Behrouz Eslami-Mossallam

Delft University of Technology

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Constantijn van der Smagt

Vrije Universiteit Amsterdam

Marco Tompitak

Leiden University

Ilya J. Finkelstein

The University of Texas at Austin

Misha Klein

Delft University of Technology

John A. Hawkins

University of California, Davis

Martin Depken

Delft University of Technology

John van Noort

Huygens Institute for History and Culture of the Netherlands

Koen van der Sanden

Netherlands Organisation for Applied Scientific Research

Stephen K. Jones

Vilnius University

Raoul Schram

Utrecht University

Cooperative Institutions

Delft University of Technology

Leiden University

The University of Texas at Austin

Vrije Universiteit Amsterdam

Lorentz Center

International Institute of Information Technology, Hyderabad

National Institute for Subatomic Physics

Center for Systems Biology

Institut de Biologie Moléculaire et Cellulaire

Columbia University

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Multiplexing Genetic and Nucleosome Positioning Codes: A Computational Approach

PLoS ONE (2016)

Behrouz Eslami-Mossallam Raoul Schram Marco Tompitak John van Noort Helmut Schiessel

Eukaryotic DNA is strongly bent inside fundamental packaging units: the nucleosomes. It is known that their positions are strongly influenced by the mechanical properties of the underlying DNA sequence. Here we discuss the possibility that these mechanical properties and the concomitant nucleosome positions are not just a side product of the given DNA sequence, e.g. that of the genes, but that a mechanical evolution of DNA molecules might have taken place. We first demonstrate the possibility of multiplexing classical and mechanical genetic information using a computational nucleosome model. In a second step we give evidence for genome-wide multiplexing in Saccharomyces cerevisiae and Schizosacharomyces pombe. This suggests that the exact positions of nucleosomes play crucial roles in chromatin function.

10.1371/journal.pone.0156905

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

Nature Communications (2022)

Behrouz Eslami-Mossallam Misha Klein Constantijn van der Smagt Koen van der Sanden Stephen K. Jones

Abstract The S. pyogenes (Sp) Cas9 endonuclease is an important gene-editing tool. Sp Cas9 is directed to target sites based on complementarity to a complexed single-guide RNA (sgRNA). However, Sp Cas9-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 Sp Cas9-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 Sp Cas9; elucidating determinants of CRISPR fidelity; and revealing pathways to increased specificity and efficiency in engineered systems.

Guide RNA

Nuclease

Complementarity (molecular biology)

10.1038/s41467-022-28994-2

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Citations (29)