CRISPR-mediated control of the bacterial initiation of replication

CRISPR-mediated control of the bacterial initiation of replication

Wiktor, J.M. (TU Delft BN/Cees Dekker Lab)
Lesterlin, Christian (University of Oxford)
Sherratt, David J. (University of Oxford)
Dekker, C. (TU Delft BN/Cees Dekker Lab)

2016-05-05

Programmable control of the cell cycle has been shown to be a powerful tool in cell-biology studies. Here, we develop a novel system for controlling the bacterial cell cycle, based on binding of CRISPR/dCas9 to the origin-of-replication locus. Initiation of replication of bacterial chromosomes is accurately regulated by the DnaA protein, which promotes the unwinding of DNA at oriC. We demonstrate that the binding of CRISPR/dCas9 to any position within origin or replication blocks the initiation of replication. Serial-dilution plating, single-cell fluorescence microscopy, and flow-cytometry experiments show that ongoing rounds of chromosome replication are finished upon CRISPR/dCas9 binding, but no new rounds are initiated. Upon arrest, cells stay metabolically active and accumulate cell mass. We find that elevating the temperature from 37 to 42°C releases the CRISR/dCas9 replication inhibition, and we use this feature to recover cells from the arrest. Our simple and robust method of controlling the bacterial cell cycle is a useful asset for synthetic biology and DNA-replication studies in particular. The inactivation of CRISPR/dCas9 binding at elevated temperatures may furthermore be of wide interest for CRISPR/Cas9 applications in genomic engineering.

http://resolver.tudelft.nl/uuid:3ca4487a-b797-42f9-bcd1-437aa8a2b679

https://doi.org/10.1093/nar/gkw214

0305-1048

Nucleic acids research, 44 (8), 3801-3810

Institutional Repository

journal article

© 2016 J.M. Wiktor, Christian Lesterlin, David J. Sherratt, C. Dekker