Analytical Chem JoVE (Open Access) Citable · DOI

CRISPR-Mediated Reorganization of Chromatin Loop Structure

Name: CRISPR-Mediated Reorganization of Chromatin Loop Structure
Uploaded: 2026-07-09
Duration: 9 min 20 s
Description: Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene ...

DOI: 10.3791/57457-v

What you'll learn

✓ Design and prepare CRISPR-dCas9 plasmids with custom guide RNAs for chromatin targeting
✓ Produce lentiviral vectors and transduce mammalian cells for stable transgene expression
✓ Measure reversible chromatin loop formation and gene expression modulation at target loci

Protocol

Biopharma Insights Chromatin looping plays a significant role in gene regulation; however, there have been no technological advances that allow for selective and reversible modification of chromatin loops. Here we describe a powerful system for chromatin loop re-organization using CRISPR-dCas9 (CLOuD9), demonstrated to selectively and reversibly modulate gene expression at targeted loci.

Difficulty

advanced

Total time

~7–10 days (plasmid prep 2 days, viral production 3–4 days, transduction and analysis 2–3 days)

Model organism

HEK293 or similar mammalian cell line

Biosafety

BSL-2

Steps

Prepare plasmids and insert guide RNAs

Clone or insert custom guide RNA sequences into CRISPR-dCas9 expression plasmids. Verify insert orientation and sequence fidelity by restriction digest and sequencing.

▶ 01:02

Produce lentiviral vector particles

Transfect HEK293T packaging cells with lentiviral plasmid mix (transfer vector, gag-pol, env) to generate infectious viral supernatant. Harvest and concentrate virus over 48–72 hours.

▶ 03:26

Transduce target cells and induce dimerization

Infect cultured mammalian cells with lentiviral particles carrying CLOuD9 components. Apply dimerization inducer (e.g., dexamethasone or rapamycin) to activate chromatin looping.

▶ 05:12

Measure reversible chromatin loop formation

Perform 3C/4C/Hi-C or chromosome conformation capture to quantify loop frequency between target loci. Verify reversibility by removing dimerization inducer and re-measuring.

▶ 07:08

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