Gene disruption and ME Limitatations.pptx

Gene disruption Gene knockouts (also known as gene deletion or gene inactivation) are a widely used genetic engineering technique that involves the targeted removal or inactivation of a specific gene within an organism's genome. Main types of gene knockouts 1. Complete: A complete gene knockout...

Gene disruption Gene knockouts (also known as gene deletion or gene inactivation) are a widely used genetic engineering technique that involves the targeted removal or inactivation of a specific gene within an organism's genome. Main types of gene knockouts 1. Complete: A complete gene knockout permanently inactivates the gene 2. Conditional: A conditional gene knockout allows for the gene to be turned off and on at specific times or in specific tissues. Conditional knockouts are particularly useful for studying developmental processes and for understanding the role of a gene in specific cell types or tissues. Gene knockouts have been widely used in many different organisms, including bacteria, yeast, fruit flies, zebrafish, and mice. In mice, gene knockouts are commonly used to study the function of specific genes in development, physiology, and cancer research. The use of gene knockouts in mouse models has been particularly valuable in the study of human diseases. For example, gene knockouts in mice have been used to study the role of specific genes in cancer, neurological disorders, immune disorders, and metabolic disorders. Multiple knockouts Knocking out two genes simultaneously in an organism is known as a double knockout (DKO). Similarly the terms triple knockout (TKO) and quadruple knockouts (QKO) are used to describe three or four knocked out genes, respectively. However, one needs to distinguish between heterozygous and homozygous KOs. In the former, only one of two gene copies (alleles) is knocked out, in the latter both are knocked out. Applications of Gene disruption Knockouts are primarily used to understand the role of a specific gene or DNA region by comparing the knockout organism to a wildtype with a similar genetic background. Knockout organisms are also used as screening tools in the development of drugs, to target specific biological processes or deficiencies by using a specific knockout, or to understand the mechanism of action of a drug by using a library of knockout organisms spanning the entire genome, such as in Saccharomyces cerevisiae. Methods of Gene disruption 1. Gene knockout by mutation 2. Gene silencing 3. DNA Shuffling - Reassortment during homologous recombination 4. Site-specific nucleases (Zinc-finger nuclease (TALEN) 5. CRISPR/Cas9 nuclease, Transcription activator-like effector 1. Gene knockout by mutation 1. Gene knockout by mutation Mutagenesis 2. Gene silencing • For gene knockout investigations, RNA interference (RNAi), a more recent method, also known as gene silencing, has gained popularity. • In RNA interference (RNAi), messenger RNA for a particular gene is inactivated using small interfering RNA (siRNA) or short hairpin RNA (shRNA). • This effectively stops the gene from being expressed. • Oncogenes like Bcl-2 and p53, as well as genes linked to neurological disease, genetic disorders, and viral infections, have all been targeted for gene silencing utilizing RNA interference (RNAi). 2. Gene silencing “RNA interference is the process in which the gene expression is inhibited by RNA molecules by neutralizing the targeted mRNA molecules.” What is RNA Interference? RNA interference is an evolutionarily conserved mechanism triggered by doublestranded RNA that uses the gene’s own DNA sequence to turn it off. This process is known as gene silencing. It is a gene regulatory mechanism that limits the level of transcript in two ways: •Suppressing transcription (Transcriptional gene silencing) •Degrading the RNA produced (post-transcriptional gene silencing) The process was discovered by two American scientists Andrew Z. Fire and Craig C. Mello in the cells of the nematode worm Caenorhabditis elegans. They introduced short segments of double-stranded RNA into the cells of C.elegans and inhibited the expression of certain genes. Mechanism of RNAi 3. DNA Shuffling - Reassortment during homologous recombination Homologous recombination is the exchange of genes between two DNA strands that include extensive regions of base sequences that are identical to one another. In eukaryotic species, bacteria, and some viruses, homologous recombination happens spontaneously and is a useful tool in genetically engineered. Homologous recombination, which takes place during meiosis in eukaryotes, is essential for the repair of double-stranded DNA breaks and promotes genetic variation by allowing the movement of genetic information during chromosomal crossing. This method involves inserting foreign DNA into a cell that has a sequence similar to the target gene while being flanked by sequences that are the same upstream and downstream of the target gene. The target gene's DNA is substituted with the foreign DNA sequence during replication when the cell detects the similar flanking regions as homologues. The target gene is "knocked out" by the exchange. By using this technique to target particular alleles in embryonic stem cells in mice, it is possible to create knockout mice. With the aid of gene targeting, numerous mouse genes have been shut down, leading to the creation of hundreds of distinct mouse models of various human diseases, such as cancer, diabetes, cardiovascular diseases, and neurological disorders. Mario Capecchi, Sir Martin J. Evans, and Oliver Smithies performed groundbreaking research on homologous recombination in mouse stem cells, and they shared the 2007 Nobel Prize in Physiology or Medicine for their findings. 4. Site-specific nucleases {Zinc-finger nuclease, Transcription activator-like effector nuclease (TALEN)} There are currently three methods in use that involve precisely targeting a DNA sequence in order to introduce a double-stranded break. Once this occurs, the cell's repair mechanisms will attempt to repair this double stranded break, often through non-homologous end joining (NHEJ), which involves directly ligating the two cut ends together. This may be done imperfectly, therefore sometimes causing insertions or deletions of base pairs, which cause frameshift mutations. These mutations can render the gene in which they occur nonfunctional, thus creating a knockout of that gene. This process is more efficient than homologous recombination, and therefore can be more easily used to create biallelic knockouts. Zinc-finger nuclease Zinc-finger nucleases consist of DNA binding domains that can precisely target a DNA sequence. Each zinc-finger can recognize codons of a desired DNA sequence, and therefore can be modularly assembled to bind to a particular sequence. These binding domains are coupled with a restriction endonuclease that can cause a double stranded break (DSB) in the DNA. Repair processes may introduce mutations that destroy functionality of the gene. Transcription activator-like effector nuclease (TALEN) Transcription activator-like effector nucleases (TALENs) also contain a DNA binding domain and a nuclease that can cleave DNA. The DNA binding region consists of amino acid repeats that each recognize a single base pair of the desired targeted DNA sequence. If this cleavage is targeted to a gene coding region, and NHEJ-mediated repair introduces insertions and deletions, a frameshift mutation often results, thus disrupting function of the gene. 5. CRISPR/Cas9 CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a genetic engineering technique that allows for precise editing of the genome. One application of CRISPR is gene knockout, which involves disabling or "knocking out" a specific gene in an organism. The process of gene knockout with CRISPR involves three main steps: 1. Designing a guide RNA (gRNA) that targets a specific location in the genome, 2. Delivering the gRNA and a Cas9 enzyme (which acts as a molecular scissors) to the target cell 3. Allowing the cell to repair the cut in the DNA. When the cell repairs the cut, it can either join the cut ends back together, resulting in a non-functional gene, or introduce a mutation that disrupts the gene's function. This technique can be used in a variety of organisms, including bacteria, yeast, plants, and animals, and it allows scientists to study the function of specific genes by observing the effects of their absence. CRISPRbased gene knockout is a powerful tool for understanding the genetic basis of disease and for developing new therapies. It is important to note that CRISPR-based gene knockout, like any genetic engineering technique, has the potential to produce unintended or harmful effects on the organism, so it should be used with caution. The coupled Cas9 will cause a double stranded break in the DNA. Following the same principle as zinc-fingers and TALENs, the attempts to repair these double stranded breaks often result in frameshift mutations that result in an nonfunctional gene. 5. CRISPR/Cas9 Although recently developed programmable editing tools, such as zinc finger nucleases and transcription activator-like effector nucleases, have significantly improved the capacity for precise genome modification, these techniques have limitations. CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 technology represents a significant improvement over these other next-generation genome editing tools, reaching a new level of targeting, efficiency, and ease of use. The CRISPR/Cas9 system allows for site-specific genomic targeting in virtually any organism. The principle of CRISPR/Cas9-mediated gene disruption. A single guide RNA (sgRNA), consisting of a crRNA sequence that is specific to the DNA target, and a tracrRNA sequence that interacts with the Cas9 protein (1), binds to a recombinant form of Cas9 protein that has DNA endonuclease activity (2). The resulting complex will cause target-specific double-stranded DNA cleavage (3). The cleavage site will be repaired by the nonhomologous end joining (NHEJ) DNA repair pathway, (4). An error-prone process that may result in insertions/deletions (INDELs) that may disrupt gene function (4). Advantages and Limitations of Gene disruption Advantages: It allows researchers to study the function of a specific gene in vivo, and to understand the role of the gene in normal development and physiology as well as in the pathology of diseases. By studying the phenotype of the organism with the knocked out gene, researchers can gain insights into the biological processes that the gene is involved in. Limitations: For example, the loss of a single gene may not fully mimic the effects of a genetic disorder, and the knockouts may have unintended effects on other genes or pathways. Additionally, gene knockouts are not always a good model for human disease as the mouse genome is not identical to the human genome, and mouse physiology is different from human physiology. Limitations and Bottlenecks in Metabolic Engineering Limitations and Bottlenecks in Metabolic Engineering Limitations and Bottlenecks in Metabolic Engineering  One of the major challenges of metabolic engineering is to identify optimal organisms and to determine targets for manipulations in individual genes, whole pathways, or even in transcriptional and translational control elements.  Ethical concerns and regulations for GMOs.  Linear model: since most of the biological systems are highly non-linear and complex.  No kinetic or regulatory terms are explicitly used..  During metabolic flux analysis, a steady state model consideration, not dynamic model.