CRISPR/Cas transcriptional modulation

1. A method of screening for one or more genes that modulate a phenotype, the method comprising:

contacting a plurality of cells with a library of structurally distinct small guide RNAs (sgRNAs) that target at least 10 4 genes, thereby generating a plurality of test cells, the plurality of test cells each comprising:

a small guide RNA (sgRNA); and

a nuclease deficient sgRNA-mediated nuclease (dCas9) that recognizes a PAM site having the sequence NGG,

wherein the dCas9 comprises

a dCas9 domain fused to a transcriptional modulator;

selecting the test cells on the basis of the phenotype;

quantitating a frequency of the structurally distinct sgRNAs within the population of selected test cells, wherein the sgRNAs that target the at least 10 4 genes that modulate the phenotype are overrepresented or underrepresented in the selected test cells, wherein:

i) the sgRNAs that target the genes are overrepresented or underrepresented within the selected test cells relative to the frequency of the corresponding sgRNAs in the sgRNA library; or

ii) the method further comprises contacting a plurality of control cells with the sgRNA library, wherein the plurality of control cells are not subject to the selecting on the basis of the phenotype, and the sgRNAs that target the genes are overrepresented or underrepresented in the selected test cells relative to a corresponding frequency in the plurality of control cells; or

iii) wherein the sgRNAs that target the genes are overrepresented or underrepresented in the selected test cells relative to the corresponding frequency in the test cells at an earlier time point in a culturing of the test cells;

wherein at least a majority of the library of structurally distinct sgRNAs are targeted to a region between −50 to +350 relative to a transcription start site of the targeted genes if the transcriptional modulator is a transcriptional repressor; and −400 to −50 bp relative to the transcription start site of the targeted genes if the transcriptional modulator is a transcriptional activator;

wherein a majority of the at least 10 4 genes are targeted with no more than 25 structurally distinct sgRNAs and

wherein the structurally distinct sgRNAs of the library comprise binding regions:

(i) beginning at the 5′ end with a guanosine nucleotide;

(ii) having a length of between 19 and 21 nucleotides;

(iii) lacking a UUU sequence; and

(iv) targeting a genomic site in which the PAM site having the sequence NGG is not followed by a G.

2. The method of claim 1 , wherein the library of sgRNAs contains at least 50,000 structurally distinct sgRNAs.

3. The method of claim 1 , wherein the selecting the test cells on the basis of the phenotype comprises selecting the test cells on the basis of protein expression, RNA expression, or protein activity, or wherein the selecting the test cells on the basis of the phenotype comprises fluorescence activated cell sorting, affinity purification of cells, selection based on cell motility or culturing the cells, thereby selecting the test cells on the basis of cellular proliferation.

4. The method of claim 1 , wherein the sgRNA is encoded by an expression cassette in the test cell, the expression cassette comprising a promoter operably linked to a polynucleotide encoding the sgRNA.

5. A method of identifying a lead compound for treatment of a phenotype, the method comprising:

performing a method according to claim 1 , thereby identifying a gene that modulates the phenotype; and

identifying or screening for a lead compound that modulates

expression of the gene or activity of a peptide encoded by the gene.

6. A method of identifying interacting genes, the method comprising:

(i) performing a method according to claim 1 , thereby identifying a plurality of structurally distinct sgRNAs that target genes that modulate the phenotype;

(ii) contacting a plurality of test cells with a library comprising a plurality of pairwise combinations of the structurally distinct sgRNAs identified in (i);

(iii) selecting the test cells based on the phenotype; and

(iv) quantitating frequency of the pairwise combinations of structurally distinct sgRNAs within the population of selected cells, wherein the pairwise combinations of structurally distinct sgRNAs that are overrepresented or underrepresented in the selected cells are predicted to target interacting genes.

7. The method of claim 6 , wherein the pair wise combinations of structurally distinct sgRNAs comprise a first member and a second member of the pair, and wherein the first member and second member target unlinked genes.

8. The method of claim 6 , wherein the pairwise combinations of structurally distinct sgRNAs that are overrepresented or underrepresented in the selected cells are overrepresented or underrepresented relative to the frequency of first and second members of each pairwise combination in (ii).

9. A method of optimizing an sgRNA, the method comprising:

performing a method according to claim 1 ,

wherein the plurality of structurally distinct sgRNAs target different regions within or next to a single gene,

wherein the most overrepresented or underrepresented sgRNAs in the selected test cells are identified as optimized sgRNAs that target the gene.

10. The method of claim 1 , wherein a majority of the library of structurally distinct sgRNAs comprise binding regions having at least one of the following:

(i) a binding region that targets a gene that is at least 1 bp apart from a gene targeted by another sgRNA of the library;

(ii) a GC percentage of between about 40% and about 60%; and

(iii) a methylated or fluorescent nucleotide.

11. The method of claim 1 , wherein a majority of the library of structurally distinct sgRNAs do not contain a nucleotide sequence of three or more repeated nucleotides, excluding a 3′ transcription termination sequence of the structurally distinct sgRNAs.

12. The method of claim 1 , wherein a majority of the library of structurally distinct sgRNAs lack a binding region having three or more nucleotide mismatches between the binding region and the targeted gene.

13. The method of claim 1 , wherein a majority of the library of structurally distinct sgRNAs are targeted to non-overlapping genes.

14. The method of claim 1 , wherein a majority of the targeted genes are targeted with 10 or fewer structurally distinct sgRNAs.

15. The method of claim 1 , wherein a majority of the library of structurally distinct sgRNAs are targeted to a region of between 0 and 350 base pairs downstream of a transcription start site of the targeted genes; and wherein the transcriptional modulator is a transcriptional repressor.

16. The method of claim 15 , wherein a majority of the library of structurally distinct sgRNAs are targeted to a region of between 25 and 100 base pairs downstream of the transcription start site of the targeted genes.

17. The method of claim 1 , wherein the library of sgRNAs contains fewer than 5×10 4 structurally distinct sgRNAs and wherein the at least 10 4 genes are targeted with less than 25 sgRNAs per targeted gene.