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Updated: Mar 5, 2026
Engineering Oncogenic Heterozygous Gain-of-Function Mutations in Human Hematopoietic Stem and Progenitor Cells
Published on: March 10, 2023
CRISPR-Cas9-guided oncogenic chromosomal translocations with conditional fusion protein expression in human
Fabio Vanoli1, Mark Tomishima1, Weiran Feng1,2
1Developmental Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065.
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.
Insights
Scientists developed a new gene editing strategy to model cancer-causing genomic rearrangements. This method efficiently selects cells with specific oncogenic translocations, like EWSR1-WT1, for cancer research.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Gene editing is used to model tumor cell genomic rearrangements.
- Nonhomologous end joining (NHEJ) is a common but inefficient repair pathway for these rearrangements.
- Cloning cells with desired rearrangements before oncogenic transformation is challenging due to low event frequency.
Purpose of the Study:
- To develop a novel gene editing strategy for efficient modeling of cancer-relevant genomic rearrangements.
- To select human mesenchymal stem cells harboring the EWSR1-WT1 oncogenic translocation.
- To create a conditionally expressible fusion transcript under endogenous promoter control.
Main Methods:
- Combined CRISPR-Cas9 technology with homology-directed repair (HDR).
- Targeted induction of double-strand breaks at specific endogenous loci.
- Selection of human mesenchymal stem cells with the EWSR1-WT1 translocation.
- Utilized Cre recombinase for conditional expression of the fusion transcript.
Main Results:
- Successfully generated human mesenchymal stem cells with the EWSR1-WT1 oncogenic translocation.
- The EWSR1-WT1 fusion transcript is expressed under the endogenous EWSR1 promoter.
- Conditional expression of the fusion transcript was achieved using Cre recombinase.
- The developed method is adaptable for generating other cancer-relevant rearrangements.
Conclusions:
- The novel strategy efficiently selects cells with specific oncogenic translocations.
- This approach facilitates the study of cancer development and progression.
- The method is versatile and can be applied to model various cancer-associated genomic rearrangements.
