A one-step CRISPR-based strategy for endogenous gene tagging in Drosophila melanogaster

The ability to tag endogenous genes to create fluorescent fusion proteins has revolutionized the studies of protein subcellular localization and dynamics and regulation in live cells. Here, we report a precise, rapid, and one-step endogenous gene tagging system in Drosophila melanogaster, which allows us to screen for engineered lines without the visible eye marker. Specifically, we developed an efficient screening strategy for the identification of the homologous integration events by employing a PCR-based method to characterize individual flies using only a small segment of their middle leg. Here, we detail design and construction of gRNA plasmids and donor plasmid, and methods for screening, and confirmation of engineered lines. Together, these protocols make tagging any endogenous protein in Drosophila more efficient and faster, enabling studies of a wide range of cellular processes in live cells within intact organisms.