Fig. 1

Gene editing of pfexo. (A) CRISPR-Cas9 editing was used to install the pfexoE415G variant codon into the endogenous locus. Using a donor plasmid and Cas9-aided homologous recombination, the region of the pfexo encoding Met409 to Glu514 was replaced with a recodonized pfexo gene (navy blue) with the point mutation at residue 415 (a cyan star) from Glu to Gly. Stop codon is represented as a red hexagon. Position of hybridisation of primers used for confirmation of the integration event by diagnostic PCR are shown as coloured arrows. A schematic of the co-transfected plasmid for expression of Cas9 nuclease and the guide RNAs is also shown. (B) Diagnostic PCR analysis of genomic DNA of the control parental B5 line, the transfected parasites prior to limiting dilution cloning and clone parasite lines expressing pfexoE415G (clone B8). Expected sizes of the various PCR amplicons are indicated on the right of the gel, whilst the left-hand lane contains double-stranded DNA ladder marker fragment (Quick-load Purple 1 kb Plus DNA ladder (New England Biolabs)). (C) Chromatograms of a region of exon 2 obtained from the PCR product amplified pfexo gene of B5 and B5-rexo-E415G-B8 lines. The codon “GAG” encoding Glu in B5 parasites is changed to “GGC” encoding Gly in B5-rexo-E415G-B8 clone