Ep 41: Discovery, Development, and Cures - The CRISPR Saga

You may think that the CRISPR-Cas genome engineering tool was developed from simple human ingenuity. And while its development certainly involved ingenuity, CRISPR was birthed in the wild. As a product of nature, it has an essential function in its native host; spoilers: its natural function involves defending its host from attacks. From the initial discovery in nature to its adoption as a gene therapy tool it took decades of twists and turns, ups, downs, and yes, ins and outs too. 

In this episode of We Love Science Podcast, Fatu and Shekerah welcome special guest, Irina Gostimskaya, as the CRISPR Chronicles continues. Together, we take a CRISPR walk through history, discussing the men and women who made it possible to bring CRISPR therapies to the world through their curiosity, perseverance, and a sprinkle of good and bad luck.

Tune in to the episode to learn about:

·        The cliffhanger that showed the world the first CRISPR sequence 

·        The real purpose of CRISPR in the wild 

·        How scientists assembled the puzzle pieces to use CRISPR for Gene editing 

·        The CRISPR patent dispute

·        Bonus Food Science Content: Learn all about Russian cuisine

For more information on this topic, visit our website: WeLoveSciencePodcast.com

Reach out to Irina: gostimskaya@gmail.com

Read Irina’s excellent CRISPR review article: CRISPR–Cas9: A History of Its Discovery and Ethical Considerations of Its Use in Genome Editing. Biochemistry Moscow 87, 777–788 (2022).

Jump directly to the next episodes in the CRISPR series:

• Ep 47:  The CRISPR Cure for Sickle Cell Disease

Other Great Episodes:

• Ep 38: The Secret Behind CRISPR Gene Editing
• Ep 35: How CRISPR Gene Therapy is Bringing Hope to the World
• Ep 28: Yogurt Making - DIY Style - A Food Science Short

Reach out to Fatu:
www.linkedin.com/in/fatubm
Twitter: @thee_fatu_b
and LoveSciencePodcast@gmail.com

Reach out to Shekerah:
www.linkedin.com/in/shekerah-primus
and LoveSciencePodcast@gmail.com

Music from Pixabay: Future Artificial Intelligence Technology 130 by TimMoor
Music from https://freemusicarchive.org/music/Scott_Holmes: Hotshot by ScottHolmesMusic

Transcript

Shekerah Primus  0:00  
What can you do with your love a science, we'll tell you

Hello, hello, everyone. Thank you for tuning in to another episode of your favorite podcast. We love science. We are your hosts. I'm Shekerah

Fatu Badiane-Markey  0:44  
And I'm Fatu, and today is another all CRISPR episode and I'm super excited.

Shekerah Primus  0:51  
Yes we are. Yes it is. The CRISPR Chronicles continue today. So today we're going to talk about how it all started and how we got to using it as a gene therapy. So we'll take a CRISPR walk through history, see what I did there. So chatting with us today is a special guest contributor, Dr. Irina Gostimskaya, who is joining us from the University of Cambridge in the UK. Irina is a scientist and the author of one of my favorite CRISPR review articles that I read when I was preparing for this series. So Irina, welcome, welcome. Thanks for joining us. I'm so happy that you could come on today.

Irina Gostimskaya  1:38  
Thank you. Thank you. Hello to both of you, and everyone listening. Thank you for having me. I'm really excited to be participating in your podcast today.

Shekerah Primus  1:50  
Excellent, Wonderful, so. Yeah, so I know that today's topic is right up your alley, right. You did a whole lot of research into this. But before we jump in to CRISPR How about we do a little bit of food science guys, what do you think? Yeah. Okay. So I know that you're originally from Russia, and I've never been to Russia and I don't think you have either Fatu. Have you been to Russia?

Fatu Badiane-Markey  2:19  
No, I have not, I have not. 

Shekerah Primus  2:21  
Yeah. Okay. So neither of us had been to Russia. So we're both really curious. I'm curious for sure. About the food there. So can you tell us a bit about what the food is like in Russia? And what is your favorite Russian like dish or dessert?

Irina Gostimskaya  2:40  
Yeah, so I was born in Russia and spent my childhood there so of course, I'm used to Russian cuisine. That's my you know, what you grow up with is what you then crave later in life often. Yeah. Well, I mean, the cuisine itself is just basically meat and vegetables. And of course, because weather is cold in Russia, vegetables, you know, only certain types we can grow not, not other types. There's a lot of potatoes and carrots. And cabbages and onions and garlic. So that those are the normal normal ingredients for Russian cuisine. Many called soups, stews, and but no spices practically no chili some black pepper if you're lucky. And people use mustard and horseradish for flavoring.  Yeah, so because I like because we're gonna gherkins and sauerkraut. So it's common for Eastern European cuisine. I buy it here in the UK as well. Those in certain shops that can find the right things for me that remind me of my childhood. And then also interestingly, also, bread is quite often rye rather than wheat because wheat doesn't grow in the in the north of Russia only in the south. It's not so common. Yes. And so rye bread I quite like that. And also some nice dairy products. I'm really happy with being able to buy here in the UK occasionally in certain shops. So Kefir it's yes, yeah. So I think of it as a hybrid between the yogurt and the beer.

Shekerah Primus  4:36  
It's like the best of both worlds right.

Irina Gostimskaya  4:48  
Yeah, apparently I was just reading about it the other day, up to 30 different microbes make it happen. So bacteria yeast. Yes, it's full of probiotics. It's the same bacteria that make yogurts and sour cream, but there's also yeast cultures. So it's an interesting one. Yeah.

Shekerah Primus  5:09  
Fatu, Maybe we need to do a kefir episode, right. We did a yogurt episode.

Exactly.

Fatu Badiane-Markey  5:17  
I was just thinking that

Irina Gostimskaya  5:18  
yogurt is mainly Lactobacteria. Bifidobacteria, Streptococcus, I think, yes, and kefir is even more bacteria and yeast. Then it's a bit more acidic and it can get even fizzy if you overferment a little bit. I Like it yes I like it and then there's tvorog so that's a soft cheese and I make fried pancakes from it. Some sort of cheese cakes but they're fried

Shekerah Primus  5:20  
Oh, interesting, fried cheese pancakes, interesting

Fatu Badiane-Markey  5:54  
That sounds really good.

Irina Gostimskaya  5:54  
And then I've been in Britain for a while now. So I'm used to fish and chips, and curries. so we have a lot of fantastic shops from India, Pakistan, Bangladesh, Nepal, you know, my favorite curry place in Manchester was a Nepalese curry actually and then Cambridge. Yeah. And then Cambridge I found the dosa place which is amazing you know dosas the crispy pancakes

from South India I think, I love the Indian cuisines so yes, fish and chips is good, but I think curries are my favorite

Shekerah Primus  6:31  
Yeah, I agree with that. you're speaking my language with the curries . Because I love curries too.

Yeah, 

So do you know how to make them? 

Irina Gostimskaya  6:45  
No.

Shekerah Primus  6:47  
That's fine.

Irina Gostimskaya  6:49  
 Initially, when I came to Britain, I thought they were too spicy. You know, I couldn't really handle a medium curry. I would go for the mild or the mildest of the mild. Yes, because I was not used to chili. And then slowly slowly, build up Resistance and addiction as well

Fatu Badiane-Markey  7:07  
I like that

Shekerah Primus  7:11  
Resistance and addiction, Oh lord those 2 go hand in hand don't they

Excellent. That was great. Thank you for sharing that with us. I'm also curious about the desserts in Russia. I know you mentioned yogurt, kefir. Do you have any like sort of sweet desserts?

Irina Gostimskaya  7:37  
Yeah, those so those fried cheesecakes that they make they're sweet normally they're quite like something in between the pancake and the cheesecake. They are quite traditional. Yeah, just normal European style cakes. You know, chocolate cake. Yeah. My mother used to bake for me. I don't really bake. I think it's really yeah it's really tempting. If you have this warm cake you know if you end up eating too much and I'm feeling guilty. I prefer buying my dessert small one at a time because otherwise I wouldn't stop.

Fatu Badiane-Markey  8:23  
That's a very practical way to do it Irina, I like that.

Shekerah Primus  8:34  
Excellent, ladies.

All right. So let's jump into CRISPR for today. So Irina, I know you're trained as a scientist as a biochemist. So can you tell us to begin how you became interested in CRISPR?

Irina Gostimskaya  8:55  
Yes, so actually, it was my COVID project. The labs were closed down for a while and I had a bit more free time because of, you know, saving it on commuting and other normal activities that people do like going out and meeting people face to face. And so I thought, Okay, I'll read a bit more about how CRISPR was discovered and linked interesting questions around it. At the time, I was just trying to find the suitable tool for in vitro DNA manipulations. But then as soon as I started reading about the history and the genetic engineering overall and about the ethical issues behind it, I just ended up reading quite quite a lot and papers and talks, and I watched interviews that are there. Online and then in the end, I wrote my own review describing the the history of CRISPR discovery and also how it can be used for genetic engineering, plus all the moral and ethical issues that come with this power.

Fatu Badiane-Markey  9:56  
Wow, that's really fantastic Irina,

Shekerah Primus  10:01  
the vaunted review 

Fatu Badiane-Markey  10:02  
Yeah.

You know, I like how it started, you know, as you know, like okay, we have the pandemic, everything's shut down. So let me read and like, study and do some more research. And then you discovered so much more about CRISPR. I like that. I really like that.

Shekerah Primus  10:20  
Yeah, 

Irina Gostimskaya  10:21  
yes, I think if it hadn't been for the pandemic, I probably would have just read the necessary practical details of how to use CRISPR cas 9 system for cutting DNA or for editing DNA. But what's a bit more time I just got interested about everything around it and then and then I was invited to write the review and I thought, Well, why don't I just write it about CRISPR cas 9 and things that are hopefully interesting to other people as well? 

Fatu Badiane-Markey  10:48  
Yeah,

Shekerah Primus  10:48  
Yeah, wonderful. I love that story. Right? That's like, one positive thing that came out of the pandemic. That's a great story. Thank you for sharing that with us. So I know that you'll be sharing some of that all of that knowledge that you learned with us today and with our listeners as well. So just a quick recap for our listeners. In the first two installments of the CRISPR Chronicles, we gave just an overview of CRISPR as a gene editing tool. We talked about how it works. So how is it able to change DNA, right? And we discussed a bit about the impact that it's already having in people's lives because of the power of this system to cure genetic diseases like sickle cell disease. So if you missed those two episodes, we will put a link in the show notes so you can easily find them. So in today's episode, we're going to take a step back to look at CRISPR's origin story, those of you listeners who have been with us for a while know that we love origin stories. So how did it all start? What were the surprises and the big aha moments in the field? And also some of the incorrect theories, right, because that's always a part of science and, you know, happens in science. And then lastly, we'll talk about who won that race to develop CRISPR into a life saving therapy or to develop CRISPR for use as a genetic engineering tool. Sound good to you guys.

Definitely.

So CRISPR is known today as a powerful tool for genome engineering. But like most tools in biology CRISPR was not created from you know, the genius of the human mind. Like some people might think, right. So in fact, it was discovered in nature just in another living organism in bacteria. To be exact. And I think a lot of people would be surprised at just how many of the tools we use today in biotechnology were first discovered just by doing basic research in these single celled organisms in bacteria. It's pretty awesome.

Fatu Badiane-Markey  13:09  
No, totally. I think that's one of the things that I really love about biology. It's like we have this sort of, you know, very, like, reuse and you know, kind of, like reapply mentality. So it's like nature has already thought of these really neat things right? And it's kind of up to us to put together the clues and figure out other ways to use some of these like really, I think novel you know, like mechanisms. Almost like detectives like little little little bio-detectives. Yeah, yeah. So I think you know, my favorite example of that is always like, luciferase from fireflies and how we use that as an indicator in science for other things. Yeah. Or like GFP, which is a fluorescent protein from jellyfish. Sowe also use that. Yeah. So so many examples. Yeah, but yeah, in the case of CRISPR Shekerah you said it was first discovered in bacteria. So who discovered it? 

Shekerah Primus  14:07  
Yeah, yeah, I love those two examples you just gave by the way, because luciferase and green fluorescent protein, those are both so cool and so fun. And young kids who are just you know, learning science they always love playing around with it and seeing the you know, the bright colors, of course, so, exactly. Those are some fun examples. Exactly. Alright, so CRISPR so, I really love this origin story, because the scientist who very first discovered CRISPR, who first published a CRISPR sequence, wasn't even studying anything close to CRISPR at the time, right? And so I always love and these kinds of things happen in science when you just sort of find something by chance, and then it turns out to be so amazing. So I know that you know a lot about this story too Irina, so how about I'll get us started with it and then you can finish telling the story How does that sound?

Irina Gostimskaya  14:56  
Yeah sure, yes. Yes.

Shekerah Primus  14:59  
Okay. Okay. So, the first time that a CRISPR sequence was reported in the literature, was back in 1987. And this was by a Japanese scientist, named Okay, I hope I'm not going to mess up his name Yoshizumi Ishino. And he was a postdoc at the time studying E. Coli. And so as we know, E. coli is a type of bacteria. And if you've ever gotten food poisoning, it could have been from E. coli because it's one of the bacteria that can cause food poisoning. So just to put the year in context, a little bit here so 1987 I was already born Yay.

Fatu Badiane-Markey  15:36  
Me too

Irina Gostimskaya  15:39  
me too

Shekerah Primus  15:45  
But that's not the context I wanted to say. Right? That's just aside we were we were in the world. But that was about a decade after Sanger sequencing was first invented. And so this was the start of the genetic sequencing era. Right. So scientists were really, really excited to have an easy way to sequence DNA. So they were sequencing as many genes and as many, like smaller genomes like bacterial and archaeal genomes, as many of those and as quickly as they could, right. So now I say quickly, because these days to prepare and send a piece of DNA out to be sequenced and receive your sequence back, it's about maybe two days depending on what you're doing. And I'm sure some places have same day service as well. Right. But back then, that took months. That took months but still, it was easier and quicker compared to what was available at the time. So it was the start of the genetic sequencing era, scientists are sequencing genes like mad. And so Dr. Ishino was studying a gene in E. Coli. Um, it was an enzyme, I believe it was a phosphatase enzyme that he was studying. And so like many other scientists, he sequenced it, right? He used this new technology to try and get some more insight into how this gene, how this enzyme was functioning. And so he was doing this research completely not even looking for any thing related to CRISPR. But he saw this really strange sequence as part of his his sequencing data. Part of his data. And it was really interesting, and he was curious about it, right. So they wrote the paper all up about the gene that they were studying, and the end. This is a really nice short, sweet paper Fatu. I'm telling you, I wish papers today were still this like short and sweet.

It's Beautiful right cause, you know, the more you learn about science, you gotta have a longer introduction to explain everything we know and then a longer discussion to you know, it's everything just gets longer and longer and more complex, but yeah, this was a really beautiful, simple short and sweet paper back then. And the CRISPR sequence literally got one single paragraph. It was the very last paragraph at the end of the paper. That's all the sequence got. They talked about. They had a figure showing what it looked like and talked about it and literally said we don't know what this is, right. This is interesting, but we don't know what it is. And I love that they did that. So he published his paper. And then he went off to start his lab. And then the field continues on. Irina, can you pick it up for us from there?

Irina Gostimskaya  18:31  
 Yes. Yes. Actually, yeah. So the last sentence of that paper says, So far, no sequence homologous to these has been found elsewhere in prokaryotes. And the biological significance of these sequences is not known.

Shekerah Primus  18:50  
Can you imagine that you just end up people with Yeah, we don't know what this is.

Fatu Badiane-Markey  18:56  
It's like a cliffhanger.

Shekerah Primus  18:57  
I know it's like a cliffhanger You're so right? You're so right.

Irina Gostimskaya  19:03  
Yeah. So they were Palindromic Repeats that they found and palindromic means reading either way, gives you the same information. So in terms of DNA, it's whether it's the top strand or the bottom strand, you would get the same sequence of letters and the repeats were interspaced with some other information they didn't know what the information was. And yeah they weren't repeats. And then the next hero of the CRISPR saga

Shekerah Primus  19:35  
I read that paper too

Irina Gostimskaya  19:36  
He started his PhD in 1992. So that's Francisco Mojica in Spain. I think he's my probably favorite scientist in this whole story. We don't pick favorites, but, ok I think he is.

Shekerah Primus  19:57  
we can have favorites. It's okay.

Irina Gostimskaya  19:58  
Yes, yes, I think so. So he worked at the University of Alicante. I think he's probably still there. He was studying some Halophilic archaea. So those are salt loving creatures. These are the microbes but they're not bacteria. And they live in salt lakes and they prefer really, really salty water so they wouldn't even grow in the sea. It's not salty enough for them. It's a very interesting phenomenon, because how can you live like that? If it's so salty, if it's practically a saturated salt, and it's, he was just interested in the biology and they were sequencing those genes that thought were responsible for the salinity resistance, and they discovered similar repeats and then it was before internet those times it could not Google search. 

Shekerah Primus  19:58  
That is a good distinction. 

Irina Gostimskaya  20:10  
So he had to go to the library and just look at this old fashioned index catalog, yeah, for repeats, and he found the paper from Japan. From Ishino. And he realized they were something very similar, although the sequences were different. So it's two different domains of life, bacteria and archaea, and have different metabolism, and they're quite distinct, and then they have something similar meaning it's important to know it's something that has been conserved through the evolution, it must have stayed around for millions of years. And it must be important so that's how Francisco Mojico got interested in that. It took many years for him to understand what the sequences were he kept going was very determined. That's why I think it's a great story. So he publishes the paper on repeats in the 90s. And then by 2000, I think some other groups started publishing on those repeats and using different terms, so it was quite messy at the time that the terms were difficult. It was difficult to understand what people were talking about people were using SRSRs short regulatory space repeats. Some other groups were using DRs direct repeats in Mojico's group actually, initially they use the term T reps, tandem repeats, but that's not strictly speaking correct because they're not tandem. tandem is back to back and they were interspaced into Yes, so they were not actually head to tail as such. They were interspaced

Shekerah Primus  22:48  
Right. Exactly. Yeah. That's the important part. Right. The interspacing.

Irina Gostimskaya  22:51  
Yeah. And then by 2001. Francisco Mojico actually came up with this CRISPR term and he first shared it with his wife and he asked her if she thought it was a good term. And she said it was better for a dog than a scientific term

She was saying yeah you can name a dog like that. But in the end She liked it. 

Shekerah Primus  23:21  
I love that response

Irina Gostimskaya  23:21  
So he emailed his collaborator in the Netherlands, Ruud Jansen and told him he thinks It's a good term and Ruud loved it. He said yeah, it's crispy.

And snappy so the term basically was accepted, became a scientific term, and then so everyone went ahead with it 

Shekerah Primus  23:51  
Yeah.

Fatu Badiane-Markey  23:53  
I love that. And Can you give us the entire meaning of the acronym of CRISPR?

Irina Gostimskaya  23:59  
Yes. So it's Clustered Regularly Interspaced Short Palindromic Repeats

Shekerah Primus  24:07  
It's a mouthful

Fatu Badiane-Markey  24:11  
and I'm always so impressed with how people come up with these acronyms. And I like that we have CRISPR. I feel like biologists always come up with like, you know, the most interesting acronyms. I really like that origin story Shekerah and Irina. So they found this interesting sort of like pattern in all these micro organisms in bacteria and archaea, and very distinct like very different organisms, which I think makes it like so really interesting. And then they realized, like somehow this is important for something, maybe for gene editing is that how they made the jump.

Shekerah Primus  25:01  
Well, that would have been nice and easy, right? But not quite science never Makes it easy. Right? I think we can all attest to this. Science just never makes it easy for us. We always gotta work for it, right? So they realized that it was important, right? But we still didn't really know much about it. So we knew how the locus was organized, you know that regular interspacing that Irina talked about. We knew how it was organized. And we knew that it was obviously important because it was found in so many different microorganisms, right? It was conserved in all of these different organisms. in 2 domains of life in bacteria and archaea. Right. So we knew that it was important, but we didn't know what the function was.  so there was still a long way to go. So as scientists tried to figure it out. They proposed and tested different theories that didn't quite pan out right and again, that is a normal part of science. So just to mention a couple of the incorrect theories that were proposed for the CRISPR sequence, one was that the CRISPR sequence participates in gene expression control especially of the surrounding genes because some papers mentioned that the CRISPR sequence was always very close to genes that were very important for bacterial functions. So they're like, oh, maybe it has something to do with gene expression control. And another one was that the CRISPR sequence was important for replicon partitioning. And so this is a critical, like really essential process during DNA replication and cell division in bacteria so that the replicated DNA is partitioned properly into the daughter cells. But so just to mention two of those, but of course, neither of those hypotheses were correct, right. We know the correct hypothesis now, and it wasn't until the early 2000s When the function of the CRISPR sequence really started to come to light right Irina.

Irina Gostimskaya  27:03  
Yes, yes. By 2003, Francisco Mojico discovered that there's a match between CRISPR sequences, and some viruses and that was around a decade later since he started the work and all the time with very little results, as I was saying before, and as soon as they, the group, they saw those results. They just went ahead quickly wrote the paper. Tried to publish it submitted the they submitted the manuscript within a couple of months after after the discovery.

Shekerah Primus  27:41  
Nice That's like record speed, right right Fatu

Fatu Badiane-Markey  27:45  
Really, yeah. Really fast. 

Irina Gostimskaya  27:47  
Yes. Yes. Really fast and then submitted to nature. Of course, one of the most prestigious journals, and they just think we've made a huge discovery, we have to be really quick. And, it gets rejected, the paper was not accepted. Because there was not much experimental proof. So it was all just bioinformatics basically. And they were asked for the experimental proof. And they tried to get it again and again and then it just wasn't working and they finally published it in 2005. And the same year a couple of other labs published their research similar research as well. And what they didn't know at the time, is that why it was so difficult to get experimental proof, the strains of E. coli that we grow in the laboratory and under the conditions that we grow them, the immune systems just get repressed you know, they don't need it and the Yeah, so you don't really see much of CRISPR in action with the conditions.

Shekerah Primus  28:47  
Right. So yeah, yeah, as I said, science makes us work for it. Biology makes us work for it. Doing research so with that story, Irina, like I'm thinking, you know, doing research is so difficult already. Right? But it can be I think the challenges with getting your work published, like the challenges that they went through can make it like twice as frustrating, right? Even even more than that, right? I think probably we've all had stories of papers that were rejected or whatever. I definitely have a story of a paper that still has not been accepted so

Fatu Badiane-Markey  29:26  
The struggle. 

Shekerah Primus  29:27  
The struggle is so real. I think all scientists can commiserate with, you know, the struggle that they went through to get their work. Published. So I'm glad that they finally got their work published. So basically, in 2005, three separate groups right, including the Mojica group, independently published papers, showing that the spacers in the CRISPR locus had matching sequences in the viruses. And that led them to propose that CRISPR was actually part of the bacterial immune system.

Fatu Badiane-Markey  30:02  
Oh, okay. So we're gonna have to take a little time out there. People might be confused, wondering why in the world do bacteria have immune systems like what are they doing with all that right? I mean, when I, I mean, they're tiny. They imfect us, right? They make us sick, other plants, other animals. What? What are they? What are they scared of like? What is it infecting them immune system? Bacteria what, Like I just, I just don't see it.

Shekerah Primus  30:36  
Yeah no that That is an excellent question. but, you know, let's not, you know, beat down on bacteria. Because, you know, they need to defend themselves as well. And they have immune systems to defend themselves against viruses. Alright, so no prejudice against bacteria. They're struggling too girl. They're strugling To survive, too it's not just us. So, yes, viruses don't only plague us and plants and our pets and animals, but they attack bacteria as well. So it's kind of wacky, I know to imagine, right? Because bacteria, as you said, they're already so tiny. We can't even see them. And we just think of them as pathogens that you know make us sick. And to think that viruses are even smaller than that, that they can infect bacteria. It's kind of mind boggling, but it's true. Right? So the specific type of virus that attacks bacteria is called a bacteriophage or just phage for short. So bacteria have several different defense mechanisms, or many I should say many different defense mechanisms to fight viral infection. And we know restriction enzymes is another one of those. Just another tool that was found in bacteria that we now use in Biology so since they are simple organisms, right compared to us as humans, you know, we're made up of trillions of cells that bacteria is only one this one single cell, right a single cell. But its immune system is simple, right? Because it's a single celled organism but it's incredibly effective. And so CRISPR is part of the bacterial immune system to fight against viruses. Yep. So that's why they need immune system. ,

Fatu Badiane-Markey  32:26
Yeah okay. I get that. Now. That's really neat, I guess like everyone's got an enemy right.

Shekerah Primus  32:32 
everyone's got an enemy right

Fatu Badiane-Markey  32:34  
They're coming for you no matter what
So going back to what you guys said so from the first discovery of CRISPR to its function as part of the bacterial immune system that took about 20 years that's quite a while quite a bit. of time.

Shekerah Primus  32:52  
Yeah, yeah. It was a wild girl. Yeah. They were struggling.

Irina Gostimskaya  32:57  
Indeed

Fatu Badiane-Markey  32:58  
So We have these 20 years, you know, that I guess fly by. Whether they're kind of like seeing CRISPR for the first time figuring out what it is, and then they have a really good idea. Of what it does. So can we talk more about exactly how it functions as the bacterial immune system? Like what exactly is it doing?

Irina Gostimskaya  33:17  
Yeah, yeah. So initially, it was suggested that the CRISPR works by the way of RNA interference. So that's RNA. Attacking RNA. There's some double stranded RNA involved, and then some protein comes and chops up the RNA. But later it was shown that it's actually DNA that gets destroyed. And that means that it can be used for genetic engineering. Yes, if it works with DNA, if it's specific if it's efficient, then we can do something with the genomes. But now we know that some CRISPR systems actually do cleave RNA as well. So there's some some work with DNA, some work with RNA, some probably can do both. And obviously, the dangerous genetic elements can be either RNA, or DNA. And both types need to be tackled by bacteria. Yes.

Shekerah Primus  34:14  
Yeah. So CRISPR has a lot of diversity. I love that. 

Irina Gostimskaya  34:17  
Yes, yes. And so the prokaryotes they copy a snippet of this genetic information that comes to attack them when they get infected. And then they add this little bit of DNA to the library of genetic elements. So the CRISPR region of the genome is like a library. And it's very similar to our immune system. So it has our immune system has a memory as well, when we have B cells and T cells fighting the pathogens in our immune system. So when those get activated by infection and begin to replicate, some of their offspring becomes long lived memory cells. So throughout our lifetime, these memory cells remember specific pathogens and then they can help us to have a strong response if the pathogen is detected again.

Fatu Badiane-Markey  35:12  
Okay, I see that's really amazing. That bacteria can accomplish almost the same thing as our bodies can, even though they're only one cell compared to our trillions of cells. That's really cool. That's really cool. I like that. I like that .

Irina Gostimskaya  35:28 
definitely, yeah. And then going back to the CRISPR discoveries, the first experimental evidence came from two French scientists, Barrangou and Horvath. They were working for a yogurt company and going back to yogurt. 

Shekerah Primus  35:44  
Yogurt Everywhere. 

Irina Gostimskaya  35:47  
Danisco, that's a Danish company. They have a obviously a big collection of bacterial strains that they use for yogurt making. And those two scientists were able to trace back the history of how the spacers get acquired by the CRISPR locus when the bacteria get bacteriophages so they have this collection they could refer to and then they could also introduce new spacers into the CRISPR locus and basically vaccinate the bacteria with those so create new strains, which are more resistant to bacteriophages. 

Shekerah Primus  36:24  
Yeah, yeah. So cool. Yeah. 

Irina Gostimskaya  36:27  
so the timeline of discoveries, if we think of it overall was 1980s 1990s. So CRISPR is discovered, but we don't really know what it does first in bacteria and then in archaea. clearly important. What it does, we don't know and then, by 2005, the function was understood and CRISPR cas-9. So cas-9 is a protein that was discovered in association with the CRISPR locus so it was known before the protein but people didn't know what it did. And the first time there was understood that cas-9 was a part of the CRISPR system was in 2005. And that was a team of scientists in France. And then in 2008, team of researchers, led by John van der Oost in the Netherlands discovered that there is a small RNA that guides cas-9 protein to the DNA that needs to be destroyed. So before the DNA is cleaved, it needs to be found. So how do we find it so there's a matching RNA that binds to that DNA and that's how cas-9 knows Okay, that's where I cleave, that's what I need to kill. That's the foreign genetic information that came to attack me so I have to somehow protect myself. So those small RNA molecules were called CRISPR, RNA crRNA. And then in 2011, Emmanuelle Charpentier discovered the second small RNA that is very important, and at the time it was thought it just needed for crisper RNA maturation, so to make the guide RNA to mature let's say you need a second RNA, there was called TracrRNA, but later was discovered this tracrRNA is also important for the final CRISPR cas-9 complex to work all together. So the cas-9 needs to bind the protein needs to bind 2 RNAs the CRISPR RNA and TracrRNA. That's how it will work. So in the end, we knew what was needed. So it's cas-9, which is a protein, 2 RNAs. And that was all the components necessary for attacking the dangerous genetic material.

Shekerah Primus  38:27  
Yes, yeah. Yeah, very cool. So once they figured all that out, that's when the race basically started. Yeah, that's when the wheels started spinning and the race started with scientists trying to figure out just how exactly the CRISPR machinery worked so that in the science world we say that's trying to determine the molecular mechanism, right, because that's where the secret lies. That's how you know how to replicate the bacterial CRISPR system in the lab to use it as a genetic engineering tool. So that's when that race started to figure to figure that out.

Fatu Badiane-Markey  39:30 
Yes right the tension heats up. And the Doudna-Charpentier team won the race

Irina Gostimskaya  39:33  
They did

Shekerah Primus  39:34  
They sure did

Irina Gostimskaya  39:35  
They could assemble the whole complex of cas9 protein and the RNAs and then the target DNA and then it all worked? Yeah, yes. And one really important thing that they did, also was to make the system even simpler. So they combined the two RNAs, crRNA, crisper RNA, and TracrRNA. And they made it into one chimeric molecule, which is a single molecule rather than two separate ones, and they called it single guide RNA. So that's, that was a really good, practical invention, basically, that they introduced. In the field of CRISPR and genetic engineering.

Shekerah Primus  40:24  
won them the Nobel Yeah.

Irina Gostimskaya  40:29  
And then, of course, everybody knows there was another group in Lithuania, that were working, you know, at the same time doing similar things and so the, it was led by Virginijus Siksnys. They did very similar things. And they were collaborating with the yogurt researchers, Barrangou and Horvath, that we mentioned earlier, but their team, they only were able to publish later than Doudna and Charpentier. They had some problems publishing as well, and then also that they didn't realize the importance of tracrRNA in the complex formation. So they, they knew the tracrRNA was important for the maturation of CRISPR RNA, but they didn't know that actually, it needs to be added to the complex for it to assemble and work so that was one little bit that was missing from their understanding of how it all works. And then, yeah, as we see, it's a very complicated story, the history of discoveries of the in the CRISPR field. Yes, there's so many labs involved so many people working in parallel, and then people getting little bits of the of the jigsaw puzzle. Yeah, they were not always coming together very well. And it was also difficult to it's very difficult to acknowledge individual contributions, you know, when when people who is it who should be rewarded? Yes. The person who discovered or the person who understood or the person who published first and it's of course

Shekerah Primus  42:07  
yeah, science has a lot of politics, some people may not know

Fatu Badiane-Markey  42:13  
It does.

Irina Gostimskaya  42:13  
Yes. And there were actually other ways of genetic engineering and there are still you know, the the recent sickle cell anemia treatments that were approved by FDA in December, just a month ago. There were two formulations actually one CRISPR based and one not CRISPR based, you know, some other way of genetic engineering and that works as well and then and they will also this zinc finger nucleases. ZFNs, and TALENS and you know there were other tools at the time available, but CRISPR was just, you know, more efficient, less expensive, and that's very important. And it just became the tool of choice and it just made this revolution as we see now in so many areas of science finds its applications and bioengineering as well. Yeah. Yes. So, the next Yeah, and then the next step, the crucial one, after scientists understood how it all works, was to make it work in eukaryotic cells. So much more complicated system. Yes, of course, preferably human cells. That's what we all want. To treat diseases Yes, yes. And then in January 2013, almost simultaneously five papers came out from five different groups all achieved the goal of modifying eukaryotic cells with CRISPR cas9 system. Two publications of those are accepted as the best. They're the widely cited one. So that's one from George Church in Harvard University and the other one from Feng Zhang in the Broad Institute so that. That's our golden standard now for human cells. modification, yes, those were the pioneering ones.

Fatu Badiane-Markey  44:10  
Yes, yeah. Yeah. I love this like it definitely is a CRISPR saga like you were saying in the beginning Irina it's like ups and downs and like cliffhangers, you know, and it's it's literally like all these really smart, you know, innovative people like on this quest for knowledge and the truth. You know, I love that it's so exciting.

Shekerah Primus  44:33  
And the money let's be honest. 

Fatu Badiane-Markey  44:38  
Ok Ok that too that too

Shekerah Primus  44:39  
Or maybe not the money but like the recognition, right the recognition, like Irina was saying

Irina Gostimskaya  44:45  
Yes the prizes, lots of prizes, lots of awards, and everything, everything. Yes, .

Fatu Badiane-Markey  44:52  
definitely

Irina Gostimskaya  44:54  
and fame of course

Shekerah Primus  44:55  
And fame, yes there's all of that. We're all humans, right? We're scientists. We're also all humans. So all of that our egos and all of that always comes into play. And so it's hard to. We're full people.

Fatu Badiane-Markey  45:07  
I like that. We're full people. So yeah, how so I guess who has gained like fame and fortune now from all of this are there patents for CRISPR. So every time you use one that you know or like for a gene therapy like someone I guess owns it, and it's somehow getting paid off of this. So I guess yeah, what has been I guess the impact you know, in in other parts of the of the real world, now that we have CRISPR and have ways of actually using it? 

Shekerah Primus  45:43  
Yeah, the real world right so people care about the cures and the money right, that's the real world. Make me better make me money. So you're right. Someone's somebody's making money. A lot of people actually are making money from CRISPR patents and the patenting situation is actually pretty complicated. It was really complicated for a while, I think it's still a little dicey. But basically, the Zhang team at the Broad Institute are the ones who won the patent dispute. So there was a dispute basically, between the Doudna-Charpentier team, they're called the CVC. And the team from the Broad Institute, and the Broad Institute won the patent for using CRISPR gene editing in eukaryotes, and that's what really matters, right, because that's where the money is using it in people so making gene therapies for humans. So the CVC the Doudna- Charpentier team. They appealed that decision and they lost and they're probably appealing again, I'm not sure. And so just to make it clear that CVC team includes of course, Doudna, Charpentier, the University of Vienna, and University of California, Berkeley, who is representing Doudna. But yeah, so the Broad Institute won the patent battle, and they sold exclusive rights to Editas. So now as Irina mentioned, now we've got this sickle cell disease CRISPR therapy that's been approved by the FDA. And so that was made by vertex pharmaceuticals and CRISPR therapeutics. And so now they have to pay Editas for the right to develop and sell that treatment. Because Editas has the patent they own the rights to that patents to use CRISPR in eukaryotic cells, right. And so it's a huge sum. The last time I read it was something like 50 to $100 million plus royalties, right so lots of people are making bank.

Fatu Badiane-Markey  47:51  
I feel like we've come a long way since like Salk and him giving away the polio vaccine because it was like you know for the greater good to now having patents on actual biotech and I'm not saying one is right or wrong either way, I think you can just say it's a sign of the times and how things have changed. And this also makes me think Shekerah, like maybe we need to get like an IP bio person on for one of our future episodes to really understand. this stuff right Yeah, but this is really incredible. Just wow, no words. That's all I got.

Irina Gostimskaya  48:34  
Also, you know, it's also interesting that Emmanuelle Charpentier, she was working in Sweden at the time. The University of Umea. It's in the north of Sweden. And the rules are very different. There from the UK and the US. So academics actually own their IP, the intellectual property that they generate. So it's a very good place to be if you're a professor who wants to be an entrepreneur, yes. So in the UK, and in the US, it has to go through the university that you're employed by, and they want a part of the wealth that is generated by your IP. While in Sweden, yes, it's a different rule. It's a completely different story there, and that's why it's the team Charpentier, Vienna, California, it's because it's Charpentier on her own as an individual. And then University of California University of Vienna, the other two collaborators were working for. So that's a very interesting one. But, of course, the sad thing is that this gene therapy is so expensive, you know, it'd be just so good if it was it doesn't have to be seemingly it's not so complicated to do, particularly with sickle cell anemia. So sickle cell disease, the price at the time when the clinical trial started, the price per patient was something like $1 million, you know, US dollars. It's crazy. And it's a shame, I think for quite some time. It's probably not going to be affordable for most people in the world, even if it all goes smoothly from now on with administering it. And yeah, let's hope that once there's more and more companies, you know, more and more companies get involved in genetic engineering with CRISPR. And there's more formulations available and there's a bit of competition and the price will go down. Yeah, it's a shame if it's, you know, if the tool is invented, and it cannot be used because of the prohibitive price that would be really sad to see for years to come possibly where we shall see, we shall see.

Shekerah Primus  50:36  
I agree. Yeah, I agree. And so the last that I heard was 2 million per patient Irina. So it seems like the price is going up right now right but

Irina Gostimskaya  50:39 
It depends on the on the actual formulation on the type of you know, Yeah, different. Different circumstances, I think and yes, it's crazy. Yeah.

Shekerah Primus  51:00  
Yeah. So I agree with you, you know, we can only hope that with time it becomes more affordable, right? That's usually the way that all technologies go they start off ridiculously expensive and then the price comes down. And of course, if no one can afford it, or very few people can afford it. These companies aren't making very much money anyway. Right. So it's in everyone's best interest to make it affordable. Whether we have government subsidies or you know, some sort of partnerships with granting groups. I don't know patient advocate groups, things like that. So it's in everyone's best interest to sort of make this treatment affordable for for people who need it. All right. So yeah. All right. So there you have it, folks, that is the story of the discovery and development of CRISPR into a genome engineering life saving tool. And on the next installment of the CRISPR Chronicles, we'll have an entire show about sickle cell disease. What is it? When was it discovered? What are the symptoms and basically what what is it like living with this life threatening debilitating disease? Sickle cell like sickle cell anemia? And then how does the CRISPR gene therapy for sickle cell disease work right, so be sure to tune in for that one that should be a very, very interesting people patient-centric episode that we'll have so thank you both Irina, and Fatu for a great discussion today. I really enjoyed it. And special thanks to our guest today, Irina Gostimskaya.

Irina Gostimskaya  52:44  
My pleasure. Yes, I really love this and yeah, thank you, to you for creating all these lovely content on your website and all these podcasts and popularizing science and you know, making it easier to understand certain complicated topics. And yeah, I'm sure you invest a lot of time and effort and it's great. Thank you for that. Great.

Shekerah Primus  53:12  
Yes, yes. Thank you so much. It is a passion project for the both of us and we have fun doing it definitely. So we we're happy to hear that people are finding value in it. So but I just want to say that you made our episode extra special.

Fatu Badiane-Markey  53:29  
Irina's like the icing on top of the cake for this one.

Irina Gostimskaya  53:40  
Thank you.

Shekerah Primus  53:41  
Excellent so Thank you for coming on to chat with us. If anyone is interested in getting in contact with you, Irina for any reason. What's the best way to reach you?

Irina Gostimskaya  53:51  
Yeah, so you know, I've written this review that I already mentioned, and that's just my email address there. So I've intentionally put my gmail address rather than any work address because you know, in science you move around and you change institutions and the email addresses change, so the Gmail is just my personal address that I keep and it's just my surname@gmail.com. So yes, the surname is quite rare so I could just get this gmail account with with.

Shekerah Primus  54:27  
Excellent, and I can attest that it works Folks because that is exactly how I reached her as you can tell, she is awesome, very personable, easy to talk to so She's wonderful. Reach out to her. And we'll definitely put links in the show notes so that people can easily find your review and reach out to you 

Irina Gostimskaya  54:46  
Thank you. 

Fatu Badiane-Markey  54:46  
Yes. And with that, we'd like to thank our listeners for supporting the podcast if you'd like to reach us, Fatu and Shekerah, you can send us an email at loveSciencepodcast@gmail.com. Until next time, bye everybody.

Shekerah Primus  55:04  
Bye everyone

Irina Gostimskaya  55:07  
Bye

I think of it as a hybrid between the yogurt and the beer.