PROJECT SUMMARY More than 300 million people worldwide are affected by a genetic health condition. Over 4,400 genetic diseases have been identified; nearly all of which are considered rare, which limits the amount of research each receives. Gene therapy is an attractive approach for treatment of genetic disease because of its broad applicability. CRISPR-Cas9 genome editing systems (CRISPR) have revolutionized gene therapy research and other fields of life science, however, no CRISPR-based treatments have reached the market and clinical application still faces important challenges. In this project, we aim to develop design automation software to help improve how genetic donor templates are packaged for genomic integration via CRISPR, thereby increasing CRISPR editing efficiency. Whereas such templates are usually delivered as unstructured (linear) single-stranded DNA, recent studies indicate genome integration efficiency is significantly improved when templates are folded into compact shapes using techniques from DNA nanotechnology. Such nanostructured genetic payloads (NGPs) for CRISPR have the potential to become an essential component of genetic therapy and personalized medicine. The long-term goal of the project is to provide researchers with software for designing more effective CRISPR treatments to improve the lives of people with genetic health problems. Our solution will also advance other application domains where DNA nanotechnology is being employed, such as nanomedicine, nanosensing and biocompatible nanomaterials, thereby supporting the mission of the National Institute of General Medical Sciences (NIGMS): improving the effectiveness of computational approaches in biomedical research. Academic software exists to facilitate design of DNA nanostructures, however, these applications either require extensive expertise or are limited to 3D wireframe designs. Design of a novel DNA nanostructure of modest complexity that is not among a small set of simple designs can require hundreds of hours of expert labor. Moreover, because NGPs are new to science, no software currently exists to automatically generate DNA nanostructures for a given set of NGP design parameters. In Aim 1 of this project, we will employ an iterative design-build-test development cycle we have used to bring other software products to market to develop novel parametric design software (PDS) able to create NGPs automatically for a given genetic template and set of design parameters. In Aim 2, we will simulate and synthesize eight NGPs and characterize them via molecular modeling and atomic force microscopy to confirm they meet design specifications. We will then test these NGPs for CRISPR editing efficiency against unstructured payload controls in vitro. In Phase II, we will enhance the PDS and use it to explore the vast space of NGP designs for those that optimize CRISPR performance via combinatorial testing across multiple cell lines, templates and insertion targets. Ultimat...