LAUSR

Direct visualization of single-nucleotide variation (SNV) in single cells is of great importance for understanding the spatial organization of genomes and their relationship with cell phenotypes. Herein, we developed a new strategy for visualizing SNVs in a nuclear genome using colocalization of dual-engineered CRISPR probes (CoDEC). By engineering the structure of sgRNA, we incorporated a hairpin in the spacer domain for improving SNV recognition specificity and a loop in the nonfunctional domain for localized signal amplification. Using guide probe-based colocalization strategy, we can successfully distinguish on-target true positive signals from the off-target false positives with high accuracy. Comparing with a proximity ligation-based assay (CasPLA), the probe colocalization strategy extended applicable target gene sites (the distance between two designed probes can be extended to around 200nt) and improved detection efficiency. This newly developed method provides a facile way for studying in situ information on SNVs in individual cells for basic research and clinical applications with single-molecule and single-nucleotide resolutions.