Fig. 1

Generation of zebrafish vcp knockout by CRISPR/Cas9 gene editing. (A) Structure and partial sequence of the vcp gene and protein. CRISPR/Cas9 gene editing modifies exon 3 of vcp leading to the insertion of 206 nucleotides, a frame shift, and finally the introduction of a premature stop codon, and thereby the premature termination of Vcp translation after 138 amino acids. (B,C) Immunoblot analysis and quantification of homozygous mutant vcp^ex3/ex3 embryo protein lysate compared to protein lysate obtained from clutchmates (indicated as WT) at 72 hpf (N = 3; mean ± S.D, p ≤ 0.0001 determined using two-tailed t-test). Error bars indicate s.d.; **** p < 0.0001. (D) Quantitative real-time PCR of vcp^ex3/ex3 and WT embryos at 72 hpf shows no alterations in vcp transcript levels in vcp^ex3/ex3 embryos (N = 3, mean ± SD, p < 0.0001 determined using two-tailed t-test). Error bars indicate s.d.; ns, not significant. (E) In-crosses of heterozygous carriers yielded offspring demonstrating the regular Mendelian genotypic ratio of 25% homozygous wild-type (vcp^+/+; WT), 50% heterozygous (vcp^+/ex3), and 25% homozygous mutant (vcp^ex3/ex3) embryos (N = 3, n = 100). (F) A Kaplan–Meier survival curve was constructed for 11 homozygous vcp^ex3/ex3 mutants and 28 control embryos, which showed a sharp increase in mortality starting at 72 hpf in vcp^ex3/ex3 mutants compared to their wild-type clutchmates (n = 39, Mantel–Cox test, p < 0.0001). (G) Lateral view of brightfield images of WT and vcp^ex3/ex3 zebrafish embryos at 24, 48, and 72 hpf. Homozygous vcp^ex3/ex3 mutants exhibit phenotypic alterations at 72 hpf, whereas vcp^ex3/ex3 mutant embryos at 24 and 48 hpf are indistinguishable from their wild-type clutchmates. ns: not significant.