An Enhanced Gene Targeting Toolkit for Drosophila: Golic+ |
| |
Authors: | Hui-Min Chen Yaling Huang Barret D Pfeiffer Xiaohao Yao Tzumin Lee |
| |
Institution: | *Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia 20147;†Department of Neurobiology, University of Massachusetts, Worcester, Massachusetts 01655 |
| |
Abstract: | 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 gene targeting during oogenesis with lethality inhibitor and CRISPR/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. |
| |
Keywords: | gene targeting homologous recombination CRISPR Drosophila repressor/miRNA-based lethality selection |
|
|