Perturb-tracing enables high-content screening of multiscale 3D genome regulators

Abstract

Three-dimensional (3D) genome organization becomes altered during development, aging, and disease, but the factors regulating chromatin topology are incompletely understood and currently there are no technologies to 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 screen of 137 selected genes (420 sgRNAs) in human cells and visualized alterations to their genome organization alongside sgRNA-paired barcode readout by BARC-FISH 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.

Repair of DNA double-strand breaks leaves heritable impairment to genome function

“…To account for the cell-to-cell variations in 3-D chromatin arrangement ( 43 – 45 ) we were looking for conditions that would allow us to address this at a single-cell, or even better, a single-allele level. To this end, we employed dual-color 3-D DNA fluorescence in situ hybridization staining (DNA FISH), an established approach to quantify distance between genomic regions ( 46 , 47 ), combined with immunofluorescence labeling of the repair protein 53BP1 as a marker of the Cas9-generated DSBs in the c-MYC ORF (Fig. 4A, B).…”

Highly multiplexed, image-based pooled screens in primary cells and tissues with PerturbView

“…FISH-based detection of sgRNAs and barcodes has recently emerged as an alternative to ISS for reading out multiplex screens 12,32,54 . These methods generally either directly detect an sgRNA with a FISH probe or detect an expanded FISH barcode often proximal to the sgRNA of interest.…”

CRISPR screens in mechanism and target discovery for AML

“…Future studies to identify T cells targets in AML models are appreciated. Recently, the combination with other technologies, such as adenine base editors [ 105 , 106 ], immunomagnetic cell sorting [ 107 ], BARC-FISH, chromatin tracing [ 108 ] and dual CRISPR-Cas9 [ 109 ], enables CRISPR screen visible and accurate, allowing the identification of new targets undetectable previously. These novel CRISPR screen methods might be applied to screen candidate therapeutic targets for AML in the future.…”