Editing transgenic DNA components by inducible gene replacement in Drosophila melanogaster

Chun Chieh Lin, Christopher J. Potter

Research output: Contribution to journalArticlepeer-review

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 homology assisted CRISPR knock-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.

Original languageEnglish (US)
Pages (from-to)1613-1628
Number of pages16
JournalGenetics
Volume203
Issue number4
DOIs
StatePublished - Aug 2016

Keywords

  • CRISPR/Cas9
  • Gene conversion
  • Genomic engineering
  • Homology-directed repair
  • Updating transgenes

ASJC Scopus subject areas

  • Genetics

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