Introduction

Coral reefs, which hold a quarter of all marine diversity, are in critical conditions due to climate change. Their populations have been dropping drastically due to factors such as increased ocean temperatures and ocean acidification, which are caused by anthropogenic activities. This has a deadly consequence for other marine organisms that rely on corals for shelter, food, and safety for spawning.

To prevent mass marine life declination, it is crucial to understand corals on a deeper level in order to find a way to help them survive the effects of climate change. Genetic engineering is one of the most promising methods of creating resistance and potentially even immunity to increasing ocean temperatures and coral bleaching; however, there are many challenges and risks to genetically altering such vital cnidarians like corals, which will be described in more detail below.

Primary Objective

To understand the potential future role of CRISPR Cas9 genetic engineering on Acropora corals.

Secondary Objective

To find a potential means of genetically engineering Acropora to increase resistance to effects of increased ocean temperatures.

Background Research

1. Gain a comprehensive understanding of the corals under the Acropora genus, including a general knowledge of:
   • Anatomy and life cycle
   • Significant symbiotic relationships
   • Impact on marine ecosystems
   • Causes of endangerment
2. Review knowledge of DNA structure and placement within cnidarian species
3. Learn about the general components and role of DNA within cells, including:
   • RNA formation
   • Polypeptide chain and protein synthesis
   • Restriction enzymes and their role within DNA technology
4. Understand the causes of Acropora coral endangerment, specifically coral bleaching, on a genetic level

Project Research

1. Research what CRISPR Cas9 genetic technology is, how it is used, its limitations, and how it has been used in the past; specifically in circumstances that involve:
   • other cnidarian species similar to Acropora corals
   • direct endosymbiotic partners of Acropora
2. Research possible solutions to coral endangerment, specifically (but not limited to) alteration on the genetic level involving heat resistance
3. Researching, gathering, and considering the feasibility, challenges and consequences of using genetic engineering in wildlife generally as a temporary or long-term solution to the effects of climate change
4. Researching and analyzing the potential of alternative solutions in comparison to genetic modification
5. Discussing issues and improvement ideas for CRISPR technology use in corals

Introduction

Human population growth, climate change and rising global temperatures are known for their drastic effects on terrestrial endangered species such as polar bears and red-eyed tree frogs. However, these changes in climate also have a deadly effect on the creatures that inhabit our ocean floors and host entire oceanic ecosystems. Despite being one of the oldest surviving life forms on Earth, the coral reef has been an endangered species in rapid decline for over 70 years. Due to the lack of research on coral reefs however, there has been little effort to save these reefs despite their vital roles in marine diversity, underwater ecosystems, and human safety. 

This project aims to discuss the role that genetic engineering could have in saving the coral reef, specifically stony corals under the genus of Acropora. It also aims to cover the ethics of genetic engineering in animals and wildlife organisms, as well as other measures that humans can take to help stall the rapid decline of Acropora and other corals in general.

Coral Bleaching

Coral bleaching is a phenomenon in most stony corals, including Acropora, which is characterized by the coral's release of zooxanthellae during unsuitably hot conditions. The loss of zooxanthellae algae is most distinct due to the fact that it takes the pigment of the coral with it, leaving the polyps bare or "bleached." Especially in Acropora corals, where the algae is the main source of energy for the polyps, algae expulsion is a sign that the coral is very near death. This is because without algae carrying out photosynthesis, the corals will starve due to insufficient nutrition and die. Due to the increasingly varying ocean temperatures (see Fig 1.2), corals have been increasingly put at risk for bleaching (see Fig. 1.1). 

The Upregulation of HSF1 and NFᴋB During Heat Shock

A 2020 study on the anemone Aiptasia (which is strikingly genetically similar to many different Acropora corals) found that certain genes were upregulated and then immediately downregulated in reaction to heat shock (Fig 3.1 - 3.3). The scientists used RNA sequencing to observe gene expression during heat stress until Aiptasia corals were completely bleached. This study disproved the long-standing theory that the upregulation of the immune gene, NFᴋB, and its activation of innate-immunity and apoptosis genes were the cause of the algae release; in reality, these genes actually returned to nearly bare minimum levels before bleaching was detectable.

The study also concluded that while the reason for the initiation of coral bleaching under early heat stress are not clear, the dramatic downregulation of immunity and heat-shock genes such as HSF1 and NFᴋB plays a huge role in the release of the aposymbiotic zooxanthellae algae (Fig 3.4). However, some evidence collected in this study can also suggest that the zooxanthellae stop releasing nutrients to the coral after initial heat shock which might cause the polyp to misinterpret it as a hostile organism.

Genome of the Anemone Aiptasia

Scientists used flow cytometry to estimate the size of the genome, then Illumina short-read sequencing to obtain the genome sequence. The resulting generated sequence was similar to the genetic material of other cnidarians 

Aiptasia is an ideal model for scientists to study the cnidarian-zooflagellate endosymbiosis because while it contains the relationship with the zooxanthellae algae, it also quickly reproduces asexually under lab conditions. It can live under conditions where there are no dinoflagellate specimens, and it can also be inhabited by different kinds of symbiodinium. It's closely related to the Acropora genus, which can help scientists understand how genetic engineering can be used in the Acropora corals, which are more commonly found in the ocean. (see Fig 2.2)

Results of the Genome Analysis

• Researchers found that the DNA structure of Aiptasia was very similar to that of non-endosymbiotic cnidarians; however, due to the fact that over 3,000 of the genes had no clear homologous result, they could not conclude that Aiptasia has a closer hereditary relationship with non-endosymbiotic cnidarians than previously thought.
• Aiptasia has a fair amount of genetic repetition (26%), making it a good candidate for CRISPR technologies (see Fig 2.1)
• Missing central-pathway intermediates, but does contain a gene that can do cystathionine-β-synthase which is important in amino acid synthesis, detoxification, and metabolism of the anemone. However, although Aiptasia and Acropora are similar, Acropora does not have this gene

A History of CRISPR Technology

CRISPR was discovered in 1987, in the immune systems of archaea and bacteria. However, only later in the early 2000s did the understanding of the role CRISPR played in the immune system finally come to light. In 2007, an experiment was conducted on lactic acid bacterium to prove CRISPR’s role in the immune system and further explain how it worked.

CRISPR is relatively new, considering that it has only been 30 years since its discovery.

In 2018, CRISPR-Cas9 was used to modify human embryo cells for resistance against HIV, but it was done with little caution and prior research which gained lots of backlash from the scientific community. Some even called for “a moratorium on inheritable genomic manipulations''. No manipulations on the human genome have been attempted since. 

In 2020, the Nobel Prize in Chemistry was awarded to Emmanuelle Charpentier and Jennifer Doudna who discovered the CRISPR/Cas9 system could be used to edit and cut DNA precisely (manipulate the genome of an organism). 

Philip Cleaves' Coral CRISPR Experiment 

In 2018, Phillip Cleaves, a postdoctoral scholar at Stanford, made efforts to genetically modify corals with the help of CRISPR. By calculating the spawning cycle of corals, Cleaves managed to successfully collect coral zygotes in Australia. This experiment has been done in effort to, as stated by Cleaves, “use CRISPR/Cas9 with the express interest to start understanding what genes are critical to coral biology.”

Target Genes 

Cleaves wanted to remove the following genes:

• Fibroblast growth factor 1a because FGF signaling, a protein that communicates with other molecules outside the cell, helps corals sense the environment which triggers larval settlement, the process where free-swimming coral larvae attach onto specific substrate and metamorphosis.
• Red fluorescent protein (RFP) converts blue light into orange-red wavelengths. Red light reaches farther into the coral tissues which contain symbiotic algae. This helps maximize photosynthesis.  
• Green fluorescent protein (GFP) protects algae from collecting too much sunlight.

GFP and RFP proteins respond to environmental perturbations. These genes have multiple copies, and therefore could target multiple copies with one sgRNA (single guide RNA).

Design of CRISPR, specifically sgRNAs

The design was to reduce off-target effects, where Cas9 cuts DNA that is similar but not identical to target. The design allowed the detection of mutations through endogenous restriction sites, a method of defense where  restriction enzymes cut specific DNA sequences. Furthermore, to prevent toxicity or delay in mutations, corals were injected with in vitro transcribed sgRNAs precomplexed with Cas9 protein instead of vector-driven expression as this offered a more direct approach.

How was CRISPR inserted into the zygotes?

The CRISPR ribonucleoprotein was injected into fertilized eggs. The targeted position was far enough upstream in the genes' coding sequences so mutations would change the reading frame, the triple codon when paired with mRNA, and knock out gene function. The targeted position needed to be also downstream to avoid functional gene products to be expressed. Less than 400 fertilized zygotes were injected with two or three sgRNA/Cas9 complexes while some received the Cas9 protein alone

Results

CRISPR was not able to cut off the red and green fluorescent proteins due to having multiple paralogs as they come from a common ancestral gene (Fig. 4.3). This is caused by CRISPR changing genes that are fairly identical to each other through DNA sequencing. One gene copy of fibroblast growth factor 1a is present in corals. Some embryos had the FGF1a significantly mutated, this proves CRISPR’s success in single-copy genes. Furthermore, the mediated mutagenesis in the sea anemone system, Aiptasia, and the Acropora shared similar mutations.

Mutation inductions in 50% of the sample continued for several cell cycles (cell duplication and division). Around 50-75% of embryos successfully developed into larvae (Fig. 4.5).

Through RFLP, it was determined that the restriction enzymes were unable to properly cut the DNA segment at its restriction site. As a result, some genes lost their restriction sites due to induced mutations. Through PCR-amplified fragments, the mutated target gene was cloned by vectors (Fig. 4.2). The results show how the larvae carried both normal and mutated alleles of the same gene. 

Phenotypes associated with CRISPR/Cas9 Mutations

The larvae injected with FGF1a sgRNA continued to undergo metamorphosis, even if the FGF signaling gene, responsible for metamorphosis, was knocked out. The larvae injected with GFP or RFP sgRNA showed no drastic changes in their fluorescence patterns aside from seven larvae, which showed complete absence of GFP or RFP fluorescence (Fig. 4.4). Despite the induced mutations in the larvae, there were no major changes in their phenotypes.

Reduced thermal tolerance in a coral carrying CRISPR-induced mutations in the gene for a heat-shock transcription factor: an experiment by Philip Cleaves

Overview

In a sea-anemone (Aiptasia) model system, the goal was to disable the target gene: Heat Shock Transcription Factor using sgRNA/Cas9 ribonucleoprotein complexes. Disabling HSF1 will show how significant the gene affects the Aiptasia’s heat tolerance. It was suggested that by effectively eliminating HSF1, the resulting mutation would reduce or disable the burst of transcription that occurs immediately after the coral faces heat stress.

Design

Two sgRNAs were made to target exon 3 and the other to target exon 9 (Fig. 5.1). Target sites were chosen to avoid possible complications like off-target effects and alternative transcription start sites (Fig. 5.1).

How was it inserted?

In November 2018, over three nights of spawning, fertilized zygotes were injected with either a Cas9 protein alone or sgRNA1/Cas9 and sgRNA2/Cas9 ribonucleoprotein complexes. Each night 100-300 individuals were injected. Out of the zygotes injected, 68-90% survived and developed normally for 12 h (Fig. 5.3).

Experiment

The larvae were exposed to 34 degrees celsius for two days, a temperature that triggers heat stress (Fig. 5.2). As a result, mutant larvae survived at 27 degrees celsius but died at 34 degrees (Fig. 5.2). Wild-type larvae showed more resistance when exposed to 34 degrees (Fig. 5.2). Some mutant larvae survived but had fewer changes in the HSF1 gene and a higher proportion of normal gene sequences (Fig. 5.2).

Limitations

Due to the insufficient research that has been done on the genome of a coral, disabling one or a few genes may not show a significant phenotypic change. It would have been preferable if several genes with multiple sgRNAs could induce a pronounced phenotype. Furthermore, microinjection is a tedious process. 

The Role of Bicarbonate Transporter SLC4ү in Stony Corals

In this study, scientists removed the bicarbonate transporting SLC4ү gene from stony corals using CRISPR Cas9 technology to observe its impact on the coral's skeletal development. Knowing that calicoblast cells use enzymes to extract water from the calcium and bicarbonate ions in order to form a skeleton through biomineralization, and that SLC4ү is only expressed in these stony coral calicoblast cells, we can conclude that this gene plays a significant role in the formation of corallite. As predicted, the removal of the gene through Cas9 sgRNA1 and sgRNA2 causes large gaps in the corallite where septa usually would have formed. 

Unexpectedly, some of the highly mutated juvenile polyps showed little external phenotype changes, while some lowly mutated polyps showed extreme changes. For the former, we can assume that this may have been caused by genetic clones in cells surrounding the mutated group; however, this cannot explain why less mutated DNA patterns would cause a larger mutation if all coral polyps from the colony are genetically identical. As the specific details as to how SLC4ү genes transport bicarbonate still remain unclear, it is impossible to determine the exact purpose of the gene.

Cattle Fetal Fibroblast CRISPR to Knock in Genes

This experiment was designed to find the expression levels of each promoter in the mammalian locus Rosa26. 

Method Summary

Researchers used CRISPR Cas/9 technology to insert four GFP vectors driven by different promoters that all drive high levels of gene expression into the ubiquitous locus Rosa26 of cattle fetuses. They constructed vectors by designing sgRNA with computer software, and then subcloning the sgRNA onto a mammal-optimized Cas9 expression plasmid. CRISPR Cas/9 was nucleofected into prepared cells. They then used PCR to mass multiply, amplify and detect magnitude of the nucleofected cells and their gene expressions.

Result Summary

The CAG promoter had the highest expression level, but cRosa26 could support many different promoter types. This suggests that cRosa26 is suitable for transgene expression with multiple different promoters, and will result in a mostly stable and high expression manner.

Rabbit ROSA: Identification and characterization of rabbit ROSA26 for gene knock-in and stable reporter gene expression

This experiment was designed to locate the rabbit orthologue of ROSA26 in the rabbit genome. 

Method Summary

Researchers utilized genomic sequence homology analysis to find the orthologue of the mouse's ROSA26 gene in the rabbit genome. They then used PCR and RACE experiments to uncover the variants of DNA that were ubiquitously expressed across many tissues.

Result Summary

PCR, RACE 3', and RACE 5' experiments revealed that the locus codes two transcript variants of lncRNA, and rbRosaV1 and rbRosaV2, all non-coding. These were expressed stably and ubiquitously.

The Ethics of Genetic Engineering

Genetic Modification in Wildlife

Ethics, and even the feasibility of genetic technology hinge entirely on the public opinion and support. Promisingly, a large percentage of the public is open to the technology that could help save biodiversity amid the climate despite negative media portrayals. However, if we were to genetically engineer wildlife in the name of conservation, we would not be the first. Previous examples of genetic engineering and scientific intervention in wildlife include:

• the black footed ferret
• the Southern Corroboree Frog

The Black-Footed Ferret

Back in the 1980s, black footed ferrets were already endangered due to plague and habitat loss. Conservationists managed to capture the 18 remaining ferrets, and put them into a captive breeding program to save the population, one of the ferrets being a female named Willa. However, only 7 of them actually bred, Willa not included, which means that the rest of the black footed ferrets today are directly related as cousins or as siblings, which means that there is a reduced amount of genetic diversity. Willa's genetic information was preserved because of her unique genetic material, and 30 years later cloned into a new ferret named Elizabeth Ann. Elizabeth is the first genetically cloned wildlife animal.

Scientists hoped that Elizabeth Ann would be able to introduce new genetic material into the rest of the ferrets, but she had a medical issue. However, they are hoping to create more clones of Willa to hopefully introduce new genetic diversity into remaining ferrets, which will help increase the resistance and adaptability of the species which will soon allow them to be reintroduced into the wild. 

Without human intervention and cloning, the ferrets would have no chance of becoming independent from humans, if not totally extinct.

The Southern Corroboree Frog

The Southern Corroboree Frog has been a highly endangered species due to the introduction of a fungal disease called Batrachochytrium dendrobatidis (Bd). If humans continued to use the traditional selective breeding, we would not be able to save the frogs. Using genetic studies, scientists were able to find the immunogenetic differences within the species to manipulate in future generations of corroboree frogs, which is more effective and much faster than breeding resistant frogs.

Preventing Unintentional Side Effects

With the introduction of a new species into an ecosystem, come consequences to its surroundings. Understandably, one of the of the largest public concerns about genetically engineering a wildlife organism such as a coral is the unintended effects that it might have on its ecosystem. Many have these reservations come from previous failed scientific intervention scenarios, such as the 1935 cane toad incident in Australia, and the slightly more recent Monsanto Cereal Crop incident. However, both of these scenarios have little to do with the effects of genetic engineering, and are completely preventable.

Cane Toads of Australia

The cane toad, Bufo marinus, was first introduced to Australia in 1935 to predate upon the destructive beetles in sugarcane crops, before agricultural pesticides were widely used. The toads quickly became invasive species due to their extreme adaptability to different environments, and their ability to secrete lethal poison into their predators' bodies. There is no way to completely exterminate them in Australia yet. Although not directly involved with genetic engineering, the cane toad is often used as an example of flawed scientific intervention with nature that had catastrophic results.

However, it is important to remember that the incident happened nearly a century ago, and scientific research and experiment policies have since developed regulations and policies that would completely prevent this kind of accident from happening. With the right amount of care, the cane toad incident of 1935 is completely preventable. Additionally, genetically engineering a pre-existing organism does not cause quite as drastic an effect as introducing an entirely new one; the genetically engineered is largely the same as before with only small modifications. 

Monsanto and Cereal Crops

Although also not related to genetic engineering, the Monsanto cereal crops is an ongoing example of why so many people object against applying "unnatural" science in the real world, especially when it involves the food chain. Monsanto is a company that sells Roundup, a herbicide that is often used by farmers of oats and grains. However, Roundup contains glyphosate, a chemical that is linked with causing cancer. Recent tests in oat cereal brands such as Cheerios and Nature Valley reveal an alarming concentration of glyphosate, which has increased concern about the use of scientific intervention in the production of organisms at the bottom of the food chain.

Few organisms consume coral reefs. Genetic engineering might possibly pose a risk to the organisms that do, but this risk can be mitigated by observing the growth of the genetically modified organism in a lab-induced ecosystem environment.

Experimentation With Genetic Modification in Animals

Experiments that involve genetic modification must consider the safety of the organism and of its environment. These factors include:

• Invasiveness of procedures 
  • Will there be any problems with coral reproduction?
  • Will the genetically modified parental organisms pass on the modifications safely to their offspring?
• Number of animals required
  • Will this affect the mortality of the animal embryos being modified?
  • How many animals will survive modification?
• Unanticipated welfare concerns
  • Will there be undesirable phenotypes?

 

In designing experiments that involve genetic modification, researchers mitigate risk and ethical concerns through: reducing the number of animals involved, to maintain a healthy population; refining practices, to minimize pain and distress; and using non-animal alternatives when possible, to mitigate risks of unexpected mutation and animal stress.

An Evolving Opinion on Scientific Intervention

The main barrier against using genetic engineering technology is the public opinion and public perception of the ethics of genetic engineering in the wild. Using genetic engineering for restoration hinges on the permission of the public, so it is important to understand the ethical circumstances behind genetic engineering. How supportive is the public of using biotechnologies?

Study 1 - Genetically engineered heat-resistant coral: An initial analysis of public opinion

The general public opinion regarding coral reefs has been increasingly worried. A study done showed that tourists responded with more negative descriptions after the 2017 coral bleaching event in comparison to the descriptions given before the event in 2013.

This study surveyed 1,148 Australians about the use of genetic engineering in corals after giving the participants basic definitions of synthetic biology and its uses, as well as giving a basic overview of what the proposed genetically remodeled coral would look like. The participants were people representing different types of educational levels, backgrounds, ages, and genders.

Results of the survey (Fig. 6.0)

Q: How much would you say you know about gene editing of coral?

• Males reported more awareness than females (34% as opposed to 24%).
• The younger the participants were the more likely to be aware of the technology.
• Surprisingly, the level of education of the participants was not correlated to awareness at all.

Q: To what extent would you be willing to support this technology?

• Older people were less likely to support the technology.
• Females were slightly less likely to support the technology than males.

Q: Why do/don't you support genetically altering corals?

• Information was sorted with multiple regression analysis, which revealed themes in peoples answers. Over 50% of participants supported the use of technology.
• The most common reason for not supporting the technology was the potentially catastrophic consequences in nature. This was followed by concern of human utilization in other means.
• Other reasons that people oppose gene editing tech is the lack of scientific evidence that it will work, the interference with "naturalness" (what's in nature isn't ours to alter), and the concern that the technology is an expensive "band-aid" solution to the underlying issue of climate change.

Study 2 - Most Americans Accept Genetic Engineering of Animals That Benefits Human Health, but Many Oppose Other Uses 

The study found that the general public is more inclined to accept genetic engineering for medical uses, such as cloning organs for transplanting, over technologies that involve bringing back animals that are extinct or for recreational purposes, like changing physical characteristics for aesthetic purposes.

Present Solutions to Slow Down and/or prevent Dying Coral Reefs in Response to Climate Change

Coral Restoration

The goal of coral restoration is to breed different species of coral in a lab and plant them on an artificial reef.  A coral nursery contains developing corals, small samples are taken to be outplanted. Those samples will be placed in a reef restoration site. This provides a way to repopulate reefs that have faced degradation. This has improved marine ecosystems, however, this may not be a long term solution. Planting corals that are not heat tolerant are constantly threated by the effects of climate change, therefore, this process may not be as sustainable. Planting corals that are more heat tolerant cannot guarantee that the corals will last as future conditions will always change. An ecosystem with a population dominated by one species of coral can easily be wiped out than an ecosystem filled with diverse species of corals. 

The main solution: Reducing our Carbon Footprint

The root of dying reefs is anthropogenic activity. We have emitted an excessive amount of carbon dioxide which has led to severe consequences of climate change. Several agreements have been made between countries to improve our carbon footprint (net-zero) which, as a result, will lower rising global temperatures. Here are some actions taken by the biggest emitters to cut carbon emissions.

Clean energy sources: A huge portion of our energy comes from burning fossil fuels. Currently, many sustainable energy sources, such as, hydro, solar and wind sources are being developed.

Planting Trees: Trees play a crucial role to reduce climate change. They consistently take carbon from the atmosphere for photosynthesis, and release oxygen. Many countries like the US aims to plant millions of trees each year. 

Promoting public transportation: With the rising cost of living, many countries have decided to cut down on transit fees/tickets to promote public-transport over individual transport.

Coral Bleaching

Fig 1.1

r

Fig 1.2

Fig.1.1 and 1.2: Minimal changes in daily high surface temperatures drastically change the number of corals at risk of coral bleaching.

The Anemone Aiptasia Genome Analysis

Charts taken directly from

Fig. 2.1

Content of the Aiptasia genome assembly.

 Fig 2.2

2.2 A: A phylogenetic tree relating the anthozoans Aiptasia to Acropora digitifera, one of the most common corals suffering from coral bleaching. B: An aposymbiotic polyp under white light. C: A symbiotic polyp viewed under white light. D: A symbiotic polyp viewed under fluorescence microscopy to view red chlorophyll of the zooxanthellae algae

Insights into coral bleaching under heat stress from analysis of gene expression in a sea anemone system

Fig 3.1

Heatmap shows the difference in return to down-regulation of genes (red is up-regulated, blue is down-regulated). It seems like in both aposymbiotic and symbiotic anemones, returning to regular levels takes about 12 hours in Group 1 and 48 hours in Group 2

Fig 3.2

Fig 3.2 shows that the spike in NFKB1 up-regulation is much higher in aposymbiotic species than it is in symbiotic.

Fig 3.3

Graph C shows an analysis of the different genes that were up-regulated and their roles within the anemone using Gene Ontology ID technology

Fig 3.4

Graphs B & C show that there is little difference in the genetic sequences between NFkB and HSF1 genes, and the upregulated unidentified genes. We can say with near certainty that these are the genes that were in high activity during the time of heat stress.

CRISPR/Cas9-mediated genome editing in a reef-building coral

Fig. 4.1

Cas9 are found in targeted exons because they are junk segments (Cas9 itself is not part of the intended outcome). Bg/ll, Pvull and Sm/l could potentially be Cas9 cleavage sites/nearby restriction-enzyme sites. + and - signs indicate the presence or absence of either Cas9 and sgRNA.

Fig. 4.2

1. Genomic DNA of individual larvae is amplified by PCR and analyzed by gel electrophoresis. Interior lanes are one larva, outside lanes are molecular-size markers. Arrows show incomplete digestion of DNA.
2. Comparison between sequencing tools
3. Mutations shown in each target gene of wild type larva. Base-pair changes in blue and deletions in dashes

Fig. 4.3

Editing of two paralogs found in a single larvae. Each paralog compares their respective alleles. Dashes represent deleted nucleotides. Inserted or altered nucleotides in blue. Red lettering shows the positions where the alleles differ within a 64-bp segment. Under GFP1 sgRNA and GFP2 sgRNA, each sequence shows a different version of the same genetic sequence. WT is the original sequence. 

Fig. 4.4

A and B are the injected larvae with normal metamorphosis. C and D are the injected larvae with no presence of GFP or RFP fluorescence.

Fig 4.5

Reduced thermal tolerance in a coral carrying CRISPR-induced mutations in the gene for a heat-shock transcription factor

Fig. 5.1

Section A: Cas9 with sgRNA 1 target site is exon 3. Cas9 with sgRNA 2 target site is exon 9.

Section B: shows percentage of larvae with mutations

Section C: Two individual larvae, both larvae show the both exons 3 and 9. Each line under an exon represents a different mutation event. Black bars represent position of sgRNA target sites; nucleotides in red represent PAM sites. Dashes are deletions of nucleotides. Blue letters are insertion/replaced nucleotides.

Fig. 5.2

Uninjected, Cas9-injected (control), and sgRNA/Cas9-injected larvae faced heat stress at 34 degrees for two days. Most uninjected and Cas9-injected larvae survived under these conditions as shown in section A, B and C. There was also little mortality for larvae injected with sgRNA + Cas9 at 27 degrees. Although, the larvae faced significant death in section E.

Fig. 5.3

Ethics on Coral Engineering: Survey Result Analysis

Fig. 6.0

Fig. 6 shows the results of a survey conducted in Australia about the general public opinion using multiple regression to find common themes in answers.

Coral reefs are critical to the well-being of the marine ecosystems, the biodiversity of the oceans, humanity, and ultimately, the entire planet. In addition to being a crucial keystone species, corals also play vital roles in ecosystem maintenance, coastal erosion, and water filtration. Despite their importance, their endangerment is often left unrecognized and understated by the public. As a result, coral reef degradation continues to accelerate exponentially as humans remain neglectful of the devastating consequences climate change has beneath the deep blue waters.

We need to take urgent action to help revitalize our oceans from the deadly effects of climate change. Current measures, such as coral restoration and clean water restoration policies, are not effective enough to sufficiently stall coral bleaching. Although it is not a permanent solution, genetic engineering holds much promise when it comes to helping preserve coral reef life for a little longer. It opens up a huge number of possibilities when it comes to expanding the coral's need for extremely specialized environmental conditions.

We can use different genetic technologies, such as cloning and CRISPR Cas9, to study the genomes of different types of corals and the purposes of individual genes through gene knock out. Many climate scientists and biologists have used CRISPR in corals for the latter; experts like Philip Cleaves have experimented with knock-out to determine, confirm, and study the purposes of genes like HSF1 and proteins such as GFP. Given this information, we can also knock in genes into parts of the genome in order to ubiquitously enhance a genetic characteristic or add a new one. In the former scenario, we would do this by utilizing computer software to design sgRNA strands that already exist within the organism. Theoretically, this would allow us to increase the expression of a gene within the genome.

Genetic engineering faces a lot of ethical concerns. The most common concerns include the impacts of the mutations on other organisms, human utilization of engineering in other means (i.e. genetic engineering organisms in "evil" ways), and interfering with the inherent "naturalness of the Earth." The first of these concerns can be addressed with adequate testing and evaluation of mutated organisms in a fake imitation of their natural environment. The concern regarding human utilzation with ulterior motives can be addressed by conducting the alterations publicly and with the approval of the various Ethics and Integrity groups under the Natural Sciences and Engineering Research Council of Canada. This council directly addresses conflict of interest, confidentiality, and responsible conduct of research in regards to Canadian legislation. As to interefering with the natural order of the Earth, it is key to remember the entire reason that corals are critically endangered: drastically increased climate change as a result of human activity. In order to help preserve the biodiversity that we have endangered with human actions, it is reasonable to consider using new, innovative technologies for the better of the Earth. 

We would also like to point out that with the rapid decline of coral population, it is clear that corals will soon become extinct if the decline continues. At this point in their population numbers, genetic engineering might become one of the only methods to extend their lives for a little longer; therefore it is critical to understand the impact of the technology on the corals before genetic engineering becomes the last resort.

Ultimately however, genetic engineering may not be a permanent solution to the issue of coral bleaching due to climate change. The final solution to helping preserve the corals is to deal with the cause of climate change -- greenhouse gas emissions and carbon footprints. In order to preserve the corals and the marine life on Earth in the long term, it is critical that we also learn to reduce our emissions. Genetic engineering is a tool that can allow us more time to discover ways to do this while maintaining endangered species, but in the end it is up to humanity to help reverse the effects of climate change in order to save endangered species such as the coral reefs.

There is still lots of research that needs to be done to study the genome of a coral and its interactions with its symbiotes to offer a stable approach to genetically modify them. But while genetic engineering in wildlife is not a perfect final solution and faces many problems, including managing the public opinion and funding, it remains still one of the most promising technologies that can help buy humanity time as we rush to restore the damage we have done. We must consider the role of genetics in the fight against climate change, because it may quickly become the only method to conserve biodiversity.

An Ideal Experiment

Due to our lack of resources at our disposal and our location, it is not realistic for us to conduct an experiment that involves genetically engineering a coral. However, if we had the resources and were to approach using CRISPR in corals, below is a generalized theoretical experiment procedure that we would take. This experiment would only be conducted presuming the following conditions:

• Permits from regulatory and ethical authorities, including the Panel on Research Ethics in Canada and the World Health Organization, have been obtained.
• A safe harbour locus on cnidarians has been discovered.
• There is a homologous site on the Aiptasia anemone.

Insights into the potential of knock-in of additional HSF1 genes in sea anemone model system using CRISPR Cas9

Problem

How might adding extra copies of the gene HSF1 to the anemone Aiptasia affect its heat tolerance in the context of endosymbiotic zooxanthellae bleaching?

Hypothesis

If the HSF1 genes are successfully integrated and accepted by the genome of Aiptasia, then its heat tolerance will increase which will lessen its rate of bleaching. This is because the additional expression of HSF1 will somewhat counterbalance the natural sudden downregulation during heat shock, which will decrease the chance of the anemone misreading the dinoflagellate symbiote as a hostile intrusion.

Variables

Main Manipulated Variable:

The presence of additional HSF1 genes in the Aiptasia anemone.

Main Responding Variable:

Range of heat tolerance of the anemone until signs of bleaching are evident.

Controlled Variables Include:

The age of the anemones, the initial genetic material of the anemones prior to adding CRISPR-induced HSF1 genes into the clones, the control group of wild-type larvae, the water conditions during the stages of development, the times and temperatures of heat shock, the species of anemone, the voltage at which electroporation is conducted at.

Background and Significance

The relationship between the dinoflagellate zooxanthellae algae and cnidarians, such as Acropora and Aiptasia, is incredibly complex. To this day, most of the endosymbiotic relationship remains in shadow. This makes it difficult for scientists to determine the causes of coral bleaching on a genetic level. Analyses from previous studies reveal that during heat shock, immunity genes and heat tolerance genes such as HSF1 are dramatically upregulated, and then deregulated to nearly minimal levels. This study aims to clarify some relationships between the two specimen and how it affects coral bleaching. It will confirm that the cause of coral bleaching is dramatic downregulation in immune genes. We will use the model anemone Aiptasia, because of its genetic similarity to the common stony corals under the genus of Acropora, we willl use CRISPR Cas9 technology to deliver additional HSF1 genes to minimize the effects of immune gene deregulation. 

Procedure

Step 1. Preparing the HSF1 gRNA

1. Using distilled water (or through the manufacturer's instructions), dilute equal amounts of forward and backward lyophilized oligonucleotides of HSF1 genes into PCR tubes.
2. Using vortex machines, spin the PCR tubes to mix the HSF1 genes.
3. Add the digestive enzyme into a guide expression vector solution, and allow digestion for an hour.
4. Use agarose gel to separate DNA fragments with gel electrophoresis
5. remove extended DNA fragments from the gel
6. begin the ligation process by mixing the HSF1 oligonucleotides with the guide expression vector. Add a reaction buffer if necessary, and spin to mix.
7. Add the DNA ligase to close the DNA, and spin the mixture to mix. Allow the mixture to fully react overnight at room temperature.
8. Add the bacteria vector into the mixture. Keep cool for an hour until the DNA ligase has fully penetrated the bacteria body, and heat shock for one minute.
9. Plate the culture onto a petri plate that contains a toxin that will isolate the bacteria that has the gHSF1 RNA.
10. Isolate the gHSF1 RNA

*note: if there are no easily obtained HSF1 oligonucleotides available since it is not a naturally occuring loose strand of gRNA, an alternative method to obtain gRNA of HSF1 is to use computer software to design several strands of it, and then to subclone the strands onto Cas9 expression plasmids. 

Step 2. Cloning zygotes

1. Obtain somatic cells from the male Aiptasia anemone, and an egg cell from the female organism.
2. Using a micropipette and microscopy equipment, physically remove the nucleus from multiple egg cells. 
3. Using electroporation, move the somatic cells into the egg cells. Electroporate once again to remove all contents of the somatic cells except for the DNA.

Step 3. Using CRISPR and Cas9

1. Use electroporation and or CRISPR to move the DNA into the genetic material of the zygote.

Step 4. Observing Development

1. Move zygotes into a tank with moving water and suitable salt concentrations.
2. Observe planulae as they settle along substrate at the bottom of the tank. 
3. As planulae develop, measure height and gamete development.
4. Move anemones away/separate tanks.

Step 5. Exposing larvae to high temperatures.

1. Gradually increase temperature by 1.5℃ - 2℃ over 8 weeks.
2. Compare heat resistance between wild-type larvae and mutant larvae.

Next Steps

Assuming successful significant increase in the thermal tolerance of the Aiptasia, public support, government consent, and sufficient financial support, the anemones would be tested to ensure that they do not have an unexpected consequence in the wild. If these tests showed no inherent harm to their imitation of the natural ecosystem environment, then we would follow up with gradual introduction and integration into a true wildlife ecosystem, such as the Eastern Atlantic, where they would continue to be closely montitored. 

Some of the tests that could be conducted on the anemones are as followed.

1. Gamete production of the genetically modified anemones in comparison to their non-modified counterparts.
2. Growth rate of the genetically modified anemones in comparison to their non-modified counterparts.
3. Using four oxygenated salt-water aquariums, create an imitation of a saltwater ecosystem using live sand. In two tanks, introduce the GMAs. In the other two, introduce the non-modified clones. Gradually introduce organisms that interact significantly with Aiptasia anemones, such as predators like butterfly fish, clownfish, cleaner shrimp, Berghia Verrucicornis sea slugs, and tuxedo urchins. Monitor the health of these organisms.

Further Discussion

If this experiment were successful, it would be an incredibly important development in the prevention of coral bleaching on massive scales. Since Aiptasia is strikingly similar to many stony coral species, including Acropora, the experiment's success would open up possibilities of other experiments involving the effects of heat shock in stony corals. This experiment is the tip of an iceberg of climate solutions and genetic technology potential.

References

(2019, October 1). Ocean acidification from human activities. Retrieved December 17, 2023, from https://www.dfo-mpo.gc.ca/videos/acidic-acides-eng.html

. (2022, October 2). . - YouTube. Retrieved December 17, 2023, from https://aquariumia.com/what-eats-coral/?expand_article=1

Addgene. (n.d.). Addgene. Retrieved February 16, 2024, from https://www.addgene.org/search/catalog/plasmids/?q=42230

All About Coral & Coral Reefs - Diet & Eating Habits. (n.d.). SeaWorld.org. Retrieved December 17, 2023, from https://seaworld.org/animals/all-about/coral-and-coral-reefs/diet/

All About Coral & Coral Reefs - Reproduction. (n.d.). SeaWorld.org. Retrieved December 16, 2023, from https://seaworld.org/animals/all-about/coral-and-coral-reefs/reproduction/

Approaches to Coral Reef Conservation. (2018, September 10). Coral Reef Alliance. Retrieved March 9, 2024, from https://coral.org/en/blog/restoration/

Are corals animals, plants, or something else? : Ocean Exploration Facts. (n.d.). NOAA Ocean Exploration. Retrieved December 17, 2023, from https://oceanexplorer.noaa.gov/facts/coral-animal.html

Are corals rocks, plants, or animals? (n.d.). Florida Keys National Marine Sanctuary. Retrieved December 16, 2023, from https://floridakeys.noaa.gov/corals/coralanimals.html

Armitage, H. (2018, April 23). CRISPR used to genetically edit coral | News Center | Stanford Medicine. Stanford Medicine. Retrieved December 31, 2023, from https://med.stanford.edu/news/all-news/2018/04/crispr-used-to-genetically-edit-coral.html?microsite=news&tab=news

Basic Information about Coral Reefs | US EPA. (2023, May 11). Environmental Protection Agency. Retrieved December 16, 2023, from https://www.epa.gov/coral-reefs/basic-information-about-coral-reefs

Basic Information about Coral Reefs | US EPA. (2023, May 11). Environmental Protection Agency. Retrieved December 17, 2023, from https://www.epa.gov/coral-reefs/basic-information-about-coral-reefs

Berry, D., & Uno, E. (2012, April 2). DNA Structure and Replication: Crash Course Biology #10. YouTube. Retrieved January 29, 2024, from https://www.youtube.com/watch?v=8kK2zwjRV0M

Berry, D., & Uno, E. (2016, February 18). What is CRISPR? YouTube. Retrieved January 29, 2024, from https://www.youtube.com/watch?v=MnYppmstxIs

BiotechLucas. (2022, May 18). Understand Electroporation In Under 3 Minutes. Youtube. Retrieved January 29, 2024, from https://www.youtube.com/watch?v=9eMjtT83iIM

Black-footed Ferret Project, Major Milestones. (n.d.). Revive & Restore. Retrieved January 28, 2024, from https://reviverestore.org/projects/black-footed-ferret/major-milestones/

Bone Grafting. (n.d.). Johns Hopkins Medicine. Retrieved December 16, 2023, from https://www.hopkinsmedicine.org/health/treatment-tests-and-therapies/bone-grafting

CAG promoter. (n.d.). Wikipedia. Retrieved February 16, 2024, from https://en.wikipedia.org/wiki/CAG_promoter

The cane toad (Bufo marinus) - fact sheet. (2021, October 10). DCCEEW. Retrieved February 14, 2024, from https://www.dcceew.gov.au/environment/invasive-species/publications/factsheet-cane-toad-bufo-marinus

Catells, L. (2017, July 5). .,. Get the glow: the secret to deep-water corals' radiance. Retrieved December 31, 2023, from https://www.nature.com/articles/nature.2017.22259

Cleaves, P. A., Strader, M. E., Bay, L. K., Pringle, J. R., & Matz, M. V. (2018, March 7). CRISPR/Cas9-mediated genome editing in a reef-building coral. PNAS. Retrieved January 6, 2024, from https://www.pnas.org/doi/10.1073/pnas.1722151115

Cleves, P. A., Tinoco, A. I., Bradford, J., Dimitri Perrin, Bay, L. K., & Pringle, J. R. (2020, November 9). Reduced thermal tolerance in a coral carrying CRISPR-induced mutations in the gene for a heat-shock transcription factor. PNAS. Retrieved January 29, 2024, from https://www.pnas.org/doi/full/10.1073/pnas.1920779117

Coastal flooding. (n.d.). Wikipedia. Retrieved December 17, 2023, from https://en.wikipedia.org/wiki/Coastal_flooding

Coral Basics | Flower Garden Banks National Marine Sanctuary. (n.d.). Flower Garden Banks. Retrieved December 17, 2023, from https://flowergarden.noaa.gov/education/coralbasics.html

Coral reef ecosystems. (n.d.). National Oceanic and Atmospheric Administration. Retrieved December 16, 2023, from https://www.noaa.gov/education/resource-collections/marine-life/coral-reef-ecosystems

Coral Reef Restoration: A guide to coral restoration method. (2021, January 18). UN Environment Programme. Retrieved January 30, 2024, from https://www.unep.org/resources/report/coral-reef-restoration-guide-coral-restoration-method

Coral Reefs. (2021, October 11). UNFCCC. Retrieved December 17, 2023, from https://unfccc.int/news/coral-reefs

Coral reefs: our underwater food factory. (2021, September 21). Economist Impact. Retrieved December 16, 2023, from https://impact.economist.com/ocean/biodiversity-ecosystems-and-resources/coral-reefs-our-underwater-food-factory

Coral Skeleton - Coral Disease & Health Consortium. (n.d.). NOAA Coral Disease and Health Consortium. Retrieved December 16, 2023, from https://cdhc.noaa.gov/coral-biology/coral-skeleton/

Coral: What Does it Eat? (2015, March 25). YouTube. Retrieved December 17, 2023, from https://www.youtube.com/watch?v=tZuxZdG6TfM

Crash Course. (2012, April 22). DNA, Hot Pockets, & The Longest Word Ever: Crash Course Biology #11. YouTube. Retrieved January 11, 2024, from https://www.youtube.com/watch?v=itsb2SqR-R0&t=247s

Deep-sea Corals | Smithsonian Ocean. (2018, April 30). Smithsonian Ocean. Retrieved December 16, 2023, from https://ocean.si.edu/ecosystems/coral-reefs/deep-sea-corals

Deep-sea Corals | Smithsonian Ocean. (2018, April 30). Smithsonian Ocean. Retrieved December 17, 2023, from https://ocean.si.edu/ecosystems/coral-reefs/deep-sea-corals

Dharamsi, J. (2023, September 29). Genetic Engineering. YouTube. Retrieved December 23, 2023, from https://www.youtube.com/watch?v=CDw4WPng2iE

Douch, A. (2017, September 7). What is the PAM? - A CRISPR Whiteboard Lesson. YouTube. Retrieved February 15, 2024, from https://www.youtube.com/watch?v=iSEEw4Vs_B4

Dr Matt & Dr Mike. (2021, 09 7). How does cloning work? - explained in 2 mins! [In this video, Dr Matt explains the process of animal cell cloning.] [Video]. Youtube. https://youtu.be/g74LQlc7h4c?si=zvuB0E5Ck4DlnmW4

Gallagher, A. (n.d.). Tough Teeth and Parrotfish Poop | Smithsonian Ocean. Smithsonian Ocean. Retrieved December 16, 2023, from https://ocean.si.edu/ocean-life/fish/tough-teeth-and-parrotfish-poop

Gel Electrophoresis. (2017, September 27). YouTube. Retrieved January 29, 2024, from https://www.youtube.com/watch?v=ZDZUAleWX78

The Genetically Engineered American Chestnut | CBAN. (2023, December 13). Canadian Biotechnology Action Network. Retrieved January 28, 2024, from https://cban.ca/background-the-genetically-engineered-american-chestnut-darling-58/

Genetically engineered heat-resistant coral: An initial analysis of public opinion. (n.d.). NCBI. Retrieved February 6, 2024, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8782467/

Genetic Research Boosts Black-footed Ferret Conservation Efforts | U.S. Fish & Wildlife Service. (2021, February 18). U.S. Fish and Wildlife Service. Retrieved January 28, 2024, from https://www.fws.gov/press-release/2021-02/genetic-research-boosts-black-footed-ferret-conservation-efforts

Gostimskaya, I. (2022, August 15). CRISPR–Cas9: A History of Its Discovery and Ethical Considerations of Its Use in Genome Editing. NCBI. Retrieved February 14, 2024, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9377665/

Gostimskaya, I. (2022, August 15). CRISPR–Cas9: A History of Its Discovery and Ethical Considerations of Its Use in Genome Editing. NCBI. Retrieved February 17, 2024, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9377665/

Great Barrier Reef. (n.d.). UNESCO World Heritage Centre. Retrieved December 16, 2023, from https://whc.unesco.org/en/list/154/

Green, E. (n.d.). Replanting Coral Reefs. Happy Earth. Retrieved January 29, 2024, from https://www.happyearthapparel.com/blogs/journal/replanting-coral-reefs

Hancock, L. (n.d.). Everything You Need to Know about Coral Bleaching—And How We Can Stop It | Pages. WWF. Retrieved December 17, 2023, from https://www.worldwildlife.org/pages/everything-you-need-to-know-about-coral-bleaching-and-how-we-can-stop-it

Henle, A. M., & Wells, A. (2019, January 24). How CRISPR lets you edit DNA - Andrea M. Henle. YouTube. Retrieved December 23, 2023, from https://www.youtube.com/watch?v=6tw_JVz_IEc

How are countries cutting emissions? And other top climate change stories | World Economic Forum. (2022, September 2). The World Economic Forum. Retrieved January 30, 2024, from https://www.weforum.org/agenda/2022/09/climate-change-latest-stories-02-september/

How can we save our dying coral reefs? (2012, September 5). BBC. Retrieved January 29, 2024, from https://www.bbc.com/future/article/20120905-save-our-dying-coral-reefs

How Corals Reproduce. (n.d.). Coral Reef Alliance. Retrieved December 16, 2023, from https://coral.org/en/coral-reefs-101/how-corals-reproduce/

How do corals eat? (n.d.). Florida Keys National Marine Sanctuary. Retrieved December 17, 2023, from https://floridakeys.noaa.gov/corals/coralseat.html

How Do Oil Spills Affect Coral Reefs? (2013, December 6). NOAA's Office of Response and Restoration. Retrieved December 17, 2023, from https://response.restoration.noaa.gov/about/media/how-do-oil-spills-affect-coral-reefs.html

How the cleaner fish and remora help keep coral reef fish healthy and clean. (n.d.). Britannica. Retrieved December 17, 2023, from https://www.britannica.com/video/180308/cleaner-fish-fishes-remora-coral-reefs

Johnson, A. (2020, November 16). Genetic engineering | Genetics | Biology | FuseSchool. YouTube. Retrieved December 23, 2023, from https://www.youtube.com/watch?v=DIM38NlkWEo

Keys Education. (2020, April 12). Coral Anatomy Virtual Lesson [Youtube Video]. Youtube. Retrieved January 27, 2024, from https://www.youtube.com/watch?v=F4rJYW-kb10

Kitts, P. (n.d.). Green fluorescent protein as a reporter of gene expression and protein localization. PubMed. Retrieved February 16, 2024, from https://pubmed.ncbi.nlm.nih.gov/8777060/

Kosch, T. A., & Kosch, T. (2022, January 17). Using genetics to conserve wildlife | Pursuit by The University of Melbourne. Pursuit. Retrieved January 28, 2024, from https://pursuit.unimelb.edu.au/articles/using-genetics-to-conserve-wildlife

KSLOF Coral Reef Education: Free Coral Life Cycle CourseLiving Oceans Foundation. (n.d.). Living Oceans Foundation. Retrieved December 16, 2023, from https://www.livingoceansfoundation.org/education/portal/course/life-cycle/

Md, S. (2023, October 21). ,. , - YouTube. Retrieved January 27, 2024, from https://www.pnas.org/doi/full/10.1073/pnas.2015737117

Mitra, S. (n.d.). reduce Your Carbon Footprint Now! 10 Shockingly Simple Ways to Save the Planet. Let's Talk Geography. Retrieved March 9, 2024, from https://letstalkgeography.com/reduce-your-carbon-footprint/

Morgan, K. (2014, April 3). Plasmids 101: The Promoter Region – Let's Go! Addgene Blog. Retrieved February 16, 2024, from https://blog.addgene.org/plasmids-101-the-promoter-region

Newbert, C. (n.d.). Coral. National Geographic Education. Retrieved December 17, 2023, from https://education.nationalgeographic.org/resource/coral/

9 Astonishing Facts about Corals: Filter Water, Mitigate Storms, Treat Cancer. (n.d.). Odd Facts. Retrieved December 17, 2023, from https://ofacts.org/invertebrates/facts-about-corals/#b_Filter_and_improve_the_quality_of_seawater

Ocean acidification in the Great Barrier Reef. (n.d.). Wikipedia. Retrieved December 17, 2023, from https://en.wikipedia.org/wiki/Ocean_acidification_in_the_Great_Barrier_Reef

Oil spills & coral reefs | ICRI. (n.d.). International Coral Reef Initiative. Retrieved December 17, 2023, from https://icriforum.org/oil-spills-coral-reefs/

PCR (Polymerase Chain Reaction). (2020, October 1). YouTube. Retrieved January 29, 2024, from https://www.youtube.com/watch?v=a5jmdh9AnS4

Primer. (n.d.). National Human Genome Research Institute. Retrieved February 17, 2024, from https://www.genome.gov/genetics-glossary/Primer

Restriction enzymes. (2015, March 25). YouTube. Retrieved January 29, 2024, from https://www.youtube.com/watch?v=U2cKywEn6KY

Riley, M. (2023, August 19). Preserving the Black-Footed Ferret Species Through Gene Cloning and Genetic Engineering – Meeteetse Museums. Meeteetse Museums. Retrieved January 28, 2024, from https://meeteetsemuseums.org/preserving-the-black-footed-ferret-species-through-gene-cloning-and-genetic-engineering/

ROSA26. (n.d.). Wikipedia. Retrieved February 20, 2024, from https://en.wikipedia.org/wiki/ROSA26

7 Fascinating Animals That Eat Corals! See Pictures - Animal Quarters. (2021, September 25). - Animal Quarters. Retrieved December 17, 2023, from https://www.animalquarters.com/animals-that-eat-coral/

Seven ways you're connected to coral reefs. (2020, February 28). UNEP. Retrieved December 17, 2023, from https://www.unep.org/news-and-stories/story/seven-ways-youre-connected-coral-reefs

Shallow Coral Reef Habitat | NOAA Fisheries. (2022, February 4). NOAA Fisheries. Retrieved December 16, 2023, from https://www.fisheries.noaa.gov/national/habitat-conservation/shallow-coral-reef-habitat

Shallow Coral Reef Habitat | NOAA Fisheries. (2022, February 4). NOAA Fisheries. Retrieved December 16, 2023, from https://www.fisheries.noaa.gov/national/habitat-conservation/shallow-coral-reef-habitat

Storlazzi, C. D., & Rey, A. (n.d.). Role of Reefs in Coastal Protection | U.S. Geological Survey. USGS.gov. Retrieved December 17, 2023, from https://www.usgs.gov/centers/pcmsc/science/role-reefs-coastal-protection

Thrope, S. (2022, May 23). QUICKLY Understand Transfection. YouTube. Retrieved January 29, 2024, from https://www.youtube.com/watch?v=5qKqEruyyMA

Timeline: CRISPR-Cas9. (n.d.). WhatisBiotechnology.org. Retrieved March 9, 2024, from https://www.whatisbiotechnology.org/index.php/timeline/science/CRISPR-Cas9

Top 8 Best Coral Reefs in the World. (n.d.). Audley Travel. Retrieved December 16, 2023, from https://www.audleytravel.com/ca/blog/2017/december/the-best-coral-reefs-in-the-world

Tourism. (n.d.). Coral Reef Alliance. Retrieved December 16, 2023, from https://coral.org/en/coral-reefs-101/why-care-about-reefs/tourism/

Understanding the Science of Ocean and Coastal Acidification | US EPA. (n.d.). Environmental Protection Agency. Retrieved December 17, 2023, from https://www.epa.gov/ocean-acidification/understanding-science-ocean-and-coastal-acidification

Wang, M., Xie, Y., Wang, Y., & Gu, L. (2022, November 11th). CRISPR/Cas9-mediated knock-in strategy at the Rosa26 locus in cattle fetal fibroblasts. Plos One, 17(11), p1-12. EBESCOhost. 10.1371/journal.pone.0276811

Webber, H. (n.d.). Ocean Currents in Australia. Redmap. Retrieved December 16, 2023, from https://www.redmap.org.au/article/ocean-currents-in-australia/

What Are Corals? Corals Tutorial. (n.d.). National Ocean Service. Retrieved December 16, 2023, from https://oceanservice.noaa.gov/education/tutorial_corals/coral01_intro.html

What is Coral Restoration, and Can it Save Coral Reefs? (2022, December 2). Coral Reef Alliance. Retrieved January 30, 2024, from https://coral.org/en/blog/what-is-coral-restoration-and-can-it-save-coral-reefs/

What is the PAM? - A CRISPR Whiteboard Lesson. (2017, September 7). YouTube. Retrieved February 17, 2024, from https://www.youtube.com/watch?v=iSEEw4Vs_B4

What Is Weathering? (n.d.). NOAA's Office of Response and Restoration. Retrieved December 17, 2023, from https://response.restoration.noaa.gov/oil-and-chemical-spills/significant-incidents/exxon-valdez-oil-spill/what-weathering.html

When carbonate formation loses equilibrium « World Ocean Review. (n.d.). World Ocean Review. Retrieved December 17, 2023, from https://worldoceanreview.com/en/wor-1/ocean-chemistry/acidification/when-carbonate-formation-loses-equilibrium/

Why are coral reefs important? (n.d.). Natural History Museum. Retrieved December 17, 2023, from https://www.nhm.ac.uk/discover/quick-questions/why-are-coral-reefs-important.html

Withers, D. (n.d.). Reefs and Pharmaceuticals. the Coral Digest. Retrieved December 16, 2023, from https://www.coraldigest.org/index.php/Pharmaceuticals

Yang, D., Song, J., Zhang, J., Xu, J., Zhu, T., Wang, Z., Lai, L., & Chen, Y. E. (2016, April 27). Identification and characterization of rabbit ROSA26 for gene knock-in and stable reporter gene expression. Sci Rep, 6. https://doi.org/10.1038/srep25161

Zooxanthellae: Corals Tutorial. (n.d.). National Ocean Service. Retrieved December 17, 2023, from https://oceanservice.noaa.gov/education/tutorial_corals/coral02_zooxanthellae.html

Ethics and Integrity. (2023, June 12). Retrieved March 12th from https://www.nserc-crsng.gc.ca/nserc-crsng/governance-gouvernance/eic-eic_eng.asp

Image Sources:

Project Banner: Coral-banner.jpg, Mrroper1987, https://commons.wikimedia.org/wiki/File:Coral-banner.jpg under Creative Commons License 4.0

Main Project Image: Coral Reef Stock photos by Vecteezy

We would like to thank our science fair coordinator, Mr. Buhler, for his continued support with our project formatting and submission. We would also like to thank Ms. Ulyott for helping mentor us in the basics of microbiology and genetics.

Thanks to Anaya Satavalekar for helping us understand some of the key concepts in the structure of DNA, as well as offering her experience and guidance in our project presentation.

Lastly, we would also like to thank our families for their unwavering support.

Special Thanks

We would like to offer special thanks to Professor Simon Donner, PhD of climate science and professor at University of British Columbia, for his deep insight on the feasibility of genetic engineering in wildlife and climate action.