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片段（称为间隔段）被捕获并用作序列特异性免疫记忆来识别和破坏匹配序列时，细菌就会“免疫”。该项目将研究多种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应用于生物技术。了解这些系统是如何被它们的自然宿主编程的，将更好地使研究人员在体内产生不同的CRISPR文库，并将有利于基于CRISPR的技术，旨在细胞历史的分子记录。本项目的教育计划包括基于课程的研究体验，本科生将系统地测量针对不同病毒的不同CRISPR类型的干扰。本课程将让学生亲身体验分子生物学和病毒感染的概念，并为学生提供快速生成和发布新数据的明确框架。这项研究的目的是了解不同的CRISPR类型如何从外源遗传元件中获得新的间隔序列。seeligeri李斯特菌及其噬菌体是一种易于处理的模型，用于研究四种不同的CRISPR类型，包括靶向rna的CRISPR- cas13系统。这种模式细菌对于研究cas13介导的干扰在VI型CRISPR免疫过程中的机制和后果非常有用。然而，建立免疫的初始间隔物获取事件背后的过程仍不清楚。该方案将采用新一代测序技术，以高灵敏度检测罕见采集事件。将对crispr相关基因进行系统诱变，以评估其对免疫的贡献。CRISPR-Cas13系统独特地靶向RNA而不是DNA，并且将检查获得的间隔序列，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，将对获得的间隔物进行生化重组，以研究间隔物整合位点选择的机制基础。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值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.