Figure 2.
Regulation of Cas9 activity in HeLa cells by miR-21-Cas9 switch. (A) Representative phase contrast and fluorescent microscopic images at 72 h after RNA transfection. Scale bar represents 200 μm. (B) Representative flow cytometry histograms of EGFP expression. x- and y-axes indicate EGFP intensity and % of max, respectively. (C) Quantification of Cas9 activity from (B). The percentage of EGFP negative cells was defined as Cas9 activity. Cas9 activity was reduced in miR-21-responsive Cas9 mRNA. (D) Verification of endogenous miR-21-dependent Cas9 silencing by using miR-21 inhibitor. The miR-21 inhibitor rescued Cas9 activity in miR-21-responsive Cas9 mRNA, whereas a negative miRNA inhibitor did not. For the negative control, cells were transfected with Cas9 mRNA and a non-targeting sgRNA (sgRNADMD). Error bars indicate the mean ± SD (n = 3).