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

• US 20150031134A1
• Filed: 09/26/2014
• Published: 01/29/2015
• Est. Priority Date: 12/12/2012
• Status: Abandoned Application

• Current Assignee: Massachusetts Institute of Technology, Rockefeller University
• Original Assignee: Massachusetts Institute of Technology, Rockefeller University, The Broad Institute, Inc.
• Inventors: Zhang, Feng, Cox, David Benjamin Turitz, Marraffini, Luciano, Bikard, David Olivier, Jiang, Wenyan
• Application Number:
  US14/497,627
• Publication Number:
  US 20150031134A1
• Time in Patent Office: Days
• US Class Current: 435/471
• 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

1-34. -34. (canceled)

35. A method of selecting one or more prokaryotic cell(s) by introducing one or more mutations in one or more prokaryotic cell(s), the method comprising:

introducing one or more vectors into the prokaryotic cell(s);

wherein the one or more vectors drive expression of one or more of;

a CRISPR enzyme, a guide sequence linked to a tracr mate sequence, a tracr sequence, and an editing template;

and all of a CRISPR enzyme, a guide sequence linked to a tracr mate sequence, and a tracr sequence, and an editing template are produced in the prokaryotic cell(s);

introducing the editing template into a target polynucleotide through recombination, wherein the editing template comprises the one or more mutations that abolish CRISPR enzyme cleavage;

allowing a CRISPR complex to bind to the target polynucleotide to effect cleavage of the target polynucleotide;

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

wherein binding of the CRISPR complex to the target polynucleotide induces cell death;

thereby allowing one or more prokaryotic cell(s) in which one or more mutations have been introduced to be selected.

36. The method of claim 35, wherein the CRISPR enzyme is a type II CRISPR system enzyme.

37. The method of claim 35, wherein the CRISPR enzyme is a Cas9.

38. The method of claim 37, wherein the Cas9 is S. pyogenes Cas9.

39. The method of claim 35 wherein the CRISPR enzyme is not endogenous to the prokaryote(s).

40. The method of claim 35 wherein the prokaryote(s) is S. pneumoniae or E. coli.

41. The method of claim 35 wherein the one or more vectors are plasmids.

42. A method of inducing cell death of one or more prokaryotic cell(s) comprising:

introducing one or more vectors into the prokaryotic cell(s);

wherein the one or more vectors drive expression of one or more of;

a CRISPR enzyme, a guide sequence linked to a tracr mate sequence, and a tracr sequence;

and all of a CRISPR enzyme, a guide sequence linked to a tracr mate sequence, and a tracr sequence, are produced in the prokaryotic cell(s);

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

wherein binding of the CRISPR complex to the target polynucleotide results in Cas9-directed cleavage at a targeted site in the prokaryote(s) and induces cell death.

43. The method of claim 42, wherein the CRISPR enzyme is a type II CRISPR system enzyme.

44. The method of claim 42, wherein the CRISPR enzyme is a Cas9.

45. The method of claim 44, wherein the Cas9 is S. pyogenes Cas9.

46. The method of claim 42, wherein the CRISPR enzyme is not endogenous to the prokaryote(s).

47. The method of claim 42 wherein the prokaryote(s) is S. pneumoniae or E. coli.

48. The method of claim 42 wherein the one or more vectors are plasmids.

49. A method of generating one mutation or multiple mutations in one or more prokaryotic cell(s) by introducing the one mutation or multiple mutations in one or more prokaryotic cell(s) comprising:

A. generating one mutation in one or more prokaryotic cell(s) by introducing the mutation in one or more prokaryotic cell(s), by a method comprising;

introducing one or more vectors into the prokaryotic cell(s);

wherein the one or more vectors drive expression of one or more of;

a CRISPR enzyme, a guide sequence linked to a tracr mate sequence, a tracr sequence, and an editing template;

and all of a CRISPR enzyme, a guide sequence linked to a tracr mate sequence, and a tracr sequence, and an editing template are produced in the prokaryotic cell(s);

allowing a CRISPR complex to bind to a target polynucleotide to effect cleavage of the target polynucleotide whereby the editing template comprising the mutation is introduced through recombination in the one or more prokaryote(s); and

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

thereby generating one or more prokaryotic cell(s) in which the mutation has been introduced;

or

B. generating multiple mutations in one or more prokaryotic cell(s) by introducing the multiple mutations in one or more prokaryotic cell(s), by a method comprising;

introducing one or more vectors into the prokaryotic cell(s);

wherein the one or more vectors drive expression of one or more of;

a CRISPR enzyme, a guide sequence linked to a tracr mate sequence, a tracr sequence, and an editing template;

and all of a CRISPR enzyme, a guide sequence linked to a tracr mate sequence, and a tracr sequence, and an editing template are produced in the prokaryotic cell(s);

allowing a CRISPR complex to bind to a target polynucleotide to effect cleavage of the target polynucleotide whereby the editing template comprising the multiple mutations is introduced through recombination in the one or more prokaryote(s); and

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

thereby generating one or more prokaryotic cell(s) in which multiple mutations have been introduced;

or

C. generating multiple mutations in one or more prokaryotic cell(s) by introducing the multiple mutations in one or more prokaryotic cell(s), by a method comprising;

introducing one or more vectors into the prokaryotic cell(s);

wherein the one or more vectors drive expression of one or more of;

a CRISPR enzyme, a guide sequence linked to a tracr mate sequence, a tracr sequence, and an editing template;

and all of a CRISPR enzyme, a guide sequence linked to a tracr mate sequence, and a tracr sequence, and an editing template are produced in the prokaryotic cell(s);

allowing a CRISPR complex to bind to a target polynucleotide to effect cleavage of the target polynucleotide whereby the editing template comprising a first mutation is introduced through recombination in the one or more prokaryote(s); and

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

thereby generating one or more prokaryotic cell(s) in which the first mutation has been introduced;

introducing one or more vectors into the prokaryotic cell(s) having the first mutation;

wherein the one or more vectors drive expression of one or more of;

a CRISPR enzyme, a guide sequence linked to a tracr mate sequence, a tracr sequence, and an editing template;

and all of a CRISPR enzyme, a guide sequence linked to a tracr mate sequence, and a tracr sequence, and an editing template are produced in the prokaryotic cell(s);

allowing a CRISPR complex to bind to a target polynucleotide to effect cleavage of the target polynucleotide whereby the editing template comprising a second mutation is introduced through recombination in the one or more prokaryote(s); and

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

thereby generating one or more prokaryotic cell(s) in which the first and the second mutation has been introduced.

50. The method of claim 49, wherein the CRISPR enzyme is a type II CRISPR system enzyme.

51. The method of claim 49, wherein the CRISPR enzyme is Cas9.

52. The method of claim 51, wherein the Cas9 is S. pyogenes Cas9.

53. The method of claim 49, wherein the CRISPR enzyme is not endogenous to the prokaryote(s).

54. The method of claim 49 wherein the prokaryote(s) is S. pneumoniae or E. coli.

55. The method of claim 49 wherein the one or more vectors are plasmids.

56. A method for introducing mutation(s) into a prokaryote comprising introducing the mutation(s) by recombineering, and selecting for prokaryote(s) having the mutation(s)

by introducing one or more vectors into the prokaryotic cell(s);

wherein the one or more vectors drive expression of one or more of;

a CRISPR enzyme, a guide sequence linked to a tracr mate sequence, a tracr sequence and an editing oligonucleotide;

and all of a CRISPR enzyme, a guide sequence linked to a tracr mate sequence, a tracr sequence, and an editing template are produced in the prokaryotic cell(s);

introducing the editing oligonucleotide comprising the mutation(s) into a target polynucleotide by recombineering,

allowing a CRISPR complex to bind to the target polynucleotide to effect cleavage of the target polynucleotide, wherein the target sequence is in prokaryote(s) not having the mutation(s) from recombineering; and

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

wherein binding of the CRISPR complex to the target polynucleotide results in Cas9-directed cleavage at a targeted site in the prokaryote(s) not having the mutation(s) and induces cell death.

57. The method of claim 56, wherein the CRISPR enzyme is a type II CRISPR system enzyme.

58. The method of claim 56, wherein the CRISPR enzyme is Cas9.

59. The method of claim 56, wherein the Cas9 is S. pyogenes Cas9.

60. The method of claim 56, wherein the CRISPR enzyme is not endogenous to the prokaryote(s).

61. The method of claim 56 wherein the prokaryote(s) is E. coli.