A platform for genome editing insect vectors of leishmaniasis (Lutzomyia longipalpis and Phlebotomus papatasi) and Chagas disease (Rhodnius prolixus) via CRISPR-Cas9 based approaches.

Chagas disease and leishmaniasis currently infect an estimated 11 million people, causing mortality in the thousands and close to 1 million disability-adjusted life years lost. Control efforts, in the context of vector management have had mixed success and new intervention approaches are needed. Research into the genetic modification of insect vectors has been revolutionised by the application of new gene editing approaches such as CRISPR-Cas9. They allow for the precise manipulation of gene targets within multiple eukaryotic species. Utilising techniques such as gene drives it is possible to achieve successful integration of exogenous DNA within the germline, spreading inherited traits through a population at a rate higher than Mendelian inheritance. Recent approaches in the context of medically important insect vectors have focused on the integration and expression of effector molecules, and the targeting of genes affecting reproductive fitness. Both approaches have the capacity to interrupt disease transmission. To date there is a dearth of published information regarding genetic modification of the insect vectors of Chagas disease or leishmaniasis. The research outlined in this thesis has contributed towards the development of CRISPR-Cas9 methodologies applied to the insect vectors of Chagas disease (triatomine bugs) and leishmaniasis (phlebotomine sand flies). We focused on the successful validation of CRISPR-Cas9 systems within the sand flies Lutzomyia longipalpis and Phlebotomus papatasi and the triatomine bug species Rhodnius prolixus. A number of gene targets were identified, which when functionally lost could elicit a phenotypic response in the insects. gRNAs targeting these phenotypic genes were incorporated into CRISPR-Cas9 DNA constructs, which were then transfected into sand fly and triatomine bug embryos. We observed phenotypic changes when targeting cuticle tanning and wing phenotype genes in sand flies. In R. prolixus bugs we utilized CRISPR-Cas9 homology directed repair approaches, potentially integrating exogenous DNA into the genome of embryos as confirmed by PCR. The integration of exogenous DNA via homology directed repair has not previously been achieved in these insects. The gene editing platform we developed here has the potential to contribute the understanding and development of novel control methods for Chagas disease and leishmaniasis, alleviating an enormous burden of human suffering.