Genome editing in the human malaria parasite Plasmodium falciparum using the CRISPR-Cas9 system

Authors:
Mehdi Ghorbal, Molly Gorman, Cameron Ross Macpherson, Rafael Miyazawa Martins, Artur Scherf, Jose-Juan Lopez-Rubio
In:
Source: Nat Biotechnol
Publication Date: (2014)
Issue: 32(8): 819-21
Research Area:
Parasitology
Basic Research
Cells used in publication:
Plasmodium falciparum
Species: unicellular
Tissue Origin:
Platform:
4D-Nucleofector™ X-Unit
Experiment
Parasites were transfected as previously described either by electroporating ring-stage parasites18 or nucleofection of schizont stages11,19. For transfections with circular DNA, 50 µg of each plasmid were used. For transfections with linear DNA, pL7-egfp or pL7-kahrp plasmid was linearized with double-cutting restriction enzyme HincII, extracted by phenol-chloroform, verified for linearization in agarose gel and 10 µg were subsequently used for transfection. Prior to transfection, the appropriate DNA was ethanol-precipitated and resuspended in 10 µl and 30 µl of Tris-EDTA buffer for schizont nucleofection and ring-electroporation, respectively. Lonza Summary: The authors adapted CRISPR-Cas9 expression system to Plasmodium falciparum and transfected it into schizont stages using the Nucleofector 4D technology. This was the first demonstration of CRISPR-Cas9 genome editing in a eukaryotic pathogen. The results show specific gene knockouts and single-nucleotide substitutions achieved in a significantly shorter time than formerly done. The success of this marker-free approach is crucial for consecutive genome manipulations for malaria research and likely for other relevant pathogens.
Abstract
Genome manipulation in the malaria parasite Plasmodium falciparum remains largely intractable and improved genomic tools are needed to further understand pathogenesis and drug resistance. We demonstrated the CRISPR-Cas9 system for use in P. falciparum by disrupting chromosomal loci and generating marker-free, single-nucleotide substitutions with high efficiency. Additionally, an artemisinin-resistant strain was generated by introducing a previously implicated polymorphism, thus illustrating the value of efficient genome editing in malaria research