Base editing enzymes

Abstract

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.