CRISPR-BASED GENOME MODIFICATION AND REGULATION

• US 20160298133A1
• Filed: 06/21/2016
• Published: 10/13/2016
• Est. Priority Date: 12/06/2012
• Status: Abandoned Application

• Current Assignee: Sigma-Aldrich Company (Sigma-Aldrich Corporation)
• Original Assignee: Sigma-Aldrich Company (Sigma-Aldrich Corporation)
• Inventors: CHEN, Fuqiang, DAVIS, Gregory D., KANG, Qiaohua, KNIGHT, Scott W.
• Application Number:
  US15/188,909
• Publication Number:
  US 20160298133A1
• 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 method for cleaving a chromosomal sequence in a cell or embryo, the method comprising introducing into the cell or embryo (a) at least one fusion protein or nucleic acid encoding at least one fusion protein, wherein the fusion protein comprises a Cas9 protein mutated in the RuvC and HNH domains so that it no longer possesses nuclease activity or a fragment thereof and a cleavage domain, and (b) at least one guide RNA or DNA encoding at least one guide RNA, wherein the at least one guide RNA guides the mutated Cas9 protein of the fusion protein to a target site in the chromosomal sequence and the cleavage domain of the fusion protein cleaves the chromosomal sequence.

2. The method of claim 1, wherein the mutation in the RuvC domain of the mutated Cas9 protein is D10A.

3. The method of claim 1, wherein the mutation in the HNH domain of the mutated Cas9 is H840A or H839A.

4. The method of claim 1, wherein fusion protein further comprises at least one additional domain chosen from a nuclear localization signal, a cell-penetrating domain, and a marker domain.

5. The method of claim 1, wherein one fusion protein or nucleic acid encoding one fusion protein and two guide RNAs or DNA encoding two guide RNAs are introduced into the cell or embryo.

6. The method of claim 1, wherein two fusion proteins or nucleic acid encoding two fusion proteins and two guide RNAs or DNA encoding two guide RNAs are introduced into the cell or embryo.

7. The method of claim 6, wherein each fusion protein comprises a different Cas9 protein or fragment thereof.

8. The method of claim 6, wherein each fusion protein comprises a different cleavage domain.

9. The method of claim 1, further comprising introducing into the cell or embryo at least one donor polynucleotide.

10. The method of claim 1, wherein the cell is a human cell, a non-human mammalian cell, a stem cell, a non-mammalian vertebrate cell, an invertebrate cell, a plant cell, or a single cell eukaryotic organism.

11. The method of claim 1, wherein the embryo is a non-human one cell animal embryo.

12. The method of claim 1, wherein the cleavage domain is derived from a type II-S endonuclease.

13. The method of claim 12, wherein the type II-S endonuclease is FokI or a derivative thereof.

14. The method of claim 1, wherein the at least one guide RNA is at least partially chemically synthesized.

15. A fusion protein comprising a CRISPR/Cas-like protein or fragment thereof and a cleavage domain, wherein the CRISPR/Cas-like protein is a Cas9 protein mutated in the RuvC and HNH domains so that it no longer possesses nuclease activity.

16. The fusion protein of claim 15, wherein the mutation in the RuvC domain of the mutated Cas9 protein is D10A.

17. The fusion protein of claim 15, wherein the mutation in the HNH domain of the mutated Cas9 is H840A or H839A.

18. The fusion protein of claim 15, wherein the fusion protein further comprises at least one additional domain chosen from a nuclear localization signal, a cell-penetrating domain, and a marker domain.

19. The fusion protein of claim 15, wherein the cleavage domain is derived from a type II-S endonuclease.

20. The fusion protein of claim 19, wherein the type II-S endonuclease is FokI or a derivative thereof.

21. A nucleic acid encoding a fusion protein, the fusion protein comprising a CRISPR/Cas-like protein or fragment thereof and a cleavage domain, wherein the CRISPR/Cas-like protein is a Cas9 protein mutated in the RuvC and HNH domains so that it no longer possesses nuclease activity.

22. The nucleic acid of claim 21, wherein the fusion protein further comprises at least one additional domain chosen from a nuclear localization signal, a cell-penetrating domain, and a marker domain.

23. The nucleic acid of claim 21, wherein the nucleic acid is operably linked to at least one promoter control sequence.

24. The nucleic acid of claim 21, wherein the nucleic acid is codon-optimized for expression in a eukaryotic cell and the fusion protein further comprises a nuclear localization signal.

25. The nucleic acid of claim 24, wherein the nucleic acid is operably linked to at least one promoter control sequence.

26. The nucleic acid of claim 25, wherein the nucleic acid is present in a vector.

27. The nucleic acid of claim 26, wherein the vector is a lentiviral vector or an adeno-associated viral vector.

28. The nucleic acid of claim 27, wherein the vector further encodes a guide RNA.

29. The nucleic acid of claim 21, wherein the cleavage domain is derived from a type II-S endonuclease.

30. The nucleic acid of claim 29, wherein the type II-S endonuclease is FokI or a derivative thereof.