Introduction
In 1987, scientists came across the concept of CRISPR when they noticed an unexpected chain of DNA sequences that were altered and modified, which was then recognised as a CRISPR in Escherichia coli in the course of gene analysis. Once CRISPR was discovered all bacterial species were distinguished by short sequences similar to other bacteria such as halophilic archaea. A few years later there was a slight relation between CRISPR and the protection of the body by the immune system that was recognised prior by other researchers.
A few years later there was a slight relation between CRISPR and the protection of the body by the immune system that was recognised prior by other researchers. The purpose of CRISPR was to be aligned to the encoded DNA CRISPR loci which are known as CRISPR-Cas. The characteristics of CRISPR loci is that the majority of the spacers in CRISPR repeats are the same as the fragments of the genes.
The CRISPR-Cas system has three phases: an adaptation that has small scale pieces of DNA similar to viruses and are joined to the CRISPR loci. (Makarova, Aravind and Koonin, 2011). The second phase is identified as the expression and processing; this consists of the processing of strands of DNA copied into new forms and cleaved by an extensive transcript of a CRISPR locus that is formed into a smaller crRNas. The final phase is the interference where the DNA and RNA are aimed at by a ribonucleoprotein that has Cas proteins.CRISPR-Cas systems again segregate three distinct types, for example type I and type III systems only depend on crRNA however type III uses trans-activating CRISPR.
Applications
Glaucoma
Crispr-Cas9 can be used to treat a very common eye disease, glaucoma which is most likely to cause blindness that is permanent. Currently there are no treatments effective enough, and no cure, however some clinical studies conducted show that you can prevent cell death, vision loss and optic nerve damage (associated with glaucoma) by decreasing the intraocular pressure. To reduce the IOP, there are two options – eye drops and surgery however with both these options the glaucoma is likely to return and also the side effects are quite severe e.g. with the eyedrops you have to remember to administer then every day, which some can forget and so the efficacy of the treatment decreases. Also, there could be potential complications with the surgery, so only the most skilled surgeons can perform it, who are in high demand and in low supply. So, a new technology using gene editing with Crispr cas9 could provide a permanent solution to the problem. However, as it turns out there are multiple genes which heighten the risk of glaucoma and Crispr cas9 which is monogenic in function and can only edit one gene that is complementary to it, as its specific, which makes the editing useless in that case (Wu et al., 2020). So instead we could use Crispr Cas 9 to target reduce IOP as we know higher levels of the pressure increase the risk of glaucoma. You can reduce the IOP by disrupting the ciliary body aqueous humour production which is controlled by the gene called ‘Aquaporin 1 gene’. The study shows that they used an injection of a virus containing the Crispr Cas 9 vector and injected it straight into the eyes of the mouse with glaucoma. So, then what happens is that the Crispr Cas 9 is then manifested which in turn cleave the ‘Aquaporin 1 gene’. Due to the fact that this gene is essential in the creation of aqueous humour, when its cleaved out, this would in turn decrease the IOP therefore lowering the risk of Glaucoma. This is just one of the applications of the Crispr Cas9 gene.
Cancer
Cancer has always been a major problem that humans encounter, it can cause complications and harm even mentally too as it affects the quality of life of patients. Even after some treatment, the side effects can still leave patients with life changing issues even though they are free from cancer. However, CRISPR-Cas9 has a high potential in the field of cancer immunotherapy (Xia et al.,2018). Immunotherapy is using the body’s own immune system in the battle against cancer. CRISPR-CAS9 being a low cost but highly efficient gene editing tool (memi et al.,2018), it promises to show great results in cancer immunotherapy.
The applications of CRISPR-Cas9 in this field are used in combination with CAR-T cells. Currently, the majority of CAR T-cell clinical trials have come from patient’s own autologous T cells but this method does have some problems as it is a time consuming and an extremely expensive process. In order to find an even more efficient and cheaper treatment to this problem, is the idea of using and creating a ‘universal CAR-T cell’ which is genetically modified T-cells from healthy donors (Mollanoori et al.,2018). To achieve this treatment, you must bypass the problem of rejection of the infused modified T cells from healthy donors known as graft-versus-host disease (GVHD), and you also have to make sure that the CAR-T cells are more targeted at the cancer cells and hence avoiding unnecessary damage to healthy cells according to (Molanoori et al.,2018). This is where CRISPR-Cas9 can be applied.
The human leukocyte antigen (HLA) molecules on the allogeneic CAR-T cell would be rejected by the body’s immune system as it would be recognised as foreign HLA molecules (Molanoori et al.,2018). This is why researchers are now trying to investigate ways of using the CRISPR-Cas9 tool to disrupt or silence both TCRS and HLA molecules in these universal T-cells and they are using this technology due to it potentially being able to edit and effect multiple genes at a time. Multiple studies show CRISPR/Cas9 causing the removal of the TCR beta chain and beta-2-microglobulin (B2m) which is an essential subunit of the HLA molecule and this is used in the production of universal CAR-T cells (Molanoori et al.,2018).
On top of that studies also showing these genetically modified CAR-T cells produced from CRISPR/Cas9 has a targeting efficiency of 90% for single gene disruption without weakening the effector function (Mollanoori et al.,2018).
After more research to ensure the safety and accuracy of this technology, CRISPR-Cas9 can be implemented in the next potential treatment for cancer.
Cystic Fibrosis
One application of CRISPR-CAS9 is the cure of a genetically inherited condition known as cystic fibrosis. Cystic fibrosis is the buildup of excess mucus in the lungs and digestive system; its symptoms include breathing difficulty and deficiencies in nutrients, as the body will have issues in taking in adequate nutrients due to the excess mucus. Due to the nature of the condition being genetically inherited, symptoms will show from a very early stage in a person’s life.
In the medical industry there is no cure for the condition, but rather there are medical drugs that mask the symptoms for a short-term period. Hence, those suffering with cystic fibrosis are generally known to have a short life expectancy.
Over the years there has been progress in the industry in finding cures for the condition, one being gene therapy. This includes replacing the defective gene, and whilst this can cure the condition, it can be difficult to deliver the gene to the target site.
The way in CRISPR-CAS9 solves the issue in a pioneering way is that it edits the defective gene instead of replacing it and allows the condition to be solved from its root cause. The process in which it works is very precise, this allows for lifelong expression and reduces the chance of mutations occurring.
The process in which the technology works is that it uses two molecules individually known as guide RNA (gRNA) this has an identical sequence as the target site, and Cas9 which is a nuclease used to cut DNA. (Marangi M, Pistritto G. 2018)
Firstly, gRNA couples with Cas9 and both travel to the target site. Next, Cas9 cuts the desired site, and the gRNA binds to the site using complementary base pairing. The Cas9 molecule then creates site specific double strand breaks (DSBs). Finally, the DSBs are repaired by the broken strands being ligated.
Although there have been many advances over the years in treating the symptoms of the condition, these have increased quality of life and life expectancy, but it is still relatively short and only masks the symptoms. The other treatment discussed was gene therapy, but this technique has issues in delivery whereby it is difficult to reach the target site. The CRISPR-Cas9 technology provides a treatment that will potentially cure the conditions from its root cause.
Crops
The CRISPR system can also be used on crops. Characteristics that can be modified for improvements include plant aesthetics, plant architecture, disease tolerance and also yield(1). I will be more focusing on how this genome editing tool kit can be used to increase yield of crops, as crop yield provides food and environments for life. The method of this is as mentioned before, a three step-procedure including the acquisition stage, expression stage and the interference stage (3). One example of a crop genome that can be edited is that of rice. In the indica rice line known as IR58025B, there is a gene called the dense and erect panicle1 (DEP1). This gene is a protein that plays a major role in the yield of rice production (in grains per panicle) and also other small factors such as nitrogen uptake and stress-tolerance, which also play a role towards yield of rice (2). This gene is then deleted by the crispr system, along with a few other genes such as GN1a and GS3, depending on what type of rice; the genes may vary. For most examples, deleting these genes increases the number of grains produced, increased size of the grains and also an increase in the density and erections of the panicles(4). Due to current world issues such as population growth, climate change and food storage, all of which are linked towards crop production, there has to be new approaches towards the subject in order to meet the growing global demand for consumption of food (6). Advantages of CRISPR is that it is more precise, faster, more efficient when editing genomes (even at multiplex levels) and even cheaper compared to other genome editing tools like ZFNs, zinc finger nucleases and TALENs, transcriptional activator-like effector nucleases(6). This procedure is known to be a friendly user tool which has development in genome edited crop plants to prevent harmful effects from climate change, establish future food security for our increasing population and also helps gain resilience against pests and abiotic stresses. (6) One disadvantage of using crispr on crop production is off-target. This is when the produced sgRNA (from the crispr) isn’t completely complementary with the DNA template it is supposed to act upon. This could lead to lack of proteins that are needed for that specific crop or it could lead to a mutation. However, the chance of the effects of off-target occuring is low (5).
2020-2-11-1581456199