Chromatin Dynamics in DNA Damage Response Directly Observed by CRISPR Imaging

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 an improved CRISPR-based genome imaging system using dCas9 combined with SunTag and split fluorophores, we visualized large centromeric and pericentromeric domains in live cells. When synchronized in S phase, the domains expanded and also exhibited highly extended fibers reaching out several microns with complex structures such as branching, bridging, and looping. The chromatin extensions colocalized with repair pathway proteins such as γH2AX, pATM, and pCHK2. When dCas9 was fused to 53BP1, the extended fibers as well as the expanded body of the domains disappeared, suggesting that the fusion forced the damaged regions to follow the NHEJ pathway by preventing BRCA1 from knocking 53BP1 off the DNA. This finding provides a novel system for the study of long-range chromatin dynamics in homology-directed repair of DNA double-strand breaks. The unique capability of CRISPR imaging to provide live cell movies allows to dissect the biophysical mechanisms of such dynamic nanostructures of chromatin.