Enhancing the C. elegans animal resource through genome editing

通过基因组编辑增强线虫动物资源

• 批准号: 10453663
• 负责人: Ann E. Rougvie
• 金额: $ 60.52万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10453663
• 关键词: ATP phosphohydrolase; Address; Alleles; Animal Model; Animals; Area; Attention; Behavior; Biology; Biomedical Research; CRISPR/Cas technology; Caenorhabditis; Caenorhabditis elegans; Candidate Disease Gene; Categories; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Collection; Communities; Databases; Deposition; Development; Disease; Dissection; Equilibrium; Event; Funding; G-Protein-Coupled Receptors; Generations; Genes; Genetic; Genome; Goals; Gold; Guide RNA; Hand; Health; Human; Human Biology; Human Genetics; Information Distribution; Institutes; Invertebrates; Ion Channel; Knock-in; Knock-out; Laboratories; Lead; Light; Methods; Microinjections; Modeling; Molecular; Molecular Biology; Molecular Genetics; Monitor; Mutation; Neurons; Oligonucleotides; Orthologous Gene; Pathway interactions; Peptide Hydrolases; Phase; Phenotype; Phosphotransferases; Physiology; Production; Proteins; Proteomics; Quality Control; Reagent; Reproducibility; Research; Research Personnel; Research Project Grants; Research Support; Resources; Scientist; Site; United States National Institutes of Health; Variant; WormBase; animal resource; base; community center; design; gene discovery; gene function; gene interaction; genetic analysis; genome editing; genome resource; human disease; interest; knockout gene; loss of function; loss of function mutation; metabolomics; mutant; programs; screening; transcriptome sequencing; trend; variant of unknown significance; web site

Project Summary/Abstract The objective of this project is to extend the C. elegans genetic toolkit available to researchers by providing publicly available gene knockout (KO) mutations in genes of high value to those interested in human biology and disease. C. elegans is a proven model for discovery of gene function and for embedding genes into functional pathways, many of which were discovered in this transparent animal and are conserved in humans. Loss-of-function mutations are the gold-standard for genetic analysis and allow inferences of gene function and interaction. A relatively recent trend is that large-scale screens (e.g., RNA-seq, proteomics, natural variation studies) generate long candidate gene lists for researchers to sort through and functionally validate, requiring loss of function mutants in many genes to be examined. Having a community-driven resource generate KOs in such genes provides reproducibility and an economy of scale that benefits all. We have developed an efficient and high throughput CRISPR-Cas9 based gene knockout (KO) project to provide a set of precisely edited gene knockout strains to the communities of C. elegans researchers and human geneticists. We seek to continue the production phase of this project, thereby transformatively increasing the efficiency and impact of C. elegans molecular genetics. We employ a highly coordinated three-site production strategy to generate KOs. Each gene edit is carefully confirmed, and validated strains are grossly phenotyped and deposited into the Caenorhabditis Genetics Center (CGC) strain collection along with detailed strain information for distribution to the community. Strains are advertised through both the CGC and WormBase websites. We have generated over 1,000 KOs to date; these KOs have supported a variety of research projects – spanning from mechanistic studies of human variants to metabolomics – funded by at least 14 NIH Institutes and Centers, demonstrating the high impact of our resource. We propose to continue to use our established pipelines to target an additional 2500 C. elegans orthologs of human genes. We will prioritize known or suspected human disease genes, druggable gene classes, as well as understudied genes that are conserved to humans but that have no actionable information. This is a multi-PI project which includes the lead-PIs of the CGC and of WormBase and a subcontractor who maintains multiple community-centered databases, including a CRISPR guide RNA selection site.

项目概要/摘要 该项目的目标是通过提供研究人员可用的线虫遗传工具包 对于那些对人类生物学感兴趣的人来说，公开的基因敲除（KO）突变具有很高的价值 和疾病。线虫是发现基因功能和将基因嵌入到细胞中的经过验证的模型 功能途径，其中许多是在这种透明动物中发现的，并且在人类中是保守的。 功能丧失突变是遗传分析的黄金标准，可以推断基因功能和 相互作用。一个相对较新的趋势是大规模筛选（例如 RNA-seq、蛋白质组学、自然变异） 研究）生成长候选基因列表，供研究人员进行分类和功能验证，需要 许多待检查基因的功能丧失突变体。拥有社区驱动的资源可在以下方面产生 KO 这些基因提供了可复制性和造福所有人的规模经济。我们开发了一种高效的 以及基于高通量CRISPR-Cas9的基因敲除（KO）项目，提供一组精确编辑的基因 向秀丽隐杆线虫研究人员和人类遗传学家社区提供敲除菌株。我们寻求继续 该项目的生产阶段，从而彻底提高线虫的效率和影响 分子遗传学。我们采用高度协调的三站点生产策略来生成 KO。每个 基因编辑经过仔细确认，并对经过验证的菌株进行粗表型分析并存入 秀丽隐杆线虫遗传学中心 (CGC) 收集菌株以及详细的菌株信息，以便分发给 社区。菌株通过 CGC 和 WormBase 网站进行宣传。我们已经生成了 迄今为止已完成超过 1,000 次 KO；这些 KO 支持了各种研究项目——从机械到 代谢组学的人类变异研究——由至少 14 个 NIH 研究所和中心资助，证明 我们资源的巨大影响力。我们建议继续利用我们已建立的管道来瞄准更多 2500 条人类基因的线虫直系同源物。我们将优先考虑已知或可疑的人类疾病基因， 可药物基因类别，以及人类保守但没有得到充分研究的基因 可操作的信息。这是一个多 PI 项目，其中包括 CGC 和 WormBase 的主要 PI 以及 维护多个以社区为中心的数据库（包括 CRISPR 引导 RNA）的分包商 选择网站。