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Series GSE142181 Query DataSets for GSE142181
Status Public on Jan 14, 2020
Title Engineering of effector domains for targeted DNA methylation with reduced off-target effects
Organism Homo sapiens
Experiment type Methylation profiling by high throughput sequencing
Summary Epigenome editing is a promising technology, potentially allowing the stable reprogramming of gene expression profiles without alteration of the DNA sequence. Targeted DNA methylation has been successfully documented by many groups for silencing of selected genes, but recent publications have raised concerns regarding its specificity. In the current work, we developed new EpiEditors for programmable DNA methylation in cells with a high efficiency and improved specificity. First, we demonstrated that the dCas9-SunTag scaffold, which has been used earlier for signal amplification, can be combined with the DNMT3A-DNMT3L effector domain allowing a strong methylation at the target genomic locus. We demonstrated that off-target activity of this system is mainly due to untargeted freely diffusing DNMT3A-DNMT3L subunits. Therefore, we generated several DNMT3A variants containing mutations, which reduced their endogenous DNA binding strength. We analyzed the genome-wide DNA methylation of selected variants and confirmed a striking reduction of untargeted methylation. For all potential applications of targeted DNA methylation, the efficiency and specificity of the treatment are the key factors. By developing highly active targeted methylation systems with strongly improved specificity, our work contributes to the future applications of this approach.
Overall design HEK293 cells were transiently transfected with dCas9-SunTag, antibody-fused catalytic domain of DNMT3A/3L (wild type / DNMT3A mutants) and sgRNA targeting the CpG island of the ISG15 gene. After 3 days, the cells were sorted using FACS to obtain only cells carrying each plasmid. MBD2 pulldown was perfomed with sonicated genomic DNA and methylation changes were analysed genome-wide by NGS.
Web link
Contributor(s) Broche J, Hofacker D, Laistner L, Adam S, Bashtrykov P, Jeltsch A
Citation(s) 31941101
Submission date Dec 17, 2019
Last update date Mar 16, 2020
Contact name Albert Jeltsch
Phone +49 685 64390
Organization name University Stuttgart
Department Institute of Biochemistry and Technical Biochemistry
Street address Allmandring 31
City Stuttgart
ZIP/Postal code 70569
Country Germany
Platforms (1)
GPL21290 Illumina HiSeq 3000 (Homo sapiens)
Samples (4)
GSM4222239 Untreated cells
GSM4222240 Ab-3A3L (WT)
GSM4222241 Ab-3A3L (K844E)
BioProject PRJNA596181
SRA SRP237924

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Supplementary file Size Download File type/resource
GSE142181_RAW.tar 173.3 Mb (http)(custom) TAR (of BED, BIGWIG)
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Raw data are available in SRA
Processed data provided as supplementary file

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