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 - 138 . (canceled)
139 . An engineered nucleic acid editing system, comprising:
a. 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, wherein said endonuclease comprises an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 23, and wherein said endonuclease is configured to be deficient in nuclease activity;
b. a base editor coupled to said endonuclease; and
c. 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 non-guide ribonucleic acid sequence configured to bind to said endonuclease.
140 . The engineered nucleic acid editing system of claim 139 , wherein said endonuclease comprises an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 23.
141 . The engineered nucleic acid editing system of claim 139 , wherein said endonuclease comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 23.
142 . The engineered nucleic acid editing system of claim 139 , wherein said endonuclease comprises an amino acid sequence of SEQ ID NO: 23.
143 . The engineered nucleic acid editing system of claim 139 , wherein said endonuclease comprises an aspartate to alanine mutation at residue 9 relative to SEQ ID NO: 23 when optimally aligned.
144 . The engineered nucleic acid editing system of claim 139 , wherein said endonuclease comprises a nickase mutation.
145 . The engineered nucleic acid editing system of claim 139 , wherein said RuvC domain lacks nuclease activity.
146 . The engineered nucleic acid editing system of claim 139 , wherein said endonuclease is configured to cleave one strand of a double-stranded target deoxyribonucleic acid.
147 . The engineered nucleic acid editing system of claim 139 , wherein said endonuclease comprises an amino acid sequence comprising less than 80% sequence identity to a Cas9 endonuclease.
148 . The engineered nucleic acid editing system of claim 139 , wherein said non-guide ribonucleic acid sequence comprises a tracr sequence.
149 . The engineered nucleic acid editing system of claim 148 , wherein said tracr sequence comprises at least 80% sequence identity to about 60 to 90 consecutive nucleotides of SEQ ID NO: 29.
150 . The engineered nucleic acid editing system of claim 148 , wherein said tracr sequence comprises at least 80% sequence identity to SEQ ID NO: 29.
151 . The engineered nucleic acid editing system of claim 139 , wherein said endonuclease is configured to bind to a protospacer adjacent motif (PAM) sequence of SEQ ID NO: 147.
152 . The engineered nucleic acid editing system of claim 139 , wherein said base editor comprises a sequence with at least 70% sequence identity to any one of SEQ ID NOs: 1-17.
153 . The engineered nucleic acid editing system of claim 139 , wherein said base editor is an adenosine deaminase.
154 . The engineered nucleic acid editing system of claim 153 , wherein said adenosine deaminase comprises a sequence with at least 80% sequence identity to any one of SEQ ID NOs: 8 or 164.
155 . The engineered nucleic acid editing system of claim 139 , wherein said base editor is a cytosine deaminase.
156 . The engineered nucleic acid editing system of claim 155 , wherein said cytosine deaminase comprises a sequence with at least 80% sequence identity to any one of SEQ ID NOs: 1-7 or 9-17.
157 . The engineered nucleic acid editing system of claim 139 , wherein said guide ribonucleic acid sequence is complementary to a prokaryotic, bacterial, archaeal, eukaryotic, fungal, plant, mammalian, or human genomic sequence.
158 . The engineered nucleic acid editing system of claim 139 , wherein said guide ribonucleic acid sequence is 15-24 nucleotides in length.
159 . The engineered nucleic acid editing system of claim 139 , wherein said endonuclease or said base editor comprises one or more nuclear localization sequences (NLSs).
160 . The engineered nucleic acid editing system of claim 139 , wherein said endonuclease is covalently coupled directly to said base editor or covalently coupled to said base editor through a linker.
161 . The engineered nucleic acid editing system of claim 139 , wherein said sequence identity is determined by a BLASTP, CLUSTALW, MUSCLE, MAFFT, or Smith-Waterman homology search algorithm.
162 . The engineered nucleic acid editing system of claim 139 , wherein said sequence identity is determined by 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.
163 . The engineered nucleic acid editing system of claim 139 , further comprising a uracil deoxyribonucleic acid glycosylase inhibitor.
164 . The engineered nucleic acid editing system of claim 163 , wherein said uracil deoxyribonucleic acid glycosylase inhibitor comprises a sequence with at least 70% sequence identity to SEQ ID NO: 18.