# An Update on Genome Editor CRISPR-Cas9: Truly, Is it a Friend or Foe?

### Abstract

As gene editing is increasingly becoming the norm and focus of molecular biology, many fundamental questions have arisen as people question the true morality behind the topic. In particular, CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats- Associated Protein 9) and its keen accuracy have become subject to much discourse. To ultimately weigh such an innovation’s assets and liabilities, this particular article will first commence to simplify the rather complicated biology behind CRISPR-Cas9 in an attempt to help readers condense the topic. Then, this article will refer to two recent literary studies conducted with CRISPR-Cas9 and the two’s fundamental outcomes as a method of justifying why Cas9 requires gradual implementation. Finally, a breakdown of CRISPR-Cas9’s pros and cons will help corroborate the ultimate conclusion that more research and time to weigh the aforementioned pros and cons is dire to the proper implementation of CRISPR-Cas9 in society. To clarify, the significance of the article is to contend and explicate why Cas9 must be researched further before being employed ubiquitously in labs worldwide.

### Introduction

The recent advances of CRISPR-Cas9 and other targeted genome editors in molecular biology has raised much speculation regarding the morality of gene editing. Specifically, one recent 2018 New York Times article[1] has reported an announcement by Chinese researcher He Jiankui that has raised the eyes of many—Researcher He has declared successful alteration of a gene in embryos for HIV (Human Immunodeficiency Viruses) resistance. He alleges to have directed CRISPR-Cas9 to CCR₅ (C-C Chemokine Receptor type 5), a gene that allows successful translation of a protein HIV needs to enter cells in ribosomes, allowing formation of such genetically-modified embryos. With researchers like He already creating “designer babies,” the question of morality regarding CRISPR-Cas9 has become more imperative with time. However, for the newcomer, another question is much more imperative—What exactly is CRISPR-Cas9 and how does it work?

To answer such a question, CRISPR-Cas9, by definition, is a complex of enzymes and genetic guides that finds and edits DNA, allowing for strands of DNA to be manipulated safely and meticulously. The genome editor originally evolved in bacteria for defense against viruses.[2] By deploying waves of DNA-cutting proteins to chop up any viral genes floating around, the bacteria (in the case it survived) incorporated tiny snippets of DNA into their own genomes so that they could take a “mugshot” of the virus for faster recognition in their next encounter. The bacteria would space out the guide RNA (gRNA), or bits of viral code, with repetitive, palindromic sequences in between, ultimately helping them protect themselves against viruses they’ve previously met.

Why are the origins of CRISPR-Cas9 so pertinent? Well, referring to one informative article by Wired[3], scientists utilized this technique by making their own gRNA that could identify a particular block of code in any living cell, for example a genetic defect or an undesirable trait. Upon injection of this gRNA with Cas9 into a targeted cell, the guide RNA would form a complex with Cas9 with one end of the RNA forming a hairpin-like curve that kept it stuck in the protein and the other end dangling out to interact with any DNA the complex came across. Once the complex was in the nucleus, the complex would bump along the entire genome, attaching every time it met a small sequence called PAM (Protospacer Adjacent Motif). PAM allows the complex to grab onto the DNA, which destabilizes the molecule and thus unzips the DNA. The gRNA would slip in to see if the DNA matched with itself. If it was a match, the gRNA triggered production of nucleases, or scissor-like appendages, to cut the DNA into two. If not, the complex moved on. Likewise, CRISPR-Cas9 enables for current scientists to snip out unwanted DNA, or even to replace it, in the case that replacement DNA is inserted into the cell along with the complex.

In the part that follows, therefore, an update with a few studies on CRISPR-Cas9 over the past two years will be provided for an ultimatum regarding Cas9’s future to be reached. Cas9 has changed dramatically over the past two years, and therefore, pros and cons may have to be reevaluated to accommodate the recent changes.

### Literature Review

Having discussed the definition and the mechanisms of CRISPR-Cas9 and since the ultimate goal of this article is to provide deeper insight into the pros and cons of Cas9, two more recent studies regarding the ethics of CRISPR-Cas9 and gene editing will now be discussed. By looking further into these two studies, readers will be able to analyze and break down the net gains and losses of the innovation through deeper investigations.

One particular recent study that brought lots of speculation is the aforementioned experiment by Chinese researcher He Jiankui. He Jiankui (age 34) began working with CRISPR technology while obtaining a doctorate in biophysics from Rice University. Later, he did postdoctoral research at Stanford only to return back to his home country China in 2012, founding two genetic-testing companies.[4]

With all his credentials, He Jiankui, in late 2018, used CRISPR-Cas9 to disable the gene CCR₅, which as stated before, creates a protein that makes it possible for HIV to infect cells. With the apparent help of a Chinese HIV/AIDS advocacy organization, he recruited couples consisting of a male with HIV and a female without the virus. Then, he used CRISPR-Cas9 to disable the CCR₅ gene in their embryos, eventually creating HIV-resistant twins.[5] From what researcher He has presented in a conference at Hong Kong, He was potentially able to disable both copies of CCR₅ in one of the twins, named “Nana.” In the other twin, dubbed “Lulu,” he claims to have only disabled one copy of the gene, meaning protection against HIV was to be limited. Also, upon further inspection of the data He had presented at the conference, Dr. Kiran Musunuru, a geneticist at the University of Pennsylvania, was able to assimilate that a clear evidence of mosaicism, or a mosaic pattern being evinced on the placenta, was present in at least one of the twins, presumably Lulu due to Dr.Musunuru’s inability to fully disable both copies of her CCR₅ gene.[4]

Despite these implications, however, the study did show that Cas9 was now able to be manipulated to meticulously find, cut, and potentially replace a targeted gene, which could be applied to more perilous and fatal genetic disorders.[6] HIV itself could easily be averted with other techniques such as the usage of caesarean sections to deliver the babies of mothers with the virus, which explains the backlash He faced after his presentation. However, for other more uncircumventable diseases, experimentation with CRISPR-Cas9 may be the only chance the child gets to live a normal, healthy life. In fact, George Daley, dean of Harvard Medical School, pointed to the notorious Huntington’s disease and Tay–Sachs disease as examples of diseases that might be averted only through gene editing.[5]

Another recent study regarding the advent of CRISPR-Cas9 is one about the concerning affiliation between the gene p53 and CRISPR-Cas9—specifically, in human pluripotent stem cells (hPSCs), as published in Nature Magazine in June 2018.[7] To contextualize the study, the gene p53’s inherent role in the cell is to activate a biochemical “first-aid kit” for any “injuries” that may occur upon the DNA strand. However, this activation has become a growing problem as CRISPR-Cas9 works by cutting both strands of the DNA molecule and removing or even replacing a specified set of DNA. However, due to the way the gene p53 works, the gene does one of two things after CRISPR-Cas9 cuts both strands of the double helix—it either mends the DNA break or initiates apoptosis. Obviously, under normal circumstances, the gene p53 plays an essential role in preventing the mitosis of mutated cells and in preservation of DNA in the cell; however, ultimately, either of the two paths p53 takes to maintain the DNA fully intercepts Cas9’s function, rendering Cas9 dysfunctional in this case.

Having said that, this particular study, further talked about by Scientific American[8], regarded the continuously arising question about whether Cas9 truly did work more efficiently with cells lacking or having dysfunctional p53 genes. Since CRISPR-Cas9 worked better without a natural inhibitor to impede its function, researchers from Novartis wanted to confirm such a notion with these human pluripotent stem cells (hPSCs) by testing CRISPR-Cas9 for increased accuracy with utilization of lab mice.[9] Upon experimentation, they attained an average insertion or deletion efficiency that was greater than 80% for CRISPR-Cas9 in cells with dysfunctional/lacking p53 genes. The reason this particular study was so groundbreaking was not because of the high efficiency displayed by Cas9, however. Rather, it was due to its result: the high efficiency of CRISPR-Cas9 revealed that double-strand breaks (DSBs) induced by Cas9 were toxic and killed most hPSCs. Beforehand, previous studies could not nor did put such a focus on the toxicity of hPSCs due to low transfection efficiency and low induction of DSBs. Likewise, this study proved to a new level the essential correlation of p53 genes and Cas9.

These two aforementioned studies likewise gave scientists worldwide deeper insight into the nascent field of genetic engineering. Specifically, experiments like those of He Jiankui on the twins could render inauspicious, permanent results on fetuses, while the strong direct correlation between p53 genes and Cas9 only make the tool more liable to cancer. Having said that, it is time to reflect upon recent research of Cas9 and come to an ultimatum on Cas9’s implications.

### Discussion/Implications

CRISPR-Cas9 gave one revolutionary path in the name of genetic engineering, making “designer babies” no longer a capricious idea of science fiction and instead the reality of today. However, Cas9 does have its advantages and disadvantages. After reviewing and weighing both, this article will explicate exactly why more research and time to consider rather than the taking of rash actions with the new innovation is essential to preventing harm to society.

One of the advantages of Cas9 is that those with inevitable genetic diseases have a chance to live a healthy life, or even just to survive. Many patients with Huntington’s, Duchenne muscular dystrophy, etc are unable to live very long due to their inherited conditions and are thus sometimes even prescribed an early death. Cas9 alleviates such a prescription by giving at least a hope, and a chance to survive and live healthy, despite all of the risks. Another perk is that Cas9 could reduce the risk of inherited medical conditions such as diabetes, cancer, and HIV and increase the health of societal members in general, potentially causing a perfectly feasible 10-20% improvement in health.[10] Furthermore, Cas9 is a more economic option compared to other gene modifiers, including morpholino oligos (MOs). To corroborate, the average cost for an MO is around $400 per oligo; however, one lane containing four Cas9 samples costs only around$0.13.[11] The sharp contrast in price between Cas9 and other genetic modifiers renders Cas9 much more readily available and accessible for the common people. Thus, even if class division between the wealthy and the impecunious were to develop as a result of using genetic tools, out of the viable tools Cas9 would be the most accessible yet accurate. Likewise, these perks render Cas9 the best and most potential candidate out of many genetic editors, further explicating reasons for implementing Cas9 into contemporary society.

However, many disadvantages also exist for Cas9, especially regarding the ethical implications that can result. One such disadvantage is that the technology is used is still at a very experimental stage in the status quo. Having been developed only in the last quarter of a century, CRISPR-Cas9 still contains many unknown risks and may even show new ramifications. For instance, as evidenced in the Novartis study above[7], the finding that the gene p53 causes for cells to die or prevents Cas9 from working demonstrates that further work on Cas9 needs to be done. Though gene p53 can simply be arguably extirpated from the DNA, a lack of or dysfunctional p53 is often associated with ovarian, colorectal, and lung cancers, meaning that such a solution may not be astute.[12] Another disadvantage is the widening of the gap between rich and poor as the rich can afford increasingly advantageous genetic additions to their children while the poor may not, ultimately widening the gap further. Such class division could fundamentally alter the structure of modern society as the inequality expands unrestrictedly. Cas9 also has more scientific implications as it disrupts the evolution of humankind. Such disruption does not affect only humans, however; the interconnected nature of the natural ecosystem causes for all animals in the foodweb to be affected as humankind undergoes artificial selection, the frequency of certain traits being determined by human choice rather than naturally. Finally, the last drawback is the ethical implications as a more intelligent, athletic, and “ideal” human is formed. This promotion of an “ideal type” not only ruins the natural evolution of humankind but also can decimate the diversity of humans. In the status quo, we respect and accept diversity as one of the unique characteristics of “humanity,” yet the proliferation of Cas9 as a genetic tool can fundamentally change this concept of humanity. Many abhor the idea of a tool altering the morals of humanity and thus contend against Cas9’s widespread usage.

Looking at the net benefits and losses, it is evident that a gradual approach with Cas9 is needed, even if the current implementation could potentially save many lives from genetic disorders. Being more cautious with new innovations tends to allow for more astute decisions to be made. Researcher He Jiankui, with his rather impetuous implementation of Cas9, produced embryos with mosaic-patterned cells, not an auspicious sign for both embryos and his reputation alike. Likewise, a gradual approach to Cas9 will be worth the wait when more knowledge on the topic is assimilated, enabling more educated decisions to be made.

### Conclusion

To recapitulate, CRISPR-Cas9 is a revolutionary gene editor that has been researched and developed heavily very recently over the last quarter of the century. CRISPR-Cas9 is a gene editor originating from bacteria that could be manipulated to take out or replace strands of DNA. Two recent studies regarding Cas9 has proved more potential and drawbacks of its usage, which ultimately brought up the net advantages and disadvantages to be gained from Cas9. Researcher He alleged to have made the first genetically modified twins in late 2018, while in early 2018 the gene p53 was scientifically proven to be affiliated with inhibition of Cas9. Narrowing the studies down, net benefits and losses were analyzed, generally benefits being an increase in health among patients and society in general and drawbacks being the lack of research upon the subject. The analysis ultimately brought up the notion that Cas9 must be implemented gradually and cautiously into society over the course of a few decades rather than imposing it in the status quo as some researchers have proposed. Yet, further research regarding Cas9 properties and implications are also needed in lieu of grave consequences that may result.

### References

1. Gina Kolata, Sui-lee Wee and Pam Belluck, “Chinese Scientist Claims to Use Crispr to Make First Genetically Edited Babies”, The New York Times. November 26, 2018, https://www.nytimes.com/2018/11/26/health/gene-editing-babies-china.html.
2. “What Are Genome Editing and CRISPR-Cas9? – Genetics Home Reference – NIH”, U.S. National Library of Medicine, October 15, 2019, https://ghr.nlm.nih.gov/primer/genomicresearch/genomeediting.
3. Megan Molteni, “Everything You Need to Know About Crispr Gene Editing”, Wired, May 12, 2017, https://www.wired.com/story/what-is-crispr-gene-editing/.
4. Gina Kolata and Pam Belluck, “Why Are Scientists So Upset About the First Crispr Babies?”, The New York Times, December 5, 2018, https://www.nytimes.com/2018/12/05/health/crispr-gene-editing-embryos.html.
5. David Cyranoski, “CRISPR-baby Scientist Fails to Satisfy Critics”, Nature News, last modified November 30, 2018, https://www.nature.com/articles/d41586-018-07573-w.
6. Dennis Normile Nov, “CRISPR Bombshell: Chinese Researcher Claims to Have Created Gene-edited Twins”, Science Magazine, November 26, 2018, https://www.sciencemag.org/news/2018/11/crispr-bombshell-chinese-researcher-claims-have-created-gene-edited-twins.
7. Robert J. Ihry, Kathleen A. Worringer, Max R. Salick, Elizabeth Frias, Daniel Ho, Kraig Theriault, Sravya Kommineni, Julie Chen, Marie Sondey, Chaoyang Ye, Ranjit Randhawa, Tripti Kulkarni, Zinger Yang, Gregory McAllister, Carsten Russ, John Reece-Hoyes, William Forrester, Gregory R. Hoffman, Ricardo Dolmetsch and Ajamete Kaykas, “P53 Inhibits CRISPR–Cas9 Engineering in Human Pluripotent Stem Cells”, Nature News, June 11, 2018.
8. Sharon Begley, “CRISPR-Edited Cells Linked to Cancer Risk in 2 Studies”, Scientific American, June 12, 2018, https://www.scientificamerican.com/article/crispr-edited-cells-linked-to-cancer-risk-in-2-studies/.
9. Robert J. Ihry, Kathleen A. Worringer, Max R. Salick, Elizabeth Frias, Daniel Ho, Kraig Theriault, Sravya Kommineni, Julie Chen, Marie Sondey, Chaoyang Ye, Ranjit Randhawa, Tripti Kulkarni, Zinger Yang, Gregory McAllister, Carsten Russ, John Reece-Hoyes, William Forrester, Gregory R. Hoffman, Ricardo Dolmetsch and Ajamete Kaykas, “P53 Inhibits CRISPR–Cas9 Engineering in Human Pluripotent Stem Cells”, Nature Medicine 24, (June 11, 2018): 939-946, https://doi.org/10.1038/s41591-018-0050-6.
10. Mark Crawford, “8 Ways CRISPR-Cas9 Can Change the World”, The American Society of Mechanical Engineers, May 31, 2017, https://www.asme.org/topics-resources/content/8-ways-crisprcas9-can-change-world.
11. Dipankan Bhattacharya, Chris A. Marfo, Davis Li, Maura Lane and Mustafa K. Khokha, “CRISPR/Cas9: An inexpensive, efficient loss of function tool to screen human disease genes in Xenopus”, Developmental Biology 408, no. 2 (December 15, 2015): 196-204, https://doi.org/10.1016/j.ydbio.2015.11.003.
12. Maximilian Vieler and Suparna Sanyal, “p53 Isoforms and Their Implications in Cancer” Cancers (Basel) 10, no. 9 (August 25, 2018), https://doi.org/10.3390/cancers10090288.

Cover Image Credit: Ernesto del Aguila III, National Human Genome Research Institute, NIH