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

Patent metadata

Field	Value
Publication number	US-10415059-B2
Application number	US-201715430218-A
Country	US
Kind code	B2
Filing date	Feb 10, 2017
Priority date	Mar 15, 2013
Publication date	Sep 17, 2019
Grant date	Sep 17, 2019

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 RNA-guided genome editing in a cell, the method comprising contacting the cell with a guide RNA (gRNA) that includes a complementarity region consisting of 17-18 nucleotides that are complementary to 17-18 consecutive nucleotides of the complementary strand of a target genomic sequence, wherein the gRNA comprises 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, or SEQ ID NO: 2408, and wherein in the presence of a S. pyogenes Cas9 nuclease, the gRNA complementarity region binds and directs the Cas9 nuclease to the target genomic sequence, and the Cas9 nuclease edits the target genomic sequence.

2. A Streptococcus pyogenes gRNA molecule that includes a complementarity region at the 5′ end of the gRNA consisting of 17-18 nucleotides that are complementary to 17-18 consecutive nucleotides of the complementary strand of a target genomic sequence, wherein the target genomic sequence is immediately 5′ of a protospacer adjacent motif, wherein the gRNA is a single gRNA or a CRISPR RNA (crRNA), and wherein in the presence of an S. pyogenes Cas9 nuclease, the gRNA complementarity region binds and directs the Cas9 nuclease to the target genomic sequences, and the Cas9 nuclease edits the target genomic sequence.

3. The gRNA of claim 2 , wherein the complementarity region of the gRNA consists of 17 nucleotides.

4. The gRNA of claim 2 , comprising a ribonucleic acid 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, or SEQ ID NO: 2408.

5. The gRNA of claim 2 , comprising a ribonucleic acid that includes one or more uracil (U) at the 3′ end of the molecule.

6. The gRNA of claim 2 , comprising a ribonucleic acid that includes one or more additional nucleotides at the 5′ end of the RNA molecule that is not complementary to the target genomic sequence.

7. The gRNA of claim 2 , comprising a ribonucleic acid that includes one, two, or three additional nucleotides at the 5′ end of the RNA molecule that are not complementary to the target genomic sequence.

8. The gRNA of claim 2 , wherein the complementarity region is complementary to 17 consecutive nucleotides of the complementary strand of a target genomic sequence.

9. The gRNA of claim 2 , wherein the complementarity region is complementary to 18 consecutive nucleotides of the complementary strand of a target genomic sequence.

10. A DNA molecule encoding the gRNA of claim 2 .

11. A vector comprising the DNA molecule of claim 10 .

12. A host cell expressing the vector of claim 11 .

13. The host cell of claim 12 , wherein the cell is a eukaryotic cell.

14. The host cell of claim 13 , wherein the cell is a mammalian cell.

15. The gRNA of claim 2 , wherein the complementarity region of the gRNA consists of 18 nucleotides.

16. The gRNA of claim 2 , and wherein the gRNA retains the ability to form a complex with a Cas9 nuclease or a catalytically inactive Cas9 (dCas9) nuclease.

17. A complex comprising:
a Streptococcus pyogenes Cas9 nuclease and
a Streptococcus pyogenes gRNA molecule that includes a complementarity region at the 5′ end of the gRNA consisting of 17-18 nucleotides that are complementary to 17-18 consecutive nucleotides of the complementary strand of a target genomic sequence, wherein the target genomic sequence is immediately 5′ of a protospacer adjacent motif,
wherein the gRNA is a single gRNA or a CRISPR RNA (crRNA), and
wherein in the presence of the S. pyogenes Cas9 nuclease, the gRNA complementary region binds and directs the Cas9 nuclease to the target genomic sequence, and the Cas9 nuclease edits the target genomic sequence.

18. The complex of claim 17 , wherein the Cas9 nuclease is a dCas9 nuclease.

19. The complex of claim 17 , wherein the complementarity region of the gRNA consists of 17 nucleotides.

20. The complex of claim 17 , wherein the gRNA comprises a ribonucleic acid 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, or SEQ ID NO: 2408.

21. The complex of claim 17 , wherein the gRNA includes one or more uracil (U) at the 3′ end of the molecule.

22. The complex of claim 17 , wherein the gRNA includes one or more additional nucleotides at the 5′ end of the gRNA that is not complementary to the target genomic sequence.

23. The complex of claim 17 , wherein the gRNA includes one,
two, or three additional nucleotides at the 5′ end of the RNA molecule that are not complementary to the target genomic sequence.

24. The complex of claim 17 , wherein the complementarity region is complementary to 17 consecutive nucleotides of the complementary strand of a target genomic sequence.

25. The complex of claim 17 , wherein the complementarity region is complementary to 18 consecutive nucleotides of the complementary strand of a target genomic sequence.

26. The complex of claim 17 , wherein the complementarity region of the gRNA consists of 18 nucleotides.

27. A DNA molecule encoding the complex of claim 17 .

28. A vector comprising the DNA molecule of claim 27 .

29. A host cell expressing the vector of claim 28 .

30. The host cell of claim 29 , wherein the cell is a eukaryotic cell.

31. The host cell of claim 30 , wherein the cell is a mammalian cell.

Assignees

• Massachusetts Gen Hospital

Inventors

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

Classifications

• C12N9/22

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title

• C12N9/1007

  Methyltransferases (general) (2.1.1.) · CPC title

• C12N9/16

  acting on ester bonds (3.1) · CPC title

• C12N15/85

  for animal cells · CPC title

• C12Y301/21004

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