CRISPR-Cas (clustered, regularly interspaced short palindromic repeats coupled with CRISPR-associated proteins) is a bacterial immunity system that protects against invading phages or plasmids. In the process of CRISPR adaptation, short pieces of DNA ('spacers') are acquired from foreign elements and integrated into the CRISPR array. So far, it has remained a mystery how spacers are preferentially acquired from the foreign DNA while the self chromosome is avoided. Here we show 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 show that the avoidance of self is mediated by the RecBCD double-stranded DNA break repair complex. Our results suggest that, in Escherichia coli, acquisition of new spacers largely depends on RecBCD-mediated processing of double-stranded DNA 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 both from high copy plasmids and from phages.
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Acknowledgements We thank N. Barkai, A. Tanay, E. Mick, S. Doron, A. Stern, T. Dagan and M. Shamir for discussion. We also thank the Skarstad group for providing the MG1655dnaC2 strain, the Michel group for providing the JJC1819 strain and D. Dar for assistance in Illumina sequencing. R.S. was supported, in part, by the Israel Science Foundation (personal grant 1303/12 and I-CORE grant 1796), the European Research Council Starting Grant programme (grant 260432), Human Frontier Science Program (grant RGP0011/2013), the Abisch-Frenkel foundation, the Pasteur-Weizmann Council, the Minerva Foundation, and by a Deutsch-Israelische Projektkooperation grant from the Deutsche Forschungsgemeinschaft. U.Q. was supported, in part, by the European Research Council Starting Grant programme (grant 336079), the Israel Science Foundation (grant 268/14) and the Israeli Ministry of Health (grant 9988-3). A.L. is grateful to the Azrieli Foundation for the award of an Azrieli Fellowship.
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