Project 3 - Mapping Long-range Allosteric Pathways in CRISPR-Cas9

项目 3 - 绘制 CRISPR-Cas9 中的长程变构途径

基本信息

批准号：
10271625
负责人：
GEORGE LISI
金额：
$ 37.74万
依托单位：
BROWN UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-06-01 至 2026-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10271625
关键词：
Active Sites Allosteric Regulation Amino Acids Binding Biochemical Biological Biological Assay Biological Models Biological Process Biomedical Engineering Biophysics CRISPR/Cas technology Cell physiology Centers of Research Excellence Chemicals Clustered Regularly Interspaced Short Palindromic Repeats Communication Community Networks Computational Biology Computer Simulation Computing Methodologies Coupling Crystallization DNA DNA Binding Data Disease Engineering Ensure Enzymes Equilibrium Gene Targeting Genome Genomics Guide RNA Investigation Knowledge Laboratories Length Link Maps Methods Modernization Modification Molecular Molecular Biology Molecular Conformation Motion Mutation Nuclear Magnetic Resonance Nucleotides Pathogenicity Pathology Pathway Analysis Pathway interactions Play Process Protein Biochemistry Proteins RNA Binding Regulation Regulator Genes Relaxation Roentgen Rays Role Signal Pathway Signal Transduction Site Specificity Structure Tertiary Protein Structure Therapeutic Work base biophysical analysis computer studies design drug discovery ds-DNA enzyme mechanism experimental study flexibility genome editing human disease improved in vivo insight interdisciplinary approach millisecond molecular dynamics mutant novel novel strategies nuclease precision medicine protein function recruit response scaffold simulation small molecule inhibitor success theories therapeutic enzyme tool

项目摘要

Project Summary Gene regulatory mechanisms are critical for proper cellular and protein function, and modern molecular biology has linked numerous pathologies to dysregulation of these processes. Although modification of the genome to correct pathogenic mutations is a promising therapeutic approach, these efforts cannot be successful without knowledge of the underlying biochemistry of protein machinery such as CRISPR- Cas9 (Cas9). Cas9 can be a customizable tool to edit and correct disease-linked (genomic) mutations, however, to fully realize these applications, novel strategies to overcome its off-target effects and poor temporal control must be investigated. Cas9 utilizes a guide RNA molecule to recruit, stabilize, and facilitate cleavage of double-stranded DNA after recognition of a well-known protospacer adjacent motif (PAM) sequence. Prior X-ray crystal structures indicate that conformational changes within the Cas9 nucleases, HNH and RuvC, are required for effective catalytic function. However, these structures offer little mechanistic information, as the target DNA and catalytic nucleases are never observed in an activated state. The conformational shift of HNH, in particular, is correlated to motions of neighboring subdomains, all of which are activated from >20 Å away by the PAM-binding domain, suggesting an allosteric mechanism. Understanding this allosteric coupling would have exciting potential for precision medicine by establishing novel paradigms to control and enhance the spatial and temporal function of Cas9. We recently identified a pathway of millisecond timescale motions spanning the HNH nuclease and reaching multiple Cas9 domains that computational results suggest is a portion of a larger allosteric network that controls Cas9 function. To investigate the reach of this allosteric network and the role of molecular motions in its mechanism, my laboratory will undertake a synergistic solution NMR and computational study to map the long-range allosteric pathway of Cas9. We will now (1) characterize allosteric mutants of HNH that are known to alter Cas9 specificty, (2) establish the biophysical roles of the neighboring REC2 and REC3 domains in propagating allosteric signals to/from HNH, and (3) characterize the conformational ensemble governing the full-length Cas9 protein. This multidisciplinary approach of NMR spin relaxation experiments, molecular dynamics simulations, and network theory, will probe multi-timescale protein motions in Cas9, revealing specific amino acids responsible for transmitting structural or dynamic information. These studies will use both full-length Cas9 and novel engineered constructs to interrogate specific domains within the 160 kDa enzyme. The structural and dynamic findings of this work will be correlated to function with new in vivo assays to provide a detailed understanding of the Cas9 allosteric mechanism.

项目概要基因调控机制对于正常的细胞和蛋白质功能至关重要，现代分子生物学已将许多病理学与这些过程的失调联系起来。基因组来纠正致病性突变是一种有前途的治疗方法，但这些努力不能在不了解 CRISPR 等蛋白质机制的基础生物化学知识的情况下也能取得成功- Cas9 (Cas9) 可以是编辑和纠正疾病相关（基因组）突变的可定制工具，然而，为了充分实现这些应用，需要克服其脱靶效应和较差的新策略必须研究 Cas9 利用引导 RNA 分子来招募、稳定和控制时间。在识别众所周知的原型间隔子相邻基序后促进双链 DNA 的切割 (PAM) 序列表明 Cas9 内的构象发生变化。核酸酶 HNH 和 RuvC 是有效催化功能所必需的，但是这些结构提供了有效的催化功能。几乎没有什么机制信息，因为靶 DNA 和催化核酸酶从未在 HNH 的激活状态尤其与邻近的运动相关。子结构域，所有这些子结构域都被 PAM 结合结构域激活距离大于 20 Å，这表明了解这种变构耦合对于精确度具有令人兴奋的潜力。通过建立新的范式来控制和增强医学的空间和时间功能我们最近发现了一条跨越 HNH 核酸酶的毫秒级运动路径。并到达多个 Cas9 结构域，计算结果表明这些结构域是更大变构的一部分控制 Cas9 功能的网络研究该变构网络的范围及其作用。分子运动的机制，我的实验室将进行协同解决核磁共振和我们现在将 (1) 描述 Cas9 长程变构途径的计算研究。已知会改变 Cas9 特异性的 HNH 变构突变体，(2) 建立邻近的 REC2 和 REC3 结构域在向/从 HNH 传播变构信号时，以及 (3) 表征控制全长 Cas9 蛋白的构象整体。核磁共振自旋弛豫实验、分子动力学模拟和网络理论的方法，将探测 Cas9 中的多时间尺度蛋白质运动，揭示负责传递的特定氨基酸这些研究将使用全长 Cas9 和新颖的工程信息。构建以询问 160 kDa 酶内的特定域的结构和动态。这项工作的结果将与新的体内测定的功能相关联，以提供详细的了解 Cas9 变构机制。