Key Laboratory of Genetics, Breeding and Reproduction of Grass-Feeding Livestock, Ministry of Agriculture(MOA), Key Laboratory of Animal Biotechnology of Xinjiang, Urumqi, Xinjiang, 830026, China.
Institute of Animal Biotechnology, Xinjiang Academy of Animal Science, Urumqi, Xinjiang, 830026, China.
Sci Rep. 2017 Aug 15;7(1):8149. doi: 10.1038/s41598-017-08636-0.
Coat color is an important characteristic and economic trait in domestic sheep. Aiming at alteration of Chinese merino sheep coat color by genome manipulation, we disrupted sheep agouti signaling protein gene by CRISPR/Cas9. A total of seven indels were identified in 5 of 6 born lambs. Each targeted lamb happened at least two kinds of modifications, and targeted lambs with multiple modifications displayed variety of coat color patterns. Three lambs with 4 bp deletion showed badgerface with black body coat color in two lambs, and brown coat color with light ventral pigmentation in another one. The black-white spotted color was observed in two lambs with 2 bp deletion. Further analysis unraveled that modifications happened in one or more than two copies of ASIP gene, and moreover, the additional spontaneous mutations of D and/or D preceding the targeting modification could also involve the formation of coat color patterns. Taken together, the entanglement of ASIP modifications by CRISPR/Cas9, spontaneous D/D mutations, and ASIP gene duplications contributed to the variety of coat color patterns in targeted lambs.
绵羊的被毛颜色是一个重要的特征和经济性状。本研究旨在通过基因组操作来改变中国美利奴羊的被毛颜色，利用 CRISPR/Cas9 技术对绵羊刺鼠相关蛋白信号蛋白（agouti signaling protein，ASIP）基因进行了敲除。在 6 只出生羔羊中，有 5 只羔羊检测到了 7 种不同的插入缺失突变。每只靶向羔羊至少发生了两种类型的修饰，并且发生多种修饰的靶向羔羊表现出多种被毛颜色图案。3 只携带 4 bp 缺失的羔羊表现出两种不同的斑驳色，两只羔羊呈现黑色体被毛色和白色面部的狸花色，另一只羔羊呈现棕色体被毛色和浅色腹部色素沉着。两只携带 2 bp 缺失的羔羊表现出黑白相间的斑点色。进一步分析表明，修饰发生在一个或多个 ASIP 基因拷贝中，此外，靶向修饰前后的 D 和/或 D 基因的自发突变也可能涉及到被毛颜色图案的形成。总之，CRISPR/Cas9 对 ASIP 的修饰、D/D 基因的自发突变以及 ASIP 基因的重复，共同导致了靶向羔羊被毛颜色图案的多样性。
