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

• US 10,415,059 B2
• Filed: 02/10/2017
• Issued: 09/17/2019
• 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, Sander, Jeffry D., Maeder, Morgan, Fu, Yanfang
• Primary Examiner(s): Babic, Christopher M
• Assistant Examiner(s): Aron, Kimberly A.
• Application Number:
  US15/430,218
• Publication Number:
  US 20170152508A1
• Time in Patent Office: 949 Days
• Field of Search: None
• 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, using truncated guide RNAs (tru-gRNAs).

Claims

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.