REETU ARVIKAR

Age 14 | Edmonton, AB

2019, 2021 Genome Alberta Award Regional Science Fair | 2019 Young Chemist Award Regional Science Fair

Edited by Luka Zivkovic

INTRODUCTION

Deoxyribonucleic Acid (DNA) is a complex molecule that guides the growth, development, function, and reproduction of every living organism. Inside the molecule, information is encoded and 4 nucleotides are paired to make up the code that carries these instructions. If you change the DNA of an organism (change its “instructions”), then you are changing the being carrying it (Kurzgesagt, 2016).

CRISPR stands for clustered regularly interspaced short palindromic repeats. CRISPR is a specific, efficient, and versatile gene-editing technology that we can harness to modify, delete, or correct precise regions of our DNA. It is a bacterium’s most effective antivirus system. This name originates from the fact that it has repeating sequences of genetic material. These segments of genes are what make up the basis of CRISPR-Cas9 technology. Cas9 is a protein that is part of the CRISPR system, it is responsible for locating, cutting, and degrading viral DNA in a specific way. CRISPR-Cas9 is a potential genetic engineering technology co-invented by Jennifer Doudna & Emmanuelle Charpentier. It can be used to cure genetic diseases by editing genomes, specifically, making changes to the DNA in cells. This technology allows cells to make precise changes to certain sections of DNA. Here is a timeline of the use of CRISPR-Cas 9:

TIMELINE OF THE USE OF CRISPR-CAS9 BETWEEN 1987 & 2020

1987 - CRISPR mechanism first published
2002 - Term “CRISPR-Cas9” published for the first time
2005 - French scientists suggested CRISPR spacer sequences can provide cell immunity against phage infection and degrade DNA
2011 - Emmanuelle Charpentier and Jennifer Doudna joined forces to investigate Cas9 enzyme
2012 - First commercialization of CRISPR-Cas9 technology
2013 (Jan) - CRISPR-Cas9 used in human genome editing
2013 (Aug) - CRISPR-Cas9 used to engineer a rat’s genome, plant genomes (rice, wheat, Arabidopsis, tobacco, and sorghum)
2015 (May) - First report of genes edited in human embryos ignited global ethical debate about gene editing technology
2015 (Sept) - UK scientists sought a license to genetically modify human embryos. The license would be used to study the role played by genes in human fertilization.
2015 (Nov) - US scientists genetically modified mosquitoes via CRISPR-Cas9 to prevent them from carrying malaria
2016 - UK scientists authorized to genetically modify human embryos using CRISPR-Cas9
2017 - Published research demonstrated using CRISPR-Cas9 to eliminate HIV infected mice
2018 - Chinese scientist announced first gene edited babies
2019 - WHO called on countries to ban experiments that would lead to more gene-edited babies
2020 (Mar) - First patient received gene editing therapy with CRISPR directly administered into the body
2020 (June) - Research published casting doubt over safety of using CRISPR-Cas9 to modify human embryos
2020 (Oct) - Novel Prize in Chemistry awarded to Emmanuelle Charpentier and Jennifer Doudna ‘for the development of a method for genome editing’.

HOW CRISPR-CAS9 WORKS

CRISPR was discovered through a basic project directed at discovering how bacteria fight viral infections. When attacked by phages, most bacteria try to resist but fail because their protection tools are too weak. In order for bacteria to rid themselves of a viral infection, bacterial cells have an adaptive/acquired immune system called CRISPR which detects viral DNA and destroys it. An adaptive immune system is a response system intended to destroy molecular forces foreign to the host to prevent death by infection of pathogens such as viruses, fungi, and parasites. Bacteriophages are an example of a virus. When they infect cells, they inject their DNA into the bacterium to then go through a process of replication inside the bacterium. The bacterium stores a part of the viral DNA in their own genetic code. When the virus attacks again, the bacterium makes an RNA copy from the CRISPR archive and uses Cas9 to precisely scan the bacterium for signs of the virus invader. It compares every letter of DNA to the sample from the archive. When it finds a 100% perfect match, it is activated and cuts out the viral DNA. This makes the injected viral DNA futile, protecting the bacterium against the attack.

**Figure 1**: Bacteriophages insert their DNA into bacteria to generally overtake the bacterium.

**Figure 2**: CRISPR archive stores a copy of integrated DNA as a “generic vaccine” (Doudna, 2015).

Integrated bits of viral DNA are copied, stored, and passed on to progeny to protect themselves, over generations, from viruses. The cells keep a record of the infection, acting as a “genetic vaccine”. After DNA is integrated, the cell makes an exact replica of the viral DNA in a molecule called RNA (orange regions in Figure 2.0). RNA is a chemical cousin of DNA and has the ability to read DNA, this allows interaction with DNA molecules that have a matching sequence. Bits of RNA from the CRISPR locus (locus is the position of a gene or mutation on a chromosome) bind to Cas9 (white regions in Figure 2.0) to form a complex that functions as a sentinel (a sentinel is an indicator of the presence of disease) in a cell. The complex runs through all of the DNA to find sites matching the sequence in the RNAs. When those specific sites of DNA (blue regions in Figure 2.0) are found, the Cas9 cleaver has access to precisely make a double-stranded break in the viral DNA helix. This complex also has the ability to be programmed to recognize particular DNA sequences and make a cut.

**Figure 3**: When cells detect a break in DNA, they can repair it two different ways (Doudna, 2015).

Cells naturally detect broken DNA and repair it when needed. When cells detect a break in DNA, it fixes that break by either pasting together ends of the broken DNA with a slight change in sequence at the site or by integrating an entirely new piece of DNA into the existing sequence. We can cure disease by programming CRISPR technology to make a break in DNA at or near a mutation. This will induce DNA repairs and the mutated DNA will be essentially erased or “deleted” (Doudna, 2015).

There are 2 ways of repairing a DNA break: homologous and non-homologous end-joining. Homologous repairs require a homologous sequence/template to guide a repair and integrates an entirely new piece of DNA into existing sequence. Non-homologous repairs can result in the additional deletion of a few base pairs and it does not need a homologous template. Breaks are directly ligated (Figure 3.0).

The CRISPR-Cas9 system is comprised of 2 parts. Cas-9, is the enzyme responsible for cutting DNA and a guide RNA is the sequence responsible for directing Cas-9 to its specific location in the DNA. Cas-9 associates with the guide RNA, forming a complex that can easily and precisely target a desired site in the DNA. The actual gene editing process begins when the complex recognizes and binds to a short segment of DNA adjacent to the target site. Initiating the unwinding of the DNA Helix. This allows the guide RNA to pair with a specific target sequence in the DNA, if the sequence is a 100% match, it makes a double-stranded break in the viral DNA helix. RNA is a chemical cousin of DNA and it has the ability to read DNA, this allows interaction with DNA molecules that have a matching sequence. The goal of this process is to make the viral DNA futile, thus protecting a bacterium against an attack (CRISPR Therapeutics).

BENEFITS

Through this, we gain the ability to correct mutations that may cause diseases such as Sickle Cell Anemia, Cystic Fibrosis, Huntington’s Disease, Type 1 Tyrosinemia, Tay-Sachs, HIV, and more. Furthermore, the earliest genetically modified animal was born in 1974. Further looking into the use of this technology for advanced human engineering, in the 1990s, there was a brief look into human embryo engineering directed towards treating maternal infertility. Babies carried genetic information from 3 humans, making them the first humans ever to have 3 genetic parents. In one experiment, scientists found that they could remove the DNA of integrated HIV virus from infected human cells. In 2016, the HIV virus was removed from rats who had the virus in almost all of their body cells. By simply injecting CRISPR into their tails, it removed more than 50% of the virus from cells all over their body. Along with HIV, CRISPR therapy can be used to cure other retroviruses that hide inside human DNA such as Herpes (Kurzgesagt, 2016). An example of CRISPR being used on mice: CRISPR was applied to mice with natural black pigmentation, by changing only one gene, their offspring sustained a lighter pink hue. This shows how we can use CRISPR to make precise changes to DNA and program it to fulfill its function (Doudna, 2015).

Along with the ones listed above, CRISPR may have the power to cure cancer. Cancer occurs when cells refuse to die and keep multiplying while concealing themselves from the immune system. CRISPR can edit immune cells to make them better “cancer hunters'' (Kurzgesagt, 2016). A modified version of Cas9 is already being built to change just a single letter. There is a high possibility that a future reality for curing cancer will look like a couple of injections of a few thousand of your own cells that were engineered in a lab. In 2016, the first clinical trial for CRISPR cancer treatment was approved. Soon enough, Chinese researchers announced that they were treating lung cancer patients using this technology. We may be able to end over 3000 genetic diseases that are caused by a single incorrect letter/mutation in DNA. With the use of this advanced technology, humankind has much higher chances of survival in the future. Every one of these experiments and trials have led to an even stronger understanding of this technology. Once a fundamental understanding has been achieved, we can use this technology to cure diseases on an unimaginable scale and potentially create humans to our liking, all to the benefit of our society.

LIMITATIONS

While there are many advantages to this marvelous technology, there are a few risks involved with using CRISPR-Cas9 for gene editing:

It is difficult to deliver it to mature cells in large quantities. Viral vectors, tools used to deliver genetic material into cells, are largely used.
Sometimes cells that take in CRISPR-Cas9 don’t have genome editing capabilities, this may make CRISPR inefficient in some scenarios.
However powerful CRISPR is, it is not infallible and incorrect/unintended edits can still happen that at times may even go unnoticed. The gene edit might be successful in terms of disabling a disease but it may also accidentally trigger unwanted changes.
Unless used on reproductive cells or early embryos, CRISPR is limited to the individual and dies with them.

CRISPR is a revolutionary tool that can be extremely useful but currently, we simply don’t know enough about our own genetics to avoid unpredictable consequences. The main point of focus for research currently is accuracy and making sure we only change what we need to.

CRISPR has relative simplicity, it reduces costs by 99% compared to traditional gene-editing techniques, experiments take only up to a few months, and almost anyone with a lab and scientific knowledge/understanding can perform it (Kurzgesagt, 2016). CRISPR also allows scientists to edit live cells to switch genes on and off and target and study particular DNA sequences. The first applications of this new technology occurred in blood because it is relatively easy to deliver tools to blood cells as compared to solid tissues (Doudna, 2015).

THE FUTURE OF CRISPR-CAS9

CRISPR technology can and most likely will be used for more than just curing genetic diseases. We can use it for the creation of modified humans (engineering humans to have enhanced properties) and designer babies. For example, stronger bones, different eye colour, height, high IQ, no baldness, perfect pitch, 20/20 vision, low risk of diseases, stronger metabolism, etc. Currently, researchers are continually discovering what genes are responsible for which of these properties in humans. Once known, this tool can be very useful. At first, CRISPR was seen as a tool to cure genetic diseases and stop the progression of diseases such as cancer and Alzheimer's. Next, it was seen as a tool to enhance features in humans, for example, enhanced metabolisms, perfect eyesight, height, muscular structure, full hair, extraordinary intelligence, and more. After this, scientists began researching CRISPR technology as a way to solve the single biggest mortality risk factor, aging. About ⅔ of people dying every day pass away due to age-related causes. The explanation behind aging is the accumulation of damage done to ourselves (i.e. DNA breaks and systems responsible for fixing those breaks wearing off). There are also genes directly affecting aging. A combination of gene editing and therapy could be used to prevent, stop the progression of, or reverse aging. There is a possibility of borrowing genes from animals who are already immune to aging. Biological aging could be something that eventually stops being a thing, we would die eventually but we might be able to spend a few thousand years here on Earth. Even further into the future, we may be able to eliminate obesity if we are in possession of a modified immune system. We could even engineer humans to be equipped for extended space travel and the ability to cope with harsh conditions (Kurzgesagt, 2016).

ETHICAL CONSIDERATIONS

While there are positive aspects to a future involving CRISPR-Cas9, there are also equally negative aspects to carefully consider. There is controversy and ethical issues, especially regarding CRISPR’s application to human embryos. This is due to the fact that there will be gradual but irreversible changes to the human gene pool. Modified human embryos could alter the genome of our entire species because engineered traits will be passed on to progeny. Slowly over generations, our entire species will be modified and “when everyone's super, no one will be (The Incredibles)”. Many argue that not using genetic modification is unethical because denying children the cure, forces them to go through preventable pain and suffering. Others argue that the children do not give consent for the changes made to their DNA. The issue is that once the first few genetically engineered kids are born, there is no turning back. Technology will progress and enhancing humans will be tempting. The ethics of using this powerful technology on humans is the reason research has been paused in the past.

While preselecting humans based on what we understand as healthy may sound ethically and morally wrong, we are already living in a world with preselected humans. The majority of pregnant women in the world get tests done during pregnancy and often terminate pregnancies that are going to lead to a lot of suffering for a child. So, we are in fact already deciding what kinds of humans get to live and what don’t.

Furthermore, imagine what strong dictatorships would do if they embraced this technology and cemented their rule forever. What would happen if everyone in the army is a “superhuman”, this tells us that this technology is also capable of severe destruction. In theory, it is doable, but these disquieting scenarios are far off into the future (Kurzgesagt, 2016).

conclusion

CRISPR-Cas9 is a tool that has been researched since the late 20th century and will most likely carry on far into the future. CRISPR-Cas9 works by storing a piece of unwanted viral DNA in a CRISPR archive, uses Cas-9 as a tool to scan for a match to that DNA, and removes it from any given live cell. Through this, we may be able to end diseases of all sorts, including major ones such as Alzheimer’s and various types of Cancer. While there are multiple benefits and limitations to this tool, it is powerful enough to extend our life expectancy by centuries and help us travel through space much beyond our current power. While it may not be wise to ban research in this field completely, approaching this powerful tool cautiously and transparently is the best method. Despite evident ethical issues, we still have a lot to gain and this might just be the first step to the natural evolution of intelligent species. While all this research is in its infancy and is not reachable in the near future, CRISPR technology is revolutionary and will definitely make a glaring difference in humanity.

REFERENCES

Being Patient. (Nov. 30, 2020). With “Genetic Scissors” Scientists Edit Alzheimer’s Prevention Into Brain Cells. Retrieved from: https://www.beingpatient.com/crispr-cas9-genetic-scissors-alzheimers/
Bird B. (Director). (November 5, 2004). The Incredibles. Pixar Animation Studios, Walt Disney Pictures.
CRISPR Therapeutics. (N.D.). CRISPR/Cas9. Retrieved from: http://www.crisprtx.com/#news
NCBI. (2002). The Adaptive Immune System. Retrieved from: https://www.ncbi.nlm.nih.gov/books/NBK21070/
Scitable. (2008). Chemical Structure of RNA. Retrieved from: https://www.nature.com/scitable/topicpage/chemical-structure-of-rna-348/
TED Ideas Worth Spreading. (September 2015). Jennifer Doudna How CRISPR lets us edit our DNA. Retrieved from: https://www.ted.com/talks/jennifer_doudna_how_crispr_lets_us_edit_our_dna/transcript
The Jackson Laboratory. (N.D.). What is CRISPR? Retrieved from: https://www.jax.org/personalized-medicine/precision-medicine-and-you/what-is-crispr
What is biotechnology? (N.D.). CRISPR-Cas9. Retrieved from: https://www.whatisbiotechnology.org/index.php/science/summary/crispr
YouTube. (May 24, 2017). Biologist Explains One Concept in 5 Levels of Difficulty - CRISPR I WIRED. Retrieved from: https://www.youtube.com/watch?v=sweN8d4_MUg
YouTube. (August 10, 2016). Genetic Engineering Will Change Everything Forever - CRISPR. Retrieved from: https://www.youtube.com/watch?v=jAhjPd4uNFY
YouTube. (August 22, 2020). How Gene Editing Is Curing Disease. Retrieved from: https://www.youtube.com/watch?v=ezfwqmKC9Uc
YouTube. (Jun 28, 2013). Three parents and a baby. Retrieved from: https://www.youtube.com/watch?v=GcubrH6HRnk

Designer DNA: CRISPR-Cas9