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

J Mol Biol. 2020 Mar 27;432(7):2204-2216. doi: 10.1016/j.jmb.2020.02.007. Epub 2020 Feb 19.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 5-Methylcytosine / chemistry*
  • Arabidopsis / genetics*
  • Arabidopsis / metabolism
  • Arabidopsis Proteins / antagonists & inhibitors
  • Arabidopsis Proteins / genetics*
  • Arabidopsis Proteins / metabolism
  • CRISPR-Associated Protein 9 / genetics*
  • CRISPR-Associated Protein 9 / metabolism
  • CRISPR-Cas Systems*
  • Epigenesis, Genetic
  • Gene Editing*
  • Nuclear Proteins / antagonists & inhibitors
  • Nuclear Proteins / genetics*
  • Nuclear Proteins / metabolism
  • Transcriptional Activation


  • Arabidopsis Proteins
  • Nuclear Proteins
  • ROS1 protein, Arabidopsis
  • 5-Methylcytosine
  • CRISPR-Associated Protein 9