Current control strategies for mitigating the impact of Leishmaniasis via vector control do not provide a panacea. New strategies for vector control are required. This research aims to develop novel molecular tools to interrupt transmission of Leishmania by the manipulation of olfactory genes, affecting host seeking, and subsequently spreading traits rapidly through insect populations by the application of gene drive technologies. CRISPR-Cas9 offers unparalleled capacity to manipulate eukaryotic genomes. The technology has been applied successfully to a variety of invertebrate species, initially to knockout genes, and more recently to insert exogenous DNA such as expression of anti-parasite peptides, and to target genes involved in fecundity. This method shows promise in the context of interrupting transmission of pathogens, and will be developed in phlebotomine sand flies. The research has three main objectives. First, to identify and rationalise endogenous gene targets for proof-of-concept CRISPR-based modification in sand flies. Endogenous non-lethal phenotypic marker genes were identified, alongside olfactory genes involved in host seeking behaviour. Three key gene families were identified as playing an important role in olfaction and host detection in sand flies. Second, to develop a suite of modification tools to deliver CRISPR-Cas9 components using plasmid
and non-plasmid based approaches, to demonstrate precise knockouts of these targeted genes. An in vitro cell line platform was developed and optimised for validation of these tools via chemo-transfection, prior to in vivo studies. Third, to attempt in vivo modification of sand flies. CRISPR tools targeting phenotypic marker genes and olfactory genes were delivered to sand fly embryos by conventional microinjection, and assessment of modifications was conducted using phenotypic observation, heteroduplex assessment, and Sanger sequence and computational methods. Overall, these objectives aimed to lay the foundations for the development of CRISPR-based tools towards novel gene drive control strategies for Leishmania vectors.