ENGINEERING AND OPTIMIZATION OF SYSTEMS, METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION WITH FUNCTIONAL DOMAINS

• US 20140256046A1
• Filed: 03/26/2014
• Published: 09/11/2014
• Est. Priority Date: 12/12/2012
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation), Massachusetts Institute of Technology, The Broad Institute, Inc.
• Original Assignee: Massachusetts Institute of Technology, The Broad Institute, Inc.
• Inventors: Zhang, Feng, Cong, Le, Platt, Randall Jeffrey, Sanjana, Neville Espi
• Granted Patent: US 8,999,641 B2
• Time in Patent Office: Days
• US Class Current: 435/455
• 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 15/1082 Preparation or screening gene libraries by chromosomal integration of polynucleotide sequences, HR-, site-specific-recombination, transposons, viral vectors
• 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 15/86 Viral vectors
• CPC Class Codes: C12N 2320/30 Special therapeutic applications
• CPC Class Codes: C12N 2740/15043 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 7/00 Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

Abstract

The invention provides for engineering and optimization of systems, methods, and compositions for manipulation of sequences and/or activities of target sequences. 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 with additional functional domains. Also provided are methods of directing CRISPR complex formation in prokaryotic and eukaryotic cells to ensure enhanced specificity for target recognition and avoidance of toxicity.

Claims

1. A method of altering expression of at least one gene product comprising introducing into a eukaryotic cell containing and expressing a DNA molecule having a target sequence and encoding the gene product 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 comprises two or more mutations and is a DNA binding protein that does not direct cleavage of the DNA molecule,

wherein the CRISPR-Cas system comprises a nucleotide sequence encoding one or more activator domains,

whereby expression of the at least one gene product is altered; and

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

2. The method of claim 1, wherein the CRISPR-Cas system further comprises a flexible linker between the Cas9 protein and the one or more activator domains.

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

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

5. The method of claim 1, wherein the guide RNA comprises a guide sequence and a tracr sequence.

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

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

8. The method of claim 1, wherein the Cas9 protein comprises two or more mutations selected from the group consisting of D10A, E762A, H840A, N854A, N863A and D986A with reference to the position numbering of a Streptococcus pyogenes Cas9 protein.

9. The method of claim 1, wherein the expression of one or more gene products is increased.

10. The method of claim 1, wherein the expression of one or more gene products is decreased.

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

12. The method of claim 1, wherein the viral vectors are selected from the group consisting of retroviral, lentiviral, adenoviral, adeno-associated and herpes simplex viral vectors.

13. A CRISPR-Cas system-mediated genome targeting method comprising introducing into a eukaryotic cell containing and expressing a DNA molecule having a target sequence and encoding at least one gene product 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 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 comprises two or more mutations and is a DNA binding protein that does not direct cleavage of the DNA molecule,

wherein the CRISPR-Cas system further comprises a nucleotide sequence encoding one or more heterologous functional domains,

wherein the one or more heterologous functional domains is an activator domain,

whereby expression of the at least one gene product is altered through the CRISPR-Cas system acting as to the DNA molecule comprising the guide RNA directing sequence-specific binding of the CRISPR-Cas system, whereby there is genome targeting; and

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

14. The method of claim 13, wherein the CRISPR-Cas system further comprises a flexible linker between the Cas9 protein and the one or more activator domains.

15. The method of claim 13, wherein the CRISPR-Cas system further comprises one or more NLS(s).

16. The method of claim 13, wherein the CRISPR-Cas system comprises a tracr sequence.

17. The method of claim 13, wherein the Cas9 protein is codon optimized for expression in the eukaryotic cell.

18. The method of claim 13, wherein the eukaryotic cell is a mammalian or human cell.

19. The method of claim 13, wherein the expression of one or more gene products is increased.

20. The method of claim 13, wherein the expression of one or more gene products is decreased.

21. The method of claim 13, wherein the Cas9 protein comprises two or more mutations selected from the group consisting of D10A, E762A, H840A, N854A, N863A and D986A with reference to the position numbering of a Streptococcus pyogenes Cas9 protein.

22. The method of claim 13, wherein the one or more vectors are viral vectors.

23. The method of claim 13, wherein the viral vectors are selected from the group consisting of retroviral, lentiviral, adenoviral, adeno-associated and herpes simplex viral vectors.

24. 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 a DNA molecule in a eukaryotic cell, wherein the DNA molecule encodes and the eukaryotic cell expresses at least one gene product, wherein the CRISPR-Cas system comprises a tracr sequence, wherein the Cas9 protein comprises two or more mutations and is a DNA binding protein that does not direct cleavage of the DNA molecule, wherein the CRISPR-Cas system further comprises a nucleotide sequence encoding one or more activator domains, whereby expression of the at least one gene product is altered;

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

25. The CRISPR-Cas system of claim 24, wherein the CRISPR-Cas system further comprises a flexible linker between the Cas9 protein and the one or more activator domains.

26. The CRISPR-Cas system of claim 24, wherein the CRISPR-Cas system further comprises one or more NLS(s).

27. The CRISPR-Cas system of claim 24, wherein the Cas9 protein is codon optimized for expression in the eukaryotic cell.

28. The CRISPR-Cas system of claim 24, wherein the eukaryotic cell is a mammalian or human cell.

29. The CRISPR-Cas system of claim 24, wherein the expression of one or more gene products is increased.

30. The CRISPR-Cas system of claim 24, wherein the expression of one or more gene products is decreased.