'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.

原核细胞拥有 CRISPR 介导的适应性免疫系统，可保护它们免受外来移动的侵害 遗传因素，例如入侵的噬菌体和病毒。该免疫系统的一个核心特征是RNA- 能够靶向非自身 DNA 或 RNA 进行序列降解的引导监视复合体 特定地点的方式。效应蛋白由单亚基 Cas 核酸酶或多亚基组成 流行的多亚基 CRISPR 监视核糖核蛋白复合物，及其内在的 顺式作用或相关反式作用解旋酶-核酸酶。该应用重点关注冷冻电镜结构和 生物化学（结构引导的界面突变）研究阐明与 dsDNA 相关的机制见解 I-F 型靶向和 III-A 型多亚基 CRISPR 系统的 ssRNA/ssDNA 靶向，以及 深入了解裂解机制以及噬菌体进化的抗 CRISPR 蛋白的裂解抑制作用。 目前，与 IIIB 型 Cmr 对应物相比，III-A 型 Csm 的表征要少得多。我们有 最近解决了来自 T. crRNA 结合的 III-A 型 Csm（标记为 CsmcrRNA）的基于冷冻电镜的结构。 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 鉴于 III 型，与自身免疫抑制起源相关的结构引导机制问题 与 I 型系统不同，系统缺乏 PAM 序列，无法破译靶标 RNA 介导的原理 ssDNA 活性的转录偶联激活，以及第二信使循环的产生 寡腺苷酸，进而激活反式作用核酸酶 Csm6 的非特异性 RNA 降解活性。 我们最近解决了基于冷冻电镜的 crRNA 结合型 I-F Csy 复合物（标记为 CsycrRNA）的结构 在部分 R 环 dsDNA 不存在和存在的情况下从铜绿假单胞菌中分离并确定识别原理 以及三元复合物形成的相关构象转变。目标 2 侧重于扩展这一点 研究 CsycrRNA 在缺乏时结合完整 R 环 dsDNA 的结构和构象转变 以及反式作用解旋酶核酸酶 Cas3 的存在。这些努力应涉及基本原则 用于 Cas3 识别和切割的非目标 DNA 链置换和 R 环定位。 我们最近解决了基于冷冻电镜的 I-F 型 CsycrRNA 结构与结合的抗 CRISPR AcrF 蛋白 1、2 和 10，从而确定 AcrF 抑制剂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 了解针对 CsycrRNA 的其他抗 CRISPR AcrF 蛋白 6、7、8 和 9 的功能， 识别双链 DNA 替代抗 CRISPR 方法（包括变构抑制）的潜力 裂解抑制。迄今为止，还没有针对 III 型 CRISPR-Cas 的抗 CRISPR 的报道 系统，但如果这些被确定，我们计划将我们的结构研究扩展到这些复合体 用于关闭 III 型 CRISPR-Cas 途径的一系列抗 CRISPR 策略的表征。