The generation of <i>smyd4</i>–deficient zebrafish using the CRISPR/Cas9 technology.

(A) Schematic diagram of the smyd4 gene structure and the sgRNA3 sequence for the exon 6 targeting site. The sgRNAs were injected into the embryos with Cas9 mRNA at the single-cell stage; (B) The targeting site of sgRNA3 was well-conserved from zebrafish to humans; (C) Sanger sequencing confirmed the insertion mutation (c.1629_1630inGAATAATACTG; p.Leu544Glufs*1); (D) A schematic diagram of the insertion mutation. The smyd4 protein contains four functional domains: two TPR domains (green), one MYND domain (yellow), and one SET domain (purple). The insertion mutation created a truncated mutant protein that lacks the entire C-terminus, which contained the key functional domain TPR2; (E) qRT-PCR confirmation that smyd4 expression is significantly decreased in MZsmyd4L544Efs*1 embryos; (F) Whole-mount in situ hybridization analysis confirmed the loss of smyd4 expression in MZsmyd4L544Efs*1 embryos at 72 hpf.