Genome engineering in the nematode C. elegans

线虫的基因组工程。 elegans

• 批准号: 10565428
• 负责人: ERIK M JORGENSEN
• 金额: $ 31.68万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-01 至 2027-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10565428
• 关键词: Acceleration; Amino Acid Motifs; Animals; Biochemical; Biological Assay; Biological Models; Biology; C. elegans genome; Caenorhabditis elegans; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Collection; Communities; Computer software; DNA; DNA Integration; DNA cassette; Disease; Engineering; Event; Excision; Freezing; Future; Gene Modified; Gene Targeting; Generations; Genes; Genetic; Genetic Diseases; Genetic Recombination; Genome; Genome engineering; Genomics; Goals; Guide RNA; Hand; Human; Human Genetics; Individual; Injections; Laboratories; Libraries; Location; Measures; Methods; Modeling; Modification; Molecular; Molecular Biology; Mutate; Nematoda; Oligonucleotides; Organism; Phenotype; Process; Proteins; Proteome; Random Allocation; Reaction; Reagent; Research Personnel; Series; Signal Pathway; Site; Software Tools; Speed; Structure; Testing; Time; Transgenes; Work; Writing; cell type; cost; cost effective; design; experience; experimental study; gene function; genetic manipulation; genome editing; genomic tools; improved; model organism; novel; rapid technique; recombinase; recombinase-mediated cassette exchange; repaired; software development; tool

Project Summary CRISPR offers the promise of total control over genes in model organisms, such as the nematode C. elegans. To make this a reality, we need functional tags on all proteins that we can use as handles to influence the biology of any cell. However, each individual edit requires unique reagents and takes experienced worm geneticists 6 weeks or more to create. To edit many genes with diverse tags one gene at a time is just not practical. The goals of this project are to make CRISPR genome modifications simple, inexpensive and with increased throughput. We propose a series of multiplexed genome engineering methods that will accelerate gene tagging in C. elegans 10- to 100-fold. First, we propose to optimize cassette exchange methods using diverse recombinases that will allow geneticists to alter one gene with many diverse tags. Second, we propose to develop a multiplexed CRISPR strategy that will allow groups to modify many genes within a single editing experiment. Third, we will develop software and reagent libraries required to modify all genes in the genome. • Aim 1. One gene: recombinase-mediated cassette exchange. We will characterize the germline activity of a diverse set of recombinases and develop cassette exchange methods for rapidly integrating transgenes or tags anywhere in the genome. • Aim 2. Many genes: multiplex CRISPR. Current methods require a unique injection cocktail for each unique gene modification. We will develop a multiplex CRISPR strategy in which the reagents for tagging many unique genes are injected simultaneously to generate many edited worm strains, each with a single edited target. • Aim 3. All genes: software and molecular reagents. To tag the proteome, reagents cannot be efficiently designed one-at-a-time, by hand. We will write software that identifies optimal tagging locations and designs the required reagents, and we will build build a cost-effective pooled molecular workflow to build genome editing reagents. C. elegans shares most of the genes mutated in human genetic diseases; as a simple, compact and rapidly developing animal, it is an attractive platform to study these genes. In the future, the genome engineering pipelines developed here could be used to insert a swappable tagging site in every protein-coding gene in the C. elegans genome, making it possible to easily add any tag to any gene. Such a strain collection would be a boon for cell biologists and geneticists, enabling new inroads in studying how cells work and how to fix them when disease processes cause them to malfunction.

项目摘要 CRISPR提供了对模式生物（如线虫）中基因的完全控制的希望 C.优雅的。为了实现这一点，我们需要所有蛋白质上的功能标签，我们可以使用这些标签作为手柄， 影响任何细胞的生物学。然而，每个单独的编辑需要独特的试剂，并需要经验丰富的 蠕虫遗传学家6周或更长的时间来创建。一次编辑一个基因， 不实用。该项目的目标是使CRISPR基因组修饰简单，廉价， 具有增加的吞吐量。我们提出了一系列多重基因组工程方法， 加快C. 10到100倍。首先，我们建议优化盒式磁带交换 使用不同的重组酶的方法，这将允许遗传学家用许多不同的标签改变一个基因。 其次，我们建议开发一种多重CRISPR策略，允许团队修改许多基因， 在一个单一的编辑实验中。第三，我们将开发软件和试剂库所需的修改全部 基因组中的基因。 ·目标1。一个基因：重组酶介导的盒交换。我们将描述生殖细胞活性 并开发用于快速整合转基因的盒式交换方法 基因组中任何地方的标签。 ·目标2。多基因：多重CRISPR。目前的方法需要一个独特的注射鸡尾酒为每一个 独特的基因改造我们将开发一种多重CRISPR策略，其中用于标记多个CRISPR的试剂将被用于标记多个CRISPR。 同时注入独特的基因，以产生许多编辑过的蠕虫菌株，每个菌株都有一个编辑过的 目标 目标3。所有基因：软件和分子试剂。为了标记蛋白质组，试剂不能被有效地 一次一个手工设计我们将编写软件，确定最佳的标记位置和设计 所需的试剂，我们将建立一个具有成本效益的汇集分子工作流程，以建立基因组编辑 试剂 C. elegans分享了人类遗传疾病中的大部分突变基因;作为一种简单、紧凑和快速的 发展中的动物，这是一个有吸引力的平台来研究这些基因。在未来，基因组工程 这里开发的管道可以用来在每一个蛋白质编码基因中插入一个可交换的标记位点， C. elegans基因组，使其可以轻松地添加任何标签到任何基因。这样的菌株收集将是 细胞生物学家和遗传学家的布恩，使研究细胞如何工作和如何修复它们的新进展 当疾病进程导致它们发生故障时。