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

Jun 27, 2023Insect science

Key genes and pathways linked to 20-hydroxyecdysone hormone signaling in silkworms found by genome-wide CRISPR screening

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Abstract

Genome-wide screening identified key genes in the silkworm's 20-hydroxyecdysone signaling pathway.

The hormone 20-hydroxyecdysone regulates essential proteins involved in cellular processes primarily in the cytoplasm and nucleus.
Pathway enrichment analysis indicates that 20-hydroxyecdysone may activate phosphorylation, impacting innate immunity and energy metabolism.
Increased tolerance to 20-hydroxyecdysone was observed in cells with knockout alleles of relevant genes.
Findings suggest that the CRISPR/Cas9-based library is effective for exploring hormone signaling pathways in insects.

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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.

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

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