Principles of target DNA cleavage and the role of Mg2+ in the catalysis of CRISPR-Cas9
Principles of target DNA cleavage and the role of Mg2+ in the catalysis of CRISPR-Cas9
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DOI：
10.1038/s41929-022-00848-6
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发表时间：
2022-10-06
期刊：
影响因子：
37.8
通讯作者：
Palermo, Giulia
中科院分区：
文献类型：
--
作者：
Nierzwicki, Lukasz;East, Kyle W.;Palermo, Giulia
作为 CRISPR-Cas9 基因组编辑技术的核心，核酸内切酶 Cas9 在 DNA 中引入了位点特异性断裂。然而，改善 Cas9 功能的精确机制信息仍然缺失。在此，将多微秒分子动力学、自由能和多尺度模拟与溶液核磁共振和DNA裂解实验相结合，以解决目标DNA裂解的催化机制。我们发现活性 HNH 核酸酶的构象紧密依赖于催化 Mg2+，揭示了其主要结构作用。这种活化的 Mg2+ 结合的 HNH 通过分子模拟、核磁共振 (NMR) 和 DNA 裂解测定得到了一致的描述，还揭示了催化 H840 的质子化状态受到活性位点突变的强烈影响。最后，从头算量子力学（密度泛函理论）/分子力学模拟和元动力学建立了催化机制，表明催化作用由H840激活并由K866完成，从而使DNA切割实验合理化。这些信息对于增强 CRISPR-Cas9 的酶功能以改善基因组编辑至关重要。
At the core of the CRISPR-Cas9 genome-editing technology, the endonuclease Cas9 introduces site-specific breaks in DNA. However, precise mechanistic information to ameliorate Cas9 function is still missing. Here, multimicrosecond molecular dynamics, free energy and multiscale simulations are combined with solution NMR and DNA cleavage experiments to resolve the catalytic mechanism of target DNA cleavage. We show that the conformation of an active HNH nuclease is tightly dependent on the catalytic Mg2+, unveiling its cardinal structural role. This activated Mg2+-bound HNH is consistently described through molecular simulations, nuclear magnetic resonance (NMR) and DNA cleavage assays, revealing also that the protonation state of the catalytic H840 is strongly affected by active site mutations. Finally, ab initio quantum mechanics (density functional theory)/molecular mechanics simulations and metadynamics establish the catalytic mechanism, showing that the catalysis is activated by H840 and completed by K866, thus rationalizing DNA cleavage experiments. This information is critical to enhancing the enzymatic function of CRISPR-Cas9 towards improved genome editing.