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DNA epigenome editing using CRISPR-Cas SunTag-directed DNMT3A [BS-Seq]

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE97812 | GEO | 2017/08/22

SECONDARY ACCESSION(S): PRJNA382984

REPOSITORIES: GEO

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DNA epigenome editing using CRISPR-Cas SunTag-directed DNMT3A [WGBS]

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.

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DNA epigenome editing using CRISPR-Cas SunTag-directed DNMT3A [RRBS]

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DNA epigenome editing using CRISPR-Cas SunTag-directed DNMT3A [RNA-Seq]

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DNA epigenome editing using CRISPR-Cas SunTag-directed DNMT3A

Project description:DNA epigenome editing using CRISPR-Cas SunTag-directed DNMT3A

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Site-specific manipulation of Arabidopsis loci using CRISPR-Cas9 SunTag systems

Project description:Understanding genomic functions requires site-specific manipulation of loci via efficient protein effector targeting systems. However, few approaches for targeted manipulation of the epigenome are available in plants. Here, we adapt the dCas9-SunTag system to engineer targeted gene activation and DNA methylation in Arabidopsis. We demonstrate that a dCas9-SunTag system utilizing the transcriptional activator VP64 drives robust and specific activation of several loci, including protein coding genes and transposable elements, in diverse chromatin contexts. In addition, we present a CRISPR-based methylation targeting system for plants, utilizing a SunTag system with the catalytic domain of the Nicotiana tabacum DRM methyltransferase, which efficiently targets DNA methylation to specific loci, including the FWA promoter, triggering a developmental phenotype, and the SUPERMAN promoter. These SunTag systems represent valuable tools for the site-specific manipulation of plant epigenomes.

2019-01-22 | GSE125230 | GEO

A modular dCas9-SunTag DNMT3A epigenome editing system overcomes pervasive off-target activity of direct fusion dCas9-DNMT3A constructs

Project description:A modular dCas9-SunTag DNMT3A epigenome editing system overcomes pervasive off-target activity of direct fusion dCas9-DNMT3A constructs

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Pan-Cancer DNA Hypermethylation Leads to Transcriptional Upregulation of Homeobox Oncogene

Project description:Cancers have long been recognized to be not only genetically but also epigenetically distinct from their tissues of origin. Although genetic alterations underlying oncogene upregulation have been well studied, to what extent epigenetic mechanisms, such as DNA methylation, can also induce oncogene expression remains unknown. Here, through pan-cancer analysis of 4,182 genome-wide profiles, including whole-genome bisulfite sequencing (WGBS) data from 30 normal tissues and 35 solid tumors, we discovered a strong correlation between gene-body hypermethylation of DNA methylation canyons (broad under-methylated regions) and overexpression of ~43% homeobox genes, many of which are also oncogenes. To gain insights into the cause-and-effect relationship, we used a newly developed dCas9-SunTag-DNMT3A system to methylate genomic sites of interest. The locus-specific hypermethylation of gene-body canyon, but not promoter, of homeobox oncogene DLX1 and POU3F3, can direct increase its gene expression. Together, our pan-cancer analysis followed by functional validation reveals DNA hypermethylation as a novel epigenetic mechanism for homeobox oncogene upregulation.

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DNA epigenome editing using CRISPR-Cas SunTag-directed DNMT3A.

Project description:BackgroundDNA methylation has widespread effects on gene expression during development. However, our ability to assign specific function to regions of DNA methylation is limited by the poor correlation between global patterns of DNA methylation and gene expression.ResultsHere, we utilize nuclease-deactivated Cas9 protein fused to repetitive peptide epitopes (SunTag) recruiting multiple copies of antibody-fused de novo DNA methyltransferase 3A (DNMT3A) (dCas9-SunTag-DNMT3A) to amplify the local DNMT3A concentration to methylate genomic sites of interest. We demonstrate that dCas9-SunTag-DNMT3A dramatically increases CpG methylation at the HOXA5 locus in human embryonic kidney (HEK293T) cells. Furthermore, using a single guide RNA, dCas9-SunTag-DNMT3A is able to methylate a 4.5-kb genomic region and repress HOXA5 gene expression. Reduced representation bisulfite sequencing and RNA-seq show that dCas9-SunTag-DNMT3A methylates regions of interest with minimal impact on the global DNA methylome and transcriptome.ConclusionsThis effective and precise tool enables site-specific manipulation of DNA methylation and may be used to address the relationship between DNA methylation and gene expression.

| S-EPMC5604343 | biostudies-literature

Reduction in Gene Expression Noise by Targeted Decondensation of Gene Loci

Project description:ATAC-seq was done on various cell lines generated from HeLa cells. The cell lines contained dCas9 constructs conjugated to the histone acetyltransferase p300, as well as a similar system with multiple p300 fragments present, per dCas9 enzyme utilizing SunTag.

2020-09-04 | GSE157399 | GEO

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