Fig. 1: Development of the dual-activator enCRISPRa system.
From: Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing
a Schematic of enCRISPRa containing three components: a dCas9-p300 fusion protein, the sgRNA with two MS2 hairpins, and the MCP-VP64 fusion protein. b Expression of MYOD upon dCas9 alone, dCas9-VP64 (V), dCas9-p300 (P), dCas9-VP64 + MCP-p300 (enCRISPRa-VP) or dCas9-p300 + MCP-VP64 (enCRISPRa-PV)-mediated enhancer activation in HEK293T cells. mRNA expression relative to nontransduced cells (control) is shown as mean ± SEM (n = 4 experiments). The differences between control and dCas9 activators were analyzed by a one-way ANOVA. #P < 0.05, ###P < 0.001. The difference between different dCas9 activators were analyzed by a one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, n.s. not significant. c Expression of HBE1, HBG1/2 and HBB upon dCas9 alone, dCas9-VP64 (V), dCas9-p300 (P), or enCRISPRa (VP and PV)-mediated activation of the HS2 enhancer in HEK293T cells. mRNA expression relative to nontransduced cells is shown as mean ± SEM (n = 4 experiments) and analyzed by a one-way ANOVA. d Expression of MYOD upon dxCas9-VPR, SunTag, SAM or enCRISPRa-mediated enhancer activation in HEK293T cells. mRNA expression relative to nontransduced cells is shown as mean ± SEM (n = 4 experiments). The differences between control and dCas9 activators were analyzed by a one-way ANOVA. #P < 0.05, ###P < 0.001. The difference between different dCas9 activators were analyzed by a one-way ANOVA. ***P < 0.001. e Expression of HBE1, HBG1/2 and HBB upon dxCas9-VPR, SunTag, SAM or enCRISPRa-mediated activation of HS2 in HEK293T cells. mRNA expression relative to nontransduced cells is shown as mean ± SEM (n = 4 experiments) and analyzed by a one-way ANOVA. f Genome-wide analysis of dCas9 binding in HEK293T cells expressing HS2-specific sgRNA (two replicates sgHS2-rep1 and sgHS2-rep2) or nontargeting sgGal4. Data points for the sgRNA target regions and the predicted off-targets are shown as green and red, respectively. The x- and y axis denote the normalized read counts (left) or mean normalized read counts from n = 2 experiments (right). Source data are provided as a Source Data file.
