by Christopher Gerry
figures by Mike MacArthur

The last few years have seen an explosion in our capacity to study the human genetic code. In particular, a technology called CRISPR/Cas9 has been at the forefront of many of these advances, capturing the imagination of scientists and the attention of the general public.

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a type of bacterial self-defense mechanism that was evolved to recognize viral invaders. After some tinkering, however, it can now be used to cut human DNA with exquisite precision. These molecular scissors allow geneticists to either inactivate stretches of the genome or rewrite the code altogether, which can greatly facilitate the study and (hopefully) treatment of human disease.

One of the scientists at the forefront of this rapidly evolving field is Dr. John Doench of the Broad Institute of Harvard and MIT. Dr. Doench joined the Broad Institute in 2009 after earning his Ph.D. in biology from MIT and performing his postdoctoral research at Harvard Medical School. As the Associate Director of the Genetic Perturbation Platform, he oversees the group’s research and development efforts, manages collaborations with scientists all over the Greater Boston area, and offers advice to those who wish to meddle with DNA.

I recently sat down with John to discuss the field’s origins, the promising future of CRISPR-based therapies, and everything in between.

The transcript below has been edited for clarity and brevity. Text in parentheses signifies post-interview editor comments.

Christopher Gerry: What’s your background? How did you get into the genetic perturbations field?

John Doench: Before there was CRISPR there was RNAi, which had tremendous promise as a technology for being able to knock down (decrease) the expression of any gene of interest. My research transitioned to using RNAi to ask questions about what genes were involved in a particular process. When CRISPR came along, it was seen as another way of doing basically the exact same thing. It made sense as soon as I saw it, and I said “Oh, I need to start working on this.”

CG: What are the types of questions that you can ask with both RNAi and CRISPR?

JD: One obvious place is in cancer research. One problem with cancer cells is they keep growing and dividing, and you want to find genes that stop them from growing and dividing. So, you can take panels of cancer cell lines, apply these techniques to knock out (inactivate) every gene in the genome, and see which one prevents cancer cells from growing.

You can also ask very specific mechanistic questions. We did a project with a group over at Brigham and Women’s Hospital where they were interested in the pathogen that causes the majority of seafood illnesses, Vibrio parahaemolyticus. We didn’t know how this thing attached to cells and started its pathogen life cycle, so we did a screen to find the proteins that are required for this pathogen to attach to a human cell and start the infection process.

CG: By a screen you mean systematically going through the genome, knocking out all the genes, and seeing whatever it is you want to see?

JD: Exactly. I might want to find the genes that cause this cell to die, that prevent this pathogen from getting in, or that prevent this drug from working.

CG: What about the differences between CRISPR and RNAi?

JD: RNAi technology reduces the messenger RNA level in a cell (which relays the information in DNA so that it can be turned into protein), but the DNA isn’t altered. Contrast that with CRISPR, where CRISPR technology is a permanent change because you’re modifying the DNA of your gene of interest (which, as a result, permanently alters the corresponding RNA and protein); there’s no going back from that (Fig. 1).

CG: Another difference is the ability to edit the genome. You’ve talked about how you can inactivate certain genes and proteins with CRISPR, but how does the genome editing process actually work?

JD: When we say “knock out” that’s generally a fairly sloppy process and one doesn’t have a lot of control over that; it’s easy to break things. But with gene editing, what we’re actually trying to do is fix things. We can use the CRISPR system to not just direct DNA cutting, but also to introduce new DNA into the cell (Fig. 2).

There are many cases in which a single DNA base change causes one to have a genetic disease like sickle cell anemia. In theory, CRISPR can correct those. You can imagine it would be like genetic surgery where you go in and have your “CRISPR treatment.” Some technician will take (bone marrow) cells out of your body, they’ll be modified in the lab, and then put back into you (Fig. 3). And then you’re cured of that disease forever and ever—it was a procedure you went through when you were five years old and then that’s it, you’re done, you’re fixed. It’s not a conventional therapy, and it still has a ways to go, especially in terms of safety and efficacy. Getting it to work efficiently is a challenge because again: breaking is easy, fixing is hard.

But then there’s also the question of which diseases to go after. Let’s take a disease like hypertrophic cardiomyopathy (HCM, which makes it hard for the heart to pump blood) where there’s a relatively high frequency of the population that has it and it’s due to any number of small mutations in one of various genes. But we’re not going to remove the heart [laughs] and edit heart cells. If we can’t do that, then we need to deliver all of the CRISPR machinery to heart cells, which isn’t really doable now either. So, it’s not like every genetic disease is going to be conquered by CRISPR in the next couple of years; picking the diseases where CRISPR is going to be most impactful will certainly be important.

CG: What about the prospect of using CRISPR for diseases of the blood? Can’t we access the stem cells in our bone marrow (where most blood cells are produced), edit them with CRISPR outside of the body, and place them back into the patient?

JD: You can even expand that idea to modify the immune system to recognize tumor cells, for example. We’re not building a new technology per se when it comes to manipulating hematopoietic (blood cell-producing) stem cells. Not to say that it’s easy, but we’re not starting from scratch here. People get bone marrow transplants; it’s a thing that we know how to do.

But an important issue relating to blood diseases and gene editing is the potential toxicity. If you’re manipulating a million cells in a dish and make the change you want to make, how can you be sure that you didn’t affect anything else? Because if it’s a one-in-a-million event and that cell goes back into the person, that person will get leukemia. That’s not to say you can’t possibly protect against a one-in-a-million event, but you’d have to confirm that this population of cells is clean enough to be put back into a person.

CG: Or maybe put them into a mouse first and see if that mouse develops cancer?

JD: Exactly. But, again, that’s very personalized, which is another way of saying that these techniques are not going to be cheap! This is genetic surgery: you’re going to have an entire team composed of the equivalent of multiple skilled surgeons and nurses taking care of you.

CG: It seems like the ultimate promise of precision medicine brought to fruition.

JD: Yeah, and we normally think about precision medicine in terms of sequencing your genome and then giving you a powerful multivitamin that has all of the right things that you—and only you—need, and it’ll tell you what your diet is…

CG: And then, after that, you’ll be completely disease-free and live until 178…

JD: And you can sit and watch TV all day [laughs]. It’ll be great.

CG: Where do you think the field is headed?

JD: Let’s first talk about its impact on the public: CRISPR therapies. I hope that 2017 isn’t a year of disappointment because there’s clearly a lot of hype around CRISPR right now. One hopes that it’s not overhyped such that when clinical applications have challenges—and they definitely will have challenges—that the pendulum doesn’t swing too far back in the other direction because CRISPR wasn’t a “magic bullet.” CRISPR is going into the clinic in 2017. Obviously, everyone hopes that it’s a smashing success, but it’ll be difficult and one hopes that there will be a commitment to longer-term building of the technology.

When it comes to basic research, that’s where CRISPR is steaming ahead with very few road bumps whatsoever, and there’s a lot of excitement and enthusiasm. People are continually working to find new CRISPR systems that do different things: some cut RNA, some mutate DNA, and some bind to DNA without cutting it. And also, there are a lot of genes out there and we still don’t know what the vast majority of them do. We don’t know what the vast majority of the ones that are expressed in any given tissue do. We don’t know the vast majority of the genes that cause disease.

There’s a lot of basic biological research that continues to need to happen, and CRISPR is going to be one of the first tools in the toolbox that anyone’s going to apply to their particular biological problem of interest.

Christopher Gerry is a third-year graduate student in the Department of Chemistry & Chemical Biology at Harvard University.

For more information:

On CRISPR: see here
On Dr. Doench’s work: see here