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

Nat Commun. 2019 Sep 20;10(1):4296. doi: 10.1038/s41467-019-12339-7.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • 5-Methylcytosine / metabolism
  • CRISPR-Cas Systems
  • Cell Cycle Proteins / metabolism
  • Cell Line, Tumor
  • Cell Proliferation
  • Clustered Regularly Interspaced Short Palindromic Repeats*
  • DNA Demethylation*
  • DNA Glycosylases / metabolism
  • DNA Methylation
  • DNA Repair*
  • DNA-(Apurinic or Apyrimidinic Site) Lyase / metabolism
  • Gene Editing
  • HEK293 Cells
  • Humans
  • Mixed Function Oxygenases / genetics
  • Oxidation-Reduction
  • Promoter Regions, Genetic
  • Proto-Oncogene Proteins / genetics
  • Proto-Oncogene Proteins / metabolism
  • RNA, Guide, CRISPR-Cas Systems / metabolism*
  • Sequence Analysis, RNA


  • Cell Cycle Proteins
  • GADD45A protein, human
  • Proto-Oncogene Proteins
  • RNA, Guide, CRISPR-Cas Systems
  • 5-Methylcytosine
  • Mixed Function Oxygenases
  • TET1 protein, human
  • DNA Glycosylases
  • DNA-(Apurinic or Apyrimidinic Site) Lyase
  • NEIL2 protein, human