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  • Published on: 7th September 2019
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History of GMOs.

Genetically modified organisms, or GMOs, are quite self-explanatory: they are organisms that are genetically modified. Some people think that GMOs are poison and that they are an unnatural result of science. However, humans have been genetically modifying organisms since prehistoric times, when farmers would plant seeds and sometimes cross two different plants in order to produce an offspring plant with the desirable characteristics of both parent plants. However, this wasn’t well characterized until the late 1850s—early 1860s, when Gregor Mendel, an Austrian monk, did experiments with pea plants and proposed the Mendelian Laws of Inheritance, which were used by European plant scientists to efficiently breed plants with desirable characteristics (American public media, 2015).

Perhaps the “evil, unnatural” process of making GMOs is more associated with modern genetic engineering. It all started in 1953, when Watson and Crick “figured out” the structure of DNA (some say that it was Rosalind Franklin who did, and that Watson and Crick stole her data). This seminal discovery led to a chain reaction of events in molecular genetics: in the 1960s, Linn and Arber discovered restriction enzymes in Escherichia coli. This enabled Boyer and Cohen in 1973 to successfully synthesize cloned recombinant DNA in the same bacterium. In 1974, the first “modern” GMO was created by Cohen, Chang, and Boyer. In 1977, Sanger created a method for DNA sequencing called Sanger chain dideoxynucleotide termination method.

Soon, the morality of the powerful tools of molecular genetics was questioned. In 1976, the National Institute of Health established guidelines for genetic modification research. The bioethics eventually reached the Supreme Court. In 1980, Diamond vs Chakrabarty led to the decision that genetically modified life can be patented (American public media, 2015). As a result, the first genetically modified mouse was made. In 1982, the US Food and Drug Administration approved “Humulin”, or human insulin synthesized by bacteria; this is an application of recombinant DNA. In 1983, a powerful tool that allowed specific DNA fragments to be copied many times, called the polymerase chain reaction (PCR) was invented by Karry Mullis. Also in 1983, the first genetically modified plants were created. In 1985, the first tests were done for genetically engineered tobacco in Belgium. In 1987, the US not only started to genetically modify tobacco but also tomatoes. Mice were modified to carry human genes. A plant was genetically modified to resist herbicide. In 1988, the plant to synthesize a drug is created via genetic modification. In 1991, the first trials for gene therapy appeared.

The 1990s is when people started to find GMOs to be evil. The USDA approved Calgene’s Favr Savr tomato, which is genetically modified to stay firm and not soften for longer periods of time, and the FDA had to proclaim to the people that GMO food isn’t “inherently” dangerous and doesn’t need special regulation, which implies that there was already some fear about the safety of GMO food. In 1993, a cow was genetically modified to produce more milk; it was approved by the FDA. In 1994, the first successful plant in vitro fertilization was performed. In 1995, the famous Bt potato and corn were invented. In 1995-96, Roundup Ready Soybeans, resistant to glyphosate (herbicide) was created, and even today there is a big backlash pertaining to the use of glyphosate and its potential health hazards. In 1996, Dolly the sheep was the first cloned animal. Because of all the negative attention attracted by GMO food, eventually in 2000 the International Biosafety Protocol was created to label GMO crops (American public media, 2015). However, 50 more nations are required to ratify it before it goes into effect.

However, GMOs aren’t as bad as they’re made out to be. Golden rice was developed to help combat Vitamin A deficiency. Also, in 2006, a pig was genetically modified to produce omega-3 fatty acids, which find their way into pills and have positive health effects. In 2010, Amflora, a genetically modified potato, is a approved; its starch can be used for making paper and adhesives. The list goes on and on.

A. How is GMO food made?

In this section, the production of modern GMO food will be discussed rather than selective breeding, because selective breeding is not as efficient. This modern method usually involves the synthesis of recombinant DNA, meaning that DNA from two different species is combined. For example, if a crop is very tasty to insects and they simply won’t let the plant grow, scientists might insert the gene for Bt, an insect toxin produced by a bacterium, into the plant genome and then grow a plant based on that new genome. That is a recombinant piece of DNA.

The first step is to extract the DNA of the gene of interest from the organism. for example, if it’s a bacterial gene, the bacterial genome can be isolated and purified; then, PCR can be used with specific primers to isolate the DNA of interest. The next step is to clone this gene (PCR will automatically do this). Third, the isolated copied gene has to be able to work in a different organism. In other words, how can it be inputted into another piece of DNA, and how can we be sure that it is expressed in the new organism? For example, if bacterial DNA is being inputted into eukaryotic DNA, bacterial promoters are recognized by sigma factors but eukaryotes don’t have sigma factors. Additionally, once the mRNA is made, bacteria use the Shine-Dalgarno sequence to find the AUG, whereas in eukaryotes, a combination of cap-binding protein activity and Kozak sequence scanning is needed to find the AUG. Restriction digestion and primers with overhanging ends can easily be utilized to add/subtract the necessary sequences and add restriction ends so that the “prepared” gene of interest can be inserted and ligated into the DNA of the organism where this gene of interest is to be expressed.

After preparing the recombinant DNA, the cells of the organism have to express it, so the DNA needs to enter the cells somehow. There are various methods of doing so. In order to grow a Bt plant, first Agrobacterium is transformed with recombinant plasmid; then, the grown-up Agrobacterium is placed in contact with plant cells, and the Agrobacterium will insert the gene of interest into the plant’s chromosomes. The plant cells are thus transformed. Another method is to use a “gene gun”, where gold particles coated with the gene of interest are shot at plant cells to get the DNA into the nucleus (Plant and Soil Sciences eLibrary pro., 2015). A similar method is to place plant cells in a solution with microfibers infused with the gene of interest, and these microfibers stab the plant cells (Plant and Soil Sciences eLibrary pro., 2015). Besides those methods, electroporation could be used, where an electrical pulse applied to plant cells opens up temporary holes in the cell wall/membrane, allowing the gene of interest to enter (Plant and Soil Sciences eLibrary pro., 2015).

The gene of interest is now inside the cells. The transgenic plant is grown, and it may be backcrossed with “elite breeding lines” so that a hybrid plant is produced with the new characteritics also the old beneficial characteristics that were not a result of genetic modification.

B. A Specific Look at Bt Plants (pbs.org, 2001)

As mentioned before, some plants are especially delicious to insects, and since they can’t control themselves, the farmers must protect the plants. Scientists have come to the rescue and made use of the fact that Bt protein, produced by Bacillus thuringiensis is toxic to insects. Moreover, using the theme of recombinant DNA, the genes coding for production of Bt protein are isolated and sticky ends are added using restriction enzymes.  Another gene to be added to the end of Bt protein gene is a herbicide resistance gene (the purpose will be clear later in the paragraph). This could perhaps be accomplished by using multiple overhanging primers containing portions of this herbicide resistance gene. Sticky ends could be added to the ends of  this Bt_Protein-Herbicide_Resistant DNA with restriction enzymes and the same ones are used to cut the Ti vector so that the BT_Protein-Herbicide_Resistance DNA can be ligated into the Ti vector. Then, the Ti plasmid is inserted (perhaps via transformation) into Agrobacterium tumefaciens. After this bacterium reproduces, parts of the Bt plant leaf are to be placed on the grown bacterial culture so that the bacteria can transfer the gene of interest to the plant cells. Later, these plant cells are grown on a separate medium. Here is where the herbicide resistance comes into play. We can spray the culture with herbicide after letting the cells grow a bit; the cells that took up the plasmid and are expressing the Bt must also be expressing the herbicide resistance genes. However, the cells that did not take up plasmid not only fail to express Bt but also the herbicide resistance genes, rendering them defenseless and dead in the presence of herbicide. This selection is used to create a population with only the plant cells containing the resistance genes. Finally, a pure stock of plant cells is made and the plant can be grown, with back crossing as necessary.

C. CRISPR method (New)

If you looked at the method of inserting Bt into plants, you can see that it is very laborious. The CRISPR method aims to shorten the general process. For the CRISPR method, you would take the gene of interest and have ssDNA oligonucleotides ordered for it (Bortesi, 2015). These ssDNAs can be inserted into a location in nuclear DNA specified by guide RNAs that you must also generate (Quetier, 2015). Research has to be done about specific promoters and also sequences like Shine-Dalgarno or Kozak, based on whether the organism being genetically modified is prokaryotic or eukaryotic, respectively. Finally, when all of this research is done, the CRISPR workflow can be run using the oligonucleotides, guide RNAs, and cells receiving the genetic modification.

D. RNAi technologies (New).

Sometimes, simply deleting or reducing the gene levels of an already present gene in a plant can confer beneficial characteristics. For example, people don’t like to eat watermelons or oranges with seeds in every bite, so it may be useful to silence the genes that lead to seed production. RNA interference (RNAi) is a method that uses small interfering RNA (siRNA) to specifically target the mRNA of the gene that you want to silence (Watson, 2005). The siRNA binds to the mRNA and with the help of specific siRNA-interacting proteins, the mRNA is cleaved and thus the protein coded for by the mRNA cannot be produced (Sherman, 2015). There are a few ways the siRNA can be used. A gene gun or the microfiber method can be used to “shoot” the siRNAs into the cells. However, the problem with this method is that the siRNAs will eventually get degraded, and once they are degraded, whatever they were supposed to silence will not be silenced anymore. To fix this problem, the gene coding for siRNA could be implanted into the cells’ genomes so that the siRNA is always produced. Additionally, antibiotic/herbicide resistance genes can be used for selection as necessary

D. How are GMO animals made?

It’s impressive to genetically modify cell lines, plants, and bacteria. However, what would be even more exciting and challenging? Genetically modifying an entire animal! The challenge with this is that an animal is multicellular and their life cycles are far longer than those of a plant.

Thus, the general paradigm for generating a GMO animal is the following: as usual, the DNA coding for the gene of interest must be extracted from the organism, and this gene is to be cloned using PCR, with sticky ends added, and important regulatory sequences (Kozak vs Shine Dalgarno, promoters) are added/subtracted as necessary. Once the gene of interest is ready to be inserted into the organism being genetically modified, it is important to remove the egg from the animal of interest and inject the gene of interest into the removed egg. Finally, the egg is implanted into a surrogate animal. That way, since all somatic cells will be derived from the sperm and the egg (including the new DNA that was just been added in), all of the cells will have the new DNA as well.

E. GMO Safety

Besides the many times irrational fears of non-scientists, there are actually some possible risks and controversies regarding GMOs. For example, foreign gene expression may alter an organism’s metabolism, growth rate, and other signaling pathways. Additionally, there is a risk of horizontal gene transfer of antibiotic/herbicide/resistance genes to other organisms like bacteria; this would worsen current resistance cases. Bt corn may kill non-harmful insects such as the monarch caterpillar, which itself is a natural pesticide. GMOs may also reduce genetic diversity; if nearby plants pick up a GMO gene and some disease comes along that specifically affects the plants with the GMO gene, then all those plants can be easily wiped out. In fact, if one is not very careful with the siRNA RNAi, the RNAi might silence the wrong gene (Casacuberta, 2014).

Thus, scientists have come up with some “GMO safety”. A GMO food is considered to be safe if it is safe for consumption by a conventional species. Also, certain experiments have been done to see if GMOs actually cause any problems. In GMO maize seeds overexpressing phyA2 compared to non-GMO counterparts, 210 metabolites were analyzed and 9/210 had significant changes (Rao, 2015). This indicates that GMOs may alter an organism’s metabolism (Rao, 2015). In contrast, when transgenic rice was compared to non-transgenic rice and each one was separately fed to rats, no adverse effects were observed (Zou, 2015).

F. Other Applications of GMOs

GMOs are not all that bad at all. Besides seemingly unnatural food, they can actually be found in a “3-parent child”. If a parent has a mitochondrial disease, since mitochondrial DNA has a different sequence than the human genome, a healthy person can donate his/her mitochondrial DNA to replace the diseased mitochondrial DNA in the mother’s mitochondria, and technically, the child resulting from this could be a “3-parent child”. GMOs can also be used to produce medicines, drugs, and vaccines (Phillips, 2008). For example, bacteria can produce insulin if they are engineered with the gene, and this helps diabetic people out since using genetically modified bacteria is more efficient than extracting the insulin from a pig or a cow (Phillips, 2008). GMOs are also widely used in research in order to figure out unknown functions of genes. A very simplistic way to do research is along the lines of “If x is missing, what happens (or doesn’t happen)?”. Finally, the most arguably unethical part of genetically modified organisms is to make a “wonder baby” with all the desirable characteristics, and that will be discussed later on.

Next Major Advances

Since one charge leveled against GMOs is how they are poisonous (for producing toxins), what if a GMO could be designed such that the toxin production is conditionally regulated based on the environment? For example, a plant could do its toxin secretion only when it senses an insect. If the toxin isn’t always produced, then that’s less “poison” produced by the plant and most likely the fruit will taste better. People would also likely feel safer about GMOs.

However, in order to truly attack the issue where a specific genetic modification would cause a change in the metabolism of an organism, perhaps a computer program could be designed such that it can predict all possible effects of a gene in a certain place to ensure safety. Such a program can also make suggestions on which genes to edit and where genes can be inserted and still be expressed without leading to detrimental changes in the cell. Once all of the possibilities are mapped out (which requires the function of all genes to be determined, and entire genomes to be sequenced and studied), then this program would be possible, and it would make GMOs almost completely risk free.

Additionally, since GMOs could hurt biodiversity, why not design a few GMOs for the same purpose to ensure biodiversity just in case there’s a species-specific blight or disease. That way, if one species is affected, then at least the other two species have a better chance of survival. For this, the minimum change to the DNA sequence such that the beneficial function is still conferred but the change is enough for the plant species to evade a blight that afflicts the same species using a slightly different GMO sequence must be determined. Perhaps computer programming can play an integral role in this work.

Lastly, sometimes targeted genome editing may not be accurate, and this can have detrimental effects on an organism. Thus, how can we achieve a 100% accurate target rate for genome editing? Since there are specific proteins that associate with different portions of DNA (especially with histone tails for example), perhaps the guide RNA and the proteins it interacts with can somehow be combined with proteins specific to the sequence being targeted to ensure that the guide RNA only guides editing to the correct gene locus.

Bioethics

Genetic modification of organisms for food is definitely ethical and necessary because eventually, with rising global temperatures and populations, it may not be possible to feed everyone and grow all food “organically” (Ranger, 2015). Additionally, genetic modification or organisms to produce enough drugs and medicines is not only ethical but necessary because it would be unethical to deplete all rare indigenous populations of original drugs that produce said drug—not only would this be causing extinction of a plant, this would drive up the drug prices, again favoring the very rich. Genetic modification of organisms for research is ethical if and only if the organisms are caused a minimal amount of pain, and that the experiment has a direct useful application.

Genetic modification could technically be used to prevent genetic diseases from being transferred from parent to offspring. In my opinion, I think that is ethical, as extra resources are needed to care for children born with defects. However, it would not be ethical to use genetic modification to create a “designer baby” because this would disproportionately favor the rich who could afford this modification, and the rich could easily rule the world and not have competition. It’s for the same reasoning why steroids are not allowed anymore. Also, even if this were just to select eye and hair color, there would be a decrease in diversity, which is detrimental if a human-specific disease suddenly arose.

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