CRISPR/Cas9-Mediated Targeting of BPV-1-Transformed Primary Equine Sarcoid Fibroblasts
Abstract
:1. Introduction
2. Materials and Methods
2.1. Sample Collection
2.2. Targeted Locus Amplification
2.3. Cell Culture
2.4. BPV-1 PCR and rtPCR
2.5. Lentivirus Production
2.6. Transduction of EqS and Control Fibroblasts
2.7. TIDE-PCR and TIDE Analysis
2.8. Proliferation Assays
2.9. Immunofluorescence
2.10. Quantitative Real-Time PCR
2.11. Quantification and Statistical Analysis
3. Results
3.1. Targeted Locus Amplification Does Not Detect BPV-Integrations into the Host Cell Genome
3.2. BPV-1 Viral Load, Long Term Passaging, and Proliferation of Primary EqS Fibroblasts
3.3. CRISPR-Mediated Targeting of BPV-1 E5, E6, and LCR Reduces BPV-1 Loads in EqS Fibroblasts
3.4. CRISPR-Mediated Targeting of Vimentin Reduces BPV-1 Loads in EqS Fibroblasts
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
- Campo, M.S. Papillomavirus and disease in humans and animals. Vet. Comp. Oncol. 2003, 1, 3–14. [Google Scholar] [CrossRef]
- Olson, C., Jr.; Cook, R.H. Cutaneous sarcoma-like lesions of the horse caused by the agent of bovine papilloma. Proc. Soc. Exp. Biol. Med. 1951, 77, 281–284. [Google Scholar] [CrossRef]
- Otten, N.; von Tscharner, C.; Lazary, S.; Antczak, D.F.; Gerber, H. DNA of bovine papillomavirus type 1 and 2 in equine sarcoids: PCR detection and direct sequencing. Arch. Virol. 1993, 132, 121–131. [Google Scholar] [CrossRef]
- Nasir, N.; Reid, S.W. Bovine papillomaviral gene expression in equine sarcoid tumours. Virus Res. 1999, 61, 171–175. [Google Scholar] [CrossRef]
- Lunardi, M.; de Alcântara, B.K.; Otonel, R.A.; Rodrigues, W.B.; Alfieri, A.F.; Alfieri, A.A. Bovine papillomavirus type 13 DNA in equine sarcoids. J. Clin. Microbiol. 2013, 51, 2167–2171. [Google Scholar] [CrossRef]
- Wenker, C.; Hoby, S.; Steck, B.L.; Ramsauer, A.S.; Blatter, S.; Tobler, K. Equine sarcoids in captive wild equids: Diagnostic and clinical management of 16 cases-a possible predisposition of the european cohort of somali wild ass (Equus africanus somaliensis)? J. Zoo. Wildl. Med. 2021, 52, 28–37. [Google Scholar] [CrossRef]
- Ragland, W.L.; Keown, G.H.; Spencer, G.R. Equine sarcoid. Equine Vet. J. 1970, 2, 2–11. [Google Scholar] [CrossRef]
- Cotchin, E. A general survey of tumours in the horse. Equine Vet. J. 1977, 9, 16–21. [Google Scholar] [CrossRef]
- Broström, H. Equine sarcoids. A clinical and epidemiological study in relation to equine leucocyte antigens (ELA). Acta Vet. Scand. 1995, 36, 223–236. [Google Scholar] [CrossRef]
- Knottenbelt, D.C. The Equine Sarcoid: Why Are There so Many Treatment Options? Vet. Clin. N. Am. Equine Pract. 2019, 35, 243–262. [Google Scholar] [CrossRef]
- Amtmann, E.; Müller, H.; Sauer, G. Equine connective tissue tumors contain unintegrated bovine papilloma virus DNA. J. Virol. 1980, 35, 962–964. [Google Scholar] [CrossRef]
- Yuan, Z.Q.; Gault, E.A.; Gobeil, P.; Nixon, C.; Campo, M.S.; Nasir, L. Establishment and characterization of equine fibroblast cell lines transformed in vivo and in vitro by BPV-1: Model systems for equine sarcoids. Virology 2008, 373, 352–361. [Google Scholar] [CrossRef]
- Yuan, Z.; Gault, E.A.; Campo, M.S.; Nasir, L. Different contribution of bovine papillomavirus type 1 oncoproteins to the transformation of equine fibroblasts. J. Gen. Virol. 2011, 92 Pt 4, 773–783. [Google Scholar] [CrossRef]
- Cullen, A.P.; Reid, R.; Campion, M.; Lörincz, A.T. Analysis of the physical state of different human papillomavirus DNAs in intraepithelial and invasive cervical neoplasm. J. Virol. 1991, 65, 606–612. [Google Scholar] [CrossRef]
- Pett, M.; Coleman, N. Integration of high-risk human papillomavirus: A key event in cervical carcinogenesis? J. Pathol. 2007, 212, 356–367. [Google Scholar] [CrossRef]
- Zapatka, M.; Borozan, I.; Brewer, D.S.; Iskar, M.; Grundhoff, A.; Alawi, M.; Desai, N.; Sültmann, H.; Moch, H.; Cooper, C.S.; et al. The landscape of viral associations in human cancers. Nat. Genet. 2020, 52, 320–330. [Google Scholar] [CrossRef]
- Hu, Z.; Yu, L.; Zhu, D.; Ding, W.; Wang, X.; Zhang, C.; Wang, L.; Jiang, X.; Shen, H.; He, D.; et al. Disruption of HPV16-E7 by CRISPR/Cas system induces apoptosis and growth inhibition in HPV16 positive human cervical cancer cells. Biomed. Res. Int. 2014, 2014, 612823. [Google Scholar] [CrossRef]
- Kennedy, E.M.; Kornepati, A.V.; Goldstein, M.; Bogerd, H.P.; Poling, B.C.; Whisnant, A.W.; Kastan, M.B.; Cullen, B.R. Inactivation of the human papillomavirus E6 or E7 gene in cervical carcinoma cells by using a bacterial CRISPR/Cas RNA-guided endonuclease. J. Virol. 2014, 88, 11965–11972. [Google Scholar] [CrossRef]
- Zhen, S.; Hua, L.; Takahashi, Y.; Narita, S.; Liu, Y.H.; Li, Y. In vitro and in vivo growth suppression of human papillomavirus 16-positive cervical cancer cells by CRISPR/Cas9. Biochem. Biophys. Res. Commun. 2014, 450, 1422–1426. [Google Scholar] [CrossRef]
- Zhen, S.; Lu, J.J.; Wang, L.J.; Sun, X.M.; Zhang, J.Q.; Li, X.; Luo, W.J.; Zhao, L. In Vitro and In Vivo Synergistic Therapeutic Effect of Cisplatin with Human Papillomavirus16 E6/E7 CRISPR/Cas9 on Cervical Cancer Cell Line. Transl. Oncol. 2016, 9, 498–504. [Google Scholar] [CrossRef]
- Yu, L.; Hu, Z.; Gao, C.; Feng, B.; Wang, L.; Tian, X.; Ding, W.; Jin, X.; Ma, D.; Wang, H. Deletion of HPV18 E6 and E7 genes using dual sgRNA-directed CRISPRCas9 inhibits growth of cervical cancer cells. Int. J. Clin. Exp. Med. 2017, 10, 9206–9213. [Google Scholar]
- Inturi, R.; Jemth, P. CRISPR/Cas9-based inactivation of human papillomavirus oncogenes E6 or E7 induces senescence in cervical cancer cells. Virology 2021, 562, 92–102. [Google Scholar] [CrossRef] [PubMed]
- Martano, M.; Corteggio, A.; Restucci, B.; De Biase, M.E.; Borzacchiello, G.; Maiolino, P. Extracellular matrix remodeling in equine sarcoid: An immunohistochemical and molecular study. BMC Vet. Res. 2016, 12, 24. [Google Scholar] [CrossRef] [PubMed]
- Knottenbelt, D.C. A suggested clinical classification for the equine sarcoid. Clin. Tech. Equine Pract. 2005, 4, 277–295. [Google Scholar] [CrossRef]
- De Vree, P.J.; de Wit, E.; Yilmaz, M.; van de Heijning, M.; Klous, P.; Verstegen, M.J.; Wan, Y.; Teunissen, H.; Krijger, P.H.; Geeven, G.; et al. Targeted sequencing by proximity ligation for comprehensive variant detection and local haplotyping. Nat. Biotechnol. 2014, 32, 1019–1025. [Google Scholar] [CrossRef]
- Smith, T.F.; Waterman, M.S. Identification of common molecular subsequences. J. Mol. Biol. 1981, 147, 195–197. [Google Scholar] [CrossRef]
- Yuan, Z.; Philbey, A.W.; Gault, E.A.; Campo, M.S.; Nasir, L. Detection of bovine papillomavirus type 1 genomes and viral gene expression in equine inflammatory skin conditions. Virus Res. 2007, 124, 245–249. [Google Scholar] [CrossRef]
- Francica, P.; Mutlu, M.; Blomen, V.A.; Oliveira, C.; Nowicka, Z.; Trenner, A.; Gerhards, N.M.; Bouwman, P.; Stickel, E.; Hekkelman, M.L.; et al. Functional Radiogenetic Profiling Implicates ERCC6L2 in Non-homologous End Joining. Cell Rep. 2020, 32, 108068. [Google Scholar] [CrossRef]
- Brinkman, E.K.; Chen, T.; Amendola, M.; van Steensel, B. Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Res. 2014, 42, e168. [Google Scholar] [CrossRef]
- Goujon, M.; McWilliam, H.; Li, W.; Valentin, F.; Squizzato, S.; Paern, J.; Lopez, R. A new bioinformatics analysis tools framework at EMBL-EBI. Nucleic Acids Res. 2010, 38, W695–W699. [Google Scholar] [CrossRef]
- Ziege, S.; Baumgärtner, W.; Wewetzer, K. Toward defining the regenerative potential of olfactory mucosa: Establishment of Schwann cell-free adult canine olfactory ensheathing cell preparations suitable for transplantation. Cell Transplant. 2013, 22, 355–367. [Google Scholar] [CrossRef] [PubMed]
- Schindelin, J.; Arganda-Carreras, I.; Frise, E.; Kaynig, V.; Longair, M.; Pietzsch, T.; Preibisch, S.; Rueden, C.; Saalfeld, S.; Schmid, B.; et al. Fiji: An open-source platform for biological-image analysis. Nat. Methods 2012, 9, 676–682. [Google Scholar] [CrossRef] [PubMed]
- Brandt, S.; Haralambus, R.; Schoster, A.; Kirnbauer, R.; Stanek, C. Peripheral blood mononuclear cells represent a reservoir of bovine papillomavirus DNA in sarcoid-affected equines. J. Gen. Virol. 2008, 89 Pt 6, 1390–1395. [Google Scholar] [CrossRef] [PubMed]
- Brandt, S.; Haralambus, R.; Shafti-Keramat, S.; Steinborn, R.; Stanek, C.; Kirnbauer, R. A subset of equine sarcoids harbours BPV-1 DNA in a complex with L1 major capsid protein. Virology 2008, 375, 433–441. [Google Scholar] [CrossRef]
- Haralambus, R.; Burgstaller, J.; Klukowska-Rötzler, J.; Steinborn, R.; Buchinger, S.; Gerber, V.; Brandt, S. Intralesional bovine papillomavirus DNA loads reflect severity of equine sarcoid disease. Equine Vet. J. 2010, 42, 327–331. [Google Scholar] [CrossRef]
- Haspeslagh, M.; Vlaminck, L.; Martens, A. The possible role of Stomoxys calcitrans in equine sarcoid transmission. Vet. J. 2018, 231, 8–12. [Google Scholar] [CrossRef]
- Bae, D.H.; Marino, M.; Iaffaldano, B.; Fenstermaker, S.; Afione, S.; Argaw, T.; McCright, J.; Kwilas, A.; Chiorini, J.A.; Timmons, A.E.; et al. Design and Testing of Vector-Producing HEK293T Cells Bearing a Genomic Deletion of the SV40 T Antigen Coding Region. Mol. Ther. Methods Clin. Dev. 2020, 18, 631–638. [Google Scholar] [CrossRef]
- The Cancer Dependency Map Consortium. Available online: https://depmap.org/portal/gene/VIM?tab=dependency&dependency=GeCKO (accessed on 10 August 2023).
- Nulton, T.J.; Olex, A.L.; Dozmorov, M.; Morgan, I.M.; Windle, B. Analysis of The Cancer Genome Atlas sequencing data reveals novel properties of the human papillomavirus 16 genome in head and neck squamous cell carcinoma. Oncotarget 2017, 8, 17684–17699. [Google Scholar] [CrossRef]
- Gysens, L.; Vanmechelen, B.; Haspeslagh, M.; Maes, P.; Martens, A. New approach for genomic characterisation of equine sarcoid-derived BPV-1/-2 using nanopore-based sequencing. Virol. J. 2022, 19, 8. [Google Scholar] [CrossRef]
- Gysens, L.; Vanmechelen, B.; Maes, P.; Martens, A.; Haspeslagh, M. Complete genomic characterization of bovine papillomavirus type 1 and 2 strains infers ongoing cross-species transmission between cattle and horses. Vet. J. 2023, 298–299, 106011. [Google Scholar] [CrossRef]
- Gaynor, A.M.; Zhu, K.W.; Dela Cruz, F.N., Jr.; Affolter, V.K.; Pesavento, P.A. Localization of Bovine Papillomavirus Nucleic Acid in Equine Sarcoids. Vet. Pathol. 2016, 53, 567–573. [Google Scholar] [CrossRef] [PubMed]
- Teifke, J.P. Morphologische und molekularbiologische Untersuchungen zur Atiologie des equinen Sarkoids [Morphologic and molecular biologic studies of the etiology of equine sarcoid]. Tierarztl. Prax. 1994, 22, 368–376. [Google Scholar] [PubMed]
- Gobeil, P.A.; Yuan, Z.; Gault, E.A.; Morgan, I.M.; Campo, M.S.; Nasir, L. Small interfering RNA targeting bovine papillomavirus type 1 E2 induces apoptosis in equine sarcoid transformed fibroblasts. Virus Res. 2009, 145, 162–165. [Google Scholar] [CrossRef] [PubMed]
- Zhu, Q.S.; Rosenblatt, K.; Huang, K.L.; Lahat, G.; Brobey, R.; Bolshakov, S.; Nguyen, T.; Ding, Z.; Belousov, R.; Bill, K.; et al. Vimentin is a novel AKT1 target mediating motility and invasion. Oncogene 2011, 30, 457–470. [Google Scholar] [CrossRef]
- Costigliola, N.; Ding, L.; Burckhardt, C.J.; Han, S.J.; Gutierrez, E.; Mota, A.; Groisman, A.; Mitchison, T.J.; Danuser, G. Vimentin fibers orient traction stress. Proc. Natl. Acad. Sci. USA 2017, 114, 5195–5200. [Google Scholar] [CrossRef] [PubMed]
- Jiu, Y.; Lehtimäki, J.; Tojkander, S.; Cheng, F.; Jäälinoja, H.; Liu, X.; Varjosalo, M.; Eriksson, J.E.; Lappalainen, P. Bidirectional Interplay between Vimentin Intermediate Filaments and Contractile Actin Stress Fibers. Cell Rep. 2015, 11, 1511–1518. [Google Scholar] [CrossRef]
- Jiu, Y.; Peränen, J.; Schaible, N.; Cheng, F.; Eriksson, J.E.; Krishnan, R.; Lappalainen, P. Vimentin intermediate filaments control actin stress fiber assembly through GEF-H1 and RhoA. J. Cell Sci. 2017, 130, 892–902. [Google Scholar] [CrossRef]
- Strouhalova, K.; Přechová, M.; Gandalovičová, A.; Brábek, J.; Gregor, M.; Rosel, D. Vimentin Intermediate Filaments as Potential Target for Cancer Treatment. Cancers 2020, 12, 184. [Google Scholar] [CrossRef]
- Hartig, R.; Shoeman, R.L.; Janetzko, A.; Tolstonog, G.; Traub, P. DNA-mediated transport of the intermediate filament protein vimentin into the nucleus of cultured cells. J. Cell Sci. 1998, 111 Pt 24, 3573–3584. [Google Scholar] [CrossRef]
- Chulanov, V.; Kostyusheva, A.; Brezgin, S.; Ponomareva, N.; Gegechkori, V.; Volchkova, E.; Pimenov, N.; Kostyushev, D. CRISPR Screening: Molecular Tools for Studying Virus-Host Interactions. Viruses 2021, 13, 2258. [Google Scholar] [CrossRef]
sgRNA | Sequence 5′-3′ | Target |
---|---|---|
NT 1 | TGATTGGGGGTCGTTCGCCA | none targeting |
VIM2 | GGTGGAGCGCGACAACCTGG | Vimentin exon 1 |
LCR | CCATCACCGTTTTTTCAAGC | BPV-1 long control region |
E5 | CATTTTGAGTGCTCCTGTAC | BPV-1 E5 |
E6 | AGGTGTTCCAGTAACAGGTG | BPV-1 E6 |
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Monod, A.; Koch, C.; Jindra, C.; Haspeslagh, M.; Howald, D.; Wenker, C.; Gerber, V.; Rottenberg, S.; Hahn, K. CRISPR/Cas9-Mediated Targeting of BPV-1-Transformed Primary Equine Sarcoid Fibroblasts. Viruses 2023, 15, 1942. https://doi.org/10.3390/v15091942
Monod A, Koch C, Jindra C, Haspeslagh M, Howald D, Wenker C, Gerber V, Rottenberg S, Hahn K. CRISPR/Cas9-Mediated Targeting of BPV-1-Transformed Primary Equine Sarcoid Fibroblasts. Viruses. 2023; 15(9):1942. https://doi.org/10.3390/v15091942
Chicago/Turabian StyleMonod, Anne, Christoph Koch, Christoph Jindra, Maarten Haspeslagh, Denise Howald, Christian Wenker, Vinzenz Gerber, Sven Rottenberg, and Kerstin Hahn. 2023. "CRISPR/Cas9-Mediated Targeting of BPV-1-Transformed Primary Equine Sarcoid Fibroblasts" Viruses 15, no. 9: 1942. https://doi.org/10.3390/v15091942
APA StyleMonod, A., Koch, C., Jindra, C., Haspeslagh, M., Howald, D., Wenker, C., Gerber, V., Rottenberg, S., & Hahn, K. (2023). CRISPR/Cas9-Mediated Targeting of BPV-1-Transformed Primary Equine Sarcoid Fibroblasts. Viruses, 15(9), 1942. https://doi.org/10.3390/v15091942