High throughput CRISPR-mediated functional validation of regulatory elements

高通量 CRISPR 介导的调控元件功能验证

• 批准号: 10240102
• 负责人: Bing Ren
• 金额: $ 157.19万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-02-01 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10240102
• 关键词: Address; Biological Process; Biology; Candidate Disease Gene; Cell Line; Chromatin; Clustered Regularly Interspaced Short Palindromic Repeats; DNA Sequence; Data; Disease; Elements; Gene Expression Regulation; Genes; Genetic Variation; Goals; Health; Human; Knowledge; Mammalian Cell; Maps; Mediating; Nucleic Acid Regulatory Sequences; Regulator Genes; Regulatory Element; Reporter; Tissues; Untranslated RNA; Validation; cell type; epigenomics; experimental study; genome editing; interest; mammalian genome; trait

Project Summary The overarching goal of the proposed study is to functionally characterize a large number of candidate functional elements in the mammalian genome. The ENCODE projects have revealed millions of putative regulatory elements across more than one hundred cell types and tissues. While these maps have significantly expanded our knowledge of non-coding sequences, there are still large gaps between having descriptive maps of functional elements and understanding the biology of these elements underlying gene regulation. These include: (a) few candidate functional elements predicted by the ENCODE experiments are functionally validated; (b) Epigenomic studies have not given/revealed information on the target genes of candidate functional elements. Therefore, it is still a challenge to interpret the biological functions of non-coding DNA sequences. To address these issues, the objective of this UM1 application is to perform large scale functional characterization of candidate functional elements in their native chromatin context. We will first identify candidate regulatory elements utilizing ENCODE data and generate reporter tagged genes of interest in cell lines utilizing a high throughput, automated platform. Second, we will interrogate candidate functional elements in their native chromatin contexts utilizing two complementary high throughput CRIPSR/Cas9 mediated genome editing approaches. We anticipate these analyses will significantly advance our knowledge of the biological functions of candidate regulatory regions and gene regulation in mammalian cells.

项目概要 拟议研究的总体目标是从功能上描述大量候选者 哺乳动物基因组中的功能元件。 ENCODE 项目揭示了数百万个假定的 超过一百种细胞类型和组织的调节元件。虽然这些地图显着 扩展了我们对非编码序列的知识，但在描述性图谱之间仍然存在很大差距 功能元件并了解这些元件的基因调控生物学。这些 包括： (a) ENCODE 实验预测的少数候选功能元件在功能上是有效的 已验证； (b) 表观基因组研究尚未给出/揭示候选靶基因的信息 功能元素。因此，解释非编码DNA的生物学功能仍然是一个挑战 序列。为了解决这些问题，该 UM1 应用程序的目标是执行大规模功能 候选功能元件在其天然染色质环境中的表征。我们首先会识别 利用编码数据生成候选调控元件并生成细胞中感兴趣的报告基因标记基因 生产线利用高吞吐量的自动化平台。其次，我们将询问候选功能元素 在其天然染色质环境中利用两个互补的高通量 CRIPSR/Cas9 介导 基因组编辑方法。我们预计这些分析将显着提高我们对 哺乳动物细胞中候选调控区和基因调控的生物学功能。

项目成果

Parallel characterization of cis-regulatory elements for multiple genes using CRISPRpath.

• DOI: 10.1126/sciadv.abi4360
• 发表时间: 2021-09-17
• 期刊: Science advances
• 影响因子: 13.6
• 作者: Ren X;Wang M;Li B;Jamieson K;Zheng L;Jones IR;Li B;Takagi MA;Lee J;Maliskova L;Tam TW;Yu M;Hu R;Lee L;Abnousi A;Li G;Li Y;Hu M;Ren B;Wang W;Shen Y
• 通讯作者: Shen Y