Development of a system for real-time measurement of DNA replication in live mammalian cells

DNA replication in mammalian cells comprises a fundamental and highly regulated process as abnormalities can lead to genome instability. Thus, monitoring DNA replication in cells could give us insights into genome duplication and related diseases. We have previously established an approach for real-time monitoring of DNA replication in individual live yeast cells (Dovrat et al, 2018). Here, we aim to establish an analogous system in mammalian cells to monitor and determine DNA replication rates in single living cells. Non-repetitive 96-mer of bacterial operators tetO and lacO arrays are inserted downstream of an early origin in the mammalian genome by the CRISPR-Cas9 system. Next, tet repressor (TetR) fused to GFP and lac repressor (LacI) fused to tdTomato are inserted into the genome using lentiviral transduction resulting in the formation of two fluorescent dots at the arrays loci. Live-cell fluorescent imaging is then used to monitor the fluorescent intensity of the dots during DNA replication enabling the measurements of replication rates in live mammalian cells. Our system can be used for the investigation of replication during cancer cells proliferation, response to DNA damage and a variety of replication-associated processes such as cohesion and replication-transcription conflicts.

Dovrat D, et al. (2018) A Live-Cell Imaging Approach for Measuring DNA Replication Rates. Cell Rep 24(1):252–258.