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 guide nucleic acid comprising:
- optionally, a spacer reverse complementary to a first region of a target nucleic acid, wherein the spacer is included in the guide nucleic acid, or the spacer is included in a second, different guide nucleic acid when not included in the guide nucleic acid;
- a scaffold configured to bind to a Cas nuclease;
- a reverse transcriptase template encoding a sequence to be reverse transcribed into a first synthesized strand to be inserted into the target nucleic acid;
- a first strand primer binding site reverse complementary to a second region of the target nucleic acid;
- and
- at least one of:
- (i) a guide nucleic acid positioning system (GPS) region and a GPS binding site that hybridizes to the GPS region, wherein the GPS region and the GPS binding site are at least 10 nucleotides in length and are at least 60% reverse complementary to each other, and wherein hybridization of the GPS region and the GPS binding site positions the first strand primer binding site closer to the second region of the target nucleic acid,
- (ii) a GPS region that hybridizes to a GPS binding site on the second guide nucleic acid, wherein the GPS region and the GPS binding site are at least 10 nucleotides in length and are at least 60% reverse complementary to each other, wherein the second region of the target nucleic acid does not include any part of the first region of the target nucleic acid, and wherein the second region of the target nucleic acid does not include any part of a reverse complement of the first region of the target nucleic acid, and wherein hybridization of the GPS region and the GPS binding site positions the first strand primer binding site closer to the second region of the target nucleic acid, or
- (iii) a modification in the reverse transcriptase template that disrupts a track of at least 4 consecutive nucleotides of the same base in the target nucleic acid.
Dependencies are inferred from the claim text.