@CireniaSketches / CNIO.
Felipe Cortés, lead researcher with the CNIO group that created the 'repairome' explains how they did it and the reasons behind this work, published in 'Science'
The human repairome refers to the set of 20,000 types of scars that remain in human DNA after being repaired following a break
The article was co-authored by Ernesto López and Israel Salguero from CNIO’s DNA Topology and Breaks Group; and Daniel Giménez from CNIO’s Chromosome Dynamics Group.
The researcher from the National Cancer Research Centre (CNIO) Felipe Cortés answers key questions about the human repairome, which has been presented in Science. The article was co-authored by Ernesto López and Israel Salguero from CNIO’s Topology and DNA Breaks Group; and Daniel Giménez from CNIO’s Chromosome Dynamics Group.
The human repairome refers to the set of 20,000 types of scars that remain in human DNA after being repaired following a break. The CNIO group has identified these scars – they are the mutations that remain in the DNA after repair – and has made them available to the global scientific community through its website.
Question: You have explained how the scars left on DNA after being repaired vary depending on which genes are missing in that DNA. And the ‘human repairome’ contains all possible scar patterns. You have created it by switching off a different gene in 20,000 different cell populations; causing breaks in the cells’ DNA; and observing their scars after repair. Who came up with the idea to undertake this ambitious research, and why?
Answer: The idea came to me in 2015, before I was at CNIO. It must be almost exactly ten years ago because it was during the holidays. The technology for mass analysis to eliminate each of our genes already existed, and to verify relatively simple effects such as growth, drug sensitivity, etc. We made some previous attempts, but it wasn’t until we joined CNIO and Ernesto, Israel and Daniel started working on the project that the circumstances were right to start in earnest.
We used a simple trick. Using CRISPR-Cas gene editing technology, we simultaneously generated lists of hundreds of repairs that occurred when each of the 20,000 genes was missing. From the beginning we knew it had to work, and that it would be a significant breakthrough. The question was whether we were going to be able to do it on a large scale. And we started with preliminary experiments to determine that.
Were other groups in the world also trying it?
In 2021, just when we had all the conditions in place, a study was published in Cell that used our same technology, but focused on a small group of genes (about 400) that were already known to play a role in DNA repair.
It was a major set-back, because top-tier journals primarily value novelty and that could limit our future publication. But in the end we decided to go ahead, because that study, by focusing on genes with known function, did not contribute much to the understanding of repair mechanisms. We believed that looking at all genes, in addition to creating a very useful reference tool for the entire scientific community, could lead to new discoveries, and this has indeed been the case.
Has it taken you as long as you thought?
Since we made this decision, the process has been very fast, considering the scale of the project: only four years from the start of the experiment to publication is really a very short space of time.
We know that there is at least one other group that has also conducted a similar study, although they have not published it yet. The results will be complementary because, as far as we know, they have been carried out in a different cell line. In our study, we are already seeing that scars change from one type of cell to another.
We have the human proteome, the interactome, the transcriptome… What weight does the repairome carry among all those ‘omics’?
It is a new layer of genomic knowledge that can be integrated with all these others. For example, it is very interesting to compare the similarity between patterns in the repairome with the known interactome. We see that factors that are part of the same complex, or with relevant functional interactions, have a similar impact on scars.
In fact, this is something we have used to identify new genes involved in repair, and this functional interaction analysis is integrated into our repairome query website.
What questions will the repairome help to answer in the short term? And beyond that?
It can be used immediately as a consultation tool. For example, if a gene is identified that is suspected to play a role in repairing breaks, or which may affect the accumulation of mutations in cancer, it could be confirmed simply by looking on the repairome website.
In the short to medium term, the data generated can be used to identify new factors and relationships between repair pathways, possible explanations for mutational patterns, and therapeutic targets for cancer treatment. In the publication, we have only selected, validated and characterised a series of examples to illustrate what the repairome can do; there is much more information there that is now available to the entire scientific community.
Conceptually, why has the cell learned to repair breaks instead of avoiding them?
There are also mechanisms to prevent them. But some will inevitably occur. It’s fascinating. The cell also actively uses breaks for processes in which it needs to generate genetic variability.
This is the case of gamete formation, which generates variability for the next generation, and the maturation of lymphocytes, which generates a sufficiently variable and extensive repertoire of receptors and antibodies that can recognise any pathogen. In these cases, types of repair are promoted that are very likely to create scars.
Why is it important to understand the mechanisms whereby breaks are repaired?
They have very relevant direct implications for health. Incorrect repair results in the accumulation of mutations and genomic instability, which are closely related to tumour development and progression, as well as ageing.
On the other hand, many of the classic anti-tumour agents are based on killing cancer cells by inducing breaks in their DNA, so the factors responsible for repairing these breaks are good targets for increasing the effectiveness of these treatments.
As there are several different ways to repair breaks, knowing the active repair mechanisms in each tumour is very significant when it comes to designing personalised treatments. Although in a more indirect way, the repair mechanisms also influence tumour immunogenicity and, therefore, immunotherapy treatments.
Finally, the targeted generation of DNA breaks is the basis of gene editing by CRISPR-Cas technologies, and in fact, it is what we have used to generate the repairome. Understanding repair mechanisms is therefore essential for the development of efficient and controlled gene editing techniques.
Why are scars on repaired DNA different depending on which genes are present in the DNA?
The genes that are or are not in the DNA will determine the proteins that are present to repair the cell breakage. There are numerous pathways to repair breaks that support, overlap and compete with each other. Each one depends on multiple proteins directly, as well as on indirect factors, such as the overall condition of the cell. The absence or presence of each of these proteins will determine which repair pathways are employed, and therefore, the final outcome of the repair.
The human repairome is available to the international scientific community. What area of research do you think will turn to this website first?
It will attract immediate attention from the fields of DNA repair and genomic instability, as well as CRISPR technologies and gene editing. We expect it will also attract those interested in tumour evolution. We believe it could gradually become a reference tool for the rest of the scientific community.
What will be the next step in your investigation?
Our idea now is to expand the results with new types of cells and specific conditions, combining the inactivation of several genes and adding more mutations to our analysis.
With a sufficiently extensive amount of data, and applying AI models, we should be able to ‘decode’ the relationship between genetic status and mutational patterns in real-world conditions that are more complex than the absence of individual genes, which is the first step we have taken now. This is hugely significant when it comes to establishing personalised treatments, predicting the evolution of tumours, and accurately controlling gene editing.
About the National Cancer Research Centre (CNIO)
The National Cancer Research Centre (CNIO) is a public research centre under the Department of Science, Innovation and Universities. It is the largest cancer research centre in Spain and one of the most important in Europe. It includes around five hundred scientists, along with support staff, who are working to improve the prevention, diagnosis and treatment of cancer.
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