Project Summary/Abstract This project aims to develop a complete toolbox for a new technique of mosaic analysis that is compatible with nearly all existing Drosophila melanogaster resources, without the need for further genetic modifications. Mosaic analysis is a powerful approach for studying molecular and cellular mechanisms of human disease. By generating homozygous cells in otherwise heterozygous animals, mosaic techniques allow tissue-specific analysis of pleiotropic genes and have played key roles in many important discoveries in biology. Current mosaic techniques in Drosophila primarily rely on the Flp/FRT site-specific recombination system and require a pair of homologous chromosomes that each contains an FRT sequence near the centromere. However, most existing genetic resources in Drosophila, such as deficiency libraries, transposon-disrupted mutant collections, and strains derived from wild natural populations, do not harbor appropriate FRT sites, preventing their efficient application in mosaic analysis. New mosaic techniques that do not depend on site-specific recombination systems are needed to unleash the full potential of these existing resources. Mosaic analysis by gRNA-induced crossing-over (MAGIC) is a new mosaic technique that does not require site-specific recombination and is compatible with unmodified chromosomes. Although the effectiveness of MAGIC has been demonstrated in the germline, the nervous system, and epithelial tissues, MAGIC reagents are presently only available for a single chromosome arm. This project will first establish an optimized MAGIC toolkit that can be used for mosaic analysis over the entire genome throughout Drosophila tissues. MAGIC screens will also be conducted to identify deficiency lines that are associated with morphological defects in neurons and epithelial cells. Specifically, four aims are proposed: (1) establish an optimized and complete MAGIC toolkit for all Drosophila chromosome arms; (2) generate strains expressing Cas9 in precursor cells of diverse tissues for MAGIC applications; (3) develop anti-CRISPR tools for safe and versatile MAGIC applications in Drosophila; and (4) screen deficiency lines by MAGIC for genes involved in neuronal and epithelial morphogenesis. The Drosophila strains and constructs developed in this project will be donated to the Bloomington Drosophila Stock Center and plasmid depositories, respectively, for easy distribution to the research community. The screen results will be made publicly available for further identification and characterization of responsible genes by interested labs. An advisory committee has been established to provide feedback to this project. Community inputs will be solicited regarding candidate precursor Cas9 lines to generate. The proposed MAGIC tools will allow exploitation of existing genetic resources in systematic gene-function analysis, genome- wide genetic screens, and tissue-specific analysis of natural variants. Attaining the goal...