Evaluation of dCas9 targeting to the TSS of various genes.

<p>Diagram of sgRNA binding sites and plots of mRNA repression in HEK 293T by dCas9 and sgRNAs targeting (A) <i>GAPDH</i>, (B) <i>PPIB</i>, (C) <i>MAPK1</i>, (D) <i>MAPK14</i>, (E) <i>TP53</i>, and (F) <i>RB1</i>. Each sgRNA is numbered by the distance from the transcriptional start site. “−”, upstream of +1; “+”, downstream of +1; +1, transcriptional start site. Labeled sites above and below the transcribed region indicate sgRNAs targeting the template or non-template DNA strands, respectively. Relative expression of each mRNA in HEK 293T cells co-transfected with dCas9 and sgRNA constructs. After three days, cells transfected with dCas9 and indicated sgRNA constructs were sorted for BFP-positive cells, analyzed by qRT-PCR. Results were normalized, linearly rescaled, and calculated for mean fold change (<i>n</i> = 3)±95% confidence interval, relative to Scramble1 negative control sgRNA. *<i>P</i><0.02, **<i>P</i><0.002 compared to Scramble1 sgRNA control by paired, one-sided t-test.