Fluorogenic CRISPR for DNA imaging in live mammalian cells

Wan, W.#; Ji, X.#; Song, H.; Zhang, Z.; Kwok, C.; Fang, X.*; Li, X.*

Cell Chemical Biology (IF=7.2), 2025, Accept.

Abstract

Spatiotemporal imaging of genomic DNA dynamics in live mammalian cells is essential for elucidating eukaryotic organization and processes relevant to health and disease.  CRISPR systems facilitate the development of live cell DNA imaging tools.  However, conventional CRISPR imaging tools typically utilize constitutively fluorescent proteins, resulting in high background noise, nonspecific nucleolar signals, and low signal-to-noise ratios.  To address this, fluorogenic CRISPR-based imaging tools have been developed.  These tools remain non-fluorescent until they bind to the target DNA, thus significantly reducing the background and enhancing the sensitivity.  This review summarizes four fluorogenic CRISPR strategies, each utilizing different fluorogenic reporters, including fluorogenic proteins, fluorogenic RNA aptamers, split fluorescent proteins, and molecular beacons.  These fluorogenic CRISPR approaches successfully monitored the subnucleus gene loci localization, dynamics, and DNA breaks and repairs.  We anticipate that this review can inspire researchers to expand the fluorogenic CRISPR for cellular DNA imaging and diverse bioapplications.