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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. [ABSTRACT FROM AUTHOR]

The ectopic overexpression of foreign genes in animal genomes is an important strategy for gain-of-function study and establishment of transgenic animal models. Previous studies showed that two loci (Rosa26 and pH11) were identified as safe harbor locus in pig genomes, which means foreign genes can be integrated into this locus for stable expression. Moreover, integration of a transgene may interfere with the endogenous gene expression of the target locus after the foreign fragments are inserted. Here, we provide a new strategy for efficient transgene knock-in in the endogenous GAPDH gene via CRISPR/Cas9 mediated homologous recombination. This strategy has no influence on the expression of the endogenous GAPDH gene. Thus, the GAPDH locus is a new alternative safe harbor locus in the pig genome for foreign gene knock-ins. This strategy is promising for agricultural breeding and biomedical model applications. [ABSTRACT FROM AUTHOR]