Optical Control of a CRISPR/Cas9 System for Gene Editing by Using Photolabile crRNA
Optical Control of a CRISPR/Cas9 System for Gene Editing by Using Photolabile crRNA
复制标题
使用不稳定性 crRNA 进行基因编辑的 CRISPR/Cas9 系统的光学控制
DOI：
10.1002/anie.202009890
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发表时间：
2020-09-08
影响因子：
16.6
通讯作者：
Tang, Xinjing
中科院分区：
文献类型：
--
作者：
Zhang, Yu;Ling, Xinyu;Tang, Xinjing
目前CRISPR/Cas9是广泛使用的高效基因编辑工具。以高时间和空间分辨率精确控制 CRISPR/Cas9 系统对于研究基因调控和编辑至关重要。在这里，我们通过在 5' 末端耦合维生素 E 和光不稳定接头来灭活 CRISPR/Cas9 系统，合成了一种新型光控 crRNA。维生素 E 修饰不会影响 Cas9/crRNA/tracrRNA 复合物的核糖核蛋白 (RNP) 形成，但会抑制 RNP 与靶 DNA 的结合。在光照射下，通过 T7E1 检测和 Sanger 测序以及 EGFP 稳定表达细胞中 EGFP 表达的基因敲低，维生素 E 笼状 crRNA 被成功激活，从而实现人类细胞中血管内皮细胞生长因子 A (VEGFA) 的光诱导基因组编辑。这种新的 crRNA 笼蔽策略可以为 CRISPR/Cas9 介导的基因编辑的时空光调节提供新方法。
Currently CRISPR/Cas9 is a widely used efficient tool for gene editing. Precise control over the CRISPR/Cas9 system with high temporal and spatial resolution is essential for studying gene regulation and editing. Here, we synthesized a novel light-controlled crRNA by coupling vitamin E and a photolabile linker at the 5 ' terminus to inactivate the CRISPR/Cas9 system. The vitamin E modification did not affect ribonucleoprotein (RNP) formation of Cas9/crRNA/tracrRNA complexes but did inhibit the association of RNP with the target DNA. Upon light irradiation, vitamin E-caged crRNA was successfully activated to achieve light-induced genome editing of vascular endothelial cell-growth factor A (VEGFA) in human cells through a T7E1 assay and Sanger sequencing as well as gene knockdown of EGFP expression in EGFP stably expressing cells. This new caging strategy for crRNA could provide new methods for spatiotemporal photoregulation of CRISPR/Cas9-mediated gene editing.