CRISPR-Cas9–guided oncogenic chromosomal translocations with conditional fusion protein expression in human mesenchymal cells

Abstract

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.CRISPR-Cas9 | chromosomal translocation | homology-directed repair | EWSR1-WT1 | DSRCT

Generation of chromosomal translocations that lead to conditional fusion protein expression using CRISPR-Cas9 and homology-directed repair

“…We first focused on one pair, gRNA EWSR1-2 and gRNA WT1-1 [25], which gave particularly good scores for target specificity (Fig. S1).…”

“…4D). Furthermore, conditional expression of the EWSR1-WT1 fusion protein was detected by Western blot analysis, and strong induction of a known target of the EWSR1-WT1 fusion, PDGF-A, was also observed [25]. …”

“…The chromosomal translocation was confirmed by fluorescence in situ hybridization, demonstrating a reciprocal translocation and unrearranged chromosomes 11 and 22 [25]. The reciprocal translocation could be generated through NHEJ giving rise to the WT1-EWSR1 fusion (Fig.…”

“…Further, once induced by Cre, fusion gene expression is regulated by the endogenous promoter (e.g., EWSR1 in the example shown here). The protocol described here generates the EWSR1-WT1 translocation in non-immortalized hMSCs, but the system has also proved to be effective in immortalized hMSCs and transformed HEK293 cells [25, 30]. Theoretically, the approach is applicable to unrelated translocations and genomic rearrangements as well as other cell lines.…”

“…Thus, the expectation was that integration of the donor by HDR would be increased by transiently impairing the NHEJ pathway. When cells were not treated with a DNA-PKcs inhibitor, we previously observed a reduction in the number of hygromycin-resistant clones, fewer of these had the correct 3’- junction, and none was positive for amplification across the translocation [25]. …”