State Key Laboratory of Chemical Oncogenomics, Key Laboratory of Chemical Biology, Shenzhen International Graduate School, Tsinghua University, Shenzhen, 518055, PR China; Department of Chemistry, Tsinghua University, Beijing, 100084, PR China.
State Key Laboratory of Chemical Oncogenomics, Key Laboratory of Chemical Biology, Shenzhen International Graduate School, Tsinghua University, Shenzhen, 518055, PR China.
Anal Chim Acta. 2023 Mar 15;1246:340896. doi: 10.1016/j.aca.2023.340896. Epub 2023 Jan 25.
Multiple circulating non-coding RNAs (ncRNAs) in serum may serve as vital biomarkers for use in diagnosing early-stage colorectal cancer (CRC). Herein, a universal platform for one-pot detection of CRC-related ncRNAs was developed based on branched rolling circle amplification and CRISPR-Cas12a (BRCACas). For the implementation of the method, primers incorporating ncRNA sequences of circulating CRC-associated RNAs (piRNA or miRNA) were designed that could specifically hybridize with circular probes to initiate the BRCA process. Thereafter, the generation of dendritic DNA products triggered Cas12a trans-cleavage activity to elicit a fluorescent signal. The proposed method, combining high BRCA reaction efficiency with powerful Cas12a trans-cleavage activity, provided greatly enhanced detection sensitivity, as reflected by limits of detection (LODs) for model piRNA (piR-54265) and model miRNA (miR21) of 0.76 fM and 0.87 fM, respectively. Notably, the proposed BRCACas platform, assaying two different types of CRC-associated ncRNAs in patient samples, produced consistent results with the conventional reverse transcription-quantitative PCR (RT-qPCR) method. Therefore, the one-pot, isothermal, and specific BRCACas platform provided excellent performance, thus demonstrating its promise as a rapid, adaptable, and practical diagnostic/prognostic cancer screening method.
多种循环非编码 RNA（ncRNA）可作为诊断早期结直肠癌（CRC）的重要生物标志物。在此，我们基于分支滚环扩增和 CRISPR-Cas12a（BRCA Cas）开发了一种用于 CRC 相关 ncRNA 的一锅检测的通用平台。对于该方法的实施，设计了包含循环 CRC 相关 RNA（piRNA 或 miRNA）的 ncRNA 序列的引物，该引物能够与环状探针特异性杂交以启动 BRCA 过程。此后，枝状 DNA 产物的生成引发 Cas12a 转切割活性以产生荧光信号。所提出的方法结合了高 BRCA 反应效率和强大的 Cas12a 转切割活性，提供了大大增强的检测灵敏度，模型 piRNA（piR-54265）和模型 miRNA（miR21）的检测限（LOD）分别为 0.76 fM 和 0.87 fM。值得注意的是，该平台在患者样本中同时检测两种不同类型的 CRC 相关 ncRNA，与传统的逆转录定量聚合酶链反应（RT-qPCR）方法具有一致的结果。因此，这种一锅、等温且特异性的 BRCA Cas 平台具有出色的性能，从而证明了其作为一种快速、适应性强且实用的癌症诊断/预后筛查方法的潜力。
