Versatile generation of precise gene edits in bovines using SEGCPN

Abstract Background Gene knockout and knock-in have been widely performed in large farm animals based on genome editing systems. However, many types of precise gene editing, including targeted deletion, gene tagging, and large gene fragment replacement, remain a challenge in large farm animals. Results Here, we established versatile self-excising gene-targeting technology in combination with programmable nucleases (SEGCPN) to efficiently generate various types of precise gene editing in bovine. First, we used this versatile method to successfully generate bovine embryos with point mutations and 11-bp deletions at the MSTN locus. Second, we successfully generated bulls with EGFP labeling at the SRY locus. Finally, we successfully generated humanized cows in which the endogenous 18-kb α-casein gene was replaced with a 2.6-kb human α-lactalbumin gene. Conclusions In summary, our new SEGCPN method offers unlimited possibilities for various types of precise gene editing in large animals for application both in agriculture and disease models.