Site-Directed Mutagenesis of Gene Sequences in Cells - Essay Example

?Site directed mutagenesis All the genes are en d in the same DNA strand that is having two single strands overlapping on each other. Each strand is made up of the oligo nucleotides A, C, T and G. These Reading frames contain different codons. In this single DNA strand are the reading frames for the different genes. These genes encodes for specific function and protein synthesis. These genes are responsible for the many diseases in the human. Gene therapy is used to manipulate the genes and correct the diseases that are of genetic origin. The genetic origin of the thousands of diseases can be found by discovering new molecular biology techniques. The ability to generate new DNA molecules and the process of inserting it into the genome to correct the diseases are the biomedical research nowadays. This type of modification of the cellular phenotype has become the back bone for the biomedical research. Knowing the position and the reading frame of the genes will help us to identify the nonsense mutants by the ochre suppressors. These non sense mutants are highlighted and this will help us for an efficient screening for silent mutants. The small oligonucleotides containing 7 nucleotides in length can be used to form stable duplexes with the single stranded DNA. (Jensen et al. 2011). These duplexes are stable even with a mismatch. This led to the introduction of oligonucleotide mutagenesis. The emergence of site directed mutagenesis as an analytical tool in the in the year 1985 was a mile stone in the field of molecular biology. (Herzog and Zolotukhin 2010). Site directed mutagenesis is widely used for the study of the protein functions. The mutation at a defined site of the DNA leads to the change of an amino acid specific to a protein and this leads to the alteration of the protein molecules. Two methods of site directed mutagenesis can be used. The first one is overlapping method and the second one is called as whole round second round PCR. They create modified DNA sequences that are used to study the importance of the specific residues in protein structure and function. Protein engineering and altered protein substrate activity can be achieved using Site directed mutagenesis. PCR based Site directed mutagenesis is the most common method where the desired mutation is carried in one of the primer and annealed to the site of interest. Two cycles of PCR are carried out for insertion of mutation. (Lloyd 2005). Fig 1: Methods available to site specifically modify a genome target. (Herzog and Zolotukhin 2010). Zinc finger nuclease (ZFN) is a dimeric protein with each monomer having a zinc finger array (contains three or four zinc fingers). They are artificial restriction enzymes that have a lot of application in plants and animals. These zinc fingers (Cys 2 Hys 2) are arranged in the ? ? ? – fold ( beta- beta- alpha folding ) which is coordinated by the Zn+ ion and has the non specific cleavage site. (Gupta et al. 2011).These zinc finger arrays are bound to the cleavage site of the type II restriction endonuclease enzyme, Fok I. Fok l is responsible for the cleavage function of the Type II endonuclease. (Gupta et al. 2011).The dimerization of the cleavage domain is much essential for the enzyme activity. If the zinc finger array is engineered to bind to some specific site of the genome, then Fok l will perform the cutting action at that site. This double stranded break can be utilized for the site directed mutagenesis for protein engineering. This break is then repaired by using either homologous recombination (HR) or non-homologous end joining (NHEJ) technique. The HR requires extensive homology between the strands whereas NHEJ does not require any homology and the repair at the targeted site usually results in the mutation. (Herzog and Zolotukhin 2010). Because of this reason NHEJ is used in the site directed mutagenesis associated with the ZFNs. ZFN mediated gene modification will produce cell lines to contain biallelic knock outs. If ZFN targeted gene modification is applied in vivo then the frequency of transmit of the mutant alleles to their offspring’s will be very high. (Herzog and Zolotukhin 2010). Zinc Finger nucleases are used for two important things. First is to create the mutation at the specific genomic sites. Second is to develop the Gene therapy in the higher plants and animals where their frequency is very low. The Zinc fingers will first bind to both the strands of the targeted sequence. They have the ability to identify 30 amino acids in the sequence using the 3 base pair nucleotide sequence. (Singleton 2010). These zinc fingers are identified by two methods. The first one is the selection process where the required fingers are found by using the library. The second one is the use of the computer generated matches. These Zinc fingers are delivered using the inducible promoter for genetic transformation. (Singleton 2010). ZFN has heavy potentials to facilitate the genome modifications. They do so by knock outs and gene replacements by homologous combinations. ZFNs are used in gene therapy and clinical applications. ZFNs can be easily engineered. ZFNs are engineered to target the gene of interest in any part of the DNA sequence with high conformity. They do the target editing and create the double stranded breaks at the strand. These strands are important for the site directed mutagenesis. Site directed mutagenesis stimulates the cell’s DNA- repair process which is very natural. They are also used to enhance the frequency of gene therapy using the homologous recombination. (Singleton 2010). Targeted mutagenesis is an important tool to investigate the basic biology of plants, animals and micro organisms. Targeted mutagenesis using the zinc finger nucleases was studied for Arabidopsis thaliana. Arabidopsis thaliana is the model plant used for the study of large scale genome analysis. This is also very important for the other plant species such as rice, maize, wheat, soya bean and tomato. A lot of researches have been performed and the genome analysis data’s are plenty. There is a need for the analysis of these data in a systematic manner. Site directed mutagenesis is used to investigate the plant genome functions and the modifications at the genetic level. Gene targeting can also be used but the efficiency of mutation was very less in this technique. ZFN genes were engineered to ABA-INSENSITIVE 4 (ABAI4) gene. (Osakabe et al., 2010). ABI4 codes for the ERF/AP2 transcription factor family that regulates the production of Abscisic acid that is responsible for abiotic stress and seed development. The engineered ZFNs cleaved the recognition sites of the ABAI4 genes. The ZFN target site in the ABAI 4 genes was [5?-NNCNNCNNC (Nx4~7) GNNGNNGNN-3? (N = A, C, G, and T). (Osakabe et al., 2010). A combination of the three zinc finger modules was designed for this gene using the triplets 5?-GNN-3?. These targets were tested using the LacZ promoter assay test and found to have high affinity for their target. Invitro DNA digestion assay was performed using the in vitro translation system with wheat germ extracts. The combination of ZFN_AAA and ZFN_TCC was found to digest the targeted sequence perfectly. In Arabidopsis thaliana the ZFN insertion and expression was performed using the heat shock protein HSP 18.2 gene promoter. A heat pulse at 40°C for 90 minutes using the 12 days old Arabidopsis plant transgenic line helped the insertion, followed by the test for the expression using the surveyor nuclease. (Osakabe et al., 2010). Surveyor nuclease is capable of identifying the mismatch pairs and the mutations present in the genetic line. The signal was positive. Thus higher mutation efficiency was achieved using the site directed mutagenesis in higher plants. Substitution mutations are also possible in this technique. ZFN mediated mutageneis was found to have greater mutation efficiency and better study rather than the Gene therapy procedures. (Osakabe et al., 2010). Figure2: Structure of Zinc finger Nuclease. Figure 3: Dimer of three finger ZFNs bound to the target cleavage site. The spacer consists of 7 base pairs. Targeted gene alteration uses the additional oligo nucleotides like single - stranded oligo nucleotides, small DNA fragments, pseudo complementary peptide nucleic acids, zinc finger nucleases and adenovirus associated vectors. The efficiency of gene repair mechanism was very low in the somatic cell line and extremely low for the stem cells. The percentages of efficiency are 0 – 20 % and 0.1% respectively. (Jensen et al. 2011). Zinc finger nucleases were also used for the somatic and germ line gene disruption studies in zebra fish. Zebra fish is the vertebrate model for understanding the developments and diseases in the vertebrates. Facile forward genetics, rapid development and transparent embryos make zebra fish, the best model for vertebrate studies. Reverse genetic approaches using the mRNA knockdown strategies, TILLING point mutations and the gene targeting methods were not successful in zebra fish. The resistance of the genome at the metazoa state for targeted gene alteration was the main drawback in this technique. It was overcome by the use of Zinc Finger Nucleases. (Doyon et al. 2008). ZFNs are able to induce targeted insertions and deletions by double stranded breaks. In this particular study golden zebra fish and no tail zebra fish were targeted. The ZFN – encoding mRNA was inserted into the 1-cell embryo stage. This method produced greater percentage of mutation than any other method. These mutations created functional loss at the phenotype level. Thus it is a very promising approach. Zinc finger proteins against the gol and ntl were designed. For the wild type studies ZFNs containing FokI was used. The disrupted ntl genes were found at the adult stages with an average of 20% mutations. Thus it is proved from this study that ZFN mediated mutations can be performed at fertilized egg level in zebra fish, the model of vertebrate animals. (Doyon et al. 2008). Mammalian cells use many genetic repair pathways to maintain the genetic stability of the genomes. For understanding these pathways, further optimization of the present targeted gene therapy. Zinc finger nucleases can be effectively used for the targeted gene therapy both in the episomal ad chromatic loci. The targeted specificity is present at the amino - terminal end of the Zinc fingers. The zinc fingers of these domains are engineered according to the specific target by the modification o the amino acid composition such that high specific target binding Zinc fingers are produced. These Zinc fingers are made to target the specific sites in the opposite direction and have the ability to recognize 18 – 24 base pairs. (Jensen et al. 2011). As targeted binding is much essential for the recognition of the genes in the mammalian genome, this technique is found to be very promising. These ZFNs when inserted into the cells, they will cause permanent mutation in the genes. This type of genetic correction is much useful for the mammalians. The somatic genetic correction efficiency was found to be around 18-30% and has the reproducing ability of the results. Using these technique somatic as well as hematopoietic stem cells was successfully targeted. (Jensen et al. 2011). In the mammalian cells, it was found that if a double stranded break is formed by any method, then gene targeting will be of several magnitudes high. In addition to the simulation of the gene targeting by homologous recombination’s, gene site specific mutations can be successfully created by the zinc finger nucleases by non-homologous end – joining ( NHEJ) technique. (Jensen et al. 2011). A work was carried out on Drosophila melanogaster’s eye color loci location using the ZFN to recognize them. It was found that they have created very small insertions and deletions in the yellow gene. This was done by the mutagenic repair of by NHEJ. This process has a great potential in creating experimental cell lines particularly for the human lines, as this cannot be created with ease. The mammalian cells use four different types of molecular pathways for the repair. They are 1. Mismatch Repair. 2. Nucleotide Excision repair 3. Base excision repair and 4. Homology-directed repair and Non-Homologous End Joining (NHEJ). (Jensen et al. 2011). Zinc finger nucleases use the homology directed repair in the mismatch repair and the nucleotide excision repair. ZFNs are found to produce high frequencies of off target cleavages because of the pairing of the ZFN. This is also due to the homo dimerization of the ZFN – Fokl domains. (Jensen et al. 2011). This off- target cleavage can be minimized by the addition of positive or negative charges to the individual ZFN construction by creating a electrostatic repulsion among the bases. These when used in the experiments found to show 40% reduction in the off target cleavages. Another option is arresting the cells at the G2/M phase of the cells. The half life of the Zinc finger nucleases can be reduced by the addition of N terminal arginine. This resulted in reduced genotoxicity of the cell without affecting the efficiency of the cells. These ZFNs are constructed using the modern modular assembly method. Among the publically available platform, the OPEN is found to provide all the details required for the development. Genetic modification and the improvement of the crop varieties are the important topics in the field of plant biotechnology. Large scale genome sequence analysis and the EST sequencing projects are going on and the need for the storage and identification of the information are increasing every day. To establish targeted modifications, the genome of the plants must be searched up. Hence, came the need of targeted mutagenesis for the study of the plant gene functions and genetic modifications of the important crops. The genetically modified crops can also be developed using this technique. The Agrobacterium mediated gene transfer is practiced for the insertion of the desired gene. This resulted in the production of very low efficiency GT. (Lloyd et al. 2005). The NHEJ based targeted technology was developed using the Arabidopsis thaliana plant. Here the subsequent repairs of the DSBs found to produce many deletions and insertions at the joining site. Zinc Finger engineering can be done in three ways. The methods are 1. Context Dependent Assembly (CoDA). 2. Oligomerized Pool Engineering (OPEN). 3. Modular Assembly. The protocols for all these above methods are present at the Zinc Finger Consortium. Some of the software’s that are available for Zinc Finger engineering are ZiFit, ZiFDB and ZFN genome. (zincfingers.org) ZiFiT - this is web accessible software that is used for the design of the engineered zinc finger arrays. This software identifies the potential target sites that ZFN can engineer. ZiFiT has additional support from the CoDAS and OPEN. ZiFDB – this is a web accessible database for Zinc finger arrays and Zinc finger engineered arrays. ZiFDB organizes the information about the zinc finger modules and the engineered zinc finger modules. Eaxh zinc finger motif recognizes a continuous 3bp DNA sequence . the modularity of the structure and the DNA binding ability makes the zinc finger motif a good scaffold for the engineered DNA binding proteins. ZFNgenome – a genome wide resource or identifying zinc finger in various organisms. This database contains the ZFN target sites of seven organisms. Homosapiens ( humans) , Danio rerio ( Zebra fish) , Drosophila melanogaster ( fruit fly), Caenorhabditis elegans (nematode) , Arabidopsis thaliana ( thale cress) , Chalmydomonas reinhardtii ( green algae) and Saccharomyces cerevisiae ( budding yeast ). (zincfingers.org). This is the first compendium of ZFN targeted sites sequenced and stored. The identification of the ZFN target site is going on in further pants and animals. Rattus norvegicus (brown rat), Tribolium castaneum (red flour beetle) and Mus musculus (mouse) are the three animal species that are being worked up with along another three plant species such as Oryza sativa(rice), Zea mays (maize) and glycine max ( soy bean). The main aim of ZFN genome database is to create a user friendly interface between the data’s submitted and the database. Thus they have an established genome browser containing the information about the annotations and the other resources that are found in the model organisms. This also enables to design software tools and experimental protocols for the Zinc finger consortium. (zincfingers.org) Figure 4: Zinc finger architecture. (Reyon et al. 2011). Oligomerized Pool Engineering (OPEN) is the techniques used for engineering Zinc finger nucleases. This is a very robust and publically available technique. OPEN was able to create functionally efficient ZFNs in the plants, humans and zebra fish. This is a selection- based method. A pre constructed pool of Zinc Finger Arrays( ZFA) were already placed in the data base and the process of selecting the right ZFA with high affinity and specificity for the target sequence is done using OPEN. (Reyon et al. 2011). In vitro sucession rates are higher for OPEN technique than the other traditional methods of modular assembly. 16 possible GNN triplets as well as TNN triplets are possible in the modules of OPEN reagents. (Reyon et al. 2011). OPEN ZFN is used in the ZFN genome to display the potential target sites. ZFN genome identifies the “pre defined” ZFN targets for any gene or region of interest. In this ZFN genome, the ZiFiT tool and the NCBI BLAST links are provided to enable further comparison of the results. (Reyon et al. 2011). The potential target sites are represented by the color codes. Blue represents the potential targets that can be acted in vivo; purple indicates less active and red color indicates the inactive sites. This site has some trouble shooting parts like preprocessing of the data and analyzing the quality of the input by quality assurance tests like selecting 5 kb segments of the genomic sequence and analyzing the each chromosome and rescanning these databases using the ZiFiT software. (Reyon et al. 2011). Thus Zinc Finger Nucleases are developed mainly to increase the gene targeting and expression percentage in the mammalian and the plant systems and to introduce the mutations at the target site based on imperfect repair using ZFNs. The use of zinc finger nucleases seems to be a very promising method for site directed mutagenesis. The engineering of ZFNs and the binding of ZFNs with the target and the role of the Fokl are some of the key points in targeted gene therapy. Zinc finger nucleases are successfully used both in vivo and in vitro. But yet the safety issues concerning the genotoxicity of ZFN require high through put screening. (Jensen et al. 2011). References: Doyon, Y., McCammon, JM., Miller, JC., Faraji, F.,Ngo, C., Katibah, GE., Amora, R., Hocking, TD., Zhang, L., Rebar, EJ., Gregory, PD., Urnov, FD., and Amache, SL. 2008, “Heritable Targeted Gene Disruption in Zebrafish Using Designed Zinc Finger Nucleases”, Nature Biotechnology, Vol.26, No.6, 702 – 708. Herzog, RW., and Zolotukhin, S. 2010, A guide to human gene therapy, World Scientific. Jensen, NM., Dalsgaard, T., Jakobsen, M., Nielsen, RR., Sorensen, CB., Bolund, L., and Jensen TG. 2011, “An update on targeted gene repair in mammalian cells: methods and mechanisms”, Journal of Biomedical Science, Vol.18, No.1, 10. Osakabe, K., Osakabe, Y., and Toki, S 2010, “site directed mutagenesis in Arabidopsis using custom designed zinc finger nucleases”, Proceedings of the National Academy of Sciences of the United States of America, Vol.107, No .26, 12034 – 12039. Singleton, P 2010, Dictionary of DNA and Genome Technology, Wiley and Sons. Reyon, D.,Kirkpatrick, JR., Sander, JD., Zhang, F., Voytas, DF., Joung, JK., Dobbs, D., and Coffman, CR. 2011, “ZFNGenome: A comprehensive resource for locating zinc finger nuclease target sites in model organisms”, BMC genomics, Vol.12, No.83. Zincfingers.org, zinc finger consortium- Scientific Background, http://www.zincfingers.org/scientific-background.htm, Last accessed on March 20, 2011. Read More