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Appropriate selection drugs were added to the medium when necessary as indicated below. Cultures were subcultured to fresh medium every 3–4 days. major 5-ASKH (MHOM/SU/73/5-ASKH), and their genetically modified derived lines reported in this study were routinely grown at 25 ☌ in monophasic M199 medium (Sigma-Aldrich, München, Germany) supplemented with 20% heat-inactivated fetal calf serum (Sigma-Aldrich), 10 mg/L hemin, 100 μM adenine, 5 μM 6-biopterin, 40 mM HEPES (pH 7.4), 2 mM L-glutamine, 100 units/ml penicillin and 100 μg/mL streptomycin (hereafter referred as complete M199 medium).

braziliensis strain PER005 (MHOM/PE/01/LH2182(PER005)) clone 2 (clone originally derived from a clinical isolate), L. This facilitates deletion of essential genes and observation of the cell biological and morphological effects on living cells in a time-dependent manner. CRISPR gene editing also allows for in situ addition of flanking loxP sites to a gene of interest and the subsequent rapamycin-inducible gene deletion by dimerisable Cre (DiCre) recombinase. Third, both single and multigene families can be targeted with this system, and it even allows simultaneous editing of multiple loci, as well as the identification of essential genes. Second, the generation of CRISPR-derived null mutants is facilitated by the use of donor DNA repair cassettes (containing antibiotic selection markers) flanked by short homology arms targeting the gene of interest (GOI), in a single transfection. This is particularly the case when a gene required for optimal in vitro survival and/or growth is targeted, since Leishmania have the remarkable ability to adapt to environmental changes by chromosome copy number variations. braziliensis by CRISPR–Cas9-mediated gene editing sets the stage for testing the role of specific genes in that parasite’s biology, including functional studies of virulence factors in relevant animal models to reveal novel therapeutic targets to combat American tegumentary leishmaniasis.įirst, CRISPR–Cas9 allows the rapid generation of gene deletion or gene disruption mutants in the promastigote stage (within 1–2 weeks depending on the species) thus minimising the occurrence of compensatory adaptations in the parasites. In summary, the feasibility of genetic manipulation of L. major HSP23 null mutants could be complemented phenotypically with transgenes from a range of trypanosomatids. The function of HSP23 is indeed conserved throughout the Trypanosomatida as L. braziliensis that matched the phenotypes reported previously for the respective L. We obtained homozygous Cas9-free HSP23- and HSP100-null mutants in L. braziliensis single-copy genes ( HSP23 and HSP100).

As proof of principle, we demonstrate the targeted replacement of a transgene ( eGFP) and two L. braziliensis that was previously developed for Old World Leishmania major and New World L. In this study, we successfully applied a cloning-free, PCR-based CRISPR–Cas9 technology in L. braziliensis through reverse genetics analyses has so far lagged behind in comparison with Old World Leishmania spp. Despite its importance, the study of the unique biology of L.

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braziliensis) is the main cause of human tegumentary leishmaniasis in the New World, a disease affecting the skin and/or mucosal tissues. The protozoan parasite Leishmania ( Viannia) braziliensis (L.

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