An RNA-mediated DNA melting mechanism for CRISPR-Cas9
Hedger, G.; Jiang, V.; Ekman, F.; Wang, Q.; Shaw, D. E.
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CRISPR-Cas9 systems, adaptive defense mechanisms in bacteria and archaea, have been widely adopted as powerful gene editing tools, revolutionizing biological and medical research. In the first steps of CRISPR-Cas9 gene editing, the Cas9 protein, in complex with RNA, facilitates DNA melting and subsequent RNA-DNA hybrid formation, but the atomic-level mechanism of this fundamental process is not fully understood. Here, we present the results of long-timescale molecular dynamics simulations in which Cas9-RNA complexes bound to double-helical DNA and promoted the formation of RNA-DNA base pairs in a unidirectional, stepwise manner. Unexpectedly, we observed a direct role for the RNA in facilitating DNA melting events through a mechanism in which RNA bases intercalated within the DNA and promoted strand separation. In addition, breathing motions within the Cas9 DNA-binding cleft contributed to the sequential formation of RNA-DNA base pairs. These simulation results, obtained for two structurally distinct Cas9 proteins, together with supporting experimental work, suggest a novel RNA-dependent mechanism for DNA melting that may be conserved in other Cas proteins.
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