Construction of CRISPR-Cas9 plasmids to dissect the PI3K/AKT/Foxo pathway in Aedes aegypti

Abstract

CRISPR-Cas9 is the most accurate genetic engineering tool that is currently in use to knockout genes. The VigLab, led by Professor George Christophides, has been playing a leadership in developing CRISPR-Cas9-based constructs which express Cas9 and sgRNAs to drive highly specific knockouts and/or knockins of target genes. The VigiLab is currently funded by the Bill and Melinda Gates Foundation to use this methodology for the production of GM mosquitoes which are refractory to Plasmodium, the malaria parasite. Under this fellowship, we aim to leverage on the methodology developed by the Vigilab to design optimized constructs to knockout important components of the PI3K/AKT/Foxo pathway in our laboratory. We have identified the PI3K/AKT/Foxo pathway as a key regulator of mosquito gut homeostasis, which controls the expression of REDOX and antiviral-related genes and influences the load and diversity of gut bacteria. The student will also learn important techniques used by the Vigilab to study the involvement of bacteria in the control of gut homeostasis in mosquitoes. Our group at Unesp has already a solid partnership with Prof. Chritophide's group at Imperial College and this be instrumental to improve the ones currently employed by our group to develop CRISPR-Cas9 mutant mosquitoes. (AU)