Despite efforts by many government authorities to stamp out the disease from national pig populations, the disease remains widespread. Here, antiviral small hairpin RNAs (shRNAs) were selected and then inserted at the porcine Rosa26 (pRosa26) locus via a CRISPR/Cas9-mediated knock-in strategy. Finally, anti-CSFV transgenic (TG) pigs were produced by somatic nuclear transfer (SCNT). Notably, in vitro and in vivo viral challenge assays further demonstrated that these TG pigs could effectively limit the replication of CSFV and reduce CSFV-associated clinical signs and mortality, and disease resistance could be stably transmitted to the F1-generation. Altogether, our work demonstrated that RNA interference (RNAi) technology combining CRISPR/Cas9 technology offered the possibility to produce TG animal with improved resistance to viral infection. The use of these TG pigs can reduce CSF-related economic losses and this antiviral strategy may be useful for future antiviral research.
Classical swine fever (CSF), caused by classical swine fever virus (CSFV), and is a highly contagious, often fatal porcine disease that causes significant economic losses. Due to the economic importance of this virus to the pig industry, the biology and pathogenesis of CSFV have been investigated extensively. Despite efforts by many government authorities to stamp out the disease from national pig populations, the disease remains widespread, and it is only a matter of time before the virus is reintroduced and the next round of disease outbreaks occurs. These findings highlight the necessity and urgency for developing effective approaches to eradicate the challenging CSFV. In this study, we successfully produced anti-CSFV TG pigs by combining RNAi technology and CRISPR/Cas9 technologies, and viral challenge results confirmed that these TG pigs could effectively limit the replication of CSFV in vivo and in vitro. Additionally, we confirmed that the disease resistance traits in the TG founders were stably transmitted to their F1-generation offspring. Altogether, our work reported the combinational application of CRISPR/Cas9 and RNA interference (RNAi) technology in the generation of anti-CSFV TG pigs, it provided an alternative strategy to change the virus. The results of this study suggested that these TG pigs offered potential benefits over commercial vaccination and reduced CSFV-related economic losses.