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 . An engineered nucleic acid editing polypeptide, comprising an adenosine deaminase, wherein said adenosine deaminase comprises a sequence with at least 60% sequence identity to at least 20 consecutive amino acids of SEQ ID NO: 50.
135 . The engineered nucleic acid editing polypeptide of claim 134 , further comprising 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, and wherein said endonuclease is configured to be deficient in nuclease activity.
136 . The engineered nucleic acid editing polypeptide of claim 135 , wherein said RuvC domain lacks nuclease activity.
137 . The engineered nucleic acid editing polypeptide of claim 135 , wherein said Class 2, type II endonuclease comprises a nickase mutation.
138 . The engineered nucleic acid editing polypeptide of claim 135 , wherein said Class 2, type II endonuclease is configured to be catalytically dead.
139 . The engineered nucleic acid editing polypeptide of claim 135 , wherein said Class 2, type II Cas endonuclease comprises an aspartate to alanine mutation at residue 9 relative to SEQ ID NO: 70, residue 13 relative to SEQ ID NOs: 71, 72, or 74, residue 12 relative to SEQ ID NO:
73, residue 17 relative to SEQ ID NO: 75, residue 23 relative to SEQ ID NO: 76, or residue 10 relative to SEQ ID NO: 597 when optimally aligned.
140 . The engineered nucleic acid editing polypeptide of claim 135 , further comprising a uracil DNA glycosylase inhibitor (UGI) coupled to said Class 2, type II endonuclease or said adenosine deaminase.
141 . The engineered nucleic acid editing polypeptide of claim 140 , wherein said uracil DNA glycosylase inhibitor comprises a sequence with at least 70%, 80%, 90% or 95% identity to any one of SEQ ID NOs: 52-56 or SEQ ID NO: 67 or a variant thereof.
142 . The engineered nucleic acid editing polypeptide of claim 134 , further comprising an endonuclease comprising a RuvC domain, wherein said endonuclease is derived from an uncultivated microorganism, wherein said endonuclease is a Class 2, type V Cas endonuclease, and wherein said endonuclease is configured to be deficient in nuclease activity.
143 . The engineered nucleic acid editing polypeptide of claim 142 , wherein said RuvC domain lacks nuclease activity.
144 . The engineered nucleic acid editing polypeptide of claim 142 , wherein said Class 2, type V endonuclease comprises a nickase mutation.
145 . The engineered nucleic acid editing polypeptide of claim 142 , further comprising a uracil DNA glycosylase inhibitor (UGI) coupled to said Class 2, type V endonuclease or said adenosine deaminase.
146 . The engineered nucleic acid editing polypeptide of claim 145 , wherein said uracil DNA glycosylase inhibitor comprises a sequence with at least 70%, 80%, 90% or 95% identity to any one of SEQ ID NOs: 52-56 or SEQ ID NO: 67 or a variant thereof.
147 . 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 is configured to comprise a mutation of a RuvC domain residue such that said RuvC domain of said endonuclease is deficient in nuclease activity; and
(b) a base editor coupled to said endonuclease, wherein said base editor comprises a sequence having at least 60% identity to at least 20 consecutive amino acids of SEQ ID NO: 50; and
(c) 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 portion comprising a ribonucleic acid sequence, wherein said ribonucleic acid sequence is configured such that said guide portion hybridizes to a target deoxyribonucleic acid sequence; and
(ii) a non-guide portion with a sequence configured such that said non-guide portion binds said endonuclease.
148 . The engineered nucleic acid editing system of claim 147 , wherein said Class 2, type II Cas endonuclease comprises a nickase mutation.
149 . The engineered nucleic acid editing polypeptide of claim 147 , wherein said Class 2, type II Cas endonuclease is configured to be catalytically dead.
150 . The engineered nucleic acid editing system of claim 147 , wherein said Class 2, type II Cas endonuclease comprises an aspartate to alanine mutation at residue 9 relative to SEQ ID NO: 70, residue 13 relative to SEQ ID NOs: 71, 72, or 74, residue 12 relative to SEQ ID NO: 73, residue 17 relative to SEQ ID NO: 75, residue 23 relative to SEQ ID NO: 76, or residue 10 relative to SEQ ID NO: 597 when optimally aligned.
151 . An engineered nucleic acid editing system, comprising:
(a) an endonuclease comprising a RuvC domain, wherein said endonuclease is derived from an uncultivated microorganism, wherein said endonuclease is a Class 2, type V Cas endonuclease, wherein said endonuclease is configured to comprises a mutation of a RuvC domain residue such that said RuvC domain of said endonuclease is deficient in nuclease activity; and
(b) a base editor coupled to said endonuclease, wherein said base editor comprises a sequence having at least 60% identity to at least 20 consecutive amino acids of SEQ ID NO: 50; and
(c) 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 portion comprising a ribonucleic acid sequence, wherein said ribonucleic acid sequence is configured such that said guide portion hybridizes to a target deoxyribonucleic acid sequence; and
(ii) a non-guide portion with a sequence configured such that said non-guide portion binds said endonuclease.
152 . The engineered nucleic acid editing system of claim 151 , wherein said RuvC domain lacks nuclease activity.
153 . The engineered nucleic acid editing system of claim 151 , wherein said Class 2, type V Cas endonuclease comprises a nickase mutation.