ENZYMES WITH RUVC DOMAINS
The present disclosure provides for endonuclease enzymes having distinguishing domain features, as well as methods of using such enzymes or variants thereof.
1 - 104 . (canceled)
105 . A method of editing a TRACT locus in a cell, comprising contacting to said cell:
(a) a ribonucleic acid- (RNA-) guided endonuclease or a nucleic acid encoding said RNA-guided endonuclease; and
(b) an engineered guide RNA or a nucleic acid encoding said engineered guide RNA, wherein said engineered guide RNA is configured to form a complex with said RNA-guided endonuclease and said engineered guide RNA comprises a spacer sequence configured to hybridize to a region of said TRAC locus,
wherein said spacer sequence comprises a sequence having at least 85% sequence identity to at least 18 consecutive nucleotides of any one of SEQ ID NOs: 5950-5965.
106 . The method of claim 105 , wherein said RNA-guided endonuclease is a class II, type II Cas endonuclease.
107 . The method of claim 105 , wherein said RNA-guided endonuclease comprises a sequence having at least 75% sequence identity to any one of SEQ ID NOs: 1-1826, 5718-5846, or 6257.
108 . The method of claim 105 , wherein said RNA-guided endonuclease comprises a RuvCIII domain comprising a sequence having at least 75% sequence identity to any one of SEQ ID NOs: 2242, 2244, 1827-2241, 2243, or 2245-3637.
109 . The method of claim 105 , wherein said RNA-guided endonuclease further comprises an HNH domain.
110 . The method of claim 109 , wherein said HNH domain comprises a sequence with at least 80% sequence identity to any one of SEQ ID NOs: 3638-5460.
111 . The method of claim 105 , wherein said engineered guide RNA comprises a sequence configured to bind said RNA-guided endonuclease.
112 . The method of claim 111 , wherein said sequence configured to bind said RNA-guided endonuclease comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 5461-5511, 5862-5886, 5888-5890, 5892-5893, 5895-5896, or 6279-6301.
113 . The method of claim 105 , wherein said cell is a human cell.
114 . The method of claim 105 , wherein said cell is a peripheral blood mononuclear cell (PBMC), a T-cell, a natural killer (NK) cell, a hematopoietic stem cell (HSCT), a B-cell, or any combination thereof.
115 . The method of claim 105 , wherein said contacting further comprises transfecting said cell with said nucleic acid encoding said RNA-guided endonuclease and said nucleic acid encoding said engineered guide RNA.
116 . The method of claim 105 , wherein said spacer sequence comprises a sequence having at least 85% sequence identity to 18-22 consecutive nucleotides of any one of SEQ ID NOs: 5950-5965.
117 . The method of claim 105 , wherein said spacer sequence comprises a sequence having at least 90% sequence identity to at least 18 consecutive nucleotides of any one of SEQ ID NOs: 5950-5965.
118 . The method of claim 105 , further comprising contacting to said cell with a deoxyribonucleic acid (DNA) repair template comprising from 5′ to 3′: a first homology arm comprising a sequence of at least 20 nucleotides 5′ to said region of said TRACT locus, a synthetic DNA sequence of at least 10 nucleotides, and a second homology arm comprising a sequence of at least 20 nucleotides 3′ to said region of said TRAC locus.
119 . The method of claim 118 , wherein said cell is a PBMC or a T-cell.
120 . The method of claim 118 , wherein said contacting to said cell further comprises transfecting said cell with said nucleic acid encoding said RNA-guided endonuclease and said nucleic acid encoding said engineered guide RNA.
121 . The method of claim 118 , wherein said DNA repair template is encoded by a vector.
122 . The method of claim 121 , wherein said vector is a plasmid, a minicircle, a CELiD, or a viral vector.
123 . The method of claim 122 , wherein said vector is a viral vector, wherein said viral vector is an adeno-associated virus (AAV) derived virion or a lentivirus.
124 . The method of claim 108 , wherein said RuvCIII domain comprises a sequence having at least 75% sequence identity to SEQ ID NO: 2242 or SEQ ID NO: 2244.
125 . The method of claim 124 , wherein said RuvCIII domain comprises a sequence having at least 90% sequence identity to SEQ ID NO: 2242 or SEQ ID NO: 2244.
126 . The method of claim 125 , wherein said RuvCIII domain comprises a sequence of SEQ ID NO: 2242 or SEQ ID NO: 2244.
127 . The method of claim 110 , wherein said HNH domain comprises a sequence having at least 80% sequence identity to SEQ ID NO: 4061 or SEQ ID NO: 4063.
128 . The method of claim 107 , wherein said RNA-guided endonuclease comprises a sequence having at least 75% sequence identity to SEQ ID NO: 421 or SEQ ID NO: 423.
129 . The method of claim 128 , wherein said RNA-guided endonuclease comprises a sequence having at least 90% sequence identity to SEQ ID NO: 421 or SEQ ID NO: 423.
130 . The method of claim 129 , wherein said RNA-guided endonuclease comprises a sequence of SEQ ID NO: 421 or SEQ ID NO: 423.
131 . The method of claim 130 , wherein said spacer sequence comprises a sequence having at least 18 consecutive nucleotides of any one of SEQ ID NOs: 5950-5965.
132 . The method of claim 131 , wherein said spacer sequence comprises a sequence having at least 18 consecutive nucleotides of any one of SEQ ID NOs: 5955 or 5963.