'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
- 来源:
- 关键词:Adaptive Immune SystemAddressArchaeaArchitectureAutoimmunityBacteriaBacteriophagesBindingBiochemicalCRISPR/Cas technologyClustered Regularly Interspaced Short Palindromic RepeatsComplexCoupledCryoelectron MicroscopyCrystallizationDNAData CollectionEscherichia coliEvaluationEventGenerationsGenetic TranscriptionGenome engineeringGuide RNAHost DefenseImmune systemIndividualInfectionInstitutionInvadedInvestigationLabelLettersLiteratureMediatingMethodologyMobile Genetic ElementsMolecularMutationNatureNew YorkNucleic AcidsNucleotidesPathway interactionsPeriodicityPositioning AttributePreparationPreventionProkaryotic CellsProteinsPseudomonas aeruginosaRNARNA DegradationRegulationReportingResearchResolutionRibonucleoproteinsSecond Messenger SystemsSignal PathwaySiteStructureSystemTitanTransactivationViralVirusarms racebasecomputerized data processingconformational conversionds-DNAgenome editinghelicaseinsightinstrumentinterfacialnucleaseoligoadenylateprogramsscaffoldstructural biology
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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{{ 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:
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 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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