Abstract：
Coat colour largely determines the market demand for several cat breeds. The KIT proto-oncogene (KIT) gene is a key gene controlling melanoblast differentiation and melanogenesis. KIT mutations usually cause varied changes in coat colour in mammalian species. In this study, we used a pair of single-guide RNAs (sgRNAs) to delete exon 17 of KIT in somatic cells isolated from two different Chinese Li Hua feline foetuses. Edited cells were used as donor nuclei for somatic cell nuclear transfer (SCNT) to generate cloned embryos presenting an average cleavage rate exceeding 85%, and an average blastocyst formation rate exceeding 9.5%. 131 cloned embryos were transplanted into four surrogates, and all surrogates carried their pregnancies to term, and delivered 4.58% (6/131) alive cloned kittens, with 1.53% (2/131) being KIT-edited heterozygotes (KITD17/+). The KITD17/+ cats presented an obvious darkness reduction in the mackerel tabby coat. Immunohistochemical analysis (IHC) of skin tissues indicated impaired proliferation and differentiation of melanoblasts caused by the lack of exon17 in feline KIT. To our knowledge, this is the first report on coat colour modification of cats through gene editing. The findings could facilitate further understanding of the regulatory role of KIT on feline coat colour and provide a basis for the breeding of cats with commercially desired coat colour.
摘要：
外套颜色在很大程度上决定了几种猫品种的市场需求。KIT原癌基因（KIT）基因是控制黑素细胞分化和黑素生成的关键基因。KIT突变通常会导致哺乳动物物种的皮毛颜色发生变化。在这项研究中,我们使用一对单向导RNA（sgRNA）在从两个不同的中国李华猫胎儿分离的体细胞中删除KIT的外显子17。编辑的细胞用作体细胞核移植（SCNT）的供体核，以产生平均卵裂率超过85％的克隆胚胎。平均囊胚形成率超过9.5%。131个克隆胚胎被移植到4个代孕体内,所有的代孕母亲都怀孕到足月，并交付了4.58%(6/131)的活克隆小猫，1.53%(2/131)是KIT编辑的杂合子(KITD17/+)。KITD17/猫在鲭鱼白斑大衣中表现出明显的黑暗减少。皮肤组织的免疫组织化学分析（IHC）表明，猫KIT中缺乏外显子17导致黑素细胞增殖和分化受损。据我们所知,这是通过基因编辑对猫进行毛色修饰的第一份报告。这些发现可以促进进一步了解KIT对猫科动物毛色的调节作用，并为具有商业期望毛色的猫的繁殖提供基础。
