一种用于军团菌毒力基因研究的多重 CRISPR 干扰工具。
A multiplex CRISPR interference tool for virulence gene interrogation in Legionella pneumophila.
机构信息
Division of Molecular and Cellular Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, 20892, USA.
出版信息
Commun Biol. 2021 Feb 4;4(1):157. doi: 10.1038/s42003-021-01672-7.
Catalytically inactive dCas9 imposes transcriptional gene repression by sterically precluding RNA polymerase activity at a given gene to which it was directed by CRISPR (cr)RNAs. This gene silencing technology, known as CRISPR interference (CRISPRi), has been employed in various bacterial species to interrogate genes, mostly individually or in pairs. Here, we developed a multiplex CRISPRi platform in the pathogen Legionella pneumophila capable of silencing up to ten genes simultaneously. Constraints on precursor-crRNA expression were overcome by combining a strong promoter with a boxA element upstream of a CRISPR array. Using crRNAs directed against virulence protein-encoding genes, we demonstrated that CRISPRi is fully functional not only during growth in axenic media, but also during macrophage infection, and that gene depletion by CRISPRi recapitulated the growth defect of deletion strains. By altering the position of crRNA-encoding spacers within the CRISPR array, our platform achieved the gradual depletion of targets that was mirrored by the severity in phenotypes. Multiplex CRISPRi thus holds great promise for probing large sets of genes in bulk in order to decipher virulence strategies of L. pneumophila and other bacterial pathogens.
无活性的 dCas9 通过空间位阻来阻止 RNA 聚合酶在特定基因上的活性,从而在 CRISPR(cr)RNA 的指导下实现基因转录抑制。这种基因沉默技术,称为 CRISPR 干扰(CRISPRi),已被用于各种细菌中,以研究单个或成对的基因。在这里,我们在病原体嗜肺军团菌中开发了一种可同时沉默多达十个基因的多路 CRISPRi 平台。通过将强启动子与 CRISPR 阵列上游的 boxA 元件结合使用,克服了前体-crRNA 表达的限制。使用针对毒力蛋白编码基因的 crRNA,我们证明 CRISPRi 不仅在无细胞培养基中生长时具有完全功能,而且在巨噬细胞感染时也具有功能,并且 CRISPRi 的基因耗竭可重现缺失菌株的生长缺陷。通过改变 CRISPR 阵列中 crRNA 编码间隔区的位置,我们的平台实现了靶基因的逐渐耗竭,表型的严重程度与之相匹配。因此,多路 CRISPRi 有望在批量探测大量基因,以解析嗜肺军团菌和其他细菌病原体的毒力策略。