Enhancing the C. elegans animal resource through genome editing

项目摘要/摘要 该项目的目标是扩展线虫遗传工具包，为研究人员提供 对人类生物学感兴趣的人具有高价值基因的公开基因敲除(KO)突变 和疾病。线虫是发现基因功能和将基因嵌入到 功能通路，其中许多是在这种透明动物中发现的，在人类中是保守的。 功能丧失突变是遗传分析的黄金标准，并允许推断基因功能和 互动。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)生成长长的候选基因列表以供研究人员排序和功能验证，需要 待检测的多个基因功能缺失突变。让社区驱动的资源在以下方面产生KO 这些基因提供了可再生性和规模经济，使所有人都受益。我们已经开发出一种高效的 并以高通量CRISPR-Cas9为基础的基因敲除(KO)项目提供了一套精确编辑的基因 基因敲除菌株对线虫研究人员和人类遗传学家群体的影响。我们寻求继续 该项目的生产阶段，从而革命性地提高了线虫的效率和影响 分子遗传学。我们采用高度协调的三地点生产战略来产生KO。每个人 基因编辑经过仔细确认，验证的菌株被大体上进行表型鉴定并存放到 线虫遗传中心(CGC)的菌株收集以及详细的菌株信息将分发给 社区。毒株通过CGC和WormBase网站进行宣传。我们已经产生了 到目前为止，已有1,000多个KO；这些KO支持了各种研究项目-从机械学到 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，展示 我们的资源所带来的巨大影响。我们建议继续使用我们现有的管道来瞄准额外的 2500个线虫人类基因的同源基因。我们将优先考虑已知或可疑的人类疾病基因， 可用药的基因类别，以及对人类保守但没有研究的基因 可采取行动的信息。这是一个多PI项目，其中包括CGC和WormBase以及 维护多个以社区为中心的数据库的分包商，包括CRISPR指南RNA 选拔地点。

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.