Fig. 2

Fig. 2

a Diagram illustrating 15 gRNAs (blue) with NGH PAM in three zebrafish genes. Five gRNAs targeting exon 1, exons 3, 4 and 5 and exons 2 and 3 of slc45a2, slc24a5 and tbxta, respectively. Different NGH PAMs for each target are detailed in Supplementary Data 1. b mRNA SpG shows high activity in NGH sites. Individual gRNAs described in panel a were injected with the SpG mRNA (240 pg gRNA and 300 pg mRNA per embryo). Stacked barplots show the percentage of albino-like/golden-like (gol-like)/class III, severe/class II, mild/class I, and phenotypically wild-type (WT) embryos 48 hpf after injection. (n) total number of injected embryos. The results were obtained from at least two independent experiments. c Phenotypes obtained after the injection of the mRNA–gRNA duplex targeting slc24a5 showing different levels of mosaicism (golden-like, severe, mild) compared to the WT. Lateral views (scale bar, 1 mm) and insets of the eyes (scale bar, 0.2 mm) of 48 hpf embryos are shown. d Phenotypes obtained after injection of the mRNA–gRNA duplex targeting tbxta in zebrafish embryos (Lateral views). Levels of mosaicism compared to wild type (WT) were evaluated at 28 hpf. Class I: Short tail (least extreme). Class II: Absence of notochord and short tail (medium level). Class III: Absence of notochord and extremely short tail (most extreme). Scale bar, 0.5 mm. e Diagram illustrating 8 gRNAs (green) with NAN PAM in three zebrafish genes. Three gRNAs targeting slc45a2 exons 1 and 5, two gRNAs targeting slc24a5 exons 1 and 3 and three gRNAs targeting tbxta exons 1 and 2. Different NAN PAMs for each target are detailed in Supplementary Data 1. f mRNA SpRY shows high activity in NAN sites. Individual gRNAs described in panel e were injected with the SpRY mRNA (240 pg gRNA and 300 pg mRNA per embryo). Stacked barplots show the percentage of albino-like/golden-like/class III, severe/class II, mild/class I, and phenotypically wild-type (WT) embryos 48 hpf after injection. (n) total number of injected embryos. The results were obtained from at least two independent experiments.