Engineering the C. Elegans Genome

 描述（由申请人提供）：无偏正向遗传筛选在模式生物遗传学中发挥核心作用。C.在过去的35年里，线虫筛选对生物学中一些最重要的发现做出了贡献：Ras-MAP激酶途径、细胞死亡途径、RNA干扰和microRNA的转录后调节都是例子。然而，只有~10%的基因具有在正向筛选中分离的等位基因。我们建议开发一种基于转座子的基因陷阱，将同时产生平衡的敲除等位基因和细胞表达模式。作为这种方法的补充，我们将设计构建体和方法来标准化和降低基于CRISPR的基因陷阱的成本。虽然C.虽然线虫可能是所有生物体中最具特征的解剖结构，但蠕虫遗传学一直被难以明确地将特定基因的表达分配给特定细胞所混淆。我们将生成蠕虫菌株和开源显微镜硬件，以帮助个人用户以及自动化系统解决分配基因表达模式的最后一步。作为一个整体，提出的方法将成为整个C。elegans研究社区目标1.随机基因陷阱我们将对果蝇的Mos1转座子进行修饰，使其在插入C. elegans基因组，即破坏该基因并用tagRFP报告基因表达模式。目标二。定向基因陷阱。我们将使用CRISPR设计和测试靶向基因陷阱的高通量策略。在此目的中产生的克隆文库也可以由各个实验室用于获得基因组中任何基因的表达模式和无效等位基因。目标3。表达式模式工具包。我们将建立线虫菌株和显微镜硬件，用于高通量记录和分析3D基因的表达模式。这些工具将被应用于基因陷阱，但也将用于C。elegans社区的细胞表达身份，特别是在蠕虫的大脑。

 DESCRIPTION (provided by applicant): Unbiased forward genetic screens play a central role in model organism genetics. C. elegans screens have contributed to some of the most important discoveries in biology in the last 35 years: Ras - MAP kinase pathways, cell death pathways, RNA interference and posttranscriptional regulation by microRNAs are examples. However, only ~10% of genes have alleles that have been isolated in forward screens. We propose to develop a transposon-based gene trap that will simultaneously generate balanced knockout alleles and cellular expression patterns. As a complement to this method, we will design constructs and methods to standardize and lower the costs of CRISPR-based gene traps. Although C. elegans has possibly the most thoroughly characterized anatomy of any organism, worm genetics has been confounded by the difficulty of unambiguously assigning expression of a specific gene to a particular cell. We will generate worm strains and open-source microscope hardware that will aid individual users as well as automated systems in solving this final step in assigning gene expression patterns. As a package, the methods will propose to develop will be a resource for the whole C. elegans research community. Aim 1. Random gene traps. We will modify the Mos1 transposon from Drosophila so that it will act as a gene trap when inserted into the C. elegans genome, that is, disrupt the gene and report the gene expression pattern with tagRFP. Aim 2. Directed gene traps. We will design and test a high-throughput strategy for targeted gene traps using CRISPR. The library of clones generated in this aim can be also be used by individual labs to obtain the expression pattern and a null allele of any gene in the genome. Aim 3. Expression pattern toolkit. We will build nematode strains and microscopy hardware for high-throughput recording and analyzing the expression patterns of genes in 3D. These tools will be applied to the gene traps but will also be of use to the C. elegans community for cell expression identities, particularly in the brain of the worm.