A New Powerful Method for Site-Specific Transgene Stabilization Based on Chromosomal Double-Strand Break Repair

Transgenic insects are a promising tool in sterile insect techniques and population replacement strategies. Such transgenic insects can be created using nonautonomous transposons, which cannot be transferred without a transposase source. In biocontrol procedures where large numbers of insects are released, there is increased risk of transgene remobilization caused by external transposase sources that can alter the characteristics of the transgenic organisms lead horizontal transgene transfer to other species. Here we describe a novel, effective method for transgene stabilization based on the introduction of directed double-strand breaks (DSB) into a genome-integrated sequence and their subsequent repair by the single-strand annealing (SSA) pathway. Due to the construct's organization, the repair pathway is predictable, such that all transposon and marker sequences can be deleted, while preserving integration of exogenous DNA in the genome. The exceptional conservation of DNA repair pathways makes this method suitable for a broad range of organisms.