Novel Tet3 enzymes for next generation epigenetic sequencing

Epigenetic regulation of gene expression is essential for cellular development and differentiation processes in higher eukaryotes. Modifications of cytidine, in particular 5-methylcytidine (5mdC) in DNA plays a central role through impacting on chromatin structure, repression of transposons and regulation of transcription. DNA methylation is actively installed by DNA methyltransferases and reversed through Tet-dioxygenase-mediated oxidation of 5mdC to 5-hydroxylmethylcytosine (5hmdC), formylcytosine (5fdC) and 5-carboxycytosine (5cadC). In order to understand the role of these epigenetic DNA modification in cellular differentiation and developmental processes, as well as disease states mapping and tracing of 5mdC and its oxidized forms is crucial. Bisulfite sequencing, the benchmark for mapping 5mdC for the last decades, suffers from degradation of the majority of genetic material through the harsh chemical treatment. Alternative, sequencing methods often utilize Tet-enzyme-mediated oxidation of 5mdC to locate 5mdC and 5hmdC in genomic DNA. Here we report the development of novel Tet3-variants for oxidation-based bisulfite free 5mdC- sequencing.