Editing DNA Methylation in the Mammalian Genome

Cell. 2016 Sep 22;167(1):233-247.e17. doi: 10.1016/j.cell.2016.08.056.

Abstract

Mammalian DNA methylation is a critical epigenetic mechanism orchestrating gene expression networks in many biological processes. However, investigation of the functions of specific methylation events remains challenging. Here, we demonstrate that fusion of Tet1 or Dnmt3a with a catalytically inactive Cas9 (dCas9) enables targeted DNA methylation editing. Targeting of the dCas9-Tet1 or -Dnmt3a fusion protein to methylated or unmethylated promoter sequences caused activation or silencing, respectively, of an endogenous reporter. Targeted demethylation of the BDNF promoter IV or the MyoD distal enhancer by dCas9-Tet1 induced BDNF expression in post-mitotic neurons or activated MyoD facilitating reprogramming of fibroblasts into myoblasts, respectively. Targeted de novo methylation of a CTCF loop anchor site by dCas9-Dnmt3a blocked CTCF binding and interfered with DNA looping, causing altered gene expression in the neighboring loop. Finally, we show that these tools can edit DNA methylation in mice, demonstrating their wide utility for functional studies of epigenetic regulation.

Publication types

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

MeSH terms

  • Animals
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Brain-Derived Neurotrophic Factor / genetics
  • CCCTC-Binding Factor
  • CRISPR-Associated Protein 9
  • Cell Line
  • Clustered Regularly Interspaced Short Palindromic Repeats*
  • CpG Islands
  • DNA (Cytosine-5-)-Methyltransferases / genetics
  • DNA (Cytosine-5-)-Methyltransferases / metabolism*
  • DNA Methylation / genetics*
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism*
  • Endonucleases / genetics
  • Endonucleases / metabolism
  • Enhancer Elements, Genetic
  • Epigenesis, Genetic*
  • Gene Editing / methods*
  • Genome
  • Mice
  • MyoD Protein / metabolism
  • Neurons / metabolism
  • Promoter Regions, Genetic
  • Protein Binding
  • Proto-Oncogene Proteins / genetics
  • Proto-Oncogene Proteins / metabolism*
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Repressor Proteins / metabolism

Substances

  • Bacterial Proteins
  • Brain-Derived Neurotrophic Factor
  • CCCTC-Binding Factor
  • Ctcf protein, mouse
  • DNA-Binding Proteins
  • MyoD Protein
  • Proto-Oncogene Proteins
  • Recombinant Fusion Proteins
  • Repressor Proteins
  • TET1 protein, mouse
  • DNA (Cytosine-5-)-Methyltransferases
  • DNA methyltransferase 3A
  • CRISPR-Associated Protein 9
  • Cas9 endonuclease Streptococcus pyogenes
  • Endonucleases