Department of Parasitology and Tropical Medicine, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan.
Department of Molecular Protozoology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan.
Front Cell Infect Microbiol. 2022 Jul 5;12:915656. doi: 10.3389/fcimb.2022.915656. eCollection 2022.
The emergence and spread of drug-resistant 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 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 , 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 mRNA expression levels are strongly associated with mefloquine resistance in several field-derived lines with various genetic backgrounds. This study provides compelling evidence that 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.
耐药疟原虫的出现和传播降低了抗疟药物的疗效，威胁到了使用青蒿素联合疗法的全球疟疾消除运动。人们已经研究了抗疟药物耐药相关基因中的氨基酸替换对药物敏感性的影响；然而，由于缺乏稳健的遗传方法，这些基因扩增的影响仍未得到探索。在这里，我们使用高效的 CRISPR/Cas9 系统在转基因 寄生虫中生成了一个额外的耐药相关基因拷贝，并研究了它们对药物的反应。将药物耐药相关基因表达盒插入基因组中，导致靶基因（编码多药耐药蛋白 1）的 mRNA 水平大致增加了两倍。基因重复事件导致对氯喹、青蒿琥酯和双氢青蒿素的耐药性，而编码内质网蛋白酶 2 和内质网蛋白酶 3 的基因组区域的重复并不影响对包括哌喹在内的抗疟药物的耐药性。我们进一步证明，在具有各种遗传背景的几个源自现场的 株系中， mRNA 表达水平与氯喹耐药性密切相关。这项研究提供了有力的证据表明 可作为监测氯喹耐药寄生虫的分子标记。使用 CRISPR/Cas9 系统将长 DNA 整合到寄生虫基因组中为评估拷贝数变异对药物反应的影响提供了有用的工具。
