Activator StrandModifications in CRISPR/Cas12a: Unlockingthe Potential for Casp-3-Targeted Biosensing and Imaging Analysisof Apoptosis

The CRISPR/Cas12a system has emerged as a powerful tool in biosensing due to its unique <i>trans</i>-cleavage activity. This study conducted an in-depth investigation of the modulatory capabilities of this system, particularly focusing on the 5′-end modifications of the activator strand, and found that introducing a hairpin structure (HP) at the 5′-end of the activator strand, which was designed based on the RESET effect, can effectively suppress the activator strand’s ability to activate the <i>trans</i>-cleavage activity of the CRISPR/Cas12a system. This suppression is independent of the HP’s relation to the activator strand and the type of linker used (DNA, RNA or peptide). Detaching the HP from the activator strand restores the system’s activity. These findings enrich the development of CRISPR/Cas12a-based biosensors, and expand their application beyond DNA-based target detection to peptide sequence-based target recognition. Based on this discovery, we constructed a sensitive biosensor for caspase-3 (Casp-3), a key executor in apoptosis, by linking the HP to the activator strand with a peptide linker containing a Casp-3 recognition site. The proposed biosensor has been validated for its sensitivity and specificity in detecting Casp-3, as well as for monitoring drug-induced apoptosis through the imaging of Casp-3 in living cells, providing a valuable tool for studying the apoptotic process, screening drugs, assessing drug efficacy, and evaluating treatment outcomes. This strategy also shows promise for detecting other peptide-based targets, broadening the horizons for early disease biomarker detection and timely therapeutic interventions.