CCCTC-binding factor (CTCF) is an architectural protein involved in the 
three-dimensional (3D) organization of chromatin. In this study, we assayed the 
3D genomic contact profiles of a large number of CTCF binding sites with 
high-resolution 4C-seq. As recently reported, our data also suggest that 
chromatin loops preferentially form between CTCF binding sites oriented in a 
convergent manner. To directly test this, we used CRISPR/Cas9 genome editing to 
delete core CTCF binding sites in three loci, including the CTCF site in the 
Sox2 super-enhancer. In all instances, CTCF and cohesin recruitment were lost, 
and chromatin loops with distal, convergent CTCF sites were disrupted or 
destabilized. Re-insertion of oppositely oriented CTCF recognition sequences 
restored CTCF and cohesin recruitment, but did not re-establish chromatin loops. 
We conclude that CTCF binding polarity plays a functional role in the formation 
of higher-order chromatin structure.