David Septian Sumanto Marpaung, Ya-Yu Chen, Murali Mohana Rao Singuru, Min-Chieh Chuang
Off-target effects of the CRISPR/Cas system refer to suboptimal activity triggered by activators containing unintended modifications beyond the intended target, which can lead to unwanted genetic changes—particularly problematic in therapeutic applications. In this study, the off-target effects of Cas12a are strategically repurposed for advanced Hg2+ detection by leveraging a structure-transformable activator. The proposed approach integrates a two-segment poly-T DNA activator with the CRISPR/Cas12a system, where the activator forms hairpin structures through thymine–Hg2+–thymine base pairing in response to mercury concentrations. Increasing Hg2+ concentrations promote the formation and stability of hairpin DNA, significantly suppressing Cas12a activity and resulting in an ON–OFF fluorescence signal modulated by trans-cleavage activity. The effect of varying thymine segment lengths in the poly-T activator was investigated to increase the hybridization burden for crRNA, ultimately enhancing detection sensitivity. The Hg2+-dependent structural transformation of the activator was characterized using circular dichroism (CD), while urea and native PAGE analyses confirmed the catalytic behavior of both cis- and trans-cleavage activities, respectively, as a function of Hg2+ concentration. The trans-cleavage efficiency (kcat/KM) decreased by 3.03-fold as Hg2+ concentration increased from 0 to 40 nM. Under optimized conditions, the biosensor demonstrated a linear detection range of 0–30 nM, a detection limit as low as 0.372 nM, and high selectivity for Hg2+ over other metal ions. Furthermore, the biosensor was successfully applied for Hg2+ detection in field water samples. These findings establish a robust foundation for exploiting the off-target effects of the CRISPR/Cas12a system for the efficient detection of heavy metals in environmental monitoring. © 2025
International Ph.D. Program in Biomedical and Materials Science, Tunghai University, Taichung, 407224, Taiwan; Department of Biosystems Engineering, Institut Teknologi Sumatera, Lampung Selatan, 35365, Indonesia; Department of Chemistry, Tunghai University, Taichung, 407224, Taiwan; Sustainability Science and Management Program, Tunghai University, Taichung, 407224, Taiwan