Fig. 4

Efficiency of 5mC demethylation induced by Casilio-ME platforms and dCas9-TET1 system. MLH1 promoter was targeted by using components of the indicated methylation editing system in the presence of MLH1-sgRNAs. Cells were collected 3 days after transfection and corresponding genomic DNA was subjected to high throughput amplicon BSeq and oxBSeq. a 5mC conversion to cytosine by TET1 and BER pathways is depicted above panels. Frequencies (mean ± S.E.M.; n = 2) of 5mC (upper panel), 5hmC (middle panel) and bisulfite converted CpG (C, 5fC, and 5caC) (lower panel) plotted against CpG positions within MLH1 promoter region are shown. The obtained levels of (5mC + 5hmC) and 5mC from two experiments were concordant. Arrows indicate locations of CpG overlapping one of the six sgRNAs target sequences. Statistical significance of differences in methylation patterns were tested. p < 0.0001, two-way ANOVA. b Box plot of frequencies of different CpG variants measured by BSeq and oxBSeq across the MLH1 promoter regions in cells transfected with the indicated 5mC demethylation system is shown. For each box plot the thick line inside the box represents the median value and the surrounding bottom and top lines represent the 25th and 75th percentiles. The whiskers represent min and max values, the x represents the mean value. **p < 0.01, and ***p < 0.0001, two-way ANOVA. c same as b but focusing on CpG 86–209 of the MLH1 promoter proximal-intron1 region. NS, not significant p > 0.05, and ***p < 0.01, two-way ANOVA