Fluorogenic CRISPR for genomic DNA imaging

Zhongxuan Zhang, Xiaoxiao Rong, Tianjin Xie, Zehao Li, Haozhi Song, Shujun Zhen, Haifeng Wang, Jiahui Wu, Samie R. Jaffrey and Xing Li ()
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Zhongxuan Zhang: Chinese Academy of Science
Xiaoxiao Rong: Chinese Academy of Science
Tianjin Xie: Chinese Academy of Science
Zehao Li: Chinese Academy of Science
Haozhi Song: Chinese Academy of Science
Shujun Zhen: Southwest University
Haifeng Wang: Tsinghua University
Jiahui Wu: University of Massachusetts
Samie R. Jaffrey: Cornell University
Xing Li: Chinese Academy of Science

Nature Communications, 2024, vol. 15, issue 1, 1-12

Abstract: Abstract Genomic DNA exhibits high heterogeneity in terms of its dynamic within the nucleus, its structure and functional roles. CRISPR-based imaging approaches can image genomic loci in living cells. However, conventional CRISPR-based tools involve expressing constitutively fluorescent proteins, resulting in high background and nonspecific nucleolar signal. Here, we construct fluorogenic CRISPR (fCRISPR) to overcome these issues. fCRISPR is designed with dCas9, an engineered sgRNA, and a fluorogenic protein. Fluorogenic proteins are degraded unless they are bound to specific RNA hairpins. These hairpins are inserted into sgRNA, resulting in dCas9: sgRNA: fluorogenic protein ternary complexes that enable fluorogenic DNA imaging. With fCRISPR, we image various genomic DNA in different human cells with high signal-to-noise ratio and sensitivity. Furthermore, fCRISPR tracks chromosomes dynamics and length. fCRISPR also allows DNA double-strand breaks (DSBs) and repair to be tracked in real time. Taken together, fCRISPR offers a high-contrast and sensitive platform for imaging genomic loci.

Date: 2024
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DOI: 10.1038/s41467-024-45163-9

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