title: Efficient editing of malaria parasite genome using the CRISPR/Cas9 system.
abstract: Malaria parasites are unicellular organisms residing inside the red blood cells, and current methods for editing the parasite genes have been inefficient. The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats and Cas9 endonuclease-mediated genome editing) system is a new powerful technique for genome editing and has been widely employed to study gene function in various organisms. However, whether this technique can be applied to modify the genomes of malaria parasites has not been determined. In this paper, we demonstrated that Cas9 is able to introduce site-specific DNA double-strand breaks in the Plasmodium yoelii genome that can be repaired through homologous recombination. By supplying engineered homologous repair templates, we generated targeted deletion, reporter knock-in, and nucleotide replacement in multiple parasite genes, achieving up to 100% efficiency in gene deletion and 22 to 45% efficiencies in knock-in and allelic replacement. Our results establish methodologies for introducing desired modifications in the P. yoelii genome with high efficiency and accuracy, which will greatly improve our ability to study gene function of malaria parasites. Importance: Malaria, caused by infection of Plasmodium parasites, remains a world-wide public health burden. Although the genomes of many malaria parasites have been sequenced, we still do not know the functions of approximately half of the genes in the genomes. Studying gene function has become the focus of many studies; however, editing genes in malaria parasite genomes is still inefficient. Here we designed several efficient approaches, based on the CRISPR/Cas9 system, to introduce site-specific DNA double-strand breaks in the Plasmodium yoelii genome that can be repaired through homologous recombination. Using this system, we achieved high efficiencies in gene deletion, reporter tagging, and allelic replacement in multiple parasite genes. This technique for editing the malaria parasite genome will greatly facilitate our ability to elucidate gene function.
authors.affiliation: State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Biology, School of Life Sciences, Xiamen University, Xiamen, Fujian, China.
authors.forename: Cui
authors.initials: C
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Biology, School of Life Sciences, Xiamen University, Xiamen, Fujian, China.
authors.forename: Bo
authors.initials: B
authors.lastname: Xiao
authors.affiliation: State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Biology, School of Life Sciences, Xiamen University, Xiamen, Fujian, China.
authors.forename: Yuanyuan
authors.initials: Y
authors.lastname: Jiang
authors.affiliation: State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Biology, School of Life Sciences, Xiamen University, Xiamen, Fujian, China.
authors.forename: Yihua
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Biology, School of Life Sciences, Xiamen University, Xiamen, Fujian, China.
authors.forename: Zhenkui
authors.initials: Z
authors.lastname: Li
authors.affiliation: State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Biology, School of Life Sciences, Xiamen University, Xiamen, Fujian, China.
authors.forename: Han
authors.initials: H
authors.lastname: Gao
authors.affiliation: State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Biology, School of Life Sciences, Xiamen University, Xiamen, Fujian, China.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Ling
authors.affiliation: State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Biology, School of Life Sciences, Xiamen University, Xiamen, Fujian, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Wei
authors.affiliation: State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Biology, School of Life Sciences, Xiamen University, Xiamen, Fujian, China.
authors.forename: Shaoneng
authors.initials: S
authors.lastname: Li
authors.affiliation: State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Biology, School of Life Sciences, Xiamen University, Xiamen, Fujian, China.
authors.forename: Mingke
authors.initials: M
authors.lastname: Lu
authors.affiliation: Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA cuihuiting@xmu.edu.cn yuanjing@xmu.edu.cn.
authors.forename: Xin-Zhuan
authors.initials: XZ
authors.lastname: Su
authors.affiliation: State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Biology, School of Life Sciences, Xiamen University, Xiamen, Fujian, China cuihuiting@xmu.edu.cn yuanjing@xmu.edu.cn.
authors.forename: Huiting
authors.initials: H
authors.lastname: Cui
authors.affiliation: State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Biology, School of Life Sciences, Xiamen University, Xiamen, Fujian, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Yuan
mesh_terms: D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D018503:Genome, Protozoan / Q000235:genetics*; D010967:Plasmodium yoelii / Q000235:genetics*