Wright, J. T.; Yendluri, S.; Thomas, N. C.; Butterfield, C. N.; Dangerfield, T. L.; Taylor, D. W.

CRISPR-Cas12a nucleases provide an attractive alternative to Cas9 due to their compact RNA scaffold, T-rich PAM requirement, and improved target specificity. However, the mechanistic features that govern activity and discrimination across Cas12a orthologs remain incompletely understood. Here, we characterize Cas12a-MG29-1, a highly active and specific nuclease identified through metagenomic mining, using cryogenic electron microscopy, mutational analysis, and kinetic modeling. The Cas12a-MG29-1 structure reveals repositioned flexible loops near the distal end of the R-loop, including reduced engagement of one loop region and additional contacts formed by a second distal loop. Structure-guided mutagenesis and loop-swap experiments indicate that distal R-loop architecture modulates target discrimination in a context-dependent manner. Single-turnover cleavage and stopped-flow measurements show that Cas12a-MG29-1 and AsCas12a form reversible R-loops with similar kinetics but differ in strand cleavage following R-loop formation. Global kinetic modeling demonstrates that Cas12a-MG29-1 exhibits accelerated non-target strand cleavage, shifting kinetic partitioning toward product formation. This faster irreversible commitment provides a mechanistic explanation for enhanced activity and specificity without altering initial target interrogation. Together, these findings identify distal R-loop interactions and catalytic commitment as key determinants of Cas12a function and provide a framework for interpreting and engineering next-generation Cas12a orthologs.