CRISPR-related methods and compositions with governing gRNAS

Patent metadata

Field	Value
Publication number	US-9834791-B2
Application number	US-201414536319-A
Country	US
Kind code	B2
Filing date	Nov 7, 2014
Priority date	Nov 7, 2013
Publication date	Dec 5, 2017
Grant date	Dec 5, 2017

Abstract

Disclosed herein are methods and compositions useful in targeting a payload to, or editing a target nucleic acid, where a governing gRNA molecule is used to target, optionally inactivate, a Cas9 molecule or a Cas9 molecule/gRNA complex.

First claim

What is claimed is:

1. A method of silencing a component of a CRISPR/Cas system in a eukaryotic cell, the method comprising:
contacting the eukaryotic cell with:
(a) a nucleic acid encoding a Cas9 molecule and a nucleic acid encoding a first guide RNA (gRNA) molecule; and
(b) a governing gRNA molecule that comprises a nucleotide sequence targeting domain that targets the nucleic acid encoding the Cas9 molecule and/or the nucleic acid encoding said first gRNA molecule,

wherein
(i) the Cas9 molecule and the first gRNA molecule form a complex that cleaves a target nucleic acid of the eukaryotic cell,
(ii) the Cas9 molecule and the governing gRNA molecule form a complex that cleaves and thereby silences the nucleic acid encoding the Cas9 molecule and/or the nucleic acid encoding the first gRNA, and
(iii) the nucleotide sequence targeting domain of the governing gRNA molecule is selected from the group consisting of:
a nucleotide sequence targeting domain that provides a Cas9 molecule-mediated cleavage event in the Cas9 molecule-amino acid coding sequence of the nucleic acid sequence;
a nucleotide sequence targeting domain that provides a Cas9 molecule-mediated cleavage event in a non-coding sequence of the nucleic acid encoding the Cas9 molecule;
a nucleotide sequence targeting domain that provides a Cas9 molecule-mediated cleavage event in an untranslated sequence of the nucleic acid encoding the Cas9 molecule;
a nucleotide sequence targeting domain that provides a Cas9 molecule-mediated cleavage event 5′ of the Cas9 molecule-coding region of the nucleic acid encoding the Cas9 molecule;
a nucleotide sequence targeting domain that provides a Cas9 molecule-mediated cleavage event 3′ of the Cas9 molecule-coding region of the nucleic acid encoding the Cas9 molecule;
a nucleotide sequence targeting domain that provides a Cas9 molecule-mediated cleavage event in the promoter region of the nucleic acid encoding the Cas9 molecule, wherein the promoter region is functionally linked to the Cas9 molecule amino acid coding region; and
a nucleotide sequence targeting domain that provides a Cas9 molecule-mediated cleavage event in an intronic sequence of the nucleic acid encoding the Cas9 molecule.

2. The method of claim 1 , wherein the Cas9 molecule is an S. pyogenes Cas9 molecule.

3. The method of claim 1 , wherein the Cas9 molecule is an S. aureus Cas9 molecule.

4. The method of claim 1 , wherein the gRNA is a chimeric gRNA.

5. The method of claim 1 , wherein the gRNA is a modular gRNA.

6. The method of claim 1 , wherein the nucleic acid encoding said Cas9 molecule is carried by a first vector.

7. The method of claim 6 , comprising contacting the cell with the nucleic acid encoding said gRNA molecule, wherein the nucleic acid encoding said gRNA molecule is carried by a second vector.

8. The method of claim 1 , further comprising the step of contacting the cell with at least one of a gRNA molecule that targets a sequence in the genome of the cell and a nucleic acid encoding a gRNA molecule that targets a sequence in the genome of the cell.

9. A method of treating a subject, the method comprising:
altering a eukaryotic cell or a target nucleic acid of a eukaryotic cell by contacting the eukaryotic cell or target nucleic acid of the eukaryotic cell with a CRISPR/Cas system comprising a first nucleic acid encoding a Cas9 and a first guide RNA (gRNA);
silencing the CRISPR/Cas system by additionally contacting the eukaryotic cell of the subject with:
a governing gRNA molecule comprising a nucleotide sequence targeting domain that targets the first nucleic acid encoding the Cas9 molecule and/or a nucleic acid encoding said first gRNA molecule,
wherein the Cas9 molecule and the governing gRNA form a complex that cleaves the first nucleic acid encoding the Cas9 molecule and/or the nucleic acid encoding said first gRNA molecule, thereby silencing alteration of the eukaryotic cell or a target nucleic acid of the eukaryotic cell while treating the subject, and

wherein the nucleotide sequence targeting domain of the governing gRNA molecule is selected from the group consisting of:
a nucleotide sequence targeting domain that provides a Cas9 molecule-mediated cleavage event in the Cas9 molecule-amino acid coding sequence of the nucleic acid sequence;
a nucleotide sequence targeting domain that provides a Cas9 molecule-mediated cleavage event in a non-coding sequence of the first nucleic acid encoding the Cas9 molecule;
a nucleotide sequence targeting domain that provides a Cas9 molecule-mediated cleavage event in an untranslated sequence of the first nucleic acid encoding the Cas9 molecule;
a nucleotide sequence targeting domain that provides a Cas9 molecule-mediated cleavage event 5′ of the Cas9 molecule-coding region of the first nucleic acid encoding the Cas9 molecule;
a nucleotide sequence targeting domain that provides a Cas9 molecule-mediated cleavage event 3′ of the Cas9 molecule-coding region of the first nucleic acid encoding the Cas9 molecule;
a nucleotide sequence targeting domain that provides a Cas9 molecule-mediated cleavage event in the promoter region of the first nucleic acid encoding the Cas9 molecule, wherein the promoter region is functionally linked to the Cas9 molecule amino acid coding region; and
a nucleotide sequence targeting domain that provides a Cas9 molecule-mediated cleavage event in an intronic sequence of the first nucleic acid encoding the Cas9 molecule.

10. The method of claim 9 , wherein the Cas9 molecule is an S. pyogenes Cas9 molecule.

11. The method of claim 9 , wherein the Cas9 molecule is an S. aureus Cas9 molecule.

12. The method of claim 9 , wherein the gRNA is a chimeric gRNA.

13. The method of claim 9 , wherein the gRNA is a modular gRNA.

14. The method of claim 9 , wherein the first nucleic acid encoding said Cas9 molecule is carried by a first vector.

15. The method of claim 14 , comprising contacting the cell with the nucleic acid encoding said gRNA molecule, wherein the nucleic acid encoding said gRNA molecule is carried by a second vector.

16. The method of claim 9 , further comprising the step of contacting the cell or subject with at least one of a gRNA molecule that targets a sequence in the genome of the cell or subject and a nucleic acid encoding a gRNA molecule that targets a sequence in the genome of the cell.

17. A method of inactivating a Cas9 of a CRISPR/Cas system in a eukaryotic cell, comprising
contacting the eukaryotic cell with:
a first nucleic acid encoding a Cas9;
a second nucleic acid encoding a first guide RNA (gRNA) targeted to a sequence in a genome of the eukaryotic cell; and
a third nucleic acid encoding a governing gRNA targeted to a nucleic acid encoding the Cas9,
wherein the contacting drives expression of the Cas9, the first gRNA and the governing gRNA,
wherein the Cas9 and the governing gRNA form a complex; and
wherein the complex is capable of cleaving the first nucleic acid, thereby inactivating the first nucleic acid and inactivating the Cas9 component of the CRISPR/Cas system.

18. The method of claim 17 , wherein the first nucleic acid is carried by a first vector.

19. The method of claim 18 , wherein the second nucleic acid is carried by a second vector.

20. The method of claim 17 , wherein the nucleic acid encoding the Cas9 and the nucleic acid encoding the governing gRNA are carried on the same vector.

21. A method of silencing a component of the CRISPR/Cas system in a eukaryotic cell

Assignees

• Editas Medicine Inc
• Broad Inst Inc
• Massachusetts Inst Technology
• Univ Iowa Res Found

Inventors

• Zhang Feng
• Palestrant Deborah
• Davidson Beverly
• Mata-Fink Jordi
• Rodriguez Edgardo
• Borisy Alexis

Classifications

• C12N2310/10

  Type of nucleic acid · CPC title

• C12N2310/20

  involving clustered regularly interspaced short palindromic repeats [CRISPR] · CPC title

• C12N15/902 Primary

  using homologous recombination · CPC title

• C12N15/111

  General methods applicable to biologically active non-coding nucleic acids · CPC title

• C12N15/63

  Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression · CPC title