CRISPR-Cas Nickase Systems, Methods And Compositions For Sequence Manipulation in Eukaryotes

• US 20140310830A1
• Filed: 12/12/2013
• Published: 10/16/2014
• Est. Priority Date: 12/12/2012
• Status: Abandoned Application

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation), Massachusetts Institute of Technology, The Broad Institute, Inc.
• Original Assignee: Feng Zhang, Le Cong
• Inventors: ZHANG, Feng, CONG, Le, RAN, Fei
• Application Number:
  US14/105,031
• Publication Number:
  US 20140310830A1
• Time in Patent Office: Days
• US Class Current: 800/18
• 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 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• 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 15/8509 for producing genetically modified animals, e.g. transgenic
• 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 2310/3519 Fusion with another nucleic acid
• CPC Class Codes: C12N 2310/531 Stem-loop; Hairpin
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

Abstract

The invention provides for 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. Also provided are methods of directing CRISPR complex formation in eukaryotic cells and methods for selecting specific cells by introducing precise mutations utilizing the CRISPR/Cas system.

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 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 CRISPR enzyme comprising at least one or more nuclear localization sequences (NLSs) in the proximity of a terminus of the CRISPR enzyme,

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 one or more mutations in a catalytic domain thereby rendering the CRISPR enzyme to a nickase that cleaves a single DNA strand, and

wherein the chimeric RNA polynucleotide sequence comprises two or more hairpins.

3. The composition of claim 1 or 2, wherein the first regulatory element is a polymerase III promoter.

4. The composition of claim 1 or 2, wherein the second regulatory element is a polymerase II promoter.

5. The composition of claim 1 or 2, wherein the CRISPR enzyme comprises one or more NLSs of sufficient strength to drive accumulation of said CRISPR enzyme in a detectable amount in the nucleus of a eukaryotic cell.

6. The composition of claim 1 or 2, wherein the tracr sequence exhibits at least 50% of sequence complementarity along the length of the tracr mate sequence when optimally aligned.

7. The composition of claim 1 or 2, wherein the CRISPR enzyme is a type 11 CRISPR system enzyme.

8. The composition of claim 1 or 2, wherein the CRISPR enzyme is a Cas9 enzyme.

9. The composition of claim 1 or 2, wherein the CRISPR enzyme is codon-optimized for expression in a eukaryotic cell.

10. The composition of claim 1 or 2, wherein the guide sequence is at least 15 nucleotides in length.

11. The composition of claim 1 or 2, wherein the chimeric RNA polynucleotide sequence comprises two, three, four or five hairpins.

12. The composition of claim 1 or 2, wherein the catalytic domain is selected from the group comprising RuvCI, RuvCII, RuvCIII or HNH domain.

13. The composition of claim 1 or 2, wherein the CRISPR enzyme comprises a mutation in a residue selected from the group consisting of D10, E762, H840, N854, N863, or D986.

14. The composition of claim 1 or 2, wherein the CRISPR enzyme comprises a mutation selected from the group comprising D10A, E762A, H840A, N854A, N863A or D986A.

29. A eukaryotic host cell comprising the composition of any of the preceding claims.

30. An organism comprising the eukaryotic host cell of claim 29.

31. A non-human organism comprising the eukaryotic host cell of claim 29.

32. A kit comprising the composition of any of claims 1 to 28 and instructions for using said kit.

33. A method of altering the expression of a genomic locus of interest in a eukaryotic cell comprising

contacting the genomic locus with the composition of any of claims 1 to 28, and

determining if the expression of the genomic locus has been altered.

2. A multiplexed CRISPR enzyme system, wherein the system comprises 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 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 CRISPR enzyme comprising at least one or more nuclear localization sequences (NLSs) in the proximity of a terminus of the CRISPR enzyme,

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

to 3′

orientation,

wherein components I and 11 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 one or more mutations in a catalytic domain thereby rendering the CRISPR enzyme to a nickase that cleaves a single DNA strand, and

wherein the chiRNA polynucleotide sequence comprises two or more hairpins, and

wherein in the multiplexed system multiple chiRNA polynucleotide sequences are used.

15. 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 eukaryotic cell, and

(b) a tracr mate sequence,

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 (NLSs) in the proximity of a terminus of the CRISPR enzyme, and

II. 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, and

wherein the CRISPR enzyme comprises one or more mutations in a catalytic do am thereby rendering the CRISPR enzyme to a nickase that cleaves a single DNA strand.

17. The composition of claim 15 or 16, wherein the first regulatory element is a polymerase III promoter.

18. The composition of claim 15 or 16, wherein the second regulatory element is a polymerase II promoter.

19. The composition of claim 15 or 16, wherein the third regulatory element is a polymerase III promoter.

20. The composition of claim 15 or 16, wherein the CRISPR enzyme comprises one or more NLSs of sufficient strength to drive accumulation of said CRISPR enzyme in a detectable amount in the nucleus of a eukaryotic cell.

21. The composition of claim 15 or 16, wherein the tracr sequence exhibits at least 50% of sequence complementarity along the length of the tracr mate sequence when optimally aligned.

22. The composition of claim 15 or 16, wherein the CRISPR enzyme is a type 11 CRISPR system enzyme.

23. The composition of claim 15 or 16, wherein the CRISPR enzyme is a Cas9 enzyme.

24. The composition of claim 15 or 16, wherein the CRISPR enzyme is codon-optimized for expression in a eukaryotic cell.

25. The composition of claim 15 or 16, wherein the guide sequence is at least 15 nucleotides in length.

26. The composition of claim 15 or 16, wherein the catalytic domain is selected from the group comprising RuvCI, RuvCII, RuvCIII or HNH domain.

27. The composition of claim 15 or 16, wherein the CRISPR enzyme comprises a mutation in a residue selected from the group consisting of D10, E762, H840, N854, N863, or D986.

28. The composition of claim 15 or 16, wherein the CRISPR enzyme comprises a mutation selected from the group comprising D10A, E762A, H840A, N854A, N863A or D986A.

16. A multiplexed CRISPR enzyme system, wherein the system comprises 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 eukaryotic cell, and

(b) a tracr mate sequence,

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 (NLSs) in the proximity of a terminus of the 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 one or more mutations in a catalytic domain thereby rendering the CRISPR enzyme to a nickase that cleaves a single DNA strand, and

wherein in the multiplexed system multiple guide sequences and a single tracr sequence is used.