Zinc finger nuclease-based double-strand breaks attenuate malaria parasites and reveal rare microhomology-mediated end joining

• Corresponding authors: Mirko Singer mirko.singer@med.uni-heidelberg.de - Friedrich Frischknecht freddy.frischknecht@med.uni-heidelberg.de

Author Affiliations

¹ Integrative Parasitology, Center for Infectious Diseases, University of Heidelberg Medical School, Im Neuenheimer Feld 324, Heidelberg, 69120, Germany
² Parasitology, Center for Infectious Diseases, University of Heidelberg Medical School, Im Neuenheimer Feld 324, Heidelberg, 69120, Germany
³ MalVa GmbH, Heidelberg, Germany
⁴ Virology, Center for Infectious Diseases, University of Heidelberg Medical School, Im Neuenheimer Feld 267, Heidelberg, 69120, Germany
⁵ German Center for Infectious Diseases, Im Neuenheimer Feld 324, Heidelberg, 69120, Germany

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Genome Biology 2015, 16:249  doi:10.1186/s13059-015-0811-1
Published: 17 November 2015

 

Abstract

Background

Genome editing of malaria parasites is key to the generation of live attenuated parasites used in experimental vaccination approaches. DNA repair in Plasmodium generally occurs only through homologous recombination. This has been used to generate transgenic parasites that lack one to three genes, leading to developmental arrest in the liver and allowing the host to launch a protective immune response. While effective in principle, this approach is not safe for use in humans as single surviving parasites can still cause disease. Here we use zinc-finger nucleases to generate attenuated parasite lines lacking an entire chromosome arm, by a timed induction of a double-strand break. Rare surviving parasites also allow the investigation of unconventional DNA repair mechanisms in a rodent malaria parasite.

Results

A single, zinc-finger nuclease-induced DNA double-strand break results in the generation of attenuated parasite lines that show varying degrees of developmental arrest, protection efficacy in an immunisation regime and safety, depending on the timing of zinc-finger nuclease expression within the life cycle. We also identify DNA repair by microhomology-mediated end joining with as little as four base pairs, resulting in surviving parasites and thus breakthrough infections.

Conclusions

Malaria parasites can repair DNA double-strand breaks with surprisingly small mini-homology domains located across the break point. Timely expression of zinc-finger nucleases could be used to generate a new generation of attenuated parasite lines lacking hundreds of genes.