In vitro Gene Editing Using Primary Cells Derived from Cas9-Expressing Pigs

Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9) technology has significantly facilitated the generation of gene-edited (GE) pigs. Although GE pigs are promising animals for agricultural and biomedical applications, the entire process of generating useful GE pigs is time- and labor-intensive. To overcome this, <italic>in vivo</italic> gene-editing techniques have been developed, where Cas9 nucelase and single guide RNA (sgRNA) are directly injected into animals; however, efficiency remains low owing to the large size of the nuclease. In this study, we generated a Cas9-expressing pig by inserting the Cas9 gene into the ROSA26 locus, resulting in its constitutive expression in various tissues. We also confirmed the pig's fertility. <italic>In vitro</italic> experiments with primary cells from the pig confirmed effective gene deletion by adding only sgRNAs. The results suggest that the Cas9-expressing pig generated in this study could serve as an effective platform for <italic>in vivo</italic> and <italic>in vitro</italic> gene editing in agricultural and biomedical research.