Insights Into Coat Color Variation: Unraveling the Role of ASIP by Gene Editing in Fine-Wool Sheep.

Sheep coat color is an important phenotypic trait that influences consumer preferences and breeding. Although the Agouti Signaling Protein (ASIP) gene has been linked to pigmentation, the mechanisms underlying its function remain unclear. In a previous study, we generated ASIP gene-edited fine-wool sheep (F0) with diverse coat colors using CRISPR/Cas9. Building on this foundation, the present study investigates coat color variation in 33 F1 offspring produced by mating F0 gene-edited rams with either F0 gene-edited ewes or wild-type white ewes. Comprehensive genotyping identified two inherited ASIP mutations-a 4-bp deletion and a 27-bp deletion with a 1-bp insertion-alongside natural variants (D5, D9) and copy number variation. These genetic differences were associated with a wide range of coat color phenotypes in the F1 population. To explore the underlying mechanisms, we conducted histological analyses (H&E staining), immunohistochemistry, melanin content quantification, qRT-PCR, and Western blotting. Results showed that dark-coated individuals exhibited reduced ASIP expression and elevated levels of TYR and TYRP1, consistent with increased eumelanin synthesis. In contrast, white-coated sheep showed higher ASIP expression and lower levels of melanogenic enzymes. Our findings provide functional validation that ASIP is a central regulator of pigmentation in sheep and reveal how the interplay between gene editing, natural mutations, and gene dosage contributes to diverse coat color outcomes. This work establishes a valuable model for dissecting coat color genetics and offers a molecular framework for future breeding strategies aimed at wool color diversification and economic trait improvement in fine-wool sheep.