Accelerated homologous recombination and subsequent genome modification in Drosophila

Development. 2013 Dec;140(23):4818-25. doi: 10.1242/dev.100933. Epub 2013 Oct 23.


Gene targeting by 'ends-out' homologous recombination enables the deletion of genomic sequences and concurrent introduction of exogenous DNA with base-pair precision without sequence constraint. In Drosophila, this powerful technique has remained laborious and hence seldom implemented. We describe a targeting vector and protocols that achieve this at high frequency and with very few false positives in Drosophila, either with a two-generation crossing scheme or by direct injection in embryos. The frequency of injection-mediated gene targeting can be further increased with CRISPR-induced double-strand breaks within the region to be deleted, thus making homologous recombination almost as easy as conventional transgenesis. Our targeting vector replaces genomic sequences with a multifunctional fragment comprising an easy-to-select genetic marker, a fluorescent reporter, as well as an attP site, which acts as a landing platform for reintegration vectors. These vectors allow the insertion of a variety of transcription reporters or cDNAs to express tagged or mutant isoforms at endogenous levels. In addition, they pave the way for difficult experiments such as tissue-specific allele switching and functional analysis in post-mitotic or polyploid cells. Therefore, our method retains the advantages of homologous recombination while capitalising on the mutagenic power of CRISPR.

Keywords: Drosophila; Functional genomics; Gene targeting; Homologous recombination.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Clustered Regularly Interspaced Short Palindromic Repeats / genetics
  • DNA / genetics
  • DNA Breaks, Double-Stranded
  • Drosophila melanogaster / genetics*
  • Gene Targeting
  • Genetic Markers
  • Genetic Vectors / genetics*
  • Homologous Recombination
  • Mutagenesis, Insertional
  • Recombination, Genetic*
  • Sequence Deletion


  • Genetic Markers
  • DNA