Programmable Protein-DNA Crosslinking for the Direct Capture and Quantification of 5-Formylcytosine

5-Formylcytosine (5fC) is an epigenetic nucleobase of mammalian genomes that occurs as intermediate of active DNA demethylation. 5fC uniquely interacts and reacts with key nuclear proteins, indicating functions in genome regulation. Transcription-activator-like effectors (TALEs) are repeat-based DNA binding proteins that can serve as probes for the direct, programmable recognition and analysis of epigenetic nucleobases. However, no TALE repeats for the selective recognition of 5fC are available, and the typically low genomic levels of 5fC represent a particular sensitivity challenge. We here advance TALE-based nucleobase targeting from recognition to covalent crosslinking. We report TALE repeats bearing the ketone-amino acid p-acetylphenylalanine (pAcF) that universally bind all mammalian cytosine nucleobases, but selectively form diaminooxy-linker-mediated dioxime crosslinks to 5fC. We identify repeat-linker combinations enabling sin-gle CpG resolution, and demonstrate the direct quantifica-tion of 5fC l...