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Related Experiment Video

Updated: Feb 22, 2026

CRISPR Epigenome Editing in Human Cells using Plasmid DNA Transfection and mRNA Nucleofection Delivery
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DNA epigenome editing using CRISPR-Cas SunTag-directed DNMT3A.

Yung-Hsin Huang1,2,3, Jianzhong Su4, Yong Lei2,3

  • 1Department of Developmental Biology, Baylor College of Medicine, Houston, TX, 77030, USA.

Genome Biology
|September 20, 2017
PubMed
Summary

Researchers developed a precise DNA methylation tool, dCas9-SunTag-DNMT3A, to modify gene expression. This method effectively targets specific genomic regions, enabling new studies on DNA methylation and gene regulation.

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Area of Science:

  • Epigenetics and Molecular Biology
  • Genomics and Gene Regulation

Background:

  • DNA methylation plays a crucial role in gene expression during development.
  • Current limitations exist in correlating global DNA methylation patterns with specific gene expression functions.

Purpose of the Study:

  • To develop a precise tool for site-specific DNA methylation.
  • To investigate the relationship between targeted DNA methylation and gene expression.

Main Methods:

  • Utilized nuclease-deactivated Cas9 fused to SunTag and DNA methyltransferase 3A (DNMT3A) (dCas9-SunTag-DNMT3A).
  • Applied the tool to induce CpG methylation at the HOXA5 locus in HEK293T cells.
  • Employed single guide RNA for targeted methylation of a 4.5-kb genomic region, followed by reduced representation bisulfite sequencing and RNA-seq.

Main Results:

  • Achieved significant CpG methylation at the HOXA5 locus using dCas9-SunTag-DNMT3A.
  • Demonstrated successful methylation of a 4.5-kb genomic region and repression of HOXA5 gene expression.
  • Confirmed site-specific methylation with minimal impact on the global DNA methylome and transcriptome.

Conclusions:

  • Developed an effective and precise tool for site-specific DNA methylation manipulation.
  • The tool facilitates the investigation of DNA methylation's role in gene expression regulation.