Cas-Rainbow: Cas12a-driven single-reaction multiplex detection system

Recently, CRISPR/Cas system has garnered considerable attention owing to its high cleavage efficiency. However, it lacks the ability to differentiate between signal sources. To address this, we have developed a multiplex detection system called Cas-Rainbow. This system combines CRISPR/Cas with rolling circle amplification (RCA) and hybridization chain reaction (HCR) to enable the detection of multiple miRNAs using reporter probes labeled with different fluorescent groups. The RCA product can trigger the formation of HCR and activate the Cas12a/crRNA complex, achieving the simultaneous detection of multiple miRNAs. Detection limits of three miRNAs (miR-155, miR-10b, and miR-21) were 33 fM, 41 fM, and 79 fM, respectively. Cas-Rainbow system could effectively identify these three miRNAs in serum, distinguishing breast cancer patients from healthy donors with accuracy of 100 %. Moreover, this system exhibited exceptional performance in logic gate circuits, showcasing its remarkable versatility and robustness. In summary, our proposed method offers a new approach for multiplex detection based on the CRISPR/Cas system.