Highly efficient correction of structural mutations of 450 kb KIT locus in kidney cells of Yorkshire pig by CRISPR/Cas9

Abstract

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.