Center for scalable knockout and multimodal phenotyping in genetically diverse human genomes

摘要 MorPhiC项目的核心使命是通过基因组学来定义每个人类基因的功能。 使用多细胞系统创建空表型的综合目录。基因缺失的影响 细胞环境和遗传背景对复杂表型的影响很大。因此 对于在不同的遗传背景中开发可扩展的敲除方法， 多细胞系统中的表型分析是人类生物学的信息。我们的生产中心将 利用我们在人类多能干细胞（hPSC）引导分化、类器官 工程，基因编辑，以及我们结合大规模CRISPR-Cas9敲除的丰富经验 hPSC分化的表型分析。我们计划进行广泛的策展和质量控制， 一组约100个hPSC细胞系，主要包括诱导多能干细胞（iPSC）细胞系和一些胚胎干细胞系。 干细胞（ESC）系，来自不同的祖先群体，以及来自男性和女性，以产生hPSC repository for distribution.我们将进一步优先考虑影响神经发育和代谢的基因， 疾病（例如，自闭症和糖尿病），用于在这些不同的hPSC系中进行敲除， 科学界。为了研究敲除表型，我们将优化三种不同的多细胞 系统，一个基于微模式的早期三胚层分化的胃样模型，一个定义的神经胶质三胚层分化模型， 培养系统和3D胰岛样类器官培养。利用这些多细胞系统， 水平的复杂性，我们将进行广泛的表型分析，在一个多层系统，以允许规模 根据所分析的基因和hPSC系背景（反映了人类遗传特性）进行分析。 背景）。将纳入原代人类胰岛进行几项表型分析以测试普遍性 hPSC系统之外。我们希望与联盟伙伴合作，优先考虑阶段的目标基因 MorPhiC项目的第一部分，制定数据和资源共享的标准，并优化联合方法， 分析。我们的生产中心有望提供丰富的敲除人类多能干细胞资源 来自不同遗传背景的细胞系，多细胞环境中广泛的敲除表型数据集， 提供多种人类生物学信息，强大且可扩展的敲除和表型分析管道沿着 相关的可转移方法，并为MorPhiC目录建立强大的用例。优化 诱变和表型分析管道沿着可扩展的方法将为全面的 MorPhiC目录生产工作在第2阶段。

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.