Using programmable DNA binding proteins to enhance targeted genome modification

• US 10,266,851 B2
• Filed: 02/20/2017
• Issued: 04/23/2019
• Est. Priority Date: 06/02/2016
• Status: Active Grant

• Current Assignee: Sigma-Aldrich Company (Sigma-Aldrich Corporation)
• Original Assignee: Sigma-Aldrich Company (Sigma-Aldrich Corporation)
• Inventors: Chen, Fuqiang
• Primary Examiner(s): Dunston, Jennifer
• Application Number:
  US15/437,148
• Publication Number:
  US 20170349913A1
• Time in Patent Office: 792 Days
• Field of Search: None
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

Abstract

Compositions and methods for using programmable DNA binding proteins to increase the efficiency and/or specificity of targeted genome modification or to facilitate the detection of specific genomic loci in eukaryotic cells.

Claims

1. A method for increasing targeted double-stranded cleavage activity and/or specificity at a target chromosomal region in a eukaryotic cell, the method comprising introducing into the eukaryotic cell:

(a) a CRISPR system having binding activity and double-stranded cleavage activity, or a nucleic acid encoding said CRISPR system, comprising (i) a catalytically active Type II CRISPR/Cas9 protein or a catalytically active Type V CRISPR/Cpf1 protein, and (ii) a guide RNA; and

(b) at least one CRISPR system having binding activity but lacking cleavage activity, or a nucleic acid encoding said CRISPR system, comprising (i) a catalytically inactive Type II CRISPR/Cas9 protein or a catalytically inactive Type V CRISPR/Cpf1 protein, and (ii) a guide RNA;

wherein the CRISPR system of subpart (a) and the at least one CRISPR system of subpart (b) are introduced into the eukaryotic cell in the absence of a donor polynucleotide;

wherein the CRISPR system of subpart (a) is targeted to a target chromosomal sequence within the target chromosomal region and the at least one CRISPR system of subpart (b) is targeted to and, excluding any off-target chromosomal binding, binds solely to a site that is proximal to the target chromosomal sequence within the target chromosomal region, such that the CRISPR systems of subparts (a) and (b) are located in spatial proximity only by the chromosomal DNA binding action of their respective guide RNA and are within about 250 base pairs of one another on the target chromosomal region, and

wherein binding of the at least one CRISPR system of subpart (b) to the site proximal to the target chromosomal sequence increases accessibility of the CRISPR system of subpart (a) to the target chromosomal sequence, thereby increasing targeted double-stranded cleavage activity and/or specificity.

2. The method of claim 1, wherein the CRISPR system of subpart (a) and the at least one CRISPR system of subpart (b) are within about 200 base pairs of one another on the target chromosomal region.

3. The method of claim 2, wherein the CRISPR system of subpart (a) and the at least one CRISPR system of subpart (b) are within about 100 base pairs of one another on the target chromosomal region.

4. The method of claim 3, wherein the CRISPR system of subpart (a) and the at least one CRISPR system of subpart (b) are within about 75 base pairs of one another on the target chromosomal region.

5. The method of claim 4, wherein the CRISPR system of subpart (a) and the at least one CRISPR system of subpart (b) are within about 50 base pairs of one another on the target chromosomal region.

6. The method of claim 5, wherein the CRISPR system of subpart (a) and the at least one CRISPR system of subpart (b) are within about 25 base pairs of one another on the target chromosomal region.

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

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

9. The method of claim 1, wherein the eukaryotic cell is a mammalian cell.

10. The method of claim 9, wherein the mammalian cell is a human cell.

11. The method of claim 1, wherein nucleic acid encoding each CRISPR system of subpart (a) and subpart (b) is mRNA or DNA.

12. The method of claim 1, wherein nucleic acid encoding each CRISPR system of subpart (a) and subpart (b) and/or encoding each guide RNA of subpart (a) and subpart (b) is part of a plasm id vector or a viral vector.

13. The method of claim 1, wherein the guide RNA of each CRISPR system of subpart (a) and subpart (b) is at least partially chemically synthesized.

14. The method of claim 1, wherein the guide RNA of each CRISPR system of subpart (a) and subpart (b) is enzymatically synthesized.

15. The method of claim 1, wherein the CRISPR system of subpart (a) comprises a catalytically active Type V CRISPR/Cpf1 protein and the at least one CRISPR system of subpart (b) comprises a catalytically inactive Type II CRISPR/Cas9 protein.

16. The method of claim 1, wherein the CRISPR system of subpart (a) comprises a catalytically active Type II CRISPR/Cas9 protein and the at least one CRISPR system of subpart (b) comprises a catalytically inactive Type II CRISPR/Cas9 protein.

17. The method of claim 1, wherein two CRISPR systems having binding activity but lacking cleavage activity are introduced into the eukaryotic cell.

18. The method of claim 1, wherein

(i) the CRISPR system of subpart (a) is a Type IIB Francisella novicida CRISPR/Cas9 protein and the at least one CRISPR system of subpart (b) is a Type IIA Streptococcus pyogenes CRISPR/Cas9 protein;

(ii) the CRISPR system of subpart (a) is a Type IIA S. pyogenes CRISPR/Cas9 protein and the at least one CRISPR system of subpart (b) is a Type IIB F. novicida CRISPR/Cas9 protein;

(iii) the CRISPR system of subpart (a) is a Type IIC Campylobacter jejuni CRISPR/Cas9 protein and the at least one CRISPR system of subpart (b) is a Type IIA S. pyogenes CRISPR/Cas9 protein;

or

(iv) the CRISPR system of subpart (a) is a Type V F. novicida CRISPR/Cpf1 protein and the at least one CRISPR system of subpart (b) is a Type IIA S. pyogenes CRISPR/Cas9 protein.