Transgene removal using an in cis programmed homing endonuclease via single-strand annealing in the mosquito Aedes aegypti.

While gene drive strategies have been proposed to aid in the control of mosquito-borne diseases, additional genome engineering technologies may be required to establish a defined end-of-product-life timeline. We previously demonstrated that single-strand annealing (SSA) was sufficient to program the scarless elimination of a transgene while restoring a disrupted gene in the disease vector mosquito Aedes aegypti. Here, we extend these findings by establishing that complete transgene removal (four gene cassettes comprising ~8-kb) can be programmed in cis. Reducing the length of the direct repeat from 700-bp to 200-bp reduces, but does not eliminate, SSA activity. In contrast, increasing direct repeat length to 1.5-kb does not increase SSA rates, suggesting diminishing returns above a certain threshold size. Finally, we show that while the homing endonuclease Y2-I-AniI triggered both SSA and NHEJ at significantly higher rates than I-SceI at one genomic locus (P5-EGFP), repair events are heavily skewed towards NHEJ at another locus (kmo), suggesting the nuclease used and the genomic region targeted have a substantial influence on repair outcomes. Taken together, this work establishes the feasibility of engineering temporary transgenes in disease vector mosquitoes, while providing critical details concerning important operational parameters.