Genome editing compositions targeting the b2m gene and methods of use

Abstract

Provided herein are compositions comprising prime editing systems, and methods of using prime editing systems to edit B2M gene. Provided herein are compositions comprising edited cells, and methods to generated edited cells. Also provided are methods of using the edited cells.

Description

GENOME EDITING COMPOSITIONS TARGETING THE B2M GENE AND METHODS OF USECROSS 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 β chain encoded by the β-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 β 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) attarget 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 β2-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 β2-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 β2-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 SEQID 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 β2-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: 327.

[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. Insome 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+2 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-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.3_8 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.17C>G edit. In some embodiments, the PAM silencing edit is a c.11C>G 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 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. 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 templatecomprising 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.

[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 selectedfrom 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, and l l90 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: 327.

[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) asequence 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) 10 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) 8 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, 956,933, 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 20 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 comprisesa 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 sequencecorresponding 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 thengRNA 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 fiirther 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 Cas9 protein; 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 fiirther 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 aTRAC 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 4-20 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: 1303 or 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 nucleotides11-17, 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 8-13 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.

[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 27 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 eachother. 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 templatecomprises 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.

[0057] In some embodiments, the first TRAC PEgRNA and / or the second TRAC PEgRNA fiirther 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 fiirther comprises the recombinase or a nucleic acid encoding the recombinase. In some embodiments, the recombinase is fiised or linked to the prime editor.

[0060] In some embodiments, the prime editing system fiirther 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 fiirther 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 fiirther 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 fiision 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%,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.

[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.l 1 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 thepopulation 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 farther 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 farther 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 farther 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 farther comprise a C.17C>G substitution or a c.l 1C>G 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 farther comprise a C.17C>G substitution or a c.l 1C>G 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 farther comprise a C.17C>G substitution or a c.l 1C>G 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 farther comprise a C.17C>G substitutionor a c.l 1C>G 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 sequence GGCTTGTCGACGACGGCGGTCTCAGTGGTGTACGGTACAAACC (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 14 positions22547458 and 22547533, b) between human chromosome 14 positions 22547458and 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 14 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 β2-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 22 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.

[0080] In some embodiments, the prime editing system farther 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 farther 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 farther 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 fasion 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 farther 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 thatcomprises 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 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.

[0088] 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.

[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. 1 depicts a schematic of a prime editing guide RNA (PEgRNA) binding to a double stranded target DNA sequence.

[0093] FIG. 2 depicts a PEgRNA architectural overview in an exemplary schematic of PEgRNA designed for a prime editor.

[0094] FIG. 3 is 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. 4 depicts 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. 4B depicts 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.DETAILED DESCRIPTION

[0097] Provided herein, in some embodiments, are compositions and methods to edit the target gene β2 -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 withthe 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 γδ 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 γδ T cell. In some embodiments, the cell is an effector T cell. In some embodiments, the cell is a thymocyte. Insome 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 γδ 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 Thelper 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 γδ 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, CD8, 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 humansubject 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 50 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 fiill-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 S. 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 unction , a protein-protein binding function, or a protein-DNA function. In some embodiments, a protein comprises multiple proteindomains. 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 fiision protein comprising a Cas9 protein domain of S. 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 fiision, 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 fiision 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,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 polynucleotidesequence. “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, 3100 or 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 85:2444,1988; 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 FASTA package (Pearson W and Lipman D, 1988, Proc. Natl. Acad. Sci. USA, 85: 2444-2448). Both of these programs are based on the Needleman-Wunsch algorithm which is used to find the 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", FEBS 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,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 farther 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 intemucleoside 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 asecond 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,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- firee 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 byPEgRNA 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,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, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 3 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, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 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 β chain of MHC Class I.

[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 effectiveamount” 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, No67, Kp03, Pa01, Nm60, BcelNTa, BcytINTd, SscINTd, SacINTd, Hin, Gin, Tn3, I3-six, CinH, ParA, y6, Bxbl, OC31, TP901, TG1, pBT1, R4, pRV1, pFC1, MR11, A118, U153, and gp29. 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.

[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 useful 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.

[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 positionin 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 maycomprise 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, forexample, 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 full-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+1 or 3x+2 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+1 or 3x+2 nucleotides in the codingregion, 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 1, 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.

[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)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, FEN1. 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.

[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,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 sequencebetween 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 ODis 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 “replacementduplex (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 No67 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 anotherCRISPR-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., FEN1), 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 S. 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 fiised 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 fiision 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

[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, Pfii, 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.

[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 POLA1 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 POLE1 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 (POLK) 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.

[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, vtoesii, 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 fiiriosus (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 UlTma).

[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; bovineleukemia 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 some embodiments, 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. An exemplary 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. An exemplary 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 DRWLSNARMTHYQALLLDTDRVQFGPWALNPATLLPLPEEGLQHNCLDILAEAHG TRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQR AELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSS 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 DRWLSNARMTHYQALLLDTDRVQFGPWALNPATLLPLPEEGLQHNCLDILAEAHG 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, T33OX, 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, T33OP, 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, T33OP, 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, T33OP, 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, T33OP, L603W, T306K, and W313F ascompared 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, T33OP, 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, T33OP, 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 ARMTHYQALLLDTDRVQFGPWALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLT DQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIAL TQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGWLTSEGKEIKNKDEIL ALLKA 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 isabout 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 IDNOs: 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 variantthereof. 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 FokI 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 I, type II, type III, type IV, type V Cas protein, or a type VI Cas protein. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (e.g., Csn1 or Csx12), Cas10, Cas10d, Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i, Csy1 , Csy2, Csy3, Csy4, Csel, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2, Csa5, Csn1, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csx11,Csfl, Csf2, CsO, Csf4, Csdl, Csd2, Cstl, Cst2, Csh1, Csh2, Csal, Csa2, Csa3, Csa4, Csa5, Type II Cas effector proteins, Type V Cas effector proteins, Type VI Cas effector proteins, CARF, DinG, Cpfl, Cas12b / C2c1, Cas12c / C2c3, Cas12b / C2c1, Cas12c / C2c3, SpCas9(K855A), eSpCas9(1.1), SpCas9-HFl, hyper accurate Cas9 variant (HypaCas9), Cas Φ, 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, Candidates 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 Streptococcusthermophilus (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 gassed, 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, Candidates 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, tesion, chimera, or any combination thereof relative to a corresponding wild-type version of the Cas protein. Insome 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 editorcomprises 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 Cas9 nickase 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 D10X amino acid substitution compared to a wild type S. 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 S. pyogenes Cas9, wherein X is any amino acid other than H.

[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.

[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 fiised to a heterologous polypeptide providing increased or decreased stability. The fiised 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, Cas9 polypeptide 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, fiision, 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 canis (Sc), Streptococcus thermophilus (St), Staphylococcus lugdunensis (Siu), 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 fragmentor 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 22 below.

[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 Cas9 protein 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 comprisesan 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 Cas9 (SpCas9) amino acid sequence usefiil 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. Insome 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 SluCas9 comprises 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 SaCas9 lacks 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 SaCas9 comprises 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 proteinamino 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., sRGN3.1, sRGN3.3. 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 (Siu), 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%, atleast 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) useful 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 Cas9 protein (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 Cas9 protein comprises an amino acid sequence set forth in any one of SEQ ID NOs: 694, 695, 697, or 698.Table 22: Exemplary Cas protein sequences

[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 (i.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 (i.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 DIO 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 DIO, 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.

[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 D10X 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 D10X 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.

[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 Cas9 protein, including any wild type Cas9, or mutant Cas9 (e.g., a dead Cas9 or Cas9 nickase), or fragment Cas9, or circular permutant Cas9, or other variant of Cas9 disclosed herein or known in the art. In some embodiments, a Cas9 variant may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acid changes compared to a reference Cas9, e.g., a wild type Cas9. In some embodiments, the Cas9 variant comprises a fragment of a reference Cas9 (e.g., a gRNA binding domain or a DNA- cleavage domain), such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of 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-basedprime 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 (i.e., located upstream of the 5' end of the protospacer). In other embodiments, the PAM can be a 3' PAM (i.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 23 below. 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 23 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

[0234] In some embodiments, a prime editor comprises a Cas9 polypeptide comprising one or mutations selected from the group consisting of: A61R, LI 11R, DI 135V, 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, R1114G, DI 135E, DI 135L, D1135N, S1136W, V1139A, D1180G, G1218K, G1218R, G1218S, E1219Q, E1219V, E1219V, Q1221H, P1249S, E1253K, N1317R, A1320V, P1321S, A1322R, I1322V, D1332G, R1332N, A1332R, R1333K, R1333P, R1335L, R1335Q, R1335V, T1337N,T1337R, S1338T, H1349R, and any combinations thereof as compared to a wildtype SpCas9 polypeptide 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 FnCas9 polypeptide 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 Cas9 polypeptide, 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 Cas9 or 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]-[1-1267]-C-terminus;

[0239] N-terminus-[1168-1368]-[optional linker]-[1-1167]-C-terminus;

[0240] N-terminus-[1068-1368]-[optional linker]-[1-1067]-C-terminus;

[0241] N-terminus-[968-1368]-[optional linker]-[1-967]-C-terminus;

[0242] N-terminus-[868-1368]-[optional linker]-[1-867]-C-terminus;

[0243] N-terminus-[768-1368]-[optional linker]-[1-767]-C-terminus;

[0244] N-terminus-[668-1368]-[optional linker]-[1-667]-C-terminus;

[0245] N-terminus-[568-1368]-[optional linker]-[1-567]-C-terminus;

[0246] N-terminus-[468-1368]-[optional linker]-[1-467]-C-terminus;

[0247] N-terminus-[368-1368]-[optional linker]-[1-367]-C-terminus;

[0248] N-terminus-[268-1368]-[optional linker]-[1-267]-C-terminus;

[0249] N-terminus-[168-1368]-[optional linker]-[1-167]-C-terminus;

[0250] N-terminus-[68-1368]-[optional linker]-[1-67]-C-terminus;

[0251] N-terminus-[10-1368]-[optional linker]-[1-9]-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]-[1-101]-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]-[1-1248]-C-terminus; or

[0257] N-terminus-[1300-1368]-[optional linker]-[1-1299]-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]-[1-102]-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]-[1-1249]-C-terminus; or

[0262] N-terminus-[1301-1368]-[optional linker]-[1-1300]-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 inSEQ 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 Cas9 (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,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 1282 would 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, and Cas9- 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 Cas9 functional 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 1300 amino acids, less than 1290 amino acids, than less than 1280 amino acids, less than 1270 amino 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 1110 amino 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 650 amino acids, less than 600 amino acids, less than 550 amino acids, or less than 500 aminoacids, 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, Cas12a, Cas12bl, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas1O, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, 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 Cas12e (CasX), a Cas12d (CasY), a Cas12bl (C2cl), a Cas13a (C2c2), a Cas12c (C2c3), a GeoCas9, a CjCas9, a Cas12g, a Cas12h, a Cas12i, a Cas13b, a Cas13c, a Cas13d, a Cas14, 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 24 below:Table 24: Exemplary Cas proteins and nomenclature

[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 maycomprise a Cas12a (Cpfl) polypeptide or functional variants thereof. In some embodiments, the Cas12a 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 Cas12a 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 Cas12a 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 (C2c1) 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 Cas12e, a Cas 12d, a Cas13, Cas 14a, Cas 14b, Cas 14c, Cas14d, Cas14e, Cas14f, Cas 14g, Cas14h, Cas14u, or a CasΦ 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 Cas12e, Cas 12d, Cas 13, Cas14a, Cas14b, Cas14c, Cas14d, Cas14e, Cas14f, Cas14g, Cas14h, Cas14u, or Cas Φ protein. In some embodiments, the Cas protein is a Cas12e, Cas 12d, Cas13, or Cas Φ nickase.Nuclear Localization Sequences

[0272] In some embodiments, a prime editor further comprises one or more nuclear localization sequence (NLS). In some embodiments, the NLS helps promote translocation of a protein into the cell nucleus. In some embodiments, a prime editor comprises a fusion protein, e.g., a fusion protein comprising a DNA binding domain and a DNA polymerase, that comprises one or more NLSs. In some embodiments, one or more polypeptides of the prime editor are fused to or linked to one or more NLSs. In some embodiments, the prime editor comprises a DNA binding domain and a DNA polymerase domain that are provided in trans, wherein the DNA binding domain and / or the DNA polymerase domain is fused or linked to one or more NLSs.

[0273] In certain embodiments, a prime editor or prime editing complex comprises at least one NLS. In some embodiments, a prime editor or prime editing complex comprises at least two NLSs. In embodiments with at least two NLSs, the NLSs can be the same NLS, or they can be different NLSs.

[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 farther comprise 2 NLSs. In other cases, a prime editor may farther comprise 3 NLSs. In one case, a primer editor can farther 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 fasion 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). Insome 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 hriRNP 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

[0280] In some embodiments, a prime editing complex comprises a fiision 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 fiision 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 H840 A)] -[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 N394KH840A)]-[linker]-[MMLV_RT(D200N)(T330P)(L603W)(T306K)(W313F)] 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 farther 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: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 16 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 sequence SGGSSGGSSGSETPGTSESATPESAGSYPYDVPDYAGSAAPAAKKKKLDGSGSGGSS GGS (SEQ ID NO: 733).

[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 fiised or linked to the C-terminal end of a DNA polymerase domain. In some embodiments, a prime editor comprises a DNA binding domain fiised 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-protein recruitment polypeptide]-COOH. In some embodiments, a prime editor comprises a fiision protein and a DNA binding domain provided in trans, wherein the fiision 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 fiision 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 fiised 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 fiision protein comprises a Cas9(H840A) nickase and a M-MLV RT that comprises amino acid substitutions D200N, T33OP, 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, T33OP, T306K, W313F, and L603W compared to a wild type M-MLV RT. The amino acid sequence of an exemplary prime editor fiision protein and its individual components in shown in Table 26. In some embodiments, a prime editor fiision protein comprises a Cas9 (R221K N394K H840A) nickase and a M-MLV RT that comprises amino acid substitutions D200N, T33OP, T306K, W313F, and L603W compared to a wild type M-MLV RT. The amino acid sequence of an exemplary Prime editor fiision protein and its individual components in shown in Table 27. In some embodiments anexemplary 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 componentsTable 27: lists exemplary prime editor and its componentsPEgRNA 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 ormore 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 farther 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 editingtemplate 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 gRN A core of a PEgRNA 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 gRNAcore. In some embodiments, the PEgRNA comprises, from 5' to 3': an editing target, a PBS, a spacer, and a gRNA 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): 95559564 (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.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 100 nucleotides. In some embodiments, the spacer is 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, or 25 nucleotides in length. In some embodiments, the spacer is from 15 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 20 nucleotides, about 8 to 20 nucleotides, about 10 to 20 nucleotides, about 12 to 20 nucleotides, about 14 to 20 nucleotides, about 16 to 20 nucleotides, or about 18 to 20 nucleotides in length. In some embodiments, the PBS is 8 to 17 nucleotides in length. Insome 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 11 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 16 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 12 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 15 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 11 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).

[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. In some 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 25 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 19 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).

[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 B2M gene 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

[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 nucleotidesubstitution 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 3 nucleotides, from 1 to 4 nucleotides, from 1 to 5 nucleotides, form 2 to 5 nucleotides, from 3 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 13 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 20 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.

[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 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, 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 6 nucleotides, 0 to 8 nucleotides, 0 to 10 nucleotides, , 2 to 4 nucleotides, 2 to 6 nucleotides, 2 to 8 nucleotides, 2 to 10 nucleotides, 2 to 12 nucleotides, 4 to 6 nucleotides, 4 to 8 nucleotides, 4 to 10 nucleotides, 4 to 12 nucleotides, 4 to 14 nucleotides, 6 to 8 nucleotides, 6 to 10 nucleotides, 6 to 12 nucleotides, 6 to 14 nucleotides, 6 to 16 nucleotides, 8 to 10 nucleotides, 8 to 12 nucleotides, 8 to 14 nucleotides, 8 to 16 nucleotides, 8 to 18 nucleotides, 10 to 12 nucleotides, 10 to 14 nucleotides, 10 to 16 nucleotides, 10 to 18 nucleotides, 10 to 20 nucleotides, 12 to 14 nucleotides, 12 to 16 nucleotides, 12 to 18 nucleotides, 12 to 20 nucleotides, 12 to 22 nucleotides, 14 to 16 nucleotides, 14 to 18 nucleotides, 14 to 20nucleotides, 14 to 22 nucleotides, 14 to 24 nucleotides, 16 to 18 nucleotides, 16 to 20 nucleotides, 16 to 22 nucleotides, 16 to 24 nucleotides, 16 to 26 nucleotides, 18 to 20 nucleotides, 18 to 22 nucleotides, 18 to 24 nucleotides, 18 to 26 nucleotides, 18 to 28 nucleotides, 20 to 22 nucleotides, 20 to 24 nucleotides, 20 to 26 nucleotides, 20 to 28 nucleotides, 20 to 30 nucleotides, 30 to 40 nucleotides, 40 to 50 nucleotides, 50 to 60 nucleotides, 60 to 70 nucleotides, 70 to 80 nucleotides, 80 to 90 nucleotides, 90 to 100 nucleotides, 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 10 nucleotides, , 2 to 4 nucleotides, 2 to 6 nucleotides, 2 to 8 nucleotides, 2 to 10 nucleotides, 2 to 12 nucleotides, 4 to 6 nucleotides, 4 to 8 nucleotides, 4 to 10 nucleotides, 4 to 12 nucleotides, 4 to 14 nucleotides, 6 to 8 nucleotides, 6 to 10 nucleotides, 6 to 12 nucleotides, 6 to 14 nucleotides, 6 to 16 nucleotides, 8 to 10 nucleotides, 8 to 12 nucleotides, 8 to 14 nucleotides, 8 to 16 nucleotides, 8 to 18 nucleotides, 10 to 12 nucleotides, 10 to 14 nucleotides, 10 to 16 nucleotides, 10 to 18 nucleotides, 10 to 20 nucleotides, 12 to 14 nucleotides, 12 to 16 nucleotides, 12 to 18 nucleotides, 12 to 20 nucleotides, 12 to 22 nucleotides, 14 to 16 nucleotides, 14 to 18 nucleotides, 14 to 20 nucleotides, 14 to 22 nucleotides, 14 to 24 nucleotides, 16 to 18 nucleotides, 16 to 20 nucleotides, 16 to 22 nucleotides, 16 to 24 nucleotides, 16 to 26 nucleotides, 18 to 20 nucleotides, 18 to 22 nucleotides, 18 to 24 nucleotides, 18 to 26 nucleotides, 18 to 28 nucleotides, 20 to 22 nucleotides, 20 to 24 nucleotides, 20 to 26 nucleotides, 20 to 28 nucleotides, 20 to 30 nucleotides, 30 to 40 nucleotides, 40 to 50 nucleotides, 50 to 60 nucleotides, 60 to 70 nucleotides, 70 to 80 nucleotides, 80 to 90 nucleotides, 90 to 100 nucleotides, 100 to 110 nucleotides, 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 nucleotidesupstream 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 someembodiments, 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 21 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 16 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 contiguousnucleotides 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 8 nucleotides, 0 to 10 nucleotides, 2 to 4 nucleotides, 2 to 6 nucleotides, 2 to 8 nucleotides, 2 to 10 nucleotides, 2 to 12 nucleotides, 4 to 6 nucleotides, 4 to 8 nucleotides, 4 to 10 nucleotides, 4 to 12 nucleotides, 4 to 14 nucleotides, 6 to 8 nucleotides, 6 to 10 nucleotides, 6 to 12 nucleotides, 6 to 14 nucleotides, 6 to 16 nucleotides, 8 to 10 nucleotides, 8 to 12 nucleotides, 8 to 14 nucleotides, 8 to 16 nucleotides, 8 to 18 nucleotides, 10 to 12 nucleotides, 10 to 14 nucleotides, 10 to 16 nucleotides, 10 to 18 nucleotides, 10 to 20 nucleotides, 12 to 14 nucleotides, 12 to 16 nucleotides, 12 to 18 nucleotides, 12 to 20 nucleotides, 12 to 22 nucleotides, 14 to 16 nucleotides, 14 to 18 nucleotides, 14 to 20 nucleotides, 14 to 22 nucleotides, 14 to 24 nucleotides, 16 to 18 nucleotides, 16 to 20 nucleotides, 16 to 22 nucleotides, 16 to 24 nucleotides, 16 to 26 nucleotides, 18 to 20 nucleotides, 18 to 22 nucleotides, 18 to 24 nucleotides, 18 to 26 nucleotides, 18 to 28 nucleotides, 20 to 22 nucleotides, 20 to 24 nucleotides, 20 to 26 nucleotides, 20 to 28 nucleotides, or 20 to 30 nucleotides 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 theposition 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 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 30 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 nucl eotide 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 γδ 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).

[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 P AM 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 nucleotides longer than the nick to edit distance. In some embodiments, for example, the nick to edit distance is 8 nucleotides, and the editing template is 10 to 15, 10 to 20, 10 to 25, 10 to 30, 10 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 80 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 aPEgRNA following 5?-spacer-gRNA core-RTT-PBS-3forientation, 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 editsresults 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”, “β2 microglobulin protein”, or “β 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 having immunomodulatory 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

[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 AAACATTTTGTCATATAAGATTCATATTTACTTCTTATACATTTGATAAAGTAAGGCATGGTTGTGGTTAATCTGGTTTATTTTTGTTCCACAAGTTAAATAAATCATAAAA 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 binding to a Cas12b-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 lowzer 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);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' 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.

[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 15 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).

[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 MS2 aptamer 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 spac...

Claims

WHAT IS CLAIMED IS:

1. A prime editing guide RNA (PEgRNA) or one or more polynucleotides encoding thePEgRNA, wherein the PEgRNA comprises: a. a spacer that is complementary to a search target sequence on a first strand of a β2-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.

2. A prime editing guide RNA (PEgRNA) or one or more polynucleotides encoding thePEgRNA, wherein the PEgRNA comprises: a. a spacer that is complementary to a search target sequence on a first strand of a β2-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: 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: 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 thePEgRNA, wherein the PEgRNA comprises:a. a spacer that is complementary to a search target sequence on a first strand of a β2-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.

4. A prime editing guide RNA (PEgRNA) or one or more polynucleotides encoding thePEgRNA, wherein the PEgRNA comprises: a. a spacer that is complementary to a search target sequence on a first strand of a β2-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: 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: 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 IDNOs: 202-204.

7. The PEgRNA of claim 1, wherein the spacer comprises SEQ ID NO: 204.

8. The PEgRNA of claim 2, wherein the spacer comprises at its 3’ end any one of SEQ IDNO: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.

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 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.

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.

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_8 deletion and a TAATGA insertion (c.3_8delinsTAATGA).

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 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.

59. The PEgRNA of claim 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.

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.

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(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.

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; 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.

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; 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.

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: 1049-1052 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. or66. 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.

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(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 24 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 20 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, 35 nucleotides in length.

72. The PEgRNA of any one of claims 1-71, wherein the PBS has a length of 17 nucleotides or less.

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

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, 76 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, 77 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. f90. 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.

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; and b. an ngRNA core capable of binding a Cas9 protein.

97. The prime editing system of claim 96, wherein the ngRNA spacer is 17-22 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:(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- 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.

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- 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.

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.

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- 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.

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 mindicates 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, and b. a reverse transcriptase.

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

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 Cas9 nickase 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.

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 comparedto 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.l 1 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 farther 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 farther 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 farther 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.

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.17C>G substitution or a c.l 1C>G 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 farther comprise a C.17C>G substitution or a c.l 1C>G 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 farther comprise a C.17C>G substitution or a c.l 1C>G 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 farther comprise a C.17C>G substitution or a c.l 1C>G 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.

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 β2- 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.

176. The PEgRNA of claim 175, wherein the spacer of the PEgRNA is from 17 to 22 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.

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, farther 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 17 to 22 nucleotides in length.

183. The prime editing system of claim 182, wherein the spacer of the ngRNA is 20 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, farther 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.

187. The prime editing system of any one of claims 175-185, farther comprising: an N-terminal extein comprising an N-terminal fragment of a prime editor fasion 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 fasion 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-terminalfragment 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.

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