By Julia Jaramillo and Kinjal Desai

In our previous article about CRISPR, we discussed some uses of this gene-editing technology in scientific research. Here, we go into a little more depth on how it works and answer your questions about the science and implications of CRISPR technology.

Genes are like words. They are made up of the letters A, G, T or C in different combinations to spell over 20,000 different genes in human beings. There are 3 billion letters in every single cell of our bodies. This is our DNA, our genetic material. Not all the letters in our DNA make words, but a fraction of them do. These words carry highly important information for the cells, such as instructions to divide, produce energy, get rid of waste, and even instructions to die if the cell gets damaged beyond repair. 

Over the past few decades, researchers have just been learning how to ‘read’ genes and have subsequently read almost the entire 3 billion letters of human DNA. This has uncovered a lot of important information about health and disease. 

One big finding is the identification of novel disease-causing mutations. A mutation is when a letter in a gene word is wrong, or the gene is misspelt. This can either have no implications for the way the gene is read, or plenty, depending on the type of mistake that is made. For instnce, you probably read that word as ‘instance’, even though it was spelt incorrectly. In this case, the misspelling didn’t affect the interpretation of the word. But what if the word ‘home’ were misspelt as ‘hole’? This changes the meaning of the word completely, even though just a single letter was replaced by another one. 

Imagine something similar happening in our DNA. If a gene has a mutation, there is the possibility of it not being able to carry out its function properly. If the gene is important for key functions of the cell, such as growth or death, mutations may lead to disease.

By sequencing DNA from healthy individuals and those with disease, we can identify mutations that might be causing the disease. To truly understand what the mutations are doing to the cells and to possibly correct the error, we need the help of gene-editing technologies like CRISPR.

Q: What is CRISPR?

A: Clustered Regularly Interspaced Short Palindromic Repeats, also known as ‘CRISPR’, has been steadily gaining media attention because of its widespread application in the biological sciences as a new tool for DNA editing. Interestingly, the CRISPR system is not new. The first hints of CRISPR arose in the late 1980s through studies of bacteria, but its biological function would remain unclear for over 20 years. 

Through the work of many scientists, CRISPR was eventually found to serve as a defense mechanism against viruses that insert their genetic material into the host. The basic components of this mechanism include a molecular guide and a CRISPR-associated protein (Cas) that acts like a pair of DNA scissors. The molecular guide recruits the Cas protein to specific regions of DNA, where it can then cut and inactivate or modify the target genes.

Using nature’s creation, scientists can now study the effects of disrupting the function of specific genes. Modifying the molecular guide to target any region of DNA allows scientists to inactivate specific genes. In the clinic, CRISPR also shows promise to ‘fix’ the DNA of individuals who have one or more disease-causing mutations. To fix the DNA, we can include a corrected gene to serve as a template when the cut DNA is put back together. 

This is a very exciting area of research; however, as Spider-Man’s uncle once said, “With great power comes great responsibility”. Scientists have recognized the ethical implications of editing an individual’s DNA. We’ll explain a little more about CRISPR and discuss the ethics of gene-editing.

CRISPR technology: How it works. With a molecular guide, CRISPR-associated protein, Cas, attaches and cuts DNA at a specific target gene. A corrected DNA template is then inserted into the targeted location, effectively correcting the DNA in that location.

Q: How does CRISPR solve potential health defects? And what are the non-medical reasons for which it is used?

A: CRISPR technology is one of many gene-editing techniques developed over the last few decades. It is one of numerous tools genetic researchers have been using to study the functions of 20,000+ human genes. CRISPR works by inactivating a very specific DNA sequence of interest. This then allows scientists to infer the function of that gene by observing how our cells behave in its absence.

As we mentioned earlier, more recent applications of CRISPR have also begun to be used to introduce very specific mutations into a DNA sequence. This gives researchers a tool to understand not only what the gene does as a unit, but how each specific letter of the gene contributes to the functioning of that gene. It also allows researchers to understand how specific mutations can lead to disease. 

Understanding exactly how a mutation acts has further given rise to the possibility of reversing the mutation in cells to see if this prevents disease development. When those studies were successful, the next idea was to try this in human embryos. This way, an infant genetically predisposed to developing a certain disease after birth could be prevented from doing so. This is the basis of gene-editing research in human embryos. 

Q: What’s the difference between Cas9 and Cpf-1? Is one better than the other? 

A: Both Cas9 and Cpf-1 are CRISPR-associated proteins, which can be programmed to make highly specific ‘cuts’ in DNA with the help of the other components of the CRISPR system, including the molecular guide. Cas9 is currently better characterised and more widely used in CRISPR technology, but Cpf-1 has recently emerged as a viable alternative to Cas9. Depending on the researcher’s goals or experimental design, they may prefer one or the other for their experiment.

Q: Can CRISPR edit genomes of an entire organism? Like all of my cells? Or can it be targeted to specific cells?

A: At present, CRISPR can only edit the genomes of cells in a dish. However, if a fertilized human embryo were to be edited prior to being implanted in a uterus and the resulting foetus was brought to term, all the cells of the infant would technically carry the gene edit.

Q: Has any mammalian embryo been modified and then brought to term? Have changed characteristics/genes been passed on to subsequent generations?

A: Yes, human embryos have been modified genetically and brought to term. In 2018, a Chinese scientist, He Jiankui, edited human embryos to make them resistant to HIV.

This research was conducted secretly and without permission of the university at which he worked. Violating national and international research guidelines, he then implanted them in the mother and two twin girls were brought to term. The infants were sequenced and found to carry the genetic modification in their DNA.

At present, it is still too early to tell if the edit will be passed on to the next generation. 

Q: Yuval Noah Harari, author of Sapiens and Homo Deus, talks a lot about the difference between Western and Asian morals and ethics with respect to gene modifications. Is the West going to be left behind? His premise is that once one group embraces it, the changes will quickly allow them to accelerate their abilities and exponentially better themselves.

A: It is difficult to foresee the long-term implications of different environments and cultural attitudes on scientific progress. However, it should be noted that the act of bringing genetically edited embryos to term drew harsh criticism by scientists across the world and a gene editing conference was organized, in part, to dissuade Dr. He from carrying out this kind of research.

It is also noteworthy that He Jiankui is a physicist and not a biologist, and further, he had the financial means to carry out this experiment without requiring external funding (which is very rare). This means his research was not subjected to the usual ethical and scientific scrutiny typical to these processes. 

He has subsequently been fired from his position at the University and charged with criminal acts by the Chinese government. 

Scientists across cultures have come together to discuss how to best prevent such a thing from happening again. This may result in stricter global ethical guidelines to match the evolving ethical dilemmas we face now.

Another point to keep in mind is that complex traits (like intelligence) are not controlled by a single gene, or even a set of genes. Not only have we not identified the genes involved in many such traits, but they would also be highly influenced by the environment, suggesting the answer to heightened intelligence is likely not as simple as a quick DNA ‘fix’.

Q: What do you think of the ethics of bioengineering the destruction of an entire species like mosquitoes? Or llamas? I hate llamas (lol).

A: The ecosystem may be too complex to attempt something like that. Species interdependence is also something to be kept in mind. CRISPR is best used for biological research in laboratories, and in the future, as a potential tool for medical intervention.

Q: How do we differentiate notions of disease vs. difference?

A: Genetic differences actually contribute to the evolutionary progress of all species. At present, researchers are focusing on using medical technology such as CRISPR to study and possibly prevent disease. Here, disease could be defined as a condition that is detrimental to  an individual’s lifespan and ability to live an independent life. The debate of using CRISPR to modify traits that impact other aspects of well-being may be far more complex genetically as well as morally. 

Q: How do we know it will always work the same way, in every individual? 

A: We don’t know that, because every time gene-editing is performed there could be potential side effects that may vary from experiment to experiment (or person to person). Not knowing the long-term implications of these ‘off-target’ effects is a big reason that gene-editing is not currently safe for use in humans.

Q: How do you deal with something new emerging due to side effects of editing?

A: Typically, we use software that predicts the list of possible side effects from a given CRISPR experiment, based on similarity of different DNA sequences to the region of DNA being targeted by the CRISPR tool.

Often, we choose the CRISPR guides based on the least off-target effects as well as ones that do not specifically tamper with another gene’s activity.

As a safety check, the entire DNA can be sequenced after a CRISPR experiment in order to assess possible off-target effects that were generated by the CRISPR experiment.  

Q: Can new mutations emerge as side effects of a CRISPR experiment?

A: This is absolutely a possibility. As discussed in the question above, the ideal CRISPR experiment is one which doesn’t affect any gene’s functioning except the intended one. However, a mutation introduced via a CRISPR experiment has the potential to remain in the cells and their progeny for generations. It is an important consideration in the improvement of the CRISPR technology.

Q: “Nature always finds a way.” How do you think using an artificial method of creating or erasing mutations will affect this? 

A: The CRISPR experiments in human embryos are currently aiming to target disease-causing mutations. However, the long-term side effects of such a technology are harder to predict.

Q: Do you think an imbalance could be created in the population – too many Einsteins, too many Mozarts, too many Frankensteins?

A: Most population imbalances tend to even out over evolutionary time. It would be both ethically and scientifically demanding to use CRISPR in humans for any reason other than reverse disease-causing mutations, and even that should be carried out with extreme caution.

Q: Who regulates the use of CRISPR technology? Is it governmental or institutional?

A: There are regulations at institutional, provincial, and federal levels governing every research institute in the world. However, scientists are currently trying to come together to ensure international scientific and ethical standards for such experiments in human embryos.

Q: Since CRISPR/Cas is an antiviral defense mechanism in bacteria, do you think we could use it against the novel coronavirus?

A: The novel coronavirus, SARS-CoV2, contains single-stranded RNA as its genetic material, unlike our double-stranded DNA. Interestingly, there is evidence that Cas proteins can cut RNA. Its use against the novel coronavirus is an area of research, but off-target, adverse events remain to be a concern for its clinical application.

Q: What are the big picture implications? The end of cancer?

A: Cancer is such a complex disease that is dependent on so many genetic and extrinsic factors, that it would be extremely difficult to correct cancer initiation by CRISPR. 

At the moment, CRISPR technology is being used to correct simpler illnesses that are more mono-causal and those which can be reversed by correcting a single faulty mutation in the DNA. 

A good example of this is the inherited and incurable disease, cystic fibrosis. It is caused by a mutation in a gene known as CFTR. If both parents carry one mutated version of this gene, there is a 25% chance that their child will have two mutated copies of this gene, leading to cystic fibrosis, which is a debilitating and ultimately fatal disease. Identifying and reversing this mutation in embryos could prevent this illness in families that carry a mutated copy of the gene.

Interestingly, people who carry only one (of two) copies of the mutated gene, do not get cystic fibrosis (although there is the chance of passing it on to their child as mentioned above) and have been reported to be more resistant to cholera. This is a perfect example of genetic variation in nature acting in often mysterious ways, and a good reason to be critical and careful to balance the unknown side effects of using CRISPR technology in medical advancement, while considering its positive impact on health and disease. 

Many thanks to Robert, Amita, Joanne, Urvi and Maitri for your questions! Questions may have been modified for clarity.

Kinjal obtained her doctoral degree from Dartmouth College with a focus on genetics and epigenetics. She is currently a postdoctoral fellow at the Hospital for Sick Children, where she studies the development of a pediatric brain cancer called medulloblastoma.
Julia recently completed her Master of Science degree at the University of Toronto. She is currently working as a research technologist at the Hospital for Sick Children. Her work predominantly focuses on an adult brain cancer called glioblastoma. 


The New York Times: Clue to why cystic fibrosis has survived

Penn Medicine: Early results from first-in-U.S. trial of CRISPR-edited immune cells for cancer patients suggest safety of approach

Washington Post: Chinese scientist who claimed to create gene-edited babies sentenced to 3 years in prison

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