A platform for the transgenic modification of sand flies (Lutzomyia longipalpis) and triatomine bugs (Rhodnius prolixus) towards deriving insect vectors refractory to disease agents mediated by CRISPR-Cas9 gene drive

Vector-borne pathogens Leishmania and Trypanosoma cruzi cause the devastating human diseases leishmaniasis and Chagas disease respectively. We will contribute to a new strategy to prevent these diseases by genetically modifying insect vectors, sand flies and triatomine bugs, leading eventually to transgenic insects that are resistant to disease agents.

We will use two different transgenic approaches, PiggyBac and CRISPR-Cas9. PiggyBac involves semi-random insertion of exogenous DNA into the genome. CRISPR-Cas9 is a highly targeted method to manipulate genes. Microinjecting insect embryos with PiggyBac plasmids will produce insects that express fluorescent markers for subsequent CRISPR-Cas9 knockout experiments. Knockout of fluorescent markers demonstrate that system components are functional and will allow us to precisely target genes of interest. We will create knockouts of endogenous genes that are either non-essential or important in sustaining infection in insect vectors. Insertion and expression of exogenous DNA will encourage inheritance to offspring at levels above Mendelian inheritance.

The findings of this study will help in the development of approaches that can interrupt the transmission of diseases by these vector-borne pathogens.