Fig. 1

A Phylogenetic bootstrap tree illustrated the evolutionary relationship of aldh3b1 across representative vertebrate species. B Alignment of the Aldh3b1 amino acid sequence revealed high similarity, including conserved active site and domain among the indicated species. C Targeted CRISPR/Cas9 mutagenesis of aldh3b1 in exon 2 resulted in 16 bp insertions, altering the amino acid sequence. Created in BioRender. Bennewitz, K. (2026) https://BioRender.com/3e7287f. D, E Quantitative analysis showed that aldh3b1 mRNA expression levels remain consistent through various developmental stages of zebrafish larvae and was predominantly expressed in the brain and eyes of adult zebrafish (n = 3 for each stage and organ). F, Galdh3b1 genotyping can be clearly distinguished on agarose gels (F) and was in line with the mendelian inheritance in the first generation of F2 (G). H Immunoblot analysis confirmed the absence of Aldh3b1 protein in aldh3b1^-/- eye. I Total ALDH enzyme activity was reduced in aldh3b1^-/- larvae using the substrate acetaldehyde (n = 3/4). J–L Phenotypic assessment of aldh3b1^-/- adult zebrafish revealed no significant changes in survival rate (J, n = 50/50), weight (K, n = 11/11), and length (L, n = 11/11) compared to aldh3b1^+/+ adult zebrafish. Dpf, days post fertilization; bp, base pair. Statistical analysis was performed using one-way ANOVA for panels (D, E), two-tailed Student’s t test for (I, K, L) and Log-rank test for panel (J). Error bars represent mean ± standard deviation (SD). In Fig. 1K, L, the center line represents the median, the bounds of the box represent the 25th and 75th percentiles, and the whiskers extend to the minimum and maximum values. Source data are provided as a Source Data file.