Fig. 2: Whole-genome analysis of the attenuated LmCen^−/− L. major.

a Sequence coverage across each of the centrin gene family members in the LmCen^−/− L. major. Note that only the targeted LmjF.22.1410 centrin gene has no sequence reads resulting from CRISPR gene editing. b1-b2 Southern blot analysis (b1) revealing the absence of the LmjF.22.1410 centrin gene in the genome of LmCen^−/− parasite compared to wildtype L. major, LmWT. The ethidium bromide-stained agarose gel (b2) showing the BglII digested genomic DNA from LmWT and LmCen^−/−. c Percent sequence coverage (Y-axis) for all protein-coding genes from chromosome 1 to 36 (X-axis) by Illumina sequencing of the whole genome of the LmCen^−/− L. major. The blue line across the X axis is composed of 8307 dots where each dot represents a gene starting from chromosome 1 (left) to chromosome 36 (right) and is placed according to the portion of the open reading frame supported by sequencing reads. Open blue circles indicate genes where misalignments of sequencing Illumina reads occurred for some multicopy genes, although these genes were verified to be intact. Red circles and line markers correspond to the 5 centrin genes across the genome in chromosomes 7, 22, 32, 34 and 36. Only the targeted centrin gene (LmjF.22.1410) has been deleted from the genome and therefore has 0% coverage. d Coverage of the pLdCN CRISPR plasmid sequence generated from whole-genome sequencing. No homologous plasmid sequences were detected in the LmCen^−/− genome except for the positions ~5000 to ~6000 corresponding to the Leishmania donovani A2 gene intergenic sequence (A2-IGS) that were incorporated into the pLdCN plasmid for expression of the Neo^R gene. Therefore, the A2-IGS genomic sequence reads can align to this portion of the plasmid although the pLdCN CRISPR plasmid is not present in LmCen^−/−.