RMgmDB - Rodent Malaria genetically modified Parasites
SummaryRMgm-5192
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*RMgm-5192| Successful modification | The 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) |
Reference 1 (PMID number) : 36622145 |
| MR4 number | |
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| Parent parasite used to introduce the genetic modification | |
| Rodent Malaria Parasite | P. berghei |
| Parent strain/line | P. berghei ANKA |
| Name parent line/clone | RMgm-4870 |
| Other information parent line | The 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 |
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| The mutant parasite was generated by | |
| Name PI/Researcher | Shinzawa N, Iwanaga S |
| Name Group/Department | Department of Molecular Protozoology, Research Institute for Microbial Diseases |
| Name Institute | Osaka University |
| City | Osaka |
| Country | Japan |
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| Name of the mutant parasite | |
| RMgm number | RMgm-5192 |
| Principal name | AP2-Sp2(mut) |
| Alternative name | |
| Standardized name | |
| Is the mutant parasite cloned after genetic modification | Yes |
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| Phenotype | |
| Asexual blood stage | Not tested |
| Gametocyte/Gamete | Not tested |
| Fertilization and ookinete | Not tested |
| Oocyst | 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. |
| Sporozoite | 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. |
| Liver stage | Not 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. Protein (function) Additional information Other mutants |
Mutated: Mutant parasite with a mutated gene| top of page | |||||||||||||||||||||||||||
| Details of the target gene | |||||||||||||||||||||||||||
| Gene Model of Rodent Parasite | PBANKA_1001800 | ||||||||||||||||||||||||||
| Gene Model P. falciparum ortholog | PF3D7_0404100 | ||||||||||||||||||||||||||
| Gene product | AP2 domain transcription factor AP2-SP2, putative | ||||||||||||||||||||||||||
| Gene product: Alternative name | ApiAP2; AP2-SP2 | ||||||||||||||||||||||||||
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| Details of the genetic modification | |||||||||||||||||||||||||||
| Short description of the mutation | AP2-Sp binding motifs mutated in the upstream region of the AP2-Sp2 gene | ||||||||||||||||||||||||||
| Inducable system used | No | ||||||||||||||||||||||||||
| Short description of the conditional mutagenesis | Not available | ||||||||||||||||||||||||||
| Additional remarks inducable system | |||||||||||||||||||||||||||
| Type of plasmid/construct | (Linear) plasmid double cross-over | ||||||||||||||||||||||||||
| PlasmoGEM (Sanger) construct/vector used | No | ||||||||||||||||||||||||||
| Modified PlasmoGEM construct/vector used | No | ||||||||||||||||||||||||||
| Plasmid/construct map | |||||||||||||||||||||||||||
| Plasmid/construct sequence | |||||||||||||||||||||||||||
| Restriction sites to linearize plasmid | |||||||||||||||||||||||||||
| Selectable marker used to select the mutant parasite | unknown | ||||||||||||||||||||||||||
| Promoter of the selectable marker | unknown | ||||||||||||||||||||||||||
| Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||||
| Selection (negative) procedure | No | ||||||||||||||||||||||||||
| Additional remarks genetic modification | Parasites 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 | |||||||||||||||||||||||||||
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Transgene: Mutant parasite expressing a transgene| top of page | |||||||||||||||||||
| Type and details of transgene | |||||||||||||||||||
| Is the transgene Plasmodium derived | Transgene: not Plasmodium | ||||||||||||||||||
| Transgene name | Cas9 from Streptococcus pyogenes | ||||||||||||||||||
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| Details of the genetic modification | |||||||||||||||||||
| Inducable system used | No | ||||||||||||||||||
| Additional remarks inducable system | |||||||||||||||||||
| Type of plasmid/construct | (Linear) plasmid double cross-over | ||||||||||||||||||
| PlasmoGEM (Sanger) construct/vector used | No | ||||||||||||||||||
| Modified PlasmoGEM construct/vector used | No | ||||||||||||||||||
| Plasmid/construct map | |||||||||||||||||||
| Plasmid/construct sequence | |||||||||||||||||||
| Restriction sites to linearize plasmid | |||||||||||||||||||
| Selectable marker used to select the mutant parasite | No selectable marker | ||||||||||||||||||
| Promoter of the selectable marker | N.A. | ||||||||||||||||||
| Selection (positive) procedure | N.A. | ||||||||||||||||||
| Selection (negative) procedure | N.A. | ||||||||||||||||||
| Additional remarks genetic modification | Cas9 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 procedure | The mutant does not contain a drug-selectable marker (hdhfr-yfcu) which has been removed by negative selection | ||||||||||||||||||
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| Other details transgene | |||||||||||||||||||
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| Promoter | |||||||||||||||||||
| Gene Model of Parasite | PBANKA_0711900 | ||||||||||||||||||
| Gene Model P. falciparum ortholog | PF3D7_0818900 | ||||||||||||||||||
| Gene product | heat shock protein 70 | ||||||||||||||||||
| Gene product: Alternative name | HSP70 | ||||||||||||||||||
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| 3'-UTR | |||||||||||||||||||
| Gene Model of Parasite | PBANKA_0711900 | ||||||||||||||||||
| Gene product | heat shock protein 70 | ||||||||||||||||||
| Gene product: Alternative name | HSP70 | ||||||||||||||||||
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| Insertion/Replacement locus | |||||||||||||||||||
| Replacement / Insertion | Replacement locus | ||||||||||||||||||
| Gene Model of Parasite | Not available | ||||||||||||||||||
| Gene product | Not available | ||||||||||||||||||
| Gene product: Alternative name | small subunit ribosomal rna gene (c-type unit) | ||||||||||||||||||
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