利用 CRISPR/Cas9 辅助基因组编辑调控马疱疹病毒-1 的复制动态。

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

机构信息

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.

出版信息

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

DOI:10.3390/v16030409

PMID:38543774

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC10975850/

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.

摘要

(1) 背景：马疱疹病毒 1 型（EHV-1）是一种在世界范围内大多数马群中流行的高度传染性病毒病原体。基因组编辑技术如 CRISPR/Cas9 已成为精确 RNA 引导的基因组修饰的强大工具；(2) 方法：我们设计了针对三个必需（ORF30、ORF31 和 ORF7）和一个非必需（ORF74）EHV-1 基因的单指导 RNA（sgRNA），并确定它们对病毒复制动力学的影响；(3) 结果：我们证明了靶向必需裂解基因的 sgRNA 降低了 EHV-1 的复制，而靶向 ORF74 的 sgRNA 则几乎没有影响。靶向 ORF30 的 sgRNA 对抑制 EHV-1 复制的效果最强，病毒基因组拷贝数和感染性子代病毒产量减少。下一代测序鉴定了在选择性 sgRNA 的特定切割位点具有缺失的变体。此外，我们评估了不同 sgRNA 之间的组合，发现靶向 ORF30 和 ORF7 的 sgRNA 双重组合比使用单个 sgRNA 更能显著抑制病毒复制，表明存在协同作用；(4) 结论：数据表明 sgRNA 引导的 CRISPR/Cas9 可用于抑制 EHV-1 的复制，表明该可编程技术可用于开发针对 EHV-1 的新型、安全且有效的治疗和预防方法。