Technology development – genetic approaches for the trypanosomatids

Genetic tools and technologies, applied to the trypanosomatids, have had substantial impacts on efforts to characterize protein function, and to identify drug resistance mechanisms and drug targets. To increase throughput, ‘loss-of-function’, ‘gain-of-function’ and tagging approaches have all been scaled up, typically to achieve genome-scale coverage Indeed, high-throughput approaches have been parallelized, whereby millions of parasites, each with a specific single protein depleted or overexpressed, can be screened in one experiment WCAIR teams have pioneered the development and application of these approaches to drug mode-of-action and drug resistance studies. A current focus is on developing gene editing technologies

We have used RNA interference loss-of-function screening to facilitate drug mechanism-of-action studies for over a decade, as reviewed in 2022 1 , whereas overexpression gain-of-function screening has emerged more recently and more readily yields direct drug-target identification

CRISPR–Cas9-based approaches have revolutionized biotechnology by enabling RNA-programmed targeting of specific chromosomal loci, and Cas9-based gene editing is increasingly impacting drug discovery efforts against parasites

Precision editing of drug targets, for example, facilitates the generation of drug-resistant strains and provides insight into structure–activity relationships Quantitative measures of drug resistance can also be important in determining whether specific mutations have had, or are likely to have, a detrimental impact in a clinical setting

We developed a tightly regulated inducible Cas9-based editing system for T brucei 2 The approach has been used for one-step double-allele knockout, or for precision gene tagging and subcellular localisation studies; we also established a transient delivery format, and the reagents have been shared with the wider research community (https://www.addgene.org/David Horn/). Precision templated base-editing has been particularly powerful for investigating drug-resistance mechanisms

Another new parasite gene editing technology is oligo targeting, which is Cas9-independent, and has now been used to edit priority drug targets in T brucei, T cruzi and Leishmania 3; the WCAIR team won the School of Life Sciences best innovation prize 2022 for this work We are currently scaling up oligo-targeting to increase throughput. The approach can now be used to generate ‘libraries’ of >1000 mutants for parallelized drug-resistance screening against priority drug targets, and to profile all possible mutations around each drug-binding pocket. Oligo-targeting subsequently has the potential to be the method of choice to address a variety of important questions, and to greatly facilitate studies on drug resistance and more.

Thus, WCAIR have developed and implemented several novel genetic approaches, innovative technologies that broadly facilitate research on the parasites we study

These developments meet a key goal for WCAIR, which is to develop new technologies that make drug discovery for neglected tropical diseases faster and smarter, and we look forward to delivering further technological advances in this area