Kinetic Basis for DNA Target Specificity of CRISPR-Cas12a

Abstract

<p id="P3">Class 2 CRISPR-Cas nucleases are programmable genome editing tools with promising applications in human health and disease. However, DNA cleavage at off-target sites that resemble the target sequence is a pervasive problem that remains poorly understood mechanistically. Here, we use quantitative kinetics to dissect the reaction steps of DNA targeting by <i>Acidaminococcus sp</i> Cas12a (also known as Cpf1). We show that Cas12a binds DNA tightly in two kinetically separable steps. Protospacer-adjacent motif (PAM) recognition is followed by rate-limiting R-loop propagation, leading to inevitable DNA cleavage of both strands. Despite functionally irreversible binding, Cas12a discriminates strongly against mismatches along most of the DNA target sequence. This result implies substantial reversibility during R-loop formation—a late transition state—and defies common descriptions of a “seed” region. Our results provide a quantitative basis for the DNA cleavage patterns measured <i>in vivo</i> and observations of greater reported target specificity for Cas12a than for the Cas9 nuclease. </p><p id="P4"> <div class="figure-container so-text-align-c"> <img alt="" class="figure" src="/document_file/6ede2026-da2e-4bf5-9b5c-1e6e3abcd13d/PubMedCentral/image/nihms-1040849-f0001.jpg"/> </div> </p><p id="P5">Strohkendl et al. dissect DNA binding and cleavage by CRISPR-Cas12a. They show that binding is functionally irreversible, yet Cas12a discriminates against mismatches with the target DNA extending beyond a seed region. These results suggest that R-loop propagation is readily reversible, enabling Cas12a to select DNA sequences more precisely than Cas9. </p>