Targeted DNA demethylation in vivo using dCas9-peptide repeat and scFv-TET1 catalytic domain fusions

Sumiyo Morita, Hirofumi Noguchi, Takuro Horii, Kazuhiko Nakabayashi, Mika Kimura, Kohji Okamura, Atsuhiko Sakai, Hideyuki Nakashima, Kenichiro Hata, Kinichi Nakashima, Izuho Hatada

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Abstract

Despite the importance of DNA methylation in health and disease, technologies to readily manipulate methylation of specific sequences for functional analysis and therapeutic purposes are lacking. Here we adapt the previously described dCas9-SunTag for efficient, targeted demethylation of specific DNA loci. The original SunTag consists of ten copies of the GCN4 peptide separated by 5-amino-acid linkers. To achieve efficient recruitment of an anti-GCN4 scFv fused to the ten-eleven (TET) 1 hydroxylase, which induces demethylation, we changed the linker length to 22 amino acids. The system attains demethylation efficiencies >50% in seven out of nine loci tested. Four of these seven loci showed demethylation of >90%. We demonstrate targeted demethylation of CpGs in regulatory regions and demethylation-dependent 1.7- to 50-fold upregulation of associated genes both in cell culture (embryonic stem cells, cancer cell lines, primary neural precursor cells) and in vivo in mouse fetuses.

Original languageEnglish
Pages (from-to)1060-1065
Number of pages6
JournalNature Biotechnology
Volume34
Issue number10
DOIs
Publication statusPublished - Oct 1 2016

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All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology
  • Molecular Medicine
  • Biomedical Engineering

Cite this

Morita, S., Noguchi, H., Horii, T., Nakabayashi, K., Kimura, M., Okamura, K., ... Hatada, I. (2016). Targeted DNA demethylation in vivo using dCas9-peptide repeat and scFv-TET1 catalytic domain fusions. Nature Biotechnology, 34(10), 1060-1065. https://doi.org/10.1038/nbt.3658