Cas9-FokI fusion proteins and uses thereof

Patent metadata

Field	Value
Publication number	US-9322037-B2
Application number	US-201414320498-A
Country	US
Kind code	B2
Filing date	Jun 30, 2014
Priority date	Sep 6, 2013
Publication date	Apr 26, 2016
Grant date	Apr 26, 2016

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.

First claim

What is claimed is:

1. 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).

2. The fusion protein of claim 1 , wherein the nuclease domain comprises a FokI DNA cleavage domain consisting essentially of amino acids 300-583, 320-583, 340-583, 360-583, or 388-583 of FokI (MFLSMVSKIRTFGWVQNPGKFENLKRVVQVFDRNSKVHNEVKNIKIPTLVKESKIQKELVA IMNQHDLIYTYKELVGTGTSIRSEAPCDAIIQATIADQGNKKGYIDNWSSDGFLRWAHALGF IEYINKSDSFVITDVGLAYSKSADGSAIEKEILIEAISSYPPAIRILTLLEDGQHLTKFDLGKNL GFSGESGFTSLPEGILLDTLANAMPKDKGEIRNNWEGSSDKYARMIGGWLDKLGLVKQGK KEFIIPTLGKPDNKEFISHAFKITGEGLKVLRRAKGSTKFTRVPKRVYWEMLATNLTDKEYV RTRRALILEILIKAGSLKIEQIQDNLKKLGFDEVIETIENDIKGLINTGIFIEIKGRFYQLKDHIL QFVIPNRGVTKQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMK VYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQT RNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGG EMIKAGTLTLEEVRRKFNNGEINF, SEQ ID NO: 328).

3. The fusion protein of claim 1 , further comprising a nuclear localization signal (NLS) domain.

4. The fusion protein of claim 3 , wherein the fusion protein comprises the structure [NLS domain]-GGS-[FokI DNA cleavage domain]-[peptide linker]-[dCas9 domain].

5. The fusion protein of claim 1 , wherein the amino acid sequence of the peptide linker is selected from the group consisting of SGSETPGTSESATPES (SEQ ID NO:16), SGSETPGTSESA (SEQ ID NO:17), and SGSETPGTSESATPEGGSGGS (SEQ ID NO:18).

6. The fusion protein of claim 1 , wherein the fusion protein is encoded by a nucleotide sequence set forth as SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12.

7. A dimer of the fusion protein of claim 1 .

8. The fusion protein of claim 1 , wherein the nuclease-inactivated Cas9 domain is bound to a gRNA.

9. A kit comprising a fusion protein of claim 1 .

10. The kit of claim 9 , further comprising one or more gRNAs and/or vectors for expressing one or more gRNAs.

Assignees

• Harvard College

Inventors

• Liu David R
• Guilinger John Paul
• Thompson David B

Classifications

• C12N9/1241

  Nucleotidyltransferases (2.7.7) · CPC title

• C07K2319/80

  containing a DNA binding domain, e.g. Lacl or Tet-repressor · CPC title

• A61K38/00

  Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00) · CPC title

• A61K47/00

  Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient · CPC title

• C12N15/907 Primary

  in mammalian cells · CPC title