Engineering and optimization of improved systems, methods and enzyme compositions for sequence manipulation

• US 8,889,418 B2
• Filed: 06/02/2014
• Issued: 11/18/2014
• 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, Ran, Fei
• Primary Examiner(s): Rao, Manjunath
• Assistant Examiner(s): HOLLAND, PAUL J
• Application Number:
  US14/293,674
• Publication Number:
  US 20140273234A1
• Time in Patent Office: 169 Days
• Field of Search: 435/6.1, 435/6.13, 435/195, 435/196, 435/199, 435/320.1, 435/220, 424/94.61, 536/23.1, 536/23.2, 536/23.7, 536/24.1
• US Class Current: 435/455
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: A61P 27/02 Ophthalmic agents
• 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 15/8213 Targeted insertion of genes into the plant genome by homologous recombination
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 2750/14143 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2800/22 Vectors comprising a coding region that has been codon optimised for expression in a respective host
• CPC Class Codes: C12N 2810/10 Vectors comprising a non-peptidic targeting moiety
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12N 9/96 Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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 compositions and methods related to components of a CRISPR complex particularly comprising a Cas ortholog enzyme.

Claims

1. A modified Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) enzyme wherein the enzyme is a Cas9 protein that is a chimeric CRISPR enzyme in that it comprises a first fragment and a second fragment, wherein each of the first and second fragments is from a different Cas9 protein from a bacteria belonging to the group consisting of Corynebacter, Sutterella, Legionella, Treponema, Filifactor, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, Sphaerochaeta, Azospirillum, Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Staphylococcus, Nitratifractor, and Campylobacter.

2. The modified CRISPR enzyme of claim 1 wherein the C terminus of the modified CRISPR enzyme is from a Streptococcus pyogenes (Spy Cas9 enzyme.

3. The modified CRISPR enzyme of claim 1 wherein the N terminus of the modified CRISPR enzyme is from a Sp Cas9 enzyme.

4. The modified CRISPR enzyme of claim 1 wherein the modified CRISPR enzyme includes a RuvC I domain of a Sp Cas9 enzyme.

5. The modified CRISPR enzyme of claim 1 wherein the modified CRISPR enzyme includes a RuvC II domain of a Sp Cas9 enzyme.

6. The modified CRISPR enzyme of claim 1 wherein the modified CRISPR enzyme includes a RuvC III domain of a Sp Cas9 enzyme.

7. The modified CRISPR enzyme of claim 1 wherein the modified CRISPR enzyme includes a HNH domain of a Sp Cas9 enzyme.

8. The modified CRISPR enzyme of claim 1 wherein the N terminus of the modified CRISPR enzyme is from a Streptococcus thermophilus (St) Cas9 enzyme.

9. The modified CRISPR enzyme of claim 1 wherein the C terminus of the modified CRISPR enzyme is from a St Cas9 enzyme.

10. The modified CRISPR enzyme of claim 1 wherein the modified CRISPR enzyme includes a RuvC I domain of a St Cas9 enzyme.

11. The modified CRISPR enzyme of claim 1 wherein the modified CRISPR enzyme includes a RuvC II domain of a Sp Cas9 enzyme.

12. The modified CRISPR enzyme of claim 1 wherein the modified CRISPR enzyme includes a RuvC III domain of a St Cas9 enzyme.

13. The modified CRISPR enzyme of claim 1 wherein the modified CRISPR enzyme includes a HNH domain of a St Cas9 enzyme.

14. The modified CRISPR enzyme of claim 1 wherein a protospacer adjacent motif (PAM) recognized by the CRISPR enzyme comprises 5′

-NGG, wherein N is any nucleotide.

15. The modified CRISPR enzyme of claim 1 wherein a protospacer adjacent motif (PAM) recognized by the CRISPR enzyme comprises 5′

-NRG, wherein N is any nucleotide and R is a purine.

16. The modified CRISPR enzyme of claim 1 wherein a protospacer adjacent motif (PAM) recognized by the CRISPR enzyme comprises 5′

-NNGRR, wherein N is any nucleotide and R is a purine.

17. The modified CRISPR enzyme of claim 1 wherein a protospacer adjacent motif (PAM) recognized by the CRISPR enzyme comprises a PAM sequence set forth in FIG. 7 or FIG. 13A-II.

18. The modified CRISPR enzyme of claim 1 wherein a protospacer adjacent motif (PAM) recognized by the CRISPR enzyme comprises 5′

-GAAA, 5′

-GAAC, 5′

-GAAG, 5′

-GAAT, 5′

-GAGA, 5′

-GAGC, 5′

-GAGG, or 5′

-GGAA.

19. The modified CRISPR enzyme of claim 1 wherein the chimera comprises a Sp_St3 chimera.

20. The modified CRISPR enzyme of claim 1 wherein the chimera comprises a St3_Sp chimera.

21. A nucleic acid molecule encoding the modified CRISPR enzyme of claim 1, wherein the nucleic acid molecule is codon-optimized for expression in a eukaryotic cell.

22. A vector comprising:

a nucleic acid molecule encoding the modified CRISPR enzyme of claim 1 and further comprising a nucleic acid molecule(s) encoding one or more nuclear localization signal(s) (NLS(s)); and

a regulatory element operable in a eukaryotic cell operably linked to the nucleic acid molecules encoding the modified CRISPR enzyme and the NLS(s).

23. An engineered, programmable, non-naturally occurring Type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) (CRISPR-Cas) system that alters expression of at least one gene product in a eukaryotic cell containing and expressing a DNA molecule having a target sequence and encoding said gene product, wherein the CRISPR-Cas system comprises the modified CRISPR enzyme of claim 1.

24. The CRISPR-Cas system of claim 23 wherein when introduced into a cell having a DNA molecule, the CRISPR-Cas system cleaves the DNA, and the CRISPR-Cas system includes DNA for insertion into a cleaved strand of the DNA molecule.

25. A method of altering expression of at least one gene product in a eukaryotic cell containing and expressing a DNA molecule having a target sequence and encoding said gene product comprising introducing into said eukaryotic 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 the modified CRISPR enzyme of claim 1,

wherein said CRISPR-Cas system further comprises one or more nuclear localization signal(s) (NLS(s)), and components (a) and (b) are located on same or different vectors of the system;

whereby said guide RNA targets the target sequence and said CRISPR enzyme cleaves the DNA molecule;

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

, wherein said modified CRISPR enzyme and said guide RNA do not naturally occur together.

26. The method of claim 25, further comprising inserting DNA into a cleaved strand of the DNA molecule.

27. A CRISP R-Cas system-mediated genome targeting method in a eukaryotic cell containing a DNA molecule having a target sequence comprising introducing into said eukaryotic cell 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 the modified CRISPR enzyme of claim 1,

wherein said CRISPR-Cas system further comprises one or more nuclear localization signal(s) (NLS(s)), and components (a) and (b) are located on same or different vectors of the system;

the method further comprising inserting DNA into a cleaved strand of the DNA molecule;

whereby there is CRISPR-Cas system-mediated genome targeting through said CRISPR-Cas system acting as to the DNA molecule comprising said guide RNA directing sequence-specific binding of the CRISPR-Cas system, whereby there is genome editing; and

, wherein said modified CRISPR enzyme and said guide RNA do not naturally occur together.

28. The method of claim 27, wherein the eukaryotic cell is a mammalian or human cell.