Mapping Long‐range Allosteric Pathways in CRISPR‐Cas9

绘制 CRISPR-Cas9 中的长程变构途径

• 批准号: 10350163
• 负责人: GEORGE LISI
• 金额: $ 26.59万
• 依托单位: BROWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-01 至 2021-08-03
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10350163
• 关键词: Mapping; Long; range; Allosteric; Pathways

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 longrange allosteric pathway of Cas9. We will (1) characterize the molecular determinants of protein motions in the HNH nuclease, (2) establish the biophysical roles of the neighboring REC2 and REC3 domains in Cas9 signal transduction and (3) characterize the interaction of the PAM sequence with its binding domain to evaluate its role as an allosteric activator. Specifically, this multidisciplinary approach of NMR spin relaxation experiments and molecular dynamics, network theory, and Eigenvector Centrality simulations will probe differential protein motions in Cas9, revealing specific amino acids responsible for transmitting structural or dynamic information to affect biological response. 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 biochemical and cellular assays to provide a detailed understanding of the Cas9 allosteric mechanism.

基因调节机制对于适当的细胞和蛋白质功能至关重要，现代分子生物学具有 将许多病理与这些过程的失调联系起来。尽管基因组的修改以纠正 致病突变是一种有前途的治疗方法，如果不了解，这些努力就无法成功 蛋白质机械的基础生物化学，例如CRISPR-CAS9（CAS9）。 CAS9可以是可自定义的编辑工具 但是，纠正与疾病相关的（基因组）突变，以充分实现这些应用 必须研究其脱离目标的效果和临时控制不佳。 CAS9利用指南RNA分子到 认识到众所周知的原始探针后，招募，稳定和促进双链DNA的裂解 基序（PAM）序列。先前的X射线晶体结构表明Cas9核内的会议变化， HNH和RUVC是有效的催化功能所必需的。但是，这些结构几乎没有机械 信息，因为在激活状态下从未观察到靶DNA和催化核。构象 特别是HNH的移位与相邻子域的运动相关，所有这些都从>20Å中激活 由PAM结合域离开，提出了变构机制。了解这种变构耦合将 通过建立新型范式来控制和增强空间和 CAS9的临时功能。我们最近确定了跨越HNH的毫秒时尺度运动的途径 核酸酶并达到多个CAS9域，计算结果表明是较大变构的一部分 控制CAS9功能的网络。研究这种变构网络的范围和分子运动的作用 在其机制中，我的实验室将进行协同解决方案NMR和计算研究以绘制纵向 Cas9的变构途径。我们将（1）表征HNH中蛋白质运动的分子决定剂 核酸酶，（2）在Cas9信号转移中建立相邻REC2和REC3域的生物物理作用 （3）表征PAM序列与其结合结构域的相互作用，以评估其作为变构的作用 激活剂。特别是，这种NMR自旋松弛实验和分子动力学的多学科方法， 网络理论和特征向量中心性模拟将探测Cas9中的差异蛋白质运动，从而揭示 负责传输结构或动态信息以影响生物学反应的特定氨基酸。这些 研究将同时使用全长Cas9和新型工程结构来询问160中的特定领域 KDA酶。这项工作的结构和动态发现将与生化和 蜂窝暗杀，以提供对CAS9变构机制的详细理解。