Figure 1
From: A surrogate reporter system for multiplexable evaluation of CRISPR/Cas9 in targeted mutagenesis
Establishment of a method for the evaluation of CRISPR/Cas-mediated targeted mutagenesis using a universal donor. (a) Schematic of linear donor fragment that contains a reporter system. The guide RNA cutting site is labelled at the 5′ end on the linear donors, as indicated. The stop codon is labelled with*. pA, polyA signal. (b) Representative flow cytometry plots of HeLa cells co-transfected with sgRNA and donors that consist of CMV-EGFP-pA (upper) or CMV-EGFP (lower) for three days. (c) FACS analysis for EGFP positivity in cells co-transfected with donors consisting of CMV-EGFP-pA and sgRNA/mCherry-expressing plasmids. The EGFP positivity has been normalised by mCherry expression using an equation of the form [Q2/(Q1 + Q2) × 100%]. Error bars indicate s.d. (n = 3), two-tailed p-values for the t-test. NS, not significant; *P < 0.05. (d) FACS analysis for EGFP positivity in cells co-transfected with donors consisting of CMV-EGFP and sgRNA/mCherry-expressing plasmids. Error bars indicate s.d. (n = 3), two-tailed p-values for the t-test. NS, not significant; ***P < 0.001. (e) FACS analysis for EGFP positivity in cells co-transfected with sgRNA and linear donors consisting of CMV-EGFP for the indicated number of days. The EGFP positivity was normalised by the transfection efficiency (mCherry), followed by subtracting EGFP positivity in the sgRNActrl group. The error bars indicate s.d. (n = 3).
