RMgmDB - Rodent Malaria genetically modified Parasites


Malaria parasiteP. berghei
MutatedGene model (rodent): PBANKA_1219500; Gene model (P.falciparum): PF3D7_0709000; Gene product: chloroquine resistance transporter (CRT)
Details mutation: a mutated CRT with amino acid change N331I
Phenotype Asexual bloodstage;
Last modified: 23 July 2021, 14:44
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene mutation
Reference (PubMed-PMID number) Reference 1 (PMID number) : 34222045
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/ResearcherIkeda M, Mita T
Name Group/DepartmentDepartment of Tropical Medicine and Parasitology, Faculty of Medicine
Name InstituteJuntendo University
Name of the mutant parasite
RMgm numberRMgm-5041
Principal namePbCRT-N331I
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Asexual blood stageSlower growth rate of asexual blood stages and reduced sensitivity to Piperaquine.
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

The mutant expresses a mutated version of CRT with amino acid change N331I

Protein (function)
The Chloroquine Resistance Transporter (CRT) is an integral membrane protein localized to the parasite's internal digestive vacuole membrane. Mutations in CRT result in a decreased accumulation of chloroquine within the digestive vacuole in the parasite as a result of increased transport of chloroquine.

Slower growth rate of asexual blood stages and reduced sensitivity to Piperaquine.

Additional information

Other mutants

  Mutated: Mutant parasite with a mutated gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1219500
Gene Model P. falciparum ortholog PF3D7_0709000
Gene productchloroquine resistance transporter
Gene product: Alternative nameCRT
Details of the genetic modification
Short description of the mutationa mutated CRT with amino acid change N331I
Inducable system usedNo
Short description of the conditional mutagenesisNot available
Additional remarks inducable system
Type of plasmid/constructCRISPR/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
Selectable marker used to select the mutant parasitehdhfr
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe plasmid pYC for the CRISPR/Cas9 gene editing of P. yoelii was kindly gifted by Dr. Yuan (Zhang et al., 2014). To customize pYC for P. berghei, the U6 promoter of P. yoelii in pYC was excluded by inverse PCR using the primers A and B, which are designed in an outward direction from the P. yoelii U6 promoter region. The U6 promoter of P. berghei (PBANKA_1354380) was amplified with the primers C and D and ligated into the pYC PCR product by using an In-Fusion HD Cloning kit (TAKARA). The resultant plasmid, pBC, was used for P. berghei genome editing.
As donor DNA, a 1,343 bp fragment containing PbCRT(N331I) was synthesized (Genewiz, Japan) and ligated to the HindIII/AflII site in pBC by an In-Fusion HD Cloning kit. The candidate guide RNA (gRNA) was designed by the Eukaryotic Pathogen CRISPR Guide RNA Design Tool (http://grna.ctegd.uga.edu/). A double-stranded DNA coding for the candidate gRNA was ligated into the BsmBI site of pBC; therefore, the gRNA was transcribed under the P. berghei U6 promoter. The resultant plasmid, pPbCRT(N331I), was electroporated into wild-type P. berghei. Transfection and subsequent parasite cloning followed a previously described protocol (Janse et al., 2006).
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