Nat Commun 2019 Sep 20; 10(1):4296
HG009900,CA034196,Leukemia Research Foundation New Investigator Grant,Jackson Laboratory Director's Innovation Fund
Here we develop 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 alters the CpG methylation landscape of promoter regions and activates methylation-silenced genes. We augment 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 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 Casilio-ME platforms. This work establishes a platform for editing DNA methylation to enable research investigations interrogating DNA methylomes.
Taghbalout, Aziz; Du, Menghan; Jillette, Nathaniel L.; Rosikiewicz, Wojciech; Rath, Abhijit; Heinen, Christopher D; Li, Sheng; and Cheng, Albert, "Enhanced CRISPR-based DNA demethylation by Casilio-ME-mediated RNA-guided coupling of methylcytosine oxidation and DNA repair pathways." (2019). Faculty Research 2019. 211.