RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5515

Malaria parasite	P. yoelii
Genotype
Genetic modification not successful
Disrupted	Gene model (rodent): PY17X_1241800; Gene model (P.falciparum): PF3D7_0523800; Gene product: food vacuole resident transporter 1 (FVRT1, PyDMT1 (Divalent metal transporter 1))
Phenotype	No phenotype has been described

Last modified: 16 June 2024, 21:20

*RMgm-5515

Successful modification	The 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	≥ 5
Reference (PubMed-PMID number)	Reference 1 (PMID number) : 38049852
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Parent parasite used to introduce the genetic modification
Rodent Malaria Parasite	P. yoelii
Parent strain/line	P. y. yoelii 17X
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/Researcher	Zhong M, Zhou B
Name Group/Department	Faculty of Synthetic Biology
Name Institute	Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences
City	Shenzhen
Country	China

Disrupted: Mutant parasite with a disrupted gene

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Details of the target gene

Gene Model of Rodent Parasite

PY17X_1241800

Gene Model P. falciparum ortholog

PF3D7_0523800

Gene product

food vacuole resident transporter 1

Gene product: Alternative name

FVRT1, PyDMT1 (Divalent metal transporter 1)

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Details of the genetic modification

Inducable system used

Additional remarks inducable system

Type of plasmid/construct used

CRISPR/Cas9 construct: integration through double strand break repair

PlasmoGEM (Sanger) construct/vector used

Modified PlasmoGEM construct/vector used

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

Promoter of the selectable marker

eef1a

Selection (positive) procedure

pyrimethamine

Selection (negative) procedure

Additional remarks genetic modification

The unsuccessful attempts to disrupt this gene indicate an essential function during asexual blood stage growth/multiplication

To clarify the physiological function of PyDMT1 in Plasmodium, we attempted to knock out PyDMT1 in P. yoelii17X by CRISPR-Cas9. Nine guide RNAs were designed to cover the Pydmt1 gene, and each guide RNA and homologous recombination arms were independently electro-transfected five times. No Pydmt1 knockout parasites were obtained in all these trials.

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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