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Fig. 3 Generation of promoter-less drish mutant using CRISPR/Cas9. (A) Schematic diagram showing the targeting sites of two gRNAs (red arrows). gRNA 1 targeted the 5′UTR region of drish gene, while gRNA 2 targeted ~800 bp upstream of the 5′UTR region. The lower diagram shows drish mutant allele (drishci51) with a 797 bp deletion and the adjacent junctional sequence. (B) qPCR analysis showed a significant reduction of drish in drish−/− mutant embryos compared to drish+/+ embryos at 3 dpf. (C, D) WISH showed a significant reduction of drish mRNA in drish−/− mutant embryo at 24 hpf. Lateral view with anterior to the left is shown. Note the absence of neural expression but presence of some endothelial expression. (E–H) Brightfield images of drish mutant and control wild-type embryos at 48 hpf. (G, H) show higher magnification view of the anterior portion of an embryo. No apparent morphological defects were observed. Scale bars: 100 μm.