Fig. 2

Fig. 2

a Timeline. Cre mRNA injections at the 1-cell stage elicit ubiquitous recombination (blue box). At 12 hpf, a heat treatment triggers transient Cas9-GFP expression causing the subsequent permanent mutagenesis of the tyr target site (green box). Analysis of GFP expression and pigmentation was conducted at 22 and 50 hpf, respectively. b Expression of GFP is detected in a ubiquitous fashion at 22 hpf. In comparison to controls (ctrl), GFP-positive embryos (GFP+) display a significant loss of black pigment along the body (c) and in the developing eye (d). Examples shown are representatives across five experiments showing the same result. A total of >150 GFP-positive individuals and their respective non-GFP-positive siblings were analyzed. Scale bars: 150 µm in b and d; 500 µm in c. e Graph showing quantification of RPE pigmentation determined using the mean of the histogram from images of control embryos expressing no fluorescent protein (Non-FP), DsRed-positive (DsRed+), and GFP-positive (GFP+) siblings as well as tyrosinase mutants (tyr^−/−) at 50 hpf. n (Non-FP, DsRed+, tyr^−/−) = 12; n (GFP+) = 18. Data were analyzed using one-way ANOVA, followed by Tukey’s post hoc test. Values are presented as mean ± SEM; ****p ≤ 0.0001, ns (non-significant) >0.9999.