Title
A kinetic model predicts SpCas9 activity, improves off-target classification, and reveals the physical basis of targeting fidelity
Author
Eslami-Mossallam, B.
Klein, M.
Smagt, C.V.D.
Sanden, K.V.D.
Jones Jr, S.K.
Hawkins, J.A.
Finkelstein, I.J.
Depken, M.
Publication year
2022
Abstract
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 char acterize 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. Chemicals / CAS endonuclease, 9055-11-2; CRISPR-Associated Protein 9; Endonucleases; RNA, Guide.
Subject
Benchmarking
Chemical binding
Classification
Experimental study
RNA
Buildings and Infrastructures
2015 Urbanisation
To reference this document use:
http://resolver.tudelft.nl/uuid:3ac193ac-484d-4277-9fad-82206add08a2
DOI
https://doi.org/10.1038/s41467-022-28994-2
TNO identifier
967440
Publisher
Nature Research
ISSN
2041-1723
Source
Nature Communications, 13 (13), 1-10
Document type
article