CRISPR-Cas9 Gene Editing

By: Amy Hammett

Introduction

CRISPR, which is short for clustered regularly interspaced short palindromic repeats, was originally discovered by Ishino from Osaka University in Japan in 1987 in the bacteria Escherichia coli. However, during the time of its discovery, it was difficult to study these particular DNA fragments, which meant their understanding of its significance was limited. Years later in 1995, a breakthrough occurred by Francisco Mojica from the University of Alicante where similar structures were discovered in the Haloferax mediterranei. Since similar structures were found in different classes or prokaryotes, its evolutionary significance was realized. Since then, further research has been done and with the studies of Rodolphe Barrangou and Philippe Horvath, it was found that the spacers that existed caused a unique acquired immune system within these prokaryotes. Since then, the project of CRISPR had begun and was transformed in ways that it could be applied to eukaryotes, such as humans. What caught the attention of several scientists was the evolved and acquired immune system of prokaryotes known as CRISPER-Cas. This interest expanded into the project CRISPR-Cas9; a method designed for gene-editing.

Procedure

The term CRISPR itself is a genomic locus containing multiple short repetitive repeats that are separated by other sequences called spacers. In simple terms, there are three steps of CRISPR-Cas9’s method: recognition, cleavage, and then repairment. Cas proteins are divided into multiple classes of Class I, which are made of several subunits of Cas-protein complexes, and Class II, which employ only one Cas-protein. Cas9 is a DNA-cutting enzyme that is employed to cut specific sections of DNA in a more detained and refined way, which could allow individuals to genetically modify a person’s traits through insertion of new genetic material or the removal of already existing genes. The enzyme consists of two regions named recognition lobe and nuclease lobe. The recognition lobe is made-up of recognition I and recognition II domains that are responsible for binding the guide RNA, and the nuclease lobe consists of RuvC and HNH domains that are used to cut single-stranded DNA along with an interacting domain called Protospacer Adjacent Motif (PAM), which is responsible for initiating the binding of targeted section of DNA. The cutting enzyme, Cas9, is guided by a “single guide RNA,” which is also known as crRNA, to the specified targeted section of the genome for modification. The guide RNA consists of two parts known as CRISPR RNA, or crRNA, and trans-activating CRISPR RNA, or tracrRNA. crRNA is responsible for specifying the target DNA sequence and tracrRNA served as a “binding scaffold for Cas-9 nuclease” (Asmamaw and Zawdie). For genetic editing, crRNA and tracrRNA can be combined to form a guide RNA, also known as sgRNA, to target any desired genetic sequence that one wishes to edit. As many researchers and scientists have continued to experiment with this technique, several questions regarding its ethicalness have surfaced due to its wide range of potentials usages.

Applications

CRISPR-Cas9 has a wide variety of applications in many areas such as the medical field, biotechnology, agriculture, and many more. One of the main aims of usage for this technology is to cure, or eliminate, diseases that are encoded in our genome. This would be done by removing and replacing the disease-causing gene with a new portion of DNA. Such diseases include, and are not limited to, sickle cell disease, cystic fibrosis, and muscular dystrophy. Not only can application of CRISPR-Cas9 eliminate genetic diseases, it can also be used to artificially regulate the activation or repression of a targeted gene by a further modified Cas-9. Additionally, the technique can aid researchers by distinguishing certain genes by combining it was marker with Cas-9. In the field of agriculture, there has been a lasting concern regarding resources, and the need for new methods of increasing food production has risen. With CRISP-Cas9, scientists have the ability to genetically modify foods to elevate nutritional values along and resistance towards disease along other things.

Issues

Even though CRISPR-Cas9 has proven to significantly useful in its applications regarding medicine, agriculture, and several other areas, much debate concerning its negative consequences and ethicalness has been brought about. This is due to its powerful nature of genetic modifications that can be applied to a wide variety of organisms, such as humans, and one of the main concerns is the misuse of the tool for things beyond its original purpose. One such concern is mentioned in the article, “Anticipating emerging biotechnology threats” by Kathleen M. Vogel and Sonia Ben Ouagrham-Gormely that was published in 2018. This article mentions the potential emergence of harmful biotechnological weapons through the use of CRISPR-Cas9 such as chimeric bioweapons, which are devices that cause the manifestation of one disease but damage the body with another disease. In another article named “Redesigning nature: to be or not to be?” published in 2017 by Jayashree Das, Pritha Dey, and Pradipta Banerjee, the article discusses controversies surrounding CRISPR-Cas9’s application towards genetic editing. While the intention of CRISPR-Cas9 is to help eradicate harmful defected genes form the human genome, it can be used in less purposeful ways. The article states “scientist have come across genes that help shape contours of the face…genes controlling features of the body is known…jump to genes controlling intelligence, and muscle and fat deposition” (Das, Dey, and Banerjee 1346). Additionally, the authors mention the term “designer babies” where children have their features genetically modified while in the mother’s womb (Das, Dey, and Banerjee 1346). This issue is also shared by the article of “Everything in Moderation, Even Hype: Learning from Vaccine Controversies to Strike a Balance with CRISPR” by Shawna Benson from 2017. Once again, CRISPR has the potential to alter genetic traits, which have been “rendered eugenic,” (Benson 819). Therefore, along with the previous article mentioned, both share the concern of genetic alteration leading to a “genetically engineered society favoring certain traits – intellectual, cognitive, emotional, physical or racial-above others” (Benson 819).           

Each of these articles discussed major issues that have the potential to arise by having CRISPR integrated into society. Even so, not all of these articles focused solely on the negative effects of CRISPR. For instance, Beson’s article primary brought attention to CRISPR’s beneficial aspects and rejected the idea of completely shutting down the project altogether. Due to CRISPR’s dual natured methodologies, debate by researchers, scientists, and ethicists have continued internationally. In addition, safety protocols and applications have also been discussed as CRISPR could easily be manipulated into creating a tool meant for harm.

Opinion

While I do believe CRISPR holds significant potential that could vastly alters the way we look at medicine and produce resources, I also believe it is equally as important to address and consider such concerns mentioned in the articles included above. With CRISPR, doctors and researchers will have the power and ability to eliminate heritable, or genetic, diseases that we once deemed uncurable, which could save and improve hundreds if not thousands of lives worldwide. This new emerging technology could also prove helpful in the issue regarding food. As the world population has surpassed eight billion people, the question of how to be able to feed that many people have been a rising concern, and with CRISPR, scientists have a new method of improving the issue by genetically modifying crops. However, this also brings the question if the positive outcomes of CRISPR are enough to overcome the negative consequences that could occur. Yes, CRISPR can basically revolutionize the medical field by correcting defective genetic diseases, but CRISPR can also cause and/or create diseases. It can also be heavily predicated that in times of tension and violence between organizations and/or foreign countries, CRISPR-Cas9 would be heavily abused to create new weapons of destruction which completely goes against its original purpose of healing and improving society. I also believe that CRISPR could transform the meaning of plastic surgery. Already plastic surgery has been used in extreme situations for the simple purpose of changing ones looks. Originally, plastic surgery was used for reconstruction to improve the function of a certain part of the body, but now, it’s largely used for cosmetic purposes. Since CRISPR’s applications could easily fall under the same blanket as cosmetic plastic surgery, who says it will not be used in such ways? These are only some concerns and ideas that I have thought of, and there are many more controversies surrounding this topic.

Works Cited

Asmamaw, Misganaw, and Belay Zawdie. “Mechanism and Applications of CRISPR/Cas-9-Mediated Genome Editing.” Biologics : targets & therapy vol. 15 353-361. 21 Aug. 2021, doi:10.2147/BTT.S326422

Benston, Shawna. “Everything in Moderation, Even Hype: Learning from Vaccine Controversies to Strike a Balance with CRISPR.” Journal of Medical Ethics, vol. 43, no. 12, BMJ, 2017, pp. 819–23, https://www.jstor.org/stable/26879628

Gostimskaya, Irina. “CRISPR-Cas9: A History of Its Discovery and Ethical Considerations of Its Use in Genome Editing.” Biochemistry. Biokhimiia vol. 87,8 (2022): 777-788. doi:10.1134/S0006297922080090

Gutmann, Amy, and Jonathan D. Moreno. “Keep CRISPR Safe: Regulating a Genetic Revolution.” Foreign Affairs, vol. 97, no. 3, Council on Foreign Relations, 2018, pp. 171–76, http://www.jstor.org/stable/44822154.

Hille, Frank, and Emmanuelle Charpentier. “CRISPR-Cas: Biology, Mechanisms and Relevance.” Philosophical Transactions: Biological Sciences, vol. 371, no. 1707, 2016, pp. 1–12. JSTOR, http://www.jstor.org/stable/26143993. Accessed 27 Oct. 2023.

Ishino, Yoshizumi et al. “History of CRISPR-Cas from Encounter with a Mysterious Repeated Sequence to Genome Editing Technology.” Journal of bacteriology vol. 200,7 e00580-17. 12 Mar. 2018, doi:10.1128/JB.00580-17

Kozubek, James. “Crispr-Cas9 Is Impossible to Stop.” Georgetown Journal of International Affairs, vol. 18, no. 2, Georgetown University Press, 2017, pp. 112–19, http://www.jstor.org/stable/26396025.

Vogel, Kathleen M., and Sonia Ben Ouagrham-Gormley. “Anticipating Emerging Biotechnology Threats: A Case Study of CRISPR.” Politics and the Life Sciences, vol. 37, no. 2, Cambridge University Press, 2018, pp. 203–19, https://www.jstor.org/stable/26677575.