Extended Data Figure 4. Transgenic P. falciparum expressing PfKelch13^C580Y mutation in two different strains of P. falciparum using distinct approaches and markers.

a, Strategy 1: CRISPR-Cas9 used to introduce a single point mutation in the P. falciparum NF54 genome as described elsewhere²⁰. Both parent and mutant strains were sequenced to ensure that the only change detected was PfKelch13^C580Y. Western blots using anti-PfKelch13 antibodies confirm that mutation does not change the level of PfKelch13 protein. b, Strategy 2 expresses a dominant negative PfKelch13^C580Y and wild type PfKelch13^WT tagged with HA, and driven by the constitutive cam promoter in P. falciparum 3D7. Western blots confirm that anti-HA antibodies detected tagged forms of PfKelch13 in transfected but not non-transfected 3D7. Comparable levels of wild type and mutant transgenes are expressed in resulting two strains of parasites (as shown). In a-b, BiP, an ER marker serves as a parasite loading control in western blots. Molecular weight standards (in kDa) are as indicated. c, Western blot indicate that antibodies to PfKelch13 specifically recognize a ~83-kDa protein in infected (IE) but not uninfected erythrocytes (U). Molecular weight standards (in kDa) are as indicated. In a-c: data are representative of three experimental replicates; raw data are in Supplementary Data 2.