Visualizing Single-Nucleotide Variations in a Nuclear Genome Using Colocalization of Dual-Engineered CRISPR Probes

Area of Science:

• Genomics
• Molecular Biology
• Cell Biology

Background:

• Direct visualization of single-nucleotide variations (SNVs) is crucial for understanding genome organization and cell phenotypes.
• Existing methods face limitations in specificity, efficiency, and target site accessibility.

Purpose of the Study:

• To develop a novel strategy for visualizing SNVs in the nuclear genome with high resolution.
• To improve the accuracy and efficiency of SNV detection in single cells.

Main Methods:

• Developed a CRISPR-based visualization strategy using colocalization of dual-engineered CRISPR probes (CoDEC).
• Engineered sgRNA with a hairpin for specificity and a loop for signal amplification.
• Utilized a guide probe-based colocalization strategy to differentiate true positive signals from false positives.

Main Results:

• CoDEC successfully visualized SNVs in situ within single cells.
• The method achieved high accuracy in distinguishing on-target signals from off-target signals.
• Compared to CasPLA, CoDEC extended applicable target gene sites and improved detection efficiency.

Conclusions:

• CoDEC offers a facile and accurate method for studying in situ SNV information at single-molecule and single-nucleotide resolutions.
• This technique has potential applications in basic research and clinical diagnostics.
• CoDEC advances the field of single-cell genomics and epigenomics.