Photo-Removable CRISPR-Cas12a Systems for Programmable Translational Modulation

Abstract Messenger RNA (mRNA) has emerged as a powerful tool for developing new therapeutics and biotechnologies due to its inherent ability to express proteins. However, the lack of precise control over protein expression remains a critical limitation for mRNA-based biomedical applications. Herein, we developed a novel photoactivatable mRNA translational modulation strategy that leverages a photoremovable Cas12a-crRNA ribonucleoprotein (RNP) complex as a programmable translational modulator. This RNP consists of Cas12a and a crRNA engineered with photocleavable (PC) linkers in its spacer region. The resulting binary complex binds specifically to the 5′-untranslated region (5′-UTR) of target mRNAs and inhibits translational initiation. Upon light irradiation, the PC-linkers are cleaved, leading to RNP dissociation and rapid restoration of protein expression. This strategy demonstrated robust phototriggered protein expression both in vitro (firefly luciferase) and in living cells (firefly luciferase, EGFP, and mCherry). Furthermore, the therapeutic potential of this approach was demonstrated in Hep3B cells, where phototriggered translation of tumor suppressor p53 mRNA successfully induced apoptosis. Our study demonstrates an innovative attempt at employing photoremovable RNP to modulate mRNA translation and provides distinct insights for rational exploration of mRNA-based precise therapeutics.