What is CRISPR‑Cas9, the Tool Behind the Gene‑Edited Babies in China?
Earlier this week, it was reported that the world’s first gene-edited designer babies had been born in China. So, we sat down with Debojyoti Chakraborty, PhD, a senior scientist whose lab at the Institute of Genomics and Integrative Biology in New Delhi is a leader in genome editing research in India, to learn all about CRISPR-Cas9, the most common gene editing tool at the juncture of science fiction and real life.
The Swaddle: What does your research focus on?
Debojyoti Chakraborty: My specialty is on developing the genome editing tool based on CRISPR-Cas9, and using it to correct mutations which have disease relevance. In particular, what we are looking at is to correct the sickle cell mutation in Indian patients via their induced pluripotent stem cells.
The Swaddle: What is CRISPR-Cas9? What does it do?
DC: Its origin is in bacteria. What happens is, bacteria are attacked by certain types of viruses known as bacteriophages. When they attack bacteria, viral DNA get integrated into the bacterial DNA, and these make more copies of the virus. In this way, the virus kills the bacteria [from the inside], because it propagates [itself]. Its own DNA is present inside the bacterial DNA [and takes over].
However, bacteria have also evolved a system of immunity against these invading viruses by this CRISPR system. You can think of it like a library where there are a lot of books. Whenever there is a new book that comes in, you put in a catalog number for that book, so that later on, you can go and identify that particular book by which catalog number it corresponds to.
“It is reality, it’s not science fiction. People are doing this. Ethical, social, legal concerns and more public awareness about the technology — about what it can do and what it cannot do — has to start.”
Through this mechanism it can bring in a tiny piece of the viral DNA, and [isolate it within] its own DNA. When the next time the virus attacks it again, this piece of viral DNA is acting as a kind of a marker, a catalog number, to prompt an immune system reaction against the invading virus – a new copy of the same book.
How the [bacterium] does that is by assembling different proteins, of which Cas9 is one of them. Cas9 and these other proteins are specializing in destroying DNA. They can cut and chop DNA into pieces. But they don’t get activated under normal conditions, otherwise they would chop up the bacteria’s own DNA. They only get activated when the viral DNA has gotten integrated into the bacteria and the same kind of virus attacks again. The marker viral DNA gets converted to RNA, which combines with Cas9 to form an enzyme-RNA complex and and chops off the viral DNA thereby not allowing it to infect the bacteria any more. Thus, every time there is a new viral attack, there is a small portion of the viral DNA which gets integrated [into the bacteria] and then that is used as immunity against the same virus once it comes back.
The Swaddle: Then how did CRISPR-Cas9 become a tool to use on the human genome?
DC: This bacterial immunity mechanism was known to scientists for quite some time. Some years ago, some smart people came and thought ‘OK, good, that means that you can actually take this Cas9 protein, and you can make RNA which targets some region of DNA in a human cell, and you can do the same thing.’ So they reconstituted this entire thing, taking Cas9 and targeting a gene in the human cell, and they saw that yes, in the human cell as well, the protein was acting in the same way; it was cutting DNA.
Once DNA is cut, in a human cell, the cell is very smart: it tries to think of a way to repair it, because it cannot afford to have mistakes in its DNA. Most of the times it does this in a quick and dirty manner and can produce small changes in the DNA where a few letters maybe lost or added. However, if done in a precise manner by providing a replacement DNA sequence that can specifically get integrated at the cut site, you can introduce whatever foreign DNA you want into that cut site. That is the basic principle of using Cas9 for therapy: that if you have some mutation in the DNA, you can actually use the Cas9 to cut at that region where the mutation exists, and then substitute a small DNA piece, which has got the corrected sequence.
The Swaddle: The most commonly used version of CRISPR-Cas9 can only reach and edit 10% of the genome. Recent research out of MIT claims to have discovered a Cas9 enzyme that can reach and edit up to 50% of the genome. What does that mean for you, as a researcher?
DC: CRISPR-Cas9 works by recognizing a small DNA sequence on the human genome, called a PAM motive.
A PAM is like a letterbox. Cas9 first goes and knocks on the PAM. And then if the right PAM is present, then the Cas9 is given access to the main door. You basically knock on the door, and it opens. If the person you really want to meet is present, only then can the Cas9 perform its function and can ‘enter the house’ to do its cutting and replacing. The first thing to take care of, in using CRISPR-Cas9 as a tool, is whether there’s a PAM in the region you want to target. If the PAM is there, then the Cas9 can bind to DNA [and do its work].
If you can reach more regions, as in the case of this new Cas-9 enzyme, it also means the Cas9 or whatever similar DNA-cutting protein, can go and accidentally bind to more places in the genome other than the target PAM. This is known as off-targeting. Off-targeting is very bad because, if you want to target just the sickle cell mutation, say, but you end up also cutting some other important gene – which is responsible for liver metabolism, let’s say – then that’s not what you can give as a therapy, because it’s potentially deleterious and dangerous.
Therapeutically, it can have a huge advantage if you can target more regions of the genome. But the concern would still be how far in the off-targeting space it can perform.
“What is important to understand here is that he has corrected the gene in normal embryos and not carriers of a disease. This means that it still falls under the purview of ‘designer baby.’”
Therefore there’s a lot of research which is involved across the world in trying to figure out if you can make this tool much more specific, and not cause off-targets. My lab is actually trying to address this problem, to make better and more specific genome editing agents.
We at IGIB are also focused on using CRISPR-Cas9 to correct the DNA mutation for sickle cell anemia in induced pluripotent stem cells that we make from patients. What we do is we take blood from patients and these blood cells we convert into pluripotent stem cells. Stem cells are basically the first cells of the body – they can give rise to any other cell type in the body. So if you can make a change in the stem cell, you can ensure that whatever cell forms from that stem cell will have that change. Also, you can make that stem cell become whatever you want to make. So in our case, after you make the change of the sickle cell mutation in the stem cell DNA, you would differentiate it, or convert it, into healthy blood cells.
We’re collaborating with doctors and others to try to find how to do this.
The Swaddle: We cover CRISPR most often as a tool that parents-to-be could. How close is that to being standard fertility treatment? Given recent developments, it seems quite close.
DC: There has been a lot of progress in the field of CRISPR research, but there has to be a lot of caution about using it for therapy. Till there is a global consensus on the safety of this technology, embryonic or germline (one that progresses into the next generation) genome editing is not permitted.
The controversy over this has recently been sparked by a Chinese scientist’s claims about producing babies that are genetically edited to make them protected from HIV infection in future. What is important to understand here is that he has corrected the gene in normal embryos and not carriers of a disease. This means that it still falls under the purview of ‘designer baby,’ where you are trying to make a potentially advantageous gene correction that might provide benefits under certain conditions — but not actually correcting any disease.
Of course, in addition to the problems of off-targeting that the babies might have been exposed to, the gene editing actually makes them more prone to some other disorders. Most importantly, HIV protection does not necessitate gene correction and there are many other ways to do that. If such research is not regulated, then people who have the access to such tools will be able to select for traits in their babies, and this is not such a nice thing.
Currently FDA-approved gene therapy trials based on CRISPR are targeting certain blood-borne disorders in patients and this is happening on a case-to-case basis. In China, they have had clinical trials that started a couple of years back. So, it is reality, it’s not science fiction. People are doing this. The safety, efficacy and follow-up, these are the issues where a lot of investment is currently needed. It’s a new thing, therefore, ethical, social, legal concerns and more public awareness about the technology about what it can do and what it cannot do, is also something that has to start. There needs to be a lot of dialogue based on this.
“CRISPR-Cas9 has a lot of potential and a lot of facility to cure possible disorders which do not have a cure at the moment. There has to be a lot of effort to develop this system better.”
The Swaddle: Is that happening? What type of regulatory scene, what discussions are going on in India regarding your research and its potential use?
DC: In India, there has been some draft guidelines that are still in the process of coming out in a final form. There are draft guidelines for using genome editing in stem cells, for example, and there are genome editing task forces that have been built by different government agencies [that provide research funding]. There are a lot of people who are beginning to take up genome editing as part of their research.
But there is clearly a distinction between using genome editing to answer biological questions: For example, if I’m studying a protein in certain types of cells that I grow in the lab, and I want to know what happens when I knock out this protein – does the cell behave in a different pattern? – then I can use CRISPR-Cas9 to knock it out very easily, and that will allow me to address the basic biology question of the function of that protein.
But when you’re doing this for therapy, or alteration, you’re having to do a lot of things in respect to safety, efficacy, and what is the long-term effect of such a protein [cas9] in the human body. Any kind of clinical trial with CRISPR-Cas9 comes with these additional questions.
The Swaddle: Are you aware of any clinical trials here?
DC: No, nothing with respect to CRISPR-Cas9, nothing in India. In the US and China, they do have this currently going on.
The Swaddle: What does popular media / the average person get wrong about CRISPR-Cas9?
DC: The main thing that often gets misunderstood is, you know, it’s a game-changing technology for sure, but one has to be very careful and be very realistic about where you can use CRISPR-Cas9 for and also what are the limitations of it. And also a little education on what rampant or unsolicited use of genome editing can lead to. Designer babies, for example, where you can get a breed of humans which are much more superior and so on. These are things which you can potentially do [using CRISPR-Cas9] but which you do not want to do.
Therefore, a lot of understanding and discussion about the harmful potential of genome editing needs to be there, in parallel with all of the positive things that genome editing has to offer. I think media often times is not very aware of the shortcomings of the technique, and that has to be parallely brought out. As with any DNA changing biological technology, it has its own drawbacks. And those drawbacks are something that can be worked on, and one can make it better and better. CRISPR-Cas9 has a lot of potential and a lot of facility to cure possible disorders which do not have a cure at the moment. There has to be a lot of effort to develop this system better.