Fig. 5

Experimental workflow for functional investigation of PCa risk-associated CTCF sites. Phase 1: Plasmids encoding guide RNAs that target sequences on each side of a PCa risk-associated CTCF site were introduced into the PCa cell line 22Rv1 along with a Cas9 expression vector (see the “Methods” section for details). The resultant cell pool was analyzed to determine deletion efficiency (red slashes represent alleles in each cell that harbor a CTCF site deletion). Single cells were then selected and expanded into clonal populations for RNA-seq analysis. Phase 2: After identifying the gene most responsive (within a ± 1-Mb window) to deletion of the region encompassing a risk-associated CTCF site, plasmids encoding guide RNAs that target the risk-associated CTCF anchor region and/or the regions encompassing the CTCF sites looped to the risk CTCF site and a Cas9 expression plasmid were introduced into 22Rv1 cells; cell pools were analyzed by PCR to check deletion frequency and by RT-qPCR to measure expression of the target gene