Summary

RMgm-1097
Malaria parasiteP. yoelii
Genotype
MutatedGene model (rodent): PY17X_0712100; Gene model (P.falciparum): Not available; Gene product: heat shock protein, putative (HSP70)
Details mutation: Silent mutations introduced into the hsp70 gene using CRISPR/Cas9 technology
PhenotypeNo phenotype has been described
Last modified: 27 July 2014, 13:50
  *RMgm-1097
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene mutation
Reference (PubMed-PMID number) Reference 1 (PMID number) : 24987097
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 Biology, School of Life
Name InstituteXiamen University
CityXiamen, Fujian
CountryChina
Name of the mutant parasite
RMgm numberRMgm-1097
Principal namen.a.
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant contains a mutated hsp70 locus.
This locus has been mutated by  the CRISPR/Cas9 genome editing system through  introduction of a double strand break (by Cas9 and a targeting single guide RNA; sgRNA), followed by repair through homologous recombination (see 'Additional information')

Protein (function)
Rodent parasites have several (three, syntenically conserved) hsp70 (related) genes. The orthology of these genes with the (four) hsp70 genes of P. falciparum is not clear

Phenotype
The phenotype has not been analysed in detail. This mutant has been generated to demonstrate gene mutation (nucleotide substitution) by the CRISPR/Cas9 genome editing system (see 'Additional information')

Additional information

The CRISPR/Cas9 genome editing system has been used to delete sera1.

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).
Target-specific DSBs introduced by the CRISPR/Cas9 system can be repaired by homologous recombination if a donor template is provided. The CRISPR/Cas9 system has been shown to be highly efficient in other organisms for generating gene knock-in (KI), KO, or allelic replacements.

CRISPR/Cas9-mediated genome editing requires expression of two components: Cas9 nuclease and a targeting single guide RNA (sgRNA), which form a complex to induce a double-strand break (DSB) at the targeted site.

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 was generated to introduce silent mutation of nucleotides into the coding region of the Pyhsp70 gene. First a donor template was generated containing three silent nucleotide substitutions, creating an AatII site into the Pyhsp70 coding region for easy restriction fragment length polymorphism (RFLP) analysis (see Fig. A). One sgRNA was designed to target the site 100 bp upstream of the AatII site (see Fig. 4). To avoid donor DNA digestion by Cas9/sgRNA effector, three nucleotide substitutions were introduced at the binding site of sgRNA in the donor template
(see Fig. B). See Figure below



FIG.: Targeted nucleotide replacement in P. yoelii heat shock protein 70 gene (Pyhsp70). (A) Schematic construct for Pyhsp70 nucleotide replacement. The homologous donor template comprises a fragment of Pyhsp70 spanning 340 bp upstream and 620 bp downstream of the Cas9 target site (red thunderbolt). (B) The donor template sequence is identical to the genomic sequence but contains three nucleotide substitutions (green lowercase letters) that create an AatII restriction site for detecting modification by restriction enzyme digestion. In addition, three nucleotides at the sgRNA-binding site in the donor sequence are mutated (blue lowercase letters). The sequence of the protospacer adjacent motif (PAM) is shown in bold type.s.

Considering potential variation in target site accessibility by the Cas9/sgRNA complex,  two sgRNAs were designed to target the 3'end of the Pysera1 exon 2, generating plasmids pYC-sera1-sgRNA1 and pYC-sera1-sgRNA2 (see below).

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 were observed microscopically 5 to 7 days after electroporation.

Other mutants

See RMgm-1096 for gene-tagging mutants that have been generated by this CRISPR/Cas9 system
See RMgm-1095 for gene-deletion (knock-out) mutants that have been generated by this CRISPR/Cas9 system


  Mutated: Mutant parasite with a mutated gene
Details of the target gene
Gene Model of Rodent Parasite PY17X_0712100
Gene Model P. falciparum ortholog Not available
Gene productheat shock protein, putative
Gene product: Alternative nameHSP70
Details of the genetic modification
Short description of the mutationSilent mutations introduced into the hsp70 gene using CRISPR/Cas9 technology
Inducable system usedNo
Short description of the conditional mutagenesisNot available
Additional remarks inducable system
Type of plasmid/constructCRISPR/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
Selectable marker used to select the mutant parasitehdhfr
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationTo generate the Cas9/sgRNA vector for targeted nucleotide replacement in the Pyhsp70 (PY17X_0712100), first approximately 1 kb of the coding region (with the Cas9/sgRNA cleavage site in the center of the region) was PCR-amplified as the donor template. Two or three nucleotide substitutions to create restriction sites without change of amino acids were introduced using synthetic oligonucleotides and PCR amplification. For each gene, one sgRNA was designed to target the site close to the restriction enzyme cutting site. To prevent binding of sgRNA to the target site in the donor template, 3 nucleotide mutations were also introduced into the sgRNA-binding site in the template.
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