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

• US 20160281072A1
• Filed: 05/20/2016
• Published: 09/29/2016
• Est. Priority Date: 12/12/2012
• Status: Active Application

• Current Assignee: Massachusetts Institute of Technology, The Broad Institute, Inc.
• Original Assignee: Massachusetts Institute of Technology, The Broad Institute, Inc.
• Inventors: ZHANG, FENG
• Application Number:
  US15/160,710
• Publication Number:
  US 20160281072A1
• Time in Patent Office: Days
• US Class Current: 1/1
• 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 non-naturally occurring or engineered composition comprising a delivery system operably configured to deliver an engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) (CRISPR-Cas) complex to a eukaryotic cell containing a DNA molecule having a target sequence adjacent to a Protospacer Adjacent Motif (PAM), the system comprising:

I. one or more regulatory elements operably linked to one or more CRISPR-Cas complex polynucleotide sequences comprising

(a) a guide sequence capable of hybridizing to the target sequence in the eukaryotic 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 Type II CRISPR enzyme,

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,

the guide sequence comprises more than about 10 nucleotides in length, and

the tracr sequence comprises more than about 30 nucleotides in length, and

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

the tracr sequence exhibits at least 50% sequence complementarity along the length of the tracr mate, and

the CRISPR enzyme and the guide RNA do not naturally occur together.

2. The composition according to claim 1, wherein the CRISPR-Cas complex polynucleotide sequences comprises a chimeric guide RNA comprising the guide sequence, the tracr sequence, and a tracr mate sequence.

3. The composition according to claim 1, wherein the expression of two or more gene products is altered.

4. The composition according to claim 1, wherein the eukaryotic cell is a mammalian cell.

5. The composition according to claim 1 wherein the eukaryotic cell is a human cell.

6. The composition according to claim 1, wherein the expression of one or more gene products is decreased.

7. The composition according to claim 1, wherein the tracr sequence comprises more than about 40 nucleotides in length.

8. The composition according to claim 1, wherein the tracr sequence comprises more than about 50 nucleotides in length

9. The composition according to claim 1, wherein the guide sequence comprises more than about 75 nucleotides in length.

10. The composition according to claim 8, wherein the guide sequence comprises more than about 75 nucleotides in length.

11. The composition according to claim 1, wherein the CRISPR enzyme comprises one or more mutations.

12. The composition according to claim 11, wherein the one or more mutations comprise one or more mutations of D10 with reference to Streptococcus pyogenes Cas9 (“

SpCas9”

), H840 SpCas9, N854 SpCas9 or N863 SpCas9.

13. The composition according to claim 12 wherein the one or more mutations comprise D10A SpCas9, H840A SpCas9, N854A SpCas9 or N863A SpCas9.

14. The composition according to claim 11 wherein the one or more mutations comprise two mutations.

15. The composition according to claim 14 wherein the mutations comprise D10A SpCas9 and H840A SpCas9, or corresponding residues of other CRISPR enzymes.

16. The composition according to claim 11, wherein the one or more mutations is in a catalytically active domain of the CRISPR enzyme comprising RuvCI, RuvCII or RuvCIII.

17. The composition according to claim 1, wherein the CRISPR complex includes one or more nuclear localization signals (NLS(s)).

18. The composition according to claim 1, wherein the CRISPR enzyme comprises a fusion of a Type II Cas9 protein and one or more effector domains.

19. The composition according to claim 18, wherein the one or more effector domains comprises one or more NLS(s).

20. The composition according to claim 19, wherein the one or more effector domains comprises a transposase domain, integrase domain, recombinase domain, resolvase domain, invertase domain, protease domain, DNA methyltransferase domain, DNA demethylase domain, histone acetylase domain, histone deacetylases domain, nuclease domain, repressor domain, activator domain, nuclear-localization signal domain, transcription-protein recruiting domain, cellular uptake activity associated domain, nucleic acid binding domain or antibody presentation domain.

21. The composition according to claim 1, which is a multiplexed composition comprising multiple guide sequences capable of hybridizing to multiple target sequences.

22. The composition according to claim 2, which is a multiplexed composition comprising multiple guide sequences capable of hybridizing to multiple target sequences.

23. The composition according to claim 1, wherein the delivery system comprises a vector system comprising one or more vectors, and wherein components I and II are located on the same or different vectors of the system;

or, wherein the delivery system comprises a nanoparticle, liposome, exosome, yeast system, microvesicle, or gene gun.

24. The composition according to claim 23, wherein the delivery system comprises a vector system comprising one or more vectors and components I and II are located on the same vector.

25. The composition according to claim 23, wherein the delivery system comprises a vector system comprising one or more vectors and the one or more vectors comprise one or more viral vectors, and the one or more viral vectors comprise one or more retrovirus, lentivirus, adenovirus, adeno-associated virus or herpes simplex virus vectors.

26. A eukaryotic cell comprising the composition or CRISPR complex of claim 1.

27. A cell culture from the cell of claim 26.

28. A eukaryotic cell translation product from the cell of claim 26.

29. A eukaryotic cell translation product from a cell of the culture of claim 27.

30. The composition of claim 1 wherein the PAM has a nucleotide sequence comprising NNAGAAW.