Efficient Recreation of t(11;22) EWSR1-FLI1+ in Human Stem Cells Using CRISPR/Cas9
Efficient methodologies for recreating cancer-associated chromosome translocations are in high demand as tools for investigating how such events initiate cancer. The CRISPR/Cas9 system has been used to reconstruct the genetics of these complex rearrangements at native loci while maintaining the arch...
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| Format: | Article |
| Language: | English |
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Elsevier
2017-05-01
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| Series: | Stem Cell Reports |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2213671117301686 |
| _version_ | 1818317144384864256 |
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| author | Raul Torres-Ruiz Marta Martinez-Lage Maria C. Martin Aida Garcia Clara Bueno Julio Castaño Juan C. Ramirez Pablo Menendez Juan C. Cigudosa Sandra Rodriguez-Perales |
| author_facet | Raul Torres-Ruiz Marta Martinez-Lage Maria C. Martin Aida Garcia Clara Bueno Julio Castaño Juan C. Ramirez Pablo Menendez Juan C. Cigudosa Sandra Rodriguez-Perales |
| author_sort | Raul Torres-Ruiz |
| collection | DOAJ |
| description | Efficient methodologies for recreating cancer-associated chromosome translocations are in high demand as tools for investigating how such events initiate cancer. The CRISPR/Cas9 system has been used to reconstruct the genetics of these complex rearrangements at native loci while maintaining the architecture and regulatory elements. However, the CRISPR system remains inefficient in human stem cells. Here, we compared three strategies aimed at enhancing the efficiency of the CRISPR-mediated t(11;22) translocation in human stem cells, including mesenchymal and induced pluripotent stem cells: (1) using end-joining DNA processing factors involved in repair mechanisms, or (2) ssODNs to guide the ligation of the double-strand break ends generated by CRISPR/Cas9; and (3) all-in-one plasmid or ribonucleoprotein complex-based approaches. We report that the generation of targeted t(11;22) is significantly increased by using a combination of ribonucleoprotein complexes and ssODNs. The CRISPR/Cas9-mediated generation of targeted t(11;22) in human stem cells opens up new avenues in modeling Ewing sarcoma. |
| first_indexed | 2024-12-13T09:32:39Z |
| format | Article |
| id | doaj.art-780919482ffd49d4a410c727e962bb2a |
| institution | Directory Open Access Journal |
| issn | 2213-6711 |
| language | English |
| last_indexed | 2024-12-13T09:32:39Z |
| publishDate | 2017-05-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Stem Cell Reports |
| spelling | doaj.art-780919482ffd49d4a410c727e962bb2a2022-12-21T23:52:26ZengElsevierStem Cell Reports2213-67112017-05-01851408142010.1016/j.stemcr.2017.04.014Efficient Recreation of t(11;22) EWSR1-FLI1+ in Human Stem Cells Using CRISPR/Cas9Raul Torres-Ruiz0Marta Martinez-Lage1Maria C. Martin2Aida Garcia3Clara Bueno4Julio Castaño5Juan C. Ramirez6Pablo Menendez7Juan C. Cigudosa8Sandra Rodriguez-Perales9Molecular Cytogenetics Group, Human Cancer Genetics Program, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid 28029, SpainMolecular Cytogenetics Group, Human Cancer Genetics Program, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid 28029, SpainMolecular Cytogenetics Group, Human Cancer Genetics Program, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid 28029, SpainHematopoietic Innovative Therapies Division, Centro Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT)-Centro Investigaciones Biomédicas Red Enfermedades Raras (CIBERER), Madrid 28040, SpainDepartment of Biomedicine, Josep Carreras Leukemia Research Institute, School of Medicine, University of Barcelona, Barcelona 08036, SpainDepartment of Biomedicine, Josep Carreras Leukemia Research Institute, School of Medicine, University of Barcelona, Barcelona 08036, SpainVIVEBioTECH, San Sebastian 20009, SpainDepartment of Biomedicine, Josep Carreras Leukemia Research Institute, School of Medicine, University of Barcelona, Barcelona 08036, SpainMolecular Cytogenetics Group, Human Cancer Genetics Program, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid 28029, SpainMolecular Cytogenetics Group, Human Cancer Genetics Program, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid 28029, SpainEfficient methodologies for recreating cancer-associated chromosome translocations are in high demand as tools for investigating how such events initiate cancer. The CRISPR/Cas9 system has been used to reconstruct the genetics of these complex rearrangements at native loci while maintaining the architecture and regulatory elements. However, the CRISPR system remains inefficient in human stem cells. Here, we compared three strategies aimed at enhancing the efficiency of the CRISPR-mediated t(11;22) translocation in human stem cells, including mesenchymal and induced pluripotent stem cells: (1) using end-joining DNA processing factors involved in repair mechanisms, or (2) ssODNs to guide the ligation of the double-strand break ends generated by CRISPR/Cas9; and (3) all-in-one plasmid or ribonucleoprotein complex-based approaches. We report that the generation of targeted t(11;22) is significantly increased by using a combination of ribonucleoprotein complexes and ssODNs. The CRISPR/Cas9-mediated generation of targeted t(11;22) in human stem cells opens up new avenues in modeling Ewing sarcoma.http://www.sciencedirect.com/science/article/pii/S2213671117301686CRISPRcancer translocationhuman stem cellsgenome engineeringEwing sarcomaMSCiPSCcancer modelingCas9disease model |
| spellingShingle | Raul Torres-Ruiz Marta Martinez-Lage Maria C. Martin Aida Garcia Clara Bueno Julio Castaño Juan C. Ramirez Pablo Menendez Juan C. Cigudosa Sandra Rodriguez-Perales Efficient Recreation of t(11;22) EWSR1-FLI1+ in Human Stem Cells Using CRISPR/Cas9 Stem Cell Reports CRISPR cancer translocation human stem cells genome engineering Ewing sarcoma MSC iPSC cancer modeling Cas9 disease model |
| title | Efficient Recreation of t(11;22) EWSR1-FLI1+ in Human Stem Cells Using CRISPR/Cas9 |
| title_full | Efficient Recreation of t(11;22) EWSR1-FLI1+ in Human Stem Cells Using CRISPR/Cas9 |
| title_fullStr | Efficient Recreation of t(11;22) EWSR1-FLI1+ in Human Stem Cells Using CRISPR/Cas9 |
| title_full_unstemmed | Efficient Recreation of t(11;22) EWSR1-FLI1+ in Human Stem Cells Using CRISPR/Cas9 |
| title_short | Efficient Recreation of t(11;22) EWSR1-FLI1+ in Human Stem Cells Using CRISPR/Cas9 |
| title_sort | efficient recreation of t 11 22 ewsr1 fli1 in human stem cells using crispr cas9 |
| topic | CRISPR cancer translocation human stem cells genome engineering Ewing sarcoma MSC iPSC cancer modeling Cas9 disease model |
| url | http://www.sciencedirect.com/science/article/pii/S2213671117301686 |
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