Rapid Generation of Marker-Free P. falciparum Fluorescent Reporter Lines Using Modified CRISPR/Cas9 Constructs and Selection Protocol
| Autor: | Takashi Imai, Severine Chevalley-Maurel, Jai Ramesar, Hans Kroeze, Chris J. Janse, Shahid M. Khan, Blandine Franke-Fayard, Guido M. de Roo, Catherin Marin Mogollon, Sabrina A.J. Veld, Fiona J. A. van Pul |
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| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Plasmodium Physiology Drug Resistance lcsh:Medicine Genome Green fluorescent protein Genome editing Medicine and Health Sciences CRISPR Clustered Regularly Interspaced Short Palindromic Repeats Malaria Falciparum lcsh:Science Genetics Gene Editing Protozoans Multidisciplinary Malarial Parasites Hematology Body Fluids Blood Anatomy Research Article Transgene 030106 microbiology Plasmodium falciparum Biology DNA construction Research and Analysis Methods Transfection Heterocyclic Compounds 4 or More Rings 03 medical and health sciences Antimalarials parasitic diseases Parasite Groups Parasitic Diseases Humans Molecular Biology Techniques Gene Molecular Biology Selectable marker Cas9 lcsh:R Organisms Biology and Life Sciences Marker Genes Isoquinolines Parasitic Protozoans 030104 developmental biology Mutation Plasmid Construction lcsh:Q Parasitology Genome Protozoan Apicomplexa Selectable Markers Cloning |
| Zdroj: | PLoS ONE PLoS ONE, Vol 11, Iss 12, p e0168362 (2016) |
| ISSN: | 1932-6203 |
| Popis: | The CRISPR/Cas9 system is a powerful genome editing technique employed in a wide variety of organisms including recently the human malaria parasite, P. falciparum. Here we report on further improvements to the CRISPR/Cas9 transfection constructs and selection protocol to more rapidly modify the P. falciparum genome and to introduce transgenes into the parasite genome without the inclusion of drug-selectable marker genes. This method was used to stably integrate the gene encoding GFP into the P. falciparum genome under the control of promoters of three different Plasmodium genes (calmodulin, gapdh and hsp70). These genes were selected as they are highly transcribed in blood stages. We show that the three reporter parasite lines generated in this study (GFP@cam, GFP@gapdh and GFP@hsp70) have in vitro blood stage growth kinetics and drug-sensitivity profiles comparable to the parental P. falciparum (NF54) wild-type line. Both asexual and sexual blood stages of the three reporter lines expressed GFP-fluorescence with GFP@hsp70 having the highest fluorescent intensity in schizont stages as shown by flow cytometry analysis of GFP-fluorescence intensity. The improved CRISPR/Cas9 constructs/protocol will aid in the rapid generation of transgenic and modified P. falciparum parasites, including those expressing different reporters proteins under different (stage specific) promoters. |
| Databáze: | OpenAIRE |
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