Summary

RMgm-4448
Malaria parasiteP. yoelii
Genotype
DisruptedGene model (rodent): PY17X_1004400; Gene model (P.falciparum): PF3D7_0405300; Gene product: liver specific protein 2, putative (LISP2)
Transgene
Transgene not Plasmodium: GFP-Luciferase
Promoter: Gene model: PBANKA_1133300; Gene model (P.falciparum): PF3D7_1357100; Gene product: elongation factor 1-alpha (eef1a)
3'UTR: Gene model: PBANKA_0719300; Gene product: bifunctional dihydrofolate reductase-thymidylate synthase, putative (dhfr/ts)
Replacement locus: Gene model: PY17X_0306600; Gene product: 6-cysteine protein (P230p)
Phenotype Liver stage;
Last modified: 18 May 2018, 12:56
  *RMgm-4448
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption, Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 29440367
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-689
Other information parent lineRMgm-689 (1971cl1) is a P.y.yoelii 17XNL mutant expressing the fusion protein GFP-Luciferase under the control of the constitutive P. berghei eef1a promoter. The mutant does not contain a drug-selectable marker.
The mutant parasite was generated by
Name PI/ResearcherVaughan AM; Kappe SHI
Name Group/DepartmentCenter for Infectious Disease Research
Name InstituteCenter for Infectious Disease Research
CitySeattle
CountryWashington
Name of the mutant parasite
RMgm numberRMgm-4448
Principal nameLISP2(-)
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 ookineteNot different from wild type
OocystNot different from wild type
SporozoiteNot different from wild type
Liver stageReduced liver stage development resulting in prolonged prepatent period.
We chose to study LISP2 because it is expressed on the middle-to-late liver stage parasitophorous vacuole membrane and deletion of P. berghei LISP2 leads to incomplete late liver stage growth arrest
To determine if P. yoelii lisp2− arrests during liver stage development, groups of BALB/cJ mice were i.v. challenged with either 1,000 marker-free GFP-luciferase-expressing 1971cl1 parent parasites (here referred to as wild type) or 1,000 lisp2− sporozoites, and time to blood stage patency was determined. All wild-type-infected mice became blood stage patent on day 3 after challenge, whereas two of seven P. yoelii lisp2−-infected mice did not become patent and the remaining mice showed severe delays to patency, becoming patent between 5 and 7 days after infection. When mice were challenged with 10,000 lisp2−sporozoites, all mice became blood stage patent from days 4 through 6, demonstrating the incomplete attenuation of P. yoelii lisp2−.
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of LISP2
The gene has been disrupted using CRISPR/cas9 genome editing (using constructs as described for mutant RMgm-1095).

Protein (function)
The protein contains a predicted Plasmodium 6-cysteine motif. Several studies analysing expression of this protein in P. berghei provide evidence for specific expression in liver stages. The protein has been named LISP2 and sequestrin.

Phenotype
Reduced liver stage development resulting in prolonged prepatent period.
We chose to study LISP2 because it is expressed on the middle-to-late liver stage parasitophorous vacuole membrane and deletion of P. berghei LISP2 leads to incomplete late liver stage growth arrest
To determine if P. yoelii lisp2− arrests during liver stage development, groups of BALB/cJ mice were i.v. challenged with either 1,000 marker-free GFP-luciferase-expressing 1971cl1 parent parasites (here referred to as wild type) or 1,000 lisp2− sporozoites, and time to blood stage patency was determined. All wild-type-infected mice became blood stage patent on day 3 after challenge, whereas two of seven P. yoelii lisp2−-infected mice did not become patent and the remaining mice showed severe delays to patency, becoming patent between 5 and 7 days after infection. When mice were challenged with 10,000 lisp2−sporozoites, all mice became blood stage patent from days 4 through 6, demonstrating the incomplete attenuation of P. yoelii lisp2−.

Additional information
The lisp2 gene was deleted using the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 technology, which allows efficient editing of the parasite genome. The advantage to this system is that transgenic parasites do not carry a drug susceptibility marker and thus can easily undergo further genetic manipulation.

Deletion of P. yoelii LISP2 was achieved based on the previously reported CRISPR/Cas9 strategy using plasmid pYC. In brief, LISP2 was deleted using double-crossover homologous recombination following a double-stranded DNA break mediated by Cas9 containing a guide RNA targeting the gene of interest. Complementary regions upstream and downstream of the open reading frame were ligated into plasmid pYC, as was the 20-nucleotide guide RNA sequence, resulting in the creation of plasmid pYC_LISP2. The pYC plasmid was transfected into the blood stage schizonts of P. yoelii line 1971cl1, a marker-free parasite that behaves as the wild type and expresses a green fluorescent protein (GFP)-luciferase fusion throughout the life cycle under the control of the elongation factor 1 alpha promoter. This led to the creation of the P. yoelii lisp2−. Two separate knockout clones from two independent transfections were initially phenotypically analyzed throughout the life cycle.

Other mutants


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PY17X_1004400
Gene Model P. falciparum ortholog PF3D7_0405300
Gene productliver specific protein 2, putative
Gene product: Alternative nameLISP2
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
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe lisp2 gene was deleted using the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 technology, which allows efficient editing of the parasite genome. The advantage to this system is that transgenic parasites do not carry a drug susceptibility marker and thus can easily undergo further genetic manipulation.

Deletion of P. yoelii LISP2 was achieved based on the previously reported CRISPR/Cas9 strategy using plasmid pYC. In brief, LISP2 was deleted using double-crossover homologous recombination following a double-stranded DNA break mediated by Cas9 containing a guide RNA targeting the gene of interest. Complementary regions upstream and downstream of the open reading frame were ligated into plasmid pYC, as was the 20-nucleotide guide RNA sequence, resulting in the creation of plasmid pYC_LISP2. The pYC plasmid was transfected into the blood stage schizonts of P. yoelii line 1971cl1, a marker-free parasite that behaves as the wild type and expresses a green fluorescent protein (GFP)-luciferase fusion throughout the life cycle under the control of the elongation factor 1 alpha promoter. This led to the creation of the P. yoelii lisp2−. Two separate knockout clones from two independent transfections were initially phenotypically analyzed throughout the life cycle.
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

  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameGFP-Luciferase
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct(Linear) plasmid double cross-over
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid
Selectable marker used to select the mutant parasitehdhfr/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedureNo
Selection (negative) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modification
Additional remarks selection procedure
Other details transgene
Promoter
Gene Model of Parasite PBANKA_1133300
Gene Model P. falciparum ortholog PF3D7_1357100
Gene productelongation factor 1-alpha
Gene product: Alternative nameeef1a
Primer information details of the primers used for amplification of the promoter sequence  Click to view information
Primer information details of the primers used for amplification of the promoter sequence  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
3'-UTR
Gene Model of Parasite PBANKA_0719300
Gene productbifunctional dihydrofolate reductase-thymidylate synthase, putative
Gene product: Alternative namedhfr/ts
Primer information details of the primers used for amplification the 3'-UTR sequences  Click to view information
Primer information details of the primers used for amplification the 3'-UTR sequences  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Insertion/Replacement locus
Replacement / InsertionReplacement locus
Gene Model of Parasite PY17X_0306600
Gene product6-cysteine protein
Gene product: Alternative nameP230p
Primer information details of the primers used for amplification of the target sequences  Click to view information
Primer information details of the 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