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| Details of the target gene |
| Gene Model of Rodent Parasite |
PY17X_0916600
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| Gene Model P. falciparum ortholog |
PF3D7_1133300
|
| Gene product | LEM3/CDC50 family protein, putative |
| Gene product: Alternative name | CDC50c |
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| Details of the genetic modification |
| Inducable system used | No |
| Additional remarks inducable system |
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| 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 |
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| Plasmid/construct sequence |
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| Restriction sites to linearize plasmid |
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| Partial or complete disruption of the gene | Complete |
| Additional remarks partial/complete disruption |
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| 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 | The unsuccessful attempts to disrupt this gene indicate an essential function during asexual blood-stage growth
CRISPR-Cas9 plasmid pYCm was used for all the parasite genetic modification. To construct vectors for gene editing, we amplified 5′ and 3′ genomic sequence (400 to 500 bp) of target genes as homologous arms using specific primers and inserted the sequences into specific restriction sites in pYCm. Oligonucleotides for single guide RNAs (sgRNAs) were mixed in pairs, denatured at 95°C for 3 min, annealed at room temperature for 5 min, and ligated into pYCm. The sgRNAs were designed to target the coding region of a gene using the online program EuPaGDT. DNA fragments encoding 6HA, 4Myc, 3V5, and GFP or mCherry were inserted between the left and right arms in frame with the gene of interest. For each gene, two sgRNAs were designed to target sites close to the C- or N-terminal part of the coding region. Infected red blood cells (iRBCs) were electroporated with 5 to 10 μg of plasmid DNA using the Lonza Nucleofector . Transfected parasites were immediately intravenously injected into a naïve mouse and were exposed to pyrimethamine (6 mg/ml) 24 hours after transfection. |
| Additional remarks selection procedure | |
Primer information: Primers used for amplification of the target sequences  Primer information: Primers used for amplification of the target sequences 
| 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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*RMgm-5055