Hogan Andrew M, Rahman A S M Zisanur, Lightly Tasia J, Cardona Silvia T
Department of Microbiology , University of Manitoba , Winnipeg , Manitoba R3T 2N2 , Canada.
Department of Medical Microbiology & Infectious Diseases , University of Manitoba , Winnipeg , Manitoba R3T 2N2 , Canada.
ACS Synth Biol. 2019 Oct 18;8(10):2372-2384. doi: 10.1021/acssynbio.9b00232. Epub 2019 Sep 20.
Genetic tools are critical to dissecting the mechanisms governing cellular processes, from fundamental physiology to pathogenesis. Members of the genus have potential for biotechnological applications but can also cause disease in humans with a debilitated immune system. The lack of suitable genetic tools to edit GC-rich genomes has hampered the exploration of useful capacities and the understanding of pathogenic features. To address this, we have developed CRISPR interference (CRISPRi) technology for gene silencing in , testing it in , , and . Tunable expression was provided by placing a codon-optimized from under control of a rhamnose-inducible promoter. As a proof of concept, the operon controlling genes necessary for phenylacetic acid degradation was targeted by plasmid-borne gRNAs, resulting in near complete inhibition of growth on phenylacetic acid as the sole carbon source. This was supported by reductions in mRNA expression. The utility of CRISPRi to probe other functions at the single cell level was demonstrated by knocking down and , which dramatically reduces polyhydroxybutyrate granule accumulation and motility, respectively. As a hallmark of the mini-CTX system is the broad host-range of integration, we putatively identified 67 genera of Proteobacteria that might be amenable to modification with our CRISPRi toolkit. Our CRISPRi toolkit provides a simple and rapid way to silence gene expression to produce an observable phenotype. Linking genes to functions with CRISPRi will facilitate genome editing with the goal of enhancing biotechnological capabilities while reducing 's pathogenic arsenal.
遗传工具对于剖析从基础生理学到发病机制的细胞过程调控机制至关重要。该属的成员具有生物技术应用潜力，但也会在免疫系统衰弱的人类中引发疾病。缺乏适用于编辑富含GC基因组的遗传工具阻碍了对其有用能力的探索以及对致病特征的理解。为解决这一问题，我们开发了用于在该属中进行基因沉默的CRISPR干扰（CRISPRi）技术，并在[具体物种1]、[具体物种2]和[具体物种3]中进行了测试。通过将来自[具体物种]的密码子优化的[基因名称]置于鼠李糖诱导型启动子的控制下，实现了可调节表达。作为概念验证，控制苯乙酸降解所需基因的[具体操纵子名称]操纵子被质粒携带的引导RNA靶向，导致在以苯乙酸作为唯一碳源时生长几乎完全受到抑制。这通过[具体基因名称]mRNA表达的降低得到了支持。通过敲低[具体基因1]和[具体基因2]分别显著减少聚羟基丁酸酯颗粒积累和运动性，证明了CRISPRi在单细胞水平探测其他功能的效用。由于mini-CTX系统的一个标志是广泛的宿主整合范围，我们推测鉴定出67个变形菌属，它们可能适合用我们的CRISPRi工具包进行修饰。我们的CRISPRi工具包提供了一种简单快速的方法来沉默基因表达以产生可观察到的表型。利用CRISPRi将基因与功能联系起来将有助于基因组编辑，目标是增强生物技术能力，同时减少该属的致病武器库。
