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 language | English |
|---|---|
| Pages (from-to) | 1060-1065 |
| Number of pages | 6 |
| Journal | Nature Biotechnology |
| Volume | 34 |
| Issue number | 10 |
| DOIs | |
| Publication status | Published - 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
Targeted DNA demethylation in vivo using dCas9-peptide repeat and scFv-TET1 catalytic domain fusions. / Morita, Sumiyo; Noguchi, Hirofumi; Horii, Takuro; Nakabayashi, Kazuhiko; Kimura, Mika; Okamura, Kohji; sakai, atsuhiko; Nakashima, Hideyuki; Hata, Kenichiro; Nakashima, Kinichi; Hatada, Izuho.
In: Nature Biotechnology, Vol. 34, No. 10, 01.10.2016, p. 1060-1065.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Targeted DNA demethylation in vivo using dCas9-peptide repeat and scFv-TET1 catalytic domain fusions
AU - Morita, Sumiyo
AU - Noguchi, Hirofumi
AU - Horii, Takuro
AU - Nakabayashi, Kazuhiko
AU - Kimura, Mika
AU - Okamura, Kohji
AU - sakai, atsuhiko
AU - Nakashima, Hideyuki
AU - Hata, Kenichiro
AU - Nakashima, Kinichi
AU - Hatada, Izuho
PY - 2016/10/1
Y1 - 2016/10/1
N2 - 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.
AB - 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.
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UR - http://www.scopus.com/inward/citedby.url?scp=84992126346&partnerID=8YFLogxK
U2 - 10.1038/nbt.3658
DO - 10.1038/nbt.3658
M3 - Article
C2 - 27571369
AN - SCOPUS:84992126346
VL - 34
SP - 1060
EP - 1065
JO - Nature Biotechnology
JF - Nature Biotechnology
SN - 1087-0156
IS - 10
ER -