CAREER: Adaptive and regulatory diversity in natural CRISPR-Cas systems

CRISPR-CAS系统是广泛存在于微生物基因组中的抗病毒免疫系统。在病毒感染期间，当入侵DNA的一段(称为间隔区)被捕获并用作序列特定的免疫记忆以识别和破坏匹配的序列时，细菌就被“免疫”了。该项目将研究不同的CRISPR系统获得免疫记忆的机制，并发现细菌用来控制这一过程的因素。CRISPR-Cas核酸酶的可编程序列特异性核酸识别和生化活性已被广泛的生物技术所利用。了解这些系统是如何由其自然宿主编程的，将使研究人员能够更好地在体内生成不同的CRISPR文库，并将有利于旨在分子记录细胞历史的CRISPR技术。该项目的这个教育计划由一个基于课程的研究经验组成，在这个经验中，本科生参与者将系统地测量不同类型的CRISPR针对不同病毒的干扰。本课程将让学生亲身体验分子生物学和病毒感染的概念，并为学生快速生成和发布新数据提供一个明确的框架。本研究的目标是了解不同类型的CRISPR如何从外来基因元素中获得新的间隔区。塞利格里李斯特菌及其噬菌体是研究四种不同CRISPR类型的简便模型，包括RNA靶向CRISPR-Cas13系统。这种模式菌在研究Cas13介导的干扰VI型CRISPR免疫过程中的机制和后果方面非常有用。然而，建立免疫力的最初间隔区获得事件背后的过程仍然不清楚。这项提议将使用下一代测序来检测高灵敏度的罕见获取事件。CRISPR相关基因将被系统地诱变，以评估它们对免疫的贡献。CRISPR-Cas13系统唯一地针对RNA而不是DNA，将对获得的间隔区进行检查，以了解该系统是否以及如何指定转录目标序列的捕获。最后，空间器的获取将进行生化重组，以调查空间器集成选址的机制基础。这一奖项反映了NSF的法定使命，并已通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

CRISPR-Cas systems are anti-viral immune systems widespread in microbial genomes. Bacteria are “immunized” during viral infection when a fragment of invader DNA (called a spacer) is captured and used as a sequence-specific immunological memory to recognize and destroy matching sequences. This project will investigate the mechanisms by which diverse CRISPR systems acquire immunological memory, and to discover factors that bacteria use to control this process. The programmable sequence-specific nucleic acid recognition and biochemical activities of CRISPR-Cas nucleases have been exploited for a wide range of biotechnologies. Understanding how these systems are programmed by their natural hosts will better enable researchers to generate diverse CRISPR libraries in vivo, and will benefit CRISPR-based technologies aimed at molecular recording of cellular histories. This education plan of this project is comprised of a course-based research experience in which undergraduate participants will systematically measure interference by diverse CRISPR types targeting different viruses. This course will give students hands-on experience with the concepts of molecular biology and viral infection, and also represents a defined framework for students to rapidly generate and publish novel data.The goal of this research is to understand how diverse CRISPR types acquire new spacers from foreign genetic elements. Listeria seeligeri and its phages is a tractable model for studying four distinct CRISPR types, including the RNA-targeting CRISPR-Cas13 system. This model bacterium has been extremely useful for investigating the mechanisms and consequences of Cas13-mediated interference during type VI CRISPR immunity. However, the processes underlying the initial spacer acquisition events that establish immunity remain unclear. This proposal will employ next generation sequencing to detect rare acquisition events with high sensitivity. CRISPR-associated genes will be systematically mutagenized to assess their contributions to immunization. The CRISPR-Cas13 system uniquely targets RNA rather than DNA, and the acquired spacers will be examined to understand whether and how this system specifies the capture of transcribed target sequences. Finally, spacer acquisition will be biochemically reconstituted to investigate the mechanistic basis of spacer integration site selection.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.