Prime editing factor variants, constructs, and methods for improving prime editing efficiency and accuracy

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 pathway while performing prime editing of the target site. Thus, the present disclosure provides a method for editing a nucleic acid molecule by prime editing, which involves contacting the nucleic acid molecule with a prime editing factor, a pegRNA, and an inhibitor of the DNA mismatch repair pathway, thereby increasing editing efficiency and / or reducing indel formation and installing one or more modifications to the nucleic acid molecule at the target site. The present disclosure further provides a polynucleotide for editing a DNA target site by prime editing, comprising a nucleic acid sequence encoding napDNAbp, a polymerase, and an inhibitor of the DNA mismatch repair pathway, where the napDNAbp and the polymerase are capable in the presence of a pegRNA that installs one or more modifications at the DNA target site with increased editing efficiency and / or reduced indel formation. The present disclosure further provides vectors, cells, and kits comprising the compositions and polynucleotides of the present disclosure. The present disclosure also provides compositions and methods for prime editing with improved editing efficiency and / or reduced indel formation by modified prime editor fusion proteins. The present disclosure further provides vectors, cells and kits comprising the compositions and polynucleotides of the disclosure.

Description

[Technical Field]

[0001] government support This invention was made with government support under Grant Nos. AI142756, AI150551, HG009490, EB022376, EB031172, GM118062, CA072720, GM138167, U01AI142756, RM1HG009490, R01EB022376, and R35GM118062 awarded by the National Institutes of Health, and Grant No. HR0011-17-2-0049 awarded by the Department of Defense. The government has certain rights in this invention.

[0002] Related Applications This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63 / 255,897, filed October 14, 2021, U.S. Provisional Patent Application No. 63 / 231,230, filed August 9, 2021, U.S. Provisional Patent Application No. 63 / 194,913, filed May 28, 2021, U.S. Provisional Patent Application No. 63 / 194,865, filed May 28, 2021, U.S. Provisional Patent Application No. 63 / 176,202, filed April 16, 2021, and U.S. Provisional Patent Application No. 63 / 136,194, filed January 11, 2021, each of which is incorporated herein by reference.

[0003] Incorporation by Reference Additionally, this application relates to the following patent applications directed to prime editing that were previously filed by one or more of the present inventors: U.S. Provisional Patent Application No. 62 / 820,813, filed March 19, 2019; U.S. Provisional Patent Application No. 62 / 858,958, filed June 7, 2019; U.S. Provisional Patent Application No. 62 / 889,996, filed August 21, 2019; U.S. Provisional Patent Application No. 62 / 922,654, filed August 21, 2019; U.S. Provisional Patent Application No. 62 / 913,553, filed October 10, 2019; U.S. Provisional Patent Application No. 62 / 922,654, filed October 10, 2019; U.S. Provisional Patent Application No. 62 / 913,553, filed October 10, 2019; Provisional Patent Application No. 62 / 973,558; U.S. Provisional Patent Application No. 62 / 931,195, filed November 5, 2019; U.S. Provisional Patent Application No. 62 / 944,231, filed December 5, 2019; U.S. Provisional Patent Application No. 62 / 974,537, filed December 5, 2019; U.S. Provisional Patent Application No. 62 / 991,069, filed March 17, 2020; U.S. Provisional Patent Application No. 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 on March 19, 2020; International PCT Application No. PCT / US2020 / 023583 filed on March 19, 2020; International PCT Application No. PCT / US2020 / 023730 filed on March 19, 2020; International PCT Application No. PCT / US2020 / 023713 filed on March 19, 2020; International PCT Application No. PCT / US2020 / 023712 filed on March 19, 2020; International PCT Application No. PCT / US2020 / 02372 filed on March 19, 2020 No. 7, 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, each of which is incorporated by reference in its entirety. [Background technology]

[0004] Recent developments in prime editing allow for the insertion, deletion, and / or replacement of genomic DNA sequences without the need for error-prone double-strand DNA breaks. See Anzalone et al., "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 the target genomic site but also encodes information for installing the desired edit. Without wishing to be bound by any particular theory, prime editing proceeds via a putative multi-step editing process: 1) the Cas9 domain binds to and nicks the target genomic DNA site specified by the spacer sequence of the pegRNA; 2) the reverse transcriptase domain uses the nicked genomic DNA as a primer to initiate 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 replacing the resulting 5' flap species via invasion by the edited 3' flap, excision of the 5' flap containing the original DNA sequence, and ligation of a 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 in the heteroduplex using the edited strand as a template for repair, completing the editing process.

[0005] Since 2019, prime editing has been applied to introduce genetic changes into a wide variety of cells and / or organisms. Given its rapid adoption, prime editing represents a powerful tool for genome editing. Despite its versatility and widespread use, prime editing efficiency can vary significantly across different editing classes, target loci, and cell types (Anzalone et al., 2019). Therefore, modifications to the prime editing system that improve the specificity and / or efficiency of the prime editing process would greatly contribute to the advancement of this technology. In particular, modifications that facilitate more efficient integration of the edited DNA strand synthesized by the prime editing factors into the target genomic site are desirable. It is also desirable to reduce the frequency of indel by-products that can form as a result of prime editing. Such further modifications to prime editing would advance this technology. Summary of the Invention

[0006] In one aspect, the present disclosure relates to the observation that the efficiency and / or specificity of prime editing is influenced by a cell's own DNA mismatch repair (MMR) DNA repair pathway. MMR is a multifactorial pathway that involves the correction of base pair 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 aspect as "pooled CRISPRi screening for prime editing outcomes," which resulted in the identification of various genetic determinants, including MMR, as affecting the efficiency and / or specificity of prime editing. Thus, in one aspect, the present disclosure provides novel prime editing systems that include means for inhibiting and / or circumventing the effects of MMR, thereby increasing the efficiency and / or specificity of prime editing. In one aspect, the present disclosure provides prime editing systems that include MMR-inhibiting proteins, such as dominant-negative variants of MMR proteins, such as, but not limited to, dominant-negative MLH1 proteins (i.e., "MLH1dn"). In another embodiment, the prime editing system includes installing one or more silent mutations near the intended edit, thereby allowing the intended edit to avoid MMR recognition even in the absence of an MMR inhibitory protein such as MLH1dn. In another aspect, the present disclosure provides novel genetic screens for identifying genetic determinants, such as MMR, that affect the efficiency and / or specificity of prime editing. In yet a further aspect, the present disclosure provides nucleic acid constructs encoding the improved prime editing systems described herein. The disclosure in another aspect also provides vectors (e.g., AAV or lentiviral vectors) comprising nucleic acids encoding the improved prime editing systems described herein. In yet another aspect, the present disclosure provides cells comprising the improved prime editing systems described herein. The present disclosure also provides components of genetic screens, including nucleic acid and / or vector constructs, guide RNAs, pegRNAs, cells (e.g., CRISPRi cells), and other reagents and / or materials for performing the genetic screens disclosed herein.In yet another aspect, the present disclosure provides compositions and kits, e.g., pharmaceutical compositions, that include the improved prime editing systems described herein and can be administered to a cell, tissue, or organism by any suitable means, such as gene therapy, mRNA delivery, virus-like particle delivery, or ribonucleoprotein (RNP) delivery. In yet another aspect, the present disclosure provides methods for using the improved prime editing systems to install one or more edits into a target nucleic acid molecule, e.g., a genomic locus. In yet another aspect, the present disclosure provides methods for treating a disease or disorder using the improved prime editing systems to correct or otherwise repair one or more genetic alterations (e.g., single nucleotide polymorphisms) in a target nucleic acid molecule, e.g., a genomic locus that contains one or more disease-causing mutations.

[0007] Thus, in various aspects, the present disclosure describes improved and modified approaches for prime editing, including inhibiting the DNA mismatch repair (MMR) system during prime editing. The inventors surprisingly found that when one or more functions of the DNA mismatch repair (MMR) system are inhibited, blocked, or otherwise inactivated during prime editing (e.g., using an MLH1dn inhibitor of MMR), the editing efficiency of prime editing can be significantly increased (e.g., at least 2-fold increase, at least 3-fold increase, at least 4-fold increase, at least 5-fold increase, at least 6-fold increase, at least 7-fold increase, at least 8-fold increase, at least 9-fold increase, at least 10-fold increase, or more). In addition, the inventors have surprisingly found that the frequency of indel formation resulting from prime editing may be significantly reduced (e.g., by about a 2-fold reduction, about a 3-fold reduction, about a 4-fold reduction, about a 5-fold reduction, about a 6-fold reduction, about a 7-fold reduction, about an 8-fold reduction, about a 9-fold reduction, or about a 10-fold reduction or less) when one or more functions of the DNA mismatch repair (MMR) system are inhibited, blocked, or otherwise inactivated during prime editing.

[0008] The present disclosure also describes, in other aspects, improved and modified approaches for prime editing, including circumventing the DNA mismatch repair (MMR) system during prime editing. The inventors have surprisingly found that, in the presence or absence of an inhibitor of MMR, the editing efficiency of prime editing may be significantly increased (e.g., 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 Also 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 increase).The inventors have surprisingly found that, in the presence or absence of an inhibitor of MMR, the frequency of indel formation may be significantly reduced (e.g., 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 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 increase).

[0009] In some embodiments, the present disclosure describes an improved prime editing system, referred to herein as "PE4," that includes PE2 plus an MLH1 dominant-negative protein (e.g., wild-type MLH1 truncated at amino acids 754-756, as further described herein). In certain embodiments, MLH1dn is expressed in trans in cells containing the PE2 fusion protein. MLH1dn and PE2 may be provided together or separately, e.g., by delivery on separate plasmids, separate vectors (e.g., AAV or lentiviral vectors), separate vector-like particles, separate ribonucleoprotein complexes (RNPs), or by delivery on the same plasmid, the same vector (e.g., AAV or lentiviral vectors), the same vector-like particle, or the same ribonucleoprotein complex (RNP). In other embodiments, MLH1dn may be fused to PE2 or otherwise associated, bound, or attached to PE2 so that they are co-delivered.

[0010] In other aspects, the present disclosure describes an improved prime editing system, designated "PE5," that includes PE3 (PE2 + second-strand nicking guide RNA) + MLH1 dominant-negative protein (e.g., wild-type MLH1 truncated at amino acids 754-756, as further described herein). In certain aspects, MLH1dn is expressed in trans in cells containing PE3 prime editing factors. MLH1dn and PE3 may be provided together or separately, e.g., by delivery on separate plasmids, separate vectors (e.g., AAV or lentiviral vectors), separate vector-like particles, separate ribonucleoprotein complexes (RNPs), or by delivery on the same plasmid, the same vector (e.g., AAV or lentiviral vectors), the same vector-like particle, or the same ribonucleoprotein complex (RNP). In other aspects, MLH1dn may be fused to PE3 or otherwise associated, bound, or attached to PE3 so that they are co-delivered.

[0011] In another aspect, the present disclosure describes an optimized PE2 prime editor architecture, referred to herein as "PEmax." PEmax is a modified form of PE2 that includes modified reverse transcriptase codon usage, SpCas9 mutations, and an NLS sequence, and is depicted in Figure 54B. Specifically, PEmax refers to a PE complex containing 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)]-[linker]-[MMLV_RT(D200N)(T330P)(L603W)]-[bipartite NLS]-[NLS]+desired PE gRNA, where the PE fusion has the amino acid sequence of SEQ ID NO:99, as shown below. [ka] [ka]

[0012] In some embodiments, PE4 may be modified to replace the PE2 fusion protein with PEmax, in which case the modified prime editing system may be referred to as "PE4max."

[0013] In some embodiments, PE5 may be modified to replace the PE3 prime editing element with PEmax, in which case the modified prime editing system may be referred to as "PE5max" and includes a second strand nicking guide RNA.

[0014] The present inventors have developed prime editing that allows for the insertion, deletion, and / or substitution of genomic DNA sequences without the need for error-prone double-stranded DNA breaks. The present disclosure now provides improved methods of prime editing that involve blocking, inhibiting, circumventing, or inactivating the MMR pathway (e.g., by inhibiting, blocking, or inactivating MMR pathway proteins, including MLH1) during prime editing, whereby, surprisingly, doing so results in increased editing efficiency and reduced indel formation. As used herein, "during" prime editing can embrace any suitable sequence of events, such that a prime editing step can be applied before, simultaneously with, or after a step of blocking, inhibiting, circumventing, or inactivating the MMR pathway (e.g., by targeted inhibition of MLH1).

[0015] In various aspects, 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 encodes information to direct Cas9 to a target genomic site and install the desired edit. Prime editing proceeds through a multistep editing process: 1) the Cas9 domain binds to and nicks the target genomic DNA site specified by the spacer sequence of the pegRNA; 2) the reverse transcriptase domain uses the nicked genomic DNA as a primer to initiate 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 replacing the resulting 5' flap via invasion by the edited 3' flap, excision of the 5' flap containing the original DNA sequence, and ligation of a 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 in the heteroduplex using the edited strand as a template for repair, completing the editing process.

[0016] Efficient integration of the desired edit requires that the newly synthesized 3' flap contains a portion of sequence homologous to the genomic DNA site. This homology allows 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 contains less sequence homology than the endogenous 5' flap, competition is expected to favor the 5' flap strand. Therefore, a potential limiting factor in the efficiency of prime editing may be the inability of the edit-containing 3' flap to effectively invade and displace the 5' flap strand. Furthermore, successful 3' flap invasion and 5' flap removal only integrates the edit into one strand of the double-stranded DNA genome. Permanent installation of the edit requires cellular DNA repair to use the edited strand as a template to replace the unedited complementary DNA strand. Although cells can be engineered to favor displacement of the unedited strand over the edited strand by using a secondary sgRNA (i.e., PE3-based) to introduce a nick in the unedited strand adjacent to the edit (step 4 above), this process still relies on a second step of DNA repair.

[0017] The present disclosure describes modified approaches to prime editing that further inhibit, block, or otherwise inactivate the DNA mismatch repair (MMR) system. In certain embodiments, one or more proteins of the MMR system can be inhibited, blocked, or otherwise inactivated, including, but not limited to, 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, EXO1, POLδ, and PCNA. The present disclosure contemplates any suitable means of inhibiting, blocking, or otherwise inactivating the DNA mismatch repair (MMR) system at the genetic level, including, but not limited to, inactivating one or more key proteins of the MMR system by introducing one or more mutations into genes encoding proteins of the MMR system, such as 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, EXO1, POLδ, and PCNA.

[0018] Thus, in one aspect, the present disclosure provides methods for editing nucleotide molecules (e.g., genomes) using prime editing while blocking, inhibiting, circumventing, or otherwise inactivating the DNA mismatch repair (MMR) system.

[0019] In another aspect, the present disclosure provides methods for editing a nucleotide molecule (e.g., a genome) using prime editing while blocking, inhibiting, bypassing, or otherwise inactivating proteins 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, EXO1, POLδ, and PCNA.

[0020] In yet another aspect, the present disclosure provides methods for editing a nucleotide molecule (e.g., a genome) using prime editing while blocking, inhibiting, avoiding, or otherwise inactivating MLH1 or a variant thereof.

[0021] In another aspect, the present disclosure provides methods for editing a nucleotide molecule (e.g., a genome) using prime editing while blocking, inhibiting, bypassing, or otherwise inactivating PMS2 (or MutL alpha) or a variant thereof.

[0022] In yet another aspect, the present disclosure provides methods for editing a nucleotide molecule (e.g., a genome) using prime editing while blocking, inhibiting, bypassing, or otherwise inactivating PMS1 (or MutL beta) or a variant thereof.

[0023] In yet another aspect, the present disclosure provides methods for editing a nucleotide molecule (e.g., a genome) using prime editing while blocking, inhibiting, bypassing, or otherwise inactivating MLH3 (or MutL gamma) or a variant thereof.

[0024] In another aspect, the present disclosure provides methods for editing a nucleotide molecule (e.g., a genome) using prime editing while blocking, inhibiting, bypassing, or otherwise inactivating MutS alpha (MSH2-MSH6) or a variant thereof.

[0025] In yet another aspect, the present disclosure provides methods for editing a nucleotide molecule (e.g., a genome) using prime editing while blocking, inhibiting, bypassing, or otherwise inactivating MSH2 or a variant thereof.

[0026] In another aspect, the present disclosure provides methods for editing a nucleotide molecule (e.g., a genome) using prime editing while blocking, inhibiting, avoiding, or otherwise inactivating MSH6 or a variant thereof.

[0027] In yet another aspect, the present disclosure provides methods for editing a nucleotide molecule (e.g., a genome) using prime editing while blocking, inhibiting, avoiding, or otherwise inactivating PCNA or a variant thereof.

[0028] In yet another aspect, the present disclosure provides methods for editing a nucleotide molecule (e.g., a genome) using prime editing while blocking, inhibiting, avoiding, or otherwise inactivating an RFC or a variant thereof.

[0029] In another aspect, the present disclosure provides methods for editing a nucleotide molecule (e.g., a genome) using prime editing while blocking, inhibiting, avoiding, or otherwise inactivating EXO1 or a variant thereof.

[0030] In yet another aspect, the present disclosure provides methods for editing a nucleotide molecule (e.g., a genome) using prime editing while blocking, inhibiting, avoiding, or otherwise inactivating POLδ or a variant thereof.

[0031] Thus, in one aspect, the present disclosure provides methods for editing nucleotide molecules (e.g., genomes) using prime editing while blocking, inhibiting, circumventing, or otherwise inactivating the DNA mismatch repair (MMR) system.

[0032] In another aspect, the present disclosure provides a method for circumventing MMR by installing one or more silent mutations near the intended editing site, thereby circumventing MMR and thereby improving the editing efficiency of prime editing. In various embodiments, the number of silent mutations installed can be one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more. Another silent mutation can be located upstream or downstream of the intended editing site (or in combination, if multiple silent mutations are involved), on the same or opposite DNA strand as the intended editing site (or in combination, if multiple silent mutations are involved). The silent mutation may be located upstream or downstream of the intended edit, 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 circumvention by silent mutation installation results in a significant increase in the editing efficiency of prime editing when one or more silent mutations are installed near the desired site for installing a genetic alteration by prime editing in the presence or absence of an inhibitor of MMR (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 5-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 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 increase).In various embodiments, the method of circumventing MMR by silent mutation installation results in a reduction in the frequency of prime-editing indel formation when one or more silent mutations are installed near a desired site for installing a genetic alteration by prime editing in the presence or absence of an inhibitor of MMR (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 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 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).

[0033] In another aspect, the disclosure provides a method for editing a nucleotide molecule (e.g., a genome), comprising contacting a target nucleotide molecule with a prime editing factor and an inhibitor of the MMR system, e.g., one or more inhibitors 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, EXO1, POLδ, 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 yet other embodiments, the inhibitor can be a variant of an MMR protein (e.g., a variant encoded by a dominant-negative mutant of a gene encoding an MMR protein that adversely affects the function or expression of a normal wild-type MMR protein, also referred to herein as a "dominant-negative mutant," "dominant-negative variant," or "dominant-negative protein," e.g., a "dominant-negative MMR protein"). In some embodiments, the inhibitor is a dominant-negative mutant of an MMR protein that inhibits the activity of a wild-type MMR protein. For example, the inhibitor can be one or more MLH1 protein variants (e.g., dominant-negative mutants) 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, EXO1, POLδ, or PCNA, e.g., a dominant-negative mutant of MLH1. In yet other embodiments, inhibitors may be targeted at the level of transcription, e.g., with siRNA or other nucleic acid agents that knock down the level of transcripts encoding 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, EXO1, POLδ, or PCNA.In yet other embodiments, the step of "contacting a target nucleotide molecule with a prime editor" may include (i) directly delivering to the cell an effective amount of a prime editor fusion protein (e.g., PE1 or PE2) complexed with a lipid delivery system; (ii) delivering to the cell an mRNA or delivery complex comprising an mRNA encoding the prime editor fusion protein and / or a suitable pegRNA; and (iii) a DNA vector (e.g., an AAV or lentiviral vector, a plasmid, or other nucleic acid delivery vector) encoding the prime editor fusion protein and / or a suitable pegRNA on one or more DNA vectors.

[0034] 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 MLH1 or a 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 editing factor 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 yet 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 the MLH1 transcript, e.g., an siRNA or other nucleic acid agent that knocks down the level of the MLH1 transcript, e.g., the transcript encoding MLH1. In yet other embodiments, the step of "contacting a target nucleotide molecule with a prime editor" may include (i) directly delivering to the cell an effective amount of a prime editor fusion protein (e.g., PE1 or PE2) complexed with a lipid delivery system; (ii) delivering to the cell an mRNA or delivery complex comprising an mRNA encoding the prime editor fusion protein and / or a suitable pegRNA; and (iii) a DNA vector (e.g., an AAV or lentiviral vector, a plasmid, or other nucleic acid delivery vector) encoding the prime editor fusion protein and / or a suitable pegRNA on one or more DNA vectors.

[0035] 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 PMS2 (or MutL alpha) or a 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 editing factor 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 yet 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 transcriptional level of PMS2 (or MutL alpha), e.g., an siRNA or other nucleic acid agent that knocks down the level of the transcript encoding ML PMS2 (or MutL alpha). In yet other embodiments, the step of "contacting a target nucleotide molecule with a prime editor" may include (i) directly delivering to the cell an effective amount of a prime editor fusion protein (e.g., PE1 or PE2) complexed with a lipid delivery system; (ii) delivering to the cell an mRNA or delivery complex comprising an mRNA encoding the prime editor fusion protein and / or a suitable pegRNA; and (iii) a DNA vector (e.g., an AAV or lentiviral vector, a plasmid, or other nucleic acid delivery vector) encoding the prime editor fusion protein and / or a suitable pegRNA on one or more DNA vectors.

[0036] 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 a 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 editing factor 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 yet 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 transcriptional level with an siRNA or other nucleic acid agent that knocks down the level of a transcript encoding PMS1 (or MutL beta), e.g., PMS1 (or MutL beta). In yet other embodiments, the step of "contacting a target nucleotide molecule with a prime editor" may include (i) directly delivering to the cell an effective amount of a prime editor fusion protein (e.g., PE1 or PE2) complexed with a lipid delivery system; (ii) delivering to the cell an mRNA or delivery complex comprising an mRNA encoding the prime editor fusion protein and / or a suitable pegRNA; and (iii) a DNA vector (e.g., an AAV or lentiviral vector, a plasmid, or other nucleic acid delivery vector) encoding the prime editor fusion protein and / or a suitable pegRNA on one or more DNA vectors.

[0037] 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 MLH3 (or MutL gamma) or a 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 editing factor 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 yet 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 transcriptional level with an siRNA or other nucleic acid agent that knocks down the level of a transcript encoding P MLH3 (or MutL gamma), e.g., MLH3 (or MutL gamma). In yet other embodiments, the step of "contacting a target nucleotide molecule with a prime editor" may include (i) directly delivering to the cell an effective amount of a prime editor fusion protein (e.g., PE1 or PE2) complexed with a lipid delivery system; (ii) delivering to the cell an mRNA or delivery complex comprising an mRNA encoding the prime editor fusion protein and / or a suitable pegRNA; and (iii) a DNA vector (e.g., an AAV or lentiviral vector, a plasmid, or other nucleic acid delivery vector) encoding the prime editor fusion protein and / or a suitable pegRNA on one or more DNA vectors.

[0038] In yet another aspect, the present disclosure provides methods for editing a nucleotide molecule (e.g., a genome) using prime editing while blocking, inhibiting, or otherwise inactivating MutS alpha (MSH2-MSH6) or a variant thereof. In another aspect, the present disclosure provides methods for editing a nucleotide molecule (e.g., a genome), comprising contacting a target nucleotide molecule with a prime editing factor 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 yet other embodiments, the inhibitor can be a dominant-negative mutant of MutS alpha (MSH2-MSH6). In yet other embodiments, the inhibitor may be targeted at the transcriptional level 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" may include (i) directly delivering to the cell an effective amount of a prime editor fusion protein (e.g., PE1 or PE2) complexed with a lipid delivery system, (ii) delivering to the cell an mRNA or delivery complex containing mRNA encoding the prime editor fusion protein and / or an appropriate pegRNA, and (iii) a DNA vector (e.g., an AAV or lentiviral vector, a plasmid, or other nucleic acid delivery vector) encoding the prime editor fusion protein and / or an appropriate pegRNA on one or more DNA vectors.

[0039] In yet another aspect, the present disclosure provides methods for editing a nucleotide molecule (e.g., a genome) using prime editing while blocking, inhibiting, or otherwise inactivating MSH2 or a variant thereof. In another aspect, the present disclosure provides methods 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 yet 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 the MSH2 transcript, e.g., with an siRNA or other nucleic acid agent that knocks down the level of the MSH2 transcript, e.g., the transcript encoding MSH2. In yet other embodiments, the step of "contacting a target nucleotide molecule with a prime editor" may include (i) directly delivering to the cell an effective amount of a prime editor fusion protein (e.g., PE1 or PE2) complexed with a lipid delivery system; (ii) delivering to the cell an mRNA or delivery complex comprising an mRNA encoding the prime editor fusion protein and / or a suitable pegRNA; and (iii) a DNA vector (e.g., an AAV or lentiviral vector, a plasmid, or other nucleic acid delivery vector) encoding the prime editor fusion protein and / or a suitable pegRNA on one or more DNA vectors.

[0040] In yet another aspect, the present disclosure provides methods for editing a nucleotide molecule (e.g., a genome) using prime editing while blocking, inhibiting, or otherwise inactivating MSH6 or a variant thereof. In another aspect, the present disclosure provides methods 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 yet 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 the MSH6 transcript, e.g., an siRNA or other nucleic acid agent that knocks down the level of the MSH6 transcript, e.g., the transcript encoding MSH6. In yet other embodiments, the step of "contacting a target nucleotide molecule with a prime editor" may include (i) directly delivering to the cell an effective amount of a prime editor fusion protein (e.g., PE1 or PE2) complexed with a lipid delivery system; (ii) delivering to the cell an mRNA or delivery complex comprising an mRNA encoding the prime editor fusion protein and / or a suitable pegRNA; and (iii) a DNA vector (e.g., an AAV or lentiviral vector, a plasmid, or other nucleic acid delivery vector) encoding the prime editor fusion protein and / or a suitable pegRNA on one or more DNA vectors.

[0041] In yet another aspect, the present disclosure provides methods for editing a nucleotide molecule (e.g., a genome) using prime editing while blocking, inhibiting, or otherwise inactivating PCNA or a variant thereof. In another aspect, the present disclosure provides methods 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 yet 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 the PCNA transcript, e.g., an siRNA or other nucleic acid agent that knocks down the level of the PCNA transcript, e.g., the transcript encoding PCNA. In yet other embodiments, the step of "contacting a target nucleotide molecule with a prime editor" may include (i) directly delivering to the cell an effective amount of a prime editor fusion protein (e.g., PE1 or PE2) complexed with a lipid delivery system; (ii) delivering to the cell an mRNA or delivery complex comprising an mRNA encoding the prime editor fusion protein and / or a suitable pegRNA; and (iii) a DNA vector (e.g., an AAV or lentiviral vector, a plasmid, or other nucleic acid delivery vector) encoding the prime editor fusion protein and / or a suitable pegRNA on one or more DNA vectors.

[0042] 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 the RFC or a 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 editing factor and an inhibitor of the 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 the RFC. In yet other embodiments, the inhibitor can be a dominant-negative mutant of the RFC. In still other embodiments, the inhibitor can be targeted at the level of the RFC transcript, e.g., an siRNA or other nucleic acid agent that knocks down the level of the RFC transcript, e.g., the transcript encoding the RFC. In yet other embodiments, the step of "contacting a target nucleotide molecule with a prime editor" may include (i) directly delivering to the cell an effective amount of a prime editor fusion protein (e.g., PE1 or PE2) complexed with a lipid delivery system; (ii) delivering to the cell an mRNA or delivery complex comprising an mRNA encoding the prime editor fusion protein and / or a suitable pegRNA; and (iii) a DNA vector (e.g., an AAV or lentiviral vector, a plasmid, or other nucleic acid delivery vector) encoding the prime editor fusion protein and / or a suitable pegRNA on one or more DNA vectors.

[0043] 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 EXO1 or a 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 editing factor and an inhibitor of EXO1. In various embodiments, the inhibitor can be a small molecule inhibitor. In other embodiments, the inhibitor can be an anti-EXO1 antibody, e.g., a neutralizing antibody that inactivates EXO1. In yet other embodiments, the inhibitor can be a dominant-negative mutant of EXO1. In still other embodiments, the inhibitor can be targeted at the level of the EXO1 transcript, e.g., an siRNA or other nucleic acid agent that knocks down the level of the EXO1 transcript, e.g., the transcript encoding EXO1. In yet other embodiments, the step of "contacting a target nucleotide molecule with a prime editor" may include (i) directly delivering to the cell an effective amount of a prime editor fusion protein (e.g., PE1 or PE2) complexed with a lipid delivery system; (ii) delivering to the cell an mRNA or delivery complex comprising an mRNA encoding the prime editor fusion protein and / or a suitable pegRNA; and (iii) a DNA vector (e.g., an AAV or lentiviral vector, a plasmid, or other nucleic acid delivery vector) encoding the prime editor fusion protein and / or a suitable pegRNA on one or more DNA vectors.

[0044] In yet another aspect, the present disclosure provides methods for editing a nucleotide molecule (e.g., a genome) using prime editing while blocking, inhibiting, or otherwise inactivating POLδ or a variant thereof. In another aspect, the present disclosure provides methods for editing a nucleotide molecule (e.g., a genome) comprising contacting a target nucleotide molecule with a prime editor and an inhibitor of POLδ. In various embodiments, the inhibitor can be a small molecule inhibitor. In other embodiments, the inhibitor can be an anti-POLδ antibody, e.g., a neutralizing antibody that inactivates POLδ. In yet other embodiments, the inhibitor can be a dominant-negative mutant of POLδ. In still other embodiments, the inhibitor can be targeted at the level of the POLδ transcript, e.g., an siRNA or other nucleic acid agent that knocks down the level of the POLδ transcript, e.g., the transcript encoding POLδ. In yet other embodiments, the step of "contacting a target nucleotide molecule with a prime editor" may include (i) directly delivering to the cell an effective amount of a prime editor fusion protein (e.g., PE1 or PE2) complexed with a lipid delivery system; (ii) delivering to the cell an mRNA or delivery complex comprising an mRNA encoding the prime editor fusion protein and / or a suitable pegRNA; and (iii) a DNA vector (e.g., an AAV or lentiviral vector, a plasmid, or other nucleic acid delivery vector) encoding the prime editor fusion protein and / or a suitable pegRNA on one or more DNA vectors.

[0045] In one aspect, the present disclosure provides methods for editing a nucleic acid molecule by prime editing. In some embodiments, the method includes contacting the nucleic acid molecule with a prime editor, a pegRNA, and an inhibitor of a DNA mismatch repair pathway, thereby installing one or more modifications to the nucleic acid molecule at a target site.

[0046] The method may increase the efficiency of prime editing and / or reduce 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 an inhibitor of the DNA mismatch repair pathway. In some embodiments, the frequency of indel formation is reduced 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 an inhibitor of the DNA mismatch repair pathway.

[0047] In some embodiments, the inhibitor of the DNA mismatch repair pathway inhibits one or more proteins in the DNA mismatch repair pathway. In some embodiments, the one or more proteins are 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, EXO1, POLδ, and PCNA. In particular embodiments, the one or more proteins is MLH1. In some embodiments, the MLH1 comprises the 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 100% (including 100%) sequence identity to SEQ ID NO:204.

[0048] The inhibitor utilized in the methods 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 (e.g., a dominant-negative variant of MLH1) that inhibits the activity of a wild-type MMR protein. In certain embodiments, the inhibitor is an antibody that inhibits the activity of one or more proteins in the DNA mismatch repair pathway. In some embodiments, the inhibitor is a small molecule that inhibits the activity of one or more proteins in 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 in the DNA mismatch repair pathway. In some embodiments, the inhibitor is a dominant-negative variant of MLH1 that inhibits MLH1.

[0049] In some embodiments, the dominant negative variants are (a) MLH1 E34A (SEQ ID NO: 222), (b) MLH1 Δ756 (SEQ ID NO: 208), (c) MLH1 Δ754-756 (SEQ ID NO: 209), (d) MLH1 E34A Δ754-756 (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 NLS SV40 (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) NLS SV40 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 100% (including 100%) sequence identity to MLH1 501-753 (SEQ ID NO: 223), or any of SEQ ID NOs: 208 to 213, 215, 216, 218, 222, or 223.

[0050] The prime editor 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, Cas12e, Cas12h, Cas12i, Cas12g, Cas12f (Cas14), Cas12f1, Cas12j (CasΦ), and Argonaute, optionally having nickase activity. In certain embodiments, the napDNAbp comprises the amino acid sequence of any one of SEQ ID NOs: 2, 4-67, or 99(PEmax), or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity to any one of SEQ ID NOs: 2, 4-67, or 99(PEmax). In certain embodiments, the napDNAbp comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 37 (e.g., napDNAbp of PE1 and PE2), or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity to 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 polymerase is a reverse transcriptase. In certain aspects, the reverse transcriptase comprises the amino acid sequence of any one of SEQ ID NOs: 69-98, or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity to any one of SEQ ID NOs: 69-98.

[0051] The napDNAbp and the polymerase of the prime editor may be linked together to form a fusion protein. In some embodiments, the napDNAbp and the polymerase of the prime editor are linked by a linker to form the fusion protein. In certain embodiments, the linker comprises the amino acid sequence of any one of SEQ ID NOs: 102, or 118-131, or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity to 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.

[0052] The components used in the methods (e.g., the prime editor, pegRNA, and / or the inhibitor of the DNA mismatch repair pathway) may be encoded on a DNA vector. In some embodiments, the prime editor, 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 an AAV or lentiviral DNA vector. In some embodiments, the AAV vector is serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0053] The prime editing factor utilized in the methods of the present disclosure may be further linked to additional components. In some embodiments, the prime editing factor as a fusion protein is further linked to an inhibitor of a DNA mismatch repair pathway by a second linker. In certain embodiments, the second linker is an autohydrolyzable linker. In certain embodiments, the second linker comprises the amino acid sequence of any one of SEQ ID NOs: 102, 118-131, or 233-236, or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity to 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.

[0054] 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 or 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; (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 include changing (1) a G:C base pair to a T:A base pair, (2) a G:C base pair to an A:T base pair, (3) a G:C base pair to a C:G base pair, (4) a T:A base pair to a G:C base pair, (5) a T:A base pair to an A:T base pair, (6) a T:A base pair to a C:G base pair, (7) a C:G base pair to a G:C base pair, (8) a C:G base pair to a T:A base pair, (9) a C:G base pair to an A:T base pair, (10) an A:T base pair to a T:A base pair, (11) an A:T base pair to a G:C base pair, or (12) an A:T base pair to a C:G base pair. In some embodiments, the one or more modifications comprise 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.

[0055] The methods of the present disclosure may be used to make corrections to one or more disease-associated genes. In some embodiments, the one or more modifications comprise 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; hypertension; 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; congenital pachyonychia; phenylketonuria; severe combined immunodeficiency; sickle cell disease; Smith-Lemli-Opitz syndrome; trinucleotide repeat disorders; prion diseases; and Tay-Sachs disease.

[0056] In another aspect, the present disclosure provides compositions for editing nucleic acid molecules by prime editing. In some embodiments, the compositions include a prime editor, a pegRNA, and an inhibitor of a DNA mismatch repair pathway, wherein the composition is capable of installing one or more modifications into the nucleic acid molecule at a target site.

[0057] The composition may increase the efficiency of prime editing and / or reduce 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 an inhibitor of the DNA mismatch repair pathway. In some embodiments, the frequency of indel formation is reduced 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 an inhibitor of the DNA mismatch repair pathway.

[0058] In some embodiments, the inhibitor of the DNA mismatch repair pathway inhibits one or more proteins in the DNA mismatch repair pathway. In some embodiments, the one or more proteins are 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, EXO1, POLδ, and PCNA. In particular embodiments, the one or more proteins is MLH1. In some embodiments, the MLH1 comprises the 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 100% (including 100%) sequence identity to SEQ ID NO:204.

[0059] 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 (e.g., a dominant-negative variant of MLH1) that inhibits the activity of a wild-type MMR protein. In certain embodiments, the inhibitor is an antibody that inhibits the activity of one or more proteins in the DNA mismatch repair pathway. In some embodiments, the inhibitor is a small molecule that inhibits the activity of one or more proteins in 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 in the DNA mismatch repair pathway. In some embodiments, the inhibitor is a dominant-negative variant of MLH1 that inhibits MLH1.

[0060] In some embodiments, the dominant negative variants are (a) MLH1 E34A (SEQ ID NO: 222), (b) MLH1 Δ756 (SEQ ID NO: 208), (c) MLH1 Δ754-756 (SEQ ID NO: 209), (d) MLH1 E34A Δ754-756 (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 NLS SV40 (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) NLS SV40 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 100% (including 100%) sequence identity to MLH1 501-753 (SEQ ID NO: 223), or any of SEQ ID NOs: 208 to 213, 215, 216, 218, 222, or 223.

[0061] The prime editor utilized in the compositions of the present disclosure comprises 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, Cas12f (Cas14), Cas12f1, Cas12j (CasΦ), and Argonaute, optionally having nickase activity. In certain embodiments, the napDNAbp comprises the amino acid sequence of any one of SEQ ID NOs: 2, 4-67, or 99(PEmax), or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity to any one of SEQ ID NOs: 2, 4-67, or 99(PEmax). In certain embodiments, the napDNAbp comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 37 (i.e., napDNAbp of PE1 and PE2), or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity to 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 aspects, the reverse transcriptase comprises the amino acid sequence of any one of SEQ ID NOs: 69-98, or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity to any one of SEQ ID NOs: 69-98.

[0062] The napDNAbp and the prime editor polymerase may be linked together to form a fusion protein. In some embodiments, the napDNAbp and the prime editor polymerase are linked by a linker to form the fusion protein. In certain embodiments, the linker comprises the amino acid sequence of any one of SEQ ID NOs: 102, 118-131, or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity to 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.

[0063] Components used in the compositions disclosed herein (e.g., a prime editor, a pegRNA, and / or an inhibitor of a DNA mismatch repair pathway) may be encoded on a DNA vector. In some embodiments, the prime editor, the pegRNA, and the inhibitor of a DNA mismatch repair pathway are encoded on one or more DNA vectors. In certain embodiments, the one or more DNA vectors comprise an AAV or lentiviral DNA vector. In some embodiments, the AAV vector is serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0064] The prime editing factor utilized in the compositions of the present disclosure may be further linked to additional components. In some embodiments, the prime editing factor as a fusion protein is further linked to an inhibitor of a DNA mismatch repair pathway by a second linker. In certain embodiments, the second linker is an autohydrolyzable linker. In certain embodiments, the second linker comprises the amino acid sequence of any one of SEQ ID NOs: 102, 118-131, or 233-236, or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity to 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.

[0065] 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 or 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; (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 include changing (1) a G:C base pair to a T:A base pair, (2) a G:C base pair to an A:T base pair, (3) a G:C base pair to a C:G base pair, (4) a T:A base pair to a G:C base pair, (5) a T:A base pair to an A:T base pair, (6) a T:A base pair to a C:G base pair, (7) a C:G base pair to a G:C base pair, (8) a C:G base pair to a T:A base pair, (9) a C:G base pair to an A:T base pair, (10) an A:T base pair to a T:A base pair, (11) an A:T base pair to a G:C base pair, or (12) an A:T base pair to a C:G base pair. In some embodiments, the one or more modifications comprise 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.

[0066] The compositions of the present disclosure may be used to make corrections to one or more disease-associated genes. In some embodiments, the one or more modifications comprise 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; hypertension; 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; congenital pachyonychia; phenylketonuria; severe combined immunodeficiency; sickle cell disease; Smith-Lemli-Opitz syndrome; trinucleotide repeat disorders; prion diseases; and Tay-Sachs disease.

[0067] In another aspect, the present disclosure provides a polynucleotide 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 a DNA mismatch repair pathway, wherein the napDNAbp and the polymerase are capable of installing one or more modifications at the DNA target site in the presence of a pegRNA.

[0068] The polynucleotide may increase the efficiency of prime editing and / or reduce 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 an inhibitor of the DNA mismatch repair pathway. In some embodiments, the frequency of indel formation is reduced 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 an inhibitor of the DNA mismatch repair pathway.

[0069] In some embodiments, the inhibitor of the DNA mismatch repair pathway inhibits one or more proteins in the DNA mismatch repair pathway. In some embodiments, the one or more proteins are 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, EXO1, POLδ, and PCNA. In particular embodiments, the one or more proteins is MLH1. In some embodiments, the MLH1 comprises the 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 100% (including 100%) sequence identity to SEQ ID NO:204.

[0070] 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 MMR protein (e.g., a dominant-negative variant of MLH1) that inhibits the activity of a wild-type MMR protein. In certain embodiments, the inhibitor is an antibody that inhibits the activity of one or more proteins in the DNA mismatch repair pathway. In some embodiments, the inhibitor is a small molecule that inhibits the activity of one or more proteins in 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 in the DNA mismatch repair pathway. In some embodiments, the inhibitor is a dominant-negative variant of MLH1 that inhibits MLH1.

[0071] In some embodiments, the dominant negative variants are (a) MLH1 E34A (SEQ ID NO: 222), (b) MLH1 Δ756 (SEQ ID NO: 208), (c) MLH1 Δ754-756 (SEQ ID NO: 209), (d) MLH1 E34A Δ754-756 (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 NLS SV40 (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) NLS SV40 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 100% (including 100%) sequence identity to MLH1 501-753 (SEQ ID NO: 223), or any of SEQ ID NOs: 208 to 213, 215, 216, 218, 222, or 223.

[0072] Prime editors utilized in the polynucleotides of the present disclosure include 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 variants 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, Cas12f (Cas14), Cas12f1, Cas12j (CasΦ), and Argonaute, optionally having nickase activity. In certain embodiments, the napDNAbp comprises the amino acid sequence of any one of SEQ ID NOs: 2, 4-67, or 99(PEmax), or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity to any one of SEQ ID NOs: 2, 4-67, or 99(PEmax). In certain embodiments, the napDNAbp comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 37 (i.e., napDNAbp of PE1 and PE2), or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity to 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 aspects, the reverse transcriptase comprises the amino acid sequence of any one of SEQ ID NOs: 69-98, or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity to any one of SEQ ID NOs: 69-98.

[0073] The napDNAbp and the prime editor polymerase may be linked together to form a fusion protein. In some embodiments, the napDNAbp and the prime editor polymerase are linked by a linker to form the fusion protein. In certain embodiments, the linker comprises the amino acid sequence of any one of SEQ ID NOs: 102, 118-131, or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity to 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.

[0074] The polynucleotides disclosed herein may comprise vectors. In some embodiments, the polynucleotides are DNA vectors. In certain embodiments, the DNA vectors are AAV or lentivirus DNA vectors. In some embodiments, the AAV vectors are serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0075] The prime editing factor encoded by the polynucleotides disclosed herein may also be further linked to additional components. In some embodiments, the prime editing factor as a fusion protein is further linked to an inhibitor of a DNA mismatch repair pathway by a second linker. In certain embodiments, the second linker comprises an autohydrolyzable linker. In certain embodiments, the second linker comprises the amino acid sequence of any one of SEQ ID NOs: 102, 118-131, or 233-236, or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity to 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.

[0076] 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 or 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; (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 include changing (1) a G:C base pair to a T:A base pair, (2) a G:C base pair to an A:T base pair, (3) a G:C base pair to a C:G base pair, (4) a T:A base pair to a G:C base pair, (5) a T:A base pair to an A:T base pair, (6) a T:A base pair to a C:G base pair, (7) a C:G base pair to a G:C base pair, (8) a C:G base pair to a T:A base pair, (9) a C:G base pair to an A:T base pair, (10) an A:T base pair to a T:A base pair, (11) an A:T base pair to a G:C base pair, or (12) an A:T base pair to a C:G base pair. In some embodiments, the one or more modifications comprise 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.

[0077] The polynucleotides of the present disclosure may be used to make corrections to one or more disease-associated genes. In some embodiments, the one or more modifications comprise 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; hypertension; 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; congenital pachyonychia; phenylketonuria; severe combined immunodeficiency; sickle cell disease; Smith-Lemli-Opitz syndrome; trinucleotide repeat disorders; prion diseases; and Tay-Sachs disease.

[0078] In another aspect, the disclosure provides a cell. In some embodiments, the cell comprises any of the polynucleotides described herein.

[0079] In another aspect, the present disclosure provides pharmaceutical compositions. In some aspects, the pharmaceutical composition comprises any of the compositions disclosed herein. In some aspects, the pharmaceutical composition comprises any of the compositions disclosed herein and a pharmaceutically acceptable excipient. In some aspects, the pharmaceutical composition comprises any of the polynucleotides disclosed herein. In some aspects, the pharmaceutical composition comprises any of the polynucleotides disclosed herein and a pharmaceutically acceptable excipient.

[0080] In another aspect, the present disclosure provides kits. In some embodiments, the kits include any of the compositions disclosed herein, a pharmaceutical excipient, and instructions for editing a DNA target site by prime editing. In some embodiments, the kits include any of the polynucleotides disclosed herein, a pharmaceutical excipient, and instructions for editing a DNA target site by prime editing.

[0081] The present disclosure also provides a method and pegRNA for prime editing that prevents changes introduced into a target nucleic acid molecule from being corrected by the MMR pathway without the need to provide an inhibitor of the MMR pathway. Surprisingly, pegRNAs designed with consecutive nucleotide mismatches compared to the endogenous sequence of the target site on the target nucleic acid, for example, pegRNAs with three or more consecutive mismatches, can avoid correction by the MMR pathway, resulting in increased prime editing efficiency and reduced indel formation compared to the introduction of a single nucleotide mismatch using prime editing. In addition, consecutive nucleotide insertions or deletions at the target site of the target nucleic acid introduced by prime editing, for example, insertions or deletions longer than 10 nucleotides, also avoid correction by the MMR pathway, resulting in increased prime editing efficiency and reduced indel formation compared to the introduction of insertions or deletions shorter than 10 nucleotides using prime editing.

[0082] Thus, in another aspect, the present disclosure provides a method for editing a nucleic acid molecule by prime editing, the method comprising contacting the nucleic acid molecule with a prime editor (e.g., PE2, PE3, or any of the other prime editors described herein) and contacting a pegRNA with a DNA synthesis template on its extension arm that contains three or more consecutive nucleotide mismatches compared 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 a change 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 mutation may be in a coding region of the target nucleic acid molecule (i.e., a portion of a gene that encodes a protein), or the silent mutation may be in a non-coding region of the target nucleic acid molecule. In some embodiments, if the silent mutation is in a coding region, the silent mutation introduces one or more alternative codons into the nucleic acid molecule that encode the same amino acid as in the unedited nucleic acid molecule. In some embodiments, when the silent mutation is in a non-coding region, the silent mutation is in a region of the nucleic acid molecule that does not affect splicing, gene regulation, RNA lifespan, or other biological properties of the target site on the nucleic acid molecule.

[0083] To achieve the benefit of avoiding correction by the MMR pathway, thereby increasing prime editing efficiency and / or reducing indel formation, any number of consecutive nucleotide mismatches can be designed into the DNA synthesis template of the pegRNA compared to the sequence of the target site. In some embodiments, the DNA synthesis template contains 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 contains 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 with the endogenous sequence of the 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 compared to the endogenous sequence of the target site on the nucleic acid molecule. In certain embodiments, the use of three or more consecutive nucleotide mismatches results in an 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 increase in prime editing efficiency compared to methods using a pegRNA comprising a DNA synthesis template containing only one consecutive nucleotide mismatch compared to the endogenous sequence of the target site on the nucleic acid molecule.In certain embodiments, the use of three or more consecutive nucleotide mismatches results in at least a 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 decrease in the frequency of indel formation compared to methods using pegRNA comprising a DNA synthesis template containing only one consecutive nucleotide mismatch compared to the endogenous sequence of the target site on a nucleic acid molecule.

[0084] In another aspect, the present disclosure provides a method for editing a nucleic acid molecule by prime editing, the method comprising contacting the nucleic acid molecule with a prime editor (e.g., PE2, PE3, or any of the other prime editors described herein) and contacting a pegRNA with a DNA synthesis template on its extension arm that contains an insertion or deletion of 10 or more consecutive nucleotides relative to the endogenous sequence of a target site on the nucleic acid molecule. In some embodiments, the DNA synthesis template for the pegRNA can be designed to introduce an insertion or deletion of more than 3 nucleotides to avoid or reduce the effects of mismatch correction by the cellular MMR pathway, thereby improving prime editing efficiency. In some embodiments, the DNA synthesis template for the pegRNA is designed to introduce one or more insertions and / or deletions of 3, 4, 5, 6, 7, 8, 9, 10, or more consecutive nucleotides to avoid or reduce the effects of mismatch correction by the cellular MMR pathway, thereby improving prime editing efficiency. In some embodiments, insertions or deletions of any length of more than 10 consecutive nucleotides can be used to achieve the advantage of avoiding 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 consecutive nucleotides relative to the endogenous sequence of the target site on the 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 consecutive nucleotides relative to the endogenous sequence of the target site on the nucleic acid molecule edited by prime editing. In some embodiments, the DNA synthesis template includes 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 consecutive nucleotides relative to the endogenous sequence of the target site on the 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 consecutive nucleotides relative to the endogenous sequence of the target site on the nucleic acid molecule edited by prime editing. In some embodiments, the DNA synthesis template comprises an insertion or deletion of 11 or more consecutive nucleotides, 12 or more consecutive nucleotides, 13 or more consecutive nucleotides, 14 or more consecutive nucleotides, 15 or more consecutive nucleotides, 16 or more consecutive nucleotides, 17 or more consecutive nucleotides, 18 or more consecutive nucleotides, 19 or more consecutive nucleotides, 20 or more consecutive nucleotides, 21 or more consecutive nucleotides, 22 or more consecutive nucleotides, 23 or more consecutive nucleotides, 24 or more consecutive nucleotides, or 25 or more consecutive nucleotides relative to the target site on the nucleic acid molecule. In certain embodiments, the DNA synthesis template comprises an insertion or deletion of 15 or more consecutive nucleotides relative to the endogenous sequence of the target site on the nucleic acid molecule.

[0085] In some embodiments, prime editing with a pegRNA designed to introduce an insertion and / or deletion of multiple consecutive nucleotides, e.g., three or more consecutive nucleotides, relative to the endogenous sequence of the target site results in an increase in prime editing efficiency of 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 prime editing with a corresponding control pegRNA (e.g., a control pegRNA that does not introduce an insertion or deletion of three or more consecutive nucleotides). 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 consecutive nucleotides relative to the endogenous sequence of the target site results in at least a 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 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 consecutive nucleotides).In some embodiments, generating an insertion or deletion of 10 or more consecutive nucleotides results in an 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 increase in primed editing efficiency compared to methods using pegRNA comprising a DNA synthesis template that comprises an insertion or deletion of less than 10 nucleotides relative to the endogenous sequence of the target site on a nucleic acid molecule. In some embodiments, creating an insertion or deletion of 10 or more nucleotides results in at least a 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 decrease in indel formation frequency compared to methods using pegRNA comprising a DNA synthesis template that comprises an insertion or deletion of less than 10 nucleotides relative to the endogenous sequence of the target site on a nucleic acid molecule.

[0086] In another aspect, the present disclosure also provides pegRNAs useful for editing nucleic acid molecules by prime editing while avoiding correction of the introduced changes in the nucleic acid molecule by the MMR pathway, thereby increasing prime editing efficiency and / or reducing indel formation. In some embodiments, the extended arm of a pegRNA provided by the present disclosure comprises three or more consecutive nucleotide mismatches with the endogenous sequence of a target site on a nucleic acid molecule. In some embodiments, at least one of the three consecutive nucleotide mismatches with the endogenous sequence of the target site is a silent mutation. In some embodiments, at least one of the consecutive nucleotide mismatches results in a change 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 mutation may be in a coding region of the target nucleic acid molecule (i.e., a portion of a gene that encodes a protein) or in a non-coding region of the target nucleic acid molecule. In some embodiments, if the silent mutation is in a coding region, the silent mutation introduces one or more alternative codons into the nucleic acid molecule that encode the same amino acid as in the unedited nucleic acid molecule. In some embodiments, when the silent mutation is in a non-coding region, the silent mutation is in a region of the nucleic acid molecule that does not affect splicing, gene regulation, RNA lifespan, or other biological properties of the target site on the nucleic acid molecule.

[0087] To achieve the advantage of avoiding correction by the MMR pathway, any number of consecutive nucleotide mismatches, three or more, can be incorporated into the extension arm of the pegRNA described herein. In some embodiments, the DNA synthesis template of the extension arm of the pegRNA contains 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 with the endogenous sequence of the 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 contains at least three consecutive nucleotide mismatches with the endogenous sequence of the target site on the nucleic acid molecule edited by prime editing. In some embodiments, the DNA synthesis template for the extension arm of the pegRNA contains 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 the target site on the nucleic acid molecule edited by prime editing. In some embodiments, the DNA synthesis template for the extension arm of the pegRNA contains 3, 4, 5, 6, 7, 8, 9, or 10 consecutive nucleotide mismatches relative to the endogenous sequence of the target site on the nucleic acid molecule edited by prime editing. In some embodiments, the DNA synthesis template for the extension arm of the pegRNA contains 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more consecutive nucleotide mismatches relative to the endogenous sequence of the 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 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 increase in primed editing efficiency compared to a pegRNA comprising a DNA synthesis template containing only one consecutive nucleotide mismatch relative to the endogenous sequence of the target site on a nucleic acid molecule. In certain embodiments, the use of three or more consecutive nucleotide mismatches results in at least a 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 decrease in the frequency of indel formation compared to a pegRNA comprising a DNA synthesis template containing only one consecutive nucleotide mismatch compared to the endogenous sequence of the target site on a nucleic acid molecule.

[0088] 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 a 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, EXO1, POLδ, and / or PCNA). In some embodiments, the one or more proteins is MLH1. In certain aspects, MLH1 comprises the 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 100% (including 100%) sequence identity to SEQ ID NO:204.

[0089] Any inhibitor of the DNA mismatch repair pathway may be used in the system described herein. In some embodiments, the inhibitor is an antibody that inhibits the activity of one or more proteins in the DNA mismatch repair pathway. In some embodiments, the inhibitor is a small molecule that inhibits the activity of one or more proteins in 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 in 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).

[0090] In certain aspects, the dominant negative variants used in the systems of the present disclosure are (a) MLH1 E34A (SEQ ID NO:222), (b) MLH1 Δ756 (SEQ ID NO:208), (c) MLH1 Δ754-756 (SEQ ID NO:209), (d) MLH1 E34A Δ754-756 (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 501-756 (SEQ ID NO:215), (i) MLH1 501-753 (SEQ ID NO:216), (j) MLH1 461-753 (SEQ ID NO:218), or (k) NLSSV40 MLH1 501-753 (SEQ ID NO: 223), or any of SEQ ID NOs: 208-213, 215, 216, 218, 222, or 223. The present disclosure also contemplates methods for performing primed editing on nucleic acid molecules in cells in which MMR activity has been completely knocked out (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 reduced indel formation) without the need to provide an inhibitor of MMR.

[0010] Accordingly, in another aspect, the present disclosure provides methods for editing a nucleic acid molecule by prime editing, the methods comprising contacting the 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 methods further comprise contacting the nucleic acid molecule with a second-strand nicking gRNA.In certain aspects, 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 as compared to methods performed in cells that do not comprise a knockout of one or more genes involving MMR. In certain aspects, the frequency of indel formation is reduced 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 as compared to methods performed in cells that do not include a knockout of one or more genes involved in MMR. In some embodiments, the one or more genes involved in MMR are 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, EXO1, POLδ, and PCNA. In particular embodiments, the one or more genes is a gene encoding MLH1 (e.g., comprising the 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 100% (including 100%) sequence identity to SEQ ID NO:204).

[0091] In another aspect, the disclosure provides methods for editing a nucleic acid molecule by prime editing, the methods comprising contacting the 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. In some embodiments, the methods further comprise contacting the nucleic acid molecule with a second-strand nicking gRNA.

[0092] In some embodiments, the prime editing efficiency is 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 some embodiments, the frequency of indel formation is 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.

[0093] In some embodiments, the inhibitor of p53 is a protein. In certain embodiments, the inhibitor of p53 is the protein i53. 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.

[0094] In another aspect, the present disclosure describes improved prime editor fusion proteins that include PEmax of SEQ ID NO: 99. The present disclosure also contemplates fusion proteins having an amino acid sequence having 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% sequence identity to SEQ ID NO:99.

[0095] The inventors have surprisingly found that the editing efficiency of prime editing can 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 or more increase) when one or more components of a canonical prime editor fusion protein (i.e., PE2) are modified. Modifications can include modified amino acid sequences of one or more components (e.g., the Cas9 component, the reverse transcriptase component, or the linker).

[0096] In other aspects, the disclosure also provides compositions and pharmaceutical compositions including PEmax, methods of prime editing using PEmax, polynucleotides and vectors encoding PEmax, and kits and cells comprising PEmax.

[0097] It should be understood that the foregoing concepts, and additional concepts described below, may be arranged in any suitable combination, as the disclosure is not limited in this respect. Furthermore, other advantages and novel features of the present disclosure will become apparent from the following detailed description of various non-limiting embodiments when considered in conjunction with the accompanying drawings.

[0098] The following drawings form part of this specification and are included to further demonstrate certain aspects of the disclosure that may be better understood by reference to one or more of these drawings in combination with the detailed description of certain aspects presented herein. [Brief explanation of the drawings]

[0099] [Figure 1]

[0023] Figure 1 provides a schematic showing that prime editing enables guide RNA-templated genome engineering. DNA prime editing intermediates that can be repaired by cellular factors are indicated by boxes.

[0100] [Figure 2] A schematic diagram of DNA repair CRISPRi screening for prime editing outcomes is provided.

[0101] [Figure 3A] Figure 3A shows the optimization of prime editing efficiency at target sites. Figure 3A provides a schematic of the optimization process. [Figure 3B] Figure 3B shows the % of reads with specific modifications at the target site in HeLa cells. [Figure 3C] Figure 3C shows the % of reads with specific modifications at the target site by blasticidin selection in HeLa cells.

[0102] [Figure 4A] Figure 4A shows primed-editing CRISPRi screening using a DNA repair library. Figure 4A provides a schematic of the screening process. [Figure 4B]Figure 4B shows the % of reads with specific modifications in bulk editing of post-screening HeLa cells.

[0103] [Figure 5A] We show that CRISPRi screening reveals that DNA mismatch repair limits prime editing efficiency. Knockdown of mismatch repair proteins (MSH2, MSH6, PMS2, and MLH1) improves the efficiency of PE2 by 3-fold and PE3 by 2-fold. [Figure 5B] We show that CRISPRi screening reveals that DNA mismatch repair limits prime editing efficiency. Knockdown of mismatch repair proteins (MSH2, MSH6, PMS2, and MLH1) improves the efficiency of PE2 by 3-fold and PE3 by 2-fold.

[0104] [Figure 6A] We 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. [Figure 6B] We 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. [Figure 6C] We 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.

[0105] [Figure 7A] We show that complete MMR knockout dramatically improves prime editing, and that in the absence of MMR, PE2 editing efficiency matches that of PE3. [Figure 7B]We show that complete MMR knockout dramatically improves prime editing, and that in the absence of MMR, PE2 editing efficiency matches that of PE3.

[0106] [Figure 8A] A schematic diagram of the mechanism of mismatch repair (MMR) is provided. In the first step, MSH2:MSH6 (MutSα) binds the mismatch and recruits MLH1:PMS2 (MutLα). The DNA nick signals MMR which strand to repair. In the second step, MutLα indiscriminately cleaves the nicked strand 5' and 3' of the mismatch. In the third step, EXO1 excises the mismatch from the nick generated by MutLα. In the fourth step, POLδ resynthesizes the excised strand, followed by LIG1 ligation.

[0107] [Figure 8B]This figure provides another schematic of the mismatch repair mechanism MMR in eukaryotic cells. The left side of the schematic shows the 5' MMR. (A) MutS homolog proteins (MSH, purple) MutSα (MSH2-MSH6) or MutSβ (MSH2-MSH3) recognize and bind to the mismatch. RPA bound to single-stranded DNA prevents EXO1 from accessing and degrading the DNA. (B) In the sliding clamp model, the mismatched MutSα / β binds ATP, activates a nucleotide switch, and becomes a sliding clamp that diffuses along the DNA. Multiple MSH clamps are loaded at a single mismatch. Interaction of EXO1 with the MSH sliding clamp overcomes the RPA barrier and activates EXO1 for 5' to 3' resection from the 5' nick. MutL homolog proteins (MLHs) (MutLα is ScMlh1-Pms1 or HsMLH1-PMS2) bind ATP and may interact with the MSH sliding clamp, but MLHs are not absolutely required for 5' MMR in vitro. In other models, MSHs may remain mismatched to allow resection, or they may load multiple MLH clamps onto the DNA near the mismatch (not shown). (C) In the sliding clamp model, the EXO1 / MSH complex dissociates after excising several hundred nucleotides. Recursive rounds of MSH-EXO1 resection create an RPA-coated resection path that extends from the 5' nick to just beyond the mismatch. MLHs may limit resection by regulating the number of MSH clamps on the DNA. (D) RFC (not shown) loads a PCNA clamp in a specific orientation onto the 3' end of the strand break or gap, and PCNA facilitates high-fidelity DNA synthesis by Polδ or ε. (E) DNA ligase I seals the nick. The right side of the schematic shows the 3' MMR. (A) MSH recognizes the mismatch. (B) In the sliding clamp model, ATP-dependent binding and nucleotide switching create an MSH sliding clamp that diffuses away from the mismatch. Interaction of the ATP-bound MLH heterodimer with the MSH sliding clamp and PCNA oriented toward the 3' end activates MLH chain-specific nicking.Alternatively, ATP-activated MSH may remain mismatched to load MLH and activate nicking (not shown). (C) Resection can be EXO1-dependent or -independent, resulting in an RPA-coated resection track. The EXO1-independent Polδ strand displacement pathway is not shown. (D) Polδ or ε with PCNA completes gap filling. (E) DNA ligase I seals the nick.

[0108] [Figure 9A] 1 provides a schematic of mismatch repair of the PE2 intermediate. MMR inhibition provides additional time for flap ligation and removes strand discrimination signals for heteroduplex repair. [Figure 9B] 1 provides a schematic of mismatch repair of the PE2 intermediate. MMR inhibition provides additional time for flap ligation and removes strand discrimination signals for heteroduplex repair. [Figure 9C] 1 provides a schematic of mismatch repair of the PE2 intermediate. MMR inhibition provides additional time for flap ligation and removes strand discrimination signals for heteroduplex repair.

[0109] [Figure 10] We demonstrate that expression of a dominant-negative MLH1 mutant enhances PE2 efficiency. The MLH1 dominant-negative mutant improves PE2 efficiency by 2-4 fold. RNF2+3 G to C does not respond to MMR inhibition.

[0110] [Figure 11A] Figure 1 shows the effect of MLH1 mutants on PE3. The MLH1 mutant reduces PE3 indels by half. [Figure 11B] Figure 1 shows the effect of MLH1 mutants on PE3. The MLH1 mutant reduces PE3 indels by half.

[0111] [Figure 12A]The improvement of the MLH1 mutant translates to other sites: Figure 12A shows that PE2 editing efficiency is increased in the MLH1 mutant, and only RNF2+3 G to C is resistant to MMR inhibition. [Figure 12B] FIG. 12B shows that the MLH1 mutant reduces the occurrence of indels by half.

[0112] [Figure 13] 1 provides a schematic showing mismatch repair of the PE3 intermediate.

[0113] [Figure 14]

[0023] Figure 1 provides a schematic showing that mismatch repair differentially resolves PE3 intermediates, with one editing-preferred intermediate being required for mismatch repair.

[0114] [Figure 15A] Screening of MLH1 mutants for smaller size and improved activity is shown. Figure 15A shows that MLH1 Δ754-756 most strongly promotes PE2 editing (hereafter referred to as MLH1dn). The MLH1 N-terminal domain approaches the efficacy of MLH1dn (hereafter referred to as MLH1dnNTD). MLH1 dominant-negative mutants may function by saturating MutS binding. [Figure 15B] Figure 15B shows that the MLH1 N-terminal domain plus NLS approaches the activity of MLH1neg. [Figure 15C] Figure 15C shows that MLH1dn fusion to PE via a self-cleaving P2A linker (PE-2A-MLH1dn) can improve prime editing efficiency. [Figure 15D] Figures 15D-F show that MMR KD phenocopies MLH1neg expression. [Figure 15E] Figures 15D-F show that MMR KD phenocopies MLH1neg expression. [Figure 15F] Figures 15D-F show that MMR KD phenocopies MLH1neg expression. [Figure 15G] Figures 15G-15H 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. [Figure 15H] Figures 15G-15H 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.

[0115] [Figure 16] This shows that MLH1dn reduces PE3 indels. A silent pegRNA is a pegRNA that does not encode editing or generate mismatches. MLH1dn only reduces PE3 indels when mismatches are generated.

[0116] [Figure 17] We show that mismatch repair of PE heteroduplexes generates diffuse indel patterns. These edits result in a broad distribution of indels in PE3, whereas inhibition of MMR with MLH1dn narrows the distribution. This suggests that MMR performs incision after mismatch recognition that contributes to the indels generated by PE3.

[0117] [Figure 18] PE3 mismatch repair intermediates are shown.

[0118] [Figure 19A] Figure 1 shows that MMR excision of the targeted locus generates an indel in PE3. [Figure 19B] Figure 1 shows that MMR excision of the targeted locus generates an indel in PE3.

[0119] [Figure 20A] We show that MMR knockdown or knockout does not affect the RNF2+3 G to C, suggesting that either the RNF2 site is not repaired by MMR or the resulting C:C mismatch is not repaired by MMR. [Figure 20B] We show that MMR knockdown or knockout does not affect the RNF2+3 G to C, suggesting that either the RNF2 site is not repaired by MMR or the resulting C:C mismatch is not repaired by MMR.

[0120] [Figure 21A] This shows that MLH1dn can be used to improve other substitution edits in RNF2. [Figure 21B] This shows that MLH1dn can be used to improve other substitution edits in RNF2. [Figure 21C] This shows that MLH1dn can be used to improve other substitution edits in RNF2.

[0121] [Figure 22A] Figure 1 shows that MLH1dn improves substitution editing at other sites, including HEK3. MLH1dn strongly enhances PE2 editing and reduces PE3 indels. [Figure 22B] Figure 1 shows that MLH1dn improves substitution editing at other sites, including HEK3. MLH1dn strongly enhances PE2 editing and reduces PE3 indels.

[0122] [Figure 23A] Figure 1 shows that MLH1dn improves substitution editing at other sites, including FANCF. MLH1dn strongly enhances PE2 editing and reduces PE3 indels. [Figure 23B] Figure 1 shows that MLH1dn improves substitution editing at other sites, including FANCF. MLH1dn strongly enhances PE2 editing and reduces PE3 indels. [Figure 23C] Figure 1 shows that MLH1dn improves substitution editing at other sites, including FANCF. MLH1dn strongly enhances PE2 editing and reduces PE3 indels. [Figure 23D]Figure 1 shows that MLH1dn improves substitution editing at other sites, including FANCF. MLH1dn strongly enhances PE2 editing and reduces PE3 indels.

[0123] [Figure 24A] Figure 24A shows that PE improvement by MHL1dn is mismatch dependent. MLH1dn increases PE2 editing by an average of 2-fold in HEK293T cells. Figure 24B shows that G to C editing (C:C mismatch) is not affected by MMR in HEK293T cells. This suggests that G to C editing has a higher baseline efficiency than other substitutions. [Figure 24B] Figure 24B shows a substantial increase in the ratio of edit:indel purity from MLH1dn used with PE3, which is also mismatch dependent.

[0124] [Figure 25A] We show that MLH1dn also improves the efficiency of small insertion and deletion editing: MMR is known to repair insertions and deletions less than 15 nucleotides in length. [Figure 25B] We show that MLH1dn also improves the efficiency of small insertion and deletion editing: MMR is known to repair insertions and deletions less than 15 nucleotides in length. [Figure 25C] We show that MLH1dn also improves the efficiency of small insertion and deletion editing: MMR is known to repair insertions and deletions less than 15 nucleotides in length. [Figure 25D] We show that MLH1dn also improves the efficiency of small insertion and deletion editing: MMR is known to repair insertions and deletions less than 15 nucleotides in length.

[0125] [Figure 26A] Figure 1 shows that MLH1dn reduced the incorporation of the pegRNA scaffold. Scaffold incorporation events at these sites occur via double-strand break (DSB) intermediates. [Figure 26B]Figure 1 shows that MLH1dn reduced the incorporation of the pegRNA scaffold. Scaffold incorporation events at these sites occur via double-strand break (DSB) intermediates.

[0126] [Figure 27] Figure 1 shows that MLH1dn does not promote substantial PE off-target editing. A slight increase in off-target (OT) editing was observed at HEK4 off-target site 3.

[0127] [Figure 28A] We show that MLH1dn does not induce detectable microsatellite instability at the biomarker locus. MMR inhibition is known to cause shortening of homopolymeric microsatellite regions. [Figure 28B] We show that MLH1dn does not induce detectable microsatellite instability at the biomarker locus. MMR inhibition is known to cause shortening of homopolymeric microsatellite regions.

[0128] [Figure 29] We show that MLH1dn provides a method to enhance prime editing efficiency at sites lacking good ngRNA, such as HEK4.

[0129] [Figure 30] We show that MLH1dn ameliorates PE at the disease site.

[0130] [Figure 31] We show that MLH1dn enhances the installation of the protective APOE Christchurch allele in mouse astrocytes, demonstrating a 50% improvement in editing efficiency and a significant reduction in indels.

[0131] [Figure 32] This indicates that HEK293T cells are impaired in MMR. The MLH1 promoter is hypermethylated in HEK293T cells, resulting in reduced MLH1 expression.

[0132] [Figure 33A] Figure 33A shows prime editing by PE2. [Figure 33B] Figure 33B shows prime editing by PE3.

[0133] [Figure 34A] Figure 34A shows PRNP+6 G to T editing. [Figure 34B] Figure 34B shows APOE +6 G to T and +10 C to A edits.

[0134] [Figure 35A] This shows that MLH1dn has a greater effect in MMR-competent cell lines such as HeLa. [Figure 35B] This shows that MLH1dn has a greater effect in MMR-competent cell lines such as HeLa.

[0135] [Figure 36A] Figure 1 shows that the improvement of MLH1dn synergizes with stabilized pegRNA. [Figure 36B] Figure 1 shows that the improvement of MLH1dn synergizes with stabilized pegRNA. [Figure 36C] Figure 1 shows that the improvement of MLH1dn synergizes with stabilized pegRNA. [Figure 36D] Figure 1 shows that the improvement of MLH1dn synergizes with stabilized pegRNA.

[0136] [Figure 37A] We demonstrate that sequential substitutions are useful as an alternative strategy to evade MMR. [Figure 37B] We demonstrate that sequential substitutions are useful as an alternative strategy to evade MMR.

[0137] [Figure 38] We show that MMR does not efficiently repair three or more consecutive substitutions, thus providing a way to circumvent MMR and increase PE efficiency.

[0138] [Figure 39A] 1 shows that MLH1neg ameliorates PE in HeLa cells. [Figure 39B] Figure 1 shows that MLH1neg ameliorates PE in HeLa cells. [Figure 39C] Figure 1 shows that MLH1neg ameliorates PE in HeLa cells.

[0139] [Figure 40A] Pooled Repair-seq CRISPRi screening reveals genetic determinants of substitutions for prime editing outcomes. Figure 40A shows that prime editing by the PE2 system is mediated by the PE2 enzyme (Streptococcus pyogenes Cas9 (SpCas9) H840A nickase fused to reverse transcriptase) and a prime editing guide RNA (pegRNA). The PE3 system uses an additional single guide RNA (sgRNA) to nick the unedited strand and achieve higher editing efficiency. PBS, primer binding site. RT template, reverse transcription template. [Figure 40B] Figure 40B provides an overview of the prime-editing Repair-seq CRISPRi screen. A library of CRISPRi sgRNAs and pre-validated prime editing sites are transduced into a CRISPRi cell line and transfected with prime editing factors targeting the editing sites. The CRISPRi sgRNA identities and prime editing sites are amplified together from genomic DNA and paired-end sequenced to link each gene perturbation to an editing outcome. SaCas9, Staphylococcus aureus Cas9. [Figure 40C-40D]Figure 40C shows the effect of each CRISPRi sgRNA on the percentage of sequencing reads reporting the intended G·C to C·G prime edit at the target editing site in the pooled CRISPRi screen. Each value represents all sequencing reads with the same CRISPRi sgRNA. Figure 40D shows the effect of CRISPRi sgRNA on editing efficiency across all screening conditions. Black dots represent individual non-targeting sgRNAs, the black line represents the average across all non-targeting sgRNAs, and the gray shading represents a kernel density estimate of the distribution of all sgRNAs. [Figure 40E] Figures 40E-40G show a comparison of the gene-level impact of CRISPRi targeting on intended G·C to C·G prime editing across different screening conditions. (Figure 40E) K562 PE2 vs. HeLa PE2. (Figure 40F) K562 PE3+50 vs. HeLa PE3+50. (Figure 40G) K562 PE2 vs. K562 PE3+50. The effect of each gene is calculated as the average log2 fold change in frequency from a non-targeting sgRNA for the two most extreme sgRNAs targeting the gene. Plotted quantities are the average of n = 2 independent biological replicates for each cell type, and bars indicate the range of values ​​spanned by replicate. Black dots represent 20 random sets of three non-targeting sgRNAs. [Figure 40F] Figures 40E-40G show a comparison of the gene-level impact of CRISPRi targeting on intended G·C to C·G prime editing across different screening conditions. (Figure 40E) K562 PE2 vs. HeLa PE2. (Figure 40F) K562 PE3+50 vs. HeLa PE3+50. (Figure 40G) K562 PE2 vs. K562 PE3+50. The effect of each gene is calculated as the average log2 fold change in frequency from a non-targeting sgRNA for the two most extreme sgRNAs targeting the gene. Plotted quantities are the average of n = 2 independent biological replicates for each cell type, and bars indicate the range of values ​​spanned by replicate. Black dots represent 20 random sets of three non-targeting sgRNAs. [Figure 40G]Figures 40E-40G show a comparison of the gene-level impact of CRISPRi targeting on intended G·C to C·G prime editing across different screening conditions. (Figure 40E) K562 PE2 vs. HeLa PE2. (Figure 40F) K562 PE3+50 vs. HeLa PE3+50. (Figure 40G) K562 PE2 vs. K562 PE3+50. The effect of each gene is calculated as the average log2 fold change in frequency from a non-targeting sgRNA for the two most extreme sgRNAs targeting the gene. Plotted quantities are the average of n = 2 independent biological replicates for each cell type, and bars indicate the range of values ​​spanned by replicate. Black dots represent 20 random sets of three non-targeting sgRNAs.

[0140] [Figure 41A] Genetic modulators of unintended prime editing events. Figures 41A-41D show representative examples of four categories of unintended prime editing events observed in CRISPRi screens. In each panel, black bars indicate the sequence of the editing event, blue bars indicate the genome sequence surrounding the target editing site, and orange bars indicate the pegRNA sequence. Blue and orange lines between the editing event and the genome or pegRNA indicate the local alignment between the resulting sequence and the associated reference sequence. Alignment mismatches are marked with an X, and insertions are marked with a downward indentation. The location of the programmed edit is marked with a gray box. Red and cyan rectangles on the genome indicate the SaCas9 protospacer and PAM, and black vertical lines indicate the location of the SaCas9 nick site. Orange, beige, gray, and red rectangles on the pegRNA indicate the primer binding site (PBS), reverse transcription template (RTT), scaffold, and spacer, respectively. [Figure 41B]Figures 41A-41D show representative examples of four categories of unintended prime editing results observed in CRISPRi screens. In each panel, black bars indicate the sequence of the editing result, blue bars indicate the genome sequence surrounding the target editing site, and orange bars indicate the pegRNA sequence. Blue and orange lines between the editing result and the genome or pegRNA indicate the local alignment between the result sequence and the relevant reference sequence. Alignment mismatches are marked with an X, and insertions are marked with a downward indentation. The location of the programmed edit is marked with a gray box. Red and cyan rectangles on the genome indicate the SaCas9 protospacer and PAM, and black vertical lines indicate the location of the SaCas9 nick site. Orange, beige, gray, and red rectangles on the pegRNA indicate the primer binding site (PBS), reverse transcription template (RTT), scaffold, and spacer, respectively. [Figure 41C] Figures 41A-41D show representative examples of four categories of unintended prime editing results observed in CRISPRi screens. In each panel, black bars indicate the sequence of the editing result, blue bars indicate the genome sequence surrounding the target editing site, and orange bars indicate the pegRNA sequence. Blue and orange lines between the editing result and the genome or pegRNA indicate the local alignment between the result sequence and the relevant reference sequence. Alignment mismatches are marked with an X, and insertions are marked with a downward indentation. The location of the programmed edit is marked with a gray box. Red and cyan rectangles on the genome indicate the SaCas9 protospacer and PAM, and black vertical lines indicate the location of the SaCas9 nick site. Orange, beige, gray, and red rectangles on the pegRNA indicate the primer binding site (PBS), reverse transcription template (RTT), scaffold, and spacer, respectively. [Figure 41D]Figures 41A-41D show representative examples of four categories of unintended prime editing results observed in CRISPRi screens. In each panel, black bars indicate the sequence of the editing result, blue bars indicate the genome sequence surrounding the target editing site, and orange bars indicate the pegRNA sequence. Blue and orange lines between the editing result and the genome or pegRNA indicate the local alignment between the result sequence and the relevant reference sequence. Alignment mismatches are marked with an X, and insertions are marked with a downward indentation. The location of the programmed edit is marked with a gray box. Red and cyan rectangles on the genome indicate the SaCas9 protospacer and PAM, and black vertical lines indicate the location of the SaCas9 nick site. Orange, beige, gray, and red rectangles on the pegRNA indicate the primer binding site (PBS), reverse transcription template (RTT), scaffold, and spacer, respectively. [Figure 41E] Figures 41E-F provide a summary of the editing outcome categories observed in the PE2 screen (Figure 41E) and the PE3+50 screen (Figure 41F) in K562 cells. Plotted quantities are the mean ± SD of all sgRNAs for each indicated gene (60 non-targeting sgRNAs, 3 sgRNAs per target gene), averaged across n=2 independent biological replicates. [Figure 41F] Figures 41E-F provide a summary of the editing outcome categories observed in the PE2 screen (Figure 41E) and the PE3+50 screen (Figure 41F) in K562 cells. Plotted quantities are the mean ± SD of all sgRNAs for each indicated gene (60 non-targeting sgRNAs, 3 sgRNAs per target gene), averaged across n=2 independent biological replicates. [Figure 41G-41H]Figures 41G-41H show a comparison of the effect of knockdown of all genes targeted in the CRISPRi screen on the frequency of reverse-transcribed sequence ligation at unintended locations (Figure 41G) or deletion (Figure 41H) from PE3+50. The effect of each gene is calculated as the average log2 fold change in frequency from a non-targeting sgRNA for the two most extreme sgRNAs targeting the gene. Plotted quantities are the average of n=2 independent biological replicates for each cell type, and bars indicate the range of values ​​spanned by replicate. Black dots represent 20 random sets of three non-targeting sgRNAs. [Figure 41I] Figure 41I shows the frequency of deletions as a function of genomic location for the programmed PE3+50 nick (dashed vertical line) in K562 screening replicate 1 across all reads of the indicated CRISPRi sgRNA set (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 deletion frequency as a function of genomic location from MSH2, MSH6, MLH1, and PMS2 sgRNAs compared to non-targeting sgRNAs (bottom). [Figure 41J] Figure 41J shows the effect of gene knockdown on the percentage of all observed deletions that remove at least 25-nt of sequence outside the programmed PE3+50 nick in the K562 screen. Each dot represents all reads for all sgRNAs targeting each gene. Black dots represent 20 sets of three random non-targeting sgRNAs.

[0141] [Figure 42A] Figure 42A shows a model of mismatch repair of a prime editing intermediate. Figure 42A shows a model of DNA mismatch repair (MMR) of a PE2 intermediate. MMR excises and replaces the strand nicked during repair of the heteroduplex substrate generated by the prime editor. Infrequent ligation of the nick before MMR recognition deprives the strand discrimination signal for MMR, resulting in unbiased degradation of the heteroduplex. [Figure 42B] Figure 42B shows a model of MMR of the PE3 intermediate. PE3 installs an additional nick in the unedited strand that can direct MMR to replace the unedited strand. Ligation of the edited strand nick leaves only the complementary strand nick for signal repair by MMR, resulting in the desired primed editing outcome. [Figure 42C] Figure 42C shows the prime editing efficiency of PE2 and PE3 prime editors at endogenous sites (HEK3, EMX1, and RUNX1) in HEK293T cells pretreated with knockdown siRNA against MSH2, MSH6, MLH1, or PMS2 transcripts. Cells were pretransfected with siRNA three days before transfection with the prime editor components and siRNA. Genomic DNA was harvested three days after transfection with the prime editor and additional siRNA and then sequenced. Bars represent the mean of n=3 independent biological replicates. [Figure 42D] Figure 42D shows prime editing efficiency in HAP1 ΔMSH2 and HAP1 ΔMLH1 cells (mean of n=3 independent biological replicates). Δ, gene knockout.

[0142] [Figure 43A] Figure 43A shows that engineered dominant-negative MMR proteins (dominant-negative variants of MSH2, MSH6, PMS2, and MLH1) enhance prime editing. Figure 43B shows improved editing at HEK2, EMX1, and RUNX1 sites by co-expressing PE2 in trans with human MMR proteins or dominant-negative variants in HEK293T cells. MMR proteins include MSH2, MSH6, PMS2, and MLH1. The dominant-negative variants are designated MSH2 K675R, MSH6 K1140R, PMS2 E41A, PMS2 E705K, MLH1 E34A, and MLH1 Δ756. All values ​​are shown from n=3 independent biological replicates. [Figure 43B]Figure 43B shows the functional annotation of the 756 aa human MLH1 protein, which includes the ATPase domain, the MSH2 interaction domain, the NLS domain, the PMS2 dimerization domain, and the endonuclease domain. [Figure 43C] Figure 43C shows enhanced editing of MLH1 variants co-expressed with PE2 in HEK3, EMX1, and RUNX1 sites in HEK293T cells. Red boxes indicate mutations that inactivate MLH1 ATPase or endonuclease function. MLH1dn, MLH1 Δ754-756. MLH1NTD-NLS, codon-optimized MLH1 1-335-NLSSV40. All values ​​are shown from n=3 independent biological replicates. [Figure 43D] Figure 43D shows a comparison of the top three dominant-negative MLH1 variants in additional prime editing. All values ​​from n=3 independent biological replicates are shown. [Figure 43E] Figure 43E shows prime editing of PE2 and MLH1dn in trans, PE2 and MLH1NTD-NLS in trans, and PE2-P2A-MLH1dn (human codon optimized) in HEK293T cells. Bars represent the average of n=3 independent biological replicates. [Figure 43F] Figure 43F compares the structures of PE2, PE3, PE4, and PE5. In particular, the PE4 editing system consists of a prime editor enzyme (nickase-Cas9-RT fusion), MLH1dn, and pegRNA. The PE5 editing system consists of a prime editor enzyme, MLH1dn, pegRNA, and a second-strand nicking sgRNA. [Figure 43G] Figure 43G shows the editing efficiency of the PE2, PE3, PE4, and PE5 lines in HEK293T cells. Bars represent the mean of n=3 independent biological replicates.

[0143] [Figure 44A]Figure 44A shows the characterization of PE4 and PE5 across various prime editing classes and cell types. Figure 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 combined mean ± SD of all individual values ​​from n=3 independent biological replicates is shown. [Figure 44B] Figure 44B shows the 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. [Figure 44C] Figure 44C shows that PE4 improves prime editing of 1 bp and 3 bp insertions and deletions compared to PE2 in HEK293T cells. Bars represent the mean of n=3 independent biological replicates. [Figure 44D] Figure 44D shows PE4 editing enhancement relative to PE2 across 33 different insertion and deletion prime edits. Bars represent the average of all individual values ​​from n=3 independent biological replicates. [Figure 44E] Figures 44E-F provide a summary of PE2 and PE4 editing efficiencies for 35 different substitutions of 1 to 5 consecutive bases at five endogenous sites in HEK293T cells. For each number of consecutive bases changed, seven pegRNAs were tested. Shown are the means ± SD of all individual values ​​from n=3 independent biological replicates. [Figure 44F] Figures 44E-F provide a summary of PE2 and PE4 editing efficiencies for 35 different substitutions of 1 to 5 consecutive bases at five endogenous sites in HEK293T cells. For each number of consecutive bases changed, seven pegRNAs were tested. Shown are the means ± SD of all individual values ​​from n=3 independent biological replicates. [Figure 44G]Figures 44G-H show that installing additional silent or benign mutations near the intended edit can enhance editing efficiency by generating heteroduplex substrates that circumvent MMR. The PAM sequence (NGG) of each target is underlined. The amino acid sequence of the target gene is centered above each DNA codon. Values ​​represent the mean ± SD of n = 3 independent biological replicates. [Figure 44H] Figures 44G-H show that installing additional silent or benign mutations near the intended edit can enhance editing efficiency by generating heteroduplex substrates that circumvent MMR. The PAM sequence (NGG) of each target is underlined. The amino acid sequence of the target gene is centered above each DNA codon. Values ​​represent the mean ± SD of n = 3 independent biological replicates. [Figure 44I] Figure 44I shows a comparison of prime editing enhancement in different cell types. The PE4 and PE5 systems enhance prime editing to a greater extent in MMR-deficient cells (MMR-) than in MMR-competent cells (MMR+). The same set of 30 pegRNAs encoding single-base substitution edits was tested in HEK293T and HeLa cells. K562 and U2OS cells were edited with 10 pegRNAs, a direct subset of the 30 pegRNAs tested in HEK293T and HeLa cells. The mean ± SD of all individual values ​​from a set of n=3 independent biological replicates is shown. P values ​​were calculated using the Mann-Whitney U test. [Figure 44J] Figure 44J shows prime editing by PE2, PE3, PE4, and PE5 in HeLa, K562, and U2OS cells. Bars represent the mean of n=3 independent biological replicates.

[0144] [Figure 45A]The effect of dominant-negative MLH1 on the purity and off-targeting of primed editing products is shown. Figure 45A shows that the pegRNA encoding the edit programs a base change in the nascent 3' DNA flap, generating a heteroduplex after flap interconversion. The non-edited pegRNA is templated by a 3' DNA flap with perfect complementarity to the genomic target site. [Figure 45B] Figure 45B shows the frequency of single base mutations or indels derived from PE3 or PE5, which have four editing-coding pegRNAs that program four non-editing pegRNAs. The short horizontal bar indicates the average of all individual values ​​from a set of n=3 independent biological replicates. [Figure 45C] Figure 45C shows the ratio of indel frequencies from PE5 to PE3, which has pegRNAs encoding four edits that program single-base mutations, or pegRNAs encoding four edits that program single-base mutations, or four non-edited pegRNAs. The short horizontal bar indicates the average of all individual values ​​from a set of n=3 independent biological replicates. [Figure 45D] Figure 45D shows the distribution of deletions in genomic target DNA created by PE3 and PE5 using pegRNAs encoding 12 substitutions at the endogenous DNMT1 and RNF2 loci in HEK293T cells. Dotted lines indicate the positions of pegRNA- and sgRNA-directed nicks. Data represent the mean ± SD of n=3 independent biological replicates. [Figure 45E] Figure 45E shows the PE5 / PE3 ratio of the frequency of deletions that remove sequences >25-nt outside the pegRNA- and sgRNA-directed nicks in HEK293T cells. Each dot represents one of 84 total pegRNAs that program substitution edits at seven loci combined (mean of n=3 independent biological replicates). [Figure 45F]Figure 45F shows the PE5 / PE3 ratio of the frequency of editing outcomes with unintended PEG RNA scaffold incorporation or unintended flap recombination in HEK293T cells. Each dot represents one of 84 total PEG RNAs that program substitution edits at seven loci combined (mean of n=3 independent biological replicates). [Figure 45G] Figure 45G shows off-target prime editing by PE2 and PE4 in HEK293T cells. Bars represent the mean of n=3 independent biological replicates. [Figure 45H] Figure 45H shows high-throughput sequencing analysis of 17 susceptibility microsatellite repeat loci used in the clinical diagnosis of MMR deficiency. HAP1 and HeLa cells are MMR-competent, while HCT116 cells are MMR-impaired. HAP1 ΔMSH2 cells underwent 60 cell divisions after 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.

[0145] [Figure 46A] Figure 46A shows that the PEmax architecture with the PE4 and PE5 editing systems enhances editing in disease-relevant gene targets and cell types. Figure 46B shows a schematic of the PE2 and PEmax editor architecture. bpNLSSV40, bipartite SV40 NLS nuclear localization signal. MMLV RT, Moloney murine leukemia virus reverse transcriptase pentamutant; codon opt., human codon optimized. [Figure 46B] Figure 46B shows that the engineered pegRNA (epegRNA) contains a 3' RNA structural motif that improves prime editing performance. [Figure 46C]Figure 46C shows the prime editing efficiency of PE4 and PE5 in combination with the PEmax architecture and epeg RNA. Seven single-base substitution edits targeting different loci were tested in HeLa and HEK293T cells. Fold changes represent the average fold increase from each edit tested. Shown are the mean ± SD of all individual values ​​from n=3 independent biological replicates. [Figure 46D] Figure 46D shows prime editing at therapeutically relevant sites in wild-type HeLa and HEK293T cells. The HBB locus was edited at the E6 codon, which is commonly mutated in sickle cell disease (E6V) patients. The CDKL5 edit is at the site where the c.1412delA mutation causes CDKL5 deficiency. epegRNA was used to edit the HBB, PRNP, and CDKL5 loci. Bars represent the mean of n=3 independent biological replicates. [Figure 46E] Figure 46E shows correction of CDKL5 c.1412delA via an A·T insertion and silent G·C to A·T editing in iPSCs derived from a patient heterozygous for the allele. Editing efficiency indicates the percentage of sequencing reads with the c.1412delA correction from the editable allele carrying the mutation. Indel frequency reflects all sequencing reads containing any indel. Bars represent the average of n=3 independent biological replicates. [Figure 46F] Figure 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.

[0146] [Figure 47A] Figure 47A shows the design and results of Repair-seq screening for substitution prime editing results. Figure 47B shows the optimization of Staphylococcus aureus (Sa)-pegRNA to install a G C to C G edit within a lentiviral-integrated HBB sequence using SaPE2 in HEK293T cells. PBS, primer binding site. Data represent the average of n=3 independent biological replicates. [Figure 47B] Figure 47B shows the design of the prime-editing Repair-seq lentiviral vector (pPC1000). For Repair-seq screening, a 453-bp region containing the CRISPRi sgRNA sequence and the prime-editing results is amplified from genomic DNA for paired-end Illumina sequencing. The CRISPRi sgRNA is sequenced in the 44-nt Illumina forward read (R1), and the prime-editing site (encompassing the +50 and -50 nick sites) is sequenced in the 263-nt Illumina reverse read (R2). Black triangles indicate the positions of the SaPE2-induced nicks programmed by the Sa-pegRNA and Sa-sgRNA. The sizes of all vector components are drawn to scale. [Figure 47C] Figure 47C shows a schematic of the PE2, PE3+50, and PE3-50 prime editing constructs using the SaPE2 protein (SaCas9 N580A fused to engineered MMLV RT). [Figure 47D] Figure 47D shows validation of the intended G C to C G edit at the lentiviral-integrated Repair-seq editing site in HeLa cells expressing dCas9-BFP-KRAB. Bars represent the mean of n=2 independent biological replicates. [Figure 47E] Figure 47E shows prime editing at Repair-seq editing sites in HeLa cells expressing dCas9-BFP-KRAB with and without blasticidin selection. SaPE2-P2A-BlastR prime editing elements were used for all conditions. Bars represent the mean of n=2. [Figure 47F] Figure 47F shows the functional annotation classes of genes targeted by the pooled CRISPRi sgRNA library used in the Repair-seq screen. [Figure 47G]Figures 47G-J show that knockdown of MSH2, MSH6, MLH1, and PMS2 increases the frequency of the intended +6 G C to C G prime edit across all Repair-seq screens. Dots represent reads from individual CRISPRi sgRNAs. [Figure 47H] Figures 47G-J show that knockdown of MSH2, MSH6, MLH1, and PMS2 increases the frequency of the intended +6 G C to C G prime edit across all Repair-seq screens. Dots represent reads from individual CRISPRi sgRNAs. [Figure 47I] Figures 47G-J show that knockdown of MSH2, MSH6, MLH1, and PMS2 increases the frequency of the intended +6 G C to C G prime edit across all Repair-seq screens. Dots represent reads from individual CRISPRi sgRNAs. [Figure 47J] Figures 47G-J show that knockdown of MSH2, MSH6, MLH1, and PMS2 increases the frequency of the intended +6 G C to C G prime edit across all Repair-seq screens. Dots represent reads from individual CRISPRi sgRNAs.

[0147] [Figure 48A] Genetic modulators of unintended prime editing outcomes are shown. Figure 48A shows a summary of PE3-50 outcomes in the HeLa CRISPRi screen. TP53BP1 knockdown dramatically reduces the formation of all unintended editing outcomes. [Figure 48B] Figure 48B supplements Figure 41H and shows further details of PE2 results in the K562 CRISPRi screen. [Figure 48C] Figure 48C shows further details of the results of PE3+50 in the K562 CRISPRi screen, supplementing the information in Figure 41G. [Fig. 48D-48I]Figures 48D-I show a comparison of the effect of gene knockdown on the frequency of the indicated outcome categories in the indicated screening conditions. Plotted quantities are the mean log2 fold change from non-targeting sgRNAs for the two most extreme sgRNAs per gene, averaged across n=2 independent biological replicates per condition. Error bars indicate the range of values ​​spanned by replicates. Black dots represent 20 random sets of three non-targeting sgRNAs. Figure 48D shows that MSH2, MLH1, and PMS2 knockdown results in a greater fold change in the installation of additional edits than intended edits in the K562 PE2 screen. Figure 48E shows that unintended ligation of reverse-transcribed sequences in the PE2 screen in K562 and HeLa cells is most increased by knockdown of Fanconi anemia genes (red) and a set of RAD51 homologs and other genes involved in homologous recombination (blue). Figure 48F shows deletions in the PE2 screen in K562 and HeLa cells, most significantly increased by RAD51 homologs and a set of other genes involved in homologous recombination (blue). Figure 48G shows that, in addition to MSH2, MLH1, and PMS2, HLTF knockdown results in a greater fold change in the installation of additional edits than the intended edits in the K562 PE3+50 screen. Figure 48H shows that tandem duplications in the HeLa and K562 PE3+50 screens are most significantly reduced by knockdown of POLD and RFC subunits. Figure 48I shows that deletions in the HeLa PE3+50 and PE3-50 screens have dramatically different genetic regulators, highlighting the differences in processing different overhang configurations.

[0148] [Figure 49A]Validation of prime-editing Repair-seq screening results. Figures 49A-49B show the alignment of Sa-pegRNAs, their template 3' DNA flaps after SaPE2 reverse transcription, and the genome target sequence (top). Compared to the Sa-pegRNA used in Repair-seq screening (Figure 49A), the Sa-pegRNA with the recoded scaffold sequence (Figure 49B) templates an extended 3' DNA flap with reduced homology to the genome target sequence. The recoded Sa-pegRNA contains two base-pair changes that preserve base-pairing interactions within the scaffold. Reverse transcription of the Sa-pegRNA scaffold can generate an incorrectly extended 3' flap that is integrated into the genome. Vertical lines indicate base pairing. Xs indicate mismatches between the incorrectly extended reverse-transcribed 3' flap and the genome sequence. Figures 49A-49B also show the frequency of editing outcome categories observed at screened editing sites from arrayed PE2 and PE3+50 experiments in HeLa CRISPRi cells (bottom). Primed editing with Sa-pegRNA used in siteRepair-seq screening (Figure 49A) or scored Sa-pegRNA (Figure 49B) results in different frequencies of unintended edit installation from near-matched scaffolds. Plotted quantities are the mean ± SD of n=4 independent biological replicates for each cell line containing MSH2 or non-targeting CRISPRi sgRNA. [Figure 49B]Figures 49A-49B show the alignment of Sa-pegRNAs, their template 3' DNA flaps after SaPE2 reverse transcription, and the genomic target sequence (top). Compared to the Sa-pegRNAs used in the Repair-seq screening (Figure 49A), Sa-pegRNAs with the recoded scaffold sequence (Figure 49B) template extended 3' DNA flaps with reduced homology to the genomic target sequence. The recoded Sa-pegRNAs contain two base-pair changes that preserve base-pairing interactions within the scaffold. Reverse transcription of the Sa-pegRNA scaffold can generate erroneously extended 3' flaps that are integrated into the genome. Vertical lines indicate base pairing. Xs indicate mismatches between the erroneously extended reverse-transcribed 3' flap and the genomic sequence. Figures 49A-49B also show the frequency of editing outcome categories observed at screened editing sites from arrayed PE2 and PE3+50 experiments in HeLa CRISPRi cells (bottom). Primed editing with Sa-pegRNAs used in siteRepair-seq screening (Figure 49A) or scored Sa-pegRNAs (Figure 49B) results in different frequencies of unintended edit installation from near-matched scaffolds. Plotted abundances are the mean ± SD of n=4 independent biological replicates for each cell line containing MSH2 or non-targeting CRISPRi sgRNA. [Figure 49C] Figure 49C shows the mechanism of DNA mismatch repair in humans. [Figure 49D] Figure 49D shows that mismatch repair of primed-edited heteroduplex intermediates could install additional unprogrammed nicks from MutLα endonuclease activity. Excision from these unprogrammed nicks and subsequent repair of the resulting intermediates may contribute to the larger and more frequent indel outgrowths observed from MMR activity. [Figure 49E]Figure 49E shows the knockdown efficiency of siRNA treatment compared to a non-targeting siRNA control in HEK293T cells. Cells were transfected with siRNA, incubated for 3 days, transfected with PE2, pegRNA, and the same siRNA, and then incubated for another 3 days before relative RNA abundance was 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. [Figure 49F] Figure 49F shows editing in HEK293T cells co-transfected with prime editor components and siRNA. Cells were not pretreated with siRNA prior to transfection with prime editor components. Bars represent the mean of n=3 independent biological replicates.

[0149] [Figure 50A] Figure 50A shows the development and characterization of dominant-negative MMR proteins that enhance prime editing. Figure 50A shows prime editing efficiency from MMR proteins or dominant-negative variants expressed in trans with PE2 or directly fused to PE2 in HEK293T cells. 32 aa linker, (SGGS)x2-XTEN-(SGGS)x2 (SEQ ID NO: 125) (SGGSSGGSSGSETPGTSESATPES SGGSSGGS (SEQ ID NO: 125) or structurally, [SGGS]-[SGGS]-[SGSETPGTSESATPES]-[SGGS]-[SGGS] (SEQ ID NO: 125)). Codon opt, human codon optimization. Data within the same graph are from experiments performed simultaneously. Data represent the mean ± SD of n=3 independent biological replicates. [Figure 50B] Figure 50B shows titration of MLH1dn and PE2 plasmid transfection doses in HEK293T cells. The maximum plasmid amounts tested were 200 ng of PE2 and 100 ng of MLH1dn. Data represent the mean ± SD of n=3 independent biological replicates. [Figure 50C]Figure 50C shows prime editing by MLH1dn co-expression in MMR-deficient HCT116 cells containing a biallelic deletion in MLH1. Bars represent the average of three replicates. [Figure 50D] Figure 50D shows a comparison of prime editing with human MLH1dn (human codon optimized) or mouse MLH1dn (mouse codon optimized) in human HEK293T cells. Bars represent the mean of n=3 independent biological replicates. [Figure 50E] Figure 50E shows a comparison of prime editing with human MLH1dn (human codon optimized) or mouse MLH1dn (mouse codon optimized) in mouse N2A cells. Bars represent the average of n=3 replicates. [Figure 50F] Figure 50F shows that MLH1 knockout in a clonal HeLa cell line significantly increases prime editing efficiency over MLH1 co-expression in clonal wild-type HeLa cells. △, knockout. Bars represent the mean of n=3 or 4 independent biological replicates. [Figure 50G] Figure 50G shows editing at the FANCF locus by PE3b and PE5b (complementary strand nicks specific to the edited sequence) in HEK293T cells. PE5b, PE3b editing system with MLH1dn co-expression. Bars represent the mean of n=3 independent biological replicates. [Figure 50H] Figure 50H shows editing at the HEK2 locus with complementary strand nicks in HEK293T cells. "None" indicates no nicks, which indicates a PE2 or PE4 editing strategy. Bars represent the average of n=3 independent biological replicates.

[0150] [Figure 51A] Figure 51 shows the characterization of PE4 and PE5 across diverse prime editing classes and cell types. Figure 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. [Figure 51B]Figure 51B provides a summary of PE4 enhancement of editing efficiency relative to PE2 for 84 single-base substitution edits across seven endogenous sites in HEK293T cells. PE4 / PE2 fold improvement may be lower for PAM edits due to higher base editing efficiency for PAM edits or higher representation of G C to C G edits (5 out of 15 in this category). Data represent the mean ± SD of n=3 independent biological replicates. [Figure 51C] Figure 51C shows the efficiency of single-base substitution prime editing to change the PAM (+5 G and +6 G bases) of the prime-edited target protospacer in HEK293T cells. Shown are four G·C to A·T, five G·C to C·G, and six G·C to T·A PAM edits across seven endogenous sites combined. Shown are the averages of all individual values ​​from n=3 independent biological replicates. [Figure 51D] Figure 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. [Figure 51E] Figure 51E shows the effect of MMR gene knockout on G C to C G editing at the RNF2 locus in HAP1 cells. Δ, gene knockout. Bars represent the mean of n=3 independent biological replicates. [Figure 51F] Figure 51F shows primed editing at integration screen editing sites by 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 average of n=5 independent biological replicates. [Figure 51G] Figure 51G provides a summary of the improved editing efficiency of PE5 over PE3 for 84 single-base substitution edits in HEK293T cells. Shown is the combined mean ± SD of all individual values ​​from n=3 independent biological replicates. [Figure 51H]Figure 51H shows PE4 enhancement of editing efficiency relative to PE2 across a range of insertion and deletion prime editing lengths in HEK293T cells. A total of 33 different prime edits at the three endogenous loci combined are shown. The average of all individual values ​​from n=3 independent biological replicates is shown. [Figure 51I] Figure 51I shows that PE5 improves editing efficiency and reduces indel by-products compared to PE3 across small insertion and deletion prime editing in HEK293T cells. [Figure 51J] Figure 51J shows PE2 and PE4 editing efficiencies at 33 different insertion- and deletion-primed edits across the three endogenous loci combined. Bars represent the average of all individual values ​​from n=3 independent biological replicates.

[0151] [Figure 52A] Characterization of the PE4 and PE5 systems and improvement of prime editing efficiency by additional silent mutations are shown. Figure 52A shows the substitution of consecutive bases by PE2 and PE4 in HEK293T cells. The top sequence shows the original, unedited genome sequence. Numbers indicate the position of the edited nucleotide relative to the PE2 nick site. Nucleotides within the SpCas9 PAM sequence (NGG) are underlined. The sequence of the intended editing product is shown below, with the edited nucleotides marked in red. Bars represent the average of n=3 independent biological replicates. [Figure 52B] Figure 52B shows that installing additional silent mutations can increase prime editing efficiency by bypassing MMR. The PE4 / PE2 fold change in editing frequency reflects the degree to which MMR activity interferes with the indicated prime editing. Edited nucleotides that create the indicated coding mutations are marked in red, and edited nucleotides that create silent mutations are marked in green. Data represent the mean ± SD of n=3 independent biological replicates. [Figure 52C]Figure 52C shows the installation of 22 single-base substitution prime edits across seven endogenous sites in HeLa cells with PE2, PE3, PE4, and PE5. Bars represent the average of n=3 independent biological replicates.

[0152] [Figure 53A] Figure 53A shows the effect of dominant-negative MLH1 on the purity and off-targeting of primed editing products. Figure 53A shows the frequency of indels in HEK293T cells treated with pegRNA, nicking sgRNA, and PE2 enzyme, RT-impaired PE2 (PE2-dRT), or nickase Cas9 (SpCas9 H840A), with and without MLH1dn. The non-edited pegRNA encodes a 3' DNA flap with perfect homology to the genomic target. Bars represent the average of n=3 independent biological replicates. [Figure 53B] Figure 53B shows the distribution of deletion results from PE3 and PE5 at endogenous loci in HEK293T cells. Twelve different pegRNAs that program single-base substitutions at each indicated locus were tested. Dotted lines indicate the positions of pegRNA and sgRNA-directed nicks. Data represent the mean ± SD of n=3 independent biological replicates. [Figure 53C] Figure 53C shows the distribution of deletion outcomes from PE3 and PE5 with editing-encoding and non-editing pegRNA in HEK293T cells. The non-editing pegRNA is templated with a 3' DNA flap that has perfect complementarity to the genomic target sequence. Data represent the mean ± SD of n=3 independent biological replicates. [Figure 53D] Figure 53D shows the frequency of all prime editing events involving unintended incorporation of pegRNA scaffold sequences or unintended flap recombination in HEK293T cells. Twelve pegRNAs, each programming a different single-base substitution, were tested at each of the seven loci shown. Each dot represents an individual pegRNA at the indicated locus (mean of n = 3 independent biological replicates). [Figure 53E]Figure 53E shows off-target prime editing by PE2 and PE4 in HEK293T cells (mean of n=3 independent biological replicates). [Figure 53F] Figure 53F shows the distribution and cumulative distribution of microsatellite repeat lengths in the indicated cell types and treatments. HAP1 and HeLa cells are MMR-competent, while HCT116 cells are MMR-impaired. HAP1 ΔMSH2 cells underwent 60 cell divisions after knockout of MSH2. HeLa cells were transiently transfected with PE2 or PE4 components and then grown for 3 days before sequencing. wt, wild type. All values ​​from n=2 independent biological replicates are shown. [Figure 53G] Figure 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 length was assayed from genomic DNA harvested from these PE2- and PE4-treated HeLa cells.

[0153] [Figure 54A] Figure 54A shows that use of the PEmax architecture with the PE4 and PE5 editing systems enhances editing in disease-relevant gene targets and cell types. Figure 54B shows a schematic of the PE2 and PEmax editor architecture. bpNLSSV40, bipartite SV40 NLS. MMLV RT, Moloney murine leukemia virus reverse transcriptase pentamutant. GS codon, Genscript human codon optimized. [Figure 54B] Figure 54B shows an engineered pegRNA (epegRNA) containing a 3' RNA structural motif that improves prime editing performance. [Figure 54C] Figure 54C shows the prime editing efficiency of PE4 and PE5 in combination with the PEmax architecture and epeg RNA. Seven single-base substitution edits targeting different loci were tested in HeLa and HEK293T cells. The fold change represents the average fold increase from each edit tested. The mean ± SD of all individual values ​​from n=3 independent biological replicates is shown. [Figure 54D] Figure 54D shows prime editing at therapeutically relevant sites in wild-type HeLa and HEK293T cells. The HBB locus was edited at the E6 codon, which is commonly mutated in sickle cell disease (E6V) patients. The CDKL5 edit is at the site where the c.1412delA mutation causes CDKL5 deficiency. epegRNA was used to edit the HBB, PRNP, and CDKL5 loci. Bars represent the mean of n=3 independent biological replicates. [Figure 54E] Figure 54E shows correction of CDKL5 c.1412delA via an A·T insertion and silent G·C to A·T editing in iPSCs derived from a patient heterozygous for the allele. Editing efficiency indicates the percentage of sequencing reads with the c.1412delA correction from the editable allele carrying the mutation. Indel frequency reflects all sequencing reads containing any indel. Bars represent the average of n=3 independent biological replicates. [Figure 54F] Figure 54F shows prime editing in primary T cells. Bars represent the mean of n=3 independent biological replicates from different healthy T cell donors.

[0154] [Figure 55A] Development of PEmax and application of PE4 and PE5 to primary cell types are shown. Figures 55A-B show screening of prime editor variants to maximize editing efficiency in HeLa cells. All prime editor architectures carry the Cas9 H840A mutation to prevent nicking of the complementary DNA strand in the target protospacer. *NLSSV40 contains a 1-aa deletion outside of the PKKKRKV (SEQ ID NO: 132) NLSSV40 consensus sequence. All individual values ​​from n=3 independent biological replicates are shown. [Figure 55B]Development of PEmax and application of PE4 and PE5 to primary cell types are shown. Figures 55A-B show screening of prime editor variants to maximize editing efficiency in HeLa cells. All prime editor architectures carry the Cas9 H840A mutation to prevent nicking of the complementary DNA strand in the target protospacer. *NLSSV40 contains a 1-aa deletion outside of the PKKKRKV (SEQ ID NO: 132) NLSSV40 consensus sequence. All individual values ​​from n=3 independent biological replicates are shown.

[0155] [Figure 56A] The development of PEmax and application of PE4 and PE5 to primary cell types are shown. Figure 56A shows a screen for prime editor variants to improve editing efficiency with the PE3 system in HeLa cells. All prime editor architectures carry the SpCas9 H840A mutation to prevent nicking of the complementary DNA strand in 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 ​​from n=3 independent biological replicates are shown. [Figure 56B] Figure 56B shows the architectures of the original PE2 editor (Anzalone et al., 2019), PE2* (Liu et al., 2021), CMP-PE-V1 (Park et al., 2021), and the prime editor variants (PEmax, CMP-PEmax) developed in this study. HN1, HMGN1; H1G, histone H1 central globular domain; codonopt, human codon optimization. [Figure 56C] Figure 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. [Figure 56D]Figure 56D shows the fold change in editing efficiency of primed editor architectures compared to the PE2 with PE3 system in HeLa cells. Shown are the means ± SD of all individual values ​​from n=3 independent biological replicates. [Figure 56E] Figure 56E shows intended edit 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-primed edits targeting different endogenous loci were tested for each condition. Shown are the means ± SD of all individual values ​​from n=3 independent biological replicates. [Figure 56F] Figure 56F shows correction of CDKL5 c.1412delA via an A·T insertion and G·C to A·T editing in iPSCs derived from a patient heterozygous for the disease allele. Editing efficiency indicates the percentage of sequencing reads with the c.1412delA correction from the editable allele carrying the mutation. Indel frequency reflects all sequencing reads containing any indels that do not map to the c.1412delA allele or the wild-type sequence. 1 μg of PE2 mRNA was used in all conditions shown. Bars represent the average of n=3 independent biological replicates. [Figure 56G] Figure 56G shows prime editing in primary T cells. Bars represent the mean of n=3 independent replicates from different T cell donors.

[0156] [Figure 57A]We demonstrate that the recorded pegRNA scaffold reduces unintended consequences from scaffold sequence incorporation. Figure 57A shows the alignment of a primed-edit Repair-seq target site with the SaPE2-generated 3' DNA flap templated by (top) the Sa-pegRNA used for Repair-seq screening or (bottom) the Sa-pegRNA with the recorded scaffold sequence. The 3' flap sequence is aligned with the template region (RT template or scaffold) of the Sa-pegRNA shown above. Red indicates the position of the intended +6 G·C to C·G edit programmed by both Sa-pegRNAs. Blue indicates positions where the genomic target sequence does not align with the 3' flap sequence templated by the Sa-pegRNA scaffold. Unintended edits from incorporation of the 3' flap containing the reverse-transcribed Sa-pegRNA scaffold sequence may occur at these blue-marked nucleotides. [Figure 57B] Figure 57B shows a summary of editing outcome categories observed in PE2 and PE3+50 experiments in HeLa CRISPRi cells. Screening pegRNA indicates the Sa-pegRNA used for prime-editing Repair-seq screening. The Sa-pegRNA with the recorded scaffold (sequence shown in Figure 54A) avoids sequence homology with Repair-seq editing sites. 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.

[0157] [Figure 58] A comparison of PE3max (PE3 editing system containing PEmax protein) and PE3 (PE3 editing system containing PE2 protein) in HeLa cells is shown (mean of n=3 independent biological replicates).

[0158] [Figure 59] We show that PE improvement by MLH1dn depends on the prime edit size: MMR most efficiently repairs substitutions and insertion and deletion errors of lengths of approximately 13 bp or less.

[0159] [Figure 60] PE4 and pegRNA enable prime editing by a single pegRNA integrant.

[0160] [Figure 61] We show that PE5 improves the installation of the protective Christchurch allele in an APOE4 mouse astrocyte model.

[0161] [Figures 62A-62C] This shows that inhibition of p53 enhances the efficiency and accuracy of PE3-primed editing. This is particularly true when the nicking sgRNA nicks upstream (negative side) of the nick directed by the pegRNA. Each point on the graph represents an individual CRISPRi gene knockdown in the Repair-seq screen. The axis indicates the log2 fold change compared to the control. Knockdown of TP53BP1 (p53 gene) increased intended editing (x-axis) and reduced three types of unintended editing (y-axis), including ligation of reverse-transcribed sequences at unintended locations (Figure 62A), unintended deletions (Figure 62B), and unintended tandem duplications (Figure 62C). [Figure 62B] This shows that inhibition of p53 enhances the efficiency and accuracy of PE3-primed editing. This is particularly true when the nicking sgRNA nicks upstream (negative side) of the nick directed by the pegRNA. Each point on the graph represents an individual CRISPRi gene knockdown in the Repair-seq screen. The axis indicates the log2 fold change compared to the control. Knockdown of TP53BP1 (p53 gene) increased intended editing (x-axis) and reduced three types of unintended editing (y-axis), including ligation of reverse-transcribed sequences at unintended locations (Figure 62A), unintended deletions (Figure 62B), and unintended tandem duplications (Figure 62C). [Figure 62C]This shows that inhibition of p53 enhances the efficiency and accuracy of PE3-primed editing. This is particularly true when the nicking sgRNA nicks upstream (negative side) of the nick directed by the pegRNA. Each point on the graph represents an individual CRISPRi gene knockdown in the Repair-seq screen. The axis indicates the log2 fold change compared to the control. Knockdown of TP53BP1 (p53 gene) increased intended editing (x-axis) and reduced three types of unintended editing (y-axis), including ligation of reverse-transcribed sequences at unintended locations (Figure 62A), unintended deletions (Figure 62B), and unintended tandem duplications (Figure 62C).

[0162] [Figure 63] We show that p53 inhibitors (i53) can enhance the efficiency and precision of PE3 prime editing. This is particularly true when the nicking sgRNA nicks upstream (negative side) of the nick directed at the pegRNA. Only the EMX1 site uses a nick on the "-" side. Figure 64 depicts various aspects of the present disclosure, including the use of CRISPRi screening to reveal cellular genes, including mismatch repair genes, that affect the outcome of prime editing; the use of engineered MLH1 in the mismatch repair (MMR) pathway to enhance the efficiency and precision of prime editing; and the demonstration that the improved prime editing systems described herein (e.g., the PE4 and PE5 systems, as well as the PEmax editor) have been shown to exhibit the same beneficial effects in many cell types.

[0163] [Figure 64] We show that CRISPRi screening reveals cellular determinants of prime genome editing, that engineered MLH1 proteins enhance prime editing efficiency and precision, and that improved prime editing systems have been characterized across edits and cell types.

[0164] [Figure 65] 1 provides a schematic showing the optimization of the PE2 protein.

[0165] [Figure 66] Figure 1 shows the fold change in frequency of intended editing using PE2 and various other PE constructs in HEK293T cells (low plasmid dose) at a range of gene targets (HEK3, EMX1, RNF2, FANCF, FUNX1, DNMT1, VEGFA, HEK4, PRNP, APOE, CXCR4, HEK3).

[0166] [Figure 67] Figure 1 shows the fold change in frequency of intended editing using PE3 and various prime editor constructs in HeLa cells at a range of gene targets (HEK3, FANCF, RUNX1, VEGFA).

[0167] [Figure 68] A comparison of prime editing in HEK293T versus HeLa using various PE constructs is shown.

[0168] [Figure 69] 1 shows the NLS architecture optimization of PE3 in HeLa cells.

[0169] [Figure 70] A schematic diagram showing the final PEmax construct corresponding to SEQ ID NO:99 is provided.

[0170] [Figure 71] Figure 1 shows that PEmax increases indels in addition to intended edits. DETAILED DESCRIPTION OF THE INVENTION

[0171] definition Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which this invention belongs. The following references provide those skilled in the art with general definitions of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them unless otherwise specified.

[0172] Cas9 The term "Cas9" or "Cas9 nuclease" refers to an RNA-guided nuclease comprising a Cas9 domain or a fragment thereof (e.g., a protein comprising an active or inactive DNA cleavage domain of Cas9 and / or a gRNA-binding domain of Cas9). A "Cas9 domain," as used herein, is a protein fragment comprising an active or inactive cleavage domain of Cas9 and / or a gRNA-binding domain of Cas9. A "Cas9 protein" is a full-length Cas9 protein. Cas9 nuclease is also sometimes referred to as casn1 nuclease or CRISPR (clustered regularly interspaced short palindromic repeats (CRISPR)). C Lustered R regularly I Interspaced S hort P alindromic RCRISPR is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements, and conjugative plasmids). CRISPR clusters contain a spacer, a sequence complementary to the preceding mobile element, and a targeting invading nucleic acid. The CRISPR cluster is transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems, proper processing of the pre-crRNA requires a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and a Cas9 domain. The tracrRNA acts as a guide for ribonuclease 3-aided processing of the pre-crRNA. Subsequently, the Cas9 / crRNA / tracrRNA endonucleolytically cleaves linear or circular dsDNA targets complementary to the spacer. The target strand that is not complementary to the crRNA is first cleaved endonucleolytically, followed by 3'-5' exonucleolytic excision. In nature, DNA binding and cleavage typically require both a protein and an RNA. However, single-guide RNAs ("sgRNAs" or simply "gRNAs") can be engineered to incorporate aspects of both the crRNA and tracrRNA into a single RNA species. See, for example, Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna JA, Charpentier E. Science 337:816-821 (2012), the entire contents of which are incorporated herein by reference. Cas9 recognizes a short motif in the CRISPR repeat (the PAM or protospacer adjacent motif) to help distinguish self from non-self.Cas9 nuclease sequences and structures are well known to those skilled in the art (e.g., "Complete genome sequence of an M1 strain of Streptococcus pyogenes." Ferretti et al., JJ, McShan WM, Ajdic DJ, Savic DJ, Savic G., Lyon K., Primeaux C., Sezate S., Suvorov AN, Kenton S., Lai HS, Lin SP, Qian Y., Jia HG, Najar FZ, Ren Q., Zhu H., Song L., White J., Yuan X., Clifton SW, Roe BA, McLaughlin RE, Proc. Natl. Acad. Sci. USA98:4658-4663(2001); "CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III." Deltcheva E., Chylinski K., Sharma See CM, Gonzales K., Chao Y., Pirzada ZA, Eckert MR, Vogel J., Charpentier E., Nature 471:602-607 (2011); and "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity." Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna JA, Charpentier E. Science 337:816-821 (2012), the entire contents of each of which are incorporated herein by reference. Cas9 orthologs have been described in a variety of species, including, but not limited to, S. pyogenes and S. thermophilus. Additional suitable Cas9 nucleases and sequences will be apparent to those of skill in the art based on the present disclosure.Such Cas9 nucleases and sequences also include Cas9 sequences from the organisms and loci disclosed in Chylinski, Rhun, and Charpentier, "The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems" (2013) RNA Biology 10:5, 726-737, the entire contents of which are incorporated herein by reference. In some embodiments, the Cas9 nuclease comprises one or more mutations that partially impair or inactivate the DNA cleavage domain.

[0173] A Cas9 domain with inactivated nucleases may be interchangeably referred to as a "dCas9" protein (representing a nuclease-inactivated Cas9). Methods for generating a Cas9 domain (or a fragment thereof) with an inactive DNA cleavage domain are known (see, e.g., Jinek et al., Science. 337:816-821 (2012); Qi et al., "Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression" (2013) Cell. 28;152(5):1173-83, the entire contents of each of which are incorporated herein by reference). For example, the DNA cleavage domain of Cas9 is known to contain two subdomains: an HNH nuclease subdomain and a RuvC1 subdomain. The HNH subdomain cleaves the strand complementary to the gRNA, while the RuvC1 subdomain cleaves the non-complementary strand. Mutations within these subdomains can silence the nuclease activity of Cas9. For example, mutations D10A and H840A completely inactivate the nuclease activity of S. pyogenes Cas9 (Jinek et al., Science. 337:816-821 (2012); Qi et al., Cell. 28;152(5):1173-83 (2013)). In some embodiments, proteins comprising fragments of Cas9 are provided. For example, in some embodiments, the protein comprises one of two Cas9 domains: (1) the gRNA-binding domain of Cas9; or (2) the DNA-cleavage domain of Cas9. In some embodiments, proteins comprising Cas9 or fragments thereof are referred to as "Cas9 variants." Cas9 variants share homology with Cas9 or fragments thereof.For example, a Cas9 variant is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, at least about 99.8% identical, or at least about 99.9% identical to a wild-type Cas9 (e.g., SpCas9 of SEQ ID NO: 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 a 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 a 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).

[0174] circular permutants As used herein, the term "circular permutant" refers to a protein or polypeptide (e.g., Cas9) that contains a circular permutation, which is a change in the structural conformation of a protein accompanied by a change in the order of amino acids appearing in the protein's amino acid sequence. In other words, a circular permutation is a protein with altered N- and C-termini compared to its wild-type counterpart, e.g., the C-terminal half of the wild-type protein becomes a new N-terminal half. Circular permutation (or CP) is essentially a topological rearrangement of a protein's primary sequence, with its N- and C-termini connected, often with a peptide linker, while simultaneously splitting the sequence at a different location to create new adjacent N- and C-termini. The result is a protein structure that may have different connections but often the same or an overall similar three-dimensional (3D) shape, potentially including improved or altered characteristics, including reduced susceptibility to proteolysis, improved catalytic activity, altered substrate or ligand binding, and / or improved thermostability. Circularly permuted proteins can occur naturally (e.g., concanavalin A and lectins). In addition, circular permutations can arise as a result of post-translational modifications or can be engineered using recombinant techniques.

[0175] Circularly permuted Cas9 The term "circularly permuted Cas9" refers to any Cas9 protein or variant thereof that is generated as a circular permutation, thereby locally rearranging its N- and C-termini. Such circularly permuted Cas9 proteins ("CP-Cas9"), or variants thereof, retain the ability to bind to DNA when complexed with a guide RNA (gRNA). See Oakes et al., "Protein Engineering of Cas9 for enhanced function," Methods Enzymol, 2014, 546:491-511, and Oakes et al., "CRISPR-Cas9 Circular Permutants as Programmable Scaffolds for Genome Modification," Cell, January 10, 2019, 176:254-267, each of which is incorporated herein by reference. The present disclosure contemplates the use of any previously known CP-Cas9 or new CP-Cas9, so long as the resulting circularly permuted protein retains the ability to bind to DNA when complexed with a guide RNA (gRNA). Exemplary CP-Cas9 proteins are SEQ ID NOS: 54-63.

[0176] CRISPR CRISPR is a family of DNA sequences (i.e., CRISPR clusters) in bacteria and archaea that represent snippets of preceding infection by viruses that have invaded prokaryotes. These snippets of DNA are used by prokaryotic cells to detect and destroy DNA from subsequent attacks by similar viruses, effectively forming a prokaryotic immune defense system in conjunction with an array of CRISPR-associated proteins (including Cas9 and its homologs) and CRISPR-associated RNAs. In nature, CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In certain types of CRISPR systems (e.g., type II CRISPR systems), proper processing of the pre-crRNA requires a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and the Cas9 protein. The tracrRNA acts as a guide for ribonuclease 3-aided processing of the pre-crRNA. Cas9 / crRNA / tracrRNA then endolytically cleaves linear or circular dsDNA targets complementary to the RNA. Specifically, the target strand not complementary to the crRNA is first endolytically cut, followed by 3'-5' exolytic trimming. In nature, DNA binding and cleavage typically require both a protein and RNA. However, single-guide RNAs ("sgRNAs" or simply "gNRAs") can be engineered to incorporate aspects of both the crRNA and tracrRNA into the guide RNA of a single RNA species. For example, see Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna JA, Charpentier E. Science 337:816-821 (2012), the entire contents of which are incorporated herein by reference. Cas9 helps distinguish self from non-self by recognizing a short motif in the CRISPR repeat (the PAM or protospacer adjacent motif).CRISPR biology and Cas9 nuclease sequences and structures are well known to those skilled in the art (e.g., "Complete genome sequence of an M1 strain of Streptococcus pyogenes." Ferretti et al., JJ, McShan WM, Ajdic DJ, Savic DJ, Savic G., Lyon K., Primeaux C., Sezate S., Suvorov AN, Kenton S., Lai HS, Lin "CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III." Deltcheva E., Chylinski See K., Sharma CM, Gonzales K., Chao Y., Pirzada ZA, Eckert MR, Vogel J., Charpentier E., Nature 471:602-607 (2011); and "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity." Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna JA, Charpentier E. Science 337:816-821 (2012), the entire contents of each of which are incorporated herein by reference. 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 the present disclosure.Such Cas9 nucleases and sequences also include Cas9 sequences from the organisms and loci disclosed in Chylinski, Rhun, and Charpentier, "The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems" (2013) RNA Biology 10:5, 726-737, the entire contents of which are incorporated herein by reference.

[0177] In certain types of CRISPR systems (e.g., type II CRISPR systems), proper processing of the pre-crRNA requires a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and the Cas9 protein. The tracrRNA acts as a guide for ribonuclease 3-aided processing of the pre-crRNA. Cas9 / crRNA / tracrRNA then endolytically cleaves linear or circular nucleic acid targets complementary to the RNA. Specifically, the target strand not complementary to the crRNA is first endolytically cut and then 3'-5' exolytically trimmed. In nature, DNA binding and cleavage typically require both a protein and an RNA. However, single-guide RNAs ("sgRNAs" or simply "gRNAs") can be engineered to incorporate aspects of both the crRNA and tracrRNA into the guide RNA of a single RNA species.

[0178] Generally, a "CRISPR system" collectively refers to the transcripts and other elements that accompany the expression of or direct the activity of CRISPR-associated ("Cas") genes, and includes sequences encoding Cas genes, tracr (transactivating CRISPR) sequences (e.g., tracrRNA or an active partial tracrRNA), tracr mate sequences (which, in the context of an endogenous CRISPR system, encompass "direct repeats" and partial direct repeats processed by the tracrRNA), guide sequences (also referred to as "spacers" in the context of an endogenous CRISPR system), or other sequences and transcripts from the CRISPR locus. The tracrRNA of the system is complementary (fully or partially) to the tracr mate sequence present on the guide RNA.

[0179] DNA synthesis template As used herein, the term "DNA synthesis template" refers to the region or portion of the extension arm of a PEG RNA that is utilized by the polymerase of a prime editor as the template strand (encoding a 3' single-stranded DNA flap that contains the desired edit and then replaces the corresponding strand of endogenous DNA at the target site through the mechanism of prime editing). The extension arm encompasses the DNA synthesis template and may be composed of DNA or RNA. In the case of RNA, the polymerase of the prime editor may be an RNA-dependent DNA polymerase (e.g., reverse transcriptase). In the case of DNA, the polymerase of the prime editor may be a DNA-dependent DNA polymerase. In various embodiments, the DNA synthesis template may include all or a portion of the "editing template" and "homology arm," as well as any 5'-end modification region e2. That is, depending on the nature of the e2 region (e.g., whether it comprises a hairpin, two-loop, or stem / loop secondary structure), the polymerase may encode none, part, or all of the e2 region. In other words, in the case of a 3' extension arm, the DNA synthesis template may comprise a portion of the extension arm extending from the 5' end of the primer binding site (PBS) to the 3' end of the gRNA core, which may serve as a template for synthesis of a single strand of DNA by a polymerase (e.g., reverse transcriptase). In the case of a 5' extension arm, the DNA synthesis template may comprise a portion of the extension arm extending from the 5' end of the PEGRNA molecule to the 3' end of the editing template. In some embodiments, the DNA synthesis template excludes the primer binding site (PBS) of a PEGRNA having either a 3' extension arm or a 5' extension arm. Certain embodiments described herein refer to an "RT template" that encompasses the editing template and homology arms, i.e., the sequence of the PEGRNA extension arms that are actually used as a template during DNA synthesis. The term "RT template" is equivalent to the term "DNA synthesis template." In certain embodiments, RT template may be used to refer to a template polynucleotide for reverse transcription, e.g., in a prime editing system, complex, or method that uses a prime editor with 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, in a prime editing system, complex, or method that uses a prime editing factor having a polymerase that is an RNA-dependent DNA polymerase or a DNA-dependent DNA polymerase.

[0180] In some embodiments, the DNA synthesis template is a single-stranded portion of a PEG RNA that is 5' to the PBS, includes a region of complementarity to the PAM strand (i.e., the non-target strand or edited strand), and contains 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 downstream of the nick site, except for one or more non-complementary nucleotides at the intended nucleotide edit position. In some embodiments, the DNA synthesis template is complementary or substantially complementary to a sequence on the non-target strand immediately downstream (i.e., directly downstream) of the nick site, except for one or more non-complementary nucleotides at the intended nucleotide edit position. In some embodiments, one or more non-complementary nucleotides at the intended nucleotide edit position are immediately downstream of the nick site. In some embodiments, the DNA synthesis template contains one or more nucleotide edits to the double-stranded target DNA sequence. In some embodiments, the DNA synthesis template contains one or more nucleotide edits to the non-target strand of the double-stranded target DNA sequence. For each PEgRNA described herein, the nick site is characteristic of the specific napDNAbp to which the gRNA core of the PEgRNA associates and is characteristic of the specific PAM required for recognition and function of the napDNAbp. For example, in the case of a PEgRNA comprising a gRNA core that associates with SpCas9, the nick site is at the phosphodiester bond between base 3 (position "-3" relative to position 1 of the PAM sequence) and base 4 (position "-4" relative to position 1 of the PAM sequence). In some embodiments, the DNA synthesis template and primer binding site are directly 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, for example, 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 of the DNA synthesis template of the PEgRNA to be incorporated into the target DNA sequence. In some embodiments, the DNA synthesis template contains two or more nucleotide edits to the double-stranded target DNA sequence.In such embodiments, each nucleotide edit is a specific nucleotide edit at a specific position within the DNA synthesis template, where each nucleotide edit is at a different specific position compared to any of the other nucleotide edits within 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. The nucleotide edit may refer to an edit on the DNA synthesis template compared to a sequence on the target strand of the target gene, or the nucleotide edit may refer to an edit encoded by the DNA synthesis template on newly synthesized single-stranded DNA that replaces the endogenous target DNA sequence on the non-target strand.

[0181] Dominant-negative variants The terms "dominant-negative variant" and "dominant-negative mutant" refer to a gene or gene product (e.g., a protein) that contains a mutation that results in a gene product that acts antagonistically to (i.e., inhibits the activity of) the wild-type gene product. Dominant-negative mutations generally result in an altered (often inactive) molecular function. 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).

[0182] Editing Template The term "editing template" refers to the portion of the extension arm that encodes the desired edit on the single-stranded 3' DNA flap synthesized by a polymerase, e.g., a DNA-dependent DNA polymerase, an RNA-dependent DNA polymerase (e.g., a reverse transcriptase). Certain embodiments described herein refer to an "RT template," which refers to both the editing template and the homologous arm together, i.e., the sequence of the PEGRNA extension arm that is actually used as a template during DNA synthesis. The term "RT editing template" is also equivalent to the term "DNA synthesis template," except that an RT editing template reflects the use of a primed editor with a polymerase that is a reverse transcriptase, while a DNA synthesis template more broadly reflects the use of a primed editor with either polymerase.

[0183] Extension arm The term "extension arm" refers to the nucleotide sequence components of a PEG RNA that include a primer binding site and a DNA synthesis template (e.g., an editing template and a homology arm) for a polymerase (e.g., a reverse transcriptase). In some embodiments, the extension arm is positioned at the 3' end of the guide RNA. In other embodiments, the extension arm is positioned at the 5' end of the guide RNA. In some embodiments, the extension arm includes a DNA synthesis template and a primer binding site. In some embodiments, the extension arm includes the following components in the 5' to 3' direction: a DNA synthesis template and a primer binding site. In some embodiments, the extension arm also includes a homology arm. In various embodiments, the extension arm includes the following components in the 5' to 3' direction: a homology arm, an editing template, and a primer binding site. In various embodiments, the extension arm includes the following components in the 5' to 3' direction: a homology arm, an editing template, and a primer binding site. Because the polymerization activity of reverse transcriptase is in the 5' to 3' direction, the preferred arrangement of the homology arms, editing template, and primer binding site is in the 5' to 3' direction so that the reverse transcriptase, once primed by the annealed primer sequence, polymerizes single-stranded DNA using the editing template as the complementary template strand.

[0184] The extension arm may also be generally described as comprising two regions: for example, a primer binding site (PBS) and a DNA synthesis template. The primer binding site binds to a primer sequence formed from the endogenous DNA strand of the target site when it becomes nicked by a prime editor complex, thereby exposing its 3' end on the nicked endogenous strand. As described herein, binding of the primer sequence to the primer binding site on the extension arm of the PEGRNA creates a double-stranded region with an exposed 3' end (i.e., 3' of the primer sequence), which then provides a substrate for a polymerase that begins polymerizing single strands of DNA from the exposed 3' end along the length of the DNA synthesis template. The sequence of the single-stranded DNA product is the complement of the DNA synthesis template. Polymerization continues 5' of the DNA synthesis template (or extension arm) until polymerization terminates. Thus, the DNA synthesis template represents the portion of the extension arm that is encoded by the polymerase of the primed editor complex into a single-stranded DNA product (i.e., a 3' single-stranded DNA flap containing the desired gene editing information), which replaces the corresponding endogenous DNA strand at the target site immediately downstream of the PE-induced nick site. Without being bound by theory, polymerization of the DNA synthesis template continues toward the 5' end of the extension arm until a termination event occurs. Polymerization may terminate in a variety of ways, including, but not limited to, (a) reaching the 5' end of the PE-gRNA (e.g., in the case of a 5' extension arm, the DNA polymerase simply runs out of template), (b) reaching an untraversable RNA secondary structure (e.g., a hairpin or stem / loop), or (c) reaching a replication termination signal, e.g., a specific nucleotide sequence that blocks or inhibits the polymerase, or a signal on a nucleic acid form such as supercoiled DNA or RNA.

[0185] fusion proteins The term "fusion protein," as used herein, refers to a hybrid polypeptide comprising 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) portion of the protein, thereby forming an "amino-terminal fusion protein" or a "carboxy-terminal fusion protein," respectively. The protein may comprise different domains, such as a nucleic acid-binding domain (e.g., the gRNA-binding domain of Cas9, which directs the protein to bind to a target site) and a nucleic acid-cleavage domain or catalytic domain of a nucleic acid-editing protein. Another example includes Cas9 or its equivalent fused to a reverse transcriptase. Any of the proteins provided herein may be produced by any method known in the art. For example, the proteins provided herein may be produced via recombinant protein expression and purification, which is particularly suitable for fusion proteins containing peptide linkers. Methods for recombinant protein expression and purification are well known and include those described by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012) (the entire contents of which are incorporated herein by reference).

[0186] Guide RNA ("gRNA") As used herein, the term "guide RNA" refers to a specific type of guide nucleic acid that most commonly associates with the Cas protein of CRISPR-Cas9 and binds to Cas9 to direct the Cas9 protein to a specific sequence in a DNA molecule (including complementarity with the protospacer sequence of the guide RNA). However, the term also embraces equivalent guide nucleic acid molecules that bind to Cas9 equivalents, homologs, orthologs, or paralogs, whether naturally occurring or non-naturally occurring (e.g., engineered or recombinant), and that otherwise program the Cas9 equivalent to localize to a specific target nucleotide sequence. Cas9 equivalents may also include other napDNAbp from any type of CRISPR system (e.g., type II, type V, type VI), including Cpf1 (type V CRISPR-Cas system), C2c1 (type V CRISPR-Cas system), C2c2 (type VI CRISPR-Cas system), and C2c3 (type V CRISPR-Cas system). 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 and structures of guide RNAs are provided herein. In addition, methods for designing suitable guide RNA sequences are also provided herein. As used herein, "guide RNA" may also be referred to as "traditional guide RNA," in contrast to modified forms of guide RNAs called "prime-edited guide RNAs" (or "PEgRNAs").

[0187] Guide RNAs or PEGRNAs may contain a variety of structural elements, including but not limited to:

[0188] Spacer sequence - a sequence in the guide RNA or PEGRNA (having a length of approximately 20 nt) that binds to the protospacer in the target DNA.

[0189] gRNA core (or gRNA scaffold or backbone sequence)—refers to the sequence within the gRNA responsible for Cas9 binding, not including the 20-bp spacer / targeting sequence used to guide Cas9 to the target DNA. In some embodiments, the gRNA core or scaffold comprises a sequence that contains one or more nucleotide changes compared to a naturally occurring CRISPR-Cas guide RNA scaffold, such as a Cas9 guide RNA scaffold. In some embodiments, the sequence of the gRNA core is designed to contain minimal or no sequence homology to the endogenous sequence of the target nucleic acid at the target site, thereby reducing unintended editing. For example, in some embodiments, one or more base pairs within the second stem loop of the Cas9 gRNA core may be “flipped” (e.g., a GU base pair and a UA base pair, as illustrated in Figure 49A) to reduce unintended editing. In some embodiments, the gRNA core contains no more than 1%, 5%, 10%, 15%, 20%, 25%, or 30% sequence homology to the sequence of the double-stranded target DNA adjacent to 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides upstream or downstream of the position(s) of one or more nucleotide edits.

[0190] Extension Arm - A single-stranded extension at the 3' or 5' end of the PEGRNA, which contains a primer binding site and a DNA synthesis template sequence that encodes a single-stranded DNA flap containing the desired genetic alteration by a polymerase (e.g., reverse transcriptase), which then incorporates onto the endogenous DNA by displacing the corresponding endogenous strand, thereby installing the desired genetic alteration.

[0191] Transcription terminator - The guide RNA or PEgRNA may comprise a transcription termination sequence 3' of the molecule. In some embodiments, the PEgRNA comprises a transcription termination sequence between the DNA synthesis template and the gRNA core.

[0192] homology As used herein, the terms "homology," "homology," or "percent homology" refer to the degree of sequence identity between an amino acid or polynucleotide sequence and a corresponding reference sequence. "Homology" can refer to polymer sequences, e.g., similar polypeptide or DNA sequences. Homology can refer to, for example, a nucleic acid sequence having 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 a nucleic acid sequence may exhibit 93%, 95%, or 98% sequence identity to a reference nucleic acid sequence. For example, a "region of homology to a genomic region" can be a region of DNA having a sequence similar to a given genomic region within a genome. The region of homology can be of any length sufficient to facilitate binding of a spacer or protospacer sequence to a genomic region. For example, the homologous region can be 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, 20 ... The amino acid sequence may comprise 00, 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.

[0193] When a percentage of sequence homology or sequence identity is specified, in the context of two nucleic acid sequences or two polypeptide sequences, the percentage of homology or sequence identity generally refers to the alignment of two or more sequences over a portion of their length when compared and aligned for maximum correspondence. If a position in the compared sequences can be occupied by the same base or amino acid, the molecules can be homologous at that position. Unless otherwise specified, sequence homology or sequence identity is measured over a specific length of a nucleic acid, polypeptide, or portion thereof. In some embodiments, homology or identity is measured over a functional or specific portion of that length.

[0194] Alignment of sequences to assess sequence homology can be performed by algorithms known in the art, such as the Basic Local Alignment Search Tool (BLAST) algorithm described in Altschul et al., J. Mol. Biol. 215:403-410, 1990. A publicly available internet interface for performing BLAST analyses is accessible from the National Center for Biotechnology Information. Further known algorithms include those described by Smith & Waterman, "Comparison of Biosequences," Adv. Appl. 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 automated implementations of these or similar algorithms. Global alignment programs may be used to align similar sequences of approximately equal size. Examples of global alignment programs include NEEDLE (available at www.ebi.ac.uk / Tools / psa / emboss_needle / ), which is part of the EMBOSS package (Rice P et al., 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. Sci. USA, 85:2444-2448).Both of these programs are based on the Needleman-Wunsch algorithm, which is used to find the optimal 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 et al. ("Current Protocols in Molecular Biology", John Wiley & Sons Inc, 1994-1998, Chapter 15, 1998).

[0195] Those skilled in the art will understand that amino acid (or nucleotide) positions may be determined in homologous sequences based on alignment, for example, "H840" in a reference Cas9 sequence may correspond to H839, or another corresponding position in a Cas9 homolog when aligned to the reference Cas9 sequence.

[0196] host cell As used herein, the term "host cell" 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 Cas9 or a Cas9 equivalent and reverse transcriptase.

[0197] Inhibition As used herein, the term "inhibit", "inhibit" or "inhibition" in the context of proteins and enzymes, for example, in the context of enzymes involved in DNA mismatch repair pathways, refers to the reduction of the activity of protein or enzyme.In some embodiments, this term refers to the level of enzyme activity, for example, the activity level of one or more enzymes in DNA mismatch repair pathways, being reduced to a level that is statistically significantly lower than the initial level, which may be, for example, the baseline level of enzyme activity.In some embodiments, this term refers to the level of enzyme activity, for example, the activity of one or more enzymes in DNA mismatch repair pathways, being reduced 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 the initial level, which may be, for example, the baseline level of enzyme activity.

[0198] Linker The term "linker" as used herein refers to a molecule that connects two other molecules or moieties. A linker can be an amino acid sequence, in the case of a linker that connects two fusion proteins. For example, Cas9 can be fused to a reverse transcriptase by an amino acid linker sequence. A linker can also be a nucleotide sequence, in the case of linking two nucleotide sequences together. For example, in this case, a conventional guide RNA is linked via a spacer or linker nucleotide sequence to the RNA extension of a prime-edited guide RNA, which may include an RT template sequence and an RT primer binding site. In other embodiments, the linker is an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker is 5-100 amino acids in length, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30-35, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-150, or 150-200 amino acids in length. Longer or shorter linkers are also contemplated. In certain embodiments, the linker is an autohydrolyzable linker (e.g., a 2A self-cleaving peptide, as further described herein). Self-hydrolyzing linkers, such as 2A self-cleaving peptides, can induce ribosome skipping during protein translation, so that the ribosome cannot form a peptide bond between two genes or gene fragments.

[0199] MLH1 The term "MLH1" refers to the gene encoding the DNA mismatch repair enzyme MLH1 (or MutL homolog 1). The protein encoded by this gene can heterodimerize with the mismatch repair endonuclease PMS2 to form MutL alpha (MutLα), which is part of the DNA mismatch repair system. MLH1 mediates protein-protein interactions during mismatch recognition, strand discrimination, and strand removal. During mismatch repair, the heterodimer MSH2:MSH6 (MutSα) forms and binds to the mismatch. MLH1 then heterodimerizes with PMS2 (MutLα) and binds to the MSH2:MSH6 heterodimer. The MutLα heterodimer then cleaves the nicked strand 5' and 3' of the mismatch, followed by excision of the mismatch from the nick generated by MutLα by EXO1. Finally, POLδ resynthesizes the cleaved strand, followed by LIG1 ligation.

[0200] An exemplary amino acid sequence of MLH1 is human isoform 1, P40692-1:>sp|P40692|MLH1_HUMAN DNA mismatch repair protein Mlh1 OS=Homo sapiens OX=9606 GN=MLH1 PE=1 SV=1: (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 100% (inclusive) sequence identity to SEQ ID NO: 204.

[0201] Another exemplary amino acid sequence of MLH1 is human isoform 2, P40692-2 (amino acids 1-241 of isoform 1 are missing): DNA mismatch repair protein Mlh1 OS=Homo sapiens OX=9606 GN=MLH1>sp|P40692-2|MLH1_HUMAN isoform 2: MNGYISNANYSVKKCIFLLFINHRLVESTSLRKAIETVYAAYLPKNTHPFLYLSLEISPQNVDVNVHPTKHEVHFLHEESILERVQQHIESKLLGSNSSRMYFTQTLLPGLAGPSGEMVKSTTSLTSSSTS GSSDKVYAHQMVRTDSREQKLDAFLQPLSKPLSSQPQAIVTEDKTDISSGRARQQDEEMLELPAPAEVAAKNQSLEGDTTKGTSEMSEKRGPTSSNPRKRHREDSDVEMVEDDSRKEMTAACTPRRRIINL TSVLSLQEEINEQGHEVLREMLHNHSFVGCVNPQWALAQHQTKLYLLNTTKLSEELFYQILIYDFANFGVLRLSEPAPLFDLAMLALDSPESGWTEEDGPKEGLAEYIVEFLKKKAEMLADYFSLEIDEEGNLIGLPLLIDNYVPPLEGLPIFILRLATEVNWDEEKECFESLSKECAMFYSIRKQYISEESTLSGQQSEVPGSIPNSWKWTVEHIVYKALRSHILPPKHFTEDGNILQLANLPDLYKVFERC (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 100% (inclusive) sequence identity to SEQ ID NO: 205.

[0202] Another exemplary amino acid sequence of MLH1 is human isoform 3, P40692-3 (amino acids 1-101 (MSFVAGVIRR...ASISTYGFRG (SEQ ID NO: 206) are replaced by MAF): DNA mismatch repair protein Mlh1 OS=Homo sapiens OX=9606 GN=>sp|P40692-2|MLH1_HUMAN isoform 2 of MLH1: (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 100% (inclusive) sequence identity to SEQ ID NO: 207.

[0203] The present disclosure contemplates targeting MLH1 and / or components of the MMR pathway that interact with MLH1, including any wild-type or naturally occurring variant of MLH1, 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 to any of SEQ ID NOs: 204, 208-213, 215, 216, 218, 222, or 223, or nucleic acid molecules encoding any MLH1 or MLH1 variant (e.g., a dominant-negative mutant of MLH1 described herein), to inhibit, block, or otherwise inactivate wild-type MLH1 function in the MMR pathway, thereby inhibiting, blocking, or otherwise inactivating the MMR pathway, e.g., during genome editing using a prime editor.

[0204] 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 an MLH1 mutant protein or expressing an MLH1 mutant nucleic acid encoding an MLH1 mutant protein in a target cell. In some embodiments, the MLH1 mutant protein interferes with the function of the wild-type MLH1 protein in the MMR pathway, thereby inactivating it. In some embodiments, the MLH1 mutant is a dominant-negative mutant. In some embodiments, the MLH1 mutant protein can bind to an MLH1-interacting protein, such as MutS.

[0205] Without being bound by theory, the MLH1 dominant-negative mutant functions by saturating MutS binding, thereby blocking MutS-wild-type MLH1 binding and interfering with the function of the wild-type MLH1 protein in the MMR pathway.

[0206] In various embodiments, the dominant negative MLH1 is, for example, MLH1 E34A having the following amino acid sequence based on SEQ ID NO: 222 (underlined and bold to indicate the E34A mutation): [ka] (SEQ ID NO: 222), 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 100% (including 100%) sequence identity to SEQ ID NO: 222.

[0207] In various other embodiments, the dominant negative MLH1 is, for example, MLH1 Δ756, based on SEQ ID NO: 208, having the following amino acid sequence (underlined and bold to indicate the Δ756 mutation at the C-terminus of the sequence): [ka] [ka] (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 100% (including 100%) sequence identity to SEQ ID NO: 208 (where "[-]" indicates the amino acid residue(s) deleted compared to the parent or wild-type sequence).

[0208] In yet another embodiment, the dominant negative MLH1 is, for example, MLH1 Δ754-Δ756 having the following amino acid sequence, based on SEQ ID NO: 209 (underlined and bold to indicate the Δ754-Δ756 mutation at the C-terminus of the sequence): [ka] (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 100% (including 100%) sequence identity to SEQ ID NO: 209 ( [ka] indicates the amino acid residue(s) deleted compared to the parent or wild-type sequence).

[0209] In yet another embodiment, the dominant negative MLH1 has the following amino acid sequence, based on, for example, SEQ ID NO: 210: MLH1 E34A Δ754-Δ756 (underlined and bold to indicate the E34A and Δ754-Δ756 mutations): [ka] [ka] (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 100% (including 100%) sequence identity to SEQ ID NO: 210.

[0210] In certain embodiments, the dominant-negative MLH1 is, for example, based on SEQ ID NO: 211 and has the following amino acid sequence: MLH1 1-335 (containing amino acids 1 to 335 of SEQ ID NO: 204); MSFVAGVIRRLDETVVNRIAAGEVIQRPANAIKEMIENCLDAKSTSIQVIVKEGGLKLIQIQDNGTGIRKEDLDIVCERFTTSKLQSFEDLASISTYGFRGEALASISHVAHVTITTKTADGKCAYRASYSDGKLKAPPKPCAGNQGTQITVEDLFYNIATRRKALKNPSEE YGKILEVVGRYSVHNAGISFSVKKQGETVADVRTLPNASTVDNIRSIFGNAVSRELIEIGCEDKTLAFKMNGYISNANYSVKCCIFLLFINHRLVESTSLRKAIETVYAAYLPKNTHPFLYLSLEISPQNVDVNVHPTKHEVHFLHEESILERVQQHIESKLL (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 100% (including 100%) sequence identity to SEQ ID NO: 211.

[0211] In another aspect, the dominant-negative MLH1 is, for example, based on SEQ ID NO:212 and having the following amino acid sequence: MLH1 1-335 E34A (containing amino acids 1-335 of SEQ ID NO:204 and the E34A mutation relative to SEQ ID NO:204): [ka] (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 100% (including 100%) sequence identity to SEQ ID NO: 212.

[0212] In yet another embodiment, the dominant negative MLH1 has the following amino acid sequence, based on, for example, SEQ ID NO: 204: MLH1 1-335 NLS SV40 (or MLH1dn NTD ) (containing amino acids 1 to 335 of SEQ ID NO: 204 and the SV40 NLS sequence): [ka] [ka] (SEQ ID NO: 213), The underlined and bolded portion refers to the SV40 NLS), 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 100% (including 100%) sequence identity to SEQ ID NO: 213.

[0213] In yet another embodiment, the dominant negative MLH1 is, for example, 代替 (Based on SEQ ID NO: 204, having the following amino acid sequence (containing amino acids 1-335 of SEQ ID NO: 204 and an alternative NLS sequence): MSFVAGVIRRLDETVVNRIAAGEVIQRPANAIKEMIENCLDAKSTSIQVIVKEGGLKLIQIQDNGTGIRKEDLDIVCERFTTSKLQSFEDLASISTYGFRGEALASISHVAHVTITTKTADGKCAYRASYSDGKLKAPPKPCAGNQGTQITVEDLFYNIATRRKALKNPSEEYGKILEVVGR YSVHNAGISFSVKKQGETVADVRTLPNASTVDNIRSIFGNAVSRELIEIGCEDKTLAFKMNGYISNANYSVKCCIFLLFINHRLVESTSLRKAIETVYAAYLPKNTHPFLYLSLEISPQNVDVNVHPTKHEVHFLHEESILERVQQHIESKLL-[Alternative NLS Sequence] (SEQ ID NO: 214) - [Alternative 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 100% (including 100%) sequence identity to SEQ ID NO: 214. The alternative NLS sequence may be any suitable NLS sequence, including but not limited to: [Table 1] In some embodiments, the NLS sequence is added to the N-terminus of the protein and begins with a methionine ("M"). In other embodiments, the NLS sequence may be added to the C-terminus of the protein or between multiple domains of a fusion protein and does not begin with a methionine (i.e., for example, the M in SEQ ID NOs: 101, 1, and 134 is not encompassed by the NLS when added to the C-terminus or between two domains of a fusion protein).

[0214] In yet another embodiment, the dominant negative MLH1 is, for example, MLH1 501-756, which corresponds to a C-terminal fragment of SEQ ID NO: 204, corresponding to amino acids 501-756 of SEQ ID NO: 204: INLTSVLSLQEEINEQGHEVLREMLHNHSFVGCVNPQWALAQHQTKLYLLNTTKLSEELFYQILIYDFANFGVLRLSEPAPLFDLAMLALDSPESGWTEEDGPKEGLAEYIVEFLKKKAEMLADYFSLEIDEEGNLIGLPLLIDNYVPPLEGLPIFILRLATEVNWDEEKECFESLSKECAMFYSIRKQYISEESTLSGQQSEVPGSIPNSWKWTVEHIVYKALRSHILPPKHFTEDGNILQLANLPDLYKVFERC (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 100% (including 100%) sequence identity to SEQ ID NO: 215.

[0215] In yet another embodiment, the dominant negative MLH1 is, for example, MLH1 501-753, which corresponds to a C-terminal fragment of SEQ ID NO: 204, corresponding to amino acids 501-753 of SEQ ID NO: 204: [ka] (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 100% (including 100%) sequence identity to SEQ ID NO: 216.

[0216] In yet another embodiment, the dominant negative MLH1 is, for example, MLH1 461-756, a C-terminal fragment of SEQ ID NO: 204, corresponding to amino acids 461-756 of SEQ ID NO: 204: KRGPTSSNPRKRHREDSDVEMVEDDSRKEMTAACTPRRRIINLTSVLSLQEEINEQGHEVLREMLHNHSFVGCVNPQWALAQHQTKLYLLNTTKLSEELFYQILIYDFANFGVLRLSEPAPLFDLAMLALDSPESGWTEEDGPKEGLAEYIVEFLKKKAEMLADYFSLEIDEEGNLIGLPLLIDNYVPPLEGLPIFILRLATEVNWDEEKECFESLSKECAMFYSIRKQYISEESTLSGQQSEVPGSIPNSWKWTVEHIVYKALRSHILPPKHFTEDGNILQLANLPDLYKVFERC (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 100% (including 100%) sequence identity to SEQ ID NO: 217.

[0217] In various embodiments, the dominant negative MLH1 is, for example, MLH1 461-753, a C-terminal fragment of SEQ ID NO: 204, corresponding to amino acids 461-753 of SEQ ID NO: 204: [ka] (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 100% (including 100%) sequence identity to SEQ ID NO: 218. In various other embodiments, the dominant negative MLH1 is a C-terminal fragment of SEQ ID NO: 204, e.g., corresponding to amino acids 461-753 of SEQ ID NO: 204, and does not contain an N-terminal NLS, e.g., NLS SV40 MLH1 461-753, which also includes: [ka] (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 100% (including 100%) sequence identity to 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.

[0218] napDNAbp As used herein, the term "nucleic acid programmable DNA binding protein" or "napDNAbp" (of which Cas9 is an example) refers to a protein that uses RNA:DNA hybridization to target and bind to specific sequences in a DNA molecule. Each napDNAbp is associated with at least one guide nucleic acid (e.g., a guide RNA) that localizes the napDNAbp to a DNA sequence that includes a DNA strand (i.e., the target strand) that is complementary to the guide nucleic acid or a portion thereof (e.g., the protospacer of the guide RNA). In other words, the guide nucleic acid "programs" the napDNAbp (e.g., Cas9 or equivalent) to localize and bind to the complementary sequence.

[0219] Without being bound by theory, the binding mechanism of the napDNAbp-guide RNA complex generally involves the napDNAbp forming an R-loop that induces unwinding of the double-stranded DNA target (thereby separating the strands in the region bound by the napDNAbp). The guide RNA spacer sequence then hybridizes to the "target strand." This displaces the "non-target strand," which is complementary to the target strand, forming the single-stranded region of the R-loop. In some embodiments, the napDNAbp contains one or more nuclease activities, which then cleave the DNA to release various types of lesions. For example, the napDNAbp may contain nuclease activities that cleave the non-target strand at a first location and / or the target strand at a second location. In response to the nuclease activity, the target DNA may be cleaved to form a "double-stranded break" in which both strands are cleaved. In other embodiments, the target DNA may be cleaved at only a single site, i.e., the DNA is "nicked" on one strand. Exemplary napDNAbps with various nuclease activities include "Cas9 nickase" ("nCas9") and inactivated Cas9 that lacks any nuclease activity ("inactivated Cas9" or "dCas9"). Exemplary sequences for these and other napDNAbps are provided herein.

[0220] Nickase As used herein, the term "nickase" may refer to a Cas9 in which one of the two nuclease domains has been inactivated. This enzyme is capable of cleaving only one strand of a target DNA. As used herein, "nickase" may refer to a napDNAbp (e.g., a Cas protein) that can cleave 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 the 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 the catalytic domain of a standard napDNAbp (e.g., a Cas protein), where the one or more mutations reduce or eliminate the 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 includes one or more mutations in the RuvC-like domain relative to the wild-type Cas9 sequence or to equivalent amino acid positions in other Cas9 variants or Cas9 equivalents. In some embodiments, the nickase is a Cas9 that includes one or more mutations in the HNH-like domain relative to the wild-type Cas9 sequence or to equivalent amino acid positions in other Cas9 variants or Cas9 equivalents. In some embodiments, the nickase is a Cas9 that includes an aspartic acid to alanine substitution (D10A) in the RuvC I catalytic domain of Cas9 relative to the standard Cas9 sequence or to equivalent amino acid positions in other Cas9 variants or Cas9 equivalents. In some embodiments, the nickase is a Cas9 that includes H840A, N854A, and / or N863A mutations relative to the standard Cas9 sequence or to equivalent amino acid positions in other Cas9 variants or Cas9 equivalents. In some embodiments, the term "Cas9 nickase" refers to a Cas9 in which one of the two nuclease domains has been inactivated. This enzyme can cleave only one strand of the target DNA. In some embodiments, the nickase is a Cas protein that is not a Cas9 nickase.

[0221] In some embodiments, the napDNAbp of the primed editing complex comprises an endonuclease with 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 nicks in each strand. For example, a nuclease-active Cas protein can generate nicks in each strand of a double-stranded target DNA. In some embodiments, the two nicks on both strands are staggered nicks generated by a napDNAbp comprising, e.g., Cas12a or Cas12b1. In some embodiments, the two nicks on both strands are at the same genomic location, generated by a napDNAbp comprising, e.g., Cas9 comprising a nuclease-active Cas. In some embodiments, the napDNAbp comprises an endonuclease that is a nickase. For example, in some embodiments, the napDNAbp comprises an endonuclease that contains one or more mutations that reduce the 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 that contains one or more mutations that abolish the nuclease activity. In various embodiments, the napDNAbp is a Cas9 protein or a variant thereof. The napDNAbp can also be a nuclease-active Cas9, a nuclease-inactive Cas9 (dCas9), or a Cas9 nickase (nCas9). In preferred embodiments, the napDNAbp is a Cas9 nickase (nCas9) that nicks only one strand. In certain embodiments, the napDNAbp is a Cas9 nickase, a Cas12a nickase, or a Cas12b1 nickase.In some aspects, the napDNAbp may be selected from the group consisting of Cas9, Cas12e, Cas12d, Cas12a, Cas12b1, Cas12b2, Cas13a, Cas12c, Cas12d, Cas12e, Cas12h, Cas12i, Cas12g, Cas12f (Cas14), Cas12f1, Cas12j (CasΦ), and Argonaute, and optionally has nickase activity such that only one strand is cleaved. In some embodiments, the napDNAbp is selected from Cas9, Cas12e, Cas12d, Cas12a, Cas12b1, Cas12b2, Cas13a, Cas12c, Cas12d, Cas12e, Cas12h, Cas12i, Cas12g, Cas12f (Cas14), Cas12f1, Cas12j (CasΦ), and Argonaute, and optionally has nickase activity such that one DNA strand is preferentially cleaved relative to the other DNA strand.

[0222] Nick site The terms "cleavage site," "nick site," and "cleavage site," used interchangeably herein in the context of prime editing, refer to a specific position between two nucleotides or two base pairs in a double-stranded target DNA sequence. In some embodiments, the location of the nick site is determined relative to the location of a specific PAM sequence. In some embodiments, the nick site is a specific position where a nick occurs when double-stranded target DNA that recognizes a specific PAM sequence is contacted with a napDNAbp, e.g., a nickase such as a Cas nickase. For each PEgRNA described herein, the nick site is characteristic of the specific napDNAbp with which the gRNA core of the PEgRNA associates and is characteristic of the specific PAM required for recognition and function of the napDNAbp. For example, in the case of a PEgRNA comprising a gRNA core that associates with SpCas9, the nick site is at a phosphodiester bond between base 3 (the "-3" position relative to position 1 of the PAM sequence) and base 4 (the "-4" position relative to position 1 of the PAM sequence).

[0223] In some embodiments, the nick site is in the target strand of the double-stranded target DNA sequence. In some embodiments, the nick site is in the non-target strand of the double-stranded target DNA sequence. In some embodiments, the nick site is in the protospacer sequence. In some embodiments, the nick site is adjacent to the protospacer sequence. In some embodiments, the nick site is downstream of a region complementary to the primer binding site of the PEgRNA, e.g., on the non-target strand. In some embodiments, the nick site is downstream of a region binding to the primer binding site of the PEgRNA, e.g., on the non-target strand. In some embodiments, the nick site is immediately downstream of a region complementary to the primer binding site of the PEgRNA, e.g., on the non-target strand. In some embodiments, the nick site is upstream of a specific PAM sequence on the non-target strand of the double-stranded target DNA, the PAM sequence being specific for recognition by the napDNAbp associated with the gRNA core of the 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, and the PAM sequence is specific for recognition by the napDNAbp associated with the gRNA core of the PE gRNA. In some embodiments, the nick site is three nucleotides upstream of the PAM sequence, and the PAM sequence is recognized by Streptococcus pyogenes Cas9 nickase, P. lavamentivorans Cas9 nickase, C. diphtheriae Cas9 nickase, N. cinerea Cas9, S. aureus Cas9, or N. lari Cas9 nickase. In some embodiments, the nick site is three nucleotides upstream of the PAM sequence, and the PAM sequence is recognized by Cas9 nickase, which comprises a nuclease-active HNH domain and a nuclease-inactive RuvC domain. In some embodiments, the nick site is two base pairs upstream of the PAM sequence, and the PAM sequence is recognized by S. thermophilus Cas9 nickase.

[0224] nucleic acid molecule The term "nucleic acid," as used herein, refers to a polymer of nucleotides. The polymer may be composed of 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-methyladenosine, 5-methylcytidine, C5 bromouridine, C5 fluorouridine, C5 iodouridine, C5 propynyluridine, C5 propynylcytidine, C5 methylcytidine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)methylguanine, 4-acetylcytidine, 5 ... The bases may include bases such as uridine, 5-(carboxyhydroxymethyl)uridine, dihydrouridine, methylpseudouridine, 1-methyladenosine, 1-methylguanosine, N6-methyladenosine, and 2-thiocytidine, chemically modified bases, biologically modified bases (e.g., methylated bases), intercalated bases, modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, 2'-O-methylcytidine, arabinose, and hexose), or modified phosphate groups (e.g., phosphorothioate and 5'-N phosphoramidite linkages).

[0225] P53 As used herein, "p53" refers to tumor protein 53. Among other functions, p53 plays a role in DNA damage and repair, particularly in its role in regulating the cell cycle, apoptosis, and genomic stability. P53 can activate DNA repair proteins when DNA is damaged. P53 can also arrest cell proliferation by holding the cell cycle at the G1 / S regulatory point of DNA damage recognition, thereby providing DNA repair proteins more time to repair DNA damage before allowing the cell to continue through the cell cycle. Thus, in some embodiments of the methods described herein, p53 is inhibited (e.g., with the p53 inhibitor protein "i53" or another p53 inhibitor) to increase the efficiency of prime editing.

[0226] PEG-RNA As used herein, the terms "prime editing guide RNA" or "PEgRNA" or "extended guide RNA" refer to specialized forms of guide RNA that have been modified to include one or more additional sequences for practicing the prime editing methods and compositions described herein. As described herein, a prime editing guide RNA includes one or more "extended regions" of nucleic acid sequence. The extended region may include, but is not limited to, a single strand of RNA or DNA. Additionally, the extended region may occur at the 3' end of an existing guide RNA. In other configurations, the extended region may occur at the 5' end of an existing guide RNA. In yet other configurations, the extended region may occur at an intramolecular region at the end of an existing guide RNA (e.g., in the gRNA core region that attaches and / or binds to the napDNAbp). The extended region comprises a "DNA synthesis template" that encodes a single-stranded DNA (by the polymerase of the prime editor), a molecule that is then (a) designed to be homologous to the endogenous target DNA to be edited, and (b) contains at least one desired nucleotide change (e.g., a transition, transversion, deletion, or insertion) to be introduced or incorporated into the endogenous target DNA. The extended region may also contain other functional sequence elements (such as, but not limited to, a "primer binding site" and a "spacer or linker" sequence), or other structural elements (such as, but not limited to, an aptamer, a stem-loop, a hairpin, a two-loop (e.g., a 3' two-loop), or an RNA-protein recruitment domain (e.g., an MS2 hairpin)). As used herein, a "primer binding site" includes a sequence that hybridizes to a single-stranded DNA sequence having a 3' end generated from the R-loop nicked DNA.

[0227] In certain embodiments, the PEG RNA has a 5' extension arm, a spacer, and a gRNA core. The 5' extension further comprises, in a 5' to 3' direction, a reverse transcriptase template, a primer binding site, and a linker. The reverse transcriptase template may also be more broadly referred to as a "DNA synthesis template," where the polymerase of the prime editor described herein is not RT but another type of polymerase.

[0228] In certain other embodiments, the PEGRNA has a 5' extension arm, a spacer, and a gRNA core. The 5' extension further comprises, in a 5' to 3' direction, a reverse transcriptase template, a primer binding site, and a linker. The reverse transcriptase template may also be more broadly referred to as a "DNA synthesis template," where the polymerase of the prime editor described herein is not RT but another type of polymerase.

[0229] In yet another embodiment, the PEGRNA comprises, from 5' to 3', 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, from 5' to 3', a "homology arm," an "editing template," and a "primer binding site." In certain embodiments, the PEGRNA comprises, from 5' to 3', a space, a DNA synthesis template, and a primer binding site. The extension arm (3) may also comprise optional modification regions at the 3' and 5' ends, which may be the same or different sequences. The 3' end of the PEGRNA may comprise a transcription terminator sequence. These sequence elements of the PEGRNA are further described and defined herein.

[0230] In yet another embodiment, the PEGRNA comprises, from 5' to 3', 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, from 3' to 5', a "homology arm," an "editing template," and a "primer binding site." The extension arm (3) may also comprise optional modification regions at the 3' and 5' ends, which may be the same or different sequences. The PEGRNA may also comprise a transcription terminator sequence at the 3' end. These sequence elements of the PEGRNA are further described and defined herein.

[0231] PE1 As used herein, "PE1" refers to a primed editor system comprising a fusion protein comprising Cas9(H840A) with the following structure: [NLS]-[Cas9(H840A)]-[linker]-[MMLV_RT(wt)]+desired PE gRNA and wild-type MMLV RT, where the PE fusion has the amino acid sequence of SEQ ID NO: 100, shown as follows: [ka] [ka]

[0232] Alternatively, PE1 may also refer to the prime editor fusion protein of SEQ ID NO: 100, i.e., without the pegRNA complexed thereto. PE1 may be complexed to the pegRNA during operation and / or use in prime editing.

[0233] PE2 As used herein, "PE2" refers to a primed editing system comprising a fusion protein comprising Cas9(H840A) and a variant MMLV RT having the following structure: [NLS]-[Cas9(H840A)]-[linker]-[MMLV_RT(D200N)(T330P)(L603W)(T306K)(W313F)]+a desired PEgRNA, wherein the PE fusion has the amino acid sequence of SEQ ID NO: 107, as shown below. [ka] [ka]

[0234] 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 to the pegRNA during operation and / or use in prime editing.

[0235] PE3 As used herein, "PE3" refers to PE2 plus a second strand nicking guide RNA that complexes with PE2 to introduce a nick on the non-edited DNA strand to direct preferential displacement of the edited strand.

[0236] PE3b As used herein, "PE3b" refers to PE3, but the second-strand nicking guide RNA is designed for temporal control by not incorporating a second-strand nick until after the desired edit has been incorporated. This is achieved by designing a gRNA with a spacer sequence that matches only the edited strand, not the original allele. Using this strategy, hereafter referred to as PE3b, a mismatch between the protospacer and the non-edited allele should not favor nicking by the sgRNA until after the editing event on the PAM strand has occurred.

[0237] PE4 As used herein, "PE4" refers to a system comprising PE2 and an MLH1 dominant-negative protein (i.e., wild-type MLH1 truncated at amino acids 754-756, as further described herein) expressed in trans. In some embodiments, PE4 refers to a fusion protein comprising PE2 and an MLH1 dominant-negative protein linked via an optional linker.

[0238] PE5 As used herein, "PE5" refers to a system comprising PE3 and an MLH1 dominant-negative protein (i.e., wild-type MLH1 lacking amino acids 754-756, as further described herein (which may be referred to as "MLH1 Δ754-756" or "MLH1dn")) expressed in trans. In some embodiments, PE5 refers to a fusion protein comprising PE3 and an MLH1 dominant-negative protein linked via an optional linker.

[0239] PEmax As used herein, "PEmax" (see Figure 54B) 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)]-[linker]-[MMLV_RT(D200N)(T330P)(L603W)]-[bipartite NLS]-[NLS]+desired PE gRNA, wherein the PE fusion has the amino acid sequence of SEQ ID NO:99.

[0240] PE4max As used herein, "PE4max" refers to PE4, but with the PE2 moiety replaced by PEmax.

[0241] PE5max As used herein, "PE5max" refers to PE5, but where the PE2 moiety of PE3 has been replaced with PEmax.

[0242] PE-short As used herein, "PE-short" refers to a PE construct fused to a C-terminal truncating reverse transcriptase and having the following amino acid sequence: [ka] [ka]

[0243] polymerase As used herein, the term "polymerase" refers to an enzyme that synthesizes a nucleotide strand, which may be used in connection with the prime editor system described herein. The polymerase may be a "template-dependent" polymerase (i.e., a polymerase that synthesizes a nucleotide strand based on the order of nucleotide bases in a template strand). The polymerase may also be a "template-independent" polymerase (i.e., a polymerase that synthesizes a nucleotide strand without the requirement of a template strand). The 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 may be a "DNA-dependent DNA polymerase" (i.e., whereby the template molecule is a strand of DNA). In such cases, the DNA template molecule may be a PEG-RNA and 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., a guide RNA component including a spacer and a gRNA core) and a DNA portion (i.e., an extension arm). In various other embodiments, the DNA polymerase may be an "RNA-dependent DNA polymerase" (i.e., according to which the template molecule is a strand of RNA). In such cases, the PEGRNA is RNA, i.e., involves RNA extension. The term "polymerase" may also refer to an enzyme that catalyzes the polymerization of nucleotides (i.e., polymerase activity). Generally, the enzyme will initiate synthesis at the 3' end of a primer annealed to a polynucleotide template sequence (e.g., a primer sequence annealed to the primer binding site of the PEGRNA) and proceed toward the 5' end of the template strand. A "DNA polymerase" catalyzes the polymerization of deoxynucleotides. As used herein with reference to a DNA polymerase, the term DNA polymerase encompasses "functional fragments thereof." A "functional fragment thereof" refers to any portion of a wild-type or mutant DNA polymerase encompassing less than the entire amino acid sequence of the polymerase, which portion retains the ability to catalyze the polymerization of polynucleotides under at least one set of conditions.Such a functional fragment may exist as a separate entity, or it may be a component of a larger polypeptide such as a fusion protein.

[0244] Prime Edit As used herein, the term "prime editing" refers to an approach to gene editing that uses a napDNAbp, a polymerase (e.g., reverse transcriptase), and a specialized guide RNA that contains a DNA synthesis template for encoding the desired new genetic information (or deleting genetic information), which is then incorporated into the target DNA sequence. A specific embodiment of prime editing is depicted in the embodiment of FIG. 1. Classical prime editing is described in the inventors' publication, Anzalone, AV et al., "Search-and-replace genome editing without double-strand breaks or donor DNA." Nature 576, 149-157 (2019), the entire contents of which are incorporated herein by reference.

[0245] Prime editing represents a platform for genome editing, a versatile and precise genome editing method that directly writes new genetic information at defined DNA sites. It uses a nucleic acid-programmable DNA-binding protein ("napDNAbp") that works in conjunction with a polymerase (i.e., in the form of a fusion protein or otherwise provided in trans with the napDNAbp). The prime editing system is programmed by a prime editing (PE) guide RNA ("PEgRNA"), which defines the target site and serves as a template for synthesis of the desired edit in the form of a replaced DNA strand by an extension (either DNA or RNA) engineered onto the guide RNA (e.g., at the 5' or 3' end of the guide RNA or internally). The replacement strand containing the desired edit (e.g., a single nucleobase substitution) shares the same sequence as (or is homologous to) the endogenous strand at the target site to be edited (immediately downstream of the nick site), except that it encompasses the desired edit. DNA repair and / or replication mechanisms replace the endogenous strand downstream of the nick site with the newly synthesized replacement strand containing the desired edit. In some cases, prime editing may be considered a "search and replace" genome editing technology, because the prime editors described herein not only search for and locate the desired target site to be edited, but also simultaneously encode a replacement strand containing the desired edit that is incorporated into the endogenous DNA strand at the corresponding target site. The prime editors disclosed herein relate, in part, to the mechanism of target-priming reverse transcription (TPRT), which can be engineered to perform precise CRISPR / Cas-based genome editing with high efficiency and genetic flexibility. TPRT is used by mobile DNA elements such as mammalian non-LTR retrotransposons and bacterial group II introns. Herein, we demonstrate how Cas protein-reverse transcriptase fusions or related systems can be used to target specific DNA sequences with a guide RNA, generate a single-stranded nick at the target site, and use the nicked DNA as a primer for reverse transcription of the engineered reverse transcriptase template incorporated into the guide RNA.However, while the concept begins with a prime editor using reverse transcriptase as the DNA polymerase component, the prime editors described herein are not limited to reverse transcriptase and may encompass the use of virtually any DNA polymerase. Indeed, although the present specification may refer to a prime editor having a "reverse transcriptase" throughout, it is expressly stated here that reverse transcriptase is only one type of DNA polymerase that may function in prime editing. Therefore, whenever the specification refers to a "reverse transcriptase," those skilled in the art should understand that any suitable DNA polymerase may be used in place of reverse transcriptase. Thus, in one aspect, a prime editor 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 on the target DNA. The specialized guide RNA also contains new genetic information in the form of an extension encoding a replacement strand of DNA containing the desired genetic alteration, which is used to replace the corresponding endogenous DNA strand at the target site. To transfer information from the PEGRNA into the target DNA, the mechanism of prime editing involves nicking the target site on one strand of DNA to expose a 3' hydroxyl group. The exposed 3' hydroxyl group can then be used to prime DNA polymerization of the extension encoding the edit on the PEGRNA directly to the target site. In various embodiments, the extension that provides a template for polymerization of the replacement strand containing the edit can be formed from RNA or DNA. In the case of RNA extension, the polymerase of the prime editor can be an RNA-dependent DNA polymerase (e.g., reverse transcriptase). In the case of DNA extension, the polymerase of the prime editor can be a DNA-dependent DNA polymerase.The newly synthesized strand formed by the primed editing elements disclosed herein (i.e., the replacement DNA strand containing the desired edit) will be homologous to (i.e., have the same sequence as) the genomic target sequence, except for the inclusion of the 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-stranded DNA flap. It will compete for hybridization with the complementary homologous endogenous DNA strand, thereby displacing the corresponding endogenous strand. In some embodiments, the system may be combined with the use of an error-prone reverse transcriptase enzyme (e.g., provided as a fusion protein with a Cas9 domain or provided in trans relative to the Cas9 domain). The error-prone reverse transcriptase enzyme may introduce alterations during synthesis of the single-stranded DNA flap. Thus, in some embodiments, an error-prone reverse transcriptase may be utilized to introduce nucleotide changes into the target DNA. Depending on the error-prone reverse transcriptase used in the system, the alterations may be random or non-random. Degradation of the hybridized intermediate (including the single-stranded DNA flap synthesized by the reverse transcriptase hybridized to the endogenous DNA strand) can involve removal of the resulting displaced flap of the endogenous DNA (e.g., by the 5'-end DNA flap endonuclease FEN1), ligation of the synthesized single-stranded DNA flap to the target DNA, and assimilation of the desired nucleotide change as a result of cellular DNA repair and / or replication processes. Because templated DNA synthesis offers single-nucleotide precision for any nucleotide modification, including insertions and deletions, the scope of this approach is very broad and can foreseeably be used in countless applications in basic research and therapeutics.

[0246] In various embodiments, prime editing operates by contacting a target DNA molecule (in which a nucleotide sequence change 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) includes an extension at the 3' or 5' end of the guide RNA, or at an intramolecular location within the guide RNA, that encodes the desired nucleotide change (e.g., a single nucleotide change, insertion, or deletion). In step (a), the napDNAbp / extended gRNA complex contacts the DNA molecule, and the extended gRNA guides the napDNAbp to bind to the target locus. In step (b), a nick is introduced (e.g., with a nuclease or chemical agent) into one of the strands of DNA at the target locus, thereby creating an accessible 3' end on one of the strands at the target locus. In some embodiments, the nick is created on the strand of DNA corresponding to the R-loop strand, i.e., the strand that is not hybridized to the guide RNA sequence, i.e., the "non-target strand." However, a nick can be introduced into either strand. That is, a nick could be introduced into the "target strand" of the R-loop (i.e., the strand hybridized to the protospacer of the extended gRNA) or the "non-target strand" (i.e., the strand that forms the single-stranded portion of the R-loop and is complementary to the target strand). In step (c), the 3' end of the DNA strand (formed by the nick) interacts with the extended portion of the guide RNA to prime reverse transcription (i.e., "RT primed to prime target"). In some embodiments, the 3' end DNA strand hybridizes to a specific RT prime sequence in the extended portion of the guide RNA, i.e., the "reverse transcriptase prime sequence" or "primer binding site" on the PEGRNA. In step (d), a reverse transcriptase (or other suitable DNA polymerase) is introduced. This synthesizes a single strand of DNA from the 3' end of the primed site toward the 5' end of the primed edited guide RNA. The DNA polymerase (eg, reverse transcriptase) may be fused to the napDNAbp or alternatively may be provided in trans relative to the napDNAbp.This forms a single-stranded DNA flap containing the desired nucleotide change (e.g., a single-base change, insertion, or deletion, or a combination thereof) that is otherwise homologous to the endogenous DNA at or adjacent to the nick site. In step (e), the napDNAbp and guide RNA are released. Steps (f) and (g) involve degradation of the single-stranded DNA flap, resulting in the desired nucleotide change being incorporated into the target locus. This process can be driven toward desired product formation by removing the corresponding 5' endogenous DNA flap, which forms once the 3' single-stranded DNA flap invades and hybridizes with the endogenous DNA sequence. Without being bound by theory, the cell's endogenous DNA repair and replication processes degrade the mismatched DNA and incorporate the nucleotide change(s) to form the desired altered product. The process can also be driven toward product formation by "second-strand nicking." This process may introduce at least one or more of the following genetic changes: transversions, transitions, deletions, and insertions.

[0247] The terms "prime editing element (PE) system" or "prime editing element (PE)" or "PE system" or "PE editing system" refer to compositions involved in the methods of genome editing using target-primed reverse transcription (TPRT) described herein, including, but not limited to, napDNAbps, reverse transcriptase (or another DNA polymerase), a fusion protein (e.g., comprising napDNAbps and reverse transcriptase, or comprising napDNAbps and a DNA polymerase), a prime editing guide RNA, and a complex comprising the fusion protein and the prime editing guide RNA, as well as auxiliary elements such as a second-strand nicking component (e.g., a second-strand sgRNA) and a 5' endogenous DNA flap removal endonuclease (e.g., FEN1) to help drive the prime editing process toward editing product formation.

[0248] 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 a primer binding site and a DNA synthesis template; however, the PEGRNA may also take the form of two individual molecules: a guide RNA and a trans-prime editor RNA template (tPERT), which essentially houses an extension arm (including, inter alia, a primer binding site and a DNA synthesis domain) and an RNA-protein recruitment domain (e.g., an MS2 aptamer or hairpin) on the same molecule, which becomes co-localized or recruited to a modified prime editor complex comprising a tPERT recruitment protein (e.g., an MS2cp protein that binds to the MS2 aptamer).

[0249] Prime editing factor The term "prime editor" refers to a fusion construct comprising a napDNAbp (e.g., Cas9 nickase) and a reverse transcriptase, which is capable of performing prime editing on a target nucleotide sequence in the presence of a PEgRNA (or "extended guide RNA"). The term "prime editor" may also refer to a fusion protein, or a fusion protein complexed with a PEgRNA and / or a fusion protein further complexed with an sgRNA that nicks the second strand. In some embodiments, a prime editor may also refer to a complex comprising a fusion protein (reverse transcriptase fused to a napDNAbp), a PEgRNA, and a regular guide RNA that is capable of directing the nicking step to the second site 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 with an inhibitor of the DNA mismatch repair pathway, such as a dominant-negative MLH1 protein. In various embodiments, an inhibitor of a DNA mismatch repair pathway, such as a dominant-negative MLH1 protein, may be provided in trans to the prime editor. In other embodiments, an inhibitor of a DNA mismatch repair pathway, such as a dominant-negative MLH1 protein, may be conjugated to the prime editor, e.g., coupled to the prime editor fusion protein via a linker.

[0250] Primer binding site The term "primer binding site" or "PBS" refers to a portion of a PEgRNA as a component of an extension arm (e.g., at the 3' end of the extension arm). The term "primer binding site" refers to a single-stranded portion of a PEgRNA as a component of an extension arm that includes 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 the nick site in the non-target strand. In some embodiments, the primer binding site is complementary to a region immediately upstream of the nick site in the non-target strand. In some embodiments, the primer binding site can bind to a primer sequence formed after nicking of the target sequence by a prime editor (e.g., the nickase component of the prime editor, e.g., the Cas9 nickase). When the prime editor cleaves one strand of the target DNA sequence (e.g., the Cas nickase component of the prime editor), a 3'-end ssDNA flap is formed, which serves as a primer sequence that anneals to the primer binding site on the PEgRNA to prime reverse transcription. In some embodiments, the PBS is complementary or substantially complementary to and can anneal to the free 3' end on the non-target strand of the double-stranded target DNA at the site of the nick. In some embodiments, the PBS annealed to the free 3' end on the non-target strand can initiate target-primed DNA synthesis.

[0251] Protospacer As used herein, the term "protospacer" refers to a sequence (~20 bp) on 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, to one strand of it, i.e., the "target strand" versus the "non-target strand" of the target DNA sequence). In some embodiments, the Cas protein component itself, e.g., a specific protospacer adjacent motif (PAM), which varies depending on the type of Cas protein, is also required for the Cas nickase component of the prime editor to function. For example, the most commonly used Cas9 nuclease from S. pyogenes recognizes the PAM sequence NGG on the non-target strand, which is found directly downstream of the target sequence on genomic DNA. Those skilled in the art will appreciate that the state of the art literature sometimes refers to the "protospacer" as the ~20-nt target-specific guide sequence on the guide RNA itself, rather than referring to it as a "spacer." Therefore, in some cases, the term "protospacer" as used herein may be used interchangeably with the term "spacer." The context surrounding the appearance of either "protospacer" or "spacer" will help inform the reader as to whether the term refers to a gRNA or a DNA target.

[0252] Protospacer adjacent motif (PAM) As used herein, the term "protospacer adjacent sequence" or "PAM" refers to a DNA sequence of approximately 2-6 base pairs that is the key targeting component of the Cas9 nuclease. Typically, the PAM sequence is located on either strand, downstream in the 5' to 3' direction from the site cut by Cas9. The standard PAM sequence (i.e., the PAM sequence associated with Streptococcus pyogenes Cas9 nuclease or SpCas9) is 5'-NGG-3', where "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. Additionally, any given Cas9 nuclease, e.g., SpCas9, can be modified to alter the nuclease's PAM specificity by allowing the nuclease to recognize alternative PAM sequences.

[0253] For example, with reference to the standard SpCas9 amino acid sequence of SEQ ID NO: 2, the PAM sequence can be modified by introducing one or more mutations, including (a) the D1135V, R1335Q, and T1337R "VQR variant," which alters the PAM specificity to NGAN or NGNG; (b) the D1135E, R1335Q, and T1337R "EQR variant," which alters the PAM specificity to NGAG; and (c) the D1135V, G1218R, R1335E, and T1337R "VRER variant," which alters the PAM specificity to NGCG. In addition, the D1135E variant of standard SpCas9 still recognizes NGG, but more selectively than the wild-type SpCas9 protein.

[0254] It will also be understood that Cas9 enzymes from different bacterial species (i.e., Cas9 orthologs) can have different PAM specificities. For example, Cas9 from Staphylococcus aureus (SaCas9) recognizes NGRRT or NGRRN. Additionally, Cas9 from Neisseria meningitis (NmCas) recognizes NNNNGATT. In another example, Cas9 from Streptococcus thermophilis (StCas9) recognizes NNAGAAW. In yet another example, Cas9 from Treponema denticola (TdCas) recognizes NAAAAC. These are examples and are not meant to be limiting. Furthermore, it will be understood that non-SpCas9s bind to a variety of PAM sequences, making them useful when a suitable SpCas9 PAM sequence is not present at the desired target cut site. Furthermore, non-SpCas9s may have other characteristics that make them more useful than SpCas9s. For example, Cas9 from Staphylococcus aureus (SaCas9) is approximately 1 kilobase smaller than SpCas9. Therefore, it can be packaged into adeno-associated virus (AAV). Further reference may be made to Shah et al., "Protospacer recognition motifs: mixed identities and functional diversity," RNA Biology, 10(5):891-899, which is incorporated herein by reference.

[0255] reverse transcriptase The term "reverse transcriptase" describes a class of polymerases characterized as RNA-dependent DNA polymerases. All known reverse transcriptases require a primer to synthesize DNA transcripts from an RNA template. Historically, reverse transcriptases have primarily been used to transcribe mRNA into cDNA, which can then be cloned onto a vector for further manipulation. Avian myoblastosis virus (AMV) reverse transcriptase was the first widely used RNA-dependent DNA polymerase (Verma, Biochim. Biophys. Acta 473:1 (1977)). The enzyme possesses 5'-3' RNA-directed DNA polymerase activity, 5'-3' DNA-directed DNA polymerase activity, and RNase H activity. RNase H is a processive 5' and 3' ribonuclease specific for the RNA strand of an RNA-DNA hybrid (Perbal, A Practical Guide to Molecular Cloning, New York: Wiley & Sons (1984)). Known viral reverse transcriptases lack the 3'-5' exonuclease activity necessary for proofreading, so transcription errors cannot be corrected by the reverse transcriptase (Saunders and Saunders, Microbial Genetics Applied to Biotechnology, London: Croom Helm (1987)). A detailed study of the activity of AMV reverse transcriptase and its associated RNase H activity was presented by Berger et al., Biochemistry 22:2365-2372 (1983). Another reverse transcriptase extensively used in molecular biology is the reverse transcriptase originating from Moloney murine leukemia virus (M-MLV). See, e.g., Gerard, GR, DNA 5:271-279 (1986) and Kotewicz, ML, et al., Gene 35:249-258 (1985). An M-MLV reverse transcriptase substantially lacking RNase H activity has also been described. See, for example, US Pat. No. 5,244,797. The present invention contemplates the use of any such reverse transcriptase or variant or mutant thereof.

[0256] In addition, the present invention contemplates the use of reverse transcriptases that are error-prone, or may be referred to as error-prone reverse transcriptases, or reverse transcriptases that do not support high-fidelity incorporation of nucleotides during polymerization. During the synthesis of a single-stranded DNA flap based on a guide RNA and an incorporated RT template, the error-prone reverse transcriptase may introduce one or more nucleotides that are mismatched with the RT template sequence, thereby introducing changes to the nucleotide sequence through error-prone polymerization of the single-stranded DNA flap. These errors introduced during the synthesis of the single-stranded DNA flap are then incorporated into the double-stranded molecule by hybridization to the corresponding endogenous target strand, removal of the displaced endogenous strand, ligation, and then another round of endogenous DNA repair and / or sequencing processes.

[0257] Reverse transcription As used herein, the term "reverse transcription" refers to the ability of an enzyme to synthesize a DNA strand (i.e., complementary DNA or cDNA) using RNA as a template. In some embodiments, reverse transcription can be "error-prone reverse transcription." This refers to the property of certain reverse transcriptases that their DNA polymerization activity is error-prone.

[0258] Proteins, peptides, and polypeptides The terms "protein," "peptide," and "polypeptide" are used interchangeably herein and refer to a polymer of amino acid residues linked together by peptide (amide) bonds. The terms refer to proteins, peptides, or polypeptides of any size, structure, or function. Typically, a protein, peptide, or polypeptide will be at least three amino acids in length. A protein, peptide, or polypeptide may refer to an individual protein or a group of proteins. One or more of the amino acids in a protein, peptide, or polypeptide may be modified by the addition of a chemical entity, such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, or a linker for conjugation, functionalization, or other modification. A protein, peptide, or polypeptide may also be a single molecule or a multimolecular complex. A protein, peptide, or polypeptide may be just a fragment of a naturally occurring protein or peptide. A protein, peptide, or polypeptide may be naturally occurring, recombinant, synthetic, or any combination thereof. Any of the proteins provided herein may be produced by any method known in the art. For example, the proteins provided herein may be produced via recombinant protein expression and purification, which is particularly suitable for fusion proteins containing peptide linkers. Methods for recombinant protein expression and purification are well known and include those described by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012) (the entire contents of which are incorporated herein by reference).

[0259] Silent mutations As used herein, the term "silent mutation" refers to a mutation in a nucleic acid molecule that does not affect the phenotype of the nucleic acid molecule or the protein it produces when encoding a protein. Silent mutations can occur in the coding region of a nucleic acid (i.e., the segment of a gene that encodes a protein) or in the non-coding region of a nucleic acid. A silent mutation in a nucleic acid sequence, such as a target DNA sequence or a DNA synthesis template sequence installed in a target sequence, can be a nucleotide change that does not affect the expression or function of the amino acid sequence encoded by the nucleic acid sequence or other functional characteristics of the target nucleic acid sequence. When silent mutations occur in a coding region, they can be synonymous mutations. A synonymous mutation refers to the substitution of one base for another in a gene such that the corresponding amino acid residue in the protein produced by the gene is not modified. This is due to the redundancy of the genetic code, which allows multiple different codons to encode the same amino acid in a particular organism. When a silent mutation is located in a non-coding region or at a junction between a coding region and a non-coding region (e.g., an intron / exon junction), it may be located in a region that does not affect any biological properties of the nucleic acid molecule (e.g., splicing, gene regulation, RNA lifespan, etc.). Silent mutations may be useful, for example, to increase the length of edits made to a target nucleotide sequence using prime editing to avoid editing correction by the MMR pathway described herein. In certain embodiments, the number of silent mutations installed may be one, two, three, four, five, six, seven, eight, nine, or ten or more. In certain other embodiments involving at least two silent mutations, the silent mutations may be installed within 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides of the intended editing site.

[0260] Spacer sequence As used herein, the term "spacer sequence" in reference to a guide RNA or PEgRNA refers to a portion of the guide RNA or PEgRNA of about 20 nucleotides (e.g., 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides) that contains a nucleotide sequence that shares the same sequence as a protospacer sequence in the target DNA sequence. The spacer sequence anneals to the complement of the protospacer sequence to form a ssRNA / ssDNA hybrid structure at the target site and a corresponding R-loop ssDNA structure on the endogenous DNA strand.

[0261] target site The term "target site" refers to a sequence on a nucleic acid molecule that is edited by a prime editor (PE) disclosed herein. A target site may refer to an endogenous sequence within the nucleic acid molecule to be edited, for example, an endogenous genomic sequence of a target genome that is identical to the sequence of the DNA synthesis template except for one or more nucleotide edits installed on the DNA synthesis template (and except that the DNA synthesis template contains uracil instead of thymine), or a corresponding endogenous sequence on a non-target strand that is complementary to the DNA synthesis template except for one or more mismatches at the position of one or more nucleotide edits installed on the DNA synthesis template. Furthermore, a target site may also refer to a sequence on a nucleic acid molecule to which a complex of a prime editor (PE) and a gRNA binds.

[0262] variant As used herein, the term "variant" should be interpreted to mean exhibiting qualities with a pattern that deviates from those found in nature; for example, a variant Cas9 is a Cas9 that contains one or more changes in amino acid residues compared to the wild-type Cas9 amino acid sequence. The term "variant" encompasses homologous proteins that have at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 99% percent identity with a reference sequence and have the same or substantially the same functional activity(ies) as the reference sequence. The term also encompasses mutants, truncations, or domains of a reference sequence that exhibit the same or substantially the same functional activity(ies) as the reference sequence.

[0263] vector The term "vector," as used herein, refers to a nucleic acid that can be modified to encode a gene of interest and that can enter a host cell and mutate or replicate within the host cell (and then transfer the replicative form of the vector into another host cell). Exemplary suitable vectors include viral vectors, such as retroviral vectors or bacteriophages and filamentous phages, and conjugative plasmids. Additional suitable vectors will be apparent to those of skill in the art based on the present disclosure.

[0264] Detailed Description of Specific Embodiments 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 performing prime editing of a target site. The inventors surprisingly found that the editing efficiency of prime editing is 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, 25-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- 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 Increased, 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 or more increase). In addition, the inventors have surprisingly found that the frequency of indel formation resulting from prime editing may be significantly reduced (e.g., by a 2-fold reduction, a 3-fold reduction, a 4-fold reduction, a 5-fold reduction, a 6-fold reduction, a 7-fold reduction, an 8-fold reduction, a 9-fold reduction, or a 10-fold reduction or less) when one or more functions of the DNA mismatch repair (MMR) system are inhibited, blocked, or otherwise inactivated during prime editing.

[0265] The present disclosure relates to the surprising discovery that the efficiency and / or specificity of prime editing is influenced by the cell's own DNA mismatch repair (MMR) 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 screening for prime editing outcomes," which led to the identification of various genetic determinants, including MMR, as affecting the efficiency and / or specificity of prime editing. Accordingly, in one aspect, the present disclosure provides novel prime editing systems that include means for inhibiting and / or circumventing the effects of MMR, thereby increasing the efficiency and / or specificity of prime editing. In one embodiment, the present disclosure provides a prime editing system that includes an MMR-inhibiting protein, such as a dominant-negative MMR protein, such as, but not limited to, a dominant-negative MLH1 protein (i.e., "MLH1dn"). In another embodiment, the prime editing system includes installing one or more silent mutations near the intended edit, thereby allowing the intended edit to bypass MMR recognition even in the absence of an MMR-inhibiting protein such as MLH1dn. In another aspect, the present disclosure provides novel genetic screens for identifying genetic determinants, such as MMR, that affect the efficiency and / or specificity of prime editing. In still a further aspect, the present disclosure provides nucleic acid constructs encoding the improved prime editing systems described herein. The disclosure in another aspect also provides vectors (e.g., AAV or lentiviral vectors) comprising nucleic acids encoding the improved prime editing systems described herein. In yet another aspect, the present disclosure provides cells comprising the improved prime editing systems described herein. The disclosure also provides components of genetic screens, including nucleic acid and / or vector constructs, guide RNAs, pegRNAs, cells (e.g., CRISPRi cells), and other reagents and / or materials for performing the genetic screens disclosed herein.In yet another aspect, the present disclosure provides compositions and kits, e.g., pharmaceutical compositions, that include the improved prime editing systems described herein and can be administered to a cell, tissue, or organism by any suitable means, such as gene therapy, mRNA delivery, virus-like particle delivery, or ribonucleoprotein (RNP) delivery. In yet another aspect, the present disclosure provides methods for using the improved prime editing systems to install one or more edits into a target nucleic acid molecule, e.g., a genomic locus. In yet another aspect, the present disclosure provides methods for treating a disease or disorder using the improved prime editing systems to correct or otherwise repair one or more genetic alterations (e.g., a single polymorphism) in a target nucleic acid molecule, e.g., a genomic locus that contains one or more disease-causing mutations.

[0266] In one embodiment, the MLH1 protein is inhibited, blocked, or otherwise inactivated. In other embodiments, other proteins of the MMR system are inhibited, blocked, or 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, EXO1, POLδ, and PCNA. Inhibition may involve inhibiting the protein with an inhibitor (e.g., an antibody or small molecule inhibitor, or a dominant-negative variant of the protein that disrupts, blocks, or otherwise inactivates the function of the protein, e.g., a dominant-negative form of MLH1). Inhibition may also involve any other suitable means, such as proteolysis (e.g., PROTAC-based degradation of MLH1), inhibition at the transcriptional level (e.g., siRNA transcriptolysis / gene silencing or microRNA-based inhibition of translation of MLH1 transcripts), or inhibition at the genetic level (i.e., installing a mutation in the MLH1 gene (or regulatory region) that inactivates or reduces expression of the MLH1 gene, or installing a mutation that inactivates, blocks, or minimizes the activity of the encoded MLH1 product). Furthermore, the present disclosure contemplates that a prime editor (e.g., delivered as a fusion protein comprising a napDNAbp and a polymerase, such as Cas9 nickase fused to a reverse transcriptase) may be administered together with any inhibitor of the DNA mismatch repair pathway.

[0267] Thus, the present disclosure provides a method for editing a nucleic acid molecule by prime editing, comprising contacting the nucleic acid molecule with a prime editing factor, a pegRNA, and an inhibitor of a DNA mismatch repair pathway, thereby installing one or more modifications to the nucleic acid molecule at the target site with increased editing efficiency and / or reduced indel formation. The present disclosure also provides a polynucleotide for editing a DNA target site by prime editing, comprising a nucleic acid sequence encoding a napDNAbp, a polymerase, and an inhibitor of a DNA mismatch repair pathway, wherein the napDNAbp and the polymerase are capable of installing one or more modifications at the DNA target site with increased editing efficiency and / or reduced indel formation in the presence of a pegRNA. The present disclosure also provides vectors, cells, and kits comprising the compositions and polynucleotides of the present disclosure, as well as methods for making such vectors, cells, and kits, and methods for delivering such compositions, polynucleotides, vectors, cells, and kits to cells in vitro, ex vivo (e.g., during cell-based therapies to modify cells outside the body), and in vivo.

[0268] MMR pathway As noted above, the present disclosure relates to the observation that the efficiency and / or specificity of prime editing is influenced 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 genome stability (see, for example, Figures 8A and 8B). Escherichia coli MutS and MutL and their eukaryotic homologs, MutSα and MutLα, respectively, are key players in MMR-associated genome maintenance. In various aspects, the present disclosure contemplates any suitable means of inhibiting, blocking, or otherwise inactivating the DNA mismatch repair (MMR) system, including, but not limited to, inactivating one or more key proteins of the MMR system at the genetic level, for example, by introducing one or more mutations into the gene(s) encoding the proteins 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-MSH3), MSH2, MSH6, PCNA, RFC, EXO1, POLδ, and PCNA. The nucleotide and amino acid sequences of such naturally occurring proteins and variants thereof are known in the art. Exemplary sequences are as follows: The present disclosure embraces the use of any inhibitor, blocker, 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 inhibiting, blocking, or otherwise inactivating one or more of the MMR protein or variants thereof results in inhibiting, blocking, or inactivating the MMR pathway.Inhibition, 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 one or more MMR proteins, such as by introducing a mutation that inactivates the MMR protein or variant thereof), the transcriptional level (e.g., by transcriptional knockdown), the translational level (e.g., by blocking translation of one or more MMR proteins from their cognate transcripts), or the protein level (e.g., by administration of an inhibitor (e.g., small molecule, antibody, dominant-negative protein variant), or targeted protein degradation (e.g., PROTAC-based degradation)).

[0269] In one aspect, the present disclosure provides improved prime editing methods that include further inhibiting the DNA mismatch repair (MMR) system during prime editing by inhibiting, blocking, or inactivating MLH1 or a variant thereof.

[0270] 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 (MMR) system. DNA repair is initiated by binding of MutS alpha (MSH2-MSH6) or MutS beta (MSH2-MSH3) to a dsDNA mismatch, followed by recruitment of MutL alpha to the heteroduplex. The assembly of a MutL-MutS-heteroduplex ternary complex in the presence of RFC and PCNA is sufficient to activate the endonuclease activity of PMS2. This introduces a single-strand break near the mismatch, thus generating a new entry point for the exonuclease EXO1 to degrade the mismatch-containing strand. DNA methylation prevents the break, thus ensuring that only the newly mutated DNA strand is corrected. MutL alpha (MLH1-PMS2) physically interacts with the clamp loader subunit of DNA polymerase III, suggesting that it may play a role in recruiting DNA polymerase III to sites of MMR. It is also involved in DNA damage signaling, a process that induces cell cycle arrest and can lead to apoptosis in cases of major DNA damage. MLH1 also heterodimerizes with MLH3 to form MutL gamma, which plays a role in mitosis.

[0271] The "standard" human MLH1 amino acid sequence is represented by SEQ ID NO:204.

[0272] MLH1 may also 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.

[0273] MLH1 may also 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.

[0274] MMR inhibitors and methods for inhibiting MMR The present disclosure provides a method for editing a nucleic acid molecule by prime editing, comprising contacting the nucleic acid molecule with a prime editor, a pegRNA, and an inhibitor of a DNA mismatch repair pathway, thereby increasing editing efficiency and / or reducing indel formation and installing one or more modifications to the nucleic acid molecule at a target site. Accordingly, the present disclosure contemplates any suitable means for inhibiting MMR. In one aspect, the present disclosure provides for administering an effective amount of an inhibitor of the MMR pathway. In various aspects, 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 genes encoding one or more MMR proteins, e.g., by introducing a mutation that inactivates the MMR protein or variants thereof), the transcriptional level (e.g., by transcriptional knockdown), the translational level (e.g., by blocking translation of one or more MMR proteins from their cognate transcripts), or the protein level (e.g., by application of inhibitors (e.g., small molecules, antibodies, dominant-negative protein partners) or targeted protein degradation (e.g., PROTAC-based degradation). The present disclosure also contemplates methods of prime editing designed to install modifications into nucleic acid molecules that avoid correction by the MMR pathway, without the need to provide an MMR inhibitor.

[0275] The present inventors have developed prime editing that allows for the insertion, deletion, or substitution of genomic DNA sequences without the need for error-prone double-stranded DNA breaks. The present disclosure now provides improved methods of prime editing that involve blocking, inhibiting, or inactivating the MMR pathway (e.g., by inhibiting, blocking, or inactivating MMR pathway proteins, including MLH1) during prime editing, whereby, surprisingly, doing so results in increased editing efficiency and reduced indel formation. As used herein, "during" prime editing can encompass any suitable sequence of events, such that a prime editing step can be applied before, simultaneously with, or after a step of blocking, inhibiting, or inactivating the MMR pathway (e.g., by targeted inhibition of MLH1).

[0276] 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 encodes information to direct Cas9 to a target genomic site and install the desired edit. Prime editing proceeds through a multistep editing process: 1) the Cas9 domain binds to and nicks the target genomic DNA site specified by the spacer sequence of the pegRNA; 2) the reverse transcriptase domain uses the nicked genomic DNA as a primer to initiate 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 replacing the resulting 5' flap via invasion by the edited 3' flap, excision of the 5' flap containing the original DNA sequence, and ligation of a 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 in the heteroduplex using the edited strand as a template for repair, completing the editing process.

[0277] Efficient integration of the desired edit requires that the newly synthesized 3' flap contains a portion of sequence homologous to the genomic DNA site. This homology allows 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 contains less sequence homology than the endogenous 5' flap, competition is expected to favor the 5' flap strand. Therefore, a potential limiting factor in the efficiency of prime editing may be the inability of the edit-containing 3' flap to effectively invade and displace the 5' flap strand. Furthermore, successful 3' flap invasion and 5' flap removal only integrates the edit into one strand of the double-stranded DNA genome. Permanent installation of the edit requires cellular DNA repair to use the edited strand as a template to replace the unedited complementary DNA strand. Although cells can be engineered to favor displacement of the unedited strand over the edited strand by using a secondary sgRNA (PE3-based) to introduce a nick in the unedited strand adjacent to the edit (step 4 above), this process still relies on a second step of DNA repair.

[0278] The present disclosure describes modified approaches to prime editing that involve further inhibiting, blocking, or otherwise inactivating the DNA mismatch repair (MMR) system. In some embodiments, an MMR inhibitor is provided to a target nucleic acid along with other components of the prime editing system; for example, an exogenous MMR inhibitor, such as an siRNA, can be provided to a cell containing the target nucleic acid. In some embodiments, a prime editing system component, e.g., pegRNA, is designed to install a modification that circumvents the MMR system into the target nucleic acid without the need to provide an inhibitor. In certain embodiments, one or more proteins of the DNA mismatch repair (MMR) system can be inhibited, blocked, or otherwise inactivated, including, but not limited to, 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, EXO1, POLδ, and PCNA. The present disclosure contemplates any suitable means of inhibiting, blocking, or otherwise inactivating the DNA mismatch repair (MMR) system at the genetic level, including, but not limited to, inactivating one or more key proteins of the MMR system by introducing one or more mutations into genes encoding proteins of the MMR system, such as 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, EXO1, POLδ, and PCNA.

[0279] Thus, in one aspect, the present disclosure provides methods for editing nucleotide molecules (e.g., genomes) using prime editing while blocking, inhibiting, or otherwise inactivating the DNA mismatch repair (MMR) system.

[0280] In another aspect, the present disclosure provides methods for editing a nucleotide molecule (e.g., a genome) using prime editing while blocking, inhibiting, or otherwise inactivating proteins 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, EXO1, POLδ, and PCNA.

[0281] In one aspect, the present disclosure provides an improved prime editing method, which includes further inhibiting the DNA mismatch repair (MMR) system during prime editing by inhibiting, blocking, or inactivating MLH1 or its variants. 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 (MMR) system. DNA repair is initiated by binding of MutS alpha (MSH2-MSH6) or MutS beta (MSH2-MSH3) to a dsDNA mismatch, followed by recruitment of MutL alpha to the heteroduplex. The assembly of a MutL-MutS-heteroduplex ternary complex in the presence of RFC and PCNA is sufficient to activate the endonuclease activity of PMS2. This introduces a single-strand break near the mismatch, thus creating a new entry point for the exonuclease EXO1 to degrade the mismatch-containing strand. DNA methylation prevents breakage, thus ensuring that only newly mutated DNA strands are corrected. MutL alpha (MLH1-PMS2) physically interacts with the clamp loader subunit of DNA polymerase III, suggesting that it may play a role in recruiting DNA polymerase III to sites of MMR. It is also involved in DNA damage signaling, a process that induces cell cycle arrest and can lead to apoptosis in the event of major DNA damage. MLH1 also heterodimerizes with MLH3 to form MutL gamma, which plays a role in cell division. The "standard" human MLH1 amino acid sequence is represented by SEQ ID NO: 204.

[0282] MLH1 may also 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.

[0283] MLH1 may also 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.

[0284] The present disclosure contemplates that any of the following MLH1 proteins may be inhibited by an inhibitor or otherwise blocked or inactivated to inhibit the MMR pathway during prime editing. Furthermore, such exemplary proteins may also be used to engineer or otherwise create dominant-negative variants, which may be used as certain inhibitors when administered in an amount effective to block, inactivate, or inhibit MMR. Without being bound by theory, it is believed that MLH1 dominant-negative mutants may saturate MutS binding. 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 to any of the following sequences: [Table 2-1] [Table 2-2]

[0285] The methods and compositions described herein utilize MLH1 mutant or truncated variants. In some embodiments, mutant and truncated variants of the human MLH1 wild-type protein are utilized.

[0286] In one aspect, the present disclosure provides truncated variants of human MLH1. In some embodiments, amino acids 754-756 of the wild-type human MLH1 protein are truncated (Δ754-756, hereinafter referred to as MLH1dn). In some embodiments, truncated variants of human MLH1 are provided that include only the N-terminal domain (amino acids 1-335) (hereinafter referred to as MLH1dn). NTD In various aspects, the following MLH1 variants are provided in the present disclosure: [Table 3-1] [Table 3-2] [Table 3-3]

[0287] In another aspect, the present disclosure provides methods for editing a nucleotide molecule (e.g., a genome) using prime editing while blocking, inhibiting, or otherwise inactivating PMS2 (or MutL alpha) or a variant thereof.

[0288] In yet another aspect, the present disclosure provides methods for...

Claims

1. A prime editor comprising: (i) a nucleic acid programmable DNA binding protein (napDNAbp); and (ii) a DNA polymerase, wherein the napDNAbp is a Cas9 nickase (nCas9) that contains an R221K amino acid substitution, an N394K amino acid substitution, and an amino acid substitution that inactivates HNH domain nuclease activity, or a corresponding amino acid substitution, compared to a wild-type Cas9 as set forth in SEQ ID NO:

2.

2. The prime editor of claim 1, wherein nCas9 is linked to a DNA polymerase in a fusion protein.

3. The fusion protein has the structure NH 2 3. The prime editor of claim 2, comprising -[napDNAbp]-[DNA polymerase]-COOH, where "]-[" indicates an optional linker sequence.

4. 4. The prime editor of any one of claims 1 to 3, wherein nCas9 comprises R221K, N394K and H840X amino acid substitutions compared to wild-type Cas9 as set forth in SEQ ID NO:2, where X is any amino acid other than histidine.

5. 5. The prime editor of claim 4, wherein nCas9 comprises an H840A amino acid substitution compared to wild-type Cas9 as set forth in SEQ ID NO:

2.

6. The prime editor of any one of claims 1 to 5, wherein nCas9 comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO:

104.

7. The prime editing element of any one of claims 1 to 6, wherein nCas9 comprises the amino acid sequence of SEQ ID NO:

104.

8. The prime editor of any one of claims 2 to 7, wherein the nCas9 and the DNA polymerase are linked by a linker in a fusion protein.

9. The prime editor of claim 8, wherein the linker comprises the sequence of SEQ ID NO:

105.

10. 10. The prime editing element of claim 1, further comprising: (i) an SV40 NLS at the N-terminus; and (ii) an SV40 NLS and / or a c-Myc NLS at the C-terminus.

11. 11. The prime editing element of claim 10, wherein the N-terminal SV40 NLS comprises SEQ ID NO:101, the C-terminal SV40 NLS comprises SEQ ID NO:140, and the C-terminal c-Myc NLS comprises SEQ ID NO:

135.

12. The prime editing element of any one of claims 1 to 11, wherein the DNA polymerase is a reverse transcriptase.

13. 13. The prime editor of claim 12, wherein the reverse transcriptase is a retroviral reverse transcriptase.

14. The prime editor of claim 12 or 13, wherein the reverse transcriptase is Moloney Murine Leukemia Virus Reverse Transcriptase (MMLV-RT) comprising an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 81 to 98.

15. The prime editing element of any one of claims 12 to 14, wherein the reverse transcriptase is Moloney murine leukemia virus reverse transcriptase (MMLV-RT) comprising an amino acid sequence of any one of SEQ ID NOs: 81 to 98.

16. The prime editor of any one of claims 12 to 15, wherein the reverse transcriptase comprises D200N, T330P, T306K, W313F, and L603W amino acid substitutions compared to MMLV-RT as set forth in SEQ ID NO:

81.

17. The prime editing factor of any one of claims 2 to 16, wherein the fusion protein comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO:

99.

18. The prime editing factor of any one of claims 2 to 17, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO:

99.

19. A prime editing factor system comprising the prime editing factor of any one of claims 1 to 18 and a prime editing guide RNA (pegRNA).

20. 20. The prime editor system of Claim 19, further comprising a second strand nicking single guide RNA (sgRNA).

21. 19. One or more polynucleotides encoding the primed editing factor of any one of claims 1 to 18.

22. A polynucleotide encoding the prime editing factor of claim 17 or 18.

23. 20. A method for editing a nucleic acid molecule comprising contacting the nucleic acid molecule with the primed editing factor system of claim 19.