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

遗传多样性人类基因组中可扩展的敲除和多模式表型中心

• 批准号: 10518021
• 负责人: Danwei Huangfu
• 金额: $ 194.25万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-15 至 2027-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10518021
• 关键词: 3-Dimensional; Address; Affect; Astrocytes; Biological Assay; CRISPR/Cas technology; Catalogs; Cell Line; Cell physiology; Collection; Communities; Complex; Data; Data Set; Diabetes Mellitus; Disease; ES Cell Line; Embryo; Endoderm Cell; Engineered Gene; Engineering; Ensure; Experimental Designs; Feedback; Female; Foundations; Future; Genes; Genetic; Genetic Variation; Genetic study; Germ Layers; Goals; Human; Human Biology; Human Genetics; Human Genome; Investigation; Islets of Langerhans; Joints; Knock-out; Knockout Mice; Link; Metabolic; Metabolic Diseases; Methods; Microglia; Mission; Modeling; Mutagenesis; Neurodegenerative Disorders; Neurodevelopmental Disorder; Neurons; Non-Insulin-Dependent Diabetes Mellitus; Organoids; Penetrance; Phase; Phenotype; Population; Production; Publications; Quality Control; Research; Research Personnel; Resource Sharing; Resources; Societies; System; Testing; Time; Underrepresented Populations; Work; autism spectrum disorder; base; cell repository; data sharing; data standards; disease phenotype; ethnic minority population; experience; genome-wide; human disease; human pluripotent stem cell; induced pluripotent stem cell; islet; knockout gene; large scale production; male; member; multimodality; phenotypic data; programs; racial minority; screening; single-cell RNA sequencing; stem cell differentiation

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.

抽象的 MorPhiC 项目的核心任务是通过 使用多细胞系统创建无效表型的综合目录。基因丢失的影响 对复杂表型的影响受到细胞环境和遗传背景的强烈影响。因此，它 对于在不同的遗传背景中开发可扩展的敲除方法至关重要，然后是稳健的 多细胞系统中的表型分析可为人类生物学提供信息。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、类器官方面的集体专业知识 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划和质量控制来选择 一组约 100 个 hPSC 系，包括大部分诱导多能干细胞 (iPSC) 系和一些胚胎细胞系 来自不同祖先群体以​​及男性和女性的干细胞 (ESC) 系，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 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，以便与 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原肠胚模型，一个定义的神经胶质三 培养系统和 3D 胰岛类器官培养。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，以允许规模化 根据分析的基因和 hPSC 系背景（反映人类遗传 背景）。原代人类胰岛将被纳入多项表型分析中，以测试其普遍性 超越 hPSC 系统。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1号，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资源 来自不同遗传背景的品系，多细胞环境中广泛的敲除表型数据集 提供有关不同人类生物学、强大且可扩展的敲除和表型分析流程的信息 相关的可转移方法，并为 MorPhiC 目录建立强大的用例。优化后的 诱变和表型分析管道以及可扩展的方法将为全面的 MorPhiC 第二阶段的目录制作工作。