'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:9906243
- 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 2023-03-31
- 项目状态:已结题
- 来源:
- 关键词:Adaptive Immune SystemAddressAntiviral AgentsArchaeaArchitectureAutoimmunityBacteriaBacteriophagesBindingBiochemicalCleaved cellClustered Regularly Interspaced Short Palindromic RepeatsComplexCoupledCryoelectron MicroscopyCrystallizationDNAData CollectionEscherichia coliEvaluationEventGenerationsGenetic TranscriptionGenome engineeringGuide RNAHost DefenseImmune systemIndividualInfectionInstitutionInvadedInvestigationLabelLettersLiteratureMediatingMethodologyMobile Genetic ElementsMolecularMutationNatureNew YorkNucleic AcidsNucleotidesPathway interactionsPeriodicityPositioning AttributePreparationPreventionProkaryotic CellsProteinsPseudomonas aeruginosaRNARNA DegradationRaceRegulationReportingResearchResolutionRibonucleoproteinsSecond Messenger SystemsSignal PathwaySiteStructureSystemTitanTransactivationVirusarmbasecomputerized 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。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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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:
10360477 - 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
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8169226 - 财政年份:2010
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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
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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
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7955161 - 财政年份:2009
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组蛋白/表观遗传学密码中的识别事件
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7955105 - 财政年份:2009
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$ 35.92万 - 项目类别:
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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:
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