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CRISPR/Cas9 Gene Editing to Make Conditional Mutants of Human Malaria Parasite P. falciparum
Published on: September 18, 2018
CRISPR/Cas9 mediated sequential editing of genes critical for ookinete motility in Plasmodium yoelii
Cui Zhang1, Han Gao1, Zhenke Yang1
1State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, School of Life Sciences, Xiamen University, Xiamen, Fujian 361102, China.
Abstract:
CRISPR/Cas9 has been successfully adapted for gene editing in malaria parasites including Plasmodium falciparum and Plasmodium yoelii. However, the reported methods were limited to editing one gene at a time. In practice, it is often desired to modify multiple genetic loci in a parasite genome. Here we describe a CRISPR/Cas9 mediated genome editing method that allows successive modification of more than one gene in the genome of P. yoelii using an improved single-vector system (pYCm) we developed previously. Drug resistant genes encoding human dihydrofolate reductase (hDHFR) and a yeast bifunctional protein (yFCU), with cytosine deaminase (CD) and uridyl phosphoribosyl transferase (UPRT) activities in the plasmid, allowed sequential positive (pyrimethamine, Pyr) and negative (5-fluorocytosine, 5FC) selections and generation of transgenic parasites free of the episomal plasmid after genetic modification. Using this system, we were able to efficiently tag a gene of interest (Pyp28) and subsequently disrupted two genes (Pyctrp and Pycdpk3) that are individually critical for ookinete motility. Disruption of the genes either eliminated (Pyctrp) or greatly reduced (Pycdpk3) ookinete forward motility in matrigel in vitro and completely blocked oocyst development in mosquito midgut. The method will greatly facilitate studies of parasite gene function, development, and disease pathogenesis.
Insights
This study introduces a new CRISPR/Cas9 gene editing method for malaria parasites, enabling multiple gene modifications in Plasmodium yoelii. This advance facilitates research into parasite function and malaria pathogenesis.
Area of Science:
- Parasitology
- Genetics
- Molecular Biology
Background:
- CRISPR/Cas9 gene editing is established in malaria parasites like Plasmodium falciparum and Plasmodium yoelii.
- Existing methods are limited to single-gene modifications.
- Simultaneous modification of multiple genetic loci is frequently desired for functional studies.
Purpose of the Study:
- To develop a CRISPR/Cas9 based genome editing method for sequential modification of multiple genes in P. yoelii.
- To utilize an improved single-vector system (pYCm) for efficient genetic manipulation.
- To enable positive and negative selections for generating genetically modified parasites free of episomal plasmids.
Main Methods:
- Employed a CRISPR/Cas9 system with drug resistance genes (hDHFR, yFCU) for sequential selection.
- Utilized pyrimethamine (Pyr) for positive selection and 5-fluorocytosine (5FC) for negative selection.
- Successfully tagged the Pyp28 gene and disrupted the Pyctrp and Pycdpk3 genes in P. yoelii.
Main Results:
- Demonstrated efficient sequential gene editing in P. yoelii using the pYCm vector system.
- Gene disruption of Pyctrp eliminated, and Pycdpk3 greatly reduced, ookinete motility in vitro.
- Disruption of these genes completely blocked oocyst development in the mosquito midgut.
Conclusions:
- The developed method allows for efficient, successive modification of multiple genes in the P. yoelii genome.
- This technique significantly aids in studying parasite gene function, development, and malaria disease pathogenesis.
- The system facilitates the generation of transgenic parasites without episomal plasmids, simplifying downstream analysis.
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