CRISPR: The Double-Edged Sword of Genetic Engineering

Catalyst Volume 10

The phrase “genetic engineering” often brings to mind a mad scientist manipulating mutant genes or a Frankenstein-like creation emerging from a test tube. Brought to the forefront by heated debates over genetically modified crops, genetic engineering has long been viewed as a difficult, risky process fraught with errors.¹

The intentional use of CRISPRs, short for clustered regularly interspaced sport palindromic repeats, turned the world of genetic engineering on its head. Pioneered by Jennifer Doudna of Berkeley² in 2012 and praised as the “Model T” of genetic engineering,³ CRISPR as a tool is both transforming what it means to edit genes and raising difficult ethical and moral questions about genetic engineering as a discipline.

CRISPR itself is no new discovery. The repeats are sequences used by bacteria and microorganisms to protect against viral infections. Upon invasion by a virus, CRISPR identifies the DNA segments from the invading virus, processes them into “spacers,” or short palindromic repeats of DNA, and inserts them back into the bacterial genome.⁴ When the bacterial DNA undergoes transcription, the resulting RNA is a single-chain molecule that acts as a guide to destroy viral material. In a way, the RNA functions as a blacklist for the bacterial cell: re-invasion attempts by the same virus are quickly identified using the DNA record and subsequently stopped. That same blacklist enables CRISPR to be a powerful engineering tool. The spacers act as easily identifiable flags in the genome, allowing for high precision when manipulating individual nucleotide sequences in genes.⁵ The old biotechnology system can be perceived as a confused traveler holding an inaccurate map, with a general location and vague person to meet. By the same analogy, CRISPR provides a mugshot of the person to meet and the precise coordinates of where to find them. Scientists have taken advantage of this precision and now use modified proteins, such as Cas-9, to activate gene expression as opposed to cutting the DNA,⁶ an innovative style of genetic engineering. Traditional genetic engineering can be a shot in the dark, but with the accuracy of CRISPR, mutations are very rare.⁷ For the first time, scientists are able to pinpoint the exact location of genes, cut the desired sequence, and leave no damage. Another benefit of CRISPR is the reincorporation of genes that have become lost, either by breeding or evolution, bringing back extinct qualities. For example, scientists have succeeded in introducing mammoth genes into living elephant cells.^8,9 Even better, CRISPR is very inexpensive, costing around $75 to edit a gene at Baylor College of Medicine,¹⁰ and accessible to anyone with biological expertise, starting with graduate students.¹¹ The term “Model T of genetic engineering” could hardly be more appropriate.

CRISPR stretches the boundaries of bioengineering. One enterprising team from China led by oncologist Dr. Lu You has already begun trials on humans. They plan on injecting cells modified using the CRISPR-Cas9 system into patients with metastatic non-small cell lung cancer--patients who otherwise have little hope of survival.¹² To prevent attacks on healthy cells, extracted critical immune mediators called T cells will be edited with the CRISPR-Cas9 system. CRISPR will identify and “snip” out a gene that encodes PD-1, a protein that acts as a check on the T-cell’s capacity to launch an immune response. Essentially, Lu’s team is creating super-T-cells, ones that have no mercy for any suspicious activity. This operation is very risky. CRISPR’s mechanisms are not thoroughly understood, and mistakes with gene editing could have drastic consequences.¹¹ In addition, the super T cells could attack in an autoimmune reaction, leading to degradation of critical organs. In an attempt to prevent such a response, Lu’s team will extract T-cells from the tumor itself, as those T-cells would likely have already specialized in attacking cancer cells. To ensure patient safety, the team will examine the effects of three different dosage regimens on ten patients, watching closely for side effects and responsiveness.

Trials involving such cutting-edge technology raise many questions. With ease of use and accessibility, CRISPR has the potential to become a tool worthy of science fiction horror. Several ethics groups have raised concerns over the inappropriate use of CRISPR: they worry that the technology could be used by amateurs and thus yield dangerous results. In spite of these concerns, China greenlit direct editing of human embryos, creating international uproar and a moratorium on further human embryo testing.¹³ But such editing could lead to new breakthroughs: CRISPR could reveal how genes regulate early embryonic development, leading to a better understanding of infertility and miscarriages.¹⁴

This double-edged nature defines CRISPR-Cas9’s increasing relevance at the helm of bioengineering. The momentum behind CRISPR and its seemingly endless applications continue to broach long-unanswered questions in biology, disease treatment, and genetic engineering. Still, with that momentum comes caution: with CRISPR’s discoveries come increasingly blurred ethical distinctions.

References

1. Union of Concerned Scientists. http://www.ucsusa.org/food_and_agriculture/our-failing-food-system/genetic-engineering/risks-of-genetic-engineering.html#.WBvf3DKZPox (accessed Sept. 30, 2016).
2. Doudna, J. Nature [Online] 2015, 7583, 469-471. http://www.nature.com/news/genome-editing-revolution-my-whirlwind-year-with-crispr-1.19063 (accessed Oct. 5, 2016).
3. Mariscal, C.; Petropanagos, A. Monash Bioeth. Rev. [Online] 2016, 102, 1-16. http://link.springer.com/article/10.1007%2Fs40592-016-0062-2 (accessed Oct. 5, 2016).
4. Broad Institute. https://www.broadinstitute.org/what-broad/areas-focus/project-spotlight/questions-and-answers-about-crispr (accessed Sept. 30, 2016).
5. Pak, E. CRISPR: A game-changing genetic engineering technique. Harvard Medical School, July 31, 2015. http://sitn.hms.harvard.edu/flash/2014/crispr-a-game-changing-genetic-engineering-technique/ (accessed Sept. 30, 2016)
6. Hendel, A. et al. Nature Biotech. 2015, 33, 985-989.
7. Kleinstiver, B. et al. Nature. 2016, 529, 490-495.
8. Shapiro, B. Genome Biology. [Online] 2015, 228. N.p. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4632474/ (accessed Nov. 1, 2016).
9. Reardon, S. Nature. [Online] 2016, 7593, 160-163. http://www.nature.com/news/welcome-to-the-crispr-zoo-1.19537 (accessed Nov. 1, 2016).
10. Baylor College of Medicine. https://www.bcm.edu/research/advanced-technology-core-labs/lab-listing/mouse-embryonic-stem-cell-core/services/crispr-service-schedule (accessed Sept. 30, 2016).
11. Ledford, H. Nature. [Online] 2015, 7554, 20-24. http://www.nature.com/news/crispr-the-disruptor-1.17673 (accessed Oct. 11, 2016).
12. Cyranoski, D. Nature. [Online] 2016, 7613, 476-477. http://www.nature.com/news/chinese-scientists-to-pioneer-first-human-crispr-trial-1.20302 (accessed Sept. 30, 2016).
13. Kolata, G. Chinese Scientists Edit Genes of Human Embryos, Raising Concerns. The New York Times, April 23, 2015. http://www.nytimes.com/2015/04/24/health/chinese-scientists-edit-genes-of-human-embryos-raising-concerns.html?_r=0 (accessed Oct. 2, 2016).
14. Stein, R. Breaking Taboo, Swedish Scientist Seeks To Edit DNA of Healthy Human Embryos. NPR, Sept. 22, 2016. http://www.npr.org/sections/health-shots/2016/09/22/494591738/breaking-taboo-swedish-scientist-seeks-to-edit-dna-of-healthy-human-embryos (accessed Sept. 22, 2016).

Corals in Hot Water, Literally

Catalyst VOlume 10

Coral reefs support more species per unit area than any other marine environment, provide over half a billion people worldwide with socio-economic benefits, and produce an estimated USD $30 billion annually.¹ Many people do not realize that these diverse ecosystems are at risk of extinction as a result of human activity--the Caribbean has already lost 80% of its coral cover in the past few decades² and some estimates report that at least 60% of all coral will be lost by 2030.¹ One of the most predominant and direct threats to the health of these fragile ecosystems is the enormous amount of carbon dioxide and methane that have spilled into the atmosphere, warming the planet and its oceans on unprecedented levels.

Corals are Cnidarians, the phylum characterized by simple symmetrical structural anatomy. Corals reproduce either asexually or sexually and create stationary colonies made up of hundreds of genetically identical polyps.³ The major reef-building corals belong to a sub-order of corals, called Scleractinia. These corals contribute substantially to the reef. framework and are key species in building and maintaining the structural complexity of the reef.³ The survival of this group is of particular concern, since mass die- offs of these corals affect the integrity of the reef. Corals form a symbiosis with tiny single-celled algae of the genus Symbiodinium. This symbiotic relationship supports incredible levels of biodiversity and is a beautifully intricate relationship that is quite fragile to sudden environmental change.³

The oceans absorb nearly half of the carbon dioxide in the atmosphere through chemical processes that occur at its surface.⁴ Carbon dioxide combines with water molecules to create a mixture of bicarbonate, calcium carbonate, and carbonic acid. Calcium carbonate is an important molecule used by many marine organisms to secrete their calcareous shells or skeletons. The increase of carbon dioxide in the atmosphere shifts this chemical equilibrium, creating higher levels of carbonic acid and less calcium carbonate.⁴ Carbonic acid increases the acidity of the ocean and this phenomenon has been shown to affect the skeletal formation of juvenile corals.⁵ Acidification weakens the structural integrity of coral skeletons and contributes to heightened dissolution of carbonate reef structure.³

The massive influx of greenhouse gases into our atmosphere has also caused the planet to warm very quickly. Corals are in hot water, literally. Warmer ocean temperatures have deadly effects on corals and stress the symbiosis that corals have with the algae that live in their tissues. Though coral can procure food by snatching plankton and other organisms with protruding tentacles, they rely heavily on the photosynthesizing organism Symbiodinium for most of their energy supply.³ Symbiodinium provides fixed carbon compounds and sugars necessary for coral skeletal growth. The coral provides the algae with a fixed position in the water column, protection from predators, and supplementary carbon dioxide.³ Symbiodinium live under conditions that are 1 to 2° C below their maximum upper thermal limit. Under warmer conditions due to climate change, sea surface temperatures can rise a few degrees above their maximum thermal limit. This means that a sudden rise in sea temperatures can stress Symbiodinium by causing photosynthetic breakdown and the formation of reactive oxygen species that are toxic to corals.³ The algae leave or are expelled from the coral tissues as a mechanism for short-term survival in what is known as bleaching. Coral will die from starvation unless the stressor dissipates and the algae return to the coral’s tissues.³

Undoubtedly, the warming of the seas is one of the most widespread threats to coral reef ecosystems. However, other threats combined with global warming may have synergistic effects that heighten the vulnerability of coral to higher temperatures. These threats include coastal development that either destroys local reefs or displaces sediment to nearby reefs, smothering them. Large human populations near coasts expel high amounts of nitrogen and phosphorous into the ecosystem, which can increase the abundance of macroalgae and reduce hard coral cover. Increased nutrient loading has been shown to be a factor contributing to a higher prevalence of coral disease and coral bleaching.⁶ Recreational fishing and other activities can cause physical injury to coral making them more susceptible to disease. Additionally, fishing heavily reduces population numbers of many species of fish that keep the ecosystem in balance.

The first documented global bleaching event in 1998 killed off an estimated 16% of the world’s reefs; the world experienced the destruction of the third global bleaching event occurred only last year.¹ Starting in mid-2015, an El Niño Southern Oscillation (ENSO) weather event spurred hot sea surface temperatures that decimated coral reefs across the Pacific, starting with Hawaii, then hitting places like American Samoa, Australia, and reefs in the Indian Ocean.⁷ The aftermath in the Great Barrier Reef is stunning; the north portion of the reef experienced an average of 67% mortality.⁸ Some of these reefs, such as the ones surrounding Lizard Island, have been reduced to coral skeletons draped in macroalgae. With climate change, it is expected that the occurrence of ENSO events will become more frequent, and reefs around the world will be exposed to greater thermal stress.¹

Some scientists are hopeful that corals may be able to acclimatize in the short term and adapt in the long term to warming ocean temperatures. The key to this process lies in the genetic type of Symbiodinium that reside in the coral tissues. There are over 250 identified types of Symbiodinium, and genetically similar types are grouped into clades A-I. The different clades of these algae have the potential to affect the physiological performance of their coral host, including responses to thermotolerance, growth, and survival under more extreme light conditions.³ Clade D symbiont types are generally more thermotolerant than those in other clades. Studies have shown a low abundance of Clade D organisms living in healthy corals before a bleaching event, but after bleaching and subsequently recovering, the coral has a greater abundance of Clade D within its tissues.^9,10 Many corals are generalists and have the ability to shuffle their symbiont type in response to stress.¹¹

However, there is a catch. Though some algal members of Clade D are highly thermotolerant, they are also known as selfish opportunists. The reason healthy, stress-free corals generally do not have a symbiosis with this clade is that it tends to hoard the energy and organic compounds it creates from photosynthesis and shares fewer products with its coral host.³

Approaches that seemed too radical a decade ago are now widely considered as the only means to save coral reefs from the looming threat of extinction. Ruth Gates, a researcher at the Hawaii Institute of Marine Biology is exploring the idea of assisted evolution in corals. Her experiments include breeding individual corals in the lab, exposing them to an array of stressors, such as higher temperatures and lower pH, and picking the hardiest survivors to transplant to reefs.¹² In other areas of the globe, scientists are breeding coral larvae in labs and then releasing them onto degraded reefs where they will hopefully settle and form colonies.

Governments and policy makers can create policies that have significant impact on the health of reefs. The creation of marine protected areas that heavily regulates or outlaws harvesting of marine species offers sanctuary to a stressed and threatened ecosystem.³ There is still a long way to go, and the discoveries being made so far about coral physiology and resilience are proving that the coral organism is incredibly complex.

The outlook on the future of healthy reefs is bleak; rising fossil fuel consumption rates mock the global goal of keeping rising temperatures below two degrees Celsius. Local stressors such as overfishing, pollution, and coastal development cause degradation of reefs worldwide. Direct human interference in the acclimatization and adaptation of corals may be instrumental to their survival. Rapid transitions to cleaner sources of energy, the creation of more marine protection areas, and rigid management of reef fish stocks may ensure coral reef survival. If humans fail in this endeavor, one of the most biodiverse and productive ecosystems on earth that has persisted for millions of years may come crashing to an end within our lifetime.

References

1. Cesar, H., L. Burke, and L. Pet-Soede. 2003. "The Economics of Worldwide Coral Reef Degradation." Arnhem, The Netherlands: Cesar Environmental Economics Consulting. http://pdf.wri.org/cesardegradationreport100203.pdf (accessed Dec 14, 2016)
2. Gardner, T.A. et al. Science 2003, 301:958–960.
3. Sheppard C., Davy S., Piling G., The Biology of Coral Reefs; Biology of Habitats Series; Oxford University Press; 1st Edition, 2009
4. Branch, T.A.et al. Trends in Ecology and Evolution 2013, 28:178-185
5. Foster, T. et al. Science Advances 2016, 2(2) e1501130
6. Vega Thurber, R.L. et al. Glob Change Biol 2013, 20:544-554
7. NOAA Coral Watch, NOAA declares third ever global coral bleaching event. Oct 8, 2015. http://www.noaanews.noaa.gov/stories2015/100815-noaa-declares-third-ever-global-coral-bleaching-event.html (accessed Dec 15, 2016)
8. ARC Centre of Excellence for Coral Reef Studies, Life and Death after the Great Barrier Reef Bleaching. Nov 29, 2016 https://www.coralcoe.org.au/media-releases/life-and-death-after-great-barrier-reef-bleaching (accessed Dec 13, 2016)
9. Jones A.M. et al. Proc. R. Soc. B 2008, 275:1359-1365
10. Silverstein, R. et al. Glob Change Biology 2014, 1:236-249
11. Correa, A.S.; Baker, A.C. Glob Change Biology 2010, 17:68-75
12. Mascarelli, M. Nature 2014, 508:444-446

Cognitive Neuroscience: A Glimpse of the Future

Catalyst Volume 10

Cognitive Neuroscience is a branch of science that addresses the processes in the brain that occur during cognitive activity. The discipline addresses how psychological and cognitive activities are caused by and correlated to the neural connections in our brain. It bridges psychology and neuroscience.

Dr. Simon Fischer-Baum, an assistant professor and researcher at Rice University,  co-directs the neuroplasticity lab at the BioScience Research Collaborative. He received his B.A. in Neuroscience and Behavior from Columbia University in 2003 and received his Ph.D. in Cognitive Sciences from Johns Hopkins University in 2010.

Dr. Fischer-Baum describes his research as the “intersection of psychology and neuroscience and computer science to some extent.” He is interested in instances of how we understand and pronounce a word once we see it. He also studies memory and how information is encoded in the brain. In his opinion, functional magnetic resonance imaging (fMRI) and other tools of cognitive neuroscience are extremely relevant to cognitive psychology despite public perception. For example, he believes that there is a “serious disconnect” as a result of the belief that the methods and findings of cognitive neuroscience do not apply to cognitive psychology. Cognitive psychologists have been attempting to discover the variation between the different levels of processing and how information travels between these levels. Cognitive neuroscience can help achieve these goals through the use of fMRIs.

fMRI shows which parts of the brain are active when the subject is performing a task. During any task, multiple regions of the brain are involved, with each region processing different types of information. For example, reading a word involves processing both visual information and meaning; when you are reading a word, multiple regions of the brain are active. However, one problem with fMRIs is that while they demonstrate what regions of the brain are active, they do not convey what function each region is carrying out.  One of the main objectives of Dr. Fischer-Baum’s work is to pioneer new methods similar to computer algorithms to decode what data from an fMRI tells us about what tasks the brain is performing. “I want to be able to take patterns of activity and decode and relate it back to the levels of representation that cognitive psychologists think are going on in research,” Dr. Fischer-Baum explains.

Recently, Dr. Fischer-Baum published a study of a patient who suffered severe written language impairments after experiencing a hemorrhagic stroke. Although this patient’s reading of familiar words improved throughout the years, he still presented difficulties in processing abstract letter identity information for individual letters. Someone who is able to utilize abstract letter representations can  recognize letters independent of case or font; in other words, this person  is able to identify letters regardless of the whether they are upper case, lower case, or a different font. In the studied patient, Dr. Fischer-Baum’s team observed contralesional reorganization. Compromised regions of the left hemisphere that contained orthography-processing regions (regions that process the set of conventions for writing a language) were organized into homologous regions in the right hemisphere. Through the use of fMRI, the research team determined that the patient’s residual reading ability was supported by functional take-over, which is when injury-damaged functions are taken over by healthy brain regions. These results were found by scanning the brain of the patient as he read and comparing the data with that of a control group of young healthy adults with normal brain functions.

While Dr. Fischer-Baum has made substantial progress in this project, the research has not been without challenges. The project began in 2013 and took three years to complete, which is a long time for Dr. Fischer-Baum’s field of study. Due to this, none of the co-authors from Rice University know each other despite all working on the project at some point in time with another. Because of the amount of time spent on the project, many of the students rotated in and out while working on various parts; the students never worked on the project at the same time as their peers. In addition, the project’s  interdisciplinary approach required the input of  many collaborators with different abilities. All of the Rice undergraduate students that worked on the project were from different majors although most were from the Cognitive Sciences Department and the Statistics Department. At times, this led to miscommunication between the different students and researchers on the project. Since the students came from different backgrounds, they had different approaches to solving problems. This led to the students at times not being harmonious during many aspects of the project.  

Another major setback occurred in bringing ideas to fruition. “You realize quickly when you begin a project that there are a million different ways to solve the problem that you are researching, and trying to decide which is the right or best way can sometimes be difficult,” Dr. Fischer-Baum said. As a result of this, there have been a lot of false starts, and it has taken a long time in order to get work off the ground. How did Dr. Fischer-Baum get past this problem? “Time, thinking, discussion, and brute force,” he chuckled. “You realize relatively quickly that you need to grind it out and put in effort in order to get the job done.”

Despite these obstacles, Dr. Fischer-Baum has also undertaken other projects in order to keep his mind busy. In one, he works with stroke patients with either reading or writing deficits to understand how written language is broken down in the mind. He studies specific patterns in the patients’ brain activity to investigate how reading and writing ability differ from each other. In another of Dr. Fischer-Baum’s projects he works with Dr. Paul Englebretson of the Linguistics Department in order to research the brain activity of blind people as they read Braille. “There is a lot of work on how the reading system works, but a lot of it is based on the perspective of reading by sight,” Dr. Fischer-Baum acknowledged. “I am very interested to see how the way we read is affected by properties of our visual system. Comparing sight and touch can show how much senses are a factor in reading.”

Ultimately, Dr. Fischer-Baum conducts his research with several goals in mind. The first is to build an approach to cognitive neuroscience that is relevant to the kinds of theories that we have in the other cognitive sciences, especially cognitive psychology. “While it feels like studying the mind and studying the brain are two sides of the same coin and that all of this data should be relevant for understanding how the human mind works, there is still a disconnect between the two disciplines,” Dr. Fischer-Baum remarked. He works on building methods in order to bridge this disconnect.

In addition to these goals for advancing the field of cognitive neuroscience, there are clinical implications as well to Dr. Fischer-Baum’s research. Gaining more insight into brain plasticity following strokes can be used to build better treatment and recovery programs. Although the research requires further development, the similarity between different regions and their adaptations following injury can lead to a better understanding of the behavioral and neural differences in patterns of recovery. Additionally, Dr. Fischer-Baum aims to understand the relationship between spontaneous and treatment-induced recovery and how the patterns of recovery of language differ as a result of the initial brain injury type and location. Through the combined use of cognitive psychology and fMRI data, the brains of different stroke patients can be mapped and the data can be used to create more successful treatment-induced methods of language recovery. By virtue of Dr. Fischer-Baum’s research, not only can cognitive neuroscience be applied to many other disciplines, but it can also significantly improve the lives of millions of people around the world.