[Combinatorial CRISPR inversions of CTCF sites in HOXD cluster reveal complex insulator function]

CTCF (CCCTC-binding factor) is a zinc-finger protein which plays a vital role in the three-dimensional (3D) genome architecture. A pair of forward-reverse convergent CTCF binding sites (CBS elements) mediates long-distance DNA interactions to form chromatin loops with the assistance of the cohesin complex, while CBS elements at the chromatin domain boundaries show reverse-forward divergent patterns and function as insulators to discriminate against DNA interaction between chromatin domains. However, there are still many unresolved problems regarding CTCF-mediated insulation function. In order to study the connections between chromatin loops and the insulation function of CBS elements, we combinatorically inverted CBS elements at the HOXD locus by using CRISPR/Cas9 DNA-fragment editing methods in the HEK293T cell line and obtained five different kinds of single-cell CRISPR clones. By performing quantitative high-resolution chromosome conformation capture copy (QHR-4C) experiments, we found that boundary CBS inversions abolish original chromatin loops and establish new loops from the opposite direction, thus shifting the insulator boundary to the new divergent CTCF sites. Furthermore, tandem CBS elements block cohesin permeated from the opposite orientation to function as insulators. RNA-seq experiments showed that alterations of local three-dimensional genome architecture would further influence gene expression of the HOXD cluster. In conclusion, a pair of divergent CBS elements function as insulators by forming chromatin loops within chromatin domains to block cohesin sliding.