Visualizing Nucleic Acid Loci with the CRISPR-Cas System: Current Approaches

The visualization of genomic loci in living cells is crucial for detecting mutations and observing the spatial proximity of DNA regions in the 3D nuclear environment. One of the primary applications of the clustered regularly interspaced short palindromic repeats–CRISPR-associated protein 9 (CRISPR/Cas9) system is the non-invasive, real-time labeling of DNA loci in living cells, enabled by its unique characteristics. Visualization of genomic loci has been made possible by the use of an endonuclease-inactive form of the Cas9 protein (dCas9) and sgRNA in combination with fluorescent molecules. However, using CRISPR/Cas9 for targeting DNA regions has certain limitations, the most significant being suboptimal signal-to-noise ratio, the need for multiplexed labeling, and the large size of the Cas fluorescent reporter sytem, which impacts the complex’s functionality and complicates its delivery. Current variations of the method using CRISPR/dCas9 overcome these limitations in different ways. This review examines the evolution of genome locus visualization methods based on CRISPR/Cas9 from the initial use of the system to the present.