Mapping the Cellular Responses to DNA Double-Strand Breaks Using On-Demand CRISPR technologies and High-resolution Fluorescence Microscopy

The integrity of the human genome is constantly challenged by environmental and cellular stresses, resulting in various DNA damage and gene mutations. Many proteins have evolved to rapidly detect, signal, and repair DNA damage inside living cells, forming an orchestrated network known as DNA damage response (DDR). Unsurprisingly, DDR defects, such as DNA repair protein mutations, are often linked to human diseases, including developmental abnormalities, accelerated aging, and common cancers. The past decades of biochemical and genetic research have generated a wealth of knowledge regarding the identities of DDR factors, their roles in genome maintenance, and how they contribute to the diseases when they go awry. However, the detailed spatiotemporal parameters by which DDR factors mediate DNA repair remain largely elusive. What timescales do DDR factors search for and bind to damaged DNA in living cells? Do DDR factors form specific structures to facilitate an accurate repair? How does DNA damage regulate other nuclear DNA activities, such as transcription? This research program aims to address these fundamental questions by investigating DDR dynamics during DNA double-strand break (DSB) repair. DSB is one of the most genotoxic DNA damage types frequently occurring in our bodies. Recently, we have established an experimental platform that allows quantitative visualization of DDR factors and on-demand DSB induction at specific genomic loci and with a second-scale temporal resolution, a capability achieved by marrying high-resolution fluorescence microscopy with the very fast (vf)CRISPR technique pioneered by our lab. Here, we will take full advantage of this novel platform and comprehensively map the DSB-induced dynamics of DDR factors, chromosome translocation, and activities of transcription and cGAS in single human cells. This study will strongly complement DSB repair research conventionally performed in test tubes and at the ensemble level, providing valuable mechanistic insights into DSB repair with unprecedented resolutions.