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New techniques that can be used to create site specific modifications in complex genomes hold huge potential for many aspects of genetic research, as well as for biotechnological and therapeutic applications. A new approach which has recently emerged is based on zinc-finger nucleases (ZFNs). ZFNs are restriction endonucleases that have been synthetically engineered to cleave double stranded DNA at a pre determined genomic site. To achieve this, they are designed to contain a DNA binding domain, which is made up of zinc finger proteins and a non-specific restriction endonuclease domain, typically the type IIS restriction enzyme FokI .
The zinc finger proteins allow the ZFN to bind to a specific nucleotide base sequence as each zinc finger domain binds to approximately 3bp of DNA. Linking several zinc finger domains together allows binding to longer DNA sequences, therefore increasing their specificity. To cleave the double stranded DNA, the restriction enzyme FokI must first form a dimer . By designing two different ZFN subunits which bind to the DNA sequence in the opposite orientation, the catalytic Fokl domain is able to dimerise. The target sequence of the two zinc finger domains is designed in sufficient length to be statistically unique, even in the most complex of genomes, therefore resulting in site specific DNA cleavage (figure 1).
ZFNs have been proven to work successfully in Arabidopsis thaliana , zebrafish , Caenorhabditis elegans, Drosophila melanogaster , rats , human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) .
Figure 1 | Structure of zinc finger nucleases
Each ZFN contains an endonuclease FokI domain joined to several zinc finger proteins. The zinc finger protein domains recognise specific DNA sequences that surround the intended cleavage site.
Mechanisms of DNA double strand break repair
All eukaryotic cells have derived effective mechanisms in which to repair double strand breaks in DNA. The two main mechanisms of DNA repair in eukaryotic cells are that of non-homologous end joining (NHEJ) and homologous directed repair (HDR) . Although these DNA repair pathways are extremely conserved, they have the possibility of being exploited .
In non-homologous end joining (NHEJ), double strand DNA breaks are directly ligated by a multicomplex of proteins including DNA ligase; this usually results in accurate DNA repair. However, if the double strand break produces overhangs that are not compatible, such as in the loss of a nucleotide, then repair by NHEJ will be mutagenic. Mutations such as deletions in the repaired DNA can lead to a frame shift and subsequent gene inactivation .
One approach of ZFNs is to utilise this DNA repair mechanism in order to add markers or tags to endogenous genes. The idea is to provide a double stranded marker or tag sequence in combination with the ZFN. The sequence is designed with overhangs complementary to those on the cleaved DNA sequence so that during repair the marker or tag sequence will be incorporated into the gene at a specific position. Alternately, if two double strand breaks are made simultaneously on the same stretch of DNA, a large deletion can be achieved (figure 2).
The other major DNA repair pathway is homology directed repair (HDR). HDR is a form of homologous recombination in which double stranded breaks are repaired by copying the genetic information from a homologous sequence, usually the sister chromatid. In HDR, the 5Ê¹ ends of the double strand break are removed; generating 3Ê¹ single stranded tails, which allows for invasion by the template DNA. . One application of this pathway is to provide an exogenous donor DNA template in combination to the ZFNs. This technique can be used to introduce modifications such as a single nucleotide change or to edit a dysfunctional gene as in gene correction . HDR can also be used to insert whole genes, if the template DNA carries the open reading frame (ORF) of a transgene or multiple trasngenes then theses can be incorporated into the DNA at the site of the break (figure 2).
Figure 2 | Genome editing using ZFNs
The two main DNA repair mechanisms and their possible outcomes, non-homologous end joining (NHEJ) and homologous directed repair (HDR). Adapted from .
Gene disruption using ZFNs
Gene disruption is one of the easiest methods of gene editing using ZFNs. Disrupting the function of a gene allows for investigation into its function. Another popular method used in research is gene knockout, which can be used to generate animal models of disease, in particular animal models of genetic disorders. Mashimo et al., 2010 used ZFNs technology to created 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 into the pronucleus of fertilised rat oocytes. The offspring that were produced carried a variety of deletion and insertion mutations, all resulting in very little or no expression of II2rg mRNA . Animal models of disease such as the X-SCID rats generated by Mashimo et al can be valuable in vivo tools which can be used during drug development or for the development of safe and effective gene therapy.
Gene correction using ZFNs
In another study, Urnov et al., 2005 designed ZFNs directed against a pathogenic mutation hotspot found within exon 5 of the interleukin-2 receptor-γ (IL2Rγ) gene, resulting in X-linked SCID. Using ZFNs in combination with a corrective donor molecule on human K562 cell lines, they found that ~20% of the population carried the corrective modification at the endogenous loci and ~7% were homozygous for the donor specified genotype .
Gene addition using ZFNs
Transgenesis is the process by which an exogenous gene is integrated into the host genome usually by vectors or nuclear injection combined with drug selection. As the transgene is usually randomly integrated, its expression can be unpredictable and unstable . The accurately placed double strand break induced by ZFNs can assist with the site specific integration of transgenes. Moehle et al., 2007 designed ZFNs directed against exon 5 of the interleukin-2 receptor-γ (IL2Rγ) gene, in combination with a DNA donor which carried a 12bp tag and a 900bp transgene into HEK293 cells. After 72 hours, ~5% of the chromatids had acquired the transgene between the ZFN recognition sites .
ZFNs have also been used in human embryonic stem cells (ESCs) and induced pluripotent stem cell (iPSC). Hockemeyer et al., 2009, used ZFNs that were specific for the OCT4 (POU5F1) gene in combination with a donor template. The donor template was designed to contain a splice acceptor and an enhanced green fluorescent protein (eGFP)-2A-puromycin cassette. They found expression of two proteins: a fusion protein which consisted of the first 132 amino acids of human OCT4 fused to eGFP (OCT4EX1-eGFP) and puromycin N-acetyltransferase, therefore generating reporter cells which can monitor the pluripotent state of human ESCs .
Therapeutic applications of ZFNs
As discussed the ability of ZFNs to manipulate the genome at pre determined sites has radically changed the field of molecular research. In addition, ZFNs have also shown great potential within the medical field. The ability to correct a disease causing allele could have huge therapeutic potential for many monogenetic disorders.
ZFN mediated gene disruption therapy has reached clinical trials for the treatment of HIV and glioblastoma. In the HIV clinical trials (clinicaltrials.gov NCT00842634 and NCT01044654), ZFNs have been designed to knockout the chemokine receptor type 5 (CCR5) gene in patient T cells. The CCR5 protein is required for certain common types of HIV infection to enter into and infect T cells . In another clinical trial for the treatment of glioblastoma (clinicaltrials.gov NCT01082926), ZFNs have been designed to disrupt the glucocorticoid receptor gene as part of cancer immunotherapy treatment.
Limitations of ZFNs
One of the main advantages of ZFN technology is that it has the potential to create a fully penetrant and heritable genetic modification; however there are still some major limitations that need to be overcome. The major limitation is off target cleavages which can be induced at related sequences elsewhere in the genome. Off target cleavages can be unpredictable and are often genotoxic. In order to overcome this issue, ZFNs can be designed with longer DNA recognition sites such as 12bp-18bp, which upon dimerisation will recognise a 24bp-23bp sequence. Another challenge when designing ZFNs is the choice delivery system (DNA, RNA or viral). Lombardo et al., 2007 found that integrase-defective lentiviral vectors (IDLV) best supported the delivery of ZFNs and donor DNA templates to a variety of different cell types .
Aside from the various limitations, ZFN technology has provided site specific genome modification a number of model organisms and human cells. Although the field is relatively new, the techniques used have unveiled a powerful and exciting range of experimental and therapeutic possibilities.