SpG and SpRY are active nucleases at minimal PAM targets in zebrafish.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.
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