A one-tube autocatalytic transcription-driven CRISPR cascade for ultrasensitive mRNA detection

The rapid identification and precise quantification of cancer biomarkers are essential for the purposes of diagnosis, classification, and therapeutic intervention. Traditional molecular diagnostic methodologies, such as polymerase chain reaction (PCR), provide considerable sensitivity; however, they depend on exponential amplification and advanced instrumentation, thereby constraining their applicability for point-of-care testing. In this study, we developed a Transcription-driven CRISPR Cascade Amplification (TCCA) for one-tube detection of mRNA at the concentration of 0.6 copies/μL within just 30 min. The target-induced assembly of a three-way junction (TWJ) facilitates the generation of a split T7 promoter, which initiates transcription and activates Cas13a collateral cleavage. The activated Cas13a subsequently cleaves cascade probes and releases new trigger strands, forming a transcription-driven cascade amplification circuit. The assay enables multiplex detection of breast cancer-associated mRNA biomarkers (HBB, KRT17, and CD55) in cell lysates, demonstrating robust performance in intricate biological matrices. Furthermore, incorporation of a multiplex OR-gated logic design enables parallel target recognition, thereby enhancing detection reliability for rapid and accurate clinical diagnostics.