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:10592389
- 负责人:
- 金额:$ 28.2万
- 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:AccelerationAddressAffectAllosteric RegulationApplied ResearchBasic ScienceBehaviorBindingBiological SciencesBiologyBiophysicsCRISPR/Cas technologyCancer PatientCatalysisCatalytic DomainCellsClustered Regularly Interspaced Short Palindromic RepeatsCollaborationsComplementary DNAComplexComputer SimulationComputing MethodologiesDNADNA BindingDNA SequenceDNA Sequence AlterationDataDependenceDevelopmentEngineeringEnzymesEventExhibitsFosteringFree EnergyGene Expression RegulationGenetic DiseasesGenomeGrantGuide RNAHuman GeneticsInvestigationIonic StrengthsIonsKineticsKnowledgeMalignant NeoplasmsMeasurementMediatingMetabolismMetalsMethodsMolecular ConformationNerve DegenerationNucleic Acid BindingNucleic AcidsOrganismOutcomeProteinsProtocols documentationQuantum MechanicsResearchRibonucleoproteinsRoleSafetySamplingScientistSeriesSignal TransductionSiteSpecificitySystemT-LymphocyteTechnologyTheoretical StudiesVariantbiophysical analysisbiophysical propertiesclinical applicationclinical efficacycomputer frameworkcomputer studiesdaltondivalent metalendonucleasefunctional improvementgenome editinggraph theoryimprovedinhibitorinnovationmolecular 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.
抽象的
CRISPR-Cas9是革新生命的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。通过启用 DNA 校正
突变,这项技术有望治疗多种人类遗传疾病,如第一个所示
使用 CRISPR-Cas9 修饰的 T 细胞治疗癌症患者。该技术基于
核酸内切酶 Cas9,与引导 RNA 结合以识别和切割互补 DNA
序列。 CRISPR-Cas9工具的不断开发和工程化开启了新的篇章
有趣的假设可以对系统进行深入研究。在这里,PI将实施
非常规的多尺度方法,结合各种最先进的理论方法,
阐明金属依赖性催化、选择性机制中的变构以及抑制
系统的。我们将追求三个具体目标,其特点是:(目标 1)DNA 切割依赖性
对除 Mg2+ 以外的替代二价金属离子的研究以及与其相关的构象效应
绑定; (目标 2)新设计的 Cas9 变体中见证的变构调节具有增强的
特异性; (目标3)通过天然存在的抗CRISPR蛋白来实现抑制机制
对基因调控的控制。为了实现这些目标,我们将利用经典采样和增强采样
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,从头算MD与图论的结合将实现协同作用
捕获瞬时亚纳秒信号传输的方法。这将揭示多远的距离
变构效应通过不断演变的催化步骤影响动力学,阐明变构的作用
有助于催化。这些多尺度方法将为生物物理提供计算框架
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。理论研究将密切进行
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实现可测试
预测。总的来说,这项研究将扩大机械知识的范围
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:
$ 28.2万 - 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:
$ 28.2万 - 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:
10378667 - 财政年份:2021
- 资助金额:
$ 28.2万 - 项目类别:
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