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DNA Methylation Editing by CRISPR-guided Excision of 5-Methylcytosine.


Devesa-Guerra I, Morales-Ruiz T, Pérez-Roldán J, Parrilla-Doblas JT, Dorado-León M, García-Ortiz MV, Ariza RR, Roldán-Arjona T

Tools for actively targeted DNA demethylation are required to increase our knowledge about regulation and specific functions of this important epigenetic modification. DNA demethylation in mammals involves TET-mediated oxidation of 5-methylcytosine (5-meC), which may promote its replication-dependent dilution and/or active removal through base excision repair (BER). However, it is still unclear whether oxidized derivatives of 5-meC are simply DNA demethylation intermediates or rather epigenetic marks on their own. Unlike animals, plants have evolved enzymes that directly excise 5-meC without previous modification. In this work, we have fused the catalytic domain of Arabidopsis ROS1 5-meC DNA glycosylase to a CRISPR-associated null-nuclease (dCas9) and analyzed its capacity for targeted reactivation of methylation-silenced genes, in comparison to other dCas9-effectors. We found that dCas9-ROS1, but not dCas9-TET1, is able to reactivate methylation-silenced genes and induce partial demethylation in a replication-independent manner. We also found that reactivation induced by dCas9-ROS1, as well as that achieved by two different CRISPR-based chromatin effectors (dCas9-VP160 and dCas9-p300), generally decreases with methylation density. Our results suggest that plant 5-meC DNA glycosylases are a valuable addition to the CRISPR-based toolbox for epigenetic editing.

Tags
Antibody
CRISPR

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Published
February, 2020

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Products used in this publication

  • Antibody cas9 icon
    C15200216-100
    CRISPR/Cas9 monoclonal antibody 4G10

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