高通量筛选揭示了通过靶向 CRISPR 前导序列抑制 Rho 终止来调节 crRNA 生物发生的 sRNAs。
High-throughput screen reveals sRNAs regulating crRNA biogenesis by targeting CRISPR leader to repress Rho termination.
机构信息
State Key Laboratory of Trauma, Burns and Combined Injury, Institute of Surgery Research, Daping Hospital, Army Medical University, Chongqing, China.
Department of Biomedical Sciences, School of Medicine and Health Sciences, University of North Dakota, Grand Forks, ND, USA.
出版信息
Nat Commun. 2019 Aug 19;10(1):3728. doi: 10.1038/s41467-019-11695-8.
Discovery of CRISPR-Cas systems is one of paramount importance in the field of microbiology. Currently, how CRISPR-Cas systems are finely regulated remains to be defined. Here we use small regulatory RNA (sRNA) library to screen sRNAs targeting type I-F CRISPR-Cas system through proximity ligation by T4 RNA ligase and find 34 sRNAs linking to CRISPR loci. Among 34 sRNAs for potential regulators of CRISPR, sRNA pant463 and PhrS enhance CRISPR loci transcription, while pant391 represses their transcription. We identify PhrS as a regulator of CRISPR-Cas by binding CRISPR leaders to suppress Rho-dependent transcription termination. PhrS-mediated anti-termination facilitates CRISPR locus transcription to generate CRISPR RNA (crRNA) and subsequently promotes CRISPR-Cas adaptive immunity against bacteriophage invasion. Furthermore, this also exists in type I-C/-E CRISPR-Cas, suggesting general regulatory mechanisms in bacteria kingdom. Our findings identify sRNAs as important regulators of CRISPR-Cas, extending roles of sRNAs in controlling bacterial physiology by promoting CRISPR-Cas adaptation priming.
CRISPR-Cas 系统的发现是微生物学领域的一个重大突破。目前，CRISPR-Cas 系统的精细调控机制仍有待确定。在这里，我们通过 T4 RNA 连接酶的邻近连接，使用小型调控 RNA (sRNA) 文库筛选靶向 I-F 型 CRISPR-Cas 系统的 sRNA，发现了 34 个与 CRISPR 基因座相关的 sRNA。在 34 个可能调控 CRISPR 的 sRNA 中，sRNA pant463 和 PhrS 增强了 CRISPR 基因座的转录，而 pant391 则抑制了它们的转录。我们确定 PhrS 通过与 CRISPR 先导序列结合抑制 Rho 依赖的转录终止，从而成为 CRISPR-Cas 的调控因子。PhrS 介导的反终止作用促进了 CRISPR 基因座的转录，产生 CRISPR RNA (crRNA)，并随后促进了 CRISPR-Cas 对噬菌体入侵的适应性免疫。此外，这种现象也存在于 I-C/-E 型 CRISPR-Cas 中，这表明在细菌王国中存在普遍的调控机制。我们的研究结果表明 sRNA 是 CRISPR-Cas 的重要调控因子，通过促进 CRISPR-Cas 的适应性启动，扩展了 sRNA 在控制细菌生理方面的作用。
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