Generation of PCBP1-deficient pigs using CRISPR/Cas9-mediated gene editing.

Classical swine fever virus (CSFV), a classic swine fever pathogen, causes severe economic losses worldwide. Poly (rC)-binding protein 1 (PCBP1), which interacts with Npro of CSFV, plays a vital role in CSFV growth. We are the first to report the generation of PCBP1-deficient pigs via gene-editing technology. The PCBP1-deficient pigs exhibited normal birth weight and reproductive-performance traits and developed normally. Viral challenge experiments indicated that primary cells isolated from F0- and F1-generation pigs exhibited significantly reduced CSFV infection. Additional mechanistic exploration further confirmed that the PCBP1 deficiency-mediated antiviral effect is related to the activation of type I interferon (IFN). Besides showing that a gene-editing strategy could be used to generate PCBP1-deficient pigs, our study introduces a valuable animal model for further investigating the infection mechanisms of CSFV that will help to develop better antiviral solutions.

Identification and Functional Analysis of the Regulatory Elements in the pHSPA6 Promoter.

Functional and expressional research of heat shock protein A6 (HSPA6) suggests that the gene is of great value for neurodegenerative diseases, biosensors, cancer, etc. Based on the important value of pigs in agriculture and biomedicine and to advance knowledge of this little-studied HSPA member, the stress-sensitive sites in porcine HSPA6 (pHSPA6) were investigated following different stresses. Here, two heat shock elements (HSEs) and a conserved region (CR) were identified in the pHSPA6 promoter by a CRISPR/Cas9-mediated precise gene editing strategy. Gene expression data showed that sequence disruption of these regions could significantly reduce the expression of pHSPA6 under heat stress. Stimulation studies indicated that these regions responded not only to heat stress but also to copper sulfate, MG132, and curcumin. Further mechanism studies showed that downregulated pHSPA6 could significantly affect some important members of the HSP family that are involved in HSP40, HSP70, and HSP90. Overall, our results provide a new approach for investigating gene expression and regulation that may contribute to gene regulatory mechanisms, drug target selection, and breeding stock selection.