Abstract / Description
UNLABELLED: produce a multitude of secondary metabolites, which have been exploited in drug discovery campaigns for more than three-quarters of a century. Our understanding of microbial physiology has been revolutionized by genome sequencing and large-scale functional studies. Technology for genome-wide investigations in species, however, has lagged behind that for other bacterial systems, hindering exploitation of unprecedented quantities of genomic data. Here, we develop a platform for clustered regularly interspaced short palindromic repeats interference sequencing (CRISPRi-seq) for spp. By performing CRISPRi-seq with 2,160 unique sgRNAs targeting all operons (432 operons) encoding membrane transporters (629 genes) representing 1.1Mb of the 6.8Mb genome for combined with hit validation, we discovered that only a small proportion (13 of 432 operons, 25 kb) contribute positively to fitness. Our work provides both a first-in-class platform for high-throughput functional genomics and a generalized blueprint for screens in species. IMPORTANCE: bacteria are prolific producers of clinically essential natural products, yet high-throughput tools to systematically interrogate their genomes remain underdeveloped. By establishing a robust CRISPRi-seq platform for functional screening in , our work closes a critical technological gap in functional genomics. Our study not only identifies a small subset of transporter operons essential for fitness but also introduces a scalable, generalizable approach for dissecting gene function. This platform will accelerate systems-level understanding of an industrially and medically important genus.