Collaborative Research: Mechanisms of RNA-directed activation of a Cas9 nuclease competent for DNA interrogation

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This project will investigate how a complex of an RNA and protein, named CRISPR-Cas9, works as a molecular scissor to change an organism's DNA. CRISPR-Cas9 is natively used by lower organisms such as bacteria to defend against viral infections. The CRISPR-Cas9 system is being adapted as a powerful tool for manipulating genomes of many organisms in a 'by-design and on-demand' fashion. On the horizon are innovations that use CRISPR-Cas9 to produce drought-resistant crops; to combat mosquito transmitted diseases such as malaria; and to cure human illness by fixing an individual's faulty DNA. While studies on CRISPR-Cas9 have advanced rapidly and are poised to revolutionize many fields in biology, details on its mechanism of action remain to be elucidated. This project will fill this knowledge gap by identifying the mechanisms by which RNA molecules convert Cas9's molecular structure into a form suitable for selecting DNA targets. Beyond advancing scientific understanding and methodologies, the project will explore collaborations with biotech companies that may yield new technological applications and promote economic developments. It also includes research training for students at all levels and curriculum innovations such as 'virtual reality' (VR) demonstrations of the Cas9-RNA complex, thus contributing to the development of STEM (Science, Technology, Engineering and Mathematics) workforce.The overall objective of the project is to elucidate the roles of specific domains of Cas9 and guide RNA in initiating and directing a cascade of conformational changes towards the state ready for DNA interrogation. The proposed work will leverage on the expertise of the PI in the functional and structural (X-ray crystallography) characterization of protein-nucleic acid complexes; and that of the Co-PI in site directed spin labeling studies of nucleic acids and protein-nucleic acid complexes. Experiments will focus on Streptococcus pyogenes Cas9, the workhorse in genome engineering, and test an overarching hypothesis that the interactions of the bridge-helix of Cas9 with specific RNA elements, initiate and induce Cas9 domain rearrangements to assemble a DNA-interrogative protein-RNA complex. Specific Aims are: Dissect the role of the bridge helix in initiating conformational changes in Cas9 (Aim 1); Identify the protein and RNA elements that displaces the nuclease lobe (Aim 2) and a helical domain (HD-III, Aim 3) in response to the bridge-helix conformational change. The results from the three aims will identify the sequential steps in the conformational cascade and will be widely applicable to similar CRISPR-Cas systems.