RMgmDB - Rodent Malaria genetically modified Parasites


Malaria parasiteP. berghei
DisruptedGene model (rodent): PBANKA_1115100; Gene model (P.falciparum): PF3D7_0515500; Gene product: gametogenesis essential protein 1, putative (GEP1)
Phenotype Gametocyte/Gamete;
Last modified: 16 June 2020, 18:22
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption
Reference (PubMed-PMID number) Reference 1 (PMID number) : 32273496
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone Not applicable
Other information parent line
The mutant parasite was generated by
Name PI/ResearcherJiang Y, Yuan J
Name Group/DepartmentState Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network
Name InstituteSchool of Life Sciences, Xiamen University
CityXiamen, Fujian
Name of the mutant parasite
RMgm numberRMgm-4814
Principal nameΔ1116300
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Asexual blood stageNot different from wild type
Gametocyte/GameteNormal gametocyte production; no exflagellation centers are formed
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

The mutant lacks expression of PBANKA_1115100 (GEP1)

Protein (function)
To identify membrane proteins critical in sensing XA or transducing XA-induced signal during gametogenesis, we identified 59 P. yoelii genes that are expressed in gametocytes and encode proteins with 1 to 22 predicted transmembrane domains (TMs) from the PlasmoDB database. We designed single guide RNA (sgRNA) to disrupt each of these genes using CRISPR/Cas9 methods16,17 and were able to successfully knockout (KO) 45 (76%) of the genes in the P. yoelii 17XNL strain, obtaining at least two cloned lines for each mutant . The remaining 14 genes (24%) were refractory to repeated deletion attempts using three independent sgRNA sequences, suggesting their essential roles for asexual blood-stage growth. The 45 gene deletion mutants proliferated asexually in mouse blood normally and were able to produce both male and female gametocytes although the gametocytemia level varied among these mutants.
Next we measured the gametogenesis of male gametocyte by counting exflagellation centers (ECs) formed in vitro after stimulation with 50 μMXAat 22 °C.Only one mutant (PY17X_1116300; GEP1) showed complete deficiency in EC formation and male gamete release and this mutant was analysed in greater detail.

The PY17X_1116300 (GEP1) gene contains four exons encoding a putative amino acid transporter protein.This is the ortolog of PBANKA_1115100 (GEP1)


Normal asexual blood stage growth/multiplication;
Normal gametocyte production; no exflagellation centers are formed.
The phenotype has not been analysed in great detail in the P. berghei gene-deletion mutant. See mutant RMgm-4813 for a P. yoelli gene-deletion mutant lacking GEP1 for detailed analyses (see also below)

Additional information
From the Abstract (based on analyses of mutant RMgm-4813)
GEP1 disruption abolishes XA-stimulated cGMP synthesis and the subsequent signaling and cellular events, such as Ca2+ mobilization, gamete formation, and gametes egress out of erythrocytes. GEP1 interacts with GCα, a cGMP synthesizing enzyme in gametocytes. Both GEP1 and GCα are expressed in cytoplasmic puncta of both male and female gametocytes. Depletion of GCα impairs XA-stimulated gametogenesis, mimicking the defect of GEP1 disruption.

Other mutants
See other mutants generated in this study

  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1115100
Gene Model P. falciparum ortholog PF3D7_0515500
Gene productgametogenesis essential protein 1, putative
Gene product: Alternative nameGEP1
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct usedCRISPR/Cas9 construct: integration through double strand break repair
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid
Partial or complete disruption of the geneComplete
Additional remarks partial/complete disruption
Selectable marker used to select the mutant parasitehdhfr/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationCRISPR/Cas9 plasmid pYCm was used for all the genetic modifications. For gene deleting, 5’-genomic and 3’-genomic segments (400 to 700 bp) of the target genes were amplified as left and right homologous arms, respectively, using gene specific primers. The PCR products were digested with appropriate restriction enzymes and the digested products were inserted into matched restriction sites of pYCm.
Oligonucleotides for sgRNAs were annealed and ligated into pYCm17. For each deletion modification, two sgRNAs were designed to disrupt the coding region of a target gene using the online program ZiFit47. For gene tagging, a 400 to 800 bp segment from N-terminal or C-terminal of the coding region and 400 to 800 bp sequences from 5’UTR or 3’UTR of a target gene were amplified and fused with a DNA fragment encoding 6HA or 4Myc in frame at N-terminal or C-terminal of the gene. For each tagging modification, two sgRNAs were designed to target sites close to the C-terminal or N-terminal of the gene coding region.
Additional remarks selection procedure
Primer information: Primers used for amplification of the target sequences  Click to view information
Primer information: Primers used for amplification of the target sequences  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Sequence Primer 3
Additional information primer 3
Sequence Primer 4
Additional information primer 4
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6