Knockout (KO) of psen2 gene. (a) Partial nucleotide and amino acid sequences of psen2 exon 4 showing a 7-bp deletion, confirmed by DNA sequencing. Deleted nucleotides are represented by dashes. CRISPR target site is highlighted in yellow and the protospacer-adjacent motif in bold. The 7-bp deletion results in a coding frameshift, with the insertion of premature stop codons (three in exon 4 marked in the figure as red asterisks, nine more in the following coding region). (b) Sequencing chromatographs of psen2+/+ and psen2−/− alleles. The red box delineates the area of the induced deletion. (c) RT-qPCR analysis on 1-year psen2+/+ and psen2−/− fish brain, showing a statistically significant decrease in psen2 mRNA level in psen2−/− mutants. Values represent the mean ± SEM. Data were generated from 3 biological replicates for each condition. Statistical significance was determined by two-tailed Student’s t-test (***, p < 0.001). (d) Representative images of psen2+/+ and psen2−/− 6-dpf larvae. Scale bar: 500 µm. (e) psen2+/+ and psen2−/− surface pigmentation at 6 months, showing an undetectable deposition of melanin in melanocytes of the psen2−/− mutant. Scale bar: 1 mm.
