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Session A2 Paper 218 Disclaimer—This paper partially fulfills a writing requirement for first year (freshman) engineering students at the University of Pittsburgh Swanson School of Engineering. This paper is a student, not a professional, paper. This paper is based on publicly available information and may not provide complete analyses of all relevant data. If this paper is used for any purpose other than these authors’ partial fulfillment of a writing requirement for first year (freshman) engineering students at the University of Pittsburgh Swanson School of Engineering, the user does so at his or her own risk. USING THE CRISPR/CAS9 GENE EDITING TOOL TO DEVELOP A CURE FOR DUCHENNE MUSCULAR DYSTROPHY Kris Keppel, [email protected], Vidic 2:00, Brendan Lettrich, [email protected], Vidic 2:00 Abstract- CRISPR, short for Clustered Regularly Interspaced Short Palindromic Repeats, is a revolutionary gene editing system that has the power to cut and paste any section of DNA and potentially cure many genetic diseases. CRISPR consists of two parts, with its partner enzyme “cas9” performing the cutting and pasting, and CRISPR being an RNA sequence that guides the enzyme to the proper location in the cell. It can be programmed to locate any gene, and can cut hundreds of bases or just a few. While CRISPR has also been applied to agriculture and other fields, the focus of this paper is on the use of this technology in the search for a cure for Duchenne muscular dystrophy (DMD), which affects one in every 3500 men. This paper will be organized into four main sections. The first two will cover background information on CRISPR and DMD, delving into topics such as the technicalities of how CRISPR works, as well as past attempts to cure DMD. The next section will investigate how CRISPR is being applied to muscular dystrophy by looking closely at one specific experiment which used a germline editing method to show muscle redevelopment in mice with DMD. Finally, the last section will cover the ethical and sustainability concerns related to CRISPR, and this will be followed with a conclusion that demonstrates why CRISPR is important to the conference and to engineering. Key Words—CRISPR/Cas9, Duchenne muscular dystrophy (DMD), ethics, gene, muscle development, mutation, RNA A REVOLUTION IN GENE EDITING The field of genetic engineering has seen many explosive discoveries over the past few decades, but few have carried with them the potential that is harbored by a technology known as Clustered Regularly Interspaced Short Palindromic Repeats, better known as CRISPR. This gene editing tool can remove any desired section of DNA, and then insert a replacement and repair the strand just like new. It has taken the field of genetics by storm due to its virtually limitless potential. CRISPR’S usage in agriculture and industry has already begun, but the focus of this paper will be on its development as a cure for genetic diseases, specifically Duchenne muscular dystrophy (DMD). For many of these diseases, DMD included, the mutation that causes the disease University of Pittsburgh, Swanson School of Engineering 1 3.3.2017 is well known, but unfortunately science has yet to discover a mechanism by which to fix it. CRISPR has the potential to be that mechanism, and the way in which it could do so will be the focal point of this paper. DUCHENNE MUSCULAR DYSTROPHY In order to fully comprehend the significance of applying CRISPR to find a cure for Duchenne muscular dystrophy, some background knowledge on the disease itself is necessary. The Muscular Dystrophy Association defines Duchenne muscular dystrophy as “a genetic disorder characterized by progressive muscle degeneration and weakness” [1]. Duchenne is one of nine types of muscular dystrophy, but it is the most common form among children. Duchenne muscular dystrophy occurs almost exclusively in boys, although there have been very rare cases in which it has been documented in girls. This is because DMD is due to a mutation in the gene that codes for the dystrophin protein, which is located on the X chromosome. Since women carry two X chromosomes, carriers of the disease have one proper form of the gene and one damaged copy, but only one is needed in order for production of dystrophin to be carried out effectively. In men, however, having only one X chromosome means that if they inherit the mutated form of the gene, they will express DMD [1]. Symptoms and Prognosis According to the Muscular Dystrophy Association, for children afflicted by DMD, muscle weakness typically begins between the ages of three and five, beginning in muscles located in the hip, thigh, and shoulder area, and later spreading to the extremities. This will continue to progress at a varying rate, but typically the child is forced into a wheelchair between the ages of seven and twelve. Outside of the physical ailment, it should be noted that DMD patients typically have no other issues, and are both cognitively and socially normal. However, as they progress into their teen years, it becomes increasingly difficult to maintain a normal life. During the teen years, the process of muscle degradation becomes more severe in the respiratory and cardiovascular systems, both of which tend to bring about more serious Kris Keppel Brendan Lettrich problems. Respiratory complications make it difficult to cough and thus leave patients prone to serious infection, and heart failure is typically the ultimate cause of death in DMD victims. Overall, the prognosis for those diagnosed with Duchenne muscular dystrophy is an average life expectancy of approximately twenty-five to thirty years, but recent advances in medicine have increased longevity of life, and there have even been some rare cases of patients living into their forties and fifties [1]. These two findings led to the conclusion that CRISPR actually served as an immune system of sorts within bacteria, working with Cas9 to help protect the organism against deadly viruses. The mechanism was simple: whenever a previously encountered virus entered the bacteria, the CRISPR DNA sequence that was produced from the first encounter recognized the virus, and produced an RNA molecule with a complementary base sequence to the viral sequence. This molecule then paired up with a Cas9 enzyme and lead it to the invader, where it could be destroyed. Following this development, scientists around the world immediately recognized the potential of this technology as a new gene-editing device for animals and potentially humans. Per Heidi Ledford of Nature magazine, the popularity of CRISPR began an exponential climb, as the number of articles published about CRISPR grew from zero in 2006 to over 600 in 2014, and its U.S. funding jumped from nonexistent in 2007 to over $80 million in 2014 [5]. Other Treatment Methods While there is no cure yet developed for DMD, there have been a few methods developed over the years that have, at the very least, provided some relief for patients. According to Dr. Jerry Mendell of Nationwide Children’s Hospital, corticosteroids are the only medications that have been shown to provide any benefit, but with them come many unpleasant side effects. Prednisone, for example, has shown the ability to prolong ambulation by about one or two years, but also causes weight gain, osteoporosis, and cushingoid appearance (swelling of the face), amongst other things [2]. Thus, treatment with corticosteroids is not often a popular choice. Per Dr. Chengzu Long of the University of TexasSouthwestern, aside from medication, other gene therapy methods similar to CRISPR, such transcription activator-like effector nucleases (TALENS) and zinc finger nucleases, have also been attempted. These methods have shown to be unreliable, however, because they have added a functional copy of the gene while also retaining the mutated gene, which leads to unreliable and often unsuccessful return of function [3]. CRISPR, meanwhile, replaces the mutated gene with a fully functional copy. How CRISPR Acts as Molecular Scissors Due to the incredibly fast rate at which the CRISPR technology was adopted, the scientific community has quickly improved the use of CRISPR, although there are some variations in how it is used. The most important component is the CRISPR RNA molecule, which includes two types of RNA: programmable guide RNA and tracer RNA. These two parts typically function as two separate molecules, but Jennifer Doudna, a researcher at the University of California, Berkeley, was able to combine these two into one complete molecule—one that is now the most commonly used form of CRISPR [6]. The other component is the Cas9 enzyme, which binds to the location determined by the guide RNA, and then performs the cutting of the target gene. The first step in the process of removing a target gene is to combine these two separate components into one main complex, which can be done in one of two ways. The original, more common way, is to simply inject both components at once into the target animal, and allow them to form the complex on their own. However, a new method has been developed by Randall Platt of the Broad Institute, who injected the Cas9 enzyme into a mouse embryo, thereby making it a permanent part of the genome [4]. The method by which the two are combined ultimately does not make a difference, but Platt’s method simplifies the process and produces many “ready-to-edit” subjects. Once the RNA and Cas9 are combined and injected into the cell, the complex immediately begins locating the specific gene to bind to. This specific location is determined by the guide RNA, a series of about twenty nucleotides, previously programmed by scientists, which correspond to one particular target sequence in the cell. Per Addgene, a company specializing in the production of genetic materials, this target sequence must meet two conditions: that it is “unique compared to the rest of the genome,” and that “the target is present immediately upstream from a Protospacer Adjacent BACKGROUND INFO ON CRISPR/CAS9 CRISPR was first discovered in E. coli bacteria at the Research Institute of Microbial Diseases in Osaka, Japan in December of 1987 [4]. While the scientists responsible for the discovery were able to detect these strange repeating sequences in E. coli DNA, along with the Cas9 enzyme, they were unable to determine their function; thus, CRISPR was disregarded as an insignificant side note for many years. In fact, it was not until 2005 when the next milestone was made in the development and understanding of this technology. It was at this time that Francisco Mojica from the University of Alicante compared CRISPR’s DNA to that of thousands of other organisms, and was astonished to find that each sequence within CRISPR was in fact a fragment of DNA from an invading virus. This was followed up in 2007 by the work of two scientists at the Danisco food company, who found that while many of their yogurt cultures were destroyed by bacteria, the only ones that were saved all contained CRISPR sequences within their genome [4]. 2 Kris Keppel Brendan Lettrich said, “In the past it was a student’s entire thesis to change one gene, CRISPR just knocked that out of the park” [6]. This statement displays how much CRISPR has simplified the process of editing a gene. Another one of CRISPR’s many advantages is the speed at which it works, and consequently the speed at which experiments or studies using it can be completed. In the past, it could take scientists up to two full years to successfully change just one gene in a population of mice. This slow pace led to an extremely lengthy process of finding cures for diseases, and greatly hindered the progress of the field of genetics as a whole. With CRISPR, this has all changed. In October 2015, researchers at Harvard University were able to successfully alter 62 different genes simultaneously in pig embryos with the use of CRISPR, and the whole process took less than a year [6]. This ability provides an opportunity to gather huge amounts of information in a relatively short amount of time, an opportunity not presented by any other technology. Lastly, CRISPR is so different from other gene therapy methods because of its low cost. Past advances in gene editing technology, such as TALENS and zinc fingers, often cost $5,000 or more to order [5]. Meanwhile, with CRISPR, the total cost of the customized RNA guide strand and Cas9 together is a mere $30 [5]. This vast difference in price, coupled with the speed, simplicity, and effectiveness of CRISPR makes it the obvious choice for scientists involved in genetic engineering. Motif (PAM)” [7]. This PAM sequence is a series of only a few bases that is vital to the cutting process, as it serves as an activator for the Cas9 enzyme, directing the bonding between it and the gene and initiating the cutting of the desired sequence. FIGURE 1 [8] CRISPR/Cas9 complex bound to specified location in gene The whole complex of the Cas9 enzyme, guide RNA, tracer RNA, and PAM sequence can be seen in Figure 1 above from Tamar Laboratory Supplies. According to Ledford, after the CRISPR/Cas9 complex finds the correct location with a proper PAM sequence, it binds to the location and “unzips” the DNA strand, so that the guide RNA sequence can bond with its corresponding base pairs. As long as there is sufficient homology between the RNA and DNA sequences, the Cas9 enzyme will begin cutting and removing the desired gene from the DNA sequence [7]. HOW CRISPR/CAS9 CAN CURE DMD With the general idea of how CRISPR acts as a gene cleaving tool, this knowledge can now be applied specifically to the gene that causes Duchenne muscular dystrophy. This investigation will delve deeper into the technical details and experimental techniques involved in working with CRISPR and DMD, and provide insight into how research experiments being conducted today might ultimately lead to a cure in the future. WHY CRISPR IS THE PREFERRED TOOL There have been many genetic engineering tools discovered over the past few decades, many of which have worked using a similar mechanism to that used by CRISPR. Thus, it is fair to ask the question, what makes CRISPR/Cas9 so special, and why is it the overwhelmingly preferred method among scientists today? The answer is that CRISPR is faster, simpler, and cheaper to use than other previously discovered technologies. One of the main reasons CRISPR is preferred is its simplicity in comparison to other tools. Essentially all that is required is for one to acquire the necessary components, most of which can be purchased very easily, and then inject them together into the target animal. While there would clearly need to be much planning put into the design of the experiment, once inserted into the subject, the technology does the rest. The best way to express just how simple this process can be, however, is through the opinions of the researchers themselves. In one article published by the New York Times Magazine, author Jennifer Kahn did just that, asking scientists working with CRISPR about the simplicity of the technology. Bruce Conklin of the Gladstone Institutes Mechanism by Which CRISPR Repairs the Damaged Dystrophin Gene Dr. Chengzu Long from the University of TexasSouthwestern became one of the first to pioneer the use of CRISPR on DMD when he injected the system into mice affected with the disease, and then measured the consequential levels of muscle development. While there have been many other similar experiments completed, approximately the same methodology is used in each, and thus the focus in this section will be directed only at Long’s work. The objective was to use the CRISPR/Cas9 system to fix the point mutation located in exon 23 of the dystrophin gene in mice, which causes Duchenne muscular dystrophy, and 3 Kris Keppel Brendan Lettrich then measure how effective the procedure was in stimulating muscle generation in the mice [3]. Long further states in his report that the exact mutation is the misplacement of a T nucleotide where a C nucleotide is supposed to be, which consequently causes a premature stop codon that halts expression of the dystrophin gene as a whole. The mutation can be seen in greater detail in Figure 2 below. zygotes, the zygotes were reimplanted into surrogate mothers, who produced the offspring that Long desired. The entire germline editing process can be summarized in Figure 3 below. FIGURE 3 [3] Summary of the germline technique used to produce desired offspring FIGURE 2 [3] Diagram of the point mutation in exon 23 that creates stop codon Once in the zygote, the CRISPR/Cas9 complex acted exactly as described earlier. The guide RNA lead the system to the proper location, where the cas9 enzyme recognized the PAM and bonds, and then began the cutting process. After it has cut the DNA and removed the faulty base, either through NHEJ or HDR, the DNA is repaired and the proper C base is put into place [3]. The first step in the procedure was to produce a population of mice with Duchenne muscular dystrophy. To do so, Long used the germline technique, whereby he crossed a female with two copies of the mutated DMD gene with a male housing the mutated DMD gene on its only X chromosome, thus ensuring that the parents would produce offspring with DMD. He did not allow these two parents to directly produce offspring, however, but rather gathered sperm and eggs from the pair before combining them to artificially form zygotes, which he labeled “mdx”. This technique allowed Long to produce many offspring, while also ensuring that each had the desired characteristic. Additionally, Long prepared zygotes of wild-type mice, which did not contain the mutated gene. This population of wild-type mice served as the control group in the experiment. After the zygotes were prepared, the next objective was to inject the proper materials into each zygote. For this experiment, the required materials were the guide RNA molecule, the cas9 enzyme, and in some cases a template RNA strand for homology-directed repair (HDR). Homology-directed repair is a more accurate way of “sewing” the DNA back together after it has been cleaved, as opposed to its counterpart, nonhomologous end-joining (NHEJ), which can at times lead to more mutations when repairing the cut DNA. In this experiment, Long chose to use both methods, and the results of the experiment later showed no significant difference in muscle regeneration between the two techniques. With the proper materials gathered, Long injected these materials into only certain zygotes for the purpose of creating four distinct groups of mice. The four groups were: wild-type mice, wild-type mice with CRISPR/Cas9, mdx mice, and mdx mice with CRISPR/Cas9. After the CRISPR/Cas9 system was injected into the desired Results of the Experiment Upon completion of the experiment, the team compared the various groups of mice based on many aspects of muscle strength and development. Specifically, the scientists performed histological analysis when the mice were between seven and nine weeks old on four different types of dystrophic muscle: quadriceps, soleus (hindlimb muscle), diaphragm (respiratory), and heart. Notably, as a subpart of the histological analysis, the scientists performed immunostaining on these areas of muscle in each of the four groups of mice, which essentially made the dystrophin protein fluoresce so that levels of the protein could be easily observed. Long compared pictures of each of the four muscle groups from a wild-type mouse, an mdx mouse, two HDRrepaired mice with 17% and 41% gene correction, respectively, and an NHEJ-repaired mouse with 83% gene correction. As expected, the wild-type mouse showed full dystrophin expression in each of the four areas, and the mdx mouse displayed virtually no dystrophin expression in each of the areas [3]. The important finding, though, was that in all three cases in which an mdx mouse had been injected with the CRISPR/Cas9 system, there was substantial muscle development, to the point that, in the cases of the HDR 41% and NHEJ 83% samples, dystrophin expression was essentially even with that of the wild-type mouse. Furthermore, even the other HDR sample with just 17% gene 4 Kris Keppel Brendan Lettrich correction showed muscle regeneration that far outweighed the level of correction at the molecular level [3]. The findings are summarized in Figure 4 below. arise when this is attempted on fully developed cells. There is hope, however, as recent studies involving the adenoassociated virus (AAV) transport system have been shown to be effective in delivering materials to somatic cells, and more importantly have been shown to be most effective in regions severely affected by DMD, such as skeletal and heart muscle [3]. In fact, in a recent experiment performed by Dr. Christopher Nelson at Duke University, the AAV system was used to successfully deliver CRISPR/Cas9 into adult mice suffering from DMD, and the results showed noticeable muscle regeneration within the mice [9]. This was another huge milestone along the path to a cure for humans. Another problem still exists, though, in that even if CRISPR can be safely transported into somatic cells, it will still be very difficult to alter enough cells in order to make a difference overall. With trillions of cells in the body, altering the gene in just a couple hundred or even thousand will not provide any real benefit to DMD patients. However, this is where the satellite cells discussed earlier will come into play. If scientists are able to direct the CRISPR/Cas9 system to the satellite cells only, this could lead to progressive muscle regeneration in all areas of the body, which could in turn reverse the effects of Duchenne muscular dystrophy. Although this is a real possibility, with all of the issues stated above, this idealistic goal is still at least a few years away from coming to fruition. Additionally, these are just the scientific issues, which will not be the only obstacle standing in the way of CRISPR. FIGURE 4 [3] Comparison of immunostaining of the dystrophin protein in four different muscle types for each group of mice This last result was arguably the most significant finding made by Long and his team. With just 17% gene correction yielding up to 60% of muscle growth, clearly there must be some way in which partial muscle development contributes to an even greater level of regeneration. Long’s proposal for this involved satellite cells, which are the skeletal system’s supply of stem cells. He hypothesized that even when a small percentage of cells contain the corrected dystrophin gene, if some of these are satellite cells, then these can stimulate growth in other muscle cells around them, causing progressive regeneration of the dystrophic muscle overall [3]. It should be noted that, as a side experiment of sorts, Long and his team also recorded the number of off-target mutations produced by the CRISPR/Cas9 system while it was repairing the dystrophin gene. After analyzing the data, it was deemed that there was no higher frequency of off-target mutations in the CRISPR modified mice when compared to control group wild-type and mdx mice. This finding was also quite notable, as one of the most common fears of the use of CRISPR is that it is, at times, dangerously inaccurate [3]. SUSTAINABILITY OF CRISPR When dealing with any emerging technology, concern must be directed not only at its current progression, but also at its future development, to ensure that it exhibits all the qualities of a sustainable product. To be considered sustainable a technology must check many boxes, but in general, according to the UN’s 1987 Brundtland Report, sustainability can be defined as “satisfying the needs of the present generation without compromising the ability of future generations to meet their own needs” [10]. Placing the focus specifically on CRISPR, however, this definition can be whittled down to a more explicit goal: ensuring the continued health of humans and the surrounding environment. Many uses of genetic engineering in the field of agriculture have generated controversy over the effects that genetically-modified products might have on the environment, as at times scientists are essentially creating new species through all the genetic modifications [11]. While there is a small chance that the use of CRISPR could have similar negative effects, it is unlikely that such targeted and small-scale genomic changes will have any ecological consequences. Another common environmental concern with technologies in just about any field of engineering is the waste products associated with the process. Again, CRISPR poses no issue when it comes to this. The components of CRISPR Future Testing with CRIPSR/Cas9 and DMD While Long’s experiment was a great success and provided much hope for the future, his germline method of editing the mice genomes is still not currently feasible in humans. Thus, there are still a fair amount of hurdles this technology must overcome before it can be used as a way to cure DMD in people. Since the germline method is not a realistic possibility, one of the biggest issues when applying this technology to humans is finding an easy way to direct the materials needed into somatic cells. There is very little issue when injecting the system into a zygote, but more problems 5 Kris Keppel Brendan Lettrich that will be injected into subjects are common biological elements, and will be destroyed in the cell not long after completing their task, consequently posing no threat to the environment or to the humans themselves. Additionally, it should be noted that production and storage of CRISPR carry no threat as well, as the materials are collected from viral DNA, and if not stored properly will simply denature [4]. While there little to no environmental risks associated with the use of the CRISPR/Cas9 system, some viable concerns have been raised over how the technology’s use could bring about some unexpected negative health effects in people. CRISPR is still early in its development, so much more testing needs to be completed to ensure that no off-target mutations occur, as these could potentially lead to severely damaging health defects. Scientists are already making great progress on this front, however, as seen by the negligible amount of mistakes made by CRISPR in Dr. Long’s experiment [3]. It is not just the off-target mutations that have raised concern, though, as many critics are beginning to worry about the unintended effects of the desired DNA cuts as well. It is well known that many genes are pleiotropic, meaning they can influence the expression of more than one phenotype [12]. This means that in cutting a strand of DNA to achieve one goal, we could accidentally lead to another change in gene expression that was not accounted for. While this change could be something insignificant, it could also cause a more dangerous change that could put someone at a higher risk for cancer, Alzheimer’s, or other diseases. With hundreds of thousands of genes in the human genome, researchers do not fully understand all of the functions of each and every gene, and thus this concern is something that could lead to real problems. This issue is already in the process of being addressed, however, as researchers like those at the National Human Genome Research Institute are currently working to discover all of these functions in order to gain a better understanding of our genetic code as a whole. With more efficient gene editing tools being produced, such as CRISPR, this research can be done much faster, and thus the day when we can fully map the function of each gene may be someday soon [13].. As long as these issues are dealt with, the CRISPR/Cas9 system should be a sustainable genetic engineering tool that can be used for the foreseeable future. Once again, however, there are still more issues that must be addressed, as the CRISPR/Cas9 system has raised a few ethical concerns as well. now the risk of gene editing is considerably higher than the reward because more research and testing still needs to be done. The CRISPR/cas9 system is no exception to this [10]. Scientists have just recently begun testing on human embryos and many scientists like Jennifer Doudna, one of CRISPR’s biggest supporters, still think that it’s too early to start human testing [6]. However, coupling the limitless potential of CRISPR with the vast quantities of money that could be profited from it, there is a very strong chance that some wealthy, powerful people could begin pushing for it to be used in practice. An Imperfect Technology According to the Journal of Clinical Research and Bioethics, the first major concern with the CRISPR/cas9 system is the risks and consequences compared with the reward. The CRISPR/cas9 system is relatively new to the scientific world and is in the process of still being developed. With that being said, if the system were put to commercial use today, the results would be very unpredictable and likely dangerous. However, the potential for the system is immense and growing each day. The CRISPR/cas9 system has the potential to cure several genetic diseases, such as DMD, diabetes, and many types of cancer, maximize the food consumers get from crops and livestock, form animal chimeras for organ transplants, and enhance the human genome. The vast potential of CRISPR has led to an explosion of interest by scientists to perfect the system. However, this can cause some scientists to disregard the safety concerns and push for its use [14]. For instance, within the last year, a group of Chinese researchers announced that they began their first trials on human embryos. This shocked the science community because most scientists believed that human testing was a few years down the road. One specific critic, Keith Joung, who studies gene editing and specializes in methods of tracking down cas9’s off-target mutations, believes that CRISPR needs to be developed more before it can be tested on human embryos. Joung learned through his studies that the frequency of these off-target mutations that result in genetic mutations range from 0.1% to 60% of the time [5]. Along with the high frequency of off-target mutations, scientists have some concern with the possibility of gene editing in the human germline, according to the Journal of Clinical Research and Bioethics. The main problem with editing the germline is that even if the CRISPR/cas9 system is used correctly and there are no mutations at the time, there is no way to determine how the edited genome will affect future generation. Another problem with using the CRISPR/cas9 system to edit the germline is there is no way to enforce informed consent. If a person agrees to having CRISPR used on them to edit a gene in their germline causing a disease, then a genetic mutation caused by the edited gene could occur in a latter generation and that person suffers the consequences of the first person’s choice. Using the ETHICAL CONCERNS WITH CRISPR As engineers continue to develop more advanced technology, ethics is becoming a large aspect of engineering. A lot of controversy spurs around gene editing in general, not just the CRISPR/cas9 system. Per Renuka Sivapatham of the Buck Institute for Research on Aging, many people feel as though it is immoral to change a person’s genome. As of right 6 Kris Keppel Brendan Lettrich CRISPR/cas9 system to edit a gene in the human germline is still being questioned by the scientific community [14]. Accessed 1.25.2017. http://www.nature.com/cr/journal/v26/n5/full/cr201628a.htm l [3] C. Long, et al. “Prevention of muscular dystrophy in mice by CRISPR/Cas9-mediated editing of germline DNA.” Science. 9.04.2014. Accessed 1.25.2017. http://science.sciencemag.org/content/345/6201/1184.full [4] M. Specter. “The Gene Hackers.” The New Yorker. 11.16.2015. Accessed 2.19.2017. http://www.newyorker.com/magazine/2015/11/16/the-genehackers [5] H. Ledford. “CRISPR, the disruptor.” Nature. 6.03.2015. Accessed 2.08.2017. http://www.nature.com/news/crisprthe-disruptor-1.17673 [6] J. Kahn. “The Crispr Quandary.” The New York Times Magazine. 11.09.2015. Accessed 2.09.2017. https://www.nytimes.com/2015/11/15/magazine/the-crisprquandary.html?_r=0 [7] “CRISPR/Cas9 Guide – Overview of CRISPR/Cas9.” AddGene. Accessed 2.16.2017. https://www.addgene.org/crispr/guide/ [8] “Edit-R CRISPR-Cas9.” Tamar Laboratory Supplies, Ltd. 2017. Accessed 2.14.2017. http://www.tamar.co.il/crisprcas9/ [9] C. Nelson, et al. “In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy.” Science.. 1.22.2016. Accessed 1.08.2017. http://science.sciencemag.org/content/351/6271/403 [10] G.H. Bruntland. Chapter 2. Our Common Future: Report of the World Commission on Environment and Development. 3.20.1987. Accessed 3.24.2017. http://www.un-document.net/ocf-02.htm [11] E. Rodriguez. “Ethical Issues in Genome Editing Using Crispr/Cas9 System.” Journal of Clinical Research and Bioethics. 3.24.2016. Accessed 1.10.2017. https://www.omicsonline.org/open-access/ethical-issues-ingenome-editing-using-crisprcas9-system-2155-96271000266.php?aid=70914 [12] “Pleiotropy.” Natural Living Center. 2011. Accessed 3.26.2017. http://www.naturallivingcenter.net/ns/DisplayMonograph.as p?StoreID=b571dewxvcs92jj200akhmccqa7w8v75&DocID =genomic-pleiotropy [13] “An Overview of the Human Genome Project.” National Human Genome Research Institute. 12.27.2012. Accessed 3.25.2017. https://www.genome.gov/12011241/thecompletion-of-the-sequence-and-remaining-goals/ [14] R. Sivapatham. “Ethical Implications of Human Genetic Engineering.” SAGE Buck Institute. 8.19.2015. Accessed 2.10.2017. http://sage.buckinstitute.org/ethical-implicationsof-human-genetic-engineering-2/ The Creation of a Superior Few A second major concern with the use of CRISPR/cas9 to edit a person’s genome is the effect it would have on society as a whole. Gene editing tools would be very expensive to use, so people tend to believe that CRISPR is only accessible to the wealthy. The gap then between the poor and the wealthy would increase because only the wealthy would be able to afford using CRISPR to cure a disease. Also, at first CRISPR is mainly used for curing genetic disease, but it has the potential of editing genes that detail other aspects of a person’s body. CRISPR could be used to enhance a person’s athletic ability, height, eye color, hair color, etc. [11]. This worries people because the price of gene editing would be very high and only available to the wealthy. Although this may seem far-fetched to some, this could eventually lead to the creation of a superior few or race of sorts. Those who could afford it could use CRISPR’s abilities to enhance themselves genetically, perhaps by developing greater intellectual capacity [11]. While the technology is years away from potentially being able to accomplish such a task, even the distant future must be considered when weighing the benefits and consequences of something as revolutionary as CRISPR. FUTURE OF CRISPR Although other gene editing tools have been used in an attempt to develop cures for genetic diseases, CRISPR has the potential to be one of the most impactful systems in the biomedical engineering field. Dr. Long’s experiment could potentially set the foundation for the endless possibilities for the CRIPSR/cas9 system. If Dr. Long is able to develop a way to cure DMD with CRISPR, it could be modified to then cure several other complex genetic diseases such as Alzheimer’s disease, cancer, AIDS/HIV and many more. However, the uses of CRISPR do not stop at just curing diseases, CRISPR could potentially be used for genetically modifying crops and livestock to reap the maximum amount of food and nutrients. Along with that, CRISPR can be used to genetically modify animals to carry human organs that include hearts, kidneys, and lungs, all of which are in high demand. In the end, the uses of CRISPR are endless and the impact it can have on the medical world is unmeasurable. SOURCES [1] “Duchenne Muscular Dystrophy (DMD).” Muscular Dystrophy Association. 2017. Accessed 2.15.2017. https://www.mda.org/disease/duchenne-muscular-dystrophy [2] J. Mendell. “Duchenne muscular dystrophy: CRISPR/Cas9 treatment.” Cell Research. 3.01.2016. 7 Kris Keppel Brendan Lettrich ADDITIONAL SOURCES D. Kostos. “CRISPR Gene Editing: The Future & Ethics of Engineering in our World.” 8.31.2016. Accessed 1.09.2017. http://www.jove.com/blog/2016/08/31/crispr-gene-editingthe-future-ethics-of-engineering-our-world J. Kozubek. “How Gene Editing Could Ruin Human Evolution.” Time Magazine. 1.09.2017. Accessed 1.24.2017. http://time.com/4626571/crispr-genemodification-evolution/ H. Ledford. “CRISPR: gene editing is just the beginning.” Nature. 3.07.2016. Accessed 1.09.2017. http://www.nature.com/news/crispr-gene-editing-is-just-thebeginning-1.19510 ACKNOWLEDGEMENTS We would like to acknowledge our writing instructor Diane Kerr and our co-chair Andja Potknojak for their assistance in creating this paper. They provided great feedback throughout the process that allowed us to make this paper the best that it can be. Additionally, we would both like to thank our respective floor mates and engineering peers for providing support and empathy for the last few months. 8