CRISPR/Cas9-Mediated Targeting of BPV-1-Transformed Primary Equine Sarcoid Fibroblasts

Anne Monod; Christoph Koch; Christoph Jindra; Maarten Haspeslagh; Denise Howald; Christian Wenker; Vinzenz Gerber; Sven Rottenberg; Kerstin Hahn

doi:10.3390/v15091942

CRISPR/Cas9-Mediated Targeting of BPV-1-Transformed Primary Equine Sarcoid Fibroblasts

Viruses. 2023 Sep 17;15(9):1942. doi: 10.3390/v15091942.

Authors

Anne Monod^{1

2}, Christoph Koch¹, Christoph Jindra³, Maarten Haspeslagh⁴, Denise Howald², Christian Wenker⁵, Vinzenz Gerber¹, Sven Rottenberg², Kerstin Hahn²

Affiliations

¹ Swiss Institute of Equine Medicine (ISME), Department of Clinical Veterinary Medicine, Vetsuisse Faculty, University of Bern, 3001 Bern, Switzerland.
² Institute of Animal Pathology, Vetsuisse Faculty, University of Bern, 3001 Bern, Switzerland.
³ Research Group Oncology, University Equine Clinic, University of Veterinary Medicine, 1210 Vienna, Austria.
⁴ Department of Large Animal Surgery, Anesthesiology and Orthopaedics, Faculty of Veterinary Medicine, Ghent University, 9820 Merelbeke, Belgium.
⁵ Zoo Basel, Binningerstrasse 40, 4054 Basel, Switzerland.

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.

Keywords: CRISPR/Cas9; Vimentin; bovine papillomavirus; episomes; equine sarcoid; genome integration.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
CRISPR-Cas Systems*
Fibroblasts
Gene Targeting
Horses
Oncogenes
Skin Neoplasms*

Grants and funding

This research was supported by the Swiss Armed Forces, Veterinary Department (headed by Colonel Stéphane Montavon), and ISME Equine Research, grant number 33-890.