Fig. 1: Generation of marker free LmCen^−/− parasite.

a The pLdCN vector used to express Cas9 and gRNAa and gRNAb in Leishmania. A2-IGS, L. donovani A2 gene intergenic sequence; rRNAP, L. donovani ribosomal RNA promoter; H, Hepatitis delta virus ribozyme; HH, Hammerhead ribozyme. b Schematic of gene deletion strategy showing gRNAa and gRNAb targeting sites in the L. major centrin gene locus (LmjF.22.1410) and the expected gene deletion sequence after transfection of the cells with a 50 nucleotide oligonucleotide donor. The primers F1-R1 and F2-R2 used to detect this deletion are indicated. c PCR analysis with primers F1-R1 and F2-R2 revealing loss of the centrin gene. Lane 1, Wildtype L. major; lane 2, L. major centrin null mutant. d Sequence analysis confirming the flanking DNA breaks joined together by the transfected 50 nucleotide oligonucleotide donor. See the supplementary information for the detailed sequence. e An immunoblot, representative of three independent experiments, with an α-LdCentrin antibody showing the re-expression of Centrin in LmCen^−/− parasites transfected with a pKSNeo-LmCEN plasmid (LmCen^−/−-AB, Addback). f LmCen^−/− was unable to induce ear cutaneous lesions in C57BL/6 mice compared to wildtype L. major or the centrin add-back parasites of LmCen^−/− showing restored virulence (green line). C57BL/6 mice (n = 5 per group) were infected intradermally (1 × 10⁶) with LmWT, LmCen^−/− or LmCen^−/−AB parasites and the ear lesion development was monitored weekly. Data is plotted as mean ± SEM. and is representative of two independent experiments. Unpaired two tailed Student’s t test was used to calculate statistical significance between LmWT and LmCen^−/− or LmCen^−/−AB and LmCen^−/− groups (**p < 0.01,***p < 0.001). g Parasite load in the infected ears of the mice (n = 5 per group). Parasite burden was determined by limiting dilution assay. Data is plotted as mean ± SEM. and is representative of two independent experiments. Unpaired two tailed Student’s t test was used to calculate statistical significance. Statistical analysis was performed by unpaired two-tailed t-test (***p < 0.001).