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Details of the target gene |
Gene Model of Rodent Parasite |
PY17X_0410700
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Gene Model P. falciparum ortholog |
PF3D7_0310100
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Gene product | calcium-dependent protein kinase 3 |
Gene product: Alternative name | CDPK3 |
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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
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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/yfcu |
Promoter of the selectable marker | eef1a |
Selection (positive) procedure | pyrimethamine |
Selection (negative) procedure | No |
Additional remarks genetic modification | Because the constitutive expression of Cas9 in the PyCas9ki parasite, only sgRNA cassette and homologous DNA templates are needed for editing a specific parasite locus. We next removed the SpCas9 coding sequence in the pYCm vector via mutagenesis, resulting in a smaller plasmid, pYCs. To test whether the endogenously expressed Cas9 protein could function in CRISPR/Cas9-mediated gene modification, we attempted to delete two genes (ctrp and cdpk3) in the genome of the PyCas9ki parasites, separately. The ctrp and cdpk3 genes in both Plasmodium berghei and P. yoelii parasites were previously disrupted, leading to complete loss or severe defect in ookinete gliding motility and absence of oocysts in the mosquito after infection. We constructed a plasmid pYCs-cdpk3 containing a 46-bp tag DNA (for PCR primers) flanked by two homologous regions of cdpk3 (0.49 kb of the 5′-flanking region and 0.53 kb of the 3′-flanking region). Two sgRNAs targeting the exon1 and exon 2 of the cdpk3, respectively, were designed and inserted into the pYCs-cdpk3 vector, generating plasmids pYCs-cdpk3-sgRNA1 and pYCs-cdpk3-sgRNA2. One day after electroporation of the plasmids into the PyCas9ki parasite, parasites were selected with Pyr supplied in drinking water. Pyr-resistant parasites were observed microscopically 5 to 6 days after electroporation. Expression of sgRNA1 and sgRNA2 transcripts was detected using RT-PCR in the transfected parasites. PCR analysis of genomic DNA from parental strain PyCas9ki and plasmid-transfected parasites indicated successful integration of left and right homologous arms at specific sites directed by both sgRNA1 and sgRNA2, but not by control sgRNA targeting irrelevant sequences. After limiting dilution cloning, two parasite clones with disrupted cdpk3 gene were obtained and confirmed by PCR genotyping. |
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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