University of Veterinary Medicine Vienna CRISPR/Cas9-Mediated Targeting of BPV-1-Transformed Primary Equine Sarcoid Fibroblasts 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. en TEXT article Bovine Papillomavirus Type-1; Vimentin Intermediate-Filaments; In-Vitro; Gene-Expression; Dna; Cells; Vivo; E6; Establishment; Inactivation 2023 2023 Monod, Anne (University of Bern) Hahn, Kerstin (University of Bern) Rottenberg, Sven (University of Bern) Gerber, Vinzenz (University of Bern) Wenker, Christian (Zoo Basel) Howald, Denise (University of Bern) Haspeslagh, Maarten (Ghent University) Koch, Christoph (University of Bern) Jindra, Christoph (University of Veterinary Medicine Vienna) MDPI