Bmpali, Bmb1 and Bmcap are necessary for uric acid granule formation in Bombyx mori.

Uric acid is the end-product of nitrogen metabolism of the silkworm and other lepidopterans. The accumulation of uric acid particles in the epidermis causes the larval silkworm to appear white and opaque. However, the mechanism of uric acid granule formation is still unclear. Silkworm epidermis color is linked to the genes responsible for uric acid particle formation. We first identified two genes in the Bombyx mori genome that encode subunits of the Bloc-1 (Biogenesis of Lysosome-related Organelles Complex-1) by homology to these genes in other eukaryotes, Bmpali and Bmb1. Mutation in these genes caused a transparent phenotype in the silkworm larvae, and the loss of BmBloc-1 subunit gene Bmcap resulted in the same phenotype. These three genes are highly conserved between human and silkworm. We discovered that Bmpali, Bmcap, and Bmb1 localize in the cytoplasm of BmN cells. Yeast two-hybrid assays demonstrated that the Bmpali physically interacts with both Bmcap and Bmb1. Investigating the roles of Bmpali, Bmb1, and Bmcap is essential for uric acid granule formation understanding in Bombyx mori. These mutants present a valuable silkworm model for studying the biogenesis of lysosome-related organelles (LROs).

Functional characterization of Bmcap in uric acid metabolism in the silkworm.

After a millennium of domestication, numerous silkworm mutants have emerged that exhibit transparent epidermis, which is caused by abnormally low levels of uric acid. We identified the Bombyx mori gene Bmcap (BMSK0003832) as the homolog of cappuccino, a subunit of the biogenesis of lysosome-related organelles complex-1 (BLOC-1) that has been extensively characterized in human, mouse, and insect species, by analyzing the amino acid sequences of putative purine metabolism genes. Using the clustered regularly interspaced palindromic repeats (CRISPR) / CRISPR-associated protein 9 system, we disrupted Bmcap, resulting in decreased uric acid levels in the silkworm epidermis and a translucent skin phenotype. In the Bmcap mutant, the purine metabolism, nitrogen metabolism, pyrimidine metabolism, and membrane system were altered compared to the wild type. Biogenesis of lysosome-related organelle complex genes play a role in the pigmentation and biogenesis of lysosome-related organelles (LROs) in platelets, melanocytes, and megakaryocytes. LROs exhibit unique morphologies and functions in various tissues and cells. Investigation of the Bmcap mutant will enhance our understanding of the uric acid metabolic pathway in silkworms, and this mutant offers a valuable silkworm model for LRO studies.

Identification of molecular subgroups in osteomyelitis induced by staphylococcus aureus infection through gene expression profiles.

BACKGROUND: Staphylococcus aureus (S. aureus) infection-induced osteomyelitis (OM) is an inflammatory bone disease accompanied by persistent bone destruction, and the treatment is challenging because of its tendency to recur. Present study was aimed to explore the molecular subgroups of S. aureus infection-induced OM and to deepen the mechanistic understanding for molecularly targeted treatment of OM. METHODS: Integration of 164 OM samples and 60 healthy samples from three datasets of the Gene Expression Omnibus (GEO) database. OM patients were classified into different molecular subgroups based on unsupervised algorithms and correlations of clinical characteristics between subgroups were analyzed. Next, The CIBERSORT algorithm was used to evaluate the proportion of immune cell infiltration in different OM subgroups. Weighted gene co-expression analysis (WGCNA) was used to identify different gene modules and explore the relationship with clinical characteristics, and further annotated OM subgroups and gene modules by the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. RESULTS: Two subgroups with excellent consistency were identified in this study, subgroup and hospital length of stay were independent predictors of OM. Compared with subgroup I, OM patients in subgroup II had longer hospital length of stay and more severe disease. Meanwhile, the infiltration proportions of monocytes and macrophages M0 were higher in patients of OM subgroup II. Finally, combined with the characteristics of the KEGG enrichment modules, the expression of osteoclast differentiation-related genes such as CTSK was upregulated in OM subgroup II, which may be closely associated with more severe OM patients. CONCLUSION: The current study showed that OM subgroup II had longer hospital length of stay and more severe disease, the osteoclast differentiation pathway and the main target CTSK contribute to our deeper understanding for the molecular mechanisms associated with S. aureus infection-induced OM, and the construction of molecular subgroups suggested the necessity for different subgroups of patients to receive individualized treatment.

Identification of Ferroptosis-Related Biomarkers for Diagnosis and Molecular Classification of Staphylococcus aureus-Induced Osteomyelitis.

Objective: Staphylococcus aureus (SA)-induced osteomyelitis (OM) is one of the most common refractory diseases in orthopedics. Early diagnosis is beneficial to improve the prognosis of patients. Ferroptosis plays a key role in inflammation and immune response, while the mechanism of ferroptosis-related genes (FRGs) in SA-induced OM is still unclear. The purpose of this study was to determine the role of ferroptosis-related genes in the diagnosis, molecular classification and immune infiltration of SA-induced OM by bioinformatics. Methods: Datasets related to SA-induced OM and ferroptosis were collected from the Gene Expression Omnibus (GEO) and ferroptosis databases, respectively. The least absolute shrinkage and selection operator (LASSO) and support vector machine-recursive feature elimination (SVM-RFE) algorithms were combined to screen out differentially expressed-FRGs (DE-FRGs) with diagnostic characteristics, and gene set enrichment analysis (GSEA) and gene set variation analysis (GSVA) were used to explore specific biological functions and pathways. Based on these key DE-FRGs, a diagnostic model was established, and molecular subtypes were divided to explore the changes in the immune microenvironment between molecular subtypes. Results: A total of 41 DE-FRGs were identified. After screening and intersecting with LASSO and SVM-RFE algorithms, 8 key DE-FRGs with diagnostic characteristics were obtained, which may regulate the pathogenesis of OM through the immune response and amino acid metabolism. The ROC curve indicated that the 8 DE-FRGs had excellent diagnostic ability for SA-induced OM (AUC=0.993). Two different molecular subtypes (subtype 1 and subtype 2) were identified by unsupervised cluster analysis. The CIBERSORT analysis revealed that the subtype 1 OM had higher immune cell infiltration rates, mainly in T cells CD4 memory resting, macrophages M0, macrophages M2, dendritic cells resting, and dendritic cells activated. Conclusion: We developed a diagnostic model related to ferroptosis and molecular subtypes significantly related to immune infiltration, which may provide a novel insight for exploring the pathogenesis and immunotherapy of SA-induced OM.

Downregulated annexin A1 expression correlates with poor prognosis, metastasis, and immunosuppressive microenvironment in Ewing's sarcoma.

OBJECTIVE: Ewing's sarcoma (ES) is a common bone malignancy in children and adolescents that severely affects the prognosis of patients. The aim of this study was to identify novel biomarkers and potential therapeutic targets for ES. METHODS: Highly prognosis-related hub genes were identified by independent prognostic analysis in the GSE17679 dataset. We then performed survival analysis, Cox regression analysis and clinical correlation analysis on the key gene and validated them with the GSE63157, GSE45544 and GSE73166 datasets. Differentially expressed genes (DEGs) were screened based on the high and low expression of key gene, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses, Gene Set Enrichment Analysis (GSEA), and Gene Set Variation Analysis (GSVA) were performed to explore the underlying mechanisms of ES, and significant module genes were established based on protein-protein interaction (PPI) networks. Furthermore, the correlations between module genes and the immune microenvironment were analyzed and the correlations between the key gene and immune infiltration levels in sarcoma were investigated using TIMER and TISIDB. Finally, the expression levels of these key genes in ES cell lines (RD-ES and A673 cells) were further validated by real-time quantitative PCR (RT-qPCR). CCK-8 and EdU assays were performed to assess the effect of ANXA1 knockdown on RD-ES cell proliferation. RESULTS: ANXA1 was identified as a key gene for ES prognosis. The overall survival (OS) time of patients with low ANXA1 expression was shorter, and the expression level of ANXA1 in the metastatic group was significantly lower than that in the primary group (P<0.01). Additionally, the abundance of 12 immune cells in the ANXA1 low-expression group was significantly lower than that in the high-expression group (all P<0.05), which may be related to the inhibition of the immune microenvironment. A PPI network was constructed based on 96 DEGs to further identify the five ANXA1-related module genes (COL1A2, MMP9, VIM, S100A11 and S100A4). The expression levels of ANXA1, COL1A2, MMP9, VIM, S100A11 and S100A4 were significantly different between ES cell lines and mesenchymal stem cells after validation in two ES cell lines (all P<0.01). Among these genes, ANXA1, COL1A2, MMP9, VIM and S100A4 were significantly associated with the prognosis of ES patients (all P<0.05). Importantly, ANXA1 knockdown significantly promoted the proliferation of RD-ES cells, which may explain the susceptibility to ES metastasis in the ANXA1 low-expression group. CONCLUSIONS: ANXA1 may serve as an independent prognostic biomarker for ES patients and is associated with metastasis and the immunosuppressive microenvironment in ES, which needs to be validated in further studies.

The RNase III enzyme Dicer1 is essential for larval development in Bombyx mori.

MicroRNAs (miRNAs) are important regulators of nearly all aspects of biological processes in eukaryotes. During the biogenesis of miRNAs, the RNase III enzyme Dicer processes double-strand precursor miRNAs into mature miRNAs and promotes the assembly of RNA-induced silencing complexes (RISCs). Dicer has been reported to participate in a wide range of physiological processes, including development and immunity, in some insect species. However, the physiological roles of Dicer in lepidopterans remain poorly understood. In this study, we investigated the function of Bombyx mori Dicer1. We first performed sequence alignment and found that the sequence of functional domains of Dicer1 are varied among Lepidoptera, Diptera, Coleoptera, Blattaria, and Orthoptera. Using a binary clustered regularly interspaced palindromic repeats (CRISPR) / CRISPR-associated protein 9 genome editing approach, we showed that BmDicer1 mutants have arrested development from the 3rd instar into the 4th instar. RNA sequencing analysis indicated that the defects in BmDicer1 mutants are due to dysregulation of genes that encode proteins involved in metabolism, protein degradation, absorption, and renin-angiotensin pathways. Analysis using quantitative real-time polymerase chain reaction showed that mutation of BmDicer1 altered expression of miRNAs and their target genes. Therefore, our study demonstrates the critical roles of BmDicer1 in miRNA biogenesis and larval development in silkworm.

The Prmt5-Vasa module is essential for spermatogenesis in Bombyx mori.

In lepidopteran insects, dichotomous spermatogenesis produces eupyrene spermatozoa, which are nucleated, and apyrene spermatozoa, which are anucleated. Both sperm morphs are essential for fertilization, as eupyrene sperm fertilize the egg, and apyrene sperm is necessary for the migration of eupyrene sperm. In Drosophila, Prmt5 acts as a type II arginine methyltransferase that catalyzes the symmetrical dimethylation of arginine residues in the RNA helicase Vasa. Prmt5 is critical for the regulation of spermatogenesis, but Vasa is not. To date, functional genetic studies of spermatogenesis in the lepidopteran model Bombyx mori has been limited. In this study, we engineered mutations in BmPrmt5 and BmVasa through CRISPR/Cas9-based gene editing. Both BmPrmt5 and BmVasa loss-of-function mutants had similar male and female sterility phenotypes. Through immunofluorescence staining analysis, we found that the morphs of sperm from both BmPrmt5 and BmVasa mutants have severe defects, indicating essential roles for both BmPrmt5 and BmVasa in the regulation of spermatogenesis. Mass spectrometry results identified that R35, R54, and R56 of BmVasa were dimethylated in WT while unmethylated in BmPrmt5 mutants. RNA-seq analyses indicate that the defects in spermatogenesis in mutants resulted from reduced expression of the spermatogenesis-related genes, including BmSxl, implying that BmSxl acts downstream of BmPrmt5 and BmVasa to regulate apyrene sperm development. These findings indicate that BmPrmt5 and BmVasa constitute an integral regulatory module essential for spermatogenesis in B. mori.

BmHen1 is essential for eupyrene sperm development in Bombyx mori but PIWI proteins are not.

In lepidopteran insects, sperm dimorphism is a remarkable feature, in which males exhibit two different types of sperms. Both sperm morphs are essential for fertilization: Eupyrene sperm carry DNA and fertilize eggs, whereas apyrene sperm, which do not have nuclei, are necessary for transport of eupyrene sperm into eggs. In this study, we showed that the gene BmHen1, which encodes a methyltransferase that modifies piRNAs, is necessary for eupyrene sperm development in the lepidopteran model insect, Bombyx mori. Loss-of-function mutants of BmHen1 of both sexes were sterile. BmHen1 female mutants laid fewer eggs than wild-type females, and the eggs laid had morphological defects. Immunofluorescence analysis of BmHen1 male mutants revealed that nuclei formation in the eupyrene sperm was defective, whereas apyrene sperm were normal. In mice, worms, and flies, the components in piRNA biogenesis pathway play an important role in gonad development; therefore, we constructed mutations in genes encoding core elements in the piRNA biogenesis pathway, Siwi, and BmAgo3. To our surprise, no obvious phenotypes were observed in the male reproduction system in the Siwi and BmAgo3 mutants, which demonstrated that sperm development in B. mori does not depend on piRNAs. As the sperm development phenotype in BmHen1 mutants mimics the phenotype of the BmPnldc1 mutants, we then performed RNA sequencing analysis of sperm bundles from both mutants. We found that the defects in eupyrene sperm resulted from dysregulation of the expression of genes involved in energy metabolism. Taken together, our findings demonstrate the crucial functions of BmHen1 in the development of eupyrene sperm and provide evidence that spermatogenesis in B. mori is PIWI-independent. Our results suggest potential targets for lepidopteran pest control and broaden our knowledge of the reproduction in this order of insects.

Synergistic value of fractional flow reserve and low­density non­calcified plaque based on coronary computed tomography angiography for the identification of lesion­specific ischemia.

Increasing evidence has suggested that plaque characteristics are closely associated with ischemia, and coronary computed tomography (CT) angiography-derived fractional flow reserve (FFRCT) based on deep machine learning algorithms has also been used to identify lesion-specific ischemia. Therefore, the aim of the present study was to explore the predictive ability of plaque characteristics in combination with deep learning-based FFRCT for lesion-specific ischemia. To meet this end, invasive FFR was used as a reference standard, with the joint aims of the early prediction of ischemic lesions and guiding clinical treatment. In the present study, the plaque characteristics, including non-calcified plaque (NCP), low-density NCP (LD-NCP), plaque length, total plaque volume (TPV), remodeling index, calcified plaque, fibrous plaque and plaque burden, were obtained using a semi-automated program. The FFRCT values were derived based on a deep machine learning algorithm. On the basis of the data obtained, differences among the values between the atopic ischemia and the non-significant lesions groups were analyzed to further determine the predictive value of independent predictors for atopic ischemia. Of the plaque features, FFRCT, LD-NCP, NCP, TPV and plaque length differed significantly when comparing between the lesion-specific ischemia and no hemodynamic abnormality groups, and LD-NCP and FFRCT were both independent predictors for ischemia. Additionally, FFRCT combined with LD-NCP showed a greater ability at discriminating ischemia compared with FFRCT or LD-NCP alone. Taken together, the findings of the present study suggest that the combination of FFRCT and LD-NCP has a synergistic effect in terms of predicting ischemia, thereby facilitating the identification of specific ischemia in patients with coronary artery disease.

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

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.

BmPMFBP1 regulates the development of eupyrene sperm in the silkworm, Bombyx mori.

Sperm deliver the male complement of DNA to the ovum, and thus play a key role in sexual reproduction. Accordingly, spermatogenesis has outstanding significance in fields as disparate as infertility treatments and pest-control, making it a broadly interesting and important focus for molecular genetics research in a wide range of species. Here we investigate spermatogenesis in the model lepidopteran insect Bombyx mori (silkworm moth), with particular focus on the gene PMFBP1 (polyamine modulated factor 1 binding protein 1). In humans and mouse, PMFBP1 is essential for spermatogenesis, and mutations of this gene are associated with acephalic spermatozoa, which cause infertility. We identified a B. mori gene labeled as "PMFBP1" in GenBank's RefSeq database and sought to assess its role in spermatogenesis. Like in mammals, the silkworm version of this gene (BmPMFBP1) is specifically expressed in testes. We subsequently generated BmPMFBP1 mutants using a transgenic CRISPR/Cas9 system. Mutant males were sterile while the fertility of mutant females was comparable to wildtype females. In B. mori, spermatogenesis yields two types of sperm, the nucleated fertile eupyrene sperm, and anucleated unfertile apyrene sperm. Mutant males produced abnormal eupyrene sperm bundles but normal apyrene sperm bundles. For eupyrene sperm, nuclei were mislocated and disordered inside the bundles. We also found the BmPMFBP1 deficiency blocked the release of eupyrene sperm bundles from testes to ejaculatory seminalis. We found no obvious abnormalities in the production of apyrene sperm in mutant males, and double-matings with apyrene-deficient sex-lethal mutants rescued the ΔBmPMFBP1 infertility phenotype. These results indicate BmPMFBP1 functions only in eupyrene spermatogenesis, and highlight that distinct genes underlie the development of the two sperm morphs commonly found in Lepidoptera. Bioinformatic analyses suggest PMFBP1 may have evolved independently in lepidoptera and mammals, and that despite the shared name, are likely not homologous genes.

Transgenic genome editing-derived antiviral therapy to nucleopolyhedrovirus infection in the industrial strain of the silkworm.

The silkworm (Bombyx mori) is a domesticated and economically important insect. During the whole growth period, silkworm suffers various pathogen infection. Nearly 80% of silk cocoon crop losses are attributed to viral diseases. The circular double-stranded DNA virus Bombyx mori nuclepolyhedrovirus (BmNPV) is the major viral pathogen responsible for massive silkworm death and huge economic losses in the sericulture industry. Up to now, almost all the new strategies for developing immunity against BmNPV are in laboratory strains because of the lack of transgenic technology in industrial silkworm strains. We previously demonstrated that modification of BmNPV genome DNA with the antivirus transgenic CRISPR/Cas9 system effectively improved the resistance of laboratory silkworm strains to viral pathogens. The industrial strains are monovoltine or bivoltine. It is very difficult to break the diapause before microinjection for genetic transformation. Here, we show that the anti-BmNPV transgenic CRISPR/Cas9 system also works in the industrial silkworm strain Jingsong. In this study, we successfully broke diapause and obtained generation-skipping embryos and constructed two TG Jingsong lines. Both lines exhibited significantly enhanced immunity to BmNPV without significant changes in most of the commercially important traits. These results demonstrate that the construction of TG silkworm lines carrying a heritable immune defense system against BmNPV could be an effective strategy to enhance the resistance of industrial silkworm strains to the most devastating DNA virus. The research opened a window for genetic modification of the important strains from laboratory strains to industrial strains.

5'-Nucleotidase Plays a Key Role in Uric Acid Metabolism of Bombyx mori.

Uric acid (UA) is the end-product in the human purine metabolism pathway. The UA that accumulates in silkworm tissues is excreted as a nitrogen waste product. Here, we first validated that Bombyx mori has a homolog of the human gene that encodes the 5'-nucleotidase (5'N) involved in purine metabolism. The B. mori gene, Bm5'N, is located upstream of other genes involved in UA metabolism in the silkworm. Disruption of Bm5'N via the CRISPR/Cas9 system resulted in decreased UA levels in the silkworm epidermis and caused a translucent skin phenotype. When Bm5'N mutant silkworms were fed with the uric acid precursor inosine, the UA levels in the epidermis increased significantly. Furthermore, the metabolomic and transcriptomic analyses of Bm5'N mutants indicated that loss of the Bm5'N affected purine metabolism and the ABC transport pathway. Taken together, these results suggest that the UA pathway is conserved between the silkworm and humans and that the Bm5'N gene plays a crucial role in the uric acid metabolism of the silkworm. Thus, the silkworm may be a suitable model for the study of UA metabolism pathways relevant to human disease.

Correction: Gtsf1 is essential for proper female sex determination and transposon silencing in the silkworm, Bombyx mori.

[This corrects the article DOI: 10.1371/journal.pgen.1009194.].

Construction of Baculovirus-Inducible CRISPR/Cas9 Antiviral System Targeting BmNPV in Bombyx mori.

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.

Gtsf1 is essential for proper female sex determination and transposon silencing in the silkworm, Bombyx mori.

Sex determination pathways are astoundingly diverse in insects. For instance, the silk moth Bombyx mori uniquely use various components of the piRNA pathway to produce the Fem signal for specification of the female fate. In this study, we identified BmGTSF1 as a novel piRNA factor which participates in B. mori sex determination. We found that BmGtsf1 has a distinct expression pattern compared to Drosophila and mouse. CRISPR/Cas9 induced mutation in BmGtsf1 resulted in partial sex reversal in genotypically female animals by shifting expression of the downstream targets BmMasc and Bmdsx to the male pattern. As levels of Fem piRNAs were substantially reduced in female mutants, we concluded that BmGtsf1 plays a critical role in the biogenesis of the feminizing signal. We also demonstrated that BmGTSF1 physically interacted with BmSIWI, a protein previously reported to be involved in female sex determination, indicating BmGTSF1 function as the cofactor of BmSIWI. BmGtsf1 mutation resulted in piRNA pathway dysregulation, including piRNA biogenesis defects and transposon derepression, suggesting BmGtsf1 is also a piRNA factor in the silkworm. Furthermore, we found that BmGtsf1 mutation leads to gametogenesis defects in both male and female. Our data suggested that BmGtsf1 is a new component involved in the sex determination pathway in B. mori.

Identification of a germline-expression promoter for genome editing in Bombyx mori.

Identification of stage- and tissue-specific cis-regulatory elements will enable more precise genomic editing. In previous studies of the silkworm Bombyx mori, we identified and characterized several tissue- and sex-specific cis-regulatory elements using transgenic technology, including a female- and fat body-specific promoter, vitellogenin, testis-specific promoters, Radial spoke head 1 (BmR1) and beta-tubulin 4 (Bmß4). Here we report a cis-regulatory element specific for a somatic and germ cell-expressed promoter, nanos (Bmnos). We investigated activities of three truncated promoter sequences upstream of the transcriptional initiation site sequences of Bmnos in vitro (nos-0.6kb, nos-1kb and nos-2kb) and in vivo (nos-2kb). In BmN cultured cells, all three lengths drove expression of the gene encoding enhanced green fluorescence protein (EGFP), although nos-2kb had the highest fluorescence activity. In transgenic silkworms, nos-2kb drove EGFP expression at the early embryonic stage, and fluorescence was concentrated in the gonads at later embryonic stages. In addition, this cis-regulatory element was not sex differentiated. The fluorescence intensity gradually weakened following the larval developmental stage in the gonads and were broadly expressed in the whole body. The nos-2kb promoter drove the Cas9 system with efficiency comparable to that of the broad-spectrum strong IE1 promoter. These results indicate that Bmnos is an effective endogenous cis-regulatory element in the early embryo and in the gonad that can be used in applications involving the clustered, regularly interspaced, short palindromic repeats (CRISPR)/Cas9 system.