CRISPR-CAS COMPONENT SYSTEMS, METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION

• US 20180327756A1
• Filed: 08/05/2016
• Published: 11/15/2018
• Est. Priority Date: 12/12/2012
• Status: Active Application

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation), Massachusetts Institute of Technology, The Broad Institute, Inc.
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation), Massachusetts Institute of Technology, The Broad Institute, Inc.
• Inventors: Zhang, Feng, Cong, Le, Cox, David Benjamin Turitz, Hsu, Patrick, Lin, Shuailiang, Ran, Fei, Platt, Randall Jeffrey, Sanjana, Neville Espi
• Application Number:
  US15/230,161
• Publication Number:
  US 20180327756A1
• Time in Patent Office: Days
• 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 15/113 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 15/70 Vectors or expression systems specially adapted for E. coli
• CPC Class Codes: C12N 15/74 Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
• CPC Class Codes: C12N 15/746 for lactic acid bacteria (Streptococcus; Lactococcus; Lactobacillus; Pediococcus; Enterococcus; Leuconostoc; Propionibacterium; Bifidobacterium; Sporolactobacillus)
• CPC Class Codes: C12N 15/79 Vectors or expression systems specially adapted for eukaryotic hosts
• 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 2320/11 for the determination of target sites, i.e. of active nucleic acids
• CPC Class Codes: C12N 2320/30 Special therapeutic applications
• CPC Class Codes: C12N 2750/14143 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2800/101 for bacteria
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: G16B 20/00 : ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
• CPC Class Codes: G16B 20/20 : Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection
• CPC Class Codes: G16B 20/30 : Detection of binding sites or motifs
• CPC Class Codes: G16B 20/50 : Mutagenesis
• CPC Class Codes: G16B 30/00 : ICT specially adapted for sequence analysis involving nucleotides or amino acids
• CPC Class Codes: G16B 30/10 : Sequence alignment; Homology search

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 single eukaryotic expression vector comprising:

I. a CRISPR-Cas complex chimeric RNA (chiRNA) polynucleotide sequence,

wherein the polynucleotide sequence comprises

(a) a guide sequence capable of hybridizing to a target mammalian genome DNA sequence in a mammalian cell, wherein the target sequence is adjacent to a DNA Protospacer Adjacent Motif (PAM),

(b) a tracr mate sequence, and

(c) a tracr sequence, and

II. a nucleotide sequence encoding a Cas9 comprising at least one or more nuclear localization signals (NLSs) in the proximity of a terminus of the Cas9,

wherein;

components I and II are each operably linked to a regulatory element for transcription thereof in the mammalian cell and the vector includes at least one regulatory element therefor,

when the vector is introduced into the mammalian cell, components I and II are transcribed in the mammalian cell,

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, there is PAM recognition, mammalian genome DNA cleavage and altered gene expression in the mammalian cell,

the CRISPR complex comprises the Cas9 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

the chimeric RNA polynucleotide sequence comprises two or more hairpins and

the guide RNA comprises a tracr sequence comprising 30 or more nucleotides in length.

3. The composition of claim 1, wherein the regulatory element comprises a polymerase III promoter.

4. The composition of claim 1, wherein the regulatory element comprises a polymerase II promoter.

5. The composition of claim 1, wherein the guide sequence comprises about or more than about 10-75 nucleotides in length, and the tracr sequence comprises about or more than about 30-50 nucleotides in length, and the tracr sequence exhibits at least 50% sequence complementarity along the length of the tracr mate.

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

7. The composition of claim 1, wherein the nucleotide sequence encoding the Cas9 is codon-optimized for expression in a eukaryotic cell.

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

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

14. The composition of claim 1, wherein the vector comprises more than one regulatory element for transcription of components I and II or components I, II and III.

19. An isolated mammalian cell comprising the composition of claim 1.

20. A non-human animal comprising the mammalian cell of claim 19.

21. A non-human organism comprising the mammalian cell of claim 19.

22. A kit comprising the composition of claim 1 and instructions for using said kit.

23. 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 claim 1, and

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

36. The composition of claim 1 wherein the vector comprises an adenovirus, a lentivirus or an adeno-associated virus.

37. The composition of claim 1, wherein said cleavage comprises cleaving two strands at the location of the target sequence.

38. The composition of claim 1, wherein said cleavage results in decreased transcription of a target gene.

39. The method of claim 23, further comprising repairing cleaved DNA by homologous recombination with an exogenous template polynucleotide, wherein said repair results in a mutation comprising an insertion, deletion, or substitution of one or more nucleotides of said target polynucleotide.

40. The method of claim 39, wherein said mutation results in one or more amino acid changes in a protein expressed from a gene comprising the target sequence.

41. The method of claim 23, comprising delivering said vector to the mammalian cell in a subject.

42. The method of claim 23, wherein comprising delivering said vector to a mammalian cell in a cell culture.

43. The method of claim 42, comprising isolating said mammalian cell from a subject prior to said delivering.

44. The method of claim 43, further comprising returning said mammalian cell and/or progeny cell(s) therefrom to said subject.

45. The composition according to claim 1 wherein the Cas9 comprises a fusion of a Cas9 protein and one or more effector domains.

46. The composition or system or vector according to claim 45, 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, transcription-protein recruiting domain, cellular uptake activity associated domain, nucleic acid binding domain or antibody presentation domain.

47. A mammalian cell comprising composition, system or vector of claim 36.

48. The method of claim 23 including cleaving two DNA strands at the location of the target sequence.

50. The method of claim 23 wherein altering expression comprises genome editing.

51. The composition of claim 1 wherein altered expression comprises genome editing.

53. The composition or system or vector according to claim 46, wherein the one or more effector domains comprises a nuclease domain.

54. The composition claim 1 as to component I there is a first regulatory element and as to component II there is a second regulatory element.

2. A multiplexed CRISPR system comprising a single eukaryotic expression vector comprising:

I. more than one CRISPR-Cas complex chimeric RNA (chiRNA) polynucleotide sequence,

wherein each polynucleotide sequence comprises

(a) a guide sequence capable of hybridizing to a target mammalian genome DNA sequence in a mammalian cell adjacent to a DNA Protospacer Adjacent Motif (PAM),

wherein each guide sequence of each of the more than one polynucleotide sequences is capable of hybridizing to a different target mammalian genome DNA sequence in the mammalian cell;

each target sequence being adjacent to a DNA Protospacer Adjacent Motif (PAM),

(b) a tracr mate sequence, and

(c) a tracr sequence, and

II. a nucleotide sequence encoding a Cas9 comprising at least one or more nuclear localization signals (NLSs) in the proximity of a terminus of the Cas9,

wherein;

each of components I and II are operably linked to a regulatory element for transcription thereof in the mammalian cell and the vector includes at least one regulatory element therefor,

when the vector is introduced into the mammalian cell, components I and II are transcribed in the mammalian cell.

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, there is PAM recognition, mammalian genome DNA cleavage and altered gene expression in the mammalian cell,

the CRISPR complex comprises the Cas9 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

each chimeric RNA polynucleotide sequence comprises two or more hairpins and

each guide RNA comprises a tracr sequence comprising 30 or more nucleotides in length.

52. The system of claim 2 wherein the DNA cleavage and altered gene expression comprises multiplexed editing within a single genome including mammalian genome DNA cleavage at more than one loci.

10. A non-naturally occurring or engineered composition comprising a single eukaryotic expression vector comprising:

I. (a) a guide sequence capable of hybridizing to a mammalian genome DNA target sequence in a mammalian cell, wherein the target sequence is adjacent to a DNA Protospacer Adjacent Motif (PAM), and

(b) a tracr mate sequence,

II. a nucleotide sequence encoding a Cas9 comprising at least one or more nuclear localization signals (NLSs) in the proximity of a terminus of the Cas9, and

III. a tracr sequence, wherein the tracr sequence comprises 30 or more nucleotides in length,

wherein components I, II and III are each operably linked to a regulatory element and the vector includes one or more regulatory elements for transcription of components I, II and III,

when the vector is introduced into the mammalian cell, components I, II and III are transcribed in the mammalian cell,

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, there is PAM recognition, mammalian genome DNA cleavage and altered gene expression in the mammalian cell,

the CRISPR complex comprises the Cas9 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

the chimeric RNA polynucleotide sequence comprises two or more hairpins and

the guide RNA comprises a tracr sequence comprising 30 or more nucleotides in length.

12. The composition of claim 10, wherein the regulatory element comprises a polymerase III promoter.

13. The composition of claim 10, wherein the regulatory element comprises a polymerase II promoter.

15. The composition of claim 10, wherein the guide sequence comprises about or more than about 10-75 nucleotides in length, and the tracr sequence comprises about or more than about 30-50 nucleotides in length, and the tracr sequence exhibits at least 50% sequence complementarity along the length of the tracr mate.

16. The composition of claim 10, wherein the tracr sequence exhibits at least 50% of sequence complementarity along the length of the tracr mate sequence.

17. The composition of claim 10, wherein the nucleotide sequence encoding the Cas9 is codon-optimized for expression in a eukaryotic cell.

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

55. The composition of claim 10, wherein as to component I there is a first regulatory element, as to component II there is a second regulatory element, and as to component III there is a third regulatory element.

11. A multiplexed CRISPR system, wherein the system comprises a single eukaryotic expression vector comprising:

I. (a) more than one guide sequence, wherein a guide sequence is capable of hybridizing to a mammalian genome DNA target sequence in a mammalian cell adjacent to a DNA Protospacer Adjacent Motif (PAM), and each guide sequence of the more than one guide sequences is capable of hybridizing to a different target mammalian genome DNA sequence in the mammalian cell;

each target sequence being adjacent to a DNA Protospacer Adjacent Motif (PAM),

(b) a tracr mate sequence,

II. a nucleotide sequence encoding a Cas9 comprising at least one or more nuclear localization signals (NLSs) in the proximity of a terminus of the Cas9, and

III. a tracr sequence, wherein the tracr sequence comprises 30 or more nucleotides in length,

each of components I, II and III are operably linked to a regulatory element for transcription thereof in the mammalian cell and the vector includes at least one regulatory element therefor,

when the vector is introduced into the mammalian cell, components I, II and III are transcribed in the mammalian cell,

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, there is PAM recognition, mammalian genome DNA cleavage and altered gene expression in the mammalian cell,

the CRISPR complex comprises the Cas9 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

each chimeric RNA polynucleotide sequence comprises two or more hairpins and

each guide RNA comprises a tracr sequence comprising 30 or more nucleotides in length.

24. A single eukaryotic expression vector comprising:

I. a. CRISPR-Cas complex chimeric RNA (chiRNA) polynucleotide sequence,

wherein the polynucleotide sequence comprises

(a) a guide sequence capable of hybridizing to a target mammalian genome DNA sequence in a mammalian cell, wherein the target sequence is adjacent to a DNA Protospacer Adjacent Motif (PAM),

(b) a tracr mate sequence, and

(c) a tracr sequence, and

II. a nucleotide sequence encoding a Cas9 comprising at least one or more nuclear localization signals (NLSs) in the proximity of a terminus of the Cas9,

wherein;

components I and II are each operably linked to a regulatory element for transcription thereof in the mammalian cell and the vector includes at least one regulatory element therefor,

when the vector is introduced into the mammalian cell, components I and II are transcribed in the mammalian cell,

when transcribed, the tracr mate sequence hybridizes to the trace sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence, there is PAM recognition, mammalian genome DNA cleavage and altered gene expression in the mammalian cell,

the CRISP complex comprises the Cas9 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

the chimeric RNA polynucleotide sequence comprises two or more hairpins and

the guide RNA comprises a tracr sequence comprising 30 or more nucleotides in length.

25. The vector of claim 24, wherein the tracr sequence is downstream of the tracr mate sequence and both are under the control of the same regulatory element.

26. The vector of claim 24, wherein component (a) further comprises two or more guide sequences, 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, there is PAM recognition, mammalian genome DNA cleavage and altered gene expression in the mammalian cell.

27. The vector of claim 26, wherein the tracr sequence is under the control of a regulatory element separate from that controlling guide sequence transcription.

28. The vector of claim 24, wherein the tracr sequence exhibits at least 50% of sequence complementarity along the length of the tracr mate sequence.

29. The vector of claim 24, wherein the nucleotide sequence encoding the Cas9 is codon-optimized for expression in a eukaryotic cell.

30. The vector of claim 24, wherein the Cas9 directs cleavage of one or two strands at the location of the target sequence.

31. The vector of claim 24, wherein the regulatory element comprises a polymerase III promoter.

32. The vector of claim 24, wherein the regulatory element comprises a polymerase II promoter.

33. The vector of claim 27, wherein comprising more than one regulatory element for transcription of components I and II.

34. The vector of claim 24, wherein the guide sequence comprises about or more than about 10-75 nucleotides in length, and the tracr sequence comprises about or more than about 30-50 nucleotides in length, and the tracr sequence exhibits at least 50% sequence complementarity along the length of the tracr mate.

35. The vector of claim 24, wherein fewer than 50% of the nucleotides of the guide sequence participate in self-complementary base-pairing.

49. The method of claim including DNA cleavage resulting in decreased transcription of a gene.