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Stem Cell Reports. 2017 May 9;8(5):1408-1420. doi: 10.1016/j.stemcr.2017.04.014.

Efficient Recreation of t(11;22) EWSR1-FLI1+ in Human Stem Cells Using CRISPR/Cas9.

Author information

1
Molecular Cytogenetics Group, Human Cancer Genetics Program, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid 28029, Spain; Department of Biomedicine, Josep Carreras Leukemia Research Institute, School of Medicine, University of Barcelona, Barcelona 08036, Spain. Electronic address: rtorresr@cnio.es.
2
Molecular Cytogenetics Group, Human Cancer Genetics Program, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid 28029, Spain.
3
Hematopoietic Innovative Therapies Division, Centro Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT)-Centro Investigaciones Biomédicas Red Enfermedades Raras (CIBERER), Madrid 28040, Spain.
4
Department of Biomedicine, Josep Carreras Leukemia Research Institute, School of Medicine, University of Barcelona, Barcelona 08036, Spain.
5
VIVEBioTECH, San Sebastian 20009, Spain.
6
Department of Biomedicine, Josep Carreras Leukemia Research Institute, School of Medicine, University of Barcelona, Barcelona 08036, Spain; Instituciò Catalana de Recerca i Estudis Avançats (ICREA), Passeig Lluis Companys, Barcelona 08010, Spain; Centro de Investigación Biomédica en Red de Cáncer (CIBER-ONC), ISCIII, Barcelona 08010, Spain.
7
Molecular Cytogenetics Group, Human Cancer Genetics Program, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid 28029, Spain. Electronic address: srodriguezp@cnio.es.

Abstract

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.

KEYWORDS:

CRISPR; Cas9; Ewing sarcoma; MSC; cancer modeling; cancer translocation; disease model; genome engineering; human stem cells; iPSC

PMID:
28494941
PMCID:
PMC5425785
DOI:
10.1016/j.stemcr.2017.04.014
[Indexed for MEDLINE]
Free PMC Article

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