Project Summary Chemical modifications to genomic DNA are ubiquitous across all kingdoms of life. They are diverse in nature including methylation, hydroxymethylation, and formylation of nucleic acid residues. It is also increasingly clear that these modifications have diverse regulatory roles. In eukaryotes, DNA modifications regulate gene expression, RNA splicing, genome organization, and gene imprinting; these molecular functions also have important downstream consequences for development, disease, and organismal interactions with environmental stimuli and chemicals. Furthermore, with the advent of genome and epigenome editing technologies, these modifications can bias the specificity and efficiency of molecular tools. Given the broad importance of DNA modifications, any experimental tools that are able to control, measure, sense, or track them would have profound impacts across biological disciplines. What is particularly needed, but not yet technologically possibly, is the ability to simultaneously sense epigenetic modifications at specific genomic locations in living and single cells. This capability would unlock a broad palette of new experimental approaches to reveal new insights into topics such as gene imprinting, causation vs. correlation of epigenetic modifications, and cell and tissue stochasticity. Furthermore, it would inform the development of new classes of affinity reagents for biochemical assays, disease detection, and diagnostics and of genome and epigenome editing technologies that are aware of and specific towards DNA modification state. To drive this new realm of studies, we will create the first molecular tool that binds DNA hydroxymethylation at specific and programmable genomic loci, and provide a broadly applicable platform for engineering many other similar molecular reagents.