Project Summary This proposal centers on uncovering intrinsic physical properties of DNA duplex (i.e., DNA shape) and elucidating their roles in CRSIPR Cas9 and Cas12a target discrimination. CRISPR (Clustered-Regularly- Interspaced-Short-Palindromic-Repeats) systems have been adapted into versatile and programmable agents for manipulating nucleic acid targets in a genome-wide fashion, unleashing a revolution in genome editing and manipulation that is still rapidly advancing. CRISPR-based technology is built upon specific recognition of nucleic acids, and a major obstacle hampering its applications in therapeutic settings is the “off-target effect”, in which uncontrolled and undesired actions of CRISPR on aberrant gene targets result in deleterious consequences. Significant improvement of CRISPR specificity is required, and this depends on further in-depth understanding of mechanism of CRISPR target discrimination. Proposed work focuses on Cas9 and Cas12a that are most widely used for engineering DNA genomes. Cas9 and Cas12a both use an effector protein-RNA complex to cleave double-stranded DNAs, and select their cognate targets based on: (i) base-pairing between the RNA guide and a segment of the DNA target-strand designated as protospacer and (ii) a short protospacer-adjacent-motif (PAM) within the target DNA. Ca9/Cas12a target discrimination rely on intrinsic DNA shape, which is determined collectively by the local “core” base-pair(s) and many other factors including (distal) peripheral sequences and topological constraints (e.g., supercoiling). However, current studies on Cas9/Cas12a target selection focus on DNA features at the core segment spanning the PAM and protospacer, and have not yet accounted for impacts of peripheral sequences beyond direct RNA/DNA pairing or DNA topological constraints. We have developed unique site-directed spin labeling methods to obtain sequence-dependent conformation (shape) of free duplexes as well as DNAs bound by proteins including Cas9 and Cas12a. Our recent work shows that DNA sequences not involved in RNA/DNA pairing can modulate Cas9-induced DNA unwinding and cleavage, and the information enables gene editing in cells with short RNA guides that are known to enhance specificity. Building on these findings, we will investigate how DNA peripheral sequences and supercoiling modulate Cas9/Cas12a target discrimination as well as affect the shape of free DNA core segments. Information learned will be incorporated into algorithms for enhancing CRISPR targeting specificity, as well as for predicting three- dimensional DNA shape from the linear one-dimensional sequence in a genome-wide setting. The project will advance understanding on DNA specific recognition that is fundamental to biology, and will contribute to development of the next generation of scientific workforce.