title: An enhanced gene targeting toolkit for Drosophila: Golic+.
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 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.
authors.affiliation: Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia 20147 Department of Neurobiology, University of Massachusetts, Worcester, Massachusetts 01655.
authors.forename: Hui-Min
authors.initials: HM
authors.lastname: Chen
authors.affiliation: Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia 20147.
authors.forename: Yaling
authors.initials: Y
authors.lastname: Huang
authors.affiliation: Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia 20147.
authors.forename: Barret D
authors.initials: BD
authors.lastname: Pfeiffer
authors.affiliation: Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia 20147.
authors.forename: Xiaohao
authors.initials: X
authors.lastname: Yao
authors.affiliation: Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia 20147 Department of Neurobiology, University of Massachusetts, Worcester, Massachusetts 01655 leet@janelia.hhmi.org.
authors.forename: Tzumin
authors.initials: T
authors.lastname: Lee
mesh_terms: D000818:Animals; D004247:DNA; D004330:Drosophila / Q000235:genetics*; D005260:Female; D018390:Gene Targeting / Q000379:methods*; D059765:Homologous Recombination; D009866:Oogenesis; D010063:Ovum; D019076:Transgenes
chemical_list: D004247:DNA
keywords: CRISPR; Drosophila; gene targeting; homologous recombination; repressor/miRNA-based lethality selection