CRISPR-Cas9: Computational Insights Toward Improved Genome Editing

Abstract

Life sciences are undergoing a transformative phase due to an emerging genome-editing technology based on the RNA-programmable CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9) system. In this system, the endonuclease Cas9 associates with a guide RNA to match and cleave complementary sequences in double stranded DNA, forming an RNA:DNA hybrid and a displaced non-target DNA strand. Although extensive structural studies are ongoing, the conformational dynamics of Cas9 and its interplay with the nucleic acids during association and DNA cleavage are largely unclear. This missing aspect hampers the precise structure-based design of CRISPR-Cas9 genome-editing tools with improved specificity. Here, we report the first biophysical study – based on extensive multi-microseconds molecular simulations integrated with structural data – revealing the conformational plasticity of Cas9 and identifying the key determinants that allow its large-scale conformational changes during nucleic acid binding and processing. We identify a remarkable conformational plasticity as an intrinsic property of the nuclease HNH domain, being a necessary factor allowing for the HNH domain repositioning during catalysis. More importantly, we disclose a key role of the non-target DNA during the process of activation of the HNH domain, showing how the non-target DNA positioning triggers local conformational changes that favor the formation of a catalytically competent Cas9. Our outcomes further suggest new and precise protein-engineering modifications, which are of fundamental importance for the rational design of more effective genome-editing tools. Overall, these novel findings constitute a reference for future experimental studies aimed at a full characterization of the dynamic features and at the improvement of biological applications of the CRISPR-Cas9 system.