BPS2026 – Ultra-high resolution DNA FISH and beyond

DNA fluorescence in situ hybridization (FISH) is a powerful technique for localizing specific genomic loci within cells and tissues. Traditional DNA FISH methods require high-temperature denaturation of double-stranded DNA for oligo probe hybridization. However, this high-temperature treatment disrupts protein molecules, posing challenges for quantitative imaging of proteins along with chromatin. Additionally, global denaturation increases non-specific background noise, preventing imaging at sub-kilobase resolution. To address these issues, we developed GOLDFISH-X, an exonuclease-based genome oligopaint via local denaturation FISH method. This technique uses Cas9 nickase to identify and cleave specific genomic targets, followed by exonuclease digestion to create local single-stranded DNA regions for probe hybridization. Consequently, GOLDFISH-X eliminates heat denaturation, preserves protein integrity, and achieves high signal-to-background ratio for non-repetitive genomic targets. GOLDFISH-X enables detection of genomic segments as short as 150 bps. Combined with very fast CRISPR for opto-controlled double-strand break (DSB) induction, we reveal the dynamics of DNA repair protein recruitment at single DSB sites. In summary, GOLDFISH-X offers an immunofluorescence-compatible platform with sub-kilobase resolution, thereby expanding the scope of high-resolution genomic imaging.