Impact of Divalent Metal Ions on Regulation of Trans-Cleavage Activity of CRISPR-Cas13a: A Combined Experimental and Computational Study.
Chembiochem
; 24(10): e202300034, 2023 05 16.
Article
in English
| MEDLINE | ID: covidwho-2308421
ABSTRACT
CRISPR-LbuCas13a has emerged as a revolutionary tool for inâ
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 inâ
vitro diagnosis.
Keywords
Full text:
Available
Collection:
International databases
Database:
MEDLINE
Main subject:
RNA
/
Manganese
Type of study:
Experimental Studies
Language:
English
Journal:
Chembiochem
Journal subject:
Biochemistry
Year:
2023
Document Type:
Article
Affiliation country:
Cbic.202300034
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