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CRISPR/Cas9 Gene Editing to Make Conditional Mutants of Human Malaria Parasite P. falciparum
Published on: September 18, 2018
Tagging to endogenous genes of Plasmodium falciparum using CRISPR/Cas9
Dexuan Kuang1, Jichen Qiao2,3, Zhou Li4
1Institute of Medical Biology, Chinese Academy of Medical Science and Peking Union Medical College, Kunming, Yunnan, 650118, China.
Background:
Plasmodium falciparum is the deadliest malaria parasite. Currently, there are seldom commercial antibodies against P. falciparum proteins, which greatly limits the study on Plasmodium. CRISPR/Cas9 is an efficient genome editing method, which has been employed in various organisms. However, the use of this technique in P. falciparum is still limited to gene knockout, site-specific mutation and generation of green fluorescent protein (GFP) reporter line with disruption of inserted sites.
Results:
We have adapted the CRISPR/Cas9 system to add commercial tag sequences to endogenous genes of P. falciparum. To add HA or HA-TY1 tags to ck2β1, ck2α and stk, pL6cs-hDHFR-ck2β1/ck2α/stk was constructed, which contained sequences of tags, specific homologous arms, and sgRNA. The P. falciparum 3D7 strain was subsequently transfected with pUF1-BSD-Cas9 and pL6cs-hDHFR-ck2β1/ck2α/stk plasmids via electroporation. After that, BSD and WR99210 drugs were added to the culture to screen parasites containing both plasmids. Twenty days after electroporation, live parasites appeared and were collected to check the tagging by PCR, DNA sequencing, Western blotting and immuno-fluorescence assays. The results showed that the tags were successfully integrated into the C-terminus of these three proteins.
Conclusions:
We have improved the method to integrate tags to Plasmodium falciparum genes using the CRISPR/Cas9 method, which lays the foundation for further study of Plasmodium falciparum at the molecular level.
Insights
Researchers improved CRISPR/Cas9 gene editing in Plasmodium falciparum, enabling the addition of commercial tags to parasite proteins. This advancement facilitates further molecular studies of the deadliest malaria parasite.
Area of Science:
- Molecular Biology
- Genetics
- Parasitology
Background:
- Plasmodium falciparum is the deadliest malaria parasite, but limited commercial antibodies hinder research.
- Current CRISPR/Cas9 applications in P. falciparum are restricted to gene knockout and reporter lines.
Purpose of the Study:
- To adapt and improve the CRISPR/Cas9 system for tagging endogenous genes in P. falciparum.
- To enable the generation of tagged P. falciparum protein lines for enhanced molecular analysis.
Main Methods:
- Constructed plasmids (pL6cs-hDHFR-ck2β1/ck2α/stk) containing tag sequences and homologous arms.
- Transfected P. falciparum 3D7 strain with Cas9 and tag plasmids via electroporation.
- Selected successfully transfected parasites using drug resistance markers (BSD and WR99210) and confirmed tag integration via PCR, sequencing, Western blotting, and immunofluorescence assays.
Main Results:
- Successfully adapted CRISPR/Cas9 to integrate commercial HA or HA-TY1 tags into the C-terminus of ck2β1, ck2α, and stk genes in P. falciparum.
- Confirmed successful tag integration through multiple validation methods, demonstrating the efficacy of the improved system.
Conclusions:
- Developed an improved CRISPR/Cas9-based method for gene tagging in P. falciparum.
- This technique provides a foundation for advanced molecular-level investigations of P. falciparum proteins and pathways.
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