CRISPR adaptation biases explain preference for acquisition of foreign DNA

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. In the bacterial immunity system CRISPR, spacer acquisition is facilitated near replication-termination regions. CRISPR recognition of foreign DNA The bacterial immunity system known as CRISPR depends on the acquisition of short sequences from invading phage genomes or plasmids; these DNAs are called spacers. The spacer acquisition process avoids incorporating host DNAs, but how host and phage DNAs are distinguished was unclear. Rotem Sorek and colleagues have found that formation of spacer DNAs requires replication-dependent DNA double-strand breaks. Host DNA contains a much higher occurrence of the octameric Chi sequence, which attenuates the nuclease activity of RecBCD and thus leads to fewer fragments. In addition, phage genomes have a greater level of replication forks that can give rise to breaks.