RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5038
Malaria parasiteP. yoelii
Genotype
Genetic modification not successful
DisruptedGene model (rodent): PY17X_0920500; Gene model (P.falciparum): PF3D7_1129400; Gene product: rRNA (cytosine-C(5))-methyltransferase, putative (NOP2, NSUN3)
PhenotypeNo phenotype has been described
Last modified: 28 February 2022, 16:40
  *RMgm-5038
Successful modificationThe gene/parasite could not be changed/generated by the genetic modification.
The following genetic modifications were attempted Gene disruption
Number of attempts to introduce the genetic modification 3
Reference (PubMed-PMID number) Reference 1 (PMID number) : 35210361
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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
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Attempts to generate the mutant parasite were performed 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
CityShanghai
CountryChina
  Disrupted: Mutant parasite with a disrupted gene
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Details of the target gene
Gene Model of Rodent Parasite PY17X_0920500
Gene Model P. falciparum ortholog PF3D7_1129400
Gene productrRNA (cytosine-C(5))-methyltransferase, putative
Gene product: Alternative nameNOP2, NSUN3
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Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct usedCRISPR/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
Partial or complete disruption of the geneComplete
Additional remarks partial/complete disruption
Selectable marker used to select the mutant parasitehdhfr/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe unsuccessful attempts to disrupt this gene indicate an essential function during asexual blood-stage growth

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).


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.
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
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Conceptual design of the database: Dr. C.J. Janse (Leiden Malaria Research Group, Leiden, The Netherlands).
Technical design and realization: S. Mededovic and A. van Wigcheren