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

Abstract

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.

The Antiviral Molecule 5-Pyridoxolactone Identified Post BmNPV Infection of the Silkworm, Bombyx mori

“…Among them, the ABC transporter pathway has been identified in multiple proteome and transcriptome studies [ 9 , 40 ]. In previous studies, knockout of the V-ATPase subunit gene BMgn016795A of the ABC transporter pathway resulted in a significant decrease in proliferation of BmNPV in BmE cells [ 8 ]. The endosomal acidification mediated by this gene may play a role in the process of BmNPV entering cells, although the mechanism is unknown.…”

“…Bombyx mori nucleopolyhedrovirus (BmNPV) baculovirus is a major pathogen of the silkworm and causes serious economic losses [ 6 , 7 ]. Genetics and molecular biology studies have generated antiviral strains and revealed immunological responses [ 8 , 9 , 10 , 11 , 12 ]. However, the resistance mechanism remains unclear.…”

Advances in the Arms Race Between Silkworm and Baculovirus

“…Identification of resistance genes and analysis of silkworm antiviral mechanisms against BmNPV merit further investigation. In future experiments, we will screen for negative regulatory factors in the immune pathway using genome-wide CRISPR ( 114 ) and identify the host proteins that bind the virus by use of inhibitors. The target genes will be knocked out via gene editing to improve silkworm resistance.…”

Identifying essential genes across eukaryotes by machine learning

“…castaneum and the silk moth Bombyx mori . We used the available RNA interference (RNAi) ( 24 ) and CRISPR ( 25 ) screen with defined lethality status as a control, while validating further predictions experimentally. Moreover, we aimed to fill the gap between human cell line screens and population studies, by integrating information from the model organisms to improve human essential gene assignments.…”