Genome-scale RNAi screens in African trypanosomes

Anti-trypanosomatid drug discovery: progress and challenges

“…RNA interference loss-of-function screening has been used for these mechanism-of-action studies for several years 126 , and has more recently facilitated identification of the proteasome as the target of a new preclinical candidate in Trypanosoma brucei and Leishmania spp. 54 and, for a separate chemical series, facilitated identification of divalent metal chelation as the mechanism underpinning toxicity 125 ; the latter compounds were deprioritized as anti-leishmanial leads as a result.…”

Paving the Way: Contributions of Big Data to Apicomplexan and Kinetoplastid Research

“…Functional screens generally rely on the generation of mutant parasites en masse followed by specific screening assays to identify subpopulations that meet pre-defined criteria, before matching genotype to phenotype. The repertoire of tools for direct and conditional gene, mRNA and protein regulation developed and optimised for Apicomplexans ( Briquet et al., 2021 ) and Kinetoplastids ( Lander and Chiurillo, 2019 ; Horn, 2022 ) is extensive. Many of these technologies have also been scaled to enable functional screens ( Figure 7 ).…”

“…There are several gene disruption approaches that target all/many genes within the genome ( Gomes et al., 2015 ; Sidik et al., 2018 ; Baker et al., 2021 ; Horn, 2022 ). Additional methods have been developed to study the mutants within a population by: quantifying relative fitness of mutants ( Gomes et al., 2015 ; Sidik et al., 2018 ), studying their localisation, and classification with high-content imaging ( Li et al., 2022 ; Smith et al., 2022 ), and isolating subpopulations with specific phenotypes ( Stanway et al., 2019 ; Harding et al., 2020 ).…”

“…As some kinetoplastids, like T. brucei , have functional, inducible RNA interference (RNAi) machinery, knockdown generation using short hairpin RNA (shRNA) is a widely used method for controlling expression. Other kinetoplastids with non-canonical RNAi mechanisms have been adapted for RNAi knockdowns ( Horn, 2022 ) and even Apicomplexans, like P. berghei , can be adapted to express a minimal, non-canonical RNAi pathway ( Hentzschel et al., 2020 ) enabling the use of RNAi to knockdown expression.…”

“…Improvements to the transfection protocols used on the cultured bloodstage form of T. brucei ( Burkard et al., 2007 ) and the introduction of double stranded breaks ( Glover and Horn, 2009 ) to further enhance efficiency has also enabled screens to be performed on this, more disease relevant, stage. To date, more than 60 screens have been performed on trypanosomes [reviewed in ( Horn, 2022 )] ranging from looking broadly at fitness ( Alsford et al., 2011 ), to identifying mechanisms of drug resistance ( Baker et al., 2011 ; Schumann Burkard et al., 2011 ) and biological processes like DNA repair ( Burkard et al., 2007 ) and cell cycle progression ( Schumann Burkard et al., 2013 ).…”