Journal
CHEMBIOCHEM
Volume -, Issue -, Pages -Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/cbic.202300034
Keywords
CRISPR; divalent metal Ions; molecular dynamics simulation; RNA detection; trans-cleavage activity
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CRISPR-LbuCas13a is a revolutionary tool for in vitro diagnosis, requiring Mg2+ for its nuclease activity. Experimental results showed that both Mn2+ and Ca2+ can replace Mg2+ as cofactors of LbuCas13a, while Ni2+, Zn2+, Cu2+, or Fe2+ inhibit its cleavage activity and Pb2+ has no significant effect.
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.
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