CRISPR Imaging Reveals Chromatin Dynamics under Replication Stress

Abstract

Direct visualization of the genomic elements in living cells is required to explore the relationship between the dynamic organization of chromatin and its functional roles. Using a CRISPR-based genome imaging system, we visualized large centromeric and pericentromeric domains in live cells. CRISPR labeling of a large domain with high density induced DNA damage response. As a result, in S phase, the domain expanded and exhibited highly extended fiber-like structure that reached out over several microns with complex features such as branching, bridging, and looping. The labeled domain colocalized with repair pathway proteins such as γH2AX, pATM, and pCHK2. PCNA clusters were found to localize where the chromatin fibers stem from the domain body. When dCas9 was fused to 53BP1, the fiber extension as well as the domain expansion disappeared, suggesting that the fusion forced the damaged region to follow the NHEJ pathway by preventing BRCA1 from knocking off 53BP1. This in turn implies that the expansion and extension of the damaged domain were the result of homology-directed repair process. This finding suggests an imaging-based system for the study of long-range chromatin dynamics in the homology-directed repair of DNA double-strand breaks in live cells.