PROJECT SUMMARY/ABSTRACT A central challenge of the post-genomic era is to comprehensively characterize the cellular role of the ~20,000 proteins encoded in the human genome. Functional tagging is a powerful strategy to characterize the cellular role of proteins. In particular, tags allow access to two key features of protein function: localization (using fluorescent tags) and interaction partners (using epitope tags and immuno-precipitation). Hence, by tagging proteins in a systematic manner, a comprehensive functional description of an organism’s proteome can be achieved. For this purpose, we have previously developed FP11 tags based on self-complementing split fluorescent proteins, which, in combination with gene editing using Cas9/sgRNA ribonucleoprotein (RNPs), enable rapid, efficient and highly scalable tagging of endogenous proteins in mammalian cell lines. While our results have paved the way for the large-scale generation of endogenously tagged human cell lines for the proteome-wide analysis of protein localization and interaction networks in a native cellular context. However, for practical generation and analysis of large-scale libraries, several major technical limitations still need to be addressed: brightness, color availability, and live imaging platforms with low photobleaching. In the proposed project, we plan to engineer improved FP11 tags and new split protein fragment tags to address these technical challenges. We will also demonstrate the powerful applications by developing a new selective plane illumination microscopy (SPIM) system to screen an endogenously tagged library.