期刊
NUCLEIC ACIDS RESEARCH
卷 44, 期 8, 页码 3801-3810出版社
OXFORD UNIV PRESS
DOI: 10.1093/nar/gkw214
关键词
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资金
- Netherlands Organisation for Scientific Research (NWO/OCW) , Frontiers of Nanoscience program
- European Research Council [247072, 16 669598]
- Wellcome Trust [SIA099204/Z/12Z]
- Leverhulme Trust [RP2013-K-017]
- NWO/OCW
- BBSRC [BB/I004785/1] Funding Source: UKRI
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 degrees 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.
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