KOMP2-Phase3 Production and Phenotyping by the DTCC Consortium PROJECT SUMMARY/ABSTRACT This application will renew funding for our consortium (DTCC) to continue to participate in Phase3 of the NIH Knockout Mouse Phenotyping Project (KOMP2). DTCC proposes to produce 600 null mutant mouse lines for genes with little to no functional annotation or phenotype by electroporation of Cas9 RNA-guided nucleases into 1-cell stage C57BL/6N zygotes and upload all production data and information to the International Mouse Phenotyping Consortium (IMPC)’s GenTaR database. DTCC will then generate age-matched and sex-balanced cohorts of 600 mutant homozygous or heterozygous mouse lines and wildtype C57BL/6N control mice for juvenile phenotyping in the Early Adult Pipeline and embryo phenotyping of homozygous nonviable mouse lines in the Embryo Pipeline. We will use categorical and continuous tests and procedures standardized by the IMPC and selected by balancing the breadth of phenotyping with a >4% abnormal phenotype hit rate across the IMPC during KOMP-Phase2 to develop an informative multi-domain pipeline. After quality assurance of our processes and quality control of our products, all data, metadata, and images will be available for upload to the Data Coordination Center (DCC). After additional quality control at the DCC, our data will be transferred to the Central Data Archive for statistical analysis and from there to the IMPC web portal for public access. All mutant mouse lines will be available upon request by the research community as live mice while production and phenotyping are in progress and thereafter as frozen germplasm and as live mice after cryorecovery from the Mutant Mouse Resource and Research Center and Canadian Mouse Mutant Resource. Additional biological resources (e.g., fixed tissue) will also be available through the web portal and all DTCC’s standardized operating procedures, expertise, and advice are available by email or videoconference. To continuously adapt and improve high-throughput design, production, and phenotyping, DTCC will conduct technology development projects to increase efficiency of production (e.g., expanding the genome editing toolbox, generating landing- pad alleles) and pilot phenotyping tests (e.g., automated home-cage monitoring, whole-body MRI) to assess their utility, capability, cost-effectiveness, and informative value to the Early Adult and Embryo Pipelines. In addition to prioritizing genes with a human ortholog and no or little functional annotation, DTCC will also actively request the scientific community to nominate genes to produce and phenotype that have likely disease associations. Grant-funded member centers of DTCC include the University of California Davis (UCD; lead institution) and The Center for Phenogenomics (TCP; subcontractor) in Toronto, Canada. We propose to leverage our extensive experience and continue to use and refine well-tested coordinated management, strategy, protocols, and processes established in KOMP2-Phase1 and 2. In KOMP2-Phase2, DTCC is on track to complete all committed deliverables and complete all value-added expected activities (e.g., accelerated CRISPR/Cas9 electroporation of C57BL/6N zygotes, increased production rate of germline mutants, completion of both production and phenotyping pipelines, and finishing of technology development projects). In addition to input from NIH Program Officers, DTCC will be guided by advice and recommendations of local scientific domain experts and the IMPC Panel of Scientific Consultants. DTCC will participate in all KOMP2 and IMPC activities, including monthly videoconferences, extant and ad hoc committees, special interest workgroups and meetings, national and international conferences, and site visits. To maximize the scientific output and impact on biomedical research, DTCC also commits to coordinate its efforts with those production, phenotyping, and technology development efforts at other KOMP2-Phase3 funded centers. By the end of 5 years, DTCC will have contributed genotype-confirmed mouse lines and validated multi-body system phenotyping data for 600 mutant mouse lines on time and within budget to contribute to the global effort to functionally annotate the mammalian genome.

KOMP2-Phase3 Production and Phenotyping by the DTCC Consortium PUBLIC HEALTH RELEVANCE (NARRATIVE STATEMENT) The normal sequence of every gene in the human genome is known. But how do abnormal changes in a gene’s sequence cause or contribute to disease? These changes, called mutations, can either be inherited (like the mutation that causes cystic fibrosis) or acquired during a person's life (like the mutations that cause many types of cancer). If researchers can learn how mutations cause diseases, they can better understand how to prevent them or develop drugs to prevent or treat them. Fortunately, the genes and biology of mice and humans are very similar and mouse genes can be easily mutated. But scientists need strains of mice with a mutation in every gene to explore how any one mutated gene causes disease. This project will create mouse models with single gene mutations for 600 human genes, especially genes with little to no known functional information. We will also analyze them with the same kind of tests used on people like vision tests, blood samples, and medical imaging to identify abnormalities (called ‘phenotypes’) that might be caused by any of the mutated genes. Our project will ask the community which genes to work on, and all the mutated mouse models and phenotype data will be available to any scientist around the world. We will also do technology development to improve how we produce and phenotype mice and share that information with the scientific community. All with a goal to improve human health.