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.
Highlights
Artificial selection in different regions of the world has strongly accelerated porcine evolution and has resulted in pig coat colour variations in contrast to their wild ancestors [5]
The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has been widely employed in genome editing, including endogenous gene disruption, targeted sites insertion, and chromosomal rearrangements, in various organisms ranging from viruses to eukaryotes since its development [11, 19, 20], with advantages including easy programmability, wide applicability, and time saving
Efficient cutting at KIT locus in porcine kidney cells by CRISPR/Cas9 To evaluate the targeting efficiencies of the designed single guide RNA (sgRNA) (Fig. 1a and Additional file 1: Table S1) at the KIT locus in the kidney cells of Yorkshire pigs, firstly, genomic DNA of cells with four copies of the KIT locus transfected with pX458-sgRNAs
Summary
Artificial selection in different regions of the world has strongly accelerated porcine evolution and has resulted in pig coat colour variations in contrast to their wild ancestors [5]. KIT (Dominant White locus) may play a major role in determining the white coat colour in the Yorkshire and Landrace pig breeds. KIT was previously mapped to chromosome 8 of pigs, encoding the proto-oncogene receptor tyrosine kinase, which plays a crucial role in the survival and migration of neural-crest–derived melanocyte precursors [2]. Four alleles have been identified at the dominant white locus: the recessive i allele for wild-type solid colour, the semi-dominant Ip allele for the patch phenotype, the fully dominant I allele for the dominant white phenotype, and IBe for the dominant belt phenotype [16]. The CRISPR/Cas system has been widely employed in genome editing, including endogenous gene disruption, targeted sites insertion, and chromosomal rearrangements, in various organisms ranging from viruses to eukaryotes since its development [11, 19, 20], with advantages including easy programmability, wide applicability, and time saving
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
