Cas9-recombinase fusion proteins and uses thereof

Patent metadata

Field	Value
Publication number	US-9388430-B2
Application number	US-201414320467-A
Country	US
Kind code	B2
Filing date	Jun 30, 2014
Priority date	Sep 6, 2013
Publication date	Jul 12, 2016
Grant date	Jul 12, 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 recombination, using Cas9 fusion proteins (e.g., nuclease-inactivated Cas9 fused to a recombinase 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 dimer or tetramer of a fusion protein wherein the fusion protein comprises two domains: (i) a nuclease-inactivated Cas9 (dCas9); and (ii) a recombinase catalytic domain;
wherein the nuclease-inactivated Cas9 domain is bound to a guide RNA (gRNA); and
wherein the dimer or the tetramer is bound to a nucleic acid.

2. The dimer or tetramer of a fusion protein of claim 1 , wherein the recombinase catalytic domain is a monomer of the recombinase catalytic domain of Hin recombinase, Gin recombinase, or Tn3 recombinase.

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

4. The dimer or tetramer of a fusion protein of claim 3 , wherein one or more domains of the fusion protein are separated by a peptide linker or a non-peptide linker.

5. The dimer or tetramer of a fusion protein of claim 4 , wherein the peptide linker comprises an XTEN linker, an amino acid sequence comprising one or more repeats of the tri-peptide GGS, or any sequence as provided in FIG. 12A (SEQ ID NOs: 14-25 and 301).

6. The dimer of the fusion protein of claim 1 , wherein each fusion protein of the dimer is bound to opposite strands of the nucleic acid.

7. A kit comprising a dimer or tetramer of a fusion protein of claim 1 .

Assignees

• Harvard College

Inventors

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

Classifications

• C07K2319/80

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

• 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

• C12N9/22 Primary

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title