Summary

RMgm-4453
Malaria parasiteP. yoelii
Genotype
DisruptedGene model (rodent): PY17X_0410700; Gene model (P.falciparum): PF3D7_0310100; Gene product: calcium-dependent protein kinase 3 (CDPK3)
Phenotype Fertilization and ookinete;
Last modified: 18 May 2018, 18:02
  *RMgm-4453
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption
Reference (PubMed-PMID number) Reference 1 (PMID number) : 29684399
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. yoelii
Parent strain/lineP. y. yoelii 17XNL
Name parent line/clone RMgm-4452
Other information parent lineThe cas9-knockin parasite (PyCas9ki): this mutant (RMgm4452) contains a C-terminal quadruple Myc (4Myc) tagged Spcas9 gene introduced into the silent sera1 gene locus. The Spcas9 gene is under control of the 5'- and 3'-UTR regions of sera1. The mutant does not contain a drug-selectable marker.
The mutant parasite was generated by
Name PI/ResearcherQian P; Yuan J
Name Group/DepartmentState Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signal Network
Name InstituteSchool of Life Sciences, Xiamen University
CityXiamen, Fujian
CountryChina
Name of the mutant parasite
RMgm numberRMgm-4453
Principal nameΔcdpk3
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNot different from wild type
Fertilization and ookineteNormal ookinete production. Severe defect in ookinete gliding motility in vitro
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of cdpk3 (and expresses SpCas9 and lacks expression of sera1). It does not contain a drug-selectable marker

Protein (function)
CDPK3 belongs to an expanded family of Ca2+ dependent protein kinases (CDPKs). CDPKs combine an amino-terminal serine/threonine kinase domain and a carboxy-terminal calmodulin-like domain, composed of four EF hands, in the same molecule. The protein plays a role in ookinetes. Ookinetes lacking expression of CDPK3 fail to bind and traverse the cell of the midgut wall.

Phenotype
Not analysed in detail. The mutant was generated by CRISPR/Cas9 technology as 'a-proof-of-principle' to show disruption of genes in a parasite stably expressing Cas9.

Severe defect in ookinete gliding motility in vitro

Additional information
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.

Other mutants


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PY17X_0410700
Gene Model P. falciparum ortholog PF3D7_0310100
Gene productcalcium-dependent protein kinase 3
Gene product: Alternative nameCDPK3
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 modificationBecause 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  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