High-Throughput Screening of PAM-Flexible Cas9 Variants for Expanded Genome Editing in the Silkworm (Bombyx mori).

Genome editing provides novel opportunities for the precise genome engineering of diverse organisms. Significant progress has been made in the development of genome-editing tools for Bombyx mori (B. mori) in recent years. Among these, CRISPR/Cas9, which is currently the most commonly used system in lepidopteran insects, recognizes NGG protospacer adjacent motif (PAM) sequences within the target locus. However, Cas9 lacks the ability to target all gene loci in B. mori, indicating the need for Cas9 variants with a larger editing range. In this study, we developed a high-throughput screening platform to validate Cas9 variants at all possible recognizable and editable PAM sites for target sequences in B. mori. This platform enabled us to identify PAM sites that can be recognized by both xCas9 3.7 and SpCas9-NG variants in B. mori and to assess their editing efficiency. Cas9 shows PAM sites every 13 base pairs in the genome, whereas xCas9 3.7 and SpCas9-NG have an average distance of 3.4 and 3.6 base pairs, respectively, between two specific targeting sites. Combining the two Cas9 variants could significantly expand the targeting range of the genome, accelerate research on the B. mori genome, and extend the high-throughput rapid screening platform to other insects, particularly those lacking suitable NGG PAM sequences.

A review on complete silk gene sequencing and de novo assembly of artificial silk.

Silk, especially spider and insect silk, is a highly versatile biomaterial with potential applications in biomedicine, materials science, and biomimetic engineering. The primary structure of silk proteins is the basis for the mechanical properties of silk fibers. Biotechnologies such as single-molecule sequencing have facilitated an increasing number of reports on new silk genes and assembled silk proteins. Therefore, this review aims to provide a comprehensive overview of the recent advances in representative spider and insect silk proteins, focusing on identification methods, sequence characteristics, and de novo design and assembly. The review discusses three identification methods for silk genes: polymerase chain reaction (PCR)-based sequencing, PCR-free cloning and sequencing, and whole-genome sequencing. Moreover, it reveals the main spider and insect silk proteins and their sequences. Subsequent de novo assembly of artificial silk is covered and future research directions in the field of silk proteins, including new silk genes, customizable artificial silk, and the expansion of silk production and applications are discussed. This review provides a basis for the genetic aspects of silk production and the potential applications of artificial silk in material science and biomedical engineering.

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.

Genome-wide CRISPR screening reveals key genes and pathways associated with 20-hydroxyecdysone signal transduction in the silkworm (Bombyx mori).

Metamorphosis is a complex developmental process involving multiple pathways and a large number of genes that are regulated by juvenile hormone (JH) and 20-hydroxyecdysone (20E). Despite important progress in understanding various aspects of silkworm biology, the hormone signaling pathway in the silkworm remains poorly understood. Genome-wide screening using clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated protein 9 (Cas9)-based libraries has recently emerged as a novel method for analyzing genome function, enabling further research into essential genes, drug targets, and virus-host interaction. Previously, we constructed a genome-wide CRISPR/Cas9-based library of the silkworm (Bombyx mori) and successfully revealed the genes involved in biotic or abiotic stress factor responses. In this study, we used our silkworm CRISPR library and large-scale genome-wide screening to analyze the key genes in the silkworm 20E signaling pathway and their mechanisms of action. Functional annotation showed that 20E regulates key proteins in processes that mainly occur in the cytoplasm and nucleus. Pathway enrichment analysis showed that 20E can activate phosphorylation and may affect innate immunity, interfere with intracellular nutrition and energy metabolism, and eventually cause cell apoptosis. The screening results were experimentally validated by generating cells with knockout alleles of the relevant genes, which had increased tolerance to 20E. Our findings provide a panoramic overview of signaling in response to 20E in the silkworm, underscoring the utility of genome-wide CRISPR mutant libraries in deciphering hormone signaling pathways and the mechanisms that regulate metamorphosis in insects.

Effect of eIF6 on the development of silk glands and silk protein synthesis of the silkworm, Bombyx mori.

The silkworm is a lepidopteran domesticated from the wild silkworm, mostly valued for its efficient synthesis of silk protein. This species' ability to spin silk has supported the 5500-year-old silk industry and the globally known "Silk Road", making the transformation of mulberry leaves into silk of great concern. Therefore, research on the silk-related genes of silkworms and their regulatory mechanisms has attracted increasing attention. Previous studies have revealed that domestic silk gland cells are endoreduplication cells, and their high-copy genome and special chromatin conformation provide conditions for the high expression of silk proteins. In this study, we systematically investigate the expression pattern of eukaryotic initiation factors (eIFs) and identified the eIF6 as a eukaryotic translation initiation factor involved in the synthesis of silk proteins. We generated an eIF6 gene deletion mutant strain of silkworm using the CRISPR/Cas9 system and investigated the function of eIF6 in silk gland development and silk protein synthesis. The results showed that deletion of eIF6 inhibited the individual development of silkworm larvae, inhibited the development of silk glands, and significantly reduced the cocoon layer ratio. Therefore, we elucidated the function of eIF6 in the development of silk glands and the synthesis of silk proteins, which is important for further elucidation of the developmental process of silk glands and the mechanism underlying the ultra-high expression of silk proteins.

FibH Gene Complete Sequences (FibHome) Revealed Silkworm Pedigree.

The highly repetitive and variable fibroin heavy chain (FibH) gene can be used as a silkworm identification; however, only a few complete FibH sequences are known. In this study, we extracted and examined 264 FibH gene complete sequences (FibHome) from a high-resolution silkworm pan-genome. The average FibH lengths of the wild silkworm, local, and improved strains were 19,698 bp, 16,427 bp, and 15,795 bp, respectively. All FibH sequences had a conserved 5' and 3' terminal non-repetitive (5' and 3' TNR, 99.74% and 99.99% identity, respectively) sequence and a variable repetitive core (RC). The RCs differed greatly, but they all shared the same motif. During domestication or breeding, the FibH gene mutated with hexanucleotide (GGTGCT) as the core unit. Numerous variations existed that were not unique to wild and domesticated silkworms. However, the transcriptional factor binding sites, such as fibroin modulator-binding protein, were highly conserved and had 100% identity in the FibH gene's intron and upstream sequences. The local and improved strains with the same FibH gene were divided into four families using this gene as a marker. Family I contained a maximum of 62 strains with the optional FibH (Opti-FibH, 15,960 bp) gene. This study provides new insights into FibH variations and silkworm breeding.

Genetically engineered Blue silkworm capable of synthesizing natural blue pigment.

Synthetic biology is an eco-friendly and sustainable approach for the production of compounds, particularly used when the production processes involve toxic reagents. In this study, we used the silk gland of silkworm to produce indigoidine, a valuable natural blue pigment that cannot be synthesized naturally in animals. We genetically engineered these silkworms by integrating the indigoidine synthetase (idgS) gene from S. lavendulae and the PPTase (Sfp) gene from B. subtilis into the silkworm genome. In the resulting Blue silkworm, indigoidine was detected at a high level in the posterior silk gland (PSG), spanning all developmental stages from larvae to adults, without affecting silkworm growth or development. This synthesized indigoidine was secreted from the silk gland and subsequently stored in the fat body, with only a small fraction being excreted by the Malpighian tubule. Metabolomic analysis revealed that Blue silkworm efficiently synthesized indigoidine by upregulating l-glutamine, the precursor of indigoidine, and succinate, which is related to energy metabolism in the PSG. This study represents the first synthesis of indigoidine in an animal and therefore opens a new avenue for the biosynthesis of natural blue pigments and other valuable small molecules.

A molecular atlas reveals the tri-sectional spinning mechanism of spider dragline silk.

The process of natural silk production in the spider major ampullate (Ma) gland endows dragline silk with extraordinary mechanical properties and the potential for biomimetic applications. However, the precise genetic roles of the Ma gland during this process remain unknown. Here, we performed a systematic molecular atlas of dragline silk production through a high-quality genome assembly for the golden orb-weaving spider Trichonephila clavata and a multiomics approach to defining the Ma gland tri-sectional architecture: Tail, Sac, and Duct. We uncovered a hierarchical biosynthesis of spidroins, organic acids, lipids, and chitin in the sectionalized Ma gland dedicated to fine silk constitution. The ordered secretion of spidroins was achieved by the synergetic regulation of epigenetic and ceRNA signatures for genomic group-distributed spidroin genes. Single-cellular and spatial RNA profiling identified ten cell types with partitioned functional division determining the tri-sectional organization of the Ma gland. Convergence analysis and genetic manipulation further validated that this tri-sectional architecture of the silk gland was analogous across Arthropoda and inextricably linked with silk formation. Collectively, our study provides multidimensional data that significantly expand the knowledge of spider dragline silk generation and ultimately benefit innovation in spider-inspired fibers.

Combined CRISPR toolkits reveal the domestication landscape and function of the ultra-long and highly repetitive silk genes.

Highly repetitive sequences play a major structural and function role in the genome. In the present study, we developed Cas9-assisted cloning and SMRT sequencing of long repetitive sequences (CACS) to sequence and manipulate highly repetitive genes from eukaryotic genomes. CACS combined Cas9-mediated cleavage of a target segment from an intact genome, Gibson assembly cloning, and PacBio SMRT sequencing. Applying CACS, we directly cloned and sequenced the complete sequences of fibroin heavy chain (FibH) genes from 17 domesticated (Bombyx mori) and 7 wild (Bombyx mandarina) silkworms. Our analysis revealed the unique fine structure organization, genetic variations, and domestication dynamics of FibH. We also demonstrated that the length of the repetitive regions determined the mechanical properties of silk fiber, which was further confirmed by Cas9 editing of FibH. CACS is a simple, robust, and efficient approach, providing affordable accessibility to highly repetitive regions of a genome. STATEMENT OF SIGNIFICANCE: Silkworm silk is the earliest and most widely used animal fiber, and its excellent performance mainly depends on the fibroin heavy chain (FibH) protein. The FibH gene is the main breakthrough in understanding the formation mechanism and improvement of silk fiber. In the study, we developed a CACS method for characterizing the fine structure and domestication landscape of 24 silkworm FibH genes. We used CRISPR/Cas9 to edit the repetitive sequence of FibH genes, revealing the relationship between FibH genes and mechanical properties of silkworm silk. Our study is helpful in modifying silk genes to manipulate other valuable highly repetitive sequences, and provides insight for silkworm breeding.

Pupal Diapause Termination and Transcriptional Response of Antheraea pernyi (Lepidoptera: Saturniidae) Triggered by 20-Hydroxyecdysone.

The pupal diapause of univoltine Antheraea pernyi hampers sericultural and biotechnological applications, which requires a high eclosion incidence after artificial diapause termination to ensure production of enough eggs. The effect of pupal diapause termination using 20-hydroxyecdysone (20E) on the eclosion incidence has not been well-documented in A. pernyi. Here, the dosage of injected 20E was optimized to efficiently terminate pupal diapause of A. pernyi, showing that inappropriate dosage of 20E can cause pupal lethality and a low eclosion incidence. The optimal ratio of 20E to 1-month-old pupae was determined as 6 µg/g. Morphological changes showed visible tissue dissociation at 3 days post-injection (dpi) and eye pigmentation at 5 dpi. Comprehensive transcriptome analysis identified 1,355/1,592, 494/203, 584/297, and 1,238/1,404 upregulated and downregulated genes at 1, 3, 6, and 9 dpi, respectively. The 117 genes enriched in the information processing pathways of "signal transduction" and "signaling molecules and interaction" were upregulated at 1 and 3 dpi, including the genes involved in FOXO signaling pathway. One chitinase, three trehalase, and five cathepsin genes related to energy metabolism and tissue dissociation showed high expression levels at the early stage, which were different from the upregulated expression of four other chitinase genes at the later stage. Simultaneously, the expression of several genes involved in molting hormone biosynthesis was also activated between 1 and 3 dpi. qRT-PCR further verified the expression patterns of two ecdysone receptor genes (EcRB1 and USP) and four downstream response genes (E93, Br-C, ßFTZ-F1, and cathepsin L) at the pupal and pharate stages, respectively. Taken together, these genes serve as a resource for unraveling the mechanism underlying pupal-adult transition; these findings facilitate rearing of larvae more than once a year and biotechnological development through efficient termination of pupal diapause in A. pernyi in approximately half a month.

Genome-Wide Identification of M14 Family Metal Carboxypeptidases in Antheraea pernyi (Lepidoptera: Saturniidae).

The M14 family metal carboxypeptidase genes play an important role in digestion and pathogenic infections in the gut of insects. However, the roles of these genes in Antheraea pernyi (Guérin-Méneville, 1855) remain to be analyzed. In the present study, we cloned a highly expressed M14 metal carboxypeptidase gene (ApMCP1) found in the gut and discovered that it contained a 1,194 bp open reading frame encoding a 397-amino acid protein with a predicted molecular weight of 45 kDa. Furthermore, 14 members of the M14 family metal carboxypeptidases (ApMCP1-ApMCP14) were identified in the A. pernyi genome, with typical Zn_pept domains and two Zn-anchoring motifs, and were further classified into M14A, M14B, and M14D subfamilies. Expression analysis indicated that ApMCP1 and ApMCP9 were mainly expressed in the gut. Additionally, we observed that ApMCP1 and ApMCP9 displayed opposite expression patterns after starvation, highlighting their functional divergence during digestion. Following natural infection with baculovirus NPV, their expression was significantly upregulated in the gut of A. pernyi. Our results suggest that the M14 family metal carboxypeptidase genes are conservatively digestive enzymes and evolutionarily involved in exogenous pathogenic infections.

Comparison of Long-Read Methods for Sequencing and Assembly of Lepidopteran Pest Genomes.

Lepidopteran species are mostly pests, causing serious annual economic losses. High-quality genome sequencing and assembly uncover the genetic foundation of pest occurrence and provide guidance for pest control measures. Long-read sequencing technology and assembly algorithm advances have improved the ability to timeously produce high-quality genomes. Lepidoptera includes a wide variety of insects with high genetic diversity and heterozygosity. Therefore, the selection of an appropriate sequencing and assembly strategy to obtain high-quality genomic information is urgently needed. This research used silkworm as a model to test genome sequencing and assembly through high-coverage datasets by de novo assemblies. We report the first nearly complete telomere-to-telomere reference genome of silkworm Bombyx mori (P50T strain) produced by Pacific Biosciences (PacBio) HiFi sequencing, and highly contiguous and complete genome assemblies of two other silkworm strains by Oxford Nanopore Technologies (ONT) or PacBio continuous long-reads (CLR) that were unrepresented in the public database. Assembly quality was evaluated by use of BUSCO, Inspector, and EagleC. It is necessary to choose an appropriate assembler for draft genome construction, especially for low-depth datasets. For PacBio CLR and ONT sequencing, NextDenovo is superior. For PacBio HiFi sequencing, hifiasm is better. Quality assessment is essential for genome assembly and can provide better and more accurate results. For chromosome-level high-quality genome construction, we recommend using 3D-DNA with EagleC evaluation. Our study references how to obtain and evaluate high-quality genome assemblies, and is a resource for biological control, comparative genomics, and evolutionary studies of Lepidopteran pests and related species.

Genetic Code Expansion System for Tight Control of Gene Expression in Bombyx mori Cell Lines.

Inducible gene expression systems are important tools for studying gene function and to control protein synthesis. With the completion of the detailed map of the silkworm (Bombyx mori) genome, the study of Bombyx mori has entered the post-genome era. While the functions of many genes have been described in detail, many coding genes remain unidentified. Except for the available tetracycline induction system, there is currently a dearth of other effective induction systems for B. mori. A genetic code expansion system can be used for protein labeling and to regulate gene expression. Here, we have established a genetic code expansion system for B. mori based on the well-researched tRNAPyl/PylRS pair from Methanosarcina mazei. We used H-Lys(Boc)-OH, which is a lysine derivative to efficiently and tightly control the expression of the reporter gene DsRed[TAG]EGFP (D[TAG]G), which encoded a H-Lys(Boc)-OH-bearing protein fused with DsRed and EGFP (here regarded as D[Boc]G) in B. mori cell lines BmE and BmNs. In D[TAG]G, the amber stop codon is recognized as the orthogonal tRNAPyl. Successful application of genetic code expansion system in silkworm cell lines will support the research into the function of silkworm genes and paves the way for the identification of new genes and protein markers in silkworm.

Precise Characterization of Bombyx mori Fibroin Heavy Chain Gene Using Cpf1-Based Enrichment and Oxford Nanopore Technologies.

To study the evolution of gene function and a species, it is essential to characterize the tandem repetitive sequences distributed across the genome. Cas9-based enrichment combined with nanopore sequencing is an important technique for targeting repetitive sequences. Cpf1 has low molecular weight, low off-target efficiency, and the same editing efficiency as Cas9. There are numerous studies on enrichment sequencing using Cas9 combined with nanopore, while there are only a few studies on the enrichment sequencing of long and highly repetitive genes using Cpf1. We developed Cpf1-based enrichment combined with ONT sequencing (CEO) to characterize the B. mori FibH gene, which is composed of many repeat units with a long and GC-rich sequence up to 17 kb and is not easily amplified by means of a polymerase chain reaction (PCR). CEO has four steps: the dephosphorylation of genomic DNA, the Cpf1 targeted cleavage of FibH, adapter ligation, and ONT sequencing. Using CEO, we determined the fine structure of B. moriFibH, which is 16,845 bp long and includes 12 repetitive domains separated by amorphous regions. Except for the difference of three bases in the intron from the reference gene, the other sequences are identical. Surprisingly, many methylated CG sites were found and distributed unevenly on the FibH repeat unit. The CEO we established is an available means to depict highly repetitive genes, but also a supplement to the enrichment method based on Cas9.

CRISPR-Mediated Endogenous Activation of Fibroin Heavy Chain Gene Triggers Cellular Stress Responses in Bombyx mori Embryonic Cells.

The silkworm Bombyx mori is an economically important insect, as it is the main producer of silk. Fibroin heavy chain (FibH) gene, encoding the core component of silk protein, is specifically and highly expressed in silk gland cells but not in the other cells. Although the silkworm FibH gene has been well studied in transcriptional regulation, its biological functions in the development of silk gland cells remain elusive. In this study, we constructed a CRISPRa system to activate the endogenous transcription of FibH in Bombyx mori embryonic (BmE) cells, and the mRNA expression of FibH was successfully activated. In addition, we found that FibH expression was increased to a maximum at 60 h after transient transfection of sgRNA/dCas9-VPR at a molar ratio of 9:1. The qRT-PCR analysis showed that the expression levels of cellular stress response-related genes were significantly up-regulated along with activated FibH gene. Moreover, the lyso-tracker red and monodansylcadaverine (MDC) staining assays revealed an apparent appearance of autophagy in FibH-activated BmE cells. Therefore, we conclude that the activation of FibH gene leads to up-regulation of cellular stress responses-related genes in BmE cells, which is essential for understanding silk gland development and the fibroin secretion process in B. mori.

Deep Sequencing Reveals the Comprehensive CRISPR-Cas9 Editing Spectrum in Bombyx mori.

Application of the clustered regularly interspaced short palindromic repeats associated 9 (CRISPR-Cas9) technology has revolutionized biology by greatly enhancing the ability to introduce mutations into DNA for research and prospective therapeutic purposes. However, the understanding of Cas9 editing outcomes is still limited. Previously, it was considered that Cas9 introduces stochastic insertions or deletions (indels) at the target site. In the current study, we performed in vivo multiplex editing, deep sequencing, and comprehensive analysis of its editing outcomes in Bombyx mori (B. mori). A total of 31161 editing events from 9 single-guide RNA (sgRNA) sites in 16 individuals were generated and analyzed, and we found that Cas9 introduces mutations with some regularity rather than via stochastic indels. The editing efficiency varies with sgRNA sequences, individuals, and orientation. Small deletions account for the vast majority of mutated sequences, followed by a small fraction of substitutions and insertions. The most likely mutations are deletions between two microhomologous sequences or single-base deletions at the cleavage site in the absence of microhomologous pairs. Insertions are formed by diverse mechanisms, including direct acquisition of free genomic fragments, duplication of broken ends, replication of adjacent sequences, or random addition of free nucleotides. The above results indicate that the Cas9 editing spectrum is reproducible and predictable. Thus, our findings enable a deeper understanding of Cas9-mediated mutagenesis and better design of genome editing experiments, as well as elucidate the DNA double-strand break repair processes in B. mori.

In-depth transcriptome unveils the cadmium toxicology and a novel metallothionein in silkworm.

Heavy metal pollution has gradually become a major global issue. It is so far reaching in part because heavy metals are absorbed by soil and affect almost all species via ecological cycles. Silkworms (Bombyx mori) are poisoned by heavy metals through a soil-mulberry-silkworm system, which inhibits larval growth and development and leads to a decrease in silk production. In the present study, we performed transcriptome sequencing of larval midgut with cadmium exposure to explore the toxicological mechanism of heavy metal, and found that the following potential pathways may be involved in cadmium infiltration: endocytosis, oxidative phosphorylation, and MAPK signaling. Moreover, we identified a novel metallothionein in silkworm, which is inhibited by cadmium exposure and able to improve heavy metal tolerance in B. mori cell lines and Escherichia coli. We also generated a transgenic silkworm strain overexpressing metallothionein and the result showed that metallothionein observably enhanced larval viability under cadmium exposure. This study used RNA sequencing to reveal a mechanism for cadmium toxicology, and identified and functionally verified BmMT, offering a new potential heavy metal-tolerant silkworm variety.

A deep insight into the transcriptome of midgut and fat body reveals the toxic mechanism of fluoride exposure in silkworm.

Fluoride generally exists in the natural environment, and has been reported to induce serious environmental hazard to animals, plants, and even humans via ecological cycle. Silkworm, Bombyx mori, which showed significant growth and reproductivity reduction when exposed to fluoride, has become a model to evaluate the toxicity of fluoride. However, the detailed mechanism underlying fluoride toxicity and corresponding transport proteins remain unclear. In this study, we performed RNA-seq of the larval midgut and fat body with fluoride exposure and normal treatment. Differential analysis showed that there were 4405 differentially expressed genes in fat body and 4430 DEGs in midgut with fluoride stress. By Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses, we identified several key pathways involved in the fluoride exposure and poisoning. We focused on the oxidative phosphorylation and MAPK signal pathway. QRT-PCR confirmed that oxidative phosphorylation process was remarkably inhibited by fluoride exposure and resulted in the blocking of ATP synthesis. The MAPK signal pathway was stimulated via phosphorylation signal transduction. Moreover, by protein structure analysis combined with the DEGs, we screen 36 potential membrane proteins which might take part in transporting fluoride. Taken together, the results of our study expanded the underlying mechanisms of fluoride poisoning on silkworm larval growth and development, and implied potential fluoride transport proteins in silkworm.

STING-dependent autophagy suppresses Nosema bombycis infection in silkworms, Bombyx mori.

Nosema bombycis is a unicellular spore-forming obligate parasite, related to fungi, and causes infections in economically important animals and are opportunistic human pathogens. However, the mechanisms of host response to N. bombycis remain unclear. STING (stimulator of interferon genes) is an adapter protein involved in the innate immune response to pathogens. In this study, a transgenic gRNA vector containing BmSTING was constructed and microinjected to generate the transgenic line BmSTINGΔ6bp/WT and BmSTINGΔ5bp/WT in silkworms. The expression of BmSTING was significantly reduced in BmSTINGΔ5bp/WT compared to non-transgenic silkworm. The mortality and LC3 (microtubule-associated protein 1 light chain 3) level in BmSTINGΔ6bp/WT and BmSTINGΔ5bp/WT was significantly decreased in the early infection stage of N. bombycis, but the transgenic silkworms died rapidly in the later stage. Furthermore, both BmSTING and LC3 were increased in BmE cell lines after infection with N. bombycis. This study highlights the role of STING-dependent pathways response to microsporidia in silkworm, Bombyx mori.

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.