The ability to deliver pathogen-resistance genes into mosquito populations has long been sought as a potential alternative for disrupting dengue or malaria transmission where funds and infrastructure are the limiting factors in effective mosquito control. While effective gene drive transgenes based on CRISPR/Cas9 have been developed for model organism Drosophila and for malaria mosquitoes, Aedes aegypti, the most medically important vector of dengue, yellow fever and chikungunya viruses, lags behind for reasons that remain largely unexplored and unknown. In this project, D. melanogaster and A. aegypti will be employed to evaluate novel hypotheses regarding how genome structure and DNA repair influence both homing gene drive and transgene removal based on single strand annealing. Following from previous work, multigeneration cage experiments will be performed on this transgene removal strategy in the context of an active gene drive in both flies and mosquitoes, followed by a wave of transgene removal (Aim 1). Next, the role of local microhomology, nuclease characterisics and DNA repair protein recruitment will be examined on both the rates of both homing gene drive and transgene removal in A. aegypti, where gene drive has lagged behind (Aim 2). Finally, the role of chromosomal position on both homing gene drive and transgene removal will be tested in the context of both synthetic targets and new haplolethal target genes (Aim 3). This innovative approach takes advantage of naturally occurring processes that are conserved throughout eukaryota to completely eliminate all transgenic sequences following potential field releases. Thus, it is anticipated that this project will have a substantial impact on National and International conversations concerning gene drive technology as a whole, and will raise expectations for what is possible in any future trial to generate pathogen-resistant mosquitoes.