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Session A1 Paper 99 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. THE POTENTIAL OF CRISPR TECHNOLOGY IN THE TREATMENT OF DUCHENNE MUSCULAR DYSTROPHY Aaron Mcvaugh, [email protected], Mahboobin, 4:00, Alexander Short, [email protected], Mahboobin, 4:00 Abstract— Duchenne muscular dystrophy (DMD) is an inherited disease caused by mutations in gene encoding for dystrophin, a protein that ensures muscle integrity. Without this protein, one’s muscles will deteriorate with time. A new genetic modification technique called CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) could help treat this disease. CRISPR works by altering the patient’s DNA and replacing the mutated gene, allowing for the protein to be produced in the body. CRISPR has an advantage over other methods of treatment for DMD because of its accuracy, simplicity, and cost, allowing for large scale production, eventually. The relevance of CRISPR technology on diseases such as DMD is very significant to society as well as the field of bioengineering. CRISPR therapy may allow people who carry deleterious gene characteristics to have children without passing on dangerous traits. There is a great potential to improve the lives of people with degenerative diseases, as well as to ensure the health of people in future generations. Our paper will also expand on how this developmental technology is interconnected with aspects of sustainability. Our paper will show the vast potential of CRISPR for a large-scale impact on society. We will express our technology through many different examples and will use various methods to explain CRISPR. One method is to utilize the results of ongoing animal testing of this technology, as well as information about the future of this technology on human patients. These and many other sources will clearly convey the importance of CRISPR. Key Words— bioengineering, CRISPR, degenerative diseases, gene encoding, genetic modification, Duchenne muscular dystrophy, dystrophin CRISPR TECHNOLOGY OFFERS NEW TREATMENT FOR DUCHENNE MUSCULAR DYSTROPHY CRISPR technology is a revolutionary form of gene editing. This new process has the potential to conquer a great number of genetic disorders and can improve the lives of not University of Pittsburgh, Swanson School of Engineering 1 Submission Date 31.03.2017 only current patients, but of those in future generations. According to, What is Biotechnology.org, CRISPR/Cas9 is a genetic engineering technology that can be used to identify and edit the DNA of destructive viruses [1]. There are two main components of this innovative technology. First, there is the Cas9 system, which is “an enzyme that can cut a double DNA strand at a very precise point, nick it, or block its gene expression.” [1]. The second component is CRISPR, which is a short strand of RNA used to guide the Cas9 enzyme to bind with a genome sequence to make a cut [1]. Due to this progression of events, scientists can disrupt the gene’s function or insert a new sequence to code for a new function [1]. This would allow scientists to identify a gene mutation and then alter the mutation using this powerful system. The CRISPR technology would cut the mutation from the DNA and replace it with a healthy sequence. This ability to virtually eliminate a genetic disorder is important in developing new ways for doctors to increase the health of their patients now and for generations to come. This new process has very intriguing potential to scientists and researchers, for use in healing people from diseases, but also because it is an ethical and sustainable process. This new technology can revolutionize gene editing and have a positive impact on the lives of many people. One specific area in which CRISPR can have a major influence is with Duchenne Muscular Dystrophy. Per the Muscular Dystrophy Association (MDA), “Duchenne muscular dystrophy (DMD) is a genetic disorder characterized by progressive muscle degeneration and weakness” [2]. DMD is caused by the absence of the dystrophin protein that aids in keeping muscle cells intact [2]. The lack of dystrophin causes muscle cells to be weak and easily damaged [2]. In patients, muscle weakness can begin as early as age three, first affecting the muscles in the hips and pelvic area [2]. Later, it affects the skeletal muscles in the arms and legs including an enlargement of the calf muscles [2]. These symptoms have major side effects on people’s lives and health. CRISPR has the potential to help cure this disease, due to its unique ability to edit genes. The system’s ability to cut and replace gene mutations can play a key role in supplanting flawed dystrophin genes in people with DMD. Even though the future of this revolutionary technology shines brightly, it currently cannot be used to cure DMD. This is because it is Aaron Mcvaugh Alexander Short still in the laboratory test phase and is currently being tested on mice and not humans. According to Science Magazine, CRISPR could potentially be used in humans to cut out a faulty exon so that the cell could then make a shortened version of dystrophin [3]. This approach, while unperfected, could play a major role in helping to cure DMD, as well as open a new realm of possible solutions to battle other perilous diseases. for genome editing in eukaryotic cells [4]. The system targeted and attacked the bad genome and removed it from the cell. This astonishing method has been consistently effective in simple life forms such as bacteria, as well as, more complex life forms such as mice. This demonstrates its reliability and effectiveness. Even from early testing, it is easy to see how CRISPR has evolved into such a large area of interest in human health care. This system has proven to be not only dependable, but also powerful in its gene editing ability. THE HISTORY OF CRISPR/CAS9 AND GENETIC MODIFICATION THE CRISPR/CAS9 MECHANISM The CRISPR Mechanism in Viral Immune Response Just like all major discoveries, the origin of the CRISPR/Cas9 system has undergone much research over the past twenty years. Per the Broad Institute, it was in 2005 when Francisco Mojica originally discovered CRISPR and its function [4]. He identified that CRISPR was an adaptive immune system for bacteria [4]. This means that bacteria have the capability to identify and kill invaders to its system. It is adaptive because it can remember previous attackers and can use the same methods to destroy them yet again. This important finding was critical in the development of CRISPR as we know it today. It was also in 2005 that the second part of the CRISPR system was discovered. Alexander Bolotin discovered a large protein associated with CRISPR which was designated as Cas9 [4]. He realized that Cas9 contained spacers that were essential for target recognition and this became known as protospacer adjacent motif (PAM) [4]. The function of Cas9 as a targeting device in the current technology stems from this discovery. This and many other of the basic functions of this current day complex system were identified within simple bacteria. In 2006, Eugene Koonin discovered that CRISPR acts as the immune system within bacteria that defends against phage DNA [4]. Koonin’s discovery was an important follow-up to the discovery by Mojica. Mojica previously identified that CRISPR defended against some type of invasion and now Koonin realized that phage DNA performed this invasion. These two discoveries, in particular, were important in determining how CRISPR can be functional in healing and protecting the DNA of species such an animals or humans. Thus, spanning over a decade, the basic functions of CRISPR have been deeply researched and thoroughly understood. The design of the current technology, and its ability to have an impact on other, more complex life forms is derived from the immune system of basic bacteria. The original purpose of CRISPR in bacteria, was to develop immunity from invading viral DNA. All cells, human and bacteria alike, have DNA that code for proteins which carry out many functions inside the cell. Viruses essentially hijack the cell and force it to reproduce until the host cell dies. Since all living organisms use similar DNA structures, viruses can attack any cell in any organism, including humans. These structures only differ slightly in their composition. Per New England BioLabs, CRISPR works as an adaptive immunity to viruses by remembering viral DNA that was originally destroyed and thus easily destroying the virus in the future [5]. This facet of CRISPR is very important to its development in genetic modification. There are three types of CRISPR immunity mechanisms, although this paper will only focus on the second type due to its importance in genetic modification. The mechanism for CRISPR in bacteria is Cas1 and Cas2, which are variants of the Cas9 protein [5]. They work to identify the viral DNA and cut it into segments of about twenty base pairs [5]. In order for the Cas proteins to identify the viral DNA, they must use a short segment of base pairs of about three to five, called a Protospacer Adjacent Motif (PAM) [5]. Per the US National Library of Medicine, a PAM is required for Cas proteins to cut at the segment of DNA [6]. This is a very important initial step in the function of CRISPR. In order for the proper strand of DNA to be cut it must be properly identified so that the wrong strand of DNA is not attacked. This process of properly and precisely identifying the bad location on a DNA strand is very important to how this system functions. It also highlights how well CRISPR can serve as a cure for genetic disorders. Along with crRNA, separate trans-activating crRNA (tracrRNA) is also formed [5]. These two RNA sequences from a duplex similar to the DNA double helix [6]. These combinations are important to setting up the system so that it can invade the organism and make the proper cuts. The crRNA then works with Cas9 to destroy the invading DNA [5]. These sequences of events are very specific, which is important so that the system can attack and destroy the proper strand of DNA. Thus, the mechanism must be split up into parts so that it can work seamlessly and cut the invading viral Translation of Early Research to the Functionality of CRISPR/Cas9 The discoveries regarding simple bacteria in the early twenty-first century were very important to the development of CRISPR. They essentially describe the basic functionality of how CRISPR is used now. In 2013, Feng Zheng and his team were the first people to successfully use CRISPR/Cas9 2 Aaron Mcvaugh Alexander Short DNA. This next section will describe the usefulness of this biological process in terms of genetic modification. mutations in the gene for dystrophin resulting in muscle degradation. Since the cause is the lack of dystrophin in the patient’s body, this disorder is a good candidate for genetic modification to signal the DNA cells to produce more dystrophin. DMD has been cured in lab mice, as well as, isolated human cells. In a study regarding DMD in mice, embryos of mdx (dystrophin deficient) mice were modified with CRISPR/Cas9 [8]. According to the academic journal, Science, mdx mice are good models to utilize for replicating human disease and have a mutation in their gene regarding in a lack of dystrophin [8]. In this study, the mice had a nonsense mutation in exon 23 in the gene for dystrophin [8]. Thus, these mice all had significant protein deficiencies. A nonsense mutation is a mutation that codes for a premature stop codon, ending the protein synthesis before a function protein is completed [8]. This means that the mice do not have the ability to produce dystrophin because the stop codon halts the process. This represented a genetic problem in the mice that needed to be repaired using CRISPR technology. Groups of both normal and mdx mice were injected with elements of CRISPR [8]. There were also two control groups for both the normal and mdx mice that were not injected [8]. Restriction fragment length polymorphisms (RFLP) were used to identify the corrected gene in the mdx mice [8]. A RFLP is a useful tool in comparing two different sets of DNA, in this case the control and modified groups, which shows if the correction was successful [8]. The resulting mdx mice showed a mix of corrected and uncorrected cells [8]. Analysis of hind limb muscle, respiratory muscle, and heart muscle was performed on both normal, and mdx corrected mice which were all at seven to nine years of age [8]. The analysis showed some of the mdx mice had a complete absence of the typical signs of DMD in the mdx mice [8]. This means that the CRISPR system correctly removed the dangerous DNA and implemented a healthy sequence. This healthy sequence then effectively contributed to the production of dystrophin which allows for proper muscle functioning and growth. This result is a very positive sign. It proves that CRISPR can be applied to more complex organisms than just bacteria. The results of this testing show that CRISPR is effective in repairing the damaged muscles caused by the effects of DMD. This was shown by conducting muscle performance testing. In the testing, the cured mdx mice exhibited enhanced muscle performance compared to the uncured mdx mice [8]. Thus, the system had positive effects, not only in the DNA coding, but also in the terrible ramifications of this disorder. This finding is important to the widespread use of this technology. The vast capability of this system in genetic modification has hope for great success in human patients. The CRISPR Mechanism Compared to Other Methods The CRISPR system is not without limitations; however, it still compares favorably to other genetic modification techniques. For example, retargeting Cas9 is simple to do, since only twenty base pairs of crRNA need to be changed [7]. Other methods of genetic modification are TALEN and zinc fingers (ZNF). ZNFs are composed of many zinc ‘fingers’ that can target three to four base pairs and combining them allow it to target a specific area. However, ZNFs are expensive and time consuming to produce. TALENs are built from amino acid arrays which target a single nucleotide and can be arranged to target the gene. While TALENs are easier to produce and cheaper then ZNFs, both do not compare as favorably to CRISPR [7]. Unlike TALENs and ZNFs, CRISPR is not man made and is relatively simple which makes it much easier and cheaper to produce. Thus, the simplistic nature of CRISPR allows it to be more effective during the process of targeting, cutting, and replacing the genes. Another favorable aspect of CRISPR is that Cas9 cuts specifically between two base pairs while TALENs cleaves anywhere in a range of twelve base pairs [7]. This is very vital because imprecisely cutting the DNA can lead to off target mutations which can be detrimental to the health of the patient receiving treatment However, an important characteristic of all genetic modification techniques is that they all make positive mutations as well as off target mutations. The percentage of desired mutations achieved compares favorably for CRISPR in relation to TALENs and zinc fingers. CRISPR has achieved percentages of about 70% while TALENs and zinc fingers range between 1% and 50% [5]. This means that CRISPR makes a higher percentage of positive genetic mutations that can benefit the patient versus other methods. TALENS and zinc fingers result in some positive mutations, but also in more harmful genetic mutations compared to CRISPR. Off target mutations usually occur in regions where there are only a few base pair differences between it and the target region. Cas9 can tolerate up to five base pair differences [5]. This means that it can tolerate these differences without causing off target mutations. Thus, research has shown that CRISPR has the capability to provide a safer and easier method of performing genetic modification. This is important to the health of patients who need reliable technology that can cure debilitating disorders. THE ROLE OF CRISPR/CAS9 IN CURING DMD Currently, CRISPR is unable to cure human patients with DMD; however, ongoing achievements in this technology give a hopeful outlook. DMD is caused by 3 Aaron Mcvaugh Alexander Short processes are still being studied to minimize the potential of mutations in both the TALENs and CRISPR system. In general, the results of the comparison test proved there is no major overall difference in the effectiveness between TALENs and CRISPR. However, it was found that in using iPSCs pathways, the TALENs approach is not as reliable as using CRISPR, specifically in targeting DNA without having a large number of genetic mutations. This is an important characteristic of CRISPR that sets it apart from other gene modifying approaches. CRISPR proves to be more effective because it does not require as many genetic mutations. This is critical because the fewer genetic mutations that occur, the less likely the patient is harmed. The results of this study serve as a pathway to more complicated testing within the human body. The next step in this process would be to transplant the corrected iPSCs cells back into the patient [9]. From there, the iPSCs cells could merge with myofibers or, more efficiently, correct surrounding cells that will aid the myofibers [9]. Even though testing these methods in human cells is promising, there are currently a few problems that researchers most resolve. The faults lie in both the exon skipping and frameshift methods. For these methods to be considered successful, the US National Library of Medicine notes that the number of corrected genes would need to be great, since 30% to 40% of body weight is being affected [10]. This is very important to scientists because they cannot move on to human trials if the results are not significant enough to have a major effect on the body. In conclusion, this comparative study found that the genetic modification method of TALENs is not as effective as using CRISPR directly. Although this testing proves that CRISPR can be utilized in different situations to work with genetic modification, there are many more humanized tests that need to be performed. Dystrophin Gene Correction in iPSCs Derived from Human Cells FIGURE 1[9] Corrected and uncorrected myofibers Currently, experiments for human DMD correction are limited to lab conditions outside of the human body. One such laboratory experiment compared the usefulness of CRISPR in curing DMD to other methods of genetic modification. One specific method of comparison relates the TALENs system to CRISPR. TALENs, as discussed earlier, is another method of genetic modification that has been found to be similarly favorable in modifying defective genes, but is not always perfect in gene repair. Due to the process of developing these technologies, experiments for human DMD correction are limited to lab conditions outside of the human body. One study, comparing these two technologies, describes multiple correction pathways for curing DMD in patient-derived induced pluripotent stem cells (iPSCs), which are stem cells derived from adult cells. Per Science Direct, this study uses cells from a DMD patient with a deletion of a DNA coding region converted into iPSCs [9]. The results of the process can be seen in Figure 1 where the dystrophin gene is produced through by the combination of different steps, including, exon skipping, frameshifting and genome editing. These three steps are used to complete gene editing in both the CRISPR, as well as the TALENs technology. Studying both technologies by the same set of methods, allows the researchers gather information more effectively to compare TALENs versus CRISPR. This comparison test begins with insertion or deletion mutations, called indel mutations, cause the reading frame to shift, disrupting not only the immediate area of the mutation, but the rest of the gene sequence [9]. Exon skipping works to restore the reading frame, resulting in a dystrophin protein that is partially functional [9]. The next step, frameshifting, attempts to restore the reading frame by making more indel mutations and shifting the frame further [9]. Finally, the system is ready to perform gene editing procedures. Although these steps can produce positive results, the fact of having mutations is not always a promising sign. However, these CRISPR in the Future: Challenges to Overcome The largest concern regarding CRISPR and other genetic modification technologies is the chance of off target mutations to the genome. As previously stated, Cas9 will tolerate differences up to five base pairs in the crRNA and will not tolerate any differences in the PAM sequence. The zero tolerance in the PAM sequence is limiting, but necessary. Thus, any use of CRISPR must also analyze potential sites for off target mutations. Even then, some areas might contain so many high potential sites that CRISPR would be ineffective. In addition, iPS cell transplantation after correction still has not been fully developed and embryo correction is also not currently possible. CRISPR is a promising tool and its development will undoubtedly open new doors for treatment for DMD and beyond. However, no single tool or method has been proven to be perfect and thus each treatment must be specifically tailored to ensure safe and positive outcomes. 4 Aaron Mcvaugh Alexander Short health of future generations. This technology can allow for children to grow, free of disorders that they would normally contract from their parents. Thus, the ability of this procedure would be life saving for many families and individuals. However, there is simply not enough justification of the CRISPR technology to allow for its use in genome editing at its current state. There is great hope that this technology can be a bridge to a new age of treating patients with not only muscular dystrophy but with other degenerative diseases. Yet, there are too many ethical concerns for this system to be put into clinical use. It is not yet proven that CRISPR will not attack the wrong DNA strand or cause other genetic mutations. Thus, because of the lack of evidence to prove otherwise, the rule and codes of ethics overpower the benefits of this unique technology, at this time. ETHICAL CONSDERATIONS OF CRISPR TECHNOLOGY The great potential for this technology is exciting to both researchers and scientists. However, since this technology is still in the research stage of development, scientists and bioengineers must discuss other factors as well. Scientists must consider the different impacts CRISPR would have on society if/when it became introduced into the market. Per the Journal of Clinical Research and Bioethics, “An important ethical issue in research is that benefits must be greater than risks. Greater attention must be placed on risks, since they may damage living beings or the environment.” [11]. These are two very important pieces of information, because even though CRISPR has great positive potential as a technology, we must find a balance between the benefits and the risks. One concern is that large genomes may contain identical or homologous DNA sequences to the targeted harmful DNA. [11]. CRISPR/Cas9 may split the unintended DNA sequences causing mutations and even cell death [4]. Due to this, improvements have been made to reduce off target mutations as scientists try to ensure that the benefits of CRISPR greatly outweigh its risks. Another source of ethical consideration is in the possibility of genome editing in human germline [11]. This is the genome that can be passed on to future generations by gametes (fertilized eggs) [11]. There is concern that the CRISPR/Cas9 system can produce mutations and side effects that may transmit to future generations [11]. This is concerning to researchers because at the current time, they are unsure if there would be side effects that would cause harm to offspring. This alarm also goes hand-in-hand with the inability of unborn children to give consent on such matters. Great care and thought should be taken because these risks could potentially affect their lives [4]. Currently, the risks of these side effects greatly outweigh the potential benefits, causing this method of CRISPR to be postponed until further studies can prove otherwise. In order for this technique to be ethically safe for future generations, scientists need to develop the system with little to no potential side effects of mutations. Even though there are still serious concerns regarding germline genome editing, very invaluable aspects to using CRISPR in germlines exist. According to BioMed Central, CRISPR can be used to improve the human condition of offspring and future generations through the eradication of deleterious mutations [12]. The article states, “It might allow people who carry such deleterious traits to have children to whom they are genetically related without the prospect of passing on problematic or dangerous conditions. To the extent these changes would persist across the generations, it could benefit not only the immediate offspring, but also all of the descendants of those who use the technology.” [12]. This potential benefit is groundbreaking in the field of health and medicine. CRISPR has the capability to affect the wellbeing of not only people of the current age, but also to protect the Sustainability and CRISPR CRISPR not only has many different ethical considerations, but it also has an important foothold in the field of sustainability. UCLA says, “sustainable development is development that meets the needs of the present without compromising the ability of future generations to meet their own needs” [13]. This definition relates to CRISPR because the entire basis of the technology is working to meet the needs of current day people. Presently, there are not many treatments for genetic disorders, such as DMD, that are effective. Some of the available treatments also hold the risk of causing off target mutations in a patient’s genome. CRISPR technology is a system that can be proven to meet the needs of people without harming their livelihood. CRISPR can also be utilized to enhance the livelihood of future generations, as well. CRISPR is being studied to provide a safer and more effective technique to dispose of harmful DNA in the genome. Based on tests, CRISPR has been more effective in restoring the dystrophin gene to end the effects of DMD than other methods such as TALENs and zinc fingers. Not only is CRISPR more beneficial, but it also comes with less chance of producing genetic mutations. This clarifies the effect CRISPR has on sustainability. This technology is being designed with the current needs of people in mind and it has the potential to service these needs with little chance of harm. It also does not compromise the needs of the future because a possible function of its design is to eliminate the traits of genetic diseases in offspring so that they do not carry on their parents’ genetic disorder. This technology was designed with not only short term goals in mind, but is also geared toward the future and the positive effect it can have on generations to come. Thwink.org defines sustainability in a different manner. It states that sustainability is “the ability to continue a defined behavior indefinitely” [14]. This definition brings up a different spin on sustainability than the previous one. The concept behind this definition is that if something is sustainable it is kept in a similar form or state for a long period 5 Aaron Mcvaugh Alexander Short of time. CRISPR technology meets this definition of sustainability because it preserves the quality of life, indefinitely once it is utilized. Genetic disorders can significantly disrupt the health of the individuals. CRISPR has the potential to cure genetic disorders through genetic modification and can eliminate the harmful effects of these terrible diseases. Many people are in need of better healthcare technology and more efficient methods to improve their overall health. The functionality of CRISPR provides a cornerstone for better technology that can improve the overall wellbeing of many patients immediately and then sustain that health over the long term. CRISPR can help to improve the health of people in the present, while also protecting offspring of the future. This dual nature of this revolutionary system clearly shows how it fits perfectly into the concept of sustainability. Sustainability evolves around improving the overall condition of life, in the present state and sustaining that improved condition, while not compromising the state of health of future generations. CRISPR can transform the world of genetic modification, improving lives in the current state, maintaining this improved state of health, and improving the health of future generations. The concept and study of sustainability is very important to the work and efforts of all engineers. The design of CRISPR and its many abilities serve as a perfect mixture of meeting current needs while not compromising future needs. Its capabilities are unique and are fitted to prove long sustainability for the health of current and future patients. Therefore, CRISPR plays an essential role in sustaining quality of life. discovery. The purpose of CRISPR to help cure DMD in humans follows the same basic steps as it would in protecting bacteria. This technology has been proven, not only in a lab, but in nature, by protecting a very simple version of life, bacteria. The CRISPR/Cas9 system is very complex and it involves many different types of systems and tools to perform various functions inside a bacterium. However, the most important function for humans is the potential to remove bad DNA. To do so, CRISPR follows a series of important steps that are crucial to its ability to perform genetic modification. CRISPR can easily be delivered to an organism’s system. Its main components include crRNA, tracRNA, Cas9, and a repair template for DNA. These components are essential to the specific functions of this technology. They are involved in targeting the harmful DNA so that the Cas9 protein can dissect the DNA. This dissection is where healthy, replacement DNA can be transferred. This method of repairing DNA sequences is specific to its purpose, as well as revolutionary in the field of biomedicine. This process also has an upper hand over the less reliable methods that can be related to producing a fair amount of genetic mutations. Thus, this system is on the verge of innovative medical technology that can outperform older and more inaccurate methods. One of the biggest potential uses of this technology is in the form of curing DMD. Even though this apparatus is in the early stages of development, it shows positive signs or a hopeful outlook. Tests on mice have shown that the possibility of using CRISPR on human organisms to cure DMD is not far into the future. When tested on mice almost half off the alleles that were corrected by the CRISPR process had no signs of DMD. Even though this value may seem low, it is a very positive sign for a technology that is in the early stages of development. This advancement gives way for CRISPR having the potential to cure more complex multicellular organisms, such as humans. This technology has the capability to save people’s lives in many different fashions. However, one very important possibility is curing DMD which is a debilitating and treacherous disorder. Early success in development, as well as the long history of CRISPR, has shown glimpses of this technology’s potential. Even though there are ethical concerns, they are only prominent because of the preliminary stages of this system. When looking past these concerns, the road ahead for CRISPR is a very promising one. Even with many more trials and tests along the way, there is a light at the end of the tunnel for a truly inspirational and exhilarating technology. CRISPR HAS THE POTENTIAL TO BE A BREAKTHROUGH TECHNOLOGY Even though CRISPR technology is still in clinical stages, it is very evident that this system has bright hope for a very prominent future. The potential to virtually wipe out medical disorders and diseases such as Duchenne Muscular Dystrophy (DMD) is very appealing to scientists in the field of medicine. CRISPR would virtually remove damaging DNA and replace it with a healthy strand. The combination of using the functions of CRISPR, as well as the Cas9 system provide a powerful one-two punch to knock out even the worst genetic disorders, such as DMD. This approach can save lives of not only current patients, but also has the possibility to work in the human germline to help prevent this disease from being passed on to offspring. A system that can perform genetic-type healing has great value in the field of medicine and bioengineering. This technology’s budding future stems from the origins of its history. Scientists over the past years have worked to perfect and improve this system in hopes of progressing it out of clinical trials. As more research has been completed, the capability of this technology has increased. However, the basic functionality of CRSIPR can be traced back to its early SOURCES [1] “What is Biotechnology?” The Biotechnology and Medicine Education Trust. 2016. Accessed 2.24.2017. http://www.whatisbiotechnology.org/science/crispr 6 Aaron Mcvaugh Alexander Short [2] “Duchenne Muscular Dystrophy (DMD)” The Muscular Dystrophy Association. 2017. Accessed 2.26.2017 https://www.mda.org/disease/duchenne-muscular-dystrophy [3] J. Kaiser. “CRISPR helps heal mice with muscular dystrophy.” Science. 12.31.2015. Accessed 1.9.2017. http://www.sciencemag.org/news/2015/12/crispr-helps-healmice-muscular-dystrophy [4] “CRISPR Timeline” Broad Institute. 2017. 2.24.2017 https://www.broadinstitute.org/what-broad/areasfocus/project-spotlight/crispr-timeline [5] A. Reis, B. Hornblower, B. Robb, G. Tzertzinis. “CRISPR/Cas9 and Targeted Genome Editing: A New Era in Molecular Biology” New England Biolabs inc. 2014. Accessed 1.10.2017. https://www.neb.com/tools-and-resources/featurearticles/crispr-cas9-and-targeted-genome-editing-a-new-erain-molecular-biology [6] M. Terns, R. Terns. “CRISPR-Based Adaptive Immune Systems” HHS Public Access. 04.29.2011. Accessed 2.28.2017. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3119747/ [7] N. Protoc. “Genome engineering using the CRISPR-Cas9 system” HHS Public Access. 10.24.2013. Accessed 2.28.2017. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3969860/ [8] C. Long, J. McAnally, J. Shelton, A. Mireault, R. BasselDuby, E. Olson. “Prevention of muscular dystrophy in mice by CRISPR/Cas9–mediated editing of germline DNA” Science. 5.9.2014 Accessed 1.10.2017 http://science.sciencemag.org/content/345/6201/1184.full [9] H. Li, N. Fujimoto. “Precise Correction of the Dystrophin Gene in Duchenne Muscular Dystrophy Patient Induced Pluripotent Stem Cells by TALEN and CRISPR-Cas9” Cell Press. 1.13.2015. Accessed 3.1.2017 http://www.sciencedirect.com/science/article/pii/S22136711 1400335X [10] A. Hotta. “Genome Editing Gene Therapy for Duchenne Muscular Dystrophy” IOS Press. 9.22.2015 Accessed 3.1.2017 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5240599/ [11] E. Rodriguez. “Ethical Issues in Genome Editing using Crispr/Cas9 System.” Journal of Clinical Research and Bioethics. 3.24.2016. Accessed 2.28.2017. https://www.omicsonline.org/open-access/ethical-issues-ingenome-editing-using-crisprcas9-system-2155-96271000266.php?aid=70914 [12] D. Carroll, R. Alta Charo. “The societal opportunities and challenges of genome editing.” BioMedCentral. 11.5.2015. Accessed 1.9.2017. https://genomebiology.biomedcentral.com/articles/10.1186/s 13059-015-0812-0 [13] “What is Sustainability” UCLA Sustainability. 2016. Accessed 3.18.2017. https://www.sustain.ucla.edu/about-us/what-is-sustainability/ [14] “Sustainability” thwink.org. 2014. Accessed 3.18.2017. http://www.thwink.org/sustain/glossary/Sustainability.htm ACKNOWLEDGEMENTS We would like to thank our writing instructor Professor Zellmann for guiding us throughout this writing process. We would also like to thank our session co-chair Mikayla Ferchaw for her continued advice and support. Her knowledge about this assignment is very important to our success and she has been a great help. We are very grateful for their support during this paper and we are thankful for their aid in creating the final portion of this paper. 7