A universal CRISPR/Cas12a nucleic acid sensing platform based on proximity extension and transcription-unleashed self-supply crRNA

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. Scheme 1 . Principle of the CRISPR/Cas12a DNA sensing platform based on proximity extension and transcription-unleashed self-supply crRNA. • A universal Cas12a-based sensing strategy was developed for rapid and sensitive detection of nucleic acids. • Since proximity recognition mechanism, the method could specifically detect a diverse range of nucleic acid sequences. • Owing to cascade enzymatic reaction and Cas12a trans-cleavage, the detection limit of the method was down to amol level.