FUNCTIONAL GENOMICS USING CRISPR-CAS SYSTEMS, COMPOSITIONS, METHODS, KNOCK OUT LIBRARIES AND APPLICATIONS THEREOF

• US 20140357530A1
• Filed: 08/19/2014
• Published: 12/04/2014
• Est. Priority Date: 12/12/2012
• Status: Abandoned Application

• Current Assignee: Massachusetts Institute of Technology, The Broad Institute, Inc.
• Original Assignee: Massachusetts Institute of Technology, The Broad Institute, Inc.
• Inventors: Zhang, Feng, Sanjana, Neville Espi, Shalem, Ophir
• Application Number:
  US14/463,253
• Publication Number:
  US 20140357530A1
• Time in Patent Office: Days
• US Class Current: 506/16
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 15/1034 Isolating an individual clone by screening libraries
• 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 15/1093 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 15/635 Externally inducible repressor mediated regulation of gene expression, e.g. tetR inducible by tetracyline
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 2750/14143 viral genome or elements thereof as genetic vector

Abstract

The present invention generally relates to compositions, methods applications 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 CRISPR-Cas system guide RNAs comprising guide sequences that are capable of targeting a plurality of target sequences in a plurality of genomic loci in a population of eukaryotic cells.

2. The library of claim 1, wherein the population of cells is a population of embryonic stem (ES) cells.

3. The library of claim 1, wherein the target sequence in the genomic locus is a non-coding sequence.

4. The library of claim 1, wherein gene function of one or more gene products is altered by said targeting.

5. The library of claim 1, wherein said targeting results in a knockout of gene function.

6. The library of claim 1, wherein the targeting is of about 100 or more sequences.

7. The library of claim 1, wherein the targeting is of about 1000 or more sequences.

8. The library of claim 1, wherein the targeting is of about 20,000 or more sequences.

9. The library of claim 1, wherein the targeting is of the entire genome.

10. The library of claim 1, wherein the targeting is of a panel of target sequences focused on a relevant or desirable pathway.

11. The library of claim 10, wherein the pathway is an immune pathway.

12. The library of claim 5, wherein targeting is of about 100 or more sequences.

13. The library of claim 5, wherein targeting is of about 1000 or more sequences.

14. The library of claim 5, wherein targeting is of about 20,000 or more sequences.

15. The library of claim 5, wherein targeting is of the entire genome.

16. The library of claim 5, wherein the targeting is of a panel of target sequences focused on a relevant or desirable pathway.

17. The library of claim 16, wherein the pathway is an immune pathway.

18. The library of claim 16, wherein the pathway is a cell division pathway.

19. The library of claim 5, wherein the knockout of gene function comprises:

introducing into each cell in the population of cells a vector system of one or more vectors comprising an engineered, non-naturally occurring CRISPR-Cas system comprising

I. a Cas protein, and

II. one or more guide RNAs,

wherein components I and II may be same or on different vectors of the system, integrating components I and II into each cell,

wherein the guide sequence targets a unique gene in each cell,

wherein the Cas protein is operably linked to a regulatory element,

wherein when transcribed, the guide RNA comprising the guide sequence directs sequence-specific binding of a CRISPR-Cas system to a target sequence in the genomic loci of the unique gene,

inducing cleavage of the genomic loci by the Cas protein, and

confirming different knockout mutations in a plurality of unique genes in each cell of the population of cells thereby generating a gene knockout cell library.

20. The library of claim 19, wherein the one or more vectors are plasmid vectors.

21. The library of claim 19, wherein the regulatory element is an inducible promoter.

22. The library of claim 19, wherein the inducible promoter is a doxycycline inducible promoter.

23. The library of claim 19, wherein the confirming of different knockout mutations is by whole exome sequencing.

24. The library of claim 19, wherein the knockout mutation is achieved in 100 or more unique genes.

25. The library of claim 19, wherein the knockout mutation is achieved in 1000 or more unique genes.

26. The library of claim 19, wherein the knockout mutation is achieved in 20,000 or more unique genes.

27. The library of claim 19, wherein the knockout mutation is achieved in the entire genome,

28. The library of claim 19, wherein the knockout of gene function is achieved in a plurality of unique genes which function in a particular physiological pathway or condition.

29. The library of claim 28, wherein the pathway or condition is an immune pathway or condition.

30. The library of claim 28, wherein the pathway or condition is a cell division pathway or condition.