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CRISPR-dCas9 mediated TET1 targeting for selective DNA demethylation at BRCA1 promoter

Samrat Roy Choudhury1, Yi Cui1, Katarzyna Lubecka2

  • 1Department of Agricultural & Biological Engineering, Bindley Bioscience Centre, Purdue University, West Lafayette, IN 47907, USA.

Oncotarget
|July 1, 2016
PubMed

Insights

Researchers developed a CRISPR-Cas9 tool to demethylate the BRCA1 gene promoter, reactivating this tumor suppressor. This epigenetic editing approach offers a new strategy for cancer research and therapy.

Area of Science:

  • Epigenetics
  • Molecular Biology
  • Cancer Genetics

Background:

  • DNA hypermethylation of tumor suppressor genes is a key event in cancer development, leading to gene silencing and promoting tumorigenesis.
  • Epigenetic silencing of genes like BRCA1 impairs cellular functions, driving cancer initiation and progression.
  • Understanding and manipulating DNA methylation patterns are crucial for cancer research.

Purpose of the Study:

  • To investigate site-specific and spatiotemporal DNA methylation alterations in the BRCA1 gene promoter.
  • To develop and test a novel CRISPR-Cas9 based tool for targeted epigenetic modification.
  • To explore the potential of reactivating tumor suppressor genes through epigenetic editing.

Main Methods:

  • Development of a programmable CRISPR-Cas9 demethylase tool by fusing deactivated Cas9 (dCas9) with the catalytic domain of Ten-Eleven Translocation 1 (TET1CD).
  • Design of single-guide RNAs (sgRNAs) to direct the dCas9-TET1CD fusion protein to specific regions of the BRCA1 promoter.
  • Analysis of DNA methylation and 5-hydroxymethylation (5-hmC) levels at targeted loci to confirm demethylation and enzyme activity.

Main Results:

  • The CRISPR-Cas9-TET1CD fusion protein successfully and selectively demethylated targeted regions within the BRCA1 promoter.
  • Targeted demethylation led to significant transcriptional up-regulation of the BRCA1 gene.
  • An increase in 5-hydroxymethylation (5-hmC) was observed at demethylated sites, confirming the catalytic activity of TET1.
  • The modular design allows for targeting other genomic regions and substituting different epigenetic modifiers.

Conclusions:

  • A novel, programmable CRISPR-Cas9 based epigenetic tool can precisely target and demethylate specific DNA regions, such as the BRCA1 promoter.
  • This approach effectively reactivates silenced tumor suppressor genes, offering a potential therapeutic strategy for cancers driven by epigenetic alterations.
  • The developed platform is versatile for investigating epigenetic regulatory pathways and developing new epigenetic therapies.
Keywords:
BRCA1CRISPR-dCas9DNA demethylationTET1gene activation

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