Abstract：
Breast cancer is reported to be one of the most lethal cancers in women, and its multi-target detection can help improve the accuracy of diagnosis. In this work, a cluster regularly interspaced short palindromic repeats (CRISPR)-Cas13a/Cas12a-based system was established for the simultaneous fluorescence detection of breast cancer biomarkers circROBO1 and BRCA1. CRISPR-Cas13a and CRISPR-Cas12a were directly activated by their respective targets, resulting in the cleavage of short RNA and DNA reporters, respectively, thus the signals of 6-carboxyfluorescein (FAM) and 6-carboxy-xrhodamine (ROX) were restored. As the fluorescence intensities of FAM and ROX were dependent on the concentrations of circROBO1 and BRCA1, respectively, synchronous fluorescence scanning could achieve one-step detection of circROBO1 and BRCA1 with detection limits of 0.013 pM and 0.26 pM, respectively. The system was highly sensitive and specific, holding high diagnostic potential for the detection of clinical samples. Furthermore, the competing endogenous RNA mechanism between circROBO1 and BRCA1 was also explored, providing a reliable basis for the intrinsic regulatory mechanism of breast cancer.
摘要：
据报道，乳腺癌是女性最致命的癌症之一，多目标检测有助于提高诊断的准确性。在这项工作中,我们建立了一个基于集群规则间隔的短回文重复(CRISPR)-Cas13a/Cas12a的系统,用于同时荧光检测cirroBO1和BRCA1的乳腺癌生物标志物.CRISPR-Cas13a和CRISPR-Cas12a被各自的靶标直接激活，导致短RNA和DNA报告基因的切割，分别,因此，6-羧基荧光素（FAM）和6-羧基-xrohodamine（ROX）的信号恢复。由于FAM和ROX的荧光强度分别取决于circROBO1和BRCA1的浓度，同步荧光扫描可以实现circROBO1和BRCA1的一步检测，检出限为0.013pM和0.26pM，分别。该系统具有高度的敏感性和特异性，对临床样本的检测具有很高的诊断潜力。此外，还探索了circROBO1和BRCA1之间的竞争内源性RNA机制，为乳腺癌的内在调控机制提供可靠依据。
