High-content image-based CRISPR screening reveals regulators of 3D genome architectures

Three-dimensional (3D) genome organization becomes altered during development, aging, and disease, but the factors regulating chromatin topology are incompletely understood and currently no technology can efficiently screen for new regulators of long-range chromatin organization. Here, we developed an image-based high-content CRISPR screening platform that combines a new FISH-based barcode readout method (BARC-FISH) with chromatin tracing. We performed a pooled loss-of-function genetic screen in human cells and visualized alterations to their genome organization from 13,000 imaging target-perturbation combinations, alongside perturbation-paired barcode readout in the same single cells. Using 1.4 million 3D positions along chromosome traces, we identified 26 regulators of chromatin architectures at different length scales. We found that the ATP-dependent helicase CHD7, the loss of which causes the congenital syndrome CHARGE, cooperates with CTCF to promote large-scale chromatin compaction. Altogether, our method enables scalable, high-throughput identification of chromatin topology regulators that will stimulate new insights into the 3D genome. One-Sentence Summary In situ CRISPR screen, cellular barcoding, and chromatin tracing identify regulators of higher-order chromatin organization. ### Competing Interest Statement S.W., B.Y. and M.H. are inventors on a patent applied for by Yale University related to this work. S.W. is a share-holder and consultant of Translura, Inc. The remaining authors declare no competing interests.