You can always be judged by your scars. This is the idea that sums up what researchers at Spanish National Cancer Research Centre (CNIO) claim is one of the new breakthroughs in basic and biomedical research, in their release of the “human REPAIRome.”
The CNIO team, led by Felipe Cortés-Ledesma, PhD, head of the CNIO’s DNA Topology and DNA Breaks group, has identified and catalogued 20,000 types of scars that remain in repaired human DNA after a double-stranded break (DSB). They’ve called the resource the human REPAIRome—and have made the data available to the global scientific community through the human REPAIRome portal.
The team claims the human REPAIRome will provide valuable basic insight and allow researchers to rapidly check out how each of the 20,000 human genes affects DNA repair. The REPAIRome could also have clinical utility, they suggest. Being able to interpret the pattern of scarring in a patient’s tumor cells could help determine the best treatment for each cancer type.
“It is an ambitious piece of work, which we hope will become a truly useful resource in cancer research and also in clinical practice,” said Cortés. “It has been a strenuous and painstaking effort because there are some 20,000 patterns, as many as there are genes in human DNA,” added Ernesto López de Alba, PhD.
Senior author Cortés, together with co-lead author López, and colleagues, reported on their work in Science, in a paper titled “A comprehensive genetic catalog of human double-strand break repair,” in which they stated, “These results exemplify the potential of REPAIRome to drive future discoveries in DSB repair, CRISPR-Cas gene editing and the etiology of cancer mutational signatures.” The work was carried out in collaboration with CNIO’s Computational Oncology and Genomic Integrity and Structural Biology groups.
REPAIRome specifically addresses the repair of one of the most serious types of DNA damage, DNA double-strand breaks (DSBs). This is the simultaneous breakage of both strands of the double helix of the DNA molecule and can be caused by an error during DNA replication or by external factors such as exposure to X-rays, sunlight (UV radiation) or drugs.
“DNA double-strand breaks are dangerous lesions because they disrupt the integrity and continuity of the genome,” the authors wrote. Their incorrect repair can cause mutations and genome rearrangements linked to diseases, including cancer onset and progression. Conversely, the team pointed out, DSB-inducing agents, as well as inhibitors targeting DSB repair factors and pathways, are widely used in cancer therapy. “DSBs also underlie the efficacy of a wide range of anticancer therapies, and their targeted induction and repair constitute the basis for gene editing technologies such as CRISPR-Cas systems,” the team continued.
Hence the biomedical importance of understanding how double-stranded breaks are repaired, as well as how to prevent repair. Understanding the human REPAIRome may then help to identify new therapeutic targets. “Understanding the molecular mechanisms of DSB repair therefore has direct implications for understanding tumor evolution and improving therapeutic and genome engineering strategies,” the investigators stated.
DSB repairs do leave a trace. Each repair leaves behind a trail of genetic alterations. Researchers speak of a ‘mutational footprint’ or, metaphorically, the scars left behind after repair. Just as the marks on the skin are different after a cut and a burn, the alterations in DNA after a repair reveal the type of damage suffered.
They also reveal other details about, for example, how the cell has repaired the break. While scars on the skin might let the trained eye recognize the stitch used, in DNA, the mutational fingerprint indicates the repair mechanisms used by the cell. The scar pattern left in a cell’s DNA varies depending on which genes are missing or present.
This point is key, because it has made the current breakthrough possible, according to the CNIO group. “Sequence outcomes at DSB target sites in the form of insertions and deletions (indels) are not random but show specific patterns that define the relative contribution of the factors and pathways involved,” the scientists explained. “Indel profiling in different contexts thus provides a potent tool for our molecular understanding of DSB repair mechanisms.” Decoding the scar to understand the original damage, and its repair, is important in many areas of research, and specifically in cancer. “This is very relevant for cancer treatment, because many cancer therapies precisely work by causing DNA breaks,” commented Cortés.
Cancer treatments often stop working because tumor cells learn to repair the breaks caused by drugs, making tumors resistant to therapy. Understanding how the cell repairs the breaks in each case can help overcome resistance.
The researchers’ achievement has been to reveal how each of our genes affects scarring. To develop the human REPAIRome CNIO researchers generated some 20,000 different cell populations, disabling a different gene in each of them. They then used CRISPR gene editing to create DNA breaks in each, and observed the imprint (scar) left on the molecule after the cell repaired the break. “… we combined genome-wide CRISPR screening and massive-parallel profiling to provide a comprehensive picture of how Cas9-induced DSB-repair patterns are affected upon the disruption of each of more than 18,000 human genes,” they stated. Cortés added, “… if you look at certain scars in the DNA of tumors, you can infer which genes are not working, and this is useful for designing specific treatments.”
One of the main advances that made the study possible was to perform this massive analysis simultaneously in all of the nearly 20,000 populations, rather than one by one. It is a specific technological development that has value in its own right and, “… can be used for future studies that aim to simultaneously analyze the effect of all human genes,” stated study co-first author Israel Salguero, PhD. Co-first author Daniel Giménez-Llorente, PhD, a researcher in the CNIO Chromosome Dynamics group, added, “… this has required a significant computational effort, including the development of new analysis and representation tools.”
The team reported the new findings made possible by REPAIRome, including include new proteins involved in both promoting and preventing DNA repair. They have also discovered a pattern of mutations related both to kidney cancer and to low oxygen (hypoxic) conditions in other tumors. This is a finding that could lead to new therapeutic approaches in the future.
The REPAIRome web portal will allow researchers around the world to rapidly check out how any human gene affects DNA repair, analyze functional correlations between genes and explore molecular pathways involved. “With REPAIRome, we provide a consultative resource for the general research community, as well as a tool to drive scientific discoveries in the DSB repair, CRISPR-Cas gene editing, and cancer genomics fields, with its corresponding biotechnological and clinical implications,” the authors stated. “The REPAIRome resource that we generated provides a rich dataset in nontransformed (RPE1) and cancer (U2OS) cell lines that, with the help of a publicly available and browsable webtool (https://repairome.bioinfo.cnio.es/), can be consulted for the potential involvement of any gene of interest in DSB repair.”
The researchers in addition hope the human REPAIRome will contribute to improving current gene-editing tools, as new CRISPR-Cas systems are based precisely on inducing breaks to cause specific changes in DNA. “A deep understanding of DSB repair mechanisms and their influence on mutational outcomes is of great interest, with broad implications for human health—including cancer development and therapy—as well as for enhancing precision of CRISPR-Cas gene-editing technologies,” they stated.
