Isonitrile-Tetrazine Click-and-Release Chemistry for Controlling RNA-Guided Nucleic Acid Cleavage.

With the increasing demand for the regulation of CRISPR systems, a considerable number of studies have been conducted to control their excessive activity levels. In this context, we propose a method that involves a bioorthogonal cleavage reaction between isonitrile and tetrazine to modulate the cleavage activity of the CRISPR system. Importantly, isonitrile demonstrates significant potential for modifying sgRNAs, making it a promising candidate for bioorthogonal reactions, a phenomenon that has not been previously reported. Our approach utilizes the 3-isocyanopropyl-carbonate group as a caging group to deactivate the CRISPR systems, while tetrazine acts as an activator to restore their activities. Through the implementation of post-synthetic modifications and click-and-release chemistry, we have successfully achieved the regulation of RNA-guided nucleic acid cleavage, which holds great promise for controlling gene editing in human cells.