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EcCRISPR Report Summary

Project ID: 336079
Funded under: FP7-IDEAS-ERC
Country: Israel

Mid-Term Report Summary - ECCRISPR (Novel roles, components, and mechanisms of the Escherichia coli CRISPR/Cas system)

Halfway through the timeline, we achieved significant parts of two aims of the project. The first is the establishment of the CRISPR-Cas system as a weapon against antibiotic resistant bacteria. The increasing threat of pathogen resistance to antibiotics requires the development of novel antimicrobial strategies. We established a proof of concept for a genetic strategy that aims to sensitize bacteria to antibiotics and selectively kill antibiotic-resistant bacteria. We used temperate phages to deliver a functional CRISPR-Cas system into the genome of antibiotic-resistant bacteria. The delivered CRISPR-Cas system destroyed both antibiotic resistance-conferring plasmids and genetically-modified lytic phages. This linkage between antibiotic-sensitization and protection from lytic phages is a key feature of the strategy. It allows programming of lytic phages to kill only antibiotic-resistant bacteria while protecting antibiotic-sensitized bacteria. Phages designed according to this strategy may be used on hospital surfaces and hand-sanitizers to facilitate replacement of antibiotic-resistant pathogens with sensitive ones.
The second accomplished aim is the elucidation of self versus foreign discrimination of DNA in the CRISPR-Cas system. In the process of CRISPR adaptation, short pieces of DNA (“spacers”) are acquired from foreign elements and integrated into the CRISPR array. It so far remained a mystery how spacers are preferentially acquired from the foreign DNA while the self chromosome is avoided. We showed that spacer acquisition is replication-dependent, and that DNA breaks formed at stalled replication forks promote spacer acquisition. Chromosomal hotspots of spacer acquisition were confined by Chi sites, which are sequence octamers highly enriched on the bacterial chromosome, suggesting that these sites limit spacer acquisition from self DNA. We further showed that the avoidance of “self” is mediated by the RecBCD dsDNA break repair complex. Our results suggest that in E. coli, acquisition of new spacers depends on RecBCD-mediated processing of dsDNA breaks occurring primarily at replication forks, and that the preference for foreign DNA is achieved through the higher density of Chi sites on the self chromosome, in combination with the higher number of forks on the foreign DNA. This model explains the strong preference to acquire spacers from both high copy plasmids and phages.
These two achievements combine both basic and applicable studies of the CRISPR-Cas system, and significantly contribute to both aspects.

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