CRISPR-mediated control of the bacterial initiation of replication

Jakub Wiktor, Christian Lesterlin, David J. Sherratt, Cees Dekker*

*Corresponding author for this work

Research output: Contribution to journalArticleScientificpeer-review

25 Citations (Scopus)
45 Downloads (Pure)


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.

Original languageEnglish
Pages (from-to)3801-3810
Number of pages10
JournalNucleic Acids Research
Issue number8
Publication statusPublished - 5 May 2016


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