Figure 1.
Repression of TP53 by targeting dCas9 to the transcriptional start site.
(A) HEK 293T cells were co-transfected with dCas9 (left) and sgRNA (right) expression plasmids. Codon-optimized dCas9 was fused to three copies of nuclear localization signal (NLS) and was co-expressed with mCherry fluorescent protein. sgRNA plasmid expresses mBFP and sgRNA off separate promoters. PCMV, CMV promoter; 2A, ribosomal slippage site; PuroR, puromycin resistance gene; IRES, internal ribosome entry site; mBFP, TagBFP fluorescent protein; BlastR, blasticidin resistance gene; PU6, U6 promoter. (B) Locations of sgRNA binding sites in the TP53 promoter and transcribed region (thick line). Each sgRNA is numbered by the distance (bp) from the transcriptional start site. “−”, upstream of +1; “+”, downstream of +1; “K”, one thousand bp; +1, transcriptional start site. Labeled sites above and below the transcribed region indicate sgRNAs targeting the template or non-template DNA strands, respectively. (C, D) Relative expression of TP53 mRNA in cells co-transfected with dCas9 and indicated sgRNA constructs. After three days, cells were (C) directly analyzed by qRT-PCR or (D) sorted for BFP-positive cells, then 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<0.01 compared to non-targeted sgRNA control by paired, one-sided t-test. (E) Immunoblot from HEK 293T cell lysate three days post-transfection with shRNA against TP53, dCas9 and sgRNAs against TP53 (e.g., dCas9/−T36), dCas9 and non-targeted sgRNA (dCas9/Scramble1), other control constructs (GFP expression vector, which served as non-specific vector control, or pSuper without any shRNA encoded), or mock (PEI only). See Figure 2E for location of sgRNA +T111. pSuper constructs were co-transfected with GFP-IRES-mCherry-2A-Puro expression constructs for selection purposes and experimental consistency (pSuper/GFP, pSuper-p53/GFP). Protein ladder (lane 2 from left) is not visible. See Figure S5 for uncropped immunoblots.
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.