(a) Wild-type, 2-dpf slc24a5b1 embryos and a series of slc24a5 CRISPR-injected embryos; anterior is to the left. Wild-type pigmentation is lost in slc24a5 (‘golden’) mutants. Injection of Cas9-encoding mRNA and an sgRNA targeting slc24a5 resulted in embryos with mosaic loss of eye pigmentation (a ʹ). (b) Model of the slc24a5 CRISPR target site with arrows denoting PCR primers used for sequence analysis of the locus. (c) Sequencing from embryos injected with 100/1,200 pg of slc24a5 sgRNA/Cas9-encoding mRNA. Deletions are denoted by dashes (-), and insertions are in bold. The underlined sequence denotes the NGG motif used by Cas9. (d) Quantitation of eye pigmentation as assessed by mean pixel gray value of the eye with varying amounts of Cas9-encoding mRNA and a constant 100 pg of slc24a5 sgRNA. Each open triangle represents an individual animal. (e,f) Quantitation of toxicity seen in injected embryos for varying amounts of Cas9-encoding mRNA and a constant 100 pg of slc24a5 sgRNA (e) or a constant 1,200 pg of Cas9 with varying slc24a5 sgRNA (f). Toxicity encompassed embryo death, edema, localized cell death and general developmental defects. In d, N = 24 for each condition; error bars denote s.e.m. In e and f, N > 77 embryos for each point.