Fig 2: 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

Fig 3: Specific and potent reactivation of silenced tumor suppressor genes by CRISPRa compared to epi-drugs, and CRISPRa-enabled genome multiplexing in Hep3B and HuH-7 HCC cells. A–F Heatmap of the 12-tumor suppressor gene (TSG) panel comparing the fold change in mRNA regulation evaluated by qRT-PCR 48 h after transient transfections (A), (B), (D) and (E), 72 h after 5-aza, or 48 h after SAHA treatments (C) and (F). The transfection conditions and the epi-drug treatments are arranged in rows, and the genes in columns. Hep3B and HuH-7 cells were either transfected with CRISPRa (SpdCas9-VPR and MS2-MCP-p65-HSF1) along with the most potent TSG-targeting gRNA, or with no gRNA (NO G) as control; or treated with epi-drugs or vehicle control. Relative gene expression was normalized to cells transfected with empty vector control and compared to CRISPRa with NO G (A), (B), (D), and (E), or normalized and compared to cells treated with vehicle control (C) and (F) for statistical analysis. Data presented as means (n = 3). A Targeted transcriptional regulation of four TSGs by CRISPRa in Hep3B cells. P values were determined by two-way ANOVA with Dunnett's multiple comparisons test (HHIP G4: ****P < 0.0001; MT1M G1: ****P < 0.0001; PZP G2: ****P < 0.0001; and TTC36 G3: **** P < 0.0001). B Targeted genome multiplexing for simultaneous transcriptional reactivation of four TSGs by CRISPRa in Hep3B cells. P values were determined by two-way ANOVA with Šídák's multiple comparisons test (HHIP: **P = 0.0092; MT1E: P = not significant; MT1M: ****P < 0.0001; PZP: ****P < 0.0001; and TTC36: ****P < 0.0001). C Untargeted transcriptional regulation of several TSGs in Hep3B cells treated with 10 µM 5-aza (MT1E: ****P < 0.0001; MT1M: ****P < 0.0001; PTGR1: ****P < 0.0001; PZP: ****P < 0.0001; and TTC36: *P = 0.0207), 1 µM SAHA, or 4 µM SAHA (HHIP: ****P < 0.0001; and MT1E: **P = 0.0089). P values were determined by two-way ANOVA with Dunnett's multiple comparisons test. D Targeted transcriptional regulation of three TSGs by CRISPRa in HuH-7 cells. P values were determined by two-way ANOVA with Šídák's multiple comparisons test. (HHIP G4: ****P < 0.0001; PZP G2: ****P < 0.0001; and TTC36 G3: ****P < 0.0001). E Targeted genome multiplexing for simultaneous transcriptional reactivation of three TSGs by CRISPRa in HuH-7 cells. P values were determined by two-way ANOVA with Šídák's multiple comparisons test (HHIP: ****P < 0.0001; MT1E: P = not significant; PZP: ****P < 0.0001; TMEM106A: P = not significant; and TTC36: ****P < 0.0001). F Untargeted transcriptional regulation of several TSGs in HuH-7 cells treated with 10 µM 5-aza (HHIP: ****P < 0.0001; PZP: ****P < 0.0001; TMEM106A: ****P < 0.0001; and TTC36: ****P < 0.0001), 1 µM SAHA, or 4 µM SAHA (BCO2: ****P < 0.0001; HHIP: ****P < 0.0001; and TTC36: **P = 0.0072). P values were determined by two-way ANOVA with Dunnett's multiple comparisons test

Fig 4: Reactivation of silenced tumor suppressor genes by CRISPRa correlates with phenotypic reprogramming in Hep3B HCC cells. A, B Transcriptional reactivation of four silenced tumor suppressor genes (TSGs), HHIP, MT1M, PZP, and TTC36, targeted individually and simultaneously (MIX 4 genes) by SpdCas9-VPR stably expressed along with the corresponding TSG-gRNA in Hep3B HCC cells. A Data shown as fold log10 change in TSG mRNA levels, evaluated by qRT-PCR, relative to SpdCas9-VPR with no gRNA (NO G). Data presented as means ± SEM (n = 3) and P values were determined by one-way ANOVA with Dunnett's multiple comparisons test (****P < 0.0001, *P = 0.0107). B Heatmap comparing the fold change in mRNA regulation evaluated by qRT-PCR. The TSG-gRNAs are arranged in rows, and the genes in columns. Data presented as means (n = 3). C–F Immunofluorescence of HHIP, MT1M, PZP, TTC36, SpdCas9-VPR, and Hoechst-stained cell-nuclei in stable Hep3B cells expressing SpdCas9-VPR alone (NO G), SpdCas9-VPR targeting HHIP with G4, MT1M with G1, PZP with G2, TTC36 with G3, or SpdCas9-VPR co-targeting all four TSGs (MIX 4 genes). G–I Phenotypic reprogramming in Hep3B cells lentivirally transduced with SpdCas9-VPR targeting and upregulating HHIP with G4, TTC36 with G3, the MIX 4 genes, or with NO G as control. G Cell proliferation assessed by a-Ki-67 immunostaining (green), superimposed on nuclear Hoechst 33258 staining (blue). Data normalized to SpdCas9-VPR NO G, presented as means ± SEM (n = 3), and P values were determined by unpaired t-test (****P < 0.0001, ***P = 0.0003). H Cell viability determined using a luminescence assay (CellTiter-Glo®). Data shown as fold change compared to SpdCas9-VPR NO G at 24, 48, and 72 h, presented as means ± SEM (n = 3), and P values were determined by unpaired t-test with Welch's correction (****P < 0.0001). I Inhibition of cell migration assessed by the Boyden chamber assay. Data normalized to SpdCas9-VPR NO G, presented as means ± SEM (n = 3), and P values were determined by unpaired t-test (*P = 0.0390, ****P < 0.0001, ***P = 0.0008)

Fig 5: Upregulation of tumor suppressor genes by CRISPRa in Hep3B HCC cells. A Schematic representation of CRISPRa consisting of SpdCas9 C-terminally fused to the tripartite transactivator VPR (VP64, p65, and Rta) and coupled with the gRNA-MS2-MCP system that recruits the bipartite transactivator p65-HSF1 for targeted epigenetic editing. As indicated for each tumor suppressor gene (TSG), gRNAs, designated as G1, G2, G3, and G4, direct CRISPRa to the forward (right arrow) or reverse (left arrow) DNA strand within the regulatory region and proximal promoter of the TSG. gRNA numbering (±) refers to the distance in base pairs from the transcription start site (TSS) of each targeted TSG. B–H Reactivation of TSGs by CRISPRa was evaluated by qRT-PCR 48 h after transient transfection. Fold change in TSG mRNA expression from transfected cells with CRISPRa and TSG-targeting gRNAs, and normal hepatocytes was normalized to control transfections with empty vector (EV) and compared to CRISPRa with no gRNA (NO G) for statistical analysis. From left to right: B HHIP: *P = 0.0390, ****P < 0.0001; C MT1M: ***P = 0.0004, ****P < 0.0001; D PZP: ***P = 0.0006, ****P < 0.0001, ***P = 0.0002, *P = 0.0359, **P = 0.0013, ****P < 0.0001; E TTC36: **P = 0.0013, **P = 0.0022, ***P = 0.0001, ***P = 0.0004, ****P < 0.0001; F MT1E: *P = 0.0349, ****P < 0.0001, ***P = 0.0002; G miR-122-5p: **P = 0.0014, *P = 0.0119, ****P < 0.0001; H PTGR1: ***P = 0.0001, ****P < 0.0001. 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, MS2 RNA aptamer, MCP MS2 coat protein, HSF1 heat shock factor 1, Chr chromosome, (+) forward DNA strand, (-) reverse DNA strand, and MIX 4G combination of all four gRNAs targeting a TSG