Genome engineering in the nematode C. elegans

项目摘要 CRISPR提供了完全控制模式生物中的基因的前景，例如线虫 线虫。为了实现这一点，我们需要所有蛋白质上的功能标签，我们可以将其用作句柄 影响任何细胞的生物学。然而，每个单独的编辑都需要独特的试剂，并且需要经验丰富的人 蠕虫遗传学家需要6周或更长时间才能制造出来。用不同的标签编辑多个基因，一次编辑一个基因 不切实际。该项目的目标是使CRISPR基因组修改简单、廉价和 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，这些方法将 将线虫的基因标记速度提高10到100倍。首先，我们建议优化盒式磁带更换 方法使用不同的重组酶，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路CRISPR策略，允许团队修改许多基因 在一次编辑实验中。第三，我们将开发软件和试剂库，以修改所有 基因组中的基因。 目的1.一个基因：重组酶介导盒交换。我们将描述生殖系活动的特征 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.多个基因：多重CRISPR。目前的方法需要一种独特的注射鸡尾酒 独特的基因修饰。我们将开发一种多重CRISPR策略，在该策略中，用于标记许多 同时注入独特的基因以产生许多编辑过的蠕虫菌株，每个都有一个编辑过的 目标。 ·目标3.所有基因：软件和分子试剂。为了标记蛋白质组，试剂不能有效地 一次一个地手工设计。我们将编写识别最佳标签位置和设计的软件 所需的试剂，我们将构建一个经济高效的分子池工作流来构建基因组编辑 试剂。 线虫分享了人类遗传病中突变的大部分基因；作为一种简单、紧凑和快速的 对于发育中的动物来说，这是一个有吸引力的研究这些基因的平台。在未来，基因组工程 这里开发的管道可以用来在每个编码蛋白质的基因中插入一个可交换的标签位点 线虫基因组，使人们可以很容易地为任何基因添加任何标签。这样的菌株集合将是一种 细胞生物学家和遗传学家的福音，使研究细胞如何工作以及如何修复它们取得了新的进展 当疾病过程导致它们出现故障时。

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.