IL324508A

IL324508A - Genome editing compositions targeting the b2m gene and methods for use

Info

Publication number: IL324508A
Application number: IL324508A
Authority: IL
Original assignee: Prime Medicine Inc
Priority date: 2023-05-16
Filing date: 2024-05-16
Publication date: 2026-01-01
Also published as: EP4713455A1; AU2024274412A1; KR20260026099A; CN121532511A; WO2024238825A1; MX2025013657A

Description

WO 2024/238825 PCT/US2024/029746 GENOME EDITING COMPOSITIONS TARGETING THE B2M GENE AND METHODS OF USE CROSS REFERENCE [0001]This application claims the benefit of U.S. Provisional Application No. 63/502,563, filed May 16, 2023, and U.S. Provisional Application No. 63/603,477, filed November 28, 2023, each of which is incorporated herein by reference in its entirety.

BACKGROUND [0002]Cell therapies provide potential treatment approaches for multiple diseases, including cancers, autoimmune diseases, and hematological disorders. For example, adoptive T cell therapy can involve the ex vivo manipulation of T cells to express T-cell receptors (TCRs) or chimeric antigen receptors (CARs) engineered to recognize a tumor specific antigen. Cell therapy can involve autologous (i.e. patient-derived) cells, e.g., T cells or hematopoietic stem cells (HSCs), which can avoid issues of immunogenicity and intolerance upon reintroduction of the ex vivo manipulated cells. However, manufacturing challenges (e.g., time, expense, poor quality/quantity of extracted T cells) for autologous cell therapies make allogeneic (i.e. donor-derived) therapies an attractive alternative. An obstacle to allogeneic cell therapies is expression of endogenous proteins on cell surface that affect compatibility of donor-derived cells. For example, human leukocyte antigens (HLAs) on the surface of allogeneic T cells or HSCs may result in rejection by the host immune system, leading to graft dysfunction and failure. While cells from HLA matched donors and immunosuppression regimens could reduce the risk of host rejection, the former is often unavailable, and the latter has significant side effects such as increased risk of infection. [0003]The human HLA proteins are heterodimers composed of an a chain encoded by variant HLA genes and a 0 chain encoded by the 0-2 microglobulin (B2M) gene. The B2M gene is located at human genome 15q.21.1 and encodes a mRNA of approximately 360 bases. Because the 0 chain is required for dimerization and structure of the HLA complex, disruption of the endogenous HLA can be achieved by knocking out or knocking down expression of the B2M gene. Therefore, the effect of allograft rejection is expected to be reduced or eliminated by genetically modifying the B2M gene to reduce or eliminate its expression. [0004]Programmable nucleases such as CRISPR-Cas9 make double-strand DNA breaks (DSBs) that can disrupt genes by inducing mixtures of insertions and deletions (indels) at WO 2024/238825 PCT/US2024/029746 target sites. DSBs, however, are associated with undesired outcomes, including complex mixtures of products and translocations. There is a need in the art for compositions and methods to precisely disrupt the B2M gene without introducing DSBs.

SUMMARY [0005]In some aspects, provided herein are methods and compositions for introducing donor DNA into target DNA with Prime Editing. [0006]In some aspects, a prime editing guide RNA (PEgRNA) or one or more polynucleotides encoding the PEgRNA comprises: a spacer that is complementary to a search target sequence on a first strand of a 02-microglobulin (B2M) gene, wherein the spacer comprises at its 3’ end SEQ ID NO: 205; a gRNA core capable of binding to a Cas9 protein; and an extension arm comprising: an editing template that comprises a region of complementarity to an editing target sequence on a second strand of the B2M gene, and a primer binding site (PBS) that comprises at its 5’ end a sequence that is a reverse complement of nucleotides 10-14 of SEQ ID NO: 205, wherein the first strand and second strand are complementary to each other, and wherein the editing template encodes one or more nucleotide changes compared to the editing target sequence. [0007]In some aspects, prime editing guide RNA (PEgRNA) or one or more polynucleotides encoding the PEgRNA comprises: a spacer that is complementary to a search target sequence on a first strand of a 02-microglobulin (B2M) gene, wherein the spacer comprises at its 3’ end SEQ ID NO: 4; a gRNA core capable of binding to a Cas9 protein; and an extension arm comprising: an editing template that comprises a region of complementarity to an editing target sequence on a second strand of the B2M gene, and a primer binding site (PBS) that comprises at its 5’ end a sequence that is a reverse complement of nucleotides 10-14 of SEQ ID NO: 4, wherein the first strand and second strand are complementary to each other, and wherein the editing template encodes one or more nucleotide changes compared to the editing target sequence. [0008]A prime editing guide RNA (PEgRNA) or one or more polynucleotides encoding the PEgRNA comprises: a spacer that is complementary to a search target sequence on a first strand of a 02-microglobulin (B2M) gene, wherein the spacer comprises at its 3’ end SEQ ID NO: 272; a gRNA core capable of binding to a Cas9 protein; and an extension arm comprising: an editing template that comprises a region of complementarity to an editing target sequence on a second strand of the B2M gene, and a primer binding site (PBS) that comprises at its 5’ end a sequence that is a reverse complement of nucleotides 10-14 of SEQ WO 2024/238825 PCT/US2024/029746 ID NO: 272, wherein the first strand and second strand are complementary to each other, and wherein the editing template encodes one or more nucleotide changes compared to the editing target sequence. [0009]In some aspects, a prime editing guide RNA (PEgRNA) or one or more polynucleotides encoding the PEgRNA comprises: a spacer that is complementary to a search target sequence on a first strand of a 02-microglobulin (B2M) gene, wherein the spacer comprises at its 3’ end SEQ ID NO: 330; a gRNA core capable of binding to a Cas9 protein; and an extension arm comprising: an editing template that comprises a region of complementarity to an editing target sequence on a second strand of the B2M gene, and a primer binding site (PBS) that comprises at its 5’ end a sequence that is a reverse complement of nucleotides 10-14 of SEQ ID NO: 330, wherein the first strand and second strand are complementary to each other, and wherein the editing template encodes one or more nucleotide changes compared to the editing target sequence. [0010]In some embodiments, the spacer is from 17-22 nucleotides in length, optionally wherein the spacer is 20 nucleotides in length. In some embodiments, the spacer comprises at its 3’ end any one of SEQ ID NOs: 202-204. In some embodiments, the spacer comprises SEQ ID NO: 204. In some embodiments, the spacer comprises at its 3’ end any one of SEQ ID NO:s 1-3. In some embodiments, the spacer comprises SEQ ID NO: 1. In some embodiments, the spacer comprises at its 3’ end any one of SEQ ID NO:s 269-271. In some embodiments, the spacer comprises SEQ ID NO: 269. In some embodiments, the spacer comprises at its 3’ end any one of SEQ ID NO:s 327-329. In some embodiments, the spacer comprises SEQ ID NO: 3TL [0011]In some embodiments, the PEgRNA further comprises one or more nucleotide changes encoded by the editing template comprises a non-synonymous edit that alters the mRNA sequence or protein sequence encoded by the B2M gene. In some embodiments, the non-synonymous edit results in one or more in-frame stop codons in the B2M gene. In some embodiments, wherein the one or more in-frame stop codons comprise a nonsense mutation in the B2M gene. In some embodiments, the non-synonymous edit comprises an insertion in the B2M gene. In some embodiments, the non-synonymous edit comprises one or more substitutions in the B2M gene. In some embodiments, the insertion comprises an insertion an in-frame stop codon in the B2M gene, optionally wherein the insertion comprises an insertion of two or more consecutive in-frame stop codons in the B2M gene. In some embodiments, the insertion is comprises a TAATAA, a TTATTA, or a TAATAG nucleotide insertion. In WO 2024/238825 PCT/US2024/029746 some embodiments, the non-synonymous edit comprises a frameshift mutation in the B2M gene. In some embodiments, the frameshift mutation is an insertion or of 3x+l or 3x+nucleotides, wherein x is an integer equal to or greater than 0. In some embodiments, the frameshift mutation is a deletion of 3x+l or 3x+2 nucleotides, wherein x is an integer equal to or greater than 0. In some embodiments, the insertion is 1, 2 or 4 nucleotides in length. In some embodiments, the deletion is 1 nucleotide in length. [0012]In some embodiments, the PEgRNA further comprises a non-synonymous edit, wherein the non-synonymous edit alters a protospacer adjacent motif (PAM) sequence that is immediately 3’ to a protospacer sequence in the second strand of the B2M gene that is complementary to the search target sequence in the first strand of the B2M gene. In some embodiments, the PAM sequence is NGG and the non-synonymous edit is a NGG->NGC edit. In some embodiments, the protospacer sequence comprises a nick site that is three nucleotides upstream of the 5’ most nucleotide of the PAM sequence, and wherein the number of nucleotides from the nick site to the position in the second strand of the B2M gene corresponding to the non-synonymous edit is 1 to 19 nucleotides, wherein the number of nucleotides does not include the 5’ most nucleotide position on the second strand corresponding to the non-synonymous edit. In some embodiments, the number of nucleotides from the nick site to the position in the second strand of the B2M gene corresponding to the non-synonymous edit is 1, 2, 7, 8, 13, 14, or 19 nucleotides. In some embodiments, the number of nucleotides from the nick site to the position in the second strand of the B2M gene corresponding to the non-synonymous edit is equal to or less than 8 nucleotides. In some embodiments, the number of nucleotides from the nick site to the position in the second strand of the B2M gene corresponding to the non-synonymous edit is 1 or 2 nucleotides. [0013]In some embodiments, the non-synonymous edit is at a chromosomal location corresponding to coding sequence position c.51, c.54, or c.50 of a wildtype B2M gene. In some embodiments, the non-synonymous edit comprises a c.54insTAATAA insertion. In some embodiments, wherein the non-synonymous edit comprises a c.51delC deletion or a c.50insG insertion. In some embodiments, the non-synonymous edit is at a chromosomal location corresponding to coding sequence position c.54, c.60, or c.66 in a wildtype B2M gene. In some embodiments, the non-synonymous edit comprises to a c.54_55insCC insertion or a c.54_55insTAAG insertion. In some embodiments, the non-synonymous edit comprises a c.54_55insTAATAA insertion. In some embodiments, the non-synonymous edit comprises a c.66_67insCC insertion or a c.66_67insTAAG insertion. In some embodiments, the non- WO 2024/238825 PCT/US2024/029746 synonymous edit comprises a c.66_67insTAATAA insertion. In some embodiments, the non- synonymous edit comprises a c.60_65deletion and a TAATAG insertion (c.60_65_delinsTAATAG). In some embodiments, wherein the non-synonymous edit is at a chromosomal location corresponding to coding sequence position c.21 or c.3 of a wildtype B2M gene. In some embodiments, the non-synonymous edit comprises a c.21insTAATAA insertion. In some embodiments, the non-synonymous edit comprises a c. 21_22insCC insertion or a c.21_22insTAAG edit. In some embodiments, the non-synonymous edit comprises a c.3_4insCC insertion or a c.3_4insTAAG insertion. In some embodiments, the non-synonymous edit comprises a c.38 deletion and a TAATGA insertion (c.3_8delinsTAATGA). In some embodiments, the non-synonymous edit is at a chromosomal location corresponding to coding sequence position c.21, c.15 or c.3 of a wildtype B2M gene. In some embodiments, wherein the non-synonymous edit comprises a c. 15_16insCC insertion or a c. 15_16insTAAG insertion. In some embodiments, the non-synonymous edit comprises a c. 15_16insTAATAA insertion. In some embodiments, the non-synonymous edit comprises a c. 3_4insCC insertion or a c. 3_4insTAAG insertion. In some embodiments, the non-synonymous edit comprises a c. 3_4insTAATAA insertion. In some embodiments, the non-synonymous edit comprises a c.3_8 deletion and a TAATGA insertion (c.3_8delinsTAATGA). [0014]In some embodiments, the PEgRNA further comprises an editing template, wherein the editing template further encodes a an additional PAM silencing edit. In some embodiments, the PAM silencing edit is a c.58G>C edit. In some embodiments, the PAM silencing edit is a C.17OG edit. In some embodiments, the PAM silencing edit is a c.11OG edit. In some embodiments, the editing template comprises at least 6, 8, or 10 contiguous nucleotides complementary with the editing target sequence, wherein the at least 6, 8, or contiguous nucleotides are upstream of the position of the 5’ most nucleotide of the one or more nucleotide changes encoded in the editing template. In some embodiments, the editing template comprises 4, 6, 8, or 10 contiguous nucleotides complementary with the editing target sequence, wherein the 4, 6, 8, or 10 contiguous nucleotides are upstream of the position of the 5’ most nucleotide of the one or more nucleotide changes encoded in the editing template. [0015]In some aspects, a prime editing guide RNA (PEgRNA), or a nucleic acid encoding the PEgRNA comprises a spacer comprising at its 3’ end SEQ ID NO: 205; a gRNA core capable of binding to a Cas9 protein; and an extension arm comprising: an editing template WO 2024/238825 PCT/US2024/029746 comprising at its 3’ end: (A) nucleotides 13-24 of SEQ ID NO: 221, (B) nucleotides 12-20 of SEQ ID NO: 227, or (C) nucleotides 7-17 of SEQ ID NO: 231, and a primer binding site (PBS) comprising at its 5’ end a sequence that is a reverse complement of nucleotides 10-of SEQ ID NO: 205. [0016]In some embodiments, a prime editing guide RNA (PEgRNA), or a nucleic acid encoding the PEgRNA comprises: a spacer comprising at its 3’ end SEQ ID NO: 205; a gRNA core capable of binding to a Cas9 protein; and an extension arm comprising: an editing template comprising at its 3’ end: (A) nucleotides 13-24 of SEQ ID NO: 221, (B) nucleotides 12-20 of SEQ ID NO: 227, or (C) nucleotides 7-17 of SEQ ID NO: 231, and a primer binding site (PBS) comprising at its 5’ end a sequence that is a reverse complement of nucleotides 10-14 of SEQ ID NO: 205. [0017]In some embodiments, the PEgRNA further comprises (i) the editing template comprises at its 3’ end nucleotides 13-24 of SEQ ID NO: 221, optionally wherein the editing template comprises SEQ ID NO: 219, 220, or (ii) the editing template comprises at its 3’ end nucleotides 12-20 of SEQ ID NO: 227, optionally wherein the editing template comprises at its 3’ end SEQ ID NO: any one of SEQ ID NOs: 224-227, or (ii) the editing template comprises at its 3’ end nucleotides 7-17 of SEQ ID NO: 231, optionally wherein the editing template comprises at its 3’ end any one of SEQ ID NOs: 229-231. [0018]In some aspects, a prime editing guide RNA (PEgRNA), or a nucleic acid encoding the PEgRNA comprises a spacer comprising at its 3’ end SEQ ID NO: 1; a gRNA core capable of binding to a Cas9 protein; and an extension arm comprising: an editing template comprising at its 3’ end: (A) nucleotides 5-16 of SEQ ID NO: 19, or (B) a sequence selected from the group consisting of SEQ ID NO:s 900, 904, 908, 912, 916, 920, and 924, a primer binding site (PBS) comprising at its 5’ end a sequence that is a reverse complement of nucleotides 10-14 of SEQ ID NO: 1. [0019]In some embodiments, the PEgRNA comprises an editing template, wherein the editing template comprises: (i) a sequence selected from the group consisting of SEQ ID NOs: 900-903, or (ii) a sequence selected from the group consisting of SEQ ID NOs: 904- 907, or (iii) a sequence selected from the group consisting of SEQ ID NOs: 908-911, or (iv) a sequence selected from the group consisting of SEQ ID NOs: 912-915, or (v) a sequence selected from the group consisting of SEQ ID NOs: 916-919, 928, and 929,or (vi) a sequence selected from the group consisting of SEQ ID NOs: 920-923, or (vii) a sequence selected WO 2024/238825 PCT/US2024/029746 from the group consisting of SEQ ID NOs: 924-927, or (viii) a sequence selected from the group consisting of SEQ ID NOs: 18-20. [0020]In some aspects, a prime editing guide RNA (PEgRNA), or a nucleic acid encoding the PEgRNA comprises a spacer comprising at its 3’ end SEQ ID NO: 269; a gRNA core capable of binding to a Cas9 protein; and an extension arm comprising: an editing template comprising at its 3’ end: (A) nucleotides 3-16 of SEQ ID NO:286, or (B) a sequence selected from the group consisting of SEQ ID NO:s 1033, 1037, 1041, 1045, 1049, 1053, and 1057, and a primer binding site (PBS) comprising at its 5’ end a sequence that is a reverse complement of nucleotides 10-14 of SEQ ID NO: 269. [0021]In some embodiments, the PEgRNA comprises an editing template, wherein the editing template comprises (i) a sequence selected from the group consisting of SEQ ID NOs: 1033-1036, or (ii) a sequence selected from the group consisting of SEQ ID NOs: 1037-1040, or (iii) a sequence selected from the group consisting of SEQ ID NOs: 1041-1044, or (iv) a sequence selected from the group consisting of SEQ ID NOs: 1045-1048, or (v) a sequence selected from the group consisting of SEQ ID NOs: 1049-1052 andl061-1063, or (vi) a sequence selected from the group consisting of SEQ ID NOs: 1053-1056, or (vi) a sequence selected from the group consisting of SEQ ID NOs: 1057-1060, or (vii) a sequence selected from the group consisting of SEQ ID NOs: 286-288. [0022]In some aspects, a prime editing guide RNA (PEgRNA), or a nucleic acid encoding the PEgRNA comprises a spacer comprising at its 3’ end SEQ ID NO: 327; a gRNA core capable of binding to a Cas9 protein; and an extension arm comprising an editing template comprising at its 3’ end: (A) nucleotides 6-16 of SEQ ID NO:344, or (B) a sequence selected from the group consisting of SEQ ID NO:s 1162, 1166, 1170, 1174, 1178, 1182,andlland a primer binding site (PBS) comprising at its 5’ end a sequence that is a reverse complement of nucleotides 10-14 of SEQ ID NO: 3TL [0023]In some embodiments, the PEgRNA further comprises an editing template, wherein the editing template comprises (i) a sequence selected from the group consisting of (i) SEQ ID NOs: 1162-1165, or (ii) a sequence selected from the group consisting of SEQ ID NOs: 1166-1169, or (iii) a sequence selected from the group consisting of SEQ ID NOs: 1170- 1173, or (iv) a sequence selected from the group consisting of SEQ ID NOs: 1174-1177, or (v) a sequence selected from the group consisting of SEQ ID NOs: 1178-1181 and 1191, or (vi) a sequence selected from the group consisting of SEQ ID NOs: 1182-1185, or (vi) a WO 2024/238825 PCT/US2024/029746 sequence selected from the group consisting of SEQ ID NOs: 1186-1190, or (vii) a sequence selected from the group consisting of SEQ ID NOs: 344-346. [0024]In some embodiments, the editing template has a length of 24 nucleotides or less, or a length of 20 nucleotides or less. In some embodiments, the editing template has a length of (i) to 20 nucleotides, (ii) 12 to 20 nucleotides, or (iii) 11 to 17 nucleotides. In some embodiments, the editing template is 16 to 24 nucleotides in length. In some embodiments, the editing template is 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 24, 25, 26, 27, 28, 29, 30, 31, 33, 35 nucleotides in length. [0025]In some embodiments, the PBS has a length of 17 nucleotides or less. In some embodiments, the PBS has a length of (i) 8 to 15 nucleotides, (ii) 8 to 14 nucleotides, or (iii) to 12 nucleotides. In some embodiments, the PBS is 8, 10, or 12 nucleotides in length. In some embodiments, the PBS comprises a sequence set forth in any one of sequence numbers 206-218. In some embodiments, the PBS comprises a sequence set forth in any one of sequence numbers 5-17. In some embodiments, the PBS comprises a sequence set forth in any one of sequence numbers 273-285. In some embodiments, the PBS comprises a sequence set forth in any one of sequence numbers 331-343. [0026]In some embodiments, the spacer, the gRNA core, the RTT, and the PBS form a contiguous sequence in a single molecule. In some embodiments, the PEgRNA comprises from 5’ to 3’, the spacer, the gRNA core, the RTT, and the PBS. In some embodiments, the gRNA core comprises SEQ ID NO: 646. In some embodiments, the gRNA core comprises SEQ ID NO: 653. [0027]In some embodiments, the PEgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 232-262. In some embodiments, the PEgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 21-29 and 930-1016. In some embodiments, the PEgRNA comprises a sequence as set forth in SEQ ID NO: 933, 937, 961, 941, 957, or 936. In some embodiments, the PEgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 289-297 and 1064-1151. In some embodiments, the PEgRNA comprises a sequence as set forth in SEQ ID NO: 1141 or 1143. In some embodiments, the PEgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 347-355 and 1192-1279. In some embodiments, the PEgRNA comprises a sequence as set forth in SEQ ID NO: 1269 or 1265. In some embodiments, the PEgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 957, 961, 965, 980, 1016, 956, WO 2024/238825 PCT/US2024/029746 933, 941, 937, 1223, 988, 984, 1225, 1151, 1095, 1091, 964, 960, 940, 1221, 945, 1219, 932, 1015, 1014, 1075, 1222, 1250, 936, 1013, 1119, 1226, and 949. [0028]In some embodiments, the PEgRNA further comprises a 3’ motif, optionally wherein the 3’ motif is connected to the 3’ end of the PBS via a linker. [0029]In some embodiments, the PEgRNA further comprises 3’ mN*mN*mN*N and/or 5’mN*mN*mN* modifications, where m indicates that the nucleotide contains a 2’-O-Me modification and a * indicates the presence of a phosphorothioate bond. In some embodiments, the PEgRNA further comprises 3’ mT*mT*mT*T and 5’mN*mN*mN* modifications, where m indicates that the nucleotide contains a 2’-O-Me modification, a * indicates the presence of a phosphorothioate bond, and a T indicates the presence of an additional uridine nucleotide. [0030]In some embodiments, human chromosome locations and coding sequence locations are as set forth in Genome Reference Consortium Human Build 38 (GrCh38). [0031]In some aspects, a prime editing system comprises the PEgRNA or the one or more polynucleotides encoding the PEgRNA. [0032]In some aspects, a prime editing system, further comprises a nick guide RNA (ngRNA), or a nucleic acid encoding the ngRNA, wherein the ngRNA comprises: a) a ngRNA spacer that is complementary to a ngRNA search target sequence on the second strand of the B2M gene; and b) an ngRNA core capable of binding a Cas9 protein. [0033]In some embodiments, the prime editing system comprises an ngRNA spacer, wherein the ngRNA spacer is 17-22 nucleotides in length, optionally wherein the ngRNA spacer is nucleotides in length. In some embodiments, the ngRNA core comprises SEQ ID NO: 646 or 653. In some embodiments, the prime editing system comprises a PEgRNA spacer, wherein the PEgRNA spacer comprises at its 3’ end SEQ ID NO: 205. In some embodiments, the ngRNA spacer comprises at its 3’ end a sequence corresponding to nucleotides 4-20, 3-20, 2- 20, or 1-20 of any one of SEQ ID NOs: 263-268, optionally wherein ngRNA spacer comprises at its 3’ end any one of SEQ ID NOs: 263-268. In some embodiments, the ngRNA spacer comprises at its 3’ end nucleotides 1-20 of SEQ ID NO: 268, optionally wherein the ngRNA comprises SEQ ID NO: 824 or 825. [0034]In some embodiments, the prime editing system comprises (i) the non-synonymous edit encoded by the editing template comprises a c.51delC deletion and the ngRNA spacer comprises at its 3’ end sequence corresponding to nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 266; or (ii) the non-synonymous edit encoded by the editing template comprises WO 2024/238825 PCT/US2024/029746 a c.50insG insertion, and the ngRNA spacer comprises at its 3’ end sequence corresponding to nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 267 or 268. [0035]In some embodiments, the ngRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 824-827. In some embodiments, the PEgRNA spacer comprises at its 3’ end SEQ ID NO: 4. In some embodiments, the ngRNA spacer comprises at its 3’ end a sequence corresponding to nucleotides 4-20, 3-20, 2-20, or 1-20 of any one of SEQ ID NOs: 1017-1024. In some embodiments, ngRNA spacer comprises any one of SEQ ID NOs: 1017-1024. [0036]In some embodiments, the prime editing system comprises (i) the editing template encodes a c.54_55insCC edit and wherein the ngRNA spacer comprises at its 3’ end sequence corresponding to nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 1018, (ii) the editing template encodes a c.66_67insCC edit and wherein the ngRNA spacer comprises at its 3’ end sequence corresponding to nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 1019, (iii) the editing template encodes a c.54_55insTAAG edit and wherein the ngRNA spacer comprises at its 3’ end sequence corresponding to nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 1020, (iv) the editing template encodes a c.66_67insTAAG edit and wherein the ngRNA spacer comprises at its 3’ end sequence corresponding to nucleotides 4-20, 3-20, 2- 20, or 1-20 of SEQ ID NO: 1021, (v) the editing template encodes a c.54_55insTAATAA edit and wherein the ngRNA spacer comprises at its 3’ end sequence corresponding to nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 1022, (vi) the template encodes a c.66_67insTAATAA edit and wherein the ngRNA spacer comprises at its 3’ end sequence corresponding to nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 1023, or (vii) the template encodes a c.60_65delinsTAATAG edit and wherein the ngRNA spacer comprises at its 3’ end sequence corresponding to nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 1024. [0037]In some embodiments, the ngRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 1025-1032. In some embodiments, the PEgRNA spacer comprises at its 3’ end SEQ ID NO: 272. In some embodiments, the ngRNA spacer comprises at its 3’ end a sequence corresponding to nucleotides 4-20, 3-20, 2-20, or 1-20 of any one of SEQ ID NOs: 1152-1156. In some embodiments, the ngRNA spacer comprises any one of SEQ ID NOs: 1152-1156. [0038]In some embodiments, the prime editing system comprises (i) the editing template encodes a c.3_4insCC edit and wherein the ngRNA spacer comprises at its 3’ end sequence WO 2024/238825 PCT/US2024/029746 corresponding to nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 1153, (ii) the editing template encodes a c.3_4insTAAG edit and wherein the ngRNA spacer comprises at its 3’ end sequence corresponding to nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 1154, (iii) the editing template encodes a c.3_4insTAATAA edit and wherein the ngRNA spacer comprises at its 3’ end sequence corresponding to nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 1155, or (iv) the editing template encodes a c.3_8delinsTAATGA edit and wherein the ngRNA spacer comprises at its 3’ end sequence corresponding to nucleotides 4- 20, 3-20, 2-20, or 1-20 of SEQ ID NO: 1156. [0039]In some embodiments, the ngRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 1157-1161. In some embodiments, the PEgRNA spacer comprises at its 3’ end SEQ ID NO: 330. In some embodiments, the ngRNA spacer comprises at its 3’ end a sequence corresponding to nucleotides 4-20, 3-20, 2-20, or 1-20 of any one of SEQ ID NOs: 1280-1284. In some embodiments, the ngRNA spacer comprises any one of SEQ ID NOs: 1280-1284. [0040]In some embodiments, the prime editing system comprises (i) the editing template encodes a c.3_4insCC edit and wherein the ngRNA spacer comprises at its 3’ end sequence corresponding to nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 1281, (ii) the editing template encodes a c.3_4insTAAG edit and wherein the ngRNA spacer comprises at its 3’ end sequence corresponding to nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 1282, (iii) the editing template encodes a c.3_4insTAATAA edit and wherein the ngRNA spacer comprises at its 3’ end sequence corresponding to nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 1283,or (iv) the editing template encodes a c.3_8delinsTAATGAedit and wherein the ngRNA spacer comprises at its 3’ end sequence corresponding to nucleotides 4- 20, 3-20, 2-20, or 1-20 of SEQ ID NO: 1284. In some embodiments, the ngRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 1285-1289. [0041]In some embodiments, the prime editing system comprises (i) the PEgRNA comprises a sequence as set forth in SEQ ID NO: 933 or 937, and the ngRNA comprises a sequence as set forth in SEQ ID NO: 1018; (ii) the PEgRNA comprises a sequence as set for the in SEQ ID NO: 961, and the ngRNA comprises a sequence as set forth in SEQ ID NO: 1020; (iii) the PEgRNA comprises a sequence as set for the in SEQ ID NO: 941, and the ngRNA comprises a sequence as set forth in SEQ ID NO: 1018; (iv) the PEgRNA comprises a sequence as set for the in SEQ ID NO: 957, and the ngRNA comprises a sequence as set forth in SEQ ID NO: 1020; (v) the PEgRNA comprises a sequence as set for the in SEQ ID NO: 936, and the WO 2024/238825 PCT/US2024/029746 ngRNA comprises a sequence as set forth in SEQ ID NO: 1018; (vi) the PEgRNA comprises a sequence as set for the in SEQ ID NO: 1141 or 1143, and the ngRNA comprises a sequence as set forth in SEQ ID NO: 1156, or (vii) the PEgRNA comprises a sequence as set for the in SEQ ID NO: 1269 or 1265, and the ngRNA comprises a sequence as set forth in SEQ ID NO: 1284. [0042]In some embodiments, the ngRNA comprises 3’ mN*mN*mN*N and/or 5’mN*mN*mN* modifications, where m indicates that the nucleotide contains a 2’-O-Me modification and a * indicates the presence of a phosphorothioate bond. In some embodiments, the ngRNA comprises 3’ mT*mT*mT*T and 5’mN*mN*mN* modifications, where m indicates that the nucleotide contains a 2’-O-Me modification, a * indicates the presence of a phosphorothioate bond, and a T indicates the presence of an additional uridine nucleotide. [0043]In some embodiments, the prime editing system further comprises a TRAC-PEgRNA or one or more polynucleotides encoding the TRAC-PEgRNA, wherein the TRAC-PEgRNA comprises: a TRAC-spacer that is complementary to a search target sequence on a first strand of T-cell receptor a constant (TRAC) gene; a TRAC-gRNA core capable of binding to a Casprotein; and a TRAC-extension arm comprising: a TRAC-editing template that comprises a region of complementarity to an editing target sequence on a second strand of the TRAC gene, and a TRAC-primer binding site (PBS) that comprises at its 5’ end the reverse complement of nucleotides p to (q-3) of the second spacer, wherein q is the length of the second spacer, and p is an integer from 1 to (q-6), wherein the first strand and second strand are complementary to each other, and wherein the editing template encodes one or more nucleotide changes compared to the editing target sequence. [0044]In some embodiments, the TRAC-spacer is 17 to 22 nucleotides in length. In some embodiments, the editing template encodes an in-frame stop codon in the TRAC gene or a frameshift mutation in the TRAC gene. In some embodiments, the editing template encodes a recombinase recognition sequence recognized by a recombinase, or the reverse complement thereof. [0045]In some embodiments, the prime editing system further comprises a first TRAC-prime editing guide RNA (PEgRNA) or one or more polynucleotides encoding the first TRAC- PEgRNA, and a second TRAC-PEgRNA or one or more polynucleotides encoding the second TRAC-PEgRNA, wherein the first TRAC-PEgRNA comprises: a) a first TRAC- spacer that is complementary to a first TRAC-search target sequence on a first strand of a WO 2024/238825 PCT/US2024/029746 TRAC gene, b) a first TRAC-gRNA core capable of binding to a Cas9 protein; and c) a first TRAC-extension arm comprising (A) a first TRAC-editing template and (B) a first TRAC- primer binding site (PBS) that comprises at its 5’ end the reverse complement of nucleotides p to (q-3) of the first TRAC-spacer, wherein q is the length of the first TRAC-spacer, and p is an integer of 1 to (q-6); wherein the second TRAC-PEgRNA comprises: a second TRAC- spacer that is complementary to a second TRAC- search target sequence on a second strand of the TRAC gene complementary to the first strand, a second TRAC-gRNA core capable of binding to a Cas9 protein; and a second TRAC- extension arm comprising (A) a second TRAC-editing template and a (B) second TRAC-PBS that comprises at its 5’ end the reverse complement of nucleotides m to (n-3) of the second TRAC-spacer, wherein n is the length of the second TRAC- spacer, and m is an integer of 1 to (n-6). [0046]In some embodiments, the first TRAC-spacer comprises at its 3’ end nucleotides 4-of a sequence selected from the group consisting of SEQ ID NO:s 1303 and 1353. In some embodiments, the second TRAC spacer comprises at its 3’ end nucleotides 4-20 of a sequence selected from the group consisting of SEQ ID NO:s 1417, 1481, and 1532. In some embodiments, the first TRAC spacer has a length of 17 to 22 nucleotides, and/or wherein the second TRAC spacer has the length of 17 to 22 nucleotides. In some embodiments, wherein the first TRAC spacer and the second TRAC spacer are each 20 nucleotides in length. In some embodiments, wherein the first TRAC-spacer comprises at its 3’ end SEQ ID NO: 13or 1353. In some embodiments, the second TRAC spacer comprises at its 3’ end SEQ ID NO 1417, 1481, or 1532. In some embodiments, the first TRAC PBS is 7-17 nucleotides in length and comprises at its 5’ end a sequence that is the reverse complement of nucleotides 11-17, 10-17, 9-17, 8-17, 7-17, 6-17, 5-17, 4-17, 3-17, 2-17, or 1-17 of the selected sequence for the first TRAC spacer. In some embodiments the second TRAC PBS is 7-17 nucleotides in length and comprises at its 5’ end a sequence that is the reverse complement of nucleotides 11-17, 10-17, 9-17, 8-17, 7-17, 6-17, 5-17, 4-17, 3-17, 2-17, or 1-17 of the selected sequence for the second TRAC spacer. [0047]In some embodiments, wherein the first and/or the second TRAC PBS is 8-nucleotides in length. In some embodiments, the first and/or the second TRAC PBS is 11, 12, or 13 nucleotides in length. [0048]In some embodiments, the first gRNA core, the second gRNA core, or both comprise SEQ ID NO: 646 or 653.

WO 2024/238825 PCT/US2024/029746 id="p-49"

[0049]In some embodiments, the first TRAC editing template comprises a region of complementarity to the second TRAC editing template. In some embodiments, the first TRAC editing template and the second TRAC editing template each encodes all or a fragment of a recombinase recognition sequence (RRS) or the reverse complement thereof, wherein the first TRAC editing template encodes at least a 5’ portion of the RRS or the reverse complement thereof, wherein the second TRAC editing template encodes at least a 3’ portion of the RRS or the reverse complement thereof, and wherein at least 10 nucleotides at the 5’ ends of the first and the second TRAC editing templates have perfect reverse complementarity to each other. In some embodiments, at least 15, 20, 25, or 30 nucleotides at the 5’ ends of the first and the second TRAC editing templates have perfect reverse complementarity to each other, optionally wherein at least 20, 21, 22, 23, 24, 25, 26, or nucleotides at the 5’ ends of the first and the second TRAC editing templates have prefect reverse complementarity to each other. [0050]In some embodiments, the first TRAC editing template encodes the RRS. In some embodiments, the second TRAC editing template encodes the RRS. In some embodiments, wherein the RRS is an attB sequence recognized by a Bxbl recombinase. In some embodiments, the RRS is an attP sequence recognized by a Bxbl recombinase. In some embodiments, the first TRAC editing template comprises an RTT #1 from Table 39 and the second TRAC editing template comprises an RTT #2 in Table 39, or wherein the first TRAC editing template comprises an RTT #2 from Table 39 and the second TRAC editing template comprises an RTT #1 in Table 39. In some embodiments, the first TRAC editing template comprises a 5’ fragment of an RTT listed in Table 39 and wherein the second TRAC editing template comprises a full length or 5’ fragment of the corresponding RTT pair and wherein at least 10 nucleotides at the 5’ ends of the first and the second TRAC editing templates have perfect reverse complementarity to each other. In some embodiments, wherein the second TRAC editing template comprises a 5’ fragment of an RTT listed in Table 39 and wherein the first TRAC editing template comprises a full length or 5’ fragment of the corresponding RTT pair and wherein at least 10 nucleotides at the 5’ ends of the first and the second TRAC editing templates have perfect reverse complementarity to each other. [0051]In some embodiments, at least 15, 20, 25, or 30 nucleotides at the 5’ ends of the first and the second TRAC editing templates have perfect reverse complementarity to each other, optionally wherein at least 20, 21, 22, 23, 24, 25, 26, or 27 nucleotides at the 5’ ends of the first and the second TRAC editing templates have prefect reverse complementarity to each WO 2024/238825 PCT/US2024/029746 other. In some embodiments, the length of the region of complementarity of the first TRAC editing template is at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% of the length of the first TRAC editing template, optionally wherein the length of the region of complementarity of the first editing template is at least 52%, at least 53%, or at least 55% of the length of the first TRAC editing template. In some embodiments, the length of the region of complementarity of the second TRAC editing template is at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% of the length of the second TRAC editing template, optionally wherein the length of the region of complementarity of the second TRAC editing template is at least 52%, at least 53%, or at least 55% of the length of the second TRAC editing template. [0052]In some embodiments, the prime editing system comprises TRAC spacers, wherein (a) the first TRAC spacer comprises SEQ ID NO: 1303, and the first TRAC PBS comprises SEQ ID NO: 1312, or (b) the first TRAC spacer comprises SEQ ID NO: 1353, and the first TRAC PBS comprises SEQ ID NO: 1361, 1362, 1363, or 1364. In some embodiments, the second TRAC spacer comprises SEQ ID NO: 1417, and the second TRAC PBS comprises SEQ ID NO:1428, or the second TRAC spacer comprises SEQ ID NO: 1481, and the second TRAC PBS comprises SEQ ID NO: 1489. In some embodiments, (a) the first TRAC spacer comprises SEQ ID NO: 1303, and the first TRAC PBS has the sequence according to SEQ ID NO: 1313 or SEQ ID NO: 1314; or the first TRAC spacer comprises SEQ ID NO: 1353, and the first TRAC PBS has the sequence according to SEQ ID NO: 1361 or SEQ ID NO: 1363; and (b) the second TRAC spacer comprises SEQ ID NO: 1417, and the second TRAC PBS has the sequence according to SEQ ID NO: 1426 or SEQ ID NO: 1428, the second TRAC spacer comprises SEQ ID NO: 1480, and the second TRAC PBS has the sequence according to SEQ ID NO: 1486 or SEQ ID NO: 1487; or the second TRAC spacer comprises SEQ ID NO: 1532, and the second TRAC PBS has the sequence according to SEQ ID NO: 1541 or 1543. [0053]In some embodiments, the first TRAC spacer comprises SEQ ID NO: 1353, and the first TRAC PBS has the sequence according to SEQ ID NO: 1361, and wherein the second TRAC spacer comprises SEQ ID NO: 1417, and the second TRAC PBS has the sequence according to SEQ ID NO: 1426. In some embodiments, the first TRAC editing template WO 2024/238825 PCT/US2024/029746 comprises SEQ ID NO: 1577 and the second editing TRAC template comprises SEQ ID NO: 1584. In some embodiments, the first editing template comprises SEQ ID NO: 1584 and the second editing template comprises SEQ ID NO: 1577. [0054]In some embodiments, the first TRAC PEgRNA comprises a 5’ TRAC PEgRNA sequence selected from any one of Tables 34 and 35, and wherein the second TRAC PEgRNA comprises a 3’ TRAC PEgRNA sequence selected from any one of Tables 36-38. In some embodiments, the first TRAC PEgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 1328, 1382, 1387, and 1413; and wherein the second TRAC PEgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 1475, 1476, 1477, 1525, 1526, 1527, 1573, and 1574. In some embodiments, the first TRAC PEgRNA comprises SEQ ID NO: 1382 and the second TRAC PEgRNA comprises SEQ ID NO: 1527. In some embodiments, the first TRAC PEgRNA comprises SEQ ID NO: 1401 and the second TRAC PEgRNA comprises SEQ ID NO: 1459. In some embodiments, the first TRAC PEgRNA comprises SEQ ID NO: 1343 and the second TRAC PEgRNA comprises SEQ ID NO: 1566. In some embodiments, the first TRAC PEgRNA comprises SEQ ID NO: 1390 and the second TRAC PEgRNA comprises SEQ ID NO: 1456. In some embodiments, the first TRAC PEgRNA comprises SEQ ID NO: 1336 and the second TRAC PEgRNA comprises SEQ ID NO: 1560. In some embodiments, the first TRAC PEgRNA comprises SEQ ID NO: 1345 and the second TRAC PEgRNA comprises SEQ ID NO: 1566. In some embodiments, the first TRAC PEgRNA comprises SEQ ID NO: 374 and the second TRAC PEgRNA comprises SEQ ID NO: 1251. [0055]In some embodiments, the first TRAC PEgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 1322, 1336, 1372, and 126; and wherein the second TRAC PEgRNA comprises a sequence selected from the group consisting of SEQ ID NOs:1442, 1456, 1501, and 1513. [0056]In some embodiments, the first TRAC PEgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 1401, 1406, 1343,, and 1345; and wherein the second TRAC PEgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 1451, 1516, 1568, 1566, and 1459. In some embodiments, the first TRAC PEgRNA comprises SEQ ID NO: 1401, and wherein the second TRAC PEgRNA comprises SEQ ID NO: 1459.

WO 2024/238825 PCT/US2024/029746 id="p-57"

[0057]In some embodiments, the first TRAC PEgRNA and/or the second TRAC PEgRNA further comprises a 3’ motif, optionally wherein the 3’ motif is connected to the 3’ end of the first PBS or the second PBS via a linker. [0058]In some embodiments, the first TRAC PEgRNA and/or the second TRAC PEgRNA further comprises 5’mN*mN*mN* and 3’ mN*mN*mN*N modifications, where m indicates that the nucleotide contains a 2’-O-Me modification and a * indicates the presence of a phosphorothioate bond. [0059]In some embodiments, the prime editing system further comprises the recombinase or a nucleic acid encoding the recombinase. In some embodiments, the recombinase is fused or linked to the prime editor. [0060]In some embodiments, the prime editing system further comprises polynucleotide or a nucleic acid encoding the polynucleotide, wherein the polynucleotide comprises (a) a donor sequence and (b) a second recombinase recognition sequence (RRS) recognized by the recombinase. In some embodiments, the donor sequence encodes a chimeric antigen receptor (CAR). In some embodiments, (i) the RRS comprises SEQ ID NO: 1590, and the second RRS comprises SEQ ID NO: 1591, or (ii) the RRS comprises SEQ ID NO: 1591, and the second RRS comprises SEQ ID NO: 1590. In some embodiments, the recombinase is Bxbl. [0061]In some embodiments, the prime editing system further comprises a prime editor or one or more polynucleotides encoding the prime editor, wherein the prime editor comprises a) a Cas9 nickase having a nuclease inactivating mutation in the HNH domain, and b) a reverse transcriptase. In some embodiments, the prime editor is a fusion protein. [0062]In some embodiments, the prime editing system further comprises an N-terminal extein comprising an N-terminal fragment of a prime editor fusion protein and an N-intein or a polynucleotide encoding the N-terminal extein; a C-terminal extein comprising a C- terminal fragment of the prime editor fusion protein and a C-intein, or a polynucleotide encoding the C-terminal extein; wherein the N-intein and the C-intein of the N-terminal and C-terminal exteins are capable of self-excision to join the N-terminal fragment and the C- terminal fragment to form the prime editor fusion protein, and wherein the prime editor fusion protein comprises a Cas9 nickase having a nuclease inactivating mutation in the HNH domain and a reverse transcriptase (RT) domain. In some embodiments, the Cas9 nickase comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NOs: 676 or 677. In some embodiments, the reverse transcriptase comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, WO 2024/238825 PCT/US2024/029746 97%, 98%, 99%, or 100% identity to SEQ ID NO: 673. In some embodiments, the sequence identities are determined by Needleman-Wunsch alignment of two protein sequences with Gap Costs set to Existence: 11 Extension: 1 where percent identity is calculated by dividing the number of identities by the length of the alignment. [0063]In some embodiments, the prime editing system comprises polynucleotides, wherein the one or more polynucleotides encoding the prime editor, the polynucleotide encoding the N-terminal extein, or the polynucleotide encoding the C-terminal extein are mRNA. [0064]In some aspects, a population of viral particles collectively comprises the one or more polynucleotides encoding the PEgRNA or the prime editing system provided herein. In some embodiments, the viral particles are AAV particles. [0065]In some aspects, an LNP comprises the prime editing system provided herein. In some embodiments, the LNP comprises the PEgRNA and optionally the ngRNA, the polynucleotide encoding the Cas9 nickase, and the polynucleotide encoding the reverse transcriptase. In some embodiments, the LNP further comprises the polynucleotide encoding the Cas9 nickase and the polynucleotide encoding the reverse transcriptase are mRNA. In some embodiments, the polynucleotide encoding the Cas9 nickase and the polynucleotide encoding the reverse transcriptase are in the same molecule. [0066]In some aspects, a method of editing a B2M gene comprises contacting the B2M gene with: (a) the PEgRNA, and a prime editor comprising a Cas9 nickase having a nuclease inactivating mutation in the HNH domain and a reverse transcriptase, (b) the prime editing system, (c) the population of viral particles, or (d) the LNP provided herein. In some embodiments, the B2M gene is in a cell. In some aspects, method of generating an engineered cell comprises introducing into a cell or a population of cells: (a) the PEgRNA, and a prime editor comprising a Cas9 nickase having a nuclease inactivating mutation in the HNH domain and a reverse transcriptase, (b) the prime editing system, (c) the population of viral particles, or (d) the LNP provided herein. In some embodiments, the cell or the population of cells are in a subject. In some embodiments, the cell or the population of cells are ex vivo, optionally wherein the cell or the population of cells are obtained from a subject or a cell bank. In some embodiments, the cell or the population of cells are human cells. In some embodiments, the cell or the population of cells are immune cells. In some embodiments, the cell or the population of cells are T cells, optionally wherein the cell or the population of cells are cytotoxic T cells.

WO 2024/238825 PCT/US2024/029746 id="p-67"

[0067]In some aspects, an engineered cell or a population of engineered cells comprises a premature stop codon in the B2M gene relative to a wildtype B2M gene. [0068]In some aspects, an engineered cell or a population of engineered cells comprises a B2M gene comprising an insertion, a deletion, a substitution, or a combination thereof compared to a wildtype B2M gene at a chromosomal location corresponding to coding sequence position c.51, c.54, or c.50 of a wildtype B2M gene. [0069]In some aspects, an engineered cell or a population of engineered cells comprises a B2M gene comprising an insertion, a deletion, a substitution, or a combination thereof compared to a wildtype B2M gene at a chromosomal location corresponding to coding sequence position c.54, c.60, or c.66 of a wildtype B2M gene, optionally wherein the B2M gene comprises an insertion, a deletion, a substitution, or a combination thereof at a chromosomal location corresponding to coding sequence position c. 58 of a wildtype B2M gene. [0070]In some aspects, an engineered cell or a population of engineered cells comprises a B2M gene comprising an insertion, a deletion, a substitution, or a combination thereof compared to a wildtype B2M gene at a chromosomal location corresponding to coding sequence position c.21 or c.3 of a wildtype B2M gene, optionally wherein the B2M gene comprises an insertion, a deletion, a substitution, or a combination thereof at a chromosomal location corresponding to coding sequence position c. 17 of a wildtype B2M gene. [0071]In some aspects, an engineered cell or a population of engineered cells comprises a B2M gene comprising an insertion, a deletion, a substitution, or a combination thereof compared to a wildtype B2M gene at a chromosomal location corresponding to coding sequence position c.21, c.15 or c.3 of a wildtype B2M gene, optionally wherein the B2M gene comprises an insertion, a deletion, a substitution, or a combination thereof at a chromosomal location corresponding to coding sequence position c.11 of a wildtype B2M gene. [0072]In some embodiments, the cell or the population of cells comprises a c.51delC deletion in the B2M gene relative to a wildtype B2M gene. In some embodiments, the cell or the population of cells comprises a c.50insG insertion in the B2M gene relative to a wildtype B2M gene. In some embodiments, the cell or the population of cells comprises a c.54_55insCC insertion in the B2M gene relative to a wildtype B2M gene. In some embodiments, the cell or the population of cells comprises a c.54_55insTAAG insertion in the B2M gene relative to a wildtype B2M gene. In some embodiments, the cell or the WO 2024/238825 PCT/US2024/029746 population of cells comprises a c.54_55insTAATAA insertion in the B2M gene relative to a wildtype B2M gene. In some embodiments, the cell or the population of cells comprises comprising a c.66_67insCC insertion in the B2M gene relative to a wildtype B2M gene, optionally wherein the cell or the population of cells further comprise a c.58G>C substitution in the B2M gene relative to a wildtype B2M gene. In some embodiments, the cell or population of cells comprises a c.66_67insTAAG insertion in the B2M gene relative to a wildtype B2M gene, optionally wherein the cell or the population of cells further comprise a c.58G>C substitution in the B2M gene relative to a wildtype B2M gene. In some embodiments, the cell or the population of cells comprises a c.66_67insTAATAA insertion in the B2M gene relative to a wildtype B2M gene, optionally wherein the cell or the population of cells further comprise a c.58G>C substitution in the B2M gene relative to a wildtype B2M gene. In some embodiments, the cell or the population of cells comprises a c.60_65deletion and a TAATAG insertion (c.60_64delinsTAATAG) in the B2M gene relative to a wildtype B2M gene, optionally wherein the cell or the population of cells further comprise a c.58G>C substitution in the B2M gene relative to a wildtype B2M gene. In some embodiments, the cell or the population of cells comprises a c.21_22insCC insertion in the B2M gene relative to a wildtype B2M gene. In some embodiments, the cell or the population of cells comprises a c.21_22insTAAG insertion in the B2M gene relative to a wildtype B2M gene. In some embodiments, the cell or the population of cells comprises a c.21_22insTAATAA insertion in the B2M gene relative to a wildtype B2M gene. In some embodiments, the cell or the population of cells comprises a c.3_4insCC insertion in the B2M gene relative to a wildtype B2M gene, optionally wherein the cell or the population of cells further comprise a C.17OG substitution or a c.l lOG substitution in the B2M gene relative to a wildtype B2M gene. In some embodiments, the cell or the population of cells comprises a c.3_4insTAAG insertion in the B2M gene relative to a wildtype B2M gene, optionally wherein the cell or the population of cells further comprise a C.17OG substitution or a c.l lOG substitution in the B2M gene relative to a wildtype B2M gene. In some embodiments, the cell or the population of cells comprises a c.3_4insTAATAA insertion in the B2M gene relative to a wildtype B2M gene, optionally wherein the cell or the population of cells further comprise a C.17OG substitution or a c.l lOG substitution in the B2M gene relative to a wildtype B2M gene. In some embodiments, the cell or the population of cells comprises a c.3_8deletion and a TAATGA insertion (c.3_8delinsTAATGA) in the B2M gene relative to a wildtype B2M gene, optionally wherein the cell or the population of cells further comprise a C.17OG substitution WO 2024/238825 PCT/US2024/029746 or a c.11OG substitution in the B2M gene relative to a wildtype B2M gene. In some embodiments, the cell or the population of cells comprises a c.l5_16insCC insertion in the B2M gene relative to a wildtype B2M gene. In some embodiments, the cell or the population of cells comprises a c. 15_16insTAAG insertion in the B2M gene relative to a wildtype B2M gene. In some embodiments, the cell or the population of cells comprises a c.l5_16insTAATAA insertion in the B2M gene relative to a wildtype B2M gene. [0073]In some embodiments, the cell or the population of cells comprise a TRAC gene that comprises a sequenceGGCTTGTCGACGACGGCGGTCTCAGTGGTGTACGGTACAAACC (SEQ ID NO: 9999) and/or GGTTTGTCTGGTCAACCACCGCGGTCTCCGTCGTCAGGATCAT (SEQ ID NO: 10000) relative to a wildtype TRAC gene. In some embodiments, the edited TRAC gene comprises an insert sequence comprising, from 5’ to 3’, GGCTTGTCGACGACGGCGGTCTCAGTGGTGTACGGTACAAACC (SEQ ID NO: 9999), a donor sequence, and GGTTTGTCTGGTCAACCACCGCGGTCTCCGTCGTCAGGATCAT (SEQ ID NO: 10000). In some embodiments, the edited TRAC gene comprises an insert sequence comprising, from 5’ to 3’, GGTTTGTCTGGTCAACCACCGCGGTCTCCGTCGTCAGGATCAT (SEQ ID NO: 10000), a donor sequence, and GGCTTGTCGACGACGGCGGTCTCAGTGGTGTACGGTACAAACC (SEQ ID NO: 9999). [0074]In some embodiments, the cell or the population of cells comprises a donor sequence, wherein the donor sequence encodes a chimeric antigen receptor (CAR), optionally wherein the donor encodes a CD 19 CAR. [0075]In some embodiments, the cell or the population of cells comprises an insert sequence, wherein the insert sequence is between a first chromosome location and a second chromosome location, wherein the first chromosome location is selected from the group consisting of human chromosome 14 positions 22547458, 22547457, 22547449, and 22547448, and wherein the second chromosome location is selected from the group consisting of human chromosome 14 positions 22547533, 22547523, 22547491, 22547528, 22547497, 22547579, 22547522, 22547485, 22547506, 22547560, 22547505, 22547529, and 22547490. In some embodiments, the insert sequence is a) between human chromosome positions22547458 and 22547533, b) between human chromosome 14 positions 22547458 WO 2024/238825 PCT/US2024/029746 and 22547522, c) between human chromosome 14 positions 22547458 and 22547529, d) between human chromosome 14 positions 22547449 and 22547533, e) between human chromosome 14 positions 22547449 and 22547522, or f) between human chromosome positions 22547449 and 22547529. [0076]In some embodiments, the human chromosome locations and coding sequence locations are as set forth in Genome Reference Consortium Human Build 38 (GrCh38). [0077]In some embodiments, the cell or the population of cells are in a subject. In some embodiments, the cell or the population of cells are ex vivo, optionally wherein the cell or the population of cells are obtained from a subject or a cell bank. In some embodiments, the cell or the population of cells are human cells. In some embodiments, the cell or the population of cells are immune cells. In some embodiments, the cell or the population of cells are T cells, optionally wherein the cell or the population of cells are cytotoxic T cells. [0078]In some aspects, a method of immunotherapy comprising administering to a subject (a) the PEgRNA, and a prime editor comprising a Cas9 nickase having a nuclease inactivating mutation in the HNH domain and a reverse transcriptase, (b) the prime editing system (c) the population of viral particles, (d) the LNP or (e) the cell or the population of cells provided herein. [0079]In some aspects, a prime editing guide RNA (PEgRNA) or one or more polynucleotides encoding the PEgRNA, the PEgRNA comprising a) a spacer that is complementary to a search target sequence on a first strand of a 02-microglobulin (B2M) gene, wherein the spacer comprises at its 3’ end a PEgRNA spacer sequence selected from any one of Tables 1-21; b) a gRNA core capable of binding to a Cas9 protein, and c) an extension arm comprising i) an editing template comprising at its 3’ end an RTT sequence selected from the same Table as the PEgRNA Spacer sequence, and ii) a primer binding site (PBS) comprising at its 5’ end a PBS sequence selected from the same Table as the PEgRNA Spacer sequence. In some embodiments, the spacer of the PEgRNA is from 17 to nucleotides in length. In some embodiments, the spacer of the PEgRNA is 20 nucleotides in length. In some embodiments, the spacer, the gRNA core, the editing template, and the PBS form a contiguous sequence in a single molecule. In some embodiments, the PEgRNA comprises from 5’ to 3’, the spacer, the gRNA core, the editing template, and the PBS. In some embodiments, the prime editing system comprises the PEgRNA or the one or more polynucleotides.

WO 2024/238825 PCT/US2024/029746 id="p-80"

[0080]In some embodiments, the prime editing system further comprises a nick guide RNA (ngRNA), or one or more polynucleotides encoding the ngRNA, wherein the ngRNA comprises: (i) an ngRNA spacer that comprises a region of complementarity to a second strand of the B2M gene; and (ii) an ngRNA core capable of binding a Cas9 protein. In some embodiments, the spacer of the ngRNA is from 17 to 22 nucleotides in length. In some embodiments, the spacer of the ngRNA is 20 nucleotides in length. In some embodiments, the ngRNA spacer comprises at its 3’ end an ngRNA Spacer sequence selected from the same Table as the PEgRNA Spacer sequence. In some embodiments, the ngRNA comprises an ngRNA sequence selected from the same Table as the PEgRNA Spacer sequence. [0081]In some embodiments, the prime editing system further comprises a prime editor comprising a Cas9 nickase having a nuclease inactivating mutation in the HNH domain, or one or more polynucleotides encoding the Cas9 nickase, and a reverse transcriptase, or one or more polynucleotides encoding the reverse transcriptase. [0082]In some embodiments, the prime editing system further comprises an N-terminal extein comprising an N-terminal fragment of a prime editor fusion protein and an N-intein or a polynucleotide encoding the N-terminal extein; and a C-terminal extein comprising a C- terminal fragment of the prime editor fusion protein and a C-intein, or a polynucleotide encoding the C-terminal extein; wherein the N-intein and the C-intein of the N-terminal and C-terminal exteins are capable of self-excision to join the N-terminal fragment and the C- terminal fragment to form the prime editor fusion protein, and wherein the prime editor fusion protein comprises a Cas9 nickase and a reverse transcriptase (RT) domain. In some embodiments, the Cas9 nickase comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NOs: 676 or 677. In some embodiments, the reverse transcriptase comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 673. In some embodiments, the sequence identities are determined by Needleman-Wunsch alignment of two protein sequences with Gap Costs set to Existence: 11 Extension: 1 where percent identity is calculated by dividing the number of identities by the length of the alignment. [0083]In some embodiments, the prime editing system further comprises a TRAC-PEgRNA pair, wherein the TRAC-PEgRNA pair comprises: a) a first TRAC-prime editing guide RNA (PEgRNA) or one or more polynucleotides encoding the first TRAC-PEgRNA, and b) a second TRAC-PEgRNA or one or more polynucleotides encoding the second TRAC- PEgRNA, wherein the first TRAC-PEgRNA comprises: i) a first TRAC-spacer that WO 2024/238825 PCT/US2024/029746 comprises at its 3’ end a 5’ TRAC-PEgRNA spacer sequence selected from any one of Tables and 35, ii) a first TRAC-gRNA core capable of binding to a Cas9 protein; and iii) a first TRAC-extension arm comprising (A) a first TRAC-editing template and (B) a first TRAC- primer binding site (PBS) that comprises at its 5’ end a 5’ TRAC-PBS sequence selected from the same Table as the first TRAC-spacer, wherein the second TRAC-PEgRNA comprises: i) a second TRAC-spacer that comprises at its 3’ end a 5’ TRAC-PEgRNA spacer sequence selected from any one of Tables 36-38, ii) a second TRAC-gRNA core capable of binding to a Cas9 protein; and iii) a second TRAC- extension arm comprising (A) a second TRAC-editing template and (B) a second TRAC- primer binding site (PBS) that comprises at its 5’ end a 5’ TRAC-PBS sequence selected from the same Table as the second TRAC- spacer. [0084]In some embodiments, the first TRAC editing template comprises a region of complementarity to the second TRAC editing template. In some embodiments, wherein the first TRAC editing template and the second TRAC editing template each encodes all or a fragment of a recombinase recognition sequence (RRS) or the reverse complement thereof, wherein the first TRAC editing template encodes at least a 5’ portion of the RRS or the reverse complement thereof, wherein the second TRAC editing template encodes at least a 3’ portion of the RRS or the reverse complement thereof, and wherein at least 10 nucleotides at the 5’ ends of the first and the second TRAC editing templates have perfect reverse complementarity to each other. [0085]In some embodiments, at least 15, 20, 25, or 30 nucleotides at the 5’ ends of the first and the second TRAC editing templates have perfect reverse complementarity to each other, optionally wherein at least 20, 21, 22, 23, 24, 25, 26, or 27 nucleotides at the 5’ ends of the first and the second TRAC editing templates have prefect reverse complementarity to each other. [0086]In some embodiments, the first TRAC editing template encodes the RRS or wherein the second TRAC editing template encodes the RRS, optionally wherein the RRS is an attB sequence recognized by a Bxbl recombinase or an attP sequence recognized by a Bxbl recombinase. [0087]In some embodiments, the first TRAC editing template comprises an RTT #1 from Table 39 and the second TRAC editing template comprises an RTT #2 in Table 39, or wherein the first TRAC editing template comprises an RTT #2 from Table 39 and the second TRAC editing template comprises an RTT #1 in Table 39.

WO 2024/238825 PCT/US2024/029746 id="p-88"

[0088]In some embodiments, the first TRAC editing template comprises SEQ ID NO: 15and the second editing TRAC template comprises SEQ ID NO: 1584, or wherein the first editing template comprises SEQ ID NO: 1584 and the second editing template comprises SEQ ID NO: 1577. [0089]In some embodiments, the first TRAC PEgRNA comprises a 5’ TRAC PEgRNA sequence selected from any one of Tables 34 and 35, and wherein the second TRAC PEgRNA comprises a 3’ TRAC PEgRNA sequence selected from any one of Tables 36-38.

INCORPORATION BY REFERENCE [0090]All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS [0091]The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which: [0092] FIG. Idepicts a schematic of a prime editing guide RNA (PEgRNA) binding to a double stranded target DNA sequence. [0093] FIG. 2depicts a PEgRNA architectural overview in an exemplary schematic of PEgRNA designed for a prime editor. [0094] FIG. 3is a schematic showing the spacer and gRNA core part of an exemplary guide RNA, in two separate molecules. The rest of the PEgRNA structure is not shown. [0095] FIG. 4depicts an exemplary schematic of a dual prime editing system for editing both strands of a double-stranded target DNA. Same color/shading indicates complementarity or identity between sequences. [0096] FIG. 4Bdepicts an exemplary schematic of dual prime editing with a replacement duplex (RD) comprising an overlap duplex (OD). Same color/shading indicates complementarity or identity between sequences.

WO 2024/238825 PCT/US2024/029746 DETAILED DESCRIPTION [0097]Provided herein, in some embodiments, are compositions and methods to edit the target gene 02-microglobulin (B2M) with prime editing. Compositions provided herein can comprise prime editors (PEs) that may use engineered guide polynucleotides, e.g., prime editing guide RNAs (PEgRNAs), that can direct PEs to specific DNA targets and can encode DNA edits on the target gene B2M that serve a variety of functions, including direct disruption of the target gene. The edits can disrupt the B2M gene by, for example, by introducing one or more stop codons, introducing a frameshift mutation (insertion or deletion), or disrupting a splice site. Also provided are compositions that comprise edited cells generated by the methods disclosed herein. [0098]The following description and examples illustrate embodiments of the present disclosure in detail. It is to be understood that this disclosure is not limited to the particular embodiments described herein and as such can vary. Those of skill in the art will recognize that there are numerous variations and modifications of this disclosure, which are encompassed within its scope. Although various features of the present disclosure can be described in the context of a single embodiment, the features can also be provided separately or in any suitable combination. Conversely, although the present disclosure can be described herein in the context of separate embodiments for clarity, the present disclosure can also be implemented in a single embodiment. Definitions [0099]Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art. [0100]The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used herein, the singular forms "a", "an" and "the " are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms "including ", "includes ", "having", "has", "with ", or variants thereof as used herein mean "comprising ". [0101]Unless otherwise specified, the words "comprising ", "comprise ", "comprises ", "having", "have ", "has", "including ", "includes ", "include ", "containing ", "contains " and "contain " are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. [0102]Reference to "some embodiments ", "an embodiment ", "one embodiment ", or "other embodiments " means that a particular feature or characteristic described in connection with WO 2024/238825 PCT/US2024/029746 the embodiments is included in at least one or more embodiments, but not necessarily all embodiments, of the present disclosure. [0103]The term "about " or "approximately " in relation to a numerical means, a range of values that fall within 10% greater than or less than the value. For example, about x means x±(10% * x). [0104]In some embodiments, the cell is a human cell. A cell may be of or derived from different tissues, organs, and/or cell types. In some embodiments, the cell is a primary cell. As used herein, the term "primary cell " means a cell isolated from an organism, e.g., a mammal, which is grown in tissue culture (i.e., in vitro) for the first time before subdivision and transfer to a subculture. In some non-limiting examples, mammalian cells, including primary cells and stem cells can be modified through introduction of one or more polynucleotides, polypeptide, and/or prime editing compositions (e.g., through transfection, transduction, electroporation and the like) and further passaged. [0105]Such modified cells include a T-cell, e.g., a primary T cell, e.g., an inflammatory T cell, a T helper cell, a cytotoxic T-cell, a CD4+ T-cell, a CD8+ T cell, a memory T cell, a regulatory T cell, a natural killer T cell, a mucosal associated invariant T cell, a y8 T cell, an alpha beta T cell, a naive T cell, or an effector T cell), formed elements of the blood (e.g., lymphocytes, bone marrow cells), precursors or progenitors thereof, differentiated or de- differentiated cell thereof, and stem cells. In some embodiments, a cell is a naive T cell (e.g., a naive CD8+ T cell). In some embodiments, the cell is a transformed T cell. In some embodiments, the cell is an immune cell (e.g., a primary immune cell) or a progenitor or a precursor thereof). In some embodiments, the cell is a T-cell, or a progenitor or a precursor thereof. In some embodiments, the cell is a human T cell, or a progenitor or a precursor thereof. In some embodiments, the cell is a T helper cell (e.g., Thl cell, Th2 cell, Th9 cell, Thl7 cell, Th22 cell, and Tfh (follicular helper) cell). In some embodiments, the cell is a cytotoxic T cell. In some embodiments, the cell is a CD8+ T cell. In some embodiments, the cell is a CD4+ T cell. In some embodiments, the cell is a memory T cell (e.g., (e.g., central memory T cell (Tcm), stem memory T cell (TSCM), effector memory T cell, Tissue resident memory T cell). In some embodiments, the cell is an effector memory T cell (e.g., Tem cells and TEMRA (CD45RA+) cells). In some embodiments, the cell is a regulatory T cell. In some embodiments, the cell is a natural killer T cell. In some embodiments, the cell is a Mucosal associated invariant T cell. In some embodiments, the cell is a y8 T cell. In some embodiments, the cell is an effector T cell. In some embodiments, the cell is a thymocyte. In WO 2024/238825 PCT/US2024/029746 some embodiments, the cell is a lymphoid cell. In some embodiments, the cell is a common lymphoid progenitor cells. In some embodiments, the cell is an early thymic progenitor cell. In some embodiments, the cell is a CD3+ cell. In some embodiments, the cell is a tumor infiltrating lymphocyte. In some embodiments, the cell is a myeloid cell. In some embodiments, the cell is a plasma cell. In some embodiments, the cell is an activated T cell. [0106]In some embodiments, the cell is a stem cell (e.g., adult stem cell, embryonic stem cell, non-embryonic stem cell), cord blood stem cell, progenitor cell, bone marrow stem cell, induced pluripotent stem cell, totipotent stem cell, a CD34+ cell, or hematopoietic stem cell). In some embodiments, the cell is a pluripotent cell (e.g., a pluripotent stem cell). In some embodiments, the cell (e.g., a stem cell) is an embryonic stem cell, tissue-specific stem cell, mesenchymal stem cell, or an induced pluripotent stem cell. In some embodiments, the cell is an induced pluripotent stem cell (iPSC). In some embodiments, the cell is a hematopoietic stem cell. In some embodiments, the cell is a hematopoietic stem and progenitor cell. In some embodiments, the cell is a multipotent progenitor cell. In some embodiments, the cell is a T- cell progenitor. In some embodiments, the cell is a T-cell precursor. In some embodiments, the cell is an embryonic stem cell (ESC). In some embodiments, the cell is a human stem cell. In some embodiments, the cell is a human pluripotent stem cell. In some embodiments, the cell is a non-embryonic stem cell. In some embodiments, the cell is an induced human pluripotent stem cell. In some embodiments, the cell is a human stem cell. In some embodiments, the cell is a human embryonic stem cell. In some embodiments, the cell is a human T-cell progenitor. In some embodiments, the cell is a human T-cell precursor. [0107]In some embodiments, a cell is not isolated from an organism but forms part of a tissue or organ of an organism, e.g., a mammal. [0108]In some embodiments, the cell is a differentiated cell. In some embodiments, the cell is differentiated from an induced pluripotent stem cell. In some embodiments, the cell is a T- cell e.g., a primary T cell, e.g., an inflammatory T cell, a T helper cell, a cytotoxic T-cell, a CD4+ T-cell, a CD8+ T cell, a memory T cell, a regulatory T cell, a natural killer T cell, a mucosal associated invariant T cell, a y8 T cell, an alpha beta T cell, a naive T cell, or an effector T cell differentiated from an iPSC, ESC, a T-cell precursor, or a T-cell progenitor. [0109]In some embodiments, the cell is a differentiated human cell. In some embodiments, the cell is differentiated from an induced human pluripotent stem cell. In some embodiments, the cell edited by prime editing can be differentiated into, or give rise to recovery of a population of cells, e.g., a T-cell e.g., a primary T cell, e.g., an inflammatory T cell, a T WO 2024/238825 PCT/US2024/029746 helper cell, a cytotoxic T-cell, a CD4+ T-cell, a CD8+ T cell, a memory T cell, a regulatory T cell, a natural killer T cell, a mucosal associated invariant T cell, a y8 T cell, an alpha beta T cell, a naive T cell, or an effector T cell. In some embodiments, the cell is in a subject, e.g., a human subject. In some embodiments, the cell is obtained from a subject prior to editing. For example, in some embodiments, the cell is obtained from a patient having a cancer, a microbial infection, a graft vs host disease, or an autoimmune disorder. Prior to editing by the methods and compositions disclosed herein the cell can be obtained from a subject through a variety of non-limiting methods. T cells can be obtained from a number of non- limiting sources, for example, peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. Methods of collecting blood cells, isolating and enriching T cells, and expanding them ex vivo may be by methods known in the art. In some embodiments, the cell can be obtained from a cell bank, a blood bank, cell culture, or any number of T cell lines available, and known to those skilled in the art. Cells may also be obtained from a tissue biopsy, surgery, blood, plasma, serum, or other biological fluid. In some embodiments, the cell can be obtained prior to editing from one or more healthy donors, from a patient having a cancer, a microbial infection, a graft versus host infection, or an autoimmune disorder. [0110]For example, a cell can be obtained (i.e., isolated or purified) prior to editing from a whole blood sample by lysing red blood cells or a fractionated blood sample, and removing peripheral mononuclear blood cells by centrifugation. The cell can be further isolated or purified using a selective purification method that isolates the cell based on cell-specific markers such as CD25, CD3, CD4, CDS, CD28, CD45RA, or CD45RO (e.g., by flow cytometry). In one embodiment, CD4+ is used as a marker to select T cells. In one embodiment, CD8+ is used as a marker to select T cells. In one embodiment, CD4+ and CD8+ are used as a marker to select regulatory T cells. [0111]In some embodiments, the edited cell produced using the methods and compositions disclosed herein are cultured, proliferated, expanded, differentiated, and or de-differentiated in vitro. [0112]In some embodiments, the cell comprises a prime editor, a PEgRNA, or a prime editing composition disclosed herein. In some embodiments, the cell further comprises an ngRNA. In some embodiments, the cell is from a human subject. In some embodiments, the cell is from a human subject, and comprises a prime editor, a PEgRNA, or a prime editing composition for editing a B2M gene. In some embodiments, the cell is from the human WO 2024/238825 PCT/US2024/029746 subject and the B2M gene has been edited by prime editing. In some embodiments, the human subject is a healthy donor. In some embodiments, the human subject has a disease, disorder, or a condition, e.g., a cancer, a microbial infection, or an autoimmune disorder.. In some embodiments, the human subject is in need of, or is undergoing, or will be undergoing an immune cell immunotherapy (e.g., a T cell therapy such as a CAR-T cell therapy). [0113]The term "substantially " as used herein may refer to a value approaching 100% of a given value. In some embodiments, the term may refer to an amount that may be at least about 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 99.99% of a total amount. In some embodiments, the term may refer to an amount that may be about 100% of a total amount. [0114]The terms "protein " and "polypeptide " can be used interchangeably to refer to a polymer of two or more amino acids joined by covalent bonds (e.g, an amide bond) that can adopt a three-dimensional conformation. In some embodiments, a protein or polypeptide comprises at least 10 amino acids, 15 amino acids, 20 amino acids, 30 amino acids or amino acids joined by covalent bonds (e.g, amide bonds). In some embodiments, a protein comprises at least two amide bonds. In some embodiments, a protein comprises multiple amide bonds. In some embodiments, a protein comprises an enzyme, enzyme precursor proteins, regulatory protein, structural protein, receptor, nucleic acid binding protein, a biomarker, a member of a specific binding pair (e.g, a ligand or aptamer), or an antibody. In some embodiments, a protein may be a full-length protein (e.g, a fully processed protein having certain biological function). In some embodiments, a protein may be a variant or a fragment of a full-length protein. For example, in some embodiments, a Cas9 protein domain comprises an H840A amino acid substitution compared to a naturally occurring 8. pyogenes Cas9 protein. A variant of a protein or enzyme, for example a variant reverse transcriptase, comprises a polypeptide having an amino acid sequence that is about 60% identical, about 70% identical, about 80% identical, about 90% identical, about 95% identical, about 96% identical, about 97% identical, about 98% identical, about 99% identical, about 99.5% identical, or about 99.9% identical to the amino acid sequence of a reference protein. [0115]In some embodiments, a protein comprises one or more protein domains or subdomains. As used herein, the term "polypeptide domain ", "protein domain ", or "domain " when used in the context of a protein or polypeptide, refers to a polypeptide chain that has one or more biological functions, e.g, a catalytic function, a protein-protein binding function, or a protein-DNA function. In some embodiments, a protein comprises multiple protein WO 2024/238825 PCT/US2024/029746 domains. In some embodiments, a protein comprises multiple protein domains that are naturally occurring. In some embodiments, a protein comprises multiple protein domains from different naturally occurring proteins. For example, in some embodiments, a prime editor may be a fusion protein comprising a Cas9 protein domain of 8. pyogenes and a reverse transcriptase protein domain of a retrovirus (e.g., a Moloney murine leukemia virus) or a variant of the retrovirus. A protein that comprises amino acid sequences from different origins or naturally occurring proteins may be referred to as a fusion, or chimeric protein. [0116]In some embodiments, a protein comprises a functional variant or functional fragment of a full-length wild-type protein. A "functional fragment " or "functional portion ", as used herein, refers to any portion of a reference protein (e.g, a wild-type protein) that encompasses less than the entire amino acid sequence of the reference protein while retaining one or more of the functions, e.g, catalytic or binding functions. For example, a functional fragment of a reverse transcriptase may encompass less than the entire amino acid sequence of a wild-type reverse transcriptase, but retains the ability under at least one set of conditions to catalyze the polymerization of a polynucleotide. When the reference protein is a fusion of multiple functional domains, a functional fragment thereof may retain one or more of the functions of at least one of the functional domains. For example, a functional fragment of a Cas9 may encompass less than the entire amino acid sequence of a wild-type Cas9, but retains its DNA binding ability and lacks its nuclease activity partially or completely. [0117]A "functional variant " or "functional mutant ", as used herein, refers to any variant or mutant of a reference protein (e.g, a wild-type protein) that encompasses one or more alterations to the amino acid sequence of the reference protein while retaining one or more of the functions, e.g, catalytic or binding functions. In some embodiments, the one or more alterations to the amino acid sequence comprises amino acid substitutions, insertions or deletions, or any combination thereof. In some embodiments, the one or more alterations to the amino acid sequence comprises amino acid substitutions. For example, a functional variant of a reverse transcriptase may comprise one or more amino acid substitutions compared to the amino acid sequence of a wild-type reverse transcriptase, but retains the ability under at least one set of conditions to catalyze the polymerization of a polynucleotide. When the reference protein is a fusion of multiple functional domains, a functional variant thereof may retain one or more of the functions of at least one of the functional domains. For example, in some embodiments, a functional fragment of a Cas9 may comprise one or more amino acid substitutions in a nuclease domain, e.g, an H840A amino acid substitution, WO 2024/238825 PCT/US2024/029746 compared to the amino acid sequence of a wild type Cas9, but retains the DNA binding ability and lacks the nuclease activity partially or completely. [0118]The term "function " and its grammatical equivalents as used herein may refer to a capability of operating, having, or serving an intended purpose. Functional may comprise any percent from baseline to 100% of an intended purpose. For example, functional may comprise or comprise about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or up to about 100% of an intended purpose. In some embodiments, the term functional may mean over or over about 100% of normal function, for example, 125%, 150%, 175%, 200%, 250%, 300%, 400%, 500%, 600%, 700% or up to about 1000% of an intended purpose. [0119]In some embodiments, a protein or polypeptides includes naturally occurring amino acids (e.g, one of the twenty amino acids commonly found in peptides synthesized in nature, and known by the one letter abbreviations A, R, N, C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y and V). In some embodiments, a protein or polypeptides includes non-naturally occurring amino acids (e.g, amino acids which is not one of the twenty amino acids commonly found in peptides synthesized in nature, including synthetic amino acids, amino acid analogs, and amino acid mimetics). In some embodiments, a protein or polypeptide is modified. [0120]In some embodiments, a protein comprises an isolated polypeptide. The term "isolated " means free or removed to varying degrees from components which normally accompany it as found in the natural state or environment. For example, a polypeptide naturally present in a living animal is not isolated, and the same polypeptide partially or completely separated from the coexisting materials of its natural state is isolated. [0121]In some embodiments, a protein is present within a cell, a tissue, an organ, or a virus particle. In some embodiments, a protein is present within a cell or a part of a cell (e.g, a bacteria cell, a plant cell, or an animal cell). In some embodiments, the cell is in a tissue, in a subject, or in a cell culture. In some embodiments, the cell is a microorganism (e.g, a bacterium, fungus, protozoan, or virus). In some embodiments, a protein is present in a mixture of analytes (e.g, a lysate). In some embodiments, the protein is present in a lysate from a plurality of cells or from a lysate of a single cell. [0122]The terms "homologous, " "homology, " or "percent homology " as used herein refer to the degree of sequence identity between an amino acid and a corresponding reference amino acid sequence or a polynucleotide sequence and a corresponding reference polynucleotide WO 2024/238825 PCT/US2024/029746 sequence. "Homology " can refer to polymeric sequences, e.g., polypeptide or DNA sequences that are similar. Homology can mean, for example, nucleic acid sequences with at least about: 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity. In other embodiments, a "homologous sequence " of nucleic acid sequences may exhibit 93%, 95% or 98% sequence identity to the reference nucleic acid sequence. For example, a "region of homology to a genomic region " can be a region of DNA that has a similar sequence to a given genomic region in the genome. A region of homology can be of any length that is sufficient to promote binding of a spacer, a primer binding site or protospacer sequence to the genomic region. For example, the region of homology can comprise at least 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 31or more bases in length such that the region of homology has sufficient homology to undergo binding with the corresponding genomic region. [0123]When a percentage of sequence homology or identity is specified, in the context of two nucleic acid sequences or two polypeptide sequences, the percentage of homology or identity generally refers to the alignment of two or more sequences across a portion of their length when compared and aligned for maximum correspondence. When a position in the compared sequence can be occupied by the same base or amino acid, then the molecules can be homologous at that position. Unless stated otherwise, sequence homology or identity is assessed over the specified length of the nucleic acid, polypeptide or portion thereof. In some embodiments, the homology or identity is assessed over a functional portion or specified portion of the length. [0124]Alignment of sequences for assessment of sequence homology can be conducted by algorithms known in the art, such as the Basic Local Alignment Search Tool (BLAST) algorithm, which is described in Altschul et al, J. Mol. Biol. 215:403- 410,1990. A publicly available, internet interface, for performing BLAST analyses is accessible through the National Center for Biotechnology Information. Additional known algorithms include those published in: Smith & Waterman, "Comparison of Biosequences ", Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, "A general method applicable to the search for similarities in the amino acid sequence of two proteins " J. Mol. Biol. 48:443, 1970; Pearson & Lipman "Improved tools for biological sequence comparison ", Proc. Natl. Acad. Sci. USA 85:2444, WO 2024/238825 PCT/US2024/029746 1988; or by automated implementation of these or similar algorithms. Global alignment programs may also be used to align similar sequences of roughly equal size. Examples of global alignment programs include NEEDLE (available at www.ebi.ac.uk/Tools/psa/emboss_needle/ ) which is part of the EMBOSS package (Rice P et al., Trends Genet., 2000; 16: 276-277), and the GGSEARCH program https://fasta.bioch.virginia.edu/fasta_www2/ , which is part of the PASTA package (Pearson W and Lipman D, 1988, Proc. Natl. Acad. Sci. USA, 85: 2444-2448). Both of these programs are based on the Needleman-Wunsch algorithm which is used to find the optimum alignment (including gaps) of two sequences along their entire length. A detailed discussion of sequence analysis can also be found in Unit 19.3 of Ausubel et al ("Current Protocols in Molecular Biology " John Wiley & Sons Inc, 1994-1998, Chapter 15, 1998). Unless otherwise specified, alignment between a query sequence and a reference sequence is performed with Needleman- Wunsch alignment with Gap Costs set to Existence: 11 Extension: 1 where percent identity is calculated by dividing the number of identities by the length of the alignment, as further described in Altschul et al.("Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402,1997) and Altschul et al, ("Protein database searches using compositionally adjusted substitution matrices", FEES J. 272:5101-5109, 2005). [0125]A skilled person understands that amino acid (or nucleotide) positions may be determined in homologous sequences based on alignment, for example, "H840" in a reference Cas9 sequence may correspond to H839, or another position in a Cas9 homolog. [0126]The term "polynucleotide " or "nucleic acid molecule " can be any polymeric form of nucleotides, including DNA, RNA, a hybridization thereof, or RNA-DNA chimeric molecules. In some embodiments, a polynucleotide comprises cDNA, genomic DNA, mRNA, tRNA, rRNA, or microRNA. In some embodiments, a polynucleotide is double stranded, e.g, a double-stranded DNA in a gene. In some embodiments, a polynucleotide is single-stranded or substantially single-stranded, e.g., single-stranded DNA or an mRNA. In some embodiments, a polynucleotide is a cell-free nucleic acid molecule. In some embodiments, a polynucleotide circulates in blood. In some embodiments, a polynucleotide is a cellular nucleic acid molecule. In some embodiments, a polynucleotide is a cellular nucleic acid molecule in a cell circulating in blood. [0127]Polynucleotides can have any three-dimensional structure. The following are nonlimiting examples of polynucleotides: a gene or gene fragment (for example, a probe, WO 2024/238825 PCT/US2024/029746 primer, EST or SAGE tag), an exon, an intron, intergenic DNA (including, without limitation, heterochromatic DNA), messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), a ribozyme, cDNA, a recombinant polynucleotide, a branched polynucleotide, a plasmid, a vector, isolated DNA, isolated RNA, sgRNA, guide RNA, a nucleic acid probe, a primer, an snRNA, a long non-coding RNA, a snoRNA, a siRNA, a miRNA, a tRNA-derived small RNA (tsRNA), an antisense RNA, an shRNA, or a small rDNA-derived RNA (srRNA). [0128]In some embodiments, a polynucleotide comprises deoxyribonucleotides, ribonucleotides or analogs thereof. In some embodiments, a polynucleotide comprises modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. [0129]In some embodiments, a polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for thymine when the polynucleotide is RNA. In some embodiments, the polynucleotide may comprise one or more other nucleotide bases, such as inosine (I), which is read by the translation machinery as guanine (G). [0130]In some embodiments, a polynucleotide may be modified. As used herein, the terms "modified " or "modification " refers to chemical modification with respect to the A, C, G, T and U nucleotides, and is indicated as mA, mC, mG, mT, and mT. In some embodiments, modifications may be on the nucleoside base and/or sugar portion of the nucleosides that comprise the polynucleotide. In some embodiments, the modification may be on the internucleoside linkage (e.g., phosphate backbone). In some embodiments, multiple modifications are included in the modified nucleic acid molecule. In some embodiments, a single modification is included in the modified nucleic acid molecule. [0131]The term "complement ", "complementary ", or "complementarity " as used herein, refers to the ability of two polynucleotide molecules to base pair with each other.Complementary polynucleotides may base pair via hydrogen bonding, which may be Watson Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding. For example, an adenine on one polynucleotide molecule will base pair to a thymine or an uracil on a second polynucleotide molecule and a cytosine on one polynucleotide molecule will base pair to guanine on a WO 2024/238825 PCT/US2024/029746 second polynucleotide molecule. Two polynucleotide molecules are complementary to each other when a first polynucleotide molecule comprising a first nucleotide sequence can base pair with a second polynucleotide molecule comprising a second nucleotide sequence. For instance, the two DNA molecules 5'-ATGC-3' and 5'-GCAT-3' are complementary, and the complement of the DNA molecule 5'-ATGC-3' is 5'-GCAT-3'. A percentage of complementarity indicates the percentage of nucleotides in a polynucleotide molecule which can base pair with a second polynucleotide molecule (e.g., 5, 6, 7, 8, 9,10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary, respectively). "Perfectly complementary " means that all the contiguous nucleotides of a polynucleotide molecule will base pair with the same number of contiguous nucleotides in a second polynucleotide molecule. "Substantially complementary " as used herein refers to a degree of complementarity that can be 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% over all or a portion of two polynucleotide molecules. In some embodiments, the portion of complementarity may be a region of 10, 15, 20, 25, 30, 35, 40, 45, 50, or more nucleotides. "Substantial complementary " can also refer to a 100% complementarity over a portion or a region of two polynucleotide molecules. In some embodiments, the portion or the region of complementarity between the two polynucleotide molecules is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% of the length of at least one of the two polynucleotide molecules or a functional or defined portion thereof. [0132]As used herein, "expression " refers to the process by which polynucleotides are transcribed into mRNA and/or the process by which polynucleotides, e.g., the transcribed mRNA, translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell. In some embodiments, expression of a polynucleotide, e.g., a gene or a DNA encoding a protein, is determined by the amount of the protein encoded by the gene after transcription and translation of the gene. In some embodiments, expression of a polynucleotide, e.g., a gene or a DNA encoding a protein, is determined by the amount of a functional form of the protein encoded by the gene after transcription and translation of the gene. In some embodiments, expression of a gene is determined by the amount of the mRNA, or transcript, that is encoded by the gene after transcription the gene. In some embodiments, expression of a polynucleotide, e.g., an mRNA, is determined by the amount of the protein encoded by the mRNA after translation of the mRNA. In some embodiments, expression of a polynucleotide, WO 2024/238825 PCT/US2024/029746 e.g., a mRNA or coding RNA, is determined by the amount of a functional form of the protein encoded by the polypeptide after translation of the polynucleotide. [0133]The term "sequencing " as used herein, may comprise capillary sequencing, bisulfite- free sequencing, bisulfite sequencing, TET-assisted bisulfite (TAB) sequencing, ACE- sequencing, high-throughput sequencing, Maxam-Gilbert sequencing, massively parallel signature sequencing, Polony sequencing, 454 pyrosequencing, Sanger sequencing, Illumina sequencing, SOLID sequencing, Ion Torrent semiconductor sequencing, DNA nanoball sequencing, Heliscope single molecule sequencing, single molecule real time (SMRT) sequencing, nanopore sequencing, shot gun sequencing, RNA sequencing, or any combination thereof. [0134]The terms "equivalent " or "biological equivalent " are used interchangeably when referring to a particular molecule, or biological or cellular material, and means a molecule having minimal homology to another molecule while still maintaining a desired structure or functionality. [0135]The term "encode " as it is applied to polynucleotides refers to a polynucleotide which is said to "encode " another polynucleotide, a polypeptide, or an amino acid if, in its native state or when manipulated by methods well known to those skilled in the art, it can be used as polynucleotide synthesis template, e.g., transcribed into an RNA, reverse transcribed into a DNA or cDNA, and/or translated to produce an amino acid, or a polypeptide or fragment thereof. In some embodiments, a polynucleotide comprising three contiguous nucleotides form a codon that encodes a specific amino acid. In some embodiments, a polynucleotide comprises one or more codons that encode a polypeptide. In some embodiments, a polynucleotide comprising one or more codons comprises a mutation in a codon compared to a wild-type reference polynucleotide. In some embodiments, the mutation in the codon encodes an amino acid substitution in a polypeptide encoded by the polynucleotide as compared to a wild-type reference polypeptide. In some embodiments, a polynucleotide encodes another polynucleotide which contains one or more desired nucleotide edits to be installed in a target DNA. For example, in some embodiments, an editing template of a PEgRNA encodes a single stranded DNA that contains one or more nucleotide changes compared to the endogenous editing target DNA sequence in a target gene, e.g., a target B2M gene, wherein the single stranded DNA is otherwise identical to the editing target sequence. Accordingly, in some embodiments, the editing template "encodes " the one or more nucleotide changes. As used herein in the context of specific nucleotide changes encoded by WO 2024/238825 PCT/US2024/029746 PEgRNA editing templates, unless indicated otherwise, the specific nucleotide changes encoded refer to the changes installed into the coding strand (sense strand) of the target gene, although the editing target sequence may be on the sense strand or antisense strand of the target gene, e.g., the B2M gene. For example, when a PEgRNA mediates Prime Editing that results in insertion of a TAATAA sequence in the sense strand of the target gene, the editing template encodes a TAATAA insertion although the single strand DNA synthesized using the editing template sequence as a template may contain TTATTA in the corresponding position and has sequence identity to the antisense strand. [0136]The term "mutation " as used herein refers to a change and/or alteration in an amino acid sequence of a protein or nucleic acid sequence of a polynucleotide. Such changes and/or alterations may comprise the substitution, insertion, deletion and/or truncation of one or more amino acids, in the case of an amino acid sequence, and/or nucleotides, in the case of nucleic acid sequence, compared to a reference amino acid or a reference nucleic acid sequence. In some embodiments, the reference sequence is a wild-type sequence. In some embodiments, a mutation in a nucleic acid sequence of a polynucleotide encodes a mutation in the amino acid sequence of a polypeptide. In some embodiments, the mutation in the amino acid sequence of the polypeptide or the mutation in the nucleic acid sequence of the polynucleotide is a mutation associated with a disease state. [0137]The term "subject " and its grammatical equivalents as used herein may refer to a human or a non-human. A subject may be a mammal. A human subject may be male or female. A human subject may be of any age. A subject may be a human embryo. A human subject may be a newborn, an infant, a child, an adolescent, or an adult. A human subject may be in need of treatment for a genetic disease or disorder. A human subject may be in need of an immune cell immunotherapy (e.g., a T cell therapy). A human subject may be in need of a CAR-T cell therapy. Alternatively, the human subject may be a healthy donor. [0138]The terms "treatment " or "treating " and their grammatical equivalents may refer to the medical management of a subject with an intent to cure, ameliorate, or ameliorate a symptom of, a disease, condition, or disorder. Treatment may include active treatment, that is, treatment directed specifically toward the improvement of a disease, condition, or disorder. Treatment may include causal treatment, that is, treatment directed toward removal of the cause of the associated disease, condition, or disorder. In addition, this treatment may include palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, condition, or disorder. Treatment may include supportive treatment, WO 2024/238825 PCT/US2024/029746 that is, treatment employed to supplement another specific therapy directed toward the improvement of the disease, condition, or disorder. In some embodiments, a condition may be pathological. In some embodiments, a treatment may not completely cure or prevent a disease, condition, or disorder. In some embodiments, a treatment ameliorates, but does not completely cure or prevent a disease, condition, or disorder. In some embodiments, a subject may be treated for 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, years, 4 years, 5 years, 6 years, indefinitely, or life of the subject. [0139]The term "ameliorate " and its grammatical equivalents means to decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease. [0140]The terms "prevent " or "preventing " means delaying, forestalling, or avoiding the onset or development of a disease, condition, or disorder for a period of time. Prevent also means reducing risk of developing a disease, disorder, or condition. Prevention includes minimizing or partially or completely inhibiting the development of a disease, condition, or disorder. In some embodiments, a composition, e.g.,a pharmaceutical composition, prevents a disorder by delaying the onset of the disorder for 12 hours, 24 hours, 2 days, 3 days, 4 days, days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, indefinitely, or life of a subject. [0141]The term "effective amount " or "therapeutically effective amount " refers to a quantity of a composition, for example a prime editing composition comprising a construct, that can be sufficient to result in a desired activity upon introduction into a subject as disclosed herein. An effective amount of the prime editing compositions can be provided to the target gene or cell, whether the cell is ex vivo or in vivo. [0142]An effective amount can be the amount to induce, for example, at least about a 2-fold change (increase or decrease) or more in the amount of target nucleic acid modulation (e.g, expression of target gene to produce functional protein) observed relative to a negative control. An effective amount or dose can induce, for example, about 2-fold decrease, about 3- fold decrease, about 4-fold decrease, about 5-fold decrease, about 6-fold decrease, about 7- fold decrease, about 8-fold decrease, about 9-fold decrease, about 10-fold decrease, about 25- fold decrease, about 50-fold decrease, or about 100-fold decrease, in target gene modulation (e.g, expression of a target B2M gene to produce functional 0 chain of MHC Class 1. [0143]The amount of target gene modulation may be measured by any suitable method known in the art. In some embodiments, the "effective amount " or "therapeutically effective WO 2024/238825 PCT/US2024/029746 amount " is the amount of a composition that is required to ameliorate the symptoms of a disease relative to an untreated patient. In some embodiments, an effective amount is the amount of a composition sufficient to introduce an alteration in a gene of interest (e.g., a B2M gene) in a cell (e.g, a cell in vitro or in vivo). [0144]In some embodiments, an effective amount can be an amount to induce, when administered to a population of cells, a certain percentage of the population of cells to have an edit in a target gene (e.g., a B2M gene). For example, in some embodiments, an effective amount can be the amount to induce, when administered to or introduced to a population of cells, installation of one or more intended nucleotide edits in the B2M gene, in at least about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% of the population of cells. [0145]As it relates to the edited cells produced by the methods and composition comprising the edited cells disclosed herein; a "therapeutically effective amount " refers to a quantity of a composition comprising the edited cells that can be sufficient to result in a desired activity upon introduction into a subject (e.g., a human subject). [0146]The term "recombinase, " as used herein, refers to a site-specific enzyme that mediates the recombination of DNA between recombinase recognition sequences, which results in the excision, integration, inversion, or exchange (e.g., translocation) of DNA fragments between the recombinase recognition sequences. Recombinases can be classified into two distinct families: serine recombinases (e.g., resolvases and invertases) and tyrosine recombinases (e.g., integrases). Examples of serine recombinases include, without limitation, Si74, N067, Kp03, PaOl, Nm60, BcelNTa, BcytINTd, SscINTd, SacINTd, Hin, Gin, Tn3,13-six, CinH, ParA, y6, Bxbl, OC31, TP901, TGI, pBTl, R4, pRVI, pFCl, MR11, Al 18, U153, andgp29. Examples of tyrosine recombinases include, without limitation, Cre, FLP, R, Lambda, HK101, HK022, and pSAM2. Recombinases have numerous applications, including the creation of gene knockouts/knock-ins and gene therapy applications, as described in international publication no. WO2020191248A1, which is hereby incorporated by reference in its entirety. The recombinases provided herein are not meant to be exclusive examples of recombinases that can be used in embodiments of the invention. The methods and compositions of the invention can be expanded by mining databases for new orthogonal recombinases or designing synthetic recombinases with defined DNA specificities.

WO 2024/238825 PCT/US2024/029746 id="p-147"

[0147]In some embodiments, the catalytic domains of a recombinase are fused to a programmable DNA binding domain of a prime editor, such as a RNA-programmable nuclease (e.g., dCas9, Cas9 nickase, or a fragment thereof), such that the recombinase domain does not comprise a nucleic acid binding domain or is unable to bind to a target nucleic acid (e.g., the recombinase domain is engineered such that it does not have specific DNA binding activity). For example, serine recombinases of the resolvase-invertase group, e.g., Tn3 and 76 resolvases and the Hin and Gin invertases, have modular structures with autonomous catalytic and DNA-binding domains. The catalytic domains of these recombinases are thus amenable to being combined with, e.g., fused to or connected to a prime editor or a component thereof. Additionally, many other natural serine recombinases having an N-terminal catalytic domain and a C-terminal DNA binding domain are known (e.g., phiC31 integrase, TnpX transposase, IS607 transposase), and their catalytic domains can be combined with, e.g., fused to or complexed with a prime editor or a component thereof for programmable recombination. [0148]Other examples of recombinases that are usefill in the methods and compositions described herein are known to those of skill in the art, and any new recombinase that is discovered or generated is expected to be able to be used in the different embodiments of the invention. [0149]The term "recombinase recognition sequence", or equivalently as "RRS" or "recombinase target sequence" or "recombinase site," as used herein, refers to a nucleotide sequence target recognized by a recombinase and which undergoes strand exchange with another DNA molecule having a a corresponding RRS (e.g., an RRS recognized by the same recombinase) that results in excision, integration, inversion, or exchange of DNA fragments between the recombinase recognition sequences. In various embodiments, a prime editing composition may install one or more recombinase sites in a target sequence, or in more than one target sequence. When more than one recombinase site is installed by prime editing, the recombinase sites can be installed at adjacent target sites or non-adjacent target sites (e.g., separate chromosomes). In various embodiments, single installed recombinase sites can be used as "landing sites" for a recombinase- mediated reaction between the genomic recombinase site and a second recombinase site within an exogenously supplied nucleic acid molecule, e.g., a plasmid or a DNA vector. This enables the targeted integration of a desired nucleic acid molecule.

WO 2024/238825 PCT/US2024/029746 id="p-150"

[0150]The term "recombine, " or "recombination, " in the context of a nucleic acid modification (e.g., a genomic modification), is used to refer to the process by which two or more nucleic acid molecules, or two or more regions of a single nucleic acid molecule, are modified by the action of a recombinase protein. Recombination can result in, inter alia, the insertion, inversion, excision, or translocation of nucleic acids, e.g., in or between one or more nucleic acid molecules. Prime Editing [0151]The term "prime editing " refers to programmable editing of a target DNA using a prime editor complexed with a PEgRNA to incorporate an intended nucleotide edit (also referred to herein as a nucleotide change) into the target DNA through target-primed DNA synthesis. A target gene of prime editing may comprise a double stranded DNA molecule having two complementary strands: a first strand that may be referred to as a "target strand " or a "non-edit strand ", and a second strand that may be referred to as a "non-target strand, " or an "edit strand. " In some embodiments, in a prime editing guide RNA (PEgRNA), a spacer sequence is complementary or substantially complementary to a specific sequence on the target strand, which may be referred to as a "search target sequence ". In some embodiments, the spacer sequence anneals with the target strand at the search target sequence. The target strand may also be referred to as the "non-Protospacer Adjacent Motif (non-PAM strand). " In some embodiments, the non-target strand may also be referred to as the "PAM strand ". In some embodiments, the PAM strand comprises a protospacer sequence and optionally a protospacer adjacent motif (PAM) sequence. In prime editing using a Cas-protein-based prime editor, a PAM sequence refers to a short DNA sequence immediately adjacent to the protospacer sequence on the PAM strand of the target gene. A PAM sequence may be specifically recognized by a programmable DNA binding protein, e.g., a Cas nickase or a Cas nuclease. In some embodiments, a specific PAM is characteristic of a specific programmable DNA binding protein, e.g., a Cas nickase or a Cas nuclease A protospacer sequence refers to a specific sequence in the PAM strand of the target gene that is complementary to the search target sequence. In a PEgRNA, a spacer sequence may have a substantially identical sequence as the protospacer sequence on the edit strand of a target gene, except that the spacer sequence may comprise Uracil (U) and the protospacer sequence may comprise Thymine (T). [0152]In some embodiments, the double stranded target DNA comprises a nick site on the PAM strand (or non-target strand). As used herein, a "nick site " refers to a specific position WO 2024/238825 PCT/US2024/029746 in between two nucleotides or two base pairs of the double stranded target DNA. In some embodiments, the position of a nick site is determined relative to the position of a specific PAM sequence. In some embodiments, the nick site is the particular position where a nick will occur when the double stranded target DNA is contacted with a nickase, for example, a Cas nickase, that recognizes a specific PAM sequence. In some embodiments, the nick site is upstream of a specific PAM sequence on the PAM strand of the double stranded target DNA. In some embodiments, the nick site is downstream of a specific PAM sequence on the PAM strand of the double stranded target DNA. In some embodiments, the nick site is upstream of a PAM sequence recognized by a Cas9 nickase, wherein the Cas9 nickase comprises a nuclease active RuvC domain and a nuclease inactive HNH domain. In some embodiments, the nick site is 3 nucleotides upstream of the PAM sequence, and the PAM sequence is recognized by a Streptococcus pyogenes Cas9 nickase, a P. lavamentivorans Cas9 nickase, a C. diphtheriae Cas9 nickase, a N. cinerea Cas9, a S. aureus Cas9, or a N. lari Cas9 nickase. In some embodiments, the nick site is 3 base pairs upstream of the PAM sequence, and the PAM sequence is recognized by a Cas9 nickase, wherein the Cas9 nickase that comprises a nuclease active RuvC domain and a nuclease inactive HNH domain. In some embodiments, the nick site is 2 nucleotides upstream of the PAM sequence, and the PAM sequence is recognized by a S. thermophilus Cas9 nickase that comprises a nuclease active RuvC domain and a nuclease inactive HNH domain. [0153]A "primer binding site " (also referred to as PBS or primer binding site sequence) is a single-stranded portion of the PEgRNA that comprises a region of complementarity to the PAM strand (i.e. the non-target strand or the edit strand). The PBS is complementary or substantially complementary to a sequence on the PAM strand of the double stranded target DNA that is immediately upstream of the nick site. In some embodiments, in the process of prime editing, the PEgRNA complexes with and directs a prime editor to bind the search target sequence on the target strand of the double stranded target DNA, and generates a nick at the nick site on the non-target strand of the double stranded target DNA. In some embodiments, the PBS is complementary to or substantially complementary to, and can anneal to, a free 3' end on the non-target strand of the double stranded target DNA at the nick site. In some embodiments, the PBS annealed to the free 3' end on the non-target strand can initiate target-primed DNA synthesis. [0154]An "editing template " of a PEgRNA is a single-stranded portion of the PEgRNA that is 5' of the PBS and which encodes a single strand of DNA. The editing template may WO 2024/238825 PCT/US2024/029746 comprise a region of complementarity to the PAM strand (i.e., the non-target strand or the edit strand), and comprises one or more intended nucleotide edits compared to the endogenous sequence of the double stranded target DNA. In some embodiments, the editing template and the PBS are immediately adjacent to each other. Accordingly, in some embodiments, a PEgRNA in prime editing comprises a single-stranded portion that comprises the PBS and the editing template immediately adjacent to each other. In some embodiments, the single stranded portion of the PEgRNA comprising both the PBS and the editing template is complementary or substantially complementary to an endogenous sequence on the PAM strand (i.e., the non-target strand or the edit strand) of the double stranded target DNA except for one or more non-complementary nucleotides at the intended nucleotide edit position(s). As used herein, regardless of relative 5'-3' positioning in other context, the relative positions as between the PBS and the editing template, and the relative positions as among elements of a PEgRNA, are determined by the 5' to 3' order of the PEgRNA as a single molecule regardless of the position of sequences in the double stranded target DNA that may have complementarity or identity to elements of the PEgRNA. In some embodiments, the editing template is complementary or substantially complementary to a sequence on the PAM strand that is immediately downstream of the nick site, except for one or more nucleotide changes (e.g., one or more non-complementary nucleotides) at the intended nucleotide edit positions. The endogenous, e.g., genomic, sequence that is complementary or substantially complementary to the editing template, except for the one or more non-complementary nucleotides at the position corresponding to the intended nucleotide edit, may be referred to as an "editing target sequence ". In some embodiments, the editing template comprises identity or substantial identity to a sequence on the target strand that is complementary to, or having the same position in the genome as, the editing target sequence, except for one or more nucleotide changes (e.g., one or more insertions, deletions, or substitutions) at the intended nucleotide edit positions. [0155]In some embodiments, the editing target sequence is in a non-coding region of the target B2M gene. In some embodiments, the editing target sequence is in a coding region of the target B2M gene. In some embodiments, the editing target sequence is in an exon of the target B2M gene.. In some embodiments, the editing target sequence is in an exon, an intron, an exon-intron injunction, or a regulatory element of the B2M gene. In some embodiments, the editing target sequence is in an open reading frame of the B2M gene. In some embodiments, the editing target sequence is in an untranslated region of the B2M gene, for WO 2024/238825 PCT/US2024/029746 example, a 3'-UTR or a 5'- UTR. In some embodiments, the editing target sequence is in a regulatory element of the B2Mgene. In some embodiments, the editing target sequence is in a promoter, an enhancer, an operator, a silencer, an insulator, a terminator, a transcription initiation sequence, a translation initiation sequence (e.g., a Kozak sequence), or any combination thereof of the B2M gene. In some embodiments, the editing target sequence is in a splice acceptor-splice donor (SA-SD) site in a B2M gene. [0156]In some embodiments, the editing template encodes a single stranded DNA, wherein the single stranded DNA comprises identity or substantial identity to the editing target sequence except for one or more nucleotide changes (e.g., one or more insertions, deletions, or substitutions) at the positions of the one or more intended nucleotide edits. In some embodiments, the methods disclosed herein result in incorporation of one or more nucleotide changes in a B2Mgene. In some embodiments, the methods disclosed herein result in introducing a mutation in a B2M gene. In some embodiments, the incorporation of the one or more nucleotide changes in the target B2Mgene introduces a mutation (e.g., a missense mutation, a nonsense mutation, a frame-shift mutation, a null mutation, a mutation that generates a premature stop codon, or a combination thereof) in the target B2M gene. In some embodiments, the incorporation of the one or more nucleotide changes introduces a frame shift mutation and/or generate one or more premature stop codons (e.g., at least 1, 2, 3, 4, 5, or more premature stop codons) in the target gene (e.g., a B2M gene). In some embodiments, the incorporation of the one or more nucleotide changes generates at least 2 premature stop codons in the target gene. In some embodiments, the incorporation of one or more nucleotide changes generates at least 2, 3,4, 5, or more consecutive premature stop codons in the target gene (e.g., B2M gene). In some embodiments, a premature stop codon is generated in exon 1, exon 2, exon 3, exon 4 or a combination thereof in a B2M gene. A "premature stop codon" is a mutation (e.g., a nonsense mutation or insertion) within a sequence of a target gene (e.g., B2M gene) that generates a stop codon at a position not normally found in the wild-type gene (e.g., wild type B2M gene). A premature stop codon may result in a truncated and/or non- functional protein compared to the hill-length protein encoded by the corresponding wildtype target gene. As used herein, a "frame shift mutation " means a mutation wherein the reading frame of codons of the coding region is altered due to the one or more nucleotide changes. For example, in some embodiments, a frameshift mutation is a insertion of 3x+l or 3x+nucleotides in the coding region, wherein x is an integer equal to or greater than 0. In some embodiments, a frameshift mutation is a deletion of 3x+l or 3x+2 nucleotides in the coding WO 2024/238825 PCT/US2024/029746 region, wherein x is an integer equal to or greater than 0. In some embodiments, a frame shift mutation results in a premature stop codon in the target gene. In some embodiments, a frameshift mutation results in a non-functional protein encoded by the target gene. As used herein, a "missense mutation " refers to a change in the type of an amino acid in the protein expresses by a target gene due to the change or substitution of a base in the corresponding target gene (e.g., B2M gene). As used herein, a "nonsense mutation " refers to a mutation wherein a sense codon that encodes an amino acid is changed to a stop codon. As used herein, a "null mutation " refers to a mutation in a target gene (e.g., B2M gene) that results in complete loss of functional protein expression from the mutated target gene. [0157]In some embodiments, a mutation is introduced in a non-coding region of the target B2M gene. In some embodiments, a mutation is introduced in a coding region of the target B2M gene. In some embodiments, a mutation is introduced in an exon of the target B2M gene. In some embodiments, a mutation is introduced in exon l, exon 2, exon 3, exon 4, or any combination thereof of the target B2M gene. In some embodiments, a mutation is introduced in an exon, an intron, an exon-intron injunction, or a regulatory element of the B2M gene. In some embodiments, a mutation is introduced in an open reading frame of the B2M gene. In some embodiments, a mutation is introduced in an untranslated region of the B2M gene, for example, a 3'-UTR or a 5'- UTR. In some embodiments, a mutation is introduced in a regulatory element of the B2M gene. In some embodiments, a mutation is introduced in a promoter, an enhancer, an operator, a silencer, an insulator, a terminator, a transcription initiation sequence, a translation initiation sequence (e.g., a Kozak sequence), or any combination thereof of the B2M gene. In some embodiments, a mutation is introduced in splice acceptor-splice donor (SA-SD) site in a B2M gene. In some embodiments, a mutation is introduced in a B2M gene, wherein the mutation generates a splice acceptor-splice donor (SA-SD) site in a B2M gene. In some embodiments, a mutation is introduced in a splice site in a B2M gene. In some embodiments, a mutation is introduced in a splice site, disrupts a splice site in a B2M gene. In some embodiments, the methods disclosed herein generate any one of the following edits in a B2M gene to generate a STOP codon: CAG to TAG; CAA to TAA; CGA to TGA; TGG to TGA; TGG to TAG; or TGG to TAA. In some embodiments, the one or more intended nucleotide edits may be introduced at a 3’-UTR, for example, in a poly adenylation (poly-A) site. In some embodiments, one or more premature, in frame stop codons (e.g., two stop codons) are inserted into the B2M gene.

WO 2024/238825 PCT/US2024/029746 id="p-158"

[0158]In some embodiments, the editing template may encode the wild-type or non-disease associated gene sequence (or its complement if the edit strand is the antisense strand of a gene). In some embodiments, the editing template may encode the wild-type or non-disease associated protein, but contain one or more synonymous mutations relative to the wild-type or non-disease associated protein coding region. Such synonymous mutations may include, for example, mutations that decrease the ability of a PEgRNA to rebind to the same target sequence once the desired edit is installed in the genome (e.g., synonymous mutations that silence the endogenous PAM sequence or that edit the endogenous protospacer). As used herein, a synonymous edit, synonymous change, or synonymous mutation in a gene refers to a nucleotide change that does not alter the protein sequence or the mRNA sequence encoded by the gene. A non-synonymous edit, non-synonymous change, or non-synonymous mutation in a gene refers to a nucleotide change that results in alteration in the protein sequence or the mRNA (e.g. by altering splice donor or acceptor sequence) sequence encoded by the gene. [0159]In some embodiments, a PEgRNA complexes with and directs a prime editor to bind to the search target sequence of the target gene. In some embodiments, the bound prime editor generates a nick on the edit strand (PAM strand) of the target gene at the nick site. In some embodiments, a primer binding site (PBS) of the PEgRNA anneals with a free 3' end formed at the nick site, and the prime editor initiates DNA synthesis from the nick site, using the free 3' end as a primer. Subsequently, a single-stranded DNA encoded by the editing template of the PEgRNA is synthesized. In some embodiments, the newly synthesized single- stranded DNA comprises one or more intended nucleotide edits (i.e., one or more nucleotide changes) compared to the endogenous target gene sequence. In some embodiments, incorporation of the one or more intended nucleotide edits in the target B2M gene introduces a mutation (e.g., a missense mutation, a nonsense mutation, a frame-shift mutation, a null mutation, a mutation that generates a premature stop codon, or a combination thereof) in the target B2M gene. In some embodiments, the one or more intended nucleotide edits introduce a frame shift mutation and/or generates one or more premature stop codons (e.g., at least 1, 2, 3, 4, 5, or more premature stop codons) in the target gene (e.g., B2M gene). In some embodiments, the one or more intended nucleotide edits generates at least 2 premature stop codons in the target gene. In some embodiments, the one or more intended nucleotide edits generates at least 2, 3,4, 5, or more consecutive premature stop codons in the target gene (e.g., a B2M gene). In some embodiments, the one or more intended nucleotide edits comprise the insertion of one or more premature, in frame stop codons (e.g., two stop codons) WO 2024/238825 PCT/US2024/029746 into the B2M gene. Accordingly, in some embodiments, the editing template of a PEgRNA is complementary to a sequence in the edit strand except for one or more mismatches at the intended nucleotide edit positions in the editing template. The endogenous, e.g., genomic, sequence that is partially complementary to the editing template may be referred to as an "editing target sequence ". Accordingly, in some embodiments, the newly synthesized single stranded DNA has identity or substantial identity to a sequence in the editing target sequence, except for one or more insertions, deletions, or substitutions at the intended nucleotide edit positions. In some embodiments, the editing template comprises at least 4 contiguous nucleotides of complementarity with the edit strand wherein the at least 4 contiguous nucleotides are located upstream of the 5’ most edit in the editing template. In some embodiments, the editing template comprises 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 contiguous nucleotides of complementarity with the edit strand wherein the at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 contiguous nucleotides are located upstream of the 5’ most edit in the editing template. [0160]In some embodiments, the newly synthesized single-stranded DNA equilibrates with the editing target on the edit strand of the target gene for pairing with the target strand of the target gene. In some embodiments, the editing target sequence of the target gene is excised by a flap endonuclease (FEN), for example, FENI. In some embodiments, the FEN is an endogenous FEN, for example, in a cell comprising the target gene. In some embodiments, the FEN is provided as part of the prime editor, either linked to other components of the prime editor or provided in trans. In some embodiments, the newly synthesized single stranded DNA, which comprises the intended nucleotide edit, replaces the endogenous single stranded editing target sequence on the edit strand of the target gene. In some embodiments, the newly synthesized single stranded DNA and the endogenous DNA on the target strand form a heteroduplex DNA structure at the region corresponding to the editing target sequence of the target gene. In some embodiments, the newly synthesized single-stranded DNA comprising the nucleotide edit is paired in the heteroduplex with the target strand of the target DNA that does not comprise the nucleotide edit, thereby creating a mismatch between the two otherwise complementary strands. In some embodiments, the mismatch is recognized by DNA repair machinery, e.g., an endogenous DNA repair machinery. In some embodiments, through DNA repair, the intended nucleotide edit is incorporated into the target gene.

WO 2024/238825 PCT/US2024/029746 id="p-161"

[0161]In some embodiments, prime editing may comprise programmable editing of a target DNA using one or more prime editors each complexed with a PEgRNA ("dual prime editing "). Dual prime editing refers to programmable editing of a double-stranded target DNA using two or more PEgRNAs, each of which is complexed with a prime editor for incorporating one or more intended nucleotide edits into the double-stranded target DNA. In some embodiments, dual prime editing incorporates one or more intended nucleotide edits into a double-stranded target DNA through excision of an endogenous DNA segment and/or replacement of the endogenous DNA segment with newly synthesized DNA via target- primed DNA synthesis. In some embodiments, dual prime editing may be used to edit a target DNA that is or is part of a target gene. [0162]In some embodiments, dual prime editing involves using two different PEgRNAs each complexed with a prime editor, wherein each of the two PEgRNAs comprises a spacer complementary or substantially complementary to a separate search target sequence. In some embodiments, each of the two PEgRNAs anneals with a separate search target sequence through its spacer. Accordingly, references to a "PAM strand ", a "non-PAM strand ", a "target strand ’, a "non-target strand ", an "edit strand " or a "non-edit strand " are relative in the context of a specific PEgRNA, e.g., one of the two PEgRNAs in dual prime editing. [0163]In some embodiments, dual prime editing involves two PEgRNAs, different from one another, each complexed with a prime editor. In some embodiments, each of the two PEgRNAs comprises a region of complementarity to a distinct search target sequence of the target DNA, wherein the two distinct search target sequences are on the two complementary strands of the target DNA. In some embodiments, the two PEgRNAs each can direct a prime editor to initiate the prime editing process on the two complementary strands of the target DNA. [0164]In some embodiments, dual prime editing involves two PEgRNAs each complexed with a prime editor. In some embodiments, a first PEgRNA comprises a first spacer complementary to a first search target sequence on a first strand of a double-stranded target DNA, e.g., a double-stranded target gene. In the context of the first PEgRNA, the first strand of the double-stranded target DNA may be referred to as a first target strand, and the complementary strand referred to as the first PAM strand. [0165]In some embodiments, a second PEgRNA comprises a second spacer complementary to a second search target sequence on a second strand of the double-stranded target DNA. In some embodiments, the first strand and the second strand of the double-stranded target DNA, WO 2024/238825 PCT/US2024/029746 e.g., a double-stranded target gene, are complementary to each other. Accordingly, in some embodiments, the second PEgRNA and the first PEgRNA bind opposite strands of the double-stranded target DNA. In the context of the second PEgRNA, the second strand of the double-stranded target DNA may be referred to as a second target strand, and the complementary strand referred to as the second PAM strand. In some embodiments, the first target strand is the same strand as the second PAM strand of the double-stranded target DNA. In some embodiments, the second target strand is the same strand as the first PAM strand of the double-stranded target DNA. [0166]In some embodiments, the first PEgRNA anneals with the first target strand of the double-stranded target DNA, through the first spacer of the first PEgRNA. In some embodiments, the first PEgRNA complexes with and directs a first prime editor to bind the double-stranded target DNA at the position corresponding to the first search target sequence. In some embodiments, the second PEgRNA anneals with the second search target sequence on the second target strand of the double-stranded target DNA, through a second spacer of the second PEgRNA. In some embodiments, the second PEgRNA complexes with and directs a second prime editor to bind the double-stranded target DNA at the position corresponding to the second search target sequence. In some embodiments, the first prime editor and the second prime editor are the same. In some embodiments, the first prime editor and the second prime editor are different. [0167]In some embodiments, the bound first prime editor generates a first nick on the first PAM strand of the double-stranded target DNA. In some embodiments, a first PEgRNA comprises a first primer binding site (PBS), also referred to herein as "primer binding site sequence ", that is complementary to the sequence of the first PAM strand of the double- stranded target DNA that is immediately upstream of the first nick site, and can anneal with the sequence of the first strand at a free 3’ end formed at the first nick site. In some embodiments, a first PEgRNA comprises a first primer binding site (PBS) that anneals to a free 3’ end formed at the first nick site and the first prime editor initiates DNA synthesis from the nick site, using the free 3’ end as a primer. In some embodiments, the first prime editor generates a first newly synthesized single-stranded DNA encoded by a first editing template of the first PEgRNA. [0168]In some embodiments, the bound second prime editor generates a second nick on the second PAM strand of the double-stranded target DNA. In some embodiments, the double- stranded target DNA, e.g., a target gene, comprises a double-stranded DNA sequence WO 2024/238825 PCT/US2024/029746 between the first nick generated by the first prime editor on the second target strand (also referred to as the first PAM strand) and the second nick generated by the second prime editor on the first target strand (also referred to as the second PAM strand), which may be referred to as an inter-nick duplex (IND). In some embodiments, the two strands of an IND are completely complementary to each other. In some embodiments, the two strands of an IND are partially complementary to each other. In some embodiments, the IND is subsequently excised from the double-stranded target DNA, e.g., the target gene. [0169]In some embodiments, a first PEgRNA comprises a first primer binding site (PBS) that is complementary to a free 3’ end of the second strand of the double-stranded target DNA formed at the first nick site. In some embodiments, the first PBS anneals with the free 3’ end formed at the first nick site, and the first prime editor initiates DNA synthesis from the first nick site, using the free 3’ end at the first nick site as a primer. In some embodiments, the first prime editor synthesizes a first new single-stranded DNA encoded by the first editing template of the first PEgRNA. In some embodiments, the second PEgRNA comprises a second PBS that is complementary to a free 3’ end of the first strand of the double-stranded target DNA formed at the second nick site. In some embodiments, the second PBS anneals with the free 3’ end formed at the second nick site, and the second prime editor initiates DNA synthesis from the nick site, using the free 3’ end at the second nick site as a primer. In some embodiments, the second prime editor synthesizes a second newly synthesized single- stranded DNA encoded by a second editing template of the second PEgRNA. [0170]In some embodiments, through DNA repair, the sequence of the first newly synthesized single-stranded DNA encoded by the first editing template and/or the sequence of the second newly synthesized single-stranded DNA encoded by the second editing template is incorporated into the double-stranded target DNA, e.g., a target gene, thereby incorporating one or more intended nucleotide edits in the double-stranded target DNA, e.g., the target gene. [0171]In some embodiments, the first editing template and the second editing template comprise a region of complementarity or substantial complementarity to each other. Accordingly, in some embodiments, the first newly synthesized single-stranded DNA and the second newly synthesized single-stranded DNA have a region of complementarity to each other. The complementary region between the first newly synthesized single-stranded DNA and the second newly synthesized single-stranded DNA may be referred to as an overlap duplex (OD). As exemplified in Fig.4A, in some embodiments, through DNA repair, the OD WO 2024/238825 PCT/US2024/029746 is incorporated in the double-stranded target DNA, e.g., the target gene, thereby incorporating one or more intended nucleotide edits encoded by the first editing template and the second editing template into the double-stranded target DNA, e.g., the target gene. In some embodiments, the OD replaces all or a portion of the IND, thereby incorporating one or more intended nucleotide edits in the double-stranded target DNA, e.g., the target gene. In some embodiments, the IND is excised or degraded, and the OD is incorporated at the place of the IND excision, followed by ligation of the nicks on both strands of the double-stranded target DNA, e.g., the target gene, thereby incorporating the one or more intended nucleotide edits in the double-stranded target DNA. [0172]In some embodiments, the first newly synthesized single-stranded DNA encoded by the first editing template further comprises a region that does not have complementarity with the second newly synthesized single-stranded DNA encoded by the second editing template (see exemplary schematic in FIG. 4B). In some embodiments, the second newly synthesized single-stranded DNA encoded by the second editing template further comprises a region that does not have complementarity with the first newly synthesized single-stranded DNA encoded by the first editing template. Accordingly, in some embodiments, the first newly synthesized single-stranded DNA encoded by the first editing template and the second newly synthesized single-stranded DNA encoded by the second editing template can anneal to each other through the partially complementary sequences to form an OD that is linked to a 5’ overhang and/or a 3’overhang. In some embodiments, the IND is removed, the OD, along with the 5’ overhang and/or the 3’ overhang, is incorporated at the place of the IND excision in the double-stranded target DNA, e.g., the target gene. Through DNA repair, the gaps corresponding to the positions of the 5’ overhang and/or the 3’ overhangs are filled and ligated, thereby incorporating the one or more intended nucleotide edits in the double- stranded target DNA, e.g., the target gene. [0173]Accordingly, in some embodiments, the IND is replaced by the sequence of (A+C), (B+C), or (A+B+C), wherein A is the region, and its complementary strand, of the first newly synthesized single-stranded DNA that is not complementary to the second newly synthesized single-stranded DNA, wherein B is the region, and its complementary strand, of the second newly synthesized single-stranded DNA that is not complementary to the first newly synthesized single-stranded DNA, and wherein C is the OD. The double-stranded sequence of (A+C), (B+C), or (A+B+C) that replaces the IND may be referred to as the "replacement WO 2024/238825 PCT/US2024/029746 duplex (RD)". In some embodiments, through DNA repair, the RD replaces the IND of a target DNA. [0174]In some embodiments, the RD or the OD may comprise a recombinase recognition sequences (RRSs), e.g., a RRS recognized by a Bxbl recombinase, a Cre recombinase, a PaOl recombinase, a Si74 recoimbinase, a N067 recombinase, a Kp03 recombinase, a Nm60 recombinase, a BcelNTa recombinase, a NcytINTd recombinase, a SscINTd recombinase, a SacINTd recombinase, or a recombinase recognition site corresponding to any recombinase disclosed herein. In some embodiments, the RD or the OD may comprise one, two, or more recombinase recognition sites corresponding to a recombinase. [0175]Replacement of the IND by the RD or the OD comprising one or more RRS sequences with dual prime editing may result in insertion of the one or more RRSs into the target gene. Depending on the number and orientation of the RRSs, they can be used as landing sites for a recombinase-mediated reaction between the RRSs. For example, a single RSS inserted into a target gene, e.g., a TRAC gene, can be used for integration of an exogenous DNA donor sequence via recombination between the inserted RRS and a second RRS within an exogenous supplied DNA donor. If two RRS sites are inserted in adjacent regions of DNA, depending on the orientation of the RRS sites, these can be used for recombinase-mediated excision or inversion of the intervening sequence, or for recombinase-mediated cassette exchange with exogenous DNA for cargo integration. Prime Editor [0176]The term "prime editor (PE)" refers to the polypeptide or polypeptide components involved in prime editing, or any polynucleotide(s) encoding the polypeptide or polypeptide components. In various embodiments, a prime editor includes a polypeptide domain having DNA binding activity and a polypeptide domain having DNA polymerase activity. In some embodiments, the prime editor further comprises a polypeptide domain having nuclease activity. In some embodiments, the polypeptide domain having DNA binding activity comprises a nuclease domain or nuclease activity. In some embodiments, the polypeptide domain having nuclease activity comprises a nickase, or a fully active nuclease. As used herein, the term "nickase " refers to a nuclease capable of cleaving only one strand of a double-stranded DNA target. In some embodiments, the prime editor comprises a polypeptide domain that is an inactive nuclease. In some embodiments, the polypeptide domain having programmable DNA binding activity comprises a nucleic acid guided DNA binding domain, for example, a CRISPR-Cas protein, for example, a Cas9 nickase, a Cpfl nickase, or another WO 2024/238825 PCT/US2024/029746 CRISPR-Cas nuclease. In some embodiments, the polypeptide domain having DNA polymerase activity comprises a template-dependent DNA polymerase, for example, a DNA- dependent DNA polymerase or an RNA-dependent DNA polymerase. In some embodiments, the DNA polymerase is a reverse transcriptase. In some embodiments, the prime editor comprises additional polypeptides involved in prime editing, for example, a polypeptide domain having 5' endonuclease activity, e.g., a 5' endogenous DNA flap endonucleases (e.g, FENI), for helping to drive the prime editing process towards the edited product formation. In some embodiments, the prime editor further comprises an RNA-protein recruitment polypeptide, for example, a MS2 coat protein. [0177]A prime editor may be engineered. In some embodiments, the polypeptide components of a prime editor do not naturally occur in the same organism or cellular environment. In some embodiments, the polypeptide components of a prime editor may be of different origins or from different organisms. In some embodiments, a prime editor comprises a DNA binding domain and a DNA polymerase domain that are derived from different species. In some embodiments, a prime editor comprises a Cas polypeptide (DNA binding domain) and a reverse transcriptase polypeptide (DNA polymerase) that are derived from different species. For example, a prime editor may comprise a 8. pyogenes Cas9 polypeptide and a Moloney murine leukemia virus (M-MLV) reverse transcriptase polypeptide. [0178]In some embodiments, polypeptide domains of a prime editor may be fused or linked by a peptide linker to form a fusion protein. In other embodiments, a prime editor comprises one or more polypeptide domains provided in trans as separate proteins, which are capable of being associated to each other through non-peptide linkages or through aptamers or recruitment sequences. For example, a prime editor may comprise a DNA binding domain and a reverse transcriptase domain associated with each other by an RNA-protein recruitment aptamer, e.g., a MS2 aptamer, which may be linked to a PEgRNA. Prime editor polypeptide components may be encoded by one or more polynucleotides in whole or in part. In some embodiments, a single polynucleotide, construct, or vector encodes the prime editor fusion protein. In some embodiments, multiple polynucleotides, constructs, or vectors each encode a polypeptide domain or portion of a domain of a prime editor, or a portion of a prime editor fusion protein. For example, a prime editor fusion protein may comprise an N-terminal portion fused to an intein-N and a C-terminal portion fused to an intein-C, each of which is individually encoded by an AAV vector. Prime Editor Nucleotide Polymerase Domain WO 2024/238825 PCT/US2024/029746 id="p-179"

[0179]In some embodiments, a prime editor comprises a nucleotide polymerase domain, e.g., a DNA polymerase domain. The DNA polymerase domain may be a wild-type DNA polymerase domain, a full-length DNA polymerase protein domain, or may be a functional mutant, a functional variant, or a functional fragment thereof. In some embodiments, the polymerase domain is a template dependent polymerase domain. For example, the DNA polymerase may rely on a template polynucleotide strand, e.g., the editing template sequence, for new strand DNA synthesis. In some embodiments, the prime editor comprises a DNA- dependent DNA polymerase. For example, a prime editor having a DNA-dependent DNA polymerase can synthesize a new single stranded DNA using a PEgRNA editing template that comprises a DNA sequence as a template. In such cases, the PEgRNA is a chimeric or hybrid PEgRNA, and comprising an extension arm comprising a DNA strand. The chimeric or hybrid PEgRNA may comprise an RNA portion (including the spacer and the gRNA core) and a DNA portion (the extension arm comprising the editing template that includes a strand of DNA). [0180]In some embodiments, the DNA polymerases can be wild type polymerases from eukaryotic, prokaryotic, archaeal, or viral organisms, and/or the polymerases may be modified by genetic engineering, mutagenesis, or directed evolution-based processes. The polymerases can be a T7 DNA polymerase, T5 DNA polymerase, T4 DNA polymerase, Klenow fragment DNA polymerase, DNA polymerase III and the like. The polymerases can be thermostable, and can include Taq, Tne, Tma, Pfu, Tfl, Tth, Stoffel fragment, VENT® and DEEPVENT® DNA polymerases, KOD, Tgo, JDF3, and mutants, variants and derivatives thereof. [0181]In some embodiments, the DNA polymerase is a bacteriophage polymerase, for example, a T4, T7, or phi29 DNA polymerase. In some embodiments, the DNA polymerase is an archaeal polymerase, for example, pol I type archaeal polymerase or a pol II type archaeal polymerase. In some embodiments, the DNA polymerase comprises a thermostable archaeal DNA polymerase. In some embodiments, the DNA polymerase comprises a eubacterial DNA polymerase, for example, Pol I, Pol II, or Pol III polymerase. In some embodiments, the DNA polymerase is a Pol I family DNA polymerase. In some embodiments, the DNA polymerase is a E.coli Pol I DNA polymerase. In some embodiments, the DNA polymerase is a Pol II family DNA polymerase. In some embodiments, the DNA polymerase is a Pyrococcus furiosus (Pfu) Pol II DNA polymerase. In some embodiments, the DNA Polymerase is a Pol IV family DNA polymerase. In some embodiments, the DNA polymerase is a E.coli Pol IV DNA polymerase.

WO 2024/238825 PCT/US2024/029746 id="p-182"

[0182]In some embodiments, the DNA polymerase comprises a eukaryotic DNA polymerase. In some embodiments, the DNA polymerase is a Pol-beta DNA polymerase, a Pol-lambda DNA polymerase, a Pol-sigma DNA polymerase, or a Pol-mu DNA polymerase. In some embodiments, the DNA polymerase is a Pol-alpha DNA polymerase. In some embodiments, the DNA polymerase is a POLAl DNA polymerase. In some embodiments, the DNA polymerase is a POLA2 DNA polymerase. In some embodiments, the DNA polymerase is a Pol-delta DNA polymerase. In some embodiments, the DNA polymerase is a POLDI DNA polymerase. In some embodiments, the DNA polymerase is a POLD2 DNA polymerase. In some embodiments, the DNA polymerase is a human POLDI DNA polymerase. In some embodiments, the DNA polymerase is a human POLD2 DNA polymerase. In some embodiments, the DNA polymerase is a POLD3 DNA polymerase. In some embodiments, the DNA polymerase is a POLD4 DNA polymerase. In some embodiments, the DNA polymerase is a Pol-epsilon DNA polymerase. In some embodiments, the DNA polymerase is a POLEI DNA polymerase. In some embodiments, the DNA polymerase is a POLE2 DNA polymerase. In some embodiments, the DNA polymerase is a POLE3 DNA polymerase. In some embodiments, the DNA polymerase is a Pol-eta (POLH) DNA polymerase. In some embodiments, the DNA polymerase is a Pol-iota (POLI) DNA polymerase. In some embodiments, the DNA polymerase is a Pol-kappa (POLK) DNA polymerase. In some embodiments, the DNA polymerase is a Revl DNA polymerase. In some embodiments, the DNA polymerase is a human Revl DNA polymerase. In some embodiments, the DNA polymerase is a viral DNA-dependent DNA polymerase. In some embodiments, the DNA polymerase is a B family DNA polymerases. In some embodiments, the DNA polymerase is a herpes simplex virus (HSV) UL30 DNA polymerase. In some embodiments, the DNA polymerase is a cytomegalovirus (CMV) UL54 DNA polymerase. [0183]In some embodiments, the DNA polymerase is an archaeal polymerase. In some embodiments, the DNA polymerase is a Family B/pol I type DNA polymerase. For example, in some embodiments, the DNA polymerase is a homolog of Pfu from Pyrococcus furiosus. In some embodiments, the DNA polymerase is a pol II type DNA polymerase. For example, in some embodiments, the DNA polymerase is a homolog of P. furiosus DP1/DP2 2-subunit polymerase. In some embodiments, the DNA polymerase lacks 5' to 3' nuclease activity. Suitable DNA polymerases (pol I or pol II) can be derived from archaea with optimal growth temperatures that are similar to the desired assay temperatures.

WO 2024/238825 PCT/US2024/029746 id="p-184"

[0184]In some embodiments, the DNA polymerase comprises a thermostable archaeal DNA polymerase. In some embodiments, the thermostable DNA polymerase is isolated or derived from Pyrococcus species (furiosus, species GB-D, woesti, abysii, horikoshii), Thermococcus species (kodakaraensis KOD1, litoralis, species 9 degrees North-7, species JDF-3, gorgonarius), Pyrodictium occultum, and Archaeoglobus fulgidus. [0185]Polymerases may also be from eubacterial species. In some embodiments, the DNA polymerase is a Pol I family DNA polymerase. In some embodiments, the DNA polymerase is an E.coli Pol I DNA polymerase. In some embodiments, the DNA polymerase is a Pol II family DNA polymerase. In some embodiments, the DNA polymerase is a Pyrococcus furiosus (Pfii) Pol II DNA polymerase. In some embodiments, the DNA Polymerase is a Pol III family DNA polymerase. In some embodiments, the DNA Polymerase is a Pol IV family DNA polymerase. In some embodiments, the DNA polymerase is an E.coli Pol IV DNA polymerase. In some embodiments, the Pol I DNA polymerase is a DNA polymerase functional variant that lacks or has reduced 5' to 3' exonuclease activity. [0186]Suitable thermostable pol I DNA polymerases can be isolated from a variety of thermophilic eubacteria, including Thermus species and Thermotoga maritima such as Thermus aquaticus (Taq), Thermus thermophilus (Tth) and Thermotoga maritima (Tma UITma). [0187]In some embodiments, a prime editor comprises an RNA-dependent DNA polymerase domain, for example, a reverse transcriptase (RT). A RT or an RT domain may be a wild type RT domain, a full-length RT domain, or may be a functional mutant, a functional variant, or a functional fragment thereof. An RT or an RT domain of a prime editor may comprise a wild- type RT, or may be engineered or evolved to contain specific amino acid substitutions, truncations, or variants. An engineered RT may comprise sequences or amino acid changes different from a naturally occurring RT. In some embodiments, the engineered RT may have improved reverse transcription activity over a naturally occurring RT or RT domain. In some embodiments, the engineered RT may have improved features over a naturally occurring RT, for example, improved thermostability, reverse transcription efficiency, or target fidelity. In some embodiments, a prime editor comprising the engineered RT has improved prime editing efficiency over a prime editor having a reference naturally occurring RT. [0188]In some embodiments, a prime editor comprises a virus RT, for example, a retrovirus RT. Non-limiting examples of virus RT include Moloney murine leukemia virus (M-MLV, MMLVRT or M-MLV RT); human T-cell leukemia virus type 1 (HTLV-1) RT; bovine WO 2024/238825 PCT/US2024/029746 leukemia virus (BLV) RT; Rous Sarcoma Virus (RSV) RT; human immunodeficiency virus (HIV) RT, M-MFV RT, Avian Sarcoma-Leukosis Virus (ASLV) RT, Rous Sarcoma Virus (RSV) RT, Avian Myeloblastosis Virus (AMV) RT, Avian Erythroblastosis Virus (AEV) Helper Virus MCAV RT, Avian Myelocytomatosis Virus MC29 Helper Virus MCAV RT, Avian Reticuloendotheliosis Virus (REV-T) Helper Virus REV-A RT, Avian Sarcoma Virus UR2 Helper Virus (UR2AV) RT, Avian Sarcoma Virus Y73 Helper Virus YAV RT, Rous Associated Virus (RAV) RT, and Myeloblastosis Associated Virus (MAV) RT, all of which may be suitably used in the methods and composition described herein. [0189]In some embodiments, the prime editor comprises a wild type M-MLV RT, a functional mutant, a functional variant, or a functional fragment thereof. [0190]In some embodiments, the prime editor comprises a reference M-MLV RT, a functional mutant, a functional variant, or a functional fragment thereof. In some embodiments, the RT domain or a RT is a M-MLV RT (e.g., wild-type M-MLV RT, a functional mutant, a functional variant, or a functional fragment thereof). In some embodiments, the RT domain or a RT is a M-MLV RT (e.g., a reference M-MLV RT, a functional mutant, a functional variant, or a functional fragment thereof). In someembodiments, a M-MLV RT, e.g., reference M-MLV RT, comprises an amino acid sequence as set forth in any one of SEQ ID NO: 672. [0191]In some embodiments, a reference M-MLV RT is a wild-type M-MLV RT. Anexemplary amino acid sequence of a reference M-MLV RT is provided in SEQ ID NO: 671. [0192]In some embodiments, the prime editor comprises a wild type M-MLV RT. Anexemplary amino acid sequence of a wild type M-MLV RT is provided in SEQ ID NO: 671. [0193]TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLK ATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRP VQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLF AFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDD LLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWL TEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNW GPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPV AYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPP DRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHG TRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQR AELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEIL WO 2024/238825 PCT/US2024/029746 ALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSS P (SEQ ID NO: 671). [0194]In some embodiments, the prime editor comprises a reference M-MLV RT. An exemplary amino acid sequence of a reference M-MLV RT is provided in SEQ ID NO: 672. [0195]TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLK ATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRP VQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLF AFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDD LLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWL TEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNW GPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPV AYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPP DRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHG TRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQR AELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEIL ALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSS P (SEQ ID NO: 672). [0196]In some embodiments, the prime editor comprises a M-MLV RT comprising one or more of amino acid substitutions P51X, S67X, E69X, L139X, T197X, D200X, H204X, F209X, E302X, T306X, F309X, W313X, T330X, L345X, L435X, N454X, D524X, E562X, D583X, H594X, L603X, E607X, or D653X as compared to the reference M-MLV RT as set forth in SEQ ID NO: 672, where X is any amino acid other than the original amino acid in the reference M-MLV RT. In some embodiments, the prime editor comprises a M-MLV RT comprising one or more of amino acid substitutions P51L, S67K, E69K, L139P, T197A, D200N, H204R, F209N, E302K, E302R, T306K, F309N, W313F, T330P, L345G, L435G, N454K, D524G, E562Q, D583N, H594Q, L603W, E607K, and D653N as compared to the reference M-MLV RT as set forth in SEQ ID NO: 672. In some embodiments, the prime editor comprises a M-MLV RT comprising one or more of amino acid substitutions D200N, T330P, L603W, T306K, and W313F as compared to the reference M-MLV RT as set forth in SEQ ID NO: 672. In some embodiments, the prime editor comprises a M-MLV RT comprising amino acid substitutions D200N, T330P, L603W, T306K, and W313F as compared to the wild type M-MMLV RT as set forth in SEQ ID NO: 672. In some embodiments, a prime editor comprising the D200N, T330P, L603W, T306K, and W313F as WO 2024/238825 PCT/US2024/029746 compared to a reference M-MLV RT as set forth in SEQ ID NO: 672. In some embodiments, the prime editor comprises a M-MLV RT comprising one or more of amino acid substitutions D200N, T330P, L603W, T306K, and W313F as compared to a wild type M-MMLV RT as set forth in SEQ ID NO: 671. In some embodiments, a prime editor may comprise amino acid substitutions D200N, T330P, L603W, T306K, and W313F as compared to a reference M- MLV RT as set forth in SEQ ID NO: 672. In some embodiments, the prime editor comprises a M-MLV RT that comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 671, 672, or 673. In some embodiments, the prime editor comprises a M-MLV RT that comprises an amino acid sequence that is selected from the group consisting of SEQ ID NOs: 671, 672, or 673or a variant or fragment thereof. In some embodiments, the prime editor comprises a M-MLV RT that comprises an amino acid sequence set forth in SEQ ID NO: 673.TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTP VSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLR EVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWR DPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAAT SELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKE TVMGQPTPKTPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKA YQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKL DPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSN ARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLT DQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIAL TQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKA LFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP (SEQ ID NO: 673). [0197]In some embodiments, an RT variant may be a functional fragment of a reference RT that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or up to 100, or up to 200, or up to 300, or up to 400, or up to 500 or more amino acid changes compared to a wild type RT, e.g., SEQ ID NO: 671. In some embodiments, the RT variant comprises a fragment of a wild type RT, e.g., SEQ ID NO: 671, such that the fragment is WO 2024/238825 PCT/US2024/029746 about 70% identical, about 80% identical, about 90% identical, about 95% identical, about 96% identical, about 97% identical, about 98% identical, about 99% identical, about 99.5% identical, or about 99.9% identical to the corresponding fragment of the wild type RT, e.g., SEQ ID NO: 671. In some embodiments, the fragment is 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% identical, 96%, 97%, 98%, 99%, or 99.5% of the amino acid length of a corresponding wildtype RT (M-MLV reverse transcriptase) (e.g., SEQ ID NO: 671). [0198]In some embodiments, an RT variant may be a functional fragment of a reference RT that has 1, 2, 3,4, 5, 6, 7, 8, 9,10,11, 12, 13, 14, 15,16,17,18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40,41,42, 43, 44, 45, 46,47,48,49, 50, or up to 100, or up to 200, or up to 300, or up to 400, or up to 500 or more amino acid changes compared to a reference RT, e.g., SEQ ID NO: 672. In some embodiments, the RT variant comprises a fragment of a reference RT, such that the fragment is about 70% identical, about 80% identical, about 90% identical, about 95% identical, about 96% identical, about 97% identical, about 98% identical, about 99% identical, about 99.5% identical, or about 99.9% identical to the corresponding fragment of a reference RT, e.g., SEQ ID NO: 672. In some embodiments, the fragment is 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% identical, 96%, 97%, 98%, 99%, or 99.5% of the amino acid length of a reference RT, e.g., a M-MLV RT, e.g., SEQ ID NO: 672. [0199]In some embodiments, the RT functional fragment is at least 100 amino acids in length. In some embodiments, the fragment is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, or up to 600 or more amino acids in length. [0200]In still other embodiments, the functional RT variant is truncated at the N-terminus or the C-terminus, or both, by a certain number of amino acids which results in a truncated variant which still retains sufficient DNA polymerase function. In some embodiments, the functional RT variant, e.g., a functional MMLV RT variant, is truncated at the C-terminus to abolish or reduce RNAase H activity and still retain DNA polymerase activity. [0201]In some embodiments, a prime editing composition or a prime editing system disclosed herein comprises a polynucleotide (e.g., a DNA, a RNA, e.g., a mRNA) that encodes a M-MLV RT. In some embodiments, the polynucleotide encodes a M-MLV RT that comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to an amino acid sequence set forth in any one of SEQ ID WO 2024/238825 PCT/US2024/029746 NOs: 671, 672, or 673. In some embodiments, the polynucleotide encodes a M-MLV RT that comprises an amino acid sequence that is selected from the group consisting of SEQ ID NOs: 671, 672, and 673. In some embodiments, the polynucleotide encodes a M-MLV RT that comprises an amino acid sequence that is set forth in SEQ ID NO: 673. [0202]In some embodiments, a prime editor comprises a eukaryotic RT, for example, a yeast, drosophila, rodent, or primate RT. In some embodiments, the prime editor comprises a Group II intron RT, for example, a. Geobacillus stearothermophilus Group II Intron (Gsl- IIC) RT or a Eubacterium rectale group II intron (Eu.re.I2) RT. In some embodiments, the prime editor comprises a retron RT. In some embodiments, a prime editor comprises a eukaryotic RT, for example, a yeast, drosophila, rodent, or primate RT. In some embodiments, the prime editor comprises a Group II intron RT, for example, a. Geobacillus stearothermophilus Group II Intron (GsI-IIC) RT or a Eubacterium rectale group II intron (Eu.re.I2) RT. In some embodiments, the prime editor comprises a retron RT. Programmable DNA Binding Domain [0203]In some embodiments, the DNA-binding domain of a prime editor is a programmable DNA binding domain. In some embodiments, a prime editor comprises a DNA binding domain that comprises an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the sequences set forth in SEQ ID NOs: 674-701. In some embodiments, the DNA binding domain comprises an amino acid sequence that has no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 differences e.g., mutations e.g., deletions, substitutions and/or insertions compared to any one of the amino acid sequences set forth in SEQ ID NOs: 674-701. In some embodiments, the DNA binding domain of a prime editor is a programmable DNA binding domain. A programmable DNA binding domain refers to a protein domain that is designed to bind a specific nucleic acid sequence, e.g., a target DNA or a target RNA. In some embodiments, the DNA-binding domain is a polynucleotide programmable DNA-binding domain that can associate with a guide polynucleotide (e.g, a PEgRNA) that guides the DNA-binding domain to a specific DNA sequence, e.g, a search target sequence in a target gene. In some embodiments, the DNA-binding domain comprises a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Associated (Cas) protein. A Cas protein may comprise any Cas protein described herein or a functional fragment or functional variant WO 2024/238825 PCT/US2024/029746 thereof. In some embodiments, a DNA-binding domain may also comprise a zinc-finger protein domain. In other cases, a DNA-binding domain comprises a transcription activator- like effector domain (TALE). In some embodiments, the DNA-binding domain comprises a DNA nuclease. For example, the DNA-binding domain of a prime editor may comprise an RNA-guided DNA endonuclease, e.g., a Cas protein. In some embodiments, the DNA- binding domain comprises a zinc finger nuclease (ZFN) or a transcription activator like effector domain nuclease (TALEN), where one or more zinc finger motifs or TALE motifs are associated with one or more nucleases, e.g., a Fok I nuclease domain. [0204]In some embodiments, the DNA-binding domain comprises a nuclease activity. In some embodiments, the DNA-binding domain of a prime editor comprises an endonuclease domain having single strand DNA cleavage activity. For example, the endonuclease domain may comprise a Fokl nuclease domain. In some embodiments, the DNA-binding domain of a prime editor comprises a nuclease having full nuclease activity. In some embodiments, the DNA-binding domain of a prime editor comprises a nuclease having modified or reduced nuclease activity as compared to a wild type endonuclease domain. For example, the endonuclease domain may comprise one or more amino acid substitutions as compared to a wild type endonuclease domain. In some embodiments, the DNA-binding domain of a prime editor has a nickase activity. In some embodiments, the DNA-binding domain of a prime editor comprises a Cas protein domain that is a nickase. In some embodiments, compared to a wild type Cas protein, the Cas nickase comprises one or more amino acid substitutions in a nuclease domain that reduces or abolishes its double strand nuclease activity but retains DNA binding activity. In some embodiments, the Cas nickase comprises an amino acid substitution in a HNH domain. In some embodiments, the Cas nickase comprises an amino acid substitution in a RuvC domain. [0205]In some embodiments, the DNA-binding domain comprises a CRISPR associated protein (Cas protein) domain. A Cas protein may be a Class 1 or a Class 2 Cas protein. A Cas protein can be a type 1, type II, type III, type IV, type V Cas protein, or a type VI Cas protein. Non-limiting examples of Cas proteins include Casi, CasIB, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (e.g., Csnl or Csxl2), CaslO, CaslOd, Casl2a/Cpfl, Casl2b/C2cl, Casl2c/C2c3, Casl2d/CasY, Casl2e/CasX, Casl2g, Casl2h, Casl2i, Csyl, Csy2, Csy3, Csy4, Csel, Cse2, Cse3, Cse4, Cse5e, Cscl, Csc2, Csa5, Csnl, Csn2, Csml, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, CsxlS, Csxll, WO 2024/238825 PCT/US2024/029746 Csfl, Csf2, CsO, Csf4, Csdl, Csd2, Cstl, Cst2, Cshl, Csh2, Csal, Csa2, Csa3, Csa4, Csa5, Type II Cas effector proteins, Type V Cas effector proteins, Type VI Cas effector proteins, CARF, DinG, Cpfl, Casl2b/C2cl, Casl2c/C2c3, Casl2b/C2cl, Casl2c/C2c3, SpCas9(K855A), eSpCas9(l.l), SpCas9-HFl, hyper accurate Cas9 variant (HypaCas9), Cas O, and homologues, modified or engineered variants, mutants, and/or functional fragments thereof. A Cas protein can be a chimeric Cas protein that is fused to other proteins or polypeptides. A Cas protein can be a chimera of various Cas proteins, for example, comprising domains of Cas proteins from different organisms. [0206]A Cas protein, e.g., Cas9, can be from any suitable organism. In some aspects, the organism is Streptococcus pyogenes (S. pyogenes). In some aspects, the organism is Staphylococcus aureus (S. aureus). In some aspects, the organism is Streptococcus thermophilus (S. thermophilus). In some embodiments, the organism is Staphylococcus lugdunensis. [0207]Non-limiting examples of suitable organism include Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Nocardiopsis dassonvillei, Streptomyces pristinae spiralis, Streptomyces viridochromo genes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, AlicyclobacHlus acidocaldarius. Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum. Lactobacillus delbrueckii. Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Pseudomonas aeruginosa, Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans , Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus, Acaryochloris marina, Leptotrichia shahii, and Francisella novicida. In some embodiments, the organism is Streptococcus pyogenes (S. pyogenes). In some embodiments, the organism is Staphylococcus aureus (S. aureus). In some embodiments, the organism is Streptococcus WO 2024/238825 PCT/US2024/029746 thermophilus (S. thermophilus). In some embodiments, the organism is Staphylococcus lugdunensis (S. lugdunensis). [0208]In some embodiments, a Cas protein can be derived from a variety of bacterial species including, but not limited to, Veillonella atypical, Fusobacterium nucleatum, Filifactor alocis, Solobacterium moorei, Coprococcus catus, Treponema denticola, Peptoniphilus duerdenii, Catenibacterium mitsuokai, Streptococcus mutans, Listeria innocua, Staphylococcus pseudintermedius, Acidaminococcus intestine, Olsenella uli, Oenococcus kitaharae, Bifidobacterium bifidum, Lactobacillus rhamnosus, Lactobacillus gasseri, Finegoldia magna, Mycoplasma mobile, Mycoplasma gallisepticum. Mycoplasma ovipneumoniae, Mycoplasma canis, Mycoplasma synoviae, Eubacterium rectale, Streptococcus thermophilus, Eubacterium dolichum, Lactobacillus coryniformis subsp. Torquens, Ilyobacter polytropus, Ruminococcus albus, Akkermansia muciniphila, Acidothermus cellulolyticus. Bifidobacterium longum. Bifidobacterium dentium, Corynebacterium diphtheria, Elusimicrobium minutum, Nitratifractor salsuginis, Sphaerochaeta globus, Fibrobacter succinogenes subsp.Succinogenes, Bacteroides fragilis, Capnocytophaga ochracea, Rhodopseudomonas palustris, Prevotella micans, Prevotella ruminicola, Flavobacterium columnare, Aminomonas paucivorans, Rhodospirillum rubrum, Candidatus Puniceispirillum marinum, Verminephrobacter eiseniae, Ralstonia syzygii, Dinoroseobacter shibae, Azospirillum, Nitrobacter hamburgensis, Bradyrhizobium, Wolinella succinogenes, Campylobacter jejuni subsp. Jejuni, Helicobacter mustelae, Bacillus cereus, Acidovorax ebreus, Clostridium perfringens, Parvibaculum lavamentivorans, Roseburia intestinalis, Neisseria meningitidis, Pasteurella multocida subsp. Multocida, Sutterella wadsworthensis, proteobacterium, Legionella pneumophila, Parasutterella excrementihominis, Wolinella succinogenes, and Francisella novicida. [0209]In some embodiments, a Cas protein, e.g., Cas9, can be a wild type or a modified form of a Cas protein. In some embodiments, a Cas protein, e.g., Cas9, can be a nuclease active variant, nuclease inactive variant, a nickase, or a functional variant or functional fragment of a wild type Cas protein. In some embodiments, a Cas protein, e.g., Cas9, can be a wild type or a modified form of a Cas protein. A Cas protein, e.g., Cas9, can be a nuclease active variant, nuclease inactive variant, a nickase, or a functional variant or functional fragment of a wild type Cas protein. In some embodiments, a Cas protein, e.g., Cas9, can comprise an amino acid change such as a deletion, insertion, substitution, fusion, chimera, or any combination thereof relative to a corresponding wild-type version of the Cas protein. In WO 2024/238825 PCT/US2024/029746 some embodiments, a Cas protein can be a polypeptide with at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or sequence similarity to a wild type exemplary Cas protein. [0210]A Cas protein, e.g., Cas9, may comprise one or more domains. Non-limiting examples of Cas domains include, guide nucleic acid recognition and/or binding domain, nuclease domains (e.g, DNase or RNase domains, RuvC, HNH), DNA binding domain, RNA binding domain, helicase domains, protein-protein interaction domains, and dimerization domains. In various embodiments, a Cas protein comprises a guide nucleic acid recognition and/or binding domain can interact with a guide nucleic acid, and one or more nuclease domains that comprise catalytic activity for nucleic acid cleavage. [0211]In some embodiments, a Cas protein, e.g., Cas9, comprises one or more nuclease domains. A Cas protein can comprise an amino acid sequence having at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nuclease domain (e.g., RuvC domain, HNH domain) of a wild-type Cas protein. In some embodiments, a Cas protein comprises a single nuclease domain. For example, a Cpfl may comprise a RuvC domain but lacks HNH domain. In some embodiments, a Cas protein comprises two nuclease domains, e.g., a Cas9 protein can comprise an HNH nuclease domain and a RuvC nuclease domain. [0212]In some embodiments, a prime editor comprises a Cas protein, e.g., Cas9, wherein all nuclease domains of the Cas protein are active. In some embodiments, a prime editor comprises a Cas protein having one or more inactive nuclease domains. One or a plurality of the nuclease domains (e.g, RuvC, HNH) of a Cas protein can be deleted or mutated so that they are no longer functional or comprise reduced nuclease activity. In some embodiments, a Cas protein, e.g., Cas9, comprising mutations in a nuclease domain has reduced (e.g, nickase) or abolished nuclease activity while maintaining its ability to target a nucleic acid locus at a search target sequence when complexed with a guide nucleic acid, e.g., a PEgRNA. [0213]In some embodiments, a prime editor comprises a Cas nickase that can bind to the target gene in a sequence-specific manner and generate a single-strand break at a protospacer within double-stranded DNA in the target gene, but not a double-strand break. For example, the Cas nickase can cleave the edit strand or the non-edit strand of the target gene, but may not cleave both. In some embodiments, a prime editor comprises a Cas nickase comprising two nuclease domains (e.g, Cas9), with one of the two nuclease domains modified to lack catalytic activity or deleted. In some embodiments, the Cas nickase of a prime editor WO 2024/238825 PCT/US2024/029746 comprises a nuclease inactive RuvC domain and a nuclease active HNH domain. In some embodiments, the Cas nickase of a prime editor comprises a nuclease inactive HNH domain and a nuclease active RuvC domain. In some embodiments, a prime editor comprises a Casnickase having an amino acid substitution in the RuvC domain e.g., an amino acid substitution that reduces or abolishes nuclease activity of the RuvC domain. In some embodiments, the Cas9 nickase comprises a DI OX amino acid substitution compared to a wild type 8. pyogenes Cas9, wherein X is any amino acid other than D. In some embodiments, a prime editor comprises a Cas9 nickase having an amino acid substitution in the HNH domain e.g., an amino acid substitution that reduces or abolishes nuclease activity of the HNH domain. In some embodiments, the Cas9 nickase comprises a H840X amino acid substitution compared to a wild type 8. pyogenes Cas9, wherein X is any amino acid other thanH. [0214]In some embodiments, a prime editor comprises a Cas protein that can bind to the target gene in a sequence-specific manner but lacks or has abolished nuclease activity and may not cleave either strand of a double stranded DNA in a target gene. Abolished activity or lacking activity can refer to an enzymatic activity less than 1%, less than 2%, less than 3%, less than 4%, less than 5%, less than 6%, less than 7%, less than 8%, less than 9%, or less than 10% activity compared to a wild-type exemplary activity (e.g, wild-type Cas9 nuclease activity). In some embodiments, a Cas protein of a prime editor completely lacks nuclease activity. A nuclease, e.g, Cas9, that lacks nuclease activity may be referred to as nuclease inactive or "nuclease dead " (abbreviated by "d"). A nuclease dead Cas protein (e.g, dCas, dCas9) can bind to a target polynucleotide but may not cleave the target polynucleotide. In some embodiments, a dead Cas protein is a dead Cas9 protein. In some embodiments, a prime editor comprises a nuclease dead Cas protein wherein all of the nuclease domains (e.g, both RuvC and HNH nuclease domains in a Cas9 protein; RuvC nuclease domain in a Cpfl protein) are mutated to lack catalytic activity, or are deleted. [0215]A Cas protein can be modified. A Cas protein, e.g, Cas9, can be modified to increase or decrease nucleic acid binding affinity, nucleic acid binding specificity, and/or enzymatic activity. Cas proteins can also be modified to change any other activity or property of the protein, such as stability. For example, one or more nuclease domains of the Cas protein can be modified, deleted, or inactivated, or a Cas protein can be truncated to remove domains that are not essential for the function of the protein or to optimize (e.g, enhance or reduce) the activity of the Cas protein.

WO 2024/238825 PCT/US2024/029746 id="p-216"

[0216]A Cas protein can be a fusion protein. For example, a Cas protein can be fused to a cleavage domain, an epigenetic modification domain, a transcriptional regulation domain, or a polymerase domain. A Cas protein can also be fused to a heterologous polypeptide providing increased or decreased stability. The fused domain or heterologous polypeptide can be located at the N-terminus, the C-terminus, or internally within the Cas protein. [0217]In some embodiments, the Cas protein of a prime editor is a Class 2 Cas protein. In some embodiments, the Cas protein is a type II Cas protein. In some embodiments, the Cas protein is a Cas9 protein, a modified version of a Cas9 protein, a Cas9 protein homolog, mutant, variant, or a functional fragment thereof. As used herein, a Cas9, Cas9 protein, Caspolypeptide or a Cas9 nuclease refers to an RNA guided nuclease comprising one or more Cas9 nuclease domains and a Cas9 gRNA binding domain having the ability to bind a guide polynucleotide, e.g., a PEgRNA. A Cas9 protein may refer to a wild type Cas9 protein from any organism or a homolog, ortholog, or paralog from any organisms; any functional mutants or functional variants thereof; or any functional fragments or domains thereof. In some embodiments, a prime editor comprises a full-length Cas9 protein. In some embodiments, the Cas9 protein can generally comprises at least about 50%, 60%, 70%, 80%, 90%, 100% sequence identity to a wild type reference Cas9 protein (e.g, Cas9 from S. pyogenes). In some embodiments, the Cas9 comprises an amino acid change such as a deletion, insertion, substitution, fusion, chimera, or any combination thereof as compared to a wild type reference Cas9 protein. [0218]In some embodiments, a Cas9 protein may comprise a Cas9 protein from Streptococcus pyogenes (Sp), Staphylococcus aureus (Sa), Streptococcus cants (Sc), Streptococcus thermophilus (St), Staphylococcus lugdunensis (Slu), Neisseria meningitidis (Nm), Campylobacter jejuni (Cj), Francisella novicida (Fn), or Treponema denticola (Td), or any Cas9 homolog or ortholog from an organism known in the art. In some embodiments, a Cas9 polypeptide is a SpCas9 polypeptide, e.g., comprising an amino acid sequence as set forth in NCBI Accession No. WP 038431314 or a fragment or variant thereof. In some embodiments, a Cas9 polypeptide is a SaCas9 polypeptide, e.g., comprising an amino acid sequence as set forth in Uniprot Accession No. J7RUA5 or a fragment or variant thereof. In some embodiments, a Cas9 polypeptide is a ScCas9 polypeptide, e.g., comprising an amino acid sequence as set forth in Uniprot Accession No. A0A3P5YA78 or a fragment or variant thereof. In some embodiments, a Cas9 polypeptide is a StCas9 polypeptide, e.g., comprising an amino acid sequence as set forth in NCBI Accession No. WP 007896501.1 or a fragment WO 2024/238825 PCT/US2024/029746 or variant thereof. In some embodiments, a Cas9 polypeptide is a SluCas9 polypeptide, e.g., comprising an amino acid sequence as set forth in any of NCBI Accession No.WP 230580236.1 or WP_250638315.1 or WP_242234150.1, WP_241435384.1,WP 002460848.1, KAK58371.1, or a fragment or variant thereof. In some embodiments, a Cas9 polypeptide is a NmCas9 polypeptide, e.g., comprising an amino acid sequence as set forth in any of NCBI Accession No. WP 002238326.1 or WP 061704949.1 or a fragment or variant thereof. In some embodiments, a Cas9 polypeptide is a CjCas9 polypeptide, e.g., comprising an amino acid sequence as set forth in any of NCBI Accession No.WP_100612036.1, WP_116882154.1, WP_116560509.1, WP_116484194.1,WP_116479303.1, WP_115794652.1, WP_100624872.1, or a fragment or variant thereof. In some embodiments, a Cas9 polypeptide is a FnCas9 polypeptide, e.g., comprising the amino acid sequence as set forth in Uniprot Accession No. A0Q5Y3 or a fragment or variant thereof. In some embodiments, a Cas9 polypeptide is a TdCas9 polypeptide, e.g., comprising the amino acid sequence as set forth in NCBI Accession No. WP 147625065.1 or a fragment or variant thereof. In some embodiments, a Cas9 polypeptide is a chimera comprising domains from two or more of the organisms described herein or those known in the art. In some embodiments, a Cas9 polypeptide is a Cas9 polypeptide from Streptococcus macacae, e.g., comprising the amino acid sequence as set forth in NCBI Accession No.WP_003079701.1 or a fragment or variant thereof. In some embodiments, a Cas9 polypeptide is a Cas9 polypeptide generated by replacing a PAM interaction domain of a SpCas9 with that of a Streptococcus macacae Cas9 (Spy-mac Cas9). Exemplary Cas sequences are provided in Table 22below. [0219]In some embodiments, a Cas9 protein comprises an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the sequences set forth in SEQ ID NOs: 674-701. In some embodiments, a Casprotein is a Cas9 nickase that comprises an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the sequences set forth in SEQ ID NOs: 675, 676, 677, 679, 680, 682, 683, 685, 686, 688, 689, 691, 692, 694, 695, 697, 698, 700, or 701. In some embodiments, a Cas9 protein comprises WO 2024/238825 PCT/US2024/029746 an amino acid sequence that is selected from the group consisting of SEQ ID NOs: 674-701. In some embodiments, a prime editor comprises a Cas9 protein that comprises an amino acid sequence that lacks a N-terminus methionine relative to an amino acid sequence set forth in any one of SEQ ID NOs: 674, 675, 678, 679, 681, 682, 684, 685, 687, 688, 690, 691, 693, 694, 696, 697, 699, or 700. In some embodiments, the prime editing compositions or prime editing systems disclosed herein comprises a polynucleotide (e.g., a DNA, or an RNA, e.g., an mRNA) that encodes a Cas9 protein that comprises an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the sequences set forth in SEQ ID NOs: 674-701. [0220]In some embodiments, a Cas9 protein comprises a Cas9 protein from Streptococcus pyogenes (Sp), e.g., as according to NC_002737.2:854751-858857 or the protein encoded by UniProt Q99ZW2, e.g., as according to SEQ ID NO: 674. In some embodiments, a prime editor comprises a Cas9 protein (e.g., a SpCas9) as according to any one of the sequences set forth in SEQ ID NOs: 674-677 or a variant thereof. In some embodiments, the Cas9 protein is a SpCas9. In some embodiments, a SpCas9 can be a wild type SpCas9, a SpCas9 variant, or a nickase SpCas9. In some embodiments, the SpCas9 lacks the N-terminus methionine relative to a corresponding SpCas9 (e.g., a wild type SpCas9, a SpCas9 variant or a nickase SpCas9). In some embodiments, a prime editor comprises a Cas9 protein, having an amino acid sequence as according to SEQ ID NO:674, not including the N-terminus methionine. In some embodiments, a wild type SpCas9 comprises an amino acid sequence set forth in SEQ ID NO: 674. In some embodiments, a prime editor comprises a Cas9 protein comprising one or more mutations (e.g., amino acid substitutions, insertions and/or deletions) relative to a corresponding wild type Cas9 protein (e.g., a wild type SpCas9). In some embodiments, a prime editor comprises a Cas9 protein comprising an amino acid sequence set forth in SEQ ID NO: 675, SEQ ID NO: 676 or SEQ ID NO: 677. Exemplary Streptococcus pyogenes Cas(SpCas9) amino acid sequence usefill in the prime editors disclosed herein are provided below in SEQ ID NOs: 674-677. [0221]In some embodiments, a prime editor comprises a Cas9 protein (e.g., a SluCas9) as according to any one of the SEQ ID NOS: 678-680 or a variant thereof. In some embodiments, a prime editor comprises a Cas9 protein from Staphylococcus lugdunensis (SluCas9) e.g., as according to any one of the SEQ ID NOs: 678-680 or a variant thereof. In WO 2024/238825 PCT/US2024/029746 some embodiments, the Cas9 protein is a SluCas9. In some embodiments, a SluCas9 can be a wild type SluCas9, a SluCas9 variant, or a nickase SluCas9. In some embodiments, the SluCas9 lacks the N-terminus methionine relative to a corresponding SluCas9 (e.g., a wild type SluCas9, a SluCas9 variant or a nickase SluCas9). In some embodiments, a prime editor comprises a Cas9 protein, having an amino acid sequence as according to SEQ ID NO: 678, not including the N-terminus methionine. In some embodiments, a wild type SluCascomprises an amino acid sequence set forth in SEQ ID NO: 678. In some embodiments, a prime editor comprises a Cas9 protein comprising one or more mutations (e.g., amino acid substitutions, insertions and/or deletions) relative to a corresponding wild type Cas9 protein (e.g., a wild type SluCas9). In some embodiments, the Cas9 protein comprising one or more mutations relative to a wild type Cas9 protein comprises an amino acid sequence set forth in SEQ ID NO: 679. Exemplary Staphylococcus lugdunensis Cas9 (SluCas9) amino acid sequence useful in the prime editors disclosed herein are provided below in SEQ ID NOs: 678-680. [0222]In some embodiments, a prime editor comprises a Cas9 protein from Staphylococcus aureus (SaCas9) e.g., as according to any of the SEQ ID NOS: 681-683, or a variant thereof. In some embodiments, a prime editor comprises a Cas9 protein from Staphylococcus aureus (SaCas9) e.g., as according to any one of the SEQ ID NOS: 681-683, or a variant thereof. In some embodiments, the Cas9 protein is a SaCas9. In some embodiments, a SaCas9 can be a wild type SaCas9, a SaCas9 variant, or a nickase SaCas9. In some embodiments, the SaCaslacks the N-terminus methionine relative to a corresponding SaCas9 (e.g., a wild type SaCas9, a SaCas9 variant or a nickase SaCas9). In some embodiments, a prime editor comprises a Cas9 protein, having an amino acid sequence as according to SEQ ID NO: 681, not including the N-terminus methionine. In some embodiments, a wild type SaCascomprises an amino acid sequence set forth in SEQ ID NO: 681. In some embodiments, a prime editor comprises a Cas9 protein comprising one or more mutations (e.g., amino acid substitutions, insertions and/or deletions relative to a corresponding wild type Cas9 protein (e.g., a wild type SaCas9). In some embodiments, the Cas9 protein comprising one or more mutations relative to a wild type Cas9 protein comprises an amino acid sequence set forth in SEQ ID NO: 682. Exemplary Staphylococcus aureus Cas9 (SaCas9) amino acid sequence useful in the prime editors disclosed herein are provided below in SEQ ID NOs: 681-683. [0223]In some embodiments, a prime editor comprises a Cas protein, e.g., a Cas9 variant, comprising modifications that allow altered PAM recognition. Exemplary Cas9 protein WO 2024/238825 PCT/US2024/029746 amino acid sequence (e.g., Cas9 variant with altered PAM recognition specificities) that are useful in the Prime editors of the disclosure are provided below in SEQ ID NOs 684-692, 699-701. In some embodiments, a prime editor comprises a Cas9 protein as according to any one of the sequences set forth in SEQ ID NOs: 684-692, 699-701 or a variant thereof. In some embodiments, the Cas9 protein is a Cas9 variant, for example, a SpCas9 variant (e.g., SpCas9-NG, SpCas9-NGA, SpRY, or SpG). In some embodiments, the Cas9 protein lacks the N-terminus methionine relative to a corresponding Cas9 protein (e.g., a Cas9 variant set forth in any one of SEQ ID NOs: 684, 685, 687, 688, 690, 691, 699, or 700). In some embodiments, a prime editor comprises a Cas9 protein (e.g., a Cas9 variant), having an amino acid sequence as according to any one of SEQ ID NOs: 684, 687, 690, or 699 not including the N-terminus methionine. In some embodiments, a prime editor comprises a Cas9 protein comprising one or more mutations (e.g., amino acid substitutions, insertions and/or deletions) relative to a corresponding Cas9 protein (e.g., a Cas9 protein set forth in any one of SEQ ID NOs: 684, 687, 690, or 699). In some embodiments, the Cas9 protein comprising one or mutations relative to a corresponding Cas9 protein comprises an amino acid sequence set forth in any one of SEQ ID NOs: 685, 686, 688, 689, 691, 692, 700, or 701. [0224]In some embodiments, a Cas9 protein is a chimeric Cas9, e.g., modified Cas9, e.g., synthetic RNA-guided nucleases (sRGNs), e.g., modified by DNA family shuffling, e.g., 8RGN3.1, 8RGN3.3. In some embodiments, the DNA family shuffling comprises, fragmentation and reassembly of parental Cas9 genes, e.g., one or more of Cas9s from Staphylococcus hyicus (Shy), Staphylococcus lugdunensis (Slu), Staphylococcus microti (Smi), and Staphylococcus pasteuri (Spa). In some embodiments, a modified sluCas9 shows increased editing efficiency and/or specificity relative to a sluCas9 that is not modified. In some embodiments, a modified Cas9, e.g., a sRGN shows at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1000% increase in editing efficiency compared to a Cas9 that is not modified. In some embodiments, a Cas9, e.g., a sRGN shows at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1000% increase in specificity compared to a Cas9 that is not modified. In some embodiments, a Cas9, e.g., a sRGN shows at least 10%, at least 20%, at least 30%, at WO 2024/238825 PCT/US2024/029746 least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1000% increase in cleavage activity compared to a Cas9 that is not modified. In some embodiments, a Cas9, e.g., a sRGN shows ability to cleave a 5'-NNGG-3' PAM-containing target. In some embodiments, a prime editor comprises a Cas9 protein (e.g., a chimeric Cas9), e.g., as according any one of the sequences set forth in SEQ ID NOs: 693-698, or a variant thereof. Exemplary amino acid sequences of Cas9 protein (e.g., sRGN) usefill in the prime editors disclosed herein are provided below in SEQ ID NOs: 693-698. In some embodiments, a prime editor comprises a Cas9 protein, that lacks a N-terminus methionine relative to SEQ ID NO: 693 or SEQ ID NO: 696. In some embodiments, a prime editor comprises a Cas9 protein comprising one or more mutations (e.g., amino acid substitutions, insertions and/or deletions) relative to a corresponding Casprotein (e.g., a Cas9 protein set forth in SEQ ID NO: 693 or SEQ ID NO: 696). In some embodiments, the Cas9 protein comprising one or mutations relative to a corresponding Casprotein comprises an amino acid sequence set forth in any one of SEQ ID NOs: 694, 695, 697, or 698. Table 22:Exemplary Cas protein sequencesSEQ ID NO: Sequence descriptionAmino acid sequence 674wild type Streptococc US Pyogenes Cas(SpCas9) MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHS IKKNLIGALLFD SGET AEATRLKRT ARRRYTRRKNRIC YLQEIF SNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAY HEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIE GDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSA RLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDL AEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQL PEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGT EELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDF YPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETI TPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLY EYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNR KVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFD DKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSD GFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLA GSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTT QKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNK VLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRK WO 2024/238825 PCT/US2024/029746 FDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRM NTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNY HHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMI AKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETN GETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKV EKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKK DLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNF LYLASHYEKLKG SP EDNEQKQLF VEQHKHYLDEIEQI SEF SK RVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAP AAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQL GGD675 SpCasH840A nickase MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHS IKKNLIGALLFDSGET AEATRLKRT ARRRYTRRKNRIC YLQEIF SNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAY HEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIE GDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSA RLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDL AEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQL PEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGT EELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDF YPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETI TPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLY EYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNR KVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFD DKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSD GFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLA GSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTT QKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNK VLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRK FDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRM NTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNY HHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMI AKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETN GETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKV EKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKK DLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNF LYLASHYEKLKG SP EDNEQKQLF VEQHKHYLDEIEQI SEF SK RVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAP AAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQL GGD676 Met (-) SpCasH840A nickase DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSI KKNLIGALLFDSGET AEATRLKRT ARRRYTRRKNRIC YLQEIF SNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAY HEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIE GDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSA WO 2024/238825 PCT/US2024/029746 RLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDL AEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQL PEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGT EELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDF YPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETI TPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLY EYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNR KVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFD DKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSD GFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLA GSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTT QKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNK VLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRK FDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRM NTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNY HHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMI AKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETN GETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKV EKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKK DLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNF LYLASHYEKLKG SP EDNEQKQLF VEQHKHYLDEIEQI SEF SK RVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAP AAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQL GGD677 Met (-) CAS(R221KN394K H840A) nickase DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSI KKNLIGALLFDSGET AEATRLKRT ARRRYTRRKNRIC YLQEIF SNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAY HEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIE GDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSA RLSKSRKLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDL AEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQL PEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGT EELLVKLKREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDF YPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETI TPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLY EYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNR KVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFD DKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSD GFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLA GSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTT QKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNK VLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRK FDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRM WO 2024/238825 PCT/US2024/029746 NTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNY HHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMI AKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETN GETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKV EKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKK DLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNF LYLASHYEKLKG SP EDNEQKQLF VEQHKHYLDEIEQI SEF SK RVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAP AAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQL GGD678 wild type Staphyloco ecus lugdunensi s (Slu)Cas9 MNQKFILGLDIGITSVGYGLIDYETKNIIDAGVRLFPEANVENN EGRRSKRGSRRLKRRRIHRLERVKKLLEDYNLLDQSQIPQSTN PYAIRVKGLSEALSKDELVIALLHIAKRRGIHKIDVIDSNDDVG NELSTKEQLNKNSKLLKDKFVCQIQLERMNEGQVRGEKNRF KTADIIKEIIQLLNVQKNFHQLDENFINKYIELVEMRREYFEGP GKGSPYGWEGDPKAWYETLMGHCTYFPDELRSVKYAYSAD LFNALNDLNNLVIQRDGLSKLEYHEKYHIIENVFKQKKKPTLK QIANEINVNPEDIKGYRITKSGKPQFTEFKLYHDLKSVLFDQSI LENEDVLDQIAEILTIYQDKDSIKSKLTELDILLNEEDKENIAQL TGYTGTHRLSLKCIRLVLEEQWYSSRNQMEIFTHLNIKPKKIN LTAANKIPKAMIDEFILSPVVKRTFGQAINLINKIIEKYGVPEDII IELARENNSKDKQKFINEMQKKNENTRKRINEIIGKYGNQNA KRLVEKIRLHDEQEGKCLYSLESPPLEDLLNNPNHYEVDHHPR SVSFDNSYHNKVLVKQSENSKKSNLTPYQYFNSGKSKLSYNQ FKQHILNLSKSQDRISKKKKEYLLEERDINKFEVQKEFINRNLV DTRYATRELTNYLKAYFSANNMNVKVKTINGSFTDYLRKVW KFKKERNHGYKHHAEDALIIANADFLFKENKKLKAVNSVLEK PEIESKQLDIQVDSEDNYSEMFIIPKQVQDIKDFRNFKYSHRVD KKPNRQLINDTLYSTRKKDNSTYIVQTIKDIYAKDNTTLKKQF DKSPEKFLMYQHDPRTFEKLEVIMKQYANEKNPLAKYHEET GEYLTKYSKKNNGPIVKSLKYIGNKLGSHLDVTHQFKSSTKK LVKLSIKPYRFDVYLTDKGYKFITISYLDVLKKDNYYYIPEQK YDKLKLGKAIDKNAKFIASFYKNDLIKLDGEIYKIIGVNSDTR NMIELDLPDIRYKEYCELNNIKGEPRIKKTIGKKVNSIEKLTTD VLGNVFTNTQYTKPQLLFKRGN679 SluCasN582A nickase MNQKFILGLDIGITSVGYGLIDYETKNIIDAGVRLFPEANVENN EGRRSKRGSRRLKRRRIHRLERVKKLLEDYNLLDQSQIPQSTN PYAIRVKGLSEALSKDELVIALLHIAKRRGIHKIDVIDSNDDVG NELSTKEQLNKNSKLLKDKFVCQIQLERMNEGQVRGEKNRF KTADIIKEIIQLLNVQKNFHQLDENFINKYIELVEMRREYFEGP GKGSPYGWEGDPKAWYETLMGHCTYFPDELRSVKYAYSAD LFNALNDLNNLVIQRDGLSKLEYHEKYHIIENVFKQKKKPTLK QIANEINVNPEDIKGYRITKSGKPQFTEFKLYHDLKSVLFDQSI LENEDVLDQIAEILTIYQDKDSIKSKLTELDILLNEEDKENIAQL TGYTGTHRLSLKCIRLVLEEQWYSSRNQMEIFTHLNIKPKKIN LTAANKIPKAMIDEFILSPVVKRTFGQAINLINKIIEKYGVPEDII IELARENNSKDKQKFINEMQKKNENTRKRINEIIGKYGNQNA KRLVEKIRLHDEQEGKCLYSLESPPLEDLLNNPNHYEVDHHPR SVSFDNSYHNKVLVKQSEASKKSNLTPYQYFNSGKSKLSYNQ WO 2024/238825 PCT/US2024/029746 FKQHILNLSKSQDRISKKKKEYLLEERDINKFEVQKEFINRNLV DTRYATRELTNYLKAYFSANNMNVKVKTINGSFTDYLRKVW KFKKERNHGYKHHAEDALIIANADFLFKENKKLKAVNSVLEK PEIESKQLDIQVDSEDNYSEMFIIPKQVQDIKDFRNFKYSHRVD KKPNRQLINDTLYSTRKKDNSTYIVQTIKDIYAKDNTTLKKQF DKSPEKFLMYQHDPRTFEKLEVIMKQYANEKNPLAKYHEET GEYLTKYSKKNNGPIVKSLKYIGNKLGSHLDVTHQFKSSTKK LVKLSIKPYRFDVYLTDKGYKFITISYLDVLKKDNYYYIPEQK YDKLKEGKAIDKNAKFIASFYKNDLIKLDGEIYKIIGVNSDTR NMIELDLPDIRYKEYCELNNIKGEPRIKKTIGKKVNSIEKLTTD VLGNVFTNTQYTKPQLLFKRGN680 Met (-) SluCasnickase NQKFILGLDIGITSVGYGLIDYETKNIIDAGVRLFPEANVENNE GRRSKRGSRRLKRRRIHRLERVKKLLEDYNLLDQSQIPQSTNP YAIRVKGLSEALSKDELVIALLHIAKRRGIHKIDVIDSNDDVG NELSTKEQLNKNSKLLKDKFVCQIQLERMNEGQVRGEKNRF KTADIIKEIIQLLNVQKNFHQLDENFINKYIELVEMRREYFEGP GKGSPYGWEGDPKAWYETLMGHCTYFPDELRSVKYAYSAD LFNALNDLNNLVIQRDGLSKLEYHEKYHIIENVFKQKKKPTLK QIANEINVNPEDIKGYRITKSGKPQFTEFKLYHDLKSVLFDQSI LENEDVLDQIAEILTIYQDKDSIKSKLTELDILLNEEDKENIAQL TGYTGTHRLSLKCIRLVLEEQWYSSRNQMEIFTHLNIKPKKIN LTAANKIPKAMIDEFILSPVVKRTFGQAINLINKIIEKYGVPEDII IELARENNSKDKQKFINEMQKKNENTRKRINEIIGKYGNQNA KRLVEKIRLHDEQEGKCLYSLESPPLEDLLNNPNHYEVDHHPR SVSFDNSYHNKVLVKQSEASKKSNLTPYQYFNSGKSKLSYNQ FKQHILNLSKSQDRISKKKKEYLLEERDINKFEVQKEFINRNLV DTRYATRELTNYLKAYFSANNMNVKVKTINGSFTDYLRKVW KFKKERNHGYKHHAEDALIIANADFLFKENKKLKAVNSVLEK PEIESKQLDIQVDSEDNYSEMFIIPKQVQDIKDFRNFKYSHRVD KKPNRQLINDTLYSTRKKDNSTYIVQTIKDIYAKDNTTLKKQF DKSPEKFLMYQHDPRTFEKLEVIMKQYANEKNPLAKYHEET GEYLTKYSKKNNGPIVKSLKYIGNKLGSHLDVTHQFKSSTKK LVKLSIKPYRFDVYLTDKGYKFITISYLDVLKKDNYYYIPEQK YDKLKEGKAIDKNAKFIASFYKNDLIKLDGEIYKIIGVNSDTR NMIELDLPDIRYKEYCELNNIKGEPRIKKTIGKKVNSIEKLTTD VLGNVFTNTQYTKPQLLFKRGN681 Staphyloco ccus aureus Cas(SaCas9) MKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVEN NEGRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSG INPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEED TGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINR FKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYY EGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNA DLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTL KQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARK EIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNL KGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKVD LSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIII ELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAK YLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRS VSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKISYETFK WO 2024/238825 PCT/US2024/029746 KHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVD TRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKF KKERNKGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQ MFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRV DKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKL KKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKY YEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPN SRNKWKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYY EVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIG VNNDLLNRIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSI KKY STDILGNL YEVKSKKHPQHIKKG682 SaCasN580A nickase MKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVEN NEGRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSG INPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEED TGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINR FKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYY EGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNA DLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTL KQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARK EIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNL KGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKVD LSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIII ELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAK YLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRS VSFDNSFNNKVLVKQEEASKKGNRTPFQYLSSSDSKISYETFK KHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVD TRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKF KKERNKGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQ MFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRV DKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKL KKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKY YEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPN SRNKWKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYY EVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIG VNNDLLNRIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSI KKY STDILGNL YEVKSKKHPQIIKKG683 Met (-) SaCasnickase KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNE GRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINP YEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTG NELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFK TSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEG PGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADL YNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLK QIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEII ENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLK GYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDL SQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIE LAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKY LIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSV SFDNSFNNKVLVKQEEASKKGNRTPFQYLSSSDSKISYETFKK WO 2024/238825 PCT/US2024/029746 HILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDT RYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKF KKERNKGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQ MFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRV DKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKL KKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKY YEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPN SRNKWKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYY EVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIG VNNDLLNRIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSI KKY STDILGNL YEVKSKKHPQHIKKG684 SpCas9- NG (VRVRFR R) MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHS IKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIF SNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAY HEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIE GDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSA RLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDL AEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQL PEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGT EELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDF YPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETI TPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLY EYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNR KVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFD DKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSD GFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLA GSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTT QKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNK VLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRK FDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRM NTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNY HHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMI AKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETN GETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESIRPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLWAKV EKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKK DLIIKLPKYSLFELENGRKRMLASARFLQKGNELALPSKYVNF LYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSK RVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAP RAFKYFDTTIDRKVYRSTKEVLDATLIHQSITGLYETRIDLSQL GGD685 spCas9-NG (H840AV RVRFRR) Nickase MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHS IKKNLIGALLFDSGET AEATRLKRT ARRRYTRRKNRIC YLQEIF SNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAY HEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIE GDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSA RLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDL WO 2024/238825 PCT/US2024/029746 AEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQL PEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGT EELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDF YPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETI TPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLY EYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNR KVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFD DKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSD GFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLA GSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTT QKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNK VLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRK FDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRM NTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNY HHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMI AKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETN GETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESIRPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLWAKV EKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKK DLIIKLPKYSLFELENGRKRMLASARFLQKGNELALPSKYVNF LYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSK RVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAP RAFKYFDTTIDRKVYRSTKEVLDATLIHQSITGLYETRIDLSQL GGD686 Met (-) SpCas9- NG Nickase DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSI KKNLIGALLFDSGET AEATRLKRT ARRRYTRRKNRIC YLQEIF SNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAY HEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIE GDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSA RLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDL AEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQL PEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGT EELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDF YPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETI TPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLY EYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNR KVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFD DKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSD GFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLA GSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTT QKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNK VLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRK FDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRM NTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNY WO 2024/238825 PCT/US2024/029746 HHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMI AKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETN GETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESIRPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLWAKV EKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKK DLIIKLPKYSLFELENGRKRMLASARFLQKGNELALPSKYVNF LYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSK RVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAP RAFKYFDTTIDRKVYRSTKEVLDATLIHQSITGLYETRIDLSQL GGD687 spCas9- NGA (VRQR) MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHS IKKNLIGALLFDSGET AEATRLKRT ARRRYTRRKNRIC YLQEIF SNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAY HEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIE GDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSA RLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDL AEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQL PEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGT EELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDF YPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETI TPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLY EYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNR KVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFD DKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSD GFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLA GSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTT QKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNK VLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRK FDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRM NTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNY HHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMI AKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETN GETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVAKV EKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKK DLIIKLPKYSLFELENGRKRMLASARELQKGNELALPSKYVNF LYLASHYEKLKG SP EDNEQKQLF VEQHKHYLDEIEQI SEF SK RVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAP AAFKYFDTTIDRKQYRSTKEVLDATLIHQSITGLYETRIDLSQL GGD688 spCas9- NGA (H840A V RQR) Nickase MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHS IKKNLIGALLFDSGET AEATRLKRT ARRRYTRRKNRIC YLQEIF SNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAY HEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIE GDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSA RLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDL AEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI WO 2024/238825 PCT/US2024/029746 LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQL PEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGT EELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDF YPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETI TPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLY EYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNR KVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFD DKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSD GFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLA GSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTT QKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNK VLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRK FDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRM NTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNY HHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMI AKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETN GETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVAKV EKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKK DLIIKLPKYSLFELENGRKRMLASARELQKGNELALPSKYVNF LYLASHYEKLKG SP EDNEQKQLF VEQHKHYLDEIEQI SEF SK RVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAP AAFKYFDTTIDRKQYRSTKEVLDATLIHQSITGLYETRIDLSQL GGD689 Met(-) spCas9- NGA Nickase DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSI KKNLIGALLFDSGET AEATRLKRT ARRRYTRRKNRIC YLQEIF SNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAY HEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIE GDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSA RLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDL AEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQL PEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGT EELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDF YPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETI TPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLY EYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNR KVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFD DKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSD GFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLA GSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTT QKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNK VLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRK FDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRM NTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNY HHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMI WO 2024/238825 PCT/US2024/029746 AKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETN GETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVAKV EKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKK DLIIKLPKYSLFELENGRKRMLASARELQKGNELALPSKYVNF LYLASHYEKLKG SP EDNEQKQLF VEQHKHYLDEIEQI SEF SK RVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAP AAFKYFDTTIDRKQYRSTKEVLDATLIHQSITGLYETRIDLSQL GGD690 SpRY Cas9 MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHS IKKNLIGALLFDSGET AERTRLKRT ARRRYTRRKNRIC YLQEIF SNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAY HEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIE GDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSA RLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDL AEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQL PEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGT EELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDF YPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETI TPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLY EYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNR KVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFD DKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSD GFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLA GSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTT QKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNK VLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRK FDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRM NTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNY HHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMI AKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETN GETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESIRPKRNSDKLIARKKDWDPKKYGGFLWPTVAYSVLWAK VEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVK KDLIIKLPKYSLFELENGRKRMLASAKQLQKGNELALPSKYV NFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFS KRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTRLGA PRAFKYFDTTIDPKQYRSTKEVLDATLIHQSITGLYETRIDLSQ LGGD691 SpRY Cas(H840A) Nickase MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHS IKKNLIGALLFDSGET AERTRLKRT ARRRYTRRKNRIC YLQEIF SNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAY HEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIE GDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSA RLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDL AEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQL WO 2024/238825 PCT/US2024/029746 PEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGT EELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDF YPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETI TPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLY EYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNR KVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFD DKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSD GFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLA GSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTT QKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNK VLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRK FDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRM NTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNY HHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMI AKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETN GETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESIRPKRNSDKLIARKKDWDPKKYGGFLWPTVAYSVLWAK VEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVK KDLIIKLPKYSLFELENGRKRMLASAKQLQKGNELALPSKYV NFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFS KRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTRLGA PRAFKYFDTTIDPKQYRSTKEVLDATLIHQSITGLYETRIDLSQ LGGD692 Met(-) SpRY CasNickase DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSI KKNLIGALLFDSGETAERTRLKRTARRRYTRRKNRICYLQEIF SNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAY HEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIE GDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSA RLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDL AEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQL PEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGT EELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDF YPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETI TPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLY EYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNR KVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFD DKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSD GFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLA GSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTT QKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNK VLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRK FDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRM NTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNY HHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMI AKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETN WO 2024/238825 PCT/US2024/029746 GETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESIRPKRNSDKLIARKKDWDPKKYGGFLWPTVAYSVLWAK VEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVK KDLIIKLPKYSLFELENGRKRMLASAKQLQKGNELALPSKYV NFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFS KRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTRLGA PRAFKYFDTTIDPKQYRSTKEVLDATLIHQSITGLYETRIDLSQ LGGD693 8RGN3.1 MNQKFILGLDIGITSVGYGLIDYETKNIIDAGVRLFPEANVENN EGRRSKRGSRRLKRRRIHRLERVKLLLTEYDLINKEQIPTSNNP YQIRVKGLSEILSKDELAIALLHLAKRRGIHNVDVAADKEETA SDSLSTKDQINKNAKFLESRYVCELQKERLENEGHVRGVENR FLTKDIVREAKKIIDTQMQYYPEIDETFKEKYISLVETRREYFE GPGQGSPFGWNGDLKKWYEMLMGHCTYFPQELRSVKYAYS ADLFNALNDLNNLIIQRDNSEKLEYHEKYHIIENVFKQKKKPT LKQIAKEIGVNPEDIKGYRITKSGTPEFTSFKLFHDLKKVVKD HAILDDIDLLNQIAEILTIYQDKDSIVAELGQLEYLMSEADKQS ISELTGYTGTHSLSLKCMNMIIDELWHSSMNQMEVFTYLNMR PKKYELKGYQRIPTDMIDDAILSPVVKRTFIQSINVINKVIEKY GIPEDIIIELARENNSDDRKKFINNLQKKNEATRKRINEIIGQTG NQNAKRIVEKIRLHDQQEGKCLYSLESIPLEDLLNNPNHYEVD HIIPRSVSFDNSYHNKVLVKQSENSKKSNLTPYQYFNSGKSKL SYNQFKQHILNLSKSQDRISKKKKEYLLEERDINKFEVQKEFIN RNLVDTRYATRELTNYLKAYFSANNMNVKVKTINGSFTDYL RKVWKFKKERNHGYKHHAEDALIIANADFLFKENKKLKAVN SVLEKPEIETKQLDIQVDSEDNYSEMFIIPKQVQDIKDFRNFKY SHRVDKKPNRQLINDTLYSTRKKDNSTYIVQTIKDIYAKDNTT LKKQFDKSPEKFLMYQHDPRTFEKLEVIMKQYANEKNPLAK YHEETGEYLTKYSKKNNGPIVKSLKYIGNKLGSHLDVTHQFK SSTKKLVKLSIKNYRFDVYLTEKGYKFVTIAYLNVFKKDNYY YIPKDKYQELKEKKKIKDTDQFIASFYKNDLIKLNGDLYKIIG VNSDDRNIIELDYYDIKYKDYCEINNIKGEPRIKKTIGKKTESIE KFTTDVLGNLYLHSTEKAPQLIFKRGL694 SRGN3.1(N585A) Nickase MNQKFILGLDIGITSVGYGLIDYETKNIIDAGVRLFPEANVENN EGRRSKRGSRRLKRRRIHRLERVKLLLTEYDLINKEQIPTSNNP YQIRVKGLSEILSKDELAIALLHLAKRRGIHNVDVAADKEETA SDSLSTKDQINKNAKFLESRYVCELQKERLENEGHVRGVENR FLTKDIVREAKKIIDTQMQYYPEIDETFKEKYISLVETRREYFE GPGQGSPFGWNGDLKKWYEMLMGHCTYFPQELRSVKYAYS ADLFNALNDLNNLIIQRDNSEKLEYHEKYHIIENVFKQKKKPT LKQIAKEIGVNPEDIKGYRITKSGTPEFTSFKLFHDLKKVVKD HAILDDIDLLNQIAEILTIYQDKDSIVAELGQLEYLMSEADKQS ISELTGYTGTHSLSLKCMNMIIDELWHSSMNQMEVFTYLNMR PKKYELKGYQRIPTDMIDDAILSPVVKRTFIQSINVINKVIEKY GIPEDIIIELARENNSDDRKKFINNLQKKNEATRKRINEIIGQTG NQNAKRIVEKIRLHDQQEGKCLYSLESIPLEDLLNNPNHYEVD HIIPRSVSFDNSYHNKVLVKQSEASKKSNLTPYQYFNSGKSKL SYNQFKQHILNLSKSQDRISKKKKEYLLEERDINKFEVQKEFIN RNLVDTRYATRELTNYLKAYFSANNMNVKVKTINGSFTDYLRKVWKFKKERNHGYKHHAEDALIIANADFLFKENKKLKAVN WO 2024/238825 PCT/US2024/029746 SVLEKPEIETKQLDIQVDSEDNYSEMFIIPKQVQDIKDFRNFKY SHRVDKKPNRQLINDTLYSTRKKDNSTYIVQTIKDIYAKDNTT LKKQFDKSPEKFLMYQHDPRTFEKLEVIMKQYANEKNPLAK YHEETGEYLTKYSKKNNGPIVKSLKYIGNKLGSHLDVTHQFK SSTKKLVKLSIKNYRFDVYLTEKGYKFVTIAYLNVFKKDNYY YIPKDKYQELKEKKKIKDTDQFIASFYKNDLIKLNGDLYKIIG VNSDDRNIIELDYYDIKYKDYCEINNIKGEPRIKKTIGKKTESIE KFTTDVLGNLYLHSTEKAPQLIFKRGL695 Met(-) 8RGN3.(N584A)Ni ckase NQKFILGLDIGITSVGYGLIDYETKNIIDAGVRLFPEANVENNE GRRSKRGSRRLKRRRIHRLERVKLLLTEYDLINKEQIPTSNNP YQIRVKGLSEILSKDELAIALLHLAKRRGIHNVDVAADKEETA SDSLSTKDQINKNAKFLESRYVCELQKERLENEGHVRGVENR FLTKDIVREAKKIIDTQMQYYPEIDETFKEKYISLVETRREYFE GPGQGSPFGWNGDLKKWYEMLMGHCTYFPQELRSVKYAYS ADLFNALNDLNNLIIQRDNSEKLEYHEKYHIIENVFKQKKKPT LKQIAKEIGVNPEDIKGYRITKSGTPEFTSFKLFHDLKKVVKD HAILDDIDLLNQIAEILTIYQDKDSIVAELGQLEYLMSEADKQS ISELTGYTGTHSLSLKCMNMIIDELWHSSMNQMEVFTYLNMR PKKYELKGYQRIPTDMIDDAILSPVVKRTFIQSINVINKVIEKY GIPEDIIIELARENNSDDRKKFINNLQKKNEATRKRINEIIGQTG NQNAKRIVEKIRLHDQQEGKCLYSLESIPLEDLLNNPNHYEVD HIIPRSVSFDNSYHNKVLVKQSEASKKSNLTPYQYFNSGKSKL SYNQFKQHILNLSKSQDRISKKKKEYLLEERDINKFEVQKEFIN RNLVDTRYATRELTNYLKAYFSANNMNVKVKTINGSFTDYL RKVWKFKKERNHGYKHHAEDALIIANADFLFKENKKLKAVN SVLEKPEIETKQLDIQVDSEDNYSEMFIIPKQVQDIKDFRNFKY SHRVDKKPNRQLINDTLYSTRKKDNSTYIVQTIKDIYAKDNTT LKKQFDKSPEKFLMYQHDPRTFEKLEVIMKQYANEKNPLAK YHEETGEYLTKYSKKNNGPIVKSLKYIGNKLGSHLDVTHQFK SSTKKLVKLSIKNYRFDVYLTEKGYKFVTIAYLNVFKKDNYY YIPKDKYQELKEKKKIKDTDQFIASFYKNDLIKLNGDLYKIIG VNSDDRNIIELDYYDIKYKDYCEINNIKGEPRIKKTIGKKTESIE KFTTDVLGNLYLHSTEKAPQLIFKRGL696 SRGN3.3 MNQKFILGLDIGITSVGYGLIDYETKNIIDAGVRLFPEANVENN EGRRSKRGSRRLKRRRIHRLERVKLLLTEYDLINKEQIPTSNNP YQIRVKGLSEILSKDELAIALLHLAKRRGIHNVDVAADKEETA SDSLSTKDQINKNAKFLESRYVCELQKERLENEGHVRGVENR FLTKDIVREAKKIIDTQMQYYPEIDETFKEKYISLVETRREYFE GPGQGSPFGWNGDLKKWYEMLMGHCTYFPQELRSVKYAYS ADLFNALNDLNNLIIQRDNSEKLEYHEKYHIIENVFKQKKKPT LKQIAKEIGVNPEDIKGYRITKSGTPEFTSFKLFHDLKKVVKD HAILDDIDLLNQIAEILTIYQDKDSIVAELGQLEYLMSEADKQS ISELTGYTGTHSLSLKCMNMIIDELWHSSMNQMEVFTYLNMR PKKYELKGYQRIPTDMIDDAILSPVVKRTFIQSINVINKVIEKY GIPEDIIIELARENNSDDRKKFINNLQKKNEATRKRINEIIGQTG NQNAKRIVEKIRLHDQQEGKCLYSLESIPLEDLLNNPNHYEVD HIIPRSVSFDNSYHNKVLVKQSENSKKSNLTPYQYFNSGKSKL SYNQFKQHILNLSKSQDRISKKKKEYLLEERDINKFEVQKEFIN RNLVDTRYATRELTSYLKAYFSANNMDVKVKTINGSFTNHLR KVWRFDKYRNHGYKHHAEDALIIANADFLFKENKKLQNTNK WO 2024/238825 PCT/US2024/029746 ILEKPTIENNTKKVTVEKEEDYNNVFETPKLVEDIKQYRDYKF SHRVDKKPNRQLINDTLYSTRMKDEHDYIVQTITDIYGKDNT NLKKQFNKNPEKFLMYQNDPKTFEKLSIIMKQYSDEKNPLAK YYEETGEYLTKYSKKNNGPIVKKIKLLGNKVGNHLDVTNKY ENSTKKLVKLSIKNYRFDVYLTEKGYKFVTIAYLNVFKKDNY YYIPKDKYQELKEKKKIKDTDQFIASFYKNDLIKLNGDLYKII GVNSDDRNIIELDYYDIKYKDYCEINNIKGEPRIKKTIGKKTESI EKFTTDVLGNLYLHSTEKAPQLIFKRGL697 SRGN3.3(N585A) Nickase MNQKFILGLDIGITSVGYGLIDYETKNIIDAGVRLFPEANVENN EGRRSKRGSRRLKRRRIHRLERVKLLLTEYDLINKEQIPTSNNP YQIRVKGLSEILSKDELAIALLHLAKRRGIHNVDVAADKEETA SDSLSTKDQINKNAKFLESRYVCELQKERLENEGHVRGVENR FLTKDIVREAKKIIDTQMQYYPEIDETFKEKYISLVETRREYFE GPGQGSPFGWNGDLKKWYEMLMGHCTYFPQELRSVKYAYS ADLFNALNDLNNLIIQRDNSEKLEYHEKYHIIENVFKQKKKPT LKQIAKEIGVNPEDIKGYRITKSGTPEFTSFKLFHDLKKVVKD HAILDDIDLLNQIAEILTIYQDKDSIVAELGQLEYLMSEADKQS ISELTGYTGTHSLSLKCMNMIIDELWHSSMNQMEVFTYLNMR PKKYELKGYQRIPTDMIDDAILSPVVKRTFIQSINVINKVIEKY GIPEDIIIELARENNSDDRKKFINNLQKKNEATRKRINEIIGQTG NQNAKRIVEKIRLHDQQEGKCLYSLESIPLEDLLNNPNHYEVD HIIPRSVSFDNSYHNKVLVKQSEASKKSNLTPYQYFNSGKSKL SYNQFKQHILNLSKSQDRISKKKKEYLLEERDINKFEVQKEFIN RNLVDTRYATRELTSYLKAYFSANNMDVKVKTINGSFTNHLR KVWRFDKYRNHGYKHHAEDALIIANADFLFKENKKLQNTNK ILEKPTIENNTKKVTVEKEEDYNNVFETPKLVEDIKQYRDYKF SHRVDKKPNRQLINDTLYSTRMKDEHDYIVQTITDIYGKDNT NLKKQFNKNPEKFLMYQNDPKTFEKLSIIMKQYSDEKNPLAK YYEETGEYLTKYSKKNNGPIVKKIKLLGNKVGNHLDVTNKY ENSTKKLVKLSIKNYRFDVYLTEKGYKFVTIAYLNVFKKDNY YYIPKDKYQELKEKKKIKDTDQFIASFYKNDLIKLNGDLYKII GVNSDDRNIIELDYYDIKYKDYCEINNIKGEPRIKKTIGKKTESI EKFTTDVLGNLYLHSTEKAPQLIFKRGL698 Met(-) SRGN3.3( N584A)Nic kase NQKFILGLDIGITSVGYGLIDYETKNIIDAGVRLFPEANVENNE GRRSKRGSRRLKRRRIHRLERVKLLLTEYDLINKEQIPTSNNP YQIRVKGLSEILSKDELAIALLHLAKRRGIHNVDVAADKEETA SDSLSTKDQINKNAKFLESRYVCELQKERLENEGHVRGVENR FLTKDIVREAKKIIDTQMQYYPEIDETFKEKYISLVETRREYFE GPGQGSPFGWNGDLKKWYEMLMGHCTYFPQELRSVKYAYS ADLFNALNDLNNLIIQRDNSEKLEYHEKYHIIENVFKQKKKPT LKQIAKEIGVNPEDIKGYRITKSGTPEFTSFKLFHDLKKVVKD HAILDDIDLLNQIAEILTIYQDKDSIVAELGQLEYLMSEADKQS ISELTGYTGTHSLSLKCMNMIIDELWHSSMNQMEVFTYLNMR PKKYELKGYQRIPTDMIDDAILSPVVKRTFIQSINVINKVIEKY GIPEDIIIELARENNSDDRKKFINNLQKKNEATRKRINEIIGQTG NQNAKRIVEKIRLHDQQEGKCLYSLESIPLEDLLNNPNHYEVD HIIPRSVSFDNSYHNKVLVKQSEASKKSNLTPYQYFNSGKSKL SYNQFKQHILNLSKSQDRISKKKKEYLLEERDINKFEVQKEFIN RNLVDTRYATRELTSYLKAYFSANNMDVKVKTINGSFTNHLR KVWRFDKYRNHGYKHHAEDALIIANADFLFKENKKLQNTNK WO 2024/238825 PCT/US2024/029746 ILEKPTIENNTKKVTVEKEEDYNNVFETPKLVEDIKQYRDYKF SHRVDKKPNRQLINDTLYSTRMKDEHDYIVQTITDIYGKDNT NLKKQFNKNPEKFLMYQNDPKTFEKLSIIMKQYSDEKNPLAK YYEETGEYLTKYSKKNNGPIVKKIKLLGNKVGNHLDVTNKY ENSTKKLVKLSIKNYRFDVYLTEKGYKFVTIAYLNVFKKDNY YYIPKDKYQELKEKKKIKDTDQFIASFYKNDLIKLNGDLYKII GVNSDDRNIIELDYYDIKYKDYCEINNIKGEPRIKKTIGKKTESI EKFTTDVLGNLYLHSTEKAPQLIFKRGL699 SpG MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHS IKKNLIGALLFDSGET AEATRLKRT ARRRYTRRKNRIC YLQEIF SNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAY HEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIE GDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSA RLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDL AEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQL PEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGT EELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDF YPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETI TPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLY EYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNR KVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFD DKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSD GFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLA GSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTT QKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNK VLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRK FDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRM NTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNY HHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMI AKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETN GETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFLWPTVAYSVLVVAK VEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVK KDLIIKLPKYSLFELENGRKRMLASAKQLQKGNELALPSKYV NFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFS KRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGA PAAFKYFDTTIDRKQYRSTKEVLDATLIHQSITGLYETRIDLSQ LGGD700 SpG(H840A) NickaseMDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHS IKKNLIGALLFDSGET AEATRLKRT ARRRYTRRKNRIC YLQEIF SNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAY HEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIE GDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSA RLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDL AEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQL PEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGT WO 2024/238825 PCT/US2024/029746 701 EELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDF YPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETI TPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLY EYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNR KVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFD DKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSD GFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLA GSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTT QKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNK VLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRK FDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRM NTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNY HHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMI AKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETN GETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFLWPTVAYSVLVVAK VEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVK KDLIIKLPKYSLFELENGRKRMLASAKQLQKGNELALPSKYV NFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFS KRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGA PAAFKYFDTTIDRKQYRSTKEVLDATLIHQSITGLYETRIDLSQ LGGDMet(-) SpG(H8A) Nickase DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSI KKNLIGALLFDSGET AEATRLKRT ARRRYTRRKNRIC YLQEIF SNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAY HEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIE GDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSA RLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDL AEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQL PEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGT EELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDF YPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETI TPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLY EYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNR KVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFD DKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSD GFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLA GSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTT QKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNK VLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRK FDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRM NTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNY HHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMI AKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETN GETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK WO 2024/238825 PCT/US2024/029746 ESILPKRNSDKLIARKKDWDPKKYGGFLWPTVAYSVLVVAK VEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVK KDLIIKLPKYSLFELENGRKRMLASAKQLQKGNELALPSKYV NFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFS KRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGA PAAFKYFDTTIDRKQYRSTKEVLDATLIHQSITGLYETRIDLSQ LGGD [0225]In some embodiments, a Cas9 protein comprises a variant Cas9 protein containing one or more amino acid substitutions. In some embodiments, a wildtype Cas9 protein comprises a RuvC domain and an HNH domain. In some embodiments, a prime editor comprises a nuclease active Cas9 protein that may cleave both strands of a double stranded target DNA sequence. In some embodiments, the nuclease active Cas9 protein comprises a functional RuvC domain and a functional HNH domain. In some embodiments, a prime editor comprises a Cas9 nickase that can bind to a guide polynucleotide and recognize a target DNA, but can cleave only one strand of a double stranded target DNA. In some embodiments, the Cas9 nickase comprises only one functional RuvC domain or one functional HNH domain. In some embodiments, a prime editor comprises a Cas9 that has a non-functional HNH domain and a functional RuvC domain. In some embodiments, the prime editor can cleave the edit strand (z.e., the PAM strand), but not the non-edit strand of a double stranded target DNA sequence. In some embodiments, a prime editor comprises a Cas9 having a non-functional RuvC domain that can cleave the target strand (z.e., the non- PAM strand), but not the edit strand of a double stranded target DNA sequence. In some embodiments, a prime editor comprises a Cas9 that has neither a functional RuvC domain nor a functional HNH domain, which may not cleave any strand of a double stranded target DNA sequence. [0226]In some embodiments, a prime editor comprises a Cas9 having a mutation in the RuvC domain that reduces or abolishes the nuclease activity of the RuvC domain. In some embodiments, the Cas9 comprises a mutation at amino acid D10 as compared to a wild type SpCas9 as set forth in SEQ ID NO: 674, or a corresponding mutation thereof. In some embodiments, the Cas9 comprises a D10A mutation as compared to a wild type SpCas9 as set forth in SEQ ID NO: 674, or a corresponding mutation thereof. In some embodiments, the Cas9 polypeptide comprises a mutation at amino acid D10, G12, and/or G17 as compared to a wild type SpCas9 as set forth in SEQ ID NO: 674, or a corresponding mutation thereof. In some embodiments, the Cas9 polypeptide comprises a D10A mutation, a G12A mutation, and/or a G17A mutation as compared to a wild type SpCas9 as set forth in SEQ ID NO: 674, or a corresponding mutation thereof.

WO 2024/238825 PCT/US2024/029746 id="p-227"

[0227]In some embodiments, a prime editor comprises a Cas9 polypeptide having a mutation in the HNH domain that reduces or abolishes the nuclease activity of the HNH domain. In some embodiments, the Cas9 polypeptide comprises a mutation at amino acid H840 as compared to a wild type SpCas9 as set forth in SEQ ID NO: 674, or a corresponding mutation thereof. In some embodiments, the Cas9 polypeptide comprises a H840A mutation as compared to a wild type SpCas9 as set forth in SEQ ID NO: 674, or a corresponding mutation thereof. In some embodiments, the Cas9 polypeptide comprises a mutation at amino acid E762, D839, H840, N854, N856, N863, H982, H983, A984, D986, and/or a A987 as compared to a wild type SpCas9 as set forth in SEQ ID NO: 674, or a corresponding mutation thereof. In some embodiments, the Cas9 polypeptide comprises a E762A, D839A, H840A, N854A, N856A, N863A, H982A, H983A, A984A, and/or a D986A mutation as compared to a wild type SpCas9 as set forth in SEQ ID NO: 674, or a corresponding mutation thereof. In some embodiments, the Cas9 polypeptide comprises a mutation at amino acid residue R221, N394, and/or H840 as compared to a wild type SpCas9 (e.g., SEQ ID NO: 674). In some embodiments, the Cas9 polypeptide comprises a R221K, N394L, and/or H840A mutation as compared to a wild type SpCas9 as set forth in SEQ ID NO: 674, or a corresponding mutation thereof. In some embodiments, the Cas9 polypeptide comprises a mutation at amino acid residue R220, N393, and/or H839 as compared to a wild type SpCas9 (e.g., SEQ ID NO: 674) lacking a N-terminal methionine, or a corresponding mutation thereof. In some embodiments, the Cas9 polypeptide comprises a R220K, N393K, and/or H839A mutation as compared to a wild type SpCas9 (as set forth in SEQ ID NO: 674) lacking a N-terminal methionine, or a corresponding mutation thereof. [0228]In some embodiments, a prime editor comprises a Cas9 having one or more amino acid substitutions in both the HNH domain and the RuvC domain that reduce or abolish the nuclease activity of both the HNH domain and the RuvC domain. In some embodiments, the prime editor comprises a nuclease inactive Cas9, or a nuclease dead Cas9 (dCas9). In some embodiments, the dCas9 comprises a H840X substitution and a Di OX mutation compared to a wild type SpCas9 as set forth in SEQ ID NO: 674or corresponding mutations thereof, wherein X is any amino acid other than H for the H840X substitution and any amino acid other than D for the DI OX substitution. In some embodiments, the dead Cas9 comprises a H840A and a D10A mutation as compared to a wild type SpCas9 as set forth in SEQ ID NO: 674, or corresponding mutations thereof.

WO 2024/238825 PCT/US2024/029746 id="p-229"

[0229]In some embodiments, the N-terminal methionine is removed from the amino acid sequence of a Cas9 nickase, or from any Cas9 variant, ortholog, or equivalent disclosed or contemplated herein. For example, methionine-minus (Met (-)) Cas9 nickases include any one of the sequences set forth in SEQ ID NOs: 676, 677, 680, 683, 686, 689, 692, 695, 698, 701, or a variant thereof having an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto. [0230]Besides dead Cas9 and Cas9 nickase variants, the Cas9 proteins used herein may also include other Cas9 variants having at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 99.9% sequence identity to any reference Casprotein, including any wild type Cas9, or mutant Cas9 (e.g., a dead Cas9 or Cas9 nickase), or fragment Cas9, or circular permutant Cas9, or other variant of Cas9 disclosed herein or known in the art. In some embodiments, a Cas9 variant may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acid changes compared to a reference Cas9, e.g., a wild type Cas9. In some embodiments, the Casvariant comprises a fragment of a reference Cas9 (e.g, a gRNA binding domain or a DNA- cleavage domain), such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of a reference Cas9, e.g., a wild type Cas9. In some embodiments, the fragment is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid length of a corresponding wild type Cas9. [0231]In some embodiments, a Cas9 fragment is a functional fragment that retains one or more Cas9 activities. In some embodiments, the Cas9 fragment is at least 100 amino acids in length. In some embodiments, the fragment is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, or at least 1300 amino acids in length. [0232]In some embodiments, a prime editor comprises a Cas protein, e.g., Cas9, containing modifications that allow altered PAM recognition. In prime editing using a Cas-protein-based WO 2024/238825 PCT/US2024/029746 prime editor, a "protospacer adjacent motif (PAM)", PAM sequence, or PAM-like motif, may be used to refer to a short DNA sequence immediately following the protospacer sequence on the PAM strand of the target gene. In some embodiments, the PAM is recognized by the Cas nuclease in the prime editor during prime editing. In certain embodiments, the PAM is required for target binding of the Cas protein. The specific PAM sequence required for Cas protein recognition may depend on the specific type of the Cas protein. A PAM can be 1,2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides in length. In some embodiments, a PAM is between 2-6 nucleotides in length. In some embodiments, the PAM can be a 5' PAM (z.e., located upstream of the 5' end of the protospacer). In other embodiments, the PAM can be a 3' PAM (z.e., located downstream of the 5' end of the protospacer).In some embodiments, the Cas protein of a prime editor recognizes a canonical PAM, for example, a SpCas9 recognizes 5'-NGG-3' PAM. In some embodiments, the Cas protein of a prime editor has altered or non-canonical PAM specificities. Exemplary PAM sequences and corresponding Cas variants are described in Table 23below. It should be appreciated that for each of the variants provided, the Cas protein comprises one or more of the amino acid substitutions as indicated compared to a wild type Cas protein sequence, for example, the Cas9 as set forth in SEQ ID NO: 674. The PAM motifs as shown in Table below are in the order of 5’ to 3’. In some embodiments, the Cas proteins of the disclosure can also be used to direct transcriptional control of target sequences, for example silencing transcription by sequence-specific binding to target sequences. In some embodiments, a Cas protein described herein may have one or mutations in a PAM recognition motif. In some embodiments, a Cas protein described herein may have altered PAM specificity. [0233]As used in PAM sequences in Table 23, "N" refers to any one of nucleotides A, G, C, and T, "R" refers to nucleotide A or G, "V" refers to nucleotide A or T; "V" refers to any one of nucleotides A or C or G, and "Y" refers to nucleotide C or T. Table 23: Cas protein variants and corresponding PAM sequences Variant PAM spCas9 (wild type) NGG, NGA, NAG, NGNGAspCas9- VRVRFRR RI 335V, Li 111R, D1135 V, G1218R, E1219F, A1322R, T1337RNG spCas9-VQR (D1135V, R1335Q, T1337R) NGAspCas9-EQR (D1135E, R1335Q, T1337R) NGAspCas9-VRER (D1135V, G1218R, R1335E, T1337R) NGCG WO 2024/238825 PCT/US2024/029746 spCas9-VRQR (D1135V, G1218R, R1335Q, T1337R) NGACas9-NG (L1111R, D1135V, G1218R, E1219F, A1322R, T1337R, R1335V)NGN SpG Cas9 (D1135L, S1136W, G1218K, E1219Q,R1335Q, T1337R)NGN SyRY Cas9(A61R, L1111R, N1317R, A1322R, andR1333P)NRN xCas9 (E480K, E543D, E1219V, K294R, Q1256K, A262T, S409I, M694I)NGN SluCa9 NNGGsRGNI, sRGN2, sRGN4, SRGN3.1, SRGN3.3 NNGGsaCas9 NNGRRT, NNGRRNsaCas9-KKH (E782K, N968K, R1015H) NNNRRTspCas9-MQKSER (D1135M, S1136Q, G1218K, E1219S, R1335E,T1337R)NGCG/NGCN spCas9-LRKIQK(D1135L, S1136R, G1218K, E1219I, R1335Q, T1337K)NGTN spCas9-LRVSQK (D1135L, S1136R, G1218V, E1219S, R1335Q, T1337K)NGTN spCas9-LRVSQL(D1135L, S1136R, G1218V, E1219S, R1335Q, T1337L)NGTN Cpfl TTTVSpy-Mac NAANmCas9 NNNNGATTStCas9 NNAGAAWTdCas9 NAAAAC id="p-234"

[0234]In some embodiments, a prime editor comprises a Cas9 polypeptide comprising one or mutations selected from the group consisting of: A61R, LI 11R, D1135V, R221K, A262T, R324L, N394K, S409I, S409I, E427G, E480K, M495V, N497A, Y515N, K526E, F539S, E543D, R654L, R661A, R661L, R691A, N692A, M694A, M694I, Q695A, H698A, R753G, M763I, K848A, K890N, Q926A, K1003A, R1060A, LI 111R, RI 114G, D1135E, D1135L, D1135N, S1136W, V1139A, D1180G, G1218K, G1218R, G1218S, E1219Q, E1219V, E1219V, Q1221H, P1249S, E1253K, N1317R, A1320V, P1321S, A1322R, 11322V, D1332G, R1332N, A1332R, R1333K, R1333P, R1335L, R1335Q, R1335V, T1337N, WO 2024/238825 PCT/US2024/029746 T1337R, S1338T, H1349R, and any combinations thereof as compared to a wildtype SpCaspolypeptide as set forth in SEQ ID NO: 674. [0235]In some embodiments, a prime editor comprises a SaCas9 polypeptide. In some embodiments, the SaCas9 polypeptide comprises one or more of mutations E782K, N968K, and R1015H as compared to a wild type SaCas9. In some embodiments, a prime editor comprises a FnCas9 polypeptide, for example, a wildtype FnCas9 polypeptide or a FnCaspolypeptide comprising one or more of mutations E1369R, E1449H, or R1556A as compared to the wild type FnCas9. In some embodiments, a prime editor comprises a Sc Cas9, for example, a wild type ScCas9 or a ScCas9 polypeptide comprises one or more of mutations I367K, G368D, I369K, H371L, T375S, T376G, and T1227K as compared to the wild type ScCas9. In some embodiments, a prime editor comprises a Stl Cas9 polypeptide, a St3 Caspolypeptide, or a SluCas9 polypeptide. [0236]In some embodiments, a prime editor comprises a Cas polypeptide that comprises a circular permutant Cas variant. For example, a Cas9 polypeptide of a prime editor may be engineered such that the N-terminus and the C-terminus of a Cas9 protein (e.g., a wild type Cas9 protein, or a Cas9 nickase) are topically rearranged to retain the ability to bind DNA when complexed with a guide RNA (gRNA). An exemplary circular permutant configuration may be N-terminus-[original C-terminus]-[original N-terminus]-C-terminus. Any of the Cas9 proteins described herein, including any variant, ortholog, or naturally occurring Casor equivalent thereof, may be reconfigured as a circular permutant variant. [0237]In various embodiments, the circular permutants of a Cas protein, e.g., a Cas9, may have the following structure: N-terminus-[original C-terminus]-[optional linker]-[original N- terminus]-C-terminus. In some embodiments, a circular permutant Cas9 comprises any one of the following structures (amino acid positions as set forth in SEQ ID NO: 674): [0238]N-terminus-[1268-1368]-[optional linker]-[l-1267]-C-terminus; [0239]N-terminus-[ 1168- 1368]-[optional linker]-[!-1167]-C-terminus; [0240]N-terminus-[1068-1368]-[optional linker]-[!-1067]-C-terminus; [0241]N-terminus-[968-1368]-[optional linker]-[l-967]-C-terminus; [0242]N-terminus-[868-1368]-[optional linker]-[l-867]-C-terminus; [0243]N-terminus-[768-1368]-[optional linker]-[l-767]-C-terminus; [0244]N-terminus-[668-1368]-[optional linker]-[l-667]-C-terminus; [0245]N-terminus-[568-1368]-[optional linker]-[l-567]-C-terminus; [0246]N-terminus-[468-1368]-[optional linker]-[l-467]-C-terminus; WO 2024/238825 PCT/US2024/029746 id="p-247"

[0247]N-terminus-[368-1368]-[optional linker]-[ 1-367]-C-terminus; [0248]N-terminus-[268-1368]-[optional linker]-[ l-267]-C-terminus; [0249]N-terminus-[168-1368]-[optional linker]-[l-167]-C-terminus; [0250]N-terminus-[68-1368]-[optional linker]-[ l-67]-C-terminus; [0251]N-terminus-[10-1368]-[optional linker]-[ l-9]-C-terminus, or the corresponding circular permutants of other Cas9 proteins (including other Cas9 orthologs, variants, etc). [0252]In some embodiments, a circular permutant Cas9 comprises any one of the following structures (amino acid positions as set forth in SEQ ID NO: 674): [0253]N-terminus-[102-1368]-[optional linker]-[! -101 ]-C-terminus; [0254]N-terminus-[ 1028-1368]-[optional linker]-[ 1-1027]-C-terminus; [0255]N-terminus-[ 1041-1368]-[optional linker]-[ 1-1043]-C-terminus; [0256]N-terminus-[1249-1368]-[optional linker]-[!-1248]-C-terminus; or [0257]N-terminus-[1300-1368]-[optional linker]-[!-1299]-C-terminus, or the corresponding circular permutants of other Cas9 proteins (including other Cas9 orthologs, variants, etc). [0258]In some embodiments, a circular permutant Cas9 comprises any one of the following structures (amino acid positions as set forth in SEQ ID NO: 674- 1368 amino acids of UniProtKB - Q99ZW2 N-terminus-[103-1368]-[optional linker]-[!-102]-C-terminus: [0259]N-terminus-[ 1029-1368]-[optional linker]-[ 1-1028]-C-terminus; [0260]N-terminus-[ 1042-1368]-[optional linker]-[ 1-1041 ]-C-terminus; [0261]N-terminus-[1250-1368]-[optional linker]-[!-1249]-C-terminus; or [0262]N-terminus-[1301-1368]-[optional linker]-[l-1300]-C-terminus, or the corresponding circular permutants of other Cas9 proteins (including other Cas9 orthologs, variants, etc). [0263]In some embodiments, the circular permutant can be formed by linking a C-terminal fragment of a Cas9 to an N-terminal fragment of a Cas9, either directly or by using a linker, such as an amino acid linker. In some embodiments, thee C-terminal fragment may correspond to the 95% or more of the C-terminal amino acids of a Cas9 (e.g., amino acids about 1300-1368 as set forth in SEQ ID No: 674 or corresponding amino acid positions thereof), or the 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% or more of the C-terminal amino acids of a Cas9 (e.g., SEQ ID NO 674 or a ortholog or a variant thereof). The N-terminal portion may correspond to 95% or more of the N-terminal amino acids of a Cas9 (e.g., amino acids about 1-1300 as set forth in WO 2024/238825 PCT/US2024/029746 SEQ ID NO: 674 or corresponding amino acid positions thereof), or 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% or more of the N terminal amino acids of a Cas9 (e.g., as set forth in SEQ ID NO: 674 or corresponding amino acid positions thereof). [0264]In some embodiments, the circular permutant can be formed by linking a C-terminal fragment of a Cas9 to an N-terminal fragment of a Cas9, either directly or by using a linker, such as an amino acid linker. In some embodiments, the C-terminal fragment that is rearranged to the N-terminus includes or corresponds to the C-terminal 30% or less of the amino acids of a Cas9 (e.g., amino acids 1012-1368 as set forth in SEQ ID NO: 674 or corresponding amino acid positions thereof). In some embodiments, the C-terminal fragment that is rearranged to the N-terminus, includes or corresponds to the C-terminal 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%,5%, 4%, 3%, 2%, or 1% of the amino acids of a Cas(e.g., as set forth in SEQ ID NO: 674 or corresponding amino acid positions thereof). In some embodiments, the C-terminal fragment that is rearranged to the N-terminus, includes or corresponds to the C-terminal 410 residues or less of a Cas9 (e.g., as set forth in SEQ ID No: 674 or corresponding amino acid positions thereof). In some embodiments, the C-terminal portion that is rearranged to the N-terminus, includes or corresponds to the C-terminal 410, 400, 390, 380, 370, 360, 350, 340, 330, 320, 310, 300, 290, 280, 270, 260, 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 residues of a Cas9 (e.g., as set forth in SEQ ID NO: 674 or corresponding amino acid positions thereof). In some embodiments, the C-terminal portion that is rearranged to the N-terminus includes or corresponds to the C-terminal 357, 341, 328, 120, or 69 residues of a Cas9 (e.g., as set forth in SEQ ID NO: 674 or corresponding amino acid positions thereof). [0265]In other embodiments, circular permutant Cas9 variants may be a topological rearrangement of a Cas9 primary structure based on the following method, which is based on S. pyogenes Cas9 of SEQ ID NO: 674: (a) selecting a circular permutant (CP) site corresponding to an internal amino acid residue of the Cas9 primary structure, which dissects the original protein into two halves: an N-terminal region and a C-terminal region; (b) modifying the Cas9 protein sequence (e.g., by genetic engineering techniques) by moving the original C-terminal region (comprising the CP site amino acid) to precede the original N- terminal region, thereby forming a new N-terminus of the Cas9 protein that now begins with the CP site amino acid residue. The CP site can be located in any domain of the Cas9 protein, WO 2024/238825 PCT/US2024/029746 including, for example, the helical-II domain, the RuvCIII domain, or the CTD domain. For example, the CP site may be located (as set forth in SEQ ID NO: 674 or corresponding amino acid positions thereof) at original amino acid residue 181, 199, 230, 270, 310, 1010,1016, 1023, 1029, 1041,1247,1249, or 1282. Thus, once relocated to the N-terminus, original amino acid 181, 199, 230, 270, 310, 1010, 1016, 1023, 1029, 1041, 1247, 1249, or 12would become the new N-terminal amino acid. Nomenclature of these CP-Cas9 proteins may be referred to as Cas9-CP181, Cas9-CP199, Cas9-CP230, Cas9-CP270, Cas9-CP310, Cas9-CP1010, Cas9-CP1016, Cas9-CP1023, Cas9-CP1029, Cas9-CP1041, Cas9-CP1247, Cas9-CP1249, andCas9- CP1282, respectively. This description is not meant to be limited to making CP variants from SEQ ID NO: 674, but may be implemented to make CP variants in any Cas9 sequence, either at CP sites that correspond to these positions, or at other CP sites entirely. This description is not meant to limit the specific CP sites in any way. Virtually any CP site may be used to form a CP-Cas9 variant. [0266]In some embodiments, a prime editor comprises a Cas9 functional variant that is of smaller molecular weight than a wild type SpCas9 protein. In some embodiments, a smaller- sized Cas9 functional variant may facilitate delivery to cells, e.g., by an expression vector, nanoparticle, or other means of delivery. In certain embodiments, a smaller-sized Casfunctional variant is a Class 2 Type II Cas protein. In certain embodiments, a smaller-sized Cas9 functional variant is a Class 2 Type V Cas protein. In certain embodiments, a smaller- sized Cas9 functional variant is a Class 2 Type VI Cas protein. [0267]In some embodiments, a prime editor comprises a SpCas9 that is 1368 amino acids in length and has a predicted molecular weight of 158 kilodaltons. In some embodiments, a prime editor comprises a Cas9 functional variant or functional fragment that is less than 13amino acids, less than 1290 amino acids, than less than 1280 amino acids, less than 12amino acids, less than 1260 amino acid, less than 1250 amino acids, less than 1240 amino acids, less than 1230 amino acids, less than 1220 amino acids, less than 1210 amino acids, less than 1200 amino acids, less than 1190 amino acids, less than 1180 amino acids, less than 1170 amino acids, less than 1160 amino acids, less than 1150 amino acids, less than 1140 amino acids, less than 1130 amino acids, less than 1120 amino acids, less than 11amino acids, less than 1100 amino acids, less than 1050 amino acids, less than 1000 amino acids, less than 950 amino acids, less than 900 amino acids, less than 850 amino acids, less than 800 amino acids, less than 750 amino acids, less than 700 amino acids, less than 6amino acids, less than 600 amino acids, less than 550 amino acids, or less than 500 amino WO 2024/238825 PCT/US2024/029746 acids, but at least larger than about 400 amino acids and retaining the one or more functions, e.g., DNA binding function, of the Cas9 protein. [0268]In some embodiments, the Cas protein may include any CRISPR associated protein, including but not limited to, Casl2a, Casl2bl, Casl, CasIB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Csel, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, homologs thereof, or modified versions thereof, and preferably comprising a nickase mutation (e.g., a mutation corresponding to the D10A mutation of the wild type Cas9 polypeptide of SEQ ID NO: 674). In various other embodiments, the napDNAbp can be any of the following proteins: a Cas9, a Cas 12a (Cpfl), a Casl2e (CasX), a Casl 2d (CasY), a Casl2bl (C2cl), a Casl3a (C2c2), a Casl2c (C2c3), a GeoCas9, a CjCas9, a Casl2g, a Casl2h, a Casl2i, a Casl3b, a Casl3c, a Casl3d, a Casl4, a Csn2, an xCas9, an SpCas9-NG, a circularly permuted Cas9, or an Argonaute (Ago) domain, or a functional variant or fragment thereof. [0269]Exemplary Cas proteins and nomenclature are shown in Table 24below: Table 24:Exemplary Cas proteins and nomenclature Legacy nomenclature Current nomenclature type 11 CRISPR-Cas enzymesCas9 sametype V CRISPR-Cas enzymesCpfl Cas 12aCasX Casl2eC2cl Casl2blCasl2b2 sameC2c3 Cas 12cCasY Casl2dC2c4 sameC2c8 sameC2c5 sameC2cl0 sameC2c9 sametype VI CRISPR-Cas enzymesC2c2 Casl 3aCasl 3d sameC2c7 Casl 3cC2c6 Casl 3b id="p-270"

[0270]In some embodiments, prime editors described herein may also comprise Cas proteins other than Cas9. For example, in some embodiments, a prime editor as described herein may WO 2024/238825 PCT/US2024/029746 100 comprise a Cas 12a (Cpfl) polypeptide or functional variants thereof. In some embodiments, the Cas 12a polypeptide comprises a mutation that reduces or abolishes the endonuclease domain of the Cas 12a polypeptide. In some embodiments, the Cas 12a polypeptide is a Cas 12a nickase. In some embodiments, the Cas protein comprises an amino acid sequence that comprises at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a naturally occurring Casl2a polypeptide. [0271]In some embodiments, a prime editor comprises a Cas protein that is a Cas 12b (C2cl) or a Cas 12c (C2c3) polypeptide. In some embodiments, the Cas protein comprises an amino acid sequence that comprises at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a naturally occurring Cas 12b (C2cl) or Cas 12c (C2c3) protein. In some embodiments, the Cas protein is a Cas 12b nickase or a Cas 12c nickase. In some embodiments, the Cas protein is a Casl2e, a Cas 12d, a Casl3, Cas 14a, Cas 14b, Cas 14c, Casl4d, Casl4e, Casl4f, Cas 14g, Casl4h, Casl4u, or a CasO polypeptide. In some embodiments, the Cas protein comprises an amino acid sequence that comprises at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a naturally-occurring Casl2e, Cas 12d, Cas 13, Casl4a, Casl4b, Casl4c, Casl4d, Casl4e, Casl4f, Casl4g, Casl4h, Casl4u, or Cas WO 2024/238825 PCT/US2024/029746 101 id="p-274"

[0274]In some instances, a prime editor may further comprise at least one nuclear localization sequence (NLS). In some cases, a prime editor may further comprise 1 NLS. In some cases, a prime editor may further comprise 2 NLSs. In other cases, a prime editor may further comprise 3 NLSs. In one case, a primer editor can further comprise more than 4, 5, 6, 7, 8, 9 or 10 NLSs. [0275]In addition, the NLSs can be expressed as part of a prime editor complex. In some embodiments, a NLS can be positioned almost anywhere in a protein's amino acid sequence, and generally comprises a short sequence of three or more or four or more amino acids. The location of the NLS fusion can be at the N-terminus, the C-terminus, or positioned anywhere within a sequence of a prime editor or a component thereof (e.g., inserted between the DNA- binding domain and the DNA polymerase domain of a prime editor fusion protein, between the DNA binding domain and a linker sequence, between a DNA polymerase and a linker sequence, between two linker sequences of a prime editor fusion protein or a component thereof, in either N-terminus to C-terminus or C-terminus to N-terminus order). In some embodiments, a prime editor is fusion protein that comprises an NLS at the N terminus. In some embodiments, a prime editor is fusion protein that comprises an NLS at the C terminus. In some embodiments, a prime editor is fusion protein that comprises at least one NLS at both the N terminus and the C terminus. In some embodiments, the prime editor is a fusion protein that comprises two NLSs at the N terminus and/or the C terminus. [0276]Any NLSs that are known in the art are also contemplated herein. The NLSs may be any naturally occurring NLS, or any non-naturally occurring NLS (e.g., an NLS with one or more mutations relative to a wild-type NLS). In some embodiments, the one or more NLSs of a prime editor comprise bipartite NLSs. In some embodiments, a nuclear localization signal (NLS) is predominantly basic. In some embodiments, the one or more NLSs of a prime editor are rich in lysine and arginine residues. In some embodiments, the one or more NLSs of a prime editor comprise proline residues. In some embodiments, a nuclear localization signal (NLS) comprises the sequence MDSLLMNRRKFLYQFKNVRWAKGRRETYLC (SEQ ID NO: 702), KRTADGSEFESPKKKRKV (SEQ ID NO: 703), KRTADGSEFEPKKKRKV (SEQ ID NO: 704), NLSKRPAAIKKAGQAKKKK (SEQ ID NO: 705), RQRRNELKRSF (SEQ ID NO: 706), or NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 707). [0277]In some embodiments, a NLS is a monopartite NLS. For example, in some embodiments, a NLS is a SV40 large T antigen NLS PKKKRKV (SEQ ID NO: 708). In WO 2024/238825 PCT/US2024/029746 102 some embodiments, a NLS is a bipartite NLS. In some embodiments, a bipartite NLS comprises two basic domains separated by a spacer sequence comprising a variable number of amino acids. In some embodiments, a NLS is a bipartite NLS. In some embodiments, a bipartite NLS consists of two basic domains separated by a spacer sequence comprising a variable number of amino acids. In some embodiments, the spacer amino acid sequence comprises the sequence KRXXXXXXXXXXKKKL (Xenopus nucleoplasmin NLS) (SEQ ID NO: 709), wherein X is any amino acid. In some embodiments, the NLS comprises a nucleoplasmin NLS sequence KRPAATKKAGQAKKKK (SEQ ID NO: 710). In some embodiments, a NLS is a noncanonical sequences such as M9 of the hnRNP Al protein, the influenza virus nucleoprotein NLS, and the yeast Gal4 protein NLS.In some embodiments, a NLS is a noncanonical sequences such as M9 of the hnRNP Al protein, the influenza virus nucleoprotein NLS, and the yeast Gal4 protein NLS. [0278]Other non-limiting examples of NLS sequences are provided in Table 25 below. In some embodiments, a bipartite NLS consists of two basic domains separated by a spacer sequence comprising a variable number of amino acids. In some embodiments, a NLS comprises an amino acid sequence that is at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 702-720. In some embodiments, a NLS comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 702-720. In some embodiments, a prime editing composition comprises a polynucleotide that encodes a NLS that comprises an amino acid sequence that is at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 702-720. In some embodiments, a prime editing composition comprises a polynucleotide that encodes a NLS that comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 702-720. [0279]Any NLSs that are known in the art are also contemplated herein. The NLSs may be any naturally occurring NLS, or any non-naturally occurring NLS (e.g., an NLS with one or more mutations relative to a wild-type NLS). In some embodiments, the one or more NLSs of a prime editor comprise bipartite NLSs. In some embodiments, the one or more NLSs of a prime editor are rich in lysine and arginine residues. In some embodiments, the one or more NLSs of a prime editor comprise proline residues. Non-limiting examples of NLS sequences are provided in Table 25 below. Table 25:Exemplary nuclear localization sequences WO 2024/238825 PCT/US2024/029746 103 Description Sequence SEQ ID NO: NLS of SV40 Large T-AG PKKKRKV 708NLS MKRTADGSEFESPKKKRKV 711NLS MDSLLMNRRKFLYQFKNVRWAKGRRETYLC 702NLS of Nucleoplasmin AVKRPAATKKAGQAKKKKLD 712NLS of EGL-13 MSRRRKANPTKLSENAKKLAKEVEN 713NLS of C-Myc PAAKRVKLD 714NLS of Tus-protein KLKIKRPVK 715NLS of polyoma large T- AGVSRKRPRP 716 NLS of Hepatitis D virus antigenEGAPPAKRAR 717 NLS of Rev protein RQARRNRRRRWRERNR 718NLS of murine p53 PPQPKKKPLDGE 719C terminal linker and NLS of an exemplary prime editor fusion protein SGGSKRTADGSEFEPKKKRKV 720 NLS KRTADGSEFEPKKKRKV 704 id="p-280"

[0280]In some embodiments, a prime editing complex comprises a fusion protein comprising a DNA binding domain (e.g, Cas9(H840A)) and a reverse transcriptase (e.g, a variant MMLV RT) having the following structure: [NLS]-[Cas9(H840A)]-[linker]- [MMLV_RT(D200N)(T330P)(L603W)(T306K)(W313F)], and a desired PEgRNA. In some embodiments, the prime editing complex comprises a prime editor fusion protein that has the amino acid sequence of SEQ ID NO: 740. Sequence of an exemplary prime editor fusion protein comprising a DNA binding domain (e.g, Cas9(H840A)) and a reverse transcriptase (e.g, a variant MMLV RT) having the following structure: [NLS]- [Cas9(H840A)]-[linker]- [MMLV_RT(D200N)(T330P)(L603W)(T306K)(W313F)] and its components are shown in Table 26. [0281]In some embodiments, a prime editing complex comprises a fusion protein comprising a DNA binding domain (e.g, Cas9((R221K N394K H840A)) and a reverse transcriptase (e.g, a variant MMLV RT) having the following structure: [NLS]- [Cas9((R221K N394K H840A)]-[linker]- [MMLV_RT(D200N)(T330P)(L603W)(T306K)(W313F)], and a desired PEgRNA. In some embodiments, the prime editing complex comprises a prime editor fusion protein that has the amino acid sequence of SEQ ID NO: 741. Sequence of an exemplary prime editor fusion protein comprising a DNA binding domain (e.g, Cas9(H840A)) and a reverse transcriptase (e.g, a variant MMLV RT) having the following structure: [NLS]- [Cas9 (R221K N394K WO 2024/238825 PCT/US2024/029746 104 H840A)]-[linker]-[MMLV_RT(D200N)(T330P)(L603W)(T306K)(W3 13F)] and its components are shown in Table 27. [0282]Polypeptides comprising components of a prime editor may be fused via peptide linkers, or may be provided in trans relevant to each other. For example, a reverse transcriptase may be expressed, delivered, or otherwise provided as an individual component rather than as a part of a fusion protein with the DNA binding domain. In such cases, components of the prime editor may be associated through non-peptide linkages or co- localization functions. In some embodiments, a prime editor further comprises additional components capable of interacting with, associating with, or capable of recruiting other components of the prime editor or the prime editing system. For example, a prime editor may comprise an RNA-protein recruitment polypeptide that can associate with an RNA-protein recruitment RNA aptamer. In some embodiments, an RNA-protein recruitment polypeptide can recruit, or be recruited by, a specific RNA sequence. Non limiting examples of RNA- protein recruitment polypeptide and RNA aptamer pairs include a MS2 coat protein and a MS2 RNA hairpin, a PCP polypeptide and a PP7 RNA hairpin, a Com polypeptide and a Com RNA hairpin, a Ku protein and a telomerase Ku binding RNA motif, and a Sm7 protein and a telomerase Sm7 binding RNA motif. In some embodiments, the prime editor comprises a DNA binding domain fused or linked to an RNA-protein recruitment polypeptide. In some embodiments, the prime editor comprises a DNA polymerase domain fused or linked to an RNA-protein recruitment polypeptide. In some embodiments, the DNA binding domain and the DNA polymerase domain fused to the RNA-protein recruitment polypeptide, or the DNA binding domain fused to the RNA-protein recruitment polypeptide and the DNA polymerase domain are co-localized by the corresponding RNA-protein recruitment RNA aptamer of the RNA-protein recruitment polypeptide. In some embodiments, the corresponding RNA- protein recruitment RNA aptamer fused or linked to a portion of the PEgRNA or ngRNA. For example, an MS2 coat protein fused or linked to the DNA polymerase and a MS2 hairpin installed on the PEgRNA for co-localization of the DNA polymerase and the RNA-guided DNA binding domain (e.g., a Cas9 nickase). In certain embodiments, components of a prime editor are directly fused to each other. In certain embodiments, components of a prime editor are associated to each other via a linker. [0283]In some embodiments, a prime editor comprises a polypeptide domain, an MS2 coat protein (MCP), that recognizes an MS2 hairpin. In some embodiments, the nucleotide sequence of the MS2 hairpin (or equivalently referred to as the "MS2 aptamer ") is: WO 2024/238825 PCT/US2024/029746 105 GCCAACATGAGGATCACCCATGTCTGCAGGGCC (SEQ ID NO: 721). In some embodiments, the amino acid sequence of the MCP is:GSASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQ NRKYTIKVEVPKVATQTVGGEELPVAGWRSYLNMELTIPIFATNSDCELIVKAMQGL LKDGNPIPSAIA ANSGIY (SEQ ID NO: 722). [0284]As used herein, a linker can be any chemical group or a molecule linking two molecules or moieties, e.g., a DNA binding domain and a polymerase domain of a prime editor. In some embodiments, a linker is an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker comprises a non-peptide moiety. The linker may be as simple as a covalent bond, or it may be a polymeric linker many atoms in length, for example, a polynucleotide sequence. In certain embodiments, the linker is a covalent bond (e.g, a carbon-carbon bond, disulfide bond, carbon-heteroatom bond, etc.). [0285]In certain embodiments, two or more components of a prime editor are linked to each other by a peptide linker. In some embodiments, a peptide linker is 5-100 amino acids in length, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30-35, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-150, or 150-200 amino acids in length. In some embodiments, the peptide linker is amino acids in length, 24 amino acids in length, 64 amino acids in length, or 96 amino acids in length. [0286]In some embodiments, the linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 723), (G)n (SEQ ID NO: 724), (EAAAK)n (SEQ ID NO: 725), (GGS)n (SEQ ID NO: 726), (SGGS)n (SEQ ID NO: 727), (XP)n (SEQ ID NO: 728), or any combination thereof, wherein n is independently an integer between 1 and 30, and wherein X is any amino acid. In some embodiments, the linker comprises the amino acid sequence (GGS)n (SEQ ID NO: 726), wherein n is 1, 3, or 7. In some embodiments, the linker comprises the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 729). In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 730). In some embodiments, the linker comprises the amino acid sequence SGGSGGSGGS (SEQ ID NO: 731). In some embodiments, the linker comprises the amino acid sequence SGGS (SEQ ID NO: 732). In other embodiments, the linker comprises the amino acid sequenceSGGSSGGSSGSETPGTSESATPESAGSYPYDVPDYAGSAAPAAKKKKLDGSGSGGSS GGS (SEQ ID NO: 733).

WO 2024/238825 PCT/US2024/029746 106 id="p-287"

[0287]In some embodiments, a linker comprises 1-100 amino acids. In some embodiments, the linker comprises the amino acid sequence GGSGGS (SEQ ID NO: 734), GGSGGSGGS (SEQ ID NO: 735), or SGGSSGGSSGSETPGTSESATPESSGGSSGGSS (SEQ ID NO: 736). [0288]In certain embodiments, two or more components of a prime editor are linked to each other by a non-peptide linker. In some embodiments, the linker is a carbon-nitrogen bond of an amide linkage. In certain embodiments, the linker is a cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic or heteroaliphatic linker. In certain embodiments, the linker is polymeric (e.g, polyethylene, polyethylene glycol, polyamide, polyester, etc.). In certain embodiments, the linker comprises a monomer, dimer, or polymer of aminoalkanoic acid. In certain embodiments, the linker comprises an aminoalkanoic acid (e.g, glycine, ethanoic acid, alanine, beta-alanine, 3- aminopropanoic acid, 4-aminobutanoic acid, 5-pentanoic acid, etc.). In certain embodiments, the linker comprises a monomer, dimer, or polymer of aminohexanoic acid (Ahx). In certain embodiments, the linker is based on a carbocyclic moiety (e.g, cyclopentane, cyclohexane). In other embodiments, the linker comprises a polyethylene glycol moiety (PEG). In certain embodiments, the linker comprises an aryl or heteroaryl moiety. In certain embodiments, the linker is based on a phenyl ring. The linker may include functionalized moieties to facilitate attachment of a nucleophile (e.g, thiol, amino) from the peptide to the linker. Any electrophile may be used as part of the linker. Exemplary electrophiles include, but are not limited to, activated esters, activated amides, Michael acceptors, alkyl halides, aryl halides, acyl halides, and isothiocyanates. [0289]Components of a prime editor may be connected to each other in any order. In some embodiments, the DNA binding domain and the DNA polymerase domain of a prime editor may be fused to form a fusion protein, or may be joined by a peptide or protein linker, in any order from the N terminus to the C terminus. In some embodiments, a prime editor comprises a DNA binding domain fused or linked to the C-terminal end of a DNA polymerase domain. In some embodiments, a prime editor comprises a DNA binding domain fused or linked to the N-terminal end of a DNA polymerase domain. In some embodiments, the prime editor comprises a fusion protein comprising the structure NH2-[DNA binding domain]- [polymerase]-COOH; or NH2-[polymerase]-[DNA binding domain]-COOH, wherein each instance of "]-[" indicates the presence of an optional linker sequence. In some embodiments, a prime editor comprises a fusion protein and a DNA polymerase domain provided in trans, wherein the fusion protein comprises the structure NH2-[DNA binding domain]-[RNA- WO 2024/238825 PCT/US2024/029746 107 protein recruitment polypeptide]-COOH. In some embodiments, a prime editor comprises a fusion protein and a DNA binding domain provided in trans, wherein the fusion protein comprises the structure NH2-[DNA polymerase domain]-[RNA-protein recruitment polypeptide]-COOH. [0290]In some embodiments, a prime editor fusion protein, a polypeptide component of a prime editor, or a polynucleotide encoding the prime editor fusion protein or polypeptide component, may be split into an N-terminal half and a C-terminal half or polypeptides that encode the N-terminal half and the C terminal half, and provided to a target DNA in a cell separately. For example, in certain embodiments, a prime editor fusion protein may be split into a N-terminal and a C-terminal half for separate delivery in AAV vectors, and subsequently translated and colocalized in a target cell to reform the complete polypeptide or prime editor protein. In such cases, separate halves of a protein or a fusion protein may each comprise a split-intein to facilitate colocalization and reformation of the complete protein or fusion protein by the mechanism of intein facilitated trans splicing. In some embodiments, a prime editor comprises a N-terminal half fused to an intein-N, and a C-terminal half fused to an intein-C, or polynucleotides or vectors (e.g., AAV vectors) encoding each thereof. When delivered and/or expressed in a target cell, the intein-N and the intein-C can be excised via protein trans-splicing, resulting in a complete prime editor fusion protein in the target cell. In some embodiments, an exemplary protein described herein may lack a methionine residue at the N-terminus. [0291]In some embodiments, a prime editor fusion protein comprises a Cas9(H840A) nickase and a wild type M-MLV RT. In some embodiments, a prime editor fusion protein comprises a Cas9(H840A) nickase and a M-MLV RT that comprises amino acid substitutions D200N, T330P, T306K, W313F, and L603W compared to a wild type M-MLV RT. In some embodiments, a prime editor fusion protein comprises a Cas9(H840A) nickase and a M-MLV RT that comprises amino acid substitutions D200N, T330P, T306K, W313F, and L603W compared to a wild type M-MLV RT. The amino acid sequence of an exemplary prime editor fusion protein and its individual components in shown in Table 26.In some embodiments, a prime editor fusion protein comprises a Cas9 (R221K N394K H840A) nickase and a M-MLV RT that comprises amino acid substitutions D200N, T330P, T306K, W313F, and L603W compared to a wild type M-MLV RT. The amino acid sequence of an exemplary Prime editor fusion protein and its individual components in shown in Table 27.In some embodiments an WO 2024/238825 PCT/US2024/029746 108 exemplary prime editor protein may comprise an amino acid sequence as set forth in any of the SEQ ID NO: 740 or SEQ ID NO: 741. [0292]In various embodiments, a prime editor fusion protein comprises an amino acid sequence that is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to PEI, PE2, or any of the prime editor fusion sequences described herein or known in the art. Table 26:lists exemplary prime editor and its componentsSEQIDNO: DESCRIPTION SEQUENCE 740 Exemplary Prime Editor [NLS]- [Cas9(H840A)]- [linker]- [MMLV_RT(D2N)(T330P)(L603W) (T306K)(W313F)] - [NLS] MKRTADGSEFESPKKKRKVDKKYSIGLDIGTNSVGWAV ITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGET AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDD SFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYP TIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIE GDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAK AILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGL TPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQ YADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKR YDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAG YIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNR EKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITP WNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHS LLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIV DLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRF NASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLF EDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGR LSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHD DSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGI LQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKG WO 2024/238825 PCT/US2024/029746 109 QKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEK LYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFL KDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYW RQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQ LVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITL KSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGT ALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKA TAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGE IVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVL VVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFL EAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGE LQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQ LFVEQHKHYLDEHEQISEFSKRVILADANLDKVLSAYN KHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDR KRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDSGG SSGGSSGSETPGTSESATPESSGGSSGGSSTZMED^F^L^ ETSKEPDVSLGSTWLSDFPQA WAETGGMGLA VRQAPLIIPLK ATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNT PLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSG LPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGI SGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQ YVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQ KQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFL GKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQ EIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGP WRRPVAYLSKKLDPVAAGWPPCLRMVAAIA VLTKDAGKLTM GQPL VILAPHA VEAL VKQPPDR WLSNARMTHYQALLLDTDR VQFGPWALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTD QPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKAL PAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIH GEIYRRRGWLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPG WO 2024/238825 PCT/US2024/029746 110 HQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSPSGGSKRTADGSEFEPKKKRKV KEY:NUCLEAR LOCALIZATION SEQUENCE (NLS) CAS9(H840A) 33-AMINO ACID LINKER M-MLV REVERSE TRANSCRIPTASE 711 - N-terminal NLS MKRTADGSEFESPKKKRKV676 -CAS9(H840A) (MET MINUS)DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDR HSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRIC YLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFG NIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALA HMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEEN PINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFG NLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLL AQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGY AGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKI EKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEE VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRK VTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLL KIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTY AHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGK TILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQG DSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKP ENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQIL KEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLS DYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSE EVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSEL WO 2024/238825 PCT/US2024/029746 ill DKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIR EVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AWGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEI GKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETG EIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKES ILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAK VEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYK EVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELAL PSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLD ElIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAEN IIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIH QSITGLYETRIDLSQLGGD736 - linker between CAS9 domain and RT domain (amino acids) SGGSSGGSSGSETPGTSESATPESSGGSSGGSS 673 -MMLVRTD200N T330PL603W T306KW313F TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGM GLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQR LLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVN KRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLR LHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLF NEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQ GTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEG QRWLTEARKETVMGQPTPKTPRQLREFLGKAGFCRLEIP GFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLT APALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPV AYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQ PLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDR VQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDL TDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVI WAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSR YAFATAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALF WO 2024/238825 PCT/US2024/029746 112 LPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITET PDTSTLLIENSSP720 - C- terminal NLS SGGSKRTADGSEFEPKKKRKV Table 27:lists exemplary prime editor and its componentsSEQ ID NO.

DESCRIPTION SEQUENCE 741 Exemplary prime editor [NLS]- [Cas9((R220K) (R393K) (H839A)]- [linker]- [MMLV_RT(D2N)(T330P)(L603W )(T306K)(W313F)] - [NLS] MKRTADGSEFESPKKKRKVDKKYSIGLDIGTNSVGWAV ITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGET AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDD SFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYP TIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIE GDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAK A.ILSA.^ILSI^S^UCLEN^LIA.^^LPGEICICN^GLFGN^LI.AI jSLGL TPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQ YADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKR YDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAG YIDGGASQEEFYKFIKPILEKMDGTEELLVKLKREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNR EKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITP WNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHS LLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIV DLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRF NASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLF EDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGR LSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHD DSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGI LQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKG QKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEK LYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFL KDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYW RQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQ LVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITL KSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGT ALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKA TAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGE IVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLV VAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLE AKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGEL QKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQL FVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNK HRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRK RYTSTKEVLDATLIHOSITGLYETRIDLSOLGGDSGGSS GGSKRTADGSEFESPKKKRKVSGGSSGGSTZTVZEDSERZ HETSKEPDVSLGSTWLSDFPQA WAETGGMGLA VRQAPLIIPL WO 2024/238825 PCT/US2024/029746 113 KATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWN TPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLS GLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMG ISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILL QYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQIC QKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREF LGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAY QEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLG PWRRPVA YLSKKLDPVAAGWPPCLRMVAAIA VLTKDAGKLT MGQPL VILAPHA VEAL VKQPPDR WLSNARMTHYQALLLDTD RVQFGPWALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLT DQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKA LPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIH GETYRRRGWLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPG HQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSPSG GSKRTADGSEFESPKKKRKVGSGPAAKR VKLD KEY:N-terminal bipartiteSV40NLS CAS9(R221K N394K H840A) SGGSx2-met-bpSV40NLS-SGGSx2 LINKER M-MLV D200N T306K W313F T330PL603WREVERSETRANSCRIPTASEC-terminal linker- NLS1 C-terminal linker-NLS2 711 - N-terminal bpSV40NLSMKRTADGSEFESPKKKRKV 677 -CAS9(R221KN394KH840A)DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDR HSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRIC YLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFG NIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALA HMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEEN PINASGVDAKAILSARLSKSRKLENLIAQLPGEKKNGLFG NLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLL AQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGY AGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLKREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKI EKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEE VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRK VTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLL KIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTY AHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGK TILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQG DSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKP ENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQIL KEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLS DYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEE VVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELD WO 2024/238825 PCT/US2024/029746 114 KAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIRE VKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNA WGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIG KATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEI VWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESIL PKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKV EKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKE VKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALP SKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDE IIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENII HLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ SITGLYETRIDLSQLGGD737 - SGGSx2- bpSV40NLS- SGGSx2 linker SGGSSGGSKRTADGSEFESPKKKRKVSGGSSGGS 673 -MMLVRTD200N T330PL603W T306KW313F TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGM GLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQR LLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVN KRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLR LHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLF NEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQG TRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQ RWLTEARKETVMGQPTPKTPRQLREFLGKAGFCRLFIPGF AEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPA LGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYL SKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLV ILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQ FGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTD QPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWA KALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAF ATAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPK RLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTS TLLIENSSP738 C-terminal linker-NLSSGGSKRTADGSEFESPKKKRKV 739 C-terminal linker- NLS2GSGPAAKRVKLD PEgRNA for editing of B2M gene [0293]The term "prime editing guide RNA", or "PEgRNA", refers to a guide polynucleotide that comprises one or more intended nucleotide edits (i.e., one or more nucleotide changes) for incorporation into the target DNA. In some embodiments, the PEgRNA associates with and directs a prime editor to incorporate the one or more intended nucleotide edits into the target gene via prime editing. "Nucleotide edit " or "intended nucleotide edit " refers to a specified deletion of one or more nucleotides at one specific position, insertion of one or WO 2024/238825 PCT/US2024/029746 115 more nucleotides at one specific position, substitution of a single nucleotide, or other alterations at one specific position to be incorporated into the sequence of the target gene. Intended nucleotide edit may refer to the edit on the editing template as compared to the sequence on the target strand of the target gene, or may refer to the edit encoded by the editing template on the newly synthesized single stranded DNA that replaces the editing target sequence, as compared to the editing target sequence. In some embodiments, incorporation of the one or more intended nucleotide edits in the target B2M gene introduces a mutation (e.g., a missense mutation, a nonsense mutation, a frame-shift mutation, a null mutation, a mutation that generates a premature stop codon, or a combination thereof) in the target B2M gene. In some embodiments, the one or more intended nucleotide edits introduce a frame shift mutation and/or generate one or more premature stop codons (e.g., at least 1, 2, 3, 4, 5, or more premature stop codons) in the target gene (e.g., B2M gene). In some embodiments, the one or more intended nucleotide edits generates at least 2 premature stop codons in the target gene. In some embodiments, the one or more intended nucleotide edits generates at least 2, 3,4, 5, or more consecutive premature stop codons in the target gene (e.g., a B2M gene). In some embodiments, the one or more intended nucleotide edits comprise insertion of one or more premature, in frame stop codons (e.g., two stop codons) into the B2M gene. In some embodiments, a PEgRNA comprises a spacer sequence that is complementary or substantially complementary to a search target sequence on a target strand of the target gene. In some embodiments, the PEgRNA comprises a gRNA core that associates with a DNA binding domain, e.g., a CRISPR-Cas protein domain, of a prime editor. In some embodiments, the PEgRNA further comprises an extended nucleotide sequence comprising one or more intended nucleotide edits compared to the endogenous sequence of the target gene, wherein the extended nucleotide sequence may be referred to as an extension arm. [0294]In certain embodiments, the extension arm comprises a primer binding site sequence (PBS) that can initiate target-primed DNA synthesis. In some embodiments, the PBS is complementary or substantially complementary to a free 3' end on the edit strand of the target gene at a nick site generated by the prime editor. In some embodiments, the extension arm further comprises an editing template that comprises one or more intended nucleotide edits to be incorporated in the target gene by prime editing. In some embodiments, the editing template is a template for an RNA-dependent DNA polymerase domain or polypeptide of the prime editor, for example, a reverse transcriptase domain. The reverse transcriptase editing WO 2024/238825 PCT/US2024/029746 116 template may also be referred to herein as an RT template, or RTT. In some embodiments, the editing template comprises partial complementarity to an editing target sequence in the target gene, e.g., an B2M gene. In some embodiments, the editing template comprises substantial or partial complementarity to the editing target sequence except at the position of the intended nucleotide edits to be incorporated into the target gene. An exemplary architecture of a PEgRNA including its components is as demonstrated in FIG. 2. [0295]In some embodiments, a PEgRNA includes only RNA nucleotides and forms an RNA polynucleotide. In some embodiments, a PEgRNA is a chimeric polynucleotide that includes both RNA and DNA nucleotides. For example, a PEgRNA can include DNA in the spacer sequence, the gRNA core, or the extension arm. In some embodiments, a PEgRNA comprises DNA in the spacer sequence. In some embodiments, the entire spacer sequence of a PEgRNA is a DNA sequence. In some embodiments, the PEgRNA comprises DNA in the gRNA core, for example, in a stem region of the gRNA core. In some embodiments, the PEgRNA comprises DNA in the extension arm, for example, in the editing template. An editing template that comprises a DNA sequence may serve as a DNA synthesis template for a DNA polymerase in a prime editor, for example, a DNA-dependent DNA polymerase.Accordingly, the PEgRNA may be a chimeric polynucleotide that comprises RNA in the spacer, gRNA core, and/or the PBS sequences and DNA in the editing template. [0296]Components of a PEgRNA may be arranged in a modular fashion. In some embodiments, the spacer and the extension arm comprising a primer binding site sequence (PBS) and an editing template, e.g., a reverse transcriptase template (RTT), can be interchangeably located in the 5' portion of the PEgRNA, the 3' portion of the PEgRNA, or in the middle of the gRNA core. In some embodiments, a PEgRNA comprises a PBS and an editing template sequence in 5' to 3' order. In some embodiments, the gRNA core of a PEgRN A of this disclosure may be located in between a spacer and an extension arm of the PEgRNA. In some embodiments, the gRNA core of a PEgRNA may be located at the 3' end of a spacer. In some embodiments, the gRNA core of a PEgRNA may be located at the 5' end of a. spacer. In some embodiments, the gRNA core of a PEgRNA may be located at the 3' end of an extension arm. In some embodiments, the gRNA core of a PEgRNA may be located at the 5' end of an extension arm. In some embodiments, the PEgRNA comprises, from 5' to 3': a spacer, a gRNA core, and an extension arm. In some embodiments, the PEgRNA comprises, from 5' to 3': a spacer, a gRNA core, an editing template, and a PBS. In some embodiments, the PEgRNA comprises, from 5׳ to 3': an extension arm, a spacer, and a gRNA WO 2024/238825 PCT/US2024/029746 117 core. In some embodiments, the PEgRNA comprises, from 5' to 3': an editing target, a PBS, a spacer, and a gRN A core. [0297]In some embodiments, a PEgRNA comprises a single polynucleotide molecule that comprises the spacer sequence, the gRNA core, and the extension arm. In some embodiments, a PEgRNA comprises multiple polynucleotide molecules, for example, two polynucleotide molecules. In some embodiments, a PEgRNA comprise a first polynucleotide molecule that comprises the spacer and a portion of the gRNA core, and a second polynucleotide molecule that comprises the rest of the gRNA core and the extension arm. In some embodiments, the gRNA core portion in the first polynucleotide molecule and the gRNA core portion in the second polynucleotide molecule are at least partly complementary to each other. In some embodiments, the PEgRNA may comprise a first polynucleotide comprising the spacer and a first portion of a gRNA core comprising, which may be also be referred to as a crRNA. In some embodiments, the PEgRNA comprise a second polynucleotide comprising a second portion of the gRNA core and the extension arm, wherein the second portion of the gRNA core may also be referred to as a trans-activating crRNA, or tracr RNA. In some embodiments, the crRNA portion and the tracr RNA portion of the gRNA core are at least partially complementary to each other. In some embodiments, the partially complementary portions of the crRNA and the tracr RNA form a lower stem, a bulge, and an upper stem, as exemplified in FIG. 3.The gRNA core of PEgRNAs (also referred to as gRNA scaffold or gRNA backbone) are analogous to the gRNA scaffold or backbone of classic CRISPR-Cas9 guide RNAs and CRISPR-Cas9 single guide RNAs (sgRNAs). The gRNA core structure and exemplary sequences as described in PCT publication WO2020191234, Nowak et al., Nucleic Acids Research 44(20): 955595(2016), Doudna et al. Annu. Rev. Biophys. 2017. 46:505-29, and any gRNA core structure and sequences known in the art are incorporated herein by reference in its entirety. [0298]In some embodiments, a spacer sequence comprises a region that has substantial complementarity to a search target sequence on the target strand of a double stranded target DNA, e.g., an B2M gene. In some embodiments, the spacer sequence of a PEgRNA is identical or substantially identical to a protospacer sequence on the edit strand of the target gene (except that the protospacer sequence comprises thymine and the spacer sequence may comprise uracil). In some embodiments, the spacer sequence is at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to a search target sequence in the target gene.

WO 2024/238825 PCT/US2024/029746 118 In some embodiments, the spacer comprises is substantially complementary to the search target sequence. [0299]In some embodiments, the length of the spacer varies from about 10 to about 1nucleotides. In some embodiments, the spacer is 16 nucleotides, 17 nucleotides, nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, nucleotides, or 25 nucleotides in length. In some embodiments, the spacer is from nucleotides to 30 nucleotides in length, 15 to 25 nucleotides in length, 18 to 22 nucleotides in length, 10 to 20 nucleotides in length, or 20 to 30 nucleotides in length. In some embodiments, the spacer is 16 to 22 nucleotides in length, e.g., about 16, 17, 18, 19, 20, 21, or 22 nucleotides in length. [0300]As used herein in a PEgRNA or a nick guide RNA sequence, or fragments thereof such as a spacer, PBS, or RTT sequence, unless indicated otherwise, it should be appreciated that the letter "T" or "thymine " indicates a nucleobase in a DNA sequence that encodes the PEgRNA or guide RNA sequence, and is intended to refer to a uracil (U) nucleobase of the PEgRNA or guide RNA or any chemically modified uracil nucleobase known in the art, such as 5-methoxyuracil. [0301]The extension arm of a PEgRNA may comprise a primer binding site (PBS) and an editing template (e.g, an RTT). The extension arm may be partially complementary to the spacer. In some embodiments, the editing template (e.g, RTT) is partially complementary to the spacer. In some embodiments, the editing template (e.g, RTT) and the primer binding site (PBS) are each partially complementary to the spacer. [0302]An extension arm of a PEgRNA may comprise a primer binding site sequence (PBS, or PBS sequence) that comprises complementarity to and can hybridize with a free 3' end of a single stranded DNA in the target gene (e.g, the B2M gene) generated by nicking with a prime editor at the nick site on the PAM strand. [0303]The length of the PBS sequence may vary depending on, e.g, the prime editor components, the search target sequence and other components of the PEgRNA. [0304]In some embodiments, the PBS is about 3 to 19 nucleotides in length, in some embodiments, the PBS is about 3 to 17 nucleotides in length. In some embodiments, the PBS is about 4 to 16 nucleotides, about 6 to 16 nucleotides, about 6 to 18 nucleotides, about 6 to nucleotides, about 8 to 20 nucleotides, about 10 to 20 nucleotides, about 12 to nucleotides, about 14 to 20 nucleotides, about 16 to 20 nucleotides, or about 18 to nucleotides in length. In some embodiments, the PBS is 8 to 17 nucleotides in length. In WO 2024/238825 PCT/US2024/029746 119 some embodiments, the PBS is 8 to 16 nucleotides in length. In some embodiments, the PBS is 8 to 15 nucleotides in length. In some embodiments, the PBS is 8 to 14 nucleotides in length. In some embodiments, the PBS is 8 to 13 nucleotides in length. In some embodiments, the PBS is 8 to 12 nucleotides in length. In some embodiments, the PBS is 8 to nucleotides in length. In some embodiments, the PBS is 8 to 10 nucleotides in length. In some embodiments, the PBS is 8 or 9 nucleotides in length. In some embodiments, the PBS is or 17 nucleotides in length. In some embodiments, the PBS is 15 to 17 nucleotides in length. In some embodiments, the PBS is 14 to 17 nucleotides in length. In some embodiments, the PBS is 13 to 17 nucleotides in length. In some embodiments, the PBS is to 17 nucleotides in length. In some embodiments, the PBS is 11 to 17 nucleotides in length. In some embodiments, the PBS is 10 to 17 nucleotides in length. In some embodiments, the PBS is 9 to 17 nucleotides in length. In some embodiments, the PBS is about 7 to nucleotides in length. In some embodiments, the PBS is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 nucleotides in length. In some embodiments, the PBS is 8 to 14 nucleotides in length. For example, the PBS can be 8, 9, 10, 11, 12, 13, or 14 nucleotides in length. In some embodiments, the PBS is 11 or 12 nucleotides in length. In some embodiments, the PBS is to 13 nucleotides in length. In some embodiments, the PBS is 11 to 14 nucleotides in length. [0305]The PBS may be complementary or substantially complementary to a DNA sequence in the edit strand of the target gene. By annealing with the edit strand at a free hydroxy group, e.g., a free 3' end generated by prime editor nicking, the PBS may initiate synthesis of a new single stranded DNA encoded by the editing template at the nick site. In some embodiments, the PBS is at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to a region of the edit strand of the target gene (e.g, the B2M gene). In some embodiments, the PBS is perfectly complementary, or 100% complementary, to a region of the edit strand of the target gene (e.g, the B2M gene). [0306]An extension arm of a PEgRNA may comprise an editing template that serves as a DNA synthesis template for the DNA polymerase in a prime editor during prime editing. [0307]The length of an editing template may vary depending on, e.g., the prime editor components, the search target sequence and other components of the PEgRNA. In some embodiments, the editing template serves as a DNA synthesis template for a reverse transcriptase, and the editing template is referred to as a reverse transcription editing template (RTT).

WO 2024/238825 PCT/US2024/029746 120 id="p-308"

[0308]The editing template (e.g, RTT), in some embodiments, is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In some embodiments, the RTT is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In some embodiments, the RTT is 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length. In some embodiments,the RTT is 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleotides in length. Insome embodiments, the RTT is 10 to 110 nucleotides in length. In some embodiments, the RTT is 10 to 109, 10 to 108, 10 to 107, 10 to 106, 10 to 105, 10 to 104, 10 to 103, 10 to 102, or 10 to 101 nucleotides in length. In some embodiments, the RTT is at least 8 and no more than 50 nucleotides in length. In some embodiments, the RTT is at least 8 and no more than nucleotides in length. In some embodiments, the RTT is about 10 to about 20 nucleotides in length. In some embodiments, the RTT is about 11, 12, 13, 14, 15, 16, 17, 18, or nucleotides in length. In some embodiments, the RTT is 11 to 17 nucleotides in length. In some embodiments, the RTT is 12 to 17 nucleotides in length. In some embodiments, the RTT is 12 to 16 nucleotides in length. In some embodiments, the RTT is 13 to 17 nucleotides in length. In some embodiments, the RTT is 11, 12, 13, 14, 15, 16, or 17 nucleotides in length. In some embodiments the RTT is 12 nucleotides in length. In some embodiments the RTT is 16 nucleotides in length. In some embodiments the RTT is 17 nucleotides in length. [0309]In some embodiments, the editing template (e.g, RTT) sequence is about 70%, 75%, 80%, 85%, 90%, 95%, or 99% complementary to the editing target sequence on the edit strand of the target gene. In some embodiments, the editing template sequence (e.g, RTT) is substantially complementary to the editing target sequence. In some embodiments, the editing template sequence (e.g, RTT) is complementary to the editing target sequence except at positions of the intended nucleotide edits to be incorporated int the target gene. In some embodiments, the editing template comprises a nucleotide sequence comprising about 85% to about 95% complementarity to an editing target sequence in the edit strand in the target gene (e.g, the B2M gene). In some embodiments, the editing template comprises about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementarity to an editing target sequence in the edit strand of the target gene (e.g, the B2M gene).

WO 2024/238825 PCT/US2024/029746 121 id="p-310"

[0310]In some embodiments, an editing template may be configured to introduce one or more recombinase recognition sequences into the target gene, e.g., the B2M gene. For example, an editing template may encode, or further encode, one or more recombinase recognition sequences (RRSs). Such editing templates, or RTTs, can enable insertion of the RRS(s) in the target gene, and the RRS(s) can be used as landing sites for recombinase mediated DNA insertion, deletion, inversion, or replacement. For example, in some embodiments, prime editing insertion of an RRS (e.g., an attB sequence) allows integration of a DNA donor sequence mediated by a recombinase that recognizes the RRS (e.g., Bxbl), wherein the DNA donor sequence also comprises an RRS recognized by the recombinase (e.g., an attP sequence). In some embodiments, prime editing insertion of two RRSs allow for deletion of the target gene sequence between the two RRSs or inversion of the target gene sequence between the two RRSs mediated by a recombinase that recognizes the two RRSs, depending on the orientation of the two RRSs. In some embodiments, prime editing insertion of two RRSs allow for cassette exchange of the target gene sequence between the two RRSs and a DNA donor sequence mediated by a corresponding recombinase, wherein the DNA donor sequence is flanked by two RRSs that are also recognized by the recombinase. [0311]Exemplary RRS sequences that can be encoded by the PEgRNA RTTs are provided in Table 32.A skilled person understands that RRSs recognized by the same recombinase can be used for targeted insertion and other recombination events, for example, by inserting an attB sequence in a target B2Mgene via prime editing, and providing a Bxbl recombinase and a circular DNA donor construct containing an attP sequence for integration of the DNA donor sequence in the B2M gene at the attB site. In some embodiments, orthogonal recognition can be achieved by altering the central dinucleotide of the RRS. For example, the central dinucleotides of Bxbl attB or attP sequences can be GT or GA, shown in bold in Table 32.In some embodiments, the central dinucleotides of a RRS can be any two nucleotides, where each nucleotide is A, T, G, or C. Additional RRSs described herein and those known in the art, as well as corresponding recombinases, are also contemplated. Table 32. Exemplary RRS sequences and corresponding recombinases SEQ ID NO DNA sequence Corresponding recombinase RRS description 1590GGCTTGTCGACGACGGCGGTCTCCGTCGT CAGGATCATBxbl Bxbl attB 1591GGTTTGTCTGGTCAACCACCGCGGTCTCA GTGGTGTACGGTACAAACCBxbl Bxbl attP WO 2024/238825 PCT/US2024/029746 122 1592GGCTTGTCGACGACGGCGGACTCCGTCG TCAGGATCATBxblBxbl attB-GA 1593GGTTTGTCTGGTCAACCACCGCGGACTCA GTGGTGTACGGTACAAACCBxblBxbl attP-GA 1598 TTCCGACGCAGTTTCCGACGAGTACGAG GACGAGGACAGACGTGCCTACCGGCAAG GTCAAGTGGTTCAACAGCGAGAAGGGCT TCGGCTTTCTCTCCCGCGACGACGGCGGSi74 Si74 attB 1599 TAGTGACGTCTGTCCGCGCAGTGATCGA GGGAGTGTGTGCTTTGCCGACTGGCAAG GTCAAGCCGGTCTGCTAGGCACAGAGAG CCGGTACAGTCCTCCCCATGCAACCCAASi74 Si74 attP 1600 GTGACCCGTTCCGCCGTCGAGAACGCCA CCTCCGTGGCGCGCATGGTGCTCACCACC GAGAGCGCCGTGGTCGACAAGCCGGCCG AGGAAGAGCCCGCGAACGGCCACCACGG CC N067 N067 attB 1601 TGATTACGATCAGTGCCCTGGGAGGCGA TTCCGGCATGGCTTATATCCAACACCACC GAGAGCGCTGTTGTCGAGCGTGTAAGCC AGGACGAGGACGAGCACGCCCACGGGCA CG N067 N067 attP 1602 TTTGCATTTAAAATCGGAGCATCATTTTT CAACAGAAACGACTATGAGCGCAATGAC CCTTGATTTACCTCGCCGCTTTCCGTGGC CAACGCTGCTGTCCGTGGCTATCCACGKp03 Kp03 attB 1603 AGAACCTTGAAAAACTATGGCTTATGCT ACCTCGCCGAATAGCTCAAATACAATGA CCCTTGAATATAGGCTATTCGGGTTTTGA AGGTATCTGGTTTTTATTCACATCACAAKp03 Kp03 attP 1604 CGAAATGGTTGGCGTTGAGGTCAATGAT TAATGTGTATAGGGTTAACATTTAAATCA GTACAATCGTAGACGCTCTACACTATTTT CTGTGTATAAAAATATCGAGAATAAACG CTT Nm60 Nm60 attB 1605 GCTGGTCGAGGAGTTAATGAAGATCTCC AACATGTTCGGCTACGGTGAGGTAAATC AGTACAATCGTCACTCGACCCTAATGTCT GCCTATAAAAACATCAAAGATGAACATT TCCG Nm60 Nm60 attP 1606GATATGGGGAAGTGAATCAGTACAACCG CCACAGTACCBceINTaBceINTa attB 1607TTCGGGTGCTGGGTTGTTGTCTCTGGACA GTGATCCATGGGAAACTACTCAGCACCABceINTa BceINTa attP 1608ATTGTTGTTGTTTTTCCAGATCCAGTTGG TCCTGTAAATATAAGCAATCCBcytINTdBcytINTd attB 1609GGTTGTATTTGTAGAACTTGACCAGTTGT TTTAGTAACATAAATACAACTBcytINTdBcytINTd attP WO 2024/238825 PCT/US2024/029746 123 1610CATTATATGTTTTTACAATCCGGGCCGCC ATACTGTAAGAACATATAATGSscINTd SscINTd attB 1611GATATGGGGAAGTGAATcaGTACAACCGC CACAGTACCSscINTd SscINTd attP 1612CGTTATAGGGTATTGCAGTACCGACCGCC ATACTGTAATACCCTATAACGSacINTd SacINTd attB 1613GATGCACTGAGCTCACCGTCCGGACCGC CATACTGACTTATGATATAAGASacINTd SacINTd attP id="p-312"

[0312]An intended nucleotide edit in an editing template of a PEgRNA may comprise various types of alterations as compared to the target gene sequence. In some embodiments, the nucleotide edit is a single nucleotide substitution as compared to the target gene sequence. In some embodiments, the nucleotide edit is a deletion as compared to the target gene sequence. In some embodiments, the nucleotide edit is an insertion as compared to the target gene sequence. In some embodiments, the editing template comprises one to ten intended nucleotide edits as compared to the target gene sequence. In some embodiments, the editing template comprises one or more intended nucleotide edits as compared to the target gene sequence. In some embodiments, the editing template comprises two or more intended nucleotide edits as compared to the target gene sequence. In some embodiments, the editing template comprises three or more intended nucleotide edits as compared to the target gene sequence. In some embodiments, the editing template comprises four or more, five or more, or six or more intended nucleotide edits as compared to the target gene sequence. In some embodiments, the editing template comprises two single nucleotide substitutions, insertions, deletions, or any combination thereof, as compared to the target gene sequence. In some embodiments, the editing template comprises three single nucleotide substitutions, insertions, deletions, or any combination thereof, as compared to the target gene sequence. In some embodiments, the editing template comprises four, five, or six single nucleotide substitutions, insertions, deletions, or any combination thereof, as compared to the target gene sequence. In some embodiments, a nucleotide substitution comprises an adenine (A)-to-thymine (T) substitution. In some embodiments, a nucleotide substitution comprises an A-to-guanine (G) substitution. In some embodiments, a nucleotide substitution comprises an A-to-cytosine (C) substitution. In some embodiments, a nucleotide substitution comprises a T-A substitution. In some embodiments, a nucleotide substitution comprises a T-G substitution. In some embodiments, a nucleotide substitution comprises a T-C substitution. In some embodiments, a nucleotide substitution comprises a G-to-A substitution. In some embodiments, a nucleotide WO 2024/238825 PCT/US2024/029746 124 substitution comprises a G-to-T substitution. In some embodiments, a nucleotide substitution comprises a G-to-C substitution. In some embodiments, a nucleotide substitution comprises a C-to-A substitution. In some embodiments, a nucleotide substitution comprises a C-to-T substitution. In some embodiments, a nucleotide substitution comprises a C-to-G substitution. [0313]In some embodiments, a nucleotide insertion is at least 1, at least 2, at least 3, at least 4, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, or at least 20 nucleotides in length. In some embodiments, a nucleotide insertion is from 1 to 2 nucleotides, from 1 to nucleotides, from 1 to 4 nucleotides, from 1 to 5 nucleotides, form 2 to 5 nucleotides, from to 5 nucleotides, from 3 to 6 nucleotides, from 3 to 8 nucleotides, from 4 to 9 nucleotides, from 5 to 10 nucleotides, from 6 to 11 nucleotides, from 7 to 12 nucleotides, from 8 to nucleotides, from 9 to 14 nucleotides, from 10 to 15 nucleotides, from 11 to 16 nucleotides, from 12 to 17 nucleotides, from 13 to 18 nucleotides, from 14 to 19 nucleotides, from 15 to nucleotides in length. In some embodiments, a nucleotide insertion is a single nucleotide insertion. In some embodiments, a nucleotide insertion comprises insertion of two nucleotides. In some embodiments, the one or more intended nucleotide edits introduce a frame shift mutation and/or generate one or more premature stop codons (e.g., at least 1, 2, 3, 4, 5, or more premature stop codons) in the target gene (e.g., B2M gene). In some embodiments, the one or more intended nucleotide edits generates at least 2 premature stop codons in the target gene. In some embodiments, the one or more intended nucleotide edits generates at least 2, 3,4, 5, or more consecutive premature stop codons in the target gene (e.g., a B2M gene). In some embodiments, the one or more intended nucleotide edits comprise insertion of one or more premature, in frame stop codons (e.g., two stop codons) into the B2M gene. The editing template of a PEgRNA may comprise one or more intended nucleotide edits, compared to the B2M gene to be edited. Position of the intended nucleotide edit(s) relevant to other components of the PEgRNA, or to particular nucleotides (e.g, mutations) in the B2M target gene may vary. In some embodiments, the nucleotide edit is in a region of the PEgRNA corresponding to or homologous to the protospacer sequence. In some embodiments, the nucleotide edit is in a region of the PEgRNA corresponding to a region of the B2M gene outside of the protospacer sequence.

WO 2024/238825 PCT/US2024/029746 125 id="p-314"

[0314]In some embodiments, the position of a nucleotide edit incorporation in the target gene mayBy "upstream " and "downstream " it is intended to define relevant positions at least two regions or sequences in a nucleic acid molecule orientated in a 5'-to-3' direction. For example, a first sequence is upstream of a second sequence in a DNA molecule where the first sequence is positioned 5’ to the second sequence. Accordingly, the second sequence is downstream of the first sequence. [0315]In some embodiments, the position of a nucleotide edit incorporation in the target gene can be determined based on position of the nick site. In some embodiments, position of an intended nucleotide edit is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, or 150 nucleotides apart from the nicksite. In some embodiments, position of an intended nucleotide edit is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, or150 nucleotides downstream of the nick site on the PAM strand (or the non-target strand, or the edit strand) of the double stranded target DNA. In some embodiments, position of the intended nucleotide edit in the editing template may be referred to by aligning the editing template with the partially complementary editing target sequence on the edit strand, and referring to nucleotide positions on the editing strand where the intended nucleotide edit is incorporated. Accordingly, in some embodiments, a nucleotide edit in an editing template is at a position corresponding to a position about 0, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, or 150 nucleotides apart from the nick site. In some embodiments, a nucleotide edit in an editing template is at a position corresponding to a position about 0 to 2 nucleotides, 0 to 4 nucleotides, 0 to nucleotides, 0 to 8 nucleotides, 0 to 10 nucleotides, , 2 to 4 nucleotides, 2 to 6 nucleotides, to 8 nucleotides, 2 to 10 nucleotides, 2 to 12 nucleotides, 4 to 6 nucleotides, 4 to nucleotides, 4 to 10 nucleotides, 4 to 12 nucleotides, 4 to 14 nucleotides, 6 to 8 nucleotides, to 10 nucleotides, 6 to 12 nucleotides, 6 to 14 nucleotides, 6 to 16 nucleotides, 8 to nucleotides, 8 to 12 nucleotides, 8 to 14 nucleotides, 8 to 16 nucleotides, 8 to 18 nucleotides, to 12 nucleotides, 10 to 14 nucleotides, 10 to 16 nucleotides, 10 to 18 nucleotides, 10 to nucleotides, 12 to 14 nucleotides, 12 to 16 nucleotides, 12 to 18 nucleotides, 12 to nucleotides, 12 to 22 nucleotides, 14 to 16 nucleotides, 14 to 18 nucleotides, 14 to 20 WO 2024/238825 PCT/US2024/029746 126 nucleotides, 14 to 22 nucleotides, 14 to 24 nucleotides, 16 to 18 nucleotides, 16 to nucleotides, 16 to 22 nucleotides, 16 to 24 nucleotides, 16 to 26 nucleotides, 18 to nucleotides, 18 to 22 nucleotides, 18 to 24 nucleotides, 18 to 26 nucleotides, 18 to nucleotides, 20 to 22 nucleotides, 20 to 24 nucleotides, 20 to 26 nucleotides, 20 to nucleotides, 20 to 30 nucleotides, 30 to 40 nucleotides, 40 to 50 nucleotides, 50 to nucleotides, 60 to 70 nucleotides, 70 to 80 nucleotides, 80 to 90 nucleotides, 90 to 1nucleotides, 100 to 110 nucleotides, 110 to 120 nucleotides, 120 to 130 nucleotides, 130 to 140 nucleotides, or 140 to 150 nucleotides apart from the nick site. In some embodiments, when referred to in the context of the PAM strand (or the non-target strand, or the edit strand), a nucleotide edit in an editing template is at a position corresponding to a position about 0 to 2 nucleotides, 0 to 4 nucleotides, 0 to 6 nucleotides, 0 to 8 nucleotides, 0 to nucleotides, , 2 to 4 nucleotides, 2 to 6 nucleotides, 2 to 8 nucleotides, 2 to 10 nucleotides, to 12 nucleotides, 4 to 6 nucleotides, 4 to 8 nucleotides, 4 to 10 nucleotides, 4 to nucleotides, 4 to 14 nucleotides, 6 to 8 nucleotides, 6 to 10 nucleotides, 6 to 12 nucleotides, to 14 nucleotides, 6 to 16 nucleotides, 8 to 10 nucleotides, 8 to 12 nucleotides, 8 to nucleotides, 8 to 16 nucleotides, 8 to 18 nucleotides, 10 to 12 nucleotides, 10 to nucleotides, 10 to 16 nucleotides, 10 to 18 nucleotides, 10 to 20 nucleotides, 12 to 14nucleotides, 12 to 16 nucleotides, 12 to 18 nucleotides, 12 to 20 nucleotides, 12 to 22nucleotides, 14 to 16 nucleotides, 14 to 18 nucleotides, 14 to 20 nucleotides, 14 to 22nucleotides, 14 to 24 nucleotides, 16 to 18 nucleotides, 16 to 20 nucleotides, 16 to 22nucleotides, 16 to 24 nucleotides, 16 to 26 nucleotides, 18 to 20 nucleotides, 18 to 22nucleotides, 18 to 24 nucleotides, 18 to 26 nucleotides, 18 to 28 nucleotides, 20 to 22nucleotides, 20 to 24 nucleotides, 20 to 26 nucleotides, 20 to 28 nucleotides, 20 to 30nucleotides, 30 to 40 nucleotides, 40 to 50 nucleotides, 50 to 60 nucleotides, 60 to 70nucleotides, 70 to 80 nucleotides, 80 to 90 nucleotides, 90 to 100 nucleotides, 100 to 1nucleotides, 110 to 120 nucleotides, 120 to 130 nucleotides, 130 to 140 nucleotides, or 140 to 150 nucleotides downstream from the nick site. The relative positions of the intended nucleotide edit(s) and nick site may be referred to by numbers. For example, in some embodiments, the nucleotide immediately downstream of the nick site on a PAM strand (or the non-target strand, or the edit strand) may be referred to as at position 0. The nucleotide immediately upstream of the nick site on the PAM strand (or the non-target strand, or the edit strand) may be referred to as at position -1. The nucleotides downstream of position 0 on the PAM strand may be referred to as at positions +1, +2, +3, +4, ... +n, and the nucleotides WO 2024/238825 PCT/US2024/029746 127 upstream of position -1 on the PAM strand may be referred to as at positions -2, -3,-4, ..., -n. Accordingly, in some embodiments, the nucleotide in the editing template that corresponds to position 0 when the editing template is aligned with the partially complementary editing target sequence by complementarity may also be referred to as position 0 in the editing template, the nucleotides in the editing template corresponding to the nucleotides at positions +1, +2, +3, +4, ..., +n on the PAM strand of the double stranded target DNA may also be referred to as at positions +1, +2, +3, +4,..., +n in the editing template, and the nucleotides in the editing template corresponding to the nucleotides at positions -1, -2, -3, -4, ..., -n on the PAM strand on the double stranded target DNA may also be referred to as at positions -1, -2, -3,-4, ..., -n on the editing template, even though when the PEgRNA is viewed as a standalone nucleic acid, positions +1, +2, +3, +4, ..., +n are 5' of position 0 and positions -1, -2, -3, -4, ...-n are 3' of position 0 in the editing template. In some embodiments, an intended nucleotide edit is at position +n of the editing template relative to position 0. Accordingly, the intended nucleotide edit may be incorporated at position +n of the PAM strand of the double stranded target DNA (and subsequently, the target strand of the double stranded target DNA) by prime editing. The corresponding positions of the intended nucleotide edit incorporated in the B2M gene may also be referred to based on the nicking position generated by a prime editor based on sequence homology and complementarity. For example, in embodiments, the number of nucleotides from the nucleotide edit to be incorporated into the B2M gene to the nick site (also referred to as the "nick-to-edit distance ", not including the nucleotides in the edit) may be determined by the position of the nick site and the position of the nucleotide(s) corresponding to the intended nucleotide edit(s), for example, by identifying sequence complementarity between the spacer and the search target sequence and sequence complementarity between the editing template and the editing target sequence. In certain embodiments, the position of the nucleotide edit can be in any position downstream of the nick site on the edit strand (or the PAM strand). As used herein, the distance between the nick site and the nucleotide edit, for example, where the nucleotide edit comprises an insertion or deletion, refers to the 5’ most position of the nucleotide edit for a nick that creates a 3’ free end on the edit strand (i.e., the "near position " of the nucleotide edit to the nick site). The nick-to-edit distance for a specific edit, e.g., a non-synonymous edit that results in a premature stop codon in the target gene, can also be referred to as the number of nucleotides from the nick site to the position of the edit (not including the 5’ most nucleotide at the edit).In some embodiments, the nick-to-edit distance is 2 to 106 nucleotides. In some WO 2024/238825 PCT/US2024/029746 128 embodiments, the nick-to-edit distance is 2 to 105, 2 to 104, 2 to 103, 2 to 102, 2 to 101, 2 to 100, 2 to 99, 2 to 98, or 2 to 97 nucleotides. In some embodiments, the nick-to-edit distance is 2 to 90, 2 to 80, 2 to 70, 2 to 60, 2 to 50, 2 to 40, or 2 to 30 nucleotides. In some embodiments, the nick-to-edit distance is 2 to 25, 2 to 20, 2 to 15, or 2 to 10 nucleotides. In some embodiments, the nick-to-edit distance is 2, 3, 4, 5, 6, or 7 nucleotides in length. In some embodiments, the nick-to-edit distance is 28 nucleotides. In some embodiments, the nick-to-edit distance is 22 nucleotides. In some embodiments, the nick-to-edit distance is nucleotides. In some embodiments, the nick-to-edit distance is 17 nucleotides. In some embodiments, the nick-to-edit distance is 16 nucleotides. In some embodiments, the nick-to- edit distance is 4 nucleotides. In some embodiments, the nick-to-edit distance is nucleotides. In some embodiments, the nick-to-edit distance is 1 to 19 nucleotides. In some embodiments, the nick-to-edit distance is 16 nucleotides. In some embodiments, the nick-to- edit distance is 1, 2, 7, 8, 13, 14, or 19 nucleotides. In some embodiments, the nick-to-edit distance is equal to or less than 8 nucleotides. In some embodiments, the nick-to-edit distance is 1 or 2 nucleotides. [0316]The RTT length and the nick-to-edit distance relate to the length of the portion of the RTT that is upstream of (i.e. 5’ to) the 5’-most edit in the RTT and is complementary to the edit strand. In some embodiments, the editing template comprises at least 4 contiguous nucleotides of complementarity with the edit strand wherein the at least 4 nucleotides contiguous are located upstream of the 5’ most edit in the editing template. In some embodiments, the editing template comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or more contiguous nucleotides of complementarity with the edit strand wherein the at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or more contiguous nucleotides are located upstream of the 5’ most edit in the editing template. In some embodiments, the editing template comprises 20-25, 25-30, 30-35, 35-40, 45-45, or 45-50 contiguous nucleotides of complementarity with the edit strand wherein the 20-25, 25-30, 30-35, 35-40, 45-45, or 45-50 or more contiguous nucleotides are located upstream of the 5’ most edit in the editing template. In some embodiments, the editing template comprises 9-14 contiguous nucleotides of complementarity with the edit strand wherein the 9-14 contiguous nucleotides are located upstream of the 5’ most edit in the editing template. In some embodiments, the editing template comprises 6-10 contiguous nucleotides of complementarity with the edit strand wherein the 6-10 contiguous nucleotides are located upstream of the 5’ most edit in the editing template. In some embodiments, the editing template comprises 10 contiguous WO 2024/238825 PCT/US2024/029746 129 nucleotides of complementarity with the edit strand wherein the 10 contiguous nucleotides are located upstream of the 5’ most edit in the editing template. In some embodiments, the editing template comprises 9 contiguous nucleotides of complementarity with the edit strand wherein the 9 contiguous nucleotides are located upstream of the 5’ most edit in the editing template. [0317]When referred to within the PEgRNA, positions of the one or more intended nucleotide edits may be referred to relevant to components of the PEgRNA. For example, an intended nucleotide edit may be 5’ or 3’ to the PBS. In some embodiments, a PEgRNA comprises the structure, from 5’ to 3’: a spacer, a gRNA core, an editing template, and a PBS. In some embodiments, the intended nucleotide edit is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides upstream to the 5’ most nucleotide of the PBS. In some embodiments, the intended nucleotide edit is 0 to 2 nucleotides, 0 to 4 nucleotides, 0 to 6 nucleotides, 0 to nucleotides, 0 to 10 nucleotides, 2 to 4 nucleotides, 2 to 6 nucleotides, 2 to 8 nucleotides, 2 to nucleotides, 2 to 12 nucleotides, 4 to 6 nucleotides, 4 to 8 nucleotides, 4 to 10 nucleotides, to 12 nucleotides, 4 to 14 nucleotides, 6 to 8 nucleotides, 6 to 10 nucleotides, 6 to nucleotides, 6 to 14 nucleotides, 6 to 16 nucleotides, 8 to 10 nucleotides, 8 to 12 nucleotides, to 14 nucleotides, 8 to 16 nucleotides, 8 to 18 nucleotides, 10 to 12 nucleotides, 10 to nucleotides, 10 to 16 nucleotides, 10 to 18 nucleotides, 10 to 20 nucleotides, 12 to 14nucleotides, 12 to 16 nucleotides, 12 to 18 nucleotides, 12 to 20 nucleotides, 12 to 22nucleotides, 14 to 16 nucleotides, 14 to 18 nucleotides, 14 to 20 nucleotides, 14 to 22nucleotides, 14 to 24 nucleotides, 16 to 18 nucleotides, 16 to 20 nucleotides, 16 to 22nucleotides, 16 to 24 nucleotides, 16 to 26 nucleotides, 18 to 20 nucleotides, 18 to 22nucleotides, 18 to 24 nucleotides, 18 to 26 nucleotides, 18 to 28 nucleotides, 20 to 22nucleotides, 20 to 24 nucleotides, 20 to 26 nucleotides, 20 to 28 nucleotides, or 20 to 30nucleotides upstream to the 5’ most nucleotide of the PBS. [0318]The corresponding positions of the intended nucleotide edit incorporated in the target gene may also be referred to based on the nicking position generated by a prime editor based on sequence homology and complementarity. For example, in some embodiments, the distance between (i.e. the number of nucleotides) the nucleotide edit to be incorporated into the target B2M gene and the nick site (also referred to as the "nick to edit distance ", wherein the number of nucleotides does not include the 5’ most nucleotide position on the second strand corresponding to the edit) may be determined by the position of the nick site and the WO 2024/238825 PCT/US2024/029746 130 position of the nucleotide(s) corresponding to the intended nucleotide edit(s), for example, by identifying sequence complementarity between the spacer and the search target sequence and sequence complementarity between the editing template and the editing target sequence. In certain embodiments, the position of the nucleotide edit can be in any position downstream of the nick site on the edit strand (or the PAM strand) generated by the prime editor, such that the distance between the nick site and the intended nucleotide edit is 0,1, 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 nucleotides in length. In some embodiments, the position of the nucleotide edit is 0, 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 nucleotides in length. In some embodiments, the position of the nucleotide edit is 0, 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 nucleotides upstream of the nick site on the edit strand. In some embodiments, the position of the nucleotide edit is 0, 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 nucleotides downstream of the nick site on the edit strand. In some embodiments, the position of the nucleotide edit is 0 base pair from the nick site on the edit strand, that is, the editing position is at the same position as the nick site. As used herein, the distance between the nick site and the nucleotide edit, for example, where the nucleotide edit comprises an insertion or deletion, refers to the 5' most position of the nucleotide edit for a nick that creates a 3’ free end on the edit strand (i.e., the "near position " of the nucleotide edit to the nick site). Similarly, as used herein, the distance between the nick site and a PAM position edit, for example, where the nucleotide edit comprises an insertion, deletion, or substitution of two or more contiguous nucleotides, refers to the 5' most position of the nucleotide edit and the 5׳ most position of the PAM sequence. [0319]In some embodiments, the editing template extends beyond a nucleotide edit to be incorporated to the target B2M gene sequence. For example, in some embodiments, the editing template comprises at least 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleotides. [0320]In some embodiments, the editing template can comprise a second edit relative to a target sequence. The second edit can be designed to mutate or otherwise silence a PAM sequence such that a corresponding nucleic acid guided nuclease or CRISPR nuclease is no longer able to cleave the target sequence (such edits referred to as "PAM silencing edits).

WO 2024/238825 PCT/US2024/029746 131 id="p-321"

[0321]Without wishing to be bound by any particular theory, PAM silencing edits may prevent the Cas, e.g., Cas9, nickase, from re-nicking the edit strand before the edit is incorporated in the target strand, therefore improving prime editing efficiency. In some embodiments, a PAM silencing edit alters the sequence of a transcript or a protein sequence encoded by the B2M gene. In some embodiments, a PAM silencing edit is a synonymous edit that does not alter the amino acid sequence or the mRNA sequence encoded by the B2M gene after incorporation of the edit. In some embodiments, a PAM silencing edit is at a position corresponding to a coding region, e.g., an exon, of a B2M gene. In some embodiments, a PAM silencing edit is at a position corresponding to a non-coding region, e.g., an intron, of a B2M gene. In some embodiments, the edits in an intron of a B2M gene is not at a position that corresponds to intron-exon junction and the edit does not affect transcript splicing. [0322]In some embodiments, the length of the editing template is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleotideslonger than the nick to edit distance. In some embodiments, for example, the nick to editdistance is 8 nucleotides, and the editing template is 10 to 15, 10 to 20, 10 to 25, 10 to 30, to 35, 10 to 40, 10 to 45, 10 to 50, 10 to 55, 10 to 60, 10 to 65, 10 to 70, 10 to 75, or 10 to nucleotides in length. In some embodiments, the nick to edit distance is 22 nucleotides, and the editing template is 24 to 28, 24 to 30, 24 to 32, 24 to 34, 24 to 36, 24 to 37, 24 to 38, 24 to 40, 24 to 45, 24 to 50, 24 to 55, 24 to 60, 24 to 65, 24 to 70, 24 to 75, 24 to 80, 24 to 85, 24 to 90, 24 to 95, 24 to 100, 24 to 105, 24 to 100, 24 to 105, or 24 to 110 nucleotides in length. [0323]In some embodiments, the editing template comprises an adenine at the first nucleobase position (e.g, for a PEgRNA following 5‘-spacer-gRNA core-RTT-PBS-3 ‘ orientation, the 5? most nucleobase is the "first base "). In some embodiments, the editing template comprises a guanine at the first nucleobase position (e.g., for a PEgRNA following 5?-spacer-gRNA core-RTT-PBS-3 ‘ orientation, the 5' most nucleobase is the "first base "). In some embodiments, the editing template comprises an uracil at the first nucleobase position (e.g, for a PEgRNA following 5‘-spacer-gRNA core-RTT-PBS-3 ‘ orientation, the 5' most nucleobase is the "first base "). In some embodiments, the editing template comprises a cytosine at the first nucleobase position (e.g., for a PEgRNA following 5?-spacer-gRNA core- RTT-PBS-3׳ orientation, the 5' most nucleobase is the "first base "). In some embodiments, the editing template does not comprise a cytosine at the first nucleobase position (e.g., for a WO 2024/238825 PCT/US2024/029746 132 PEgRNA following 5?-spacer-gRNA core-RTT-PBS-3 ‘ orientation, the 5' most nucleobase is the "first base "). [0324]The editing template of a PEgRNA may encode a new single stranded DNA (e.g.,by reverse transcription) to replace an editing target sequence in the target gene. In some embodiments, the editing target sequence in the edit strand of the target gene is replaced by the newly synthesized strand, and the nucleotide edit(s) are incorporated in the region of the target gene. In some embodiments, the target gene is an B2M gene. In some embodiments, the editing template of the PEgRNA encodes a newly synthesized single stranded DNA that comprises a mutation or a nucleotide alteration compared to a wild type B2M gene sequence. In some embodiments, the newly synthesized DNA strand replaces the editing target sequence in the target B2M gene, wherein the editing target sequence (or the endogenous sequence complementary to the editing target sequence on the target strand of the B2M gene) comprises a wild type B2M gene. [0325]In some embodiments, the newly synthesized single stranded DNA encoded by the editing target sequence replaces the editing target sequence, and introduces a mutation in the editing target sequence of the B2M gene. [0326]In some embodiments, the editing template comprises one or more intended nucleotide edits compared to the sequence on the target strand of the B2M gene that is complementary to the editing target sequence. In some embodiments, the editing template encodes a single stranded DNA that comprises one or more intended nucleotide edits compared to the editing target sequence. In some embodiments, the single stranded DNA replaces the editing target sequence by prime editing, thereby incorporating the one or more intended nucleotide edits. In some embodiments, incorporation of the one or more intended nucleotide edits introduces the mutation in the editing target sequence compared to wild type nucleotides at corresponding positions in the B2M gene. [0327]In some embodiments, the editing target sequence comprises a mutation that is located between positions 44,711,517 -44,718,145 of human chromosome 15 according to GRCh38. [0328]For example, in some embodiments, incorporation of the one or more intended nucleotide edits results in one or more codons that are different from a wild type codon. In some embodiments, incorporation of the one or more intended nucleotide edits results in one or more codons that encode one or more amino acids different from the wild type B2M protein. In some embodiments, incorporation of the one or more intended nucleotide edits WO 2024/238825 PCT/US2024/029746 133 results in one or more in frame premature stop codons, leading to a truncated polypeptide compared to the wild-type B2M protein. By a "B2M protein ", "P2 microglobulin protein ", or "P chain of MHC Class I" is meant a protein having at least about 85% amino acid sequence identity to NCBI Accession No. P61769.1 or fragment thereof and havingimmunomodulatory activity. An exemplary amino acid sequence of wild type B2M protein is provided in SEQ ID NO: 742 (NCBI Accession No. P61769.1. By "B2M gene " is meant a nucleic acid encoding a B2M protein. Exemplary B2M nucleic acid sequences are publicly available, for example, at UCSC human genome database, Gene ENSG00000166710.23, which sequence is incorporated herein by reference in its entirety. Exemplary mRNA/cDNA sequence of wild type B2M protein is provided in SEQ ID NO: 743. [0329] Wild-type B2M protein sequence (SEQ ID NO: 742) MSRSVALAVLALLSLSGLEAIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVD LLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKW DRDM id="p-330"

[0330] Wild-type B2M mRNA/CDNA sequence (SEQ ID NO: 743) ATTCCTGAAGCTGACAGCATTCGGGCCGAGATGTCTCGCTCCGTGGCCTTAGCTG TGCTCGCGCTACTCTCTCTTTCTGGCCTGGAGGCTATCCAGCGTACTCCAAAGATT CAGGTTTACTCACGTCATCCAGCAGAGAATGGAAAGTCAAATTTCCTGAATTGCT ATGTGTCTGGGTTTCATCCATCCGACATTGAAGTTGACTTACTGAAGAATGGAGA GAGAATTGAAAAAGTGGAGCATTCAGACTTGTCTTTCAGCAAGGACTGGTCTTTC TATCTCTTGTACTACACTGAATTCACCCCCACTGAAAAAGATGAGTATGCCTGCC GTGTGAACCATGTGACTTTGTCACAGCCCAAGATAGTTAAGTGGGATCGAGACA TGTAAGCAGCATCATGGAGGTTTGAAGATGCCGCATTTGGATTGGATGAATTCCA AATTCTGCTTGCTTGCTTTTTAATATTGATATGCTTATACACTTACACTTTATGCA CAAAATGTAGGGTTATAATAATGTTAACATGGACATGATCTTCTTTATAATTCTA CTTTGAGTGCTGTCTCCATGTTTGATGTATCTGAGCAGGTTGCTCCACAGGTAGCT CTAGGAGGGCTGGCAACTTAGAGGTGGGGAGCAGAGAATTCTCTTATCCAACAT CAACATCTTGGTCAGATTTGAACTCTTCAATCTCTTGCACTCAAAGCTTGTTAAG ATAGTTAAGCGTGCATAAGTTAACTTCCAATTTACATACTCTGCTTAGAATTTGG GGGAAAATTTAGAAATATAATTGACAGGATTATTGGAAATTTGTTATAATGAATG AAACATTTTGTCATATAAGATTCATATTTACTTCTTATACATTTGATAAAGTAAGG WO 2024/238825 PCT/US2024/029746 134 CATGGTTGTGGTTAATCTGGTTTATTTTTGTTCCACAAGTTAAATAAATCATAAAA CTTGA [0331]A guide RNA core (also referred to herein as the gRNA core, gRNA scaffold, or gRNA backbone sequence) of a PEgRNA may contain a polynucleotide sequence that binds to a DNA binding domain (e.g., Cas9) of a prime editor. The gRNA core may interact with a prime editor as described herein, for example, by association with a DNA binding domain, such as a DNA nickase of the prime editor. [0332]One of skill in the art will recognize that different prime editors having different DNA binding domains from different DNA binding proteins may require different gRNA core sequences specific to the DNA binding protein. In some embodiments, the gRNA core is capable of binding to a Cas9-based prime editor. In some embodiments, the gRNA core is capable of binding to a Cpfl-based prime editor. In some embodiments, the gRNA core is capable of bi ndi ng to a Casl2b-based prime editor. [0333]In some embodiments, the gRNA core comprises regions and secondary structures involved in binding with specific CRISPR Cas proteins. For example, in a Cas9 based prime editing system, the gRNA core of a PEgRNA may comprise one or more regions of a base paired "lower stem " adjacent to the spacer sequence and a base paired "upper stem " following the lower stem, where the lower stem and upper stem may be connected by a "bulge " comprising unpaired RNAs. The gRNA core may further comprise a "nexus " distal from the spacer sequence, followed by a hairpin structure, e.g, at the 3׳ end, as exemplified in FIG. 3. In some embodiments, the gRNA core comprises modified nucleotides as compared to a wild type gRNA core in the lower stem, upper stem, and/or the hairpin. For example, nucleotides in the lower stem, upper stem, an/or the hairpin regions may be modified, deleted, or replaced. In some embodiments, RNA nucleotides in the lower stem, upper stem, an/or the hairpin regions may be replaced with one or more DNA sequences. In some embodiments, the gRNA core comprises unmodified or wild type RNA sequences in the nexus and/or the bulge regions. In some embodiments, the gRNA core does not include long stretches of A-T pairs, for example, a GUUUU-AAAAC pairing element. In some embodiments, a prime editing system comprises a prime editor and a PEgRN A, wherein the prime editor comprises a SpCas9 nickase variant thereof, and the gRNA core of the PEgRNA comprises the sequence:GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU GAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO: 646); WO 2024/238825 PCT/US2024/029746 135 GUUUGAGAGCUAGAAAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUU GAAAAAGUGGGACCGAGUCGGUCC (SEQ ID NO: 648), or GUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCG UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO: 649). In some embodiments, the gRNA core comprises the sequence GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU GAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO: 646). Any gRNA core sequences known in the art are also contemplated in the prime editing compositions described herein. [0334]In some embodiments, the PEgRNA and/or ngRNA comprises a gRNA core that comprises a nucleic acid sequence selected from the Table 28 below. In some embodiments, the PEgRNA and/or ngRNA comprises a gRNA core that comprises a nucleic acid sequence that has at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 653, 646, 652, 647, 649,654, or 648. In some embodiments, the PEgRNA and/or ngRNA comprises a gRNA core that comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 653, 646, 652, 647, 649, 654, or 648.Table: 28: lists exemplary nucleic acid sequences of gRNA core (gRNA scaffold). The sequences in Table 28 below 7 are annotated with SEQ ID NO as required by ST.26 standard. Although all the sequences provided in Table 28 are RNA sequences, "T" is used instead of a"U" in the sequences for consistency with the ST.26 standard. SEQ ID NO Sequence 646 GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCA ACTTGAAAAAGTGGCACCGAGTCGGTGC647 GTTTAAGAGCGGGGAAATCCGCAAGTTTAAATAAGGCTAGTCCGTTAT CAGCGTGAAAACGCGGCACCGAGTCGGTGC648 GTTTGAGAGCTAGAAATAGCAAGTTTAAATAAGGCTAGTCCGTTATCA ACTTGAAAAAGTGGGACCGAGTCGGTCC649 GTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTA GTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC650 GTTITAGTACTCTGGAAACAGAATCTACTAAAACAAGGCAAAATGCCG TGTTTATCTCGTCAACTTGTTGGCGAGA651 GTTTTAGTACTCTGGAAACAGAATCTACTGAAACAAGACAATATGTCG TGTTTATCCCATCAATTTATTGGTGGGA652 GTTTAAGAGCTAGAAATAGCAAGTTTAAATAAGGCTAGTCCGTTATCA ACTTGAAAAAGTGGCACCGAGTCGGTGC653 GTTTAAGAGCTAGAAATAGCAAGTTTAAATAAGGCTAGTCCGTTATCA GCGTGAAAACGCGGCACCGAGTCGGTGC WO 2024/238825 PCT/US2024/029746 136 654 GTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTA GTCCGTTATCAGCGTGAAAACGCGGCACCGAGTCGGTGC656 GTTTTAGAGCTATACGTAGCAAGTTAAAATAAGGCTAGTCCGTTATCA ACTTTACGAAGTGGCACCGAGTCGGTGC657 GTTTTAGAGCTATACGTAGCAAGTTAAAATAAGGCTAGTCCGTTATCA ACTTTACGAAGTGGGACCGAGTCGGTCC658 GTTTTAGAGCTAGCTCATGAAAATGAGCTAGCAAGTTAAAATAAGGCT AGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCC660 GTTTTAGAGCTATGCTGGAAACAGCATAGCAAGTTAAAATAAGGCTA GTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC id="p-335"

[0335]In some embodiments, a PEgRNA comprises a linker. In some embodiments, the secondary structure or a 3’ motif is linked to one or more other component of a PEgRNA via a linker. For example, in some embodiments, the secondary structure is at the 3’ end of the PEgRNA (e.g., a RTT, or a PBS) and is linked to the 3’ end of a PBS via a linker. For example, in some embodiments, a 3’ motif is at the 3’ end of the PEgRNA and is linked to the 3’ end of a PEgRNA (e.g., a RTT or a PBS) via a linker. In some embodiments, the secondary structure or a 5’ motif is at the 5’ end of the PEgRNA and is linked to the 5’ end of a spacer via a linker. In some embodiments, the linker is a nucleotide linker that is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. In some embodiments, the linker is 5 to 10 nucleotides in length. In some embodiments, the linker is 10 to 20 nucleotides in length. In some embodiments, the linker is to 25 nucleotides in length. In some embodiments, the linker is 8 nucleotides in length. [0336]In some embodiments, the linker is designed to minimize base pairing between the linker and another component of the PEgRNA. In some embodiments, the linker is designed to minimize base pairing between the linker and the spacer. In some embodiments, the linker is designed to minimize base pairing between the linker and the PBS. In some embodiments, the linker is designed to minimize base pairing between the linker and the editing template. In some embodiments, the linker is designed to minimize base pairing between the linker and the sequence of the RNA secondary structure. In some embodiments, the linker is optimized to minimize base pairing between the linker and another component of the PEgRNA, in order of the following priority: spacer, PBS, editing template and then scaffold. In some embodiments, base paring probability is calculated using ViennaRNA 2.0 ,as described in Lorenz, R. et al. ViennaRNA package 2.0. Algorithms Mol. Biol. 6, incorporated by reference in its entirety herein, under standard parameters (37 °C, 1 M NaCl, 0.05 M MgC12).

WO 2024/238825 PCT/US2024/029746 137 id="p-337"

[0337]A PEgRNA may also comprise optional modifiers, e.g., 3' end modifier region and/or an 5' end modifier region. In some embodiments, a PEgRNA comprises at least one nucleotide that is not part of a spacer, a gRNA core, or an extension arm. The optional sequence modifiers could be positioned within or between any of the other regions shown, and not limited to being located at the 3' and 5' ends. In certain embodiments, the PEgRNA comprises secondary RNA structure, such as, but not limited to, aptamers, hairpins, stem/loops, toeloops, and/or RNA-binding protein recruitment domains (e.g., the MSaptamer which recruits and binds to the MS2cp protein). In some embodiments, a PEgRNA comprises a short stretch of uracil at the 5' end or the 3' end. For example, in some embodiments, a PEgRNA comprising a 3' extension arm comprises a "UUU" sequence at the 3' end of the extension arm. In some embodiments, a PEgRNA comprises a toeloop sequence at the 3' end. In some embodiments, the PEgRNA comprises a 3' extension arm and a toeloop sequence at the 3' end of the extension arm. In some embodiments, the PEgRNA comprises a 5' extension arm and a toeloop sequence at the 5' end of the extension arm. In some embodiments, the PEgRNA comprises a toeloop element having the sequence 5'- GAAANNNNN-3‘, wherein N is any nucleobase. In some embodiments, the secondary RNA structure is positioned within the spacer. In some embodiments, the secondary structure is positioned within the extension arm. In some embodiments, the secondary structure is positioned within the gRNA core. In some embodiments, the secondary structure is positioned between the spacer and the gRNA core, between the gRNA core and the extension arm, or between the spacer and the extension arm. In some embodiments, the secondary structure is positioned between the PBS and the editing template. In some embodiments the secondary structure is positioned at the 3' end or at the 5' end of the PEgRNA. In some embodiments, the PEgRNA comprises a transcriptional termination signal at the 3' end of the PEgRNA. In addition to secondary RNA structures, the PEgRNA may comprise a chemical linker or a poly(N) linker or tail, where "N" can be any nucleobase. In some embodiments, the chemical linker may function to prevent reverse transcription of the gRNA core. [0338]In some embodiments, a prime editing system or composition further comprises a nick guide polynucleotide, such as a nick guide RNA (ngRNA). In some embodiments, a ngRNA comprises a spacer (referred to as a ngRNA spacer or ng spacer) and a gRNA core, wherein the spacer of the ngRNA comprises a region of complementarity to the edit strand, and wherein the gRNA core can interact with a Cas, e.g., Cas9, of a prime editor. Without wishing to be bound by any particular theory, an ngRNA may bind to the edit strand and WO 2024/238825 PCT/US2024/029746 138 direct the Cas nickase to generate a nick on the non-edit strand (or target strand). In some embodiments, the nick on the non-edit strand directs endogenous DNA repair machinery to use the edit strand as a template for repair of the non-edit strand, which may increase efficiency of prime editing. In some embodiments, the non-edit strand is nicked by a prime editor localized to the non-edit strand by the ngRNA. Accordingly, also provided herein are PEgRNA systems comprising at least one PEgRNA and at least one ngRNA. [0339]A prime editing system comprising a PEgRNA (or one or more polynucleotide encoding the PEgRNA) and a prime editor protein (or one or more polynucleotides encoding the prime editor), may be referred to as a PE2 prime editing system and the corresponding editing approach referred to as PE2 approach or PE2 strategy. A PE2 system does not contain a ngRNA. A prime editing system comprising a PEgRNA (or one or more polynucleotide encoding the PEgRNA), a prime editor protein (or one or more polynucleotides encoding the prime editor), and a ngRNA (or one or more polynucleotides encoding the ngRNA) may be referred to as a "PE3" prime editing system. In some embodiments, an ngRNA spacer sequence is complementary to a portion of the edit strand that includes the intended nucleotide edit, and may hybridize with the edit strand only after the edit has been incorporated on the edit strand. Such ngRNA may be referred to a "PE3b" ngRNA, and the prime editing system a PE3b prime editing system. [0340]In some embodiments, a PEgRNA or a nick guide RNA (ngRNA) can be chemically synthesized, or can be assembled or cloned and transcribed from a DNA sequence, e.g., a plasmid DNA sequence, or by any RNA oligonucleotide synthesis method known in the art. In some embodiments, DNA sequence that encodes a PEgRNA (or ngRNA) may be designed to append one or more nucleotides at the 5? end or the 3' end of the PEgRNA (or nick guide RNA) encoding sequence to enhance PEgRNA transcription. For example, in some embodiments, a DNA sequence that encodes a PEgRNA (or nick guide RNA) (or an ngRNA) may be designed to append a nucleotide G at the 5' end. Accordingly, in some embodiments, the PEgRNA (or nick guide RNA) may comprise an appended nucleotide G at the 5' end. In some embodiments, a DNA sequence that encodes a PEgRNA (or nick guide RNA) may be designed to append a sequence that enhances transcription, e.g., a Kozak sequence, at the 5' end. In some embodiments, a DNA sequence that encodes a PEgRNA (or nick guide RNA) may be designed to append the sequence CACC or CCACC at the 5' end. Accordingly, in some embodiments, the PEgRNA (or nick guide RNA) may comprise an appended sequence CACC or CCACC at the 5' end. In some embodiments, a DNA sequence that encodes a WO 2024/238825 PCT/US2024/029746 139 PEgRNA (or nick guide RNA) may be designed to append the sequence TTT, TTTT, TTTTT, TTTTTT, q’j’jTTTj׳ a! the 3' end. Accordingly, in some embodiments, the PEgRNA (or nick guide RNA) may comprise an appended sequence UUU, UUUU, UUUUU, UUUUUU, or UUUUUUU at the 3' end. In some embodiments, a PEgRNA or ngRNA may include a modifying sequence at the 3' end having the sequence AACAUUGACGCGUCUCUACGUGGGGGCGCG (SEQ ID NO: 745). In some embodiments, a PEgRNA or a ngRNA comprises the sequence TTTT at the 3' end. In someenc ] jn ׳ 3 ؛ a ךיךיךיךיךיךיךי embodiments, a PEgRNA or a ngRNA comprises the sequencesome embodiments, a PEgRNA or a ngRNA comprises a 3’ terminator sequence (e.g., TTTT;) at the 3’ end. In some embodiments, a PEgRNA or a ngRNA comprises a transcription adaptation sequence (e.g., TTTTTTT) at the 3’ end. The sequences in TTTT and TTTTTTT are RNA sequences, "T" is used instead of a "U" in the sequences for consistency with the ST.26 standard. [0341]In some embodiments, the ng search target sequence is located on the non-target strand, within 10 base pairs to 100 base pairs of an intended nucleotide edit incorporated by the PEgRNA on the edit strand. In some embodiments, the ng target search target sequence is within 10 bp, 20 bp, 30 bp, 40 bp, 50 bp, 60 bp, 70 bp, 80 bp, 90 bp, 91 bp, 92 bp, 93 bp, bp, 95 bp, 96 bp, 97 bp, 98 bp, 99 bp, or 100 bp of an intended nucleotide edit incorporated by the PEgRNA on the edit strand. In some embodiments, the 5' ends of the ng search target sequence and the PEgRNA search target sequence are within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 bp apart from each other. In some embodiments, the 5' ends of the ng search target sequence and the PEgRNA search target sequence are within 10 bp, 20 bp, 30 bp, 40 bp, 50 bp, 60 bp, bp, 80 bp, 90 bp, 91 bp, 92 bp, 93 bp, 94 bp, 95 bp, 96 bp, 97 bp, 98 bp, 99 bp, or 100 bp apart from each other. [0342]In some embodiments, an ng spacer sequence is complementary to, and may hybridize with the second search target sequence only after an intended nucleotide edit has been incorporated on the edit strand, by the editing template of a PEgRNA. In some embodiments, such a prime editing system maybe referred to as a "PE3b" prime editing system or composition. In some embodiments, the ngRNA comprises a spacer sequence that matches only the edit strand after incorporation of the nucleotide edits, but not the endogenous target gene sequence on the edit strand. Accordingly, in some embodiments, an intended nucleotide edit is incorporated within the ng search target sequence.

WO 2024/238825 PCT/US2024/029746 140 id="p-343"

[0343]Exemplary combinations of PEgRNA components, e.g., spacer, PBS, and edit template/RTT, exemplary full-length PEgRNAs, as well as combinations of PEgRNA and corresponding ngRNA(s) are provided in Tables 1-21. Tables 1-21 each contain three columns. The left column is the sequence number. The middle column provides the sequence of the component, labeled with a SEQ ID NO where required by ST.26 standard. Although all the sequences provided in Tables 1-21 are RNA sequences, "T" is used instead of a "U" in the sequences for consistency with the ST.26 standard. The right column contains a description of the sequence. [0344]The disclosed RTT and hill-length PEgRNA are designed to disrupt the coding sequence of the B2M gene. The RTTs and hill-length PEgRNAs in Tables 1-21 encode edits that, when integrated into the B2M gene, result in one or more premature in frame stop codons compared to a wild-type B2M gene sequence. The RTTs and PEgRNAs can thus be used to reduce or eliminate expression of B2M protein encoded by the B2M gene. [0345]The PEgRNAs exemplified in Tables 1-21 comprise: (a) a spacer comprising at its 3’ end a sequence corresponding to a listed PEgRNA spacersequence; (b) a gRNA core capable of complexing with a Cas9 protein, and (c) an extension arm comprising: (i) an editing template comprising at its 3’ end any RTT sequence from the same table as the PEgRNA spacer, and (ii) a prime binding site (PBS) comprising at its 5’ end any PBS sequence from the same table as the PEgRNA spacer. The spacers in Tables 1-21 each corresponds to a NGG PAM sequence, wherein N refers to any one of nucleotide A, G, C, or T. Accordingly, in some embodiments, the PEgRNA spacers and PEgRNAs exemplified in Tables 1-21 can be used with a prime editor comprising any appropriate Cas9 protein capable of recognizing an NGG PAM, e.g., a SpCas9 H840A nickase. Additional exemplary Casvariants that can recognize an NGG PAM sequence are provided in Table 23. The PEgRNA spacer can be, for example, 17-22 nucleotides in length. In some embodiments, the PEgRNA spacer is 20 nucleotides in length. [0346]The editing template can be referred to as a reverse transcription template (RTT). The disclosed RTTs and full-length PEgRNAs in Tables 1-21 are designed to disrupt the B2M gene by introducing premature stop codons and frame shift mutations into the B2M gene. For example, certain exemplary RTTs disclosed in Tables 1-21 and corresponding full-length PEgRNAs are designed to insert two consecutive in frame stop codons (e.g., TAATAA) into the B2M gene. Alternatively, other exemplaryRTT disclosed in Tables 1-21 and full-length PEgRNA are designed to introduce a frameshift mutation into the B2M gene by a single WO 2024/238825 PCT/US2024/029746 141 nucleotide insertion c.50insG or a single nucleotide deletion c.51delC. The edits encoded or comprised by the RTTs and the full length PEgRNAs are indicated in the Description column of Tables 1-21 .In some of Tables 1-21, RTTs are further annotated with a * followed by a number code. As described below, a PE3b ngRNA spacer annotated with the same * and number code as an RTT in the same Table has perfect complementarity to the edit strand post-edit by a PEgRNA containing the RTT. In some embodiments, the RTT comprises at least 4 contiguous nucleotides of complementarity with the edit strand, wherein the at least contiguous nucleotides are located upstream of the 5’ most edit in the RTT. In some embodiments, the RTT comprises at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous nucleotides of complementarity with the edit strand, wherein the at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous nucleotides are located upstream of the 5’ most edit in the RTT. In some embodiments, the RTT comprises at least contiguous nucleotides of complementarity with the edit strand, wherein the at least contiguous nucleotides are located upstream of the 5’ most edit in the RTT. In some embodiments, the RTT comprises at least 8 contiguous nucleotides of complementarity with the edit strand, wherein the at least 8 contiguous nucleotides are located upstream of the 5’ most edit in the RTT. In some embodiments, the RTT comprises at least 10 contiguous nucleotides of complementarity with the edit strand, wherein the at least 10 contiguous nucleotides are located upstream of the 5’ most edit in the RTT. In some embodiments, the RTT comprises at least 4, 5, 6, 7, 8, 9, or 10 contiguous nucleotides of complementarity with the edit strand, wherein the at least 4, 5, 6, 7, 8, 9, or 10 contiguous nucleotides are located upstream of the 5’ most edit in the RTT. [0347]The PBS can be, for example, 5 to 17 nucleotides in length. [0348]The PEgRNA provided in Tables 1-21 can comprise, from 5’ to 3’, the spacer, the gRNA core, the edit template, and the PBS. The 3’ end of the edit template can be contiguous with the 5’ end of the PBS. The PEgRNA can comprise multiple RNA molecules or can be a single RNA molecule. Any PEgRNA exemplified in Tables 1-21 may comprise, or further comprise, a 3’ motif at the 3’ end of the extension arm, such as a universal motif, a sequence specific motif, or a series of 1, 2, 3, 4, 5, 6, 7 or more U nucleotides. In some embodiments, a universal or structural 3’ motif that is capable of forming a tertiary structure on its own such as a hairpin, a pseudoknot, or other RNA structure is used. In some embodiments, a sequence specific motif is used that is designed to hybridize with a portion of the RTT while not WO 2024/238825 PCT/US2024/029746 142 covering the PBS. Whether a universal or sequence specific motif is used, it can be connected to the 3’ of the PBS via a linker sequence. In some embodiments, the PEgRNA comprises U nucleotides at its 3’ end. The 4 consecutive U nucleotides in exemplary PEgRNAs in Tables 1-21 are included in the sequences. Without being bound by theory, such 3’ motifs are believed to increase PEgRNA stability. The PEgRNA may be chemically synthesized and may alternatively or additionally comprise one or more chemical modifications, such as phosphorothioate (PS) bond(s), 2’-O-methylated (2’-Ome) nucleotides, or a combination thereof. In some embodiments, the PEgRNA comprise 3’ mN*mN*mN*N and 5’mN*mN*mN* modifications, where m indicates that the nucleotide contains a 2’-O-Me modification and a * indicates the presence of a phosphorothioate bond. PEgRNA sequences exemplified in Tables 1-21 may alternatively be adapted for expression from a nucleic acid template with a U6 promoter, for example, by including a 5’ terminal G if the spacer of the PEgRNA begins with another nucleotide, by including 6 or 7 U nucleotides at the 3’ end of the extension arm, or both. Such transcription-adapted sequences may further comprise a universal or sequence specific motif between the PBS and the 3’ terminal U series. [0349]In some embodiments, the gRNA core for the PEgRNA comprises a sequence selected from SEQ ID NOs: 646, 647, 648, 649, 650, 651, 652, 653, 654, 656, 657, 658, 660. In some embodiments, the gRNA core comprises a canonical guide RNA scaffold sequence recognized by SpCas9, as set forth in SEQ ID NO: 646. In some embodiments, the gRNA core sequence comprises one or more modifications compared to a canonical SpCas9 guide RNA scaffold sequence as set forth in SEQ ID NO: 646. In some embodiments, the gRNA core comprises SEQ ID NO: 652. In some embodiments, the gRNA core comprises SEQ ID NO: 647. In some embodiments, the gRNA core comprises SEQ ID NO: 648. In some embodiments, the gRNA core comprises SEQ ID NO: 649. In some embodiments, the gRNA core comprises SEQ ID NO: 650. In some embodiments, the gRNA core comprises SEQ ID NO: 651. In some embodiments, the gRNA core comprises SEQ ID NO: 652. In some embodiments, the gRNA core comprises SEQ ID NO: 653. In some embodiments, the gRNA core comprises SEQ ID NO: 654. In some embodiments, the gRNA core comprises SEQ ID NO: 656. In some embodiments, the gRNA core comprises SEQ ID NO: 657. In some embodiments, the gRNA core comprises SEQ ID NO: 658. In some embodiments, the gRNA core comprises SEQ ID NO: 660. Full-length PEgRNAs that contain a non-canonical gRNA core sequence are indicated with SEQ ID NOs in the right column of Tables 1-21.

WO 2024/238825 PCT/US2024/029746 143 id="p-350"

[0350]Any of the PEgRNAs of Tables 1-21 can be used in a Prime Editing system further comprising a nick guide RNA (ngRNA). Such ngRNA can comprise a spacer and a gRNA core capable of complexing with a Cas9 protein. It can be advantageous to select a ngRNA spacer that has a PAM sequence compatible with the Cas9 protein used in the Prime Editor, thus avoiding the need to use two different Cas9 proteins. The ngRNA is capable of directing a complexed Cas9 protein to bind the edit strand of the B2M gene; thus, a complexed Casnickase containing a nuclease inactivating mutation in the HNH domain will nick the non- edit strand. A PE3 ngRNA spacer has perfect complementarity to the edit strand both pre- and post-edit; a PE3b ngRNA spacer has perfect complementarity to the edit strand post-edit. A PE3b spacer annotated with a * followed by a number code has perfect complementarity to the edit strand post-edit with a PEgRNA containing an RTT from the same Table and annotated with the same number code. [0351]Specific nucleotide changes encoded by the PEgRNAs are annotated in Tables 1-by referring to locations in the coding sequence of a wildtype B2M gene, where c.xx or c.xx_yy refer to position xx or between positions xx and yy in the coding sequence of a wildtype B2M gene. For example, annotation c.54_55insTAAG (or c.54insTAAG, or C.54TAAG insertion) means an insertion of nucleotide sequence TAAG between positions corresponding to positions 54 and 55 of the coding sequence in a wildtype B2M gene; c.60_65delinsTAATAG means deletion of nucleotides corresponding to nucleotides 60 to in the coding sequence a wildtype B2M gene, and insertion of nucleotide sequence TAATAG at the deletion site. Unless otherwise specified, chromosome locations and coding sequence positions are as set forth in Genome Reference Consortium Human Build 38 (GrCH38). [0352]Table 1 provides exemplary Prime Editing guide RNAs (PEgRNAs) and components designed to disrupt the B2M gene by encoding the insertion of two in frame stop codons or a frameshift mutation into the B2M gene. The PEgRNAs in Table 1 can be used with any Prime Editor containing an appropriate Cas9 protein capable of recognizing an NGG PAM sequence, wherein N refers to any one of nucleotide A, G, C, or T. Exemplary Cas9 variants that can recognize an NGG PAM sequence are provided in Table 23. [0353]The PEgRNAs exemplified in Table 1 comprise: (a) a spacer comprising at its 3’ end a sequence corresponding to sequence number 4; (b) a gRNA core capable of complexing with a Cas9 protein, and (c) an extension arm comprising: (i) an editing template comprising at its 3’ end a sequence corresponding to any one of sequence numbers 18, 19, 20, and 900- 929 and (ii) a prime binding site (PBS) comprising at its 5’ end a sequence corresponding to WO 2024/238825 PCT/US2024/029746 144 sequence number 5. The PEgRNA spacer can be, for example, 17-22 nucleotides in length. In some embodiments, the PEgRNA spacer is 17-20 nucleotides in length, and can comprise the sequence corresponding to any one of sequence numbers 1-4. In some embodiments, the PEgRNA spacer comprises sequence number 1. The editing template can be referred to as a reverse transcription template (RTT). The editing templates exemplified in Table 1 are designed to disrupt the B2M gene. The disrupting edit can be introduced into any suitable position in the B2M gene, e.g., in the coding region of the B2M gene, for example, at a position corresponding to positions c.54, c.66, or c.60 of the coding sequence of a wildtype B2M gene. The editing template can encode an insertion of one or more in frame stop codons, e.g., two consecutive in frame stop codons into the B2M gene, e.g., a C.54TAATAA insertion, as exemplified by an editing template that comprise at its 3’ end the sequence corresponding to sequence number 18, 19, or 20; a c.54_55TAATAA insertion as exemplified by an editing template that comprise at its 3’ end the sequence corresponding to any one of sequence numbers 916-919 and 928-929; or a c.66_67insTAATAA insertion as exemplified by an editing template that comprise at its 3’ end the sequence corresponding to any one of in sequence numbers 920-923. Alternatively, the editing template can encode a frameshift mutation in the B2M gene, e.g., a c.66_67insCC insertion as exemplified by an editing template that comprise at its 3’ end the sequence corresponding to any one of sequence numbers 900-903; a c.66_67insCC insertion as exemplified by an editing template that comprise at its 3’ end the sequence corresponding to any one of sequence numbers 904- 907; a c.54_55insTAAG insertion as exemplified by an editing template that comprise at its 3’ end the sequence corresponding to any one of sequence numbers 908-911; a c.66_67insTAAG insertion as exemplified by an editing template that comprise at its 3’ end the sequence corresponding to any one of sequence numbers 912-915; or a c.60_65deletion_TAATAG insertion (c.60_65_delinsTAATAG) as exemplified by an editing template that comprise at its 3’ end the sequence corresponding to any one of sequence numbers 924-927. The edit that disrupts the B2M gene expression can be proximal relative to the nick position (i.e. three nucleotides upstream of the 5’ most nucleotide of the PAM sequence). For example, the nick-to-edit distance for the disrupting edit (i.e. non-synonymous edit that disrupts B2M coding sequence) can be 2 nucleotides, as exemplified by an editing template that comprise at its 3’ end the sequence corresponding to any one of sequence numbers 900-903, 908-911, 916-919, and 928-929. In such cases where the non-synonymous edit is with the position corresponding to the PAM sequence (i.e. the nick-to-edit distance of WO 2024/238825 PCT/US2024/029746 145 the non-synonymous edit is 0 to 2), the edit can also be a PAM-silencing edit, e.g., a NGG- >NGC edit, that alters the PAM sequence. Without being bound by theory, a PAM-silencing edit can prevent the corresponding nucleic acid guided nuclease or CRISPR nuclease or nickase in the Prime Editor from cleaving the target sequence once the PAM-silencing edit is incorporated, thereby improving editing efficiency. The edit that disrupts the B2M gene expression can also be distal relative to the nick position. For example, the nick-to-edit distance for the disrupting edit (i.e. non-synonymous edit that disrupts B2M coding sequence) can be 8 nucleotides, as exemplified by an editing template that comprise at its 3’ end the sequence corresponding to any one of sequence numbers 924-927, or 14 nucleotides as exemplified by an editing template that comprise at its 3’ end the sequence corresponding to any one of sequence numbers 904-907, 912-915, and 920-923. In cases where the non- synonymous disrupting edit is more distal from the nick site, the editing template can further encode a synonymous or non-synonymous PAM-silencing edit, for example, a c.58G->C (NGG->NGC PAM silencing) edit. B2M disrupting edits and PAM-silencing edits are indicated for the exemplary editing templates in the right column of Table 1. In some embodiments, the RTT comprises at least 4 contiguous nucleotides of complementarity with the edit strand, wherein the at least 4 contiguous nucleotides are located upstream of the 5’ most edit in the RTT. In some embodiments, the RTT comprises at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous nucleotides of complementarity with the edit strand, wherein the at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous nucleotides are located upstream of the 5’ most edit in the RTT. In some embodiments, the RTT comprises at least 6 contiguous nucleotides of complementarity with the edit strand, wherein the at least 6 contiguous nucleotides are located upstream of the 5’ most edit in the RTT. In some embodiments, the RTT comprises at least 8 contiguous nucleotides of complementarity with the edit strand, wherein the at least 8 contiguous nucleotides are located upstream of the 5’ most edit in the RTT. In some embodiments, the RTT comprises at least 10 contiguous nucleotides of complementarity with the edit strand, wherein the at least contiguous nucleotides are located upstream of the 5’ most edit in the RTT. In some embodiments, the RTT comprises at least 4, 5, 6, 7, 8, 9, or 10 contiguous nucleotides of complementarity with the edit strand, wherein the at least 4, 5, 6, 7, 8, 9, or 10 contiguous nucleotides are located upstream of the 5’ most edit in the RTT.

WO 2024/238825 PCT/US2024/029746 146 id="p-354"

[0354]The PBS can be, for example, 5 to 17 nucleotides in length and can comprise the sequence corresponding to any one of sequence numbers 5-17. [0355]The PEgRNA can comprise, from 5’ to 3’, the spacer, the gRNA core, the edit template, and the PBS. The 3’ end of the edit template can be contiguous with the 5’ end of the PBS. The PEgRNA can comprise multiple RNA molecules or can be a single RNA molecule. Exemplary PEgRNAs provided in Table 1 can comprise a sequence corresponding to any one of sequence numbers 21- 29 and 930-1016. Any PEgRNA exemplified in Table may comprise, or further comprise, a 3’ motif at the 3’ end of the extension arm, such as a universal motif, a sequence specific motif, or a series of 1, 2, 3, 4, 5, 6, 7 or more U nucleotides. In some embodiments, a universal or structural 3’ motif that is capable of forming a tertiary structure on its own such as a hairpin, a pseudoknot, or other RNA structure is used. In some embodiments, a sequence specific motif is used that is designed to hybridize with a portion of the RTT while not covering the PBS. Whether a universal or sequence specific motif is used, it can be connected to the 3’ of the PBS via a linker sequence. In some embodiments, the PEgRNA comprises 4 U nucleotides at its 3’ end. Without being bound by theory, such 3’ motifs are believed to increase PEgRNA stability. The PEgRNA may alternatively or additionally comprise one or more chemical modifications, such as phosphorothioate (PS) bond(s), 2’-O-methylated (2’-Ome) nucleotides, or a combination thereof. In some embodiments, the PEgRNA comprise 3’ mN*mN*mN*N and 5’mN*mN*mN* modifications, where m indicates that the nucleotide contains a 2’-O-Me modification and a * indicates the presence of a phosphorothioate bond. PEgRNA sequences exemplified in Table 1 may alternatively be adapted for expression from a nucleic acid template with a U6 promoter, for example, by including a 5’ terminal G if the spacer of the PEgRNA begins with another nucleotide, by including 6 or 7 U nucleotides at the 3’ end of the extension arm, or both. Such transcription-adapted sequences may further comprise a universal or sequence specific motif between the PBS and the 3’ terminal U series.. [0356]Any of the PEgRNAs of Table 1 can be used in a Prime Editing system further comprising a nick guide RNA (ngRNA). Such ngRNA can comprise a spacer comprising at its 3’ end a sequence corresponding to nucleotides 4-20 of any ngRNA spacer listed in Table I and a gRNA core capable of complexing with a Cas9 protein. For example, the sequence in the spacer of the ngRNA can comprise nucleotides 4-20, 3-20, 2-20, or 1-20 of any one of sequence numbers 1017-1024. In some embodiments, the spacer of the ngRNA is a ngRNA WO 2024/238825 PCT/US2024/029746 147 spacer listed in Table 1. It can be advantageous to select a ngRNA spacer that has a PAM sequence compatible with the Cas9 protein used in the Prime Editor, thus avoiding the need to use two different Cas9 proteins. The ngRNA is capable of directing a complexed Casprotein to bind the edit strand (the PAM strand) of the B2M gene; thus, a complexed Casnickase containing a nuclease inactivating mutation in the HNH domain will nick the non- edit strand. A PE3 ngRNA spacer has perfect complementarity to the edit strand both pre- and post-edit; a PE3b ngRNA spacer has perfect complementarity to the edit strand post-edit. A PE3b ngRNA spacer in Table 1 annotated with the same * and number code as an RTT in Table 1 has perfect complementarity to the edit strand post-edit by a PEgRNA containing the RTT. [0357]Exemplary ngRNAs provided in Table 1 can comprise a sequence corresponding to any one of sequence numbers 1025-1032. A PE3 ngRNA has perfect complementarity to the edit strand both pre- and post-edit; a PE3b ngRNA has perfect complementarity to the edit strand post-edit. A PE3b ngRNA in Table 1 annotated with the same * and number code as an RTT in Table 1 has perfect complementarity in its spacer to the edit strand post-edit by a PEgRNA containing the RTT. [0358]Any ngRNA exemplified in Table 1 may comprise, or further comprise a series of 1, 2, 3, 4, 5, 6, 7 or more U nucleotides. The ngRNA may alternatively or additionally comprise one or more chemical modifications, such as phosphorothioate (PS) bond(s), 2’-O-methylated (2’-Ome) nucleotides, or a combination thereof. In some embodiments, the ngRNA comprise 3’ mN*mN*mN*N and 5’mN*mN*mN* modifications, where m indicates that the nucleotide contains a 2’-O-Me modification and a * indicates the presence of a phosphorothioate bond. ngRNA sequences exemplified in Table 1 may alternatively be adapted for expression from a nucleic acid template with a U6 promoter, for example, by including a 5’ terminal G if the spacer of the ngRNA begins with another nucleotide, by including 6 or 7 U nucleotides at the 3’ end of the extension arm, or both. [0359]Table 2 provides exemplary Prime Editing guide RNAs (PEgRNAs) and components designed to disrupt the B2M gene by encoding the insertion of two in frame stop codons into the B2M gene, for example, c.l2InsTAATAA. [0360]The PEgRNAs exemplified in Table 2 comprise: (a) a spacer comprising at its 3’ end a sequence corresponding to sequence number 33; (b) a gRNA core capable of complexing with a Cas9 protein, and (c) an extension arm comprising: (i) an editing template comprising at its 3’ end a sequence corresponding to any one of sequence numbers 47-49, and (ii) a WO 2024/238825 PCT/US2024/029746 148 prime binding site (PBS) comprising at its 5’ end a sequence corresponding to sequence number 34. The PEgRNA spacer can be, for example, 17-22 nucleotides in length. In some embodiments, the PEgRNA spacer is 17-20 nucleotides in length, and can comprise the sequence corresponding to any one of sequence numbers 30-33. In some embodiments, the PEgRNA spacer comprises sequence number 30. The editing template can be referred to as a reverse transcription template (RTT). The PEgRNAs exemplified in Table 2 are designed to disrupt the B2M gene by encoding the insertion of two in frame stop codons or the complement thereof into the B2M gene, e.g., a C.12TAATAA insertion. For example, the editing template can comprise at its 3’ end the sequence corresponding to sequence number 47, 48, or 49. The PBS can be, for example, 5 to 17 nucleotides in length and can comprise the sequence corresponding to any one of sequence numbers 34-46. [0361]The PEgRNA can comprise, from 5’ to 3’, the spacer, the gRNA core, the edit template, and the PBS. The 3’ end of the edit template can be contiguous with the 5’ end of the PBS. The PEgRNA can comprise multiple RNA molecules or can be a single RNA molecule. Exemplary PEgRNAs provided in Table 2 can comprise a sequence corresponding to any one of sequence numbers 50, 51, 52, 53, 54, 55, 56, 57, or, 58. Any PEgRNA exemplified in Table 2 may comprise, or further comprise, a 3’ motif at the 3’ end of the extension arm, such as a universal motif, a sequence specific motif, or a series of 1, 2, 3, 4, 5, 6, 7 or more U nucleotides. In some embodiments, a universal or structural 3’ motif that is capable of forming a tertiary structure on its own such as a hairpin, a pseudoknot, or other RNA structure is used. In some embodiments, a sequence specific motif is used that is designed to hybridize with a portion of the RTT while not covering the PBS. Whether a universal or sequence specific motif is used, it can be connected to the 3’ of the PBS via a linker sequence. In some embodiments, the PEgRNA comprises 4 U nucleotides at its 3’ end. Without being bound by theory, such 3’ motifs are believed to increase PEgRNA stability. The PEgRNA may alternatively or additionally comprise one or more chemical modifications, such as phosphorothioate (PS) bond(s), 2’-O-methylated (2’-Ome) nucleotides, or a combination thereof. In some embodiments, the PEgRNA comprise 3’ mN*mN*mN*N and 5’mN*mN*mN* modifications, where m indicates that the nucleotide contains a 2’-O-Me modification and a * indicates the presence of a phosphorothioate bond. PEgRNA sequences exemplified in Table 2 may alternatively be adapted for expression from a nucleic acid template with a U6 promoter, for example, by including a 5’ terminal G if the spacer of the PEgRNA begins with another nucleotide, by including 6 or 7 U nucleotides at WO 2024/238825 PCT/US2024/029746 149 the 3’ end of the extension arm, or both. Such transcription-adapted sequences may further comprise a universal or sequence specific motif between the PBS and the 3’ terminal U series. The modifications included in the selection of full length PEgRNAs included in Table are annotated in column 3 of Table 2. [0362]Table 3 provides exemplary Prime Editing guide RNAs (PEgRNAs) and components designed to disrupt the B2M gene by encoding the insertion of two in frame stop codons into the B2M gene, for example, c.46InsTAATAA. [0363]The PEgRNAs exemplified in Table 3 comprise: (a) a spacer comprising at its 3’ end a sequence corresponding to sequence number 62; (b) a gRNA core capable of complexing with a Cas9 protein, and (c) an extension arm comprising: (i) an editing template comprising at its 3’ end a sequence corresponding to any one of sequence numbers 76-78, and (ii) a prime binding site (PBS) comprising at its 5’ end a sequence corresponding to sequence number 63. The PEgRNA spacer can be, for example, 17-22 nucleotides in length. In some embodiments, the PEgRNA spacer is 17-20 nucleotides in length, and can comprise the sequence corresponding to any one of sequence numbers 59-62. In some embodiments, the PEgRNA spacer comprises sequence number 59. The editing template can be referred to as a reverse transcription template (RTT). The RTTs exemplified in Table 3 are designed to disrupt the B2M gene by encoding the insertion of two in frame stop codons or the complement thereof into the B2M gene, e.g., a C.46TAATAA insertion. For example, the editing template can comprise at its 3’ end the sequence corresponding to sequence number 76, 77, or 78. The PBS can be, for example, 5 to 17 nucleotides in length and can comprise the sequence corresponding to any one of sequence numbers 63-75. [0364]The PEgRNA can comprise, from 5’ to 3’, the spacer, the gRNA core, the edit template, and the PBS. The 3’ end of the edit template can be contiguous with the 5’ end of the PBS. The PEgRNA can comprise multiple RNA molecules or can be a single RNA molecule. Exemplary PEgRNAs provided in Table 3 can comprise a sequence corresponding to any one of sequence numbers 79, 80, 81, 82, 83, 84, 85, 86, or 87. Any PEgRNA exemplified in Table 3 may comprise, or further comprise, a 3’ motif at the 3’ end of the extension arm, such as a universal motif, a sequence specific motif, or a series of 1, 2, 3, 4, 5, 6, 7 or more U nucleotides. In some embodiments, a universal or structural 3’ motif that is capable of forming a tertiary structure on its own such as a hairpin, a pseudoknot, or other RNA structure is used. In some embodiments, a sequence specific motif is used that is designed to hybridize with a portion of the RTT while not covering the PBS. Whether a WO 2024/238825 PCT/US2024/029746 150 universal or sequence specific motif is used, it can be connected to the 3’ of the PBS via a linker sequence. In some embodiments, the PEgRNA comprises 4 U nucleotides at its 3’ end. Without being bound by theory, such 3’ motifs are believed to increase PEgRNA stability. The PEgRNA may alternatively or additionally comprise one or more chemical modifications, such as phosphorothioate (PS) bond(s), 2’-O-methylated (2’-Ome) nucleotides, or a combination thereof. In some embodiments, the PEgRNA comprise 3’ mN*mN*mN*N and 5’mN*mN*mN* modifications, where m indicates that the nucleotide contains a 2’-O-Me modification and a * indicates the presence of a phosphorothioate bond. PEgRNA sequences exemplified in Table 3 may alternatively be adapted for expression from a nucleic acid template with a U6 promoter, for example, by including a 5’ terminal G if the spacer of the PEgRNA begins with another nucleotide, by including 6 or 7 U nucleotides at the 3’ end of the extension arm, or both. Such transcription-adapted sequences may further comprise a universal or sequence specific motif between the PBS and the 3’ terminal U series. The modifications included in the selection of full length PEgRNAs included in Table are annotated in column 3 of Table 3. [0365]Table 4 provides exemplary Prime Editing guide RNAs (PEgRNAs) and components designed to disrupt the B2M gene by encoding the insertion of two in frame stop codons into the B2M gene, for example, c.51InsTAATAA. [0366]The PEgRNAs exemplified in Table 4 comprise: (a) a spacer comprising at its 3’ end a sequence corresponding to sequence number 91; (b) a gRNA core capable of complexing with a Cas9 protein, and (c) an extension arm comprising: (i) an editing template comprising at its 3’ end a sequence corresponding to any one of sequence numbers 105-107, and (ii) a prime binding site (PBS) comprising at its 5’ end a sequence corresponding to sequence number 92. The PEgRNA spacer can be, for example, 17-22 nucleotides in length. In some embodiments, the PEgRNA spacer is 17-20 nucleotides in length, and can comprise the sequence corresponding to any one of sequence numbers 88, 89, 90, or 91. In some embodiments, the PEgRNA spacer comprises sequence number 88. The editing template can be referred to as a reverse transcription template (RTT). The RTTs exemplified in Table 4 are designed to disrupt the B2M gene by encoding the insertion of two in frame stop codons or the complement thereof into the B2M gene, e.g., a C.51TAATAA insertion. For example, the editing template can comprise at its 3’ end the sequence corresponding to sequence number 105, 106 or 107. The PBS can be, for example, 5 to 17 nucleotides in length and can comprise the sequence corresponding to any one of sequence numbers 92-104.

WO 2024/238825 PCT/US2024/029746 151 id="p-367"

[0367]The PEgRNA can comprise, from 5’ to 3’, the spacer, the gRNA core, the edit template, and the PBS. The 3’ end of the edit template can be contiguous with the 5’ end of the PBS. The PEgRNA can comprise multiple RNA molecules or can be a single RNA molecule. Exemplary PEgRNAs provided in Table 4 can comprise a sequence corresponding to any one of sequence numbers 108, 109, 110, 111, 112, 113, 114, 115, or 116. Any PEgRNA exemplified in Table 4 may comprise, or further comprise, a 3’ motif at the 3’ end of the extension arm, such as a universal motif, a sequence specific motif, or a series of 1, 2, 3, 4, 5, 6, 7 or more U nucleotides. In some embodiments, a universal or structural 3’ motif that is capable of forming a tertiary structure on its own such as a hairpin, a pseudoknot, or other RNA structure is used. In some embodiments, a sequence specific motif is used that is designed to hybridize with a portion of the RTT while not covering the PBS. Whether a universal or sequence specific motif is used, it can be connected to the 3’ of the PBS via a linker sequence. In some embodiments, the PEgRNA comprises 4 U nucleotides at its 3’ end. Without being bound by theory, such 3’ motifs are believed to increase PEgRNA stability. The PEgRNA may alternatively or additionally comprise one or more chemical modifications, such as phosphorothioate (PS) bond(s), 2’-O-methylated (2’-Ome) nucleotides, or a combination thereof. In some embodiments, the PEgRNA comprise 3’ mN*mN*mN*N and 5’mN*mN*mN* modifications, where m indicates that the nucleotide contains a 2’-O-Me modification and a * indicates the presence of a phosphorothioate bond. PEgRNA sequences exemplified in Table 4 may alternatively be adapted for expression from a nucleic acid template with a U6 promoter, for example, by including a 5’ terminal G if the spacer of the PEgRNA begins with another nucleotide, by including 6 or 7 U nucleotides at the 3’ end of the extension arm, or both. Such transcription-adapted sequences may further comprise a universal or sequence specific motif between the PBS and the 3’ terminal U series. The modifications included in the selection of full length PEgRNAs included in Table are annotated in column 3 of Table 4. [0368]Table 5 provides exemplary Prime Editing guide RNAs (PEgRNAs) and components designed to disrupt the B2M gene by encoding the insertion of two in frame stop codons into the B2M gene, for example, c.4InsTAATAA. [0369]The PEgRNAs exemplified in Table 5 comprise: (a) a spacer comprising at its 3’ end a sequence corresponding to sequence number 120; (b) a gRNA core capable of complexing with a Cas9 protein, and (c) an extension arm comprising: (i) an editing template comprising at its 3’ end a sequence corresponding to sequence number 134-136, and (ii) a prime binding WO 2024/238825 PCT/US2024/029746 152 site (PBS) comprising at its 5’ end a sequence corresponding to sequence number 121. The PEgRNA spacer can be, for example, 17-22 nucleotides in length. In some embodiments, the PEgRNA spacer is 17-20 nucleotides in length, and can comprise the sequence corresponding to any one of sequence numbers 117, 118, 119, or 120. In some embodiments, the PEgRNA spacer comprises sequence number 117. The editing template can be referred to as a reverse transcription template (RTT). The RTTs exemplified in Table 5 are designed to disrupt the B2M gene by encoding the insertion of two in frame stop codons or the complement thereof into the B2M gene, e.g., a C.4TAATAA insertion. For example, the editing template can comprise at its 3’ end the sequence corresponding to sequence number 134, 135, or 136. The PBS can be, for example, 5 to 17 nucleotides in length and can comprise the sequence corresponding to any one of sequence numbers 121-133. [0370]The PEgRNA can comprise, from 5’ to 3’, the spacer, the gRNA core, the edit template, and the PBS. The 3’ end of the edit template can be contiguous with the 5’ end of the PBS. The PEgRNA can comprise multiple RNA molecules or can be a single RNA molecule. Exemplary PEgRNAs provided in Table 5 can comprise a sequence corresponding to any one of sequence numbers 137, 138, 139, 140, 141, 142, 143, 144, or 145. Any PEgRNA exemplified in Table 5 may comprise, or further comprise, a 3’ motif at the 3’ end of the extension arm, such as a universal motif, a sequence specific motif, or a series of 1, 2, 3, 4, 5, 6, 7 or more U nucleotides. In some embodiments, a universal or structural 3’ motif that is capable of forming a tertiary structure on its own such as a hairpin, a pseudoknot, or other RNA structure is used. In some embodiments, a sequence specific motif is used that is designed to hybridize with a portion of the RTT while not covering the PBS. Whether a universal or sequence specific motif is used, it can be connected to the 3’ of the PBS via a linker sequence. In some embodiments, the PEgRNA comprises 4 U nucleotides at its 3’ end. Without being bound by theory, such 3’ motifs are believed to increase PEgRNA stability. The PEgRNA may alternatively or additionally comprise one or more chemical modifications, such as phosphorothioate (PS) bond(s), 2’-O-methylated (2’-Ome) nucleotides, or a combination thereof. In some embodiments, the PEgRNA comprise 3’ mN*mN*mN*N and 5’mN*mN*mN* modifications, where m indicates that the nucleotide contains a 2’-O-Me modification and a * indicates the presence of a phosphorothioate bond. PEgRNA sequences exemplified in Table 5 may alternatively be adapted for expression from a nucleic acid template with a U6 promoter, for example, by including a 5’ terminal G if the spacer of the PEgRNA begins with another nucleotide, by including 6 or 7 U nucleotides at WO 2024/238825 PCT/US2024/029746 153 the 3’ end of the extension arm, or both. Such transcription-adapted sequences may further comprise a universal or sequence specific motif between the PBS and the 3’ terminal U series. The modifications included in the selection of full length PEgRNAs included in Table are annotated in column 3 of Table 5. [0371]Table 6 provides exemplary Prime Editing guide RNAs (PEgRNAs) and components designed to disrupt the B2Mgene by encoding the insertion of two in frame stop codons into the B2Mgene, for example, c.4InsTAATAA. [0372]The PEgRNAs exemplified in Table 6 comprise: (a) a spacer comprising at its 3’ end a sequence corresponding to sequence number 149; (b) a gRNA core capable of complexing with a Cas9 protein, and (c) an extension arm comprising: (i) an editing template comprising at its 3’ end a sequence corresponding to any one of sequence numbers 163-165, and (ii) a prime binding site (PBS) comprising at its 5’ end a sequence corresponding to sequence number 150. The PEgRNA spacer can be, for example, 17-22 nucleotides in length. In some embodiments, the PEgRNA spacer is 17-20 nucleotides in length, and can comprise the sequence corresponding to any one of sequence numbers 146, 147, 148, or 149. In some embodiments, the PEgRNA spacer comprises sequence number 146. The editing template can be referred to as a reverse transcription template (RTT). The RTTs exemplified in Table are designed to disrupt the B2Mgene by encoding the insertion of two in frame stop codons or the complement thereof into the B2Mgene, e.g., a C.4TAATAA insertion. For example, the editing template can comprise at its 3’ end the sequence corresponding to sequence number 163, 164, or 165. The PBS can be, for example, 5 to 17 nucleotides in length and can comprise the sequence corresponding to any one of sequence numbers 150-162. [0373]The PEgRNA can comprise, from 5’ to 3’, the spacer, the gRNA core, the edit template, and the PBS. The 3’ end of the edit template can be contiguous with the 5’ end of the PBS. The PEgRNA can comprise multiple RNA molecules or can be a single RNA molecule. Exemplary PEgRNAs provided in Table 6 can comprise a sequence corresponding to any one of sequence numbers 166, 167, 168, 169, 170, 171, 172, 173, or 174. Any PEgRNA exemplified in Table 6 may comprise, or further comprise, a 3’ motif at the 3’ end of the extension arm, such as a universal motif, a sequence specific motif, or a series of 1, 2, 3, 4, 5, 6, 7 or more U nucleotides. In some embodiments, a universal or structural 3’ motif that is capable of forming a tertiary structure on its own such as a hairpin, a pseudoknot, or other RNA structure is used. In some embodiments, a sequence specific motif is used that is designed to hybridize with a portion of the RTT while not covering the PBS. Whether a WO 2024/238825 PCT/US2024/029746 154 universal or sequence specific motif is used, it can be connected to the 3’ of the PBS via a linker sequence. In some embodiments, the PEgRNA comprises 4 U nucleotides at its 3’ end. Without being bound by theory, such 3’ motifs are believed to increase PEgRNA stability. The PEgRNA may alternatively or additionally comprise one or more chemical modifications, such as phosphorothioate (PS) bond(s), 2’-O-methylated (2’-Ome) nucleotides, or a combination thereof. In some embodiments, the PEgRNA comprise 3’ mN*mN*mN*N and 5’mN*mN*mN* modifications, where m indicates that the nucleotide contains a 2’-O-Me modification and a * indicates the presence of a phosphorothioate bond. PEgRNA sequences exemplified in Table 6 may alternatively be adapted for expression from a nucleic acid template with a U6 promoter, for example, by including a 5’ terminal G if the spacer of the PEgRNA begins with another nucleotide, by including 6 or 7 U nucleotides at the 3’ end of the extension arm, or both. Such transcription-adapted sequences may further comprise a universal or sequence specific motif between the PBS and the 3’ terminal U series. The modifications included in the selection of full length PEgRNAs included in Table are annotated in column 3 of Table 6. [0374]Table 7 provides exemplary Prime Editing guide RNAs (PEgRNAs) and components designed to disrupt the B2M gene by encoding the insertion of two in frame stop codons into the B2M gene, for example, c.4InsTAATAA. [0375]The PEgRNAs exemplified in Table 7 comprise: (a) a spacer comprising at its 3’ end a sequence corresponding to sequence number 178; (b) a gRNA core capable of complexing with a Cas9 protein, and (c) an extension arm comprising: (i) an editing template comprising at its 3’ end a sequence corresponding to any one of sequence numbers 192-194, and (ii) a prime binding site (PBS) comprising at its 5’ end a sequence corresponding to sequence number 179. The PEgRNA spacer can be, for example, 17-22 nucleotides in length. In some embodiments, the PEgRNA spacer is 17-20 nucleotides in length, and can comprise the sequence corresponding to any one of sequence numbers 175, 176, 177, or 178. In some embodiments, the PEgRNA spacer comprises sequence number 175. The editing template can be referred to as a reverse transcription template (RTT). The RTTs exemplified in Table are designed to disrupt the B2M gene by encoding the insertion of two in frame stop codons or the complement thereof into the B2M gene, e.g., a C.4TAATAA insertion. For example, the editing template can comprise at its 3’ end the sequence corresponding to sequence number 192, 193, or 194. The PBS can be, for example, 5 to 17 nucleotides in length and can comprise the sequence corresponding to any one of sequence numbers 179-191.

WO 2024/238825 PCT/US2024/029746 155 id="p-376"

[0376]The PEgRNA can comprise, from 5’ to 3’, the spacer, the gRNA core, the edit template, and the PBS. The 3’ end of the edit template can be contiguous with the 5’ end of the PBS. The PEgRNA can comprise multiple RNA molecules or can be a single RNA molecule. Exemplary PEgRNAs provided in Table 7 can comprise a sequence corresponding to any one of sequence numbers 195, 196, 197, 198, 199, 200, or 201. Any PEgRNA exemplified in Table 7 may comprise, or further comprise, a 3’ motif at the 3’ end of the extension arm, such as a universal motif, a sequence specific motif, or a series of 1, 2, 3, 4, 5, 6, 7 or more U nucleotides. In some embodiments, a universal or structural 3’ motif that is capable of forming a tertiary structure on its own such as a hairpin, a pseudoknot, or other RNA structure is used. In some embodiments, a sequence specific motif is used that is designed to hybridize with a portion of the RTT while not covering the PBS. Whether a universal or sequence specific motif is used, it can be connected to the 3’ of the PBS via a linker sequence. In some embodiments, the PEgRNA comprises 4 U nucleotides at its 3’ end. Without being bound by theory, such 3’ motifs are believed to increase PEgRNA stability. The PEgRNA may alternatively or additionally comprise one or more chemical modifications, such as phosphorothioate (PS) bond(s), 2’-O-methylated (2’-Ome) nucleotides, or a combination thereof. In some embodiments, the PEgRNA comprise 3’ mN*mN*mN*N and 5’mN*mN*mN* modifications, where m indicates that the nucleotide contains a 2’-O-Me modification and a * indicates the presence of a phosphorothioate bond. PEgRNA sequences exemplified in Table 7 may alternatively be adapted for expression from a nucleic acid template with a U6 promoter, for example, by including a 5’ terminal G if the spacer of the PEgRNA begins with another nucleotide, by including 6 or 7 U nucleotides at the 3’ end of the extension arm, or both. Such transcription-adapted sequences may further comprise a universal or sequence specific motif between the PBS and the 3’ terminal U series. The modifications included in the selection of full length PEgRNAs included in Table are annotated in column 3 of Table 7. [0377]Table 8 provides exemplary Prime Editing guide RNAs (PEgRNAs) and components designed to disrupt the B2M gene by introducing one or more premature in frame stop codons. The PEgRNAs in Table 8 can be used with any Prime Editor containing an appropriate Cas9 protein capable of recognizing an NGG PAM sequence, wherein N refers to any one of nucleotide A, G, C, or T. Exemplary Cas9 variants that can recognize an NGG PAM sequence are provided in Table 23. The PEgRNAs exemplified in Table 8 comprise: (a) a spacer comprising at its 3’ end a sequence corresponding to sequence number 205; (b) a WO 2024/238825 PCT/US2024/029746 156 gRNA core capable of complexing with a Cas9 protein, and (c) an extension arm comprising: (i) an editing template comprising at its 3’ end (A) nucleotides 13-24 of sequence number 221, (B) nucleotides 12-20 of sequence number 227, or (C) nucleotides 7-17 of sequence number 231, (ii) a prime binding site (PBS) comprising at its 5’ end a sequence corresponding to sequence number 206. [0378]The PEgRNA spacer can be, for example, 17-22 nucleotides in length. In some embodiments, the PEgRNA spacer is 17-20 nucleotides in length, and can comprise the sequence corresponding to any one of sequence numbers 202, 203, 204, or 205. [0379]The editing template can be referred to as a reverse transcription template (RTT). The RTTs exemplified in Table 8 are designed to disrupt the B2M gene by introducing one or more in frame premature stop codons in the coding sequence of the B2M gene. For example, the editing template can encode an insertion of two consecutive stop codons or the complement thereof the two consecutive stop codons. In some embodiments, the insertion comprises a c.54insTAATAA edit. An exemplary RTT can comprise at its 3’ end the sequence corresponding to nucleotides 13-24, 12-24, 11-24, 10-24, 9-24, 8-24, 7-24, 6-24, 5- 24, 4-24, 3-24, 2-24, or 1-24 of sequence number 221. In some embodiments, the RTT comprises at its 3’ end the sequence corresponding to sequence number 219, 220, or 221. Alternatively, the editing template can encode one or more frame shift mutations relative to the wildtype B2M gene. For example, the frame shift mutation can be a c.51delC deletion or the complement thereof. An exemplary RTT can comprise at its 3’ end the sequence corresponding to nucleotides 12-20, 11-20, 10-20, 9-20, 8-20, 7-20, 6-20, 5-20, 4-20, 3-20, 2- 20, or 1-20 of sequence number 227. In some embodiments, the RTT comprises at its 3’ end the sequence corresponding to sequence number 222, 223, 224, 225, 226, or 227.Alternatively, the frame shift mutation can be a c.50insG insertion or the complement thereof. An exemplary RTT can comprise at its 3’ end the sequence corresponding to nucleotides 7- 17, 6-17, 5-17, 4-17, 3-17, 2-17, or 1-17 of sequence number 231. In some embodiments, the RTT comprises at its 3’ end the sequence corresponding to sequence numbe228, 229, 230, or 231. Nucleotide changes introduced by the edits encoded in the RTTs and PEgRNAs are indicated in the right column of Table 8. In some embodiments, the RTT comprises at least contiguous nucleotides of complementarity with the edit strand, wherein the at least contiguous nucleotides are located upstream of the 5’ most edit in the RTT. [0380]The PBS can be, for example, 5 to 17 nucleotides in length and can comprise the sequence corresponding to any one of sequence numbers 206-218.

WO 2024/238825 PCT/US2024/029746 157 id="p-381"

[0381]The PEgRNA can comprise, from 5’ to 3’, the spacer, the gRNA core, the edit template, and the PBS. The 3’ end of the edit template can be contiguous with the 5’ end of the PBS. The PEgRNA can comprise multiple RNA molecules or can be a single RNA molecule. Exemplary PEgRNAs provided in Table 8 can comprise a sequence corresponding to any one of sequence numbers 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, or 262. Any PEgRNA exemplified in Table 8 may comprise, or further comprise, a 3’ motif at the 3’ end of the extension arm, such as a universal motif, a sequence specific motif, or a series of 1, 2, 3, 4, 5, 6, 7 or more U nucleotides. In some embodiments, a universal or structural 3’ motif that is capable of forming a tertiary structure on its own such as a hairpin, a pseudoknot, or other RNA structure is used. In some embodiments, a sequence specific motif is used that is designed to hybridize with a portion of the RTT while not covering the PBS. Whether a universal or sequence specific motif is used, it can be connected to the 3’ of the PBS via a linker sequence. In some embodiments, the PEgRNA comprises 4 U nucleotides at its 3’ end. Without being bound by theory, such 3’ motifs are believed to increase PEgRNA stability. The PEgRNA may alternatively or additionally comprise one or more chemical modifications, such as phosphorothioate (PS) bond(s), 2’-O-methylated (2’- Ome) nucleotides, or a combination thereof. In some embodiments, the PEgRNA comprise 3’ mN*mN*mN*N and 5’mN*mN*mN* modifications, where m indicates that the nucleotide contains a 2’-O-Me modification and a * indicates the presence of a phosphorothioate bond. PEgRNA sequences exemplified in Table 8 may alternatively be adapted for expression from a nucleic acid template with a U6 promoter, for example, by including a 5’ terminal G if the spacer of the PEgRNA begins with another nucleotide, by including 6 or 7 U nucleotides at the 3’ end of the extension arm, or both. Such transcription-adapted sequences may further comprise a universal or sequence specific motif between the PBS and the 3’ terminal U series. The modifications included in the selection of full length PEgRNAs included in Table are annotated in column 3 of Table 8. [0382]Any of the PEgRNAs of Table 8 can be used in a Prime Editing system further comprising a nick guide RNA (ngRNA). Such ngRNA can comprise a spacer comprising at its 3’ end a sequence corresponding to nucleotides 4-20 of any ngRNA spacer listed in Table and a gRNA core capable of complexing with a Cas9 protein. For example, the sequence in the spacer of the ngRNA can comprise nucleotides 4-20, 3-20, 2-20, or 1-20 of any one of sequence numbers 263-268. In some embodiments, the spacer of the ngRNA is a ngRNA WO 2024/238825 PCT/US2024/029746 158 spacer listed in Table 8. It can be advantageous to select a ngRNA spacer that has a PAM sequence compatible with the Cas9 protein used in the Prime Editor, thus avoiding the need to use two different Cas9 proteins. The ngRNA is capable of directing a complexed Casprotein to bind the edit strand of the B2M gene; thus, a complexed Cas9 nickase containing a nuclease inactivating mutation in the HNH domain will nick the non-edit strand. A PEngRNA spacer has perfect complementarity to the edit strand both pre- and post-edit; a PE3b ngRNA spacer has perfect complementarity to the edit strand post-edit. A PE3b ngRNA spacer in Table 8 annotated with the same * and number code as an RTT in Table 8 has perfect complementarity to the edit strand post-edit by a PEgRNA containing the RTT. [0383]Exemplary ngRNAs provided in Table 8 can comprise a sequence corresponding to any one of sequence numbers 824-827. A PE3 ngRNA has perfect complementarity to the edit strand both pre- and post-edit; a PE3b ngRNA has perfect complementarity to the edit strand post-edit. A PE3b ngRNA in Table 8 annotated with the same * and number code as an RTT in Table 8 has perfect complementarity in its spacer to the edit strand post-edit by a PEgRNA containing the RTT. [0384]Any ngRNA exemplified in Table 8 may comprise, or further comprise a series of 1, 2, 3, 4, 5, 6, 7 or more U nucleotides. The ngRNA may alternatively or additionally comprise one or more chemical modifications, such as phosphorothioate (PS) bond(s), 2’-O-methylated (2’-Ome) nucleotides, or a combination thereof. In some embodiments, the ngRNA comprise 3’ mN*mN*mN*N and 5’mN*mN*mN* modifications, where m indicates that the nucleotide contains a 2’-O-Me modification and a * indicates the presence of a phosphorothioate bond. ngRNA sequences exemplified in Table 8 may alternatively be adapted for expression from a nucleic acid template with a U6 promoter, for example, by including a 5’ terminal G if the spacer of the ngRNA begins with another nucleotide, by including 6 or 7 U nucleotides at the 3’ end of the extension arm, or both. [0385]Table 9 provides exemplary Prime Editing guide RNAs (PEgRNAs) and components designed to disrupt the B2M gene by encoding the insertion of two in frame stop codons or a frameshift mutation into the B2M gene. The PEgRNAs in Table 9 can be used with any Prime Editor containing an appropriate Cas9 protein capable of recognizing an NGG PAM sequence, wherein N refers to any one of nucleotide A, G, C, or T. Exemplary Cas9 variants that can recognize an NGG PAM sequence are provided in Table 23. [0386]The PEgRNAs exemplified in Table 9 comprise: (a) a spacer comprising at its 3’ end a sequence corresponding to sequence number 272; (b) a gRNA core capable of complexing WO 2024/238825 PCT/US2024/029746 159 with a Cas9 protein, and (c) an extension arm comprising: (i) an editing template comprising at its 3’ end a sequence corresponding to any one of sequence numbers 286-288 and 1033- 1063, and (ii) a prime binding site (PBS) comprising at its 5’ end a sequence corresponding to sequence number 273. The PEgRNA spacer can be, for example, 17-22 nucleotides in length. In some embodiments, the PEgRNA spacer is 17-20 nucleotides in length, and can comprise the sequence corresponding to any one of sequence numbers 269, 270, 271, or 272. In some embodiments, the PEgRNa spacer comprises sequence number 269. The editing template can be referred to as a reverse transcription template (RTT). The RTTs exemplified in Table 9 are designed to disrupt the B2M. The disrupting edit can be introduced into any suitable position in the B2M gene, e.g., in the coding region of the B2M gene, for example, at a position corresponding to positions c.21, or c.3 of the coding sequence of a wildtype B2M gene. The editing template can encode an insertion of one or more in frame stop codons, e.g., two consecutive in frame stop codons into the B2M gene, e.g., a c.21insTAATAA insertion as exemplified by an editing template that comprise at its 3’ end the sequence corresponding to any one of sequence numbers 286-288, 1049-1052, and 1061-1063; or a c.3_4insTAATAA insertion as exemplified by an editing template that comprise at its 3’ end the sequence corresponding to any one of sequence numbers 1053-1056. Alternatively, the editing template can encode a frameshift mutation in the B2M gene, e.g., a c.21_22insCC insertion as exemplified by an editing template that comprise at its 3’ end the sequence corresponding to any one of sequence numbers 1033-1036; a c.3_4insCC insertion as exemplified by an editing template that comprise at its 3’ end the sequence corresponding to any one of sequence numbers 1037-1040; a c.21_22insTAAG insertion as exemplified by an editing template that comprise at its 3’ end the sequence corresponding to any one of sequence numbers 1041-1044; a c.3_4insTAAG insertion as exemplified by an editing template that comprise at its 3’ end the sequence corresponding to any one of sequence numbers 1045- 1048; or a c.3_8deletion_TAATGA insertion (c.60_65_delinsTAATGA) as exemplified by an editing template that comprise at its 3’ end the sequence corresponding to any one of sequence numbers 1057-1060 . [0387]The edit that disrupts the B2M gene expression can be proximal relative to the nick position (i.e. three nucleotides upstream of the 5’ most nucleotide of the PAM sequence). For example, the nick-to-edit distance for the disrupting edit (i.e. non-synonymous edit that disrupts B2M coding sequence) can be 1 nucleotide, as exemplified by an editing template that comprise at its 3’ end the sequence corresponding to any one of sequence numbers 286- WO 2024/238825 PCT/US2024/029746 160 288, 1033-1036, 1041-1044, 1049-1052, and 1061-1063. In such cases where the non- synonymous edit is with the position corresponding to the PAM sequence (i.e. the nick-to- edit distance of the non-synonymous edit is 0 to 2), the edit can also be a PAM-silencing edit, e.g., an NGG->NGC edit, that alters the PAM sequence. Without being bound by theory, a PAM-silencing edit can prevent the corresponding nucleic acid guided nuclease or CRISPR nuclease or nickase in the Prime Editor from cleaving the target sequence once the PAM- silencing edit is incorporated, thereby improving editing efficiency. The edit that disrupts the B2M gene expression can also be distal relative to the nick position. For example, the nick- to-edit distance for the disrupting edit (i.e. non-synonymous edit that disrupts B2M coding sequence) can be 19 nucleotides, as exemplified by an editing template that comprise at its 3’ end the sequence corresponding to any one of sequence numbers 1037-1040, 1045-1048, and 1053-1056. In cases where the non-synonymous disrupting edit is more distal from the nick site, the editing template can further encode a synonymous or non-synonymous PAM- silencing edit, for example, a c.17C->G (NGG->NGC PAM silencing) edit. B2M disrupting edits and PAM-silencing edits are indicated for the exemplary editing templates in the right column of Table 9. In some embodiments, the RTT comprises at least 4 contiguous nucleotides of complementarity with the edit strand, wherein the at least 4 contiguous nucleotides are located upstream of the 5’ most edit in the RTT. In some embodiments, the RTT comprises at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21,22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous nucleotides of complementarity with the edit strand, wherein the at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous nucleotides are located upstream of the 5’ most edit in the RTT. In some embodiments, the RTT comprises at least 6 contiguous nucleotides of complementarity with the edit strand, wherein the at least 6 contiguous nucleotides are located upstream of the 5’ most edit in the RTT. In some embodiments, the RTT comprises at least 8 contiguous nucleotides of complementarity with the edit strand, wherein the at least 8 contiguous nucleotides are located upstream of the 5’ most edit in the RTT. In some embodiments, the RTT comprises at least 10 contiguous nucleotides of complementarity with the edit strand, wherein the at least 10 contiguous nucleotides are located upstream of the 5’ most edit in the RTT. In some embodiments, the RTT comprises at least 4, 5, 6, 7, 8, 9, or 10 contiguous nucleotides of complementarity with the edit strand, wherein the at least 4, 5, 6, 7, 8, 9, or 10 contiguous nucleotides are located upstream of the 5’ most edit in the RTT.

WO 2024/238825 PCT/US2024/029746 161 id="p-388"

[0388]The PBS can be, for example, 5 to 17 nucleotides in length and can comprise the sequence corresponding to any one of sequence numbers 273-285. [0389]The PEgRNA can comprise, from 5’ to 3’, the spacer, the gRNA core, the edit template, and the PBS. The 3’ end of the edit template can be contiguous with the 5’ end of the PBS. The PEgRNA can comprise multiple RNA molecules or can be a single RNA molecule. Exemplary PEgRNAs provided in Table 9 can comprise a sequence corresponding to any one of sequence numbers 289- 297 and 1064-1151. Any PEgRNA exemplified in Table 9 may comprise, or further comprise, a 3’ motif at the 3’ end of the extension arm, such as a universal motif, a sequence specific motif, or a series of 1, 2, 3, 4, 5, 6, 7 or more U nucleotides. In some embodiments, a universal or structural 3’ motif that is capable of forming a tertiary structure on its own such as a hairpin, a pseudoknot, or other RNA structure is used. In some embodiments, a sequence specific motif is used that is designed to hybridize with a portion of the RTT while not covering the PBS. Whether a universal or sequence specific motif is used, it can be connected to the 3’ of the PBS via a linker sequence. In some embodiments, the PEgRNA comprises 4 U nucleotides at its 3’ end. Without being bound by theory, such 3’ motifs are believed to increase PEgRNA stability. The PEgRNA may alternatively or additionally comprise one or more chemical modifications, such as phosphorothioate (PS) bond(s), 2’-O-methylated (2’-Ome) nucleotides, or a combination thereof. In some embodiments, the PEgRNA comprise 3’ mN*mN*mN*N and 5’mN*mN*mN* modifications, where m indicates that the nucleotide contains a 2’-O-Me modification and a * indicates the presence of a phosphorothioate bond. PEgRNA sequences exemplified in Table 9 may alternatively be adapted for expression from a nucleic acid template with a U6 promoter, for example, by including a 5’ terminal G if the spacer of the PEgRNA begins with another nucleotide, by including 6 or 7 U nucleotides at the 3’ end of the extension arm, or both. Such transcription-adapted sequences may further comprise a universal or sequence specific motif between the PBS and the 3’ terminal U series. [0390]Any of the PEgRNAs of Table 9 can be used in a Prime Editing system further comprising a nick guide RNA (ngRNA). Such ngRNA can comprise a spacer comprising at its 3’ end a sequence corresponding to nucleotides 4-20 of any ngRNA spacer listed in Table and a gRNA core capable of complexing with a Cas9 protein. For example, the sequence in the spacer of the ngRNA can comprise nucleotides 4-20, 3-20, 2-20, or 1-20 of any one of sequence numbers 1152-1156. In some embodiments, the spacer of the ngRNA is a ngRNA WO 2024/238825 PCT/US2024/029746 162 spacer listed in Table 9. It can be advantageous to select a ngRNA spacer that has a PAM sequence compatible with the Cas9 protein used in the Prime Editor, thus avoiding the need to use two different Cas9 proteins. The ngRNA is capable of directing a complexed Casprotein to bind the edit strand (the PAM strand) of the B2M gene; thus, a complexed Casnickase containing a nuclease inactivating mutation in the HNH domain will nick the non- edit strand. A PE3 ngRNA spacer has perfect complementarity to the edit strand both pre- and post-edit; a PE3b ngRNA spacer has perfect complementarity to the edit strand post-edit. A PE3b ngRNA spacer in Table 9 annotated with the same * and number code as an RTT in Table 9 has perfect complementarity to the edit strand post-edit by a PEgRNA containing the RTT. [0391]Exemplary ngRNAs provided in Table 9 can comprise a sequence corresponding to any one of sequence numbers 1157-1161. A PE3 ngRNA has perfect complementarity to the edit strand both pre- and post-edit; a PE3b ngRNA has perfect complementarity to the edit strand post-edit. A PE3b ngRNA in Table 9 annotated with the same * and number code as an RTT in Table 9 has perfect complementarity in its spacer to the edit strand post-edit by a PEgRNA containing the RTT. [0392]Any ngRNA exemplified in Table 9 may comprise, or further comprise a series of 1, 2, 3, 4, 5, 6, 7 or more U nucleotides. The ngRNA may alternatively or additionally comprise one or more chemical modifications, such as phosphorothioate (PS) bond(s), 2’-O-methylated (2’-Ome) nucleotides, or a combination thereof. In some embodiments, the ngRNA comprise 3’ mN*mN*mN*N and 5’mN*mN*mN* modifications, where m indicates that the nucleotide contains a 2’-O-Me modification and a * indicates the presence of a phosphorothioate bond. ngRNA sequences exemplified in Table 9 may alternatively be adapted for expression from a nucleic acid template with a U6 promoter, for example, by including a 5’ terminal G if the spacer of the ngRNA begins with another nucleotide, by including 6 or 7 U nucleotides at the 3’ end of the extension arm, or both. [0393]Table 10 provides exemplary Prime Editing guide RNAs (PEgRNAs) and components designed to disrupt the B2M gene by encoding the insertion of two in frame stop codons into the B2M gene, for example, c.21InsTAATAA. [0394]The PEgRNAs exemplified in Table 10 comprise: (a) a spacer comprising at its 3’ end a sequence corresponding to sequence number 301; (b) a gRNA core capable of complexing with a Cas9 protein, and (c) an extension arm comprising: (i) an editing template comprising at its 3’ end a sequence corresponding to any one of sequence numbers 315-317, and (ii) a WO 2024/238825 PCT/US2024/029746 163 prime binding site (PBS) comprising at its 5’ end a sequence corresponding to sequence number 302. The PEgRNA spacer can be, for example, 17-22 nucleotides in length. In some embodiments, the PEgRNA spacer is 17-20 nucleotides in length, and can comprise the sequence corresponding to any one of sequence numbers 298, 299, 300, or 301. In some embodiments, the PEgRNA spacer comprises sequence number 298. The editing template can be referred to as a reverse transcription template (RTT). The RTTs exemplified in Table are designed to disrupt the B2M gene by encoding the insertion of two in frame stop codons or the complement thereof into the B2M gene, e.g., a C.21TAATAA insertion. For example, the editing template can comprise at its 3’ end the sequence corresponding to sequence number 315, 316, or 317. The PBS can be, for example, 5 to 17 nucleotides in length and can comprise the sequence corresponding to any one of sequence numbers 302- 314. [0395]The PEgRNA can comprise, from 5’ to 3’, the spacer, the gRNA core, the edit template, and the PBS. The 3’ end of the edit template can be contiguous with the 5’ end of the PBS. The PEgRNA can comprise multiple RNA molecules or can be a single RNA molecule. Exemplary PEgRNAs provided in Table 10 can comprise a sequence corresponding to any one of sequence numbers 318, 319, 320, 321, 322, 323, 324, 325, or, 326. Any PEgRNA exemplified in Table 10 may comprise, or further comprise, a 3’ motif at the 3’ end of the extension arm, such as a universal motif, a sequence specific motif, or a series of 1, 2, 3, 4, 5, 6, 7 or more U nucleotides. In some embodiments, a universal or structural 3’ motif that is capable of forming a tertiary structure on its own such as a hairpin, a pseudoknot, or other RNA structure is used. In some embodiments, a sequence specific motif is used that is designed to hybridize with a portion of the RTT while not covering the PBS. Whether a universal or sequence specific motif is used, it can be connected to the 3’ of the PBS via a linker sequence. In some embodiments, the PEgRNA comprises 4 U nucleotides at its 3’ end. Without being bound by theory, such 3’ motifs are believed to increase PEgRNA stability. The PEgRNA may alternatively or additionally comprise one or more chemical modifications, such as phosphorothioate (PS) bond(s), 2’-O-methylated (2’- Ome) nucleotides, or a combination thereof. In some embodiments, the PEgRNA comprise 3’ mN*mN*mN*N and 5’mN*mN*mN* modifications, where m indicates that the nucleotide contains a 2’-O-Me modification and a * indicates the presence of a phosphorothioate bond. PEgRNA sequences exemplified in Table 10 may alternatively be adapted for expression from a nucleic acid template with a U6 promoter, for example, by including a 5’ terminal G if WO 2024/238825 PCT/US2024/029746 164 the spacer of the PEgRNA begins with another nucleotide, by including 6 or 7 U nucleotides at the 3’ end of the extension arm, or both. Such transcription-adapted sequences may further comprise a universal or sequence specific motif between the PBS and the 3’ terminal U series. The modifications included in the selection of full length PEgRNAs included in Table are annotated in column 3 of Table 10. [0396]Table 11 provides exemplary Prime Editing guide RNAs (PEgRNAs) and components designed to disrupt the B2M gene by encoding the insertion of two in frame stop codons or a frameshift mutation into the B2M gene. The PEgRNAs in Table 11 can be used with any Prime Editor containing an appropriate Cas9 protein capable of recognizing an NGG PAM sequence, wherein N refers to any one of nucleotide A, G, C, or T. Exemplary Cas9 variants that can recognize an NGG PAM sequence are provided in Table 23. [0397]The PEgRNAs exemplified in Table 11 comprise: (a) a spacer comprising at its 3’ end a sequence corresponding to sequence number 330; (b) a gRNA core capable of complexing with a Cas9 protein, and (c) an extension arm comprising: (i) an editing template comprising at its 3’ end a sequence corresponding to any one of sequence numbers 344-346 and 1162- 1191, and (ii) a prime binding site (PBS) comprising at its 5’ end a sequence corresponding to sequence number 331. The PEgRNA spacer can be, for example, 17-22 nucleotides in length. In some embodiments, the PEgRNA spacer is 17-20 nucleotides in length, and can comprise the sequence corresponding to any one of sequence numbers 327, 328, 329, or 330. In some embodiments, the PEgRNA spacer comprises sequence number 327. The editing template can be referred to as a reverse transcription template (RTT). The RTTs exemplified in Table 11 are designed to disrupt the B2M gene. The disrupting edit can be introduced into any suitable position in the B2M gene, e.g., in the coding region of the B2M gene, for example, at a position corresponding to positions c.21, c.15, or c.3 of the coding sequence of a wildtype B2M gene. The editing template can encode an insertion of one or more in frame stop codons, e.g., two consecutive in frame stop codons into the B2M gene, e.g., a c.21insTAATAA insertion, as exemplified by an editing template that comprise at its 3’ end the sequence corresponding to any one of sequence numbers 344-346; a c.l5_16insTAATAA insertion as exemplified by an editing template that comprise at its 3’ end the sequence corresponding to any one of sequence numbers 1178-1181 and 1191; or a c.3_4insTAATAA insertion as exemplified by an editing template that comprise at its 3’ end the sequence corresponding to any one of in sequence numbers 1182-1185. Alternatively, the editing template can encode a frameshift mutation in the B2M gene, e.g., a c.l5_16insCC insertion WO 2024/238825 PCT/US2024/029746 165 as exemplified by an editing template that comprise at its 3’ end the sequence corresponding to any one of sequence numbers 1162-1165; a c.3_4insCC insertion as exemplified by an editing template that comprise at its 3’ end the sequence corresponding to any one of sequence numbers 1166-1169; a c.l5_16insTAAG insertion as exemplified by an editing template that comprise at its 3’ end the sequence corresponding to any one of sequence numbers 1170-1173; a c.3_4insTAAG insertion as exemplified by an editing template that comprise at its 3’ end the sequence corresponding to any one of sequence numbers 1174- 1177; or a c.3_8deletion_TAATGA insertion (c.3_8delinsTAATGA) as exemplified by an editing template that comprise at its 3’ end the sequence corresponding to any one of sequence numbers 1186-1190. [0398]The edit that disrupts the B2M gene expression can be proximal relative to the nick position (i.e. three nucleotides upstream of the 5’ most nucleotide of the PAM sequence). For example, the nick-to-edit distance for the disrupting edit (i.e. non-synonymous edit that disrupts B2M coding sequence) can be 1 nucleotides, as exemplified by an editing template that comprise at its 3’ end the sequence corresponding to any one of sequence numbers 1162- 1165, 1170-1173, 1178-1181, and 1191. In such cases where the non-synonymous edit is with the position corresponding to the PAM sequence (i.e. the nick-to-edit distance of the non-synonymous edit is 0 to 2), the edit can also be a PAM-silencing edit, e.g., a NGG- >NGC edit, that alters the PAM sequence. Without being bound by theory, a PAM-silencing edit can prevent the corresponding nucleic acid guided nuclease or CRISPR nuclease or nickase in the Prime Editor from cleaving the target sequence once the PAM-silencing edit is incorporated, thereby improving editing efficiency. [0399]The edit that disrupts the B2M gene expression can also be distal relative to the nick position. For example, the nick-to-edit distance for the disrupting edit (i.e. non-synonymous edit that disrupts B2M coding sequence) can be 13 nucleotides, as exemplified by an editing template that comprise at its 3’ end the sequence corresponding to any one of sequence numbers 1166-1169, 1174-1177, or 1182-1185. In cases where the non-synonymous disrupting edit is more distal from the nick site, the editing template can further encode a synonymous or non-synonymous PAM-silencing edit, for example, a c.l 1C->G (NGG- >NGC PAM silencing) edit. B2M disrupting edits and PAM-silencing edits are indicated for the exemplary editing templates in the right column of Table 11. In some embodiments, the RTT comprises at least 4 contiguous nucleotides of complementarity with the edit strand, wherein the at least 4 contiguous nucleotides are located upstream of the 5’ most edit in the WO 2024/238825 PCT/US2024/029746 166 RTT. In some embodiments, the RTT comprises at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous nucleotides of complementarity with the edit strand, wherein the at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous nucleotides are located upstream of the 5’ most edit in the RTT. In some embodiments, the RTT comprises at least 6 contiguous nucleotides of complementarity with the edit strand, wherein the at least 6 contiguous nucleotides are located upstream of the 5’ most edit in the RTT. In some embodiments, the RTT comprises at least 8 contiguous nucleotides of complementarity with the edit strand, wherein the at least 8 contiguous nucleotides are located upstream of the 5’ most edit in the RTT. In some embodiments, the RTT comprises at least 10 contiguous nucleotides of complementarity with the edit strand, wherein the at least 10 contiguous nucleotides are located upstream of the 5’ most edit in the RTT. In some embodiments, the RTT comprises at least 4, 5, 6, 7, 8, 9, or 10 contiguous nucleotides of complementarity with the edit strand, wherein the at least 4, 5, 6, 7, 8, 9, or 10 contiguous nucleotides are located upstream of the 5’ most edit in the RTT. [0400]. The PBS can be, for example, 5 to 17 nucleotides in length and can comprise the sequence corresponding to any one of sequence numbers 331-343. [0401]The PEgRNA can comprise, from 5’ to 3’, the spacer, the gRNA core, the edit template, and the PBS. The 3’ end of the edit template can be contiguous with the 5’ end of the PBS. The PEgRNA can comprise multiple RNA molecules or can be a single RNA molecule. Exemplary PEgRNAs provided in Table 11 can comprise a sequence corresponding to any one of sequence numbers 347- 355 and 1192-1279. Any PEgRNA exemplified in Table 11 may comprise, or further comprise, a 3’ motif at the 3’ end of the extension arm, such as a universal motif, a sequence specific motif, or a series of 1, 2, 3, 4, 5, 6, 7 or more U nucleotides. In some embodiments, a universal or structural 3’ motif that is capable of forming a tertiary structure on its own such as a hairpin, a pseudoknot, or other RNA structure is used. In some embodiments, a sequence specific motif is used that is designed to hybridize with a portion of the RTT while not covering the PBS. Whether a universal or sequence specific motif is used, it can be connected to the 3’ of the PBS via a linker sequence. In some embodiments, the PEgRNA comprises 4 U nucleotides at its 3’ end. Without being bound by theory, such 3’ motifs are believed to increase PEgRNA stability. The PEgRNA may alternatively or additionally comprise one or more chemical modifications, such as phosphorothioate (PS) bond(s), 2’-O-methylated (2’-Ome) WO 2024/238825 PCT/US2024/029746 167 nucleotides, or a combination thereof. In some embodiments, the PEgRNA comprise 3’ mN*mN*mN*N and 5’mN*mN*mN* modifications, where m indicates that the nucleotide contains a 2’-O-Me modification and a * indicates the presence of a phosphorothioate bond. PEgRNA sequences exemplified in Table 11 may alternatively be adapted for expression from a nucleic acid template with a U6 promoter, for example, by including a 5’ terminal G if the spacer of the PEgRNA begins with another nucleotide, by including 6 or 7 U nucleotides at the 3’ end of the extension arm, or both. Such transcription-adapted sequences may further comprise a universal or sequence specific motif between the PBS and the 3’ terminal U series.. [0402]Any of the PEgRNAs of Table 11 can be used in a Prime Editing system further comprising a nick guide RNA (ngRNA). Such ngRNA can comprise a spacer comprising at its 3’ end a sequence corresponding to nucleotides 4-20 of any ngRNA spacer listed in Table and a gRNA core capable of complexing with a Cas9 protein. For example, the sequence in the spacer of the ngRNA can comprise nucleotides 4-20, 3-20, 2-20, or 1-20 of any one of sequence numbers 1280-1284. In some embodiments, the spacer of the ngRNA is a ngRNA spacer listed in Table 11. It can be advantageous to select a ngRNA spacer that has a PAM sequence compatible with the Cas9 protein used in the Prime Editor, thus avoiding the need to use two different Cas9 proteins. The ngRNA is capable of directing a complexed Casprotein to bind the edit strand (the PAM strand) of the B2M gene; thus, a complexed Casnickase containing a nuclease inactivating mutation in the HNH domain will nick the non- edit strand. A PE3 ngRNA spacer has perfect complementarity to the edit strand both pre- and post-edit; a PE3b ngRNA spacer has perfect complementarity to the edit strand post-edit. A PE3b ngRNA spacer in Table 11 annotated with the same * and number code as an RTT in Table 11 has perfect complementarity to the edit strand post-edit by a PEgRNA containing the RTT. [0403]Exemplary ngRNAs provided in Table 11 can comprise a sequence corresponding to any one of sequence numbers 1285-1289. A PE3 ngRNA has perfect complementarity to the edit strand both pre- and post-edit; a PE3b ngRNA has perfect complementarity to the edit strand post-edit. A PE3b ngRNA in Table 11 annotated with the same * and number code as an RTT in Table 11 has perfect complementarity in its spacer to the edit strand post-edit by a PEgRNA containing the RTT. [0404]Any ngRNA exemplified in Table 11 may comprise, or further comprise a series of 1, 2, 3, 4, 5, 6, 7 or more U nucleotides. The ngRNA may alternatively or additionally comprise WO 2024/238825 PCT/US2024/029746 168 one or more chemical modifications, such as phosphorothioate (PS) bond(s), 2’-O-methylated (2’-Ome) nucleotides, or a combination thereof. In some embodiments, the ngRNA comprise 3’ mN*mN*mN*N and 5’mN*mN*mN* modifications, where m indicates that the nucleotide contains a 2’-O-Me modification and a * indicates the presence of a phosphorothioate bond. ngRNA sequences exemplified in Table 11 may alternatively be adapted for expression from a nucleic acid template with a U6 promoter, for example, by including a 5’ terminal G if the spacer of the ngRNA begins with another nucleotide, by including 6 or 7 U nucleotides at the 3’ end of the extension arm, or both. [0405]Table 12 provides exemplary Prime Editing guide RNAs (PEgRNAs) and components designed to disrupt the B2M gene by encoding the insertion of two in frame stop codons into the B2M gene, for example, c.2InsTAATAA. [0406]The PEgRNAs exemplified in Table 12 comprise: (a) a spacer comprising at its 3’ end a sequence corresponding to sequence number 359; (b) a gRNA core capable of complexing with a Cas9 protein, and (c) an extension arm comprising: (i) an editing template comprising at its 3’ end a sequence corresponding to any one of sequence numbers 372-375, and (ii) a prime binding site (PBS) comprising at its 5’ end a sequence corresponding to sequence number 360. The PEgRNA spacer can be, for example, 17-22 nucleotides in length. In some embodiments, the PEgRNA spacer is 17-20 nucleotides in length, and can comprise the sequence corresponding to any one of sequence numbers 356, 357, 358, or 359. In some embodiments, the PEgRNA spacer comprises sequence number 356. The editing template can be referred to as a reverse transcription template (RTT). The RTTs exemplified in Table are designed to disrupt the B2M gene by encoding the insertion of two in frame stop codons or the complement thereof into the B2M gene, e.g., a C.2TAATAA insertion. For example, the editing template can comprise at its 3’ end the sequence corresponding to sequence number 373, 374, or 375. The PBS can be, for example, 5 to 17 nucleotides in length and can comprise the sequence corresponding to any one of sequence numbers 360- 372. [0407]The PEgRNA can comprise, from 5’ to 3’, the spacer, the gRNA core, the edit template, and the PBS. The 3’ end of the edit template can be contiguous with the 5’ end of the PBS. The PEgRNA can comprise multiple RNA molecules or can be a single RNA molecule. Exemplary PEgRNAs provided in Table 12 can comprise a sequence corresponding to any one of sequence numbers 376, 377, 378, 379, 380, 381, 382, 383, or 384. Any PEgRNA exemplified in Table 12 may comprise, or further comprise, a 3’ motif at WO 2024/238825 PCT/US2024/029746 169 the 3’ end of the extension arm, such as a universal motif, a sequence specific motif, or a series of 1, 2, 3, 4, 5, 6, 7 or more U nucleotides. In some embodiments, a universal or structural 3’ motif that is capable of forming a tertiary structure on its own such as a hairpin, a pseudoknot, or other RNA structure is used. In some embodiments, a sequence specific motif is used that is designed to hybridize with a portion of the RTT while not covering the PBS. Whether a universal or sequence specific motif is used, it can be connected to the 3’ of the PBS via a linker sequence. In some embodiments, the PEgRNA comprises 4 U nucleotides at its 3’ end. Without being bound by theory, such 3’ motifs are believed to increase PEgRNA stability. The PEgRNA may alternatively or additionally comprise one or more chemical modifications, such as phosphorothioate (PS) bond(s), 2’-O-methylated (2’- Ome) nucleotides, or a combination thereof. In some embodiments, the PEgRNA comprise 3’ mN*mN*mN*N and 5’mN*mN*mN* modifications, where m indicates that the nucleotide contains a 2’-O-Me modification and a * indicates the presence of a phosphorothioate bond. PEgRNA sequences exemplified in Table 12 may alternatively be adapted for expression from a nucleic acid template with a U6 promoter, for example, by including a 5’ terminal G if the spacer of the PEgRNA begins with another nucleotide, by including 6 or 7 U nucleotides at the 3’ end of the extension arm, or both. Such transcription-adapted sequences may further comprise a universal or sequence specific motif between the PBS and the 3’ terminal U series. The modifications included in the selection of full length PEgRNAs included in Table are annotated in column 3 of Table 12. [0408]Table 13 provides exemplary Prime Editing guide RNAs (PEgRNAs) and components designed to disrupt the B2M gene by encoding the insertion of two in frame stop codons into the B2M gene, for example, c.MInsTAATAA. [0409]The PEgRNAs exemplified in Table 13 comprise: (a) a spacer comprising at its 3’ end a sequence corresponding to sequence number 388; (b) a gRNA core capable of complexing with a Cas9 protein, and (c) an extension arm comprising: (i) an editing template comprising at its 3’ end a sequence corresponding to any one of sequence numbers 402-404, and (ii) a prime binding site (PBS) comprising at its 5’ end a sequence corresponding to sequence number 389. The PEgRNA spacer can be, for example, 17-22 nucleotides in length. In some embodiments, the PEgRNA spacer is 17-20 nucleotides in length, and can comprise the sequence corresponding to any one of sequence numbers 385, 387, 387, or 388. In some embodiments, the PEgRNA spacer comprises sequence number 385. The editing template can be referred to as a reverse transcription template (RTT). The RTTs exemplified in Table WO 2024/238825 PCT/US2024/029746 170 13 are designed to disrupt the B2M gene by encoding the insertion of two in frame stop codons or the complement thereof into the B2M gene, e.g., a C.14TAATAA insertion. For example, the editing template can comprise at its 3’ end the sequence corresponding to sequence number 402, 403, or 404. The PBS can be, for example, 5 to 17 nucleotides in length and can comprise the sequence corresponding to any one of sequence numbers 389- 401. [0410]The PEgRNA can comprise, from 5’ to 3’, the spacer, the gRNA core, the edit template, and the PBS. The 3’ end of the edit template can be contiguous with the 5’ end of the PBS. The PEgRNA can comprise multiple RNA molecules or can be a single RNA molecule. Exemplary PEgRNAs provided in Table 13 can comprise a sequence corresponding to any one of sequence numbers 405, 406, 407, 408, 409, 410, 411, 412, or 413. Any PEgRNA exemplified in Table 13 may comprise, or further comprise, a 3’ motif at the 3’ end of the extension arm, such as a universal motif, a sequence specific motif, or a series of 1, 2, 3, 4, 5, 6, 7 or more U nucleotides. In some embodiments, a universal or structural 3’ motif that is capable of forming a tertiary structure on its own such as a hairpin, a pseudoknot, or other RNA structure is used. In some embodiments, a sequence specific motif is used that is designed to hybridize with a portion of the RTT while not covering the PBS. Whether a universal or sequence specific motif is used, it can be connected to the 3’ of the PBS via a linker sequence. In some embodiments, the PEgRNA comprises 4 U nucleotides at its 3’ end. Without being bound by theory, such 3’ motifs are believed to increase PEgRNA stability. The PEgRNA may alternatively or additionally comprise one or more chemical modifications, such as phosphorothioate (PS) bond(s), 2’-O-methylated (2’- Ome) nucleotides, or a combination thereof. In some embodiments, the PEgRNA comprise 3’ mN*mN*mN*N and 5’mN*mN*mN* modifications, where m indicates that the nucleotide contains a 2’-O-Me modification and a * indicates the presence of a phosphorothioate bond. PEgRNA sequences exemplified in Table 13 may alternatively be adapted for expression from a nucleic acid template with a U6 promoter, for example, by including a 5’ terminal G if the spacer of the PEgRNA begins with another nucleotide, by including 6 or 7 U nucleotides at the 3’ end of the extension arm, or both. Such transcription-adapted sequences may further comprise a universal or sequence specific motif between the PBS and the 3’ terminal U series. The modifications included in the selection of full length PEgRNAs included in Table are annotated in column 3 of Table 13.

WO 2024/238825 PCT/US2024/029746 171 id="p-411"

[0411]Table 14 provides exemplary Prime Editing guide RNAs (PEgRNAs) and components designed to disrupt the B2M gene by encoding the insertion of two in frame stop codons into the B2M gene, for example, c.41InsTAATAA. [0412]The PEgRNAs exemplified in Table 14 comprise: (a) a spacer comprising at its 3’ end a sequence corresponding to sequence number 417; (b) a gRNA core capable of complexing with a Cas9 protein, and (c) an extension arm comprising: (i) an editing template comprising at its 3’ end a sequence corresponding to any one of sequence numbers 431-433, and (ii) a prime binding site (PBS) comprising at its 5’ end a sequence corresponding to sequence number 418. The PEgRNA spacer can be, for example, 17-22 nucleotides in length. In some embodiments, the PEgRNA spacer is 17-20 nucleotides in length, and can comprise the sequence corresponding to any one of sequence numbers 414, 415, 416, or 417. In some embodiments, the PEgRNA spacer comprises sequence number 414. The editing template can be referred to as a reverse transcription template (RTT). The RTTs exemplified in Table are designed to disrupt the B2M gene by encoding the insertion of two in frame stop codons or the complement thereof into the B2M gene, e.g., a C.41TAATAA insertion. For example, the editing template can comprise at its 3’ end the sequence corresponding to sequence number 431, 432, or 433. The PBS can be, for example, 5 to 17 nucleotides in length and can comprise the sequence corresponding to any one of sequence numbers 418- 430. [0413]The PEgRNA can comprise, from 5’ to 3’, the spacer, the gRNA core, the edit template, and the PBS. The 3’ end of the edit template can be contiguous with the 5’ end of the PBS. The PEgRNA can comprise multiple RNA molecules or can be a single RNA molecule. Exemplary PEgRNAs provided in Table 14 can comprise a sequence corresponding to any one of sequence numbers 434, 435, 436, 437, 438, 439, 440, 441, or 442. Any PEgRNA exemplified in Table 14 may comprise, or further comprise, a 3’ motif at the 3’ end of the extension arm, such as a universal motif, a sequence specific motif, or a series of 1, 2, 3, 4, 5, 6, 7 or more U nucleotides. In some embodiments, a universal or structural 3’ motif that is capable of forming a tertiary structure on its own such as a hairpin, a pseudoknot, or other RNA structure is used. In some embodiments, a sequence specific motif is used that is designed to hybridize with a portion of the RTT while not covering the PBS. Whether a universal or sequence specific motif is used, it can be connected to the 3’ of the PBS via a linker sequence. In some embodiments, the PEgRNA comprises 4 U nucleotides at its 3’ end. Without being bound by theory, such 3’ motifs are believed to WO 2024/238825 PCT/US2024/029746 172 increase PEgRNA stability. The PEgRNA may alternatively or additionally comprise one or more chemical modifications, such as phosphorothioate (PS) bond(s), 2’-O-methylated (2’- Ome) nucleotides, or a combination thereof. In some embodiments, the PEgRNA comprise 3’ mN*mN*mN*N and 5’mN*mN*mN* modifications, where m indicates that the nucleotide contains a 2’-O-Me modification and a * indicates the presence of a phosphorothioate bond. PEgRNA sequences exemplified in Table 14 may alternatively be adapted for expression from a nucleic acid template with a U6 promoter, for example, by including a 5’ terminal G if the spacer of the PEgRNA begins with another nucleotide, by including 6 or 7 U nucleotides at the 3’ end of the extension arm, or both. Such transcription-adapted sequences may further comprise a universal or sequence specific motif between the PBS and the 3’ terminal U series. The modifications included in the selection of full length PEgRNAs included in Table are annotated in column 3 of Table 14. [0414]Table 15 provides exemplary Prime Editing guide RNAs (PEgRNAs) and components designed to disrupt the B2M gene by encoding the insertion of two in frame stop codons into the B2M gene, for example, c.75InsTAATAA. [0415]The PEgRNAs exemplified in Table 15 comprise: (a) a spacer comprising at its 3’ end a sequence corresponding to sequence number 446; (b) a gRNA core capable of complexing with a Cas9 protein, and (c) an extension arm comprising: (i) an editing template comprising at its 3’ end a sequence corresponding to any one of sequence numbers 460-462, and (ii) a prime binding site (PBS) comprising at its 5’ end a sequence corresponding to sequence number 447. The PEgRNA spacer can be, for example, 17-22 nucleotides in length. In some embodiments, the PEgRNA spacer is 17-20 nucleotides in length, and can comprise the sequence corresponding to any one of sequence numbers 443, 444, 445, or 446. In some embodiments, the PEgRNA spacer comprises sequence number 443. The editing template can be referred to as a reverse transcription template (RTT). The RTTs exemplified in Table are designed to disrupt the B2M gene by encoding the insertion of two in frame stop codons or the complement thereof into the B2M gene, e.g., a C.75TAATAA insertion. For example, the editing template can comprise at its 3’ end the sequence corresponding to sequence number 460, 461, or 462. The PBS can be, for example, 5 to 17 nucleotides in length and can comprise the sequence corresponding to any one of sequence numbers 447- 459. [0416]The PEgRNA can comprise, from 5’ to 3’, the spacer, the gRNA core, the edit template, and the PBS. The 3’ end of the edit template can be contiguous with the 5’ end of WO 2024/238825 PCT/US2024/029746 173 the PBS. The PEgRNA can comprise multiple RNA molecules or can be a single RNA molecule. Exemplary PEgRNAs provided in Table 15 can comprise a sequence corresponding to any one of sequence numbers 463, 464, 465, 466, 467, 468, 469, 470, or 471. Any PEgRNA exemplified in Table 15 may comprise, or further comprise, a 3’ motif at the 3’ end of the extension arm, such as a universal motif, a sequence specific motif, or a series of 1, 2, 3, 4, 5, 6, 7 or more U nucleotides. In some embodiments, a universal or structural 3’ motif that is capable of forming a tertiary structure on its own such as a hairpin, a pseudoknot, or other RNA structure is used. In some embodiments, a sequence specific motif is used that is designed to hybridize with a portion of the RTT while not covering the PBS. Whether a universal or sequence specific motif is used, it can be connected to the 3’ of the PBS via a linker sequence. In some embodiments, the PEgRNA comprises 4 U nucleotides at its 3’ end. Without being bound by theory, such 3’ motifs are believed to increase PEgRNA stability. The PEgRNA may alternatively or additionally comprise one or more chemical modifications, such as phosphorothioate (PS) bond(s), 2’-O-methylated (2’- Ome) nucleotides, or a combination thereof. In some embodiments, the PEgRNA comprise 3’ mN*mN*mN*N and 5’mN*mN*mN* modifications, where m indicates that the nucleotide contains a 2’-O-Me modification and a * indicates the presence of a phosphorothioate bond. PEgRNA sequences exemplified in Table 15 may alternatively be adapted for expression from a nucleic acid template with a U6 promoter, for example, by including a 5’ terminal G if the spacer of the PEgRNA begins with another nucleotide, by including 6 or 7 U nucleotides at the 3’ end of the extension arm, or both. Such transcription-adapted sequences may further comprise a universal or sequence specific motif between the PBS and the 3’ terminal U series. The modifications included in the selection of full length PEgRNAs included in Table are annotated in column 3 of Table 15. [0417]Table 16 provides exemplary Prime Editing guide RNAs (PEgRNAs) and components designed to disrupt the B2M gene by encoding the insertion of two in frame stop codons into the B2M gene, for example, c.77InsTAATAA. [0418]The PEgRNAs exemplified in Table 16 comprise: (a) a spacer comprising at its 3’ end a sequence corresponding to sequence number 475; (b) a gRNA core capable of complexing with a Cas9 protein, and (c) an extension arm comprising: (i) an editing template comprising at its 3’ end a sequence corresponding to any one of sequence numbers 489-491, and (ii) a prime binding site (PBS) comprising at its 5’ end a sequence corresponding to sequence number 476. The PEgRNA spacer can be, for example, 17-22 nucleotides in length. In some WO 2024/238825 PCT/US2024/029746 174 embodiments, the PEgRNA spacer is 17-20 nucleotides in length, and can comprise the sequence corresponding to any one of sequence numbers 472, 473, 474, or 475. In some embodiments, the PEgRNA spacer comprises sequence number 472. The editing template can be referred to as a reverse transcription template (RTT). The RTTs exemplified in Table are designed to disrupt the B2M gene by encoding the insertion of two in frame stop codons or the complement thereof into the B2M gene, e.g., a C.77TAATAA insertion. For example, the editing template can comprise at its 3’ end the sequence corresponding to sequence number 489, 490, or 491. The PBS can be, for example, 5 to 17 nucleotides in length and can comprise the sequence corresponding to any one of sequence numbers 476- 488. [0419]The PEgRNA can comprise, from 5’ to 3’, the spacer, the gRNA core, the edit template, and the PBS. The 3’ end of the edit template can be contiguous with the 5’ end of the PBS. The PEgRNA can comprise multiple RNA molecules or can be a single RNA molecule. Exemplary PEgRNAs provided in Table 16 can comprise a sequence corresponding to any one of sequence numbers 492, 493, 494, 495, 496, 497, 498, 499, or 500. Any PEgRNA exemplified in Table 16 may comprise, or further comprise, a 3’ motif at the 3’ end of the extension arm, such as a universal motif, a sequence specific motif, or a series of 1, 2, 3, 4, 5, 6, 7 or more U nucleotides. In some embodiments, a universal or structural 3’ motif that is capable of forming a tertiary structure on its own such as a hairpin, a pseudoknot, or other RNA structure is used. In some embodiments, a sequence specific motif is used that is designed to hybridize with a portion of the RTT while not covering the PBS. Whether a universal or sequence specific motif is used, it can be connected to the 3’ of the PBS via a linker sequence. In some embodiments, the PEgRNA comprises 4 U nucleotides at its 3’ end. Without being bound by theory, such 3’ motifs are believed to increase PEgRNA stability. The PEgRNA may alternatively or additionally comprise one or more chemical modifications, such as phosphorothioate (PS) bond(s), 2’-O-methylated (2’- Ome) nucleotides, or a combination thereof. In some embodiments, the PEgRNA comprise 3’ mN*mN*mN*N and 5’mN*mN*mN* modifications, where m indicates that the nucleotide contains a 2’-O-Me modification and a * indicates the presence of a phosphorothioate bond. PEgRNA sequences exemplified in Table 16 may alternatively be adapted for expression from a nucleic acid template with a U6 promoter, for example, by including a 5’ terminal G if the spacer of the PEgRNA begins with another nucleotide, by including 6 or 7 U nucleotides at the 3’ end of the extension arm, or both. Such transcription-adapted sequences may further WO 2024/238825 PCT/US2024/029746 175 comprise a universal or sequence specific motif between the PBS and the 3’ terminal U series. The modifications included in the selection of full length PEgRNAs included in Table are annotated in column 3 of Table 16. [0420]Table 17 provides exemplary Prime Editing guide RNAs (PEgRNAs) and components designed to disrupt the B2M gene by encoding the insertion of two in frame stop codons into the B2M gene, for example, c.62InsTAATAA. [0421]The PEgRNAs exemplified in Table 17 comprise: (a) a spacer comprising at its 3’ end a sequence corresponding to sequence number 504; (b) a gRNA core capable of complexing with a Cas9 protein, and (c) an extension arm comprising: (i) an editing template comprising at its 3’ end a sequence corresponding to any one of sequence numbers 518-520, and (ii) a prime binding site (PBS) comprising at its 5’ end a sequence corresponding to sequence number 505. The PEgRNA spacer can be, for example, 17-22 nucleotides in length. In some embodiments, the PEgRNA spacer is 17-20 nucleotides in length, and can comprise the sequence corresponding to any one of sequence numbers 501, 502, 503, or 504. In some embodiments, the PEgRNA spacer comprises sequence number 501. The editing template can be referred to as a reverse transcription template (RTT). The RTTs exemplified in Table are designed to disrupt the B2M gene by encoding the insertion of two in frame stop codons or the complement thereof into the B2M gene, e.g., a C.62TAATAA insertion. For example, the editing template can comprise at its 3’ end the sequence corresponding to sequence number 518, 519, or 520. The PBS can be, for example, 5 to 17 nucleotides in length and can comprise the sequence corresponding to any one of sequence numbers 505- 517. [0422]The PEgRNA can comprise, from 5’ to 3’, the spacer, the gRNA core, the edit template, and the PBS. The 3’ end of the edit template can be contiguous with the 5’ end of the PBS. The PEgRNA can comprise multiple RNA molecules or can be a single RNA molecule. Exemplary PEgRNAs provided in Table 17 can comprise a sequence corresponding to any one of sequence numbers 521, 522, 523, 524, 525, 526, 527, 528, or 529. Any PEgRNA exemplified in Table 17 may comprise, or further comprise, a 3’ motif at the 3’ end of the extension arm, such as a universal motif, a sequence specific motif, or a series of 1, 2, 3, 4, 5, 6, 7 or more U nucleotides. In some embodiments, a universal or structural 3’ motif that is capable of forming a tertiary structure on its own such as a hairpin, a pseudoknot, or other RNA structure is used. In some embodiments, a sequence specific motif is used that is designed to hybridize with a portion of the RTT while not covering the WO 2024/238825 PCT/US2024/029746 176 PBS. Whether a universal or sequence specific motif is used, it can be connected to the 3’ of the PBS via a linker sequence. In some embodiments, the PEgRNA comprises 4 U nucleotides at its 3’ end. Without being bound by theory, such 3’ motifs are believed to increase PEgRNA stability. The PEgRNA may alternatively or additionally comprise one or more chemical modifications, such as phosphorothioate (PS) bond(s), 2’-O-methylated (2’- Ome) nucleotides, or a combination thereof. In some embodiments, the PEgRNA comprise 3’ mN*mN*mN*N and 5’mN*mN*mN* modifications, where m indicates that the nucleotide contains a 2’-O-Me modification and a * indicates the presence of a phosphorothioate bond. PEgRNA sequences exemplified in Table 17 may alternatively be adapted for expression from a nucleic acid template with a U6 promoter, for example, by including a 5’ terminal G if the spacer of the PEgRNA begins with another nucleotide, by including 6 or 7 U nucleotides at the 3’ end of the extension arm, or both. Such transcription-adapted sequences may further comprise a universal or sequence specific motif between the PBS and the 3’ terminal U series. The modifications included in the selection of full length PEgRNAs included in Table are annotated in column 3 of Table 17. [0423]Table 18 provides exemplary Prime Editing guide RNAs (PEgRNAs) and components designed to disrupt the B2M gene by encoding the insertion of two in frame stop codons into the B2M gene, for example, c.90InsTAATAA. [0424]The PEgRNAs exemplified in Table 18 comprise: (a) a spacer comprising at its 3’ end a sequence corresponding to sequence number 533; (b) a gRNA core capable of complexing with a Cas9 protein, and (c) an extension arm comprising: (i) an editing template comprising at its 3’ end a sequence corresponding to any one of sequence numbers 547-549, and (ii) a prime binding site (PBS) comprising at its 5’ end a sequence corresponding to sequence number 534. The PEgRNA spacer can be, for example, 17-22 nucleotides in length. In some embodiments, the PEgRNA spacer is 17-20 nucleotides in length, and can comprise the sequence corresponding to any one of sequence numbers 530, 531, 532, or 533. In some embodiments, the PEgRNA spacer comprises sequence number 530. The editing template can be referred to as a reverse transcription template (RTT). The RTTs exemplified in Table are designed to disrupt the B2M gene by encoding the insertion of two in frame stop codons or the complement thereof into the B2M gene, e.g., a C.90TAATAA insertion. For example, the editing template can comprise at its 3’ end the sequence corresponding to sequence number 547, 548, or 549. The PBS can be, for example, 5 to 17 nucleotides in WO 2024/238825 PCT/US2024/029746 177 length and can comprise the sequence corresponding to any one of sequence numbers 534- 546. [0425]The PEgRNA can comprise, from 5’ to 3’, the spacer, the gRNA core, the edit template, and the PBS. The 3’ end of the edit template can be contiguous with the 5’ end of the PBS. The PEgRNA can comprise multiple RNA molecules or can be a single RNA molecule. Exemplary PEgRNAs provided in Table 18 can comprise a sequence corresponding to any one of sequence numbers 550, 551, 552, 553, 554, 555, 556, 557, or 558. Any PEgRNA exemplified in Table 18 may comprise, or further comprise, a 3’ motif at the 3’ end of the extension arm, such as a universal motif, a sequence specific motif, or a series of 1, 2, 3, 4, 5, 6, 7 or more U nucleotides. In some embodiments, a universal or structural 3’ motif that is capable of forming a tertiary structure on its own such as a hairpin, a pseudoknot, or other RNA structure is used. In some embodiments, a sequence specific motif is used that is designed to hybridize with a portion of the RTT while not covering the PBS. Whether a universal or sequence specific motif is used, it can be connected to the 3’ of the PBS via a linker sequence. In some embodiments, the PEgRNA comprises 4 U nucleotides at its 3’ end. Without being bound by theory, such 3’ motifs are believed to increase PEgRNA stability. The PEgRNA may alternatively or additionally comprise one or more chemical modifications, such as phosphorothioate (PS) bond(s), 2’-O-methylated (2’- Ome) nucleotides, or a combination thereof. In some embodiments, the PEgRNA comprise 3’ mN*mN*mN*N and 5’mN*mN*mN* modifications, where m indicates that the nucleotide contains a 2’-O-Me modification and a * indicates the presence of a phosphorothioate bond. PEgRNA sequences exemplified in Table 18 may alternatively be adapted for expression from a nucleic acid template with a U6 promoter, for example, by including a 5’ terminal G if the spacer of the PEgRNA begins with another nucleotide, by including 6 or 7 U nucleotides at the 3’ end of the extension arm, or both. Such transcription-adapted sequences may further comprise a universal or sequence specific motif between the PBS and the 3’ terminal U series. The modifications included in the selection of full length PEgRNAs included in Table are annotated in column 3 of Table 18. [0426]Table 19 provides exemplary Prime Editing guide RNAs (PEgRNAs) and components designed to disrupt the B2M gene by encoding the insertion of two in frame stop codons into the B2M gene, for example, c.35InsTAATAA. [0427]The PEgRNAs exemplified in Table 19 comprise: (a) a spacer comprising at its 3’ end a sequence corresponding to sequence number 562; (b) a gRNA core capable of complexing WO 2024/238825 PCT/US2024/029746 178 with a Cas9 protein, and (c) an extension arm comprising: (i) an editing template comprising at its 3’ end a sequence corresponding to any one of sequence numbers 576-578, and (ii) a prime binding site (PBS) comprising at its 5’ end a sequence corresponding to sequence number 563. The PEgRNA spacer can be, for example, 17-22 nucleotides in length. In some embodiments, the PEgRNA spacer is 17-20 nucleotides in length, and can comprise the sequence corresponding to any one of sequence numbers 559, 560, 561, or 562. In some embodiments, the PEgRNA spacer comprises sequence number 559. The editing template can be referred to as a reverse transcription template (RTT). The RTTs exemplified in Table are designed to disrupt the B2M gene by encoding the insertion of two in frame stop codons or the complement thereof into the B2M gene, e.g., a C.35TAATAA insertion. For example, the editing template can comprise at its 3’ end the sequence corresponding to sequence number 576, 577, or 578. The PBS can be, for example, 5 to 17 nucleotides in length and can comprise the sequence corresponding to any one of sequence numbers 563- 575. [0428]The PEgRNA can comprise, from 5’ to 3’, the spacer, the gRNA core, the edit template, and the PBS. The 3’ end of the edit template can be contiguous with the 5’ end of the PBS. The PEgRNA can comprise multiple RNA molecules or can be a single RNA molecule. Exemplary PEgRNAs provided in Table 19 can comprise a sequence corresponding to any one of sequence numbers 579, 580, 581, 582, 583, 584, 585, 586, or 587. Any PEgRNA exemplified in Table 19 may comprise, or further comprise, a 3’ motif at the 3’ end of the extension arm, such as a universal motif, a sequence specific motif, or a series of 1, 2, 3, 4, 5, 6, 7 or more U nucleotides. In some embodiments, a universal or structural 3’ motif that is capable of forming a tertiary structure on its own such as a hairpin, a pseudoknot, or other RNA structure is used. In some embodiments, a sequence specific motif is used that is designed to hybridize with a portion of the RTT while not covering the PBS. Whether a universal or sequence specific motif is used, it can be connected to the 3’ of the PBS via a linker sequence. In some embodiments, the PEgRNA comprises 4 U nucleotides at its 3’ end. Without being bound by theory, such 3’ motifs are believed to increase PEgRNA stability. The PEgRNA may alternatively or additionally comprise one or more chemical modifications, such as phosphorothioate (PS) bond(s), 2’-O-methylated (2’- Ome) nucleotides, or a combination thereof. In some embodiments, the PEgRNA comprise 3’ mN*mN*mN*N and 5’mN*mN*mN* modifications, where m indicates that the nucleotide contains a 2’-O-Me modification and a * indicates the presence of a phosphorothioate bond.

WO 2024/238825 PCT/US2024/029746 179 PEgRNA sequences exemplified in Table 19 may alternatively be adapted for expression from a nucleic acid template with a U6 promoter, for example, by including a 5’ terminal G if the spacer of the PEgRNA begins with another nucleotide, by including 6 or 7 U nucleotides at the 3’ end of the extension arm, or both. Such transcription-adapted sequences may further comprise a universal or sequence specific motif between the PBS and the 3’ terminal U series. The modifications included in the selection of full length PEgRNAs included in Table are annotated in column 3 of Table 19. [0429]Table 20 provides exemplary Prime Editing guide RNAs (PEgRNAs) and components designed to disrupt the B2M gene by encoding the insertion of two in frame stop codons into the B2M gene, for example, c.InsTAATAA. [0430]The PEgRNAs exemplified in Table 20 comprise: (a) a spacer comprising at its 3’ end a sequence corresponding to sequence number 591; (b) a gRNA core capable of complexing with a Cas9 protein, and (c) an extension arm comprising: (i) an editing template comprising at its 3’ end a sequence corresponding to any one of sequence numbers 605-607, and (ii) a prime binding site (PBS) comprising at its 5’ end a sequence corresponding to sequence number 592. The PEgRNA spacer can be, for example, 17-22 nucleotides in length. In some embodiments, the PEgRNA spacer is 17-20 nucleotides in length, and can comprise the sequence corresponding to any one of sequence numbers 588, 589, 590, or 591. In some embodiments, the PEgRNA spacer comprises sequence number 588. The editing template can be referred to as a reverse transcription template (RTT). The RTTs exemplified in Table are designed to disrupt the B2M gene by encoding the insertion of two in frame stop codons or the complement thereof into the B2M gene, e.g., a C.8TAATAA insertion. For example, the editing template can comprise at its 3’ end the sequence corresponding to sequence number 605, 606, or 607. The PBS can be, for example, 5 to 17 nucleotides in length and can comprise the sequence corresponding to any one of sequence numbers 592- 604. [0431]The PEgRNA can comprise, from 5’ to 3’, the spacer, the gRNA core, the edit template, and the PBS. The 3’ end of the edit template can be contiguous with the 5’ end of the PBS. The PEgRNA can comprise multiple RNA molecules or can be a single RNA molecule. Exemplary PEgRNAs provided in Table 20 can comprise a sequence corresponding to any one of sequence numbers 608, 609, 610, 611, 612, 613, 614, 615, or 616. Any PEgRNA exemplified in Table 20 may comprise, or further comprise, a 3’ motif at the 3’ end of the extension arm, such as a universal motif, a sequence specific motif, or a WO 2024/238825 PCT/US2024/029746 180 series of 1, 2, 3, 4, 5, 6, 7 or more U nucleotides. In some embodiments, a universal or structural 3’ motif that is capable of forming a tertiary structure on its own such as a hairpin, a pseudoknot, or other RNA structure is used. In some embodiments, a sequence specific motif is used that is designed to hybridize with a portion of the RTT while not covering the PBS. Whether a universal or sequence specific motif is used, it can be connected to the 3’ of the PBS via a linker sequence. In some embodiments, the PEgRNA comprises 4 U nucleotides at its 3’ end. Without being bound by theory, such 3’ motifs are believed to increase PEgRNA stability. The PEgRNA may alternatively or additionally comprise one or more chemical modifications, such as phosphorothioate (PS) bond(s), 2’-O-methylated (2’- Ome) nucleotides, or a combination thereof. In some embodiments, the PEgRNA comprise 3’ mN*mN*mN*N and 5’mN*mN*mN* modifications, where m indicates that the nucleotide contains a 2’-O-Me modification and a * indicates the presence of a phosphorothioate bond. PEgRNA sequences exemplified in Table 20 may alternatively be adapted for expression from a nucleic acid template with a U6 promoter, for example, by including a 5’ terminal G if the spacer of the PEgRNA begins with another nucleotide, by including 6 or 7 U nucleotides at the 3’ end of the extension arm, or both. Such transcription-adapted sequences may further comprise a universal or sequence specific motif between the PBS and the 3’ terminal U series. The modifications included in the selection of full length PEgRNAs included in Table are annotated in column 3 of Table 20. [0432]Table 21 provides exemplary Prime Editing guide RNAs (PEgRNAs) and components designed to disrupt the B2M gene by encoding the insertion of two in frame stop codons into the B2M gene, for example, c.95InsTAATAA. [0433]The PEgRNAs exemplified in Table 21 comprise: (a) a spacer comprising at its 3’ end a sequence corresponding to sequence number 620; (b) a gRNA core capable of complexing with a Cas9 protein, and (c) an extension arm comprising: (i) an editing template comprising at its 3’ end a sequence corresponding to any one of sequence numbers 634-636, and (ii) a prime binding site (PBS) comprising at its 5’ end a sequence corresponding to sequence number 621. The PEgRNA spacer can be, for example, 17-22 nucleotides in length. In some embodiments, the PEgRNA spacer is 17-20 nucleotides in length, and can comprise the sequence corresponding to any one of sequence numbers 617, 618, 619, or 620. In some embodiments, the PEgRNA spacer comprises sequence number 617. The editing template can be referred to as a reverse transcription template (RTT). The RTTs exemplified in Table are designed to disrupt the B2M gene by encoding the insertion of two in frame stop WO 2024/238825 PCT/US2024/029746 181 codons or the complement thereof into the B2M gene, e.g., a c.95TAATAA insertion. For example, the editing template can comprise at its 3’ end the sequence corresponding to sequence number 634, 635, or 636. The PBS can be, for example, 5 to 17 nucleotides in length and can comprise the sequence corresponding to any one of sequence numbers 621- 633.[0434] The PEgRNA can comprise, from 5’ to 3’, the spacer, the gRNA core, the edit template, and the PBS. The 3’ end of the edit template can be contiguous with the 5’ end of the PBS. The PEgRNA can comprise multiple RNA molecules or can be a single RNA molecule. Exemplary PEgRNAs provided in Table 21 can comprise a sequence corresponding to any one of sequence numbers 637, 638, 639, 640, 641, 642, 643, 644, or 645. Any PEgRNA exemplified in Table 21 may comprise, or further comprise, a 3’ motif at the 3’ end of the extension arm, such as a universal motif, a sequence specific motif, or a series of 1, 2, 3, 4, 5, 6, 7 or more U nucleotides. In some embodiments, a universal or structural 3’ motif that is capable of forming a tertiary structure on its own such as a hairpin, a pseudoknot, or other RNA structure is used. In some embodiments, a sequence specific motif is used that is designed to hybridize with a portion of the RTT while not covering the PBS. Whether a universal or sequence specific motif is used, it can be connected to the 3’ of the PBS via a linker sequence. In some embodiments, the PEgRNA comprises 4 U nucleotides at its 3’ end. Without being bound by theory, such 3’ motifs are believed to increase PEgRNA stability. The PEgRNA may alternatively or additionally comprise one or more chemical modifications, such as phosphorothioate (PS) bond(s), 2’-O-methylated (2’- Ome) nucleotides, or a combination thereof. In some embodiments, the PEgRNA comprise 3’ mN*mN*mN*N and 5’mN*mN*mN* modifications, where m indicates that the nucleotide contains a 2’-O-Me modification and a * indicates the presence of a phosphorothioate bond. PEgRNA sequences exemplified in Table 21 may alternatively be adapted for expression from a nucleic acid template with a U6 promoter, for example, by including a 5’ terminal G if the spacer of the PEgRNA begins with another nucleotide, by including 6 or 7 U nucleotides at the 3’ end of the extension arm, or both. Such transcription-adapted sequences may further comprise a universal or sequence specific motif between the PBS and the 3’ terminal U series. The modifications included in the selection of full length PEgRNAs included in Table are annotated in column 3 of Table 21. [0435]A ngRNA protospacer may be in close proximity to the PEgRNA spacer, or may be upstream or downstream of the PEgRNA spacer. In some embodiments, the distance WO 2024/238825 PCT/US2024/029746 182 generated by the PEgRNA nick site and the ngRNA nick site (referred to as the nick-to-nick distance) is about 3 to about 100 nucleotides. In some embodiments, the distance generated by the PEgRNA nick site and the ngRNA nick site (referred to as the nick-to-nick distance) is about 4-90, 4-80, 4-70, 4-60, 4-50, 4-40, 4-30,4-20, or 4-10 nucleotides. In some embodiments, the distance generated by the PEgRNA nick site and the ngRNA nick site (referred to as the nick-to-nick distance) is about 10-20, 20-30, 30-40,40-50, 50-60, 60-70, 70-80,80-90, or 90-100 nucleotides. In some embodiments, the nick-to-nick distance is about 4-88 nucleotides. In some embodiments, the nick-to-nick distance is about 4-72 nucleotides. In some embodiments, the nick-to-nick distance is about 4-61 nucleotides. In some embodiments, the nick-to-nick distance is about 61-72 nucleotides. In some embodiments, the nick-to-nick distance is about 61-88 nucleotides. In some embodiments, the nick-to-nick distance is about 72-88 nucleotides. In some embodiments, the nick-to-nick distance is about 4-7 nucleotides. In some embodiments, the nick-to-nick distance is 4, 5, 6, or 7 nucleotides. In some embodiments, the nick-to-nick distance is about 41-96 nucleotides. In some embodiments, the nick-to-nick distance is about 41-82 nucleotides. In some embodiments, the nick-to-nick distance is about 41-44 nucleotides. In some embodiments, the nick-to-nick distance is about 44-82 nucleotides. In some embodiments, the nick-to-nick distance is about 44-96 nucleotides. In some embodiments, the nick-to-nick distance is about 82-nucleotides. In some embodiments, the nick-to-nick distance is 41, 44, 82, or 96 nucleotides. In some embodiments, the intended nucleotide edit is incorporated within about 1-nucleotides of the position corresponding to the PAM of the ng search target sequence. [0436]The gRNA core of a PEgRNA or ngRN A can be any gRNA scaffold sequence that is capable of interacting with a Cas protein that recognizes the corresponding PAM of the PEgRNA or ngRNA. In some embodiments, gRNA core of a PEgRNA or a ngRNA comprises a nucleic acid sequence selected from SEQ ID Nos: 646-654, 656-658, or 660. [0437]In some embodiments, a PEgRNA (or ngRNA) comprises an additional secondary structure at the 5’ end. In some embodiments, a PEgRNA (or ngRNA) comprises an additional secondary structure at the 3’ end. [0438]In some embodiments, the secondary structure comprises a pseudoknot. In some embodiments, the secondary structure comprises a pseudoknot derived from a virus. In some embodiments, the secondary structure comprises a pseudoknot of a Moloney murine leukemia virus (M-MLV) genome (a mpknot). In some embodiments, the secondary structure comprises a nucleotide sequence selected from the group consisting of WO 2024/238825 PCT/US2024/029746 183 GGGUCAGGAGCCCCCCCCCUGAACCCAGGAUAACCCUCAAAGUCGGGGGGCAA CC (SEQ ID No: 746), GUCAGGGUCAGGAGCCCCCCCCCUGAACCCAGGAUAACCCUCAAAGUCGGGGG GCAACCC (SEQ ID No: 747), GGGUCAGGAGCCCCCCCCCUGAACCCAGGAAAACCCUCAAAGUCGGGGGGCAA CCC (SEQ ID No: 748), GGGUCAGGAGCCCCCCCCCUGCACCCAGGAAAACCCUCAAAGUCGGGGGGCAA CCC (SEQ ID No: 749), GGGUCAGGAGCCCCCCCCCUGCACCCAGGAUAACCCUCAAAGUCGGGGGGCAA CCC (SEQ ID No: 750), GUCAGGGUCAGGAGCCCCCCCCCUGAACCCAGGAAAACCCUCAAAGUCGGGGG GCAACCC (SEQ ID No: 751), GUCAGGGUCAGGAGCCCCCCCCCUGCACCCAGGAAAACCCUCAAAGUCGGGGG GCAACCC (SEQ ID No: 752), and GGGUCAGGAGCCCCCCCCCUGAACCCAGGAUAACCCUCAAAGUCGGGGGGC (SEQ ID No: 753), or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith. In some embodiments, the secondary structure comprises a nucleotide sequence of GGGUCAGGAGCCCCCCCCCUGAACCCAGGAUAACCCUCAAAGUCGGGGGGC (SEQ ID No: 753), or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith.[0439] In some embodiments, the secondary structure comprises a quadruplex. In some embodiments, the secondary structure comprises a G-quadruplex. In some embodiments, the secondary structure comprises a nucleotide sequence selected from the group consisting of gq2 (UGGUGGUGGUGGU) (SEQ ID No: 754), stk40 (GGGACAGGGCAGGGACAGGG) (SEQ ID No: 755), apc2 (GGGUCCGGGUCUGGGUCUGGG) (SEQ ID No: 756), stard(GGGCAGGGUCUGGGCUGGG) (SEQ ID No: 757), tnsl(GGGCUGGGAUGGGAAAGGG) (SEQ ID No: 758), ceacam(GGGCUCUGGGUGGGCCGGG) (SEQ ID No: 759), ercl (GGGCUGGGCUGGGCAGGG) (SEQ ID No: 760), pitpnm3(GGGUGGGCUGGGAAGGG) (SEQ ID No: 761), rlf (GGGAGGGAGGGCUAGGG) (SEQ ID No: 762), ube3c(GGGCAGGGCUGGGAGGG) (SEQ ID No: 763), tafl5(GGGUGGGAGGGCUGGG) (SEQ ID No: 764), and xml WO 2024/238825 PCT/US2024/029746 184 (GCGUAACCUCCAUCCGAGUUGCAAGAGAGGGAAACGCAGUCUC) (SEQ ID No: 765), or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith.[0440] In some embodiments, the secondary structure comprises a P4-P6 domain of a Group I intron. In some embodiments, the secondary structure comprises the nucleotide sequence of GGAAUUGCGGGAAAGGGGUCAACAGCCGUUCAGUACCAAGUCUCAGGGGAAA CUUUGAGAUGGCCUUGCAAAGGGUAUGGUAAUAAGCUGACGGACAUGGUCCU AACCACGCAGCCAAGUCCUAAGUCAACAGAUCUUCUGUUGAUAUGGAUGCAGU UCA (SEQ ID No: 766), or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith.[0441] In some embodiments, the secondary structure comprises a riboswitch aptamer. In some embodiments, the secondary structure comprises a riboswitch aptamer derived from a prequeosine-1 riboswitch aptamer. In some embodiments, the secondary structure comprises a modified prequeosine-1 riboswitch aptamer. In some embodiments, the secondary structure comprises a nucleotide sequence selected from the group consisting of: UUGACGCGGUUCUAUCUAGUUACGCGUUAAACCAACUAGAAA (SEQ ID No: 767), UUGACGCGGUUCUAUCUACUUACGCGUUAAACCAACUAGAAA (SEQ ID No: 768), CGCGAGUCUAGGGGAUAACGCGUUAAACUUCCUAGAAGGCGGUU (SEQ ID No: 769), CGCGGAUCUAGAUUGUAACGCGUUAAACCAUCUAGAAGGCGGUU (SEQ ID No: 770), CGCGUCGCUACCGCCCGGCGCGUUAAACACACUAGAAGGCGGUU (SEQ ID No: 771), and CGCGGUUCUAUCUAGUUACGCGUUAAACCAACUAGAA (SEQ ID No: 772), or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith. In some embodiments, the secondary structure comprises a nucleotide sequence selected from the group consisting of UUGACGCGGUUCUAUCUAGUUACGCGUUAAACCAACUAGAAA (SEQ ID No: 767), CGCGAGUCUAGGGGAUAACGCGUUAAACUUCCUAGAAGGCGGUU (SEQ ID No: 769), CGCGGAUCUAGAUUGUAACGCGUUAAACCAUCUAGAAGGCGGUU (SEQ ID No: 770), CGCGUCGCUACCGCCCGGCGCGUUAAACACACUAGAAGGCGGUU (SEQ ID No: 771), and CGCGGUUCUAUCUAGUUACGCGUUAAACCAACUAGAA (SEQ ID No: 772), or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith. In some embodiments, the secondary structure comprises a WO 2024/238825 PCT/US2024/029746 185 nucleotide sequence of and CGCGGUUCUAUCUAGUUACGCGUUAAACCAACUAGAA (SEQ ID No: 772), or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith.[0442] In some embodiments, the PEgRNA comprises a RNA secondary structure and/or a linker disclosed in Nelson et al. Engineered pegRNAs improve prime editing efficiency. Nat Biotechnol. (2021), the entirety of which is incorporated herein by reference.[0443] In some embodiments, a PEgRNA is transcribed from a nucleotide encoding the PEgRNA, for example, a DNA plasmid encoding the PEgRNA. In some embodiments, the PEgRNA comprises a self-cleaving element. In some embodiments, the self-cleaving element improves transcription and/or processing of the PEgRNA when transcribed form the nucleotide encoding the PEgRNA. In some embodiments, the PEgRNA comprises a hairpin or a RNA quadruplex. In some embodiments, the PEgRNA comprises a self-cleaving ribozyme element, for example, a hammerhead, a pistol, a hatchet, a hairpin, a VS, a twister, or a twister sister ribozyme. In some embodiments, the PEgRNA comprises a HDV ribozyme. In some embodiments, the PEgRNA comprises a hairpin recognized by Csy4. In some embodiments, the PEgRNA comprises an ENE motif. In some embodiments, the PEgRNA comprises an element for nuclear expression (ENE) from MALATI Inc RNA. In some embodiments, the PEgRNA comprises an ENE element from Kaposi ’s sarcoma- associated herpesvirus (KSHV). In some embodiments, the PEgRNA comprises a 3’ box of a Ul snRNA. In some embodiments, the PEgRNA forms a circular RNA.[0444] In some embodiments, the PEgRNA comprises a RNA secondary structure or a motif that improves binding to the DNA-RNA duple or enhances PEgRNA activity. In some embodiments, the PEgRNA comprises a sequence derived from a native nucleotide element involved in reverse transcription, e.g., initiation of retroviral transcription. In some embodiments, the PEgRNA comprises a sequence of, or derived from, a primer binding site of a substrate of a reverse transcriptase, a polypurine tract (PPT), or a kissing loop. In some embodiments, the PEgRNA comprises a dimerization motif, a kissing loop, or a GNRA tetraloop - tetraloop receptor pair that results in circularization of the PEgRNA. In some embodiments, the PEgRNA comprises a RNA secondary structure of a motif that results in physical separation of the spacer and the PBS of the PEgRNA, thereby prevents occlusion of the spacer and improves PEgRNA activity. In some embodiments, the PEgRNA comprises a secondary structure or motif, e.g., a 5’ or 3’ extension in the spacer region that form a toehold or hairpin, wherein the secondary structure or motif competes favorably against annealing WO 2024/238825 PCT/US2024/029746 186 between the spacer and the PBS of the PEgRNA, thereby prevents occlusion of the spacer and improves PEgRNA activity.[0445] In some embodiments, a PEgRNA comprises the sequenceGGCCGGCA UGGUCCCAGCCUCCUCGCUGGCGCCGGCUGGGCAACA UGCUUCGGC AUGGCGAAUGGGAC (SEQ ID No: 773) at the 3’ end. In some embodiments, a PEgRNA comprises the structure [spacer]-[gRNA core]-[editing template]-[PBS]-GGCCGGCA UGGUCCCAGCCUCCUCGCUGGCGCCGGCUGGGCAACA UGCUUCGGC AUGGCGAAUGGGAC (SEQ ID NO: 773/ or [spacer]-[gRNA core]-[editing template]- [PBS]-GGCCGGCA UGGUCCCAGCCUCCUCGCUGGCGCCGGCUGGGCAACA UGCUUCGGC AUGGCGAAUGGGAC-(U)n (SEQ ID NO: 785), wherein n is an integer between 3 and 7. The structure derived from hepatitis D virus (HDV) is italicized.[0446] In some embodiments, the PEgRNA comprises the sequenceGGUGGGAGACGUCCCACC (SEQ ID No: 774) at the 5’ end and/or the sequence UGGGAGACGUCCCACC (SEQ ID NO: 786/ at the 3’ end. In some embodiments, the PEgRNA comprises the following structure (M-MLV kissing loop):GGUGGGAGACGUCCCACC (SEQ ID NO: 774)-[spacer]-[gRNA core]-[editing template]- [PBS]-UGGGAGACGUCCCACC (SEQ ID NO: 786/ or GGUGGGAGACGUCCCACC (SEQ ID NO: 774/-[spacer]-[gRNA core]-[editing template]-[PBS]-UGGGAGACGUCCCACC-(U)n (SEQ ID NO: 787), wherein n is an integer between 3 and 7. The kissing loop structure is italicized.[0447] In some embodiments, the PEgRNA comprises the sequenceGAGCAGCAUGGCGUCGCUGCUCAC (SEQ ID No: 775) at the 5’ end and/or the sequence CCAUCAGUUGACACCCUGAGG (SEQ ID No: 776) at the 3’ end. In some embodiments, the PEgRNA comprises the following structure (VS ribozyme kissing loop):[0448] GAGCAGCAUGGCGUCGCUGCUCAC (SEQ ID NO; 775/-[spacer]-[gRNA core]- [editing template]-[PBS]- CCAUCAGUUGACACCCUGAGG (SEQ ID NO; 776/ or GAGCAGCAUGGCGUCGCUGCUCAC (SEQ ID NO: 775)-[spacer]-[gRNA core]-[editing template]-[PBS]- CCAUCAGUUGACACCCUGAGG-(^ (SEQ ID NO: 788), wherein n is an integer between 3 and 7. (VS ribozyme kissing loop).[0449] In some embodiments, the PEgRNA comprises the sequenceGCAGACCUAAGUGGUGACAUAUGGUCUG (SEQ ID No: 777) at the 5’ end and/or the sequence CAUGCGAUUAGAAAUAAUCGCAUG (SEQ ID No: 778) at the 3’ end. In some WO 2024/238825 PCT/US2024/029746 187 embodiments, the PEgRNA comprises the following structure (tetraloop and receptor): GCAGACCUAAGUGGUGACAUAUGGUCUG (SEQ ID NO: 777)-[spacer]-IgRNA core]- [editing template]-[PBS]- CAUGCGAUUAGAAAUAAUCGCAUG (SEQ ID NO: 778), or GCAGACCUAAGUGGUGACAUAUGGUCUG (SEQ ID NO: 777)-[spacer]-IgRNA core]- [editing template]-[PBS]- CAUGCGAUUAGAAAUAAUCGCAUG-(U)n (SEQ ID NO: 789), wherein n is an integer between 3 and 7. The tetraloop/tetraloop receptor structure is italicized. [0450]In some embodiments, the PEgRNA comprises the sequenceGGCCGGCAUGGUCCCAGCCUCCUCGCUGGCGCCGGCUGGGCAACAUGCUUCGG CAUGGCGAAUGGGAC (SEQ ID No: 773) or UCUGCCAUCAAAGCUGCGACCGUGCUCAGUCUGGUGGGAGACGUCCCACCGGC CGGCAUGGUCCCAGCCUCCUCGCUGGCGCCGGCUGGGCAACAUGCUUCGGCAU GGCGAAUGGGAC (SEQ ID No: 779). [0451]In some embodiments, a PEgRNA comprises a gRNA core that comprises a modified direct repeat compared to the sequence of a naturally occurring CRISPR-Cas guide RNA scaffold, for example, a Cas9 gRNA scaffold. In some embodiments, the PEgRNA comprises a "flip and extension (F+E)" gRNA core, wherein one or more base pairs in a direct repeat is modified. In some embodiments, the PEgRNA comprises a first direct repeat (the first paring element or the lower stem), wherein a Uracil is changed to a Adenine (such that in the stem region, a U-A base pair is changed to a A-U base pair). In some embodiments, the PEgRNA comprises a first direct repeat wherein the fourth U-A base pair in the stem is changed to a A- U base pair. In some embodiments, the PEgRNA comprises a first direct repeat wherein one or more U-A base pair is changed to a G-C or C-G base pair. For example, in some embodiments, the PEgRNA comprises a first direct repeat comprising a modification to a GUUUU-AAAAC pairing element, wherein one or more of the U-A base pairs is changed to a A-U base pair, a G-C base pair, or a C-G base pair. In some embodiments, the PEgRNA comprises an extended first direct repeat. [0452]In some embodiments, a PEgRNA comprises a gRNA core comprises the sequence GUUUUAGAGCUAUACGUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU UACGAAGUGGCACCGAGUCGGUGC (SEQ ID No: 656) or GUUUUAGAGCUAUACGUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU UACGAAGUGGGACCGAGUCGGUCC (SEQ ID No: 657).

WO 2024/238825 PCT/US2024/029746 188 id="p-453"

[0453]In some embodiments, a PEgRNA comprises a gRNA core comprising the sequence GUUUUAGAGCUAGCUCAUGAAAAUGAGCUAGCAAGUUAAAAUAAGGCUAGUC CGUUAUCAACUUGAAAAAGUGGGACCGAGUCGGUCC (SEQ ID No: 658).[0454] In some embodiments, a PEgRNA comprises a gRNA core comprising the sequence GUUUGAGAGCUAGAAAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUU GAAAAAGUGGGACCGAGUCGGUCC (SEQ ID No: 648). [0455]In some embodiments, a PEgRNA comprises a gRNA core comprising the sequence GUUUAAGAGCUAGAAAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUU GAAAAAGUGGCACCGAGUCGGUGC (SEQ ID No: 652). In some embodiments, a PEgRNA comprise a gRNA core comprising the sequence GUUUUAGAGCUAUGCUGGAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCG UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID No: 660). [0456]In some embodiments, a PEgRNA comprise a gRNA core comprising the sequence GUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCG UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID No: 649). [0457]A PEgRNA and/or an ngRNA of this disclosure, in some embodiments, may include modified nucleotides, e.g., chemically modified DNA or RNA nucleobases, and may include one or more nucleobase analogs (e.g, modifications which might add functionality, such as temperature resilience). In some embodiments, PEgRNAs and/or ngRNAs as described herein may be chemically modified. The phrase "chemical modifications, " as used herein, can include modifications which introduce chemistries which differ from those seen in naturally occurring DNA or RNAs, for example, covalent modifications such as the introduction of modified nucleotides, (e.g, nucleotide analogs, or the inclusion of pendant groups which are not naturally found in DNA or RNA molecules). [0458]In some embodiments, the PEgRNAs provided in the disclosure may further comprise nucleotides added to the 5’ of the PEgRNAs. In some embodiments, the PEgRNA further comprises 1, 2, or 3 additional nucleotides added to the 5’ end. The additional nucleotides can be guanine, cytosine, adenine, or uracil. In some embodiments, the additional nucleotide at the 5’ end of the PEgRNA is a guanine or cytosine. In some embodiments, the additional nucleotides can be chemically or biologically modified. [0459]In some embodiments, the PEgRNAs provided in the disclosure may further comprise nucleotides to the 3’ of the PEgRNAs. In some embodiments, the PEgRNA further comprises 1, 2, or 3 additional nucleotides to the 3’ end. The additional nucleotides can be guanine, WO 2024/238825 PCT/US2024/029746 189 cytosine, adenine, or uracil. In some embodiments, the additional nucleotides at the 3’ end of the PEgRNA is a polynucleotide comprising at least 1 uracil. In some embodiments, the additional nucleotides can be chemically or biologically modified. [0460]In some embodiments, a PEgRNA or ngRNA is produced by transcription from a template nucleotide, for example, a template plasmid. In some embodiments, a polynucleotide encoding the PEgRNA or ngRNA is appended with one or more additional nucleotides that improves PEgRNA or ngRNA function or expression, e.g., expression from a plasmid that encodes the PEgRNA or ngRNA. In some embodiments, a polynucleotide encoding a PEgRNA or ngRNA is appended with one or more additional nucleotides at the 5’ end or at the 3’ end. In some embodiments, the polynucleotide encoding the PEgRNA or ngRNA is appended with a guanine at the 5’ end, for example, if the first nucleotide at the 5’ end of the spacer is not a guanine. In some embodiments, a polynucleotide encoding the PEgRNA or ngRNA is appended with nucleotide sequence CACC at the 5’ end. In some embodiments, the polynucleotide encoding the PEgRNA or ngRNA is appended with an additional nucleotide adenine at the 3’ end, for example, if the last nucleotide at the 3’ end of the PBS is a Thymine. In some embodiments, the polynucleotide encoding the PEgRNA or ngRNA is appended with additional nucleotide sequence TTTTTT, TTTTTTT, TTTTT, or TTTT at the 3’ end. In some embodiments, the PEgRNA or ngRNA comprises the appended nucleotides from the transcription template. In some embodiments, the PEgRNA or ngRNA further comprises one or more nucleotides at the 5’ end or the 3’ end in addition to spacer, PBS, and RTT sequences, in some embodiments, the PEgRNA or ngRNA further comprises a guanine at the 5’ end, for example, when the first nucleotide at the 5’ end of the spacer is not a guanine. In some embodiments, the PEgRNA or ngRNA further comprises nucleotide sequence CACC at the 5’ end. In some embodiments, the PEgRNA or ngRNA further comprises an adenine at the 3’ end, for example, if the last nucleotide at the 3’ end of the PBS is a thymine. In some embodiments, the PEgRNA or ngRNA further comprises nucleotide sequence UUUUUUU, UUUUUU, UUUUU, or UUUU at the 3’ end. [0461]In some embodiments, the PEgRNAs and/or ngRNAs provided in this disclosure may have undergone a chemical or biological modifications. Modifications may be made at any position within a PEgRNA or ngRNA, and may include modification to a nucleobase or to a phosphate backbone of the PEgRNA or ngRNA. In some embodiments, chemical modifications can be a structure guided modifications. In some embodiments, a chemical modification is at the 5’ end and/or the 3’ end of a PEgRNA. In some embodiments, a WO 2024/238825 PCT/US2024/029746 190 chemical modification is at the 5’ end and/or the 3’ end of a ngRNA. In some embodiments, a chemical modification may be within the spacer sequence, the extension arm, the editing template sequence, or the primer binding site of a PEgRNA. In some embodiments, a chemical modification may be within the spacer sequence or the gRNA core of a PEgRNA or a ngRNA. In some embodiments, a chemical modification may be within the 3’ most nucleotides of a PEgRNA or ngRNA. In some embodiments, a chemical modification may be within the 3’ most end of a PEgRNA or ngRNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more chemically modified nucleotides at the 3' end. In some embodiments, a PEgRNA or ngRNA comprises 3 contiguous chemically modified nucleotides at the 3' end. In some embodiments, a chemical modification may be within the 5’ most end of a PEgRNA or ngRNA. In some embodiments, a PEgRNA or ngRNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more chemically modified nucleotides at the 3’ end. In some embodiments, a PEgRNA or ngRNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more chemically modified nucleotides at the 5’ end. In some embodiments, a PEgRNA or ngRNA comprises 1, 2, 3, 4, or 5 or more chemically modified nucleotides at the 3’ end. In some embodiments, a PEgRNA or ngRNA comprises 1, 2, 3, 4, or 5 more chemically modified nucleotides at the 5’ end. In some embodiments, a PEgRNA or ngRNA comprises 1, 2, or 3 or more chemically modified nucleotides at the 3’ end. In some embodiments, a PEgRNA or ngRNA comprises 1, 2, or 3 more chemically modified nucleotides at the 5’ end. In some embodiments, a PEgRNA or ngRNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more contiguous chemically modified nucleotides at the 3’ end. In some embodiments, a PEgRNA or ngRNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more contiguous chemically modified nucleotides at the 5’ end. In some embodiments, a PEgRNA or ngRNA comprises 1, 2, 3, 4, or 5 contiguous chemically modified nucleotides at the 3’ end. In some embodiments, a PEgRNA or ngRNA comprises 1, 2, 3, 4, or 5 contiguous chemically modified nucleotides at the 5’ end. In some embodiments, a PEgRNA or ngRNA comprises 1, 2, or 3 contiguous chemically modified nucleotides at the 3’ end. In some embodiments, a PEgRNA or ngRNA comprises 1, 2, or 3 contiguous chemically modified nucleotides at the 5’ end. In some embodiments, a PEgRNA or ngRNA comprises 3 contiguous chemically modified nucleotides at the 3’ end. In some embodiments, a PEgRNA or ngRNA comprises 1, 2, 3, 4, 5, or more chemically modified nucleotides near the 3’ end. In some embodiments, a PEgRNA or ngRNA comprises 3 contiguous chemically modified nucleotides at the 3’ end. In some embodiments, a PEgRNA or ngRNA comprises 3 contiguous chemically modified nucleotides at the 5’ end. In some embodiments, a PEgRNA or ngRNA comprises 1, 2, 3, 4, WO 2024/238825 PCT/US2024/029746 191 , or more chemically modified nucleotides near the 3’ end. In some embodiments, a PEgRNA or ngRNA comprises 1, 2, 3, 4, 5, or more contiguous chemically modified nucleotides near the 3’ end. In some embodiments, a PEgRNA or ngRNA comprises 1, 2, 3, 4, 5, or more chemically modified nucleotides near the 3’ end, where the 3’ most nucleotide is not modified, and the 1, 2, 3,4, 5, or more chemically modified nucleotides precede the 3’ most nucleotide in a 5’-to-3 ’ order. In some embodiments, a PEgRNA or ngRNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more chemically modified nucleotides near the 3’ end, where the 3’ most nucleotide is not modified, and the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more chemically modified nucleotides precede the 3’ most nucleotide in a 5’-to-3 ’ order. [0462]In some embodiments, a PEgRNA or ngRNA comprises one or more chemical modified nucleotides in the gRNA core. As exemplified in FIG. 3,the gRNA core of a PEgRNA may comprise one or more regions of a base paired lower stem, a base paired upper stem, where the lower stem and upper stem may be connected by a bulge comprising unpaired RNAs. The gRNA core may further comprise a nexus distal from the spacer sequence. In some embodiments, the gRNA core comprises one or more chemically modified nucleotides in the lower stem, upper stem, and/or the hairpin regions. In some embodiments, all of the nucleotides in the lower stem, upper stem, and/or the hairpin regions are chemically modified. [0463]A chemical modification to a PEgRNA or ngRNA can comprise a 2'-O- thionocarbamate-protected nucleoside phosphoramidite, a 2׳-O-methyl (M), a 2׳-O-methyl 3'phosphorothioate (MS), or a 2׳-O-methyl 3'thioPACE (MSP), or any combination thereof. In some embodiments, a chemically modified PEgRNA and/or ngRNA can comprise a '-O- methyl (M) RNA, a 2׳-O-methyl 3'phosphorothioate (MS) RNA, a 2׳-O-methyl 3'thioPACE (MSP) RNA, a 2’-F RNA, a phosphorothioate bond modification, any other chemical modifications known in the art, or any combination thereof. A chemical modification may also include, for example, the incorporation of non-nucleotide linkages or modified nucleotides into the PEgRNA and/or ngRNA (e.g., modifications to one or both of the 3' and 5' ends of a guide RNA molecule). Such modifications can include the addition of bases to an RNA sequence, complexing the RNA with an agent (e.g., a protein or a complementary nucleic acid molecule), and inclusion of elements which change the structure of an RNA molecule (e.g., which form secondary structures).

WO 2024/238825 PCT/US2024/029746 192 Prime Editing Compositions [0464]Disclosed herein, in some embodiments, are compositions, systems, and methods using a prime editing composition. The term "prime editing composition " or "prime editing system " refers to compositions involved in the method of prime editing as described herein. A prime editing composition may include a prime editor, e.g., a prime editor fusion protein, and a PEgRNA. A prime editing composition may further comprise additional elements, such as second strand nicking ngRNAs. Components of a prime editing composition may be combined to form a complex for prime editing, or may be kept separately, e.g., for administration purposes. [0465]In some embodiments, a prime editing composition comprises a prime editor fusion protein complexed with a PEgRNA and optionally complexed with a ngRNA. In some embodiments, the prime editing composition comprises a prime editor comprising a DNA binding domain and a DNA polymerase domain associated with each other through a PEgRNA. For example, the prime editing composition may comprise a prime editor comprising a DNA binding domain and a DNA polymerase domain linked to each other by an RNA-protein recruitment aptamer RNA sequence, which is linked to a PEgRNA. In some embodiments, a prime editing composition comprises a PEgRNA and a polynucleotide, a polynucleotide construct, or a vector that encodes a prime editor fusion protein. [0466]In some embodiments, a prime editing composition comprises a PEgRNA, a ngRNA, and a polynucleotide, a polynucleotide construct, or a vector that encodes a prime editor fusion protein. In some embodiments, a prime editing composition comprises multiple polynucleotides, polynucleotide constructs, or vectors, each of which encodes one or more prime editing composition components. In some embodiments, the PEgRNA of a prime editing composition is associated with the DNA binding domain, e.g., a Cas9 nickase, of the prime editor. In some embodiments, the PEgRNA of a prime editing composition complexes with the DNA binding domain of a prime editor and directs the prime editor to the target DNA. [0467]In some embodiments, a prime editing composition comprises one or more polynucleotides that encode prime editor components and/or PEgRNA or ngRNAs. In some embodiments, a prime editing composition comprises a polynucleotide encoding a fusion protein comprising a DNA binding domain and a DNA polymerase domain. In some embodiments, a prime editing composition comprises (i) a polynucleotide encoding a fusion protein comprising a DNA binding domain and a DNA polymerase domain, and (ii) a WO 2024/238825 PCT/US2024/029746 193 PEgRNA or a polynucleotide encoding the PEgRNA. In some embodiments, a prime editing composition comprises (i) a polynucleotide encoding a fusion protein comprising a DNA binding domain and a DNA polymerase domain, (ii) a PEgRNA or a polynucleotide encoding the PEgRNA, and (iii) an ngRNA or a polynucleotide encoding the ngRNA. In some embodiments, a prime editing composition comprises (i) a polynucleotide encoding a DNA binding domain of a prime editor, e.g., a Cas9 nickase, (ii) a polynucleotide encoding a DNA polymerase domain of a prime editor, e.g., a reverse transcriptase, and (iii) a PEgRNA or a polynucleotide encoding the PEgRNA. In some embodiments, a prime editing composition comprises (i) a polynucleotide encoding a DNA binding domain of a prime editor, e.g., a Cas9 nickase, (ii) a polynucleotide encoding a DNA polymerase domain of a prime editor, e.g., a reverse transcriptase, (iii) a PEgRNA or a polynucleotide encoding the PEgRNA, and (iv) an ngRNA or a polynucleotide encoding the ngRNA. [0468]In some embodiments, the polynucleotide encoding the DNA biding domain or the polynucleotide encoding the DNA polymerase domain further encodes an additional polypeptide domain, e.g., an RNA-protein recruitment domain, such as a MS2 coat protein domain. In some embodiments, a prime editing composition comprises (i) a polynucleotide encoding a N-terminal half of a prime editor fusion protein and an intein-N and (ii) a polynucleotide encoding a C-terminal half of a prime editor fusion protein and an intein-C. In some embodiments, a prime editing composition comprises (i) a polynucleotide encoding a N-terminal half of a prime editor fusion protein and an intein-N (ii) a polynucleotide encoding a C-terminal half of a prime editor fusion protein and an intein-C, (iii) a PEgRNA or a polynucleotide encoding the PEgRNA, and/or (iv) an ngRNA or a polynucleotide encoding the ngRNA. In some embodiments, a prime editing composition comprises (i) a polynucleotide encoding a N-terminal portion of a DNA binding domain and an intein-N, (ii) a polynucleotide encoding a C-terminal portion of the DNA binding domain, an intein-C, and a DNA polymerase domain. In some embodiments, the DNA binding domain is a Cas protein domain, e.g., a Cas9 nickase. In some embodiments, the prime editing composition comprises (i) a polynucleotide encoding a N-terminal portion of a DNA binding domain and an intein- N, (ii) a polynucleotide encoding a C-terminal portion of the DNA binding domain, an intein- C, and a DNA polymerase domain, (iii) a PEgRNA or a polynucleotide encoding the PEgRNA, and/or (iv) a ngRNA or a polynucleotide encoding the ngRNA. [0469]In some embodiments, a prime editing system comprises one or more polynucleotides encoding one or more prime editor polypeptides, wherein activity of the prime editing system WO 2024/238825 PCT/US2024/029746 194 may be temporally regulated by controlling the timing in which the vectors are delivered. For example, in some embodiments, a polynucleotide encoding the prime editor and a polynucleotide encoding a PEgRNA may be delivered simultaneously. For example, in some embodiments, a polynucleotide encoding the prime editor and a polynucleotide encoding a PEgRNA may be delivered sequentially. [0470]In some embodiments, a polynucleotide encoding a component of a prime editing system may further comprise an element that is capable of modifying the intracellular half- life of the polynucleotide and/or modulating translational control. In some embodiments, the polynucleotide is a RNA, for example, an mRNA. In some embodiments, the half-life of the polynucleotide, e.g., the RNA may be increased. In some embodiments, the half-life of the polynucleotide, e.g., the RNA may be decreased. In some embodiments, the element may be capable of increasing the stability of the polynucleotide, e.g., the RNA. In some embodiments, the element may be capable of decreasing the stability of the polynucleotide, e.g., the RNA. In some embodiments, the element may be within the 3' UTR of the RNA. In some embodiments, the element may include a polyadenylation signal (PA). In some embodiments, the element may include a cap, e.g., an upstream mRNA or PEgRNA end. In some embodiments, the RNA may comprise no PA such that it is subject to quicker degradation in the cell after transcription. [0471]In some embodiments, the element may include at least one AU-rich element (ARE). The AREs may be bound by ARE binding proteins (ARE-BPs) in a manner that is dependent upon tissue type, cell type, timing, cellular localization, and environment. In some embodiments the destabilizing element may promote RNA decay, affect RNA stability, or activate translation. In some embodiments, the ARE may comprise 50 to 150 nucleotides in length. In some embodiments, the ARE may comprise at least one copy of the sequence AUUUA. In some embodiments, at least one ARE may be added to the 3' UTR of the RNA. In some embodiments, the element may be a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE). In further embodiments, the element is a modified and/or truncated WPRE sequence that is capable of enhancing expression from the transcript. In some embodiments, the WPRE or equivalent may be added to the 3' UTR of the RNA. In some embodiments, the element may be selected from other RNA sequence motifs that are enriched in either fast- or slow-decaying transcripts. In some embodiments, the polynucleotide, e.g., a vector, encoding the PE or the PEgRNA may be self-destroyed via WO 2024/238825 PCT/US2024/029746 195 cleavage of a target sequence present on the polynucleotide, e.g., a vector. The cleavage may prevent continued transcription of a PE or a PEgRNA. [0472]Polynucleotides encoding prime editing composition components can be DNA, RNA, or any combination thereof. In some embodiments, a polynucleotide encoding a prime editing composition component is an expression construct. In some embodiments, a polynucleotide encoding a prime editing composition component is a vector. In some embodiments, the vector is a DNA vector. In some embodiments, the vector is a plasmid. In some embodiments, the vector is a virus vector, e.g., a retroviral vector, adenoviral vector, lentiviral vector, herpesvirus vector, or an adeno-associated virus vector (AAV). [0473]In some embodiments, polynucleotides encoding polypeptide components of a prime editing composition are codon optimized by replacing at least one codon (e.g., about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. In some embodiments, a polynucleotide encoding a polypeptide component of a prime editing composition are operably linked to one or more expression regulatory elements, for example, a promoter, a 3' UTR, a 5' UTR, or any combination thereof. In some embodiments, a polynucleotide encoding a prime editing composition component is a messenger RNA (mRNA). In some embodiments, the mRNA comprises a Cap at the 5' end and/or a poly A tail at the 3' end. [0474]The PEgRNAs described herein can be used in combination in a Prime Editing composition with one or more additional PEgRNAs that are designed to edit a different location in the same target gene or to edit a different gene. For example, the PEgRNAs described herein can be used in a multiplexed Prime Editing composition with one or more additional PEgRNAs that are designed to edit a gene encoding a T cell receptor subunit. Without being bound by theory, multiplexed disruption of MHC class I protein expression and T cell receptor expression in an engineered cell, e.g., immune cell, may reduce the allogenic reaction both of a host immune system and of the T cell receptor to host cells. Therefore, multiplexed disruption of B2M and T cell receptor gene(s), e.g., TRAC, may be used to generate engineered immune cells with reduced risk of graft versus host disease and graft rejection. [0475]In some embodiments, a prime editing system comprise a PEgRNA designed to edit a B2M gene or a nucleic acid encoding the PEgRNA as described herein. In some embodiments, the Prime Editing system further comprises a PEgRNA or a nucleic acid WO 2024/238825 PCT/US2024/029746 196 encoding the PEgRNA that targets a TRAC locus. In some embodiments, the PEgRNA that targets the TRAC locus encodes one or more edits that, when integrated into the TRAC locus, reduces or eliminates expression of the TRAC gene. For example, in some embodiments, the PEgRNA targeting the TRAC locus encodes one or more in frame premature stop codons, nonsense mutations, missense mutations, and/or frameshift mutations that result in decreased or eliminated expression of the TRAC gene. In some embodiments, a PEgRNA that targets a TRAC locus can encode, or further encode one or more recombinase recognition sites (RRSs) or its complement thereof. RRSs introduced into the TRAC gene may be used as landing sites for insertion of exogenous donor sequences in a target gene, e.g., the TRAC gene, in the presence of a recombinase that recognizes the RRS. In some embodiments, the prime editing system further comprises a recombinase that recognizes the RRS introduced into the TRAC gene by prime editing, or a nucleic acid encoding the recombinase. In some embodiments, the prime editing system further comprises a polynucleotide comprising (a) a donor sequence that can be used to introduce chimeric antigen receptor (CAR) into the TRAC locus, and (b) a corresponding RRS recognized by the recombinase. [0476]In some embodiments, the system further comprises a second PEgRNA or a nucleic acid encoding the second PEgRNA, wherein the second PEgRNA comprises: a. a second spacer that is complementary to a search target sequence on a first strand of T-cell receptor a constant (TRAC) gene, b. a second gRNA core capable of binding to a Cas9 protein, and c. a second extension arm comprising: i. a second editing template that comprises a region of complementarity to an editing target sequence on a second strand of the TRAC gene, and ii. a second primer binding site (PBS) that comprises at its 5’ end the reverse complement of nucleotides p to (q-3) of the second spacer, wherein q is the length of the second spacer, and p is an integer from 1 to (q-6), wherein the first strand and second strand are complementary to each other, and wherein the editing template encodes one or more nucleotide changes compared to the editing target sequence. [0477]In some embodiments, the second spacer is 17 to 22 nucleotides in length. [0478]In some embodiments, the editing template encodes an in-frame stop codon in the TRAC gene or a frameshift mutation in the TRAC gene. [0479]In some embodiments, the editing template encodes a recombinase recognition sequence recognized by a recombinase, or the reverse complement thereof. [0480]In some embodiments, the system further comprises a PEgRNA pair, wherein the PEgRNA pair comprises: a. a first prime editing guide RNA (PEgRNA) or one or more WO 2024/238825 PCT/US2024/029746 197 polynucleotides encoding the first PEgRNA, and b. a second PEgRNA or one or more polynucleotides encoding the second PEgRNA, wherein the first PEgRNA comprises: i. a first spacer that is complementary to a first search target sequence on a first strand of a TRAC gene, ii. a first gRNA core capable of binding to a Cas9 protein; and iii. a first extension arm comprising (A) a first editing template and (B) a first primer binding site (PBS) that comprises at its 5’ end the reverse complement of nucleotides p to (q-3) of the first spacer, wherein q is the length of the first spacer, and p is an integer of 1 to (q-6); wherein the second PEgRNA comprises: i. a second spacer that is complementary to a second search target sequence on a second strand of the TRAC gene complementary to the first strand, ii. a second gRNA core capable of binding to a Cas9 protein; and iii. a second extension arm comprising a second editing template and a second PBS that comprises at its 5’ end the reverse complement of nucleotides m to (n-3) of the second spacer, wherein n is the length of the first spacer, and m is an integer of 1 to (q-6). [0481]In some embodiments, the first spacer has a length of 17 to 22 nucleotides, or wherein the second spacer has the length of 17 to 22 nucleotides. In some embodiments, the first spacer and the second spacer each have a length of 20 nucleotides. In some embodiments, the first spacer is selected from the group consisting of SEQ ID NOs: 1303 and 1353. In some embodiments, the second spacer is selected from the group consisting of SEQ ID NOs: 1417, 1481 and 1532. [0482]In some embodiments, first TRAC PBS is 7-17 nucleotides in length and comprises at its 5’ end a sequence that is the reverse complement of nucleotides 11-17,10-17, 9-17, 8-17, 7-17, 6-17, 5-17, 4-17, 3-17, 2-17, or 1-17 of the selected sequence for the first TRAC spacer. In some embodiments, the second TRAC PBS is 7-17 nucleotides in length and comprises at its 5’ end a sequence that is the reverse complement of nucleotides 11-17,10-17, 9-17, 8-17, 7-17, 6-17, 5-17, 4-17, 3-17, 2-17, or 1-17 of the selected sequence for the second TRAC spacer. In some embodiments, the first and/or the second TRAC PBS is 11, 12, or nucleotides in length. [0483]In some embodiments, the first TRAC gRNA core, the second gRNA core, or both comprise SEQ ID NO: 646 or 653. [0484]In some embodiments, the first TRAC editing template comprises a region of complementarity to the second TRAC editing template. [0485]In some embodiments, the first TRAC editing template and the second TRAC editing template each encodes all or a fragment of a recombinase recognition sequence (RRS) or the WO 2024/238825 PCT/US2024/029746 198 reverse complement thereof, wherein the first TRAC editing template encodes at least a 5’ portion of the RRS or the reverse complement thereof, wherein the second TRAC editing template encodes at least a 3’ portion of the RRS or the reverse complement thereof, and wherein at least 10 nucleotides at the 5’ ends of the first and the second TRAC editing templates have perfect reverse complementarity to each other. [0486]In some embodiments, at least 15, 20, 25, or 30 nucleotides at the 5’ ends of the first and the second TRAC editing templates have perfect reverse complementarity to each other, optionally wherein at least 20, 21, 22, 23, 24, 25, 26, or 27 nucleotides at the 5’ ends of the first and the second TRAC editing templates have prefect reverse complementarity to each other. In some embodiments, the first editing template and the second editing template each encodes all or a fragment of a recombinase recognition sequence recognized by a recombinase, or the reverse complement thereof. [0487]In some embodiments, the first editing template and/or the recombinase recognition sequence encodes the recombinase recognition sequence or the reverse complement thereof. [0488] [0489]In some embodiments, Prime Editing composition or system further comprises a comprise a pair of TRAC-PEgRNAs comprising a 5’ TRAC PEgRNA (also referred to as a first TRAC PEgRNA) and a 3’ TRAC PEgRNA (also referred to as a second TRAC PEgRNA). The 5’ TRAC PEgRNA can comprise a 5’ TRAC spacer selected from any one of the 5’ TRAC PEgRNA spacer sequences in Tables 34-35 and a PBS selected from the same Table as the 5’ TRAC spacer. The 3’ TRAC PEgRNA can comprise a 3’ TRAC spacer selected from any one of the 3’ TRAC PEgRNA spacer sequences in Tables 36-38 and a PBS selected from the same Table as a 3’ TRAC spacer. [0490]In some embodiments, Prime Editing composition or system further comprises a comprise a pair of TRAC-PEgRNAs comprising a 5’ TRAC PEgRNA (also referred to as a first TRAC PEgRNA) and a 3’ TRAC PEgRNA (also referred to as a second TRAC PEgRNA), wherein the 5’ TRAC PEgRNA can be any one of 5’ TRAC PEgRNA sequences in Tables 34-35, and wherein the 3’ TRAC PEgRNA can any one of 3’ TRAC PEgRNA sequences in Tables in Tables 36-38. [0491]The TRAC PEgRNAs in Tables 34-38 can be used with any Prime Editor containing an appropriate Cas9 protein capable of recognizing an NGG PAM sequence, wherein N refers to any one of nucleotide A, G, C, or T. Exemplary Cas9 variants that can recognize an NGG PAM sequence are provided in Table 23. It can be advantageous to select TRAC-PEgRNA WO 2024/238825 PCT/US2024/029746 199 spacers that has a PAM sequence compatible with the Cas9 protein used in the Prime Editor for the B2M targeting PEgRNA, thus avoiding the need to use two different Cas9 proteins. [0492]The 5’ TRAC PEgRNA spacers and the 3’ TRAC PEgRNA spacers exemplified in Tables 34-38 can be, for example, 17 to 22 nucleotides in length. Each spacer within a single table correspond to the same PAM sequence and nick site when used with a compatible Casnickase. In some embodiments, the 5’ TRAC PEgRNA spacer and/or the 3’ TRAC PEgRNA spacer are 20 nucleotides in length. The TRAC PBS of the 5’ TRAC PEgRNA and the 3’ TRAC PEgRNA can be, for example, 5 to 19 nucleotides in length. In some embodiments, the TRAC PBS is 8 to 17 nucleotides in length. In some embodiments, the TRAC PBS is 8 to nucleotides in length. In some embodiments, the TRAC PBS is 8 to 13 nucleotides in length. In some embodiments, the TRAC PBS is 8, 10, or 12 nucleotides in length. In some embodiments, the RTT of the 5’ TRAC PEgRNA (the 5’ TRAC RTT) comprise a region of complementarity to the RTT of the 3’ TRAC PEgRNA (the 3’ TRAC RTT). The 5’ TRAC PEgRNA is capable of complexing with a prime editor comprising a Cas9 nickase (e.g. a Cas9 having an inactivated HNH nuclease domain) to generate a first nick, and the 3’ TRAC PEgRNA is capable of complexing with a prime editor comprising a Cas9 nickase (e.g. a Cas9 having an inactivated HNH nuclease domain) to generate a second nick on the TRAC gene. Accordingly, contacting the TRAC gene with the Prime Editing system comprising the TRAC-PEgRNA pair can result in deletion of the sequence between the first nick and the second nick (the deleted region referred to as the IND) and insertion of the replacement duplex (RD) encoded by the 5’ RTT and the 3’ RTT. The 5’ RTT and the 3’ RTT can be completely complementary to each other throughout their entire length, or they can be partially complementary, e.g., the region of complementarity can be at the 5’ ends of the 3’ RTT and the 5’ RTT. The region of complementarity (also referred to herein as the overlapping duplex or the OD) between the 5’ RTT and the 3’ RTT can have various lengths and GC content. In some embodiments, the OD is about 15 to 38 base pairs in length. In some embodiments, the OD is 18 to 38 base pairs in length. In some embodiments, the OD is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, or 38 base pairs in length. In some embodiments, the OD is 20, 21, 22, 23, 24, 25, 26, 27 or more base pairs in length. In some embodiments, the OD has a GC content of at least about 27%. In some embodiments, the OD has a GC content of about 30% to about 85%. In some embodiments, the OD has a GC content of about 40% to about 70%. In some embodiments, the OD has a GC content of about 63% to about 70%. The RD can also have WO 2024/238825 PCT/US2024/029746 200 various lengths and GC content. In some embodiments, when the 5’ RTT and the 3’ RTT are completely complementary to each other throughout their entire length, the OD and the RD have the same length and GC content. In some embodiments, the OD and the RD have different lengths and GC content. In some embodiments, the ratio of OD/RD is at least about 20%. In some embodiments, the ratio of OD/RD is about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% or more. In some embodiments, the ratio of OR/RD is about 52% or more. In some embodiments, the ratio of OR/RD is about 53% or more. In some embodiments, the ratio of OR/RD is about 55% or more. In some embodiments, the OD and the RD have the same sequence and length. [0493]Exemplary TRAC RTT pairs are provided in Table 39. Each RTT#1 and RTT#2 can be used as the basis of the RTTs in a PEgRNA pair, with either RTT#1 or RTT#2 serving as the basis for the 5’ RTT and the other as the basis for the 3’ RTT. Each RTT#1 in Table has a region of complementarity to the RTT#2. In some embodiments, a pair of TRAC RTTs having the same length is selected. In some embodiments, a TRAC PEgRNA pair will comprise the full length of RTT# 1 and RTT#2 and will form an OD along their complete lengths. In other embodiments, at least one of the TRAC PEgRNA pair will comprise less than the full-length sequence of the RTT#1 and/or RTT#2 from a single RTT pair. Such TRAC RTT pairs will form an OD only at the 5’ ends of the RTTs (corresponding to the 3’ ends of the newly synthesized strands). In some such embodiments, the first TRAC editing template comprises a 5’ fragment of a TRAC RTT listed in Table 39 and wherein the second TRAC editing template comprises a full length or 5’ fragment of the corresponding RTT pair and wherein at least 10 nucleotides at the 5’ end of the first and second TRAC editing templates have perfect reverse complementarity to each other. In other such embodiments, the second TRAC editing template comprises a 5’ fragment of an RTT listed in Table 39 and wherein the first TRAC editing template comprises a full length or 5’ fragment of the corresponding RTT pair and wherein at least 10 nucleotides at the 5’ end of the first and second TRAC editing templates have perfect reverse complementarity to each other. In any embodiments, at least 15, 20, 25, 30, 35 or more nucleotides at the 5’ end of the first and second TRAC editing templates can have perfect reverse complementarity to each other. In some embodiments, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37 or more nucleotides at the 5’ end of the first and the second WO 2024/238825 PCT/US2024/029746 201 TRAC editing templates have perfect complementarity to each other. In some embodiments, 20, 21, 22, 23, 24, 25, 26, 27 or more nucleotides at the 5’ end of the first and second TRAC editing templates can have perfect reverse complementarity to each other. In some embodiments, 20, 21, 22, 27 or more nucleotides at the 5’ end of the first and second TRAC editing templates can have perfect reverse complementarity to each other. The RTT pairing information (RTT1 or RTT2) and corresponding recombinase of TRAC PEgRNA sequences are indicated in the third column of Tables 34-38. For example, 5’ PEgRNA (Bxbl_38nt_RTTl) describes a 5’ PEgRNA sequence wherein the RTT has the sequence of Bxbl_38nt_RTT#l as provided in Table 39. [0494]The exemplary RTTs in Table 39 encode attB sequences or portions thereof that are recognized by Bxbl recombinase. In some embodiments, the first TRAC RTT and the second TRAC RTT can also encode one or more of any other recombinase recognition sequences (RRSs) disclosed herein or known in the art. Exemplary RSS sequences are provided in Table 32. [0495]TRAC RTTs designed to encode one or more RRS sequence can result in incorporation of the RRS sequence(s) in the target TRAC gene, which can be recognized by a corresponding recombinase. When contacted with the recombinase, the edited TRAC gene can undergo recombination with an exogenous DNA sequence that comprises a corresponding RRS sequence, e.g., attP sequence, that can be recognized by the same recombinase. In some embodiments, the exogenous DNA sequence comprises a donor sequence linked to the corresponding RRS sequence, and recombinase mediated recombination can result in integration of the donor sequence in the target TRAC gene. Exemplary RRS sequence pairs, e.g., attB-attP pairs, are provided in Table 32, where the orthogonal pairing relationship between the attB and the corresponding attP sequences are indicated by their identifiers in the first column. [0496]The exemplary TRAC RTTs provided in Table 39 encode attB sequences or fragments thereof. A PEgRNA or a dual prime editing PEgRNA pair that can comprise an RTT or an RTT pair exemplified in Table 39, or a fragment thereof, and can be used in a prime editing composition that further comprises an exogenous DNA sequence comprising a donor sequence and a corresponding attP sequence for integration of the donor sequence in TRAC. However, PEgRNA sequences that comprise RTT or RTT pairs that encode an attP sequence (or a fragment thereof) and the TRAC spacer and PBS sequences described here in are also contemplated. Such PEgRNA, or a pair of such PEgRNA, can be used in a prime WO 2024/238825 PCT/US2024/029746 202 editing composition that further comprises an exogenous DNA sequence comprising a donor sequence and a corresponding attB sequence for integration of the donor sequence in TRAC. [0497]In some embodiments, at least 15, 20, 25, or 30 nucleotides at the 5’ ends of the first and the second TRAC editing templates have perfect reverse complementarity to each other, optionally wherein at least 20, 21, 22, 23, 24, 25, 26, or 27 nucleotides at the 5’ ends of the first and the second TRAC editing templates have prefect reverse complementarity to each other. [0498]In some embodiments, the length of the region of complementarity of the first TRAC editing template is at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% of the length of the first TRAC editing template, optionally wherein the length of the region of complementarity of the first editing template is at least 52%, at least 53%, or at least 55% of the length of the first TRAC editing template. [0499]In some embodiments, the length of the region of complementarity of the second TRAC editing template is at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% of the length of the second TRAC editing template, optionally wherein the length of the region of complementarity of the second TRAC editing template is at least 52%, at least 53%, or at least 55% of the length of the second TRAC editing template. [0500]In some embodiments, the prime editing system comprises (a) the first TRAC spacer comprises SEQ ID NO: 1303, and the first TRAC PBS comprises SEQ ID NO: 1312, or (b) the first TRAC spacer comprises SEQ ID NO: 1353, and the first TRAC PBS comprises SEQ ID NO: 1361, 1362, 1363, or 1364. [0501]In some embodiments, the prime editing system comprises (a) the second TRAC spacer comprises SEQ ID NO: 1417, and the second TRAC PBS comprises SEQ ID NO:1428, or (b) the second TRAC spacer comprises SEQ ID NO: 1481, and the second TRAC PBS comprises SEQ ID NO: 1489. [0502]In some embodiments, the prime editing system comprises (a) the first TRAC spacer comprises SEQ ID NO: 1303, and the first TRAC PBS has the sequence according to SEQ ID NO: 1313 or SEQ ID NO: 1314; or the first TRAC spacer comprises SEQ ID NO: 1353, and the first TRAC PBS has the sequence according to SEQ ID NO: 1361 or SEQ ID NO: 1363; WO 2024/238825 PCT/US2024/029746 203 and (b) the second TRAC spacer comprises SEQ ID NO: 1417, and the second TRAC PBS has the sequence according to SEQ ID NO: 1426 or SEQ ID NO: 1428, the second TRAC spacer comprises SEQ ID NO: 1480, and the second TRAC PBS has the sequence according to SEQ ID NO: 1486 or SEQ ID NO: 1487; or the second TRAC spacer comprises SEQ ID NO: 1532, and the second TRAC PBS has the sequence according to SEQ ID NO: 1541 or 1543. [0503]In some embodiments, the first TRAC spacer comprises SEQ ID NO: 1353, and the first TRAC PBS has the sequence according to SEQ ID NO: 1361, and wherein the second TRAC spacer comprises SEQ ID NO: 1417, and the second TRAC PBS has the sequence according to SEQ ID NO: 1426. [0504]In some embodiments, the first TRAC editing template comprises SEQ ID NO: 15and the second editing TRAC template comprises SEQ ID NO: 1584. [0505]In some embodiments, wherein the first editing template comprises SEQ ID NO: 15and the second editing template comprises SEQ ID NO: 1577. [0506]In some embodiments, the first TRAC PEgRNA comprises a 5’ TRAC PEgRNA sequence selected from any one of Tables 34 and 35, and wherein the second TRAC PEgRNA comprises a 3’ TRAC PEgRNA sequence selected from any one of Tables 36-38. [0507]In some embodiments, the first TRAC PEgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 1328, 1382, 1387, and 1413; and wherein the second TRAC PEgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 1475, 1476, 1477, 1525, 1526, 1527, 1573, and 1574. [0508]In some embodiments, the first TRAC PEgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 1322, 1336, 1372, and 126; and wherein the second TRAC PEgRNA comprises a sequence selected from the group consisting of SEQ ID NOs:1442, 1456, 1501, and 1513. [0509]In some embodiments, the first TRAC PEgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 1401, 1406, 1343,, and 1345; and wherein the second TRAC PEgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 1451, 1516, 1568, 1566, and 1459. [0510]In some embodiments, the first TRAC PEgRNA and/or the second TRAC PEgRNA further comprises a 3’ motif, optionally wherein the 3’ motif is connected to the 3’ end of the first PBS or the second PBS via a linker.

WO 2024/238825 PCT/US2024/029746 204 id="p-511"

[0511]In some embodiments, the first TRAC PEgRNA and/or the second TRAC PEgRNA further comprises 5’mN*mN*mN* and 3’ mN*mN*mN*N modifications, where m indicates that the nucleotide contains a 2’-O-Me modification and a * indicates the presence of a phosphorothioate bond. [0512]In some embodiments, the prime editing system further comprises a recombinase or a nucleic acid encoding the recombinase. In some embodiments, the recombinase is fused or linked to the prime editor. In some embodiments, the prime editing system further comprises comprising a polynucleotide or a nucleic acid encoding the polynucleotide, wherein the polynucleotide comprises (a) a donor sequence and (b) a second recombinase recognition sequence (RRS) recognized by the recombinase. In some embodiments, the donor sequence comprises an open reading frame that encodes a polypeptide. In some embodiments, the donor sequence encodes a chimeric antigen receptor (CAR). [0513]In some embodiments, the RRS comprises SEQ ID NO: 1590, and the second RRS comprises SEQ ID NO: 1591. In some embodiments, the RRS comprises SEQ ID NO: 1591, and the second RRS comprises SEQ ID NO: 1590. In some embodiments, wherein the recombinase is Bxbl. [0514]In some embodiments, the prime editing system further comprises comprising a prime editor or one or more polynucleotides encoding the prime editor, wherein the prime editor comprises: a) a Cas9 nickase having a nuclease inactivating mutation in the HNH domain, and b) a reverse transcriptase. [0515]In some embodiments, the prime editor is a fusion protein. [0516]In some embodiments, the prime editing system further comprises an N-terminal extein comprising an N-terminal fragment of a prime editor fusion protein and an N-intein or a polynucleotide encoding the N-terminal extein; a C-terminal extein comprising a C- terminal fragment of the prime editor fusion protein and a C-intein, or a polynucleotide encoding the C-terminal extein; wherein the N-intein and the C-intein of the N-terminal and C-terminal exteins are capable of self-excision to join the N-terminal fragment and the C- terminal fragment to form the prime editor fusion protein, and wherein the prime editor fusion protein comprises a Cas9 nickase having a nuclease inactivating mutation in the HNH domain and a reverse transcriptase (RT) domain. [0517]In some embodiments, the Cas9 nickase comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NOs: 676 or 677. In some embodiments wherein the reverse transcriptase comprises an amino acid WO 2024/238825 PCT/US2024/029746 205 sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 673. In some embodiments, the sequence identities are determined by Needleman-Wunsch alignment of two protein sequences with Gap Costs set to Existence: Extension: 1 where percent identity is calculated by dividing the number of identities by the length of the alignment. In some embodiments, the one or more polynucleotides encoding the prime editor, the polynucleotide encoding the N-terminal extein, or the polynucleotide encoding the C-terminal extein are mRNA. [0518]Exemplary TRAC-PEgRNA pairs and components are provided in Tables 34-38. [0519]In some embodiments, the prime editing system further comprises a TRAC-PEgRNA pair, wherein the TRAC-PEgRNA pair comprises: a first TRAC-prime editing guide RNA (PEgRNA) or one or more polynucleotides encoding the first TRAC-PEgRNA, and a second TRAC-PEgRNA or one or more polynucleotides encoding the second TRAC-PEgRNA, wherein the first TRAC-PEgRNA comprises: a first TRAC-spacer that comprises at its 3’ end a 5’ TRAC-PEgRNA spacer sequence selected from any one of Tables 34 and 35, a first TRAC-gRNA core capable of binding to a Cas9 protein; and a first TRAC-extension arm comprising (A) a first TRAC-editing template and (B) a first TRAC- primer binding site (PBS) that comprises at its 5’ end a 5’ TRAC-PBS sequence selected from the same Table as the first TRAC-spacer, wherein the second TRAC-PEgRNA comprises: a second TRAC- spacer that comprises at its 3’ end a 3’ TRAC-PEgRNA spacer sequence selected from any one of Tables 36-38, a second TRAC-gRNA core capable of binding to a Cas9 protein; and a second TRAC- extension arm comprising (A) a second TRAC-editing template and (B) a second TRAC- primer binding site (PBS) that comprises at its 5’ end a 3’ TRAC-PBS sequence selected from the same Table as the second TRAC-spacer. In some embodiments, the first TRAC editing template comprises a region of complementarity to the second TRAC editing template. In some embodiments, wherein the first TRAC editing template and the second TRAC editing template each encodes all or a fragment of a recombinase recognition sequence (RRS) or the reverse complement thereof, wherein the first TRAC editing template encodes at least a 5’ portion of the RRS or the reverse complement thereof, wherein the second TRAC editing template encodes at least a 3’ portion of the RRS or the reverse complement thereof, and wherein at least 10 nucleotides at the 5’ ends of the first and the second TRAC editing templates have perfect reverse complementarity to each other. In some embodiments, wherein at least 15, 20, 25, or 30 nucleotides at the 5’ ends of the first and the second TRAC editing templates have perfect reverse complementarity to each other, WO 2024/238825 PCT/US2024/029746 206 optionally wherein at least 20, 21, 22, 23, 24, 25, 26, or 27 nucleotides at the 5’ ends of the first and the second TRAC editing templates have prefect reverse complementarity to each other. In some embodiments, the first TRAC editing template encodes the RRS or wherein the second TRAC editing template encodes the RRS, optionally wherein the RRS is an attB sequence recognized by a Bxbl recombinase or an attP sequence recognized by a Bxbl recombinase. In some embodiments, wherein the first TRAC editing template comprises an RTT #1 from Table 39 and the second TRAC editing template comprises an RTT #2 in Table 39, or wherein the first TRAC editing template comprises an RTT #2 from Table 39 and the second TRAC editing template comprises an RTT #1 in Table 39. In some embodiments, the first TRAC editing template comprises SEQ ID NO: 1577 and the second editing TRAC template comprises SEQ ID NO: 1584, or wherein the first editing template comprises SEQ ID NO: 1584 and the second editing template comprises SEQ ID NO: 1577. In some embodiments, the first TRAC PEgRNA comprises a 5’ TRAC PEgRNA sequence selected from any one of Tables 34 and 35, and wherein the second TRAC PEgRNA comprises a 3’ TRAC PEgRNA sequence selected from any one of Tables 36-38. [0520]The sequences in Tables 36-38 below are annotated with SEQ ID NO as required by ST.26 standard. Although all the sequences provided in Tables 36-38 are RNA sequences, "T" is used instead of a "U" in the sequences for consistency with the ST.26 standard.

Table 34 SEQ ID NO SEQUENCE DESCRIPTION 1300 CTGGGTTGGGGCAAAGA (SEQ ID NO: 1300) ' TRAC PEgRNA spacer (GGG PAM) 1301 CCTGGGTTGGGGCAAAGA (SEQ ID NO: 1301)5'TRAC PEgRNA spacer (GGG PAM) 1302 GCCTGGGTTGGGGCAAAGA (SEQ ID NO: 1302)5'TRACPEgRNA spacer (GGG PAM) 1303 AGCCTGGGTTGGGGCAAAGA (SEQ ID NO: 1303)5'TRAC PEgRNA spacer (GGG PAM) 1304 CAGCCTGGGTTGGGGCAAAGA (SEQ ID NO: 1304)5'TRACPEgRNA spacer (GGG PAM) WO 2024/238825 PCT/US2024/029746 207 1305 CCAGCCTGGGTTGGGGCAAAGA (SEQ ID NO: 1305)5'TRAC PEgRNA spacer (GGG PAM)1306 TTGCC 5' TRAC PBS1307 TTGCCC 5' TRAC PBS1308 TTGCCCC 5' TRAC PBS1309 TTGCCCCA 5' TRAC PBS1310 TTGCCCCAA 5' TRAC PBS1311 TTGCCCCAAC (SEQ ID NO: 1311) 5' TRAC PBS1312 TTGCCCCAACC (SEQ ID NO: 1312) 5' TRAC PBS1313 TTGCCCCAACCC (SEQ ID NO: 1313) 5' TRAC PBS1314 TTGCCCCAACCCA (SEQ ID NO: 1314) 5' TRAC PBS1315 TTGCCCCAACCCAG (SEQ ID NO: 1315) 5' TRAC PBS1316 TTGCCCCAACCCAGG (SEQ ID NO: 1316) 5' TRAC PBS1317 TTGCCCCAACCCAGGC (SEQ ID NO: 1317) 5' TRAC PBS1318 TTGCCCCAACCCAGGCT (SEQ ID NO: 1318) 5' TRAC PBS1319 TTGCCCCAACCCAGGCTG (SEQ ID NO: 1319) 5' TRAC PBS1320 TTGCCCCAACCCAGGCTGG (SEQ ID NO: 1320) 5' TRAC PBS 1321AGCCTGGGTTGGGGCAAAGAGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCACGACGGAGACCGC CGTCGTCGACAAGCCTTGCCCC (SEQ ID NO: 1321) ’ TRAC pegRNA (Bxbl 29nt RTT1) 1322 AGCCTGGGTTGGGGCAAAGAGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCACGACGGAGACCGC CGTCGTCGACAAGCCTTGCCCCAACC (SEQ ID NO: 1322) ’ TRAC pegRNA (Bxbl 29nt RTT 1) 1324 AGCCTGGGTTGGGGCAAAGAGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCGACGACGGAGACCG CCGTCGTCGACAAGCCTTGCCCCAACC (SEQ ID NO: 1324) ’ TRAC pegRNA (Bxbl 30nt RTT 1) 1326 AGCCTGGGTTGGGGCAAAGAGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCGACGACGGAGACCG CCGTCGTCGACAAGCCTTGCCCCAACCCA (SEQ ID NO: 1326) ’ TRAC pegRNA (Bxbl 30nt RTT 1) 1328 AGCCTGGGTTGGGGCAAAGAGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCGACGACGGAGACCG CCGTCGTCGACAAGCCTTGCCCCAACCTTTT (SEQ ID NO: 1328) ’ TRAC pegRNA (Bxbl_30nt_RTT 1); contains 3' end modification TTTT 1329 AGCCTGGGTTGGGGCAAAGAGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCATGATCCTGACGAC GGAGACCGCCGTCGTCGACAAGCCTTGCCCC (SEQ ID NO: 1329) ’ TRAC pegRNA (Bxbl 38nt RTT 1) WO 2024/238825 PCT/US2024/029746 208 1330 AGCCTGGGTTGGGGCAAAGAGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCGACGACGGAGACCG CCGTCGTCGACAAGCCTTGCCCCAACCCAGG (SEQ ID NO: 1330) ’ TRAC pegRNA (Bxbl 30nt RTT1) 1333 AGCCTGGGTTGGGGCAAAGAGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCGACGACGGAGACCG CCGTCGTCGACAAGCCTTGCCCCAACCCATTTT (SEQ ID NO: 1333) ’ TRAC pegRNA (Bxbl_30nt_RTT 1); contains 3' end modification TTTT 1334 AGCCTGGGTTGGGGCAAAGAGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCGACGACGGAGACCG CCGTCGTCGACAAGCCTTGCCCCAACCCAGGCT (SEQ ID NO: 1334) ’ TRAC pegRNA (Bxbl 30nt RTT 1) 1335 AGCCTGGGTTGGGGCAAAGAGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCATGATCCTGACGAC GGAGACCGCCGTCGTCGACAAGCCTTGCCCCAA (SEQ ID NO: 1335) ’ TRAC pegRNA (Bxbl 38nt RTT 1) 1336 AGCCTGGGTTGGGGCAAAGAGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCATGATCCTGACGAC GGAGACCGCCGTCGTCGACAAGCCTTGCCCCAACC (SEQ ID NO: 1336) ’ TRAC pegRNA (Bxbl 38nt RTT 1) 1337 AGCCTGGGTTGGGGCAAAGAGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCGACGACGGAGACCG CCGTCGTCGACAAGCCTTGCCCCAACCCAGGTTTT (SEQ ID NO: 1337) ’ TRAC pegRNA (Bxbl_30nt_RTT 1); contains 3' end modification TTTT 1339 AGCCTGGGTTGGGGCAAAGAGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCGACGACGGAGACCG CCGTCGTCGACAAGCCTTGCCCCAACCCAGGCTTTTT (SEQ ID NO: 1339) ’ TRAC pegRNA (Bxbl_30nt_RTT 1); contains 3' end modification TTTT 1340 AGCCTGGGTTGGGGCAAAGAGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCATGATCCTGACGAC GGAGACCGCCGTCGTCGACAAGCCTTGCCCCAATTTT (SEQ ID NO: 1340) ’ TRAC pegRNA (Bxbl_38nt_RTT 1); contains 3' end modification TTTT 1341 AGCCTGGGTTGGGGCAAAGAGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCATGATCCTGACGAC GGAGACCGCCGTCGTCGACAAGCCTTGCCCCAACCCA (SEQ ID NO: 1341) ’ TRAC pegRNA (Bxbl 38nt RTT 1) WO 2024/238825 PCT/US2024/029746 209 1343 AGCCTGGGTTGGGGCAAAGAGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCATGATCCTGACGAC GGAGACCGCCGTCGTCGACAAGCCTTGCCCCAACCTT TT (SEQIDNO: 1343) ’ TRAC pegRNA (Bxbl_38nt_RTT 1); contains 3' end modification TTTT 1344 AGCCTGGGTTGGGGCAAAGAGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCATGATCCTGACGAC GGAGACCGCCGTCGTCGACAAGCCTTGCCCCAACCCA GG (SEQ ID NO: 1344) ’ TRAC pegRNA (Bxbl 38nt RTT 1) 1345 AGCCTGGGTTGGGGCAAAGAGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCATGATCCTGACGAC GGAGACCGCCGTCGTCGACAAGCCTTGCCCCAACCCA TTTT (SEQ ID NO: 1345) ’ TRAC pegRNA (Bxbl_38nt_RTT 1); contains 3' end modification TTTT 1346 AGCCTGGGTTGGGGCAAAGAGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCATGATCCTGACGAC GGAGACCGCCGTCGTCGACAAGCCTTGCCCCAACCCA GGCT (SEQIDNO: 1346) ’ TRAC pegRNA (Bxbl 38nt RTT 1) 1348 AGCCTGGGTTGGGGCAAAGAGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCATGATCCTGACGAC GGAGACCGCCGTCGTCGACAAGCCTTGCCCCAACCCA GGTTTT (SEQ ID NO: 1348) ’ TRAC pegRNA (Bxbl_38nt_RTT 1); contains 3' end modification TTTT 1349 AGCCTGGGTTGGGGCAAAGAGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCATGATCCTGACGAC GGAGACCGCCGTCGTCGACAAGCCTTGCCCCAACCCA GGCTTTTT (SEQ ID NO: 1349) ’ TRAC pegRNA (Bxbl_38nt_RTT 1); contains 3' end modification TTTT Table 35 SEQ ID NO SEQUENCE DESCRIPTION 1350 GACTCCAGCCTGGGTTG (SEQ ID NO: 1350)5'TRACPEgRNA spacer (GGG PAM) 1351 GGACTCCAGCCTGGGTTG (SEQ ID NO: 1351)5'TRAC PEgRNA spacer (GGG PAM) 1352 TGGACTCCAGCCTGGGTTG (SEQ ID NO: 1352)5'TRAC PEgRNA spacer (GGG PAM) WO 2024/238825 PCT/US2024/029746 210 1353 CTGGACTCCAGCCTGGGTTG (SEQ ID NO: 1353)5'TRAC PEgRNA spacer (GGG PAM) 1354 TCTGGACTCCAGCCTGGGTTG (SEQ ID NO: 1354)5'TRAC PEgRNA spacer (GGG PAM) 1355 ATCTGGACTCCAGCCTGGGTTG (SEQ ID NO: 1355)5'TRACPEgRNA spacer (GGG PAM)1356 CCCAG 5' TRAC PBS1357 CCCAGG 5' TRAC PBS1358 CCCAGGC 5' TRAC PBS1359 CCCAGGCT 5' TRAC PBS1360 CCCAGGCTG 5' TRAC PBS1361 CCCAGGCTGG (SEQ ID NO: 1361) 5' TRAC PBS1362 CCCAGGCTGGA (SEQ ID NO: 1362) 5' TRAC PBS1363 CCCAGGCTGGAG (SEQ ID NO: 1363) 5' TRAC PBS1364 CCCAGGCTGGAGT (SEQ ID NO: 1364) 5' TRAC PBS1365 CCCAGGCTGGAGTC (SEQ ID NO: 1365) 5' TRAC PBS1366 CCCAGGCTGGAGTCC (SEQ ID NO: 1366) 5' TRAC PBS1367 CCCAGGCTGGAGTCCA (SEQ ID NO: 1367) 5' TRAC PBS1368 CCCAGGCTGGAGTCCAG (SEQ ID NO: 1368) 5' TRAC PBS1369 CCCAGGCTGGAGTCCAGA (SEQ ID NO: 1369) 5' TRAC PBS1370 CCCAGGCTGGAGTCCAGAT (SEQ ID NO: 1370) 5' TRAC PBS 1371CTGGACTCCAGCCTGGGTTGGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCACGACGGAGACCGC CGTCGTCGACAAGCCCCCAGG (SEQ ID NO: 1371) ’ TRAC pegRNA (Bxbl 29nt RTT1) 1372 CTGGACTCCAGCCTGGGTTGGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCACGACGGAGACCGC CGTCGTCGACAAGCCCCCAGGCTGG (SEQ ID NO: 1372) ’ TRAC pegRNA (Bxbl 29nt RTT 1) 1374 CTGGACTCCAGCCTGGGTTGGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCGACGACGGAGACCG CCGTCGTCGACAAGCCCCCAGGCTGGA (SEQ ID NO: 1374) ’ TRAC pegRNA (Bxbl 30nt RTT 1) 1375 CTGGACTCCAGCCTGGGTTGGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCGACGACGGCGGTCT CCGTCGTCAGGATCATCCCAGGCTGGA (SEQ ID NO: 1375) ’ TRAC pegRNA (Bxbl 30nt RTT2) 1377 CTGGACTCCAGCCTGGGTTGGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCGACGACGGAGACCG CCGTCGTCGACAAGCCCCCAGGCTGGAGT (SEQ ID NO: 1377) ’ TRAC pegRNA (Bxbl 30nt RTT 1) WO 2024/238825 PCT/US2024/029746 211 1378 CTGGACTCCAGCCTGGGTTGGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCGACGACGGCGGTCT CCGTCGTCAGGATCATCCCAGGCTGGAGT (SEQ ID NO: 1378) ’ TRAC pegRNA (Bxbl 30nt RTT2) 1379 CTGGACTCCAGCCTGGGTTGGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCATGATCCTGACGAC GGAGACCGCCGTCGTCGACAAGCCCCCAGG (SEQ ID NO: 1379) ’ TRAC pegRNA (Bxbl 38nt RTT 1) 1382 CTGGACTCCAGCCTGGGTTGGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCGACGACGGAGACCG CCGTCGTCGACAAGCCCCCAGGCTGGATTTT (SEQ ID NO: 1382) ’ TRAC pegRNA (Bxbl_30nt_RTT 1); contains 3' end modification TTTT 1383 CTGGACTCCAGCCTGGGTTGGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCGACGACGGCGGTCT CCGTCGTCAGGATCATCCCAGGCTGGATTTT (SEQ ID NO: 1383) ’ TRAC pegRNA (Bxbl_30nt_RTT 2); contains 3' end modification TTTT 1384 CTGGACTCCAGCCTGGGTTGGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCGACGACGGAGACCG CCGTCGTCGACAAGCCCCCAGGCTGGAGTCC (SEQ ID NO: 1384) ’ TRAC pegRNA (Bxbl 30nt RTT 1) 1386 CTGGACTCCAGCCTGGGTTGGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCATGATCCTGACGAC GGAGACCGCCGTCGTCGACAAGCCCCCAGGCT (SEQ ID NO: 1386) ’ TRAC pegRNA (Bxbl 38nt RTT 1) 1387 CTGGACTCCAGCCTGGGTTGGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCGACGACGGAGACCG CCGTCGTCGACAAGCCCCCAGGCTGGAGTTTTT (SEQ ID NO: 1387) ’ TRAC pegRNA (Bxbl_30nt_RTT 1); contains 3' end modification TTTT 1388 CTGGACTCCAGCCTGGGTTGGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCGACGACGGCGGTCT CCGTCGTCAGGATCATCCCAGGCTGGAGTTTTT (SEQ ID NO: 1388) ’ TRAC pegRNA (Bxbl_30nt_RTT 2); contains 3' end modification TTTT 1389 CTGGACTCCAGCCTGGGTTGGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCGACGACGGAGACCG CCGTCGTCGACAAGCCCCCAGGCTGGAGTCCAG (SEQ ID NO: 1389) ’ TRAC pegRNA (Bxbl 30nt RTT 1) WO 2024/238825 PCT/US2024/029746 212 1390 CTGGACTCCAGCCTGGGTTGGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCATGATCCTGACGAC GGAGACCGCCGTCGTCGACAAGCCCCCAGGCTGG (SEQIDNO: 1390) ’ TRAC pegRNA(Bxbl 38nt RTT1) 1393 CTGGACTCCAGCCTGGGTTGGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCGACGACGGAGACCG CCGTCGTCGACAAGCCCCCAGGCTGGAGTCCTTTT (SEQIDNO: 1393) ’ TRAC pegRNA (Bxbl_30nt_RTT 1); contains 3' end modification TTTT 1394 CTGGACTCCAGCCTGGGTTGGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCATGATCCTGACGAC GGAGACCGCCGTCGTCGACAAGCCCCCAGGCTGGA (SEQIDNO: 1394) ’ TRAC pegRNA (Bxbl 38nt RTT 1) 1395 CTGGACTCCAGCCTGGGTTGGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCGGCTTGTCGACGAC GGCGGTCTCCGTCGTCAGGATCATCCCAGGCTGGA (SEQIDNO: 1395) ’ TRAC pegRNA (Bxbl 38nt RTT2) 1396 CTGGACTCCAGCCTGGGTTGGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCATGATCCTGACGAC GGAGACCGCCGTCGTCGACAAGCCCCCAGGCTTTTT (SEQIDNO: 1396) ’ TRAC pegRNA (Bxbl_38nt_RTT 1); contains 3' end modification TTTT 1398 CTGGACTCCAGCCTGGGTTGGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCGACGACGGAGACCG CCGTCGTCGACAAGCCCCCAGGCTGGAGTCCAGTTTT (SEQIDNO: 1398) ’ TRAC pegRNA (Bxbl_30nt_RTT 1); contains 3' end modification TTTT 1399 CTGGACTCCAGCCTGGGTTGGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCATGATCCTGACGAC GGAGACCGCCGTCGTCGACAAGCCCCCAGGCTGGAG T (SEQIDNO: 1399) ’ TRAC pegRNA (Bxbl 38nt RTT 1) 1400 CTGGACTCCAGCCTGGGTTGGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCGGCTTGTCGACGAC GGCGGTCTCCGTCGTCAGGATCATCCCAGGCTGGAGT (SEQ ID NO: 1400) ’ TRAC pegRNA(Bxbl 38nt RTT 2) 1401 CTGGACTCCAGCCTGGGTTGGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCATGATCCTGACGAC GGAGACCGCCGTCGTCGACAAGCCCCCAGGCTGGTTT T (SEQ ID NO: 1401) ’ TRAC pegRNA (Bxbl_38nt_RTT 1); contains 3' end modification TTTT WO 2024/238825 PCT/US2024/029746 213 1403 CTGGACTCCAGCCTGGGTTGGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCATGATCCTGACGAC GGAGACCGCCGTCGTCGACAAGCCCCCAGGCTGGAT TTT (SEQ ID NO: 1403) ’ TRAC pegRNA (Bxbl_38nt_RTT 1); contains 3' end modification TTTT 1404 CTGGACTCCAGCCTGGGTTGGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCGGCTTGTCGACGAC GGCGGTCTCCGTCGTCAGGATCATCCCAGGCTGGATT TT (SEQ ID NO: 1404) ’ TRAC pegRNA (Bxbl_38nt_RTT 2); contains 3' end modification TTTT 1405 CTGGACTCCAGCCTGGGTTGGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCATGATCCTGACGAC GGAGACCGCCGTCGTCGACAAGCCCCCAGGCTGGAG TCC (SEQ ID NO: 1405) ’ TRAC pegRNA (Bxbl 38nt RTT 1) 1406 CTGGACTCCAGCCTGGGTTGGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCATGATCCTGACGAC GGAGACCGCCGTCGTCGACAAGCCCCCAGGCTGGAG Tutt (SEQ ID NO: 1406) ’ TRAC pegRNA (Bxbl_38nt_RTT 1); contains 3' end modification TTTT 1407 CTGGACTCCAGCCTGGGTTGGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCGGCTTGTCGACGAC GGCGGTCTCCGTCGTCAGGATCATCCCAGGCTGGAGT TTTT (SEQ ID NO: 1407) ’ TRAC pegRNA (Bxbl_38nt_RTT 2); contains 3' end modification TTTT 1408 CTGGACTCCAGCCTGGGTTGGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCATGATCCTGACGAC GGAGACCGCCGTCGTCGACAAGCCCCCAGGCTGGAG TCCAG (SEQ ID NO: 1408) ’ TRAC pegRNA (Bxbl 38nt RTT 1) 1411 CTGGACTCCAGCCTGGGTTGGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCATGATCCTGACGAC GGAGACCGCCGTCGTCGACAAGCCCCCAGGCTGGAG TCCTTTT (SEQ ID NO: 1411) ’ TRAC pegRNA (Bxbl_38nt_RTT 1); contains 3' end modification TTTT 1412 CTGGACTCCAGCCTGGGTTGGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCATGATCCTGACGAC GGAGACCGCCGTCGTCGACAAGCCCCCAGGCTGGAG TCCAGTTTT (SEQ ID NO: 1412) ’ TRAC pegRNA (Bxbl_38nt_RTT 1); contains 3' end modification TTTT 1413CTGGACTCCAGCCTGGGTTGGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCACGACGGAGACCGC ’ TRAC pegRNA (Bxbl 29nt RTT WO 2024/238825 PCT/US2024/029746 214 CGTCGTCGACAAGCCCCCAGGCTGGTTTT (SEQ ID NO: 1413)1); contains 3’ end modification TTTT Table 36 SEQ ID NO SEQUENCE DESCRIPTION 1414 GTCTCTCAGCTGGTACA (SEQ ID NO: 1414)3'TRAC PEgRNA spacer (CGG PAM) 1415 AGTCTCTCAGCTGGTACA (SEQ ID NO: 1415)3'TRACPEgRNA spacer (CGG PAM) 1416 GAGTCTCTCAGCTGGTACA (SEQ ID NO: 1416)3'TRAC PEgRNA spacer (CGG PAM) 1417 AGAGTCTCTCAGCTGGTACA (SEQ ID NO: 1417)3'TRACPEgRNA spacer (CGG PAM) 1418 TAGAGTCTCTCAGCTGGTACA (SEQ ID NO: 1418)3'TRAC PEgRNA spacer (CGG PAM) 1419 TTAGAGTCTCTCAGCTGGTACA (SEQ ID NO: 1419)3'TRACPEgRNA spacer (CGG PAM)1420 ACCAG 3' TRAC PBS1421 ACCAGC 3' TRAC PBS1422 ACCAGCT 3' TRAC PBS1423 ACCAGCTG 3' TRAC PBS1424 ACCAGCTGA 3' TRAC PBS1425 ACCAGCTGAG (SEQ ID NO: 1425) 3' TRAC PBS1426 ACCAGCTGAGA (SEQ ID NO: 1426) 3' TRAC PBS1427 ACCAGCTGAGAG (SEQ ID NO: 1427) 3' TRAC PBS1428 ACCAGCTGAGAGA (SEQ ID NO: 1428) 3' TRAC PBS1429 ACCAGCTGAGAGAC (SEQ ID NO: 1429) 3' TRAC PBS1430 ACCAGCTGAGAGACT (SEQ ID NO: 1430) 3' TRAC PBS1431 ACCAGCTGAGAGACTC (SEQ ID NO: 1431) 3' TRAC PBS1432 ACCAGCTGAGAGACTCT (SEQ ID NO: 1432) 3' TRAC PBS1433 ACCAGCTGAGAGACTCTA (SEQ ID NO: 1433) 3' TRAC PBS1434 ACCAGCTGAGAGACTCTAA (SEQ ID NO: 1434) 3' TRAC PBS 1435AGAGTCTCTCAGCTGGTACAGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCACGGCGGTCTCCGTC GTCAGGATCATACCAGCTGA (SEQ ID NO: 1435) 3’TRAC pegRNA(Bxbl 30nt RTT2) WO 2024/238825 PCT/US2024/029746 215 1437AGAGTCTCTCAGCTGGTACAGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCACGACGGCGGTCTC CGTCGTCAGGATCATACCAGCTGA (SEQ ID NO: 1437) 3’TRACpegRNA(Bxbl 29nt RTT2) 1438AGAGTCTCTCAGCTGGTACAGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCACGGCGGTCTCCGTC GTCAGGATCATACCAGCTGATTTT (SEQ ID NO: 1438) 3’TRAC pegRNA (Bxbl_30nt_RTT 2); contains 3' end modification TTTT 1441 AGAGTCTCTCAGCTGGTACAGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCGACGACGGCGGTCT CCGTCGTCAGGATCATACCAGCTGAGA (SEQ ID NO: 1441) 3’TRAC pegRNA(Bxbl 30nt RTT2) 1442 AGAGTCTCTCAGCTGGTACAGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCACGACGGCGGTCTC CGTCGTCAGGATCATACCAGCTGAGAGA (SEQ ID NO: 1442) 3' pegRNA (Bxbl 29nt RTT2) 1445 AGAGTCTCTCAGCTGGTACAGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCGACGACGGCGGTCT CCGTCGTCAGGATCATACCAGCTGAGAGA (SEQ ID NO: 1445) 3’TRAC pegRNA(Bxbl 30nt RTT2) 1446 AGAGTCTCTCAGCTGGTACAGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCGACGACGGAGACCG CCGTCGTCGACAAGCCACCAGCTGAGAGA (SEQ ID NO: 1446) 3’TRAC pegRNA(Bxbl 30nt RTT1) 1447 AGAGTCTCTCAGCTGGTACAGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCGACGACGGCGGTCT CCGTCGTCAGGATCATACCAGCTGAGATTTT (SEQ ID NO: 1447) 3’TRAC pegRNA (Bxbl_30nt_RTT 2); contains 3' end modification TTTT 1448 AGAGTCTCTCAGCTGGTACAGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCGACGACGGCGGTCT CCGTCGTCAGGATCATACCAGCTGAGAGACT (SEQ ID NO: 1448) 3’TRAC pegRNA(Bxbl 30nt RTT2) 1450 AGAGTCTCTCAGCTGGTACAGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCGACGACGGCGGTCT CCGTCGTCAGGATCATACCAGCTGAGAGATTTT (SEQ ID NO: 1450) 3’TRAC pegRNA (Bxbl_30nt_RTT 2); contains 3' end modification TTTT1451AGAGTCTCTCAGCTGGTACAGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA3’TRAC pegRNA WO 2024/238825 PCT/US2024/029746 216 AAAAGTGGCACCGAGTCGGTGCGACGACGGAGACCG CCGTCGTCGACAAGCCACCAGCTGAGAGATTTT (SEQ ID NO: 1451) (Bxbl_30nt_RTT 1); contains 3' end modification TTTT 1452 AGAGTCTCTCAGCTGGTACAGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCGGCTTGTCGACGAC GGCGGTCTCCGTCGTCAGGATCATACCAGCTGA (SEQ ID NO: 1452) 3’TRAC pegRNA(Bxbl 38nt RTT2) 1454 AGAGTCTCTCAGCTGGTACAGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCGACGACGGCGGTCT CCGTCGTCAGGATCATACCAGCTGAGAGACTTTTT (SEQ ID NO: 1454) 3’TRAC pegRNA (Bxbl_30nt_RTT 2); contains 3' end modification TTTT 1455 AGAGTCTCTCAGCTGGTACAGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCGGCTTGTCGACGAC GGCGGTCTCCGTCGTCAGGATCATACCAGCTGAGA (SEQ ID NO: 1455) 3’TRAC pegRNA(Bxbl 38nt RTT2) 1456 AGAGTCTCTCAGCTGGTACAGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCGGCTTGTCGACGAC GGCGGTCTCCGTCGTCAGGATCATACCAGCTGAGAGA (SEQ ID NO: 1456) 3’TRAC pegRNA(Bxbl 38nt RTT2) 1457 AGAGTCTCTCAGCTGGTACAGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCGGCTTGTCGACGAC GGCGGTCTCCGTCGTCAGGATCATACCAGCTGATTTT (SEQ ID NO: 1457) 3’TRAC pegRNA (Bxbl_38nt_RTT 2); contains 3' end modification TTTT 1459 AGAGTCTCTCAGCTGGTACAGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCGGCTTGTCGACGAC GGCGGTCTCCGTCGTCAGGATCATACCAGCTGAGATT TT (SEQ ID NO: 1459) 3’TRAC pegRNA (Bxbl_38nt_RTT 2); contains 3' end modification TTTT 1460 AGAGTCTCTCAGCTGGTACAGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCGGCTTGTCGACGAC GGCGGTCTCCGTCGTCAGGATCATACCAGCTGAGAGA CT (SEQ ID NO: 1460) 3’TRAC pegRNA(Bxbl 38nt RTT2) 1461 AGAGTCTCTCAGCTGGTACAGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCGGCTTGTCGACGAC GGCGGTCTCCGTCGTCAGGATCATACCAGCTGAGAGA TTTT (SEQ ID NO: 1461) 3’TRAC pegRNA (Bxbl_38nt_RTT 2); contains 3' end modification TTTT WO 2024/238825 PCT/US2024/029746 217 1462 AGAGTCTCTCAGCTGGTACAGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCATGATCCTGACGAC GGAGACCGCCGTCGTCGACAAGCCACCAGCTGAGAG A (SEQ ID NO: 1462) 3’TRACpegRNA(Bxbl 38nt RTT1) 1463 AGAGTCTCTCAGCTGGTACAGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCATGATCCTGACGAC GGAGACCGCCGTCGTCGACAAGCCACCAGCTGAGAG ATTTT (SEQ ID NO: 1463) 3’TRAC pegRNA(Bxbl 38nt RTT1) 1464 AGAGTCTCTCAGCTGGTACAGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCGGCTTGTCGACGAC GGCGGTCTCCGTCGTCAGGATCATACCAGCTGAGAGA CTTTTT (SEQ ID NO: 1464) 3’TRAC pegRNA (Bxbl_38nt_RTT 2); contains 3' end modification TTTT 1465 AGAGTCTCTCAGCTGGTACAGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCATGATCCTGACGAC GGAGACCGCCGTCGTCGACAAGCCACCAGCTGAGAG ATTTTTTTT (SEQ ID NO: 1465) 3’TRAC pegRNA (Bxbl_38nt_RTT 1); contains 3' end modification TTTT 1469 AGAGTCTCTCAGCTGGTACAGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCCCGTGACCTACATGC TCGAAGGGCGTATGCGCCACGAAGACCAGCTGAGAG ATTTT (SEQ ID NO: 1469) 3’TRAC pegRNA (Bxbl_39nt_RTT 2); contains 3' end modification TTTT 1474 AGAGTCTCTCAGCTGGTACAGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCATGATCCTGACGAC GGAGACCGCCGTCGTCGACAAGCCACCAGCTGATTTT (SEQ ID NO: 1474) 3’TRAC pegRNA(Bxbl_8ntRTTl).contains 3' end modification TTTT 1475 AGAGTCTCTCAGCTGGTACAGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCGACGACGGAGACCG CCGTCGTCGACAAGCCACCAGCTGAGATTTT (SEQ ID NO: 1475) 3’TRAC pegRNA (Bxbl_30nt RTT1), contains 3’ end modification TTTT 1476 AGAGTCTCTCAGCTGGTACAGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCACGACGGCGGTCTC CGTCGTCAGGATCATACCAGCTGAGAGATTTT (SEQ ID NO: 1476) 3’TRAC pegRNA (Bxbl_29nt RTT2), contains 3’ end modification TTTT WO 2024/238825 PCT/US2024/029746 218 1477 AGAGTCTCTCAGCTGGTACAGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCACGACGGCGGTCTC CGTCGTCAGGATCATACCAGCTGAGATTTT (SEQ ID NO: 1477) 3’TRAC pegRNA ((Bxbl_29nt RTT2), contains 3’ end modification TTTT Table 37 SEQ ID NO SEQUENCE DESCRIPTION 1478 GGTACACGGCAGGGTCA (SEQ ID NO: 1478)3'TRAC PEgRNA spacer (GGG PAM) 1479 TGGTACACGGCAGGGTCA (SEQ ID NO: 1479)3'TRACPEgRNA spacer (GGG PAM) 1480 CTGGTACACGGCAGGGTCA (SEQ ID NO: 1480)3'TRAC PEgRNA spacer (GGG PAM) 1481 GCTGGTACACGGCAGGGTCA (SEQ ID NO: 1481)3'TRACPEgRNA spacer (GGG PAM) 1482 AGCTGGTACACGGCAGGGTCA (SEQ ID NO: 1482)3'TRAC PEgRNA spacer (GGG PAM) 1483 CAGCTGGTACACGGCAGGGTCA (SEQ ID NO: 1483)3'TRACPEgRNA spacer (GGG PAM)1484 CCCTG 3' TRAC PBS1485 CCCTGC 3' TRAC PBS1486 CCCTGCC 3' TRAC PBS1487 CCCTGCCG 3' TRAC PBS1488 CCCTGCCGT 3' TRAC PBS1489 CCCTGCCGTG (SEQ ID NO: 1489) 3' TRAC PBS1490 CCCTGCCGTGT (SEQ ID NO: 1490) 3' TRAC PBS1491 CCCTGCCGTGTA (SEQ ID NO: 1491) 3' TRAC PBS1492 CCCTGCCGTGTAC (SEQ ID NO: 1492) 3' TRAC PBS1493 CCCTGCCGTGTACC (SEQ ID NO: 1493) 3' TRAC PBS1494 CCCTGCCGTGTACCA (SEQ ID NO: 1494) 3' TRAC PBS1495 CCCTGCCGTGTACCAG (SEQ ID NO: 1495) 3' TRAC PBS1496 CCCTGCCGTGTACCAGC (SEQ ID NO: 1496) 3' TRAC PBS1497 CCCTGCCGTGTACCAGCT (SEQ ID NO: 1497) 3' TRAC PBS1498 CCCTGCCGTGTACCAGCTG (SEQ ID NO: 1498) 3' TRAC PBS WO 2024/238825 PCT/US2024/029746 219 1499GCTGGTACACGGCAGGGTCAGTTTTAGAGCTAGAAA TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG AAAAAGTGGCACCGAGTCGGTGCACGACGGCGGTCT CCGTCGTCAGGATCATCCCTGC (SEQ ID NO: 1499) 3’TRAC pegRNA(Bxbl 29nt RTT2) 1500GCTGGTACACGGCAGGGTCAGTTTTAGAGCTAGAAA TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG AAAAAGTGGCACCGAGTCGGTGCGACGACGGCGGTC TCCGTCGTCAGGATCATCCCTGCC (SEQ ID NO: 1500) 3’TRAC pegRNA(Bxbl 30nt RTT2) 1501 GCTGGTACACGGCAGGGTCAGTTTTAGAGCTAGAAA TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG AAAAAGTGGCACCGAGTCGGTGCACGACGGCGGTCT CCGTCGTCAGGATCATCCCTGCCGTG (SEQ ID NO: 1501) 3’TRAC pegRNA(Bxbl 29nt RTT2) 1502 GCTGGTACACGGCAGGGTCAGTTTTAGAGCTAGAAA TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG AAAAAGTGGCACCGAGTCGGTGCGACGACGGCGGTC TCCGTCGTCAGGATCATCCCTGCCGT (SEQ ID NO: 1502) 3’TRAC pegRNA(Bxbl 30nt RTT2) 1503 GCTGGTACACGGCAGGGTCAGTTTTAGAGCTAGAAA TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG AAAAAGTGGCACCGAGTCGGTGCGACGACGGCGGTC TCCGTCGTCAGGATCATCCCTGCCTTTT (SEQ ID NO: 1503) 3’TRAC pegRNA (Bxbl_30nt_RTT 2); contains 3' end modification TTTT 1504 GCTGGTACACGGCAGGGTCAGTTTTAGAGCTAGAAA TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG AAAAAGTGGCACCGAGTCGGTGCGACGACGGCGGTC TCCGTCGTCAGGATCATCCCTGCCGTGT (SEQ ID NO: 1504) 3’TRAC pegRNA(Bxbl 30nt RTT2) 1505 GCTGGTACACGGCAGGGTCAGTTTTAGAGCTAGAAA TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG AAAAAGTGGCACCGAGTCGGTGCGACGACGGCGGTC TCCGTCGTCAGGATCATCCCTGCCGTTTTT (SEQ ID NO: 1505) 3’TRAC pegRNA (Bxbl_30nt_RTT 2); contains 3' end modification TTTT 1506 GCTGGTACACGGCAGGGTCAGTTTTAGAGCTAGAAA TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG AAAAAGTGGCACCGAGTCGGTGCGACGACGGCGGTC TCCGTCGTCAGGATCATCCCTGCCGTGTAC (SEQ ID NO: 1506) 3’TRAC pegRNA(Bxbl 30nt RTT2) 1507 GCTGGTACACGGCAGGGTCAGTTTTAGAGCTAGAAA TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG AAAAAGTGGCACCGAGTCGGTGCGGCTTGTCGACGA CGGCGGTCTCCGTCGTCAGGATCATCCCTGC (SEQ ID NO: 1507) 3’TRAC pegRNA(Bxbl 38nt RTT2) 1508GCTGGTACACGGCAGGGTCAGTTTTAGAGCTAGAAA TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG AAAAAGTGGCACCGAGTCGGTGCGACGACGGCGGTC 3’TRAC pegRNA (Bxbl_30nt_RTT 2); contains 3' end WO 2024/238825 PCT/US2024/029746 220 TCCGTCGTCAGGATCATCCCTGCCGTGTTTTT (SEQ ID NO: 1508)modification TTTT 1509 GCTGGTACACGGCAGGGTCAGTTTTAGAGCTAGAAA TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG AAAAAGTGGCACCGAGTCGGTGCGGCTTGTCGACGA CGGCGGTCTCCGTCGTCAGGATCATCCCTGCC (SEQ ID NO: 1509) 3’TRAC pegRNA(Bxbl 38nt RTT2) 1510 GCTGGTACACGGCAGGGTCAGTTTTAGAGCTAGAAA TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG AAAAAGTGGCACCGAGTCGGTGCGGCTTGTCGACGA CGGCGGTCTCCGTCGTCAGGATCATCCCTGCCG (SEQ ID NO: 1510) 3’TRAC pegRNA(Bxbl 38nt RTT2) 1511 GCTGGTACACGGCAGGGTCAGTTTTAGAGCTAGAAA TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG AAAAAGTGGCACCGAGTCGGTGCGACGACGGCGGTC TCCGTCGTCAGGATCATCCCTGCCGTGTACTTTT (SEQ ID NO: 1511) 3’TRAC pegRNA (Bxbl_30nt_RTT 2); contains 3' end modification TTTT 1512 GCTGGTACACGGCAGGGTCAGTTTTAGAGCTAGAAA TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG AAAAAGTGGCACCGAGTCGGTGCGGCTTGTCGACGA CGGCGGTCTCCGTCGTCAGGATCATCCCTGCCGT (SEQ ID NO: 1512) 3’TRAC pegRNA(Bxbl 38nt RTT2) 1513 GCTGGTACACGGCAGGGTCAGTTTTAGAGCTAGAAA TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG AAAAAGTGGCACCGAGTCGGTGCGGCTTGTCGACGA CGGCGGTCTCCGTCGTCAGGATCATCCCTGCCGTG (SEQ ID NO: 1513) 3’TRAC pegRNA(Bxbl 38nt RTT2) 1514 GCTGGTACACGGCAGGGTCAGTTTTAGAGCTAGAAA TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG AAAAAGTGGCACCGAGTCGGTGCGGCTTGTCGACGA CGGCGGTCTCCGTCGTCAGGATCATCCCTGCCTTTT (SEQ ID NO: 1514) 3’TRAC pegRNA (Bxbl_38nt_RTT 2); contains 3' end modification TTTT 1515 GCTGGTACACGGCAGGGTCAGTTTTAGAGCTAGAAA TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG AAAAAGTGGCACCGAGTCGGTGCGGCTTGTCGACGA CGGCGGTCTCCGTCGTCAGGATCATCCCTGCCGTGT (SEQ ID NO: 1515) 3’TRAC pegRNA(Bxbl 38nt RTT2) 1516 GCTGGTACACGGCAGGGTCAGTTTTAGAGCTAGAAA TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG AAAAAGTGGCACCGAGTCGGTGCGGCTTGTCGACGA CGGCGGTCTCCGTCGTCAGGATCATCCCTGCCGTTTT (SEQ ID NO: 1516) 3’TRAC pegRNA (Bxbl_38nt_RTT 2); contains 3' end modification TTTT 1517GCTGGTACACGGCAGGGTCAGTTTTAGAGCTAGAAA TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG AAAAAGTGGCACCGAGTCGGTGCGGCTTGTCGACGA 3’TRAC pegRNA(Bxbl 38nt RTT2) WO 2024/238825 PCT/US2024/029746 221 CGGCGGTCTCCGTCGTCAGGATCATCCCTGCCGTGTA (SEQIDNO: 1517) 1518 GCTGGTACACGGCAGGGTCAGTTTTAGAGCTAGAAA TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG AAAAAGTGGCACCGAGTCGGTGCGGCTTGTCGACGA CGGCGGTCTCCGTCGTCAGGATCATCCCTGCCGTTTT T (SEQIDNO: 1518) 3’TRAC pegRNA (Bxbl_38nt_RTT 2); contains 3' end modification TTTT 1519 GCTGGTACACGGCAGGGTCAGTTTTAGAGCTAGAAA TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG AAAAAGTGGCACCGAGTCGGTGCGGCTTGTCGACGA CGGCGGTCTCCGTCGTCAGGATCATCCCTGCCGTGTA C (SEQIDNO: 1519) 3’TRAC pegRNA(Bxbl 38nt RTT2) 1520 GCTGGTACACGGCAGGGTCAGTTTTAGAGCTAGAAA TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG AAAAAGTGGCACCGAGTCGGTGCGGCTTGTCGACGA CGGCGGTCTCCGTCGTCAGGATCATCCCTGCCGTGTT TT (SEQIDNO: 1520) 3’TRAC pegRNA (Bxbl_38nt_RTT 2); contains 3' end modification TTTT 1521 GCTGGTACACGGCAGGGTCAGTTTTAGAGCTAGAAA TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG AAAAAGTGGCACCGAGTCGGTGCGGCTTGTCGACGA CGGCGGTCTCCGTCGTCAGGATCATCCCTGCCGTGTT TTT (SEQIDNO: 1521) 3’TRAC pegRNA (Bxbl_38nt_RTT 2); contains 3' end modification TTTT 1522 GCTGGTACACGGCAGGGTCAGTTTTAGAGCTAGAAA TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG AAAAAGTGGCACCGAGTCGGTGCGGCTTGTCGACGA CGGCGGTCTCCGTCGTCAGGATCATCCCTGCCGTGTA TTTT (SEQ ID NO: 1522) 3’TRAC pegRNA (Bxbl_38nt_RTT 2); contains 3' end modification TTTT 1523 GCTGGTACACGGCAGGGTCAGTTTTAGAGCTAGAAA TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG AAAAAGTGGCACCGAGTCGGTGCGGCTTGTCGACGA CGGCGGTCTCCGTCGTCAGGATCATCCCTGCCGTGTA CTTTT (SEQ ID NO: 1523) 3’TRAC pegRNA (Bxbl_38nt_RTT 2); contains 3' end modification TTTT 1524 GCTGGTACACGGCAGGGTCAGTTTTAGAGCTAGAAA TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG AAAAAGTGGCACCGAGTCGGTGCATGATCCTGACGA CGGAGACCGCCGTCGTCGACAAGCCCCCTGCCTTTT (SEQ ID NO: 1524) 3’TRAC pegRNA (Bxbl 38nt_RTTl); contains 3' end modification TTTT 1525 GCTGGTACACGGCAGGGTCAGTTTTAGAGCTAGAAA TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG AAAAAGTGGCACCGAGTCGGTGCGACGACGGAGACC GCCGTCGTCGACAAGCCCCCTGCCTTTT (SEQ ID NO: 1525) 3’TRAC pegRNA (Bxbl 30nt_RTTl); contains 3' end modification TTTT WO 2024/238825 PCT/US2024/029746 222 1526 GCTGGTACACGGCAGGGTCAGTTTTAGAGCTAGAAA TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG AAAAAGTGGCACCGAGTCGGTGCGACGACGGAGACC GCCGTCGTCGACAAGCCCCCTGCCGTTTTT (SEQ ID NO: 1526) 3’TRAC pegRNA (Bxbl 30nt_RTTl); contains 3' end modification TTTT 1527 GCTGGTACACGGCAGGGTCAGTTTTAGAGCTAGAAA TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG AAAAAGTGGCACCGAGTCGGTGCACGACGGCGGTCT CCGTCGTCAGGATCATCCCTGCCGTTTT (SEQ ID NO: 1527) 3’TRAC pegRNA (Bxbl 29nt_RTT2); contains 3' end modification TTTT 1528GCTGGTACACGGCAGGGTCAGTTTTAGAGCTAGAAA TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG AAAAAGTGGCACCGAGTCGGTGCACGACGGCGGTCT CCGTCGTCAGGATCATCCCTGCCG (SEQ ID NO: 1528) 3’TRAC pegRNA (Bxbl 29nt RTT2) Table 38 SEQ ID NO SEQUENCE DESCRIPTION 1529 CTCAGCTGGTACACGGC (SEQ ID NO: 1529)3'TRACPEgRNA spacer (AGG PAM) 1530 TCTCAGCTGGTACACGGC (SEQ ID NO: 1530)3'TRAC PEgRNA spacer (AGG PAM) 1531 CTCTCAGCTGGTACACGGC (SEQ ID NO: 1531)3'TRACPEgRNA spacer (AGG PAM) 1532 TCTCTCAGCTGGTACACGGC (SEQ ID NO: 1532)3'TRAC PEgRNA spacer (AGG PAM) 1533 GTCTCTCAGCTGGTACACGGC (SEQ ID NO: 1533)3'TRAC PEgRNA spacer (AGG PAM) 1534 AGTCTCTCAGCTGGTACACGGC (SEQ ID NO: 1534)3'TRACPEgRNA spacer (AGG PAM)1535 GTGTA 3' TRAC PBS1536 GTGTAC 3' TRAC PBS1537 GTGTACC 3' TRAC PBS1538 GTGTACCA 3' TRAC PBS1539 GTGTACCAG 3' TRAC PBS1540 GTGTACCAGC (SEQ ID NO: 1540) 3' TRAC PBS1541 GTGTACCAGCT (SEQ ID NO: 1541) 3' TRAC PBS1542 GTGTACCAGCTG (SEQ ID NO: 1542) 3' TRAC PBS WO 2024/238825 PCT/US2024/029746 223 1543 GTGTACCAGCTGA (SEQ ID NO: 1543) 3' TRAC PBS1544 GTGTACCAGCTGAG (SEQ ID NO: 1544) 3' TRAC PBS1545 GTGTACCAGCTGAGA (SEQ ID NO: 1545) 3' TRAC PBS1546 GTGTACCAGCTGAGAG (SEQ ID NO: 1546) 3' TRAC PBS1547 GTGTACCAGCTGAGAGA (SEQ ID NO: 1547) 3' TRAC PBS1548 GTGTACCAGCTGAGAGAC (SEQ ID NO: 1548) 3' TRAC PBS1549 GTGTACCAGCTGAGAGACT (SEQ ID NO: 1549) 3' TRAC PBS 1550 TCTCTCAGCTGGTACACGGCGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCGACGACGGCGGTCT CCGTCGTCAGGATCATGTGTACCAGCT (SEQ ID NO: 1550) 3’TRAC pegRNA(Bxbl 30nt RTT2) 1551 TCTCTCAGCTGGTACACGGCGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCGACGACGGCGGTCT CCGTCGTCAGGATCATGTGTACCAGCTGA (SEQ ID NO: 1551) 3’TRAC pegRNA(Bxbl 30nt RTT2) 1552 TCTCTCAGCTGGTACACGGCGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCGACGACGGAGACCG CCGTCGTCGACAAGCCGTGTACCAGCTGA (SEQ ID NO: 1552) 3’TRAC pegRNA(Bxbl 30nt RTT1) 1553 TCTCTCAGCTGGTACACGGCGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCGACGACGGCGGTCT CCGTCGTCAGGATCATGTGTACCAGCTTTTT (SEQ ID NO: 1553) 3’TRAC pegRNA (Bxbl_30nt_RTT 2); contains 3' end modification TTTT 1554 TCTCTCAGCTGGTACACGGCGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCGACGACGGCGGTCT CCGTCGTCAGGATCATGTGTACCAGCTGAGA (SEQ ID NO: 1554) 3’TRAC pegRNA(Bxbl 30nt RTT2) 1555 TCTCTCAGCTGGTACACGGCGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCGACGACGGCGGTCT CCGTCGTCAGGATCATGTGTACCAGCTGATTTT (SEQ ID NO: 1555) 3’TRAC pegRNA (Bxbl_30nt_RTT 2); contains 3' end modification TTTT 1556 TCTCTCAGCTGGTACACGGCGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCGACGACGGAGACCG CCGTCGTCGACAAGCCGTGTACCAGCTGATTTT (SEQ ID NO: 1556) 3’TRAC pegRNA (Bxbl_30nt_RTT 1); contains 3' end modification TTTT 1557TCTCTCAGCTGGTACACGGCGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCGGCTTGTCGACGAC3’TRAC pegRNA WO 2024/238825 PCT/US2024/029746 224 GGCGGTCTCCGTCGTCAGGATCATGTGTACCAG (SEQ ID NO: 1557)(Bxbl 38nt RTT 2) 1558 TCTCTCAGCTGGTACACGGCGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCGACGACGGCGGTCT CCGTCGTCAGGATCATGTGTACCAGCTGAGAGA (SEQ ID NO: 1558) 3’TRAC pegRNA(Bxbl 30nt RTT2) 1559 TCTCTCAGCTGGTACACGGCGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCGACGACGGCGGTCT CCGTCGTCAGGATCATGTGTACCAGCTGAGATTTT (SEQIDNO: 1559) 3’TRAC pegRNA (Bxbl_30nt_RTT 2); contains 3' end modification TTTT 1560 TCTCTCAGCTGGTACACGGCGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCGGCTTGTCGACGAC GGCGGTCTCCGTCGTCAGGATCATGTGTACCAGCT (SEQIDNO: 1560) 3’TRAC pegRNA(Bxbl 38nt RTT2) 1561 TCTCTCAGCTGGTACACGGCGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCGGCTTGTCGACGAC GGCGGTCTCCGTCGTCAGGATCATGTGTACCAGTTTT (SEQIDNO: 1561) 3’TRAC pegRNA (Bxbl_38nt_RTT 2); contains 3' end modification TTTT 1562 TCTCTCAGCTGGTACACGGCGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCGACGACGGCGGTCT CCGTCGTCAGGATCATGTGTACCAGCTGAGAGATTTT (SEQIDNO: 1562) 3’TRAC pegRNA (Bxbl_30nt_RTT 2); contains 3' end modification TTTT 1563 TCTCTCAGCTGGTACACGGCGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCGACGACGGCGGTCT CCGTCGTCAGGATCATGTGTACCAGCTGATTTTTTTT (SEQIDNO: 1563) 3’TRAC pegRNA (Bxbl_30nt_RTT 2); contains 3' end modification TTTT 1564 TCTCTCAGCTGGTACACGGCGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCGGCTTGTCGACGAC GGCGGTCTCCGTCGTCAGGATCATGTGTACCAGCTGA (SEQIDNO: 1564) 3’TRAC pegRNA(Bxbl 38nt RTT2) 1565 TCTCTCAGCTGGTACACGGCGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCATGATCCTGACGAC GGAGACCGCCGTCGTCGACAAGCCGTGTACCAGCTG A (SEQIDNO: 1565) 3’TRAC pegRNA(Bxbl 38nt RTT1) 1566TCTCTCAGCTGGTACACGGCGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCGGCTTGTCGACGAC 3’TRAC pegRNA(Bxbl 38nt RTT WO 2024/238825 PCT/US2024/029746 225 GGCGGTCTCCGTCGTCAGGATCATGTGTACCAGCTTT TT (SEQ ID NO: 1566)2); contains 3' end modification TTTT 1567 TCTCTCAGCTGGTACACGGCGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCGGCTTGTCGACGAC GGCGGTCTCCGTCGTCAGGATCATGTGTACCAGCTGA GA (SEQ ID NO: 1567) 3’TRAC pegRNA(Bxbl 38nt RTT2) 1568 TCTCTCAGCTGGTACACGGCGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCGGCTTGTCGACGAC GGCGGTCTCCGTCGTCAGGATCATGTGTACCAGCTGA TTTT (SEQ ID NO: 1568) 3’TRAC pegRNA (Bxbl_38nt_RTT 2); contains 3' end modification TTTT 1569 TCTCTCAGCTGGTACACGGCGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCATGATCCTGACGAC GGAGACCGCCGTCGTCGACAAGCCGTGTACCAGCTG ATTTT (SEQ ID NO: 1569) 3’TRAC pegRNA (Bxbl_38nt_RTT 1); contains 3' end modification TTTT 1570 TCTCTCAGCTGGTACACGGCGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCGGCTTGTCGACGAC GGCGGTCTCCGTCGTCAGGATCATGTGTACCAGCTGA GAGA (SEQ ID NO: 1570) 3’TRAC pegRNA(Bxbl 38nt RTT2) 1571 TCTCTCAGCTGGTACACGGCGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCGGCTTGTCGACGAC GGCGGTCTCCGTCGTCAGGATCATGTGTACCAGCTGA GATTTT (SEQ ID NO: 1571) 3’TRAC pegRNA (Bxbl_38nt_RTT 2); contains 3' end modification TTTT 1572 TCTCTCAGCTGGTACACGGCGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCGGCTTGTCGACGAC GGCGGTCTCCGTCGTCAGGATCATGTGTACCAGCTGA GAGATTTT (SEQ ID NO: 1572) 3’TRAC pegRNA (Bxbl_38nt_RTT 2); contains 3' end modification TTTT 1573 TCTCTCAGCTGGTACACGGCGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCGACGACGGAGACCG CCGTCGTCGACAAGCCGTGTACCAGCTTTTT (SEQ ID NO: 1573) 3’TRAC pegRNA (Bxbl_30nt_RTT 1); contains 3' end modification TTTT 1574 TCTCTCAGCTGGTACACGGCGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCACGACGGCGGTCTC CGTCGTCAGGATCATGTGTACCAGCTGATTTT (SEQ ID NO: 1574) 3’TRAC pegRNA (Bxbl_29nt_RTT 2); contains 3' end modification TTTT WO 2024/238825 PCT/US2024/029746 226 Table 39. SEQID NO Sequence Description 1575ACGACGGAGACCGCCGTCGTCGACAAGCCBxbl_29nt_RTT# 1576GACGACGGAGACCGCCGTCGTCGACAAGCCBxbl_30nt_RTT# 1577ATGATCCTGACGACGGAGACCGCCGTCGTCGACAA GCCBxbl_38nt_RTT# 1582ACGACGGCGGTCTCCGTCGTCAGGATCATBxbl_29nt_RTT# 1583GACGACGGCGGTCTCCGTCGTCAGGATCATBxbl_30nt_RTT# 1584GGCTTGTCGACGACGGCGGTCTCCGTCGTCAGGATC ATBxbl_38nt_RTT# 1589GACGACGGAGACCGCCGTCGTCGACAAGCCBxbl_30nt_RTT# id="p-521"

[0521]The donor sequence for integration into the TRAC gene can encode any desired sequence, e.g., a chimeric antigen receptor (CAR), a T cell receptor (TCR), a B cell receptor, a NK cell receptor, a cytokine receptor, a chemokine receptor, a cytokine, a chemokine, a signaling protein, an antigen binding domain, an antibody, a kill switch, a reporter protein, a dominant negative mutant of a immune cell signaling protein, or a functional fragment or variant thereof. Without being bound by theory, compared to other strategies for donor integration, e.g., CAR integration, in immune cells such as semi-random integration via lentivirus or transposons, prime editing-and-recombinase-mediated integration in the TRAC gene as described herein is targeted to specific positions in the TRAC gene and reduces the risk of unintended disruption or activation of non-target genes. Without being bound by theory, compared to other targeted integration strategies such as nuclease and homology directed repair (HDR), prime editing-and-recombinase-mediated integration in the TRAC gene as described herein is unlikely to involve double strand breaks (DSBs) that are exposed to cellular repair mechanisms, and can result in improved integration efficiency and/or reduced risk associated with DSBs, e.g., genome rearrangements, chromothripsis, or unintended gene disruption or activation such as p53 activation. [0522]Recombination of the exogenous DNA sequence comprising an RRS at the prime edited TRAC gene comprising a corresponding RSS can result in integration of the donor sequence in the TRAC gene flanked by the recombination products of the RSS sequences.

WO 2024/238825 PCT/US2024/029746 227 For example, in some embodiments, the TRAC PEgRNA pairs install an attB sequence in the TRAC gene, and the exogenous DNA sequence comprises a donor sequence and a corresponding attP sequence. After recombinase mediated recombination, the TRAC gene comprises the following insert sequence from 5’ to 3’: attL-donor sequence-attR. In some embodiments, the PEgRNA or PEgRNA pairs install an attP sequence in the TRAC gene, and the exogenous DNA sequence comprises a donor sequence and a corresponding attB sequence. After recombinase mediated recombination, the TRAC gene comprises the following insert sequence from 5’ to 3’: attR-donor sequence-attL. Accordingly, provided herein are edited cells that comprise in the TRAC gene an insert sequence, wherein the insert sequence comprises (i) 5’-attL-donor sequence-attR-3 ‘ or (ii) 5’-attR-donor sequence-attL-3 ‘. In some embodiments, the edited cells, e.g., T cells provide herein comprise in the TRAC gene an insert sequence, wherein the insert sequence comprises 5’-attL-donor sequence-attR- 3’. [0523]Position of the insert sequence depends on the TRAC PEgRNA spacers designed for integration of the attB (or attP) sequence encoded by the TRAC RTTs. In some embodiments, the insert sequence in the edited cell is between the first nick site according to the first TRAC PEgRNA and the second nick site according to the second TRAC PEgRNA of a dual prime editing TRAC PEgRNA pair (i.e. the region corresponding to the IND according to the TRAC PEgRNA pair). In some embodiments, the insert sequence is immediately downstream of the first nick site. In some embodiments, the insert sequence is immediately upstream of the first nick site. Nick site locations according to exemplary TRAC PEgRNA spacers provided herein in human chromosome 14 are provided in Table 40 below. Unless otherwise specified, chromosome locations and coding sequence positions (e.g. c.xxxx indicates position xxxx in the coding sequence of a certain gene) are as set forth in Genome Reference Consortium Human Build 38 (GrCH38). Accordingly, a cell, e.g., a T cell edited with the TRAC PEgRNA provided in Tables 34-38 and contacted with (i) a recombinase and (ii) a donor sequence linked to an attP sequence may comprise in the TRAC gene an insert sequence between the nick position of any 5’ TRAC spacer in Table 40 and the nick position of any 3’ TRAC spacer in Table 40, depending on the 5’ TRAC spacer and the 3’ TRAC spacer used in the first TRAC PEgRNA and the second TRAC PEgRNA, wherein the insert sequence comprises 5’-attL-donor sequence-attR-3 ‘. In some embodiments, the edited cell comprises the insert sequence between human chromosome 14 positions 22547458 and 22547533. In some embodiments, the edited cell comprises the insert sequence between WO 2024/238825 PCT/US2024/029746 228 human chromosome 14 positions 22547458 and 22547522. In some embodiments, the edited cell comprises the insert sequence between human chromosome 14 positions 22547458 and 22547529. In some embodiments, the edited cell comprises the insert sequence between human chromosome 14 positions 22547449 and 22547533. In some embodiments, the edited cell comprises the insert sequence between human chromosome 14 positions 22547449 and 22547522. In some embodiments, the edited cell comprises the insert sequence between human chromosome 14 positions 22547449 and 22547529. [0524]Exemplary sequences of attB-attP sequence pairs are provided in Table 32. In some embodiments, the attB sequence isGGCTTGTCGACGACGGCGGTCTCCGTCGTCAGGATCAT (SEQ ID NO: 1590). In some embodiments, the attP sequence isGGTTTGTCTGGTCAACCACCGCGGTCTCAGTGGTGTACGGTACAAACC (SEQ ID NO: 1591). In some embodiments, the attL sequence is GGCTTGTCGACGACGGCGGTCTCAGTGGTGTACGGTACAAACC (SEQ ID NO: 9999). In some embodiments, the attR sequence is GGTTTGTCTGGTCAACCACCGCGGTCTCCGTCGTCAGGATCAT (SEQ ID NO: 10000). In some embodiments, the recombinase is a Bxbl recombinase. In some embodiments, the Bxbl recombinase comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: WOOL Table 40 ’ spacer TRAC SEQ ID NO 5’ nick site chromosome position 1300-1305 (Table 34 TRAC PEgRNAs) 225474581350-1355 (Table 35 PEgRNAs) 22547449 3’ spacer SEQ ID NO 3’ nick site chromosome position !414-1419 (Table 36 PEgRNAs) 225475331478-1483 (Table 37 TRAC PEgRNAs) 225475221529-1534 (Table 38 TRAC PEgRNAs) 22547529 WO 2024/238825 PCT/US2024/029746 229 id="p-525"

[0525]In some embodiments, a prime editingsystem further comprising a recombinase described herein or a nucleic acid encoding the recombinase. [0526]In some embodiments, the recombinase is fused or linked to the prime editor. [0527]In some embodiments, the system further comprising a polynucleotide or a nucleic acid encoding the polynucleotide, wherein the polynucleotide comprises (a) a donor sequence and (b) a second recombinase recognition sequence recognized by the recombinase. [0528]In some embodiments, the donor sequence comprises an open reading frame that encodes a polypeptide. [0529]In some embodiments, the donor sequence encodes a chimeric antigen receptor (CAR). [0530]In some embodiments, the recombinase is Bxbl. [0531]In some embodiments, (i) the recombinase recognition sequence comprises SEQ ID NO: 1590, and the second recombinase recognition sequence comprises SEQ ID NO: 1591, or (ii) the recombinase recognition sequence comprises SEQ ID NO: 1591, and the second recombinase recognition sequence comprises SEQ ID NO: 1590. id="p-532"

[0532]Unless otherwise indicated, references to nucleotide positions in human chromosomes are as set forth in human genome assembly consortium Human build 38 (GRCh38), GenBank accession GCF 000001405.3 8.

Prime editing composition comprising recombinases [0533]In some embodiments, the prime editing composition provided herein comprises one or more recombinases, or one or more polynucleotides encoding the recombinases, wherein the PEgRNA of the prime editing composition encode insertion of one or more recombinase recognition sequences (RRSs) in the target gene, e.g., the B2M gene or the TRAC gene. [0534]In some embodiments, the recombinase is a tyrosine recombinase. In some embodiments, the recombinase is a serine recombinase. Examples of tyrosine recombinases include, without limitation, Cre, FLP, R, Lambda, HK101, HK022, andpSAM2.Examples of serine recombinases include, without limitation, Si74, N067, Kp03, PaOl, Nm60, BcelNTa, BcytINTd, SscINTd, SacINTd, Hin, Gin, Tn3,13-six, CinH, ParA, y6, Bxbl, OC31, TP901, TGI, pBTl, R4, pRVI, pFCl, MR11, Al 18, U153, and gp29.Exemplary serine recombinase sequences and corresponding recombinases are provided in Table 32. Amino acid sequences of the recombinases are in Table 33 below ("*" indicates a stop codon): WO 2024/238825 PCT/US2024/029746 230 Table 33.Exemplary serine recombinase sequences Recombinase SEQID NO: Amino acid sequence Bxbl 10001 MRALVVIRLSRVTDATTSPERQLESCQQLCAQRGW DVVGVAEDLDVSGAVDPFDRKRRPNLARWLAFEE QPFDVIVAYRVDRLTRSIRHLQQLVHWAEDHKKLV VSATEAHFDTTTPFAAVVIALMGTVAQMELEAIKE RNRSAAHFNIRAGKYRGSLPPWGYLPTRVDGEWRL VPDPVQRERILEVYHRVVDNHEPLHLVAHDLNRRG VLSPKDYFAQLQGREPQGREWSATALKRSMISEAM LGYATLNGKTVRDDDGAPLVRAEPILTREQLEALR AELVKTSRAKPAVSTPSLLLRVLFCAVCGEPAYKFA GGGRKHPRYRCRSMGFPKHCGNGTVAMAEWDAF CEEQVLDLLGDAERLEKVWVAGSDSAVELAEVNA ELVDLTSLIGSPAYRAGSPQREALDARIAALAARQE ELEGLEARPSGWEWRETGQRFGDWWREQDTAAK NTWLRSMNVRLTFDVRGGLTRTIDFGDLQEYEQHL RLGSVVERLHTGMS Si74 10002 MQPNLRYLACLRLSADSDGSTSIEWQRGVIRHHVSS PHLSGVLVGEAEDTDVSGSLSPFKRPKLGKWLTAK ADEFDVIIAAKMDRLTRRSMHFNELLEWAQQNGKF IVCVEEGFDLSTPQGKMMARMTAVFAEAEWDTIQA RILNGVQTRLENRSWLVGAPPTGYRIKTVEGGKRKI LEIDQDFYPYVEEIFRRIREGQSTHRIARDFNGRSVL TWGDHLRKLKGEEPKGTQWQATIINKFIRSSWVPG LYTYKGEAVLDDQGDPVILPETPLATMDEWTDLVD RIKPAPKPEGATGGSRNSAKSLLSGVAHCGECGAPF TSLMDSGYKRKDGTKVPGHRRYRCSNKFKGGDCK NGSYVRADVLDSWVDQAIRDSIGQEDMYERAGKG PSQARELQETKARLAKLEADYESGKYDGEGQDESY WRMNKNLSAKVAHLAKQEAERANPTFKATGKKYGEVWEAKDQEDRRDFLRTYGVKVFVWGEGADKK WO 2024/238825 PCT/US2024/029746 231 Recombinase SEQID NO: Amino acid sequence DRGYAMNLGDIKTMAEELFPNRDRARFKLVHTHNAPEGYLSKIGIAVGLLKYGHPLEVKLRSPENS* N067 10003 MPESPPRALIVIRLSKVTDATTSPERQLAECRAICEK RGYEVVGVAEDLDVSGAVDPFDRKKRPHLARWLH GEHLDDNGEPVPFEVIVVYRVDRLTRSVRHLQKLV AWADDHKKLVVSATEAHFDTSLPFSAVLIALIGTVA EMELAGISERNASAARHNIQAGRYRGSTPPWGYVP SNDTGEWRLVRDKEQADVINEVARRVIEGEPLQKI AHDLTRRGILTPKDNFAKQRGREIKGREWSVTQLK RSLLSEAMLGHAVSGGAAVRNDDGSPVVRSEPILSR EIYDRVAAELSSRAKRGEPNKRTSSLLLGVISCGNPC LHKQQGHECPEGCSGTCDEPVYKFNGGSHSQFPRY RCRTMTRAYKCGNRTIRADQADAQVERTILALLGS SERLERVWDAGEDHSAELADINDELVDLTSQLGVG AFRAGTPQRAKLDARIASLAERQAQLSSEAVVPAG WKLLPTGELFGDWWSRQDLTARNVWLRSMGVRA RFKRDDKTLYIDLGNLNDLISGLKPGGTAQRVRGG LQAMERNGIQGMVFSDADSEVMPAPAAGYMWIQP VEGVWVYTSEALLAAAAERQALRREKIEDEFAYGP GDFDEDWD* Kp03 10004 MRQVITYLRFSSKPQERGDSIRRQKGLFERWLKDNP DAKVVDEFSDEGASAYHGHHLKGDFGRMLQNIQD GKYLSGQTVLLVESETRLNRQKARNTENLVDLITG KGVDVICLESGKIYTSTNIDDLDTSIQLKIAAHIAHQ QSKEKSIKVSAAWEHRAQLALEGKQQLTKNVPGWI DPDTRKLNEHSSTVVTIFDLLLSGESLHNIARYLQA NNIKSFSRREKANGFSVHSVRTILRSESTVGTLAASK RNDRPAIPNYYEPAIDVATFNKAQEILSKNRVGRAP ASDNPITINLFKGIIRCQCGASVHPTGVKATYQGVY RCNNVPDGRCNVPTIKRKPFDKWMLDNIVGFLERD DGNNTDKRKAEIEYQISLVTSKLKKATTLLLELDDV WO 2024/238825 PCT/US2024/029746 232 Recombinase SEQID NO: Amino acid sequence TELKEQVKELNIQRSNLQSELDELNQRETLSDKPLH HLSEIDLTTKAGRVEAQLILSKFVQSIELQREMIIITL RNGTVIGKSRDLSPVLSQDLMKQVVSSPSPTDIDMF SVITSDEEFRKSGKQVTKRS* PaOl 10005 MPSAFSYVRFSSGKQAKGSSEHRQRAMLGQWLEQ HPSFTLSDLRFEDLGRSGFSGEHLDHGLGQLLAAID SGAIKSGDVILVEAVDRIGRLEPLEMLPLFSRIVKAG VSVITLEDGHVYDRSSVNETSLFLLVAKIQQAHEYS NRLSRRINASYTARREKAKAGLGIKRETPVWLTTD GQLVPHVAPHIAQAFQDYADGLGERRICRKLRESG LEEFSKTNATTVRRWLKNRTAIGYWNDIPDVYPHV VDPALFYQVQQRLDAPKVDRAKPSAHYLTGLVKC AVCGRNYNYKQRKHTDPAMLCTSRARLAGEGCSN SKTYPVIVLDQVRKLTSLPFLQHAMESASSQADPSS QRLAVIDGEIGELSRKISEATKALLVLGFTPEIQESLE QLKTAREALEEERATLLLPQAEKLTTAQLEAFSNGL LDDEPMKLNHVLQTAGYSMVVHPDGSIDVDGKRF VYEGASRKEKVYKLRLIGEDKQWSLPILTPQMATY KSLFMAAVRLPGDPSEEELRRFEEAKHSER* Nm60 10006 MSRPTGLTIDIYLRKSRKDLEEEKKASESGETYDTLE RHRRTLFAVAKKERHNIANIYEEVVSGESVSERPQI QAMLRNLETSHIEGVLVMDLDRLGRGDMLDQGLL DRAFRYSGAKILTPTEVYDPESETWELVFGIKSLVA REELKAITRRMQRGRVASAGEGKSISKVPPYGYLRD ENLRLYPDPETAWVVKKMFEMMRDGHGRIAVAQE LDKLGIKPPNDKRRSWAPSSITAIIKNEVYLGTIIWG QVKYSKRNGKYKKTKLPRSKWTIKENAHEPIVSRE LFEAANRAHTGRWRPSTNATKTLSNPLAGVLKCDV CGFTMLYQPRPNRPNDFIRCTQPTCQAVQKGATLA LVEQHILDGLKQFAQELELQTEVPELDNDKDIAVKK SLVGNKQEEIAQLETQKSKLHDLLERGIYDVDTFLE WO 2024/238825 PCT/US2024/029746 233 Recombinase SEQID NO: Amino acid sequence RQQNLNNRINGLQDDIRNIESEIKKEEVRNSSVLNLL PQLQTVISEYENADTESKNRLLKSVLEKATYLRKKE WTKRDQFIIQLYPKI* BceINTa 10007 MYPYDVPDYAGSYRPESLDVCIYLRKSRKDVEEER RAIEEGSSYNALERHRKRLFAIAKAENHNIIDIFEEV ASGESIQERPQMQQLLRKLEGNEIDGVLVIDLDRLG RGDMLDAGMIDRAFRYSSTKIITPTDVYDPDDESWE LVFGIKSLISRQELKSITKRLQNGRIDSVKEGKHIGK KPPYGYLKDENLRLYPDPEKAWIVKKIFELMCDGK GRQMIAAELDRLGIDPPVTKRGAWDSSTITSIIKNEV YTGVIVWGKFKHKKRNGKYTRHKNPQEKWIMYEN AHEPIISKELFDAANEAHSSRHKPAVITSKKLTNPLA GILKCKLCGYTMLIQTRKDRPHNYLRCNNPACKGK QKQSVFNLVEEKLLYSLQQIVDEYQAQKVEEVEID DSKLISFKEKAIISKEKELKELQAQKGNLHDLLEQGI YTVEIFLERQKNLVERITSIENDIEVLQKEIETEQIKE HNKTEFIPALKTVIESYHKTTNIELKNQLLKTILSTVT YYRHPDWKTNEFEIQVYFKIS* BcytINTd 10008 MYPYDVPDYAGSAVGIYIRVSTQEQASEGHSIESQK KKLASYCEIQGWDDYRFYIEEGISGKNTNRPKLKLL MEHIEKGKINILLVYRLDRLTRSVIDLHKLLNFLQEH GCAFKSATETYDTTTANGRMSMGIVSLLAQWETEN MSERIKLNLEHKVLVEGERVGAIPYGFDLSDDEKLV KNEKSAILLDMVERVENGWSVNRIVNYLNLTNNDR NWSPNGVLRLLRNPALYGATRWNDKIAENTHEGIIS KERFNRLQQILADRSIHHRRDVKGTYIFQGVLRCPV CDQTLSVNRFIKKRKDGTEYCGVLYRCQPCIKQNK YNLAIGEARFLKALNEYMSTVEFQTVEDEVIPKKSE REMLESQLQQIARKREKYQKAWASDLMSDDEFEK LMVETRETYDECKQKLESCEDPIKIDETYLKEIVYM WO 2024/238825 PCT/US2024/029746 234 Recombinase SEQID NO: Amino acid sequence FHQTFNDLESEKQKEFISKFIRTIRYTVKEQQPIRPDKSKTGKGKQKVIITEVEFYQS* SscINTd 10009 MNEKNLEIGAAYIRVSTDDQTELSPDAQLRVILEAA KKDGIIIPQEFVFMEDRGRSGRRADNRPEFQRMISTA RQNPSPFRYLYLWKFSRFARNQEESAFYKGILRKKC GVTIKSVSEPIMEGMFGRLVEMIIEWSDEFYSVNLS GEVLRGMTQKALEHGYQLTPCLGYDAVGHGRPYV INEEQYQIVEFIHRSFFDGKDMTWIAREANRRGYHT RRGNPFDTRAVRIILTNSFYVGLVKWNDVTFQGTH ECRESVTSVFSANQERLNRIHRPRGRRQASSCKHWL SGLLKCSICGASLGYNQTKDLTKRGHAFQCWKYTK GIHPGSCSVSSLKAEAAVLESLQMILETGEVEYTYE QREKHLDDNKLTLIQKSLERLDTKELRIREAYESGID TLDEFKTNKARLQRERDQLMEELEELHSQEEPEDVP GKEILIERIQNVYDLLQSPDVDNDDKGNAVRSIIKKI VYIKESKTFCFYYYV* SacINTd 10010 MKEKVSERKTGAIYIRVSTDKQEELSPDAQLRLLLD YAKKDSIDVPKEYIFQDNGISGRKANKRPAFQNMIA LAKSKEHPIDTIIVWKFSRFARNQEESIVYKSLLKKN NVDVVSVSEPLIDGPFGSLIERIIEWMDEYYSIRLSGE VMRGMTQNAMRGHYQSDAPIGYTSPGDKKPPVINP DTVQIPLMIKDMFLSGSTQLQIARKLNDSGYRTKRG NLWDARGVRYVLENPFYIGKSRWNYTERGRRLKP ADEVIYADGNWEALWDEDTFKEIQKRLALNMRKS KSRDISAAKHWLSGLLICSSCGGTLAFGGAHNMRG FQCWKYSKGFCSESHYISTGPIEKMVLEYLEAVMHS PALSYTVISSSSVDASSKLSDLERQLQKIDAKEKRIK AAYLNEIDTLEEYKANKTALEEERRTVEKEIEELTLS DVKYSKEDLDKKMKQNISDLLRVLRDESADYIQKG NMMRNVVDHIVFNRKNTSLDVFLKLW* WO 2024/238825 PCT/US2024/029746 235 id="p-535"

[0535]In some embodiments, the prime editing composition further comprises a DNA polynucleotide for recombination with the target gene containing the RRS(s) introduced by prime editing. In some embodiments, the DNA polynucleotide comprises (a) a donor sequence and (b) one or more RRSs recognized by the recombinase. In some embodiments, the prime editing composition comprises a recombinase that recognizes a first RRS introduced into the B2M gene by prime editing, and further comprises a DNA polynucleotide comprising (a) a donor sequence and (b) a second RRS recognized by the same recombinase. In some embodiments, the first RRS is an attB sequence, and the second RRS is an attP sequence. [0536]The DNA polynucleotide can be in any configuration that allows for recombination mediated by the recombinase and the RRSs. For example, the DNA polynucleotide can be a plasmid, a circular construct, or a minicircle. [0537]The donor sequence can be any sequence for targeted integration into a target gene, e.g., the B2M gene or the TRAC gene. In some embodiments, the donor sequence comprises an expression cassette comprising a gene or an open reading frame (ORF) driven by one or more promoters. In some embodiments, the donor sequence comprises multiple expression cassettes each driven by one or more promoters. In some embodiments, the donor sequence comprises an expression cassette comprising two or more genes or ORFs driven by a promoter, wherein the two or more genes or ORFs are separated by a sequence encoding a self-cleaving peptide, e.g., a 2A peptide. In some embodiments, the donor sequence does not contain a promoter, but contains a splice acceptor sequence preceding the start codon of the transgene ORF. In some embodiments, a transgene or transgene ORF in the donor sequence encodes a polypeptide. In some embodiments, the polypeptide is involved binding, regulation, or activity of an immune cell, e.g., a T cell, and may function in combination or synergistically with disruption of the target gene, e.g., the B2M gene or the TRAC gene, by prime editing. In some embodiments, the polypeptide is a cellular receptor or a portion thereof, e.g., a receptor that directs specific antigen binding. For example, the polypeptide may be a chimeric antigen receptor (CAR), a T cell receptor (TCR), a B cell receptor, a NK cell receptor, or a functional portion thereof. Exemplary CARs include a CD 19 CAR, CDCAR, BAFF CAR, B7H2 CAR, GD2 CAR, MSLN CAR, HER2 CAR, EGFR CAR, CDCAR, BCMA CAR, or other CARs that are therapeutically relevant. Exemplary TCRs include NY-ESO-1, AHNAK(S2580F), ERBB2(H473Y), HPV-E6, MAGE-A3/A6, E7, HA- 1, or other TCRs that are therapeutically relevant. Exemplary NK cell receptors CD 16, WO 2024/238825 PCT/US2024/029746 236 NKG2D, DNAM1, NKp46, NKp44, NKp30, LFAI, CD27, or other NK cell receptors that are therapeutically relevant. In some embodiments, the polypeptide is a cellular receptor or a functional portion thereof, e.g., a receptor that modulates immune cell activity. In some embodiments, the polypeptide is a cytokine receptor, e.g., IL2RA, IL2RB, IL7R, IL12R, IL18R, or IFNGR, or a functional portion thereof. In some embodiments, the polypeptide is a chemokine receptor, e.g., CCR2, CCR4, CCR5, CCR6, CXCR1, CXCR2, CXCR3, CXCR4, or a functional portion thereof. In some embodiments, the polypeptide is an immune checkpoint protein, e.g., PD1, CTLA4, CISH, TIGIT, TIM3, or LAG3. In some embodiments, the polypeptide is a cytokine, e.g., IL2, IL15, IL7, TNF, or IFNG. In some embodiments, the polypetide is a chemokine, e.g., CCL2, CCL5, CCL17, CCL20, CCL22, CXCL2, CXCL5, CXCL6, CXCL9, CXCL10, CXCL11, or CXCL12. In some embodiments, the polypeptide is a dominant-negative mutant of a protein involved in immune cell signaling, e.g. T cell signaling. For example, the polypeptide may be a dominant negative mutant of TGFBR, IL10R, IL35R, FOXP3, or STATS. In some embodiments, the polypeptide is a transcription factor involved in immune cell signaling, e.g. T cell signaling. For example, the polypeptide may be BLIMP1, IL2RA, IL2RB, BCL6, ID2, C-MYB, IL6R, TCF1, EOMES, STAT1, STAT3, STAT4, STATS, TBET, NFkB, GATA-3, IRF-4. In some embodiments, the polypeptide may be a signaling protein, e.g., LCK, SYK, or ZAP70. In some embodiments, the polypeptide is a reporter protein, e.g., a fluorescence protein. In some embodiments, the polypeptide is an epitope tag. In some embodiments, the polypeptide is a "suicide protein " or a "kill switch " that allows for specific elimination of cells containing the polypeptide, e.g., for specific elimination of edited cells after administration. For example, the polypeptide may be iCasp9, HSV-TK, or an epitope for antibody-mediated elimination (e.g., CD20 for rituximab-mediated elimination). [0538]The recombinase (or polynucleotides encoding the recombinase) and/or the DNA polynucleotide may be provided to a cell containing the target gene, e.g., the B2M gene or the TRAC gene, simultaneously or sequentially. In some embodiments, the recombinase or polynucleotides encoding the recombinase and/or the DNA polynucleotide may be provided 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days after contacting the cell with a prime editor (or polynucleotide encoding the prime editor) and the PEgRNAs. In some embodiments, the recombinase or polynucleotides encoding the recombinase and/or the DNA polynucleotide may be provided at the same time a prime editor (or polynucleotide encoding the prime editor) and the PEgRNAs. In some embodiments, the recombinase is fused to, or connected WO 2024/238825 PCT/US2024/029746 237 to via a linker, e.g., a peptide linker, the prime editor, e.g., a prime editor fusion protein. The prime editor or any component thereof and the recombinase, can be encoded by DNA vectors (e.g. AAV vectors) or mRNAs.Pharmaceutical eampositiaus [0539]Disclosed herein are pharmaceutical compositions comprising any of the prime editing composition components, for example, prime editors, fusion proteins, polynucleotides encoding prime editor polypeptides, PEgRNAs, ngRNAs, and/or prime editing complexes described herein. Also disclosed herein are pharmaceutical compositions comprising the edited cells described herein. The pharmaceutical compositions of the present disclosure can be used to treat a disease, disorder, or a condition. [0540]The term "pharmaceutical composition ", as used herein, refers to a composition formulated for pharmaceutical use. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises additional agents, e.g., for specific delivery, increasing half-life, or other therapeutic compounds. [0541]In some embodiments, a pharmaceutically-acceptable carrier comprises any vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g, lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the compound from one site (e.g., the delivery site) of the body, to another site (e.g, organ, tissue or portion of the body). A pharmaceutically acceptable carrier is "acceptable " in the sense of being compatible with the other ingredients of the formulation and not injurious to the tissue of the subject (e.g, physiologically compatible, sterile, physiologic pH, etc.) [0542]Formulations of the pharmaceutical compositions described herein can be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient(s) into association with an excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, shaping and/or packaging the product into a desired single- or multi-dose unit. Pharmaceutical formulations can additionally comprise a pharmaceutically acceptable excipient, which, as used herein, includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired.

WO 2024/238825 PCT/US2024/029746 238 Methods of Editing [0543] The methods and compositions disclosed herein can be used to edit a target gene of interest by prime editing.[0544] In some embodiments, the prime editing method comprises contacting a target gene, e.g, a B2M gene, with a PEgRNA and a prime editor (PE) polypeptide described herein. In some embodiments, the target gene is double stranded, and comprises two strands of DNA complementary to each other. In some embodiments, the contacting with a PEgRNA and the contacting with a prime editor are performed sequentially. In some embodiments, the contacting with a prime editor is performed after the contacting with a PEgRNA. In some embodiments, the contacting with a PEgRNA is performed after the contacting with a prime editor. In some embodiments, the contacting with a PEgRNA, and the contacting with a prime editor are performed simultaneously. In some embodiments, the PEgRNA and the prime editor are associated in a complex prior to contacting a target gene.[0545] In some embodiments, contacting the target gene with the prime editing composition results in binding of the PEgRNA to a target strand of the target gene, e.g., a B2M gene. In some embodiments, contacting the target gene with the prime editing composition results in binding of the PEgRNA to a search target sequence on the target strand of the target gene upon contacting with the PEgRNA. In some embodiments, contacting the target gene with the prime editing composition results in binding of a spacer sequence of the PEgRNA to a search target sequence with the search target sequence on the target strand of the target gene upon said contacting of the PEgRNA.[0546] In some embodiments, contacting the target gene with the prime editing composition results in binding of the prime editor to the target gene, e.g., the target B2M gene, upon the contacting of the PE composition with the target gene. In some embodiments, the DNA binding domain of the PE associates with the PEgRNA. In some embodiments, the PE binds the target gene, e.g., a B2M gene, directed by the PEgRNA. Accordingly, in some embodiments, the contacting of the target gene result in binding of a DNA binding domain of a prime editor of the target B2M gene directed by the PEgRNA.[0547] In some embodiments, contacting the target gene with the prime editing composition results in a nick in an edit strand of the target gene, e.g., a B2M gene by the prime editor upon contacting with the target gene, thereby generating a nicked on the edit strand of the target gene. In some embodiments, contacting the target gene with the prime editing composition results in a single-stranded DNA comprising a free 3’ end at the nick site of the WO 2024/238825 PCT/US2024/029746 239 edit strand of the target gene. In some embodiments, contacting the target gene with the prime editing composition results in a nick in the edit strand of the target gene by a DNA binding domain of the prime editor, thereby generating a single-stranded DNA comprising a free 3' end at the nick site. In some embodiments, the DNA binding domain of the prime editor is a Cas domain. In some embodiments, the DNA binding domain of the prime editor is a Cas9. In some embodiments, the DNA binding domain of the prime editor is a Casnickase. [0548]In some embodiments, contacting the target gene with the prime editing composition results in hybridization of the PEgRNA with the 3' end of the nicked single-stranded DNA, thereby priming DNA polymerization by a DNA polymerase domain of the prime editor. In some embodiments, the free 3' end of the single-stranded DNA generated at the nick site hybridizes to a primer binding site sequence (PBS) of the contacted PEgRNA, thereby priming DNA polymerization. In some embodiments, the DNA polymerization is reverse transcription catalyzed by a reverse transcriptase domain of the prime editor. In some embodiments, the method comprises contacting the target gene with a DNA polymerase, e.g., a reverse transcriptase, as a part of a prime editor fusion protein or prime editing complex (in cis), or as a separate protein (in trans). [0549]In some embodiments, contacting the target gene with the prime editing composition generates an edited single stranded DNA that is coded by the editing template of the PEgRNA by DNA polymerase mediated polymerization from the 3' free end of the single- stranded DNA at the nick site. In some embodiments, the editing template of the PEgRNA comprises one or more intended nucleotide edits compared to endogenous sequence of the target gene, e.g., a B2M gene. In some embodiments, the intended nucleotide edits are incorporated in the target gene, by excision of the 5’ single stranded DNA of the edit strand of the target gene generated at the nick site and DNA repair. In some embodiments, the intended nucleotide edits are incorporated in the target gene by excision of the editing target sequence and DNA repair. In some embodiments, excision of the 5’ single stranded DNA of the edit strand generated at the nick site is by a flap endonuclease. In some embodiments, the flap nuclease is FEN 1. In some embodiments, the method further comprises contacting the target gene with a flap endonuclease. In some embodiments, the flap endonuclease is provided as a part of a prime editor fusion protein. In some embodiments, the flap endonuclease is provided in trans.

WO 2024/238825 PCT/US2024/029746 240 id="p-550"

[0550]In some embodiments, contacting the target gene with the prime editing composition generates a mismatched heteroduplex comprising the edit strand of the target gene that comprises the edited single stranded DNA, and the unedited target strand of the target gene. Without being bound by theory, the endogenous DNA repair and replication may resolve the mismatched edited DNA to incorporate the nucleotide change(s) to form the desired edited target gene (e.g., edited B2M gene). [0551]In some embodiments, the method further comprises contacting the target gene, e.g., a B2M gene, with a nick guide (ngRNA) disclosed herein. In some embodiments, the ngRNA comprises a spacer that binds a second search target sequence on the edit strand of the target gene. In some embodiments, the contacted ngRNA directs the PE to introduce a nick in the target strand of the target gene. In some embodiments, the nick on the target strand (non-edit strand) results in endogenous DNA repair machinery to use the edit strand to repair the non- edit strand, thereby incorporating the intended nucleotide edit in both strand of the target gene and modifying the target gene. In some embodiments, the ngRNA comprises a spacer sequence that is complementary to, and may hybridize with, the second search target sequence on the edit strand only after the intended nucleotide edit(s) are incorporated in the edit strand of the target gene. [0552]In some embodiments, the target gene is contacted by the ngRNA, the PEgRNA, and the PE simultaneously. In some embodiments, the ngRNA, the PEgRNA, and the PE form a complex when they contact the target gene. In some embodiments, the target gene is contacted with the ngRNA, the PEgRNA, and the prime editor sequentially. In some embodiments, the target gene is contacted with the ngRNA and/or the PEgRNA after contacting the target gene with the PE. In some embodiments, the target gene is contacted with the ngRNA and/or the PEgRNA before contacting the target gene with the prime editor. [0553]In some embodiments, the target gene, e.g., a B2M gene, is in a cell. Accordingly, also provided herein are methods of modifying or editing a cell, such as a human cell, a human primary cell, a human iPSC-derived cell, and/or a human T-cell or a precursor thereof, or a progenitor thereof. [0554]In some embodiments, the prime editing method comprises introducing a PEgRNA, a prime editor, and/or a ngRNA into the cell that has the target gene. In some embodiments, the prime editing method comprises introducing into the cell that has the target gene with a prime editing composition comprising a PEgRNA, a prime editor polypeptide, and/or a ngRNA. In some embodiments, the PEgRNA, the prime editor polypeptide, and/or the ngRNA form a WO 2024/238825 PCT/US2024/029746 241 complex prior to the introduction into the cell. In some embodiments, the PEgRNA, the prime editor polypeptide, and/or the ngRNA form a complex after the introduction into the cell. The prime editors, PEgRNA and/or ngRNAs, and prime editing complexes may be introduced into the cell by any delivery approaches described herein or any delivery approach known in the art, including ribonucleoprotein (RNPs), lipid nanoparticles (LNPs), viral vectors, non-viral vectors, mRNA delivery, and physical techniques such as cell membrane disruption by a microfluidics device. The prime editors, PEgRNA and/or ngRNAs, and prime editing complexes may be introduced into the cell simultaneously or sequentially. [0555]In some embodiments, the prime editing method comprises introducing into the cell a PEgRNA or a polynucleotide encoding the PEgRNA, a prime editor polynucleotide encoding a prime editor polypeptide, and optionally an ngRNA or a polynucleotide encoding the ngRNA. In some embodiments, the method comprises introducing the PEgRNA or the polynucleotide encoding the PEgRNA, the polynucleotide encoding the prime editor polypeptide, and/or the ngRNA or the polynucleotide encoding the ngRNA into the cell simultaneously. In some embodiments, the method comprises introducing the PEgRNA or the polynucleotide encoding the PEgRNA, the polynucleotide encoding the prime editor polypeptide, and/or the ngRNA or the polynucleotide encoding the ngRNA into the cell sequentially. In some embodiments, the method comprises introducing the polynucleotide encoding the prime editor polypeptide into the cell before introduction of the PEgRNA or the polynucleotide encoding the PEgRNA and/or the ngRNA or the polynucleotide encoding the ngRNA. In some embodiments, the polynucleotide encoding the prime editor polypeptide is introduced into and expressed in the cell before introduction of the PEgRNA or the polynucleotide encoding the PEgRNA and/or the ngRNA or the polynucleotide encoding the ngRNA into the cell. In some embodiments, the polynucleotide encoding the prime editor polypeptide is introduced into the cell after the PEgRNA or the polynucleotide encoding the PEgRNA and/or the ngRNA or the polynucleotide encoding the ngRNA are introduced into the cell. The polynucleotide encoding the prime editor polypeptide, the PEgRNA or the polynucleotide encoding the PEgRNA, and/or the ngRNA or the polynucleotide encoding the ngRNA, may be introduced into the cell by any delivery approaches described herein or any delivery approach known in the art, for example, by RNPs, LNPs, viral vectors, non-viral vectors, mRNA delivery, and physical delivery. In some embodiments, the polynucleotide is a DNA polynucleotide. In some embodiments, the polynucleotide is a RNA polynucleotide, e.g., mRNA polynucleotide.

WO 2024/238825 PCT/US2024/029746 242 id="p-556"

[0556]In some embodiments, the polynucleotide encoding the prime editor polypeptide, the polynucleotide encoding the PEgRNA, and/or the polynucleotide encoding the ngRNA integrate into the genome of the cell after being introduced into the cell. In some embodiments, the polynucleotide encoding the prime editor polypeptide, the polynucleotide encoding the PEgRNA, and/or the polynucleotide encoding the ngRNA are introduced into the cell for transient expression. Accordingly, also provided herein are cells modified by prime editing. [0557]The engineered cells provided herein comprise edits in the target genes, e.g., the B2M gene. In some embodiments, provided herein are engineered cell or a population of engineered cells comprising a B2M gene comprising an insertion, a deletion, a substitution, or a combination thereof compared to a wildtype B2M gene at a chromosomal location corresponding to coding sequence position c.51, c.54, or c.50 of a wildtype B2M gene. [0558]In some embodiments, provided herein are engineered cell or a population of engineered cells comprising a B2M gene comprising an insertion, a deletion, a substitution, or a combination thereof compared to a wildtype B2M gene at a chromosomal location corresponding to coding sequence position c.54, c.60, or c.66 of a wildtype B2M gene, optionally wherein the B2M gene comprises an insertion, a deletion, a substitution, or a combination thereof at a chromosomal location corresponding to coding sequence position c.58 of a wildtype B2M gene. [0559]In some embodiments, provided herein are engineered cell or a population of engineered cells comprising a B2M gene comprising an insertion, a deletion, a substitution, or a combination thereof compared to a wildtype B2M gene at a chromosomal location corresponding to coding sequence position c.21 or c.3 of a wildtype B2M gene, optionally wherein the B2M gene comprises an insertion, a deletion, a substitution, or a combination thereof at a chromosomal location corresponding to coding sequence position c. 17 of a wildtype B2M gene. [0560]In some embodiments, provided herein are engineered cell or a population of engineered cells comprising a B2M gene comprising an insertion, a deletion, a substitution, or a combination thereof compared to a wildtype B2M gene at a chromosomal location corresponding to coding sequence position c.21, c.15 or c.3 of a wildtype B2M gene, optionally wherein the B2M gene comprises an insertion, a deletion, a substitution, or a combination thereof at a chromosomal location corresponding to coding sequence position c.11 of a wildtype B2M gene..

WO 2024/238825 PCT/US2024/029746 243 id="p-561"

[0561]In some embodiments, the cell or the population of cells comprise a c.51delC deletion in the B2M gene relative to a wildtype B2M gene. [0562]In some embodiments, the cell or the population of cells comprise a c.50insG insertion in the B2M gene relative to a wildtype B2M gene. [0563]In some embodiments, the cell or the population of cells comprise a c.54_55insCC insertion in the B2M gene relative to a wildtype B2M gene. [0564]In some embodiments, the cell or the population of cells comprise a c.54_55insTAAG insertion in the B2M gene relative to a wildtype B2M gene. [0565]In some embodiments, the cell or the population of cells comprise a c.54_55insTAATAA insertion in the B2M gene relative to a wildtype B2M gene. [0566]In some embodiments, the cell or the population of cells comprise a c.66_67insCC insertion in the B2M gene relative to a wildtype B2M gene, optionally wherein the cell or the population of cells further comprise a c.58G>C substitution in the B2M gene relative to a wildtype B2M gene. [0567]In some embodiments, the cell or the population of cells comprise a c.66_67insTAAG insertion in the B2M gene relative to a wildtype B2M gene, optionally wherein the cell or the population of cells further comprise a c.58G>C substitution in the B2M gene relative to a wildtype B2M gene. [0568]In some embodiments, the cell or the population of cells comprise a c.66_67insTAATAA insertion in the B2M gene relative to a wildtype B2M gene, optionally wherein the cell or the population of cells further comprise a c.58G>C substitution in the B2M gene relative to a wildtype B2M gene. [0569]In some embodiments, the cell or the population of cells comprise a c.60_65deletion and a TAATAG insertion (c.60_64delinsTAATAG) in the B2M gene relative to a wildtype B2M gene, optionally wherein the cell or the population of cells further comprise a c.58G>C substitution in the B2M gene relative to a wildtype B2M gene. [0570]In some embodiments, the cell or the population of cells comprise a c.21_22insCC insertion in the B2M gene relative to a wildtype B2M gene. [0571]In some embodiments, the cell or the population of cells comprise a c.21_22insTAAG insertion in the B2M gene relative to a wildtype B2M gene. [0572]In some embodiments, the cell or the population of cells comprise a c.21_22insTAATAA insertion in the B2M gene relative to a wildtype B2M gene.

WO 2024/238825 PCT/US2024/029746 244 id="p-573"

[0573]In some embodiments, the cell or the population comprise a c.3_4insCC insertion in the B2M gene relative to a wildtype B2M gene, optionally wherein the cell or the population of cells further comprise a C.17OG substitution or a c.l lOG substitution in the B2M gene relative to a wildtype B2M gene. [0574]In some embodiments, the cell or the population of cells comprise a c.3_4insTAAG insertion in the B2M gene relative to a wildtype B2M gene, optionally wherein the cell or the population of cells further comprise a C.17OG substitution or a c.l lOG substitution in the B2M gene relative to a wildtype B2M gene. [0575]In some embodiments, the cell or the population of cells comprise a c.3_4insTAATAA insertion in the B2M gene relative to a wildtype B2M gene, optionally wherein the cell or the population of cells further comprise a C.17OG substitution or a c.l lOG substitution in the B2M gene relative to a wildtype B2M gene. [0576]In some embodiments, the cell or the population of cells comprise a c.3_8deletion and a TAATGA insertion (c.3_8delinsTAATGA) in the B2M gene relative to a wildtype B2M gene, optionally wherein the cell or the population of cells further comprise a C.17OG substitution or a c.l lOG substitution in the B2M gene relative to a wildtype B2M gene. [0577]In some embodiments, the cell or the population of cells comprise a c.l5_16insCC insertion in the B2M gene relative to a wildtype B2M gene. [0578]In some embodiments, the cell or the population of cells comprise a c. 15_16insTAAG insertion in the B2M gene relative to a wildtype B2M gene. [0579]In some embodiments, the cell or the population of cells comprise a c.l5_16insTAATAA insertion in the B2M gene relative to a wildtype B2M gene. id="p-580"

[0580]In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a non-human primate cell, bovine cell, porcine cell, rodent or mouse cell. In some embodiments, the cell is a human cell. [0581]In some embodiments, the cell is a T-cell, e.g., a primary T cell, e.g., an inflammatory T cell, a T helper cell, a cytotoxic T-cell, a CD4+ T-cell, a CD8+ T cell, a memory T cell, a regulatory T cell, a natural killer T cell, a mucosal associated invariant T cell, a y8 T cell, an alpha beta T cell, a naive T cell, or an effector T cell), formed elements of the blood (e.g., lymphocytes, bone marrow cells), precursors or progenitors thereof, differentiated or de- differentiated cell thereof, and stem cells. In some embodiments, a cell is a naive T cell (e.g., a naive CD8+ T cell). In some embodiments, the cell is a transformed T cell. In some WO 2024/238825 PCT/US2024/029746 245 embodiments, the cell is an immune cell (e.g., a primary immune cell) or a progenitor or a precursor thereof). In some embodiments, the cell is a T-cell, or a progenitor or a precursor thereof. In some embodiments, the cell is a human T cell, or a progenitor or a precursor thereof. In some embodiments, the cell is a T helper cell (e.g., Thl cell, Th2 cell, Th9 cell, Thl7 cell, Th22 cell, and Tfh (follicular helper) cell). In some embodiments, the cell is a cytotoxic T cell. In some embodiments, the cell is a CD8+ T cell. In some embodiments, the cell is a CD4+ T cell. In some embodiments, the cell is a memory T cell (e.g., (e.g., central memory T cell (TCM), stem memory T cell (TSCM), effector memory T cell, Tissue resident memory T cell). In some embodiments, the cell is an effector memory T cell (e.g., TEM cells and TEMRA (CD45RA+) cells). In some embodiments, the cell is a regulatory T cell. In some embodiments, the cell is a natural killer T cell. In some embodiments, the cell is a Mucosal associated invariant T cell. In some embodiments, the cell is a y8 T cell. In some embodiments, the cell is an effector T cell In some embodiments, the cell is a thymocyte. In some embodiments, the cell is a lymphoid cell. In some embodiments, the cell is a common lymphoid progenitor cells. In some embodiments, the cell is an early thymic progenitor cell. In some embodiments, the cell is a CD3+ cell. In some embodiments, the cell is a tumor infiltrating lymphocyte. In some embodiments, the cell is a myeloid cell. In some embodiments, the cell is a plasma cell. In some embodiments, the cell is an activated T cell. [0582]In some embodiments, the cell is a stem cell (e.g., adult stem cell, embryonic stem cell, non-embryonic stem cell), cord blood stem cell, progenitor cell, bone marrow stem cell, induced pluripotent stem cell, totipotent stem cell, a CD34+ cell, or hematopoietic stem cell). In some embodiments, the cell is a pluripotent cell (e.g., a pluripotent stem cell). In some embodiments, the cell (e.g., a stem cell) is an embryonic stem cell, tissue-specific stem cell, mesenchymal stem cell, or an induced pluripotent stem cell. In some embodiments, the cell is an induced pluripotent stem cell (iPSC). In some embodiments, the cell is a hematopoietic stem cell. In some embodiments, the cell is a hematopoietic stem and progenitor cell. In some embodiments, the cell is a multipotent progenitor cell. In some embodiments, the cell is a T- cell progenitor. In some embodiments, the cell is a T-cell precursor. In some embodiments, the cell is an embryonic stem cell (ESC). In some embodiments, the cell is a human stem cell. In some embodiments, the cell is a human pluripotent stem cell. In some embodiments, the cell is a non-embryonic stem cell. In some embodiments, the cell is an induced human pluripotent stem cell. In some embodiments, the cell is a human stem cell. In some WO 2024/238825 PCT/US2024/029746 246 embodiments, the cell is a human embryonic stem cell. In some embodiments, the cell is a human T-cell progenitor. In some embodiments, the cell is a human T-cell precursor. [0583]In some embodiments, a cell is not isolated from an organism but forms part of a tissue or organ of an organism, e.g., a mammal. [0584]In some embodiments, the cell is a differentiated cell. In some embodiments, the cell is differentiated from an induced pluripotent stem cell. In some embodiments, the cell is a T- cell e.g., a primary T cell, e.g., an inflammatory T cell, a T helper cell, a cytotoxic T-cell, a CD4+ T-cell, a CD8+ T cell, a memory T cell, a regulatory T cell, a natural killer T cell, a mucosal associated invariant T cell, a y8 T cell, an alpha beta T cell, a naive T cell, or an effector T cell differentiated from an iPSC, ESC, a T-cell precursor, or a T-cell progenitor. [0585]In some embodiments, the cell is a differentiated human cell. in some embodiments, the cell is differentiated from an induced human pluripotent stem cell. In some embodiments, the cell edited by prime editing can be differentiated into, or give rise to recovery of a population of cells, e.g., a T-cell e.g., a primary T cell, e.g., an inflammatory T cell, a T helper cell, a cytotoxic T-cell, a CD4+ T-cell, a CD8+ T cell, a memory T cell, a regulatory T cell, a natural killer T cell, a mucosal associated invariant T cell, a y8 T cell, an alpha beta T cell, a naive T cell, or an effector T cell. In some embodiments, the cell is in a subject, e.g., a human subject. In some embodiments, the cell is obtained from a subject prior to editing. For example, in some embodiments, the cell is obtained from a patient having a cancer, a microbial infection, or an autoimmune disorder. Prior to editing by the methods and compositions disclosed herein the cell can be obtained from a subject through a variety of non-limiting methods. T cells can be obtained from a number of non- limiting sources, for example, peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. Methods of collecting blood cells, isolating and enriching T cells, and expanding them ex vivo may be by methods known in the art. In some embodiments, the cell can be obtained from a cell bank, a blood bank, cell culture, or any number of T cell lines available, and known to those skilled in the art. Cells may also be obtained from a tissue biopsy, surgery, blood, plasma, serum, or other biological fluid. In some embodiments, the cell can be obtained prior to editing from one or more healthy donors, from a patient having a cancer, a microbial infection, a graft versus host infection, or an autoimmune disorder. [0586]For example, a cell can be obtained (i.e., isolated or purified) prior to editing from a whole blood sample by lysing red blood cells and removing peripheral mononuclear blood WO 2024/238825 PCT/US2024/029746 247 cells by centrifugation. The cell can be further isolated or purified using a selective purification method that isolates the cell based on cell-specific markers such as CD25, CD3, CD4, CDS, CD28, CD45RA, or CD45RO (e.g., by flow cytomotery). In one embodiment, CD4+ is used as a marker to select T cells. In one embodiment, CD8+ is used as a marker to select T cells. In one embodiment, CD4+ and CD8+ are used as a marker to select regulatory T cells. [0587]In some embodiments, the target gene edited by prime editing is in a chromosome of the cell. In some embodiments, the intended nucleotide edits incorporate in the chromosome of the cell and are inheritable by progeny cells. In some embodiments, the intended nucleotide edits introduced to the cell by the prime editing compositions and methods are such that the cell and progeny of the cell also include the intended nucleotide edits. In some embodiments, the cell is autologous, allogeneic, or xenogeneic to a subject. In some embodiments, the cell is from or derived from a subject. In some embodiments, the cell is from or derived from a human subject. In some embodiments, the cell is introduced back into the subject, e.g., a human subject, after incorporation of the intended nucleotide edits by prime editing. [0588]In some embodiments, the method provided herein comprises introducing the prime editor polypeptide or the polynucleotide encoding the prime editor polypeptide, the PEgRNA or the polynucleotide encoding the PEgRNA, and/or the ngRNA or the polynucleotide encoding the ngRNA into a plurality or a population of cells that comprise the target gene. In some embodiments, the population of cells is of the same cell type. In some embodiments, the population of cells is of the same tissue or organ. In some embodiments, the population of cells is heterogeneous. In some embodiments, the population of cells is homogeneous. In some embodiments, the population of cells is from a single tissue or organ, and the cells are heterogeneous. In some embodiments, the introduction into the population of cells is ex vivo. In some embodiments, the introduction into the population of cells is in vivo, e.g, into a human subject. [0589]In some embodiments, the target gene is in a genome of each cell of the population. In some embodiments, introduction of the prime editor polypeptide or the polynucleotide encoding the prime editor polypeptide, the PEgRNA or the polynucleotide encoding the PEgRNA, and/or the ngRNA or the polynucleotide encoding the ngRNA results in incorporation of one or more intended nucleotide edits in the target gene in at least one of the cells in the population of cells. In some embodiments, introduction of the prime editor WO 2024/238825 PCT/US2024/029746 248 polypeptide or the polynucleotide encoding the prime editor polypeptide, the PEgRNA or the polynucleotide encoding the PEgRNA, and/or the ngRNA or the polynucleotide encoding the ngRNA results in incorporation of the one or more intended nucleotide edits in the target gene in a plurality of the population of cells. In some embodiments, introduction of the prime editor polypeptide or the polynucleotide encoding the prime editor polypeptide, the PEgRNA or the polynucleotide encoding the PEgRNA, and/or the ngRNA or the polynucleotide encoding the ngRNA results in incorporation of the one or more intended nucleotide edits in the target gene in each cell of the population of cells. In some embodiments, introduction of the prime editor polypeptide or the polynucleotide encoding the prime editor polypeptide, the PEgRNA or the polynucleotide encoding the PEgRNA, and/or the ngRNA or the polynucleotide encoding the ngRNA results in incorporation of the one or more intended nucleotide edits in the target gene in sufficient number of cells such that the disease or disorder is treated, prevented or ameliorated. [0590]In some embodiments, editing efficiency of the prime editing compositions and method described herein can be measured by calculating the percentage of edited target genes in a population of cells introduced with the prime editing composition. In some embodiments, the editing efficiency is determined after 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, hours, 48 hours, 3 days, 4 days, 5 days, 7 days, 10 days, or 14 days of exposing a target gene (e.g., a B2M gene within the genome of a cell) to a prime editing composition. In some embodiments, editing efficiency of the prime editing compositions and method described herein can be measured by calculating the percentage of edited target genes in a population of cells introduced with the prime editing composition. In some embodiments, the editing efficiency is determined after 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 weeks of exposing a target gene (e.g., a B2M gene within the genome of a cell) to a prime editing composition. In some embodiments, the population of cells introduced with the prime editing composition is ex vivo. In some embodiments, the population of cells introduced with the prime editing composition is in vitro. In some embodiments, the population of cells introduced with the prime editing composition is in vivo. In some embodiments, the prime editing methods di sclosed herein have an editing efficiency of at least about 1%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99% relative to a suitable control. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least 25% relative to a WO 2024/238825 PCT/US2024/029746 249 suitable control. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least 35% relative to a suitable control, prime editing method disclosed herein has an editing efficiency of at least 30% relative to a suitable control. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least 45% relative to a suitable control. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least 50% relative to a suitable control . In some embodiments, editing efficiency of prime the prime editing compositions and method described herein can be measured by calculating the percentage of edited target genes in a population of cells after in vivo engraftment of the edited cells. In some embodiments, the editing efficiency is determined after 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 weeks of engraftment. In some embodiments, the editing efficiency is determined after 8 or 16 weeks of engraftment. In some embodiments, prime editing is able to maintain in edited cells at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or more than 95% of editing efficiency after 8 or weeks post engraftment. [0591]In some embodiments, the methods disclosed herein have an editing efficiency of at least about 1%, at least about 5%, at least about 7.5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% of editing a primary cell (as measured in a population of primary cells) relative to a suitable control. [0592]In some embodiments, the methods disclosed herein have an editing efficiency of at least about 5%, at least about 7.5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% of editing a target cell (e.g., a human primary cell, human IPSC, or human T cell or a progenitor or a precursor thereof) relative to a corresponding control target cell. In some embodiments, the target cell is a human cell (e.g., a human primary cell, human iPSC, or human T cell, or a progenitor or a precursor thereof). [0593]In some embodiments, the prime editing compositions provided herein are capable of incorporated one or more intended nucleotide edits without generating a significant proportion of indels. The term "indel(s)", as used herein, refers to the insertion or deletion of WO 2024/238825 PCT/US2024/029746 250 a nucleotide base within a polynucleotide, for example, a target gene. Such insertions or deletions can lead to frame shift mutations within a coding region of a gene. Indel frequency of editing can be calculated by methods known in the art. In some embodiments, indel frequency can be calculated based on sequence alignment such as the CRISPResso algorithm as described in Clement et al., Nat. Biotechnol. 37(3): 224-226 (2019), which is incorporated herein in its entirety. In some embodiments, the prime editing methods disclosed herein can have an indel frequency of less than 30%, less than 20%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1.5%, or less than 1%. [0594]In some embodiments, any number of indels is determined after at least 1 hour, at least 2 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least hours, at least 3 days, at least 4 days, at least 5 days, at least 7 days, at least 10 days, or at least 14 days of exposing a target gene (e.g., a B2M gene within the genome of a cell) to a prime editing composition. [0595]In some embodiments, the prime editing compositions provided herein are capable of incorporated one or more intended nucleotide edits efficiently without generating a significant proportion of indels. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 1% and an indel frequency of less than 1% in a target cell, e.g, a human T cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 1% and an indel frequency of less than 0.5% in a target cell, e.g, a human T cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 1% and an indel frequency of less than 0.1% in a target cell, e.g, a human T cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 5% and an indel frequency of less than 1% in a target cell, e.g, a human T cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 5% and an indel frequency of less than 0.5% in a target cell, e.g, a human T cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 5% and an indel frequency of less than 0.1% in a target cell, e.g, a human T cell. [0596]In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 7.5% and an indel frequency of less than 1% in a target cell, e.g, a human T-cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 7.5% and an indel frequency of less than 0.5% in a target WO 2024/238825 PCT/US2024/029746 251 cell, e.g., a human human T-cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 7.5% and an indel frequency of less than 0.1% in a target cell, e.g., a human T-cell. [0597]In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 10% and an indel frequency of less than 1% in a target cell, e.g., a human T-cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 10% and an indel frequency of less than 0.5% in a target cell, e.g., a human T-cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 10% and an indel frequency of less than 0.1% in a target cell, e.g., a human T-cell. [0598]In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 15% and an indel frequency of less than 1% in a target cell, e.g., a human T-cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 15% and an indel frequency of less than 0.5% in a target cell, e.g., a human T-cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 15% and an indel frequency of less than 0.1% in a target cell, e.g., a human T-cell. [0599]In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 20% and an indel frequency of less than 1% in a target cell, e.g., a human T-cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 20% and an indel frequency of less than 0.5% in a target cell, e.g., a human T-cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 20% and an indel frequency of less than 0.1% in a target cell, e.g., a human T-cell. [0600]In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 30% and an indel frequency of less than 1% in a target cell, e.g., a human T-cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 30% and an indel frequency of less than 0.5% in a target cell, e.g., a human T-cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 30% and an indel frequency of less than 0.1% in a target cell, e.g., a human T-cell. [0601]In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 40% and an indel frequency of less than 1% in a target cell, e.g., a WO 2024/238825 PCT/US2024/029746 252 human T-cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 40% and an indel frequency of less than 0.5% in a target cell, e.g., a human T-cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 40% and an indel frequency of less than 0.1% in a target cell, e.g., a human T-cell. [0602]In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 50% and an indel frequency of less than 1% in a target cell, e.g., a human T-cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 50% and an indel frequency of less than 0.5% in a target cell, e.g., a human T-cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 50% and an indel frequency of less than 0.1% in a target cell, e.g., a human T-cell. [0603]In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 60% and an indel frequency of less than 1% in a target cell, e.g., a human T-cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 60% and an indel frequency of less than 0.5% in a target cell, e.g., a human T-cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 60% and an indel frequency of less than 0.1% in a target cell, e.g., a human T-cell. [0604]In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 70% and an indel frequency of less than 1% in a target cell, e.g., a human T-cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 70% and an indel frequency of less than 0.5% in a target cell, e.g., a human T-cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 70% and an indel frequency of less than 0.1% in a target cell, e.g., a human T-cell. [0605]In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 80% and an indel frequency of less than 1% in a target cell, e.g., a human T-cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 80% and an indel frequency of less than 0.5% in a target cell, e.g., a human T-cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 80% and an indel frequency of less than 0.1% in a target cell, e.g., a human T-cell.

WO 2024/238825 PCT/US2024/029746 253 id="p-606"

[0606]In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 90% and an indel frequency of less than 1% in a target cell, e.g., a human T-cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 90% and an indel frequency of less than 0.5% in a target cell, e.g., human T-cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 90% and an indel frequency of less than 0.1% in a target cell, e.g., a human T-cell. [0607]In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 95% and an indel frequency of less than 1% in a target cell, e.g., a human T-cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 95% and an indel frequency of less than 0.5% in a target cell, e.g., a human T-cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 95% and an indel frequency of less than 0.1% in a target cell, e.g., a human T-cell. [0608]In some embodiments, the prime editing compositions provided herein are capable of incorporating one or more intended nucleotide edits efficiently without generating a significant proportion of indels. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 1% and an indel frequency of less than 10% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 1% and an indel frequency of less than 7.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 1% and an indel frequency of less than 5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 1% and an indel frequency of less than 2.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 1% and an indel frequency of less than 1% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 1% and an indel frequency of less than 0.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 1% and an indel frequency of less than 0.1% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 5% and an indel frequency of less than 1% in a population of target cells. In some embodiments, the prime editing methods WO 2024/238825 PCT/US2024/029746 254 disclosed herein have an editing efficiency of at least about 5% and an indel frequency of less than 0.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 5% and an indel frequency of less than 0.1% in a population of target cells. [0609]In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 7.5% and an indel frequency of less than 10% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 7.5% and an indel frequency of less than 7.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 7.5% and an indel frequency of less than 5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 7.5% and an indel frequency of less than 2.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 7.5% and an indel frequency of less than 1% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 7.5% and an indel frequency of less than 0.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 7.5% and an indel frequency of less than 0.1% in a population of target cells. [0610]In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 10% and an indel frequency of less than 10% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 10% and an indel frequency of less than 7.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 10% and an indel frequency of less than 5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 10% and an indel frequency of less than 2.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 10% and an indel frequency of less than 1% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 10% and an indel frequency of less than 0.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein WO 2024/238825 PCT/US2024/029746 255 have an editing efficiency of at least about 10% and an indel frequency of less than 0.1% in a population of target cells. [0611]In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 15% and an indel frequency of less than 10% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 15% and an indel frequency of less than 7.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 15% and an indel frequency of less than 5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 15% and an indel frequency of less than 2.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 15% and an indel frequency of less than 1% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 15% and an indel frequency of less than 0.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 15% and an indel frequency of less than 0.1% in a population of target cells. [0612]In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 15% and an indel frequency of less than 10% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 15% and an indel frequency of less than 7.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 15% and an indel frequency of less than 5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 15% and an indel frequency of less than 2.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 20% and an indel frequency of less than 1% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 20% and an indel frequency of less than 0.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 20% and an indel frequency of less than 0.1% in a population of target cells.

WO 2024/238825 PCT/US2024/029746 256 id="p-613"

[0613]In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 30% and an indel frequency of less than 10% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 30% and an indel frequency of less than 7.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 30% and an indel frequency of less than 5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 30% and an indel frequency of less than 2.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 30% and an indel frequency of less than 1% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 30% and an indel frequency of less than 0.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 30% and an indel frequency of less than 0.1% in a population of target cells. [0614]In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 40% and an indel frequency of less than 10% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 40% and an indel frequency of less than 7.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 40% and an indel frequency of less than 5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 40% and an indel frequency of less than 2.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 40% and an indel frequency of less than 1% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 40% and an indel frequency of less than 0.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 40% and an indel frequency of less than 0.1% in a population of target cells. [0615]In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 50% and an indel frequency of less than 10% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an WO 2024/238825 PCT/US2024/029746 257 editing efficiency of at least about 50% and an indel frequency of less than 7.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 50% and an indel frequency of less than 5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 50% and an indel frequency of less than 2.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 50% and an indel frequency of less than 1% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 50% and an indel frequency of less than 0.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 50% and an indel frequency of less than 0.1% in a population of target cells. [0616]In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 60% and an indel frequency of less than 10% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 60% and an indel frequency of less than 7.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 60% and an indel frequency of less than 5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 60% and an indel frequency of less than 2.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 60% and an indel frequency of less than 1% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 60% and an indel frequency of less than 0.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 60% and an indel frequency of less than 0.1% in a population of target cells. [0617]In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 70% and an indel frequency of less than 10% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 70% and an indel frequency of less than 7.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 70% and an indel frequency of less than 5% in a WO 2024/238825 PCT/US2024/029746 258 population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 70% and an indel frequency of less than 2.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 70% and an indel frequency of less than 1% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 70% and an indel frequency of less than 0.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 70% and an indel frequency of less than 0.1% in a population of target cells. [0618]In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 80% and an indel frequency of less than 10% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 80% and an indel frequency of less than 7.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 80% and an indel frequency of less than 5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 80% and an indel frequency of less than 2.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 80% and an indel frequency of less than 1% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 80% and an indel frequency of less than 0.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 80% and an indel frequency of less than 0.1% in a population of target cells. [0619]In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 90% and an indel frequency of less than 10% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 90% and an indel frequency of less than 7.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 90% and an indel frequency of less than 5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 90% and an indel frequency of less than 2.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein WO 2024/238825 PCT/US2024/029746 259 have an editing efficiency of at least about 90% and an indel frequency of less than 1% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 90% and an indel frequency of less than 0.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 90% and an indel frequency of less than 0.1% in a population of target cells. [0620]In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 95% and an indel frequency of less than 10% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 95% and an indel frequency of less than 7.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 95% and an indel frequency of less than 5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 95% and an indel frequency of less than 2.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 95% and an indel frequency of less than 1% as measured in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 95% and an indel frequency of less than 0.5% as measured in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 95% and an indel frequency of less than 0.1% as measured in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 90% and an indel frequency of less than 10% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 90% and an indel frequency of less than 7.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 90% and an indel frequency of less than 5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 90% and an indel frequency of less than 2.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 90% and an indel frequency of less than 1% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of WO 2024/238825 PCT/US2024/029746 260 at least about 90%. In some embodiments, the population of target cell comprises a population of human primary cells, human iPSCs, or human T-cell. [0621]In some embodiments, a target cell (or a population thereof), is a T cell, e.g., a primary T cell, e.g., an inflammatory T cell, a T helper cell, a cytotoxic T-cell, a CD4+ T- cell, a CD8+ T cell, a memory T cell, a regulatory T cell, a natural killer T cell, a mucosal associated invariant T cell, a y8 T cell, an alpha beta T cell, a naive T cell, or an effector T cell), formed elements of the blood (e.g., lymphocytes, bone marrow cells), precursors or progenitors thereof, differentiated or de-differentiated cell thereof, and stem cells. In some embodiments, the target cell is any one of cells described herein. [0622]In some embodiments, any number of indels is determined after at least 1 hour, at least 2 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least hours, at least 3 days, at least 4 days, at least 5 days, at least 7 days, at least 10 days, or at least 14 days of exposing a target gene (e.g, a B2M gene within the genome of a cell) to a prime editing composition. In some embodiments, the editing efficiency is determined after I hour, 2 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 3 days, 4 days, 5 days, 7 days, days, or 14 days of exposing a target gene (e.g., a B2M gene within the genome of a cell) to a prime editing composition. [0623]In some embodiments, the prime editing composition described herein result in less than 50%, less than 40%, less than 30%, less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, less than 0.09%, less than 0.08%, less than 0.07%, less than 0.06%, less than 0.05%, less than 0.04%, less than 0.03%, less than 0.02%, or less than 0.01% off-target editing in a chromosome that includes the target gene. In some embodiments, off-target editing is determined after at least 1 hour, at least 2 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 3 days, at least 4 days, at least days, at least 7 days, at least 10 days, or at least 14 days of exposing a target gene (e.g, a nucleic acid within the genome of a cell) to a prime editing composition. [0624]In some embodiments, the prime editing methods described herein result in less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or less than 0.5% large deletion in edited cells. In some WO 2024/238825 PCT/US2024/029746 261 embodiments, the prime editing methods described herein result in less than 4% large deletion in edited cells. In some embodiments, the prime editing methods described herein result in less than 3%large deletion in edited cells. In some embodiments, the prime editing methods described herein result in less than 2% large deletion in edited cells. In some embodiments, the prime editing methods described herein result in less than l%large deletion in edited cells. In some embodiments, the prime editing methods described herein does not result in detectable level of large deletion in edited cells. [0625]In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 90% and an indel frequency of less than 10% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 90% and an indel frequency of less than 7.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 90% and an indel frequency of less than 5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 90% and an indel frequency of less than 2.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 90% and an indel frequency of less than 1% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 90%. In some embodiments, the population of target cell comprises a population of human primary cells, human iPSCs, or human T-cell. [0626]In some embodiments, the prime editing compositions (e.g., PEgRNAs and prime editors as described herein) and prime editing methods disclosed herein can be used to edit a target B2M gene. In some embodiments, the target B2M gene is a wild type B2M gene. In some embodiments, the compositions and methods disclosed herein introduce a mutation in a coding region of the target B2M gene. In some embodiments, the mutation is introduced in an exon of the target B2M gene. In some embodiments, the one or more intended nucleotide edits are incorporated in a non-coding region of the target B2M gene. In some embodiments, the one or more intended nucleotide edits are introduced in a coding region of the target B2M gene. In some embodiments, the one or more intended nucleotide edits are introduced in an exon of the target B2M gene. In some embodiments, the one or more intended nucleotide edits are introduced in exon l, exon 2, exon 3, exon 4, or any combination thereof of the target B2M gene. In some embodiments, the one or more intended nucleotide edits are incorporated in an exon, an intron, an exon-intron injunction, or a regulatory element of the WO 2024/238825 PCT/US2024/029746 262 B2M gene. In some embodiments, the one or more intended nucleotide edits are incorporated in an open reading frame of the B2M gene. In some embodiments, the one or more intended nucleotide edits are incorporated in an untranslated region of the B2M gene, for example, a 3'-UTR or a 5'- UTR. In some embodiments, the one or more intended nucleotide edits are incorporated in a regulatory element of the B2M gene. In some embodiments, the one or more intended nucleotide edits is incorporated in a promoter, an enhancer, an operator, a silencer, an insulator, a terminator, a transcription initiation sequence, a translation initiation sequence (e.g., a Kozak sequence), or any combination thereof of the B2M gene. In some embodiments, the one or more intended nucleotide edits are incorporated in splice acceptor- splice donor (SA-SD) site in a B2M gene. In some embodiments, incorporation of the one or more intended nucleotide edits generate a splice acceptor-splice donor (SA-SD) site in a B2M gene. In some embodiments, the one or more intended nucleotide edits are incorporated in a splice site in a B2M gene. In some embodiments, incorporation of a one or more intended nucleotide edits, disrupts a splice site in a B2M gene. In some embodiments, the methods disclosed herein generate any one of the following edits in a B2M gene to generate a premature Stop codon: CAG to TAG; CAA to TAA; CGA to TGA; TGG to TGA; TGG to TAG; or TGG to TAA. In some embodiments, the one or more intended nucleotide edits may be introduced at a 3’-UTR, for example, in a poly adenylation (poly-A) site. In some embodiments, the one or more intended nucleotide edits comprise insertion of one or more premature, in frame stop codons (e.g., two stop codons) into the B2M gene.In some embodiments, the prime editing method comprises contacting a target B2M gene with a prime editing composition comprising a prime editor, a PEgRNA, and/or a ngRNA. In some embodiments, contacting the target B2M gene with the prime editing composition results in incorporation of one or more intended nucleotide edits in the target B2M gene. In some embodiments, the incorporation is in a region of the target B2M gene that corresponds to an editing target sequence in the B2Mgene. In some embodiments, the one or more intended nucleotide edits comprises a single nucleotide substitution, an insertion, a deletion, or any combination thereof, compared to the endogenous sequence of the target B2Mgene. In some embodiments, incorporation of the one or more intended nucleotide edits results in introduction of one or more mutations in the target B2M gene. In some embodiments, contacting the target B2M gene with the prime editing composition results in incorporation of one or more intended nucleotide edits in the target B2M gene, which introduces the mutation in the editing target sequence (or a double stranded region comprising the editing target WO 2024/238825 PCT/US2024/029746 263 sequence and the complementary sequence to the editing target sequence on a target strand) in the target B2M gene. In some embodiments, the target B2M gene is in a target cell. Accordingly, in one aspect provided herein is a method of editing a target cell comprising a target B2M gene that results in expression of a B2M polypeptide that comprises one or more mutations relative to a wild B2M polypeptide. In another aspect provided herein is a method of editing a target cell comprising a target B2M gene that results in reduced or eliminated expression of functional TCRa chain protein. In some embodiments, the methods of the present disclosure comprise introducing a prime editing composition comprising a PEgRNA, a prime editor polypeptide, and/or a ngRNA into the target cell that has the target B2M gene to edit the target B2M gene, thereby generating an edited cell. In some embodiments, the target B2M gene is a wild type B2M gene, e.g., a wild type human B2M gene. In some embodiments, the target cell can be any one of cells described herein. [0627]In some embodiments, components of a prime editing composition described herein are provided to a target cell in vitro. In some embodiments, components of a prime editing composition described herein are provided to a target cell ex vivo. In some embodiments, components of a prime editing composition described herein are provided to a target cell in vivo. In some embodiments, the cell edited by prime editing can be differentiated into, or give rise to recovery of a population of cells. In some embodiments, the target cell is an ex vivo cell. In some embodiments, the target cell is an ex vivo cell obtained from a human subject. In some embodiments, the target cell is in a subject, e.g., a human subject. [0628]In some embodiments, incorporation of the one or more intended nucleotide edits in the target B2M gene decreases expression and/or function of a TCRa chain protein. In some embodiments, expression and/or function of a B2M protein may be measured when expressed in a target cell. In some embodiments, incorporation of the one or more intended nucleotide edits in the target B2M gene leads to a fold change in a level of B2M gene expression, B2M mRNA expression, or a combination thereof. In some embodiments, a change (e.g., a decrease) in the level of B2M gene expression, B2M mRNA expression, or a combination thereof can comprise a fold change of, e.g., 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8- fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80- fold, 90-fold, 100-fold or more as compared to expression in a suitable control cell. In some embodiments, a change (e.g., a decrease) in the level of a TCRa chain protein function can comprise a fold change of, e.g., 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90- WO 2024/238825 PCT/US2024/029746 264 fold, 100-fold or more as compared to expression in a suitable control cell. In some embodiments, incorporation of the one or more intended nucleotide edits in the target B2M gene decreases expression of a B2M gene, B2M mRNA and/or B2M protein e.g., by at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more as compared to the expression in a suitable control cell. In some embodiments, incorporation of the one or more intended nucleotide edits in the target B2M gene results in complete loss of expression of a B2M gene, a B2M mRNA and/or a B2M protein as compared to the expression in a suitable control cell. As used herein "complete loss of expression " means that the expression of MHCI beta chain protein is substantially not detected. [0629]In some embodiments, incorporation of the one or more intended nucleotide edits in the target B2M gene decreases function of B2M protein e.g., by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more as compared to that in a suitable control cell. In some embodiments, incorporation of the one or more intended nucleotide edits in the target B2M gene results in complete loss of function of the B2M protein as compared to that in a suitable control cell. As used herein "complete loss of function " means that the function of B2M protein is substantially not detected. [0630]In some embodiments, incorporation of the one or more intended nucleotide edits in the target B2M gene increases expression of a truncated B2M protein e.g., by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more as compared to expression of a full length wild type B2M protein. [0631]In some embodiments, incorporation of the one or more intended nucleotide edits in the target B2M gene decreases expression of a T cell receptor e.g., by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more as compared to that in a suitable control cell. In some embodiments, incorporation of the one or more intended nucleotide edits in the target B2M gene results in complete loss of expression of a T cell receptor e.g., by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more as compared to that in a suitable control cell. In some embodiments, incorporation of the one or more intended nucleotide edits in the target B2M gene decreases function of a T cell receptor e.g., by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more as compared to that in a suitable control cell. In some embodiments, incorporation of the one or more intended nucleotide edits in the target B2M gene results in complete loss of function of a T cell receptor as compared to that in a suitable control cell. In some embodiments, WO 2024/238825 PCT/US2024/029746 265 incorporation of the one or more intended nucleotide edits in the target B2M gene changes (e.g., increases or decreases) activity of a T cell e.g., by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more as compared to that in a suitable control cell. In some embodiments, function or activity of a TCR comprises cytolytic activity, antigen dependent proliferation, and/ or helper activity including secretion of cytokines. In some embodiments, function or activity of a TCR (e.g., expressed on a naive, memory, or memory- type T cell) may also include antigen-dependent proliferation. [0632]In some embodiments, incorporation of the one or more intended nucleotide edits result reduced alloreactivity (e.g., reduced by at least about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more) in a subject upon administration, as compared to that upon administering a suitable control cell.In some embodiments, a suitable control cell is a cell that comprises a wild type B2M gene, or is a cell not introduced or contacted with a prime editing composition described herein. In some embodiments, a suitable control cell is a cell from a healthy subject. [0633]In some embodiments, a B2M expression change can be measured by a B2M functional assay. In some embodiments, protein expression can be measured using a protein assay. In some embodiments, protein expression can be measured using antibody testing. In some embodiments, an antibody can comprise anti- TCRa chain. In some embodiments, protein expression can be measured using ELISA, mass spectrometry, Western blot, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), high performance liquid chromatography (HPLC), electrophoresis, or any combination thereof. In some embodiments, a protein assay can comprise SDS-PAGE and densitometric analysis of a Coomassie Blue- stained gel. [0634]In some embodiments, a decrease in B2M expression can be measured by a decrease in B2M mRNA expression. Expression can be measured by any known method, such as quantitative PCR (qPCR), including but not limited to PCR, real-time PCR (e.g., Sybr-green), and/or hot PCR. In some cases, expression can be measured by detecting the level of mRNA transcripts of the genes. For example, expression can be measured by Northern blotting, nuclease protection assays (e.g., RNase protection assays), reverse transcription PCR, quantitative PCR (e.g., real-time PCR such as real-time quantitative reverse transcription PCR), in situ hybridization (e.g., fluorescent in situ hybridization (FISH)), dot-blot analysis, differential display, serial analysis of gene expression, subtractive hybridization, microarrays, nanostring, and/or sequencing (e.g., next-generation sequencing).

WO 2024/238825 PCT/US2024/029746 266 Methods for targeting the B2M locus [0635]in some embodiments, provided herein are methods for editing a B2M gene, comprising: i. contacting a B2M gene, or a cell comprising a B2M gene with an effective amount of a prime editing composition, or a pharmaceutical composition comprising a prime editing composition as described herein; ii. introducing in a cell comprising a B2M gene an effective amount of a prime editing composition, or a pharmaceutical composition comprising a prime editing composition as described herein; or iii. administering to a subject an effective amount of a prime editing composition, or a pharmaceutical composition comprising a prime editing composition as described herein. In some embodiments, provided herein are methods for generating an edited cell, comprising: introducing an effective amount of a prime editing composition, or a pharmaceutical composition comprising a prime editing composition as described herein in a cell comprising a B2M gene. In some embodiments, provided herein are methods for treating a subject in need thereof; comprising administering to the subject i. an effective amount of a prime editing composition, ii. a pharmaceutical composition comprising a prime editing composition as described herein, or iii. an effective amount of composition comprising a population of edited cells generated by the methods of the disclosure. In some embodiments, provided herein are methods to prepare a cell or a population of cell (e.g., a T cell or a population of T cells) for immunotherapy, comprising contacting a cell or a population of cells with an effective amount of a prime editing composition, or a pharmaceutical composition comprising a prime editing composition as described herein. In some embodiments, contacting reduces the alloreactivity induced by the contacted cell or the contacted population of cells upon administering to a subject as compared to that upon administering a suitable control cell or a population of suitable control cells. In some embodiments, provided herein are methods to reduce alloreactivity of a cell or a population of cells upon administering to a subject as compared to that upon administering a suitable control cell or a population of suitable control cells, comprising: contacting the cell or the population of cells with an effective amount of of a prime editing composition, or a pharmaceutical composition comprising a prime editing composition as described herein. [0636]In some embodiments, methods disclosed herein result in incorporation of one or more intended nucleotide edits in B2M gene. In some embodiments, methods disclosed herein result in introducing a mutation in the B2M gene. In some embodiments, the target gene (e.g., B2M gene) comprise an editing target sequence. In some embodiments, methods disclosed herein result in incorporation of one or more intended nucleotide edits in the target WO 2024/238825 PCT/US2024/029746 267 gene (i.e., B2M gene) that introduces a mutation in the editing target sequence (or a double stranded region comprising the editing target sequence and the complementary sequence to the editing target sequence on a target strand) of the target gene. [0637]In some embodiments, the method provided herein comprises administering to a subject an effective amount of a prime editing composition, for example, a PEgRNA, a prime editor, and/or a ngRNA. In some embodiments, the method comprises administering to the subject an effective amount of a prime editing composition described herein, for example, polynucleotides, vectors, or constructs that encode prime editing composition components, or RNPs, LNPs, and/or polypeptides comprising prime editing composition components. Prime editing compositions can be administered to target the B2M gene in a subject, e.g, a human subject, suffering from, having, susceptible to, or at risk for a disease or condition, e.g., a cancer, a microbial infection, an autoimmune disorder. Prime editing compositions can be administered to target the B2M gene in a subject, e.g., a human subject, in need of an immunotherapy (e.g., a T cell based therapy, e.g., a CAR-T therapy). Identifying a subject in need of such treatment can be in the judgment of a subject or a health care professional and can be subjective (e.g., opinion) or objective (e.g., measurable by a test or diagnostic method). In some embodiments, the method comprises directly administering prime editing compositions provided herein to a subject. [0638]In some embodiments, the method provided herein comprises contacting a B2M gene, or a cell comprising a B2M gene, with an effective amount of a prime editing composition, for example, a PEgRNA, a prime editor, and/or a ngRNA. In some embodiments, the method comprises contacting an effective amount of a prime editing composition described herein, for example, polynucleotides, vectors, or constructs that encode prime editing composition components, or RNPs, LNPs, and/or polypeptides comprising prime editing composition components. [0639]In some embodiments, the method provided herein comprises introducing an effective amount of a prime editing composition, for example, a PEgRNA, a prime editor, and/or a ngRNA in a cell comprising a B2M gene. In some embodiments, the method comprises introducing an effective amount of a prime editing composition described herein, for example, polynucleotides, vectors, or constructs that encode prime editing composition components, or RNPs, LNPs, and/or polypeptides comprising prime editing composition components.

WO 2024/238825 PCT/US2024/029746 268 id="p-640"

[0640]The prime editing compositions described herein can be delivered with in any form as described herein, e.g., as LNPs, RNPs, polynucleotide vectors such as viral vectors, or mRNAs. The prime editing compositions can be formulated with any pharmaceutically acceptable carrier described herein or known in the art for administering directly to a subject. Components of a prime editing composition or a pharmaceutical composition thereof may be administered to the subject, contacted to a B2M gene, or introduced in a cell comprising a B2M gene simultaneously or sequentially. For example, in some embodiments, the method comprises administering, contacting with or introducing a prime editing composition, or pharmaceutical composition thereof, comprising a complex that comprises a prime editor fusion protein and a PEgRNA and/or a ngRNA. In some embodiments, the method comprises administering, contacting with, or introducing a polynucleotide or vector encoding a prime editor simultaneously with a PEgRNA and/or a ngRNA. In some embodiments, the method comprises administering, contacting with, or introducing a polynucleotide or vector encoding a prime editor before a PEgRNA and/or a ngRNA. [0641]In some embodiments, provided herein are methods to generate a population of edited cell. In some embodiments, a population of cells comprising a B2M gene may be contacted with a prime editing composition (e.g., a PEgRNA, a prime editor, and optionally an ngRNA as described herein) disclosed herein. In some embodiments, a prime editing composition (e.g., a PEgRNA, a prime editor, and optionally an ngRNA as described herein) disclosed herein may be introduced in a population of cells. [0642]In some embodiments, the editing template may comprise one or more synonymous mutations relative to the wild-type B2M gene. Such synonymous mutations may include, for example, mutations that decrease the ability of a PEgRNA to rebind to the same target sequence once the desired edit is installed in the genome (e.g., synonymous mutations that silence the endogenous PAM sequence or that edit the endogenous protospacer).Accordingly, one or more synonymous mutations may include a synonymous PAM silencing edit. [0643]Suitable routes of administrating the prime editing compositions to a subject include, without limitation: topical, subcutaneous, transdermal, intradermal, intralesional, intraarticular, intraperitoneal, intravesical, transmucosal, gingival, intradental, intracochlear, transtympanic, intraorgan, epidural, intrathecal, intramuscular, intravenous, intravascular, intraosseus, periocular, intratumoral, intracerebral, and intracerebroventricular administration. In some embodiments, the compositions described are administered WO 2024/238825 PCT/US2024/029746 269 intraperitoneally, intravenously, or by direct injection or direct infusion. In some embodiments, the compositions described are administered by direct injection or infusion into a subject. In some embodiments, the compositions described herein are administered by direct injection. [0644]In some embodiments, the method comprises administering to a subject an effective amount of the edited cells (e.g., a population of edited cells) or a composition comprising the edited cells (e.g., a population of edited cells) described herein that are generated by the methods of the disclosure (e.g., with a prime editing composition). In some embodiments, the edited cells are allogeneic. In some embodiments, allogeneic cells are or have been contacted ex vivo with a prime editing composition or pharmaceutical composition thereof and are introduced into a subject in need thereof. In some embodiments, the edited cells are autologous to the subject. In some embodiments, cells are removed from a subject and contacted ex vivo with a prime editing composition or pharmaceutical composition thereof to generate edited cells and the edited cells are re-introduced into the subject. [0645]In some embodiments, cells are contacted ex vivo with one or more components of a prime editing composition. In some embodiments, the ex vzvo-contacted cells are introduced into the subject, and the subject is administered in vivo with one or more components of a prime editing composition. For example, in some embodiments, cells are contacted ex vivo with a prime editor and introduced into a subject. In some embodiments, the subject is a human subject. In some embodiments, the subject is then administered with a PEgRNA and/or a ngRNA, or a polynucleotide encoding the PEgRNA and/or the ngRNA. In some embodiments, the ex vivo contacted cells are allogenic. In some embodiments, the ex vivo contacted cells are autologous. [0646]In some embodiments, cells contacted with the prime editing composition are determined for incorporation of the one or more intended nucleotide edits in the genome before re-introduction into the subject. In some embodiments, the cells are enriched for incorporation of the one or more intended nucleotide edits in the genome before re- introduction into the subject. In some embodiments, the edited cells are primary cells. In some embodiments, the edited cells are progenitor cells. In some embodiments, the edited cells are stem cells. In some embodiments, the edited cells are induced pluripotent stem cells . In some embodiments, the edited cells are primary human cells. In some embodiments, the edited cells are human progenitor cells. In some embodiments, the edited cells are human stem cells. In some embodiments, the edited cells are human induced pluripotent stem cells .

WO 2024/238825 PCT/US2024/029746 270 In some embodiments, the cell is a neuron. In some embodiments, the cell is a neuron from basal ganglia. In some embodiments, the cell is a neuron from basal ganglia of a subject. In some embodiments, the cell is a neuron in the basal ganglia of a subject. In some embodiments, the edited cells are T-cells. In some embodiments, the edited cells are a T-cell, e.g., a primary T cell, e.g., an inflammatory T cell, a T helper cell, a cytotoxic T-cell, a CD4+ T-cell, a CD8+ T cell, a memory T cell, a regulatory T cell, a natural killer T cell, a mucosal associated invariant T cell, a y8 T cell, an alpha beta T cell, a naive T cell, or an effector T cell), formed elements of the blood (e.g., lymphocytes, bone marrow cells), precursors or progenitors thereof, differentiated or de-differentiated cell thereof, and stem cells. In some embodiments, the edited cells are transformed T cells. In some embodiments, the edited cell is a naive T cell (e.g., a naive CD8+ T cell). In some embodiments, the edited cell is an immune cell (e.g., a primary immune cell) or a progenitor or a precursor thereof). In some embodiments, the edited cell is a T-cell, or a progenitor or a precursor thereof. In some embodiments, the edited cell is a human T cell, or a progenitor or a precursor thereof. In some embodiments, the edited cell is a T helper cell (e.g., Thl cell, Th2 cell, Th9 cell, Thlcell, Th22 cell, and Tfh (follicular helper) cell). In some embodiments, the edited cell is a cytotoxic T cell. In some embodiments, the edited cell is a CD8+ T cell. In some embodiments, the edited cell is a CD4+ T cell. In some embodiments, the edited cell is a memory T cell (e.g., (e.g., central memory T cell (TCM), stem memory T cell (TSCM), effector memory T cell, Tissue resident memory T cell). In some embodiments, the edited cell is an effector memory T cell (e.g., TEM cells and TEMRA (CD45RA+) cells). In some embodiments, the cell is a regulatory T cell. In some embodiments, the edited cell is a natural killer T cell. In some embodiments, the edited cell is a Mucosal associated invariant T cell. In some embodiments, the edited cell is a y8 T cell. In some embodiments, the edited cell is an effector T cell. In some embodiments, the edited cell is a thymocyte. In some embodiments, the edited cell is a lymphoid cell. In some embodiments, the edited cell is a common lymphoid progenitor cells. In some embodiments, the edited cell is an early thymic progenitor cell. In some embodiments, the edited cell is a CD3+ cell. In some embodiments, the edited cell is a tumor infiltrating lymphocyte. In some embodiments, the edited cell is a myeloid cell. In some embodiments, the edited cell is a plasma cell. In some embodiments, the edited cell is an activated T cell. [0647]In some embodiments, the edited cell is a stem cell (e.g., adult stem cell, embryonic stem cell, non-embryonic stem cell), cord blood stem cell, progenitor cell, bone marrow stem WO 2024/238825 PCT/US2024/029746 271 cell, induced pluripotent stem cell, totipotent stem cell, a CD34+ cell, or hematopoietic stem cell). In some embodiments, the edited cell is a pluripotent cell (e.g., a pluripotent stem cell). In some embodiments, the edited cell (e.g., a stem cell) is an embryonic stem cell, tissue- specific stem cell, mesenchymal stem cell, or an induced pluripotent stem cell. In some embodiments, the edited cell is an induced pluripotent stem cell (iPSC). In some embodiments, the edited cell is a hematopoietic stem cell. In some embodiments, the edited cell is a hematopoietic stem and progenitor cell. In some embodiments, the edited cell is a multipotent progenitor cell. In some embodiments, the edited cell is a T-cell progenitor. In some embodiments, the edited cell is a T-cell precursor. In some embodiments, the edited cell is an embryonic stem cell (ESC). In some embodiments, the edited cell is a human stem cell. In some embodiments, the edited cell is a human pluripotent stem cell. In some embodiments, the edited cell is a non-embryonic stem cell. In some embodiments, the edited cell is an induced human pluripotent stem cell. In some embodiments, the edited cell is a human stem cell. In some embodiments, the edited cell is a human embryonic stem cell. In some embodiments, the edited cell is a human T-cell progenitor. In some embodiments, the edited cell is a human T-cell precursor. [0648]In some embodiments, an edited cell is not isolated from an organism but forms part of a tissue or organ of an organism, e.g., a mammal. [0649]In some embodiments, the edited cell is a differentiated cell. In some embodiments, the edited cell can be further differentiated. In some embodiments, the edited cell is differentiated from an induced pluripotent stem cell. In some embodiments, the edited cell is a T-cell e.g., a primary T cell, e.g., an inflammatory T cell, a T helper cell, a cytotoxic T-cell, a CD4+ T-cell, a CD8+ T cell, a memory T cell, a regulatory T cell, a natural killer T cell, a mucosal associated invariant T cell, a y8 T cell, an alpha beta T cell, a naive T cell, or an effector T cell differentiated from an iPSC, ESC, a T-cell precursor, or a T-cell progenitor. [0650]In some embodiments, the edited cell is a differentiated human cell. In some embodiments, the edited cell is differentiated from an induced human pluripotent stem cell. In some embodiments, the edited cell edited by prime editing can be differentiated into, or give rise to recovery of a population of cells, e.g., a T-cell e.g., a primary T cell, e.g., an inflammatory T cell, a T helper cell, a cytotoxic T-cell, a CD4+ T-cell, a CD8+ T cell, a memory T cell, a regulatory T cell, a natural killer T cell, a mucosal associated invariant T cell, a y8 T cell, an alpha beta T cell, a naive T cell, or an effector T cell. In some embodiments, the edited cell is in a subject, e.g., a human subject. In some embodiments, the WO 2024/238825 PCT/US2024/029746 272 edited cell is ex vivo, in vitro or in vivo. In some embodiments, the edited cell is an isolated edited cell. [0651]In some embodiments, the edited cell is a T cell. In some embodiments, the edited or engineered T cell comprises a premature stop codon in a B2M gene relative to a wildtype B2M gene. In some embodiments, the edited or engineered T cell comprises a B2M gene comprising a c.51delC edit relative to a wildtype B2M gene. In some embodiments, the edited or engineered T cell comprises a B2M gene comprising a c.50insG edit relative to a wildtype B2M gene. In some embodiments, the edited or engineered T cell comprises a B2M gene comprising a c.54insTAATAA edit relative to a wildtype B2M gene. [0652]In some embodiments, the edited cells are an ex vivo cells. In some embodiments, the edited cells are an ex vivo cells obtained from a human subject. In some embodiments, the edited cells are in a subject, e.g., a human subject. The prime editing composition or components thereof may be introduced into a cell by any delivery approaches as described herein, including LNP administration, RNP administration, electroporation, nucleofection, transfection, viral transduction, microinjection, cell membrane disruption and diffusion, or any other approach known in the art. [0653]The cells edited with prime editing can be introduced into the subject by any route known in the art. In some embodiments, the edited cells are administered to a subject by direct infusion. In some embodiments, the edited cells are administered to a subject by intravenous infusion. In some embodiments, the edited cells are administered to a subject as implants. [0654]The pharmaceutical compositions, prime editing compositions, and cells, as described herein, can be administered in effective amounts. In some embodiments, the effective amount depends upon the mode of administration. In some embodiments, the effective amount depends upon the stage of the condition, the age and physical condition of the subject, the nature of concurrent therapy, if any, and like factors well-known to the medical practitioner. [0655]The specific dose administered can be a uniform dose for each subject. Alternatively, a subject ’s dose can be tailored to the approximate body weight of the subject. Other factors in determining the appropriate dosage can include the disease or condition to be treated or prevented, the severity of the disease, the route of administration, and the age, sex and medical condition of the patient. [0656]In embodiments wherein components of a prime editing composition are administered sequentially, the time between sequential administration can be at least 1 hour, at least 2 WO 2024/238825 PCT/US2024/029746 273 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 3 days, at least 4 days, at least 5 days, at least 7 days, at least 10 days, or at least days. [0657]In some embodiments, a method of monitoring treatment progress is provided. In some embodiments, the method includes the step of determining a level of diagnostic marker, for example, expression of B2M protein, expression of T cell receptor, or measurement associated with a change in T-cell function and/or activity, in a subject who has been administered an effective amount of a prime editing composition described herein. The level of the diagnostic marker determined in the method can be compared to known levels of the marker in either healthy normal controls or in other afflicted subjects to establish the subject ’s disease status.Edited cells and used thereof [0658]In one aspect, the disclosure provides methods for generating edited cells. In one aspect, the disclosure provides an isolated cell or a population of cells (e.g., an isolated T cell or a population of T cells) generated by the methods disclosed herein. In one aspect, the disclosure provides compositions comprising a population of the edited cells. In one aspect, the disclosure provides a method for treating a subject in need thereof, comprising administering to the subject an effective amount of a composition comprising a population of the edited cells described herein. In some embodiments, the edited cells are autologous to the subject. Some embodiments comprise autologous cell immunotherapy, wherein the cells (e.g., T cells) are obtained from a subject having a disease prior to prime editing. The obtained cells are subjected to prime editing to edit a B2M gene using the methods disclosed herein. In some embodiments, the edited cells can be further modified to insert a chimeric antigen receptor, and effectively re-direct against a desired antigen. Accordingly, in some embodiments, the cells are obtained from a subject in need of an immune cell immunotherapy (e.g., a CAR-T cell therapy). In some embodiments, the autologous cells obtained from a subject prior to editing are cultured prior to editing. In some embodiments, the autologous cells obtained from a subject for ex vivo prime editing, are cultured, and edited shortly after they are obtained. In some embodiments, the autologous cells obtained from a subject and stored for future use (e.g., future prime editing). [0659]Allogenic cell therapies can have similar clinical potential as autologous cell therapy, and can allow treatment of many patients with cells derived from healthy donors, thereby reducing manufacturing costs associated with autologous therapies. In some embodiments, WO 2024/238825 PCT/US2024/029746 274 the edited cells are allogenic to the subject. The present disclosure provides prime editing systems, and methods to achieve efficient and reliable precise editing of B2M gene. Editing of the B2M gene using the methods disclosed herein may generate edited cells that are devoid of or expresses low amounts of an endogenous T cell receptor, for example, a TCR alpha chain (such a via prime editing of B2M gene). Additionally, the edited cells generated using the methods and compositions of the disclosure may exhibit reduced the alloreactivity, and may prevent graft vs host disease upon administration to a subject in need. [0660]In some embodiments, the edited cells exhibit reduced alloreactivity as compared to a suitable control cells. In some embodiments, administering an effective amount of a composition comprising a population of the edited cells of the disclosure induces a reduced alloreactivity (e.g., by at least about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more) to the edited cells in the subject compared to that induced upon administering of composition comprising a population of suitable control cells. In one aspect, provided herein are methods to reduce alloreactivity to a cell, comprising contacting a cell with an effective amount of a prime editing composition disclosed herein. In some embodiments, the edited cells are non-alloreactive cells. [0661]In some embodiments, the cells are obtained from one or more healthy subjects or donors, and are subjected to prime editing of B2M gene. In some embodiments, the edited cells are further modified to insert a chimeric antigen receptor to redirect the edited cells to specifically bind a desired antigen. In some embodiments, a CAR-T cell i.e., a cell comprising a chimeric antigen receptor is contacted with the prime editing compositions disclosed herein for editing of a B2M gene. [0662]In some embodiments, the edited cells are expanded ex vivo prior to administering to a subject. In some embodiments, the edited cells are expanded in vivo, for example, after administering the edited cells to a subject. In some embodiments, the edited cells can be used as a medicament. In some embodiments, the medicament can be used to treat subjects having, diagnosed with, or at risk of developing a disease, disorder, or a condition, e.g., a cancer, a microbial infection such as a pathogenic infection, a bacterial infection, a viral infection, an autoimmune disorder, or a graft versus host disease. In some embodiments, the edited cells can be used in manufacture of a medicament for a treatment of a disease, disorder, or a condition, e.g., cancer, a microbial infection, such as a pathogenic infection, a bacterial infection, a viral infection, an autoimmune disorder, or a graft versus host disease. In some embodiments, the subject can be undergoing or in need of an immunosuppressive therapy.

WO 2024/238825 PCT/US2024/029746 275 id="p-663"

[0663]In one aspect, provided herein are methods of treatment of a subject in need thereof. In one aspect, provided herein are methods of treatment or prevention of a disease, disorder, or condition in a subject. In some embodiments, the subject is diagnosed with, has, or is at a risk of developing a disease, disorder, or a condition. In some embodiments, the subject is in need of, or undergoing, or will be undergoing an immunotherapy, for example, an autologous immune cell immunotherapy, an allogenic immune cell immune therapy. In some embodiments, the immune cell immunotherapy is a CAR-T cell therapy. In some embodiments, the disease, disorder, or a condition is responsive to immunotherapy, for example, an autologous immune cell immunotherapy, an allogenic immune cell immune therapy. In some embodiments, the methods disclosed herein comprise administering an effective amount of a prime editing composition disclosed herein, the edited cells disclosed herein, or a composition comprising the edited cells disclosed herein to the subject. [0664]Nonlimiting examples of a disease, disorder or condition include neoplasia, T-cell acute lymphoblastic leukemia (T-ALL), mycosis fungoides (MF), Sezary syndrome (SS), Peripheral T/NK cell lymphoma, Anaplastic large cell lymphoma ALK+, Primary cutaneous T cell lymphoma, T cell large granular lymphocytic leukemia, Angioimmunoblastic T/NK cell lymphoma, Hepatosplenic T cell lymphoma, Primary cutaneous CD30 + lymphoproliferative disorders, Extranodal NK/T cell lymphoma, Adult T cell leukemia/lymphoma, T cell prolymphocytic leukemia, Subcutaneous panniculitis like T- cell lymphoma, Primary cutaneous gamma delta T-cell lymphoma, Aggressive NK cell leukemia, and Enteropathy associated T cell lymphoma. [0665]Nonlimiting examples of a disease, disorder or condition include a cancer. In some embodiments, the cancer is a solid tumor, hematological cancer, or soft tissue cancer. In some embodiments, the cancer cell is selected from the group consisting of bladder cancer, epithelial cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, esophageal cancer, gastrointestinal cancer, leukemia, liver cancer, lung cancer, lymphoma, myeloma, ovarian cancer, prostate cancer, sarcoma, stomach cancer, thyroid cancer, acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, anal canal, rectal cancer, ocular cancer, cancer of the neck, gallbladder cancer, pleural cancer, oral cancer, cancer of the vulva, colon cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumor, Hodgkin lymphoma, kidney cancer, mesothelioma, mastocytoma, melanoma, multiple myeloma, myeloma, nasopharynx cancer, non-Hodgkin lymphoma, pancreatic cancer, peritoneal cancer, renal cancer, skin cancer, small intestine cancer, stomach cancer, testicular WO 2024/238825 PCT/US2024/029746 276 cancer, and thyroid cancer. In some embodiments, the cancer cell is selected from the group consisting of gastrointestinal cancer, breast cancer, lymphoma, and prostate cancer. [0666]Non-limiting examples of a disease, disorder, or a condition include an inflammatory or an autoimmune disease, for example, in some embodiments, the disease is acute disseminated encephalomyelitis, acute motor axonal neuropathy, Addison's disease, adiposis dolorosa, adult-onset still's disease, alopecia areata, ankylosing spondylitis, anti-glomerular basement membrane nephritis, anti-neutrophil cytoplasmic antibody-associated vasculitis, anti-n-methyl-d- aspartate receptor encephalitis, antiphospholipid syndrome, anti synthetase syndrome, aplastic anemia, autoimmune angioedema, autoimmune encephalitis, autoimmune enteropathy, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome, autoimmune neutropenia, autoimmune oophoritis, autoimmune orchitis, autoimmune pancreatitis, autoimmune polyendocrine syndrome, autoimmune polyendocrine syndrome type 2, autoimmune polyendocrine syndrome type 3, autoimmune progesterone dermatitis, autoimmune retinopathy, autoimmune thrombocytopenic purpura, autoimmune thyroiditis, autoimmune urticaria, autoimmune uveitis, halo concentric sclerosis, behqef s disease, bickerstaff s encephalitis, bullous pemphigoid, celiac disease, chronic fatigue syndrome, chronic inflammatory demyelinating polyneuropathy, churg-strauss syndrome, cicatricial pemphigoid, cogan syndrome, cold agglutinin disease, complex regional pain syndrome, crest syndrome, crohn's disease, dermatitis herpetiformis, dermatomyositis, diabetes mellitus type 1, discoid lupus erythematosus, endometriosis, enthesitis, enthesitis-related arthritis, eosinophilic esophagitis, eosinophilic fasciitis, epidermolysis bullosa acquisita, erythema nodosum, essential mixed cryoglobulinemia, evans syndrome, felty syndrome, fibromyalgia, gastritis, gestational pemphigoid, giant cell arteritis, goodpasture syndrome, graves' disease, graves ophthalmopathy, guillain-barre syndrome, hashimoto's encephalopathy, hashimoto thyroiditis, henoch-schonlein purpura, hidradenitis suppurativa, idiopathic dilated cardiomyopathy, idiopathic inflammatory demyelinating diseases, IgA nephropathy, IgG4- related systemic disease, inclusion body myositis, inflamatory bowel disease, intermediate uveitis, interstitial cystitis, juvenile arthritis, kawasaki's disease, lambert-eaton myasthenic syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, ligneous conjunctivitis, linear IgA disease, lupus nephritis, lupus vasculitis, lyme disease (chronic), meniere's disease, microscopic colitis, microscopic polyangiitis, mixed connective tissue disease, mooren's ulcer, morphea, mucha-habermann disease, multiple sclerosis, myasthenia gravis, WO 2024/238825 PCT/US2024/029746 277 myocarditis, myositis, neuromyelitis optica, neuromyotonia, opsoclonus myoclonus syndrome, optic neuritis, ord's thyroiditis, palindromic rheumatism, paraneoplastic cerebellar degeneration, parry romberg syndrome, parsonage-turner syndrome, pediatric autoimmune neuropsychiatric disorder associated with streptococcus, pemphigus vulgaris, pernicious anemia, pityriasis lichenoides et varioliformis acuta, poems syndrome, polyarteritis nodosa, polymyalgia rheumatica, polymyositis, postmyocardial infarction syndrome, postpericardiotomy syndrome, primary biliary cirrhosis, primary immunodeficiency, primary sclerosing cholangitis, progressive inflammatory neuropathy, psoriasis, psoriatic arthritis, pure red cell aplasia, pyoderma gangrenosum, raynaud ’s phenomenon, reactive arthritis, relapsing polychondritis, restless leg syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, rheumatoid vasculitis, sarcoidosis, schnitzler syndrome, scleroderma, sjogren's syndrome, stiff person syndrome, subacute bacterial endocarditis, susac's syndrome, sydenham chorea, sympathetic ophthalmia, systemic lupus erythematosus, systemic scleroderma, thrombocytopenia, tolosa-hunt syndrome, transverse myelitis, ulcerative colitis, undifferentiated connective tissue disease, urticaria, urticarial vasculitis, vasculitis, or vitiligo. [0667]Non-limiting examples of a disease, disorder or a condition includes a microbial infection, or a pathogenic infection, for example, a bacterial infection, or a viral infection. [0668]The administration of the edited cells or population of edited cells can consist of the administration of 104- 109 cells per kg body weight, preferably 105 to 106 cells/kg body weight including all integer values of cell numbers within those ranges. The edited cells or population of edited cells or a composition comprising them can be administrated in one or more doses. In another embodiment, said effective amount of a composition comprising the population of edited cells can be administrated as a single dose. In another embodiment, said effective amount can be administrated as more than one dose over a period time. Timing of administration is within the judgment of managing physician and depends on the clinical condition of the patient. While individual needs vary, determination of optimal ranges of effective amounts of a given cell type for a particular disease or conditions within the skill of the art. An effective amount of a composition comprising a population of edited cells means an amount which provides a therapeutic or prophylactic benefit. The dosage administrated will be dependent upon the age, health and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment and the nature of the effect desired. In another embodiment, said effective amount of edited cells or composition comprising those cells are WO 2024/238825 PCT/US2024/029746 278 administrated parenterally. Said administration can be an intravenous administration. Said administration can be directly done by injection. [0669]In some embodiments, the cell, the target cell, or the edited cell can be a CAR-T cell. A CAR-T cell is a T cell that expresses a chimeric antigen receptor. In some embodiments, the cell of the disclosure (e.g., the cell, the target cell, or the edited cell) further comprises an exogenous transgene, for example, that encodes an exogenous T cell receptor or a functional fragment thereof, or a chimeric antigen receptor (CAR) or a functional fragment thereof. In some embodiments, the methods of the disclosure further comprise introducing an exogenous transgene in the cell (e.g., the target cell, the cell comprising a B2M gene, the edited cell). [0670]As used herein, a "transgene " refers to an exogenous DNA sequence that is introduced into the genome of a cell, including an edited cell. As used herein, an "exogenous T cell receptor" or "exogenous TCR" refers to a TCR whose sequence is introduced into the genome of a cell (e.g., a T cell) that can confer specificity for a specific epitope or antigen (e.g., an epitope or antigen preferentially present on the surface of a cancer cell or other disease-causing cell or particle). In some embodiments, exogenous T cell receptors can comprise alpha and beta chains or, alternatively, may comprise gamma and delta chains. Exogenous TCRs useful in the disclosure may have specificity to any antigen or epitope of interest. As used herein, a "chimeric antigen receptor" or "CAR" refers to an engineered receptor that confers or grafts specificity for a target antigen onto a cell (e.g., a T cell). The term "CAR" as used herein and generally used in the art, refers to a recombinant fusion protein that has an antigen-specific extracellular domain coupled to an intracellular domain that directs the cell to perform a specialized function upon binding of a target antigen to the extracellular domain. The terms "artificial T- cell receptor," "chimeric T-cell receptor," and "chimeric immunoreceptor" may each be used interchangeably herein with the term "chimeric antigen receptor." [0671]The modification of T cells to express a CAR, and precise editing of a B2M gene is accomplished by using a prime editing systems and methods disclosed herein. Prime editing of B2M gene in combination with a CAR insertion is a usefill strategy to generate edited cells for cell therapy (e.g., autologous T cells or allogenic T cells). In some embodiments, the exogenous transgene e.g., a CAR can be introduced into the cell, for example, by using a plasmid, a minicircle vector, a linearized double stranded DNA construct, or a viral vector. For example, applicable methods for delivery the nucleic acid molecule encoding the chimeric antigen receptor (and the nucleic acid(s) encoding the base editor) can be found in WO 2024/238825 PCT/US2024/029746 279 International Application No. PCT/US2009/040040 and US Patent Nos.8,450,1 12;, each of which is incorporated herein in its entirety. Additionally, those methods and vectors described herein for delivering the nucleic acid encoding the prime editing systems are applicable to deli vering the nucleic acid encoding the chimeric antigen receptor. [0672]In some embodiments, about or at least about 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of a population of cells transduced with a polynucleotide encoding a chimeric antigen receptor (CAR) of the present disclosure (e.g., those listed above) surface-express the CAR. In one aspect, provided herein are edited cells comprising a chimeric antigen and an edited B2M gene. In some embodiments, the edited B2M gene is generated by prime editing. In some embodiments, provided herein is a CAR-T cell with prime edits in B2M gene, such that the CAR-1' cell has reduced or negligible or no expression of endogenous B2M protein. [0673]In some embodiments, the edited cells of the disclosure comprise a transgene encoding a CAR, and one or more intended nucleotide edits in a B2M gene. In some embodiments, the edited cells of the disclosure express a CAR and comprise one or more intended nucleotide edits in a B2M gene. In some embodiments, the edited cell of the disclosure induces an immune response against a target antigen upon administration to a subject. The edited cells of the disclosure (e.g., comprising a transgene that encodes a CAR, or expression a CAR) may be specifically directed towards a target antigen, e.g., to target antigen that may be presented by a cell in a host. In some embodiments, the immune response encompasses cytotoxicity. In some embodiments, the edited cell of the disclosure exhibit enhanced cytotoxic response against a target antigen as compared to a suitable control cell. In some embodiments, the edited cell exhibits an enhanced cytotoxic response by at least 1.1- fold, 1.5-fold, 2- fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold or more compared to a suitable control cell. In some embodiments, the edited cell can kill at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 500% or at least 1000% more target cells that express a target antigen than a suitable control cell. In some embodiments, the edited cell can induce higher memory response. In some embodiments, the edited cell can induce lower levels of inflammatory cytokines than a suitable control cell, that is, the edited cell does not cause a cytokine storm response. In some embodiments, the edited cell is administered to an allogenic subject, wherein the edited cell has no rejection by the subject.

WO 2024/238825 PCT/US2024/029746 280 In some embodiments, the edited cell e.g., an allogenic edited cell induces negligible or minimum rejection by the subject. In some embodiments, the edited cell has fratricide resistance. [0674]A chimeric antigen receptor typically comprises at least an extracellular ligand- binding domain or moiety and an intracellular domain that comprises one or more signaling domains and/or co-stimulatory domains. In some embodiments, the extracellular ligand- binding domain or moiety is in the form of a single-chain variable fragment (scFv) derived from a monoclonal antibody, which provides specificity for a particular epitope or antigen (e.g., an epitope or antigen preferentially present on the surface of a cell, such as a cancer cell or other disease-causing cell or particle). In some embodiments, the scFv is attached via a linker sequence. In various embodiments, the extracellular ligand-binding domain is specific for any antigen or epitope of interest. In some embodiments, the scFv is murine, humanized, or fully human. [0675]The extracellular domain of a chimeric antigen receptor can also comprise an autoantigen (see, Payne et al. (2016), Science 353 (6295): 179-184), that can be recognized by autoantigen-specific B cell receptors on B lymphocytes, thus directing the cell (e.g., a T cell to specifically target and kill autoreactive B lymphocytes in antibody-mediated autoimmune diseases. Such CARs can be referred to as chimeric autoantibody receptors (CAARs), and their use is encompassed by the disclosure. [0676]The extracellular domain of a chimeric antigen receptor can also comprise a naturally- occurring ligand for an antigen of interest, or a fragment of a naturally-occurring ligand which retains the ability to bind the antigen of interest. [0677]The intracellular stimulatory domain can include one or more cytoplasmic signaling domains that transmit an activation signal to the immune effector cell following antigen binding. Such cytoplasmic signaling domains can include, without limitation, CD3. The intracellular stimulatory domain can also include one or more intracellular co-stimulatory domains that transmit a proliferative and/or cell-survival signal after ligand binding. As used herein, a "co-stimulatory domain" refers to a polypeptide domain which transmits an intracellular proliferative and/or cell-survival signal upon activation. Activation of a co- stimulatory domain may occur following homodimerization of two co-stimulatory domain polypeptides. Activation may also occur, for example, following activation of a construct comprising the co-stimulatory domain (e.g., a chimeric antigen receptor or an inducible regulatory construct). Generally, a co-stimulatory domain can be derived from a WO 2024/238825 PCT/US2024/029746 281 transmembrane co-stimulatory receptor, particularly from an intracellular portion of a co- stimulatory receptor. Such intracellular co-stimulatory domains can be any of those known in the art and can include, without limitation, CD27, CD28, CDS, 4-1BB (CD137), 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen- 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3 and a ligand that specifically binds with CD83, Ni, N6, or any combination thereof. Exemplary T cell signaling domains are known in the art. Non-limiting examples include the CD3, CDS, CD28, CD27, CD154, GITR (TNFRSF18), CD134 (OX40), and CD 137 (4-IBB) signaling domains. In some embodiments, the intracellular signaling domain of the chimeric antigen receptor comprises a primary signaling domain. In some embodiments, the chimeric antigen receptor comprises the primary signaling domain and a secondary, or co-stimulatory, signaling domain. In some embodiments, the primary signaling domain comprises one or more immunoreceptor tyrosine-based activation motifs, or ITAMs. In some embodiments, the primary signaling domain comprises more than one IT AM. ITAMs incorporated into the chimeric antigen receptor may be derived from ITAMs from other cellular receptors. In some embodiments, the primary signaling domain comprising an IT AM may be derived from subunits of the TCR complex, such as CD3 gamma, CD3 delta, CD3 epsilon, or CD3 zeta . In some embodiments, the primary signaling domain comprising an ITAM may be derived from FcR y, FcR beta , CDS, CD22, CD79a, CD79b, or CD66d. [0678]A chimeric antigen receptor can further include additional structural elements, including a transmembrane domain that is attached to the extracellular ligand-binding domain via a hinge or spacer sequence. The transmembrane domain of the chimeric antigen receptors described herein spans the CAR-T cell ’s lipid bilayer cellular membrane and separates the extracellular binding domain and the intracellular signaling domain. In some embodiments, the transmembrane domain may be derived from a non- human transmembrane domain and, in some embodiments, humanized. By "humanized " is meant having the sequence of the nucleic acid encoding the transmembrane domain optimized such that it is more reliably or efficiently expressed in a human subject. [0679]The transmembrane domain can be derived from any membrane-bound or transmembrane protein. For example, the transmembrane polypeptide can be a subunit of the T-cell receptor e.g., polypeptide constituting CD3 complex), IL2 receptor p55 (a chain), p(0 chain) or 7 chain, subunit chain of Fc receptors (e.g., Fey receptor III) or CD proteins such as the CD8 alpha chain. Additional examples of such proteins include, but are not limited to, subunits of the T cell receptor (TCR) complex, PD1, or any of the Cluster of Differentiation WO 2024/238825 PCT/US2024/029746 282 proteins, or other proteins, that are expressed in the immune effector cell and that have a transmembrane domain. Transmembrane domains for use in the disclosed CARs can include at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CDS, CDS, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. In some embodiments, the transmembrane domain is derived from CD4, CDS , CD28 and CD3. Alternatively the transmembrane domain can be synthetic and can comprise predominantly hydrophobic residues such as leucine and valine. The chimeric antigen receptor is designed, in some embodiments, to comprise a spacer between the transmembrane domain and the extracellular domain, the intracellular domain, or both. Such spacers can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or amino acids in length. In some embodiments, the spacer can be 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acids in length. In still other embodiments the spacer can be between 100 and 500 amino acids in length. The spacer can be any polypeptide that links one domain to another and are used to position such linked domains to enhance or optimize chimeric antigen receptor function. [0680]The hinge region refers to any oligo- or polypeptide that functions to link the transmembrane domain to the extracellular ligand-binding domain. For example, a hinge region may comprise up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids. Hinge regions may be derived from all or part of naturally occurring molecules, such as from all or part of the extracellular region of CD8, CD4 or CD28, or from all or part of an antibody constant region. Alternatively, the hinge region may be a synthetic sequence that corresponds to a naturally occurring hinge sequence, or may be an entirely synthetic hinge sequence. In particular examples, a hinge domain can comprise a part of a human CD8 alpha chain, FcyRIIIa receptor or IgGl. [0681]In some embodiments, the CAR has specificity for a target antigen that is a disease associated antigen. In some embodiments, the CAR specifically binds a target antigen or a neoantigen expressed by a cancer cell (e.g., expressed or presented on the surface of a cancer cell). In some embodiments, the target antigen or a neoantigen is from an oncogene or tumor suppressor gene (e.g., a mutated tumor suppressor gene). In some embodiments, the target antigen comprises a T cell epitope. In some embodiments, the target antigen recognized and bound by the extracellular domain is a protein or peptide, a nucleic acid, a lipid, or a polysaccharide. Target antigens can be heterologous, such as those expressed in a pathogenic bacteria or virus. Target antigens can also be synthetic; for example, some individuals have WO 2024/238825 PCT/US2024/029746 283 extreme allergies to synthetic latex and exposure to this antigen can result in an extreme immune reaction. In some embodiments, the target antigen is autologous, and is expressed on a diseased or otherwise altered cell. For example, in some embodiments, the target antigen is expressed in a neoplastic cell. In some embodiments, the neoplastic cell is a malignant T- or NK-cell. In some embodiments, the malignant T- or NK-cell is a malignant precursor T- or NK-cell. In some embodiments, the malignant T- or NK-cell is a malignant mature T- or NK- cell. Nonlimiting examples of neoplasia include T-cell acute lymphoblastic leukemia (T- ALL), mycosis fungoides (MF), Sezary syndrome (SS), Peripheral T/NK cell lymphoma, Anaplastic large cell lymphoma ALK+, Primary cutaneous T cell lymphoma, T cell large granular lymphocytic leukemia, Angioimmunoblastic T/NK cell lymphoma, Hepatosplenic T cell lymphoma, Primary cutaneous CD30 + lymphoproliferative disorders, Extranodal NK/T cell lymphoma, Adult T cell leukemia/lymphoma, T cell prolymphocytic leukemia, Subcutaneous panniculitis like T- cell lymphoma, Primary cutaneous gamma delta T-cell lymphoma, Aggressive NK cell leukemia, and Enteropathy associated T cell lymphoma. [0682]In some embodiments, the cancer is a solid tumor, hematological cancer, or soft tissue cancer. In some embodiments, the cancer cell is selected from the group consisting of bladder cancer, epithelial cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, esophageal cancer, gastrointestinal cancer, leukemia, liver cancer, lung cancer, lymphoma, myeloma, ovarian cancer, prostate cancer, sarcoma, stomach cancer, thyroid cancer, acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, anal canal, rectal cancer, ocular cancer, cancer of the neck, gallbladder cancer, pleural cancer, oral cancer, cancer of the vulva, colon cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumor, Hodgkin lymphoma, kidney cancer, mesothelioma, mastocytoma, melanoma, multiple myeloma, myeloma, nasopharynx cancer, non-Hodgkin lymphoma, pancreatic cancer, peritoneal cancer, renal cancer, skin cancer, small intestine cancer, stomach cancer, testicular cancer, and thyroid cancer. In some embodiments, the cancer cell is selected from the group consisting of gastrointestinal cancer, breast cancer, lymphoma, and prostate cancer. [0683]In some embodiments, the exogenous TCR or CAR specifically binds to a target antigen expressed by a pathogen e.g., a bacterium, virus, fungus, yeast, parasite (e.g., single- celled or multicellular eukaryotic parasite), or other microorganism. [0684]In some embodiments, the exogenous TCR or CAR specifically binds to a target antigen associated with a disease (e.g., an inflammatory or autoimmune disease). In some embodiments, the antigen comprises a T cell epitope. In some embodiments, the disease is WO 2024/238825 PCT/US2024/029746 284 acute disseminated encephalomyelitis, acute motor axonal neuropathy, Addison's disease, adiposis dolorosa, adult-onset still's disease, alopecia areata, ankylosing spondylitis, anti- glomerular basement membrane nephritis, anti-neutrophil cytoplasmic antibody-associated vasculitis, anti-n-methyl-d- aspartate receptor encephalitis, antiphospholipid syndrome, anti synthetase syndrome, aplastic anemia, autoimmune angioedema, autoimmune encephalitis, autoimmune enteropathy, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome, autoimmune neutropenia, autoimmune oophoritis, autoimmune orchitis, autoimmune pancreatitis, autoimmune polyendocrine syndrome, autoimmune polyendocrine syndrome type 2, autoimmune polyendocrine syndrome type 3, autoimmune progesterone dermatitis, autoimmune retinopathy, autoimmune thrombocytopenic purpura, autoimmune thyroiditis, autoimmune urticaria, autoimmune uveitis, halo concentric sclerosis, behqef s disease, bickerstaff s encephalitis, bullous pemphigoid, celiac disease, chronic fatigue syndrome, chronic inflammatory demyelinating polyneuropathy, churg-strauss syndrome, cicatricial pemphigoid, cogan syndrome, cold agglutinin disease, complex regional pain syndrome, crest syndrome, crohn's disease, dermatitis herpetiformis, dermatomyositis, diabetes mellitus type 1, discoid lupus erythematosus, endometriosis, enthesitis, enthesitis-related arthritis, eosinophilic esophagitis, eosinophilic fasciitis, epidermolysis bullosa acquisita, erythema nodosum, essential mixed cryoglobulinemia, evans syndrome, felty syndrome, fibromyalgia, gastritis, gestational pemphigoid, giant cell arteritis, goodpasture syndrome, graves' disease, graves ophthalmopathy, guillain-barre syndrome, hashimoto's encephalopathy, hashimoto thyroiditis, henoch-schonlein purpura, hidradenitis suppurativa, idiopathic dilated cardiomyopathy, idiopathic inflammatory demyelinating diseases, IgA nephropathy, IgG4- related systemic disease, inclusion body myositis, inflamatory bowel disease, intermediate uveitis, interstitial cystitis, juvenile arthritis, kawasaki's disease, lambert-eaton myasthenic syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, ligneous conjunctivitis, linear IgA disease, lupus nephritis, lupus vasculitis, lyme disease (chronic), meniere's disease, microscopic colitis, microscopic polyangiitis, mixed connective tissue disease, mooren's ulcer, morphea, mucha-habermann disease, multiple sclerosis, myasthenia gravis, myocarditis, myositis, neuromyelitis optica, neuromyotonia, opsoclonus myoclonus syndrome, optic neuritis, ord's thyroiditis, palindromic rheumatism, paraneoplastic cerebellar degeneration, parry romberg syndrome, parsonage-turner syndrome, pediatric autoimmune neuropsychiatric disorder associated with streptococcus, pemphigus vulgaris, pernicious WO 2024/238825 PCT/US2024/029746 285 anemia, pityriasis lichenoides et varioliformis acuta, poems syndrome, polyarteritis nodosa, polymyalgia rheumatica, polymyositis, postmyocardial infarction syndrome, postpericardiotomy syndrome, primary biliary cirrhosis, primary immunodeficiency, primary sclerosing cholangitis, progressive inflammatory neuropathy, psoriasis, psoriatic arthritis, pure red cell aplasia, pyoderma gangrenosum, raynaud ’s phenomenon, reactive arthritis, relapsing polychondritis, restless leg syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, rheumatoid vasculitis, sarcoidosis, schnitzler syndrome, scleroderma, sjogren's syndrome, stiff person syndrome, subacute bacterial endocarditis, susac's syndrome, sydenham chorea, sympathetic ophthalmia, systemic lupus erythematosus, systemic scleroderma, thrombocytopenia, tolosa-hunt syndrome, transverse myelitis, ulcerative colitis, undifferentiated connective tissue disease, urticaria, urticarial vasculitis, vasculitis, or vitiligo. In various embodiments, the CAR-T cells have low levels of tonic signaling. In embodiments, the tonic signaling is about or less than about 10, 9, 8, 7, 6, 5, 4, 3, 2, 0.5, or 0.1 times the tonic signaling in a reference cell. Non-limiting examples of a reference cell is a T cell not expressing a CAR or a T cell expressing a reference CAR.Delivery [0685]Prime editing compositions described herein can be delivered to a cellular environment with any approach known in the art. Components of a prime editing composition can be delivered to a cell by the same mode or different modes. For example, in some embodiments, a prime editor can be delivered as a polypeptide or a polynucleotide (DNA or RNA) encoding the polypeptide. In some embodiments, a PEgRNA can be delivered directly as an RNA or as a DNA encoding the PEgRNA. [0686]In some embodiments, a prime editing composition component is encoded by a polynucleotide, a vector, or a construct. In some embodiments, a prime editor polypeptide, a PEgRNA and/or a ngRNA is encoded by a polynucleotide. In some embodiments, the polynucleotide encodes a prime editor fusion protein comprising a DNA binding domain and a DNA polymerase domain. In some embodiments, the polynucleotide encodes a DNA polymerase domain of a prime editor. In some embodiments, the polynucleotide encodes a DNA polymerase domain of a prime editor. In some embodiments, the polynucleotide encodes a portion of a prime editor protein, for example, a N-terminal portion of a prime editor fusion protein connected to an intein-N. In some embodiments, the polynucleotide encodes a portion of a prime editor protein, for example, a C-terminal portion of a prime editor fusion protein connected to an intein-C. In some embodiments, the polynucleotide WO 2024/238825 PCT/US2024/029746 286 encodes a PEgRNA and/or a ngRNA. In some embodiments, the polypeptide encodes two or more components of a prime editing composition, for example, a prime editor fusion protein and a PEgRNA. [0687]In some embodiments, the polynucleotide encoding one or more prime editing composition components is delivered to a target cell is integrated into the genome of the cell for long-term expression, for example, by a retroviral vector. In some embodiments, the polynucleotide delivered to a target cell is expressed transiently. For example, the polynucleotide may be delivered in the form of a mRNA, or a non-integrating vector (non- integrating virus, plasmids, minicircle DNAs) for episomal expression. [0688]In some embodiments, a polynucleotide encoding one or more prime editing system components can be operably linked to a regulatory element, e.g., a transcriptional control element, such as a promoter. In some embodiments, the polynucleotide is operably linked to multiple control elements. Depending on the expression system utilized, any of a number of suitable transcription and translation control elements, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. may be used in the expression vector (e.g., U6 promoter, Hl promoter). [0689]In some embodiments, the polynucleotide encoding one or more prime editing composition components is a part of, or is encoded by, a vector. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a non-viral vector. [0690]Non-viral vector delivery systems can include DNA plasmids, RNA (e.g., a transcript of a vector described herein), naked nucleic acid, and nucleic acid complexed with a delivery vehicle, such as a liposome. In some embodiments, the polynucleotide is provided as an RNA, e.g., a mRNA or a transcript. Any RNA of the prime editing systems, for example a guide RNA or a base editor-encoding mRNA, can be delivered in the form of RNA. In some embodiments, one or more components of the prime editing system that are RNAs is produced by direct chemical synthesis or may be transcribed in vitro from a DNA. In some embodiments, a mRNA that encodes a prime editor polypeptide is generated using in vitro transcription. Guide polynucleotides (e.g., PEgRNA or ngRNA) can also be transcribed using in vitro transcription from a cassette containing a T7 promoter, followed by the sequence "GG", and guide polynucleotide sequence. In some embodiments, the prime editor encoding mRNA, PEgRNA, and/or ngRNA are synthesized in vitro using an RNA polymerase enzyme (e.g., T7 polymerase, T3 polymerase, SP6 polymerase, etc.). Once synthesized, the RNA can directly contact a target B2M gene or can be introduced into a cell using any suitable WO 2024/238825 PCT/US2024/029746 287 technique for introducing nucleic acids into cells (e.g, microinjection, electroporation, transfection). In some embodiments, the prime editor-coding sequences, the PEgRNAs, and/or the ngRNAs are modified to include one or more modified nucleoside e.g., using pseudo-U or 5-Methyl-C. [0691]Methods of non-viral delivery of nucleic acids can include lipofection, electroporation, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, nanoparticles, cell penetrating peptides and associated conjugated molecules and chemistry, naked DNA, artificial virions, cell membrane disruption by a microfluidics device, and agent-enhanced uptake of DNA. Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides can be used. Delivery can be to cells (e.g., in vitro or ex vivo administration) or target tissues (e.g., in vivo administration). The preparation of lipid:nucleic acid complexes, including targeted liposomes such as immunolipid complexes, can be used. [0692]Viral vector delivery systems can include DNA and RNA viruses, which can have either episomal or integrated genomes after delivery to the cell. RNA or DNA viral based systems can be used to target specific cells and trafficking the viral payload to an organelle of the cell. Viral vectors can be administered directly (in vivo) or they can be used to treat cells in vitro, and the modified cells can optionally be administered after delivery (ex vivo). [0693]In some embodiments, the viral vector is a retroviral, lentiviral, adenoviral, adeno- associated viral or herpes simplex viral vector. Retroviral vectors can include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof. In some embodiments, the retroviral vector is a lentiviral vector. In some embodiments, the retroviral vector is a gamma retroviral vector. In some embodiments, the viral vector is an adenoviral vector. In some embodiments, the viral vector is an adeno- associated virus ("AAV") vector. [0694]In some embodiments, polynucleotides encoding one or more prime editing composition components are packaged in a virus particle. Packaging cells can be used to form virus particles that can infect a target cell. Such cells can include 293 cells, (e.g., for packaging adenovirus), and y2 cells or PA317 cells (e.g., for packaging retrovirus). Viral vectors can be generated by producing a cell line that packages a nucleic acid vector into a viral particle. The vectors can contain the minimal viral sequences required for packaging and subsequent integration into a host. The vectors can contain other viral sequences being WO 2024/238825 PCT/US2024/029746 288 replaced by an expression cassette for the polynucleotide(s) to be expressed. The missing viral functions can be supplied in trans by the packaging cell line. For example, AAV vectors can comprise ITR sequences from the AAV genome which are required for packaging and integration into the host genome. In some embodiment, the polynucleotides are a DNA polynucleotide. In some embodiment, the polynucleotides are an RNA polynucleotide; e.g., an mRNA polynucleotide. [0695]In some embodiments, the AAV vector is selected for tropism to a particular cell, tissue, organism. In some embodiments, the AAV vector is pseudotyped, e.g., AAV5/8. In some embodiments, polynucleotides encoding one or more prime editing composition components are packaged in a first AAV and a second AAV. In some embodiments, the polynucleotides encoding one or more prime editing composition components are packaged in a first rAAV and a second rAAV. [0696]In some embodiments, dual AAV vectors are generated by splitting a large transgene expression cassette in two separate halves (5' and 3' ends that encode N-terminal portion and C-terminal portion of, e.g., a prime editor polypeptide), where each half of the cassette is no more than 5kb in length, optionally no more than 4.7 kb in length, and is packaged in a single AAV vector. In some embodiments, the full-length transgene expression cassette is reassembled upon co-infection of the same cell by both dual AAV vectors. In some embodiments, a portion or fragment of a prime editor polypeptide, e.g., a Cas9 nickase, is fused to an intein. The portion or fragment of the polypeptide can be fused to the N-terminus or the C-terminus of the intein. In some embodiments, a N-terminal portion of the polypeptide is fused to an intein-N, and a C-terminal portion of the polypeptide is separately fused to an intein-C. In some embodiments, a portion or fragment of a prime editor fusion protein is fused to an intein and fused to an AAV capsid protein. In some embodiments, intein-N may be fused to the N-terminal portion of a first domain described herein, and intein-C may be fused to the C-terminal portion of a second domain described herein for the joining of the N-terminal portion to the C-terminal portion, thereby joining the first and second domains. In some embodiments, the first and second domains are each independently chosen from a DNA binding domain or a DNA polymerase domain. The intein, nuclease and capsid protein can be fused together in any arrangement (e.g., nuclease-intein-capsid, intein- nuclease-capsid, capsid-intein-nuclease, etc.). In some embodiments, a polynucleotide encoding a prime editor fusion protein is split in two separate halves, each encoding a portion of the prime editor fusion protein and separately fused to an intein. In some embodiments, WO 2024/238825 PCT/US2024/029746 289 each of the two halves of the polynucleotide is packaged in an individual AAV vector of a dual AAV vector system. In some embodiments, each of the two halves of the polynucleotide is no more than 5kb in length, optionally no more than 4.7 kb in length. In some embodiments, the full-length prime editor fusion protein is reassembled upon co-infection of the same cell by both dual AAV vectors, expression of both halves of the prime editor fusion protein, and self-excision of the inteins. In some embodiments, the in vivo use of dual AAV vectors results in the expression of full-length full-length prime editor fusion proteins. In some embodiments, the use of the dual AAV vector platform allows viable delivery of transgenes of greater than about 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0 kb in size. [0697]In some embodiments, an intein is inserted at a splice site within a Cas protein. In some embodiments, insertion of an intein disrupts a Cas activity. As used herein, "intein" refers to a self-splicing protein intron (e.g., peptide), e.g., which ligates flanking N-terminal and C-terminal exteins (e.g., fragments to be joined). In some embodiments, an intein may comprise a polypeptide that is able to excise itself and join exteins with a peptide bond (e.g., protein splicing). In some embodiments, an intein of a precursor gene comes from two genes (e.g., split intein). In some embodiments, an intein may be a synthetic intein. Non-limiting examples of intein pairs that may be used in accordance with the present disclosure include: dnaE-n and dnaE-c. a 4-hydroxytamoxifen (4-HT)-responsive intein, an iCas molecule, a Ssp DnaX intein, Ter DnaE3 intein, Ter ThyX intein, Rma DnaB intein, Cfa DnaE intein, Ssp GyrB intein, and Rma DnaB intein. In some embodiments, intein fragments may be fused to the N terminal and C-terminal portion of a split Cas protein respectively for joining the fragments of split Cas9. [0698]In some embodiments, the split Cas9 system may be used in general to bypass the packing limit of the viral delivery vehicles. In some embodiments, a split Cas9 may be a Type II CRISPR system Cas9. In some embodiments, a first nucleic acid encodes a first portion of the Cas9 protein having a first split-intein and wherein the second nucleic acid encodes a second portion of the Cas9 protein having a second split-intein complementary to the first split-intein and wherein the first portion of the Cas9 protein and the second portion of the Cas9 protein are joined together to form the Cas9 protein. In some embodiments, the first portion of the Cas9 protein is the N-terminal fragment of the Cas9 protein and the second portion of the Cas9 protein is the C-terminal fragment of the Cas9 protein. In some embodiments, a split site may be selected which are surface exposed due to the sterical need for protein splicing.

WO 2024/238825 PCT/US2024/029746 290 id="p-699"

[0699]In some embodiments, a Cas protein may be split into two fragments at any C, T, A, or S. In some embodiments, a Cas9 may be intein split at residues 203-204, 280-292, 292- 364, 311-325, 417-438, 445-483, 468-469, 481-502, 513-520, 522-530, 565-637, 696-707, 713-714, 795-804, 803-810, 878-887, and 1153-1154. In some embodiments, protein is divided into two fragments at SpCas9 T310, T313, A456, S469, or C574. In some embodiments, split Cas9 fragments across different split pairs yield combinations that provided the complete polypeptide sequence activate gene expression even when fragments are partially redundant. In some embodiments, a functional Cas9 protein may be reconstituted from two inactive split-Cas9 peptides in the presence of gRNA by using a split-intein protein splicing strategy. In some embodiment, the split Cas9 fragments are fused to either a N- terminal intein fragment or a C-terminal intein fragment, which can associate with each other and catalytically splice the two split Cas9 fragments into a functional reconstituted Casprotein. In some embodiments, a split-Cas9 can be packaged into self-complementary AAV. In some embodiments, a split-Cas9 comprises a 2.5 kb and a 2.2 kb fragment of S. pyogenes Cas9 coding sequences. [0700]In some embodiments, a split-Cas9 architecture reduces the length and/or size of the coding sequences of a viral vector, e.g., AAV. [0701]A target cell can be transiently or non-transiently transfected with one or more vectors described herein. A cell can be transfected as it naturally occurs in a subject. A cell can be taken or derived from a subject and transfected. A cell can be derived from cells taken from a subject, such as a cell line. In some embodiments, a cell transfected with one or more vectors described herein can be used to establish a new cell line comprising one or more vector- derived sequences. In some embodiments, a cell transiently transfected with the compositions of the disclosure (such as by transient transfection of one or more vectors, or transfection with RNA), and modified through the activity of a prime editor, can be used to establish a new cell line comprising cells containing the modification but lacking any other exogenous sequence. Any suitable vector compatible with the host cell can be used with the methods of the disclosure. Non-limiting examples of vectors include pXTl, pSG5, pSVK3, pBPV, pMSG, andpSVLSV40. [0702]In some embodiments, a prime editor protein can be provided to cells as a polypeptide. In some embodiments, the prime editor protein is fused to a polypeptide domain that increases solubility of the protein. In some embodiments, the prime editor protein is formulated to improve solubility of the protein.

WO 2024/238825 PCT/US2024/029746 291 id="p-703"

[0703]In some embodiment, a prime editor polypeptide is fused to a polypeptide permeant domain to promote uptake by the cell. In some embodiments, the permeant domain is a including peptide, a peptidomimetic, or a non-peptide carrier. For example, a permeant peptide may be derived from the third alpha helix of Drosophila melanogaster transcription factor Antennapaedia, referred to as penetratin, which comprises the amino acid sequence RQIKIWFQNRRMKWKK (SEQ ID NO: 790). As another example, the permeant peptide can comprise the HIV-1 tat basic region amino acid sequence, which may include, for example, amino acids 49-57 of naturally-occurring tat protein. Other permeant domains can include poly-arginine motifs, for example, the region of amino acids 34-56 of HIV-1 rev protein, nona-arginine, and octa-arginine. The nona-arginine (R9) sequence can be used. The site at which the fusion can be made may be selected in order to optimize the biological activity, secretion or binding characteristics of the polypeptide. [0704]In some embodiments, a prime editor polypeptide is produced in vitro or by host cells, and it may be further processed by unfolding, e.g, heat denaturation, DTT reduction, etc. and may be further refolded. In some embodiments, a prime editor polypeptide is prepared by in vitro synthesis. Various commercial synthetic apparatuses can be used. By using synthesizers, naturally occurring amino acids can be substituted with unnatural amino acids. In some embodiments, a prime editor polypeptide is isolated and purified in accordance with recombinant synthesis methods, for example, by expression in a host cell and the lysate purified using HPLC, exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification technique. [0705]In some embodiments, a prime editing composition, for example, prime editor polypeptide components and PEgRNA/ngRNA are introduced to a target cell by nanoparticles. In some embodiments, the prime editor polypeptide components and the PEgRNA and/or ngRNA form a complex in the nanoparticle. Any suitable nanoparticle design can be used to deliver genome editing system components or nucleic acids encoding such components. In some embodiments, the nanoparticle is inorganic. In some embodiments, the nanoparticle is organic. In some embodiments, a prime editing composition is delivered to a target cell, e.g., a hepatocyte, in an organic nanoparticle, e.g., a lipid nanoparticle (LNP) or polymer nanoparticle. [0706]In some embodiments, LNPs are formulated from cationic, anionic, neutral lipids, or combinations thereof. In some embodiments, neutral lipids, such as the fusogenic phospholipid DOPE or the membrane component cholesterol, are included to enhance WO 2024/238825 PCT/US2024/029746 292 transfection activity and nanoparticle stability. In some embodiments, LNPs are formulated with hydrophobic lipids, hydrophilic lipids, or combinations thereof. Lipids may be formulated in a wide range of molar ratios to produce an LNP. Any lipid or combination of lipids that are known in the art can be used to produce an LNP. Exemplary lipids used to produce LNPs are provided in Table 29 below. [0707]In some embodiments, components of a prime editing composition form a complex prior to delivery to a target cell. For example, a prime editor fusion protein, a PEgRNA, and/or a ngRNA can form a complex prior to delivery to the target cell. In some embodiments, a prime editing polypeptide (e.g., a prime editor fusion protein) and a guide polynucleotide (e.g, a PEgRNA or ngRNA) form a ribonucleoprotein (RNP) for delivery to a target cell. In some embodiments, the RNP comprises a prime editor fusion protein in complex with a PEgRNA. RNPs may be delivered to cells using known methods, such as electroporation, nucleofection, or cationic lipid-mediated methods, or any other approaches known in the art. In some embodiments, delivery of a prime editing composition or complex to the target cell does not require the delivery of foreign DNA into the cell. In some embodiments, the RNP comprising the prime editing complex is degraded over time in the target cell. Exemplary lipids for use in nanoparticle formulations and/or gene transfer are shown in Table 29 below 7. Table 29: Exemplary lipids for nanoparticle formulation or gene transfer Lipid Abbreviation Feature 1,2-Dioleoyl-sn-glycero-3 -phosphatidylcholine DOPC Helper1,2-Dioleoyl-sn-glycero-3 -phosphatidylethanolamine DOPE HelperCholesterol HelperN41-(2,3-Dioleyloxy)prophyliN,N,N-trimethylammonium chlorideDOTMA Cationic 1,2-Dioleoyloxy-3 -trimethylammonium-propaneDioctadecylamidoglycylspermineDOGS Cationic N-(3-Aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)- 1- propanaminium bromideGAP-DLRIE Cationic Cetyltrimethylammonium bromide CTAB Cationic6-Lauroxyhexyl omithinate LHON Cationicl-(2,3-Dioleoyloxypropyl)-2,4,6-trimethylpyridinium 2Oc Cationic2,3 -Dioley loxy-N-P(spenninecarboxamido-ethy 1 J- N,Ndimethyl-1-propanatninium trifluoroacetate DOSPA Cationic 1,2-Dioley 1 -3-trimethylamtnonium-propane DOPA CationicN-(2-Hydroxyethyl)-N,N-dimethy 1 -2,3-bis(tetradecyloxy)-l - propanaminium bromideMDRIE Cationic Dimyristooxypropyl dimethyl hydroxyethyl ammonium bromideDMRI Cationic WO 2024/238825 PCT/US2024/029746 293 Lipid Abbreviation Feature 3P־[N־(N’, N’-Dimethylaminoethane)-carbamoyl]cholesterol DC-Chol CationicBis-guanidium-tren-cholesterol BGTC Cationic1,3 -Diodeoxy-2-(6-carboxy-spermy 1 )-propylamide DOSPER CationicDimethyloctadecylammonium bromide DDAB CationicDioctadecylamidoglicylspermidin DSL Cationicrac-[(2,3-Dioctadecyloxypropyl)(2-hydroxyethyl)]- dimethylammonium chlorideCLIP-1 Cationic rac-[2(2,3-Dihexadecyloxypropyloxymethyloxy) ethyl]trimethylammoniun bromideCLIP-6 Cationic Ethyldimyristoylphosphatidylcholine EDMPC Cationicl,2-Distearyloxy-N,N-dimethyl-3-aminopropane DSDMA Cationic1,2-Dimyristoyl-trimethylammonium propane DMTAP CationicO,O'-Dimyristyl-N-lysyl aspartate DMKE Cationic1,2-Distearoyl-sn-glycero-3-ethylpho sphocholine DSEPC CationicN-Palmitoyl D-erythro-sphingosyl carbamoyl-spenmine CCS CationicN-t-Butyl-N0-tetradecyl-3-tetradecylaminopropionamidine diC14- amidineCationic Octadecenolyoxy[ethyl-2-heptadecenyl-3 hydroxyethyl] imidazolinium chlorideDOTIM Cationic N1 -Cholesteryloxycarbonyl-3 ,7-diazanonane- 1,9-diamine CDAN Cationic2-(3-Bis(3-amino-propyl)-amino]propylamino)- Nditetradecylcarbamoylme-ethyl-acetamideRPR209120 Cationic 1,2-dilinoleyloxy-3 -dimethylaminopropane DLinDMA Cationic2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane DLin-KC2- DMACationic dilinoleyl-methyl-4-dimethylaminobutyrate DLin-MC3- DMACationic id="p-708"

[0708]Exemplary polymers for use in nanoparticle formulations and/or gene transfer are shown in Table 30below. Table 30: Exemplary polymer for nanoparticle formulation or gene transfer Polymer AbbreviationPoly(ethylene)glycol PEGPolyethylenimine PEIDithiobis (succinimidylpropionate) DSPDimethyl-3 ,3 '-dithiobispropionimidate DTBPPolyethylene imine)biscarbamate PEICPoly(L-lysine) PLLHistidine modified PLLPoly (N -vinylpyrrolidone) PVPPoly(propylenimine) PPIPoly(amidoamine) PAMAMPoly(amidoethylenimine) SS PAEITriethylenetetramine TETAPoly(P-aminoester) WO 2024/238825 PCT/US2024/029746 294 Poly(4-hydroxy-L-proline ester) PHPPoly(allylamine)Poly (a- [ 4-aminobutyl] -L-gly colic acid) PAGAPoly(D,L-lactic-co-glycolic acid) PLGAPoly (N-ethyl-4-vinylpyridinium bromide)Poly(phosphazene)s PPZPoly(phosphoester)s PPEPoly(phosphoramidate)s PPAPoly(N-2-hydroxypropylmethacrylamide) pHPMAPoly (2-(dimethylamino)ethyl methacrylate) pDMAEMAPoly(2-aminoethyl propylene phosphate) PPE-EAChitosanGalactosylated chitosanN-dodacylated chitosamHistoneCollagenDextran-spermine D-SPM id="p-709"

[0709]Exemplary delivery methods for polynucleotides encoding prime editing composition components are shown in Table 31 below. Table 31: Exemplary polynucleotide delivery methods Delivery Vector/Mode Delivery into Non- Dividing Ceils Duration of Expression Genome Integration Type of Molecule Delivered Physical (e.g., electroporation, particle gun. Calcium phosphate transfection) YES Transient NO Nucleic Acids and Proteins Viral Retrovirus NO Stable YES RNALenti virus YES Stable YES/NO with modification RNA Adenovirus YES Transient NO DNAAdeno- Associated Virus (AAV) YES Stable NO DNA Vaccinia Virus YES Very TransientNO DNA Herpes Simplex VirusYES Stable NO DNA Non-Viral Cationic YES Transient Depends on what is delivered Nucleic acids and Proteins WO 2024/238825 PCT/US2024/029746 295 Delivery Vector/Mode Delivery into Non- Dividing Cells Duration of Expression Genome Integration Type of Molecule Delivered Polymeric NanoparticlesYES Transient NO Nucleic AcidsBiological Attenuated BacteriaYES Transient NO Nucleic AcidsNon-Viral Delivery Vehicles Engineered BacteriophagesYES Transient NO Nucleic AcidsMammalian Virus-like Particles YES Transient NO Nucleic Acids Biological liposomes: Erythrocyte Ghosts and Exosomes YES Transient NO Nucleic Acids id="p-710"

[0710]The prime editing compositions of the disclosure, whether introduced as polynucleotides or polypeptides, can be provided to the cells for about 30 minutes to about hours, e.g., 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours 4 hours, 5 hours, 6 hours, hours, 8 hours, 12 hours, 16 hours, 18 hours, 20 hours, or any other period from about minutes to about 24 hours, which can be repeated with a frequency of about every day to about every 4 days, e.g., every 1.5 days, every 2 days, every 3 days, or any other frequency from about every day to about every four days. The compositions may be provided to the subject cells one or more times, e.g., one time, twice, three times, or more than three times, and the cells allowed to incubate with the agent(s) for some amount of time following each contacting event e.g., 16-24 hours. In cases in which two or more different prime editing system components, e.g., two different polynucleotide constructs are provided to the cell (e.g, different components of the same prime editing system, or two different guide nucleic acids that are complementary to different sequences within the same or different target genes), the compositions may be delivered simultaneously (e.g., as two polypeptides and/or nucleic acids). Alternatively, they may be provided sequentially, e.g, one composition being provided first, followed by a second composition. [0711]The prime editing compositions and pharmaceutical composition s of the disclosure, whether introduced as polynucleotides or polypeptides, can be administered to subjects in need thereof for about 30 minutes to about 24 hours, e.g, 1 hour, 1.5 hours, 2 hours, 2.hours, 3 hours, 3.5 hours 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 16 hours, 18 WO 2024/238825 PCT/US2024/029746 296 hours, 20 hours, or any other period from about 30 minutes to about 24 hours, which can be repeated with a frequency of about every day to about every 4 days, e.g., every 1.5 days, every 2 days, every 3 days, or any other frequency from about every day to about every four days. The compositions may be provided to the subject one or more times, e.g., one time, twice, three times, or more than three times. In cases in which two or more different prime editing system components, e.g., two different polynucleotide constructs are administered to the subject (e.g., different components of the same prime editing system, or two different guide nucleic acids that are complementary to different sequences within the same or different target genes), the compositions may be administered simultaneously (e.g., as two polypeptides and/or nucleic acids). Alternatively, they may be provided sequentially, e.g., one composition being provided first, followed by a second composition. Table 1 Sequ ence Num her Sequence Description ACTCTCTCTTTCTGGCCTGG (SEQ ID NO: 1) SpacerCTCTCTCTTTCTGGCCTGG (SEQ ID NO: 2) SpacerTCTCTCTTTCTGGCCTGG (SEQ ID NO: 3) SpacerCTCTCTTTCTGGCCTGG (SEQ ID NO: 4) SpacerGGCCA PBSGGCCAG PBSGGCCAGA PBSGGCCAGAA PBSGGCCAGAAA PBSGGCCAGAAAG (SEQ ID NO: 10) PBSGGCCAGAAAGA (SEQ ID NO: 11) PBSGGCCAGAAAGAG (SEQ ID NO: 12) PBSGGCCAGAAAGAGA (SEQ ID NO: 13) PBSGGCCAGAAAGAGAG (SEQ ID NO: 14) PBSGGCCAGAAAGAGAGA (SEQ ID NO: 15) PBSGGCCAGAAAGAGAGAG (SEQ ID NO: 16) PBSGGCCAGAAAGAGAGAGT (SEQ ID NO: 17) PBSATAGCCTCTTATTACA (SEQ ID NO: 18) RTT (c.54InsTAATAA edit) 19CTGGATAGCCTCTTATTACA (SEQ ID NO: 19)RTT (c.54InsTAATAA edit) 20CACGCTGGATAGCCTCTTATTACA (SEQ ID NO: 20) RTT (c.54InsTAATAA edit) 900 CCTCGGCA (SEQ ID NO: 900) RTT (c.54_55insCC, NGG>NGC PAM silencing edit)*l WO 2024/238825 PCT/US2024/029746 297 901 AGCCTCGGCA (SEQ ID NO: 901) RTT (c.54_55insCC, NGONGC PAM silencing edit)*l 902 ATAGCCTCGGCA (SEQ ID NO: 902) RTT (c.54_55insCC, NGONGC PAM silencing edit)*l 903 GGATAGCCTCGGCA (SEQ ID NO: 903) RTT (c.54_55insCC, NGONGC PAM silencing edit)*l 904CACGGGCTGGATAGGCTCCA (SEQ ID NO: 904) RTT (c.66_67insCC, C.58OC, NGONGC PAM silencing edit)*2 905CTCACGGGCTGGATAGGCTCCA (SEQ ID NO: 905) RTT (c.66_67insCC, C.58OC, NGONGC PAM silencing edit)*2 906GACTCACGGGCTGGATAGGCTCCA (SEQ ID NO: 906) RTT (c.66_67insCC, C.58OC, NGONGC PAM silencing edit)*2 907GAGACTCACGGGCTGGATAGGCTCCA (SEQ ID NO: 907) RTT (c.66_67insCC, C.58OC, NGONGC PAM silencing edit)*2 908 CCTCCTTACA (SEQ ID NO: 908) RTT (c.54_55insTAAG, NGONGC PAM silencing edit)*3 909 AGCCTCCTTACA (SEQ ID NO: 909) RTT (c.54_55insTAAG, NGONGC PAM silencing edit)*3 910 ATAGCCTCCTTACA (SEQ ID NO: 910) RTT (c.54_55insTAAG, NGONGC PAM silencing edit)*3 911 GGATAGCCTCCTTACA (SEQ ID NO: 911) RTT (c.54_55insTAAG, NGONGC PAM silencing edit)*3 912CACGCTTACTGGATAGGCTCCA (SEQ ID NO: 912) RTT (c.66_67insTAAG, C.58OC, NGONGC PAM silencing edit)*4 913CTCACGCTTACTGGATAGGCTCCA (SEQ ID NO: 913) RTT (c.66_67insTAAG, C.58OC, NGONGC PAM silencing edit)*4 914GACTCACGCTTACTGGATAGGCTCCA (SEQ ID NO: 914) RTT (c.66_67insTAAG, C.58OC, NGONGC PAM silencing edit)*4 915GAGACTCACGCTTACTGGATAGGCTCCA (SEQ ID NO: 915) RTT (c.66_67insTAAG, C.58OC, NGONGC PAM silencing edit)*4 916 CCTCTTATTACA (SEQ ID NO: 916) RTT (c.54_55insTAATAA, NGONGC PAM silencing edit)*5 WO 2024/238825 PCT/US2024/029746 298 917 AGCCTCTTATTACA (SEQ ID NO: 917) RTT (c.54_55insTAATAA, NGONGC PAM silencing edit)*5 918 ATAGCCTCTTATTACA (SEQ ID NO: 918) RTT (c.54_55insTAATAA, NGONGC PAM silencing edit)*5 919 GGATAGCCTCTTATTACA (SEQ ID NO: 919) RTT (c.54_55insTAATAA, NGONGC PAM silencing edit)*5 920CACGTTATTACTGGATAGGCTCCA (SEQ ID NO: 920) RTT (c.66_67insTAATAA, C.58OC, NGONGC PAM silencing edit)*6 921CTCACGTTATTACTGGATAGGCTCCA (SEQ ID NO: 921) RTT (c.66_67insTAATAA, C.58OC, NGONGC PAM silencing edit)*6 922GACTCACGTTATTACTGGATAGGCTCCA (SEQ ID NO: 922) RTT (c.66_67insTAATAA, C.58OC, NGONGC PAM silencing edit)*6 923GAGACTCACGTTATTACTGGATAGGCTCCA (SEQ ID NO: 923) RTT (c.66_67insTAATAA, C.58OC, NGONGC PAM silencing edit)*6 924 CACGCTATTAAGGCTCCA (SEQ ID NO: 924) RTT (c.60_65delinsTAATAG, C.58OC, NGONGC PAM silencing edit)*7 925CTCACGCTATTAAGGCTCCA (SEQ ID NO: 925) RTT (c.60_65delinsTAATAG, C.58OC, NGONGC PAM silencing edit)*7 926GACTCACGCTATTAAGGCTCCA (SEQ ID NO: 926) RTT (c.60_65delinsTAATAG, C.58OC, NGONGC PAM silencing edit)*7 927GAGACTCACGCTATTAAGGCTCCA (SEQ ID NO: 927) RTT (c.60_65delinsTAATAG, C.58OC, NGONGC PAM silencing edit)*7 928 GATAGCCTCTTATTACA (SEQ ID NO: 928) RTT (c.54_55insTAATAA, NGONGC PAM silencing edit)*5 929 !GGATAGCCTCTTATTACA (SEQ ID NO: 929) RTT (c.54_55insTAATAA, NGONGC PAM silencing edit)*5 ACTCTCTCTTTCTGGCCTGGGTTTAAGAGCT AGAAATAGCAAGTTTAAATAAGGCTAGTCC GTTATCAGCGTGAAAACGCGGCACCGAGTC GGTGCATAGCCTCTTATTACAGGCCAGAAA (SEQ ID NO: 21) PEgRNA (c.54InsTAATAA edit, contains gRNA core SEQ ID NO: 653) WO 2024/238825 PCT/US2024/029746 299 ACTCTCTCTTTCTGGCCTGGGTTTAAGAGCT AGAAATAGCAAGTTTAAATAAGGCTAGTCC GTTATCAGCGTGAAAACGCGGCACCGAGTC GGTGCCTGGATAGCCTCTTATTACAGGCCA GAAA (SEQ ID NO: 22) PEgRNA (c.54InsTAATAA edit, contains gRNA core SEQ ID NO: 653) ACTCTCTCTTTCTGGCCTGGGTTTAAGAGCT AGAAATAGCAAGTTTAAATAAGGCTAGTCC GTTATCAGCGTGAAAACGCGGCACCGAGTC GGTGCCACGCTGGATAGCCTCTTATTACAG GCCAGAAA (SEQ ID NO: 23) PEgRNA (c.54InsTAATAA edit, contains gRNA core SEQ ID NO: 653) ACTCTCTCTTTCTGGCCTGGGTTTAAGAGCT AGAAATAGCAAGTTTAAATAAGGCTAGTCC GTTATCAGCGTGAAAACGCGGCACCGAGTC GGTGCATAGCCTCTTATTACAGGCCAGAAA GAG (SEQ ID NO: 24) PEgRNA (c.54InsTAATAA edit, contains gRNA core SEQ ID NO: 653) ACTCTCTCTTTCTGGCCTGGGTTTAAGAGCT AGAAATAGCAAGTTTAAATAAGGCTAGTCC GTTATCAGCGTGAAAACGCGGCACCGAGTC GGTGCCTGGATAGCCTCTTATTACAGGCCA GAAAGAG (SEQ ID NO: 25) PEgRNA (c.54InsTAATAA edit, contains gRNA core SEQ ID NO: 653) ACTCTCTCTTTCTGGCCTGGGTTTAAGAGCT AGAAATAGCAAGTTTAAATAAGGCTAGTCC GTTATCAGCGTGAAAACGCGGCACCGAGTC GGTGCCACGCTGGATAGCCTCTTATTACAG GCCAGAAAGAG (SEQ ID NO: 26) PEgRNA (c.54InsTAATAA edit, contains gRNA core SEQ ID NO: 653) ACTCTCTCTTTCTGGCCTGGGTTTAAGAGCT AGAAATAGCAAGTTTAAATAAGGCTAGTCC GTTATCAGCGTGAAAACGCGGCACCGAGTC GGTGCATAGCCTCTTATTACAGGCCAGAAA GAGAGA (SEQ ID NO: 27) PEgRNA (c.54InsTAATAA edit, contains gRNA core SEQ ID NO: 653) ACTCTCTCTTTCTGGCCTGGGTTTAAGAGCT AGAAATAGCAAGTTTAAATAAGGCTAGTCC GTTATCAGCGTGAAAACGCGGCACCGAGTC GGTGCCTGGATAGCCTCTTATTACAGGCCA GAAAGAGAGA (SEQ ID NO: 28) PEgRNA (c.54InsTAATAA edit, contains gRNA core SEQ ID NO: 653) ACTCTCTCTTTCTGGCCTGGGTTTAAGAGCT AGAAATAGCAAGTTTAAATAAGGCTAGTCC GTTATCAGCGTGAAAACGCGGCACCGAGTC GGTGCCACGCTGGATAGCCTCTTATTACAG GCCAGAAAGAGAGA (SEQ ID NO: 29) PEgRNA (c.54InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 930 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCCTCGGCAGGCCAGAA (SEQ ID NO: 930) PEgRNA (c.54_55insCC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)* 1 931 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT PEgRNA (c.54_55insCC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)* 1 WO 2024/238825 PCT/US2024/029746 300 CGGTGCAGCCTCGGCAGGCCAGAA (SEQ ID NO: 931) 932 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCATAGCCTCGGCAGGCCAGAA (SEQ ID NO: 932) PEgRNA (c.54_55insCC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)* 1 933 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGGATAGCCTCGGCAGGCCAGAA (SEQ ID NO: 933) PEgRNA (c.54_55insCC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)* 1 934 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCCTCGGCAGGCCAGAAAG (SEQ ID NO: 934) PEgRNA (c.54_55insCC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)* 1 935 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCAGCCTCGGCAGGCCAGAAAG (SEQ ID NO: 935) PEgRNA (c.54_55insCC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)* 1 936 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCATAGCCTCGGCAGGCCAGAAAG (SEQ ID NO: 936) PEgRNA (c.54_55insCC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)* 1 937 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGGATAGCCTCGGCAGGCCAGAAA G (SEQ ID NO: 937) PEgRNA (c.54_55insCC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)* 1 938 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCCTCGGCAGGCCAGAAAGAG (SEQ ID NO: 938) PEgRNA (c.54_55insCC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)* 1 939 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCAGCCTCGGCAGGCCAGAAAGAG (SEQ ID NO: 939) PEgRNA (c.54_55insCC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)* 1 940 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCATAGCCTCGGCAGGCCAGAAAGA G (SEQ ID NO: 940) PEgRNA (c.54_55insCC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)* 1 941ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTCPEgRNA (c.54_55insCC, NGONGC PAM silencing WO 2024/238825 PCT/US2024/029746 301 CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGGATAGCCTCGGCAGGCCAGAAA GAG (SEQ ID NO: 941) edit, contains gRNA core SEQ ID NO: 646)* 1 942 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCACGGGCTGGATAGGCTCCAGGCC AGAA (SEQ ID NO: 942) PEgRNA (c.66_67insCC, C.58OC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*2 943 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCTCACGGGCTGGATAGGCTCCAGG CCAGAA (SEQ ID NO: 943) PEgRNA (c.66_67insCC, C.58OC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*2 944 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGACTCACGGGCTGGATAGGCTCCA GGCCAGAA (SEQ ID NO: 944) PEgRNA (c.66_67insCC, c.58G>C, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*2 945 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGAGACTCACGGGCTGGATAGGCTC CAGGCCAGAA (SEQ ID NO: 945) PEgRNA (c.66_67insCC, C.58OC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*2 946 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCACGGGCTGGATAGGCTCCAGGCC AGAAAG (SEQ ID NO: 946) PEgRNA (c.66_67insCC, C.58OC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*2 947 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCTCACGGGCTGGATAGGCTCCAGG CCAGAAAG (SEQ ID NO: 947) PEgRNA (c.66_67insCC, C.58OC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*2 948 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGACTCACGGGCTGGATAGGCTCCA GGCCAGAAAG (SEQ ID NO: 948) PEgRNA (c.66_67insCC, C.58OC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*2 949 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGAGACTCACGGGCTGGATAGGCTC CAGGCCAGAAAG (SEQ ID NO: 949) PEgRNA (c.66_67insCC, C.58OC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*2 950 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCACGGGCTGGATAGGCTCCAGGCC AGAAAGAG (SEQ ID NO: 950) PEgRNA (c.66_67insCC, C.58OC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*2 WO 2024/238825 PCT/US2024/029746 302 951 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCTCACGGGCTGGATAGGCTCCAGG CCAGAAAGAG (SEQ ID NO: 951) PEgRNA (c.66_67insCC, C.58OC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*2 952 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGACTCACGGGCTGGATAGGCTCCA GGCCAGAAAGAG (SEQ ID NO: 952) PEgRNA (c.66_67insCC, C.58OC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*2 953 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGAGACTCACGGGCTGGATAGGCTC CAGGCCAGAAAGAG (SEQ ID NO: 953) PEgRNA (c.66_67insCC, c.58G>C, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*2 954 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCCTCCTTACAGGCCAGAA (SEQ ID NO: 954) PEgRNA (c.54_55insTAAG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*3 955 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCAGCCTCCTTACAGGCCAGAA (SEQ ID NO: 955) PEgRNA (c.54_55insTAAG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*3 956 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCATAGCCTCCTTACAGGCCAGAA (SEQ ID NO: 956) PEgRNA (c.54_55insTAAG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*3 957 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGGATAGCCTCCTTACAGGCCAGAA (SEQ ID NO: 957) PEgRNA (c.54_55insTAAG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*3 958 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCCTCCTTACAGGCCAGAAAG (SEQ ID NO: 958) PEgRNA (c.54_55insTAAG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*3 959 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCAGCCTCCTTACAGGCCAGAAAG (SEQ ID NO: 959) PEgRNA (c.54_55insTAAG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*3 960 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT PEgRNA (c.54_55insTAAG, NGONGC PAM silencing WO 2024/238825 PCT/US2024/029746 303 CGGTGCATAGCCTCCTTACAGGCCAGAAAG (SEQIDNO: 960)edit, contains gRNA core SEQ ID NO: 646)*3 961 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGGATAGCCTCCTTACAGGCCAGAA AG(SEQIDNO: 961) PEgRNA (c.54_55insTAAG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*3 962 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCCTCCTTACAGGCCAGAAAGAG (SEQIDNO: 962) PEgRNA (c.54_55insTAAG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*3 963 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCAGCCTCCTTACAGGCCAGAAAGA G (SEQ ID NO: 963) PEgRNA (c.54_55insTAAG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*3 964 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCATAGCCTCCTTACAGGCCAGAAAG AG (SEQ ID NO: 964) PEgRNA (c.54_55insTAAG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*3 965 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGGATAGCCTCCTTACAGGCCAGAA AGAG(SEQIDNO: 965) PEgRNA (c.54_55insTAAG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*3 966 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCACGCTTACTGGATAGGCTCCAGG CCAGAA (SEQ ID NO: 966) PEgRNA (c.66_67insTAAG, C.58OC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*4 967 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCTCACGCTTACTGGATAGGCTCCA GGCCAGAA (SEQ ID NO: 967) PEgRNA (c.66_67insTAAG, C.58OC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*4 968 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGACTCACGCTTACTGGATAGGCTC CAGGCCAGAA (SEQ ID NO: 968) PEgRNA (c.66_67insTAAG, C.58OC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*4 969 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT PEgRNA (c.66_67insTAAG, C.58OC, NGONGC PAM silencing edit, contains WO 2024/238825 PCT/US2024/029746 304 CGGTGCGAGACTCACGCTTACTGGATAGGCTCCAGGCCAGAA (SEQ ID NO: 969)gRNA core SEQ ID NO: 646)*4 970 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCACGCTTACTGGATAGGCTCCAGG CCAGAAAG (SEQ ID NO: 970) PEgRNA (c.66_67insTAAG, C.58OC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*4 971 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCTCACGCTTACTGGATAGGCTCCA GGCCAGAAAG (SEQ ID NO: 971) PEgRNA (c.66_67insTAAG, C.58OC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*4 972 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGACTCACGCTTACTGGATAGGCTC CAGGCCAGAAAG (SEQ ID NO: 972) PEgRNA (c.66_67insTAAG, c.58G>C, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*4 973 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGAGACTCACGCTTACTGGATAGGC TCCAGGCCAGAAAG (SEQ ID NO: 973) PEgRNA (c.66_67insTAAG, C.58OC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*4 974 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCTCACGCTTACTGGATAGGCTCCA GGCCAGAAAGAG (SEQ ID NO: 974) PEgRNA (c.66_67insTAAG, C.58OC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*4 975 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGACTCACGCTTACTGGATAGGCTC CAGGCCAGAAAGAG (SEQ ID NO: 975) PEgRNA (c.66_67insTAAG, C.58OC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*4 976 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGAGACTCACGCTTACTGGATAGGC TCCAGGCCAGAAAGAG (SEQ ID NO: 976) PEgRNA (c.66_67insTAAG, C.58OC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*4 977 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCCTCTTATTACAGGCCAGAA (SEQ ID NO: 977) PEgRNA (c.54_55insTAATAA, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*5 WO 2024/238825 PCT/US2024/029746 305 978 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCAGCCTCTTATTACAGGCCAGAA (SEQIDNO: 978) PEgRNA (c.54_55insTAATAA, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*5 979 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCATAGCCTCTTATTACAGGCCAGAA (SEQIDNO: 979) PEgRNA (c.54_55insTAATAA, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*5 980 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGGATAGCCTCTTATTACAGGCCAG AA (SEQIDNO: 980) PEgRNA (c.54_55insTAATAA, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*5 981 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCCTCTTATTACAGGCCAGAAAG (SEQIDNO: 981) PEgRNA (c.54_55insTAATAA, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*5 982 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCAGCCTCTTATTACAGGCCAGAAAG (SEQIDNO: 982) PEgRNA (c.54_55insTAATAA, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*5 983 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCATAGCCTCTTATTACAGGCCAGAA AG(SEQIDNO: 983) PEgRNA (c.54_55insTAATAA, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*5 984 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGGATAGCCTCTTATTACAGGCCAG AAAG (SEQ ID NO: 984) PEgRNA (c.54_55insTAATAA, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*5 985 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCCTCTTATTACAGGCCAGAAAGAG (SEQIDNO: 985) PEgRNA (c.54_55insTAATAA, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*5 986 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCAGCCTCTTATTACAGGCCAGAAAG AG (SEQ ID NO: 986) PEgRNA (c.54_55insTAATAA, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*5 987 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT PEgRNA (c.54_55insTAATAA, NGONGC PAM silencing WO 2024/238825 PCT/US2024/029746 306 CGGTGCATAGCCTCTTATTACAGGCCAGAAAGAG(SEQIDNO: 987)edit, contains gRNA core SEQ ID NO: 646)*5 988 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGGATAGCCTCTTATTACAGGCCAG AAAGAG (SEQ ID NO: 988) PEgRNA (c.54_55insTAATAA, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*5 989 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCACGTTATTACTGGATAGGCTCCA GGCCAGAA (SEQ ID NO: 989) PEgRNA (c.66_67insTAATAA, C.58OC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*6 990 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCTCACGTTATTACTGGATAGGCTC CAGGCCAGAA (SEQ ID NO: 990) PEgRNA (c.66_67insTAATAA, c.58G>C, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*6 991 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGACTCACGTTATTACTGGATAGGC TCCAGGCCAGAA (SEQ ID NO: 991) PEgRNA (c.66_67insTAATAA, C.58OC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*6 992 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGAGACTCACGTTATTACTGGATAG GCTCCAGGCCAGAA (SEQ ID NO: 992) PEgRNA (c.66_67insTAATAA, C.58OC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*6 993 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCACGTTATTACTGGATAGGCTCCA GGCCAGAAAG (SEQ ID NO: 993) PEgRNA (c.66_67insTAATAA, C.58OC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*6 994 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCTCACGTTATTACTGGATAGGCTC CAGGCCAGAAAG (SEQ ID NO: 994) PEgRNA (c.66_67insTAATAA, C.58OC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*6 995 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGACTCACGTTATTACTGGATAGGC TCCAGGCCAGAAAG (SEQ ID NO: 995) PEgRNA (c.66_67insTAATAA, C.58OC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*6 WO 2024/238825 PCT/US2024/029746 307 996 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGAGACTCACGTTATTACTGGATAG GCTCCAGGCCAGAAAG (SEQ ID NO: 996) PEgRNA (c.66_67insTAATAA, C.58OC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*6 997 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCACGTTATTACTGGATAGGCTCCA GGCCAGAAAGAG (SEQ ID NO: 997) PEgRNA (c.66_67insTAATAA, C.58OC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*6 998 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCTCACGTTATTACTGGATAGGCTC CAGGCCAGAAAGAG (SEQ ID NO: 998) PEgRNA (c.66_67insTAATAA, c.58G>C, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*6 999 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGACTCACGTTATTACTGGATAGGC TCCAGGCCAGAAAGAG (SEQ ID NO: 999) PEgRNA (c.66_67insTAATAA, C.58OC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*6 1000 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGAGACTCACGTTATTACTGGATAG GCTCCAGGCCAGAAAGAG (SEQ ID NO: 1000) PEgRNA (c.66_67insTAATAA, C.58OC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*6 1001 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCACGCTATTAAGGCTCCAGGCCAG AA (SEQ ID NO: 1001) PEgRNA (c.60_65delinsTAATAG, C.58OC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*7 1002 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCTCACGCTATTAAGGCTCCAGGCC AGAA (SEQ ID NO: 1002) PEgRNA (c.60_65delinsTAATAG, C.58OC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*7 1003 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGACTCACGCTATTAAGGCTCCAGG CCAGAA (SEQ ID NO: 1003) PEgRNA (c.60_65delinsTAATAG, C.58OC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*7 WO 2024/238825 PCT/US2024/029746 308 1004 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGAGACTCACGCTATTAAGGCTCCA GGCCAGAA (SEQ ID NO: 1004) PEgRNA (c.60_65delinsTAATAG, C.58OC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*7 1005 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCACGCTATTAAGGCTCCAGGCCAG AAAG (SEQ ID NO: 1005) PEgRNA (c.60_65delinsTAATAG, C.58OC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*7 1006 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCTCACGCTATTAAGGCTCCAGGCC AGAAAG (SEQ ID NO: 1006) PEgRNA (c.60_65delinsTAATAG, c.58G>C, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*7 1007 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGACTCACGCTATTAAGGCTCCAGG CCAGAAAG (SEQ ID NO: 1007) PEgRNA (c.60_65delinsTAATAG, C.58OC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*7 1008 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGAGACTCACGCTATTAAGGCTCCA GGCCAGAAAG (SEQ ID NO: 1008) PEgRNA (c.60_65delinsTAATAG, C.58OC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*7 1009 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCACGCTATTAAGGCTCCAGGCCAG AAAGAG (SEQ ID NO: 1009) PEgRNA (c.60_65delinsTAATAG, C.58OC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*7 1010 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCTCACGCTATTAAGGCTCCAGGCC AGAAAGAG (SEQ ID NO: 1010) PEgRNA (c.60_65delinsTAATAG, C.58OC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*7 1011 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGACTCACGCTATTAAGGCTCCAGG CCAGAAAGAG (SEQ ID NO: 1011) PEgRNA (c.60_65delinsTAATAG, C.58OC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*7 WO 2024/238825 PCT/US2024/029746 309 1012 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGAGACTCACGCTATTAAGGCTCCA GGCCAGAAAGAG (SEQ ID NO: 1012) PEgRNA (c.60_65delinsTAATAG, C.58OC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*7 1013 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGATAGCCTCTTATTACAGGCCAGA AAGAG (SEQ ID NO: 1013) PEgRNA (c.54_55insTAATAA, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*5 1014 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGATAGCCTCTTATTACAGGCCAGA AAG(SEQIDNO: 1014) PEgRNA (c.54_55insTAATAA, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*5 1015 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGATAGCCTCTTATTACAGGCCAGA A (SEQ ID NO: 1015) PEgRNA (c.54_55insTAATAA, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*5 1016 ACTCTCTCTTTCTGGCCTGGGTTTTAGAGCT AGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCTGGATAGCCTCTTATTACAGGCCA GAAAGAG (SEQ ID NO: 1016) PEgRNA (c.54_55insTAATAA, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*5 1017GGCCGAGATGTCTCGCTCCG (SEQ ID NO: 1017) ngRNA spacer 1018TCACGCTGGATAGCCTCGGC (SEQ ID NO: 1018) ngRNA spacer*! 1019TCACGGGCTGGATAGGCTCC (SEQ ID NO: 1019) ngRNA spacer*2 1020ACGCTGGATAGCCTCCTTAC (SEQ ID NO: 1020) ngRNA spacer* 3 1021ACGCTTACTGGATAGGCTCC (SEQ ID NO: 1021) ngRNA spacer*4 1022GCTGGATAGCCTCTTATTAC (SEQ ID NO: 1022) ngRNA spacer* 5 1023GTTATTACTGGATAGGCTCC (SEQ ID NO: 1023) ngRNA spacer* 6 1024ACTCACGCTATTAAGGCTCC (SEQ ID NO: 1024) ngRNA spacer*7 1025 GGCCGAGATGTCTCGCTCCGGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGT CCGTTATCAACTTGAAAAAGTGGCACCGAG TCGGTG (SEQ ID NO: 1025) ngRNA (contains ngRNA spacer SEQ ID NO: 10and gRNA core SEQ ID NO:646) 1026TCACGCTGGATAGCCTCGGCGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTngRNA* 1 (contains ngRNA spacer SEQ ID NO: 1018 WO 2024/238825 PCT/US2024/029746 310 CCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTG (SEQ ID NO: 1026)and gRNA core SEQ ID NO:646) 1027 TCACGGGCTGGATAGGCTCCGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGT CCGTTATCAACTTGAAAAAGTGGCACCGAG TCGGTG (SEQ ID NO: 1027) ngRNA*2 (contains ngRNA spacer SEQ ID NO: 10and gRNA core SEQ ID NO:646) 1028 ACGCTGGATAGCCTCCTTACGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGT CCGTTATCAACTTGAAAAAGTGGCACCGAG TCGGTG (SEQ ID NO: 1028) ngRNA* 3 (contains ngRNA spacer SEQ ID NO: 10and gRNA core SEQ IDNO:646) 1029 ACGCTTACTGGATAGGCTCCGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGT CCGTTATCAACTTGAAAAAGTGGCACCGAG TCGGTG (SEQ ID NO: 1029) ngRNA*4 (contains ngRNA spacer SEQ ID NO: 10and gRNA core SEQ IDNO:646) 1030 GCTGGATAGCCTCTTATTACGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGT CCGTTATCAACTTGAAAAAGTGGCACCGAG TCGGTG (SEQ ID NO: 1030) ngRNA* 5 (contains ngRNA spacer SEQ ID NO: 10and gRNA core SEQ IDNO:646) 1031 GTTATTACTGGATAGGCTCCGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGT CCGTTATCAACTTGAAAAAGTGGCACCGAG TCGGTG (SEQ ID NO: 1031) ngRNA* 6 (contains ngRNA spacer SEQ ID NO: 10and gRNA core SEQ IDNO:646) 1032 ACTCACGCTATTAAGGCTCCGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGT CCGTTATCAACTTGAAAAAGTGGCACCGAG TCGGTG (SEQ ID NO: 1032) ngRNA*7 (contains ngRNA spacer SEQ ID NO: 10and gRNA core SEQ IDNO:646) Table 2 Seque nce Numb er Sequence Description 30GGCCGAGATGTCTCGCTCCG (SEQ ID NO: 30) Spacer 31GCCGAGATGTCTCGCTCCG (SEQ ID NO: 31) Spacer 32CCGAGATGTCTCGCTCCG (SEQ ID NO: 32) Spacer 33CGAGATGTCTCGCTCCG (SEQ ID NO: 33) SpacerAGCGA PBSAGCGAG PBSAGCGAGA PBSAGCGAGAC PBS WO 2024/238825 PCT/US2024/029746 311 Seque nee Numb er Sequence Description 38 AGCGAGACA PBSAGCGAGACAT (SEQ ID NO: 39) PBSAGCGAGACATC (SEQ ID NO: 40) PBSAGCGAGACATCT (SEQ ID NO: 41) PBSAGCGAGACATCTC (SEQ ID NO: 42) PBSAGCGAGACATCTCG (SEQ ID NO: 43) PBS 44AGCGAGACATCTCGG (SEQ ID NO: 44) PBS 45AGCGAGACATCTCGGC (SEQ ID NO: 45) PBS 46AGCGAGACATCTCGGCC (SEQ ID NO: 46) PBS 47AAGGCCACTTATTAGG (SEQ ID NO: 47) RTT (c.l2insTAATAA edit) 48AGCTAAGGCCACTTATTAGG (SEQ ID NO: 48) RTT (c.l2insTAATAA edit) 49GCACAGCTAAGGCCACTTATTAGG (SEQ ID NO: 49) RTT (c.l2insTAATAA edit) GGCCGAGATGTCTCGCTCCGGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCAAG GCCACTTATTAGGAGCGAGACA (SEQ ID NO: 50) PEgRNA (c.l2InsTAATAA edit, contains gRNA core SEQ ID NO: 653) GGCCGAGATGTCTCGCTCCGGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCAGCT AAGGCCACTTATTAGGAGCGAGACA (SEQ ID NO: 51) PEgRNA (c.l2InsTAATAA edit, contains gRNA core SEQ ID NO: 653) GGCCGAGATGTCTCGCTCCGGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCGCAC AGCTAAGGCCACTTATTAGGAGCGA GACA (SEQ ID NO: 52) PEgRNA (c.l2InsTAATAA edit, contains gRNA core SEQ ID NO: 653) WO 2024/238825 PCT/US2024/029746 312 Seque nee Numb er Sequence Description GGCCGAGATGTCTCGCTCCGGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCAAG GCCACTTATTAGGAGCGAGACATCT (SEQ ID NO: 53) PEgRNA (c.l2InsTAATAA edit, contains gRNA core SEQ ID NO: 653) GGCCGAGATGTCTCGCTCCGGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCAGCT AAGGCCACTTATTAGGAGCGAGACA TCT (SEQ ID NO: 54) PEgRNA (c.l2InsTAATAA edit, contains gRNA core SEQ ID NO: 653) GGCCGAGATGTCTCGCTCCGGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCGCAC AGCTAAGGCCACTTATTAGGAGCGA GACATCT (SEQ ID NO: 55) PEgRNA (c.l2InsTAATAA edit, contains gRNA core SEQ ID NO: 653) GGCCGAGATGTCTCGCTCCGGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCAAG GCCACTTATTAGGAGCGAGACATCT CGG (SEQ ID NO: 56) PEgRNA (c.l2InsTAATAA edit, contains gRNA core SEQ ID NO: 653) GGCCGAGATGTCTCGCTCCGGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCAGCT AAGGCCACTTATTAGGAGCGAGACA TCTCGG (SEQ ID NO: 57) PEgRNA (c.l2InsTAATAA edit, contains gRNA core SEQ ID NO: 653) GGCCGAGATGTCTCGCTCCGGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCGCAC AGCTAAGGCCACTTATTAGGAGCGA GACATCTCGG (SEQ ID NO: 58) PEgRNA (c.l2InsTAATAA edit, contains gRNA core SEQ ID NO: 653) WO 2024/238825 PCT/US2024/029746 313 Table 3 Seque nce Numb er Sequence Description 59CTCGCGCTACTCTCTCTTTC (SEQ ID NO: 59) Spacer 60TCGCGCTACTCTCTCTTTC (SEQ ID NO: 60) Spacer 61CGCGCTACTCTCTCTTTC (SEQ ID NO: 61) Spacer 62GCGCTACTCTCTCTTTC (SEQ ID NO: 62) SpacerAGAGA PBSAGAGAG PBSAGAGAGA PBSAGAGAGAG PBSAGAGAGAGT PBSAGAGAGAGTA (SEQ ID NO: 68) PBSAGAGAGAGTAG (SEQ ID NO: 69) PBSAGAGAGAGTAGC (SEQ ID NO: 70) PBSAGAGAGAGTAGCG (SEQ ID NO: 71) PBSAGAGAGAGTAGCGC (SEQ ID NO: 72) PBS 73AGAGAGAGTAGCGCG (SEQ ID NO: 73) PBS 74AGAGAGAGTAGCGCGA (SEQ ID NO: 74) PBS 75AGAGAGAGTAGCGCGAG (SEQ ID NO: 75) PBS 76CAGGCCAGATTATTAA (SEQ ID NO: 76) RTT (c.46insTAATAA edit) 77CCTCCAGGCCAGATTATTAA (SEQ ID NO: 77) RTT (c.46insTAATAA edit) 78ATAGCCTCCAGGCCAGATTATTAA (SEQ ID NO: 78) RTT (c.46insTAATAA edit) WO 2024/238825 PCT/US2024/029746 314 Seque nee Numb er Sequence Description CTCGCGCTACTCTCTCTTTCGTTTAA GAGCTAGAAATAGCAAGTTTAAATA AGGCTAGTCCGTTATCAGCGTGAAA ACGCGGCACCGAGTCGGTGCCAGGC CAGATTATTAAAGAGAGAGT (SEQ ID NO: 79) PEgRNA (c.46InsTAATAA edit, contains gRNA core SEQ ID NO: 653) CTCGCGCTACTCTCTCTTTCGTTTAA GAGCTAGAAATAGCAAGTTTAAATA AGGCTAGTCCGTTATCAGCGTGAAA ACGCGGCACCGAGTCGGTGCCCTCC AGGCCAGATTATTAAAGAGAGAGT (SEQ ID NO: 80) PEgRNA (c.46InsTAATAA edit, contains gRNA core SEQ ID NO: 653) CTCGCGCTACTCTCTCTTTCGTTTAA GAGCTAGAAATAGCAAGTTTAAATA AGGCTAGTCCGTTATCAGCGTGAAA ACGCGGCACCGAGTCGGTGCATAGC CTCCAGGCCAGATTATTAAAGAGAG ACT (SEQ ID NO: 81) PEgRNA (c.46InsTAATAA edit, contains gRNA core SEQ ID NO: 653) CTCGCGCTACTCTCTCTTTCGTTTAA GAGCTAGAAATAGCAAGTTTAAATA AGGCTAGTCCGTTATCAGCGTGAAA ACGCGGCACCGAGTCGGTGCCAGGC CAGATTATTAAAGAGAGAGTAGC (SEQ ID NO: 82) PEgRNA (c.46InsTAATAA edit, contains gRNA core SEQ ID NO: 653) CTCGCGCTACTCTCTCTTTCGTTTAA GAGCTAGAAATAGCAAGTTTAAATA AGGCTAGTCCGTTATCAGCGTGAAA ACGCGGCACCGAGTCGGTGCCCTCC AGGCCAGATTATTAAAGAGAGAGTA GC (SEQ ID NO: 83) PEgRNA (c.46InsTAATAA edit, contains gRNA core SEQ ID NO: 653) CTCGCGCTACTCTCTCTTTCGTTTAA GAGCTAGAAATAGCAAGTTTAAATA AGGCTAGTCCGTTATCAGCGTGAAA ACGCGGCACCGAGTCGGTGCATAGC CTCCAGGCCAGATTATTAAAGAGAG AGTAGC (SEQ ID NO: 84) PEgRNA (c.46InsTAATAA edit, contains gRNA core SEQ ID NO: 653) CTCGCGCTACTCTCTCTTTCGTTTAA GAGCTAGAAATAGCAAGTTTAAATA AGGCTAGTCCGTTATCAGCGTGAAA ACGCGGCACCGAGTCGGTGCCAGGC CAGATTATTAAAGAGAGAGTAGCGC G (SEQ ID NO: 85) PEgRNA (c.46InsTAATAA edit, contains gRNA core SEQ ID NO: 653) WO 2024/238825 PCT/US2024/029746 315 Seque nce Numb er Sequence Description CTCGCGCTACTCTCTCTTTCGTTTAA GAGCTAGAAATAGCAAGTTTAAATA AGGCTAGTCCGTTATCAGCGTGAAA ACGCGGCACCGAGTCGGTGCCCTCC AGGCCAGATTATTAAAGAGAGAGTA GCGCG (SEQIDNO: 86) PEgRNA (c.46InsTAATAA edit, contains gRNA core SEQ ID NO: 653) CTCGCGCTACTCTCTCTTTCGTTTAA GAGCTAGAAATAGCAAGTTTAAATA AGGCTAGTCCGTTATCAGCGTGAAA ACGCGGCACCGAGTCGGTGCATAGC CTCCAGGCCAGATTATTAAAGAGAG AGTAGCGCG (SEQ ID NO: 87) PEgRNA (c.46InsTAATAA edit, contains gRNA core SEQ ID NO: 653) Table 4 Seque nce Numb er Sequence Description 88GCTACTCTCTCTTTCTGGCC (SEQ ID NO: 88) Spacer 89CTACTCTCTCTTTCTGGCC (SEQ ID NO: 89) Spacer 90TACTCTCTCTTTCTGGCC (SEQ ID NO: 90) Spacer 91ACTCTCTCTTTCTGGCC (SEQ ID NO: 91)SpacerCAGAA PBSCAGAAA PBSCAGAAAG PBSCAGAAAGA PBSCAGAAAGAG PBSCAGAAAGAGA (SEQ ID NO: 97) PBSCAGAAAGAGAG (SEQ ID NO: 98) PBSCAGAAAGAGAGA (SEQ ID NO: 99) PBS100 CAGAAAGAGAGAG (SEQ ID NO: 100) PBS 101CAGAAAGAGAGAGT (SEQ ID NO: 101) PBS WO 2024/238825 PCT/US2024/029746 316 Seque nee Numb er Sequence Description 102CAGAAAGAGAGAGTA (SEQ ID NO: 102) PBS 103CAGAAAGAGAGAGTAG (SEQ ID NO: 103) PBS 104CAGAAAGAGAGAGTAGC (SEQ ID NO: 104) PBS 105GCCTCCAGTTATTAGC (SEQ ID NO: 105) RTT (c.51insTAATAA edit) 106GATAGCCTCCAGTTATTAGC (SEQ ID NO: 106) RTT (c.51insTAATAA edit) 107GCTGGATAGCCTCCAGTTATTAGC (SEQ ID NO: 107) RTT (c.51insTAATAA edit) 108 GCTACTCTCTCTTTCTGGCCGTTTAA GAGCTAGAAATAGCAAGTTTAAATA AGGCTAGTCCGTTATCAGCGTGAAA ACGCGGCACCGAGTCGGTGCGCCTC CAGTTATTAGCCAGAAAGAG (SEQ ID NO: 108) PEgRNA (c.51InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 109 GCTACTCTCTCTTTCTGGCCGTTTAA GAGCTAGAAATAGCAAGTTTAAATA AGGCTAGTCCGTTATCAGCGTGAAA ACGCGGCACCGAGTCGGTGCGATAG CCTCCAGTTATTAGCCAGAAAGAG (SEQ ID NO: 109) PEgRNA (c.51InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 110 GCTACTCTCTCTTTCTGGCCGTTTAA GAGCTAGAAATAGCAAGTTTAAATA AGGCTAGTCCGTTATCAGCGTGAAA ACGCGGCACCGAGTCGGTGCGCTGG ATAGCCTCCAGTTATTAGCCAGAAA GAG (SEQ ID NO: 110) PEgRNA (c.51InsTAATAA edit, contains gRNA core SEQ ID NO: 653) ill GCTACTCTCTCTTTCTGGCCGTTTAA GAGCTAGAAATAGCAAGTTTAAATA AGGCTAGTCCGTTATCAGCGTGAAA ACGCGGCACCGAGTCGGTGCGCCTC CAGTTATTAGCCAGAAAGAGAGA (SEQ ID NO: 111) PEgRNA (c.51InsTAATAA edit, contains gRNA core SEQ ID NO: 653) WO 2024/238825 PCT/US2024/029746 317 Seque nce Numb er Sequence Description 112 GCTACTCTCTCTTTCTGGCCGTTTAA GAGCTAGAAATAGCAAGTTTAAATA AGGCTAGTCCGTTATCAGCGTGAAA ACGCGGCACCGAGTCGGTGCGATAG CCTCCAGTTATTAGCCAGAAAGAGA GA (SEQ ID NO: 112) PEgRNA (c.51InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 113 GCTACTCTCTCTTTCTGGCCGTTTAA GAGCTAGAAATAGCAAGTTTAAATA AGGCTAGTCCGTTATCAGCGTGAAA ACGCGGCACCGAGTCGGTGCGCTGG ATAGCCTCCAGTTATTAGCCAGAAA GAGAGA (SEQ ID NO: 113) PEgRNA (c.51InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 114 GCTACTCTCTCTTTCTGGCCGTTTAA GAGCTAGAAATAGCAAGTTTAAATA AGGCTAGTCCGTTATCAGCGTGAAA ACGCGGCACCGAGTCGGTGCGCCTC CAGTTATTAGCCAGAAAGAGAGAGT A (SEQ ID NO: 114) PEgRNA (c.51InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 115 GCTACTCTCTCTTTCTGGCCGTTTAA GAGCTAGAAATAGCAAGTTTAAATA AGGCTAGTCCGTTATCAGCGTGAAA ACGCGGCACCGAGTCGGTGCGATAG CCTCCAGTTATTAGCCAGAAAGAGA GAGTA (SEQ ID NO: 115) PEgRNA (c.51InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 116 GCTACTCTCTCTTTCTGGCCGTTTAA GAGCTAGAAATAGCAAGTTTAAATA AGGCTAGTCCGTTATCAGCGTGAAA ACGCGGCACCGAGTCGGTGCGCTGG ATAGCCTCCAGTTATTAGCCAGAAA GAGAGAGTA (SEQ ID NO: 116) PEgRNA (c.51InsTAATAA edit, contains gRNA core SEQ ID NO: 653) Table 5 Seque nce Numb er Sequence Description 117TTCCTGAAGCTGACAGCATT (SEQ ID NO: 117) Spacer 118TCCTGAAGCTGACAGCATT (SEQ ID NO: 118) Spacer WO 2024/238825 PCT/US2024/029746 318 Seque nce Numb er Sequence Description 119CCTGAAGCTGACAGCATT (SEQ ID NO: 119) Spacer 120CTGAAGCTGACAGCATT (SEQ ID NO: 120) Spacer121 GCTGT PBS122 GCTGTC PBS123 GCTGTCA PBS124 GCTGTCAG PBS125 GCTGTCAGC PBS126 GCTGTCAGCT (SEQ ID NO: 126) PBS127 GCTGTCAGCTT (SEQ ID NO: 127) PBS128 GCTGTCAGCTTC (SEQ ID NO: 128) PBS129 GCTGTCAGCTTCA (SEQ ID NO: 129) PBS130 GCTGTCAGCTTCAG (SEQ ID NO: 130) PBS 131GCTGTCAGCTTCAGG (SEQ ID NO: 131) PBS 132GCTGTCAGCTTCAGGA (SEQ ID NO: 132) PBS 133GCTGTCAGCTTCAGGAA (SEQ ID NO: 133) PBS 134AGATTATTACATCTCGGCCCGAAT (SEQ ID NO: 134) RTT (c.4insTAATAA edit) 135AGCGAGATTATTACATCTCGGCCCGAAT(SEQIDNO: 135) RTT (c.4insTAATAA edit) 136ACGGAGCGAGATTATTACATCTCGGCCCGAAT (SEQ ID NO: 136) RTT (c.4insTAATAA edit) 137 TTCCTGAAGCTGACAGCATTGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCAGAT TATTACATCTCGGCCCGAATGCTGT CAGC (SEQ ID NO: 137) PEgRNA (c.4InsTAATAA edit, contains gRNA core SEQ ID NO: 653) WO 2024/238825 PCT/US2024/029746 319 Seque nee Numb er Sequence Description 138 TTCCTGAAGCTGACAGCATTGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCAGCG AGATTATTACATCTCGGCCCGAATG CTGTCAGC (SEQ ID NO: 138) PEgRNA (c.4InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 139 TTCCTGAAGCTGACAGCATTGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCACGG AGCGAGATTATTACATCTCGGCCCG AATGCTGTCAGC (SEQ ID NO: 139) PEgRNA (c.4InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 140 TTCCTGAAGCTGACAGCATTGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCAGAT TATTACATCTCGGCCCGAATGCTGT CAGCTTC (SEQ ID NO: 140) PEgRNA (c.4InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 141 TTCCTGAAGCTGACAGCATTGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCAGCG AGATTATTACATCTCGGCCCGAATG CTGTCAGCTTC (SEQ ID NO: 141) PEgRNA (c.4InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 142 TTCCTGAAGCTGACAGCATTGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCACGG AGCGAGATTATTACATCTCGGCCCG AATGCTGTCAGCTTC (SEQ ID NO: 142) PEgRNA (c.4InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 143 TTCCTGAAGCTGACAGCATTGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCAGAT TATTACATCTCGGCCCGAATGCTGT CAGCTTCAGG (SEQ ID NO: 143) PEgRNA (c.4InsTAATAA edit, contains gRNA core SEQ ID NO: 653) WO 2024/238825 PCT/US2024/029746 320 Seque nce Numb er Sequence Description 144 TTCCTGAAGCTGACAGCATTGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCAGCG AGATTATTACATCTCGGCCCGAATG CTGTCAGCTTCAGG (SEQ ID NO: 144) PEgRNA (c.4InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 145 TTCCTGAAGCTGACAGCATTGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCACGG AGCGAGATTATTACATCTCGGCCCG AATGCTGTCAGCTTCAGG (SEQ ID NO: 145) PEgRNA (c.4InsTAATAA edit, contains gRNA core SEQ ID NO: 653) Table 6 Seque nce Numb er Sequence Description 146TCCTGAAGCTGACAGCATTC (SEQ ID NO: 146) Spacer 147CCTGAAGCTGACAGCATTC (SEQ ID NO: 147) Spacer 148CTGAAGCTGACAGCATTC (SEQ ID NO: 148) Spacer 149TGAAGCTGACAGCATTC (SEQ ID NO: 149) Spacer150 TGCTG PBS151 TGCTGT PBS152 TGCTGTC PBS153 TGCTGTCA PBS154 TGCTGTCAG PBS155 TGCTGTCAGC (SEQ ID NO: 155) PBS156 TGCTGTCAGCT (SEQ ID NO: 156) PBS157 TGCTGTCAGCTT (SEQ ID NO: 157) PBS158 TGCTGTCAGCTTC (SEQ ID NO: 158) PBS159 TGCTGTCAGCTTCA (SEQ ID NO: 159) PBS WO 2024/238825 PCT/US2024/029746 321 Seque nee Numb er Sequence Description 160TGCTGTCAGCTTCAG (SEQ ID NO: 160) PBS 161TGCTGTCAGCTTCAGG (SEQ ID NO: 161) PBS 162TGCTGTCAGCTTCAGGA (SEQ ID NO: 162) PBS 163AGATTATTACATCTCGGCCCGAA (SEQ ID NO: 163) RTT (c.4insTAATAA edit) 164AGCGAGATTATTACATCTCGGCCCG AA (SEQ ID NO: 164) RTT (c.4insTAATAA edit) 165ACGGAGCGAGATTATTACATCTCGGCCCGAA (SEQ ID NO: 165) RTT (c.4insTAATAA edit) 166 TCCTGAAGCTGACAGCATTCGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCAGAT TATTACATCTCGGCCCGAATGCTGT CAG (SEQ ID NO: 166) PEgRNA (c.4InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 167 TCCTGAAGCTGACAGCATTCGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCAGCG AGATTATTACATCTCGGCCCGAATG CTGTCAG (SEQ ID NO: 167) PEgRNA (c.4InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 168 TCCTGAAGCTGACAGCATTCGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCACGG AGCGAGATTATTACATCTCGGCCCG AATGCTGTCAG (SEQ ID NO: 168) PEgRNA (c.4InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 169 TCCTGAAGCTGACAGCATTCGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCAGAT TATTACATCTCGGCCCGAATGCTGT CAGCTT (SEQ ID NO: 169) PEgRNA (c.4InsTAATAA edit, contains gRNA core SEQ ID NO: 653) WO 2024/238825 PCT/US2024/029746 322 Seque nce Numb er Sequence Description 170 TCCTGAAGCTGACAGCATTCGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCAGCG AGATTATTACATCTCGGCCCGAATG CTGTCAGCTT (SEQ ID NO: 170) PEgRNA (c.4InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 171 TCCTGAAGCTGACAGCATTCGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCACGG AGCGAGATTATTACATCTCGGCCCG AATGCTGTCAGCTT (SEQ ID NO: 171) PEgRNA (c.4InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 172 TCCTGAAGCTGACAGCATTCGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCAGAT TATTACATCTCGGCCCGAATGCTGT CAGCTTCAG (SEQ ID NO: 172) PEgRNA (c.4InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 173 TCCTGAAGCTGACAGCATTCGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCAGCG AGATTATTACATCTCGGCCCGAATG CTGTCAGCTTCAG (SEQ ID NO: 173) PEgRNA (c.4InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 174 TCCTGAAGCTGACAGCATTCGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCACGG AGCGAGATTATTACATCTCGGCCCG AATGCTGTCAGCTTCAG (SEQ ID NO: 174) PEgRNA (c.4InsTAATAA edit, contains gRNA core SEQ ID NO: 653) Table 7 Seque nce Numb er Sequence Description 175AAGGACCAGAGCGGGAGGGT (SEQ ID NO: 175) Spacer WO 2024/238825 PCT/US2024/029746 323 Seque nce Numb er Sequence Description 176AGGACCAGAGCGGGAGGGT (SEQ ID NO: 176) Spacer 177GGACCAGAGCGGGAGGGT (SEQ ID NO: 177) Spacer 178GACCAGAGCGGGAGGGT (SEQ ID NO: 178) Spacer179 CTCCC PBS180 CTCCCG PBS181 CTCCCGC PBS182 CTCCCGCT PBS183 CTCCCGCTC PBS184 CTCCCGCTCT (SEQ ID NO: 184) PBS185 CTCCCGCTCTG (SEQ ID NO: 185) PBS186 CTCCCGCTCTGG (SEQ ID NO: 186) PBS187 CTCCCGCTCTGGT (SEQ ID NO: 187) PBS188 CTCCCGCTCTGGTC (SEQ ID NO: 188) PBS 189CTCCCGCTCTGGTCC (SEQ ID NO: 189) PBS 190CTCCCGCTCTGGTCCT (SEQ ID NO: 190) PBS 191CTCCCGCTCTGGTCCTT (SEQ ID NO: 191) PBS 192CAGTAATAACGTGAGTCTCTCCTAC C (SEQ ID NO: 192) RTT (c.4insTAATAA edit) 193TATCCAGTAATAACGTGAGTCTCTCCTACC (SEQ ID NO: 193) RTT (c.4insTAATAA edit) 194AGGCTATCCAGTAATAACGTGAGTC TCTCCTACC (SEQ ID NO: 194) RTT (c.4insTAATAA edit) 195 AAGGACCAGAGCGGGAGGGTGTTT AAGAGCTAGAAATAGCAAGTTTAAA TAAGGCTAGTCCGTTATCAGCGTGA AAACGCGGCACCGAGTCGGTGCCAG TAATAACGTGAGTCTCTCCTACCCTC CCGCTC (SEQ ID NO: 195) PEgRNA (c.4InsTAATAA edit, contains gRNA core SEQ ID NO: 653) WO 2024/238825 PCT/US2024/029746 324 Seque nce Numb er Sequence Description 196 AAGGACCAGAGCGGGAGGGTGTTT AAGAGCTAGAAATAGCAAGTTTAAA TAAGGCTAGTCCGTTATCAGCGTGA AAACGCGGCACCGAGTCGGTGCTAT CCAGTAATAACGTGAGTCTCTCCTA CCCTCCCGCTC (SEQ ID NO: 196) PEgRNA (c.4InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 197 AAGGACCAGAGCGGGAGGGTGTTT AAGAGCTAGAAATAGCAAGTTTAAA TAAGGCTAGTCCGTTATCAGCGTGA AAACGCGGCACCGAGTCGGTGCAG GCTATCCAGTAATAACGTGAGTCTC TCCTACCCTCCCGCTC (SEQ ID NO: 197) PEgRNA (c.4InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 198 AAGGACCAGAGCGGGAGGGTGTTT AAGAGCTAGAAATAGCAAGTTTAAA TAAGGCTAGTCCGTTATCAGCGTGA AAACGCGGCACCGAGTCGGTGCCAG TAATAACGTGAGTCTCTCCTACCCTC CCGCTCTGG (SEQ ID NO: 198) PEgRNA (c.4InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 199 AAGGACCAGAGCGGGAGGGTGTTT AAGAGCTAGAAATAGCAAGTTTAAA TAAGGCTAGTCCGTTATCAGCGTGA AAACGCGGCACCGAGTCGGTGCTAT CCAGTAATAACGTGAGTCTCTCCTA CCCTCCCGCTCTGG (SEQ ID NO: 199) PEgRNA (c.4InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 200 AAGGACCAGAGCGGGAGGGTGTTT AAGAGCTAGAAATAGCAAGTTTAAA TAAGGCTAGTCCGTTATCAGCGTGA AAACGCGGCACCGAGTCGGTGCCAG TAATAACGTGAGTCTCTCCTACCCTC CCGCTCTGGTCC (SEQ ID NO: 200) PEgRNA (c.4InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 201 AAGGACCAGAGCGGGAGGGTGTTT AAGAGCTAGAAATAGCAAGTTTAAA TAAGGCTAGTCCGTTATCAGCGTGA AAACGCGGCACCGAGTCGGTGCTAT CCAGTAATAACGTGAGTCTCTCCTA CCCTCCCGCTCTGGTCC (SEQ ID NO: 201) PEgRNA (c.4InsTAATAA edit, contains gRNA core SEQ ID NO: 653) Table 8 Seq uen Sequence Description WO 2024/238825 PCT/US2024/029746 325 ce nu mbe r 202 ACTCACGCTGGATAGCCTCC (SEQ ID NO: 202) spacer203 CTCACGCTGGATAGCCTCC (SEQ ID NO: 203) spacer204 TCACGCTGGATAGCCTCC (SEQ ID NO: 204) spacer205 CACGCTGGATAGCCTCC (SEQ ID NO: 205) spacer206 GGCTA PBS207 GGCTAT PBS208 GGCTATC PBS209 GGCTATCC PBS210 GGCTATCCA PBS211 GGCTATCCAG (SEQ ID NO: 211) PBS212 GGCTATCCAGC (SEQ ID NO: 212) PBS213 GGCTATCCAGCG (SEQ ID NO: 213) PBS214 GGCTATCCAGCGT (SEQ ID NO: 214) PBS215 GGCTATCCAGCGTG (SEQ ID NO: 215) PBS216 GGCTATCCAGCGTGA (SEQ ID NO: 216) PBS217 GGCTATCCAGCGTGAG (SEQ ID NO: 217) PBS218 GGCTATCCAGCGTGAGT (SEQ ID NO: 218) PBS219 CTGGCCTGTAATAAGA (SEQ ID NO: 219) RTT (c.54InsTAATAA edit)220 CTTTCTGGCCTGTAATAAGA (SEQ ID NO: 220) RTT (c.54InsTAATAA edit)221 CTCTCTTTCTGGCCTGTAATAAGA (SEQ ID NO: 221) RTT (c.54InsTAATAA edit)222 TCTCTTTCTGGCTGGA (SEQ ID NO: 222) RTT (c.51delC edit)*l223 TCTGGCTGGA (SEQ ID NO: 223) RTT (c.51delC edit)*l224 TTTCTGGCTGGA (SEQ ID NO: 224) RTT (c.51delC edit)*l225 TCTTTCTGGCTGGA (SEQ ID NO: 225) RTT (c.51delC edit)*l226 TCTCTCTTTCTGGCTGGA (SEQ ID NO: 226) RTT (c.51delC edit)*l227 ACTCTCTCTTTCTGGCTGGA (SEQ ID NO: 227) RTT (c.51delC edit)*l228 CTGGGCCTGGA (SEQ ID NO: 228) RTT (c.50insG edit)*2229 TTCTGGGCCTGGA (SEQ ID NO: 229) RTT (c.50insG edit)*2230 CTTTCTGGGCCTGGA (SEQ ID NO: 230) RTT (c.50insG edit)*2 WO 2024/238825 PCT/US2024/029746 326 231 CTCTTTCTGGGCCTGGA (SEQ ID NO: 231) RTT (c.50insG edit)*2232 ACTCACGCTGGATAGCCTCCGTTTTAGAGCTAGAA ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAAC TTGAAAAAGTGGCACCGAGTCGGTGCTCTCTTTCTG GCTGGAGGCTATCCAGCGTTTT (SEQ ID NO: 232) PEgRNA (c.51delC edit, contains gRNA core SEQ ID NO: 646)* 1233 ACTCACGCTGGATAGCCTCCGTTTTAGAGCTAGAA ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAAC TTGAAAAAGTGGCACCGAGTCGGTGCTCTGGCTGG AGGCTATCCAGCGTTTT (SEQ ID NO: 233) PEgRNA (c.51delC edit, contains gRNA core SEQ ID NO: 646)* 1234 ACTCACGCTGGATAGCCTCCGTTTTAGAGCTAGAA ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAAC TTGAAAAAGTGGCACCGAGTCGGTGCTTTCTGGCT GGAGGCTATCCAGCGTTTT (SEQ ID NO: 234) PEgRNA (c.51delC edit, contains gRNA core SEQ ID NO: 646)* 1235 ACTCACGCTGGATAGCCTCCGTTTTAGAGCTAGAA ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAAC TTGAAAAAGTGGCACCGAGTCGGTGCTCTTTCTGG CTGGAGGCTATCCAGCGTTTT (SEQ ID NO: 235) PEgRNA (c.51delC edit, contains gRNA core SEQ ID NO: 646)* 1236 ACTCACGCTGGATAGCCTCCGTTTTAGAGCTAGAA ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAAC TTGAAAAAGTGGCACCGAGTCGGTGCTCTCTCTTTC TGGCTGGAGGCTATCCAGCGTTTT (SEQ ID NO: 236) PEgRNA (c.51delC edit, contains gRNA core SEQ ID NO: 646)* 1237 ACTCACGCTGGATAGCCTCCGTTTTAGAGCTAGAA ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAAC TTGAAAAAGTGGCACCGAGTCGGTGCACTCTCTCT TTCTGGCTGGAGGCTATCCAGCGTTTT (SEQ ID NO: 237) PEgRNA (c.51delC edit, contains gRNA core SEQ ID NO: 646)* 1 238 ACTCACGCTGGATAGCCTCCGTTTTAGAGCTAGAA ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAAC TTGAAAAAGTGGCACCGAGTCGGTGCCTTTCTGGG GCTGGAGGCTATCCAGCGTTTT (SEQ ID NO: 238) PEgRNA (c.50insG edit, contains gRNA core SEQ ID NO: 646)*2239 ACTCACGCTGGATAGCCTCCGTTTTAGAGCTAGAA ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAAC TTGAAAAAGTGGCACCGAGTCGGTGCCTGGGCCTG GAGGCTATCCTTTT (SEQ ID NO: 239) PEgRNA (c.50insG edit, contains gRNA core SEQ ID NO: 646)*2240 ACTCACGCTGGATAGCCTCCGTTTTAGAGCTAGAA ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAAC TTGAAAAAGTGGCACCGAGTCGGTGCTTCTGGGCC TGGAGGCTATCCTTTT (SEQ ID NO: 240) PEgRNA (c.50insG edit, contains gRNA core SEQ ID NO: 646)*2241 ACTCACGCTGGATAGCCTCCGTTTTAGAGCTAGAA ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAAC TTGAAAAAGTGGCACCGAGTCGGTGCCTTTCTGGG CCTGGAGGCTATCCTTTT (SEQ ID NO: 241) PEgRNA (c.50insG edit, contains gRNA core SEQ ID NO: 646)*2242 ACTCACGCTGGATAGCCTCCGTTTTAGAGCTAGAA ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAAC TTGAAAAAGTGGCACCGAGTCGGTGCCTCTTTCTG GGCCTGGAGGCTATCCTTTT (SEQ ID NO: 242) PEgRNA (c.50insG edit, contains gRNA core SEQ ID NO: 646)*2243 ACTCACGCTGGATAGCCTCCGTTTTAGAGCTAGAA ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACPEgRNA (c.50insG edit, contains gRNA WO 2024/238825 PCT/US2024/029746 327 TTGAAAAAGTGGCACCGAGTCGGTGCCTGGGCCTGGAGGCTATCCAGTTTT (SEQ ID NO: 243)core SEQ ID NO: 646)*2244 ACTCACGCTGGATAGCCTCCGTTTTAGAGCTAGAA ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAAC TTGAAAAAGTGGCACCGAGTCGGTGCTTCTGGGCC TGGAGGCTATCCAGTTTT (SEQ ID NO: 244) PEgRNA (c.50insG edit, contains gRNA core SEQ ID NO: 646)*2245 ACTCACGCTGGATAGCCTCCGTTTTAGAGCTAGAA ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAAC TTGAAAAAGTGGCACCGAGTCGGTGCCTTTCTGGG CCTGGAGGCTATCCAGTTTT (SEQ ID NO: 245) PEgRNA (c.50insG edit, contains gRNA core SEQ ID NO: 646)*2246 ACTCACGCTGGATAGCCTCCGTTTTAGAGCTAGAA ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAAC TTGAAAAAGTGGCACCGAGTCGGTGCCTCTTTCTG GGCCTGGAGGCTATCCAGTTTT (SEQ ID NO: 246) PEgRNA (c.50insG edit, contains gRNA core SEQ ID NO: 646)*2247 ACTCACGCTGGATAGCCTCCGTTTTAGAGCTAGAA ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAAC TTGAAAAAGTGGCACCGAGTCGGTGCCTGGGCCTG GAGGCTATCCAGCGTTTT (SEQ ID NO: 247) PEgRNA (c.50insG edit, contains gRNA core SEQ ID NO: 646)*2248 ACTCACGCTGGATAGCCTCCGTTTTAGAGCTAGAA ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAAC TTGAAAAAGTGGCACCGAGTCGGTGCTTCTGGGCC TGGAGGCTATCCAGCGTTTT (SEQ ID NO: 248) PEgRNA (c.50insG edit, contains gRNA core SEQ ID NO: 646)*2249 ACTCACGCTGGATAGCCTCCGTTTTAGAGCTAGAA ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAAC TTGAAAAAGTGGCACCGAGTCGGTGCCTCTTTCTG GGCCTGGAGGCTATCCAGCGTTTT (SEQ ID NO: 249) PEgRNA (c.50insG edit, contains gRNA core SEQ ID NO: 646)*2250 ACTCACGCTGGATAGCCTCCGTTTTAGAGCTAGAA ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAAC TTGAAAAAGTGGCACCGAGTCGGTGCCTGGGCCTG GAGGCTATCCAGCGTGTTTT (SEQ ID NO: 250) PEgRNA (c.50insG edit, contains gRNA core SEQ ID NO: 646)*2251 ACTCACGCTGGATAGCCTCCGTTTTAGAGCTAGAA ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAAC TTGAAAAAGTGGCACCGAGTCGGTGCTTCTGGGCC TGGAGGCTATCCAGCGTGTTTT (SEQ ID NO: 251) PEgRNA (c.50insG edit, contains gRNA core SEQ ID NO: 646)*2252 ACTCACGCTGGATAGCCTCCGTTTTAGAGCTAGAA ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAAC TTGAAAAAGTGGCACCGAGTCGGTGCCTTTCTGGG CCTGGAGGCTATCCAGCGTGTTTT (SEQ ID NO: 252) PEgRNA (c.50insG edit, contains gRNA core SEQ ID NO: 646)*2253 ACTCACGCTGGATAGCCTCCGTTTTAGAGCTAGAA ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAAC TTGAAAAAGTGGCACCGAGTCGGTGCCTCTTTCTG GGCCTGGAGGCTATCCAGCGTGTTTT (SEQ ID NO: 253) PEgRNA (c.50insG edit, contains gRNA core SEQ ID NO: 646)*2 254 ACTCACGCTGGATAGCCTCCGTTTAAGAGCTAGAA ATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAGC GTGAAAACGCGGCACCGAGTCGGTGCCTGGCCTGT AATAAGAGGCTATCCA (SEQ ID NO: 254) PEgRNA (c.54insTAATAA edit, contains gRNA core SEQ ID NO: 653) WO 2024/238825 PCT/US2024/029746 328 255 ACTCACGCTGGATAGCCTCCGTTTAAGAGCTAGAA ATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAGC GTGAAAACGCGGCACCGAGTCGGTGCCTTTCTGGC CTGTAATAAGAGGCTATCCA (SEQ ID NO: 255) PEgRNA (c.54insTAATAA edit, contains gRNA core SEQ ID NO: 653)256 ACTCACGCTGGATAGCCTCCGTTTAAGAGCTAGAA ATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAGC GTGAAAACGCGGCACCGAGTCGGTGCCTCTCTTTC TGGCCTGTAATAAGAGGCTATCCA (SEQ ID NO: 256) PEgRNA (c.54insTAATAA edit, contains gRNA core SEQ ID NO: 653)257 ACTCACGCTGGATAGCCTCCGTTTAAGAGCTAGAA ATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAGC GTGAAAACGCGGCACCGAGTCGGTGCCTGGCCTGT AATAAGAGGCTATCCAGCG (SEQ ID NO: 257) PEgRNA (c.54insTAATAA edit, contains gRNA core SEQ ID NO: 653)258 ACTCACGCTGGATAGCCTCCGTTTAAGAGCTAGAA ATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAGC GTGAAAACGCGGCACCGAGTCGGTGCCTTTCTGGC CTGTAATAAGAGGCTATCCAGCG (SEQ ID NO: 258) PEgRNA (c.54insTAATAA edit, contains gRNA core SEQ ID NO: 653)259 ACTCACGCTGGATAGCCTCCGTTTAAGAGCTAGAA ATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAGC GTGAAAACGCGGCACCGAGTCGGTGCCTCTCTTTC TGGCCTGTAATAAGAGGCTATCCAGCG (SEQ ID NO: 259) PEgRNA (c.54insTAATAA edit, contains gRNA core SEQ ID NO: 653)260 ACTCACGCTGGATAGCCTCCGTTTAAGAGCTAGAA ATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAGC GTGAAAACGCGGCACCGAGTCGGTGCCTGGCCTGT AATAAGAGGCTATCCAGCGTGA (SEQ ID NO: 260) PEgRNA (c.54insTAATAA edit, contains gRNA core SEQ ID NO: 653)261 ACTCACGCTGGATAGCCTCCGTTTAAGAGCTAGAA ATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAGC GTGAAAACGCGGCACCGAGTCGGTGCCTTTCTGGC CTGTAATAAGAGGCTATCCAGCGTGA (SEQ ID NO: 261) PEgRNA (c.54insTAATAA edit, contains gRNA core SEQ ID NO: 653)262 ACTCACGCTGGATAGCCTCCGTTTAAGAGCTAGAA ATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAGC GTGAAAACGCGGCACCGAGTCGGTGCCTCTCTTTC TGGCCTGTAATAAGAGGCTATCCAGCGTGA (SEQ ID NO: 262) PEgRNA (c.54insTAATAA edit, contains gRNA core SEQ ID NO: 653)999ACTCACGCTGGATAGCCTCCGTTTAAGAGCTAGAA ATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAGC GTGAAAACGCGGCACCGAGTCGGTGCCTGGCCTGT AATAAGAGGCTATCCAGCGTGATTTT PEgRNA (c.54insTAATAA edit, contains gRNA core SEQ ID NO: 653)263 GGCCGAGATGTCTCGCTCCG (SEQ ID NO: 263) ngRNA spacer264 TCCTGAAGCTGACAGCATTC (SEQ ID NO: 264) ngRNA spacer265 TTCCTGAAGCTGACAGCATT (SEQ ID NO: 265) ngRNA spacer266 TACTCTCTCTTTCTGGCTGG (SEQ ID NO: 266) ngRNA spacer*! WO 2024/238825 PCT/US2024/029746 329 267 CTCTCTCTTTCTGGGCCTGG (SEQ ID NO: 267) ngRNA spacer*2268 CTACTCTCTCTTTCTGGGCC (SEQ ID NO: 268) ngRNA spacer*2824 CTACTCTCTCTTTCTGGGCCGTTTTAGAGCTAGAAA TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACT TGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 824) ngRNA (contains ngRNA spacer SEQ ID NO: 268 and gRNA core SEQ ID NO: 646)825 CTACTCTCTCTTTCTGGGCGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTT GAAAAAGTGGCACCGAGTCGGTGCTTTT (SEQ ID NO: 825) ngRNA (contains ngRNA spacer SEQ ID NO: 268 and gRNA core SEQ ID NO: 646)826 TACTCTCTCTTTCTGGCTGGGTTTTAGAGCTAGAAA TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACT TGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 826) ngRNA (contains ngRNA spacer SEQ ID NO: 266 and gRNA core SEQ ID NO: 646)827 CTCTCTCTTTCTGGGCCTGGGTTTTAGAGCTAGAAA TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACT TGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 827) ngRNA (contains ngRNA spacer SEQ ID NO: 267 and gRNA core SEQ ID NO: 646)999GGCCGAGATGTCTCGCTCCGGTTTTAGAGCTAGAA ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAAC TTGAAAAAGTGGCACCGAGTCGGTGCTTTT ngRNA (contains ngRNA spacer SEQ ID NO: 263 and gRNA core SEQ ID NO: 646)999TCCTGAAGCTGACAGCATTCGTTTTAGAGCTAGAA ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAAC TTGAAAAAGTGGCACCGAGTCGGTGCTTTT ngRNA (contains ngRNA spacer SEQ ID NO: 264 and gRNA core SEQ ID NO: 646)999TTCCTGAAGCTGACAGCATTGTTTTAGAGCTAGAA ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAAC TTGAAAAAGTGGCACCGAGTCGGTGCTTTT ngRNA (contains ngRNA spacer SEQ ID NO: 265 and gRNA core SEQ ID NO: 646)999TACTCTCTCTTTCTGGCTGGGTTTTAGAGCTAGAAA TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACT TGAAAAAGTGGCACCGAGTCGGTGCTTTT ngRNA* 1 (contains ngRNA spacer SEQ ID NO: 266 and gRNA core SEQ ID NO: 646)999CTCTCTCTTTCTGGGCCTGGGTTTTAGAGCTAGAAA TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACT TGAAAAAGTGGCACCGAGTCGGTGCTTTT ngRNA*2 (contains ngRNA spacer SEQ ID NO: 267 and gRNA core SEQ ID NO: 646) WO 2024/238825 PCT/US2024/029746 330 Table 9 Sequen ce numbe r Sequence Description 269 GAGTAGCGCGAGCACAGCTA (SEQ ID NO: 269)Spacer 270 AGTAGCGCGAGCACAGCTA (SEQ ID NO: 270)Spacer 271 GTAGCGCGAGCACAGCTA (SEQ ID NO: 271) Spacer272 TAGCGCGAGCACAGCTA (SEQ ID NO: 272) Spacer273 CTGTG PBS274 CTGTGC PBS275 CTGTGCT PBS276 CTGTGCTC PBS277 CTGTGCTCG PBS278 CTGTGCTCGC (SEQ ID NO: 278) PBS279 CTGTGCTCGCG (SEQ ID NO: 279) PBS280 CTGTGCTCGCGC (SEQ ID NO: 280) PBS281 CTGTGCTCGCGCT (SEQ ID NO: 281) PBS282 CTGTGCTCGCGCTA (SEQ ID NO: 282) PBS283 CTGTGCTCGCGCTAC (SEQ ID NO: 283) PBS284 CTGTGCTCGCGCTACT (SEQ ID NO: 284) PBS285 CTGTGCTCGCGCTACTC (SEQ ID NO: 285) PBS286 GTGGCCTTATAATAAG (SEQ ID NO: 286) RTT (c.21insTAATAA edit)287 CTCCGTGGCCTTATAATAAG (SEQ ID NO: 287)RTT (c.21insTAATAA edit)288 CTCGCTCCGTGGCCTTATAATAAG (SEQ ID NO: 288)RTT (c.21insTAATAA edit)1033 CTTACCG (SEQ ID NO: 1033) RTT (c.21_22insCC, NGONGC PAM silencing edit)1034 GCCTTACCG (SEQ ID NO: 1034) RTT (c.21_22insCC, NGONGC PAM silencing edit)1035 TGGCCTTACCG (SEQ ID NO: 1035) RTT (c.21_22insCC, NGONGC PAM silencing edit)1036 CGTGGCCTTACCG (SEQ ID NO: 1036) RTT (c.21_22insCC, NGONGC PAM silencing edit)1037 GATGCCTCTCGCTCCGTGGGCTTAG (SEQ ID NO: 1037)RTT (c.3_4insCC, C.17OG, NGONGC PAM silencing edit)*l WO 2024/238825 PCT/US2024/029746 331 1038 GAGATGCCTCTCGCTCCGTGGGCTTAG (SEQIDNO: 1038)RTT (c.3_4insCC, C.17OG, NGONGC PAM silencing edit)*l1039 CCGAGATGCCTCTCGCTCCGTGGGCTTAG (SEQIDNO: 1039)RTT (c.3_4insCC, C.17OG, NGONGC PAM silencing edit)*l1040 GGCCGAGATGCCTCTCGCTCCGTGGGCTTA G (SEQ ID NO: 1040)RTT (c.3_4insCC, C.17OG, NGONGC PAM silencing edit)*l1041 CTTATAAGG (SEQ ID NO: 1041) RTT (c.21_22insTAAG, NGONGC PAM silencing edit)1042 GCCTTATAAGG (SEQ ID NO: 1042) RTT (c.21_22insTAAG, NGONGC PAM silencing edit)1043 TGGCCTTATAAGG (SEQ ID NO: 1043) RTT (c.21_22insTAAG, NGONGC PAM silencing edit)1044 CGTGGCCTTATAAGG (SEQ ID NO: 1044) RTT (c.21_22insTAAG, NGONGC PAM silencing edit)1045 GATGTAAGTCTCGCTCCGTGGGCTTAG (SEQ ID NO: 1045)RTT (c.3_4insTAAG, C.17OG, NGONGC PAM silencing edit)*21046 GAGATGTAAGTCTCGCTCCGTGGGCTTAG (SEQ ID NO: 1046)RTT (c.3_4insTAAG, C.17OG, NGONGC PAM silencing edit)*21047 CCGAGATGTAAGTCTCGCTCCGTGGGCTTA G (SEQ ID NO: 1047)RTT (c.3_4insTAAG, C.17OG, NGONGC PAM silencing edit)*21048 GGCCGAGATGTAAGTCTCGCTCCGTGGGCT TAG (SEQ ID NO: 1048)RTT (c.3_4insTAAG, C.17OG, NGONGC PAM silencing edit)*21049 CTTATAATAAG (SEQ ID NO: 1049) RTT (c.21_22insTAATAA, NGONGC PAM silencing edit)1050 GCCTTATAATAAG (SEQ ID NO: 1050) RTT (c.21_22insTAATAA, NGONGC PAM silencing edit)1051 TGGCCTTATAATAAG (SEQ ID NO: 1051) RTT (c.21_22insTAATAA, NGONGC PAM silencing edit)1052 CGTGGCCTTATAATAAG (SEQ ID NO: 1052) RTT (c.21_22insTAATAA, NGONGC PAM silencing edit) WO 2024/238825 PCT/US2024/029746 332 1053 GATGTAATAATCTCGCTCCGTGGGCTTAG (SEQIDNO: 1053)RTT (c.3_4insTAATAA, C.17OG, NGONGC PAM silencing edit) *31054 GAGATGTAATAATCTCGCTCCGTGGGCTTA G (SEQ ID NO: 1054)RTT (c.3_4insTAATAA, C.17OG, NGONGC PAM silencing edit) *31055 CCGAGATGTAATAATCTCGCTCCGTGGGCT TAG (SEQ ID NO: 1055)RTT (c.3_4insTAATAA, C.17OG, NGONGC PAM silencing edit) *31056 GGCCGAGATGTAATAATCTCGCTCCGTGGG CTTAG (SEQ ID NO: 1056)RTT (c.3_4insTAATAA, C.17OG, NGONGC PAM silencing edit) *31057 GATGTAATGATCCGTGGGCTTAG (SEQ ID NO: 1057)RTT (c.3_8delinsTAATGA, C.17OG, NGONGC PAM silencing edit) *41058 GAGATGTAATGATCCGTGGGCTTAG (SEQ ID NO: 1058)RTT (c.3_8delinsTAATGA, C.17OG, NGONGC PAM silencing edit) *41059 CCGAGATGTAATGATCCGTGGGCTTAG (SEQIDNO: 1059)RTT (c.3_8delinsTAATGA, C.17OG, NGONGC PAM silencing edit) *41060 GGCCGAGATGTAATGATCCGTGGGCTTAG (SEQ ID NO: 1060)RTT (c.3_8delinsTAATGA, C.17OG, NGONGC PAM silencing edit) *41061 CCTTATAATAAG (SEQ ID NO: 1061) RTT (c.21_22insTAATAA, NGONGC PAM silencing edit)1062 GTGGCCTTATAATAAG (SEQ ID NO: 1062) RTT (c.21_22insTAATAA, NGONGC PAM silencing edit)1063 TCCGTGGCCTTATAATAAG (SEQ ID NO: 1063)RTT (c.21_22insTAATAA, NGONGC PAM silencing edit)289 GAGTAGCGCGAGCACAGCTAGTTTAAGAG CTAGAAATAGCAAGTTTAAATAAGGCTAGT CCGTTATCAGCGTGAAAACGCGGCACCGAG TCGGTGCGTGGCCTTATAATAAGCTGTGCT CG (SEQ ID NO: 289) PEgRNA (c.21InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 290 GAGTAGCGCGAGCACAGCTAGTTTAAGAG CTAGAAATAGCAAGTTTAAATAAGGCTAGT CCGTTATCAGCGTGAAAACGCGGCACCGAG PEgRNA (c.21InsTAATAA edit, contains gRNA core SEQ ID NO: 653) WO 2024/238825 PCT/US2024/029746 333 TCGGTGCCTCCGTGGCCTTATAATAAGCTGTGCTCG (SEQ ID NO: 290)291 GAGTAGCGCGAGCACAGCTAGTTTAAGAG CTAGAAATAGCAAGTTTAAATAAGGCTAGT CCGTTATCAGCGTGAAAACGCGGCACCGAG TCGGTGCCTCGCTCCGTGGCCTTATAATAA GCTGTGCTCG (SEQ ID NO: 291) PEgRNA (c.21InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 292 GAGTAGCGCGAGCACAGCTAGTTTAAGAG CTAGAAATAGCAAGTTTAAATAAGGCTAGT CCGTTATCAGCGTGAAAACGCGGCACCGAG TCGGTGCGTGGCCTTATAATAAGCTGTGCT CGCGC (SEQ ID NO: 292) PEgRNA (c.21InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 293 GAGTAGCGCGAGCACAGCTAGTTTAAGAG CTAGAAATAGCAAGTTTAAATAAGGCTAGT CCGTTATCAGCGTGAAAACGCGGCACCGAG TCGGTGCCTCCGTGGCCTTATAATAAGCTG TGCTCGCGC (SEQ ID NO: 293) PEgRNA (c.21InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 294 GAGTAGCGCGAGCACAGCTAGTTTAAGAG CTAGAAATAGCAAGTTTAAATAAGGCTAGT CCGTTATCAGCGTGAAAACGCGGCACCGAG TCGGTGCCTCGCTCCGTGGCCTTATAATAA GCTGTGCTCGCGC (SEQ ID NO: 294) PEgRNA (c.21InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 295 GAGTAGCGCGAGCACAGCTAGTTTAAGAG CTAGAAATAGCAAGTTTAAATAAGGCTAGT CCGTTATCAGCGTGAAAACGCGGCACCGAG TCGGTGCGTGGCCTTATAATAAGCTGTGCT CGCGCTAC (SEQ ID NO: 295) PEgRNA (c.21InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 296 GAGTAGCGCGAGCACAGCTAGTTTAAGAG CTAGAAATAGCAAGTTTAAATAAGGCTAGT CCGTTATCAGCGTGAAAACGCGGCACCGAG TCGGTGCCTCCGTGGCCTTATAATAAGCTG TGCTCGCGCTAC (SEQ ID NO: 296) PEgRNA (c.21InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 297 GAGTAGCGCGAGCACAGCTAGTTTAAGAG CTAGAAATAGCAAGTTTAAATAAGGCTAGT CCGTTATCAGCGTGAAAACGCGGCACCGAG TCGGTGCCTCGCTCCGTGGCCTTATAATAA GCTGTGCTCGCGCTAC (SEQ ID NO: 297) PEgRNA (c.21InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 1064 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCTTACCGCTGTGCTC (SEQ ID NO: 1064) PEgRNA (c.21_22insCC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)1065 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGCCTTACCGCTGTGCTC (SEQ ID NO: 1065) PEgRNA (c.21_22insCC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)1066 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTCPEgRNA (c.21_22insCC, NGONGC PAM WO 2024/238825 PCT/US2024/029746 334 CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCTGGCCTTACCGCTGTGCTC (SEQ ID NO: 1066) silencing edit, contains gRNA core SEQ ID NO:646)1067 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCGTGGCCTTACCGCTGTGCTC (SEQ ID NO: 1067) PEgRNA (c.21_22insCC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)1068 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCTTACCGCTGTGCTCGC (SEQ ID NO: 1068) PEgRNA (c.21_22insCC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)1069 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGCCTTACCGCTGTGCTCGC (SEQ ID NO: 1069) PEgRNA (c.21_22insCC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)1070 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCTGGCCTTACCGCTGTGCTCGC (SEQ ID NO: 1070) PEgRNA (c.21_22insCC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)1071 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCGTGGCCTTACCGCTGTGCTCGC (SEQ ID NO: 1071) PEgRNA (c.21_22insCC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)1072 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCTTACCGCTGTGCTCGCGC (SEQ ID NO: 1072) PEgRNA (c.21_22insCC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)1073 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGCCTTACCGCTGTGCTCGCGC (SEQ ID NO: 1073) PEgRNA (c.21_22insCC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)1074 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCTGGCCTTACCGCTGTGCTCGCGC (SEQ ID NO: 1074) PEgRNA (c.21_22insCC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)1075 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCGTGGCCTTACCGCTGTGCTCGCG C (SEQ ID NO: 1075) PEgRNA (c.21_22insCC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646) WO 2024/238825 PCT/US2024/029746 335 1076 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGATGCCTCTCGCTCCGTGGGCTTA GCTGTGCTC (SEQ ID NO: 1076) PEgRNA (c.3_4insCC, C.17OG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)* 11077 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGAGATGCCTCTCGCTCCGTGGGCT TAGCTGTGCTC (SEQ ID NO: 1077) PEgRNA (c.3_4insCC, c.CG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)* 11078 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCCGAGATGCCTCTCGCTCCGTGGG CTTAGCTGTGCTC (SEQ ID NO: 1078) PEgRNA (c.3_4insCC, C.17OG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)* 11079 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGGCCGAGATGCCTCTCGCTCCGTG GGCTTAGCTGTGCTC (SEQ ID NO: 1079) PEgRNA (c.3_4insCC, C.17OG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)* 11080 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGATGCCTCTCGCTCCGTGGGCTTA GCTGTGCTCGC (SEQ ID NO: 1080) PEgRNA (c.3_4insCC, C.17OG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)* 11081 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGAGATGCCTCTCGCTCCGTGGGCT TAGCTGTGCTCGC (SEQ ID NO: 1081) PEgRNA (c.3_4insCC, C.17OG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)* 11082 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCCGAGATGCCTCTCGCTCCGTGGG CTTAGCTGTGCTCGC (SEQ ID NO: 1082) PEgRNA (c.3_4insCC, C.17OG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)* 11083 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGGCCGAGATGCCTCTCGCTCCGTG GGCTTAGCTGTGCTCGC (SEQ ID NO: 1083) PEgRNA (c.3_4insCC, C.17OG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)* 11084 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGATGCCTCTCGCTCCGTGGGCTTA GCTGTGCTCGCGC (SEQ ID NO: 1084) PEgRNA (c.3_4insCC, C.17OG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)* 11085 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT PEgRNA (c.3_4insCC, C.17OG, NGONGC PAM silencing edit, WO 2024/238825 PCT/US2024/029746 336 CGGTGCGAGATGCCTCTCGCTCCGTGGGCTTAGCTGTGCTCGCGC (SEQ ID NO: 1085)contains gRNA core SEQ ID NO: 646)* 11086 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCCGAGATGCCTCTCGCTCCGTGGG CTTAGCTGTGCTCGCGC (SEQ ID NO: 1086) PEgRNA (c.3_4insCC, c.CG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)* 11087 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGGCCGAGATGCCTCTCGCTCCGTG GGCTTAGCTGTGCTCGCGC (SEQ ID NO: 1087) PEgRNA (c.3_4insCC, C.17OG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)* 1 1088 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCTTATAAGGCTGTGCTC (SEQ ID NO: 1088) PEgRNA (c.21_22insTAAG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)1089 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGCCTTATAAGGCTGTGCTC (SEQ ID NO: 1089) PEgRNA (c.21_22insTAAG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)1090 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCTGGCCTTATAAGGCTGTGCTC (SEQ ID NO: 1090) PEgRNA (c.21_22insTAAG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)1091 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCGTGGCCTTATAAGGCTGTGCTC (SEQ ID NO: 1091) PEgRNA (c.21_22insTAAG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)1092 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCTTATAAGGCTGTGCTCGC (SEQ ID NO: 1092) PEgRNA (c.21_22insTAAG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)1093 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGCCTTATAAGGCTGTGCTCGC (SEQ ID NO: 1093) PEgRNA (c.21_22insTAAG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646) WO 2024/238825 PCT/US2024/029746 337 1094 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCTGGCCTTATAAGGCTGTGCTCGC (SEQ ID NO: 1094) PEgRNA (c.21_22insTAAG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)1095 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCGTGGCCTTATAAGGCTGTGCTCG C (SEQ ID NO: 1095) PEgRNA (c.21_22insTAAG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)1096 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCTTATAAGGCTGTGCTCGCGC (SEQ ID NO: 1096) PEgRNA (c.21_22insTAAG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)1097 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGCCTTATAAGGCTGTGCTCGCGC (SEQ ID NO: 1097) PEgRNA (c.21_22insTAAG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)1098 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCTGGCCTTATAAGGCTGTGCTCGCG C (SEQ ID NO: 1098) PEgRNA (c.21_22insTAAG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)1099 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCGTGGCCTTATAAGGCTGTGCTCG CGC(SEQIDNO: 1099) PEgRNA (c.21_22insTAAG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)1100 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGATGTAAGTCTCGCTCCGTGGGCT TAGCTGTGCTC (SEQ ID NO: 1100) PEgRNA (c.3_4insTAAG, C.17OG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*21101 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGAGATGTAAGTCTCGCTCCGTGGG CTTAGCTGTGCTC (SEQ ID NO: 1101) PEgRNA (c.3_4insTAAG, C.17OG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*21102 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT PEgRNA (c.3_4insTAAG, C.17OG, NGONGC PAM silencing edit, WO 2024/238825 PCT/US2024/029746 338 CGGTGCCCGAGATGTAAGTCTCGCTCCGTGGGCTTAGCTGTGCTC (SEQ ID NO: 1102)contains gRNA core SEQ ID NO: 646)*21103 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGGCCGAGATGTAAGTCTCGCTCCG TGGGCTTAGCTGTGCTC (SEQ ID NO: 1103) PEgRNA (c.3_4insTAAG, c.CG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*21104 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGATGTAAGTCTCGCTCCGTGGGCT TAGCTGTGCTCGC (SEQ ID NO: 1104) PEgRNA (c.3_4insTAAG, C.17OG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*21105 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGAGATGTAAGTCTCGCTCCGTGGG CTTAGCTGTGCTCGC (SEQ ID NO: 1105) PEgRNA (c.3_4insTAAG, C.17OG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*21106 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCCGAGATGTAAGTCTCGCTCCGTG GGCTTAGCTGTGCTCGC (SEQ ID NO: 1106) PEgRNA (c.3_4insTAAG, C.17OG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*21107 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGGCCGAGATGTAAGTCTCGCTCCG TGGGCTTAGCTGTGCTCGC (SEQ ID NO: 1107) PEgRNA (c.3_4insTAAG, C.17OG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*2 1108 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGATGTAAGTCTCGCTCCGTGGGCT TAGCTGTGCTCGCGC (SEQ ID NO: 1108) PEgRNA (c.3_4insTAAG, C.17OG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*21109 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGAGATGTAAGTCTCGCTCCGTGGG CTTAGCTGTGCTCGCGC (SEQ ID NO: 1109) PEgRNA (c.3_4insTAAG, C.17OG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*21110 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCCGAGATGTAAGTCTCGCTCCGTG GGCTTAGCTGTGCTCGCGC (SEQ ID NO: 1110) PEgRNA (c.3_4insTAAG, C.17OG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*2 1111 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGGCCGAGATGTAAGTCTCGCTCCG PEgRNA (c.3_4insTAAG, C.17OG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*2 WO 2024/238825 PCT/US2024/029746 339 TGGGCTTAGCTGTGCTCGCGC (SEQ ID NO: 1111)1112 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCTTATAATAAGCTGTGCTC (SEQ ID NO: 1112) PEgRNA (c.21_22insTAATAA, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)1113 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGCCTTATAATAAGCTGTGCTC (SEQ ID NO: 1113) PEgRNA (c.21_22insTAATAA, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)1114 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCTGGCCTTATAATAAGCTGTGCTC (SEQ ID NO: 1114) PEgRNA (c.21_22insTAATAA, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)1115 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCGTGGCCTTATAATAAGCTGTGCT C (SEQ ID NO: 1115) PEgRNA (c.21_22insTAATAA, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)1116 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCTTATAATAAGCTGTGCTCGC (SEQ ID NO: 1116) PEgRNA (c.21_22insTAATAA, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)1117 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGCCTTATAATAAGCTGTGCTCGC (SEQ ID NO: 1117) PEgRNA (c.21_22insTAATAA, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)1118 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCTGGCCTTATAATAAGCTGTGCTCG C (SEQ ID NO: 1118) PEgRNA (c.21_22insTAATAA, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)1119 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCGTGGCCTTATAATAAGCTGTGCT CGC(SEQIDNO: 1119) PEgRNA (c.21_22insTAATAA, NGONGC PAM silencing edit, contains WO 2024/238825 PCT/US2024/029746 340 gRNA core SEQ ID NO: 646)1120 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCTTATAATAAGCTGTGCTCGCGC (SEQIDNO: 1120) PEgRNA (c.21_22insTAATAA, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)1121 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGCCTTATAATAAGCTGTGCTCGCG C (SEQIDNO: 1121) PEgRNA (c.21_22insTAATAA, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)1122 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCTGGCCTTATAATAAGCTGTGCTCG CGC (SEQIDNO: 1122) PEgRNA (c.21_22insTAATAA, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)1123 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCGTGGCCTTATAATAAGCTGTGCT CGCGC (SEQ ID NO: 1123) PEgRNA (c.21_22insTAATAA, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)1124 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGATGTAATAATCTCGCTCCGTGGG CTTAGCTGTGCTC (SEQ ID NO: 1124) PEgRNA (c.3_4insTAATAA, C.17OG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*31125 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGAGATGTAATAATCTCGCTCCGTG GGCTTAGCTGTGCTC (SEQ ID NO: 1125) PEgRNA (c.3_4insTAATAA, C.17OG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*31126 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCCGAGATGTAATAATCTCGCTCCG TGGGCTTAGCTGTGCTC (SEQ ID NO: 1126) PEgRNA (c.3_4insTAATAA, C.17OG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*31127 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGGCCGAGATGTAATAATCTCGCTC PEgRNA (c.3_4insTAATAA, C.17OG, NGONGC PAM silencing edit, WO 2024/238825 PCT/US2024/029746 341 CGTGGGCTTAGCTGTGCTC (SEQ ID NO: 1127)contains gRNA core SEQ ID NO: 646)*31128 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGATGTAATAATCTCGCTCCGTGGG CTTAGCTGTGCTCGC (SEQ ID NO: 1128) PEgRNA (c.3_4insTAATAA, C.17OG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*31129 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGAGATGTAATAATCTCGCTCCGTG GGCTTAGCTGTGCTCGC (SEQ ID NO: 1129) PEgRNA (c.3_4insTAATAA, C.17OG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*31130 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCCGAGATGTAATAATCTCGCTCCG TGGGCTTAGCTGTGCTCGC (SEQ ID NO: 1130) PEgRNA (c.3_4insTAATAA, C.17OG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*31131 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGGCCGAGATGTAATAATCTCGCTC CGTGGGCTTAGCTGTGCTCGC (SEQ ID NO: 1131) PEgRNA (c.3_4insTAATAA, C.17OG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*31132 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGATGTAATAATCTCGCTCCGTGGG CTTAGCTGTGCTCGCGC (SEQ ID NO: 1132) PEgRNA (c.3_4insTAATAA, C.17OG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*31133 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGAGATGTAATAATCTCGCTCCGTG GGCTTAGCTGTGCTCGCGC (SEQ ID NO: 1133) PEgRNA (c.3_4insTAATAA, C.17OG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*31134 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCCGAGATGTAATAATCTCGCTCCG TGGGCTTAGCTGTGCTCGCGC (SEQ ID NO: 1134) PEgRNA (c.3_4insTAATAA, C.17OG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*31135 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGGCCGAGATGTAATAATCTCGCTC PEgRNA (c.3_4insTAATAA, C.17OG, NGONGC PAM silencing edit, WO 2024/238825 PCT/US2024/029746 342 CGTGGGCTTAGCTGTGCTCGCGC (SEQ ID NO: 1135)contains gRNA core SEQ ID NO: 646)*31136 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGATGTAATGATCCGTGGGCTTAGC TGTGCTC (SEQ ID NO: 1136) PEgRNA (c.3_8delinsTAATGA, C.17OG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*41137 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGAGATGTAATGATCCGTGGGCTTA GCTGTGCTC (SEQ ID NO: 1137) PEgRNA (c.3_8delinsTAATGA, C.17OG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*41138 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCCGAGATGTAATGATCCGTGGGCT TAGCTGTGCTC (SEQ ID NO: 1138) PEgRNA (c.3_8delinsTAATGA, C.17OG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*41139 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGGCCGAGATGTAATGATCCGTGGG CTTAGCTGTGCTC (SEQ ID NO: 1139) PEgRNA (c.3_8delinsTAATGA, C.17OG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*41140 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGATGTAATGATCCGTGGGCTTAGC TGTGCTCGC (SEQ ID NO: 1140) PEgRNA (c.3_8delinsTAATGA, C.17OG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*41141 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGAGATGTAATGATCCGTGGGCTTA GCTGTGCTCGC (SEQ ID NO: 1141) PEgRNA (c.3_8delinsTAATGA, C.17OG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*41142 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCCGAGATGTAATGATCCGTGGGCT TAGCTGTGCTCGC (SEQ ID NO: 1142) PEgRNA (c.3_8delinsTAATGA, C.17OG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*41143 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGGCCGAGATGTAATGATCCGTGGG CTTAGCTGTGCTCGC (SEQ ID NO: 1143) PEgRNA (c.3_8delinsTAATGA, C.17OG, NGONGC PAM silencing edit, WO 2024/238825 PCT/US2024/029746 343 contains gRNA core SEQ ID NO: 646)*41144 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGATGTAATGATCCGTGGGCTTAGC TGTGCTCGCGC (SEQ ID NO: 1144) PEgRNA (c.3_8delinsTAATGA, C.17OG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*41145 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGAGATGTAATGATCCGTGGGCTTA GCTGTGCTCGCGC (SEQ ID NO: 1145) PEgRNA (c.3_8delinsTAATGA, C.17OG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*41146 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCCGAGATGTAATGATCCGTGGGCT TAGCTGTGCTCGCGC (SEQ ID NO: 1146) PEgRNA (c.3_8delinsTAATGA, C.17OG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*41147 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGGCCGAGATGTAATGATCCGTGGG CTTAGCTGTGCTCGCGC (SEQ ID NO: 1147) PEgRNA (c.3_8delinsTAATGA, C.17OG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*41148 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCCTTATAATAAGCTGTGCTC (SEQ ID NO: 1148) PEgRNA (c.21_22insTAATAA, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)1149 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGTGGCCTTATAATAAGCTGTGCTC (SEQ ID NO: 1149) PEgRNA (c.21_22insTAATAA, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)1150 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCCTTATAATAAGCTGTGCTCGC (SEQ ID NO: 1150) PEgRNA (c.21_22insTAATAA, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)1151 GAGTAGCGCGAGCACAGCTAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCTCCGTGGCCTTATAATAAGCTGTG CTC (SEQ ID NO: 1151) PEgRNA (c.21_22insTAATAA, NGONGC PAM silencing edit, contains WO 2024/238825 PCT/US2024/029746 344 gRNA core SEQ ID NO: 646)1152 GGCCGAGATGTCTCGCTCCG (SEQ ID NO: 1152)ngRNA spacer 1153 CCGAGATGCCTCTCGCTCCG (SEQ ID NO: 1153)ngRNA spacer*! 1154 GAGATGTAAGTCTCGCTCCG (SEQ ID NO: 1154)ngRNA spacer*2 1155 GATGTAATAATCTCGCTCCG (SEQ ID NO: 1155)ngRNA spacer* 3 1156 GGCCGAGATGTAATGATCCG (SEQ ID NO: 1156)ngRNA spacer*4 1157 GGCCGAGATGTCTCGCTCCGGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTG (SEQ ID NO: 1157) ngRNA (contains ngRNA spacer SEQ ID NO: 11and gRNA core SEQ ID NO:646)1158 CCGAGATGCCTCTCGCTCCGGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTG (SEQ ID NO: 1158) ngRNA* 1 (contains ngRNA spacer SEQ ID NO: 1153 and gRNA core SEQ ID NO:646)1159 GAGATGTAAGTCTCGCTCCGGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTG (SEQ ID NO: 1159) ngRNA*2 (contains ngRNA spacer SEQ ID NO: 1154 and gRNA core SEQ ID NO:646)1160 GATGTAATAATCTCGCTCCGGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTG (SEQ ID NO: 1160) ngRNA* 3 (contains ngRNA spacer SEQ ID NO: 1155 and gRNA core SEQ ID NO:646)1161 GGCCGAGATGTAATGATCCGGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTG (SEQ ID NO: 1161) ngRNA*4 (contains ngRNA spacer SEQ ID NO: 1156 and gRNA core SEQ ID NO:646) Table 10 Seque nce Numb er Sequence Description 298AGGGTAGGAGAGACTCACGC (SEQ ID NO: 298) Spacer 299GGGTAGGAGAGACTCACGC (SEQ ID NO: 299) Spacer 300GGTAGGAGAGACTCACGC (SEQ ID NO: 300) Spacer 301GTAGGAGAGACTCACGC (SEQ ID NO: 301) Spacer WO 2024/238825 PCT/US2024/029746 345 Seque nce Numb er Sequence Description 302 TGAGT PBS303 TGAGTC PBS304 TGAGTCT PBS305 TGAGTCTC PBS306 TGAGTCTCT PBS307 TGAGTCTCTC (SEQ ID NO: 307) PBS308 TGAGTCTCTCC (SEQ ID NO: 308) PBS309 TGAGTCTCTCCT (SEQ ID NO: 309) PBS310 TGAGTCTCTCCTA (SEQ ID NO: 310) PBS311 TGAGTCTCTCCTAC (SEQ ID NO: 311) PBS 312TGAGTCTCTCCTACC (SEQ ID NO: 312) PBS 313TGAGTCTCTCCTACCC (SEQ ID NO: 313) PBS 314TGAGTCTCTCCTACCCT (SEQ ID NO: 314) PBS 315CTATCCAGTAATAACG (SEQ ID NO: 315) RTT (c.21insTAATAA edit) 316GAGGCTATCCAGTAATAACG (SEQ ID NO: 316) RTT (c.21insTAATAA edit) 317CCTGGAGGCTATCCAGTAATAACG (SEQ ID NO: 317) RTT (c.21insTAATAA edit) 318 AGGGTAGGAGAGACTCACGCGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCCTAT CCAGTAATAACGTGAGTCTCT (SEQ ID NO: 318) PEgRNA (c.21InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 319 AGGGTAGGAGAGACTCACGCGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCGAG GCTATCCAGTAATAACGTGAGTCTC T (SEQ ID NO: 319) PEgRNA (c.21InsTAATAA edit, contains gRNA core SEQ ID NO: 653) WO 2024/238825 PCT/US2024/029746 346 Seque nee Numb er Sequence Description 320 AGGGTAGGAGAGACTCACGCGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCCCTG GAGGCTATCCAGTAATAACGTGAGT CTCT (SEQ ID NO: 320) PEgRNA (c.21InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 321 AGGGTAGGAGAGACTCACGCGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCCTAT CCAGTAATAACGTGAGTCTCTCCT (SEQ ID NO: 321) PEgRNA (c.21InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 322 AGGGTAGGAGAGACTCACGCGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCGAG GCTATCCAGTAATAACGTGAGTCTC TCCT (SEQ ID NO: 322) PEgRNA (c.21InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 323 AGGGTAGGAGAGACTCACGCGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCCCTG GAGGCTATCCAGTAATAACGTGAGT CTCTCCT (SEQ ID NO: 323) PEgRNA (c.21InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 324 AGGGTAGGAGAGACTCACGCGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCCTAT CCAGTAATAACGTGAGTCTCTCCTA CC (SEQ ID NO: 324) PEgRNA (c.21InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 325 AGGGTAGGAGAGACTCACGCGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCGAG GCTATCCAGTAATAACGTGAGTCTC TCCTACC (SEQ ID NO: 325) PEgRNA (c.21InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 326 AGGGTAGGAGAGACTCACGCGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCCCTG GAGGCTATCCAGTAATAACGTGAGT CTCTCCTACC (SEQ ID NO: 326) PEgRNA (c.21InsTAATAA edit, contains gRNA core SEQ ID NO: 653) WO 2024/238825 PCT/US2024/029746 347 Table 11 Sequen ce Numbe r Sequence Description 327 CGCGAGCACAGCTAAGGCCA (SEQ ID NO: 327)Spacer 328 GCGAGCACAGCTAAGGCCA (SEQ ID NO: 328)Spacer 329 CGAGCACAGCTAAGGCCA (SEQ ID NO: 329) Spacer330 GAGCACAGCTAAGGCCA (SEQ ID NO: 330) Spacer331 CCTTA PBS332 CCTTAG PBS333 CCTTAGC PBS334 CCTTAGCT PBS335 CCTTAGCTG PBS336 CCTTAGCTGT (SEQ ID NO: 336) PBS337 CCTTAGCTGTG (SEQ ID NO: 337) PBS338 CCTTAGCTGTGC (SEQ ID NO: 338) PBS339 CCTTAGCTGTGCT (SEQ ID NO: 339) PBS340 CCTTAGCTGTGCTC (SEQ ID NO: 340) PBS341 CCTTAGCTGTGCTCG (SEQ ID NO: 341) PBS342 CCTTAGCTGTGCTCGC (SEQ ID NO: 342) PBS343 CCTTAGCTGTGCTCGCG (SEQ ID NO: 343) PBS344 CGCTCCGTGTAATAAG (SEQ ID NO: 344) RTT (c.21insTAATAA edit)345 GTCTCGCTCCGTGTAATAAG (SEQ ID NO: 345)RTT (c.21insTAATAA edit)346 AGATGTCTCGCTCCGTGTAATAAG (SEQ ID NO: 346)RTT (c.21insTAATAA edit)1162 CGTGCCG (SEQ ID NO: 1162) RTT (c.l5_16insCC, NGONGC PAM silencing edit)1163 TCCGTGCCG (SEQ ID NO: 1163) RTT (c.l5_16insCC, NGONGC PAM silencing edit)1164 GCTCCGTGCCG (SEQ ID NO: 1164) RTT (c.l5_16insCC, NGONGC PAM silencing edit)1165 TCGCTCCGTGCCG (SEQ ID NO: 1165) RTT (c.l5_16insCC, NGONGC PAM silencing edit)1166 GATGCCTCTCGCTGCGTGG (SEQ ID NO: 1166)RTT (c.3_4insCC, C.11OG, NGONGC PAM silencing edit)*l WO 2024/238825 PCT/US2024/029746 348 1167 GAGATGCCTCTCGCTGCGTGG (SEQ ID NO: 1167)RTT (c.3_4insCC, c.HOG, NGONGC PAM silencing edit)*l1168 CCGAGATGCCTCTCGCTGCGTGG (SEQ ID NO: 1168)RTT (c.3_4insCC, c.HOG, NGONGC PAM silencing edit)*l1169 GGCCGAGATGCCTCTCGCTGCGTGG (SEQ ID NO: 1169)RTT (c.3_4insCC, c.HOG, NGONGC PAM silencing edit)*l1170 CGTGTAAGG (SEQ ID NO: 1170) RTT (c.l5_16insTAAG, NGONGC PAM silencing edit)1171 TCCGTGTAAGG (SEQ ID NO: 1171) RTT (c.l5_16insTAAG, NGONGC PAM silencing edit)1172 GCTCCGTGTAAGG (SEQ ID NO: 1172) RTT (c.l5_16insTAAG, NGONGC PAM silencing edit)1173 TCGCTCCGTGTAAGG (SEQ ID NO: 1173) RTT (c.l5_16insTAAG, NGONGC PAM silencing edit)1174 GATGTAAGTCTCGCTGCGTGG (SEQ ID NO: 1174)RTT (c.3_4insTAAG, c.HOG, NGONGC PAM silencing edit)*21175 GAGATGTAAGTCTCGCTGCGTGG (SEQ ID NO: 1175)RTT (c.3_4insTAAG, c.HOG, NGONGC PAM silencing edit)*21176 CCGAGATGTAAGTCTCGCTGCGTGG (SEQ ID NO: 1176)RTT (c.3_4insTAAG, c.HOG, NGONGC PAM silencing edit)*21177 GGCCGAGATGTAAGTCTCGCTGCGTGG (SEQ ID NO: 1177)RTT (c.3_4insTAAG, c.HOG, NGONGC PAM silencing edit)*21178 CGTGTAATAAG (SEQ ID NO: 1178) RTT (c.l5_16insTAATAA, NGONGC PAM silencing edit)1179 TCCGTGTAATAAG (SEQ ID NO: 1179) RTT (c.l5_16insTAATAA, NGONGC PAM silencing edit)1180 GCTCCGTGTAATAAG (SEQ ID NO: 1180) RTT (c.l5_16insTAATAA, NGONGC PAM silencing edit)1181 TCGCTCCGTGTAATAAG (SEQ ID NO: 1181) RTT (c.l5_16insTAATAA, NGONGC PAM silencing edit) WO 2024/238825 PCT/US2024/029746 349 1182 GATGTAATAATCTCGCTGCGTGG (SEQ ID NO: 1182)RTT (c.3_4insTAATAA, c.llOG, NGONGC PAM silencing edit) *31183 GAGATGTAATAATCTCGCTGCGTGG (SEQ ID NO: 1183)RTT (c.3_4insTAATAA, c.llOG, NGONGC PAM silencing edit) *31184 CCGAGATGTAATAATCTCGCTGCGTGG (SEQ ID NO: 1184)RTT (c.3_4insTAATAA, c.llOG, NGONGC PAM silencing edit) *31185 GGCCGAGATGTAATAATCTCGCTGCGTGG (SEQ ID NO: 1185)RTT (c.3_4insTAATAA, c.llOG, NGONGC PAM silencing edit) *31186 GATGTAATGATGCGTGG (SEQ ID NO: 1186) RTT (c.3_8delinsTAATGA, c.llOG, NGONGC PAM silencing edit) *41187 GAGATGTAATGATGCGTGG (SEQ ID NO: 1187)RTT (c.3_8delinsTAATGA, c.llOG, NGONGC PAM silencing edit) *41188 CCGAGATGTAATGATGCGTGG (SEQ ID NO: 1188)RTT (c.3_8delinsTAATGA, c.llOG, NGONGC PAM silencing edit) *41189 GGCCGAGATGTAATGATGCGTGG (SEQ ID NO: 1189)RTT (c.3_8delinsTAATGA, c.llOG, NGONGC PAM silencing edit) *41190 ATGTAATGATGCGTGG (SEQ ID NO: 1190) RTT (c.3_8delinsTAATGA, c.llOG, NGONGC PAM silencing edit) *41191 CCGTGTAATAAG (SEQ ID NO: 1191) RTT (c.l5_16insTAATAA, NGONGC PAM silencing edit)347 CGCGAGCACAGCTAAGGCCAGTTTAAGAG CTAGAAATAGCAAGTTTAAATAAGGCTAGT CCGTTATCAGCGTGAAAACGCGGCACCGAG TCGGTGCCGCTCCGTGTAATAAGCCTTAGC TG (SEQ ID NO: 347) PEgRNA (c.21InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 348 CGCGAGCACAGCTAAGGCCAGTTTAAGAG CTAGAAATAGCAAGTTTAAATAAGGCTAGT CCGTTATCAGCGTGAAAACGCGGCACCGAG TCGGTGCGTCTCGCTCCGTGTAATAAGCCT TAGCTG (SEQ ID NO: 348) PEgRNA (c.21InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 349 CGCGAGCACAGCTAAGGCCAGTTTAAGAG CTAGAAATAGCAAGTTTAAATAAGGCTAGT CCGTTATCAGCGTGAAAACGCGGCACCGAG PEgRNA (c.21InsTAATAA edit, WO 2024/238825 PCT/US2024/029746 350 TCGGTGCAGATGTCTCGCTCCGTGTAATAAGCCTTAGCTG (SEQ ID NO: 349)contains gRNA core SEQ ID NO: 653)350 CGCGAGCACAGCTAAGGCCAGTTTAAGAG CTAGAAATAGCAAGTTTAAATAAGGCTAGT CCGTTATCAGCGTGAAAACGCGGCACCGAG TCGGTGCCGCTCCGTGTAATAAGCCTTAGC TGTGC (SEQ ID NO: 350) PEgRNA (c.21InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 351 CGCGAGCACAGCTAAGGCCAGTTTAAGAG CTAGAAATAGCAAGTTTAAATAAGGCTAGT CCGTTATCAGCGTGAAAACGCGGCACCGAG TCGGTGCGTCTCGCTCCGTGTAATAAGCCT TAGCTGTGC (SEQ ID NO: 351) PEgRNA (c.21InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 352 CGCGAGCACAGCTAAGGCCAGTTTAAGAG CTAGAAATAGCAAGTTTAAATAAGGCTAGT CCGTTATCAGCGTGAAAACGCGGCACCGAG TCGGTGCAGATGTCTCGCTCCGTGTAATAA GCCTTAGCTGTGC (SEQ ID NO: 352) PEgRNA (c.21InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 353 CGCGAGCACAGCTAAGGCCAGTTTAAGAG CTAGAAATAGCAAGTTTAAATAAGGCTAGT CCGTTATCAGCGTGAAAACGCGGCACCGAG TCGGTGCCGCTCCGTGTAATAAGCCTTAGC TGTGCTCG (SEQ ID NO: 353) PEgRNA (c.21InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 354 CGCGAGCACAGCTAAGGCCAGTTTAAGAG CTAGAAATAGCAAGTTTAAATAAGGCTAGT CCGTTATCAGCGTGAAAACGCGGCACCGAG TCGGTGCGTCTCGCTCCGTGTAATAAGCCT TAGCTGTGCTCG (SEQ ID NO: 354) PEgRNA (c.21InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 355 CGCGAGCACAGCTAAGGCCAGTTTAAGAG CTAGAAATAGCAAGTTTAAATAAGGCTAGT CCGTTATCAGCGTGAAAACGCGGCACCGAG TCGGTGCAGATGTCTCGCTCCGTGTAATAA GCCTTAGCTGTGCTCG (SEQ ID NO: 355) PEgRNA (c.21InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 1192 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCGTGCCGCCTTAGCT (SEQ ID NO: 1192) PEgRNA (c.l5_16insCC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)1193 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCTCCGTGCCGCCTTAGCT (SEQ ID NO: 1193) PEgRNA (c.l5_16insCC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)1194 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGCTCCGTGCCGCCTTAGCT (SEQ ID NO: 1194) PEgRNA (c.l5_16insCC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)1195 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTCPEgRNA (c.l5_16insCC, NGONGC PAM WO 2024/238825 PCT/US2024/029746 351 CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCTCGCTCCGTGCCGCCTTAGCT (SEQ ID NO: 1195) silencing edit, contains gRNA core SEQ ID NO:646)1196 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCGTGCCGCCTTAGCTGT (SEQ ID NO: 1196) PEgRNA (c.l5_16insCC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)1197 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCTCCGTGCCGCCTTAGCTGT (SEQ ID NO: 1197) PEgRNA (c.l5_16insCC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)1198 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGCTCCGTGCCGCCTTAGCTGT (SEQ ID NO: 1198) PEgRNA (c.l5_16insCC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)1199 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCTCGCTCCGTGCCGCCTTAGCTGT (SEQ ID NO: 1199) PEgRNA (c.l5_16insCC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)1200 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCGTGCCGCCTTAGCTGTGC (SEQ ID NO: 1200) PEgRNA (c.l5_16insCC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)1201 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCTCCGTGCCGCCTTAGCTGTGC (SEQ ID NO: 1201) PEgRNA (c.l5_16insCC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)1202 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGCTCCGTGCCGCCTTAGCTGTGC (SEQ ID NO: 1202) PEgRNA (c.l5_16insCC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)1203 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCTCGCTCCGTGCCGCCTTAGCTGTG C (SEQ ID NO: 1203) PEgRNA (c.l5_16insCC, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)1204 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGATGCCTCTCGCTGCGTGGCCTTA GCT (SEQ ID NO: 1204) PEgRNA (c.3_4insCC, C.11OG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)* 1 WO 2024/238825 PCT/US2024/029746 352 1205 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGAGATGCCTCTCGCTGCGTGGCCT TAGCT (SEQ ID NO: 1205) PEgRNA (c.3_4insCC, c.llOG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)* 11206 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCCGAGATGCCTCTCGCTGCGTGGC CTTAGCT (SEQ ID NO: 1206) PEgRNA (c.3_4insCC, c.llOG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)* 11207 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGGCCGAGATGCCTCTCGCTGCGTG GCCTTAGCT (SEQ ID NO: 1207) PEgRNA (c.3_4insCC, c.llOG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)* 11208 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGATGCCTCTCGCTGCGTGGCCTTA GCTGT (SEQ ID NO: 1208) PEgRNA (c.3_4insCC, c.llOG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)* 11209 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGAGATGCCTCTCGCTGCGTGGCCT TAGCTGT (SEQ ID NO: 1209) PEgRNA (c.3_4insCC, c.llOG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)* 11210 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCCGAGATGCCTCTCGCTGCGTGGC CTTAGCTGT (SEQ ID NO: 1210) PEgRNA (c.3_4insCC, c.llOG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)* 11211 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGGCCGAGATGCCTCTCGCTGCGTG GCCTTAGCTGT (SEQ ID NO: 1211) PEgRNA (c.3_4insCC, c.llOG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)* 11212 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGATGCCTCTCGCTGCGTGGCCTTA GCTGTGC (SEQ ID NO: 1212) PEgRNA (c.3_4insCC, c.llOG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)* 11213 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGAGATGCCTCTCGCTGCGTGGCCT TAGCTGTGC (SEQ ID NO: 1213) PEgRNA (c.3_4insCC, c.llOG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)* 11214 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT PEgRNA (c.3_4insCC, c.llOG, NGONGC PAM silencing edit, WO 2024/238825 PCT/US2024/029746 353 CGGTGCCCGAGATGCCTCTCGCTGCGTGGCCTTAGCTGTGC (SEQ ID NO: 1214)contains gRNA core SEQ ID NO: 646)* 11215 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGGCCGAGATGCCTCTCGCTGCGTG GCCTTAGCTGTGC (SEQ ID NO: 1215) PEgRNA (c.3_4insCC, c.llOG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)* 11216 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCGTGTAAGGCCTTAGCT (SEQ ID NO: 1216) PEgRNA (c.l5_16insTAAG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)1217 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCTCCGTGTAAGGCCTTAGCT (SEQ ID NO: 1217) PEgRNA (c.l5_16insTAAG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)1218 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGCTCCGTGTAAGGCCTTAGCT (SEQ ID NO: 1218) PEgRNA (c.l5_16insTAAG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)1219 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCTCGCTCCGTGTAAGGCCTTAGCT (SEQ ID NO: 1219) PEgRNA (c.l5_16insTAAG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)1220 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCGTGTAAGGCCTTAGCTGT (SEQ ID NO: 1220) PEgRNA (c.l5_16insTAAG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)1221 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCTCCGTGTAAGGCCTTAGCTGT (SEQ ID NO: 1221) PEgRNA (c.l5_16insTAAG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)1222 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGCTCCGTGTAAGGCCTTAGCTGT (SEQ ID NO: 1222) PEgRNA (c.l5_16insTAAG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646) WO 2024/238825 PCT/US2024/029746 354 1223 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCTCGCTCCGTGTAAGGCCTTAGCTG T (SEQ ID NO: 1223) PEgRNA (c.l5_16insTAAG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)1224 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCGTGTAAGGCCTTAGCTGTGC (SEQ ID NO: 1224) PEgRNA (c.l5_16insTAAG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)1225 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCTCCGTGTAAGGCCTTAGCTGTGC (SEQ ID NO: 1225) PEgRNA (c.l5_16insTAAG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)1226 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGCTCCGTGTAAGGCCTTAGCTGTG C (SEQ ID NO: 1226) PEgRNA (c.l5_16insTAAG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)1227 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCTCGCTCCGTGTAAGGCCTTAGCTG TGC (SEQ ID NO: 1227) PEgRNA (c.l5_16insTAAG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)1228 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGATGTAAGTCTCGCTGCGTGGCCT TAGCT (SEQ ID NO: 1228) PEgRNA (c.3_4insTAAG, C.11OG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*21229 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGAGATGTAAGTCTCGCTGCGTGGC CTTAGCT (SEQ ID NO: 1229) PEgRNA (c.3_4insTAAG, C.11OG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*21230 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCCGAGATGTAAGTCTCGCTGCGTG GCCTTAGCT (SEQ ID NO: 1230) PEgRNA (c.3_4insTAAG, C.11OG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*21231 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT PEgRNA (c.3_4insTAAG, C.11OG, NGONGC PAM silencing edit, WO 2024/238825 PCT/US2024/029746 355 CGGTGCGGCCGAGATGTAAGTCTCGCTGCGTGGCCTTAGCT (SEQ ID NO: 1231)contains gRNA core SEQ ID NO: 646)*21232 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGATGTAAGTCTCGCTGCGTGGCCT TAGCTGT (SEQ ID NO: 1232) PEgRNA (c.3_4insTAAG, c.llOG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*21233 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGAGATGTAAGTCTCGCTGCGTGGC CTTAGCTGT (SEQ ID NO: 1233) PEgRNA (c.3_4insTAAG, c.llOG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*21234 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCCGAGATGTAAGTCTCGCTGCGTG GCCTTAGCTGT (SEQ ID NO: 1234) PEgRNA (c.3_4insTAAG, c.llOG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*21235 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGGCCGAGATGTAAGTCTCGCTGCG TGGCCTTAGCTGT (SEQ ID NO: 1235) PEgRNA (c.3_4insTAAG, c.llOG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*21236 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGATGTAAGTCTCGCTGCGTGGCCT TAGCTGTGC (SEQ ID NO: 1236) PEgRNA (c.3_4insTAAG, c.llOG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*21237 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGAGATGTAAGTCTCGCTGCGTGGC CTTAGCTGTGC (SEQ ID NO: 1237) PEgRNA (c.3_4insTAAG, c.llOG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*21238 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCCGAGATGTAAGTCTCGCTGCGTG GCCTTAGCTGTGC (SEQ ID NO: 1238) PEgRNA (c.3_4insTAAG, c.llOG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*21239 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGGCCGAGATGTAAGTCTCGCTGCG TGGCCTTAGCTGTGC (SEQ ID NO: 1239) PEgRNA (c.3_4insTAAG, c.llOG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*21240 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCGTGTAATAAGCCTTAGCT (SEQ ID NO: 1240) PEgRNA (c.l5_16insTAATAA, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646) WO 2024/238825 PCT/US2024/029746 356 1241 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCTCCGTGTAATAAGCCTTAGCT (SEQ ID NO: 1241) PEgRNA (c.l5_16insTAATAA, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)1242 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGCTCCGTGTAATAAGCCTTAGCT (SEQ ID NO: 1242) PEgRNA (c.l5_16insTAATAA, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)1243 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCTCGCTCCGTGTAATAAGCCTTAGC T (SEQ ID NO: 1243) PEgRNA (c.l5_16insTAATAA, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)1244 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCGTGTAATAAGCCTTAGCTGT (SEQ ID NO: 1244) PEgRNA (c.l5_16insTAATAA, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)1245 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCTCCGTGTAATAAGCCTTAGCTGT (SEQ ID NO: 1245) PEgRNA (c.l5_16insTAATAA, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)1246 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGCTCCGTGTAATAAGCCTTAGCTG T (SEQ ID NO: 1246) PEgRNA (c.l5_16insTAATAA, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)1247 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCTCGCTCCGTGTAATAAGCCTTAGC TGT (SEQ ID NO: 1247) PEgRNA (c.l5_16insTAATAA, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)1248 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCGTGTAATAAGCCTTAGCTGTGC (SEQ ID NO: 1248) PEgRNA (c.l5_16insTAATAA, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646) WO 2024/238825 PCT/US2024/029746 357 1249 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCTCCGTGTAATAAGCCTTAGCTGTG C (SEQ ID NO: 1249) PEgRNA (c.l5_16insTAATAA, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)1250 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGCTCCGTGTAATAAGCCTTAGCTG TGC (SEQ ID NO: 1250) PEgRNA (c.l5_16insTAATAA, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)1251 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCTCGCTCCGTGTAATAAGCCTTAGC TGTGC (SEQ ID NO: 1251) PEgRNA (c.l5_16insTAATAA, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)1252 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGATGTAATAATCTCGCTGCGTGGC CTTAGCT (SEQ ID NO: 1252) PEgRNA (c.3_4insTAATAA, C.11OG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*31253 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGAGATGTAATAATCTCGCTGCGTG GCCTTAGCT (SEQ ID NO: 1253) PEgRNA (c.3_4insTAATAA, C.11OG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*31254 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCCGAGATGTAATAATCTCGCTGCG TGGCCTTAGCT (SEQ ID NO: 1254) PEgRNA (c.3_4insTAATAA, C.11OG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*31255 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGGCCGAGATGTAATAATCTCGCTG CGTGGCCTTAGCT (SEQ ID NO: 1255) PEgRNA (c.3_4insTAATAA, C.11OG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*31256 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGATGTAATAATCTCGCTGCGTGGC CTTAGCTGT (SEQ ID NO: 1256) PEgRNA (c.3_4insTAATAA, C.11OG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*3 WO 2024/238825 PCT/US2024/029746 358 1257 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGAGATGTAATAATCTCGCTGCGTG GCCTTAGCTGT (SEQ ID NO: 1257) PEgRNA (c.3_4insTAATAA, c.llOG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*31258 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCCGAGATGTAATAATCTCGCTGCG TGGCCTTAGCTGT (SEQ ID NO: 1258) PEgRNA (c.3_4insTAATAA, c.llOG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*31259 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGGCCGAGATGTAATAATCTCGCTG CGTGGCCTTAGCTGT (SEQ ID NO: 1259) PEgRNA (c.3_4insTAATAA, c.llOG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*31260 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGATGTAATAATCTCGCTGCGTGGC CTTAGCTGTGC (SEQ ID NO: 1260) PEgRNA (c.3_4insTAATAA, c.llOG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*31261 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGAGATGTAATAATCTCGCTGCGTG GCCTTAGCTGTGC (SEQ ID NO: 1261) PEgRNA (c.3_4insTAATAA, c.llOG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*31262 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCCGAGATGTAATAATCTCGCTGCG TGGCCTTAGCTGTGC (SEQ ID NO: 1262) PEgRNA (c.3_4insTAATAA, c.llOG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*31263 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGGCCGAGATGTAATAATCTCGCTG CGTGGCCTTAGCTGTGC (SEQ ID NO: 1263) PEgRNA (c.3_4insTAATAA, c.llOG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*31264 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGATGTAATGATGCGTGGCCTTAGC T (SEQ ID NO: 1264) PEgRNA (c.3_8delinsTAATGA, c.llOG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*4 WO 2024/238825 PCT/US2024/029746 359 1265 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGAGATGTAATGATGCGTGGCCTTA GCT (SEQIDNO: 1265) PEgRNA (c.3_8delinsTAATGA, c.llOG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*41266 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCCGAGATGTAATGATGCGTGGCCT TAGCT (SEQ ID NO: 1266) PEgRNA (c.3_8delinsTAATGA, c.llOG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*41267 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGGCCGAGATGTAATGATGCGTGGC CTTAGCT (SEQ ID NO: 1267) PEgRNA (c.3_8delinsTAATGA, c.llOG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*41268 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGATGTAATGATGCGTGGCCTTAGC TGT (SEQ ID NO: 1268) PEgRNA (c.3_8delinsTAATGA, c.llOG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*41269 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGAGATGTAATGATGCGTGGCCTTA GCTGT (SEQ ID NO: 1269) PEgRNA (c.3_8delinsTAATGA, c.llOG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*41270 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCCGAGATGTAATGATGCGTGGCCT TAGCTGT (SEQ ID NO: 1270) PEgRNA (c.3_8delinsTAATGA, c.llOG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*41271 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGGCCGAGATGTAATGATGCGTGGC CTTAGCTGT (SEQ ID NO: 1271) PEgRNA (c.3_8delinsTAATGA, c.llOG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*41272 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGATGTAATGATGCGTGGCCTTAGC TGTGC (SEQ ID NO: 1272) PEgRNA (c.3_8delinsTAATGA, c.llOG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*4 WO 2024/238825 PCT/US2024/029746 360 1273 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGAGATGTAATGATGCGTGGCCTTA GCTGTGC (SEQ ID NO: 1273) PEgRNA (c.3_8delinsTAATGA, c.llOG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*41274 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCCGAGATGTAATGATGCGTGGCCT TAGCTGTGC (SEQ ID NO: 1274) PEgRNA (c.3_8delinsTAATGA, c.llOG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*41275 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCGGCCGAGATGTAATGATGCGTGGC CTTAGCTGTGC (SEQ ID NO: 1275) PEgRNA (c.3_8delinsTAATGA, c.llOG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*41276 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCATGTAATGATGCGTGGCCTTAGCT GT (SEQ ID NO: 1276) PEgRNA (c.3_8delinsTAATGA, c.llOG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*41277 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCCGTGTAATAAGCCTTAGCTGT (SEQ ID NO: 1277) PEgRNA (c.l5_16insTAATAA, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)1278 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCCCGTGTAATAAGCCTTAGCT (SEQ ID NO: 1278) PEgRNA (c.l5_16insTAATAA, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)1279 CGCGAGCACAGCTAAGGCCAGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGCATGTAATGATGCGTGGCCTTAGCT GTGC (SEQ ID NO: 1279) PEgRNA (c.3_8delinsTAATGA, c.llOG, NGONGC PAM silencing edit, contains gRNA core SEQ ID NO: 646)*41280 GGCCGAGATGTCTCGCTCCG (SEQ ID NO: 1280)ngRNA spacer 1281 CCGAGATGCCTCTCGCTGCG (SEQ ID NO: 1281)ngRNA spacer*! 1282 GAGATGTAAGTCTCGCTGCG (SEQ ID NO: 1282)ngRNA spacer*2 WO 2024/238825 PCT/US2024/029746 361 1283 GATGTAATAATCTCGCTGCG (SEQ ID NO: 1283)ngRNA spacer* 3 1284 GGCCGAGATGTAATGATGCG (SEQ ID NO: 1284)ngRNA spacer*4 1285 GGCCGAGATGTCTCGCTCCGGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTG (SEQ ID NO: 1285) ngRNA (contains ngRNA spacer SEQ ID NO: 12and gRNA core SEQ ID NO:646)1286 CCGAGATGCCTCTCGCTGCGGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTG (SEQ ID NO: 1286) ngRNA* 1 (contains ngRNA spacer SEQ ID NO: 1281 and gRNA core SEQIDNO:646)1287 GAGATGTAAGTCTCGCTGCGGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTG (SEQ ID NO: 1287) ngRNA*2 (contains ngRNA spacer SEQ ID NO: 1282 and gRNA core SEQIDNO:646)1288 GATGTAATAATCTCGCTGCGGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTG (SEQ ID NO: 1288) ngRNA* 3 (contains ngRNA spacer SEQ ID NO: 1283 and gRNA coreSEQIDNO:646)1289 GGCCGAGATGTAATGATGCGGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTG (SEQ ID NO: 1289) ngRNA*4 (contains ngRNA spacer SEQ ID NO: 1284 and gRNA core SEQIDNO:646) Table 12 Seque nce Numb er Sequence Description 356GTCTTTTCCCGATATTCCTC (SEQ ID NO: 356) Spacer 357TCTTTTCCCGATATTCCTC (SEQ ID NO: 357) Spacer 358CTTTTCCCGATATTCCTC (SEQ ID NO: 358) Spacer 359TTTTCCCGATATTCCTC (SEQ ID NO: 359) Spacer360 GAATA PBS361 GAATAT PBS362 GAATATC PBS363 GAATATCG PBS364 GAATATCGG PBS365 GAATATCGGG (SEQ ID NO: 365) PBS WO 2024/238825 PCT/US2024/029746 362 Seque nee Numb er Sequence Description 366 GAATATCGGGA (SEQ ID NO: 366) PBS367 GAATATCGGGAA (SEQ ID NO: 367) PBS368 GAATATCGGGAAA (SEQ ID NO: 368) PBS 369GAATATCGGGAAAA (SEQ ID NO: 369) PBS 370GAATATCGGGAAAAG (SEQ ID NO: 370) PBS 371GAATATCGGGAAAAGA (SEQ ID NO: 371) PBS 372GAATATCGGGAAAAGAC (SEQ ID NO: 372) PBS 373AGTTTATTAACCTGAG (SEQ ID NO: 373) RTT (c.2insTAATAA edit) 374TTGGAGTTTATTAACCTGAG (SEQ ID NO: 374) RTT (c.2insTAATAA edit) 375ATCTTTGGAGTTTATTAACCTGAG (SEQ ID NO: 375) RTT (c.2insTAATAA edit) 376 GTCTTTTCCCGATATTCCTCGTTTAA GAGCTAGAAATAGCAAGTTTAAATA AGGCTAGTCCGTTATCAGCGTGAAA ACGCGGCACCGAGTCGGTGCAGTTT ATTAACCTGAGGAATATCGG (SEQ ID NO: 376) PEgRNA (c.2InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 377 GTCTTTTCCCGATATTCCTCGTTTAA GAGCTAGAAATAGCAAGTTTAAATA AGGCTAGTCCGTTATCAGCGTGAAA ACGCGGCACCGAGTCGGTGCTTGGA GTTTATTAACCTGAGGAATATCGG (SEQ ID NO: 377) PEgRNA (c.2InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 378 GTCTTTTCCCGATATTCCTCGTTTAA GAGCTAGAAATAGCAAGTTTAAATA AGGCTAGTCCGTTATCAGCGTGAAA ACGCGGCACCGAGTCGGTGCATCTT TGGAGTTTATTAACCTGAGGAATAT CGG (SEQ ID NO: 378) PEgRNA (c.2InsTAATAA edit, contains gRNA core SEQ ID NO: 653) WO 2024/238825 PCT/US2024/029746 363 Seque nee Numb er Sequence Description 379 GTCTTTTCCCGATATTCCTCGTTTAA GAGCTAGAAATAGCAAGTTTAAATA AGGCTAGTCCGTTATCAGCGTGAAA ACGCGGCACCGAGTCGGTGCAGTTT ATTAACCTGAGGAATATCGGGAA (SEQ ID NO: 379) PEgRNA (c.2InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 380 GTCTTTTCCCGATATTCCTCGTTTAA GAGCTAGAAATAGCAAGTTTAAATA AGGCTAGTCCGTTATCAGCGTGAAA ACGCGGCACCGAGTCGGTGCTTGGA GTTTATTAACCTGAGGAATATCGGG AA (SEQ ID NO: 380) PEgRNA (c.2InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 381 GTCTTTTCCCGATATTCCTCGTTTAA GAGCTAGAAATAGCAAGTTTAAATA AGGCTAGTCCGTTATCAGCGTGAAA ACGCGGCACCGAGTCGGTGCATCTT TGGAGTTTATTAACCTGAGGAATAT CGGGAA (SEQ ID NO: 381) PEgRNA (c.2InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 382 GTCTTTTCCCGATATTCCTCGTTTAA GAGCTAGAAATAGCAAGTTTAAATA AGGCTAGTCCGTTATCAGCGTGAAA ACGCGGCACCGAGTCGGTGCAGTTT ATTAACCTGAGGAATATCGGGAAAA G (SEQ ID NO: 382) PEgRNA (c.2InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 383 GTCTTTTCCCGATATTCCTCGTTTAA GAGCTAGAAATAGCAAGTTTAAATA AGGCTAGTCCGTTATCAGCGTGAAA ACGCGGCACCGAGTCGGTGCTTGGA GTTTATTAACCTGAGGAATATCGGG AAAAG (SEQ ID NO: 383) PEgRNA (c.2InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 384 GTCTTTTCCCGATATTCCTCGTTTAA GAGCTAGAAATAGCAAGTTTAAATA AGGCTAGTCCGTTATCAGCGTGAAA ACGCGGCACCGAGTCGGTGCATCTT TGGAGTTTATTAACCTGAGGAATAT CGGGAAAAG (SEQ ID NO: 384) PEgRNA (c.2InsTAATAA edit, contains gRNA core SEQ ID NO: 653) WO 2024/238825 PCT/US2024/029746 364 Table 13 Seque nce Numb er Sequence Description 385CTCAGGTACTCCAAAGATTC (SEQ ID NO: 385) Spacer 386TCAGGTACTCCAAAGATTC (SEQ ID NO: 386) Spacer 387CAGGTACTCCAAAGATTC (SEQ ID NO: 387) Spacer 388AGGTACTCCAAAGATTC (SEQ ID NO: 388) Spacer389 TCTTT PBS390 TCTTTG PBS391 TCTTTGG PBS392 TCTTTGGA PBS393 TCTTTGGAG PBS394 TCTTTGGAGT (SEQ ID NO: 394) PBS395 TCTTTGGAGTA (SEQ ID NO: 395) PBS396 TCTTTGGAGTAC (SEQ ID NO: 396) PBS397 TCTTTGGAGTACC (SEQ ID NO: 397) PBS398 TCTTTGGAGTACCT (SEQ ID NO: 398) PBS 399TCTTTGGAGTACCTG (SEQ ID NO: 399) PBS 400TCTTTGGAGTACCTGA (SEQ ID NO: 400) PBS 401TCTTTGGAGTACCTGAG (SEQ ID NO: 401) PBS 402TAAACCTGTTATTAAA (SEQ ID NO: 402) RTT (c.UinsTAATAA edit) 403TGAGTAAACCTGTTATTAAA (SEQ ID NO: 403) RTT (c.UinsTAATAA edit) 404GACGTGAGTAAACCTGTTATTAAA (SEQ ID NO: 404) RTT (c.UinsTAATAA edit) WO 2024/238825 PCT/US2024/029746 365 Seque nee Numb er Sequence Description 405 CTCAGGTACTCCAAAGATTCGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCTAAA CCTGTTATTAAATCTTTGGAG (SEQ ID NO: 405) PEgRNA (c.UInsTAATAA edit, contains gRNA core SEQ ID NO: 653) 406 CTCAGGTACTCCAAAGATTCGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCTGAG TAAACCTGTTATTAAATCTTTGGAG (SEQ ID NO: 406) PEgRNA (c.UInsTAATAA edit, contains gRNA core SEQ ID NO: 653) 407 CTCAGGTACTCCAAAGATTCGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCGACG TGAGTAAACCTGTTATTAAATCTTTG GAG (SEQ ID NO: 407) PEgRNA (c.UInsTAATAA edit, contains gRNA core SEQ ID NO: 653) 408 CTCAGGTACTCCAAAGATTCGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCTAAA CCTGTTATTAAATCTTTGGAGTAC (SEQ ID NO: 408) PEgRNA (c.UInsTAATAA edit, contains gRNA core SEQ ID NO: 653) 409 CTCAGGTACTCCAAAGATTCGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCTGAG TAAACCTGTTATTAAATCTTTGGAGT AC (SEQ ID NO: 409) PEgRNA (c.UInsTAATAA edit, contains gRNA core SEQ ID NO: 653) 410 CTCAGGTACTCCAAAGATTCGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCGACG TGAGTAAACCTGTTATTAAATCTTTG GAGTAC (SEQ ID NO: 410) PEgRNA (c.UInsTAATAA edit, contains gRNA core SEQ ID NO: 653) 411 CTCAGGTACTCCAAAGATTCGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCTAAA CCTGTTATTAAATCTTTGGAGTACCT G (SEQ ID NO: 411) PEgRNA (c.UInsTAATAA edit, contains gRNA core SEQ ID NO: 653) WO 2024/238825 PCT/US2024/029746 366 Seque nce Numb er Sequence Description 412 CTCAGGTACTCCAAAGATTCGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCTGAG TAAACCTGTTATTAAATCTTTGGAGT ACCTG (SEQ ID NO: 412) PEgRNA (c.UInsTAATAA edit, contains gRNA core SEQ ID NO: 653) 413 CTCAGGTACTCCAAAGATTCGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCGACG TGAGTAAACCTGTTATTAAATCTTTG GAGTACCTG (SEQ ID NO: 413) PEgRNA (c.UInsTAATAA edit, contains gRNA core SEQ ID NO: 653) Table 14 Seque nce Numb er Sequence Description 414TCACGTCATCCAGCAGAGAA (SEQ ID NO: 414) Spacer 415CACGTCATCCAGCAGAGAA (SEQ ID NO: 415) Spacer 416ACGTCATCCAGCAGAGAA (SEQ ID NO: 416) Spacer 417CGTCATCCAGCAGAGAA (SEQ ID NO: 417) Spacer418 TCTGC PBS419 TCTGCT PBS420 TCTGCTG PBS421 TCTGCTGG PBS422 TCTGCTGGA PBS423 TCTGCTGGAT (SEQ ID NO: 423) PBS424 TCTGCTGGATG (SEQ ID NO: 424) PBS425 TCTGCTGGATGA (SEQ ID NO: 425) PBS426 TCTGCTGGATGAC (SEQ ID NO: 426) PBS427 TCTGCTGGATGACG (SEQ ID NO: 427) PBS WO 2024/238825 PCT/US2024/029746 367 Seque nee Numb er Sequence Description 428TCTGCTGGATGACGT (SEQ ID NO: 428) PBS 429TCTGCTGGATGACGTG (SEQ ID NO: 429) PBS 430TCTGCTGGATGACGTGA (SEQ ID NO: 430) PBS 431CTTTCCATTTTATTAC (SEQ ID NO: 431) RTT (c.41insTAATAA edit) 432TTGACTTTCCATTTTATTAC (SEQ ID NO: 432) RTT (c.41insTAATAA edit) 433AAATTTGACTTTCCATTTTATTAC (SEQ ID NO: 433) RTT (c.41insTAATAA edit) 434 TCACGTCATCCAGCAGAGAAGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCCTTT CCATTTTATTACTCTGCTGGA (SEQ ID NO: 434) PEgRNA (c.41InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 435 TCACGTCATCCAGCAGAGAAGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCTTGA CTTTCCATTTTATTACTCTGCTGGA (SEQ ID NO: 435) PEgRNA (c.41InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 436 TCACGTCATCCAGCAGAGAAGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCAAAT TTGACTTTCCATTTTATTACTCTGCT GGA (SEQ ID NO: 436) PEgRNA (c.41InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 437 TCACGTCATCCAGCAGAGAAGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCCTTT CCATTTTATTACTCTGCTGGATGA (SEQ ID NO: 437) PEgRNA (c.41InsTAATAA edit, contains gRNA core SEQ ID NO: 653) WO 2024/238825 PCT/US2024/029746 368 Seque nce Numb er Sequence Description 438 TCACGTCATCCAGCAGAGAAGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCTTGA CTTTCCATTTTATTACTCTGCTGGAT GA (SEQ ID NO: 438) PEgRNA (c.41InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 439 TCACGTCATCCAGCAGAGAAGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCAAAT TTGACTTTCCATTTTATTACTCTGCT GGATGA (SEQ ID NO: 439) PEgRNA (c.41InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 440 TCACGTCATCCAGCAGAGAAGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCCTTT CCATTTTATTACTCTGCTGGATGACG T (SEQ ID NO: 440) PEgRNA (c.41InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 441 TCACGTCATCCAGCAGAGAAGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCTTGA CTTTCCATTTTATTACTCTGCTGGAT GACGT (SEQ ID NO: 441) PEgRNA (c.41InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 442 TCACGTCATCCAGCAGAGAAGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCAAAT TTGACTTTCCATTTTATTACTCTGCT GGATGACGT (SEQ ID NO: 442) PEgRNA (c.41InsTAATAA edit, contains gRNA core SEQ ID NO: 653) Table 15 Seque nce Numb er Sequence Description 443TTCCTGAATTGCTATGTGTC (SEQ ID NO: 443) Spacer 444TCCTGAATTGCTATGTGTC (SEQ ID NO: 444) Spacer WO 2024/238825 PCT/US2024/029746 369 Seque nce Numb er Sequence Description 445CCTGAATTGCTATGTGTC (SEQ ID NO: 445) Spacer 446CTGAATTGCTATGTGTC (SEQ ID NO: 446) Spacer447 ACATA PBS448 ACATAG PBS449 ACATAGC PBS450 ACATAGCA PBS451 ACATAGCAA PBS452 ACATAGCAAT (SEQ ID NO: 452) PBS453 ACATAGCAATT (SEQ ID NO: 453) PBS454 ACATAGCAATTC (SEQ ID NO: 454) PBS455 ACATAGCAATTCA (SEQ ID NO: 455) PBS 456ACATAGCAATTCAG (SEQ ID NO: 456) PBS 457ACATAGCAATTCAGG (SEQ ID NO: 457) PBS 458ACATAGCAATTCAGGA (SEQ ID NO: 458) PBS 459ACATAGCAATTCAGGAA (SEQ ID NO: 459) PBS 460AAACCCAGATTATTAC (SEQ ID NO: 460) RTT (c.75insTAATAA edit) 461GATGAAACCCAGATTATTAC (SEQ ID NO: 461) RTT (c.75insTAATAA edit) 462GATGGATGAAACCCAGATTATTAC (SEQ ID NO: 462) RTT (c.75insTAATAA edit) 463 TTCCTGAATTGCTATGTGTCGTTTAA GAGCTAGAAATAGCAAGTTTAAATA AGGCTAGTCCGTTATCAGCGTGAAA ACGCGGCACCGAGTCGGTGCAAACC CAGATTATTACACATAGCAA (SEQ ID NO: 463) PEgRNA (c.75InsTAATAA edit, contains gRNA core SEQ ID NO: 653) WO 2024/238825 PCT/US2024/029746 370 Seque nee Numb er Sequence Description 464 TTCCTGAATTGCTATGTGTCGTTTAA GAGCTAGAAATAGCAAGTTTAAATA AGGCTAGTCCGTTATCAGCGTGAAA ACGCGGCACCGAGTCGGTGCGATGA AACCCAGATTATTACACATAGCAA (SEQ ID NO: 464) PEgRNA (c.75InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 465 TTCCTGAATTGCTATGTGTCGTTTAA GAGCTAGAAATAGCAAGTTTAAATA AGGCTAGTCCGTTATCAGCGTGAAA ACGCGGCACCGAGTCGGTGCGATGG ATGAAACCCAGATTATTACACATAG CAA (SEQ ID NO: 465) PEgRNA (c.75InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 466 TTCCTGAATTGCTATGTGTCGTTTAA GAGCTAGAAATAGCAAGTTTAAATA AGGCTAGTCCGTTATCAGCGTGAAA ACGCGGCACCGAGTCGGTGCAAACC CAGATTATTACACATAGCAATTC (SEQ ID NO: 466) PEgRNA (c.75InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 467 TTCCTGAATTGCTATGTGTCGTTTAA GAGCTAGAAATAGCAAGTTTAAATA AGGCTAGTCCGTTATCAGCGTGAAA ACGCGGCACCGAGTCGGTGCGATGA AACCCAGATTATTACACATAGCAAT TC (SEQ ID NO: 467) PEgRNA (c.75InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 468 TTCCTGAATTGCTATGTGTCGTTTAA GAGCTAGAAATAGCAAGTTTAAATA AGGCTAGTCCGTTATCAGCGTGAAA ACGCGGCACCGAGTCGGTGCGATGG ATGAAACCCAGATTATTACACATAG CAATTC (SEQ ID NO: 468) PEgRNA (c.75InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 469 TTCCTGAATTGCTATGTGTCGTTTAA GAGCTAGAAATAGCAAGTTTAAATA AGGCTAGTCCGTTATCAGCGTGAAA ACGCGGCACCGAGTCGGTGCAAACC CAGATTATTACACATAGCAATTCAG G (SEQ ID NO: 469) PEgRNA (c.75InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 470 TTCCTGAATTGCTATGTGTCGTTTAA GAGCTAGAAATAGCAAGTTTAAATA AGGCTAGTCCGTTATCAGCGTGAAA ACGCGGCACCGAGTCGGTGCGATGA AACCCAGATTATTACACATAGCAAT TCAGG (SEQ ID NO: 470) PEgRNA (c.75InsTAATAA edit, contains gRNA core SEQ ID NO: 653) WO 2024/238825 PCT/US2024/029746 371 Seque nce Numb er Sequence Description 471 TTCCTGAATTGCTATGTGTCGTTTAA GAGCTAGAAATAGCAAGTTTAAATA AGGCTAGTCCGTTATCAGCGTGAAA ACGCGGCACCGAGTCGGTGCGATGG ATGAAACCCAGATTATTACACATAG CAATTCAGG (SEQ ID NO: 471) PEgRNA (c.75InsTAATAA edit, contains gRNA core SEQ ID NO: 653) Table 16 Seque nce Numb er Sequence Description 472TCCTGAATTGCTATGTGTCT (SEQ ID NO: 472) Spacer 473CCTGAATTGCTATGTGTCT (SEQ ID NO: 473) Spacer 474CTGAATTGCTATGTGTCT (SEQ ID NO: 474) Spacer 475TGAATTGCTATGTGTCT (SEQ ID NO: 475) Spacer476 CACAT PBS477 CACATA PBS478 CACATAG PBS479 CACATAGC PBS480 CACATAGCA PBS481 CACATAGCAA (SEQ ID NO: 481) PBS482 CACATAGCAAT (SEQ ID NO: 482) PBS483 CACATAGCAATT (SEQ ID NO: 483) PBS484 CACATAGCAATTC (SEQ ID NO: 484) PBS485 CACATAGCAATTCA (SEQ ID NO: 485) PBS 486CACATAGCAATTCAG (SEQ ID NO: 486) PBS 487CACATAGCAATTCAGG (SEQ ID NO: 487) PBS 488CACATAGCAATTCAGGA (SEQ ID NO: 488) PBS WO 2024/238825 PCT/US2024/029746 372 Seque nee Numb er Sequence Description 489GAAACCCTTATTAAGA (SEQ ID NO: 489) RTT (c.77insTAATAA edit) 490GGATGAAACCCTTATTAAGA (SEQ ID NO: 490) RTT (c.77insTAATAA edit) 491GGATGGATGAAACCCTTATTAAGA (SEQ ID NO: 491) RTT (c.77insTAATAA edit) 492 TCCTGAATTGCTATGTGTCTGTTTAA GAGCTAGAAATAGCAAGTTTAAATA AGGCTAGTCCGTTATCAGCGTGAAA ACGCGGCACCGAGTCGGTGCGAAAC CCTTATTAAGACACATAGCA (SEQ ID NO: 492) PEgRNA (c.77InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 493 TCCTGAATTGCTATGTGTCTGTTTAA GAGCTAGAAATAGCAAGTTTAAATA AGGCTAGTCCGTTATCAGCGTGAAA ACGCGGCACCGAGTCGGTGCGGATG AAACCCTTATTAAGACACATAGCA (SEQ ID NO: 493) PEgRNA (c.77InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 494 TCCTGAATTGCTATGTGTCTGTTTAA GAGCTAGAAATAGCAAGTTTAAATA AGGCTAGTCCGTTATCAGCGTGAAA ACGCGGCACCGAGTCGGTGCGGATG GATGAAACCCTTATTAAGACACATA GCA (SEQ ID NO: 494) PEgRNA (c.77InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 495 TCCTGAATTGCTATGTGTCTGTTTAA GAGCTAGAAATAGCAAGTTTAAATA AGGCTAGTCCGTTATCAGCGTGAAA ACGCGGCACCGAGTCGGTGCGAAAC CCTTATTAAGACACATAGCAATT (SEQ ID NO: 495) PEgRNA (c.77InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 496 TCCTGAATTGCTATGTGTCTGTTTAA GAGCTAGAAATAGCAAGTTTAAATA AGGCTAGTCCGTTATCAGCGTGAAA ACGCGGCACCGAGTCGGTGCGGATG AAACCCTTATTAAGACACATAGCAA TT (SEQ ID NO: 496) PEgRNA (c.77InsTAATAA edit, contains gRNA core SEQ ID NO: 653) WO 2024/238825 PCT/US2024/029746 373 Seque nce Numb er Sequence Description 497 TCCTGAATTGCTATGTGTCTGTTTAA GAGCTAGAAATAGCAAGTTTAAATA AGGCTAGTCCGTTATCAGCGTGAAA ACGCGGCACCGAGTCGGTGCGGATG GATGAAACCCTTATTAAGACACATA GCAATT (SEQ ID NO: 497) PEgRNA (c.77InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 498 TCCTGAATTGCTATGTGTCTGTTTAA GAGCTAGAAATAGCAAGTTTAAATA AGGCTAGTCCGTTATCAGCGTGAAA ACGCGGCACCGAGTCGGTGCGAAAC CCTTATTAAGACACATAGCAATTCA G (SEQ ID NO: 498) PEgRNA (c.77InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 499 TCCTGAATTGCTATGTGTCTGTTTAA GAGCTAGAAATAGCAAGTTTAAATA AGGCTAGTCCGTTATCAGCGTGAAA ACGCGGCACCGAGTCGGTGCGGATG AAACCCTTATTAAGACACATAGCAA TTCAG (SEQ ID NO: 499) PEgRNA (c.77InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 500 TCCTGAATTGCTATGTGTCTGTTTAA GAGCTAGAAATAGCAAGTTTAAATA AGGCTAGTCCGTTATCAGCGTGAAA ACGCGGCACCGAGTCGGTGCGGATG GATGAAACCCTTATTAAGACACATA GCAATTCAG (SEQ ID NO: 500) PEgRNA (c.77InsTAATAA edit, contains gRNA core SEQ ID NO: 653) Table 17 Seque nce Numb er Sequence Description 501ACCCAGACACATAGCAATTC (SEQ ID NO: 501) Spacer 502CCCAGACACATAGCAATTC (SEQ ID NO: 502) Spacer 503CCAGACACATAGCAATTC (SEQ ID NO: 503) Spacer 504CAGACACATAGCAATTC (SEQ ID NO: 504) Spacer505 TTGCT PBS WO 2024/238825 PCT/US2024/029746 374 Seque nce Numb er Sequence Description 506 TTGCTA PBS507 TTGCTAT PBS508 TTGCTATG PBS509 TTGCTATGT PBS510 TTGCTATGTG (SEQ ID NO: 510) PBS511 TTGCTATGTGT (SEQ ID NO: 511) PBS512 TTGCTATGTGTC (SEQ ID NO: 512) PBS513 TTGCTATGTGTCT (SEQ ID NO: 513) PBS514 TTGCTATGTGTCTG (SEQ ID NO: 514) PBS 515TTGCTATGTGTCTGG (SEQ ID NO: 515) PBS 516TTGCTATGTGTCTGGG (SEQ ID NO: 516) PBS 517TTGCTATGTGTCTGGGT (SEQ ID NO: 517) PBS 518ATTTCCTGTAATAAAA (SEQ ID NO: 518) RTT (c.62insTAATAA edit) 519TCAAATTTCCTGTAATAAAA (SEQ ID NO: 519) RTT (c.62insTAATAA edit) 520AAAGTCAAATTTCCTGTAATAAAA (SEQ ID NO: 520) RTT (c.62insTAATAA edit) 521 ACCCAGACACATAGCAATTCGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCATTT CCTGTAATAAAATTGCTATGT (SEQ ID NO: 521) PEgRNA (c.62InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 522 ACCCAGACACATAGCAATTCGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCTCAA ATTTCCTGTAATAAAATTGCTATGT (SEQ ID NO: 522) PEgRNA (c.62InsTAATAA edit, contains gRNA core SEQ ID NO: 653) WO 2024/238825 PCT/US2024/029746 375 Seque nee Numb er Sequence Description 523 ACCCAGACACATAGCAATTCGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCAAA GTCAAATTTCCTGTAATAAAATTGC TATGT (SEQ ID NO: 523) PEgRNA (c.62InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 524 ACCCAGACACATAGCAATTCGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCATTT CCTGTAATAAAATTGCTATGTGTC (SEQ ID NO: 524) PEgRNA (c.62InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 525 ACCCAGACACATAGCAATTCGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCTCAA ATTTCCTGTAATAAAATTGCTATGTG TC (SEQ ID NO: 525) PEgRNA (c.62InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 526 ACCCAGACACATAGCAATTCGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCAAA GTCAAATTTCCTGTAATAAAATTGC TATGTGTC (SEQ ID NO: 526) PEgRNA (c.62InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 527 ACCCAGACACATAGCAATTCGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCATTT CCTGTAATAAAATTGCTATGTGTCTGG (SEQ ID NO: 527) PEgRNA (c.62InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 528 ACCCAGACACATAGCAATTCGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCTCAA ATTTCCTGTAATAAAATTGCTATGTG TCTGG (SEQ ID NO: 528) PEgRNA (c.62InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 529 ACCCAGACACATAGCAATTCGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCAAA GTCAAATTTCCTGTAATAAAATTGC TATGTGTCTGG (SEQ ID NO: 529) PEgRNA (c.62InsTAATAA edit, contains gRNA core SEQ ID NO: 653) WO 2024/238825 PCT/US2024/029746 376 Table 18 Seque nce Numb er Sequence Description 530AAGTCAACTTCAATGTCGGA (SEQ ID NO: 530) Spacer 531AGTCAACTTCAATGTCGGA (SEQ ID NO: 531) Spacer 532GTCAACTTCAATGTCGGA (SEQ ID NO: 532) Spacer 533TCAACTTCAATGTCGGA (SEQ ID NO: 533) Spacer534 GACAT PBS535 GACATT PBS536 GACATTG PBS537 GACATTGA PBS538 GACATTGAA PBS539 GACATTGAAG (SEQ ID NO: 539) PBS540 GACATTGAAGT (SEQ ID NO: 540) PBS541 GACATTGAAGTT (SEQ ID NO: 541) PBS542 GACATTGAAGTTG (SEQ ID NO: 542) PBS 543GACATTGAAGTTGA (SEQ ID NO: 543) PBS 544GACATTGAAGTTGAC (SEQ ID NO: 544) PBS 545GACATTGAAGTTGACT (SEQ ID NO: 545) PBS 546GACATTGAAGTTGACTT (SEQ ID NO: 546) PBS 547TCATCCATAATAATCC (SEQ ID NO: 547) RTT (c.90insTAATAA edit) 548GGTTTCATCCATAATAATCC (SEQ ID NO: 548) RTT (c.90insTAATAA edit) 549TCTGGGTTTCATCCATAATAATCC (SEQ ID NO: 549) RTT (c.90insTAATAA edit) WO 2024/238825 PCT/US2024/029746 377 Seque nee Numb er Sequence Description 550 AAGTCAACTTCAATGTCGGAGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCTCAT CCATAATAATCCGACATTGAA (SEQ ID NO: 550) PEgRNA (c.90InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 551 AAGTCAACTTCAATGTCGGAGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCGGTT TCATCCATAATAATCCGACATTGAA (SEQ ID NO: 551) PEgRNA (c.90InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 552 AAGTCAACTTCAATGTCGGAGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCTCTG GGTTTCATCCATAATAATCCGACAT TGAA (SEQ ID NO: 552) PEgRNA (c.90InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 553 AAGTCAACTTCAATGTCGGAGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCTCAT CCATAATAATCCGACATTGAAGTT (SEQ ID NO: 553) PEgRNA (c.90InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 554 AAGTCAACTTCAATGTCGGAGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCGGTT TCATCCATAATAATCCGACATTGAA GTT(SEQIDNO: 554) PEgRNA (c.90InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 555 AAGTCAACTTCAATGTCGGAGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCTCTG GGTTTCATCCATAATAATCCGACATTGAAGTT (SEQ ID NO: 555) PEgRNA (c.90InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 556 AAGTCAACTTCAATGTCGGAGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCTCAT CCATAATAATCCGACATTGAAGTTGAC (SEQ ID NO: 556) PEgRNA (c.90InsTAATAA edit, contains gRNA core SEQ ID NO: 653) WO 2024/238825 PCT/US2024/029746 378 Seque nce Numb er Sequence Description 557 AAGTCAACTTCAATGTCGGAGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCGGTT TCATCCATAATAATCCGACATTGAAGTTGAC (SEQ ID NO: 557) PEgRNA (c.90InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 558 AAGTCAACTTCAATGTCGGAGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCTCTG GGTTTCATCCATAATAATCCGACAT TGAAGTTGAC (SEQ ID NO: 558) PEgRNA (c.90InsTAATAA edit, contains gRNA core SEQ ID NO: 653) Table 19 Seque nce Numb er Sequence Description 559TTTGACTTTCCATTCTCTGC (SEQ ID NO: 559) Spacer 560TTGACTTTCCATTCTCTGC (SEQ ID NO: 560) Spacer 561TGACTTTCCATTCTCTGC (SEQ ID NO: 561) Spacer 562GACTTTCCATTCTCTGC (SEQ ID NO: 562) Spacer563 GAGAA PBS564 GAGAAT PBS565 GAGAATG PBS566 GAGAATGG PBS567 GAGAATGGA PBS568 GAGAATGGAA (SEQ ID NO: 568) PBS569 GAGAATGGAAA (SEQ ID NO: 569) PBS570 GAGAATGGAAAG (SEQ ID NO: 570) PBS571 GAGAATGGAAAGT (SEQ ID NO: 571) PBS 572GAGAATGGAAAGTC (SEQ ID NO: 572) PBS WO 2024/238825 PCT/US2024/029746 379 Seque nee Numb er Sequence Description 573GAGAATGGAAAGTCA (SEQ ID NO: 573) PBS 574GAGAATGGAAAGTCAA (SEQ ID NO: 574) PBS 575GAGAATGGAAAGTCAAA (SEQ ID NO: 575) PBS 576TCATCCATAATAAGCA (SEQ ID NO: 576) RTT (c.35insTAATAA edit) 577CACGTCATCCATAATAAGCA (SEQ ID NO: 577) RTT (c.35insTAATAA edit) 578TACTCACGTCATCCATAATAAGCA (SEQ ID NO: 578) RTT (c.35insTAATAA edit) 579 TTTGACTTTCCATTCTCTGCGTTTAA GAGCTAGAAATAGCAAGTTTAAATA AGGCTAGTCCGTTATCAGCGTGAAA ACGCGGCACCGAGTCGGTGCTCATC CATAATAAGCAGAGAATGGA (SEQ ID NO: 579) PEgRNA (c.35InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 580 TTTGACTTTCCATTCTCTGCGTTTAA GAGCTAGAAATAGCAAGTTTAAATA AGGCTAGTCCGTTATCAGCGTGAAA ACGCGGCACCGAGTCGGTGCCACGT CATCCATAATAAGCAGAGAATGGA (SEQ ID NO: 580) PEgRNA (c.35InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 581 TTTGACTTTCCATTCTCTGCGTTTAA GAGCTAGAAATAGCAAGTTTAAATA AGGCTAGTCCGTTATCAGCGTGAAA ACGCGGCACCGAGTCGGTGCTACTC ACGTCATCCATAATAAGCAGAGAAT GGA(SEQIDNO: 581) PEgRNA (c.35InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 582 TTTGACTTTCCATTCTCTGCGTTTAA GAGCTAGAAATAGCAAGTTTAAATA AGGCTAGTCCGTTATCAGCGTGAAA ACGCGGCACCGAGTCGGTGCTCATC CATAATAAGCAGAGAATGGAAAG (SEQ ID NO: 582) PEgRNA (c.35InsTAATAA edit, contains gRNA core SEQ ID NO: 653) WO 2024/238825 PCT/US2024/029746 380 Seque nce Numb er Sequence Description 583 TTTGACTTTCCATTCTCTGCGTTTAA GAGCTAGAAATAGCAAGTTTAAATA AGGCTAGTCCGTTATCAGCGTGAAA ACGCGGCACCGAGTCGGTGCCACGT CATCCATAATAAGCAGAGAATGGAA AG (SEQ ID NO: 583) PEgRNA (c.35InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 584 TTTGACTTTCCATTCTCTGCGTTTAA GAGCTAGAAATAGCAAGTTTAAATA AGGCTAGTCCGTTATCAGCGTGAAA ACGCGGCACCGAGTCGGTGCTACTC ACGTCATCCATAATAAGCAGAGAAT GGAAAG (SEQ ID NO: 584) PEgRNA (c.35InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 585 TTTGACTTTCCATTCTCTGCGTTTAA GAGCTAGAAATAGCAAGTTTAAATA AGGCTAGTCCGTTATCAGCGTGAAA ACGCGGCACCGAGTCGGTGCTCATC CATAATAAGCAGAGAATGGAAAGT CA (SEQ ID NO: 585) PEgRNA (c.35InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 586 TTTGACTTTCCATTCTCTGCGTTTAA GAGCTAGAAATAGCAAGTTTAAATA AGGCTAGTCCGTTATCAGCGTGAAA ACGCGGCACCGAGTCGGTGCCACGT CATCCATAATAAGCAGAGAATGGAA AGTCA (SEQ ID NO: 586) PEgRNA (c.35InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 587 TTTGACTTTCCATTCTCTGCGTTTAA GAGCTAGAAATAGCAAGTTTAAATA AGGCTAGTCCGTTATCAGCGTGAAA ACGCGGCACCGAGTCGGTGCTACTC ACGTCATCCATAATAAGCAGAGAAT GGAAAGTCA (SEQ ID NO: 587) PEgRNA (c.35InsTAATAA edit, contains gRNA core SEQ ID NO: 653) Table 20 Seque nce Numb er Sequence Description 588CGTGAGTAAACCTGAATCTT (SEQ ID NO: 588) Spacer 589GTGAGTAAACCTGAATCTT (SEQ ID NO: 589) Spacer WO 2024/238825 PCT/US2024/029746 381 Seque nce Numb er Sequence Description 590TGAGTAAACCTGAATCTT (SEQ ID NO: 590) Spacer 591GAGTAAACCTGAATCTT (SEQ ID NO: 591) Spacer592 ATTCA PBS593 ATTCAG PBS594 ATTCAGG PBS595 ATTCAGGT PBS596 ATTCAGGTT PBS597 ATTCAGGTTT (SEQ ID NO: 597) PBS598 ATTCAGGTTTA (SEQ ID NO: 598) PBS599 ATTCAGGTTTAC (SEQ ID NO: 599) PBS600 ATTCAGGTTTACT (SEQ ID NO: 600) PBS601 ATTCAGGTTTACTC (SEQ ID NO: 601) PBS 602ATTCAGGTTTACTCA (SEQ ID NO: 602) PBS 603ATTCAGGTTTACTCAC (SEQ ID NO: 603) PBS 604ATTCAGGTTTACTCACG (SEQ ID NO: 604) PBS 605TACTCCATAATAAAAG (SEQ ID NO: 605) RTT (c.8insTAATAA edit) 606CAGGTACTCCATAATAAAAG (SEQ ID NO: 606) RTT (c.8insTAATAA edit) 607TCCTCAGGTACTCCATAATAAAAG (SEQ ID NO: 607) RTT (c.8insTAATAA edit) 608 CGTGAGTAAACCTGAATCTTGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCTACT CCATAATAAAAGATTCAGGTT (SEQ ID NO: 608) PEgRNA (c.8InsTAATAA edit, contains gRNA core SEQ ID NO: 653) WO 2024/238825 PCT/US2024/029746 382 Seque nee Numb er Sequence Description 609 CGTGAGTAAACCTGAATCTTGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCCAGG TACTCCATAATAAAAGATTCAGGTT (SEQ ID NO: 609) PEgRNA (c.8InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 610 CGTGAGTAAACCTGAATCTTGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCTCCT CAGGTACTCCATAATAAAAGATTCA GGTT(SEQIDNO: 610) PEgRNA (c.8InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 611 CGTGAGTAAACCTGAATCTTGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCTACT CCATAATAAAAGATTCAGGTTTAC (SEQ ID NO: 611) PEgRNA (c.8InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 612 CGTGAGTAAACCTGAATCTTGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCCAGG TACTCCATAATAAAAGATTCAGGTT TAC (SEQ ID NO: 612) PEgRNA (c.8InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 613 CGTGAGTAAACCTGAATCTTGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCTCCT CAGGTACTCCATAATAAAAGATTCAGGTTTAC (SEQ ID NO: 613) PEgRNA (c.8InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 614 CGTGAGTAAACCTGAATCTTGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCTACT CCATAATAAAAGATTCAGGTTTACTCA (SEQ ID NO: 614) PEgRNA (c.8InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 615 CGTGAGTAAACCTGAATCTTGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCCAGG TACTCCATAATAAAAGATTCAGGTT TACTCA (SEQ ID NO: 615) PEgRNA (c.8InsTAATAA edit, contains gRNA core SEQ ID NO: 653) WO 2024/238825 PCT/US2024/029746 383 Seque nce Numb er Sequence Description 616 CGTGAGTAAACCTGAATCTTGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCTCCT CAGGTACTCCATAATAAAAGATTCAGGTTTACTCA (SEQ ID NO: 616) PEgRNA (c.8InsTAATAA edit, contains gRNA core SEQ ID NO: 653) Table 21 Seque nce Numb er Sequence Description 617CAGTAAGTCAACTTCAATGT (SEQ ID NO: 617) Spacer 618AGTAAGTCAACTTCAATGT (SEQ ID NO: 618) Spacer 619GTAAGTCAACTTCAATGT (SEQ ID NO: 619) Spacer 620TAAGTCAACTTCAATGT (SEQ ID NO: 620) Spacer621 TTGAA PBS622 TTGAAG PBS623 TTGAAGT PBS624 TTGAAGTT PBS625 TTGAAGTTG PBS626 TTGAAGTTGA (SEQ ID NO: 626) PBS627 TTGAAGTTGAC (SEQ ID NO: 627) PBS628 TTGAAGTTGACT (SEQ ID NO: 628) PBS629 TTGAAGTTGACTT (SEQ ID NO: 629) PBS630 TTGAAGTTGACTTA (SEQ ID NO: 630) PBS 631TTGAAGTTGACTTAC (SEQ ID NO: 631) PBS 632TTGAAGTTGACTTACT (SEQ ID NO: 632) PBS 633TTGAAGTTGACTTACTG (SEQ ID NO: 633) PBS WO 2024/238825 PCT/US2024/029746 384 Seque nee Numb er Sequence Description 634CCATCCGACTAATAAA (SEQ ID NO: 634) RTT (c.95insTAATAA edit) 635TCATCCATCCGACTAATAAA (SEQ ID NO: 635) RTT (c.95insTAATAA edit) 636GGTTTCATCCATCCGACTAATAAA (SEQ ID NO: 636) RTT (c.95insTAATAA edit) 637 CAGTAAGTCAACTTCAATGTGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCCCAT CCGACTAATAAATTGAAGTTG (SEQ ID NO: 637) PEgRNA (c.95InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 638 CAGTAAGTCAACTTCAATGTGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCTCAT CCATCCGACTAATAAATTGAAGTTG (SEQ ID NO: 638) PEgRNA (c.95InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 639 CAGTAAGTCAACTTCAATGTGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCGGTT TCATCCATCCGACTAATAAATTGAA GTTG (SEQ ID NO: 639) PEgRNA (c.95InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 640 CAGTAAGTCAACTTCAATGTGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCCCAT CCGACTAATAAATTGAAGTTGACT (SEQ ID NO: 640) PEgRNA (c.95InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 641 CAGTAAGTCAACTTCAATGTGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCTCAT CCATCCGACTAATAAATTGAAGTTGACT (SEQ ID NO: 641) PEgRNA (c.95InsTAATAA edit, contains gRNA core SEQ ID NO: 653) WO 2024/238825 PCT/US2024/029746 385 Seque nee Numb er Sequence Description 642 CAGTAAGTCAACTTCAATGTGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCGGTT TCATCCATCCGACTAATAAATTGAA GTTGACT (SEQ ID NO: 642) PEgRNA (c.95InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 643 CAGTAAGTCAACTTCAATGTGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCCCAT CCGACTAATAAATTGAAGTTGACTT AC (SEQ ID NO: 643) PEgRNA (c.95InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 644 CAGTAAGTCAACTTCAATGTGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCTCAT CCATCCGACTAATAAATTGAAGTTGACTTAC (SEQ ID NO: 644) PEgRNA (c.95InsTAATAA edit, contains gRNA core SEQ ID NO: 653) 645 CAGTAAGTCAACTTCAATGTGTTTA AGAGCTAGAAATAGCAAGTTTAAAT AAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGCGGTT TCATCCATCCGACTAATAAATTGAA GTTGACTTAC (SEQ ID NO: 645) PEgRNA (c.95InsTAATAA edit, contains gRNA core SEQ ID NO: 653) Exemplary Embodiments [0712]In certain aspects, provided herein are the following exemplary embodiments: [0713]Embodiment 1. A prime editing guide RNA (PEgRNA) or one or more polynucleotides encoding the PEgRNA, wherein the PEgRNA comprises: a) a spacer that is complementary to a search target sequence on a first strand of a p2-microglobulin (B2M) gene, wherein the spacer comprises at its 3’ end SEQ ID NO: 205; b) a gRNA core capable of binding to a Cas9 protein; and c) an extension arm comprising: i) an editing template that comprises a region of complementarity to an editing target sequence on a second strand of the B2M gene, and ii) a primer binding site (PBS) that comprises at its 5’ end a sequence that is a reverse complement of nucleotides 10-14 of SEQ ID NO: 205, wherein the first strand and second strand are complementary to each other, and wherein the editing template encodes one or more nucleotide changes compared to the editing target sequence.

WO 2024/238825 PCT/US2024/029746 386 id="p-714"

[0714]Embodiment 2. A prime editing guide RNA (PEgRNA) or one or more polynucleotides encoding the PEgRNA, wherein the PEgRNA comprises: a) a spacer that is complementary to a search target sequence on a first strand of a p2-microglobulin (B2M) gene, wherein the spacer comprises at its 3’ end SEQ ID NO: 4; b) a gRNA core capable of binding to a Cas9 protein; and c) an extension arm comprising: ii) an editing template that comprises a region of complementarity to an editing target sequence on a second strand of the B2M gene, and ii) a primer binding site (PBS) that comprises at its 5’ end a sequence that is a reverse complement of nucleotides 10-14 of SEQ ID NO: 4, wherein the first strand and second strand are complementary to each other, and wherein the editing template encodes one or more nucleotide changes compared to the editing target sequence. [0715]Embodiment 3. A prime editing guide RNA (PEgRNA) or one or more polynucleotides encoding the PEgRNA, wherein the PEgRNA comprises: a) a spacer that is complementary to a search target sequence on a first strand of a p2-microglobulin (B2M) gene, wherein the spacer comprises at its 3’ end SEQ ID NO: 272; b) a gRNA core capable of binding to a Cas9 protein; and c) an extension arm comprising: i) an editing template that comprises a region of complementarity to an editing target sequence on a second strand of the B2M gene, and ii) a primer binding site (PBS) that comprises at its 5’ end a sequence that is a reverse complement of nucleotides 10-14 of SEQ ID NO: 272, wherein the first strand and second strand are complementary to each other, and wherein the editing template encodes one or more nucleotide changes compared to the editing target sequence. [0716]Embodiment 4. A prime editing guide RNA (PEgRNA) or one or more polynucleotides encoding the PEgRNA, wherein the PEgRNA comprises: a) a spacer that is complementary to a search target sequence on a first strand of a p2-microglobulin (B2M) gene, wherein the spacer comprises at its 3’ end SEQ ID NO: 330; b) a gRNA core capable of binding to a Cas9 protein; and c) an extension arm comprising: 1) an editing template that comprises a region of complementarity to an editing target sequence on a second strand of the B2M gene, and ii) a primer binding site (PBS) that comprises at its 5’ end a sequence that is a reverse complement of nucleotides 10-14 of SEQ ID NO: 330, wherein the first strand and second strand are complementary to each other, and wherein the editing template encodes one or more nucleotide changes compared to the editing target sequence. [0717]Embodiment 5. The PEgRN A of any one of embodiments 1-4, wherein the spacer is from 17-22 nucleotides in length, optionally wherein the spacer is 20 nucleotides in length.

WO 2024/238825 PCT/US2024/029746 387 id="p-718"

[0718]Embodiment 6. The PEgRNA of embodiment 1, wherein the spacer comprises at its3’ end any one of SEQ ID NOs: 202-204. [0719]Embodiment 7. The PEgRNA of embodiment 1, wherein the spacer comprises SEQ ID NO: 204. [0720]Embodiment 8. The PEgRNA of embodiment 2, wherein the spacer comprises at its3’ end any one of SEQ ID NO:s 1-3. [0721]Embodiment 9. The PEgRNA of embodiment 2, wherein the spacer comprises SEQ ID NO: 1. [0722]Embodiment 10. The PEgRNA of embodiment 3, wherein the spacer comprises at its 3’ end any one of SEQ ID NO:s 269-271. [0723]Embodiment 11. The PEgRNA of embodiment 3, wherein the spacer comprises SEQ ID NO: 269. [0724]Embodiment 12. The PEgRNA of embodiment 4, wherein the spacer comprises at its 3’ end any one of SEQ ID NO:s 327-329. [0725]Embodiment 13. The PEgRNA of embodiment 4, wherein the spacer comprises SEQ ID NO: 327. [0726]Embodiment 14. The PEgRNA of any one of embodiments 1-13, wherein the one or more nucleotide changes encoded by the editing template comprises a non-synonymous edit that alters the mRNA sequence or protein sequence encoded by the B2M gene. [0727]Embodiment 15. The PEgRNA of embodiment 14, wherein the non-synonymous edit results in one or more in-frame stop codons in the B2M gene. [0728]Embodiment 16. The PEgRNA of embodiment 15, wherein the one or more in-frame stop codons comprise a nonsense mutation in the B2M gene. [0729]Embodiment 17. The PEgRNA of embodiment 14, wherein the non-synonymous edit comprises an insertion in the B2M gene. [0730]Embodiment 18. The PEgRNA of embodiment 14, wherein the non-synonymous edit comprises one or more substitutions in the B2M gene. [0731]Embodiment 19. The PEgRNA of embodiment 17, wherein the insertion comprises an insertion an in-frame stop codon in the B2M gene, optionally wherein the insertion comprises an insertion of two or more consecutive in-frame stop codons in the B2M gene. [0732]Embodiment 20. The PEgRNA of embodiment 19, wherein the insertion is comprises a TAATAA, a TTATTA, or a TAATAG nucleotide insertion.

WO 2024/238825 PCT/US2024/029746 388 id="p-733"

[0733]Embodiment 21. The PEgRNA of embodiment 14, wherein the non-synonymous edit comprises a frameshift mutation in the B2M gene. [0734]Embodiment 22. The PEgRNA of embodiment 21, wherein the frameshift mutation is an insertion or of 3x+l or 3x+2 nucleotides, wherein x is an integer equal to or greater than 0. [0735]Embodiment 23. The PEgRNA of embodiment 21, wherein the frameshift mutation is a deletion of 3x+l or 3x+2 nucleotides, wherein x is an integer equal to or greater than 0. [0736]Embodiment 24. The PEgRNA of embodiment 22, wherein the insertion is 1, 2 or nucleotides in length. [0737]Embodiment 25. The PEgRNA of embodiment 23, wherein the deletion is nucleotide in length. [0738]Embodiment 26. The PEgRNA of any one of embodiments 14-25, wherein the non- synonymous edit alters a protospacer adjacent motif (PAM) sequence that is immediately 3’ to a protospacer sequence in the second strand of the B2M gene that is complementary to the search target sequence in the first strand of the B2M gene. [0739]Embodiment 27. The PEgRNA of embodiment 26, wherein the PAM sequence is NGG and the non-synonymous edit is a NGG->NGC edit. [0740]Embodiment 28. The PEgRNA of embodiment 26 or 27, wherein the protospacer sequence comprises a nick site that is three nucleotides upstream of the 5’ most nucleotide of the PAM sequence, and wherein the number of nucleotides from the nick site to the position in the second strand of the B2M gene corresponding to the non-synonymous edit is 1 to nucleotides, wherein the number of nucleotides does not include the 5’ most nucleotide position on the second strand corresponding to the non-synonymous edit. [0741]Embodiment 29. The PEgRNA of embodiment 28, wherein the number of nucleotides from the nick site to the position in the second strand of the B2M gene corresponding to the non-synonymous edit is 1, 2, 7, 8, 13, 14, or 19 nucleotides. [0742]Embodiment 30. The PEgRNA of embodiment 29, wherein the number of nucleotides from the nick site to the position in the second strand of the B2M gene corresponding to the non-synonymous edit is equal to or less than 8 nucleotides. [0743]Embodiment 31. The PEgRNA of embodiment 30, wherein the number of nucleotides from the nick site to the position in the second strand of the B2M gene corresponding to the non-synonymous edit is 1 or 2 nucleotides.

WO 2024/238825 PCT/US2024/029746 389 id="p-744"

[0744]Embodiment 32. The PEgRNA of any one of embodiments 1, 5-7 and 14-31, wherein the non-synonymous edit is at a chromosomal location corresponding to coding sequence position c.51, c.54, or c.50 of a wildtype B2M gene. [0745]Embodiment 33. The PEgRNA of embodiment 32, wherein the non-synonymous edit comprises a c.54insTAATAA insertion. [0746]Embodiment 34. The PEgRNA of embodiment 32, wherein the non-synonymous edit comprises a c.51delC deletion or a c.insG insertion. [0747]Embodiment 35. The PEgRNA of any one of embodiments 2, 5, 8-9 and 14-31, wherein the non-synonymous edit is at a chromosomal location corresponding to coding sequence position c.54, c.60, or c.66 in a wildtype B2M gene. [0748]Embodiment 36. The PEgRNA of embodiment 35, wherein the non-synonymous edit comprises to a c.54_55insCC insertion or a c.54_55insTAAG insertion. [0749]Embodiment 37. The PEgRNA of embodiment 35, wherein the non-synonymous edit comprises a c.54_55insTAATAA insertion. [0750]Embodiment 38. The PEgRNA of embodiment 35, wherein the non-synonymous edit comprises a c.66_67insCC insertion or a c.66_67insTAAG insertion. [0751]Embodiment 39. The PEgRNA of embodiment 32, wherein the non-synonymous edit comprises a c.66_67insTAATAA insertion. [0752]Embodiment 40. The PEgRNA of embodiment 35, wherein the non-synonymous edit comprises a c.60_65deletion and a TAATAG insertion (c.60_65_delinsTAATAG). [0753]Embodiment 41. The PEgRNA of embodiment 3, 5, 10-11 and 14-31, wherein the non-synonymous edit is at a chromosomal location corresponding to coding sequence position c.21 or c.3 of a wildtype B2M gene. [0754]Embodiment 42. The PEgRNA of embodiment 41, wherein the non-synonymous edit comprises a c.21insTAATAA insertion. [0755]Embodiment 43. The PEgRNA of embodiment 41, wherein the non-synonymous edit comprises a c. 21_22insCC insertion or a c.21_22insTAAG edit. [0756]Embodiment 44. The PEgRNA of embodiment 41, wherein the non-synonymous edit comprises a c.3_4insCC insertion or a c.3_4insTAAG insertion. [0757]Embodiment 45. The PEgRNA of embodiment 41, the non-synonymous edit comprises a c.3_8 deletion and a TAATGA insertion (c.3_8delinsTAATGA).

WO 2024/238825 PCT/US2024/029746 390 id="p-758"

[0758]Embodiment 46. The PEgRNA of any one of embodiments 4-5 and 12-31, wherein the non-synonymous edit is at a chromosomal location corresponding to coding sequence position c.21, c.15 or c.3 of a wildtype B2M gene. [0759]Embodiment 47. The PEgRNA of embodiment 46, wherein the non-synonymous edit comprises a c.21insTAATAA insertion. [0760]Embodiment 48. The PEgRNA of embodiment 46, wherein the non-synonymous edit comprises a c. 15 16insCC insertion or a c. l5 16insTAAG insertion. [0761]Embodiment 49. The PEgRNA of embodiment 46, wherein the non-synonymous edit comprises a c. 15_16insTAATAA insertion. [0762]Embodiment 50. The PEgRNA of embodiment 46, wherein the non-synonymous edit comprises a c. 3_4insCC insertion or a c. 3_4insTAAG insertion. [0763]Embodiment 51. The PEgRNA of embodiment 46, wherein the non-synonymous edit comprises a c. 3_4insTAATAA insertion. [0764]Embodiment 52. The PEgRNA of embodiment 46, wherein the non-synonymous edit comprises a c.3_8 deletion and a TAATGA insertion (c.3_8delinsTAATGA). [0765]Embodiment 53. The PEgRNA of any one of embodiments 1-52, wherein the editing template further encodes a an additional PAM silencing edit. [0766]Embodiment 54. The PEgRNA of embodiment 53, wherein the PAM silencing edit is a c.58G>C edit. [0767]Embodiment 55. The PEgRNA of embodiment 53, wherein the PAM silencing edit is ac.170G edit. [0768]Embodiment 56. The PEgRNA of embodiment 53, wherein the PAM silencing edit is ac.HOG edit. [0769]Embodiment 57. The PEgRNA of any one of embodiments 1-56, wherein the editing template comprises at least 4 contiguous nucleotides complementary w7ith the editing target sequence, wherein the at least 4 contiguous nucleotides are upstream of the position of the 5’ most nucleotide of the one or more nucleotide changes encoded in the editing template. [0770]Embodiment 58. .The PEgRNA of embodiment 57, wherein the editing template comprises at least 6, 8, or 10 contiguous nucleotides complementary with the editing target sequence, wherein the at least 6, 8, or 10 contiguous nucleotides are upstream of the position of the 5’ most nucleotide of the one or more nucleotide changes encoded in the editing template.

WO 2024/238825 PCT/US2024/029746 391 id="p-771"

[0771]Embodiment 59. The PEgRNA of embodiment 57, wherein the editing template comprises 4, 6, 8, or 10 contiguous nucleotides complementary with the editing target sequence, wherein the 4, 6, 8, or 10 contiguous nucleotides are upstream of the position of the 5’ most nucleotide of the one or more nucleotide changes encoded in the editing template. [0772]Embodiment 60. A prime editing guide RNA (PEgRNA), or a nucleic acid encoding the PEgRNA, wherein the PEgRNA comprises: a) a spacer comprising at its 3’ end SEQ ID NO: 205; b) a gRNA core capable of binding to a Cas9 protein; and c) an extension arm comprising: i) an editing template comprising at its 3’ end: (A) nucleotides 13-24 of SEQ ID NO: 221, (B) nucleotides 12-20 of SEQ ID NO: 227, or (C) nucleotides 7-17 of SEQ ID NO: 231, and ii) a primer binding site (PBS) comprising at its 5’ end a sequence that is a reverse complement of nucleotides 10-14 of SEQ ID NO: 205. [0773]Embodiment 61. The PEgRNA of any one of embodiments 1,5-7, 14-34, 53-60, wherein: (i) the editing template comprises at its 3’ end nucleotides 13-24 of SEQ ID NO: 221, optionally wherein the editing template comprises SEQ ID NO: 219, 220, or (ii) the editing template comprises at its 3’ end nucleotides 12-20 of SEQ ID NO: 227, optionally wherein the editing template comprises at its 3’ end SEQ ID NO: any one of SEQ ID NOs: 224-227, or (ii) the editing template comprises at its 3’ end nucleotides 7-17 of SEQ ID NO: 231, optionally wherein the editing template comprises at its 3’ end any one of SEQ ID NOs: 229-231. [0774]Embodiment 62. A prime editing guide RNA (PEgRNA), or a nucleic acid encoding the PEgRNA, wherein the PEgRNA comprises: a) a spacer comprising at its 3’ end SEQ ID NO: 1; b) a gRN A core capable of binding to a Cas9 protein; and c) an extension arm comprising: i) an editing template comprising at its 3’ end: (A) nucleotides 5-16 of SEQ ID NO: 19, or (B) a sequence selected from the group consisting of SEQ ID NO:s 900, 904, 908, 912, 916, 920, and 924, ii) a primer binding site (PBS) comprising at its 5’ end a sequence that is a reverse complement of nucleotides 10-14 of SEQ ID NO: 1. [0775]Embodiment 63. The PEgRNA of any one of embodiments 2, 5, 8-9, 14-31, 35-40, 53-59 and 62, wherein the editing template comprises: (i) a sequence selected from the group consisting of SEQ ID NOs: 900-903, or (ii) a sequence selected from the group consisting of SEQ ID NOs: 904-907, or (iii) a sequence selected from the group consisting of SEQ ID NOs: 908-911, or (iv) a sequence selected from the group consisting of SEQ ID NOs: 912- 915, or (v) a sequence selected from the group consisting of SEQ ID NOs: 916-919, 928, and 929,or (vi) a sequence selected from the group consisting of SEQ ID NOs: 920-923, or (vii) a WO 2024/238825 PCT/US2024/029746 392 sequence selected from the group consisting of SEQ ID NOs: 924-927, or (viii) a sequence selected from the group consisting of SEQ ID NOs: 18-20. [0776]Embodiment 64. A prime editing guide RNA (PEgRNA), or a nucleic acid encoding the PEgRNA, wherein the PEgRNA comprises: a) a spacer comprising at its 3’ end SEQ ID NO: 269; b) a gRNA core capable of binding to a Cas9 protein; and c) an extension arm comprising: i) an editing template comprising at its 3’ end: (A) nucleotides 3-16 of SEQ ID NO:286, or (B) a sequence selected from the group consisting of SEQ ID NO:s 1033, 1037, 1041, 1045, 1049, 1053, and 1057, and ii) a primer binding site (PBS) comprising at its 5’ end a sequence that is a reverse complement of nucleotides 10-14 of SEQ ID NO: 269. [0777]Embodiment 65. The PEgRNA of any one of embodiments 3, 5, 10-11,14-31, 41-45, 53-59 and 64, wherein the editing template comprises : (i) a sequence selected from the group consisting of SEQ ID NOs: 1033-1036, or (ii) a sequence selected from the group consisting of SEQ ID NOs: 1037-1040, or (ill) a sequence selected from the group consisting of SEQ ID NOs: 1041-1044, or (iv) a sequence selected from the group consisting of SEQ ID NOs: 1045-1048, or (v) a sequence selected from the group consisting of SEQ ID NOs: 1049-10and 1061-1063, or (vi) a sequence selected from the group consisting of SEQ ID NOs: 1053- 1056, or (vi) a sequence selected from the group consisting of SEQ ID NOs: 1057-1060, or (vii) a sequence selected from the group consisting of SEQ ID NOs: 286-288. [0778]Embodiment 66. A prime editing guide RNA (PEgRNA), or a nucleic acid encoding the PEgRNA, wherein the PEgRNA comprises: a) a spacer comprising at its 3’ end SEQ ID NO: 327; b) a gRNA core capable of binding to a Cas9 protein; and c) an extension arm comprising: i) an editing template comprising at its 3’ end: (A) nucleotides 6-16 of SEQ ID NO:344, or (B) a sequence selected from the group consisting of SEQ ID NO:s 1162, 1166, 1170, 1174, 1178, 1182, and 1190 and ii) a primer binding site (PBS) comprising at its 5’ end a sequence that is a reverse complement of nucleotides 10-14 of SEQ ID NO: 327. [0779]Embodiment 67. The PEgRNA of any one of claims 4-5, 12-31, 46-59 and 66, wherein the editing template comprises: (i) a sequence selected from the group consisting of (i) SEQ ID NOs: 1162-1165, or (ii) a sequence selected from the group consisting of SEQ ID NOs: 1166-1169, or (iii) a sequence selected from the group consisting of SEQ ID NOs: 1170-1173, or (iv) a sequence selected from the group consisting of SEQ ID NOs: 1174- 1177, or (v) a sequence selected from the group consisting of SEQ ID NOs: 1178-1181 and 1191, or (vi) a sequence selected from the group consisting of SEQ ID NOs: 1182-1185, or WO 2024/238825 PCT/US2024/029746 393 (vi) a sequence selected from the group consisting of SEQ ID NOs: 1186-1190, or (vii) a sequence selected from the group consisting of SEQ ID NOs: 344-346. [0780]Embodiment 68. The PEgRNA of any one of embodiments 1-67, wherein the editing template has a length of 24 nucleotides or less, or a length of 20 nucleotides or less. [0781]Embodiment 69. The PEgRNA of embodiment 68, wherein the editing template has a length of (i) 10 to 20 nucleotides, (ii) 12 to 20 nucleotides, or (iii) 11 to 17 nucleotides. [0782]Embodiment 70. The PEgRNA of embodiment 68, wherein the editing template is to 24 nucleotides in length. [0783]Embodiment 71. The PEgRNA of any one of embodiments 1-67, wherein the editing template is 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 24, 25, 26, 27, 28, 29, 30, 31, 33, 35 nucleotides in length. [0784]Embodiment 72. The PEgRNA of any one of embodiments 1-71, wherein the PBS has a length of 17 nucleotides or less. [0785]Embodiment 73. The PEgRNA of embodiment 72, wherein the PBS has a length of (i) to 15 nucleotides, (ii) 8 to 14 nucleotides, or (iii) 8 to 12 nucleotides. [0786]Embodiment 74. The PEgRNA of embodiment 30, wherein the PBS is 8, 10, or nucleotides in length. [0787]Embodiment 75. The PEgRNA of any one of embodiments 1,5-7, 14-34, 53-61 and 68-74, wherein the PBS comprises a sequence set forth in any one of sequence numbers 206- 218. [0788]Embodiment 76. The PEgRNA of any one of embodiments 2, 5, 8-9, 14-31, 35-40, 53-59, 62-63 and 68-74, wherein the PBS comprises a sequence set forth in any one of sequence numbers 5-17. [0789]Embodiment 77. The PEgRNA of any one of embodiments 3, 5, 10-11, 14-31, 41-45, 53-59, 64-65 and 68-74, wherein the PBS comprises a sequence set forth in any one of sequence numbers 273-285. [0790]Embodiment 78. The PEgRNA of any one of embodiments 4-5, 12-31, 46-59 and 66- 74, wherein the PBS comprises a sequence set forth in any one of sequence numbers 331- 343. [0791]Embodiment 79. The PEgRNA of any one of embodiments 1-78, wherein the spacer, the gRNA core, the RTT, and the PBS form a contiguous sequence in a single molecule. [0792]Embodiment 80. The PEgRNA of embodiment 79, comprising from 5’ to 3’, the spacer, the gRNA core, the RTT, and the PBS.

WO 2024/238825 PCT/US2024/029746 394 id="p-793"

[0793]Embodiment 81. The PEgRNA of any one of embodiments 1-80, wherein the gRNA core comprises SEQ ID NO: 646. [0794]Embodiment 82. The PEgRNA of any one of embodiments 1-80, wherein the gRNA core comprises SEQ ID NO: 653. [0795]Embodiment 83. The PEgRNA of any one of embodiments 1,5-7, 14-34, 53-61, 68-and 79-82 comprising a sequence selected from the group consisting of SEQ ID NOs: 232- 262. [0796]Embodiment 84. The PEgRNA of any one of embodiments 2, 5, 8-9, 14-31, 35-40, 53-59, 62-63, 68-74, 76 and 79-82 comprising a sequence selected from the group consisting of SEQ ID NOs: 21-29 and 930-1016. [0797]Embodiment 85. The PEgRNA of embodiment 84, comprising a sequence as set forth in SEQ ID NO: 933, 937, 961, 941, 957, or 936. [0798]Embodiment 86. The PEgRNA of any one of embodiments 3, 5, 10-11, 14-31, 41-45, 53-59, 64-65, 68-74, 77 and 79-82 comprising a sequence selected from the group consisting of SEQ ID NOs: 289-297 and 1064-1151. [0799]Embodiment 87. The PEgRNA of claim 86, comprising a sequence as set forth in SEQ ID NO: 1141, or 1143. [0800]Embodiment 88. The PEgRNA of any one of embodiments 4-5, 12-31, 46-59 and 66- 74, 78-82 comprising a sequence selected from the group consisting of SEQ ID NOs: 347- 355 and 1192-1279. [0801]Embodiment 89. The PEgRNA of embodiment 88, comprising a sequence as set forth in SEQ ID NO: 1269 or 1265. [0802]Embodiment 90. The PEgRNA of any one of embodiments 1-74, comprising a sequence selected from the group consisting of SEQ ID NOs: 957, 961, 965, 980, 1016, 956, 933, 941, 937, 1223, 988, 984, 1225, 1151, 1095, 1091, 964, 960, 940, 1221, 945, 1219, 932, 1015, 1014, 1075, 1222, 1250, 936, 1013, 1119, 1226, and 949. [0803]Embodiment 91. The PEgRNA of any one of embodiments 1-90, further comprising a 3’ motif, optionally wherein the 3’ motif is connected to the 3’ end of the PBS via a linker. [0804]Embodiment 92. The PEgRNA of any one of the preceding embodiments, farther comprising 3’ mN*mN*mN*N and/or 5’mN*mN*mN* modifications, where m indicates that the nucleotide contains a 2’-O-Me modification and a * indicates the presence of a phosphorothioate bond.

WO 2024/238825 PCT/US2024/029746 395 id="p-805"

[0805]Embodiment 93. The PEgRNA of any one of the preceding embodiments, further comprising 3’ mT*mT*mT*T and 5’mN*mN*mN* modifications, where m indicates that the nucleotide contains a 2’-O-Me modification, a * indicates the presence of a phosphorothioate bond, and a T indicates the presence of an additional uridine nucleotide. [0806]Embodiment 94. The PEgRNA of any one of the preceding embodiments, wherein human chromosome locations and coding sequence locations are as set forth in Genome Reference Consortium Human Build 38 (GrCh38). [0807]Embodiment 95. A prime editing system comprising the PEgRNA or the one or more polynucleotides encoding the PEgRNA of any one of the preceding embodiments. [0808]Embodiment 96. A prime editing system of embodiment 95, further comprising a nick guide RNA (ngRNA), or a nucleic acid encoding the ngRNA, wherein the ngRNA comprises: a) a ngRNA spacer that is complementary to a ngRNA search target sequence on the second strand of the B2M gene; and b) an ngRNA core capable of binding a Cas9 protein. [0809]Embodiment 97. The prime editing system of embodiment 96, wherein the ngRNA spacer is 17-22 nucleotides in length, optionally wherein the ngRNA spacer is 20 nucleotides in length. [0810]Embodiment 98. The prime editing system of embodiment 96 or 97, wherein the ngRNA core comprises SEQ ID NO: 646 or 653. [0811]Embodiment 99. The prime editing system of any one of embodiments 96-98, wherein the PEgRNA spacer comprises at its 3’ end SEQ ID NO: 205. [0812]Embodiment 100. The prime editing system of embodiment 99, wherein the ngRNA spacer comprises at its 3’ end a sequence corresponding to nucleotides 4-20, 3-20, 2-20, or 1- of any one of SEQ ID NOs: 263-268, optionally wherein ngRNA spacer comprises at its 3’ end any one of SEQ ID NOs: 263-268. [0813]Embodiment 101. The prime editing system of embodiment 100, wherein the ngRNA spacer comprises at its 3’ end nucleotides 1-20 of SEQ ID NO: 268, optionally wherein the ngRNA comprises SEQ ID NO: 824 or 825. [0814]Embodiment 102. The prime editing system of embodiment 99, wherein: (i) the non- synonymous edit encoded by the editing template comprises a c.5 1 delC deletion and the ngRNA spacer comprises at its 3’ end sequence corresponding to nucleotides 4-20, 3-20, 2- 20, or 1-20 of SEQ ID NO: 266; or (ii) the non-synonymous edit encoded by the editing template comprises a c.insG insertion, and the ngRNA spacer comprises at its 3’ end sequence corresponding to nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 267 or 268.

WO 2024/238825 PCT/US2024/029746 396 id="p-815"

[0815]Embodiment 103. The prime editing system of any one of embodiments 99-102, wherein the ngRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 824-827. [0816]Embodiment 104. The prime editing system of any one of embodiments 96-98, wherein the PEgRNA spacer comprises at its 3’ end SEQ ID NO: 4. [0817]Embodiment 105. The prime editing system of embodiment 104, wherein the ngRNA spacer comprises at its 3’ end a sequence corresponding to nucleotides 4-20, 3-20, 2-20, or 1- of any one of SEQ ID NOs: 1017-1024. [0818]Embodiment 106. The prime editing system of embodiment 104, wherein ngRNA spacer comprises any one of SEQ ID NOs: 1017-1024. [0819]Embodiment 107. The prime editing system of embodiment 104, wherein: (i) the editing template encodes a c.54 55insCC edit and wherein the ngRNA spacer comprises at its 3’ end sequence corresponding to nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 1018, (11) the editing template encodes a c.66 67insCC edit and wherein the ngRNA spacer comprises at its 3’ end sequence corresponding to nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 1019, (iii) the editing template encodes a c.54_55insTAAG edit and wherein the ngRNA spacer comprises at its 3’ end sequence corresponding to nucleotides 4-20, 3-20, 2- 20, or 1-20 of SEQ ID NO: 1020, (iv) the editing template encodes a c.66_67insTAAG edit and wherein the ngRNA spacer comprises at its 3’ end sequence corresponding to nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 1021, (v) the editing template encodes ac.54 55insTAATAA edit and wherein the ngRNA spacer comprises at its 3’ end sequence corresponding to nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 1022, [0820](vi) the template encodes a c.66__67insTAATAA edit and wherein the ngRNA spacer comprises at its 3’ end sequence corresponding to nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 1023, or (vii) the template encodes a c.60_65delinsTAATAG edit and wherein the ngRNA spacer comprises at its 3’ end sequence corresponding to nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 1024. [0821]Embodiment 108. The prime editing system of any one of embodiments 104-107, wherein the ngRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 1025-1032. [0822]Embodiment 109. The prime editing system of any one of embodiments 96-98, wherein the PEgRNA spacer comprises at its 3’ end SEQ ID NO: 272.

WO 2024/238825 PCT/US2024/029746 397 id="p-823"

[0823]Embodiment 110. The prime editing system of embodiment 109, wherein the ngRNA spacer comprises at its 3’ end a sequence corresponding to nucleotides 4-20, 3-20, 2-20, or 1- of any one of SEQ ID NOs: 1152-1156. [0824]Embodiment 111. The prime editing system of embodiment 109, wherein ngRNA spacer comprises any one of SEQ ID NOs: 1152-1156. [0825]Embodiment 112. The prime editing system of claim 109, wherein: (i) the editing template encodes a c.3 4insCC edit and wherein the ngRNA spacer comprises at its 3’ end sequence corresponding to nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 1153, (ii) the editing template encodes a c.3_4insTAAG edit and wherein the ngRNA spacer comprises at its 3’ end sequence corresponding to nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 1154, (iii) the editing template encodes a c.3_4insTAATAA edit and wherein the ngRNA spacer comprises at its 3’ end sequence corresponding to nucleotides 4-20, 3-20, 2-20, or 1- of SEQ ID NO: 1155, or (iv) the editing template encodes a c.3_8delinsTAATGA edit and wherein the ngRNA spacer comprises at its 3’ end sequence corresponding to nucleotides 4- 20, 3-20, 2-20, or 1-20 of SEQ ID NO: 1156. [0826]Embodiment 113. The prime editing system of any one of embodiments 109-112, wherein the ngRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 1157-1161. [0827]Embodiment 114. The prime editing system of any one of embodiments 96-98, wherein the PEgRNA spacer comprises at its 3’ end SEQ ID NO: 330. [0828]Embodiment 115. The prime editing system of embodiment 114, wherein the ngRNA spacer comprises at its 3’ end a sequence corresponding to nucleotides 4-20, 3-20, 2-20, or 1- of any one of SEQ ID NOs: 1280-1284. [0829]Embodiment 116. The prime editing system of embodiment 114, wherein ngRNA spacer comprises any one of SEQ ID NOs: 1280-1284. [0830]Embodiment 117. The prime editing system of embodiment 114, wherein: (i) the editing template encodes a c.3_4insCC edit and wherein the ngRNA spacer comprises at its 3’ end sequence corresponding to nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 1281, (ii) the editing template encodes a c.3_4insTAAG edit and wherein the ngRNA spacer comprises at its 3’ end sequence corresponding to nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 1282, (iii) the editing template encodes a c.3_4insTAATAA edit and wherein the ngRNA spacer comprises at its 3’ end sequence corresponding to nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 1283,or (iv) the editing template encodes a WO 2024/238825 PCT/US2024/029746 398 c.3 8delmsTAATGAedit and wherein the ngRNA spacer comprises at its 3’ end sequence corresponding to nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 1284. [0831]Embodiment 118. The prime editing system of any one of embodiments 115-117, wherein the ngRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 1285-1289. [0832]Embodiment 119. The prime editing system of any one of embodiments 96-118, wherein: (i) the PEgRNA comprises a sequence as set forth in SEQ ID NO: 933 or 937, and the ngRNA comprises a sequence as set forth in SEQ ID NO: 1018; (ii) the PEgRNA comprises a sequence as set for the in SEQ ID NO: 961, and the ngRNA comprises a sequence as set forth in SEQ ID NO: 1020; (iii) the PEgRNA comprises a sequence as set for the in SEQ ID NO: 941, and the ngRNA comprises a sequence as set forth in SEQ ID NO: 1018; (iv) the PEgRNA comprises a sequence as set for the in SEQ ID NO: 957, and the ngRNA comprises a sequence as set forth in SEQ ID NO: 1020; (v) the PEgRNA comprises a sequence as set for the in SEQ ID NO: 936, and the ngRNA comprises a sequence as set forth in SEQ ID NO: 1018; (vi) the PEgRNA comprises a sequence as set for the in SEQ ID NO: 1141, or 1143, and the ngRNA comprises a sequence as set forth in SEQ ID NO: 1156, or (vii) the PEgRNA comprises a sequence as set for the in SEQ ID NO: 1269 or 1265, and the ngRNA comprises a sequence as set forth in SEQ ID NO: 1284. [0833]Embodiment 120. The prime editing system of any one of embodiments 96-119, wherein the ngRNA comprises 3’ mN*mN*mN*N and/or 5’mN*mN*mN* modifications, where m indicates that the nucleotide contains a 2’-O-Me modification and a * indicates the presence of a phosphorothioate bond. [0834]Embodiment 121. The prime editing system of any one of embodiments 96-120, wherein the ngRNA comprises 3’ mT*mT*mT*T and 5’mN*mN*mN* modifications, where m indicates that the nucleotide contains a 2’-O-Me modification, a * indicates the presence of a phosphorothioate bond, and a T indicates the presence of an additional uridine nucleotide. [0835]Embodiment 122. The prime editing system of any one of embodiments 95-121, wherein the system further comprises a TRAC-PEgRNA or one or more polynucleotides encoding the TRAC-PEgRNA, wherein the TRAC-PEgRNA comprises: a) a TRAC-spacer that is complementary to a search target sequence on a first strand of T-cell receptor a constant (TRAC) gene; b) a TRAC-gRNA core capable of binding to a Cas9 protein; and c) a TRAC-extension arm comprising: i) a TRAC-editing template that comprises a region of complementarity to an editing target sequence on a second strand of the TRAC gene, and ii) a WO 2024/238825 PCT/US2024/029746 399 TRAC-primer binding site (PBS) that comprises at its 5’ end the reverse complement of nucleotides p to (q-3) of the second TRAC spacer, wherein q is the length of the second TRAC spacer, and p is an integer from 1 to (q-6), wherein the first strand and second strand are complementary to each other, and wherein the editing template encodes one or more nucleotide changes compared to the editing target sequence. [0836]Embodiment 123. The prime editing system of embodiment 122, wherein the TRAC- spacer is 17 to 22 nucleotides in length. [0837]Embodiment 124. The prime editing system of embodiment 122 or 123, wherein the editing template encodes an in-frame stop codon in the TRAC gene or a frameshift mutation in the TRAC gene. [0838]Embodiment 125. The prime editing system of any one of embodiments 122-124, wherein the editing template encodes a recombinase recognition sequence recognized by a recombinase, or the reverse complement thereof. [0839]Embodiment 126. The prime editing system of any one of embodiments 95-121, wherein the system further comprises a TRAC-PEgRNA pair, wherein the TRAC-PEgRNA pair comprises: a) a first TRAC-prime editing guide RNA (PEgRNA) or one or more polynucleotides encoding the first TRAC-PEgRNA, and b) a second TRAC-PEgRNA or one or more polynucleotides encoding the second TRAC-PEgRNA, wherein the first TRAC- PEgRNA comprises: i) a first TRAC-spacer that is complementary to a first TRAC-search target sequence on a first strand of a TRAC gene, ii) a first TRAC-gRNA core capable of binding to a Cas9 protein; and iii) a first TRAC-extension ami comprising (A) a first TRAC- editing template and (B) a first TRAC- primer binding site (PBS) that comprises at its 5’ end the reverse complement of nucleotides p to (q-3) of the first TRAC-spacer, wherein q is the length of the first TRAC-spacer, and p is an integer of 1 to (q-6); wherein the second TRAC- PEgRNA comprises: i) a second TRAC-spacer that is complementary to a second TRAC- search target sequence on a second strand of the TRAC gene complementary to the first strand, ii) a second TRAC-gRNA core capable of binding to a Cas9 protein; and iii) a second TRAC- extension arm comprising a second TRAC-editing template and a second TRAC-PBS that comprises at its 5’ end the reverse complement of nucleotides m to (n-3) of the second TRAC-spacer, wherein n is the length of the second TRAC- spacer, and m is an integer of to (n-6).

WO 2024/238825 PCT/US2024/029746 400 id="p-840"

[0840]Embodiment 127. The prime editing system of embodiment 126, wherein the first TRAC-spacer comprises at its 3’ end nucleotides 4-20 of a sequence selected from the group consisting of SEQ ID NO:s 1303 and 1353. [0841]Embodiment 128. The prime editing system of embodiment 126 or 127, wherein the second TRAC spacer comprises at its 3’ end nucleotides 4-20 of a sequence selected from the group consisting of SEQ ID NO:s 1417, 1481, and 1532. [0842]Embodiment 129. The prime editing system of embodiment 128, wherein the first TRAC spacer has a length of 17 to 22 nucleotides, and/or wherein the second TRAC spacer has the length of 17 to 22 nucleotides. [0843]Embodiment 130. The prime editing system of Embodiment 129, wherein the first TRAC spacer and the second TRAC spacer are each 20 nucleotides in length. [0844]Embodiment 131. The prime editing system of embodiment 128, wherein the first TRAC-spacer comprises at its 3’ end SEQ ID NO: 1303 or 1353. [0845]Embodiment 132. The prime editing system of embodiment 131, wherein the second TRAC spacer comprises at its 3’ end SEQ ID NO 1417, 1481, or 1532. [0846]Embodiment 133. The prime editing system of any one of embodiments 122-132, wherein the first TRAC PBS is 7-17 nucleotides in length and comprises at its 5’ end a sequence that is the reverse complement of nucleotides 11-17,10-17, 9-17, 8-17, 7-17, 6-17, 5-17, 4-17, 3-17, 217־, or 1-17 of the selected sequence for the first TRAC spacer. [0847]Embodiment 134. The prime editing system of any one of embodiments 122-133, wherein the second TRAC PBS is 7-17 nucleotides in length and comprises at its 5’ end a sequence that is the reverse complement of nucleotides 11-17, 10-17, 9-17, 8-17, 7-17, 6-17, 5-17, 4-17, 3-17, 2-17, or 1-17 of the selected sequence for the second TRAC spacer. [0848]Embodiment! 35. The prime editing system of embodiment 133 or 134, wherein the first and/or the second TRAC PBS is 8-13 nucleotides in length. [0849]Embodiment 136. The prime editing system of embodiment 135, wherein the first and/or the second TRAC PBS is 11, 12, or 13 nucleotides in length. [0850]Embodiment 137. The prime editing system of any one of embodiments 122-136, wherein the first gRNA core, the second gRNA core, or both comprise SEQ ID NO: 646 or 653. [0851]Embodiment 138. The prime editing system of any one of embodiments 122-1wherein the first TRAC editing template comprises a region of complementarity to the second TRAC editing template.

WO 2024/238825 PCT/US2024/029746 401 id="p-852"

[0852]Embodiment 139. The prime editing system of embodiment 138, wherein the first TRAC editing template and the second TRAC editing template each encodes all or a fragment of a recombinase recognition sequence (RRS) or the reverse complement thereof, wherein the first TRAC editing template encodes at least a 5’ portion of the RRS or the reverse complement thereof, wherein the second TRAC editing template encodes at least a 3’ portion of the RRS or the reverse complement thereof, and wherein at least 10 nucleotides at the 5’ ends of the first and the second TRAC editing templates have perfect reverse complementarity to each other. [0853]Embodiment 140. The prime editing system of embodiment 139, wherein at least 15, 20, 25, or 30 nucleotides at the 5’ ends of the first and the second TRAC editing templates have perfect reverse complementarity to each other, optionally wherein at least 20, 21, 22, 23, 24, 25, 26, or 27 nucleotides at the 5’ ends of the first and the second TRAC editing templates have prefect reverse complementarity to each other. [0854]Embodiment 141. The prime editing system of embodiment 139 or 140, wherein the first TRAC editing template encodes the RRS. [0855]Embodiment 142. The prime editing system of any one of embodiments 139-141, wherein the second TRAC editing template encodes the RRS. [0856]Embodiment 143. The prime editing system of any one of embodiments 139-142, wherein the RRS is an attB sequence recognized by a Bxbl recombinase. [0857]Embodiment 144. The prime editing system of any one of embodiments 139-143, wherein the RRS is an attP sequence recognized by a Bxbl recombinase. [0858]Embodiment 145. The prime editing system of any one of embodiments 122-144, wherein the first TRAC editing template comprises an RTT #1 from Table 39 and the second TRAC editing template comprises an RTT #2 in Table 39, or wherein the first TRAC editing template comprises an RTT #2 from Table 39 and the second TRAC editing template comprises an RTT #1 in Table 39. [0859]Embodiment 146. The prime editing system of any one of embodiments 122-144, wherein the first TRAC editing template comprises a 5’ fragment of an RTT listed in Table and wherein the second TRAC editing template comprises a full length or 5’ fragment of the corresponding RTT pair and wherein at least 10 nucleotides at the 5’ ends of the first and the second TRAC editing templates have perfect reverse complementarity to each other. [0860]Embodiment 147. The prime editing system of any one of embodiments 122-146, wherein the second TRAC editing template comprises a 5’ fragment of an RTT listed in WO 2024/238825 PCT/US2024/029746 402 Table 39 and wherein the first TRAC editing template comprises a full length or 5’ fragment of the corresponding RTT pair and wherein at least 10 nucleotides at the 5’ ends of the first and the second TRAC editing templates have perfect reverse complementarity to each other. [0861]Embodiment 148. The prime editing system of embodiment 146 or 147, wherein at least 15, 20, 25, or 30 nucleotides at the 5’ ends of the first and the second TRAC editing templates have perfect reverse complementarity to each other, optionally wherein at least 20, 21, 22, 23, 24, 25, 26, or 27 nucleotides at the 5’ ends of the first and the second TRAC editing templates have prefect reverse complementarity to each other. [0862]Embodiment 149. The prime editing system of embodiment 146 or 147, wherein the length of the region of complementarity of the first TRAC editing template is at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% of the length of the first TRAC editing template, optionally wherein the length of the region of complementarity of the first TRAC editing template is at least 52%, at least 53%, or at least 55% of the length of the first TRAC editing template. [0863]Embodiment 150. The prime editing system of embodiment 146 or 147, wherein the length of the region of complementarity of the second TRAC editing template is at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% of the length of the second TRAC editing template, optionally wherein the length of the region of complementarity of the second TRAC editing template is at least 52%, at least 53%, or at least 55% of the length of the second TR AC editing template. [0864]Embodiment 151. The prime editing system of any one of embodiments 122-150, wherein: (a) the first TRAC spacer comprises SEQ ID NO: 1303, and the first TRAC PBS comprises SEQ ID NO: 1312, or (b) the first TRAC spacer comprises SEQ ID NO: 1353, and the first TRAC PBS comprises SEQ ID NO: 1361, 1362, 1363, or 1364. [0865]Embodiment 152. The prime editing system of any one of embodiments 122-151, wherein: (a) the second TRAC spacer comprises SEQ ID NO: 1417, and the second TRAC PBS comprises SEQ ID NO: 1428, or (b) the second TRAC spacer comprises SEQ ID NO: 1481, and the second TRAC PBS comprises SEQ ID NO:1489.

WO 2024/238825 PCT/US2024/029746 403 id="p-866"

[0866]Embodiment 153. The prime editing system of any one of embodiments 122-150, wherein: (a) the first TRAC spacer comprises SEQ ID NO: 1303, and the first TRAC PBS has the sequence according to SEQ ID NO: 1313 or SEQ ID NO: 1314; or the first TRAC spacer comprises SEQ ID NO: 1353, and the first TRAC PBS has the sequence according to SEQ ID NO: 1361 or SEQ ID NO: 1363; and (b) the second TRAC spacer comprises SEQ ID NO: 1417, and the second TRAC PBS has the sequence according to SEQ ID NO: 1426 or SEQ ID NO: 1428, the second TRAC spacer comprises SEQ ID NO: 1480, and the second TRAC PBS has the sequence according to SEQ ID NO: 1486 or SEQ ID NO: 1487; or the second TRAC spacer comprises SEQ ID NO: 1532, and the second TRAC PBS has the sequence according to SEQ ID NO: 1541 or 1543. [0867]Embodiment 154. The prime editing system of embodiment 153, wherein the first TRAC spacer comprises SEQ ID NO: 1353, and the first TRAC PBS has the sequence according to SEQ ID NO: 1361, and wherein the second TRAC spacer comprises SEQ ID NO: 1417, and the second TRAC PBS has the sequence according to SEQ ID NO: 1426. [0868]Embodiment 155. The prime editing system of embodiment 153 or 154, wherein the first TRAC editing template comprises SEQ ID NO: 1577 and the second TRAC editing template comprises SEQ ID NO: 1584. [0869]Embodiment 156. The prime editing system of embodiment 153 or 154, wherein the first editing template comprises SEQ ID NO: 1584 and the second TRAC editing template comprises SEQ ID NO: 1577. [0870]Embodiment 157. The prime editing system of embodiment 138, wherein the first TRAC PEgRNA comprises a 5’ TR AC PEgRNA sequence selected from any one of Tables and 35, and wherein the second TRAC PEgRNA comprises a 3’ TRAC PEgRNA sequence selected from any one of Tables 36-38. [0871]Embodiment 158. The prime editing system of embodiment 138, wherein the first TRAC PEgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 1328, 1382, 1387, and 1413; and wherein the second TRAC PEgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 1475, 1476, 1477, 1525, 1526, 1527, 1573, and 1574. [0872]Embodiment 159. The prime editing system of embodiment 138, wherein the first TRAC PEgRNA comprises SEQ ID NO: 1382 and the second TRAC PEgRNA comprises SEQ ID NO: 1527.

WO 2024/238825 PCT/US2024/029746 404 id="p-873"

[0873]Embodiment 160. The prime editing system of embodiment 138, wherein the first TRAC PEgRNA comprises SEQ ID NO: 1401 and the second TRAC PEgRNA comprises SEQIDNO: 1459. [0874]Embodiment 161. The prime editing system of embodiment 138, wherein the first TRAC PEgRNA comprises SEQ ID NO: 1343 and the second TRAC PEgRNA comprises SEQ ID NO: 1566. [0875]Embodiment 162. The prime editing system of embodiment 138, wherein the first TRAC PEgRNA comprises SEQ ID NO: 1390 and the second TRAC PEgRNA comprises SEQIDNO: 1456. [0876]Embodiment 163. The prime editing system of embodiment 138, wherein the first TRAC PEgRNA comprises SEQ ID NO: 1336 and the second TRAC PEgRNA comprises SEQIDNO: 1560. [0877]Embodiment 164. The prime editing system of embodiment 138, wherein the first TRAC PEgRNA comprises SEQ ID NO: 1345 and the second TRAC PEgRNA comprises SEQ ID NO: 1566. [0878]Embodiment 165. The prime editing system of embodiment 138, wherein the first TRAC PEgRNA comprises SEQ ID NO: 374 and the second TRAC PEgRNA comprises SEQIDNO: 1251. [0879]Embodiment 166. The prime editing system of embodiment 138, wherein the first TRAC PEgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 1322, 1336, 1372, and 126; and wherein the second TRAC PEgRNA comprises a sequence selected from the group consisting of SEQ ID NOs:1442, 1456, 1501, and 1513. [0880]Embodiment 167. The prime editing system of embodiment 138, wherein the first TRAC PEgRNA comprises a sequence selected from the group consisting of SEQ ID NOs :1401, 1406, 1343,, and 1345; and ,wherein the second TRAC PEgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 1451, 1516, 1568, 1566, and 1459. [0881]Embodiment 168. The prime editing system of embodiment 138, wherein the first TRAC PEgRNA comprises SEQ ID NO: 1401, and wherein the second TRAC PEgRNA comprises SEQ ID NO: 1459. [0882]Embodiment 169. The prime editing system of any one of embodiments 122-168, wherein the first TRAC PEgRNA and/or the second TRAC PEgRNA further comprises a 3’ WO 2024/238825 PCT/US2024/029746 405 motif, optionally wherein the 3’ motif is connected to the 3’ end of the first TRAC PBS or the second TRAC PBS via a linker. [0883]Embodiment 170. The prime editing system of any one of embodiments 122-169, wherein the first TRAC PEgRNA and/or the second TRAC PEgRNA further comprises 5’mN*mN*mN* and 3’ mN*mN*mN*N modifications, where m indicates that the nucleotide contains a 2’-O-Me modification and a * indicates the presence of a phosphorothioate bond. [0884]Embodiment 171. The prime editing system of any one of embodiments 122-170, further comprising the recombinase or a nucleic acid encoding the recombinase. [0885]Embodiment 172. The prime editing system of embodiment 171, wherein the recombinase is fused or linked to the prime editor. [0886]Embodiment 173. The prime editing system of embodiment 171 or 172, further comprising a polynucleotide or a nucleic acid encoding the polynucleotide, wherein the polynucleotide comprises (a) a donor sequence and (b) a second recombinase recognition sequence (RRS) recognized by the recombinase. [0887]Embodiment 174. The prime editing system of embodiment 173, wherein the donor sequence comprises an open reading frame that encodes a polypeptide. [0888]Embodiment 175. The prime editing system of embodiment 173, wherein the donor sequence encodes a chimeric antigen receptor (CAR). [0889]Embodiment 176. The prime editing system of any one of embodiments 173-175, wherein (i) the RRS comprises SEQ ID NO: 1590, and the second RRS comprises SEQ ID NO: 1591, or (ii) the RRS comprises SEQ ID NO: 1591, and the second RRS comprises SEQ ID NO: 1590. [0890]Embodiment 177. The prime editing system of any one of embodiments 173-175, wherein the recombinase is Bxbl. [0891]Embodiment 178. The prime editing system of any one of embodiments 95-177, further comprising a prime editor or one or more polynucleotides encoding the prime editor, wherein the prime editor comprises: a) a Cas9 nickase having a nuclease inactivating mutation in the HNH domain, and b) a reverse transcriptase. [0892]Embodiment 179. The prime editing system of embodiment 178, wherein the prime editor is a fusion protein.

WO 2024/238825 PCT/US2024/029746 406 id="p-893"

[0893]Embodiment 180. The prime editing system of any one of embodiments 95-177, further comprising: an N-terminal extein comprising an N-terminal fragment of a prime editor fusion protein and an N-intein or a polynucleotide encoding the N-terminal extein; a C-terminal extein comprising a C-terminal fragment of the prime editor fusion protein and a C-intein, or a polynucleotide encoding the C-terminal extein; wherein the N-intein and the C- intein of the N-terminal and C-terminal exteins are capable of self-excision to join the N- terminal fragment and the C-terminal fragment to form the prime editor fusion protein, and wherein the prime editor fusion protein comprises a Cas9 nickase having a nuclease inactivating mutation in the HNH domain and a reverse transcriptase (RT) domain. [0894]Embodiment 181. The prime editing system of any one of embodiments 178-180, wherein the Cas9 nickase comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NOs: 676 or 677. [0895]Embodiment 182. The prime editing system of any one of embodiments 178-181, wherein the reverse transcriptase comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 673. [0896]Embodiment 183.The prime editing system of embodiment 181 or 182, wherein the sequence identities are determined by Needleman-Wunsch alignment of two protein sequences with Gap Costs set to Existence: 11 Extension: 1 where percent identity is calculated by dividing the number of identities by the length of the alignment. [0897]Embodiment 184. The prime editing system of any one of embodiments 178-183, wherein the one or more polynucleotides encoding the prime editor, the polynucleotide encoding the N-terminal extein, or the polynucleotide encoding the C-terminal extein are mRNA. [0898]Embodiment 185. A population of viral particles collectively comprising the one or more polynucleotides encoding the PEgRNA of any one of embodiments 1-94 or the prime editing system of any one of embodiments 95-184. [0899]Embodiment 186. The population of viral particles of embodiment 185, wherein the viral particles are AAV particles. [0900]Embodiment 187. An LNP comprising the prime editing system of any one of embodiments 95-184. [0901]Embodiment 188. The LNP of embodiment 187, comprising the PEgRNA and optionally the ngRNA, the polynucleotide encoding the Cas9 nickase, and the polynucleotide encoding the reverse transcriptase.

WO 2024/238825 PCT/US2024/029746 407 id="p-902"

[0902]Embodiment 189. The LNP of embodiment 188, wherein the polynucleotide encoding the Cas9 nickase and the polynucleotide encoding the reverse transcriptase are mRNA. [0903]Embodiment 190. The LNP of embodiment 188 or 189, wherein the polynucleotide encoding the Cas9 nickase and the polynucleotide encoding the reverse transcriptase are in the same molecule. [0904]Embodiment 191. A method of editing a B2M gene, the method comprising contacting the B2M gene with: (a) the PEgRNA of any one of embodiments 1-94, and a prime editor comprising a Cas9 nickase having a nuclease inactivating mutation in the HNH domain and a reverse transcriptase, (b) the prime editing system of any one of embodiments 95-184, (c) the population of viral particles of embodiment 185 or 186, or (d) the LNP of any one of embodiments 187-190. [0905]Embodiment 192. The method of embodiment 191, wherein the B2M gene is in a cell. [0906]Embodiment 193. A method of generating an engineered cell, the method comprising introducing into a cell or a population of cells: (a) the PEgRNA of any one of embodiments 1-94, and a prime editor comprising a Cas9 nickase having a nuclease inactivating mutation in the HNH domain and a reverse transcriptase, (b) the prime editing system of any one of embodiments 95-184, (c) the population of viral particles of embodiment 185 or 186, or (d) the LNP of any one of embodiments 187-190. [0907]Embodiment 194. A method of generating an engineered cell, the method comprising introducing into a cell or a population of cells (a) the prime editing system of any one of embodiments 122-184, (b) the population of viral particles of embodiment 185 or 186, or (c) the LNP of any one of embodiments 187-190. [0908]Embodiment 195. The method of any one of embodiments 192-194, wherein the cell or the population of cells are in a subject. [0909]Embodiment 196. The method of any one of embodiments 192-194, wherein the cell or the population of cells are ex vivo, optionally wherein the cell or the population of cells are obtained from a subject or a cell bank. [0910]Embodiment 197. The method of any one of embodiments 192-196, wherein the cell or the population of cells are human cells. [0911]Embodiment 198. The method of embodiment 197, wherein the cell or the population of cells are immune cells or stem cells.

WO 2024/238825 PCT/US2024/029746 408 id="p-912"

[0912]Embodiment 199. The method of embodiment 198, wherein the cell or the population of cells are T cells or hematopoietic stem cells (HSCs), optionally wherein the cell or the population of cells are cytotoxic T cells. [0913]Embodiment 200. A cell or a population of cells generated by the method of any one of embodiments 192-199. [0914]Embodiment 201. An engineered cell or a population of engineered cells comprising a premature stop codon in the B2M gene relative to a wildtype B2M gene. [0915]Embodiment 202. An engineered cell or a population of engineered cells comprising a B2M gene comprising an insertion, a deletion, a substitution, or a combination thereof compared to a wildtype B2M gene at a chromosomal location corresponding to coding sequence position c.51, c.54, or c.50 of a wildtype B2M gene. [0916]Embodiment 203. An engineered cell or a population of engineered cells comprising a B2M gene comprising an insertion, a deletion, a substitution, or a combination thereof compared to a wildtype B2M gene at a chromosomal location corresponding to coding sequence position c.54, c.60, or c.66 of a wildtype B2M gene, optionally wherein the B2M gene comprises an insertion, a deletion, a substitution, or a combination thereof at a chromosomal location corresponding to coding sequence position c. 58 of a wildtype B2M gene. [0917]Embodiment 204. An engineered cell or a population of engineered cells comprising a B2M gene comprising an insertion, a deletion, a substitution, or a combination thereof compared to a wildtype B2M gene at a chromosomal location corresponding to coding sequence position c.21 or c.3 of a wildtype B2M gene, optionally wherein the B2M gene comprises an insertion, a deletion, a substitution, or a combination thereof at a chromosomal location corresponding to coding sequence position c. 17 of a wildtype B2M gene. [0918]Embodiment 205. An engineered cell or a population of engineered cells comprising a B2M gene comprising an insertion, a deletion, a substitution, or a combination thereof compared to a wildtype B2M gene at a chromosomal location corresponding to coding sequence position c.21, c.15 or c.3 of a wildtype B2M gene, optionally wherein the B2M gene comprises an insertion, a deletion, a substitution, or a combination thereof at a chromosomal location corresponding to coding sequence position c.11 of a wildtype B2M gene. [0919]Embodiment 206. The cell or the population of cells of embodiment 201 comprising a c.51delC deletion in the B2M gene relative to a wildtype B2M gene.

WO 2024/238825 PCT/US2024/029746 409 id="p-920"

[0920]Embodiment 207. The cell or the population of cells of embodiment 201 comprising a c.50insG insertion in the B2M gene relative to a wildtype B2M gene. [0921]Embodiment 208. The cell or the population of cells of embodiment 201 comprising a c.54_55insCC insertion in the B2M gene relative to a wildtype B2M gene. [0922]Embodiment 209. The cell or the population of cells of embodiment 201 comprising a c.54_55insTAAG insertion in the B2M gene relative to a wildtype B2M gene. [0923]Embodiment 210. The cell or the population of cells of embodiment 201 comprising a c.54_55insTAATAA insertion in the B2M gene relative to a wildtype B2M gene. [0924]Embodiment 211. The cell or the population of cells of embodiment 201 comprising a c.66_67insCC insertion in the B2M gene relative to a wildtype B2M gene, optionally wherein the cell or the population of cells further comprise a c.58G>C substitution in the B2M gene relative to a wildtype B2M gene. [0925]Embodiment 212. The cell or the population of cells of embodiment 201 comprising a c.66_67insTAAG insertion in the B2M gene relative to a wildtype B2M gene, optionally wherein the cell or the population of cells further comprise a c.58G>C substitution in the B2M gene relative to a wildtype B2M gene. [0926]Embodiment 213. The cell or the population of cells of embodiment 201 comprising a c.66_67insTAATAA insertion in the B2M gene relative to a wildtype B2M gene, optionally wherein the cell or the population of cells further comprise a c.58G>C substitution in the B2M gene relative to a wildtype B2M gene. [0927]Embodiment 214. The cell or the population of cells of embodiment 201 comprising a c.60_65deletion and a TAATAG insertion (c.60_64delinsTAATAG) in the B2M gene relative to a wildtype B2M gene, optionally wherein the cell or the population of cells further comprise a c.58G>C substitution in the B2M gene relative to a wildtype B2M gene. [0928]Embodiment 215. The cell or the population of cells of embodiment 201 comprising a c.21_22insCC insertion in the B2M gene relative to a wildtype B2M gene. [0929]Embodiment 216. The cell or the population of cells of embodiment 201 comprising a c.21_22insTAAG insertion in the B2M gene relative to a wildtype B2M gene. [0930]Embodiment 217. The cell or the population of cells of embodiment 201 comprising a c.21_22insTAATAA insertion in the B2M gene relative to a wildtype B2M gene. [0931]Embodiment 218. The cell or the population of cells of embodiment 201 comprising a c.3_4insCC insertion in the B2M gene relative to a wildtype B2M gene, optionally wherein WO 2024/238825 PCT/US2024/029746 410 the cell or the population of cells further comprise a C.17OG substitution or a c.l 1OG substitution in the B2M gene relative to a wildtype B2M gene. [0932]Embodiment 219. The cell or the population of cells of embodiment 201 comprising a c.3_4insTAAG insertion in the B2M gene relative to a wildtype B2M gene, optionally wherein the cell or the population of cells further comprise a C.17OG substitution or a c.l 1OG substitution in the B2M gene relative to a wildtype B2M gene. [0933]Embodiment 220. The cell or the population of cells of embodiment 201 comprising a c.3_4insTAATAA insertion in the B2M gene relative to a wildtype B2M gene, optionally wherein the cell or the population of cells further comprise a C.17OG substitution or a c.l 1OG substitution in the B2M gene relative to a wildtype B2M gene. [0934]Embodiment 221. The cell or the population of cells of embodiment 201 comprising a c.3_8deletion and a TAATGA insertion (c.3_8delinsTAATGA) in the B2M gene relative to a wildtype B2M gene, optionally wherein the cell or the population of cells further comprise a C.17OG substitution or a c.l 1OG substitution in the B2M gene relative to a wildtype B2M gene. [0935]Embodiment 222. The cell or the population of cells of embodiment 201 comprising a c.l5_16insCC insertion in the B2M gene relative to a wildtype B2M gene. [0936]Embodiment 223. The cell or the population of cells of embodiment 201 comprising a c. 15_16insTAAG insertion in the B2M gene relative to a wildtype B2M gene. [0937]Embodiment 224. The cell or the population of cells of embodiment 201 comprising a c.l5_16insTAATAA insertion in the B2M gene relative to a wildtype B2M gene. [0938]Embodiment 225. The cell or the population of cells of any one of embodiments 201- 224 comprising a TRAC gene that comprises a sequenceGGCTTGTCGACGACGGCGGTCTCAGTGGTGTACGGTACAAACC (SEQ ID NO: 9999) and/or GGTTTGTCTGGTCAACCACCGCGGTCTCCGTCGTCAGGATCAT (SEQ ID NO: 10000) relative to a wildtype TRAC gene. [0939]Embodiment 226. The cell or the population of cells of embodiment 225, wherein the edited TRAC gene comprises an insert sequence comprising, from 5’ to 3’, GGCTTGTCGACGACGGCGGTCTCAGTGGTGTACGGTACAAACC (SEQ ID NO: 9999), a donor sequence, and GGTTTGTCTGGTCAACCACCGCGGTCTCCGTCGTCAGGATCAT (SEQ ID NO: 10000).

WO 2024/238825 PCT/US2024/029746 411 id="p-940"

[0940]Embodiment 227. The cell or the population of cells of embodiment 225, wherein the edited TRAC gene comprises an insert sequence comprising, from 5’ to 3’, GGTTTGTCTGGTCAACCACCGCGGTCTCCGTCGTCAGGATCAT (SEQ ID NO: 10000), a donor sequence, and GGCTTGTCGACGACGGCGGTCTCAGTGGTGTACGGTACAAACC (SEQ ID NO: 9999). [0941]Embodiment 228. The cell or the population of cells of embodiment 226 or 227, wherein the donor sequence encodes a chimeric antigen receptor (CAR), optionally wherein the donor encodes a CD 19 CAR. [0942]Embodiment 229. The cell or the population of cells of any one of embodiments 226- 228, wherein the insert sequence is between a first chromosome location and a second chromosome location, wherein the first chromosome location is selected from the group consisting of human chromosome 14 positions 22547458, 22547457, 22547449, and 22547448, and wherein the second chromosome location is selected from the group consisting of human chromosome 14 positions 22547533, 22547523, 22547491, 22547528, 22547497, 22547579, 22547522, 22547485, 22547506, 22547560, 22547505, 22547529, and 22547490. [0943]Embodiment 230. The cell or the population of cells of any one of embodiments 226- 229, wherein the insert sequence is a) between human chromosome 14 positions225474and 22547533, b) between human chromosome 14 positions 22547458 and 22547522, c) between human chromosome 14 positions 22547458 and 22547529, d) between human chromosome 14 positions 22547449 and 22547533, e) between human chromosome positions 22547449 and 22547522, or f) between human chromosome 14 positions 225474and 22547529. [0944]Embodiment 231. The cell or the population of cells of any one of embodiments 202- 229, wherein the human chromosome locations and coding sequence locations are as set forth in Genome Reference Consortium Human Build 38 (GrCh38). [0945]Embodiment 232. The cell or the population of cells of any one of embodiments 200- 231, wherein the cell or the population of cells are in a subject. [0946]Embodiment 233. The cell or the population of cells of any one of embodiments 200- 231, wherein the cell or the population of cells are ex vivo, optionally wherein the cell or the population of cells are obtained from a subject or a cell bank.

WO 2024/238825 PCT/US2024/029746 412 id="p-947"

[0947]Embodiment 234. The cell or the population of cells of any one of embodiment 200- 233, wherein the cell or the population of cells are human cells. [0948]Embodiment 235. The cell or the population of cells of embodiment 234, wherein the cell or the population of cells are immune cells or stem cells. [0949]Embodiment 236. The cell or the population of cells of embodiment 235, wherein the cell or the population of cells are T cells or hematopoietic stem cells (HSCs), optionally wherein the cell or the population of cells are cytotoxic T cells. [0950]Embodiment 237. A method of immunotherapy comprising administering to a subject the (a) the PEgRNA of any one of claims 1-94, and a prime editor comprising a Cas9 nickase having a nuclease inactivating mutation in the HNH domain and a reverse transcriptase, (b) the prime editing system of any one of embodiments 95-184, (c) the population of viral particles of embodiment 185 or 186 (d) the LNP of any one of embodiments 187-190, or I the cell or the population of cells of any one of embodiments 200-235. [0951]Embodiment 238. A prime editing guide RNA (PEgRNA) or one or more polynucleotides encoding the PEgRNA, the PEgRNA comprising: a) a spacer that is complementary to a search target sequence on a first strand of a 02-microglobulin (B2M) gene, wherein the spacer comprises at its 3’ end a PEgRNA spacer sequence selected from any one of Tables 1-21; b) a gRNA core capable of binding to a Cas9 protein, and c) an extension arm comprising: i) an editing template comprising at its 3’ end an RTT sequence selected from the same Table as the PEgRNA Spacer sequence, and ii) a primer binding site (PBS) comprising at its 5’ end a PBS sequence selected from the same Table as the PEgRNA Spacer sequence. [0952]Embodiment 239. The PEgRNA of embodiment 238, wherein the spacer of the PEgRNA is from 17 to 22 nucleotides in length. [0953]Embodiment 240. The PEgRNA of embodiment 239, wherein the spacer of the PEgRNA is 20 nucleotides in length. [0954]Embodiment 241. The PEgRNA of any one of embodiments 238-240, wherein the spacer, the gRNA core, the editing template, and the PBS form a contiguous sequence in a single molecule. [0955]Embodiment 242. The PEgRNA of embodiment 241, comprising from 5’ to 3’, the spacer, the gRNA core, the editing template, and the PBS. [0956]Embodiment 243. A prime editing system comprising the PEgRNA or the one or more polynucleotides of embodiments 238-242.

WO 2024/238825 PCT/US2024/029746 413 id="p-957"

[0957]Embodiment 244. The prime editing system of embodiment 243, further comprising a nick guide RNA (ngRNA), or one or more polynucleotides encoding the ngRNA, wherein the ngRNA comprises: (i) an ngRNA spacer that comprises a region of complementarity to a second strand of the B2M gene; and (ii) an ngRNA core capable of binding a Cas9 protein. [0958]Embodiment 245. The prime editing system of embodiment 244, wherein the spacer of the ngRNA is from 17 to 22 nucleotides in length. [0959]Embodiment 246. The prime editing system of embodiment 245, wherein the spacer of the ngRNA is 20 nucleotides in length. [0960]Embodiment 247. The prime editing system of any one of embodiment 238-245, wherein the ngRNA spacer comprises at its 3’ end an ngRNA Spacer sequence selected from the same Table as the PEgRNA Spacer sequence. [0961]Embodiment 248. The prime editing system of any one of embodiments 238-245, wherein the ngRNA comprises an ngRNA sequence selected from the same Table as the PEgRNA Spacer sequence. [0962]Embodiment 249. The prime editing system of any one of embodiments 238-248, further comprising: a prime editor comprising a Cas9 nickase having a nuclease inactivating mutation in the HNH domain, or one or more polynucleotides encoding the Cas9 nickase, and a reverse transcriptase, or one or more polynucleotides encoding the reverse transcriptase. [0963]Embodiment 250. The prime editing system of any one of embodiments 238-245, further comprising: an N-terminal extein comprising an N-terminal fragment of a prime editor fusion protein and an N-intein or a polynucleotide encoding the N-terminal extein; and a C-terminal extein comprising a C-terminal fragment of the prime editor fusion protein and a C-intein, or a polynucleotide encoding the C-terminal extein; wherein the N-intein and the C- intein of the N-terminal and C-terminal exteins are capable of self-excision to join the N- terminal fragment and the C-terminal fragment to form the prime editor fusion protein, and wherein the prime editor fusion protein comprises a Cas9 nickase and a reverse transcriptase (RT) domain. [0964]Embodiment 251. The prime editing system of embodiment 249 or 250, wherein the Cas9 nickase comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NOs: 676 or 677. [0965]Embodiment 252. The prime editing system of any one of embodiments 249-251, wherein the reverse transcriptase comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 673.

WO 2024/238825 PCT/US2024/029746 414 id="p-966"

[0966]Embodiment 253. The prime editing system of embodiments 251 or 252, wherein the sequence identities are determined by Needleman-Wunsch alignment of two protein sequences with Gap Costs set to Existence: 11 Extension: 1 where percent identity is calculated by dividing the number of identities by the length of the alignment. [0967]Embodiment 254. The prime editing system of any one of claims 238-248, wherein the system further comprises a TRAC-PEgRNA pair, wherein the TRAC-PEgRNA pair comprises: a) a first TRAC-prime editing guide RNA (PEgRNA) or one or more polynucleotides encoding the first TRAC-PEgRNA, and b) a second TRAC-PEgRNA or one or more polynucleotides encoding the second TRAC-PEgRNA, wherein the first TRAC- PEgRNA comprises: i) a first TRAC-spacer that comprises at its 3’ end a 5’ TRAC-PEgRNA spacer sequence selected from any one of Tables 34 and 35, ii) a first TRAC-gRNA core capable of binding to a Cas9 protein; and iii) a first TRAC-extension arm comprising (A) a first TRAC-editing template and (B) a first TRAC- primer binding site (PBS) that comprises at its 5’ end a 5’ TRAC-PBS sequence selected from the same Table as the first TRAC- spacer, wherein the second TRAC-PEgRNA comprises: i) a second TRAC-spacer that comprises at its 3’ end a 3’ TRAC-PEgRNA spacer sequence selected from any one of Tables 36-38, ii) a second TRAC-gRNA core capable of binding to a Cas9 protein; and iii) a second TRAC- extension arm comprising (A) a second TRAC-editing template and (B) a second TRAC- primer binding site (PBS) that comprises at its 5’ end a 3’ TRAC-PBS sequence selected from the same Table as the second TRAC-spacer. [0968]Embodiment 255. The prime editing system of embodiment 254, wherein the first TRAC editing template comprises a region of complementarity to the second TRAC editing template. [0969]Embodiment 256. The prime editing system of embodiment 255, wherein the first TRAC editing template and the second TRAC editing template each encodes all or a fragment of a recombinase recognition sequence (RRS) or the reverse complement thereof, wherein the first TRAC editing template encodes at least a 5’ portion of the RRS or the reverse complement thereof, wherein the second TRAC editing template encodes at least a 3’ portion of the RRS or the reverse complement thereof, and wherein at least 10 nucleotides at the 5’ ends of the first and the second TRAC editing templates have perfect reverse complementarity to each other. [0970]Embodiment 257. The prime editing system of embodiment 256, wherein at least 15,20,25, or 30 nucleotides at the 5’ ends of the first and the second TRAC editing templates have perfect reverse complementarity to each other, optionally wherein at least 20,21,22,23,24,25,26, or 27 WO 2024/238825 PCT/US2024/029746 415 nucleotides at the 5’ ends of the first and the second TRAC editing templates have prefect reverse complementarity to each other. [0971]Embodiment 258. The prime editing system of embodiment 256 or 257, wherein the first TRAC editing template encodes the RRS or wherein the second TRAC editing template encodes the RRS, optionally wherein the RRS is an attB sequence recognized by a Bxbl recombinase or an attP sequence recognized by a Bxbl recombinase. [0972]Embodiment 259. The prime editing system of any one of embodiments 254-258, wherein the first TRAC editing template comprises an RTT #1 from Table 39 and the second TRAC editing template comprises an RTT #2 in Table 39, or wherein the first TRAC editing template comprises an RTT #2 from Table 39 and the second TRAC editing template comprises an RTT #1 in Table 39. [0973]Embodiment 260. The prime editing system of embodiment 259, wherein the first TRAC editing template comprises SEQ ID NO: 1577 and the second editing TRAC template comprises SEQ ID NO: 1584, or wherein the first TRAC editing template comprises SEQ ID NO: 1584 and the second TRAC editing template comprises SEQ ID NO: 1577.Embodiment 261. The prime editing system of embodiment 254, wherein the first TRAC PEgRNA comprises a 5’ TRAC PEgRNA sequence selected from any one of Tables 34 and 35, and wherein the second TRAC PEgRNA comprises a 3’ TRAC PEgRNA sequence selected from any one of Tables 36-38.Examples [0974]The following examples are provided for illustrative purposes only and are not intended to limit the scope of the claims provided herein. Example 1: PEgRNA screen for editing the B2M gene [0975]187 PEgRNAs with different spacer, PBS, and RTT combinations were designed to introduce two consecutive stop codons in the B2M gene coding sequence to disrupt B2M expression. The PEgRNAs were screened with the following experimental protocol modified based on a pooled screening protocol format using lentivirus libraries (Kim, H.K., Yu, G., Park, J. et al. Predicting the efficiency of prime editing guide RNAs in human cells. Nat Biotechnol (2020)). [0976]Specifically, lentiviral vectors were designed and constructed such that each lentiviral vector contains a sequence that encodes a single PEgRNA and a corresponding target sequence. The lentiviral vectors were used to transduce HEK293T cells at a low MOI to allow for ~1 virus integrant per infected cell. The cells were grown under antibiotic selection to enrich for cells containing the editing construct. After selection, the enriched cells were transfected with a plasmid encoding a prime editor fusion protein. 3 days after transfection, WO 2024/238825 PCT/US2024/029746 416 genomic DNA was harvested, and the target sequences were amplified and sequenced to examine editing efficiency. [0977]Accordingly, the oligo nucleotide library contains oligo nucleotides each has the following structure, in a 5' to 3' order: homology to a U6 promoter, a PEgRNA spacer sequence, either a partial scaffold fragment with internal BsmBI site or a full scaffold sequence (depending on the library design), a 7-nt polyT terminator, a 12-nt barcode 1, the DNA target site, a 12-nt barcode 2, and a 3' primer binding site. By having the target site flanked by two barcodes, matched at the stage of synthesis, crossover of PCR during amplification from gDNA were identified and removed from sequence analysis.

The oligo library was amplified using the following primers:Forward primer:CGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCReverse primer:TTATTACAGGGACAGCAGAGATCCAGTTTATCGATNNNNNNATCGCGGTACGCC AAGCT id="p-978"

[0978]Gibson assembly: Lentiviral vectors were constructed using Gibson assembly method as described below. An acceptor vector was digested with Esp3I or BsmBI. The linearized product was purified and used for Gibson assembly with a 1:1 -1:7 molar ratio to the oligo library. Electrocompetent NEB stable cells were transformed. The next day, the bacteria cells were harvested, and DNA was extracted with Qiagen plasmid plus midiprep using manufacturer ’s standard protocol. Clones were sequenced with next generation sequencing to ensure that the library as designed is represented in the clones. id="p-979"

[0979]Make lentivirus: Lentivirus was prepared with a 3-plasmid system: On day 0, HEK293T cells were plated in a T150 flask. On day 1, 18 ug of lenti transfer vector, 18 ug of psPAX2, 12 ug of pMD2.g, and opti-mem up to 1250 uL were mixed. A separate solution containing lipofectamine 2000 was freshly prepared. Then, the two solutions were mixed and incubated. After 48 hours of incubation (day 3), the supernatant media was collected. 5x peg solution was added to the lentivirus solution to a final concentration of lx, the solution was mixed well, and the mixture was left at 4 °C overnight. The next day, the precipitated lentivirus) was pelleted and resuspended and stored at -80 °C.

WO 2024/238825 PCT/US2024/029746 417 id="p-980"

[0980]Full scale tissue culture transduction, transfection׳ . Cells were plated in a T150 flask on day -1. On day 0, infect cells were predetermined with virus concentration condition to achieve -30% survival after antibiotic selection. The cells were transfected on day 6. On days 8-13, cells were harvested and genomic DNA was extracted for NGS prep. Forward and reverse primers as provided below are used to determine the optimal gDNA concentration to use per PCR reaction. Unique barcoding primers were used to uniquely identify editing at each target sequence. Sequencing results were analyzed, after removing reads that 1) do not contain barcodes or 2) contain crossover events (i.e. mismatch between the barcodes and the spacer sequences), with CRISPRESSO as described in Clement, K. et al., Nat. Biotechnol. 37, 224—226 (2019).

Table 41: Forward primer SEQ ID NO Forward primer sequence 1616dsw50ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNCTTGTGGAA AGGACGAAACACC1617dsw51ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNCTTGTGGA AAGGACGAAACACC1618dsw52ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCTTGTGG AAAGGACGAAACACC1619dsw53ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNCTTGTG GAAAGGACGAAACACC Table 42: Reverse Primer SEQ ID NO Reverse primer sequence 1620dsw5TGGAGTTCAGACGTGTGCTCTTCCGATCTNGGACAGCAGAGAT CCAGTTTATCGAT1621dsw6TGGAGTTCAGACGTGTGCTCTTCCGATCTNNGGACAGCAGAGA TCCAGTTTATCGAT1622dsw7TGGAGTTCAGACGTGTGCTCTTCCGATCTNNNGGACAGCAGAG ATCCAGTTTATCGAT id="p-981"

[0981]letter "N" in Tables 41 and 42indicate any one of nucleotides A, G, C, or T that are used in designing the primers avoid crossover events.

WO 2024/238825 PCT/US2024/029746 418 id="p-982"

[0982]PEgRNA sequences and the results are summarized in Table 43.The genomic DNA was sequenced and analyzed twice, and sequencing results from both times are reported (Sequencing #1 and Sequencing #2).

WO 2024/238825 PCT/US2024/029746 419 Table 43 N 4k M 6s 0 ׳ a cm o CD CD CD o CD Ov CD CD 1-H CD CD CD CD 1-H Ov CD cj CD CN CD CD CD 1-H CD CD CD CD CD CN cq vi o 1-H CD CD CD 0 o s y o CZ2 6s M 2 ׳y cj 1-H Ov CN CD 1-H 1-H cn o o CD 00 m o CN CD CD Ov cq rn m CD CD CD CN CD CD CD CD CD CD CD 00 CD CD CN CD CD CD M 6s u 0 ׳ a M CD CD CD 1-H CD CD o 1-H CD 1-H CD CD CD CD CD 1-H m CD CD CD 1-H CD CD 1-H VO CD CD CD cn 00 CN CD CN CD CD CD 0 o s y o CZ2 M a •y cj VO 1-H CD VO 1-H CD CD 00 in o CD CD in rj 1-H CD CD CD 00 o 1-H CD CD 1-H CD CD CD CD VO CD CD CN CD CD CD דכ |e 61 A H O' CZ2 1-H CN CN CN m CN in CN VO CN CN 00 CN Ov CN o m 1-H m CN m m m »n m m m in 00 m Ov o 1-H CN 43 CZ2 M Ov Ov Ov CN CN CN in in in Ov Ov Ov CN CN CN m m m Ov Ov Ov CN O' M o GO Q £ H Ov Ov Ov CN CN CN in in in 00 m 00 m 00 m 1-H 1-H 1-H o ׳y o ׳y o o fl o y ؛ ן H סנ a 1—1 I Hסנ a 1—1 I Hסנ a 1—1 I Hסנ a 1—1 I Hסנ a 1—1 I Hסנ a 1—1 Hסנ a 1—1 H סנ a 1—1 Hסנ a 1—1 Hסנ.s Hסנ.s Hסנ.s I Hסנ.s I Hסנ.s I Hסנ.s Hסנ.s Hסנ.s Hסנ.s I Hסנ.s I Hסנ.s I Hסנ.s I Hסנ.s in Q in Q in Q in Q in Q in Q ך 0 Q ך 0 Q ך 0 Q CS 1-H Q CS 1-H Q CS 1-H Q CS 1-H Q CS 1-H Q CS 1-H Q CS 1-H Q CS 1-H Q CS 1-H Q Q Q Q Q *"e 00 Ov CD CN 00 Ov CD CN 00 Ov CD CN 00 Ov 00 Ov 00 Ov r- ؟ r ^o |g1׳ fl W co 1-H 1-H 1-H 1-H 1-H 1-H 1-H 1-H 1-H 1-H CD 1-H 1-H CD 1-H 1-H CD 1-H 1-H CD 1-H 1-H CD 1-H 1-H CD 1-H 1-H CD 1-H 1-H CD 1-H 1-H CD 1-H סץ m סץ m סץ m סץ m WO 2024/238825 PCT/US2024/029746 420 m cq o 1-H o o 1-H o o ס 1-H ס ס 1-H ס ס ،N ס ס ،N סס 1-H ס ס 1-H ס 1-H ס 1-H ס ס ס m ס ס ס ס 1-H ס cj ס ס o o סץ m o cq o o 1-H 1-H o 00 o o o o ס ס 00 ،N ס m m ססץ סס cn ס 1-H ،N ס m ס ס 00 ،N סס סס ס ס ס ס ס ס ס o o o o o o o CN o 1-H o o cq o o ס ،N ס ס ،N ס ס 1-H ס ס m סס ،N ס ס 00 ס סס ססר סרס in ס ס ס 1-H סץס ס o o o o o 00 סץ in o 00 cq o o o ס ס m ס in ס ،N ס ס 1-H in 1-H סססס סס ס ס ס ס ס ס ס o o o o o o o m in >ס 00 00 00 ס 1-H סץ ס 1-H ס 1-H 1-H 1-H 1-H 1-H ،N 1-H cn 1-H 1-H 1-H סר 1-H 1-H m 00 m סץ m ס 1-H 1-H 1-H 9 1-H m 1-H 1-H m 1-H 00 סץ o 1-H cq m m m סץ סץ סץ ،N ،N ،N סר סר סרסץ סץ סץ ،N ،N ،N m m m סץ סץ סץ cq cq o o m m m סץ סץ סץסץ סץסץ סץסץ סץ ،N ס 1-H ،N ס 1-H ،N ס 1-H m (M m (M m (M 00 (M 00 (M 00 (M 1-H m 1-H m 1-H m »n »n »n in in I סנ g S Q I סנ g S Q H סנ g S Q ן H סנ g ס ן H סנ g ס ן H סנ a 1-H m ס ן H סנ a 1-H m ס ן H סנ a 1-H m ס ן H סנ a 1-H m ס ן H סנ a 1-H m ס ן H סנ a 1-H m ס ן H סנ a 1-H סר ס ן H סנ a 1-H סר ס ן H סנ a 1-H סר ס H סנ a ס H 1סנ a ס H סנ a ס H 1סנ a ס H סנ a ס H 1סנ a ס H 1סנ Q H סנ Q H 1סנ Q H סנ Q H סנ Q H 1סנ Q H סנ Q H 1סנ Q r- 00 £ 00 m ס 1-H ס 1-H ס 1-H m ס 1-H ס 1-H ס 1-H סר ס 1-H ס 1-H ס 1-H m m m >ס m m m m >ס m m m m m m m m m סץ m סץ m סץ m סץ m 00 00 00 00 00 00 00 00 00 1-H 1-H 1-H 1-H 1-H 1-H 1-H 1-H 1-H 1-H 1-H 1-H 1-H 1-H 1-H 1-H 1-H 1-H 1-H 1-H 1-H 1-H 1-H WO 2024/238825 PCT/US2024/029746 421 cn o o 1-H in 1-H o CN CD CD VO m CD 1-H CD m m o Ov CN CD CD VO Ov CD rn cq cj m 1-H 1-H CD m 1-H m CD CD CD 1-H 1-H 1-H m CN CD Ov m CD 00 m o 1-H CN CD CD VO CD 1-H CD CD o o o o cn o in o CD m o m CD CD 1-H CN CD 1-H cj Ov VO CD Ov m CD Ov Ov cq cj m סץ in o 00 m in 00 o cn in in CN Ov cj 00 vo cj 00 סץ 1-H o cn VO VO o o o 1-H o CN CD CD m m o in o 1-H m o m CN CD CD in o ؟ r 1-H CD CD סץ in 1-H CN CD CD CD (N m 1-H CD Ov m CD m o 1-H CN CD CD 1-H CN CD 1-H CD o o o o o Ov CD 1-H m 1-H CD 1-H CD CD CD m o m 1-H m o CN CD VO 1-H 1-H 00 rj 1-H CN CD CN Ov VO Ov CN CD Ov 1-H cj VO VO cq 00 cn 1-H VO VO VO vi cn 1-H 1-H cq 1-H cn 1-H 1-H in סץ VO Ov Ov 00 סץ Ov Ov CD CD CN 1-H CD CN in CN m m CN VO m CN in CN 00 m CN Ov m CN CD VO CN 1-H VO CN CN VO CN Ov 00 CN CD Ov CN 1-H Ov CN CN Ov CN m Ov CN Ov CN m Ov CN VO Ov CN cq in in in Ov Ov Ov CN CN m m Ov Ov Ov CN CN CN m m m Ov Ov Ov CN CN CN m m in o VO o VO o VO m m m 00 00 00 סץ סץ CD 1-H CN CD 1-H CN CD 1-H CN m CN m CN m CN VO CN VO CN VO CN CN CN CN CD 00 CN CD 00 CN CD 00 CN m 00 CN m 00 CN סנ Q H 1סנ Q H סנ Q H 1סנ Q H סנ Q H סנ Q H סנ Q H סנ Q H 1סנ Q H סנ Q H 1סנ Q I H סנ a 1—1 in Q I H סנ a 1—1 in Q I H סנ a 1—1 in Q H סנ a 1—1 in Q H סנ a 1—1 in Q H סנ a 1—1 in Q I H סנ a 1—1 in Q I H סנ a 1—1 in Q I H סנ a 1—1 in Q I H סנ a 1-H CN Q I H סנ a 1-H CN Q I H סנ a 1-H CN Q I H סנ a 1-H CN Q I H סנ a 1-H CN Q I H סנ a 1-H CN Q H סנ a 1-H CN Q H סנ a 1-H CN Q in m in cq Ov m סץ Ov CN Ov m סץ CN Ov m סץ Ov CN CD CN CN 1-H CN CN Ov CN CD CN CN 1-H CN CN Ov CN CD CN CN 1-H CN CN VO 00 CN 00 CN 00 00 CN VO 00 CN 00 CN 00 00 CN VO 00 CN 00 CN VO 1-H VO 1-H VO 1-H VO 1-H in 1-H m 1-H m 1-H m 1-H m 1-H m 1-H m 1-H CN CD CN CN CD CN CN CD CN CN CD CN CN CD CN CN CD CN CN CD CN CN CD CN CN CD CN Ov VO CN Ov VO CN Ov VO CN Ov VO CN Ov VO CN Ov VO CN Ov VO CN Ov VO CN WO 2024/238825 PCT/US2024/029746 422 ס 00 1-H 1-H o in o 1-H cn o 00 cq o m o o m o CD o 1-H vo CD CD CD m m 1-H Ov CD m Ov CD סר o vo »ר cj CD CD CD vo CD cj CD 1-H CD CD CD 1-H VO o cq cq 1-H 1-H 1-H o o o 1-H o Ov o VO Ov CD CD m CD CD CD CD CD o CD CD CD CD CD CD CD סר m o CD סר סרס vo CD CD CD CD CD in eq 1-H 1-H m o 1-H o 00 eq 1-H m cq o m in o 1-H o o m o CD CN VO 1-H 1-H CD CD 1-H 1-H CD En o En Ov Ov CD CN CD CD CD 00 cn CD CD CD CD CD cn vo o m 1-H cn rj 1-H Ov "ו cj cq 1-H 1-H 00 1-H VO cj CD CD CD CD 1-H CD CD 9 cn CD CD CD vo CD CD CD m o CD CD CD Ov cq cn סץ cn o cq m 1-H cq m cq cq m m cq m m in cq m VO CN m m cn סץ cn o סר m 1-H סר m CN סר m m סר m n ؛ m סר סר m cn cn cn סץ cn o 00 m 1-H 00 m CN 00 m m 00 m m in Ov Ov Ov cq cq cq in in in Ov Ov Ov CN CN CN in in סר Ov Ov Ov CN CN CN in in in m 00 CN VO o VO o m VO o m Ov o m Ov o m Ov o m CN cn CN cn CN cn סר m m סר m m סר m m 00 m m 00 m m 00 m m 1-H cn 1-H cn 1-H cn 3 m 3 m 3 m m m m o cn o cn o cn סנ a 1-H CN Q I H סנ a 1-H CN Q I H סנ a 1-H CN Q I H סנ a 1-H CN Q I H סנ a 1-H CN Q I H סנ a 1-H CN Q I H סנ a 1-H CN Q H סנ a 1-H CN Q H סנ a 1-H CN Q H סנ a 1-H CN Q H סנ a 1-H CN Q H סנ a 1-H CN Q H סנ a 1-H CN Q H סנ a 1-H CN Q H סנ a 1-H CN Q H סנ a 1-H CN Q H סנ a 1-H CN Q H סנ a 1-H CN Q H סנ a 1-H CN Q H סנ a 1-H CN Q H סנ a 1-H CN Q H סנ a 1-H CN Q H סנ a 1-H CN Q H סנ a 1-H CN Q H סנ a 1-H CN Q H סנ a 1-H CN Q H סנ a 1-H CN Q H סנ a 1-H CN Q 00 00 CN in cn cn 1-H m in cn cn 1-H m in cn cn 1-H m m in cn cn m in cn cn m in cn cn cn cn cn in cn cn cn cn in cn cn cn cn in cn Ov VO cq 00 Ov cq 00 Ov cq 00 Ov cq 00 Ov cq 00 Ov cq 00 Ov cq 00 Ov cq 00 Ov CN 00 Ov CN m m m m m m m m m סר m סר m סר m סר m סר m סר m סר m סר m סר m WO 2024/238825 PCT/US2024/029746 423 ס ס ס ס cn ס ס ס m ס סס o o o o o m cn o o o in o o ،N cq o o o o ،N סץ o o o ס ס ס ס ס 1-H סס ס ס ס o o o o o CN o o o o o o o o o o 1-H o o CD o ס ס ס ס m סס ס ס ס ס o o o o ץ 0 o 00 eq סר o o o o o o o o o 1-H m סץ o o o ס ס ס ס ס ס ס ס ס ס o o o o o o o o o o o o o o o o o o m ס ס ס 00 ססץ סס 1-H 1-H 1-H ،N cn m in m m cn 00 m סץ m o 1-H cq m in VD 00 סץ o 1-H cq סץ סץ סץ סץ ،N ،N ،N m m m סץ סץ סץ ،N ،N ،N in in in סץ סץ סץ cq cq cq m m m סץ m m m m סץ m סץ m סץ m סץ סץ m סץ סץ m סץ סץ m ،N ،N cq cq cq cq סר CN סר CN סר CN 00 CN 00 CN 00 CN 1-H סר 1-H in 1-H in in in in in in in ס ן סנ a 1-H ס ן סנ a 1-H ס ן סנ a 1-H ס ן H סנ a 1-H ס ן H סנ a 1-H ס ן H סנ a 1-H ס ן H סנ a 1-H ס ן H סנ a 1-H ס ן H סנ a 1-H ס ן H סנ a 1-H ס I H סנ a 1-H Q I H סנ a 1-H Q H סנ a 1-H Q H סנ a 1-H Q H סנ a 1-H Q H סנ a 1-H Q H סנ a 1-H Q H סנ a 1-H Q 1 H סנ .s in Q H סנ .s in Q H סנ .s in Q 1 H סנ .s in Q H סנ .s in Q H סנ .s in Q 1 H סנ .s in Q H סנ .s in Q H סנ .s in Q H סנ Q ،N ס m ס ס ،N ס m ס ס ،N ס m ס ס 1-H m CN m m m 1-H m CN m m m 1-H m CN m m m o 1-H ،N o 1-H ،N o 1-H ،N סץ m 00 m m 00 m m 00 m m 00 m m 00 m m 00 m m 00 m m 00 m m 00 m 1-H 1-H 1-H 1-H 1-H 1-H 1-H 1-H 1-H 9 9 9 9 9 9 9 9 WO 2024/238825 PCT/US2024/029746 424 ס ססץ ס o o cq o o o 1-H m o o 1-H CN CD CD CD סץ CD 1-H 1-H CD CD m CD CD CD CN CD CD m סץ m CD 1-H CD CD CD m m rj CD CN CD CD ס ס ס o o o o o סץ cq o CN VD CD CD m in cn m cn סץ in 1-H CD 1-H o 00 eq 1-H CD CD CD CD 1-H CD CD CD CD CD CD m CD CD CD ס ס£ ס o o o o cq m o o CN CD CD CD m סץ סץ CD CD o CD CD CD 9 rj in CD CD CD CD CD CD CD CD ס 1-H ס סס o o o o o 1-H CD 1-H m o CD cn cn ץ 0 "ו cj CD 00 1-H סץ in 1-H CD CD CD CD VD CD CD CD CD VD CD 1-H CD CN CD m in 1-H ץ 0 m סץ ץ 0 00 סץסץ סץ o o m 1-H cq m CN CN m m cq m ni m n ؛ cq n ؛ CN m m 00 cq m סץ CN m o m m 1-H m m CN m m m m m in m m m m m m in m 00 m m סץ m o 00 m סץ סץ CN cq cq m m m סץ סץ סץ CN CN CN m m m סץ סץ סץ CN CN CN m m m סץ סץ ס ס m m m 00 00 00 00 סץ CD m סץ CD m סץ CD m CN m CN m CN m m m m m m m 00 m m 00 m m 00 m m 1-H m 1-H m 1-H m in in in m m ן סנ eg in ס ן סנ eg in ס ן H סנ Q H סנ .s in Q H סנ .s in Q H סנ Q H סנ .s in Q H סנ .s in Q I H M ،g CN Q I H M ،g CN VD Q I H M ،g CN Q H M ،g CN Q H M ،g CN VD Q H M ،g CN Q H M ،g CN Q H M ،g CN VD Q H M ،g CN Q H M a סץ Q H M a סץ Q H M a סץ Q H M a סץ Q H M a סץ Q H M a סץ Q H M a סץ Q H M a סץ Q H M a סץ Q H »נ .s in m Q H »נ .s in m Q ס סץ 1-H סץסץ o סץ 1-H סץסץ o סץ 1-H סץ 00 m סץ m CD CN m 00 m סץ m CD CN m 00 m סץ m CD CN m in 00 m סץ m in 00 m סץ m in 00 m סץ m m in 1-H o m 1-H o m 1-H o m 1-H o m 1-H o n ؛ 1-H o m 1-H o m 1-H o m 1-H o m o m m o m m o m m o m m o m m o m m o m m o m m o m m סץ m m סץ m m WO 2024/238825 PCT/US2024/029746 425 ס ס 1-H O o o Ov cj o o o o o ،N o o o 00 o 1-H VO vi o 3 CD o o o o Ov cq VO o ס ס 00 1-H o o m o o cj 1-H o o m o VO o m 00 rj m o סץ m 1-H 00 o m m o in o cq o in 1-H סץ 00 in m VO Ov m in m o cq cq VO ס ס m O o o Ov cq rn o o o o o o o o 1-H 00 cn 00 in tn CD Ov o o CD o o 00 1-H o ס ס in 1-H o Ov >ץ 1-H o o o 1-H o Ov o 1-H 1-H 00 1-H o (N m 1-H o 1-H VO o in o m סץ 1-H m m in Ov VO cq m o cq 1-H cj VO cq 1-H 00 m (N 00 in m 00 m m m 00 m 00 m 00 m 00 o VO Ov o VO o 1-H VO 1-H 1-H cq tn 1-H m cn 00 m Ov m VO o 1-H cq tn 3 m Ov ،N ،N ،N m m m Ov Ov Ov cq cq ،N m m m Ov Ov Ov cq cq cq m m m m ס in o m o m tn m tn m tn m VO Ov m VO Ov m VO Ov m סץ סץ m סץ סץ m סץ סץ m ،N o ،N o ،N o m cq VO m cq VO m cq VO 00 cq VO 00 cq VO 00 cq VO 1-H m 1-H m 1-H m ן H) eg in ס ן a) eg !n m Q a) eg in m Q H a) eg in m Q a) eg in m Q H a) eg in m Q H a) eg in m Q H ; a) eg Q H a) eg Q H ; a) eg Q H «) .8 Q H «) .8 Q H «) .8 Q H «) .8 Q H «) .8 Q H «) .8 Q I H «) .8 in סץ Q I H «) .8 in סץ Q I H «) .8 in סץ Q I H «) .8 in סץ Q I H «) .8 in סץ Q I H «) .8 in סץ Q 1 H «) .8 in סץ Q H «) .8 in סץ Q 1 H «) .8 in סץ Q 00 in in in 00 m m in 00 m m o VO VO o VO o VO m o o o m o o o m m m VO m VO m m m VO m VO m m m VO m VO in in סץ in in סץ m m סץ m m סץ m m סץ m m סץ m m 00 00 m 00 00 m 00 00 m 00 00 m 00 00 m 00 00 m 00 00 m 00 00 m 00 00 m 1-H VO 1-H VO 1-H VO 1-H VO 1-H VO 1-H VO 1-H VO 1-H VO 1-H VO WO 2024/238825 PCT/US2024/029746 426 Example 2: Prime Editing of the B2M gene in HEK293T cells [0983]22 PEgRNAs each having the spacer sequence SEQ ID NO:202 were designed to introduce a c.50insG or a c.51delC frameshift mutation in the coding sequence of B2M and result in a premature stop codon and disrupt B2M expression. Two ngRNAs were also designed to test the efficiency of PE3b (both ngRNA spacers having perfect complementarity with the PAM strand after the nucleotide edit is incorporated) editing strategy. [0984]The PEgRNAs and ngRNAs were chemically synthesized. The PEgRNAs and ngRNAs each contained a 3’ UUUU end adaptation as indicated in the sequences, and further contained chemical modifications 3’ mN*mN*mN*N and 5’mN*mN*mN*N modifications, where m indicates that the nucleotide contains a 2’-O-Me modification and a * indicates a phosphorothioate bond. [0985]HEK293T cells were transfected with the PEgRNA (and ngRNA where applicable) and polynucleotide encoding a prime editor fusion protein. 72 hours after transfection, genomic DNA was extracted by aspirating media, adding QuickExtract DNA Extraction solution (Lucigen, #QR9050) and incubating according to manufacturer ’s protocol for high- throughput sequencing. [0986]The following primers were used: Table 44 PPrimer nameSequence BB2M ForwardACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTGGGCA CGCGTTTAATATAAG BB2M ReverseTGGAGTTCAGACGTGTGCTCTTCCGATCTAGATCCAGCCCTGG ACTAGC id="p-987"

[0987]Sequence numbers of the PEgRNAs, ngRNAs and corresponding components, as well as editing efficiency and indel frequency are summarized in Tables 45 (PE2 editing strategy) and 46 (PE3 editing strategy with ngRNAs). The number of contiguous nucleotides in the editing template that are 1) complementary to the editing target sequence in the B2M gene and 2) upstream of the position of the 5’ most nucleotide of the edit encoded in the editing template (referred to in Table 45 as Homology arm length) is also provided. Each PEgRNA (or PEgRNA-ngRNA pair) was tested with two or three technical replicates, as indicated in Tables 45 and 46. not tested Table 45 PEgRNA SEQID No Spacer SEQID No PBS SEQ ID No RTTSEQ ID No Edit encoded Homology arm length Editing% Repl Editing% Rep2 Editing% Rep3 Indels% rep 1 Indels% rep 2 Indels% rep 3 239 202 209 228 c.50insG 3 0.01 0.01 - 4.74 1.88 -240 202 209 229 c.50insG 5 1.88 0.87 - 2.75 1.12 -241 202 209 230 c.50insG 7 0.23 2.36 - 0.1 1.43 -242 202 209 231 c.50insG 9 2.58 0.91 - 2.93 1.28 -243 202 211 228 c.50insG 3 0 0 - 0 0.13 -244 202 211 229 c.50insG 5 1.69 1.12 - 1.48 1.25 -245 202 211 230 c.50insG 7 3.22 2.51 - 1.22 1.24 -247 202 213 228 c.50insG 3 0.09 0.07 - 2.64 1.81 -248 202 213 229 c.50insG 5 8.31 6.66 - 3.76 2.85 -238 202 213 230 c.50insG 7 9.38 14.62 - 1.71 2.83 -249 202 213 231 c.50insG 9 1.99 7.23 - 0.61 2.86 -250 202 215 228 c.50insG 3 0.01 0.02 - 0.73 2 -251 202 215 229 c.50insG 5 3.44 6.73 - 1.37 3.01 -252 202 215 230 c.50insG 7 4.52 14.45 - 0.62 2.42 -253 202 215 231 c.50insG 9 1.47 6.1 - 0.69 2.12 -233 202 213 223 c.51delC 5 3.23 4.92 3.28 3.74 5.33 2.65234 202 213 224 c.51delC 7 20.85 19.5 18.99 1.87 1.44 1.39235 202 213 225 c.51delC 9 20.81 19.15 18.96 1.47 1.39 1.47232 202 213 222 c.51delC 11 17.52 19.86 19.21 1.22 1.41 1.2236 202 213 226 c.51delC 13 19.86 21.66 21.58 1.55 1.82 1.62237 202 213 227 c.51delC 15 19.88 20.16 20.44 1.74 2.18 2.38 WO 2024/238825 PCT/US2024/029746 427 Table 46 PEgRNA SEQ ID No PBS SEQ ID No RTT SEQ ID No Edit encoded ngRNA spacer SEQ ID No ngRNA SEQ ID No Editing% Repl Editing% Rep2 Editing% Rep3 Indels% rep 1 Indels% rep 2 Indels% rep 3 239 209 228 c.SOinsG 267 827 0.00 0.07 - 1.37 2.57 -240 209 229 c.SOinsG 267 827 1.41 8.32 - 0.98 4.25 -241 209 230 c.SOinsG 267 827 2.59 11.72 - 0.79 3.30 -242 209 231 c.SOinsG 267 827 0.76 7.38 - 0.33 2.44 -243 211 228 c.SOinsG 267 827 0.00 0.05 - 0.29 3.01 -244 211 229 c.SOinsG 267 827 3.39 7.68 - 1.12 3.00 -245 211 230 c.SOinsG 267 827 4.23 13.61 - 0.74 2.71 -246 211 231 c.SOinsG 267 827 2.05 12.19 - 0.54 2.72 -247 213 228 c.SOinsG 267 827 0.13 0.12 - 0.33 4.01 -248 213 229 c.SOinsG 267 827 4.98 20.31 - 0.87 4.95 -238 213 230 c.SOinsG 267 827 7.93 31.63 - 0.86 4.20 -249 213 231 c.SOinsG 267 827 3.74 22.24 - 0.71 3.96 -250 215 228 c.SOinsG 267 827 0.00 0.17 - 0.26 2.39 -251 215 229 c.SOinsG 267 827 3.20 21.53 - 0.70 4.88 -252 215 230 c.SOinsG 267 827 4.95 28.80 - 0.50 2.85 -253 215 231 c.SOinsG 267 827 1.09 18.44 - 0.28 3.47 -239 209 228 c.SOinsG 268 824 0.00 0.02 - 0.55 2.09 -240 209 229 c.SOinsG 268 824 1.31 4.37 - 0.44 1.99 -241 209 230 c.SOinsG 268 824 1.93 10.46 - 0.48 2.64 -242 209 231 c.SOinsG 268 824 0.89 4.50 - 0.47 1.41 -243 211 228 c.SOinsG 268 824 0.02 0.05 - 0.32 1.83 -244 211 229 c.SOinsG 268 824 0.63 4.35 - 0.33 1.83 -245 211 230 c.SOinsG 268 824 5.31 15.24 - 0.77 2.42 -246 211 231 c.SOinsG 268 824 1.14 8.81 - 0.15 1.90 - WO 2024/238825 PCT/US2024/029746 428 247 213 228 c.SOinsG 268 824 0.03 0.11 - 0.33 2.61 -248 213 229 c.SOinsG 268 824 3.03 18.94 - 0.60 4.86 -238 213 230 c.SOinsG 268 824 5.21 24.47 - 0.78 3.76 -249 213 231 c.SOinsG 268 824 0.94 14.68 - 0.25 2.87 -250 215 228 c.SOinsG 268 824 0.00 0.01 - 0.17 0.41 -251 215 229 c.SOinsG 268 824 1.75 17.96 - 0.40 4.17 -252 215 230 c.SOinsG 268 824 4.64 19.30 - 0.39 2.35 -253 215 231 c.SOinsG 268 824 0.54 17.62 - 0.17 4.20 -233 213 223 c.SldelC 266 826 6.09 5.25 4.51 3.56 2.39 1.98234 213 224 c.SldelC 266 826 31.68 31.50 24.56 1.25 1.46 0.92235 213 225 c.SldelC 266 826 34.72 31.44 29.34 1.95 1.49 1.25232 213 222 c.SldelC 266 826 31.08 31.90 24.09 1.34 1.29 1.02236 213 226 c.SldelC 266 826 36.62 36.44 31.77 1.87 1.95 1.55237 213 227 c.SldelC 266 826 45.69 45.93 40.09 2.10 2.80 3.01 WO 2024/238825 PCT/US2024/029746 430 id="p-988"

[0988]3 PEgRNAs each having the spacer sequence SEQ ID NO: 202 were further tested for Prime Editing interruption of the B2M gene. The 3 PEgRNAs each encoded a c.50insG frameshift mutation, a c.51delC frameshift mutation, or a c.54insTAATAA insertion of two consecutive stop codons. The PEgRNA and ngRNA sequences are as indicated in Tables and 48 below and further contained chemical modifications 3’ mN*mN*mN*N and 5’mN*mN*mN*N modifications, where m indicates that the nucleotide contains a 2’-O-Me modification and a * indicates a phosphorothioate bond.The PEgRNAs and ngRNAs were chemically synthesized, HEK293T cells were transfected, editing efficiency and indel frequency were measured as described above in this Example. The results are summarized in Tables 47 and 48. Each PEgRNA (or PEgRNA-ngRNA pair) was tested with two technical replicates, as indicated in Tables 47(PE2 editing strategy) and 48(PE3 editing strategy). "X": editing not detected. ngRNAs that are PE3b ngRNAs (spacers having perfect complementarity with the PAM strand after the nucleotide edit is incorporated) are indicated with "*". Table 47 PEgRNA SEQ ID PEgRNA spacer SEQ ID PBS SEQ ID RTT SEQ ID Editing % Repl Editing % Rep2 Indels % rep Indels % rep 9993 202 216 219 3.34 2.37 0.67 0.52232 202 213 222 22.81 X 1.85 2.92238 202 213 230 6.68 9.61 1.05 1.46 Table 48 PEgRN A SEQ ID PBS SEQ ID RTT SEQ ID ngRNA SEQ ID ngRNA spacer SEQ ID Editing % Repl Editing % Rep2 Indels % rep Indels % rep 9993 216 219 9994 263 1.70 4.57 0.88 2.269993 216 219 9995 264 2.15 2.82 1.37 1.759993 216 219 9996 265 2.06 3.79 1.62 3.63232 213 222 9994 263 11.38 10.58 7.95 5.64232 213 222 9995 264 19.69 21.66 11.56 15.07232 213 222 9996 265 18.35 11.05 12.58 9.53232 213 222 9997 266* 23.49 14.83 0.28 0.44238 213 230 9994 263 6.56 9.04 4.60 6.49238 213 230 9995 264 8.98 13.11 5.79 12.14238 213 230 9996 265 6.31 7.07 5.52 6.69238 213 230 9998 267* 11.04 13.53 0.32 0.40 WO 2024/238825 PCT/US2024/029746 431 Example 3: Prime Editing of the B2M gene in HepG2 cells [0989]263 PEgRNAs were designed to target and disrupt the expression of the B2M gene. Spacers having the sequence of SEQ ID NO: 1, SEQ ID NO: 269, or SEQ ID NO: 3T1 were used for the PEgRNAs. The PEgRNAs each encode an edit that insert two consecutive stop codons or introduces a frameshift mutation that results in a premature stop codon in the B2M gene. Edits encoded by the PEgRNAs are provided in Tables 1-21 and also in the data summary Tables 49 and 50 below in the "Edits encoded " column. Each of the PEgRNAs also encode an NGG->NGC PAM silencing edit. In Tables 49and 50,where only one edit is indicated, the edit also results in the PAM silencing. Where two edits are indicated for one PEgRNA, the G->C or C->G nucleotide substitution is the PAM silencing edit. The nick-to- edit distance (i.e. number of nucleotides from the position of the edit to the position the nick site) for the stop codon insertion or frameshift mutation encoded by each PEgRNA are also indicated in the Tables. [0990]17 ngRNAs, including 1 PE3 ngRNA and 16 PE3b ngRNAs, were also designed to test the efficiency of PE3 editing strategy. PEgRNAs and ngRNAs were chemically synthesized and included end modifications 3’ mN*mN*mN*N and 5’mN*mN*mN*, where a "m" indicates that the nucleotide contains a 2’-O-Me modification and a * indicates the presence of a phosphorothioate bond. [0991]HepG2 cells were cultured in EMEM with 10% FBS and 1% P/S. For 96 well-plates, the cells were seeded at 10,000 cells per well 24 hours prior to transfection. Seeded cells were transfected with Lipofectamine MessengerMAX reagent according to manufacturer ’s protocol with PEgRNAs (and ngRNAs where appliable) and plasmid DNA encoding a prime editor fusion protein. 72 hours after transfection, genomic DNA was extracted by aspirating media, adding QuickExtract DNA Extraction solution and incubating according to manufacturer ’s protocol for high-throughput sequencing using NextSeq. The following sequencing primers were used for clonal amplification: Forward primer - GCTGGGCACGCGTTTAATAT Reverse primer - GGGCCACCAAGGAGAACTT id="p-992"

[0992]The PCR samples were then barcoded and pooled according to manufacturer ’s protocol for NextSeq analysis. [0993]PEgRNA sequences and the results are summarized in Tables 49 (PE2 editing strategy) and 50 (PE3 editing strategy) . PE3b ngRNAs and corresponding ngRNA spacers WO 2024/238825 PCT/US2024/029746 432 are indicated with a Two control samples were included in this experiment: a negative non-transfection control ("NTC") and a positive control using Prime Medicine ’s internal control PEgRNA targeting the HEK3 gene ("HEK3-CTT"). The control rows are repeated in both Tables 49 and 50 for convenience, "n/a": not applicable. not included.

Table 49 PEgRNA SEQID NO PEgRNA spacer SEQID NO RTT SEQ ID NO RTT length PBS SEQ ID NO PBS length Edits Encoded Homology arm length Nick-to- edit distance Editing% Indels% HEK3- CTT- - - - - - 17.74 0.17NTC n/a n/a n/a n/a n/a n/a n/a0.00 0.00930 1 900 8 8 8 c.54_55insCC 4 2 2.34 0.36931 1 901 10 8 8 c.54_55insCC 6 2 10.29 0.63932 1 902 12 8 8 c.54_55insCC 8 2 21.01 0.81933 1 903 14 8 8 c.54_55insCC 10 2 36.51 0.92934 1 900 8 10 10 c.54_55insCC 4 2 2.31 0.19935 1 901 10 10 10 c.54_55insCC 6 2 11.28 0.01936 1 902 12 10 10 c.54_55insCC 8 2 19.00 0.83937 1 903 14 10 10 c.54_55insCC 10 2 32.35 0.98938 1 900 8 12 12 c.54_55insCC 4 2 3.41 0.08939 1 901 10 12 12 c.54_55insCC 6 2 0.22 0.58940 1 902 12 12 12 c.54_55insCC 8 2 23.01 0.85941 1 903 14 12 12 c.54_55insCC 10 2 36.43 0.89942 1 904 20 8 8 c.66_67insCC, c.58G>C 4 14 0.79 0.28943 1 905 22 8 8 c.66_67insCC, c.58G>C 6 14 4.73 0.67944 1 906 24 8 8 c.66_67insCC, c.58G>C 8 14 13.08 0.94945 1 907 26 8 8 c.66_67insCC, c.58G>C 10 14 22.36 0.43946 1 904 20 10 10 c.66_67insCC, c.58G>C 4 14 0.46 0.32947 1 905 22 10 10 c.66_67insCC, c.58G>C 6 14 3.85 0.31948 1 906 24 10 10 c.66_67insCC, c.58G>C 8 14 10.73 0.69949 1 907 26 10 10 c.66_67insCC, c.58G>C 10 14 18.00 0.87950 1 904 20 12 12 c.66_67insCC, c.58G>C 4 14 0.11 0.41 WO 2024/238825 PCT/US2024/029746 433 WO 2024/238825 PCT/US2024/029746 434 o o so o SO 1-* 9 o o 1-* OS o in o o OS OS o so o so o 00 so o m so o 3 o m o OS o o o 1-* m o 04 o o o o 1-* o OS o in o o 1-* en en o 1-* so 1-* so o OS in so so m OS OS in o o in in o 1-* in m 00 1-* in in 1-* in S in o in o o o o o S 1-* o סך OS m so o 04 1-* so m o סך -* 1 o 1-H 1-H 04 04 04 04 04 04 04 04 04 04 04 04 1-H 1-H 1-H 1-H 1-H 1-H 1-H 1-H 1-H 1-H 1-H 04 04 SO 00 o 1-* 't so 00 o 1-* 't so 00 o 1-* 't so 00 o 1-* 't so 00 o 1-* 't so 00 o 1-* so 00 o 1-* 't so u 6 00 in d o o a '0i SO so d u 6 00 in d o o ،s '0i so so d u 6 00 in d o o a '0i so so d ס s :י in d ס 5 H s :י in d ס s ל in d ס 5 H s :י in d ס s ל in d ס 5 H s :י in d ס s :י in d ס 5 H s :י in d ס s :י in d ס 5 H s ל in d ס s :י in d ס 5 H s :י in d u 6 00 in d ס 5 H .9 ^°ו so so d u 6 00 in d ס a ^°ו so so d u 6 00 in d ס 5 H .g ^°ו so so d u 6 00 in d ס a ^°ו so so d u 6 00 in d ס 5 H .g ^°ו so so d u 6 00 in d ס a ^°ו so so d u 6 00 in d ס 5 H .g ^°ו so so d u 6 00 in d ס a ^°ו so so d u 6 00 in d ס 5 H .g ^°ו so so d u 6 00 in d ס a ^°ו so so d u 6 00 in d ס 5 H .g ^°ו so so d s :י in d .5 in :י in d 04 04 04 00 00 00 00 o 1-* o 1-* o 1-* o 1-* 04 04 04 04 00 00 00 00 o 1-* o 1-* o 1-* o 1-* 04 04 04 00 00 04 04 04 00 00 00 00 o 1-* o 1-* o 1-* o 1-* 04 04 04 04 00 00 00 00 o 1-* o 1-* o 1-* o 1-* 04 04 04 00 00 04 S so o 1-* 1-H so o 1-* 1-H so o 1-* 1-H so 04 s so 00 04 s so 00 S so 00 04 1-H in o OS so o OS o OS 00 o OS OS o OS o OS OS 00 o OS OS o OS o OS OS 00 o OS OS o OS o OS OS 04 OS m OS OS in OS 04 OS m OS OS in OS m OS OS in OS so OS OS 1-* 1-* 1-* 1-* 1-* 1-* 1-* 1-* 1-* 1-* 1-* 1-* 1-* 1-* 1-* 1-* 1-* 1-* 1-* 1-* 1-* 1-* 1-* 1-* 1-* 1-* 1-* 1-* 1-* in os 04 in os m in os in os in in os so in os n ؛ os 00 in os OS in os o so OS so OS 04 so OS m so OS 3 OS in so OS SO so OS so OS 00 so OS OS so OS o OS 1-* OS 04 OS m OS OS in OS SO OS OS 00 OS 979 1 18 16 8 8 c.54_55insTAATAA 8 2 17.33 0.47980 1 919 18 8 8 c.54_55insTAATAA 10 2 37.39 0.56981 1 916 12 10 10 c.54_55insTAATAA 4 2 1.52 0.25982 1 917 14 10 10 c.54_55insTAATAA 6 2 6.25 0.97983 1 18 16 10 10 c.54_55insTAATAA 8 2 11.53 0.36984 1 919 18 10 10 c.54_55insTAATAA 10 2 30.03 0.50985 1 916 12 12 12 c.54_55insTAATAA 4 2 1.87 0.26986 1 917 14 12 12 c.54_55insTAATAA 6 2 10.63 1.16987 1 18 16 12 12 c.54_55insTAATAA 8 2 16.68 1.29988 1 919 18 12 12 c.54_55insTAATAA 10 2 30.20 0.33989 1 920 24 8 8c.66_67insTAATAA, c.58G>C14 0.29 0.07 990 1 921 26 8 8c.66_67insTAATAA, c.58G>C14 0.01 0.39 991 1 922 28 8 8c.66_67insTAATAA, c.58G>C14 3.01 0.68 992 1 923 30 8 8c.66_67insTAATAA, c.58G>C14 12.05 0.57 993 1 920 24 10 10c.66_67insTAATAA, c.58G>C14 0.20 0.39 994 1 921 26 10 10c.66_67insTAATAA, c.58G>C14 1.33 1.11 995 1 922 28 10 10c.66_67insTAATAA, c.58G>C14 2.03 0.41 996 1 923 30 10 10c.66_67insTAATAA, c.58G>C14 7.76 1.04 997 1 920 24 12 12c.66_67insTAATAA, c.58G>C14 0.01 0.12 998 1 921 26 12 12c.66_67insTAATAA, c.58G>C14 0.18 0.09 999 1 922 28 12 12c.66_67insTAATAA, c.58G>C14 0.47 0.21 1000 1 923 30 12 12c.66_67insTAATAA, c.58G>C14 2.29 0.04 WO 2024/238825 PCT/US2024/029746 435 1001 1 924 18 8 8c.60_65delinsTAATAG, c.58G>C8 3.40 0.10 1002 1 925 20 8 8c.60_65delinsTAATAG, c.58G>C8 11.05 0.23 1003 1 926 22 8 8c.60_65delinsTAATAG, c.58G>C8 8.60 1.10 1004 1 927 24 8 8c.60_65delinsTAATAG, c.58G>C8 9.81 0.51 1005 1 924 18 10 10c.60_65delinsTAATAG, c.58G>C8 2.08 0.20 1006 1 925 20 10 10c.60_65delinsTAATAG, c.58G>C8 6.72 0.68 1007 1 926 22 10 10c.60_65delinsTAATAG, c.58G>C8 4.64 0.82 1008 1 927 24 10 10c.60_65delinsTAATAG, c.58G>C8 7.28 1.43 1009 1 924 18 12 12c.60_65delinsTAATAG, c.58G>C8 0.32 0.11 1010 1 925 20 12 12c.60_65delinsTAATAG, c.58G>C8 2.25 0.17 1011 1 926 22 12 12c.60_65delinsTAATAG, c.58G>C8 2.49 0.51 1012 1 927 24 12 12c.60_65delinsTAATAG, c.58G>C8 4.28 0.191013 1 928 17 12 12 c.54_55insTAATAA 9 2 18.94 1.711014 1 928 17 10 10 c.54_55insTAATAA 9 2 19.80 0.691015 1 928 17 8 8 c.54_55insTAATAA 9 2 21.00 0.711016 1 929 19 12 12 c.54_55insTAATAA 11 2 37.15 0.711064 269 1033 7 276 8 c.21_22insCC 4 1 2.41 2.191065 269 1034 9 276 8 c.21_22insCC 6 1 2.67 0.731066 269 1035 11 276 8 c.21_22insCC 8 1 7.73 0.181067 269 1036 13 276 8 c.21_22insCC 10 1 13.00 0.481068 269 1033 7 278 10 c.21_22insCC 4 1 4.93 0.361069 269 1034 9 278 10 c.21_22insCC 6 1 5.16 0.12 WO 2024/238825 PCT/US2024/029746 436 WO 2024/238825 PCT/US2024/029746 437 00 s© ס 1-H 1-* ס in ס 04 סי-* 00 ס 1-H 1-* 04 ס 00 m o 04 o o o o o m o m o 04 o s© o o in Os o 3 o o 04 s© o o 1-* 04 Os o m o 1-* in o Os o n o ס S© in os in in s© סר ס 1-H Os Os Os ס in 00 m 04 in m 00 o 00 S© m m סך י-* o S© o s© 04 so 1-* o m Os 04 S© סך 04 Os Os S in m 04 s© 1-* in 04 m 04 m 1-* 1-* י-* י-* 1-* 1-* Os Os Os Os os Os Os Os os Os Os Os 1-* 1-* 1-* 1-* 1-* 1-* 1-* 1-* 1-* 1-* 00 ס -* 1 't s© 00 ס -* 1 't s© 00 o 1-* 't s© 00 o 1-* 't s© 00 o 1-* 't s© 00 o 1-* 't S© 00 o 1-* 't s© ס ס 9 . 1 ^ 04 d ס ס ،g ^1 04 d ס ס .9 C4 ^1 04 d ס ס ،g ^1 04 d ס ס .9 C4 ^1 04 d ס ס 5 . 04 1 ^ 04 d ס A ססס ס 9 . * סר d ס d u u .9 * m d ס A o d u u .9 * m d ס d u u .9 * m d ס A o d u u .9 * m d ס d u u .9 * m d ס A o d u u .9 * m d ס d u u .9 * m d ס A o d u u .9 * m d ס d u u .9 * m d ס A o d u u .9 * m d ס o u u .9 * m d ס 5 H .9 04 d ס 9 . 04 d ס 5 H .9 04 d ס 9 . 04 d ס 5 H .9 04 d ס 9 . 04 d ס 5 H .9 04 d ס 9 . 04 d ס 5 H .9 04 d ס 9 . 04 d ס -* 1 ס -* 1 04 04 04 04 00 00 00 00 o 1-* o 1-* o 1-* o 1-* 04 04 04 04 00 00 00 00 o 1-* o 1-* o 1-* o 1-* 04 04 00 00 ס 00 ס 00 ס 00 ס 00 s© S© S© S© 00 00 00 00 o 04 o 00 o 04 o 00 S© S© S© S© 00 00 00 00 o 04 o 00 1-* m ס Os 1-* סר in 04 04 Os 04 m in 04 04 Os 04 m in 04 04 Os 04 m os 1-* m in os 1-* m in Os 1-* in סר ס י-*יי© סר ס י-*סר סר ס -* 1 ס -* 1 in סר ס י-* S© סר ס י-* m ס י-* 00 m o 1-* Os m o 1-* o o 1-* m o 1-* 00 m o 1-* Os m o 1-* o o 1-* m o 1-* 00 m o 1-* Os m o 1-* o o 1-* 1-* o 1-* 04 o 1-* m o 1-* o 1-* 1-* o 1-* 04 o 1-* m o 1-* o 1-* 1-* o 1-* 04 o 1-* Os s© Os יי© ©ץ יי© Os יי© Os יי© Os s© Os s© Os s© Os s© Os s© Os s© Os s© Os s© Os s© Os s© Os s© Os s© Os s© Os s© Os s© Os s© Os s© Os s© Os s© Os s© Os s© Os s© Os s© סס -* 1 י-* ס -* 1 04 ס -* 1 סרס י-*ס -* 1 in ס -* 1 S© o 1-* o 1-* o 1-* Os o 1-* o 00 o 1-* 1-* 00 o 1-* 04 00 o 1-* m 00 o 1-* $ o 1-* in 00 o 1-* s© 00 o 1-* 00 o 1-* 00 00 o 1-* Os 00 o 1-* o Os o 1-* Os o 1-* 04 Os o 1-* m Os o 1-* Os o 1-* in Os o 1-* s© Os o 1-* Os o 1-* 1098 269 1043 13 280 12 c.21_22insTAAG 8 1 13.69 0.501099 269 1044 15 280 12 c.21_22insTAAG 10 1 0.00 0.261100 269 1045 27 276 8 c.3_4insTAAG, C.17OG 4 19 0.07 0.081101 269 1046 29 276 8 c.3_4insTAAG, C.17OG 6 19 0.52 1.511102 269 1047 31 276 8 c.3_4insTAAG, C.17OG 8 19 1.23 3.561103 269 1048 33 276 8 c.3_4insTAAG, C.17OG 10 19 3.70 0.261104 269 1045 27 278 10 c.3_4insTAAG, C.17OG 4 19 0.02 0.271105 269 1046 29 278 10 c.3_4insTAAG, C.17OG 6 19 0.33 1.081106 269 1047 31 278 10 c.3_4insTAAG, C.17OG 8 19 1.48 0.251107 269 1048 33 278 10 c.3_4insTAAG, C.17OG 10 19 4.99 2.111108 269 1045 27 280 12 c.3_4insTAAG, C.17OG 4 19 0.03 0.321109 269 1046 29 280 12 c.3_4insTAAG, C.17OG 6 19 0.23 0.161110 269 1047 31 280 12 c.3_4insTAAG, C.17OG 8 19 1.33 0.371111 269 1048 33 280 12 c.3_4insTAAG, C.17OG 10 19 5.55 0.681112 269 1049 11 276 8 c.21_22insTAATAA 4 1 1.39 0.521113 269 1050 13 276 8 c.21_22insTAATAA 6 1 1.27 1.221114 269 1051 15 276 8 c.21_22insTAATAA 8 1 5.17 0.191115 269 1052 17 276 8 c.21_22insTAATAA 10 1 12.48 0.141116 269 1049 11 278 10 c.21_22insTAATAA 4 1 1.93 0.081117 269 1050 13 278 10 c.21_22insTAATAA 6 1 2.48 0.961118 269 1051 15 278 10 c.21_22insTAATAA 8 1 8.02 0.361119 269 1052 17 278 10 c.21_22insTAATAA 10 1 18.79 0.541120 269 1049 11 280 12 c.21_22insTAATAA 4 1 1.17 0.181121 269 1050 13 280 12 c.21_22insTAATAA 6 1 2.81 0.121122 269 1051 15 280 12 c.21_22insTAATAA 8 1 6.45 1.091123 269 1052 17 280 12 c.21_22insTAATAA 10 1 11.33 1.061124 269 1053 29 276 8 c.3_4insTAATAA, C.17OG 4 19 0.01 0.001125 269 1054 31 276 8 c.3_4insTAATAA, C.17OG 6 19 0.22 0.15 WO 2024/238825 PCT/US2024/029746 438 1126 269 1055 33 276 8 c.3_4insTAATAA, C.17OG 8 19 0.58 0.601127 269 1056 35 276 8 c.3_4insTAATAA, C.17OG 10 19 3.55 0.131128 269 1053 29 278 10 c.3_4insTAATAA, C.17OG 4 19 0.01 0.161129 269 1054 31 278 10 c.3_4insTAATAA, C.17OG 6 19 0.19 0.281130 269 1055 33 278 10 c.3_4insTAATAA, C.17OG 8 19 1.12 0.211131 269 1056 35 278 10 c.3_4insTAATAA, C.17OG 10 19 7.49 0.141132 269 1053 29 280 12 c.3_4insTAATAA, C.17OG 4 19 0.01 0.091133 269 1054 31 280 12 c.3_4insTAATAA, C.17OG 6 19 0.47 0.991134 269 1055 33 280 12 c.3_4insTAATAA, C.17OG 8 19 0.82 1.061135 269 1056 35 280 12 c.3_4insTAATAA, C.17OG 10 19 4.22 2.351136 269 1057 23 276 8c.3_8delinsTAATGA, c.CG13 0.66 0.92 1137 269 1058 25 276 8c.3_8delinsTAATGA, c.CG13 0.75 0.31 1138 269 1059 27 276 8c.3_8delinsTAATGA, c.CG13 0.59 0.29 1139 269 1060 29 276 8c.3_8delinsTAATGA, c.CG13 1.03 0.31 1140 269 1057 23 278 10c.3_8delinsTAATGA, C.17OG13 0.87 0.61 1141 269 1058 25 278 10c.3_8delinsTAATGA, c.CG13 1.61 0.63 1142 269 1059 27 278 10c.3_8delinsTAATGA, c.CG13 0.98 0.14 1143 269 1060 29 278 10c.3_8delinsTAATGA, c.CG13 2.31 1.95 1144 269 1057 23 280 12c.3_8delinsTAATGA, c.CG13 0.83 0.35 1145 269 1058 25 280 12c.3_8delinsTAATGA, c.CG13 1.93 0.41 1146 269 1059 27 280 12c.3_8delinsTAATGA, c.CG13 1.53 0.90 1147 269 1060 29 280 12c.3_8delinsTAATGA, C.17OG13 2.18 0.81 WO 2024/238825 PCT/US2024/029746 439 WO 2024/238825 PCT/US2024/029746 440 in in o in o so in o in o 04 in 1-* in so 1-* o os o o m m o os o o so o 04 o o o o o o 00 so o o 00 o o m o 04 in o OS o OS o o m o 00 o o o סך -* 1 o o m m in SO OS o so in so 1-* 3 OS in 04 m 04 SO so in in m OS so סך o 1-* o in 00 in OS so o o 04 1-* OS OS o in in o m 00 so m so o 04 os m o 1-* 1-* 1-* 1-* 1-* 1-* 1-* 1-* 1-* 1-* 1-* 1-* 1-* 1-* 1-* 1-* 1-* m m m m m m m m m m m m in OS in 't so 00 o 1-* 't so 00 o 1-* 't SO 00 o 1-* 't so 00 o 1-* 't so 00 o 1-* 't so 00 o 1-* .g ^1 04 d ،g 04 ^1 04 d .g ^1 04 d ،g ^1 04 d u u .9 so ^1 in o u u g in d u u .9 so ^1 in d u u g in d u u .9 so ^1 in d u u g in d u u .9 so ^1 in d u u g ^1 in d u u ،s so ^1 in d u u g ^1 in d u u ،s so ^1 in d u u g ^1 in o ס A u o o U סנ .g * m d ס d o U סנ .g * m d ס A u d o U סנ .g * m d ס d o U סנ .g * m d ס A u d o U סנ .g * m d ס d o U סנ .g * m d ס A u d o U סנ .g * m d ס d o U סנ .g * m d ס A u d o U סנ .g * m d ס d o U סנ .g * m d ס A u d o U סנ .g * m d ס o o U סנ .g * m d 00 00 o 1-* 00 00 00 00 00 o 1-* o 1-* o 1-* o 1-* 04 04 04 04 00 00 00 00 o 1-* o 1-* o 1-* o 1-* 04 04 04 04 so so 00 so m m m m m m so m m so m m so m m so m m 00 m m 00 m m 00 m m 00 m m m m m m m m so m m so m m so m m so m m 00 m m 00 m m 00 m m 00 m m 04 so 04 OS o OS 1-* m o OS 1-* m o OS 1-* m OS m in OS m in OS m in SO o 1-* so 00 so o 1-* m so o 1-* 04 so 1-* m so 1-* 1-* in so 1-* 04 so 1-* m so 1-* 1-* in so 1-* 04 so 1-* m so 1-* 1-* in so 1-* so so 1-* so 1-* 00 so 1-* os so 1-* SO so 1-* so 1-* 00 so 1-* os so 1-* SO so 1-* so 1-* 00 so 1-* os so 1-* OS so OS so OS so OS so 04 m 04 m 04 m 04 m 04 m 04 m 04 m 04 m 04 m 04 m 04 m 04 m 04 m 04 m 04 m 04 m 04 m 04 m 04 m 04 m 04 m 04 m 04 m 04 m 00 1-* OS 1-* o in 1-* 1-* in 1-* 04 os 1-* m os 1-* 1-* in os 1-* so OS 1-* os 1-* 00 os 1-* os OS 1-* o o o 04 o m o o in o so o o 00 o os o o 04 04 04 m 04 04 in 04 1216 327 1170 9 334 8 c. 1516insTAAG 4 1 1.41 0.231217 327 1171 11 334 8 c. 1516insTAAG 6 1 14.87 0.551218 327 1172 13 334 8 c. 1516insTAAG 8 1 15.62 1.631219 327 1173 15 334 8 c. 1516insTAAG 10 1 22.15 1.851220 327 1170 9 336 10 c. 1516insTAAG 4 1 1.35 0.691221 327 1171 11 336 10 c. 1516insTAAG 6 1 22.60 0.281222 327 1172 13 336 10 c. 1516insTAAG 8 1 19.46 0.951223 327 1173 15 336 10 c. 1516insTAAG 10 1 31.08 0.521224 327 1170 9 338 12 c. 1516insTAAG 4 1 1.35 0.161225 327 1171 11 338 12 c. 1516insTAAG 6 1 28.69 0.251226 327 1172 13 338 12 c. 1516insTAAG 8 1 18.13 0.081227 327 1173 15 338 12 c. 1516insTAAG 10 1 16.33 0.641228 327 1174 21 334 8 c.3_4insTAAG, c.llOG 4 13 0.02 0.371229 327 1175 23 334 8 c.3_4insTAAG, c.llOG 6 13 0.38 1.421230 327 1176 25 334 8 c.3_4insTAAG, c.llOG 8 13 0.48 0.091231 327 1177 27 334 8 c.3_4insTAAG, c.llOG 10 13 4.26 1.211232 327 1174 21 336 10 c.3_4insTAAG, c.llOG 4 13 0.02 0.071233 327 1175 23 336 10 c.3_4insTAAG, c.llOG 6 13 0.18 0.751234 327 1176 25 336 10 c.3_4insTAAG, c.llOG 8 13 0.66 0.241235 327 1177 27 336 10 c.3_4insTAAG, c.llOG 10 13 4.94 0.311236 327 1174 21 338 12 c.3_4insTAAG, c.llOG 4 13 0.01 0.141237 327 1175 23 338 12 c.3_4insTAAG, c.llOG 6 13 0.24 0.081238 327 1176 25 338 12 c.3_4insTAAG, c.llOG 8 13 0.28 1.801239 327 1177 27 338 12 c.3_4insTAAG, c.llOG 10 13 2.03 0.191240 327 1178 11 334 8 c.l5_16insTAATAA 4 1 0.87 0.101241 327 1179 13 334 8 c. 1516insTAATAA 6 1 4.85 0.461242 327 1180 15 334 8 c.l5_16insTAATAA 8 1 6.30 0.251243 327 1181 17 334 8 c. 1516insTAATAA 10 1 6.18 0.25 WO 2024/238825 PCT/US2024/029746 441 1244 327 1178 11 336 10 c.l5_16insTAATAA 4 1 1.21 0.191245 327 1179 13 336 10 c. 1516insTAATAA 6 1 13.61 0.121246 327 1180 15 336 10 c.l5_16insTAATAA 8 1 11.15 0.281247 327 1181 17 336 10 c. 1516insTAATAA 10 1 12.35 1.481248 327 1178 11 338 12 c.l5_16insTAATAA 4 1 1.45 0.051249 327 1179 13 338 12 c. 1516insTAATAA 6 1 16.57 0.811250 327 1180 15 338 12 c.l5_16insTAATAA 8 1 19.30 0.551251 327 1181 17 338 12 c. 1516insTAATAA 10 1 15.40 1.261252 327 1182 23 334 8 c.3_4insTAATAA, c.llOG 4 13 0.00 0.141253 327 1183 25 334 8 c.3_4insTAATAA, c.llOG 6 13 0.22 0.291254 327 1184 27 334 8 c.3_4insTAATAA, c.llOG 8 13 0.94 0.491255 327 1185 29 334 8 c.3_4insTAATAA, c.llOG 10 13 4.36 0.251256 327 1182 23 336 10 c.3_4insTAATAA, c.llOG 4 13 0.01 1.161257 327 1183 25 336 10 c.3_4insTAATAA, c.llOG 6 13 0.44 0.161258 327 1184 27 336 10 c.3_4insTAATAA, c.llOG 8 13 1.45 0.091259 327 1185 29 336 10 c.3_4insTAATAA, c.llOG 10 13 7.27 0.351260 327 1182 23 338 12 c.3_4insTAATAA, c.llOG 4 13 0.02 0.101261 327 1183 25 338 12 c.3_4insTAATAA, c.llOG 6 13 0.11 0.251262 327 1184 27 338 12 c.3_4insTAATAA, c.llOG 8 13 0.67 0.211263 327 1185 29 338 12 c.3_4insTAATAA, c.llOG 10 13 2.83 0.411264 327 1186 17 334 8c.3_8delinsTAATGA, c.llOG7 0.88 1.10 1265 327 1187 19 334 8c.3_8delinsTAATGA, c.llOG7 8.59 0.65 1266 327 1188 21 334 8c.3_8delinsTAATGA, c.llOG7 1.56 0.80 1267 327 1189 23 334 8c.3_8delinsTAATGA, c.llOG7 1.54 0.37 1268 327 1186 17 336 10c.3_8delinsTAATGA, c.llOG7 2.21 1.36 WO 2024/238825 PCT/US2024/029746 442 1269 327 1187 19 336 10c.3_8delinsTAATGA, c.HOG7 13.50 0.89 1270 327 1188 21 336 10c.3_8delinsTAATGA, c.HOG7 1.55 1.71 1271 327 1189 23 336 10c.3_8delinsTAATGA, c.iiog7 2.02 2.09 1272 327 1186 17 338 12c.3_8delinsTAATGA, c.iiog7 1.07 2.11 1273 327 1187 19 338 12c.3_8delinsTAATGA, c.HOG7 6.53 1.09 1274 327 1188 21 338 12c.3_8delinsTAATGA, c.HOG7 1.19 0.35 1275 327 1189 23 338 12c.3_8delinsTAATGA, c.HOG7 0.85 0.04 1276 327 1190 16 336 10c.3_8delinsTAATGA, c.HOG7 1.78 0.171277 327 1191 12 336 10 c.l5_16insTAATAA 5 1 6.21 0.151278 327 1191 12 334 8 c. 1516insTAATAA 5 1 5.46 0.301279 327 1190 16 338 12c.3_8delinsTAATGA, c.HOG7 0.57 0.51 Table 50 PEgRNA SEQ ID NO PEgRNA Spacer SEQ ID NO RTT SEQ ID NO RTT length PBS SEQ ID NO PBS length Homology arm length Nick-to-edit distance ngRNA spacer SEQ ID No ngRNA SEQ ID No Editing % Indels% HEK3-CTT - - - - - - - - 17.74 0.17NTC n/a n/a n/a n/a n/a n/a n/a n/a0.00 0.00985 1 916 12 12 12 4 2 1022* 1030* 0.12 0.53997 1 920 24 12 12 4 14 1023* 1031* 0.21 0.16977 1 916 12 8 8 4 2 1022* 1030* 0.21 0.74981 1 916 12 10 10 4 2 1022* 1030* 0.28 1.341009 1 924 18 12 12 4 8 1024* 1032* 0.46 0.16962 1 908 10 12 12 4 2 1020* 1028* 0.50 0.53 WO 2024/238825 PCT/US2024/029746 443 998 1 921 26 12 12 6 14 1023* 1031* 0.78 0.29954 1 908 10 8 8 4 2 1020* 1028* 0.83 0.72970 1 912 22 10 10 4 14 1021* 1029* 0.92 0.22974 1 913 24 12 12 6 14 1021* 1029* 1.16 0.26975 1 914 26 12 12 8 14 1021* 1029* 1.52 0.17938 1 900 8 12 12 4 2 1018* 1026* 1.59 1.49950 1 904 20 12 12 4 14 1019* 1027* 1.65 0.17989 1 920 24 8 8 4 14 1023* 1031* 1.72 0.32930 1 900 8 8 8 4 2 1018* 1026* 1.74 2.36934 1 900 8 10 10 4 2 1018* 1026* 2.02 2.06999 1 922 28 12 12 8 14 1023* 1031* 2.26 0.33966 1 912 22 8 8 4 14 1021* 1029* 2.33 0.34993 1 920 24 10 10 4 14 1023* 1031* 2.61 0.67990 1 921 26 8 8 6 14 1023* 1031* 3.69 0.95976 1 915 28 12 12 10 14 1021* 1029* 3.89 0.11958 1 908 10 10 10 4 2 1020* 1028* 3.93 0.97946 1 904 20 10 10 4 14 1019* 1027* 4.29 0.681010 1 925 20 12 12 6 8 1024* 1032* 4.31 0.671000 1 923 30 12 12 10 14 1023* 1031* 4.44 0.27971 1 913 24 10 10 6 14 1021* 1029* 4.83 0.72994 1 921 26 10 10 6 14 1023* 1031* 4.92 0.91942 1 904 20 8 8 4 14 1019* 1027* 5.00 0.551011 1 926 22 12 12 8 8 1024* 1032* 5.29 1.15972 1 914 26 10 10 8 14 1021* 1029* 5.73 0.441005 1 924 18 10 10 4 8 1024* 1032* 5.81 0.511001 1 924 18 8 8 4 8 1024* 1032* 6.27 0.93967 1 913 24 8 8 6 14 1021* 1029* 6.48 0.941012 1 927 24 12 12 10 8 1024* 1032* 6.57 1.09973 1 915 28 10 10 10 14 1021* 1029* 6.62 0.30 WO 2024/238825 PCT/US2024/029746 444 968 1 914 26 8 8 8 14 1021* 1029* 8.49 0.46951 1 905 22 12 12 6 14 1019* 1027* 8.74 2.31969 1 915 28 8 8 10 14 1021* 1029* 8.77 0.36947 1 905 22 10 10 6 14 1019* 1027* 9.13 1.94943 1 905 22 8 8 6 14 1019* 1027* 11.74 2.85978 1 917 14 8 8 6 2 1022* 1030* 12.60 1.111007 1 926 22 10 10 8 8 1024* 1032* 12.70 2.47982 1 917 14 10 10 6 2 1022* 1030* 13.64 1.19996 1 923 30 10 10 10 14 1023* 1031* 13.98 0.651006 1 925 20 10 10 6 8 1024* 1032* 14.82 1.97986 1 917 14 12 12 6 2 1022* 1030* 15.44 1.321008 1 927 24 10 10 10 8 1024* 1032* 15.52 2.67948 1 906 24 10 10 8 14 1019* 1027* 15.92 2.00995 1 922 28 10 10 8 14 1023* 1031* 16.27 1.06959 1 909 12 10 10 6 2 1020* 1028* 16.37 1.52952 1 906 24 12 12 8 14 1019* 1027* 17.16 2.56944 1 906 24 8 8 8 14 1019* 1027* 18.25 2.66955 1 909 12 8 8 6 2 1020* 1028* 18.37 1.62991 1 922 28 8 8 8 14 1023* 1031* 20.02 1.51983 1 18 16 10 10 8 2 1022* 1030* 20.10 0.99963 1 909 12 12 12 6 2 1020* 1028* 20.41 1.64953 1 907 26 12 12 10 14 1019* 1027* 20.46 3.181004 1 927 24 8 8 10 8 1024* 1032* 22.09 3.691003 1 926 22 8 8 8 8 1024* 1032* 22.37 4.41987 1 18 16 12 12 8 2 1022* 1030* 22.37 1.05949 1 907 26 10 10 10 14 1019* 1027* 23.29 3.151002 1 925 20 8 8 6 8 1024* 1032* 25.31 2.961013 1 928 17 12 12 9 2 1022* 1030* 25.36 1.20945 1 907 26 8 8 10 14 1019* 1027* 28.28 3.72 WO 2024/238825 PCT/US2024/029746 445 979 1 18 16 8 8 8 2 1022* 1030* 28.67 1.48992 1 923 30 8 8 10 14 1023* 1031* 28.73 1.89988 1 919 18 12 12 10 2 1022* 1030* 32.57 1.18964 1 910 14 12 12 8 2 1020* 1028* 35.60 1.49960 1 910 14 10 10 8 2 1020* 1028* 40.01 1.721014 1 928 17 10 10 9 2 1022* 1030* 40.55 1.861015 1 928 17 8 8 9 2 1022* 1030* 41.23 2.021016 1 929 19 12 12 11 2 1022* 1030* 43.14 2.00931 1 901 10 8 8 6 2 1018* 1026* 48.01 2.41980 1 919 18 8 8 10 2 1022* 1030* 49.33 1.94984 1 919 18 10 10 10 2 1022* 1030* 50.71 1.82939 1 901 10 12 12 6 2 1018* 1026* 50.77 2.88935 1 901 10 10 10 6 2 1018* 1026* 51.40 2.87940 1 902 12 12 12 8 2 1018* 1026* 56.01 3.03956 1 910 14 8 8 8 2 1020* 1028* 56.06 2.53965 1 911 16 12 12 10 2 1020* 1028* 57.97 2.32932 1 902 12 8 8 8 2 1018* 1026* 59.42 2.69936 1 902 12 10 10 8 2 1018* 1026* 60.85 2.79957 1 911 16 8 8 10 2 1020* 1028* 65.77 2.25941 1 903 14 12 12 10 2 1018* 1026* 66.18 3.05961 1 911 16 10 10 10 2 1020* 1028* 68.23 2.97937 1 903 14 10 10 10 2 1018* 1026* 68.85 2.60933 1 903 14 8 8 10 2 1018* 1026* 75.61 2.811124 269 1182 23 276 8 4 13 1155* 1160* 0.01 0.161128 269 1182 23 278 10 4 13 1155* 1160* 0.02 0.171132 269 1182 23 280 12 4 13 1155* 1160* 0.02 0.081104 269 1174 21 278 10 4 13 1154* 1159* 0.03 0.201100 269 1174 21 276 8 4 13 1154* 1159* 0.03 0.201108 269 1174 21 280 12 4 13 1154* 1159* 0.03 0.19 WO 2024/238825 PCT/US2024/029746 446 WO 2024/238825 PCT/US2024/029746 447 LV^ 1 ־ 06 9^01 £101 1 17 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IHl־ 6£ 9 ־ 10 *1911 *9911 £ 01 01 8£2: £2: 6811 692: £Hl־ 179 9 ־ 10 9201 £101 1 8 8 9£2: 11 17911 692: 9901־ 6£ 9 ־ 178 *1911 *9911 £ 01 21 082: £2: 6811 692: £Hl־ £9 9 ־ £9 *0911 *9911 £1 01 01 8£2: 62: 9811 692: l£ll־ 6£ 9 ־ £1 9201 £101 1 8 01 8£2: 91 0811 692: 8111־ 8£ 9 ־ 20 *1911 *9911 £ 9 21 082: 61 £811 692: 91711־ 6£ 17 ־ £8 9201 £101 1 8 8 9£2: £1 Z£ll 692: 0601:£־ 2 17 ־ 6£ *8911 *£911 £1 9 8 9£2: 12: £911 692: ££01־ £17 17 ־ £2 *1911 *9911 £ 8 21 082: 12: 8811 692: 9HI־ 178 17 ־ £1 *8911 *£911 £1 01 21 082: 92: 6911 692: £801 1232 327 1174 21 336 10 4 13 1282* 1287* 0.15 0.351240 327 1178 11 334 8 4 1 1017 1025 0.31 1.171279 327 1190 16 338 12 3 7 1284* 1289* 0.37 0.441212 327 1166 19 338 12 4 13 1281* 1286* 0.42 0.211253 327 1183 25 334 8 6 13 1283* 1288* 0.46 0.291261 327 1183 25 338 12 6 13 1283* 1288* 0.49 0.341237 327 1175 23 338 12 6 13 1282* 1287* 0.50 0.181224 327 1170 9 338 12 4 1 1017 1025 0.55 1.291248 327 1178 11 338 12 4 1 1017 1025 0.59 1.991208 327 1166 19 336 10 4 13 1281* 1286* 0.61 0.331220 327 1170 9 336 10 4 1 1017 1025 0.63 1.391192 327 1162 7 334 8 4 1 1017 1025 0.67 2.601214 327 1168 23 338 12 8 13 1281* 1286* 0.69 0.171200 327 1162 7 338 12 4 1 1017 1025 0.70 1.201204 327 1166 19 334 8 4 13 1281* 1286* 0.70 0.301278 327 1191 12 334 8 5 1 1017 1025 0.73 2.991196 327 1162 7 336 10 4 1 1017 1025 0.73 2.271229 327 1175 23 334 8 6 13 1282* 1287* 0.74 0.221244 327 1178 11 336 10 4 1 1017 1025 0.82 2.221264 327 1186 17 334 8 4 7 1284* 1289* 0.85 0.311216 327 1170 9 334 8 4 1 1017 1025 0.85 2.111233 327 1175 23 336 10 6 13 1282* 1287* 0.86 0.301257 327 1183 25 336 10 6 13 1283* 1288* 0.87 0.251274 327 1188 21 338 12 8 7 1284* 1289* 0.88 0.021262 327 1184 27 338 12 8 13 1283* 1288* 0.92 0.181275 327 1189 23 338 12 10 7 1284* 1289* 0.92 0.191276 327 1190 16 336 10 3 7 1284* 1289* 0.94 0.951272 327 1186 17 338 12 4 7 1284* 1289* 0.94 0.121277 327 1191 12 336 10 5 1 1017 1025 0.96 2.76 WO 2024/238825 PCT/US2024/029746 450 1238 327 1176 25 338 12 8 13 1282* 1287* 1.14 0.131194 327 1164 11 334 8 8 1 1017 1025 1.34 1.101270 327 1188 21 336 10 8 7 1284* 1289* 1.47 0.081215 327 1169 25 338 12 10 13 1281* 1286* 1.56 0.341267 327 1189 23 334 8 10 7 1284* 1289* 1.60 0.291230 327 1176 25 334 8 8 13 1282* 1287* 1.69 0.211268 327 1186 17 336 10 4 7 1284* 1289* 1.79 0.521254 327 1184 27 334 8 8 13 1283* 1288* 1.86 0.491234 327 1176 25 336 10 8 13 1282* 1287* 1.99 0.281258 327 1184 27 336 10 8 13 1283* 1288* 1.99 0.541206 327 1168 23 334 8 8 13 1281* 1286* 2.28 0.471271 327 1189 23 336 10 10 7 1284* 1289* 2.41 0.311266 327 1188 21 334 8 8 7 1284* 1289* 2.54 0.221239 327 1177 27 338 12 10 13 1282* 1287* 2.73 0.231207 327 1169 25 334 8 10 13 1281* 1286* 2.95 0.441243 327 1181 17 334 8 10 1 1017 1025 3.02 0.671195 327 1165 13 334 8 10 1 1017 1025 3.03 1.051193 327 1163 9 334 8 6 1 1017 1025 3.20 1.551213 327 1167 21 338 12 6 13 1281* 1286* 3.83 0.401242 327 1180 15 334 8 8 1 1017 1025 3.92 1.291199 327 1165 13 336 10 10 1 1017 1025 3.97 1.151198 327 1164 11 336 10 8 1 1017 1025 4.23 1.541263 327 1185 29 338 12 10 13 1283* 1288* 4.39 0.431210 327 1168 23 336 10 8 13 1281* 1286* 4.58 0.981241 327 1179 13 334 8 6 1 1017 1025 5.32 1.381211 327 1169 25 336 10 10 13 1281* 1286* 5.79 1.031255 327 1185 29 334 8 10 13 1283* 1288* 6.18 0.541209 327 1167 21 336 10 6 13 1281* 1286* 6.20 0.551247 327 1181 17 336 10 10 1 1017 1025 6.36 1.54 WO 2024/238825 PCT/US2024/029746 451 1197 327 1163 9 336 10 6 1 1017 1025 6.44 2.531227 327 1173 15 338 12 10 1 1017 1025 6.58 0.541246 327 1180 15 336 10 8 1 1017 1025 6.76 2.691273 327 1187 19 338 12 6 7 1284* 1289* 7.17 0.211226 327 1172 13 338 12 8 1 1017 1025 7.26 0.941259 327 1185 29 336 10 10 13 1283* 1288* 7.95 0.721203 327 1165 13 338 12 10 1 1017 1025 7.95 1.321231 327 1177 27 334 8 10 13 1282* 1287* 8.85 0.671205 327 1167 21 334 8 6 13 1281* 1286* 8.87 0.711202 327 1164 11 338 12 8 1 1017 1025 9.71 2.571249 327 1179 13 338 12 6 1 1017 1025 10.24 1.521235 327 1177 27 336 10 10 13 1282* 1287* 10.51 0.911201 327 1163 9 338 12 6 1 1017 1025 10.57 2.621222 327 1172 13 336 10 8 1 1017 1025 10.72 1.331265 327 1187 19 334 8 6 7 1284* 1289* 10.82 0.571218 327 1172 13 334 8 8 1 1017 1025 11.07 1.661217 327 1171 11 334 8 6 1 1017 1025 11.40 1.771250 327 1180 15 338 12 8 1 1017 1025 11.95 2.311251 327 1181 17 338 12 10 1 1017 1025 12.13 1.561245 327 1179 13 336 10 6 1 1017 1025 12.41 2.831225 327 1171 11 338 12 6 1 1017 1025 14.55 1.581219 327 1173 15 334 8 10 1 1017 1025 16.51 1.271221 327 1171 11 336 10 6 1 1017 1025 17.76 2.631269 327 1187 19 336 10 6 7 1284* 1289* 18.80 0.641223 327 1173 15 336 10 10 1 1017 1025 20.19 1.70 WO 2024/238825 PCT/US2024/029746 452 WO 2024/238825 PCT/US2024/029746 453 Example 4: Prime Editing of the B2M gene in T cells [0994]Prime Editing at the B2M gene was examined in human T cells. Two PEgRNA- ngRNA pairs were tested. The PEgRNAs and ngRNAs were chemically synthesized. The PEgRNAs and ngRNAs each contained a 3’ UUUU end adaptation as indicated in the sequences, and further contained chemical modifications 3’ mN*mN*mN*N and 5’mN*mN*mN*N modifications, where m indicates that the nucleotide contains a 2’-O-Me modification and a * indicates a phosphorothioate bond. [0995]T cells were cultured in ImmunoCult XF T cell Expansion medium (StemCell Technologies) containing 1% L-glutamine, 1% Penicillin/Streptomycin (Lonza), and 1IU/mL IL-2 (PeproTech). T cells were activated with DynaBeads Human T-Activator CD3/CD28 (ThermoFisher) at a 1:1 bead:cell ratio for 48 hours prior to electroporation. After 48 hours of stimulation, DynaBeads were magnetically removed, and T cells were washed once with PBS prior to resuspension in electroporation buffer. T cells were resuspended in MaxCyte electroporation buffer containing polynucleotide encoding a prime editor fusion protein, PEgRNA, and ngRNA for electroporation. Following electroporation, T cells were transferred to an empty well-plate and allowed to recover at 37 °C, 5% CO2 for minutes. Subsequently, cells were suspended in basal medium (without cytokines) and incubated at 37 °C, 5% CO2 for 20 minutes before being suspended in complete T cell medium. Genomic DNA was extracted using Lucigen QuickExtraction Solution according to manufacturer ’s protocol for sequence analysis. [0996]Prime Editing efficiency and indel frequency were assessed by amplicon sequencing of using the primers listed in Table 44 for B2M amplification. These primers bear Illumina forward and reverse adapters, allowing amplicons generating in a first round of PCR (PCRI) to serve as templates for a second round of PCR (PCR2) with unique Illumina barcoding primer pairs. PCR2 products were pooled by common amplicons, purified by gel electrophoresis, and quantified by Qubit (ThermoFisher). Libraries were sequenced on an Illumina MiSeq instrument using Illumna MiSeq control software. Reads were demultiplexed by the Illumina MiSeq control software and analyzed using CRISPResso 2.

Three technical replicates were included for PEgRNA-ngRNA pairs tested. The results are summarized in Table 51.

WO 2024/238825 PCT/US2024/029746 454 Table 51 PEgRNA SEQ ID NO ngRNA SEQ ID NO %Editin grepl %Editin grepl %Editin grep 3 %Indel s rep 1 %Indel s rep 1 %Indel s rep 3 232 826 87.45 88.83 84.15 2.24 2.75 2.72238 827 2.95 3.48 2.39 0.00 0.00 0.00 Example 5: Multiplex Prime Editing of the B2M gene and the TRAC gene in T cells id="p-997"

[0997]The ability of multiplexed editing using dual Prime Editing of a TRAC locus and single PEgRNA editing of B2M was examined. PEgRNA according to SEQ ID NO: 232 and a ngRNA according to SEQ ID NO: 825 were used to electroporate T cells along with dual Prime Editing PEgRNA pairs targeting the TRAC gene. 5’ TRAC PEgRNAs having the sequences of SEQ ID NO:s 1401 and 1343, and 3’ TRAC PEgRNAs having the sequences of SEQ ID NO:s: 1459 and 1568 were tested for targeted integration of a 38bp attB sequence GGCTTGTCGACGACGGCGGTCTCCGTCGTCAGGATCAT at the TRAC locus. [0998]To test attB-attP-Bxbl mediated integration of donor sequence, polynucleotides encoding a prime editor fusion protein, a Bxbl integrase enzyme, as well as a DNA nanoplasmid harboring the coding sequence of a CD19-CAR and a GFP reporter were used to electroporate the T cells. The nanoplasmid contained corresponding attP sequence GGTTTGTCTGGTCAACCACCGCGGTCTCAGTGGTGTACGGTACAAACC and a sequence encoding a CD 19 CAR connected to a sequence encoding GFP reporter by a T2A sequence. [0999]The PEgRNAs and ngRNAs each contained a 3’ UUUU end adaptation as indicated in the sequences, and further contained chemical modifications 3’ mN*mN*mN*N and 5’mN*mN*mN*N modifications, where m indicates that the nucleotide contains a 2’-O-Me modification and a * indicates a phosphorothioate bond. [1000] Human T cells were cultured and electroporated with the Prime Editor, TRAC 5’ PEgRNA, TRAC 3’ PEgRNA, B2M PEgRNA, and B2M ngRNA components as described in Example 4. For recombinase mediated donor integration, the Bxbl integrase DNA donor components were also electroporated using Maxcyte as described in Example 4. [1001] Assessment of Prime Editing by amplicon sequencing: Prime Editing efficiency and indels frequency were assessed by amplicon sequencing as described in Example 4 using the primers listed in Table 44for B2M and Table 52and TRAC amplification, respectively.

WO 2024/238825 PCT/US2024/029746 455 Table 52 PrimerNameSequence TRACForwardACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNATGAGACCGTGACTTGCCAG TRACReverseTGGAGTTCAGACGTGTGCTCTTCCGATCTTGCTGTTGTTGAAGGCGTTT id="p-1002"

[1002] Assessment of protein loss and cargo expression by flow cytometry: CD3 protein loss, B2M protein loss, and transgene expression were evaluated via flow cytometry detection of B2M, CD3, GFP, and CD19-CAR. The following antibodies and proteins were used: PE- conjugated anti-B2M (clone 2M2; BioLegend), BV605-conjugated anti-CD3 (clone SK7; BD Biosciences), Atto 647N-conjugated recombinant human CD19 (BioTechne). GFP signals were assessed based on flow cytometry fluorescence without antibody staining. SYTOX Blue dead cell stain (ThermoFisher). Protein expression was evaluated via flow cytometry detection by normalizing to average CD3 or B2M expression of mock (electroporation only) controls. Gating schema of the follow cytometry excluded dead cells to avoid auto-fluorescent signals. In addition, SYTOX Blue dead cell stain (ThermoFisher) was used to detect cell death.. No appreciable cell death or appreciable difference in cell death between edited and mock control cells were detected in the flow cytometry. [1003] Assessment of cargo integration by ddPCR: Targeted integration of DNA cargo at the TRAC locus after contacting with the Bxbl mRNA and Nanoplasmid donor was quantified using ddPCR. Three sets of primers and probes (Table 53) were designed to detect unedited TRAC gene (ddPCR WT%), attB integration only (ddPCR attB%), and attL sequence as the result of Bxbl mediated cargo integration (ddPCR attL%). Each sample was run as a duplexed assay consisting of an internal reference primer + probe set and an experimental primer + probe set. Reactions were set up in duplicate using the ddPCR Supermix for Probes (Bio-Rad) with 50 ng genomic DNA per assay. Droplets were generated and analyzed using the QX200 Droplet Digital PCR system (Bio-Rad). [1004] Table 53. ddPCR Primers Primer / probe name Sequence TRACddPCRuni ver sal_F orward CTTGCCAGCCCCACAGAG TRACddPCRWTReverse CTGTGGGACAAGAGGATCAG WO 2024/238825 PCT/US2024/029746 456 TRAC_ddPCR_attB_Reverse ATGATCCTGACGACGGAGA TRAC_ddPCR_attL_Reverse TGTACCGTACACCACTGAGA TRAC_ddPCR_universal_Probe (FAM)TTGTCCATCACTGGCATCTGGACTCCA TRACddPCRcontrolF orward CTCTTTATGGCCCTTGTCACT TRAC_ddPCR_control_Reverse CCATCACACATAGACCCTGC TRAC ddPCR control Probe (Cy5)CATTGGACTCTAGAATGAAGCCAGGCA Internal reference_Forward CCCGATATTCCTCAGGTACT Internal referenceReverse GGATGAAACCCAGACACATAG Internal reference Probe (TAM) CGTCATCCAGCAGAGAATGGAAAG id="p-1005"

[1005] Two separate experiments were performed, each experiment including 2 biological replicates using CD3+ T cells from two healthy human donors. Sequence numbers of the B2M PEgRNA and ngRNA, the TRAC-editing PEgRNAs, the efficiency of multiplexed editing of TRAC and B2M, CD3 protein loss, and CD 19 CAR expression and GFP reporter expression are summarized in Tables 54 and 55. n/a: not measured. not applicable.

Table 54 Editing components ’ TRAC PEgRN ASEQ ID NO 3’ TRAC PEgRN ASEQ ID NO B2M editing % B2M indels % B2M protein loss % TRAC attB % TRAC indels % CD3 protein loss % GFP% CAR+ % ddPCR WT % ddPCR attB % ddPCR attL % TRAC 5’ pegRNA,TRAC 3’ pegRNA,B2M pegRNA, B2M ngRNA, CD19CAR-GFPcargo 1401 1459 80.36 2.97 83.5 n/a n/a 70.34 58.8 58.1 8.05 16.11 55.87 TRAC 5’ pegRNA, TRAC 3’ pegRNA, B2M pegRNA, B2M ngRNA, CD19CAR-GFP cargo 1343 1568 82.81 3.02 85.32 n/a n/a 71.76 56.5 52.1 15.1 15.96 52.28 Mock (electroporation only)n/a n/a 0.01 0 - 0 0 - 0.96 0.1 98.98 1.11 0.95 B2M pegRNA, B2M ngRNA,n/a n/a 85.13 3.03 88.5 0 0 0.51 0.26 0.8 97.18 1.26 1.06TRAC 5' pegRNA, TRAC 3' pegRNA1401 1459 0.01 0 0 69.51 13.96 71.25 0.67 0.15 12.64 86.34 1.2TRAC 5' pegRNA, TRAC 3' pegRNA B2M pegRNA, B2M ngRNA1401 1459 79.7 2.81 83.5 63.79 9.2 72.79 0.8 0.22 12.87 83.17 0.99 CD19CAR-GFP cargo only1401 1459 0.2 0 0.41 n/a n/a 72.88 56.5 52.1 16.83 30.69 52.48 WO 2024/238825 PCT/US2024/029746 457 Table 55 Editing components ' TRAC PEgRN ASEQ ID NO 3' TRAC PEgRN ASEQ ID NO B2M editing % B2M indels % B2M protein loss % TRAC attB % TRAC indels % CD3 protein loss % GFP% CAR+ % ddPCR WT % ddPCR attB % ddPCR attL % TRACS' pegRNA, TRAC 3' pegRNA, B2M pegRNA, B2M ngRNA 1401 1459 85.98 3.31 91.4 71.62 16.91 88.8 n/a n/a n/a n/a n/a WO 2024/238825 PCT/US2024/029746 458

Claims

1. WO 2024/238825 PCT/US2024/029746 459 WHAT IS CLAIMED IS:1. A prime editing guide RNA (PEgRNA) or one or more polynucleotides encoding the PEgRNA, wherein the PEgRNA comprises:a. a spacer that is complementary to a search target sequence on a first strand of a 02-microglobulin (B2M) gene, wherein the spacer comprises at its 3’ end SEQIDNO: 205;b. a gRNA core capable of binding to a Cas9 protein; andc. an extension arm comprising:i. an editing template that comprises a region of complementarity to an editing target sequence on a second strand of the B2M gene, andii. a primer binding site (PBS) that comprises at its 5’ end a sequence that is a reverse complement of nucleotides 10-14 of SEQ ID NO: 205, wherein the first strand and second strand are complementary to each other, and wherein the editing template encodes one or more nucleotide changes compared to the editing target sequence.

2. A prime editing guide RNA (PEgRNA) or one or more polynucleotides encoding the PEgRNA, wherein the PEgRNA comprises:a. a spacer that is complementary to a search target sequence on a first strand of a 02-microglobulin (B2M) gene, wherein the spacer comprises at its 3’ end SEQ ID NO: 4;b. a gRNA core capable of binding to a Cas9 protein; andc. an extension arm comprising:i. an editing template that comprises a region of complementarity to an editing target sequence on a second strand of the B2M gene, andii. a primer binding site (PBS) that comprises at its 5’ end a sequence that is a reverse complement of nucleotides 10-14 of SEQ ID NO: 4, wherein the first strand and second strand are complementary to each other, and wherein the editing template encodes one or more nucleotide changes compared to the editing target sequence.

3. A prime editing guide RNA (PEgRNA) or one or more polynucleotides encoding the PEgRNA, wherein the PEgRNA comprises: WO 2024/238825 PCT/US2024/029746 460 a. a spacer that is complementary to a search target sequence on a first strand of a p2-microglobulin (B2M) gene, wherein the spacer comprises at its 3’ end SEQ ID NO: 272;b. a gRNA core capable of binding to a Cas9 protein; andc. an extension arm comprising:i. an editing template that comprises a region of complementarity to an editing target sequence on a second strand of the B2M gene, andii. a primer binding site (PBS) that comprises at its 5’ end a sequence that is a reverse complement of nucleotides 10-14 of SEQ ID NO: 272,wherein the first strand and second strand are complementary to each other, and wherein the editing template encodes one or more nucleotide changes compared to the editing target sequence.

4. A prime editing guide RNA (PEgRNA) or one or more polynucleotides encoding the PEgRNA, wherein the PEgRNA comprises:a. a spacer that is complementary to a search target sequence on a first strand of a 02-microglobulin (B2M) gene, wherein the spacer comprises at its 3’ end SEQ ID NO: 330;b. a gRNA core capable of binding to a Cas9 protein; andc. an extension arm comprising:i. an editing template that comprises a region of complementarity to an editing target sequence on a second strand of the B2M gene, andii. a primer binding site (PBS) that comprises at its 5’ end a sequence that is a reverse complement of nucleotides 10-14 of SEQ ID NO: 330,wherein the first strand and second strand are complementary to each other, and wherein the editing template encodes one or more nucleotide changes compared to the editing target sequence.

5. The PEgRNA of any one of claims 1-4, wherein the spacer is from 17-22 nucleotides in length, optionally wherein the spacer is 20 nucleotides in length.

6. The PEgRNA of claim 1, wherein the spacer comprises at its 3’ end any one of SEQ ID NOs: 202-204.

7. The PEgRNA of claim 1, wherein the spacer comprises SEQ ID NO: 204. WO 2024/238825 PCT/US2024/029746 461

8. The PEgRNA of claim 2, wherein the spacer comprises at its 3’ end any one of SEQ ID NO:s 1-3.

9. The PEgRNA of claim 2, wherein the spacer comprises SEQ ID NO: 1.

10. The PEgRNA of claim 3, wherein the spacer comprises at its 3’ end any one of SEQ ID NO:s 269-271.

11. The PEgRNA of claim 3, wherein the spacer comprises SEQ ID NO: 269.

12. The PEgRNA of claim 4, wherein the spacer comprises at its 3’ end any one of SEQ ID NO:s 327-329.

13. The PEgRNA of claim 4, wherein the spacer comprises SEQ ID NO: 327.

14. The PEgRNA of any one of claims 1-13, wherein the one or more nucleotide changes encoded by the editing template comprises a non-synonymous edit that alters the mRNA sequence or protein sequence encoded by the B2M gene.

15. The PEgRNA of claim 14, wherein the non-synonymous edit results in one or more in-frame stop codons in the B2M gene.

16. The PEgRNA of claim 15, wherein the one or more in-frame stop codons comprise a nonsense mutation in the B2M gene.

17. The PEgRNA of claim 14, wherein the non-synonymous edit comprises an insertion in the B2M gene.

18. The PEgRNA of claim 14, wherein the non-synonymous edit comprises one or more substitutions in the B2M gene.

19. The PEgRNA of claim 17, wherein the insertion comprises an insertion an in-frame stop codon in the B2M gene, optionally wherein the insertion comprises an insertion of two or more consecutive in-frame stop codons in the B2M gene.

20. The PEgRNA of claim 19, wherein the insertion is comprises a TAATAA, a TTATTA, or a TAATAG nucleotide insertion.

21. The PEgRNA of claim 14, wherein the non-synonymous edit comprises a frameshift mutation in the B2M gene.

22. The PEgRNA of claim 21, wherein the frameshift mutation is an insertion or of 3x+l or 3x+2 nucleotides, wherein x is an integer equal to or greater than 0.

23. The PEgRNA of claim 21, wherein the frameshift mutation is a deletion of 3x+l or 3x+2 nucleotides, wherein x is an integer equal to or greater than 0.

24. The PEgRNA of claim 22, wherein the insertion is 1, 2 or 4 nucleotides in length.

25. The PEgRNA of claim 23, wherein the deletion is 1 nucleotide in length. WO 2024/238825 PCT/US2024/029746 462

26. The PEgRNA of any one of claims 14-25, wherein the non-synonymous edit alters a protospacer adjacent motif (PAM) sequence that is immediately 3’ to a protospacer sequence in the second strand of the B2M gene that is complementary to the search target sequence in the first strand of the B2M gene.

27. The PEgRNA of claim 26, wherein the PAM sequence is NGG and the non- synonymous edit is a NGG->NGC edit.

28. The PEgRNA of claim 26 or 27, wherein the protospacer sequence comprises a nick site that is three nucleotides upstream of the 5’ most nucleotide of the PAM sequence, and wherein the number of nucleotides from the nick site to the position in the second strand of the B2M gene corresponding to the non-synonymous edit is 1 to nucleotides, wherein the number of nucleotides does not include the 5’ most nucleotide position on the second strand corresponding to the non-synonymous edit.

29. The PEgRNA of claim 28, wherein the number of nucleotides from the nick site to the position in the second strand of the B2M gene corresponding to the non-synonymous edit is 1, 2, 7, 8, 13, 14, or 19 nucleotides.

30. The PEgRNA of claim 29, wherein the number of nucleotides from the nick site to the position in the second strand of the B2M gene corresponding to the non-synonymous edit is equal to or less than 8 nucleotides.

31. The PEgRNA of claim 30, wherein the number of nucleotides from the nick site to the position in the second strand of the B2M gene corresponding to the non-synonymous edit is 1 or 2 nucleotides.

32. The PEgRNA of any one of claimsl, 5-7 and 14-31, wherein the non-synonymous edit is at a chromosomal location corresponding to coding sequence position c.51, c.54, or c.50 of a wildtype B2M gene.

33. The PEgRNA of claim 32, wherein the non-synonymous edit comprises a c.54insTAATAA insertion.

34. The PEgRNA of claim 32, wherein the non-synonymous edit comprises a c.51delC deletion or a c.50insG insertion.

35. The PEgRNA of any one of claims 2, 5, 8-9 and 14-31, wherein the non-synonymous edit is at a chromosomal location corresponding to coding sequence position c.54, c.60, or c.66 in a wildtype B2M gene.

36. The PEgRNA of claim 35, wherein the non-synonymous edit comprises to a c.54_55insCC insertion or a c.54_55insTAAG insertion. WO 2024/238825 PCT/US2024/029746 463

37. The PEgRNA of claim 35, wherein the non-synonymous edit comprises a c.54_55insTAATAA insertion.

38. The PEgRNA of claim 35, wherein the non-synonymous edit comprises a c.66_67insCC insertion or a c.66_67insTAAG insertion.

39. The PEgRNA of claim 32, wherein the non-synonymous edit comprises a c.66_67insTAATAA insertion.

40. The PEgRNA of claim 35, wherein the non-synonymous edit comprises a c.60_65deletion and a TAATAG insertion (c.60_65_delinsTAATAG).

41. The PEgRNA of claim 3, 5, 10-11 and 14-31, wherein the non-synonymous edit is at a chromosomal location corresponding to coding sequence position c.21 or c.3 of a wildtype B2M gene.

42. The PEgRNA of claim 41, wherein the non-synonymous edit comprises a c.21insTAATAA insertion.

43. The PEgRNA of claim 41, wherein the non-synonymous edit comprises a c. 21_22insCC insertion or a c.21_22insTAAG edit.

44. The PEgRNA of claim 41, wherein the non-synonymous edit comprises a c.3_4insCC insertion or a c.3_4insTAAG insertion.

45. The PEgRNA of claim 41, the non-synonymous edit comprises a c.3_8 deletion and a TAATGA insertion (c.3_8delinsTAATGA).

46. The PEgRNA of any one of claims 4-5 and 12-31, wherein the non-synonymous edit is at a chromosomal location corresponding to coding sequence position c.21, c.15 or c.3 of a wildtype B2M gene.

47. The PEgRNA of claim 46, wherein the non-synonymous edit comprises a c.21insTAATAA insertion.

48. The PEgRNA of claim 46, wherein the non-synonymous edit comprises a c. 15_16insCC insertion or a c. 15_16insTAAG insertion.

49. The PEgRNA of claim 46, wherein the non-synonymous edit comprises a c. 15_16insTAATAA insertion.

50. The PEgRNA of claim 46, wherein the non-synonymous edit comprises a c. 3_4insCC insertion or a c. 3_4insTAAG insertion.

51. The PEgRNA of claim 46, wherein the non-synonymous edit comprises a c. 3_4insTAATAA insertion.

52. The PEgRNA of claim 46, wherein the non-synonymous edit comprises a c.3_deletion and a TAATGA insertion (c.3_8delinsTAATGA). WO 2024/238825 PCT/US2024/029746 464

53. The PEgRNA of any one of claims 1-52, wherein the editing template further encodes an additional PAM silencing edit.

54. The PEgRNA of claim 53, wherein the PAM silencing edit is a c.58G>C edit.

55. The PEgRNA of claim 53, wherein the PAM silencing edit is a c.17C>G edit.

56. The PEgRNA of claim 53, wherein the PAM silencing edit is a c.l 1C>G edit.

57. The PEgRNA of any one of claims 1-56, wherein the editing template comprises at least 4 contiguous nucleotides complementary with the editing target sequence, wherein the at least 4 contiguous nucleotides are upstream of the position of the 5’ most nucleotide of the one or more nucleotide changes encoded in the editing template.

58. The PEgRNA of claim 57, wherein the editing template comprises at least 6, 8, or contiguous nucleotides complementary with the editing target sequence, wherein the at least 6, 8, or 10 contiguous nucleotides are upstream of the position of the 5’ most nucleotide of the one or more nucleotide changes encoded in the editing template.

59. The PEgRNA of claim 57, wherein the editing template comprises 4, 6, 8, or contiguous nucleotides complementary with the editing target sequence, wherein the 4, 6, 8, or 10 contiguous nucleotides are upstream of the position of the 5’ most nucleotide of the one or more nucleotide changes encoded in the editing template.

60. A prime editing guide RNA (PEgRNA), or a nucleic acid encoding the PEgRNA, wherein the PEgRNA comprises:a. a spacer comprising at its 3’ end SEQ ID NO: 205;b. a gRNA core capable of binding to a Cas9 protein; andc. an extension arm comprising:i. an editing template comprising at its 3’ end: (A) nucleotides 13-24 of SEQ ID NO: 221, (B) nucleotides 12-20 of SEQ ID NO: 227, or (C) nucleotides 7-17 of SEQ ID NO: 231, andii. a primer binding site (PBS) comprising at its 5’ end a sequence that is a reverse complement of nucleotides 10-14 of SEQ ID NO: 205.

61. The PEgRNA of any one of claims 1,5-7, 14-34, 53-60, wherein:(i) the editing template comprises at its 3’ end nucleotides 13-24 of SEQ ID NO: 221, optionally wherein the editing template comprises SEQ ID NO: 219, 220,or WO 2024/238825 PCT/US2024/029746 465 (ii) the editing template comprises at its 3’ end nucleotides 12-20 of SEQ ID NO: 227, optionally wherein the editing template comprises at its 3’ end SEQ ID NO: any one ofSEQIDNOs: 224-227, or(ii) the editing template comprises at its 3’ end nucleotides 7-17 of SEQ ID NO: 231, optionally wherein the editing template comprises at its 3’ end any one of SEQ ID NOs: 229-231.

62. A prime editing guide RNA (PEgRNA), or a nucleic acid encoding the PEgRNA, wherein the PEgRNA comprises:a. a spacer comprising at its 3 ’ end SEQ ID NO: 1; b. a gRNA core capable of binding to a Cas9 protein; andc. an extension arm comprising:i. an editing template comprising at its 3’ end: (A) nucleotides 5-16 of SEQ ID NO: 19, or (B) a sequence selected from the group consisting of SEQ ID NO:s 900, 904, 908, 912, 916, 920, and 924,ii. a primer binding site (PBS) comprising at its 5’ end a sequence thatis a reverse complement of nucleotides 10-14 of SEQ ID NO: 1.

63. The PEgRNA of any one of claims 2, 5, 8-9, 14-31, 35-40, 53-59 and 62, wherein the editing template comprises:(i) a sequence selected from the group consisting of SEQ ID NOs: 900-903, or(ii) a sequence selected from the group consisting of SEQ ID NOs: 904-907, or(iii) a sequence selected from the group consisting of SEQ ID NOs: 908-911, or(iv) a sequence selected from the group consisting of SEQ ID NOs: 912-915, or(v) a sequence selected from the group consisting of SEQ ID NOs: 916-919, 928, and 929,or(vi) a sequence selected from the group consisting of SEQ ID NOs: 920-923, or(vii) a sequence selected from the group consisting of SEQ ID NOs: 924-927, or(viii) a sequence selected from the group consisting of SEQ ID NOs: 18-20.

64. A prime editing guide RNA (PEgRNA), or a nucleic acid encoding the PEgRNA, wherein the PEgRNA comprises:a. a spacer comprising at its 3’ end SEQ ID NO: 269;b. a gRNA core capable of binding to a Cas9 protein; andc. an extension arm comprising: WO 2024/238825 PCT/US2024/029746 466 i. an editing template comprising at its 3’ end: (A) nucleotides 3-16 of SEQ ID NO:286, or (B) a sequence selected from the group consisting of SEQ ID NO:s 1033, 1037, 1041, 1045, 1049, 1053, and 1057, andii. a primer binding site (PBS) comprising at its 5’ end a sequence that is a reverse complement of nucleotides 10-14 of SEQ ID NO: 269.

65. The PEgRNA of any one of claims 3, 5, 10-11, 14-31, 41-45, 53-59 and 64, wherein the editing template comprises:(i) a sequence selected from the group consisting of SEQ ID NOs: 1033-1036, or(ii) a sequence selected from the group consisting of SEQ ID NOs: 1037-1040, or (iii) a sequence selected from the group consisting of SEQ ID NOs: 1041-1044, or (iv) a sequence selected from the group consisting of SEQ ID NOs: 1045-1048, or (v) a sequence selected from the group consisting of SEQ ID NOs: 1049-10andl061-1063, or(vi) a sequence selected from the group consisting of SEQ ID NOs: 1053-1056, or (vi) a sequence selected from the group consisting of SEQ ID NOs: 1057-1060, or (vii) a sequence selected from the group consisting of SEQ ID NOs: 286-288. or

66. A prime editing guide RNA (PEgRNA), or a nucleic acid encoding the PEgRNA, wherein the PEgRNA comprises:a. a spacer comprising at its 3’ end SEQ ID NO: 327;b. a gRNA core capable of binding to a Cas9 protein; andc. an extension arm comprising:i. an editing template comprising at its 3’ end: (A) nucleotides 6-16 of SEQ ID NO:344, or (B) a sequence selected from the group consisting of SEQ ID NO:s 1162, 1166, 1170, 1174, 1178, 1182, and 1190;andii. a primer binding site (PBS) comprising at its 5’ end a sequence that is a reverse complement of nucleotides 10-14 of SEQ ID NO: 327.

67. The PEgRNA of any one of claims 4-5, 12-31,46-59 and 66, wherein the editing template comprises:(i) a sequence selected from the group consisting of (i) SEQ ID NOs: 1162-1165, or (ii) a sequence selected from the group consisting of SEQ ID NOs: 1166-1169, or WO 2024/238825 PCT/US2024/029746 467 (iii) a sequence selected from the group consisting of SEQ ID NOs: 1170-1173, or (iv) a sequence selected from the group consisting of SEQ ID NOs: 1174-1177, or (v) a sequence selected from the group consisting of SEQ ID NOs: 1178-1181 and 1191, or(vi) a sequence selected from the group consisting of SEQ ID NOs: 1182-1185, or(vi) a sequence selected from the group consisting of SEQ ID NOs: 1186-1190, or (vii) a sequence selected from the group consisting of SEQ ID NOs: 344-346.

68. The PEgRNA of any one of claims 1-67, wherein the editing template has a length of nucleotides or less, or a length of 20 nucleotides or less.

69. The PEgRNA of claim 68, wherein the editing template has a length of (i) 10 to nucleotides, (ii) 12 to 20 nucleotides, or (iii) 11 to 17 nucleotides.

70. The PEgRNA of claim 68, wherein the editing template is 16 to 24 nucleotides in length.

71. The PEgRNA of any one of claims 1-67, wherein the editing template is 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 24, 25, 26, 27, 28, 29, 30, 31, 33, nucleotides in length.

72. The PEgRNA of any one of claims 1-71, wherein the PBS has a length of nucleotides or less.

73. The PEgRNA of claim 72, wherein the PBS has a length of (i) 8 to 15 nucleotides, (ii) to 14 nucleotides, or (iii) 8 to 12 nucleotides.

74. The PEgRNA of claim 30, wherein the PBS is 8, 10, or 12 nucleotides in length.

75. The PEgRNA of any one of claims 1,5-7, 14-34, 53-61 and 68-74, wherein the PBS comprises a sequence set forth in any one of sequence numbers 206-218.

76. The PEgRNA of any one of claims 2, 5, 8-9, 14-31, 35-40, 53-59, 62-63 and 68-74, wherein the PBS comprises a sequence set forth in any one of sequence numbers 5- 17.

77. The PEgRNA of any one of claims 3, 5, 10-11, 14-31, 41-45, 53-59, 64-65 and 68-74, wherein the PBS comprises a sequence set forth in any one of sequence numbers 273- 285.

78. The PEgRNA of any one of claims 4-5, 12-31,46-59 and 66-74, wherein the PBS comprises a sequence set forth in any one of sequence numbers 331-343.

79. The PEgRNA of any one of claims 1-78, wherein the spacer, the gRNA core, the RTT, and the PBS form a contiguous sequence in a single molecule. WO 2024/238825 PCT/US2024/029746 468

80. The PEgRNA of claim 79, comprising from 5’ to 3’, the spacer, the gRNA core, the RTT, and the PBS.

81. The PEgRNA of any one of claims 1-80, wherein the gRNA core comprises SEQ ID NO: 646.

82. The PEgRNA of any one of claims 1-80, wherein the gRNA core comprises SEQ ID NO: 653.

83. The PEgRNA of any one of claims 1,5-7, 14-34, 53-61, 68-75 and 79-82 comprising a sequence selected from the group consisting of SEQ ID NOs: 232-262.

84. The PEgRNA of any one of claims 2, 5, 8-9, 14-31, 35-40, 53-59, 62-63, 68-74, and 79-82 comprising a sequence selected from the group consisting of SEQ ID NOs: 21-29 and 930-1016.

85. The PEgRNA of claim 84, comprising a sequence as set forth in SEQ ID NO: 933, 937, 961, 941, 957, or 936.

86. The PEgRNA of any one of claims 3, 5, 10-11, 14-31, 41-45, 53-59, 64-65, 68-74, and 79-82 comprising a sequence selected from the group consisting of SEQ ID NOs: 289-297 and 1064-1151.

87. The PEgRNA of claim 86, comprising a sequence as set forth in SEQ ID NO: 1141 or 1143.

88. The PEgRNA of any one of claims 4-5, 12-31,46-59 and 66-74, 78-82 comprising a sequence selected from the group consisting of SEQ ID NOs: 347-355 and 1192- 1279.

89. The PEgRNA of claim 88, comprising a sequence as set forth in SEQ ID NO: 1269 or 1265.f

90. The PEgRNA of any one of claims 1-74, comprising a sequence selected from the group consisting of SEQ ID NOs: 957, 961, 965, 980, 1016, 956, 933, 941, 937, 1223, 988, 984, 1225, 1151, 1095, 1091, 964, 960, 940, 1221, 945, 1219, 932, 1015, 1014, 1075, 1222, 1250, 936, 1013, 1119, 1226, and 949.

91. The PEgRNA of any one of claims 1-90, further comprising a 3’ motif, optionally wherein the 3’ motif is connected to the 3’ end of the PBS via a linker.

92. The PEgRNA of any one of the preceding claims, further comprising 3’ mN*mN*mN*N and/or 5’mN*mN*mN* modifications, where m indicates that the nucleotide contains a 2’-O-Me modification and a * indicates the presence of a phosphorothioate bond. WO 2024/238825 PCT/US2024/029746 469

93. The PEgRNA of any one of the preceding claims, further comprising 3’ mT*mT*mT*T and 5’mN*mN*mN* modifications, where m indicates that the nucleotide contains a 2’-O-Me modification, a * indicates the presence of a phosphorothioate bond, and a T indicates the presence of an additional uridine nucleotide.

94. The PEgRNA of any one of the preceding claims, wherein human chromosome locations and coding sequence locations are as set forth in Genome Reference Consortium Human Build 38 (GrCh38).

95. A prime editing system comprising the PEgRNA or the one or more polynucleotides encoding the PEgRNA of any one of the preceding claims.

96. A prime editing system of claim 95, further comprising a nick guide RNA (ngRNA), or a nucleic acid encoding the ngRNA, wherein the ngRNA comprises: a. a ngRNA spacer that is complementary to a ngRNA search target sequence on the second strand of the B2M gene; andb. an ngRNA core capable of binding a Cas9 protein.

97. The prime editing system of claim 96, wherein the ngRNA spacer is 17-nucleotides in length, optionally wherein the ngRNA spacer is 20 nucleotides in length.

98. The prime editing system of claim 96 or 97, wherein the ngRNA core comprises SEQ ID NO: 646 or 653.

99. The prime editing system of any one of claims 96-98, wherein the PEgRNA spacer comprises at its 3’ end SEQ ID NO: 205.

100. The prime editing system of claim 99, wherein the ngRNA spacer comprises at its 3’ end a sequence corresponding to nucleotides 4-20, 3-20, 2-20, or 1-20 of any one of SEQ ID NOs: 263-268, optionally wherein ngRNA spacer comprises at its 3’ end any one of SEQ ID NOs: 263-268.

101. The prime editing system claim 100, wherein the ngRNA spacer comprises at its 3’ end nucleotides 1-20 of SEQ ID NO: 268, optionally wherein the ngRNA comprises SEQ ID NO: 824 or 825.

102. The prime editing system of claim 99, wherein: WO 2024/238825 PCT/US2024/029746 470 (i) the non-synonymous edit encoded by the editing template comprises a c.51delC deletion and the ngRNA spacer comprises at its 3’ end sequence corresponding to nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 266; or(ii) the non-synonymous edit encoded by the editing template comprises a c.50insG insertion, and the ngRNA spacer comprises at its 3’ end sequence corresponding to nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 267 or 268.

103. The prime editing system of any one of claims 99-102, wherein the ngRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 824-827.

104. The prime editing system of any one of claims 96-98, wherein the PEgRNA spacer comprises at its 3’ end SEQ ID NO: 4.

105. The prime editing system of claim 104, wherein the ngRNA spacer comprises at its 3’ end a sequence corresponding to nucleotides 4-20, 3-20, 2-20, or 1-20 of any one of SEQ ID NOs: 1017-1024.

106. The prime editing system of claim 104, wherein ngRNA spacer comprises any one of SEQ ID NOs: 1017-1024.

107. The prime editing system of claim 104, wherein:(i) the editing template encodes a c.54_55insCC edit and wherein the ngRNA spacer comprises at its 3’ end sequence corresponding to nucleotides 4-20, 3-20, 2-20, or 1- of SEQ ID NO: 1018,(ii) the editing template encodes a c.66_67insCC edit and wherein the ngRNA spacer comprises at its 3’ end sequence corresponding to nucleotides 4-20, 3-20, 2-20, or 1- of SEQ ID NO: 1019,(iii) the editing template encodes a c.54_55insTAAG edit and wherein the ngRNA spacer comprises at its 3’ end sequence corresponding to nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 1020,(iv) the editing template encodes a c.66_67insTAAG edit and wherein the ngRNA spacer comprises at its 3’ end sequence corresponding to nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 1021,(v) the editing template encodes a c.54_55insTAATAA edit and wherein the ngRNA spacer comprises at its 3’ end sequence corresponding to nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 1022,(vi) the template encodes a c.66_67insTAATAA edit and wherein the ngRNA spacer comprises at its 3’ end sequence corresponding to nucleotides 4-20, 3-20, 2-20, or 1- of SEQ ID NO: 1023, or WO 2024/238825 PCT/US2024/029746 471 (vii) the template encodes a c.60_65delinsTAATAG edit and wherein the ngRNA spacer comprises at its 3’ end sequence corresponding to nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 1024.

108. The prime editing system of any one of claims 104-107, wherein the ngRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 1025-1032.

109. The prime editing system of any one of claims 96-98, wherein the PEgRNA spacer comprises at its 3’ end SEQ ID NO: 272.

110. The prime editing system of claim 109, wherein the ngRNA spacer comprises at its 3’ end a sequence corresponding to nucleotides 4-20, 3-20, 2-20, or 1-20 of any one of SEQ ID NOs: 1152-1156.

111. The prime editing system of claim 109, wherein ngRNA spacer comprises any one of SEQ ID NOs: 1152-1156.

112. The prime editing system of claim 109, wherein:(i) the editing template encodes a c.3_4insCC edit and wherein the ngRNA spacer comprises at its 3’ end sequence corresponding to nucleotides 4-20, 3-20, 2-20, or 1- of SEQ ID NO: 1153,(ii) the editing template encodes a c.3_4insTAAG edit and wherein the ngRNA spacer comprises at its 3’ end sequence corresponding to nucleotides 4-20, 3-20, 2-20, or 1- of SEQ ID NO: 1154,(iii) the editing template encodes a c.3_4insTAATAA edit and wherein the ngRNA spacer comprises at its 3’ end sequence corresponding to nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 1155, or(iv) the editing template encodes a c.3_8delinsTAATGA edit and wherein the ngRNA spacer comprises at its 3’ end sequence corresponding to nucleotides 4-20, 3-20, 2-20, or 1 -20 of SEQ ID NO: 1156.

113. The prime editing system of any one of claims 109-112, wherein the ngRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 1157-1161.

114. The prime editing system of any one of claims 96-98, wherein the PEgRNA spacer comprises at its 3’ end SEQ ID NO: 330.

115. The prime editing system of claim 114, wherein the ngRNA spacer comprises at its 3’ end a sequence corresponding to nucleotides 4-20, 3-20, 2-20, or 1-20 of any one of SEQ ID NOs: 1280-1284.

116. The prime editing system of claim 114, wherein ngRNA spacer comprises any one of SEQ ID NOs: 1280-1284. WO 2024/238825 PCT/US2024/029746 472

117. The prime editing system of claim 114, wherein:(i) the editing template encodes a c.3_4insCC edit and wherein the ngRNA spacer comprises at its 3’ end sequence corresponding to nucleotides 4-20, 3-20, 2-20, or 1- ofSEQIDNO: 1281,(ii) the editing template encodes a c.3_4insTAAG edit and wherein the ngRNA spacer comprises at its 3’ end sequence corresponding to nucleotides 4-20, 3-20, 2-20, or 1- ofSEQIDNO: 1282,(iii) the editing template encodes a c.3_4insTAATAA edit and wherein the ngRNA spacer comprises at its 3’ end sequence corresponding to nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 1283,or(iv) the editing template encodes a c.3_8delinsTAATGAedit and wherein the ngRNA spacer comprises at its 3’ end sequence corresponding to nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 1284.

118. The prime editing system of any one of claims 115-117, wherein the ngRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 1285-1289.

119. The prime editing system of any one of claims 96-118, wherein:(i) the PEgRNA comprises a sequence as set forth in SEQ ID NO: 933 or 937, and the ngRNA comprises a sequence as set forth in SEQ ID NO: 1018;(ii) the PEgRNA comprises a sequence as set for the in SEQ ID NO: 961, and the ngRNA comprises a sequence as set forth in SEQ ID NO: 1020;(iii) the PEgRNA comprises a sequence as set for the in SEQ ID NO: 941, and the ngRNA comprises a sequence as set forth in SEQ ID NO: 1018;(iv) the PEgRNA comprises a sequence as set for the in SEQ ID NO: 957, and the ngRNA comprises a sequence as set forth in SEQ ID NO: 1020;(v) the PEgRNA comprises a sequence as set for the in SEQ ID NO: 936, and the ngRNA comprises a sequence as set forth in SEQ ID NO: 1018;(vi) the PEgRNA comprises a sequence as set for the in SEQ ID NO: 1141 or 1143, and the ngRNA comprises a sequence as set forth in SEQ ID NO: 1156,or(vii) the PEgRNA comprises a sequence as set for the in SEQ ID NO: 1269 or 1265, and the ngRNA comprises a sequence as set forth in SEQ ID NO: 1284.

120. The prime editing system of any one of claims 96-119, wherein the ngRNA comprises 3’ mN*mN*mN*N and/or 5’mN*mN*mN* modifications, where m WO 2024/238825 PCT/US2024/029746 473 indicates that the nucleotide contains a 2’-O-Me modification and a * indicates the presence of a phosphorothioate bond.

121. The prime editing system of any one of claims 96-120, wherein the ngRNA comprises 3’ mT*mT*mT*T and 5’mN*mN*mN* modifications, where m indicates that the nucleotide contains a 2’-O-Me modification, a * indicates the presence of a phosphorothioate bond, and a T indicates the presence of an additional uridine nucleotide.

122. The prime editing system of any one of claims 95-121, further comprising a prime editor or one or more polynucleotides encoding the prime editor, wherein the prime editor comprises:a. a Cas9 nickase having a nuclease inactivating mutation in the HNH domain, andb. a reverse transcriptase.

123. The prime editing system of claim 122, wherein the prime editor is a fusion protein.

124. The prime editing system of any one of claims 95-121, further comprising:an N-terminal extein comprising an N-terminal fragment of a prime editor fusion protein and an N-intein or a polynucleotide encoding the N-terminal extein;a C-terminal extein comprising a C-terminal fragment of the prime editor fusion protein and a C-intein, or a polynucleotide encoding the C-terminal extein;wherein the N-intein and the C-intein of the N-terminal and C-terminal exteins are capable of self-excision to join the N-terminal fragment and the C-terminal fragment to form the prime editor fusion protein, and wherein the prime editor fusion protein comprises a Cas9 nickase having a nuclease inactivating mutation in the HNH domain and a reverse transcriptase (RT) domain.

125. The prime editing system of any one of claims 122-124, wherein the Cas9 nickase comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NOs: 676 or 677.

126. The prime editing system of any one of claims 122-125, wherein the reverse transcriptase comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 673.

127. The prime editing system of claim 125 or 126, wherein the sequence identities are determined by Needleman-Wunsch alignment of two protein sequences with Gap Costs set to Existence: 11 Extension: 1 where percent identity is calculated by dividing the number of identities by the length of the alignment. WO 2024/238825 PCT/US2024/029746 474

128. The prime editing system of any one of claims 122-127, wherein the one or more polynucleotides encoding the prime editor, the polynucleotide encoding the N- terminal extein, or the polynucleotide encoding the C-terminal extein are mRNA.

129. A population of viral particles collectively comprising the one or more polynucleotides encoding the PEgRNA of any one of claims 1-94 or the prime editing system of any one of claims 95-128.

130. The population of viral particles of claim 129, wherein the viral particles are AAV particles.

131. An LNP comprising the prime editing system of any one of claims 95-128.

132. The LNP of claim 131, comprising the PEgRNA and optionally the ngRNA, the polynucleotide encoding the Cas9 nickase, and the polynucleotide encoding the reverse transcriptase.

133. The LNP of claim 132, wherein the polynucleotide encoding the Cas9 nickase and the polynucleotide encoding the reverse transcriptase are mRNA.

134. The LNP of claim 132 or 133, wherein the polynucleotide encoding the Casnickase and the polynucleotide encoding the reverse transcriptase are in the same molecule.

135. A method of editing a B2M gene, the method comprising contacting the B2M gene with: (a) the PEgRNA of any one of claims 1-94, and a prime editor comprising a Cas9 nickase having a nuclease inactivating mutation in the HNH domain and a reverse transcriptase, (b) the prime editing system of any one of claims 95-128, (c) the population of viral particles of claim 129 or 130, or (d) the LNP of any one of claims 131-134.

136. The method of claim 135, wherein the B2M gene is in a cell.

137. A method of generating an engineered cell, the method comprising introducing into a cell or a population of cells: (a) the PEgRNA of any one of claims 1-94, and a prime editor comprising a Cas9 nickase having a nuclease inactivating mutation in the HNH domain and a reverse transcriptase, (b) the prime editing system of any one of claims 95-128, (c) the population of viral particles of claim 129 or 130, or (d) the LNP of any one of claims 131-134.

138. The method of claim 136 or 137, wherein the cell or the population of cells are in a subject. WO 2024/238825 PCT/US2024/029746 475

139. The method of claim 136 or 137, wherein the cell or the population of cells are ex vivo, optionally wherein the cell or the population of cells are obtained from a subject or a cell bank.

140. The method of any one of claims 136-139, wherein the cell or the population of cells are human cells.

141. The method of claim 140, wherein the cell or the population of cells are immune cells or stem cells.

142. The method of claim 141, wherein the cell or the population of cells are T cells or hematopoietic stem cells (HSCs), optionally wherein the cell or the population of cells are cytotoxic T cells.

143. A cell or a population of cells generated by the method of any one of claims 136- 142.

144. An engineered cell or a population of engineered cells comprising a premature stop codon in the B2M gene relative to a wildtype B2M gene.

145. An engineered cell or a population of engineered cells comprising a B2M gene comprising an insertion, a deletion, a substitution, or a combination thereof compared to a wildtype B2M gene at a chromosomal location corresponding to coding sequence position c.51, c.54, or c.50 of a wildtype B2M gene.

146. An engineered cell or a population of engineered cells comprising a B2M gene comprising an insertion, a deletion, a substitution, or a combination thereof compared to a wildtype B2M gene at a chromosomal location corresponding to coding sequence position c.54, c.60, or c.66 of a wildtype B2M gene, optionally wherein the B2M gene comprises an insertion, a deletion, a substitution, or a combination thereof at a chromosomal location corresponding to coding sequence position c. 58 of a wildtype B2M gene.

147. An engineered cell or a population of engineered cells comprising a B2M gene comprising an insertion, a deletion, a substitution, or a combination thereof compared to a wildtype B2M gene at a chromosomal location corresponding to coding sequence position c.21 or c.3 of a wildtype B2M gene, optionally wherein the B2M gene comprises an insertion, a deletion, a substitution, or a combination thereof at a chromosomal location corresponding to coding sequence position c. 17 of a wildtype B2M gene.

148. An engineered cell or a population of engineered cells comprising a B2M gene comprising an insertion, a deletion, a substitution, or a combination thereof compared WO 2024/238825 PCT/US2024/029746 476 to a wildtype B2M gene at a chromosomal location corresponding to coding sequence position c.21, c.15 or c.3 of a wildtype B2M gene, optionally wherein the B2M gene comprises an insertion, a deletion, a substitution, or a combination thereof at a chromosomal location corresponding to coding sequence position c.11 of a wildtype B2M gene..

149. The cell or the population of cells of claim 144 comprising a c.51delC deletion in the B2M gene relative to a wildtype B2M gene.

150. The cell or the population of cells of claim 144 comprising a c.50insG insertion in the B2M gene relative to a wildtype B2M gene.

151. The cell or the population of cells of claim 144 comprising a c.54_55insCC insertion in the B2M gene relative to a wildtype B2M gene.

152. The cell or the population of cells of claim 144 comprising a c.54_55insTAAG insertion in the B2M gene relative to a wildtype B2M gene.

153. The cell or the population of cells of claim 144 comprising a c.54_55insTAATAA insertion in the B2M gene relative to a wildtype B2M gene.

154. The cell or the population of cells of claim 144 comprising a c.66_67insCC insertion in the B2M gene relative to a wildtype B2M gene, optionally wherein the cell or the population of cells further comprise a c.58G>C substitution in the B2M gene relative to a wildtype B2M gene.

155. The cell or the population of cells of claim 144 comprising a c.66_67insTAAG insertion in the B2M gene relative to a wildtype B2M gene, optionally wherein the cell or the population of cells further comprise a c.58G>C substitution in the B2M gene relative to a wildtype B2M gene.

156. The cell or the population of cells of claim 144 comprising a c.66_67insTAATAA insertion in the B2M gene relative to a wildtype B2M gene, optionally wherein the cell or the population of cells further comprise a c.58G>C substitution in the B2M gene relative to a wildtype B2M gene.

157. The cell or the population of cells of claim 144 comprising a c.60_65deletion and a TAATAG insertion (c.60_64delinsTAATAG) in the B2M gene relative to a wildtype B2M gene, optionally wherein the cell or the population of cells further comprise a c.58G>C substitution in the B2M gene relative to a wildtype B2M gene.

158. The cell or the population of cells of claim 144 comprising a c.21_22insCC insertion in the B2M gene relative to a wildtype B2M gene. WO 2024/238825 PCT/US2024/029746 477

159. The cell or the population of cells of claim 144 comprising a c.21_22insTAAG insertion in the B2M gene relative to a wildtype B2M gene.

160. The cell or the population of cells of claim 144 comprising a c.21_22insTAATAA insertion in the B2M gene relative to a wildtype B2M gene.

161. The cell or the population of cells of claim 144 comprising a c.3_4insCC insertion in the B2M gene relative to a wildtype B2M gene, optionally wherein the cell or the population of cells further comprise a C.17OG substitution or a c.l lOG substitution in the B2M gene relative to a wildtype B2M gene.

162. The cell or the population of cells of claim 144 comprising a c.3_4insTAAG insertion in the B2M gene relative to a wildtype B2M gene, optionally wherein the cell or the population of cells further comprise a C.17OG substitution or a c.l lOG substitution in the B2M gene relative to a wildtype B2M gene.

163. The cell or the population of cells of claim 144 comprising a c.3_4insTAATAA insertion in the B2M gene relative to a wildtype B2M gene, optionally wherein the cell or the population of cells further comprise a C.17OG substitution or a c.l lOG substitution in the B2M gene relative to a wildtype B2M gene.

164. The cell or the population of cells of claim 144 comprising a c.3_8deletion and a TAATGA insertion (c.3_8delinsTAATGA) in the B2M gene relative to a wildtype B2M gene, optionally wherein the cell or the population of cells further comprise a C.17OG substitution or a c.l lOG substitution in the B2M gene relative to a wildtype B2M gene.

165. The cell or the population of cells of claim 144 comprising a c.l5_16insCC insertion in the B2M gene relative to a wildtype B2M gene.

166. The cell or the population of cells of claim 144 comprising a c. 15_16insTAAG insertion in the B2M gene relative to a wildtype B2M gene.

167. The cell or the population of cells of claim 144 comprising a c.l5_16insTAATAA insertion in the B2M gene relative to a wildtype B2M gene.

168. The cell or the population of cells of any one of claims 145-167, wherein the human chromosome locations and coding sequence locations are as set forth in Genome Reference Consortium Human Build 38 (GrCh38).

169. The cell or the population of cells of any one of claims 143-168, wherein the cell or the population of cells are in a subject. WO 2024/238825 PCT/US2024/029746 478

170. The cell or the population of cells of any one of claims 143-168, wherein the cell or the population of cells are ex vivo, optionally wherein the cell or the population of cells are obtained from a subject or a cell bank.

171. The cell or the population of cells of any one of claims 143-168, wherein the cell or the population of cells are human cells.

172. The cell or the population of cells of claim 171, wherein the cell or the population of cells are immune cells or stem cells.

173. The cell or the population of cells of claim 172, wherein the cell or the population of cells are T cells or hematopoietic stem cells (HSCs), optionally wherein the cell or the population of cells are cytotoxic T cells.

174. A method of immunotherapy comprising administering to a subject the (a) the PEgRNA of any one of claims 1-94, and a prime editor comprising a Cas9 nickase having a nuclease inactivating mutation in the HNH domain and a reverse transcriptase, (b) the prime editing system of any one of claims 95-128, (c) the population of viral particles of claim 129 or 130, (d) the LNP of any one of claims 131-134, or (e) the cell or the population of cells of any one of claims 143-172.

175. A prime editing guide RNA (PEgRNA) or one or more polynucleotides encoding the PEgRNA, the PEgRNA comprising:a) a spacer that is complementary to a search target sequence on a first strand of a 02- microglobulin (B2M) gene, wherein the spacer comprises at its 3’ end a PEgRNA spacer sequence selected from any one of Tables 1-21;b) a gRNA core capable of binding to a Cas9 protein, andc) an extension arm comprising:i) an editing template comprising at its 3’ end an RTT sequence selected from the same Table as the PEgRNA Spacer sequence, andii) a primer binding site (PBS) comprising at its 5’ end a PBS sequence selected from the same Table as the PEgRNA Spacer sequence.

176. The PEgRNA of claim 175, wherein the spacer of the PEgRNA is from 17 to nucleotides in length.

177. The PEgRNA of claim 176, wherein the spacer of the PEgRNA is 20 nucleotides in length.

178. The PEgRNA of any one of claims 175-177, wherein the spacer, the gRNA core, the editing template, and the PBS form a contiguous sequence in a single molecule. WO 2024/238825 PCT/US2024/029746 479

179. The PEgRNA of claim 178, comprising from 5’ to 3’, the spacer, the gRNA core, the editing template, and the PBS.

180. A prime editing system comprising the PEgRNA or the one or more polynucleotides of claims 175-179.

181. The prime editing system of claim 180, further comprising a nick guide RNA (ngRNA), or one or more polynucleotides encoding the ngRNA, wherein the ngRNA comprises:(i) an ngRNA spacer that comprises a region of complementarity to a second strand of the B2M gene; and(ii) an ngRNA core capable of binding a Cas9 protein.

182. The prime editing system of claim 181, wherein the spacer of the ngRNA is from to 22 nucleotides in length.

183. The prime editing system of claim 182, wherein the spacer of the ngRNA is nucleotides in length.

184. The prime editing system of any one of claims 175-182, wherein the ngRNA spacer comprises at its 3’ end an ngRNA Spacer sequence selected from the same Table as the PEgRNA Spacer sequence.

185. The prime editing system of any one of claims 175-182, wherein the ngRNA comprises an ngRNA sequence selected from the same Table as the PEgRNA Spacer sequence.

186. The prime editing system of any one of claims 175-185, further comprising:a prime editor comprising a Cas9 nickase having a nuclease inactivating mutation in the HNH domain, or one or more polynucleotides encoding the Casnickase, anda reverse transcriptase, or one or more polynucleotides encoding the reverse transcriptase.

187. The prime editing system of any one of claims 175-185, further comprising:an N-terminal extein comprising an N-terminal fragment of a prime editor fusion protein and an N-intein or a polynucleotide encoding the N-terminal extein; anda C-terminal extein comprising a C-terminal fragment of the prime editor fusion protein and a C-intein, or a polynucleotide encoding the C-terminal extein;wherein the N-intein and the C-intein of the N-terminal and C-terminal exteins are capable of self-excision to join the N-terminal fragment and the C-terminal WO 2024/238825 PCT/US2024/029746 480 fragment to form the prime editor fusion protein, and wherein the prime editor fusion protein comprises a Cas9 nickase and a reverse transcriptase (RT) domain.

188. The prime editing system of claim 186 or 187, wherein the Cas9 nickase comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NOs: 676 or 677.

189. The prime editing system of any one of claims 186-188, wherein the reverse transcriptase comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 673.

190. The prime editing system of claim 188 or 189, wherein the sequence identities are determined by Needleman-Wunsch alignment of two protein sequences with Gap Costs set to Existence: 11 Extension: 1 where percent identity is calculated by dividing the number of identities by the length of the alignment.