CRISPR-Cas12a has been widely used in genome editing and nucleic acid detection. In both of these applications, Cas12a cleaves target DNA in a divalent metal ion-dependent manner. However, when and how metal ions contribute to the cleavage reaction is unclear. Here, using a single-molecule FRET assay, we reveal that these metal ions are necessary for stabilising cleavage-competent conformations and that they are easily exchangeable, suggesting that they are dynamically coordinated.