Groupe de Biologie de Synthèse, Institut Pasteur, Paris 75015, France.
Groupe de Biologie de Synthèse, Institut Pasteur, Paris 75015, France.
Methods. 2020 Feb 1;172:61-75. doi: 10.1016/j.ymeth.2019.07.024. Epub 2019 Aug 1.
The catalytic null mutant of the Cas9 endonuclease from the bacterial CRISPR immune system, known as dCas9, can be guided by a small RNA to bind DNA sequences of interest and block gene transcription in a strategy known as CRISPRi. This powerful gene silencing method has already been used in a large number of species and in high throughput screens. Here we provide detailed design rules, methods and novel vectors to perform CRISPRi experiments in S. aureus and in E. coli, using the well characterized dCas9 protein from S. pyogenes. In particular, we describe a vector based on plasmid pC194 which is broadly used in Firmicutes, as well as a vector based on the very broad host-range rolling circle plasmid pLZ12, reported to replicate in both Firmicutes and Proteobacteria. A potential caveat of adapting dCas9 tools to various bacterial species is that dCas9 was shown to be toxic when expressed too strongly. We describe a method to optimize the expression level of dCas9 in order to avoid toxicity while ensuring strong on-target repression activity. We demonstrate this method by optimizing a pLZ12 based vector originally developed for S. aureus so that it can work in E. coli. This article should provide all the resources required to perform CRISPRi experiments in a broad range of bacterial species.
细菌 CRISPR 免疫系统中的 Cas9 内切酶的催化无效突变体，称为 dCas9，可以被小 RNA 引导，与感兴趣的 DNA 序列结合，并阻断 CRISPRi 策略中的基因转录。这种强大的基因沉默方法已经在许多物种和高通量筛选中得到应用。在这里，我们提供了详细的设计规则、方法和新型载体，用于在金黄色葡萄球菌和大肠杆菌中进行 CRISPRi 实验，使用来自化脓性链球菌的经过充分表征的 dCas9 蛋白。特别是，我们描述了一种基于质粒 pC194 的载体，该载体在厚壁菌门中广泛使用，以及一种基于广泛宿主范围的滚环质粒 pLZ12 的载体，据报道该质粒可以在厚壁菌门和变形菌门中复制。将 dCas9 工具适应于各种细菌物种的一个潜在问题是，当表达太强时，dCas9 被证明是有毒的。我们描述了一种优化 dCas9 表达水平的方法，以避免毒性，同时确保强靶标抑制活性。我们通过优化最初为金黄色葡萄球菌开发的基于 pLZ12 的载体来证明这种方法，使其能够在大肠杆菌中工作。本文应提供在广泛的细菌物种中进行 CRISPRi 实验所需的所有资源。
