Fig. 1

Strategy to introduce the k13-C580Y, fd-D193Y and arps10-V127M alleles. A k13-C580Y mutagenesis. Plasmid pCas9_pfk13g and linearized plasmid pMA_k13MT were co-transfected into the 3D7 parasite; pCas9_pfk13g contains expression cassettes for Cas9 and k13 specific guide RNA for generating a double-strand break in k13. The k13-C580Y mutation was introduced by homology-directed DNA repair using k13HR1 and k13HR2 sequences in pMA_k13MT. The k1/k2 primer pair was used to identify the wild-type genotype (product size = 481 bp) of the unmodified parasite and the k1/k3 pair was used to identify the modified gene (product size = 492 bp). B Genotype analysis by PCR amplification of the modified parasite after cloning. Two transgenic parasite clones (K^MTC1 and K^MTC2) were isolated and tested by PCR analysis in comparison with 3D7 (C) DNA sequence analysis of K^MTC1. The k13-C580Y mutated codon is highlighted in red (TGT(C) ◊ TaT(Y)). D fd-D193Y mutagenesis. The pCas9_pffd and pMK_fdMT plasmid pair was used for Pffd gene modification. The f1/f2 and f1/f3 primer pairs were used to identify unmodified parasites (product size = 704 bp) and modified parasites (product size = 711 bp), respectively. For the sequence analysis, the f4/f5 primer pair was used to amplify and read the sequence. E The genotype analysis of the fd-D193Y mutated parasites. E (upper) PCR result for clonal parasites (F^MTC1 and F^MTC2) with single fd-D193Y mutation. E (lower) PCR result for cloned parasite (K^MTF^MT) with double mutation k13^C580Yfd^D193Y. F DNA sequence analysis result confirming the fd-D193Y mutation: the sequence result for the F^MTC1 parasite (F, upper) and K^MTF^MT (F, lower) with the fd-D193Y mutated codon highlighted in red (GAC(D) ◊ tAt(Y)). G arps10-V127M mutagenesis. The pCas9_pfarps10 and linearized pGemT_arps10MT plasmids were used to modify the arps10 gene in 3D7 and K^MTF^MT parasites. The a1/a2 (product size = 462 bp) and a3/a2 (product size = 459 bp) primer pairs were used to identify unmodified parasite and modified parasite, respectively. The a4/a2 primer pairs were used to amplify and read the sequence for sequence analysis. H PCR genotypic analysis: (H, upper) PCR result for two clonal lines of A^MT. (H, lower) PCR result for one clonal line of K^MTF^MTA^MT. I DNA sequence analysis confirming arps10-V127M mutation. Data are shown for A^MT (I, upper) and K^MTF^MTA^MT (I, lower) clonal lines with the arps10-V127M mutated codon highlighted in red (GTG(V) ◊ aTG(M)). Pfk13-g, Pffd-g or Pfarps10-g: Pfk13, Pffd or Pfarps10 specific guide RNA gene cassette, cas9: Cas9 gene cassette, hdhfr-yfcu: drug selectable marker, HR; Homologous region, K^MT = k13^C580Y mutation, F^MT = fd^D193Y mutation, A^MT = arps10^V127M mutation