Vigouroux Antoine, Bikard David
Synthetic Biology, Institut Pasteur, Paris, France.
Microbial Morphogenesis and Growth, Institut Pasteur, Paris, France.
Microbiol Mol Biol Rev. 2020 Apr 1;84(2). doi: 10.1128/MMBR.00077-19. Print 2020 May 20.
CRISPR-Cas systems have been engineered as powerful tools to control gene expression in bacteria. The most common strategy relies on the use of Cas effectors modified to bind target DNA without introducing DNA breaks. These effectors can either block the RNA polymerase or recruit it through activation domains. Here, we discuss the mechanistic details of how Cas effectors can modulate gene expression by blocking transcription initiation or acting as transcription roadblocks. CRISPR-Cas tools can be further engineered to obtain fine-tuned control of gene expression or target multiple genes simultaneously. Several caveats in using these tools have also been revealed, including off-target effects and toxicity, making it important to understand the design rules of engineered CRISPR-Cas effectors in bacteria. Alternatively, some types of CRISPR-Cas systems target RNA and could be used to block gene expression at the posttranscriptional level. Finally, we review applications of these tools in high-throughput screens and the progress and challenges in introducing CRISPR knockdown to other species, including nonmodel bacteria with industrial or clinical relevance. A deep understanding of how CRISPR-Cas systems can be harnessed to control gene expression in bacteria and build powerful tools will certainly open novel research directions.
CRISPR-Cas系统已被设计成控制细菌基因表达的强大工具。最常见的策略依赖于使用经过修饰的Cas效应物来结合目标DNA，而不引入DNA断裂。这些效应物既可以阻断RNA聚合酶，也可以通过激活结构域招募它。在这里，我们讨论Cas效应物如何通过阻断转录起始或作为转录障碍来调节基因表达的机制细节。CRISPR-Cas工具可以进一步设计，以实现对基因表达的微调控制或同时靶向多个基因。使用这些工具时的一些注意事项也已被揭示，包括脱靶效应和毒性，这使得了解工程化CRISPR-Cas效应物在细菌中的设计规则变得很重要。另外，某些类型的CRISPR-Cas系统靶向RNA，可用于在转录后水平阻断基因表达。最后，我们回顾了这些工具在高通量筛选中的应用，以及将CRISPR敲低技术引入其他物种（包括具有工业或临床相关性的非模式细菌）的进展和挑战。深入了解如何利用CRISPR-Cas系统控制细菌中的基因表达并构建强大的工具，必将开辟新的研究方向。
