CRISPR-Cas13 immunity in Listeria seeligeri

摘要 CRISPR系统是原核生物适应性免疫系统，它使用RNA引导的Cas核酸酶来 识别和破坏含有序列互补性的噬菌体核酸 核糖核酸。不同细菌所拥有的CRISPR系统可以非常多样化，并使用不同的 中和感染噬菌体的策略。CRISPR系统在传统模型中没有得到很好的表现 细菌。因此，它们的功能通常是通过在大肠杆菌中异源过表达来研究的。 因此，我们对人类共同进化所产生的复杂相互作用的了解有限。 CRISPR-CAS系统及其自然宿主和感染它们的噬菌体。我的研究 实验室致力于建立自然模型来研究CRISPR-CAS之间的界面 免疫、细菌寄主生理学和噬菌体感染。而六种CRISPR类型中的大多数使用CA 为了识别和切割噬菌体DNA，VI型CRISPR系统使用核酸酶Cas13来 取而代之的是切断RNA。我已经开发了一种VI型CRISPR系统的天然细菌宿主，李斯特菌 和它的一组噬菌体，作为研究这个系统如何保护 抗感染。一旦Cas13与靶病毒RNA结合，它就会被激活为非特异性核糖核酸酶， 导致噬菌体和细菌RNA的广泛切割以及噬菌体的流产 生命周期。因此，具有VI型免疫力的感染细胞不能溶解，也不能产生病毒后代，但 停止生长，进入休眠状态。这项提案的目标是(I)确定哪些成绩单 被Cas13裂解触发进入休眠；(Ii)了解L.seigeri细胞如何在休眠中存活 状态，并在噬菌体被消灭后复活自己，以及(Iii)发现和 塞利格里乳杆菌中控制Cas13活性的内源调控机制及其特性 噬菌体。这项研究产生的结果将提供对分子的基本见解 以RNA为靶标的CRISPR系统的生物学，并协助将其发展为生物技术工具。最后， Cas13诱导的细胞休眠类似于一种称为持续的现象，在这种现象中 病原菌亚群停止生长并对杀菌剂产生瞬时耐受性 在人类感染期间使用抗生素。因此，这里提出的研究可以揭示一般的 持久性细菌在抗生素暴露下存活并重新进入生长周期的机制， 这将是治疗干预的有吸引力的靶点。

ABSTRACT CRISPR systems are prokaryotic adaptive immune systems that use RNA-guided Cas nucleases to recognize and destroy bacteriophage nucleic acids containing sequence complementarity to the guide RNA. CRISPR systems harbored by different bacteria can be extremely diverse and use different strategies to neutralize infecting phages. CRISPR systems are not well-represented in traditional model bacteria. As such, their function has typically been studied by heterologous overexpression in E. coli. Accordingly, we have limited knowledge of the complex interactions that arose from the co-evolution of CRISPR-Cas systems with their natural hosts and the phages that infect them. Research in my laboratory focuses on establishing natural models to investigate the interfaces between CRISPR-Cas immunity, bacterial host physiology, and phage infection. While most of the six CRISPR types use Cas DNases to recognize and cleave phage DNA, the type VI CRISPR system uses the nuclease Cas13 to cut RNA instead. I have developed a natural bacterial host of the type VI CRISPR system, Listeria seeligeri, and a collection of its phages as a tractable model for studying how this system protects against infection. Once Cas13 engages target viral RNA, it becomes activated as a non-specific RNase, resulting in widespread cleavage of both phage and bacterial RNA and the abortion of the phage lifecycle. Thus, infected cells with type VI immunity do not lyse, and fail to produce viral progeny, but stop growing and become dormant. The goals of this proposal are to (i) determine which transcripts cleaved by Cas13 trigger entry into dormancy; (ii) understand how L. seeligeri cells survive the dormant state, and resuscitate themselves once the phage has been eliminated, and (iii) discover and characterize endogenous regulatory mechanisms controlling Cas13 activity in L. seeligeri and its phages. The results generated by this research will provide fundamental insights into the molecular biology of RNA-targeting CRISPR systems and aid in their development as biotechnology tools. Finally, Cas13-induced cellular dormancy bears similarity to a phenomenon termed persistence, in which subpopulations of pathogenic bacteria stop growing and become transiently tolerant of bactericidal antibiotics during human infection. Therefore, the studies proposed here could reveal general mechanisms by which persistent bacteria survive antibiotic exposure and re-enter the growth cycle, which would represent attractive targets for therapeutic intervention.