African sleeping sickness and related livestock diseases cost developing economies an estimated $4 billion each year, yet researchers still test parasite genes one at a time in large groups of infected animals. A new technique called DRiF-Seq changes this: it tracks thousands of genetically modified trypanosomes simultaneously in a single animal, slashing the number of mice or cattle needed for a given experiment by more than 100-fold while actually improving statistical reliability. The problem is that most labs still use the old, animal-heavy approach. This project will train researchers from multiple countries—spanning medical, veterinary, and fundamental biology—in the hands-on production, processing, and analysis of mutant libraries. The team will also create detailed protocols so other labs can adopt the method independently. If successful, this training programme will rapidly cut animal use in trypanosome research worldwide. More broadly, it provides a concrete template for applying parallelised genetic screening to other infectious diseases, potentially transforming how infection biology experiments are designed and reducing animal numbers across the field.
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Human African trypanosomiasis, caused by subspecies of Trypanosoma brucei, is a deadly neglected tropical disease of sub-Saharan Africa. Related diseases in cattle (caused predominantly by T. congolense and T. vivax) place an estimated ~$4 billion burden on developing economies in Africa, Asia and South America. No vaccine currently exists, and drug resistance is a known issue. As such, the function of trypanosome genes and their role in disease is of great scientific interest for understanding parasite biology, identifying virulence factors and tackling drug resistance. Typically, functional genetic studies in trypanosome research infect genetically identical single mutants into cohorts of animals and, as a result, use a large (and growing) number of experimental animals. There is great opportunity for reduction in animal usage in infection research by exploiting next-generation sequencing and organism barcoding to test pathogen gene mutant phenotypes in parallel. Building directly on on-going NC3Rs funding, we have demonstrated that replacing traditional gene-by-gene approaches with parallelisation can achieve a very substantial reduction in animal usage in trypanosome functional genetics. Our high-throughput phenotyping technology (DRiF-Seq) generates and quantitatively tracks 1000s of mutants in individual animals with greater statistical power than gene-by-gene approaches. This increases robustness and reliability, measures mutant-mutant variation and potentially achieves >100-fold reduction of animal usage for specific tests. Here we propose to accelerate the adoption of parallelisation in trypanosome research by transfer of skills in DRiF-Seq to a number of key early-adopter labs. These partner labs encompass multiple disciplines and applications (medical, veterinary, and basic biology), are trans-national, and have animal experiments that could immediately be reduced by parallelisation. They are also very well placed to encourage uptake of methods to further labs in infection biology. In this proposal, we will provide direct, hands-on training in the developers’ lab in the methods of mutant library production, processing and analysis, while also creating comprehensive protocols documentation for know-how transfer that can be followed elsewhere. This will greatly accelerate uptake of this specific reduction method in trypanosomes to maximise the 3Rs impact of on-going work, but also provides an example that should stimulate adoption of similar parallelisation approaches more widely in infection research.
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