A mechanistic model improves off-target predictions and reveals the physical basis of SpCas9 fidelity

Eslami-Mossallam, B.; Klein, M.; Smagt, C. v. d.; Sanden, K. v. d.; Jones, S. K.; Hawkins, J. A.; Finkelstein, I. J.; Depken, M.

doi:10.1101/2020.05.21.108613

bioRxiv · 10.1101/2020.05.21.108613

A mechanistic model improves off-target predictions and reveals the physical basis of SpCas9 fidelity

Eslami-Mossallam, B.·Klein, M.·Smagt, C. v. d.·Sanden, K. v. d.·Jones, S. K.·Hawkins, J. A.·Finkelstein, I. J.·Depken, M.

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Abstract

The S. pyogenes (Sp) Cas9 endonuclease is an important gene-editing tool. SpCas9 is directed to target sites via a single guide RNA (sgRNA). However, SpCas9 also binds and cleaves genomic off-target sites that are partially matched to the sgRNA. Here, we report a microscopic kinetic model that simultaneously captures binding and cleavage dynamics for SpCas9 and Sp-dCas9 in free-energy terms. This model not only outperforms state-of-the-art off-target prediction tools, but also details how Sp-Cas9s structure-function relation manifests itself in binding and cleavage dynamics. Based on the biophysical parameters we extract, our model predicts SpCas9s open, intermediate, and closed complex configurations and indicates that R-loop progression is tightly coupled with structural changes in the targeting complex. We show that SpCas9 targeting kinetics are tuned for extended sequence specificity while maintaining on-target efficiency. Our extensible approach can characterize any CRISPR-Cas nuclease - benchmarking natural and future high-fidelity variants against SpCas9; elucidating determinants of CRISPR fidelity; and revealing pathways to increased specificity and efficiency in engineered systems.

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Eslami-Mossallam, B., Klein, M., Smagt, C. v. d., Sanden, K. v. d., Jones, S. K., Hawkins, J. A., Finkelstein, I. J., Depken, M.. 2020-05-22. A mechanistic model improves off-target predictions and reveals the physical basis of SpCas9 fidelity. https://doi.org/10.1101/2020.05.21.108613

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