Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated CRISPR-associated sequence (CAS) proteins constitute a novel antiviral defence system that is widespread in prokaryotes. Repeats are separated by spacers, some of them homologous to sequences in mobile genetic elements. Although the whole process involved remains uncharacterized, it is known that new spacers are incorporated into CRISPR loci of the host during a phage challenge, conferring specific resistance against the virus. Moreover, it has been demonstrated that such interference is based on small RNAs carrying a spacer. These RNAs would guide the defence apparatus to foreign molecules carrying sequences that match the spacers. Despite this essential role, the spacer uptake mechanism has not been addressed. A first step forward came from the detection of motifs associated with spacer precursors (protospacers) of Streptococcus thermophilus, revealing a specific recognition of donor sequences in this species. Here we show that the conservation of proto-spacer adjacent motifs (PAMs) is a common theme for the most diverse CRISPR systems. The PAM sequence depends on the CRISPR-CAS variant, implying that there is a CRISPR-type-specific (motif-directed) choice of the spacers, which subsequently determines the interference target. PAMs also direct the orientation of spacers in the repeat arrays. Remarkably, observations based on such polarity argue against a recognition of the spacer precursors on transcript RNA molecules as a general rule. INTRODUCTIONProkaryotes belonging to the most varied groups contain a peculiar type of repetitive DNA, recognized in 2000 as a family (Mojica et al., 2000), distinguished by the regular spacing of the recurrent motif, and consequently defined as short regularly spaced repeats (SRSR). A majority are partially palindromic, a feature that was later incorporated in the present denomination of CRISPR (clustered regularly interspaced short palindromic repeats), proposed by Jansen and co-workers in agreement with our group (Jansen et al., 2002). Repeat units are 24-47 bp long, and alternate with unique sequences (spacers) of similar size (27-72 bp). Despite considerable divergence, CRISPR can be classified based on sequence similarity (Kunin et al., 2007), defining 12 major groups (henceforth referred in the text as CRISPR-n, where n is the group identification number). Arrays of the same CRISPR are sometimes immediately followed by a conserved AT-rich sequence (Mojica et al., 2000) known as the leader (Jansen et al., 2002). In arrays with a degenerated terminal repeat, the leader is typically located on the opposite edge. Although their role remains undetermined, various reports suggest that leaders promote transcription towards the repeats (Brouns et al., 2008;Lillestøl et al., 2006;Mandin et al., 2007;Tang et al., 2002Tang et al., , 2005Willkomm et al., 2005). Also, the preferential incorporation of new spacers at the leaderproximal side of the array is consistent with a participation in the reco...

CRISPR (clustered regularly interspaced short palindromic repeats) and CAS (CRISPR-associated sequence) proteins are constituents of a novel genetic barrier that limits horizontal gene transfer in prokaryotes by means of an uncharacterized mechanism. The fundamental discovery of small RNAs as the guides of the defence apparatus arose as a result of Escherichia coli studies. However, a survey of the system diversity in this species in order to further contribute to the understanding of the CRISPR mode of action has not yet been performed. Here we describe two CRISPR/CAS systems found in E. coli, following the analysis of 100 strains representative of the species' diversity. Our results substantiate different levels of activity between loci of both CRISPR types, as well as different target preferences and CRISPR relevances for particular groups of strains. Interestingly, the data suggest that the degeneration of one CRISPR/CAS system in E. coli ancestors could have been brought about by self-interference.

Prokaryotes immunize themselves against transmissible genetic elements by the integration (acquisition) in clustered regularly interspaced short palindromic repeats (CRISPR) loci of spacers homologous to invader nucleic acids, defined as protospacers. Following acquisition, mono-spacer CRISPR RNAs (termed crRNAs) guide CRISPR-associated (Cas) proteins to degrade (interference) protospacers flanked by an adjacent motif in extrachomosomal DNA. During acquisition, selection of spacer-precursors adjoining the protospacer motif and proper orientation of the integrated fragment with respect to the leader (sequence leading transcription of the flanking CRISPR array) grant efficient interference by at least some CRISPR-Cas systems. This adaptive stage of the CRISPR action is poorly characterized, mainly due to the lack of appropriate genetic strategies to address its study and, at least in Escherichia coli, the need of Cas overproduction for insertion detection. In this work, we describe the development and application in Escherichia coli strains of an interference-independent assay based on engineered selectable CRISPR-spacer integration reporter plasmids. By using this tool without the constraint of interference or cas overexpression, we confirmed fundamental aspects of this process such as the critical requirement of Cas1 and Cas2 and the identity of the CTT protospacer motif for the E. coli K12 system. In addition, we defined the CWT motif for a non-K12 CRISPR-Cas variant, and obtained data supporting the implication of the leader in spacer orientation, the preferred acquisition from plasmids harboring cas genes and the occurrence of a sequential cleavage at the insertion site by a ruler mechanism.

SummaryCRISPR-Cas systems adapt their immunological memory against their invaders by integrating short DNA fragments into clustered regularly interspaced short palindromic repeat (CRISPR) loci. While Cas1 and Cas2 make up the core machinery of the CRISPR integration process, various class I and II CRISPR-Cas systems encode Cas4 proteins for which the role is unknown. Here, we introduced the CRISPR adaptation genes cas1, cas2, and cas4 from the type I-D CRISPR-Cas system of Synechocystis sp. 6803 into Escherichia coli and observed that cas4 is strictly required for the selection of targets with protospacer adjacent motifs (PAMs) conferring I-D CRISPR interference in the native host Synechocystis. We propose a model in which Cas4 assists the CRISPR adaptation complex Cas1-2 by providing DNA substrates tailored for the correct PAM. Introducing functional spacers that target DNA sequences with the correct PAM is key to successful CRISPR interference, providing a better chance of surviving infection by mobile genetic elements.

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