br Experimental Procedures br Author Contributions br Acknow

Experimental Procedures
Author Contributions
Acknowledgments We thank A. Villanueva, C. Saura, C. Cruz, A. Fernández, E. Nadal, M. Martin, and R. Iggo for reagents and helpful advice, A. Welm and Y. DeRose for sharing PDX models, V. Peg, X. Serres, J. Balmaña, J. Pérez, and C. Hierro for providing samples, S. Hernández-Ortega, R. Gil, L. Barberá, and G. Boigues, the Pathology Department of the University Hospital of Bellvitge, the IDIBELL animal facility, histology service and UB-SCT, for technical support, and members of the laboratory for useful discussions and reading of the manuscript. This work was supported by grants to E. González Suárez by the Spanish Ministry of Economy and Competitivity MINECO and from the Health Institute Carlos III (ISCIII) SAF2008-01975, SAF2011-22893, SAF2014-55997, PIE13/00022, co-funded by FEDER funds/European Regional Development Fund (ERDF – a way to build Europe), by a Career Catalyst Grant from the Susan G Komen FoundationCCR13262449 and by funds to A. Prat from ISCIII-PI13/01718, by a Career Catalyst Grant from the Susan G. Komen Foundation, by Banco Bilbao Vizcaya Argentaria (BBVA) Foundation, and by a Sociedad Española de Oncología Médica (SEOM) grant. The PDX from VHIO were supported by a “GHD-pink” research support via the FERO Foundation to V. Serra. V.S. is recipient of ISCIII grants (PI13/01714 and CP14/0028). M.P., J.G.M., and P.P. were recipients of FPU/FPI grants from the MINECO.
Introduction Chromosome translocations are common genomic events in cancer and are the initiating event in many leukemias and sarcomas (http://cgap.nci.nih.gov/Chromosomes/Mitelman). Cancer-associated chromosomal translocations generate novel chromosomes, placing NVP-TNKS656 cost in new linkage relationships that can result in the generation of fusion genes or the overexpression of proto-oncogenes. Given their contribution to cancer pathogenesis, chromosomal translocations have been widely studied to better understand the mechanisms involved in their formation and the downstream molecular consequences (Chen et al., 2010; Guryanova and Levine, 2013; Rodriguez-Perales et al., 2015). Genetically modified cells and animal models are essential tools for studying both the function of chromosomal translocations and the initiating oncogenic events in cancer. Targeted chromosomal translocations have been generated de novo in human and mouse models using technologies based on P1 Cre-loxP (Forster et al., 2005; Van Deursen et al., 1995), zinc-finger nucleases (ZFN) (Brunet et al., 2009), and transcription activator-like effector nucleases (TALENs) (Piganeau et al., 2013); these approaches generate two derivative chromosomes while maintaining the spatial architecture and regulatory elements of the genomic rearrangement. The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system (Jinek et al., 2012) has been adapted to induce specific double-strand breaks (DSBs) in the genomes of many species (Cong et al., 2013; Mali et al., 2013). This technology has facilitated and accelerated genome engineering through the targeting of specific locations in the genome guided by single-guide RNA (sgRNA) (Jinek et al., 2012). A key advantage of the CRISPR/Cas9 genome editing over more traditional gene-targeting strategies is its high efficiency, which renders common selection procedures unnecessary (Li et al., 2014; Yang et al., 2013). The CRISPR/Cas9 system was recently adapted to induce chromosomal translocations in vitro (Torres et al., 2014b) and in vivo in mouse models (Blasco et al., 2014; Maddalo et al., 2014). These approaches were based in the simultaneous use of two sgRNAs, resulting in two targeted DSBs that when resolved by the DNA repair machinery can generate specific chromosomal translocations. However, although CRISPR/Cas9 technology considerably increases the efficiency of chromosomal translocations, identification of a bona fide targeted clone requires screening of many clones, especially with stem cells.