CRISPR/Cas9-mediated deletion in tdTomato knock-in cells. (A) Schematic diagram of the target sites (blue) in tdTomato knock-in cells and primers (red arrow) for PCR assay. Green arrowheads indicate predicted cleavage site by Cas9. (B) Fluorescence microscopic observation of the expression level of tdTomato. The Cas9-GFP, tdTomato sgRNA or empty sgRNA expression construct was introduced into the tdTomato-expressing cells, and the resulting fluorescence was imaged under identical conditions. Red fluorescence images of a representative area are shown. (C) Intracellular fluorescence intensity levels in different CRISPR conditions. Each dot represents an individual cell. AX3 reveals the fluorescence level of parental strain without tdTomato. (D) PCR amplification using primers flanking the target site. Lane 1 presents the tdTomato knock-in cell as a control, whereas rest of the lanes represent eight individual clones expressing both Cas9 and sgRNA. Gel image was cropped, but no other bands were present.