Activating Lobule VI PCTH+-Med Pathway in Cerebellum Blocks the Acquisition of Methamphetamine Conditioned Place Preference in Mice.

Cerebellum has been implicated in drug addiction; however, its underlying cellular populations and neuronal circuitry remain largely unknown. In the current study, we identified a neural pathway from tyrosine hydroxylase (TH)-positive Purkinje cells (PCTH+) in cerebellar lobule VI to calcium/calmodulin-dependent protein kinase II (CaMKII)-positive glutamatergic neurons in the medial cerebellar nucleus (MedCaMKII), forming the lobule VI PCTH+-MedCaMKII pathway in male mice. In naive male mice, inhibition of PCTH+ neurons activated Med neurons. During conditioned place preference (CPP) training, exposure to methamphetamine (METH) inhibited lobule VI PCTH+ neurons while excited MedCaMKII neurons in mice. Silencing MedCaMKII using a tetanus toxin light chain (tettox) suppressed the acquisition of METH CPP in mice but resulted in motor coordination deficits in naive mice. In contrast, activating lobule VI PCTH+ terminals within Med inhibited the activity of Med neurons and subsequently blocked the acquisition of METH CPP in mice without affecting motor coordination, locomotor activity, and sucrose reinforcements in naive mice. Our findings identified a novel lobule VI PCTH+-MedCaMKII pathway within the cerebellum and explored its role in mediating the acquisition of METH-preferred behaviors.

High-throughput and genome-scale targeted mutagenesis using CRISPR in a nonmodel multicellular organism, Bombyx mori.

Large-scale genetic mutant libraries are powerful approaches to interrogating genotype-phenotype correlations and identifying genes responsible for certain environmental stimuli, both of which are the central goal of life science study. We produced the first large-scale CRISPR-Cas9-induced library in a nonmodel multicellular organism, Bombyx mori We developed a piggyBac-delivered binary genome editing strategy, which can simultaneously meet the requirements of mixed microinjection, efficient multipurpose genetic operation, and preservation of growth-defect lines. We constructed a single-guide RNA (sgRNA) plasmid library containing 92,917 sgRNAs targeting promoters and exons of 14,645 protein-coding genes, established 1726 transgenic sgRNA lines following microinjection of 66,650 embryos, and generated 300 mutant lines with diverse phenotypic changes. Phenomic characterization of mutant lines identified a large set of genes responsible for visual phenotypic or economically valuable trait changes. Next, we performed pooled context-specific positive screens for tolerance to environmental pollutant cadmium exposure, and identified KWMTBOMO12902 as a strong candidate gene for breeding applications in sericulture industry. Collectively, our results provide a novel and versatile approach for functional B. mori genomics, as well as a powerful resource for identifying the potential of key candidate genes for improving various economic traits. This study also shows the effectiveness, practicality, and convenience of large-scale mutant libraries in other nonmodel organisms.

Synaptotagmin 1 Suppresses Colorectal Cancer Metastasis by Inhibiting ERK/MAPK Signaling-Mediated Tumor Cell Pseudopodial Formation and Migration.

Although synaptotagmin 1 (SYT1) has been identified participating in a variety of cancers, its role in colorectal cancer (CRC) remains an enigma. This study aimed to demonstrate the effect of SYT1 on CRC metastasis and the underlying mechanism. We first found that SYT1 expressions in CRC tissues were lower than in normal colorectal tissues from the CRC database and collected CRC patients. In addition to this, SYT1 expression was also lower in CRC cell lines than in the normal colorectal cell line. SYT1 expression was downregulated by TGF-ß (an EMT mediator) in CRC cell lines. In vitro, SYT1 overexpression repressed pseudopodial formation and reduced cell migration and invasion of CRC cells. SYT1 overexpression also suppressed CRC metastasis in tumor-bearing nude mice in vivo. Moreover, SYT1 overexpression promoted the dephosphorylation of ERK1/2 and downregulated the expressions of Slug and Vimentin, two proteins tightly associated with EMT in tumor metastasis. In conclusion, SYT1 expression is downregulated in CRC. Overexpression of SYT1 suppresses CRC cell migration, invasion, and metastasis by inhibiting ERK/MAPK signaling-mediated CRC cell pseudopodial formation. The study suggests that SYT1 is a suppressor of CRC and may have the potential to be a therapeutic target for CRC.

Histone crotonylation of peripheral blood mononuclear cells is a potential biomarker for diagnosis of colorectal cancer.

BACKGROUND: Blood-based tests have public appeal in screening cancers due to their minimally invasive nature, ability to integrate with other routine blood tests, and high compliance. This study aimed to investigate whether certain epigenetic modulation of peripheral blood mononuclear cells (PBMCs) could be a biomarker of colorectal cancer (CRC). RESULTS: Western blotting of histones in the PBMCs from 40 colorectal cancer patients and 40 healthy controls was performed to identify the crotonylation sites of proteins. The correlation of crotonylation with tumor staging and diagnostic efficacy were analyzed. Crotonylation of H2BK12 (H2BK12cr) was identified significantly upregulated in the PBMCs of CRC patients compared to healthy controls, and were closely related to distant metastasis (P = 0.0478) and late TNM stage (P = 0.0201). Receiver operator characteristic curve (ROC) analysis demonstrated that the area under curve (AUC) of H2BK12cr was 0.8488, the sensitivity was 70%, and the specificity was 92.5%. The H2BK12cr parameter significantly increased the diagnostic effectiveness of CRC compared with the commercial carcinoembryonic antigen assays. CONCLUSIONS: The H2BK12cr level in PBMCs of CRC patients has a potential to be a biomarker for distinguishing CRC patients from healthy controls with the advantages of easy operation and high diagnostic efficacy.

mPFC DUSP1 mediates adolescent cocaine exposure-induced higher sensitivity to drug in adulthood.

Adolescent cocaine abuse increases the risk for developing addiction in later life, but the underlying molecular mechanism remains poorly understood. Here, we establish adolescent cocaine-exposed (ACE) male mouse models. A subthreshold dose of cocaine (sdC) treatment, insufficient to produce conditioned place preference (CPP) in adolescent mice, induces CPP in ACE mice during adulthood, along with more activated CaMKII-positive neurons, higher dual specificity protein kinase phosphatase-1 (Dusp1) mRNA, lower DUSP1 activity, and lower DUSP1 expression in CaMKII-positive neurons in the medial prefrontal cortex (mPFC). Overexpressing DUSP1 in CaMKII-positive neurons suppresses neuron activity and blocks sdC-induced CPP in ACE mice during adulthood. On the contrary, depleting DUSP1 in CaMKII-positive neurons activates more neurons and further enhances sdC-induced behavior in ACE mice during adulthood. Also, ERK1/2 might be a downstream signal of DUSP1 in the process. Our findings reveal a role of mPFC DUSP1 in ACE-induced higher sensitivity to the drug in adult mice. DUSP1 might be a potential pharmacological target to predict or treat the susceptibility to addictive drugs caused by adolescent substance use.

Specific enterotype of gut microbiota predicted clinical effect of methotrexate in patients with rheumatoid arthritis.

OBJECTIVE: The most used drug for the treatment of rheumatoid arthritis (RA) remains methotrexate (MTX). Unfortunately, up to 50% of patients do not achieve a clinically adequate outcome. Here we study whether the gut microbiota patterns can aid in the prediction of MTX efficacy for RA. METHOD: To dissect gut microbiome profiles of RA patients (n = 145), 16S rRNA gene sequencing was performed. Dirichlet multinomial mixture (DMM) clustering was used to identify enterotypes at genus level. The relationships between enterotypes and clinical measures (such as lymphocyte subsets and cytokines detected by flow cytometry) were explored. Then, enterotype stability was evaluated by the stratification of the RA patient cohort (n = 66) in Shanghai, China, using the same method. Finally, the enterotype-based gut microbial human index classifier was applied to another independent RA patient cohort (n = 27) to identify the factors associated with MTX clinical response. RESULTS: Our analysis revealed that the RA patients always displayed two different dysbiotic microbiota patterns: RA E1 comprised predominantly Prevotella and RA E2 comprised predominantly Bacteroides. Among all of the lymphocyte subsets and cytokines, only the number of CD8+ T cells showed a significant difference between RA E1 and RA E2. These results were validated in the RA patient cohort in Shanghai, China. Significant associations of RA E1 with clinical response to subsequent MTX treatment were confirmed by another independent RA patient cohort. CONCLUSION: Together, the enterotype-based gut microbial human index (EGMI) classifier was useful to precisely and effectively identify enterotypes of individual RA patients, which could effectively evaluate MTX clinical responses.

Low-dose IL-2 improved clinical symptoms by restoring reduced regulatory T cells in patients with refractory rheumatoid arthritis: A randomized controlled trial.

Background: Regulatory T cells (Tregs) have been found to play crucial roles in immune tolerance. However, the status of Tregs in refractory rheumatoid arthritis (RA) is still unclear. Moreover, low-dose interleukin-2 (IL-2) has been reported to selectively promote the expansion of Tregs. This study investigated the status of CD4+ Tregs and low-dose IL-2 therapy in patients with refractory RA. Methods: The absolute number of CD4+CD25+FOXP3+ Treg (CD4 Treg), CD4+IL17+ T (Th17), and other subsets in peripheral blood (PB) from 41 patients with refractory RA and 40 healthy donors was characterized by flow cytometry combined with an internal microsphere counting standard. Twenty-six patients with refractory RA were treated with daily subcutaneous injections of 0.5 million IU of human IL-2 for five consecutive days. Then, its effects on CD4 Treg and Th17 cells in PB were analyzed. Results: A decrease in the absolute number of PB CD4 Tregs rather than the increase in the number of Th17 was found to contribute to an imbalance between Th17 and CD4 Tregs in these patients, suggesting an essential role of CD4 Tregs in sustained high disease activity. Low-dose IL-2 selectively increased the number of CD4 Tregs and rebalanced the ratio of Th17 and CD4 Tregs, leading to increased clinical symptom remission without the observed side effects. Conclusions: An absolute decrease of PB CD4 Tregs in patients with refractory RA was associated with continuing disease activation but not the increase of Th17 cells. Low-dose IL-2, a potential therapeutic candidate, restored decreased CD4 Tregs and promoted the rapid remission of patients with refractory RA without overtreatment and the observed side effects. Clinical trial registration: http://www.chictr.org.cn/showproj.aspx?proj=13909, identifier ChiCTR-INR-16009546.

Genome-wide CRISPR-Cas9 screening in Bombyx mori reveals the toxicological mechanisms of environmental pollutants, fluoride and cadmium.

Fluoride and cadmium, two typical environmental pollutants, have been extensively existed in the ecosystem and severely injured various organisms including humans. To explore the toxicological properties and the toxicological mechanism of fluoride and cadmium in silkworm, we perform a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) -based functional genomic screen, which can directly measure the genetic requirement of genes in response to the pollutants. Our screen identifies 751 NaF-resistance genes, 753 NaF-sensitive genes, 757 CdCl2-resistance genes, and 725 CdCl2-sensitive genes. The top-ranked resistant genes are experimentally verified and the results show that their loss conferred resistance to fluoride or cadmium. Functional analysis of the resistant- and sensitive-genes demonstrates enrichment of multiple signaling pathways, among which the MAPK signaling pathway and DNA damage and repair are both required for fluoride- or cadmium-induced cell death, whereas the Toll and Imd signaling pathway and Autophagy are fluoride- or cadmium-specific. Moreover, we confirm that these pathways are truly involved in the toxicological mechanism in both cultured cells and individual tissues. Our results supply potential targets for rescuing the biohazards of fluoride and cadmium in silkworm, and reveal the feasible toxicological mechanism, which highlights the role of functional genomic screens in elucidating the toxicity mechanisms of environmental pollutants.

The novel insight into the outcomes of CRISPR/Cas9 editing intra- and inter-species.

The CRISPR/Cas (clustered regularly interspaced short palindromic repeat technology/CRISPR-associated protein) is a widely used and powerful research tool in biosciences and a promising therapeutic agent for treating genetic diseases. Mutations induced by Cas9 are generally considered stochastic and unpredictable, thus hindering its applications where precise genetic alternations are required. Here, through deep sequencing and analysis of genome editing outcomes of multiple sites in four distinct species, we found that Cas9-induced mutations are coincident in mutation types but are significantly different in indel patterns among species. In human and mouse cells, indels were almost evenly distributed at both ends of the cleavage sites. However, the indels mainly appeared at the upstream of cleavage sites in Bombyx mori, while they predominantly occurred downstream of the cleavage sites in the zebrafish Danio rerio. We also found that within a species, indel patterns are sequence dependent, wherein deletions between two adjacent micro-homology sequences were the most frequently observed mutations in the repair spectrum. These results suggested the species differences in DNA repair processes during Cas9-induced gene editing, and the important role of sequence structure at the target site in predicting the gene editing outcome.

Genome-wide CRISPR screening reveals genes essential for cell viability and resistance to abiotic and biotic stresses in Bombyx mori.

High-throughput genetic screens are powerful methods to interrogate gene function on a genome-wide scale and identify genes responsible to certain stresses. Here, we developed a piggyBac strategy to deliver pooled sgRNA libraries stably into cell lines. We used this strategy to conduct a screen based on genome-wide clustered regularly interspaced short palindromic repeat technology (CRISPR)-Cas9 in Bombyx mori cells. We first constructed a single guide RNA (sgRNA) library containing 94,000 sgRNAs, which targeted 16,571 protein-coding genes. We then generated knockout collections in BmE cells using the piggyBac transposon. We identified 1006 genes that are essential for cell viability under normal growth conditions. Of the identified genes, 82.4% (829 genes) were homologous to essential genes in seven animal species. We also identified 838 genes whose loss facilitated cell growth. Next, we performed context-specific positive screens for resistance to biotic or nonbiotic stresses using temperature and baculovirus separately, which identified several key genes and pathways from each screen. Collectively, our results provide a novel and versatile platform for functional annotations of B. mori genomes and deciphering key genes responsible for various conditions. This study also shows the effectiveness, practicality, and convenience of genome-wide CRISPR screens in nonmodel organisms.

High-Throughput Screening Identifies Two Novel Small Molecule Enhancers of Recombinant Protein Expression.

As a primary strategy for production of biological drugs, recombinant proteins produced by transient transfection of mammalian cells are essential for both basic research and industrial production. Here, we established a high-throughput screening platform for improving the expression levels of recombinant proteins. In total, 10,011 small molecule compounds were screened through our platform. After two rounds of screening, we identified two compounds, Apicidin and M-344, that significantly enhanced recombinant protein expression. Both of the selected compounds were histone deacetylase inhibitors, suggesting that the two small molecules increased the expression levels of recombinant proteins by promoting histone acetylation. Moreover, both molecules showed low cytotoxicity. Therefore, our findings suggest that these small molecules may have wide applications in the future.

Deep Insight into the Transcriptome of the Single Silk Gland of Bombyx mori.

The silk gland synthesizes and secretes a large amount of protein and stores liquid silk protein at an extremely high concentration. Interestingly, silk proteins and serine protease inhibitors are orderly arranged in the silk gland lumen and cocoon shells. Silk fiber formation and the spinning mechanism have not been fully elucidated. Therefore, we conducted a comparative transcriptome analysis of seven segments of the single silk gland to characterize internal changes in the silk gland during the 5th instar of mature larvae. In total, 3121 differentially expressed genes were identified in the seven segments. Genes highly expressed in the middle silk gland (MSG) were mainly involved in unsaturated fatty acid biosynthesis, fatty acid metabolism, apoptosis-fly, and lysosome pathways, whereas genes highly expressed in the posterior silk gland (PSG) were mainly involved in ribosome, proteasome, citrate cycle, and glycolysis/gluconeogenesis pathways. Thus, the MSG and PSG differ greatly in energy source use and function. Further, 773 gradually upregulated genes (from PSG to MSG) were involved in energy metabolism, silk protein synthesis, and secretion, suggesting that these genes play an important role in silk fiber formation. Our findings provide insights into the mechanism of silk protein synthesis and transport and silk fiber formation.

Programmable targeted epigenetic editing using CRISPR system in Bombyx mori.

DNA methylation has been proven to play roles in regulating gene expression, cell fate, disease determination, and chromatin architecture organization in mammals and plants, and is a significant component of epigenetic modification. Compared to mammals or plants, the status and function of DNA methylation are poorly understood in insects, which is partially due to the lack of efficient manipulation tools. In this study, we show that fusion protein of catalytically inactive Cas9 (dCas9) with TET1 can efficiently demethylate genomic DNA of silkworm Bombyx mori, in a programmable target region specific manner. We first developed an all-in-one vector to maximize the targeting efficiency of dCas9-TET1. Then we selected 3 endogenous genes that were previously found to harbor methylated DNA, and designed gRNAs within the methylated region. Co-transfection of dCas9-TET1 and gRNA successfully erased methylation marks near the targeting region, with efficiencies from about 17.50% to 40.00%. Furthermore, targeted demethylation on gene body resulted in increased mRNA transcription level. Unlike the previously widely used decitabine, a methylation inhibitor, dCas9-TET1 is more effective and specific, and has no unwanted impact on whole-genome methylation. DCas9-TET1 provides a powerful tool for investigating the functional significance of DNA methylation in a locus-specific manner, and for exploring the unknown links between methylation and development in insects.

Programmable activation of Bombyx gene expression using CRISPR/dCas9 fusion systems.

The recently developed clustered regularly interspaced short palindromic repeats (CRISPR)-based techniques have made it possible to reprogram target gene expression without cloning complementary DNA or disturbing genomic sequence in mammalian cells and several multicellular organisms. We previously showed that CRISPR-associated protein 9 (Cas9) and CRISPR from Prevotella and Francisella 1 (Cpf1) could induce target mutations, deletions, inversions, and duplications both singly and multiplex in silkworm, Bombyx mori. However, it remains unknown whether the CRISPR activation (CRISPRa) system can be used in B. mori. In this study, we investigated the CRISPRa system, in which a nuclease dead Streptococcus pyogenes Cas9 (SpCas9) is fused to two transcription activation domains, including VP64 (a tetramer of the herpes simplex VP16 transcriptional activator domain), and VPR (a tripartite activator, composed of VP64, p65, and Rta). The results showed that both dCas9-VP64 and dCas9-VPR systems could be used in B. mori cells, of which the latter showed significantly higher activity. The dCas9-VPR system showed considerable activity on all five tested target genes, and further analysis revealed that the up-regulation of genes was negatively correlated to their basal expression level. We also observed that this system could be used to upregulate a range of target genes. Taken together, our findings demonstrate that CRISPRa can be a powerful tool to study gene functions in B. mori and perhaps other non-drosophila insects.

Programmable Single and Multiplex Base-Editing in Bombyx mori Using RNA-Guided Cytidine Deaminases.

Genome editing using standard tools (ZFN, TALEN, and CRISPR/Cas9) rely on double strand breaks to edit the genome. A series of new CRISPR tools that convert cytidine to thymine (C to T) without the requirement for DNA double-strand breaks was developed recently and quickly applied in a variety of organisms. Here, we demonstrate that CRISPR/Cas9-dependent base editor (BE3) converts C to T with a high frequency in the invertebrate Bombyx mori silkworm. Using BE3 as a knock-out tool, we inactivated exogenous and endogenous genes through base-editing-induced nonsense mutations with an efficiency of up to 66.2%. Furthermore, genome-scale analysis showed that 96.5% of B. mori genes have one or more targetable sites that can be edited by BE3 for inactivation, with a median of 11 sites per gene. The editing window of BE3 reached up to 13 bases (from C1 to C13 in the range of gRNA) in B. mori Notably, up to 14 bases were substituted simultaneously in a single DNA molecule, with a low indel frequency of 0.6%, when 32 gRNAs were co-transfected. Collectively, our data show for the first time that RNA-guided cytidine deaminases are capable of programmable single and multiplex base editing in an invertebrate model.

Tissue-specific genome editing of laminA/C in the posterior silk glands of Bombyx mori.

The RNA-guided CRISPR/Cas9 system has been shown to be a powerful tool for genome editing in various organisms. A comprehensive toolbox for multiplex genome editing has been developed for the silkworm, Bombyx mori, a lepidopteran model insect of economic importance. However, as previous methods mainly relied on delivery of transient Cas9/guide RNA (gRNA), they could not be used in loss-of-function studies of essential genes. Here, we report a simple and versatile tissue-specific genome editing strategy. We perform a proof-of-principle demonstration by establishing and crossing two transgenic B. mori lines, one expressing Cas9 protein in the posterior silk glands (PSGs) and the other constitutively expressing BmlaminA/C (BmLMN) gRNA. All BmLMN alleles in the PSG cells were edited precisely at the target genome region, resulting in diverse mutations. mRNA expression of BmLMN was reduced by up to 75%, and only very low levels of BmLaminA/C protein were detected. Knockout of BmLMN produced obvious defects in gland cell development and cocoon production. In this study, we developed an efficient strategy for spatially controlled genome editing, providing unprecedented opportunities for investigating the function of essential/lethal genes in B. mori, with potential application for other insects.

Increasing the yield of middle silk gland expression system through transgenic knock-down of endogenous sericin-1.

Various genetically modified bioreactor systems have been developed to meet the increasing demands of recombinant proteins. Silk gland of Bombyx mori holds great potential to be a cost-effective bioreactor for commercial-scale production of recombinant proteins. However, the actual yields of proteins obtained from the current silk gland expression systems are too low for the proteins to be dissolved and purified in a large scale. Here, we proposed a strategy that reducing endogenous sericin proteins would increase the expression yield of foreign proteins. Using transgenic RNA interference, we successfully reduced the expression of BmSer1 to 50%. A total 26 transgenic lines expressing Discosoma sp. red fluorescent protein (DsRed) in the middle silk gland (MSG) under the control of BmSer1 promoter were established to analyze the expression of recombinant. qRT-PCR and western blotting showed that in BmSer1 knock-down lines, the expression of DsRed had significantly increased both at mRNA and protein levels. We did an additional analysis of DsRed/BmSer1 distribution in cocoon and effect of DsRed protein accumulation on the silk fiber formation process. This study describes not only a novel method to enhance recombinant protein expression in MSG bioreactor, but also a strategy to optimize other bioreactor systems.

An integrated CRISPR Bombyx mori genome editing system with improved efficiency and expanded target sites.

Genome editing enabled unprecedented new opportunities for targeted genomic engineering of a wide variety of organisms ranging from microbes, plants, animals and even human embryos. The serial establishing and rapid applications of genome editing tools significantly accelerated Bombyx mori (B. mori) research during the past years. However, the only CRISPR system in B. mori was the commonly used SpCas9, which only recognize target sites containing NGG PAM sequence. In the present study, we first improve the efficiency of our previous established SpCas9 system by 3.5 folds. The improved high efficiency was also observed at several loci in both BmNs cells and B. mori embryos. Then to expand the target sites, we showed that two newly discovered CRISPR system, SaCas9 and AsCpf1, could also induce highly efficient site-specific genome editing in BmNs cells, and constructed an integrated CRISPR system. Genome-wide analysis of targetable sites was further conducted and showed that the integrated system cover 69,144,399 sites in B. mori genome, and one site could be found in every 6.5 bp. The efficiency and resolution of this CRISPR platform will probably accelerate both fundamental researches and applicable studies in B. mori, and perhaps other insects.

Genome editing of BmFib-H gene provides an empty Bombyx mori silk gland for a highly efficient bioreactor.

Evolution has produced some remarkable creatures, of which silk gland is a fascinating organ that exists in a variety of insects and almost half of the 34,000 spider species. The impressive ability to secrete huge amount of pure silk protein, and to store proteins at an extremely high concentration (up to 25%) make the silk gland of Bombyx mori hold great promise to be a cost-effective platform for production of recombinant proteins. However, the extremely low production yields of the numerous reported expression systems greatly hindered the exploration and application of silk gland bioreactors. Using customized zinc finger nucleases (ZFN), we successfully performed genome editing of Bmfib-H gene, which encodes the largest and most abundant silk protein, in B. mori with efficiency higher than any previously reported. The resulted Bmfib-H knocked-out B. mori showed a smaller and empty silk gland, abnormally developed posterior silk gland cells, an extremely thin cocoon that contain only sericin proteins, and a slightly heavier pupae. We also showed that removal of endogenous Bmfib-H protein could significantly increase the expression level of exogenous protein. Furthermore, we demonstrated that the bioreactor is suitable for large scale production of protein-based materials.

Highly efficient multiplex targeted mutagenesis and genomic structure variation in Bombyx mori cells using CRISPR/Cas9.

Bombyx mori is an economically important insect and a model organism for studying lepidopteran and arthropod biology. Using a highly efficient CRISPR/Cas9 system, we showed that this system could mediate highly efficient targeted genome editing of a single gene locus, large chromosomal deletion or inversion, and also multiplex genome editing of 6 genes simultaneously in BmNs cell line derived from B. mori. The simplicity and high efficiency of our system provide unprecedented possibilities for researchers to implement precise and sophisticated manipulation of a chosen B. mori gene in BmNs cells easily in a limited time course, and perhaps new opportunities for functional genomics of B. mori and other lepidopteran insects.