Live Imaging of DNA/mRNA with CRISPR/Cas9
DNA visualization is an important application in understanding a variety of cellular processes, such as replication, transcription, and recombination, and the interactions between DNA and associated proteins and RNA. Two techniques are commonly used for DNA imaging, fluorescence in situ hybridization (FISH) and fluorescent tagging of DNA-binding proteins. FISH uses fluorescently tagged nucleic acid probes to bind and visualize DNA. While this technique offers the flexibility to target specific sequences through base pairing of the nucleic acid probes, it cannot be used for live imaging because of the requirement for sample fixation. Conversely, proteins tagged with a fluorescent label can be used for live imaging, but are limited by their fixed target sequences, restricting their use mostly to repetitive DNA sequences, such as telomeres.
New advances in CRISPR/Cas9 technology offer the benefits of both live imaging and easy target sequence customization and flexibility. Inactivated dCas9 can be tagged with fluorophores for imaging both repetitive DNA elements and protein-encoding genes, enabling us to observe chromatin organization throughout the cell cycle. In addition to live DNA imaging, the CRISPR/Cas9 system can be used for live RNA imaging as well. Modifications to the gRNA sequence allow for mRNA recognition and tracking. Using CRISPR-mediated RNA imaging techniques, researchers have been able to visualize the accumulation of ACTB, CCNA2 and TRFC mRNAs in RNA granules. These new applications improve existing methodologies for live imaging within cells allowing for the study of dynamic cellular processes involving DNA and RNA.