BASE EDITING ENZYMES
The present disclosure provides for endonuclease enzymes having distinguishing domain features, as well as methods of using such enzymes or variants thereof.
1 - 133 . (canceled)
134 . A method for modifying a nucleic acid molecule, the method comprising contacting said nucleic acid molecule with an engineered complex comprising a base editor, wherein said base editor comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 386.
135 . The method of claim 134 , wherein said base editor comprises an adenosine deaminase.
136 . The method of claim 135 , wherein said adenosine deaminase comprises an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 50-51, 385-443, or 448-475.
137 . The method of claim 135 , wherein said nucleic acid molecule comprises an adenine, and modifying said nucleic acid molecule comprises converting said adenine to guanine.
138 . The method of claim 135 , wherein said base editor comprises the amino acid sequence of SEQ ID NO: 386.
139 . The method of claim 134 , wherein said engineered complex further comprises an endonuclease comprising a RuvC domain and an HNH domain, wherein said endonuclease is derived from an uncultivated microorganism, wherein said endonuclease is a class 2, type II Cas endonuclease.
140 . The method of claim 139 , wherein said endonuclease comprising said RuvC domain and said HNH domain is covalently coupled directly to said base editor or covalently coupled to said base editor through a linker.
141 . The method of claim 139 , wherein said endonuclease comprises a sequence with at least 80% sequence identity to SEQ ID NO: 597.
142 . The method of claim 141 , wherein said endonuclease comprises an aspartate to alanine mutation at residue 10 relative to SEQ ID NO: 597 when optimally aligned.
143 . The method of claim 139 , wherein said endonuclease comprises a sequence with at least 80% sequence identity to any one of SEQ ID NOs: 70-78.
144 . The method of claim 139 , wherein said endonuclease is configured to be deficient in nuclease activity.
145 . The method of claim 139 , wherein said endonuclease cleaves one strand of said nucleic acid molecule.
146 . The method of claim 139 , wherein said endonuclease comprises a nickase mutation.
147 . The method of claim 139 , wherein said RuvC domain lacks nuclease activity.
148 . The method of claim 139 , wherein said endonuclease is configured to bind a protospacer adjacent motif (PAM) sequence comprising SEQ ID NO: 598.
149 . The method of claim 139 , wherein said endonuclease is catalytically dead.
150 . The method of claim 139 , wherein a full length amino acid sequence of said endonuclease has less than 80% sequence identity to a full length amino acid sequence of an S. pyogenes Cas9 endonuclease.
151 . The method of claim 139 , wherein said endonuclease or said base editor comprises one or more nuclear localization sequences (NLSs).
152 . The method of claim 139 , wherein said engineered complex further comprises an engineered guide ribonucleic acid structure configured to bind to said endonuclease and said nucleic acid molecule.
153 . The method of claim 152 , wherein said engineered guide ribonucleic acid structure comprises a sequence with at least 80% sequence identity to SEQ ID NO: 489.
154 . The method of claim 152 , wherein said engineered guide ribonucleic acid structure comprises (i) a guide portion that hybridizes to said nucleic acid molecule and (ii) a non-guide portion that binds to said endonuclease.
155 . The method of claim 154 , wherein said engineered guide ribonucleic acid structure comprises one ribonucleic acid molecule comprising (i) said guide portion and (ii) said non-guide portion.
156 . The method of claim 154 , wherein said guide portion is 15-24 nucleotides in length.
157 . The method of claim 134 , wherein said engineered complex further comprises a uracil deoxyribonucleic acid glycosylase inhibitor.
158 . The method of claim 157 , wherein said uracil deoxyribonucleic acid glycosylase inhibitor comprises a sequence with at least 70% sequence identity to any one of SEQ ID NOs: 52-56 or SEQ ID NO: 67.
159 . The method of claim 134 , wherein said nucleic acid molecule is a eukaryotic, plant, fungal, mammalian, rodent, or human nucleic acid molecule.
160 . The method of claim 134 , 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.