Ultrasensitive and high-specific microRNA detection using hyper-branching rolling circle amplified CRISPR/Cas13a biosensor

Accurate detection of trace microRNAs (miRNA) is highly valuable to provide an indication for early-stage diseases diagnosis. Recently, RNA-guided and RNA-targeting CRISPR/Cas13a has been proven to be a versatile RNA biosensing component due to its high-fidelity and unique target-activated collateral cleavage activity. Here, we developed a novel hyper-branching rolling circle amplified CRISPR/Cas13a biosensor (termed HyperCas) for ultra-sensitive and highly specific miRNA detection. HyperCas takes full advantage of high efficiency of HRCA reaction as well as robust collateral cleavage activity of Cas13a, which ensures ultra-high signal amplification efficiency. The experimental results confirmed HyperCas can dramatically differentiate highly homologous microRNA-17 (miR-17) family members, and achieved an ultralow limit-of-detection (LOD) of 200 aM, which is 3-orders of magnitude lower than direct Cas13a mediated miRNA detection. Compared to conventional HRCA assay, HyperCas also showed 500-fold superior detection sensitivity. Moreover, HyperCas can accurately analyze miR-17 from cancer cell extracts and keep good detection ability for human serum samples, suggesting its great promise for early diseases diagnosis.