Cas9 effector-mediated regulation of transcription, differentiation and gene editing/labeling

Abstract

The present disclosure relates to methods of and systems for modifying the transcriptional regulation of stem or progenitor cells to promote their differentiation or reprogramming of somatic cells. Further, the labeling and editing of human genomic loci in live cells with three orthogonal CRISPR/Cas9 components allow multicolor detection of genomic loci with high spatial resolution, which provides an avenue for barcoding elements of the human genome in the living state.

We claim: 
     
         1 . A method, comprising:
 a) providing;
 i) at least one stem cell comprising at least one specific genomic target; and 
 ii) a first and second lentiviral vectors, wherein said first lentiviral vector encodes a nuclease deficient Cas9-effector domain fusion protein and said second lentiviral vector comprises at least one sgRNA gene complementary with said specific genomic sequence; 
   b) expressing said first and second lentiviral vectors in said at least one stem cell wherein a nuclease deficient Cas9-effector domain fusion protein/sgRNA complex is formed; and   c) regulating transcription of said specific genomic target with said nuclease deficient Cas9-effector domain/sgRNA complex.   
     
     
         2 . The method of  claim 1 , wherein said at least one stem cell comprises a progenitor cell. 
     
     
         3 . The method of  claim 1 , wherein said regulating transcription is selected from the group consisting of enhancement of transcriptional activity and repression of transcriptional activity. 
     
     
         4 . The method of  claim 1 , wherein said specific genomic target is selected from the group consisting of a genomic region and a gene. 
     
     
         5 . The method of  claim 1 , wherein said first and second lentiviral vectors comprise a single vector. 
     
     
         6 . The method of  claim 1 , wherein said at least one sgRNA gene targets an intrachromosomal genomic sequence. 
     
     
         7 . The method of  claim 1 , wherein said at least one sgRNA gene targets an interchromosomal genomic sequence. 
     
     
         8 . The method of  claim 1 , wherein said regulating transcription results in phenotypic change of said at least one stem cell. 
     
     
         9 . The method of  claim 1 , wherein said effector domain is selected from the group consisting of a histone modification domain, a DNA modification domain and a RNA modification domain. 
     
     
         10 . The method of  claim 1 , wherein said regulating transcription modulates differentiation of said at least one stem cell. 
     
     
         11 . The method of  claim 1 , wherein said first and second lentiviral vectors comprise a promoter selected from the group consisting of a constitutive promoter and an inducible promoter. 
     
     
         12 . A method, comprising:
 a) providing;
 i) at least one cell comprising at least one specific genomic target; and 
 ii) a first and second lentiviral vectors, wherein said first lentiviral vector encodes at least one catalytically active Cas9-fluorescent protein fusion protein and said second lentiviral vector comprises at least one truncated sgRNA gene complementary with said specific genomic sequence; 
   b) expressing said first and second lentiviral vectors in said at least one stem cell wherein at least one catalytically active Cas9-fluorescent protein fusion protein/truncated sgRNA complex is formed; and   c) labeling said at least one specific genomic target with said catalytically active Cas9-fluorescent protein fusion protein/sgRNA complex.   
     
     
         13 . The method of  claim 12 , wherein said fluorescent protein is selected from the group consisting of a green fluorescent protein, a red fluorescent protein and a blue fluorescent protein. 
     
     
         14 . The method of  claim 12 , wherein each of said at least one specific genomic targets are labeled with a different colored fluorescent protein. 
     
     
         15 . The method of  claim 12 , wherein said at least one specific genomic target is selected from the group consisting of an intrachromosomal genomic sequence and an interchromosomal genomic sequence. 
     
     
         16 . The method of  claim 12 , further comprising imaging each of said labeled specific genomic targets. 
     
     
         17 . The method of  claim 16 , further comprising determining a distance between each of said labeled specific genomic targets on said image. 
     
     
         18 . The method of  claim 17 , further comprising constructing a chromsomal map comprising said labeled specific genomic targets on the basis of said distance. 
     
     
         19 . The method of  claim 18 , wherein said distance is selected from the group consisting of an interchromosomal distance and an intrachromosomal distance. 
     
     
         20 . The method of  claim 12 , wherein said at least one cell is a cell culture.