Linf_S9F1_Cas9-T7 whole genome sequencing

The L. donovani complex is responsible for visceral leishmaniasis, a vector-borne disease transmitted through the bite of female phlebotomine sand flies. This disease affects many mammals which can serve as reservoirs with transmission widespread across Asia, Africa, the Americas, and the Mediterranean basin, including South of France. In human, visceral leishmaniasis poses a fatal threat if left untreated. Investigating the pathogenesis is crucial, necessitating a suitable strain to explore the underlying mechanisms. While most genetic engineering studies focus on cutaneous leishmaniasis through manipulation of L. mexicana, no strain within the L. donovani complex has been adequately characterized for genetic engineering studies on visceral leishmaniasis. In this study, we screened four strains of the L. donovani complex. The growth rate of promastigote forms, the ability to differentiate into axenic amastigotes, and the capacity to infect macrophages were key factors in selecting L. infantum S9F1 (MHOM/MA/67/ITMAP263) as a promising strain for both in vitro and in vivo studies. To further characterize this strain, we determined the IC50 values of drugs, used as selection markers for genetic engineering, through MTT assays. Subsequently, we adapted and tested the CRISPR-Cas9 system to tag and delete genes of interest. Overall, our work has enabled the establishment of a versatile toolkit for genetic engineering studies, facilitating a deeper understanding of the pathogenesis of visceral leishmaniasis.