Kathy Wang ‘14, THURJ Staff

One of the most remarkable, game-changing technologies to be uncovered in biology has just emerged in the past year: CRISPR/Cas, a new technique – adapted from a bacterial immune system – for editing human genomes. During an interview with THURJ, Kiran Musunuru, Assistant Professor of Stem Cell and Regenerative Biology at Harvard University, says of the emerging technology, “I’ve never seen anything like it in science.”

Musunuru, who is also an inpatient attending cardiologist at Brigham and Women’s Hospital and a research associate at the Broad Institute, works to pave the way to curing heart diseases by identifying genetic variants associated with the diseases. With his research, he hopes to inform the development of future therapies, which would render patients’ own cells disease-free through genetic modification.

Modifying genomes isn’t a particularly new idea—scientists have been modifying and deleting genes in model organisms for decades. But now, with the discovery and adoption of CRISPR/Cas, it takes weeks instead of years to create complex genetically modified mice, and researchers have successfully used the technology to create genetically modified monkeys. This may even mean that there areno longer any significant technical barriers to making designer babies.

A biotechnology company based on CRISPR/Cas technology, Editas Medicine, was founded last year, and has already secured $43 million in funding since its launch last November. The company is developing CRISPR/Cas to correct faulty genes in cells within a human patient’s existing organs, with the goal of curing genetic diseases without transplantation.
This new human genome editing technology will quickly change the way we think about our ability to modify nature, about disease, and about the limitations of our own bodies. The public will have to grapple with the question: How will the definition of illness change if any gene can be “mutated” or “corrected” to perform more optimally within our own bodies, and in our offspring?

Here, Professor Musunuru shares his perspectives on the history, biology, technical barriers, and upcoming potential of CRISPR/Cas with THURJ.

From the Interview:*

THURJ: Why is there so much interest in developing new genome editing technologies? 

KM: Human cells have traditionally been difficult to genetically modify compared to mouse embryonic stem cells, which are then used to generate knockdown genetically modified mice. Fortunately, this disadvantage of working with human cells has been mitigated by the emergence of genome editing technologies. A variety of tools have now been developed.

The double-strand break in DNA allows you to greatly increase the efficiency of genetic modification. It’s part and parcel of what actually happens in homologous recombination. Homologous recombination always has to start with a double-strand break, just as part of its nature. And usually,Musunuru the double-strand has to happen by chance. So, you can imagine it will be a very rare event. And you have to use a lot of tricks to make up for the fact that it’s a very, very rare event, things like antibiotic selection, and so forth, to pull out just the cells that you want that have the event the question. By using a genome editing tool, by consciously going in and generating a double-strand break, you’re jump-starting the process. The cell has multiple repair mechanisms by which it can repair that double-strand break. Depending on what you want to do, whether you want to knock out a gene, or knock in a DNA variant, or knock in a whole cassette, you take advantage of one or the other of these repair mechanisms. And, because they’re all started by a double-strand break, the efficiency becomes much, much higher if you’re able to introduce that double-strand break at will. And so that’s really the whole game of genome editing. Using a genome editing tool increases the efficiency by orders of magnitude.

The very first of these tools, known as the Zinc (Zn) finger nucleases, have been around, at least in concept, for at least a few decades. But putting them into practice has been more complicated. A Zn finger is a DNA-binding domain that binds 3 nucleotides on a gene. You can make a designer protein that can target a particular site of the genome, wherever you want, and introduce a double-stranded break. As it turns out, putting together Zn finger nucleases is not quite as simple as putting together Legos.

But the entire field changed when the 2nd generation technology emerged over the years 2009 to 2012. This technology has an entirely different DNA-binding domain, from a group of plant pathogens. These DNA binding domains come from proteins called TAL effector nucleases. And they evolved in a very distinctive way, whereby they have an array of repeats of fixed size. They’ve evolved such that each of those repeat domains binds or recognizes one DNA base pair. And these proteins have anywhere from 10 to 30 of these repeats. So, it can recognize and bind DNA sequences anywhere from 10 to 30 base pairs in length. And unlike Zn finger nucleases, they turn out to be extremely easy to engineer. And you can make the DNA binding domain and attach it to a nuclease domain, and make a combination of them, and then cut wherever you want in the genome, and the outcome is exactly the same. There was a lot of excitement in the scientific community about having a new set of tools, that are much easier to use and accessible to really any laboratory with some background in modifying genes.

And then, in early 2013, yet another wave of genome editing technology became available, that uses an entirely different system, from bacteria. These are systems that actually evolved a sort of adaptive immune system, although it’s weird to think of bacteria having adaptive immune systems. It turns out in some sense they do: they have the capacity to take pieces of DNA that were introduced into their cells by foreign invaders, convert them into RNA, and store them in the bacterial genome. Bacteria evolved a system whereby they can actually take those RNA sequences and combine them with a Cas protein, and use that to essentially scan any DNA that comes into contact with a match to that RNA sequence. Then, a double-strand break is generated at the matched sequence. So you can imagine why this would be useful to the bacterium. It has been invaded by a virus, it survived the viral invasion, and it kept fingerprints of the viral DNA around via this system. Then it uses the Cas protein and those RNA sequences to recognize any future introduction of that same DNA, and then immediately cleave it. So that’s what I mean by an adaptive immune system. This system has become known as CRISPR/Cas. CRISPR is the RNA component, and Cas is the protein component.

If CRISPR/Cas is a system to make double-strand breaks in DNA, then why not take it out of bacteria and see if you can get it to work in mammalian cells. And this in fact is what happened in early 2013. The CRISPR/Cas system, using a part protein called Cas9, from a particular bacterial species, Streptococcus pyrogenes, was adapted by several groups, simultaneously, for use in mammalian cells, and somewhat miraculously, it works! Subsequent work over the next few months showed that not only does it work, it actually works really well.

Most of the time, the CRISPR/Cas system outperforms the other two in terms of efficiency. Now there’s another advantage to the CRISPR/Cas system. Unlike Zn fingers and TALENs, where you have to make the proteins from scratch any time you want to cleave at a new sequence of DNA, the protein component in CRISPR/Cas is the same. It is the RNA component that you have to change. If you want to retune the CRISPR/Cas system to recognize a different sequence in the genome, all you need to do is change 20 nucleotides in that RNA molecule, and almost magically, it will bind and cleave the new site. Using CRISPR/Cas, all you need to do is make changes to the RNA sequence, and that turns out to be far easier to do than making proteins. That lends itself very nicely to a whole variety of applications.

THURJ: What challenges still exist in implementing the technology?

KM: Now the one big concern about CRISPR/Cas compared to the others is the possibility of off-target effects. Because you designed a system that is intended to make a double-strand break in DNA, there’s always the danger that the system will also make double- strand breaks in places elsewhere in the genome— places you don’t want the breaks to be. The common perception from what data has been published suggests that CRISPRs are less clean with respect to off-target effects than TALENs. It’s not surprising. With strength comes weakness, right? Increased on-target efficiency brings along the increased risk of mutations in other places in the genome where you don’t want them.

For one, it can cause bad things to happen to the cell. For example, if the mutation falls in an oncogene, or tumor suppressor gene, you can imagine really messing up that cell. When you’re doing genetic studies, and you’re using genome editing to make isogenic cell lines, but it turns out that you have introduced other mutations other than the ones you’ve intended, then you could potentially confound your experiments. You might see differences that aren’t actually due to your intended genetic mutation, but possibly due to the off-target effects.

So you have to be careful, but off-target effects are not by any means a deal breaker. You have to make an experimental design that takes off-target effects into account. For example, you probably never want to do an experiment where it’s just one wild-type cell line or clone and one mutant cell line/clone. You probably want to use several of each. That way, if there are off-target effects, it’s unlikely that one cell line has the same off-target effects as another. So if you see consistent differences between cell lines, you can feel pretty confident that it’s due to the genetic alteration.

THURJ: What is your interest in using and developing these technologies?

KM: I’m a geneticist by trade, so I’m very interested in genes that have been discovered from studies in either human populations or individuals who have unusual clinical disorders or syndromes. I’m interested in using stem cells as a model to understand the functional consequences of the genetic variation or the alteration in genes. And this is not by any means a new approach. It’s been done for decades, but primarily with the mouse as a model. What’s new now with the development of CRISPR/Cas is the ability to use human pluripotent stem cells as a model. There are several advantages.

The advantage of human pluripotent stem cells is that they have normal human genomes, and you can pas- sage them in culture for quite a long time, while still maintaining a normal genome, and they’ll still retain their ability to differentiate to different cell types. Also, to study noncoding DNA regions, it becomes important to use a human system. Now, another advantage is that they’re pluripotent, which means that you can differentiate them into a whole wide variety of different tissue types: everything from cardiomyoctes to hepatocytes to neurons. Really, you’re only limited by what protocols are available.

The fact that you now have tools that greatly increase efficiency, now make working with human pluripotent stem cells quite feasible. Now, it’s more and more common to successfully do genetic modification in human cells. Now, you can say, “I want to knock down a gene,” and you can do it in just a few weeks. Or, if you want to put in a genetic variant, you can do it in a few weeks. Now, you can have genetically matched stem cells that are essentially the same because they came from the same source, except that you’ve made one genetic alteration. You take those cells, and differentiate them into whichever cell type you think is of most relevance to whatever particular phenotype you’re studying. So you might, for example, have cardiomyocyte lines, one of which is wild-type, one of which is mutated in a gene that might be important for the contraction pattern of the heart cells, and actually be able to study them in a dish, and say, “Wow, I’ve modified the gene. I’ve changed the property of the cells,” and be actually able to figure out what happens.

THURJ: What are some other applications? 

KM: One of the more recent applications was—this was actually done by two different groups and published just last month—you can make genome-wide libraries that have CRISPR/Cas that target every gene in the genome, or most of the genes in the genome. Just as people have been doing whole-genome RNAi studies, where you knock down one particular gene in each individual cell or clone, you can then study those cells and look for whatever effects you want, and pick out those cells, and figure out which genes were modulated by your RNAi. Now you can do that with the CRISPR/Cas system in a genome-wide fashion. This has been demonstrated; it works nicely, arguably in superior fashion to RNAi. And it’s cleaner in the sense that while with RNAi you’ll never knock a gene down 100%, whereas with CRISPR/Cas, you’ll potentially have 100% knockdown of gene function. And that’s what happens with the application of these libraries. You get a library of cells in which different genes are knocked down, and you can figure out which cells have the properties you want, and figure out which genes are responsible for those properties.

Another application is, because you have a fixed Cas9 protein component, once you put in that protein, if you put in multiple RNAs, then that Cas9 protein can bind to any one of those individual RNAs, and that complex can then go find that magic sequence in the genome and generate the break there. So, if you have multiple RNA sequences that you introduce along with the Cas9, then you can actually knock out several genes at once.

You can do other fun things. For example, if you want to delete a locus or a piece of the chromosome, you can generate two CRISPR RNAs, put them in, and then Cas9 will use those CRISPRs, make breaks on either side of that locus, and at some frequency, [you] will lose all the DNA between the cuts, so you can fully delete not just a small portion of DNA, but eventually a very big portion of the DNA, as large as kilobases, hundreds of kilobases, potentially. This has really been a game changer in a number of respects, because it’s made it much easier to make the system that targets whatever sites you want. You can multiplex it to do multiple things at once to the same cells, and you can make large genome-wide libraries to do whole genome screens.

The other aspect of genome editing technology that is really starting to catch fire now is that it is not limited to cells. What’s remarkable is that it works really well in single cell embryos, and probably across many species. Although no one has publicly announced that they’re doing it yet, people are probably working on human embryos.

What has been demonstrated is that the CRISPR/Cas system works extremely well on mouse embryos, and this has been a game changer. I’m not sure if anyone actually expected it, or saw it coming. Normally, to make a knockout mouse, you have to go through a very elaborate process that takes at least a year. Whereas with CRISPR/Cas, what’s remarkable is that it’s so efficient that you can put it in, inject the CRISPR/Cas system into a mouse single cell embryo, or a rat embryo, or as it turns out, a monkey embryo, and the CRISPR/Cas will introduce an alteration, whether it’s a knockout, or it’s even been shown to introduce sequences into the genome.

Because it’s happened in a single cell embryo, you can implant that embryo into a foster mother. And you let that embryo come to term: That takes about 3 weeks. It works so efficiently that many of the mice that you obtain through the method already have full gene knockouts. So, instead of taking more than a year to generate a full knockout mouse, it’s now down to a month. You can imagine that it’s a huge game changer. And this is not just something with which to do gene knockouts. It’s versatility. You can use it to make conditional knockout mice in just a month. It’s amazing. It’s certainly going to be useful for mice. But it’s going to be useful for a number of species we’ve never been able to target before. Rats we’ve been able to target in the past, but now we can do it much more regularly. Really, lots and lots of species: pigs, chimpanzees, and dogs. Really, there’s no reason to think that this won’t work in any particular species.

It does raise the specula that down the road, someone might, for better or for worse, decide that this might be a useful thing to do in human embryos, and the fact of the matter is, I’m certain it would work in humans. The technology to make designer babies…It exists, it’s there, and it would not actually be technically challenging to do that. It’s been talked about a lot in science fiction, but the time has arrived. Any scientist with some familiarity [with] the technology and [who] has just some familiarity with working with human embryos has access to surrogate mothers, [and] could make a genetically modified human baby.

THURJ: Are you concerned about how the public will react to the technology?

KM: So far, there don’t seem to be any negative repercussions, perhaps because no one has actually used it yet. And even though the scientific community is very, very excited about this technology, I’m not sure it’s percolated out to the lay public yet. It will. It will, because eventually it’s going to be impossible to ignore the fact that, you know, you’re making genetically modified monkeys, and then, it’s a very small step from a monkey to a human. I would expect over the next year or so there is going to be a lot more attention paid to this by the public. And it’s hard to know what the reaction is going to be. It’s going to be very mixed, right?

There are going to be proponents who will say, “Wow! This is a way that we can cure genetic disorders!” And then there are going to be the others who say “Wow! This is a way that we can enhance our baby.” And depending on your perspective, that might be a positive thing, and that might not. Some people say this has opened the door to designer babies, and that’s not a good thing, right? We should not mess with nature. We should let nature take its course. We should not be going in and changing the stuff of life in a controlled way, and giving our babies really any genetically determined trait we want: eye color, hair color, possibly other traits that are at least somewhat genetically determined: personality, athleticism. You know, none of this is very far out of reach, when you’re talking about this or that technology. It’s going to happen. There’s no question. It’s just a question of where, who, and under what circumstances, but there’s really not much of a technical barrier anymore.

THURJ: If genome editing is to be used to treat patients, how do you think that might be implemented?

KM: There’s at least one company that has already been formed to try to develop their therapeutic applications for the technology: It’s a company called Editas Medicine. It just started a few months ago, here in Cambridge, MA actually. There are others, potentially in the works, and there’s a lot of excitement about the technology. The aim would be to use this technology to modify genes within the human body in such a way as to improve the health of human beings. If you had, for example, a faulty gene in the liver that is causing some medical condition—a very serious condition that could only previously be cured by a liver transplant to get rid of the faulty cells with the gene—now you could imagine using the genome editing technology, delivering it in some fashion, by injection or whatnot, so that it gets to the liver and basically fixes that gene. Then, you wouldn’t have to transplant the liver, because it’s now being fixed from within. I think there’s a way to go before that thing happens, but certainly, there’s a lot of interest. I wouldn’t be surprised to see applications of this in the next 10 years.

THURJ: What challenges still exist in implementing the technology?

KM: I think there are quite a few unanswered questions. Can you get to the tissue you want to? Part of it’s going to be the efficacy of CRISPR/Cas. We don’t know yet how it will work in different tissue types. We know that it works in embryos. But that doesn’t necessarily mean it’s going to work well in the liver, or work well in hematopoietic stem cells, or skin cells, or […] particular neurons in the brain.

And then delivery is another issue. It’s going to be much easier to deliver to the skin than it is to [deliver] to the central nervous system, as you might expect. So that’s a barrier that has to be overcome.

And of course, there’s always the safety issue. As I mentioned before, the off-target effects. You can imagine how in human beings, that risk is significant because you could be, for example, trying to cure a genetic disease in the liver. But if even just a few cells happen to pick up off-target effects—mutations in oncogenes or tumor suppressor genes—then you might be laying the seeds for the formation of cancer.

These are all issues that have to be addressed. That doesn’t mean that they invalidate the technology. Every therapeutic approach has its weaknesses, has its adverse effects. It’s always going to be a balance of whether the benefits of the therapy greatly outweigh the risks of it. So we’ll just have to see how things play out. And there’s so much energy going into this field, so many investigators working on this in a variety of different ways, that it’s already happening. We’re seeing strategies by which to reduce the off-target effects. Some very clever strategies. So I feel pretty confident that this is an issue that will be squarely addressed and dealt with as we go forward.

You have to keep in mind, the first use of CRISPR/Cas in mammalian cells was in January 2013. Now we’re still in January 2014. So, a year since the technology emerged. I’ve never seen anything like it in science: Something that has emerged and [been] so rapidly adopted and worked on by so many labs that, every week, a few more papers are published. Not just, you know, small little papers, a little tweaking of the technology, but major innovations in the technology. It’s an exciting time.

* This interview has been edited and condensed for clarity.









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