Fluorogenic CRISPR for DNA imaging in live mammalian cells

Wenjin Wan; Xin Ji; Haozhi Song; Zhongxuan Zhang; Chun Kit Kwok; Xiaohong Fang; Xing Li

doi:10.1016/j.chembiol.2025.10.013

Fluorogenic CRISPR for DNA imaging in live mammalian cells

Wenjin Wan (Co-first Author)
, Xin Ji (Co-first Author)
, Haozhi Song
, Zhongxuan Zhang
, Chun Kit Kwok
, Xiaohong Fang^*
, Xing Li^*

^*Corresponding author for this work

Research output: Journal Publications and Reviews › RGC 21 - Publication in refereed journal › peer-review

1 Citation (Scopus)

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 greatly 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. © 2025 Published by Elsevier Ltd.

Original language	English
Pages (from-to)	33-44
Number of pages	12
Journal	Cell Chemical Biology
Volume	33
Issue number	1
Online published	5 Jan 2026
DOIs	https://doi.org/10.1016/j.chembiol.2025.10.013
Publication status	Published - 15 Jan 2026

Funding

This work was supported by National Natural Science Foundation of China (no. T2521004 , 32494800 ; 32271515 , 32311530120 , 32471343 , and 32222089 ), National Key Research Program (no. 2023YFC2604302 ), National Key Scientific Program of China (no. 2022YFA1304503), Biological Breeding-National Science and Technology Major Project (no. 2023ZD04046 ), High-level Talent Program for Innovate Talent, Hunan Province (no. 2024RC4015 ), Beijing Natural Science Foundation (no. L255013 ), Initiative Scientific Research Program , Institute of Zoology (no. 2024IOZ0202 ), State Key Laboratory of Animal Biodiversity Conservation , Integrated Pest Management (no. SKLA2508 ), Yuelushan Laboratory Breeding Program (no. YLS-2025-ZY01002 ) and Research Grants Council (RGC) of the Hong Kong Special Administrative Region (no. CityU 11101525 , RFS2425-1S02 , CityU 11100123 , CityU 11100222 , and CityU 11100421 ). We thank Xing Li lab members for their useful help and comments.

Research Keywords

CRISPR
DNA imaging
fluorogenic CRISPR
fluorogenic probe
high contrast imaging

RGC Funding Information

RGC-funded

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title = "Fluorogenic CRISPR for DNA imaging in live mammalian cells",

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 greatly 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. {\textcopyright} 2025 Published by Elsevier Ltd.",

keywords = "CRISPR, DNA imaging, fluorogenic CRISPR, fluorogenic probe, high contrast imaging",

author = "Wenjin Wan and Xin Ji and Haozhi Song and Zhongxuan Zhang and Kwok, \{Chun Kit\} and Xiaohong Fang and Xing Li",

year = "2026",

month = jan,

day = "15",

doi = "10.1016/j.chembiol.2025.10.013",

language = "English",

volume = "33",

pages = "33--44",

journal = "Cell Chemical Biology",

issn = "2451-9448",

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TY - JOUR

T1 - Fluorogenic CRISPR for DNA imaging in live mammalian cells

AU - Wan, Wenjin

AU - Ji, Xin

AU - Song, Haozhi

AU - Zhang, Zhongxuan

AU - Kwok, Chun Kit

AU - Fang, Xiaohong

AU - Li, Xing

PY - 2026/1/15

Y1 - 2026/1/15

N2 - 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 greatly 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. © 2025 Published by Elsevier Ltd.

AB - 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 greatly 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. © 2025 Published by Elsevier Ltd.

KW - CRISPR

KW - DNA imaging

KW - fluorogenic CRISPR

KW - fluorogenic probe

KW - high contrast imaging

UR - https://www.scopus.com/pages/publications/105027409692

UR - https://www.scopus.com/record/pubmetrics.uri?eid=2-s2.0-105027409692&origin=recordpage

U2 - 10.1016/j.chembiol.2025.10.013

DO - 10.1016/j.chembiol.2025.10.013

M3 - RGC 21 - Publication in refereed journal

C2 - 41494531

SN - 2451-9448

VL - 33

SP - 33

EP - 44

JO - Cell Chemical Biology

JF - Cell Chemical Biology

IS - 1

ER -

Fluorogenic CRISPR for DNA imaging in live mammalian cells

Abstract

Funding

Research Keywords

RGC Funding Information

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GRF: Developing L-RNA-Based Fluorescence Light-Up Aptamers for the Detection and Imaging of Functional G-Quadruplex Structures

RFS: Developing RNA Structure-Targeting Bi-/Multi-Functional Laptamer-Based Chimeras for Gene Regulation

GRF: Developing Novel Short Peptides to Target G-quadruplex Structure and Modulate Gene Function

Cite this

Fluorogenic CRISPR for DNA imaging in live mammalian cells

Abstract

Funding

Research Keywords

RGC Funding Information

Access Output

Other Access Options

Permanent Link

Other Files and Links

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Projects

GRF: Developing L-RNA-Based Fluorescence Light-Up Aptamers for the Detection and Imaging of Functional G-Quadruplex Structures

RFS: Developing RNA Structure-Targeting Bi-/Multi-Functional Laptamer-Based Chimeras for Gene Regulation

GRF: Developing Novel Short Peptides to Target G-quadruplex Structure and Modulate Gene Function

Cite this