Helmholtz Institute for RNA-based Infection Research, Helmholtz Centre for Infection Research, Würzburg D-97080, Germany.
Medical Faculty, University of Würzburg, Würzburg D-97080, Germany.
Proc Natl Acad Sci U S A. 2024 Feb 6;121(6):e2311323121. doi: 10.1073/pnas.2311323121. Epub 2024 Jan 31.
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 for genome-wide sRNA screens. By assessing the abundance of different protospacer-adjacent motifs, we identify the 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 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.
微生物组中心的干预措施受到我们对其许多组成物种的基因功能理解不完整的限制。这尤其适用于小 RNA（sRNA），它们作为微生物组物种的重要调节剂而出现，但往往会被传统的功能基因组学方法所忽略。在这里，我们在丰富的微生物群成员 中建立了 CRISPR 干扰（CRISPRi），用于进行全基因组 sRNA 筛选。通过评估不同的间隔邻近基序的丰度，我们确定了 B14 Cas12a 作为这些细菌中 CRISPR 筛选的合适核酸酶，并生成了诱导型 Cas12a 表达系统。使用荧光素酶报告菌株，我们推断出指导设计规则，并利用这些知识组装了用于自动 gRNA 设计的计算管道。通过将产生的引导文库进行表型筛选，我们发现 sRNA BatR 通过调节参与 细胞表面结构的基因来增加对胆汁盐的敏感性。我们的研究为揭示这些主要人类肠道共生体的遗传潜力奠定了基础，更广泛地说，为鉴定细菌 sRNA 的隐藏功能奠定了基础。
