RMgmDB - Rodent Malaria genetically modified Parasites


Malaria parasiteP. berghei
MutatedGene model (rodent): PBANKA_0112100; Gene model (P.falciparum): PF3D7_0613800; Gene product: AP2 domain transcription factor, putative (ApiAP2)
Details mutation: A phenylalanine 1823 is replaced with with serine (F1823S)
Phenotype Asexual bloodstage;
Last modified: 25 August 2020, 15:22
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) : 32788672
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/ResearcherAkkaya M, Pierce SK
Name Group/DepartmentLaboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases
Name InstituteNational Institutes of Health
Name of the mutant parasite
RMgm numberRMgm-4856
Principal namePbA-AP2(S)
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Asexual blood stagePbANKA(S) induces ECM (like wild type PbANKA(F) parasites
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

The mutant expresses a mutated PBANKA_0112100, in which T-to-C transition at position 5468 is introduced. This mutation results resulted in a change of a phenylalanine (F) into a serine (S) (5,467 TTT to TCT). 

Protein (function)
See RMgm-4823 for details
PBANKA_0112100 is an essential transcription factor and therefore no viable knock out could be generated.

We compared infections by the mutant parasites PbNK65(F) (RMgm-4823) and PbANKA(S) in C57BL/6 mice to infections with their WT counterparts PbNK65(S) and PbANKA(F)

Evidence is presented that:
- PbNK65(F) does not induce ECM
- PbANKA(S) induces ECM (like wild type PbANKA(F) parasites

Additional information
PBANKA_0112100 is an essential transcription factor and therefore no viable knock out could be generated. See also RMgm-4823 for more details for these studies

From the Abstract: 
The single nucleotide polymorphysim (SNP) in the ApiAP2 gene PBANKA_0112100 is neither necessary nor sufficient to induce ECM and thus cannot account for parasite strain-specific (ANKA versus NK65) differences in ECM phenotypes.

From the paper: 
In contrast, a highly related strain to PbANKA, PbNK65, causes severe anemia in the absence of any detectable brain pathology (ECM). A comparison of high coverage genomic sequences of PbANKA and PbNK65 revealed that these two strains differ by only 21 single nucleotide polymorphisms (SNPs) in their coding regions. Thus, remarkably, a small number of SNPs may account for the dramatically different disease outcomes of infection with PbANKA versus PbNK65. The majority of the genes containing SNPs are of unknown function, however, two SNPs were identified in genes encoding proteins belonging to ApiAP2 transcriptional factor (TF) family namely PBANKA_0112100 and PBANKA_1415700. The SNP in PBANKA_1415700 was located immediately before the stop codon and hence unlikely to induce structural alterations that would lead to functional differences in the expressed ApiAP2. On the other hand, the SNP in PBANKA_0112100 was located in the predicted DNA-binding domain of ApiAP2 resulting in a substitution of a Serine (S) to Phenylalanine (F) at amino acid 1823 in the expressed protein, two biochemically distinct amino acids. Therefore, this SNP had the potential to influence the function of ApiAP2.
Our previous genetic and functional analysis using this SNP (RMgm-4823) in order to dissect out the function of this transcription factor revealed that the SNP in the DNA binding region of PBANKA_0112100 alters the expression of 46 Plasmodium genes. Among these 46 genes 39 belong to either BIR(22/46), fam-a (7/46), fam-b ( 8/46) or fam-c (2/46) gene families all of which are known to be involved in virulence and evasion related functions

Other mutants


  Mutated: Mutant parasite with a mutated gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0112100
Gene Model P. falciparum ortholog PF3D7_0613800
Gene productAP2 domain transcription factor, putative
Gene product: Alternative nameApiAP2
Details of the genetic modification
Short description of the mutationA phenylalanine 1823 is replaced with with serine (F1823S)
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/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationFor editing the AP2 (PBANKA_0112100) gene in Plasmodium berghei ANKA, replacing phenylalanine 1823 with serine (F1823S), we used the clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein 9 (Cas9) system (CRISPR/Cas9). A single plasmid system containing pYC plasmid express all the essential components of CRISPR-Cas9; Cas9 endonuclease expressed as a fusion protein with drug selection marker hDHFR, gRNA:tracrRNA chimera driven by PyU6 promoter and a homology template for repair of double stranded break and concomitant introduction of desired mutation. A guide sequence of 20 nucleotides (5′ GCTGAATTAAAACCCCAAAG 3′), with protospacer adjacent motif (5′ AGG 3′), was selected by manual curation for targeting the Cas9 endonuclease to result in the desired editing (5,467 TTT to TCT) in the AP2 gene. The 900 nucleotides of synthetic sequence (given in Supplementary Information) containing the mutated guide region and the desired single nucleotide polymorphism (SNP) (5,467 TTT to TCT) and shield mutations to overcome repeated restriction of the modified genomic locus, was sub-cloned in the pYC plasmid using NcoI and XhoI restriction enzyme sites. The resulting plasmid, pYC_ANKAAP2NK65, was used for the transfection of the P. berghei ANKA parasites
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