A versatile CRISPR/Cas12a-based biosensing platform coupled with a target-protected transcription strategy

Besides the critical role in gene editing, CRISPR/Cas system also brings a new signal amplification mechanism to the development of next generation biosensing technologies. Herein, we have developed a versatile CRISPR/Cas12a sensing platform by combining a target protection-based transcription amplification strategy with the Cas12a-based signal amplification mechanism, which allows for the sensitive detection of both nucleic acid and non-nucleic acid targets. In this design, a rationally designed transcription template sequence is able to avoid Exonuclease I (Exo I) degradation only in the existence of the target-mediated binding events including either nucleic acid hybridization or protein-based affinity interactions. This target binding-induced protection effect can facilitate the subsequent transcription amplification to generate crRNA and activate the subsequent Cas12a trans-cleavage signal amplification mechanism to yield target dosage-responsive fluorescence signal. In contrast, if the target is absent, the protection-free transcription template will be completely digested by Exo I, thus no fluorescence response is produced. This new strategy well eliminates the T7 polymerase-associated non-specific transcription background and realizes the sensitive detection of various kinds of biomolecules including microRNA, protein, as well as exosome, broadening the application scenarios of CRISPR/Cas system in the field of bioanalysis and biosensing.