Molecular and cellular characterization of essential human genes.
人类必需基因的分子和细胞特征。
基本信息
- 批准号:10708906
- 负责人:
- 金额:$ 163.13万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 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:
- 关键词:AcuteAllelesAuxinsBar CodesBiologicalBiological ProcessCardiac MyocytesCardiovascular DiseasesCatalogsCell DeathCell Differentiation processCell LineCell LineageCell membraneCellsChemicalsChromatinChronicClustered Regularly Interspaced Short Palindromic RepeatsCodeCodsDataDefectDevelopmentDiseaseDoseEncyclopedia of DNA ElementsEngineeringEssential GenesFoundationsGene ExpressionGenerationsGenesGeneticGerm LayersGoalsHumanHuman DevelopmentHuman GenomeHuman Genome ProjectIn VitroInternationalKnock-inKnock-outKnowledgeLocationMediatingMethodsMitoticModelingMolecularMusOrganoidsPhasePhenotypePlant Growth RegulatorsPluripotent Stem CellsProcessProductionProliferatingProteinsResourcesRoleSmall Interfering RNASystemWestern BlottingWritingcell typeconditional knockoutembryonic stem cellfitnessgenetic approachgenetic manipulationhuman diseasehuman modelinduced pluripotent stem cellinsightinterestknock-downknockout genelive cell imagingmolecular phenotypepluripotencyresearch and developmentresponsescale upscreeningstem
项目摘要
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空
可以模拟早期人类发育的细胞系统中的等位基因和广泛的人类发育的细胞系统中的等位基因。
疾病我们将建立一个数据生产研发中心,以应对
RFA-HG-21-029:细胞中的α等位基因的分子表型(MorPhiC)第1阶段,旨在
为每个人类基因最终建立一个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
能够快速消耗目标蛋白。该方法允许蛋白质的时间控制
水平来研究无效等位基因的后果。我们将利用超灵敏的降解决定子
在低剂量的生长素作用下,
小化学植物激素。我们将结合联合收割机基于CRISPR的靶向基因座工程,
纯合敲入目标基因末端的一个小的生长素诱导降解决定子(mAID),
在开放获取的人诱导多能干细胞中产生零等位基因的化学可控开关
(hiPSC)细胞,其可以分化成各种细胞谱系和多细胞类器官以建模
人类发展和疾病。值得注意的是,每个AIDS降解决定子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
都是共用的
所提出的方法是通用的,并且可以快速地去除由各种不同的蛋白质编码的靶蛋白。
人类基因的种类。我们的战略将是特别有利和关键的研究零
表型的必需基因,不能研究慢性消耗使用遗传方法
(such作为CRISPR KO),因为敲入导致细胞死亡。因此,为了突出
我们的策略是,我们优先通过CRISPR介导的敲入过程创建无效等位基因,
在250个必需基因中条形码化AID降解决定子。我们选择了与人类疾病有关的基因,
国际小鼠表型鉴定协会(IMPC)。我们建议将
细胞表型(存活、增殖、有丝分裂功能和分化)和分子生物学特性
表型,包括基因表达和选择无效等位基因的染色质可及性。这
信息将提供独特的见解,这些发育关键的生物功能,
基因.它将强调建立化学诱导降解决定子系统作为一种生物学方法的实用性。
MorPhiC联盟目标的可推广策略。创建的条形码和条件
无效等位基因资源将提供一个独特的机会来暂时控制无效等位基因的时间,
多能干细胞状态和各种终末分化的细胞类型或多细胞类器官系统
可以从多能干细胞中产生。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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Mazhar Adli其他文献
Mazhar Adli的其他文献
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{{ truncateString('Mazhar Adli', 18)}}的其他基金
Chemo-mediated transcriptional reprogramming in ovarian cancer
卵巢癌中化疗介导的转录重编程
- 批准号:
10346704 - 财政年份:2022
- 资助金额:
$ 163.13万 - 项目类别:
Chemo-mediated transcriptional reprogramming in ovarian cancer
卵巢癌中化疗介导的转录重编程
- 批准号:
10709477 - 财政年份:2022
- 资助金额:
$ 163.13万 - 项目类别:
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:
10517781 - 财政年份:2022
- 资助金额:
$ 163.13万 - 项目类别:
Identifying the Drivers and Targeting Chemo Resistance in Ovarian Cancer
确定卵巢癌的驱动因素并针对化疗耐药性
- 批准号:
10166791 - 财政年份:2017
- 资助金额:
$ 163.13万 - 项目类别:
Identifying the Drivers and Targeting Chemo Resistance in Ovarian Cancer
确定卵巢癌的驱动因素并针对化疗耐药性
- 批准号:
9330488 - 财政年份:2017
- 资助金额:
$ 163.13万 - 项目类别:
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