CRISPR—no, it’s not a new cutting-edge kitchen gadget for low fat diets, rather it was first recognized by Osaka University researcher Yushizumi Ishino in 1987, who found sequences of interrupted repeats in microbes was unusual because repeated sequences are typically arranged consecutively along DNA. Evidence then came that CRISPR is an adaptive immune system. When a microbe is invaded by a virus, the first stage of the immune response is to capture viral DNA and insert it into a CRISPR locus in the form of a spacer. These spacers can be cut out, but damages the DNA.
Mazhar Adli, PhD, and his team have developed a technique to prevent genes from carrying out their function without causing the extensive DNA damage the current approach requires. This is important because silencing genes allows scientists to understand what individual genes do and identify the ones that cause disease. Gene silencing also may one day let doctors better treat and even cure genetic diseases with abnormal gene activity.
Our genes – the blueprints for life – are segments of the long double strands of DNA in our cells. To silence genes using the current approach, the CRISPR system cuts both strands. Doing this too often causes the cells to die –– a major limitation for CRISPR. Even cuts that are not fatal to cells can have unintended effects that result from the body’s efforts to repair the broken DNA.
Adli’s approach, on the other hand, avoids cutting the DNA altogether. Instead, it takes advantage of the fact that DNA is made of four main building blocks: cytosine, adenine, guanine and thymine. Adli’s method lets scientists use CRISPR to convert one building block into another to artificially create what are called stop codons – the “off” switches that naturally occur at the end of genes. Turn cytosine into thymine, for example, and the whole gene is silenced, meaning there is no protein production from that gene. ABILITY caught up with Dr. Adli to dig deeper into this innovative system that is revolutionizing the new era of science.
Carol Brown: Can you tell us the quick background on CRIPSR [pronounced “krisper”]?
Mazhar Adli: About 30 years ago, Japanese scientists discovered this weird sequence in bacteria, and they called them “Clustered Regularly Interspaced Short Palindromic Repeats”. So CRIPSR.
Brown: Makes me think of food.
Adli: (laughs) Thirty years ago we didn’t know anything about it, and in 2005 people started to realize that some of these genetic sequences in between those palindromic sequences actually doesn’t belong to bacteria, it belongs to some viruses. That of course immediately sparked an idea that somehow the bacteria is able to store a portion of virus genetic information into its own genome. And if the bacteria is doing this, evolutionarily it has to provide some sort of advantage to bacteria. One of the ideas is that somehow, maybe this is an immune system. In a way, you are carrying a photograph of your enemy in your own pocket. If you encounter that enemy again, you can protect yourself better. People started to work on it. How does bacteria use the CRIPSR system as an immunity against viruses?
In 2012 the first major paper was published describing that bacteria actually use this—in this CRIPSR, there’s a protein called a Cas9 protein, and it uses short RNA. Part of its short RNA belongs to the virus, so it’s kind of a complementary sequence to the virus DNA. And this is how bacteria recognizes a virus. As soon as it recognizes this short RNA, it can cleave the DNA from the virus. This gives greater protection to the bacteria, so they can survive better with the viruses.
This sparked the idea that, what if you put this protein and this short RNA in mammalian cells? Can this be used so we can go and cleave specific regions in the mammalian genome? And they showed it also works in the mammalian system. Since you have an enzyme now and RNA, a very short, RNA molecule, you can custom-design it. This gives you the ability to send this protein to anywhere you want in the genome to create these breaks or double-stem breaks in the DNA.
Brown: You’re talking about cleaving. That’s not your approach?
Adli: Right, this is not what we are doing. This is how traditional CRIPSR works. Basically, it goes and cleaves the DNA. After it cleaves the DNA, the cell has to repair this DNA, and when it makes the repair, it makes some random mutations. In a way, it’s kind of genetic scars that are left behind. Some of these scars cause mutations and cause what we call knock-out. Basically, the gene cannot produce a protein because of these random mutations. This is how the traditional CRIPSR works. When people say “CRIPSR-mediated gene silencing,” this is what they mean, the silencing as a result of these DNA breaks and due to these random mutations left behind from the breaks.
What we are doing is completely different. First of all, the traditional CRIPSR works very efficiently with the DNA double-strand breaks and with the scars, but the problem is two-fold. One, we cannot predict what’s going to happen to that DNA. It’s a completely random insertion and deletion at those sites. Second, in our genome, we usually have more than one copy of a gene. In normal cells we have at least two copies. But in the research environment, in cancers and other situations, we have multiple copies of a gene, and when there is a DNA the cell thinks something catastrophic is happening, that the cell is dying and is undergoing what we call apoptosis. It’s a programmed cell death.
So it’s not because we silence the gene or because the gene is no longer present, it just due to excessive DNA damage due to the activity of CRIPSR. So we wanted to overcome this challenge. We are not making any double-stem breaks, we are changing the genetic code. Do you have any biology background?
Brown: You said the word “biology”? What does that mean? (laughter) My undergrad is in biology, but that doesn’t mean I know much today.
Adli: I don’t know how simple I should go?
Brown: Good point. I’d guess the simpler the better.