May, N. H. | bioRxiv Science

bioRxiv2025

Genome-wide CRISPR base-editing screening defines drug response networks in Leishmania

We present a CRISPR/Cas9 cytosine base editing library for genome-wide loss-of-function screening in L. mexicana and apply it to dissect the genetics of Leishmania drug response. The results, accessible at https://www.LeishBASEeditDB.net, revealed novel response biomarkers for SbIII, miltefosine, amphotericin B, pentamidine, and the arylmethylaminosteroid 1c. We identified hundreds of loci linked to altered drug responses, including opposing effects among paralogs, cross-resistance, and collateral sensitivity. Among 41 validated candidates, we identified sterol defects in two novel amphotericin B markers, discovered a regulator of intracellular miltefosine transporter complex localization, and uncovered evidence that flagellar-associated defects reduce drug sensitivity. Parallel viability and motility screens revealed, for the first time, genome-wide fitness contributions of unique and multi-copy genes in Leishmania promastigotes. Our approach enables powerful reverse genetic screens across Leishmania species, advancing drug mechanism studies and guiding combination therapy designs. The library is available for others to screen a multitude of additional loss-of-function phenotypes.

microbiology↗

bioRxiv2024

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

The ability to analyse the function of all genes in a genome is highly desirable, yet challenging in Leishmania due to a repetitive genome, limited DNA repair mechanisms and lack of RNA interference in most species. While our introduction of a cytosine base editor (CBE) demonstrated potential to overcome these limitations (Engstler and Beneke (2023)), challenges remained, including low transfection efficiency, variable editing rates across species, parasite growth effects, and competition between deleterious and non-deleterious mutations. Here, we present an optimized approach addressing these issues. We identified a T7 RNAP promoter variant ensuring high editing rates across Leishmania species without compromising growth. A revised CBE single-guide RNAs (sgRNAs) scoring system was developed to prioritize STOP codon generation. Additionally, a triple-expression construct was created for stable integration of CBE sgRNA expression cassettes into a Leishmania safe harbor locus using AsCas12a ultra-mediated DNA double-strand breaks, increasing transfection efficiency by [~]400-fold to one transfectant per 70 transfected cells. Using this improved system for a small-scale proof-of-principle pooled screen, we successfully confirmed the essential and fitness-associated functions of CK1.2, CRK2, CRK3, AUK1/AIRK, TOR1, IFT88, IFT139, IFT140 and RAB5A in L. mexicana, demonstrating a significant improvement over our previous method. Lastly, we show the utility of co-expressing AsCas12a ultra, T7 RNAP and CBE for hybrid CRISPR gene replacement and base editing within the same cell line. Overall, these improvements will broaden the range of possible gene editing applications in Leishmania species and will enable a variety of loss-of-function screens in the near future.

molecular biology↗