CLASS II, TYPE V CRISPR SYSTEMS
Described herein are methods, compositions, and systems derived from uncultivated microorganisms useful for gene editing.
1 .- 99 . (canceled)
100 . An engineered nuclease system comprising:
(a) an endonuclease comprising a RuvCI, RuvCII, or RuvCIII domain having at least 75% sequence identity to a RuvCI, RuvCII, or RuvCIII domain of any one of SEQ ID NOs: 1-15; and
(b) an engineered guide ribonucleic acid configured to form a complex with said endonuclease, wherein said engineered guide ribonucleic acid comprises a spacer sequence configured to hybridize to a target nucleic acid sequence.
101 . The engineered nuclease system of claim 100 , wherein said endonuclease comprises a WED II domain having at least 75% sequence identity to a WED II domain of any one of SEQ ID NOs: 1-15.
102 . The engineered nuclease system of claim 100 , wherein said endonuclease is not a Cas12a endonuclease.
103 . The engineered nuclease system of claim 100 , wherein said endonuclease comprises an amino acid sequence having at least 75% sequence identity to any one of SEQ ID NOs: 1-15.
104 . The engineered nuclease system of claim 103 , wherein said endonuclease comprises the amino acid sequence of any one of SEQ ID NOs: 1-15.
105 . The engineered nuclease system of claim 100 , wherein said endonuclease comprises one or more nuclear localization sequences (NLSs) proximal to an N- or C-terminus of said endonuclease.
106 . The engineered nuclease system of claim 105 , wherein said NLS comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 36-51.
107 . The engineered nuclease system of claim 100 , wherein said engineered guide ribonucleic acid comprises a nucleic acid sequence having at least 80% sequence identity to non-degenerate the sequence of any one of SEQ ID NOs: 16-21 and 30-35.
108 . The engineered nuclease system of claim 107 , wherein said engineered guide ribonucleic acid comprises the nucleic acid sequence of any one of SEQ ID NOs: 16-21 and 30-35.
109 . The engineered nuclease system of claim 100 , further comprising a single- or double-stranded deoxyribonucleic acid repair template.
110 . A method of modifying a target nucleic acid locus, said method comprising delivering to said target nucleic acid locus an engineered nuclease system comprising:
(a) an endonuclease comprising a RuvCI, RuvCII, or RuvCIII domain having at least 75% sequence identity to a RuvCI, RuvCII, or RuvCIII domain of any one of SEQ ID NOs: 1-15; and
(b) an engineered guide ribonucleic acid configured to form a complex with said endonuclease and wherein said complex modifies said target nucleic acid locus.
111 . The method of claim 110 , wherein said endonuclease comprises a WED II domain having at least 75% sequence identity to a WED II domain of any one of SEQ ID NOs: 1-15.
112 . The method of claim 110 , wherein said endonuclease is not a Cas12a endonuclease.
113 . The method of claim 110 , wherein said endonuclease comprises an amino acid sequence having at least 75% sequence identity to any one of SEQ ID NOs: 1-15.
114 . The method of claim 113 , wherein said endonuclease comprises the amino acid sequence of any one of SEQ ID NOs: 1-15.
115 . The method of claim 110 , wherein said endonuclease comprises one or more nuclear localization sequences (NLSs) proximal to an N- or C-terminus of said endonuclease.
116 . The method of claim 110 , wherein said engineered guide ribonucleic acid comprises a nucleic acid sequence having at least 80% sequence identity to non-degenerate the sequence of any one of SEQ ID NOs: 16-21 and 30-35.
117 . The method of claim 116 , wherein said engineered guide ribonucleic acid comprises the nucleic acid sequence of any one of SEQ ID NOs: 16-21 and 30-35.
118 . The method of claim 110 , wherein said target nucleic acid locus is within a cell.
119 . The method of claim 118 , wherein said cell is a prokaryotic cell, a bacterial cell, a eukaryotic cell, a fungal cell, a plant cell, an animal cell, a mammalian cell, a rodent cell, a primate cell, a human cell, or a primary cell.