Background <p>The CRISPR/Cas12a system has revolutionized nucleic acid detection through trigger-activated nonspecific <i>trans</i>-cleavage activity. Enhancing this activity is vital for improving the sensitivity of CRISPR/Cas biosensing systems. Although various chemical enhancers have been shown to affect Cas12a ribonucleoprotein (RNP), the underlying mechanisms remain poorly understood. Investigating how these enhancers alter the structure of Cas12a RNPs is essential for elucidating the enhancement mechanisms involved.</p> Results <p>This study focuses on elucidating the structural changes in Cas12a RNPs induced by various chemical enhancers via AlphaFold 3, an emerging and powerful tool for analyzing structural changes in protein‒nucleic acid complexes. We validated the ability of AlphaFold 3 to simulate structural changes in Cas12a RNPs activated by triggers and subsequently analyzed the effects of specific enhancers, such as reducing agents (e.g., Dithiothreitol, namely DTT), divalent cations (e.g., Mg²⁺, Mn²⁺), and bovine serum albumin (BSA). Our findings revealed that DTT, simulated with a cysteine-to-serine Cas12a mutant, caused significant structural changes in Cas12a RNP, as evidenced by notable shifts in the distance between key residues (Val377 to Gln1136) and a high root mean square deviation (RMSD)(RMSD &gt; 2). Conversely, divalent cations and BSA did not cause substantial structural changes, resulting in only minor shifts in residue distance and a low RMSD (RMSD &lt; 2).</p> Conclusions <p>These results demonstrate that DTT enhances Cas12a activity by inducing significant structural rearrangements, whereas divalent cations and BSA-induced enhancements do not involve substantial structural modifications.</p> Graphical Abstract <p>Summary of structural changes from inactivated Cas12a RNP to activated Cas12a RNP and from activated Cas12a RNP to activated Cas12a RNP with an enhancer. The units of both the RMSD and distance are Å.</p> <p></p>