CRISPR-Cas9 Knockdown of a Sex Determination Gene in Aedes albopictus

Aedes mosquitoes are prominent transmitters of neglected tropical diseases such as dengue, Zika, Yellow Fever, and lymphatic filariasis among others. Based on Liu et al.'s paper, Nix has been identified as a sex-determining gene for the Aedes mosquito. The aim of this project is to investigate the role of Nix in regulating the sex determination pathway in C6/36 cell line and conduct Nix gene editing through the CRISPR/Cas9 system to alter mosquito sex. Altering mosquito sex determination would constitute a breakthrough in mosquito control, and would prevent vector-borne diseases from spreading. Specific patterns of expression of the genes dsx and fru influence the development of male and female characteristics in insects. In order to investigate whether Nix plays a role in the regulation of the sex determination pathway in C6/36 cells and in the RNA splicing of the doublesex and fruitless RNA transcripts (fru and dsx are genes regulated by Nix in the sex determination pathway), RNA expression of dsx and fru gene will be examined after the knockout of Nix in C6/36 cells. Increasing activity of genes related to female development may indicate a transition from a masculinized to a more feminine appearance. Our hypothesis suggests that by deactivating Nix, there would be an increase in the activity of both dsx and fru, indicating a potential shift towards feminization in Aedes mosquitoes. This understanding could significantly contribute to strategies aimed at controlling mosquito populations, such as Sterile Insect Technique (SIT), and reducing the spread of vector-borne diseases.