Predicted off-target sites in the TRIM29-KO pigs.

While production traits have seen accelerated genetic improvement through advanced breeding technologies, disease resilience phenotypes continue to pose significant challenges in livestock breeding system. Current gene editing technologies provide an effective and biosafe strategy to enhance livestock disease resilience through precise manipulation of host antiviral genes. In this study, we successfully generated disease-resilient pigs exhibiting broad-spectrum antiviral activity against multiple viruses, with no observed adverse effects on pig health. The E3 ubiquitin ligase TRIM29 functions as a negative regulator of type I interferon (IFN) signaling, thus representing a potential antiviral target. Knockdown of TRIM29 in PK15 cells significantly enhanced antiviral immunity against pseudorabies virus (PRV) and vesicular stomatitis virus (VSV) by augmenting type I IFN production. Translationally, we generated Trim29 knockout (KO) mice and confirmed their enhanced antiviral ability to both PRV and VSV infections. Subsequently, we produced TRIM29-KO pigs via gene editing coupled with somatic cell nuclear transfer. Compared to wild-type controls, the TRIM29-KO pigs exhibited significantly enhanced resilience to PRV infection, which was associated with elevated type I IFN levels in vivo. Furthermore, alveolar macrophages derived from TRIM29-KO pigs showed reduced susceptibility to infection with PRV, VSV, and transmissible gastroenteritis virus (TGEV), highlighting their potential broad-spectrum antiviral activity against multiple viral pathogens.