Center for Molecular Spectroscopy and Dynamics

3. 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 centromeric and pericentromeric domains in live cells containing repetitive sequences. CRISPR labeling of the domains in high density induced DNA damage response. As a result, 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. Such behavior was more prominent in S phase. The labeled domain colocalized with repair pathway proteins such as γH2AX, pATM, and pCHK2. PCNA formed clusters 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 occurred as a result of homology-directed repair process. This finding suggests the use of modified CRISPR systems for genome imaging to avoid unnecessary DNA damage response. Such behavior can be further explored to study long-range chromatin dynamics during the homology-directed repair of DNA double-strand breaks in live cells.