Genome engineering in the nematode C. elegans

线虫的基因组工程。 elegans

基本信息

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
项目类别：
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

项目摘要

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）中的基因为了使这一点成为现实，我们需要在所有蛋白质上添加功能标签，以便我们可以将其用作句柄。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建具有不同标签的许多基因，一次只需要一个基因。该项目的目标是使 CRISPR 基因组修饰变得简单、廉价且不切实际。我们提出了一系列多重基因组工程方法，可以提高通量。将线虫中的基因标记加速 10 至 100 倍首先，我们建议优化盒交换。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第三，我们将开发修改所有内容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个基因使用独特的注射混合物。我们将开发一种多重 CRISPR 策略，其中的试剂可用于标记许多基因。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：软件和分子试剂要标记蛋白质组，试剂无法有效地进行。一次设计一个，我们将编写软件来识别最佳标记位置和设计。所需的试剂，我们将建立一个具有成本效益的混合分子工作流程来构建基因组编辑试剂。秀丽隐杆线虫具有人类遗传病中大部分突变的基因，是一种简单、紧凑且快速的突变基因；发育中的动物，是未来研究这些基因的一个有吸引力的平台。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签位点线虫基因组，使得可以轻松地向任何基因添加任何标签，这样的菌株集合将是一个。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。