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. 2023 Apr 29;15:73. doi: 10.1186/s13148-023-01482-0

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© The Author(s) 2023

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 6 — 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