Schematic of the zinc finger nucleases as suggested by Miller et al. The ringed struture is formed by the conserved amino acid residues which include the Cys and His ligands, the negatively charged As11 and the hydrophobic Tyr6, Phe17 and Leu23. The black circles in this image were proposed to be the most probable DNA binding sites.
Berg (1988) proposed a model for the structure of the nuclease suggesting that it consist of a β-sheet containing two cysteine residues which form a loop and an α-helix containing a loop made up of two histidine residues and that these structural units are held together by the zinc ion. The exact way in which the zinc finger nucleases interacted with DNA remained undefined until it was reported by Pavletich and Pabo (1991) that the α-helix binds to the DNA major grove by hydrogen bond interactions of the amino acid residues in positions 1, 3 and 6 to three bases on the DNA strand. The crystal structures of ZFNs have shown that the binding sites of these nucleases are Zif268 in mice, GLI in humans and TTK in Drosophila (Berg and Schwabe, 1995). It was later revealed that while the first interaction was taking place, the amino acid at position 2 of the helix was also interacting with the other DNA strand (Klug, 2010) although this interaction does not contribute to the ZFN activity on the DNA. The interactions between the DNA phosphate backbone and the first of the two histidine zinc ligands are also essential in fixing the orientation of the ZNFs to the binding site (Berg and Schwabe, 1995).
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Although many classes of biological proteins contain structural zinc ions, the Cys-Cys- His-His zinc finger domains remain the most popular and well known class (Berg and Godwin, 1997). The binding to two Zinc finger nucleases (ZFNs) to the target sequence in the appropriate direction results in dimerization of the nuclease domains and the introduction of a double-stranded break (DSB) (Ochiai et al, 2012). These DSBs can be specialised to be site specific to enable genetic modification (Hauschild-Quintern et al, 2012). Since their discovery, zinc finger nucleases have been used to carry out mutagenesis in various plants and organisms and have a success rate of over 10 percent in target modification (Carroll, 2011). This percentage is even lower in plant models. To induce ZFN mediated genetic engineering, a plasmid or Mrna encoding the specially designed ZFN is introduced into cells or embryos by microinjection of Agrobacterium transfection. The DNA or Mrna is then translated after which, the ZFN bind to the target sequence, resulting in DNA cleavage by FokI dimerization. After this cleavage, double-strand break repair is initiated (Hauschild-Quintern et al, 2012).
Osakabe et al, in 2010 demonstrated site directed mutagenesis in Arabidopsis by targeting an endogenous gene ABA-INSENSITIVE4 which encodes a member of the ERF/AP2 transcription factor family and regulates abscisic acid. They identified four ZFN target sites in the target gene and designed three zinc finger arrays for 5'-GGAGGAGGA-3' and 5'-GTGGCGGCG-3' targeting ABI4 using zinc finger modules for 5'-GNN-3'. They introduced the ZFN expression vector pP1.2gfbPhsZFN_AB14 by Agrobacterium into the Arabidopsis genome and the heat shock promoter gene, HSP18.2 was used to drive the ZFN expression. They then selected for transgenic lines and subjected the plants to heat shock to induce the expression of ZFNs. Surveyor nuclease assay were used to identify mutations in the cloned PCR produces of the gene. In this study, they showed that ZFNs can be used to induce site directed mutagenesis in Arabidopsis.
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Figure 1: A schematic representation of the Arabidopsis ABI4 gene showing the point of the abi4 mutation. The grey bars indicate the target sites for the ZFN monomers and the arrows show the putative cleavage sites.
Zinc finger mediated genetic manipulation has been successfully carried out in plants as well. Petolino et al (2010) transformed tobacco using a target construct consisting of a reporter gene expression cassette flanked by ZFN binding sites and a construct containing a ZFN gene expression cassette. The target construct was pDAS5380 which consist of CCR5 ZFN binding site and GFP coding region and a gene sequence that encodes a ZFN that cleaves the CCR5 gene sequence called pDAS5381. GFP and GUS were used as reporter genes to quantify gene expression. The tobacco leaves were transformed using Agrobacterium containing either pDAS5380 or pDAS5381. T0 plants were self- pollinated and T1 seedlings were screened for reporter gene expression and zygosity. The T1 seedlings were germinated and homozygous plants were selected and cross pollinated. They successfully deleted the transgene flanked by zinc finger nuclease mediated cleavage site from a stable transformed plant by crossing with another plant expressing a corresponding ZFN gene.
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Ochiai et al, in 2012, inserted a GFP reporter cassette into the HpEts1 locus of sea urchins, strain Hemicentrotus pulcherrimus. They achieved this by injecting a pair of ZFNs with a targeting donor construct into the sea urchin embryos and observed them by in vivo quantitative imaging using confocal laser scanning microscopy (CLSM). They selected the HpEtS1L (5'-GGGGTTGACG-3') and HpEts1R (5'-GATGATACT-3') ZFNs which at located upstream of the stop codon of the HpEts1 gene responsible for the differentiation of PMC. In vivo quantification of GFP gene reporter expression was carried out by studying the fluorescence intensity of GFP at a single cell resolution in the embryos which allows real time analysis of endogenous gene expression. They found that there were variations in HpETs1 expression among primary mesenchyme cells suggesting that ZFN mediated transgene insertion can be used to study gene expression levels. They were able to demonstrate that insertion of a transgene into the HpEts1 locus using ZFNs could be achieved in sea orchids.
More recently, Sood et al in February 2013 used CompoZr and CoDA zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) to target nine genes in zebrafish to produce loss of function alleles. In their experiments, they used CompoZr ZFNs designed by Sigma-Aldrich and CoDA ZFNs engineered using the ZiFit software program to produce mutant zebrafish for nine genes. Equal concentrations of the CompoZr and CoDA ZFNs mRNAs were mixed and injected in multiple doses into zebrafish embryos at the 1 cell stage using microinjections. Zebrafish are effective vertebrae models because it is relatively easy to introduce new genomic material into embryos by microinjection. In this research, wild type zebrafish of strains Ekkwill (EK), Tubingen (Tu) and AB were used. To examine the activity of the ZFNs at the target site, eight to ten embryos were collected and DNA was collected from these embryos and used to perform a PCR reaction. They were able to generate multiple mutant zebrafish lines for seven of the genes they investigated.
The ability of ZFNs to activate cellular DNA repair by creating DSB in target genes is what makes them stand out as tools in genome engineering and has made them a centre of focus for scientists since the 1980s. ZFNs in the presence of designed donor DNA can be used to initiate homologous recombination between the locus of interest and the donor DNA (Sollu et al, 2010). Targeted genome cleavage by sequence specific zinc finger nucleases has been successfully used to carry out site directed mutagenesis and reverse genetics in zebrafish, mice, rats, Drosophila melanogaster, Arabidopsis thaliana and induced pluripotent human cells (Urnov et al, 2010). When zinc finger nucleases produce DSBs at a site in the genome, sequences in the DNA change as a result of either homologous recombination (HR) with the donor DNA or by inaccurate nonhomologous end joining (NHEJ) (Bozas et al, 2009). When performing site specific mutagenesis, it is important to suppress NHEJ and this can be achieved by producing single strand breaks rather than double strand breaks in the DNA (Gabsalilow et al, 2013). NHEJ can be avoided by using DNA nicking endonucleases and converting the FokI domain into a nickase by inactivating the catalytic centre of one of the FokI monomer thereby reducing unwanted mutagenesis caused by NHEJ. The efficiency of the homologous recombination that can be achieved by using ZFNs can be increased significantly by a specific DSB in the target gene locus. It has been reported however, that unwanted mutations can occur if the error-prone NHEJ repairs are carried out at the DSB rather than the desired HDR (Gabsalilow et al, 2013). However, DNA nicking prevents this unwanted result and ensures accurate gene correction or insertion.
Selecting for the outcome of the DNA repair induced by ZFNs allows for the production of gene knockouts or transgene insertion (Hauschild-Quintern et al, 2012). In ZFN mediated genetic engineering, the plasmid DNA encoding the specific ZFN is introduced into embryos by microinjection or bacterial transfection. Over the past few decades, the genome of various plants and animals and even the human genome been completely sequenced, providing us with more information about their genetic make-up. The information is particularly important when using zinc finger nucleases because the target sequence needs to be identified before the ZFNs can be designed to cleave it.
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Zinc finger nucleases are important tools in genetic engineering and provide a promising future for treating options for genetic disorders (Alwin et al, 2005). ZFNs provide an alternative to other genetic manipulation methods which rely on random integration of the transgene into the genome of the target organism. The targeted mutagenesis that is possible with ZFNs can be utilised to induce precise genetic changes at specific locations in living cells (Alwin et al, 2005). Manipulating the genotype of organisms allow the study of gene function in the pathology of inherited diseases as well as the production of desired phenotypes. The ability of zinc finger nucleases to be custom made means they can be engineered to cleavage any sequence in the genome of an organism and this can only be achieved when the information on the potential target sequence is known. Zinc finger nucleases have been effectively used to specifically target gene deletion, correction, disruption and chromosomal rearrangement and have become a promising technology is studying gene function, expression and possible gene therapy in genetic diseases. An illustration of this can be seen in the study carried out by Overlack et al (2012) who looked at Human Usher Syndrome (USH). USH causes deaf blindness and is genetically heterogenous with currently no effective treatments. The most aggressive form of the disease is called USH1 which is caused by a loss of expression of USH1 genes. They designed ZFNs for the p.R31X mutation in the Ush1c gene and concluded that cleavage of the sequence resulted in rescue DNA induced gene repair of the disease causing mutation leading to a recovery of protein expression. This provides a potential gateway for the treatment of this disease and other related diseases with similar pathology and shows how the underlying genetic defects can be corrected using zinc finger nucleases.
The complete genomic sequences for a number organisms particularly important in experimental research have been determined and this information has been utilised by genetic, biochemical and biological research to understand gene function and expression (Bibikova et al, 2002). An adequate understanding of the genome sequence is essential in site directed mutagenesis because the whole process involves directing the mutation to chosen genomic target sequences. Sequencing the whole genome and exomes of organisms has replaced positional cloning techniques in identifying disease causing genes although determining which mutations are responsible for the pathological development of these diseases remains a time consuming effort (Sood et al, 2013). It has been reported that artificial zinc finger proteins can be used to inhibit the binding of viral replication proteins to their replication origin in plants and recently, it has been suggest that these nucleases inhibit DNA replication of the human HPV-18 in mammalian cells (Mino et al, 2013). It has also been reported that HIV 1 resistance in CD4+ T cells can be induced by ZFN mediated DBS in human CCR5 coding region (Wayengera, 2011). This suggests a possible future application of ZFNs in therapeutics as an antiviral regimen.
Gene correction using ZFNs can be applied in monogenic disorders such as X-linked severe combined immune deficiency (SCID), haemophilia, sickle-cell anaemia as a possible treatment option (Klug, 2010). These ZFNs can be engineered to place or correct the defective gene and eliminate the disease phenotype. In the case of SCID, the defective gene interleukin-2 receptor common y-chain (IL2Ry) was modified in a study using a point mutation which resulted in a correction of the mutant allele (Hauschild-Quintern et al, 2012). Disruption of the CCR5 gene in hematopoietic stem progenitor cells using ZFNs has also been reported. The ability to manipulate the genome of plants and animals has been a long sought after tool for scientists as it could signify the end of genetic diseases and possibly the most important tool for disease treatment at our disposal.