PDF(Pubmed)
Abstract：
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 Streptococcus pyogenes Cas9 (SpCas9). In addition, we obtained derivatives of lead inhibitors that could penetrate the cell membrane and inhibit SpCas9 in cellulo. Two of these compounds, SP2 and SP24, were able to improve the specificity of SpCas9 in cellulo 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.
摘要：
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，基因组编辑导致的脱靶效应和DNA损伤损伤仍然是其全面临床实施的主要障碍。CRISPR-Cas系统的蛋白质和化学抑制剂可以减少脱靶效应和DNA损伤。在这里，我们描述了几种可以特异性抑制化脓性链球菌Cas9（SpCas9）活性的铅化学抑制剂的鉴定。此外,我们获得了铅抑制剂的衍生物，可以穿透细胞膜并抑制细胞中的SpCas9。其中两种化合物，SP2和SP24能够在低微摩尔浓度下提高SpCas9在细胞中的特异性。此外，微尺度热电泳（MST）分析显示，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活性。一起来看,SP24是SpCas9的新型化学抑制剂，具有增强利用SpCas9的疗法的潜力。
