Biochemical study demonstrates how metal ions regulate Cas12a cleavage and substrate affinity, highlighting mechanisms to optimize CRISPR-based diagnostics.
Key Points
To elucidate how various metal ion cofactors influence the enzymatic cleavage activity, specificity, and regulatory mechanisms of Cas12a.
Evaluated the impact of diverse metal ions on Cas12a cleavage activity and binding affinity toward single-stranded DNA (ssDNA).
Performed active-site structural alignment and site-directed mutagenesis to identify the structural basis of metal-dependent catalysis.
Select metal ions substantially enhanced Cas12a cleavage activity and reaction specificity by increasing enzyme affinity for ssDNA substrates.
Manganese ions (Mn²⁺) uniquely triggered catalytic domain cleavage of ssDNA independently without requiring the intact, full-length Cas12a protein.