Cas9-FokI fusion proteins and uses thereof
AssigneePresident and Fellows of Harvard College
InventorsDavid R. Liu, John Paul Guilinger, David B. Thompson
Life of this patent
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Abstract
Some aspects of this disclosure provide compositions, methods, and kits for improving the specificity of RNA-programmable endonucleases, such as Cas9. Also provided are variants of Cas9, e.g., Cas9 dimers and fusion proteins, engineered to have improved specificity for cleaving nucleic acid targets. Also provided are compositions, methods, and kits for site-specific nucleic acid modification using Cas9 fusion proteins (e.g., nuclease-inactivated Cas9 fused to a nuclease catalytic domain). Such Cas9 variants are useful in clinical and research settings involving site-specific modification of DNA, for example, genomic modifications.
Claims10
A fusion protein comprising two domains:
- (i) a nuclease-inactivated Cas9 (dCas9) domain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:
- 5, wherein the dCas9 comprises a D10A and/or a H840A mutation and lacks nuclease activity;
- and
- (ii) a nuclease domain that is at least 90% identical to SEQ ID NO:
- 6,
- wherein the two domains are separated by a peptide linker selected from the group consisting of (GGS)3, (GGS)6, SGSETPGTSESATPES (SEQ ID NO:16), SGSETPGTSESA (SEQ ID NO:17), SGSETPGTSESATPEGGSGGS (SEQ ID NO:18), VPFLLEPDNINGKTC (SEQ ID NO:19), GSAGSAAGSGEF (SEQ ID NO:20), SIVAQLSRPDPA (SEQ ID NO:21), MKIIEQLPSA (SEQ ID NO:22), VRHKLKRVGS (SEQ ID NO:23), GHGTGSTGSGSS (SEQ ID NO:24), MSRPDPA (SEQ ID NO:25), and GGSM (SEQ ID NO:301).
Dependencies are inferred from the claim text.