The Cas9 endonuclease can be programmed by guide RNA to introduce sequence-specific breaks in genomic DNA. Thus, Cas9-based approaches present a range of novel options for genome manipulation and precision editing. African trypanosomes are parasites that cause lethal human and animal diseases. They also serve as models for studies on eukaryotic biology, including ‘divergent’ biology. Genome modification, exploiting the native homologous recombination machinery, has been important for studies on trypanosomes but often requires multiple rounds of transfection using selectable markers that integrate at low efficiency. We report a system for delivering tetracycline inducible Cas9 and guide RNA to Trypanosoma brucei. In these cells, targeted DNA cleavage and gene disruption can be achieved at close to 100% efficiency without further selection. Disruption of aquaglyceroporin (AQP2) or amino acid transporter genes confers resistance to the clinical drugs pentamidine or eflornithine, respectively, providing simple and robust assays for editing efficiency. We also use the new system for homology-directed, precision base editing; a single-stranded oligodeoxyribonucleotide repair template was delivered to introduce a single AQP2 - T791G/L264R mutation in this case. The technology we describe now enables a range of novel programmed genome-editing approaches in T. brucei that would benefit from temporal control, high-efficiency and precision.

Heme is an iron-coordinated porphyrin that is universally essential as a protein cofactor for fundamental cellular processes, such as electron transport in the respiratory chain, oxidative stress response, or redox reactions in various metabolic pathways. Parasitic kinetoplastid flagellates represent a rare example of organisms that depend on oxidative metabolism but are heme auxotrophs. Here, we show that heme is fully dispensable for the survival of Phytomonas serpens, a plant parasite. Seeking to understand the metabolism of this heme-free eukaryote, we searched for hemecontaining proteins in its de novo sequenced genome and examined several cellular processes for which heme has so far been considered indispensable. We found that P. serpens lacks most of the known hemoproteins and does not require heme for electron transport in the respiratory chain, protection against oxidative stress, or desaturation of fatty acids. Although heme is still required for the synthesis of ergosterol, its precursor, lanosterol, is instead incorporated into the membranes of P. serpens grown in the absence of heme. In conclusion, P. serpens is a flagellate with unique metabolic adaptations that allow it to bypass all requirements for heme.cytochromes | respiration | sterols | protist