title: CRISPR-mediated control of the bacterial initiation of replication.
abstract: 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.
authors.affiliation: Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, 2628CJ Delft, The Netherlands.
authors.forename: Jakub
authors.initials: J
authors.lastname: Wiktor
authors.affiliation: Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK.
authors.forename: Christian
authors.initials: C
authors.lastname: Lesterlin
authors.affiliation: Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK.
authors.forename: David J
authors.initials: DJ
authors.lastname: Sherratt
authors.affiliation: Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, 2628CJ Delft, The Netherlands c.dekker@tudelft.nl.
authors.forename: Cees
authors.initials: C
authors.lastname: Dekker
mesh_terms: D064130:CRISPR-Associated Proteins / Q000378:metabolism; D064113:CRISPR-Cas Systems; D002876:Chromosomes, Bacterial; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004261:DNA Replication; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D018741:Replication Origin; D013696:Temperature
chemical_list: D064130:CRISPR-Associated Proteins