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Fig. 1 | Genome Biology

Fig. 1

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

Fig. 1

Zinc-finger nuclease (ZFN)-mediated double-strand breaks (DSBs) and potential repair in the context of the Plasmodium life cycle. a Parts of the life cycle relevant for this study with genome copy number of various stages indicated. Gene names of the used promoters are depicted in red at the point of their respective activation. b Design of SpZFN and LsZFN transgenic parasite lines. Chr12a and Chr12b are the sequences used for homologous integration into the genome. The selection marker hDHFR (human dihydrofolate reductase; yellow) is expressed as a fusion protein with eGFP (enhanced green fluorescent protein; green) under the constitutively active ef1α promoter. The target sequence of the ZFNs is present within the egfp gene. ZFNL and ZFNR are expressed under the control of the two promoters of csp and trap in SpZFN and the promoter of lisp2 in LsZFN. Both zfn genes are fused with a 2A skip peptide (red) in LsZFN. c ZFN-induced DSB and possible post-DSB outcomes are depicted. Homologous recombination (HR) can restore the original locus only if the genome copy number is >1 and restores ZFN binding sites, since no sufficient homology regions are flanking the break site. Potential microhomology-mediated end joining (MMEJ) can repair the DSB, resulting in small gene deletions that can be detected by loss of fluorescence. Parasites that fail to repair the DSB are expected to die

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