Coupling Cas9 to artificial inhibitory domains enhances CRISPR-Cas9 target specificity
Coupling Cas9 to artificial inhibitory domains enhances CRISPR-Cas9 target specificity
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DOI：
10.1126/sciadv.aay0187
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发表时间：
2020-02-01
期刊：
影响因子：
13.6
通讯作者：
Niopek, Dominik
中科院分区：
文献类型：
--
作者：
Aschenbrenner, Sabine;Kallenberger, Stefan M.;Niopek, Dominik
CRISPR-Cas核酸酶的有限靶特异性对其在研究和治疗中的应用提出了挑战。在这里，我们提出了一种简单而新颖的策略，通过将Cas9偶联到人工抑制结构域来增强CRISPR-Cas9基因组编辑的特异性。应用数学建模和实验的组合，我们首先确定了CRISPR-Cas9活性谱与Cas9特异性的关系。然后，我们使用人工弱化的抗CRISPR（Acr）蛋白与Cas9共表达或直接融合，以将其活性微调到选定的水平，从而实现ON-和OFF-靶编辑事件的有效动力学绝缘。我们证明了在哺乳动物细胞中使用易于有效OFF靶向的多种单向导RNA进行高度特异性的基因组编辑。最后，我们证明了我们的策略与不同的递送模式兼容，包括瞬时转染和腺相关病毒载体。总之，我们提供了一种高度通用的方法，通过动力学绝缘来减少CRISPR-Cas脱靶效应。
The limited target specificity of CRISPR-Cas nucleases poses a challenge with respect to their application in research and therapy. Here, we present a simple and original strategy to enhance the specificity of CRISPR-Cas9 genome editing by coupling Cas9 to artificial inhibitory domains. Applying a combination of mathematical modeling and experiments, we first determined how CRISPR-Cas9 activity profiles relate to Cas9 specificity. We then used artificially weakened anti-CRISPR (Acr) proteins either coexpressed with or directly fused to Cas9 to fine-tune its activity toward selected levels, thereby achieving an effective kinetic insulation of ON- and OFF-target editing events. We demonstrate highly specific genome editing in mammalian cells using diverse single-guide RNAs prone to potent OFF-targeting. Last, we show that our strategy is compatible with different modes of delivery, including transient transfection and adeno-associated viral vectors. Together, we provide a highly versatile approach to reduce CRISPR-Cas OFF-target effects via kinetic insulation.