dCas9 induces chromatin accessibility at previously inaccessible genomic loci.

Abstract

<p><b>a</b>. mESCs were co-transfected with a Tol2 transposase (TPase), a Tol2 transposon-flanked dCas9 expression cassette, and a Tol2 transposon-flanked sgRNA cassette to yield stable expression of dCas9 and a sgRNA targeted to a region with inaccessible chromatin. <b>b</b>. 16/16 loci in previously inaccessible chromatin had statistically significant increases in DNase hypersensitivity (y-axis) upon sgRNA targeting as measured by DNase-qPCR (gray dots). DNase hypersensitivity at each locus is normalized to its level in the absence of sgRNA (blue dot), and the average normalized DNase hypersensitivity in the presence of gRNA for all loci is shown (red dot), which is statistically significantly increased over–sgRNA control. At least two replicates were performed for all conditions, and a two-tailed Student’s t-test used to calculate significance. <b>c</b>. DNase-qPCR measurement of DNase hypersensitivity (y-axis) is shown +/-150 bp from the sgRNA site (x-axis) at four targeted loci. DNase-qPCR values at each datapoint are normalized to hypersensitivity in the absence of sgRNA, and all loci are oriented such that the 20 bp sgRNA sequence is immediately to the left of 0 and the NGG PAM sequence is immediately to the right of 0. Three replicates were performed for all experiments.</p