A wide range of endemic and epidemic viruses, including classic swine fever virus (CSFV), pseudorabies virus (PRV) and others, are among the most economically important pathogens in pigs and have severely affected the national economy, human health and animal welfare and productivity. The RSAD2 exhibits antiviral activity against various DNA and RNA viruses. In this study, we successfully accomplished site-specific insertion of the porcine RSAD2 gene (pRSAD2) at the porcine ROSA26 (pROSA26) locus, generating pRSAD2 gene knock-in (pRSAD2-KI) PK-15 cells and porcine foetal fibroblasts (PFFs) via CRISPR/Cas9 technology. Gene expression analysis confirmed that pRSAD2-KI cells stably and efficiently overexpressed the pRSAD2 gene. Furthermore, viral challenge studies in vitro indicated that site-specific integration of the pRSAD2 gene not only effectively reduced CSFV infection but also PRV infection. More importantly, we ultimately successfully produced a pRSAD2-KI pig that constitutively overexpressed the pRSAD2, viral challenge results indicated that fibroblasts isolated from the pRSAD2-KI pig reduced CSFV infection. Taken together, these results suggest that CRISPR/Cas9-mediated knock-in strategy can be used for producing pRSAD2-KI pigs.

中文翻译：

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通过CRISPR / Cas9技术生成pRSAD2基因敲入猪。

包括经典猪瘟病毒（CSFV），伪狂犬病病毒（PRV）等在内的多种地方性和流行性病毒是猪中经济上最重要的病原体之一，严重影响了国民经济，人类健康以及动物福利和生产力。RSAD2具有针对各种DNA和RNA病毒的抗病毒活性。在这项研究中，我们成功地在猪ROSA26（pROSA26）基因座上完成了猪RSAD2基因（pRSAD2）的位点特异性插入，产生了pRSAD2基因敲入（pRSAD2-KI）PK-15细胞和猪胎儿成纤维细胞（PFFs）通过CRISPR / Cas9技术。基因表达分析证实pRSAD2-KI细胞稳定有效地过表达pRSAD2基因。此外，体外病毒攻击研究表明，pRSAD2基因的位点特异性整合不仅有效减少了CSFV感染，而且有效减少了PRV感染。更重要的是，我们最终成功地生产了pRSAD2-KI猪，该猪组成性地过表达pRSAD2，病毒攻击结果表明，从pRSAD2-KI猪中分离出的成纤维细胞减少了CSFV感染。综上所述，这些结果表明，CRISPR / Cas9介导的敲入策略可用于生产pRSAD2-KI猪。