CRISPR-Cas9 Knock-out of Cactus Immunity Gene in Aedes albopictus Mosquitoes

The Aedes genus of mosquitoes are significant vectors for multiple neglected tropical diseases (NTDs) such as dengue fever, yellow fever, and lymphatic filariasis. Aedes albopictus is specifically native to the tropical and subtropical areas of Southeast Asia and is considered to be a potential vector for various bacterial, viral, and microfilarial transmission. Editing genes responsible for mosquito immunity potentially prevent the spread of these diseases by increasing immune activation in mosquitoes against the pathogens that cause the diseases. The Toll pathway plays an important role in mosquitoes' innate immune defense against fungal, bacterial, viral, and microfilaria infections. Degradation of Cactus, a negative regulator located in the Toll pathway, upregulates gene expression for antimicrobial peptides (AMPs) such as defensin and cecropin, which are the predominant and immune-inducible AMPs in mosquitoes. We hypothesize that CRISPR-mediated knock-out of Cactus gene could yield an upregulation of immune response. The study aims to transfect the C6/36 mosquito cell line with a pAc-sgRNA-Cas9 plasmid and a knock-in puromycin selection cassette. Successfully transfected cells are selected in puromycin medium. CRISPR on-target and off-target regions are assessed by PCR and Sanger sequencing. Expression differences of immune genes in Cactus knock-out cells are detected by RT-qPCR. Relative expression level, calculated in 2^-ΔΔCT, is analyzed using one-way ANOVA among clones for each gene of interest.