Ahsan Mohd, Saha Aakash, Ramos Delisa, Strohkendl Isabel, Skeens Erin, Lisi George P, Taylor David W, Palermo Giulia
Department of Bioengineering, University of California Riverside, 900 University Avenue, Riverside, CA 52512, United States.
Department of Molecular Biosciences, University of Texas at Austin, Austin, TX, USA.
bioRxiv. 2025 Aug 26:2025.08.25.672025. doi: 10.1101/2025.08.25.672025.
Cas9 is a metal-dependent nuclease that has revolutionized gene editing across diverse cells and organisms exhibiting varying ion uptake, metabolism, and concentrations. However, how divalent metals impact its catalytic function, and consequently its editing efficiency in different cells, remains unclear. Here, extensive molecular simulations, Markov State Models, biochemical and NMR experiments, demonstrate that divalent metals - Mg, Ca, and Co - promote activation of the catalytic HNH domain by binding within a dynamically forming divalent metal binding pocket (DBP) at the HNH-RuvC interface. Mutations in DBP residues disrupt HNH activation and impair the coupled catalytic activity of both nucleases, identifying this cryptic DBP as a key regulator of Cas9's metal-dependent activity. The ionic strength thereby promotes Cas9's conformational activation, while its catalytic activity is metal-specific. These findings are critical to improving the metal-dependent function of Cas9 and its use for genome editing in different cells and organisms.
Cas9是一种依赖金属的核酸酶，它彻底改变了在具有不同离子摄取、代谢和浓度的各种细胞和生物体中的基因编辑。然而，二价金属如何影响其催化功能，进而影响其在不同细胞中的编辑效率，仍不清楚。在这里，广泛的分子模拟、马尔可夫状态模型、生化和核磁共振实验表明，二价金属——镁、钙和钴——通过在HNH-RuvC界面处动态形成的二价金属结合口袋（DBP）内结合来促进催化HNH结构域的激活。DBP残基的突变会破坏HNH激活并损害两种核酸酶的偶联催化活性，将这个隐秘的DBP确定为Cas9金属依赖性活性的关键调节因子。离子强度从而促进Cas9的构象激活，而其催化活性具有金属特异性。这些发现对于改善Cas9的金属依赖性功能及其在不同细胞和生物体中的基因组编辑应用至关重要。
