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

A Taghbalout, M Du, N Jillette, W Rosikiewicz… - Nature …, 2019 - nature.com
Nature communications, 2019nature.com
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 …
Abstract
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.
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