Functional analysis of KIT gene structural mutations causing porcine dominant white phenotype by using genome edited mouse models

Dominant white phenotype in pigs is considered to be caused by two structural mutations in KIT gene, including a 450-kb duplication encompassing the entire KIT gene, and a splice mutation (G > A) at the first base in intron 17, which leads to the deletion of exon 17 in mature KIT mRNA, and the production of KIT protein lacking a critical catalytic domain of kinase. However, this speculation has not yet been validated by functional studies. Here, by using CRISPR/Cas9 technology, we created two mouse models mimicing the structural mutations of KIT gene in dominant white pigs, including the splice mutation mouse model KITD17/+ with exon 17 of one allele of KIT gene deleted, and duplication mutation mouse model KITdup/+ with one allele of KIT gene coding sequence (CDS) duplicated. We found that each mutation individually can not cause dominant white phenotype. Splice mutation homozygote is lethal and heterozygous mice present piebald coat. Inconsistent with previous speculation, we found KIT gene duplication mutation did not confer the patched phenotype, and had no obvious impact on coat color. Interestingly, combination of these two mutations lead to dominant white phenotype. Further molecular analysis revealed that combination of these two structural mutations could inhibit the kinase activity of the KIT protein, thus reduce the phosphorylation level of PI3K and MAPK pathway associated proteins, which may be related to the observed impaired migration of melanoblasts during embryonic development, and eventually lead to dominant white phenotype. Our study provides a further insight into the underlying genetic mechanisms of porcine dominant white coat colour.