The Fusion Oncogene FUS-CHOP Drives Sarcomagenesis of High-Grade Spindle Cell Sarcomas in Mice

Figure 5

Generation of endogenous FUS-CHOP translocation with CRISPR technology. (a) Schematic of generating chromosomal rearrangements with CRISPR/Cas9 technology. Fus and Chop introns were targeted in 3T3 cells with sgRNAs for Cas9-mediated cleavage and formation of double-stranded breaks. Repair of these breaks resulted in endogenous translocations in a proportion of cells, which were single cell sorted and expanded for characterization and screening for translocation status via PCR. (b) Surveyor assay for sgFus and sgChop validation. (c) Detection of translocation by PCR shows translocation products only when both Fus and Chop sgRNAs are used. (d) Sanger sequencing of Fus-Chop junction PCR. (e) Western blot shows colocalization of Fus and Chop antibodies in the single cell clone (FC lane, orange arrow). (f) Soft agar assay for transformation shows robust colony formation by 3T3-FC cells.