Site-specific nuclease single-cell assay targeting gene regulatory elements to silence gene expression

Abstract

This invention relates to a single cell assay for determining the effect of chromosomal contact on the transcriptional activity of genes of interest in a cell and to methods of silencing gene expression in a cell by way of perturbing gene regulatory elements which are engaged in chromosomal contact.

1 . A single cell assay for determining the effect of chromosomal contact on the transcriptional activity of at least one gene of interest in a cell, comprising:
 (i) at least one site-specific nuclease for inducing a double stranded break in at least one region of chromatin or DNA engaged in chromosomal contact in the cell;   (ii) an immunofluorescent probe or a recombinant protein expressing a fluorescent label for detecting the site of the double stranded break; and   (iii) at least one fluorescent oligonucleotide probe capable of hybridising to at least one target mRNA sequence produced by the transcription of at least one gene of interest,   wherein the presence or absence of fluorescence and the relative intensity of fluorescence of the at least one fluorescent oligonucleotide probe hybridised to the target mRNA sequence is indicative of the effect of the chromosomal contact on the transcriptional activity of the at least one gene of interest.   
     
     
         2 . The single cell assay of  claim 1 , wherein transcriptional activity of the at least one gene of interest may be abrogated by a mechanism selected from the group consisting of:
 (i) recruitment of proteins involved in a repair process of the double stranded break, which when bound to the chromatin or DNA obstruct the chromosomal contact,   (ii) enhancement of mobility of the region of chromatin or DNA containing the double stranded break, which results in a reduction in capacity of a chromatin or DNA loop to engage in chromosomal contact; and   (iii) loss of structural integrity of a chromatin or DNA loop, which results in abrogation of chromosomal contact.   
     
     
         3 . The single cell assay of  claim 1 , wherein the chromosomal contact may be an intergenic contact, an intragenic contact or both intergenic and intragenic contact. 
     
     
         4 . The single cell assay of  claim 1 , wherein the double stranded break perturbs the chromatin or DNA engaged in chromosomal contact and perturbs transcriptional activity of the at least one gene of interest. 
     
     
         5 . The single cell assay of  claim 4 , wherein the region of chromatin or DNA that is perturbed comprises a gene or regulatory element selected from the group consisting of: (i) an enhancer and/or a promoter, (ii) a site within a chromatin or DNA loop that engages in intra- or inter-chromosomal contact, and (iii) a regulatory site within a chromatin or DNA loop which determines the loop structure. 
     
     
         6 . The single cell assay of  claim 1 , wherein the site specific nuclease is selected from the group consisting of a meganuclease, a zinc finger nuclease, a TALE nuclease, a BUD1 nuclease, and a CrispR/Cas9 nuclease. 
     
     
         7 . The single cell assay of  claim 1 , wherein the site-specific nuclease is delivered to the cell by transfection of the cell with a vector encoding the site-specific nuclease and wherein the site-specific nuclease is endogenously expressed in the cell or wherein the site-specific nuclease is exogenously expressed and delivered to the cell. 
     
     
         8 . The single cell assay of  claim 1 , wherein the immunofluorescent probe is an antibody which binds to at least one of the proteins involved in the cellular repair process of a double strand break in the cell. 
     
     
         9 . The single cell assay of  claim 1 , wherein alocation of the hybridized fluorescent oligonucleotide probe in the cell is indicative of achromosomal location of gene expression for the target mRNA sequences and alocation of the hybridised immunofluorescent probe is indicative of the location of the double stranded break. 
     
     
         10 . The single cell assay of  claim 1 , wherein location of the hybridized fluorescent oligonucleotide probe in the cell is observable by a technique selected from the group consisting of:
 diffraction limited imaging techniques, sub-diffraction limit image resolution and imaging in three dimensions,   wherein detection of chromosomal location of gene expression for the target mRNA sequences and diffraction limited or sub-diffractive location of the hybridised immunofluorescent probe is indicative of location of the double stranded break.   
     
     
         11 . The single cell assay of  claim 1 , wherein the cell is a eukaryotic cell or a prokaryotic cell. 
     
     
         12 . The single cell assay of  claim 5 , wherein the gene or regulatory element is an enhancer and/or a promoter. 
     
     
         13 . The single cell assay of  claim 5 , wherein the gene or regulatory element is a site within a chromatin or DNA loop that engages in intra- or inter-chromosomal contact. 
     
     
         14 . The single cell assay of  claim 5 , wherein the gene or regulatory element is a regulatory site within a chromatin or DNA loop which determines the loop structure. 
     
     
         15 . The single cell assay of  claim 6 , wherein the site specific nuclease is a meganuclease. 
     
     
         16 . The single cell assay of  claim 6 , wherein the site specific nuclease is a zinc finger nuclease. 
     
     
         17 . The single cell assay of  claim 6 , wherein the site specific nuclease is a TALE nuclease. 
     
     
         18 . The single cell assay of  claim 6 , wherein the site specific nuclease is a BUD1 nuclease. 
     
     
         19 . The single cell assay of  claim 6 , wherein the site specific nuclease is a CrispR/Cas9 nuclease.