Figure 3
From: A CRISPR-based approach for targeted DNA demethylation
Optimization of dCas9-based demethylation system. (a) Quantitative real-time PCR (qRT-PCR) data showed RANKL gene expression changes after transfection of different molar ratios of dCas9-Tet1-CD and MS2-Tet1-CD at equal total moles in HEK-293FT cells. X axis, molar ratios of transfected components individually, in total amount of 0.5 pmol per well in a 12-well plate format. (b) qRT-PCR data examined the changes in RANKL mRNA after transfection of different amounts of the system components to eliminate random demethylation effects in HEK-293FT cell line. X axis, listed molar quantities of transfected components (pmol/well in the format of 12-well plate). (c) Assessment of mRNA transcription of RANKL gene along the time course after transfection of the dCas9-based demethylation system (days 2–8) in HEK-293FT cells. (d) Dual luciferase-based NF-κB reporter assays were performed in HEK-293FT cells expressing exogenous RANK or not. They were co-transfected with sgRNA2.0-guided demethylation, with the luciferase activities (in relative units) normalized to that of the -sgRNA group. Blank, cells transfected with the expression vectors only; -sgRNA, cells co-transfected with dCas9- and MS2-Tet1-CD but not any sgRNAs targeting RANKL sites; TM, Tet1-CD with H1652Y/D1654A mutations. Data were shown after normalization to the controls (blank group) (means±s.e.m., n=3).
