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Dynamics changes of CRISPR-Cas9 system induced by high fidelity mutations

Abstract

CRISPR-Cas9 as a powerful genome editing tool has widely been applied in biological fields. Ever since the discovery of CRISPR-Cas9 as an adaptive immune system, it has been gradually modified to perform precise genome editing in eukaryotic cells by creating double-strand breaks. Though being robust and efficient, current CRISPR-Cas9 system faces a major flaw: the off-target effect, which has not been well understood. Several Cas9 mutants show significant improvement, with very low off-target effect, however relatively lower cleavage efficiency for on-target sequences as well. In this study, the dynamics of the wild-type Cas9 from S. pyogenes and the high fidelity (HF) Cas9 mutant has been explored using molecular dynamics simulations. It turns out that the mutations cause reduction of electrostatic interactions between Cas9 and R-loop. Consequently, the flexibility of the tDNA/sgRNA heteroduplex is reduced, which may explain the reason of less tolerance of mismatches in the heteroduplex region. The mutations also affect the protein dynamics and the correlation networks among Cas9 domains. In mutant Cas9, weakened communications between two catalytic domains, as well as the mutations induced slight opening of the conformation, account for the lower on-target cleavage efficiency, and probably lower off-target as well. These findings would facilitate more precise Cas9 engineering in future.

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Publication details

The article was received on 04 Jul 2018, accepted on 11 Oct 2018 and first published on 11 Oct 2018


Article type: Paper
DOI: 10.1039/C8CP04226H
Citation: Phys. Chem. Chem. Phys., 2018, Accepted Manuscript
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    Dynamics changes of CRISPR-Cas9 system induced by high fidelity mutations

    L. Zheng, J. Shi and Y. Mu, Phys. Chem. Chem. Phys., 2018, Accepted Manuscript , DOI: 10.1039/C8CP04226H

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