Enhanced CRISPR-based DNA demethylation by Casilio-ME-mediated RNA-guided coupling of methylcytosine oxidation and DNA repair pathways

<jats:title>Abstract</jats:title><jats:p>Here we develop a methylation editing toolbox, <jats:italic>Casilio-ME</jats:italic>, that enables not only RNA-guided methylcytosine editing by targeting TET1 to genomic sites, but also by co-delivering TET1 and protein factors that couple methylcytosine oxidation to DNA repair activities, and/or promote TET1 to achieve enhanced activation of methylation-silenced genes. Delivery of TET1 activity by <jats:italic>Casilio-ME1</jats:italic> robustly alters the CpG methylation landscape of promoter regions and activates methylation-silenced genes. We augment <jats:italic>Casilio-ME1</jats:italic> to simultaneously deliver the TET1-catalytic domain and GADD45A (<jats:italic>Casilio-ME2</jats:italic>) or NEIL2 (<jats:italic>Casilio-ME3</jats:italic>) to streamline removal of oxidized cytosine intermediates to enhance activation of targeted genes. Using two-in-one effectors or modular effectors, <jats:italic>Casilio-ME2</jats:italic> and <jats:italic>Casilio-ME3</jats:italic> remarkably boost gene activation and methylcytosine demethylation of targeted loci. We expand the toolbox to enable a stable and expression-inducible system for broader application of the <jats:italic>Casilio-ME</jats:italic> platforms. This work establishes a platform for editing DNA methylation to enable research investigations interrogating DNA methylomes.</jats:p>