Cas9-mediated gene-editing in the malaria mosquito anopheles stephensi by ReMOT Control

Innovative tools are essential for advancing malaria control and depend on an understanding of molecular mechanisms governing transmission of malaria parasites by Anopheles mosquitoes. CRISPR/Cas9- based gene disruption is a powerful method to uncover underlying biology of vector-pathogen interactions and can itself form the basis of mosquito control strategies. However, embryo injection methods used to genetically manipulate mosquitoes (especially Anopheles) are difficult and inefficient, particularly for nonspecialist laboratories. Here, we adapted the ReMOT Control (Receptor-mediated Ovary Transduction of Cargo) technique to deliver Cas9 ribonucleoprotein complex to adult mosquito ovaries, generating targeted and heritable mutations in the malaria vector Anopheles stephensi without injecting embryos. In Anopheles, ReMOT Control gene editing was as efficient as standard embryo injections. The application of ReMOT Control to Anopheles opens the power of CRISPR/Cas9 methods to malaria laboratories that lack the equipment or expertise to perform embryo injections and establishes the flexibility of ReMOT Control for diverse mosquito species.