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

Author:

Monod Anne12,Koch Christoph1ORCID,Jindra Christoph3,Haspeslagh Maarten4ORCID,Howald Denise2,Wenker Christian5,Gerber Vinzenz1ORCID,Rottenberg Sven2,Hahn Kerstin2ORCID

Affiliation:

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.

Funder

Swiss Armed Forces, Veterinary Department

Publisher

MDPI AG

Subject

Virology,Infectious Diseases

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