Abstract
ABSTRACTWe 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.
Highlights
We developed a novel antiviral strategy by combining transposonbased 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 show here that a TG CRISPR/Cas9 system can be used as an anti-BmNPV strategy in a real-world biological system
The piggyBac-based TG plasmid constructed to target me53 and ie-1 contained three different types of expression cassettes: (i) Cas9 expressed under the control of the IE1 promoter, (ii) four guide RNA (gRNA) separately driven by the B. mori U6 promoter, and (iii) IE1-driven enhanced green fluorescent protein (EGFP) as the selecting marker
Summary
We developed a novel antiviral strategy by combining transposonbased 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. Moving from a time- and labor-consuming traditional breeding approach, numerous transgenic (TG) strategies have been developed in modern sericulture to improve BmNPV resistance, their practical use remains to be established These TG methods are divided into two main types: overexpression of endogenous or exogenous antiviral genes [3, 4] and inhibition of viral genes through RNA interference (RNAi) Several approaches have been developed to improve the specificity of the guide RNA (gRNA)Cas tool and accelerate its use for therapeutic applications [19,20,21,22], but more in vivo tests need to be conducted for a better understanding and nonbiased assessment of its applicability It is important and of general interest to demonstrate the efficacy of CRISPR/Cas system-mediated antiviral therapy in a legitimate, natural host-virus interaction
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