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The overall purpose of this research is to go into depth about what is crispr, the human benefit, the ethical dilemma behind the technology, and what this technology means for future generations. This research paper will touch on several different subjects related to the emerging new technology by going into its history, current use, and future ways of use. This technology is still in the infant stage and has not been used to treat disease or the human genome widespread, as of yet. From my interpretations the overall human benefit and ethical background of the technology is not yet determined.
The first objective of my first paper is to go into depth about the background of crispr, what the different mechanism of crispr is, and how it all works together to perform the task. My findings pointed me to the ability of crispr to not only edit the human genome but any organism including animals and plants. The significance of this was to learn the important parts of crispr that make it function. This was important to give a history of the device so the reader can see why and how it is used the way it is today. The conclusion of my first paper is that crispr is an old technology with new updates and that the protein cas9 plays a significant part in the editing process.
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The second objective of my second paper is about the human benefits and what it can do for the general population in a positive way. The findings I found on the human benefits led me to learn about how crispr can go in and edit specific genes to fight genetic mutations, cancer, and diseases. The significance of this to my paper is to show the real life benefits that can be provided to humans and to demonstrate successful trials that prove the technology is advancing.
The third objective of my third paper was to show the ethical dilemma that will come with being able to edit the human genome and germline cells. My findings led me to learn about how different agencies are already set up to monitor and control how animals are edited and used, and most likely something similar will be set up for humans. The significance of this section to my paper is to discuss the main topic, the direction the technology will go in, and to show the aspects on how crispr can be used for good and corrupt reasons.
- The History of CRISPR
Crispr is an emerging biotechnology that has endless possibilities. This technology is not new, but the use of it is just now being explored. Crispr has the ability to transform human life and the experience in dramatic ways. Crispr is an acronym that stands for clusters of regularly interspaced short palindromic repeats, and it is short for crispr-cas9. The term crispr was coined in two thousand and two by Jansen and Mojica (Hsu). The cas9 portion of crispr is a protein that is used as an enzyme that cuts the DNA. The cas9 is an RNA-guided endonuclease and can be targeted to any genome site to do the DNA cutting (Hsu). The idea was borrowed from seeing natural defenses in the body work from mechanisms used by bacteria. Bacteria uses RNA and various cas9 proteins to stop attacks by chopping and destroying the DNA of foreign bodies (Hsu). The recombinant DNA technology or at least the idea has been around since the nineteen seventies (Hsu). This new discovery allowed scientists to study the human genome and its genes, thus opening the door to fighting disease, age, and anything else. Crispr at its core is a biotechnology that can be used to edit genes essentially. This genome engineering specifically refers to targeting the genome, eukaryotic cells, and mammalian cells (Hsu). Genome engineering would give us the ability to change animals, drug development, food production, and offer genetic variation (Hsu). Scientists would have the ability to manipulate and change anything to enhance human needs. The crispr technology would mean the ability to delete, insert, and modify DNA. One of the many breakthroughs that brought this technology to the forefront and expedited the process to develop it fully was the development of gene targeting. The gene targeting is done by homologous recombination, which is a “knock in” and “knockout” method (Hsu). The homologous recombination is very specific with targeting but the desired recombination events are infrequent (Hsu). This has only been tested in animal models so far, but shows that the next step is still to get the recombination down to a precise accuracy. The problem is being assessed and a new method is to use the target cas9 and introduce short guide RNA instead of large bulky proteins (Hsu). The use of synthetic biology can be used to design and build, but in many areas not just the human genome. The family of cas9 proteins is characterized by two signature nuclease domains, those being RuvC and HNH (Hsu). RuvC is a full domain and split into three subdomains, and the HNH is a single nuclease domain.
- The Human Benefit of Using CRISPR
Above all, Crispr offers new hope for people living with untreatable diseases or diseases that do not have many options for treatment. Crispr not only can edit the human genome but can also personalize medicine to be used for a certain individual fitting their bodies’ exact needs. This would result in the side effects of medicine to decrease and increase healing time among patients, and also speed up new drug discoveries. Crispr would offer the ability to cure or even eradicate certain human disease and cancers, because it would open the doors for new treatments and disease manageability. Many current crispr trials are focusing on cancers, muscular genetic mutations, such as duchenne muscular dystrophy, and HIV/AIDS. Since this technology is still very new, it should only be offered to those who have exhausted all other treatment options. For some monogenic disease the cas9 can be used to delete duplicated genes or replace the gene to work again (Hsu). Crispr itself can be used to mutate somatic tissues and engineer therapeutic cells for HIV. Clinical trials successfully used ZF nucleases to combat HIV infection by knockout of the CCR5 receptor. In all patients, HIV DNA levels decreased, and in one out of four patients, HIV RNA became undetectable (Tebas et al., 2014). These results show that programmable nucleases can and do work. With the HIV virus crispr can be used to edit t cells to detect and destroy the virus, instead of being destroyed by it. Chimeric antigen receptor (CAR) T cells can be modified ex vivo and reinfused into a patient to specifically target certain cancers (Couzin-Frankel, 2013). Chinese scientist even did a trial where they took out the protein pd-1, which stops a cells immune response preventing or stopping cancer (Xinbing). Testing on mice does show that genetic disease can be reversed using crispr and also that crispr be used as a prevention tool. Recently, hydrodynamic delivery of plasmid DNA encoding Cas9 and sgRNA along with a repair template into the liver of an adult mouse model of tyrosinemia was shown to be able to correct the mutant Fah gene and rescue expression of the wild-type Fah protein in ∼1 out of 250 cells (Yin et al., 2014). All of these possibilities can become a reality in the future if scientists can get the delivery system to be more specific. Successful clinical translation will depend on appropriate and constructive delivery systems to target specific disease tissues (Hsu). These small trials are just a small scale of the number of genetic diseases and cancers that have to be tested or treated. To achieve high levels of therapeutic efficacy and simultaneously address a broad spectrum of genetic disorders, crispr will need to be significantly improved (Hsu). The delivery system is not at that stage of development yet to take on a broad spectrum of diseases. The technology is still in an infant stage, so biotechnologists/scientists cannot predict what long term effects of changing a person’s genome could mean. It is favorable to do a long term solution over repeated short term solutions, but the implications still remain unclear for long term effects (Hsu). Safety and physiological effects will need to be noted before this technology could become widespread use amongst the general public. Crispr goes beyond just disease and genetic immunity, but also into helping modify our food. Crispr could be used to increase the muscle mass of animals, render farmed animals less susceptible to disease, enhance nutritional content, or create hornless cattle that are easier to handle (Caplan). Crispr can be used to genetic crops to be resistant to environmental deprivation or pathogenic infection (Hsu). This could put food in places that have a problematic time growing food or have famine problems among the population. The ease of design and testing of Cas9 may also facilitate the treatment of highly rare genetic variants through personalized medicine (Hsu). The development of personalized drugs would cut down on patients becoming resistant to the drugs they are or at least increase the time they don’t become resistant. Agriculturally important plants have been genetically manipulated to make these less susceptible to disease and pests, more productive, and more resilient to changing climates (Caplan). Crispr could open up the future to different fuel alternatives with a new and improved version of ethanol.
- The Ethical Dilemma and Concern
Equally important, the technology crispr offers many new possibilities for humans and the future of medicine. So many questions still remain unclear and the long term impact has yet to be determined, and with that comes numerous ethical issues. With most new technology it comes with an ethical dilemma, and especially since this one will be able to change the human genome for both disease and cosmetic purposes. The most serious debate and ethical concerns come from the ability of crispr to edit germ line cells, not only will those cells be passed down to future generations but also along with any genetic change that occurred. Germ line cells and eventually embryos will be able to be edited anyway an individual likes opening up the door for “designer babies”. Opening the door for cosmetic editing would give people and usually the ones who could afford to make their child “better” in several ways than a baby born without any genetic modification(s). The ethical concerns that arise come from crispr is the question should this technology be opened to the public for cosmetic purposes or leave it just for medical reasons. The crispr technology would give us the ability to be immune to a range of disease, thus allowing us to live longer healthy lies. Crispr could theoretically directly target harmful human mutations. The concern is that public misunderstanding and mistrust of GMOs will hinder scientific progress and valid uses of crispr (Caplan). GMOs are currently looked at by some of the population as not being natural, and not in positive manner. The monotheistic following religious public could look at furthering the crispr technology into germ line cells and embryos as a way that scientist are playing God. Currently crispr modified animals and insects are regulated by the he Coordinated Framework for the Regulation of Biotechnology, which was created in 1986, the Food and Drug Administration, US Department of Agriculture, and the Environmental Protection Agency (Caplan). Crispr with its ability to edit the genome of any organism, can make insects or animals to become infertile or carry a specific disease. Scientist are currently working with the Aedes aegypti mosquito which transmits the dengue fever, to introduce a gene that will block the transmission of it from the female mosquitos (Caplan). If these genetically modified animals were to get introduced into the environment, entire ecosystems could be thrown off balance. Crispr is opening the door for genetically modified organisms to be introduced into the environment and how that would affect natural animal populations over long term is yet to be determined. Another issue would be the studying of a genetically modified humans and who oversees the child or is that child kept in a lab, because he/she still is technically a person. Crispr could also be used for negative impact in the hands of bio terrorist. The ease and efficiency of CRISPR raises the concern that anyone with the appropriate equipment could engineer a vaccine-resistant flu virus, which could kill millions around the globe (Caplan). This leads to crispr having to be regulated not only domestically here in the United States but also internationally, thus becoming a broad apparatus to control. All of this concludes toward the question that arises with many new scientific breakthroughs of how far is too far?
In the final analysis, crispr opens up an entire new world of possibilities for the human life. This technology could dramatically improve human life for everyone across all fields drastically in areas such as cancer immunotherapy, biofuel, pollution techniques, disease modification, and medicine production. The ethical debates will be a very severe debate in the upcoming decade as the crispr technology advances. The foremost concerns will arise from the what, why, and when is crispr allowed to be used. The core debate will be about the entity who will oversee this, because eventually it is an individual’s own genome and genetic succession line. I for one am excited for the future development and advancement of crispr to see how far we can go with this technology.
- Applications of cas9 as A Genome Engineering Platform
(A) The Cas9 nuclease cleaves DNA via its RuvC and HNH nuclease domains, each of which nicks a DNA strand to generate blunt-end DSBs. Either catalytic domain can be inactivated to generate nickase mutants that cause single-strand DNA breaks.
(B) Two Cas9 nickase complexes with appropriately spaced target sites can mimic targeted DSBs via cooperative nicks, doubling the length of target recognition without sacrificing cleavage efficiency.
(C) Expression plasmids encoding the Cas9 gene and a short sgRNA cassette driven by the U6 RNA polymerase III promoter can be directly transfected into cell lines of interest.
(D) Purified Cas9 protein and in vitro transcribed sgRNA can be microinjected into fertilized zygotes for rapid generation of transgenic animal models.
(E) For somatic genetic modification, high-titer viral vectors encoding CRISPR reagents can be transduced into tissues or cells of interest.
(F) Genome-scale functional screening can be facilitated by mass synthesis and delivery of guide RNA libraries.
(G) Catalytically dead Cas9 (dCas9) can be converted into a general DNA-binding domain and fused to functional effectors such as transcriptional activators or epigenetic enzymes. The modularity of targeting and flexible choice of functional domains enable rapid expansion of the Cas9 toolbox.
(H) Cas9 coupled to fluorescent reporters facilitates live imaging of DNA loci for illuminating the dynamics of genome architecture.
(I) Reconstituting split fragments of Cas9 via chemical or optical induction of heterodimer domains, such as the cib1/cry2 system from Arabidopsis, confers temporal control of dynamic cellular processes.
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