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.

项目概要 CRISPR 有望完全控制模型生物（例如线虫）中的基因 C.线虫。为了实现这一点，我们需要在所有蛋白质上添加功能标签，我们可以将其用作句柄 影响任何细胞的生物学。然而，每次单独的编辑都需要独特的试剂，并且需要经验丰富的人员 蠕虫遗传学家需要6周或更长时间才能创造出来。编辑具有不同标签的许多基因一次只需要一个基因 不实用。该项目的目标是使 CRISPR 基因组修饰变得简单、廉价且 随着吞吐量的增加。我们提出了一系列多重基因组工程方法，将 将线虫中的基因标记加速 10 至 100 倍。首先，我们建议优化盒式磁带交换 使用多种重组酶的方法将允许遗传学家用许多不同的标签来改变一个基因。 其次，我们建议开发一种多重 CRISPR 策略，允许群体修改许多基因 在单个编辑实验中。第三，我们将开发修改所有所需的软件和试剂库 基因组中的基因。 • 目标1. 一个基因：重组酶介导的盒式交换。我们将表征种系活性 一组不同的重组酶并开发用于快速整合转基因的盒式交换方法 或基因组中任何位置的标签。 • 目标2。多种基因：多重CRISPR。目前的方法需要为每种方法提供独特的注射混合物 独特的基因修饰。我们将开发一种多重 CRISPR 策略，其中用于标记许多基因的试剂 同时注入独特的基因以产生许多经过编辑的蠕虫菌株，每个菌株都有一个经过编辑的 目标。 • 目标3。所有基因：软件和分子试剂。为了标记蛋白质组，试剂不能有效地 一次一个地手工设计。我们将编写识别最佳标记位置和设计的软件 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来构建基因组编辑 试剂。 秀丽隐杆线虫拥有人类遗传疾病中大部分突变基因；作为一种简单、紧凑且快速的 正在发育的动物，它是研究这些基因的一个有吸引力的平台。未来，基因组工程 这里开发的管道可用于在每个蛋白质编码基因中插入可交换的标记位点 线虫基因组，可以轻松地将任何标签添加到任何基因。这样的菌株收集将是 对细胞生物学家和遗传学家来说是福音，在研究细胞如何工作以及如何修复细胞方面取得新进展 当疾病过程导致它们发生故障时。