Mapping Long‐range Allosteric Pathways in CRISPR‐Cas9

基因调控机制对于细胞和蛋白质的正常功能至关重要，而现代分子生物学已经 将许多病理与这些过程的失调联系在一起。虽然基因组的修改是为了纠正 致病突变是一种很有前途的治疗方法，如果不知道 蛋白质机械的基本生物化学，如CRISPR-Cas9(Cas9)。Cas9可以是一个可定制的编辑工具 然而，为了充分实现这些应用，并纠正与疾病相关的(基因组)突变，新的策略可以 克服其脱靶效应和较差的时间控制必须进行研究。Cas9利用一种引导RNA分子 识别邻近的已知蛋白间隔区后，重新招募、稳定和促进双链DNA的切割 基序(PAM)序列。先前的X射线晶体结构表明，Cas9核酸酶内的构象变化， HNH和RuvC是有效催化作用所必需的。然而，这些结构几乎没有提供机械性。 信息，因为目标DNA和催化核酸酶永远不会在激活状态下观察到。构象 特别是，HNH的移动与相邻子域的运动相关，所有这些运动都是从&>20ä激活的 被PAM结合结构域带走，暗示了变构机制。理解这种变构偶联将会 通过建立新的范例来控制和增强空间和 Cas9的时间功能。我们最近发现了一条跨越HNH的毫秒时间尺度运动路径 核酸酶和到达多个Cas9结构域的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可达性和分子运动的作用 在其机制上，我的实验室将进行协同解核磁共振和计算研究，以绘制远程 Cas9的变构途径。我们将(1)描述HNH中蛋白质运动的分子决定因素 核酸酶，(2)确定邻近的REC2和REC3结构域在Cas9信号转导中的生物物理作用 以及(3)表征PAM序列与其结合域的相互作用以评价其作为变构的作用 激活剂。具体地说，这种核磁共振自旋弛豫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，揭示 负责传递结构或动态信息以影响生物反应的特定氨基酸。这些 研究将使用全长Cas9和新的工程构建来询问160个特定的结构域 KDA酶。这项工作的结构和动态发现将与生化和功能相关 细胞分析，以提供对Cas9变构机制的详细了解。

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.