High-efficiency gene editing inAnopheles sinensisusing ReMOT control

Abstract CRISPR/Cas9-mediated gene editing provides an effective method for deciphering the molecular mechanisms underlying mosquito development and mosquito-borne disease transmission, as well as for exploring genetic control strategies. However, delivering the Cas9 ribonucleoprotein complex by embryo injection to produce genetic modifications is challenging, is mostly confined to model mosquitoes and specialized laboratories, and has low editing efficiency. Here, we established an effective Receptor-Mediated Ovary Transduction of Cargo (ReMOT) control method, enabling the introduction of heritable mutations into Anopheles sinensis , the major malaria vector in China and Southeast Asia, via the injection of female adult mosquitoes. Injection of a mixture of P2C-DsRed and saponin resulted in red fluorescence in the ovaries, with a 100% success rate. Using this system, we knocked-out the pigment synthesis genes, Aswhite and Asyellow , using injected wild-type (WT) females mated with WT males, resulting in the highest efficiency of gene editing among mosquitoes under the same mating conditions. Furthermore, the gene-editing efficiency was increased by at least 2.1-fold using injected WT females mated with mutant males. This improved ReMOT control method exhibits high editing efficiency, with important benefits in terms of functional genomics research and genetic control strategies in An. sinensis . Moreover, this represents a convenient method for gene manipulation in laboratories that are unable to perform embryo injection or that lack embryo-injection equipment.