Optical Control of a CRISPR/Cas9 System for Gene Editing by Using Photolabile crRNA. (8th September 2020)

• Record Type: Journal Article
• Authors: Zhang, Yu; Ling, Xinyu; Su, Xiaoxuan; Zhang, Shilin; Wang, Jing; Zhang, Pingjing; Feng, Wenjian; Zhu, York Yuanyuan; Liu, Tao; Tang, Xinjing
• Is Part Of: Angewandte Chemie. Volume 132:Number 47(2020)
• Page Start: 21081
• Page End: 21085
• Publication Date: 2020-09-08
• Subjects: caged crRNA -- CRISPR/Cas9 -- gene editing -- photomodulation -- vitamin E; Chemistry -- Periodicals; 540
• DOI: 10.1002/ange.202009890
• Languages: English
• ISSNs: 0044-8249

Abstract

Abstract: 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. Abstract : Caged crRNA was readily developed through a terminus modification with a photolabile linker and vitamin E for construction of a caged CRISPR/Cas9 system. The system was successfully used for photomodulation of VEGFA gene editing and control of EGFP gene silencing through the temporary inhibition of the interaction between the target DNA and Cas9/crRNA/tracrRNA complex.