Background: Mosquito-borne diseases such as malaria, dengue, and chikungunya, cause over 700 million infections and one million deaths each year. To complement traditional vector control methods, novel genetic systems are being developed to drive genetic modifications into wild mosquitoes. These systems copy the mechanisms of natural homing endonucleases to cut and repair DNA, enabling desired modifications to propagate at super-Mendelian rates. To see if exposure to homing endonucleases during genetic crosses has any unintended effects, we engineered transgenic mosquitoes that express two such genome-editing endonucleases, Cas9 and Cre recombinase, and are used for developing anti-Plasmodium effector lines. We used this strain in genetic crosses over multiple generations, to replace the genetic background of our effector line with that of the wild-type. At the same time, we isolated a phenotypically wild-type mosquito line that was exposed to these endonucleases as an internal control. Unexpectedly, this internal control showed reduced fitness. Using an integrated multi-omics approach, including comparative genome sequencing, microbiome analysis, and metabolomic profile comparisons, we identified numerous off-target mutations and a metabolomic shift in this line compared to wild-type mosquitoes that were not exposed to the endonucleases or our effector line.