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Ep 38: The Secret Behind CRISPR Gene Editing

• Shekerah Primus & Fatu Badiane-Markey • Season 3 • Episode 6

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Humans have ~6 billion bases of DNA in each cell. But every gene has a specific and unique sequence that serves as a kind of genetic address. Before any gene can be edited, the editor must first find the gene's location within the genome. Luckily CRISPR-Cas has a built-in GPS component that makes finding a gene address easy, and switching out one genetic address for another is as easy as typing a street address into your Google Maps navigation app...well almost as easy.

In this episode, show hosts Fatu and Shekerah deliver the second installment of the CRISPR Chronicles series.  We discuss the secret sauce that makes CRISPR-Cas gene editing powerful, flexible, and easy to use—it's the gene editing genie that the scientific community had been waiting for.  CRISPR-Cas gene editing has two main components (RNA and protein), each of which plays a critical role. The merger of these 2 components into a unified system is what makes CRISPR-Cas gene editing superior to its predecessors.
 
Zinc Finger Nucleases (ZFNs) and TAL Effector Nucleases (TALENs) were the gene editing tools of choice before CRISPR-Cas9 was discovered. Unfortunately, the design, production, and implementation of ZFNs and TALENs were difficult and expensive, which prevented their rapid adoption on a global scale as has occurred with CRISPR-Cas9 gene editing.
Tune in to the episode to learn:

  • How CRISPR-Cas9 gene editing works
  • What is the secret sauce that sets this technique apart?
  • Which CRISPR component is the GPS, and which is the scissor?
  • How the system can be used to create mutations and to fix mutations
  • Bonus Food Science Content: Hear about the best food to eat in Kenya

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


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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

Shekerah Primus  0:07  
What can you do with your love of science? We'll tell you

Hello, hello. Thank you for tuning in to another episode of your favorite podcast "We Love Science", the show where we discuss all the things that you can do with your love of science. We are your hosts, I'm Shekerah

Fatu Badiane-Markey  0:51  
and I'm Fatu and welcome welcome back everyone.

Shekerah Primus  0:55  
Yes, welcome welcome indeed, it's good to be back Fatu. This is really for us right this this welcome for us because we haven't recorded in a while but our listeners don't know that. Yeah, we took a little break. It's cold and wintry outside. Now by me anyway, it's cold and wintry. I know that you've got better weather. Where You are in Kenya.

Fatu Badiane-Markey  1:24  
Yes I do

Shekerah Primus  1:25  
Yes. But um, it's good to be back recording with you Fatu

Fatu Badiane-Markey  1:28  
Yeah.

Shekerah Primus  1:31  
So you're in a different country now. And you know, I'm dying to know all about the food. So tell us how are you and what have you been eating over in Kenya? Tell us about the food?

Fatu Badiane-Markey  1:50  
Yeah, no problem Shekerah. So just a quick for anyone who may have missed it. I relocated a couple of months ago now like already to Nairobi, Kenya for a new job and it's been fan tabulous full stop, exclamation point next sentence. I'm having such a good time here. The weather is a little bit warmer, not as warm as I would like, but a little bit warmer. It's between like around like 60 and 80 degrees. Like any given day. We just I think closed up on the first rainy season of the year. So I hope I won't have to deal with rain anymore for at least the next few months

Shekerah Primus  2:35  
So jealous! Let me just say let me just say it is 30 degrees in New Jersey right now. So jealous

Fatu Badiane-Markey  2:47  
Yeah. Let me just say I am not missing the North East cold at all. I will be a little sad like you know when that first snow comes in, it's like beautiful and you're just like enjoying it, but I'm not gonna miss cleaning off my car. I'm not gonna miss you know having to like sit there in the cold and like shovel a sidewalk girl. I'm over it.

Shekerah Primus  3:16  
Don't worry. I'll send you pictures of it all so you can see my pain.

Fatu Badiane-Markey  3:23  
Awesome. But so back to Kenya and the food. So I'll introduce I guess like a couple of, I guess what I would call maybe like the national dishes or dishes that are I guess like most common throughout Kenya. So the first one is Ugali, which is like a staple. And it's made usually from kind of like a starch so it can be made from like, corn meal or something along those lines. And I would describe it almost as like, not quite a porridge. Because it's like a little bit firmer than that but it's sort of like a steamed you know, like, like starchy side that you can eat with almost anything and because it like you know can hold like a shape. You usually can also then like cut it you know it'll be served in like a little like loaf, and then you can like cut it out and it's pretty good. I enjoy that. And then the other food that I really like because I love any type of grilled meat. They have a type of grilling here called Nyama Choma, which is like which is all kinds of grilled meats. And the way that it's usually served which is also pretty cool. Like when you go to the restaurants, they heat up your plate. So literally you have a plate that I think is like some kind of like metal type, you know, like thing like so literally it's like sitting on the grill and they put it in front of you and it's like steaming hot. And then they put the meat and it's like sizzling away. Oh, it's really good. Yeah, so that was basically what I ate. Yeah, my first weekend here I went with a lovely coworker and she took me to a really nice restaurant. And when I say they have all types of grilled me and I think part of this is probably just like at the restaurants for show but you can get things like ostrich and like alligator and like you know, all kinds of grilled meats. Yeah. I tried Ostrich It was very good. Did not try alligator because that was a bit too much for me. I would I would recommend Yeah, I'd recommend so if you find yourself in Kenya or Nairobi more specifically, definitely find a good Nyama Choma place pretty easy to find you know and you will not be disappointed if you're into like grilling, meats, and yumminess and then you know all kinds of sides like potatoes salad you know, like, Yeah

Shekerah Primus  5:59  
Super yummy. So does Ostrich taste like chicken?

Fatu Badiane-Markey  6:08  
this is the Ostrich you're asking about right? 

Shekerah Primus  6:11  
yeah does Ostrich taste like, I feel like a lot of things taste like chicken.

Fatu Badiane-Markey  6:16  
I don't think it tastes like chicken. It's like a it's like a really tender like juicy meat. I had like ostrich meat balls. And it was just like it hit the spot, but I don't think it tastes like chicken and I don't. I don't it's like it's more of a what I had was like it looked to me like a dark meat like a dark cut of meat. Yeah. And it was just like really seasoned really flavorful. I think ostrich meat is also supposed to be like, really healthy. I think it's like, you know, because ostriches are always running around, you it's supposed to be really lean also. They're not just like, you know, sitting around like chickens. So yeah. It was good. It was good.

Shekerah Primus  6:55  
Okay, but the chicken is good. 

Fatu Badiane-Markey  6:59  
The chicken is good. For real for real. 100% I've been doing a lot of chicken. Yeah, and I am not disappointed and I'm not mad.

Shekerah Primus  7:10  
Okay, well, you gotta give me the chicken you know the recipe or whatever the secret is over there in Kenya to make the chicken so delicious.

Fatu Badiane-Markey  7:20  
Yeah, I don't I just think it's just better quality chicken like I don't know what it yeah I literally just like season it up with some veggies and it is a good dinner that hits the spot. The other thing I really liked is there's this one supermarket where they I think make like sausages in house so they do chicken sausage and beef and their chicken sausage is the bomb.com they're it's like perfectly seasoned, super juicey. You can not dry out this sausage. In fact, like I don't even know what is in it. But every time I can I buy it. And it's so good.

Shekerah Primus  8:02  
Excellent. Yeah. All right. Well I gotta come taste that Kenyan chicken for sure girl. So be ready. All right. So let's talk CRISPR . Okay, so today we're continuing our CRISPR Chronicles series. And in the first installment, we gave an overview of CRISPR as a gene editing tool. And then we touched a little bit on the impact that it's already having in people's lives, because of the power of the system to cure previously incurable genetic diseases. So if you missed that episode, we'll be sure to put a link in the show notes. So you can go back and check it out. But in today's episode, we're continuing the series and we're going to talk about how CRISPR actually works right so how are scientists able to fix mistakes in DNA sequence in such a specific way? So you may have heard the term genetic scissors used in relation to CRISPR and maybe it conjured up some images of an actual giant pair of scissors or maybe a teeny tiny pair of scissors, I don't know. So, while that's obviously not technically accurate, it's not actual scissors, right? It's a reasonable analogy, because the CRISPR system is able to cut DNA just like a pair of scissors cuts paper or a piece of cloth. So let's so first let's talk about the components of the CRISPR system. And for now, we'll specifically focus on CRISPR-Cas9, because that is the most widely used CRISPR system that you hear about and cas9 is basically the best known CRISPR system. And, as the name suggests, the CRISPR-Cas9 system as it is used for genetic engineering is made up of two main components, the CRISPR part and the cas9 part. So the CRISPR part is an RNA components and the cas9 is a protein component, and specifically the protein is a nuclease. 

Fatu Badiane-Markey  10:23  
Okay So that makes sense. 

CRISPR is such an amazing tool, but I think it's always been something that's even for me been really hard to understand. So what do you mean for like the different components? 

Shekerah Primus  10:41  
Yeah, Yeah. So the different components really is where I feel the magic of CRISPR comes in, right? Those two things, how they come together is what got scientists so excited about it. The way that it works is so much simpler than previous genome engineering techniques that were available. And we'll touch on those other techniques in a little bit. But so for CRISPR, the protein component, which is cas9 that's what actually cuts the DNA. And so a protein that cuts DNA is called a nuclease. So cas9 is just another type of nucleus. Right? And that's, that's not really a revolutionary development, right? Because nucleases had been known for decades. Right? So this goes back to like the 1960s and 70s when restriction enzymes were discovered, and those are kind of nuclease, right? Restriction enzymes are a type of protein that can cut DNA.

Fatu Badiane-Markey  11:48  
Okay, so that makes a lot of sense. So the cas9 nuclease not that revolutionary, we've seen it before. Been there, done that. So does that mean the RNA part is like the game changer? 

Shekerah Primus  12:04  
Exactly Yeah, the RNA component is what got everyone's hearts racing. Palms sweaty. People, people were getting excited. Right and that's because RNA is such a simple molecule, compared to proteins. Right. So again, for our non scientist, listeners, hey, thanks for listening. Just know that RNA is what we call a chemical cousin of DNA. So it's very similar to DNA. And what's significant about it is that it can bind to DNA in a sequence specific manner. So just as DNA can base pair with itself, RNA can also pair up with DNA, and again in a sequence specific manner. 

Fatu Badiane-Markey  12:56  
Okay, so I'm picking up on this sequence specific manner part. Can you explain that a little bit more for our audience what that means?

Shekerah Primus  13:07  
Yes, yes, yes. Yes, it is. Very, very significant. The sequence specific manner part, because so think about this our DNA, we as humans, we've got about 6 billion, that's billion with a B, 6 billion base pairs of DNA in each cell, right. It's a lot of DNA. But every gene has a specific and unique DNA sequence. And you have to be able to find that sequence if you want to do something to that gene, right. So think about the DNA sequence of a gene as its address. That's like the address of the gene. Now if you have a piece of RNA that can bind to that particular sequence, that means that the RNA can act kind of like GPS, right? Because it can find that gene sequence and bind to it. So it's similar to using GPS when you're driving right as long as you know the address, no matter how large that city is, no matter how far away that address is, the GPS can find it right. So similarly, once you know the sequence of the gene you want to target you can use the RNA component of the CRISPR system to find that gene. So in this way, the RNA component of CRISPR can guide the cas9 nuclease, the protein component of CRISPR to the right spot in the genome, where it wants cas9 to cut the DNA.

Fatu Badiane-Markey  14:38  
Okay, got it. That's actually really cool. And I think that's part of the confusion I always had with CRISPR is really visualizing how it works. So that makes a lot of sense so thank you for all those analogies Shekerah. I feel like in my mind, and I honestly anytime or at least I guess in grad school whenever I tried to do stuff with CRISPR like way back in the day I could never get it to work. I just really kind of magic happened. Yeah, that I just did not have and so my my results were always like 00 Girl Yeah, I got CRISPR caused me some trauma for real. 

Shekerah Primus  15:23  
Oh, no, it all seems like magic. Right? Yeah. 

Fatu Badiane-Markey  15:26  
It does, it does. So You know, when you mentioned that scientists were like super hype about this when they figured out that the CRISPR system has this RNA guide. Is it because like you said you can so RNA can base pair with DNA that acts like a GPS to find the specific DNA sequence that they want to study or manipulate or whatever, or is there another reason why you know, it caught so much attention from scientists?

Shekerah Primus  15:56  
Yeah, yeah, that is an excellent question. So, yes, and yes. RNA can base pair with DNA and it's a simple molecule. That's wonderful. That's what they love. But the other reason that everyone was so excited, was because the alternative gene editing techniques that are available before CRISPR came along, did not use RNA as a guide. But instead, they used protein to find the DNA sequence. So those techniques were called Zinc Finger Nucleases and TAL Effector Nucleases. And you see nucleases in both of those names, which tells you these are proteins that can cut DNA so zinc finger nucleases and TALENs. And both of those techniques depended on protein for DNA sequence recognition. And proteins are way, way, way, way more complex structures, molecule, more complex molecules than RNA is. So they are way more difficult to design than RNA. They are way more difficult to synthesize like way, way more difficult to synthesize than RNA, and they're more difficult to validate. So basically, what that means is that Zinc Finger Nucleases and TALENs. They were cumbersome systems, they were cumbersome and they were expensive. And we all know that those two things, you know, cumbersome and expensive. Those two things are barriers for any technology to becoming widely adopted worldwide. Yeah, right which is basically what CRISPR has become world widely adopted.

Fatu Badiane-Markey  17:45  
Yeah, no, that's, that's 100 100% True. So now I get it. I can see why scientists were so happy when CRISPR was first discovered. So the RNA was really like the secret sauce. That's where all that's exciting. Yeah, so exciting. And one of the things that I love about biology is how, as scientists we can sort of take these things that are just, you know, happy in the natural world doing their own thing. And then really adopt them to our own needs. And in some cases, that ends up really changing people's lives, right. Just so amazing. Yeah. So back to how CRISPR works, though. So the RNA component is like the GPS that guides the cas9 nucleus to the specific DNA sequence that needs to be cut. And then what happens? 

Shekerah Primus  18:40  
Yeah, so, so then the cas9 is there, it's where it needs to be. The nuclease is gonna cut the DNA at that position, and the original cas9 so since its original discovery, we have lots of different derivative cas9s that do different things, but the original cas9 nuclease cuts both strands of the strands of the DNA. And when both strands of the DNA is cut, that's called a DNA double strand break. So that's both strands of the double helix, right? And then once both strands are cut, one of two things can happen. So either scientists can allow the cell to try and repair the cut in the DNA, just by normal cellular mechanisms that are used to repair these kinds of things when they happen. And that process is called non homologous end joining. And unfortunately, and very strangely, I think, to me, this is a very error prone process. So you know, I don't know if it's that the when this process evolved, it did not evolve with enough safeguards so that it would be robust. It's just a very error prone process. And so what that means is that it's very likely that during this repair a mutation will result in that spot of the DNA and so that is how scientists use CRISPR to more quickly create a mutation in a gene in lab animals to study the function of genes and lab animals. And in fact, that's how I did it, right. That's what I did when I use CRISPR in grad school to just direct it to spot where I wanted it to cut it, cut and then the normal cellular mechanism tries to repair it, and made mutations for me. So I got mutant animals that I could study. So that's the first thing that could happen. The second thing that can happen is that scientists can provide a template DNA sequence for the cell to use in repairing that cut DNA. So the use of a DNA template in this way is called homology directed repair or HDR and HDR is a much much more robust technique than the non homologous end joining. So this is one way that scientists can fix a mutation that's already present in a gene because they're literally giving the cells a new piece of DNA sequence, which tells the cell this is how I want you to repair the cut. In that DNA at that position.  So yeah, in a nutshell, that is how CRISPR works. You either can create a mutation or you can fix a mutation in that way using the CRISPR system. 

Fatu Badiane-Markey  21:30  
That's so cool, Shekerah, so cool. And honestly like for me really cleared up how it worked. Honestly, I was like, scratching my head forever about this. Like

Shekerah Primus  21:41  
Stop it, ya'll she's just playing.

Fatu Badiane-Markey  21:47  
I'm not I read the papers, I looked at the diagrams and it was just like not computing. But somehow this is how it works. I think my thing was like I was stuck on the RNA part and I like missed the nuclease part. You know what I mean? So like, I only I own I understood like okay, it gets to where it needs to go. And then what magic happens yeah, and I just, I don't know, it's just like, never made sense in my head. But um, one of the cool things that you also just mentioned is, I remember when I was in grad school, my advisor told me about a paper where they used CRISPR-cas9 to kind of like mimic how you can get mutations in the cell to cause cancer. And so for the cancer that I was studying, which is Ewing sarcoma, they recreated that like DNA break, and then in like, one out of like 1000 cells or whatever they ended up like actually making the cancer cell because this non homologous end joining like just so happened to happen, you know, occur in the way that led to the mutation that led to the cancer Yeah, to show like, Hey, this is this is like how it works. And I'm just like, thats really cool like a proof of principle type of thing. Yeah,

Shekerah Primus  23:04  
I know. I know. It's so weird to me, though. I, I always think about it. And I think it's so strange that it's such an error prone process. Yeah, right. Get a cut in the DNA. And yeah, the cell is trying to fix it. That's normal. But then it's it's incredibly inefficient. And it just makes mistakes and you can just get a mutation from that and end up with cancer, basically. Yeah. Yeah, it tells a lot about staying away from radiation. You know put on that Sunscreen, don't give any chance for your DNA to get you know, cut and degraded because you never know what could happen. The cell could just you know, mess up and whoops, you got a cancer cell. 

Fatu Badiane-Markey  23:43  
Yeah, little PSA take care of ourselves folks. 

Thank you Shekerah. I really am enjoying this CRISPR series. I think it's so fascinating. And you know, I think the other cool thing about it is like, if we just think about from the time that CRISPR was discovered, to when it was you know, kind of like adapted and implemented into something that we could use in research to when it was actually used as a tool right for treating people. That timeline is like so quick. This is happening within our lifetimes, you know? Amazing. It's really amazing. 

Shekerah Primus  24:23  
Yeah, it's a good time to be alive.

Fatu Badiane-Markey  24:26  
I love like, you know, seeing the history of like science in process.

Shekerah Primus  24:34  
History of Science in process I love that

Fatu Badiane-Markey  24:37  
yes right it's like it's happening now so cool.

Shekerah Primus  24:42  
And actually, our very next CRISPR Chronicles episode is going to be about the history of science in process. . So we're gonna do a really, really nice milestone type episode where we go through all of the different like, really big, important CRISPR discoveries over the decades that helped to push the field forward from that initial like origin story the very first time CRISPR was published in the literature, to to, I guess, to the Nobel Prize, right when they finally figured out how to put all the components together to do gene editing in basically any organism to to change the genome. So that's going to be a really nice sort of historical perspective. Next time, and we are expecting to have a special guest contributor as well for that episode, so that should be really exciting. So yeah, thank you all for listening. This was great. We always have fun doing these episodes. And if you guys want to reach out to us, please reach out to us through email at lovesciencepodcast@gmail.com. And just you know, there's also a companion blog CRISPR series going on, as well on our website. Welovesciencepodcast.com. So go check it out. There's a lot more information for you to find there. I tried to make the posts kind of fun and simple. There are some cartoons and drawings and things and I try to make it sort of story based. So there's some stories in there. So I hope you guys enjoy reading it and learn something as well about CRISPR so until next time, bye everybody