Foreign DNA acquisition by the I-F CRISPR-Cas system requires all components of the interference machinery.

CRISPR immunity depends on acquisition of fragments of foreign DNA into CRISPR arrays. For type I-E CRISPR-Cas systems two modes of spacer acquisition, naïve and primed adaptation, were described. Naïve adaptation requires just two most conserved Cas1 and Cas2 proteins; it leads to spacer acquisition from both foreign and bacterial DNA and results in multiple spacers incapable of immune response. Primed adaptation requires all Cas proteins and a CRISPR RNA recognizing a partially matching target. It leads to selective acquisition of spacers from DNA molecules recognized by priming CRISPR RNA, with most spacers capable of protecting the host. Here, we studied spacer acquisition by a type I-F CRISPR-Cas system. We observe both naïve and primed adaptation. Both processes require not just Cas1 and Cas2, but also intact Csy complex and CRISPR RNA. Primed adaptation shows a gradient of acquisition efficiency as a function of distance from the priming site and a strand bias that is consistent with existence of single-stranded adaption intermediates. The results provide new insights into the mechanism of spacer acquisition and illustrate surprising mechanistic diversity of related CRISPR-Cas systems.

The Cas6e ribonuclease is not required for interference and adaptation by the E. coli type I-E CRISPR-Cas system.

CRISPR-Cas are small RNA-based adaptive prokaryotic immunity systems protecting cells from foreign DNA or RNA. Type I CRISPR-Cas systems are composed of a multiprotein complex (Cascade) that, when bound to CRISPR RNA (crRNA), can recognize double-stranded DNA targets and recruit the Cas3 nuclease to destroy target-containing DNA. In the Escherichia coli type I-E CRISPR-Cas system, crRNAs are generated upon transcription of CRISPR arrays consisting of multiple palindromic repeats and intervening spacers through the function of Cas6e endoribonuclease, which cleaves at specific positions of repeat sequences of the CRISPR array transcript. Cas6e is also a component of Cascade. Here, we show that when mature unit-sized crRNAs are provided in a Cas6e-independent manner by transcription termination, the CRISPR-Cas system can function without Cas6e. The results should allow facile interrogation of various targets by type I-E CRISPR-Cas system in E. coli using unit-sized crRNAs generated by transcription.