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Rhodnius prolixus is currently the model vector of choice for studying Chagas disease transmission, a debilitating disease caused by Trypanosoma cruzi parasites. However, transgenesis and gene editing protocols to advance the field are still lacking. Here, we tested protocols for the maternal delivery of CRISPR-Cas9 (clustered regularly spaced palindromic repeats/Cas-9 associated) elements to developing R. prolixus oocytes and strategies for the identification of insertions and deletions (indels) in target loci of resulting gene-edited generation zero (G0) nymphs. We demonstrate successful gene editing of the eye color markers Rp-scarlet and Rp-white, and the cuticle color marker Rp-yellow, with highest effectiveness obtained using Receptor-Mediated Ovary Transduction of Cargo (ReMOT Control) with the ovary-targeting BtKV ligand. These results provide proof of concepts for generating somatic mutations in R. prolixus and potentially for generating germ line-edited lines in triatomines, laying the foundation for gene editing protocols that could lead to the development of novel control strategies for vectors of Chagas disease. 

The piRNA pathway is a specialized small RNA interference that in mosquitoes is mechanistically distant from analogous biology in the Drosophila model. Current genetic engineering methods, such as targeted genome manipulation, have a high potential to tease out the functional complexity of this intricate molecular pathway. However, progress in utilizing these methods in arthropod vectors has been geared mostly toward the development of new vector control strategies rather than to study cellular functions. Herein we propose that genetic engineering methods will be essential to uncover the full functionality of PIWI/piRNA biology in mosquitoes and that extending the applications of genetic engineering on other aspects of mosquito biology will grant access to a much larger pool of knowledge in disease vectors that is just out of reach. We discuss motivations for and impediments to expanding the utility of genetic engineering to study the underlying biology and disease transmission and describe specific areas where efforts can be placed to achieve the full potential for genetic engineering in basic biology in mosquito vectors. Such efforts will generate a refreshed intellectual source of novel approaches to disease control and strong support for the effective use of approaches currently in development. 

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.