'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。
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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$ 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:
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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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