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

Project Summary Abstract The overall goal of this proposal is to generatemaintainand distribute a new collection ofcustom engineered Drosophila melanogaster stocks that will allow versatile manipulation of genes that are highly conserved in humans but currently lack an available mutant alleleUtilizing cutting edge CRISPR technologythis collection or andapos kitandaposwill represent a powerful new set of tools that will benefit virtually all Drosophila geneticistsas such it is likely that there will be a high demand for this resource for many years to comeMany of the most important advances in our understanding of human development have come from studies using the fruit fly as an animal model systemSince many parallels exist between Drosophila and mammals in terms of the underlying molecular mechanisms controlling biological processesknowledge gained from research in Drosophila can be either directly applied or readily adapted to understanding human biology and diseaseDuring Phase I of this projectwe optimized the conditions for CRISPR Casmediated insertion of a custom andquot CRIMICandquotcassette and correctly targetedout ofgenes attemptedThis high rate of successindicates that we are perfectly poised to scale up our efforts to a larger set of genesIn this Phase II proposalwe aim to generate CRIMIC targeted alleles for an additionalconserved genes that currently lack an available mutant alleleThe 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 genebut can also serve as a docking site for further engineeringsuch as transcript or protein tagging or creating point mutations that can be used to model human disease pathologyIn coordination with other existing collections and the ongoing Gene Disruption ProjectGDPour Specific Aims are toAimSelectgene targets and constructgRNA donor pairs AimGenerate and validate a collection oftargeted gene stocks AimCreate and maintain an online searchable database for targeted genes Harnessing the benefits of new technologiesthis collection will fully complement existing collections and the ongoing GDP effortOnce generatedthese combined resources will cover the vast majority genes in the Drosophila genome that are directly relevant to human disease Project Narrative The main goal of this project is to create a new resource for the Drosophilaa fruit flyresearch community that will enable more efficient study of new genes required for normal development and functionSpecificallywe 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 modificationIn particularthese transgenic flies will allow flexible and precise engineering of the targeted gene and allow new and more powerful studies of gene functionSince a large number of genes in Drosophila are highly conserved in mammals and often provide critical insights into human gene functionthis resource will greatly benefit both basic and applied research