An RNA interference (RNAi) toolkit and its utility for functional genetic analysis ofLeishmania (Viannia)

Abstract

RNA interference (RNAi) is a powerful tool whose efficacy against a broad range of targets enables functional genetic tests individually or systematically. However, the RNAi pathway has been lost in evolution by a variety of eukaryotes including most Leishmania sp. RNAi was retained in species of the Leishmania subgenus Viannia, and here we describe the development, optimization, and application of RNAi tools to the study of L. (Viannia) braziliensis. We developed vectors facilitating generation of long-hairpin or stem-loop (StL) RNAi knockdown constructs, using Gateway site-specific recombinase technology. A survey of applications of RNAi in L. braziliensis included genes interspersed within multigene tandem arrays such as QDPR, a potential target or modulator of antifolate sensitivity. Other tests include genes involved in cell differentiation and amastigote proliferation (A600), and essential genes of the intraflagellar transport (IFT) pathway. We tested a range of stem lengths targeting the L. braziliensis hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and reporter firefly luciferase (LUC) genes and found that the efficacy of RNAi increased with stem length, and fell off greatly below about 128 nt. We used the StL length dependency to establish a useful hypomorphic approach not possible with other gene ablation strategies, with shorter IFT140 stems yielding viable cells with compromised flagellar morphology. We showed that co-selection for RNAi against adenine phosphoryl transferase (APRT1) using 4-aminopyrazolpyrimidine (APP) could increase the efficacy of RNAi against reporter constructs, a useful tool that may facilitate improvements in future work. Thus, for many genes, RNAi provides a useful tool for studying Leishmania gene function with some unique advantages.

Co-expression of the AsCas12a ultra variant, a T7 RNA Polymerase and a cytosine base editor greatly increases transfection and editing rates inLeishmaniaspecies

“…Classical pooled CRISPR transfection screening formats are unlikely to be executed successfully, unless additional selective measures are used. While, bar-seq screens (Baker et al, 2021;Beneke et al, 2019;Beneke and Gluenz, 2020) and RNAi methods (de Paiva et al, 2015;Lye et al, 2022) offer potential solutions, they come with clear limitations. Most notably, the use of RNAi is restricted to Leishmania species within the Viannia subgenus, and bar-seq screens pose logistical challenges due to the necessity of generating thousands of individually created mutants.…”

“…Without other means of selection, such as the selection of drug resistance-associated edits, this complicates further the targeting of dispersed multi-copy genes and applications of pooled CRISPR transfection formats. While RNA interference (RNAi) could offer an alternative (de Paiva et al, 2015;Lye et al, 2022), its use is confined to species within the Viannia subgenus, restricting its applicability in the majority of Leishmania species (Lye et al, 2010;Ullu et al, 2004).…”

Introducing the CRISPR/Cas9 cytosine base editor toolbox ‘LeishBASEedit’ – Gene editing and high-throughput screening inLeishmaniawithout requiring DNA double-strand breaks, homologous recombination or donor DNA

“…Currently, bar-seq screening has been the only reported method for large-scale loss-of-function screening in Leishmania and presents the only alternative to RNAi screens, which unlike to Trypanosoma brucei (65)(66)(67)(68), cannot be performed in most Leishmania species (69,70) (apart from Leishmania spp. of the Viannia subgenus (71,72)). However, for a bar-seq screen, each barcoded mutant needs to be created individually by replacing target genes with drug selectable marker cassettes (20,21), making them extremely labour intensive and most likely "one-offs" on a genomewide scale.…”