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Genome editing technologies allow scientists to modify an organism’s DNA via the addition, removal, or rearrangement of genetic material at specific genomic locations. These types of techniques could potentially be used to cure genetic disorders such as hemophilia and sickle cell anemia. One popular and widely used DNA-editing research tool that could lead to safe and effective cures for genetic disorders is the CRISPR-Cas9 system. CRISPR-Cas9 stands for Clustered Regularly Interspaced...
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The CRISPR-Cas system serves as a bacterial defense mechanism against invading genetic elements such as viruses and plasmids, forming the foundation for its adaptation as a powerful genome-editing tool. Originally discovered in prokaryotes, this system has been repurposed to revolutionize genetic engineering across a wide range of organisms, including plants, animals, and humans. The core component, Cas9, is an endonuclease derived from Streptococcus pyogenes, capable of introducing...
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Related Experiment Video

Updated: Mar 17, 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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Epigenetic Engineering of K562 Cells: Dual-Vector Episomal Strategy for Stable Targeted DNA Methylation using dCas9-DNMT3A and -HDAC1 Fusion Proteins

Published on: October 31, 2025

562

A CRISPR-based approach for targeted DNA demethylation.

Xingxing Xu1, Yonghui Tao2, Xiaobo Gao2

  • 1Key Laboratory of Systems Biology, CAS Center for Excellence in Molecular Cell Science, Innovation Center for Cell Signaling Network; Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences , Shanghai, China.

Cell Discovery
|July 28, 2016
PubMed
Summary

Scientists developed a new CRISPR-Cas based method for targeted DNA demethylation. This novel system precisely removes DNA methylation, offering potential for controlling gene expression and clinical applications.

Keywords:
CRISPRRANKLdemethylation

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Area of Science:

  • Epigenetics and Molecular Biology
  • Gene Regulation
  • Mammalian Cell Biology

Background:

  • DNA methylation is a critical epigenetic mechanism regulating gene expression in mammals.
  • Dysregulation of DNA methylation is implicated in numerous physiological and pathological processes.
  • Understanding and manipulating DNA methylation is key to controlling gene function.

Purpose of the Study:

  • To develop a novel, targeted DNA demethylation method using the CRISPR-Cas system.
  • To investigate the efficacy and specificity of this new demethylation system.
  • To explore the potential applications of targeted DNA demethylation in gene expression control.

Main Methods:

  • Engineered single guide RNAs (sgRNAs) with MS2 RNA elements (sgRNA2.0) to recruit Tet1 catalytic domain (Tet-CD).
  • Fused Tet-CD with dCas9 or MS2 coat proteins for targeted delivery to specific gene loci.
  • Utilized the dCas9/sgRNA2.0-Tet-CD system for targeted DNA demethylation and gene expression analysis.

Main Results:

  • Demonstrated significant upregulation of target genes (e.g., RANKL, MAGEB2, MMP2) following demethylation.
  • Observed a strong correlation between promoter CpG demethylation and gene transcription.
  • Showcased efficient demethylation with minimal off-target effects.

Conclusions:

  • The novel CRISPR-Cas based system enables precise, targeted DNA demethylation.
  • This method effectively controls gene expression by modulating DNA methylation.
  • The system holds promise for mechanistic studies and therapeutic interventions in diseases linked to DNA methylation.