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

Understanding the genetics of drug response in the protozoan Leishmania is critical for treatment strategies but is hindered by the parasites lack of RNAi and non-homologous end-joining. Here, we addressed this using CRISPR/Cas9 cytosine base editing for genome-wide loss-of-function screening in L. mexicana. The resulting datasets, accessible at www.LeishBASEeditDB.net, revealed numerous novel resistance and sensitivity biomarkers across five compounds: SbIII, miltefosine, amphotericin B, pentamidine, and the experimental arylmethylaminosteroid 1c. Key findings include transporter-linked cross-resistance, opposing drug responses among paralogs, and collateral sensitivities between sterol and sphingolipid metabolism. Among 41 validated candidates, we identified sterol defects in two novel amphotericin B resistance markers, discovered a regulator of tubulovesicular localization of the miltefosine transporter complex, and uncovered evidence for flagellar-mediated drug uptake. Parallel genome-wide fitness and motility screens mapped essential genes and revealed persister-like phenotypes. Our approach enables powerful reverse genetic screens across Leishmania species, advancing drug mechanism studies and guiding combination therapy designs.