Yoon Hanwool, Zhao Li Na, Warshel Arieh
Department of Chemistry, University of Southern California, 418 SGM Building, 3620 McClintock Ave, Los Angeles, California 90089-1062, United States.
ACS Catal. 2019 Feb 1;9(2):1329-1336. doi: 10.1021/acscatal.8b04324. Epub 2018 Dec 28.
Elucidating the nature of the gene editing mechanism of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is an important task in view of the role of this breakthrough to the advancement of human medicine. In particular, it is crucial to understand the catalytic mechanism of Cas9 (one of the CRISPR associated proteins) and its role in confirming accurate editing. Thus, we focus in this work on an attempt to analyze the catalytic mechanism of Cas9. Considering the absence of detailed structural information on the active form of Cas9, we use an empirical valence bond (EVB) which is calibrated on the closely related mechanism of T4 endonuclease VII. The calibrated EVB is then used in studying the reaction of Cas9, while trying several structural models. It is found that the catalytic activation requires a large conformational change, where K848 or other positively charged group moves from a relatively large distance toward the scissile phosphate. This conformational change leads to the change in position of the Mg ion and to a major reduction in the activation barrier for the catalytic reaction. Our finding provides an important clue on the nature of the catalytic activation of CAS9 and thus should help in elucidating a key aspect of the gene editing process. For example, the approach used here should be effective in exploring the nature of off target activation and its relationship to the energetics of the unwinding process. This strategy may offer ways to improve the selectivity of Cas9.
鉴于CRISPR（成簇规律间隔短回文重复序列）基因编辑机制这一突破对人类医学进步的作用，阐明其本质是一项重要任务。特别是，了解Cas9（CRISPR相关蛋白之一）的催化机制及其在确保精确编辑中的作用至关重要。因此，我们在这项工作中专注于分析Cas9的催化机制。考虑到缺乏关于Cas9活性形式的详细结构信息，我们使用了一种经验价键（EVB），它是根据T4内切核酸酶VII的密切相关机制校准的。然后，校准后的EVB用于研究Cas9的反应，同时尝试几种结构模型。研究发现，催化激活需要一个大的构象变化，其中K848或其他带正电荷的基团从相对较远的距离向可切割的磷酸基团移动。这种构象变化导致镁离子位置的改变，并使催化反应的活化能垒大幅降低。我们的发现为Cas9催化激活的本质提供了重要线索，因此应该有助于阐明基因编辑过程的一个关键方面。例如，这里使用的方法应该有效地探索脱靶激活的本质及其与解旋过程能量学的关系。这种策略可能提供提高Cas9选择性的方法。
