title: CRISPR/Cas9 mediated sequential editing of genes critical for ookinete motility in Plasmodium yoelii.
abstract: CRISPR/Cas9 has been successfully adapted for gene editing in malaria parasites including Plasmodium falciparum and Plasmodium yoelii. However, the reported methods were limited to editing one gene at a time. In practice, it is often desired to modify multiple genetic loci in a parasite genome. Here we describe a CRISPR/Cas9 mediated genome editing method that allows successive modification of more than one gene in the genome of P. yoelii using an improved single-vector system (pYCm) we developed previously. Drug resistant genes encoding human dihydrofolate reductase (hDHFR) and a yeast bifunctional protein (yFCU), with cytosine deaminase (CD) and uridyl phosphoribosyl transferase (UPRT) activities in the plasmid, allowed sequential positive (pyrimethamine, Pyr) and negative (5-fluorocytosine, 5FC) selections and generation of transgenic parasites free of the episomal plasmid after genetic modification. Using this system, we were able to efficiently tag a gene of interest (Pyp28) and subsequently disrupted two genes (Pyctrp and Pycdpk3) that are individually critical for ookinete motility. Disruption of the genes either eliminated (Pyctrp) or greatly reduced (Pycdpk3) ookinete forward motility in matrigel in vitro and completely blocked oocyst development in mosquito midgut. The method will greatly facilitate studies of parasite gene function, development, and disease pathogenesis.
authors.affiliation: State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, School of Life Sciences, Xiamen University, Xiamen, Fujian 361102, China.
authors.forename: Cui
authors.initials: C
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, School of Life Sciences, Xiamen University, Xiamen, Fujian 361102, China.
authors.forename: Han
authors.initials: H
authors.lastname: Gao
authors.affiliation: State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, School of Life Sciences, Xiamen University, Xiamen, Fujian 361102, China.
authors.forename: Zhenke
authors.initials: Z
authors.lastname: Yang
authors.affiliation: State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, School of Life Sciences, Xiamen University, Xiamen, Fujian 361102, China.
authors.forename: Yuanyuan
authors.initials: Y
authors.lastname: Jiang
authors.affiliation: State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, School of Life Sciences, Xiamen University, Xiamen, Fujian 361102, China.
authors.forename: Zhenkui
authors.initials: Z
authors.lastname: Li
authors.affiliation: State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, School of Life Sciences, Xiamen University, Xiamen, Fujian 361102, China.
authors.forename: Xu
authors.initials: X
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, School of Life Sciences, Xiamen University, Xiamen, Fujian 361102, China.
authors.forename: Bo
authors.initials: B
authors.lastname: Xiao
authors.affiliation: State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, School of Life Sciences, Xiamen University, Xiamen, Fujian 361102, China; Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Xin-Zhuan
authors.initials: XZ
authors.lastname: Su
authors.affiliation: State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, School of Life Sciences, Xiamen University, Xiamen, Fujian 361102, China. Electronic address: cuihuiting@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 Signaling Network, School of Life Sciences, Xiamen University, Xiamen, Fujian 361102, China. Electronic address: yuanjing@xmu.edu.cn.
authors.forename: Jing
authors.initials: J
authors.lastname: Yuan
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D064113:CRISPR-Cas Systems; D009033:Culicidae / Q000469:parasitology; D017353:Gene Deletion; D000072669:Gene Editing; D015870:Gene Expression; D017930:Genes, Reporter; D008124:Locomotion; D010957:Plasmids / Q000235:genetics; D010967:Plasmodium yoelii / Q000502:physiology*; D015800:Protozoan Proteins / Q000235:genetics
chemical_list: D015800:Protozoan Proteins
keywords: Cas9; Genetic modifications; Pycdpk3; Pyctrp; Rodent malaria; yFCU