Spatiotemporal control of gene editing in zebrafish embryos. (a) Temporally resolved light-mediated gene editing in zebrafish embryos microinjected with PC-gAlbino and Cas9 protein and globally exposed to the 365 nm light at indicated developmental stages. The total number of embryos exhibiting each knockout phenotype category was counted for each treatment group (N = 11–41). The representative images of each knockout phenotype are shown on the left side. Dark–no UV irradiation, only ambient light. Scale bar 1 mm. (b) Automated image analysis of impaired pigment formation in slc45a2 mutated embryos using Fiji ImageJ software. Results are expressed as individual data points overlaid with the mean ± SD (N = 11–49); ***p < 0.001, ns–not significant (p > 0.05) versus wild-type embryos according to one-way ANOVA analysis combined with Tukey’s (Holm-Sidak) posthoc test. (c) Mismatch-based mutation detection PCR assay of slc45a2 gene in embryos microinjected with Cas9 protein and PC-gAlbino and globally irradiated with 365 nm UV light at various developmental stages. (N = 5 embryos per sample). (d) Representative photographs of zebrafish embryos microinjected with PC-gAlbino and Cas9 protein and either exposed to 405 nm laser light at 12-somite stage locally in the eye area (bottom image) or kept in the ambient light (dark) as a negative control (upper image). (e) Mismatch-based mutation detection assay of slc45a2 gene in embryos microinjected with Cas9 protein and PC-gAlbino, and locally irradiated in the eye area using 405 nm laser. The assay was run on the head and tail parts separately for each individual fish, and 3–5 embryos were combined for each treatment group.