Figure 5: CRISPR-Cas9-mediated pfcpsf3 editing. | Nature Communications

Figure 5: CRISPR-Cas9-mediated pfcpsf3 editing.

From: A potent antimalarial benzoxaborole targets a Plasmodium falciparum cleavage and polyadenylation specificity factor homologue

Figure 5

(a) Dd2 parental (WT) parasites were transfected with a dual-plasmid strategy in which one plasmid encoded the sgRNA, Cas9 nuclease and a hdhfr selectable marker, and the other plasmid encoded a donor sequence containing either the T406I or Y408S PfCPSF3 mutations (in red), as well as synonymous mutations in the sgRNA-binding region. (b) Electropherograms showing unmodified Dd2 and genome-edited parasites. Grey boxes highlight nucleotides that differ from WT parasites. PfCPSF3 single-letter amino acid substitutions are indicated in red. (c) Susceptibility to AN3661 of the parental line (Dd2 WT), parasites selected in vitro (Dd2-Ra, Dd2-Rb and Dd2-Rc), and genetically modified lines (Dd2 transf. F1, F3, E1, C4, C4 cl.4, C4 cl.7 and D3; replicates: Dd2 WT, 27; Dd2-Ra, 9; Dd2 transf. F1, 4; Dd2 transf. F3, 4; Dd2 transf. E1, 4; Dd2-Rb, 11; Dd2 transf. C4, 5; Dd2 transf. C4 cl.4, 2; Dd2 transf. C4 cl.7, 2; Dd2 transf. D3, 9; Dd2-Rc, 3.). Assay details are in Supplementary Table 1. Bar graphs represent mean±s.e.m. IC50 values. The black bar denotes PfCPSF3 WT, blue bars T406I, red bars Y408S and green bar T409A. Significance was determined using a two-tailed unpaired t-test, comparing transfected parasites with the parental Dd2 strain. ****P<0.0001.

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