作为与各种疾病相关的重要生物标志物,尿嘧啶-DNA糖基化酶(UDG)检测对于疾病诊断至关重要,治疗选择,和预后评估。近年来,CRISPR-Cas12a反式切割的单链DNA探针的信号放大效应为构建高灵敏度的生物传感器提供了可用的策略。然而,其优越的反式切割活性已成为构建生物传感器的“双刃剑”,该传感器可以放大目标信号,同时还可以放大泄漏信号,导致失控。因此,结构简单的结构,极低的背景,基于CRISPR-Cas12a的高灵敏度生物传感器是该领域亟待解决的瓶颈问题。这里,我们应用CRISPR-Cas12a与DNA杂交反应来开发一种简单的,快速,低背景,和高灵敏度的UDG活性检测方法。它没有PAM限制,检出限低至2.5×10-6U/mL。据我们所知,该方法是最灵敏的UDG检测方法之一。我们还使用该系统来分析肿瘤细胞中的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转染到细胞中验证了细胞内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-Cas12a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 × 10-6 U/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.