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The most devastating disease affecting the global citrus industry is Huanglongbing (HLB), caused by the pathogen Candidatus Liberibacter asiaticus. HLB is primarily spread by the insect vector Diaphorina citri (Asian Citrus Psyllid). To counteract the rapid spread of HLB by D. citri, traditional vector control strategies such as insecticide sprays, the release of natural predators, and mass introductions of natural parasitoids are used. However, these methods alone have not managed to contain the spread of disease. To further expand the available tools for D. citri control through generating specific modifications of the D. citri genome, we have developed protocols for CRISPR-Cas9-based genetic modification. Until now, genome editing in D. citri has been challenging due to the general fragility and size of D. citri eggs. Here we present optimized methods for collecting and preparing eggs to introduce the Cas9 ribonucleoprotein (RNP) into early embryos and alternative methods of injecting RNP into the hemocoel of adult females for ovarian transduction. Using these methods, we have generated visible somatic mutations, indicating their suitability for gene editing in D. citri. These methods represent the first steps toward advancing D. citri research in preparation for future genetic-based systems for controlling HLB. 

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 non-specialist laboratories. Here, we adapted the ReMOT Control ( Re ceptor- m ediated O vary T ransduction of C argo) 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.