Human African trypanosomiasis (HAT), American trypanosomiasis and leishmaniasis affect >20 million people per annum. Related parasites cause animal African Trypanosomiasis (AAT), with ~70 million cases of livestock disease per year, creating a great economic burden on rural communities in Africa, Asia and South America. Many labs internationally use functional genetics in HAT and AAT models to identify genes important for infection establishment, survival in the host, transmission, and drug resistance. However, testing of trypanosome gene function in disease models usually involves gene-by-gene approaches of low statistical power, use laboratory-adapted strains that do not represent the behaviour of field isolates, and require large numbers of experimental animals. A substantial reduction of usage could be achieved by replacing traditional methods with parallelisation.
We have designed a method for highly-parallel phenotyping of mutants, Direct RNAi-Fragment Sequencing (DRiF-Seq), that is compatible with parasite strains that capture human disease biology. Pilot data from a set of 145 genes shows the method can be used in animal models to produce robust, quantitative measures of mutant fitness. By following 1000s of individual mutants in a single infection, in vivo DRiF-Seq also captures clonal variation and is sensitive at important points in infection where traditional methods are not, but requires fewer animals than testing a single gene mutant. This Project Grant will demonstrate and validate the applicability of in vivo DRiF-Seq to test gene sets in infection models of HAT such that it can be widely adopted (Reduction). We will also expand in vivo DRiF-Seq to genome scale in trypanosomes causing HAT and the most important AAT species - effectively eliminating new experimental animal usage in basic loss-of-fitness estimates, which make up a large proportion of current usage (Reduction/Replacement).