Hibshman Grace N, Taylor David W
Interdisciplinary Life Sciences Graduate Programs, University of Texas at Austin, Austin, TX, United States; Department of Molecular Biosciences, University of Texas at Austin, Austin, TX, United States.
Methods Enzymol. 2025;712:41-53. doi: 10.1016/bs.mie.2025.01.004. Epub 2025 Mar 6.
CRISPR-Cas9 has transformed genome editing through its programmability and versatility. Its DNA cleavage activity involves dynamic conformational changes during gRNA binding, DNA recognition, R-loop formation, and endonuclease activation. Understanding these molecular transitions is critical for improving the specificity and efficiency of Cas9, but this remains challenging precisely due to these rapid structural rearrangements. Early structural studies provided foundational insights but were limited to static states under catalytically inactive conditions. Cryo-EM has since enabled visualization of the dynamic nature of active Cas9, by enriching for specific conformations. This chapter introduces a kinetics-informed cryo-EM approach to capture the stepwise activation of Cas9 in real time. With thorough kinetic analyses, such as stopped-flow measurements of R-loop formation, we describe how to identify optimal timepoints to visualize key conformational states with cryo-EM. Integration of kinetic and structural data enables precise mapping of the conformational landscape of Cas9 and other dynamic enzymes, advancing our understanding of their molecular mechanisms and providing a framework for engineering enhanced variants.
CRISPR-Cas9凭借其可编程性和多功能性改变了基因组编辑。其DNA切割活性在gRNA结合、DNA识别、R环形成和核酸内切酶激活过程中涉及动态构象变化。理解这些分子转变对于提高Cas9的特异性和效率至关重要，但正是由于这些快速的结构重排，这仍然具有挑战性。早期的结构研究提供了基础见解，但仅限于催化无活性条件下的静态状态。自那以后，冷冻电镜通过富集特定构象，能够可视化活性Cas9的动态性质。本章介绍一种基于动力学的冷冻电镜方法，以实时捕捉Cas9的逐步激活过程。通过全面的动力学分析，如R环形成的停流测量，我们描述了如何确定最佳时间点，用冷冻电镜可视化关键构象状态。动力学和结构数据的整合能够精确绘制Cas9和其他动态酶的构象图谱，增进我们对其分子机制的理解，并为设计增强变体提供框架。
