Synthetic Biology Group, BioQuant Center, University of Heidelberg, Heidelberg, Germany.
Digital Health Center, Berlin Institute of Health (BIH) and Charité, Berlin, Germany.
Nat Chem Biol. 2020 Jul;16(7):725-730. doi: 10.1038/s41589-020-0518-9. Epub 2020 Apr 13.
Anti-CRISPR (Acr) proteins are powerful tools to control CRISPR-Cas technologies. However, the available Acr repertoire is limited to naturally occurring variants. Here, we applied structure-based design on AcrIIC1, a broad-spectrum CRISPR-Cas9 inhibitor, to improve its efficacy on different targets. We first show that inserting exogenous protein domains into a selected AcrIIC1 surface site dramatically enhances inhibition of Neisseria meningitidis (Nme)Cas9. Then, applying structure-guided design to the Cas9-binding surface, we converted AcrIIC1 into AcrIIC1X, a potent inhibitor of the Staphylococcus aureus (Sau)Cas9, an orthologue widely applied for in vivo genome editing. Finally, to demonstrate the utility of AcrIIC1X for genome engineering applications, we implemented a hepatocyte-specific SauCas9 ON-switch by placing AcrIIC1X expression under regulation of microRNA-122. Our work introduces designer Acrs as important biotechnological tools and provides an innovative strategy to safeguard CRISPR technologies.
抗 CRISPR (Acr) 蛋白是控制 CRISPR-Cas 技术的有力工具。然而，现有的 Acr 库仅限于天然存在的变体。在这里，我们对广谱 CRISPR-Cas9 抑制剂 AcrIIC1 进行了基于结构的设计，以提高其对不同靶标的效果。我们首先表明，将外源蛋白结构域插入到选定的 AcrIIC1 表面位点可极大地增强对脑膜炎奈瑟菌（Nme）Cas9 的抑制作用。然后，我们应用结构导向设计到 Cas9 结合表面，将 AcrIIC1 转化为 AcrIIC1X，这是一种有效的金黄色葡萄球菌（Sau）Cas9 抑制剂，它是一种广泛应用于体内基因组编辑的同源物。最后，为了证明 AcrIIC1X 在基因组工程应用中的实用性，我们通过将 AcrIIC1X 的表达置于 microRNA-122 的调控下，实现了肝细胞特异性 SauCas9 开启开关。我们的工作介绍了设计 Acr 作为重要的生物技术工具，并提供了一种创新策略来保护 CRISPR 技术。
