Gino Lucas Turra

• Compared to canonical model organisms, the genetic toolbox of Kinetoplastid parasites have a considerable gap in the transgenic techniques available. The implementation of the CRISPR/Cas9 technology is poised to transform the way we perform genetic manipulations and offers a new and exciting horizon for molecular parasitology. In this study, we use the Kinetoplastid parasite Leishmania tarentolae as a model organism. This unicellular eukaryote is an attractive model for both basic and applied research. Understanding Leishmania’s basic biology is valuable to underpin differences to the host that might help to treat infectious diseases. Furthermore, it also provides new examples of non-conserved mechanisms that will help to understand the fundamental principles of the biology of eukaryotes and their evolution. In this work, the CRISPR/Cas9 system was used to study mitochondrial protein import. Here I show the efficacy of CRISPR/Cas9 to generate knockout and knockin mutants. Proof- of- concept gene PF16 was used to generate knockout immotile parasites and knockin fluorescent mutants fused with mCherry. The APRT gene was also knocked out showing resistance to APP. In addition, I generated endogenous mutants of a constituent of the mitochondrial import machineries, the sulfhydryl oxidoreductase Erv. I showed that the KISS domain and cysteine 17 are dispensable for survival dismissing that their functions correlate with the essential operation/s of Erv. I report that the ERV gene and the intervening sequences of its shuttle pair cysteines are refractory to ablation and modification, respectively, indicating that they are essential for survival. I also generated Erv interactomes using full-length and mutant (ErvΔKISS) baits showing candidates with hitherto unknown functions that might be related to Erv function. I also tested the glmS riboswitch and generate endogenous mutants with CRISPR/Cas9. We asked if it was possible in Leishmania to obtain knockdown mutants with this technique. The evidence of this study indicates that the system is inefficient in provoking a knockdown phenotype for the genes characterized. An alternative negative marker was also developed in this work. I propose the APRT gene as a novel and efficient counter-selectable marker as compared to the current yFCU and TK genes. The implementation of this system could lead to first shuffling experiments that are not feasible in Leishmania further highlighting the value of this model organism.