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基于 CRISPR/Cas 介导的聚集诱导发光物的“一对一到多对”点亮式核酸检测。

CRISPR/Cas-mediated "one to more" lighting-up nucleic acid detection using aggregation-induced emission luminogens.

机构信息

State Key Laboratory of Food Science and Resources, School of Food Science and Technology, Nanchang University, Nanchang, China.

Beijing Engineering and Technology Research Center of Food Additives, Beijing Technology & Business University, Beijing, China.

出版信息

Nat Commun. 2024 Oct 3;15(1):8560. doi: 10.1038/s41467-024-52931-0.

Abstract

CRISPR diagnostics are effective but suffer from low signal transduction efficiency, limited sensitivity, and poor stability due to their reliance on the trans-cleavage of single-stranded nucleic acid fluorescent reporters. Here, we present CrisprAIE, which integrates CRISPR/Cas reactions with "one to more" aggregation-induced emission luminogen (AIEgen) lighting-up fluorescence generated by the trans-cleavage of Cas proteins to AIEgen-incorporated double-stranded DNA labeled with single-stranded nucleic acid linkers and Black Hole Quencher groups at both ends (Q-dsDNA/AIEgens-Q). CrisprAIE demonstrates superior performance in the clinical nucleic acid detection of norovirus and SARS-CoV-2 regardless of amplification. Moreover, the diagnostic potential of CrisprAIE is further enhanced by integrating it with spherical nucleic acid-modified AIEgens (SNA/AIEgens) and a portable cellphone-based readout device. The improved CrisprAIE system, utilizing Q-dsDNA/AIEgen-Q and SNA/AIEgen reporters, exhibits approximately 80- and 270-fold improvements in sensitivity, respectively, compared to conventional CRISPR-based diagnostics. We believe CrisprAIE can be readily extended as a universal signal generation strategy to significantly enhance the detection efficiency of almost all existing CRISPR-based diagnostics.

摘要

CRISPR 诊断方法虽然有效,但由于依赖于单链核酸荧光报告物的转切割,因此存在信号转导效率低、灵敏度有限和稳定性差的问题。在这里,我们提出了 CrisprAIE,它将 CRISPR/Cas 反应与“一个到多个”聚集诱导发射发光团(AIEgen)的照明荧光相结合,由 Cas 蛋白的转切割产生,将 AIEgen 掺入到双链 DNA 中,双链 DNA 标记有单链核酸接头和两端的黑洞猝灭基团(Q-dsDNA/AIEgens-Q)。CrisprAIE 无论是否进行扩增,在临床核酸检测诺如病毒和 SARS-CoV-2 方面都表现出优异的性能。此外,通过将其与球形核酸修饰的 AIEgen(SNA/AIEgen)和便携式基于手机的读取设备集成,进一步增强了 CrisprAIE 的诊断潜力。利用 Q-dsDNA/AIEgen-Q 和 SNA/AIEgen 报告物改进的 CrisprAIE 系统,与传统的基于 CRISPR 的诊断相比,灵敏度分别提高了约 80 倍和 270 倍。我们相信,CrisprAIE 可以很容易地扩展为通用的信号产生策略,显著提高几乎所有现有基于 CRISPR 的诊断的检测效率。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/eae3/11450156/5fc8b3b3f954/41467_2024_52931_Fig1_HTML.jpg

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