"DSN-mismatched CRISPR"sensor for highly selective and sensitive detection of under-expressed miR-let-7a

Several microRNAs (miRNAs) are expressed at lower levels in specific tumors, e.g., miR-let-7a in non -small cell lung cancer (NSCLC). This makes it challenging to analyze their lower abundance versus specifically elevated miRNAs. Here, we describe a novel fluorescent biosensor for the highly selective and sensitive detection of miRlet-7a constructed by combining miRNA screening assisted by a duplex -specific nuclease (DSN) with CRISPRCas12a system signal amplification. We meticulously designed a mismatch in the first three to four bases at the 5 '-end of the capture DNA to improve the signal-to-noise ratio of the CRISPR-Cas12a system. Within this "DSN-mismatched CRISPR" fluorescence strategy, miR-let-7a was accurately screened by DSN-assisted cleavage, and the mismatched capture DNA unbound to target miRNA could trigger the CRISPR-Cas12a system to produce a mass of trans -cleave fluorescence signals. This "turn-off" approach was suitable for detecting decreased levels of miRNAs. This approach can not only discriminate the single -base mismatched let -7 family but also reach a limit of detection at 64.17 fM as well as be quantified from 100 fM to 500 pM. The miR-let-7a levels were then measured in clinical serum samples from healthy volunteers and patients with NSCLC. This study holds promise for the development of a universal under -expressed miRNA assay for early diagnosis and treatment of cancers.