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

• US 20140186958A1
• Filed: 12/12/2013
• Published: 07/03/2014
• 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: Fei Ran
• Inventors: Zhang, Feng, Cong, Le, Platt, Randall Jeffrey, Sanjana, Neville Espi, Ran, Fei
• Granted Patent: US 8,993,233 B2
• Time in Patent Office: Days
• US Class Current: 435/462
• 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 non-naturally occurring or engineered composition comprising a vector system comprising one or more vectors comprising

I. a first regulatory element operably linked to a CRISPR-Cas system chimeric RNA (chiRNA) polynucleotide sequence, wherein the polynucleotide sequence comprises

(a) a guide sequence capable of hybridizing to a target sequence in a cell,

(b) a tracr mate sequence, and

(c) a tracr sequence, and

II. a second regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme comprising at least one or more nuclear localization sequences,

wherein (a), (b) and (c) are arranged in a 5′

to 3′

orientation,

wherein components I and II are located on the same or different vectors of the system,

wherein when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence,

wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence,

wherein the CRISPR enzyme comprises two or more mutations, such that the enzyme has altered nuclease compared with the wild type enzyme, and

wherein the enzyme-coding sequence further encodes one or more heterologous functional domains.

5. The composition of any preceding claim, wherein the CRISPR enzyme comprises one or more mutations in two or more catalytically active domains.

6. The composition of any preceding claim, wherein the CRISPR enzyme has reduced or abolished nuclease activity compared with the wild type enzyme.

7. The composition of any preceding claim, wherein the two mutations are D10A SpCas9 in a first catalytically active domain and H840A SpCas9 in a second catalytically active domain or corresponding residues of other CRISPR enzymes.

8. The composition of any preceding claim, wherein the CRISPR enzyme comprises two or more mutations in a residue selected from the group consisting of D10, E762, H840, N854, N863, or D986.

9. The composition of any preceding claim, wherein the CRISPR enzyme comprises two or more mutations selected from the group comprising D10A, E762A, H840A, N854A, N863A or D986A.

10. The composition of any preceding claim, wherein the CRISPR enzyme is a DNA binding protein that does not direct cleavage of either strand at the location of the target sequence.

11. The composition of any preceding claim, wherein each of the two or more mutations is in a catalytically active domain of the CRISPR enzyme selected from the group comprising RuvCI, RuvCII, RuvCIII or HNH domain.

12. The composition of any preceding claim, further comprising at least two or more nuclear localization sequences.

13. The composition of any preceding claim, wherein the functional domain is a transcriptional activation domain

14. The composition of claim 13, wherein the transcriptional activation domain is VP64.

15. The composition of any preceding claim, wherein the enzyme coding sequence encodes one or more heterologous functional domains fused to the CRISPR enzyme.

16. The composition of any preceding claim, wherein the enzyme coding sequence encodes two or more heterologous functional domains.

17. The composition of any preceding claim, wherein the enzyme coding sequence further comprises one or more linker sequences between any two domains.

18. The composition of any preceding claim, wherein the functional domain binds DNA.

19. The composition of any preceding claim, wherein the functional domain affects transcription of the target nucleic acid.

20. The composition of any preceding claim, wherein the cell is a prokaryotic or eukaryotic cell.

21. The composition of any preceding claim, wherein the cell is a eukaryotic cell.

22. The composition of claim 21, wherein the eukaryotic cell is a mammalian cell.

23. The composition of claim 22, wherein the mammalian cell is a human cell.

24. The composition of any preceding claim, wherein the CRISPR enzyme is codon optimized for expression in a eukaryotic cell.

25. The composition of any preceding claim, wherein the CRISPR enzyme is a type II CRISPR enzyme.

26. The composition of any preceding claim, wherein the CRISPR enzyme is a Cas9 enzyme.

27. The composition of any preceding claim, wherein the Cas9 enzyme is from an organism selected from the group comprising of genus Streptococcus, Campylobacter, Nitratifractor, Staphylococcus, Parvibaculum, Roseburia, Neisseria, Gluconacetobacter, Azospirillum, Sphaerochaeta, Lactobacillus, Eubacterium or Corynebacter.

28. The composition of any preceding claim, wherein the vectors of the system are viral vectors selected from the group comprising of a lentiviral vector, an adenoviral vector or an AAV vector.

29. A method of modulating gene expression at a genomic locus of interest in a cell by contacting the cell with the composition of any of the preceding claims.

2. A multiplexed CRISPR enzyme system composition comprising a vector system comprising one or more vectors comprising

I. a first regulatory element operably linked to a CRISPR-Cas system chimeric RNA (chiRNA) polynucleotide sequence, wherein the polynucleotide sequence comprises

(a) a guide sequence capable of hybridizing to a target sequence in a cell,

(b) a tracr mate sequence, and

(c) a tracr sequence, and

II. a second regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme comprising at least one or more nuclear localization sequences,

wherein (a), (b) and (c) are arranged in a 5′

to 3′

orientation,

wherein components I and II are located on the same or different vectors of the system,

wherein when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence,

wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence,

wherein the CRISPR enzyme comprises two or more mutations, such that the enzyme has altered nuclease activity compared with the wild type enzyme,

wherein the enzyme-coding sequence further encodes one or more heterologous functional domains, and

wherein in the multiplexed system composition multiple chiRNA polynucleotide sequence are used.

3. A non-naturally occurring or engineered composition comprising a vector system comprising one or more vectors comprising

I. a first regulatory element operably linked to

(a) a guide sequence capable of hybridizing to a target sequence in a cell, and

(b) at least one or more tracr mate sequences,

II. a second regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme, and

III. a third regulatory element operably linked to a tracr sequence,

wherein components I, II and III are located on the same or different vectors of the system,

wherein when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence,

wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence,

wherein the CRISPR enzyme comprises two or more mutations, such that the enzyme has altered nuclease compared with the wild type enzyme, and

wherein the enzyme-coding sequence further encodes one or more heterologous functional domains.

4. A multiplexed CRISPR enzyme system composition comprising a vector system comprising one or more vectors comprising

I. a first regulatory element operably linked to

(a) a guide sequence capable of hybridizing to a target sequence in a cell, and

(b) at least one or more tracr mate sequences,

II. a second regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme, and

III. a third regulatory element operably linked to a tracr sequence,

wherein components I, II and III are located on the same or different vectors of the system,

wherein when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence,

wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence,

wherein the CRISPR enzyme comprises two or more mutations, such that the enzyme has altered nuclease compared with the wild type enzyme,

wherein the enzyme-coding sequence further encodes one or more heterologous functional domains, and

wherein in the multiplexed system composition multiple guide sequences capable of hybridizing to multiple target sequences are used.