RNA-Guided Targeting of Genetic and Epigenomic Regulatory Proteins to Specific Genomic Loci

Abstract

Methods and constructs for RNA-guided targeting of transcriptional activators to specific genomic loci.

What is claimed is: 
     
         1 . A fusion protein comprising catalytically inactive CRISPR associated 9 (Cas9) protein linked to a heterologous functional domain. 
     
     
         2 . The fusion protein of  claim 1 , wherein the heterologous functional domain is a transcriptional activation domain. 
     
     
         3 . The fusion protein of  claim 2 , wherein the transcriptional activation domain is from VP64 or NF-κB p65. 
     
     
         4 . The fusion protein of  claim 1 , wherein the catalytically inactive Cas9 protein is from  S. pyogenes.    
     
     
         5 . The fusion protein of  claim 1 , wherein the catalytically inactive Cas9 protein comprises mutations at D10A and H840A. 
     
     
         6 . The fusion protein of  claim 1 , wherein the heterologous functional domain is linked to the N terminus or C terminus of the catalytically inactive Cas9 protein, with an optional intervening linker, wherein the linker does not interfere with activity of the fusion protein. 
     
     
         7 . The fusion protein of  claim 1 , further comprising one or both of a nuclear localization sequence and one or more epitope tags on the N-terminus, C-terminus, or in between the catalytically inactive CRISPR associated 9 (Cas9) protein and the heterologous functional domain, optionally with one or more intervening linkers. 
     
     
         8 . The fusion protein of  claim 7 , wherein the one or more epitope tags is selected from the group consisting of c-myc, 6His, and FLAG tags. 
     
     
         9 . A nucleic acid encoding the fusion protein of  claim 1 . 
     
     
         10 . A nucleic acid encoding the fusion protein of  claim 2 . 
     
     
         11 . A nucleic acid encoding the fusion protein of  claim 3 . 
     
     
         12 . A nucleic acid encoding the fusion protein of  claim 4 . 
     
     
         13 . An expression vector comprising the nucleic acid of  claim 9 . 
     
     
         14 . An expression vector comprising the nucleic acid of  claim 10 . 
     
     
         15 . An expression vector comprising the nucleic acid of  claim 11 . 
     
     
         16 . An expression vector comprising the nucleic acid of  claim 12 .