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CRISPR

Many people have heard of CRISPR, a revolutionary technology developed over the past few decades. Many scientists have been discussing the ethics behind this technology, but most people still need to learn the precise information surrounding this powerful tool. CRISPR stands for “Clustered Regularly Interspaced Short Palindromic Repeats.” This acronym refers to the genome of bacteria or archaea (prokaryotes) with short repeats of the same genetic code. However, between these repeats, there are “spacers” that the Cas enzyme, an “endonuclease” that cuts the DNA in specific places, uses to hold parts of DNA from viruses and bacteriophages to help defend from future infections. This new technology allows gene editing and enables scientists to modify genetic material to cure diseases such as sickle cell disease, muscle dystrophy, and blindness. Scientists look forward to combating future diseases — even cancer, with CRISPR. CRISPR is an evolutionary defense mechanism consisting of a Cas enzyme and Guide RNA. Initially found in E. coli, the system fights off bacteriophages, viruses that infect bacteria. When the bacteriophage injects its foreign genetic material (DNA or RNA) for the first time, the E. coli cuts off a small part of the DNA using the Cas enzyme. Crispr stores this in spacers in between repeats. These repeats serve as a library of old infections similar to Memory T and Memory B cells in the human immune system (cell-mediated and antibody-mediated lymphocytes that fight off our infections). When the bacteria are infected with the same bacteriophage, the Cas enzyme uses the spacer as its guide RNA to disable the genetic material of the bacteriophage. Crispr utilizes a Cas enzyme and an RNA guide, which can be changed depending on the gene the scientist wants to find. The repeats are then formed into crRNA, combined with a case enzyme to form the effector complex. The RNA guides Cas as it searches through DNA until it finds a match for the specific guide RNA. Cas is a precise genetic nuclease that can cut the gene at specific spots. When the cell tries to fix the cut through new bases, likely, the cell will not add the original base pairs, and the gene will be disabled. However, scientists can make this process more precise by adding a custom DNA sequence that pairs with the cut polynucleotide. Non-Homologous End Joining (NHEJ) is a natural cell repair when the enzyme performs a double-strand break (DSB). NHEJ is the process that cells use to repair the DNA; this process is exceptionally inaccurate and can lead to mutation, such as insertions or deletions. Scientists can also perform Homology directed repair (HDR), which requires a donor template that researchers can create for a more precise repair. However, NHEJ can occur anywhere in the cell cycle, while HDR is usually used during the G2 or Synthesis stage, which means that HDR is ineffective in post-mitotic cells such as neurons.



Classification of Cas enzymes


The different types of enzymes are all used differently in three stages: adaptation, Biogenesis, and interference. The adaptation usually finishes using Cas 1 or Cas 2 systems. Enzymes recognize the Viral DNA through a Protospacer Adjacent Motif (PAM), which is usually on the viral DNA and varies between enzymes. Biogenesis is when CRISPR Cas enzymes process and transcribe the crRNA, the guide RNA. Enzymes such as Cas 6 are used in Biogenesis. Class 1 enzymes have multiple proteins and enzymes in their effector complexes. In contrast, Class 2 enzymes only have a single protein in their effector complexes. Both of these classes are used in the interference stage where Class 1 uses a (CASCADE) and Class 2 uses a single effector protein.



Cas 9

Cas 9 is a class II single effector endonuclease that acts as a precise gene scissor. Cas 9 has been one of the most promising enzymes as it makes cuts that are “blunt ends,” which are convenient for gene editing. Although similar, cas12 makes sticky ends which staggers the DNA.


Current Uses

Some of the current uses of CRISPR include CRISPR-based screens, which help identify drug targets to find which gene a certain drug affects and is much more effective than the past option of RNAi (RNA interference), which would be proven insufficient to affect the phenotype in some cases.


There have also been studies on the cure of Type one diabetes, an auto-immune response where the body attacks the beta cells in our pancreas. Beta cells are necessary to produce insulin; without them, the body cannot survive. Current treatments include transplanting these cells into humans. A study has shown positive pre-clinical mesenchymal stem cell (MSC) results. However, results plummeted, and many trials resulted in clinical failure. A study conducted on MSCs in Diabetes shows that there are many restrictions to this treatment, and CRISPR could help increase the effectiveness of the MSC treatment.


Another use of CRISPR in the medical field has been shown with monogenic disorders, diseases caused by errors in single genes, such as Sickle Cell Disease and Beta-Thalassemia. Even though further testing is needed, results show that patients have high levels of fetal hemoglobin expression and are independent of transfusions. These blood-related disorders avoided delivery challenges since treatments could be injected into the bloodstream.


In Duchenne’s Muscle Dystrophy, patients have mutations of the dystrophin gene; without it, their muscles weaken and degenerate. Studies have shown through MDX Mice that have a similar mutation in their DMD (dystrophin gene) that it is curable using CRISPR Cas9. The scientists used the Adeno-associated virus (AAV) to inject the systems into the mice. We can use MSCs or iPSCs and differentiate them as skeletal muscle cells to inject into patients.


Cures for HIV and retroviruses have also been tested using CRISPR. CRISPR-Cas13 has been used since the protein modified RNA instead of DNA. Lastly, CRISPR has impacted the agriculture and bioenergy industry, and gene editing technology can potentially change the future of many fields. In the agricultural industry, gene editing can create drought or disease resistance crops which can help reduce the global hunger index and food insecurity. Organisms such as algae have gone under CRISPR treatment to double biofuel production.



Ethics

While CRISPR can be seen as a solution to problems that have plagued us throughout human history, it also has significant drawbacks. CRISPR can eliminate anomalies in society, but that ignores the fact that these anomalies are a part of who we are. If we use CRISPR to create stronger and more intelligent beings, the bloodlines seen as “biologically inferior” will be wiped out. Another issue is classifying what classifies being biologically inferior. Eventually, unwanted features will be edited to create “superior” beings. This slippery slope must be brought up in the conversation about using CRISPR in the future. While many may believe that the public can watch CRISPR to avoid these fears, who will stop those hiding it from the public eye? Literature is littered with dystopian and utopian societies that try to create super soldiers. For example, Putin talked to a youth group about CRISPR and said this at one point: “We might use it to make better and stronger soldiers that don’t feel pain.”And when biochemist Jennifer Doudna helped create CRISPR, she had a nightmare where Adolf Hitler told her “I want to understand your new technology.” In fact, in 2015, Chinese researchers announced that they had edited human embryos. In 2018, He Juanki declared the birth of twin girls with a modified gene that could bestow resistance to HIV. Experiments like these are being planned in many countries. Some people argue that humanity should look to more ethical options to edit genes, such as preimplantation genetic diagnosis. Others argue that the cure CRISPR offers exceeds any consequences. Inevitably, the argument comes down to one question: Does the ethical implications of CRISPR outweigh the promise of a society free of biological inferiority.


Delivery of CRISPR has also been a huge challenge for scientists trying to use CRISPR as a cure for genetic diseases, Adeno-associated Viruses have been studied as potential carriers of CRISPR since these viruses do not affect humans negatively. These viruses would inject Cas9 into the cells, similar to how normal viruses infect cells. Furthermore, these viruses had serotypes that targeted specific tissues and cells. However, these viruses are extremely small, and Cas9 Enzymes are relatively large. CRISPR Cas9 has a relatively high chance of off targeting in genes. Many sequences in the genome can be every similar or even the same as the target DNA and can cause the enzyme to cut other genes which could potentially cause cell death.


Conclusion

As with our technological advances, many things, such as CRISPR, shouldn’t be taken lightly. Even though gene editing has been a revolutionary discovery in the past decade, there have been many setbacks and challenges. Ethics and morals have been highly debated about CRISPR. Even though regulations have been set on gene editing, CRISPR has still been in a “gray zone” where the federal government hasn’t said anything about CRISPR. Furthermore, designing babies and modifying the human genome are extremely dangerous for children and all of humanity. Playing with intelligence and other traits can potentially wipe out an inferior human population. Furthermore, using CRISPR is still a huge risk. Even though it might be the panacea to many gene-related disorders, CRISPR still needs more testing and research before it can be used commonly.



Citations

Yang H, Ren S, Yu S, Pan H, Li T, Ge S, Zhang J, Xia N. Methods Favoring Homology-Directed Repair Choice in Response to CRISPR/Cas9 Induced-Double Strand Breaks. Int J Mol Sci. 2020 Sep 4;21(18):6461. doi: 10.3390/ijms21186461. PMID: 32899704; PMCID: PMC7555059.

Frangoul H, Altshuler D, Cappellini MD, Chen YS, Domm J, Eustace BK, Foell J, de la Fuente J, Grupp S, Handgretinger R, Ho TW, Kattamis A, Kernytsky A, Lekstrom-Himes J, Li AM, Locatelli F, Mapara MY, de Montalembert M, Rondelli D, Sharma A, Sheth S, Soni S, Steinberg MH, Wall D, Yen A, Corbacioglu S. CRISPR-Cas9 Gene Editing for Sickle Cell Disease and β-Thalassemia. N Engl J Med. 2021 Jan 21;384(3):252-260. doi: 10.1056/NEJMoa2031054. Epub 2020 Dec 5. PMID: 33283989.

Modell AE, Lim D, Nguyen TM, Sreekanth V, Choudhary A. CRISPR-based therapeutics: current challenges and future applications. Trends Pharmacol Sci. 2022 Feb;43(2):151-161. doi: 10.1016/j.tips.2021.10.012. Epub 2021 Dec 21. PMID: 34952739; PMCID: PMC9726229.

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Long C, Amoasii L, Bassel-Duby R, Olson EN. Genome Editing of Monogenic Neuromuscular Diseases: A Systematic Review. JAMA Neurol. 2016 Nov 1;73(11):1349-1355. doi: 10.1001/jamaneurol.2016.3388. PMID: 27668807; PMCID: PMC5695221. https://www.synthego.com/blog/crispr-systems

Hille F, Charpentier E. CRISPR-Cas: biology, mechanisms and relevance. Philos Trans R Soc Lond B Biol Sci. 2016 Nov 5;371(1707):20150496. doi: 10.1098/rstb.2015.0496. PMID: 27672148; PMCID: PMC5052741. Liu, Z., Dong, H., Cui, Y. et al. Application of different types of CRISPR/Cas-based systems in bacteria. Microb Cell Fact 19, 172 (2020). https://doi.org/10.1186/s12934-020-01431-z

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