GENE EDITING. IS IT THE FUTURE?
Mohith Krishna Shekar
Is gene editing the new way to treat diseases that can harm organisms (humans, animals and etc.)?
I think gene editing is the new way to treat certain diseases in organisms. The genetic information encoded in the DNA is responsible for the morphology and physiology of an organism. If we could modify DNA, we can change how cells perform or how the human body functions. This technology could strengthen our systems to fight off hereditary diseases like cystic fibrosis, type 1 Diabetes Mellitus and cancers. Intervening the immune response system could help treat acquired diseases like AIDS. By altering the DNA in the germ cells (before birth) genetic defects can be prevented at the embryonic stage. Gene editing can also be used for breeding animals, creating non-allergic food, eradicating pests, etc.
Although genome editing looks like a promising method of treatment, it has some disadvantages like deleting good genes, unpredictable outcomes. Many question how ethical is it to alter germline cells or alter traits. With deeper research and Code of ethics, gene editing can be the future of treatment.
I plan to research the topic by looking at scientific websites, articles and interviews.
-What is gene editing:
It is a procedure that Scientists use to change the DNA of an organism. Gene editing involves changing, inserting, erasing and replacing DNA in the genome of an organism
- What types of gene editing are there:
1. Restriction Enzymes: the Original Genome Editor
Gene editing started in the 1970s. Restriction enzymes recognize specific patterns of nucleotide sequences and cut them like scissors. As a result, it houses a place for new DNA substances. Restriction enzymes are not used for gene editing these days, because they are restricted by the nucleotide patterns they identify, but still remain commonly used for molecular cloning.
2. Zinc Finger Nucleases (ZFNs): Increased Recognition Potential
When the world revolutionized with more technology and more needs people came back to the thought of gene editing as they need more things these proteins can read. Then the discovery of zinc finger nucleases (ZFN) in the 1980s tried to tackle this issue.
The ZFN is made of two parts: an engineered nuclease (Fokl) fused to zinc finger DNA-binding domains. The zinc-finger DNA-binding region identifies a 3-base pair site on DNA which can be combined to understand longer sequences of DNA. However, the specificity of the DNA sites increased. But ZFN was not perfect. Aside from the negativity, ZFNs showed a strong stand in the field of medicine. Scientists used ZFN mainly to impair CCR5 on human T-cells (a significant receptor for HIV) Using ZFN editing, scientists discovered that autologous CD4+ T-cells were safe to handle and were an invigorating potential for HIV treatment. Also, ZFN editing is used to edit tumour-infiltrating lymphocytes as a strategy for metastatic melanoma.
3. TALENs Gene Editing: Single Nucleotide Resolution
The new TALENS arose in 2011 as the need for gene editing became a reality. TALENs was an improvement over ZFN. Transcription activator-like effector nucleases (TALENs) are built like ZFNs. TALENS and ZNFs use the Fokl nuclease to understand DNA strands and require dimerization to work, but the DNA bonding is different. TALENs work by using transcription activator-like effectors (TALEs) and arrays that are in front of each other that have 33-35 amino acid repeats. The repetition in the amino acids makes single-nucleotide recognition which increases the targeting ability and understandability compared to ZFNs.
4. CRISPR (Cas9) Gene Editing: CRISPR gene editing was initially discovered in 2012 by Professor Jennifer Doudna, a biochemist (University of California, Berkeley) and Professor Emmanuelle Charpentier, a microbiologist and a biochemist (Sorbonne University). They weren't the first to discover CRISPR but they were the first to describe the use of CRISPR to edit genomes. This incredible work led them to win the Nobel prize in chemistry last year. Jennifer Doudna was seeing how bacteria fight against viral infections. And she found out many bacteria have an adaptive immune system called CRISPR. In 2013, Feng Zhang explained how CRISPR can be used to edit eukaryotic DNA further.
According to Jennifer Doudna and Charpentier, CRISPR - Clustered Regularly Interspaced Short Palindromic Repeats. CRISPR is a family of DNA series located in the genomes of prokaryotic organisms such as bacteria and archaea. The protein Cas9 is an enzyme that behaves like a pair of molecular scissors that cuts strands of DNA.
CRISPR technology was modified from the natural defence mechanisms of bacteria and archaea (the domain of single-celled microorganisms). Those organisms utilize CRISPR-derived RNA and multiple Cas proteins, including Cas9, to foil attacks by viruses and other foreign bodies. They do this by cutting and destroying the DNA of a foreign invader (like a virus). Scientists are then left with 2 ends of DNA. Then they are capable of programming the Cas9 proteins and insert DNA sequences that fit on both ends of the DNA. Whenever there is damage, DNA tries to repair itself by adding the cells on the ends so that they can connect to each other or by creating a sequence. Scientists use this opportunity to insert the DNA which has changed so that it can interlock.
How does it work?
CRISPR: Crispr is actually a natural process in your body that is long functions as a bacterial immune system. This was initially found defending single-cell bacteria and archaea against invading viruses. Crisper uses 2 main components. The First is short sections of repetitive DNA ( deoxyribonucleic acid) sequences called Clustered Regularly Interspaced Short Palindromic Repeats or simply CRISPR. The second part is Cas or CRISPR-associated proteins which cut DNA like scissors. When a virus invades a bacteria, Cas proteins cut out a segment of the viral DNA to insert into the bacterium CRISPR region, capturing a chemical snapshot of the infection. Those viral codes of DNA are then copied into short pieces of RNA (ribonucleic acid). This molecule plays many vital roles in our cells, but in the case of CRISPR, the RNA binds to the protein cell called Cas9. As a result, this acts as a guard for a celebrity on duty to find a specific person latching onto floating genetic material and searching for a match of the virus. If the virus invades again, it will recognize it immediately and the Cas9 destroys the viral DNA.
Many bacteria have this defence mechanism like Halophiles, E.coli and Clostridium. But in 2012 scientists found how to hack CRISPR to target not only viral DNA but also any DNA in almost every organism. With the right tools, this viral immune system becomes a precise gene-editing tool, which can change DNA and genes almost as easily as fixing a typo on the computer. To change the DNA strands the Cas9 targets and removes, scientists create a guide RNA to match the gene they want to edit and then attach it to the Cas9 protein cell. Like the viral RNA in the CRISPR immune system, the guide RNA makes the Cas9 go towards the targeted gene. Then the proteins molecular scissors cut the DNA. Just by injecting Cas9 bound to a short piece of custom guide RNA, scientists can basically edit any gene in the genome. After the DNA is cut the DNA tries to correct itself. Proteins called nucleases trim the broken edges of the DNA and join them back together. But this repair process called nonhomologous end joining is likely to have a mistake and can lead to absent or extra bases. As a result, the gene is shut down. But if scientists add a separate order of template DNA, cellular proteins can produce a different DNA repair process called homology-directed repair. This repair process is used as a manual to guide the repair process which can repair a defective gene or even insert an entirely new one.
One step further:
CRISPR Cas9 is incredible as it could rewrite DNA easily but with it, people who get treated by this form of Gene editing may carry on some unwanted changes in their DNA. Since CRISPR Cas9 can delete and rewrite, there is a lot of room for errors as one mistake can lead to one’s eyes being disabled to one getting a disease. Like large chromosomal segments might even be deleted or rearranged using CRISPR.
However, we don’t need to fear as there is a new way that is more accurate, easier and more effective. It’s called base editing. Base editing uses the programable searching of the Cas9 cell, but instead of cutting the DNA, it directly changes it from one base to another without disrupting the rest of the cells and genes. You could think of CRISPR Cas9 like scissors while base editors are more like pencils and erasers. The first cytosine base editor (CBE), which chemically changes a cytosine–guanine (C–G) base pair to a thymine–adenine (T–A) base pair at a targeted genomic area, was made in 2016 by chemical biologists David Liu and Alexis Komor at Harvard University in Cambridge, Massachusetts. Another researcher in Davids laboratory, Nicole Gaudelli, made the first adenine base editor (ABE) in 2017. The adenine base editor chemically converts A–T to G–C base pairs.
“Base editing gives very, very good efficiency, about 40–50% efficiency for cell lines,” says Huang, a geneticist at ShanghaiTech University in China. “That’s very high efficiency compared with traditional genome editing,” which is only one-tenth as efficient, he says.
Alexis Kormor took advantage of a naturally occurring enzyme called APOBEC1. This enzyme, which is part of the cytidine deaminase family, chemically converts C to a U (uracil) then to a T that occurs in RNA. Komor combined APOBEC1 to a Cas9 nuclease that is unable to create DNA double-strand breaks. When the guide RNA directs the APOBEC1–Cas9 protein to the targeted DNA strand using CRISPR, the deaminase converts C to U. The cell’s DNA-repair system then fixes the resulting U–G mismatch by turning it into a U–A base pair, and then to a T–A pair.
We could use base editing for many genetic diseases that have single base mutations like sickle cell anemia, progeria, muscular dystrophy or Tay-Sachs disease and etc. For example, million suffer from Sickle cell anemia just because they have a single A to T point mutation in both their copies of their hemoglobin gene. And also children born with Progeria have a T at a single position in their genome where we have a C. These children age rapidly and pass away at about age 14 just because of that single point mutation. But with Base editing, this could all change. We could change that T cell into a C cell so they wouldn’t age rapidly and we could do the same with every genetic disease.
Base Pairs” T=A G=C
T= Thymine A= Adenine G= Guanine C= Cytosine
Genome editing can be done on two types of cell lines as described by the pioneers. Somatic cells in our body are non-heritable genes carrying cells that carry information for the functioning of a body. These do not pass on to the next generations. On the contrary, germline cells are inheritable genes that carry specific information and occur in the gametes. Most of the research published is targeted at somatic cell genome editing.
There are basically two ways how scientists are introducing edited genes into an organism.
1.In vivo gene therapy: This method involves directly introducing cells into the organism using a vector.
2.Ex vivo gene therapy: In this technique, scientists harvest cells from the patient and return them to the patient after genetic modification.
What are the benefits of gene editing?:
CRISPR gene editing has been used in various fields as a treatment for various diseases in humans and to introduce a new species in plants etc.
CRISPR can treat various genetic diseases like Albinism. Angelman syndrome, Ankylosing spondylitis, Apert syndrome, Charcot-Marie-Tooth disease, Congenital adrenal hyperplasia, Cystic fibrosis, Sickle cell anemia and much more according to the scientist Jennifer Doudna.
Sickle Cell Anemia:
This is a cruel genetic disease that produces a defective form of hemoglobin (the protein needed for red blood cells to carry oxygen and deliver it to the body). The defective hemoglobin cells turn the blood cells into sickle-shaped forms. These irregular cells block the bloodstream causing organ damage and sometimes extreme spikes of pain. Victoria Gray from The US was diagnosed with sickle cell anemia when she was born. Sickle cell anemia is the most common inherited blood disorder and it’s hard to treat. Scientists know the exact cell change in their body which is a single A to T point mutation in both their copies of their hemoglobin gene. Many treatments are drugs and risky surgeries but for some patients, it’s still not enough to get this disease out of their body. Victoria Gray was exploring the possibility of a bone marrow transplant but her doctors suggested something else. She jumped up to the chance and became the first American who got treated with a gene-editing technique called CRISPR. CRISPR made the revolutionary arrize of treatment and now it’s working better than any doctor could have predicted. The doctors injected with her own genetically modified 2 billion bone marrow cells into Victoria’s body which appeared to be erasing almost every symptom of her disorder. She turned out perfectly fine after 2 years and was living a normal life with no pain and symptoms of Sickle cell anemia. Her red blood cells turned back to normal and she is now living a healthy life.
Other People who got treated with CRISPR:
In 2019 and late 2020 two people were treated with CRISPR for beta-thalassemia. Beta-thalassemia is a blood disorder like sickle cell anemia where beta chain synthesis is affected. It lowers the levels of hemoglobin which results in the low level of oxygen being given to your body. The side effects of beta-thalassemia are weakness, paleness, bone issues that make facial changes, gallbladder and liver problems, swollen kidneys, diabetes type 1, delayed growth, hypothyroidism, and heart-related issues.
Eliminating HIV-1 :
CRISPR-Cas9 Gene editing coupled with LASER ART - According to Dr. Kamel Kahlili, Temple University and Dr. Howard E. Gendelman, University of Nebraska Medical centre, using CRISPR-Cas9 which can create breaks in a DNA and induce repairs by point mutation and inserted DNA sequence can result in an effective knockout of genes affected by HIV. Along with LASER ART(LONG ACTING SLOW-EFFECTIVE RELEASE ANTIRETROVIRAL THERAPY), CRISPR gene editing showed to be a promising method of treatment for HIV-1 in their experiments using Humanized mice.
Improving the quality of life:
Professor Jennifer Doudna says that Gene editing can impact the quality of life in the elderly although they have not yet reached the means to increase the span of life. Genome editing can significantly cut down infections and provide immunity against cancer which in turn can bring a great improvement in the quality of life.
Protecting endangered species and bringing back extinct species:
CRISPR Genome editing can be used as a tool to protect and increase the population of endangered species and bring back some extinct species according to Jennifer Doudna.
Gene editing is used in the development of transgenic foods (genetically modified food):
Genetically modified (GM) foods are foods derived from organisms whose genes are modified. Currently, GM foods in use are mostly from plants. These crops have been developed to improve the yield by increasing resistance to drought, insects, diseases and tolerance to herbicides. GM crops are cheaper as their yield is higher and more reliable. Example: Corn, soybean, potato, canola, apple etc.
Genetically modified animals: After many years of continuous research, thousands of dollars and several unsuccessful attempts Van Eenennaam designed a line of CRISPR cattle as per the requirements of the beef industry. In 2020, a cattle named Cosmo that underwent CRISPR gene editing as an embryo in favour of giving birth to male offspring was born. This species of cattle could improve the efficiency of beef production because male cattle are more fuel-efficient at converting hay into beef. Cattle grown today contribute a lot of greenhouse gases to our atmosphere every day. By editing the cows to produce more male offspring, we could get the same amount of beef with fewer amounts of cows which in turn will reduce the amount of carbon footprint on the whole industry. To make this happen scientists used CRISPR to insert the SRY gene (the gene for male development) onto Cosmo’s 17th chromosome which is a non-sex chromosome. That means if he has calves that have the SRY gene on the 17th chromosome, they might develop male characteristics even if they don’t inherit the male Y chromosome.
Creating model organisms for biomedical research:
CRISPR technology is widely used to create a model organism for research purposes. A typical model organism matures rapidly, easy to manipulate, has a short span of life, can produce a large number of offspring and has a sequenced genome which is well understood. Example: Mouse, Drosophila melanogaster etc
According to Jennifer Doudna, CRISPR technology can promote gene drive. By editing a gene and introducing a new sequence of DNA, it can bring about a drive of that particular genetic trait rapidly. Gene drives dramatically increase the likelihood of survival of those edited genes by spreading quickly through generations and override natural selection.
Andrea Crisanti, a geneticist at Imperial College London says that gene drive technology can change evolutionary paths and can cause extinction. For example, it can help eradicate nuisance species like malaria-causing mosquitoes.
Even though gene editing seems like the best treatment for genetic diseases and is easy to use it comes with some side effects and issues like any other medical treatment.
When editing genomes we may be able to edit them properly but we don’t know how our body will react. CRISPR gene editing is a very successful tool, but say there was a patient who got treated with CRISPR for sickle cell anemia, their genes could get mutations and some genomes could be deleted or could be inserted with a new strand which messes up the gene making the DNA strands ineffective thus resulting in the genome to shut down. This could be very dangerous as the genomes determine everything in our body and how we look. If the mutation changed our melanocortin gene (the gene that determines our skin colour), if we were white before our skin colour would change to yellow or tan or etc.
Further, CRISPR has treated many patients with genetic diseases but it’s not that accurate. It has shown that CRISPR fails 15% of the time in medical studies. At the University of Illinois, people found a failure in the Cas9 targeting system as the DNA strand which was cut didn’t detach from the DNA blocking it from the DNA to repair itself resulting in the genome shutting down.
Safety is also a big concern when people gene edit because there is a possibility that there could be off-target effects (gene edits that are in the wrong place) and mosaicism (when some cells have the edited part but others don’t). A lot of researchers have spoken about this like the people who were at the International Summit on Human Gene Editing. They said “until germline genome editing is deemed safe through research, it should not be used for clinical reproductive purposes; the risk cannot be justified by the potential benefit. Many researchers are concerned if gene editing will start humanity on a tough journey as people might use it for non-therapeutic and enhancement purposes.
In our body, one particular gene might carry more than one function or information. For example, in sickle cell trait (a mild form of the disease with single-gene deletion) the patient is known to be protected from malaria because of the genes they have. If such patients undergo CRISPR gene therapy, they are prone to dreadful malarial disease(spread by mosquitoes). Hence such therapies might do harm in other ways as they save from one disease.
Gene editing can bring the extinction of species and create potential ecological and environmental imbalance. It is difficult to understand the importance of each gene in any species unless there is deeper research.
Gene editing is a very controversial topic when it comes to the uses because there are two ways you can actually use it. One being somatic cell editing and the second being germline editing. Somatic cell editing is much safer because if genes had mutation we could fix them and they won't cause very dangerous threats to the patient. But when it comes to germline editing (editing sperm, eggs and embryos) it gets more dangerous as the person's life is at stake.
CRISPR has been used for the treatment of many diseases but it has been to treat babies at an embryonic stage too. He Jiankui is a Chinese scientist who edited the embryo in a woman before she gave birth. He edited a cell that made the babies immune to HIV because their father had AIDS. Later on, the woman gave birth to twins named Lulu and Nana. But Jiankui did this illegally with no consent from the Chinese government or the babies. He edited the germline cell which is passed onto future generations. The scientist tried to create a nonspecific sequence alteration to the CCR5 gene. The CCR5 gene is a protein cell on top of white blood cells which is involved in the immune system. The CCR5 cell acts as a receptor for chemokines. Since he was trying to strengthen the immunity to HIV and AIDS he had to target the CCR5Δ32 gene. He Jiankui did an uncharted territory gene edit because CRISPR isn’t the best tool to edit genes. CRISPR can create a risk of an edit which can cause a mutation that will have side effects that we can’t predict. Because of this action, Jiankui was sentenced to jail for 3 years and 3 million yuan or $589,882.50 in Canadian currency.
The worldwide criticism against He Jiankui and his work is believed to be basically because he didn’t take any consent for germ cell gene editing neither from any committee nor the babies. And no one knows if the babies are safe. He potentially put the embryos at risk with less researched technology.
CRISPR gene editing has opened the doors to create babies with desired characteristics physically and intellectually. The pioneer says that it is possible to design a baby but they haven’t reached there yet. There is no limitation as to where one has to stop using CRISPR.
After the world was shocked by the use of CRISPR on germline cells, many countries, WHO and certain scientific communities have come forward to create a line of control on its use. According to Jamie Metzl, a WHO member, (author of a book on genetic engineering), when we are talking about the life we need regulations at both national and international levels. We should create a balance between the need and misuse to set limitations and go step by step. WHO has put forward certain laws to control gene editing. Many countries have deemed germ cell editing as illegal.
William Hurlbut (Bioethicist, Physician) has an opinion that we need to carefully review the balance between nature and life, be respectful to it while regulating gene editing. It should be addressed to all diversities of humans without creating social problems and ecological imbalances.
Jennifer Doudna says that although CRISPR is relatively easy to use, it is very hard to do it well because it’s dangerous. She encourages scientists to engage with people in discussions about setting regulations and remove any distrust in science.
Since I have conducted a researched-based project, I have not collected data.
To conclude my hypothesis was correct. I think gene editing is the new way to treat certain diseases in organisms because it is effective at getting rid of the disease and we can use gene editing in many ways. Scientists have used gene editing in humans already to cure sickle cell anemia and many more diseases. The opportunities are endless as CRISPR is evolving from just removing specific genome strands to changing the base pairs in seconds. With this technology right around the corner, I think more people have to invest in this research because this could be the new treatment for diseases. Genetic engineering will change everything. 3000 genetic diseases are caused by single-base mutations and with CRISPR Cas9 gene editing we can cure those genetic diseases. Even though gene editing might seem very dangerous if done correctly and by following the code of ethics we can edit genes with no harm. Scientists are exploring germ-line editing which will open up a whole new reality from us being immune to most diseases without getting genetically modified (Our parents get germ-line edited and the edited gene will pass on to us). But with that stretch come some obstacles. Germ-line editing is very dangerous as one mutation can bring serious illness or malfunction in the body. With this technology, we can proceed onwards and create a new way of life. We can eventually change our characteristics like physical appearances or how we behave. With CRISPR gene editing we can cure COVID-19 as it is a big threat. If we edited the genes that Covid attacks we can get rid of the patient's symptoms and eventually the virus itself. Thus gene editing is a great way of treatment for genetic diseases making life much better.
What is gene editing:
Types of gene editing:
Ways gene editing is beneficial:
Things that are getting genetically modified:
Sickel Cell anemia:
Youtube Video for pictures and research:
I'd like to acknowledge my parents especially my mom helping me with the science fair. My mom was always there when I got stuck and helped me go forward. My dad came up with awesome ideas to make my presentation better and both my mom and dad gave me great advice. My mom also helped me understand the scientific terminology like nucleases, chromosomes, etc because she is a doctor.