Fig. 3

dCas9-TET1 induces locus-dependent DNA demethylation in DLD1 and HCT116 cells. A Schema illustrating the structure of the chimeric CRISPR-dCas9 construct fused to the catalytic domain (TET1, top) or the catalytically inactive domain (TET1-IM, bottom) of TET1. The position of the nuclear localization signal (NLS) and the mutations of the dCas9 or the TET1 catalytic domain are indicated. B Expression levels of chimeric dCas9-TET1, dCas9-TET1-IM and β-Tubulin proteins obtained by western blot analyses in control transduced or Cas9 transduced DLD1 and HCT116 cells. The approximate size of the protein products is indicated. C Line plot illustrating overall 5mC levels observed for a robust cancer-associated differentially hypermethylated region in control and tumour samples and in CRC cell lines. Data represent the average methylation status of the indicated CpG sites for the aforementioned categories. Significantly hypermethylated CpG sites observed in the differential methylation comparisons are highlighted in red. The genomic position of the gRNAs designed to modulate the DNA methylation status of this region is indicated. D Barplots representing the percentage of DNA methylation observed for the CpG sites included in the modulated differentially methylated region in DLD1 and HCT116 cells in the context of control gRNA (grey) or gRNAs targeting this DMR (blue) in cells transduced with dCas9-TET1- or dCas9-TET1-IM-related chimeras. Data represent mean ± standard deviation of at least 3 independent experiments, and two-sided Welch’s t tests were applied for the different statistical comparisons versus each corresponding control condition. ***p value < 0.001; n.s.—nonsignificant