Kinetic mechanism for fidelity of CRISPR-Cas9 variants

Abstract

CRISPR-Cas9 is a nuclease creating DNA breaks at sites with sufficient complementarity to the RNA guide. Notably, Cas9 does not require exact RNA-DNA complementarity and can cleave off-target sequences. Various high-accuracy Cas9 variants have been developed, but the precise mechanism of how these variants achieve higher accuracy remains unclear. Here, we develop a kinetic model of Cas9 substrate selection and cleavage. We parameterize the model using datasets available in the literature, including both high-throughput substrate binding and cleavage data and Forster resonance energy transfer measurements of the Cas9 HNH domain transitions. Based on the observed transition statistics, we predict that the Cas9 substrate recognition and cleavage mechanism must allow for HNH domain transitions independent of substrate binding. Additionally, we show that the enhancement in Cas9 substrate specificity must be due to changes in kinetics rather than changes in substrate binding affinities. Furthermore, the fitted model produces quantitatively realistic cleavage error predictions for substrates with protospacer adjacent motif (PAM)-distal mismatches. Finally, we use our model to identify kinetic parameters for HNH domain transitions that can be perturbed to enable high-accuracy cleavage while maintaining cleavage speeds. Our results refine the biophysical mechanism of Cas9 cleavage to inform future routes for its engineering.