Using RNA-guided FokI nucleases (RFNs) to increase specificity for RNA-guided genome editing

• US 10,138,476 B2
• Filed: 01/25/2017
• Issued: 11/27/2018
• Est. Priority Date: 03/15/2013
• Status: Active Grant

• Current Assignee: The General Hospital Corporation (Mass General Brigham, Inc.)
• Original Assignee: The General Hospital Corporation (Mass General Brigham, Inc.)
• Inventors: Joung, J. Keith, Tsai, Shengdar
• Primary Examiner(s): Holland, Paul J
• Application Number:
  US15/415,431
• Publication Number:
  US 20170327805A1
• Time in Patent Office: 671 Days
• CPC Class Codes: C07K 14/005 from viruses
• CPC Class Codes: C07K 14/195 from bacteria
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/01 containing a localisation/targetting motif
• CPC Class Codes: C07K 2319/80 containing a DNA binding domain, e.g. Lacl or Tet-repressor
• CPC Class Codes: C12N 15/01 Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 15/1031 mutagenesis by gene assembly, e.g. assembly by oligonucleotide extension PCR
• 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 15/85 for animal cells
• 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 2710/00033 Use of viral protein as therapeutic agent other than vaccine, e.g. apoptosis inducing or anti-inflammatory
• CPC Class Codes: C12N 2770/00033 Use of viral protein as therapeutic agent other than vaccine, e.g. apoptosis inducing or anti-inflammatory
• CPC Class Codes: C12N 2800/80 Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
• CPC Class Codes: C12N 9/0071 acting on paired donors with incorporation of molecular oxygen (1.14)
• CPC Class Codes: C12N 9/1007 Methyltransferases (general) (2.1.1.)
• CPC Class Codes: C12N 9/16 acting on ester bonds (3.1)
• 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 114/11 : with 2-oxoglutarate as one donor, and incorporation of one atom each of oxygen into both donors (1.14.11)
• CPC Class Codes: C12Y 201/01 : Methyltransferases (2.1.1)
• 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)

Abstract

Methods for increasing specificity of RNA-guided genome editing, e.g., editing using CRISPR/Cas9 systems.

Claims

1. A composition comprising:

a nucleic acid encoding an RNA-guided FokI Nuclease (RFN) fusion protein comprising a FokI catalytic domain sequence fused to the amino terminus of a catalytically inactive Streptococcus pyogenes CRISPR-associated 9 (dCas9) protein comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO;

5, wherein said catalytically inactive S. pyogenes Cas9 has point mutations at amino acid residues corresponding to positions (i) D10, E762, H983, or D986, and (ii) H840 or N863 of S. pyogenes Cas9, an intervening linker from 2 to 30 amino acids; and

a nucleic acid encoding two guide RNAs that direct said RFN fusion protein to a first target genomic sequence and a second target genomic sequence, wherein the guide RNAs that direct said RFN fusion protein to said first target genomic sequence and said second target genomic sequence are spaced 10 to 20 nucleotides apart, and said first target genomic sequence comprises a PAM recognition sequence positioned upstream of said first target genomic sequence and said second target genomic sequence comprises a PAM recognition sequence positioned downstream of said second target genomic sequence.

2. The composition of claim 1, wherein said guide RNAs are:

(a) two single guide RNAs, wherein one single guide RNA targets a first strand, and a second guide RNA targets a complementary strand, and FokI cuts each strand resulting in a pair of nicks on opposite DNA strands, thereby creating a double-stranded break, or

(b) a tracrRNA and two crRNAs, wherein one crRNA targets a first strand, and a second crRNA targets a complementary strand, and FokI cuts each strand resulting in a pair of nicks on opposite DNA strands, thereby creating a double-stranded break.

3. The composition of claim 1, wherein each of said two guide RNAs include a complementarity region that is complementary to 17-20 nucleotides of said first target genomic sequence and said second target genomic sequence.

4. The composition of claim 1, wherein an indel mutation is induced between said first target genomic sequence and said second target genomic sequence.

5. The composition of claim 1, wherein said first target genomic sequence and said second target genomic sequence are spaced 13-17 nucleotides apart.

6. The composition of claim 1, wherein said intervening linker comprises Gly4Ser.

7. The composition of claim 1, wherein said FokI catalytic domain comprises amino acid residues 388-583 or amino acid residues 408-583 of the amino acid sequence of SEQ ID NO:

4.

8. The composition of claim 1, wherein said point mutations are:

(i) D10A or D10N; and

(ii) H840A, H840Y or H840N.

9. The composition of claim 1, wherein said RNA-guided FokI Nuclease fusion protein comprises the amino acid sequence of SEQ ID NO:

26.

10. A composition comprising:

an RNA-guided FokI Nuclease (RFN) fusion protein comprising a FokI catalytic domain sequence fused to the amino terminus of a catalytically inactive Streptococcus pyogenes CRISPR-associated 9 (dCas9) protein comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO;

5, wherein said catalytically inactive S. pyogenes Cas9 has point mutations at amino acid residues corresponding to positions (i) D10, E762, H983, or D986, and (ii) H840 or N863 of S. pyogenes Cas9, an intervening linker from 2 to 30 amino acids; and

two guide RNAs that direct said RFN fusion protein to a first target genomic sequence and a second target genomic sequence, wherein the guide RNAs that direct said RFN fusion protein to said first target genomic sequence and said second target genomic sequence are spaced 10 to 20 nucleotides apart, and said first target genomic sequence comprises a PAM recognition sequence positioned upstream of said first target genomic sequence and said second target genomic sequence comprises a PAM recognition sequence positioned downstream of said second target genomic sequence.

11. The composition of claim 10, wherein said guide RNAs are:

(a) two single guide RNAs, wherein one single guide RNA targets a first strand, and a second guide RNA targets a complementary strand, and FokI cuts each strand resulting in a pair of nicks on opposite DNA strands, thereby creating a double-stranded break, or

(b) a tracrRNA and two crRNAs, wherein one crRNA targets a first strand, and a second crRNA targets a complementary strand, and FokI cuts each strand resulting in a pair of nicks on opposite DNA strands, thereby creating a double-stranded break.

12. The composition of claim 10, wherein each of said two guide RNAs include a complementarity region that is complementary to 17-20 nucleotides of said first target genomic sequence and said second target genomic sequence.

13. The composition of claim 10, wherein an indel mutation is induced between said first target genomic sequence and said second target genomic sequence.

14. The composition of claim 10, wherein said first target genomic sequence and said second target genomic sequence are spaced 13-17 nucleotides apart.

15. The composition of claim 10, wherein said intervening linker comprises Gly4Ser.

16. The composition of claim 10, wherein said FokI catalytic domain comprises amino acid residues 388-583 or amino acid residues 408-583 of the amino acid sequence of SEQ ID NO:

4.

17. The composition of claim 10, wherein said point mutations are:

(i) D10A or D10N; and

(ii) H840A, H840Y or H840N.

18. The composition of claim 10, wherein said RNA-guided FokI Nuclease fusion protein comprises the amino acid sequence of SEQ ID NO:

26.

19. A method for RNA guided genome editing, said method comprising expressing in said cell or contacting said cell with an RNA-guided FokI Nuclease (RFN) fusion protein comprising a FokI catalytic domain sequence fused to the amino terminus of a catalytically inactive Streptococcus pyogenes CRISPR-associated 9(dCas9) protein comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:

5, wherein said catalytically inactive S. pyogenes Cas9has point mutations at amino acid residues corresponding to positions (i) D10, E762, H983, or D986, and (ii) H840 or N863 of S. pyogenes Cas9, an intervening linker from 2 to 30amino acids; and

two guide RNAs that direct said RFN fusion protein to a first target genomic sequence and a second target genomic sequence, wherein the guide RNAs that direct said RFN fusion protein to said first target genomic sequence and said second target genomic sequence are spaced 10 to 20 nucleotides apart, and said first target genomic sequence comprises a PAM recognition sequence positioned upstream of said first target genomic sequence and said second target genomic sequence comprises a PAM recognition sequence positioned downstream of said second target genomic sequence.

20. A kit comprising:

an RNA-guided FokI Nuclease (RFN) fusion protein comprising a FokI catalytic domain sequence fused to the amino terminus of a catalytically inactive Streptococcus pyogenes CRISPR-associated 9 (dCas9) protein comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO;

5, wherein said catalytically inactive S. pyogenes Cas9 has point mutations at amino acid residues corresponding to positions (i) D10, E762, H983, or D986, and (ii) H840 or N863 of S. pyogenes Cas9, an intervening linker from 2 to 30 amino acids; and

two guide RNAs that direct said RFN fusion protein to a first target genomic sequence and a second target genomic sequence, wherein the guide RNAs that direct said RFN fusion protein to said first target genomic sequence and said second target genomic sequence are spaced 10 to 20 nucleotides apart, and said first target genomic sequence comprises a PAM recognition sequence positioned upstream of said first target genomic sequence and said second target genomic sequence comprises a PAM recognition sequence positioned downstream of said second target genomic sequence.

21. The kit of claim 20, wherein said guide RNAs are:

(a) two single guide RNAs, wherein one single guide RNA targets a first strand, and a second guide RNA targets a complementary strand, and FokI cuts each strand resulting in a pair of nicks on opposite DNA strands, thereby creating a double-stranded break, or

(b) a tracrRNA and two crRNAs, wherein one crRNA targets a first strand, and a second crRNA targets a complementary strand, and FokI cuts each strand resulting in a pair of nicks on opposite DNA strands, thereby creating a double-stranded break.

22. The kit of claim 20, wherein each of said two guide RNAs include a complementarity region that is complementary to 17-20 nucleotides of said first target genomic sequence and said second target genomic sequence.

23. The kit of claim 20, wherein an indel mutation is induced between said first target genomic sequence and said second target genomic sequence.

24. The kit of claim 20, wherein said first target genomic sequence and said second target genomic sequence are spaced 13-17 nucleotides apart.

25. The kit of claim 20, wherein said intervening linker comprises Gly4Ser.

26. The kit of claim 20, wherein said FokI catalytic domain comprises amino acid residues 388-583 or amino acid residues 408-583 of the amino acid sequence of SEQ ID NO:

4.

27. The kit of claim 20, wherein said point mutations are:

(i) D10A or D10N; and

(ii) H840A, H840Y or H840N.

28. The kit of claim 20, wherein said RNA-guided FokI Nuclease fusion protein comprises the amino acid sequence of SEQ ID NO:

26.