Project Description

Role of modulation of TLR unmethylated cPG island

Due to the specificity of toll-like receptors -and other innate immune receptors- they cannot change with evolution. Those receptors recognize the threat associated molecules such as pathogens or cell stress and are extremely specific to them so they can never be stimulated by self-molecules that are normally expressed in the physiological conditions. Some features in the pathogens are conserved to some extent and they include; cell-surface lipopolysaccharide (LPS), lipopeptides, lipoproteins and lipoarabinomannan; which are proteins such as flagellin from bacterial flagella, viral double-stranded RNA or the unmethylated CpG islands of viral and bacterial DNA, and also of the CpG islands which can be found in the eukaryotic DNA. The stereotypic inflammatory response caused by the activation of toll-like receptors raised the theories that endogenous activators of toll-like receptors may participate in the autoimmune diseases. The following is how different antigens could stimulate the TLRs; The CpG islands stimulate TLR9. The pattern recognition receptor (PRR) TLR 9 which is expressed only in B cells and pasmactoid dendritic cells in humans recognizes the CpG PAMP, which is only expressed in B cells and plasmacytoid dendritic cells (pDCs) in humans. Variable sequences were found to stimulate TLR9 with variations in the number and location of CpG dimers and the precise base sequences flanking the CpG dimers; this let us find out about 5 unofficial categories of CpG ODN based on their secondary structures, sequences and their effect on human peripheral blood mononuclear cells (PBMCs). The five categories are Class A (Type D), Class B (Type K), Class C, Class P, and Class S. It is very important to realize that during the discovery process, the "categories" were defined only when it has become evident that ODN with certain characteristics could elicit specific responses. Role of crispr in targeted methylation Correlations between epigenetic marks and gene expression pattern form a strong base for the epigenetic studies so far. Precise epigenetic modifications and gene regulation are going on in the meanwhile due to technologies that have been specially developed for epigenome editing. Epigenetic modification is of a reversible nature, like DNA methylation, that exploited in the field of cancer therapy, yet this was done non-selectively by epigenetic inhibitors. A novel approach to selectively alter gene expression trough editing at a specific loci. CRISPR-Cas9-based tool for specific DNA methylation consisting of deactivated Cas9 (dCas9) nuclease and catalytic domain of the DNA methyltransferase DNMT3A targeted by co–expression of a guide RNA to any 20 bp DNA sequence followed by the NGG trinucleotide.targeted CpG methylation in a ?35 bp wide region by the fusion protein. In order to enable methylation of a larger part of the promoter, guide RNAs are made multiple to target dCas9-DNMT3A multiple adjacent sites. According to the literature it was found that e.coli is in close relation to CRC; so we have searched the e.coli genome looking for motifs to use in our project using " motif finder tool". We found that our motif occurs 20 times in the e.coli genes. Among those genes was colibactin gene cluster. According to (Christine et. al ) there is a close relationship between CRC and colibactin gene cluster as they are responsible for DNA mutations. The gene cluster includes 18 genes, we found that clbj gene recorded the most frequent motif number, with a score of three times within the gene. This enlightened us with an idea, which is to make sure that we down-regulate the colibactin gene using the same methylation way that we are going to use to regulate the immune system.