Abstract
The silkworm Bombyx mori is an economically important insect. The sericulture industry is seriously affected by pathogen infections. Of these pathogens, Bombyx mori nucleopolyhedrovirus (BmNPV) causes approximately 80% of the total economic losses due to pathogen infections. We previously constructed a BmNPV-specific CRISPR/Cas9 silkworm line with significantly enhanced resistance to BmNPV. In order to optimize the resistance properties and minimize its impact on economic traits, we constructed an inducible CRISPR/Cas9 system for use in transgenic silkworms. We used the 39k promoter, which is induced by viral infection, to express Cas9 and the U6 promoter to express four small guide RNA targeting the genes encoding BmNPV late expression factors 1 and 3 (lef-1 and lef-3, respectively), which are essential for viral DNA replication. The system was rapidly activated when the silkworm was infected and showed considerably higher resistance to BmNPV infection than the wild-type silkworm. The inducible system significantly reduced the development effects due to the constitutive expression of Cas9. No obvious differences in developmental processes or economically important characteristics were observed between the resulting transgenic silkworms and wild-type silkworms. Adoption of this accurate and highly efficient inducible CRISPR/Cas9 system targeting BmNPV DNA replication will result in enhanced antivirus measures during sericulture, and our work also provides insights into the broader application of the CRISPR/Cas9 system in the control of infectious diseases and insect pests.
Highlights
We successfully developed an antiviral strategy using a transgenic CRISPR/Cas9 system in silkworms to direct cleavage of Bombyx mori nucleopolyhedrovirus (BmNPV) genomic DNA [25,26]
We constructed a baculovirus-inducible CRISPR/Cas9 geneIE-1 protein binds promoterFirst, and we activates of the latethe protein editing system for to usethe in 39k silkworms
To more accurately evaluate the antiviral efficiency of the TG silkworm lines, we investigated the relative number of DNA copies of BmNPV genes to monitor viral proliferation in TG and WT silkworms
Summary
The silkworm Bombyx mori is the foundation of the silk industry [1]. Silk production is impacted by biotic stresses from microbial pathogens. Infections with B. mori nucleopolyhedrovirus (BmNPV) cause more than 80% of the total economic losses due to pathogen infections [2]. BmNPV is a member of the Baculoviridae family of insect viruses. It possesses an approximately 130-kilobase, circular, double-stranded DNA genome with about
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