SummaryRMgm-5108
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Successful modification | The parasite was generated by the genetic modification |
The mutant contains the following genetic modification(s) | Gene tagging |
Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 34537287 |
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 | Not applicable |
Other information parent line | |
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The mutant parasite was generated by | |
Name PI/Researcher | Deligianni E, Siden Kiamos I |
Name Group/Department | Institute of Molecular Biology and Biotechnology |
Name Institute | Foundation for Research and Technology - Hellas |
City | Heraklion |
Country | Greece |
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Name of the mutant parasite | |
RMgm number | RMgm-5108 |
Principal name | Pbflag:act II |
Alternative name | |
Standardized name | |
Is the mutant parasite cloned after genetic modification | Yes |
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Phenotype | |
Asexual blood stage | Not different from wild type |
Gametocyte/Gamete | Not different from wild type |
Fertilization and ookinete | Not different from wild type |
Oocyst | Not different from wild type |
Sporozoite | Not different from wild type |
Liver stage | Not different from wild type |
Additional remarks phenotype | Mutant/mutation Other mutants |
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Details of the target gene | |||||||||||||||||||||||||||
Gene Model of Rodent Parasite | PBANKA_1030100 | ||||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_1412500 | ||||||||||||||||||||||||||
Gene product | actin II | ||||||||||||||||||||||||||
Gene product: Alternative name | actin2 | ||||||||||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||||||||||
Name of the tag | FLAG | ||||||||||||||||||||||||||
Details of tagging | C-terminal | ||||||||||||||||||||||||||
Additional remarks: tagging | |||||||||||||||||||||||||||
Commercial source of tag-antibodies | |||||||||||||||||||||||||||
Type of plasmid/construct | CRISPR/Cas9 construct: integration through double strand break repair | ||||||||||||||||||||||||||
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 | hdhfr | ||||||||||||||||||||||||||
Promoter of the selectable marker | unknown | ||||||||||||||||||||||||||
Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||||
Selection (negative) procedure | No | ||||||||||||||||||||||||||
Additional remarks genetic modification | In order to introduce the CRISPR/Cas9 gene-editing elements into P. berghei we adapted the system of Ribozyme-Guide-Ribozyme (RGR) that was designed for P. yoelii. The construct was generated by one round of restriction digestion and ligation. To simplify the procedure, we exploited the fact that, in P. berghei, genetic mutations can be achieved using linearized DNA molecules consisting of a resistance gene flanked by two large (250–1000bp) regions homologous to the target locus. Thus, we developed a strategy where the homology directed repair (HDR) template was provided as a linear PCR fragment. In the donor template, a silent mutation was inserted into the protospacer-adjacent motif (PAM), preventing Cas9 from cleaving the edited gene. An existing plasmid from our laboratory, encoding actin II tagged with the FLAG epitope at the amino terminus was used as a template for HDR. A two–step PCR approach was used to generate the Pbflag:act II mutant. In the first reaction (PCR1) the 5’ and 3’ flanking sequences were generated using the primer pairs A2U4F/A2PAMR and A2PAMF /A2yoe1R respectively. The silent mutation that prevents Cas9 cleavage, following repair of the DSB, was introduced using the primers A2PAMR and A2PAMF. These primers were designed with an overlapping region of 27bp thus in an overlapping PCR, the complementary areas annealed resulting to a fragment of 1928bp. The entire fusion product was subsequently successfully amplified with primers A2U5SF and A2yoe2R, yielding a 1245 bp product. The HDR template was sequenced to verify the absence of unintended mutations. The pSL1433 plasmid was obtained from Addgene Plasmid #129523)(see also M.P. Walker, S.E. Lindner Ribozyme-mediated, multiplex CRISPR gene editing and CRISPRi in Plasmodium yoelii J. Biol. Chem., 294 (24) (2019), pp. 9555-9566) while the RGR sequence was synthesized by Genescipt and provided in the pUC57 vector and then inserted into pSL1433 using EcoRI/NheI. The HDR template and the pSL1433 plasmid carrying the RGR sequence were introduced into purified schizonts of P. berghei by standard methods of transfection/electroporation. Three independent and successful experiments were carried out and the successful insertion was confirmed by PCR using the primer pairs FlagF/A2yoe1R. Following transfection and constant pyrimethamine pressure, parasitemia was observed after 7 days, similar to what was observed for P. yoelii. The plasmid persisted after removal of pyrimethamine pressure as confirmed by PCR, with primers pSL1433F/pSL1433R, although it was lost after cloning. The parasites were cloned by serial dilution and one clone was selected for further analysis. While a band for the wild type allele was observed in the uncloned population, the band was absent in the clonal population. | ||||||||||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||||||||||
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