Malaria is an infectious cause of immense human-morbidity and mortality world-over . About 250 million cases of malaria are reported annually. Despite presence of effective Artemisinin-based combination chemotherapy for treating clinical malaria, the disease still claims over 1 million lives annually, most-children under the ages of 5 years [2, 3]. Attempts to eradicate malaria through controlling the binomics of its vector-the female anopheles mosquito through use of insecticides are contravened by fears of toxicity and potential risk of evolution of resistance to DDT, the would be ideal agent . As a result, malaria continues to cause not just individual morbidity and mortality, but significant economic losses. Up-to 1.3 % decline in gross domestic product (GDP) is experienced within countries with high levels of transmission. Overall, within malaria endemic regions of the tropics and sub-tropics, clinical malaria is responsible for up to: 40 % of public health expenditures, 30 % to 50 % of inpatient hospital admissions, and 60 % of outpatient health clinic visits [1–4].
Malaria is caused by species of protozoa belonging to the genus Plasmodium[1, 5]. There are well over 100 different species of plasmodia and the parasite is capable of infecting many animal species such as reptiles, birds, and various mammals. Nonetheless, only five Plasmodium species: P. falciparum, P. vivax, P. malariae, P. ovale, and P. knowlesi have been recognized to infect and cause clinical-malaria in humans . Plasmodium knowlesi, a species that was previously only known to naturally infect macaques, has in recent years been recognized to cause zoonotic malaria in humans [5, 7]. The Plasmodia species falciparum and vivax are the most common, causing over 70 % of all cases of clinical malaria globally .
Considering that falciparum is the most deadly plasmodia species, majority efforts to devise a malaria preventive vaccine have focused on it . A safe and effective P.falciparum targeting subunit malaria vaccine however remains to be demonstrated [8, 9]. Indeed, some have argued that the complexity of the malaria parasite precludes the successful development of a sub-unit vaccine, thereby resorting to use of whole-live-attenuated P. falciparum as vaccine-candidates [10, 11].
The life-cycle of plasmodia has both a mosquito- and host-based sub-division. The hallmark of clinical malaria in man is, however, defined by invasion of red blood cells (RBCs or erythrocytes) by the parasites [12, 13]. P. vivax and -falciparum, each utilize unique receptors present on the surface membrane of erythrocytes for their invasion. On one hand, the duffy antigen/receptor for chemokine (DARC) is the receptor for merozoites of Plasmodium vivax and Plasmodium knowlesi; and for chemokines[14–17]. A single T to C substitution at nucleotide −46 in the exon of the DARC gene (darc) is common among Duffy-negative blacks with a silent FY*B allele. The same leads to impairment of the promoter activity in erythroid cells by disrupting a binding site for the GATA1 erythroid transcription factor , thereby resulting into RBC-resistance to invasion by P.vivax merozoites. On the other hand, sialic acid (SLC4A1) residues of the O-linked glycans of the major intrinsic membrane protein of erythrocytes, Glycophorin A, are the major receptors for P.falciparum invasion of RBCs[19, 20].
Given the prevailing challenges to the development of an effective malaria vaccine, we hypothesized that target mutagenesis of the well characterized host RBC-receptors for P. falciparum and P. vivax, may reduce global incidence of malaria. Mercereau-Puijalon & Ménard  have recently reported work to suggest that absolute dependence on the presence of Duffy on the red cell for P. vivax infection and development into the red cell is not true, since in some parts of the world, P. vivax infects and causes disease in Duffy-negative people. Elsewhere, targeted gene disruption studies of PfRh-1 and −2 genes of P. falciparum ligands for SLC4A1-residues by Triglia T et al., and Sahar T et al., have previously yielded mutants incapable of sialic acid-dependent invasion of human erythrocytes. As is the case for evidence to challenge DARC as the only erythrocyte-receptor for P.vivax merozoites, therefore, those P.falciparum parasites that are mutated in PfRH- 1 and 2 proteins are known to invade Sialic acids defective-RBCs normally, by using ligand-receptor interactions pathways that are independent of SLC4A1-residues, and are neuraminidase-resistant [22, 23]. Arguably, such plasmodia mutants capable of using alternate receptors to invade RBCs are likely to be still rare, and their selective adaptability poor.
Zinc finger nucleases - ZFNs - which are artificial, hybrid restriction enzymes [reviewed in ref. 24, 25], have recently become a powerful tool for primary edition of host genomes as a strategy to halt pathogen infectivity [24, 25]. Perez E et al., Holt N et al. , and Wilen CB et al.  have previously demonstrated the establishment of HIV-1 resistance in CD4+ T cells through generation of a double-strand break (DSB) at predetermined sites in the CCR5 coding region upstream of the natural CCR5D32 mutation using engineered ZFNs targeting human CCR5. As an initial step towards the experimentation of a similar approach against malaria, we aimed to identify zinc finger arrays (ZFAs) that are precursors of zinc finger nucleases (ZFNs) to be used for mutating wild-type host-RBC- receptors for merozoites of the most prevalent malaria parasites: P.-vivax and - falciparum.