RagA is essential for Leishmania.

A. Strategy used to generate the single allele or double allele L. donovani RagA disruption mutants. A gRNA was designed to target the first half of the RagA coding sequence. L. donovani 1S2D cells were transfected with a CRISPR vector (pLdCNld131620) expressing this RagA specific gRNA, followed by transfection of the bleomycin selection marker donor with 25 nt flanking sequences to integrate into the Cas9 cut site. B. PCR analysis of the surviving phleomycin resistant clones showing the bleomycin resistance gene was inserted into the target site of one RagA allele as expected, but the 1118 bp WT RagA band was still detected in all these phleomycin resistant clones. Note: the middle band is the annealing product during PCR between the 1118 bp WT RagA band and the 1658 bp disruption band. C. Microscope images showing the disruption of both RagA alleles is lethal for L. donovani. The RagA+/- partial mutant cells expressing the RagA targeting gRNA were cloned in a 96 well plate and cell growth was monitored by microscopy. The image for RagA +/- cells was taken one week after cloning; The image for RagA-/- cells was taken four weeks after cloning.