Now you see me, now you don’t: Using CRISPR/Cas9 to delete engineered genes in the yellow fever mosquito

The yellow fever mosquito, Aedes aegypti, is a human disease vector responsible for over 400 million dengue virus infections each year. Aedes genetic control approaches, such as homing gene drives, use cellular DNA repair to achieve higher than normal rates of gene inheritance to reduce mosquito-borne disease. Previously, we proposed the use of the single-strand annealing (SSA), a less common DNA repair mechanism capable of deleting entire genes from the genome, as a gene drive reversal approach. Here, we use CRISPR/Cas9 genome editing in Aedes aegypti to trigger multi-gene deletion by SSA repair and learn how CRISPR target site influences gene editing outcomes. Clusters of synthetic guide RNAs were developed with target regions located at various distances from the engineered direct repeats, with SSA readily occurring with double-strand breaks >1kb away from repeated sequences. We also observed a correlation between overall CRISPR editing activity and frequency of gene deletion by SSA. Further, we notice that the presence of repetitive DNA surrounding the target site drives specific and repeatable deletion sizes. This work furthers our understanding of DNA repair in Aedes aegypti and expands the molecular toolkit to reverse-engineer gene drive technology. We hope what we learn about Aedes SSA repair can help inform future gene drive control approaches and make their applications more acceptable to the public and regulators.