CRISPR-Cas13 immunity in Listeria seeligeri

摘要 CRISPR系统是原核适应性免疫系统，其使用RNA引导的Cas核酸酶来 识别并破坏含有与指导物互补的序列的噬菌体核酸 核糖核酸不同细菌所携带的CRISPR系统可能极其多样化，并且使用不同的 策略来中和感染性的病毒。CRISPR系统在传统模型中没有很好的代表性 细菌因此，它们的功能通常通过在大肠杆菌中异源过表达来研究。杆菌 因此，我们对生物共同进化所产生的复杂相互作用的了解有限。 CRISPR-Cas系统与它们的天然宿主以及感染它们的病毒。研究在我 该实验室专注于建立自然模型，以研究CRISPR-Cas之间的界面。 免疫、细菌宿主生理学和噬菌体感染。虽然六种CRISPR类型中的大多数使用Cas DNA酶识别和切割噬菌体DNA，VI型CRISPR系统使用核酸酶Cas13， 而不是切割RNA。我已经开发了VI型CRISPR系统的天然细菌宿主李斯特菌 seeligeri，以及它的一个集合，作为一个易于处理的模型，用于研究这个系统如何保护 抵抗感染一旦Cas13与靶病毒RNA结合，它就被激活为非特异性RNA酶， 导致噬菌体和细菌RNA的广泛切割以及噬菌体的败育 生命周期因此，具有VI型免疫力的感染细胞不裂解，并且不能产生病毒子代，但 停止生长进入休眠状态本提案的目标是（i）确定哪些成绩单 Cas13切割触发进入休眠;（ii）了解L. seeligeri细胞在休眠状态下存活 状态，并复苏自己一旦噬菌体已被消除，和（iii）发现和 表征控制L. seeligeri及其 - 是的这项研究产生的结果将为分子生物学提供基本的见解。 RNA靶向CRISPR系统的生物学，并帮助其作为生物技术工具的发展。最后， Cas13诱导的细胞休眠与称为持久性的现象相似，其中 病原菌的亚群停止生长，并变得暂时耐受杀菌剂。 抗生素在人类感染中的作用因此，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， 这将代表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.