Mutagenetic phenotype of various nuclear localization signal (NLS)-fused Cas9 proteins and N-Cas9-C mRNA for two sites of the tyr gene. Lateral views of wild-type (WT) (A and E) and tyr-target 1- (B and C) or tyr-target 2- injected (F and G) embryos at 48 hpf (hr postfertilization). The phenotypes observed were WT (A and E), mosaic hypopigmentation (B and F), and null-like (C and G). Proportions of embryos with each phenotype (D and H). Scale bars: 0.2 mm.

Mutagenetic phenotype of various nuclear localization signal (NLS)-fused Cas9 proteins and N-Cas9-C mRNA for two sites of the gol gene. (A–C) and (E–G) show lateral views of wild-type (WT) (A and E), gol-target 1 (B and C), and gol-target 2 (F and G) at 48 hpf (hr postfertilization). The phenotypes observed were WT (A and E), mosaic retinal pigmented epithelium (RPE) (B and F), and unpigmented RPE (C and G). (D and H) Proportions of embryos with each phenotype. Scale bars: 0.2 mm.

Multiplex knockout efficiency of various nuclear localization signal (NLS)-fused Cas9 proteins and N-Cas9-C mRNA. Survival rates of zebrafish embryos at 24 and 48 hr postfertilization (hpf) (A) and T7 Endonuclease I (T7E1) assays of the rates of mutagenesis for each site (B). The phenotypes observed were wild-type (WT) (C), severe mosaic hypopigmentation (D), and null-like (E). (F) Proportions of embryos with each phenotype. Scale bars: 0.2 mm.