Specificity and Regulatory Mechanism of Metal Ions in Cas12a Enzymatic Cleavage Activity.

Metal ions play key roles in both physiological and pathological processes. The activity of nearly all nucleases depends on metal ions as cofactors. CRISPR-Cas system is widely used in gene editing and nucleic acid detection, and its ongoing development and frontier research have attracted significant attention. This study aims to explore and elucidate the effects of various metal ions on Cas12a-catalysed cleavage reactions including their specificity and regulatory mechanisms. Here, we found that certain metal ions significantly affected both the cleavage activity and the reaction specificity of Cas12a. Metal ions mediate enzyme-substrate binding by increasing the affinity between Cas12a and single-stranded DNA (ssDNA). In particular, unlike other metal ions, Mn2+ can achieve cleavage of ssDNA by only activating the catalytic domain without requiring the entire Cas12a. Structural alignment and site-directed mutagenesis of the active site revealed the structural basis of Cas12a catalysis and elucidated how metal ions influence its catalytic reaction in terms of specificity and regulatory mechanism. These results provide a theoretical foundation and offer innovative perspectives for the application of Cas12a-based enzyme technology.