The white coat colour of Yorkshire and Landrace pig breeds is caused by the dominant white I allele of KIT, associated with 450-kb duplications and a splice mutation (G > A) at the first base in intron 17. To test whether genome editing can be employed to correct this structural mutation, and to investigate the role of KIT in the control of porcine coat colour, we designed sgRNAs targeting either intron 16 or intron 17 of KIT, and transfected Cas9/sgRNA co-expression plasmids into the kidney cells of Yorkshire pigs. The copy number of KIT was reduced by about 13%, suggesting the possibility of obtaining cells with corrected structural mutations of the KIT locus. Using single cell cloning, from 24 successfully expanded single cell clones derived from cells transfected with sgRNA targeting at intron 17, we obtained 3 clones with a single copy of KIT without the splice mutation. Taken together, the 12.5% (3/24) efficiency of correction of structural mutations of 450 kb fragments is highly efficient, providing a solid basis for the generation of genome edited Yorkshire pigs with a normal KIT locus. This provides an insight into the underlying genetic mechanisms of porcine coat colour.

中文翻译：

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通过CRISPR / Cas9高效纠正约克郡猪肾细胞中450 kb KIT基因座的结构突变

约克郡和长白猪品种的白大衣颜色是由KIT的显性白色I等位基因引起的，与等位基因内含子17的第一个碱基有450 kb的重复和剪接突变（G> A）。为了纠正这一结构突变，并研究KIT在控制猪毛颜色中的作用，我们设计了靶向KIT内含子16或内含子17的sgRNA，并将Cas9 / sgRNA共表达质粒转染到人肾细胞中。约克郡猪。KIT的拷贝数减少了约13％，表明有可能获得具有KIT基因座已纠正结构突变的细胞。使用单细胞克隆，从24个成功扩增的单细胞克隆中，这些克隆衍生自以sgRNA靶向内含子17转染的细胞 我们获得了三个具有单拷贝KIT且无剪接突变的克隆。综上所述，校正450 kb片段的结构突变的效率为12.5％（3/24），非常高效，为生成具有正常KIT基因座的基因组编辑的约克夏猪提供了坚实的基础。这提供了对猪大衣颜色的潜在遗传机制的了解。