CRISPR-Cas9 and PiggyBac Mediated Genetic Modification of Sand Fly Vectors Targeting Olfactory and Non-Lethal Phenotypic Genes

Leishmania spp. are protozoan parasites transmitted by female sand flies (Diptera) that cause a spectrum of devastating human pathologies affecting millions. Control of leishmaniases has proved immensely difficult: vector control strategies remain challenging and available chemotherapeutics imperfect. Gene editing of insect vectors offers prospects to interrupt disease transmission; for example by introducing antiparasite effectors or heritable modifications of genes implicated in fecundity. Here we present convergent evidence for successful gene editing using both CRISPR-Cas9 and PiggyBac approaches in two medically important sand fly species (Lutzomyia longipalpis and Phlebotomus papatasi). Targeted mutagenesis in G0 and G1 generations is supported by three independent lines of evidence, namely observable phenotypic changes, PCR-based detection assays, and confirmatory in silico algorithmic analysis of gene sequences which together provide a robust demonstration of targeted genome editing.