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

• US 9,567,604 B2
• Filed: 03/14/2014
• Issued: 02/14/2017
• 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:
  US14/213,723
• Publication Number:
  US 20140295557A1
• Time in Patent Office: 1,068 Days
• Field of Search: None
• US Class Current: 1/1
• 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 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.