Using truncated guide RNAs (tru-gRNAs) to increase specificity for RNA-guided genome editing

Patent metadata

Field	Value
Publication number	US-9567604-B2
Application number	US-201414213723-A
Country	US
Kind code	B2
Filing date	Mar 14, 2014
Priority date	Mar 15, 2013
Publication date	Feb 14, 2017
Grant date	Feb 14, 2017

Abstract

Methods for increasing specificity of RNA-guided genome editing, e.g., editing using CRISPR/Cas9 systems, using truncated guide RNAs (tru-gRNAs).

First claim

What is claimed is:

1. A method of increasing specificity of Streptococcus pyogenes CRISPR/Cas9 (Cas9) RNA-guided genome editing in a cell, the method comprising contacting the cell with a guide RNA that includes a complementarity region at the 5′ end of the guide RNA consisting of 17-18 nucleotides that are complementary to 17-18 consecutive nucleotides of the complementary strand of a selected target genomic sequence, wherein the target sequence is immediately 5′ of a protospacer adjacent motif (PAM), and wherein the guide RNA is
(i) a single guide RNA that includes at the 5′ end of the single guide RNA a complementarity region consisting of 17-18 nucleotides that are complementary to 17-18 consecutive nucleotides of the complementary strand of the selected target genomic sequence on a double-stranded DNA molecule, or
(ii) a crRNA that includes at the 5′ end of the crRNA a complementarity region consisting of 17-18 nucleotides that are complementary to 17-18 consecutive nucleotides of the complementary strand of the selected target genomic sequence, and a tracrRNA,
wherein in the presence of a S. pyogenes Cas9 genome editing enzyme, the guide RNA complementarity region binds and directs the Cas9 genome editing enzyme to the target genomic sequence, thereby increasing specificity of RNA-guided genome editing in a cell.

2. The method of claim 1 , wherein the crRNA is SEQ ID NO: 2407 and the tracrRNA is SEQ ID NO: 8; the crRNA is SEQ ID NO: 2404 and the tracrRNA is SEQ ID NO: 2405; or the crRNA is SEQ ID NO: 2408 and the tracrRNA is SEQ ID NO: 2406.

3. The method of claim 1 , wherein the tracrRNA is selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 2405, SEQ ID NO: 2406, SEQ ID NO: 2409, SEQ ID NO: 2410, SEQ ID NO: 2411 and SEQ ID NO: 2412.

4. The method of claim 1 , wherein the guide RNA is (i) the single guide RNA that includes at the 5′ end of the single RNA the complementarity region consisting of 17-18 nucleotides that are complementary to 17-18 consecutive nucleotides of the complementary strand of the selected target genomic sequence.

5. The method of claim 1 , wherein the guide RNA is a ribonucleic acid selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 2404, SEQ ID NO: 2407 and SEQ ID NO: 2408.

6. The method of claim 1 , wherein the complementarity region is complementary to 17 consecutive nucleotides of the complementary strand of the selected target genomic sequence.

7. The method of claim 1 , wherein the complementarity region is complementary to 18 consecutive nucleotides of the complementary strand of the selected target genomic sequence.

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

9. A method of inducing a break in a target region of a double-stranded DNA molecule in a cell, the method comprising expressing in or introducing into the cell:
a S. pyogenes CRISPR/Cas9 nuclease or nickase; and
a guide RNA that includes a complementarity region at the 5′ end of the guide RNA consisting of 17-18 nucleotides that are complementary to 17-18 consecutive nucleotides of the complementary strand of a double-stranded DNA molecule, wherein the target region sequence is immediately 5′ of a protospacer adjacent motif (PAM), and wherein the guide RNA complementarity region binds and directs the Cas9 nuclease or nickase to the target region sequence of a double-stranded DNA molecule, and wherein the guide RNA is
(i) a single guide RNA that includes at the 5′ end of the single guide RNA a complementarity region consisting of 17-18 nucleotides that are complementary to 17-18 consecutive nucleotides of the complementary strand of a selected target genomic sequence on a double stranded DNA molecule, or
(ii) a crRNA that includes at the 5′ end of the crRNA a complementarity region consisting of 17-18 nucleotides that are complementary to 17-18 consecutive nucleotides of the complementary strand of a selected target genomic sequence, and a tracrRNA; thereby inducing a break in the target region of a double-stranded DNA molecule in a cell.

10. The method of claim 9 , wherein the guide RNA comprises a ribonucleic acid selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 2404, SEQ ID NO: 2407 and SEQ ID NO: 2408.

11. The method of claim 9 , wherein the complementarity region is complementary to 17 consecutive nucleotides of the complementary strand of the selected target region of a double-stranded DNA molecule.

12. The method of claim 9 , wherein the complementarity region is complementary to 18 consecutive nucleotides of the complementary strand of a selected target region of the double-stranded DNA molecule.

13. The method of claim 9 , wherein the target region is in a target genomic sequence.

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

15. The method of claim 9 , wherein the crRNA is SEQ ID NO: 2407 and the tracrRNA is SEQ ID NO: 8; the crRNA is SEQ ID NO: 2404 and the tracrRNA is SEQ ID NO: 2405; or the mRNA is SEQ ID NO: 2408 and the tracrRNA is SEQ ID NO: 2406.

16. The method of claim 9 , wherein the tracrRNA is selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 2405, SEQ ID NO: 2406, SEQ ID NO: 2409, SEQ ID NO: 2410, SEQ ID NO: 2411 and SEQ ID NO: 2412.

17. A method of modifying a target region of a double-stranded DNA molecule in a cell, the method comprising expressing in or introducing into the cell:
a S. pyogenes CRISPR dCas9-heterologous functional domain fusion protein (dCas9-HFD); and a guide RNA that includes a complementarity region at the 5′ end of the guide RNA consisting of 17-18 nucleotides that are complementary to 17-18 consecutive nucleotides of the complementary strand of a selected target sequence present on a double-stranded DNA molecule, wherein the target sequence is immediately 5′ of a protospacer adjacent motif (PAM), and wherein the guide RNA is:
(i) a single guide RNA that includes a complementarity region at the 5′ end of the single guide RNA consisting of 17-18 nucleotides that are complementary to 17-18 consecutive nucleotides of the complementary strand of a selected target genomic sequence on a double stranded DNA molecule, or
(ii) a crRNA that includes at the 5′ end of the crRNA a complementarity region consisting of 17-18 nucleotides that are complementary to 17-18 consecutive nucleotides of the complementary strand of a selected target genomic sequence, and a tracrRNA, and
wherein the guide RNA complementarity region binds and directs the dCas9-HFD to the target region of the double-stranded DNA molecule, thereby modifying the target region of a double-stranded DNA molecule in a cell.

Assignees

• Massachusetts Gen Hospital

Inventors

• Joung J Keith
• Sander Jeffry D
• Maeder Morgan
• Fu Yanfang

Classifications

• C07K2319/00

  Fusion polypeptide · CPC title

• C12Y114/11

  with 2-oxoglutarate as one donor, and incorporation of one atom each of oxygen into both donors (1.14.11) · CPC title

• C12Y201/01

  Methyltransferases (2.1.1) · CPC title

• C12N15/01

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

• C12Y301/00

  Hydrolases acting on ester bonds (3.1) · CPC title