Methods and compositions for directed genome editing
AssigneeRewrite Therapeutics, Inc.
InventorSchaked Omer Halperin
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Abstract
Provided herein are compositions and methods for increasing editing efficiency of a target nucleic acid. A composition may comprise a guide nucleic acid, a Cas9 nickase, or a reverse transcriptase. The reverse transcriptase may be fused to the Cas9 nickase. The reverse transcriptase may heterodimerize with the Cas9 nickase. The reverse transcriptase may bind to a guide nucleic acid. The reverse transcriptase may be engineered to increase processivity. The guide nucleic acid may be engineered to facilitate synthesis or editing of a sequence. The guide nucleic acid, Cas9 nickase, and reverse transcriptase may be engineered to fit within AAV vectors. The guide nucleic acid may comprise a region that binds to another region on the guide nucleic acid to improve gene editing.
Claims28
A composition comprising a Cas 9 nickase and a reverse transcriptase, or one or two polynucleotides encoding the Cas 9 nickase and reverse transcriptase, wherein:
- (i) the composition comprises a first polypeptide chain comprising the Cas nickase or a segment of the Cas nickase, and a second polypeptide chain comprising the reverse transcriptase, or the one or two polynucleotides encoding the polypeptide chains, wherein the polypeptide chains comprise leucine zippers that bind one another, or
- (ii) the composition comprises a first polypeptide chain comprising a first segment of the Cas nickase, and a second polypeptide chain comprising a second segment of the Cas nickase and the reverse transcriptase, or the one or two polynucleotides encoding the polypeptide chains, wherein the polypeptide chains comprise inteins that bind one another, the Cas nickase comprises an amino acid sequence at least 80% identical to SEQ ID NO:
- 32, the first and second polypeptide chains respectively comprise amino acids 1-1124 and 1125-1368 of the Cas nickase, 1-1129 and 1130-1368 of the Cas nickase, 1-1139 and 1140-1368 of the Cas nickase, 1-1167 and 1168-1368 of the Cas nickase, 1-1172 and 1173-1368 of the Cas nickase, or 1-1202 and 1203-1368 of the Cas nickase, and the Cas nickase comprises a mutation at amino acid position 1030 or after amino acid position 1030 with regard to SEQ ID NO:
- 32, the mutation comprising a point mutation to a cysteine, threonine, alanine, or serine, or an insertion of a cysteine, threonine, alanine, or serine at the C-terminal half of the Cas9 nickase or
- (iii) the reverse transcriptase comprises a Moloney leukemia virus reverse transcriptase (mlvRT) comprising an amino acid sequence at least 80% identical to SEQ ID NO:
- 13 or at least 80% identical to a functional fragment thereof comprising at least 400 amino acids, and a point mutation at amino acid position Q84, L139, Q221, V223, T664, or L671 with regard to SEQ ID NO:
- 13.
Dependencies are inferred from the claim text.