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Rapid and efficient generation of GFP-knocked-in Drosophila by the CRISPR-Cas9-mediated genome editing |
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| Authors: | Hirono Kina Takashi Yoshitani Kazuko Hanyu-Nakamura Akira Nakamura |
| Affiliation: | 1. Department of Germline Development, Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto, Japan School of Pharmacy, Kumamoto University, Kumamoto, Japan;2. Department of Germline Development, Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto, Japan These authors contributed equally to this work. HK and TY are equal contribution to the work.;3. Department of Germline Development, Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto, Japan |
| Abstract: | 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 (GFPKI) 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 GFPKI 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. |
| Keywords: | CRISPR-Cas9 Drosophila genome editing germ cells GFP |