Figure 2. EGFP-tk integration and expression in cells expressing TMEM135-CCDC67 fusion breakpoint transcript.

(A) gRNA mediated cleavage of pCMV-TMEM135^int13-CCDC67^int9. In vitro cleavage assays were performed on PVUI linearized pCMV-TMEM135^int13-CCDC67^int9 vector using recombinant Cas9, S. pyogenes and in vitro transcribed gRNA− or gRNA+ as indicated. The cleavage generated 4317 and 3206 bp fragments of pCMV-TMEM135^int13-CCDC67^int9 vector for gRNA−, and 4414 and 3109 bp for gRNA+, indicated by arrows. (B) Genome integration and expression of TMEM135^int13-CCDC67^int9 breakpoint in PC3 and DU145 cells. Top panel: PCR products of TMEM135^int13-CCDC67^int9 breakpoint from the genome of indicated cells; Second from the top: PCR products of genomic β-actin from the genome of indicated cells. Third from the top: RT-PCR products of TMEM135^int13-CCDC67^int9 breakpoint from the mRNA of the indicated cells. Bottom panel: RT-PCR products of β-actin from the mRNA of the indicated cells. PC3 BP denotes PC3 cells transfected with pCMV-TMEM135^int13-CCDC67^int9; DU145 BP denotes DU145 cells transfected with pCMV-TMEM135^int13-CCDC67^int9; PC3 CMV denotes PC3 transfected with pCMVscript; DU145 CMV denotes DU145 cells transfected with pCMVscript. Primer sequences are listed in Supplementary table 2. (C) Infection of PC3 or DU145 cells containing TMEM135-CCDC67 breakpoint led to expression of EGFP-tk. PC3 or DU145 cells transformed with pCMV-TMEM135^int13-CCDC67^int9 were infected with pAD5-Cas9^D10A-gRNA^TMEM135int13-gRNA^CCDC67int9 and pAD-TMEM135^int13-EGFP-tk-CCDC67^int9 (Ad-TC). Expression of Cas9^D10A-RFP is indicated by red fluorescence, while expression EGFP-tk is indicated by green. PC3 or DU145 cells transformed with pCMVscript were used as controls. Selected images were shown. (D) Quantification of EGFP-tk integration/expression by flow cytometry as of (C).