Impact of Divalent Metal Ions on Regulation of Trans-cleavage Activity of CRISPR-Cas13a: A Combined Experimental and Computational Study.

Abstract

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 combined 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. Importantly, molecular dynamics simulations confirmed that calcium, magnesium and manganese hydrated ions have 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.