Investigating the metal-dependent function, allostery and inhibition of CRISPR-Cas9
研究 CRISPR-Cas9 的金属依赖性功能、变构和抑制
基本信息
- 批准号:10378667
- 负责人:
- 金额:$ 28.26万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2021
- 资助国家:美国
- 起止时间:2021-04-01 至 2026-03-31
- 项目状态:未结题
- 来源:
- 关键词:AddressAffectAllosteric RegulationApplied ResearchBasic ScienceBehaviorBindingBiological SciencesBiologyBiophysicsCRISPR/Cas technologyCancer PatientCatalysisCatalytic DomainCellsClustered Regularly Interspaced Short Palindromic RepeatsCollaborationsComplementary DNAComplexComputersComputing MethodologiesDNADNA BindingDNA SequenceDNA Sequence AlterationDataDependenceDevelopmentEngineeringEnzymesEventExhibitsFosteringFree EnergyGene Expression RegulationGenetic DiseasesGenomeGrantGuide RNAHuman GeneticsInvestigationIonic StrengthsIonsKineticsKnowledgeMalignant NeoplasmsMeasurementMediatingMetabolismMetalsMethodsMolecular ConformationNerve DegenerationNucleic Acid BindingNucleic AcidsOrganismOutcomeProteinsProtocols documentationQuantum MechanicsResearchRibonucleoproteinsRoleSafetySamplingScientistSeriesSignal TransductionSiteSpecificitySystemT-LymphocyteTechnologyTheoretical StudiesVariantbasebiophysical analysisbiophysical propertiesclinical applicationclinical efficacycomputer frameworkcomputer studiesdaltondivalent metalendonucleasegenome editinggraph theoryimprovedimproved functioninginhibitorinnovationmolecular dynamicsmolecular mechanicsnanosecondnetwork modelsnovelnucleasepreventtooltransmission processuptakeviral detection
项目摘要
Abstract
CRISPR-Cas9 is the core of a transformative genome editing technology that is innovating life
science with cutting-edge impact in basic and applied sciences. By enabling the correction of DNA
mutations, this technology promises to treat a myriad of human genetic diseases, as shown for the first
cancer patients treated with CRISPR-Cas9–modified T-cells. This technology is based on the
endonuclease Cas9, which associates with guide RNAs to recognize and cleave complementary DNA
sequences. Ceaseless development and engineering of CRISPR-Cas9 tools has opened novel
intriguing hypotheses that grant in-depth investigations of the system. Here, the PI will implement
unconventional multiscale approaches, combining a variety of state-of-the-art theoretical methods, to
clarify the metal-dependent catalysis, the allostery in the selectivity mechanisms, as well as the inhibition
of the system. We will pursue three specific aims, characterizing: (Aim 1) the DNA cleavage dependency
on alternative divalent metal ions other than Mg2+ and the conformational effects associated with their
binding; (Aim 2) the allosteric modulation witnessed in newly engineered Cas9 variants with enhanced
specificity; (Aim 3) the inhibition mechanism by naturally occurring anti-CRISPR proteins to implement
control over gene regulation. Toward these aims, we will leverage classical and enhanced sampling
molecular dynamics (MD) simulations, high-level ab-initio MD (using the Car-Parrinello and Born-
Oppenheimer approaches) and mixed quantum mechanics/molecular mechanics (QM/MM)
approaches. Moreover, combination of ab-initio MD with graph theory will implement a synergistic
approach capturing instantaneous sub-nanosecond signaling transfers. This will reveal how long-range
allosteric effects impact the dynamics through evolving catalytic steps, elucidating the role of allostery
in aiding catalysis. These multiscale approaches will offer a computational framework for the biophysical
analysis of not only CRISPR-Cas9, but can also be extended to emerging CRISPR systems that are
promising for genome editing and viral detection. Theoretical studies will be performed in close
collaboration with experimental scientists, providing kinetic measurements and biophysical
characterization, assisting in the interpretation of the experimental data and enabling testable
predictions. Overall, this proposed research will expand the repertoire of mechanistic knowledge
regarding the CRISPR-Cas9 function and lay the framework for novel engineering rationales toward
improved genome editing.
摘要
CRISPR-Cas9是一种变革性基因组编辑技术的核心,
在基础科学和应用科学领域具有前沿影响的科学。通过纠正DNA
突变,这项技术有望治疗无数的人类遗传疾病,如第一个显示的那样。
用CRISPR-Cas9修饰的T细胞治疗的癌症患者。这项技术是基于
内切核酸酶Cas9,其与引导RNA结合以识别和切割互补DNA
序列的CRISPR-Cas9工具的不断开发和工程化为人类开启了新的
有趣的假设,授权深入调查的系统。在这里,PI将实现
非传统的多尺度方法,结合各种最先进的理论方法,
阐明了金属依赖性催化、选择性机制中的变构以及抑制作用,
该系统我们将追求三个具体目标,表征:(目标1)DNA切割依赖性
对Mg 2+以外的替代性二价金属离子及其构象效应的影响
(目的2)在新工程化的Cas9变体中观察到的变构调节,
(目的3)通过天然存在的抗CRISPR蛋白实现的抑制机制
控制基因调控。为了实现这些目标,我们将利用经典和增强的采样
分子动力学(MD)模拟,高级从头算MD(使用Car-Parrinello和Born-
奥本海默方法)和混合量子力学/分子力学(QM/MM)
接近。此外,结合从头算分子动力学与图论将实现协同
方法捕捉瞬时的亚纳秒信号传输。这将揭示远程
变构效应通过进化的催化步骤影响动力学,阐明了变构的作用
帮助催化。这些多尺度方法将为生物物理学提供一个计算框架。
不仅可以分析CRISPR-Cas9,还可以扩展到新兴的CRISPR系统,
有望用于基因组编辑和病毒检测。理论研究将在密切
与实验科学家合作,提供动力学测量和生物物理
表征,协助解释实验数据,并使可测试的
预测。总的来说,这项拟议的研究将扩大机械知识的剧目
关于CRISPR-Cas9功能,并为新的工程原理奠定框架,
改进的基因组编辑。
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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Giulia Palermo其他文献
Giulia Palermo的其他文献
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{{ truncateString('Giulia Palermo', 18)}}的其他基金
Investigating the metal-dependent function, allostery and inhibition of CRISPR-Cas9
研究 CRISPR-Cas9 的金属依赖性功能、变构和抑制
- 批准号:
10186224 - 财政年份:2021
- 资助金额:
$ 28.26万 - 项目类别:
Investigating the metal-dependent function, allostery and inhibition of CRISPR-Cas9
研究 CRISPR-Cas9 的金属依赖性功能、变构和抑制
- 批准号:
10797907 - 财政年份:2021
- 资助金额:
$ 28.26万 - 项目类别:
Investigating the metal-dependent function, allostery and inhibition of CRISPR-Cas9
研究 CRISPR-Cas9 的金属依赖性功能、变构和抑制
- 批准号:
10592389 - 财政年份:2021
- 资助金额:
$ 28.26万 - 项目类别:
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