Authors

Discussion

Trypanosomatids cause the neglected tropical diseases sleeping sickness, Chagas disease and the leishmaniases. New and improved genetic technologies would facilitate studies on these lethal parasites. Scalable precision editing methods, for example, could be used to improve our understanding of potential mutations associated with drug resistance. This is a current priority given that several new anti-trypanosomal drugs, with known targets, are currently in clinical development. We report the development of a simple oligo targeting method for rapid and precision editing of priority drug targets in otherwise wild type trypanosomatids. In Trypanosoma brucei, approx. 50-b single-stranded oligodeoxynucleotides were found to be optimal, multiple base edits could be incorporated, and editing efficiency was substantially increased when mismatch repair was suppressed. Resistance-associated edits were introduced in T. brucei cyclin dependent kinase 12 (CRK12) and cleavage and polyadenylation specificity factor 3, in the Trypanosoma cruzi proteasome b5 subunit, and in Leishmania donovani CRK12. We further implemented oligo targeting for site saturation mutagenesis, targeting a specific codon in T. brucei CRK12, which revealed fourteen resistance-conferring edits encoding six distinct amino acids. The outputs confirm on-target drug activity, reveal a variety of resistance-associated mutations, and facilitate rapid assessment of potential impacts on drug efficacy.