https://phaidra.vetmeduni.ac.at/o:2619 CRISPR/Cas9-Mediated Targeting of BPV-1-Transformed Primary Equine Sarcoid Fibroblasts Monod, Anne Anne Monod University of Bern Hahn, Kerstin Kerstin Hahn 0000-0003-3602-4234 University of Bern Rottenberg, Sven Sven Rottenberg University of Bern Gerber, Vinzenz Vinzenz Gerber 0000-0002-7834-4482 University of Bern Wenker, Christian Christian Wenker Zoo Basel Howald, Denise Denise Howald University of Bern Haspeslagh, Maarten Maarten Haspeslagh 0000-0001-6381-590X Ghent University Koch, Christoph Christoph Koch 0000-0002-4574-6952 University of Bern Jindra, Christoph Christoph Jindra University of Veterinary Medicine Vienna MDPI text eng 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. Bovine Papillomavirus Type-1; Vimentin Intermediate-Filaments; In-Vitro; Gene-Expression; Dna; Cells; Vivo; E6; Establishment; Inactivation http://creativecommons.org/licenses/by/4.0/ https://phaidra.vetmeduni.ac.at/api/object/o:2619/download 10.3390/v15091942 https://phaidra.vetmeduni.ac.at/o:605 application/pdf Viruses 15(9) (2023) Viruses 15 9 1.02 MB 2023