Engineered TALE Repeats for Enhanced Imaging‐Based Analysis of Cellular 5‐Methylcytosine

Transcription‐activator‐like effectors (TALEs) are repeat‐based, programmable DNA‐binding proteins that can be engineered to recognize sequences of canonical and epigenetically modified nucleobases. Fluorescent TALEs can be used for the imaging‐based analysis of cellular 5‐methylcytosine (5 mC) in repetitive DNA sequences. This is based on recording fluorescence ratios from cell co‐stains with two TALEs: an analytical TALE targeting the cytosine (C) position of interest through a C‐selective repeat that is blocked by 5 mC, and a control TALE targeting the position with a universal repeat that binds both C and 5 mC. To enhance this approach, we report herein the development of novel 5 mC‐selective repeats and their integration into TALEs that can replace universal TALEs in imaging‐based 5 mC analysis, resulting in a methylation‐dependent response of both TALEs. We screened a library of size‐reduced repeats and identified several 5 mC binders. Compared to the 5 mC‐binding repeat of natural TALEs and to the universal repeat, two repeats containing aromatic residues showed enhancement of 5 mC binding and selectivity in cellular transcription activation and electromobility shift assays, respectively. In co‐stains of cellular SATIII DNA with a corresponding C‐selective TALE, this selectivity results in a positive methylation response of the new TALE, offering perspectives for studying 5 mC functions in chromatin regulation by in situ imaging with increased dynamic range.