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CRISPR-Mediated Reorganization of Chromatin Loop Structure
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Dissecting Locus-Specific Chromatin Interactions by CRISPR CAPTURE.

Giovanni A Botten1,2, Michael Lee1,2, Jian Xu3,4

  • 1Children's Medical Center Research Institute, University of Texas Southwestern Medical Center, Dallas, TX, USA.

Methods in Molecular Biology (Clifton, N.J.)
|November 25, 2022
PubMed
Summary
This summary is machine-generated.

Researchers developed a CRISPR-based tool called CAPTURE to identify the molecular components and interactions of cis-regulatory elements (CREs) in the genome. This method helps understand how gene expression is controlled in specific tissues.

Keywords:
Biotinylated dCas9CAPTURECAPTURE-3C-seqCAPTURE-ProteomicsCRISPRChromatin loopingEnhancerGene regulationNoncoding genomecis-regulatory elements

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Area of Science:

  • Genomics
  • Molecular Biology
  • Epigenetics

Background:

  • Tissue-specific gene expression relies on cis-regulatory elements (CREs) and trans-acting factors.
  • Current methods lack detailed understanding of the in situ molecular composition of most CREs.
  • Investigating CREs is crucial for understanding gene regulation.

Purpose of the Study:

  • To present a detailed protocol for the CRISPR-based CAPTURE (CRISPR affinity purification in situ of regulatory elements) toolbox.
  • To enable the interrogation of locus-specific chromatin complexes and configurations.
  • To identify the in situ regulatory composition of CREs.

Main Methods:

  • Utilizing an in vivo biotinylated nuclease-deficient Cas9 (dCas9) protein.
  • Employing programmable single-guide RNAs (sgRNAs) for targeting specific CREs.
  • Implementing the CAPTURE toolbox for affinity purification of CRE-associated macromolecular complexes and chromatin looping.

Main Results:

  • The CAPTURE toolbox successfully identifies CRE-associated macromolecular complexes.
  • The method allows for the detection of chromatin looping interactions.
  • Provides a detailed protocol for interrogating the molecular composition of chromatin complexes at specific genomic loci.

Conclusions:

  • The CRISPR-based CAPTURE method offers a powerful approach to decipher the molecular intricacies of CREs.
  • This technique advances the understanding of gene regulation by characterizing the in situ composition and configuration of regulatory elements.
  • The detailed protocol facilitates broader application of CAPTURE in mammalian genomics research.