CRISPR/Cas9-Mediated Disruption of the lef8 and lef9 to Inhibit Nucleopolyhedrovirus Replication in Silkworms

Yujia Liu; Xiaoqian Zhang; Dongbin Chen; Dehong Yang; Chenxu Zhu; Linmeng Tang; Xu Yang; Yaohui Wang; Xingyu Luo; Manli Wang; Yongping Huang; Zhihong Hu; Zulian Liu

doi:10.3390/v14061119

CRISPR/Cas9-Mediated Disruption of the lef8 and lef9 to Inhibit Nucleopolyhedrovirus Replication in Silkworms

Viruses. 2022 May 24;14(6):1119. doi: 10.3390/v14061119.

Authors

Yujia Liu^{1

2}, Xiaoqian Zhang¹, Dongbin Chen¹, Dehong Yang^{1

2}, Chenxu Zhu¹, Linmeng Tang^{1

2}, Xu Yang^{1

2}, Yaohui Wang¹, Xingyu Luo^{1

2}, Manli Wang³, Yongping Huang¹, Zhihong Hu³, Zulian Liu¹

Affiliations

¹ Key Laboratory of Insect Developmental and Evolutionary Biology, Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai 200032, China.
² University of Chinese Academy of Sciences, Beijing 100049, China.
³ State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China.

Abstract

Bombyx mori nucleopolyhedrovirus (BmNPV) is a pathogen that causes severe disease in silkworms. In a previous study, we demonstrated that by using the CRISPR/Cas9 system to disrupt the BmNPV ie-1 and me53 genes, transgenic silkworms showed resistance to BmNPV infection. Here, we used the same strategy to simultaneously target lef8 and lef9, which are essential for BmNPV replication. A PCR assay confirmed that double-stranded breaks were induced in viral DNA at targeted sequences in BmNPV-infected transgenic silkworms that expressed small guide RNAs (sgRNAs) and Cas9. Bioassays and qPCR showed that replication of BmNPV and mortality were significantly reduced in the transgenic silkworms in comparison with the control groups. Microscopy showed degradation of midgut cells in the BmNPV-infected wild type silkworms, but not in the transgenic silkworms. These results demonstrated that transgenic silkworms using the CRISPR/Cas9 system to disrupt BmNPV lef8 and lef9 genes could successfully prevent BmNPV infection. Our research not only provides more alternative targets for the CRISPR antiviral system, but also aims to provide new ideas for the application of virus infection research and the control of insect pests.

Keywords: BmNPV; CRISPR/Cas9; antiviral therapy; transgenic silkworm.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Animals, Genetically Modified
Bombyx*
CRISPR-Cas Systems
Nucleopolyhedroviruses* / genetics

Supplementary concepts

Bombyx mori nucleopolyhedrovirus

Grants and funding

This study was supported by grants from the National Science Foundation of China (31830093, 32100381), Strategic Priority Research Program of Chinese Academy of Sciences (XDB11010600), and Zhejiang provincial science and Technology Department (grant number 2017R01001). The funders had no role in study design, data collection, analysis, decision to publish, or preparation of the manuscript.