RT Journal Article SR Electronic T1 Casilio-ME: Enhanced CRISPR-based DNA demethylation by RNA-guided coupling methylcytosine oxidation and DNA repair pathways JF bioRxiv FD Cold Spring Harbor Laboratory SP 641993 DO 10.1101/641993 A1 Aziz Taghbalout A1 Menghan Du A1 Nathaniel Jillette A1 Wojciech Rosikiewicz A1 Abhijit Rath A1 Christopher D. Heinen A1 Sheng Li A1 Albert W. Cheng YR 2019 UL http://biorxiv.org/content/early/2019/05/19/641993.abstract AB We have developed a methylation editing toolbox, Casilio-ME, 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 Casilio-ME1 robustly altered the CpG methylation landscape of promoter regions and activated methylation-silenced genes. We augmented Casilio-ME1 to simultaneously deliver the TET1-catalytic domain and GADD45A (Casilio-ME2) or NEIL2 (Casilio-ME3) to streamline removal of oxidized cytosine intermediates to enhance activation of targeted genes. Using two-in-one effectors or modular effectors, Casilio-ME2 and Casilio-ME3 remarkably boosted gene activation and methylcytosine demethylation of targeted loci. We expanded the toolbox to enable a stable and expression-inducible system for broader application of the Casilio-ME platforms. This work establishes an advanced platform for editing DNA methylation to enable transformative research investigations interrogating DNA methylomes.