Figure 1

Figure 1

Generation and verification of CRISPR-Cas9 eif2ak2 (pkr) and pkz mutations in zebrafish (a) Structure of zebrafish eif2ak2 (pkr) and pkz genes and proteins. The protein domains including double stranded RNA-binding domains (dsRB), Z-DNA/RNA binding domains (zα) and kinase domains are aligned to the corresponding exons. The CRISPR/Cas9 gene editing targets were exon 2 in zebrafish eif2ak2 gene and exon 1 in zebrafish pkz gene; sgRNA target sequences are also displayed (orange, PAM lower case). The CRISPR/Cas9-induced changes in the WT eif2ak2 gene (34-base insertion) to generate PKR-KO, and WT pkz gene (14-base deletion) to generate the PKZ-KO mutant strains are displayed. After the generation of the PKZ-KO mutant strain, the WT eif2ak2 gene in this strain was also mutated, resulting in the PKR-PKZ-KO mutant strain (displayed below). The mutated eif2ak2 gene in the PKR-PKZ-KO strain exhibits a different mutation (7-base deletion with 1-base insertion) relative to the mutated eif2ak2 gene in PKR-KO mutant. Inserted and deleted sequences are highlighted in green (deleted sequences are represented by “-“). (b) Results from genotyping of homozygous WT, PKR-KO, PKZ-KO and PKR-PKZ-KO zebrafish groups. This involved PCR amplification of eif2ak2 (pkr) and pkz genes, in each mutant group (left and right gel images, respectively, with expected sizes of WT alleles indicated). Each gel consists of the same layout: Lane 1: 1kb Molecular Marker, Lanes 2–9 each represent a DNA extracted from single whole larva, Lanes 2–3: WT Larvae, Lanes 4–5: PKR-KO mutants, Lanes 6–7 PKZ-KO mutants, Lanes 8–9 PKR-PKZ-KO mutants. Mutant eif2ak2 (pkr) alleles were detected in PKR-KO and PKR-PKZ-KO larvae exhibiting 188-bp and 148-bp amplicons, respectively (left gel). The mutant pkz allele was detected in in PKZ-KO and PKR-PKZ-KO larvae, both exhibiting 151-bp amplicons (right gel). Higher quality figures for the whole manuscript are available in the PDF version.