MOE Key Laboratory of Gene Function and Regulation and Guangzhou Key Laboratory of Healthy Aging Research, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China.
International Cooperation Base of Pesticide and Green Synthesis (Hubei), Key Laboratory of Pesticide & Chemical Biology (CCNU), Ministry of Education, Department of Chemistry, Central China Normal University, Wuhan 430079, China.
Cells. 2022 Nov 11;11(22):3574. doi: 10.3390/cells11223574.
Genome editing tools based on CRISPR-Cas systems can repair genetic mutations in situ; however, off-target effects and DNA damage lesions that result from genome editing remain major roadblocks to its full clinical implementation. Protein and chemical inhibitors of CRISPR-Cas systems may reduce off-target effects and DNA damage. Here we describe the identification of several lead chemical inhibitors that could specifically inhibit the activity of Cas9 (SpCas9). In addition, we obtained derivatives of lead inhibitors that could penetrate the cell membrane and inhibit SpCas9 . Two of these compounds, SP2 and SP24, were able to improve the specificity of SpCas9 at low-micromolar concentration. Furthermore, microscale thermophoresis (MST) assays showed that SP24 might inhibit SpCas9 activity by interacting with both the SpCas9 protein and the SpCas9-gRNA ribonucleoprotein complex. Taken together, SP24 is a novel chemical inhibitor of SpCas9 which has the potential to enhance therapies that utilize SpCas9.
基于 CRISPR-Cas 系统的基因组编辑工具可以原位修复基因突变；然而，基因组编辑产生的脱靶效应和 DNA 损伤仍然是其全面临床应用的主要障碍。CRISPR-Cas 系统的蛋白质和化学抑制剂可以降低脱靶效应和 DNA 损伤。在这里，我们描述了几种先导化学抑制剂的鉴定，这些抑制剂可以特异性抑制 Cas9（SpCas9）的活性。此外，我们还获得了能够穿透细胞膜并抑制 SpCas9 的先导抑制剂衍生物。这两种化合物 SP2 和 SP24 能够以低微摩尔浓度提高 SpCas9 的特异性。此外，微量热泳动 (MST) 分析表明，SP24 可能通过与 SpCas9 蛋白和 SpCas9-gRNA 核糖核蛋白复合物相互作用来抑制 SpCas9 的活性。总之，SP24 是一种新型的 SpCas9 化学抑制剂，具有增强利用 SpCas9 的治疗方法的潜力。
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