FUNCTIONAL GENOMICS USING CRISPR-CAS SYSTEMS, COMPOSITIONS, METHODS, SCREENS AND APPLICATIONS THEREOF

• US 20160272965A1
• Filed: 12/17/2015
• Published: 09/22/2016
• Est. Priority Date: 06/17/2013
• Status: Active Grant

• Current Assignee: Massachusetts Institute of Technology, The Broad Institute, Inc.
• Original Assignee: Massachusetts Institute of Technology, The Broad Institute, Inc.
• Inventors: Shalem, Ophir, Doench, John, Biewener Hartenian, Ella Nicole, Zhang, Feng, Sanjana, Neville Espi, Root, David
• Granted Patent: US 10,781,444 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• 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 15/1089 Design, preparation, screening or analysis of libraries using computer algorithms
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• 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 15/907 in mammalian cells
• CPC Class Codes: C12N 2310/20 involving clustered regularly interspaced short palindromic repeats [CRISPRs]
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

Abstract

The present invention generally relates to libraries, compositions, methods, applications, kits and screens used in functional genomics that focus on gene function in a cell and that may use vector systems and other aspects related to Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas systems and components thereof. 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 genome wide library comprising a plurality of unique CRISPR-Cas system guide sequences that are capable of targeting a plurality of target sequences in genomic loci, wherein said targeting results in a knockout of gene function.

2. A method of knocking out in parallel every gene in a genome, the method comprising

contacting a population of cells with a composition comprising a vector system comprising one or more packaged vectors comprising

a) a first regulatory element operably linked to a CRISPR-Cas system chimeric RNA (chiRNA) polynucleotide sequence that targets a DNA molecule encoding a gene product,

wherein the polynucleotide sequence comprises

(a) a guide sequence capable of hybridizing to a target sequence,

(b) a tracr mate sequence, and

(c) a tracr sequence, and

b) a second regulatory element operably linked to a Cas protein and a selection marker,

wherein components (a) and (b) are located on same or different vectors of the system,

wherein each cell is transfected with a single packaged vector,

selecting for successfully transfected cells,

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 a target sequence in the genomic loci of the DNA molecule encoding the gene product,

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

wherein the guide sequence is selected from the library of claim 1,

wherein the guide RNAs target the genomic loci of the DNA molecule encoding the gene product and the CRISPR enzyme cleaves the genomic loci of the DNA molecule encoding the gene product and whereby each cell in the population of cells has a unique gene knocked out in parallel.

3. The method of claim 2 wherein the cell is a eukaryotic cell, including an animal or a plant cell.

4. The method of claim 2 wherein the vector is a lentivirus, a adenovirus or a AAV.

5. The method of claim 2 wherein the first regulatory element is a Pol III promoter selected from a H1 promoter and a U6 promoter.

6. The method of claim 2 wherein the second regulatory element is a Pol II promoter selected from a doxycycline inducible promoter, a cell-type specific promoter and an EFS promoter.

7. The method of claim 2 wherein the vector system comprises one vector.

8. The method of claim 2 wherein the CRISPR enzyme is Cas9.

9. A method of selecting individual cell knock outs that survive under a selective pressure, the method comprising

contacting a population of cells with a composition comprising a vector system comprising one or more packaged vectors comprising

a) a first regulatory element operably linked to a CRISPR-Cas system chimeric RNA (chiRNA) polynucleotide sequence that targets a DNA molecule encoding a gene product,

wherein the polynucleotide sequence comprises

(a) a guide sequence capable of hybridizing to a target sequence,

(b) a tracr mate sequence, and

(c) a tracr sequence, and

b) a second regulatory element operably linked to a Cas protein and a selection marker,

wherein components (a) and (b) are located on same or different vectors of the system,

wherein each cell is transfected with a single packaged vector,

selecting for successfully transfected cells,

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 a target sequence in the genomic loci of the DNA molecule encoding the gene product,

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

wherein the guide sequence is selected from the library of claim 1,

wherein the guide RNAs target the genomic loci of the DNA molecule encoding the gene product and the CRISPR enzyme cleaves the genomic loci of the DNA molecule encoding the gene product, whereby each cell in the population of cells has a unique gene knocked out in parallel,

applying the selective pressure,

and selecting the cells that survive under the selective pressure.

10. The method of claim 9 wherein the selective pressure is application of a drug, FACS sorting of cell markers or aging.

11. The method of claim 9 wherein the cell is a eukaryotic cell.

12. The method of claim 9 wherein the vector is a lentivirus, a adenovirus or a AAV.

13. The method of claim 9 wherein the first regulatory element is a Pol III promoter selected from a H1 promoter and a Pol III promoter selected from a H1 promoter and a U6 promoter.

14. The method of claim 9 wherein the second regulatory element is a Pol II promoter selected from an EFS promoter, a doxycycline inducible promoter and a cell-type specific promoter.

15. The method of claim 9 wherein the vector system comprises one vector.

16. The method of claim 9 wherein the CRISPR enzyme is Cas9.

17. A method of identifying the genetic basis of one or more medical symptoms exhibited by a subject, the method comprising

obtaining a biological sample from the subject and isolating a population of cells having a first phenotype from the biological sample;

contacting the cells having the first phenotype with a composition comprising a vector system comprising one or more packaged vectors comprising

a) a first regulatory element operably linked to a CRISPR-Cas system chimeric RNA (chiRNA) polynucleotide sequence that targets a DNA molecule encoding a gene product,

wherein the polynucleotide sequence comprises

(a) a guide sequence capable of hybridizing to a target sequence,

(b) a tracr mate sequence, and

(c) a tracr sequence, and

b) a second regulatory element operably linked to a Cas protein and a selection marker,

wherein components (a) and (b) are located on same or different vectors of the system,

wherein each cell is transfected with a single packaged vector,

selecting for successfully transfected cells,

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 a target sequence in the genomic loci of the DNA molecule encoding the gene product,

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

wherein the guide sequence is selected from the library of claim 1,

wherein the guide RNAs target the genomic loci of the DNA molecule encoding the gene product and the CRISPR enzyme cleaves the genomic loci of the DNA molecule encoding the gene product, whereby each cell in the population of cells has a unique gene knocked out in parallel,

applying the selective pressure,

selecting the cells that survive under the selective pressure,

determining the genomic loci of the DNA molecule that interacts with the first phenotype and identifying the genetic basis of the one or more medical symptoms exhibited by the subject.

18. The method of claim 17 wherein the selective pressure is application of a drug, FACS sorting of cell markers or aging.

19. The method of claim 17 wherein the cell is a eukaryotic cell.

20. The method of claim 17 wherein the vector is a lentivirus, a adenovirus or a AAV.

21. The method of claim 17 wherein the first regulatory element is a Pol III promoter selected from a H1 promoter and a U6 promoter.

22. The method of claim 17 wherein the second regulatory element is a Pol II promoter selected from a doxycycline inducible promoter, a cell-type specific promoter and an EFS promoter.

23. The method of claim 17 wherein the vector system comprises one vector.

24. The method of claim 17 wherein the CRISPR enzyme is Cas9.

25. A kit comprising the library of claim 1.

26. The kit of claim 25, wherein the kit comprises a single container comprising vectors comprising the library of claim 1.

27. The kit of claim 25, wherein the kit comprises a single container comprising plasmids comprising the library of claim 1.

28. A kit comprising a panel comprising a selection of unique CRISPR-Cas system guide sequences from the library of claim 1, wherein the selection is indicative of a particular physiological condition.

29. The library or method of any one of the preceding claims, wherein the targeting is of about 100 or more sequences, about 1000 or more sequences or about 20,000 or more sequences or the entire genome.

30. The library or method of any one of the preceding claims, wherein a panel of target sequences is focused on a relevant or desirable pathway, such as an immune pathway or cell division.

31. The library of any one of the preceding claims, wherein the library comprises the guide sequences of Table 1 as provided in the compact discs created Apr. 11, 2014, as filed in connection with U.S. applications 61/960,777 and 61/995,636.

57. The library of claim 1, wherein the targeting is of about 100 or more sequences, about 1000 or more sequences or about 20,000 or more sequences or the entire genome.

58. The library of claim 1, wherein a panel of target sequences is focused on a relevant or desirable pathway, such as an immune pathway or a cell division pathway.

59. The library of claim 1, wherein the library is a plasmid library selected from the group consisting of:

GeCKO1—

library of vectors (lentiCRISPRv2) each encoding a selected guide sequences—

ATCC Deposit No. PTA121339;

GeCKO2—

library of vectors each encoding a selected guide sequence of half library A (human)—

ATCC Deposit No. PTA121340;

GeCKO2—

library of vectors each encoding a selected guide sequences of half library B (human)—

ATCC Deposit No. PTA121341;

GeCKO2—

library of vectors each encoding a selected guide sequence of half library A (mouse)—

ATCC Deposit No. PTA121342; and

GeCKO2—

library of vectors each encoding a selected guide sequence of half library B (mouse)—

ATCC Deposit No. PTA121343.

60. The library, method or kit of claim 59, further comprising a vector encoding sequences for Streptococcus pyogenes Cas9.

32. A genomic library comprising a selected plurality of unique CRISPR-Cas system guide sequences that are capable of targeting a plurality of target sequences in genomic loci of a plurality of genes, wherein said targeting results in a knockout of gene function, wherein the unique CRISPR-Cas system guide sequences are selected by an algorithm that predicts the efficacy of the guide sequences based on the primary nucleotide sequence of the guide sequence and/or by a heuristic that ranks the guide sequences based on off target scores.

33. The genomic library of claim 32, wherein the guide sequences target constitutive exons downstream of a start codon of the gene.

34. The genomic library of claim 32, wherein the guide sequences target either a first or a second exon of the gene.

35. The genomic library of claim 32, wherein the guide sequences target a non-transcribed strand of the genomic loci of the gene.

36. The genomic library of claim 32, wherein a plurality of target sequences in each gene are targeted.

37. The genomic library of claim 32, wherein the plurality of genes are human genes and the library comprises guide sequences of Tables 3, 4 and/or 5.

38. The genomic library of claim 32, wherein the plurality of genes are mouse genes and the library comprises guide sequences of Tables 7, 8 and/or 9.

39. The genomic library of claim 32, wherein the library comprises sub libraries.

53. A kit comprising the library of claim 32.

54. The kit of claim 53, wherein the kit comprises a single container comprising vectors comprising the library of claim 32.

55. The kit of claim 53, wherein the kit comprises a single container comprising plasmids comprising the library of claim 32.

56. A kit comprising a panel comprising a selection of unique CRISPR-Cas system guide sequences from the library of claim 32, wherein the selection is indicative of a particular physiological condition.

40. A method of knocking out in parallel every gene in a human genome, the method comprising

contacting a population of cells with a composition comprising a vector system comprising one or more packaged vectors comprising

a) a first regulatory element operably linked to a CRISPR-Cas system chimeric RNA (chiRNA) polynucleotide sequence that targets a DNA molecule encoding a gene product,

wherein the polynucleotide sequence comprises

(a) a guide sequence capable of hybridizing to a target sequence,

(b) a tracr mate sequence, and

(c) a tracr sequence, and

b) a second regulatory element operably linked to a Cas protein and a selection marker,

wherein components (a) and (b) are located on same or different vectors of the system,

wherein each cell is transduced with a single packaged vector,

selecting for successfully transduced cells,

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 a target sequence in the genomic loci of the DNA molecule encoding the gene product,

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

wherein the guide sequence is selected from libraries comprising guide sequences of Tables 3, 4 and/or 5,

wherein the guide sequence targets the genomic loci of the DNA molecule encoding the gene product and the CRISPR enzyme cleaves the genomic loci of the DNA molecule encoding the gene product and whereby each cell in the population of cells has a unique gene knocked out in parallel.

42. The method of claim 40, wherein the method further comprises extracting DNA and determining the depletion or enrichment of the guide sequences by deep sequencing.

43. The method of claim 40, wherein the vector is a lentivirus, an adenovirus or an AAV.

44. The method of claim 40, wherein the first regulatory element is a Pol III promoter selected from a H1 promoter and a U6 promoter.

45. The method of claim 40, wherein the second regulatory element is a Pol II promoter selected from a doxycycline inducible promoter, a cell-type specific promoter and an EFS promoter.

46. The method of claim 40, wherein the vector system comprises one vector.

47. The method of claim 40, wherein the CRISPR enzyme is Cas9.

48. The method of claim 40, wherein the cell is transduced with a multiplicity of infection (MOI) of 0.3-0.75.

49. The method of claim 40, wherein the MOI is 0.3 or 0.4.

50. The method of claim 46, wherein the vector is a lentiviral vector comprising a codon optimized NLS, a codon optimized P2A bicistronic linker sequence and an optimally placed U6 driven guide RNA cassette.

51. The method of claim 40, wherein the vector system comprises two vectors.

52. The method of claim 51, wherein each of the two vectors comprises a different selection marker.

41. A method of knocking out in parallel every gene in a mouse genome, the method comprising

contacting a population of cells with a composition comprising a vector system comprising one or more packaged vectors comprising

a) a first regulatory element operably linked to a CRISPR-Cas system chimeric RNA (chiRNA) polynucleotide sequence that targets a DNA molecule encoding a gene product,

wherein the polynucleotide sequence comprises

(a) a guide sequence capable of hybridizing to a target sequence,

(b) a tracr mate sequence, and

(c) a tracr sequence, and

b) a second regulatory element operably linked to a Cas protein and a selection marker,

wherein components (a) and (b) are located on same or different vectors of the system,

wherein each cell is transduced with a single packaged vector,

selecting for successfully transduced cells,

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 a target sequence in the genomic loci of the DNA molecule encoding the gene product,

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

wherein the guide sequence is selected from libraries comprising guide sequences of Tables 7, 8 and/or 9,

wherein the guide sequence targets the genomic loci of the DNA molecule encoding the gene product and the CRISPR enzyme cleaves the genomic loci of the DNA molecule encoding the gene product and whereby each cell in the population of cells has a unique gene knocked out in parallel.

61. A plasmid library selected from the group consisting of:

GeCKO1—

sgRNA plasmids of ATCC Deposit No. PTA121339;

GeCKO2—

sgRNA plasmids of half library A (human) of ATCC Deposit No. PTA121340;

GeCKO2—

sgRNA plasmids of half library B (human) of ATCC Deposit No. PTA121341;

GeCKO2—

sgRNA plasmids of half library A (mouse) of ATCC Deposit No. PTA121342; and

GeCKO2—

sgRNA plasmids of half library B (mouse) of ATCC Deposit No. PTA121343.

62. The plasmid library of claim 61, wherein the vectors containing the sgRNA plasmids are delivered with a vector encoding sequences for Streptococcus pyogenes Cas9.

63. The plasmid library of claim 61, wherein the vectors containing the sgRNA plasmids further comprise polynucleotide sequences encoding for Streptococcus pyogenes Cas9.