Fusions of Cas9 domains and nucleic acid-editing domains
AssigneePresident and Fellows of Harvard College
InventorsDavid R. Liu, Alexis Christine Komor
Life of this patent
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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.
Claims15
A complex comprising a fusion protein and a single guide RNA (sgRNA), wherein the complex deaminates a target cytosine (C) in a target DNA molecule, the fusion protein comprising, in order, from N- to C-terminus:
- (i) a catalytic deaminase domain of an apolipoprotein B mRNA-editing complex 1 (APOBEC1) deaminase;
- and
- (ii) a Clustered Regularly Interspaced Short Palindromic Repeat-associated protein 9 (Cas9) domain, comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:
- 37, wherein the Cas9 domain of (ii) comprises a first mutation corresponding to the D10A mutation in the RuvC subdomain of SEQ ID NO:
- 37 and a second mutation corresponding to the H840A mutation in the HNH subdomain of SEQ ID NO:
- 37;
- and
- wherein the Cas9 domain is in a complex with a single guide RNA (sgRNA) that comprises a guide sequence complementary to one strand of the target DNA molecule, and the target C is located on the strand of the target DNA molecule that is not complementary to the guide sequence of the sgRNA.
A complex comprising a fusion protein and a single guide RNA (sgRNA), wherein the complex deaminates a target cytosine (C) in a target DNA molecule, the fusion protein comprising, in order, from N- to C-terminus:
- (i) a catalytic deaminase domain of an apolipoprotein B mRNA-editing complex 1 (APOBEC1) deaminase;
- and
- (ii) a Clustered Regularly Interspaced Short Palindromic Repeat-associated protein 9 (Cas9) domain comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:
- 2, wherein the Cas9 domain of (ii) comprises a first mutation corresponding to a D10A mutation in the RuvC subdomain of SEQ ID NO:
- 2 and a second mutation corresponding to the H839A mutation in the HNH subdomain of SEQ ID NO:
- 2;
- and
- wherein the Cas9 domain is in a complex with a single guide RNA (sgRNA) that comprises a guide sequence complementary to one strand of the target DNA molecule, and the target C is located on the strand of the target DNA molecule that is not complementary to the guide sequence of the sgRNA.
Dependencies are inferred from the claim text.