Chromatin organization plays a critical role in regulating gene expression. Chromatin compaction represses gene expression by physically restricting the transcriptional machinery’s access to DNA, while spatial proximity between enhancers and promoters—often mediated by chromatin loops—is essential for gene activation. To investigate the regulatory mechanisms underlying loop formation and chromatin compaction, as well as their effects on gene expression, we developed CRISPR-CLIP, a novel programmable platform for engineering chromatin loops and inducing chromatin compaction in live cells. CRISPR-CLIP functions by tethering pairs of genomic loci targeted by single-guide RNAs (sgRNAs) to induce chromatin looping. Each arm of the system is guided by an sgRNA bound by fluorescent RNA coat proteins, allowing fluorescently labeled genomic loci to be brought into close proximity and facilitating loop formation. In addition, multiple CRISPR-CLIP complexes can act cooperatively to drive chromatin compaction. We applied CRISPR-CLIP to target four pairs of genomic loci located on different chromosomes in live U2OS cells, with distances ranging from several kilobases (kb) to megabases (Mb), successfully bringing these distal loci into proximity. Furthermore, we designed CLIP-sgRNAs to target 836 copies of a repetitive sequence within a mesoscale chromatin domain spanning approximately 17 Mb on the q arm of chromosome 19, where CRISPR-CLIP effectively induced chromatin compaction and led to gene repression within the targeted region. These results establish CRISPR-CLIP as a powerful tool for engineering chromatin organization in live cells and highlight its potential as a therapeutic platform for gene regulation and disease treatment.
Chung et al. (Sun,) studied this question.