| RRC ID |
46326
|
| Author |
Morita S, Noguchi H, Horii T, Nakabayashi K, Kimura M, Okamura K, Sakai A, Nakashima H, Hata K, Nakashima K, Hatada I.
|
| Title |
Targeted DNA demethylation in vivo using dCas9-peptide repeat and scFv-TET1 catalytic domain fusions.
|
| Journal |
Nat Biotechnol
|
| 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.
|
| Volume |
34(10)
|
| Pages |
1060-1065
|
| Published |
2016-10-1
|
| DOI |
10.1038/nbt.3658
|
| PII |
nbt.3658
|
| PMID |
27571369
|
| MeSH |
Animals
Basic-Leucine Zipper Transcription Factors / genetics*
CRISPR-Associated Proteins / genetics*
Catalysis
Clustered Regularly Interspaced Short Palindromic Repeats / genetics*
CpG Islands / genetics
DNA Methylation / genetics*
Gene Targeting / methods
Genetic Engineering / methods
Mice
Mixed Function Oxygenases / genetics*
Protein Domains
Proto-Oncogene Proteins / genetics*
Saccharomyces cerevisiae Proteins / genetics*
Single-Chain Antibodies / genetics*
|
| IF |
36.553
|
| Times Cited |
162
|
|
WOS Category
|
BIOTECHNOLOGY & APPLIED MICROBIOLOGY
|
| Resource |
| Human and Animal Cells |
A549(RCB0098)
293T(RCB2202) |
