Site-Specific and Enzymatic Cross-Linking of SgRNA Enables Wavelength-Selectable Photoactivated Control of CRISPR Gene Editing

Overview

Journal J Am Chem Soc

Publisher American Chemical Society

Specialty Chemistry

Date 2022 Mar 8

PMID 35257575

Authors

Dongyang Zhang

Luping Liu

Shuaijiang Jin

Ember Tota

Zijie Li

Xijun Piao

Xuan Zhang

Xiang-Dong Fu

Neal K Devaraj

Affiliations

Soon will be listed here.

Abstract

Chemical cross-linking enables rapid identification of RNA-protein and RNA-nucleic acid inter- and intramolecular interactions. However, no method exists to site-specifically and covalently cross-link two user-defined sites within an RNA. Here, we develop RNA-CLAMP, which enables site-specific and enzymatic cross-linking (clamping) of two selected guanine residues within an RNA. Intramolecular clamping can disrupt normal RNA function, whereas subsequent photocleavage of the cross-linker restores activity. We used RNA-CLAMP to clamp two stem loops within the single-guide RNA (sgRNA) of the CRISPR-Cas9 gene editing system via a photocleavable cross-linker, completely inhibiting gene editing. Visible light irradiation cleaved the cross-linker and restored gene editing with high spatiotemporal resolution. Design of two photocleavable linkers responsive to different wavelengths of light allowed multiplexed photoactivation of gene editing in mammalian cells. This photoactivated CRISPR-Cas9 gene editing platform benefits from undetectable background activity, provides a choice of activation wavelengths, and has multiplexing capabilities.

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