RMgmDB - Rodent Malaria genetically modified Parasites


Malaria parasiteP. berghei
TaggedGene model (rodent): PBANKA_1030100; Gene model (P.falciparum): PF3D7_1412500; Gene product: actin II (actin2)
Name tag: FLAG
PhenotypeNo phenotype has been described
Last modified: 16 November 2021, 15:38
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene tagging
Reference (PubMed-PMID number) Reference 1 (PMID number) : 34537287
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/ResearcherDeligianni E, Siden Kiamos I
Name Group/DepartmentInstitute of Molecular Biology and Biotechnology
Name InstituteFoundation for Research and Technology - Hellas
Name of the mutant parasite
RMgm numberRMgm-5108
Principal namePbflag:act II
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Asexual blood stageNot different from wild type
Gametocyte/GameteNot different from wild type
Fertilization and ookineteNot different from wild type
OocystNot different from wild type
SporozoiteNot different from wild type
Liver stageNot different from wild type
Additional remarks phenotype

The mutant expresses a C-terminal FLAG-tagged version of actin II (actin2). The FLAG-tag has been introduced by a CRISPR/Cas9 protocol using linear donor template and ribozymes for sgRNA generation (see below for details).

Protein (function)
Actin, a cytoskeletal protein, has many diverse functions in eukaryotic cells ranging from roles in cell motility, cell division, vesicle trafficking to functions in cell signaling and regulation of transcription. A critical property of actin is its ability to form filamentous polymers (F-actin), and a plethora of proteins are involved in the highly dynamic regulation of F-actin formation . Actins are highly conserved proteins that often exist in multiple isoforms in the eukaryotic cell and their expression is regulated both spatially and temporally during development. The number of conventional actin genes varies among eukaryotic organisms. A few single cell eukaryotes, such as Saccharomyces cerevisiae, Toxoplasma gondii, and Trypanosoma brucei encode a single actin gene, which results in lethality when targeted with gene ablation approaches. Many organisms, however, have several conventional actin genes. Apicomplexan parasites all encode one major actin isoform, here termed Actin I. All apicomplexan parasites also contain a number of actin-related and actin-like proteins. Plasmodium species species stand out in that they all encode a second conventional actin, termed Actin II.

No phenotype was observed indicating that the FLAG-tag did not affect the function of actin II. Evidence is shown that FLAG-tagged actin II is expressed in activated male gametocytes (Western analysis).

Additional information
From the abstract: 
'Here, we successfully established a simplified CRISPR/Cas9 system for the malaria model parasite Plasmodium berghei. The homologous directed repair (HDR) template is provided as a linear template with homologous arms of 600-700bp while the CRISPR elements sgRNA and Cas9 are encoded from a single plasmid utilizing the Ribozyme-Guide-Ribozyme (RGR) expression strategy. Our approach eliminates the need for negative selection markers since the plasmid cannot be incorporated into the genome. As a test case we inserted the FLAG encoding sequence into the ACT2 locus using this new approach'.

Other mutants

  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1030100
Gene Model P. falciparum ortholog PF3D7_1412500
Gene productactin II
Gene product: Alternative nameactin2
Details of the genetic modification
Name of the tagFLAG
Details of taggingC-terminal
Additional remarks: tagging
Commercial source of tag-antibodies
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 markerunknown
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationIn order to introduce the CRISPR/Cas9 gene-editing elements into P. berghei we adapted the system of Ribozyme-Guide-Ribozyme (RGR) that was designed for P. yoelii. The construct was generated by one round of restriction digestion and ligation. To simplify the procedure, we exploited the fact that, in P. berghei, genetic mutations can be achieved using linearized DNA molecules consisting of a resistance gene flanked by two large (250–1000bp) regions homologous to the target locus. Thus, we developed a strategy where the homology directed repair (HDR) template was provided as a linear PCR fragment.
In the donor template, a silent mutation was inserted into the protospacer-adjacent motif (PAM), preventing Cas9 from cleaving the edited gene.
An existing plasmid from our laboratory, encoding actin II tagged with the FLAG epitope at the amino terminus was used as a template for HDR. A two–step PCR approach was used to generate the Pbflag:act II mutant. In the first reaction (PCR1) the 5’ and 3’ flanking sequences were generated using the primer pairs A2U4F/A2PAMR and A2PAMF /A2yoe1R respectively. The silent mutation that prevents Cas9 cleavage, following repair of the DSB, was introduced using the primers A2PAMR and A2PAMF. These primers were designed with an overlapping region of 27bp thus in an overlapping PCR, the complementary areas annealed resulting to a fragment of 1928bp. The entire fusion product was subsequently successfully amplified with primers A2U5SF and A2yoe2R, yielding a 1245 bp product. The HDR template was sequenced to verify the absence of unintended mutations.
The pSL1433 plasmid was obtained from Addgene Plasmid #129523)(see also M.P. Walker, S.E. Lindner
Ribozyme-mediated, multiplex CRISPR gene editing and CRISPRi in Plasmodium yoelii J. Biol. Chem., 294 (24) (2019), pp. 9555-9566) while the RGR sequence was synthesized by Genescipt and provided in the pUC57 vector and then inserted into pSL1433 using EcoRI/NheI. The HDR template and the pSL1433 plasmid carrying the RGR sequence were introduced into purified schizonts of P. berghei by standard methods of transfection/electroporation.
Three independent and successful experiments were carried out and the successful insertion was confirmed by PCR using the primer pairs FlagF/A2yoe1R. Following transfection and constant pyrimethamine pressure, parasitemia was observed after 7 days, similar to what was observed for P. yoelii. The plasmid persisted after removal of pyrimethamine pressure as confirmed by PCR, with primers pSL1433F/pSL1433R, although it was lost after cloning. The parasites were cloned by serial dilution and one clone was selected for further analysis. While a band for the wild type allele was observed in the uncloned population, the band was absent in the clonal population.
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