From functional genomics of vero cells to CRISPR-based genomic deletion for improved viral production rates,Biotechnology and Bioengineering

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From functional genomics of vero cells to CRISPR-based genomic deletion for improved viral production rates
Biotechnology and Bioengineering ( IF 3.5 ) Pub Date : 2022-07-23 , DOI: 10.1002/bit.28190
Marie-Angélique Sène ₁ , Yu Xia ₁ , Amine A Kamen ₁

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Despite their wide use in the vaccine manufacturing field for over 40 years, one of the main limitations to recent efforts to develop Vero cells as high-throughput vaccine manufacturing platforms is the lack of understanding of virus-host interactions during infection and cell-based virus production in Vero cells. To overcome this limitation, this manuscript uses the recently generated reference genome for the Vero cell line to identify the factors at play during influenza A virus (IAV) and recombinant vesicular stomatitis virus (rVSV) infection and replication in Vero host cells. The best antiviral gene candidate for gene editing was selected using Differential Gene Expression analysis, Gene Set Enrichment Analysis and Network Topology-based Analysis. After selection of the ISG15 gene for targeted CRISPR genomic deletion, the ISG15 genomic sequence was isolated for CRISPR guide RNAs design and the guide RNAs with the highest knockout efficiency score were selected. The CRISPR experiment was then validated by confirmation of genomic deletion via PCR and further assessed via quantification of ISG15 protein levels by western blot. The gene deletion effect was assessed thereafter via quantification of virus production yield in the edited Vero cell line. A 70-fold and an 87-fold increase of total viral particles productions in ISG15^−/− Vero cells was achieved for, respectively, IAV and rVSV while the ratio of infectious viral particles/total viral particles also significantly increased from 0.0316 to 0.653 for IAV and from 0.0542 to 0.679 for rVSV-GFP.

中文翻译：

从 Vero 细胞的功能基因组学到基于 CRISPR 的基因组删除，以提高病毒生产率

尽管 Vero 细胞在疫苗制造领域广泛使用了 40 多年，但最近开发 Vero 细胞作为高通量疫苗制造平台的主要限制之一是缺乏对感染过程中病毒与宿主相互作用的了解以及基于细胞的病毒Vero 细胞生产。为了克服这一限制，本手稿使用最近生成的 Vero 细胞系参考基因组来识别甲型流感病毒 (IAV) 和重组水泡性口炎病毒 (rVSV) 在 Vero 宿主细胞中感染和复制期间发挥作用的因素。使用差异基因表达分析、基因集富集分析和基于网络拓扑的分析来选择用于基因编辑的最佳抗病毒候选基因。选择ISG15基因进行靶向CRISPR基因组删除后，分离ISG15基因组序列进行CRISPR向导RNA设计，并选择敲除效率得分最高的向导RNA。然后通过 PCR 确认基因组缺失来验证 CRISPR 实验，并通过蛋白质印迹定量 ISG15 蛋白水平进一步评估。此后通过量化编辑的 Vero 细胞系中的病毒产量来评估基因删除效果。 IAV 和 rVSV 的 ISG15 ^−/− Vero 细胞中总病毒颗粒产量分别增加了 70 倍和 87 倍，而感染性病毒颗粒/总病毒颗粒的比率也从 0.0316 显着增加到 0.653。 rVSV-GFP 的 IAV 为 0.0542 至 0.679。

更新日期：2022-07-23

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