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

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 <i>piggyBac</i> 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 <i>Bombyx mori</i> 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 <i>piggyBac</i> 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 <i>B. mori</i> 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.