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

The ability to analyse the function of all genes in a genome has obvious appeal. However, this has been challenging inLeishmaniadue to a repetitive genome architecture, limited DNA repair mechanisms and the absence of RNA interference machinery in most species. While our previous introduction of a cytosine base editor (CBE) tool inLeishmaniashowcased the potential for bypassing these limits (Engstler and Beneke (2023)), challenges remained in achieving high transfection efficiencies, overcoming species-specific editing rates, minimizing effects on parasite growth and eliminating competition between deleterious and non-deleterious mutations. Here, we present an optimized approach to address these limitations. Firstly, we identified a T7 RNAP promoter variant that ensures high editing rates acrossLeishmaniaspecies without adversely affecting parasite growth. Secondly, we adjusted the scoring of CBE single-guide RNAs (sgRNAs) to prioritize those ensuring STOP codon generation. Thirdly, we developed a triple-expression construct enabling the integration of CBE sgRNA expression cassettes into aLeishmaniasafe harbor locus via AsCas12a ultra-mediated DNA double-strand breaks. This facilitates the generation of stable CBE sgRNA expression cell lines and increases transfection rates by ∼400-fold, resulting in up to one transfectant per 70 transfected cells. Lastly, we show how the co-expression of AsCas12a ultra, T7 RNAP and CBE can be utilized for hybrid CRISPR gene replacement and base editing approaches in the same cell line. Overall, we believe that these improvements will broaden the range of possible gene editing applications inLeishmaniaspecies and will enable a variety of loss-of-function screens in the future.