RMgmDB - Rodent Malaria genetically modified Parasites


Malaria parasiteP. berghei
TaggedGene model (rodent): PBANKA_0102400; Gene model (P.falciparum): PF3D7_0603600; Gene product: male development protein MD4, putative (AT-rich interactive domain-containing protein, putative, gARID)
Name tag: GFP
Phenotype Gametocyte/Gamete;
Last modified: 1 April 2024, 09:52
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) : 38554111
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone RMgm-4870
Other information parent lineThe mutant (RMgm-4870) contains a gene encoding Cas9 from Streptococcus pyogenes, introduced into the c/d-ssu-rrna gene. This Cas9 nuclease does not cleave double-stranded DNA in the absence of sgRNA and is thus suitable for generating the parasite which expresses it constitutively. It does not contain a drug-selectable marker that has been removed by negative selection
The mutant parasite was generated by
Name PI/ResearcherNishi T, Iwanaga S, Yuda M
Name Group/DepartmentDepartment of Medicine
Name InstituteMie University
Name of the mutant parasite
RMgm numberRMgm-5374
Principal namegARID::GFP
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Asexual blood stageNot different from wild type
Gametocyte/GameteNo GFP expression in asexual stage parasites. GFP expression in both male and female gametocytes. No GFP expression in ookinetes.
Fertilization and ookineteNot different from wild type
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

The mutant expresses a C-trminal, GFP-tagged version of gARID.

Protein (function)
The garid gene (PBANKA_0102400) is a target gene of AP2-G, and its transcription is upregulated via conditional induction of AP2-G in P. falciparum and P. berghei. Garid encodes a protein that contains an ARID at its N-terminus and a monopartite nuclear localization signal near its C-terminus. The ARID is a helix–turn–helix motif-based DNA binding domain conserved in a wide variety of eukaryotic species from fungi to vertebrates. In mammals, proteins with this domain are classified into 7 subfamilies, namely ARID1 through ARID5, JARID1, and JARID2. Of these, only ARID3 and ARID5 preferentially interact with AT-rich sequences, and the others have no clear sequence preference.

No GFP expression in asexual stage parasites. GFP expression in both male and female gametocytes. No GFP expression in ookinetes (see also mutant RMgm-5373, expressing  a C-terminal, mNeon Green-tagged version of gARID).

Additional information
Analysis of a mutant lacking expression of gARID (RMgm-5372) showed the following:
Normal growth/multiplication of asexual blood stages. Female gametocytes were observed, but no male gametocytes were observed in garid(−). No exflagellation was detected in garid(−) after the activation of gametogenesis. No male gametocytes were observed in garid(−). No exflagellation was detected in garid(−) after the activation of gametogenesis. No zygote/ookinete formation. No ookinete and oocyst formation.

To further confirm the absence of male gametocytes in garid(−), garid was knocked out in the gametocyte reporter line 820cl1 (garid[−]820), which have GFP and RFP expression cassettes under the control of male- and female-specific promoters, respectively. FACS analysis detected no GFP-positive parasites in the garid(−)820 parasites compared with the parental 820cl1 wherein approximately 3% of the parasites showed a GFP signal. This confirmed that disruption of garid results in the complete loss of male production.

When garid(−) was crossed with parasites that produce infertile males (p48/45[−]), no zygotes were observed, which was consistent with the absence of male gametocytes in garid(−). In contrast, when garid(−) was crossed with parasites that produce infertile females (p47[−]), parasites could undergo fertilization at a ratio similar to that when p48/45(−) and p47(−) were crossed. However, the ratio of conversion from female to banana-shaped ookinetes was only 19%, which was significantly smaller than the ratio for crossing p48/45(−) and p47(−), which was 40%. This result suggested that although female gametocytes of garid(−) showed normal morphology, they exhibit an abnormality that causes delay or arrest of ookinete development after fertilization.

ChIP-seq analysis of gARID showed that it forms a complex with gSNF2 (PBANKA_0312700, PF3D7_0216000), a core subunit of the SWI/SNF chromatin remodeling complex, associating with the male cis-regulatory element. Moreover, ChIP-seq of gARID in gsnf2-knockout parasites revealed an association of gARID with another cis-regulatory element, which is indicated to play a role in both male and female development.

Notably, the genome-wide peak pattern of the ChIP-seq of gARID was almost identical to that of gSNF2, previously reported. gSNF2 is a core subunit of SWI/SNF2 chromatin remodeling complex expressed in gametocytes, and gsnf2 knockout parasite (gsnf2[−]) showed impaired male gametocyte development. However, the knockout phenotypes are of not the same; gsnf2(−) produces immature male gametocytes, whereas garid(−) completely loses the ability to differentiate into male gametocytes. In addition, female gametocyte development was affected in garid(−) but not in gsnf2(−). Consistent with the differences in their phenotype, the results in the differential expression analyses for garid(−) vs. wild type and gsnf2(−) vs. wild type were also different.

A transgenic line expressing GFP-tagged gARID (gARID::GFP) was generated with the CRISPR/Cas9 system using Pbcas9, and then, gsnf2 was disrupted in the gARID::GFPC (gARID::GFPC_gsnf2(−)); mutant RMgm-5375). Analysis is of this mutant provided evidence that gARID is recruited by gSNF2 as a subunit of the SWI/SNF complex on the TGTCT motifs. In a previous study, it was shown that the female-specific transcriptional activator, PFG, is responsible for recognizing the TGTAYRTACA motifs. Therefore, gARID is recruited to the TGTAYRTACA motifs by PFG independent of gSNF2. In addition to the ten-base motif, the TGCACA motif was also enriched around the peak summits identified in the ChIP-seq using gARID::GFPC_gsnf2(−) suggesting that it could be another important gARID-associated sequence motif.

To thoroughly investigate the property of gARID binding regions other than those on the known motifs, we knocked out both gsnf2 and pfg in gARID::GFPC using CRISPR/Cas9 (gARID::GFPC_gsnf2(−)_pfg(−))

To investigate the function of the TGCACA motif as a cis-regulatory element and the relationship between the TGTCT and TGCACA motifs in male gametocyte development,  reporter assays were performed using endogenous loci. As a target for the reporter assays, the following two genes were selected: calm (PBANKA_1421000, a putative calmodulin gene) as a gene significantly down-regulated in garid(−) but only slightly in gsnf2(−) and rsph9 (PBANKA_1431500, a gene encoding a putative radial spoke head protein 9 homolog, a component of radial spokes which control axonemal dynein activity) as a gene downregulated in both garid(−) and gsnf2(−) . First, each of these genes was tagged with gfp to assess their expression using FACS (CALM::GFP and RSPH9::GFP. Then,  the TGTCT and TGCACA motifs were mutated, either or both, within the peak regions located upstream of calm and rsph9 to assess the roles of these two cis-regulatory elements in the activation of male genes.
Evidence is presented that that the TGCACA motif functions as an independent cis-regulatory element for the activation of downstream genes. Furthermore, the effect of disrupting the TGTCT motifs and both of the two motifs in the reporter assays was similar to the level of downregulation in gsnf2(−) and garid(−), respectively. This result is consistent with the implication that the differences between the phenotypes of garid(−) and gsnf2(−) could be due to the function of gARID on the TGCACA motif .

Other mutants

  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0102400
Gene Model P. falciparum ortholog PF3D7_0603600
Gene productmale development protein MD4, putative
Gene product: Alternative nameAT-rich interactive domain-containing protein, putative, gARID
Details of the genetic modification
Name of the tagGFP
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 markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe transgenic parasites were generated by the CRISPR/Cas9 system using the parasites expressing Cas9 (RMgm-4870). The Cas9-expressing parasite Pbcas9 has a cas9 cassette at the p230p locus. The hsp70 promoter controls the expression of Cas9, and Pbcas9 constitutively expresses Cas9 throughout the asexual blood cycle. Donor DNA for transfection was constructed by overlap PCR, cloned into pBluescript KS (+) using the XhoI and BamHI sites by In-Fusion cloning, and then amplified by PCR from the constructed plasmid. sgRNA vectors were constructed as previously described. Target sites of sgRNA were designed using the online tool CHOPCHOP (https://chopchop.cbu.uib.no/).
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