Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences (SB RAS), 630090 Novosibirsk, Russia.
Department of Natural Sciences, Novosibirsk State University, 630090 Novosibirsk, Russia.
Int J Mol Sci. 2022 Jan 20;23(3):1129. doi: 10.3390/ijms23031129.
The Cas9 endonuclease is an essential component of the CRISPR-Cas-based genome editing tools. The attainment of high specificity and efficiency of Cas9 during targetted DNA cleavage is the main problem that limits the clinical application of the CRISPR-Cas9 system. A deep understanding of the Cas9 mechanism and its structural-functional relationships is required to develop strategies for precise gene editing. Here, we present the first attempt to describe the solution structure of Cas9 from using hydrogen-deuterium exchange mass spectrometry (HDX-MS) coupled to molecular dynamics simulations. HDX data revealed multiple protein regions with deuterium uptake levels varying from low to high. By analysing the difference in relative deuterium uptake by apoCas9 and its complex with sgRNA, we identified peptides involved in the complex formation and possible changes in the protein conformation. The REC3 domain was shown to undergo the most prominent conformational change upon enzyme-RNA interactions. Detection of the HDX in two forms of the enzyme provided detailed information about changes in the Cas9 structure induced by sgRNA binding and quantified the extent of the changes. The study demonstrates the practical utility of HDX-MS for the elucidation of mechanistic aspects of Cas9 functioning.
Cas9 内切酶是基于 CRISPR-Cas 的基因组编辑工具的重要组成部分。在靶向 DNA 切割过程中实现 Cas9 的高特异性和高效率是限制 CRISPR-Cas9 系统临床应用的主要问题。为了开发精确基因编辑的策略，需要深入了解 Cas9 的机制及其结构功能关系。在这里，我们首次尝试使用氢氘交换质谱 (HDX-MS) 结合分子动力学模拟来描述 使用 Cas9 的溶液结构。HDX 数据显示了多个具有从低到高氘摄取水平的蛋白质区域。通过分析 apoCas9 与其与 sgRNA 的复合物之间相对氘摄取的差异，我们鉴定了参与复合物形成和可能的蛋白质构象变化的肽段。REC3 结构域在酶-RNA 相互作用时经历最显著的构象变化。检测酶的两种形式中的 HDX 提供了有关 sgRNA 结合诱导的 Cas9 结构变化的详细信息，并量化了变化的程度。该研究证明了 HDX-MS 用于阐明 Cas9 功能的机制方面的实际效用。
