Modulation of Equid Herpesvirus-1 Replication Dynamics In Vitro Using CRISPR/Cas9-Assisted Genome Editing

Rabab T Hassanien; Côme J Thieulent; Mariano Carossino; Ganwu Li; Udeni B R Balasuriya

doi:10.3390/v16030409

Modulation of Equid Herpesvirus-1 Replication Dynamics In Vitro Using CRISPR/Cas9-Assisted Genome Editing

Viruses. 2024 Mar 6;16(3):409. doi: 10.3390/v16030409.

Authors

Rabab T Hassanien^{1

2

3}, Côme J Thieulent^{1

2}, Mariano Carossino^{1

2}, Ganwu Li⁴, Udeni B R Balasuriya^{1

2}

Affiliations

¹ Louisiana Animal Disease Diagnostic Laboratory, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803, USA.
² Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803, USA.
³ Virology Department, Animal Health Research Institute, Agriculture Research Center (ARC), Dokki, Giza 12618, Egypt.
⁴ Department of Veterinary Diagnostics and Production Animal Medicine, Iowa State University, Ames, IA 50011, USA.

Abstract

(1) Background: equid alphaherpesvirus-1 (EHV-1) is a highly contagious viral pathogen prevalent in most horse populations worldwide. Genome-editing technologies such as CRISPR/Cas9 have become powerful tools for precise RNA-guided genome modifications; (2) Methods: we designed single guide RNAs (sgRNA) to target three essential (ORF30, ORF31, and ORF7) and one non-essential (ORF74) EHV-1 genes and determine their effect on viral replication dynamics in vitro; (3) Results: we demonstrated that sgRNAs targeting essential lytic genes reduced EHV-1 replication, whereas those targeting ORF74 had a negligible effect. The sgRNAs targeting ORF30 showed the strongest effect on the suppression of EHV-1 replication, with a reduction in viral genomic copy numbers and infectious progeny virus output. Next-generation sequencing identified variants with deletions in the specific cleavage site of selective sgRNAs. Moreover, we evaluated the combination between different sgRNAs and found that the dual combination of sgRNAs targeting ORF30 and ORF7 significantly suppressed viral replication to lower levels compared to the use of a single sgRNA, suggesting a synergic effect; (4) Conclusion: data demonstrate that sgRNA-guided CRISPR/Cas9 can be used to inhibit EHV-1 replication in vitro, indicating that this programmable technique can be used to develop a novel, safe, and efficacious therapeutic and prophylactic approach against EHV-1.

Keywords: CRISPR/Cas9; EHV-1; antiviral; equid alphaherpesvirus-1; gene editing; viral replication.

MeSH terms

Animals
CRISPR-Cas Systems
Gene Editing* / methods
Genome, Viral
Herpesvirus 1, Equid* / genetics
Horses
RNA, Guide, CRISPR-Cas Systems

Substances

RNA, Guide, CRISPR-Cas Systems

Grants and funding

This research was funded by USAID (Higher Education Initiative, Graduate Scholarships for Professional [GSP]), Postdoctoral Research Program for R.T.H, as a Visiting Research Scholar in Animal Disease Diagnostic Laboratory (LADDL) and Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA, USA in the period from 17 January 2022 to 15 July 2022. This study was also supported by USAID GSP and self-generated research funds (PG008671) from U.B.R.B., Louisiana Animal Disease Diagnostic Laboratory (LADDL) and Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA, USA.