Site-specific genome editing in Plasmodium falciparum using engineered zinc-finger nucleases

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Abstract

Malaria afflicts over 200 million people worldwide, and its most lethal etiologic agent, Plasmodium falciparum, is evolving to resist even the latest-generation therapeutics. Efficient tools for genome-directed investigations of P. falciparum-induced pathogenesis, including drug-resistance mechanisms, are clearly required. Here we report rapid and targeted genetic engineering of this parasite using zinc-finger nucleases (ZFNs) that produce a double-strand break in a user-defined locus and trigger homology-directed repair. Targeting an integrated egfp locus, we obtained gene-deletion parasites with unprecedented speed (2 weeks), both with and without direct selection. ZFNs engineered against the parasite gene pfcrt, responsible for escape under chloroquine treatment, rapidly produced parasites that carried either an allelic replacement or a panel of specified point mutations. This method will enable a diverse array of genome-editing approaches to interrogate this human pathogen.

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Figure 1: 2A-linked ZFNs drive efficient disruption of egfp in P. falciparum.
Figure 2: ZFNs mediate efficient gene replacement of egfp.
Figure 3: ZFN-driven allelic replacement of pfcrt.
Figure 4: ZFN editing of pfcrt with and without CQ selection.

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Acknowledgements

We thank L. Symington (Columbia University) for helpful discussions, and the sequencing core facility and staff in the Lewis-Sigler Institute for Integrative Genomics at Princeton University. M.L. is funded by a US National Institutes of Health Director's New Innovators Award (1DP2OD001315) and receives support from the Center for Quantitative Biology (P50 GM071508). D.A.F. gratefully acknowledges support from the US National Institutes of Health (R01 AI50234 and AI079709).

Author information

J.S., M.C.S.L., B.Z., A.H.L., M.L., F.D.U. and D.A.F. designed the experiments, which were performed by J.S., M.C.S.L., A.H.L. and A.E.W. B.Z., J.R.P., L.Z., E.J.R., P.D.G. and F.D.U. designed and provided the zinc-finger nucleases. J.S., M.C.S.L., A.H.L., F.D.U. and D.A.F. wrote the manuscript, with input from all authors.

Correspondence to David A Fidock.

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Competing interests

B.Z., J.R.P., L.Z., E.J.R., P.D.G., and F.D.U. are employees of Sangamo BioSciences, which designed and provided the ZFNs used in this study.

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Supplementary Figures 1 and 2 and Supplementary Tables 1–3 (PDF 2386 kb)

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Straimer, J., Lee, M., Lee, A. et al. Site-specific genome editing in Plasmodium falciparum using engineered zinc-finger nucleases. Nat Methods 9, 993–998 (2012) doi:10.1038/nmeth.2143

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