Targeted engineering of the Drosophila genome

The application of phi C31 phage integrase in Drosophila for unidirectional and site-specific DNA integration was pioneered by Groth et al. in 2004,1 and quickly triggered a wave of innovative tools taking advantage of these unique properties of phi C31. Three recent papers have further developed novel approaches that combine the phi C31-mediated DNA integration with the homologous recombination (HR)-based gene targeting(2,3) for the purpose of efficient and targeted modifications of Drosophila genomic loci. Despite significant differences, the general strategies are similar in principle in the SIRT (site-specific integrase mediated repeated targeting) approach by Gao et al.(4) the IMAGO (integrase-mediated approach for gene knock-out) approach by Choi et al. 5 and the genomic engineering approach developed by our group. 6 All three use HR-based gene targeting to first implant a single or a pair of phi C31-attP recombination sites into the target locus. Flies carrying such targeted insertions of attP sites can then be used as "founder lines", in which modified DNA sequences ("knock-in DNA") can be repeatedly and efficiently inserted back into the target locus via phi C31-mediated integration. Thus, by carrying out the targeting experiments only once, one can then directly and efficiently modify the target locus into virtually any desired knock-in allele. Here we give a brief overview of the SIRT, IMAGO and genomic engineering approaches and propose a revised genomic engineering scheme in which a single ends-out targeting event will generate founder lines suitable for both recombinase-mediated cassette exchange (RMCE) and single-site based integration of knock-in DNA.