Abstract
Visceral leishmaniasis (VL) caused by parasites of the L. donovani complex is the most severe form of leishmaniasis. Transmission is widespread in Asia, the Americas and the Mediterranean region (WHO). Currently, most genetic studies are performed on L. mexicana responsible for cutaneaous leishmaniasis. Here, we sought to establish a strain of the L. donovani complex that could be genetically modified to study VL. We selected and screened four strains of the L. donovani complex. Growth rate of the promastigote forms, ability to differentiate into axenic amastigotes and to infect macrophages were discriminating factors in the selection of L. infantum S9F1 (MHOM/MA/67/ITMAP263) as a promising strain for in vitro and in vivo studies. To further characterize this strain, we used MTT assays to determine the IC50 values of drugs used as selection markers for genetic engineering. We adapted the CRISPR-Cas9 system previously established in L. mexicana (Beneke et al. R Soc Open Sci,2017) which enables tagging and knockout of non-essential genes. We have adapted the system combining diCRE and CRISPR-cas9 that we previously developed (Yagoubat A et al. Cell Microbiol 2020) to perform inducible knock-out of essential genes. We also tested and characterized the highly efficient ribonucleoprotein-based CRISPR-Cas9 strategy (Soares Medeiros LC et al.mBIO 2017) where Cas9 and SgRNA are produced in vitro and complexed prior to transfection, and which enables marker-free edits. We would like to share the strain with anyone in the community interested in deciphering the molecular mechanisms of VL.