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J Mol Biol. 2020 Feb 19. pii: S0022-2836(20)30157-1. doi: 10.1016/j.jmb.2020.02.007. [Epub ahead of print]

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

Author information

1
Maimónides Biomedical Research Institute of Córdoba (IMIBIC), 14071, Córdoba, Spain; Department of Genetics, University of Córdoba, 14071, Córdoba, Spain; Reina Sofía University Hospital, 14071, Córdoba, Spain.
2
Maimónides Biomedical Research Institute of Córdoba (IMIBIC), 14071, Córdoba, Spain; Department of Genetics, University of Córdoba, 14071, Córdoba, Spain; Reina Sofía University Hospital, 14071, Córdoba, Spain. Electronic address: ge2roarm@uco.es.

Abstract

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.

KEYWORDS:

DNA demethylation; DNA glycosylases; TET dioxygenases; epigenetics

PMID:
32087201
DOI:
10.1016/j.jmb.2020.02.007

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