Engineering the C. Elegans Genome

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.优雅然而，任何单独的编辑从开始到结束需要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基因组修饰变得简单， 快速，并增加吞吐量。我们提出了一系列多重基因组工程方法， 将加速C中的基因标记。elegans的一个到两个数量级。首先，我们建议发展 基因盒交换方法将允许遗传学家用许多标签改变一个基因或敲除 战略布局其次，我们建议开发一种多重CRISPR策略，允许群体修改 许多基因在一个单一的编辑实验。第三，我们将开发能够修改 基因组中的所有基因，以分配给社区。 目标1.一个基因：重组酶介导的盒交换。我们将开发盒式磁带交换机 将转基因快速整合到基因组中确定的基因座的方法。 目标2.许多基因：多重CRISPR菌株。为了使许多基因的有效和容易的编辑成为可能， 我们将创建方法和试剂，用于并行执行许多CRISPR编辑。 目标3：所有基因：标记基因集合。我们将创建一个具有成本效益的汇集工作流程， 基因组编辑试剂，然后使用这些试剂标记1000个神经元表达的基因 关于GFP C.秀丽线虫与人类遗传疾病中的大多数突变基因相同，使其成为人类遗传疾病的主要模型。 研究这些基因在简单、紧凑和快速发育的动物中的功能。未来该 这里开发的基因组工程管道可以用来标记C. elegans基因组中的各种功能不同的标签。这样的菌株收集将是细胞的布恩。 生物学家和遗传学家，在研究生物体如何工作以及如何修复发生的问题上取得了新的进展。 在疾病中扭曲。

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.