CRISPR-CAS SYSTEMS AND METHODS FOR ALTERING EXPRESSION OF GENE PRODUCTS

• US 20140227787A1
• Filed: 04/18/2014
• Published: 08/14/2014
• Est. Priority Date: 12/12/2012
• Status: Active Grant

• Current Assignee: Massachusetts Institute of Technology, The Broad Institute, Inc.
• Original Assignee: Massachusetts Institute of Technology, The Broad Institute, Inc.
• Inventors: Zhang, Feng
• Granted Patent: US 8,945,839 B2
• Time in Patent Office: Days
• US Class Current: 435/456
• CPC Class Codes: A61K 38/43 Enzymes; Proenzymes; Derivatives thereof
• CPC Class Codes: A61K 38/46 Hydrolases (3)
• CPC Class Codes: A61K 38/47 acting on glycosyl compounds (3.2), e.g. cellulases, lactases
• CPC Class Codes: C12N 15/00 Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor mutants or genetically engineered microorganisms, per se C12N1/00, C12N5/00, C12N7/00; new plants per se A01H; plant reproduction by tissue culture techniques A01H4/00; new animals per se A01K67/00; use of medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases, gene therapy A61K48/00
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 15/1082 Preparation or screening gene libraries by chromosomal integration of polynucleotide sequences, HR-, site-specific-recombination, transposons, viral vectors
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• 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 15/902 using homologous recombination
• CPC Class Codes: C12N 15/907 in mammalian cells
• 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 2800/30 Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT
• CPC Class Codes: C12N 2800/80 Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
• CPC Class Codes: C12N 2800/90 Vectors containing a transposable element
• CPC Class Codes: C12N 9/14 Hydrolases (3)
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12N 9/52 derived from bacteria or Archaea
• CPC Class Codes: C12N 9/96 Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
• CPC Class Codes: C12Q 1/68 : involving nucleic acids

Abstract

The invention provides for systems, methods, and compositions for altering expression of target gene sequences and related gene products. Provided are vectors and vector systems, some of which encode one or more components of a CRISPR complex, as well as methods for the design and use of such vectors. Also provided are methods of directing CRISPR complex formation in eukaryotic cells and methods for utilizing the CRISPR-Cas system.

Claims

1. A method of directing sequence-specific binding to one or more polynucleotide loci comprising a target sequence in a eukaryotic cell by introducing into the cell an engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) system comprising one or more vectors comprising:

a) a first regulatory element operable in a eukaryotic cell operably linked to at least one nucleotide sequence encoding a CRISPR-Cas system guide RNA that hybridizes with the target sequence, and

b) a second regulatory element operable in a eukaryotic cell operably linked to a nucleotide sequence encoding a Type-II Cas9 protein,

wherein components (a) and (b) are located on same or different vectors of the system,

wherein the Cas9 protein is a mutated Cas9 protein substantially lacking all DNA cleavage activity,

whereby the guide RNA targets and hybridizes with the target sequence and the CRISPR-Cas system specifically binds to the polynucleotide loci; and

, wherein the Cas9 protein and the guide RNA do not naturally occur together.

2. The method of claim 1, wherein two or more catalytic domains of the Cas9 protein are mutated.

3. The method of claim 2, wherein a RuvC1 catalytic domain of the Cas9 protein comprises the D10A mutation and the HNH catalytic domain comprises the H840A mutation.

4. The method of claim 1, wherein the CRISPR-Cas system binds to two or more polynucleotide loci.

5. The method of claim 1, wherein the CRISPR-Cas system further comprises one or more nuclear localization signal(s) (NLS(s)).

6. The method of claim 1, wherein the CRISPR-Cas system comprises a trans-activating cr (tracr) sequence.

8. The method of claim 1, wherein the Cas9 protein is codon optimized for expression in the eukaryotic cell.

9. The method of claim 1, wherein the eukaryotic cell is a mammalian or human cell.

10. The method of claim 1, wherein the one or more vectors are viral vectors.

11. The method of claim 10, wherein the one or more viral vectors are selected from the group consisting of retroviral, lentiviral, adenoviral, adeno-associated and herpes simplex viral vectors.

12. An engineered, non-naturally occurring CRISPR-Cas system comprising one or more vectors comprising:

a) a first regulatory element operable in a eukaryotic cell operably linked to at least one nucleotide sequence encoding a CRISPR-Cas system guide RNA that hybridizes with a target sequence of one or more DNA molecules in a eukaryotic cell that contains the DNA molecule, wherein the DNA molecule encodes and the eukaryotic cell expresses at least one gene product,

b) a second regulatory element operable in a eukaryotic cell operably linked to a nucleotide sequence encoding a Type-II Cas9 protein,

wherein components (a) and (b) are located on same or different vectors of the system,

wherein the Cas9 protein is a mutated Cas9 protein substantially lacking all DNA cleavage activity,

whereby the guide RNA targets and hybridizes with the target sequence and the CRISPR-Cas system specifically binds to the DNA molecule, and, wherein the Cas9 protein and the guide RNA do not naturally occur together.

13. The system of claim 12, wherein two or more catalytic domains of the Cas9 protein are mutated.

14. The system of claim 13, wherein a RuvC1 catalytic domain of the Cas9 protein comprises a D10A mutation and a HNH catalytic domain of the Cas9 protein comprises a H840A mutation.

15. The system of claim 12, wherein the CRISPR-Cas system binds to two or more DNA molecules.

16. The system of claim 12, wherein the CRISPR-Cas system further comprises one or more NLS(s).

17. The system of claim 12, wherein the CRISPR-Cas system comprises a tracr sequence.

19. The system of claim 12, wherein the Cas9 protein is codon optimized for expression in the eukaryotic cell.

20. The system of claim 12, wherein the eukaryotic cell is a mammalian or human cell.

21. The system of claim 12, wherein the one or more vectors are viral vectors.

22. The system of claim 21, wherein the one or more viral vectors are selected from the group consisting of retroviral, lentiviral, adenoviral, adeno-associated and herpes simplex viral vectors.

23. An engineered, programmable, non-naturally occurring Type II CRISPR-Cas system comprising a Cas9 protein and at least one guide RNA that targets and hybridizes to a target sequence of one or more DNA molecules in a eukaryotic cell, wherein the Cas9 protein is a mutated Cas9 protein substantially lacking all DNA cleavage activity, wherein the CRISPR-Cas system specifically binds to the DNA molecule, and wherein the Cas9 protein and the guide RNA do not naturally occur together.

24. The CRISPR-Cas system of claim 23, wherein two or more catalytic domains of the Cas9 protein are mutated.

25. The CRISPR-Cas system of claim 24, wherein a RuvC1 catalytic domain of the Cas9 protein comprises a D10A mutation and a HNH catalytic domain of the Cas9 protein comprises a H840A mutation.

26. The CRISPR-Cas system of claim 23, wherein the CRISPR-Cas system binds to two or more DNA molecules.

27. The CRISPR-Cas system of claim 23, wherein the CRISPR-Cas system further comprises one or more NLS(s).

28. The CRISPR-Cas system of claim 23, wherein the CRISPR-Cas system comprises a tracr sequence.

29. The CRISPR-Cas system of claim 23, wherein the Cas9 protein is codon optimized for expression in the eukaryotic cell.

30. The CRISPR-Cas system of claim 23, wherein the eukaryotic cell is a mammalian or human cell.