Imaging the unimaginable: leveraging signal generation of CRISPR-Cas for sensitive genome imaging.

Fluorescence in situ hybridization (FISH) is the gold standard for visualizing genomic DNA in fixed cells and tissues, but it is incompatible with live-cell imaging, and its combination with RNA imaging is challenging. Consequently, due to its capacity to bind double-stranded DNA (dsDNA) and design flexibility, the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (CRISPR-Cas9) technology has sparked enormous interest over the past decade. In this review, we describe various nucleic acid (NA)- and protein-based (amplified) signal generation methods that achieve imaging of repetitive and single-copy sequences, and even single-nucleotide variants (SNVs), next to highly multiplexed as well as dynamic imaging in live cells. With future progress in the field, the CRISPR-(d)Cas9-based technology promises to break through as a next-generation cell-imaging technique. Adoption of clustered regularly interspaced short palindromic repeats (CRISPR)-based imaging requires consideration of (amplified) signal generation complexity, an appropriate cell delivery mechanism, and the capacity for single-copy sequence imaging. Single-copy sequence imaging is achieved both with and without signal amplification, whereas SNP or single-nucleotide variant (SNV) detection requires signal amplification. CRISPR-based imaging with specificity at the SNV and SNP levels was achieved in both live and fixed cells. The versatility of paired RNA aptamer-based signal generation enables highly multiplexed live-cell imaging of six repetitive sequences. CRISPR-based imaging was used in a clinical setting, allowing the detection of Patau syndrome (in the form of Chr13 trisomy) in patient-derived cells. [ABSTRACT FROM AUTHOR]