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Genetics. 2015 Mar;199(3):683-94. doi: 10.1534/genetics.114.173716. Epub 2015 Jan 2.

An enhanced gene targeting toolkit for Drosophila: Golic+.

Author information

1
Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia 20147 Department of Neurobiology, University of Massachusetts, Worcester, Massachusetts 01655.
2
Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia 20147.
3
Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia 20147 Department of Neurobiology, University of Massachusetts, Worcester, Massachusetts 01655 leet@janelia.hhmi.org.

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.

KEYWORDS:

CRISPR; Drosophila; gene targeting; homologous recombination; repressor/miRNA-based lethality selection

PMID:
25555988
PMCID:
PMC4349064
DOI:
10.1534/genetics.114.173716
[Indexed for MEDLINE]
Free PMC Article

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