Proteome-scale tagging and functional screening in mammalian cells by ORFtag [Cut & Run]
The comprehensive assessment of protein function is a key aim in modern biology yet has proven difficult with existing approaches. Here, we introduce ORFtag, a versatile tool for the massively parallel protein tagging and functional screening at proteome scale. ORFtag integrates a cassette bearing a promoter, the coding sequence of a protein tag, and a splice-donor site, which allows the tag to be expressed and spliced to genomic protein-coding exons and thus enables endogenous protein tagging at minimal expenditure and effort. We demonstrate the versatility of ORFtag by three functional screens for transcriptional activators and repressors and mediators of mRNA translation and stability. Overall design: anti-V5 CUT&RUN experiments in Zfp574-AID-V5 mESC cell line