CLASS II, TYPE V CRISPR SYSTEMS
Described herein are methods, compositions, and systems derived from uncultivated microorganisms useful for gene editing.
1 .- 164 . (canceled)
165 . A method of disrupting a TRAC locus in a cell, said method comprising introducing to said cell:
(a) an endonuclease or a polynucleotide encoding said endonuclease, wherein said endonuclease is configured to be selective for a protospacer adjacent motif (PAM) sequence comprising 5′-YYn-3′ (SEQ ID NO:3871), and wherein said endonuclease comprises a WED II domain and a PAM-interacting domain having at least 80% sequence identity to a WED II domain and a PAM-interacting domain, respectively, of the amino acid sequence of SEQ ID NO: 215; and
(b) an engineered guide ribonucleic acid or a polynucleotide encoding said engineered guide ribonucleic acid, wherein said engineered guide ribonucleic acid is configured to form a complex with said endonuclease, wherein said engineered guide ribonucleic acid comprises a spacer sequence configured to hybridize to a region of said TRAC locus, and wherein said engineered guide ribonucleic acid comprises a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 4378, 4394, 4404, 4370-4377, 4379-4393, 4395-4403, or 4405-4423.
166 . The method of claim 165 , wherein said engineered guide ribonucleic acid comprises a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 4378, 4394, or 4404.
167 . The method of claim 166 , wherein said engineered guide ribonucleic acid comprises the nucleotide sequence of any one of SEQ ID NOs: 4378, 4394, or 4404.
168 . The method of claim 165 , wherein said region of said TRAC locus comprises a sequence having at least 20-22 consecutive nucleotides of any one of SEQ ID NOs: 4316-4369.
169 . The method of claim 165 , wherein said TRAC locus comprises a sequence having at least 90% sequence identity to the sequence of any one of SEQ ID NOs: 4316-4369.
170 . The method of claim 169 , wherein said TRAC locus comprises the sequence of any one of SEQ ID NOs: 4316-4369.
171 . The method of claim 165 , wherein said WED II domain comprises an amino acid sequence having at least 80% sequence identity to amino acid residues 561-632 of SEQ ID NO: 215.
172 . The method of claim 171 , wherein said WED II domain comprises amino acid residues 561-632 of SEQ ID NO: 215.
173 . The method of claim 165 , wherein said PAM-interacting domain comprises an amino acid sequence having at least 80% sequence identity to amino acid residues 633-730 of SEQ ID NO: 215.
174 . The method of claim 173 , wherein said PAM-interacting domain comprises amino acid residues 633-730 of SEQ ID NO: 215.
175 . The method of claim 165 , wherein said endonuclease comprises a RuvC domain comprising an amino acid sequence having at least 80% sequence identity to a RuvCI domain, a RuvCII domain, and a RuvCIII domain of the amino acid sequence of SEQ ID NO: 215.
176 . The method of claim 165 , wherein said endonuclease comprises catalytic residues G578-W579, K583, K641, D886, E976, or D1229.
177 . The method of claim 165 , wherein said endonuclease comprises at least one of the following mutations: S168R, E172R, or N577R when an amino acid sequence of said endonuclease is aligned to the amino acid sequence of SEQ ID NO: 215.
178 . The method of claim 165 , wherein said endonuclease comprises mutations S168R and E172R when an amino acid sequence of said endonuclease is aligned to the amino acid sequence of SEQ ID NO: 215.
179 . The method of claim 165 , wherein said endonuclease comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 215.
180 . The method of claim 179 , wherein said endonuclease comprises the amino acid sequence of SEQ ID NO: 215.
181 . The method of claim 165 , wherein said endonuclease comprises the amino acid sequence of SEQ ID NO: 215 and wherein said engineered guide ribonucleic acid sequence comprises the nucleotide sequence of any one of SEQ ID NOs: 4378, 4394, or 4404.
182 . The method of claim 165 , further comprising contacting said TRAC locus with a single-stranded or a double-stranded deoxyribonucleic acid repair template.
183 . The method of claim 165 , wherein said cell is a eukaryotic cell, a T-cell, a hematopoietic stem cell, or a precursor thereof.
184 . The method of claim 165 , further comprising introducing to said cell a donor nucleic acid comprising a cargo sequence flanked on a 3′ or 5′ end by sequence having at least 80% identity to any one of SEQ ID NOs: 4424 or 4425.