CRISPR-RELATED METHODS AND COMPOSITIONS WITH GOVERNING gRNAS

• US 20150232881A1
• Filed: 11/07/2014
• Published: 08/20/2015
• Est. Priority Date: 11/07/2013
• Status: Active Grant

• Current Assignee: Editas Medicine, Inc., Massachusetts Institute of Technology, The Broad Institute, Inc., University of Iowa Research Foundation
• Original Assignee: Editas Medicine, Inc.
• Inventors: Glucksmann, Alexandra, Tartaglia, Louis Anthony, Mata-Fink, Jordi, Borisy, Alexis, Palestrant, Deborah, Czechowicz, Agnieszka Dorota
• Granted Patent: US 9,834,791 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• 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 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 15/1137 against enzymes viral enzymes C12N15/1131; receptors C12N15/1138
• 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 15/902 using homologous recombination
• CPC Class Codes: C12N 2310/10 Type of nucleic acid
• CPC Class Codes: C12N 2310/20 involving clustered regularly interspaced short palindromic repeats [CRISPRs]
• CPC Class Codes: C12N 2800/80 Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

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.

Claims

1. A gRNA molecule that targets a nucleic acid sequence that encodes a Cas9 molecule.

2. The gRNA molecule of claim 1, wherein the nucleic acid sequence comprises one or more of:

a sequence encoding the amino acid sequence of the Cas9 molecule, a sequence encoding the amino acid sequence of the Cas9 molecule comprising non-translated sequence, or a sequence encoding the amino acid sequence of the Cas9 molecule comprising non-transcribed sequence.

3. The gRNA molecule of claim 1, wherein the Cas9 molecule is an enzymatically active Cas9 (eaCas9) molecule.

4. The gRNA molecule of claim 1, wherein the Cas9 molecule is an enzymatically inactive Cas9 (eiCas9) molecule.

6. The gRNA molecule of claim 1, wherein the gRNA molecule comprises a targeting domain configured to provide a Cas9 molecule-mediated cleavage event in the nucleic acid sequence that encodes the Cas9 molecule.

7. The gRNA molecule of claim 1, wherein the gRNA molecule comprises:

a targeting domain configured to provide a Cas9 molecule-mediated cleavage event in the Cas9 molecule-amino acid coding sequence of the nucleic acid sequence;

a targeting domain configured to provide a Cas9 molecule-mediated cleavage event in a non-coding sequence of the nucleic acid sequence;

a targeting domain configured to provide a Cas9 molecule-mediated cleavage event in an untranslated sequence of the nucleic acid sequence;

a targeting domain configured to provide a Cas9 molecule-mediated cleavage event 5′

of the Cas9 molecule-coding region of the nucleic acid sequence;

a targeting domain configured to provide a Cas9 molecule-mediated cleavage event 3′

of the Cas9 molecule-coding region of the nucleic acid sequence;

a targeting domain configured to provide a Cas9 molecule-mediated cleavage event in the promoter region of the nucleic acid sequence, wherein the promoter region is functionally linked to the Cas9 molecule amino acid coding region;

or

a targeting domain configured to provide a Cas9 molecule-mediated cleavage event in Cas9 molecule intronic sequence of the nucleic acid sequence.

14. The gRNA molecule of claim 1, wherein the Cas9 molecule is an S. pyogenes Cas9 molecule.

15. The gRNA molecule of claim 1, wherein the Cas9 molecule is an S. aureus Cas9 molecule.

16. The gRNA molecule of claim 1, wherein the gRNA is a chimeric gRNA.

17. The gRNA molecule of claim 1, wherein the gRNA is a modular gRNA.

62. A pharmaceutical preparation comprising

the gRNA molecule of claim 1.

63. A cell comprising

the gRNA molecule of claim 1.

70. The cell of claim 63, further comprising:

a template nucleic acid.

87. A method of altering a cell or a target nucleic acid of a cell, comprising contacting the cell with an effective amount of

the gRNA molecule of claim 1.

90. A method of treating a subject, the method comprising contacting a cell or a subject, with an effective amount of

the gRNA molecule of claim 1.

101. A reaction mixture comprising a cell and the gRNA molecule of claim 1.

102. A kit comprising

the gRNA molecule of claim 1.

5. (canceled)

8-13. -13. (canceled)

18-19. -19. (canceled)

20. A nucleic acid comprising a sequence that encodes a) a gRNA molecule that targets a nucleic acid sequence that encodes a Cas9.

21. The nucleic acid of claim 20, wherein the nucleic acid further comprises b) a nucleic acid sequence that encodes the Cas9 molecule.

33. The nucleic acid of claim 21, wherein component a) and component b) are provided on the same nucleic acid molecule.

34. The nucleic acid of claim 21, wherein component a) and component b) are provided on different nucleic acid molecules.

42. The nucleic acid of claim 21, further comprising:

c) a third nucleic acid sequence that encodes a second gRNA molecule comprising a targeting domain which is complementary with a target nucleic acid, wherein the second gRNA does not target b).

46. A vector comprising a nucleic acid of claim 20.

22-32. -32. (canceled)

35-41. -41. (canceled)

43-45. -45. (canceled)

47-48. -48. (canceled)

49. A composition, comprising:

a) a gRNA molecule that targets a nucleic acid sequence that encodes a Cas9 molecule or a nucleic acid that encodes a gRNA molecule that targets a nucleic acid sequence that encodes a Cas9 molecule.

50. The composition of claim 49, comprising one or more of;

b) a nucleic acid sequence that encodes the Cas9 molecule;

c) a second gRNA molecule or a nucleic acid encoding the second gRNA molecule; and

d) a template nucleic acid.

51-61. -61. (canceled)

64-69. -69. (canceled)

71-86. -86. (canceled)

88-89. -89. (canceled)

91-100. -100. (canceled)