CRISPR-BASED GENOME MODIFICATION AND REGULATION

• US 20170073705A1
• Filed: 11/03/2016
• Published: 03/16/2017
• 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:
  US15/342,976
• Publication Number:
  US 20170073705A1
• 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. An isolated endonuclease comprising at least one nuclear localization signal, at least one nuclease domain, and at least one domain that interacts with a guide RNA to target the endonuclease to a specific nucleotide sequence for cleavage.

2. The isolated endonuclease of claim 1, wherein the endonuclease is derived from a Cas9 protein.

3. The isolated endonuclease of claim 1, wherein the endonuclease is modified to lack at least one functional nuclease domain.

4. The isolated endonuclease of claim 1, further comprising a cell-penetrating domain, a marker domain, or both.

5. The isolated endonuclease of claim 1, which is part of a protein-RNA complex comprising the guide RNA.

6. The isolated endonuclease of claim 5, wherein the guide RNA is a single molecule comprising a 5′

region that is complementary to a target site.

7. An isolated nucleic acid encoding the endonuclease of claim 1.

8. The isolated nucleic acid of claim 7, wherein the nucleic acid is codon optimized for translation in mammalian cells.

9. The isolated nucleic acid of claim 7, wherein the nucleic acid is codon optimized for translation in human cells.

10. The isolated nucleic acid of claim 7, which is operably linked to a promoter control sequence.

11. A vector comprising the isolated nucleic acid of claim 10.

12. The vector of claim 11, further comprising a sequence encoding a guide RNA that is operably linked to a promoter control sequence.

13. A method for modifying a chromosomal sequence in a eukaryotic cell, the method comprising:

a) introducing into the eukaryotic cell or embryo (i) at least one RNA-guided endonuclease comprising at least one nuclear localization signal or nucleic acid encoding at least one RNA-guided endonuclease comprising at least one nuclear localization signal, (ii) at least one guide RNA or DNA encoding at least one guide RNA, and, optionally, (iii) at least one donor polynucleotide; and

b) culturing the eukaryotic cell such that each guide RNA directs an RNA-guided endonuclease to a targeted site in the chromosomal sequence where the RNA-guided endonuclease introduces a double-stranded break in the targeted site, and the double-stranded break is repaired by a DNA repair process such that the chromosomal sequence is modified.

14. The method of claim 13, wherein the at least one RNA-guided endonuclease is derived from a Cas9 protein.

15. The method of claim 13, wherein the nucleic acid encoding the at least one RNA-guided endonuclease is codon optimized for expression in the eukaryotic cell.

16. The method of claim 15, wherein the nucleic acid encoding the at least one RNA-guided endonuclease is mRNA.

17. The method of claim 15, wherein the nucleic acid encoding the at least one RNA-guided endonuclease is DNA.

18. The method of claim 17, wherein the DNA is part of a vector that further comprises a sequence encoding the at least one guide RNA.

19. The method of claim 13, wherein the eukaryotic cell is a human cell, a non-human mammalian cell, a one cell animal embryo, a non-mammalian vertebrate cell, an invertebrate cell, a plant cell, or a single cell eukaryotic organism.