Department of Systems Biology, Columbia University, New York, NY, USA.
Department of Biological Sciences, Columbia University, New York, NY, USA.
Mol Syst Biol. 2020 Jul;16(7):e9427. doi: 10.15252/msb.20199427.
Programmable gene activation enables fine-tuned regulation of endogenous and synthetic gene circuits to control cellular behavior. While CRISPR-Cas-mediated gene activation has been extensively developed for eukaryotic systems, similar strategies have been difficult to implement in bacteria. Here, we present a generalizable platform for screening and selection of functional bacterial CRISPR-Cas transcription activators. Using this platform, we identified a novel CRISPR activator, dCas9-AsiA, that could activate gene expression by more than 200-fold across genomic and plasmid targets with diverse promoters after directed evolution. The evolved dCas9-AsiA can simultaneously mediate activation and repression of bacterial regulons in E. coli. We further identified hundreds of promoters with varying basal expression that could be induced by dCas9-AsiA, which provides a rich resource of genetic parts for inducible gene activation. Finally, we show that dCas9-AsiA can be ported to other bacteria of clinical and bioindustrial relevance, thus enabling bacterial CRISPRa in more application areas. This work expands the toolbox for programmable gene regulation in bacteria and provides a useful resource for future engineering of other bacterial CRISPR-based gene regulators.
可编程基因激活使精细调节内源性和合成基因回路以控制细胞行为成为可能。虽然 CRISPR-Cas 介导的基因激活已在真核系统中得到广泛开发，但类似的策略在细菌中很难实施。在这里，我们提出了一个用于筛选和选择功能性细菌 CRISPR-Cas 转录激活剂的可推广平台。使用这个平台，我们鉴定了一种新型的 CRISPR 激活子 dCas9-AsiA，它可以在定向进化后通过超过 200 倍的基因组和质粒靶标以及具有不同启动子的基因表达进行激活。经过进化的 dCas9-AsiA 可以同时在大肠杆菌中调节细菌调控子的激活和抑制。我们进一步鉴定了数百个具有不同基础表达水平的启动子，这些启动子可以被 dCas9-AsiA 诱导，这为可诱导基因激活提供了丰富的遗传部件资源。最后，我们表明 dCas9-AsiA 可以被转移到其他具有临床和生物工业相关性的细菌中，从而使细菌的 CRISPRa 能够在更多的应用领域中使用。这项工作扩展了细菌可编程基因调控的工具包，并为未来基于 CRISPR 的其他细菌基因调控器的工程提供了有用的资源。
