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Fig. 4 Generation of EYS-KO iPSCs with CRISPR/Cas9 system. (A) Target sequence of CRSPR/Cas9 gene editing is located in exon 4 of the EYS gene. Sanger sequencing of edited iPSCs shows single base deletion (c.621 delT). (B) Bright-field view of EYS-KO retinal organoids on day 180. Lower panels are higher-magnification images of the dotted boxes in the upper panels. Scale bars: 200 μm. (C) Western blot analysis of EYS and RCVRN in control and EYS-KO retinal organoids on day 180. Full-length pictures of the blots are presented in Supplemental Figure 8, G–L. (D) Representative immunofluorescence images of GRK7 and OS marker PRPH2 in control and EYS-KO retinal organoids on day 180. Lower panels are higher-magnification images of the dotted boxes in the upper panels. White arrows indicate the OS region. Scale bars: 10 μm. (E) Quantitative analysis of merged immunoreactivity of PRPH2 and GRK7 in control and EYS-KO retinal organoids in D. The y axis indicates the number of reactive dots per field. Data represent mean ± SEM (n = 3 organoids). An unpaired, 2-tailed t test was used for statistical comparison (*P < 0.05).