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Details of the target gene |
Gene Model of Rodent Parasite |
PY17X_1453700
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Gene Model P. falciparum ortholog |
PF3D7_1236600
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Gene product | p25-alpha family protein, putative |
Gene product: Alternative name | |
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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 | (Linear) plasmid double cross-over |
PlasmoGEM (Sanger) construct/vector used | No |
Modified PlasmoGEM construct/vector used | No
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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 |
Promoter of the selectable marker | eef1a |
Selection (positive) procedure | pyrimethamine |
Selection (negative) procedure | No |
Additional remarks genetic modification | A 20 bp sequence of guide RNA (gRNA) was designed upstream of the protospacer-adjacent motif (PAM), using the online CRISPR guide RNA/DNA design tool for eukaryotic pathogen (EuPaGDT; http://grna.ctegd.uga.edu), and a pair of complementary oligonucleotides were synthesized for each target site (Sangon Biotech, China). The CRISPR/Cas9 plasmid pYC, a gift from professor Jing Yuan (Xiamen University), was used for all the genetic modifications. Since the PyU6 promoter requires a guanosine nucleotide to initiate transcription, we append an extra “G” base to the beginning of the guide sequence to facilitate U6 transcription initiation if the first base of the guide sequence is not “G”. In addition, the oligonucleotides were designed to generate overhangs to be used for cloning into BsmBI-digested pYC plasmid. The cloned gRNA was placed under the PyU6 promoter and fused with a tracrRNA, which generated a sgRNA. For gene deleting, 5' -genomic and 3' -genomic segments of the target genes were amplified as left and right homologous arms, respectively, using gene-specific primers. The PCR products were digested with using HindIII and AflII restriction enzymes, and then inserted into matched restriction sites of pYC plasmid, which has the human dihydroreductase gene as drug selectable marker. Thus, the transfected parasite with this plasmid can be selected using pyrimethamine
In short: We constructed a plasmid pYC-Py05543 (Pyp25α) containing a 46 bp insert DNA flanked by two homologous regions of Py05543 (403 bp of the 5'-flanking region and 453 bp of the 3'-flanking region) to target the 3'-end of the Py05543 exon 2. One day after electroporation of the plasmid pYC-Py05543 into the P. yoelii 17XNL strain, parasites were selected with pyrimethamine. |
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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