Ultra-sensitive biosensor based on CRISPR-Cas12a and Endo IV coupled DNA hybridization reaction for uracil DNA glycosylase detection and intracellular imaging.
Ultra-sensitive biosensor based on CRISPR-Cas12a and Endo IV coupled DNA hybridization reaction for uracil DNA glycosylase detection and intracellular imaging.
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DOI：
10.1016/j.bios.2023.115118
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发表时间：
2023-02
影响因子：
12.6
通讯作者：
Kejun Dong;Wan Shu;Jiarui Zhang;Shuangshuang Cheng;Jun Zhang;Rong-wei Zhao;Teng Hua;Wei Zhang;Hongbo Wang
中科院分区：
文献类型：
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作者：
Kejun Dong;Wan Shu;Jiarui Zhang;Shuangshuang Cheng;Jun Zhang;Rong-wei Zhao;Teng Hua;Wei Zhang;Hongbo Wang
尿嘧啶-DNA糖基化酶（UDG）作为一种重要的生物标志物，其检测对于疾病的诊断、治疗方案的选择和预后评估具有重要意义。近年来，CRISPR-Cas 12 a反式切割单链DNA探针的信号放大效应为构建高灵敏度生物传感器提供了一种可行的策略。然而，其上级的反式切割活性却成为构建生物传感器的“双刃剑”，在放大靶信号的同时，也放大了泄漏信号，造成失控。因此，构建结构简单、极低背景、高灵敏度的CRISPR-Cas 12 a生物传感器是该领域亟待解决的瓶颈问题。在这里，我们将CRISPR-Cas 12 a与DNA杂交反应相结合，开发了一种简单，快速，低背景，高灵敏度的UDG活性检测方法。它不受PAM的限制，检测限低至2.5 × 10− 6 U/mL。据我们所知，该方法是检测UDG最灵敏的方法之一。我们还使用该系统分析肿瘤细胞中的UDG活性（LOD：1细胞/uL）并评估筛选UDG抑制剂的能力。此外，我们通过将生物传感器固定到细胞上，验证了细胞内UDG活性成像的可能性。我们相信这种新型传感器具有良好的临床应用前景，将有效拓宽CRISPR-Cas 12 a的应用空间。
As an essential biomarker associated with various diseases, Uracil-DNA Glycosylase (UDG) detection is vital for disease diagnosis, treatment selection, and prognosis assessment. In recent years, the signal amplification effect of the CRISPR-Cas12a trans-cleaved single-stranded DNA probe has provided an available strategy for constructing highly sensitive biosensors. However, its superior trans-cleavage activity has become a “double-edged sword” for building biosensors that can amplify the target signal while also amplifying the leakage signal, causing out of control. Therefore, the construction of structurally simple, extremely low-background, highly sensitive CRISPR-Cas12a-based biosensors is an urgent bottleneck problem in the field. Here, we applied CRISPR-Cas12a with a DNA hybridization reaction to develop a simple, rapid, low background, and highly sensitive method for UDG activity detection. It has no PAM restriction and the detection limit is as low as 2.5 × 10−6U/mL. As far as we know, this method is one of the most sensitive methods for UDG detection. We also used this system to analyze UDG activity in tumor cells (LOD: 1 cell/uL) and to evaluate the ability to screen for UDG inhibitors. Furthermore, we verified the possibility of intracellular UDG activity imaging by transfecting the biosensors to the cells. We believe this novel sensor has good clinical application prospects and will effectively broaden the application space of CRISPR-Cas12a.