Recent advancements in CRISPR-Cas toolbox for imaging applications.

The imaging of chromatin, genomic loci, RNAs, and proteins is very important to study their localization, interaction, and coordinated regulation. Recently, several clustered regularly interspaced short palindromic repeats (CRISPR) based imaging methods have been established. The refurbished tool kits utilizing deactivated Cas9 (dCas9) and dCas13 have been established to develop applications of CRISPR-Cas technology beyond genome editing. Here, we review recent advancements in CRISPR-based methods that enable efficient imaging and visualization of chromatin, genomic loci, RNAs, and proteins. RNA aptamers, Pumilio, SuperNova tagging system, molecular beacons, halotag, bimolecular fluorescence complementation, RNA-guided endonuclease labeling, and oligonucleotide-based imaging methods utilizing fluorescent proteins, organic dyes, or quantum dots have been developed to achieve improved fluorescence and signal-to-noise ratio for the imaging of chromatin or genomic loci. RNA-guided RNA targeting CRISPR systems (CRISPR/dCas13) and gene knock-in strategies based on CRISPR/Cas9 mediated site-specific cleavage and DNA repair mechanisms have been employed for efficient RNA and protein imaging, respectively. A few CRISPR-Cas-based methods to investigate the coordinated regulation of DNA-protein, DNA-RNA, or RNA-protein interactions for understanding chromatin dynamics, transcription, and protein function are also available. Overall, the CRISPR-based methods offer a significant improvement in elucidating chromatin organization and dynamics, RNA visualization, and protein imaging. The current and future advancements in CRISPR-based imaging techniques can revolutionize genome biology research for various applications.