title: Targeted DNA demethylation in vivo using dCas9-peptide repeat and scFv-TET1 catalytic domain fusions.
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.
authors: Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Japan.
authors: Sumiyo
authors: S
authors: Morita
authors: Department of Stem Cell Biology and Medicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
authors: Hirofumi
authors: H
authors: Noguchi
authors: Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Japan.
authors: Takuro
authors: T
authors: Horii
authors: Department of Maternal-Fetal Biology, National Research Institute for Child Health and Development, Tokyo, Japan.
authors: Kazuhiko
authors: K
authors: Nakabayashi
authors: Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Japan.
authors: Mika
authors: M
authors: Kimura
authors: Department of Systems BioMedicine, National Research Institute for Child Health and Development, Tokyo, Japan.
authors: Kohji
authors: http://orcid.org/0000-0001-9747-5069
authors: K
authors: Okamura
authors: Department of Stem Cell Biology and Medicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
authors: Atsuhiko
authors: A
authors: Sakai
authors: Department of Stem Cell Biology and Medicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
authors: Hideyuki
authors: H
authors: Nakashima
authors: Department of Maternal-Fetal Biology, National Research Institute for Child Health and Development, Tokyo, Japan.
authors: Kenichiro
authors: K
authors: Hata
authors: Department of Stem Cell Biology and Medicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
authors: Kinichi
authors: K
authors: Nakashima
authors: Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Japan.
authors: Izuho
authors: I
authors: Hatada
mesh_terms: D000818:Animals; D050976:Basic-Leucine Zipper Transcription Factors / Q000235:genetics*; D064130:CRISPR-Associated Proteins / Q000235:genetics*; D002384:Catalysis; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D018899:CpG Islands / Q000235:genetics; D019175:DNA Methylation / Q000235:genetics*; D018390:Gene Targeting / Q000379:methods; D005818:Genetic Engineering / Q000379:methods; D051379:Mice; D006899:Mixed Function Oxygenases / Q000235:genetics*; D000072417:Protein Domains; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics*; D057127:Single-Chain Antibodies / Q000235:genetics*
chemical_list: D050976:Basic-Leucine Zipper Transcription Factors; D064130:CRISPR-Associated Proteins; C501391:GCN4 protein, S cerevisiae; D011518:Proto-Oncogene Proteins; D029701:Saccharomyces cerevisiae Proteins; D057127:Single-Chain Antibodies; D006899:Mixed Function Oxygenases; C462539:TET1 protein, human