The 12th International Symposium on 3D Genomics

89 / 2026-04-06 17:31:46

CRISPR live-cell imaging reveals chromatin dynamics and enhancer interactions at multiple non-repetitive loci

Live-cell imaging,CRISPR PRO-LiveFISH,epigenetic,UBP-labeled sgRNA

Existing methods to visualize dynamic changes in the three-dimensional genome, promoter−enhancer interactions and the influence of epigenetic modifications in non-repetitive loci are limited. Here we introduce CRISPR PRO-LiveFISH (Pooled gRNAs with Orthogonal bases LiveFISH), which combines orthogonal bases from expanded genetic alphabet technology and rational single guide RNA (sgRNA) design to efficiently label multiple non-repetitive loci in living cells. The optimized method allows simultaneous imaging of up to six genomic loci and uses as few as 10 sgRNAs for non-repetitive loci imaging without signal amplification. We demonstrate the method in diverse cell types, including primary cells, and apply it to reveal enhancer−promoter dynamics and a correlation between genomic dynamics and epigenetic states. We also show that PCDHα−enhancer interactions may persist despite spatial mobility and that BRD4 maintains super-enhancer contacts regulating MYC oncogene expression in cancer cells. CRISPR PRO-LiveFISH can be applied to diverse studies of chromatin dynamics and genome organization in living cells.