RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5192
Malaria parasiteP. berghei
Genotype
MutatedGene model (rodent): PBANKA_1001800; Gene model (P.falciparum): PF3D7_0404100; Gene product: AP2 domain transcription factor AP2-SP2, putative (ApiAP2; AP2-SP2)
Details mutation: AP2-Sp binding motifs mutated in the upstream region of the AP2-Sp2 gene
Transgene
Transgene not Plasmodium: Cas9 from Streptococcus pyogenes
Promoter: Gene model: PBANKA_0711900; Gene model (P.falciparum): PF3D7_0818900; Gene product: heat shock protein 70 (HSP70)
3'UTR: Gene model: PBANKA_0711900; Gene product: heat shock protein 70 (HSP70)
Replacement locus: Gene model: Not available; Gene product: Not available (small subunit ribosomal rna gene (c-type unit))
Phenotype Oocyst; Sporozoite;
Last modified: 5 April 2022, 15:58
  *RMgm-5192
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene mutation, Introduction of a transgene
Reference (PubMed-PMID number) Not published (yet)
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone RMgm-4870
Other information parent lineThe mutant (RMgm-4870) contains a gene encoding Cas9 from Streptococcus pyogenes, introduced into the c/d-ssu-rrna gene. This Cas9 nuclease does not cleave double-stranded DNA in the absence of sgRNA and is thus suitable for generating the parasite which expresses it constitutively. It does not contain a drug-selectable marker that has been removed by negative selection
The mutant parasite was generated by
Name PI/ResearcherShinzawa N, Iwanaga S
Name Group/DepartmentDepartment of Molecular Protozoology, Research Institute for Microbial Diseases
Name InstituteOsaka University
CityOsaka
CountryJapan
Name of the mutant parasite
RMgm numberRMgm-5192
Principal nameAP2-Sp2(mut)
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot tested
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystOocysts formed as in wild type parasites, but the number of sporozoites collected from the midguts was approximately five times lower in the mutants than in the wild type. RT-qPCR analysis showed that transcripts of AP2-Sp2 decreased significantly. These results indicate that AP2-Sp activates AP2-Sp2 directly through the seven binding sites and that this direct regulation is essential for the normal progression of sporogony.
SporozoiteOocysts formed as in wild type parasites, but the number of sporozoites collected from the midguts was approximately five times lower in the mutants than in the wild type. RT-qPCR analysis showed that transcripts of AP2-Sp2 decreased significantly. These results indicate that AP2-Sp activates AP2-Sp2 directly through the seven binding sites and that this direct regulation is essential for the normal progression of sporogony.
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation

The mutants expresses a mutated ap2-sp2 gene (PBANKA_1001800) with the ap2-sp (PBANKA_1329800) binding motifs mutated in the upstream region of the AP2-Sp2 gene.
In addition, the mutant contains a gene encoding Cas9 from Streptococcus pyogenes, introduced into the c/d-ssu-rrna gene. This Cas9 nuclease does not cleave double stranded DNA in the absence of sgRNA and is thus suitable for generating the parasite which express it constitutively. It does not contain a drug-selectable marker which has been removed by negative selection.

The AP2-Sp2 gene harbors multiple AP2-Sp peaks in its upstream region, with nine binding motifs under the peaks. We intended to mutate all of these motifs using a double CRISPR system by targeting those nearest to each end of the peak region, but protospacer adjacent motif (PAM) sequences were not present around the motif at the 1 3′ end of the peak region. Thus, the third motif from this end was targeted by gRNA. Two motifs near the 3′-end of the peak region of one of the clones were not mutated probably because of crossover homologous recombination between the second and third motifs. 

Published in: bioRxiv preprint doi: https://doi.org/10.1101/2022.02.23.481739

Protein (function)
AP2-Sp belongs to the Apetala2 (AP2) family of genes which encode transcription factors (TFs). The AP2 family TFs were first identified in plants. Plant AP2 family TFs are named for their possession of at least one common DNA-binding domain of approximately 60 amino acids, the AP2 domain. Bioinformatic analyses have revealed the presence of 26 AP2-related genes in the Plasmodium genome. The predicted Plasmodium AP2-related genes encode proteins with one to four AP2 domains. AP2-Sp contains a single AP2 domain. See also mutant RMgm-399 lacking expression of AP2-Sp. Phenotype analyses of this mutant indicate an essential role of AP2-Sp for sporozoite formation and indicate that  AP2-Sp is a transcription factor that binds to promoter regions of genes that are expressed during sporozoite development and activates expression of genes, including genes involved in sporozoite infectivity.

The Cas9 nuclease does not cleave double stranded DNA in the absence of sgRNA and is thus suitable for generating the parasite which express it constitutively.

Phenotype
Oocysts formed as in wild type parasites, but the number of sporozoites collected from the midguts was approximately five times lower in the mutants than in the wild type. RT-qPCR analysis showed that transcripts of AP2-Sp2 decreased significantly. These results indicate that AP2-Sp activates AP2-Sp2 directly through the seven binding sites and that this direct regulation is essential for the normal progression of sporogony.

Additional information
From the Abstract
'AP2-Sp is a transcription factor (TF) essential for the formation of sporozoites or sporogony, which takes place in oocysts in the midgut of infected mosquitoes. To understand the role of this TF in the transcriptional regulatory system of this stage, we performed ChIP-seq analyses using whole mosquito midguts containing late oocysts as starting material and explored its genome-wide target genes. We identified 697 target genes.The ChIP-seq analyses also showed that AP2-Sp induces all TFs reported to be transcribed at this stage, including AP2-Sp2, AP2-Sp3, and SLARP.'

Other mutants


  Mutated: Mutant parasite with a mutated gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1001800
Gene Model P. falciparum ortholog PF3D7_0404100
Gene productAP2 domain transcription factor AP2-SP2, putative
Gene product: Alternative nameApiAP2; AP2-SP2
Details of the genetic modification
Short description of the mutationAP2-Sp binding motifs mutated in the upstream region of the AP2-Sp2 gene
Inducable system usedNo
Short description of the conditional mutagenesisNot available
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 parasiteunknown
Promoter of the selectable markerunknown
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationParasites with mutated AP2-Sp binding motifs were prepared with the CRISPR/Cas9 system using P. berghei parasites constitutively expressing Cas9 (pbcas9). A DNA fragment corresponding to the upstream region of the gene was synthesized with all motifs mutated (two point mutations per motif). Sequences for homologous recombination were added to both sides of the fragment by overlapping PCR and used as donor DNA. Two guide RNA (gRNA) sequences were designed, corresponding to the motifs on opposite sides of the synthesized fragment. The gRNA target sequences were subcloned into a plasmid for double CRISPR gRNA expression. Mature schizonts were transfected with donor DNA and the gRNA plasmid, and mutants were selected with pyrimethamine for 1.5 d, beginning 30 hours after transfection. Parasite clones were obtained by limiting dilution, and correct exchange of the original locus with the donor DNA fragment by double-crossover homologous recombination was verified by sequencing.

We created parasites in which the AP2-Sp binding motifs were mutated in the upstream region of the AP2-Sp2 gene and examined whether AP2-Sp2 was directly activated by AP2-Sp.
The AP2-Sp2 gene harbors multiple AP2-Sp peaks in its upstream region, with nine binding motifs under the peaks. We intended to mutate all of these motifs using a double CRISPR system by targeting those nearest to each end of the peak region, but protospacer adjacent motif (PAM) sequences were not present around the motif at the 1 3′ end of the peak region. Thus, the third motif from this end was targeted by gRNA. Two motifs near the 3′-end of the peak region of one of the clones were not mutated probably because of crossover homologous recombination between the second and third motifs.
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 nameCas9 from Streptococcus pyogenes
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 parasiteNo selectable marker
Promoter of the selectable markerN.A.
Selection (positive) procedureN.A.
Selection (negative) procedureN.A.
Additional remarks genetic modificationCas9 from Streptococcus pyogenes was used in this study. This Cas9 nuclease does not cleave double stranded DNA in the absence of sgRNA and is thus suitable for generating the parasite which express it constitutively.To introduce the expression cassette of Cas9 nuclease in the genome of P. berghei, the pcssu-Cas9-hy plasmid was constructed. The pcssu-Cas9-hy contained not only the Cas9 expression cassette but also the hdhfr–yfcu expression cassette as the positive/negative selection marker. The human dihydrofolate reductase gene, i.e., hdhfr confers pyrimethamine resistance to the parasites and was used as a positive selectable marker. The yfcu gene is a fusion gene of yeast cytosine deaminase and uridyl-phosphoribosyltransferase. The parasite that expresses the yfcu gene is killed by 5-FC, and the yfcu can thus be used as a negative selection marker. The Cas9 cassette and the hdhfr–yfcu cassette contained the 3′UTR of hsp70, which was used for the termination of transcription of both genes. To generate the hdhfr–yfcu fusion gene cassette, hdhfr with the ef1α promoter was amplified by PCR using pSK-125 as a template and the primers Pef1α-F and hdhfr-R, while yfcu with the 3′UTR of hsp70 was amplified from pfgRNA15 with the primers yfcu-F and hsp3UTR-R. These two amplified fragments were fused by PCR. In the fused fragment, the termination codon of hdhfr was eliminated to generate the fusion gene. The resulting hdhfr–yfcu cassette was cloned into the SalI/BamHI sites of pSK-1, resulting in the pSK-1-hy plasmid. The Cas9 expression cassette was excised by NheI/SalI from the pfCas9 plasmid15 and cloned into the pSK-1-hy plasmid, resulting in the pSK-1-Cas9-hy plasmid. To integrate the Cas9 and hdhf-yfcu cassettes into the genomic locus of the rRNA Ctype subunit (cssu) on chromosome 5, two partial sequences, HDR1 and 2, of the cssu were amplified and cloned into the KpnI/XhoI site and the BamHI/NotI site of pSK-1-Cas9-hy, resulting in the pcssu-Cas9-hy plasmid.
Additional remarks selection procedureThe mutant does not contain a drug-selectable marker (hdhfr-yfcu) which has been removed by negative selection
Other details transgene
Promoter
Gene Model of Parasite PBANKA_0711900
Gene Model P. falciparum ortholog PF3D7_0818900
Gene productheat shock protein 70
Gene product: Alternative nameHSP70
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_0711900
Gene productheat shock protein 70
Gene product: Alternative nameHSP70
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 Not available
Gene productNot available
Gene product: Alternative namesmall subunit ribosomal rna gene (c-type unit)
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