CRISPR-mediated tyr knockout to establish a visual knock-in assay. a Schematic illustration of CRISPR/Cas9-mediated gene editing of tyr. First to knock out (KO) gene function with a 25 nt deletion within the first exon and then knock-in (KI) rescue of this gene using a repair template. b Target sites and T7E1 assays of tyr and tyr25del/25del loci. PAMs are marked with red. Ctl represents PCR products without T7E1 digestion. WT denotes PCR products from uninjected embryos with T7E1 digestion. Tyr or tyr25del/25del denotes PCR products from injected embryos with T7E1 digestion. c T-cloning and Sanger sequencing identify tyr25del/25del in F2 zebrafish. Upper row shows wild type (WT) sequence. Open reading frame codons are demarked in green frame. 25 bp deletion in homozygous tyr mutants is marked with blue in upper row, which leads to a frameshift mutation (marked with red in lower row). d Lateral views of larvae at 2 dpf (scale bar = 1 mm) and adult (scale bar = 10 mm): wild type (upper row) and tyr25de/l25del (lower row).