Before attempting the tasks that comprise this curriculum unit, AP Statistics students must develop a solid understanding of CRISPR-Cas9 technology and its versatile applications. The information below will be considered the basis for this understanding.
What is CRISPR?
The discovery of CRISPR took place in 1987 when Japanese molecular biologist Yoshizumi Ishino and his fellow scientists at Osaka University studied a gene belonging to Escherichia coli (commonly known as E. coli), a microorganism that is found in the intestines of mammals. The scientists noticed that the gene exhibited a striking pattern consisting of five short repeating DNA segments followed by short nonrepeating DNA sequences they called spacers. By the late 1990s, similar patterns were discovered in other prokaryotes. In 2002, this pattern was assigned the acronym CRISPR, which stands for Clustered Regularly Interspaced Short Palindromic Repeats, by a team of scientists at Utrecht University in The Netherlands. It was not until 2007 that food scientists researching Streptococcal bacteria highlighted the importance of the CRISPR sequences. These sequences play a crucial role in the immune systems of bacteria.1 Bacteria must defend against viruses by generating enzymes. After these enzymes eliminate the viruses, other proteins store remnants of the virus’s genetic code in the spacer DNA. The bacteria uses this genetic code in order to prevent future viruses from invading. In the event of a new viral infection, the bacteria releases Cas9 enzymes, which store the genetic code. The Cas9 enzymes can then detect whether or not the virus’ RNA is complementary to the stored genetic code. If the RNA matches the stored genetic code, then the Cas9 enzymes destroy the virus by dissembling the DNA.2 In 2012, a major breakthrough occurred when scientists found that they could manipulate the Cas9 enzyme by injecting it with preconstructed RNA. When fed this fake RNA, the Cas9 enzyme sought out matching genetic codes and cut them. After conducting experiments with test tube molecules, Jennifer Doudna, Emmanuelle Charpentier, and Martin Jinek, discovered that Cas9 enzyme could be used to splice any DNA sequence at the location of one’s choice. A system known as CRISPR-Cas9 has been developed to execute this task.3
The CRISPR-Cas9 system is comprised of two molecules, the Cas9 protein and a guide RNA (gRNA). The gRNA is constructed by the scientists in advance and consists of about twenty nitrogenous base pairs. The gRNA is responsible for leading the Cas9 protein to the target location of the DNA. Since the gRNA has bases that complement the nitrogenous bases of the target DNA sequence, it attaches to the target DNA. The Cas9 protein acts like scissors and cuts out the target DNA. The cell containing the DNA then recognizes that the DNA has been spliced and uses its natural mechanisms to repair the cuts or accept a more desirable DNA sequence. CRISPR is preferred to other methods of gene-editing because it produces the Cas9 protein, and it has the ability to edit multiple genes simultaneously. Some alternatives require scientists to design an enzyme to splice the DNA at a target location.4
Health Conditions Being Addressed By CRISPR-Cas9 Technology
Researchers are currently using CRISPR-Cas9 to cure a number of diseases that are monogenic, meaning that they stem from a mutation in only one gene. Among these diseases is Huntington’s Disease, which causes neurons in the brain to degenerate. The list of conditions that can develop as a result of nerve cell degeneration is incredibly long. Those who suffer from Huntington’s disease are at an increased risk for obsessive compulsive disorder, depression, anxiety, and suicide.5 They experience muscle problems that can interfere with their ability to communicate, swallow, or move their eyes. They also might lack skills that are necessary to thrive in an academic or work environment such as organization, emotional regulation, self-awareness, self-control, memorization, and perceptiveness. Children of a parent diagnosed with Huntington’s Disease are at a 50% risk of inheriting the disease.6 Xiao-Jiang Li, a scientist at Emory University in Georgia, used CRISPR-Cas9 to eradicate defective huntingtin protein in the mutated genes of mice.7
Another disease sweeping the CRISPR-Cas9 scene is sickle cell anemia, which occurs when one has an excess of abnormally-shaped red blood cells (sickle cells, which deviate from the circular shape of normal red blood cells) that restrict the flow of blood and oxygen. On average, red blood cells live for about four months, but sickle cells typically do not live longer than a month. The shortage of red blood cells in one’s body can result in fatigue, abdominal or chest pain, jaundice, retinal damage, and a risk of stroke. A lack of functioning red blood cells can impact the immune system’s ability to defend the body against viruses. Sickle cell anemia is caused by a mutation in the ß-globin gene, which prevents the body from producing enough hemoglobin. Hemoglobin is the compound that enables blood cells to transfer oxygen from the lungs to other parts of the body.8 A treatment called CTX001 has successfully eliminated sickle cell anemia in laboratory mice. CTX001 is a therapy that involves the use of CRISPR-Cas9 to modify the ß-globin gene to increase production of hemoglobin.9 If CTX001 proves to be effective in humans, then this could provide a solution to the treatment shortage for sickle cell anemia. CRISPR-Cas9 is increasingly viewed as a potential therapy for sickle cell patients because, right now, the only ways for a patient to battle sickle anemia are to receive a blood transfusion or bone marrow transplant.10 However, transplants are not widely or readily available.
The number of people presently awaiting an organ transplant in the United States exceeds 113,000. According to the Health Resources and Services Administration, twenty individuals in need of a transplant die every day.11 For the past few years, xenotransplantation, the process of giving animal organs to humans, has been contemplated as a solution to the human donor shortage problem. Specifically, scientists have been studying the possibility of transplanting pig organs into humans because pigs reproduce in large numbers.12 Unfortunately, the human body is extremely resistant to pig organs, and as a result, might be susceptible to the porcine endogenous retrovirus (PERV). Geneticists at a company called eGenesis were able to deactivate the PERV in a sample of pig embryos. These embryos were subsequently implanted into female pigs, whose offspring did not inherit the virus.13
Use of CRISPR to Improve Crops
CRISPR is currently being used to improve the quality of agricultural produce. Bacterial spot disease is a condition affecting the marketability of tomatoes. Tomatoes infected with bacterial spot disease have unattractive brown spots surrounded by yellow or white rings on their fruits and stems.14 Scientists are using CRISPR to produce tomatoes that are resistant to bacterial spot disease and contain larger amounts of Vitamin C.15 In Brazil and Ireland, scientists are utilizing CRISPR to produce a variety of tomato infused with the capsaicin gene, which gives peppers their spicy taste. A naturally spicy tomato could replace the need for chili peppers in salsa, since chili peppers are not as abundant.16
Another disease that is interfering with the ability of companies to sell their produce is citrus greening disease, which is caused by a flying insect called the Asian citrus psyllid. In fact, in 2017, Florida suffered from a 75% decline in its production of oranges as a result of the disease.17 Although citrus greening disease does not harm humans or animals in any way, it causes trees to grow fruits that are malformed, tart, green, and smaller in size than fruits of unaffected trees. The disease also shortens the lifespans of most fruit trees and causes them to display fewer leaves.18 The United States Department of Agriculture has allocated almost 500 million dollars towards eliminating the disease so that the citrus industry of Florida can thrive again.19 Recovering the orange crop has been challenging, particularly because citrus trees take three to five years before they bear fruit. CRISPR has provided a solution to the problem of citrus greening. In 2014, scientists from the University of Florida Institute of Food and Agricultural Sciences Citrus Research and Education Center announced that they were able to use CRISPR-Cas9 to modify the citrus genome and make it resistant to citrus greening.20
Tropic Biosciences, a United Kingdom-based organization, has utilized CRISPR to successfully produce naturally decaffeinated coffee beans. The most effective method of decaffeinating coffee beans necessitates moistening the beans in water before transferring them to a stainless steel extractor. Liquid carbon dioxide is then dispersed over the bean at a high pressure to dissolve the caffeine molecules. The carbon dioxide is essential for preserving the flavor notes in the beans. The carbon dioxide is then transferred to another vessel called an absorption chamber, where it is converted to a gas. In turn, the caffeine is solidified and collected and the beans are roasted.21 CRISPR-Cas9 might eliminate the need for this expensive and time-consuming process and increase the production of decaffeinated coffee worldwide. At present, only twelve percent of the world’s coffee is decaffeinated.22
The cacao tree, an evergreen cultivated in tropical rainforests, is the source of pod-shaped fruits containing cocoa beans. Unfortunately, fungi thrive in tropical environments, too, and climate change is contributing to their presence. At least a fifth of cocoa pods worldwide are deemed unusable before they are harvested for chocolate production because they have been infected by fungal diseases. The majority of the affected cacao trees are in West Africa, which accounts for 68% of the world’s cocoa production. The most common condition infecting the cacao tree is black pod rot, which is caused by the fungus Phytophthora and results in brown spots on the tree’s fruit. This fungus tends to appear during the rainy season, when excess moisture impacts the soil acidity necessary for the cacao tree to survive. Controlling the spread of black pod infection currently involves spraying the trees with copper-based fungicides, but these chemicals might no longer be necessary.23 It has been discovered that the gene TcNPR3 plays a key role in helping the tree defend against Phytophthora.24 Researchers at Pennsylvania State University have theorized that deleting this gene using CRISPR-Cas9 will allow the cacao tree to resist pod rot. They are in the process of growing trees to investigate the accuracy of their hypothesis.
Individuals who suffer from celiac disease do not respond well to gluten, a protein found in wheat and barley. An estimated 2.5 million Americans with the autoimmune disease have not been formally diagnosed and risk damaging the lining of their small intestine if they consume foods rich in gluten. Those diagnosed with celiac disease are four times as likely as their counterparts to suffer from bowel cancer and are more vulnerable to coronary artery disease. If patients do not follow a gluten-free diet, they put themselves at risk for conditions such as osteoporosis, Type I diabetes, infertility, vitamin deficiencies, lactose intolerance, epilepsy, and multiple sclerosis.25 Food scientists are utilizing CRISPR-Cas9 to produce varieties of wheat that contain smaller amounts of gluten. Researchers in the Netherlands and in the United Kingdom are going as far as to use CRISPR-Cas9 to get rid of epitopes from gluten proteins that are responsible for immunoreactivity.26
Use of CRISPR-Cas9 to Generate Biofuels
According to the Department of Energy, fossil fuels including oil, natural gas, and coal accounted for 80% of energy consumption in the United States in 2017.27 Coal mining can pollute rivers and lakes with acidic water containing large amounts of arsenic, copper, and lead.28 Oil spills can contaminate water in oceans, destroying animal and plant species. Acidic water can deplete the ocean’s supply of calcium carbonate, a substance that crustaceans and other marine creatures depend on to grow their shells.29 Fossil fuel emissions contribute immensely to global warming because they release gases like carbon dioxide that trap heat in Earth’s atmosphere when burned. The environmental problems caused by fossil fuels have incited a pressing need for renewable energy sources such as biofuels. During photosynthesis, algae such as kelp and cyanobacteria have the ability to convert carbon dioxide and sunlight to energy and store the energy as oil.30 The number of algae strains exceeds 100,000, which makes it an enticing alternative to fossil fuels.31 The use of algae as a renewable energy source has been slowed by the limited production of lipids in several strains. A team of researchers from the company Synthetic Genomics have employed CRISPR-Cas9 to develop a strain of algae “that produces twice as much lipid as its wild parent.”32
In addition to generating biofuels, CRISPR-Cas9 might make traditional plastic use a thing of the past. Traditional plastic is a polymer synthesized from fossil fuels. Its durable, odorless, bendable, lightweight, recyclable nature makes it convenient for a number of purposes including storing food and building car parts. Of the 448 million tons of plastic produced annually, two-fifths are used as packaging material meant to be trashed soon after procurement.33 The United Nations reports that “79 percent of waste generated from plastic accumulates in landfills or in the environment, while only 9 percent is recycled and 12 percent is incinerated.”34 When plastic use increases, the environmental repercussions can be severe. First off, before plastics are manufactured, chemicals are added to their efficiency. Among these chemicals are phosphates and asbestos fibers, which make the plastics heat and flame-resistant; colorants made of iron oxide, titanium dioxide, or cadmium; and lead compounds intended to stabilize polymers. These chemicals can seep out of the plastic and enter water and the human body if it appears in food sources.35 Secondly, plastic is non-biodegradable, so it does not decay naturally. Instead, it just disintegrates into microplastics that are harming oceans and their ecosystems. In 2018, microplastics were detected in 93 percent of bottled water samples from several nations including the United States. Each year, 100,000 marine animals die from plastic consumption, as their stomach bacteria cannot break up the material into smaller units during digestion.36
Several individuals and organizations have been working to resolve the problems fueled by plastic. Dutch inventor and founder of The Ocean Cleanup Boyen Slat fundraised over 30 million dollars to establish an ocean clean-up system for removing plastic debris from the Pacific Ocean, which he is testing in San Francisco. The Coca-Cola company is aiding in the effort to reduce plastic waste worldwide by switching to recyclable, compostable packaging by 2030.37 Scientists are dependent upon production of monomers called omega-hydroxy fatty acids for bioplastics. A team of researchers from the Polytechnic Institute of New York University, led by Dr. Richard Gross, have found a way to produce omega-hydroxy fatty acids using Candida tropicalis yeast. This yeast has the ability to yield large supplies of omega-hydroxy fatty acids by transforming fatty acids of plant oils. Once these fatty acids are combined into a single molecule, the result is a biodegradable plastic that is useful for packing materials. Gross is hopeful that once this polymer is broken down, it can even function as a biofuel for military vehicles.38 With the knowledge that yeast can maximize output of omega-hydroxy fatty acids, lab scientists might be able to use CRISPR-Cas9 to isolate the gene responsible for production of these monomers.
Cons of CRISPR-Cas9 Technology
Although CRISPR-Cas9 has several applications that could revolutionize the food and medical industries, it is a relatively new technology, so we really do not know much about how it will affect future generations. Individuals who do not support the use of CRISPR-Cas9 for gene-editing are concerned about “off-target effects” that might result when the Cas9 enzyme snips the wrong part of the genome. Since similar nitrogenous base sequences can be found within one’s DNA, it is possible for the Cas9 enzyme to travel to the incorrect site. Even if the enzyme correctly identifies the target site, the long-term consequences of editing the genome are unknown. Chinese researcher He Jiankui made the news in 2018 for using CRISPR-Cas9 technology to remove the CCR5 gene from the embryos of twin girls and implant the modified embryos into their mother. He removed the gene to fulfill the wishes of their father, who did not want his daughters to suffer the way he did from HIV.39 Jiankui’s work was regarded as highly unethical by critics for a number of reasons. In order to circumvent policies banning HIV-infected individuals from using assisted reproductive technologies, he substituted normal blood samples for infected blood samples. Moreover, he “forged ethics review documents during recruitment of participants.”40 He did not consider other functions of the CCR5 gene that are perhaps essential to one’s well-being. Although the CCR5 gene encodes a protein that is not resistant to HIV, it might play a role in protecting individuals against other infections including the West Nile Virus.41 Therefore, if one or both of the twins contracts the West Nile Virus due to complications from his experiment, he can be held liable and imprisoned for a maximum of ten years.42
Not only might CRISPR-Cas9 allow scientists edit a human’s genome to reduce the risk of developing a fatal illness, but it might give parents the option of choosing which physical features they want their children to possess. If individuals utilize CRISPR-Cas9 technology for superficial reasons such as editing the genes of an embryo to give a newborn a certain eye color, hair color, or skin color, then this might fuel another eugenics movement. Eugenics is the breeding of individuals with desirable characteristics and was promoted by Adolf Hitler during World War 2 in order to create a superior Aryan race. By implementing a marriage loan program that allowed Aryan couples to avoid paying a quarter of the loan amount for each child they had together, the Nazis convinced Aryan couples to have more children. While this benefitted Aryan couples, many groups of people were negatively impacted by the eugenics movement. Individuals who were not of the Aryan race, blind, hearing-impaired, epileptic, manic-depressive, or schizophrenic were subject to sterilization laws. The Law for the Prevention of Hereditarily Diseased Offspring, enacted in 1934, led to sterilization of 300,000 to 400,000 individuals via vasectomy or ovarian tubal ligation. In 1939, Hitler introduced a law enabling doctors to kill patients who were psychologically ill and did not respond to treatment. He argued that asylum patients were an economic burden to society, so the Nazis used carbon monoxide gas chambers and lethal injections to kill them.43 Hitler’s quest to create his ideal race continued as Jews were placed into concentration camps. Josef Mengele, a doctor at Auschwitz, supervised experiments during which harmful chemicals were used to try to change the eye colors of camp prisoners to blue.44 If genetic editing of human embryos using CRISPR-Cas9 becomes more prevalent, then parents who decide to use the technology might select physical traits for their children in accordance with the social groups that are the most advantaged. For example, research has shown that height and income are positively correlated. Data from one study showed that an individual (male or female) with a height of six feet is “predicted to earn nearly $166,000 more over the course of a 30-year career than someone who clocks in at 5 feet 5 inches.”45 Furthermore, “nearly all Fortune 500 CEOs are over six-foot two-inches tall, even though people over six-foot-two make up only 3.9% of the world’s population.”46 Consequently, parents might have their children’s genes edited so that they grow up to be tall. If using gene-editing to equip a child with specific physical traits becomes a common practice among parents, this might create an insensitive society that does not embrace or tolerate diversity.
The economic implications of using CRISPR-Cas9 to edit the human genome might also be severe. Even though CRISPR-Cas9 is considered to be less expensive than alternatives, costs to edit genes using the technology are still high. At the Harvard University Stem Cell Institute, it costs $19,100 to alter one nucleotide base pair (referred to a single-point mutation) in a DNA strand.47 This is roughly thirty percent of the median annual household income in the United States. At the Yale University Genome Editing Center, the same process costs $15,000 plus up to $2,000 for genotyping.48 Therefore, if individuals want to use the technology, they will have to pay an amount that is at least twenty-five percent of the United States median household income. This means that at present, only the wealthiest individuals can afford to take advantage of CRISPR-Cas9 technology.
Among the groups of people who might receive an unfair advantage as a result of CRISPR-Cas9 are athletes. In 2007, Lee Sweeney, a physiologist at the University of Pennsylvania, was able to extract IGF-1, a gene that strengthens muscles. He injected mice, which he nicknamed “Schwarzenegger mice” with IGF-1 and found that their endurance level far exceeded that of mice in a control group. Mice in the treatment group were quicker runners than mice in the control group and did not experience weight gain even after consuming foods high in fat. Professional athletes seeking a boost in strength and energy contacted Sweeney after learning that he bred the Schwarzenegger mice. Once he became aware that athletes could use his innovation in an attempt to defeat competitors in sporting events (a practice known as gene-doping), Sweeney joined the World Anti-Doping Agency. This establishment prohibited the use of genetic modifications to enhance an individual’s athletic performance. In the past, professional athletes have undergone injections of erythropoietin (EPO), a protein that stimulates production of red blood cells, to increase their energy supply. Blood and urine tests can now confirm the presence of excess EPO in an athlete’s system. CRISPR-Cas9, on the other hand, might make it possible for athletes to get away with gene-doping since their DNA would naturally contain excess EPO.49
A Need for Clear Regulation of CRISPR-Cas9 Technology
It is very difficult to predict the consequences that CRISPR-Cas9 has for society, particularly when there are very few written laws regulating the use of CRISPR-Cas9 technology. Under the Consolidated Appropriations Act, the United States Congress has banned allocation of Food and Drug Administration (FDA) funds towards germline editing, or modifying the genes of an embryo. However, federal funds can be used for editing of somatic cells (cells other than reproductive cells). The reason for this difference is attributed to the fact that edits made to somatic cells cannot be transferred to subsequent generations. Even though the government has placed restrictions on funding, federal law does not explicitly ban gene-editing of an embryo.50
Where Do Adults in the United States Stand on the Gene-Editing Front?
The Pew Research Center conducted a poll in 2018 to determine how adults in the United States feel regarding various aspects of gene-editing technology. According to the poll, 72% of adults believe that it is okay to alter a baby’s genome to treat a congenital illness, while 27% feel that doing so would be a misuse of medical technology. Only 33% of American adults expressed support for gene-editing when informed that development of gene-editing technology would require scientists to perform tests on human embryos. Respondents in the sample who identified as “highly religious,” meaning they participate in religious services at least once a week and pray at least once a day, expressed more skepticism towards gene-editing. When the Pew Research data scientists broke down the data by religious affiliation, they discovered that the group with the largest percentage of respondents (88%) in opposition to embryonic testing consisted of white evangelical Christians. This might be in part to the fact that the Bible affirms that God is the ultimate creator. Therefore, it is possible that these voters believe that changing the genes of an individual would be usurping God’s power. The largest group in favor of using embryonic testing to speed up the development of gene-editing technology consists of Atheists (79%).
Generally speaking, American adults remain concerned about the potentially negative consequences of gene-editing technology. A majority (58%) anticipate that the technology will exacerbate the issue of income inequality since it will only be accessible to the most affluent individuals. Moreover, 54% of American adults believe that unethical use of the technology is an inevitability. Only 18% of respondents expressed confidence that gene-editing technology would lead to positive developments. 62% of adults are not convinced that medical researchers have a keen awareness of the positive and negative consequences of altering a baby’s genome.51
The Pew Research Center’s statistics give us a rough idea of how adults are responding to the prospect of gene-editing. Still, how teenagers within various demographics feel about the possibility remains a mystery and is important to investigate, considering CRISPR-Cas9 technology might be more advanced and widespread by the time they enter the workforce. It is my hope that as students complete the activities in this unit plan, they will uncover this mystery. As they learn about the potential benefits and drawbacks of CRISPR-Cas9 technology previously described and explore the variation in attitudes towards gene-editing through peer-to-peer interactions, they will discover that regulating the use of this technology is a cumbersome process. When the world consists of people who have such different life experiences, goals, and perspectives, it can be difficult to come to a consensus on a lot of issues facing humanity. In the real world, problems do not always have concrete or unique solutions. Individuals might have to think outside the box, and students should be given opportunities to perform mathematical tasks that reflect this reality.