The human genome contains millions of DNA regulatory elements,such as enhancers and insulators,most of which have not been tested experimentally.The CTCF/cohesin complex plays a central role in insulator function and higher-order chromatin organization of mammalian genomes.The clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated nuclease 9(Cas9)programed with a synthetic single-guide RNA(sgRNA)emerges as a method for genome editing in virtually any organisms.Here we report an efficient technology of targeted DNA fragment inversion in human and mouse genomes by CRISPR/Cas9 with two sgRNAs.In conjunction with chromosome conformation capture and related 4C-seq and Hi-C methods,we find directional CTCF binding to the paired CBSs with a specific combination of forward-reverse orientations determines the formation of specific DNA-looping interactions between enhancers and promoters in mammalian cells.This mechanism of CTCF-determined looping directions has important implications regarding chromosomal architecture and the insulator functions of genome-wide CBSs in genome folding and gene regulation.