(a) Schematic of the plasmids used for genome editing. pCas9-eba175 sgRNA-T and pCas9-pUC19 sgRNA-T encode Cas9 and the bsd selection marker as described in Figure 2a, and produce the eba-175 (test) and pUC19 (control) sgRNA-T respectively, from a T7 promoter-driven cassette. (b) The eba-175 locus and sgRNA-T target site are illustrated, together with the expected editing outcome and location of diagnostic PCR primers to assess locus modification (not drawn to scale). (c) PCR and (d) Southern blot analyses of the pUC19- and eba175- sgRNA-T transfected parasite populations to screen for the expected editing outcome. (e,f) Independent triplicate experiments were analyzed as in c,d above at the population level by PCR (e) and Southern blot (f). DNA isolated from one pUC19-sgRNA-T and two eba175-sgRNA-T transfections (indicated by asterisks) were selected for Southern blot analysis.