CRISPR-LbuCas13a has emerged as a revolutionary tool for in vitro diagnosis. Similar to other Cas effectors, LbuCas13a requires Mg 2 + 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 Mn 2 + and Ca 2 + could replace Mg 2 + as cofactors of LbuCas13a. In contrast, Ni 2 + , Zn 2 + , Cu 2 + , or Fe 2 + inhibits the cis-and trans-cleavage activity, while Pb 2 + 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 Mg 2 + and Mn 2 + can further enhance the transcleavage activity to allow amplified RNA detection, revealing its potential advantage for in vitro diagnosis.