Fig. 2. Establishment of a cell model to probe Dnmt3a dynamics in single cells. (a) The endogenous Dnmt3a in HeLa cells was knocked out by the CRISPR/Cas9 system and replaced with the fusion protein Dnmt3a–EGFP. (b) Experimental design: the Dnmt3a–EGFP could rescue and restore the methylation profile in KO cells; however, aberrant DNA methylation would recur after exposure to TCE, which can be correlated with the altered Dnmt3a–EGFP dynamics, if any. (c) TCE concentration-dependent DNA demethylation in model cells was determined by immunoassay (n = 3 independent replicates). (d) Time-dependent DNA demethylation induced by TCE was also determined (n = 3 independent replicates).