CRISPR-based genome modification and regulation

• US 10,745,716 B2
• Filed: 06/21/2016
• Issued: 08/18/2020
• Est. Priority Date: 12/06/2012
• Status: Active Grant

• Current Assignee: Sigma-Aldrich Company (Sigma-Aldrich Corporation)
• Original Assignee: Sigma-Aldrich Company (Sigma-Aldrich Corporation)
• Inventors: Chen, Fuqiang, Davis, Gregory D.
• Primary Examiner(s): Dunston, Jennifer
• Application Number:
  US15/188,924
• Publication Number:
  US 20160298135A1
• Time in Patent Office: 1,519 Days
• 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 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 modifying a chromosomal sequence in a eukaryotic cell, the method comprising:

introducing into the eukaryotic cell

(i) at least one RNA-guided endonuclease or nucleic acid encoding at least one RNA-guided endonuclease, wherein the at least one RNA-guided endonuclease is a clustered regularly interspersed short palindromic repeats (CRISPR)/CRISPR associated (Cas) (CRISPR-Cas) type II system protein,

wherein the nucleic acid encoding the CRISPR-Cas type II system protein is codon optimized for expression in the eukaryotic cell,

wherein the CRISPR-Cas type II system protein is a Streptococcus pyogenes Cas9 protein including at least one nuclear localization signal consisting of SEQ ID NO;

1 or SEQ ID NO;

2 covalently attached to the C-terminal amino acid of the Cas9 protein sequence; and

(ii) at least one engineered guide RNA or DNA encoding at least one engineered guide RNA, each guide RNA comprising

(1) a first region at the 5′

end that base pairs with a target site in the chromosomal sequence, and

(2) a second region that forms a secondary structure which interacts with the at least one RNA-guided endonuclease; and

, optionally,

(iii) at least one donor polynucleotide;

whereby the at least one guide RNA guides the at least one RNA-guided endonuclease to the target site in the chromosomal sequence where the RNA-guided endonuclease introduces a double-stranded break, the target site in the chromosomal sequence is immediately followed by a protospacer adjacent motif (PAM), and repair of the double-stranded break by a DNA repair process leads to modification of the chromosomal sequence.

2. The method of claim 1, wherein the optional donor polynucleotide comprises a donor sequence that has at least one nucleotide change relative to the chromosomal sequence near the target site in the chromosomal sequence.

3. The method of claim 2, wherein the donor sequence is flanked by sequences having substantial sequence identity to sequences on either side of the target site in the chromosomal sequence.

4. The method of claim 2, wherein the donor sequence further comprises a targeted cleavage site that is recognized by the at least one RNA-guided endonuclease.

5. The method of claim 1, wherein the nucleic acid encoding the at least one RNA-guided endonuclease is mRNA.

6. The method of claim 1, wherein the nucleic acid encoding the at least one RNA-guided endonuclease is DNA.

7. The method of claim 1, wherein the eukaryotic cell is a human cell, a nonhuman mammalian cell, or a plant cell.

8. The method of claim 1, wherein the eukaryotic cell is in vitro.

9. The method of claim 1, wherein the eukaryotic cell is in vivo.

10. The method of claim 1, wherein the optional donor polynucleotide is not introduced into the eukaryotic cell, and repair of the double-stranded break by a non-homologous end-joining repair process results in inactivation of the chromosomal sequence.

11. The method of claim 1, wherein the optional donor polynucleotide is introduced into the eukaryotic cell, and repair of the double-stranded break results in a change of at least one nucleotide in the chromosomal sequence.

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

13. The method of claim 1, wherein only (i) and (ii) are introduced into the eukaryotic cell.

14. The method of claim 1, wherein (i), (ii), and (iii) are introduced into the eukaryotic cell.

15. The method of claim 1, wherein the Cas9 protein is from Streptococcus pyogenes strain MGAS15252 and comprises SEQ ID NO:

9.

16. The method of claim 1, wherein the at least one nuclear localization signal covalently attached to the C-terminal amino acid of the Cas9 protein sequence consists of SEQ ID NO:

1.

17. The method of claim 1, wherein the at least one nuclear localization signal covalently attached to the C-terminal amino acid of the Cas9 protein sequence consists of SEQ ID NO:

2.

18. The method of claim 1, wherein the nucleic acid encoding the at least one RNA-guided endonuclease is mRNA and the at least one guide RNA is comprised of two non-covalently bound RNA molecules.

19. The method of claim 1, wherein the nucleic acid encoding the at least one RNA-guided endonuclease is mRNA and the at least one guide RNA is comprised of a single RNA molecule.

20. The method of claim 1, wherein the nucleic acid encoding the at least one RNA-guided endonuclease is DNA, the at least one guide RNA is encoded by DNA, and the at least one guide RNA is comprised of a single RNA molecule.

21. The method of claim 1, wherein the optional donor polynucleotide is double stranded DNA.

22. The method of claim 1, wherein the optional donor polynucleotide is single stranded DNA.