Search

CRISPR-Cas adaptive immune systems uptake short "spacer" sequences from foreign DNA and incorporate them into the host genome to serve as templates for CRISPR RNAs that guide interference against future infections. Adaptation in CRISPR systems is mediated by Cas1-Cas2 complexes that catalyze integration of prespacer substrates into the CRISPR array. Many DNA targeting systems also require Cas4 endonucleases for functional spacer acquisition. Cas4 selects prespacers containing a protospacer adjacent motif (PAM) and removes the PAM prior to integration, both of which are required to ensure host immunization. Cas1 has also been shown to function as a nuclease in some systems, but a role for this nuclease activity in adaptation has not been demonstrated. We identified a type I-G Cas4/1 fusion with a nucleolytically active Cas1 domain that can directly participate in prespacer processing. The Cas1 domain is both an integrase and a sequence-independent nuclease that cleaves the non-PAM end of a prespacer, generating optimal overhang lengths that enable integration at the leader side. The Cas4 domain sequence specifically cleaves the PAM end of the prespacer, ensuring integration of the PAM end at the spacer side. The two domains have varying metal ion requirements. While Cas4 activity is Mn 2+ dependent, Cas1 preferentially uses Mg 2+ over Mn 2+ . The dual nuclease activity of Cas4/1 eliminates the need for additional factors in prespacer processing making the adaptation module self-reliant for prespacer maturation and directional integration., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

CRISPR-Cas is an adaptive immune system that protects prokaryotes against foreign nucleic acids. Prokaryotes gain immunity by acquiring short pieces of the invading nucleic acid termed prespacers and inserting them into their CRISPR array. In type II-A systems, Cas1 and Cas2 proteins insert prespacers always at the leader-repeat junction of the CRISPR array. Among type II-A CRISPR systems, three distinct groups (G1, G2, and G3) exist according to the extent of DNA sequence conservation at the 3' end of the leader. However, the mechanisms by which these conserved motifs interact with their cognate Cas1 and Cas2 proteins remain unclear. Here, we performed in vitro integration assays, finding that for G1 and G2, the insertion site is recognized through defined mechanisms, at least in members examined to date, whereas G3 exhibits no sequence-specific insertion. G1 first recognized a 12-bp sequence at the leader-repeat junction and performed leader-side insertion before proceeding to spacer-side insertion. G2 recognized the full repeat sequence and could perform independent leader-side or spacer-side insertions, although the leader-side insertion was faster than spacer-side. The prespacer morphology requirements for Cas1-Cas2 varied, with G1 stringently requiring a 5-nucleotide 3' overhang and G2 being able to insert many forms of prespacers with variable efficiencies. These results highlight the intricacy of protein-DNA sequence interactions within the seemingly similar type II-A integration complexes and provide mechanistic insights into prespacer insertion. These interactions can be fine-tuned to expand the Cas1-Cas2 toolset for inserting small DNAs into diverse DNA targets., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Van Orden et al.)

Prokaryotes deploy CRISPR-Cas-based RNA-guided adaptive immunity to fend off mobile genetic elements such as phages and plasmids. During CRISPR adaptation, which is the first stage of CRISPR immunity, the Cas1-2 integrase complex captures invader-derived prespacer DNA and specifically integrates it at the leader-repeat junction as spacers. For this integration, several variants of CRISPR-Cas systems use Cas4 as an indispensable nuclease for selectively processing the protospacer adjacent motif (PAM) containing prespacers to a defined length. Surprisingly, however, a few CRISPR-Cas systems, such as type I-E, are bereft of Cas4. Despite the absence of Cas4, how the prespacers show impeccable conservation for length and PAM selection in type I-E remains intriguing. Here, using in vivo and in vitro integration assays, deep sequencing, and exonuclease footprinting, we show that Cas1-2/I-E-via the type I-E-specific extended C-terminal tail of Cas1-displays intrinsic affinity for PAM containing prespacers of variable length in Escherichia coli Although Cas1-2/I-E does not prune the prespacers, its binding protects the prespacer boundaries from exonuclease action. This ensures the pruning of exposed ends by exonucleases to aptly sized substrates for integration into the CRISPR locus. In summary, our work reveals that in a few CRISPR-Cas variants, such as type I-E, the specificity of PAM selection resides with Cas1-2, whereas the prespacer processing is co-opted by cellular non-Cas exonucleases, thereby offsetting the need for Cas4., (© 2019 Yoganand et al.)