Genome-wide CRISPR screening of genes and pathways for insect cell responding to abnormal environmental pH.

Cells are bathed in the extracellular fluids in which the extracellular pH (pHe) is maintained to a narrow range, and abnormal pHe is related to multiple diseases. However, the genes and signaling pathways underlying cell response to abnormal pHe remain unclear. Identification of genes responsive to extreme pHe challenge has great value in both basic research and medicine.

Complete BmFib-L knockout reveals its indispensable role in silk fiber formation.

Silkworm (Bombyx mori), belonging to the order Lepidoptera, is an important model insect for economic and scientific research. The capacity of the silkworm to secrete robust silk renders it a valuable economic resource, while its biological characteristics offer insights into a number of scientific disciplines. Despite the extensive research conducted to elucidate the mechanisms of silk secretion, many aspects remain unclear.

Overview and Evolution of Insect Fibroin Heavy Chain (FibH).

The gene, crucial for silk spinning in insects, encodes a protein that significantly influences silk fiber mechanics. Due to its large size and repetitive sequences, limited known sequences of insect FibH impede comprehensive understanding. Here, we analyzed 114 complete gene sequences from Lepidoptera (71 moths, 24 butterflies) and 13 Trichoptera, revealing single-copy in most species, with 2-3 copies in Hesperinae and Heteropterinae (subfamily of skippers).

Effects of poly (ADP-ribose) polymerase 1 (PARP1) on silk proteins in the silkworm, Bombyx mori.

Animal silk is economically important, while silk secretion is a complex and subtle mechanism regulated by many genes. We identified the poly (ADP-ribose) polymerase (PARP1) gene of the silkworm and successfully cloned its coding sequence (CDS) sequence. Using clustered regularly interspaced short palindromic repeat (CRISPR/Cas9) technology, we screened single guide RNA (sgRNA) with high knockout efficiency by cellular experiments and obtained PARP1 mutants by knocking out the PARP1 gene of the silkworm at the individual level.

Ectopic expression of BmeryCA in Bombyx mori increases silk yield and mechanical properties by altering the pH of posterior silk gland.

The silk glands are the specialized tissue where silk protein synthesis, secretion, and conformational transitions take place, with pH playing a critical role in both silk protein synthesis and fiber formation. In the present study, we have identified erythrocyte carbonic anhydrase (BmeryCA) belonging to the α-CA class in the silk gland, which is a Zn dependent metalloenzyme capable of efficiently and reversibly catalyzing the hydrated reaction of CO to HCO, thus participating in the regulation of acid-base balance. Multiple sequence alignments revealed that the active site of BmeryCA was highly conserved.

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

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 () 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.

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.

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.

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.

Gene Complete Sequences (FibHome) Revealed Silkworm Pedigree.

The highly repetitive and variable fibroin heavy chain () gene can be used as a silkworm identification; however, only a few complete sequences are known. In this study, we extracted and examined 264 gene complete sequences (FibHome) from a high-resolution silkworm pan-genome. The average lengths of the wild silkworm, local, and improved strains were 19,698 bp, 16,427 bp, and 15,795 bp, respectively.

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.

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.

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.

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.

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

The pupal diapause of univoltine 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 . Here, the dosage of injected 20E was optimized to efficiently terminate pupal diapause of , showing that inappropriate dosage of 20E can cause pupal lethality and a low eclosion incidence.

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.

Genetic Code Expansion System for Tight Control of Gene Expression in 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 () genome, the study of has entered the post-genome era. While the functions of many genes have been described in detail, many coding genes remain unidentified.

Precise Characterization of 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.

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

The silkworm is an economically important insect, as it is the main producer of silk. Fibroin heavy chain () 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 gene has been well studied in transcriptional regulation, its biological functions in the development of silk gland cells remain elusive.

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

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.

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 CdCl-resistance genes, and 725 CdCl-sensitive genes.

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.

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.

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.