ABSTRACT The core mission of the MorPhiC program is to define the function of every human gene through the creation of a comprehensive catalog of null phenotypes using multicellular systems. The impact of gene loss on complex phenotypes is strongly influenced by the cellular context and the genetic background. Therefore, it is essential to develop scalable knockout methods in diverse genetic backgrounds followed by robust phenotyping assays in multicellular systems that are informative of human biology. Our Production Center will leverage our collective expertise in human pluripotent stem cell (hPSC) guided differentiation, organoid engineering, gene editing, and our extensive experience combining large-scale CRISPR-Cas9 knockout phenotyping with hPSC differentiation. We plan to conduct extensive curation and quality control to select a panel of ~100 hPSC lines, including mostly induced pluripotent stem cell (iPSC) lines and some embryonic stem cell (ESC) lines, from diverse ancestral populations, and from males and females to generate an hPSC repository for distribution. We will further prioritize genes affected in neurodevelopmental and metabolic disorders (e.g., autism and diabetes) for conducting knockouts in these diverse hPSC lines for sharing with the scientific community. For investigation of knockout phenotypes, we will optimize three distinct multicellular systems, a micropattern-based gastruloid model for early tri-germ-layer differentiation, a defined neuro-glial tri- culture system, and a 3D pancreatic islet-like organoid culture. Using these multicellular systems with different levels of complexity, we will then conduct extensive phenotyping assays in a multitiered system to allow scaled analysis both in terms of the genes analyzed and the hPSC line background (reflective of the human genetic background). Primary human islets will be included for several phenotyping assays to test the generalizability beyond the hPSC systems. We expect to work with consortium partners to prioritize the target genes for Phase 1 of the MorPhiC project, develop standards for data and resource sharing, and optimize methods for joint analyses. Our Production Center is expected to deliver a rich resource of knockout human pluripotent stem cell lines from diverse genetic backgrounds, extensive knockout phenotyping datasets in multicellular contexts that are informative of diverse human biology, robust and scalable knockout and phenotyping pipelines along with associated transferable methods, and establish strong use cases for the MorPhiC catalog. The optimized mutagenesis and phenotyping pipelines along with the scalable methods will pave the way for a full-scale MorPhiC catalog production effort in Phase 2.

PROJECT NARRATIVE (RELEVANCE) The impact of gene loss on complex phenotypes is strongly influenced by the cellular context and the genetic background. Working with consortium partners, we will prioritize the target genes for Phase 1 of the MorPhiC project, develop standards for data and resource sharing, and optimize methods for joint analyses. Our Production Center is expected to deliver a rich resource of knockout human pluripotent stem cell lines from diverse genetic backgrounds, extensive knockout phenotyping datasets in multicellular contexts that are informative of diverse human biology, robust and scalable knockout and phenotyping pipelines along with associated transferable methods and establish strong use cases for the MorPhiC catalog.