CRISPR-related methods and compositions with governing gRNAs

Patent metadata

Field	Value
Publication number	US-10640788-B2
Application number	US-201916256615-A
Country	US
Kind code	B2
Filing date	Jan 24, 2019
Priority date	Nov 7, 2013
Publication date	May 5, 2020
Grant date	May 5, 2020

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 composition comprising a governing gRNA molecule that comprises a nucleotide sequence targeting domain that targets: a nucleic acid encoding a S. aureus Cas9 molecule or a S. pyogenes Cas9 molecule; and/or a nucleic acid encoding a gRNA molecule that targets the nucleic acid encoding the S. aureus or S. pyogenes Cas9 molecule.

2. The composition of claim 1 , wherein the nucleotide sequence targeting domain is 15 to 30 nucleotides in length.

3. The composition of claim 1 , 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 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.

4. The composition of claim 1 , wherein the nucleotide sequence targeting domain of the governing gRNA molecule is 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.

5. The composition of claim 1 , wherein the nucleotide sequence targeting domain of the governing gRNA molecule is 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.

6. The composition of claim 1 , wherein the nucleotide sequence targeting domain of the governing gRNA molecule is 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.

7. The composition of claim 1 , wherein the nucleotide sequence targeting domain of the governing gRNA molecule is 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.

8. The composition of claim 1 , wherein the nucleotide sequence targeting domain of the governing gRNA molecule is 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.

9. The composition of claim 1 , wherein the nucleotide sequence targeting domain of the governing gRNA molecule is 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.

10. The composition of claim 1 , wherein the nucleotide sequence targeting domain of the governing gRNA molecule is 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.

11. The composition of claim 1 , wherein the governing gRNA targets a naturally occurring sequence in the nucleic acid encoding the Cas9.

12. A composition comprising a governing gRNA molecule or a second nucleic acid encoding a governing gRNA molecule, the governing gRNA comprising a nucleotide sequence targeting domain that targets: a nucleic acid encoding a S. aureus or S. pyogenes Cas9 molecule; and/or a nucleic acid encoding a gRNA molecule that targets the nucleic acid encoding the S. aureus or S. pyogenes Cas9 molecule.

13. The composition of claim 12 , wherein the nucleotide sequence targeting domain is 15 to 30 nucleotides in length.

14. The composition of claim 12 , 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 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.

15. The composition of claim 12 , wherein the nucleotide sequence targeting domain of the governing gRNA molecule is 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.

16. The composition of claim 12 , wherein the nucleotide sequence targeting domain of the governing gRNA molecule is 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.

17. The composition of claim 12 , wherein the nucleotide sequence targeting domain of the governing gRNA molecule is 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.

18. The composition of claim 12 , wherein the nucleotide sequence targeting domain of the governing gRNA molecule is 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.

19. The composition of claim 12 , wherein the nucleotide sequence targeting domain of the governing gRNA molecule is 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 molecu

Assignees

• Editas Medicine Inc
• Broad Inst Inc
• The Univ Of Iowa Research Foundation
• Massachusetts Inst Technology
• Univ Of Iowa Reseach Foundation

Inventors

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

Classifications

• C12N2310/20

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

• C12N15/1137 Primary

  against enzymes (viral enzymes C12N15/1131; receptors C12N15/1138) · CPC title

• C12N15/111

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

• C12N15/902 Primary

  using homologous recombination · CPC title

• C12N15/63

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