Identification of the CKM Gene as a Potential Muscle-Specific Safe Harbor Locus in Pig Genome.

Genetically modified pigs have shown considerable application potential in the fields of life science research and livestock breeding. Nevertheless, a barrier impedes the production of genetically modified pigs. There are too few safe harbor loci for the insertion of foreign genes into the pig genome. Only a few loci (pRosa26, pH11 and Pifs501) have been successfully identified to achieve the ectopic expression of foreign genes and produce gene-edited pigs. Here, we use CRISPR/Cas9-mediated homologous directed repair (HDR) to accurately knock the exogenous gene-of-interest fragments into an endogenous CKM gene in the porcine satellite cells. After porcine satellite cells are induced to differentiate, the CKM gene promoter simultaneously initiates the expression of the CKM gene and the exogenous gene. We infer preliminarily that the CKM gene can be identified as a potential muscle-specific safe harbor locus in pigs for the integration of exogenous gene-of-interest fragments.

Identification of ACTB Gene as a Potential Safe Harbor Locus in Pig Genome.

Transgenic pigs play an important role in biomedicine and agriculture. The "safe harbor" locus maintains consistent foreign gene expression in cells and is important for transgenic pig generation. However, as only several safe harbor loci(Rosa26, pH11 and Pifs501) have been identified in pigs, meeting the needs of the insertion of various foreign genes is difficult. In this study, we develop a novel strategy for the efficient knock-in of gene-of-interest fragments into endogenous beta-actin(ACTB) gene via CRISPR/Cas9 mediated homologous recombination with normal expression of ACTB. Thus, we provide an alternative strategy to integrate exogenous genes into the pig genome that can be applied to agricultural breeding and biomedical models.