CLASS 2, TYPE V CRISPR SYSTEMS
Described herein are methods, compositions, and systems derived from uncultivated microorganisms useful for gene editing.
1 . An engineered nuclease system comprising:
a) an endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 57, 31, 1-30, 32-56, 58-325, 420-431, 476-624, 629, 1065-1090, 1114-1118, 1746-1752, and 1842-1872; and
b) an engineered guide polynucleotide configured to form a complex with the endonuclease and to hybridize to a target nucleic acid sequence.
2 . The engineered nuclease system of claim 1 , wherein the endonuclease comprises a sequence having at least 90% or 100% sequence identity to any one of SEQ ID NOs: 57, 31, 1-30, 32-56, 58-325, 420-431, 476-624, 629, 1065-1090, 1114-1118, 1746-1752, and 1842-1872.
3 . (canceled)
4 . The engineered nuclease system of claim 1 , wherein the engineered guide polynucleotide comprises a crRNA and a tracrRNA.
5 . The engineered nuclease system of claim 1 , wherein the engineered guide polynucleotide comprises a sequence having at least 90% or 100% sequence identity to any one of SEQ ID NOs: 333-335, 355-357, 410-411, 346-347, 368-369, 412-413, 326-332, 336-345, 348-354, 358-367, 414-419, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 647-766, 1091-1113, 1119-1120, 1697-1731, 1807-1836, 1876, and 1887-1894.
6 . (canceled)
7 . The engineered nuclease system of claim 1 , wherein the engineered guide polynucleotide is a single guide nucleic acid or a dual guide nucleic acid.
8 . (canceled)
9 . The engineered nuclease system of claim 1 , wherein the engineered guide polynucleotide is RNA.
10 . The engineered nuclease system of claim 1 , wherein;
(a) the endonuclease binds non-covalently to the engineered guide polynucleotide;
(b) the endonuclease is covalently linked to the engineered guide polynucleotide; or
(c) the endonuclease is fused to the engineered guide polynucleotide.
11 - 12 . (canceled)
13 . The engineered nuclease system of claim 1 , further comprising a DNA methyltransferase.
14 . The engineered nuclease system of claim 13 , wherein the DNA methyltransferase binds non-covalently to the endonuclease.
15 . The engineered nuclease system of claim 13 , wherein the DNA methyltransferase is fused to the endonuclease in a single polypeptide.
16 . The engineered nuclease system of claim 13 , wherein the DNA methyltransferase comprises Dmnt3A or Dnmt3L.
17 . An engineered nuclease system comprising:
(i) a) an endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 6-14; and
b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 333-335 and 355-357;
(ii) a) an endonuclease comprising a sequence having at least 80% sequence identity to ID NO: 15; and
b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 410-411;
(iii) a) an endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 16-29; and
b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 346-347, 368-369, and 412-413;
(iv) a) an endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 57, 31, 1-30, 32-56, 58-150, 420-431, 476-624, and 629; and
b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 326-332, 336-345, 348-354, 358-367, 414-419, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 647-766, and 1697-1731; or
(v) a) an endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1065-1090, 1114-1118, and 1746-1752; and
b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1091-1113, 1119-1120, and 1876.
18 - 31 . (canceled)
32 . A method for modifying a target nucleic acid sequence comprising contacting the target nucleic acid sequence using the engineered nuclease system of claim 1 .
33 - 37 . (canceled)
38 . A method of modifying a target nucleic acid sequence in a mammalian cell comprising contacting the mammalian cell using the engineered nuclease system of claim 1 .
39 . (canceled)
40 . A method of modifying TRAC, comprising contacting TRAC using the engineered nuclease system of claim 1 , wherein:
a) the endonuclease comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 57, 31, 1-30, 32-56, 58-15030-150, 420-431, 476-624, and 629; and
b) the engineered guide polynucleotide is configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 767-798.
41 . (canceled)
42 . A method of modifying APOA1, comprising contacting APOA1 using the engineered nuclease system of claim 1 , wherein:
a) the endonuclease comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 57, 31, 1-30, 32-56, 58-15030-150, 420-431, 476-624, and 629; and
b) the engineered guide polynucleotide is configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 831-904, 979-1021, 1219-1237, 1491-1506, and 1663-1669.
43 . (canceled)
44 . A method of modifying AAVS1, comprising contacting APOA1 using the engineered nuclease system of claim 1 , wherein:
a) the endonuclease comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 57, 31, 1-30, 32-56, 58-150, 420-431, 476-624, and 629; and
b) the engineered guide polynucleotide is configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1257-1324, 1523-1562, 1677-1686, 1753-1779, and 1807-1836.
45 . (canceled)
46 . A method of modifying an albumin gene, comprising contacting the albumin gene using the engineered nuclease system of claim 1 , wherein:
a) the endonuclease comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 57, 31, 1-30, 32-56, 58-15030-150, 420-431, 476-624, and 629; and
b) an engineered guide polynucleotide is configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1121-1169, 1393-1441, 1603-1632, 1887, 1889, 1891, and 1892-1894.
47 . (canceled)
48 . A nucleic acid encoding the engineered nuclease system of claim 1 .
49 . A cell comprising the engineered nuclease system of claim 1 .
50 - 63 . (canceled)