Site Directed Mutagenesis By Engineered Zinc Finger Nucleus Biology Essay

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A zinc finger refers to a secondary structural motif of certain proteins. The zinc finger folds in a manner that allows it to coordinate or hold a zinc metal. There are several zinc finger motifs; the most common is the Cys2His2 zinc finger that consists of an antiparallel β-sheet and an α-helix coordinated by 2 His and 2 Cys residues that bind a zinc atom. The zinc finger proteins are involved in many reactions like mediating protein-protein interactions, RNA binding, but they are most known to be involved DNA sequence-specific binding. Two or more zinc fingers comprise the DNA-binding domain. Each zinc finger domain binds to three bases on the DNA. If there are more zinc fingers, then more bases are bound. Bound regions by the zinc fingers are usually three bp apart, and bind to the major groove of DNA.

The capacity of the zinc finger proteins to recognize highly specific DNA sequences for binding was exploited by researchers to produce, design, or engineer zinc finger nucleases (ZFN) that can cut the target DNA at specific sites. Usually, these zinc finger nucleases have similar motifs as that of Cys2His2 zinc finger protein. In order to cut a specific target site on the DNA, the engineered zinc finger protein was fused with the cleavage site of FokI endonuclease, which is a restriction enzyme. FokI has a strict requirement for dimerization to the DNA; this is the reason why two different ZFNs are designed in opposing direction to bind the FokI restriction site (Figure 1). One ZNF can have several zinc fingers but only one nuclease domain.

Many engineered ZFNs are targeted to attach to different DNA regions. After the ZFNs specifically bind the target DNA regions, the Fok1 endonuclease will introduce double-strand breaks in the DNA. Site-directed mutagenesis is possible when the double-stranded breaks are rejoined or repaired by recombination (Carroll, Morton, Beumer, & Segal, 2006). Gene mutations are created after cleavage with ZFN is followed by inaccurate repair, which is much prone to error and can result in null alleles. To introduce desired mutations, the double-strand breaks should be repaired with homologous recombination using donor DNA designed for a certain mutation.

Figure 1. The heterodimerization of two different ZFNs on double-stranded DNA ascertains that the cleavage is site-specific (Davis & Stokoe, 2010).

Papers describing the use of the technology

This work was among the earliest that studied the application of ZFNs in treatment of disease. Zinc finger nucleases were designed against X-linked severe combined immune deficiency (SCID) mutation in the IL2RG gene (Figure 2) (Urnov, et al., 2005). The IL2RG or human interleukin 2 receptor gamma sub-unit is involved in lymphocyte production. To find the position of the mutation, the authors mapped the IL2RG gene, and identified the positions where mutations that cause SCID can be found. Zinc finger proteins were produced by traditional gene cloning techniques. After the genes that coded for the designed ZFNs were multiplied in bacteria, the genes were inserted into expression vectors that also expressed the reporter green fluorescence protein (GFP).

The results of the site-directed repair were visualized with the help of GFP. These showed that 18% of the cells were modified. Furthermore, 7% of the cells were genetically modified on both X chromosomes. Messenger RNA and proteins were quantified and showed that these were correlated with the genetic modification. Based on cell types, the T cells were more responsive to the genetic modification (Urnov, et al., 2005). Their early results pointed immediately to the possible use of the ZFNs in disease therapy.

Figure 2. Map of the IL2RG gene showing the region that contained the mutation for SCID (Urnov, et al., 2005).

A more current study on correcting mutations on the IL2RG locus was carried out to produce knock-out rats for the IL2RG (Mashimo, et al., 2010). ZFNs were employed because it was difficult to produce knockout rats using germline mutations from somatic or embryonic stem cells. More advanced techniques were used to produce mRNAs that coded for site-specific ZFNs. The mRNAs were microinjected into the pronucleus of mouse oocytes where the ZFN genes are expressed and are translated to produce the functional ZFNs (Figure 3). After the rats developed from the transformed embryos, the DNA was extracted, amplified, sequenced, and analyzed for differences from the normal DNA sequence. The DNA of rats arising from the transformed embryos showed deletions and insertions in many regions, which proved that the ZFNs in this instance were not specific for the IL2RG locus only. However, some offspring also showed absence of SCID, which proved that the ZFNs also homed in on the original target DNA sites.

Figure 3. Scheme for injecting mRNAs that code for site-specific ZFN targets on the IL2RG locus. Figure from Mashimo, et al., 2010.

Lombardo and co-workers reported in 2007 that they used an integrase mutant to co-deliver two ZFNs and donor DNA containing a silent point mutation in the human IL2RG locus. The delivery system was successful in introducing ZFNs and donor DNA to different human cell types, including embryonic stem cells. During the repair of the double-stranded breaks in the DNA due to the action of ZFNs, three events resulted: gene disruption, gene correction, and addition of the transgene to the target area (Lombardo, et al., 2007). The results of the experiments verified the observation that gene disruption was due to nonhomologous end-joining (NHEJ) while targeted mutation was due to homologous DNA recombination. The integration of ZFNs and donor DNA by viral delivery system was identified to be a major goal of site-directed mutagenesis.

Zinc finger nucleases were used for gene disruption in zebrafish (Danio rerio) in order to introduce somatic and germline mutation (Doyon, et al., 2008). This study was the first report on the delivery of ZFN-coding mRNA into fertilized eggs/embryos. ZFNs were designed to target the golden and no tail/Brachyury genes. mRNAs that coded for specific ZFNs were introduced by injection to 1-cell embryos. A high percentage of animals have phenotypes with the expected loss-of-function and gene mutations at the positions targeted by the specific ZFN. The no tail (ntl) alleles were disrupted, and these alleles were transmitted to progenies.

The authors of this article first tested several ZFNs for feasibility before the actual experiment on gene disruption was performed using yeast-based assays (Figure 4A). The genome data of zebrafish was searched for loci that will be tested. The no tail locus (ntl) was identified in the DNA so with the ZFN binding sites (Figure 4B). This site was identified to be an "off-target site," therefore, top five potential loci were analyzed using different engineered ZFNs. The cleavage by ZFNs were determined by PCR to amplify chromosomal regions and then nucleotide sequencing of the PCR products, endonuclease study and loss of restriction fragment length polymorphism. The selection of ZFNs that target the ntl were based on the results of these processes. Embryos injected with the ZFN targeting the ntl showed the expected phenotype (Figure 4C).

Female zebrafish that were injected with the ZFNs were outcrossed to check if the ZFN -induced mutations are heritable by the offsprings. The progenies or offspring showed the expected germline frequency for the mutation. The results for the F1 generation (founders) showed that the mutation was inherited because the offspring carried heterozygous copies of the gene. Further crossing of the founders were performed when the fish reached maturity. The F2 populations showed a mix of mutant fish (no tails), heterozygous individuals, and non-mutant phenotypes. The engineered ZFNs did not have any other effects on the DNA because the design was highly specific for the ntl only.

Figure 4. Protocol and results of testing gene deletion using ZFNs targeted to the no tail locus of zebrafish (Doyon, et al., 2008).

4. The ZFN technology was also employed in the genome editing of plants (Tovkach, Zeevi, & Tzfira, 2009). In this study, biochemical and in planta methods were employed for designing and testing of the digestion capacity of zinc finger nucleases. The zinc finger proteins were produced with a cloning vector that also carried the FokI endonuclease domain. After PCR amplification, the product was inserted into an expression cassette followed by insertion and cloning of a ZFN. Binary vectors were constructed and transferred to Agrobacterium tumefaciens which was then introduced into Arabidopsis cultures. Assays were conducted and so with DNA analysis of the transformed plants (Figure 5). Results showed that it is possible to clone and assemble tested ZFNs and corresponding targets into Agrobacterium and use the technology for plant genome editing.

Figure 5. Functional assays and vector systems for analysis of designed ZFNs in plants. Figure from Tovkach, Zeevi, & Tzfira, 2009.

Part 2

How do ZFNs carry out site-directed mutagenesis and how is the use of genome sequence data essential to use this technique (80%).

The zinc finger nuclease technique arose from the observation that zinc finger proteins can bind to DNA sequences with very high specificity. This specificity led to the development of zinc finger nucleases, which are basically restriction enzymes with a difference. The zinc finger nucleases can be designed to target a single gene on any region in the DNA, cut that region to a desired size, and repair that region or insert new DNA bases to produce novel genes. The technique is applied to introduce mutations and even for the manipulation of non-coding but regulatory regions in the DNA. The technique and its potential applications has been given so much attention in recent years because of the potential applications in the prevention and treatment of diseases, production of novel genes/proteins, and on studies in understanding gene function.

Site-directed mutagenesis

Currently, zinc finger nuclease have evolved to become potent and effective tools to carry out site-directed mutagenesis to study gene function and alter gene expression. In order to accomplish this, the zinc finger protein is appended to the nuclease domain of the FokI endonuclease to produce the zinc finger nuclease. ZFNs are effective molecular scissors due to their highly specific binding to DNA regions, and the non-selective nuclease activity of FokI. To produce the double strand breaks in the DNA, the zinc finger domains will bind triplet DNA sequences first. Since the ZNF are designed in arrays of several zinc fingers and just one endonuclease domain, then it is possible that the zinc finger will bind to a relatively long stretch of the DNA (approximately 30 base regions). The FokI endonuclease domain will then position near the region that has to be cut, but it is a requirement that two FokI endonuclease domains should dimerize.

Early studies show that if the FokI endonuclease is a monomer, it will not produce even a single lesion in the DNA strand. To achieve the dimerization, a ZFN is designed for each of the two DNA complementary strands but in antiparallel orientation that converge on the complementary strands of the region that is to be cut or removed (please refer back to Figure 1 for the diagram of ZFN binding). When the region of interest is removed, the ends are rejoined via non-homologous end joining which can result in the loss of function of the gene that was deleted or edited. Insertion of other gene sequences that come from donor DNA is also possible via homologous recombination, which leads to the production of new genes or restoration of gene functions. Whichever recombination or DNA repair mechanism is employed, ZFN technology is a very useful tool in genetic modification, repair, mutation, and understanding gene function (for review see Porteus & Carrol, 2005).

Importance of DNA sequence data in identifying ZNF binding regions

The use of ZFNs makes it possible to target any area in the DNA, including regions that code for transcription factors, structural genes, housekeeping genes, developmental genes and even non-coding regions. The requirement for designing any ZFN for any region in the DNA sequence is therefore dependent on the nucleotide sequence data, because the specific binding of ZFN comes from the recognition of specific DNA sequences. First of all, in studies that use ZFNs, the main objective is always to manipulate the genome by altering it through loss of function mutation or insertion of new genetic sequences. Therefore, the region in the DNA where one can find the gene of interest has to be sequenced or identified. This is before the zinc fingers can be designed to bind the triplet sequences. The sequence of both DNA strands must be known because the ZFNs are not designed to bind complementary strands. It must be noted that the amount of sequence data for many model genomes has helped a lot in decreasing the amount of new sequencing that is being done. Researchers only need to consult the sequence databases for organisms like Arabidopsis thaliana, Drosophila melanogaster, and Zebrafish to come up with designs for ZFNs.

Incorrect DNA sequence data leads to non-specific binding and cytotoxic effects

Several of the difficulties met with ZFN binding are related to the binding with non-specific or redundant DNA sequences. ZFNs can produce high levels of cytotoxicity when the ZFNs that is introduced will cause DNA double-strand breaks in non-target regions in the DNA. When the binding is non-specific (this is also called off-target binding), it is possible to produce gene deletions and insertions that can alter the function of certain genes resulting in adverse effects like inhibition of or too much gene expression. This is where it becomes necessary to identify highly specific target areas that are recognized by the engineered ZFN, and that these target areas are not duplicated elsewhere in the genome. This means that the ZFNs have to be highly precise in recognition of the binding sites, and this has been the focus in improving the precision of the ZFNs. Precision of ZFN binding is a consequence of having analyzed the genomic data for organism and the DNA region for manipulation.

Under biological conditions, attempts to improve binding focused on improvement of the properties that will influence or improve the recognition of DNA binding sites. Increased specificity of binding will lessen the number of cleavage in the off-target sites and will result in reduction of the cytotoxic effects (Cornu, et al., 2008). Since it is the number and quality of the ZFNs that determines the number of binding sites, ZFNs have been designed to have more arrays or zinc fingers. The optimum number of DNA bases for binding is three (triplet), but more specific binding can be achieved with the fingers will bind four bases. However, there is a disadvantage of designing the ZFNs to bind four or more DNA bases, because the chances of finding targets will be reduced.

Knowledge of sequence data improves chances for heterodimerization of nuclease domain

It was also found that if the designed ZFNs have nuclease variants the prefer heterodimerization, rather than homodimerization, then the toxicity will be reduced. This will entail searching the sequence databases for DNA targets that have anti-parallel heterodimeric sequences. Linker regions must also be well characterized because their sequence and length will affect the activity of the nuclease. Cytotoxicity of ZFNs can be decreased if the half-life of the ZFN is reduced.

Aside from their use in the improvement of ZFN specificity and reduction of the cytotoxic effects of ZFNs, genomic data is also important for the testing of the zinc fingers nucleases (Carroll, Morton, Beumer, & Segal, 2006). Caroll and co-workers gave the following recommendations when looking for plausible ZFN targets. First, the DNA sequence of the desired target is obtained and compiled. Longer DNA sequences will improve the chances of finding binding sites for ZFNs. In addition, the triplets necessary for the binding of the zinc finger must be available on the selected DNA sequence. Therefore, if there are more sequences available, the chances of finding the desired triplets are increased. After finding triplet sites in the targets, it is necessary to calculate the specificity of the designed ZFN. Then the amino acids that needs to be in the zinc fingers has to be designed. The zinc fingers have to be designed oriented "backwards" to the DNA sequence. The DNA sequence for the zinc fingers are synthesized, then the proteins are expressed in an expression vector. In newer studies, the zinc finger proteins are expressed in vivo when mRNA coding for the ZFN are injected directly to nuclei or oocytes.

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