Zhang Xue-Mei, Qiu Mei-Yu, Liao Li, Han Bing, Deng Ya-di, Zhang Ning, Hai La-Ti, Liang Long, Liu Ming-Jun
Key Laboratory of Genetics, Breeding and Reproduction of Grass-Feeding Livestock, Key Laboratory of Animal Biotechnology of Xinjiang, Ministry of Agriculture (MOA), Key Laboratory of Animal Biotechnology of Xinjiang, Urumqi, China.
Institute of Animal Biotechnology, Xinjiang Academy of Animal Science, Urumqi, China.
FASEB J. 2025 Jul 31;39(14):e70807. doi: 10.1096/fj.202500355R.
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 (D, D) 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.
绵羊的毛色是影响消费者偏好和育种的重要表型特征。尽管刺鼠信号蛋白（ASIP）基因与色素沉着有关，但其功能的潜在机制仍不清楚。在之前的一项研究中，我们使用CRISPR/Cas9技术培育出了具有不同毛色的ASIP基因编辑细毛羊（F0）。在此基础上，本研究调查了33只F1后代的毛色变异情况，这些后代是由F0基因编辑公羊与F0基因编辑母羊或野生型白色母羊交配产生的。全面的基因分型确定了两个遗传的ASIP突变——一个4碱基对缺失和一个27碱基对缺失并伴有1碱基对插入——以及自然变异（D、D）和拷贝数变异。这些遗传差异与F1群体中广泛的毛色表型相关。为了探究潜在机制，我们进行了组织学分析（苏木精-伊红染色）、免疫组织化学、黑色素含量定量、qRT-PCR和蛋白质免疫印迹分析。结果表明，毛色较深的个体ASIP表达降低，酪氨酸酶（TYR）和酪氨酸酶相关蛋白1（TYRP1）水平升高，这与真黑色素合成增加一致。相反，白色被毛的绵羊ASIP表达较高，黑色素生成酶水平较低。我们的研究结果提供了功能验证，即ASIP是绵羊色素沉着的核心调节因子，并揭示了基因编辑、自然突变和基因剂量之间的相互作用如何导致不同的毛色结果。这项工作建立了一个用于剖析毛色遗传学的有价值模型，并为未来旨在实现细毛羊毛色多样化和经济性状改善的育种策略提供了分子框架。
