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
• 负责人: Giulia Palermo
• 金额: $ 28.26万
• 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/10378667
• 关键词: 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; Computers; 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; base; biophysical analysis; biophysical properties; clinical application; clinical efficacy; computer framework; computer studies; dalton; divalent metal; endonuclease; genome editing; graph theory; improved; improved functioning; 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.

摘要 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方面具有前沿影响的科学。通过使DNA得以纠正 突变，这项技术有望治疗无数的人类遗传病，就像第一次展示的那样 CRISPR-Cas9修饰的T细胞治疗癌症患者。这项技术基于 内切酶Cas9，它与引导RNA结合，识别和切割互补DNA 序列。CRISPR-CAS9工具的不断开发和工程开创了新的 对该系统进行深入研究的耐人寻味的假设。在这里，PI将实现 非传统的多尺度方法，结合了各种最先进的理论方法， 阐明金属依赖催化、变构在选择性机理中的作用以及抑制作用 对系统的影响。我们将追求三个具体目标，其特征是：(目标1)DNA切割依赖 镁离子以外的其他二价金属离子及其构象效应 结合；(目标2)在新设计的Cas9变种中观察到的变构调节 特异性；(目标3)抑制机制由天然产生的抗CRISPR蛋白来实现 对基因调控的控制。为了达到这些目标，我们将利用经典抽样和增强抽样 分子动力学(MD)模拟，高水平从头算分子动力学(使用CAR-Parrinello和Born- Oppenheimer方法)和混合量子力学/分子力学(QM/MM) 接近了。此外，从头算MD与图论的结合将实现协同 捕获瞬时亚纳秒信号传输的方法。这将揭示出多长的距离 变构效应通过进化的催化步骤影响动力学，阐明变构的作用 在辅助催化方面。这些多尺度方法将为生物物理学提供一个计算框架 不仅对CRISPR-CAS9的分析，而且还可以扩展到新兴的CRISPR系统，这些系统 在基因组编辑和病毒检测方面很有希望。理论研究将近距离进行。 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性 预测。总体而言，这项拟议的研究将扩大机械知识的保留范围 关于CRISPR-Cas9的功能，并为新的工程原理奠定了框架，以 改进了基因组编辑。