What is CRISPR? A bioengineer explains

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Over the past decade, CRISPR has taken the biomedical world and life sciences by storm for its ability to easily and precisely edit DNA. Here, Stanford University bioengineer Stanley Qi explains how CRISPR works, why it’s such an important tool, and how it could be used in the future - including current developments in using CRISPR to edit the epigenome, which involves altering the chemistry of DNA instead of the DNA sequence itself.

"CRISPR is not merely a tool for research. It’s becoming a discipline, a driving force, and a promise that solves long-standing challenges from basic science, engineering, medicine, and the environment," said Qi, an associate professor in the Department of Bioengineering and institute scholar at Sarafan ChEM-H. "Together, we can think innovatively about how to match needs with technologies to solve the most challenging problems."

In 2019, Victoria Gray was the first person in the U.S. to receive CRISPR treatment for a genetic disease (sickle cell anemia). Now, CRISPR-based therapies are approved in the U.S. and the U.K. What is next?

Were you surprised when the 2020 Nobel Prize in chemistry went to CRISPR’s developers?

Besides treatment for diseases, what are other real-world applications for CRISPR technology?

Your group demonstrated that it’s possible to shrink CRISPR. Why is this significant?



The short answer: CRISPR is an immune system used by microbes to find and eliminate unwanted invaders.

Qi: CRISPR stands for "clustered interspaced short palindromic repeats." Biologists use the term to describe the "genetic appearance" of a system that was discovered in microbes - including bacteria and archaea - as early as 1987. For a long time, no one really understood what it did, but around 2005, researchers discovered CRISPR is an immune system. It’s used by microbes to help protect themselves from invading viruses. To stop the invaders, the microbes use CRISPR to recognize and eliminate specific trespassers.

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The short answer: When a virus or other invader enters a bacterial cell, the bacterium incorporates some of the trespasser’s DNA into its own genome so it can find and eliminate the virus during future infections.

Qi: It’s similar to the human immune system. When a virus infects us, we generate an immune memory in the form of antibodies - lots of them. Then, when the same virus infects us again, these antibodies quickly recognize the invaders and eliminate them.

When a virus infects a bacterial cell, CRISPR helps establish a memory - a genetic one. The bacterium takes a piece of the virus’s genome and inserts the DNA into its own genome. From that newly acquired DNA sequence, CRISPR creates a new "guide RNA," a sequence that helps CRISPR find the invader via sequence complementarity (i.e., A binds to T and C binds to G). So, the next time when the virus infects that bacteria cell, the guide RNA rapidly recognizes the virus DNA sequence, binds to it, and destroys it.

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The short answer: Gene therapy can mean using CRISPR as a macromolecule drug to either fix a mutated gene or regulate a defective gene to treat a disease. Cell therapy means using CRISPR to make your body’s cells attack toxic cells or regenerate beneficial cells.

Qi: Gene therapy can mean two things: One is to fix a mutated gene, and the other is to regulate a gene’s expression into protein products. Our current understanding of gene therapy is still rapidly advancing, and the challenge is managing therapy safely and cheaply. Furthermore, we’re only looking at the simplest genetic diseases. For example, sickle cell anemia is a disease we know a lot about, and it’s often caused by a single mutation. So, we can configure CRISPR to fix it. But many more diseases are caused by widespread mutations, multiple mutations, and even multiple genes. In the future, gene therapy could go beyond a single mutation, and I am optimistic that in the next decades, gene therapy will become a pillar of medicine.

Cell therapy is a little different. For example, when people try to treat leukemia, a type of white blood cell tumor, sometimes chemotherapy drugs can’t completely get rid of the tumor cells. In the past two decades, scientists have found that if they retrieve some of the patient’s T cells, which fight infections, these cells can be engineered as better fighters to recognize and eliminate tumorous cells. When the modified T cells are injected back into the patient, they can attack the tumors. However, cells are quite complicated. Sometimes, they go out of control when injected back into the patient, killing healthy cells along with the tumor cells. At other times, they may fail to work because they are suppressed by the tumor cells. CRISPR offers a powerful tool to enhance the efficacy and safety of these immune cells so that they are completely under our control for best clinical benefits.

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The short answer: CRISPR is much easier to program than other tools.

Qi: Before CRISPR, most gene-editing tools were a single protein. By changing the peptide sequence of these proteins, scientists could alter their targets. To change the target, you need to completely redesign the protein’s sequence and then test if it even works, which is tedious, unpredictable, and time-consuming. These gene-editing tools were theoretically interesting, but they were difficult to use for large-scale studies and therapeutics.

Compared to that, CRISPR is elegant because the target recognition sequence is mostly encoded within an RNA rather than a protein, and redesigning this sequence is one of the simplest things you can do in molecular biology. It makes genome editing similar to operating a GPS: If you want to go to destination A, you just type the address, and to change to destination B, you just enter the new location. So, this tool dramatically reduces the burdens, cost, timing, while increasing the precision and accuracy of a gene-editing system.

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The short answer: CRISPR can precisely modify a piece of DNA or its chemistry (so-called epigenetics) in the human body, making it a potential tool for clinical uses in the biomedical sciences.

Qi: CRISPR is a molecule and tool desired by everyone who works in the life sciences, biomedical research, and clinical settings. Its high precision is unparalleled and enables many uses including gene therapy.

My dream has been to develop new biotechnologies and apply them to diseases without a cure. Genetic diseases make up a big part of this category. Traditional medicines - small molecule drugs, surgery, and other methods - don’t work for these types of diseases. But CRISPR molecules have become highly promising as treatments because they allow us to precisely modify a piece of DNA in the human body. This could lead not only to relief but also to a cure.

Indeed, recent FDA approval of the first CRISPR drug, Casgevy, in treating sickle cell anemia and beta thalassemia speaks to its safety and potential for other diseases. Sickle cell anemia is a disease in which people have a mutation in their red blood cells. Normally, there’s no treatment other than frequent blood transfusions or bone marrow transplants from a matched donor, which are expensive and damaging to a patient’s overall health. Using CRISPR, it’s possible to perform a one-time treatment to permanently correct the mutation. There are more than 8,000 genetic diseases like that, which can be potentially considered.

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The short answer: In about a decade, scientists went from wondering if this technology would even work in human cells to getting the first CRISPR drug approved uses in the clinic.

Qi: In 2010, I was working on CRISPR as a bioengineering graduate student at the University of California, Berkeley, under Adam Arkin, a synthetic biologist and bioengineer, and collaborated with Jennifer Doudna, a biochemist and structural biologist. In the early days, CRISPR’s practical usefulness was not very publicly recognized. At that time, many counterarguments said CRISPR was just a bacterial system and most of these simply don’t work in human cells - which, to be fair, is true.

But after Jennifer Doudna and Emmanuelle Charpentier published their seminal 2012 paper on Cas9 - one type of CRISPR that cuts DNA using a single protein and an engineered single guide RNA - the research and published papers grew exponentially. Firstly, because it’s a system that everyone in the life sciences wants. Secondly, using CRISPR is super easy, flexible, and robust. It’s not like other technologies that take multiple years and millions of dollars to set up - CRISPR only takes a couple of weeks and a bit more than a few hundred dollars to set up now.

A lot of researchers significantly contributed to the rapid development. For example, within three years following its initial demonstration, structural biologists solved the high-resolution, three-dimensional structure of what Cas9 and other CRISPR proteins look like. Bioinformaticians have revealed many new species of Cas molecules beyond Cas9, many of which have novel functions. Biochemists engineered CRISPR to understand how fast and tightly it binds to DNA. Bioengineers, including me, engineered the proteins to make them work more efficiently and more specifically so they can work better in the human body for gene therapies. Also, clinical researchers started to use the tool to address particular diseases.

Furthermore, the applications of CRISPR went beyond gene editing. Epigenetic editing is an exciting development, although we still await clinical benefits. It was used for targeting the human 3-dimensional genome, visualizing the DNA dynamics, or even targeting another set of molecules, RNA, for gene regulation.

I don’t think I’m exaggerating to say that, essentially, CRISPR has been tested as a potential treatment option for every disease that we have clear knowledge about. CRISPR can’t solve all’of them, but because this tool is so powerful, easy to use, and so far-reaching, it has allowed everyone to combine their expertise with CRISPR.

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7. In 2019, Victoria Gray was the first person in the U.S. to receive CRISPR treatment for a genetic disease (sickle cell anemia). Now, CRISPR-based therapies are approved in the U.S. and the U.K. What is next?

The short answer: This is very exciting. Future CRISPR drugs will address more incurable diseases, which provide a test case for CRISPR’s efficacy and safety in different organs and patients.

: I’m super excited to see CRISPR becoming a drug to treat a disease as a one-time cure. When CRISPR first came out, there were concerns about whether these bacterial molecules could be used safely in humans and whether it was safe to cut and edit human DNA. While there are still questions regarding long-term effects (beyond the period of clinical trials in tested patients) it is very encouraging that CRISPR is safe and effective.

The next step is to expand the scope of CRISPR drugs. Medicine isn’t made in one day. Different diseases are caused by different mechanisms. There are already more than dozens of CRISPR clinical trials for different diseases in the liver, immune cells, eyes, and muscles. Furthermore CRISPR epigenetic editing is expanding the scope of disease to treat more types of muscular dystrophy, retina disorders, and brain diseases.

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8. Were you surprised when the 2020 Nobel Prize in chemistry went to CRISPR’s developers?

The short answer: Not at all. But I hope the award doesn’t lead people to think CRISPR research is finished - it’s still growing, and there’s much more to explore in basic research, medicine, and beyond.

Qi: I’m not surprised at all. Even before 2020, researchers had been discussing when the Nobel Committee would recognize CRISPR. So, when it happened, I was super excited.

Jennifer Doudna (University of California, Berkeley) and Emmanuelle Charpentier (Max Planck Unit for the Science of Pathogens) received the Nobel Prize in Chemistry only seven years after CRISPR was first reported as a molecular system for modifying the human genome.

I hope that giving the Nobel Prize to CRISPR won’t give people the impression that the genome editing field is done. This is a field that’s still growing in every corner of life sciences. Besides being explored as medicine in humans, it is expanding its influence in plants, microbes, and difficult-to-engineer organisms such as fungi. There are so many questions - about how we can use CRISPR for safely controlling the genome, how to use it for novel and innovative research, and how to make it a clinical product - that still need to be explored.

These are exciting frontiers of further increasing the safety of CRISPR-based therapies and expanding the scope of diseases treatable by this technology.

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9. Besides treatment for diseases, what are other real-world applications for CRISPR technology?

The short answer: Some other uses are diagnostics, manufacturing, sustainability, and ecological engineering.

Qi: CRISPR can be used for diagnostics. It has been developed as a way to sensitively detect pathogens in the environment that are affecting our bodies.

There are also opportunities in manufacturing, such as making products that we care about using organisms like yeast and bacteria. Imagine that we could use CRISPR to engineer new microbes that could boost production - like 10x more beer, for instance. And also, beer that tastes much better and can be catered to different people’s wants and needs.

Sustainability is also a big application for CRISPR via bioengineering. Creating sustainable, carbon-neutral methods of energy or food production is a challenge. Genome engineering may offer better manufacturing protocols through microbes that reduce greenhouse gases, plastic, and food waste.

Finally, we get to ecological engineering. For example, people are trying to eliminate certain invading or pathogenic mosquito species using CRISPR, but in my opinion, its long-term safety and impact still need careful evaluation. Other people are trying to revive extinct species. Recently, scientists announced they were trying to revive a woolly mammoth that can live in the Arctic cold.

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10. What are your views on some of the ethical concerns surrounding CRISPR?

The short answer: My research group often thinks about the ethics of CRISPR. Some ethically questionable areas are disease prevention and eliminating pesky species, and some definite unethical areas are enhancement and creating designer babies.

Qi: The ethical side of CRISPR is something my research group thinks about every day. One of the fundamental principles of ethics is to do no harm. Sure, we want to do something great and helpful to people, but at the same time, we have to consider if we’re harming other people. Using that principle, we can consider a few cases.

One example is a designer baby, which is a scary topic. That is regarded as unethical because this may create a new human species. When the germ cells - sperm and egg cells - are edited, this not only affects that single person, but also the children that person could have in the future.

Another concern is in the division of treatment, which has three categories: cure, prevention, and enhancement. Curing someone’s disease is great. Prevention, which means someone is at risk of developing a problem, is a gray area. If someone has a high chance of getting an infectious disease, should we use gene therapy to permanently modify their DNA to reduce their risk? That question really depends on if we have other options. The last category - enhancement - is likely unethical. People talk about the possibility of targeting a gene to grow more muscle or make people smarter or better looking. But if research goes into this category, only some people may be able to afford it. This could amplify the imbalance of socioeconomic status. Another facet to consider is medical necessity. Is the therapy really necessary, or are there other ways to solve the problem through currently available drugs, diet, exercise, etc.’

Beyond medicine, some scientists may want to use CRISPR for ecological reasons, for example, eliminating mosquitoes. From my viewpoint, that’s controversial because I think every species exists for a reason. If we try to eliminate mosquitoes, we might have a chain reaction that affects other life forms in the environment and can be irreversible. I hope in the future we can make this technology reversible like installing a switch so that if we make something that turns out to be less than ideal, we still have some way to reset it.

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11. Your group demonstrated that it’s possible to shrink CRISPR. Why is this significant?

The short answer: It’s tricky to deliver CRISPR molecules into cells. Shrinking the size of the molecule helps it easily traverse inside of cells and get to its DNA target.

Qi: CRISPR is such a magic molecule, but that magic only works if CRISPR gets inside cells and touches the DNA. The question is obvious: How can we even make CRISPR get inside the cell?

Human cells are designed to resist any invading DNA. So the human body has many strategies to prevent foreign DNA from getting in.

Many delivery methods scientists used have limited power. We can use retooled viruses to deliver clinical products into cells, but they have a small capacity - the Cas9 version of CRISPR usually doesn’t fit inside the virus. Therefore, the currently approved CRISPR drug requires isolating patient cells, modifying them, and putting them back in. This process is costly and slow. If we want CRISPR to become a broadly useful medicine, then we need to make the molecule as small as possible.

That’s why we made this miniature CRISPR, which we call CasMINI , which is only half the size of Cas9. We also saw that it is easier to enter cells and works better than other CRISPR molecules because it can get inside more efficiently. This miniature CRISPR can revolutionize the way that we can perform editing in the body. Our hope is to address these technical barriers then test how miniature CRISPR can be delivered to different parts of the human body to treat various genetic diseases.

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The short answer: We’re trying to use CRISPR to control gene function rather than editing genes to treat diseases.

Qi: I’m excited about exploring how to treat diseases without modifying human DNA through epigenome editing. It’s a different way of thinking about gene therapy. Unlike gene editing, epigenome editing is reversible, safer, and promising for complex diseases that can not be easily targeted by gene editing.

To enable epigenome editing, we developed the first nuclease-deactivated dCas9 in living cells, to programmably target and control gene expression, without altering the DNA sequence. For example, if a person doesn’t have enough properly working proteins, we can use epigenome editing to increase the gene expression over a long term to make more proteins to compensate for this deficiency problem, thus restoring the function to normal in patients.

In other cases, someone may have a gene mutation that produces a toxic product, such as in many muscular dystrophies or neurological degenerative diseases. Rather than using CRISPR to modify DNA, we can use our epigenome editing technology to permanently silence the gene without modifying the DNA. I am excited to test this solution in the clinic as I believe this offers a safer strategy for treatment without altering DNA.

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The short answer: There are limitations to gene editing, but new technologies are trying to expand the power of CRISPR.

Qi: One major limitation is we’ve been using it for only 10 years. Often, time is the best test of all technologies. Only by collecting data over enough time in all scenarios will we be able to understand everything about these technologies, like how safe they are over the long term.

In testing in human subjects with patients, even though we didn’t see off-target effects or immune responses, there are still question marks. We still need to constantly improve our understanding, as well as CRISPR’s accuracy and precision in different human tissues and different patients, when treating a problem.

Also, right now, CRISPR is mostly used as molecular scissors to cut DNA. But sometimes, the problem gene’s affected function isn’t caused by a DNA mutation. Sometimes, it’s a gene turning on or off abnormally that causes the problem. So in that case, CRISPR shouldn’t be used as molecular scissors to cut DNA, but rather as a switch to restore the gene to work properly. Epigenetic editing tools can well address such challenges.

CRISPR is like a powerful hammer. But the question is: Where is the nail? What is the most suitable nail to work on? For example, as of today, we still don’t know for sure which gene causes Alzheimer’s disease in many patients. To use CRISPR, we need to know which gene to target and which cell is the destination. We also need to know when to perform the treatment - sometimes treatment can only be done in an early stage of a person’s life.

Another big issue is the high costs associated with the current CRISPR medicine. How to reduce cost is a major question. I’m glad that there are active conversations between academia and industrial partners to have multiple experts in the same room to come up with the best solution.

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The short answer: It could help improve the quality of life as we age, engineer useful organisms, and even serve as a universal vaccine against viruses.

Qi: I’m excited by CRISPR possibly helping anti-aging, but less in the sense of making people live longer. No one can escape aging, and it’s a huge burden to our healthcare system and decreases the quality of life. My hope is that in the future, CRISPR isn’t just being used to save lives, but also to improve the quality of life when people age.

I also hope CRISPR can become a way to engineer a lot of useful life forms. For example, there are microbes that can capture solar energy and convert it to electricity, and maybe those could be used to produce sustainable energy. Additionally, we could engineer food that’s more nutritious, prevents obesity, and so on.

Another application could be vaccines. Even now, infectious diseases, like COVID-19, have dramatically changed everyone’s lives, which is unbelievable. So another dream is to develop cheap and safe genetic vaccines to fight all viruses, since that’s their original role in bacteria. And maybe, in the future, we could receive a small dose of CRISPR that could completely kill any new virus. It’s not easy, but given that this genetic system was designed as an antiviral system, there’s a chance this could work.

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15. How far are we from actually achieving those idealistic future goals?

The short answer: We’re close to some goals but may be far from some other idealistic goals.

Qi: When it comes to CRISPR and achieving those big dreams we have for it, we’re at different stages. For some goals, it might feel like we’re just starting out, but for others, we’re getting pretty close. For example, I’m really excited about how we’re starting to use CRISPR in real-life treatments for diseases, such as sickle cell anemia. This is a big step forward! I am also very excited about CRISPR epigenetic editing, a way to turn genes on or off without changing DNA sequence, which is getting ready for its big moment in clinical trials.

The reason we’ve come this far is thanks to a lot of people who believe in the power of safely editing our genes to make us healthier and are working hard every day to make that a reality. It’s their passion and the demand for these solutions that keep pushing us forward. I’m optimistic that many of the things we’re dreaming about with CRISPR could become real, sooner rather than later.

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