Beijing Institute of Life Sciences, Chinese Academy of Science, 100101, Beijing, China.
Department of Respiratory and Critical Care Medicine, The Affiliated Hospital of Southwest Medical University, 646000, Luzhou, Sichuan, China.
Nat Commun. 2024 Jan 31;15(1):934. doi: 10.1038/s41467-024-45163-9.
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.
基因组 DNA 在其核内的动态、结构和功能作用方面表现出高度的异质性。基于 CRISPR 的成像方法可以在活细胞中对基因组位点进行成像。然而，传统的基于 CRISPR 的工具涉及到持续表达荧光蛋白，这会导致高背景和非特异性核仁信号。在这里，我们构建了荧光 CRISPR（fCRISPR）来克服这些问题。fCRISPR 由 dCas9、工程 sgRNA 和荧光蛋白组成。除非与特定的 RNA 发夹结合，否则荧光蛋白会被降解。这些发夹插入 sgRNA 中，形成 dCas9:sgRNA:荧光蛋白三元复合物，从而实现荧光 DNA 成像。利用 fCRISPR，我们可以在不同的人类细胞中以高信噪比和灵敏度对各种基因组 DNA 进行成像。此外，fCRISPR 还可以跟踪染色体的动态和长度。fCRISPR 还可以实时跟踪 DNA 双链断裂（DSB）和修复。总之，fCRISPR 为基因组位点成像提供了一个高对比度和高灵敏度的平台。
