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

by Selleckchem | Oct 26 2023

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.

Comments:

The passage you provided describes a study that aimed to understand the hormone signaling pathway involved in metamorphosis in the silkworm (Bombyx mori) using a genome-wide CRISPR/Cas9-based library. Metamorphosis is a complex process regulated by juvenile hormone (JH) and 20-hydroxyecdysone (20E), but the specific genes and mechanisms involved in the silkworm's hormone signaling pathway were not well understood.

To investigate this, the researchers constructed a genome-wide CRISPR/Cas9-based library of the silkworm, which allowed them to analyze the function of different genes on a large scale. They used this library to screen for key genes involved in the silkworm's 20E signaling pathway and to study their mechanisms of action.

Through functional annotation, the researchers found that 20E regulates key proteins mainly in the cytoplasm and nucleus of cells. Pathway enrichment analysis revealed that 20E can activate phosphorylation, potentially affecting innate immunity, intracellular nutrition and energy metabolism, and leading to cell apoptosis (programmed cell death).

To validate the screening results, the researchers generated cells with knockout alleles (where the gene is disrupted or "knocked out") of the identified genes. They observed that these cells showed increased tolerance to 20E, supporting the role of these genes in the hormone signaling pathway.

Overall, this study provides a comprehensive understanding of the signaling response to 20E in the silkworm and highlights the usefulness of genome-wide CRISPR mutant libraries in unraveling hormone signaling pathways and the mechanisms involved in insect metamorphosis.