Is It Time For CRISPR 2.0?
Remember the very early days of the digital photo editor Adobe Photoshop? When the technology first debuted in 1989, it was state of the art, but the features were rudimentary compared to today’s high powered program. The tools of 90s-era Photoshop didn’t have the sensitivity or the precision of the version we use today.
Compare that with CRISPR, the gene editor that scientists have used to modify DNA in bacteria, fungi, rice, wheat, fish, and even human embryos. When CRISPR arrived on the scene in the early 2000s, you could say it was like early digital photo editing software. It was a breakthrough that could change the way we treat genetic disorders and diseases caused by mutated DNA. But it only contained one tool—a pair of molecular scissors that can cut out the mutated portion of DNA’s double helix.
It hasn’t stayed that way for long—in fact, enterprising scientists have already expanded what CRISPR can do. In a pair of studies out this week, researchers from the Broad Institute of MIT and Harvard have added two new features to CRISPR’s toolbar. Dr. David Liu explains a new base-editing technique that works like a molecular pencil to edit a single point on the DNA’s double helix without having to make a cut, from a study out this week in the journal Nature. And Dr. Feng Zhang discusses a separate study out in the journal Science this week, which describes a way to make temporary changes to the genetic code by using CRISPR to target RNA.
Dr. David Liu is Vice-Chair of the Faculty at the Broad Institute, professor at Harvard University, and investigator of Howard Hughes Medical Institute.
Dr. Feng Zhang is a core member at the Broad Institute of the Massachusetts Institute of Technology and Harvard University.
IRA FLATOW: This is Science Friday, I’m Ira Flatow. Remember the early days of digital photo editors, like Adobe Photoshop? And at that time, the technology was the state of the art. But the images you could create, well, they’re laughable now, compared to today’s standards.
The tools of programs like a 90s era Photoshop, remember those? You had the pencil, the lasso, the eraser. They didn’t have the sensitivity or the precision of the versions we have today.
And that’s kind of like what’s happening right now with CRISPR, the gene editor that scientists have used to modify DNA in bacteria, fungi, rice, wheat, fish, even human embryos. When CRISPR arrived on the scene in the early 2000s, you could say it was like Photoshop 1.0. It was an amazing breakthrough that could arrange the way we treat genetic disorders and diseases caused by mutated DNA. But it only contained one tool, a pair of molecular scissors that could cut out the mutated portions of the DNA’s double helix.
But like Photoshop, it didn’t stay that way for long. Enterprising scientists have already expanded what CRISPR can do. And in a pair of studies this week, researchers have added two new features to the toolbar– a new editing technique that works like a molecular pencil to edit a single point on the double helix without having to make a cut. And secondly, a kind of genetic eraser, a way to make temporary changes to the genetic code by using CRISPR to target RNA instead the DNA, not having to cut the DNA.
Joining me to discuss what we could call CRISPR 2.0 are my guests Feng Zhang, who’s a core member of the Broad Institute of MIT and Harvard and David Liu, vice chair of the faculty at the Broad Institute, professor at Harvard and investigator of Howard Hughes Medical Institute. Welcome to Science Friday.
DAVID LIU: Thank you. I’m a huge fan, and so it’s an honor.
FENG ZHANG: Thank you.
IRA FLATOW: Well, thank you, Dr. Liu. Let me ask you first. So first we have the scissors, and now we have a molecular pencil. What makes this gene editing technique more like a pencil than, say, a pair of scissors?
DAVID LIU: Standard genome editing methods, including the CRISPR-Cas9 tool that you mentioned, make double stranded cuts in DNA. And that’s especially useful, when the goal is to insert or delete DNA bases. But when the goal is simply to fix a point mutation, which actually accounts for thousands and thousands of the human genetic variants that are thought to cause certain diseases, then a different approach that we call based editing offers a more efficient and a cleaner solution.
In base editing, we directly rearrange the atoms in one base to instead resemble a different base, without cutting the DNA double helix. And that has some advantages, in terms of both efficiency and avoiding undesired byproducts of the edit.
IRA FLATOW: So in other words, you don’t have to– you leave the original as an original? So you sort of have the original template, but you can edit the tiny little pieces of it?
DAVID LIU: Correct. We don’t have to bring in a new piece of DNA that we hope to incorporate into the site of the targeting. Instead, we literally bring a molecular machine to the target base pair and rearrange the atoms in that base pair, so that they instead resemble a different base pair.
IRA FLATOW: So how important is that to combatting diseases?
DAVID LIU: Well, there are more than 50,000 human genetic changes that are currently associated with disease. And the majority of those changes– currently about 32,000– are point mutations, the simple swap of one DNA base pair for another. So from a minimalist perspective, we thought that it would be particularly efficient and avoid unnecessary byproducts, if we could directly convert the mutated base pairs back into the original base pairs, without having to cut the double helix and provoke the response to that cut, which often involves inserting or deleting DNA bases.
Now sometimes, you want to insert or delete DNA bases. And in those cases, the scissors are really the best tool for the job. But if you just want to cleanly convert one base pair to another base pair, this approach called base editing offers some real advantages.
IRA FLATOW: It’s like in any other kind of mechanics. The right tool for the right job.
DAVID LIU: That’s right.
IRA FLATOW: Dr. Zhang, you were one of the scientists behind the original application of CRISPR to DNA targets. But now you’ve expanded that to include RNA. What does using CRISPR with RNA do better or different than what it does with DNA?
FENG ZHANG: So are our genes are encoded in the DNA of our genome. But in order for these genes to do things in cells, they have to become protein. And RNA is the messenger that goes from DNA to protein.
And one of the things that we thought would be great to do is to be able to edit RNA, so that we don’t have to rely on machinery in the cell to be able to edit DNA. Because the previous method using the scissors, once you cut it, you are left to what a cell is able to do to reglue the DNA back together and allow you to make the edit that you want. And so by editing the RNA, we can put in all of the machinery that are required to make the edit into the cell. So that it doesn’t matter what type of cell you’re trying to manipulate, the machinery that we’re putting in is able to make that change.
And one of the nice things about editing RNA is that RNA is not permanent. After a while, it can get degraded by the cell. And this provides a way to reverse the treatment. It’s possible that as our understanding of biology increases over time, we may want to have new and better treatments. And this allows the patient to perhaps have a more easily upgradable therapy.
IRA FLATOW: So in some cases, it might be better to edit the DNA, in some cases better to edit the RNA.
FENG ZHANG: Exactly. I think they are quite complementary tools. Each one will likely have different diseases that are more suited for it.
IRA FLATOW: So it’s sort of like having it make a drug for you without having to give the drug, you just have to edit the RNA to make it for you.
FENG ZHANG: That’s the hope.
IRA FLATOW: Yeah. And Dr. Liu, how do you make CRISPR so that it doesn’t cut the DNA? In other words, how do we turn it from a scissors into a pencil?
DAVID LIU: So the first thing that one needs to do is of course deactivate its ability to cut. And researchers early on, including Feng Zhang’s group, reported how you could introduce simple mutations into the CRISPR-Cas9 itself that would prevent it from cutting DNA. We then attach an enzyme that directly converts one DNA base to another base.
Now in the case of the new base editor that we just reported recently, this was especially difficult. Because it turns out that nature doesn’t provide any enzyme naturally that rearranges the atoms in A to instead resemble G, which was the goal of our study. So we actually evolved such an enzyme in the laboratory ourselves, starting from an enzyme that does a related reaction on RNA. And so we had to go through quite a bit of evolution and engineering of the resulting enzyme in bacteria before we evolved variants that were very good at replacing As with a base that looks like G in DNA. And then we engineered it to work with this non-cutting form of CRISPR and finally added back a little widget that enables us to trick the cell into fixing the other DNA strand.
So DNA comes in base pairs, because it’s double stranded. So our new machine converts the A in one strand to something that looks like G and then tricks the cell into converting the T on the other strand into a C instead. And so the result is the conversion of an AT base pair to a GC base pair.
IRA FLATOW: And are you more successful? Do you have a higher success rate of doing this, using the new synthetic technique than using the natural technique from before?
DAVID LIU: So again, with the caveat that I think CRISPR-Cas9 as scissors has as its strength not converting individual base pairs but rather doing other things. But if you try to use the scissors to introduce a single base pair change, it tends to be not very efficient. And it’s also prone to a number of byproducts. You can often end up with far more stochastic insertions and deletions of bases than you do clean conversion of the initial base pair to your target.
So if you use base editors in general, we see quite a bit more efficient conversion with very few other byproducts. So in our hands, it’s proven so far to be both more efficient and more clean. Again though, I think CRISPR-Cas9 as scissors are particularly well-suited for other applications, like deleting a big chunk of DNA or inserting DNA, things which these base editors are not able to do.
IRA FLATOW: Does the DNA and the RNA ever get– for lack of a better word, ever get confused about what it should be or what you’re trying to do to it?
DAVID LIU: Well, so one of the disappointing findings, which turned into a real challenge for the development of our adenine-based editor, is the fact that the enzymes that do this reaction on RNA were very restricted to working only on RNA. And I think in general, with only some exceptions, but in general, the enzymes in the cell that operate on DNA keep their Ps and Qs straight and only operate on DNA. And those that operate on RNA tend to only operate on RNA. And so this is why I think the developments in Feng’s lab and in our lab are really very complimentary and will both help maximize the scope of these kinds of approaches.
IRA FLATOW: Dr. Zhang, going back to my Photoshop analogy here, talking about the RNA work. Would you say it was sort of like a molecular eraser or sort of like an Undo button or a Search and Replace function like you would have?
FENG ZHANG: It’s kind of like a Search and Replace function. So to build this RNA editor, we used a different type of CRISPR system. There are many different kinds of CRISPRs. And Cas9 has been used for DNA.
Here we used a different Cas, called Cas13, to be able to search and identify a letter on the RNA. And using this Cas13, we can then bring a machine over to the RNA that carries that mutation, to be able to swap out also an adenine for the letter I, which can be interpreted by machinery in a cell as G, to undo the genetic mutation.
IRA FLATOW: So listening to both of you talk now, I’m getting the idea that you can now swap out any of the letters that you want. And that’s part of the news here.
DAVID LIU: It’s not quite the case yet. We hope we’ll get there eventually. But there are four DNA letters, and therefore there’s 12 ways you can swap one letter for another letter, 4 times 3.
Last year we reported a class of base editor that could turn Cs into Ts or Gs into As. And a couple of days ago, we reported the editor that could convert As into Gs or Ts into Cs. So that’s only four in total of the possible 12 ways to convert one base to another base. But those four types of changes, which are collectively called the transitions, those four transitions happen to account for together almost 2/3 of the known pathogenic mutations. That is, the point mutations associated with human disease.
IRA FLATOW: What kind of diseases are we talking about here?
DAVID LIU: Diseases ranging from cystic fibrosis to sickle cell anemia to hereditary hemochromatosis, which is a certain blood disease that results in excessive iron buildup, phenol ketonuria, ALS, Duchenne’s muscular dystrophy, genetic epilepsies, genetic deafnesses, genetic blindnesses. These are all examples of diseases that are well-understood to be caused by the kind of point mutations that in theory we can now reverse. Although much additional work is needed before these technologies are ready to be transitioned into patients.
IRA FLATOW: This is Science Friday from PRI, Public Radio International. In case you just joined us, I’m talking with researchers working with DNA, RNA and CRISPR technology, Feng Zhang and David Liu, both of the Broad Institute. Dr. Zhang, can you give us an example of a disease we might treat with your new technique, with the RNA?
FENG ZHANG: One of the reasons that we wanted to develop RNA editing system is to try to treat diseases in the brain. With the DNA scissors system, it turns out that if we wanted to make a very accurate, precise correction in brain cells, the brain cells don’t have all the machinery turned on to do that effectively. By treating the RNA, it opens up the opportunity to target genetic mutations that may cause autism spectrum disorder, different forms of epilepsy. Of course the technology still early, and we’re still working on making the system more efficient to figure out how to put it into the brain. But those are some of the things that we’re really hoping to be able to address.
IRA FLATOW: And how close– I mean, is that the point we’re at now– I’ll ask both of you– just finding the right delivery mechanism, the delivery system to make the changes in the genetic material?
FENG ZHANG: There are more–
DAVID LIU: Certainly I think– sorry, go ahead.
FENG ZHANG: Go ahead, David.
DAVID LIU: Well certainly, I think delivery is one of the major challenges facing not just the genome editing field but really the explosion of exciting developments that raise the possibility of using macromolecules– large molecules like proteins and RNA and DNA– turning those into human therapeutics. Researchers have had some success doing that. But I think one of the real hidden benefits of this major focus on genome editing is that it’s inspired a lot of laboratories, including both of ours, to look into novel, creative, and hopefully more effective ways to achieve the delivery of these important machines.
IRA FLATOW: Dr. Zhang, did you want to jump in on that?
FENG ZHANG: So in addition to figuring out how to put it into the body, there are also other safety related things that we have to figure out. For example, how– these proteins are from bacteria. And by putting them into the human body, does it cause a strong immune response? And if so, then we’ll have to figure out ways to be able to reduce that.
And also understanding how efficient it is, how much to put into the body, the dosage to give to the patient. Those are all some of the questions that we’re working on addressing.
IRA FLATOW: You both must be very excited with this line of research.
DAVID LIU: It’s pretty exciting. I think if you had told me even just five years ago that we would be able to go into a living cell and change one base to a different base in a programmable manner, I probably would have told you you were reading too much science fiction. But it turns out that the science fiction is science reality now.
IRA FLATOW: It’s more and more everyday. And we want to wish you both good luck. And thank you both for taking time to be with us today.
DAVID LIU: Thanks very much. And congratulations, Feng, on your terrific work.
FENG ZHANG: Thank you very much.
IRA FLATOW: And Feng Zhang is a core member at the Broad Institute of MIT and Harvard. And David Liu is vice chair of the faculty at the Broad Institute and professor at Harvard, an investigator of Howard Hughes Medical Institute. We’re going to take a break and come back and talk about the technologies that are coming, they’re coming real soonish.
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