CRISPR-BASED GENOME MODIFICATION AND REGULATION

• US 20160298137A1
• Filed: 06/21/2016
• Published: 10/13/2016
• Est. Priority Date: 12/06/2012
• Status: Abandoned Application

• Current Assignee: Sigma-Aldrich Co. LLC (Merck & Co., Inc.)
• Original Assignee: Sigma-Aldrich Co. LLC (Merck & Co., Inc.)
• Inventors: CHEN, Fuqiang, DAVIS, Gregory D., KANG, Qiaohua, KNIGHT, Scott W.
• Application Number:
  US15/188,931
• Publication Number:
  US 20160298137A1
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: A61K 38/00 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 Class Codes: C07K 14/463 from amphibians
• CPC Class Codes: C07K 2319/09 containing a nuclear localisation signal
• CPC Class Codes: C07K 2319/10 containing a tag for extracellular membrane crossing, e.g. TAT or VP22
• CPC Class Codes: C07K 2319/81 containing a Zn-finger domain for DNA binding
• CPC Class Codes: C07K 7/06 having 5 to 11 amino acids
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 15/11 DNA or RNA fragments; Modified forms thereof DNA or RNA not used in recombinant technology, C07H21/00; Non-coding nucleic acids having a biological activity
• CPC Class Codes: C12N 15/63 Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• CPC Class Codes: C12N 15/67 General methods for enhancing the expression
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 2310/20 involving clustered regularly interspaced short palindromic repeats [CRISPRs]
• CPC Class Codes: C12N 2310/3513 Protein; Peptide
• CPC Class Codes: C12N 2750/14143 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2800/22 Vectors comprising a coding region that has been codon optimised for expression in a respective host
• CPC Class Codes: C12N 2800/80 Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
• CPC Class Codes: C12N 7/00 Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12N 9/96 : Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
• CPC Class Codes: C12Y 301/00 : Hydrolases acting on ester bonds (3.1)
• CPC Class Codes: C12Y 301/21004 : Type II site-specific deoxyribonuclease (3.1.21.4)
• CPC Class Codes: Y02A 50/30 : Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The present invention provides RNA-guided endonucleases, which are engineered for expression in eukaryotic cells or embryos, and methods of using the RNA-guided endonuclease for targeted genome modification in in eukaryotic cells or embryos. Also provided are fusion proteins, wherein each fusion protein comprises a CRISPR/Cas-like protein or fragment thereof and an effector domain. The effector domain can be a cleavage domain, an epigenetic modification domain, a transcriptional activation domain, or a transcriptional repressor domain. Also provided are methods for using the fusion proteins to modify a chromosomal sequence or regulate expression of a chromosomal sequence.

Claims

1. A composition comprising at least two RNA-guided nickase systems, each RNA-guided nickase system comprising (i) a RNA-guided endonuclease that is modified to cleave one strand of a double-stranded sequence and (ii) a guide RNA comprising a first region having complementarity to a target site in a chromosomal sequence and a second region that interacts with the RNA-guided endonuclease, wherein the target sites of two RNA-guided endonucleases are in close proximity but on opposite strands of the chromosomal sequence.

2. The composition of claim 1, wherein each RNA-guided endonuclease is derived from a clustered regularly interspersed short palindromic repeats (CRISPR)/CRISPR-associated (Cas) (CRISPR/Cas) type II system protein.

3. The composition of claim 2, wherein the CRISPR/Cas type II system protein is a Cas9 protein.

4. The composition of claim 3, wherein the Cas9 protein comprises a mutation in the RuvC or HNH domain.

5. The composition of claim 1, wherein each RNA-guided endonuclease is identical.

6. The composition of claim 1, wherein each RNA-guided endonuclease is different.

7. The composition of claim 1, wherein each RNA-guided endonuclease further comprises at least one additional domain chosen from a nuclear localization signal, a cell-penetrating domain, or a marker domain.

8. The composition of claim 1, wherein the target site is a Rosa26 locus, a HPRT locus, or an AAVS1 locus.

9. The composition of claim 1, wherein each target site in the chromosomal sequence is immediately followed by a protospacer adjacent motif (PAM).

10. The composition of claim 1, wherein the guide RNA is at least partially chemically synthesized.

11. A nucleic acid encoding the composition of claim 1.

12. The nucleic acid of claim 11, wherein the nucleic acid encoding each RNA-guided endonuclease is mRNA and the nucleic acid encoding each guide RNA is DNA.

13. The nucleic acid of claim 11, wherein the nucleic acid encoding each RNA-guided endonuclease is DNA and the nucleic acid encoding each guide RNA is DNA.

14. A vector comprising the nucleic acid of claim 13, wherein the nucleic acid encoding each RNA-guided endonuclease is operably linked to a promoter control sequence for expression in a eukaryotic cell, and the nucleic acid encoding each guide RNA is operably linked to a promoter control sequence for expression in a eukaryotic cell.

24. The kit of claim 14, wherein the nucleic acid encoding each RNA-guided endonuclease is DNA and the nucleic acid encoding each guide RNA is DNA.

25. The kit of claim 24, wherein the nucleic acid encoding each RNA-guided endonuclease is operably linked to a promoter control sequence for expression in a eukaryotic cell, and the nucleic acid encoding each guide RNA is operably linked to a promoter control sequence for expression in a eukaryotic cell.

15. A kit comprising at least two RNA-guided nickase systems or nucleic acid encoding said systems, each RNA-guided nickase system comprising (i) a RNA-guided endonuclease that is modified to cleave one strand of a double-stranded sequence and (ii) a guide RNA comprising a first region having complementarity to a target site in a chromosomal sequence and a second region that interacts with the RNA-guided endonuclease, wherein the target sites of two RNA-guided endonucleases are in close proximity but on opposite strands of the chromosomal sequence

16. The kit of claim 15, further comprising at least one donor polynucleotide.

17. The kit of claim 15, wherein each RNA-guided endonuclease is derived from a clustered regularly interspersed short palindromic repeats (CRISPR)/CRISPR-associated (Cas) (CRISP R/Cas) type II system protein.

18. The kit of claim 17, wherein the CRISPR/Cas type II system protein is a Cas9 protein.

19. The kit of claim 18, wherein the Cas9 protein comprises a mutation in the RuvC or HNH domain.

20. The kit of claim 15, wherein each RNA-guided endonuclease is identical.

21. The kit of claim 15, wherein each RNA-guided endonuclease is different.

22. The kit of claim 15, wherein each RNA-guided endonuclease further comprises at least one additional domain chosen from a nuclear localization signal, a cell-penetrating domain, or a marker domain

23. The kit of claim 15, wherein the nucleic acid encoding each RNA-guided endonuclease is mRNA and the nucleic acid encoding each guide RNA is DNA.

26. The kit of claim 16, wherein the donor polynucleotide comprises a donor sequence.

27. The kit of claim 26, wherein the donor sequence is flanked by sequences having substantial sequence identity to sequences on either side of the target sites in the chromosomal sequence.

28. The kit of claim 26, wherein the donor sequence is flanked by short overhangs that are compatible with overhangs generated by the RNA-guided endonuclease.

29. The kit of claim 15, wherein the guide RNA is at least partially chemically synthesized.