Mutant/mutation
Several mutants are generated that contain point mutations in K13 associated with artemisinin resistance in P. falciparum. The mutants also express mCherry under control of the constitutive hsp70 promoter.

Protein (function)
Polymorphisms in the propeller domain of the kelch protein K13 of P. falciparum (PF3D7_1343700) have been associated with artemisinin resistance. Reverse genetic approaches have been successfully used to show that the PF K13 mutations M476I, R539T, I543T, Y493H and C580Y can confer dihydroartemisinin resistance in vitro.

Additional information

To generate PB mutant parasites carrying orthologous PF K13 mutations, we attempted to introduce PB equivalents of five PF K13 mutations (M476I, Y493H, R539T, I543T and C580Y) that by reverse genetics have been previously shown to confer enhanced PF survival in in vitro RSAs. We also introduced the equivalent of the F446I mutation that is predominant in Southern China along the Myanmar border. These mutations are all validated determinants of reduced PF susceptibility to ARTs. Structural homology modelling revealed that PB and PF K13 (PBANKA_1356700 and PF3D7_1343700, respectively) are highly conserved (~84% sequence identity overall) at the C-terminal propeller domain, especially where resistance-conferring mutations  localize. PB K13 carries 12 extra amino acids, resulting in 738 amino acids for PB compared to 726 for PF. However, modelling suggests that the extra amino acids in PB do not change the overall propeller structure of K13 or the amino acid identity at the orthologous positions of the mutations examined in this study. Using a CRISPR/Cas9 system, we designed Cas9 plasmids carrying single guide RNAs (sgRNAs) to target the PB K13 locus with corresponding homology repair templates. The repair templates carried the mutations of interest as well as silent mutations that inactivated the protospacer adjacent motif (PAMs) and introduced restriction sites for restriction fragment length polymorphism (RFLP) analyses. Electroporation of the plasmids pG1004 (C592Y), pG1005 (I555T) and pG1006 (R551T) into the K13 wild-type PB 1804cl1 line yielded edited parasites (G2022(C592Y.1*), G2023(C592Y.2*), G2024(I555T*) and G2025(R551T*) with calculated 13.4%, 18.5%, 7.7% and 30.0% efficiencies respectively by RFLP analysis. Intriguingly, bulk DNA sequencing of these transformed parasites revealed that only the G2025(R551*) line carried sequence traces for the R551T amino acid substitution and accompanying silent mutations while the rest had traces only of the silent mutations. These data suggested that the C592Y and I555T mutations either result in extremely slow growing parasites or are entirely lethal in PB. We attempted to clone the G2025(R551T*) line by limiting dilution, but this could not be achieved, possibly due to the low mutant population (30.0 %) combined with a potentially slow growth rate of the mutants compared to wild-type parasites. Meanwhile, transfection of the PB 1804cl1 line with pG983 (F458I), pG984 (Y505H) and pG1008 (M488I) successfully introduced these mutations in PB K13, yielding the G1957(F458I*), G1979(Y505H*) and G1989(M488I*) lines with >93% efficiencies as confirmed by RFLP analysis. These three lines G1957(F458I*), G1979(Y505H*), G1989(M488I*) and the G2025(R551T*) AS 64 mg/kg challenged line were all cloned by limiting dilution

Other mutants