CRISPR-Cas12a effects RNA-guided cleavage of dsDNA in , after which it remains catalytically active and non-specifically cleaves ssDNA in . Native host-defence by Cas12a employs cleavage, which can be repurposed for the genome editing of other organisms, and cleavage can be used for DNA detection. Cas12a orthologues have high structural similarity and a conserved mechanism of DNA cleavage, yet highly different efficacies when applied for genome editing or DNA detection. By comparing three well characterised Cas12a orthologues (FnCas12a, LbCas12a, and AsCas12a), we sought to determine what drives their different and cleavage, and how this relates to their applied function. We integrated DNA cleavage kinetics with molecular dynamics simulations, plasmid interference in , and genome editing in human cell lines. We report large differences in cleavage kinetics between orthologues, which may be driven by dynamic REC2-NUC interactions. We generated and tested REC2 and NUC mutants, including a hitherto unstudied 'NUC loop', integrity of which is critical for the function of Cas12 orthologues. In total, our and survey of Cas12a orthologues highlights key properties that drive their function in biotechnology applications.