Search

Key to CRISPR-Cas adaptive immunity is maintaining an ongoing record of invading nucleic acids, a process carried out by the Cas1-Cas2 complex that integrates short segments of foreign genetic material (spacers) into the CRISPR locus. It is hypothesized that Cas1 evolved from casposases, a novel class of transposases. We show here that the Methanosarcina mazei casposase can integrate varied forms of the casposon end in vitro, and recapitulates several properties of CRISPR-Cas integrases including site-specificity. The X-ray structure of the casposase bound to DNA representing the product of integration reveals a tetramer with target DNA bound snugly between two dimers in which single-stranded casposon end binding resembles that of spacer 3'-overhangs. The differences between transposase and CRISPR-Cas integrase are largely architectural, and it appears that evolutionary change involved changes in protein-protein interactions to favor Cas2 binding over tetramerization; this in turn led to preferred integration of single spacers over two transposon ends.

Reverse transcriptases (RTs) closely related to those encoded by group II introns but lacking the intron RNA structure have been found associated with type III clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems, a prokaryotic immune system against invading viruses and foreign genetic elements. Two models have been proposed to explain the origin and evolutionary relationships of these RTs: (i) the >single point of origin> model, according to which these RTs originated from a single acquisition event in bacterial, with the various protein domains (RT, RT-Cas1, and Cas6-RT-Cas1 fusions) corresponding to single points in evolution; and (ii) the >various origins> model, according to which, independent acquisition events in different evolutionary episodes led to these fusions. We tested these alternative hypotheses, by analyzing and integrating published datasets of RT sequences associated with CRISPR-Cas systems and inferring phylogenetic trees by maximum likelihood (ML) methods. The RTs studied could be grouped into 13 clades, mostly in bacteria, in which they probably evolved. The various clades appear to form three independent lineages in bacteria and a recent lineage in archaea. Our data show that the Cas6 domain was acquired twice, independently, through RT-Cas1 fusion, in the bacterial lineages. Taken together, there more evidence to support the >various origins> hypothesis., This work was supported by research grants BIO2014-51953-P and BIO2017-82244-P from the Plan Nacional de I+D+i, Biotechnology Program from the Spanish Ministerio de Economía, Industria y Competitividad including ERDF (European Regional Development Fund). AG-D was supported by a FPU predoctoral fellowship from the Ministerio de Economía, Industria y Competitividad.

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins provide acquired genetic immunity against the entry of mobile genetic elements (MGEs). The immune defense provided by various subtypes of the CRISPR-Cas system has been confirmed and is closely associated with the formation of immunological memory in CRISPR arrays, called CRISPR adaptation or spacer acquisition. However, whether type II-C CRISPR-Cas systems are also involved in spacer acquisition remains largely unknown. This study explores and provides some definitive evidence regarding spacer acquisition of the type II-C CRISPR-Cas system from Riemerella anatipestifer (RA) CH-2 (RA-CH-2). Firstly, introducing an exogenous plasmid into RA-CH-2 triggered spacer acquisition of RA CRISPR-Cas system, and the acquisition of new spacers led to plasmid instability in RA-CH-2. Furthermore, deletion of cas1 or cas2 of RA-CH-2 abrogated spacer acquisition and subsequently stabilized the exogenous plasmid, suggesting that both Cas1 and Cas2 are required for spacer acquisition of RA-CH-2 CRISPR-Cas system, consistent with the reported role of Cas1 and Cas2 in type I-E and II-A systems. Finally, assays for studying Cas1 nuclease activity and the interaction of Cas1 with Cas2 contributed to a better understanding of the adaptation mechanism of RA CRISPR-Cas system. This is the first experimental identification of the naïve adaptation of type II-C CRISPR-Cas system.

The type I-F CRISPR adaptive immune system in Pseudomonas aeruginosa (PA14) consists of two CRISPR loci and six CRISPR-associated (cas) genes. Type I-F systems rely on a CRISPR RNA (crRNA)-guided surveillance complex (Csy complex) to bind foreign DNA and recruit a trans-acting nuclease (i.e., Cas2/3) for target degradation. In most type I systems, Cas2 and Cas3 are separate proteins involved in adaptation and interference, respectively. However, in I-F systems, these proteins are fused into a single polypeptide. Here we use biochemical and structural methods to show that two molecules of Cas2/3 assemble with four molecules of Cas1 (Cas2/32:Cas14) into a four-lobed propeller-shaped structure, where the two Cas2 domains form a central hub (twofold axis of symmetry) flanked by two Cas1 lobes and two Cas3 lobes. We show that the Cas1 subunits repress Cas2/3 nuclease activity and that foreign DNA recognition by the Csy complex activates Cas2/3, resulting in bidirectional degradation of DNA targets. Collectively, this work provides a structure of the Cas1–2/3 complex and explains how Cas1 and the target-bound Csy complex play opposing roles in the regulation of Cas2/3 nuclease activity.

Multiple organisms face the threat of viral infections. To combat phage invasion, bacteria and archaea have evolved an adaptive mechanism of protection against exogenic mobile genetic elements, called CRISPR-Cas. In this defense strategy, phage infection is memorized via acquisition of a short invader sequence, called a spacer, into the CRISPR locus of the host genome. Upon repeated infection, the ‘vaccinated’ host expresses the spacer as a precursor RNA, which is processed into a mature CRISPR RNA (crRNA) that guides an endonuclease to the matching invader for its ultimate destruction. Recent efforts have uncovered molecular details underlying the crRNA biogenesis and interference steps. However, until recently the step of adaptation had remained largely uninvestigated. In this minireview, we focus on recent publications that have begun to reveal molecular insights into the adaptive step of CRISPR-Cas immunity, which is required for the development of the heritable memory of the host against viruses.