Fig. 1. Overview of the main steps of the cataract gene evaluation pipeline. Each step corresponds to a section of the protocol with the same title.

Fig. 2. CRISPR-Cas9 RNP dose optimization. A. Zebrafish embryos at 2dpf. Embryos injected with CRISPR-Cas9 RNP against tyr gene (bottom) had observable pigment loss compare to uninjected wildtype zebrafish (top). B and C. Optimization of tyr gRNA concentration (UI: n = 11, 0.5 μM: n = 9, 1.5 μM: n = 9, 4.5 μM: n = 16, 13.5 μM: n = 6). D and E. Optimization of Cas9 concentration (UI: n = 16, 4.5 μM/0.75 μg/μl: n = 57, 4.5 μM/1μg/μl: n = 33, 13.5 μM/0.75 μg/μl: n = 9, 13.5 μM/1μg/μl: n = 11). The pigment areas on the retina (B, D) and body (C, E) of zebrafish injected with a series of different concentrations of tyr gRNA or Cas9 were measured and compared (UI: uninjected; **p < 0.01, ***p < 0.001, ****p < 0.0001; ns: not significant; one-way ANOVA and Tukey’s multiple comparisons test; Mean ± SEM).

Fig. 3. CRISPR-Cas9 RNP delivery route optimization. A and B. Illustrations of cell injection (A) and yolk injection (B). The zebrafish embryos were cell-injected (n = 19) or yolk-injected (n = 30) with tyr RNP. The pigment areas on the retina (C) and body (D) of zebrafish in the cell injection group and yolk injection group were measured and compared. (*p < 0.05, ns: not significant; unpaired two-tailed t-test; Mean ± SEM).

Fig. 4. Editing efficiency analysis for individual gRNA. 5 injected larvae were randomly selected at 4dpf from each gRNA injected group, and the target region of individual embryos was amplified and analyzed for indel percentage.

Fig. 5. Zebrafish cataract assessment criteria (pitting).

Fig. 6. Zebrafish cataract assessment criteria (central mass).

Fig. 7. Normal developing lens images with maturing lens fiber cells.