Novel control strategies for Lutzomyia longipalpis sand flies through CRISPR-Cas9 genetic modification of olfaction and Wolbachia transinfection

Leishmania, (a protozoan) transmitted by Sandfly insect vectors, is responsible for a huge burden of human suffering (Leishmaniasis). Current control strategies for mitigating the impact of Leishmaniasis via vector control do not provide a panacea. New strategies for control are required. Two novel methods will be explored in this project: (i) CRISPR/Cas9 genetic modification to disrupt olfaction and modify host seeking behaviour, (leading to the interruption of human biting and so parasite transmission). (ii) wolbachia transinfection to generate sandflies refractory to infection by Leishmania parasites. Several genetic techniques have been developed to edit DNA, however they are not precise in manipulation of specific genes of interest. In contrast, the CRISPR/Cas9 system provides precise targeting, both to knockout specific genes and insert anti-parasite genes in insect vectors. Briefly, a CRISPR/Cas9 system comprises an endocuclease (molecular cutting machinery) which can be directed to any chosen locus (by guide RNA) inducing a gene knockout. A refined version of the system can allow integration (knock in) or further manipulation of genes. The initial goal of this project is to build and demonstrate a functional CRISPR/Cas9 plasmid construct for Lutzomyia longipalpis and Phlebotomus papatasi with a knock out element. This element will initially target non-lethal genes to produce phenotypic change such as eye colour, to confirm expression of construct components and the ability to induce targeted knockouts. Next, olfactory candidate genes that influence host seeking behaviour will be identified using a rationalised approach, including literature and BLAST based searches to identify orthologs in closely related diptera with conserved sequence function and non-lethality. Secondarily a refined CRISPR/Cas9 construct will be developed allowing for "knock in" of specific exogenous genes. Knock-out and knock-in genetic constructs will be transfected into sandflies, and behavioural change experiments conducted. Finally, I will develop tools for a gene drive system to ensure the modifications will be inherited by progeny at a higher rate than mendelian inheritance to spread traits rapidly through insect populations. For the second novel control method, tools for wolbachia transinfection will be developed. Wolbachia is maternally inherited bacteria infecting arthropods where it has inhibitory effects on the transmission of many human pathogens: dengue, chikungunya, yellow fever, filarial nematodes and Plasmodium sp. However inhibitory effects in sandflies and Leishmania parasites are unknown. I will develop a stable line of wolbachia-infected sandflies (via microinjection), verified by Fluorescent in situ hybridisation (FISH), using probes with appropriate fluorescent labels. Subsequently, stabley transfected sandflies will be infected with Leishmania to study the impact of the wolbachia on Leishmania development and transmission. Several techniques will be used to assess the impact on transmission including dissections of the proboscis and salivary glands, forced salivation to determine parasite burden, along with other quantitative assessments including qPCR. The current project fits with the MRC remit incorporating interdisciplinary skills spanning whole organism physiology (including biochemistry and bioengineering) of disease vectors and the development of innovative tools for genetic manipulation. The project has the potential to contribute to new strategies applied to leishmaniasis to alleviate an enormous burden of human suffering.
Skills: Molecular biology and bioinformatics skills, Bioassay development, Microinjection, Advanced transgenic techniques, Fluorescent imaging techniques, Handling of Cat3 organisms.
Key words: CRISPR/Cas9, Gene drive, genetic modification, insect vectors, olfactory genes, host seeking, wolbachia transinfection, microinjection, behavioural experiments, transgenics, sandflies.