Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 114, Issue 14, Pages 3696-3701Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1700622114
Keywords
CRISPR-Cas9; chromosomal translocation; homology-directed repair; EWSR1-WT1; DSRCT
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Funding
- NIH/National Cancer Center [P30 CA008748]
- Alex's Lemonade Stand Innovation Award [NIH R01CA185660, R35GM118175]
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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 translocation EWSR1-WT1 found in the aggressive desmoplastic small round cell tumor. The expression of the fusion transcript is under the control of the endogenous EWSR1 promoter and, importantly, can be conditionally expressed using Cre recombinase. This method is easily adapted to generate any cancer-relevant rearrangement.
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