CLASS II, TYPE II CRISPR SYSTEMS
The present disclosure provides for endonuclease enzymes as well as methods of using such enzymes or variants thereof.
1 - 113 . (canceled)
114 . A method of modifying a target nucleic acid locus, said method comprising contacting said target nucleic acid locus with:
a) an endonuclease comprising a RuvC domain and an HNH domain, wherein said endonuclease comprises a sequence having at least 70% sequence identity to SEQ ID NO: 2; and
b) an engineered guide ribonucleic acid structure configured to form a complex with said endonuclease, wherein said engineered guide ribonucleic acid structure comprises:
i) a guide ribonucleic acid sequence configured to hybridize to a portion of said target nucleic acid locus; and
ii) a ribonucleic acid sequence configured to bind to said endonuclease comprising a sequence having at least 80% sequence identity to nonvariable nucleotides of any one of SEQ ID NOs: 203, 202, 613, or 614,
wherein said complex modifies said target nucleic acid locus.
115 . The method of claim 114 , wherein said endonuclease is an archaeal endonuclease.
116 . The method of claim 114 , wherein said endonuclease is a class 2, type II Cas endonuclease.
117 . The method of claim 114 , wherein said endonuclease further comprises one or more of: an arginine-rich region comprising an RRxRR motif, a domain with PF14239 homology, a recognition (REC) domain, a bridge helix (BH) domain, a wedge (WED) domain, or a PAM interacting (PI) domain.
118 . The method of claim 117 , wherein said arginine-rich region, said domain with PF14239 homology, said recognition (REC) domain, said bridge helix (BH) domain, said wedge (WED) domain, or said PAM interacting (PI) domain comprises a sequence having at least 85% sequence identity to an arginine-rich region comprising an RRxRR motif, a domain with PF14239 homology, a recognition (REC) domain, a bridge helix (BH) domain, a wedge (WED) domain, or a PAM interacting (PI) domain, respectively, of any one of SEQ ID NOs: 1-198, 221-459, 463-612, or 617-668.
119 . The method of claim 114 , wherein said endonuclease comprises one or more nuclear localization sequences (NLSs) proximal to an N-terminus or a C-terminus of said endonuclease.
120 . The method of claim 114 , wherein said endonuclease comprises a sequence having at least 90% sequence identity to SEQ ID NO: 2.
121 . The method of claim 120 , wherein said endonuclease comprises a sequence of SEQ ID NO: 2.
122 . The method of claim 114 , wherein said endonuclease comprises a sequence having less than 80% sequence identity to a SpCas9 endonuclease.
123 . The method of claim 114 , wherein said guide ribonucleic acid sequence is complementary to a eukaryotic, a fungal, a plant, a mammalian, or a human genomic sequence.
124 . The method of claim 114 , wherein said guide ribonucleic acid sequence is 15-24 nucleotides in length.
125 . The method of claim 114 , wherein said ribonucleic acid sequence configured to bind to said endonuclease comprises a sequence having at least 90% sequence identity to nonvariable nucleotides of any one of SEQ ID NOs: 203, 202, 613, or 614
126 . The method of claim 125 , wherein said ribonucleic acid sequence configured to bind to said endonuclease comprises a sequence having at least 95% sequence identity to nucleotides 23-157 of SEQ ID NO: 203, nucleotides 23-93 of SEQ ID NO: 202, nucleotides 23-145 of SEQ ID NO: 613, or nucleotides 23-157 of SEQ ID NO: 614.
127 . The method of claim 126 , wherein said ribonucleic acid sequence configured to bind to said endonuclease comprises a sequence having nucleotides 23-157 of SEQ ID NO: 203, nucleotides 23-93 of SEQ ID NO: 202, nucleotides 23-145 of SEQ ID NO: 613, or nucleotides 23-157 of SEQ ID NO: 614.
128 . The method of claim 114 , wherein said endonuclease and said ribonucleic acid sequence configured to bind to said endonuclease are derived from distinct bacterial species within a same phylum.
129 . The method of claim 114 , wherein said engineered guide ribonucleic acid structure comprises any one of SEQ ID NOs: 203, 202, 613, or 614.
130 . The method of claim 121 , wherein said engineered guide ribonucleic acid structure comprises any one of SEQ ID NOs: 203, 202, 613, or 614.
131 . The method of claim 114 , wherein said engineered guide ribonucleic acid structure comprises a single ribonucleic acid polynucleotide comprising said guide ribonucleic acid sequence and said ribonucleic acid sequence configured to bind to said endonuclease.
132 . The method of claim 114 , wherein said sequence identity is determined by said BLASTP homology search algorithm using parameters of a wordlength (W) of 3, an expectation (E) of 10, and a BLOSUM62 scoring matrix setting gap costs at existence of 11, extension of 1, and using a conditional compositional score matrix adjustment.
133 . The method of claim 114 , further comprising contacting said target nucleic acid locus with a single- or double-stranded deoxyribonucleic acid repair template comprising from 5′ to 3′: a first homology arm comprising a sequence 5′ to said target nucleic acid locus, a synthetic deoxyribonucleic acid sequence, and a second homology arm comprising a sequence 3′ to said target nucleic acid locus.
134 . The method of claim 114 , wherein said modifying comprises binding, nicking, cleaving, or marking said target nucleic acid locus.
135 . The method of claim 114 , wherein said target nucleic acid locus comprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).
136 . The method of claim 114 , wherein said target nucleic acid locus is within a cell.
137 . The method of claim 136 , wherein said cell is a eukaryotic cell, an animal cell, a mammalian cell, a rodent cell, a primate cell, or a human cell.