Zinc Finger Nucleases and Site-Directed Mutagenesis in Cells of Plants - Essay Example

?Zinc Finger Nucleases and Site-Directed Mutagenesis Zinc Finger Proteins Certain proteins known as the zinc finger proteins show a distinctive secondary structure wherein a zinc metal is coordinated by a set of specific amino acid residues. The zinc fingers are the most abundant DNA binding protein motifs that are encoded by the genomes of eukaryotes (Branden & Tooze, 1999). Two percent of the genome is estimated to encode zinc finger proteins. The name “zinc finger’ was coined after its discovery in the Xenopus protein transcription factor IIIA, when the zinc-binding domain appeared to grasp the region of the 5S RNA gene (Miller, McLachlan, & Klug, 1986). It is now well known that there are many naturally occurring zinc finger motifs, the most common of which is the Cys2His2 or C2H2 zinc finger. This particular motif is made up of 2 antiparallel ?-sheets and an ?-helix that are coordinated by 2 Histidine (his) and 2 Cysteine (cys) residues binding a zinc atom (Figure 1). This binding, plus an inner structural hydrophobic core, stabilizes the secondary structure of the protein. Figure 1. The classic zinc finger protein is made up of approximately 30 amino acids where two cysteine and two histidine residues bind to a zinc atom. The zinc finger proteins are known mediators of metabolic interactions including protein-protein interactions and RNA binding. However, the most known are their roles in DNA sequence-specific binding. From early studies on the classical C2H2 zinc finger, it was found that the zinc finger differed in principle from the other DNA-binding proteins because several zinc fingers can be linked linearly to recognize DNA sequences of varying lengths (Klug, 2005). In contrast, other DNA-binding proteins utilize the symmetry of the double helix. Usually, more than one zinc finger domain participates in binding the DNA. Each zinc finger domain binds to three nucleotide (nt) bases on the major groove of the DNA. The ?-helix, also known as the recognition helix, binds to three or more bases of with specific sequences on the DNA. Since zinc finger proteins could have more than one zinc finger motif, the DNA contacts of adjacent or arrayed zinc fingers could overlap (Iuchi, 2005). Regions that are bound by the zinc fingers are usually spaced at 3 – 5 bp apart. The modular design of the zinc fingers allows it to interact with the DNA at different regions, and allows them to be involved in many DNA interaction reactions. Thus, it is not surprising that the zinc fingers are the most encoded motif in the genome and are very abundant in nature. The highly sequence-specific DNA binding property of zinc fingers offered a lot of potential as a tool for regulating gene expression or for manipulating the genome. From the initial basic studies arose the design and engineering of zinc finger proteins for binding specific regions in the DNA, and synthesis of zinc finger nucleases for cutting the DNA at target sites and introduction of changes to the DNA (Davis & Stokoe, 2010; Durai, et. al, 2005). Zinc finger arrays can be engineered to bind long stretches of known regions in the genome and with the ultimate goal of finding cures to notable diseases. Some applications of zinc finger arrays are the inhibition of HIV-1 expression (Reynolds, et al., 2003) and the disruption of herpes simplex virus infection (Papworth, et al., 2003). To emphasize the importance of the zinc finger proteins, an online database was established that compiles information on known (naturally occurring) and engineered zinc fingers and zinc finger arrays (Fu, et al., 2009). Zinc Finger Nucleases A zinc finger nuclease (ZFN) is an engineered restriction enzyme that consists of a zinc finger array designed to recognize specific nucleotide sequences in the DNA, and a non-specific nuclease domain. Usually, the zinc fingers in the array have similar motifs as that of Cys2His2 zinc finger protein. The engineered zinc finger is fused with the cleavage domain of the restriction enzyme FokI endonuclease. Since FokI will cut the DNA only when it has dimerized to the DNA, then two different ZFNs must be designed to cut the DNA. Each ZFN has its own binding site, but they need to converge at the region where the FokI nuclease domain can dimerize (Figure 2). One ZNF can have several zinc fingers but only one nuclease domain. Zinc fingers can be engineered to target any gene or region in the DNA, cut that region, and modify that region by inserting new DNA bases. ZFNS are used to introduce mutations to produce new genes, to remove or delete genes, and to study gene function. ZFNs can also manipulate the regulatory and non-coding DNA sequences. Among the possible applications of ZFNs are designing new therapies for diseases, and the design of plants and animals for improved productivity and functions. ZFNs can be engineered to attach to any region in the DNA, which makes the knowledge of the genome sequence very important because it will identify the exact site for cleavage or modification. Figure 2. A representation of the binding of two ZFNs to specific DNA sequences. In the figure, each ZFN has four zinc fingers that bind to 12 nts on the DNA, with an effective 24 bp recognition site that is unique. Five bp separate the ZFN sites, which is cut by the endonuclease represented by the oval N. Figure from Wu, Kandavelou, & Chandrasegaran, 2007 as adapted from Urnov, et al., 2005. Site-Directed Mutagenesis Mediated by Zinc Finger Nucleases After the ZFNs specifically bind the target DNA regions, the Fok1 endonuclease will introduce double-strand breaks (DSB) in the DNA. Cell death could occur if the DSB is not repaired. Site-directed mutagenesis is possible when the double-stranded breaks are repaired by recombination or non-homologous end-joining (NHEJ) (Carroll, et al., 2006). This type of repair, (NHEJ), is prone to many errors, and could result in null alleles. To introduce desired mutations, the double-strand breaks should be repaired with homologous recombination (HR) using donor DNA designed for a certain mutation. Homologous recombination is not a very common occurrence in mammalian systems, but it can be stimulated by intentional or targeted double strand breaks in the DNA (Alwin, et al., 2005). Donor DNA can be introduced and utilized to insert gene sequences via homologous recombination (Figure 3). This action will result to the transcription and expression of novel genes or restoration/modification of the original gene function (for review see Porteus & Carrol, 2005). Figure 3. Gene targeting as mediated by zinc finger nucleases. Zinc finger nucleases targeted to gene of interest (Gene X) will produce disruption of the gene sequence, via double strand break in the DNA. If the repair is via non-homologous end joining (NHEJ), a pool of mutants will be produced. The resulting mutants may have insertions or deletions of bases and frameshift mutations. These changes can result in loss or alteration of gene function. To generate a desired mutation, ZFNs are also utilized, but in addition, homologous DNA is introduced together with the ZFN. The donor DNA contains the desired modification after the target DNA DSB has been induced. Homologous recombination is stimulated with the presence of the donor DNA, resulting in the desired repaired or modified DNA mutant. Figure is adapted and caption is paraphrased from Wu, Kandavelou, & Chandrasegaran, (2007). As summarized by Wu, Kandavelou, & Chandrasegaran (2007), ZFN-mediated gene targeting or site-directed mutagenesis can be achieved by first identifying the ZFN target sites. The site can be found on a locus in a specific chromosome (Carroll, et al., 2006). The DNA sequence of the loci should be known, so with the gene itself, because the design and specificity of the zinc fingers are dependent on these. Zinc fingers with known binding to the chosen target sites can also be selected from the database (Durai, et al., 2005). After the ZFNs have been synthesized using phage display or other techniques, these can be delivered into the cells either alone or with DNA donor. ZFNs will induce double strand breaks and produce a pool of mutants if delivered alone. When a DNA donor is included upon the introduction of ZFNs, desired mutants are produced by homologous recombination. Mutagenesis can be monitored by evaluating the changes in function or by the use of reporter gene assays. Southern blots may also be necessary to check that the donor DNA fragment did not integrate to another region in the genome (Urnov, et al., 2005). Importance of Genome Sequence Data With ZFN technology, any region in the DNA can be targeted for mutation or for alteration of gene sequence. These regions could code for structural genes, transcription factors, developmental genes, and even the promoters and the untranscribed regions in the DNA. However, all these sites can be targeted for ZFN binding only if the genomic DNA sequence is known. Knowledge of the sequence of target regions is very important because it will determine the design of the zinc fingers that will initiate contact. If the sequence is incorrect, then the zinc fingers will not be able to bond, or worse, they will bind to the wrong bases and will induce DSB in non-target regions. Therefore, the objective of utilizing ZFNs in the first place will not be achieved. It is critical that before any manipulation is performed, the target loci have to be sequenced first. Then the target bases should be identified first before the design and synthesis of the zinc fingers can proceed. It is also necessary to know the sequence of the parallel strand because the other member of the ZFN pair will not bind the same region on the complementary strand. The available sequence data of the genomes of model organisms have been very helpful in studying gene function with ZFNs. Another importance of knowing the sequence data is the reduction in the probability of binding non-target regions in the DNA. It has been observed that when ZFNs bind non-specific or redundant DNA sequences, high levels of levels of cytotoxicity are produced. Non-specific or off-target binding will also produce deletions, insertions, and frameshift mutations with adverse gene expression effects or loss-of-function of genes essential for metabolism. Engineered ZFNs must be able to recognize and bind highly specific target areas. These targets should be unique and are not duplicated elsewhere in the genome. Highly precise binding is a consequence of accurate sequence data. Improvement of the binding of the zinc finger nucleases are focused on the enhancement of the traits geared toward the recognition of DNA binding sites. With higher binding specificity, the binding at off-target sites and cleavage at these sites will be reduced, and so are the cytotoxic effects (Cornu, et al., 2008). ZFNs have been designed to have more arrays or zinc fingers in an effort to increase specificity. More specific binding can be achieved with the fingers will bind four bases, instead of the usual triplet. However, since designing the ZFNs to bind four or more DNA bases reduces the chances of finding targets, this aspect needs to be studied more. Another approach to improve the target site specificity is to employ a structure-based approach (Szczepak, et al., 2007). Protein modeling abetted with energy calculations showed that specificity was increased when the nuclease dimerization energy requirement was decreased, and when the homodimerization of the zinc fingers was prevented. With higher specificity, less cytotoxic effects were observed. Variations in the nuclease structure design that aimed to produce nuclease variants with a preference for heterodimerization also result in reduction in cytotoxic effects. The design of the nuclease variants will entail a search of 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 a requisite when testing the efficiency of the zinc fingers nucleases (Carroll, et al., 2006). Caroll and co-workers (2006) recommended that when searching for plausible ZFN targets, the DNA sequence of the desired target must be obtained and compiled. Longer DNA sequences will improve the chances of finding binding sites for ZFNs. The base triplet sequences necessary for the binding of the zinc finger must be available on the selected DNA sequence. Therefore, if more DNA regions have been sequenced, then 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. This will be followed by the design of the primary amino acids that need to be in the zinc fingers. The zinc fingers have to be oriented “backwards” to the DNA sequence. The DNA sequences for the zinc fingers are synthesized, and 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. Since their discovery and after their design and synthesis protocols became available, zinc finger nucleases have been employed in many studies on gene function, regulation, and modification. An exciting application is their use in treatment of diseases. Among the earliest studies was the design of zinc finger nucleases against X-linked severe combined immune deficiency (SCID) mutation in the IL2RG gene (Urnov, et al., 2005). The human interleukin 2 receptor gamma sub-unit (IL2RG) protein has an important function in the production of lymphocytes, which are essential for the immune response. The IL2RG gene was mapped in order to locate the disease-causing mutation resulting in SCID. After the mutation was found, zinc finger nucleases were produced by traditional gene cloning and expression techniques. The genes were expressed in expression vectors and ligated to the reporter green fluorescence protein (GFP). The ZFNs were then introduced to human cells with the objective of repairing the mutation. The results showed that the introduction of the ZFN modified the cells such that the mutation was corrected. Since T-cells were more responsive to the introduced modification, the potential of ZFNs in treating SCID is high (Urnov, et al., 2005). Lately, another study was conducted to correct the mutation on the same IL2RG locus with the objective of producing gene knock-outs for mice (Mashimo, et al., 2010). ZFNs were explored as a possible means to produce knock-out rats rather than from germline mutations of stem cells or somatic cells. Instead of gene cloning and vector expression to produce ZFNs, the authors injected mRNA that coded for site-specific ZFNs into mice embryos. The DNA of the progeny rats of the embryos that were transformed were extracted and the sequences were obtained. The results showed the presence of genetic modifications in many regions in the DNA. This showed that the ZFNs that were transcribed from the injected mRNA were not specific for the target locus only. Gene expression can also be disrupted with zinc finger nucleases. This was shown in zebrafish (Danio rerio) where somatic and germline mutations were introduced with ZFNs (Doyon, et al., 2008). Similar to what was reported in rats, mRNA that encoded specific ZFN was injected into fertilized eggs/embryos of zebrafish. The ZFNs were engineered to produce mutation in the gene that controlled the expression of tail in zebrafish. Results of the experiment showed that the resulting offspring were tailless due to the disruption in the gene sequences. The stability of the mutation was checked by outcrossing the female tailless fish to normal fish. The cross-breeding resulted in offspring that were heterozygous for the trait. This showed that the mutation induced by the ZFN was stable and heritable. Further crossing experiment to the next generation showed an F2 population with mutant fish (without tails), non-mutants (homozygous) and heterozygous fish which carried the gene deletion. Compared to the cited experiment on rats, the engineered ZFNs for zebrafish was highly specific for the gene target because genome analysis showed the absence of other mutations. The ZFN technology is also applicable for the modification of plant genomes like Arabidopsis (Osakabe, Osakabe, & Toki, 2010;Tovkach, Zeevi, & Tzfira, 2009), Zea mays (Shukla, et al., 2009), and tobacco (Townsend, et al., 2009). Biochemical and in planta methods can be used to design and test the capacity of zinc finger nucleases for restriction digestion. The zinc finger proteins can be produces with a cloning vector where the FokI endonuclease domain has been pre-inserted. After PCR amplification, the PCR product is purified and introduced into an expression cassette to produce the designed ZFN. Introduction into the plant genome can be carried out with binary vectors and Agrobacterium tumefaciens. Engineered ZFNs are valuable tools for genetic engineering of plant traits that are important in increasing world food supply, and for producing plant-based products like biofuels. References 1. Alwin, S., Gere, M., Guhl, E., Effertz, K., Barbas, C., Segal, D., et al. (2005). Custom zinc finger nucleases for use in human cells. Molecular Therapy, 12(4):610-7. 2. Branden, C., & Tooze, J. (1999). Introduction to Protein Structure: Second Edition. New York: Garland Publishing. 3. 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