'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”

• 批准号: 9906243
• 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
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-08 至 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9906243
• 关键词: Adaptive Immune System; Address; Antiviral Agents; Archaea; Architecture; Autoimmunity; Bacteria; Bacteriophages; Binding; Biochemical; Cleaved cell; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Coupled; Cryoelectron Microscopy; Crystallization; DNA; Data Collection; Escherichia coli; Evaluation; Event; Generations; Genetic Transcription; Genome engineering; Guide RNA; Host Defense; Immune system; Individual; Infection; Institution; Invaded; Investigation; Label; Letters; Literature; Mediating; Methodology; Mobile Genetic Elements; Molecular; Mutation; Nature; New York; Nucleic Acids; Nucleotides; Pathway interactions; Periodicity; Positioning Attribute; Preparation; Prevention; Prokaryotic Cells; Proteins; Pseudomonas aeruginosa; RNA; RNA Degradation; Race; Regulation; Reporting; Research; Resolution; Ribonucleoproteins; Second Messenger Systems; Signal Pathway; Site; Structure; System; Titan; Transactivation; Virus; arm; base; computerized data processing; conformational conversion; ds-DNA; genome editing; helicase; insight; instrument; interfacial; nuclease; oligoadenylate; programs; scaffold; structural biology

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.

原核细胞具有CRISPR介导的适应性免疫系统，可保护它们免受外来移动的影响 遗传因素，如入侵的噬菌体和病毒。这种免疫系统的一个中心特征是RNA-- 能够针对非自身DNA或RNA进行序列降解的制导监视复合体--以及 特定于站点的方式。效应蛋白由单亚基的CaS核酸酶或更多的 流行的多亚基CRISPR监测核糖核蛋白复合体及其固有的 顺式作用或相关的反式作用的解旋酶-核酸酶。这项应用主要针对低温电磁结构和 生物化学(结构导向界面突变)研究以阐明与dsDNA相关的机制 I-F型靶向和III-A型多亚单位CRISPR系统的ssRNA/ssDNA靶向，以及 对切割机制的洞察，以及噬菌体进化的抗CRISPR蛋白对切割的抑制。 目前，与IIIB Cmr对应的类型相比，III-A型CSM的特征要差得多。我们有 最近从T.解决了crRNA结合的III-A型CSM(标记为CsmcrRNA)的低温EM结构。 鱼腥草及其与靶RNA的络合物。在目标1中，我们建议将这些研究扩大到 与自身免疫抑制起源有关的结构引导的机制问题 与I型不同的系统缺乏PAM序列，无法破译靶向RNA介导的潜在原理 转录偶联激活单链DNA活性，以及第二信使环的产生 寡腺苷，进而激活反式核酸酶Csm6的非特异性RNA降解活性。 我们最近解决了crRNA结合型I-F CSY络合物(标记为CsycrRNA)的低温EM结构。 铜绿假单胞菌在不存在和存在部分R环dsDNA的情况下的识别原理 以及相关的三元络合物形成上的构象转变。目标2专注于扩展这一点 缺失CsycrRNA与全R-环dsDNA结合的结构和构象转变研究 以及反式解旋酶-核酸酶Cas3的存在。这些努力应解决基本原则 非靶DNA链置换和R环定位，用于识别和切割Cas3。 我们最近解决了基于冷冻EM的I-F型CsycrRNA与结合的抗CRISPR ACRF蛋白的结构 1、2和10，从而识别ACRF抑制者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械论 了解针对CsycrRNA的额外抗CRISPR ACRF蛋白6、7、8和9的功能， 确定替代的抗CRISPR方法的可能性，包括变构抑制dsDNA 卵裂抑制。迄今为止，还没有针对III型CRISPR-CAS的抗CRISPR的报道 系统，但如果这些系统被确定，我们计划将我们的结构研究扩展到这些复合体，以 阻断III型CRISPR-Cas通路的一系列抗CRISPR策略的特征。