COMPOSITIONS AND METHODS FOR NUCLEIC ACID MODIFICATIONS
The present disclosure provides nucleases and compositions, methods, and systems thereof for nucleic acid modification. More particularly, the present disclosure provides compositions and system comprising a nuclease comprising an amino acid sequence having at least 70% identity to any of SEQ ID NOs: 1-1096 and at least one gRNA.
1 .- 20 . (canceled)
21 . An engineered nuclease, wherein the engineered nuclease comprises an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 666, and at least one amino acid substitution at one or more of positions relative to SEQ ID NO: 416 selected from the group consisting of position 178, position 286, position 541, position 546, position 552, and position 873.
22 . The engineered nuclease of claim 21 , wherein the engineered nuclease comprises an amino acid substitution relative to SEQ ID NO: 416 selected from the group consisting of 178R, 286A, 541R, 546R, 552R, and 873L.
23 . The engineered nuclease of claim 21 , wherein the amino acid sequence of the engineered nuclease has at least 95% sequence identity to SEQ ID NO: 666.
24 . The engineered nuclease of claim 21 , wherein the engineered nuclease comprises the amino acid sequence of any one of SEQ ID NO: 662, SEQ ID NO: 663, SEQ ID NO: 664, SEQ ID NO: 665, SEQ ID NO: 667, SEQ ID NO: 668, SEQ ID NO: 669, SEQ ID NO: 670, SEQ ID NO: 671, and SEQ ID NO: 672.
25 . The engineered nuclease of claim 21 , wherein the amino acid sequence of the engineered nuclease has the substitution(s) E178R; N286A; N541R; D546R; K552R; F873L; E178R/D546R; E178R/K552R; D546R/K552R; E178R/D546R/K552R; E178R/K552R/N286A; or N541R/F873L relative to SEQ ID NO: 416.
26 . The engineered nuclease of claim 21 , wherein the amino acid sequence of the engineered nuclease comprises one or more of SEQ ID NOs: 1169-1174.
27 . The engineered nuclease of claim 22 , wherein the amino acid sequence of the engineered nuclease comprises the substitutions 178R and 546R.
28 . The engineered nuclease of claim 21 , wherein the amino acid sequence of the engineered nuclease comprises SEQ ID NO: 666.
29 . A fusion protein comprising the engineered nuclease of claim 21 .
30 . The fusion protein of claim 29 , wherein the fusion protein comprises a nuclear localization sequence.
31 . A kit comprising the engineered nuclease of claim 21 and at least one guide ribonucleic acid (gRNA).
32 . The kit of claim 31 , wherein the at least one gRNA comprises a first region configured to interact with the engineered nuclease and a second region configured to hybridize with a portion of a target nucleic acid.
33 . A cell comprising the engineered nuclease of claim 21 .
34 . The cell of claim 33 , wherein the cell is a mammalian cell.
35 . The cell of claim 33 , wherein the mammalian cell is a human cell.
36 . A nucleic acid encoding the engineered nuclease of claim 21 .
37 . A vector comprising the nucleic acid of claim 36 .
38 . A method of modifying a target nucleic acid, comprising contacting the target nucleic acid with the engineered nuclease of claim 21 .
39 . The method of claim 38 , wherein the target nucleic acid is present in a cell.
40 . The method of claim 39 , wherein the cell is a mammalian cell.