Abstract

Many chronic diseases have a genetic component that influences one’s risk of developing that disorder. Until recently, it was assumed nothing could be done to edit a person’s genes to reduce or eliminate their risk of disease. That changed when researchers discovered CRISPR-Cas9, a piece of biological machinery that can introduce manufactured DNA strands into the human genome. It has two components: CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), a unique RNA strand that guides the CRISPR-Cas9 complex to a targeted site on a person’s DNA, and Cas9, a protein that cuts the original DNA strand open and inserts the manufactured DNA strand (which holds the sequence of interest). CRISPR-Cas9 offers extraordinary possibilities for modern medicine through its potential to treat various genetic disorders. While scientists are only just beginning to test CRISPR-Cas9 on humans, many people have already begun to consider the variety of ways in which they can use this discovery to change their bodies. Most are excited to use gene editing to treat diseases, but others are thinking about changing the eye color of their baby, or even introducing changes to the human germline, where edits will be continually passed on to future generations. It is evident that ethical dilemmas may evolve with the introduction of CRISPR-Cas9 into society, and thus, it is important to consider the ways in which it will be available to communities and individuals and the extent to which regulation should be placed on this discovery to limit its unethical use.

Keywords

Bioethics, Genetics, Health Care

Introduction

Humans have suffered from single-gene disorders (also known as Mendelian disorders), including cystic fibrosis, muscular dystrophy, and sickle cell disease, and up until a few decades ago, some of these genetic disorders were considered unpreventable or incurable. However, with the recent discovery of CRISPR-Cas9, which was derived from the prokaryotic adaptive immune system (Garcia-Robledo, Barrera, & Tobón, 2020), scientists may have found a way to cure single-gene disorders through gene editing. Several gene-editing techniques already exist, including zinc-finger nucleases (ZNFs) and transcription activator–like effector nucleases (TALENs), but CRISPR-Cas9 is able to edit larger DNA sequences. In addition, CRISPR-Cas9 is simpler and more efficient than most other gene-editing mechanisms because the same machinery can be used for each gene-editing event, whereas other techniques require a new protein to be engineered for every target gene of interest (Schacker & Seimetz, 2019). This discovery has extraordinary potential to improve public health and take a large step toward a world of health equity through the primary prevention of single-gene disorders. While gene editing can be used to reduce the prevalence of genetic disease, scientists have additionally considered the possibility of using CRISPR-Cas9 to alter phenotypic traits of individuals, which may not be for the purposes of health, but rather for other, possibly less important reasons, such as altering a person’s physical appearance. Thus, to what extent can gene editing be considered ethical? In this article, we will examine the biology and pathophysiology of single-gene disorders and CRISPR-Cas9, explore the biomedical potential for increasing health equity through the treatment of single-gene disorders, consider limitations and challenges associated with CRISPR-Cas9, and analyze concerns associated with gene editing to understand where its use might be considered unethical.

Biological Mechanisms of CRISPR-Cas9 Gene Editing

All single-gene disorders occur as a result of a single, mutated gene, which is a sequence of nucleotides within a DNA strand. These disorders differ by the gene that gets mutated. For example, people with cystic fibrosis have a mutated CFTR (cystic fibrosis transmembrane conductance regulator) gene. An individual can receive the mutated gene in one of two ways. The first method is through mutation as a result of faulty DNA replication. The second method is the passing of mutated genes from parent to offspring.

CRISPR-Cas9 edits eukaryotic DNA using nucleotides and proteins to destroy the mutated gene and replace it with the corrected, unmutated form. It consists of two main subunits: a nuclease protein (Cas9) and an RNA (CRISPR). The CRISPR RNA primarily functions as a guide that directs the Cas9 protein to the site where the DNA will be edited. This feature distinguishes CRISPR-Cas9 from other gene-editing mechanisms, which use a protein as a guide instead of RNA. After binding at the target site, the Cas9 protein cuts the DNA and destroys the unwanted (mutated) portion, unless a disease was caused by a deletion mutation, in which case there is no need to destroy any DNA (Bozorg Qomi, Asghari, & Mojarrad, 2019).

At this point, the faulty piece of DNA has been removed from the rest of the strand, and CRISPR-Cas9 can repair the gap in the individual’s DNA through one of two ways. The first, rather faulty method is non-homologous end joining (NHEJ), and it is characterized by joining the ends of a double-strand break (DSB) of DNA. This method can result in the formation of a new frameshift mutation because it often is unable to precisely connect the two ends of a DSB. On the contrary, homology-directed repair (HDR) is considered the more precise method for repairing DNA. This method uses similar sequences found elsewhere in the genome of the same nucleus to repair the DNA. The similar sequences can be found on the sister chromatid or on a different part of the chromosome where the sequence is the same. With the HDR method, repair can be 100% accurate if the sequence used is completely homologous to the DNA that was deleted/damaged by the Cas9 protein (Shrivastav, De Haro, & Nickoloff, 2008). In addition, HDR allows scientists to edit a sequence or insert an exogenous (foreign) DNA sequence into the genome.

CRISPR-Cas9 machinery can be delivered into an organism’s genome through one of two delivery methods: ex vivo gene therapy and in vivo gene therapy. Ex vivo gene therapy is the process of removing cells from the body, editing their DNA, and then transplanting them back into the organism’s body. Ex vivo therapy is particularly useful for ensuring precise control of variables, such as dose, and it is primarily used for cases related to cancer immunotherapy and viral infection inhibition. In vivo therapy, on the contrary, is the process of directly inserting the CRISPR-Cas9 therapy into disease-affected cells/organs in the body, and it is primarily used for treating diseases that stem from genetic mutations. This process allows for gene therapy to occur inside the body, and thus, it may require less operation than ex vivo therapy (Blenke, Evers, Mastrobattista, & van der Oost, 2016; Song, 2017).

Biomedical Applications of CRISPR-Cas9

The discovery of CRISPR-Cas9 has proven itself capable of several biomedical phenomena, and with research ongoing, there are even more treatments yet to be discovered. Although CRISPR-Cas9 has only slightly been tested on humans, it has been effective in editing the genomes of mice and zebrafish (Hwang et al., 2013). What makes CRISPR-Cas9 different from similar gene-editing techniques is its ability to bind to a specific sequence and insert a completely new sequence of DNA. This feature of CRISPR-Cas9 allows experts to treat a variety of single-gene disorders that were previously considered untreatable.

Cancer is often caused by mutations in tumor suppressor genes, which are sequences that suppress cell division to prevent cancer. These mutations can be due to loss-of-function or a deletion. CRISPR-Cas9 has the ability to target mutations and edit these sequences to repair the function of tumor suppressor genes, and thus prevent the formation of a malignant tumor (Pellagatti, Dolatshad, Valletta, & Boultwood, 2015). The first clinical trial to use CRISPR-Cas9 to treat cancer occurred in China in 2016, during which scientists genetically engineered T-cells ex vivo to treat metastatic non–small cell lung cancer after all standard cancer treatments were unsuccessful (Cyranoski, 2016). This field of research could have very large implications of the global burden of cancer, a disease that has affected the lives of millions.

Another study discovered the ability of CRISPR-Cas9 to correct a mutation that causes cystic fibrosis, a condition that affects the lungs and digestive system of individuals. In this case, CRISPR-Cas9 is used to edit the CFTR gene, which causes cystic fibrosis. This experiment was performed on cultured human stem cells that have the ability to undergo ex vivo gene therapy (Pellagatti et al., 2015). In another experiment, researchers performed ex vivo gene editing on the CFTR gene in mice models and were able to successfully deliver the gene back into the mice (Yui et al., 2012).

Furthermore, CRISPR-Cas9 has been used effectively to treat blood disorders such as β-thalassemia and sickle cell disease (Stein, 2019). In treating β-thalassemia, CRISPR-Cas9 is used to delete the hemoglobin beta gene responsible for this disorder, and HDR is used to repair the gene back to its correct sequence so that it can function properly. This is just another example of how CRISPR-Cas9 can be used to treat diseases by editing genetic mutations (Pellagatti et al., 2015). These findings especially have implications on increasing health equity, given that these blood disorders disproportionately affect some racial/ethnic communities more than others (Yusuf et al., 2011).

With so many potential applications of CRISPR-Cas9 in treating biomedical diseases and conditions, this gene-editing machinery has the potential to revolutionize the field of public health as it continues to be researched and tested in clinical trials. Because of the recency of the discovery of CRISPR-Cas9, clinical research on this tool has been somewhat limited, and scientists are continuing to discover new treatment methods for genetic disorders. As CRISPR-Cas9 is being used to treat genetic mutations, it is still important to consider the limitations and challenges associated with this gene-editing machinery.

Limitations/Challenges of CRISPR-Cas9

Although CRISPR-Cas9 has the potential to help millions of people, it is still important to analyze the costs and limitations associated with this gene-editing technique. One clear limitation of CRISPR-Cas9 is its inability to target sequences longer than about twenty base pairs (Carroll, 2012). This means that CRISPR-Cas9 is limited in the diseases that it can treat. For example, it cannot be used to edit abnormal chromosome diseases, such as trisomy 21 (also known as Down’s syndrome), because it would be impossible to edit an entire chromosome.

Another limitation of CRISPR-Cas9 is its potential to create new mutations, either from off-target effects or from DSBs. Off-target effects are a result of the Cas9 protein targeting an unintended locus due to its nonspecific activity (Reyes & Lanner, 2017). Although off-target effects are unpredictable, researchers have already begun to discover methods for minimizing off-target effects of CRISPR-Cas9 gene editing (Naeem, Majeed, Hoque, & Ahmad, 2020). In addition, CRISPR-Cas9 requires DSBs to edit DNA sequences, and when DSBs are brought back together, indel mutations may occur. Similarly, for off-target effects, researchers have discovered ways to mitigate the occurrence of indel mutations as a result of DSBs (Yoshimi et al., 2020). These two mechanisms (off-target effects and DSBs) may result in the formation of new mutations that could have even worse phenotypic effects than those of the targeted gene. Although the occurrence of mutation in gene editing is uncommon, it can have large consequences that may outweigh the benefits of CRISPR-Cas9 in some cases.

Given that there are several limitations/challenges to using CRISPR-Cas9 for gene editing, scientists have already begun researching newer and more innovative gene-editing methods. For example, scientists at the Merkin Institute for Transformative Technologies in Healthcare have discovered prime editing, a type of genetic machinery that can edit genes “without requiring double-strand breaks” and has “much lower off-target editing than Cas9” (Anzalone et. al, 2019). New discoveries, like prime editing, allow for the treatment of even more diseases and have a greater potential for improving public health. Overall, gene editing offers incredible possibilities in the world of medicine and public health, and although there may be some limitations, scientists are steadfastly approaching newer and improved versions of gene-editing machinery.

Ethical Concerns of Gene Editing

Given the power and ability of CRISPR-Cas9 gene-editing technology to revolutionize the fields of medicine and genetics, there must be consideration of what would constitute unethical use of CRISPR-Cas9 and whether it should be regulated and/or prohibited.

One ethical concern of CRISPR-Cas9 research is the creation of germline mutations in human embryos (Mulvihill et al., 2017). When mutations are created in the germline, they will be continually passed onto future generations, with the potential of creating health impacts that last for eternity. Many scientists have argued for a moratorium on this type of research due to its long-lasting effects.

Furthermore, many are concerned about the commercialization of CRISPR-Cas9 associated with its eventual clinical use (Mulvihill et al., 2017). As CRISPR-Cas9 is not simply a product of nature, but rather a manufactured product made of materials found in nature, it can be patented. In addition, there is little development of governmental regulation related to gene editing because it is a new phenomenon and has barely been tested on humans (Caplan, Parent, Shen, & Plunkett, 2015). Thus, the absence of regulation on CRISPR-Cas9 makes it easier for others to use gene editing unethically. Gene editing could also be sold commercially as a service, similar to the DNA testing industry, where companies like 23andMe sell genetic testing as a service to customers, often without the involvement of health professionals.

One final concern is whether the clinical benefits of CRISPR-Cas9 would be equitably distributed to all populations or whether they will only be accessible to some. Given the existence of health disparities across many different populations and health conditions, the potential inequitable distribution of CRISPR-Cas9 could exacerbate these disparities while missing an opportunity to minimize them. This concern is especially frustrating given discussions by members of the public regarding the use of CRISPR-Cas9 to alter phenotypic traits that have nothing to do with a person’s health. It is clear that the use of CRISPR-Cas9 for health benefits must be prioritized over using it for reasons unrelated to health, such as physical appearance. Given the severe ethical implications of using CRISPR-Cas9 for gene editing, there must be some type of regulation, especially because research efforts are ongoing and clinical implementation is incoming.

Conclusion

All in all, CRISPR-Cas9 is a groundbreaking discovery that has extraordinary potential for improving public health. We can use CRISPR-Cas9 to treat several genetic disorders that were previously considered untreatable, possibly to the extent of eradication. While CRISPR-Cas9 is powerful, it has limitations, and thus, it is important to consider future discoveries that can do what CRISPR-Cas9 cannot, such as prime editing. Given that gene-editing research is on the rise and the public is becoming increasingly aware of its abilities, we must continue to evaluate the implications of this discovery as it nears entry into clinical care delivery systems. CRISPR-Cas9 is a very powerful piece of genetic machinery, and although it has the potential to improve the health of many, it can also do a lot of harm if it is too loosely regulated and used beyond the lines of what is ethical.

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