Abstract LB-031: Generation of inducible oncogenic chromosomal translocations using CRISPR-Cas9 genomic editing

Specific, reciprocal chromosomal translocations are found in numerous cancers, typically leading to the formation and expression of fusion genes with oncogenic properties. It has been demonstrated that by inducing double-stranded breaks (DSBs) in genomic DNA, using either zinc fingers nucleases (ZF), transcription activator-like effector nucleases (TALENs), or the CRISPR-Cas9 system, it is possible to faithfully recapitulate such chromosomal translocations in human cell lines. A potential pitfall is that the induction of the chromosomal translocation generates a constitutively active oncogenic fusion product, which can be detrimental to cell growth or viability in certain heterologous cell types. To overcome these obstacles, we have combined homology directed repair (HDR) with CRISPR-Cas9 genome editing to generate conditionally inducible chromosomal translocations. As proof of principle, we have used this approach to engineer human cell lines to carry the molecular hallmark present in Ewing Sarcoma, the t(11;22)(q24;q12) translocation. Our approach allows for the de novo generation and capture of the desired chromosomal translocation in the context of a conditional allele. Whole genome sequencing (WGS) and fluorescence in situ hybridization (FISH) confirmed the accuracy of our genomic editing approach, whilst analysis of mRNA and protein levels confirmed the selective inducibility and generation of biologically functional EWSR1-FLI1 fusion oncogene. Therefore, our approach establishes an innovative platform for constructing isogenic and conditionally inducible biologically relevant models for a variety of cancers driven by chromosomal translocations. Citation Format: Lee Spraggon, Luciano Martelotto, Julija Hmeljak, Emily Slotkin, Lu Wang, Jorge Reis-Filho, Marc Ladanyi. Generation of inducible oncogenic chromosomal translocations using CRISPR-Cas9 genomic editing. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-031.