Article Figures & Data
Figures
- FIG 1
Gene editing of pfap2μ. (A) CRISPR-Cas9 editing was used to install the pfap2μI592T variant codon (red) (left) and the pfap2μS160N variant codon (blue) (right) into the endogenous locus. The pictured homologous repair constructs were modified to also introduce the I592I wild-type codon in the context of silent mutations ablating the Cas9 PAM site, as a control for their impact on phenotype. Primers used for genotype mapping anneal outside the homologous repair template, as depicted (P1 and P2) (see Table S1 in the supplemental material). The recodonized sequence near alternate I592 codons (red) also includes a SpeI restriction site, and the recodonized sequence near S160 codons (blue) ablates an SnaBI restriction site for downstream PCR-RFLP mapping. (B) Clones of parasite lines expressing AP2μI592T and AP2μI592I (left) and AP2μS160N (right) were generated by limiting dilution and confirmed by PCR-RFLP genotype mapping (with SpeI or SnaBI restriction endonuclease) and Sanger sequencing. Amplification of the locus with P1 and P2 produces a 2,247-bp fragment (Table S1); for I592I transgenic parasites, SpeI digestion of an amplicon containing the transgenic locus liberates a 301-bp fragment. The native amplicon is not cleaved (3D7 lanes). For S160 transgenic parasites, SnaBI does not cleave the transgenic amplicon, while wild-type (WT) amplicons liberate a 789-bp fragment. MW, fragment length in kilobases.
- FIG 2
RSA4 h survival estimates for pfap2μ variant transgenic parasite lines (clones 1 and 2) exposed to artemisinin. Shown is RSA4 h percent survival of mutant and parental parasite lines compared to the untreated control, following a 4-h pulse of 700 nM dihydroartemisinin. The Cam3.II family of parasite lines harbors pfk13 mutations, as indicated; REV indicates that wild-type K13 is encoded (10). The mean of data from at least four biological replicates is shown for each line, each performed in technical duplicate, with the standard error. Each technical replicate enumerates 100,000 gate-stopping events. P values were derived by a Mann-Whitney U test, comparing each parasite line to 3D7. ***, P < 0.005; NS, nonsignificant.
- FIG 3
Gene editing of pfubp1. (A) CRISPR-Cas9 editing was used to install the pfubp1Val3275Phe P. falciparum orthologue of the previously described P. chabaudi mutation into the endogenous locus in 3D7, marked with an asterisk. The same scheme and homology regions were used to install pfubp1Val3306Phe, marked with a number sign. Installation of either mutation introduces a HindIII restriction site, as marked. Primers P3 and P4 (see Table S1 in the supplemental material) were used to perform PCR-RFLP mapping of transgenic clones. B indicates the position of the “Borrmann hot spot” of variation associated with reduced artemisinin susceptibility (13, 14). Coordinates above the gene are in base pairs. “in. 2” denotes the position of the second intron in the gene. (B) Clones of parasite lines expressing UBP1Val3275Phe were generated by limiting dilution and confirmed by PCR-RFLP genotype mapping (with primers P3 and P4 and HindIII restriction digestion in lanes marked with a plus sign) and by Sanger sequencing. (C) Clones of parasite lines expressing UBP1Val3306Phe were generated and confirmed as described above for Val3275Phe mutants.
- FIG 4
RSA4 h survival estimates for pfubp1 variant transgenic parasite lines (clones 1 and 2) exposed to artemisinin. RSA survival of mutant and parental parasite lines was compared to that of the untreated control, following a 4-h pulse of 700 nM dihydroartemisinin. The mean of data from at least four biological replicates is shown for each line, each performed in technical duplicate, with the standard error. Each technical replicate enumerates 100,000 gate-stopping events. P values were derived by using the Mann-Whitney U test, comparing each parasite line to 3D7. ***, P < 0.005; NS, nonsignificant.
Tables
- TABLE 1
Comparison of the in vitro susceptibility of pfap2μ variant transgenic parasite lines with that of parental 3D7 exposed to dihydroartemisinin or other antimalarial drugs for 48 h
↵a EC50 values are the means ± SEM from at least four biological replicates (different cultures and different plates), each performed in technical duplicate. Boldface type indicates a P value of <0.01 (Mann-Whitney U test), compared to the EC50 estimate for the 3D7 parental line.
- TABLE 2
Comparison of the in vitro susceptibility of pfubp1 variant transgenic parasite lines with that of parental 3D7 exposed to dihydroartemisinin or other antimalarial drugs for 48 h
↵a See the footnote to Table 1.
Supplemental material
- Supplemental file 1 -
Supplemental Tables S1 and S2 and Figure S1
PDF, 124K
- Supplemental file 1 -
