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Related Concept Videos

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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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The basic reaction of homologous recombination (HR) involves two chromatids that contain DNA sequences sharing a significant stretch of identity. One of these sequences uses a strand from another as a template to synthesize DNA in an enzyme-catalyzed reaction. The final product is a novel amalgamation of the two substrates. To ensure an accurate recombination of sequences, HR is restricted to the S and G2 phases of the cell cycle. At these stages, the DNA has been replicated already and the...
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Related Experiment Video

Updated: Nov 5, 2025

In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing
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High-Fidelity CRISPR/Cas9-Based Gene-Specific Hydroxymethylation.

Xingbo Xu1,2, Elisabeth M Zeisberg3,4

  • 1Department of Cardiology and Pneumology, University Medical Center of Göttingen, Georg-August University, Göttingen, Germany. xingbo.xu@med.uni-goettingen.de.

Methods in Molecular Biology (Clifton, N.J.)
|May 19, 2021
PubMed
Summary

Gene silencing in diseases can be reversed by targeting specific genes. This study introduces a CRISPR/Cas9 gene editing method using TET3 to induce gene-specific hydroxymethylation and reactivate silenced genes, offering potential for gene therapy.

Keywords:
CRISPR/dCas9Rasal1TET3gene reactivationhydroxymethylationmethylation

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An Engineered Split-TET2 Enzyme for Chemical-inducible DNA Hydroxymethylation and Epigenetic Remodeling
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An Engineered Split-TET2 Enzyme for Chemical-inducible DNA Hydroxymethylation and Epigenetic Remodeling

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Area of Science:

  • Epigenetics and Gene Regulation
  • Molecular Biology
  • Biotechnology

Background:

  • Aberrant promoter hypermethylation causes gene silencing, contributing to diseases like cancer and fibrosis.
  • TET enzymes (TET1, TET2, TET3) mediate active DNA demethylation by converting 5-methylcytosine to 5-hydroxymethylcytosine.

Purpose of the Study:

  • To explore the potential of gene-specific hydroxymethylation for reactivating single silenced genes.
  • To present guidelines for designing sgRNA and conducting in vitro functional assessments for gene-specific hydroxymethylation targeting.

Main Methods:

  • Utilized a spCas9 variant fused with the TET3 catalytic domain.
  • Employed CRISPR/Cas9 gene technology for targeted gene hydroxymethylation.
  • Focused on single guide RNA (sgRNA) design and in vitro functional assessments.

Main Results:

  • Successfully mediated gene-specific hydroxymethylation using the engineered spCas9-TET3 fusion protein.
  • Demonstrated subsequent reactivation of the targeted silenced gene.
  • Established foundational guidelines for this novel gene-targeting approach.

Conclusions:

  • Gene-specific hydroxymethylation via CRISPR/Cas9-TET3 fusion is a viable strategy for gene reactivation.
  • This approach holds significant promise for developing novel gene therapies for pathologies linked to gene silencing.