Figure 1

Figure 1

Generation and validation of aldh9a1b^−/‐ zebrafish line using CRISPR/Cas9 technology (A) Neighbor‐joining phylogenetic bootstrap tree of amino acid sequence showed the evolutionary relationship of aldh9a1 and its isoforms in representative vertebrate animal species. B) aldh9a1b mRNA was mostly expressed in the liver, while least expressed in the spleen in aldh9a1b^+/+ adult zebrafish, n = 5/6. C) aldh9a1b CRISPR knockout line was designed to target exon 6 and successfully generated a 10 bp deletion validated in cDNA sequencing. The 10 bp deletion resulted in an artificial stop codon indicated with a star. D) aldh9a1b^+/+, aldh9a1b^+/−, and aldh9a1b^−/− zebrafish can be distinguished on a genotyping‐PCR gel. The blue, red, and yellow arrows indicated PCR products of cDNA from aldh9a1b^+/+, aldh9a1b^+/−, and aldh9a1b^−/− zebrafish. E) mRNA levels of aldh9a1b were significantly decreased in livers and muscles of aldh9a1b^−/− mutants, n = 6/5. F) aldh9a1b^−/− mutants showed decreased Aldh enzyme activity using the substrate acetaldehyde at 5dpf, n = 4, 120 larvae per clutch. G) Survival rate was not significantly changed between aldh9a1b^+/+ and aldh9a1b^−/‐ zebrafish over the age of 1–15 months, n = 51 and n = 53. H,I) Adult aldh9a1b^−/− mutants displayed a normal body weight (H) and body length (I) compared to aldh9a1b^+/+ zebrafish, n = 11. mRNA Expression was quantified by RT‐qPCR and normalized to b2m. Each data point in this figure represented 20 larvae or one adult fish. The bars indicate mean ± SD values. Statistical analysis was performed by one‐way ANOVA, Student's t‐test, and log‐rank test. ns = not significant, ^*p < 0.05, ^**p < 0.01, ^***p < 0.001. b2m, 𝛽2 microglobulin; Bp, base pair; dpf, days post fertilization.