Article written by Rosa Martínez Corral.
In less than a decade, the field of genome engineering has been revolutionised by a series of techniques that now allow to accurately, efficiently and economically modify virtually any point in the genome of any organism. It all started in the early 1990s, when a pattern of repetitive sequences was observed in the DNA of certain bacteria. It was subsequently seen that many other types of bacteria also possess these patterns and, after years of research, these random observations have led to a revolution with strong implications both for research and therapy.
These repetitive sequences were part of a bacterial genomic region now known as CRISPR, or Clustered Regularly Interspaced Short Palindromic Repeats. They consist of repetitive DNA sequences that flank DNA fragments from past viral infections. Faced with a new infection by the same virus, the viral DNA fragment generates a complementary RNA molecule. This RNA guides its associated Cas proteins towards the viral genome, and these degrade the DNA of the virus. Therefore, this is a bacterial immune system that, like ours, retains a memory of past infections.
But not only that, the CRISPR-Cas system has also proved a useful tool in the laboratory, leading to a series of powerful techniques to modify the DNA of organisms. In the most widely-used version, an RNA molecule complementary to the region of the genome to be modified is designed in such a way that it guides a Cas protein to make a cut in that specific location. The cell then repairs itself, and depending on the aim and thus how the technique is applied, a mutation can either be repaired or introduced. The first is desirable, for instance, in the case where a genetic defect exists. On the other hand, in order to understand what the function of a genomic region is, scientists often make deleterious mutations to inactivate it and see what the consequence is.
The CRISPR-Cas system is, therefore, a simple and easily scalable tool that allows specific changes to be made to multiple parts of the genome, needing little time and few resources, something unprecedented in the field of genome engineering. For example, to inactivate a gene in a model organism, it was often necessary to cause random mutations and then select those of interest, involving a long and costly process. But with the CRISPR-Cas system this is no longer necessary. It has even been used to make genetic modifications in human embryos, opening new doors to the study of human development and the therapy of inherited genetic diseases.
But there are still problems to be solved. The most important of these is the fact that the system is not perfect and it can make cuts in unintended regions of the genome. It is also necessary to improve the methods that allow the Cas protein and RNA guide to be effective only in specific tissues, particularly if it is to be used as a therapeutic tool.
Francisco M. Mojica is a microbiologist from Alicante, Spain, where he did his PhD and where he still teaches. He was the first to publish the workings of the CRIPSR-Cas system which has in recent years taken biomedicine by storm due to its many potential applications. Mojica, who recently received the Jaime I award for basic research, came to the PRBB to explain the history of CRISPR and what it means.
How did you discover CRISPR?
It was 1993, during my PhD. I was studying how halophilic archae survive the high salinities of their habitats. Analysing their DNA, I found some sequences that seemed to be repeated up to 600 times in a row, with spaces in between, which was very weird. After publishing this, we saw someone else had found them in a completely unrelated microorganism, E. coli, a bacteria species that lives in our bodies, and we thought these repeats had to be important. So, after my PhD I set to study them more in-depth. After 10 years, I found out the ‘spaces’ between the repeats where actually sequences from different virus that infected the bacteria, and that bacteria with a specific ‘spacer X’ were immune to ‘virus X’, while those without that spacer could be attacked by the virus.
It was August, I was on holidays next to the Salinas de Santa Pota in Alicante – just where the archaea I studied 10 years earlier had been first isolated from! I’m not a fan of too much sun and decided to go to the lab with the aircon and do the DNA analyses. It was then, alone in the lab, that I realised this had to be the bacteria ‘immunity system’. I immediately went to my wife: she’s not a scientists but said “looking at your face I know this must be important!”. I told her one day, this would get a Nobel Prize. But the publishers weren’t so excited. The article was rejected from Nature and four other journals. But we knew better. In the cover letter we said this finding “could have tremendous applications in biotechnology, biology and clinical sciences”. Although I never imagined what was to come…
Where is CRISPR present, and how does it work?
It’s pretty much in all unicellular organisms: archaea, bacteria, but also in plants and even viruses! To understand how it works, let’s say it’s like if bacteria, for example, took pictures of the viruses that attacked them and kept a photoalbum of them. Then when they are attacked by a virus, if they have a picture of this virus, they will be resistant to it. The actual way it works is that when the bacteria is infected by virus X, the ‘spacer X’ (the photo) is activated, binds to the genome of virus X and calls the Cas (CRISPR associated) proteins, which then cut the genome of the virus X at the specific site where the spacer X is bound.
And why has it become suddenly so important?
That specificity and ability to cut make it a brilliant tool for genome editing! It didn’t occur to me at the time, because I was thinking only of bacteria, but once Jennifer Doudna and Emmanuelle Charpentier showed the system was able to function in vitro, and the Feng Zhang’s lab made it work in mammalian cells, applications kept on coming and the publications about CRISPR have grown exponentially!
Can you give us a couple of examples of these applications?
The most talked-about is genome editing. You see, in viruses, if their genome is targeted by a spacer and cut, they die. But in eukaryotic cells, for example in humans, there are systems to repair a cut DNA. So one could create a ‘spacer’ that contains a change you want to introduce in a gene – for example, to correct a mutation – and then you introduce it, together with the CRISPR-Cas system, into the cell to be corrected. The spacer will find its complementary sequence and call the Cas protein, which will make a cut. And then the cell repair system will mend that cut, and copy again the missing DNA sequence – only that, when copying, it will introduce the change you have sent in the spacer. This could be used to correct mutations that cause diseases, or to excise an HIV sequence from a cell, as a recent paper showed!
But there are many other potential applications; using it to study the function of genes, or making bacteria that are resistant to several viruses, which can be good for some biotechnology applications.
How has the CRISPR revolution affected you?
Lots of people with genetic problems now call me to ask how I can help them! I’m not a doctor, so I can’t really tell them much, except that this will take years… but truth is things are going so fast! The 1st clinical trial of CRISPR in humans has already started. They will modify T cells from patients to make them able to attack cancer cells. It’s really amazing. It makes me feel so proud of having been part of this.
Have you changed your focus of research to concentrate on potential applications of CRISPR?
No – I’m a microbiologist. I’m still interested in understanding how this system works. The part that is used as a tool is perfectly characterised, but we still don’t not known how bacteria acquire this immunity, and how they distinguish between the virus DNA and their own DNA. I would like to find out, to know for the pleasure of knowing. If there are practical applications, great. But it’s not my aim.
The CRISPR story is a good example of how basic research can lead to unexpected advances in practical terms…
Yes! And sometimes open, non-directed basic research may have more amazing outcomes than that targeted for a specific aim. For example, the CRISPR-Cas is a whole immune system, with the ability to adapt, to ‘take pictures’ of new viruses. Imagine we could transplant it to a person and that it worked for them like it does in bacteria. It would be immunisation à la carte for any living system! But for this to happen, we would first need to understand how it works in bacteria, why they don’t attack themselves… That’s my job.
One of the most recent posts in the “Health is Global” blog, written by Adelaida Sarukhan tries to dismantle some misleading believes that create some doubts on people about whether or not to vaccinate their children.
As Sarukhan says, “The anti-vaccine movement gained momentum more than a decade ago with the publication of a study (with 12 children) showing an association between the MMR vaccine and autism. Since then, the paper has been retracted (and its senior author discredited) because of data manipulation, and a dozen of large-scale studies (a recent one included more than 90.000 children) have conclusively shown that there is no link between the vaccine and autism. Nevertheless, the anti-vaxxers persist with a series of arguments for which there is no scientific evidence but that generate hesitancy among a worrying increase in the number of affluent and educated people who, due to the success of vaccination itself, have forgotten that not so long ago children were dying from diseases such as diphtheria, polio or measles.”
She then goes on to give some scientific evidence against some of those ill-advised arguments:
1. “Vaccines contain toxic substances such as aluminium and mercury”
2. “Too many vaccines can overload the child’s immune system”
3. “Natural immunity is better”
4. “Vaccines cause autoimmune disorders, asthma and allergies”
In the full post here you can read her convincing arguments to make your case in favour of vaccines. You can also find more about vaccines in the “Science uncovered” sections of Ellipse #82 here and #87 here (in both cases, go to page 6!).
“Health is Global” is the blog of The Barcelona Institute for Global Health, ISGlobal, an alliance between academic, government, and philanthropic institutions – including the CREAL at the PRBB – which tries to address the challenges in global health.