A one-pot CRISPR-RCA strategy for ultrasensitive and specific detection of circRNA

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.