Evaluation of sgRNA Target Sites for CRISPR-Mediated Repression of TP53
Figure 2
Evaluation of dCas9 targeting to the TSS of various genes.
Diagram of sgRNA binding sites and plots of mRNA repression in HEK 293T by dCas9 and sgRNAs targeting (A) GAPDH, (B) PPIB, (C) MAPK1, (D) MAPK14, (E) TP53, and (F) RB1. 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 (n = 3)±95% confidence interval, relative to Scramble1 negative control sgRNA. *P<0.02, **P<0.002 compared to Scramble1 sgRNA control by paired, one-sided t-test.