The science of hereditary has changed hugely since the first experiments done by Mendel in his pea garden. What he discovered then was the existence and fundamental importance of the gene and its function in inheritance, predicting the patterns of behaviour during the formation of gametes. Although there was no knowledge at the time of chromosomes and the occurrence of meiosis, Mendel had provided some order and instruction to biology that not only started to explain the passing on of information between parent and offspring but was an idea from which Darwin’s theory of evolution could corroborate with and support. Mendel’s research was relatively narrow, looking into the basic units of heredity and their transmittance to the next generation. Darwin looked at an altered version of this question, but on the widest possible scale – how could this form and change an organism’s features over thousands of years? This would later lead on to the understanding of the genotype and its effect on the phenotype of an organism, a concept that is the basis of gene editing and its uses. (Mukurjee, 2018)
Along with these fundamental scientific findings, the beginnings of the ideas behind the tools needed for genetic editing were the studies done by scientist Paul Berg on the SV40 virus. The virus had a simple structure of a set of seven genes with an outer layer of protein, and so was very small and compact, and therefore east to work with. Berg found that instead of infecting and then killing the host cell, the SV40 could insert its own DNA into the host cell’s chromosome and therefore kept being reproduced. Berg saw that if he could find a way to unclasp the loop structure of the virus and insert a ‘foreign’ DNA sequence, the viral genome could be delivered into human cells, altering its hereditary information – these were the beginnings of the CRISPR technology. (Challenges in Gene Therapy, 2017)
Bacterial enzymes were looked into to cut open the loop of genes, then to insert the foreign DNA into the viral genome. These enzymes are used in bacteria as protection against viruses as once the viral DNA is cut, it is rendered harmless to the host cell.
After these discoveries, Berg and fellow biochemist Janet Mertz worked on joining different pieces of DNA together, naming it ‘recombinant DNA’. They mixed the cutting enzyme EcoR1 and cut plasmid DNA molecules and re-joined them to create recombinant plasmid molecules. This concept was transferred to the technology behind genetic editing – the beginning of CRISPR.
How the CRISPR technology works
CRISPR-Cas9 is new technology that allows scientists and medical researchers to edit parts of the genome by altering sections of the DNA sequence, so the resulting protein is either no longer made or a non-functioning version is formed. It is currently the simplest but most precise method of gene editing, and although it is not a completely safe mechanism, with scientists still facing some major implications during its use, the main workings of the technology have been finalised.
It consists of two main parts: the enzyme, Cas-9, and the length of guide RNA. The enzyme attached acts as the ‘biological scissors’ and cuts out the two strands of mutated or faulty DNA to be removed or replaced. The guide RNA molecule is a specifically designed length of RNA of around 20 base codes that guides the enzymes to the correct part of the DNA to be removed, and then binds to it in the right place before the cutting. The base codes have to be complementary to those of the DNA which needs removing so only that specific site is cut out.
Once the faulty length of DNA has been removed, the correct version of the gene can be inserted in at the site of removal in order for the cell to work properly again. This mechanism makes up the basis of the principle of gene editing. (Challenges in Gene Therapy, 2017)
Uses of CRISPR Technology
The discovery and advancement of the CRISPR/Cas9 technology has propelled the research into personalised medicine and therapeutic treatments into a completely new area, opening up new and exciting possibilities for diagnostics and healthcare in a number of ways. The main two ways is it currently used is for drug discovery and for long-term regenerative medicine.
Researchers can use the CRISPR technology in drug discovery to identify new drug therapy targets through genome screening. They can establish the genes and therefore the proteins that cause a disease by intentionally activating or inhibiting certain genes, therefore showing the targets for the potential drugs.
Unlike gene editing tools used previously, the CRISPR system is much more accurate when switching off certain genes as there have been fewer undesired side effects from the process, ensuring that the researchers are provided with a better prediction of what will happen in clinical trials. Many diseases aren’t just monogenic and arise due to an error in an enzyme-catalysed pathway, or by interactions between different genes. CRISPR–Cas9 therefore cannot only be used to identify the causes of monogenic diseases, but also the combinations of the genes involved in those that aren’t, to offer a more effective approach to other treatments. (yourgenome.org, 2018)
It can also be used for long-term treatment of particular diseases. Although this gene editing technology has many potential uses, it is still a very new concept and in the experimental stages, and so its use is banned in most countries. The DNA changes made are passed down to future generations, potentially with unforeseen side-effects, and therefore its use on humans is not perceived as safe. There are two different types of genetic therapy – somatic cell gene therapy and germ-line gene therapy. Germ-line gene therapy is currently illegal to use in humans as the changes are passed down to future generations, and the technology is not yet fine-tuned enough for us to take this risk. However, there is a potential in it being used as a long-term treatment, and somatic cell gene therapy has already been used in some cases to cure those affected with fatal diseases. In the next section, I will explore these deeper.
Germ-line genetic therapy – Huntington’s
Huntington’s disease is an inherited disorder caused by the mutation of a single gene – the Huntingtin (HTT) gene – which results in parts of the brain becoming damaged over time. This is because the protein is needed by neurones in the brain and for the body’s development before birth.
When the huntingtin gene is transcribed, a length of mRNA is made that acts as the template for cells to make the HTT protein. If the gene is faulty, the primary structure of the huntingtin protein it produces has too many repetitions of the CAG nucleotide sequence, producing mutated Huntingtin (mHTT). This in turn appears to damage neurones in certain areas of the brain by increasing their decay rate.
It is thought that mutated huntingtin gene activates the enzyme caspase, an enzyme that is a catalyst for the process of the apoptosis (programmed cell death). This means that it is not the decrease in production of the normal HTT gene, but the increase in production of the mutated version that causes the disease. Because of this, using CRISPR technology would be an ideal solution to curing Huntington’s as it would remove the faulty gene and replace it with the normal HTT gene. This change would then be inherited and so would stop the continuation of the mutation, and therefore the disease.
As it is a dominant gene, the offspring of the affected individual will have a 50% chance of inheriting the disease. This means that genetic screening can be done at any point, including in the womb, to see if a child has a chance of inheriting the disease, in turn making it a possibility for doctors to use gene editing in the embryo to correct the mutation before the child is born. (nature.com, 2017)
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