To acquire the ability to recognize and destroy virus and plasmid invaders, prokaryotic CRISPR-Cas systems capture fragments of DNA within the host CRISPR locus. Our results indicate that the process of adaptation by a Type II-A CRISPR-Cas system in Streptococcus thermophilus requires Cas1, Cas2, and Csn2. Surprisingly, we found that Cas9, previously identified as the nuclease responsible for ultimate invader destruction, is also essential for adaptation. Cas9 nuclease activity is dispensable for adaptation. In addition, our studies revealed extensive, unbiased acquisition of the selftargeting host genome sequence by the CRISPR-Cas system that is masked in the presence of active target destruction.Supplemental material is available for this article.Received December 17, 2014; revised version accepted January 15, 2015.CRISPR-Cas systems provide prokaryotes with adaptive immunity against invaders such as viruses/phages and plasmids (Terns and Terns 2011;Barrangou and Marraffini 2014;Heler et al. 2014;van der Oost et al. 2014). CRISPR-Cas systems target invaders using information stored in CRISPRs: loci that contain alternating units of an identical repeat (repeats) and short invader-derived sequences (spacers) (Fig. 1A). CRISPR transcripts are processed to a battery of CRISPR RNAs (crRNAs) that each contains a unique invader guide sequence (and common repeat sequence). The crRNAs associate with Cas proteins to form effector complexes that recognize and degrade invading nucleic acids to effect immunity (Terns and Terns 2011;Barrangou and Marraffini 2014;Heler et al. 2014;van der Oost et al. 2014). Diverse CRISPR-Cas systems are prevalent in bacteria and archaea and are categorized into three compositionally distinct groups (Types I-III), with multiple subtypes within each group (Haft et al. 2005;Makarova et al. 2011).The initial step of capturing short fragments of invasive DNA into CRISPR loci (''adaptation'' or ''spacer acquisition'') is the least understood aspect of the CRISPR immune pathway. Adaptation appears to be a rare event but generates subpopulations of organisms that can survive infection. It has been proposed that the mechanism involves identification of ''foreign'' sequences for incorporation into the CRISPR (Datsenko et al. 2012;Yosef et al. 2012;Diez-Villasenor et al. 2013;Nunez et al. 2014), although host genome sequences have also been observed in CRISPRs at very low frequencies (Stern et al. 2010;Jiang et al. 2013;Paez-Espino et al. 2013). Selection of invader DNA fragments (protospacers) by the adaptation machinery requires the presence of a short (3-to 7-base-pair An important goal toward understanding CRISPR adaptation is identifying the proteins (Cas and non-Cas) responsible for novel spacer acquisition in CRISPR loci in diverse CRISPR-Cas systems. Genetic studies indicate that overexpression of Cas1 and Cas2-the only Cas proteins universal to all CRISPR-Cas systems-is sufficient to induce adaptation in the absence of other Cas proteins in Type I systems such as that found in Escherichia col...

CRISPR-Cas systems are RNA-based immune systems that protect prokaryotes from invaders such as phages and plasmids. In adaptation, the initial phase of the immune response, short foreign DNA fragments are captured and integrated into host CRISPR loci to provide heritable defense against encountered foreign nucleic acids. Each CRISPR contains a ∼100–500 bp leader element that typically includes a transcription promoter, followed by an array of captured ∼35 bp sequences (spacers) sandwiched between copies of an identical ∼35 bp direct repeat sequence. New spacers are added immediately downstream of the leader. Here, we have analyzed adaptation to phage infection in Streptococcus thermophilus at the CRISPR1 locus to identify cis-acting elements essential for the process. We show that the leader and a single repeat of the CRISPR locus are sufficient for adaptation in this system. Moreover, we identified a leader sequence element capable of stimulating adaptation at a dormant repeat. We found that sequences within 10 bp of the site of integration, in both the leader and repeat of the CRISPR, are required for the process. Our results indicate that information at the CRISPR leader-repeat junction is critical for adaptation in this Type II-A system and likely other CRISPR-Cas systems.

CRISPR–Cas systems provide heritable immunity against viruses by capturing short invader DNA sequences, termed spacers, and incorporating them into the CRISPR loci of the prokaryotic host genome. Here, we investigate DNA elements that control accurate spacer uptake in the type II-A CRISPR locus of Streptococcus thermophilus. We determined that purified Cas1 and Cas2 proteins catalyze spacer integration with high specificity for CRISPR repeat junctions. We show that 10 bp of the CRISPR leader sequence is critical for stimulating polarized integration preferentially at the repeat proximal to the leader. Spacer integration proceeds through a two-step transesterification reaction where the 3′ hydroxyl groups of the spacer target both repeat borders on opposite strands. The leader-proximal end of the repeat is preferentially targeted for the first site of integration through recognition of sequences spanning the leader-repeat junction. Subsequently, second-site integration at the leader-distal end of the repeat is specified by multiple determinants including a length-defining mechanism relying on a repeat element proximal to the second site of integration. Our results highlight the intrinsic ability of type II Cas1/Cas2 proteins to coordinate directional and site-specific spacer integration into the CRISPR locus to ensure precise duplication of the repeat required for CRISPR immunity.

In this issue of Molecular Cell, Nuñez et al. (2016) report that site-specific integration of foreign DNA into CRISPR loci by the Cas1-Cas2 integrase complex is promoted by a host factor, IHF (integration host factor), that binds and bends CRISPR leader DNA.

Streptococcus thermophilus is an important organism for production of cultured dairy foods, but it is susceptible to lytic phages which can lead to failed products. Consequently, mechanisms for phage resistance are an active area of research.