Poster abstracts
Poster number 49 submitted by Sydney Willey
Imaging the human genome in living cells using TO-bPNA+ triplex hybrid stems and CRISPRainbow
Sydney Willey (Department of Biological Chemistry and Pharmacology), Yufeng Liang (Department of Chemistry and Biochemistry), Yu-Chieh Chung (Department of Biological Chemistry and Pharmacology), Dennis Bong (Department of Chemistry and Biochemistry), Li-Chun Tu (Department of Biological Chemistry and Pharmacology)
Abstract:
The human genome is highly packed into the nucleus, requiring a significant amount of organization and regulation throughout the cell cycle, cell differentiation, and DNA damage repair. Much of the organization and localization of chromosomes is not well understood. The CRISPRainbow and CRISPR-Sirius systems can be used to visualize specific loci in living cells in real time and to track movements of genomic organization. These systems use guide RNAS (gRNAs) with hairpin loops that bind to RNA coat proteins genetically labeled with fluorescent proteins. However, it is limited by a high background signal and large amounts of steric bulk. Small molecule fluorophores, such as thiazole orange bifacial peptide nucleic acid (TO-bPNA+), pose advantages over fluorescent proteins in brightness and size. In this research, we develop a novel CRISPR-based imaging system combining bPNA+ and CRISPRainbow, by engineering the gRNA hairpin loop to contain a U4xU4 domain that binds to TO-bPNA+. Using this system, we can visualize target genomic loci in live cells with an improved signal-to-noise ratio and reduced steric bulk, allowing for better visualization of loci for tracking genomic dynamics and organization. Also, this system only requires the addition of TO-bPNA+ to the media prior to imaging which makes it easier to deliver and use than traditional methods. This improved CRISPR-based imaging system allows for the study of genomic organization and dynamics with high resolution in living cells over time. Future studies of chromatin dynamics in diseased cells could lead to the development of new diagnostic and therapeutic strategies.
Keywords: CRISPR, fluorescence microscopy, live-cell imaging