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 for agricultural and biomedical applications, the entire process of generating useful GE pigs is time- and labor-intensive. To overcome this, in vivo gene-editing techniques have been developed, where Cas9 nuclease and single guide RNA (sgRNA) are directly injected into animals; however, their 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. In vitro experiments with primary cells from the pig confirmed effective gene deletion by adding only sgRNAs. These results suggest that the Cas9-expressing pig generated in this study could serve as an effective platform for in vivo and in vitro gene editing in agricultural and biomedical research.