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



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.

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Figure 1: Targeted P. falciparum genome editing using sgRNA:Cas9.
Figure 2: Marker-free nucleotide editing using sgRNA:Cas9 in P. falciparum.

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We thank L. Mancio-Silva (Instituto de Medicina Molecular, Lisboa) and N. Siegel (Institute for Molecular Infection Biology, Wuerzburg) for critically reading the manuscript and PlasmoDB for the invaluable malaria-database support. F. Zhang laboratory (MIT, Boston) for deposition of pX330 in Addgene. A.B. Vaidya (Drexel University, Philadelphia) for pUF1 plasmid. D. Fidock laboratory (Columbia University, New York) for NF54EGFP strain. C. Buchrieser (Institut Pasteur, Paris) for lending Amaxa 4D electroporator (Lonza) and P. Escoll Guerrero (Institut Pasteur, Paris) for assistance. A. Nacer (Institut Pasteur, Paris) for help with immunofluorescence experiments. GenoScreen team, especially H. Blanquart and A. Gourbeyre, for sequencing. This work was supported by the Agence Nationale de la Recherche (ANR 11 JSV3 004 01 PlasmoPiggyBac), ERC Advanced Grant (PlasmoEscape 250320) and the French Parasitology consortium ParaFrap (ANR-11-LABX0024). Me.G. and Mo.G. were funded by ANR 11 JSV3 004 01 and J.-J.L.-R. by the Institut National de la Santé et de la Recherche Médicale (INSERM).

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J.-J.L.-R. devised the research experimental design with contributions from Me.G. Mo.G., Me.G. and J.-J.L.-R. performed the experiments. R.M.M. prepared the libraries for next-generation sequencing. C.R.M. conducted the bioinformatic analysis. A.S. provided funding. J.-J.L.-R. and Me.G. wrote the manuscript with contributions from all the authors.

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Correspondence to Jose-Juan Lopez-Rubio.

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The authors declare no competing financial interests.

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Supplementary Figures 1–4, Supplementary Tables 1 and 2, and Supplementary Results (PDF 1964 kb)

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Ghorbal, M., Gorman, M., Macpherson, C. et al. Genome editing in the human malaria parasite Plasmodium falciparum using the CRISPR-Cas9 system. Nat Biotechnol 32, 819–821 (2014).

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