Kinetic Mechanism for Fidelity of CRISPR-Cas9 Variants

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 parametrized by data from the literature, including single-molecule Förster resonance energy transfer (FRET) measurements. Based on observed FRET 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. 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.