Photoswitchable intein for light control of covalent protein binding and cleavage.

Precise control of covalent protein binding and cleavage in mammalian cells is crucial for manipulating cellular processes but remains challenging due to dark background, poor stability, low efficiency, or requirement of unnatural amino acids in current optogenetic tools. We introduce a photoswitchable intein (PS Intein) engineered by allosterically modulating a small autocatalytic gp41-1 intein with tandem Vivid photoreceptor. PS Intein exhibits superior functionality and low background in cells compared to existing tools. PS Intein-based systems enable light-induced covalent binding, cleavage, and release of proteins for regulating gene expression and cell fate. The high responsiveness and ability to integrate multiple inputs allow for intersectional cell targeting using cancer- and tumor microenvironment-specific promoters. PS Intein tolerates various fusions and insertions, facilitating its application in diverse cellular contexts. This versatile technology offers efficient light-controlled protein manipulation, providing a powerful tool for adding functionalities to proteins and precisely controlling protein networks in living cells.