Molecular and cellular characterization of essential human genes.

人类必需基因的分子和细胞特征。

• 批准号: 10708906
• 负责人: Mazhar Adli
• 金额: $ 163.13万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-22 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10708906
• 关键词: Acute; Alleles; Auxins; Bar Codes; Biological; Biological Process; Cardiac Myocytes; Cardiovascular Diseases; Catalogs; Cell Death; Cell Differentiation process; Cell Line; Cell Lineage; Cell membrane; Cells; Chemicals; Chromatin; Chronic; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Cods; Data; Defect; Development; Disease; Dose; Encyclopedia of DNA Elements; Engineering; Essential Genes; Foundations; Gene Expression; Generations; Genes; Genetic; Germ Layers; Goals; Human; Human Development; Human Genome; Human Genome Project; In Vitro; International; Knock-in; Knock-out; Knowledge; Location; Mediating; Methods; Mitotic; Modeling; Molecular; Mus; Organoids; Phase; Phenotype; Plant Growth Regulators; Pluripotent Stem Cells; Process; Production; Proliferating; Proteins; Resources; Role; Small Interfering RNA; System; Western Blotting; Writing; cell type; conditional knockout; embryonic stem cell; fitness; genetic approach; genetic manipulation; human disease; human model; induced pluripotent stem cell; insight; interest; knock-down; knockout gene; live cell imaging; molecular phenotype; pluripotency; research and development; response; scale up; screening; stem

We propose to generate barcoded and conditional null alleles in a cellular system that can model early human development and a broad range of human diseases. We will establish a data production research and development center in response to the RFA-HG-21-029: Molecular Phenotypes of Null Alleles in Cells (MorPhiC) Phase 1, which aims to establish a catalog of molecular and cellular phenotypes of null alleles for ultimately every human gene, using in vitro multicellular systems. Our center will utilize a chemically inducible and reversible system that enables the rapid depletion of target proteins. The approach permits temporal control of protein levels to study the consequences of null alleles. We will utilize a super sensitive degron that rapidly degrades the target protein of interest in response to a low dose of auxin, a cell membrane diffusible small chemical plant hormone. We will combine CRISPR-based targeted locus engineering to homozygous knock-in a mini auxin-inducible degron (mAID) at the end of the targe gene to create a chemically controllable switch to create null-alleles in an open-access human induced pluripotent stem (hiPSC) cell, which can be differentiated into various cell lineages and multicellular organoids to model human development and diseases. Notably, each AID-degron will also contain gene-specific barcodes, allowing tracking the fate of hundreds of thousands of null alleles when these engineered null alleles are pooled. The proposed approach is generalizable and can rapidly deplete target proteins codded by various classes of human genes. Our strategy will be particularly advantageous and critical to study the null phenotypes of essential genes, which cannot be studied by chronic depletion using genetic approaches (such as CRISPR KO) because the knock-in results in cell death. Therefore, to highlight the utility of our strategy, we prioritize creating null alleles by CRISPR mediated knock-in process to introduce barcoded AID degron in 250 essential genes. We chose genes implicated in human diseases and subviable phenotypes in the International Mouse Phenotyping Consortium (IMPC). We propose to catalog the cellular phenotypes (survival, proliferation, mitotic function, and differentiation) and molecular phonotypes, including gene expression and chromatin accessibility for select null alleles. This information will provide unique insights into the biological function of these developmentally critical genes. It will highlight the utility of establishing the chemically inducible degron system as a generalizable strategy for the goals of the MorPhiC consortium. The created barcoded and conditional null allele resource will provide a unique opportunity to temporally control the timing of null alleles in pluripotent stem state and various terminally differentiated cell types or multicellular organoid systems that can be generated from the pluripotent stem cells.

我们建议生成条形码和条件 null 细胞系统中的等位基因可以模拟早期人类发育和广泛的人类 疾病。我们将建立数据生产研发中心以应对 RFA-HG-21-029：细胞中无效等位基因的分子表型 (MorPhiC) 第 1 阶段，旨在 最终为每个人类基因建立无效等位基因的分子和细胞表型目录， 使用体外多细胞系统。我们的中心将利用化学诱导和可逆系统 从而能够快速消耗靶蛋白。该方法允许对蛋白质进行时间控制 水平来研究无效等位基因的后果。我们将利用超敏感的降解决定子，快速 响应低剂量的生长素（一种细胞膜扩散性），降解目标蛋白 小化工厂激素。我们将结合基于 CRISPR 的靶向基因座工程 在目标基因末端纯合敲入迷你生长素诱导降解决定子（mAID），以创建 化学可控开关在开放获取的人类诱导多能干中产生无效等位基因 (hiPSC) 细胞，可以分化成各种细胞谱系和多细胞类器官来建模 人类发展与疾病。值得注意的是，每个 AID-degron 还将包含基因特异性条形码， 当这些工程化的无效等位基因时，允许跟踪数十万个无效等位基因的命运 被汇集起来。 所提出的方法具有通用性，可以快速耗尽各种编码的靶蛋白 人类基因的类别。我们的策略对于研究零值将特别有利和关键 必需基因的表型，不能通过使用遗传方法进行慢性消耗来研究 （例如 CRISPR KO），因为敲入会导致细胞死亡。因此，为了突出实用性 我们的策略是，我们优先通过 CRISPR 介导的基因敲入过程创建无效等位基因，以引入 250 个必需基因中的 AID 降解决定子带有条形码。我们选择与人类疾病有关且可存活的基因 国际小鼠表型联盟 (IMPC) 中的表型。我们建议编目 细胞表型（存活、增殖、有丝分裂功能和分化）和分子 表型，包括所选无效等位基因的基因表达和染色质可及性。这 信息将为这些发育关键的生物功能提供独特的见解 基因。它将强调建立化学诱导降解决定子系统作为 MorPhiC 联盟目标的通用战略。创建的条形码和条件 空等位基因资源将提供一个独特的机会来暂时控制空等位基因的时间 多能干状态和各种终末分化细胞类型或多细胞类器官系统 可以由多能干细胞产生。