ENDONUCLEASE SYSTEMS
The present disclosure provides for endonuclease enzymes as well as methods of using such enzymes or variants thereof.
1 - 81 . (canceled)
82 . An engineered nuclease system comprising:
a) an endonuclease comprising a RuvC domain and an HNH domain; and
b) an engineered guide ribonucleic acid structure capable of forming a complex with said endonuclease comprising:
i) a guide ribonucleic acid sequence capable of hybridizing to a target deoxyribonucleic acid sequence; and
ii) a ribonucleic acid sequence capable of binding to said endonuclease, wherein said endonuclease comprises a sequence having at least 70% sequence identity to any one of SEQ ID NOs: 1 or 19-28.
83 . The engineered nuclease system of claim 82 , wherein said endonuclease is an archaeal endonuclease.
84 . The engineered nuclease system of claim 82 , wherein said endonuclease is a class 2, type II Cas endonuclease.
85 . The engineered nuclease system of claim 82 , 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.
86 . The engineered nuclease system of claim 85 , 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, or 19-28.
87 . The engineered nuclease system of claim 82 , wherein said endonuclease comprises a sequence having less than 80% sequence identity to a SpCas9 endonuclease.
88 . The engineered nuclease system of claim 82 , wherein said endonuclease comprises one or more nuclear localization sequences (NLSs) proximal to an N-terminus or a C-terminus of said endonuclease.
89 . The engineered nuclease system of claim 88 , wherein said NLS comprises a sequence selected from any one of SEQ ID NOs: 2-18.
90 . The engineered nuclease system of claim 82 , wherein said ribonucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 29 or 30.
91 . The engineered nuclease system of claim 82 , wherein said sequence identity is determined by aBLASTP 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.
92 . The engineered nuclease system of claim 82 , further comprising a single- or double-stranded deoxyribonucleic acid repair template comprising from 5′ to 3′: a first homology arm comprising a sequence of at least 20 nucleotides 5′ to said target deoxyribonucleic acid sequence, a synthetic deoxyribonucleic acid sequence of at least 10 nucleotides, and a second homology arm comprising a sequence of at least 20 nucleotides 3′ to said target deoxyribonucleic acid sequence.
93 . The engineered nuclease system of claim 82 , wherein said guide ribonucleic acid sequence is complementary to a eukaryotic, a fungal, a plant, a mammalian, or a human genomic sequence.
94 . The engineered nuclease system of claim 82 , wherein said guide ribonucleic acid sequence is 15-24 nucleotides in length.
95 . The engineered nuclease system of claim 82 , wherein said engineered guide ribonucleic acid structure comprises a single ribonucleic acid polynucleotide comprising said guide ribonucleic acid sequence and said ribonucleic acid sequence.
96 . 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 any one of SEQ ID NOs: 1 or 19-28; 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, wherein said complex modifies said target nucleic acid locus.
97 . The method of claim 96 , wherein said ribonucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 29 or 30.
98 . The method of claim 96 , wherein said modifying comprises binding, nicking, cleaving, or marking said target nucleic acid locus.
99 . The method of claim 96 , wherein said target nucleic acid locus comprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).
100 . The method of claim 96 , wherein said target nucleic acid locus is within a cell.
101 . The method of claim 100 , wherein said cell is a eukaryotic cell, an animal cell, a mammalian cell, a rodent cell, a primate cell, or a human cell.