negative variant of MLH1 that inhibits MLHI .
227. The prime editor system of claim 202, wherein the prime editor is PE2 of SEQ ID NO:
107 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or up to 100% sequence identity with SEQ ID NO: 107, and the inhibitor is a dominant negative variant of MLH1 that inhibits MLHI.
228. The prime editor system of claim 227, wherein the dominant negative variant of MLH1 is (a) MLH1 E34A (SEQ ID NO: 222), (b) MLH1 A756 (SEQ ID NO: 208), (c) MLH1 A754-(SEQ ID NO: 209), (d) MLH1 E34A A754-756 (SEQ ID NO: 210), (e) MLE11 1-335 (SEQ ID
NO: 211), (f) MLIT1 1-335 E34A (SEQ ID NO: 212), (g) MLIII 1-335 NLSSµ14 (SEQ
ID NO:
213), (h) MLH1 501-756 (SEQ ID NO: 215), (i) MLH1 501-753 (SEQ ID NO: 216), (j)1VILH1 461-753 (SEQ I) NO: 218), or (k)NLSsv40MLHI 501-753 (SEQ ID NO: 223), or a polypeptide comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to and including 100% sequence identity with any of SEQ ID NOs: 208-213, 215, 216, 218, 222, or 223.
229. The prime editor system of claim 202, wherein the prime editor is PE2 of SEQ ID NO:
107 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or up to 100% sequence identity with SEQ ID NO: 107 and the inhibitor is a dominant negative variant of MLHI that inhibits MLH1.
230. The prime editor system of claim 202, wherein the DNA polymerase is a reverse transcriptase.
231. The prime editor system of claim 230, wherein the reverse transcriptase is a retrovirus reverse transcriptase.
232. The prime editor system of claim 230, wherein the reverse transcriptase lacks RNase activity.
233. The prime editor system of claim 230, wherein the reverse transcriptase is a Moloney-Murine Leukemia Virus reverse transcriptase (MMLV-RT).
234. The prime editor system of claim 233, wherein the MMLV-RT comprises an amino acid sequence having at least 85% identity with a sequence selected from the group consisting of:
SEQ ID NOs: 89 and 701-716.
235. The prime editor system of claim 202, wherein the napDNAbp is a CRISPR
associated (Cas) nuclease.
236. The prime editor system of claim 235, wherein the napDNAbp comprises a Cas9 nuclease domain.
237. The prime editor system of claim 236, wherein the Cas9 nuclease domain is a nickase comprising a H840X substitution or a corresponding substitution as compared to a wild type Slreplococcus pyogenes Cas9 as set forth in SEQ II) NO: 18, wherein X is any amino acid other than histidine.
238. The prime editor system of any one of claims 202-237, further comprising a pegRNA that is capable of complexing with the napDNAbp of the prime editor and programming the napDNAbp to bind a target DNA. sequence.
239. A nucleic acid molecule encoding the prime editor system of any one of claims 202-238, or a component thereof.
240. A method for precisely installing a nucleotide edit of at least 15 bp in length in a double stranded target DNA sequence under conditions sufficient to evade the DNA
mismatch repair pathway, the method comprising: contacting the double stranded target DNA
sequence with a prime editor comprising a nucleic acid programmable DNA binding protein (napDNAbp), a DNA polymcrasc, and a prime editing guide RNA (PEgRNA), wherein the PEgRNA
comprises a spacer that hybridizes to a first strand of the double stranded target DNA
sequence, an extension arm that hybridizes to a second strand of the double stranded target DNA sequence, a DNA synthesis template comprising the nucleotide edit, and a gRNA core that interacts with the napDNAbp, and wherein the PEgRNA directs the prime editor to install the nucleotide edit in the double stranded target DNA sequence.
241. The method of claim 240, wherein the nucleotide edit is a deletion of at least 15 bp in length.
242. The inethod of claim 240, wherein the nucleotide edit is an insertion of at least 15 bp in length.
243. The method of claim 240, wherein the nucleotide edit is a least 16 bp, 17 bp, 18 bp, 19 bp, 20 bp, 2=l bp, 22 bp, 23 bp, 24 bp, or 25 bp in length.
244. A. method for editing a nucleic acid molecule by prime editing cornprising: contacting a nucleic acid molecule with a prime editor and a pegRNA, thereby installing one or more modifications to the nucleic acid molecule at a target site, wherein the nucleic acid molecule is in a cell comprising a knockout of one or more genes involved in the DNA
mismatch repair (MIVIR) pathway.
245. The method of claim 244, wherein the method further comprises contacting the nucleic acid molecule with a second strand nicking gRNA.
246. The method of claim 244, wherein the prirne editing efficiency is increased by at least 1.5-fold, at least 2.0 fold, at least 2.5-fold, at least 3.0-fold, at least 3.5-fold, at least 4.0-fold, at least 4.5-fold, at least 5.0-fold, at least 5.5-fold, at least 6.0-fold, at least 6.5-fold, at least 7.0-fo1d, at least 7.5-fold, at least 8.0-fold, at least 8.5-fold, at least 9.0-fold, at least 9.5-fold, at least 10.0 fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, at least 20-fold, at least 21-fold, at least 22-fold, at least 23-fold, at least 24-fold, at least 25-fold, at least 26-fold, at least 27-fold, at least 28-fold, at least 29-fold, at least 30-fold, at least 3 I-fold, at least 32-fold, at least 33-fold, at least 34-fold, at least 35-fold, at least 36-fold, at least 37-fold, at least 38-fold, at least 39-fold, at least 40-fold, at least 41-fold, at least 42-fold, at least 43-fold, at least 44-fold, at least 45-fold, at least 46-fold, at least 47-fold, at least 48-fold, at least 49-fold, at least 50-fold, at least 51-fold, at least 52-fold, at least 53-fold, at least 54-fold, at least 55-fold, at least 56-fold, at least 57-fold, at least 58-fold, at least 59-fold, at least 60-fold, at least 61-fold, at least 62-fold, at least 63-fold, at least 64-fold, at least 65-fold, at least 66-fold, at least 67-fold, at least 68-fold, at least 69-fold, at least 70-fold, at least 71-fold, at least 72-fold, at least 73-fold, at least 74-fold, or at least 75-fold relative to a method performed in a cell that does not comprise a knockout of one or more genes involved in NEAR.
247. The method of claim 244, wherein the frequency of indel formation is decreased by at least 1.5-fold, at least 2.0 fold, at least 2.5-fold, at least 3.0-fold, at least 3.5-fold, at least 4.0-fold, at least 4.5-fold, at least 5.0-fold, at least 5.5-fold, at least 6.0-fold, at least 6.5-fold, at least 7.0-fold, at least 7.5-fold, at least 8.0-fold, at least 8.5-fold, at least 9.0-fold, at least 9.5-fold, at least 10.0 fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, at least 20-fold, at least 21-fold, at least 22-fold, at least 23-fold, at least 24-fold, at least 25-fold, at least 26-fold, at least 27-fold, at least 28-fold, at least 29-fold, at least 30-fold, at least 31-fo1d, at least 32-fold, at least 33-fold, at least 34-fold, at least 35-fold, at least 36-fold, at least 37-fo1d, at least 38-fold, at least 39-fo1d, at least 40-fold, at least 41-fold, at least 42-fold, at least 43-fold, at least 44-fold, at least 45-fold, at least 46-fold, at least 47-fold, at least 48-fold, at least 49-fold, at least 50-fold, at least 51-fold, at least 52-fold, at least 53-fold, at least 54-fold, at least 55-fold, at least 56-fold, at least 57-fold, at least 58-fold, at least 59-fold, at least 60-fold, at least 61-fold, at least 62-fold, at least 63-fold, at least 64-fold, at least 65-fold, at least 66-fold, at least 67-fold, at least 68-fold, at least 69-fold, at least 70-fold, at least 71-fold, at least 72-fold, at least 73-fold, at least 74-fold, or at least 75-fold relative to a method perforrned in a cell that does not comprise a knockout of one or more genes involved in MMR.
248. The method of claim 244, wherein the one or more genes involved in MMR is selected from the group consisting of genes encoding the proteins MLHI, PMS2 (or MutL
alpha), PMS1 (or MutL beta), MLF1.3 (or MutL gamma), MutS alpha (MSFI2-MSH6), MutS
beta (MSH2-MSH3), MSH2, MSH6, PCNA, RFC, EX01, POLo, and PCNA.
249. The method of claim 248, wherein the one or more genes is the gene encoding IVILH1.
250. The method of claim 249, wherein IVILH1 comprises an amino acid sequence of SEQ ED
NO: 204, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to and including 100% sequence identity with SEQ ID NO: 204.
251. The method of claim 244, wherein the prime editor comprises a napDNAbp and a poly merase.
252. The rnethod of claim 251, wherein the napDNAbp is a nuclease active Cas9 domain, a nuclease inactive Cas9 domain, or a Cas9 nickase domain or variant thereof.
253. The method of claim 251, wherein the napDNAbp is selected from the group consisting of: Cas9, Cas12e, Cas12d, Cas12a, Cas12b1, Cas13a, Cas12c, Cas12b2, Cas13a, Cas12c, Cas12d, Cas12e, Cas12h, Cas12i, Cas12g, Cas12f (Cas14), Casl2f1, Cas12j (Cas(1)), and Argonaute and optionally has a nickase activity.
254. The method of claim 251, wherein the napDNAbp comprises an amino acid sequence of any one of SEQ ID NOs: 2, 4-67, or 99 (PEmax) or an ainino acid sequence having at least an 80%, 85%, 90%, 95%, or 99% sequence identity with any one of SEQ lD NOs: 2, 4-67, or 99 (P:Emax).
255. The method of claim 251, wherein the napDNAbp comprises an amino acid sequence of SEQ ID NO: 2 (i.e., the napDNAbp of PE1 and PE2) or an amino acid sequence having at least an 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 2.
256. The method of claim 251, wherein the polymerase is a DNA-dependent DNA
polymerase or an RNA-dependent DNA polymerase.
257. The method of claim 251, wherein the polymerase is a reverse transcriptase.
258. The method of claim 257, wherein the reverse transcriptase comprises an amino acid sequence of any one of SEQ ID NOs: 69-98 or an amino acid sequence having at least an 80%, 85%, 90%, 95%, or 99% sequence identity with any one of SEQ ID NOs: 69-98.
259. The method of claim 251, wherein the napDNAhp and the polymerase of the prime editor are joined by a linker to form a fusion protein.
260. The method of claim 259, wherein the linker comprises an amino acid sequence of any one of SEQ ID NOs: 102 or 118-131, or an arnino acid sequence having at least an 80%, 85%, 900/o, 95%, or 99% sequence identity with any one of SEQ ID NOs: 102 or 118-131.
261. The method of claim 259, wherein the linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 38, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length.
262. The method of claim 244, wherein the one or more modifications to the nucleic acid molecule installed at the target site comprises one or more transitions, one or more transversions, one or more insertions, one or more deletions, or one more inversions, and optionally are less than 15 bp.
263. The method of claim 262, wherein the one or more transitions are selected from the group consisting of: (a) T to C; (h) A to G; (c) C to T; and (d) G to A.
264. The method of claim 262, wherein the one or more transversions are selected from the group consisting of: (a) T to A; (b) T to G; (c) C to G; (d) C to A; (e) A to T; (f) A. to C; (g) G to C; and (h) G to T.
265. The method of claim 244, wherein the one or more modifications comprises changing (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 a 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
bascpair.
266. The method of claim 244, wherein the one or more modifications comprises 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.
267. The method claim 244, wherein the one or more modifications comprises a correction to a disease-associated gene.
268. The method of claim 267, 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.
269. A method for editing a nucleic acid molecule by prilne editing comprising: contacting a nucleic acid molecule with a prime editor, a pegRNA, and an inhibitor of p53, thereby installing one or more modifications to the nucleic acid molecule at a target site.
270. The method of claim 269, wherein the method further comprises contacting the nucleic acid molecule with a second strand nicking gRNA..
271. The method of claim 269, wherein the prime editing efficiency is increased by at least 1.5-fold, at least 2.0-fold, at least 2.5-fold, at least 3.0-fold, at least 3.5-fold, at least 4.0-fold, at least 4.5-fold, at least 5.0-fold, at least 5.5-fold, at least 6.0-fold, at least 6.5-fold, at least 7.0-fold, at least 7.5-fold, at least 8.0-fold, at least 8.5-fold, at least 9.0-fold, at least 9.5-fold, at least 10.0-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fo1d, at least 20-fold, at least 21-fold, at least 22-fold, at least 23-fold, at least 24-fold, at least 25-fold, at least 26-fold, at least 27-fold, at least 28-fold, at least 29-fold, at least 30-fold, at least 31-fold, at least 32-fold, at least 33-fold, at least 34-fold, at least 35-fold, at least 36-fold, at least 37-fold, at least 38-fold, at least 39-fold, at least 40-fo1d, at least 41-fold, at least 42-fold, at least 43-fold, at least 44-fold, at least 45-fold, at least 46-fold, at least 47-fold, at least 48-fold, at least 49-fold, at least 50-fold, at least 51-fold, at least 52-fold, at least 53-fold, at least 54-fold, at least 55-fold, at least 56-fold, at least 57-fold, at least 58-fold, at least 59-fold, at least 60-fold, at least 61-fold, at least 62-fold, at least 63-fold, at least 64-fold, at least 65-fold, at least 66-fold, at least 67-fold, at least 68-fold, at least 69-fold, at least 70-fold, at least 71-fold, at least 72-fold, at least 73-fold, at least 74-fold, or at least 75-fold, in the presence of the inhibitor of p53.
272. The method of claim 269, wherein the frequency of indel formation is decreased by at least 1.5-fold, at least 2.0-fold, at least 2.5-fold, at least 3.0-fold, at least 3.5-fold, at least 4.0-fold, at least 4.5-fold, at least 5.0-fold, at least 5.5-fold, at least 6.0-fold, at least 6.5-fold, at least 7.0-fold, at least 7.5-fold, at least 8.0-fold, at least 8.5-fold, at least 9.0-fold, at least 9.5-fold, at least 10.0-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, at =least 20-fold, at least 21-fold, at least 22-fold, at least 23-fold, at least 24-fold, at least 25-fold, at least 26-fold, at least 27-fold, at least 28-fold, at least 29-fold, at least 30-fold, at least 31-fold, at least 32-fold, at least 33-fold, at least 34-fold, at least 35-fold, at least 36-fold, at least 37-fold, at least 38-fold, at least 39-fold, at least 40-fold, at least 41-fold, at least 42-fold, at least 43-fold, at least 44-fold, at least 45-fold, at least 46-fold, at least 47-fold, at least 48-fold, at least 49-fold, at least 50-fold, at least 51-fold, at least 52-fold, at least 53-fold, at least 54-fold, at least 55-fold, at least 56-fold, at least 57-fold, at least 58-fold, at least 59-fold, at least 60-fold, at least 61-fold, at least 62-fold, at least 63-fold, at least 64-fold, at least 65-fold, at least 66-fold, at least 67-fold, at least 68-fold, at least 69-fold, at least 70-fold, at least 71-fold, at least 72-fold, at least 73-fo1d, at least 74-fold, or at least 75-fold, in the presence of the inhibitor of p53.
273. The method of claim 269, wherein the inhibitor of p53 is a protein.
274. The method of claim 273, wherein the protein is 153.
275. The method of claim 269, wherein the inhibitor of p53 is an antibody that inhibits the activity of p53.
276. The method of claim 269, wherein the inhibitor of p53 is a small molecule that inhibits the activity of p53.
277. The method of claim 269, wherein the inhibitor of p53 is a small interfering RNA
(siRNA) or a small non-coding micro:RNA that inhibits the activity of p53.
278. The method of claim 269, wherein the prime editor comprises a napDNAbp and a polymerase.
279. The method of claim 278, wherein the napDNAbp is a nuclease active Cas9 domain, a nuclease inactive Cas9 domain, or a Cas9 nickase domain or variant thereof
280. The method of claim 278, wherein the napDNAbp is selected from the group consisting of: Cas9, Cas12e, Cas12d, Cas12a, Cas12b1, Cas13a, Cas12c, Cas12b2, Cas13a, Cas12c, Cas12d, Cas12e, Cas12h, Casi2i, Cas12g, Cas12f (Cas14), Casl2f1, Cas12j (Cas(1)), and Argonaute and optionally has a nickase activity.
281. The method of claim 278, wherein the napDNAbp comprises an amino acid sequence of any one of SEQ ID NOs: 2, 4-67, or 104, or an amino acid sequence having at least an 80%, 85%, 90%, 95%, or 99% sequence identity with any one of SEQ ID NOs: 2, 4-67, or 104.
282. The method of claim 278, wherein the napDNAbp comprises an amino acid sequence of SEQ JD NO: 2 or SEQ ID NO: 37 (i.e., the napDNAbp of PE1 and PE2) or an amino acid sequence having at least an 80%, 85%, 90%, 95%, or 99% sequence identity witb SEQ ID
=NO: 2 or SEQ ID NO: 37.
283. The method of claim 278, wherein the polymerase is a DNA-dependent DNA
polymerase or an RNA-dependent DNA polymerase.
284. The method of claim 278, wherein the polymerase is a reverse transcriptase.
285. The method of claim 284, wherein the reverse transcriptase comprises an amino acid sequence of any one of SEQ ID NOs: 69-98 or an amino acid sequence having at least an 80%, 85 A, 90%, 95%, or 99% sequence identity with any one of SEQ ID NOs: 69-98.
286. The method of claim 278, wherein the napDNAbp and the polymerase of the prime editor are joined by a linker to form a fusion protein.
287. The method of claim 286, wherein the linker comprises an amino acid sequence of any one of SEQ ID NOs: 102 or 118-131, or an amino acid sequence having at least an 80%, 85%, 90%, 95%, or 99% sequence identity with any one of SEQ ID NOs: 102 or 118-
288. The method of claim 286, wherein the linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 38, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length.
289. The method of claim 269, wherein the prime editor, the pegRNA, and the inhibitor of p53 are encoded on one or more DNA vectors.
290. The method of claim 289, wherein the one or more DNA vectors comprise AAV
or lentivirus DNA vectors.
291. The method of claim 290, wherein the AAV vector is serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
292. The method of claim 286, wherein the prime editor as a fusion protein is further joined by a second linker to the inhibitor of p53.
293. The method of claim 292, wherein the second linker comprises a self-hydrolyzing linker.
294. The method of claim 292, wherein the second linker comprises an amino acid sequence of any one of SEQ ID NOs: 102, 118-131, or 233-236, or an amino acid sequence having at least an 80%, 85%, 90%, 95%, or 99% sequence identity with any one of S:EQ ID
NOs: 102, 118-131, or 233-236.
295. The method of claim 292, wherein the second linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length.
296. The method of claim 269, wherein the one or more modifications to the nucleic acid molecule installed at the target site comprises one or more transitions, one or more transversions, one or more insertions, one or more deletions, or one more inversions, and optionally wherein the one or more modifications are less than 15 bp.
297. The method of claim 296, wherein the one or more transitions are selected from the group consisting of: (a) T to C; (b) A to G; (c) C to T; and (d) G to A.
298. The method of claim 296, wherein the one or more transversions are selected from the group consisting of: (a) T to A; (b) T to G; (c) C to G; (d) C to A; (e) A to T; (f) A to C; (g) G to C; and (h) G to T.
299. The method of claim 269, wherein the one or more inodifications comprises changing (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 a C:G basepair, (4) a '11: A basepair to a G:C basepair, (5) a 'I':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.
300. The method of claim 269, wherein the one or more modifications comprises 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.
301. The method claim 269, wherein the one or more modifications comprises a correction to a disease-associated gene.
302. The method of claim 301, 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.
303. The method of claim 301, wherein the disease-associated gene is associated with a monogenic disorder selected from the group consisting of: Adenosine Deaminase (ADA) Deficiency, Alpha-1 Antitrypsin Deficiency, Cystic Fibrosis, Duchenne Muscular Dystrophy, Galactosemia, Hemochromatosis, Huntington's Disease, :Maple Syrup Urine Disease, Marfan Syndrome, Neurofibromatosis Type 1, Pachyonychia Congcnita, Phenylketonuria, Severe Combined Immunodeficiency, Sickle Cell Disease, Smith-Lemli-Opitz Syndrome, a trinucleotide repeat disorder, a prion disease, and Tay-Sachs Disease.
304. The method of any of the preceding claims, wherein the nucleic acid molecule is in a cell.
305. The method of claim 304, wherein the cell is a mammalian cell, a non-human primate cell, or a human cell.
306. The method of claim 304, wherein the cell is ex vivo.
307. The method of claim 304, wherein the cell is in a subject, optionally wherein the subject is human.
308. A method for treating a disease in a subject in need thereof, the method comprising administering to the subject: (i) a prime editor, (ii) a pegRN=A, and (iii) an inhibitor of a DNA
mismatch repair pathway, wherein the prime editor comprises a nucleic acid programmable DNA binding protein (napDNAbp) and a DNA polymerase, wherein the pegRNA comprises a spacer sequence, a gRNA core, and an extension arrn comprising a DNA synthesis template and a primer binding site (PBS), wherein the spacer sequence comprises a region of complementarity to a target strand of a double stranded target DNA sequence in the subject, wherein the gRNA core associates with the napDNAbp, wherein the DNA synthesis template comprises a region of complementarity to the non-target strand of the double-stranded target :DNA sequence and one or more nucleotide edits compared to the target strand double-stranded target DNA sequence;
wherein the primer binding site comprises a region of complementarity to a non-target strand of the double-stranded target DNA sequence, wherein the prime editor and the pegRNA install the one or more nucleotide edits in the double stranded target DNA, wherein installation of the one or more nucleotide edits corrects one or more mutations in the double stranded target DNA associated with the disease, thereby treating the disease in the subject.
309. A method for treat a disease in a subject in need thereof, the method comprising administering to the subject: (i) a prime editor and (ii) a pegRNA wherein the prime editor comprises a nucleic acid programmable DNA binding protein (napDNAbp) and a DNA

polymerase, wherein the pegRNA comprises a spacer sequence, a gRNA core, and an extension arm comprising a DNA synthesis template and a primer binding site (PBS), wherein the spacer sequence comprises a region of complementarity to a target strand of a double stranded target DNA sequence in the subject, wherein the gRNA core associates with the napDNAbp, wherein the DNA synthesis template comprises a region of complementarity to the non-target strand of the double-stranded target DNA sequence and comprises three or more consecutive nucleotide mismatches relative to the endogenous sequence of the double stranded target DNA sequence, wherein the three or more consecutive nucleotide mismatches comprise (i) an insertion, deletion, or substitution of x nucleotides that corrects a mutation (e.g. a disease associated mutation) and (ii) an insertion, deletion, or substitution of y nucleotides directly adjacent to the x nucleotides, wherein the insertion, deletion, or substitution of y nucleotides is a silent mutation, wherein (x+y) is an integer no less than 3, wherein y is an integer no less than 1, and wherein inclusion of the silent mutation(s) increases the efficiency, reduces unintended indel frequency, and/or improves editing outcome purity by prime editing, wherein the primer binding site comprises a region of complementarity to a non-target strand of the double-stranded target DNA sequence, wherein the prime editor and the pegRNA install the insertion, deletion, or substitution of x nucleotides in the double stranded target DNA, wherein installation insertion, deletion, or substitution of x nucleotides corrects one or more mutations in the double stranded target DNA associated with the disease, thereby treating the disease in the subject.
310. The method of claim 308 or 309, wherein the subject is a human.
311. A fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) domain and a domain comprising an RNA-dependent DNA polymerase activity, wherein the fusion protein comprises an amino acid sequence of SEQ
ID NO: 99, or an amino acid sequence have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity with SEQ
ID NO: 99
312. A fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) domain and a domain comprising an RNA-dependent DNA polyrnerase activity, wherein the napDNAbp comprises an amino acid sequence of SEQ lD NO:
104, or an amino acid sequence have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity with SEQ
ID NO: 104.
313. A fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) domain and a domain comprising an RNA-dependent DNA polymerase activity, wherein the domain comprising an RNA-dependent DNA polymerase activity comprises an amino acid sequence of SEQ ID NO: 98, or an amino acid sequence have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity with SEQ JD NO: 98.
314. The fusion protein of any of claims 311-313, further comprising a linker that joins the napDNAbp and the domain comprising the RNA-dependent :DNA polymerase activity.
315. The fusion protein of claim 314, wherein the linker comprises SEQ ID NO:
105.
316. The fusion protein of claim 311, wherein the napDNAbp is a Cas9 nickase.
317. The fusion protein of claim 316, wherein the Cas9 nickase comprises an substitution and at least one substitution at R221 or N394 relative to SEQ ID
NO: 37.
318. A complex comprising a fusion protein of any one of claims 311-317 and a PEgRNA, wherein the PEgRNA directs the fusion protein to a target DNA sequence for prime editing.
319. The complex of claim 318, wherein the PEgRNA comprises a guide RNA and a nucleic acid extension arm at the 3' or 5' end of the guide RNA.
320. The complex of claim 319, wherein the PEgRNA is capable of binding to a napDNAbp and directing the napDNAbp to the target DNA sequence.
321. A polynucleotide encoding the fusion protein of any of claims 311-317.
322. A. vector comprising the polynucleotide of claim 321, wherein expression of the fusion protein is under the control of a promoter.
323. The vector of claim 322, wherein the promoter is a U6 promoter.
324. A cell comprising the fusion protein of any of claims 311-317 and a PEgRNA bound to the napDNAbp of the fusion protein.
325. A cell comprising a complex of any one of claims 318-320.
326. A pharmaceutical composition comprising: (i) a fusion protein of any of claims 311-317, the complex of claims 318-320, the polynucleotide of claim 321, or the vector of claims 322-323; and (ii) a pharmaceutically acceptable excipient.
327. A method for editing a nucleic acid molecule by prime editing comprising:
contacting a nucleic acid molecule with a modified prime editor of any one of claims 311-317 and a pegRNA, thereby installing one or more modifications to the nucleic acid molecule at a target site.
328. The method of claim 327, wherein the method further comprises contacting the nucleic acid molecule with a second strand nicking gRNA.
329. The method of claim 327, wherein the prime editing efficiency is increased by at least 1.5-fold, at least 2.0 fold, at least 2.5-fold, at least 3.0-fold, at least 3.5-fold, at least 4.0-fo1d, at least 4.5-fold, at least 5.0-fold, at least 5.5-fold, at least 6.0-fold, at least 6.5-fold, at least 7.0-fold, at least 7.5-fold, at least 8.0-fold, at least 8.5-fold, at least 9.0-fold, at least 9.5-fold, or at least 10.0 fold relative to prime editing with PE2.