SIO459: Understanding CRISPR with Dr. Eric Jaffe

Transcript

This transcript was generated automatically and may contain errors — the audio is the record.

0:00:13 Thomas: Hello, and welcome to Serious Inquiries Only. This is episode 459. your host, Thomas Smith, and it is time for part two with Dr. Eric Jaffe. Now that he's laid all that foundation for us in the prior episode, it's time to teach us about CRISPR. Just what it is and how it works, but how it was discovered, sort of, if that's the right word, and a little bit of controversy and intrigue around that, and why it's such an important innovation and what it can and can't do. Can't do. I said last time, this is something that I personally find challenging because biology is not my strong suit, but having an enthusiastic teacher like Dr. Eric Jaffe helps a lot. I is a little bit of repetition in this episode because we recorded these at sort of disparate times. And it's very complicated and hard for someone like me to keep in my mind because biology is just not my strong suit. So there is some repetition here, but I think it's probably and necessary. Like I barely notice because it's stuff that I have a bit of a hard time grasping personally, so I appreciated the repetition, but just wanted to give you the heads up there that we do sort of retread a little bit of ground before getting into all the CRISPR stuff. Right. Well, as usual, you can support the show and avoid the ad break at patreon.com/seriouspod. so much to those who support the Show and Make It Happen. Really do appreciate it. Couldn't do it without you. With that said, let's get on to CRISPR with Dr. Eric Jaffe. And Eric, welcome back to the show for, I don't know, nth time. It's so fun having you each and every time. How are you doing today?

0:01:40 Eric: I'm doing well, and today should be a really fun one because we're talking about one of the most important developments in biology in the past 20, 30 years.

0:01:47 Thomas: Yeah, we're going to talk about CRISPR. And I feel like there was a ton of coverage of it. What? Like, I dunno, three, four years ago maybe. And I dunno. Sense of time is weird after COVID, but it feels like like that kind of dropped off completely. Like I don't hear anything about it anymore. I know it's still out there, but I wonder why that is.

0:02:03 Eric: Think it's an unfortunate kind of byproduct of the way development cycles happen in biology because when there isn't like a transformational pandemic happening where you need to kind of rush those processes, things can be in testing. It takes a long time to validate and kind of really explore the contours of what we're going to use this new technology for. So there's that like initial burst of coverage that talks about the possibilities and then that trickle as the realities start to really be realized.

0:02:30 Thomas: Well, I I am so excited. Know I you're very excited about doing this episode because I think you have a lot to say about CRISPR. You going to take us through the basics of, you what it is or how we, I don' t know, invented it, if that's the right word.

0:02:43 Eric: Exactly what I'm trying to do. So full disclosure, CRISPR is something I used in the lab all the time. It's something that pretty much every single biologist is going to at least encounter on a near daily basis at this time, but it didn't always start that way. And so like what I'd like to start this episode with kind of really putting a fine point on if you've ever seen people complaining about, like, why are we studying frogs in Madagascar when we should be doing all of this important stuff? CRISPR is the counterpoint for that because it started as a why would we study these random sequences? And then it turned into the most powerful tool we have in biology currently.

0:03:18 Thomas: Well, Well, I can't wait to hear that story. However, I would never say why are we studying and whatever, because first off, we got to see what's turning the frogs gay. I think that's very important. Alex Jones, look, he's not right about everything, but we do need to figure out what's turning the frog's gay so that we can turn more of them. Get a second.

0:03:34 Eric: For the record, wait till Alex Jones realizes that some frogs can actually change genders. So there's this thing called parthenogenesis and the frogs can actually turn female

0:03:40 Thomas: My God. Turning the frigging frogs trans. Yeah, no, they already were actually, uh, God made the frogs trans everybody. So don't don't blam science, blame God. But Uh, anyway, if that's your view but also I just want to say like I will study what they study and I always find those arguments so annoying they'll be like why are we spending blah x on whatever to go to the moon and then you compare the budget and it's actually nothing it doesn't matter so aside you presented it like you know this uh technology is it what should I call it I you know I always forget like what is the word or acronym crisper is it a describing a technique or like a machine you know like what is it sure

0:04:18 Eric: Yeah that is such a good question that's actually where I was going to start um would call this a technology so there was actually and we'll get to this at the end of our little history lesson but there was a giant patent fight over this technology that like really made headlines so I think it's fair to call it a technology exactly

0:04:34 Thomas: Well anyway um seemed like you were about to tell us that it that was the resultof some obscure you you or studies of something that led to this technology so I'm really curious to hear that story yeah

0:04:47 Eric: And let's start with what crisper is so it was a really good question you just said and what we're going to talk about when we talk about CRISPR is it's C-R-I-S-P-R, right? It's an acronym.

0:04:57 Thomas: They don't out a spell. Yeah. Oh no.

0:04:59 Eric: What that stands for is Clustered Regularly Interspersed Short Palindromic Repeats. And make sure we're all on the same page when we're talking about those palindromes, we're taking about like a man, a plan, a canal, Panama, right, those things that can be said forward and backwards in the same way. So this lab in Japan in the late 1980s found, was that bacterial genomes had this really interesting thing that looked kind of like junk DNA. They'd have these clustered, regularly interspersed base pairs, right, sequences of about 30 base pairs that were palindromes that seemed like they weren't producing any protein, right? Typically, what we use our DNA for is to make copies of this other molecule called RNA, and that will turn into protein. And it's protein that actually gives us our traits, what makes us who we are, right? The reason why genes are generally important is because they code for proteins. These pieces of DNA that they found did not seem like it coded for any protein. It seemed like they were these very random sequences that had these palindromes on either side of it, right. It would go like palindrome, random piece of DNA, that they they couldn't figure out what it's doing, and then another palindrome. And it would go like that for a bunch of hundreds and hundreds of base pairs at a time. Like many things in science, they thought, I couldn't get a grant out of this. I have no idea what's happening. I'm going to shelve this for a while. And for about a decade or so, they weren't really followed up on. And then a graduate student working at a lab in Spain made this their project. What is going on on with these. And what he found was in between those regular palindromes, right, the things that were more ordered, the random pieces of DNA were short little pieces of dna that kind of matched the same sequence that you would find in different viruses that infect bacteria, what we call bacteriophages.

0:06:51 Eric: I know you've done at least one episode on immunology. So as a teacher, I'm going to try and see if we can come to this together. Have you seen anything similar where where you have these like regular pieces of the genome that then gets variable sequences based on the types of viruses you encounter in human DNA. Can you think of any system that kind of works similar to that?

0:07:10 Thomas: Well, I mean, you're just, uh, trying to try me to something, but, um, strikes me as like, so you have like little snippets of something in between.

0:07:17 Eric: Exactly. You have little snippets of viral DNA in between regular DNA.

0:07:20 Thomas: So, it's either like a, like a vaccine or like a just an antibody or something? Is it something like that?

0:07:27 Eric: It's very similar to the way our B cells do genetic recombination to produce antibodies, right?

0:07:32 Thomas: Yeah.

0:07:32 Eric: And so these scientists being very smart people saw that similar pattern where it's like, this is very similar to our adaptive immune system. So they started calling it the bacterial adaptive immunity, right? Now we went from this random bit of sequence. Now we know it's doing a very important function in the bacteria, but it's still not quite relevant for humans yet. About a decade after that, another group started to realize that what you can do is you can use the bacterial immunity that works slightly different than the way ours does. Us. The way ours does is we produce antibodies, right? And those antibodies are proteins that recognize pieces of the virus and then neutralize it. They either market for destruction or they destroy the piece of protein they're recognizing themselves. What this bacterial DNA was doing was it was taking that DNA from the virus, incorporating it into its genome. Then the next time that virus infects the bacteria, it's got a copy of that viral DNA in its library, right and so then it sees that dna again and it goes oh wait I recognize this dna I have it in my own genome and it will produce what we call rna rna is very similar to dna but it's like the temporary cousin it's what you use on the day-to-day basis while your dna is the permanent this is who I am the rna's your day-to-day instruction a good analogy I can think of is like, if DNA is your band's entire discography, right? It's what you have on wax in your masters. It is repeatable for everyone, anywhere, at any time. RNA is like the set list that you bring on tour with you, right. It might change day by day. It Might not have every gene, every song in your discography. But it has the ones you need to have that concert at that time and place.

0:09:15 Thomas: And it has a lot of the newer albums, even though no one wants to hear that.

0:09:17 Eric: That Exactly. Right. So you got a newer virus. And it's like, we don't want to play that virus that we got from 1985, we want to get the one from 1993.

0:09:24 Thomas: Play Hotel California. Yeah.

0:09:26 Eric: And then there's the one virus asking it to play Freebird, but that's a whole other thing. Point being, right, is that it uses these pieces of RNA to match onto that same sequence of viral DNA, and then it recruits a protein that we call caspase. Caspase, whenever you hear that A -S -E at the end of a molecule, is telling you it's an enzyme. It does a chemical reaction. And what What this caspase does specifically is it cuts up that DNA into like little short pieces. So what the bacteria was doing, it was recognizing the DNA in the virus, getting a matching sequence of RNA that it had stored in its library from a prior infection, and then targeting that virus for destruction. Here's the key. RNA is just a sequence of bases in the same way that DNA is. So eventually, really smart scientists started realizing that you can use this system and can make your RNA whatever you want it to be. You can make that sequence that you're going to use whatever you wants. And in particular, you can even have it target human DNA instead of virus DNA. And you have that protein targeting human DNA, you now have a tool to both remove DNA and even alter and change it, as we'll get into. That's the real key that happened in about 2012. A researcher in California, Jennifer Doudna and her lab, along with a French researcher, Emmanuelle, and I'm going to butcher this name, Charpentier, both kind of collaborated to be able to take the system out of the bacteria, give it its own RNA that they created in the lab, and then in a test tube, targeted human genomes, right? So now you're not altering the viral DNA in that test to you're altering the human DNA.

0:11:10 Thomas: What's the bacterial? I think I didn't quite fully understand the bacterial part of the so we're looking in the friggin frogs who are gay or or whatever. And we're like, there's bacteria in there or the kind of DNA it has is bacteria. I don't, what's bacterial DNA?

0:11:23 Eric: So, yes. This is a totally separate thing. The frog was just an example. The bacteria is what was being studied. And bacteria, just like humans, have their own contained cells that they want to keep alive.

0:11:27 Thomas: Gotcha. Ah, I got stuck on the frogs. Okay. No, no. No problem. And

0:11:35 Eric: Yeah. Sorry. OK, let me make that a little more clear. So it's The Bacterial Defense System Against Viruses Infecting Bacteria.

0:11:43 Thomas: So I think what question is, is this kind of defense system something we already have, or in our bodies, or Is it that we're just using this to change our dna yeah okay

0:11:54 Eric: Is very much the latter. We use antibodies, right? We make proteins that target viral proteins, and that's our main mechanism of turning on our immune system. We have a whole bunch of other specialized cells that we can recruit. We don't need to deal with each virus at an individual molecular level. We can deal with it at a cellular level because we have those specialized immune cells, right. Bacteria just has itself to rely on. So it can't wait for those immune system cells, those B cells, those T cells, those macrophages to come over and fight its fight. Got to take it to the virus at the point of when that virus is injecting its DNA. And so what it does is it incorporates that DNA into its genome. So that the next time that virus tries infect it, will have a copy and it will create a piece of RNA that is matching to the viral DNA. So let's say the viral DNA is ATT, right? Obviously, it's going to be a lot longer, but I'm going to use three bases to show the principle. Every base pair in DNA and RNA has a partner. You have the letter C, because of the way chemistry works, a cytosine, the letter C, can only bond with a base pair that's a guanine ,the letter G. So C and G are what we call base pairs of each other. They're complementary. A can only bind with thymine. It will always bind with T. if you have a DNA sequence, that's ATC. Making an RNA sequence, and I'm going to do one thing wrong just for the ease of it. If you make an RNA sequenced, that's TAG, you are now complementary, right? That A is binding with the T, the T is binding within A and the C is binding what the G. They're matching up because of chemistry. And now you've neutralized that DNA if you're the bacteria. So that's what CRISPR was was used as. It's like a matching

0:13:38 Eric: System.

0:13:38 Thomas: How so that that neutralizes

0:13:40 Eric: What What it does is it neutralizes it by recruiting another thing. And this is actually, we always say CRISPR. We don't actually use that clustered regularly interspaced palindromic repeats when we do CRISPR in the lab. What we actually use is the protein it recruits called Cas9. And Cas space, which is this type of protein that breaks down DNA, will then get recruited by that RNA. So it's basically like bringing it over saying, I found the viral DNA. You can tell because I'm matching it like Velcro, right? My base pairs are complementary. So this is viral DNA, it brings that protein over and then like scissors, it cuts up that DNA and degrades it.

0:13:40 Thomas: It crazy

0:14:18 Eric: That's in the bacteria. Real genius of what Jennifer Doudna and her group in California did was they realized that you can take any piece of RNA that you want, including ones that are complementary to human pieces of DNA. If you supply that that caspase protein that humans don't normally produce, but you bring it in from a bacteria, you combine that piece of RNA and that caspace, right, now have the ability to target any piece of DNA in the human genome and degrade it. Because that protein is going to make a cut anywhere it recognizes that about 20 base pair sequence. Does that make sense?

0:14:55 Thomas: When you say in the human genome I mean we've talked about before how I thought every like all of our everything has our whole genome are at what level are you targeting here like oh I target my whole body and everything's different or like in fetal tissue you in development we can do like where what level do you can you do that yeah

0:15:14 Eric: That is a phenomenal question. And it's part of the genius and versatility of CRISPR. Because the answer depends on what your question and what you're trying to do is, right? If I'm trying to really change a genetic defect, I'm probably going to want to CRISPR do that Cas9 at the one cell stage, because then every cell after that division will have that genomic edit. But if I'm to fix something in an adult tissue, I's not trying to alter every gene. I'm just trying to alter the cells in that tissue, right. So what you can maybe be do is you can make a virus having that Cas9 and that piece of DNA you want to target. And then you make that virus specific to the tissue type that you want infect. So you can do that because every cell has different proteins that they express on their cell membrane. So if you choose the right collection of proteins to target with your virus, you can then have specificity in where you're delivering the CRISPR. But it's a really good question. And that's one of the big challenges is getting the right balance of like when to make the changes in edits to have the most effect and how to the what we call off -target effects that we'll get to in a little bit. The main point is right now at Jennifer Doudna, right, and her lab, we had the ability to take a piece of human DNA, put it in a test tube, combine it with a synthetic piece of RNA that we we create in the lab for whatever sequence we want, it also that protein, and then in a test tube, we can alter human genomes, right? Chromosomes in those cells in that test tube will now be forever changed in exactly the way you want them to be. Was already a huge deal. Final piece of the puzzle was a researcher at MIT called Dr. Zhang in his lab, then did the the first ever editing CRISPR in human mammalian cells. And that was the final piece of the puzzle. Because now we have the ability to, at a cellular level, edit and treat human DNA like a Word document.

0:15:34 Thomas: Crazy finding

0:17:10 Eric: And this was in 2013, about 30 years after that initial, hmm, that's interesting discovery. That's really what starts kind of the next revolution of CRISPR. CRISPR is, I don't think it's overselling it to call it the single most important biology discovery of the 21st century. And reason why is it's something that you can use in the lab and clinically. In the 20th century, and this is not like I took a poll of every biologist, but I think most biologists would agree with me. There were like five major things that really opened the floodgates of biology.

0:17:44 Thomas: The mosquitoes and the amber

0:17:46 Eric: We will get to mosquitoes, don't worry, you're going to really like the end of this podcast episode, because I know you have a thing about mosquitoes. And we will talk about how to get rid of all mosquitoes on Earth.

0:17:54 Thomas: Not a fan yeah let's do it yeah

0:17:56 Eric: Yeah, we are going to talk about the mosquito sterilization plan at the end of this podcast.

0:18:00 Thomas: let'S crisp let'S those bastards

0:18:02 Eric: But the main point is, in the 20th century, we had about five major, major discoveries, right? We had the ability to sequence DNA, which is important because you want to know what is the genetic code. Had the ability to make antibodies specific and clones of each other, which is important both for treating diseases, but also for researching how proteins work. If you want to label a protein and follow its course throughout the cell, if you wantto detect and recognize whether that protein is there to begin with, you need to use these antibodies that we developed. Want to look at the DNA, youneed to use those sequencing tools. Third big thing we did was cloning. And I don't mean cloning like Dolly the sheep. I mean, using bacteria like little protein factories to make the things that you need. Basically, bacteria can produce these extra chromosomes called plasmids, and you can use bacteria as like a farm for these extra pieces of DNA and then put it into the cell. That could be a bacterial cell or a human cell. So that's kind of how we used to do DNA editing before we had CRISPR, which is why it was such a big thing. Last one that we had that was truly foundational was a really fun thing called a polymerase chain reaction. Polymerase chain reaction, or PCR, is what lets us generate specific copies of DNA. It's what we use to identify people in criminal cases. It is what we use to study new diseases and viruses as they emerge because it allows you to identify what things it is related to. So PCR is something that we use in the lab every day. Is of this lineage where it is foundational in way that we approach biology. It affects pretty much everything that everyone does in their labs on a daily basis at this point. And the reason why is what CRISPR allows you to do is edit specific genes exactly how you would like them to do. No longer do we have to rely on kind of like random mutations from like using, you radiation to alter DNA or using those extra pieces of bacterial chromosomes. Now we can use your own DNA and study it by making changes directly to those pieces of DNA.

0:20:03 Thomas: Your own dna it's like well I feel like I need my my dna to

0:20:06 Eric: Yes. That's why we haven't done any human testing, but the powerful tool, what I mean by your own DNA, is that you're not introducing those extra pieces of chromosome, those plasmids, right? So any changes you make, you don't have to constantly maintain over and over again, day after day, right. It's not like a medicine where you'd have to take it once every three days. Once you make the change you need, it is now a part of who you are. Other things that make it really powerful. Had things that let you edit DNA before, but in the past, it either required you making those plasmids, doing cloning and bacteria, and then putting in those proteins or those plasmid, those extra pieces of chromosome into the cells, or it required using something called talons or zinc finger nucleus. And both of those use proteins to recognize and edit the DNA. Whereas CRISPR, its real powerful part is that it uses RNA. And here's why that's so important. Picture like you're building something, right? And you're build something where you need a custom -fitted piece, like either a custom piece of metal or a custom piece of plastic, right. Before we had 3D printers, you'd have to like find a company that makes these things, right, you

0:21:15 Eric: Have to contract that out, you would have to have detailed drawings and specs that you would send to them. Then they would send it back to you you could test it and maybe it would work maybe it wouldn't and you'd have to keep iterating that process right is more like you have a 3d printer in your house and you just have to type some stuff into a computer code and you get the mold you need if that one doesn't work you try the next one 30 minutes later so it's really put the speed and efficiency with which we edit dna into hyperdrive now a result of that crisper has actually had like a billion dollar patent dispute between the group that put it in the test tube, right? So that group from Jennifer Doudna's lab in UC Berkeley, the ones that brought it from in vivo in living things to in vitro in the test tube and the Zhang lab who took their work in the tests tube and brought it back into the cells. And the patent dispute was, which was was the key step? Was it taking it out of the bacteria into the test tube? Or was it taking it out the test cube into the cell? What we ended up with is kind of a split decision.

0:22:19 Thomas: Why don't they just split it oh

0:22:20 Eric: The Zhang lab got the patent. So when I was making my CRISPR orders, right, when I was designing the things that I would do for CRISPR, I would fill out my order sheets through the Zhang lab at MIT. The group with Charpentier and Jennifer Doudna got the Nobel Prize. So they kind of split the baby there. And I'm not sure that either one is truly happy with that decision.

0:22:37 Thomas: That's pretty good good good resume building yeah huh

0:22:45 Eric: The last thing I want to say about the kind of history of CRISPR before we really start diving into like what it does and how it works is the real great thing that CRISPR did besides a really good case for like that basic research is it shined a light on how we apportion credit in biology. Might not have noticed this, but every time I've mentioned a researcher in this past couple of minutes, I've mentioned that it was the lab or the group, not an individual name.

0:23:12 Eric: And the reason why is because there was this article that was published called the Heroes of CRISPR. The Heroes of CRISPR shined a light on all of the principal investigators, the leaders of the lab that would have brought these things to light, And right? I don't mean this as shade on principal investigators. My mentor was the most important person for my scientific journey. She provided immeasurable help in experimental design in depth of knowledge and expertise and you of altering and changing my plans but I was the one that was coming up with plans and for the experiment right designing my models and mechanisms and it's the same with CRISPR we give the PIs a lot of credit and it is deserved but we very rarely bring up the postdocs right the graduate students the people that are actually doing the research on a daily basis and CRISPR was a flashpoint for this re -examination of who we give credit to, right?

0:23:11 Thomas: Well good

0:24:03 Eric: So it did do a lot of really good things for that. Now, let's talk about why everyone was so interested in getting credit over CRISPR. What does CRISPR allow you to do? CRISPR allows you to is take the wheel on what we call the central dogma of biology, right. What I mean by the central doggma of biology is the way our bodies work is governed by one kind of very central workflow. We start start with our DNA, right? This is why DNA makes us who we are. We start with our dystography. Then turn that into RNA, right, that's the set list on a day to day basis. That's what our cells actually need to accomplish any given situation. And that RNA then turns into protein. Protein is the actual concert itself. It's what that finished product is, and what actually gets made and produced at the end of this. Another analogy I use is if DNA is the menu, RNA RNA is like the order sheet that the waiter is filling out. And the food is the protein that actually feeds the customer, right?

0:24:03 Thomas: Might not be important but why is this called a dogma I always think of dogmas as like kind of arbitrary things that people decided this feels like it's pretty I feel like biology wouldn't be able to like decide a different dogma one day like from the biology pope all

0:25:13 Eric: Was not around when they coined the term, but my guess is it's because it's like that foundational, right, you really cannot do biology without that DNA to RNA to protein workflow. Everything we study in biology builds off that whether it's the theory of evolution, evolution whether it's you science of biomedicine all of that flows from that simple we start with dna dna then gets turned into rna through a process called transcription that rna gives us the instructions for protein what we're doing in CRISPR is we're altering the dna so that the protein will be different and those different proteins are what changes the traits in the actual cell or organism that you're studying so

0:25:25 Thomas: We'll call it something else okay moving on yeah

0:25:56 Eric: Well, here's where it gets even funny because it's biology, right? It's a central dogma with tons and tons of exceptions. So for instance, AIDS, the HIV virus is a huge exception to the central dog. Because it doesn't go from DNA to RNA to protein. It goes from RNA to DNA toRNA to protein in a process called reverse transcription. We are not here to talk about HIV. So let's stick with that dogma of just DNA RNA proteins, right. Do with CRISPR is, let's say I'm studying my gene grainy grainy headlight 3, and I want to make changes to that gene. First thing I'm going to do is I'm gonna get a sequence of that gene, I'm right? Gunna get every base pair that's associated with that gene and then I'm gong to upload that into a computer software that looks for 3 base pairs. Pairs. Three base pairs have to be N, then GG. So it can be any base pair you want as long as the last two of those three are a guanine and a guaning, a G and a G. is the only clue to CRISPR that says I need to cut there, right? I take 20 base pairs upstream, right, the first 20 base pairs right before that, what we call NGG, that P -A -M, or particle motif, right?

0:26:03 Thomas: Don't you're just describing like where got your word document and then you got something that you can control f and you need to be able to recognize it so that you replace it with something is that essentially what's happening

0:27:18 Eric: Let, yes, let me run with that analogy real quick. Let's say every gene has its own page of words in our Word document, Even right. If you have billions and billions of words, right, thousands of chapters in this Word document, the that you will have 20 consecutive letters that are exactly the same is still pretty low just by the power of permutations, right? If you find 20 bases that are specific to your sequence, the odd that you'll be able to target your gene specifically is pretty high, right. You take those 20 bases that right next to the three bases bases NGG, and you make what's called a guide RNA. Now this is about $2 and you can make them overnight. So they are very, very cheap and easy to make. And you get a little piece of RNA that now has your 20 bases that's specific to your gene with that NGG. Now, is so cheap that let's say I'm not entirely sure that this is the 20 base pairs I want to use. I'm going to order or five more. And one of those five guide RNAs is going to successfully target my gene, right? And it's only going to target my Gene because it's got those 20 base pairs, right.

0:27:40 Thomas: Yeah so you're tagging tagging the thing you're trying to edit basically okay

0:28:30 Eric: So, now I am taking those 20 based pairs, and am still doing that cloning process where I have to bring it into a bacteria and turn it into something that I can introduce into my own cloned mammalian cells, right? So let's say that these are cells that I'm growing on a petri dish, not necessarily in a mouse embryo yet, to keep it simple. Now have a piece of RNA that has the 20 bases that's specific to my gene and that PAM, right. The thing I'm cloning is also going to have that caspase protein, that cas9 that actually does the cutting of DNA. Now I'm going to take that plasmid, that piece of DNA that has my guide RNA, the thing specific to MyGene and the caspases, the thing that cuts the DNA, and I'm going to infect it into my cells, right? Piece of DNA is now going to go into the nucleus. That Cas9 and those guide RNAs are going to be produced, and it's going to recognize those same 20 base pairs on my piece of DNA. And it's going to bind onto it just like it would in the bacterial immunity because it's got those complementary base pairs, right. So let's say the 20 bases that I targeted were all all A's, right? Guide RNA is going to be all T's and it's going to make that base pair. It's going to settle down. Then it's Going to recruit that protein over and say, this is viral DNA. We need to cut this. Now you and I know it's not viral DNA, but your cells don't.

0:29:49 Eric: So it's going to take that cast space and it'S going to cut that DNA right where you targeted. Now you have a piece of DNA, just like with scissors, where you have what we call a double -stranded break, right? It's got a gap in between the first part of the DNA on the left of where you made the cut and on the right where you make the cut. You can imagine, your DNA doesn't like having gaps in it. It doesn't just like having random holes. It likes being one continuous sequence. But it's lost the ability to have an ability to refine that information. Because normally, if you want that cell to be able to repair itself, it needs to have base pairs that it can bind to, right? Would know that if there's an AAA, I need to put TTT down. If you're cutting both strands of the DNA, you don't have any template to use.

0:29:48 Thomas: I won't tell him oh

0:30:34 Eric: It's going to do the next best thing. It's going put a random piece of DNA in there, glue it back together and go, well, good enough. Just hope. Here's the problem. We go from that DNA to RNA to of protein, the way it works is each DNA, right? Every three base pairs, every three letters of DNA gives us one amino acid of protein. Right? If my DNA bases read AAA, that will code for one type of amino acid. If my base pairs code for GGG, that codes for another type of amino asset. And it's those amino acids that give proteins their properties. If you're changing the DNA and and you're taking out some of those letters, you're now changing the function of that protein, right? It gets even more destructive. It reads the bases three at a time, you are not necessarily adding or removing in multiples of three. You can introduce what we call a frameshift mutation. And with a frameschift mutation, you're not just changing the one amino acid where you've changed the letters of, of you're changing every single amino acid in that protein afterwards. And the reason why is because let's say you have a string of three letter words, I right? Have a sentence that says the cat ate the rat. Can change one base pair, one letter in that sentence and still be fine, right? If I change the E for an A in the, it'll read a little funky, but most people would be be able to put together that it's still saying the cat ate the rat, right? If I remove that E and now it's TH, cat ate, the rat that C from cat is now part of the, the, that lost the E, right? So

0:30:41 Thomas: Wow and just hope okay yeah

0:32:12 Eric: It's really THC.

0:32:13 Thomas: It's just offsetting and still grouping everything by three it's going to be really confusing kind

0:32:17 Eric: Exactly. And not only that certain groups of three code for what we call a stop codon, because eventually your body needs to know when to stop producing this protein, right, when to no longer use the RNA to make amino acids. And that stop codon is much more likely to come early if you've now changed the order of all the amino acids, so what you're really doing with CRISPR is you're targeting the gene that you want, and then you're doing what you call an insertion or deletion, you're making that frame shift, and you're forcing that gene to be completely disrupted in what we call knockdown. Reason that's so important is if I want to to know what my gene is doing, the gene that I'm studying, the easiest way to know what it's doing is to remove its ability to function and then see what's going wrong in the cell. So what CRISPR lets you do is very, very easily knock out these proteins and then see what'S going wrong. That's already a pretty powerful tool, right? We can actually make it even more powerful. The next thing I wanted to do in my PhD project is I studied spina bifida, right, in particular, the gene that I studied, grainy head like three, actually has mutations in the human population of patients with spina bifida. Unlike my inbred mice, humans don't just have one mutation. Right. Have many, many mutations. And had like three. Which is the gene? That I studied in mice causes mutations when it's mutated. These humans have mutations in grainy. He had like three, that's clearly causing spina bifida. Have so many other mutations that it might not be that the gene that you think is causing the mutation or the phenotype is the one actually causing the phenotype. You actually have to model those mutations in the lab to make sure that they're actually disrupting protein function. What CRISPR allowed me to do was not just knock out the protein, remove the protein. But you could use a modified version of CRISPR. And this is called nickase. And the difference between this Cas9, this Cas -based protein, and the original one in bacteria, is it doesn't make a double -stranded break. It only breaks one of the strands of DNA with the other strand left over. And when your cell does that, when it has that type of break, it will typically use the unbroken sequence as like a, okay, I know something's supposed to go here. And I actually know what the right answer is, because there's that correct sequence right next to it, right? By using those rules of base pairing, it can just fill in the gap that was left over. Does that make sense?

0:34:47 Thomas: Sounds like it's just taking advantage of sort of these rules of how dna what like fixes itself or or repairs or whatever in order to accomplish different things

0:34:56 Eric: It's of the way DNA has a buddy system, right? The way A always binds with T and C always binds mit G. So if you remove both strands of the double helix, you don't have one of the buddies to provide that partner, right? If you only make a cut on one ofthe strands, you have the buddies on the other side of that helix to provide a template. That's already good. If you're a scientist trying to do really cool stuff, you can provide your own copy of the correct sequence only with the mutations that you want to include. So what you would do is you would make a single strand break in the DNA. And then I provide a copy of grainy head like three that I'm providing. And again, this is just a cheap piece of RNA you can get for like $5 in

0:35:40 Thomas: Then you have the hardware story yeah so

0:35:42 Eric: Overnight delivery. At the IDT oligos store. But But the main point is, right, I'm now giving them a copy that has the gene that I'm looking for with the exact mutation in those human patients. So now I have cells that are expressing not no copies of grainy head like three. They're specifically expressing the copy of the gene that is shown in spina bifida patients. Where that can get really powerful is now let's take this 10, 15 years down the road, right? Say you have a very specific subtype of cancer or a very specifically specific subtype of a genetic disease. We now have the ability with CRISPR to really personalize medicine. A big thing people are doing is they're removing immune system cells from patients, using CRISPR on those immune system cells, and then re -injecting those immune system selves into the people. Because let's say, you want to target cancer. You now have immune system cells that are recombined in their genetics in the exact way you need to target those cancer cells specifically.

0:36:44 Thomas: Isn't this solved everything when can I ask that question I don't know if you have more grainy

0:36:47 Eric: Don't know if you remember from the dinosaur episode, but the reason why is where CRISPR is limited is specifically in our knowledge of the biology and those layers of regulation, right? So

0:36:58 Thomas: Yeah it's a big Big -ass Word document, and we don't know exactly how changing it affects things?

0:37:05 Eric: And not only that, I'm saying that it does have a very high degree of specificity, and that's true. It is very, very specific, and we do have a lot of control over it. But think about it this way. Let's say you are running an AI language acquisition program on that same Word document, right? It's going to do very, sehr gut. Every once in a while, if you're telling it to look for those 20 base pair sequences and what they mean, it's going to get 19 of the 20 base pairs right, or it's going to take base pairs instead of 20 base pairs, right? Another way to think about this is, let's say you're trying to sort people into three different rooms, right, you have three different heights of doors that you're using to sort people.

0:37:44 Eric: You have one that's like six feet tall, one that's five feet tall and one that's four feet tall. Should be able very easily to visually get the kids, the really tall people in the short adults. Right, but every once in a while, you're going to have a tall adult that's stooping their head in underneath the five foot door. You're going to have an adult that's really, really confused going into the kid door, kids, you can never fully control. So they're going to go into the bigger doors, right? And it helps to sort visually and you will do 99 % of it. But you're still going to have some of those off target effects, right, you're still gonna have people in the wrong places. And just like with CRISPR, because it's not a machine, it is just the laws of chemistry. And those laws of chemistry aren't perfect, you're going to have what we call off targets where the wrong gene is mutated. You didn't get the thing that you wanted to have happen, have happen. You always want to make sure that you're screening your cells after. And what you do for that is you're basically taking those sequences and you're looking for your genetic sequence. But if you're asking why We're not doing this in medicine yet, it's because of those worries of off target effects. So a really famous story about this is there was a researcher in China and this was not an approved study. This was highly, highly unethical. He wanted to use CRISPR to generate a vaccine against HIV. The way he wanted to do that is there's this one immune system gene where if you remove 25 base pairs in that immune system gene, HIV, as we might know, targets those T cells, those immune system cells. And if you mutate a gene in one of those cells, HIV doesn't have the ability to enter and infect the cell anymore. So he told the mom that he was going to do an experimental vaccine with these kids. But what he was doing wasn't actually a vaccine. He was editing your DNA to remove those 25 bases. What they found when the kids were born was that he didn't actually get the edit that he wanted to do. He didn't actually remove those 25 bases that made them immune from HIV. But he did alter their DNA and about 30 other places in the genome. As you can imagine, you don't want to just randomly edit 30 places in the genome.

0:39:51 Thomas: Was that because he's a shit scientist or what? I mean, he obviously doesn't have, like, ethics, but if someone who was an amazing scientist and knew how to do this very well did that, would it at least have worked, or is it just too unpredictable?

0:40:07 Eric: The reason he's a shit scientist is because a good scientist understands the limitations of a technique in a study and account for that. Right. I would say he was a shit scientist because he didn't account for any off targets and he thought that he would get a hundred percent efficiency in his editing. That's the clinical stuff, but let's talk about why CRISPR is so useful and foundational in the lab. I'm going to talk about a couple of the ways that we can use CRISPR on a daily basis. The main thing that you can use CRISPR for really easily is what we call genetic screens. Screens work in one of two ways. There's forward genetic screens and reverse genetic screens. CRISPR is really, really, really powerful at those forward genetic screens. What a forward genetic screen is, is you randomly mutate every gene in the body, and then you grow out the cells, you grow up the embryos and you see which ones are broken and how they're broken, right? Let's say I'm studying how our cells migrate, right, what I would do is I would try to alter every single gene in that cell and then grow them from a single cell colony so that each population of cells has only one gene mutated and then see which one's are unable to migrate. It used to be a very annoying process that wasn't very powerful, and you would have to use what we call siRNA. siRNA works on that same principle of that bacterial immunity. It takes the RNA, and it's kind of what we call complementary, so it has the base pairs for the RNA you're trying to get rid of, and it binds it and then targets it for destruction. That's one way to remove function of a gene, but that's at the RNA level. It means you have to constantly redo it every couple of days to make sure that it's stably being knocked down because you're not actually changing the DNA. You're changing those individual meal tickets, right? You're

0:41:57 Eric: Changing individual set lists. You're not changing the discography. CRISPR lets you change the discographies that you can just treat them as their own population. And you don't have to do all of those other things that might stress the cell out during your experiments. That's one thing it lets you do is it lets you screen for different genes by knocking out every single gene in the genome and then seeing which ones give you the desired mutation. That's what we call forward genetics, where you look for the change that you want, and then you sequence the DNA and see what changes. What makes CRISPR so powerful is you don't only have to do forward genetics with it though, right? Let's say I want to study our old friend Sonic Hedgehog in my dinosaur building pursuit. I can now target every gene in the Sonic Hedgehog pathway specifically, knock out, remove the function of those individual genes. And now when I say Sonic Hedgehog is needed to build a wing, I can tell you exactly which gene in Sonic Hedgehog is most important to building a wing because it will be that gene specifically that either does or doesn't develop a wing in my embryo when I use CRISPR. That's the real benefit, is the ease and the specificity, right? You can target every gene in the genome, know which gene you're targeting, and do it very cheaply and quickly. So

0:43:13 Thomas: I got a question. When, if ever, would we do this in like a live full-grown person, like, would that ever be something we would want to do where but I mean, would that even work? So like I'm a 37 year old man here and I have some, I imagine it because you have some sort of, you know, genetic, uh, I don't know, disease or whatever it is, like something where it's like, okay, the only cure for this is fixing your DNA. But does that even still work? Cause like I'm all developed. I'm all, you know?

0:43:22 Eric: When we are confident that we can eliminate those off targets, the you would need to do it with — it's like a genetic counselor that would then go like, okay, you have a Tay-Sachs risk allele. And this is going to be less easy to do now with our recent Supreme Court. But basically, you would have to...

0:43:54 Thomas: No, let'S just say, just give me sci-fi hypotheticals. Is there like a condition or something that you can think of where, let's say you're given right now a magical 100 % CRISPR accuracy machine. What's like an application of that?

0:44:09 Eric: Great question.

0:44:10 Thomas: In an adult, like a full, I'm just curious about that. Because it's one thing, I mean, it's pretty easy — I get it — to modify, you know, like fetal tissue and grow your own super human baby. That's easy. No, but like that seems easier to start when, you know, there's fewer cells and just like, but, but a full grown adult, is that even possible with this technology hypothetically?

0:44:27 Eric: So It would be — there's optimizing the off targets and making sure it's specific, but what you're really trying to optimize on that end is the delivery. So when I would give CRISPR to a plate of cells, not every cell would get the change that you needed. In fact, very, very few of them would. It's a numbers game. So what you would need there is you need some type of either virus or delivery system that efficiently targets the cells you want and with 100% efficiency makes the changes that you need. It is theoretically possible to design either a virus or similar to like the little vesicles we use in the mRNA vaccine, making things that target specific cells, bring in that CRISPR-Cas9 along with the guide RNAs, and then you would make the changes you need. The easier one would be — let's say, I'm a Jewish person, right? Let's say I marry another Jewish person, and we both have the Tay-Sachs risk allele. The easiest thing to do would be going into a genetics counselor, going, both of us have the Tay-Sachs risk allele. They would go, okay, here's what I suggest you do. We're going to do this process through IVF. We are going to genetically screen each embryo. And if we see this Tay-Sachs risk allele, we are going to CRISPR that away and

0:45:44 Eric: Make sure that they are healthy babies.

0:45:45 Thomas: But like, is there any, anything where you're like, okay, I'm a mutant in the X-Men universe and, that Senator wants to unmutinize me. Can I change my whole genome or something? You know, like, is there anything, or is that just always too late in the human process?

0:46:01 Eric: You would need some type of virus that has like 100% efficiency in all cells.

0:46:05 Thomas: Well, I'm awarding you that. Like, are there any, I just don't understand how anything works. Like what if I have some sort of, like, are their diseases or conditions where that's the issue and it could be fixed by like changing your DNA as a full ass adult.

0:46:12 Eric: So, Oh, yeah. Okay. Let's say I suffer from sickle cell anemia, right? Cell anemia is caused by a misfolding in your hemoglobin protein, it's a one amino acid change from the normal healthy condition, right. You have one of those groups of three letters, one of the letters is different, it causes that to make a different amino acid, right? The way you would CRISPR that better, although really you're Cas9-ing it better, because again, CRISPR isn't really the thing we're using, it's the Cas, is you would make a guide RNA for that allele, right, you make that 20 bases that you want targeting sickle cell, the sickle-cell gene, hemoglobin. You then also have what you call an HR donor. The HR donor is that piece of DNA you give into the cell that has the correct sequence or the change that you want to make, right? You take both of those things and you package it in like a virus and you make that virus target your blood cells. Well, not your blood cells, but your hematopoietic system, right, your bone marrow, your things that produce your blood cells. You then have that virus infect your hematopoietic system and deliver that Cas9 to the cells. And let's say it's 100% efficient, right. That CRISPR is then going to deliver those guide RNAs. They're going to recognize the sequence of DNA that is your sickle cell anemia gene and recruit that Cas9, that Cas space that you also brought into the cell. And it's going to say, we found the sickle cells. Here's our mission, right? Put the drill on this asteroid and get going, Armageddon style. Cas is going to go on to the chromosome. It's going to edit that DNA. It's gonna use the HR donor that you supplied, and it's gonna rewrite that chromosome so it has a healthy version of that gene. Now when your cells are making the protein hemoglobin, they don't have that misfolded version. They have the right sequence that gives you the right RNA that gives you the correct sequence of amino acids, and now it's folding correctly. And you've cured that human of sickle cell anemia.

0:48:13 Thomas: Let's do it.

0:48:14 Eric: Yes. Problem is, well, I do one quick bridge, because it's actually a perfect segue. The reason why you might not want to do that is sickle cell anemia actually has an adaptive advantage depending on your population.

0:48:15 Thomas: No, that's cool. So Oh, right. Right. Yeah.

0:48:26 Eric: Sickle-cell anemia is bad if you're a homozygote, but if you are a heterozygotic for it, you have resistance.

0:48:32 Thomas: Do I need to censor that? I don't know. Is that, I don't want to get kids.

0:48:36 Eric: Homozygote means both of your, so we get one chromosome from our mom, one chromosome from our dad, right? Homozygote is just like homosexual is same -sex love. Homozigote means both chromosomes are the same. Heterozygotes means both chromosomes are different. If you're a heterozygote, if you've got one healthy copy, one sickle cell copy, you actually have resistance from malaria without the disease. So another thing we're using CRISPR to do is try and solve that problem. This is going to be a good one to end on because it shows both the power and my fears about CRISPR. I just talked about how CRISPR lets you alter DNA. So this. If I put in a gene that made mosquitoes sterile, male mosquitoes sterile, and then I also brought into that same piece of CRISPR that I'm introducing into the chromosome, the whole suite of instructions to make CRISPR and the guide RNA that targets that same sterilization gene, the first time you infect the mosquito DNA, it's going to incorporate that gene into its chromosome. Chromosome. Now it's going to start producing that gene, and it's going to alter the other chromosome now because that same sequence of DNA is going to target the only unchanged chromosome left. It'll no longer alter the CRISPR-changed one because you've already altered the DNA. So it's gonna target with that CRISPR it's now producing in its own body, the healthy chromosome. So now you have a mosquito with two sterile copies of the chromosome. Does

0:50:04 Thomas: So let's do it. Let's get rid of mosquitoes now.

0:50:07 Eric: But here's the real key now. Can that mosquito give birth to anything other than a sterile male?

0:50:11 Thomas: Oh, I dunno.

0:50:13 Eric: It can't, because once it gets one copy of that sterile CRISPR chromosome from the male mosquito, what's its chromosome going to do with that CRISPR-Cas9? It's going to get rewritten again. So you're bypassing that 50 -50 chance.

0:50:24 Thomas: So this is like a way harder way of just slapping every mosquito individually.

0:50:29 Eric: It's way easier because the mosquitoes are slapping themselves molecularly.

0:50:32 Thomas: Well, but if they can't reproduce at all, then so aren't you just like, Oh, okay. So it's, so not as though you're trying to change all of mosquito-dom to be less deadly. You're just getting rid of them all. Love it. Let's do it. I'm done already. Where do I sign? I would check. Write the check.

0:50:44 Eric: Exactly. And the key there, right, right that normally, right? They wouldn't be able to compete and they go away. The key here is that you have to give those sterile versions because you're bypassing normal Mendelian genetics. So any mosquito that has one copy will automatically have two copies of it. And so there is no normal inheritance patterns because you are gaming the system. You are playing with loaded dye. Here is my worries. That sounds great on paper,

0:51:14 Thomas: Yep. I agree. Let's go. Let's get to it.

0:51:16 Eric: The climate is this interconnected global system.

0:51:19 Thomas: We're already ruining it anyway. I, this is, I don't, sorry. I want to kill all the mosquitoes. I don't care.

0:51:23 Eric: I tend to agree with you, but where it gets thorny is we're gonna have countries making those decisions on individual bases for things that aren't individual sovereignty issues, right? Right.

0:51:35 Thomas: That's a good point.

0:51:36 Eric: Mosquitoes are global things, not national things. But we're going to have national decisions on these. And it goes for a lot of my worries about Christmas. Right. Is why, like, it really worries me that we have the state of politics the way it currently is, because we're starting to reach certain thresholds in our development of biotechnology. And we don't have a populace or a governmental body that can reconcile with the regulations that we need. Easily. Easily.

0:52:04 Thomas: Anything that can change an entire population of, like, a certain animal or whatever is definitely alarming. I mean, like not everything is ubiquitous as mosquitoes. I guess insects are maybe like kind of an exception. I mean normally when it's like larger animals, you can kind of track and like understand where they are and what they affect. But when it is this like ubiquitous freaking insect that's everywhere, I guess that's a different story.

0:52:25 Eric: Exactly. And just in general, when we talk about ecology, I tend to go more on the side of being very cautious and conservative because of those cascading effects. So it's a real issue that we have tools that are now more powerful than our stewardship.

0:52:42 Thomas: Yeah, that's been true since like 1945. So.

0:52:46 Thomas: Yeah. Is this a good place to call it?

0:52:49 Eric: I think the eradication of mosquitoes is about as good a capstone as it and

0:52:52 Thomas: Well, let's get on. Let's get on our crisping gloves and just go CRISPR those bastards. I think it's worth the risk. Look, we're going to end the world some way anyway. I feel like if we're gonna accidentally end the World due to climate change or whatever it might be, might as well not have mosquitoes while we do it. It'll just be more comfortable. So that's my vote

0:53:10 Eric: To be honest we're killing the insect populations anyway from that aforementioned climate change. So oh

0:53:14 Thomas: But not the right ones, you know? I feel like we're not killing the right insects. Am

0:53:19 Eric: Yeah, no, we're getting rid of the ones that give us avocados, not the ones that give us malaria

0:53:23 Thomas: Yeah, god, their eggs or something they lay out, boy, that's got to be a big insect yeah All right, well, uh, Eric Jaffe, I think that there's going to be plenty more to talk about at later dates. Thanks so much for taking us through this.

0:53:28 Eric: Our pollinators yeah appreciate Thank you, as always