The overall goal of this proposal is to generate, maintain, and distribute a new collection of 1,500 custom engineered Drosophila melanogaster stocks that will allow versatile manipulation of genes that are highly conserved in humans but currently lack an available mutant allele. Utilizing cutting edge CRISPR technology, this collection or 'kit' will represent a powerful new set of tools that will benefit virtually all Drosophila geneticists; as such it is likely that there will be a high demand for this resource for many years to come. Many of the most important advances in our understanding of human development have come from studies using the fruit fly 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 and disease. During Phase I of this project, we optimized the conditions for CRISPR/Cas9 mediated insertion of a custom CRIMIC cassette and correctly targeted 55 out of 63 genes attempted. This high rate of success (87%), indicates that we are perfectly poised to scale up our efforts to a larger set of genes. In this Phase II proposal, we aim to generate CRIMIC targeted alleles for an additional 1,500 conserved genes that currently lack an available mutant allele. The primary benefit of such a collection is that the CRIMIC insertion not only generates a strong loss-of-function or null allele of the targeted gene, but can also serve as a docking site for further engineering, such as transcript or protein tagging or creating point mutations that can be used to model human disease pathology. In coordination with other existing collections and the ongoing Gene Disruption Project (GDP), our Specific Aims are to:
Aim 1. Select 1,800 gene targets and construct 1,800 gRNA/donor pairs Aim 2. Generate and validate a collection of 1,500 targeted gene stocks Aim 3. Create and maintain an online searchable database for targeted genes Harnessing the benefits of new technologies, this collection will fully complement existing collections and the ongoing GDP effort. Once generated, these combined resources will cover the vast majority genes in the Drosophila genome that are directly relevant to human disease.
The main goal of this project is to create a new resource for the Drosophila (a fruit fly) research community that will enable more efficient study of new genes required for normal development and function. Specifically, we propose to create a large set of transgenic flies with loss-of-function mutations in genes for which no such mutations were previously available and that would allow subsequent genomic modification. In particular, these transgenic flies will allow flexible and precise engineering of the targeted gene and allow new and more powerful studies of gene function. Since a large number of genes in Drosophila are highly conserved in mammals and often provide critical insights into human gene function, this resource will greatly benefit both basic and applied research.