Figure 3

Figure 3

Generation and phenotyping of fech^−/+ fish and fech^−/− larvae. (A) Schematic representation of the organization of the zebrafish fech gene. Untranslated areas and open reading frames are depicted by white and black boxes, respectively. The target location for the sgRNA is denoted by the red arrowhead. The PAM and single-guide RNA (sgRNA) target sequences are indicated by red and blue letters, respectively. (B–D) Illustration of the insertion of 13 bp nucleotides into the target site and the introduction of a premature stop codon (*) using CRISPR/Cas9 gene editing. The inserted amino acid sequence is highlighted in yellow. (E) Genotyping of fech^−/+ and fech^−/− using PCR and agarose gel electrophoresis. (F) Confirmation of the mutation via RT-qPCR using fech target-site-specific primers in 7 dpf WT and fech^−/− larvae. (G) Detection of red fluorescence in 24 hpf fech^−/− larvae during the screening process.