Engineering multi-activator-encoded DNA nanonet to accelerate CRISPR-Cas12a activation for rapid and sensitive...

Despite CRISPR-associated (Cas) nucleases have emerged as a versatile and highly programmable tool for biosensing and molecular diagnostics, the efficient manipulation of targeted CRISPR-Cas12a activation requires further improvement. Herein, we engineered a target-response DNA nanodevice called multi-activator-encoded DNA nanonet (MAIDA) which displayed efficient manipulation of CRISPR-Cas12a trans-activity for apurinic/apyrimidinic endonuclease 1 (APE1) activity monitoring. The MAIDA nanodevice was constructed by multi-activator loops (MA loops) encoded with three activator sequences and target-response loops (TR loops) encoded with three abasic sites to generate interlocked DNA nanonet. Notably, the activator sequences on MA loop were pre-hybridized with TR loop, which not only generate AP sites but also inhibit the CRISPR-Cas12a activation in the initial state. When APE1 is present, the AP sites on the MAIDA nanodevice were recognized and cleaved to the release of MA loops, which could activate the trans-cleavage of CRISPR-Cas12a and then output the signal through electrochemiluminescence (ECL) biosensor. Finally, the experimental results demonstrate that the MA loops increase the ECL response of CRISPR-Cas12a by 1.5-fold compared with the conventional single-linear activators, and the limit of detection (LOD) of APE1 by the proposed biosensor is 1.46 × 10^-10 U/μL. The MAIDA nanodevice promoted the efficient manipulation of targeted CRISPR-Cas12a activation with high sensitivity and selectivity, which provided a promising tool for enhancing DNA-based sensing and analytical applications.