Automated Forward Genetic Analysis of Adaptive Immunity

适应性免疫的自动正向遗传分析

基本信息

批准号：
10209864
负责人：
BRUCE A BEUTLER
金额：
$ 196.36万
依托单位：
UT SOUTHWESTERN MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-06-20 至 2026-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10209864
关键词：
Affect Alleles Amino Acids Antibody Response B-Lymphocytes Binding Biological Assay CRISPR/Cas technology Candidate Disease Gene Categories Cell physiology Cells Code Computer software Data Databases Defect Development Elements Ethylnitrosourea Etiology Fingerprint Flow Cytometry Funding Genes Genetic Screening Genetic Transcription Genetic study Genome Germ-Line Mutation Immune Immune System Diseases Immune system Immunity Immunization Immunoglobulin M Immunologics Immunology Individual Induced Mutation Knock-out Knowledge Laboratories Link Lymphocyte Lymphoid Measurement Measures Meiosis Messenger RNA Metabolism Methods Molecular Mus Mutagenesis Mutant Strains Mice Mutate Mutation Names Nonsense-Mediated Decay Paper Pathway interactions Patients Phenotype Proteins Publications Publishing RNA Processing RNA Splicing Recurrence Regulation Research Personnel Role Signal Transduction Signaling Protein Source Supporting Cell Surveys Testing Time Transducers Transfer RNA Translations Update Validation Vesicle Work adaptive immune response adaptive immunity causal variant computerized tools cytokine gene discovery gene product genetic analysis genetic pedigree immune function improved insight interest loss of function mutation mutation screening novel phenotypic data protein complex protein folding receptor response screening tool trafficking vesicle transport

项目摘要

PROJECT SUMMARY During the past four years, we have made outstanding progress in mutagenizing the mouse germline genome while keeping immunity under close surveillance. Now four years into the five-year project, we have thoroughly examined viable hypomorphic mutations in more than half of all protein-encoding genes. We have declared with high confidence that 1,298 mutations in 638 genes are causative of phenotypes in FACS and/or antibody response screens. Many of these genes were novel in that their necessity for immune function had been unknown, and were re-targeted by CRISPR/Cas9 to verify causation. 154 of the 638 candidate genes (24%; almost all of them novel) were either knocked out or modified with the ENU allele using CRISPR/Cas9 editing, expanded into pedigrees, and retested for causation of phenotypes detected in screening and automated mapping. 148 of the 154 genes (96%) were verified as the source of phenotype(s) declared. An additional 156 CRISPR/Cas9 projects are active at this time, some close to completion. Some of our discoveries have been published; many more are works in progress. However, all results of FACS assays on all mutations, whether declared causative of phenotype or not, have been de-restricted for public viewing on Mutagenetix, together with tools that enable search, examination of the original phenotypes and meiotic mapping data, filtering by P-value, and direction and magnitude of individual phenotypic effects. This will enable other laboratories to pursue mechanisms of immunological phenotypes alongside us. Knowledge of genes with non-redundant function in the development and activation of adaptive immune responses is fundamental to immunology and we plan to pursue screening further. We also plan deeper studies of the mechanism(s) behind phenotypes of particular interest. Of the phenotypes named for study in our earlier proposal, we have come to understand those caused by mutations in Trp53bp1, Ampd3, Rnps1, Prkd2, and Snrnp40, and have published papers describing mechanism. Additional phenotypes (not named in the original proposal) caused by mutations in Rabl3, Gpr89, Pdia6, Ncstn, Lmbr1l, Stk4, Pacs1, Wdr37, and Mfsd1 have also been elucidated and published or submitted for publication. We now propose to examine newly verified phenotypes, all the while creating novel phenotypes for study by ourselves and others. Our work is now guided by a tool (Similarity Heatmap) that measures relatedness of phenotypes. In flow cytometry screening, a minimum of 34 measurements are made from each mouse. The results constitute a phenotypic “fingerprint” amenable to tests of statistical similarity. Mutations in some genes yield results very similar to mutations in other genes, and we can sometimes infer that multiple genes operate within a single complex of proteins or enzymatic pathway. We have also written software (Candidate Explorer) to evaluate phenotypes in advance of declaring causation, telling us the likelihood of validation should we attempt to re-target any gene in question. We restrict our efforts to the most likely novel candidates, confident that all true causative relationships will ultimately manifest as saturation advances.

项目概要在过去的四年里，我们在小鼠种系基因组诱变方面取得了显着进展在密切监视免疫力的同时，五年项目已经进行了四年，我们已经彻底做到了这一点。我们已经宣布，我们已经对一半以上的蛋白质编码基因进行了可行的亚等位突变检查。高可信度表明 638 个基因中的 1,298 个突变是 FACS 和/或抗体表型的原因其中许多基因都是新颖的，因为它们对于免疫功能是必需的。未知，并通过 CRISPR/Cas9 重新定位以验证 638 个候选基因中的 154 个（24%；几乎所有这些都是新颖的）要么被敲除，要么被使用 CRISPR/Cas9 编辑的 ENU 等位基因修饰，扩展到谱系，并重新测试筛选和自动化中检测到的表型的因果关系 154 个基因中的 148 个（96%）被验证为所声明的表型的来源。 CRISPR/Cas9 项目目前很活跃，我们的一些发现已经接近完成。已发表；还有更多工作正在进行中，但所有突变的 FACS 检测结果均已公布。已宣布是否与表型有关，已取消 Mutagenetix 上公众观看的限制，以及能够搜索、检查原始表型和减数分裂图数据、按 P 值过滤的工具，以及个体表型效应的方向和程度，这将使其他实验室能够进行研究。与我们一起了解具有非冗余功能的基因的免疫表型机制。适应性免疫反应的发展和激活是免疫学的基础，我们计划我们还计划对特定表型背后的机制进行更深入的研究。在我们之前的提案中命名为研究的表型中，我们已经了解了那些引起的兴趣。由 Trp53bp1、Ampd3、Rnps1、Prkd2 和 Snrnp40 的突变引起，并发表了描述由 Rabl3、Gpr89 突变引起的其他表型（在原始提案中未命名） Pdia6、Ncstn、Lmbr1l、Stk4、Pacs1、Wdr37 和 Mfsd1 也已阐明并发表或提交我们现在建议检查新验证的表型，同时创造新的表型。我们的工作现在由一个测量工具（相似性热图）指导。在流式细胞术筛选中，每个表型至少进行 34 次测量。结果构成了可用于统计相似性测试的表型“指纹”。一些基因产生的结果与其他基因的突变非常相似，我们有时可以推断出多个基因在单一的蛋白质复合物或酶途径中运作我们还编写了软件。（候选探索者）在宣布因果关系之前评估表型，告诉我们可能性如果我们尝试重新定位任何有问题的基因，我们会将我们的努力限制在最有可能的新基因上。候选人相信所有真正的因果关系最终都会表现为饱和度的提高。