Mechanism of RNA-independent cleavage of CRISPR-Cas9

Purpose: 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.