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. 2017 May 19;45(13):e118. doi: 10.1093/nar/gkx309

Figure 3.

Figure 3.

Regulation of Cas9 activity in iPS cells by 4x miR-302-Cas9 switch and improved sgRNA. (A) Representative phase contrast and fluorescent microscopic images at 72 h after RNA transfection. iPS-EGFP cells formed colonies (yellow dashed lines). 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). A miR-302 inhibitor rescued Cas9 activity expressed from miR-302-responsive Cas9 mRNA, whereas a negative miRNA inhibitor did not. To improve this system, 4x miR-302-responsive Cas9 mRNA (4 copies of miR-302 target site were tandemly inserted into 5΄-UTR) and sgRNA2GFP (removal of GG from 5΄ terminal) were used. Negative control is Cas9–sgRNADMD that targets DMD gene. Error bars indicate the mean ± SD (n = 3).