'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:
  • 金额:
    $ 35.92万
  • 依托单位:
  • 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。

项目成果

期刊论文数量(2)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
Structural basis for self-cleavage prevention by tag:anti-tag pairing complementarity in type VI Cas13 CRISPR systems.
  • DOI:
    10.1016/j.molcel.2020.12.033
  • 发表时间:
    2021-03-04
  • 期刊:
  • 影响因子:
    16
  • 作者:
    Wang B;Zhang T;Yin J;Yu Y;Xu W;Ding J;Patel DJ;Yang H
  • 通讯作者:
    Yang H
Cofactor-assisted dicing: insights from structural snapshots.
辅因子辅助切割:结构快照的见解。
  • DOI:
    10.1038/s41422-022-00716-9
  • 发表时间:
    2022
  • 期刊:
  • 影响因子:
    44.1
  • 作者:
    Du,Jiamu;Patel,DinshawJ
  • 通讯作者:
    Patel,DinshawJ
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DINSHAW J PATEL其他文献

DINSHAW J PATEL的其他文献

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{{ truncateString('DINSHAW J PATEL', 18)}}的其他基金

Structure-Activity Based Mechanistic Insights into Cleavage Chemistry by Self-Cleaving Nucleolytic Ribozymes
基于结构-活性的自裂解核酶裂解化学的机理见解
  • 批准号:
    10684151
  • 财政年份:
    2022
  • 资助金额:
    $ 35.92万
  • 项目类别:
'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
  • 财政年份:
    2019
  • 资助金额:
    $ 35.92万
  • 项目类别:
STRUCTURAL BIOLOGY OF RNA-MEDIATED PROCESSES AND EPIGENETIC REGULATION
RNA介导过程的结构生物学和表观遗传调控
  • 批准号:
    8361614
  • 财政年份:
    2011
  • 资助金额:
    $ 35.92万
  • 项目类别:
STRUCTURAL BIOLOGY OF RNA SILENCING AND EPIGENETIC REGULATION
RNA 沉默和表观遗传调控的结构生物学
  • 批准号:
    8169226
  • 财政年份:
    2010
  • 资助金额:
    $ 35.92万
  • 项目类别:
BYPASS FIDELITY OF OXIDATIVE DAMAGE LESIONS BY Y-FAMILY DNA POLYMERASE
Y 家族 DNA 聚合酶绕过氧化损伤损伤的保真度
  • 批准号:
    7955159
  • 财政年份:
    2009
  • 资助金额:
    $ 35.92万
  • 项目类别:
EUBACTERIAL ARGONAUTE COMPLEXES BOUND TO GUIDE DNA AND TARGET RNA
真细菌 Argonaute 复合物结合引导 DNA 和目标 RNA
  • 批准号:
    7955161
  • 财政年份:
    2009
  • 资助金额:
    $ 35.92万
  • 项目类别:
STRUCTURE OF AN AMINO ACID-SENSING RIBOSWITCH
氨基酸感应核开关的结构
  • 批准号:
    7955160
  • 财政年份:
    2009
  • 资助金额:
    $ 35.92万
  • 项目类别:
RECOGNITION EVENTS IN THE HISTONE/EPIGENETICS CODE
组蛋白/表观遗传学密码中的识别事件
  • 批准号:
    7955105
  • 财政年份:
    2009
  • 资助金额:
    $ 35.92万
  • 项目类别:
RECOGNITION EVENTS IN RNA INTERFERENCE
RNA 干扰中的识别事件
  • 批准号:
    7955106
  • 财政年份:
    2009
  • 资助金额:
    $ 35.92万
  • 项目类别:
RECOGNITION EVENTS IN THE HISTONE/EPIGENETICS CODE
组蛋白/表观遗传学密码中的识别事件
  • 批准号:
    7721242
  • 财政年份:
    2008
  • 资助金额:
    $ 35.92万
  • 项目类别:

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