Summary

RMgm-4388
Malaria parasiteP. yoelii
Genotype
DisruptedGene model (rodent): PY17X_0907300; Gene model (P.falciparum): PF3D7_1143100; Gene product: AP2 domain transcription factor AP2-O, putative (ApiAP2; AP2-O)
Phenotype Fertilization and ookinete;
Last modified: 16 December 2017, 21:14
  *RMgm-4388
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption
Reference (PubMed-PMID number) Reference 1 (PMID number) : 29233900
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. yoelii
Parent strain/lineP. y. yoelii 17XNL
Name parent line/clone Not applicable
Other information parent line
The mutant parasite was generated by
Name PI/ResearcherZhang C; Yuan J
Name Group/DepartmentState Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, Schoo
Name InstituteXiamen University
CityXiamen, Fujian
CountryChina
Name of the mutant parasite
RMgm numberRMgm-4388
Principal nameΔPyap2-o
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 ookineteNormal numbers of gametocytes are produced. Only ookinetes with aberrant morphology are produced.
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of AP2-O
The gene has been disrupted using CRISPR/cas9 genome editing (using constructs as described for mutant RMgm-1095).

Protein (function)
Most sequence-specific transcription factor families found in other eukaryotes seem to be absent from Plasmodium. Instead, an expansion of a protein family containing one or more apetala2 (AP2) DNA-binding domains was observed across the phylum apicomplexa. In total, 27 members of this family have been found in the human malaria parasite Plasmodium falciparum (although a possible 28th member of the family may be present. In total, 26 of these have syntenic orthologs in rodent malaria species, each with its unique stage-specific expression profile.
The paper presents a systematic knockout (KO) screen targeting the ApiAP2 family in the rodent malaria parasite P.yoelii

Phenotype
Normal numbers of gametocytes are produced. Only ookinetes with aberrant morphology are produced.

Additional information
The gene has been disrupted using CRISPR/cas9 genome editing (using constructs as described for mutant RMgm-1095).

From the paper: 'To investigate the functions of the PyApiAP2 gene family in parasite development, we attempted to disrupt 24 of the 26 PyApiAP2 genes in the parasite genome, excluding the orthologs of Pbap2-sp and Pbap2-l, whose functions were described when the project was initiated. We were able to knock out 12 of the 24 genes, including three PyApiAP2 genes (PY17X_1317000, PY17X_1417400, and PY17X_0523100) whose orthologs in P. berghei were either resistant to disruption or not attempted.
We carefully evaluated the morphologies of asexual/sexual stages in ICR mice and sexual stages in A. stephensi mosquitoes for the 12 gene KO mutants. We also successfully tagged six PyApiAP2 proteins (PyAP2-G, PyAP2-G3, PyAP2-O2, PyAP2-O3, PyAP2-O4, and PyAP2-O5) with 6XHA tags and PyAP2-O with mCherry to investigate protein expression and localization in the parasites. There were 12 PyApiAP2 genes (PY17X_0104500, PY17X_1231600, PY17X_1209100, PY17X_0941600, PY17X_0911000, PY17X_1361700, PY17X_1456200, PY17X_0111100, PY17X_0113700, PY17X_0838600, PY17X_0934300, and PY17X_1405400) that could not be disrupted even after 4 to 12 independent transfections and selections. The orthologs of these 12 genes in P. berghei also resisted disruption attempts and are likely to be essential for parasite viability or affect the growth of these rodent parasites in the mouse'.

Other mutants
see ApiAP2


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PY17X_0907300
Gene Model P. falciparum ortholog PF3D7_1143100
Gene productAP2 domain transcription factor AP2-O, putative
Gene product: Alternative nameApiAP2; AP2-O
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct usedCRISPR/Cas9 construct: integration through double strand break repair
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid
Partial or complete disruption of the genePartial
Additional remarks partial/complete disruption
Selectable marker used to select the mutant parasitehdhfr
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe gene has been disrupted using CRISPR/cas9 genome editing (using constructs as described for mutant RMgm-1095).
To construct the vectors to disrupt the PyApiAP2 genes, we first amplified the 5'- and 3'-flanking genomic regions (400 to 700 bp) as left and right homologous arms. The left and right arms were inserted into the restriction sites (HindIII/KpnI and NcoI for the left arm and XhoI and AflII/EcoRI for the right arm) in the pYC plasmid (RMgm-1095). Sequences for single guide RNAs (sgRNAs) were similarly annealed and ligated into the pYC plasmid.
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