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Zinc Finger Nucleases are designed proteins that fuse a zinc finger DNA-binding domain to the non-specific nuclease domain of the FokI restriction endonuclease that cuts DNA in predetermined sites (Kim et al, 1996). Zinc finger proteins are a type of DNA-binding proteins that are able to target specific nucleotide sequence, bind it and are important in expression of the genome (Brown, 2006). It is estimated that 2% of all mammalian genome code for Zinc Finger Proteins. They are common in eukaryotes with different versions; Cys2His2 fingers being the most widely used and the first to be studied amongst the Zinc Finger proteins. The Cys2His2 fingers comprises of about 12 or more amino acid residues which forms a β sheet followed by an α-helix, including 2 cysteine and 2 histidine residues. The α-helix and the β sheet form the fingers, holding a Zinc atom, coordinated by the 2 cysteine and 2 histidine residues. Each finger recognises 3 base pairs of DNA via a single α-helix (Pavletich and Pabo, 1991) (Figure 1b). Other types of Zinc finger proteins differ in structure of the fingers. Some lack the β sheet component and consist of one or more α-helix. Examples of other Zinc Finger proteins are CCHC-type, Zn2/Cys6 etc. When binding with DNA, the α-helix makes contact with the major groove while the β sheet interacts with the sugar-phosphate backbone with the Zinc atom holding both fingers in appropriate positions relative to one another (Brown, 2006). Over the last decade, Zinc finger proteins have been fused to restriction endonucleases to form an important biological tool in gene targeting, called Zinc Finger Nucleases (ZFN), which precisely targets genomic edits resulting in targeted gene deletions (knock-outs), integrations, or modifications (Kim et al, 1996).
FokI is an enzyme of the bacterial type IIS restriction endonuclease, isolated from Flavobacterium okeanokoites, consisting of a DNA binding domain at the N-terminus and a non-specific DNA cleavage domain at the C-terminus (Hiroyuki and Susumu, 1981). It binds to duplex DNA at the 5'-GGATG-3':3'-CATCC-5' recognition site with its DNA binding domain and cleaves ~1-20 nucleotides away from its recognition site. FokI exists as a monomer in solution and needs to dimerize in order to cause a double stranded break in DNA (Bitinaite et al, 1998). This also goes to suggest that 2 Zinc Finger Nucleases need dimerization of the nuclease domain in order to produce a Double stranded break [Figure 1a]. Standard ZFNs fuse the cleavage domain to the C-terminus of each zinc finger domain. Zinc finger domains can be engineered to target specific DNA sequences and this enables zinc-finger nucleases to target unique sequences within a genome (Bibikova et al, 2002). For effective cleavage at the site, both inverted copies of ZFN must be in close proximity or not more than 4-6 base pairs apart to cause a DSB (Mani et al, 2005) [Figure 1a].
Figure 1. Diagram of a zinc finger nuclease (ZFN) dimer bound to its target. Adapted from Urnov et al. (2010)
Double stranded breaks occur naturally in cells and can cause chromosomal rearrangement or cell apoptosis. When they occur, they arouse the cell's natural DNA-repair mechanism, either by Homologous Recombination (HR) or Non-Homologous End Joining (NHEJ) (Sonoda et al, 2006). Repair of double stranded breaks is crucial for maintaining genetic integrity and according to, Heidenreich et al. (2003), NHEJ is an error-prone repair mechanism which induces site-directed mutations (i.e. deletions and insertions) at the site of the Double Stranded Break in vertebrate cells. In NHEJ, the ends of the break are simply ligated together by DNA ligase IV (Morton et al, 2006). If the site of the break is clean, both stands are re-joined without any mutation, however if the break causes overhangs, bases maybe added before re-joining of the DNA strands which causes a mutation not found in the original section of the DNA (Weterings and van Gent, 2004) [Figure 2a]. In Homologous Recombination however, sister-chromatids are usually used as a template to repair damaged DNA. It is often called the 'copy and paste' mechanism because the sister chromatid has the same sequence as the damaged DNA and when used as a template, the repair will be as identical as the original DNA (without mutations). On rare occasions, homologous chromosomes or introduced DNA segments maybe used as the repair template, in which case will introduce new sequences (insertions and/or deletions) different from the original DNA (Durai et al, 2005) [Figure 2b]. Since Zinc Finger Nucleases prompt double stranded breaks at user-specific site and in turn, stimulate the cell's repair mechanism by HR or NHEJ, molecular biologists have exploited the use of the ZFN to induce site-directed mutagenesis in insects, plants and animals.
Figure 2. Schematic diagram of Homologous Recombination (HR) and Non-Homologous End Joining (NHEJ). A) Figure of NHEJ adapted from Morton et al. (2006), B) Figure of HR adapted from Weterings and van Gent DC (2004).
ZFN have been used to confer resistance to HIV-1 infection in CD4+ T-cells by genome editing (Perez et al, 2008). The seven transmembrane chemokine receptor, CCR5, is a major co-receptor found on the surface of white blood cells and used as an entry point for HIV-1 virus to infect host cells (Deng et al, 1996). In some human populations, a mutated version of the gene, CCR5-Δ32, results in deletions of some segments of the coding region of the CCR5 gene which has been found to confer resistance to HIV-1 infection in homozygous carriers (Huang et al, 1996). These deletions in the coding region of the gene make the receptor non-functional preventing the HIV-1 Virus infection. Blocking the expression of the CCR5 gene in patients has been a target for clinical therapy (Mosier et al, 1999). Perez et al. (2008) used a genetic approach by introducing engineered Zinc Finger Nucleases to primary human CD4+ T-cells to induce a double stranded break in the coding region of the CCR5 gene, causing deletions and insertions at the target site and conferring protection against the HIV-1 infection in a mouse model.
For the experiment, the ZFNs composed of 2 Zinc-finger proteins, each with four Zinc finger motifs, recognising 24 base pairs, fused to the nuclease domain of the FokI restriction endonuclease. The target of the ZFN was the DNA sequence coding the first transmembrane domain of the CCR5 co-receptor, which was believed to be important in the functionality of the receptor. It was predicted that mutations induced by NHEJ repair mechanism at the target site of the DSB upstream of the Δ32 mutation would cause the non-functionality and permanent disruption of the receptor as seen in the naturally occurring CCR5- Δ32 mutant. Sequence alignment revealed ZFN-induced insertions and deletions within the target site of the receptor. This caused the non-expression of the gene and in turn conferred resistance against the HIV-1 infection. These result showed the use of ZFN in genomic editing for the establishment of HIV-1 resistance in CD4+ T-cells.
Zinc Finger Nucleases have also been used by plant biotechnologists to genetically modify genes for the improvement of crop plants. To investigate gene function and modification in plants, site directed mutagenesis has been experimentally used via gene transfer. Osakabe et al. (2010) reported the use of engineered ZFN to inactivate a target gene in Arabidopsis thaliana, a model plant in which its genomic sequence has been elucidated. The report showed that ZFN can successfully cleave and induce heritable mutation at a target site in the ABA-INSENSITIVE (ABI4) gene that encodes the ERF/AP2 transcription factor, which plays an important role in regulating Abscisic acid (ABA), a plant hormone produced in response to environmental stress and seed development.
For the experiment, a combination of 2 Zinc Finger Proteins each with three Zinc Finger motifs, recognising 18 base pairs, fused to the FokI restriction endonuclease were designed to target the ABI4 gene in Arabidopsis. The ZFN expression cassette driven by a heat shock promoter was introduced into the Arabidopsis plant via Agrobacterium mediated transformation. The transgenic plants were treated to heat shock and its genomic DNA screened for mutations at the target site. Sequence alignment of the mutant plant treated with heat pulse revealed deletions and substitutions at the target site of the ZFN-induced break. The report also showed that the mutations induced by the ZFNs can be heritable to other generations. Progeny seeds from the mutant Arabidopsis were collected and scored for the presence of the ZFN-induced mutation within the ABI4 gene. The seeds were propagated with the leaves screened for the mutated ABI4 gene. Sequence analysis showed a single base deletion mutation at the cleavage site of the ZFN-induced mutation which was consistent with that of the parent mutant plant. The mutation was also found in the T3 (3rd generation) seeds of the T2 mutant plant. The experiment was also phenotypically tested. Since the ABI4 gene is important in ABA regulation and sugar signalling (Gazzarrini and McCourt, 2001), ABI4 mutants showed resistance to high concentration of sugar during seed development (Finkelstein and Gibson, 2002). When ZFN-induced mutants were grown in a medium of ABA and high concentration of glucose, they showed the same phenotype as those of the ABI4 mutants which also suggests the success of using ZFNs to induce site directed mutagenesis in the target ABI4 gene of Arabidopsis plant which could be heritable. This method of using ZFN has also been used to engineer herbicide tolerance in Zea mays (Shukla et al, 2009) and other crop plants like rice, wheat and tomato.
In another report by Doyon et al. (2008), they described the disruption of target somatic and germline genes in Zebra fish (Danio rerio) using engineered ZFN. This was done by injecting designed ZFN-encoding mRNA into fertilized egg of Zebra fish inducing a DSB at the target site, introduced distinct mutations in the gene locus and causing a loss-of-function phenotype [Figure 3]. The target genes tested were the golden (gol) and no tail (ntl) gene. For both experiments, four Zinc finger proteins of the Cys2His2 type were fused to the cleavage domain of FokI restriction endonuclease. The designed target for the gol gene locus was exon 4 and 9. The ZFN expression construct was transcribed and the resulting mRNA injected into Zebra fish embryos heterozygous for gol gene which have dark eye pigmentation. The phenotypic result of the clones showed unpigmented cells in the eyes of the ZFN-mRNA injected embryos suggesting mutations in the target locus of the somatic cells induced by DSB-NHEJ repair mechanism and loss-of-function of the gol gene. To verify the phenotypic results, the gol locus of the injected embryo was genotyped and at the ZFN target site, mutations ranging from 7-65 bp deletions and 4-12 bp insertions were found. These showed that injecting ZFN-encoding mRNA into embryos of Zebra fish will induce gene mutation at the target site triggered by ZFN-induced mutagenesis.
The ntl gene was also a target for experimentation in Zebra fish reported by Doyon et al. (2008). The ntl gene codes a transcription factor of which null homozygous mutation of the gene would show a lack of notochord and tail in zebra fish (Schulte-Merker et al, 1994). The designed target was the exon 2 of the ntl locus which would result in a null allele when mutated. ZFN-encoding mRNA was injected into embryos of Zebra fish. The result was consistent with injected embryo expressing phenotype like those of a null ntl mutation with truncated posterior tails. Similar to gol gene mutants, insertions and deletions were found at the precise target site of the ZFN induced by NHEJ repair mechanism. It was also shown however, that mutations induced were heritable to the next generation.
Figure 3. Diagram of Targeted mutagenesis in zebra fish adapted from Woods and Schier, (2008).
The use of ZFN to induce target mutagenesis has also been reported in insects. Bibikova et al. (2002), designed a pair of ZFN (Cys2His2) to recognize and cleave a target site in the yellow (y) gene of Drosophila melanogaster, of which when expressed in developing larvae, leads to mutations in the target site of the y gene. The y gene is required for pigmentation of the cuticle for both larvae and adult which makes visual analysis of the phenotype easily recognizable for a mutated gene. For the experiment, three Zinc finger proteins, recognizing 9 bp, linked to FokI cleavage domain were constructed to target the exon 2 of the y gene on the X chromosome of the insect. The 2 ZFN (yA and yB) were separately introduced into the Drosophila melanogaster genome by P-element-mediated transformation using a heat shock promoter. To express both ZFNs, individuals carrying the nucleases (yA and yB) were crossed with the progeny heat shocked. Yellow mosaics/patches were found on the abdominal cuticle and bristle of the male transformed larvae. Lower frequencies were found in female transformed larvae which suggested a possibility of homologous recombination repair mechanism at the cleavage site. Sequence analysis of the y gene from the heat shocked male larvae revealed small insertion (duplications), deletions and frame shifts at the target site consistent with NHEJ repair mechanism of a DSB.
Species with manipulable genomes are important to help investigate the role of genes in biology and disease. The ability to add or delete gene segments enables a detailed study of gene function. It also enables efficient and precise genetic modification. The ZFP region provides a ZFN with the ability to bind a discrete base sequence (Pavletich and Pabo, 1991). Without knowledge of this sequence, the ZFN cannot be designed effectively as the ZFP recognises specific triplet code within a genome. Zinc fingers can be manipulated to recognise a wide range of sequences. The protein and DNA must interact at the correct positions across the entire recognition site to ensure efficient binding (Urnov et al, 2010). This would permit cleavage by ZFN to be directed to different genomic sequences without the need to alter those sequences in advance. Complete genomic sequence has been determined for a number of experimental organisms, like the ones described here. Knowledge of these sequences does not lead directly to understanding of the underlying gene functions therefore genetic approaches would be greatly facilitated by the ability to direct mutations to chosen genomic targets within a genome (Bibikova et al, 2002). The sequence specificity of the ZFN is only as good as the ZFP used to engineer them. The specificity of DNA cleavage within a genome is conferred by the zinc fingers, each of which interacts with a particular triplet of DNA base pairs (Woods and Schier, 2008). Increasing the number of zinc fingers enhances the target specificity of ZFNs and can reduce off-target cleavage of DNA (Porteus and Baltimore, 2003). To further improve the specificity, a number of different techniques have been employed to increase the affinity and specificity of ZFNs. A variant of FokI restriction endonuclease termed 'Sharkey' was engineered by directed evolution to enhance the cleavage activity of the ZFN (Guo et al, 2010). According to the report, a 3-6 fold improvement was achieved by targeted mutagenesis using ZFN with the Sharkey cleavage domain relative to the wild type. Likewise, by improving the structural design of the ZFN to heterodimerise upon binding DNA, it will in turn increase the efficiency of the cleavage and reduce off-target cleavage by homodimerisation (Miller et al, 2007).
Zinc finger combinations that recognise many different DNA sequences have been identified but range of effective targets may be limiting (Bibikova et al, 2002). The fundament barriers to using Cys2His2 Zinc finger motifs to design DNA binding molecules for targeted gene mutation with specific cleavage have been overcome by engineering ZFN. These ZFNs have been shown to be successful in reverse genetics in many model organisms by carrying out sophisticated gene-function studies (Urnov et al, 2010). Increase in the specificity and affinity of these biological tools has been achieved by directed evolution and architectural design. A novel ZFN has also been engineered using the cleavage domain of the restriction enzyme PuvIII to improve specificity (Schierling et al, 2012). The next step is to extend the power of this technology to any eukaryote and also editing genes in an adult organism.