Functional assessment of enhancer-gene interactions in vivo
体内增强子-基因相互作用的功能评估
基本信息
- 批准号:10331859
- 负责人:
- 金额:$ 24.17万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2020
- 资助国家:美国
- 起止时间:2020-04-07 至 2024-01-31
- 项目状态:已结题
- 来源:
- 关键词:3-DimensionalAddressAdoptedAffectArchitectureAreaBiological ModelsBiologyCRISPR/Cas technologyCategoriesCellsChromatin StructureCodeDNADevelopmentDiseaseDistalDistantEducational workshopElementsEmbryoEnhancersEnsureEnvironmentEvolutionFacultyGene ExpressionGene Expression RegulationGene TargetingGenesGenetic TranscriptionGenomeGenomicsGoalsHeart DiseasesHumanHuman GenomeHuman Genome ProjectKnock-inKnock-in MouseKnock-outLeadershipLimb DevelopmentLimb structureLinkLocationMalignant NeoplasmsMapsMediatingMentorsMentorshipMethodsMusMutationOrganismPatternPhasePhenotypePlayPositioning AttributeProteinsRegulationResearchResearch ProposalsResearch TrainingResolutionRodentRoleSHH geneSecureSeriesSpecificityTechnologyTestingTissuesTrainingUntranslated RNAWorkcareer developmentcohortembryo tissueexperimental studygene interactiongenome analysisgenome editinggenome-widegenome-wide analysisgenomic locushuman diseasein vivoinsightmammalian genomenovelpromoterresponseskillstechnology developmenttranscriptome sequencing
项目摘要
PROJECT SUMMARY
Transcriptional enhancers are a predominant category of functional elements in the non-coding portion of the
human genome, far outnumbering the ~20,000 protein-coding genes. Mutations affecting enhancers have
been implicated in human disease, and comprehensively understanding the genome-wide architecture and
function of enhancers remains a major unsolved challenge arising from The Human Genome Project. Despite
substantial progress in mapping of these elements (e.g., by the ENCODE consortium), the in vivo target genes
of enhancers are generally unknown, and the mechanisms of their long range regulation during development
are not well explored. Recently, I developed a novel method that allows manipulation of enhancers at their
endogenous genomic location in mice using CRISPR/Cas9 genome editing (Kvon et al., Cell, 2016). In this
application, I propose to better understand the mechanisms of gene regulation by distant-acting enhancers
through in vivo mouse studies, exploiting this highly efficient CRISPR/Cas9 genome editing technology to
create enhancer knock-out and knock-in mice and employing novel methods to map enhancer-promoter
interactions. I will address the following questions regarding distal enhancer function in the genome: 1) Which
genes do different classes of enhancers regulate? 2) Is there enhancer-promoter specificity for distant-acting
enhancers? and 3) What are the consequences of enhancer loss or replacement on an organism's function?
Mentored phase: First, I propose to adapt CRISPR/Cas9 genome editing for studying long-range enhancer-
gene interactions in vivo using mouse embryonic limb as a model system. I will create a series of enhancer
knock-outs to identify their target gene(s) and a series of enhancer knock-ins to study enhancer-promoter
specificity. Second, I will adopt and optimize the Capture-C technology to identify interaction partners of
enhancers directly in mouse tissues. Independent phase: I will use methods developed in the mentored
phase to systematically map target genes for important developmental enhancers in vivo and to gain a detailed
understanding of the mechanisms governing long-range enhancer-promoter interactions on a genomic scale. I
will also use elucidated enhancer-promoter interactions to study basic principles of long-range enhancer
regulation in the mammalian genome using CRISPR/Cas9 technology. This will enable me to develop several
long-term research directions, focused on the role of enhancer-gene interactions in human evolution, disease,
and development. The main areas of research training will include: 1) Further advancing the use of
CRISPR/Cas9 in mice, 2) Capture-C technology development in mouse tissues, and 3) computational genome
analysis. My mentor (Dr. Len Pennacchio) and co-mentor (Dr. Axel Visel) are leaders in these fields. My career
development activities will focus on skills in key areas of my research, attending courses and workshops,
developing leadership and mentorship skills, and securing a faculty position. To ensure progress in my goals I
have also established a scientific advisory board consisting of my mentors, and Drs. D. Dickel, and E. Rubin.
项目总结
转录增强子是转录因子非编码部分的主要功能元件。
人类基因组,远远超过约20,000个蛋白质编码基因。影响增强剂的突变有
与人类疾病有牵连,并全面了解全基因组结构和
增强子的功能仍然是人类基因组计划中一个尚未解决的重大挑战。尽管
在定位这些元件(例如,由ENCODE联盟)、体内靶基因方面取得了实质性进展
增强剂的作用机制以及它们在发育过程中的长程调控机制通常是未知的
都没有得到很好的探索。最近,我开发了一种新的方法,允许在他们的
使用CRISPR/Cas9基因组编辑对小鼠进行内源性基因组定位(Kvon等人,Cell,2016)。在这
应用,我建议更好地理解远程作用增强子的基因调控机制。
通过活体小鼠研究,利用这种高效的CRISPR/Cas9基因组编辑技术
创建增强子敲除和敲入小鼠,并使用新方法映射增强子-启动子
互动。我将回答关于基因组远端增强子功能的以下问题:1)哪一个
不同类别的增强子调控基因?2)是否存在远距离作用的增强-启动子专一性?
增强剂?3)增强子丢失或替换对生物体功能有何影响?
指导阶段:首先,我建议采用CRISPR/Cas9基因组编辑来研究远程增强子-
以小鼠胚胎肢体为模型系统进行体内基因互作研究。我会制作一系列的增强剂
敲除以确定其目的基因(S)和一系列增强子敲入以研究增强子-启动子
专一性。其次,我将采用并优化Capture-C技术来识别
增强剂直接在小鼠组织中。独立阶段:我将使用在指导中开发的方法
在体内系统定位重要发育促进剂的靶基因并获得详细的
在基因组水平上理解长程增强子-启动子相互作用的机制。我
还将使用已阐明的增强子-启动子相互作用来研究长程增强子的基本原理
使用CRISPR/Cas9技术对哺乳动物基因组进行调控。这将使我能够开发出几个
长期研究方向,重点是增强子-基因相互作用在人类进化、疾病、
和发展。研究培训的主要领域将包括:1)进一步推动使用
小鼠的CRISPR/Cas9,2)小鼠组织中的Capture-C技术的发展,以及3)计算基因组
分析。我的导师(Len Pennacchio博士)和共同导师(Axel Visel博士)是这些领域的领导者。我的职业生涯
发展活动将侧重于我研究的关键领域的技能,参加课程和研讨会,
培养领导和指导技能,并确保获得教职。为了确保我的目标取得进展,我
我还建立了一个由我的导师、D.Dickel博士和E.Rubin博士组成的科学顾问委员会。
项目成果
期刊论文数量(0)
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会议论文数量(0)
专利数量(0)
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Evgeny Kvon其他文献
Evgeny Kvon的其他文献
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{{ truncateString('Evgeny Kvon', 18)}}的其他基金
Deciphering the mechanism of long-range gene regulation in vivo
破译体内长程基因调控机制
- 批准号:
10473041 - 财政年份:2022
- 资助金额:
$ 24.17万 - 项目类别:
Functional assessment of enhancer-gene interactions in vivo
体内增强子-基因相互作用的功能评估
- 批准号:
9545036 - 财政年份:2017
- 资助金额:
$ 24.17万 - 项目类别:
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