Helmholtz Institute for RNA-based Infection Research (HIRI), Helmholtz Centre for Infection Research (HZI), Würzburg, Germany.
Institute of Molecular Infection Biology (IMIB), University of Würzburg, Würzburg, Germany.
Methods Mol Biol. 2024;2741:101-116. doi: 10.1007/978-1-0716-3565-0_6.
Small RNAs (sRNAs) are versatile regulators universally present in species across the prokaryotic kingdom, yet their functional characterization remains a major bottleneck. Gene inactivation through random transposon insertion has proven extremely valuable in discovering hidden gene functions. However, this approach is biased toward long genes and usually results in the underrepresentation of sRNA mutants. In contrast, CRISPR interference (CRISPRi) harnesses guide RNAs to recruit cleavage-deficient Cas nucleases to specific DNA loci. The ensuing steric hindrance inhibits RNA polymerase assembly at-or migration along-predefined genes, allowing for targeted knockdown screens without major length bias. In this chapter, we provide a detailed protocol for CRISPRi-based functional screening of bacterial sRNAs. Using the abundant microbiota species Bacteroides thetaiotaomicron as a model, we describe the design and generation of a guide library targeting the full intergenic sRNA repertoire of this organism and its application to identify sRNA knockdown-associated fitness effects. Our protocol is generic and thus suitable for the systematic assessment of sRNA-associated phenotypes in a wide range of bacterial species and experimental conditions. We expect CRISPRi-based functional genomics to boost sRNA research in understudied bacterial taxa, for instance, members of the gut microbiota.
小 RNA（sRNA）是一种普遍存在于原核生物界各种物种中的多功能调节剂，但它们的功能特征仍然是一个主要的瓶颈。通过随机转座子插入进行基因失活已被证明在发现隐藏基因功能方面非常有价值。然而，这种方法偏向于长基因，通常会导致 sRNA 突变体的代表性不足。相比之下，CRISPR 干扰（CRISPRi）利用向导 RNA 招募无切割活性的 Cas 核酸酶到特定的 DNA 位点。由此产生的空间位阻抑制 RNA 聚合酶在预定基因上的组装或迁移，从而可以在没有主要长度偏差的情况下进行靶向敲低筛选。在本章中，我们提供了一种基于 CRISPRi 的细菌 sRNA 功能筛选的详细方案。我们以丰富的微生物物种拟杆菌属为模型，描述了针对该生物体全基因间 sRNA 谱的向导库的设计和生成，并将其应用于鉴定 sRNA 敲低相关的适应性效应。我们的方案是通用的，因此适用于在广泛的细菌物种和实验条件下系统评估 sRNA 相关表型。我们预计基于 CRISPRi 的功能基因组学将促进对肠道微生物群等研究较少的细菌分类群中的 sRNA 研究。
