Genetic Engineering

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Contents

Introduction

What Is Genetic Engineering?

Genetic engineering, or genetic modification, is the process of altering specific genes in a specific organism [1]. The goal of this process is to achieve a reproductive organism which exhibits traits dictated by the altered genes. Genetic engineering is used by scientists to enhance or modify the characteristics of an individual organism [2].

Over the years, this process has been observed naturally, which spans multiple generations. However, with advancements in technological tools, scientists now have more control over the process as it yields results much quicker. Additionally, these processes can theoretically be applied to any organism, mostly pertaining to plants and animals [3].

Genetic Engineering vs Gene Therapy

These are two gene-altering techniques which involve the same base element yet different results. As explained by their names, gene therapy seeks to remedy any genetic abnormalities whereas genetic engineering seeks to achieve modifications of genes to exhibit desired traits[4]. The difference, in essence, is the purpose of altering a specific gene. The means are the same yet the end result is different.

In the ‘90’s, a 4-year-old girl became the first patient to be treated by gene therapy. The girl suffered from a rare disease which resulted in a lack of disease-fighting enzymes. She was then treated with genetically engineered cells, rich with white blood cells, through recombinant DNA technology. Within hours, the engineered cells which were inserted into the girl started producing the required enzyme and restored the girl’s immune system [5].

Similarly, according to Edward O. Wilson, a Harvard entomologist, it will soon be possible to not only cure illnesses in humans but also enhance their abilities[6]. A hypothetical example of genetic engineering consists of replacing cells containing adaptive traits with ‘better’ or ‘stronger’ cells. This will, in theory, lead to enhanced communication skills or maybe certain thinking abilities. Due to ethical limitations, the days of genetically engineering humans are a bit far off, however, other organisms such as animals are currently being modified for productivity.

A Brief History

Charles Darwin & Theory of Evolution by Natural Selection

The theory of evolution by natural selection is arguably the beginnings of genetic engineering. In 1859, Charles Darwin formally introduced the idea of evolution which had been hypothesized for years [7]. Darwin explained how species have changed over generations and how each organism is an assembly of unique genes. Natural selection, according to Darwin, is the reason why species developed different physical characteristics through time [8].

Traits are responsible for changes in physical characteristics of living organisms. Humans contain these molecules known as chromosomes. Within these chromosomes are genes, which are responsible for the functioning of cells and the organisms’ various different physical characteristics. These characteristics are known as traits which are exhibited by each living organism as their own unique characteristics.

In the process of evolution, when two organisms reproduce, they offload some traits onto the offspring. As a result, the offspring exhibits characteristics from both parent organisms and risks the chances of random changes to genes, known as mutations. These changes could be a result of an accident or external influence as the physical traits of the gene are altered yielding different results[9].

Mutations that occur naturally via adaptive traits eventually lead to a new species from an existing population. This speciation, on the other hand, can be achieved artificially via genetic modification techniques. The opportunity to alter genes externally lead to a revolution in the biotech industry resulting in the introduction of tools and techniques such as CRISPR [10].

Mendel's Experiments

Mendelian Inheritance Explained[11]

Gregor Mendel, a monk known as the father of genetics, expanded on the understanding of genetics and its effects over generations. Through his experiments he was able to identify the physical traits which genes exhibit in each generation. He concluded through his experiments on pea plants, that plant height, pod shape, pod colour, seed shape, seed colour, flower position and flower colour were the seven traits that could be potentially influenced. Since cross-breeding was a known method of genetic modification in the 1800’s, he crossbred plants and published the results. He found that there are essentially two types of traits: Recessive and Dominant. These traits were apparent because he observed two different types of off springs as a result of breeding[1]. Thus, he provided solid evidence of how different genes, when crossbred, can yield hybrid generations and can eventually reveal a new species.

Discovering DNA

Francis Crick (left) and James Watson (right)[2]

The discovery of the double helix is accredited to two 19th century scientists: Francis Crick and James Watson[3]. The double helix, otherwise known as a double-stranded DNA, is the shape of DNA chains which store our genetic information. These chains consist of base pairings formed by sugar (deoxyribose), a phosphate group and one of four nitrogenous bases: Adenine (A), Thymine (T), Guanine (G) and Cytosine (C)[4].

When a group of these base pairings match through their nitrogenous bases, they form chains of DNA. This discovery was also possible with the efforts of an American biologist, Rosalind Franklin, who exposed the DNA structures with the use of X-ray technology. It wasn’t until 1951, when Francis and James discovered the correct base pairings, that the true shape of our DNA was discovered[5].

Since the discovery of the DNA, the pathway to gene modification became more intriguing with hypothetical possibilities of changing the information coded in DNAs. It was only a matter of time before the scientists figured out a way to change DNA compositions which would change genes and ultimately, entire organisms.

The First Recombinant DNA Molecule

In 1972, Paul Berg published the results of his experiments on DNA modification[6]. Through his technique he was able to isolate genes and use his method of ‘cut-and-splice’ to alter DNA strands. This breakthrough was the first proper evidence of gene editing in living organisms where a gene from one organism was ‘cut’ and ‘pasted’ into another organism. The emergence of hybrid DNA strands allowed scientists to look into animal agriculture with focus on animal welfare, disease resistance and biomedicine. Further, the possibilities of changing gene compositions led to improvements in biofuel as well as the food industry.

Evolution & Why It Matters

Darwin's Theory of Evolution[7]

According to Darwin, "species change over time as a result of changes in heritable physical or behavioral traits" [1]. Consequently, changes that allow an organism to better adapt to its environment will facilitate survival and contribute to the survival of healthy offspring. Notably, evolution occurs across species, not individuals. This means that traits that are expressed across an entire species and positively contribute to the propagation of said species are examples of a confirmed evolutionary, genetic mutation [2].

Darwin's theory of evolution has two main points. According to Brian Richmond, a curator of origins at the American Museum of Natural History in New York City , those points are:

1. "All life on Earth is connected and related to each other"[3].

2. The diversity of life is a product of "modifications of populations by natural selection where some traits were favored in the environment over others"[4].

Natural selection, which postulates the saying "survival of the fittest", refers the "fittest" merely to traits that encourage survival and reproduction. In the case of modern day humans, for instance, it is the primary contributing factor to their role as a dominant species. Neanderthals, who were the preceding ape species to modern day homo sapiens, were widespread approximately 400,000 years ago. This dominance subsequently shifted with the migration of Homo sapiens from Africa to Europe approximately 45,000 years ago. Neanderthals possessed traits akin to that of the stereotypical, classic ape: large noses to humidify air, large physical stature, and stout bodies for heat conservation [5]. Homo sapiens, in contrast, possessed much different traits: the ability to craft and utilize tools, develop trade networks, and a more developed brain which could synthesize creative thoughts and logical processes. Consequently, Homo sapiens could adapt to changes in weather and the availability of resources; Neanderthals could not [6]. Evidently, these traits which were expressed among Homo sapiens garnered positive results for their species, manifesting in healthier offspring and the development of social networks. The extinction of Neanderthals, though its cause is hotly debated, ensued soon after. Interestingly, however, interbreeding between the two species is documented through the analysis of the current human genome. It is therefore postulated that positive traits garnered from Neanderthal genes, such as heat conservation and rigid bone structure, have contributed to the further propagation of the Homo sapien species [7].

This example of evolution exemplifies the impact of widespread positive traits on a species and the role of genetic mutations which lead to these traits. It connects directly to the advent of current genetic engineering because evolution is essentially long-term, trial-and-error based genome change which occurs over entire species over hundreds of thousands of years. Evolutionary theory is crucial to understanding genetic engineering, because this new technology has the potential capacity to expedite the mutation of positive traits and defy the laws of evolution by applying it to single organisms in a drastically shorter period of time.

CRISPR

How CRISPR Works [8]

Researcher Yoshizumi Ishino first discovered regularly interspaced repeats at Osaka University in 1987. It belonged to the gut microbe E. coli. In a specific gene, they discovered five identical sequences of DNA . Further studies in the 1990's revealed that these sequences were peculiarly accompanied by a collection of genes in close proximity, which encoded enzymes that could cut and manipulate strands of DNA [1].

CRISPR, which stands for clustered regularly interspaced palondromic repeats, are a series of genetic repeats found in bacteria's DNA. When viruses called bacteriophages attack bacteria, they inject and infect the host cell and foster the replication of its own, viral cells through the bacteria's "cellular machinery" [2]. CRISPR's special capabilities extend to copying a portion of the bacteriophage DNA if they survive an attack and using it to inhibit viral growth [3].

If applied to other biological life forms, the possibilities for CRISPR are seemingly endless. Because the genetic code encapsulates trait expression, whether physical or otherwise, the manipulation of this code can theoretically cure disease, alter aesthetic abnormalities, and change the activity of specific genes in plants and animals, including humans[4].
CRISPR at Work [5]

The foremost genetic editing technology in practice today, CRISPR-Cas9, contains two elements: the CRISPR gene, which contains the palindromic repeats necessary for encryption, and the CRISPR-associated protein 9 (CAS9) [6].

1. A single-stranded molecule that reads genetic information from DNA, RNA (ribonucleic acid), is synthesized via a process called transcription which copies DNA into RNA [7].

2. RNA guides the Cas9 protein to the site of designated editing activity.

3. The Cas9 protein, which has the ability to "cut" DNA, locks onto the designated strand and unzips it. CRISPR, which accompanies the RNA and Cas9 protein, consists of healthy DNA repeats to replace the targeted, unzipped strand. This results in either the discard of mutated or faulty genes or the replacement of those genes with optimal ones.

Gene Yeo, an RNA biologist at UC San Diego, compares the Cas9 protein to a "Swiss Army knife with just a knife"; they have bolted on chemicals and proteins to turn it into a "multi-functional tool"[8].

The CRISPR Patent Case

In 2018, the U.S. Court of Appeals for the Federal Court "heard several oral arguments in the case between UC Berkeley and the Broad Institute" focused on the future of CRISPR-Cas9 gene editing technology [9]. The case was presided over by a three-judge panel. Use of the technology was first described in the Science paper by a team at UC Berkeley in 2012. It was first applied to prokaryotic cells. A few months later, it was applied to eukaryotic cells by Dr. Feng Zhang at the Broad Institute in Massachusetts, after which he applied for patents; this is notable because application to eukaryotic cells is crucial in animal and human testing.

In 2013, Berkeley filed an interference claim against the Broad Institute's patent application, now cited as a "priority contest", to the U.S. Patent Trial and Appeal Board (PTAB)[10]. The ensuing case involved the research, financial, and ethical implications of controlling the technology.

The 45-minute hearing subsequently concluded with no winner, and both parties hopeful that they would secure the patent to control research. The final decision will determine the progress of research and potential monetary gains [11]. The stalemate that currently exists is harmful to the research effort at large, because it makes it more difficult to bring CRISPR and its tools to the market. The potential result will give either one or both of the institutions the ability to license the technology to interested companies, which could produce an exclusive monopoly and hinder future research progress.

The CRISPR patent case is especially unique because it treats the technology as intellectual property. Whoever has priority access to the technology will arguably have dominion over all new findings and fiscal opportunities, similar to that of any new, innovative product that you can file a patent for. The perception of CRISPR and its associated technologies as merely a patented product is a curious one; it reduces CRISPR to a by-product of the free market, when its true value cannot really be quantified by any figure noted by currency. Further developments in the patent case will determine whether CRISPR-based institutions will apply it like a business or work toward the interest of benevolent research; a hybrid of the two is the most likely outcome.

Other Technologies

TALEN

Transcription activator-like effector nuclease (TALEN®) is a gene editing technology combines a TAL DNA-binding factor to a DNA cleavage, or cutting factor. [12]

In short, TALEN is very similar to CRISPR, in that it uses synthetic restriction enzymes to cut DNA at specific sites. Similarly, this is could be for several reasons: to replace with a healthy gene, to remove faulty genes, or to remove genes that exist in conditional relationships with others. Specifically, restriction enzymes can be engineered specially to identify and cut any desired DNA strand. The components of TAL effectors are secreted by a special bacteria called Xanthomonas [13]. Thus far, TAL effectors have been utilized in some fairly complex human experiments:

1. The technology has also been "utilized experimentally" to manipulate genes attributed to disease expression. For example, it has been used in vitro to treat the individual genetic defects that lead to inheritable diseases such as sickle cell anemia [14].

2. TALEN has been used in genetic editing to engineer "human embryonic stem cells and induced pluripotent stem cells", which come from somatic cells. These experiments have occurred in vitro, as well, most likely to improve certain genes or eliminate faulty ones. Ultimately, their goal is to use this technology for drug discovery and the practice of innovative technology such as cell xenotransplantation [15].

Zinc-Finger Nucleases

Similar to TALEN and CRISPR, "zinc-finger nucleases are a class of DNA-binding proteins that targeted editing of the genome by creating double-strand breaks in DNA at user-specified locations" [16]. The process of gene editing with zinc-finger nuclease is nearly identical to CRISPR, in which two parts, being the DNA-binding domain and DNA-cleaving domain, form a zinc-finger protein, creating a unique set of "genomic scissors" for gene editing purposes [17]. Specifically, zinc-finger nuclease experiments have been conducted to execute the following applications [18] :

1. Functional genomics and target validation - identifying and manipulating cell lines with "gene knockout" technology; in other words, replacing faulty genes which improve cell line functionality.

2. Cell-based screening - the ability to manipulate cell lines by using "reporters and promoters with endogenous genes" (genes that are synthesized within cell tissue).

3. Cell-line optimization - "creation of cell lines that produce higher yields of proteins or antibodies."

Like TALEN and CRISPR, zinc-finger nuclease can be utilized to either repair or improve the genome. The distinction of this technology lies in its application. Opposed to work in current living cells or in vitro, ZFN (zinc-finger nuclease) is expected to be applied ex-vivo using patients' own stem cells. Once the cell has been edited, the cells could be "expanded in culture and reinserted into the patient to produce differentiated cells with corrected function" [19]. This could be seminal in curing disease, improving quality of life, or enhancing a patient's mental or physical attributes by a considerable margin from an early age. The performance of such experiments ex-vivo would also be an important step in contemporary, regulated human trials, which will be essential in bringing the technology mainstream. By combining the genetic potential of stem cells with this cutting-edge technology, the possibilities are truly limitless with human application.

CRISPR in Agriculture

CRISPR at Cold Spring Harbour Labs[20]

CRISPR has the potential to revolutionize the agricultural sector by allowing humans to control the evolutionary process of plant DNA. Challenges facing the agricultural sector such as drought, pests, and increasing production demands can be alleviated with the modification to gene traits. This evolution of gene-modifying technologies significantly reduces research costs while being more precise and accurate. The democratization of gene editing will allow smaller individuals and organizations to disrupt entrenched monopolies such as Monsanto’s Round Up Ready strains of seeds [1].
Balance of Gene Expression and Crop Yield[2]


Effect of Modification of SP5G Gene [3]

Cold Spring Harbour Laboratory Tomato Plant Experiments

In 2016, Sebastian Soyk and researchers at the Cold Spring Harbour Laboratory were able to use CRISPR/Cas9 to modify tomato plants to sprout flowers earlier. Researchers were able to identify and modify a flowering repressor gene called Self-Pruning 5G (SP5G). Base pairs in the gene were modified to reduce the expression of flowering repressors. The experiment was a success and the tomato plant flowered earlier than normal. Following their success, Soyk used CRISPR to generate null mutations of SP5G, effectively making the gene non-functional. This resulted in tomatoes flowering two weeks earlier than unmodified plants. [4].

The implications of Soyk’s research is that existing farmers can increase tomato yields without added effort. Farmers in higher latitudes who are restricted to shorter growing seasons can now plant tomato crops with the modified SP5G gene. His experiments can be applied to other crops such as soybeans or wheat improving the efficiency to the entire agricultural industry. Crops with modified SP5G genes can also be modified with other genes that increase fruit size or are disease resistant. Large agricultural corporations such as Monsanto have the opportunity and resources to invest in CRISPR modified crop seeds and patent them just as they did with Roundup Ready crop strains [5]

Too Many Good Genes

Soyk also researched the interactions of two differently mutated tomato plants who were bred together. During the domestication of the tomato plant, a gene responsible for the proliferation of cell mutation called MADS-box gene caused tomato fruits to grow larger in size. This beneficial trait was bred by humans in tomato crops since it allowed for improved crop yield.

In more recent years, researchers identified another mutation in the MADS-box gene that caused tomato plants to proliferate more flowers. This separate mutation ultimately leads to high crop yield since there are more flowers that lead to fruits.

These two tomato varieties were bred with the hopes of developing a strain that produced both larger and more tomatoes than standard crops. Combining these varieties caused negative epistasis, the interaction between two genes, which led to undesired branching and sterility. The balance of vegetative traits like leaf size and root depth was usurped by reproductive qualities like flower growth and fruit size. Crossbred plants with both mutations in the MADS-box gene yielded less fruit due to the imbalance of reproductive and vegetative traits caused by the negative epistasis [6]. While the combination of traits is beneficial in theory, it caused tomato plants to try producing more fruits than it was capable of which ultimately lowered yields compared to standard crops.

With CRISPR, researchers are able to fine-tune the expression of individual genes to create a balanced crop. CRISPR allows researchers to modify individual base pairs to either enhance or restrict the expression of genes. Balancing gene expression can minimize negative epistasis by adding just enough of a trait without limiting the viability of a plant. Soyk was able to modify individual base pairs in both mutated genes to reduce their expression. He was able to balance the epistasis leading to a strain of tomato that produced slightly larger and slightly more tomatoes compared with standard crops without limiting their viability[7].


CRISPR Crop Regulation

Countries Producing GMOs [8]

Given the potential of CRISPR to revolutionize the agricultural sector, regulatory bodies are the gatekeepers to determine whether crops are widely adopted. Regulators must strike a balance of ensuring CRISPR modified crops are environmentally safe and non-toxic while not stifling research. The number of published scientific papers involving CRISPR has increased 1,453% from 2011 to 2016[9]. Funding to research labs may vanish if strict regulations make CRISPR crops not commercially viable. A country’s agricultural sector may stagnate if their strict regulations prevent breakthroughs in leniently regulated countries to be adopted.

United States

On March 28, 2018, the U.S. Secretary of Agriculture Sonny Perdue issued a statement clarifying the U.S. Department of Agriculture’s (USDA) regulation on the use of CRISPR for agricultural plants. Under its current regulation, the USDA does not regulate “plants that could otherwise have been developed through traditional breeding techniques”[10]. Since CRISPR can modify a single base pair in DNA and traditional breeding relies on mutations of a base pair in DNA, the USDA views this new technique indistinguishable to traditional methods.

The governments lax regulation on CRISPR modified plants allows organizations to bring new strains to market with much less restriction compared to GMOs. Genetically engineered plants for commercial use are regulated by the USDA, Environmental Protection Agency (EPA), and FDA to ensure they do not pose a significant risk to consumers or the environment. Crops that contain transferred traits from other species are subject to strict regulation to minimize potential risks. These risks include the potential for toxicity in human, trait exchange with wild crops, and environmental effects to other organisms[11].

Europe

On July 25th the Court of Justice of the European Union in Luxembourg ruled that CRISPR modified crops are considered genetically modified organisms (GMOs). This ruling means CRISPR modified crops will be imposed the same many hurdles and approval processes the European Union imposes on GMOs[12].

The 2001 directive that laid out regulations on GMO commercialization was based on techniques that involved inserting large segments of DNA from one organism to another. This ruling poses a major setback to CRISPR edited crops as many scientists were hoping the technique would be exempted from GMO regulations since the technique has many similarities to traditional breeding techniques. Breeders and scientists advocated that CRISPR should be considered mutagenesis, like irradiation, since individual segments of DNA would be switched.

Health Canada regulates novel food using scientific data [13]

Stefan Jansson, a plant physiologist at Umeå University in Sweden, believes genetically edited crops will be non-existent in European research because funding will cease as CRISPR edited crops are no longer commercially viable [14].

Canada

Canada takes a wholistic view of modified foods by not focussing on the method used to create new crops but rather if it results in a novel food. The Canadian government views novel foods as:

  • Foods resulting from a process not previously used for food.
  • Products that do not have a history of safe use as a food.
  • Foods that have been modified by genetic manipulation, also known as genetically modified foods, GM foods, genetically engineered foods or biotechnology-derived foods [15].

Health Canada regulates novel foods by assessing their safety through the submission of detailed scientific data. Canada, like the United States, has taken a relatively lenient stance on GMOs. Canada is the third largest producer of GMOs heavily producing GM crops such as maize, soybean, and canola. New genetic engineering techniques like CRISPR will not be regulated differently compared to traditional techniques since regulations are based on whether foods are novel [16].

Biofuels

Traditional Biofuel

Biofuels are produced by the synthesis of organic materials and covers approximately 10% of total world energy demand. Recently, advances in technology have allowed the extraction of biofuels from materials such as wood and cash crops [17]. A study in 2008 determined that 80% of biofuel production in the developed world was attributed to residential use, with 18% attributed to industrial use [18]. Biofuels have been a growing sector of the energy market for several years; by 2025, their global market share is expected to reach $54.8 billion dollars [19]. Traditional biofuels are primarily separated into two distinct types.

Biofuel Synthesis Diagram [20]

1. Bioethanol - renewable alcohol fuel made from the sugars found in grains (eg. corn, barley) [21]

2. Biodiesel - fuel made from vegetable oils, fats, or greases. By exposing these byproducts to heat and pressure, biofuel synthesis can occur.

Biodiesel is non-toxic, biodegradable, and produces lower levels (up to 60% less) of pollution than petroleum-based diesel fuel; in fact, it is usually sold as a blend in petroleum-based fuel[22].

There is a growing demand for biodiesel because of its improved impact on the environment. Biodiesel is synthesized via a method called transesterification. In this process, crops such as wheat are exposed to heat and pressure, whereby the parts of the crop are separated and turn oil into viable fuel. The process ultimately leaves behind two products: glycerin, a byproduct, and methyl esters, which is another word for biodiesel [23]. Accordingly, opposed to fossil fuels, most studies have found that producing first-generation biofuels from feedstocks results in drastically lower emissions (20-60% reduction) [24].

According to the Intergovernmental Panel on Climate Change, depleting fossil fuel reserves will "become a pressing issue in 50-100 years" [25]. The genetic engineering of biofuel is important because it represents a significant portion of the clean energy sector. Despite criticisms of biofuel synthesis and its consumption of energy, gene editing technologies such as CRISPR would be able to exponentially increase the efficacy of biofuel production by manipulating genes which inhibit plant growth and limit traits that are beneficial for energy production. Scientific research of biofuels could see significantly larger increases in biofuel synthesis in proportion to the energy their processes consume.

Algae-based Biofuel & CRISPR

The synthesis of algae-based biofuel has been a growing market to reduce the costs of land usage associated with cash crop biofuels. Microalgae are contribute to over half of "global primary production" [26] and their capacity to capture CO2 obliterates that of conventional agricultural crops [27]. Therefore, synthesis of these algae are extremely efficient in biofuel synthesis. Algae are also naturally occurring all over the world, and therefore easy to access and cultivate, thereby allowing growth in copious quantities [28]. They are genetically diverse, and over 100,000 different species belong to the algae family [29]. Genetic diversity is correspondingly crucial in researching new opportunities to harvest energy.

The common trait all algae share is their ability to synthesize different kinds and levels of oil using the process of photosynthesis [30]. These fats, or lipids, can be extracted from algae such as pond scum. Basically a two-part process, the hexane solvent method (combined with pressing the algae) "can extract up to 95% of the oil from algae" bodies [31]. The process is simple: an industrial press is applied to squeeze out the oil, and the algae that remains is treated with hexane to extract the remaining oil [32].
Freshwater Algae, or "Pond Scum", are ideal candidates for biodiesel synthesis [33]
Traditional methods of biofuel synthesis are then used to convert algal energy into biofuels.

Two major players in the algae-based biofuel market have been energy giant Exxonmobil and their partner, Synthetic Genomics. In 2017, they announced that after nearly a decade of research, they had utilized genetic engineering with CRISPR by tweaking a particular gene in a certain species of algae. Subsequently, they were able to ensure that the algae produce double the lipid output while still maintaining their usual growth rate [34]. The partners report from the Synthetic Genomics website that this development and the subsequent algae strain modification stimulates oil production from 20 to 40%[35]. A publication in Nature Biotechnology in the same year reported: "We identified 20 transcription factors as putative negative regulators of lipid production by using RNA-seq analysis of N. gaditana during nitrogen deprivation. Application of a CRISPR–Cas9 reverse-genetics pipeline enabled insertional mutagenesis of 18 of these 20 transcription factors"[36]. This concept is vital because of the concept of conditionality between genes; previously, knocking out certain genes would influence other genes which inhibited regular growth. This posed a considerable challenge because continuous growth and regeneration are necessary to make algae-based biofuel a sustainable source of energy. The implication of this study is that knocking out specific genes which do not inhibit growth will be essential in making these processes more efficient, and thus more sustainable. Such developments may work in tandem with recent work from UCLA, UC Berkeley, and UC San Diego, who have sequenced the genome of a specific green algae called "Chromochloris zofingiensis"; the algae is "a prolific producer of triacylglycerols," an essential ingredient in biofuel synthesis [37]. Understanding the nature of the algae's genome and its functionality will be an essential component in making algae a major contributor to the energy market and potentially reversing the effects of climate change.

Algae have been recognized as an alternative with great potential, but several concerns still remain. These challenges include strain identification and improvement (oil productivity and plant immunity), nutrient and resource allocation and use, and the research of complementary products that protect and cultivate the current strains [38]. The fact that an energy giant such as Exxonmobil is a leader in algae-based biofuel research has also garnered concern from many experts. This is because Exxonmobil and its subsidiaries are currently involved in several major lawsuits involving their products' impact on climate change and the extent of their knowledge of this matter [39]. Additionally, the question of whether large-scale algae synthesis is scalable is a concern. In the larger cultures, the biomass density of the algae defeats desired growth rates due to the unequal distribution of light, and algae, like any other plant require sufficient light and water to develop [40]. If this consumption in improportionate to their output, then large-scale algae-based biofuel synthesis may not be a profitable nor sustainable project.

Animal Agriculture

Selective Breeding

Selective Breeding[41]

Livestock is a major area which is impacted by gene editing. Animals are important contributors to human needs as they provide food resources for our consumption. These resources include milk and meat by-products such as leather and food for consumption.

One of the main objectives of genetic engineering in livestock is to increase disease resistance, animal productivity and the product quality. Until recently, a traditional method of altering offspring was commonly known as selective breeding. Selective breeding is the mating process of a male and female to produce offspring with specific characteristics and traits [1]. In essence, the goal of selective breeding is the reduction of production costs and increase of output values in regards to livestock. For example, it is very common to find chicken and cattle with significantly greater weight and milk and egg production in comparison with the ones we cultivated one hundred years ago.

There are essentially two types of breeding: Inbreeding and Outbreeding [2]. Inbreeding is the mating of two closely-related animals, especially over many generations. However, inbreeding is nothing but disadvantageous due to a high chance of attracting recessive traits. Outbreeding, on the other hand, is the mating of two unrelated organisms; a major advantage is the production of hybrid species with desirable traits.

There are several disadvantages to selective breeding, such as impreciseness of results and lack of control in the process. With selective breeding, it is not possible to target a specific trait; rather, it is reliant on the natural reproduction process. Moreover, it can take years from the mating process until the outcome is apparent and thus, it is costly as well. Last but not the least, animals are often harmed during or as a result of the process. For example, increased milk production in cows comes from huge udders which are painful for cows to carry [3]. Another example is the hyper-production of chicken's eggs; to make eggshells, chickens need calcium. If enough calcium is not available, they suffer from bone fractures [4].

Animal Welfare

Recombinetics & Animal Welfare[5]

The treatment of animals in farms by use of traditional methods has always raised animal rights concerns. In particular, the case of cows with horns had been labelled as ‘dangerous’ due to their threat to farm workers around them. Such nuisances in regards to animal bodily features forced biotech companies to look into gene editing animals for better body features, or none at all.

A biotech firm in Minnesota, Recombinetics, has experimented with molecular level modifications in animals to create farm animals with useful properties[1]. In particular, they were able to reproduce cattle without horns in response to concerns from local farmers. This experiment came as a breakthrough in animal gene editing with first observable evidence.

Ethical battles aside, the company has raised over $31 million from investors while attracting them with successful experimental results in animal body modifications[2]. Previously, the company has successfully genetically engineered pigs to not reach the age of maturity which taints its meat. As a result, they have been able to successfully sustain the supply of pork without concerns of stock expiring.

Disease Resistance

A challenge for the agricultural industry has been to procure plants from diseases that eat up their yields. The nearly $6 billion worth of plant production has seen increasing numbers of infectious plant diseases such as black rot pathogen, powdery mildew, crown gall disease and various others[3]. Similar to the animal agriculture case, scientists have looked to CRISPR, in particular CRISPR-Cas9 enzyme, in order to solve various issues in regards to genetic modifications of plants[4].

Dr. Junqi Song, an AgriLife Researcher and plant pathologist, along with his team treated the late blight disease in tomatoes and potatoes[5]. With CRISPR, Song was able to introduce a specific set of genetic regulators. These regulators, when inserted, enable the pre-existing disease fighting enzymes to better protect their cells against malicious pathogens. Pathogens such as phytophthora infestens, which leads to late blight disease in crops, has been observed as a prevalent disease in Texan crops [6].

Furthermore, Genus plc: A biotech firm, has been in the industry since 1933 and contributed by selling products manufactured using cattle and pig modifications. Reporting a net income of £34.4 million in 2017, the company was responsible for genetically engineering pigs to be immune to animal diseases which could potentially cost up to £1.5bn in animal by-products and deaths of such animals[7]. The researchers at Genus were able to edit a small strand of DNA in pigs thus establishling complete immunity in pigs. The change in the animal’s DNA exhibits no sign of off-target impact which was considered a huge success.

Biomedical

Xenotransplantation [8]

Xenotransplantation: The process of transferring organs from one species to another has been around since the early 1900’s[9]. Eventually, in 1984, the first case of transplant from a mammal to a human was witnessed when a baboon’s heart was transplanted into an infant girl. Over the years, this process has been reversed as well, manifested by the transplantation of human organs into animals. However, ever since the first case of xenotransplantation, there has been no successful cases of organ transplantation in humans from animals.

The success rates for this procedure is heavily dependent on the side-effects caused by microbes with the exchange of tissues. To overturn this, scientists have looked to CRISPR to explore ways of growing human organs in pigs, by eliminating pig genes and inserting human ones[10]. Companies, such as eGenesis, are investing heavily in research for stripping away viral genes and eliminating porcine DNA- the incompatible pig tissue[11]. Even though a lot more research needs to be done, the pathways and possibilities are clear for biomedical scientists in regards to human and animal welfare.

Ethical Implications

The democratization of gene editing through CRISPR has many ethical implications. Countries and individuals have different stances on what we should and should not do with this technology. Countries with strict regulation may stagnate their development compared to countries with less regard to potential negative consequences. CRISPR has the potential to bring back extinct species, make superhumans, and even wipe out entire invasive species, but should individuals play with these possibilities? Humans have a track record of over-abusing useful technologies without understanding their consequences such as the use of Polychlorinated biphenyls (PCBs) and plastics [12]. CRISPR is a technology that has the potential to cure deadly diseases and lessen world hunger, but it can also disfigure generations of humans and unintentionally damage species and environments.

Ethics of Editing Human DNA

US Public Opinion Survey Results [13]
Gene Editing in Humans Ethical Framework [14]


Gene editing in humans can be broken down into a four-category framework. Somatic refers to editing cells in the body where the DNA is not passed onto future generations. Nearly all cells in the body are somatic and have two identical copies of chromosomes.

In contrast, germline editing results in changes that alter future generations. Gametes are an organism’s reproductive cells and only contain one set of chromosomes. Male gametes are called sperm, and female gametes are called eggs or ova. Germline editing has many more ethical implications relative to somatic because the modifications will affect future offspring of the individual. Since gene editing is relatively new and epigenetics is hard to predict, a modification to improve one human trait may have several unintended consequences. In some countries, such as Canada, germline edits to humans are illegal even if they are used to cure diseases [15].

Therapy refers to editing used to treat or prevent diseases and disorders. Cosmetic serves no medical purpose and serves to improve outward appearances.

A survey conducted to observe US public opinion on gene editing found that 64% of respondents were in favour of somatic therapy and 65% for germline therapy. In contrast, only 26% found germline cosmetic applications to be acceptable and 39% for somatic enhancements. [16] The survey found the largest contributing factor to drive attitudes was whether the individual was religious. Among those who reported low religious guidance, 75% of which expressed some support for therapy applications and 45% for cosmetic enhancement, while those who reported high levels of religious guidance expressed 50% support for treatment and 28% for cosmetic enhancements[17].


Current Challenges

Before CRISPR or other gene editing techniques are implemented in humans, several technical issues should be minimized to prevent unintended consequences that could serious danger. 1,621 disorders have been identified that are caused by a single gene mutation making them the ideal candidate to be cured by CRISPR[18]. While it may be tempting to try curing these diseases, the technology can cause unintended edits and the complexity of humans may cause unidentified negative epigenetics.

Off-target editing

Off-target editing refers to cleavages that occur in the DNA that was not intended. High fidelity CRISPR techniques use single guide RNA (sgRNA) that contain around 20 nucleotides to complement the target DNA segment. Once the (sgRNA) finds the complementary portion of DNA, the cas9 portion cleaves the DNA to allow an edited segment to be inserted.

Since the humane genome contains around 3 billion base pairs[19], there is a sizable chance the 20 nucleotide sgRNA will find a complementary segment and cleave an unintended segment causing off-target edits. Off-target edits create changes that are not understood and can severely affect an organism’s viability. More precise techniques are being developed to use larger segments of sgRNA to prevent off-target editing.

Mosaicism in Embryo vs Uniform Embryo [20]

Xiao-Hui Zhang in his academic article, “Off-target Effects in CRISPR/Cas9-mediated Genome Engineering” noted off-target issues that are harder to control. He found that cleavages in DNA occurred even when there were three to five mismatches in the sgRNA [21]. Even if the sgRNA is designed to be more precise, a "good enough" match can be found and cleavages will occur. Researchers must design methods to test the success of CRISPR techniques and identify if any off target edits have occurred.

Mosaicism

Mosaicism occurs when not all cells in an edited organism contain the edited gene. This issue occurs when an embryo is a single cell and the CRISPR technique is implemented. The cell can replicate before the CRISPR process is complete causing only one of the cells to inherit the modified gene, while the other cell does not. If an embryo was modified to prevent a disease, mosaicism can cause the child to still have the disease [22].

A solution to reduce mosaicism is to use the CRISPR technique as soon as an embryo is fertilized and to end the operation after a few hours. This reduces the chance of mosaicism as the cell does not have the opportunity to replicate during the CRISPR process.

Potential Side Effects

Like any technology, gene editing technology is prone to human error. In the explanations of How Crispr Works, it was detailed how fragile this process can be depending on the actor. Previously, scientists thought that CRISPR-Cas9 only cleaves DNA if it exactly matches the correct DNA strand identified by RNA. A researcher at Delft University of Technology [23], however, has determined that DNA that resembles the target strand can also be cut by Cas9, despite containing several different letters. Moreover, since viruses mutate constantly, they can therefore change their genetic make-up to avoid detection. CRISPR Cas9 works in tandem with these mutations by tracking the evolution of a virus to better protect the patient against foes. Although this may be a functional side effect in theory, it is not functional for the purposes of human application. Experiments have shown that CRISPR-Cas9 is more likely to cut certain non-matching sequences than others [24]. This means utilization of the technology could potentially cut genes that exist in conditional relationships with other healthy, functional genes by accident. If this off-target cleavage occurs in the case of zinc-finger nuclease, the production of enough double-strand breaks to overwhelm the repair machinery may occur and, as a consequence, yield chromosomal rearrangements and/or cell death [25]. This could potentially lead to further problems in the genome or amplifying the issues that the technology is trying to remedy. In the case of donor DNA, there is also a risk of a negative immune response in the patient, similar to that of an incompatible blood transfusion. Regardless, scientists believe the response to this would be quick and easy to treat, and that more specific target identification with these technologies will be imperative moving forward.

Future Uses

Healthcare

HIV Treatment and Resistance

The Cas9 Mouse: these mice have been genetically modified to already contain the Cas9 protein for genome-editing purposes [26]

HIV, or human immunodeficiency virus, is an infection which is the precursor to the AIDS medical condition. It is a retrovirus that can be transmitted through blood or sexual contact, and inflicts progressive harm to vital organs and the immune system[27]. The AIDS scare during the 1980's was spurred from the discovery of unusually aggressive lung infections and cancers in five young men in New York and California in 1981 [28]. The disease subsequently spread rapidly and has gone on to affect 36.7 million people worldwide, 1 in 7 of which are not even aware that they are infected [29].

In 2016, Kamel Khalili from Temple University successfully eliminated HIV genes from the genomes of mice and rats infected with the virus. His team engineered the animals to incorporate specific HIV genes into nearly every cell in their body, "from the brain, to their blood cells" [30]. Khalili used CRISPR during these experiments; his team was able to "snip out" the viral genes which had been infected by injecting the CRISPR gene into their tails [31]. Thus, the team was able to eliminate 50% of the viral cells [32]. This scientific breakthrough has combined the self-healing abilities of the body with gene editing technology. HIV is particularly deadly and its impact is vast because of its spread through sexual contact and prevalence in the developing world. The deceptive nature of the virus means that many do not have the access to medicine or technology for treatment. For instance, approximately 70% of people living with HIV globally were aware that they were virus carriers in 2016; the remaining 30% (over 11 million people), however, are a potential threat to themselves and their communities unless they can access medical services [33]. Because HIV can be transmitted from mother to child, advanced gene treatment and raising awareness could potentially eradicate the disease entirely with large-scale implementation and research.

World Lunger Cancer Rates 2014 [34]
Furthermore, in China, CRISPR gene editing has been used on 86 patients since 2015 to treat cancer and HIV patients; doctors reported to journalists at the Wall Street Journal that "some patients' health had improved" [35]. However, about 15 deaths have occurred during these trials, which raises the issue of ethics involving gene editing and regulation in developed nations such as the United States. The Chinese Ministry of Health needs to approve all gene therapy operations, but due to the nature of the political system and China's emphasis on growth and innovation, it is probable that regulations are "relatively relaxed" in comparison with North America [36].

Cancer

"The Emperor of All Maladies", or cancer, has plagued human societies for thousands of years[37]. Cancer treatment will undoubtedly be the primary target for CRISPR and associated genetic editing technologies going forward, and substantial efforts have already begun. In January 2018, doctors at the University of Pennsylvania reported they would be using CRISPR to modify human immune cells to efficiently identify and kill cancer cells [38]. This is a significant deviation from the expected, current train of thought, being the manipulation of already present cancer cells. Improving immune efficiency will be a long-term goal for gene editing researchers to implement; the development of immune strength will potentially transform the possibilities for all medicinal purposes in the future. To enhance the treatment, the UPenn scientists intend to delete two genes from the patients' T-cells - one of the genes to be removed manifests as a “checkpoint” molecule, PD-1, "that cancer cells exploit to put brakes on the immune system" [39]. By doing so, researchers are ultimately changing the chemical composition of the human genome permanently. If successful, potential patients in the future will possess enhanced immune system capacities for as long as they live, which could be the first step in anti-aging, drastically elongated lifespans, and long-term healthcare reform.

In contrast, manipulating the genome to serve cancer inhibiting purposes could also backfire. Researchers have determined that gene p53 in humans plays a major role in tumor prevention by killing cells with damaged DNA. Consequently, strengthening this gene could be an important factor in battling cancer. However, p53's function also includes acting as a natural defense to foreign invaders, and may see CRISPR Cas9 as an aggressor. When DNA is replaced, p53 enters the fray and "self destructs" the gene; therefore, if certain genetic manipulations do occur and p53 destroys these genes, it could expose the body to a host of other cancers. [40]. This hypothesis works in conjunction with the conditional relationships which genes often operate in; influencing one part of the genome can have serious consequences on the function of other traits which could cause more harm than good.

Cystic Fibrosis

Cystic fibrosis is a genetic disease which is caused by a single defective gene which promotes a "thick, sticky buildup of mucus in the lungs, pancreas, and other organs", resulting in subsequent lung infections and chronic breathing difficulties [41]. This constant barrage to the immune system results in drastically reduced lifespans for patients, for which a cure is not yet available [42]. Cystic fibrosis could be a perfect candidate for gene editing since it is attributed mainly to a single defective gene. This gene, called CFTR, allows the normal transport of water and "charged ions in and out of cells" [43]. The damaged gene does not allow for this optimal flow, so this compromised process will promote bacteria build-up and results in a chronic respiratory condition where airways are blocked [44]. Theoretically, CRISPR Cas9 could be used to both remove and replace this single gene. Scientists have hypothesized that by manipulating and expanding the culture of intestinal stem cells in patients over long periods of time, genetically and phenotypically healthy CFTR genes can be developed [45].

Once again, application of the technology could allow the body to essentially heal itself. However, the nature of this damaged gene is diverse, meaning that several versions of CRISPR therapy will need to be developed to combat different genetic defects[46]. Similarly, immune system rejection could be an issue; if a patient receives the incorrect treatment or induced mutation, it could cause further problems associated with their current disease, or spur the development of new ones.

Eradicating Invasive Species and Gene Drives

Gene Drives[47]

The globalization of humans introduced dozens of species into regions where they have never existed. For example, the Asian carp was introduced from China into North America and Europe where they have no natural predators. Their large appetite and aggressiveness have devastated native species[48].

CRISPR coupled with gene drives has the potential to eradicate invasive species or disease carriers like mosquitoes. In normally reproducing organisms, there is a 50% chance a specific gene will be passed on by one of its parents. If a species with a CRISPR modified gene is introduced into the wild, only a small portion of the population will inherit the gene and it is possible that edited gene will become extinct. Gene drives have the potential to ensure all future generations will inherit a specific gene and proliferate it to the rest of the population.

Gene drives work by using CRISPR to insert specific genes into a species. Gene drive species contain the altered gene, the gene responsible for the Cas9 enzyme, and several guide RNAs. During reproduction with a wild species, the guide RNA from the gene drive directs the Cas9 enzyme to make a cut in the wild DNA and that portion of DNA is replaced with the altered gene. Gene drives effectively ensure future offspring will carry the edited genes[49]. Gene drives only have the potential to affect an entire population when the species has a quick reproduction time. For example, a single gene drive mosquito can lay 200 eggs[50]. The future 200 offspring can create 40,000 mosquitoes with gene drives and in the next cycle 8,000,000 and so on. However, if a gene drive were in a human, it would take several decades to produce offspring with gene drives. Offspring with gene drives would take another several decades before they can reproduce. Organisms with long reproductive cycles are unlikely to proliferate gene drives as compared to organisms with short cycles.

Gene drives in the natural environment must be used with extreme caution. Once a gene drive is released, it is essentially permanent and most of the species' population will carry the edited gene. If a gene drive is not released carefully, an entire species may have genes that were unintentionally edited. While gene drives can be dangerous they can also eradicate diseases. Mosquitoes can be edited to be resistant to malaria parasites possibly eradicating the disease entirely by removing the vector. Gene drives can also be used on invasive species so only female offspring are produced. Over time, the lack of male species will destroy the invasive species population.

Designer Babies

Modifying Human Intelligence?[51]

Due to loose regulation, human CRISPR trials have begun in China as recently as August 2016; a team led by Lu You, an oncologist at Sichuan University’s West China Hospital in Chengdu,started testing on lung cancer patients by injecting them with genetically-modified cell material [1]. This is a telling implication of the exciting future ahead for CRISPR and associated technologies. It is quite possible that mainstream application, especially at the in vitro level, could change the course of human history.

The engineering and conception of designer babies may be hugely controversial at first glance, but it is important to note that events have already begun to usher in this new era. In September 2016, for example, Dr. John Zhang utilized a technique that incorporates DNA from three separate parents, effectively conceiving a "designer baby". This experiment was conducted due to the fact that one parent had a family history of Leigh syndrome, which is a fatal disorder which affects the developing nervous system. The illness was responsible for the death of her first two children, and this parent wanted to give her third child the best chance for survival. Consequently, the idea was implemented and Dr. Zhang

Designer Babies[2]

has been working toward methods such as these that will prevent mitochondrial disease. In this case, "before the fertilised eggs start dividing into early-stage embryos, each nucleus is removed. The nucleus from the donor’s fertilised egg is discarded and replaced by that from the mother’s fertilised egg" [1].

The removal of faulty genes and absence of disease will evidently be the first goal of scientists before moving onward to other human factors. The timeline for this development is uncertain, and many analysts agree that mainstream human trials could be several decades away. Nevertheless, it is both concerning and galvanizing that technology's influence on the human genome could create entire hereditary lines free of disease or deformity. Many of these approaches have been extended to progenitor cell types, including embryonic stem cells [2], meaning the ability to alter genetic potential could be limitless depending on effectiveness. In theory, possibilities for aesthetic and mental attributes using stem cells could be considerably vast. Aesthetic features such as hair thickness, bone structure, muscle density, and eye color could be altered by influencing just a few genes. Additionally, intelligence, emotional awareness, critical thinking skills, and athletic intuition could be induced by either introducing the genes of a host or knocking out faulty genes which inhibit these attributes. At the Karolinska Institute in Stockholm, Fredrik Lanner has already begun to edit human genomes beginning at the embryos [3]. The ethical implications of such work are just as questionable as the positive implications of engineering genetically superior offspring. With China firmly in the lead due to loose regulations, a larger disparity between nations' population efficiency and survival rates could spur conflict. Children born with enhanced physical and mental capabilities would indefinitely affect the social hierarchy from small communities to a large scale. A study conducted in 2003 [4] determined that such phenomena are already present in our culture, showing that over a 30-year career, "a six-foot tall person would [earn] $166,000 more than someone five-foot five-inches". Thus, forced genetic mutations to further aesthetic potential could have a profound effect on our human perception of fairness and contemporary human values. The concept of human development could take on an entirely different form, as well, and the general attitude toward fate, religion, and morality would also be due for a tune-up if this technology is utilized on a massive scale. Thus, laws could see considerable change and potentially favor those with enhanced abilities and subsequently, opportunities. Such an occurrence on an institutional level could see an increase in wealth disparity and the exclusive access of gene editing to those in positions of power. By determining the genetic make-up of certain human beings from conception, a plethora of possibilities and a gateway to innumerable ethical dilemmas is swung wide open.

The debate is far from over, but the gene editing development could take some time and has several parties ready to regulate. Dr. David King, for instance, is the founder of Human Genetic Alert, which is an independent watchdog group opposed to certain outcomes of genetic engineering[5]. King provides an important antithesis to the popular debate by placing himself firmly in the camp of caution before implementation. The ethical debate will continue for the following decades, which struggles profusely with the balance between the constant improvement of life and the potential fallout this could create.

Authors

Matthew Cheng Miguel Soler Arfa Malik
Mca149@sfu.ca Msoler@sfu.ca Arfam@sfu.ca
Beedie School of Business
Simon Fraser University
Burnaby, BC, Canada
Beedie School of Business
Simon Fraser University
Burnaby, BC, Canada
Beedie School of Business
Simon Fraser University
Burnaby, BC, Canada

References

  1. https://www.newscientist.com/article/2107219-exclusive-worlds-first-baby-born-with-new-3-parent-technique/
  2. https://www.stemcell.com/human-pluripotent-stem-cell-genome-editing-using-the-arcitect-crispr-cas9-system.html
  3. https://www.nature.com/news/gene-editing-research-in-human-embryos-gains-momentum-1.19767
  4. http://timothy-judge.com/Height%20paper--JAP%20published.pdf
  5. https://futurism.com/expert-argues-that-gene-editing-will-widen-economic-class-gap/
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