The extraordinary genome-editing tool CRISPR/Cas12a has also been utilized as a powerful sensing technology owing to its highly-specificity and isothermal signal amplification. Nevertheless, the widespread application of Cas12a-based sensing methods in nucleic acid detection is limited by the targeting range and high undesired background. Herein, we established a universal Cas12a-based nucleic acid sensing strategy by using proximity extension and transcription-unleashed self-suppling of crRNA. The target was recognized and bound to a pair of adjacent probes, and then triggered the proximity-induced primer extension and transcription amplification to produce numerous crRNAs. The amplified abundant crRNAs assembled with Cas12a and dsDNA activators containing PAM to form a ternary complex, which trans-cleaved ssDNA-FQ reporters continuously to generate a strong fluorescent signal. Thus, the cascade enzymatic amplification was performed and subsequently applied for detecting target DNA down to 41.7 amol with a low nonspecific background. The application of this strategy in RNA detection has also been demonstrated, and it is expected to provide a universal and sensitive sensing platform for molecular diagnosis applications.
Keywords: CRISPR/Cas12a; Fluorescence; Nucleic acid sensing; PAM; Proximity binding; Trans-cleavage.
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