SummaryRMgm-1096
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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) : 24987097 |
MR4 number | |
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Parent parasite used to introduce the genetic modification | |
Rodent Malaria Parasite | P. yoelii |
Parent strain/line | P. y. yoelii 17XNL |
Name parent line/clone | Not applicable |
Other information parent line | |
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The mutant parasite was generated by | |
Name PI/Researcher | Zhang, C; Yuan, J |
Name Group/Department | State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Biology, School of Life |
Name Institute | Xiamen University |
City | Xiamen, Fujian |
Country | China |
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Name of the mutant parasite | |
RMgm number | RMgm-1096 |
Principal name | pYC-Py03652-gfp |
Alternative name | |
Standardized name | |
Is the mutant parasite cloned after genetic modification | Yes |
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Phenotype | |
Asexual blood stage | PY03652-GFP expression in blood stages (localized to the parasite periphery as punctate dots). |
Gametocyte/Gamete | Not tested |
Fertilization and ookinete | Not tested |
Oocyst | Not tested |
Sporozoite | Not tested |
Liver stage | Not tested |
Additional remarks phenotype | Mutant/mutation The CRISPR/Cas9 genome editing system has been used to tag PY17X_0946500 with GFP. The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats and Cas9 endonuclease-mediated genome editing) system The CRISPR/Cas9 system was originated from a prokaryotic RNA programmable nuclease that can introduce a double-strand break (DSB) at a specific site on a chromosome through heterologous expression of two components: Cas9 nuclease and a targeting single guide RNA (sgRNA).
One day after electroporation of the plasmids into the P. yoelii 17XNL strain, parasites were selected with pyrimethamine (Pyr) supplied in drinking water. Pyr-resistant parasites with integration of donor template into the 3' end of PY17X_0946500 were obtained 5 days after electroporation. sgRNA's : |
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Details of the target gene | |||||||||||||||||||||||||||
Gene Model of Rodent Parasite | PY17X_0946500 | ||||||||||||||||||||||||||
Gene Model P. falciparum ortholog | Not available | ||||||||||||||||||||||||||
Gene product | early transcribed membrane protein | ||||||||||||||||||||||||||
Gene product: Alternative name | ETRAMP | ||||||||||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||||||||||
Name of the tag | GFP | ||||||||||||||||||||||||||
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 | eef1a | ||||||||||||||||||||||||||
Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||||
Selection (negative) procedure | No | ||||||||||||||||||||||||||
Additional remarks genetic modification | To reduce the size of the plasmid construct and to overcome the problem of limited selectable markers available for P. yoelii, an expression plasmid was constructed that contains the human dihydrofolate reductase (hdhfr)-2A peptide-gfp genes under the P. berghei eef1a (Pbeef1a) promoter and showed bicistronic expression of both genes after introduction into the P. yoelii 17XNL strain. The viral “ribosome skip” 2A peptide has been shown to coordinate coexpression of two individual genes under a single promoter in P. falciparum Because Cas9 is a nuclease functioning within the nucleus, two nuclear localization signals (NLSs) were attached to the 5' and 3' of the Cas9 gene to direct the protein to the nucleus. In mammalian systems, sgRNA is synthesized by RNA polymerase III, and transcription is driven by a U6 small nuclear RNA (snRNA) promoter. By searching the P. yoelii genome database, a U6 snRNA homolog was identified and a 350-base-pair (bp) segment upstream of the transcriptional start site of U6 snRNA was cloned to function as a promoter. A Cas9-sgRNA plasmid was constructed containing both the hdhfr-2A-SpCas9 and PyU6-sgRNA cassettes with cloning sites for the insertion of donor template sequences (for homologous recombination at target sequences in the genome). Next a construct (pYC-Py03652-gfp) was generated containing a 710-bp C-terminal region of the Py03652 gene (PY17X_0946500) followed by the gfp gene and a 779-bp 3' untranslated region (3' UTR) of the Py03652 gene. To prevent binding and cleavage of the integrated DNA by the Cas9/sgRNA complex, we introduced five silent nucleotide substitutions at the sgRNA target site in the donor template. See Figure below. One sgRNA was designed to target the site close to the C-terminal part of the coding region. sgRNA's : GTCTTTATGTGGGTAACTTAAGG (genomic target sequence); GTCTTTATGTGGGTAACTTA (sgRNA target sequence) See below for primer sequences used to amplify the targeting regions of PY17X_0946500 | ||||||||||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||||||||||
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