CRISPR-Cas9-guided oncogenic chromosomal translocations with conditional fusion protein expression in human mesenchymal cells.
Gene editing techniques have been extensively used to attempt to model recurrent genomic rearrangements found in tumor cells. These methods involve the induction of double-strand breaks at endogenous loci followed by the identification of breakpoint junctions within a population, which typically arise by nonhomologous end joining. The low frequency of these events, however, has hindered the cloning of cells with the desired rearrangement before oncogenic transformation. Here we present a strategy combining CRISPR-Cas9 technology and homology-directed repair to allow for the selection of human mesenchymal stem cells harboring the oncogenic translocationEWSR1-WT1found in the aggressive desmoplastic small round cell tumor. The expression of the fusion transcript is under the control of the endogenousEWSR1promoter and, importantly, can be conditionally expressed using Cre recombinase. This method is easily adapted to generate any cancer-relevant rearrangement.
Developmental Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065.
Fabio
F
Vanoli
Developmental Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065.
Mark
M
Tomishima
Developmental Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065.|Louis V. Gerstner, Jr. Graduate School of Biomedical Sciences, Memorial Sloan Kettering Cancer Center, New York, NY 10065.
Weiran
W
Feng
Genome Dynamics of the Immune System, INSERM, UMR 1163, Imagine Institute, Paris, France.
Khadija
K
Lamribet
Genome Dynamics of the Immune System, INSERM, UMR 1163, Imagine Institute, Paris, France.
Loelia
L
Babin
Genome Dynamics of the Immune System, INSERM, UMR 1163, Imagine Institute, Paris, France.
Erika
E
Brunet
Developmental Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065; m-jasin@ski.mskcc.org.|Louis V. Gerstner, Jr. Graduate School of Biomedical Sciences, Memorial Sloan Kettering Cancer Center, New York, NY 10065.
Maria
M
Jasin
D064113:CRISPR-Cas Systems; D053903:DNA Breaks, Double-Stranded; D000072669:Gene Editing / Q000379:methods*; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism; D015514:Oncogene Proteins, Fusion / Q000235:genetics; D011401:Promoter Regions, Genetic; D034802:RNA-Binding Protein EWS / Q000235:genetics*; D014178:Translocation, Genetic; D025721:WT1 Proteins / Q000235:genetics*
C500986:EWSR1 protein, human; D015514:Oncogene Proteins, Fusion; D034802:RNA-Binding Protein EWS; D025721:WT1 Proteins; C583751:WT1 protein, human
CRISPR-Cas9; DSRCT; EWSR1–WT1; chromosomal translocation; homology-directed repair