Efficient Recreation of t(11;22) EWSR1-FLI1(+) in Human Stem Cells Using CRISPR/Cas9
Por:
Torres-Ruiz, R, Martinez-Lage, M, Martin, MC, Garcia, A, Bueno, C, Castano, J, Ramirez, JC, Menendez, P, Cigudosa, JC and Rodriguez-Perales, S
Publicada:
9 may 2017
Resumen:
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.
Filiaciones:
:
CNIO, Human Canc Genet Program, Mol Cytogenet Grp, Madrid 28029, Spain
Univ Barcelona, Sch Med, Josep Carreras Leukemia Res Inst, Dept Biomed, E-08036 Barcelona, Spain
Martinez-Lage, M:
CNIO, Human Canc Genet Program, Mol Cytogenet Grp, Madrid 28029, Spain
Martin, MC:
CNIO, Human Canc Genet Program, Mol Cytogenet Grp, Madrid 28029, Spain
Garcia, A:
Ctr Invest Biomed Red Enfermedades Raras CIBERER, Ctr Invest Energet Medioambientales & Tecnol CIEM, Hematopoiet Innovat Therapies Div, Madrid 28040, Spain
:
Univ Barcelona, Sch Med, Josep Carreras Leukemia Res Inst, Dept Biomed, E-08036 Barcelona, Spain
Castano, J:
Univ Barcelona, Sch Med, Josep Carreras Leukemia Res Inst, Dept Biomed, E-08036 Barcelona, Spain
Ramirez, JC:
VIVEBioTECH, San Sebastian 20009, Spain
:
Univ Barcelona, Sch Med, Josep Carreras Leukemia Res Inst, Dept Biomed, E-08036 Barcelona, Spain
ICREA, Barcelona 08010, Spain
ISCIII, Ctr Invest Biomed Red Canc CIBER ONC, Barcelona 08010, Spain
Cigudosa, JC:
CNIO, Human Canc Genet Program, Mol Cytogenet Grp, Madrid 28029, Spain
Rodriguez-Perales, S:
CNIO, Human Canc Genet Program, Mol Cytogenet Grp, Madrid 28029, Spain
Green Published, gold
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