Fig. 5

The krCRISPR system enabled analysis of the effects of DNMT1 mutation on DNA methylation. a Schematic of the Rescue1 and Rescue2 plasmid design. Rescue1 plasmid contains a puromycin resistant gene and a GFP gene, while Rescue 2 plasmid contains a zeocin resistant gene and RFP gene. Transfection of Rescue2 plasmid into the knockout-Resuce1 cells will result in replacement of Rescue1 plasmid by Rescue2 plasmid under zeocin selection. b Flow cytometry analysis showed that the GFP positive cells were gradually replaced by RFP positive cells over time (n = 3, error bars showed mean ± SEM). c Distribution of the four DNMT1 mutations. d Schematic of the experimental workflow. e and f Two examples of Rescue plasmid replacement. The Rescue1 plasmid was gradually replaced by Rescue2 plasmid. A PmlI restriction site for A570V and an XmaI restriction site for H97R were introduced into the Rescue2 plasmids respectively. At day 0, 2 and 15, the plasmid DNA was isolated from cells and PCR-amplified for RFLP analysis. Gene mutations are labeled in red letter; the restriction sites are underlined. Black triangles indicate the Rescue1 plasmid; red triangles indicate the Rescue2 plasmid. g The effects of individual mutations on DNA methylation were measured by LUMA. The exogenous DNMT1 genes encoded by Rescue plasmids were shown below. H97R did not influence DNA methylation level. Y511C, A570V and P507L decreased DNA methylation level (n = 3, error bars showed mean ± SEM)