The Structure of DNA & the Quest for Introducing the Specific Changes in the DNA

Published: 2021-09-29 19:25:11
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The quest for introducing the specific changes in the DNA sequence began when DNA was discovered. DNA is the building block of all life in every organism. DNA is made up of two polynucleotide strands where the two strands spiral together to form a double helix structure which is held together by hydrogen bonds between the bases. Each base can only join with one particular base which is called complementary base pairing. The base adenine always pairs with the base thymine: the base guanine always pairs with the base cytosine. There are 64 possible base triplet combinations. The order in which the amino acids are connected together determines the structure.
The primary structure determines the function of a protein and so all the protein properties. The study of genetics has progressed over time, and new discoveries are made every day. Gregor Mendel is hailed as the founding father of modern genetics. His discoveries were dismissed because his ideas conflicted with the already existing ideas of inheritance. He initially examined pea plants to study the patterns in which traits are handed down from parents to offspring and observed that the pea plant itself inherited traits . Muller discovered that genes are artificially mutable . Mutations are changes to the base sequence of DNA or RNA. The order of DNA bases in a gene determines the order of amino acids in a particular protein. Mutations can be either natural, beneficial or harmful. if a mutation occurs in a gene, the primary structure of the protein it codes for could be altered. Some amino acids are coded for by more than one triplet. The mutation produces a triplet that codes for a different amino acid, but it does not change as a neutral effect does not affect an organism. However, if a protein function is affected it can be beneficial or harmful effect on the whole organism. A mutation can be beneficial because it can have an advantage on the organism where it can increase its chances of survivals. Mutations that are beneficial to the organism are passed on to future generations by the process of natural selection. Though a mutation can be harmful because it can confer a disadvantage on the organism, which can decrease its chance on survival. There are five types of mutations.
Substitution is where one base is swapped for another. Deletion is where one base is removed, and insertion is where one base is added. Duplication is where one or more bases are repeated whilst inversion is where a sequence of bases are reversed. All these mutations can disrupt the gene and cause it to produce a defective protein. Sickle cell disease and WHIM syndrome are both examples of genetic diseases caused by simple substitution mutations whilst Huntington’s disease result from insertions or deletions in DNA. Humans constantly suffer DNA damage it occurs when we are exposed to electromagnetic radiation. Scientists are able to fix the double-strand breaks because the cells have evolved a response and a repair system to fix the break. Scientists are able to fix these mutations but must alter the genotype. It is possible to either repair the faulty gene, replace the faulty gene or add a normal gene leaving a faulty gene in place. In the 1960s, a revolution was made involving recombinant DNA.
Recombinant DNA is DNA involving inserting genes from organism into another. This is known as genetic engineering. Vectors are carriers which are used to transfer the required gene into new cells. It must be able to target the cells, be able to incorporate the desired gene into the host’s DNA and not create any side effects. Bacteria is used as a vector because only one copy of every gene is likely to be expressed and it is easier to introduce the gene into organism. Plasmids have a simple growth requirement and can reproduce at their own rate within cells. Genetic engineering is to obtain the wanted gene, clone the gene to obtain many copies using Polymerase Chain Reaction (PCR), insert a copy of the gene into a vector, the vector inserts the gene into the cell and the cell is identified and cloned. The process of PCR replicates DNA (amplifies it) to make millions of identical copies.
Traditional DNA profiling requires 1 microgram of DNA (equivalent to the DNA content of around 10,000 human cells . Viruses can be used as vector too because the gene is easily inserted and targets specific cells. The gene is not always incorporated into host DNA; it may evoke immune response and the cells may be destroyed.Gene editing is the insertion, deletion, or replacement of DNA at a specific site in the genome of an organism or cell . Gene editing has happened naturally throughout time. Researchers were studying a rare hereditary disease known as WHIM syndrome and had come across a patient, Kim, whose condition they simply could not explain.
WHIM is a painful and deadly immunodeficiency disease, and this leaves patients susceptible to infection, which causes uncontrollable warts that cover the patient’s skin and can eventually progress to cancer. She had been diagnosed with the disorder, but it disappeared from the immune system. She had been diagnosed with WHIM since birth and had been hospitalised multiple times. She had classic signs of the disease, but the scientists were surprised to discover that she had been symptom-free for over twenty years without any medical treatment, Kim had been cured. Scientists conducted experiments to understand how Kim had suddenly recovered from her life-threatening illness . The mutated gene responsible for Kim’s condition was still present in cells taken from skin cells but in her blood cells, the mutation was absent. Analysing the DNA taken from Kim’s blood cells in more detail, the scientists found the whole of the mutated gene, called CXCR4 on chromosome 2.
After running a series of tests, scientists concluded that a single cell in her body must have experienced an uncommon and usually catastrophic event called chromothripsis is in which a chromosome suddenly shatters and is then repaired, and this allowed the cell to be free of the gene causing WHIM syndrome. Scientists determined that the cell must have been a hematopoietic stem cell, a type of stem cell from which every kind of blood cell in the body originates and that has unlimited potential to create a new cell. That cell had passed along its rearranged chromosome to all its daughter cells, eventually repopulating Kim’s entire immune system with healthy new white blood cells that were free of the CXCR4 mutation. Nucleases are enzymes that cut apart nucleic acids; some cut RNA, others cut DNA. Endonucleases cut RNA or DNA somewhere within the strands, as opposed to exonucleases, which cut exclusively from the ends.
The I-Scel endonuclease was one of the most specific endonucleases known at the time, requiring a perfect match of eighteen consecutive DNA bases for it to cut a given segment. These next generations gene editing systems had three critical requirements. They had to recognise a specific DNA sequence, to be able to cut that DNA sequence, and to be easily reprogrammable to target and cut different DNA sequences. The first two criteria were necessary for generating a double-strand break, and the third was necessary for the tool to be broadly useful. I-Scel excelled at the first two but failed miserably at the third. To build a programmable DNA cutting system, bioengineers figured they would need to either retool I-Scel to target and cut new kinds of sequence or find a completely new nuclease enzyme that had already evolved to cut different DNA sequences. Scientist’s failed to redesign the I-Scel endonuclease because the majority of these enzymes recognised sequences that were only a few bases long, too short to be useful.
In 1996, Professor Srinivasan Chandrasegaran of Johns Hopkins University, realised that instead of building nucleases from scratch, finding new ones in nature, or redesigning I-SceI, he could take approach by selecting fragments of proteins that existed naturally and combining them . Nucleases would be able to recognize and cut a specific sequence of DNA. Chandrasegaran selected a module from a bacterial nuclease called FokI that could introduce breaks in DNA but had no particular sequence preference. To do the targeting, he harnessed naturally occurring proteins called zinc finger proteins. These zinc finger proteins were built of multiple repeated segments, with each segment recognizing a specific DNA sequence. Zinc finger nuclease (ZFN) are transcription factors; each finger module recognises three to four bases of sequence and with a combination of modules can target any DNA sequence.
The efficiency is pretty low even though it is pretty high specificity and time consuming. It is limited to one edit at a time. However, ZFNs were never used outside of the laboratories because ZFN is pretty expensive and it was difficult to synthesize it in the lab. The first version was discovered in 2009 and came from studies of novel types of proteins found in Xanthomonas, a pathogenic plant-infecting bacterium. These were called transcription activator–like effectors, or TALENs, these proteins are similar to zinc finger proteins . They are built of multiple repeating segments in which each segment recognises a given area of DNA. Each segment in TALENs recognises just a single base of DNA. Soon after this was discovered, three laboratories fused TALEs to the same DNA-cutting module used in ZFNS and created TALE nucleases, or TALENs. TALENs were effective inside cells, and after researchers made improvements in their design and construction. TALENs were easier to build and implement than ZFNs. TALENs have very few off-target effects due to each module targeting one nucleotide but was still pretty low in efficiency. It still allowed more specific targeting of genes.
CRISPR was hailed by Science magazine as their 2015 breakthrough of the year. Jon Moore, chief officer of Horizon Discovery, says “the targets we’re finding with CRISPR-Cas9 are going to guide the drugs coming out in the 2020s . A world without disease seems far-fetched but eventually could become reality. Gene editing is a technology able to provide a solution to human health challenges. CRISPR -Cas9 is a gene editing technology designed to locate and carefully edit DNA sequences of organisms . CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is the system designed to locate the DNA sequence. CRISPR was discovered in 1980 but it took a while for scientists to realise the ability to edit the DNA organisms but were able to discover CRISPR taken from bacterial cells.
Phages are built out of a protein exterior, called a capsid, inside of which genetic material is packaged. Phage can effectively deliver the genetic material into bacterial cells, where it can multiply and spread. A single phage can wipe out an entire bacterial population in a matter of hours. He had discovered viruses lurking in the acidic waters of hot springs, that were infectious despite temperatures that rose to over 170 degrees Fahrenheit. These viruses were known to infect single-celled microorganisms known as archaea. Cas 9 is the restriction enzyme used to cut into the DNA and edit it. The guide RNA (Grna) directs the Cas 9 to the target site. It binds to the specific location in the DNA sequence. It activates the enzyme and cuts the DNA sequence at a specific site. As all these technologies are nucleases they carry a risk of undetected off-target effects .
DNA repair mechanisms activate to repair the damage of a double stranded break The non-homologous end joining (NHEJ) pathway is useful to disrupt the function of a gene. It will bind the double stranded break back together. Homology directed repair (HDR) pathway is useful once a targeted gene is replaced with a different sequence. The DNA template is used to repair the broken sequence via HDR. However, these mechanisms often leads to mutations (deletion, insertion and substitution), which may inactivate the entire gene. Mutations are desirable because sometimes it is good to inactivate some genes. However, if the CRISPR-Cas9 happens to cut vital genes such as tumour suppressor genes which it is not supposed to cut, it may inactivate these genes and result in cancer.
Researchers are searching to minimize the off-target effects. Another reason is victims of the past. Many people have died or become disabled from government, pharmaceutical or industrial company products or harmful decisions which were flawed. No biological process is ever 100% precise. There will always be the risk changing the nucleotides that you are targeting. Recently, Nature Methods retracted the paper, and declared that there was insufficient data to support the claim of unexpected off-target effects due to CRISPR . Off target effects been a major concern about moving CRISPR from animal experiments to people. the CRISPR-Cas9 system will become the system of choice, making all others obsolete and results suggest that they are the most efficient with low mutation rates. Jennifer Doudna and the University of Berkeley are fighting against MIT for the patent. Currently, MIT holds the patent. Both MIT and Berkeley have pivotal roles in the ongoing development of this new technology . There are rapid investments in the start-up firms.

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