Fig. 4.

PfEBA165 frameshift correction results in down-regulated transcription. a Schematic showing genome editing strategy. P. falciparum 3D7 parasites were transfected with plasmids containing Cas9 expression cassettes and sgRNAs targeting near the two PfEBA165 frameshifts present in the endogenous gene, along with linear PCR donor templates containing corrected versions of these frameshifts. Cas9 mediated homologous recombination would result in correction of either one or both frameshifts, depending on whether one or both sgRNA containing plasmids were transfected. The plasmids contained the human dihydrofolate reductase (hDHFR) gene for selection, Cas9 and sgRNA expression were driven by endogenous calmodulin (cam) and U6 promoters, respectively, b Sanger sequencing of cloned double transfectant parasite line (C10) confirms correction of both frameshifts, with correction for 5′ frameshift shown in top row and 3′ frameshift shown below. Blue boxes outline sgRNA target sequences and green boxes outline PAM sites within the target sequence. The donor template incorporated changes that eliminate the PAM site, preventing re-cleavage of corrected genes, but do not affect the translated amino acid sequence. c Quantitative RT-PCR of PfEBA165 expression in 3D7 (parental line). YB4 (cloned transfectant with the 3′ frameshift corrected) and C10 and D3 (cloned transfectant lines with both the 5′ and 3′ frameshifts corrected). To control for differences in relative abundance of mature schizonts in the parasite culture before RNA extraction, PfEBA165 transcript levels were normalized to Pfcyp87 (stable expression throughout cell cycle) and expressed relative to PfAMA1 (late-stage specific expression). Data points represent mean values of three biological replicates. Error bars represent standard deviation and statistical significance was determined using a one-way ANOVA with Dunnet’s multiple comparison test for each erythrocyte category. **p < 0.01. Source Data are provided as a Source Data File