Enhanced specificity mutations perturb allosteric signaling in CRISPR-Cas9.
Enhanced specificity mutations perturb allosteric signaling in CRISPR-Cas9.
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DOI：
10.7554/elife.73601
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发表时间：
2021-12-15
期刊：
影响因子：
7.7
通讯作者：
Palermo G
中科院分区：
文献类型：
--
作者：
Nierzwicki L;East KW;Morzan UN;Arantes PR;Batista VS;Lisi GP;Palermo G
CRISPR-Cas9（成簇的规则间隔短回文重复序列和相关的Cas9蛋白）是一种具有变革性基因组编辑能力的分子工具。在分子水平上，复杂的变构信号对于DNA切割是至关重要的，但其在Cas9内切核酸酶的特异性增强中的作用知之甚少。在这里，多微秒分子动力学与溶液NMR和图论衍生的模型相结合，以探测关键的特异性增强突变的变构作用。我们发现，负责增加Cas9特异性的突变改变了催化HNH结构域的变构结构，影响了从DNA识别区到切割催化位点的信号传输。具体而言，K855 A突变强烈破坏HNH结构域的变构连接，对信号传递施加最高的扰动，而K810 A和K848 A对变构通信产生更温和的影响。这种变构信号的差异扰动与生化研究中观察到的特异性增强的顺序（K855 A> K848 A ~ K810 A）相关，其中突变实现最高特异性，最强烈地扰动信号传递。这些发现表明，从DNA识别到切割的变构通信的改变对于增加Cas9的特异性至关重要，并且可以通过突变研究靶向变构热点以改善系统的功能。
CRISPR-Cas9 (clustered regularly interspaced short palindromic repeat and associated Cas9 protein) is a molecular tool with transformative genome editing capabilities. At the molecular level, an intricate allosteric signaling is critical for DNA cleavage, but its role in the specificity enhancement of the Cas9 endonuclease is poorly understood. Here, multi-microsecond molecular dynamics is combined with solution NMR and graph theory-derived models to probe the allosteric role of key specificity-enhancing mutations. We show that mutations responsible for increasing the specificity of Cas9 alter the allosteric structure of the catalytic HNH domain, impacting the signal transmission from the DNA recognition region to the catalytic sites for cleavage. Specifically, the K855A mutation strongly disrupts the allosteric connectivity of the HNH domain, exerting the highest perturbation on the signaling transfer, while K810A and K848A result in more moderate effects on the allosteric communication. This differential perturbation of the allosteric signal correlates to the order of specificity enhancement (K855A > K848A ~ K810A) observed in biochemical studies, with the mutation achieving the highest specificity most strongly perturbing the signaling transfer. These findings suggest that alterations of the allosteric communication from DNA recognition to cleavage are critical to increasing the specificity of Cas9 and that allosteric hotspots can be targeted through mutational studies for improving the system’s function.