Designer Receptors for Nucleotide‐Resolution Analysis of Genomic 5‐Methylcytosine by Cellular Imaging

We report programmable receptors for the imaging‐based analysis of 5‐methylcytosine (5mC) in user‐defined DNA sequences of single cells. Using fluorescent transcription‐activator‐like effectors (TALEs) that can recognize sequences of canonical and epigenetic nucleobases through selective repeats, we imaged cellular SATIII DNA, the origin of nuclear stress bodies (nSB). We achieve high nucleobase selectivity of natural repeats in imaging and demonstrate universal nucleobase binding by an engineered repeat. We use TALE pairs differing in only one such repeat in co‐stains to detect 5mC in SATIII sequences with nucleotide resolution independently of differences in target accessibility. Further, we directly correlate the presence of heat shock factor 1 with 5mC at its recognition sequence, revealing a potential function of 5mC in its recruitment as initial step of nSB formation. This opens a new avenue for studying 5mC functions in chromatin regulation in situ with nucleotide, locus, and cell resolution. It needs two TALEs to tango: Programmable receptors for the imaging‐based analysis of 5‐methylcytosine (5mC) in DNA sequences of single cells are reported. Staining of SATIII DNA with pairs of 5mC‐sensitive and 5mC‐insensitive transcription‐activator‐like effectors (TALEs) allows the analysis of 5mC at a target nucleotide position and its direct correlation with the recruitment of transcription factor HSF1 as the initial step of nuclear stress body formation.