利用光可裂解向导 RNA 实现 Cas9 失活。
Cas9 deactivation with photocleavable guide RNAs.
机构信息
Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD 21205, USA.
Department of Biophysics and Biophysical Chemistry, Johns Hopkins University, Baltimore, MD 21205, USA.
出版信息
Mol Cell. 2021 Apr 1;81(7):1553-1565.e8. doi: 10.1016/j.molcel.2021.02.007. Epub 2021 Mar 3.
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 (pcRNAs) that endow Cas9 nucleases and base editors with a built-in mechanism for light-based deactivation. pcRNA enabled the fastest (<1 min) and most complete (<1% residual indels) approach for Cas9 deactivation. It also exhibited significantly enhanced specificity with wild-type Cas9. Time-resolved deactivation revealed that 12-36 h of Cas9 activity or 2-4 h 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.
精确控制 CRISPR-Cas9 将提高其安全性和适用性。受控制的 CRISPR 抑制是一种很有前途的方法，但由于抑制剂的单独传递、不完全失活和缓慢的动力学而变得复杂。为了克服这些障碍，我们设计了光可裂解的向导 RNA（pcRNA），为 Cas9 核酸酶和碱基编辑器提供了内置的基于光的失活机制。pcRNA 使 Cas9 失活的速度最快（<1 分钟）且完全（<1%的残留插入缺失）。它还表现出与野生型 Cas9 相比显著提高的特异性。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 小时足以实现高编辑效率。pcRNA 通过消除持续的损伤和修复循环，对于研究细胞对 DNA 损伤的反应非常有用，否则这些循环会使反应轨迹不同步。总之，pcRNA 扩展了 CRISPR 工具包，用于精确的基因组编辑以及 DNA 损伤和修复的研究。
Zotero 插件