Generation of conditional oncogenic chromosomal translocations using CRISPR–Cas9 genomic editing and homology-directed repair

Lee Spraggon, Luciano G. Martelotto, Julija Hmeljak, Tyler D. Hitchman, Jiang Wang, Lu Wang, Emily K. Slotkin, Pang-Dian Fan, Jorge S. Reis-Filho, Marc Ladanyi

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17 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)102-112
Number of pages11
JournalJournal of Pathology
Volume242
Issue number1
DOIs
Publication statusPublished - May 2017

Keywords

  • chromosomal translocations
  • CRISPR–Cas9
  • desmoplastic small round cell tumour (DSRCT)
  • Ewing sarcoma
  • genomic editing
  • homology-directed repair
  • oncogene

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