Journal
JOVE-JOURNAL OF VISUALIZED EXPERIMENTS
Volume -, Issue 139, Pages -Publisher
JOURNAL OF VISUALIZED EXPERIMENTS
DOI: 10.3791/57457
Keywords
Genetics; Issue 139; Chromatin loops; chromatin folding; gene expression; CRISPR; dCas9; gene regulation; reversible dimerization; dynamic chromosomal rearrangement
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Funding
- NSFGRF [DGE-114747]
- NDSEGF [FA9550-11-C-0028]
- National Cancer Institute [1F99CA222541-01]
- Burroughs Wellcome Fund
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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.
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