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Stem Cell Reports. A protein, a ‘molecular staple’ and CRISPR to generate an Ewing sarcoma model


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The technique makes it possible to generate translocations in the genome in an efficient and accurate manner

The model facilitates the study of mechanisms responsible for certain pathologies

A team from the Spanish National Cancer Research Centre (CNIO) has optimized a system capable of generating a cellular model of Ewing sarcoma. The technique, based on CRISPR and described in the pages of Stem Cell Reports, makes it possible to generate cellular models to analyse the mechanisms underlying the origin and progression of this and other diseases, as well as the search for new treatments.

CRISPR, the famous genomic editing technique, not only serves to cure diseases but also to recreate them in cellular models to study the molecular events that give rise to them. These models are crucial to study new diagnostic and therapeutic pathways. In the paper published in the journal Stem Cell Reports, the authors present a significant technological development capable of recreating Ewing sarcoma in adult and embryonic human stem cells.

“The idea is to have a system that enables us to generate a model that is as accurate as possible to what is happening in a tumour,” says Sandra Rodríguez Perales, from the Molecular Cytogenetics and Genomic Engineering Unit and leader of the research project.

In this way, with a model that reproduces the origins of the disease, it will be possible to analyse the underlying mechanisms and molecular bases of each pathology. In the case of Ewing sarcoma, the trigger of the disease is a translocation between chromosomes 11 and 22, which gives rise to the fusion of two genes, resulting in a new oncogene.

The authors had already used CRISPR to induce this alteration and generate a model of this disease, but they had encountered a low level of efficacy and other methodological difficulties when applying the technique to human stem cells. “When we were working with cell lines, everything went smoothly, but when we applied it to stem cells, we came across a lot of problems,” explains Raúl Torres Ruiz, co-author of the paper. 

To improve the results and to refine the technique, they compared three strategies to generate this translocation in the most efficient way possible using CRISPR. After several experiments, they noted that by combining the use of a sgRNA-Cas9 ribonucleoprotein complex generated in the laboratory (in place of a plasmid expression) and of a DNA “staple” – a short sequence that connects the ends of two chromosomes that breaks the CRISPR system and therefore facilitates translocation–? the success rate increased by up to a multiple of seven. This suggests, according to the authors, that we are faced with “a solid tool to induce targeted translocations”.

All the improvements implemented during the study have enabled the authors to generate this model in induced pluripotent stem cells (iPSC), which have enormous potential from a scientific point of view, since they constitute an ideal cellular model for the study of the development of various pathologies, among them the initial stages of oncogenic processes. All this will allow the study of the mechanistic bases of pathologies such as Ewing sarcoma.

In addition, it may not only be useful for this sarcoma, it is also “a valid approximation for other pathologies,” says Rodríguez Perales. “This strategy – the authors conclude- will facilitate the creation of cancer models from human stem cells and accurate genome editing to search for new drugs or cellular therapies, thus accelerating the transfer of research results from the bench to the clinic.”

This research has been funded by the Spanish National Research and Development Plan, Instituto de Salud Carlos III and FEDER (PI14/01884 and PI12/00425), and Lady TATA Memorial Trust.

Reference article

Efficient recreation of t(11;22) EWSR1-FLI1+ in human stem cells using CRISPR-Cas9. Raul Torres-Ruiz, Marta Martinez-Lage, Maria C. Martin, Aida Garcia, Clara Buen, Julio Castaño, Juan C. Ramirez, Pablo Menendez, Juan C. Cigudosa and Sandra Rodriguez-Perales (Stem Cell Reports 2017). DOI: 10.1016/j.stemcr.2017.04.014

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