Shanghai Institute for Advanced Immunochemical Studies and School of Life Science and Technology, ShanghaiTech University, 201210, Shanghai, China.
Key Laboratory of Organofluorine Chemistry, Center for Excellence in Molecular Synthesis, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 345 Ling-Ling Road, 200032, Shanghai, China.
Commun Biol. 2022 Mar 17;5(1):237. doi: 10.1038/s42003-022-03188-0.
CRISPR-based genome engineering tools are associated with off-target effects that constitutively active Cas9 protein may instigate. Previous studies have revealed the feasibility of modulating Cas9-based genome- and base-editing tools using protein or small-molecule CRISPR inhibitors. Here we screened a set of small molecule compounds with irreversible warhead, aiming to identifying small-molecule modulators of CRISPR-Cas9. It was found that selective inhibitors of nuclear export (SINEs) could efficiently inhibit the cellular activity of Cas9 in the form of genome-, base- and prime-editing tools. Interestingly, SINEs did not function as direct inhibitors to Cas9, but modulated Cas9 activities by interfering with the nuclear export process of Cas9 mRNA. Thus, to the best of our knowledge, SINEs represent the first reported indirect, irreversible inhibitors of CRISPR-Cas9. Most importantly, an FDA-approved anticancer drug KPT330, along with other examined SINEs, could improve the specificities of CRISPR-Cas9-based genome- and base editing tools in human cells. Our study expands the toolbox of CRISPR modulating elements and provides a feasible approach to improving the specificity of CRISPR-Cas9-based genome engineering tools.
基于 CRISPR 的基因组编辑工具与持续激活的 Cas9 蛋白可能引发的脱靶效应有关。先前的研究已经揭示了使用蛋白质或小分子 CRISPR 抑制剂来调节基于 Cas9 的基因组和碱基编辑工具的可行性。在这里，我们筛选了一组具有不可逆弹头的小分子化合物，旨在鉴定 CRISPR-Cas9 的小分子调节剂。结果发现，核输出选择性抑制剂（SINEs）可以有效地抑制 Cas9 在基因组、碱基和引物编辑工具形式中的细胞活性。有趣的是，SINEs 不作为 Cas9 的直接抑制剂发挥作用，而是通过干扰 Cas9 mRNA 的核输出过程来调节 Cas9 的活性。因此，据我们所知，SINEs 代表了第一个报道的 CRISPR-Cas9 的间接、不可逆抑制剂。最重要的是，一种 FDA 批准的抗癌药物 KPT330 以及其他检查过的 SINEs，可以提高 CRISPR-Cas9 为基础的基因组和碱基编辑工具在人类细胞中的特异性。我们的研究扩展了 CRISPR 调节元件的工具箱，并提供了一种可行的方法来提高基于 CRISPR-Cas9 的基因组工程工具的特异性。
