RMgmDB - Rodent Malaria genetically modified Parasites


Malaria parasiteP. yoelii
TaggedGene model (rodent): PY17X_0938600; Gene model (P.falciparum): PF3D7_1111000; Gene product: tRNA m5C-methyltransferase, putative (NSUN2)
Name tag: 6xHA
PhenotypeNo phenotype has been described
Last modified: 28 February 2022, 16:41
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) : 35210361
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. yoelii
Parent strain/lineP. y. yoelii 17XNL
Name parent line/clone Not applicable
Other information parent line
The mutant parasite was generated by
Name PI/ResearcherLiu M, Zhang Q
Name Group/DepartmentUnit of Molecular Parasitology, Research Center for Translational Medicine
Name InstituteEast 13 Hospital, Tongji University School of Medicine
Name of the mutant parasite
RMgm numberRMgm-5040
Principal namePynsun2-6xHA
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Asexual blood stageNot tested
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

The mutant expresses a C-terminal 6xHA-tagged version of NSUN2 (no evidence for expression is reported).
Published in: bioRxiv preprint doi: https://doi.org/10.1101/2021.06.06.447275

Protein (function)
5-methylcytosine (m5C) is an important epitranscriptomic modification involved in mRNA stability and translation efficiency in various biological processes, suggesting that cytosine methylation is a powerful regulator of cellular processes at the epitranscriptomic level. Specific methyltransferases (m5C writers) in various species include NSUN2 in humans.
Malaria parasites have genes that encode homologs of human and plant NSUN2 methyltransferases (m5C “writers”).

To identify potential mRNA m5C methyltransferases in P. yoelii, we searched for orthologs of hNSUN2 and identified four candidate sequences: PY17X_0804600, PY17X_0938600, PY17X_0920500, PY17X_1447800 (named PyNSUN1–PyNSUN4, respectively). Corresponding searches of the www.plasmoDB.org database identified  PF3D7_0704200, PF3D7_1111000, PF3D7_1129400, and PF3D7_1230600 (PfNSUN1–PfNSUN4) as candidate RNA methyltransferases in P. falciparum. The NSUN1 and NSUN2 sequences have close affinities to orthologs in humans, Arabidopsis thaliana, Saccharomyces cerevisiae, and Oryza sativa

Not reported

Additional information

To study the role of these putative m5C methyltransferases in Plasmodium, we performed experiments to disrupt Pynsun2 in the P. yoelii 17XNL line  by CRISPR-Cas9 gene editing. Functional disruption attempts of the sun1, sun3, and sun4 homologs were performed similarly. After a minimum of three independent transfection attempts with 2 to 3 different single guide RNA (sgRNA) sequences for each individual homolog  we obtained knock-out lines for two of the four genes (Pynsun1, Pynsun2).

In contrast to Pynsun2 knock-out line (RMgm-5036), gametocyte production in Pynsun1 knock-out line (RMgm-5037) was not significantly reduced

From the Abstract:
'5-methylcytosine (m5C) is an important epitranscriptomic modification involved in mRNA  stability and translation efficiency in various biological processes. However, it remains unclear if m5C modification contributes to the dynamic regulation of the transcriptome during the developmental cycles of Plasmodium parasites. Here, we characterize the landscape of m5C mRNA modifications at single nucleotide resolution in the asexual replication stages and gametocyte sexual stages of rodent (P. yoelii) and human (P. falciparum) malaria parasites. While different representations of m5C-modified mRNAs are associated with the different stages, the abundance of the m5C marker is strikingly enhanced in the transcriptomes of gametocytes. Our results show that m5C modifications confer stability to the Plasmodium transcripts and that a Plasmodium ortholog of NSUN2 is a major mRNA m5C methyltransferase in malaria parasites. Upon knock-out of P. yoelii nsun2 (pynsun2), marked reductions of m5C modification were observed in a panel of gametocytogenesis-associated transcripts. These reductions correlated with impaired gametocyte production in rodent and human malaria parasites. Restoration of the nsun2 gene in the knock-out parasites rescued the gametocyte production phenotype as well as m5C modification of the gametocytogenesis-associated transcripts.' 

Other mutants


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PY17X_0938600
Gene Model P. falciparum ortholog PF3D7_1111000
Gene producttRNA m5C-methyltransferase, putative
Gene product: Alternative nameNSUN2
Details of the genetic modification
Name of the tag6xHA
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/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationP. yoelii genetic modifications were performed using the CRISPR/Cas9 plasmid pYCm.
For PyNSUN2 tagging, a 400 to 530 800 bp fragment from C-terminal of the CDS and 400 to 800 bp segment from the 3'-UTR 531 were amplified and fused with a DNA sequence 6HA in frame at C-terminal of the gene
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