Prime editing with modification-tolerant polymerases

Abstract

Provided are prime editing systems with modification-tolerant polymerases and petRNAs and / or pegRNA with chemically modified primer binding sites (PBS) and / or nucleotide polymerase template (NPT).

Description

Attorney Docket. No. 767854: UM9-316PCPRIME EDITING WITH MODIFICATION-TOLERANT POLYMERASESRELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 690,531, filed September 4, 2024, the entire disclosure of which is incorporated herein by reference.BACKGROUND

[0002] Correction of genetic mutations in vivo has broad potential therapeutic application for a range of human genetic diseases. Prime editors (PE) composed of a nCas9 fused to an engineered nucleotide polymerase (NP) have enabled precise nucleotide changes, sequence insertions and deletions. Anzalone et al., “Search-and-replace genome editing without doublestrand breaks or donor DNA” Nature 576: 149-157 (2019).

[0003] However, early generations of prime editors employing M-MLV RT, designated PE2, have reduced editing activity with pegRNAs employing nucleotide modifications in the primer binding site (PBS) and reverse transcriptase template (RTT).

[0004] Accordingly, there exists a need in the art for improved prime editing systems that better tolerate nucleotide modifications.SUMMARY

[0005] In certain aspects, provided herein is a prime editing system, comprising: i) a Cas9 nickase protein or a polynucleotide sequence encoding the Cas9 nickase protein; ii) a modification-tolerant nucleotide polymerase (NP) protein or a polynucleotide sequence encoding the modification-tolerant NP protein; and one of: iii A) a prime editor template RNA (petRNA) comprising a primer binding site (PBS), and a nucleotide polymerase template (NPT), and ivA) a single guide RNA (sgRNA); or iiiB) a prime editing guide RNA (pegRNA) comprising a PBS, an NPT, and a sgRNA portion; wherein each of the PBS and NPT comprise at least one nucleotide modification.

[0006] In some embodiments, every nucleotide in the PBS is modified.

[0007] In some embodiments, every nucleotide in the NPT is modified.

[0008] In some embodiments, every nucleotide in the PBS and NPT is modified.

[0009] In some embodiments, the nucleotide modification is selected from a modification of a ribose group, a phosphate group, a nucleobase, or a combination thereof.80966915. v1Attorney Docket. No. 767854: UM9-316PC

[0010] In some embodiments, the modification of the ribose group is independently selected from the group consisting of 2'-(9-methyl, 2’-fluoro, 2’-deoxy, 2’-< -(2-methoxyethyl) (MOE), 2’-NH2 (2’-amino), 4’-thio, a bicyclic nucleotide, a locked nucleic acid (LNA), a 2’- (5)-constrained ethyl (S-cEt), a constrained MOE, a 2'-(9,4'-C-aminomethylene bridged nucleic acid (2',4'-BNANC), and a 2’-F-ANA.

[0011] In some embodiments, the modification of the ribose group is 2'-(9-methyl.

[0012] In some embodiments, at least 40% of the ribose groups in the PBS is modified.

[0013] In some embodiments, every ribose group in the PBS is modified.

[0014] In some embodiments, at least 40% of the ribose groups in the NPT is modified.

[0015] In some embodiments, every ribose group in the NPT is modified.

[0016] In some embodiments, the modification of the phosphate group is independently selected from the group consisting of a phosphorothioate, phosphonoacetate (PACE), phosphoramidate, thiophosphonoacetate (thioPACE), amide, triazole, phosphonate, or phosphotriester modification.

[0017] In some embodiments, the modification of the nucleobase group is independently selected from the group consisting of 2-thiouridine, 4-thiouridine, N6-methyladenosine, pseudouridine, 2,6-diaminopurine, inosine, thymidine, 5-methylcytosine, 5-substituted pyrimidine, isoguanine, isocytosine, and halogenated aromatic groups.

[0018] In some embodiments, the sgRNA or sgRNA portion comprises at least one nucleotide modification.

[0019] In some embodiments, the modification-tolerant NP is selected from a variant of EC48-RT, TF1-RT, or MMLV-RT.

[0020] In some embodiments, the modification-tolerant NP comprises at least 80% identity (i.e., 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to the amino acid sequence of SEQ ID NO: 17. In some embodiments, the modification-tolerant NP comprises the amino acid sequence of SEQ ID NO: 17. In some embodiments, the modification-tolerant NP consists of the amino acid sequence of SEQ ID NO: 17.

[0021] In some embodiments, the modification-tolerant NP comprises at least 80% identity (i.e., 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to the amino acid sequence of SEQ ID NO: 18. In some embodiments, the modification-tolerant NP comprises the amino acid sequence of SEQ ID NO: 18. In some embodiments, the modification-tolerant NP consists of the amino acid sequence of SEQ ID NO: 18.

[0022] In some embodiments, the modification-tolerant NP comprises at least 80% identity (i.e., 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to the amino acid280966915. v1Attorney Docket. No. 767854: UM9-316PC sequence of SEQ ID NO: 19. In some embodiments, the modification-tolerant NP comprises the amino acid sequence of SEQ ID NO: 19. In some embodiments, the modification-tolerant NP consists of the amino acid sequence of SEQ ID NO: 19.

[0023] In some embodiments, the modification-tolerant NP comprises one or more amino acid substitutions selected from I33V, E54L, Q75R, M214A, Q293R or Q293K, K321P, K380R and N447K relative to the NP of SEQ ID NO: 18 or SEQ ID NO: 19.

[0024] In some embodiments, the modification-tolerant NP comprises one or both amino acid substitutions of I33V and M214A relative to the NP of SEQ ID NO: 18 or SEQ ID NO: 19.

[0025] In some embodiments, the modification-tolerant NP comprises the amino acid substitution of I33V relative to the NP of SEQ ID NO: 18 or SEQ ID NO: 19.

[0026] In some embodiments, the modification-tolerant NP comprises the amino acid substitution of M214A relative to the NP of SEQ ID NO: 18 or SEQ ID NO: 19.

[0027] In some embodiments, the modification-tolerant NP comprises amino acid substitutions of I33V and M214A relative to the NP of SEQ ID NO: 18 or SEQ ID NO: 19.

[0028] In some embodiments, the modification-tolerant NP comprises one or more amino acid substitutions selected from K188A, K290R, N299A, K320A or K320R, and K321A relative to the NP of SEQ ID NO: 19.

[0029] In some embodiments, the modification-tolerant NP comprises amino acid substitutions K188A, K290R, N299A, K320A or K320R, and K321A relative to the NP of SEQ ID NO: 19.

[0030] In some embodiments, the modification-tolerant NP comprises at least 80% identity (i.e., 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to the amino acid sequence of any one of SEQ ID NOs: 51-56. In some embodiments, the modification-tolerant NP comprises the amino acid sequence of any one of SEQ ID NOs: 51-56. In some embodiments, the modification-tolerant NP consists of the amino acid sequence of any one of SEQ ID NOs: 51-56.

[0031] In some embodiments, the modification-tolerant NP comprises at least 80% identity (i.e., 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to the amino acid sequence of SEQ ID NO: 20. In some embodiments, the modification-tolerant NP comprises the amino acid sequence of SEQ ID NO: 20. In some embodiments, the modification-tolerant NP consists of the amino acid sequence of SEQ ID NO: 20.

[0032] In some embodiments, the Cas9 nickase protein comprises at least 80% identity (i.e., 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 57.380966915. v1Attorney Docket. No. 767854: UM9-316PC

[0033] In some embodiments, the Cas9 nickase protein and the modification-tolerant NP protein are linked together via an amino acid linker.

[0034] In some embodiments, the Cas9 nickase protein is linked to one or more MS2 binding proteins via an amino acid linker.

[0035] In some embodiments, one or more MS2 binding proteins are inlaid within the Cas9 nickase protein.

[0036] In some embodiments, the one or more MS2 binding proteins comprises at least 90% identity to the amino acid sequence of SEQ ID NO: 23.

[0037] In some embodiments, the modification-tolerant NP protein is inlaid within the Cas9 nickase protein.

[0038] In some embodiments, the modification-tolerant NP protein is linked to one or more MS2 binding proteins via an amino acid linker.

[0039] In some embodiments, the MS2 binding protein is linked to at the N-terminus of the Cas9 nickase and / or the modification-tolerant NP protein.

[0040] In some embodiments, the MS2 binding protein comprises at least 90% identity to the amino acid sequence of SEQ ID NO: 22.

[0041] In some embodiments, the Cas9 nickase protein and / or the modification-tolerant NP further comprise an N-terminal and / or C-terminal nuclear localization signal (NLS).

[0042] In some embodiments, the NLS comprises any one or more of the NLS amino acid sequences of Table 2.

[0043] In some embodiments, the amino acid linker comprises or consists of any one of the amino acid linker sequences of Table 7.

[0044] In some embodiments, the petRNA comprises at least one MS2 hairpin.

[0045] In some embodiments, the MS2 hairpin is linked to the NPT via a linker.

[0046] In some embodiments, the linker is a non-nucleotide linker.

[0047] In some embodiments, the non-nucleotide linker is a Ci-Cio alkyl.

[0048] In some embodiments, the non-nucleotide linker is a Ci alkyl.

[0049] In some embodiments, the non-nucleotide linker is a C2 alkyl.

[0050] In some embodiments, the non-nucleotide linker is a C3 alkyl.

[0051] In some embodiments, the non-nucleotide linker is a C4 alkyl.

[0052] In some embodiments, the non-nucleotide linker is a C5 alkyl.

[0053] In some embodiments, the non-nucleotide linker is a Ce alkyl.

[0054] In some embodiments, the non-nucleotide linker is selected from the group consisting of ethylene glycol and polyethylene glycol (PEG).480966915. v1Attorney Docket. No. 767854: UM9-316PC

[0055] In some embodiments, the PEG comprises or consists of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 ,17 ,18, 19, or 20 ethylene glycol units.

[0056] In some embodiments, the PEG comprises triethylene glycol or tetraethylene glycol.

[0057] In some embodiments, the PEG is a hexaethylene glycol (HEG).

[0058] In some embodiments, HEG comprises the following structure:

[0059] In some embodiments, the PEG is 2XHEG.

[0060] In some embodiments, the PEG is 2XHEG comprising the following structure:

[0061] In some embodiments, the linker is an abasic nucleotide.

[0062] In some embodiments, the linker is a 2’-O-(2-methoxyethyl) (MOE) modified nucleotide.

[0063] In one aspect, the disclosure provides a prime editing system, comprising: i) a fusion protein comprising a Cas9 nickase protein linked to a modification-tolerant nucleotide polymerase (NP) protein, or a polynucleotide encoding the fusion protein; and one of: iiA) a prime editor template RNA (petRNA) comprising a primer binding site (PBS), and a nucleotide polymerase template (NPT), and iiiA) a single guide RNA (sgRNA); or iiB) a prime editing guide RNA (pegRNA) comprising a PBS, an NPT, and a sgRNA portion; wherein each of the PBS and NPT comprise at least one nucleotide modification.

[0064] In some embodiments, every nucleotide in the PBS is modified.

[0065] In some embodiments, every nucleotide in the NPT is modified.

[0066] In some embodiments, every nucleotide in the PBS and NPT is modified.

[0067] In some embodiments, the nucleotide modification is selected from a modification of a ribose group, a phosphate group, a nucleobase, or a combination thereof

[0068] In some embodiments, the modification of the ribose group is independently selected from the group consisting of 2'-O-methyl, 2’-fluoro, 2’-deoxy, 2’-O-(2-methoxyethyl)580966915. v1Attorney Docket. No. 767854: UM9-316PC(MOE), 2’-NH2 (2’-amino), 4’-thio, a bicyclic nucleotide, a locked nucleic acid (LNA), a 2’- (5)-constrained ethyl (S-cEt), a constrained MOE, a 2'-(9,4'-C-aminomethylene bridged nucleic acid (2',4'-BNANC), and a 2’-F-ANA.

[0069] In some embodiments, the modification of the ribose group is 2'-(9-methyl.

[0070] In some embodiments, at least 40% of the ribose groups in the PBS is modified.

[0071] In some embodiments, every ribose group in the PBS is modified.

[0072] In some embodiments, at least 40% of the ribose groups in the NPT is modified.

[0073] In some embodiments, every ribose group in the NPT is modified.

[0074] In some embodiments, the modification of the phosphate group is independently selected from the group consisting of a phosphorothioate, phosphonoacetate (PACE), phosphoramidate, thiophosphonoacetate (thioPACE), amide, triazole, phosphonate, or phosphotriester modification.

[0075] In some embodiments, the modification of the nucleobase group is independently selected from the group consisting of 2-thiouridine, 4-thiouridine, N6-methyladenosine, pseudouridine, 2,6-diaminopurine, inosine, thymidine, 5-methylcytosine, 5-substituted pyrimidine, isoguanine, isocytosine, and halogenated aromatic groups.

[0076] In some embodiments, the sgRNA or sgRNA portion comprises at least one nucleotide modification.

[0077] In some embodiments, the modification-tolerant NP is selected from a variant of EC48-RT, TF1-RT, or MMLV-RT.

[0078] In some embodiments, the modification-tolerant NP comprises at least 80% identity (i.e., 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to the amino acid sequence of SEQ ID NO: 17.

[0079] In some embodiments, the modification-tolerant NP comprises at least 80% identity (i.e., 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to the amino acid sequence of SEQ ID NO: 18.

[0080] In some embodiments, the modification-tolerant NP comprises at least 80% identity (i.e., 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to the amino acid sequence of SEQ ID NO: 19.

[0081] In some embodiments, the modification-tolerant NP comprises at least 80% identity (i.e., 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to the amino acid sequence of SEQ ID NO: 20.680966915. v1Attorney Docket. No. 767854: UM9-316PC

[0082] In some embodiments, the Cas9 nickase protein comprises at least 80% identity (i.e., 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to the amino acid sequence of SEQ ID NO: 1,SEQ ID NO: 2, or SEQ ID NO: 57.

[0083] In some embodiments, the Cas9 nickase protein and the modification-tolerant NP protein are linked together via an amino acid linker.

[0084] In some embodiments, the Cas9 nickase protein is linked to one or more MS2 binding proteins via an amino acid linker.

[0085] In some embodiments, one or more MS2 binding proteins are inlaid within the Cas9 nickase protein.

[0086] In some embodiments, the one or more MS2 binding proteins comprises at least 90% identity to the amino acid sequence of SEQ ID NO: 23.

[0087] In some embodiments, the modification-tolerant NP protein is inlaid within the Cas9 nickase protein.

[0088] In some embodiments, the modification-tolerant NP protein is linked to one or more MS2 binding proteins via an amino acid linker.

[0089] In some embodiments, the MS2 binding protein is linked to at the N-terminus of the Cas9 nickase and / or the modification-tolerant NP protein.

[0090] In some embodiments, the MS2 binding protein comprises at least 90% identity to the amino acid sequence of SEQ ID NO: 22.

[0091] In some embodiments, the Cas9 nickase protein and / or the modification-tolerant NP further comprise an N-terminal and / or C-terminal nuclear localization signal (NLS).

[0092] In some embodiments, the NLS comprises any one or more of the NLS amino acid sequences of Table 2.

[0093] In some embodiments, the amino acid linker comprises or consists of any one of the amino acid linker sequences of Table 7.

[0094] In some embodiments, the petRNA comprises at least one MS2 hairpin.

[0095] In some embodiments, the MS2 hairpin is linked to the NPT via a linker.

[0096] In some embodiments, the linker is selected from the group consisting of ethylene glycol and polyethylene glycol (PEG).

[0097] In some embodiments, the PEG is a hexaethylene glycol (HEG).780966915. v1Attorney Docket. No. 767854: UM9-316PC

[0098] In some embodiments, HEG comprises the following structure:

[0099] In some embodiments, the PEG is 2XHEG.

[0100] In some embodiments, the PEG is 2XHEG comprising the following structure:

[0101] In some embodiments, the fusion protein comprises at least 90% identity (i.e., 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to any one of the amino acid sequences of SEQ ID NOs: 41-48, 58, or 59.

[0102] In some embodiments, the polynucleotide sequence encoding the Cas9 nickase protein is an mRNA.

[0103] In some embodiments, the polynucleotide sequence encoding the modification- tolerant NP protein is an mRNA.

[0104] In some embodiments, the polynucleotide sequence encoding the fusion protein is an mRNA.

[0105] In some embodiments, the polynucleotide sequence encoding the Cas9 nickase protein, the polynucleotide sequence encoding fusion protein, and / or the polynucleotide sequence encoding the modification-tolerant NP protein is within a vector.

[0106] In some embodiments, the vector is a viral vector.

[0107] In some embodiments, the viral vector is an adeno-associated virus (AAV) vector or a lentivirus (LV) vector.

[0108] In one aspect, the disclosure provides a method of delivering the prime editing system described herein to a cell, the method comprising incubating the prime editing system with the cell.

[0109] In one aspect, the disclosure provides a method of editing a target gene in a cell of a subject, comprising administering to the subject the prime editing system described herein.880966915. v1Attorney Docket. No. 767854: UM9-316PC

[0110] In one aspect, the disclosure provides a modification-tolerant nucleotide polymerase (NP), comprising one or more amino acid substitutions selected from 133 V, E54L, Q75R, M214A, Q293R or Q293K, K321P, K380R and N447K relative to the NP of SEQ ID NO: 18 or SEQ ID NO: 19.

[0111] In some embodiments, the modification-tolerant NP comprises one or both amino acid substitutions of I33V and M214A relative to the NP of SEQ ID NO: 18 or SEQ ID NO: 19.

[0112] In some embodiments, the modification-tolerant NP comprises the amino acid substitution of I33V relative to the NP of SEQ ID NO: 18 or SEQ ID NO: 19.

[0113] In some embodiments, the modification-tolerant NP comprises the amino acid substitution of M214A relative to the NP of SEQ ID NO: 18 or SEQ ID NO: 19.

[0114] In some embodiments, the modification-tolerant NP comprises amino acid substitutions of I33V and M214A relative to the NP of SEQ ID NO: 18 or SEQ ID NO: 19.

[0115] In one aspect, the disclosure provides a modification-tolerant nucleotide polymerase (NP), comprising one or more amino acid substitutions selected from K188A, K290R, N299A, K320A or K320R, and K321 A relative to the NP of SEQ ID NO: 19.

[0116] In some embodiments, the modification-tolerant NP comprises amino acid substitutions K188A, K290R, N299A, K320A or K320R, and K321A relative to the NP of SEQ ID NO: 19.

[0117] In one aspect, the disclosure provides a modification-tolerant nucleotide polymerase (NP), comprising at least 80% identity (i.e., 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to the amino acid sequence of any one of SEQ ID NOs: 51-56.

[0118] In some embodiments, the modification-tolerant NP comprises the amino acid sequence of SEQ ID NO: 51. In some embodiments, the modification-tolerant NP consists of the amino acid sequence of SEQ ID NO: 51.

[0119] In some embodiments, the modification-tolerant NP comprises the amino acid sequence of SEQ ID NO: 52. In some embodiments, the modification-tolerant NP consists of the amino acid sequence of SEQ ID NO: 52.

[0120] In some embodiments, the modification-tolerant NP comprises the amino acid sequence of SEQ ID NO: 53. In some embodiments, the modification-tolerant NP consists of the amino acid sequence of SEQ ID NO: 53.

[0121] In some embodiments, the modification-tolerant NP comprises the amino acid sequence of SEQ ID NO: 54. In some embodiments, the modification-tolerant NP consists of the amino acid sequence of SEQ ID NO: 54.980966915. v1Attorney Docket. No. 767854: UM9-316PC

[0122] In some embodiments, the modification-tolerant NP comprises the amino acid sequence of SEQ ID NO: 55. In some embodiments, the modification-tolerant NP consists of the amino acid sequence of SEQ ID NO: 55.

[0123] In some embodiments, the modification-tolerant NP comprises the amino acid sequence of SEQ ID NO: 56. In some embodiments, the modification-tolerant NP consists of the amino acid sequence of SEQ ID NO: 56.BRIEF DESCRIPTION OF THE DRAWINGS

[0124] FIG. 1A - FIG. IB shows an exemplary embodiment of a PE system comprising a split effector and an exemplary embodiment of an sPE system comprising a split guide RNA (gRNA). The diagram in FIG. 1A illustrates an exemplary split effector Prime Editor (sPE) system comprising an untethered nCas9 and an NP template. The left figure shows the sPE system with a pegRNA and the right figure shows the sPE system with a linear petRNA (LPET). The diagram in FIG. IB illustrates a split petRNA comprises an untethered single guide RNA (sgRNA) and a prime editor template RNA (petRNA) molecule, an RNA molecule that encodes a primer binding site (PBS), a nucleotide polymerase template (NPT), and a stem loop (MS2 stem loop).

[0125] FIG. 2A - FIG. 2B shows editing activity at three genomic target sties (FANCF, PRNP, and HBB). FIG. 2A shows editing activity of sPE with LPET with 2’O-Methyl modified (2’OMe) PBS and RNA reverse transcriptase template (RTT), or with a fully modified (all 2’OMe) PBS + RTT, at 3 genomic loci. FIG. 2B show editing activity of sPE with LPET with 2’OMe PBS / DNA RTT, or with a fully modified (all 2’OMe) PBS / RTT, at 3 genomic loci. For the experiments in both panels, lug Cas9 mRNA, lug MCP-MMLV RT (PE2 variant) mRNA, lOOpmol sgRNA, lOOpmol nicking sgRNA, and lOOpmol of LPET or editing template were electroporated into 50,000 HEK293T cells. Genomic DNA (gDNA) was harvested 72-96 hours post-electroporation and editing was analyzed by next-generation sequencing (NGS). All LPETs and sgRNAs were end-modified with three 2’OMe and PS linkages at both the 3’ and 5’ ends. N = 2.

[0126] FIG. 3 shows editing activity of sPE with LPET and pegRNA consisting of unmodified, 2’OMe PBS / RNA RTT, and fully modified (all 2’OMe) PBS / RTT at the FANCF genomic locus. 0.5pg Cas9 mRNA, 0.5 ug MCP-MMLV RT (PE2 variant) mRNA, 50pmol sgRNA, 50pmol nicking sgRNA and 50pmol of LPET or editing template were electroporated into 50,000 HEK293T cells. gDNA was harvested 96 hours post-electroporation and editing1080966915. v1Attorney Docket. No. 767854: UM9-316PC was analyzed by NGS. All LPETs, pegRNAs, and sgRNAs were end-modified with three 2’0Me and PS linkages at both the 3’ and 5’ ends. N = 1-2.

[0127] FIG. 4 shows editing activity of editing activity of sPE and PE2 (fused) with pegRNA consisting of unmodified, 2’0Me PBS / RNA RTT, and fully modified (2’0Me) PBS / RTT at the FANCF genomic locus. 0.5ug Cas9 mRNA, 0.5 ug MCP-MMLV RT (PE2 variant) mRNA or 0.5ug of fused PE2 mRNA, 50pmol nicking sgRNA and 50pmol of pegRNA were electroporated in 50,000 HEK293T cells. gDNA was harvested 96 hours postelectroporation and editing was analyzed by NGS. All pegRNAs were end-modified with three 2’0Me and PS linkages at both the 3’ and 5’ ends. N = 2.

[0128] FIG. 5A - FIG. 5B shows editing activity at the FANCF genomic target. FIG. 5A shows editing activity of sPE, PE2 (fused), N-term RT-PE2, and PE2-deltaRNaseH with pegRNA consisting of unmodified, 2’0Me PBS / RNA RTT, and fully modified (all 2’0Me) PBS / RTT at the FANCF genomic locus. FIG. 5B shows data for fully modified pegRNA shown separately from FIG. 5A. 0.5ug Cas9 mRNA, 0.5 ug MCP-MMLV RT (PE2 variant) mRNA or 0.5ug of fused PE2 mRNA, 50pmol sgRNA, 50pmol nicking sgRNA and 50pmol of LPET or editing template were electroporated in 50,000 HEK293T cells. gDNA was harvested 96 hours post-electroporation and editing was analyzed by NGS. All LPETs, pegRNAs, and sgRNAs were end-modified with three 2’0Me and PS linkages at both the 3’ and 5’ ends. N = 2.

[0129] FIG. 6 shows editing activity of prime editors PE2 (NP - MMLV), PE6D (NP - evolved MMLV), PE6A (NP -EC48RT), PE6B (NP -evolved TF1-RT) and PE6C (NP - evolved TF1-RT) in split format with LPET consisting of 2’0Me PBS / RNA RTT, 2’0Me PBS / DNA RTT, and fully modified (all 2’0Me) PBS / RTT) at the PRNP genomic locus. 0.5ug Cas9 mRNA, 0.5 ug MCP-MMLV RT (PE2 variant) mRNA, 50pmol sgRNA, 50pmol nicking sgRNA and 50pmol of LPET or editing template were electroporated into 50,000 HEK293T cells. gDNA was harvested 96 hours post-electroporation and editing was analyzed by NGS. All LPETs, pegRNAs, and sgRNAs were end-modified with three 2’0Me and PS linkages at both the 3’ and 5’ ends. N = 2.

[0130] FIG. 7A - FIG. 7B shows editing activity at the FANCF genomic target. FIG. 7A shows editing activity of prime editors PE2 (MMLV), PE6D (MMLV), PE6A (EC48RT), PE6B (TF1-RT) and PE6C (TF1-RT) in fused format with LPET consisting of 2’0Me PBS / RNA RTT, 2’0Me PBS / DNA RTT, and fully modified (all 2’0Me) PBS / RTT; or with pegRNA consisting of RNA PBS / RTT, 2’0Me PBS / RNA RTT, and fully modified (all 2’0Me) PBS / RTT at the FANCF genomic locus. FIG. 7B shows data for fully modified1180966915. v1Attorney Docket. No. 767854: UM9-316PCPBS+RTT for LPET and pegRNA shown separately from FIG. 7A. 0.5ug Cas9 mRNA, 0.5 ug MCP-MMLV RT (PE2 variant) mRNA, 50pmol sgRNA, 50pmol nicking sgRNA and 50pmol of LPET or editing template were electroporated into 50,000 HEK293T cells. gDNA was harvested 96 hours post-electroporation and editing was analyzed by NGS. All LPETs, pegRNAs, and sgRNAs were end-modified with three 2’0Me and PS linkages at both the 3’ and 5’ ends. N = 2.

[0131] FIG. 8 shows editing activity of prime editors PE2 (MMLV), PE6D (MMLV), PE6A (EC48RT), PE6B (TF1-RT) and PE6C (TF1-RT) in split format with LPET consisting of 2’0Me PBS / RNA RTT, 2’0Me PBS / DNA RTT, and fully modified (all 2’0Me) PBS / RTT at the PRNP genomic locus. 0.5ug Cas9 mRNA, 0.5 ug MCP-MMLV RT (PE2 variant) mRNA, 50pmol sgRNA, 50pmol nicking sgRNA and 50pmol of LPET or editing template were electroporated into 50,000 HEK293T cells. gDNA was harvested at 72 or 96 hours postelectroporation and editing was analyzed by NGS. All LPETs, pegRNAs, and sgRNAs were end-modified with three 2’0Me and PS linkages at both the 3’ and 5’ ends. N = 2.

[0132] FIG. 9 shows editing activity of prime editors PE2 (MMLV), PE6D (MMLV), PE6A (EC48RT), PE6B (TF1-RT), PE6C (TF1-RT) and PE2-deltaRNaseH (MMLV) in fused format (Nickase-Polymerase) with pegRNA consisting of RNA PBS / RTT, 2’0Me PBS / RNA RTT, and fully modified (all 2’0Me) PBS / RTT at the HBB genomic locus. 0.25ug Effector mRNA, 25pmol pegRNA, 8.33pmol nicking sgRNA were electroporated into 50,000 HEK293T cells. gDNA was harvested 96 hrs post-electroporation and editing was analyzed by NGS. All LPETs, pegRNAs, and sgRNAs are end-modified with three 2’0Me and PS linkages at both the 3’ and 5’ ends. N = 3.

[0133] FIG. 10 shows editing activity of prime editors PE2 (MMLV), PE6D (MMLV), PE6A (EC48RT), PE6B (TF1-RT), PE6C (TF1-RT) and PE2-deltaRNaseH (MMLV) in fused format (Nickase-Polymerase) with pegRNA consisting of RNA PBS / RTT, 2’0Me PBS / RNA RTT, and fully modified (all 2’0Me) PBS / RTT at the PRNP genomic locus. 0.25ug Effector mRNA and 25pmol pegRNA were electroporated into 50,000 HEK293T cells. gDNA was harvested 96 hrs post-electroporation and editing was analyzed by NGS. All LPETs, pegRNAs, and sgRNAs are end-modified with three 2’0Me and PS linkages at both the 3’ and 5’ ends. N = 3.

[0134] FIG. 11 shows editing activity of prime editors PE2 (MMLV), PE6D (MMLV), PE6A (EC48RT), PE6B (TF1-RT), PE6C (TF1-RT) and PE2-deltaRNaseH (MMLV) in fused format (Nickase-Polymerase) with pegRNA consisting of RNA PBS / RTT, 2’0Me PBS / RNA RTT, and fully modified (all 2’0Me) PBS / RTT at the PRNP genomic locus. 0.25ug Effector1280966915. v1Attorney Docket. No. 767854: UM9-316PC mRNA, 8.33pmol Nicking sgRNA and 25pmol pegRNA were electroporated into 50,000 HEK293T cells. gDNA was harvested 96 hrs post-electroporation and editing was analyzed by NGS. All LPETs, pegRNAs, and sgRNAs are end-modified with three 2’0Me and PS linkages at both the 3’ and 5’ ends. N = 3.

[0135] FIG. 12 shows editing activity of prime editors PE2 (MMLV), PE6D (MMLV), PE6A (EC48RT), PE6B (TF1-RT), PE6C (TF1-RT) and PE2-deltaRNaseH (MMLV) in split format (MCP-Polymerase) with LPET consisting of RNA PBS / RTT, 2’0Me PBS / RNA RTT, fully modified (all 2’0Me) PBS / RTT and fully modified (all 2’0Me) PBS / RTT with lOnt of MS2 2’0Me at 5’ end at the FANCF genomic locus. 0.5ug nCas9 and MCP-polymerase mRNA each, 50pmol sgRNA, and 50pmol LPET were electroporated into 50,000 HEK293T cells. gDNA was harvested 96 hrs post-electroporation and editing was analyzed by NGS. All LPETs, pegRNAs, and sgRNAs are end-modified with three 2’0Me and PS linkages at both the 3’ and 5’ ends. N = 3.

[0136] FIG. 13 shows editing activity of prime editors PE2 (MMLV), PE6D (MMLV), PE6A (EC48RT), PE6B (TF1-RT), PE6C (TF1-RT) and PE2-deltaRNaseH (MMLV) in split format (MCP-Polymerase) with LPET consisting of RNA PBS / RTT, 2’0Me PBS / RNA RTT, fully modified (all 2’0Me) PBS / RTT and fully modified (all 2’0Me) PBS / RTT with lOnt of MS2 2’0Me at 5’ end at the FANCF genomic locus. 0.5ug nCas9 and MCP-polymerase mRNA each, 50pmol sgRNA, 50pmol Nicking sgRNA and 50pmol LPET were electroporated into 50,000 HEK293T cells. gDNA was harvested 96 hrs post-electroporation and editing was analyzed by NGS. All LPETs, pegRNAs, and sgRNAs are end-modified with three 2’0Me and PS linkages at both the 3’ and 5’ ends. N = 3.

[0137] FIG. 14 shows editing activity of prime editors PE2 (MMLV), PE6D (MMLV), PE6A (EC48RT), PE6B (TF1-RT), PE6C (TF1-RT) and PE2-deltaRNaseH (MMLV) in split format (MCP-Polymerase) with LPET consisting of RNA PBS / RTT, 2’0Me PBS / RNA RTT, fully modified (all 2’0Me) PBS / RTT and fully modified (all 2’0Me) PBS / RTT with lOnt of MS2 2’0Me at 5’ end at the HBB genomic locus. 0.5ug nCas9 and MCP-polymerase mRNA each, 50pmol sgRNA, 50pmol Nicking sgRNA and 50pmol LPET were electroporated into 50,000 HEK293T cells. gDNA was harvested 96 hrs post-electroporation and editing was analyzed by NGS. All LPETs, pegRNAs, and sgRNAs are end-modified with three 2’0Me and PS linkages at both the 3’ and 5’ ends. N = 2-3.

[0138] FIG. 15 shows editing activity of prime editors PE2 (MMLV), PE6D (MMLV), PE6A (EC48RT), PE6B (TF1-RT), PE6C (TF1-RT) and PE2-deltaRNaseH (MMLV) in split format (MCP-Polymerase) with LPET consisting of RNA PBS / RTT, 2’0Me PBS / RNA RTT,1380966915. v1Attorney Docket. No. 767854: UM9-316PC fully modified (all 2’0Me) PBS / RTT and fully modified (all 2’0Me) PBS / RTT with lOnt of MS2 2’0Me at 5’ end at the PRNP genomic locus. 0.5ug nCas9 and MCP -polymerase mRNA each, 50pmol sgRNA, 50pmol Nicking sgRNA and 50pmol LPET were electroporated into 50,000 HEK293T cells. gDNA was harvested 96 hrs post-electroporation and editing was analyzed by NGS. All LPETs, pegRNAs, and sgRNAs are end-modified with three 2’0Me and PS linkages at both the 3’ and 5’ ends. N = 3.

[0139] FIG. 16 shows editing activity of prime editors PE6B (TF1-RT), PE6C (TF1-RT) and Mutants in PE6C polymerase as specified in fused format (Nickase-Polymerase) with pegRNA consisting of fully modified (all 2’0Me) PBS / RTT at the PRNP genomic locus. 0.25ug Effector mRNA and 25pmol pegRNA were electroporated into 50,000 HEK293T cells. gDNA was harvested 96 hrs post-electroporation and editing was analyzed by NGS. All LPETs, pegRNAs, and sgRNAs are end-modified with three 2’0Me and PS linkages at both the 3’ and 5’ ends. N = 2.

[0140] FIG. 17 shows editing activity of prime editor PE6C (TF1-RT) and mutants in PE6C polymerase as specified in fused format (Nickase-Polymerase) with pegRNA consisting of fully modified (all 2’0Me) PBS / RTT at the PRNP genomic locus. 0.25ug Effector mRNA and 25pmol pegRNA were electroporated into 50,000 HEK293T cells. gDNA was harvested 96 hrs post-electroporation and editing was analyzed by NGS. All LPETs, pegRNAs, and sgRNAs are end-modified with three 2’0Me and PS linkages at both the 3’ and 5’ ends. N = 2.

[0141] FIG. 18 shows editing activity of prime editor PE6C (TF1-RT) and mutants in PE6C polymerase as specified in fused format (Nickase-Polymerase) with pegRNA consisting of fully modified (all 2’0Me) PBS / RTT at the PRNP genomic locus. 0.25ug Effector mRNA and 25pmol pegRNA were electroporated into 50,000 HEK293T cells. gDNA was harvested 96 hrs post-electroporation and editing was analyzed by NGS. All LPETs, pegRNAs, and sgRNAs are end-modified with three 2’0Me and PS linkages at both the 3’ and 5’ ends. N = 2.

[0142] FIG. 19 shows editing activity of prime editor PE6C (TF1-RT) and mutants in PE6C polymerase as specified in fused format (Nickase-Polymerase) with pegRNA consisting of RNA PBS / RTT, 2’0Me PBS / RNA RTT, fully modified (all 2’0Me) PBS / RTT, 2’0Me RTT PBS RNA, 2’0Me RTT PBS RNA+PS and RTT 2’0Me PBS RNA / DNA alternating at the FANCF genomic locus. 0.25ug Effector mRNA and 25pmol pegRNA(s) were electroporated into 50,000 HEK293T cells. gDNA was harvested 96 hrs post-electroporation1480966915. v1Attorney Docket. No. 767854: UM9-316PC and editing was analyzed by NGS. All LPETs, pegRNAs, and sgRNAs are end-modified with three 2’0Me and PS linkages at both the 3’ and 5’ ends. N = 3.

[0143] FIG. 20 shows editing activity of prime editor PE6C+I33 V+M214A as specified in fused format (Nickase-Polymerase) with pegRNA consisting of multiple PBS and RTT modifications at the FANCF genomic locus. 0.25ug Effector mRNA and 25pmol pegRNA(s) were electroporated into 50,000 HEK293T cells. gDNA was harvested 96 hrs postelectroporation and editing was analyzed by NGS. All LPETs, pegRNAs, and sgRNAs are end-modified with three 2’0Me and PS linkages at both the 3’ and 5’ ends. N = 3.

[0144] FIG. 21A - FIG. 21B show a schematic representation of scaffold readthrough and non-nucleotide blocker in LPET. FIG. 21A shows a generalized representation of sPE with LPET which can result into readthrough into MS2 scaffold leading to impure flap generation containing MS2 scaffold and eventual incorporation into genome resulting into unintended edits with scaffold readthrough (unintended edits with solid line bracket, intended edits with dashed line brackets). FIG. 21B depicts a generalized representation of sPE with LPET containing a non-nucleotide blocker between MS scaffold and RTT, resulting in complete stop of polymerase readthrough into MS2 scaffold and only intended edit incorporation into genome.

[0145] FIG. 22A - FIG. 22B show a schematic representation of scaffold readthrough and non-nucleotide blocker in pegRNA. FIG. 22A depicts a generalized representation of PE with pegRNA which can result into readthrough into sgRNA scaffold leading to impure flap generation containing sgRNA scaffold and eventual incorporation into genome resulting into united edits with scaffold readthrough (unintended edits with solid line bracket, intended edits with dashed line brackets). FIG. 22B depicts a generalized representation of PE with pegRNA containing a non-nucleotide blocker between sgRNA scaffold and RTT, resulting in complete stop of polymerase readthrough into sgRNA scaffold and only intended edit incorporation into genome.

[0146] FIG. 23A shows fraction of edited reads with MS2 sequence in genome and FIG.23B depicts editing activity of prime editors PE6C (TF1-RT, left bar for each data point) and PE6B (TF1-RT, right bar for each data point) as specified in split format (Nickase + MCP- Polymerase) with 2’0Me PBS and RTT modified LPET consisting of non-nucleotide blockers, lxC6, 2xC6, IxTEG, IxHEG and 2xHEG at the FANCF genomic locus. 0.5ug Effector mRNA, nCas9 and MCP-polymerase each, 50pmol LPET, sgRNA and nicking sgRNA each, were electroporated into 50,000 HEK293T cells. gDNA was harvested 96 hrs1580966915. v1Attorney Docket. No. 767854: UM9-316PC post-electroporation and editing was analyzed by NGS. All LPETs, pegRNAs, and sgRNAs are end-modified with three 2’0Me and PS linkages at both the 3’ and 5’ ends. N = 3.

[0147] FIG. 24 shows indel activity of prime editors PE6C (TF1-RT, left bar for each data point) and PE6B (TF1-RT, right bar for each data point) as specified in split format (Nickas + MCP-Polymerase) with 2’0Me PBS and RTT modified LPET consisting of non-nucleotide blockers, lxC6, 2xC6, IxTEG, IxHEG and 2xHEG at the FANCF genomic locus. 0.5ug Effector mRNA, nCas9 and MCP-polymerase each, 50pmol LPET, sgRNA and nicking sgRNA each, were electroporated into 50,000 HEK293T cells. gDNA was harvested 96 hrs post-electroporation and editing was analyzed by NGS. All LPETs, pegRNAs, and sgRNAs are end-modified with three 2’0Me and PS linkages at both the 3’ and 5’ ends. N = 3.

[0148] FIG. 25 shows percentage of edited reads ending at each nucleotide position of MS2 stemloop of total reads with scaffold readthrough with prime editor PE6C (TF1-RT) in split format (Nickase + MCP-Polymerase) with 2’0Me PBS and RTT modified LPET consisting of non-nucleotide blockers, lxC6 (1 x C6 alkyl chain), 2xC6 (2 x C6 alkyl chain), IxTEG, IxHEG and 2xHEG at the FANCF genomic locus. Each number on X axis represent nucleotide position of MS2 detected in genome at edit site, where 0 is the last nucleotide of RTT. First nucleotide of MS2 is shown together with last nucleotide of RTT (0+1) as it is same as in the genome and cannot be distinguished as coming from MS2 readthrough. 0.5ug Effector mRNA, nCas9 and MCP-polymerase each, 50pmol LPET, sgRNA and nicking sgRNA each, were electroporated into 50,000 HEK293T cells. gDNA was harvested 96 hrs post-electroporation and editing was analyzed by NGS. All LPETs, pegRNAs, and sgRNAs are end-modified with three 2’0Me and PS linkages at both the 3’ and 5’ ends. N = 3.

[0149] FIG. 26 shows editing efficiency with prime editor PE6C (TF1-RT) in split format (Nickase + MCP-Polymerase) with 2’0Me PBS and RTT modified LPET consisting of non- nucleotide blockers IxHEG and different modification pattern of MS2 stemloop and end modification at the FANCF genomic locus. 0.5ug Effector mRNA, nCas9 and MCP- polymerase each, 50pmol LPET, sgRNA and nicking sgRNA each, were electroporated into 50,000 HEK293T cells. gDNA was harvested 96 hrs post-electroporation and editing was analyzed by NGS. All LPETs, pegRNAs, and sgRNAs are end-modified with three 2’0Me and PS linkages at both the 3’ and 5’ ends. N = 3. In the figure, from left to right, are MS2- HEG-RTT=PB2 (end mod), MS2 (3’ 3x2OMe)-HEG-RTT-PBS, MS2 (3’ 3x2OMe)-HEG- RTT-PBS-C3, MS2 (3’ 3x2OMe + PS)-HEG-RTT-PBS, (5’ 5x2OMe) MS2 (3’ 5x2OMe)- HEG-RTT-PBS, (5’ 6x2OMe) MS2 (3’ 6x2OMe)-HEG-RTT-PBS, (5’ 7x2OMe) MS2 (3’ 7x2OMe)-HEG-RTT-PBS, (5’ 9x2OMe) MS2 (3’ 8x2OMe)-HEG-RTT-PBS, MS2 (5’1680966915. v1Attorney Docket. No. 767854: UM9-316PC6x2OMe) (l lx2’F) (3’ 6x2OMe)-HEG-RTT-PBS, MS2 (5’ 9x2OMe) (6x2’F) (3’ 8x2OMe)- HEG-RTT-PBS.

[0150] FIG. 27 shows editing activity of prime editors PE6C (TF1-RT), PE6C+I33V+M214A and PE6C+I33V+M214A with PEMax linkers and NLS polymerase as specified in fused format (Nickase-Polymerase) with pegRNA consisting of PBS modified 2’0Me only (left bar) and fully modified (all 2’0Me) PBS / RTT (right bar) at the PCSK9 genomic locus. 1.5ng Effector mRNA and 1.5pmol pegRNA were electroporated into 50,000 HEPA1-6 cells. gDNA was harvested 96 hrs post-electroporation and editing was analyzed by NGS. All LPETs, pegRNAs, and sgRNAs are end-modified with three 2’0Me and PS linkages at both the 3’ and 5’ ends. N = 3.

[0151] FIG. 28 shows editing efficiency with prime editors PE6B (TF1-RT) PE6C (TF1- RT), with evolved RNaseH domain (EvPol), without RNaseH domain (EvPolARNaseH) and with Wildtype RNaseH domain (EvPol WT RNaseH) in split format (Nickase + MCP- Polymerase) with 2’0Me PBS and RTT modified LPET at the PRNP genomic locus. 0.5ug Effector mRNA, nCas9 and MCP-polymerase each, 50pmol LPET, sgRNA and nicking sgRNA each, were electroporated into 50,000 HEK293T cells. gDNA was harvested 96 hrs post-electroporation and editing was analyzed by NGS. All LPETs, pegRNAs, and sgRNAs are end-modified with three 2’0Me and PS linkages at both the 3’ and 5’ ends. N = 3.

[0152] FIG. 29 shows editing efficiency with prime editors PE6C (TF1-RT) or Wild-Type polymerase TF1-RT (WT) in split format (Nickase + MCP-Polymerase) with 2’0Me PBS and RTT modified LPET at the (FIG. 29A) FANCF (FIG. 29B) HBB (FIG. 29C) PRNP genomic locus. 0.5ug Effector mRNA, nCas9 and MCP-polymerase each, 50pmol LPET, sgRNA and nicking sgRNA each, were electroporated into 50,000 HEK293T cells. gDNA was harvested 96 hrs post-electroporation and editing was analyzed by NGS. All LPETs, pegRNAs, and sgRNAs are end-modified with three 2’0Me and PS linkages at both the 3’ and 5’ ends. N = 3.

[0153] FIG. 30 shows editing efficiency with prime editor PE6C (TF1-RT), mutation reversal to wild-type in PE6C, and wild-type polymerase TF1-RT (WT) in split format (Nickase + MCP-Polymerase) with 2’0Me PBS or PBS and RTT modified LPET at the (FIG. 30A) FANCF and (FIG. 30B) PRNP genomic locus. 0.5ug Effector mRNA, nCas9 and MCP- polymerase each, 50pmol LPET, sgRNA and nicking sgRNA each, were electroporated into 50,000 HEK293T cells. gDNA was harvested 96 hrs post-electroporation and editing was analyzed by NGS. All LPETs, pegRNAs, and sgRNAs are end-modified with three 2’0Me and PS linkages at both the 3’ and 5’ ends. N = 3.1780966915. v1Attorney Docket. No. 767854: UM9-316PCDETAILED DESCRIPTION OF THE DISCLOSURE

[0154] The present invention relates to the field of genomic engineering. In particular, described herein are prime editing systems employing a modification-tolerant nucleotide polymerase (NP) protein paired with a pegRNA or petRNA comprising one or more nucleotide modifications. The pegRNA or petRNA molecules harboring nucleotide modifications are more resistant to degradation following in vivo administration, making the prime editing system described herein particularly useful for therapeutic applications. Incorporation of nucleotide modifications or non-nucleotide linkers into the pegRNA or petRNA (such as hexaethylene glycol (HEG)) also decreases polymerase readthrough, wherein the polymerase adds guide RNA scaffold sequence at the prime edited genomic site.

[0155] To facilitate the understanding of this invention, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention. Terms such as “a”, “an” and “the” are not intended to refer to only a singular entity but also plural entities and also includes the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the invention, but their usage does not delimit the invention, except as outlined in the claims.

[0156] The term “catalytically impaired Cas9 nickase” or “nCas9”, as used herein refers to a mutated Cas9 which renders the nuclease able to cleave only one strand of deoxyribonucleic acid backbone. Depending on the position of the mutation within the Cas9 protein sequence either the target or non-target strand is cleaved. In the case of a prime editor the non-target strand is selectively cleaved.

[0157] The term “engineered reverse transcriptase” as used herein, refers to a protein that converts RNA into DNA and contains specific mutations that effect its activity efficiency. One example of a reverse transcriptase is a Moloney murine leukemia virus reverse transcriptase (M- MLV RT).

[0158] The term “reverse transcriptase template” or “RTT” as used herein refers to a ribonucleic acid sequence that is utilized as a substrate for a reverse transcriptase protein that is part of the prime editor protein complex as contemplated herein. Such templates provide the necessary information to edit a DNA sequence to support conversions including, but not limited to, base conversions, sequence insertions or sequence deletions.1880966915. v1Attorney Docket. No. 767854: UM9-316PC

[0159] The term "nucleotide polymerase template" or “NPT” as used herein refers to a deoxyribonucleic or a ribonucleic acid sequence and modifications thereof, that is utilized as a nucleic acid for a nucleotide polymerase protein (e.g., RNA polymerase or DNA polymerase) that is part of the prime editor complex as contemplated herein. Such templates provide the necessary information to edit a DNA sequence to support conversions including, but not limited to, base conversions, sequence insertions or sequence deletions.

[0160] The term “primer binding site” or “PBS” as used herein, refers to a specific nucleic acid sequence within the pegRNA or the petRNA that is complementary to the 3’ end of the nicked DNA strand. This allows annealing of the free 3’ end of the genomic DNA for extension by the nucleotide polymerase based on the template sequence encoded in the pegRNA or the petRNA.

[0161] The term, “prime editing guide RNA molecule” or “pegRNA molecule” as used herein, refers to a Cas9 guide RNA molecule that encodes the crRNA-tracrRNA (i.e., single guide RNA (sgRNA) fused to a primer binding site (PBS) and a nucleotide polymerase template (NPT) nucleic acid sequence. The primer binding site hybridizes to a desired genomic sequence released by the binding and cleavage of the Cas9 nickase. The 3’ end of the genomic sequence is extended by the nucleotide polymerase based on the nucleotide polymerase template sequence.

[0162] The term, “prime editor template RNA” or “petRNA molecule” as used herein, refers to an RNA molecule that encodes a primer binding site (PBS) and a nucleotide polymerase template (NPT). The petRNA may also encode stem loops. The petRNA may also be linear or circularized. Unlike the pegRNA, the petRNA does not include the guide RNA component.

[0163] The term “editing” or “gene editing” as used herein, refers to a genetic manipulation of a DNA sequence. Such a manipulation includes, but is not limited to, a base conversion, a sequence insertion and / or a sequence deletion. The term “group I catalytic intron” as used herein, refers to large self-splicing ribozymes which self-catalyze an excision from ribonucleotides including, but not limited to, mRNA, tRNA and rRNA. See, Figure 19. Nielsen et al., "Group I introns: Moving in new directions" RNA Biol. 6(4):375-83 (2009); and Cech T., "Self-splicing of group I introns" Annu. Rev. Biochem. 59:543-568 (1990). Their core secondary structure included paired regions. Woodson S, "Structure and assembly of group I introns" Curr. Opin. Struct. Biol. 15(3):324-330 (2005). These paired regions selfassembly into domains: i) the P4-P6 domain formed from stacking of P5, P4, P6 and P6a helices; and ii) the P3-P9 domain formed from the P8, P3, P7 and P9 helices). Cate et al.,1980966915. v1Attorney Docket. No. 767854: UM9-316PC"Crystal structure of a group I ribozyme domain: principles of RNA packing". Science. 273 (5282): 1678-1685 (1996). Group I introns often have long open reading frames inserted in loop regions.

[0164] The term “prime editing” as used herein, is a genome editing technology by which the genome of living organisms may be modified. Prime editing manipulates the genetic information of a targeted DNA site to essentially “rewrite” the coded sequences.

[0165] The term “prime editor” or “PE” as used herein, is a fusion protein comprising a catalytically impaired Cas9 endonuclease that can nick DNA and is fused to an engineered nucleotide polymerase enzyme. The petRNA comprising a PBS, an NPT along with a single guide RNA (sgRNA), are capable of programming the nCas9 to recognize a target site with the encoded crRNA-tracrRNA (as does a conventional single guide RNA). The resulting nicked genomic DNA can be extended by the nucleotide polymerase based on the petRNA template sequence to contain a new sequence. Once one strand is recoded, cellular DNA repair pathways can cause conversion of the local DNA sequence to match the new sequence. Such manipulation includes, but is not limited to, insertions, deletions, and base-to-base conversions without the need for double strand breaks (DSBs) or donor DNA templates. For example, such prime editing may be performed by a Cas9 CRISPR platform programmed with a petRNA and an sgRNA, such as a catalytically impaired Cas9 nickase platform with an appropriate nucleotide polymerase.

[0166] The term “conversion” as used herein, refers to any manipulation of a nucleic acid sequence that converts a mutated sequence into a wildtype sequence, or a wildtype sequence into a mutated sequence. For example, a converted sequence includes, but is not limited to, a base pair conversion, a nucleic acid sequence insertion or a nucleic acid sequence deletion. The term “editing-related indels” as used herein, refers to the generation of off-target and / or unintended nucleotide sequence insertions created by a prime editor.

[0167] The term “split-intein prime editor protein” refers to a prime editor protein that has been split into amino-terminal (PE2-N) and carboxy -terminal (PE2-C) segments, which are then fused into a full length PE by a trans-splicing intein. This configuration imparts flexibility to the prime editor thereby facilitating a packaging into an adeno-associated virus (AAV).

[0168] As used herein, the term “CRISPRs” or “Clustered Regularly Interspaced Short Palindromic Repeats” refers to an acronym for DNA loci that contain multiple, short, direct repetitions of base sequences. Each repetition contains a series of bases followed by 30 or so base pairs known as "spacer" sequence. The spacers are short segments of DNA from a virus2080966915. v1Attorney Docket. No. 767854: UM9-316PC and may serve as a 'memory' of past exposures to facilitate an adaptive defense against future invasions. Doudna et al. Genome editing. The new frontier of genome engineering with CRISPR-Cas9” Science 346(6213): 1258096 (2014).

[0169] As used herein, the term “Cas” or “CRISPR-associated (cas)” refers to genes often associated with CRISPR repeat-spacer arrays.

[0170] As used herein, the term “Cas9” refers to a nuclease from type II CRISPR systems, an enzyme specialized for generating double-strand breaks in DNA, with two active cutting sites (the HNH and RuvC domains), one for each strand of the double helix. tracrRNA and spacer RNA may be combined into a "single-guide RNA" (sgRNA) molecule that, mixed with Cas9, could find and cleave DNA targets through Watson-Crick pairing between the guide sequence within the sgRNA and the target DNA sequence, Jinek et al. A programmable dual- RNA-guided DNA endonuclease in adaptive bacterial immunity” Science 337(6096):816-821 (2012).

[0171] As used herein, the term “catalytically active Cas9” refers to an unmodified Cas9 nuclease comprising full nuclease activity.

[0172] The term “nickase” as used herein, refers to a nuclease that cleaves only a single DNA strand, either due to its natural function or because it has been engineered to cleave only a single DNA strand. Cas9 nickase variants that have either the RuvC or the HNH domain mutated provide control over which DNA strand is cleaved and which remains intact. Jinek et al., “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity” Science 337(6096):816-821 (2012) and Cong et al. Multiplex genome engineering using CRISPR / Cas systems” Science 339(6121):819-823 (2013). The term, “trans-activating crRNA”, “tracrRNA” as used herein, refers to a small trans- encoded RNA. For example, CRISPR / Cas (clustered, regularly interspaced short palindromic repeats / CRISPR-associated proteins) constitutes an RNA-mediated defense system, which protects against viruses and plasmids. This defensive pathway has three steps. First a copy of the invading nucleic acid is integrated into the CRISPR locus. Next, CRISPR RNAs (crRNAs) are transcribed from this CRISPR locus. The crRNAs are then incorporated into construct complexes, where the crRNA guides the complex to the invading nucleic acid and the Cas proteins degrade this nucleic acid. There are several pathways of CRISPR activation, one of which requires a tracrRNA, which plays a role in the maturation of crRNA. TracrRNA is complementary to the repeat sequence of the pre-crRNA, forming an RNA duplex. This is cleaved by RNase III, an RNA-specific ribonuclease, to form a crRNA / tracrRNA hybrid. This hybrid acts as a guide for the endonuclease Cas9, which cleaves the invading nucleic acid.2180966915. v1Attorney Docket. No. 767854: UM9-316PC

[0173] The term “protospacer adjacent motif’ or “PAM” as used herein, refers to a DNA sequence that may be required for a Cas9 / sgRNA to form an R-loop to interrogate a specific DNA sequence through Watson-Crick pairing of its guide RNA with the genome. The PAM specificity may be a function of the DNA-binding specificity of the Cas9 protein (e.g., a “protospacer adjacent motif recognition domain” at the C-terminus of Cas9).

[0174] The terms “protospacer adjacent motif recognition domain”, “PAM Interacting Domain” or “PID” as used herein, refers to a Cas9 amino acid sequence that comprises a binding site to a DNA target PAM sequence.

[0175] The term “binding site” as used herein, refers to any molecular arrangement having a specific tertiary and / or quaternary structure that undergoes a physical attachment or close association with a binding component. For example, the molecular arrangement may comprise a sequence of amino acids. Alternatively, the molecular arrangement may comprise a sequence a nucleic acids. Furthermore, the molecular arrangement may comprise a lipid bilayer or other biological material.

[0176] As used herein, the term “sgRNA” refers to single guide RNA used in conjunction with CRISPR associated systems (Cas). sgRNAs are a fusion of crRNA and tracrRNA and contain nucleotides of sequence complementary to the desired target site. Jinek et al, “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity” Science 337(6096):816- 821 (2012) Watson-Crick pairing of the sgRNA with the target site permits R-loop formation, which in conjunction with a functional PAM permits DNA cleavage or in the case of nuclease- deficient Cas9 allows binds to the DNA at that locus. In some embodiments, the sgRNA or sgRNA portion of the disclosure comprises at least one nucleotide modification as described herein.

[0177] As used herein, the term “orthogonal” refers to targets that are non-overlapping, uncorrelated, or independent. For example, if two orthogonal Cas9 isoforms were utilized, they would employ orthogonal sgRNAs that only program one of the Cas9 isoforms for DNA recognition and cleavage. Esvelt et al., “Orthogonal Cas9 proteins for RNA-guided gene regulation and editing” Nat Methods 10(11): 1116-1121 (2013). For example, this would allow one Cas9 isoform (e.g. S. pyogenes Cas9 or SpyCas9) to function as a nuclease programmed by a sgRNA that may be specific to it, and another Cas9 isoform (e.g. N meningitidis Cas9 or NmeCas9) to operate as a nuclease-dead Cas9 that provides DNA targeting to a binding site through its PAM specificity and orthogonal sgRNA. Other Cas9s include S. aureus Cas9 or SauCas9 and A. naeslundii Cas9 or AnaCas9.2280966915. v1Attorney Docket. No. 767854: UM9-316PC

[0178] The term “truncated” as used herein, when used in reference to either a polynucleotide sequence or an amino acid sequence means that at least a portion of the wild type sequence may be absent. In some cases, truncated guide sequences within the sgRNA or crRNA may improve the editing precision of Cas9. Fu, et al. “Improving CRISPR-Cas nuclease specificity using truncated guide RNAs” Nat Biotechnol. 2014 Mar;32(3):279-284 (2014).

[0179] The term “base pairs” as used herein, refer to specific nucleobases (also termed nitrogenous bases), that are the building blocks of nucleotide sequences that form a primary structure of both DNA and RNA. Double-stranded DNA may be characterized by specific hydrogen bonding patterns. Base pairs may include, but are not limited to, guanine-cytosine and adenine-thymine base pairs.

[0180] The term “specific genomic target” as used herein, refers to any pre-determined nucleotide sequence capable of binding to a Cas9 protein contemplated herein. The target may include, but may be not limited to, a nucleotide sequence complementary to a programmable DNA binding domain or an orthogonal Cas9 protein programmed with its own guide RNA, a nucleotide sequence complementary to a single guide RNA, a protospacer adjacent motif recognition sequence, an on-target binding sequence and an off-target binding sequence.

[0181] As used herein, the term “edit” “editing” or “edited” refers to a method of altering a nucleic acid sequence of a polynucleotide (e.g., for example, a wild type naturally occurring nucleic acid sequence or a mutated naturally occurring sequence) by selective deletion of a specific genomic target or the specific inclusion of new sequence through the use of an exogenously supplied DNA template. Such a specific genomic target includes, but may be not limited to, a chromosomal region, mitochondrial DNA, a gene, a promoter, an open reading frame or any nucleic acid sequence.

[0182] The term “effective amount” as used herein, refers to a particular amount of a pharmaceutical composition comprising a therapeutic agent that achieves a clinically beneficial result (i.e., for example, a reduction of symptoms). Toxicity and therapeutic efficacy of such compositions can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, and it can be expressed as the ratio LD50 / ED50. Compounds that exhibit large therapeutic indices are preferred. The data obtained from these cell culture assays and additional animal studies can be used in formulating a range of dosage for human use. The dosage of such compounds lies2380966915. v1Attorney Docket. No. 767854: UM9-316PC preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.

[0183] The terms "reduce, " "inhibit, " "diminish, " "suppress, " "decrease, " “prevent” and grammatical equivalents (including “lower, ” “smaller, ” etc.) when in reference to the expression of any symptom in an untreated subject relative to a treated subject, mean that the quantity and / or magnitude of the symptoms in the treated subject is lower than in the untreated subject by any amount that is recognized as clinically relevant by any medically trained personnel. In one embodiment, the quantity and / or magnitude of the symptoms in the treated subject is at least 10% lower than, at least 25% lower than, at least 50% lower than, at least 75% lower than, and / or at least 90% lower than the quantity and / or magnitude of the symptoms in the untreated subject.

[0184] The term "attached" as used herein, refers to any interaction between a medium (or carrier) and a drug. Attachment may be reversible or irreversible. Such attachment includes, but is not limited to, covalent bonding, ionic bonding, Van der Waals forces or friction, and the like.

[0185] The term “derived from” as used herein, refers to the source of a sample, a compound or a sequence. In one respect, a sample, a compound or a sequence may be derived from an organism or particular species. In another respect, a sample, a compound or sequence may be derived from a larger complex or sequence.

[0186] The term “protein” as used herein, refers to any of numerous naturally occurring extremely complex substances (as an enzyme or antibody) that consist of amino acid residues joined by peptide bonds, contain the elements carbon, hydrogen, nitrogen, oxygen, usually sulfur. In general, a protein comprises amino acids having an order of magnitude within the hundreds.

[0187] The term “peptide” as used herein, refers to any of various amides that are derived from two or more amino acids by combination of the amino group of one acid with the carboxyl group of another and are usually obtained by partial hydrolysis of proteins. In general, a peptide comprises amino acids having an order of magnitude with the tens.

[0188] The term “polypeptide”, as used herein, refers to any of various amides that are derived from two or more amino acids by combination of the amino group of one acid with the carboxyl group of another and are usually obtained by partial hydrolysis of proteins. In general, a peptide comprises amino acids having an order of magnitude with the tens or larger.2480966915. v1Attorney Docket. No. 767854: UM9-316PC

[0189] The term "pharmaceutically" or "pharmacologically acceptable", as used herein, refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human.

[0190] The term, "pharmaceutically acceptable carrier", as used herein, includes any and all solvents, or a dispersion medium including, but not limited to, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils, coatings, isotonic and absorption delaying agents, liposome, commercially available cleansers, and the like. Supplementary bioactive ingredients also can be incorporated into such carriers.

[0191] "Nucleic acid sequence" and "nucleotide sequence", as used herein, refer to an oligonucleotide or polynucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin which may be single- or double-stranded, and represent the sense or antisense strand.

[0192] The term "an isolated nucleic acid”, as used herein, refers to any nucleic acid molecule that has been removed from its natural state (e.g., removed from a cell and is, in a preferred embodiment, free of other genomic nucleic acid).

[0193] The terms "amino acid sequence" and "polypeptide sequence" as used herein, are interchangeable and to refer to a sequence of amino acids.

[0194] The term "portion" when used in reference to a nucleotide sequence refers to fragments of that nucleotide sequence. The fragments may range in size from 5 nucleotide residues to the entire nucleotide sequence minus one nucleic acid residue. When used in reference to an amino acid sequence refers to fragments of that amino acid sequence. The fragment may range in size from 2 amino acid residues to the entire amino acid sequence minus one amino acid residue.

[0195] As used herein, the terms "complementary" or "complementarity" are used in reference to "polynucleotides" and "oligonucleotides" (which are interchangeable terms that refer to a sequence of nucleotides) related by the base-pairing rules. For example, the sequence "C-A-G-T, " is complementary to the sequence "G-T-C-A." Complementarity can be "partial" or "total." "Partial" complementarity is where one or more nucleic acid bases is not matched according to the base pairing rules. "Total" or "complete" complementarity between nucleic acids is where each and every nucleic acid base is matched with another base under the base pairing rules. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of2580966915. v1Attorney Docket. No. 767854: UM9-316PC particular importance in amplification reactions, as well as detection methods which depend upon binding between nucleic acids.

[0196] As used herein, the term "primer" refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is induced, (i.e., in the presence of nucleotides and an inducing agent such as DNA polymerase and at a suitable temperature and pH). The primer is preferably single stranded for maximum efficiency in amplification but may alternatively be double stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products. Preferably, the primer is an oligodeoxy-ribonucleotide. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the inducing agent. The exact lengths of the primers will depend on many factors, including temperature, source of primer and the use of the method.

[0197] DNA molecules are said to have "5' ends" and "3' ends" because mononucleotides are reacted to make oligonucleotides in a manner such that the 5' phosphate of one mononucleotide pentose ring is attached to the 3' oxygen of its neighbor in one direction via a phosphodiester linkage. Therefore, an end of an oligonucleotide is referred to as the "5' end" if its 5' phosphate is not linked to the 3' oxygen of a mononucleotide pentose ring. An end of an oligonucleotide is referred to as the "3' end" if its 3' oxygen is not linked to a 5' phosphate of another mononucleotide pentose ring. As used herein, a nucleic acid sequence, even if internal to a larger oligonucleotide, also may be said to have 5' and 3' ends. In either a linear or circular DNA molecule, discrete elements are referred to as being "upstream" or 5' of the "downstream" or 3' elements. This terminology reflects the fact that transcription proceeds in a 5' to 3' fashion along the DNA strand. The promoter and enhancer elements which direct transcription of a linked gene are generally located 5' or upstream of the coding region. However, enhancer elements can exert their effect even when located 3' of the promoter element and the coding region. Transcription termination and polyadenylation signals are located 3' or downstream of the coding region.

[0198] As used herein, the term "an oligonucleotide having a nucleotide sequence encoding a gene" means a nucleic acid sequence comprising the coding region of a gene, i.e. the nucleic acid sequence which encodes a gene product. The coding region may be present in a cDNA, genomic DNA or RNA form. When present in a DNA form, the oligonucleotide may be single-stranded (i.e., the sense strand) or double-stranded. Suitable control elements2680966915. v1Attorney Docket. No. 767854: UM9-316PC such as enhancers / promoters, splice junctions, polyadenylation signals, etc. may be placed in close proximity to the coding region of the gene if needed to permit proper initiation of transcription and / or correct processing of the primary RNA transcript. Alternatively, the coding region utilized in the expression vectors of the present invention may contain endogenous enhancers / promoters, splice junctions, intervening sequences, polyadenylation signals, etc. or a combination of both endogenous and exogenous control elements.

[0199] As used herein, a “non-nucleotide linker” refers to a linker that is neither a nucleotide or a single covalent bond and that is capable of reducing readthrough of a nucleotide polymerase as described herein into an sgRNA or MS2 stemloop. Exemplary non- nucleotide linkers include, but are not limited to, ethylene glycol-based linkers (such as PEG) and alkyl based linkers.RNA-guided nucleases

[0200] RNA-guided nucleases according to the present disclosure include, without limitation, naturally occurring Type II CRISPR nucleases such as Cas9, as well as other nucleases derived or obtained therefrom. Exemplary Cas9 nucleases that may be used in the present disclosure include, but are not limited to, S. pyogenes Cas9 (SpCas9), S. aureus Cas9 (SaCas9), N. meningitidis Cas9 (NmCas9), C. jejuni Cas9 (CjCas9), and Geobacillus Cas9 (GeoCas9). In functional terms, RNA-guided nucleases are defined as those nucleases that: (a) interact with (e.g., complex with) a gRNA; and (b) together with the gRNA, associate with, and optionally cleave or modify, a target region of a DNA that includes (i) a sequence complementary to the targeting domain of the gRNA and, optionally, (ii) an additional sequence referred to as a “protospacer adjacent motif, ” or “PAM, ” which is described in greater detail below. As the following examples will illustrate, RNA-guided nucleases can be defined, in broad terms, by their PAM specificity and cleavage activity, even though variations may exist between individual RNA-guided nucleases that share the same PAM specificity or cleavage activity. Skilled artisans will appreciate that some aspects of the present disclosure relate to systems, methods and compositions that can be implemented using any suitable RNA-guided nuclease having a certain PAM specificity and / or cleavage activity. For this reason, unless otherwise specified, the term RNA-guided nuclease should be understood as a generic term, and not limited to any particular type (e.g., Cas9 vs. Cpfl), species (e.g., S. pyogenes vs. S. aureus) or variation (e.g., full-length vs. truncated or split; naturally occurring PAM specificity vs. engineered PAM specificity).2780966915. v1Attorney Docket. No. 767854: UM9-316PC

[0201] Various RNA-guided nucleases may require different sequential relationships between PAMs and protospacers. In general, Cas9s recognize PAM sequences that are 5' of the protospacer as visualized relative to the top or complementary strand.

[0202] In addition to recognizing specific sequential orientations of PAMs and protospacers, RNA-guided nucleases generally recognize specific PAM sequences. S. aureus Cas9, for example, recognizes a PAM sequence of NNGRRT, wherein the N sequences are immediately 3' of the region recognized by the gRNA targeting domain. S. pyogenes Cas9 recognizes NGG PAM sequences. It should also be noted that engineered RNA-guided nucleases can have PAM specificities that differ from the PAM specificities of similar nucleases (such as the naturally occurring variant from which an RNA-guided nuclease is derived, or the naturally occurring variant having the greatest amino acid sequence homology to an engineered RNA-guided nuclease). Modified Cas9s that recognize alternate PAM sequences are described below.

[0203] RNA-guided nucleases are also characterized by their DNA cleavage activity: naturally occurring RNA-guided nucleases typically form DSBs in target nucleic acids, but engineered variants have been produced that generate only SSBs (discussed above; see also Ran 2013, incorporated by reference herein), or that do not cut at all.

[0204] The RNA-guided nuclease Cas9 may be a variant of Cas9 with altered activity. Exemplary variant Cas9 nucleases include, but are not limited to, a Cas9 nickase (nCas9, Table 1), a catalytically dead Cas9 (dCas9), a hyper accurate Cas9 (HypaCas9) (Chen et al. Nature, 550(7676), 407-410 (2017)), a high fidelity Cas9 (Cas9-HF) (KI einstiver et al. Nature 529(7587), 490-495 (2016)), an enhanced specificity Cas9 (eCas9) (Slaymaker et al. Science 351(6268), 84-88 (2016)), and an expanded PAM Cas9 (xCas9) (Hu et al. Nature doi: 10.1038 / nature26155 (2018)).Table 1: SpyCas9 H840A Nickase Sequence2880966915. v1Attorney Docket. No. 767854: UM9-316PC2980966915. v1Attorney Docket. No. 767854: UM9-316PC3080966915. v1Attorney Docket. No. 767854: UM9-316PC

[0205] The RNA-guided nucleases may be combined with the chemically modified guide RNAs of the present disclosure to form a genome-editing system. The RNA-guided nucleases may be combined with the chemically modified guide RNAs to form an RNP complex that may be delivered to a cell where genome-editing is desired. The RNA-guided nucleases may be expressed in a cell where genome-editing is desired with the chemically modified guide RNAs delivered separately. For example, the RNA-guided nucleases may be expressed from a polynucleotide such as a vector or a synthetic mRNA. The vector may be a viral vector, including, be not limited to, an adeno-associated virus (AAV) vector or a lentivirus (LV) vector. A Cas9 fusion polypeptide (Cas9 fusion protein) may have multiple (1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, etc.) fusion partners in any combination. As an illustrative example, a Cas9 fusion protein can have a heterologous sequence that provides an activity (e.g., for transcription modulation such as a nucleotide polymerase protein, target modification, modification of a protein associated with a target nucleic acid, etc.) and can also have a subcellular localization sequence (e.g., 1 or more NLSs, Table 2).Table 2: Sequences of the NLS protein and other tags3180966915. v1Attorney Docket. No. 767854: UM9-316PC

[0206] In some cases, such a Cas9 fusion protein might also have a tag for ease of tracking and / or purification (e.g., green fluorescent protein (GFP), YFP, RFP, CFP, mCherry, tdTomato, and the like; a histidine tag, e.g., a 6*His tag; a hemagglutinin (HA) tag; a FLAG tag; a Myc tag; and the like). As another illustrative example, a Cas9 protein can have one or more NLSs (e.g., two or more, three or more, four or more, five or more, 1, 2, 3, 4, or 5 NLSs). In some cases, a fusion partner (or multiple fusion partners) (e.g., an NLS, a tag, a fusion partner providing an activity, etc.) is located at or near the C-terminus of Cas9. In some cases, a fusion partner (or multiple fusion partners) (e.g., an NLS, a tag, a fusion partner providing an activity, etc.) is located at the N-terminus of Cas9. In some cases, a Cas9 has a fusion partner (or multiple fusion partners) (e.g., an NLS, a tag, a fusion partner providing an activity, etc.) at both the N-terminus and C-terminus.

[0207] As used herein, the term “inlaid” refers to a first protein domain (e.g., a modification-tolerant nucleotide polymerase (NP) and / or MS2 binding protein (i.e., MCP or MCP domain) that is inserted between two amino acids of a second protein domain (e.g., a Cas9 protein domain). Exemplary Cas9 N-terminus and C-terminus portions of an inlaid Cas9 are described in Table 3 and Table 4. For a given inlaid Cas9, the polypeptide fusion comprises, from N-terminus to C-terminus, the N-terminus portion of the Cas9 (e.g., i-S355 in Table 3), the non-Cas9 domain (e.g., one or more MCP domains or a modification -tolerant polymerase), and the C-terminus portion of the Cas9 (e.g., i-S355 in Table 4). The designation “i-X#” in Table 3 and 4 below signifies the inlaid amino acid position within Cas9, e.g., i- S355 means that a non-Cas9 domain is inlaid after amino acid S355 in Cas9.Table 3: Sequences of the N-terminus portion of the SpyCas9 H840A Nickase3280966915. v1Attorney Docket. No. 767854: UM9-316PC3380966915. v1Attorney Docket. No. 767854: UM9-316PC3480966915. v1Attorney Docket. No. 767854: UM9-316PC3580966915. v1Attorney Docket. No. 767854: UM9-316PCTable 4: Sequences of the C-terminus portion of the SpyCas9 H840A Nickase3680966915. v1Attorney Docket. No. 767854: UM9-316PC

[0208] In certain embodiments, the modification-tolerant NP protein is inlaid within the Cas9 nickase protein. In certain embodiments, one or more MCP domains is inlaid within the Cas9 nickase protein.

[0209] In certain embodiments, the Cas9 nickase protein comprises, from N-terminus to C-terminus, the amino acid sequence of SEQ ID NO: 3 (N-terminus portion of the Cas9 nickase of i-S355), a modification-tolerant NP protein described herein or one or more MCP3780966915. v1Attorney Docket. No. 767854: UM9-316PC domains described herein, and the amino acid sequence of SEQ ID NO: 10 (C -terminus portion of the Cas9 nickase of i-S355).

[0210] In certain embodiments, the Cas9 nickase protein comprises, from N-terminus to C-terminus, the amino acid sequence of SEQ ID NO: 4 (N-terminus portion of the Cas9 nickase of i-E1026), a modification-tolerant NP protein described herein or one or more MCP domains described herein, and the amino acid sequence of SEQ ID NO: 11 (C-terminus portion of the Cas9 nickase of i-E1026).

[0211] In certain embodiments, the Cas9 nickase protein comprises, from N-terminus to C-terminus, the amino acid sequence of SEQ ID NO: 5 (N-terminus portion of the Cas9 nickase of i-N1054), a modification-tolerant NP protein described herein or one or more MCP domains described herein, and the amino acid sequence of SEQ ID NO: 12 (C-terminus portion of the Cas9 nickase of i-N1054).

[0212] In certain embodiments, the Cas9 nickase protein comprises, from N-terminus to C-terminus, the amino acid sequence of SEQ ID NO: 6 (N-terminus portion of the Cas9 nickase of i-G1247), a modification-tolerant NP protein described herein or one or more MCP domains described herein, and the amino acid sequence of SEQ ID NO: 13 (C-terminus portion of the Cas9 nickase of i-G1247).

[0213] In certain embodiments, the Cas9 nickase protein comprises, from N-terminus to C-terminus, the amino acid sequence of SEQ ID NO: 7 (N-terminus portion of the Cas9 nickase of i-D1299), a modification-tolerant NP protein described herein or one or more MCP domains described herein, and the amino acid sequence of SEQ ID NO: 14 (C-terminus portion of the Cas9 nickase of i-D1299).

[0214] In certain embodiments, the Cas9 nickase protein comprises, from N-terminus to C-terminus, the amino acid sequence of SEQ ID NO: 8 (N-terminus portion of the Cas9 nickase of i-E827), a modification-tolerant NP protein described herein or one or more MCP domains described herein, and the amino acid sequence of SEQ ID NO: 15 (C-terminus portion of the Cas9 nickase of i-E827).

[0215] In certain embodiments, the Cas9 nickase protein comprises, from N-terminus to C-terminus, the amino acid sequence of SEQ ID NO: 9 (N-terminus portion of the Cas9 nickase of i-delta(S793-R905)), a modification-tolerant NP protein described herein or one or more MCP domains described herein, and the amino acid sequence of SEQ ID NO: 16 (C- terminus portion of the Cas9 nickase of i-delta(S793-R905)).

[0216] Prime editors enable deletion, insertion, and base substitution without double-strand breaks. Anzalone et al., “Search-and-replace genome editing without double-strand breaks or3880966915. v1Attorney Docket. No. 767854: UM9-316PC donor DNA” Nature 576: 149-157 (2019). However, this known fusion of a Cas9 nickase (nCas9; PE2) and a Moloney murine leukemia virus nucleotide polymerase (M-MLV RT) is >6.3 kb. This size is beyond the packaging capacity of a single adeno-associated virus (AAV).

[0217] Production of such a large protein in recombinant form in high yield to accommodate ribonucleoprotein (RNP) delivery can also be challenging. Some split Cas9 fusion construct strategies have been tested for the delivery of genome editing tools, including split inteins and MS2 or SunTag tethers. However, most of those split Cas9 fusion construct approaches have not yet been applied to prime editors. Wang et al., “CRISPR-Based Therapeutic Genome Editing: Strategies and In Vivo Delivery by AAV Vectors” Cell 181 : 136-150 (2020): Truong et al., “Development of an intein-mediated split-Cas9 system for gene therapy” Nucleic Acids Res 43:6450-6458 (2015); Maji et al., “Multidimensional chemical control of CRISPR-Cas9” Nat Chem Biol 13:9-11 (2017)” Liu et al., “A chemicalinducible CRISPR-Cas9 system for rapid control of genome editing” Nat Chem Biol 12:980- 987 (2016): Li et al., “SWISS: multiplexed orthogonal genome editing in plants with a Cas9 nickase and engineered CRISPR RNA scaffolds” Genome Biol 21 : 141 (2020): Konermann et al., “Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex” Nature 517:583-588 (2015)” Wang et al., “sgBE: a structure- guided design of sgRNA architecture specifies base editing window and enables simultaneous conversion of cytosine and adenosine” Genome Biol 21 :222 (2020); Jiang et al., “BE-PLUS: a new base editing tool with broadened editing window and enhanced fidelity” Cell Res 28:855-861 (2018).

[0218] These previously reported PE systems may also include a conjugated RNA that consists of a single guide RNA (sgRNA), a 3’ extension containing the NP template (NPT) sequence and a primer binding site (PBS), referred to herein as a prime editor guide RNA (e.g., pegRNA). Despite their usefulness, such pegRNAs are prone to misfolding due to inevitable inappropriate base pairing between the PBS and a spacer, as well as potential NPT- scaffold binding interactions. Finally, the 3 ’-terminal extension in the pegRNA is exposed to the cytosol and is therefore susceptible to degradation by nucleases, which may compromise the integrity of the pegRNA. Therefore, efforts to reduce pegRNA misfolding and instability are needed.Fused Prime Editor Constructs

[0219] Fused prime editor constructs are composed of a polypeptide domain having DNA binding activity (e.g., a DNA binding domain, such as a Cas9 nickase or dCas9) and a polypeptide domain having DNA polymerase activity (e.g., a DNA polymerase domain, such3980966915. v1Attorney Docket. No. 767854: UM9-316PC as the modification-tolerant nucleotide polymerases described herein). The DNA polymerase domain is linked to the DNA binding domain to form a single polypeptide sequence. In some embodiments, the DNA polymerase domain is directly attached to the DNA binding domain without an intervening linker. In some embodiments, the DNA polymerase domain is linked to the DNA binding domain via an intervening amino acid linker. In some embodiments, the DNA polymerase domain is linked to the N-terminus of the DNA binding domain. In some embodiments, the DNA polymerase domain is linked to the C-terminus of the DNA binding domain. In some embodiments, the DNA polymerase domain is inlaid within the DNA binding domain.Conventional Split And Modular Prime Editor Constructs

[0220] Previously reported split prime editor fusion constructs include, but are not limited to, an MS2-PE2 and SunTag-PE2 fusion constructs. MS2-PE2 comprises an MS2 coat protein (MCP) fused to the N-terminus of an M-MLV RT protein. Multiple cognate MS2-pegRNAs were engineered by incorporating MS2 stem-loops into different positions of the sgRNA. Additionally, a split SunTag fusion construct was created by fusing an scFv protein fragment to an N-terminus of M-MLV RT protein. Subsequently, the SunTag scFv-RT fusion construct was recruited by either GCN4-nCas9 or nCas9-GCN4. These two PE2 fusion constructs are generally referred to as SunTag-PE2 (GCN4-nCas9) and PE2-SunTag (nCas9-GCN4) based on domain order of elements.

[0221] The MS2, SunTag and sPE platforms have been designated in the art as a prime editor (PE3) format. The PE3 format differs from PE2 by inclusion of an additional sgRNA that directs nicking of the unedited strand, thereby biasing repair. The respective nCas9-, RT- , pegRNA-, and nicking sgRNA-expressing plasmids were co-transfected into a HEK293T- derived mCherry reporter lentivector-transduced cell line with a premature TAG stop codon that can be reverted to wild type codon, yielding a red fluorescence signal. Liu et al., "Improved prime editors enable pathogenic allele correction and cancer modelling in adult mice" Nat Commun 12:21 (2021). The most potentMS2- and SunTag-tethered configurations were comparable in editing efficiency to a PE3 fusion construct.Split Prime Editor Constructs

[0222] In a split primer editor (sPE), the CRISPR nuclease is not linked to the nucleotide polymerase. Rather, each protein is a separate molecule. The CRISPR nuclease is recruited to the genomic site of interest via a pegRNA or a separate guide RNA (such as an sgRNA). The4080966915. v1Attorney Docket. No. 767854: UM9-316PC nucleotide polymerase may be recruited to the CRISPR nuclease via a recruitment domain, such as an MCP on the nucleotide polymerase and an MS2 hairpin on the pegRNA, a separate guide RNA, or a petRNA.Modification-tolerant nucleotide polymerase (NP)

[0223] The prime editing systems described herein utilize a modification-tolerant nucleotide polymerase (NP). The modification-tolerant NP retains sufficient editing activity when paired with a chemically modified pegRNA or petRNA. In particular, one or both of the PBS and the NPT of the pegRNA or petRNA have at least one nucleotide modification. Traditional prime editing systems utilizing MMLV RT suffer from greatly reduced editing activity when a nucleotide modification is introduced in the PBS or NPT. The modification- tolerant NPs described herein, used in either a fused or split prime editing system, resolve this issue.

[0224] In some embodiments, every nucleotide in the PBS is modified. In some embodiments, every nucleotide in the NPT is modified. In some embodiments, every nucleotide in the PBS and NPT is modified.

[0225] In some embodiments, the nucleotide modification is selected from a modification of a ribose group, a phosphate group, a nucleobase, or a combination thereof.

[0226] In some embodiments, the modification of the ribose group is independently selected from the group consisting of 2'-(9-methyl, 2’ -fluoro, 2’ -deoxy, 2’-< -(2- methoxy ethyl) (MOE), 2’-NH2 (2’-amino), 4’-thio, a bicyclic nucleotide, a locked nucleic acid (LNA), a 2’-(5)-constrained ethyl (S-cEt), a constrained MOE, a 2'-(9,4'-C-aminomethylene bridged nucleic acid (2',4'-BNANC), and a 2’-F-ANA. In some embodiments, the modification of the ribose group is 2'-(9-methyl.

[0227] In some embodiments, at least 40% (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%) of the ribose groups in the PBS is modified. In some embodiments, every ribose group in the PBS is modified.

[0228] In some embodiments, at least 40% (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%) of the ribose groups in the NPT is modified. In some embodiments, every ribose group in the NPT is modified.

[0229] In some embodiments, the modification of the phosphate group is independently selected from the group consisting of a phosphorothioate, phosphonoacetate (PACE), phosphoramidate, thiophosphonoacetate (thioPACE), amide, triazole, phosphonate, or phosphotriester modification.4180966915. v1Attorney Docket. No. 767854: UM9-316PC

[0230] In some embodiments, the modification of the nucleobase group is independently selected from the group consisting of 2-thiouridine, 4-thiouridine, N6-methyladenosine, pseudouridine, 2,6-diaminopurine, inosine, thymidine, 5-methylcytosine, 5-substituted pyrimidine, isoguanine, isocytosine, and halogenated aromatic groups.

[0231] In some embodiments, the sgRNA or sgRNA portion comprises at least one nucleotide modification.

[0232] Exemplary modification-tolerant NPs that may be used in a prime editor system are variants of EC48-RT, TF1-RT, or MMLV-RT. Said variants are employed in prime editors designated PE6A, PE6B, PE6C, and PE6D.

[0233] In some embodiments, the modification-tolerant NP comprises at least 80% identity (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to the amino acid sequence of SEQ ID NO: 17.

[0234] In some embodiments, the modification-tolerant NP comprises at least 80% identity (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to the amino acid sequence of SEQ ID NO: 18.

[0235] In some embodiments, the modification-tolerant NP comprises at least 80% identity (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to the amino acid sequence of SEQ ID NO: 19.

[0236] In some embodiments, the modification-tolerant NP comprises at least 80% identity (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to the amino acid sequence of SEQ ID NO: 20.

[0237] The exemplary modification-tolerant NP amino acid sequences are recited below in Table 5. The amino acid sequence of the M-MLV RT used in traditional prime editing systems is provided in Table 6.Table 5: Sequence of Modification-tolerant nucleotide polymerase (NP)4280966915. v1Attorney Docket. No. 767854: UM9-316PC4380966915. v1Attorney Docket. No. 767854: UM9-316PC4480966915. v1Attorney Docket. No. 767854: UM9-316PCTable 6: Sequence of the Moloney murine leukemia virus reverse transcriptase (M-MLV RT)MCP domain for Cas9 nickase and modification-tolerant polymerases.

[0238] One or more MS2 Coat Proteins (MCPs) may be linked to one or both of the Cas9 nickase and modification tolerant NP described herein. The MCP domain binds to the MS2 polynucleotide hairpin sequence, which is incorporated into the pegRNA and petRNA described herein. The term MCP and MS2 binding protein are used herein interchangeably. Exemplary MCP amino acid sequences are provided in Table 7 below. Exemplary MS2 hairpin sequences are provided in Table 8 below.Table 7: Sequences of the MS2 coat protein (MCP monomer)4580966915. v1Attorney Docket. No. 767854: UM9-316PCTable 8: Example sequences of MS2 stemloop.

[0239] In some embodiments, the Cas9 nickase protein is linked to one or more MS2 binding proteins via an amino acid linker. In some embodiments, the one or more MS2 binding proteins are linked to at the N-terminus of the Cas9 nickase. In some embodiments, the one or more MS2 binding proteins are linked to at the C-terminus of the Cas9 nickase. In some embodiments, the one or more MS2 binding proteins are inlaid within the Cas9 nickase protein. In some embodiments, the one or more MS2 binding proteins comprises at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%. 96%, 97%, 98%, 99%, or 100% identity) to the amino acid sequence of SEQ ID NO: 22. In some embodiments, the one or more MS2 binding proteins comprises at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%. 96%, 97%, 98%, 99%, or 100% identity) to the amino acid sequence of SEQ ID NO: 23.

[0240] In some embodiments, the modification-tolerant NP protein is linked to one or more MS2 binding proteins via an amino acid linker. In some embodiments, the one or more MS2 binding proteins are linked to at the N-terminus of the modification-tolerant NP protein. In some embodiments, the one or more MS2 binding proteins are linked to at the C-terminus of the modification-tolerant NP protein. In some embodiments, the one or more MS2 binding proteins comprises at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%. 96%, 97%, 98%, 99%, or 100% identity) to the amino acid sequence of SEQ ID NO: 22. In some embodiments, the one or more MS2 binding proteins comprises at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%. 96%, 97%, 98%, 99%, or 100% identity) to the amino acid sequence of SEQ ID NO: 23.4680966915. v1Attorney Docket. No. 767854: UM9-316PCSplit PetRNA

[0241] The prime editor template RNA or petRNA molecule as used herein, refers to an RNA molecule that encodes a primer binding site (PBS) and a nucleotide polymerase template (NPT), that is unattached to the single guide RNA (sgRNA). The petRNA may also encode stem loops. The petRNA may also be linear or circularized. Modifications to the petRNA can enable the prime editing potential of modular prime editing systems. The chemically modified petRNA molecules of the disclosure possess improved in vivo stability, improved genome editing efficacy, and / or reduced immunotoxicity relative to unmodified or minimally modified guide RNAs.

[0242] In certain aspects, petRNA a comprises a primer binding site, a nucleotide polymerase template (NPT), at least one MS2 hairpin, and at least one chemically modified nucleotide. In certain embodiments, the one or more modified nucleotides comprise a modification of a ribose group, a phosphate group, a nucleobase, or a combination thereof. In certain embodiments, the modification of the ribose group is selected from 2'-(9-methyl, 2’- fluoro, 2’-deoxy, 2’-< -(2-methoxyethyl) (MOE), or 2’-NH2. In certain embodiments, the modification of the phosphate group comprises a phosphorothioate, phosphonoacetate (PACE), thiophosphonoacetate (thioPACE), amide, triazole, phosphonate, or phosphotriester modification.

[0243] In certain embodiments, the modified phosphate group comprises at least one phosphorothioate internucleotide linkage. In certain embodiments, the modified phosphate group comprises between 1 and 30 phosphorothioate intemucleotide linkages (i.e., 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, or 30 phosphorothioate intemucleotide linkages).

[0244] In certain embodiments, the modified phosphate group comprises at least one phosphorothioate intemucleotide linkage on the primer binding site (PBS). In certain embodiments, the modified phosphate group comprises exactly two phosphorothioate intemucleotide linkages on the PBS. In certain embodiments, the modified phosphate group comprises exactly three phosphorothioate intemucleotide linkages on the PBS. In certain embodiments, the modified phosphate group comprises between 1 and 10 phosphorothioate intemucleotide linkage on the PBS (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 phosphorothioate intemucleotide linkages on the PBS).

[0245] In certain embodiments, the modification of the nucleobase group is selected from 2-thiouridine, 4-thiouridine, N6-methyladenosine, pseudouridine, 2, 6-di aminopurine, inosine,4780966915. v1Attorney Docket. No. 767854: UM9-316PC thymidine, 5-methylcytosine, 5-substituted pyrimidine, isoguanine, isocytosine, or halogenated aromatic groups.

[0246] In certain embodiments, said petRNA comprises one MS2 hairpin. In other embodiments, the petRNA comprises two MS2 hairpins. In other embodiments, the petRNA comprises three MS2 hairpins. In other embodiments, the petRNA comprises four MS2 hairpins.

[0247] In other embodiments, the petRNA does not comprise an MS2 sequence, but contains another nucleotide sequence or non-nucleotide structure that achieves covalent or noncovalent association with the other components of the prime editing complex.

[0248] In certain embodiments, the at least one MS2 hairpin is chemically modified. In certain embodiments, the one or more modified nucleotides of the MS2 hairpin comprises a modification of a ribose group, a phosphate group, a nucleobase, or a combination thereof. In certain embodiments, the modified MS2 hairpin comprises a phosphate group comprising a phosphorothioate, phosphonoacetate (PACE), thiophosphonoacetate (thioPACE), amide, triazole, phosphonate, or phosphotriester modification.

[0249] In certain embodiments, the modified MS2 hairpin comprises a phosphate group comprising at least one phosphorothioate intemucleotide linkage. In certain embodiments, the phosphate group comprises three, ten, or twenty-three phosphorothioate intemucleotide linkages. In certain embodiments, the phosphate group comprises between 1 and 30 phosphorothioate intemucleotide linkages (i.e., 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, or 30 phosphorothioate intemucleotide linkages).

[0250] In certain embodiments, the phosphorothioate intemucleotide linkages are located on the N terminus. In other embodiments, the phosphorothioate intemucleotide linkages are located on the C terminus.

[0251] In certain embodiments, the modification of the nucleobase group is selected from 2-thiouridine, 4-thiouridine, N6-methyladenosine, pseudouridine, 2, 6-di aminopurine, inosine, thymidine, 5-methylcytosine, 5-substituted pyrimidine, isoguanine, isocytosine, or halogenated aromatic groups.

[0252] In certain embodiments, the petRNA comprises a fully modified MS2 hairpin (i.e. 100% chemically modified MS2 hairpin).4880966915. v1Attorney Docket. No. 767854: UM9-316PCLinkers

[0253] Linkers were used to ligate components of the module prime editing system to each other. These include amino acid linkers to fuse the one or more MS2 coat proteins to each other and / or to other components of the modular prime editing system.

[0254] Exemplary linkers include, but are not limited to, an ethylene glycol chain, an alkyl chain, a polypeptide, a polysaccharide, a block copolymer, and the like (Table 9).Table 9: Amino Acid Linker Sequences

[0255] In certain embodiments, the fusion protein comprised at least one MS2 binding protein inlaid within the Cas9 nickase, wherein the one or more MS2 binding proteins are attached to the Cas9 nickase via one or more linkers.

[0256] In certain embodiments, the one or more MS2 binding proteins are attached to the Cas9 nickase via two linkers, wherein a first linker is on the N-terminus of the Cas9 nickase, and a second linker is on the C-terminus of the Cas9 nickase.PetRNA linkers

[0257] Linkers were used to attach the one or more MS2 and NPT-PBS sequences to study their effect on editing activities of the petRNA.

[0258] In one aspect, the disclosure provides a petRNA a comprising a primer binding site, a nucleotide polymerase template (NPT), and at least one MS2 hairpin, wherein the MS2 is linked to the NPT using a linker.4980966915. v1Attorney Docket. No. 767854: UM9-316PC

[0259] In an embodiment, the MS2 is linked to the NPT using a linker. In an embodiment, the linker is selected from the group consisting of ethylene glycol and polyethylene glycol (PEG). In an embodiment, the PEG is a hexaethylene glycol (HEG). In an embodiment, the HEG comprises the following structure:

[0260] In an embodiment, the PEG is 2XHEG. In an embodiment, the PEG is 2XHEG comprising the following structure:

[0261] In an embodiment, the linker is a spacer made of one or more additional nucleotides. For example, the MS2 may be linked to the NPT using a stretch of the 2'-Omethyl modified nucleotides. In other embodiments, the nucleotides may be modified in such a way as to reduce readthrough (for example, they may contain abasic nucleotides, bulky nucleotides, noncanonical backbones or charged groups that prevent polymerase readthrough.)Adeno-Associated Viruses

[0262] Adeno-associated viruses (AAV) are small viruses that infect humans and some other primate species. AAVs are small (20 nm) replication-defective, nonenveloped viruses and have linear single-stranded DNA (ssDNA) genome of approximately 4.8 kilobases (kb). Naso et al. "Adeno-Associated Virus (AAV) as a Vector for Gene Therapy" BioDrugs 31(4):317-334 (2017); and Wu et al., "Effect of Genome Size on AAV Vector Packaging" Molecular Therapy 18 (1): 80-86 (2010). AAVs are not currently known to cause disease. The viruses cause a very mild immune response. Several additional features make AAV an attractive candidate for creating viral vectors for gene therapy, and for the creation of isogenic human disease models. Grieger et al., "Adeno-associated Virus as a Gene Therapy Vector: Vector Development, Production and Clinical Applications"; Adeno-associated virus as a gene therapy vector: vector development, production and clinical applications. In: Advances in Biochemical Engineering / Biotechnology. 99. pp. 119-145 (2005). Gene therapy vectors5080966915. v1Attorney Docket. No. 767854: UM9-316PC using AAV can infect both dividing and quiescent cells and persist in an extrachromosomal state without integrating into the genome of the host cell, although in the native virus integration of virally carried genes into the host genome does occur. Deyle et al., "Adeno- associated virus vector integration". Current Opinion in Molecular Therapeutics. 11(4):442- 447 (2009).

[0263] Development of AAVs as gene therapy vectors eliminated the genomic integration capacity by removal of the rep and cap genes. The modified vector has a promoter to drive transcription of the carried gene which is inserted between inverted terminal repeats (ITRs). AAV-based gene therapy vectors consequently form episomal concatemers in the host cell nucleus. In non-dividing cells, these concatemers remain intact for the life of the host cell. In dividing cells, AAV DNA is lost through cell division, since the episomal DNA is not replicated along with the host cell DNA. Surosky et al., " Adeno-associated virus Rep proteins target DNA sequences to a unique locus in the human genome" Journal of Virology 71(10):7951-7959 (1997).

[0264] The AAV genome is built of single-stranded deoxyribonucleic acid (ssDNA), either positive- or negative-sensed, which is about 4.7 kilobase long. The genome comprises ITRs at both ends of the DNA strand, and two open reading frames (ORFs) encoding the rep and cap proteins. The rep ORF is composed of four overlapping genes encoding Rep proteins required for the AAV life cycle. The cap ORF is composed of overlapping nucleotide sequences of capsid proteins (e.g., VP1, VP2 and VP3) which interact to fouli a capsid with icosahedral symmetry. Carter BJ, "Aden-associated virus and adeno-associated virus vectors for gene delivery". In: Lassie DD, Templeton NS (eds.). Gene Therapy: Therapeutic Mechanisms and Strategies. New York City: Marcel Dekker, Inc. pp. 41-59 (2000).

[0265] AAV inverted terminal repeat (ITR) sequences usually comprise about 145 bases each and are believed required for efficient multiplication of the AAV genome. Bohenzky et al., "Sequence and symmetry requirements within the internal palindromic sequences of the adeno-associated virus terminal repeat" Virology 166(2):316-327 (1988). ITRs also have a hairpin structure which contributes to self-priming that allows a primase-independent synthesis of the second DNA strand. The ITRs were also shown to be required for host cell DNA integration / removal, efficient encapsidation and deoxyribonuclease resistance. Wang et al., "Rescue and replication signals of the adeno-associated virus 2 genome" Journal of Molecular Biology 250(5):573-580 (1995); Weitzman et al., "Adeno-associated virus (AAV) Rep proteins mediate complex formation between AAV DNA and its integration site in human DNA" PNAS USA 91(13): 5808-5812 (1994); and Zhou et al, "In vitro packaging of adeno-5180966915. v1Attorney Docket. No. 767854: UM9-316PC associated virus DNA". Journal of Virology 72(4):3241-3247 (1998). With regard to gene therapy, ITRs are configured in cis next to the therapeutic gene, in contrast the structural (cap) and packaging (rep) proteins which can be delivered in trans. Nony et al., "Novel cis-acting replication element in the adeno-associated virus type 2 genome is involved in amplification of integrated rep-cap sequences" Journal of Virology 75(20):9991-9994 (2001); Nony et al., "Evidence for packaging of rep-cap sequences into adeno-associated virus (AAV) type 2 capsids in the absence of inverted terminal repeats: a model for generation of rep-positive AAV particles" Journal of Virology 77(1):776-781 (2003); Philpott et al., "Efficient integration of recombinant adeno-associated virus DNA vectors requires a p5-rep sequence in cis" Journal of Virology 76(11 ): 5411-5421 (June 2002); and Tullis et al., "Efficient replication of adeno-associated virus type 2 vectors: a cis-acting element outside of the terminal repeats and a minimal size". Journal of Virology 74(24): 11511 - 11521 (2000).Pharmaceutical Delivery Systems

[0266] The present invention contemplates several delivery systems for PE systems that provide for roughly uniform distribution, have controllable rates of release. A variety of different media are described below that are useful in creating such delivery systems. It is not intended that any one medium or carrier is limiting to the present invention. Note that any medium or carrier may be combined with another medium or carrier.

[0267] Carriers or mediums contemplated by this invention comprise a material selected from the group comprising gelatin, collagen, cellulose esters, dextran sulfate, pentosan polysulfate, chitin, saccharides, albumin, fibrin sealants, synthetic polyvinyl pyrrolidone, polyethylene oxide, polypropylene oxide, block polymers of polyethylene oxide and polypropylene oxide, polyethylene glycol, acrylates, acrylamides, methacrylates including, but not limited to, 2-hydroxyethyl methacrylate, poly(ortho esters), cyanoacrylates, gelatin- resorcin-aldehyde type bioadhesives, polyacrylic acid and copolymers and block copolymers thereof.A. Ribonucleoprotein (RNP) Nucleotransfection

[0268] In one embodiment, the present invention contemplates mRNA delivery of the PE system. Although it is not necessary to understand the mechanism of an invention, it is believed that delivery of two smaller modular PE mRNAs (e.g., a Cas9 / RT mRNA and a pegRNA or petRNA) would improve overall stability and large scale manufacturing efficiency as opposed to full length split PE fusion constructs that are approximately 6-7kb length.5280966915. v1Attorney Docket. No. 767854: UM9-316PCCommercial translation of a full length split PE fusion construct is also problematic due to its small size. Consequently, RNP compositions comprising sPE RNA systems (e.g., nSpy Cas9 RNA + MCP-fused nucleotide polymerase) provides both manufacturing and clinical advantages. In one embodiment, an RNP composition comprising sPE RNA systems are administered using ribonucleotransfection.To efficiently transport CRISPR-Cas into target tissues / cells require overcoming several extra- and intra-cellular barriers, therefore largely limiting the applications of CRISPR-based therapeutics in vivo. Suggested delivery platforms include, but are not limited to, plasmids, RNAs and ribonucleoproteins (RNPs).

[0269] RNPs are composed of a large Cas protein and a short gRNA. gRNA can bind to DNA via Watson — Crick base pairing or the Cas protein can be conjugated to polypeptides, proteins, and PEI. These features can also be used for loading RNP. In addition, RNP can be loaded via electrostatic interactions with positively charged materials due to its negative net charge. These positively charged materials can be cationic lipids, PEI, polypeptides, and metal-organic frameworks (MOFs). Vesicles from cells can also be used to deliver RNP. It has been reported that PEI can coat a complex of Cas9 RNP and DNA nanoclews for enhanced endosomal escape. PEI-coated DNA nanoclews were shown to efficiently transfect a Cas9 RNP targeting EGFP into U2OS cells for EGFP knockout in vitro. Furthermore, the PEI- coated DNA nanoclews could also disrupt EGFP in U2OS.EGFP xenograft tumors in vivo after intratumoral injection. Recently, a nanocapsule was developed for Cas9 RNP delivery. Due to the heterogeneous surface charges of RNP, the RNP was first coated with both cationic and anionic monomers via electrostatic interactions. An imidazole-containing monomer (e.g., glutathione (GSH)-degradable crosslinker) and PEG can be absorbed to the surface of the RNP via hydrogen bonding and van der Waals interactions. Then, GSH-cleavable nanocapsules were formed around the RNP via in situ free-radical polymerization. In addition, targeting ligands, for example CPPs, can be added into the nanocapsule by conjugation to PEG. It was demonstrated that the GSH cleavable nanocapsule could protect Cas9 RNP in the endosome after cellular uptake and could be quickly cleaved by GSH after escape into the cytoplasm for subsequent genome editing. After local injection of Cas9 RNP nanocapsules, robust gene editing was observed in retinal pigment epithelium (RPE) and muscle. Because the net charge of RNP is negative, cationic liposomes or LNPs can be directly used for RNP transfection. It was demonstrated that the Cas9 protein (+22 net charges) can be rendered highly anionic by fusion to a negatively charged GFP (-30 net charges) or complexation with a gRNA. Alternatively, the positively charged PEI has also been developed for RNP delivery.5380966915. v1Attorney Docket. No. 767854: UM9-316PCFor example, Cas9 RNP was loaded onto GO-PEG-PEI via physisorption and n-stacking interactions. Xu et al., "Rational designs of in vivo CRISPR-Cas delivery systems" Adv Drug Deliv Rev (2021).

[0270] RNP delivery for genome editing in live cells may be performed with Lipofectamine® RNAiMAX lipid transfection reagent and elements of a PE system. For example, pegRNAs / petRNAs are mixed with purified Cas9 / RT proteins at an equimolar ratio in Opti-MEMTM to from an RNP complex (e.g, - 10 min at room temperature). These RNPs can then betransfected into live cells using, for example, DMEM with 10% FBS. RNP nucleotransfection may be performed by electroporation using, for example, a Lonza 96-well ShuttleTM System (Lonza, Basel, Switzerland) optionally in the presence of Alt-R® Cas9 Electroporation Enhancer (Integrated DNA Technologies, Inc). Vakulskas et al., "A high- fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human haematopoietic stem and progenitor cells" Nat Med. 24(8): 1216-1224 (2018).

[0271] The prime editing efficiency of a number of genes was compared between current PE systems and sPE in HEK293T cells using either conventional mRNA delivery or RNP- mediated nucleofection. For example, the genes included FANCF, VEGFA and HEK3.B. Microparticles

[0272] One embodiment of the present invention contemplates a medium comprising a microparticle. Preferably, microparticles comprise liposomes, nanoparticles, microspheres, nanospheres, microcapsules, and nanocapsules. Preferably, some microparticles contemplated by the present invention comprise poly(lactide-co-glycolide), aliphatic polyesters including, but not limited to, poly-glycolic acid and poly-lactic acid, hyaluronic acid, modified polysacchrides, chitosan, cellulose, dextran, polyurethanes, polyacrylic acids, psuedo- poly(amino acids), polyhydroxybutrate-related copolymers, polyanhydrides, polymethylmethacrylate, polyethylene oxide), lecithin and phospholipids.C. Liposomes

[0273] One embodiment of the present invention contemplates liposomes capable of attaching and releasing therapeutic agents described herein. Liposomes are microscopic spherical lipid bilayers surrounding an aqueous core that are made from amphiphilic molecules such as phospholipids. For example, a liposome may trap a therapeutic agent between the hydrophobic tails of the phospholipid micelle. Water soluble agents can be entrapped in the core and lipid-soluble agents can be dissolved in the shell-like bilayer.5480966915. v1Attorney Docket. No. 767854: UM9-316PCLiposomes have a special characteristic in that they enable water soluble and water insoluble chemicals to be used together in a medium without the use of surfactants or other emulsifiers. Liposomes can form spontaneously by forcefully mixing phosopholipids in aqueous media. Water soluble compounds are dissolved in an aqueous solution capable of hydrating phospholipids. Upon formation of the liposomes, therefore, these compounds are trapped within the aqueous liposomal center. The liposome wall, being a phospholipid membrane, holds fat soluble materials such as oils. Liposomes provide controlled release of incorporated compounds. In addition, liposomes can be coated with water soluble polymers, such as polyethylene glycol to increase the pharmacokinetic half-life. One embodiment of the present invention contemplates an ultra high-shear technology to refine liposome production, resulting in stable, unilamellar (single layer) liposomes having specifically designed structural characteristics. These unique properties of liposomes, allow the simultaneous storage of normally immiscible compounds and the capability of their controlled release.

[0274] In some embodiments, the present invention contemplates cationic and anionic liposomes, as well as liposomes having neutral lipids. Preferably, cationic liposomes comprise negatively-charged materials by mixing the materials and fatty acid liposomal elements and allowing them to charge-associate. Clearly, the choice of a cationic or anionic liposome depends upon the desired pH of the final liposome mixture. Examples of cationic liposomes include lipofectin, lipofectamine, and lipofectace.

[0275] One embodiment of the present invention contemplates a medium comprising liposomes that provide controlled release of at least one therapeutic agent. Preferably, liposomes that are capable of controlled release: i) are biodegradable and non-toxic; ii) carry both water and oil soluble compounds; iii) solubilize recalcitrant compounds; iv) prevent compound oxidation; v) promote protein stabilization; vi) control hydration; vii) control compound release by variations in bilayer composition such as, but not limited to, fatty acid chain length, fatty acid lipid composition, relative amounts of saturated and unsaturated fatty acids, and physical configuration; viii) have solvent dependency; iv) have pH-dependency and v) have temperature dependency.

[0276] The compositions of liposomes are broadly categorized into two classifications. Conventional liposomes are generally mixtures of stabilized natural lecithin (PC) that may comprise synthetic identical-chain phospholipids that may or may not contain glycolipids. Special liposomes may comprise: i) bipolar fatty acids; ii) the ability to attach antibodies for tissue-targeted therapies; iii) coated with materials such as, but not limited to lipoprotein and carbohydrate; iv) multiple encapsulation and v) emulsion compatibility.5580966915. v1Attorney Docket. No. 767854: UM9-316PC

[0277] Liposomes may be easily made in the laboratory by methods such as, but not limited to, sonication and vibration. Alternatively, compound-delivery liposomes are commercially available. For example, Collaborative Laboratories, Inc. are known to manufacture custom designed liposomes for specific delivery requirements.Table 10: Sequences of the full construct including linkers.5680966915. v1Attorney Docket. No. 767854: UM9-316PC5780966915. v1Attorney Docket. No. 767854: UM9-316PC5880966915. v1Attorney Docket. No. 767854: UM9-316PC5980966915. v1Attorney Docket. No. 767854: UM9-316PC6080966915. v1Attorney Docket. No. 767854: UM9-316PC6180966915. v1Attorney Docket. No. 767854: UM9-316PC6280966915. v1Attorney Docket. No. 767854: UM9-316PC6380966915. v1Attorney Docket. No. 767854: UM9-316PC6480966915. v1Attorney Docket. No. 767854: UM9-316PC6580966915. v1Attorney Docket. No. 767854: UM9-316PC6680966915. v1Attorney Docket. No. 767854: UM9-316PC6780966915. v1Attorney Docket. No. 767854: UM9-316PC6880966915. v1Attorney Docket. No. 767854: UM9-316PC6980966915. v1Attorney Docket. No. 767854: UM9-316PC7080966915. v1Attorney Docket. No. 767854: UM9-316PC7180966915. v1Attorney Docket. No. 767854: UM9-316PC7280966915. v1Attorney Docket. No. 767854: UM9-316PCTable 11: SgRNA common scaffold and variable spacer sequences.7380966915. v1Attorney Docket. No. 767854: UM9-316PCTable 12: Sequences of editing templates used in the Figures.7480966915. v1Attorney Docket. No. 767854: UM9-316PC7580966915. v1Attorney Docket. No. 767854: UM9-316PCTable 13: Sequences of sgRNA used in the Figures.7680966915. v1Attorney Docket. No. 767854: UM9-316PCTable 14: Sequences of pegRNA used in the Figures.7780966915. v1Attorney Docket. No. 767854: UM9-316PC7880966915. v1Attorney Docket. No. 767854: UM9-316PCTable 15: Example sequences of editing templates for reducing readthrough into sgRNA or MS2 stemloop.EXAMPLESExample 1: Reduced prime editing with traditional prime editors and modified pegRNA and petRNA

[0278] Genome editing efficiency with prime editing systems employing the traditional M- MLV reverse transcriptase (RT) is reduced when nucleotide modifications are employed in the reverse transcriptase template (RTT) or the primer binding site (PBS). Editing efficiencies are particularly reduced with fully modified RTT and PBS sequences. However, nucleotide modification of pegRNA and petRNA is important to improve the stability of the molecules for in vivo therapeutic use.7980966915. v1Attorney Docket. No. 767854: UM9-316PC

[0279] To start, the editing efficiency of different prime editors using M-MLV RT was determined. A split effector system with an LPET was first tested in which the PBS was fully modified with 2’0Me modifications, both the PBS and RTT were fully modified with 2’-0Me modifications, or the PBS was fully modified with 2’-0Me modifications while the RTT was modified with DNA nucleotides instead of RNA. Editing was performed at three different genomic loci (FANCF, PRNP, and HBB). As shown in FIG. 2A - FIG. 2B, editing efficiency dropped markedly when both the PBS and RTT were fully modified with 2’0Me modifications. This was observed at all three tested targets. Moreover, editing efficiency was lower than expected when only the PBS was modified. These results were recapitulated in an additional experiment using the same split prime editor in combination with LPETs or pegRNA targeting the FANCF target (see FIG. 3).

[0280] Next, a fused prime editor was employed (wherein the Cas9 nuclease is linked to the RT). Three different pegRNAs were tested targeting FANCF, one with only unmodified RNA nucleotides in the PBS and RTT, one with only a modified PBS, and one with both the PBS and RTT modified. As shown in FIG. 4, reduced editing efficiency was observed in the fused prime editor system as well.

[0281] Finally, different prime editor architectures were tested with the modified pegRNA. Namely, a prime editor with the RNaseH domain of the RT deleted (“PE2-deltaRNaseH”) and a prime editor with the RT place on the N-terminus of Cas9 (“Nterm RT-PE2”) rather than the C-terminus (“PE2”). As shown in FIG. 5 A - FIG. 5B, even with different prime editor architectures, editing efficiency was reduced with modified PBS and RTT sequences.

[0282] The data of Example 1 indicates that reduced editing efficiency may be attributable to the ability of M-MLV RT to decode 2’OMe-modified templates.Example 2: Improved prime editing with modification-tolerant polymerases and modified pegRNA and petRNA

[0283] To enhance prime editing with modified pegRNA and petRNA, different polymerases were tested. A group of polymerases, designated PE6 polymerases, were identified. Specifically, polymerases from prime editors PE6A (NP - EC48-RT), PE6B (NP - evolved TF 1 -RT), PE6C (NP - evolved TF 1 -RT) and PE6D (NP - evolved M-MLV RT) were tested in a split prime editor and fused prime editor format.

[0284] As shown in FIG. 6, each of the spilt prime editors employing one of PE6A, PE6B, PE6C, or PE6D at least retained a substantial level of editing activity in the fully modified PBS / RTT LPET experiments relative to the unmodified PBS / RTT experiments. The PE6B8080966915. v1Attorney Docket. No. 767854: UM9-316PC and PE6C prime editors even displayed editing activity with fully modified PBS / RTT that exceed the modified LPET PBS / RTT.

[0285] The PE6 polymerases were next tested in a fused prime editor system. As shown in FIG. 7A - FIG. 7B, each of the fused prime editors employing one of PE6A, PE6B, PE6C, or PE6D at least retained a substantial level of editing activity in the fully modified PBS / RTT pegRNA experiments relative to the unmodified PBS / RTT experiments.

[0286] The spilt prime editor experiment was repeated with a comparison to LPETs having DNA modification of the RTT. As shown in FIG. 8A - FIG. 8B, editing activity improved with the 2’0Me modified PBS / RTT at both 72 hours post transfection and 96 hours post transfection.

[0287] Additional data was generated with fused prime editors at the HBB genomic locus. As shown in FIG. 9, the PE6 prime editor with the evolved M-MLV RT NP tolerated fully modified 2’0Me templates in fused format with high editing activity. The same PE6 polymerases were used at the PRNP genomic locus without (FIG. 10) and with (FIG. 11) a nicking sgRNA. The shows that the PE6 prime editor with the evolved M-MLV RT NP tolerated fully modified 2’0Me templates in fused format at the PRNP locus as well.

[0288] Additional data was next generated with split primer editors with and without nicking sgRNAs. FIG. 12 and FIG. 13 show editing activity at the FANCF locus. FIG. 14 shows editing activity at the HBB locus. FIG. 15 shows editing activity at the PRNP locus. In each instance, the PE6 prime editor with the evolved M-MLV RT NP tolerated fully modified 2’0Me templates in the split format with high editing activity.Example 3: Reduced readthrough by modification-tolerant polymerases with modified pegRNA and petRNA

[0289] The PE6 prime editors with the evolved M-MLV RT NP described herein have the ability to efficiently reverse transcribe structured editing templates, but this can increase readthrough into the sgRNA region of pegRNA, resulting in incorporation of unintended edits at the target (Doman et al., Cell, 2023. 186(18): 3983-4002). To impede such readthrough into the sgRNA region of pegRNA, a non-nucleotide linker like Hexathylene glycol (HEG) was introduced between RTT and sgRNA (Table 15). The use of such non-nucleotide blockers is expected to significantly reduce readthrough into the sgRNA region when using the processive polymerases described above, while retaining precise editing activity. Such non-nucleotide linkers can also be used in petRNA (such as LPETs) to reduce readthrough into the MS2 stemloop (Table 15).8180966915. v1Attorney Docket. No. 767854: UM9-316PCExample 4: Modification-tolerant polymerases with fully modified pegRNA and petRNA

[0290] It was observed that in the fused PE format, unlike sPE with LPET, fully modified pegRNA (PBS+RTT 2’OMe) did not improve editing compared to PBS only modified pegRNA at some locus like PRNP (FIG. 10). It was hypothesized that though TF 1 -RT derived polymerases are tolerant to 2’OMe RTT, they are not as tolerant to 2’0Me as 2’OH RNA. Therefore, further engineer TF1-RT derived polymerases was conducted to improve 2’0Me tolerance and editing activity with fully modified pegRNA. First beneficial mutations for M- MLV RT, which shares structural and sequence homology to TF1-RT, were investigated. It was hypothesized that mutations that improve thermostability, interaction with Primer / Template duplex, cDNA synthesis efficiency, dNTP affinity, solubility, reduced sensitivity to inhibitors and impart other beneficial properties to the polymerase can lead to improved 2’0Me tolerance. Thus, the mutations listed in Table 16 were tested in addition to the PE6C mutations in fused format with fully modified pegRNA at PRNP genomic locus (FIG. 16). Of the mutations tested, I33V, E54L, Q75R, M214A, Q293R or K, K321P, K380R and N447K improved editing with fully modified pegRNA. Next, it was hypothesized that DNA:2’0Me-RNA Primer / Template duplex may require different optimal interaction with residues in the polymerases compared to DNA:2’0H-RNA Primer / Template duplex. Thus, the Alphafold predicted structure of TF1-RT was analyzed to identify amino acid residues within 10-15 A of Primer / Template and mutated it to either improve interaction with the Primer / Template duplex or reduce steric hindrance from 2’ sugar modified template (Table 17). These mutations were tested with PE6C mutations in the fused format at PRNP genomic locus with fully modified pegRNA (PBS+RTT 2’0Me) (FIG. 17). Of the mutations tested, K188A, N299A, K320A, K320R, K321A and K290R improved editing with fully modified pegRNA. It was hypothesized the mutations identified in FIG. 16 and FIG. 17 can be combined and can even further improve editing efficiency with fully modified pegRNA. As a proof of concept and example, I33V and M214A were selected to combine as double mutant and tested it at PRNP genomic locus with unmodified pegRNA, PBS 2’0Me pegRNA and fully modified pegRNA with PBS+RTT 2’0Me (FIG. 18). Double mutant I33V+M214A significantly improved editing efficacy compared to either single mutants or PE6C, especially with fully modified pegRNA which outcompeted unmodified or PBS only modified pegRNA.

[0291] Even with engineered TF1-RT for 2’0Me tolerance like I33V+M214A double mutant, some pegRNAs like at FANCF genomic loci, with PBS only 2’0Me modified and8280966915. v1Attorney Docket. No. 767854: UM9-316PCPBS+RTT 2’0Me modified, did not improve editing efficiency compared to unmodified pegRNA (FIG. 19). It was hypothesized that such reduced editing efficiency could be due to self-inhibitory effect of pegRNA, further stabilized by 2’0Me modification. Thus, to elevate such effect it was systematically introduced, in PBS with RTT being 2’0Me, 2’OH RNA modification to reduce the Tm of spacer / PBS duplex, protect it with phosphorothioate (PS) linkages and alternated A-form (RNA) and B-form (DNA) modifications in PBS to distort it’s structure and reduce binding to the spacer and tested at FANCF genomic locus (FIG. 19). Introducing RNA with PS linkages or alternating A-form and B-form modification both significantly improved editing efficiency compared to either unmodified pegRNA, PBS 2’0Me pegRNA or PBS+RTT 2’0Me pegRNA. Such effect was even more prevalent when using engineered double mutant I33V+M214A compared to PE6C TF1-RT. Such modification pattern can be applied to other genomic loci pegRNAs as well and modifications like 2’0Me (instead of RNA) and 2’F-ANA (instead of DNA) can be alternated to even further improved editing efficiency. The PBS modification was further optimized to reduce self- inhibitory effect of PBS and improving its stability by alternating 2’0Me and DNA with or without PS linkages, reducing sequence length of PBS with complete 2’0Me modification, and adding PS linkages with RNA and DNA alternating PBS modification and tested at FANCF genomic locus with double mutant I33V+M214A previously described (FIG. 20). It was observed that PBS modification with 2’0Me and DNA alternating pegRNA (pegRNA 8 and 9 of FIG. 20) significantly improved editing efficiency compared to all other modification patterns. Additionally, reducing the length, to a certain extent, of PBS with 2’0Me modification (pegRNA 12 and 13 of FIG. 20) also improved editing efficiency compared to full length PBS with 2’0Me (pegRNA 2 and 3 of FIG. 20). Additional PS linkages were well tolerated with DNA and RNA or DNA and 2’0Me alternating patterns (pegRNA 9 and 10 of FIG. 20). Lastly, it was shown that RTT 2’0Me modification improves editing with improved modification pattern of PBS (pegRNA 6 and 7 of FIG. 20). Thus, a pegRNA modification pattern which together with modification tolerant polymerases improves prime editing efficiency significantly was developed.Example 5: Modified pegRNA and petRNA to prevent read-through

[0292] Polymerases in PE can read into scaffold of editing template beyond RTT, sgRNA scaffold for pegRNA, MS2 scaffold for LPET or PCV2 scaffold for click editing, resulting in impure edits with scaffold incorporation in the genome. We hypothesized that using a nonnucleotide blocker between scaffold and RTT can serve as a universal strategy to reduce8380966915. v1Attorney Docket. No. 767854: UM9-316PC scaffold readthrough and improve purity of editing (FIG. 21 and FIG. 22). As a proof of concept, lxC6, 2xC6, lx Tetra-Ethylene Glycol (TEG), lx Hexa-Ethylene Glycol (HEG) and 2xHEG were tested in sPE with PBS+RTT 2’0Me LPET, at FA NCF genomic locus (FIG. 23, FIG. 24, FIG. 25) All the blockers tested virtually eradicating scaffold integration into genome (FIG. 23A) and maintained editing efficiency (FIG. 23B) compared to LPET without non-nucleotide blocker. Additionally, we also observed that use of non-nucleotide blockers significantly reduced indels compared to LPET without non-nucleotide blocker (FIG. 24). We also determined the length of MS2 scaffold incorporation at the edit site (FIG. 25). Without non-nucleotide blocker, we saw up to full length (23nt) MS2 stem loop incorporation at the edit site in the genome but with you use of non-nucleotide blocker, we did not see any significant MS2 incorporation.

[0293] Once we determined that PBS and RTT LPET can be fully chemically modified sPE system, we sought to chemically modify MS2 Stem Loop which remains majorly to be 2’0H-RNA, susceptible to nuclease mediated degradation and immune stimulation. We hypothesized that MS2 Stem Loop can be amenable to sugar modification in stem while maintaining A-form like helix. We tested multiple modification patterns of MS2 consisting of 2’0Me and 2’F-RNA in LPET with PBS+RTT 2’0Me and HEG non-nucleotide blocker with MCP-PE6C (TF1-RT) at FANCF genomic locus (FIG. 26). We observed that editing efficiency increased with increasing 2’0Me modification of stem until 6 basepairs (12nts of stem) while other modification maintained editing efficiency compared to only end modified MS2 stem loop. We show that MS2 stem loop is amenable to 2’ sugar modification. Use of PS, LNA and similar modifications can further improve MS2 stability. Modifications currently being tested are presented in Table 18.

[0294] As a proof of concept and example of application of some of the discoveries described here we tested modified pegRNAs with engineered modification tolerant polymerases at therapeutically relevant site, PCKS9, implied in familial hypercholesteremia (FIG. 27). We show that fully modified PBS+RTT 2’0Me pegRNA with modification tolerant polymerase double mutant I33V+M214A can efficiently edit at PCSK9 locus compared to PBS only modified and PE6C polymerase.

[0295] Lastly, we sought to determine what domains and mutations of the evolved and engineered TF1-RT majorly imparts 2’0Me tolerance and ability to efficiently reverse transcribe GC rich and structured RTT templates. We hypothesized that RNaseH domain can lead to improved primer / template interaction for 2’0Me modified template and GC rich or structured RTT. To determine the role of RNaseH domain, we tested MCP-PE6B and MCP-8480966915. v1Attorney Docket. No. 767854: UM9-316PCPE6C, with evolved RNaseH domain (A363 V, K413E, S492N), without RNaseH domain and with wild type (without mutations) RNaseH domain, at PRNP genomic locus with PBS+RTT 2’0Me LPET (FIG. 28). We show that RNaseH domain is essential for 2’0Me tolerance and GC rich / structured RTT, while the mutations in RNaseH domain are not essential. Next, we hypothesized that the common mutations in PE6B and PE6C impart its 2’0Me tolerance. We tested WT TF1-RT and PE6C in sPE system with PBS 2’OMe / RTT RNA and PBS+RTT 2’0Me LPET at 3 genomic loci, FANCF, HBB and PRNP (FIG. 29A-FIG. 29C). We observed that WT TF1-RT has reduced tolerance for 2’0Me RTT, especially at sites with GC rich / structured RTT like PRNP and HBB. Together with FIG. 28 and FIG. 29A-FIG. 29C, we show that mutations common between PE6C and PE6B but not present in RNaseH domain are essential for 2’0Me tolerance and ability to reverse transcribe GC rich / structured RTT.

[0296] To better understand the polymerase mutations that are involved in altering polymerase activity on chemically modified sequences, individual mutations in the polymerase incorporated in primer editor PE6C were reverted back to wild type. It was observed that mutations common in the polymerase of PE6B and PE6C are important for 2’OMe mediated editing activity while mutations in the polymerase of PE6C are important for 2’OMe mediated editing activity with a structured and / or GC rich template (see, FIG. 30A and FIG. 30B). The shared mutations in the polymerases of PE6B and PE6C are P70T, G72V, S87G, M102I, K106R, K118R, H28V, L158Q, F269L, A363V, K413E, and S492N. The mutations in the polymerase of PE6C only are S188K, I260L, S297Q, R288Q.

[0297] Table 16 - List of mutations from M-MLV RT tested in TF1-RT8580966915. v1Attorney Docket. No. 767854: UM9-316PC

[0298] Table 17 - List of mutations to improve interaction with Primer / template duplex and reduce steric hinderance tested in TF1-RT.8680966915. v1Attorney Docket. No. 767854: UM9-316PC

[0299] Table 18 - List of MS2 modification templates8780966915. v1Attorney Docket. No. 767854: UM9-316PC8880966915. v1Attorney Docket. No. 767854: UM9-316PC

[0300] Table 19 - List of pegRNAs used in the figures8980966915. v1Attorney Docket. No. 767854: UM9-316PC9080966915. v1Attorney Docket. No. 767854: UM9-316PC9180966915. v1Attorney Docket. No. 767854: UM9-316PC

[0301] Table 20 - List of LPETs used in the figures9280966915. v1Attorney Docket. No. 767854: UM9-316PC9380966915. v1Attorney Docket. No. 767854: UM9-316PC9480966915. v1Attorney Docket. No. 767854: UM9-316PC

[0302] Table 21 - List of sgRNAs used in the figures9580966915. v1Attorney Docket. No. 767854: UM9-316PC

[0303] Table 22 - List of NGS Amplicons used in the figures9680966915. v1Attorney Docket. No. 767854: UM9-316PC9780966915. v1

Claims

Attorney Docket. No. 767854: UM9-316PCCLAIMS1. A prime editing system, comprising: i) a Cas9 nickase protein or a polynucleotide sequence encoding the Cas9 nickase protein; ii) a modification-tolerant nucleotide polymerase (NP) protein or a polynucleotide sequence encoding the modification-tolerant NP protein; and one of iii A) a prime editor template RNA (petRNA) comprising a primer binding site (PBS), and a nucleotide polymerase template (NPT), and ivA) a single guide RNA (sgRNA); or iiiB) a prime editing guide RNA (pegRNA) comprising a PBS, an NPT, and a sgRNA portion; wherein each of the PBS and NPT comprise at least one nucleotide modification.

2. The prime editing system of claim 1, wherein every nucleotide in the PBS is modified.

3. The prime editing system of claim 1, wherein every nucleotide in the NPT is modified.

4. The prime editing system of claim 1, wherein every nucleotide in the PBS and NPT is modified.

5. The prime editing system of claim 1, wherein the nucleotide modification is selected from a modification of a ribose group, a phosphate group, a nucleobase, or a combination thereof6. The prime editing system of claim 5, wherein the modification of the ribose group is independently selected from the group consisting of 2'-(9-methyl, 2’-fluoro, 2’-deoxy, -O- (2-methoxyethyl) (MOE), 2’-NH2 (2’-amino), 4’-thio, a bicyclic nucleotide, a locked nucleic acid (LNA), a 2’-(5)-constrained ethyl (S-cEt), a constrained MOE, a 2'-( ,4'-C- aminomethylene bridged nucleic acid (2',4'-BNANC), and a 2’-F-ANA.98 966915. v1Attorney Docket. No. 767854: UM9-316PC7. The prime editing system of claim 5, wherein the modification of the ribose group is 2'-O-m ethyl.

8. The prime editing system of claim 5, wherein at least 40% of the ribose groups in the PBS is modified.

9. The prime editing system of claim 5, wherein every ribose group in the PBS is modified.

10. The prime editing system of claim 5, wherein at least 40% of the ribose groups in the NPT is modified.

11. The prime editing system of claim 5, wherein every ribose group in the NPT is modified.

12. The prime editing system of claim 5, wherein the modification of the phosphate group is independently selected from the group consisting of a phosphorothioate, phosphonoacetate (PACE), phosphoramidate, thiophosphonoacetate (thioPACE), amide, triazole, phosphonate, or phosphotriester modification.

13. The prime editing system of claim 5, wherein the modification of the nucleobase group is independently selected from the group consisting of 2-thiouridine, 4-thiouridine, N6-methyladenosine, pseudouridine, 2,6-diaminopurine, inosine, thymidine, 5- methylcytosine, 5-substituted pyrimidine, isoguanine, isocytosine, and halogenated aromatic groups.

14. The prime editing system of claim 1, wherein the sgRNA or sgRNA portion comprises at least one nucleotide modification.

15. The prime editing system of claim 1, wherein the modification-tolerant NP is selected from a variant of EC48-RT, TF1-RT, or MMLV-RT.99 966915. v1Attorney Docket. No. 767854: UM9-316PC16. The prime editing system of claim 1, wherein the modification-tolerant NP comprises at least 80% identity to the amino acid sequence of SEQ ID NO: 17.

17. The prime editing system of claim 1, wherein the modification-tolerant NP comprises at least 80% identity to the amino acid sequence of SEQ ID NO: 18.

18. The prime editing system of claim 1, wherein the modification-tolerant NP comprises at least 80% identity to the amino acid sequence of SEQ ID NO: 19.

19. The prime editing system of claim 1, wherein the modification-tolerant NP comprises one or more amino acid substitutions selected from I33V, E54L, Q75R, M214A, Q293R or Q293K, K321P, K380R and N447K relative to the NP of SEQ ID NO: 18 or SEQ ID NO:19.

20. The prime editing system of claim 1, wherein the modification-tolerant NP comprises one or both amino acid substitutions of I33V and M214A relative to the NP of SEQ ID NO: 18 or SEQ ID NO: 19.

21. The prime editing system of claim 1, wherein the modification-tolerant NP comprises the amino acid substitution of I33V relative to the NP of SEQ ID NO: 18 or SEQ ID NO: 1922. The prime editing system of claim 1, wherein the modification-tolerant NP comprises the amino acid substitution of M214A relative to the NP of SEQ ID NO: 18 or SEQ ID NO: 19.

23. The prime editing system of claim 1, wherein the modification-tolerant NP comprises amino acid substitutions of I33V and M214A relative to the NP of SEQ ID NO: 18 or SEQ ID NO: 19.

24. The prime editing system of claim 1, wherein the modification-tolerant NP comprises one or more amino acid substitutions selected from K188A, K290R, N299A, K320A or K320R, and K321 A relative to the NP of SEQ ID NO: 19.100 966915. v1Attorney Docket. No. 767854: UM9-316PC25. The prime editing system of claim 1, wherein the modification-tolerant NP comprises amino acid substitutions K188A, K290R, N299A, K320A or K320R, and K321 A relative to the NP of SEQ ID NO: 19.

26. The prime editing system of claim 1, wherein the modification-tolerant NP comprises at least 80% identity to the amino acid sequence of any one of SEQ ID NOs: 51-56.

27. The prime editing system of claim 1, wherein the modification-tolerant NP comprises at least 80% identity to the amino acid sequence of SEQ ID NO: 20.

28. The prime editing system of any one of claims 1-27, wherein the Cas9 nickase protein comprises at least 80% identity to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 57.

29. The prime editing system of claim 1, wherein the Cas9 nickase protein and the modification-tolerant NP protein are linked together via an amino acid linker.

30. The prime editing system of claim 1, wherein the Cas9 nickase protein is linked to one or more MS2 binding proteins via an amino acid linker.

31. The prime editing system of claim 1, wherein one or more MS2 binding proteins are inlaid within the Cas9 nickase protein.

32. The prime editing system of claim 30 or 31, wherein the one or more MS2 binding proteins comprises at least 90% identity to the amino acid sequence of SEQ ID NO: 23.

33. The prime editing system of claim 1, wherein the modification-tolerant NP protein is inlaid within the Cas9 nickase protein.

34. The prime editing system of claim 1, wherein the modification-tolerant NP protein is linked to one or more MS2 binding proteins via an amino acid linker.

35. The prime editing system of claim 30, wherein the MS2 binding protein is linked to at the N-terminus of the Cas9 nickase and / or the modification-tolerant NP protein.101 966915. v1Attorney Docket. No. 767854: UM9-316PC36. The prime editing system of claim 30, wherein the MS2 binding protein comprises at least 90% identity to the amino acid sequence of SEQ ID NO: 22.

37. The prime editing system of claim 1, wherein the Cas9 nickase protein and / or the modification-tolerant NP further comprise an N-terminal and / or C-terminal nuclear localization signal (NLS).

38. The prime editing system of claim 37, wherein the NLS comprises any one or more of the NLS amino acid sequences of Table 2.

39. The prime editing system of claim 30, wherein the amino acid linker comprises or consists of any one of the amino acid linker sequences of Table 7.

40. The prime editing system of claim 1, wherein the petRNA comprises at least one MS2 hairpin.

41. The prime editing system of claim 40, wherein the MS2 hairpin is linked to the NPT via a linker.

42. The prime editing system of claim 41, wherein the linker is a non-nucleotide linker.

43. The prime editing system of claim 42, wherein the non-nucleotide linker is a Ci-Cio alkyl.

44. The prime editing system of claim 42, wherein the non-nucleotide linker is a C3 alkyl.

45. The prime editing system of claim 42, wherein the non-nucleotide linker is a Ce alkyl.

46. The prime editing system of claim 42, wherein the non-nucleotide linker is selected from the group consisting of ethylene glycol and polyethylene glycol (PEG).

47. The prime editing system of claim 46, wherein the PEG is a hexaethylene glycol (HEG).102 966915. v1Attorney Docket. No. 767854: UM9-316PC48. The prime editing system of claim 47, wherein HEG comprises the following structure:

49. The prime editing system of claim 46, wherein the PEG is 2XHEG.

50. The prime editing system of claim 46, wherein the PEG is 2XHEG comprising the following structure:

51. The prime editing system of claim 42, wherein the linker is an abasic nucleotide.

52. The prime editing system of claim 42, wherein the linker is a 2’-O-(2-methoxyethyl) (MOE) modified nucleotide.

53. A prime editing system, comprising: i) a fusion protein comprising a Cas9 nickase protein linked to a modification-tolerant nucleotide polymerase (NP) protein, or a polynucleotide encoding the fusion protein; and one of: iiA) a prime editor template RNA (petRNA) comprising a primer binding site (PBS), and a nucleotide polymerase template (NPT), and iii A) a single guide RNA (sgRNA); or103 966915. v1Attorney Docket. No. 767854: UM9-316PC iiB) a prime editing guide RNA (pegRNA) comprising a PBS, an NPT, and a sgRNA portion; wherein each of the PBS and NPT comprise at least one nucleotide modification.

54. The prime editing system of claim 53, wherein every nucleotide in the PBS is modified.

55. The prime editing system of claim 53, wherein every nucleotide in the NPT is modified.

56. The prime editing system of claim 53, wherein every nucleotide in the PBS and NPT is modified.

57. The prime editing system of claim 53, wherein the nucleotide modification is selected from a modification of a ribose group, a phosphate group, a nucleobase, or a combination thereof58. The prime editing system of claim 57, wherein the modification of the ribose group is independently selected from the group consisting of 2'-( -methyl, 2’-fluoro, 2’-deoxy, -O- (2-methoxyethyl) (MOE), 2’-NH2 (2’-amino), 4’-thio, a bicyclic nucleotide, a locked nucleic acid (LNA), a 2’-(5)-constrained ethyl (S-cEt), a constrained MOE, a 2'-O,4'-C- aminomethylene bridged nucleic acid (2',4'-BNANC), and a 2’-F-ANA.

59. The prime editing system of claim 57, wherein the modification of the ribose group is 2'-O-m ethyl.

60. The prime editing system of claim 57, wherein at least 40% of the ribose groups in the PBS is modified.

61. The prime editing system of claim 57, wherein every ribose group in the PBS is modified.

62. The prime editing system of claim 57, wherein at least 40% of the ribose groups in the NPT is modified.104 966915. v1Attorney Docket. No. 767854: UM9-316PC63. The prime editing system of claim 57, wherein every ribose group in the NPT is modified.

64. The prime editing system of claim 57, wherein the modification of the phosphate group is independently selected from the group consisting of a phosphorothioate, phosphonoacetate (PACE), phosphoramidate, thiophosphonoacetate (thioPACE), amide, triazole, phosphonate, or phosphotriester modification.

65. The prime editing system of claim 57, wherein the modification of the nucleobase group is independently selected from the group consisting of 2-thiouridine, 4-thiouridine, N6-methyladenosine, pseudouridine, 2,6-diaminopurine, inosine, thymidine, 5- methylcytosine, 5-substituted pyrimidine, isoguanine, isocytosine, and halogenated aromatic groups.

66. The prime editing system of claim 53, wherein the sgRNA or sgRNA portion comprises at least one nucleotide modification.

67. The prime editing system of claim 53, wherein the modification-tolerant NP is selected from a variant of EC48-RT, TF1-RT, or MMLV-RT.

68. The prime editing system of claim 53, wherein the modification-tolerant NP comprises at least 80% identity to the amino acid sequence of SEQ ID NO: 17.

69. The prime editing system of claim 53, wherein the modification-tolerant NP comprises at least 80% identity to the amino acid sequence of SEQ ID NO: 18.

70. The prime editing system of claim 53, wherein the modification-tolerant NP comprises at least 80% identity to the amino acid sequence of SEQ ID NO: 19.

71. The prime editing system of claim 53, wherein the modification-tolerant NP comprises one or more amino acid substitutions selected from I33V, E54L, Q75R, M214A, Q293R or Q293K, K321P, K380R and N447K relative to the NP of SEQ ID NO: 18 or SEQ ID NO: 19.105 966915. v1Attorney Docket. No. 767854: UM9-316PC72. The prime editing system of claim 53, wherein the modification-tolerant NP comprises one or both amino acid substitutions of 133 V and M214A relative to the NP of SEQ ID NO: 18 or SEQ ID NO: 19.

73. The prime editing system of claim 53, wherein the modification-tolerant NP comprises the amino acid substitution of 133 V relative to the NP of SEQ ID NO: 18 or SEQ ID NO: 19.

74. The prime editing system of claim 53, wherein the modification-tolerant NP comprises the amino acid substitution of M214A relative to the NP of SEQ ID NO: 18 or SEQ ID NO: 19.

75. The prime editing system of claim 53, wherein the modification-tolerant NP comprises amino acid substitutions of I33V and M214A relative to the NP of SEQ ID NO: 18 or SEQ ID NO: 19.

76. The prime editing system of claim 53, wherein the modification-tolerant NP comprises one or more amino acid substitutions selected from K188A, K290R, N299A, K320A or K320R, and K321 A relative to the NP of SEQ ID NO: 19.

77. The prime editing system of claim 53, wherein the modification-tolerant NP comprises amino acid substitutions K188A, K290R, N299A, K320A or K320R, and K321 A relative to the NP of SEQ ID NO: 19.

78. The prime editing system of claim 53, wherein the modification-tolerant NP comprises at least 80% identity to the amino acid sequence of any one of SEQ ID NOs: 51- 56.

79. The prime editing system of claim 53, wherein the modification-tolerant NP comprises at least 80% identity to the amino acid sequence of SEQ ID NO: 20.106 966915. v1Attorney Docket. No. 767854: UM9-316PC80. The prime editing system of claim 53, wherein the Cas9 nickase protein comprises at least 80% identity to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 57.

81. The prime editing system of claim 53, wherein the Cas9 nickase protein and the modification-tolerant NP protein are linked together via an amino acid linker.

82. The prime editing system of claim 53, wherein the Cas9 nickase protein is linked to one or more MS2 binding proteins via an amino acid linker.

83. The prime editing system of claim 53, wherein one or more MS2 binding proteins are inlaid within the Cas9 nickase protein.

84. The prime editing system of claim 82, wherein the one or more MS2 binding proteins comprises at least 90% identity to the amino acid sequence of SEQ ID NO: 23.

85. The prime editing system of claim 53, wherein the modification-tolerant NP protein is inlaid within the Cas9 nickase protein.

86. The prime editing system of claim 53, wherein the modification-tolerant NP protein is linked to one or more MS2 binding proteins via an amino acid linker.

87. The prime editing system of claim 82, wherein the MS2 binding protein is linked to at the N-terminus of the Cas9 nickase and / or the modification-tolerant NP protein.

88. The prime editing system of claim 82, wherein the MS2 binding protein comprises at least 90% identity to the amino acid sequence of SEQ ID NO: 22.

89. The prime editing system of claim 53, wherein the Cas9 nickase protein and / or the modification-tolerant NP further comprise an N-terminal and / or C-terminal nuclear localization signal (NLS).

90. The prime editing system of claim 89, wherein the NLS comprises any one or more of the NLS amino acid sequences of Table 2.107 966915. v1Attorney Docket. No. 767854: UM9-316PC91. The prime editing system of claim 81, wherein the amino acid linker comprises or consists of any one of the amino acid linker sequences of Table 7.

92. The prime editing system of claim 53, wherein the petRNA comprises at least one MS2 hairpin.

93. The prime editing system of claim 92, wherein the MS2 hairpin is linked to the NPT via a linker.

94. The prime editing system of claim 93, wherein the linker is a non-nucleotide linker.

95. The prime editing system of claim 94, wherein the non-nucleotide linker is a Ci-Cio alkyl.

96. The prime editing system of claim 94, wherein the non-nucleotide linker is a C3 alkyl.

97. The prime editing system of claim 94, wherein the non-nucleotide linker is a Ce alkyl.

98. The prime editing system of claim 94, wherein the non-nucleotide linker is selected from the group consisting of ethylene glycol and polyethylene glycol (PEG).

99. The prime editing system of claim 98, wherein the PEG is a hexaethylene glycol (HEG).

100. The prime editing system of claim 99, wherein HEG comprises the following structure:

101. The prime editing system of claim 98, wherein the PEG is 2XHEG.108 966915. v1Attorney Docket. No. 767854: UM9-316PC102. The prime editing system of claim 98, wherein the PEG is 2XHEG comprising the following structure:

103. The prime editing system of claim 93, wherein the linker is an abasic nucleotide.

104. The prime editing system of claim 93, wherein the linker is a 2’-O-(2-methoxyethyl) (MOE) modified nucleotide.

105. The prime editing system of claim 53, wherein the fusion protein comprises at least 80% identity to an amino acid sequence of any one of SEQ ID NOs: 41-48, 58, or 59.

106. The prime editing system of claim 1 or 53, wherein the polynucleotide sequence encoding the Cas9 nickase protein is an mRNA.

107. The prime editing system of claim 1 or 53, wherein the polynucleotide sequence encoding the modification-tolerant NP protein is an mRNA.

108. The prime editing system of claim 1 or 53, wherein the polynucleotide sequence encoding the Cas9 nickase protein and / or the polynucleotide sequence encoding the modification-tolerant NP protein is within a vector.

109. The prime editing system of claim 108, wherein the vector is a viral vector.

110. The prime editing system of claim 109, wherein the viral vector is an adeno- associated virus (AAV) vector or a lentivirus (LV) vector.109 966915. v1Attorney Docket. No. 767854: UM9-316PC111. A method of delivering the prime editing system of claim 1 or 53 to a cell, the method comprising incubating the prime editing system with the cell.

112. A method of editing a target gene in a cell of a subject, comprising administering to the subject the prime editing system of claim 1 or 53.

113. A modification-tolerant nucleotide polymerase (NP), comprising one or more amino acid substitutions selected from I33V, E54L, Q75R, M214A, Q293R or Q293K, K321P, K380R and N447K relative to the NP of SEQ ID NO: 18 or SEQ ID NO: 19.

114. The modification-tolerant NP of claim 113, comprising one or both amino acid substitutions of I33V and M214A relative to the NP of SEQ ID NO: 18 or SEQ ID NO: 19.

115. The modification-tolerant NP of claim 113, wherein the modification-tolerant NP comprises the amino acid substitution of 133 V relative to the NP of SEQ ID NO: 18 or SEQ ID NO: 19.

116. The modification-tolerant NP of claim 113, wherein the modification-tolerant NP comprises the amino acid substitution of M214A relative to the NP of SEQ ID NO: 18 or SEQ ID NO: 19.

117. The modification-tolerant NP of claim 113, wherein the modification-tolerant NP comprises amino acid substitutions of I33V and M214A relative to the NP of SEQ ID NO: 18 or SEQ ID NO: 19.

118. A modification-tolerant nucleotide polymerase (NP), comprising one or more amino acid substitutions selected from K188A, K290R, N299A, K320A or K320R, and K321 A relative to the NP of SEQ ID NO: 19.

119. The modification-tolerant NP of claim 118, wherein the modification-tolerant NP comprises amino acid substitutions K188A, K290R, N299A, K320A or K320R, and K321 A relative to the NP of SEQ ID NO: 19.110 966915. v1Attorney Docket. No. 767854: UM9-316PC120. A modification-tolerant nucleotide polymerase (NP), comprising at least 80% identity to the amino acid sequence of any one of SEQ ID NOs: 51-56.111 966915. v1

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