Identification of a Cryptic Binding Site in CRISPR-Cas9 for Targeted Inhibition
Identification of a Cryptic Binding Site in CRISPR-Cas9 for Targeted Inhibition
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DOI：
10.1021/acs.jcim.3c00256
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发表时间：
2023-05
影响因子：
5.6
通讯作者：
Niubing Zhang;Zhicheng Zuo
中科院分区：
文献类型：
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作者：
Niubing Zhang;Zhicheng Zuo
对CRISPR-Cas9基因组编辑的精确控制的需求产生了对抗CRISPR分子的需求。最近，第一类小分子Cas9抑制剂已经被鉴定，验证了使用直接作用的小分子调节CRISPR-Cas9活性的可行性。然而，CRISPR-Cas9上配体结合位点的位置以及配体结合如何导致Cas9功能抑制仍然是谜。在这里，我们建立了一个综合的计算协议，包括大量的结合位点定位，分子对接，分子动力学模拟，自由能计算。最终，从动力学轨迹中发现了隐藏在其羧基末端结构域（CTD）内的Cas9配体结合位点，该结构域识别前间区序列邻近基序（PAM）。使用顶部抑制剂BRD 0539作为探针，我们证明了配体结合诱导显著的CTD结构重排朝向PAM DNA接合的不胜任构象。所揭示的BRD 0539抑制Cas9的分子机制与实验数据非常一致。这项研究为现有配体的效力改进和新型小分子制动器的合理发现提供了结构和机制基础，以开发更安全的CRISPR-Cas9技术。
The need for precision control of CRISPR-Cas9 genome editing has created a demand for anti-CRISPR molecules. Recently, the first class of small-molecule Cas9 inhibitors has been identified, verifying the feasibility of regulating CRISPR-Cas9 activity using direct-acting small molecules. However, it remains enigmatic as to the location of the ligand binding site(s) on CRISPR-Cas9 and how the ligand binding leads to Cas9 functional inhibition. Here, we established an integrative computational protocol, including massive binding site mapping, molecular docking, molecular dynamics simulations, and free energy calculations. Ultimately, a Cas9 ligand binding site was discovered from the dynamics trajectories that is hidden within its carboxyl-terminal domain (CTD), a domain recognizing the protospacer adjacent motif (PAM). Using the top inhibitor BRD0539 as a probe, we demonstrated that the ligand binding induces significant CTD structural rearrangements toward an incompetent conformation for PAM DNA engagement. The revealed molecular mechanism of BRD0539 inhibiting Cas9 is in well agreement with the experimental data. This study provides a structural and mechanistic basis for the potency improvement of existing ligands and the rational discovery of novel small-molecule brakes for developing safer CRISPR-Cas9 technologies.