title: Editing Transgenic DNA Components by Inducible Gene Replacement in Drosophila melanogaster.
abstract: Gene conversions occur when genomic double-strand DNA breaks (DSBs) trigger unidirectional transfer of genetic material from a homologous template sequence. Exogenous or mutated sequence can be introduced through this homology-directed repair (HDR). We leveraged gene conversion to develop a method for genomic editing of existing transgenic insertions in Drosophila melanogaster The clustered regularly-interspaced palindromic repeats (CRISPR)/Cas9 system is used in the H: omology A: ssisted C: RISPR K: nock-in (HACK) method to induce DSBs in a GAL4 transgene, which is repaired by a single-genomic transgenic construct containing GAL4 homologous sequences flanking a T2A-QF2 cassette. With two crosses, this technique converts existing GAL4 lines, including enhancer traps, into functional QF2 expressing lines. We used HACK to convert the most commonly-used GAL4 lines (labeling tissues such as neurons, fat, glia, muscle, and hemocytes) to QF2 lines. We also identified regions of the genome that exhibited differential efficiencies of HDR. The HACK technique is robust and readily adaptable for targeting and replacement of other genomic sequences, and could be a useful approach to repurpose existing transgenes as new genetic reagents become available.
authors.affiliation: The Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
authors.forename: Chun-Chieh
authors.initials: CC
authors.lastname: Lin
authors.affiliation: The Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 cpotter@jhmi.edu.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Potter
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics; D064113:CRISPR-Cas Systems / Q000235:genetics; D053903:DNA Breaks, Double-Stranded; D029721:Drosophila Proteins / Q000235:genetics*; D004331:Drosophila melanogaster / Q000235:genetics; D005785:Gene Conversion / Q000235:genetics*; D000072669:Gene Editing; D006434:Hemocytes / Q000378:metabolism; D009132:Muscles / Q000378:metabolism; D009457:Neuroglia / Q000378:metabolism; D009474:Neurons / Q000378:metabolism; D059767:Recombinational DNA Repair / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics*; D019076:Transgenes / Q000235:genetics*
chemical_list: D029721:Drosophila Proteins; C557617:GAL4 protein, Drosophila; D014157:Transcription Factors
keywords: CRISPR/Cas9; gene conversion; genomic engineering; homology-directed repair; updating transgenes