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Rapid and efficient generation of GFP ‐knocked‐in Drosophila by the CRISPR ‐Cas9‐mediated genome editing
Author(s) -
Kina Hirono,
Yoshitani Takashi,
HanyuNakamura Kazuko,
Nakamura Akira
Publication year - 2019
Publication title -
development, growth and differentiation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.864
H-Index - 66
eISSN - 1440-169X
pISSN - 0012-1592
DOI - 10.1111/dgd.12607
Subject(s) - crispr , cas9 , plasmid , biology , green fluorescent protein , genome , genome editing , guide rna , mcherry , genetics , computational biology , microbiology and biotechnology , gene
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 sg RNA ‐expressing transgenic lines. The injection of the mixture of a sg RNA /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.