Genetic engineering has become readily approachable since the development of the latest gene editing tool CRISPR/Cas9 in 2012. This pioneering technology holds the promise of modifying genomes of any organism in the world, curing genetic diseases and reshaping the biosphere to benefit the environment (Baltimore et al, 2015). However, there are number of ethical and regulative implications which need to be considered before CRISPR Cas9 can be permanently implemented into clinical trials, medicine and commercial use.
Science often takes its inspiration from nature, which is also true for the development of the latest gene editing tool. CRISPR/Cas9 has been adapted from bacterial immune response against invading viruses. The CRISPR/Cas9 mechanism is shown in figure 1. Upon identification of the virus, bacteria synthesises a DNA fragment identical to that of the virus. The new piece of DNA is then integrated into bacterial DNA, which forms a pattern of repeats called Clusters of Regularly Interspaced Short Palindromic Repeats (CRISPR). The bacterial cell then produces a copy of that sequence in a form of RNA, which is fragmented into smaller units. Each unit contains one of the sequences derived from the virus as well as the sequence coming from the repeat. These RNA molecules combine with the second RNA forming a protein complex called Cas9. Cas9 contains a short RNA sequence complementary to that of the virus. The RNA sequence identifies its target within the viral genome and attaches to its complementary DNA molecule. This activates the Cas9 enzyme to cut the target double strand of DNA, which then prompts DNA-repair enzymes to fix the break. However, this process is prone to errors, which often results in mutation that either disables or modifies the targeted gene, thus disabling the virus. The CRISPR/Cas9 mechanism has been implemented to work similarly in the laboratory. Scientists are able to create a small guide RNA sequence, which attaches to both: a specific DNA sequence of interest, as well as the Cas9 enzyme. Just like in bacteria, the RNA recognises the desired sequence of DNA, while the Cas9 slices the DNA at a precise site. Researchers take an advantage of cell’s own DNA repair mechanism, which allows them to make changes to existing DNA by either: deleting, adding, or replacing fragments of genetic material (Jinek et al, 2012).
The CRISPRs were first reported in 1987 in Escherichia coli, but their function remained unknown until 2005, due to the absence of relevant DNA sequence data to compare to and support their observations at the time. Since the development of genomics, the mysterious repeated sequences were being detected in a growing number of genetic material of bacteria and archaea. (Ishino et al, 2018). In 2005, scientists first proposed that CRISPR sequences were likely used by bacteria as a defence mechanism against viruses (Mojica et al, 2005), but the CRISPR/Cas9 function mechanism was not fully understood until 2008 (Brouns et al, 2008). Fast forward four years, Jennifer Doudna and Emmanuelle Charpentier published an article describing the use of CRISPR/Cas9 as means of cutting and editing DNA (Jinek et al, 2012). New research and application ideas for use of CRISPR/ Cas9 technology have been expanding ever since.
CRISPR Cas9 in medicine
CRISPR/ Cas9 allows to target and delete, add or change composition of a single gene of interest with the use of short sequence of RNA. This enables scientists to identify function of a particular gene, but also to modify it (Hsu, Lander and Zhang, 2014). This proves particularly useful in identification, but also in potential treatment of many genetic disorders. Over hundreds of thousands of human diseases are caused by genetic mutations, meaning that gene editing tools such CRISPR Cas9 may prove extremely useful in understanding and potentially treating some of them (Wang et al, 2018). In fact, a first clinical trial in Europe involving CRISPR/Cas9 gene therapy has recently commenced in Germany in hope of finding treatment for blood disorder β-thalassemia. Patients suffer from β-thalassemia, due to repressed production of haemoglobin (red blood cells). The trial involves removing blood cells from the patients and editing them with CRISPR Cas9 to cleave the BCL11A gene, responsible for blocking the production of foetal haemoglobin, normally only formed during infancy. The modified blood is then infused back to the patients to hopefully re-start production of foetal haemoglobin and alleviate the symptoms of β-thalassemia (Sürün, von Melchner and Schnütgen, 2018). In 2017, CRISPR/Cas9 has also been used by scientist to correct a gene mutation responsible for Hyperthropic Cardiomyopathy (HCM) in human embryos. HCM is a heart condition causing stiffness of the cardiac tissues, which results in weakness, frequent chest pains and if left untreated, cardiac arrest. The research involved injecting 54 human single-cell embryos with CRISPR/Cas9 at the desired gene mutation site. One day after fertilisation, 36 of the injected embryos showed no signs of harmful mutations in the gene. For 13 of the remaining embryos chances of inheriting HCM mutations dropped by 50%. None of the embryos were developed (Ma et al, 2017). Another trial involving CRISPR Cas9 began in September 2018 and is being conducted by the University of Pennsylvania. The study is focusing on three types of cancer: melanoma, sarcoma and multiple myeloma and is set to target the NY-ESO-1 antigen. NY-ESO-1 is a tumour cancer antigen associated with many poor-prognosis types of skin cancer. The study is attempting to utilise this antigen in hope of developing a potential target for cancer immunotherapy (U.S. National Library of Medicine, 2018).
CRISPR Cas9 tackles antibiotic resistance
There is a critical need for new treatment of infectious diseases which are one of the leading causes of death worldwide. An increasing number of antibiotics are becoming ineffective, due to drug-resistant pathogens, meaning previously easily treatable conditions are requiring hospitalisation and prolonged treatment (de la Fuente-Nunez et al, 2017). Long before CRISPR Cas9 has been utilised as a gene editing tool, its primary function was to defend bacteria from invading viruses. In theory, the same principle could also be applied to target bacterial DNA inside infected human cells making it an alternative form of treatment for bacterial infections. The majority of current studies focus on finding the treatments for bacterial infections such as M. tuberculosis, Escherichia coli. There are currently two ways in which scientists are trying to tackle bacterial pathogens. The first method focuses on designing a CRISPR guide RNA that would allow to cut pathogen’s DNA to completely disable it (Doerflinger et al, 2017). The second approach concentrates on finding specific bacterial genes responsible for antibiotic resistance and disabling them using CRISPR Cas9 system in hope of sensitising them to antibiotics (Goren et al, 2017). The main obstacle, currently preventing CRISPR Cas9 system from being used in place of antibiotics is its delivery into the host bacteria. In order to target the bacteria, CRISPR Cas9 needs to be genetically encoded into a bacteria infecting virus, called vector, which act as a CRISPR Cas9 carrier. The vectors are first delivered to host cells infected by bacteria. The virus then attacks the bacteria and insert their DNA, which contains CRISPR Cas9 edited targets. The CRISPR Cas9 edited targets then slice the desired section of bacterial DNA (Greene, 2018). However, this way of delivering CRISPR Cas9 into the host bacteria is associated with high risks of mutations and can potentially cause cancer. Additionally, a small size of the viruses means a limited loading capacity for the CRISPR Cas9 targets. Thus, new delivery approaches need to be developed to allow implementation of CRISPR-mediated antibacterial therapy (Wang et al, 2018).
Genome engineering in agriculture
Editing genes of the crops with the use of CRISPR Cas9 seems like another exciting prospect considering growing population and food demands. Jaganathan et al (2018) states that the global food production will need to increase by at least 60% by 2050 to meet the growing demands. Until recently, selective breeding has been the only way of improving the quality and yield of crops. However, the prospect of CRISPR Cas9 gene editing to target any desirable trait of interest in plants may offer the solution to this problem. To this date, the plants such as wheat, corn, tomatoes and rice have already been modified to improve yield, but also provide resistance against pathogens. The modification also took a fraction of the time, compared to traditional breeding approaches (Gao, 2018).
Ethical implications
Despite many potential treatment applications for CRISPR Cas9 technology, there are many implications that prevent it from being readily used in clinical setting at present. Thus far, current uses of CRISPR Cas9 have been limited to medicinal gene therapy research. Other potential applications such as treating genetic illnesses in human embryos, or further exploration of medicinal gene editing are currently restricted and treated with extreme caution across the world (Kim, 2017). However, the CRISPR Cas9 is becoming more readily available to everyone, due to its simplicity and low cost. The growth of CRISPR Cas9 can be compared to the development of XXI century technology and social media, which once popularised, became normalised by the society, despite its many dangers and ethical implications. This means an ethics stance on CRISPR Cas9 as well as regulations involving gene therapy are also likely to change in the near future. One of the main challenges of CRISPR Cas9 is that any changes made to the genome are permanent and inheritable. However, current application technique of CRISPR Cas9 in humans is not error proof, due to the high risk of producing off-target mutations (Kosicki et al, 2018). Off-target mutations are caused by cutting unintended DNA sequences in the wrong part of the genome. This causes mutations, which may lead to change of the function of the gene (Kang et al, 2017). This could potentially cause many adverse events during clinical trial, causing patients’ suffering, or even death and can be hard to predict and track by the trial investigators. However, many critically ill patients are still likely to enrol, in hopes of finding treatment for terminal conditions. This type of clinical research is fraught with ethical difficulties, due to concerns about patients’ ability to give an informed and autonomous consent for research participation (Luce et al, 2004).Another controversy surrounding CRISPR Cas9 is germline editing and the moral status of editing human embryos. One of the issues is informed consent. Many believe it is impossible to obtain an informed consent for germline CRISPR Cas9 gene therapy. This is because, the gene therapy does not directly affect the decision makers (parents), but the future generation (embryo). However, this principle is also true for IVF treatment, which is accepted in medical practice. Germline editing also raises the question of affordability. The general public fears, that genome editing will only be accessible to rich. This could potentially widen the economic class gap. Taking it a step further, some worry that editing human embryos may lead to the new era of “designer babies”, where parents are able to choose advantageous traits of their future babies. This could potentially cause class division of individuals based on their genome quality (Cribbs and Perera, 2017). CRISPR Cas9 technology involving germline editing is not efficient, nor safe enough to be used in clinical settings as of yet (Reyes and Lanner, 2017). However, a scientist named Jiankui He recently announced he edited genes of two twin girls, who were born a few weeks ago. The revelation was heavily criticised by the Organisational Committee of the Second International Summit on Human Genome Editing held on the 29th of November. The committee agreed that it was “irresponsible to proceed with any clinical use of germline editing” (The National Academies of Sciences Engineering Medicine, 2018).
Future of CRISPR Cas9
CRISPR Cas9 is still a novel technique that has only been adapted to a laboratory environment in 2012. Nevertheless, given the rapid advances in the applications of CRISPR in the past six years, the future impact of gene editing seems inevitable. Companies such as CRISPR Therapeutics, Editas Medicine and Intellia Therapeutics are already researching future treatments for conditions such as Cystic fibrosis, type 1 Diabetes, as well as various eye diseases (Philippidis, 2018). Another important research is currently being carried out by the Capital Medical University in cooperation with Peking University. The scientists are looking at utilising CRISPR Cas9 in hopes of stopping the human immunodeficiency type 1 virus (HIV-1). The initial results suggest that using CRISPR Cas9 to tackle those genes may be an effective approach to cure the disease in the future (Deng et al, 2018). The future of CRISPR technology in agriculture also seems promising in the USA, where the US Department of Agriculture (USDA) announced that CRISPR- edited crops will not be regulated. This means that plants edited with CRISPR Cas9 will be entering the US market in the near future (Chaudhary et al, 2018). The future of CRISPR edited foods does not look so bright for the countries of European Union, where Europe’s highest court ruled that gene edited crops will now be subject to strict genetically modified regulations (Callaway, 2018). The repertoire of CRISPR Cas9 applications in the future is constantly expanding. However, scientists at the Wellcome Sanger Institute recently discovered that CRISPR Cas9 gene editing in humans causes greater health implications than previously believed. This is due to CRISPR Cas9 causing mutations, at a greater than previously assumed distance from the target site. The study also revealed that the current ways of examining DNA changes failed to detect this genetic damage (Kosicki et al, 2018). Therefore, the near future of gene editing research will likely focus on overcoming these issues before CRISPR Cas9 is used in clinical settings.
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