Programmable gene activation enables fine-tuned regulation of endogenous and synthetic gene circuits to control cellular behavior. While CRISPR-Cas-mediated gene activation have 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 up to 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.

中文翻译：

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细菌中可编程和便携式CRISPR-Cas转录激活

可编程的基因激活可对内源和合成基因电路进行微调调节，以控制细胞行为。尽管针对真核系统已广泛开发了CRISPR-Cas介导的基因激活，但难以在细菌中实施类似的策略。在这里，我们提供了一个通用的平台，用于筛选和选择功能性细菌CRISPR-Cas转录激活因子。使用这个平台，我们鉴定了一种新的CRISPR激活剂dCas9-AsiA，在定向进化后，它可以通过具有不同启动子的基因组和质粒靶标激活多达200倍的基因表达。进化的dCas9-AsiA可同时介导大肠杆菌中细菌调节子的激活和抑制。。我们进一步鉴定了数百种具有不同基础表达的启动子，这些启动子可以被dCas9-AsiA诱导，而dCas9-AsiA为诱导型基因激活提供了丰富的遗传部分资源。最后，我们表明dCas9-AsiA可以移植到具有临床和生物工业相关性的其他细菌上，从而使细菌CRISPRa在更多的应用领域中得以应用。这项工作扩展了细菌中可编程基因调控的工具箱，并为将来基于其他基于CRISPR的细菌基因调控器的工程设计提供了有用的资源。