Children's Hospital, Zhejiang University School of Medicine, National Clinical Research Center for Child Health, Hangzhou, China.
Department of Obstetrics, Shanghai First Maternity and Infant Hospital, Obstetrics and Gynecology Hospital, School of Medicine, Tongji University, Shanghai 201204, China.
Anal Methods. 2024 May 23;16(20):3256-3262. doi: 10.1039/d4ay00693c.
Accurate and precise detection of circular RNA (circRNA) is imperative for its clinical use. However, the inherent challenges in circRNA detection, arising from its low abundance and potential interference from linear isomers, necessitate innovative solutions. In this study, we introduce, for the first time, the application of the CRISPR/Cas12a system to establish a one-pot, rapid (30 minutes to 2 hours), specific and ultrasensitive circRNA detection strategy, termed RETA-CRISPR (reverse transcription-rolling circle amplification (RT-RCA) with the CRISPR/Cas12a). This method comprises two steps: (1) the RT-RCA process of circRNA amplification, generating repeat units containing the back-splicing junction (BSJ) sequences; and (2) leveraging the protospacer adjacent motif (PAM)-independent Cas12a/crRNA complex to precisely recognize target sequences with BSJ, thereby initiating the collateral cleavage activity of Cas12a to generate a robust fluorescence signal. Remarkably, this approach exhibits the capability to detect circRNAs at a concentration as low as 300 aM. The sensor has been successfully employed for accurate detection of a potential hepatocellular carcinoma biomarker hsa_circ_0001445 (circRNA1445) in various cell lines. In conclusion, RETA-CRISPR seamlessly integrates the advantages of exponential amplification reaction and the robust collateral cleavage activity of Cas12a, positioning it as a compelling tool for practical CRISPR-based diagnostics.
准确和精确地检测环状 RNA (circRNA) 对于其临床应用至关重要。然而，circRNA 检测所面临的固有挑战，包括其低丰度和潜在的线性异构体干扰，需要创新的解决方案。在这项研究中，我们首次引入了 CRISPR/Cas12a 系统的应用，建立了一种一锅、快速（30 分钟至 2 小时）、特异性和超灵敏的 circRNA 检测策略，称为 RETA-CRISPR（逆转录-滚环扩增（RT-RCA）与 CRISPR/Cas12a）。该方法包括两个步骤：（1）circRNA 扩增的 RT-RCA 过程，产生包含反向剪接连接（BSJ）序列的重复单元；（2）利用无 PAM 的 Cas12a/crRNA 复合物精确识别具有 BSJ 的靶序列，从而启动 Cas12a 的旁切活性，产生强大的荧光信号。值得注意的是，该方法能够以低至 300 aM 的浓度检测 circRNAs。该传感器已成功用于各种细胞系中潜在肝癌生物标志物 hsa_circ_0001445（circRNA1445）的准确检测。总之，RETA-CRISPR 无缝集成了指数扩增反应和 Cas12a 强大的旁切活性的优势，使其成为一种有吸引力的基于 CRISPR 的实用诊断工具。
