A CRISPR/molecular beacon hybrid system for live-cell genomic imaging

Area of Science:

• Molecular Biology
• Genomics
• Cell Biology

Background:

• Existing CRISPR-based live-cell genomic imaging relies on fluorescent protein reporters, which can limit sensitivity and continuous monitoring.
• There is a need for improved imaging techniques to study dynamic genomic processes in living cells.

Purpose of the Study:

• To develop a novel fluorophore-based live-cell genomic imaging system using CRISPR technology.
• To enable sensitive and continuous visualization of specific genomic loci and chromatin dynamics.

Main Methods:

• Developed the CRISPR/MB system, combining a nuclease-deactivated Cas9 (dCas9), a molecular beacon (MB), and an engineered single-guide RNA (sgRNA) with a unique MB target sequence (sgRNA-MTS).
• Co-expressed dCas9 and sgRNA-MTS to target specific genomic loci.
• Delivered MBs that hybridize to the sgRNA-MTS, illuminating the targeted locus via fluorophore emission.

Main Results:

• Demonstrated the feasibility of CRISPR/MB for quantifying genomic loci in live cells.
• Successfully monitored chromatin dynamics using the CRISPR/MB system.
• Achieved dual-color imaging by employing two orthogonal MB/MTS pairs, showcasing multiplexing capability.

Conclusions:

• The CRISPR/MB system offers a flexible and sensitive platform for live-cell genomic imaging.
• This approach overcomes limitations of fluorescent protein reporters, enabling continuous monitoring of chromatin activities.
• The system's adaptability with various fluorophore/quencher and MB/MTS combinations suggests broad applicability in genomic research.