An enhanced gene targeting toolkit for Drosophila: Golic+
Department of Neurobiology; Tzumin Lee Lab; Graduate School of Biomedical Sciences, Neuroscience Program
Genetics and Genomics | Neuroscience and Neurobiology
Ends-out gene targeting allows seamless replacement of endogenous genes with engineered DNA fragments by homologous recombination, thus creating designer "genes" in the endogenous locus. Conventional gene targeting in Drosophila involves targeting with the preintegrated donor DNA in the larval primordial germ cells. Here we report G: ene targeting during O: ogenesis with L: ethality I: nhibitor and C: RISPR/Cas (Golic+), which improves on all major steps in such transgene-based gene targeting systems. First, donor DNA is integrated into precharacterized attP sites for efficient flip-out. Second, FLP, I-SceI, and Cas9 are specifically expressed in cystoblasts, which arise continuously from female germline stem cells, thereby providing a continual source of independent targeting events in each offspring. Third, a repressor-based lethality selection is implemented to facilitate screening for correct targeting events. Altogether, Golic+ realizes high-efficiency ends-out gene targeting in ovarian cystoblasts, which can be readily scaled up to achieve high-throughput genome editing.
DOI of Published Version
Genetics. 2015 Mar;199(3):683-94. doi: 10.1534/genetics.114.173716. Epub 2015 Jan 2. Link to article on publisher's site
Chen, Hui-Min; Huang, Ya-ling; Pfeiffer, Barret D.; Yao, Xiaohao; and Lee, Tzumin, "An enhanced gene targeting toolkit for Drosophila: Golic+" (2015). GSBS Student Publications. 1889.