The groundbreaking 2013 discovery that CRISPR-Cas9 can be programmed to efficiently edit genomes in complex organisms sparked a revolution in genetic engineering that is still rapidly unfolding. This project will test a hypothesis on how CRISPR-Cas9 discriminates between correct vs. incorrect DNA targets, and advance the capability of a biophysical technique known as site-directed spin labeling to the level of interrogating biomolecules one at a time under physiological conditions. The research is expected to yield mechanistic understanding that will aid further development of genome editing tools, and to advance technology that can benefit the broader scientific community. The project will also contribute to training of the STEM workforce via research engagement and curriculum development at both graduate and undergraduate levels, and will support outreach to the public on issues related to genome editing that have profound societal impacts.
Clustered-Regularly-Interspaced-Short-Palindromic-Repeats (CRISPR) and CRISPR-associated (Cas) proteins provide adaptive immunity for bacteria and archaea. In type II CRISPR, a single Cas9 protein is activated by small RNA(s) to cleave DNA duplexes at specific sites. A key step in Cas9 target acquisition is unwinding of the DNA duplex to form a stable R-loop structure in which the RNA guide-segment is base-paired with the target-strand of the DNA protospacer. Studies have established a sequential unwinding model for Cas9 target acquisition, and these mechanistic insights have been instrumental in driving developments of CRISPR-based technology. However, understanding of mechanisms of Cas9 function is far from complete. In this project, Aim 1 will map the boundary of Cas9-induced DNA unwinding in bulk solution using a combination of spin-labeling and fluorescence spectroscopy; Aim 2 will examine the interdependency between Cas9 target cleavage and variations in the boundary of DNA unwinding; and Aim 3 will develop spin-labeling methods for investigating Cas9-induced DNA unwinding at the level of single molecules. The work will advance fundamental understanding of CRISPR-Cas9 function by testing a "two-state equilibrium" hypothesis on target discrimination, and will yield new biophysical techniques that broadly impact studies on nucleic acids and protein-nucleic acid complexes.
This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.
