SBIR-STTR Award

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.

Thesaurus Terms:
Address;Alleles;Animal Model;Applied Research;Arm;Base;Basic Science;Biological Models;Biological Process;Clustered Regularly Interspaced Short Palindromic Repeats;Collection;Communities;Complement;Custom;Design;Development;Dna;Dna Transposable Elements;Docking;Drosophila Genus;Drosophila Melanogaster;Engineering;Evaluation;Excision;Flexibility;Fly;Fusion Protein Expression;Gene Complementation;Gene Expression Profiling;Gene Function;Gene Targeting;Generations;Genes;Genetic;Genetic Complementation Test;Genetic Recombination;Genome;Genome Sequencing;Genomics;Goals;Homologous Recombination;Human;Human Biology;Human Development;Injection Of Therapeutic Agent;Insight;Integrase;Interest;Knowledge;Letters;Loss Of Function;Loss Of Function Mutation;Mammals;Mediating;Messenger Rna;Methods;Modification;Molecular;Molecular Genetics;Mutagenesis;Mutant;Mutation;New Technology;Novel;Novel Strategies;Pattern;Phase;Phosphorus;Point Mutation;Proteins;Protocols Documentation;Public Health Relevance;Reagent;Research;Research Personnel;Research Study;Resources;Screening;Site;Source;Staging;Success;System;Technology;Testing;Time;Tool;Transcript;Transgenic Organisms;

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.

Public Health Relevance Statement:
Project Narrative 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.

Project Terms:
Alleles; Animal Model; Applied Research; Basic Science; Biological Models; Biological Process; Collection; Communities; Complement; cost; CRISPR/Cas technology; Custom; Databases; design; Development; Disease; DNA; Docking; Drosophila genome; Drosophila genus; Drosophila melanogaster; Engineering; Event; flexibility; fly; gene function; Gene Targeting; Genes; Genetic; Genome; Genomics; Goals; Guide RNA; Human; Human Biology; Human Development; human disease; Human Genetics; insight; Introns; Knowledge; Lead; Letters; loss of function; loss of function mutation; Mammals; Mediating; Modeling; Modification; Molecular; mutant; Mutation; new technology; next generation sequencing; Pathogenicity; Pathology; Phase; Point Mutation; Production; Proteins; relational database; repaired; Research; Research Personnel; Resources; scale up; Site; success; System; Technology; tool; Transcript; Transgenic Organisms; Update; virtual; web site; Work