Fig. 1

G0 Mutation of s1pr2 by Single- or Four-Guide Cas9 RNP

(A) Embryos were cell-injected at the one-cell stage with single-guide or four-guide Cas9 RNP targeting s1pr2 and scored for phenotypes at 1 day post fertilization (dpf) and 4 dpf. Embryos were first scored as dysmorphic or not (lower panel). Non-dysmorphic embryos were then scored for the miles apart cardia bifida phenotype as defined in Figure S1. The percentage with strong (blue), intermediate (red), or no (green; wild-type [WT]) phenotype is indicated. The number of non-dysmorphic embryos scored is shown at bottom. ^∗∗∗∗p < 0.0001; ns, not significant; Fisher's exact test for Strong versus other phenotype. None of 76 embryos injected with control sgRNA exhibited cardia bifida; 5% were dysmorphic. The figure represents pooled data from two independent experiments.

(B) Representative Tg(myl7:GFP) G0 embryo with a Strong cardia bifida phenotype (bottom) after injection of a four-guide Cas9 RNP mix. A control clutchmate is shown at the top. Tg(myl7:GFP) labels the heart fields (green). Scale bar, 250 μm.

(C and D) Embryos injected with a four-guide Cas9 RNP mix targeting s1pr2 were collected at 1 dpf. DNA was prepared from two pools of 3 embryos (two separate experiments); s1pr2 amplicons were cloned and 30 were sequenced. (C) Wild-type (top line) and ten representative mutant sequences from injected embryos. Arrows denote predicted site of double-stranded breaks corresponding to each of the four guides used in (A). Sequences predicted to hybridize with CRISPR sgRNA spacers are highlighted with alternating green and yellow shading. (D) Summary of characteristics of 30 s1pr2 alleles analyzed.