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 goals of this project will
provide the Drosophila community important tools and information that can greatly enhance the value of
existing Drosophila resources for understanding human disease.