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

摘要 形态程序的核心任务是通过定义每个人类基因的功能 使用多细胞系统创建一个完整的无效表型目录。基因缺失的影响 复杂表型受细胞背景和遗传背景的强烈影响。因此，它 对于在不同的遗传背景下开发可扩展的基因敲除方法至关重要，紧随其后的是强大的 多细胞系统中的表型分析为人类生物学提供了信息。我们的生产中心将 利用我们在人类多能干细胞(HPSC)引导分化方面的集体专业知识，器官 工程学、基因编辑和我们结合大规模CRISPR-Cas9基因敲除的丰富经验 HPSC分化表型。我们计划进行广泛的策划和质量控制，以选择一个 约100个hPSC系，包括大多数诱导多能干细胞(IPSC)系和一些胚胎 干细胞(ESC)系，来自不同的祖先群体，来自男性和女性，以产生hPSC 用于分发的存储库。我们将进一步优先考虑影响神经发育和代谢的基因 在这些不同的hPSC系中进行基因敲除的障碍(例如，自闭症和糖尿病)，以便与 科学界。为了研究基因敲除表型，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原肠模型，一个定义的神经胶质三层. 培养系统和三维胰岛样器培养。使用这些具有不同功能的多细胞系统 复杂程度，然后我们将在多层系统中进行广泛的表型分析，以允许 分析所分析的基因和hPSC系背景(反映人类基因 背景)。原生人类胰岛将被包括在几个表型分析中，以测试泛化能力 在hPSC系统之外。我们希望与联盟合作伙伴合作，为阶段确定目标基因的优先顺序 1.形态工程，制定数据和资源共享标准，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的资源 来自不同遗传背景的品系，多细胞环境中广泛的基因敲除表型数据集 提供了丰富的人类生物学信息、健壮且可扩展的基因敲除和表型鉴定管道以及 相关联的可移植方法，并为形态目录建立强大的用例。经过优化的 突变和表型流水线以及可扩展的方法将为全面 第二阶段的形态目录制作工作。

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.