Department of Bioengineering, McGill University, McConnell Engineering Building, Room 363, 3480 Rue University, Montreal, QC, H3A 2K6, Canada.
Human Health Therapeutics, National Research Council of Canada, Montreal, QC, Canada.
Sci Rep. 2020 Jul 22;10(1):12166. doi: 10.1038/s41598-020-68934-y.
Cell-derived influenza vaccines provide better protection and a host of other advantages compared to the egg-derived vaccines that currently dominate the market, but their widespread use is hampered by a lack of high yield, low cost production platforms. Identification and knockout of innate immune and metabolic restriction factors within relevant host cell lines used to grow the virus could offer a means to substantially increase vaccine yield. In this paper, we describe and validate a novel genome-wide pooled CRISPR/Cas9 screening strategy that incorporates a reporter virus and a FACS selection step to identify and rank restriction factors in a given vaccine production cell line. Using the HEK-293SF cell line and A/PuertoRico/8/1934 H1N1 influenza as a model, we identify 64 putative influenza restriction factors to direct the creation of high yield knockout cell lines. In addition, gene ontology and protein complex enrichment analysis of this list of putative restriction factors offers broader insights into the primary host cell determinants of viral yield in cell-based vaccine production systems. Overall, this work will advance efforts to address the public health burden posed by influenza.
与目前占据市场主导地位的鸡胚来源疫苗相比，细胞衍生的流感疫苗提供了更好的保护和许多其他优势，但由于缺乏高产、低成本的生产平台，其广泛应用受到阻碍。鉴定和敲除相关宿主细胞系中固有免疫和代谢限制因子，可能是大幅提高疫苗产量的一种手段。在本文中，我们描述并验证了一种新颖的全基因组 pooled CRISPR/Cas9 筛选策略，该策略结合了报告病毒和 FACS 选择步骤，以鉴定和对给定疫苗生产细胞系中的限制因子进行排序。我们使用 HEK-293SF 细胞系和 A/Puerto Rico/8/1934 H1N1 流感作为模型，鉴定出 64 种潜在的流感限制因子，以指导高产敲除细胞系的创建。此外，对该潜在限制因子列表进行基因本体论和蛋白质复合物富集分析，为基于细胞的疫苗生产系统中病毒产量的主要宿主细胞决定因素提供了更广泛的见解。总的来说，这项工作将推进解决流感对公共卫生造成的负担。
