Dynamic Genomic Imaging and Tracking in Living Cells by a DNA Origami-Based CRISPR‒dCas9 System.

The clustered regularly interspaced short palindromic repeat (CRISPR)-associated system has displayed promise in visualizing the dynamics of target loci in living cells, which is important for studying genome regulation. However, developing a cell-friendly and rapid transfection method for achieving dynamic and long-term genomic imaging in living cells with high specificity and accuracy is still challenging. Herein, a robust and versatile method is presented that employs a barrel-shaped DNA nanostructure (TUBE) modified with aptamers for loading, protecting, and delivering CRISPR-Cas9 to visualize specific genomic loci in living cells. This approach enables dynamic tracking of target genomic regions (Chr3q29, a repetitive region of chromosome 3) throughout the mitotic process and captures variations in their spatial distribution and quantity accurately. Distinct dynamic behaviors between the Chr3q29 and telomeres are observed, which are linked to their unique chromosomal positions and levels of mobility. High-resolution multicolor labeling of the target genes is achieved, with a high degree of colocalization between the enhanced green fluorescent protein and cyanine-5 channels, facilitating precise imaging of target loci. This method not only supports dynamic genomic imaging but also enables multiplexed tracking, providing a powerful visualization tool for studying cellular processes and genetic interactions in real time within living cells.