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