Cas9 deactivation with photocleavable guide RNAs.
Cas9 deactivation with photocleavable guide RNAs.
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DOI：
10.1016/j.molcel.2021.02.007
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发表时间：
2021-04-01
期刊：
影响因子：
16
通讯作者：
Ha T
中科院分区：
文献类型：
--
作者：
Zou RS;Liu Y;Wu B;Ha T
精确控制CRISPR-Cas9将提高其安全性和适用性。控制CRISPR抑制是一种很有前途的方法，但由于单独的抑制剂递送、不完全失活和缓慢的动力学，使其变得复杂。为了克服这些障碍，我们设计了可光切割引导rna (pcRNA)，使Cas9核酸酶和碱基编辑器具有内置的光基失活机制。pcRNA是Cas9失活最快（<1分钟）和最完整（<1%残留索引）的方法。它对野生型Cas9也表现出显著增强的特异性。时间分辨失活表明，Cas9活性12-36小时或碱基编辑器活性2-4小时足以达到较高的编辑效率。pcRNA通过消除持续的损伤和修复周期来研究细胞对DNA损伤的反应是有用的，否则会使反应轨迹不同步。总之，pcRNA扩展了CRISPR工具箱，用于精确的基因组编辑和DNA损伤和修复的研究。邹等人通过在导RNA中引入可光切割的片段，开发了对Cas9和碱基编辑器失活的光介导控制。停用在几秒钟内发生，并接近完成。这种修饰天然增强了特异性。定时失活通过在细胞群中同步终止DNA损伤，促进了DNA修复的研究。
Precise control of CRISPR-Cas9 would improve its safety and applicability. Controlled CRISPR inhibition is a promising approach, but is complicated by separate inhibitor delivery, incomplete deactivation, and slow kinetics. To overcome these obstacles, we engineered photocleavable guide RNAs (pcRNA) that endow Cas9 nucleases and base editors with a built-in mechanism for light-based deactivation. pcRNA enabled the fastest (<1 minute) and most complete approach (<1% residual indels) for Cas9 deactivation. It also exhibited significantly enhanced specificity with wild type Cas9. Time-resolved deactivation revealed that 12-36 hours of Cas9 activity or 2-4 hours of base editor activity was sufficient to achieve high editing efficiency. pcRNA is useful for studies of the cellular response to DNA damage by abolishing sustained cycles of damage and repair that would otherwise desynchronize response trajectories. Together, pcRNA expands the CRISPR toolbox for precision genome editing and studies of DNA damage and repair. Zou et al. developed light-mediated control over Cas9 and base editor deactivation by introducing a photocleavable moiety into guide RNA. Deactivation occurs within seconds and approaches completeness. This modification natively enhanced specificity. Timed deactivation facilitated studies of DNA repair by synchronizing the termination of DNA damage within a cell population.
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