Engineering the C. Elegans Genome

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。然而，任何单独的编辑从头到尾都需要6周的时间。编辑多个 基因是不切实际的。该项目的目标是使CRISPR基因组修改变得简单和 速度快，并能提高吞吐量。我们提出了一系列多重基因组工程方法， 将使线虫的基因标记速度提高一到两个数量级。首先，我们建议发展 盒式交换方法将允许遗传学家改变一个带有多个标签或敲除的基因 战略。其次，我们建议开发一种多路复用CRISPR策略，允许团体修改 在一个编辑实验中包含多个基因。第三，我们将开发能够修改的试剂库 将基因组中的所有基因分配给社区。 目的1.一个基因：重组酶介导盒交换。我们将开发一种盒式磁带交换器 一种在基因组中的特定位置快速整合转基因的方法。 目的2.多基因：多重CRISPR菌株。要实现对许多基因的高效和轻松编辑，请访问 一旦，我们将创建用于并行执行多个CRISPR编辑的方法和代理。 目的3.所有基因：标记基因收集。我们将为构建具有成本效益的池化工作流 基因组编辑试剂，然后使用这些试剂内源性标记1000个神经元表达的基因 使用绿色荧光蛋白。 线虫分享了人类遗传病中突变的大部分基因，使其成为研究 在一种简单、紧凑和快速发育的动物身上研究这些基因的功能。在未来， 这里开发的基因组工程流水线可以用来标记C。 秀丽的基因组具有各种不同的功能标签。这样的菌种收集将是细胞的福音 生物学家和遗传学家，使研究生物体如何工作以及如何修复 在疾病中走偏了。

Project Summary CRISPR offers the promise of total control over genes in model organisms, such as the nematode C. elegans. However, any individual edit takes on the order of 6 weeks from beginning to end. To edit many genes is just not practical. The goals of this project are to make CRISPR genome modifications simple and fast, and to increase the throughput. We propose a series of multiplexed genome engineering methods that will accelerate gene tagging in C. elegans by one to two orders of magnitude. First, we propose to develop cassette exchange methods that will allow geneticists to alter one gene with many tags or knockout strategies. 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 reagent libraries capable of modifying all genes in the genome for distribution to the community. Aim 1. One gene: recombinase-mediated cassette exchange. We will develop a cassette exchange method for rapidly integrating transgenes at a defined locus in the genome. Aim 2. Many genes: multiplex CRISPR strain. To enable efficient and easy editing of many genes at once, we will create methods and reagents for performing many CRISPR edits in parallel. Aim 3. All genes: tagged-gene collection. We will create a cost-effective pooled workflow for building genome editing reagents, then use these reagents to endogenously tag 1000 neuronally expressed genes with GFP. C. elegans shares most of the genes mutated in human genetic diseases, making it a major model for studying the function of these genes in a simple, compact, and rapidly developing animal. In the future, the genome engineering pipelines developed here could be used to tag every protein-coding gene in the C. elegans genome with a variety of functionally distinct tags. Such a strain collection would be a boon for cell biologists and geneticists, enabling new inroads in studying how organisms work and how to fix what goes awry in disease.