A kinetic model predicts SpCas9 activity, improves off-target classification, and reveals the physical basis of targeting fidelity

Nat Commun. 2022 Mar 15;13(1):1367. doi: 10.1038/s41467-022-28994-2.


The S. pyogenes (Sp) Cas9 endonuclease is an important gene-editing tool. SpCas9 is directed to target sites based on complementarity to a complexed single-guide RNA (sgRNA). However, SpCas9-sgRNA also binds and cleaves genomic off-targets with only partial complementarity. To date, we lack the ability to predict cleavage and binding activity quantitatively, and rely on binary classification schemes to identify strong off-targets. We report a quantitative kinetic model that captures the SpCas9-mediated strand-replacement reaction in free-energy terms. The model predicts binding and cleavage activity as a function of time, target, and experimental conditions. Trained and validated on high-throughput bulk-biochemical data, our model predicts the intermediate R-loop state recently observed in single-molecule experiments, as well as the associated conversion rates. Finally, we show that our quantitative activity predictor can be reduced to a binary off-target classifier that outperforms the established state-of-the-art. Our approach is extensible, and 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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, N.I.H., Extramural

MeSH terms

  • CRISPR-Associated Protein 9* / genetics
  • CRISPR-Associated Protein 9* / metabolism
  • CRISPR-Cas Systems*
  • Endonucleases / metabolism
  • Gene Editing
  • RNA, Guide, Kinetoplastida / genetics


  • RNA, Guide
  • CRISPR-Associated Protein 9
  • Endonucleases