Optogenetics enables precise spatiotemporal control of intracellular processes by light and has become a powerful approach for manipulating cell signaling and gene expression. While chemical induction systems and synthetic biology-based tools such as Tet-regulated promoters and CRISPRa/CRISPRi provide versatile means to control gene expression, their temporal resolution and spatial specificity are limited by diffusion and degradation of chemical stimuli. Optogenetic strategies based on light-induced dimerization (LID) systems overcome these limitations and have enabled optical control of transcription factors and endogenous gene expression. However, most existing approaches focus on regulating individual genes or promoter elements, and methods to directly manipulate entire endogenous transcriptional networks in their physiological context remain limited. Here, we describe an optogenetic system, Opto-p53, which enables light-induced activation of endogenous p53 transcriptional activity by reconstituting its functional domains via a blue-light-inducible dimerization system. This protocol provides detailed procedures for cell line construction, light stimulation, imaging, and data analysis, offering a versatile platform to investigate p53-dependent transcriptional responses under controlled activation conditions.
