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Fig. 5 | Clinical Epigenetics

Fig. 5

From: Epigenetic reactivation of tumor suppressor genes with CRISPRa technologies as precision therapy for hepatocellular carcinoma

Fig. 5

Maximizing reactivation of highly downregulated tumor suppressor genes in Hep3B and HuH-7 HCC cells. A Schematic representation depicting the CRISPRa toolbox developed for epigenetic editing. SpdCas9 C-terminally fused to VPR, i.e., SpdCas9-VPR (dark blue); gRNA-MS2-MCP-p65-HSF1 recruiting system (light blue); SpdCas9 C-terminally fused to TET1 catalytic domain, i.e., SpdCas9-TET1-CD (dark pink); gRNA-MS2-MCP-TET1-CD recruiting system (light pink); and SpdCas9 (yellow). B–H Fold change in MT1M (B), HHIP (C, D), PZP (E, F), and TTC36 (G, H) mRNA expression evaluated by qRT-PCR 96 h after transient transfection in Hep3B and HuH-7 cells. Cells were transfected with combinations of CRISPRa along with the most potent tumor suppressor gene-targeting gRNA/s, or with no gRNA (NO G) as control. Relative gene expression was normalized and compared to cells transfected with empty vector control (EV) for statistical analysis. From left to right: B MT1M: ****P < 0.0001, ***P = 0.0002; C HHIP: **** P < 0.0001, ***P = 0.0008; D HHIP: ****P < 0.0001, ***P = 0.0002; E PZP: ***P = 0.0002, ****P < 0.0001, **P = 0.0022, ***P = 0.0003, ***P = 0.0001; F PZP: ****P < 0.0001; G TTC36: ****P < 0.0001, *P = 0.0190, **P = 0.0030; H TTC36: ****P < 0.0001, **P = 0.0088, ***P = 0.0007. Data presented as means ± SEM (n = 3), and P values were determined by unpaired t-test. SpdCas9 Streptococcus pyogenes deactivated Cas9 protein adopted for epigenome engineering, VPR VP64, p65, Rta, MS2 RNA aptamer, MCP MS2-coat protein, HSF1 heat shock factor 1, TET1-CD Ten-Eleven Translocation methylcytosine dioxygenase 1-catalytic domain, MIX 4G combination of four gRNAs

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