Morphological defects observed in Rgma and Neo1 LOF embryos. A–I, Bright field images of 32 hpf larvae that were uninjected (A) or injected with the following molecular tools: std MO (B), rgma MO1 (C), rgma MO2 (D), neo1a MO2 (E), neo1a^δ1133-FLAG (F), rgma gRNA exon 2 + 3 and HF-nCas9 (rgma^ex2+3) (G), neo1a gRNA exon 3 + 8 and HF-nCas9 (neo1a^ex3+8) (H), and neo1b gRNA exon 3 + 8 and HF-nCas9 (neo1b^ex3+8) (I). Scale bar, 250 μm. J, Distribution of WT and LOF phenotypes in 32 hpf embryos treated as in A–I, with the exception of the last dataset (far right column) showing phenotypic distribution for multiplexed neo1a^ex3+8 and neo1b^ex3+8. WT, mild, moderate, and severe phenotypes exhibited 0, 1, 2–3, and 4 or more of the following features, respectively: a shortened anterior–posterior axis, poorly defined brain morphological landmarks, misshaped somites, small eyes, and decreased pigmentation (the latter is indicative of developmental delay). Uninjected: WT = 87%, mild = 6%, moderate = 6%, severe = 0%, n = 63; std MO: WT = 88%, mild = 8%, moderate = 4%, severe = 0%, n = 48; rgma MO1: WT = 58, mild = 9%, moderate = 33%, severe = 0%, n = 99; rgma MO2: WT = 5%, mild = 4%, moderate = 84%, severe = 7%, n = 57; neo1a MO2: WT = 0%, mild = 2%, moderate = 7%, severe = 91%, n = 56; neo1a^δ1133-FLAG: WT = 15%, mild = 10%, moderate = 44%, severe = 31%, n = 39; rgma^ex2+3: WT = 26%, mild = 17%, moderate = 29%, severe = 28%, n = 69; neo1a^ex3+8/neo1b^ex3+8: WT = 7%, mild = 10%, moderate = 67%, severe = 17%, n = 42. Data represent pooled results from 3 independent experiments.