Recent advancements in CRISPR-Cas toolbox for imaging applications

Area of Science:

• Molecular Biology
• Genomics
• Biotechnology

Background:

• Accurate visualization of chromatin, genomic loci, RNAs, and proteins is crucial for studying their localization, interactions, and regulation.
• Clustered regularly interspaced short palindromic repeats (CRISPR) technology has expanded beyond genome editing to enable novel imaging applications.

Purpose of the Study:

• To review recent advancements in CRISPR-based methods for efficient imaging and visualization of chromatin, genomic loci, RNAs, and proteins.
• To highlight the development of refurbished toolkits utilizing deactivated Cas9 (dCas9) and dCas13 for these imaging applications.

Main Methods:

• CRISPR-based imaging methods utilizing RNA aptamers, Pumilio, SuperNova tagging, molecular beacons, halotag, bimolecular fluorescence complementation, and RNA-guided endonuclease in situ labeling.
• Oligonucleotide-based imaging utilizing fluorescent proteins, organic dyes, or quantum dots for enhanced fluorescence and signal-to-noise ratio.
• CRISPR/dCas13 systems for RNA targeting and CRISPR/Cas9 for gene knock-in strategies for protein imaging.

Main Results:

• Development of diverse CRISPR-based tools for improved imaging of chromatin, genomic loci, RNAs, and proteins.
• CRISPR/dCas13 systems enable efficient RNA visualization, while CRISPR/Cas9 facilitates protein imaging through gene knock-in strategies.
• Methods are available to investigate DNA-protein, DNA-RNA, and RNA-protein interactions, aiding the study of chromatin dynamics and transcription.

Conclusions:

• CRISPR-based imaging methods significantly improve the elucidation of chromatin organization, RNA visualization, and protein imaging.
• These techniques offer powerful approaches to study molecular interactions and coordinated regulation within the cell.
• Advancements in CRISPR-based imaging are poised to revolutionize genome biology research and its applications.