Sex separation methods are critical for genetic sexing systems in commercial insect production and sterile insect techniques. Integration of selectable marker genes into a sex chromosome is particularly useful in insects with a heterogametic sex determination system. Here, we describe targeted gene integration of fluorescent marker expression cassettes into a randomly amplified polymorphic DNA (RAPD) marker region in the W chromosome of the lepidopteran model insect Bombyx mori using transcriptional activator-like effector nuclease (TALEN)-mediated genome editing. This silkworm strain shows ubiquitous female-specific red or green fluorescence from the embryonic to adult stages. Furthermore, we developed a binary, female-specific, embryonic lethality system combining the TALEN and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology. This system includes one strain with TALEN-mediated, W-specific Cas9 expression driven by the silkworm germ cell-specific nanos (nos) promoter and another strain with U6-derived single-guide RNA (sgRNA) expression targeting transformer 2 (tra2), an essential gene for silkworm embryonic development. Filial 1 (F1) hybrids exhibit complete female-specific lethality during embryonic stages. Our study provides a promising approach for B. mori genetic sexing and sheds light on developing sterile insect techniques in other insect species, especially in lepidopteran pests with WZ/ZZ sex chromosome systems.

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.