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

Aziz Taghbalout, Menghan Du, Nathaniel Jillette, Wojciech Rosikiewicz, Abhijit Rath, Christopher D. Heinen, Sheng Li, Albert W. Cheng

May 22, 2019

Received date 20th May 19

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.

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This is an abstract of a preprint hosted on an independent third party site. It has not been peer reviewed but is currently under consideration at Nature Communications.

Nature Communications

Nature Research, Springer Nature