Li Yanan, Huang Di, Pei Yiran, Wu Yonghua, Xu Ru, Quan Fenglei, Gao Hua, Zhang Junli, Hou Hongwei, Zhang Kaixiang, Li Jinghong
School of Pharmaceutical Sciences, Key Laboratory of Targeting Therapy and Diagnosis for Critical Diseases, Collaborative Innovation Center of New Drug Research and Safety Evaluation, State Key Laboratory of Esophageal Cancer Prevention & Treatment, Zhengzhou University, Zhengzhou450001, China.
China National Tobacco Quality Supervision & Test Center, Zhengzhou450001, China.
Anal Chem. 2023 Feb 7;95(5):2992-3001. doi: 10.1021/acs.analchem.2c04867. Epub 2023 Jan 26.
Site-specific imaging of target genes using CRISPR probes is essential for understanding the molecular mechanisms of gene function and engineering tools to modulate its downstream pathways. Herein, we develop CRISPR/Cas9-mediated signal amplification by exchange reaction (CasSABER) for programmable in situ imaging of low and nonrepetitive regions of the target gene in the cell nucleus. The presynthesized primer-exchange reaction (PER) probe is able to hybridize multiple fluorophore-bearing imager strands to specifically light up dCas9/sgRNA target-bound gene loci, enabling in situ imaging of fixed cellular gene loci with high specificity and signal-to-noise ratio. In combination with a multiround branching strategy, we successfully detected nonrepetitive gene regions using a single sgRNA. As an intensity-codable and orthogonal probe system, CasSABER enables the adjustable amplification of local signals in fixed cells, resulting in the simultaneous visualization of multicopy and single-copy gene loci with similar fluorescence intensity. Owing to avoiding the complexity of controlling in situ mutistep enzymatic reactions, CasSABER shows good reliability, sensitivity, and ease of implementation, providing a rapid and cost-effective molecular toolkit for studying multigene interaction in fundamental research and gene diagnosis.
使用CRISPR探针进行靶基因的位点特异性成像对于理解基因功能的分子机制以及调控其下游通路的工程工具至关重要。在此，我们开发了通过交换反应进行CRISPR/Cas9介导的信号放大（CasSABER），用于对细胞核中靶基因的低重复和非重复区域进行可编程原位成像。预合成的引物交换反应（PER）探针能够与多条携带荧光团的成像链杂交，以特异性地照亮与dCas9/sgRNA靶标结合的基因位点，从而实现对固定细胞基因位点的原位成像，具有高特异性和信噪比。结合多轮分支策略，我们使用单个sgRNA成功检测到了非重复基因区域。作为一种强度可编码且正交的探针系统，CasSABER能够在固定细胞中对局部信号进行可调放大，从而以相似的荧光强度同时可视化多拷贝和单拷贝基因位点。由于避免了控制原位多步酶促反应的复杂性，CasSABER具有良好的可靠性、灵敏度和易操作性，为基础研究和基因诊断中研究多基因相互作用提供了一种快速且经济高效的分子工具包。
