Figure 1.

The genetic modification of MDR1 Y976F in Plasmodium cynomolgi Berok K4A7 using CRISPR-Cas9 and its corresponding association with in vitro sensitivity to 5 antimalarial drugs. A, An all-in-one approach [8] was used for the CRISPR-Cas9 strategy for editing the mdr1 locus, consisting of a Streptococcus pyogenes Cas9 gene with a Plasmodium vivax calmodulin promotor, a U6-driving gRNA, a human dhfr selection cassette, and an mdr1-specific donor template for homology-directed repair. Donors coding for MDR1 Y976F and silent binding-site mutations were generated by site-directed mutagenesis of the wild-type P. cynomolgi mdr1 donor sequence. B, Sequencing results showing the introduction of individual Pcymdr1 mutations in recombinant parasites (Y976F), with wild-type amino and nucleic acid sequences for reference. The mutation Y976F was introduced into Pcymdr1, along with silent binding-site mutations, and a separate control BSmut line was produced with the same silent binding-site mutations and wild-type Y976. C, The geometric mean (3 biological replicates) of IC₅₀ values are shown for chloroquine, artesunate, mefloquine, piperaquine, and amodiaquine against P. cynomolgi K4A7 Pcymdr1 Y976F and the BSmut control. Significance was determined by ordinary 2-way analysis of variance and a Šídák multiple comparisons test. Abbreviations: Cas9, CRISPR-associated protein 9; CRISPR, clustered regularly interspaced short palindromic repeats; IC₅₀, 50% inhibitory concentration; ns, not significant; UTR, untranslated region.