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. 2015 Oct;43(10):838–848.e3. doi: 10.1016/j.exphem.2015.06.002

Figure 1.

Figure 1

Fluorescent reporter system for CRISPR-Cas9 activity. (A) Alignment of BFP and eGFP showing the mutations that are required for gene editing to convert BFP into eGFP (bold), the location of gRNA-27 target site (underlined), and its PAM (overlined). (B) Representative dot plot showing the change in fluorescence from BFP-expressing cells to eGFP after CRISPR-Cas9 mediated gene editing in iPS cells. (C) Gene-editing efficiency of BFP into eGFP in HEK 293 cells (black) and OX1-19 iPS cells (grey bars) using gRNA-27 and donor templates, as indicated. Templates included plasmids containing the WT eGFP sequence (WT plasmid) or a mutated eGFP sequence with the PAM altered to prevent cleavage without altering the amino acid sequence (Mut plasmid), as well as single-stranded oligonucleotides with the WT (WT ssODN) or PAM mutant (Mut ssODN) sequences. Experiments are presented as mean ± standard deviation of three independent experiments normalized to transfection efficiency measured by DsRed cotransfected plasmid for 293 cells, and ±standard deviation of an experiment performed in triplicate for the iPS cells. Statistical significance was calculated by Student's t test. ***p < 0.001; ****p < 0.0001.