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Abstract：
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 (VIM), 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.
摘要：
马类肉瘤(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。根据南方印迹,未发现BPV-1基因组整合在宿主细胞基因组中，这表明EqS发病机制不是插入诱变的结果。因此，CRISPR/Cas9暗示了一种用于选择性靶向BPV-1游离体或遗传锚定的可疑宿主因子的有趣工具。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的专有附加型持久性。为了研究CRISPR/Cas9介导的BPV-1游离体编辑，建立并表征了原代马成纤维细胞培养物。在EqS成纤维细胞培养中，CRISPR介导的游离E5和E6癌基因以及BPV-1长控制区的靶向是成功的，并导致BPV-1负荷的显着减少。此外，马波形蛋白(VIM)的缺失，在EqS中高度表达，显著减少了BPV-1游离体的数量。我们的结果表明，基于CRISPR/Cas9的基因靶向可以作为一种工具，帮助进一步解开EqS发病机理的生物学。
