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CRISPR/Cas9-Mediated Knockout of the Dicer and Ago2 Genes in BHK-21 Cell Promoted Seneca Virus A Replication and Enhanced Autophagy

文献类型： 外文期刊

作者： Wu, Xiaoyan 1 ; Wang, Shuo 3 ; Li, Chen 2 ; Shi, Jianli 2 ; Peng, Zhe 2 ; Liu, Chang 2 ; Han, Hong 2 ; Ma, Yingru 4 ; Zheng, Limei 4 ; Xu, Shaojian 2 ; Du, Wei 1 ; Li, Jun 1 ; Zhang, Fan 1 ;

作者机构： 1.Shandong Normal Univ, Coll Life Sci, Key Lab Anim Resistance Biol Shandong Prov, Jinan, Peoples R China

2.Shandong Acad Agr Sci, Div Swine Dis, Shandong Prov Key Lab Anim Dis Control & Breeding, Inst Anim Sci & Vet Med, Jinan, Peoples R China

3.Shandong Univ, Sch Life Sci, Shandong Prov Key Lab Anim Cells & Dev Biol, Qingdao, Peoples R China

4.Qingdao Agr Univ, Coll Vet Med, Qingdao, Peoples R China

关键词： CRISPR; Cas9; Ago2; dicer; Senecavirus A; viral replication; autophagy

期刊名称：FRONTIERS IN CELLULAR AND INFECTION MICROBIOLOGY （ 影响因子：6.073； 五年影响因子：6.34 ）

ISSN： 2235-2988

年卷期： 2022 年 12 卷

页码：

收录情况： SCI

摘要： RNA interference (RNAi) is a major form of antiviral defense in host cells, and Ago2 and Dicer are the major proteins of RNAi. The Senecavirus A (SVA) is a reemerging virus, resulting in vesicular lesions in sows and a sharp decline in neonatal piglet production. In this study, CRISPR/Cas9 technology was used to knock out Ago2 and Dicer genes in BHK-21 cell lines used for SVA vaccine production. Cell clones with homozygous frameshift mutations of Ago2 and Dicer genes were successfully identified. The two knockout cell lines were named BHK-Dicer(Delta-) and BHK-Ago2(Delta-). Results showed that the two genes' knockout cell lines were capable of stable passage and the cell growth rate did not change significantly. The replication rate and virus titers of SVA were significantly increased in knockout cell lines, indicating that RNAi could inhibit SVA replication. In addition, compared with normal cells, autophagy was significantly enhanced after SVA-infected knockout cell lines, while there was no significant difference in autophagy between the knockout and normal cell lines without SVA. The results confirmed that SVA could enhance the autophagy in knockout cells and promote viral replication. The two knockout cell lines can obtain viruses with high viral titers and have good application prospects in the production of SVA vaccine. At the same time, the RNAi knockout cell lines provide convenience for further studies on RNAi and SVA resistance to RNAi, and it lays a foundation for further study of SVA infection characteristics and screening of new therapeutic drugs and drug targets.

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