Project Summary:  The term epigenetics was coined in the 1940s by Conrad Waddington and defines a mechanism that is above the gene level, changing the expression of identical genes so they can be expressed differentially in different cells (Waddington, 2012). Epigenetics now involve a wide range of alterations resulting in DNA methylation, histone post-translational modifications, and alterations in nucleosome positioning (Hwang et al., 2017). This study is intended for the validation of a genetic tool, the purpose of modifying the epigenome of the mitochondrial pol g gene as an approach for research in cancer, neuro-degradative conditions and DNA Mitochondrial POLG gene. Methylation of DNA mitochondrial POLG gene will be done by using a modified CRISPR-CAS9 system. This system has been modified to take advantage of the specificity of the guidance RNA and at the same time, by fusing the methylase gene to the CAS9, achieve methylation of the targeted gene. The aims of this study, therefore, includes: generation and validation of a genetic tool to manipulate the epigenetics of DAN mitochondrion POLG gene, to perform   the sequences of RNA will be cloned and validation specific sequences for the Polymerase gamma (POLG). Then, tissue culture transfection of mammalian cells with dCAS9-methylase plasmid system into mammalian cells, DNA isolation and assessment of the methylations by qPCR amplification and confirmation of altered gene expression via western blot. The information gathered from this study will be useful for the understanding the molecular mechanism of various disease like cancer.

1.1 Background to the investigation:1.1.1 Epigenetic:     The term epigenetics was coined in the 1940s by Conrad Waddington and defines a mechanism that is above the gene level, changing the expression of identical genes so they can be expressed differentially in different cells (Waddington, 2012). Epigenetics now encompasses a wide range of alterations resulting in DNA methylation, histone post-translational modifications, and alterations in nucleosome positioning (Hwang et al., 2017)  DNA methylation usually lead to gene repression and methylation of DNA is achieved by DNA methyltransferase (DNMTs). This enzyme catalyzes will transfer of a methyl group from S-adenosyl-l-methionine (SAM) to the 5-position carbon in cytosines within DNA to generate 5-methylcytosine (Hwang et al., 2017). The methyl transfer process has been recently proved to be reversible through a process known as demethylation (Miller and Sweatt, 2007).   Figure 1: the process of DNA Methylation and Demethylation (Hwang et al., 2017)
1.1.2 Post-translational histone modifications:  Histones have core domain and several unstructured tails on the amino end harboring sites for post-translational modifications such as methylation , acylation , ubiquitylation, sumoylation, phosphorylation and poly (ADP) ribosylation. These modifications are catalyzed by enzymes known as writers. Conversely, erasers are enzymes involved in the removal of the added marks (Hwang et al., 2017). Figure 2 shows the modification of histone 3 (H3). 
Figure 2: Histone (H3) modification on the histone tail.  1.2 Diseases and epigenetics:
1.2.1 Neuro-degradative conditions:   The modification of histone is not only associated with memory modulation but also with the initiation of neuron cells degradation and eventual death. This leads to memory loss in most neuro-degradative conditions. Studies have shown that ischaemia activates REST which the silences the target gene by recruiting HDAC1 and HDAC2 enzymes in neurons which then causes the onset of neuron death (Calderone et al., 2003).1.2.2 Cancer:  In previous studies, high DNA methylation has been observed in pluripotent cells correlating with low POLGA expression and low the DNA mitochondrial number. However, in terminally differentiated astrocytes, methylation of 2 exons in (POLGA) gene has been identified to decrease as the copy number of mtDNA increases. Surprisingly, cancer cells like the glioblastoma multiforme HSR-GBM1 cells which failed to differentiate were not able to demethylate exon 2 of POLGA (Lee et al., 2015). DNA methylation of exon 2 was significantly decreased which promoted mtDNA replication and cell differentiation. The findings of this study point out the function of DNA methylation in regulation of the copy number of mtDNA. This is an important determinant in cell differentiation. Likewise, it determines the depletion of cancer cells changes and the methylation at exon 2 of the POLGA and influences the tumorigenesis of those cells. Furthermore, the formation of tumors is associated with the reinstatement of the original copy number of mtDNA suggesting that cells have an inherent set point of the mtDNA copy number (Lee et al., 2016).
1.2.3 Mitochondrial POLG gene and Disease:   Mutations in mitochondrial POLG gene causes a wide range of autosomal disorders associated with mtDNA maintenance. Most of the mutations in POLG gene mutations are associated with recessive diseases. However, alterations in the expression of the gene in response to environmental factors induced methylations has been shown to increase chances of cancer, diabetes, and neuro degradative conditions. Different disease conditions occur in response to the methylation of POLG gene in mitochondria of different cells. For instance, the occurrence of neuro-degradative diseases like Alzheimer is due to methylation of POLG gene in neuron mitochondria. The current study is therefore aimed at utilizing the ability of molecular DNA editing tools to come up with a more effective approach to research in molecular biology to resolve the problem of cancer and other diseases associated with the epigenetic modification of different gene. The current study will focus on the methylation modification of mtDNA to understand the molecular mechanism of pathogenesis and progression of cancer among other diseases. Essential information derived from this study will provide insight into the progression of normal cells to a cancerous state. The study will apply the defective CRISPR-CAS9 system to target the methylation of the POLG gene and assess for the methylation by restriction digestion enzyme and qPCR.1.3 dCAS9 Methylase, a tool for specific gene methylation.  The development of genome manipulation tools has enhanced epigenetic studies in both prokaryotic and mammalian eukaryotic cells. Some of the tools widely employed in Molecular biology to study epigenetics include CRISPR-Cas9 system, Zinc Finger Nucleases (ZFN) and, transcription activator-like effector nucleases (TALENs). These genome editing tools have been widely applied in the targeted modification of any gene sequence (Doyle et al., 2013). The genome of a cell can be modified using these techniques in cell cultures or in the whole organism (Carlson et al., 2012). Most classical methods for genome analysis remain very essential in untangling key cell processes, however, the complexity associated with gene regulation has remained a mystery to these tools. In an effort to find a solution to this inadequacy of the classical genome analysis tools, alterations have been introduced in the genome editing tools to enable the  targeted modification histone tails like methylation, demethylation, acetylation, cytosine methylation and hydroxymethylation (Falahi et al., 2013).   Today, epigenome editing tools have made it possible to investigate the relevance of some site targeted modification of a specific genomic region. These epigenetic modifications are reversible in nature, for instance, the DNA methylation and demethylation. The reversible nature has been studied in cancer therapy to provide insight into the remodeling of the aberrant epigenetic nature of cancerous cells (Heerboth et al., 2014). The shortcoming of this approach is that it lacks selectivity and can only be applied at the global level and not to particular . Epigenetic editing has therefore played a central role in gene therapy, stem cell differentiation and drug design (Rius and Lyko, 2012).   The basic structure of epigenetic editing tool (CRISPR-Cas9 system) is made up of two components: a DNA-binding domain and nuclease domain. Epigenetic editing studies have widely applied the CRISPR-Cas9 because of its inherent ability to target multiple sites on the genome-guided by the guide RNA (gRNA). A phenomenon referred to as Multiplexing.(Cong et al., 2013). The functional domain of CRISPR-CAS9 system is a nuclease enzyme that performs the site-directed modifications on the genome. Posttranslational modification of histone tails and DNA molecule modification by methylation of cytosine are vital epigenetic mechanisms for gene regulation. DNA methylation is an important a more stable epigenetic mark which can cause long-term effect on gene expression making it a more preferred epigenetic mark for editing epigenome (Bernstein et al., 2015). The catalytic domain of DNA methyltransferase 3A (DNMT3a) presents a targeting tool for the targeted methylation of mitochondrial DNA polymerase gamma (DNA Pol g). A defective CRISPR-CAS9 (dCAS9-gRNA-Methylase) system can be applied to introduce methylation in mitochondrion (DNA POLG) gene.. This study will be applied a modified version of the editing system (Dcas9-DMNT3a) to optimize the methylation of (DNA POLG) gene.
1.4 Aims: The aims of the proposed research are:
1- This study is aimed at validating a genetic tool to manipulate the specific methylation of mitochondrial DNA polymerase (pol g) gene by an epigenetic regulation.2- The sequences of DNA will be cloned and validation specific sequences for the Polymerase gamma (POLG).
3- To perform tissue culture transfection of mammalian cells with dCAS9-methylase plasmid system.
4- To isolate DNA and assess the specific gene methylations by qPCR amplification.
5- To validate methylation studies by protein expression analysis (e.g. by western blot).

1.5 Experimental approach: 
1.5.1Vector constructionA lentiviral vector will be used for the transfection of the dCAS9-methylase as well as the specific guide RNA sequences. These will be designed for targeting potential methylation sites of the POLG gene. The representation of the dCAS9-methylase is shown in figure. 
 Figure 3: the construct of the dcas9 and methyltransferase in the vectorThe sequence of the POLG gene is identified from the genome by browsing the human genome sequence, using the “gene browser” tool from Figure 3 shows the genomic target of the CpGs islands in the human POLG gene. 
1.5.2 Adapting the CRISPR-CAS9 for methylation editing: generation of the dCAS9-Methylase:  The Cas9-D10A nuclease plasmid was modified with the insertion of the H840A mutation into the Cas9 gene as described elsewhere (deficient CAS9, dCAS9), (Chen et al., 2005). The DNMT3A catalytic domain (amino acids P602-V912) was amplified by PCR and then fused to the dCAS9 gene and the final structure of the construct is presented in figure 2 and figure 5. 
Figure 5: Schematic representation of the dCAS9-Methylase system and the plasmid structures of the guidance RNA plasmid (Adapted from Anon, 2017)  

1.5.3 Cell Transfection:   For the transfection of the cell, methods applied by Vojta et al. (2016) will be applied with slight modifications. Briefly, human embryonic kidney cell lines HEK293 will be maintained in Dulbecco’s Modified Eagle Medium (Lonza, Verviers, Belgium) supplemented with 10% heat-inactivated fetal bovine serum (Biosera, Ringmer, UK), 4 mM L–glutamine (Lonza), 100 U/ml penicillin and 100 g/ml streptomycin (Lonza). Cells will be incubated at 37◦C in a humidified 5% CO2–containing an atmosphere. The cells will then be seeded into a 24–well plate and transfected at 70–90% confluence using Lipofectamine 3000 (Life Technologies, Carlsbad, California, USA) following the protocols provided by the manufacturer. Transfection will be done using 100 ng of plasmids co–expressing dCas9-DNMT3A and a chimeric sgRNA, in triplicates.

1.5.4 Assessment of methylation:   The assessment of methylation will be performed by restriction enzyme digestion followed by qPCR. First, the genomic DNA of the cells will be collected using a Qiagen DNA isolation extraction kit (Qiagen), following manufacturer’s instructions. Once concentration is determined, equal amounts of DNA will be subjected to restriction digestion with the enzyme HpaII (New England Biolab) as shown in figure 6.  Figure 6: Strategy for assessment of CpG methylation by restriction enzymes HpaII
If methylations occur, HpaII should not cut the CpG islands and PCR can be performed. If methylation is absent, then HpaII will cut the site and the PCR will be negative. Then, using flanking primers to the targeted sequences will be used in a Quantitative PCR using SYBR Green (Qiagen) as fluorescence detector (See figure 7 for details). 
Figure 7: Strategy for identification methylation changes in the POLG promoter region b q PCR
 Several set of primers will be designed to flank potential methylation sites (CpG islands in the form of CCGG sequences).

1.5.5 Validation with Western blotPOLG expression will be analyzed by western blot determination. First, HEK293 cells will be co–transfected with 100 ng of a plasmid expressing the specific guidance RNA against POLG and the dCas9-DNMT3a. Cell lysates will be homogenized in RIPA buffer containing 150 mM NaCl, 1% NP–40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris–HCl pH 8 and 2x completed protease inhibitor cocktail (Roche, Basel, Switzerland). Then 25 µg of protein extract will be analyzed on a SDS-PAGE. Protein separated on the polyacrylamide gel electrophoresis will then be transferred onto nitrocellulose membrane. For the detection of POLG expression, the membrane will be blocked with 5% nonfat dry milk in 1x phosphate buffer saline: 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 2 mM KH2PO4, pH 7.4 for 30 min. Then, the membrane will be incubated with anti-POLG antibody 1:2000 (ab204448; Abcam) in 5% milk-PBS containing 0.2% Tween 20, overnight. Then the western will be developed by incubation with anti-rabbit HRP-conjugated secondary antibody 1:2000 (Dako) for 1 hour. Housekeeping GAPDH will be tested to confirm equal loading. The antibody-tagged protein bands will be visualized by an enhanced chemiluminescence (ECL) system (Pierce Biotechnology, Waltham, MA, USA) following the manufacturer’s instructions (Schneider et al., 2012).