RMgmDB - Rodent Malaria genetically modified Parasites
SummaryRMgm-5036
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Last modified: 28 February 2022, 16:39
*RMgm-5036| Successful modification | The parasite was generated by the genetic modification |
| The mutant contains the following genetic modification(s) | Gene disruption |
| Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 35210361 |
| MR4 number | |
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| Parent parasite used to introduce the genetic modification | |
| Rodent Malaria Parasite | P. yoelii |
| Parent strain/line | P. y. yoelii 17XNL |
| Name parent line/clone | Not applicable |
| Other information parent line | |
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| The mutant parasite was generated by | |
| Name PI/Researcher | Liu M, Zhang Q |
| Name Group/Department | Unit of Molecular Parasitology, Research Center for Translational Medicine |
| Name Institute | East 13 Hospital, Tongji University School of Medicine |
| City | Shanghai |
| Country | China |
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| Name of the mutant parasite | |
| RMgm number | RMgm-5036 |
| Principal name | Pynsun2-KO |
| Alternative name | |
| Standardized name | |
| Is the mutant parasite cloned after genetic modification | Yes |
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| Phenotype | |
| Asexual blood stage | Comparative RNA-BisSeq analysis on the schizont stages of 17XNL wild-type (WT) and Pynsun2 knock-out parasites demonstrated a marked decrease in the density of m5C modifications in mRNA transcripts. By RNA-BisSeq, 7409 m5C sites were detected in 527 mRNAs of the WT parasites whereas this number was reduced to 1845 m5C sites in 265 mRNAs of the knock-outs |
| Gametocyte/Gamete | Experiments with the knock-out and WT lines demonstrated that disruptions of Pynsun2 resulted in dramatically reduced gametocyte production. The disruption of Pynsun2 affected the production of male and female gametocytes similarly while it showed no detectable effect on the propagation of asexual blood-stage parasites. |
| Fertilization and ookinete | Consistent with the effect of gene disruption on gametocyte production, in vitro ookinete conversion rates to mature forms were significantly reduced in the knock-outs relative to those of the WT parasites. Also, the numbers of midgut oocysts at day 7 and salivary gland sporozoites at day 14 were markedly decreased in Pynsun2 knock-out parasites, suggesting that Pynsun2 knock-out effects may impair the development of parasite stages in the mosquito |
| Oocyst | Consistent with the effect of gene disruption on gametocyte production, in vitro ookinete conversion rates to mature forms were significantly reduced in the knock-outs relative to those of the WT parasites. Also, the numbers of midgut oocysts at day 7 and salivary gland sporozoites at day 14 were markedly decreased in Pynsun2 knock-out parasites, suggesting that Pynsun2 knock-out effects may impair the development of parasite stages in the mosquito |
| Sporozoite | Consistent with the effect of gene disruption on gametocyte production, in vitro ookinete conversion rates to mature forms were significantly reduced in the knock-outs relative to those of the WT parasites. Also, the numbers of midgut oocysts at day 7 and salivary gland sporozoites at day 14 were markedly decreased in Pynsun2 knock-out parasites, suggesting that Pynsun2 knock-out effects may impair the development of parasite stages in the mosquito |
| Liver stage | Not tested |
| Additional remarks phenotype | Mutant/mutation 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
Phenotype Experiments with the knock-out and WT lines demonstrated that disruptions of Pynsun2 resulted in dramatically reduced gametocyte production. The disruption of Pynsun2 affected the production of male and female gametocytes similarly while it showed no detectable effect on the propagation of asexual blood-stage parasites. Consistent with the effect of gene disruption on gametocyte production, in vitro ookinete conversion rates to mature forms were significantly reduced in the knock-outs relative to those of the WT parasites. Also, the numbers of midgut oocysts at day 7 and salivary gland sporozoites at day 14 were markedly decreased in Pynsun2 knock-out parasites, suggesting that Pynsun2 knock-out effects may impair the development of parasite stages in the mosquito. 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, 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
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Disrupted: Mutant parasite with a disrupted gene| top of page | |||||||||||||||||||||||||
| Details of the target gene | |||||||||||||||||||||||||
| Gene Model of Rodent Parasite | PY17X_0938600 | ||||||||||||||||||||||||
| Gene Model P. falciparum ortholog | PF3D7_1111000 | ||||||||||||||||||||||||
| Gene product | tRNA m5C-methyltransferase, putative | ||||||||||||||||||||||||
| Gene product: Alternative name | NSUN2 | ||||||||||||||||||||||||
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| Details of the genetic modification | |||||||||||||||||||||||||
| Inducable system used | No | ||||||||||||||||||||||||
| Additional remarks inducable system | |||||||||||||||||||||||||
| Type of plasmid/construct used | CRISPR/Cas9 construct: integration through double strand break repair | ||||||||||||||||||||||||
| PlasmoGEM (Sanger) construct/vector used | No | ||||||||||||||||||||||||
| Modified PlasmoGEM construct/vector used | No | ||||||||||||||||||||||||
| Plasmid/construct map | |||||||||||||||||||||||||
| Plasmid/construct sequence | |||||||||||||||||||||||||
| Restriction sites to linearize plasmid | |||||||||||||||||||||||||
| Partial or complete disruption of the gene | Complete | ||||||||||||||||||||||||
| Additional remarks partial/complete disruption | |||||||||||||||||||||||||
| Selectable marker used to select the mutant parasite | hdhfr/yfcu | ||||||||||||||||||||||||
| Promoter of the selectable marker | eef1a | ||||||||||||||||||||||||
| Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||
| Selection (negative) procedure | No | ||||||||||||||||||||||||
| Additional remarks genetic modification | P. yoelii genetic modifications were performed using the CRISPR/Cas9 plasmid pYCm. To obtain gene knock-outs, 5' and 3'-genomic fragments (400 to 700 bp) of target genes were amplified using the corresponding primers. The PCR products were restriction-digested and cloned into matched sites of the pYCm vector. SgRNA oligonucleotides were annealed and inserted into the pYCm vector. At least two sgRNAs were designed to disrupt the CDS of a target gene for each deletion modification using the online program EuPaGDT. For PyNSUN2 tagging, a 400 to 800 bp fragment from C-terminal of the CDS and 400 to 800 bp segment from the 3'-UTR were amplified and fused with a DNA sequence 6HA in frame at C-terminal of the gene. There were at least two sgRNAs designed to target sites close to the C-terminal of the gene CDS region for incorporation of the insert | ||||||||||||||||||||||||
| Additional remarks selection procedure | |||||||||||||||||||||||||
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Primer information: Primers used for amplification of the target sequences
 Primer information: Primers used for amplification of the target sequences
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