Impact of Divalent Metal Ions on Regulation of Trans‐Cleavage Activity of CRISPR‐Cas13a: A Combined Experimental and Computational Study

CRISPR‐LbuCas13a has emerged as a revolutionary tool for in vitro diagnosis. Similar to other Cas effectors, LbuCas13a requires Mg2+ to maintain its nuclease activity. However, the effect of other divalent metal ions on its trans‐cleavage activity remains less explored. Herein, we addressed this issue by combining experimental and molecular dynamics simulation analysis. In vitro studies showed that both Mn2+ and Ca2+ could replace Mg2+ as cofactors of LbuCas13a. In contrast, Ni2+, Zn2+, Cu2+, or Fe2+ inhibits the cis‐ and trans‐cleavage activity, while Pb2+ does not affect it. Importantly, molecular dynamics simulations confirmed that calcium, magnesium, and manganese hydrated ions have a strong affinity to nucleotide bases, thus stabilizing the conformation of crRNA repeat region and enhancing the trans‐cleavage activity. Finally, we showed that combination of Mg2+ and Mn2+ can further enhance the trans‐cleavage activity to allow amplified RNA detection, revealing its potential advantage for in vitro diagnosis. This work explored the mechanism by which divalent metal ions affect the trans‐cleavage activity of LbuCas13a through molecular dynamics simulations combined with in vitro experiments. These results provide new insight into the role of divalent metal ions in LbuCas13a and practical application for amplified RNA detection using ion combination.