Figure 5

Germline transmission of genomic DNA editing and phenotypes of F1 offspring: Plots: Rod opsin levels assayed in individual eyes by dot blot assay at 14 dpf. Each data point represents a different F1 animal. Each X-axis point represents offspring of a single mating, for which the F0 parents are indicated as being WT and/or F0 animals (named Male 1, Male 2, etc.). Each plot represents samples analyzed on a separate dot blot, and all animals on each plot were modified using the same sgRNA, indicated at the bottom of the plot. (A–I) Phenotypes of F1 animals assessed by confocal microscopy. (A) Wildtype. (B–I) genetically modified (genotypes indicated on panels, for frame-conserving mutations the altered amino acid sequence is shown, with inserted amino acid residues shown in red). (B,C) frame-conserving mutations with minimal phenotype. (D–F) frame-conserving indels with significant RD phenotypes. (G–I) frame shifting indels. (A–C) High magnification panels: frame-conserving indels generating minimal or no RD phenotype do not alter rhodopsin localization, which is largely confined to outer segments (OS) and wheat germ agglutinin-positive inner segment (IS) membranes (arrowheads). (D–F) High magnification panels: frame-conserving indels generating significant RD phenotypes alter rhodopsin localization in inner segments causing a punctate distribution (D, arrowhead) or diffuse labeling consistent with ER retention (E,F, arrowheads). Green: anti-rhodopsin (B630N – A-I or 514–18 A-D high mag). Red: anti-rod transducin. Blue: Hoechst 33342. Bars = 20 µm (low mag) or 5 µm (high mag). RPE: retinal pigment epithelium. OS: outer segments. IS: inner segments. ONL: outer nuclear layer. INL: inner nuclear layer.