Fig 2. Generation of the RagC single amino acid substitution (R231C) mutant and RagC disruption mutant by CRISPR/Cas9.

A. Strategy used to generate the RagC R231C mutant and RagC gene disrupted strains. A gRNA was designed to target a site (green) close to the R231C polymorphism (red) identified in the RagC gene of the Sri Lankan cutaneous L. donovani isolate. L. donovani 1S2D promastigotes were transfected with a CRISPR vector (pLdCNld366140) expressing this RagC specific gRNA, followed by transfection of the donor repair template which contained either: the targeted point mutation (C/T, red) and an additional six nucleotides resulting in silent mutations (purple) to protect the repaired genome from subsequent Cas9 cleavage, or a bleomycin selection marker (black). Genomic DNA from these L. donovani cells clones was subjected to PCR and sequencing analysis. B. Partial sequence of the oligo donor repair induced mutations resulting in a single amino acid substitution in RagC protein (R231C, shown in red) and inactivation of the gRNA targeting site (shown in green, interspaced with disrupting silent mutations in purple). C. Direct sequencing of a PCR product amplified from a L. donovani clone showing both alleles of the RagC gene have been edited to the sequence of the oligo donor (see A & B) repair template. D. PCR analysis of RagC double allele gene disrupted mutants. PCR analysis of three phleomycin resistant clones with primers F2 and R2 show the Bleomycin resistance gene has been inserted into the target site as expected resulting in a 987 bp band, and no 447 bp WT F2R2 band was detected in these RagC disruption mutants.