Evaluation of the Effect of Gene Duplication by Genome Editing on Drug Resistance in Plasmodium falciparum

The emergence and spread of drug-resistant Plasmodium falciparum have compromised antimalarial efficacy and threatened the global malaria elimination campaign using artemisinin combination therapies. The impacts of amino acid substitutions in antimalarial drug resistance-associated genes on drug susceptibility have been investigated; however, the effects of amplification of those genes remain unexplored due to the lack of robust genetic approaches. Here, we generated transgenic P. falciparum parasites with an additional copy of a drug resistance-associated gene using the highly efficient CRISPR/Cas9 system and investigated their drug response. Insertion of a drug resistance-associated gene expression cassette in the genome resulted in a roughly twofold increase of mRNA levels of the target gene mdr1 , which encodes multidrug resistance protein 1. The gene duplication event contributed to resistance to mefloquine, lumefantrine, and dihydroartemisinin, while the duplication of a genomic region encoding plasmepsin 2 and plasmepsin 3 did not affect resistance to antimalarial drugs, including piperaquine. We further demonstrated that mdr1 mRNA expression levels are strongly associated with mefloquine resistance in several field-derived P. falciparum lines with various genetic backgrounds. This study provides compelling evidence that mdr1 could serve as a molecular marker for the surveillance of mefloquine-resistant parasites. Long DNA integration into parasite genomes using the CRISPR/Cas9 system provides a useful tool for the evaluation of the effect of copy number variation on drug response.