Construction and application of the conditionally essential gene knockdown library in to screen potential antimicrobial targets and virulence genes via Mobile-CRISPRi-seq.

is a ubiquitous human pathogen, and its clinical treatment faces two major challenges: multidrug resistance and the pathogenesis of hypervirulent . The discovery and study of conditionally essential (CE) genes that can function as potential antimicrobial targets has always been a research concern due to their restriction in the development of novel antibiotics. However, the lack of essential functional genomic data has hampered the study of the mechanisms of essential genes related to antimicrobial susceptibility. In this study, we developed a pooled CE genes mobile clustered regularly interspaced short palindromic repeat (CRISPR) interference screening method (Mobile-CRISPRi-seq) for to identify genes that play critical roles in antimicrobial fitness and host immunity . Targeting 870 predicted CE genes in , Mobile-CRISPRi-seq uncovered the depletion of tetrahydrofolate synthesis pathway genes and under trimethoprim pressure. Our screening also identified genes and related to polymyxin and β-lactam susceptibility by applying a screening strategy based on Mobile-CRISPRi-seq and comparative genomics. Furthermore, using a mouse infection model and Mobile-CRISPRi-seq, multiple virulence genes were identified, and among these genes, , and were demonstrated to contribute to the pathogenesis of . This study provides a simple, rapid, and effective platform for screening potential antimicrobial targets and virulence genes in , and this broadly applicable system can be expanded for high-throughput functional gene study in multiple pathogenic bacteria, especially in gram-negative bacteria. IMPORTANCE The discovery and investigation of conditionally essential (CE) genes that can function as potential antimicrobial targets has always been a research concern because of the restriction of antimicrobial targets in the development of novel antibiotics. In this study, we developed a pooled CE gene-wide mobile clustered regularly interspaced short palindromic repeat (CRISPR) interference sequencing (Mobile-CRISPRi-seq) strategy in to identify genes that play critical roles in the fitness of antimicrobials and host immunity . The data suggest a robust tool to screen for loss-of-function phenotypes in a pooled gene knockdown library in , and Mobile-CRISPRi-seq may be expanded to multiple bacteria for screening and identification of genes with crucial roles in the fitness of antimicrobials and hosts.