Duplex-Specific Nuclease-Assisted CRISPR-Cas12a Strategy for MicroRNA Detection Using a Personal Glucose Meter.

A CRISPR-Cas system holds great promise as a next-generation biosensing technology for molecular diagnostics. Nevertheless, the current CRISPR-Cas12a-based detection strategies always need bulky instruments or auxiliary devices to obtain a quantitative signal output, which restrains its point-of-care testing application. Herein, we proposed a duplex-specific nuclease-assisted CRISPR-Cas12a strategy to detect microRNA (miRNA) with a personal glucose meter. The target miRNA was first converted into an amplified initiator DNA via duplex-specific nuclease. Afterward, the initiator DNA activated the collateral cleavage activity of CRISPR-Cas12a to cleave the single-strand DNA (ssDNA) linker on sucrase-ssDNA-modified magnetic beads, which led to the release of sucrase. The released sucrase was collected and then utilized to catalyze sucrose to glucose, which could be quantitatively detected by a personal glucose meter. The change in the glucose signal directly reflected the concentration of miRNA, which avoided expensive equipment for signal quantification. Two different miRNAs (miRNA21 and miRNA205) could be detected by simply changing the sequence of the template strand (H strand). The developed strategy showed high sensitivity with a limit of detection (LOD) of 2.4 and 1.1 pM for miRNA21 and miRNA205, respectively. In addition, good selectivity and anti-interference ability were achieved using this method, which enabled it promising for miRNA detection at the point-of-care.