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

Patent metadata

Field	Value
Publication number	US-10544433-B2
Application number	US-201816003973-A
Country	US
Kind code	B2
Filing date	Jun 8, 2018
Priority date	Mar 15, 2013
Publication date	Jan 28, 2020
Grant date	Jan 28, 2020

Abstract

Many studies have shown that CRISPR-Cas nucleases can tolerate up to five mismatches and still cleave; it is hard to predict the effects of any given single or combination of mismatches on activity. Taken together, these nucleases can show significant off-target effects but it can be challenging to predict these sites. Described herein are methods for increasing the specificity of genome editing using the CRISPR/Cas system, e.g., using RNA-guided Foki Nucleases (RFNs), e.g., Fokl-Cas9 or Foki-dCas9-based fusion proteins.

First claim

What is claimed is:

1. A method for increasing specificity of RNA-guided genome editing in a cell, the 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 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 said two 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 method 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 method 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 method of claim 1 , wherein an indel mutation is induced between said first target genomic sequence and said second target genomic sequence.

5. The method of claim 1 , wherein the specificity of RNA-guided genome editing in a cell is increased as compared to genome editing with a native Cas9.

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

7. The method of claim 1 , wherein said intervening linker comprises Gly 4 Ser.

8. The method 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.

9. The method of claim 1 , wherein said point mutations are: (i) D10A or D10N; and (ii) H840A, H840Y or H840N.

10. The method of claim 1 , wherein said RNA-guided FokI Nuclease fusion protein comprises the amino acid sequence of SEQ ID NO: 26.

Assignees

• Massachusetts Gen Hospital

Inventors

• Joung J Keith
• Tsai Shengdar

Classifications

• C12Y201/01

  Methyltransferases (2.1.1) · CPC title

• C12N15/01

  Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor · CPC title

• C12N9/96

  Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates · CPC title

• C12Y301/21004

  Type II site-specific deoxyribonuclease (3.1.21.4) · CPC title

• C12N9/1007

  Methyltransferases (general) (2.1.1.) · CPC title