We developed a novel antiviral strategy by combining transposon-based transgenesis and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system for the direct cleavage of Bombyx mori nucleopolyhedrovirus (BmNPV) genome DNA to promote virus clearance in silkworms. We demonstrate that transgenic silkworms constitutively expressing Cas9 and guide RNAs targeting the BmNPV immediate early-1 (ie-1) and me53 genes effectively induce target-specific cleavage and subsequent mutagenesis, especially large (∼7-kbp) segment deletions in BmNPV genomes, and thus exhibit robust suppression of BmNPV proliferation. Transgenic animals exhibited higher and inheritable resistance to BmNPV infection than wild-type animals. Our approach will not only contribute to modern sericulture but also shed light on future antiviral therapy.IMPORTANCE Pathogen genome targeting has shown its potential in antiviral research. However, transgenic CRISPR/Cas9 system-mediated viral genome targeting has not been reported as an antiviral strategy in a natural animal host of a virus. Our data provide an effective approach against BmNPV infection in a real-world biological system and demonstrate the potential of transgenic CRISPR/Cas9 systems in antiviral research in other species.
Glutathione S-transferase (GST) is a gene family generally associated with detoxification and plays an important role in detoxifying exogenous compounds. The silkworm Bombyx mori is an important economic animal for silk production. However, it is liable to infection by a number of viruses and chemical agents that can contaminate its food and growing environment. Here we conducted a comparative study of strain- and tissue-specific expressions of GST in the silkworm under infections by Bombyx mori nuclear polyhedrosis virus (BmNPV) and Bombyx mori densonucleosis virus (BmDNV) and under the stress of pesticides (insecticide and herbicide). BmDNV induced an increase of GST at 24 h and a decrease at 48 and 72 h in the midgut of the DNV-susceptible strain and a decrease at 24 h and increase at 48 and 72 h in the midgut of the DNV-tolerant strain. BmDNV induced a significant increase of GST from 24 to 72 h in the fat body of both DNV-susceptible and DNV-tolerant strains. In contrast, BmNPV induced a significant decrease of GST in both the fat body and midgut of the NPV-susceptible strain and induced increase of GST from 24 to 48 h in the midgut and at 72 h in the fat body of the NPV-tolerant strain. All of these results suggest that BmGST activity varies with the strain, tissue, feeding behavior, and developmental stage of the silkworm. On treating silkworm larvae with pesticides (insecticide and herbicide), expression of the BmGSTS2 gene increased noticeably in the midgut and reached a peak at 6 to 12 h. The mRNA level of BmGSTS2 in the midgut increased during administration of the chemicals, suggesting that the induction of BmGSTS2 is part of the defense mechanism against exogenous chemicals.
Circadian rhythms, which are ubiquitous and adaptive, occur across all species, from microbes to humans, in which they organize and modify behavior and physiology. timeless (tim) is a canonical clock gene. The core composition of the Drosophila melanogaster endogenous circadian clock has been extensively investigated; however, in lepidopteran insects, including Bombyx mori, the mechanism is complicated and little is known regarding the participation of tim in the negative feedback loop responsible for behavioral activities. To arrive at a comprehensive understanding of the role of tim in the B. mori endogenous circadian clock, we exploited the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 gene editing system. We attempted to elucidate the functions of tim in the circadian clock of B. mori using Bmtim mutants. The knockouts affected two circadian behavioral activities: adult emergence and embryo hatching rhythms. Quantitative real-time polymerase chain reaction results confirmed that tim-knockouts induced relative reductions in the expression levels, and thereby the oscillation amplitudes, of Bmper and Bmclk messenger RNAs during both the photophase and scotophase. Additionally, the daily rhythmic expression of Bmdbt was upregulated in the photophase and downregulated in the scotophase in a tim-knockout. Our study reveals that tim is integral to the B. mori circadian clock and may be involved in regulating eclosion and hatching rhythms.
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