RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4870
Malaria parasiteP. berghei
Genotype
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;
Last modified: 10 September 2020, 17:17
  *RMgm-4870
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 32759952
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone Not applicable
Other information parent line
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-4870
Principal namepbcas9
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
Oocystslightly reduced oocyst numbers
SporozoiteNot different from wild type
Liver stageNot different from wild type
Additional remarks phenotype

Mutant/mutation
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

Protein (function)
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.

Phenotype
No obvious effect on development of all life cycle stages due to constitutive expression of Cas9 and integration of its expression cassette at the c-ssu-rrna locus.

Additional information
The mutant does not contain a drug-selectable marker which has been removed by negative selection.

Whole genome sequencing revealed that the constitutive expression of Cas9 did not cause unexpected mutations in the parasite genome.

Unexpected recombination events were observed when this parasite was used to modify genes with circular donor DNA plasmids:
From the paper: 
'When the target genomic locus is cleaved by the Cas9–sgRNA complex, the molecules responsible for the repair of the doublestrand break are recruited, elevating the recombination activity specifically at the locus. Thus, the observed additional recombination was probably caused by the increase in recombination activity at the target genomic locus. Similar additional recombination was found in P. falciparum when  nonessential genes for erythrocytic development were engineered by the CRISPR/Cas9 system using a plasmid that contained the donor template. Conversely, when an essential gene for erythrocytic development was mutated in our previous study, such recombination was never found despite using a circular plasmid carrying the donor template. However, the additional recombination might be overlooked in this case, because the transgenic parasites in which this recombination occurred died due to the  functional disruption of the essential gene. Thus, whenever a circular plasmid is used for delivering the donor template, an additional recombination will occur between the integrated donor template and another copy of the plasmid as the donor template, resulting in failure or reduced efficiency of the genetic modification. Therefore, this is a serious technical problem of the CRISPR/Cas9 system when using circular plasmid DNA  as the donor template.'

Based on these observations the authors developed linear donor templates ('To solve the technical problem of integrated circular donor DNA, we used a linear donor template DNA for subsequent genetic modifications.')
 

From the Abstract:

'The use of a linear donor template prevented unexpected recombination; in addition, constitutive expression of Cas9 enabled immediate cleavage of the target locus after transfection, allowing efficient integration of the donor template. Furthermore, due to the absence of the cNHEJ pathway, there were no off-target mutations in the resultant parasites. In addition, this developed method could be applied for multiple genetic modifications on different chromosomes and for large scale chromosomal deletion in the subtelomeric region.'


Other mutants


  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