CRISPRi-based phenotypic screening in bacteria: technologies, applications, and prospects

Abstract CRISPR interference (CRISPRi) has emerged as a powerful tool for high-throughput phenotypic screening in bacteria. Its programmability, scalability, and compatibility with non-model bacteria enable systematic genotype–phenotype mapping across diverse biological contexts. This review outlines the current methodologies of CRISPRi-based screening, including sgRNA library construction and screening formats, and highlights a wide range of observable phenotypes—such as gene essentiality, auxotrophy, autotrophy, drug susceptibility, and phage susceptibility. We also discuss recent advancements in strategies for delivering the CRISPRi system to non-model bacteria. Finally, we propose future directions for expanding the utility of CRISPRi-based screening. Together, these insights provide a comprehensive overview of CRISPRi as a versatile platform for bacterial functional genomics and offer perspectives on its continued evolution.