Impact of Divalent Metal Ions on Regulation of Trans‐Cleavage Activity of CRISPR‐Cas13a: A Combined Experimental and Computational Study
Zhiyuan Feng, Yangfang Yun, Xiang Li, Jingjing Zhang
Abstract
Abstract 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 trans‐cleavage activity to allow amplified RNA detection, revealing its potential advantage for in vitro diagnosis.