Cas variants for gene editing
AssigneePresident and Fellows of Harvard College
InventorsDavid R. Liu, Alexis Christine Komor
Life of this patent
Estimating the term…
Abstract
Some aspects of this disclosure provide strategies, systems, reagents, methods, and kits that are useful for the targeted editing of nucleic acids, including editing a single site within the genome of a cell or subject, e.g., within the human genome. In some embodiments, fusion proteins of Cas9 and nucleic acid editing enzymes or enzyme domains, e.g., deaminase domains, are provided. In some embodiments, methods for targeted nucleic acid editing are provided. In some embodiments, reagents and kits for the generation of targeted nucleic acid editing proteins, e.g., fusion proteins of Cas9 and nucleic acid editing enzymes or domains, are provided.
Claims44
A fusion protein comprising, in order, from N- to C-terminus:
- (i) a catalytic deaminase domain of an apolipoprotein B mRNA-editing complex (APOBEC) family deaminase comprising an amino acid sequence that is at least 95% identical to SEQ ID NO:
- 10, SEQ ID NO:
- 11, SEQ ID NO:
- 12, SEQ ID NO:
- 13, SEQ ID NO:
- 14, SEQ ID NO:
- 15, SEQ ID NO:
- 16, SEQ ID NO:
- 17, SEQ ID NO:
- 18, SEQ ID NO:
- 19, SEQ ID NO:
- 20, SEQ ID NO:
- 21, SEQ ID NO:
- 22, SEQ ID NO:
- 23, or SEQ ID NO:
- 24;
- and
- (ii) a nuclease-inactive Clustered Regulatory Interspaced Short Palindromic Repeat-associated protein 9 (dCas9) domain, wherein the dCas9 domain comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:
- 2,
- wherein the catalytic deaminase domain of (i) and the dCas9 domain of (ii) are fused via a linker comprising the amino acid sequence SGSETPGTSESATPES (SEQ ID NO:
- 93).
A fusion protein comprising, in order, from N- to C-terminus:
- (i) a catalytic deaminase domain of an apolipoprotein B mRNA-editing complex (APOBEC) family deaminase comprising an amino acid sequence that is at least 95% identical to SEQ ID NO:
- 10, SEQ ID NO:
- 11, SEQ ID NO:
- 12, SEQ ID NO:
- 13, SEQ ID NO:
- 14, SEQ ID NO:
- 15, SEQ ID NO:
- 16, SEQ ID NO:
- 17, SEQ ID NO:
- 18, SEQ ID NO:
- 19, SEQ ID NO:
- 20, SEQ ID NO:
- 21, SEQ ID NO:
- 22, SEQ ID NO:
- 23, or SEQ ID NO:
- 24;
- and
- (ii) a nuclease-inactive Clustered Regulatory Interspaced Short Palindromic Repeat-associated protein 9 (dCas9) domain, wherein the dCas9 domain comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:
- 37,
- wherein the catalytic deaminase domain of (i) and the dCas9 domain of (ii) are fused via a linker comprising the amino acid sequence SGSETPGTSESATPES (SEQ ID NO:
- 93).
Dependencies are inferred from the claim text.