Crispr/cas transcriptional modulation

Abstract

Methods, compositions, and kits are provided for CRISPR/Cas mediated transcriptional modulation.

1 . A method of screening for one or more genetic elements that modulate a phenotype, the method comprising:
 contacting a plurality of cells with a library of structurally distinct small guide RNAs (sgRNAs) that target a plurality of genetic elements, 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),
 wherein the dCas9 comprises
 a dCas9 domain fused to a transcriptional modulator; or 
 a dCas9 domain fused to an epitope fusion domain, 
 
 
   selecting the test cells on the basis of the phenotype; and   quantitating the frequency of the structurally distinct sgRNAs within the population of selected cells, wherein the sgRNAs that target genetic elements that modulate the phenotype are overrepresented or underrepresented in the selected cells.   
     
     
         2 - 65 . (canceled) 
     
     
         66 . A composition comprising a library of at least 50,000 structurally distinct small guide RNAs (sgRNAs) that target at least 10 4  genes,
 wherein the structurally distinct sgRNAs of the library comprise binding regions:   (i) lacki a UUU sequence; and   (ii) target a genomic site comprising a PAM site having the sequence NGG, wherein the sequence NGG is not followed by a G.   
     
     
         67 . The composition of  claim 66 , wherein the structurally distinct sgRNAs of the library comprise binding regions:
 (iii) begin at the 5′ end with a guanosine nucleotide; and   (iv) have a length of between 19 and 21 nucleotides.   
     
     
         68 . The composition of  claim 66 , wherein the sgRNA is encoded by an expression cassette comprising a promoter operably linked to a polynucleotide encoding the sgRNA. 
     
     
         69 . The composition of  claim 68 , wherein the promoter is inducible. 
     
     
         70 . The composition of  claim 66 , 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.   
     
     
         71 . The composition of  claim 66 , 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. 
     
     
         72 . The composition of  claim 66 , 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. 
     
     
         73 . The composition of  claim 66 , wherein a majority of the library of structurally distinct sgRNAs are targeted to non-overlapping genes. 
     
     
         74 . The composition of  claim 66 , wherein a majority of the targeted genes are targeted with 10 or fewer structurally distinct sgRNAs. 
     
     
         75 . The composition of  claim 66 , 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. 
     
     
         76 . The composition of  claim 66 , 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. 
     
     
         77 . The composition of  claim 66 , wherein the library of sgRNAs contains fewer than 5×10 4  structurally distinct sgRNAs. 
     
     
         78 . The composition of  claim 66 , wherein a majority of the at least 10 4  genes are targeted with no more than 25 structurally distinct sgRNAs. 
     
     
         79 . A method of identifying interacting genetic elements, the method comprising:
 (i) screening for one or more genetic elements that modulate a phenotype by:
 (ia) contacting a plurality of cells with a library of structurally distinct small guide RNAs (sgRNAs) that target a plurality of genetic elements, 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),
 wherein the dCas9 comprises
 a dCas9 domain fused to a transcriptional modulator; or 
 a dCas9 domain fused to an epitope fusion domain, 
 
 
 (ib) selecting the test cells on the basis of the phenotype; and 
 (ic) quantitating the frequency of the structurally distinct sgRNAs within the population of selected cells, wherein the sgRNAs that target genetic elements that modulate the phenotype are overrepresented or underrepresented in the selected cells 
 (id) identifying a plurality of structurally distinct sgRNAs that target genetic elements that modulate the phenotype; 
   (ii) contacting a plurality of interaction test cells with a library comprising a plurality of pairwise combinations of the structurally distinct sgRNAs identified in (id);   (iii) selecting the test cells on the basis of the phenotype; and   (iv) quantitating the 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 genetic elements.