CRISPR: From Humble Beginnings to Changing Humanity

A Science Story

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What started off as a study of the immune system of unique bacteria turned into CRISPR, a powerful gene-editing technology that can accurately and efficiently edit our DNA. What should we do with this technology? How far should we go? How far WILL we go? If you don't know what CRISPR is, now is the time to learn.

Show Notes/Transcription Below

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Show Notes/Transcription

When I was sitting in my genetics class in 2017, I was amazed by the things CRISPR could do. I learned about it in detail. It took up about a quarter of the entire semester. What’s crazy is that if I had gone to school 5 years earlier, it wouldn’t have even been part of the curriculum.

You may have heard of CRISPR, or maybe you haven’t. If you haven’t, it’s inevitable that you would eventually hear about it. That’s because it’s one of the most important biological discoveries ever, but many people who aren’t scientists have never heard of it.

Dr. Eric Lander of MIT and Harvard says that “it is hard to recall a revolution that has swept biology more swiftly than CRISPR.” Today we are going to learn about the story of how many scientists put together the pieces of a puzzle that could possibly change humanity forever.

DNA and how it works.

Before getting into CRISPR, lets get a quick brain refresh on how DNA works. DNA contains the building blocks for humans. There are about 20 thousand different genes that make you who you are. These genes are made up of strings of different base pairs that can be thought of as a chemical computer code. This code is translated into proteins that go and do various tasks in the body to keep us running.

There is for sure a lot more to it, but I think that should be good for now.

What Is CRISPR?

Also, to aid you in this journey I’ll give a broad overview of CRISPR as well. CRISPR is like a genetic map that tells an enzyme called Cas9 where to cut DNA. With it, we can cut out certain parts of our DNA. CRISPR (spell) is an acronym for “clustered regularly interspaced palindromic repeats”

I don’t want to spoil anything, so I’ll leave it at that.

Unlike a lot of the stories behind science, CRISPR’s history is relatively short, only about 20 years. That is, if we don’t take into account all of the research and technology that went into the modern study of genetics.

It All Started In Spain

A Spanish researcher by the name of Francisco Mojica was the first to find the CRISPR location in the DNA of bacteria in 1993. He grew up on the beach and started studying a native bacteria named Haloferax mediterrane.

Mojica examined some of the bacteria’s DNA fragments and found an interesting shape that also had a repeated sequence. With no idea what this meant, the perplexed scientist spent the next 10 years of his career being dedicated to solving this mystery.

Soon after, he spotted the repeats in many other bacteria and came to the conclusion that since it is conserved it must be important. By 2000 he found the sequence pattern in 20 different microbes. Researchers also found other genes associated with CRISPR such as the one that makes the enzyme Cas9 that we talked about earlier.

So, to make sure you’re on track, and honestly myself as well, we are in the early 2000s and we know what CRISPR is along with the genes that are associated with it. It has been found in many different bacteria so we know that it must be important, but we don’t quite know what it does yet.

The Search For CRISPR Matches In Other Organisms

Mojica was becoming an expert in the emerging CRISPR field. He was consistently searching for matches to certain regions of the CRISPR DNA found in his bacteria. There are online databases where you can do this if you ever want a fun way to spend your evening.

He tried this a few times earlier with no luck, but the databases are constantly becoming updated as more and more organisms are studied. One day, he tried again and found a surprising match. Some of the sequence matched a common virus that infects the bacteria E coli.

To give Mojica even more credit, he was doing this all manually. In a week, he had manually typed in about 4500 different sequences online to search for matches. I don’t know about you but there is no way my attention span would last that long.

CRISPR Sequences Match Common Viruses

He also started to realize that the bacteria that had parts of the viral DNA were also immune to those specific viruses. It was at this point that Mojica realized that CRISPR must encode the instructions for bacteria to protect them against specific infections.

The repeats that are part of the acronym are small pieces of DNA from viruses the bacteria have encountered. It’s a little something the bacteria uses to remember the nasty virus by so that it can fight it the next time it comes around. It’s kind of like a database the bacteria can pull from to defend itself.

Then, when a virus comes into the bacteria that matches one of those repeat units in it’s DNA, the bacteria finds the match and sends an enzyme called Cas9 to go chop up the virus. Cas9 is an enzyme that acts like a scissors to the virus’s DNA.

This is truly a beautiful act of nature. All of these bacteria already had a defense to common viruses encoded as a part of themselves. The finding was stunning, and I can imagine Mojica going out with his friends and celebrating with maybe more than a few drinks. Or going to trivia night since, you know, he is a scientist.

Here is another recap to keep us on track. At this point we know that CRISPR is a part of a gene that just repeats itself. These repeats in the bacterial gene match genes of common viruses that the bacteria is immune to. Because of this information, we can infer that CRISPR encodes the bacteria’s defense system against these viruses.

Then began the journey of getting his findings known. Mojica wrote a paper detailing his findings and sent it to the journal called nature, where it was rejected. A few others rejected it as well, saying that it lacked novelty and importance.

Finally, after 18 months of rejections, revisions and reviews, the Journal of Molecular evolution finally accepted the paper and it appeared in public on February 1, 2005. Other researchers had heard about what Mojica was doing and a few other papers started to pop-up, detailing different parts of CRISPR. These also faced the same rejection and trouble getting out to the public.

In case you didn’t know how research gets published, it’s good to know that there are quite a few gate keepers. If your research isn’t able to be accepted by a journal, your career could possibly go nowhere. These scientists, and all scientists really, were taking risks by venturing out into never before seen territory.

CRISPR And Its Cooperation With Cas9

There was a scientist named Phillippe Horvath who was researching bacteria used in making dairy products such as cheese and yogurt. The goal of the research was to learn more about the viruses that frequently attacked their bacteria and also how to overcome these attacks. Understanding how the bacteria protected themselves was of interest scientifically but also economically.

Horvath learned about CRISPR at a lactic acid bacteria conference. A conference about lactic acid bacteria isn’t necessarily something I would willingly sign up for, but I guess you can learn some pretty useful stuff there if you’re a biologist.

He independently found a clear correlation between sequences within CRISPR and viral protection in the bacteria. Horvath also did a lot of research on actually infecting bacteria with viruses. The ones that survived all had CRISPR sequences specific to the viral infection.

After this finding, Horvath and his science buddies studied the role of an enzyme that works with CRISPR called Cas9. It turns out that the Cas9 protein plays an active role in the bacteria’s immune system by going after the virus, but they didn’t know how. This was found in 2007.

And finally, this same group of scientists found that when a virus was able to kill the bacteria, it would only have this ability if the virus had some of its DNA mutated, meaning that the CRISPR/Cas9 system has a specific DNA match with its target, which in this case is the virus’s DNA.

So, this is all great for bacteria. This point in the story is really just us figuring out how bacteria fight off viruses. Nature came up with that way, and it worked. But we figure out the amazing ways nature works all the time. However, not all discoveries about nature have the possibility to change humanity like CRISPR.

Figuring What Each Part of CRISPR Does

It’s time to get into how we move towards humans. A microbial evolution expert named Eugene Koonin and a microbiologist with extra grant money named John van der Oost teamed up. Koonin had already begun work classifying CRISPR

What they did was really interesting. They started taking pieces of the CRISPR/Cas9 system from E.coli and then injected that into E.coli without the CRISPR gene so that they could see what each part did. From this they could tell which parts were crucial and which ones didn’t do as much.

In 2008 they found some very important enzymes that tell us a lot about CRISPR. They noticed that CRISPR is transcribed into RNA that guides the enzyme Cas9 to the target DNA. RNA is pretty much a copy of DNA that the cell actually uses for various purposes.

They then tested this even more in another interesting way. They designed 4 different viruses and then 4 different CRISPR sequences to match the viruses. What happened you may asked? The bacteria brutally murdered the viruses, just as the scientists had planned. It’s really crazy what can be done with modern genetics.

At this point, it wasn’t known exactly what part of the DNA process was targeted by CRISPR, just that the process is disrupted. DNA is almost constantly replicating, being transcribed into messages to be used by the cell. That message is then translated into proteins, and many other things. There were a lot of potential targets for CRISPR.

CRISPR Clips DNA

There were many guesses for where CRISPR does its work, and all but the hypothesis of Luciano Marraffini were wrong. He immediately saw the importance of CRISPR being part of the bacterial immune system and started telling everyone he could about it.

He was eager to move away from his home in Chicago to go work on CRISPR with one of the handful of groups doing research, but his wife just got a good job and he had to stay in Chicago.

It’s easy to forget that the people behind these discoveries are just people too. They have spouses, families, failures, and mistakes. Most stories are a highlight reel of a few scientists that got recognition, but there is much more to the story.

Since moving was out of the picture, Marraffini persuaded a biochemist at Northwestern named Erik Sontheimer to work with him on CRISPR. We knew indirectly that CRISPR targets DNA, but they were the first to do work that showed CRISPR specifically targeted DNA.

They realized that CRISPR was programmable as well. Bacteria normally program it with information from invading viruses to protect them in the future, but it begs the question, what are the broader applications?

‘From a practical standpoint,’’ they declared, ‘‘the ability to direct the specific addressable destruction of DNA that contains any given 24- to 48-nucleotide target sequence could have considerable functional utility, especially if the system can function outside of its native bacterial or archaeal context.’

The paper detailing their finding was published in 2008 the same year as some research we talked about earlier. There was a lot of research exploding at this time. Along with this, they filed a patent to try and get the rights to use this on animals, but ultimately they didn’t have enough research on its use.

Although scientists knew that CRISPR targets DNA, the problem was that it was so efficient at destroying the DNA that we couldn’t really understand what was going on.

The breakthrough in figuring out how CRISPR acts on DNA came when another group involved with the dairy industry was working with a bacteria called Streptococcus thermophilus. In this strain, CRISPR only gave partial protection against an outside viral attack.

This allowed them to examine different stages of the CRISPR process which showed that Cas9 was the enzyme responsible for cutting the invading DNA at very specific points. They came out with their findings in a research paper in 2010

I would recommend watching from 00:00:20-00:01:30

I think it would be helpful for both you and me to wrap up what scientists have found by this point in the story. So CRISPR is just an acronym for a part of certain bacteria’s DNA that matches regions of the DNA for common viruses.

There is an enzyme called Cas9 that uses RNA from the CRISPR DNA to search out those viral matches and then cuts both strands of the viruses DNA. Remember that RNA is just a message made from DNA that is usable. So this whole system serves as the immune system for some bacteria.

We are getting into the weeds so for me it helps to have these more simple summaries of what is going on. And there is a lot going on here. This podcast could go for hours if I got into the genetic techniques or all of the individual discoveries. It’s amazing that a lot of this is happening simultaneously and how fast these discoveries were being made.

Programming CRISPR To Make Cuts Where We Want It To

All of the discoveries leading to where we are now relied on improving technology to give the scientists better and better data. A new technique would come out or a new software, for example, which would allow the scientists to save time and explore new areas that were impossible previously. Genetics is a quickly changing field and sort of like the Wild West of science, among other areas of course.

In July, 2011 a scientist named Dr. Siksnys and his colleagues were able to clone the entire CRISPR/Cas9 DNA and insert it into a strain of E. coli that didn’t have it. It turns out that this was able to make the E. coli resistant to viral attack which means that everything CRISPR needs to works is held inside that DNA.

After the whole process was documented in bacteria, scientists switched to testing CRISPR/Cas9 in test tubes so that they could mess around with different variables and get an even more in-depth look at the process. Sometimes, relying on a little bacteria for everything muddies the waters a bit.

In 2012 Dr. Siksnys started doing this and found specific areas where Cas9 would cut and started changing the CRISPR sequence so that it would make changes where he wanted it to. Essentially, Siksnys found that CRISPR could be programmed to have Cas9 make precise cuts to DNA.

He sent his paper to be published, and like many others, it was rejected for a lack of importance. In hind sight, the editor admitted that it was a very important discovery.

Around the time of Siksnys discovery, A scientist who had been working with CRISPR for a while with a few discoveries under her belt named Emmanuelle Charpentier met another scientist named Jennifer Doudna at an American Society for Microbiology meeting.

These scientists and their conferences, I tell ya. Doudna was a world-renowned structural biologist and RNA expert at the University of California at Berkeley.

She started using this expertise with Charpentier to show similar findings as Dr. Siksnys, which was that CRISPR could be programmed. They even came out with a paper very similar to that of Siksnys about two months later.

Since Siksnys beat them to the punch, Charpentier and Doudna came out with a paper that expanded on the original idea later in 2012. So, the work of these scientists gave a blueprint to how we can practically program CRISPR to edit DNA in a test tube where we wanted it.

The Beginning Of Gene Editing In Mammalian Cells

The elephant in the room at this point was whether or not CRISPR could be used to edit the human genome and then whether or not we should do this. In the 80’s and 90’s, we had already found ways to edit the human genome, but the processes were slow and inefficient so it was that feasible on a large scale.

Feng Zhang moved at the age of 11 from China to Des Moines, Iowa. One Saturday, he got hooked on molecular biology during a small introduction course. By the age of 16, he was working 20 hours a week at a local gene-therapy lab.

He then went to Harvard where he became interested in the brain when a classmate underwent severe depression. Later, he pursued a PH.D. in chemistry under his mentor who was a neurobiologist. They developed a revolutionary technology called optogenetics Without getting too deep into the details, essentially they found a way to use light to activate gene expression which helped neurons fire better.

After this chapter, he wanted to expand his molecular toolbox and started exploring different gene editing technologies. After hearing a talk given about CRISPR in 2011, he, like many other scientists, was hooked.

He was so hooked that the next day he booked a flight to Miami so that he could be a part of a scientific meeting about CRISPR. He spent all his spare time researching CRISPR.

The First Experiments

He had his mind set on coming up with a way to use the CRISPR/Cas9 system to edit human cells. In his early work, he decreased luminescence in human embryonic kidney cells by altering the CRISPR sequence to specifically target a gene called luciferase.

This was a nice way to see that he could be successful because the gene is responsible for some luminescence, in other words it lights up. If it is knocked out, you can easily notice this because it won’t light up as much.

He found that human cells could use this system found in bacteria, although not nearly as efficient as bacteria used it. This isn’t surprising given that many bacterial processes within cells don’t work at all in humans.

Luckily, with all of the research coming out there were many different variations of CRISPR and its components to test to find one that worked better. He found that depending on which organism Cas9 was from, it acted differently in human cells. Remember that CRISPR is sort of like the directions that tells the enzyme Cas9 where to cut DNA.

The First Complete CRISPR System For Mammalians

By the middle of 2012, Zhang had pieced together a system from multiple bacteria that seemed to work pretty well. His system targeted 16 different sites in human DNA with efficiency and high accuracy. What he was doing is cutting out specific pieces of DNA and then another piece would fuse the DNA back together.

Zhang kept improving the system and even went as far as to use it in live mice. He showed that you could target anti-cancer parts of the mouse genome and delete them. When this was done, the mice would develop cancer in a matter of weeks.

You may be thinking this is a bit sinister and we should be working towards curing cancer, not causing it. However, this was extremely important because a lot of our cancer research comes from studying it in mice. Now, we could see how certain genes affected cancer growth and how we could fix it. In science, mice are much more than little pests that infest your lab, but rather crucial for scientific progress. I think we should all thank the next mouse we see.

Zhang eventually submitted a paper and it appeared in the Journal of Science in the beginning of 2013 and it became the most cited paper in the field.

Onward Progress With Mammalian Gene Editing

Other scientists were also working on editing the human genome. A month after Zhang submitted his paper, another scientist named George Church also submitted a paper. He was an expert in genomics and synthetic biology and collaborated with Zhang.

Dr. Church was no stranger to controversy. He had previous proposals to revive wooly mammoths and Neanderthals which was aided by his work in developing ways to read and write our genome on a large scale. He wanted to continue this trend by editing mammalian cells.

Some of his ideas were to genetically edit malaria carrying mosquitoes so that they would die as the DNA spread and to genetically edit pigs for organ transplants so that there would be no transfer of disease. Both of these seem like they would be awesome ideas, but there were arguments against it with concerns of disrupting entire ecosystems along with environmental concerns.

Research kept moving forward with new avenues being explored every day.

CRISPR Gains Massive Popularity

By this point in early 2013, CRISPR was going viral. Google searches on the topic skyrocketed, a trend that continues to this day. If you want to look at something interesting, go to google trends and type in CRISPR, remember that it is spelled CRISPR.

Then set the settings to about 10 years. IF it were a stock and you invested in the beginning, you’d be a trillionare today and probably a quadrillionare in not too long, if that even exists.

By 2014, the genomes of many organisms had been edited, yeast, nematode, fruit fly, Zebrafish, mouse, and monkey. The elephant in the room was and still is what can be done to humans, and also what should be done to humans.

Well, scientists have started to test CRISPR on human embryos. As of 2015, this has been done more than a few times, but some of the largest science organizations around the world made the decision that only changes that won’t be passed down can be made.

However, in 2016 we saw the first clinical trials using CRISPR/Cas9 as a cancer therapy. It focuses on using the patient’s own T-cells to target cancer.

So, What Do We Do?

Now, the whole scientific world is aware of CRISPR and more and more research is being done to push it forward. It is up to all of science to decide what we do with this powerful technology. It has been used in a few cases to cure terrible diseases formed when babies are in the wombby deleting the genes causing the problem. However, many are afraid that we can go too far and start creating designer babies.

Want your child to be 6”2’ with brown hair, blue eyes, prominent cheek bones and a predisposition to be good at math? Coming right up!

I think that there is a line that we need to draw to make it clear what we can and can’t do. Obviously, if we can halt diseases, let’s do that, but maybe stop far short of changing the child’s physical and mental characteristics. We can also use this power in other ways such as to change agriculture so that crops are stronger and have much more yield.

The story of CRISPR teaches us a lot. It is yet another story of a few dedicated scientists and cooperation between countries and disciplines that changed the world in ways that we don’t even know yet. As it gained more and more popularity, the number of scientists willing to take a risk and study CRISPR skyrocketed. It also teaches a valuable lesson in that sometimes, the most important findings come from the most unusual places.

What started off as a study of the immune system of certain bacteria turned into a technology that can edit the DNA of humans which may be one of the biggest discoveries ever. This may be a bit of a stretch, but to me it shows that no matter how insignificant, don’t take any part of your own life for granted. Know that the next person you meet or the next opportunity you take advantage of may have the power to change your life.

References

https://www.broadinstitute.org/what-broad/areas-focus/project-spotlight/crispr-timeline

https://www.broadinstitute.org/files/news/pdfs/PIIS0092867415017055.pdf

https://www.sciencefocus.com/science/who-really-discovered-crispr-emmanuelle-charpentier-and-jennifer-doudna-or-the-broad-institute/

https://www.sciencenewsforstudents.org/article/explainer-how-crispr-works

http://www.crisprupdate.com/crispr-timeline/