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Viruses2024; 16(3); 409; doi: 10.3390/v16030409

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

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 ; (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 , indicating that this programmable technique can be used to develop a novel, safe, and efficacious therapeutic and prophylactic approach against EHV-1.
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Publication Date: 2024-03-06 PubMed ID: 38543774PubMed Central: PMC10975850DOI: 10.3390/v16030409Google Scholar: Lookup
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  • Journal Article

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

Overview

  • This research investigates how CRISPR/Cas9 genome editing can be used to control the replication of Equid Alphaherpesvirus-1 (EHV-1), a contagious virus affecting horses, by targeting specific viral genes and assessing the effects on viral replication in vitro.

Background

  • EHV-1 is a widespread and highly contagious virus that infects horse populations globally.
  • The virus causes significant health issues in horses, making it important to find new ways to control or prevent infection.
  • CRISPR/Cas9 technology allows for precise RNA-guided editing of genetic material, making it a promising tool to modify viral genomes and study their biology or develop antiviral strategies.

Methods

  • Researchers designed single guide RNAs (sgRNAs) targeting four specific genes of EHV-1:
    • Three essential genes involved in viral replication: ORF30, ORF31, and ORF7.
    • One non-essential gene: ORF74, to compare effects on viral replication.
  • The effects of these sgRNAs on viral replication dynamics were tested in vitro, meaning experiments were conducted in controlled lab cell culture systems.
  • Next-generation sequencing was used to analyze genetic changes in the virus after CRISPR/Cas9 editing.
  • Combinations of sgRNAs targeting different genes were also tested to evaluate potential synergistic effects on viral suppression.

Results

  • sgRNAs targeting essential lytic genes (ORF30, ORF31, ORF7) effectively reduced EHV-1 replication, while targeting the non-essential gene ORF74 had little to no impact.
  • The most pronounced reduction was observed with sgRNAs targeting ORF30, which led to:
    • A decrease in viral genomic copy numbers inside infected cells, indicating less viral replication.
    • A reduction in the amount of infectious virus produced, demonstrating impaired virus propagation.
  • Sequencing results showed that CRISPR/Cas9 caused deletions at the target sites, confirming that the editing disrupted gene function.
  • When combining sgRNAs against two essential genes (ORF30 and ORF7), viral replication was suppressed to even lower levels than with single sgRNAs alone, implying a synergistic antiviral effect.

Conclusion

  • The study demonstrated that CRISPR/Cas9 guided by specific sgRNAs can inhibit EHV-1 replication in vitro effectively.
  • This programmable gene-editing strategy shows promise as a new therapeutic and preventive approach that could be safe and effective against EHV-1 infection.
  • CRISPR/Cas9 genome editing may thus be an innovative tool to develop antiviral treatments or vaccines for managing equine herpesvirus infections.

Cite This Article

APA
Hassanien RT, Thieulent CJ, Carossino M, Li G, Balasuriya UBR. (2024). Modulation of Equid Herpesvirus-1 Replication Dynamics In Vitro Using CRISPR/Cas9-Assisted Genome Editing. Viruses, 16(3), 409. https://doi.org/10.3390/v16030409

Publication

Viruses
ISSN: 1999-4915
NlmUniqueID: 101509722
Country: Switzerland
Language: English
Volume: 16
Issue: 3
PII: 409

Researcher Affiliations

Hassanien, Rabab T
  • Louisiana Animal Disease Diagnostic Laboratory, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803, USA.
  • Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803, USA.
  • Virology Department, Animal Health Research Institute, Agriculture Research Center (ARC), Dokki, Giza 12618, Egypt.
Thieulent, Côme J
  • Louisiana Animal Disease Diagnostic Laboratory, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803, USA.
  • Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803, USA.
Carossino, Mariano
  • Louisiana Animal Disease Diagnostic Laboratory, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803, USA.
  • Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803, USA.
Li, Ganwu
  • Department of Veterinary Diagnostics and Production Animal Medicine, Iowa State University, Ames, IA 50011, USA.
Balasuriya, Udeni B R
  • Louisiana Animal Disease Diagnostic Laboratory, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803, USA.
  • Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803, USA.

MeSH Terms

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

Conflict of Interest Statement

The authors declare no conflict of interest.

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