Type III CRISPR-Cas: beyond the Cas10 effector complex

Abstract

Type III CRISPR-Cas loci encode some of the most abundant, yet complex, immune systems of prokaryotes. They are composed of a Cas10 complex that uses an RNA guide to recognize transcripts from bacteriophage and plasmid invaders. Target recognition triggers three activities within this complex: ssDNA degradation, synthesis of cyclic oligoadenylates (cOA) that act as second messengers to activate CARF-domain effectors, and cleavage of target RNA. This review covers recent research in type III CRISPR-Cas systems that looked beyond the activity of the canonical Cas10 complexes towards: (i) ancillary nucleases and understanding how they provide defense by sensing cOA molecules; (ii) ring nucleases and their role in regulating cOA production; and (iii) CRISPR-associated proteases, including the function of the Craspase complex in a transcriptional response to phage infection.

Keywords: CARF-domain effectors; CRISPR immunity; Cas7-11; ancillary nucleases; cyclic oligoadenylates (cOA); ring nucleases.

Figure 1.. Type III-A CRISPR-Cas immunity.

Cas10 complexes (type III-A, Csm; III-B, Cmr) are activated upon annealing of the crRNA guide and a complementary target RNA, usually a bacteriophage or plasmid transcript (RNAP; RNA polymerase). The HD domain of Cas10 catalyzes the cleavage of ssDNA, including (but not limited to) the non-template strand of the target transcription bubble. On the other hand, the Palm domain of Cas10 converts ATP to cyclic oligoadenylates (cOA), the most common being cA₄ and cA₆. cOA binds to CARF domains present in type III-associated ancillary effectors. For the most common effectors associated with type III-A and III-B systems, Csm6 and Csx1, respectively, the CARF domain is fused to a HEPN domain with RNase activity. Ligand binding by the CARF domain leads to the activation of the HEPN domain and the degradation of both host and invader transcripts. This activity causes a growth arrest of the host and prevents the replication of the invader. Recently characterized CARF effectors with different effector domains have other activities. Finally, ring nucleases hydrolyze cOA molecules to linear adenylates to deactivate Csm6 and Csx1.

Figure 2.. Structures of Type III CRISPR-associated effectors, bound to cOA substrates, covered in this review.

Monomer units of homomeric complexes are shown in green, blue, and light yellow. The cOA ligand is shown in magenta. The described activity and PDB identifier are shown. (A) Thermococcus onnurineus Csm6. (B) Thermus thermophilus Can1. (C) Sulfobacillus thermosulfidooxidans Can2. (D) Treponema succinifaciens Card1. (E) Pseudomonas aeruginosa NucC. (F) Sulfurihydrogenibium sp. CalpL.

Figure 3.. Structures of cOA ring nucleases, bound to their substrates, covered in this review.

Monomers of the different dimeric complexes are shown in red and orange. The cOA ligand is shown in magenta. The PDB identifier is shown. (A) Saccharolobus solfataricus Crn1. (B) Sulfolobus islandicus rod-shaped virus 1 AcrIII-1 (Crn2). (C) Archaeoglobus fulgidus Crn3.

Figure 4.. Regulation of immune gene expression by accessory proteases during the type III CRISPR-Cas response.

(A) CalpL protease. CalpL forms a tripartite complex with the anti-σ factor CalpT and the σ factor CalpS. Sequence-specific recognition by the Cas10 complex of an invader’s transcript triggers synthesis of cA₄, which is bound by the CalpL SAVED domain to activate its protease activity. This results in the cleavage of CalpT, which liberates CalpS, presumably to regulate the expression of immune genes. In addition, it was speculated that the proteolysis products of CalpT or of other yet unknown CalpL substrates could induce cell toxicity. (B) Craspase. Type III-E CRISPR loci encode a single crRNA-guided protein, Cas7-11, which binds to the Csx29 protease to form the “Craspase” complex. Annealing of the crRNA with its target promotes a conformational change that activates Csx29, which cleaves the anti-σ factor Csx30. This results in the release of the inhibition on the σ factor CASP-σ which is free to activate the transcription if immune genes such as cas1. Furthermore, through an unknown mechanism, the proteolytic fragments of Csx30 can cause cell toxicity as an additional layer of defense.