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 - 103 . (canceled)
104 . An engineered nuclease system comprising:
(a) an endonuclease comprising a RuvC_III domain and an HNH domain, wherein said endonuclease is derived from an uncultivated microorganism, and wherein said endonuclease is a class 2, type II Cas endonuclease; and
(b) an engineered guide ribonucleic acid structure configured to form a complex with said endonuclease comprising:
i. a guide ribonucleic acid sequence configured to hybridize to a target deoxyribonucleic acid sequence; and
ii. a tracr ribonucleic acid sequence configured to bind to said endonuclease.
105 . The engineered nuclease system of claim 104 , wherein said RuvC_III domain comprises a sequence with at least 80% sequence identity to any one of SEQ ID NOs: 45-88 or SEQ ID NOs: 246-262.
106 . The engineered nuclease system of claim 104 , wherein said endonuclease is not a Cas9 endonuclease, a Cas14 endonuclease, a Cas12a endonuclease, a Cas12b endonuclease, a Cas 12c endonuclease, a Cas12d endonuclease, a Cas12e endonuclease, a Cas13a endonuclease, a Cas13b endonuclease, a Cas13c endonuclease, or a Cas 13d endonuclease.
107 . The engineered nuclease system of claim 104 , wherein said endonuclease has less than 80% identity to a Cas9 endonuclease.
108 . The engineered nuclease system of claim 104 , wherein said HNH domain further comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 89-132 or SEQ ID NOs: 263-279.
109 . The engineered nuclease system of claim 104 , wherein said tracr ribonucleic acid sequence comprises a sequence with at least 80% sequence identity to about 60 to 90 consecutive nucleotides selected from any one of SEQ ID NOs: 138-148 or SEQ ID NO: 280.
110 . The engineered nuclease system of claim 104 , wherein said endonuclease is configured to bind to a protospacer adjacent motif (PAM) sequence selected from the group comprising SEQ ID NOs: 149-158.
111 . The engineered nuclease system of claim 104 , wherein said engineered guide ribonucleic acid structure comprises one ribonucleic acid polynucleotide comprising said guide ribonucleic acid sequence and said tracr ribonucleic acid sequence.
112 . The engineered nuclease system of claim 104 , wherein said guide ribonucleic acid sequence is complementary to a eukaryotic, fungal, plant, mammalian, or human genomic sequence.
113 . The engineered nuclease system of claim 104 , wherein said guide ribonucleic acid sequence is 15-23 nucleotides in length.
114 . The engineered nuclease system of claim 104 , wherein said endonuclease comprises one or more nuclear localization sequences (NLSs) proximal to an N- or C-terminus of said endonuclease.
115 . The engineered nuclease system of claim 104 , further comprising
(c) a single- or double-stranded DNA 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 DNA sequence of at least 10 nucleotides, and a second homology arm comprising a sequence of at least 20 nucleotides 3′ to said target sequence.
116 . The engineered nuclease system of claim 104 , wherein said endonuclease comprises SEQ ID NOs: 1-44 or SEQ ID NOs: 229-245 or a variant thereof having at least 80% identity thereto.
117 . The engineered nuclease system of claim 104 , wherein said endonuclease comprises at least one peptide motif selected from the group consisting of SEQ ID NO: 223-225.
118 . The engineered nuclease system of claim 105 , wherein said sequence identity is determined by a 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.
119 . A nucleic acid comprising an engineered nucleic acid sequence optimized for expression in an organism, wherein said nucleic acid encodes an endonuclease comprising a RuvC_III domain and an HNH domain, wherein said endonuclease is a class 2, type II Cas endonuclease, and wherein said endonuclease is derived from an uncultivated microorganism.
120 . The nucleic acid of claim 119 , wherein said RuvC_III domain has at least 70% sequence identity to any one of SEQ ID NOs: 45-88 or SEQ ID NOs: 246-262.
121 . The nucleic acid of claim 119 , wherein said HNH domain has at least 80% sequence identity to any one of SEQ ID NOs: 89-132 or SEQ ID NOs 263-279.
122 . A vector comprising a nucleic acid sequence encoding a class 2, type II Cas endonuclease comprising a RuvC_III domain and an HNH domain, wherein said endonuclease is derived from an uncultivated microorganism.
123 . A method for binding, cleaving, marking, or modifying a double-stranded deoxyribonucleic acid polynucleotide, comprising:
(a) contacting said double-stranded deoxyribonucleic acid polynucleotide with a class 2, type II Cas endonuclease in complex with an engineered guide ribonucleic acid structure configured to bind to said endonuclease and said double-stranded deoxyribonucleic acid polynucleotide;
(b) wherein said double-stranded deoxyribonucleic acid polynucleotide comprises a protospacer adjacent motif (PAM); and
(c) wherein said PAM comprises a sequence selected from the group consisting of SEQ ID NOs: 149-158.