CAS9 proteins including ligand-dependent inteins

Patent metadata

Field	Value
Publication number	US-10077453-B2
Application number	US-201515329925-A
Country	US
Kind code	B2
Filing date	Jul 30, 2015
Priority date	Jul 30, 2014
Publication date	Sep 18, 2018
Grant date	Sep 18, 2018

Abstract

Some aspects of this disclosure provide compositions, methods, systems, and kits for controlling the activity of RNA-programmable endonucleases, such as Cas9, or for controlling the activity of proteins comprising a Cas9 variant fused to a functional effector domain, such as a nuclease, nickase, recombinase, deaminase, transcriptional activator, transcriptional repressor, or epigenetic modifying domain. For example, the inventive proteins provided comprise a ligand-dependent intein, the presence of which inhibits one or more activities of the protein (e.g., gRNA binding, enzymatic activity, target DNA binding). The binding of a ligand to the intein results in self-excision of the intein, restoring the activity of the protein.

First claim

What is claimed is:

1. A Cas9 protein comprising an intein.

2. The Cas9 protein of claim 1 , wherein an activity of the Cas9 protein is disrupted by the intein, and wherein the disrupted activity is restored upon excision of the intein from the Cas9 protein.

3. The Cas9 protein of claim 1 , wherein the Cas9 protein is capable of binding a guide RNA (gRNA) prior to excision of the intein.

4. The Cas9 protein of claim 1 , wherein the Cas9 protein has no or minimal gRNA binding activity prior to excision of the intein.

5. The Cas9 protein of claim 4 , wherein the Cas9 protein is a Cas9 protein comprising three domains: (i) a dCas9 domain; (ii) a ligand-dependent intein; and (iii) a nucleic acid-editing domain.

6. The Cas9 protein of claim 5 , wherein the nucleic acid-editing domain is a deaminase domain.

7. The Cas9 protein of claim 4 , wherein the Cas9 protein comprises: (i) a dCas9 domain; (ii) a ligand-dependent intein; and (iii) a transcriptional activator domain, or a transcriptional repressor domain.

8. The Cas9 protein of claim 4 , wherein the Cas9 protein comprises: (i) a dCas9 domain; (ii) a ligand-dependent intein; and (iii) an epigenetic modifier domain.

9. The Cas9 protein of claim 8 , wherein the epigenetic modifier is selected from the group consisting of histone demethylase, histone methyltransferase, hydroxylase, histone deacetylase, and histone acetyltransferase.

10. The Cas9 fusion protein of claim 9 , wherein the epigenetic modifier comprises the LSD1 histone demethylase or TET1 hydroxylase.

11. The Cas9 protein of claim 1 , wherein the Cas9 protein comprises a nuclease-inactivated Cas9 (dCas9) domain.

12. The Cas9 protein of claim 11 , wherein the Cas9 protein comprises: (i) a dCas9 domain; (ii) a ligand-dependent intein; and (iii) a recombinase catalytic domain.

13. The Cas9 protein of claim 1 , wherein the Cas9 protein is a Cas9 nickase.

14. The Cas9 protein of claim 1 , wherein the intein is a ligand-dependent intein.

15. The Cas9 protein of claim 1 , wherein the intein comprises a ligand-binding domain.

16. The Cas9 protein of claim 1 , wherein one or more domains of the Cas9 protein are separated by a peptide linker or a non-peptide linker.

17. A method for site-specific DNA cleavage comprising:
(a) contacting a Cas9 protein of claim 1 with a ligand, wherein binding of the ligand to the intein induces self-excision of the intein; and
(b) contacting a DNA with the Cas9 protein, wherein the Cas9 protein is associated with a gRNA;
wherein self-excision of the intein from the Cas9 protein in step (a) allows the RNA-guided nuclease to cleave the DNA, thereby producing cleaved DNA.

18. A method of DNA editing, the method comprising contacting a DNA molecule with
(a) a Cas9 protein of claim 5 ; and
(b) a gRNA targeting the Cas9 protein of (a) to a target nucleotide sequence of the DNA strand;
wherein the DNA molecule is contacted with the Cas9 protein and the gRNA in an amount effective and under conditions suitable for the deamination of a nucleotide base.

19. A method for transcriptional activation, comprising contacting a DNA molecule comprising a gene with
(a) a Cas9 protein of claim 7 ; and
(b) a gRNA targeting the Cas9 protein of (a) to a target nucleotide sequence of the DNA strand;
wherein the DNA molecule is contacted with the Cas9 protein and the gRNA in an amount effective and under conditions suitable for the transcriptional activation of the gene.

20. A method for epigenetic modification, comprising contacting a DNA molecule with
(a) a Cas9 protein of claim 8 ; and
(b) a gRNA targeting the Cas9 protein of (a) to a target nucleotide sequence of the DNA strand;

wherein the DNA molecule is contacted with the Cas9 protein and the gRNA in an amount effective and under conditions suitable for the epigenetic modification of the DNA.

21. A method for site-specific recombination between two DNA molecules, comprising:
(a) contacting a first DNA with a first Cas9 protein of claim 12 , wherein the dCas9 domain of the first Cas9 protein binds a first gRNA that hybridizes to a region of the first DNA;
(b) contacting the first DNA with a second Cas9 protein of claim 12 , wherein the dCas9 domain of the second Cas9 protein binds a second gRNA that hybridizes to a second region of the first DNA;
(c) contacting a second DNA with a third Cas9 protein of claim 12 , wherein the dCas9 domain of the third Cas9 protein binds a third gRNA that hybridizes to a region of the second DNA; and
(d) contacting the second DNA with a fourth Cas9 protein of claim 12 , wherein the dCas9 domain of the fourth Cas9 protein binds a fourth gRNA that hybridizes to a second region of the second DNA;
wherein the binding of the Cas9 proteins in steps (a)-(d) results in the tetramerization of the recombinase catalytic domains of the Cas9 proteins, under conditions such that the DNAs are recombined.

Assignees

• Harvard College

Inventors

• Liu David R
• Davis Kevin

Classifications

• C12N9/78

  acting on carbon to nitrogen bonds other than peptide bonds (3.5) · CPC title

• C12N9/0071

  acting on paired donors with incorporation of molecular oxygen (1.14) · CPC title

• C12N15/907 Primary

  in mammalian cells · CPC title

• C07K14/721

  Steroid/thyroid hormone superfamily, e.g. GR, EcR, androgen receptor, oestrogen receptor · CPC title

• C12Y114/11

  with 2-oxoglutarate as one donor, and incorporation of one atom each of oxygen into both donors (1.14.11) · CPC title