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.
The World Health Organization predicts that depressive disorders will be the greatest contributor to the global burden of disease by 2030. Major depression is thought to comprise a heterogeneous group of diseases caused by genetic, epigenetic and environmental factors. In humans, detrimental early life events, such as maternal neglect or abuse during childhood, are associated with increased risk of emotional disorders including major depression that may persist into adulthood. In fact, experimental and clinical studies have shown that the immaturity and plasticity of the central nervous system during childhood make it particular sensitive to stress at a young age, which may cause significant and permanent changes in brain structure and function.
On the other hand, recent clinical and experimental data suggest that the pathophysiology of several neuropsychiatric disorders, including depressive syndromes, involves activation of the immune system in response to inflammatory agents. In fact, pro-inflammatory cytokines alter tryptophan metabolism, affecting the activity of serotonin, a neurotransmitter with a key role in the modulation of mood. Therefore, the tryptophan metabolic route becomes imbalanced during depression, enhancing an alternative metabolic pathway, the kynurenine synthesis pathway, and decreasing the availability of tryptophan to be metabolized into serotonin. This metabolic change has been directly associated to the development of depressive symptoms in humans and in experimental animal models.
In a recent study published in Progress in Neuro-Psychopharmacology & Biological Psychiatry, we have shown that maternal separation indeed induces both neuroinflammation and long-lasting emotional alterations in mice. The study was developed during Irene Gracia-Rubio’s PhD training at the GReNeC and done in collaboration with other research teams: the group led by Roser Nadal in UAB for maternal behaviour evaluation, and also with Oscar Pozo and Josep Marcos, researchers of the Neuroscience Program at IMIM (Hospital del Mar Research Institute) for the analysis of the kynurenine pathways. To have the opportunity to work with all these researchers in a collaborative project has been a very positive experience.
Our aim was to explore the interplay between depressive symptoms in behavioural models, neuroinflammation, and alterations in the tryptophan-kynurenine pathway since these mechanisms could lead to the discovery of new therapy approaches.
For that, we set up different behavioural models to induce conditions of early life adversity in male and female mice. Although most studies are done only in males, we decided to study female mice since the risk of suffering depression is double in women than in men.
We used two conditions: the maternal separation paradigm in mice as a model of early life neglect, and the standard rearing condition (the ‘control’), in which offspring remained with their mothers for 21 days. We then looked at the effect of both conditions on emotional behaviour during adolescence and into adulthood.
To test these effects, we performed a range of tests of anxiety, depressive symptoms and other emotional-related behaviours. To test anxiety, we used the elevated plus maze, a test that evaluates the capability of a rodent to explore new and stressful environments. Anxiety-like behaviour is reflected by an attenuated exploratory behaviour in mice. For testing depressive-like symptoms, we used the tail-suspension test, a model to evaluate despair behaviour, in this case, the time spent immobile when a mouse is confronted to an inescapable stressful situation.
At the physiological level, we looked for signs of neuroinflammation in different brain areas, and analysed metabolites of the tryptophan-kynurenine pathway to explore the link between depressive symptoms and inflammatory reactions.
Our results showed that adverse events during early life in mice increase risk of long-lasting emotional alterations during adolescence and into adulthood. These emotional disturbances were particularly severe in females. Behavioural impairments, including depressive symptoms, were associated with neuroinflammatory reactions in the two brain regions evaluated (prefrontal cortex and hippocampus).
In conclusion, these findings support the preeminent role of neuroinflammation in emotional disorders. Our results lead us to propose that detrimental early life events such as maternal neglect reproduce most of the behavioural alterations associated with depressive symptoms in mice. These alterations seem to be long lasting since adult mice also showed these emotional alterations. We also found that females were more sensitive to adverse conditions than males since the detrimental effects observed were more intense and persisted longer in time in female mice. Our study also supports the notion that the imbalance of the tryptophan-kynurenine metabolism and the association of neuroinflammatory reactions underlie these emotional impairments under our experimental conditions.
Future investigations will explore the influence of maternal separation and neuroinflammation in other psychiatric disorders, in particular psychotic and drug use disorders.
Gracia-Rubio I, Moscoso-Castro M, Pozo OJ, Marcos J, Nadal R, Valverde O. Maternal separation induces neuroinflammation and long-lasting emotional alterations in mice. Prog Neuropsychopharmacol Biol Psychiatry. 65: 104-17, 2016. DOI: 10.1016/j.pnpbp.2015.09.003.