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 proce...