Real-Time Imaging of Specific Genomic Loci With CRISPR/dCas9 in Human Cells Using CRISPRainbow

Area of Science:

• Molecular Biology
• Genomics
• Cell Biology

Background:

• Proper genome organization is crucial for cellular function and stability.
• Disruptions in genome architecture are linked to diseases.
• Existing techniques like FISH and Hi-C require cell fixation, limiting dynamic studies.

Purpose of the Study:

• To develop a method for real-time, single-cell imaging of chromatin organization dynamics.
• To overcome limitations of fixed-cell techniques in studying genome architecture.
• To enable simultaneous tracking of multiple genomic loci in living cells.

Main Methods:

• Repurposing clustered regularly interspaced short palindromic repeats (CRISPR)/dead Cas9 (dCas9) for live-cell imaging.
• Utilizing the CRISPRainbow system for simultaneous targeting of up to seven genomic loci.
• Employing spectrally distinct fluorescent markers and single-guide RNA (sgRNA) with RNA aptamers for labeling.

Main Results:

• CRISPRainbow allows simultaneous multicolor imaging of up to seven genomic loci in living human cells.
• The system enables real-time tracking of genome dynamics without cell fixation.
• Signal amplification is achieved by targeting repetitive sequences, and endogenous DNA is targeted directly.

Conclusions:

• CRISPRainbow provides a powerful tool for studying real-time chromatin organization and genome dynamics at the single-cell level.
• This technique complements existing methods by offering live-cell validation.
• Advances in understanding genome heterogeneity and dynamics are facilitated by this approach.