Mutant/mutation
The mutant expresses two zinc-finger nucleases specific for eGFP (ZFNL, ZFNLR) under control of the liver-stage specific promoter lisp2 and expresses GFP under control of the constitutive eef1a promoter.
Both zfn genes were separated by the self-cleaving 2A skip peptide from Thosea asigna virus, which has been shown to lead to efficient self-cleavage resulting in expression of two genes in P. falciparum.
To avoid HR between both zfn genes, the zfnL was codon-modified to zfnLcm to have the lowest possible homology with zfnR. This was accomplished by first codon-optimizing zfnL for P. berghei codon usage and then manually changing all codons still identical to zfnR whenever possible.
A silent point mutation is introduced in egfp (mgfp)
The transgene casette is integrated into a silent locus on chromosome 12 (see RMgm-1023).

Protein (function)
The aim is to introduce double-strand breaks (DSB) through well-timed and tightly regulated expression of zinc-finger nucleases (ZFNs), that in the absence of the NHEJ machinery or a plasmid repair template for HR — cannot repair and thus will result in cell death.
Genetic modification in Plasmodium utilises transfection of plasmid DNA and its exclusive integration via homologous recombination (HR) in blood stage parasites. In contrast, the related apicomplexan parasite Toxoplasma gondii mainly employs non-homologous end joining (NHEJ) as the primary DNA repair pathway. In this species genes involved in the NHEJ pathway, including Ku70/80, were readily identified. A T. gondii parasite line lacking Ku80 can only perform HR, thus allowing efficient targeted gene modification via HR. Genes involved in NHEJ have so far not been identified in any Plasmodium species. Also, recent data suggest that alternative DNA repair can occur in P. falciparum

Phenotype
Several mice infected with Ls2ZFN sporozoites developed a blood stage infection, indicating that expression of the ZFNs in late liver stages did not lead to (complete) cell death. It is proposed that parasites excaped DSB by HR or non-timely ZFN expression. See also RMgm-1358, RMgm-1359 and RMgm-1360 for mutants expressing the ZFNs under control of sporozoite specific promoters resulting in a  strongly reduced infectivity of sporozoites.
In the paper the genotype of parasites of 'breakthrough blood-infections' is analysed and evidence is provided for DNA repair by microhomology-mediated end joining with as little as four base pairs, resulting in surviving parasites.

Additional information

Other mutants
See RMgm-1357, RMgm-1358, RMgm-1359, RMgm-1360 for GFP-expressing mutants that express the two ZFNs under control of different promoters