CRISPR-based screening of small RNA modulators of bile susceptibility in Bacteroides thetaiotaomicron

• 成果类型: Article
• 署名作者: Prezza, Gianluca; Liao, Chunyu; Reichardt, Sarah; Beisel, Chase L.; Westermann, Alexander J.
• 署名单位: Helmholtz Association; Helmholtz-Center for Infection Research; University of Wurzburg; University of Wurzburg; University of Wurzburg
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-10507
• DOI: 10.1073/pnas.2311323121
• 发表日期: 2024-01-29
• 关键词: translation; bacteria; targets; cpf1

摘要

Microbiota- centric interventions are limited by our incomplete understanding of the gene functions of many of its constituent species. This applies in particular to small RNAs (sRNAs), which are emerging as important regulators in microbiota species yet tend to be missed by traditional functional genomics approaches. Here, we establish CRISPR interference (CRISPRi) in the abundant microbiota member Bacteroides thetaiotaomicron for genome-wide sRNA screens. By assessing the abundance of different protospacer- adjacent motifs, we identify the Prevotella bryantii B14 Cas12a as a suitable nuclease for CRISPR screens in these bacteria and generate an inducible Cas12a expression system. Using a luciferase reporter strain, we infer guide design rules and use this knowledge to assemble a computational pipeline for automated gRNA design. By subjecting the resulting guide library to a phenotypic screen, we uncover the sRNA BatR to increase susceptibility to bile salts through the regulation of genes involved in Bacteroides cell surface structure. Our study lays the groundwork for unlocking the genetic potential of these major human gut mutualists and, more generally, for identifying hidden functions of bacterial sRNAs.