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J Vis Exp. 2018 Sep 14;(139). doi: 10.3791/57457.

CRISPR-Mediated Reorganization of Chromatin Loop Structure.

Author information

1
Department of Dermatology, Program in Epithelial Biology, Stanford University School of Medicine; Program in Cancer Biology, Stanford University School of Medicine.
2
Department of Dermatology, Program in Epithelial Biology, Stanford University School of Medicine.
3
Canary Center for Cancer Early Detection, Department of Radiology, Stanford University School of Medicine.
4
Department of Biology, Bridgewater State University.
5
System Biosciences.
6
Department of Dermatology, Program in Epithelial Biology, Stanford University School of Medicine; Program in Cancer Biology, Stanford University School of Medicine; Veterans Affairs Healthcare System; kevwang@stanford.edu.

Abstract

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene expression, but whether looping is responsible for or a result of alterations in gene expression is still unknown. Until recently, how chromatin looping affects the regulation of gene activity and cellular function has been relatively ambiguous, and limitations in existing methods to manipulate these structures prevented in-depth exploration of these interactions. To resolve this uncertainty, we engineered a method for selective and reversible chromatin loop re-organization using CRISPR-dCas9 (CLOuD9). The dynamism of the CLOuD9 system has been demonstrated by successful localization of CLOuD9 constructs to target genomic loci to modulate local chromatin conformation. Importantly, the ability to reverse the induced contact and restore the endogenous chromatin conformation has also been confirmed. Modulation of gene expression with this method establishes the capacity to regulate cellular gene expression and underscores the great potential for applications of this technology in creating stable de novo chromatin loops that markedly affect gene expression in the contexts of cancer and development.

PMID:
30272647
PMCID:
PMC6235177
DOI:
10.3791/57457
[Indexed for MEDLINE]
Free PMC Article

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