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'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.

原代细胞具有CRISPR介导的适应性免疫系统，可以保护它们免受外来移动的攻击。遗传因素，如入侵的细菌和病毒。这种免疫系统的核心特征是RNA- 能够靶向非自身DNA或RNA以在序列中降解的引导监视复合物，具体的方式。效应蛋白由单亚基Cas核酸酶或多亚基Cas核酸酶组成。普遍存在的多亚基CRISPR监视核糖核蛋白复合物，连同它们的内在顺式作用或相关的反式作用解旋酶-核酸酶。该应用程序的重点是冷冻EM结构和生物化学（结构引导的界面突变）研究，以阐明与dsDNA相关的机制见解 I-F型靶向和III-A型多亚基CRISPR系统靶向ssRNA/ssDNA，以及裂解机制的见解，以及噬菌体进化的抗CRISPR蛋白的裂解抑制。目前，III-A型Csm相对于其IIIB型Cmr对应物的特征要少得多。我们有最近解决的crRNA结合的III-A型Csm（标记的CsmcrRNA）的基于cryo-EM的结构来自T. onnurneus及其与靶RNA的复合物。在目标1中，我们建议扩展这些研究，以解决结构指导的机制问题与自身免疫抑制的起源，鉴于III型与I型系统不同的是，I型系统缺乏PAM序列，以破译靶RNA介导的靶分子的基本原理。转录偶联激活ssDNA活性，以及产生第二信使环寡聚腺苷酸，这反过来又激活反式作用核酸酶Csm 6的非特异性RNA降解活性。我们最近已经解决了crRNA结合的I-F型Csy复合物（标记的CsycrRNA）的基于cryo-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中分离，并鉴定识别原则以及三元复合物形成的相关构象转变。目标2的重点是扩展这一点 CsycrRNA的结构及其与全R环双链DNA结合时构象变化的研究和反式作用解旋酶-核酸酶Cas 3的存在。这些努力应涉及基本原则，非靶DNA链置换和R环定位，用于通过Cas 3识别和切割。我们最近已经解决了具有结合的抗CRISPR AcrF蛋白的I-F型CsycrRNA的基于冷冻-EM的结构 1、2和10，从而确定AcrF抑制因子用于靶向和阻断的替代策略 dsDNA识别机制的不同特征。目标3侧重于基于结构的机制理解靶向CsycrRNA的另外的抗CRISPR AcrF蛋白6、7、8和9的功能，确定替代抗CRISPR方法的潜力，包括dsDNA的变构抑制卵裂抑制迄今为止，还没有关于靶向III型CRISPR-Cas的抗CRISPR的报道。系统，但如果这些被确定，我们计划将我们的结构研究扩展到这些复合物，本发明提供了用于关闭III型CRISPR-Cas途径的抗CRISPR策略范围的表征。