Methods to introduce site specific, stable modifications in complex genomes hold great potential, not only for the study of gene function but also for biotechnological and therapeutic applications (Sollu et al., 2010). A promising new approach is based on zinc-finger nucleases (ZFNs), artificially constructed endonucleases that are designed to make a double strand break in a pre-determined genomic target sequence. This can then be followed by the generation of desired modifications during subsequent DNA repair.
ZFNs are engineered to contain a DNA binding domain, composed of zinc finger proteins, and a non-specific endonuclease domain derived from the FokI restriction enzyme (Urnov et al., 2010). The zinc finger protein region provides a ZFN with the ability to bind to a discrete base sequence. Each zinc finger domain consists of Ì´ 30 amino acids which fold into a ββα structure, this is stabilised by chelation of a zinc ion by the conserved Cys2-His2 residues (Durai et al., 2005). Each domain recognises and binds to approximately 3bp of DNA. Binding to longer sequences is achieved by linking several of these zinc fingers in tandem to form zinc finger proteins. As the catalytic FokI domain must dimerise to induce a double strand break (Vanamee et al., 2001), two different ZFN subunits are designed that bind the sequence of interest in the opposite orientation and with the correct spacing. The combined target sequence is sufficient in length to be statistically unique, even in complex genomes (Sollu et al., 2010) (figure 1).
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ZFNs have been proven to work successfully in Arabidopsis thaliana (Zhang et al., 2010), Caenorhabditis elegans, Drosophila melanogaster (Carroll et al., 2008), zebrafish (Doyon et al., 2008), rats (Mashimo et al., 2010) and human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) (Zou et al., 2009).
Figure 1 | Structure of zinc finger nucleases (ZFNs) Each ZFN contains a catalytic FokI domain joined by an amino acid linker to an array of zinc fingers (four are shown), that have been designed to specifically recognise sequences that flank the cleavage site.
Mechanisms of DNA double strand break repair
All eukaryotic cells have effective mechanisms to repair double strand breaks in DNA. The two primary repair pathways are non-homologous end joining (NHEJ) and homologous directed repair (HDR) (Jackson and Bartek, 2009). These highly conserved pathways can be exploited to generate a defined genetic outcome across a wide range of cell types (Urnov et al., 2010).
In NHEJ, the two broken ends are simply ligated back together. If the double strand break is complex, creating ends that are not compatible then repair by NHEJ will be mutagenic; the repaired DNA will contain small insertions or deletions at the site of the break, resulting in gene inactivation (Durai et al., 2005). If a double stranded oligonucleotide is provided with overhangs (sticky ends) complementary to those left by the ZFNs, it will be ligated into the chromosome, this approach can be used to add tags to endogenous genes. Alternately, two simultaneous double strand breaks made on the same chromosome can lead to a deletion of the entire intervening stretch (Lee et al., 2010) (figure 2).
The other major repair pathway is HDR, a form of homologous recombination that faithfully copies the genetic information from a DNA molecule of related sequence. In HDR the 5Ê¹ ends of the double strand break are resected to generate 3Ê¹ single stranded tails, allowing strand invasion by donor DNA, which serves as a template for DNA replication (Durai et al., 2005). In normal double strand break repairs the DNA donor is the sister-chromatid, therefore the template is identical to the damaged DNA, resulting in a perfect form of repair. In gene targeting an exogenous donor DNA template is provided (usually an episomal or linear extrachromosomal donor) in combination to the ZFNs. If the donor DNA specifies solely a single nucleotide change, such as a restriction fragment length polymorphism (RFLP) encoding a novel allele, this will result in gene correction, that subtly edits the endogenous allele (Urnov et al., 2005). HDR can also be used for the addition of genes, if the donor provided carries an open reading frame (ORF), a transgene or even multiple trasngenes at the position corresponding to the site of the break, the sequence will be transferred to the chromosome (Moehle et al., 2007) (figure 2).
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Figure 2 | Types of genome editing made possible using ZFNs. The two primary repair pathways: non-homologous end joining (NHEJ) and homologous directed repair (HDR) with the different outcomes that can result from the introduction of a site specific DNA double strand break. Adapted from (Urnov et al., 2010).
Gene edition using ZFNs - gene disruption and gene correction
The simplest means of gene editing is gene disruption, which takes advantage of errors introduced during DNA repair to disrupt or abolish the function of a gene or genomic region. Gene knockout (KO) is an affective tool for analysing gene function and generating model animals that recapitulate genetic disorders. Using ZFN technology, Mashimo et al., 2010 created knockout rats with X-linked Server Combined Immunodeficiency (X-SCID). They injected mRNAs encoding ZFNs designed to target the rat interleukin 2 receptor gamma (II2rg) locus, where orthologous human and mouse mutations cause X-SCID, into the pronucleus of fertilised rat oocytes. They found that the offspring carried a variety of deletion/insertion mutations, most of which were expressed as frameshift or splicing errors, resulting in no or very little expression of II2rg mRNA. The ZFN modified founders faithfully transmitted their genetic changes to the next generation along with the SCID phenotype (Mashimo et al., 2010). The X-SCID rats generated in studies such as this can be valuable in vivo tools for pre-clinical testing during drug development or gene therapy as well as model systems for examining the treatment of xenotransplanted malignancies.
Another approach, gene correction allows the transfer of single nucleotide changes from a DNA donor to the chromosome following a ZFN induced double strand break. Urnov et al., 2005 designed ZFNs directed against the X-linked SCID mutation hotspot in the interleukin-2 receptor-γ (IL2Rγ) gene. Using the ZFNs on K562 cell lines, they found that ~20% of the population carried a modification at the endogenous loci and about 7% of the cells were homozygous for the donor specified genotype, which was accurately reflected at the mRNA and protein levels. The modified cells were found to be stable for extended periods in cell culture while transcriptionally and translationally manifesting their new genotype (Urnov et al., 2005).
Transgenesis of human cells is used in functional genomics, proteomics and protein structure-function studies, and is routinely accomplished by random integration combined with drug selection. Expression of a randomly integrated transgene can be unpredictable and tends to be unstable over time due to epigenetic effects (DeKelver et al., 2010). The precisely placed double strand break induced by ZFNs can stimulate integration of long DNA stretches into a predetermined genomic location, resulting in site-specific gene addition. Moehle et al., 2007 introduced ZFNs directed against the interleukin-2 receptor-γ (IL2Rγ) gene (exon 5), in combination with a DNA donor carrying a 12bp tag and a 900bp open reading frame (ORF), flanked by locus specific homology arms into HEK293 cells. After 72 hours, ~5% of the chromatids had acquired the transgene between the ZFN recognition sites (Moehle et al., 2007).
ZFNs have also been used in human EPCs and iPSC to efficiently target a drug resistance marker to a specific gene. Hockemeyer et al., 2009, used ZFNs specific for the OCT4 (POU5F1) locus and a donor constructs containing a splice acceptor (SA) followed by an enhanced green fluorescent protein (eGFP)-2A-puromycin cassette. They reported expression of two proteins, a fusion protein comprising the first 132 amino acids of human OCT4 fused to eGFP (OCT4EX1-eGFP) and puromycin N-acetyltransferase, both under the control of the endogenous OCT4 promoter, therefore generating reporter cells which can monitor the pluripotent state of human ESCs (Hockemeyer et al., 2009).
Therapeutic applications of ZFNs
Site specific manipulation of the genome by ZFNs has revolutionised biology and holds great promise for molecular medicine (Lombardo et al., 2007). For example a corrected allele of a disease causing gene could be curative in several monogenetic diseases. Alternatively, the knockout of a gene encoding a virus receptor could be shown to eliminate rather than merely reduce infection.
ZFN mediated gene disruption is the first ZFN based approach that has been taken to clinical trails, specifically for the treatment of glioblastoma (NCT01082926) and HIV (NCT00842634 and NCT01044654). In glioblastoma phase I clinical trials, the glucocorticoid receptor gene is disrupted by ZFNs as part of a T cell based cancer immunotherapy (Urnov et al., 2010). In the HIV trials, ZFNs targeting the chemokine (C-C motif) receptor type 5 (CCR5) gene have been delivered via adenoviral vector to isolated T cells from subjects. The CCR5 protein is required for certain common types of HIV infection to enter into and infect T cells. The ZFN mediated CCR5 knockout T cells then are returned to the subject. (Perez et al., 2008). An advantage of using ZFN technology is that it creates a fully penetrant, heritable gene knockout that will persist for the lifetime of that cell and its progeny, therefore removing the need for persistent therapeutic exposure.
Limitations of ZFNs
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A potential limitation of the ZFN targeting approach is off-target DNA breaks induced at related sequences elsewhere in the genome, which may cause unpredictable genotoxic. To overcome this, ZFNs can be designed to with longer DNA recognition sites such as 12bp-18bp, which upon dimerisation of the FokI nuclease domain will recognise a 24bp-23bp sequence (such sites are rare even in complex genomes). This alongside bioinformatic tools such as SELEX (systematic evolution of ligands by exponential enrichment) can be used determine the specificity for a ZFN DNA binding domain and generate a rank order of potential off-target site with highest similarity (Tuerk et al., 1990). Another challenge when designing ZFNs is the choice delivery system (DNA, RNA or viral), the ideal method has proven to be dependent on cell type. Lombardo et al., 2007 found that integrase-defective lentiviral vectors (IDLV) support functional delivery of both ZFNs and donor DNA templates to a variety of cell types, including haematopoietic progenitors and embryonic stem cells (Lombardo et al., 2007).
Aside from the various limitations, ZFN technology has allowed site specific genome editing to become established in human cells and a number of model organisms, opening the door to a powerful range of new experimental and therapeutic possibilities.