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
• 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
• 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

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是一种正在创新生活的变革性基因组编辑技术的核心 在基础科学和应用科学中具有尖端影响的科学。通过启用DNA的校正 突变，这项技术有望治疗无数的人类遗传疾病，如第一次所示 用CRISPR-CAS9修饰的T细胞治疗的癌症患者。这项技术基于 核酸内切酶Cas9，它与指南RNA相关以识别并清除互补的DNA 序列。 CRISPR-CAS9工具的不断开发和工程开业 有趣的假设对系统进行了深入研究。在这里，PI将实施 非常规多尺度方法，将各种最先进的理论方法与 阐明金属依赖性催化，选择性机理的变构以及抑制作用 系统。我们将追求三个特定目标，描述：（目标1）DNA裂解依赖性 除Mg2+以外的其他二价金属离子以及与其相关的构象作用 结合； （AIM 2）在新设计的Cas9变体中见证的变构调制具有增强 特异性； （AIM 3）通过自然发生的抗Crispr蛋白实施的抑制作用机制 控制基因调节。针对这些目标，我们将利用古典和增强的抽样 分子动力学（MD）模拟，高级AB-Initio MD（使用CAR-PARRINELLO和BORN- Oppenheimer方法）和混合量子力学/分子力学（QM/mm） 方法。此外，Ab-Initio MD与图理论的组合将实施协同作用 捕获瞬时次纳秒信号传输的方法。这将揭示多长时间 变构效应通过不断发展的催化步骤影响动态，从而阐明了变构的作用 在有助于催化中。这些多尺度方法将为生物物理提供一个计算框架 不仅对CRISPR-CAS9的分析，而且还可以扩展到新兴的CRISPR系统 基因组编辑和病毒检测的有希望。理论研究将在 与实验科学家合作，提供动力学测量和生物物理 表征，有助于解释实验数据并启用可测试 预测。总体而言，这项拟议的研究将扩大机械知识的曲目 关于CRISPR-CAS9功能，并为新颖的工程原理奠定了框架 改进的基因组编辑。