Fig. 6: Generation of TYRP1 knockout (KO) Chahua chickens.

A Classification of chicks from mating of male recipients (PCM) and WT Chahua (YAU) hens. Chicks derived from male PCM recipients show a polydactyly (Po/po⁺) and occasionally a crested (Cr/cr⁺) phenotype, as indicated by the white dashed circles and red arrows, whereas chicks derived from TYRP1 KO Chahua PGCs do not carry either mutation. Heterozygous KO males (Z⁺/Z^TYRP1-73bp) show WT chick down coloration (left), and hemizygous KO females (Z^TYRP1-73bp/W) show brown chick down coloration with reduced eumelanin production (right). B WT and TYRP1-KO Chahua chickens in the G2 generation. Adult homozygous KO males and hemizygous KO females with a 73 bp deletion (c, d in lower panels) exhibit a brown plumage color with reduced eumelanin, whereas WT chickens (a, b in upper panels) lacking the 73 bp deletion or heterozygous males with the deletion exhibit the WT plumage color. Individual feathers from KO chickens (right) show a significant reduction in the production of eumelanin compared to WT chickens (left). The black pigmented areas change to a reddish-gray color. Pheomelanin remains largely unaffected. C PCR-based genotyping of the 73 bp deletion in exon 2 of TYRP1. The exon 2 target region was amplified using primers FD and RV. WT chickens show a single 371 bp band in females and either a 371 bp or 371/298 bp heterozygous band in males. In contrast, individuals with brown plumage show a single band of 298 bp in both sexes (right panel). D Sequences of the exon 2 target region between WT and KO alleles. The sequence chromatogram shows the WT allele (top panel) and the KO allele with a 73 bp deletion (bottom panel). The two sgRNA sequences (sgRNA#1 and sgRNA#2) are underlined in red and the PAM sequences are underlined in green. The disrupted positions marked by the two red arrows in the WT sequence result in a KO allele with a 73 bp deletion mutation in the TYRP1 target region, leading to a TGA stop codon (highlighted by the red square) at c.200–202, resulting in a shorter mRNA of 129 bp compared to the WT mRNA of 1611 bp. E Predicted amino acid sequences of TYRP1 for WT and KO individuals. The WT TYRP1 protein consists of 536 residues. The predicted truncated TYRP1 protein from the KO allele with a 73 bp deletion consists of 42 residues.