'Class I and III Multi-subunit CRISPR-Cas Surveillance Complexes: Recognition, Cleavage, Autoimmunity and Inhibition’

“I 类和 III 类多亚基 CRISPR-Cas 监视复合物：识别、切割、自身免疫和抑制”

基本信息

批准号：
10360477
负责人：
DINSHAW J PATEL
金额：
$ 35.92万
依托单位：
SLOAN-KETTERING INST CAN RESEARCH
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-04-08 至 2024-03-31
项目状态：
已结题

项目摘要

Prokaryotic cells possess CRISPR-mediated adaptive immune systems that protect them from foreign mobile genetic elements, such as invading phages and viruses. A central feature of this immune system is RNA- guided surveillance complexes capable of targeting non-self DNA or RNA for degradation in a sequence- and site-specific manner. The effector proteins are composed of either single-subunit Cas nucleases or the more prevalent multi-subunit CRISPR surveillance ribonucleoprotein complexes, together with either their intrinsic cis-acting or associated trans-acting helicase-nucleases. This application focuses on cryo-EM structural and biochemical (structure-guided interfacial mutational) studies to elucidate mechanistic insights related to dsDNA targeting by type I-F and ssRNA/ssDNA targeting by type III-A multi-subunit CRISPR systems, together with insights into cleavage mechanisms, as well as cleavage inhibition by phage-evolved anti-CRISPR proteins. Currently, the type III-A Csm is much less well characterized relative to its type-IIIB Cmr counterpart. We have recently solved cryo-EM based structures of crRNA-bound type III-A Csm (labeled CsmcrRNA) from T. onnurneus and its complexes with target RNA. In Aim 1 we propose to extend these studies to address structure-guided mechanistic issues related to the origins of autoimmunity suppression given that type III systems unlike type I lack a PAM sequence, to decipher the principles underlying target RNA-mediated transcription-coupled activation of ssDNA activity, as well as the generation of second messenger cyclic oligoadenyates, which in turn activate the nonspecific RNA degradation activity of trans-acting nuclease Csm6. We have recently solved cryo-EM based structures of crRNA-bound type I-F Csy complex (labeled CsycrRNA) from P. aeruginosa in the absence and presence of partial R-loop dsDNA and identified recognition principles and associated conformational transitions on ternary complex formation. Aim 2 focuses on extending this research to structures and conformational transitions of CsycrRNA on binding full R-loop dsDNA in the absence and presence of trans-acting helicase-nuclease Cas3. These efforts should address the principles underlying non-target DNA strand displacement and R-loop positioning for recognition and cleavage by Cas3. We have recently solved cryo-EM based structures of type I-F CsycrRNA with bound anti-CRISPR AcrF proteins 1, 2 and 10, thereby identifying alternate strategies utilized by AcrF suppressors for targeting and blocking different features of the dsDNA recognition machinery. Aim 3 focuses on a structure-based mechanistic understanding of the function of additional anti-CRISPR AcrF proteins 6, 7, 8 and 9 targeted to CsycrRNA, with the potential for identifying alternate anti-CRISPR approaches, including allosteric inhibition, for dsDNA cleavage suppression. To date, there have been no reports of anti-CRISPRs that target type III CRISPR-Cas systems, but should these be identified, we plan to extend our structural studies to these complexes towards characterization of the range of anti-CRISPR strategies for shutting down the type III CRISPR-Cas pathway.

实质性细胞具有CRISPR介导的适应性免疫系统，可保护它们免受外国移动的影响遗传因素，例如入侵噬菌体和病毒。该免疫系统的主要特征是RNA- 能够靶向非自身DNA或RNA的指导监视复合物以序列和特定地点的方式。效应子蛋白由单人物Cas核酸酶或更多普遍的多生育CRISPR监测核糖核蛋白复合物以及它们的内在顺式作用或相关的反式旋转酶核酸酶。该应用的重点是冷冻EM结构和生化（结构引导的界面突变）研究，以阐明与DSDNA相关的机械见解按I型I-F和SSRNA/SSDNA靶向IIII-A型Multi-Acrispr Systems，以及洞悉裂解机制，以及通过噬菌体发展的抗Crispr蛋白的裂解。当前，III-A型CSM相对于IIIIB型CMR对应物的特征远不那么好。我们有最近从T。 Onnurneus及其与靶RNA的复合物。在AIM 1中，我们建议扩展这些研究以解决鉴于III型，结构引导的机械问题与自身免疫抑制的起源有关与I型不同的系统缺乏PAM序列，以破译目标RNA介导的原理 ssDNA活性的转录耦合激活以及第二质体环状的产生寡载体，这又激活了反式核酸酶CSM6的非特异性RNA降解活性。我们最近解决了CRRNA结合的I型I型CSY复合物（标记的Csycrrna）的基于冷冻EM的结构在不存在和存在部分R环DsDNA的情况下，从铜绿假单胞菌识别出识别原理以及对三元复合物形成的相关构象转变。 AIM 2专注于扩展这一点在不存在的情况下，对结合完整的R环DsDNA的结构和构象过渡的研究以及跨作用解旋酶核酸酶Cas3的存在。这些努力应解决基本原则非目标DNA链位移和R环定位，以通过CAS3识别和切割。我们最近解决了I型型csycrrna的基于冷冻EM的结构 1、2和10，从而确定ACRF抑制器用于靶向和阻止的替代策略 DSDNA识别机械的不同功能。 AIM 3专注于基于结构的机械了解其他抗危机ACRF蛋白6、7、8和9的功能，其针对csycrrna的功能识别DSDNA的替代抗危机方法（包括变构抑制）的潜力切割抑制。迄今为止，尚无针对III型CRISPR-CAS的反侵略性的报道系统，但如果要确定这些，我们计划将我们的结构研究扩展到这些复合物对关闭III型CRISPR-CAS途径的抗Crispr策略范围的表征。