Discussion

 Leishmaniasis is a group of diseases caused by different species of protozoan parasites belonging to the genus Leishmania, which produce extensive morbidity and mortality in humans. Although there are some drugs available to treat these diseases, several problems associated with the uses of these drugs such as the high toxicity and the appearance of resistance are commonly reported. Thus, the development of new techniques, which are scalable to the entire genome, to understand the mechanisms of action and resistance of new and old drugs are necessary. We generated a new strategy to study the whole ORFome of Leishmania parasites using CRISPR-Cas9. To test our approach, first we designed a vector compatible with illumina sequencing to express the gRNA in L. infantum parasites expressing Cas9. As a proof of principle, we chose 4 candidates genes: miltefosine transporter (MT), aquaglyceroporin 1 (AQP1), nucleoside transporter (NT1) and thymidine kinase (TK), the disruption of which were shown to lead to the acquisition of resistance to the drugs miltelfosine, trivalent antimony SbIII, tubercidin, and 5-Fluorouracil, respectively. For each gene we designed a total of 6 gRNA (24 in total) that were pooled. After validating the diversity of this small library, the pool of gRNA was transfected in Cas9-expressing L. infantum promastigotes that were then independently selected for each drug using concentrations equivalent to 2.5x and 5x the IC₅₀. In all cases we obtained parasites growing under drug pressure while mock-transfected parasites failed to grow. Sequencing the gRNA vector recovered from these parasites confirmed the targeting of the expected gene for each drug: MT-targeting gRNA for miltefosine-selected parasites, AQP1-targeting gRNA were recovered from SbIII-selected parasites, and so on. Gene deletions were confirmed by PCR, even for genes located on polyploid chromosomes. Following this proof of principle, we have now synthesized 48.000 gRNA. This library will be transfected in Leishmania which will be selected with drugs for whole genome loss of function screens.