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：
Giulia Palermo
金额：
$ 28.2万
依托单位：
UNIVERSITY OF CALIFORNIA RIVERSIDE
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：
https://reporter.nih.gov/project-details/10592389
关键词：
Acceleration Address Affect Allosteric Regulation Applied Research Basic Science Behavior Binding Biological Sciences Biology Biophysics CRISPR/Cas technology Cancer Patient Catalysis Catalytic Domain Cells Clustered Regularly Interspaced Short Palindromic Repeats Collaborations Complementary DNA Complex Computer Simulation Computing Methodologies DNA DNA Binding DNA Sequence DNA Sequence Alteration Data Dependence Development Engineering Enzymes Event Exhibits Fostering Free Energy Gene Expression Regulation Genetic Diseases Genome Grant Guide RNA Human Genetics Investigation Ionic Strengths Ions Kinetics Knowledge Malignant Neoplasms Measurement Mediating Metabolism Metals Methods Molecular Conformation Nerve Degeneration Nucleic Acid Binding Nucleic Acids Organism Outcome Proteins Protocols documentation Quantum Mechanics Research Ribonucleoproteins Role Safety Sampling Scientist Series Signal Transduction Site Specificity System T-Lymphocyte Technology Theoretical Studies Variant biophysical analysis biophysical properties clinical application clinical efficacy computer framework computer studies dalton divalent metal endonuclease functional improvement genome editing graph theory improved inhibitor innovation molecular dynamics molecular mechanics nanosecond network models novel nuclease prevent tool transmission process uptake viral 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通过实现 DNA 校正，在基础科学和应用科学领域具有前沿影响力。突变，这项技术有望治疗多种人类遗传疾病，如第一个所示癌症患者接受 CRISPR-Cas9 修饰的 T 细胞治疗。核酸内切酶 Cas9，与引导 RNA 结合以识别和切割互补 DNA CRISPR-Cas9工具的不断开发和工程开启了新的领域。有趣的假设可以对系统进行深入研究，PI 将在此实施。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我们将追求三个具体目标，其特征是：（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结合；（目标 2）新设计的 Cas9 变体具有增强的变构调节能力特异性；（目标3）通过天然存在的抗CRISPR蛋白来实现的抑制机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）此外，从头开始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远距离传输的情况。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 系统有望密切进行基因组编辑和病毒检测的理论研究。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总体而言，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。