Engineered CRISPRa enables programmable eukaryote-like gene activation in bacteria
Engineered CRISPRa enables programmable eukaryote-like gene activation in bacteria
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DOI：
10.1038/s41467-019-11479-0
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发表时间：
2019-08-26
影响因子：
16.6
通讯作者：
Wang, Baojun
中科院分区：
文献类型：
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作者：
Liu, Yang;Wan, Xinyi;Wang, Baojun
核酸酶缺陷型 CRISPR/Cas 的转录调控是常规基因表达控制的常用且有价值的工具。 CRISPR 对细菌的干扰可以可靠且高效地实现。然而，CRISPR 激活 (CRISPRa) 的选择在灵活性和活性方面仍然有限，因为它们依赖于 sigma(70) 启动子。在这里，我们报道了一种基于 sigma(54) 依赖性启动子的类真核细菌 CRISPRa 系统，该系统支持长距离，因此支持高动态范围的多输入调节。我们的 CRISPRa 装置可以激活非模型细菌中具有生物技术相关性的 sigma(54) 依赖性启动子。它还支持多个层面的正交基因调控。将我们的 CRISPRa 与 dxCas9 相结合，进一步扩展了 DNA 靶向的灵活性，并将动态范围增强到能够构建级联 CRISPRa 电路的机制中。在应用方面，我们构建了一个可重复使用的扫描平台，可以轻松优化代谢途径，而无需重建文库。因此，这种类真核生物 CRISPRa 系统是一种强大且多功能的合成生物学工具，适用于各种研究和工业应用。
Transcriptional regulation by nuclease-deficient CRISPR/Cas is a popular and valuable tool for routine control of gene expression. CRISPR interference in bacteria can be reliably achieved with high efficiencies. Yet, options for CRISPR activation (CRISPRa) remained limited in flexibility and activity because they relied on sigma(70) promoters. Here we report a eukaryote-like bacterial CRISPRa system based on sigma(54)-dependent promoters, which supports long distance, and hence multi-input regulation with high dynamic ranges. Our CRISPRa device can activate sigma(54)-dependent promoters with biotechnology relevance in non-model bacteria. It also supports orthogonal gene regulation on multiple levels. Combining our CRISPRa with dxCas9 further expands flexibility in DNA targeting, and boosts dynamic ranges into regimes that enable construction of cascaded CRISPRa circuits. Application-wise, we construct a reusable scanning platform for readily optimizing metabolic pathways without library reconstructions. This eukaryote-like CRISPRa system is therefore a powerful and versatile synthetic biology tool for diverse research and industrial applications.