Dynamics of CDKN1A in Single Cells Defined by an Endogenous Fluorescent Tagging Toolkit

Observing the endogenous abundance, localization, and dynamics of proteins in mammalian cells is crucial to understanding their function and behavior. Currently, there is no systematic approach for the fluorescent tagging of endogenous loci. Here, we used Cas9-catalyzed DNA breaks, short homology arms, and a family of donor plasmids to establish endogenous Fluorescent tagging (eFlut): a low-cost and efficient approach to generating endogenous proteins with fluorescent labels. We validated this protocol on multiple proteins in several cell lines and species and applied our tools to study the cellcycle inhibitor CDKN1A in single cells. We uncover heterogeneity in the timing and rate of CDKN1A induction post-DNA damage and show that this variability is post-transcriptionally regulated, depends on cell-cycle position, and has long-term consequences for cellular proliferation. The tools developed in this study should support widespread study of the dynamics and localization of diverse proteins in mammalian cells.