Functional genomics using CRISPR-Cas systems, compositions, methods, screens and applications thereof
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.
1. A genome-wide screening method comprising knocking out in parallel a plurality of genes in a genome by a method which comprises:
introducing into a population of cells comprised in a single composition:
I. a plurality of CRISPR-Cas system guide polynucleotide sequences, each comprising a guide sequence that targets a DNA molecule encoding a gene product, and
II. a CRISPR-Cas protein or a polynucleotide sequence encoding the CRISPR-Cas protein,
wherein each guide sequence directs sequence-specific binding of a CRISPR complex to a target sequence in a genomic locus of the DNA molecule encoding the gene product,
wherein the CRISPR complex comprises a CRISPR-Cas protein complexed with a guide sequence that is hybridized to the target sequence,
wherein the CRISPR-Cas protein cleaves the genomic locus,
whereby each cell in the population of cells has a unique gene knocked out in parallel, and wherein each cell in the population of cells has a single CRISPR-Cas system guide polynucleotide sequence introduced therein,
wherein the CRISPR-Cas protein is a Cas9 protein, and
wherein the method further comprises selecting the population of cells for a phenotype by treating a population of cancer cells with a chemotherapy and obtaining cancer cells having resistance to the chemotherapy, performing sequencing to profile the depletion or enrichment of the CRISPR-Cas system guide polynucleotide sequences in selected cancer cells, and identifying one or more gene products associated with the resistance to the chemotherapy.
2. The method of claim 1 , wherein the cell is a eukaryotic cell.
3. The method of claim 2 , wherein the eukaryotic cell is an animal or a plant cell.
4. The method of claim 2 , wherein the eukaryotic cell is a human cell.
5. The method of claim 1 , wherein the CRISPR-Cas system guide polynucleotide sequence and the CRISPR-Cas protein are comprised in one or more vectors.
6. The method of claim 5 , wherein the CRISPR-Cas system guide polynucleotide sequence and the CRISPR-Cas protein are comprised in one vector.
7. The method of claim 6 , wherein the vector is a lentivirus, a adenovirus, or a AAV.
8. The method of claim 5 , wherein the one or more vectors is a lentivirus, an adenovirus, or an adeno-associated virus (AAV).
9. The method of claim 1 , wherein the targeting is of about 100 or more sequences.
10. The method of claim 1 , wherein the Cas9 protein is Streptococcus pyogenes Cas9.
11. The method of claim 1 , wherein the Cas9 is Staphylococcus aureus Cas9.
12. The method of claim 1 , wherein the targeting is of about 1,000 or more sequences.
13. The method of claim 1 , wherein the targeting is of about 20,000 or more sequences.
14. The method of claim 1 , wherein the targeting is of about the entire genome.
15. The method of claim 1 , wherein the method comprises performing sequencing to profile the depletion or enrichment of the CRISPR-Cas system guide polynucleotide sequences in the selected cells and using the depletion or enrichment of the CRISPR-Cas system guide polynucleotide sequences to identify one or more gene products associated with the phenotype.