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.
On December 12th Vadim Gladyshev from the Harvard Medical School, Boston, USA, gave a conference in a packed room at the PRBB invited by Roderic Guigó from the CRG. Gladyshev investigates the molecular basis for natural changes in longevity and the biological mechanisms involved in aging.
The first part of the conference focused on the mechanisms of aging. Gladyshev’s main question was: why and how do things go wrong with age?
At the beginning he introduced several aging theories that have contributed most significantly to the aging debate in the research community. Some of them were built in the 50s based on 19th century insights, whereas others are very recent. According to him, these theories are very different, each of them touching on a particular aspect of the aging process and, within that context, each has its merit, but all are incomplete.
He continued with his own view about aging. He suggested that imperfectness of biological processes leads to inevitable damage accumulation – called deleteriome – causing aging. His research group is now characterizing properties of cumulative damage and its impact on the aging process. They also study cancer as a disease of aging.
While the mechanisms of aging and the process of lifespan control may seem highly related topics, he maintained that they are different areas. To explain the difference, he used a metaphor of a river, where a lifespan would be equivalent to the time needed for the water to flow from the mountain to the ocean. According to him, the route of the river can be changed to make the journey longer, just like lifespan of humans can be extended. However, the fact that the river flows because of gravity can’t be changed, just like we cannot change the fact that the aging process occurs because of imperfectness. So the cause of aging is different from the determinants of longevity.
The second part of the conference was about mechanisms of lifespan control, trying to answer the questions: why do cells and organisms live as long as they do? and how does Nature adjust lifespan?
Gladyshev’s research team uses multiple approaches to address this question. One methodology involves studying the genes of exceptionally long-lived mammals, such as the naked mole rat, the Brandt’s bat and the bowhead whale. The Brandt’s bat (Myotis brandtii) is found throughout most of Europe and parts of Asia, and it often lives more than 40 years.
The naked mole rat (Heterocephalus glaber) is a burrowing animal commonly found in East Africa, well-adapted to their underground existence. They are characterized by small eyes, short and thin legs, hairless body (hence the common name) and wrinkled pink or yellowish skin. Their large front teeth are used to dig. This animal can live up to 31 years, the record for the longest living rodent.
Gladyshev’s group recently sequenced and analyzed the genomes of these animals, and they discovered some of the adaptations that contribute to their long lifespans. They also identified general gene expression and metabolic changes that associate with longer life.
In addition to the evolutionary study of long-lived animals, Gladyshev’s lab focuses on cell types that have different lifespan and in long-lived mouse models. They also do analysis across species and cell culture-based profiling in order to find unique and common mechanisms of longevity. Longevity signatures (based on gene expression) identify candidate interventions for lifespan extension. Ultimately, the researchers would like to find treatments or some other approaches which would help extend life span and diminish the consequences of age-related diseases.
At the end of the talk the public showed great interest on Gladyshev’s research, posing many questions about aging in yeast, epigenetic drift in aging and the relation between lifespan and maturity. In a fruitful and interesting conversation, some in the audience also suggested research approaches such as studying aging in single cells or focusing on the physics of aging. We’ll have to wait for Gladyshev’s next talk to see if some of these suggestions gave their fruits!
A report by Mari Carmen Cebrián
In her latest post, Mar Albà, head of the Evolutionary genomics lab at the UPF-IMIM, explains her group’s research into new genes and their role in mammalian-specific adaptations. You can read the paper she refers to in bioRxiv, the preprint server for biology!
“Many human genes have counterparts in distant species such as plants or bacteria. This is because they share a common origin, they were invented a long time ago in a primitive cell. However, there are some genes that do not have counterparts in other species, or only in a few of them. These genes have been born much more recently. Although they may have appeared by accident, some have acquired useful functions and been preserved by natural selection. We have recently compiled thousands of mammalian-specific gene families and asked which functions they perform. We have found an enrichment in proteins from the immune system, milk, skin and the germ cells. The most recent genes, however, are rarely functionally characterized. The results of this work provide new insights into how new genes originate and what they are selected for.
Read our paper at bioRxiv and tell us what you think!
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.
Cedric Notredame, a group leader at the CRG, tells us in his “Slow bioinformatics blog” his personal and interesting story behind the development of T-coffee, a method for multiple sequence alignment which he developed during his PhD and which is currently widely used.
“For those who have no clue what T-Coffee does, it is a multiple sequence aligner. It means that it takes a bunch of biological sequences – typically proteins – that have evolved from a common ancestor by accumulating mutations, insertions and deletions…”
If you want to know the real story behind the T-coffee success, read Notredame’s blog here!
The World Antibiotic Awareness Week took place last 14-20 November, and the Antibiotic Resistance Initiative ISGlobal team took the chance to explain to the world what are the main difficulties on the fight against antibiotic resistance – a serious problem that threatens our ability to treat infectious diseases and poses a serious risk to the progress made in global health in the past decades. They summarise the issues in four battlefronts:
1- New antibiotics
3- Mechanisms of antibiotic resistance
You can read the whole report here.
The González lab at the Institute of Evolutionary Biology (CSIC-UPF), which focuses on understanding how organisms adapt to the environment, is seeking a lab technician to join their research team. You can read more about this position – with a starting date around February 2017 – here.
You can read a bit about the lab’s citizen science project “Melanogaster: Catch the fly!” in this post.
And here you can see a post about a recent publication of the lab where they discovered several naturally occurring independent transposable element insertions in the promoter region of a cold-stress response gene in the fruitfly Drosophila melanogaster.
Ribosome profiling is a sequencing technique that detects regions in mRNAs that are being translated. Using this technique, researchers have observed mysterious patterns of translation in many transcripts believed to be non-coding (lncRNAs, or long non-coding RNAs). The patterns are very similar to those observed in protein-coding genes but the translated proteins are generally smaller. Aside from their sequence, we know nothing about these peptides. Are they functional? Do they reflect some background noise of the translation machinery?
Núria López-Bigas started her lab on Computational Oncogenomics at the GRIB, within the PRBB, ten years ago. After a very successful decade, we are sad to see her leaving. We wish her all the best in her lab’s new adventure, and we hope the very fruitful interactions she has started with the different groups at the park will continue to prosper.
In her last post on her blog, Núria says thanks to the GRIB, the UPF, the PRBB community and the PRBB Intervals programme… We want to say, thanks to you Núria, for the great research you have done and for being such an open, collaborative and supportive person, both within the scientific community at the park and with outreach events for the general public! You will be missed. Good luck and see you soon!