Fig 1.
Strategy for introducing plasmid-free CRISPR/Cas9 edits to the Plasmodium falciparum gene pfatp4.
Synchronized ring-stage parasites at 17% parasitemia in fresh donor RBCs were nucleofected with Cas9 protein, guide RNA, and template ssODN. Cultures were kept under drug pressure with 500 nM SJ733 starting on day two post transfection. After drug-resistant parasites emerged from culture, genomic DNA was isolated with standard phenol-chloroform extraction methods for library preparation. The presence and penetrance of the targeted CRISPR edits were confirmed using Sanger sequencing and whole genome NGS.
Fig 2.
Sanger and NGS sequencing coverage of targeted CRISPR mutations at the pfatp4 locus for ACP-B6-L350H and ACP-B6-P412T with clonal wild type parent strain ACP-B6. Red bars delineate the respective 20 nt guide RNA target sites and PAM sites required for each edit. NGS coverage at each location is indicated by blue columns. (a) Sequencing data of targeted locus 1002–1072 in pfatp4 from strain ACP-B6-L350H showing SJ733 resistance-conferring SNPs in L350 and four other synonymous mutations introduced by CRISPR. Sequences of wild type pfatp4 and repair template ssODN L350H are shown in alignment. The two silent mutations in ssODN L350H located 39 and 42 nt away were not incorporated into ACP-B6-L350H. (b) Sequencing data of targeted locus 1206–1276 in pfatp4 from strain ACP-B6-P412T showing the SJ733 resistance-conferring SNP and silent mutations introduced by CRISPR. Sequences of wild type pfatp4 and repair template ssODN P412T are shown in alignment.
Fig 3.
Characterization of drug resistance.
Dose-response curves and EC50 values for the antimalarial SJ733 on the parent strain ACP-B6 and the mutants ACP-B6-L350H and ACP-B6-P412T. The growth inhibition assay was conducted by seeding synchronized ring-stage parasites from each strain at 0.8% parasitemia in media supplemented with SJ733 at concentrations ranging from 3.16 nM to 100 μM and allowing for growth over 72 hours. Parasites were fixed with 1% paraformaldehyde and stained with 50 nM YOYO-1. Final parasitemia was assessed by flow cytometry and values were normalized to DMSO-only controls. Values reported are mean ± standard error (n = 3). The inset shows parasitemia of each culture after 72 hours of growth in the presence of DMSO only.