Editing Transgenic DNA Components by Inducible Gene Replacement in Drosophila melanogaster. Issue 4 (1st August 2016)

• Record Type: Journal Article
• Authors: Lin, Chun-Chieh; Potter, Christopher J
• Is Part Of: Genetics. Volume 203:Issue 4(2016)
• Page Start: 1613
• Page End: 1628
• Publication Date: 2016-08-01
• Subjects: gene conversion -- CRISPR/Cas9 -- homology-directed repair -- genomic engineering -- updating transgenes; Genetics -- Periodicals; 576.5
• DOI: 10.1534/genetics.116.191783
• Languages: English
• ISSNs: 0016-6731

Abstract

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.