Zhang Yuxuan, Zou Wentao, Zhou Yueyang, Chen Jiaqi, Hu Youtian, Wu Fang
Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Center for Systems Biomedicine, Shanghai Jiao Tong University, Shanghai, 200240, China.
Genome Biol. 2025 Mar 28;26(1):75. doi: 10.1186/s13059-025-03521-w.
Regulation of the target DNA cleavage activity of CRISPR/Cas has naturally evolved in a few bacteria or bacteriophages but is lacking in higher species. Thus, identification of bioactive agents and mechanisms that can suppress the activity of Cas9 is urgently needed to rebalance this new genetic pressure.
Here, we identify four specific inhibitors of Cas9 by screening 4607 compounds that could inhibit the endonuclease activity of Cas9 via three distinct mechanisms: substrate-competitive and protospacer adjacent motif (PAM)-binding site-occupation; substrate-targeting; and sgRNA-targeting mechanisms. These inhibitors inhibit, in a dose-dependent manner, the activity of Streptococcus pyogenes Cas9 (SpyCas9), Staphylococcus aureus Cas9 (SauCas9), and SpyCas9 nickase-based BE4 base editors in in vitro purified enzyme assays, bacteria, mammalian cells, and mice. Importantly, pamoic acid and carbenoxolone show DNA-topology selectivity and preferentially inhibit the cleavage of linear DNA compared with a supercoiled plasmid.
These pharmacologically selective inhibitors and new mechanisms offer new tools for controlling the DNA-topology selective activity of Cas9.
CRISPR/Cas靶DNA切割活性的调控在一些细菌或噬菌体中自然进化而来，但在高等生物中却不存在。因此，迫切需要鉴定能够抑制Cas9活性的生物活性剂和机制，以重新平衡这种新的遗传压力。
在这里，我们通过筛选4607种化合物，鉴定出四种Cas9的特异性抑制剂，这些化合物可通过三种不同机制抑制Cas9的核酸内切酶活性：底物竞争和原间隔序列相邻基序（PAM）结合位点占据；靶向底物；以及靶向sgRNA机制。在体外纯化酶测定、细菌、哺乳动物细胞和小鼠中，这些抑制剂以剂量依赖的方式抑制化脓性链球菌Cas9（SpyCas9）、金黄色葡萄球菌Cas9（SauCas9）以及基于SpyCas9切口酶的BE4碱基编辑器的活性。重要的是，帕莫酸和甘草次酸显示出DNA拓扑选择性，与超螺旋质粒相比，它们优先抑制线性DNA的切割。
这些具有药理学选择性的抑制剂和新机制为控制Cas9的DNA拓扑选择性活性提供了新工具。
