Project description:We demonstrate that dCas9-SunTag-DNMT3A dramatically increased CpG methylation at the HOXA5 locus in human embryonic kidney 293T cells (HEK293T). Furthermore, using a single sgRNA, dCas9-SunTag-DNMT3A was able to methylate a 4.5 kb genomic region and repress HOXA5 gene expression. Reduced representation bisulfite sequencing (RRBS) and RNA-seq showed that dCas9-SunTag-DNMT3A methylated regions of interest with minimal impact on the global DNA methylome and transcriptome.
Project description:Here, we demonstrate that dCas9-SunTag-DNMT3A dramatically increased CpG methylation at the HOXA5 locus in human embryonic kidney 293T cells (HEK293T). Furthermore, using a single sgRNA, dCas9-SunTag-DNMT3A was able to methylate a 4.5 kb genomic region and repress HOXA5 gene expression. Reduced representation bisulfite sequencing (RRBS) and RNA-seq showed that dCas9-SunTag-DNMT3A methylated regions of interest with minimal impact on the global DNA methylome and transcriptome.
Project description:We demonstrate that dCas9-SunTag-DNMT3A dramatically increased CpG methylation at the HOXA5 locus in human embryonic kidney 293T cells (HEK293T). Furthermore, using a single sgRNA, dCas9-SunTag-DNMT3A was able to methylate a 4.5 kb genomic region and repress HOXA5 gene expression. Reduced representation bisulfite sequencing (RRBS) and RNA-seq showed that dCas9-SunTag-DNMT3A methylated regions of interest with minimal impact on the global DNA methylome and transcriptome.
Project description:We demonstrate that dCas9-SunTag-DNMT3A dramatically increased CpG methylation at the HOXA5 locus in human embryonic kidney 293T cells (HEK293T). Furthermore, using a single sgRNA, dCas9-SunTag-DNMT3A was able to methylate a 4.5 kb genomic region and repress HOXA5 gene expression. Reduced representation bisulfite sequencing (RRBS) and RNA-seq showed that dCas9-SunTag-DNMT3A methylated regions of interest with minimal impact on the global DNA methylome and transcriptome.
Project description:DNA methylation is a key epigenetic modification involved in regulating gene expression and maintaining genomic integrity. Somatic patterns of DNA methylation are largely static, apart from focal dynamics at gene regulatory elements. To further advance our understanding of the role of DNA methylation in human development and disease, we inactivated all three catalytically active DNA methyltransferases in human embryonic stem cells (ESCs) using CRISPR/Cas9 genome editing. Disruption of DNMT3A or DNMT3B individually, as well as of both enzymes in tandem, creates viable, pluripotent cell lines with distinct effects on their DNA methylation landscape as assessed by whole-genome bisulfite sequencing. Surprisingly, in contrast to mouse, deletion of DNMT1 resulted in rapid cell death in human ESCs. To overcome the immediate lethality, we generated a doxycycline (DOX) responsive tTA-DNMT1* rescue line and readily obtained homozygous DNMT1 mutant lines. However, DOX-mediated repression of the exogenous DNMT1* initiates rapid, global loss of DNA methylation, followed by extensive cell death, demonstrating that DNA methylation is essential for human ESCs cultured in standard conditions. In summary, our data provide a comprehensive characterization of DNMT mutant ESCs, including single base genome-wide maps of their targets. RRBS methylation profiling of DNMT3A/3B DKO human ES cells
Project description: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.