Nucleotide substitution by tandem nick (TN) of the reporter gene. (A) Schematic of the nucleotide substitution reporter assay. Representative data from flow cytometry and epifluorescent images of 293T EGFPcC>G reporter cells are also presented. (B) Diagram of the EGFPcC>G reporter and sgRNAs. (C) Diagram of the repair templates. (D) Nucleotide substitution efficiency as measured by flow cytometry (mean ± SD, N = 3) is presented. The indicated sgRNAs were expressed with Cas9D10A. The indicated repair templates were introduced into the cells as plasmid donors. Also see Supplemental Figure S1.