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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
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 technolog...
Autores principales: | , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Frontiers Media S.A.
2022
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9093602/ https://www.ncbi.nlm.nih.gov/pubmed/35573771 http://dx.doi.org/10.3389/fcimb.2022.865744 |
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author | Wu, Xiaoyan Wang, Shuo Li, Chen Shi, Jianli Peng, Zhe Liu, Chang Han, Hong Ma, Yingru Zheng, Limei Xu, Shaojian Du, Wei Li, Jun Zhang, Fan |
author_facet | Wu, Xiaoyan Wang, Shuo Li, Chen Shi, Jianli Peng, Zhe Liu, Chang Han, Hong Ma, Yingru Zheng, Limei Xu, Shaojian Du, Wei Li, Jun Zhang, Fan |
author_sort | Wu, Xiaoyan |
collection | PubMed |
description | 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(Δ-) and BHK-Ago2(Δ-). 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. |
format | Online Article Text |
id | pubmed-9093602 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-90936022022-05-12 CRISPR/Cas9-Mediated Knockout of the Dicer and Ago2 Genes in BHK-21 Cell Promoted Seneca Virus A Replication and Enhanced Autophagy Wu, Xiaoyan Wang, Shuo Li, Chen Shi, Jianli Peng, Zhe Liu, Chang Han, Hong Ma, Yingru Zheng, Limei Xu, Shaojian Du, Wei Li, Jun Zhang, Fan Front Cell Infect Microbiol Cellular and Infection Microbiology 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(Δ-) and BHK-Ago2(Δ-). 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. Frontiers Media S.A. 2022-04-27 /pmc/articles/PMC9093602/ /pubmed/35573771 http://dx.doi.org/10.3389/fcimb.2022.865744 Text en Copyright © 2022 Wu, Wang, Li, Shi, Peng, Liu, Han, Ma, Zheng, Xu, Du, Li and Zhang https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Cellular and Infection Microbiology Wu, Xiaoyan Wang, Shuo Li, Chen Shi, Jianli Peng, Zhe Liu, Chang Han, Hong Ma, Yingru Zheng, Limei Xu, Shaojian Du, Wei Li, Jun Zhang, Fan CRISPR/Cas9-Mediated Knockout of the Dicer and Ago2 Genes in BHK-21 Cell Promoted Seneca Virus A Replication and Enhanced Autophagy |
title | CRISPR/Cas9-Mediated Knockout of the Dicer and Ago2 Genes in BHK-21 Cell Promoted Seneca Virus A Replication and Enhanced Autophagy |
title_full | CRISPR/Cas9-Mediated Knockout of the Dicer and Ago2 Genes in BHK-21 Cell Promoted Seneca Virus A Replication and Enhanced Autophagy |
title_fullStr | CRISPR/Cas9-Mediated Knockout of the Dicer and Ago2 Genes in BHK-21 Cell Promoted Seneca Virus A Replication and Enhanced Autophagy |
title_full_unstemmed | CRISPR/Cas9-Mediated Knockout of the Dicer and Ago2 Genes in BHK-21 Cell Promoted Seneca Virus A Replication and Enhanced Autophagy |
title_short | CRISPR/Cas9-Mediated Knockout of the Dicer and Ago2 Genes in BHK-21 Cell Promoted Seneca Virus A Replication and Enhanced Autophagy |
title_sort | crispr/cas9-mediated knockout of the dicer and ago2 genes in bhk-21 cell promoted seneca virus a replication and enhanced autophagy |
topic | Cellular and Infection Microbiology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9093602/ https://www.ncbi.nlm.nih.gov/pubmed/35573771 http://dx.doi.org/10.3389/fcimb.2022.865744 |
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