Rapid and efficient generation of GFP-knocked-in Drosophila by the CRISPR-Cas9-mediated genome editing

The CRISPR-Cas9 technology has been a powerful means to manipulate the genome in a wide range of organisms. A series of GFP knocked-in (GFP^KI) Drosophila strains have been generated through CRISPR-Cas9-induced double strand breaks coupled with homology-directed repairs in the presence of donor plasmids. They visualized specific cell types or intracellular structures in both fixed and live specimen. We provide a rapid and efficient strategy to identify KI lines. This method requires neither co-integration of a selection marker nor prior establishment of sgRNA-expressing transgenic lines. The injection of the mixture of a sgRNA/Cas9 expression plasmid and a donor plasmid into cleavage stage embryos efficiently generated multiple independent KI lines. A PCR-based selection allows to identify KI fly lines at the F1 generation (approximately 4 weeks after injection). These GFP^KI strains have been deposited in the Kyoto Drosophila stock center, and made freely available to researchers at non-profit organizations. Thus, they will be useful resources for Drosophila research.