Toward a Complete Set of Germline Null and Modifiable Mutations in Drosophila

描述（由申请人提供）：本提案的总体目标是产生，维持和分发一个新的工程黑腹果蝇种群集合，这将允许对目前没有突变等位基因的基因进行多功能操作。使用尖端的CRISPR技术，这个集合或“工具包”将代表一套强大的新工具，这将使几乎所有果蝇遗传学家受益，并且很可能在许多年内对该资源有很高的需求。在我们对人类发展的理解中，许多最重要的进展都来自于将果蝇作为动物模型系统的研究。由于果蝇和哺乳动物在控制生物过程的潜在分子机制方面存在许多相似之处，因此从果蝇研究中获得的知识可以直接应用于或很容易适应于理解人类生物学。将果蝇与其他模型系统区分开来的一个关键因素是，在过去100年的研究中积累了大量的遗传和分子工具。要研究任何给定的基因，两个基本试剂是功能丧失突变和基因的标记版本。目前可用的获得基因突变或标记等位基因的方法仅覆盖基因组的一部分，或者效率低下，费力且耗时。事实上，目前苍蝇基因组中有超过2000个基因缺乏突变等位基因，没有办法有效地产生功能丧失突变或标记等位基因。为了解决这个问题，我们建议生成一个新的目标集合，以补充现有的集合，并提供下游操作的最终灵活性，例如生成点和功能丧失突变，蛋白质标记和基因表达谱。我们计划将该项目分为两个阶段：第一阶段将作为原理证明并优化CRISPR介导靶向的条件，而第二阶段将生成针对2000个目前没有突变的基因的集合。为实现这一目标，我们将：开发和优化CRISPR-Cas9介导的卡带靶向。目标2。评估crispr介导的跨基因组盒式靶向。第一阶段的成功将为crispr介导的卡带靶向制定稳健的协议，并为扩大我们在第二阶段产生完整收集的努力奠定基础。利用新技术的好处，这个集合将是果蝇研究工具包的另一个关键补充。由于该提案获得了许多杰出果蝇研究人员的强烈热情，该申请包含了一套11封支持信。

DESCRIPTION (provided by applicant): The overall goal of this proposal is to generate, maintain, and distribute a new collection of engineered Drosophila melanogaster stocks that will allow versatile manipulation of genes for which there is currently no mutant allele available. Using cutting edge CRISPR technology, this collection or 'kit' will represent a powerful new set of tools that will benefit virtually all Drosophila geneticists, and it is likely that there will b a high demand for this resource for many years. Many of the most important advances in our understanding of human development have come from studies using Drosophila as an animal model system. Since many parallels exist between Drosophila and mammals in terms of the underlying molecular mechanisms controlling biological processes, knowledge gained from research in Drosophila can be either directly applied or readily adapted to understanding human biology. One key factor that sets Drosophila apart from other model systems is the huge wealth of genetic and molecular tools that have accumulated in the past 100 years of research. To study any given gene, two essential reagents are loss-of-function mutations and a tagged version of the gene. Currently available methods to obtain mutant or tagged alleles of genes cover only a portion of the genome or are inefficient, laborious, and time consuming. Indeed, there are more than 2000 genes in the fly genome that currently lack a mutant allele with no means of efficiently generating either a loss-of-function mutant or a tagged allele. To address this problem, we propose to generate a new targeted collection that will complement existing collections and offer the ultimate flexibility of downstream manipulation, such as generating both point and loss-of-function mutations, protein tagging, and gene expression profiling. We plan to split this project into two phases: Phase I will serve as a proof of principle and optimize the conditions for CRISPR- mediated targeting while Phase II will generate a collection targeting 2000 genes currently without a mutation. To achieve this goal, we will: Aim 1. Develop and optimize CRISPR-Cas9 mediated cassette targeting. Aim 2. Evaluate CRISPR-mediated cassette targeting across the genome. Success in Phase I will develop robust protocols for CRISPR-mediated cassette targeting and set the stage for expanding our effort to generate a complete collection during Phase II. Harnessing the benefits of new technologies, this collection will be another key addition to the Drosophila research tool kit. As a sign of the strong enthusiasm this proposal has received from many prominent Drosophila researchers, a set of 11 letters of support are included with this application.