CRISPR-mediated germline mutagenesis for genetic sterilization ofAnopheles gambiaemales

Abstract

ABSTRACTRapid spread of insecticide resistance among anopheline mosquitoes threatens malaria elimination efforts, necessitating development of alternative vector control technologies. Sterile Insect Technique (SIT) has been successfully implemented in multiple insect pests to suppress field populations by the release of large numbers of sterile males, yet it has proven difficult to adapt toAnophelesvectors. Here we outline adaptation of a CRISPR-based genetic sterilization system to selectively ablate male sperm cells in the malaria mosquitoAnopheles gambiae. We achieve robust mosaic biallelic mutagenesis of zero population growth(zpg, a gene essential for differentiation of germ cells) in F1 individuals after intercrossing a germline-expressing Cas9 transgenic line to a line expressingzpg-targeting gRNAs. Approximately 95% of mutagenized males display complete genetic sterilization, and cause similarly high levels of infertility in their female mates. Using a fluorescence reporter that allows detection of the germline leads to a 100% accurate selection of spermless males, improving the system. These males cause a striking reduction in mosquito population size when released at field-like frequencies in competition cages against wild type males. These findings demonstrate that such a genetic system could be adopted for SIT against important malaria vectors.