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. Ultimately, we aim to commercialize NGP software and design services to accelerate CRISPR research.
Public Health Relevance Statement: PROJECT NARRATIVE A new method of editing the human genome called CRISPR has the potential to treat the hundreds of millions of people with a genetic health condition by allowing defective sequences in their DNA to be overwritten with DNA having corrected sequence. New evidence indicates this editing process is more efficient and less toxic if the corrected DNA is first stitched into a compact package using smaller DNA fragments. In this project, we will develop software that automatically determines the set of DNA fragments, which are unique to each editing situation, that are needed to create such compact packages for the purpose of devising improved genome editing therapies.
Project Terms: Acceleration; Affect; inhibitor; Automation; Biological Sciences; Biologic Sciences; Bioscience; Life Sciences; Biomedical Research; Capital; Cell Line; CellLine; Strains Cell Lines; cultured cell line; Cells; Cell Body; DNA; Deoxyribonucleic Acid; Single-Stranded DNA; ssDNA; Engineering; Face; faces; facial; Flow Cytometry; Flow Cytofluorometries; Flow Cytofluorometry; Flow Microfluorimetry; Flow Microfluorometry; flow cytophotometry; gene therapy; DNA Therapy; Gene Transfer Clinical; Genetic Intervention; gene repair therapy; gene-based therapy; genetic therapy; genomic therapy; Genetic Engineering; Genetic Engineering Biotechnology; Genetic Engineering Molecular Biology; Recombinant DNA Technology; genetically engineered; Genome; Human Genome; human whole genome; Goals; Health; Human; Modern Man; In Vitro; Marketing; Methods; Mission; Persons; Nucleic Acids; Oligonucleotides; Oligo; oligos; Legal patent; Patents; Research; Research Personnel; Investigators; Researchers; Science; Computer software; Software; Software Design; Designing computer software; Software Tools; Computer Software Tools; software toolkit; Specificity; Genetic Template; Testing; Vendor; Writing; Measures; Medical Research; improved; Clinical; repair; repaired; Specified; Specific qualifier value; Phase; Variation; Variant; Medical; Licensing; Force Microscopy; Scanning Force Microscopy; Atomic Force Microscopy; Phase 3 Clinical Trials; phase III protocol; Phase III Clinical Trials; Genetic; Tubular formation; Tubular; Shapes; Hour; Protocols documentation; Protocol; Techniques; System; 3-Dimensional; 3-D; 3D; three dimensional; Services; biomaterial compatibility; biocompatibility; Performance; molecular modeling; Molecular Modeling Nucleic Acid Biochemistry; Molecular Modeling Protein/Amino Acid Biochemistry; Molecular Models; Base Pairing; Structure; novel; Positioning Attribute; Position; software development; develop software; developing computer software; Nanotechnology; nano tech; nano technology; nano-technological; nanotech; nanotechnological; Genomics; Effectiveness; nanomedicine; nano medicinal; nano medicine; nanomedicinal; Nanostructures; nano-sized structures; nano-structures; National Institute of General Medical Sciences; NIGMS; Therapeutic Studies; Therapy Research; Small Business Innovation Research Grant; SBIR; Small Business Innovation Research; Process; Modification; Development; developmental; self assembly; designing; design; nano materials; nanomaterials; nano-sized sensors; nanosensing; nanosensors; nanovessel; nanocarrier; scale up; combinatorial; clinical applicability; clinical application; iterative design; commercial application; commercialization; arm; operations; operation; DNA-based therapeutics; therapeutic DNA; CRISPR; CRISPR/Cas system; Clustered Regularly Interspaced Short Palindromic Repeats; Geometry; personalization of treatment; personalized therapy; personalized treatment; personalized medicine; genomic editing; genome editing; CRISPR approach; CRISPR based approach; CRISPR method; CRISPR methodology; CRISPR technique; CRISPR technology; CRISPR tools; CRISPR-CAS-9; CRISPR-based method; CRISPR-based technique; CRISPR-based technology; CRISPR-based tool; CRISPR/CAS approach; CRISPR/Cas method; CRISPR/Cas9; CRISPR/Cas9 technology; Cas nuclease technology; Clustered Regularly Interspaced Short Palindromic Repeats approach; Clustered Regularly Interspaced Short Palindromic Repeats method; Clustered Regularly Interspaced Short Palindromic Repeats methodology; Clustered Regularly Interspaced Short Palindromic Repeats technique; Clustered Regularly Interspaced Short Palindromic Repeats technology; CRISPR/Cas technology; knockin; Knock-in; genetic condition; genetic disorder; Genetic Diseases; gene-editing therapy; genome editing based therapy; genome editing therapy; genome editing treatment; genome editing-based therapeutics; therapeutic editing; therapeutic genome editing; genetic payload; bio-informatics tool; bioinformatics tool; in silico; design, build, test; manufacture
