Swiss Institute of Equine Medicine (ISME), Department of Clinical Veterinary Medicine, Vetsuisse Faculty, University of Bern, 3001 Bern, Switzerland.
Institute of Animal Pathology, Vetsuisse Faculty, University of Bern, 3001 Bern, Switzerland.
Viruses. 2023 Sep 17;15(9):1942. doi: 10.3390/v15091942.
Equine sarcoids (EqS) are fibroblast-derived skin tumors associated with bovine papillomavirus 1 and 2 (BPV-1 and -2). Based on Southern blotting, the BPV-1 genome was not found to be integrated in the host cell genome, suggesting that EqS pathogenesis does not result from insertional mutagenesis. Hence, CRISPR/Cas9 implies an interesting tool for selectively targeting BPV-1 episomes or genetically anchored suspected host factors. To address this in a proof-of-concept study, we confirmed the exclusive episomal persistence of BPV-1 in EqS using targeted locus amplification (TLA). To investigate the CRISPR/Cas9-mediated editing of BPV-1 episomes, primary equine fibroblast cultures were established and characterized. In the EqS fibroblast cultures, CRISPR-mediated targeting of the episomal E5 and E6 oncogenes as well as the BPV-1 long control region was successful and resulted in a pronounced reduction of the BPV-1 load. Moreover, the deletion of the equine Vimentin , which is highly expressed in EqS, considerably decreased the number of BPV-1 episomes. Our results suggest CRISPR/Cas9-based gene targeting may serve as a tool to help further unravel the biology of EqS pathogenesis.
马肉瘤（EqS）是与牛乳头瘤病毒 1 和 2（BPV-1 和 -2）相关的成纤维细胞来源的皮肤肿瘤。基于 Southern 印迹，未发现 BPV-1 基因组整合到宿主细胞基因组中，这表明 EqS 发病机制不是由插入诱变引起的。因此，CRISPR/Cas9 为选择性靶向 BPV-1 附加体或遗传锚定的可疑宿主因子提供了一种有趣的工具。为了在概念验证研究中解决这个问题，我们使用靶向基因座扩增（TLA）证实了 EqS 中 BPV-1 的独特附加体持续存在。为了研究 CRISPR/Cas9 介导的 BPV-1 附加体编辑，建立并表征了原代马成纤维细胞培养物。在 EqS 成纤维细胞培养物中，CRISPR 介导的对附加体 E5 和 E6 癌基因以及 BPV-1 长控制区的靶向是成功的，导致 BPV-1 负荷显著降低。此外，高度表达于 EqS 中的马波形蛋白的缺失也大大减少了 BPV-1 附加体的数量。我们的结果表明，基于 CRISPR/Cas9 的基因靶向可能是一种工具，可以帮助进一步揭示 EqS 发病机制的生物学。
