PROJECT SUMMARY The aim of this proposal is to continue to develop a toolkit designed to facilitate the functional annotation of human genes and disease associated variants through genetic studies in Drosophila melanogaster. We initiated this project three years ago through support of an R24 funded by ORIP. The Drosophila genome contains ~8,500 genes that are evolutionarily conserved in vertebrates including human. To model human diseases, we typically start by creating a severe loss-of-function mutation of a fly gene that is likely to be an ortholog of the human gene that is known or suspected to be pathogenic. We insert a SA-T2A-GAL4-polyA artificial exon into an early intron common to all transcripts of the gene of interest (GOI) using CRISPR mediated homologous recombination. This typically creates a strong loss-of-function allele that expresses the GAL4 transactivator in the same spatial and temporal pattern as the mutated gene. Hence, a UAS-nuclear or membrane GFP permits us to determine the cell types in which the gene is expressed through co-staining with known cell identity markers or based on cellular morphology. Importantly, GAL4 often allows us to rescue the phenotypes associated with the loss-of-function allele by driving a UAS-fly or human cDNA. If the human cDNA rescues we can test human variants of interest for functionality in flies, an approach that has already greatly helped in the identification of many new human diseases in the past few years. These experiments also allow detailed functional analyses to better understand the pathogenic mechanisms and to test FDA approved or experimental drugs. We have also produced a library of just over 2,000 T2A-GAL4 stocks and ~3,000 UAS- human cDNAs lines to perform these experiments systematically. We assembled a library of 33,000 full length human cDNAs from different sources, generated and sequenced ~4,000 plasmids containing the UAS-human cDNA for transformation in the fly. Nearly 3,000 of these constructs have been inserted in the fly genome in defined loci using the ΦC31 integrase, and transgenic stocks have been established. The UAS constructs are available from the Drosophila Genomics Resource Center (DGRC) and the stocks are available from the Bloomington and Kyoto stock centers. Here we propose to expand the UAS-human cDNA collection and clone the remaining 4,000 human cDNAs of the 8,500 conserved genes and establish an additional 3,000 transgenic stocks for distribution. We also propose to generate 1,000 SA-T2A-GAL4-polyA insertions in homologous fly genes using a new method that we developed to accelerate the testing of the UAS-human cDNAs by the research community and promote the systematic study of human disease associated genes. Our goal is to provide molecular, genetic and transgenic resources to the fly research community and human geneticists to accelerate the discovery of human diseases and help unravel human gene function.

PROJECT NARRATIVE We propose to generate a library of 4,000 epitope tagged UAS-human cDNA constructs of genes that are evolutionarily conserved between Drosophila and human, and produce ~3,000 transgenic strains with these constructs. We will also generate 1,000 severe loss-of-function mutations for a subset of these genes using a new CRISPR technology that we developed that allows efficient integration of GAL4. This often permits rescue of the mutant phenotype by crossing in the UAS-human-cDNA, and these reagents will therefore provide a very useful resource to study human gene function and disease in flies.