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生成、维护和分发一批新的工程化黑腹果蝇种群，这些种群将允许对目前没有可用突变等位基因的基因进行通用操作。使用最先进的CRISPR技术，这个集合或“试剂盒”将代表一套强大的新工具，将使几乎所有的果蝇遗传学家受益，并且很可能会有B对这种资源的高需求多年。我们对人类发育的理解中许多最重要的进展都来自于使用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或容易地适用于理解人类生物学。 果蝇与其他模式系统不同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，两个基本的试剂是功能丧失突变和基因的标记版本。目前获得基因的突变或标记等位基因的可用方法仅覆盖基因组的一部分，或者效率低、费力且耗时。事实上，果蝇基因组中有2000多个基因目前缺乏突变等位基因，无法有效地产生功能丧失突变体或标记等位基因。为了解决这个问题，我们建议生成一个新的靶向集合，它将补充现有的集合，并提供下游操作的最终灵活性，例如生成点突变和功能丧失突变，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介导的靶向条件，而第二阶段将产生一个针对目前没有突变的2000个基因的集合。为了实现这一目标，我们将：目标1。开发和优化CRISPR-Cas9介导的盒靶向。目标2.评估跨基因组的CRISPR介导的盒靶向。第一阶段的成功将为CRISPR介导的盒靶向开发强大的方案，并为扩大我们在第二阶段产生完整集合的努力奠定基础。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。为了表明这一提议得到了许多著名果蝇研究人员的强烈支持，本申请中包括了一组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.