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Related Experiment Video

Updated: Feb 25, 2026

Epigenetic Engineering of K562 Cells: Dual-Vector Episomal Strategy for Stable Targeted DNA Methylation using dCas9-DNMT3A and -HDAC1 Fusion Proteins
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Targeted DNA methylation in human cells using engineered dCas9-methyltransferases.

Tina Xiong1, Glenna E Meister2,3,4, Rachael E Workman5

  • 1Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD, 21218, USA.

Scientific Reports
|July 29, 2017
PubMed
Summary

Researchers developed a novel DNA methyltransferase (MTase) tool using a split MTase and dCas9. This system enables precise, programmable DNA methylation at targeted genomic sites in mammalian cells, advancing epigenetics research.

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Area of Science:

  • Genomics
  • Epigenetics
  • Molecular Biology

Background:

  • Mammalian gene expression is regulated by DNA methylation patterns.
  • Targeted DNA methyltransferases (MTases) are crucial for biological research but require precise design rules.
  • Off-target DNA methylation can lead to unintended biological effects.

Purpose of the Study:

  • To develop a programmable DNA methylation tool for precise targeting in mammalian genomes.
  • To establish design rules for site-specific DNA methylation using engineered MTases.
  • To investigate the efficiency and programmability of a novel dCas9-fused split MTase system.

Main Methods:

  • An artificially split CpG methyltransferase (sMTase) was engineered.
  • One sMTase fragment was fused to catalytically-inactive Cas9 (dCas9).
  • The dCas9-sMTase system was programmed with sgRNAs for targeted CpG site methylation in mammalian cells.

Main Results:

  • Precise mapping of RNA-programmed DNA methylation relative to PAM sites was achieved.
  • Efficient and predictable DNA methylation (up to ~70%) was observed at targeted sites.
  • Multiplexing sgRNAs allowed targeting of multiple genomic loci simultaneously.

Conclusions:

  • The dCas9-sMTase system provides a programmable tool for de novo DNA methylation.
  • This technology can be used to study DNA methylation dynamics (initiation, spreading, inheritance).
  • Potential therapeutic applications include gene silencing via targeted DNA methylation.