Knockout of the MISO Gene in the Plasmodium Falciparum Vector Anopheles Gambiae Using the CRISPR-Cas9 System

Abstract

In the course of this thesis I have sought to engineer a line of Anopheles gambiae mosquitoes, the primary vector of the African malaria parasite Plasmodium falciparum, with the Mating Induced Stimulator of Oogenesis (MISO) gene functionally knocked out of the genome. A MISO knockout line (misoKO) will enhance our ability to detect subtle phenotypes and improve the significance of earlier findings regarding MISO influence on egg production, egg development, and tolerance to Plasmodium falciparum infection. I have designed and implemented a piggyBac transgenic system to create a CRISPR/Cas9 mediated knockout of the reproductive gene MISO in Anopheles gambiae. I have isolated one mutant, misoA3ΔA76, which will likely be a hypomorphic allele of MISO. Mutant misoA3ΔA76 features a 290bp deletion of 49 amino acids constituting a removal of 25% of the amino acids in the total MISO sequence. MISO is a highly unstructured protein with no known domains besides an N-terminal secretion sequence. The misoA3ΔA76 mutant’s deletion removes 49 amino acids from the N-terminal unstructured region of the protein, including the putative secretion peptide sequence. The MISO knockout lines we are generating will revolutionize the ability of Professor Flaminia Catteruccia’s research group to interrogate the function of MISO protein activity.