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

Abstract

The spatial organization of chromatin critically impacts many essential genomic functions, from the regulation of gene expression to the replication of the genome. Changes in chromatin organization are associated with aging and a wide range of diseases including cancer. Despite the fundamental importance of correct chromatin organization and its effects on cellular behavior, very little is known regarding the precise folding arrangement of chromatin in various cell states, nor the molecular mechanisms that control chromatin organization in the cell nucleus. Current approaches are severely limited by a lack of experimental tools to directly trace chromatin folding, and to efficiently screen for molecular regulators of chromatin organization in the genome. To address these needs, we previously developed an image-based 3D genomics technique termed chromatin tracing, which enables direct 3D tracing of chromatin folding along individual chromosomes in single cells. Recently, we further developed Multiplexed Imaging of Nucleome Architectures (MINA), which enables simultaneous measurements of multiscale chromatin folding, associations of genomic regions with nuclear lamina and nucleoli, and copy numbers of numerous RNA species in the same cells in mammalian tissue. Here we report our latest technology development - an image-based high-content CRISPR screening platform - to systematically uncover new regulators of 3D genome organization. We performed a pooled loss-of-function screen of hundreds of selected genes in human cells, identified target genes by inventing a cellular barcoding technique termed BARC-FISH, and visualized genome organization by chromatin tracing in the same single cells. Using 1.4 million imaged 3D-positions along chromosome traces, we identified tens of novel regulators of chromatin architectures at different length scales. Loss of the ATP-dependent helicase CHD7, which causes the congenital syndrome CHARGE, promotes multi-scale chromatin decompaction. This method enables scalable, high-throughput identification of chromatin topology regulators in diverse contexts.