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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. 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