Clonal Analysis in Drosophila Tissues With an Enhanced MAGIC Transgenesis Method.

Mosaic animals are highly valuable for investigating complex biological processes and cell lineages in vivo. Traditional mosaic techniques in Drosophila, such as the FRT/Flp system, rely on exogenous site-specific recombination sequences, preventing their application to unmodified mutant chromosomes or wild-derived strains. Mosaic analysis by gRNA-induced crossing-over (MAGIC) overcomes this limitation by utilizing the CRISPR/Cas9 system to generate targeted double-strand breaks (DSBs) that induce somatic homologous recombination in precursor cells. Here, we describe a comprehensive protocol for applying MAGIC with a newly developed, genome-wide MAGIC kit. This protocol utilizes optimized gRNA-markers with the Qtg2.1 scaffold for high-efficiency clone induction, alongside improved fluorescent labeling strategies for both positive MAGIC (pMAGIC) and negative MAGIC (nMAGIC). The procedure details the genetic crossing schemes, temporal induction of clones, and tissue processing for diverse Drosophila cell types. This method enables convenient mosaic analysis across all chromosomes and allows for the study of pericentromeric genes, deficiency chromosomes, and species-specific alleles in interspecific hybrids. Key features • Recombinase-independent: Generates somatic mosaic clones using CRISPR/Cas9 without requiring pre-inserted FRT sequences on the test chromosome. • Genome-wide application: Includes a complete toolkit of pMAGIC and nMAGIC gRNA-markers optimized for all Drosophila chromosomal arms (X, 2L, 2R, 3L, 3R, and 4). • Optimized labeling: Employs destabilized Gal80 for brighter pMAGIC clones and tub-3xHA-BFP/IFP for unambiguous visualization of nMAGIC clones. • Broad compatibility: Applicable to a wide variety of tissues (e.g., neurons, glia, imaginal discs, polyploid tissues) and complex genetic backgrounds, including pericentromeric mutations and deficiencies.