Simultaneous detection for breast cancer-associated cell-free DNA and microRNA using multiplex CRISPR Cas12a/Cas13a system

Cell-free DNAs (cfDNAs) and microRNAs (miRNAs) have garnered significant attention in the biomedical field due to their abnormal expression levels associated with a wide range of diseases, including cancers. However, their concentration in bodily fluids is typically low and diagnosing a specific disease solely by measuring a single nucleic acid biomarker presents considerable challenges. In this study, we have achieved more accurate diagnosis of breast cancer by presenting a multiplex sensing system for breast cancer-derived cfDNA and miRNA, leveraging CRISPR-Cas12a and CRISPR-Cas13a sensing strategies. In this system, CRISPR-Cas12a and CRISPR-Cas13a are selectively activated by the target breast cancer gene 1 (BRCA1) and miRNA-10b, respectively, resulting in trans-cleavage reactions on FAM-tagged ssDNA and ROX-tagged ssRNA immobilized on the surface of gold nanorods (AuNRs). Subsequently, the quenched fluorescent probes transition to dequenching states, enabling the measurement of fluorescent signals according to FAM and ROX signifies the presence of BRCA1 and miRNA-10b, respectively. The proposed simultaneous multiple detection system demonstrates high sensitivity and rapid detection (< 1 h) for both cfDNA and miRNA, achieved through straightforward enzymatic cleavage reactions. We hope that the proposed proof-of-concept multiplex detection system for both nucleic acids will pave the way for an early and accurate diagnosis of numerous diseases.