title: Generation of conditional oncogenic chromosomal translocations using CRISPR-Cas9 genomic editing and homology-directed repair.
abstract: Chromosomal rearrangements encoding oncogenic fusion proteins are found in a wide variety of malignancies. The use of programmable nucleases to generate specific double-strand breaks in endogenous loci, followed by non-homologous end joining DNA repair, has allowed several of these translocations to be generated as constitutively expressed fusion genes within a cell population. Here, we describe a novel approach that combines CRISPR-Cas9 technology with homology-directed repair to engineer, capture, and modulate the expression of chromosomal translocation products in a human cell line. We have applied this approach to the genetic modelling of t(11;22)(q24;q12) and t(11;22)(p13;q12), translocation products of the EWSR1 gene and its 3' fusion partners FLI1 and WT1, present in Ewing's sarcoma and desmoplastic small round cell tumour, respectively. Our innovative approach allows for temporal control of the expression of engineered endogenous chromosomal rearrangements, and provides a means to generate models to study tumours driven by fusion genes. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
authors.affiliation: Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, NY, USA.
authors.forename: Lee
authors.identifier: 0000-0003-3438-4180
authors.initials: L
authors.lastname: Spraggon
authors.affiliation: Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, NY, USA.
authors.forename: Luciano G
authors.initials: LG
authors.lastname: Martelotto
authors.affiliation: Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, NY, USA.
authors.forename: Julija
authors.initials: J
authors.lastname: Hmeljak
authors.affiliation: Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, NY, USA.
authors.forename: Tyler D
authors.initials: TD
authors.lastname: Hitchman
authors.affiliation: Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, NY, USA.
authors.forename: Jiang
authors.initials: J
authors.lastname: Wang
authors.affiliation: Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, NY, USA.
authors.forename: Lu
authors.initials: L
authors.lastname: Wang
authors.affiliation: Department of Pediatrics, Memorial Sloan Kettering Cancer Center, New York, NY, USA.
authors.forename: Emily K
authors.initials: EK
authors.lastname: Slotkin
authors.affiliation: Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, NY, USA.
authors.forename: Pang-Dian
authors.initials: PD
authors.lastname: Fan
authors.affiliation: Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, NY, USA.|Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA.
authors.forename: Jorge S
authors.initials: JS
authors.lastname: Reis-Filho
authors.affiliation: Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, NY, USA.|Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA.
authors.forename: Marc
authors.initials: M
authors.lastname: Ladanyi
mesh_terms: D019677:Artificial Gene Fusion / Q000379:methods; D002880:Chromosomes, Human, Pair 11 / Q000235:genetics; D002892:Chromosomes, Human, Pair 22 / Q000235:genetics; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D004273:DNA, Neoplasm / Q000235:genetics; D058405:Desmoplastic Small Round Cell Tumor / Q000235:genetics*; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics; D006801:Humans; D015514:Oncogene Proteins, Fusion / Q000235:genetics; D059767:Recombinational DNA Repair / Q000235:genetics*; D012512:Sarcoma, Ewing / Q000235:genetics*; D014178:Translocation, Genetic; D014407:Tumor Cells, Cultured
chemical_list: D004273:DNA, Neoplasm; D015514:Oncogene Proteins, Fusion
keywords: CRISPR-Cas9; Ewing sarcoma; chromosomal translocations; desmoplastic small round cell tumour (DSRCT); genomic editing; homology-directed repair; oncogene