Pharmaceutical Sciences
Permanent URI for this collectionhttps://hdl.handle.net/20.500.12503/29938
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Browsing Pharmaceutical Sciences by Author "Chen, Xiongping"
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Item Characterize the Pre-catalytic State of CRISPR/Cas9(2020) Liu, Jin; Chen, XiongpingPurpose: CRISPR-Cas9 has been widely used as a gene-editing tool, but its catalytic mechanism remains elusive. Cas9 has two catalytic domains, HNH domain and RuvC domain. In our previous study, we have successfully identified the pre-catalytic state of the HNH domain. The purpose of this study is to identify the pre-catalytic state of the RuvC domain, which may provide an understanding of the Cas9 catalytic mechanism and strategies for Cas9 engineering. Methods: Here we use molecular dynamic simulations to discover the pre-catalytic state of Cas9. The initial structure was obtained from our previous simulations, where the HNH domain was in the pre-catalytic state. We placed two Mg2+ ions and non-target DNA strand at the RuvC domain and performed molecular dynamics simulations to capture the pre-catalytic state of the RuvC domain. Results: Our molecular dynamics simulations revealed a pre-catalytic state of Cas9 that both target DNA strand and non-target DNA strand are posed to be catalyzed by Cas9. Conclusions: In this study, we identified the first atomic-level structure of CRISPR/Cas9 with the pre-catalytic state in both catalytic domains.Item Mechanism of RNA-independent cleavage of CRISPR-Cas9(2020) Liu, Jin; Chen, Xiongping; Hayatshahi, Sayyed; Wang, Duen-ShianPurpose: CRISPR(Clustered-Regularly-interspaced-short-palindromic-repeats)-Cas9 (CRISPR-associated-protein-9) uncovered a new path toward gene therapy. However, non-specific cleavage of Cas9 raises concerns on human therapeutic applications so that it is critical to understand and minimize those non-specifics cuttings. Recent in vitro studies showed that Cas9 cleavage occurred even without the guidance from the guide RNA (gRNA) in the presence of Mn2+, implying the serious issue of off-target effect of Cas9. The purpose of this study is to elucidate the mechanism of the RNA-independent cleavage of CRISPR-Cas9, which may provide insights for the improvement of Cas9 specificity. Method: Based on our previously captured structure of catalytically-active Cas9-gRNA-dsDNA complex, we performed molecular dynamic (MD) simulations on Cas9 complexed with and without gRNA, respectively. We also compared the simulations in the presence of Mn2+vs. Mg2+. All MD simulations were performed using AMBER package with GPU acceleration. Result: We expect our MD simulations to demonstrate the different coordination environments of Mn2+ and Mg2+ in the presence or absence of gRNA, elucidating a novel mechanism for Cas9 off-target effects. Conclusion: In this study, we expect to identify the mechanism of RNA-independent cleavage of CRISPR-Cas9, shedding light on the development of new Cas9 variants to reduce off-target effects.